Application of metformin in preparation of medicine for treating bronchial pulmonary dysplasia of premature infants

Abstract

The invention discloses application of metformin to preparation of a medicine for preventing and / or treating bronchial lung dysplasia of a premature infant. It is found that the metformin can significantly reduce the death rate of bronchial pulmonary dysplasia (BPD) model young rats, reduce lung tissue macrophage and neutrophil infiltration, inhibit lung inflammation damage, prevent pulmonary alveolar septum widening and collagenous fiber deposition, reduce vascular exudation, promote lung tissue microangiogenesis and inhibit lung inflammation damage. And the survival rate and the BPD phenotype of the BPD young rats are obviously improved.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and in particular to use of metformin in preparing a medicine for treating bronchopulmonary dysplasia in premature infants. Background Art

[0002] Bronchopulmonary dysplasia (BPD) is a common pulmonary complication in premature infants. In recent decades, the level of perinatal care has been greatly improved worldwide, and more and more extremely low birth weight infants have survived. However, the incidence of BPD has increased instead of decreased. BPD is not only the main cause of chronic respiratory diseases in infants and young children, but also significantly increases the incidence of long-term respiratory diseases in surviving infants, such as respiratory tract infections in young children, asthma in adolescents, and chronic obstructive pulmonary disease (COPD), bringing a heavy burden to families and society.

[0003] BPD is a multifactorial disease. In addition to premature birth, its high-risk factors also include mechanical ventilation, hyperoxia exposure, perinatal infection, and genetic factors. In recent years, much progress has been made in the pathogenesis of BPD, such as abnormal expression of IL-1 receptor, WNT / β-catenin, and TGF-β / BMP signaling pathways, but these studies have not been successfully transformed into effective clinical prevention and treatment programs, and glucocorticoids are still widely used in children with difficult extubation. However, a large amount of evidence-based medical evidence shows that the use of glucocorticoids is significantly associated with long-term neurological developmental damage in premature infants. It can be seen that the prevention and treatment of BPD in clinical practice is still very scarce. Summary of the invention

[0004] The main purpose of the present invention is to propose the use of metformin in preparing a medicine for bronchopulmonary dysplasia in premature infants.

[0005] In one embodiment, in the drug for preventing and / or treating bronchopulmonary dysplasia of prematurity, metformin may be a pharmaceutically acceptable salt thereof.

[0006] In one embodiment, the use comprises use in the preparation of a medicament having at least one of the following effects:

[0007] Reduce the infiltration of macrophages and neutrophils in lung tissue, inhibit lung inflammatory damage, prevent alveolar septum widening and collagen fiber deposition, reduce vascular exudation and promote microangiogenesis in lung tissue.

[0008] In one embodiment, the dosage of metformin or a pharmaceutically acceptable salt thereof in the medicament is 0.1-4 mg / Kg.

[0009] The present invention proposes metformin, a drug widely used in the treatment of type 2 diabetes, which can significantly reduce the mortality rate of BPD mice, reduce the infiltration of macrophages and neutrophils in lung tissue, inhibit lung inflammatory damage, prevent alveolar septal widening and collagen fiber deposition, reduce vascular exudation, and promote microangiogenesis in lung tissue, thereby significantly improving the survival rate and BPD phenotype of BPD mice. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the structures shown in these drawings without paying creative work.

[0011] Figure 1 A is the logic diagram of the experiment of the present invention;

[0012] Figure 1 B is a bar graph of fibrin deposition quantification in the present invention;

[0013] Figure 2 Quantitative graph of animal body weight (A) and survival rate (B) in the BPD animal model of the present invention;

[0014] Figure 3 The photographs of lung morphology indicators of the animal model of the present invention;

[0015] Figure 4 This is a photo of inflammatory cell infiltration in the lungs of the animal model of the present invention;

[0016] Figure 5 This is a photo of the distribution of collagen fibers and elastic fibers in the lungs of the animal model of the present invention;

[0017] Figure 6 This is a quantitative graph of the anti-inflammatory phenotype of macrophages at different concentrations of metformin in cell experiments.

[0018] The realization of the purpose, functional features and advantages of the present invention will be further explained in conjunction with embodiments and with reference to the accompanying drawings. DETAILED DESCRIPTION

[0019] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0020] It should be noted that if there are descriptions involving "first", "second", etc. in the embodiments of the present invention, the descriptions of "first", "second", etc. are only used for descriptive purposes and cannot be understood as indicating or implying their relative importance or implicitly indicating the number of technical features indicated. Therefore, the features limited to "first" and "second" may explicitly or implicitly include at least one of the features. In addition, if "and / or" or "and / or" appears in the full text, its meaning includes three parallel schemes. Taking "A and / or B" as an example, it includes scheme A, or scheme B, or a scheme that satisfies both A and B. In addition, the technical solutions between the various embodiments can be combined with each other, but it must be based on the ability of ordinary technicians in this field to implement. When the combination of technical solutions is contradictory or cannot be implemented, it should be deemed that such a combination of technical solutions does not exist and is not within the scope of protection required by the present invention.

[0021] Metformin is a drug widely used in the treatment of type 2 diabetes. It has good blood sugar-lowering effects and can slightly reduce weight. It is also safe and well tolerated. Long-term use does not increase the risk of hyperlactatemia or lactic acidosis, provided that contraindications are well understood. Therefore, metformin can be used as a basic therapeutic drug for controlling hyperglycemia. In addition, metformin has a long history of use in the treatment of type 2 diabetes, and its safety and reliability have been demonstrated.

[0022] In the specific course of glucose control, metformin can be taken during meals or immediately after meals. The principle of dosage adjustment is to start with a small dose and gradually increase the dose. The minimum recommended dose of metformin for efficacy is 500 mg / d. If there are no obvious gastrointestinal adverse reactions, the dose can be gradually increased. The optimal effective dose is 2000 mg / d. Metformin has fewer adverse reactions and a lower probability of adverse reactions. In addition, adverse reactions of metformin often occur in the early stages of treatment. Most patients with adverse reactions can usually relieve themselves. Gradually increasing the dose or switching to a sustained-release formulation can improve tolerance. The efficacy of metformin and its adverse reactions have nothing to do with the patient's body mass index.

[0023] The present invention proposes the use of metformin in the preparation of a drug for preventing and / or treating postnatal pulmonary vascular developmental disorders in premature infants.

[0024] Bronchopulmonary dysplasia (BPD) is a common pulmonary complication in premature infants. In recent decades, the level of perinatal care has been greatly improved worldwide, and more and more extremely low birth weight infants have survived. However, the incidence of BPD has increased instead of decreased. BPD is not only the main cause of chronic respiratory diseases in infants and young children, but also significantly increases the incidence of long-term respiratory diseases in surviving infants, such as respiratory tract infections in young children, asthma in adolescents, and chronic obstructive pulmonary disease (COPD), bringing a heavy burden to families and society.

[0025] BPD is a multifactorial disease. In addition to premature birth, its high-risk factors also include mechanical ventilation, hyperoxia exposure, perinatal infection, and genetic factors. In recent years, much progress has been made in the pathogenesis of BPD, such as abnormal expression of IL-1 receptor, WNT / β-catenin, and TGF-β / BMP signaling pathways, but these studies have not been successfully transformed into effective clinical prevention and treatment programs, and glucocorticoids are still widely used in children with difficult extubation. However, a large amount of evidence-based medical evidence shows that the use of glucocorticoids is significantly associated with long-term neurological developmental damage in premature infants. It can be seen that the prevention and treatment of BPD in clinical practice is still very scarce.

[0026] At present, although there are patents and reports on metformin improving lung diseases, the applicable populations of the above-mentioned patents and reports are all adults or adolescents, which are essentially different from the populations and diseases applicable to the present invention. The applicable population of the present invention is premature infants, whose pharmacokinetics and pharmacology are quite different from those of adults; in addition, the applicable disease of the present invention is bronchopulmonary dysplasia, which is caused by premature birth and postnatal ventilator-related injuries, leading to the retardation of the normal lung development process of premature infants, and is essentially different from adult lung diseases in terms of pathogenesis.

[0027] Specifically, although metformin has been reported to be used in related diseases such as pulmonary fibrosis and bronchiectasis, the lung development of premature infants is delayed, so the physiological function of the lungs of premature infants cannot meet the needs of their normal physiological activities; and due to the special nature of premature infants, their body functions are often more fragile than those of normal infants, and the medication for infants and young children is significantly different from that for adults or adolescents; the reason is that in addition to the difference in body functions between infants and young children and adults or adolescents, the differences in their pharmacokinetics and metabolic pathways in the body are the main reasons why the medication regimens for the same disease are so different. Therefore, the existing technical means cannot prove that metformin can be used to treat BPD in premature infants.

[0028] Accordingly, the birth of premature infants is generally accompanied by the assistance of ventilators, which can easily induce BPD, and BPD can easily induce other diseases during the growth and development of premature infants. However, since it is impossible to directly inhibit or treat the onset of BPD through existing drug combinations, how to treat bronchopulmonary dysplasia in premature infants remains difficult.

[0029] The present invention found that a pregnant woman with diabetes gave birth prematurely, and the premature baby was transferred to an incubator and assisted breathing by a ventilator, which induced BPD; the pregnant woman chose breastfeeding, and then the healing speed of the BPD manifestations of the premature baby was significantly faster than that of other premature babies with BPD; the pregnant woman was questioned and it was found that the pregnant woman took a diabetes treatment drug with metformin as the main ingredient after giving birth, and metformin was found in the pregnant woman's breast milk, with a concentration of about 1-40 mg / L.

[0030] Therefore, the present invention verifies that metformin can significantly reduce the mortality rate of BPD mice, and finds that it is possible to use metformin to treat and / or prevent bronchopulmonary dysplasia in premature infants.

[0031] Metformin can significantly reduce the mortality rate of BPD pups. Specifically, metformin can reduce the infiltration of macrophages and neutrophils in lung tissue, inhibit lung inflammatory damage, prevent alveolar septum widening and collagen fiber deposition, reduce vascular exudation, and promote microangiogenesis in lung tissue, significantly improving the survival rate and BPD phenotype of BPD pups.

[0032] In order to verify the specific effect of metformin on BPD model mice, the following is a specific example:

[0033] Construction of BPD mouse model:

[0034] The present invention randomly divides newborn mice (Wistar) into two groups within 12 hours after birth and exposes them to air (Room air, RA) or 100% hyperoxia, respectively. On the first day after birth, each group is divided again: a control group, which receives normal saline as a placebo control; and a treatment group, which receives metformin (manufacturer Sigma-Aldrich, product number D150959) for treatment. There are 6 pups in each group, and the pups receive subcutaneous injections of normal saline or metformin 100 microliters every day from the 1st to the 9th day after birth for 9 consecutive days. The pups are breastfed by surrogate mothers, and the mothers are replaced daily to prevent oxygen poisoning. The oxygen concentration is monitored every day, the animal status is observed, the body weight and mortality rate are recorded, and the animals are killed and samples are collected on the 10th day; the experimental design is as follows: Figure 1 A, the specific grouping is shown in Table 1;

[0035] Table 1. Construction of BPD rat model

[0036]

[0037] For the convenience of subsequent description, in the following text and in the accompanying drawings, with reference to Table 1, the relevant abbreviations are as follows:

[0038] There are four groups in Table 1, which are: the control group in the air group is (RA-), the treatment group in the air group is (RA+), the control group in the 100% hyperoxia group is (O2-), and the treatment group in the 100% hyperoxia group is (O2+).

[0039] Specimen collection:

[0040] On the 10th day after birth, the animals were anesthetized with ketamine (50 mg / Kg) and xylazine (50 mg / Kg) and euthanized. The abdominal skin was cut open to expose the abdominal artery and vein, and the abdominal artery and vein were cut to kill by bleeding. After the blood was drained, some animals were randomly selected from each of the four groups to open the chest cavity, and the lung tissue was directly taken, which was quickly frozen in liquid nitrogen and then stored in a -80° refrigerator for Real-time PCR detection; another batch of experimental animals had their neck skin cut open, the muscle tissue around the trachea separated, the trachea exposed, and the trachea was intubated using a 24G arterial indwelling needle. The surgical thread was tied and fixed, and 4% paraformaldehyde was connected to fix in situ at 25 cmH2O for 6 minutes. The tracheal tube was removed, and the chest cavity was opened along the middle of the sternum. The lung tissue was seen to be expanded. The lung tissue and heart were separated and placed in an EP tube containing 4% paraformaldehyde in a 4°C refrigerator for 24 hours. The sections were embedded in paraffin and used for HE staining and immunohistochemistry.

[0041] Detection of fibrin deposition in lung tissue

[0042] Fibrin deposition in lung tissue homogenates was quantified by Western blotting. Tissue samples were dissolved in reducing sample buffer (containing 10 mM Tris pH 7.5, 2% SDS, 5% glycerol, 5% β-mercaptoethanol, and 0.4 mg / ml bromophenol blue) and then subjected to SDS-PAGE electrophoresis and transferred to PVDF membranes (Immobilon-FL; Millipore). The 56 kDa fibrin α chain was detected using monoclonal antibody 59D8 (Oklahoma Medical Research Foundation, diluted 1:1,000), incubated with infrared-labeled goat anti-mouse secondary antibody (IRDye 800CW; Licor Biosciences, diluted 1:5,000), and detected using an infrared detection system (Odyssey infrared imaging system, Licor Biosciences). Fibrin deposition was quantified using a baby rat fibrin standard.

[0043] like Figure 1As shown in B, the fibrin deposition in the lung tissue of the 100% hyperoxia BPD model increased significantly. Different doses of metformin (10-250 mg / Kg) can significantly reduce the fibrin deposition in the lung tissue of BPD, among which 25 mg / Kg has the most significant effect. Therefore, 25 mg / Kg was used as the intervention dose of metformin in subsequent experiments.

[0044] See also Figure 2 By recording the weight and mortality of BPD mice, the present invention found that metformin can significantly improve the weight loss caused by hyperoxia and increase the survival rate of BPD mice.

[0045] Lung morphology assessment:

[0046] Formalin-fixed, paraffin-embedded, 4-μm-thick lung tissue sections were subjected to immunohistochemical staining, including anti-von Willebrand factor (vWF; A0082; Dako Cytomation, dilution 1:4,000), anti-smooth muscle actin (ASMA; A2547; Sigma-Aldrich, 1:20,000), anti-ED-1 (a marker for monocytes and macrophages; dilution 1:2), anti-myeloperoxidase (MPO; RB-373-A1; Thermo Fisher Scientific, dilution 1:1,500), or anti-type III collagen (COL3A1; ab7778; Abcam, dilution 1:3,200), followed by staining with EnVision-HRP (Dako) and NovaRed as a chromogenic substrate and counterstaining with hematoxylin. In addition, lung tissue sections were stained with Hart's staining to observe elastic fibers, i.e., incubated in a solution containing 20 ml of catechol-fumonisin solution (X877.2; Carl Roth), 200 ml of 70% ethanol, and 4 ml of hydrochloric acid for 24 h. After four washes with double distilled water, the sections were counterstained with a solution containing 0.5 g of tartrazine (86310; Sigma-Aldrich), 200 ml of double distilled water, and 0.5 ml of acetic acid.

[0047] Alveolar capillary density was assessed by counting the number of vessels per field on vWF-stained lung tissue sections at 200× magnification. At least 10 representative fields were studied for each experimental animal, and the results were expressed as the relative number of vessels per field. Alveolar crest density analysis was performed using immunohistochemically stained ASMA lung tissue sections to assess the number of secondary alveolar buds in 10 non-overlapping fields per animal at 400× magnification and calibrated by area. Pulmonary arteriolar wall thickness was assessed by measuring the average of at least 10 vessels with a diameter greater than or equal to 30 μm on ASMA-stained lung tissue sections at 1000× magnification. Arteriolar wall thickness was calculated by the following formula: Percent wall thickness = 2× wall thickness / outer diameter × 100. Inflammatory cell counts were calculated by counting the number of ED-1-positive monocytes or MPO-positive neutrophils in the same area, and the results were expressed as the number of cells per square millimeter. Collagen and elastic fibers were analyzed by the percentage of positive area using image analysis software. All morphometric analyses excluded regions containing large vessels or bronchi. Quantitative morphometric analyses were performed using ImageJ software from the National Institutes of Health by two researchers who were blinded to the experimental group assignments.

[0048] By counting blood vessels in the same field of view, Figure 3 A and Figure 3 C, The pulmonary vascular density of BPD mice was significantly reduced, while the vascular density was significantly increased after metformin treatment ( Figure 3 I). It can be inferred that metformin can significantly improve the pulmonary vascular development of BPD mice.

[0049] ASMA can also be expressed in the muscular layer of arterioles. The present invention uses ASMA staining and measures the thickness of the ASMA-positive layer of the pulmonary arterioles, which represents the thickness of the muscular layer of the arterioles, to evaluate the remodeling of the muscular layer of the pulmonary arterioles and reflect the level of pulmonary hypertension. Figure 3 E and Figure 3 G, it was found that hyperoxia can significantly increase the remodeling of pulmonary arterioles, resulting in a phenotype of pulmonary hypertension, while metformin treatment can improve this phenotype ( Figure 3 H, 3K). ASMA can also be expressed in secondary alveolar buds. By counting ASMA-positive cells, the secondary alveolar buds can be counted to evaluate the development of secondary alveoli; refer to Figure 3 J, it can be found that the number of secondary alveolar buds in BPD lung tissue is significantly reduced, that is, metformin can significantly increase the density of secondary alveolar buds in BPD lung tissue and promote the development of secondary alveoli.

[0050] Reference Figure 4, also using immunohistochemical technology, the present invention counted the macrophages (ED1 positive) and neutrophils (MPO positive) in the lung tissue, and found that hyperoxia promoted the infiltration of inflammatory cells in the lung tissue, while metformin could significantly reduce the infiltration of inflammatory cells in the lung tissue; in addition, the present invention also found that the thickening of the alveolar septum caused by hyperoxia could also be improved by metformin; the above results suggest that metformin can significantly reduce the lung inflammatory damage caused by hyperoxia.

[0051] Reference Figure 5 The present invention quantified the deposition of collagen fibers and elastic fibers in lung tissue and found that hyperoxia can significantly promote the deposition of collagen fibers and elastic fibers in lung tissue, while metformin can effectively reduce such deposition, thereby improving lung compliance and resistance.

[0052] Notes on actual dosage:

[0053] In the above steps, the dosage of metformin is 25 mg / Kg. According to the body surface area conversion, the dosage converted to humans is about 4 mg / Kg, which is about half of the dosage of diabetes medication (500 mg / 70 Kg). Figure 6 , 10uM and 250uM metformin can inhibit the inflammatory macrophage phenotype; the present invention also verifies the effect of different doses of metformin on the pro-inflammatory / anti-inflammatory phenotype of macrophages in a cell culture model. Metformin below 250uM exhibits an anti-inflammatory phenotype, which is converted into a blood drug concentration of about 32mg / L, which is also half of the diabetes medication dose (oral metformin drug conversion rate is about 50%, adult circulating blood volume is about 5L, and blood drug concentration is about 50mg / L). Considering that 10uM metformin also exhibits an anti-inflammatory effect in in vitro experiments, combined with the description of the above-mentioned discovery process of the present invention, the actual concentration of metformin in breast milk is converted, based on the daily feeding volume of premature infants of about 200mL and the weight of premature infants of about 2Kg, the amount of metformin ingested by premature infants from breast milk is about 1-40mg per day, so it is speculated that metformin is clinically effective in treating BPD, and the concentration range is about 0.1-4mg / Kg.

[0054] The above description is only an exemplary embodiment of the present invention, and does not limit the patent scope of the present invention. All equivalent structural changes made by using the contents of the present invention specification and drawings under the technical concept of the present invention, or directly / indirectly applied in other related technical fields are included in the patent protection scope of the present invention.

Claims

1. Use of metformin in the preparation of a medicament for preventing and / or treating bronchopulmonary dysplasia in premature infants.

2. The use according to claim 1, It is characterized in that In the drug for preventing and / or treating bronchopulmonary dysplasia in premature infants, metformin may be a pharmaceutically acceptable salt thereof.

3. The use according to claim 1, It is characterized in that The use includes use in preparing a medicament having at least one of the following effects: Reduce the infiltration of macrophages and neutrophils in lung tissue, inhibit lung inflammatory damage, prevent alveolar septum widening and collagen fiber deposition, reduce vascular exudation and promote microangiogenesis in lung tissue.

4. The use according to claim 1, It is characterized in that The dosage of metformin or its pharmaceutically acceptable salt in the drug is 0.1-4 mg / Kg 。

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