CN112274520A

CN112274520A - Application of Rudesiwei in preparation of medicine for treating idiopathic pulmonary fibrosis

Info

Publication number: CN112274520A
Application number: CN202011322200.0A
Authority: CN
Inventors: 杨诚; 周红刚; 李霄鹤
Original assignee: Tianjin Jikun Pharmaceutical Technology Co ltd
Current assignee: Tianjin Jikun Pharmaceutical Technology Co ltd
Priority date: 2020-11-23
Filing date: 2020-11-23
Publication date: 2021-01-29

Abstract

The embodiment of the invention relates to application of Rudexiluwei in preparation of a medicament for treating idiopathic pulmonary fibrosis. The Ruidexiwei in the invention has good efficacy on idiopathic pulmonary fibrosis, has no adverse reaction, and can improve forced vital capacity, inspiratory airway resistance, expiratory airway resistance and dynamic compliance of patients with idiopathic pulmonary fibrosis; reducing lung collagen content in patients with idiopathic pulmonary fibrosis; reduce the area of pulmonary fibrosis or prevent the increase of the area of pulmonary fibrosis.

Description

Application of Rudesiwei in preparation of medicine for treating idiopathic pulmonary fibrosis

Technical Field

The invention relates to the field of biological medicines, in particular to application of Rudexiluwei in preparation of a medicine for treating idiopathic pulmonary fibrosis.

Background

With the increasing environmental pollution problem, the incidence of pulmonary diseases continues to rise. Pulmonary Fibrosis (PF) is a disease characterized by persistent damage to the alveoli, fibroblast proliferation and massive extracellular matrix (ECM) deposition, leading to varying degrees of inflammation and fibrosis of the alveoli and the lung interstitium, and thus to destruction of the lung structure and respiratory failure, and is also called Interstitial Lung Disease (ILD) or Diffuse Parenchymal Lung Disease (DPLD).

The causes of pulmonary fibrosis include physical factors, chemical factors, biological factors and the like, and also include pulmonary fibrosis with unknown causes, such as idiopathic pulmonary fibrosis. Idiopathic Pulmonary Fibrosis (IPF) belongs to the Idiopathic Interstitial Pneumonia (IIP) group in the family of Interstitial Lung Diseases (ILDs), and unlike pulmonary fibrosis of known cause, IPF is one of the most common and most severe chronic interstitial lung diseases of unknown etiology. Pulmonary fibrosis with known causes such as pulmonary fibrosis caused by coronavirus, and the pathogenesis and mechanism mainly include that an organism generates an over-strong immune response to the coronavirus to cause the activation of various immune cells such as lung lymphocytes and the like, so that a large number of cytokines are secreted, and the pulmonary fibrosis is caused by mediating the immune response; and IPF is originated from abnormal repair after recurrent or persistent alveolar epithelial injury, under multiple continuous injuries of known or unknown endogenous and exogenous injury factors, damaged lung epithelial cells start an injury repair mechanism, so that autophagy of cells is reduced, apoptosis is increased, epithelial regeneration repair is insufficient, residual cells are subjected to mesenchymal-like transformation, a fibrosis-promoting phenotype is presented, a large amount of fibrosis-promoting factors are secreted, a fibrosis-promoting microenvironment is formed, fibroblasts are abnormally activated and proliferated, excessive extracellular matrix deposition is generated, fibrous scars and alveoli are formed, an alveolar structure is damaged, the irreversible continuous decline of lung functions is caused, and finally respiratory failure and death are caused. IPF is clinically manifested as progressive dyspnea with irritating dry cough, the condition is constantly progressing, median survival time is about 2.8 years, 5-year survival rate is less than 50%, and patients mostly die from respiratory failure and secondary lung infection.

The only approved drugs currently available for the effective treatment of IPF are pirfenidone and nintedanib. Although these drugs can slow the decline of lung function, they cannot reverse the progress of the disease, and a significant portion of patients have poor response to the treatment, and their specific pharmacological mechanisms have not been fully elucidated. Therefore, a new potential drug target is explored, and the development of the drug which is confirmed in curative effect, relatively safe and reasonable in price for pulmonary fibrosis has important social significance and medical significance.

Reddesivir (Remdesivir) is a competitive inhibitor of nucleoside RNA-dependent RNA polymerase (RdRp) and its nucleotide triphosphate product Remdesivir-TP competes with RdRp for substrate ATP and thus interferes with viral RNA synthesis. The Retgosivir has certain inhibiting effect on the filaggous virus, the arenavirus and the coronavirus, but at present, no reports related to the Retgosivir for slowing and treating the pulmonary fibrosis caused by the non-coronavirus and no reports related to the Retgosivir for slowing and treating the idiopathic pulmonary fibrosis are provided. The structural formula of the Reidesciclovir is as follows:

the information disclosed in this background section is only for enhancement of understanding of the general background of the invention and should not be taken as an acknowledgement or any form of suggestion that this information forms the prior art already known to a person skilled in the art.

Disclosure of Invention

Object of the Invention

The invention aims to provide a new application of Reidcisvir, namely an application of Reidcisvir in preparing a medicament for treating idiopathic pulmonary fibrosis.

Solution scheme

In order to achieve the purpose of the present invention, the embodiment of the present invention provides an application of reidecivir or a pharmaceutically acceptable salt, ester, or hydrate thereof in preparing a medicament for treating idiopathic pulmonary fibrosis.

The embodiment of the invention also provides a medicament for treating idiopathic pulmonary fibrosis, which comprises the effective component of the Reidcisvir or the pharmaceutically acceptable salt, ester and hydrate thereof and the pharmaceutically acceptable auxiliary materials.

The embodiment of the invention also provides a method for treating idiopathic pulmonary fibrosis, which comprises the following steps: a step of administering an effective dose of redexivir or a pharmaceutically acceptable salt, ester, hydrate thereof to a subject in need of treatment of idiopathic pulmonary fibrosis.

In one possible implementation, the treating idiopathic pulmonary fibrosis disease includes one or more of: (1) improving lung function; (2) reducing lung collagen content; (3) reduce the area of pulmonary fibrosis or prevent the increase of the area of pulmonary fibrosis.

In one possible implementation, the improved lung function is improved forced vital capacity, inspiratory airway resistance, expiratory airway resistance, lung dynamic compliance.

In one possible implementation, the idiopathic pulmonary fibrosis disease is caused by bleomycin.

In one possible implementation, the drug for treating idiopathic pulmonary fibrosis is selected from one or more of the following dosage forms: tablet, capsule, pill, suppository, aerosol, oral liquid, granule, powder, injection, syrup, medicated liquor, tincture, distillate, and pellicle. The method for preparing the active ingredients into the medicament in the embodiment of the invention can be prepared by adopting a method known by a person skilled in the art, such as: the active ingredient may be diluted or encapsulated in a carrier such that it releases the active ingredient rapidly, slowly or slowly after administration to a subject,

in one possible implementation mode, the pharmaceutically acceptable auxiliary materials comprise one or more of a carrier, an excipient and a diluent; some examples of suitable carriers, excipients and diluents include: one or more of lactose, dextrose, sucrose, sorbitol, mannitol, starch, resin, acacia gum, 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.

In one possible implementation, the pharmaceutically acceptable auxiliary materials further include: lubricants, wetting agents, emulsifying and suspending agents, preservatives, sweetening or flavoring agents and the like.

In one possible implementation, the mode of administration of the drug for treating idiopathic pulmonary fibrosis comprises: one or more of oral administration, injection, implantation, external application, spraying and inhalation.

In one possible implementation, the subject is selected from a mammal; optionally, the subject is selected from mouse, rat, dog, rabbit, pig, monkey.

In one possible implementation, the effective dose is 0.1-50 mg/kg/d; alternatively 10-20 mg/kg/d.

The term "treating" as used herein includes its generally accepted meaning which includes arresting, preventing, inhibiting, ameliorating, and slowing, stopping or reversing the development of the resulting symptoms or the desired pathology. As such, the invention encompasses both therapeutic and prophylactic administration.

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

Advantageous effects

The embodiment of the invention provides application of Reidesciclovir or pharmaceutically acceptable salts, esters and hydrates thereof in preparing a medicament for treating idiopathic pulmonary fibrosis, wherein the Reidesciclovir or pharmaceutically acceptable salts, esters and hydrates thereof in the invention has good efficacy on idiopathic pulmonary fibrosis, has no adverse reaction, and can improve forced vital capacity, inspiratory airway resistance, expiratory airway resistance and dynamic compliance of patients with idiopathic pulmonary fibrosis; reducing lung collagen content in patients with idiopathic pulmonary fibrosis; reducing or preventing an increase in the area of pulmonary fibrosis; provides good application prospect for treating, relieving or improving pulmonary fibrosis diseases.

Drawings

One or more embodiments are illustrated by the corresponding figures in the drawings, which are not meant to be limiting. The word "exemplary" is used exclusively 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 results of body weight changes during administration to each group of mice in example 1 of the present invention, and FIG. 1B is a graph showing the results of hydroxyproline content in lung tissue after administration to each group of mice in example 1 of the present invention is completed; FIG. 1C is a graph showing quantitative statistics of fibrosis in lung tissue after the end of dosing for each group of mice in example 1 of the present invention; FIG. 1D is a photograph showing the staining of lung tissue sections after completion of administration to each group of mice in example 1 of the present invention. Wherein: NaCl represents a normal saline group, BLM represents a bleomycin group, Nintedanib-100mpk represents a positive drug group, Remdesivir-10mpk represents a Rudexilavir low dose group, and Remdesivir-20mpk represents a Rudexilavir high dose group. Represents the significant difference (P-value) between the administered group and the bleomycin group, wherein: p <0.05, x: p < 0.01.

FIG. 2A is a graph showing the results of forced vital capacity after completion of administration to each group of mice in example 1 of the present invention; FIG. 2B is a graph showing the results of inspiratory airway resistance after completion of administration for each group of mice in example 1 of the present invention; FIG. 2C is a graph showing the results of expiratory airway resistance after completion of administration in each group of mice in example 1 of the present invention; FIG. 2D is a graph showing the results of lung dynamic compliance after the end of dosing for each group of mice in example 1 of the present invention. Wherein: NaCl represents a normal saline group, BLM represents a bleomycin group, Nintedanib-100mpk represents a positive drug group, Remdesivir-10mpk represents a Rudexilavir low dose group, and Remdesivir-20mpk represents a Rudexilavir high dose group. Represents the significant difference (P-value) between the administered group and the bleomycin group, wherein: p <0.05, x: p <0.01, x: p <0.001, x: p < 0.0001. # represents a significant difference (P-value) between the redciclovir dosing group and the nintedanib dosing group, wherein #: p <0.05, # #: p < 0.01.

Fig. 3A-3H are graphs showing the results of the expression of pulmonary fibrosis markers after the end of dosing in each group of mice in example 1 of the present invention: wherein: FIG. 3A shows the ratio of the fibrosis marker proteins ColI to GAPDH; FIG. 3B shows the ratio of the fibrotic marker proteins SMA to GAPDH; FIG. 3C shows the change in RNA expression of the fibrosis marker fibrinectin; FIG. 3D shows the RNA expression changes of the fibrosis marker Col 1; FIG. 3E is a graph showing changes in RNA expression of the fibrosis marker α -SMA; FIG. 3F shows the expression distribution (upper panel) and expression statistics (lower panel) of fibrosis marker fibrinectin in tissue sections; FIG. 3G shows the expression distribution (upper panel) and expression statistics (lower panel) of the fibrosis marker Col1 in tissue sections; FIG. 3H shows the expression distribution (upper panel) and expression statistics (lower panel) of the fibrosis marker α -SMA in tissue sections; NaCl represents a normal saline group, BLM represents a bleomycin group, Nintedanib-100mpk represents a positive drug group, Remdesivir-10mpk represents a Rudexilavir low dose group, and Remdesivir-20mpk represents a Rudexilavir high dose group. Represents the significant difference (P-value) between the administered group and the bleomycin group, wherein: p <0.05, x: p <0.01, x: p < 0.001.

Detailed Description

In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below, and it is obvious that the described embodiments are a part of the embodiments of the present invention, but not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Furthermore, in the following detailed description, numerous specific details are set forth in order to provide a better understanding of the present 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, and the like that are well known to those skilled in the art are not described in detail in order to not unnecessarily obscure the present invention.

Throughout the specification and claims, unless explicitly stated otherwise, the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element or component but not the exclusion of any other element or component.

Example 1: retciclovir for slowing idiopathic pulmonary fibrosis

1. Preparing an animal model: the modelling of idiopathic pneumonia with bleomycin is currently the most used model of idiopathic pulmonary fibrosis. Male C57BL/6J wild type mice (week old 8-10 weeks) were anesthetized by intraperitoneal injection with chloral hydrate at a concentration of 10% by mass in a volume of 0.5ml/100g (body weight) and 2U/kg bleomycin was injected intratracheally and invasively. The specific implementation mode is as follows: the mouse is weighed and recorded after anaesthetizing, the mouse is fixed on an operation table, the neck is disinfected by 70% alcohol, a wound with the length of about 1cm is vertically cut on the neck of the mouse by a scalpel, a micro forceps is used for separating tissues to expose an air pipe, a syringe is inserted into the air pipe from the annular gap of the cartilage of the air pipe to the centripetal end, then a bleomycin physiological saline solution with the volume corresponding to the body weight of the bleomycin physiological saline solution is slowly injected according to the measurement of 2U/kg, and the animal is immediately erected and rotated left and right to enable the liquid medicine to be uniformly distributed in the lung.

2. Grouping of mice: the mice were divided into 5 groups, namely a normal saline group, a bleomycin group, a positive drug group, a ridciclovir low-dose group and a ridciclovir high-dose group, and each group had 5 mice.

3. Mice divided dosing situation:

the normal saline group is a sham operation group which injects normal saline (0.9% NaCl) into the trachea of the mice, and when the sham operation treatment is carried out for 1-14 days, the normal saline group is intraperitoneally injected with the normal saline (0.9% NaCl) with the same volume as that of the drug of the mice in the Rudexiwei group every day as a control;

mice in the bleomycin group, the positive drug group, the Reideciclovir low-dose group and the Reideciclovir high-dose group are molded by adopting a method of intratracheal invasive injection of 2U/kg bleomycin; wherein: the low-dose group and the high-dose group of the Reidesvir are respectively prepared by that 10mg/kg/d (low-dose group) or 20mg/kg/d (high-dose group) of the Reidesvir physiological saline solution is given to the mice by intraperitoneal injection every day when the bleomycin is treated for 1 to 14 days; the bleomycin group mice were injected intraperitoneally daily with normal saline (0.9% NaCl) of the same volume as that used in the ridciclovir group mice as a control; the positive drug group mice were gavaged daily with 100mg/kg/d nintedanib as a control in the same volume as the drug administered to the ruidsivir group mice. The administration mode of the Rudexiwei and the Nindanib is consistent with the current clinical administration mode, and the corresponding dosage is converted by referring to the optimal dosage reported in clinic or literature.

4. The detection method comprises the following steps: and (3) carrying out lung function detection, lung collagen content detection and fibrosis severity detection 14 days after the bleomycin treatment.

Detecting the content of lung collagen: namely hydroxyproline content determination, which means that a mouse is sacrificed on the 14 th day of bleomycin injection, the right lung of the mouse is separated, the mouse is placed into a 5ml ampere bottle and is dried in a 120 ℃ oven, the pH is adjusted to 6.5-8.0 after hydrolysis under the action of hydrochloric acid, residues are filtered, PBS is added to adjust the total volume to 10ml, 50 mul of sample is taken, 350 mul of deionized water is added, 200 mul of chloramine T (chloramine T) solution is added for incubation for 20 minutes at room temperature, 200 mul of perchloric acid (perchloric acid) is added for incubation for 5 minutes at room temperature, and 200 mul of P-dimethylaminobenzaldehyde (P-DMAB) is added for incubation for 20 minutes at 65 ℃. And (3) taking 200 mu L to a 96-well plate to measure the light absorption value of the sample at 570nm, 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 the entire right lung, W, was converted to Cs × 8 (dilution of the sample measured) × total volume of the sample by the following formula.

Detecting the fibrosis severity: the lung tissue sections were stained and the area of pulmonary fibrosis was calculated.

And (3) lung function detection: on day 14 of bleomycin injection, mice (0.5ml/100g) were anesthetized by intraperitoneal injection with 10% chloral hydrate, fixed in the operating table in the supine position, the neck fur was cut open, the trachea was exposed, and was bluntly isolated, an incision was cut at the proximal head of the trachea, the trachea was inserted at the trachea joint of the cannula and fixed with cotton thread, the mice were transferred to the stereograph platform, the ventilator and trachea joint were connected, and pulmonary function index parameters of the mice, including Forced Vital Capacity (FVC), inspiratory airway resistance (inhalation airway resistance), expiratory airway resistance (exhalation airway resistance) and dynamic lung compliance (dynamic compliance), were recorded.

Detecting pulmonary fibrosis markers, namely detecting the protein level expression change of the pulmonary fibrosis markers in lung tissues of various groups of mice by using a Western Blot experiment, detecting the RNA expression level change of the pulmonary fibrosis markers by using a real-time fluorescence quantitative PCR experiment, and detecting the distribution positioning and the expression quantity change of the pulmonary fibrosis markers in lung tissue slices by using an immunohistochemical analysis experiment.

5. And (3) detection results:

1) detection results of lung collagen content: the body weight of the mice in the Rudexi West group does not obviously decrease and slowly increases after self administration, the body weight of the mice in the bleomycin group does not rise after the body weight decreases, the body weight of the mice in the positive medicine group obviously decreases at the initial stage, and the body weight of the mice in the positive medicine group also obviously rises after administration (as shown in figure 1A); in addition, the hydroxyproline content in lung tissues of mice in the reidesavir group was significantly reduced and normalized relative to the bleomycin group, indicating that the reidesavir can reduce the bleomycin-induced collagen content (as shown in fig. 1B).

2) Fibrosis severity test results: the mice lung tissue sections were stained with H & E, Massson (scale: 100 μm), and quantitative statistics of fibrosis on the lung tissue sections revealed that both the degree and area of pulmonary fibrosis in the mice of the Reidesvir group were significantly lower than those in the bleomycin group (as shown in FIGS. 1C and 1D).

3) Lung function test results: compared with bleomycin mice, the ReideWest mice have increased Forced Vital Capacity (FVC) and dynamic lung compliance (dynamic compliance), decreased inspiratory airway resistance (inspiratory airway resistance) and expiratory airway resistance (expiratory airway resistance), and are superior to positive drug groups, wherein the difference between ReideWest and positive drug Nidinib in forced vital capacity, inspiratory airway resistance and dynamic lung compliance is significant. The mice in the high-dose group of the Reidesvir and the control group reach equivalent levels, so that the Reidesvir can improve the lung function of the mice with the idiopathic pulmonary fibrosis. (as shown in FIGS. 2A-D)

4) Pulmonary fibrosis marker detection results: pulmonary fibrosis is a progressive interstitial lung disease characterized by more than one of dermal cell injury, myofibroblast activation and Collagen deposition, with the main pathological features being pulmonary fibroblast activation and extracellular matrix deposition, where the key markers of pulmonary fibroblast activation are smooth muscle actin (α -SMA), the major constituent proteins of extracellular matrix including Collagen type I (Collagen I, Col1) and Fibronectin (Fibronectin); in the research, a Western Blot experiment is used for detecting the protein level expression change of the pulmonary fibrosis marker in lung tissues of various groups of mice, a real-time fluorescence quantitative PCR experiment is used for detecting the RNA expression level change of the pulmonary fibrosis marker, and an immunohistochemical analysis experiment is used for detecting the distribution positioning and the expression quantity change of the pulmonary fibrosis marker in lung tissue slices. After the administration of each group of mice is finished, the fibrosis marker proteins alpha-SMA and ColI of the mice in the Rudesevir group are obviously reduced and are superior to those in the positive medicine group (figure 3A and figure 3B). Simultaneous fluorescent quantitative PCR and immunohistochemistry on mouse lung tissue also demonstrated that ridciclovir reduced the expression of marker proteins α -SMA, ColI and Fn (fig. 3C, 3D, 3E, 3F, 3G, 3H).

Finally, it should be noted that: the above examples are only intended to illustrate the technical solution of the present invention, but not to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some technical features may be equivalently replaced; and such modifications or substitutions do not depart from the spirit and scope of the corresponding technical solutions of the embodiments of the present invention.

Claims

1. Application of Reidesciclovir or pharmaceutically acceptable salts, esters and hydrates thereof in preparing medicines for treating idiopathic pulmonary fibrosis.

2. Use according to claim 1, characterized in that: the treatment of idiopathic pulmonary fibrosis disease includes one or more of: (1) improving lung function; (2) reducing lung collagen content; (3) reduce the area of pulmonary fibrosis or prevent the increase of the area of pulmonary fibrosis.

3. Use according to claim 1, characterized in that: the lung function improvement comprises the improvement of forced vital capacity, inspiratory airway resistance, expiratory airway resistance and lung dynamic compliance.

4. Use according to claim 1, characterized in that: the idiopathic pulmonary fibrosis disease is caused by bleomycin.

5. Use according to claim 1, characterized in that: the medicament for treating the idiopathic pulmonary fibrosis disease is selected from the following group: tablet, capsule, pill, suppository, aerosol, oral liquid, granule, powder, injection, syrup, medicated liquor, tincture, distillate, and pellicle.

6. Use according to claim 1, characterized in that: the administration modes of the medicament for treating the idiopathic pulmonary fibrosis disease comprise: one or more of oral administration, injection, implantation, external application, spraying and inhalation.

7. Use according to claim 1, characterized in that: the effective dose of the effective component of the medicine for treating the idiopathic pulmonary fibrosis or pharmaceutically acceptable salts, esters and hydrates thereof is 0.1-50 mg/kg/d; alternatively 10-20 mg/kg/d.