CN115192589A

CN115192589A - Application of neoisoliquiritin in preparation of psoriasis treatment medicine

Info

Publication number: CN115192589A
Application number: CN202210819826.5A
Authority: CN
Inventors: 黄芳媛; 王晨光; 刘颖
Original assignee: Dikean Hangzhou Biotechnology Co ltd
Current assignee: Dikean Hangzhou Biotechnology Co ltd
Priority date: 2022-07-13
Filing date: 2022-07-13
Publication date: 2022-10-18

Abstract

The invention belongs to the technical field of biological medicines, and discloses application of novel isoliquiritin in preparing a medicament for treating psoriasis. The invention proves that the new isoliquiritin as the psoriasis treatment medicine can obviously improve the symptoms of rat skin lesions of an imiquimod induced psoriasis model through pharmacodynamic experiments, the erythema scale and inflammatory infiltration are obviously improved, the PASI score is obviously reduced, the pathological changes of each layer of skin at the lesion part are obvious, and the effects are realized by inhibiting the JAK1-STAT3 pathway activation through further research.

Description

Application of neoisoliquiritin in preparation of psoriasis treatment medicine

Technical Field

The invention relates to the technical field of biological medicines, in particular to application of neoisoliquiritin in preparing a medicament for treating psoriasis.

Background

The Neoisoliquiritin is an active substance extracted from liquorice and has the English name of Neoisoliquiritin and the molecular formula of C21H22O9, the molecular weight of 418.395 and CAS:7014-39-3, the structural formula is shown as follows:

psoriasis is a chronic inflammatory skin disease, which is characterized by epidermal keratinization, parakeratosis, inflammatory cell infiltration, blood vessel growth and expansion as main pathological features, and squamous erythema as a typical skin lesion. At present, clinically, external drugs for treating psoriasis are glucocorticoid, antibiotic, calcineurin inhibitor and the like, and the drugs have large side effect or poor curative effect and are easy to rebound after the treatment. Therefore, the development of a novel external anti-psoriasis medicament with less toxic and side effects and better curative effect has important significance.

Disclosure of Invention

Since few studies on pharmacological activities of the novel isoliquiritin monomeric compounds have been made, the disclosures are concentrated on androgen inhibition. At present, no document report of the new isoliquiritin in the aspect of treating skin diseases such as psoriasis and the like exists.

The invention aims to overcome the defects of the background technology and provides application of novel isoliquiritin in preparing a medicament for treating psoriasis. The inventor discovers through pharmacodynamic research that the novel isoliquiritigenin can obviously improve psoriasis-like symptoms of an imiquimod induced psoriasis model mouse, the PASI score is obviously reduced, and further researches discover that the effects are realized by inhibiting JAK1-STAT3 pathway activation.

Further, in some embodiments of the present invention, the amount of neoisoliquiritin in the medicament is 0.001wt% to 10wt%.

Further, in some embodiments of the present invention, the amount of neoisoliquiritin in the medicament is 0.01wt% to 3wt%.

Further, in some embodiments of the present invention, the amount of neoisoliquiritin in the medicament is 0.5wt% to 2wt%.

Further, in some embodiments of the present invention, the amount of neoisoliquiritin in the low-dose and high-dose groups of the neoisoliquiritin drug is 0.5wt% and 2wt%, respectively, and the other component in the drug is water.

Further, in some embodiments of the present invention, the psoriasis treatment medicament is an external preparation.

Further, in some embodiments of the invention, the psoriasis treatment medication is a cream, ointment, gel, or aqua.

Further, in some embodiments of the present invention, the psoriasis treatment medicament is an external preparation, including but not limited to cream, ointment, gel, aqua, and the like.

Drawings

FIG. 1 shows that neoisoliquiritin significantly improves the symptoms of skin lesions on the back of rats in psoriasis models;

figure 2 is that neoisoliquiritin significantly reduced the psoriasis model rat PASI score, P <0.05, P <0.01 compared to the model control group;

FIG. 3 is the pathological observation result of the skin lesion;

FIG. 4 is a representative photograph of immunohistochemistry at the site of skin lesion (40X);

FIG. 5 is a histogram of quantitative analysis of protein expression of JAK1-STAT3 signal pathway in skin lesion sites, compared with a normal control group, ^# P<0.05， ^## P<0.01; p compared to model control group<0.05，**P<0.01；

FIG. 6 is a graph showing the effect of neoisoliquiritin on psoriasis treatment in patients with informal clinical trials.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail below with reference to the accompanying drawings and embodiments. Additional aspects and advantages of the invention will be set forth in part in the description which follows and, in part, will be obvious from the description, or may be learned by practice of the invention. It is to be understood that the following description is only illustrative of the present invention and is not to be construed as limiting the present invention.

The terms "comprises," "comprising," "includes," "including," "has," "having," "contains," "containing," or any other variation thereof, as used herein, are intended to cover a non-exclusive inclusion. For example, a composition, process, method, article, or apparatus that comprises a list of elements is not necessarily limited to only those elements but may include other elements not expressly listed or inherent to such composition, process, method, article, or apparatus.

The conjunction "consisting of 8230comprises" excludes any non-specified elements, steps or components. If used in a claim, the phrase is intended to claim as closed, meaning that it does not contain materials other than those described, except for the conventional impurities associated therewith. When the phrase "consisting of 8230is present in a clause of the claim body, rather than immediately after the subject matter, it defines only the elements described in that clause; other elements are not excluded from the claims as a whole.

When an amount, concentration, or other value or parameter is expressed as a range, preferred range, or as a range of upper preferable values and lower preferable values, this is to be understood as specifically disclosing all ranges formed from any pair of any upper range limit or preferred value and any lower range limit or preferred value, regardless of whether ranges are separately disclosed. For example, when the range "1 to 5" is disclosed, the described range should be interpreted to include the ranges "1 to 4", "1 to 3", "1 to 2 and 4 to 5", "1 to 3 and 5", and the like. When a range of values is described herein, unless otherwise stated, the range is intended to include the endpoints thereof and all integers and fractions within the range.

The singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. "optional" or "any" means that the subsequently described event or events may or may not occur, and that the description includes instances where the event occurs and instances where it does not.

Approximating language, as used herein in the specification and claims, is intended to modify a quantity, such that the invention is not limited to the specific quantity, but includes equivalent parts that are acceptable for use in a generic sense without departing from the spirit and scope of the invention. Accordingly, the use of "about" to modify a numerical value means that the invention is not limited to the precise value. In some instances, the approximating language may correspond to the precision of an instrument for measuring the value. In the present description and claims, range limitations may be combined and/or interchanged, including all sub-ranges contained therein if not otherwise stated.

The indefinite articles "a" and "an" preceding an element or component of the invention are not intended to limit the number requirement (i.e., the number of occurrences) of the element or component. Thus, "a" or "an" should be read to include one or at least one, and the singular form of an element or component also includes the plural unless the number clearly indicates only the singular.

Furthermore, the description below of the terms "one embodiment," "some embodiments," "an example," "a specific example," or "some examples," etc., means that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the invention. In this specification, the schematic representations of the terms used above are not necessarily for the same embodiment or example. Further, the technical features of the embodiments of the present invention may be combined with each other as long as they do not conflict with each other.

In a specific embodiment of the present invention, the amounts of neoisoliquiritin in the low-dose and high-dose groups of the neoisoliquiritin drug are 0.5wt% and 2wt%, respectively, and the other component in the drug is water.

Example 1

Construction of imiquimod-induced psoriasis rat model

The rat psoriasis-like skin lesion animal model induced by imiquimod is similar to the expression of human psoriasis in the aspects of skin lesion morphology, histopathological structure, immune cell function, cytokine expression and the like. Therefore, the compound is used as a test animal model for evaluating the medicine for treating psoriasis.

Taking 48 SPF SD rats with half male and female, weight of 180-220g, purchased from Shanghai Jihui laboratory animal feeding company Limited [ SCXK (Shanghai) 2017-0012 ], and animal quality certification numbers are: 20170012014802. raised in animal experimental research center of Zhejiang university of traditional Chinese medicine, license for use of facility in barrier environment [ SYXK (Zhe) 2021-0012 ], raising environment: temperature: 23 ± 1 ℃, humidity: 50-70%, illumination: 150 to 200lx,12 hours of alternating light and shade (early 7; noise: <50dB; an environment automatic control system is arranged in the water dispenser, and water can be freely drunk. Rats were depilated with depilatory cream to remove back hair, applying a suitable amount of 5% imiquimod cream once a day for one week continuously per cage of 4 rats. The successful molding is shown by the appearance of scurf, keratinocyte hyperplasia and skin ulceration. The method referred to the Psoriasis Area and Severity Index (PASI) criteria was modified according to the characteristics of the rat Psoriasis model and scored from three aspects:

TABLE 1 PASI scoring criteria

According to the above standards, the total score of each rat was calculated and evenly divided into a model control group, a new isoliquiritin low dose group, a new isoliquiritin high dose group and a calcipotriol group (positive control), 8 of them were taken as normal control groups.

Example 2

The new isoliquiritin can remarkably relieve the symptoms of psoriasis skin lesions

Each test group was given sufficient feed and water daily, free diet. The activity and mental status of the animals were observed and recorded daily, and the skin lesion sites were kept photographed (0d, 3d,7d, 14d) for a total of 14 days during the experiment. Erythema, scaling, and infiltration of the skin lesions of rats were observed and photographed.

The rats have no infection, death and the like in the psoriasis molding process. The skin of the back skin preparation area of the normal control group rat is smooth and has no red swelling, scale, erosion and the like; the model control group rats showed invasive erythema, thick silvery-white scales and marked erythema and bleeding spots in the skin preparation area, and the erythema and scales gradually increased with the passage of time.

After the new isoliquiritin low and high dose groups (the dosage of the new isoliquiritin in the medicine in the low dose group and the high dose group is 0.5wt% and 2wt%, and other components in the medicine are water) and the calcipotriol group are administrated, the symptoms of erythema and scale are obviously improved, especially when the medicine is administrated for 14 days, the skin of a rat in the high dose group only has a small amount of bleeding points, the scale and ulceration symptoms almost disappear, and the improvement condition is better than that of the calcipotriol group.

The photographs of the skin lesion during the experiment are shown in figure 1. The experimental results show that the new isoliquiritin can obviously improve symptoms of erythema, scale, cutin thickening and the like of psoriasis-like skin lesions induced by imiquimod.

Example 3

Novel isoliquiritin significantly reduces PASI score of model rats

When administration was carried out at 0d,3d,7d,14d, the PASI score at the skin lesion site of the rat was scored from three sides of plaque hypertrophy, erythema and squamous debris with reference to the method of PASI standard (same as example 1), and statistical analysis was carried out.

As can be seen from the PASI scoring results, the rats in each group before administration have no difference in erythema, scales, plaque uplift and total scoring points; after 3 days of administration, compared with a model control group, the PASI score of the new isoliquiritin high-dose group is remarkably reduced (P < 0.05), and the new isoliquiritin low-dose group and the positive control group have no remarkable difference (P > 0.05); after 7 days of administration, the PASI scores of the new isoliquiritin low and high dose groups were significantly decreased (P <0.05, P-straw 0.01) and those of the calcipotriol group were decreased without significant difference (P > 0.05) compared to the model control group; after 14 days of administration, the PASI scores of the low and high dose groups and the calcipotriol group of the novel isoliquiritin were significantly decreased (P <0.05 and P < -0.01).

The results of the experimental scoring are shown in figure 2. The experimental results show that the new isoliquiritin can significantly reduce the PASI score of the imiquimod-induced psoriasis rats.

Example 4

Novel isoliquiritin obviously improves pathological conditions of injured parts of model rats

After the experiment, the rats were sacrificed for pathological observation. And performing conventional HE dyeing on the skin lesion part. The method comprises the following steps: (1) baking the slices in an oven at 60 ℃ for 2h; (2) xylene I and xylene II are respectively 15min; (3) 10min each of the absolute ethyl alcohol I and the absolute ethyl alcohol II; (4) 95% ethanol, 80% ethanol and 70% ethanol for 2min respectively; (5) washing with tap water for 1min; (6) dyeing with hematoxylin dyeing solution for 5min; (7) flushing with running water for 2min; (8) carrying out alcohol differentiation for 5s by using 0.5% hydrochloric acid; (9) flushing with running water for 1min; (10) returning blue for 20s by 0.5% diluted ammonia water; (11) washing for 3min by running water; (12) dyeing with 0.5% eosin dye liquor for 2min; (13) flushing with running water for 1min; (14) 30s of 80% ethanol and 95% ethanol respectively; (15) absolute ethyl alcohol I and absolute ethyl alcohol II are respectively used for 2min; (16) xylene I and xylene II are respectively carried out for 5min; (17) neutral gum blocking. As a result: the nucleus is blue and the cytoplasm and intercellular substance are pink.

Pathological observation results of skin lesion parts: the normal control group has complete and clear skin tissue epidermis structure, obvious horned layer, thinner multi-layer flat epithelial layer and more regular and compact cell arrangement; collagen fibers in the dermis are staggered, and cytoplasm is red-dyed uniformly; connective tissue and adipose layer structures are clearly visible; the muscle fibers of the subcutaneous muscle layer are arranged regularly, the volume and the size are uniform, the shape of the nucleus is normal, and the nucleoli are distributed around more; no obvious pathological changes are seen. After imiquimod modeling, the multiple layers of flattened epithelial layers can be thickened, cells are disorderly arranged, and the outer layers of the epidermis are covered with a layer of crust skin; collagen fibers in the superficial dermis are necrotic, a small amount of single round and deeply-dyed lymphocytes are infiltrated in the necrotic area, more oblong fibroblasts and fusiform fibroblasts are proliferated, and more erythrocytes are exuded among fibrous tissues when blood emerges in the dermis. After the neoisoliquiritin and the calcipotriol cream are used for treating, the thickening of the stratified squamous epithelium of the skin, the disorder of cell arrangement, the collagen fiber necrosis of the superficial part of the dermis, the lymphocyte infiltration in the superficial part of the dermis, the intradermal hemorrhage and the like are all improved, the neoisoliquiritin can obviously inhibit the local skin injury of rats induced by imiquimod, the high-dose effect is especially obvious, and the effect of the neoisoliquiritin is better than that of the calcipotriol.

The pathological observation result of the skin lesion part is shown in figure 3. The experimental result shows that the new isoliquiritin can obviously improve the pathological condition of the psoriasis skin lesion.

Example 5

Novel isoliquiritin regulates JAK1-STAT3 pathway to play a therapeutic role

Recent studies have shown that signal sensors and activator of transcription 3 (STAT 3) in keratinocytes, which are central regulators of inflammation and immune response, are key participants in the pathogenesis of psoriasis and psoriasis-like inflammation, and activation thereof directly accelerates the progress of psoriasis, JAK1-STAT3 is considered as an important signaling pathway for inflammatory response in psoriatic lesion tissues, and STAT3 has been recognized as a new target for psoriasis treatment.

And detecting the expression of the relevant indexes of the skin lesion part by an immunohistochemical staining method. Cutting wax blocks into 4-micron slices, baking for 2h at 60 ℃, dewaxing paraffin wax slices, washing, sealing with 3% hydrogen peroxide solution for 25min after antigen retrieval to block endogenous peroxidase, organizing pen drawing circles, sealing serum, then dropwise adding JAK1, P-STAT3, keratin17, BCL2 and MCL1 antibody diluent, incubating overnight at 4 ℃, washing with PBS, dropwise adding secondary antibody, incubating for 50min at 37 ℃, diaminobenzidine (DAB) color development, hematoxylin counterstaining cell nuclei, and sealing after transparence. The hematoxylin-stained cell nucleus is blue, and the positive expression of DAB is brown yellow. Sections were scanned with a digital section scanner and analyzed for positive expression Integral Optical Density (IOD) using IPP6.0 software.

The detection result of the skin lesion site immunohistochemical related indexes shows that after IMQ modeling, the expressions of JAK1, P-Stat3 and Keratin17 are obviously increased (P < 0.01), and the expressions of BCL2 and MCL1 are obviously reduced (P < 0.01). After the administration of low and high dose of new isoliquiritin treatment, the expressions of JAK1, P-STAT3 and Keratin17 are obviously reduced (P < 0.01), and the expressions of BCL2 and MCL1 are obviously increased (P <0.05, P < -0.01). The new isoliquiritin can obviously reduce the expression of JAK1, P-STAT3 and Keratin17 of psoriasis rats and increase the expression of BCL2 and MCL 1.

The detection results of immunohistochemical related indexes are shown in figures 4-5. The experimental immunohistochemical result shows that the novel isoliquiritin can inhibit the activation of a JAK1-STAT3 passage and increase the expression of anti-apoptosis proteins BCL2 and MCL1 by regulating the expression of JAK1, P-STAT3, keratin17, BCL2 and MCL1 proteins in the JAK1-STAT3 passage, thereby playing a therapeutic role.

Example 6

Test of hyperkeratosis of rat tail scales

Psoriatic epidermis is characterized by hyperproliferation and hypoplasia. The cornification of the rat tail scale epidermis lacks the process of granular layer formation and has similar characteristics to psoriatic parakeratosis. And the keratinization process of the rat tail scales is similar to that of the epidermis of human psoriasis, so the rat tail scales are used as a test animal model for evaluating a medicament for treating psoriasis. The invention respectively uses the new isoliquiritin bulk drug high-dose groups (other components in the drug are water) to carry out corresponding experiments, the experimental results are shown in the following table, and the results show that the new isoliquiritin can promote the formation of the epidermal granular layer of the tail scales of the mice.

Table 2 effect of neoisoliquiritin on the formation of mouse tail scale epidermal granular layer (male, n =3,

)

note: comparison with the Normal control group, p ^* <0.05,p ^** <0.01。

Table 3 effect of neoisoliquiritin on the formation of epidermal granular layer of tail scales in mice (female, n =3,

)

note: comparison with the Normal control group, p ^* <0.05,p ^** <0.01。

Example 7

Experiment on vaginal epithelium proliferation of female mice

The mouse vaginal epithelium in the estrogen cycle proliferates actively, mitotically proliferates, and cell conversion is accelerated, so that the characteristic of the psoriasis that the epidermis proliferates too fast can be simulated. If the medicine can inhibit the mitosis of the vaginal epithelium of the mouse, the suggestion is that the hyperplasia of the psoriasis epidermis can be inhibited. The experimental result shows that the neoisoliquiritin can obviously inhibit the mitosis of mouse vaginal epithelial cells, and the mitosis index is similar to that of calcipotriol group.

TABLE 4 Effect of Neoisoliquiritin on mouse vaginal epidermal cell mitosis

Note: comparison with model groups, p ^* <0.05,p ^** <0.01。

Example 8

Informal clinical experiment

In order to further research the effect of the neoisoliquiritin on treating psoriasis, the neoisoliquiritin is used as a core effective component to prepare a spray (with the dosage of 1%) and informal clinical experiments are carried out. The experiment was performed on a total of 10 psoriasis patients aged 20-60 years. The patients are not treated by any oral or external medicines within one month before the visit. Patients all use the spray of the invention, the use period is 1-3 months, the patients have no adverse reaction, and the spray has obvious effect after treatment.

Typical cases are as follows: a patient with a yellow people, female, 33 years old, has diagnosed psoriasis for more than 10 years. The skin lesions are distributed at a plurality of positions of elbows, finger joints, toes and the like, the symptoms of scale erythema, inflammatory infiltration and pruritus are obvious, and the psoriasis vulgaris is clinically diagnosed as the psoriasis vulgaris through a skin mirror and pathological sections. No oral or external medicine is used for treatment within 1 month before the visit. The spray is used for external treatment, and is used once in the morning and once in the evening. After one week of use, the inflammation of the skin lesion part is reduced, the scales are reduced, the successive area of the skin lesion is reduced, the quantity of the skin lesion is reduced, the skin lesion basically disappears after three months, and the skin of the skin lesion part is recovered to be normal. The patient in this example was evaluated for curative effect as a cure. Local and systemic adverse reactions do not appear during the administration period. The picture of the patient is shown in figure 6.

It will be understood by those skilled in the art that the foregoing is only a preferred embodiment of the present invention, and is not intended to limit the invention, and that any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the scope of the present invention.

Claims

1. Application of neoisoliquiritin in preparing medicine for treating psoriasis is provided.

2. The use of neoisoliquiritin according to claim 1 for the preparation of a medicament for the treatment of psoriasis, wherein the amount of neoisoliquiritin in the medicament is 0.001 to 10wt%.

3. The use of neoisoliquiritin according to claim 1 for the preparation of a medicament for the treatment of psoriasis, wherein the amount of neoisoliquiritin in the medicament is 0.01 to 3% by weight.

4. The use of neoisoliquiritin according to claim 1 for the preparation of a medicament for the treatment of psoriasis, wherein the amount of neoisoliquiritin in the medicament is 0.5 to 2% by weight.

5. The use of neoisoliquiritin according to claim 1 for the preparation of a medicament for the treatment of psoriasis, wherein the medicament for the treatment of psoriasis is an external preparation.

6. The use of neoisoliquiritin according to claim 1 for the preparation of a medicament for the treatment of psoriasis, wherein the medicament for the treatment of psoriasis is a cream, ointment, gel or aqua.