CN113908154A - Application of nitazoxanide and its metabolite tizoxanide in anti-psoriasis and psoriasis-like skin inflammation - Google Patents

Abstract

Translated fromChinese

本发明公开了硝唑尼特及其体内代谢物替唑尼特在抗银屑病及银屑病样皮肤炎症中的应用，本发明通过研究发现硝唑尼特(式I)具有抑制西方饮食饲料诱导的ApoE(‑/‑)小鼠皮肤银屑病样皮肤炎症性改变的作用，显著减轻脱毛及皮肤溃疡症状。硝唑尼特吸收后在体内迅速完全代谢为替唑尼特(式II)发挥作用，进一步通过细胞学实验证明硝唑尼特和替唑尼特均具有抑制人类永生化表皮细胞HaCaT细胞增殖的作用。因此，硝唑尼特及其体内代谢物替唑尼特可以应用在制备抗银屑病及银屑病样皮肤炎症药物中。本发明为银屑病及银屑病样皮肤炎症的治疗提供了新的有效的技术手段，具有广阔的应用前景。

The invention discloses the application of nitazoxanide and its metabolite tizoxanide in anti-psoriasis and psoriasis-like skin inflammation. Through research, the invention finds that nitazoxanide (formula I) has the function of inhibiting western diet Feed-induced psoriasis-like skin inflammatory changes in ApoE(‑/‑) mice significantly reduced hair loss and skin ulcer symptoms. After nitazoxanide is absorbed, it is rapidly and completely metabolized into tizoxanide (formula II) to play a role in the body. Further cytological experiments proved that both nitazoxanide and tizoxanide have the ability to inhibit the proliferation of human immortalized epidermal cells HaCaT cells. effect. Therefore, nitazoxanide and its metabolite tizoxanide in vivo can be used in the preparation of anti-psoriasis and psoriasis-like skin inflammation drugs. The invention provides new and effective technical means for the treatment of psoriasis and psoriasis-like skin inflammation, and has broad application prospects.

Description

Application of nitazoxanide and in-vivo metabolite tizoxanide in resisting psoriasis and psoriasis-like skin inflammation

Technical Field

The invention relates to the field of biological medicines, and in particular relates to nitazoxanide and a structure modifier thereof, and application of nitazoxanide in-vivo metabolites tizoxanide and a structure modifier of tizoxanide in resisting psoriasis and psoriasis-like skin inflammation.

Background

Psoriasis is commonly called as psoriasis, is a chronic inflammatory skin disease caused by various factors, has long course of disease, is easy to relapse and seriously affects the quality of life. The psoriasis is characterized by epidermal hyperplasia, hypertrophy of epidermal acanthosis, parakeratosis, inflammatory cell infiltration, etc. At present, mild to moderate psoriasis is usually treated by using local glucocorticoid, and moderate to severe psoriasis usually needs systemic treatment, and the treatments have hormone side effects and the problem of drug toxicity of the systemic treatment, so that the development of novel anti-psoriasis and anti-psoriasis-like skin inflammation medicaments is urgently needed.

Nitazoxanide is an antiparasitic drug for treating cryptosporidium, has good oral absorption and less adverse reaction, is completely metabolized into tizoxanide after being absorbed in vivo to play a role, and cannot be detected in vivo.

Disclosure of Invention

The purpose of the invention is as follows: aiming at the problems in the prior art, the invention provides the application of Nitazoxanide (ortho [ N- (5-nitrothiazol-2-yl) carbamyl ] phenol acetate) or Tizoxanide (Tizoxanide) in preparing the medicines for resisting psoriasis and psoriasis-like skin inflammation, is used for developing novel medicines for resisting psoriasis and psoriasis-like skin inflammation and solves the problems of side effects of the existing hormone medicines and the medicine toxicity of systemic treatment.

The invention also provides a pharmaceutical composition for preventing and/or treating psoriasis and psoriasis-like skin inflammation.

The technical scheme is as follows: in order to achieve the technical purpose, the invention provides the application of nitazoxanide and pharmaceutically acceptable salts thereof in preparing medicines for resisting psoriasis and psoriasis-like skin inflammation; wherein the chemical structural formula of nitazoxanide is shown as formula I:

the in vivo metabolite Tizoxanide (Tizoxanide) of nitazoxanide and the application of the pharmaceutically acceptable salt thereof in preparing psoriasis and psoriasis-like skin inflammation resistance; wherein the chemical structural formula of tizoxanide is shown as formula II:

the nitazoxanide and the pharmaceutically acceptable salt thereof have an inhibiting effect on the proliferation of human immortalized epidermal cell HaCaT cells and are applied to the preparation of the medicines for resisting psoriasis and psoriasis-like skin inflammation.

The tizoxanide and the pharmaceutically acceptable salt thereof have an inhibiting effect on human immortalized epidermal cell HaCaT cell proliferation and are applied to preparation of medicines for resisting psoriasis and psoriasis-like skin inflammation.

The compound nitazoxanide or tizoxanide can be used as a medicinal salt or a medicinal composition consisting of a solvate, and the salt is formed by the compound and metal ions or pharmaceutically acceptable amine or ammonium ions.

Wherein the metal ion comprises sodium, potassium or calcium ion and the amine comprises ethylenediamine or tromethamine.

The pharmaceutical composition for preventing and/or treating psoriasis and psoriasis-like skin inflammation contains the zoxanide or the tizoxanide or the pharmaceutically acceptable salt or solvate thereof as an active ingredient and a pharmaceutically acceptable carrier.

Wherein the composition of the compound and the carrier is capsule, powder, tablet, granule, pill, injection, syrup, oral liquid, inhalant, ointment, suppository or patch.

According to the invention, a western diet feed is established to induce an ApoE (-/-) mouse psoriasis-like model, and nitazoxanide is administered in an oral way, so that the nitazoxanide is found to remarkably improve the skin injury of model animals, relieve the depilation symptom, reduce the incidence of skin injury and improve the skin injury score and pathological changes. The oral administration of nitazoxanide is completely metabolized into a metabolite Tizoxanide (Tizoxanide) in vivo, so the effect of the nitazoxanide on improving skin injury is derived from the effect of Tizoxanide, and the effect of the nitazoxanide and Tizoxanide on the proliferation of human immortalized epidermal cell HaCaT cells is determined to verify the supposition, and both the nitazoxanide and the Tizoxanide have the inhibiting effect on the proliferation of the human immortalized epidermal cell HaCaT cells. Therefore, the invention further provides the application of nitazoxanide and tizoxanide and pharmaceutically acceptable salts thereof or compounds modified on the basis of the nitazoxanide and tizoxanide in preparing medicines for resisting psoriasis and psoriasis-like skin inflammation.

The invention provides a method for preparing nitazoxanide, which is a clinical medicine, and in the research of the effect of nitazoxanide on the metabolism of mouse lipid, the invention surprisingly discovers that the oral administration of nitazoxanide can obviously reduce the inflammatory lesion of mouse skin induced by western diet feed. The mouse skin inflammatory lesion induced by western diet feed is a psoriasis-like skin lesion model, so the invention provides the application of nitazoxanide and a structure modifier thereof, and nitazoxanide in-vivo metabolites tizoxanide and a structure modifier of tizoxanide in resisting psoriasis and psoriasis-like skin inflammation.

Has the advantages that: compared with the prior art, the invention has the following advantages:

the invention provides the application of nitazoxanide and in-vivo metabolite tizoxanide thereof in preparing medicaments for resisting psoriasis and psoriasis-like skin inflammation for the first time, and finds a novel medicament for resisting psoriasis and psoriasis-like skin inflammation. The invention uses the effects of tizoxanide and nitazoxanide on the proliferation of human immortalized epidermal cell HaCaT cells, and the use of nitazoxanide and nitazoxanide has obvious improvement effect on psoriasis-like skin pathology. Nitazoxanide is a clinical antiparasitic drug, has good oral absorption and less adverse reaction, provides a new effective technical means for treating psoriasis and psoriasis-like skin inflammation, and has wide application prospect.

Drawings

FIG. 1 photographs of Western diet feed-induced skin damage in ApoE (-/-) mice. 15 ApoE (-/-) mice were fed Western diet (Western diet, WD), and after 15 weeks, photographs of the animals developed skin lesions. Wild Type (WT) C57BL/6 mice were fed Normal basal diet (Normal diet, ND) for 15 weeks without skin changes.

FIG. 2 is a graph showing the occurrence of skin lesions in various groups of animals as a function of time. Normal basal diet (Normal diet, ND) fed Wild Type (WT) C57BL/6 mice for 15 weeks was a control. ApoE (-/-) + WD group ApoE (-/-) mice were fed Western diet feed (Western diet, WD); ApoE (-/-) + WD +100mg/kg nitazoxanide (Nit) is Western diet feed (Western diet, WD) fed to ApoE (-/-) mice and 100mg/kg nitazoxanide is administered by intragastric administration every day; ApoE (-/-) + WD +200mg/kg nitazoxanide (Nit) was administered to ApoE (-/-) mice on Western diet (Western diet, WD) with 200mg/kg of nitazoxanide administered by gavage daily. All groups had 15 animals per group and the experimental period was 15 weeks. When the skin damage changes, the number of the skin damage is counted no matter how heavy or light the skin damage changes.

FIG. 3 comparison of scores for skin lesions in various groups of animals. Normal basal diet (Normal diet, ND) fed Wild Type (WT) C57BL/6 mice for 15 weeks was a control. ApoE (-/-) + WD group ApoE (-/-) mice were fed Western diet feed (Western diet, WD); ApoE (-/-) + WD +100mg/kg nitazoxanide (Nit) is Western diet feed (Western diet, WD) fed to ApoE (-/-) mice and 100mg/kg nitazoxanide is administered by intragastric administration every day; ApoE (-/-) + WD +200mg/kg nitazoxanide (Nit) was administered to ApoE (-/-) mice on Western diet (Western diet, WD) with 200mg/kg of nitazoxanide administered by gavage daily. All groups had 15 animals per group and the experimental period was 15 weeks. Wilcoxon rank-sum test was used. P <0.05vs WT + ND; # P <0.05vs ApoE (-/-) + WD. Skin damage assessment: the appearance of 1) erythema/hemorrhage, 2) scarring/dryness, 3) edema, 4) desquamation/erosion was scored as 0 (no), 1 (mild), 2 (moderate) and 3 (severe). The sum of the individual scores was used as the dermatitis score.

FIG. 4 shows the pathological staining of skin tissue HE of each group of animals. Normal basal diet (Normal diet, ND) fed Wild Type (WT) C57BL/6 mice for 15 weeks was a control. ApoE (-/-) + WD group ApoE (-/-) mice were fed Western diet feed (Western diet, WD); ApoE (-/-) + WD +100mg/kg nitazoxanide (Nit) is Western diet feed (Western diet, WD) fed to ApoE (-/-) mice and 100mg/kg nitazoxanide is administered by intragastric administration every day; ApoE (-/-) + WD +200mg/kg nitazoxanide (Nit) was administered to ApoE (-/-) mice on Western diet (Western diet, WD) with 200mg/kg of nitazoxanide administered by gavage daily. The experimental time was 15 weeks. It can be seen that the skin HE staining of ApoE (-/-) mice fed with Western diet feed (Western diet, WD) for 15 weeks showed significant hyperplasia of the outer skin, infiltration of inflammatory cells and crusting of the outer skin, and that the pathology was significantly improved in the group given 100mg/kg oral nitazoxanide and 200 mg/kg.

FIG. 5 Nitazoxanide and tizoxanide inhibit human immortalized epidermal cell HaCaT cell proliferation. Cell proliferation was achieved by EdU-488 (Beyoclick)^TM) And (5) measuring by using a cell proliferation detection kit. HaCaT cells incubated with nitazoxanide (1, 5, 10. mu.M) and tizoxanide (1, 5, 10. mu.M) for 24h significantly inhibited cell proliferation. P<0.01vs control.n 6 Nitazoxanide, Nitazoxanide; tizoxanide, Tizoxanide.

Detailed Description

The invention will be further described with reference to specific embodiments, and the advantages and features of the invention will become apparent as the description proceeds. These examples are illustrative only and do not limit the scope of the present invention in any way. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention, and that such changes and modifications may be made without departing from the spirit and scope of the invention.

Example 1

Effect of nitazoxanide on inducing psoriasis-like changes of ApoE (-/-) mice by western diet feed

1 materials and methods

1.1 Experimental animals:

ApoE (-/-) mice, males, 8 weeks of age, body weight: 19-21 g; c57BL/6 mice, male, 8 weeks old, body weight: 19-21g, purchased from Beijing Huafukang Biotech limited.

1.2 feed:

normal standard feed (Normal Diet) was purchased from Australian cooperative feed Co., Ltd, Beijing, Ke.

Western Diet feed (Western Diet, H10141) was purchased from Beijing Huafukang Biotech Ltd containing (g) per kg: 195.00 parts of casein; 3.00 parts of methionine; 50.00 parts of corn starch; maltodextrin 100.00; sucrose 341.00; cellulose 50.00; 10.00 parts of corn oil; anhydrous cream 200.00; mineral blend M100135.00; 4.00 parts of calcium carbonate; vitamin mix V100110.00; choline bitartrate 2.00; 1.50 parts of cholesterol; antioxidant TBHQ 0.04.

1.3 reagent:

nitazoxanide (Nitazoxanide): shanghai hadamard reagent, CAS number: 55981-09-4.

Sodium carboxymethylcellulose (CMC-Na): tianjin, Fuchen chemical reagent factory.

1.4 Experimental methods:

establishment of Western diet feed induced ApoE (-/-) mouse psoriasis sample change model

The mice are fed with free drinking water and standard mouse food for one week. After one week of stable conditions ApoE (-/-) mice began feeding western diet, C57BL/6 wild type mice were fed standard mouse chow and daily weighing recorded the food intake of each group of mice. In addition, the treatment group was a group in which mice were administered western diet and simultaneously administered gavage, and the administration components were nitazoxanide (100mg/kg) and nitazoxanide (200 mg/kg); the ApoE (-/-) model group and the wild type control group were gavaged with 0.5% sodium carboxymethylcellulose as a drug solution for 6 days per week, and the body weight of the mice was weighed and recorded daily, and photographed for the appearance of the skin. After 15 weeks the mice were anesthetized and sacrificed, the skin tissue of the lesion site was taken, fixed with paraformaldehyde, and HE pathological staining was performed.

2. Results

Western Diet feeding ApoE (-/-) mice to induce changes in skin psoriasis is a method of preparing psoriasis models, as described in the literature (Shi Z, Wu X, Yu S, Huynh M, Jena PK, Nguyen M, Wan YY, Hwang ST. short-Term Expo sure to a Western Diet industries by Promoting Accumulation of IL 17A-Producing γ δ T cells. J Invest Dermatols.2020; 140(9):1815 1823.); psoriasis is mainly characterized by abnormal proliferation of skin glial cells, but cannot be completely keratinized and continuously drops off.

In addition, in the experiment of the embodiment, the nitazoxanide is completely metabolized into tizoxanide to play a role after being absorbed in vivo, and the prototype drug nitazoxanide cannot be detected in vivo, so that the tizoxanide experiment is not needed when the nitazoxanide is administered to animals.

Effect of nitazoxanide on inducing psoriasis-like changes of ApoE (-/-) mice by western diet feed

15 ApoE (-/-) mice were fed Western diet (Western diet, WD), and after 15 weeks, photographs of the animals developed skin lesions. Wild Type (WT) C57BL/6 mice were fed Normal basal diet (Normal diet, ND) for 15 weeks without skin changes. A typical photograph is shown in FIG. 1.

The skin lesions of all animals were counted as skin lesions regardless of severity. FIG. 2 is a graph of the incidence of skin damage in various groups of animals as a function of time. It can be seen that the incidence of skin damage is significantly increased in ApoE (-/-) mice fed with Western diet feed (Western diet, WD), while the incidence of skin damage is significantly reduced by nitazoxanide (100mg/kg, 200 mg/kg).

The skin lesions of the animals with skin lesions in each group were further scored and evaluated for skin lesions: 1) erythema/hemorrhage, 2) scarring/dryness, 3) edema, 4) desquamation/erosion, each scored as 0 (none), 1 (mild), 2 (moderate) and 3 (severe), with the sum of the individual scores being taken as the dermatitis score. As shown in FIG. 3, comparing the scores of skin lesions in the animals of each group, it can be seen that the administration of nitazoxanide (100mg/kg, 200mg/kg) significantly improved the score of skin lesions in ApoE (-/-) mice fed Western diet feed (Western diet, WD).

Further performing HE pathological staining on skin tissues of each group of animals, showing that the HE staining of the skin of ApoE (-/-) mice fed with Western diet feed (Western diet, WD) for 15 weeks shows remarkable hyperplasia of the outer skin, inflammatory cell infiltration and crust formation outside the outer skin, and the oral administration of nitazoxanide at a dose of 100mg/kg and 200mg/kg remarkably improves the skin pathology (see figure 4), which indicates that the nitazoxanide can improve the psoriasis-like skin inflammation and can be used as a potential medicament for treating the psoriasis-like skin inflammation.

Example 2

Tezoxanide and nitazoxanide effect on human immortalized epidermal cell HaCaT cell proliferation

1 materials and methods

1.1 cells

HepG2 human hepatoma cells, purchased from qian xin boat biotechnology limited, shanghai, cat #: ZQ0022

1.2 reagents

Tizoxanide (Tizoxanide), available from MedChemExpress, cat #: HY-12687/CS-3893

EdU-488(BeyoClick^TM) Purchased in Biyun.

1.3 Experimental methods

Cell proliferation was achieved by EdU-488 (Beyoclick)^TM) And (5) measuring by using a cell proliferation detection kit.

The method comprises the following steps:

a. 9000 cells/well number were seeded in 96-well plates. And (3) culturing the cells for 24h, and adding nitazoxanide or tizoxanide with corresponding concentration for drug treatment for 24h after the cells reach 60%.

b. EdU was diluted to 20. mu.M working solution with cell culture medium.

c. Add equal volume of preheated EdU working solution at 37 ℃ to 96-well plates and incubate for 5 hours in 37 ℃ incubator.

d. The incubation was removed, washed with PBS and fixed with 4% paraformaldehyde for 15 minutes at room temperature.

e. The fixative was removed and washed 3 times with PBS for 3-5 minutes each.

f. The wash was removed and incubated with 0.3% Triton X-100 for 10-15 minutes at room temperature.

g. The permeate was removed and washed 2 times with PBS, 3-5 minutes each.

h. The Click Reaction Solution was formulated at a ratio of 430:20:1:50 in the order of Click Reaction Buffer: CuSO4: Azide 488: Click Additive Solution, 50 microliters per well. Incubate for 30 minutes at room temperature in the dark.

i. The Click reaction solution was aspirated and washed 3 times with washing solution for 3 to 5 minutes each.

j. Incubate 1X Hoechst 33342 with light at room temperature for 10 min.

k. The Hoechst 33342 solution was aspirated and washed 3 times with wash solutions for 3-5 minutes each.

Pictures were taken with a fluorescence microscope.

2 results

Incubation of HaCaT cells, human epidermal cells, for 24 hours with nitazoxanide (1, 5, 10 μ M) and tizoxanide (1, 5, 10 μ M) significantly inhibited cell proliferation. Representative photographs are shown in FIGS. 5A and C, and statistical results are shown in FIGS. 5B and D. The important characteristic of psoriasis is hyperproliferation of skin keratinocytes without complete keratinization, and continuous desquamation and peeling, and the experiment proves that nitazoxanide and tizoxanide can be used for treating psoriasis by inhibiting glial cell hyperproliferation. In conclusion, both nitazoxanide and tizoxanide have the function of inhibiting the proliferation of glial cells, which indicates that both nitazoxanide and tizoxanide can be effective on psoriasis; however, in the case of whole animal medication, nitazoxanide is completely metabolized to tizoxanide after absorption, that is, it is in the form of tizoxanide when it acts in vivo, and both are effective in terms of pharmaceutical action.

Claims (9)

1. The application of nitazoxanide and pharmaceutically acceptable salts thereof in preparing medicines for resisting psoriasis and psoriasis-like skin inflammation is disclosed, wherein the chemical structural formula of the nitazoxanide is shown as a formula I:

2. the use of tizoxanide and its pharmaceutically acceptable salts in the preparation of medicaments for treating psoriasis and psoriasis-like skin inflammation, wherein the chemical structural formula of tizoxanide is shown in formula II:

3. the use according to claim 1, characterized in that nitazoxanide and the pharmaceutically acceptable salts thereof are used for preparing the anti-psoriasis and psoriasis-like skin inflammation medicament by having an inhibitory effect on the proliferation of human immortalized epidermal cells HaCaT cells.

4. The use according to claim 2, characterized in that the tizoxanide and the pharmaceutically acceptable salts thereof have an inhibitory effect on human immortalized epidermal cell HaCaT cell proliferation and are used for preparing the anti-psoriasis and psoriasis-like skin inflammation drugs.

5. Use according to claim 1 or 2, characterized in that the compound nitazoxanide or tizoxanide is used as a pharmaceutically acceptable salt or as a pharmaceutical composition consisting of solvates.

6. The use according to claim 1 or 2, wherein the pharmaceutically acceptable salt is a salt of the compound with a metal ion or a pharmaceutically acceptable amine or ammonium ion.

7. Use according to claim 6, wherein the metal ions preferably comprise sodium, potassium or calcium ions and the amine comprises ethylenediamine or tromethamine.

8. A pharmaceutical composition for the prevention and/or treatment of psoriasis and psoriasis-like skin inflammation comprising as active ingredient zonisamide according to claim 1 or tizoxanide according to claim 2 or a pharmaceutically acceptable salt or solvate thereof and a pharmaceutically acceptable carrier.

9. The pharmaceutical composition of claim 8, wherein the combination of the compound and the carrier is in the form of a capsule, powder, tablet, granule, pill, injection, syrup, oral liquid, inhalant, ointment, suppository, or patch.

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