CN115887471B

CN115887471B - Application of adefovir in preparing medicine for treating skin fibrosis diseases

Info

Publication number: CN115887471B
Application number: CN202211366550.6A
Authority: CN
Inventors: 杨诚; 谷小婷; 李霄鹤; 张建伟; 陈莉; 郭晓薇; 高劭妍; 肖婷; 周红刚
Original assignee: Nankai University
Current assignee: Nankai University
Priority date: 2022-11-01
Filing date: 2022-11-01
Publication date: 2024-02-13
Anticipated expiration: 2042-11-01
Also published as: CN115887471A

Abstract

The invention provides application of adefovir or pharmaceutically acceptable salts, esters and hydrates thereof in preparing medicines for treating skin fibrosis diseases. The invention shows that the adefovir has the effects of slowing down and treating the skin fibrosis, has good application prospect in the aspect of developing medicaments for resisting the skin fibrosis diseases, and provides a new medicament development direction for treating, relieving and improving the skin fibrosis diseases.

Description

Application of adefovir in preparing medicine for treating skin fibrosis diseases

Technical Field

The invention belongs to the technical field of biological medicines, and relates to a new application of adefovir, in particular to an application of adefovir in preparing a medicament for treating skin fibrosis diseases.

Background

Skin fibrosis is a connective tissue disease, a pathological consequence of abnormal repair of deep damaged dermis of skin, and can occur in various pathological processes, such as immune disease scleroderma (SSc), keloid (keloid) caused by abnormal wound healing, hypertrophic Scars (HS), etc. The appearance of skin fibrosis can cause psychological trouble to the patient, and the problems of pain, dysfunction and the like caused by the skin fibrosis seriously affect the life quality of the patient.

The skin is composed of epidermis, dermis and subcutaneous tissue, the epidermis is composed of multiple layers of cells, the dermis is mainly composed of an elastic microfiber network of fibrin (collagen and elastin), bottom adhesion molecules (fibronectin) and proteoglycans interweave, and various types of cells such as fibroblasts, endothelial cells, mast cells and the like are scattered therein. Fibroblasts are considered to be the most important effector cells in the pathogenesis of skin fibrosis, and skin injury, inflammation, infection and immune dysfunction cause imbalance in fibroblast activation, hypersecretion of extracellular matrix, and invasion of surrounding normal skin tissues, accompanied by inflammatory factor infiltration and cytokine production in large amounts, thereby causing skin thickening. Hypertrophic scars and keloids occur after skin injuries (e.g., surgery, chemical burns, scratches, mosquito bites, etc.), and also occur naturally after allergic reactions. The hypertrophic scar is limited to the original wound site and spontaneously subsides, and a large number of contractile myofibroblasts attach to the extracellular matrix through focal adhesion-like structures. Keloids extend beyond the wound edge to the surrounding skin, the inflammatory response is strong, dermal fibroblasts proliferate actively, and angiogenesis is active. Epidemiological data shows that about 1 million people per year have scars after surgery, and that burn wounds that fail to heal within 21 days have a risk of deterioration of the hyperplastic scars of 70% or more. Keloid morbidity is higher in the colored population than in the white population, with 6-16% morbidity.

Skin fibrosis is a refractory pathology, intraperitoneal injection of steroid hormones has been the gold standard of treatment since the mid 1960 s, but the effect of this treatment is mainly to relieve symptoms, often accompanied by side effects such as peripheral normal skin, fat and muscle atrophy, and osteoporosis. Existing prevention and treatment strategies are mainly focused on reducing inflammation, including scar repair surgery, pressure/freezing/radiation/laser treatment, with high recurrence rate and greater adverse reactions. There are also emerging therapies, including mesenchymal stem cell therapy, autologous fat transfer, local interferon injection, intralesional botulinum toxin injection, etc., which require more research in clinical practice. At present, less satisfactory treatments are coming into clinical practice, as the pathological mechanisms of the different types of skin fibrosis have not yet been elucidated. Therefore, a new potential drug target point is explored, and the development of the drug which has the advantages of confirmed curative effect on skin fibrosis, relative safety and reasonable price has important social and medical significance.

Remdesivir (Remdesivir) is a competitive inhibitor of nucleoside RNA-dependent RNA polymerase (RdRp), whose nucleotide triphosphate product Remdesivir-TP competes with RdRp for substrate ATP and thus can interfere with viral RNA synthesis. The adefovir has a certain inhibiting effect on filoviruses, arenaviruses and coronaviruses, but until the present, no report exists that the adefovir can slow down and treat skin fibrosis caused by non-coronaviruses. The structural formula of the Rede-Sivir is as follows:

disclosure of Invention

In view of the above, the invention aims to provide a new application of the adefovir, in particular to application of the adefovir or the adefovir in preparing medicines for treating skin fibrosis diseases, and the invention shows that the adefovir has the effects of slowing down and treating the skin fibrosis, has good application prospect in the aspect of developing medicines for resisting the skin fibrosis diseases, and provides a new medicine development direction for treating, relieving and improving the skin fibrosis diseases.

In order to achieve the above purpose, the technical scheme of the invention is realized as follows:

in one aspect, the invention provides application of adefovir or pharmaceutically acceptable salts, esters and hydrates thereof in preparing medicaments for treating skin fibrosis diseases.

Further, the skin fibrosis disease is bleomycin-induced skin fibrosis and/or keloid.

Furthermore, the adefovir can improve the related physiological indexes caused by skin fibrosis diseases.

Further, the improvement of the physiological index caused by the skin fibrosis disease is at least one of reduction of the thickness of the dermis layer, reduction of collagen deposition, reduction of the degree of skin fibrosis, reduction of fibrosis-related protein expression and reduction of scar tissue weight.

In another aspect, the invention also provides a medicament for treating skin fibrosis diseases, which comprises the adefovir as an active ingredient or pharmaceutically acceptable salts, esters and hydrates thereof.

Further, the composition also comprises pharmaceutically acceptable auxiliary materials.

Pharmaceutically acceptable auxiliary materials in the invention comprise one or more of a carrier, an excipient and a diluent; some examples of suitable carriers, excipients, and diluents include: lactose, dextrose, sucrose, sorbitol, mannitol, starch, resins, acacia, calcium phosphate, alginate, tragacanth, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water syrup (water syrup), methylcellulose, methyl and propyl parabens, talc, magnesium stearate and liquid paraffin.

Pharmaceutically acceptable auxiliary materials in the invention also comprise: lubricants, wetting agents, emulsifying and suspending agents, preservatives, sweeteners or flavoring agents and the like.

In the application or the medicine, the medicine dosage form is one or more of tablets, capsules, pills, suppositories, aerosols, oral liquid preparations, granules, powder, injections, syrups, wines, tinctures, dew and films.

The method of preparing the active ingredient into the medicine according to the present invention may be prepared by methods well known to those skilled in the art, for example: the active ingredient may be diluted with or encapsulated in a carrier such that it releases the active ingredient immediately, slowly or delay after administration to a subject.

The application or the medicine of the invention is taken orally, injected, implanted, externally applied, sprayed and inhaled.

The effective dose of the Rede-Sivir or pharmaceutically acceptable salts, esters and hydrates thereof serving as an effective ingredient of the application or the medicament is 0.1-50mg/kg/d.

Further, the effective dose of the adefovir or the pharmaceutically acceptable salt, ester and hydrate thereof as the effective ingredient is 10-20mg/kg/d.

The term "treatment" as used herein includes its generally accepted meaning including preventing, inhibiting, ameliorating, and slowing, halting or reversing the progression of the symptoms or intended lesions produced. As such, the present invention encompasses both therapeutic and prophylactic administration.

The term "effective amount" as used herein refers to an amount or dose of an active ingredient that provides a desired effect to a patient being diagnosed or treated by single or multiple administrations of the active ingredient to the patient. The effective amount can be determined by the attending diagnostician as a person skilled in the art by known techniques and by observations made in similar circumstances. In determining an effective amount or dosage of an active ingredient to be administered, the attending diagnostician should consider a variety of factors, including, but not limited to: species of mammal; volume, age, and general health; specific diseases involved; the extent or severity of the disease involved; response of the individual patient; the particular compound being administered; mode of administration; the bioavailability properties of the administered formulation; the selected dosing regimen; concomitant use of drug therapy; as well as other related situations.

Compared with the prior art, the application of the adefovir in preparing the medicine for treating the skin fibrosis diseases has the following advantages:

the invention provides a new application of adefovir, namely an application of adefovir or its pharmaceutically acceptable salts, esters and hydrates in preparing medicines for treating skin fibrosis diseases. The adefovir provided by the invention has good effects on slowing down and treating skin fibrosis, can improve the dermis thickness of patients with skin fibrosis, reduce collagen deposition, lighten the skin fibrosis degree and reduce scar tissue amount, provides a new medicament for treating, relieving or improving skin fibrosis diseases, and has good application prospects.

Drawings

One or more embodiments are illustrated by way of example and not limitation in the figures of the accompanying drawings. The word "exemplary" is used herein to mean "serving as an example, embodiment, or illustration. Any embodiment described herein as "exemplary" is not necessarily to be construed as preferred or advantageous over other embodiments.

FIG. 1A is a graph showing the hydroxyproline content of skin tissues of mice in each group in example 1 of the present invention;

FIG. 1B is a statistical chart of HE staining and dermis thickness of skin tissue of mice in each group in example 1 of the present invention;

FIG. 1C is a graph showing quantitative statistics of Masson staining and collagen content of skin tissue after completion of administration in each group of mice in example 1 of the present invention;

FIG. 1D is a graph showing quantitative statistics of sirius red staining and collagen content of skin tissues after the end of administration in each group of mice in example 1 of the present invention;

FIG. 1E is a graph showing the statistics of the expression level of the fibrosis marker protein alpha-SMA, colI, fn in skin tissues after the end of administration in each group of mice in example 1 of the present invention;

FIG. 2A is a schematic representation of the invention following the end of dosing of groups in a keloid xenograft model of example 2;

FIG. 2B is a graph showing the weight statistics of scars after the end of dosing for each group in the keloid xenograft model of example 2 of the present invention;

FIG. 2C is a statistical chart showing the expression levels of fibrotic markers α -SMA, col1 α1, col3 α1, and Fn in keloids after the end of administration in the keloid xenograft model of example 2 of the present invention.

Detailed Description

Unless defined otherwise, technical terms used in the following examples have the same meaning as commonly understood by one of ordinary skill in the art to which the present invention pertains. The test reagents used in the following examples, unless otherwise specified, are all conventional biochemical reagents; the experimental methods are conventional methods unless otherwise specified.

In addition, numerous specific details are set forth in the following description in order to provide a better illustration of the invention. It will be understood by those skilled in the art that the present invention may be practiced without some of these specific details. In some embodiments, materials, elements, methods, means, etc. well known to those skilled in the art are not described in detail in order to highlight the gist of the present invention.

The present invention will be described in detail with reference to the following examples and drawings.

Example 1

Experiment of the effect of Rede Sivir on bleomycin-induced skin fibrosis in mice

1. Preparation of bleomycin-induced skin fibrosis animal model:

preparation of bleomycin-induced skin fibrosis animal model: the bleomycin-induced skin fibrosis model is the currently commonly used skin fibrosis model. Male C57BL/6J wild-type mice (8-10 weeks old) were dehaired and subcutaneously injected with 100. Mu. L0.5UBLM solution. The specific implementation mode is as follows: mice were divided into control (saline), model (BLM) and adefovir treatment (low and high doses). All mice were dehaired to the same area within a range of about 2.0cm x 2.0cm on the backs, and then 100 μl of physiological saline (0.9% NaCl) solution was injected subcutaneously on the backs of the mice in the NaCl group, likewise 100 μl of 0.5u concentration BLM solution was injected subcutaneously on the backs of the BLM group mice, 100 μl of 0.5ublm and 12.5 μΜ,25 μΜ bleomycin mixture was injected on the backs of the experimental group mice once daily, and after each group of mice was anesthetized on day 21, skin specimens of the back injection area were obtained, and skin pathology changes of the mice were analyzed.

2. Grouping of mice:

the mice were divided into 4 groups, namely, a control group (physiological saline group), a bleomycin model group (BLM group), a low dose of Ruidexivir treatment group and a high dose of Ruidexivir treatment group, each group comprising 6 mice.

3. Group dosing of mice:

the control group (physiological saline group) is that the back of the mice is subcutaneously injected with 100 μl of physiological saline (0.9% NaCl) solution with the same volume as that of the mice in the Rede-Sivir treatment group every day as a control;

mice in the bleomycin model group (BLM group) were subcutaneously injected with 100 μl of 0.5U concentration BLM solution for molding;

the low dose treatment group of adefovir was injected with 100 μl of 0.5ublm and 12.5 μm adefovir mixture at the back of mice;

the high dose treatment group of adefovir was injected with 100 μl of a mixture of 0.5ublm and 25 μm adefovir on the back of mice;

once daily, 21 consecutive days of injection, the corresponding experimental dose of adefovir is converted with reference to the optimal dose reported in the clinic or literature.

4. The detection method comprises the following steps:

skin tissues were taken after 21 consecutive days of injection for detection of the mouse skin fibrosis level, collagen content, and fibroblast activation marker gene in skin tissues.

1) Skin collagen content detection: namely, the hydroxyproline content is measured, after 21 continuous days of injection, the skin tissue (10 mg) of the mice is taken, placed in a 5ml ampere bottle, dried in an oven at 120 ℃, hydrolyzed under the action of hydrochloric acid, the PH is adjusted to 6.5-8.0, residues are filtered, PBS is added to adjust the total volume to 10ml, 50 mu L of sample is taken, 350 mu L of deionized water is added, 200 mu L of chloramine T (ChloramineT) solution is added, the mixture is incubated for 20 minutes at room temperature, 200 mu L of perchloric acid (perchloric acid) is added, the mixture is incubated for 5 minutes at room temperature, and 200 mu L of P-dimethylaminobenzaldehyde (P-DMAB) is added, and the mixture is incubated for 20 minutes at 65 ℃. And measuring the absorbance of the sample at 570nm in 200 mu L to 96 well plates, drawing a standard curve by using the reading of the standard substance, and further obtaining the hydroxyproline concentration Cs of the measured sample according to a formula obtained by the standard curve. The amount of hydroxyproline contained in skin tissue, w=cs×8 (dilution of the sample measured) is converted to the total volume of the sample.

2) Fibrosis level detection: skin tissue sections were pathologically stained and dermis layer thickness and collagen deposition level were calculated.

Pathological staining: skin samples were fixed with 10% formalin, dehydrated, paraffin embedded and cut into 5 μm thick sections. After dewaxing in xylene and rehydration using an alcohol series, the sections were stained with hematoxylin and eosin (H & E), masson trichromat and sirius red. Images were randomly taken using an upright transmission fluorescence microscope and analyzed by Image-ProPlus6.0.

3) Detection of skin fibrosis markers the present study uses real-time fluorescent quantitative PCR experiments to detect changes in the expression level of RNA of skin fibrosis markers.

5. Detection result:

1) Skin collagen content detection results: the hydroxyproline content of the bleomycin model group is obviously increased, the hydroxyproline content of the skin tissue of the mice in the Ruidexivir treatment group is obviously reduced, and the dosage is dependent. This suggests that helcoside Wei Neng inhibits bleomycin-induced collagen synthesis in skin (as shown in fig. 1A).

2) Pathological staining: h & E, massson staining and sirius red staining (scale: 100 μm) were performed on the skin tissue sections of the mice, and quantitative statistics of skin thickness and collagen density were performed on the skin tissue sections, which revealed that the skin thickness and collagen density of the mice in the Rede-sivir treatment group were significantly lower than those of the mice in the bleomycin model group (see FIG. 1B, FIG. 1C, and FIG. 1D).

3) Skin fibrosis marker detection results: skin fibrotic lesions exhibit excessive and dense deposition of collagen fibril bundles within the dermis, which converts into myofibroblasts that are highly proliferative and highly collagen synthesizing. Myofibroblast activation, increased collagen synthesis and decreased degradation, produces an excess of extracellular matrix (extracellular matrix, ECM), with smooth muscle actin (α -SMA) as a key marker of activation, the major constituent proteins of extracellular matrix including collagen type I (collagani, col 1) and Fibronectin (Fibronectin); the present study utilizes real-time fluorescent quantitative PCR experiments to detect changes in the RNA expression levels of the skin fibrosis markers. Experimental results showed that the fibrosis marker protein α -SMA, colI, fn was significantly reduced in mice of the adefovir treatment group (fig. 1E).

1A, 1B, 1C, 1D and 1E, control represents a physiological saline group, BLM represents a bleomycin model group, remdesivir-12.5 mu M represents a low dose treatment group of Remdesivir, and Remdesivir-25 mu M represents a high dose treatment group of Remdesivir; * Represents a significant difference (P-value) between the treatment group and the model group, where: p <0.05,: p <0.01.

Example 2

Experiment of the Effect of Rede Siwei on scar weight and fibrosis related Gene expression in a keloid xenograft mouse model

1. Preparation of keloid xenograft mouse model:

the freshly removed skin tissue is peeled off in an ultra clean bench to remove excess adipose tissue, only the epidermis and dermis layers, and the keloid tissue is cut into slices of about 5X 5mm, with the weight of each tissue mass being 0.08-0.1 g. BALB/c nude mice were anesthetized, cut back approximately 0.5cm, and peeled subcutaneously to form a pouch, and the subcutaneous pouch was implanted with a tissue mass. And establishing a keloid xenograft model about 14 days after operation. The grafts of mice were divided into three groups and were given in situ injections of saline (0.9% nacl) or adefovir for a low and high dose treatment, for two weeks for a total of 6 treatments. The materials are obtained after treatment, the weight of the transplanted scar is evaluated, and further gene expression analysis is carried out.

2. Grouping of mice:

the treatment groups were divided into 3 groups, namely a control group (normal saline group) of Rede-West low-dose treatment group and a Rede-West high-dose treatment group, and 3 grafts were transplanted into each group.

3. Group dosing of mice:

the control group (physiological saline group) was mice injected with 50. Mu.l of physiological saline (0.9% NaCl) solution in situ as a control;

the low dose treatment group of rituximab was injected with 50 μl of physiological saline and 12.5 μM of a mixture of rituximab in the back of mice;

the high dose of adefovir is a mixture of 50 μl saline and 25 μM adefovir injected into the back of mice;

for two weeks, 6 treatments were total. The administration mode of the Ruidexivir is consistent with the current clinical administration mode, and the corresponding dose is converted by referring to the optimal dose reported in clinic or literature.

4. The detection method comprises the following steps:

after 6 treatments, excised transplanted keloid tissues were weighed and further analyzed for fibrosis-related gene expression.

1) Keloid gravimetric analysis: the excised transplanted keloid tissue was analyzed by weighing, taking the ratio of the reduced weight after keloid administration to the initial weight as a reference value.

2) Fibrosis-related gene expression analysis: the present study utilizes a real-time fluorescent quantitative PCR assay to detect changes in the expression level of the skin fibrosis marker RNA in keloids.

5. Detection result:

1) Keloid gravimetric analysis results: tissue weight and volume of the treated group given adefovir decreased (as shown in figures 2A, 2B).

2) Fibrosis-related gene expression analysis results: qPCR analysis showed that adefovir dose-dependently reduced expression of α -SMA, col1 α1, col3 α1 and Fn in keloid xenograft tissues (fig. 2C).

In FIGS. 2A, 2B and 2C, control represents a normal saline group, remdesivir-12.5. Mu.M represents a low dose treatment group of Remdesivir, and Remdesivir-25. Mu.M represents a high dose treatment group of Remdesivir; * Represents a significant difference (P-value) between the treatment group and the model group, where: p <0.05,: p <0.01,: p <0.001,: p <0.0001.

The foregoing description of the preferred embodiments of the invention is not intended to be limiting, but rather is intended to cover all modifications, equivalents, alternatives, and improvements that fall within the spirit and scope of the invention.

Claims

1. Application of Ruidexivir or pharmaceutically acceptable salts, esters and hydrates thereof in preparing medicines for treating skin fibrosis diseases.

2. The use according to claim 1, characterized in that: the skin fibrosis disease is bleomycin-induced skin fibrosis and/or keloid.

3. The use according to claim 1, characterized in that: the physical index of the adefovir for improving skin fibrosis diseases is at least one of reducing dermis layer thickness, reducing collagen deposition, reducing skin fibrosis degree, reducing fibrosis related protein expression and reducing scar tissue weight.

4. A use according to any one of claims 1 to 3, characterized in that: the pharmaceutical dosage form is one of tablet, capsule, pill, suppository, aerosol, granule, powder, injection, syrup, medicated wine, tincture, distillate, and pellicle.

5. A use according to any one of claims 1 to 3, characterized in that: the administration route is one of oral administration, injection, implantation and external application.