CN106924271A - Ginseng saponin C-K is used to prepare treatment psoriasis - Google Patents

Abstract

The invention provides the use of ginsenoside CK and its pharmaceutically acceptable solvate or hydrate in the preparation of medicines for treating psoriasis, and the kit containing the ginsenoside CK for treating psoriasis , a pharmaceutical composition, and a method for preparing the pharmaceutical composition.

Description

Ginsenoside C-K is used to prepare medicine for treating psoriasis

Technical field:

The invention provides the application of ginsenoside C-K and its pharmaceutically acceptable solvate or hydrate in the preparation of medicines for treating psoriasis, and the kit containing said ginsenoside C-K for treating psoriasis , a pharmaceutical composition, and a method for preparing the pharmaceutical composition.

Background technique:

"Psoriasis", also known as psoriasis, is a chronic, relapsing, inflammatory disease of the skin with clear edges, erythema, thickened skin, and covered with scales; it is stubborn and easy to relapse. Psoriasis can affect the whole body of the patient, but it is more common on the scalp, extensor sides of the extremities, and the back. The clinical symptoms showed that the patient had erythema, dry scales appeared repeatedly on the erythema, and there were bleeding spots after the scales fell off.

The incidence of psoriasis varies greatly around the world. The incidence rate of Andes Indians and Australian aborigines in America is 0%, while the incidence rate of some populations in northern Europe and Russia is 11%. In the United States, 2.2-2.6% of people suffer from it. China still lacks accurate statistics on this disease, and it is currently reported as 0.2-1.5%. The overall incidence worldwide is about 3%. Even employment and income are problematic as the quality of life of psoriasis patients is severely affected.

The skin is the front line of defense against intrusion or damage. It is composed of many epithelial and immune components, and these immune components constitute the skin-associated lymphoid tissue (SALT). Under pathological conditions, there are strong external stimuli or chronic repeated stimulation. Or due to genetic defects in the body, abnormal reactions will occur. Psoriasis vulgaris is a chronic inflammatory disease of the skin mediated by T cells and dendritic cells that is well understood and recognized by everyone.

Studies have proved that psoriasis is not only affected by genes, but also related to environmental factors. The known environmental factors that can cause the onset of psoriasis include infection, stress, excessive obesity, some drugs, smoking, excessive drinking, Even climate and weather can induce or aggravate the disease in some patients.

The above-mentioned many factors form the typical pathological changes of the skin of patients with psoriasis. The accelerated proliferation of keratinocytes leads to thickening of the skin, the normal differentiation process of keratinocytes cannot occur, the normal granular layer is lost, and the nuclei still retain keratin in the surface epithelium. Type 16 keratin exists throughout the epidermis, and a large amount of keratin exists in the dermis. Neutrophils accumulate in the epidermis, and there are a large number of monocytes in the dermis, mainly myeloid-derived dendritic cells and T cells, while the local skin erythema of psoriasis patients is due to a large number of dilated skin blood vessels .

At present, the commonly used drugs for the treatment of psoriasis can be divided into systemic drugs and topical external drugs. Generally speaking, systemic drugs are often used for patients with moderate and severe psoriasis, while systemic drugs for external use are often used for mild or some moderate patients. Medication, most clinicians are accustomed to using methotrexate (MTX) for the first-line systemic treatment of psoriasis or psoriatic arthritis in patients with psoriasis, and another commonly used drug is cyclosporine Mycocin A, because it can prevent T cell activation and cytokine expression. These two drugs have almost become the gold standard of drugs for the treatment of psoriasis. However, because these drugs are originally cytotoxic anticancer drugs or immunosuppressants, long-term use will inevitably produce serious side effects.

In recent years, due to the joint efforts of many researchers and clinicians, many new therapeutic biological agents and oral systemic targeted small molecule drugs have emerged. Among them, TNF antagonists are the most prominent, such as Etanercept and anti-TNF antibodies aldalimumab, infliximab and other biological products have brought good news to patients with moderate to severe psoriasis; new oral anti-psoriasis small molecule drugs such as: dimethyl transbutadiene Elenate (dimethyl fumaric acid) and apremilast, an inhibitor of phosphodiesterase type 4, can reduce the release of inflammatory cytokines (such as IL-12, IL-23 and TNF), induce anti-inflammatory cytokines (such as IL -10) Generation.

Clinically, Tofacitinib can be used as an oral preparation for patients with moderate and severe psoriasis.

For patients with mild psoriasis, that is, patients with less than 10% skin lesion area, topical administration is usually used. For moderate to severe patients, while using ultraviolet light and systemic therapy, topical medication can also be used as adjuvant therapy. Current topical medications for psoriasis include:

Glucocorticoids: are currently the most commonly used external preparations for patients with psoriasis. Different strengths and dosage forms can be selected according to the size of the skin lesion and the progress of the disease. Glucocorticoids can produce anti-inflammation, anti-proliferation, immunosuppression and vasoconstriction to treat psoriasis, but local use can cause side effects of glucocorticoids, and long-term use will produce tolerance, and after drug withdrawal will lead to side effects. rebound.

Vitamin D3 analogue: its representative drug is calcipotriol, which is currently a commonly used external drug for psoriasis. Its main function is to bind to vitamin D receptors, inhibit the proliferation of keratinocytes, and induce their differentiation. The current gold standard for the treatment of psoriasis. The drug has a slower onset than glucocorticoids, but a longer intermission period than glucocorticoids, but the drug has a transient stimulation to the periphery of the skin lesion and a transient increase in feces and serum.

Vitamin A derivatives such as tazarotene. It stimulates the normalization of keratinocytes, reduces hyperproliferation, and reduces the expression of inflammatory markers. This drug is a commonly used topical drug for psoriasis and has a very good curative effect, but it has a stimulating effect on the skin lesions and surrounding areas. It is strictly forbidden to use this drug during pregnancy, and it should be used with caution in children.

The immunosuppressants tacrolimus and pimecrolimus, both of which belong to calcineurin, are effective in the treatment of psoriasis on the face and flexor sides. Side effects of the drug are skin irritation and itching, and the FDA has issued a black box report on controversial lymphatic disorders, so use with caution.

Tar drugs: This product is indeed effective for some patients with psoriasis, but it will cause coloring of clothes and smell of coal tar. Animal experiments have a risk of carcinogenicity, although it has not been confirmed in humans. Children should be used with caution.

Anthralins: No coal tar-like unpleasant smell and coloring of clothes.

Salicylic acid: It has a certain curative effect, and there is no contraindication dose prescribed by the FDA, but it should be used with caution in large areas, especially for those with liver and kidney insufficiency.

The above drugs are used in combination or intermittently, but under the premise of considering their physical and chemical properties and mechanism of action, better curative effect may be achieved.

Brief description of the invention

The application provides a use of ginsenoside C-K for treating psoriasis. The advantages of using ginsenosides C-K to treat psoriasis are:

Ginsenosides C-K have anti-inflammatory effects, which are effective for psoriasis and rheumatoid arthritis;

Ginsenosides C-K can inhibit the excessive proliferation of keratinocytes and induce their differentiation;

Ginsenosides C-K can inhibit the proliferation and expansion of blood vessels;

Ginsenoside C-K can inhibit the aggregation of some inflammatory cells and the release of inflammatory factors;

The side effects of ginsenoside C-K are very small, even if the dosage far exceeds that of psoriasis, there is no side effect.

Therefore, for the treatment of psoriasis, the method of the present invention is very effective and has no side effects.

According to an invention of the present application, the present application provides the use of ginsenoside C-K and pharmaceutically acceptable solvates or hydrates thereof in the preparation of medicines for treating psoriasis.

According to a preferred embodiment of the present application, the psoriasis comprises psoriasis vulgaris.

According to an invention of the application, the application provides a kit, characterized in that, the kit contains ginsenoside C-K or a pharmaceutically acceptable solvate or hydrate thereof, and the kit is used for the treatment of silver Psoriasis.

According to an invention of the present application, the present application provides a pharmaceutical composition, characterized in that the pharmaceutical composition comprises ginsenoside C-K or a pharmaceutically acceptable solvate or hydrate thereof, and a pharmaceutically acceptable carrier, and the pharmaceutical composition is used for treating psoriasis.

According to an invention of the present application, the present application provides a preparation method of a pharmaceutical composition containing ginsenoside C-K, characterized in that, according to ginsenoside C-K or its pharmaceutically acceptable solvate or hydrate and pharmaceutically The pharmaceutical composition is prepared by mixing acceptable carriers, and the pharmaceutical composition is used for treating psoriasis.

Description of drawings

The accompanying drawings, which are incorporated herein and form a part of this specification, illustrate the invention and, together with the description, further serve to explain the principles of the invention and to enable those skilled in the relevant art to make and use the invention. Embodiments of the present invention are described with reference to the drawings by way of example only.

Figure 1 and Figure 2 show the results of immunohistochemical staining of ginsenoside C-K and blank control for HaCaT cell differentiation; Figure 1 shows the staining results of cells after 48 hours of treatment with 20 μg/ml of ginsenoside C-K; Figure 2 Staining results of blank control cells after 48 hours are shown.

Detailed description of the invention

definition

"Ginsenoside" (Ginsenoside) is a sterol compound triterpene saponin, which is the active ingredient in ginseng. Ginsenosides have a similar basic structure, which is a sterane steroid containing four rings arranged by 30 carbon atoms. Ginsenosides are structurally divided into dammarane type and oleanane type (C type). Dammarane-type ginsenosides are divided into two types: panaxadiol-type (A type) and panaxatriol-type (B-type). Panaxadiol-type (A-type) and panaxatriol-type (B-type) sapogenins belong to tetracyclic triterpenes, while aranane-type (C-type) sapogenins belong to pentacyclic triterpenes.

"Ginsenoside CK" (20(S)-Ginsenoside CK), which belongs to saponins, is a metabolite of diol type (type A) ginsenoside in the human intestine. The molecular formula of ginsenoside CK is C ₃₆ H ₆₂ O ₈ , the molecular weight is 622.87, and it is also referred to as CK in this application. The molecular structure of CK is shown in the following formula:

Modern research on ginsenoside C-K shows that ginsenoside C-K has various activities such as anti-cancer, liver protection, and immune system regulation.

The compounds described herein include anhydrous and unsolvated forms of the compounds, as well as hydrates and solvates of the compounds. The term "solvate" in this application refers to a molecule comprising the compound and one or more molecules of a pharmaceutically acceptable solvent such as ethanol. Where the solvent is water, the solvate is a hydrate.

The term "pharmaceutical composition" means a mixture containing a therapeutically effective amount of one or more of the compounds and pharmaceutically acceptable solvates or hydrates thereof, and other pharmaceutically acceptable carriers. The purpose of preparing the compound into a pharmaceutical composition is to more conveniently administer it to a subject.

The pharmaceutical composition described in the present application can be tablets, pills, capsules, granules, powders, suppositories, powders, ointments, patches, injections, solutions, suspensions, sprays, lotions, drops, wipes, etc. agent.

The term "kit" in this application can be used interchangeably with "kit". The present application discloses a kit comprising a therapeutically effective amount of the therapeutic agent or pharmaceutical composition. According to some embodiments of the present application, the kit further comprises one or more other therapeutic agents.

The term "administration", which can be used interchangeably with "administration" or "administration", refers to administering a certain dose of a compound or pharmaceutical composition to a subject through a suitable administration method.

The "administration method" includes but not limited to oral administration, intravenous administration, intrarespiratory administration, sublingual administration, topical administration, intramuscular administration, intraocular administration, transdermal absorption, gastrointestinal administration, etc. Any administration methods known in the art such as external administration, intraperitoneal administration, vaginal administration, buccal administration, rectal administration, etc. Those skilled in the art will understand that the mode of administration to a subject depends on many factors, including the location of the disease, the age of the subject, the severity of the disease, and the ingredients of the pharmaceutical composition, among others.

The compounds or pharmaceutical compositions described herein can be administered at any time. For example, the compound or pharmaceutical composition can be administered to the subject before, during or after the onset of the disease.

The compounds or pharmaceutical compositions described in this application can be administered once or multiple times. When administering multiple times, it can be administered at any time interval.

The compounds or pharmaceutical compositions described in this application can be administered alone or in combination with other suitable drugs. The combined administration may be, for example, simultaneous or sequential administration with other drugs. When administered sequentially, it may be at any interval.

The term "effective amount" or "therapeutically effective amount" of the compound refers to an amount sufficient to obtain the desired therapeutic effect after administration to a subject. The therapeutic effect is such as effective inhibition of psoriatic conditions. Those skilled in the art will understand that the appropriate dosage of the compound depends on many factors, including but not limited to the weight, age and sex of the subject to be treated, the time of administration, the severity of the disease to be treated, and the like.

The terms "treat" or "treatment" refer to the fact that a disease, disorder or condition in a subject can be "prevented", "prevented", "inhibited", "improved" or "cured" after administration of the drug.

The terms "subject", "subject" or "patient" are used interchangeably in this application and have a broad meaning including human or non-human mammals such as dogs, cats, mice, rats, sheep, pigs , goats, cows, non-human primates; or birds, such as chickens, ducks, geese; including other vertebrates, invertebrates, as well as in vitro tissues, prokaryotic cells or eukaryotic cells, etc. According to some embodiments of the present application, the subject is a mammal. According to some embodiments of the present application, the subject is a primate. According to some embodiments of the present application, the subject is a human.

The term "pharmaceutically acceptable carrier" is a pharmaceutically acceptable component or medium, including but not limited to solvents, excipients, diluents, adjuvants, fillers and the like. Common pharmaceutically acceptable carriers include physiological saline, buffers, sugars, gelatin, starch, Ringer's solution, cellulose, and the like. The pH of the carrier is usually 3-11, preferably 5-9, more preferably 7-8.

The term "half inhibitory concentration (IC ₅₀ )", or "half inhibition rate", refers to the concentration of the inhibitor when the activity of the target substance is inhibited to half during the process of inhibiting the target substance. In general, the stronger the inhibitory ability of an inhibitor, the lower the _IC50 . According to some embodiments of the present application, the _IC50 of the compound described herein is the concentration of the compound when 11β-HSD1 activity is inhibited by 50%.

Unless otherwise stated in this application or clearly contradicted by the context, all methods described in this application can be performed in any suitable order according to the understanding of those skilled in the art.

All patents, patent applications, and literature references cited in this application are hereby incorporated by reference in their entirety into this application to the same extent as if each were individually incorporated by reference. In the event of a conflict between this application and the documents provided herein, the content of this application shall control.

detailed description

This application describes preferred implementation modes and examples, and those skilled in the art can make appropriate changes to the implementation modes and examples described in this application on the basis of reading this application. Therefore, the content claimed in this application includes all equivalent modifications and changes to the subject matter in the claims of this application within the scope permitted by law.

Example 1 : CK Antiproliferative effect on human epidermal cells in vitro

This example illustrates that ginsenosides C-K (also referred to herein as CK for short) have the effect of inhibiting the proliferation of human epidermal cells in vitro. In this example, the CK was administered to the cells by dissolving in dimethyl sulfoxide, the human epidermal cells were human immortalized epidermal cells (HaCat cells), and MTT (3-(4,5-dimethylthiazole -2)-2,5-diphenyl tetrazolium bromide salt, trade name: thiazolium blue) method to detect the survival and growth of cells.

A CK sample (commercially available, Shanghai Standard Biotech Co., Ltd. purity 92%, Lot 3690/20548) was dissolved in dimethyl sulfoxide to obtain a solution with a concentration of 10 mg/ml. Then use PBS for gradient dilution to obtain diluted samples with concentrations of 1000 μg/ml, 100 μg/ml, 10 μg/ml, 1 μg/ml, 0.1 μg/ml, and 0.01 μg/ml. Cisplatin for injection (freeze-dried type) (commercially available, Qilu Pharmaceutical Co., Ltd., batch number 1WA2A1411073B) was used as a positive control drug, and it was prepared at the same concentration as the sample. The diluted samples were added to a flat-bottomed 96-well plate, and human immortalized epidermal cells (HaCat cells, subcultured by Shanghai Research Institute of Pharmaceutical Industry) in logarithmic growth phase were added and cultured for 48 hours. The relative cell viability was detected by the MTT method, and was calculated based on the light absorption value at 492 nm and based on the following formula.

Table 1: Growth inhibitory effect on HaCat cell lines in vitro

compound HaCaT cell IC50(μg/ml) CK >100 Cisplatin 3.12

The experimental results in Table 1 prove that ginsenoside CK has no obvious cytotoxic effect on HaCat cells, that is, it has no obvious effect on inhibiting the proliferation of HaCat cells.

Example 2 : CK Effects on the differentiation of human epidermal cells

This example illustrates the effect of ginsenosides C-K (CK) on the differentiation of human epidermal cells. In this example, the CK was administered to the cells by dissolving in dimethyl sulfoxide, the human epidermal cells were immortalized human epidermal cells (HaCat cells), and the differentiation of the cells was detected by immunohistochemical staining.

HaCat cells were cultured in DMEM medium (containing 10% FBS), and when reaching 80% of the culture flask area, after digestion with 0.25% trypsin, the cell concentration was adjusted to 8×10 ⁴ /ml and transferred into 6-well plates (plate Sterilized glass slides were added in the medium), and the concentration of ginsenoside CK was set to 20 μg/ml, respectively added to the corresponding wells, and a blank control was set, placed in a 37°C cell culture incubator, and applied at 0h and 48h of culture respectively. Anti-keratin 18/19 antibody (provided by the Department of Pathology and Anatomy of the Second Military Medical University of the People's Liberation Army) was used for immunohistochemical staining.

The staining results are shown in Figures 1 and 2. Figure 1 shows the staining results of cells treated with 20 μg/ml ginsenoside C-K for 48 hours; Figure 2 shows the staining results of blank control cells after 48 hours. It can be seen from the figure that brown granules appeared in HaCat cells treated with ginsenoside C-K in Figure 1, indicating that HaCat cells differentiated to form keratin, while there was no keratin formation in the blank control treatment in Figure 2.

Example 3 : CK neovascularization ( Angiogenesis of chick embryo allantoic membrane ) Has inhibitory effect

This example shows that ginsenosides C-K have inhibitory effect on chick embryo neovascularization (chick embryo allantoic membrane angiogenesis). In this embodiment, negative control substance-buffered saline (abbreviated as PBS), positive control substance-sorafenib hydrochloride (abbreviated as Sorafenib), and ginsenosides C-K were administered to chicken embryos respectively.

Sterilize the surgical instruments and filter papers used. Take fresh eggs and incubate continuously for 6 days at 37°C and 50% humidity. On the 6th day, it was identified whether the chicken embryos were viable, and the surviving eggs were taken out for window opening. First use light to locate the position of the allantoic membrane and make a mark, then make a small hole at the end of the egg air sac, place the egg with the allantoic membrane facing upwards, and use the suction ear ball to gently inhale the air sac to collapse the allantoic membrane and the egg. The shell is separated, and then a small window of 1 cm ² is opened above the allantoic membrane with a blade. Buffered saline (PBS for short), sorafenib (4 μg/μl, DMSO as solvent) and ginsenoside CK (20 μg/μl, DMSO as solvent) with a concentration of 1 μg/μl and 2 μg/μl were dropped on the filter paper and air-dried , avoiding the large blood vessels, and gently place the filter paper on the allantoic membrane. Seal the small window with transparent tape, continue to incubate for 48 hours, remove the transparent tape, expand the window and take pictures with a camera. Neovascularization was counted according to the branching points of the vessels in each picture. The results are shown in Table 2.

Calculation method of relative inhibition rate:

In the above relative inhibition rate calculation formula, the control group is the abbreviation of negative control group (or called PBS group).

Table 2: Inhibitory effect of CK on neovascularization of chicken embryos

Positive control substance-Sorafenib is a well-known targeted anticancer drug, which has obvious inhibitory effect on angiogenesis; as can be seen from the results in Table 2, the anti-angiogenic effect of ginsenosides C-K is also very obvious, and its The effect is almost equivalent to that of Sorafenib.

Example 4 : CK Effects on mouse tail squamous epidermal cells

This example shows that ginsenosides C-K can promote the formation of the granular layer of mouse tail cells, and there is a certain dose-dependent relationship.

Adopt 70 mice, be divided into 7 groups randomly, namely normal control group, blank cream group, positive control-calcipotriol group (commercially available, Irish Leo Pharmaceutical Co., Ltd., production batch number EH8304, 15g: 0.75mg, i.e. Contain 0.75mg calcipotriol in 15g emulsifiable paste), test emulsifiable paste ginsenoside C-K 0.5%, 1%, 2%, 4% four dosage groups. Except for the normal control group, before administration, the animals in each group lightly brushed the base of the tail 0.5cm 100 times with a nylon brush, and then smeared the administration (100 μl) on the same site, administered once a day, for 7 consecutive days, and the normal control group Give saline. The mice were killed 1 hour after the last administration, and a strip of skin at the administration site was taken and fixed with 10% formaldehyde solution. Embedded in paraffin, stained with HE, and observed the tail scales of each mouse under a light microscope. Where the epidermis of the scales between the two hair follicle openings has granular layers arranged in rows, it is called scales with granular layers. Count the number of scales with granular layers per 100 scales, and the results are expressed as a percentage. The results are shown in Table 3, and the t-test was used, and the P value representing statistical significance was calculated.

Table 3: The effect of ginsenoside CK on the formation of granular layer of mouse tail scale epidermis (n=10, )

Note: Compared with the normal control group, P*<0.05, P**<0.01; compared with the blank cream group, P#<0.05, P##<0.01.

Table 3 shows that under the test conditions, compared with the normal control group, after 7 days of continuous administration of the blank cream group, the calcipotriol group and the administration groups of different doses of ginsenoside C-K cream, the number of scales in the granular layer is extremely high. obviously increase. And compared with the blank cream group in the calcipotriol group and the administration groups of different doses of ginsenoside C-K cream, the number of scales with granular layer also increased significantly. This result shows that ginsenoside C-K cream can promote the formation of the granular layer of mouse tail cells, and there is a dose-dependent relationship.

Example 5 : CK Effects on guinea pig psoriasis model induced by propranolol hydrochloride

Adopt 70 guinea pigs, except the normal control group, all the other animals are evenly smeared on the skin of the back of both ears with 5% propranolol hydrochloride emulsion (about 200 μ l), twice a day, continuously smeared for four weeks, to obtain propranolol hydrochloride induced guinea pig silver Psoriasis model. After four weeks, it was divided into blank cream group, calcipotriol (same as above) group, test cream-ginsenoside C-K0.5%, 1%, 2%, 4% four doses (same as above) group. In the normal control group, the ears of normal guinea pigs were smeared with normal saline; in the other groups, different drugs (about 200 μl) were smeared on the backs of the ears of both sides of the guinea pigs, twice a day, for 7 consecutive days. The state of the animals was observed during the experiment, and the guinea pigs were sacrificed 1 hour after the last administration. The results are shown in Table 4. The t test was used, and the P value representing statistical significance was calculated.

Table 4: Ginsenoside CK on the ear epidermal thickness induced by propranolol hydrochloride for 7 days in guinea pig psoriasis model ( n=10)

Note: One-Way ANOVA statistical method was used for analysis of variance. Compared with the normal control group, P*<0.05, P**<0.01; compared with the blank cream group, P ^# <0.05, P ^## <0.01.

From the epidermal thickness data in Table 4, it can be seen that under the experimental conditions, the epidermal thickness of the blank cream group was as high as 221.65 μm, which was very significantly different from the normal control group, suggesting that this batch of animal models was successful. Compared with the blank cream group, each dose group of ginsenoside C-K had obvious inhibitory effect on the ear epidermal thickness induced by propranolol hydrochloride in guinea pig psoriasis model given for 7 days.

Claims (5)

1. The use of ginsenoside C-K or its pharmaceutically acceptable solvate or hydrate in the preparation of medicine for treating psoriasis. 2. The use according to claim 1, characterized in that the psoriasis comprises psoriasis vulgaris. 3. A kit, characterized in that the kit contains ginsenoside C-K or a pharmaceutically acceptable solvate or hydrate thereof, and the kit is used for treating psoriasis. 4. A pharmaceutical composition, characterized in that, the pharmaceutical composition comprises ginsenoside C-K or a pharmaceutically acceptable solvate or hydrate thereof, and a pharmaceutically acceptable carrier, the pharmaceutical composition is used for Treat psoriasis. 5. A preparation method of a pharmaceutical composition containing ginsenoside C-K, characterized in that it is prepared by mixing ginsenoside C-K or a pharmaceutically acceptable solvate or hydrate thereof with a pharmaceutically acceptable carrier, the Said pharmaceutical composition is used for treating psoriasis.