CN112915193B

CN112915193B - Application of KP-1 in preparation of medicine for treating chronic lung diseases

Info

Publication number: CN112915193B
Application number: CN202110244998.XA
Authority: CN
Inventors: 周丽丽; 谭慧诗
Original assignee: Southern Hospital Southern Medical University
Current assignee: Southern Hospital Southern Medical University
Priority date: 2021-03-05
Filing date: 2021-03-05
Publication date: 2022-09-13
Anticipated expiration: 2041-03-05
Also published as: CN112915193A

Abstract

The invention relates to the field of medicines, in particular to application of KP-1 polypeptide in preparing a medicine for treating chronic lung diseases. The invention discovers that the small molecular polypeptide (KP-1) has no obvious toxic or side effect in mouse animal experiments, has very good inhibition effect on pulmonary fibrosis induced by bleomycin, can obviously reduce the deposition of interstitial collagen of the lung of a mouse, obviously reduces both type I collagen and fibronectin, and obviously reduces the expression of key factors of a TGF-beta signal pathway, so that the KP-1 polypeptide is expected to be used for treating and/or preventing the pulmonary fibrosis.

Description

Application of KP-1 in preparation of medicine for treating chronic lung diseases

Technical Field

The invention relates to the field of medicines, in particular to application of KP-1 (polypeptide) in preparing a medicine for treating chronic lung diseases.

Background

Chronic lung disease, historically known as bronchopulmonary dysplasia, is a group of pulmonary disorders that arise from a variety of causes. Clinically common chronic lung diseases mainly comprise clinical common diseases such as recurrent respiratory tract infection, allergic rhinitis, chronic cough, bronchial asthma, chronic bronchitis, pulmonary interstitial fibrosis and the like. Pulmonary fibrosis, a chronic interstitial lung disease, is the ultimate outcome of various chronic lung diseases. The pathological features of pulmonary fibrosis are mainly inflammatory injury of alveolar epithelial cells, excessive proliferation of fibroblasts, abnormal deposition of extracellular matrix (ECM), and abnormal repair and remodeling of lung tissues, thereby leading to progressive reduction of lung function and finally death due to respiratory failure.

Currently, treatments for pulmonary fibrosis include: 1) anti-inflammatory therapy, which is performed by using macrolide antibiotics, glucocorticoid and immunosuppressant, wherein the glucocorticoid has anti-inflammatory function, can relieve alveolitis and slow down the progress of pulmonary fibrosis; the macrolide antibiotics have anti-inflammatory and immunoregulatory functions, and can inhibit activity of alveolar macrophage nuclear factor, inhibit expression of lung tissue interleukin-1 beta and transforming growth factor-beta, and remarkably relieve alveolitis symptoms when being treated by hormones such as erythromycin and the like. 2) The anti-fibrosis treatment can be performed by using colchicine, pirfenidone, angiotensin converting enzyme inhibitor and the like, and the colchicine can effectively inhibit the collagen deposition of the lung and reduce the generation of pulmonary fibrosis factors. Pirfenidone inhibits collagen synthesis of transforming growth factor-beta, reduces production of pulmonary extracellular matrix, and blocks mitosis of platelet derived factors, thereby inhibiting the progression of pulmonary fibrosis. 3) Antioxidant therapy, mainly including nicotinic acid, ambroxol, taurine, etc., the nicotinic acid and taurine can regulate the expression of lung cytokines, thereby reducing collagen deposition; ambroxol helps to reduce lung cell damage by scavenging oxygen free radicals in the lung, and inhibits the generation and release of tumor necrosis factor-alpha in the lung, slowing down the process of fibrosis.

However, the pathogenesis of pulmonary fibrosis is not clear, and multiple factors and multiple signal pathway interactions are involved, and there is a certain correlation with factors such as abnormal expression of cytokines, damage and repair of epithelial cells, neovascularization, abnormal expression of matrix metalloproteinases, or oxidative stress. At present, the treatment still has no breakthrough progress, the progress of the course of the pulmonary fibrosis is delayed, the incidence rate of the pulmonary interstitial fibrosis is reduced, and the method is a great challenge and great demand for preventing and treating the respiratory system diseases. Therefore, the method has very important scientific value for research on pulmonary fibrosis occurrence mechanism and effective drug prevention and treatment.

Disclosure of Invention

The present invention is directed to solving at least one of the problems of the prior art. The inventor finds that the KP-1 polypeptide can greatly relieve the pulmonary fibrosis of a bleomycin-induced pulmonary fibrosis model mouse, so that the deposition of interstitial collagen of the lung of the mouse is obviously reduced, the type I collagen and the fibronectin are both obviously reduced, and the expression of key factors of a TGF-beta signal pathway is obviously reduced, therefore, the KP-1 polypeptide is expected to be used for treating and/or preventing the pulmonary fibrosis.

The technical scheme of the invention is shown as follows.

The invention provides an application of KP-1 polypeptide in preparing a medicament for preventing and/or treating chronic lung diseases, wherein the amino acid sequence of the KP-1 polypeptide is shown in SEQ ID NO. 1.

According to some embodiments of the invention, the chronic lung disease is a chronic lung disease characterized by fibrosis.

According to some embodiments of the invention, the chronic lung disease is a drug-induced interstitial lung disease.

According to some embodiments of the invention, the chronic lung disease is pulmonary fibrosis.

According to some embodiments of the invention, the medicament further comprises other active ingredients for treating pulmonary fibrosis; the other active ingredients for treating pulmonary fibrosis can be colchicine, pirfenidone, angiotensin converting enzyme inhibitor, macrolide antibiotics, glucocorticoid, immunosuppressant, etc.

According to some embodiments of the invention, the KP-1 polypeptide is administered at a dose of 0.5-2 mg/kg/d, preferably 1 mg/kg/d.

According to some embodiments of the invention, the medicament may further comprise a pharmaceutical excipient. The pharmaceutic adjuvant is a conventional pharmaceutic carrier in the field, and can be any suitable physiologically or pharmaceutically acceptable pharmaceutic adjuvant; preferably, pharmaceutically acceptable disintegrants, diluents, lubricants, binders, wetting agents, flavoring agents, suspending agents, surfactants or preservatives are included. The disintegrating agent can be corn starch, potato starch, cross-linked polyvinylpyrrolidone, sodium carboxymethyl starch, low-substituted hydroxypropyl cellulose, cross-linked sodium carboxymethyl cellulose, calcium carboxymethyl cellulose, or alginic acid. The diluent may be lactose, sucrose, mannitol, corn starch, potato starch, calcium phosphate, calcium citrate or crystalline cellulose. The lubricant can be superfine silica gel powder, magnesium stearate, calcium stearate, stearic acid, talcum powder or anhydrous silica gel. The binder may be acacia, gelatin, dextrin, hydroxypropyl cellulose, methyl cellulose or polyvinylpyrrolidone. The wetting agent may be sodium lauryl sulfate. The flavoring agent may be aspartame, stevioside, sucrose, maltitol or citric acid. The suspending agent can be acacia, gelatin, methylcellulose, sodium carboxymethylcellulose, hydroxymethylcellulose or aluminum stearate gel. The surfactant may be lecithin, sorbitan monooleate or glyceryl monostearate. The preservative may be methyl paraben or propyl paraben.

According to some embodiments of the present invention, the dosage form of the drug is various dosage forms conventional in the art, preferably in solid, semi-solid or liquid form, and may be an aqueous solution, a non-aqueous solution or a suspension, more preferably a tablet, a capsule, a soft capsule, a granule, a pill, an oral liquid, a dry suspension, a drop pill, an injection or an infusion.

According to some embodiments of the present invention, the mode of administration of the drug may be a mode of administration conventional in the art, including but not limited to injection or oral administration. The injection can be intravenous injection, intramuscular injection, intraperitoneal injection, intradermal injection or subcutaneous injection.

The term "administered dose" as used herein is an amount capable of alleviating or delaying the progression of a disease, degenerative or injurious condition. Depending on the particular disease being treated, as well as other factors including age, weight, health, severity of symptoms, route of administration, frequency of treatment, and whether other medications are concomitant during treatment.

The term "prevention" as used herein refers to the prevention or reduction of the development of chronic lung disease or pulmonary fibrosis following use in the presence of a possible chronic lung disease or pulmonary fibrosis factor.

The term "treating" as used herein refers to reducing the extent of, or curing to normalize, or slowing the progression of chronic lung disease or pulmonary fibrosis.

The invention has the beneficial effects that:

the inventor discovers a new application of the KP-1 polypeptide in preventing and/or treating pulmonary fibrosis related pulmonary diseases through scientific research, and experiments show that the KP-1 polypeptide has no obvious toxic or side effect, has a very good inhibition effect on bleomycin-induced pulmonary fibrosis, can obviously reduce the lung interstitial collagen deposition of mice, obviously reduces both type I collagen and fibronectin, and obviously reduces the expression of key factors of a TGF-beta signal pathway, so that the KP-1 polypeptide is expected to be used for treating and/or preventing pulmonary fibrosis.

Drawings

FIG. 1 is a graph of lung Masson staining of different groups of mice;

FIG. 2 is a graph of immunostaining of fibronectin, lung tissue, from different groups of mice;

FIG. 3 is an immunoblot of lung p-Smad3 and its downstream fibrosis indicator protein levels in different groups of mice.

Detailed Description

The technical solutions of the present invention are further described below with reference to specific examples, but the present invention is not limited to these specific embodiments. The test methods used in the examples are all conventional methods unless otherwise specified; the materials, reagents and the like used are commercially available reagents and materials unless otherwise specified.

KP-1 polypeptide was synthesized by Nanjing Kinshire Biotech, Inc.

Example 1 inhibitory effect of KP-1 polypeptides on pulmonary fibrosis in the mouse Bleomycin (BLM) model 1, experimental animals: c57 mouse, male, body weight 20-22g, SPF grade.

Animals were weighed and numbered, 18 healthy mice weighing 20-22g were selected and randomly divided into 3 groups of 6 animals each. Including blank control group, model group and experimental group.

2. Moulding and grouping

Bleomycin (BLM) is a glycopeptide antibiotic used for the treatment of cancer. Mainly through producing chelate with heavy metal including iron, the latter reacts with oxygen to produce superoxide radical and hydroxyl radical, which causes DNA chain breakage, promotes lipid peroxidation and oxidation of other cell molecules, and produces oxidative stress. Bleomycin is clinically accompanied by adverse reactions of pulmonary fibrosis, and an animal model of pulmonary fibrosis generated by bleomycin has pathological characteristics of clinical patients, so bleomycin is considered as a classical modeling method when being applied to research of pulmonary fibrosis in a large amount since 1974.

1) Blank control group: after the mouse is anesthetized with 1% pentobarbital sodium at the weight of 5 mL/kg at room temperature, the four limbs of the anesthetized mouse are stretched and fixed on an operating table by using an adhesive tape, and the front teeth of the mouse are also fixed by using rubber bands, so that the skin in front of the neck is sufficiently stretched and the operation is convenient. The hair in the area before the neck of the mouse is cut off, the skin in front of the trachea is fully exposed, and the iodophor disinfects the skin in front of the neck for 3 times of trachea instillation: a longitudinal incision of about 0.5cm in length is made along the anterior median line of the neck, about 0.5cm above the sternum angle, and the anterior muscular layer of the trachea is bluntly separated with ophthalmologic forceps to expose the trachea. 50 μ L of physiological saline was intratracheally instilled in the same manner. After the liquid is dripped into the trachea, the mouse is quickly erected and quickly rotated by taking the body as the central axis, so that the physiological saline is uniformly distributed in the two lungs through centrifugal force. And observing whether blood and liquid seep around the mouse trachea, and if so, removing the exudates completely. The anterior cervical skin was aligned, sutured horizontally, and the skin was again disinfected with iodophor. And (4) observing whether abnormal conditions such as swelling, bleeding and the like exist around the incision, and if abnormal breathing and cyanosis of the mice occur, removing the suture in time and carrying out corresponding treatment. After partial sterilization, the markers were verified and placed in the corresponding cages.

2) Model group: the anesthesia and disinfection are performed as above. The four limbs of the anesthetized mouse are stretched and fixed on an operating table by using adhesive tapes, and the front teeth of the mouse are also fixed by using rubber bands, so that the skin in front of the neck is fully stretched and straightened, and the operation is convenient. The hair in the area before the neck of the mouse is cut off, the skin in front of the trachea is fully exposed, and the iodophor disinfects the skin in front of the neck for 3 times of trachea instillation: a longitudinal incision of about 0.5cm in length is made along the anterior median line of the neck, about 0.5cm above the sternum angle, and the anterior muscular layer of the trachea is bluntly separated with ophthalmologic forceps to expose the trachea. Calculating the dosage of the BLM solution according to 5mg/kg, sucking the solution with a corresponding volume by using a pipette, adding the solution into a sterile centrifuge tube, sucking the medicine in the centrifuge tube by using a syringe, fixing an air tube by using ophthalmological forceps on one hand, slowly puncturing the air tube in the direction of the heart by using the other hand-held syringe along the direction parallel to the air tube, pushing about one needle point inclined plane length towards the direction of horizontally sticking the air tube forwards, slightly drawing back the syringe to ensure that the needle channel is smooth and is in the air tube, and then dripping the medicine into the air tube. After the liquid is dripped into the trachea, the mouse is quickly erected and quickly rotated by taking the body as the central axis, so that the medicine is uniformly distributed in the two lungs through centrifugal force. And observing whether blood and liquid seep around the mouse trachea, and if so, removing the exudates completely. The anterior cervical skin was aligned, sutured horizontally, and the skin was again disinfected with iodophor. And (4) observing whether abnormal conditions such as swelling, bleeding and the like exist around the incision, and if abnormal breathing, cyanosis and the like of the mouse occur, removing the suture in time and performing corresponding treatment. After partial sterilization, the markers were verified and placed in the corresponding cages.

3) Experimental groups: the anesthesia and disinfection are performed as above. The four limbs of the anesthetized mouse are stretched and fixed on an operating table by using adhesive tapes, and the front teeth of the mouse are also fixed by using rubber bands, so that the skin in front of the neck is fully stretched and straightened, and the operation is convenient. The hair in the area before the neck of the mouse is cut off, the skin in front of the trachea is fully exposed, and the iodophor disinfects the skin in front of the neck for 3 times of trachea instillation: a longitudinal incision of about 0.5cm in length is made along the anterior median line of the neck, from about 0.5cm above the sternum angle, and the anterior muscular layer of the trachea is bluntly separated with ophthalmologic forceps to expose the trachea. Calculating the dosage of the BLM solution according to 5mg/kg, sucking the solution with a corresponding volume by using a pipette, adding the solution into a sterile centrifuge tube, sucking the medicine in the centrifuge tube by using a syringe, fixing an air tube by using ophthalmological forceps on one hand, slowly puncturing the air tube in the direction of the heart by using the other hand-held syringe along the direction parallel to the air tube, pushing about one needle point inclined plane length towards the direction of horizontally sticking the air tube forwards, slightly drawing back the syringe to ensure that the needle channel is smooth and is in the air tube, and then dripping the medicine into the air tube. After the liquid is dripped into the trachea, the mouse is quickly erected and quickly rotated by taking the body as the central axis, so that the medicine is uniformly distributed in the two lungs through centrifugal force. And observing whether blood and liquid seep around the mouse trachea, and if so, removing the exudates completely. The skin in front of the neck is aligned and sewed horizontally, and the skin is disinfected again by iodophor. And (4) observing whether abnormal conditions such as swelling, bleeding and the like exist around the incision, and if abnormal breathing, cyanosis and the like of the mouse occur, removing the suture in time and performing corresponding treatment. After partial sterilization, the markers were verified and placed in the corresponding cages.

And (4) finishing molding: after 28 days, the mice were anesthetized with 1% sodium pentobarbital (about 0.1-0.2 m1) by intraperitoneal injection, and the mice were waited for complete anesthesia. Opening the abdominal cavity, finding an abdominal aorta, cutting open and bleeding, opening the thoracic cavity, injecting 0.1-0.2 mL of PBS (phosphate buffer solution) solution into the right atrium by using an injector, slightly perfusing and flushing heart and lung tissues, taking the left lung for preparing tissue slices, and storing the right lung in liquid nitrogen for extracting tissue proteins.

3. Pharmaceutical intervention

KP-1 polypeptide water soluble powder is diluted with sterile 0.01mol/L glacial acetic acid solution to working concentration of 11.36 mg/mL. Experimental each component was raised in cages. The blank control group was observed only. The experimental group is implanted with Alzet micro-osmotic pump for 1 week after the tracheal injection, and KP-1 polypeptide with 1mg/kg/d dose is applied for 3 weeks. The model group was controlled by administering only an equivalent amount of 0.01mol/L glacial acetic acid.

4. Observation index

Pathologically, the extracellular collagen deposition in tissue is usually observed by Masson staining. More specifically, tissue fibrosis is often qualitatively, quantitatively observed and evaluated using immunostaining and immunoblotting methods (Western blotting) for fibronectin and type I collagen.

After 4 weeks of rearing, the lungs were harvested and the tissues were fixed with 10% neutral buffered formaldehyde and frozen with liquid nitrogen. After dehydration, embedding, slicing and flaking, the formaldehyde-fixed tissues are respectively stained with sirius red and immunostained with type I collagen and fibronectin. Proteins were extracted after freezing the homogenate and the levels of p-Smad3, p-Smad2 and their downstream fibrosis marker proteins were detected by immunoblotting (Western Blot).

5. Results of the experiment

(1) Deposition of collagen in mouse lung

As shown in fig. 1, which is a Masson staining graph of lungs of different groups of mice, it can be seen that lung tissue structure of the blank control group (CTL) is normal, and in lungs of BLM-induced pulmonary fibrosis model group (BLM) mice, Masson staining shows that a large amount of collagen is deposited in a lung interstitial region, while in the experimental group (BLM/KP1), it can be seen that KP-1 reduces accumulation and deposition of lung interstitial collagen, which indicates that KP-1 polypeptide can improve collagen deposition of pulmonary fibrosis mice.

(2) Changes in mouse Lung tissue fibronectin

As shown in fig. 2, which is an immunostaining graph of fibronectin in lung tissues of mice of different groups, it can be seen that the lung tissue structure of the blank control group (CTL) is normal, and the immunostaining result of fibronectin in the lungs of the BLM-induced pulmonary fibrosis model group (BLM) mice shows that fibronectin accumulation exists in the lung interstitial region, while the experimental group (BLM/KP1) can see that KP-1 reduces the deposition of interstitial fibronectin, indicating that KP-1 polypeptide can significantly reduce the level of fibronectin in the lung tissues of the pulmonary fibrosis mice.

(3) Mouse lung fibrosis index change

FIG. 3 shows immunoblots of levels of lung p-Smad3 and its downstream fibrosis indicator protein in different groups of mice; the A picture is used for detecting the expressions of fibronectin, collagen I and p-Smad3 protein in the lung through Western blot immunoblotting, Western blot results show that the fibronectin, the collagen I and the p-Smad3 protein in a pulmonary fibrosis model group (BLM) are obviously increased by BLM induction, and an experimental group (BLM/KP1) proves that KP-1 can well inhibit the three indexes; the B picture is the result of semi-quantitative analysis, and after statistical analysis, the difference of the fibrosis index of the model group (BLM) is shown to have statistical significance compared with the difference of the blank control group (Ctrl), and the difference of the experimental group (BLM/KP1) is shown to have statistical significance compared with the difference of the model group (BLM).

In conclusion, KP-1 can significantly reduce the deposition of pulmonary interstitial collagen of BLM mice, significantly reduce the expression levels of pulmonary interstitial fibronectin and type I collagen of BLM mice, and significantly inhibit abnormally activated TGF-beta signaling pathway in a pulmonary fibrosis model. Therefore, KP-1 can be a new drug for effectively inhibiting the progress of pulmonary fibrosis.

It will be appreciated by those skilled in the art that the use of the present invention is not limited to the specific applications described above. The invention is also not limited to the preferred embodiments thereof with respect to the specific elements and/or features described or depicted herein. It should be understood that the invention is not limited to the disclosed embodiment or embodiments, but is capable of numerous rearrangements, modifications and substitutions without departing from the scope of the invention as set forth and defined by the following claims.

SEQUENCE LISTING

<110> southern hospital of southern medical university

Application of <120> KP-1 in preparation of medicine for treating chronic lung diseases

<130> 111

<160> 1

<170> PatentIn version 3.5

<210> 1

<211> 29

<212> PRT

<213> Artificial sequence (Artificial sequence)

<400> 1

Phe Gln Gly Thr Phe Pro Asp Gly Phe Leu Trp Ala Val Gly Ser Ala

1 5 10 15

Ala Tyr Gln Thr Glu Gly Gly Trp Gln Gln His Gly Lys

20 25

Claims

The application of the KP-1 polypeptide in the preparation of the drugs for preventing and/or treating chronic lung diseases is characterized in that the amino acid sequence of the KP-1 polypeptide is shown in SEQ ID NO:1, and the chronic lung diseases are pulmonary fibrosis.
2. The use according to claim 1, wherein the KP-1 polypeptide is administered in a dose of 0.5-2 mg/kg/d.
3. The use according to claim 1, wherein the medicament further comprises a pharmaceutical excipient; the pharmaceutic adjuvant comprises a pharmaceutically acceptable disintegrant, a diluent, a lubricant, an adhesive, a wetting agent, a flavoring agent, a suspending agent or a preservative.
4. Use according to claim 1, wherein the medicament is in the form of a solid, semi-solid or liquid.
5. The use according to claim 1, wherein the medicament is in the form of tablets, capsules, granules, oral liquid or injections.
6. The use according to claim 1, wherein the medicament is administered by injection or orally.