Drug for bronchopulmonary dysplasia and use method therefor

Abstract

Provided in the present invention are an immunosuppressant and a method for treating and / or preventing bronchopulmonary dysplasia (BPD) in a subject, and use thereof. The immunosuppressant comprises an immunoglobulin inhibitor, a small molecule immunosuppressant, or a cell-type immunosuppressant, and the subject is preferably a premature newborn.

Description

Drugs for bronchopulmonary dysplasia and their usage

[0001] Cross-reference to related applications

[0002] This application claims priority to Chinese Patent Application No. 202411580195.1, filed on November 6, 2024, the entire contents of which are incorporated herein by reference. Technical Field

[0003] This invention relates to the prevention and treatment of diseases. Specifically, this invention relates to the use of immunosuppressants in the prevention and treatment of bronchopulmonary dysplasia, especially in newborns, and related treatment methods. Background Technology

[0004] Bronchopulmonary dysplasia (BPD) is a chronic lung disease that occurs in premature infants. It is caused by prenatal and postnatal factors that impair the normal development of immature lungs, including prematurity, genetic factors, growth restriction, mechanical trauma, oxygen toxicity, infection, and inflammation. Its main histopathological features are simplified alveolar structure and abnormal pulmonary vascularization.

[0005] With advancements in perinatal medicine, improvements in neonatal intensive care technology, and the use of prenatal glucocorticoids and exogenous surfactant therapy, the survival rate of premature infants has significantly increased, and the incidence of preterm complications has decreased. However, the incidence of bronchopulmonary dysplasia (BPD) is on the rise. Due to varying definitions of BPD, reported incidence rates range from 10% to 89%, and the incidence of BPD is negatively correlated with birth weight and gestational age. Recent reports from South Korea indicate a BPD incidence of 42.7% in very low birth weight (ELBW) newborns and 28.9% in very low birth weight (VLBW) newborns. In China, reported BPD incidence rates are 74.2% for gestational ages below 25 weeks, 51.9% for 26-27 weeks, and 33.4% for 28-29 weeks.

[0006] Bipolar disorder (BPD) is a disease that persists from fetus to adulthood (see Am J Respir Crit Care Med, 2019, 200(6):659-660.). Due to advancements in healthcare, BPD patients are surviving longer, and the long-term impact of BPD on humans is becoming increasingly apparent. Surviving BPD patients have a greater risk of developing persistent respiratory diseases such as asthma, respiratory infections, impaired exercise tolerance, early-onset emphysema, pulmonary hypertension, and chronic obstructive pulmonary disease. Furthermore, the persistent complications of BPD have been found to be expanding, including an increased risk of cerebral palsy and developmental delays, lower IQ scores, impaired executive function, behavioral challenges, delayed verbal and receptive language development, and an increased risk of growth failure—multi-organ system diseases that can persist into adulthood, resulting in impaired physical and psychological quality of life. Therefore, the treatment of BPD spans multiple disciplines across neonates, infants, children, adolescents, and adults, with a high rate of recurrent hospitalizations, placing a significant economic and psychological burden on society and families.

[0007] To date, prevention and treatment strategies for preterm birth defects (BPD), including prenatal steroid administration, postnatal surfactant administration, and mechanical ventilation, have had limited effectiveness in reducing the incidence of BPD. Existing medications such as caffeine and dexamethasone have limited efficacy and have been shown to be associated with severe, long-term neurodevelopmental disorders. Currently, there is still a lack of fundamental treatments for BPD. Therefore, there is an urgent clinical need to develop new prevention and treatment strategies for BPD, which are crucial for improving the quality of life of premature infants and their children, adolescents, and adults. Summary of the Invention

[0008] This invention provides a drug for treating bronchopulmonary dysplasia and its application. Validated using a hyperoxia-induced preterm pig BPD model, it proposes for the first time a novel immunosuppressive therapy for the prevention and treatment of neonatal bronchopulmonary dysplasia. Based on the principle of immunosuppressive regulation, this invention provides the application of immunosuppressants, including macromolecular drugs, small molecule drugs, and cellular drugs, for the prevention and treatment of BPD in subjects such as newborns.

[0009] In some aspects, the present invention provides the use of immunosuppressants in the preparation of medicaments for treating and / or preventing bronchopulmonary dysplasia (BPD) or related symptoms in subjects. Specifically, the immunosuppressant is selected from immunoglobulin inhibitors, small molecule immunosuppressants, and cellular immunosuppressants. The BPD can be mild, moderate, or severe. Related symptoms of BPD include, but are not limited to, lower respiratory tract infections, wheezing episodes, and recurrent cough.

[0010] In some implementations, the subjects are newborns (e.g., premature newborns), infants, children, adolescents, or adults.

[0011] In some embodiments, the subject is preterm at birth, for example, with a gestational age of less than 35 weeks, less than or approximately 32 weeks, or less than or approximately 30 weeks, less than or approximately 29 weeks, less than or approximately 28 weeks, less than or approximately 27 weeks, less than or approximately 26 weeks, less than or approximately 25 weeks, less than or approximately 24 weeks, less than or approximately 23 weeks, or a gestational age of less than or approximately 22 weeks. Preferably, the gestational age is less than or approximately 32 weeks. When treatment and / or prevention are performed, the subject can be in the neonatal, infant, child, adolescent, or adult stage. In some embodiments, the subject is less than 3 years, less than 2 years, less than 12 months, less than 6 months, less than 3 months, less than 2 months, or less than 1 month old.

[0012] In some embodiments, the immunoglobulin inhibitor is intravenous immunoglobulin (IVIG) or gamma globulin. When used in humans, the immunoglobulin inhibitor is preferably human-derived. The immunoglobulin inhibitor can be a commercially available finished product or a laboratory-prepared product. Commercially available IVIG is typically a colorless or pale yellow clear liquid, administered directly or diluted intravenously.

[0013] In some embodiments, the small molecule immunosuppressant is a JAK inhibitor, such as ruxolitinib or an analogue thereof, or a stereoisomer, enantiomer, or pharmaceutically acceptable salt thereof.

[0014] In some embodiments, the small molecule immunosuppressant is an IRAK4 inhibitor, such as Zabedosertib or its analogues, stereoisomers, enantiomers, or pharmaceutically acceptable salts thereof.

[0015] In some embodiments, the cellular immunosuppressant is a mesenchymal stem cell (MSC) or an immunomodulatory cell. For example, the MSC is selected from umbilical cord-derived mesenchymal stem cells, umbilical cord blood-derived mesenchymal stem cells, adipose-derived mesenchymal stem cells, peripheral blood-derived mesenchymal stem cells, and mesenchymal stem cells differentiated from pluripotent stem cells. The immunomodulatory cell can be selected from Treg cells and myeloid-derived suppressor cells (MDSCs).

[0016] In some embodiments, the immunosuppressants are formulated as pharmaceuticals or pharmaceutical compositions. Pharmaceuticals or pharmaceutical compositions comprising the immunosuppressants disclosed herein also comprise pharmaceutically acceptable carriers.

[0017] In some embodiments, the drug or pharmaceutical composition is formulated for administration to the subject via intravenous infusion, intramuscular injection, oral administration, nebulization, or intratracheal injection. The drug or pharmaceutical composition can be prepared in various forms, such as solutions, suspensions, solids (tablets, pills, etc.), and aerosols (sprays or inhalers, etc.). Preferably, the drug or pharmaceutical composition is prepared in a form suitable for neonatal administration.

[0018] In some embodiments, the prevention or treatment further includes assessing the effectiveness by detecting respiratory function indicators, lung histopathology, immunohistochemical indicators, and / or lung imaging in the subject. In some embodiments, the effectiveness is assessed by one or more of radial alveolar counts, alveolitis scores, collagen fiber area ratios, and Ashcroft fibrosis scores.

[0019] In some other aspects, this disclosure provides methods for treating and / or preventing bronchopulmonary dysplasia (BPD) or related symptoms in a subject, including administering an effective amount of an immunosuppressant to the subject. Specifically, the immunosuppressant may be selected from immunoglobulin-type, small molecule immunosuppressants, and cellular immunosuppressants. In some embodiments, the subject is a preterm neonate, or a child or adult with BPD. In some embodiments, the neonate is born at a gestational age of less than 35 weeks, preferably less than or about 32 weeks, or the neonate's PMA (corrected gestational age) is less than or about 36 weeks.

[0020] In some implementations, a single dose of IVIG is from 200 mg / kg body weight to 2000 mg / kg body weight, preferably from 200 mg / kg body weight to 1000 mg / kg body weight.

[0021] In some implementations, a single dose of a JAK inhibitor, such as ruxolitinib, is 1 mg / kg body weight to 5 mg / kg body weight, preferably 2 mg / kg body weight to 3 mg / kg body weight.

[0022] In some implementations, a single dose of an IRAK4 inhibitor, such as Zabedosertib, is 1 mg / kg body weight to 5 mg / kg body weight, preferably 2 mg / kg body weight to 3 mg / kg body weight.

[0023] In some implementations, the dose of mesenchymal stem cells administered to the subject is 1 × 10⁻⁶. 6 -3×10 7 Cells / kg body weight, preferably 1×10⁻⁶ 6 -2×10 7 Cells per kg of body weight.

[0024] In some implementations, the drug is administered between day 2 and day 56 of the newborn's life, for example, between day 14 and day 32. The administration may be a single or multiple administrations.

[0025] In some implementations, the drug may be combined with other therapeutic agents. These other therapeutic agents may include standard treatments for BPD (e.g., oxygen therapy, assisted respiratory support), corticosteroids, bronchodilators, anticholinergics, vasodilators, diuretics, antihypertensives, acetazolamide, antibiotics, antiviral agents, surfactants, caffeine, etc.

[0026] This disclosure also provides pharmaceutical compositions comprising the immunosuppressants described herein and a carrier / excipient that is pharmaceutically acceptable or suitable for administration to a subject.

[0027] In some respects, this disclosure provides immunosuppressants or pharmaceutical compositions as disclosed herein for the prevention (treatment and / or prevention) of bronchopulmonary dysplasia or related symptoms in subjects. As described above, the immunosuppressants may be selected from immunoglobulin inhibitors, small molecule immunosuppressants, and cellular immunosuppressants. Attached Figure Description

[0028] Figure 1 shows the imaging results of the control group and the treatment group after IVIG treatment of preterm pigs with BPD. A) The control group showed disordered lung markings and diffuse patchy increased density shadows; B) The increased density shadows in the lungs of the treatment group were significantly reduced compared with the control group.

[0029] Figure 2 is a schematic diagram of the H&E staining results of preterm pigs with BPD after IVIG treatment. A) In the control group, the alveoli were of varying sizes, with alternating alveolar cavities of enlargement and atelectasis, thickened alveolar septa, and inflammatory cell infiltration visible in the alveolar cavities and septa; B) The lung tissue structure in the treatment group was improved compared to the control group, with normal alveolar structure visible, and reduced inflammatory cell infiltration in the alveolar cavities and alveolar walls compared to the control group.

[0030] Figure 3 shows the Masson staining results of preterm pigs with BPD after IVIG treatment. A) In the control group, the alveolar septa were thickened, with more blue collagen fiber deposits and some alveolar surfaces covered by collagen fibers; B) In the treatment group, the degree of alveolar septal thickening was improved, and there were relatively fewer collagen fiber deposits.

[0031] Figure 4 shows the pathological statistical results of preterm pigs with bronchiectasis (BPD) after IVIG treatment. A) Compared with the control group, the number of radial alveoli was significantly increased in the treatment group (P < 0.001); B) The Szapiel score of alveolitis in the treatment group was significantly decreased compared with the control group (P < 0.001); C) The proportion of collagen fiber area in the treatment group was significantly decreased compared with the control group (P < 0.001); D) The Ashcroft fibrosis score in the treatment group was significantly decreased compared with the control group (P < 0.001). ns: no significant difference, *: P < 0.05, **: P < 0.01, ***: P < 0.001, ****: P < 0.0001.

[0032] Figure 5 shows the imaging results of the control group and the treatment group after hyperoxia-induced BPD in preterm pigs and IVIG treatment. A) The control group showed disordered lung markings and diffuse patchy high-density shadows in both lungs; B) Compared with the control group, the treatment group showed a significant reduction and alleviation of diffuse high-density shadows in both lungs.

[0033] Figure 6 shows the H&E staining results of preterm pigs after IVIG treatment for BPD induced by hyperoxia. Compared with the control group (A), the treatment group (B) showed normal alveolar structure, more intact alveolar septa, and improvements in alveolar enlargement, alveolar number, atelectasis, and alveolitis.

[0034] Figure 7 shows the Masson staining results of preterm pigs treated with IVIG after hyperoxia-induced bronchopulmonary dysplasia (BPD). Compared with the control group (A), the treatment group (B) had thinner alveolar septa and significantly reduced collagen fiber deposition.

[0035] Figure 8 shows the pathological statistical results of preterm pigs treated with IVIG during hyperoxia-induced bronchopulmonary dysplasia (BPD). A) The radial alveolar count in the treatment group was significantly increased compared with the control group (P < 0.0001); B) The Szapiel score of alveolitis in the treatment group was significantly decreased compared with the control group (P < 0.001); C) The proportion of collagen fiber area in the treatment group was significantly decreased compared with the control group (P < 0.0001); D) The Ashcroft fibrosis score in the treatment group was significantly decreased compared with the control group (P < 0.0001). ns: No significant difference, *: P < 0.05, **: P < 0.01, ***: P < 0.001, ****: P < 0.0001.

[0036] Figure 9 shows the imaging results of the control group and the treatment group after ruxolitinib was administered to preterm pigs for prevention and treatment of BPD induced by hyperoxia. A) Diffuse patchy high-density shadows in both lungs in the control group; B) Compared with the control group, the JAKi group had clearer lung markings and significantly reduced diffuse high-density shadows in both lungs.

[0037] Figure 10 shows the H&E staining results of preterm pigs after administration of ruxolitinib for prevention and treatment of BPD induced by hyperoxia. Compared with the control group (A), the JAKi group (B) showed normal alveolar structure, more intact alveolar septa, and significantly improved alveolar number, alveolar cavity, and alveolar wall inflammatory cell infiltration compared with the control group.

[0038] Figure 11 shows the Masson staining results of preterm pigs after administration of ruxolitinib for prevention and treatment of BPD induced by hyperoxia. A) In the control group, alveolar septal thickening was observed, with more blue collagen fibers and red muscle fibers, and some alveolar surfaces were covered by collagen fibers; B) In the JAKi group, the degree of alveolar septal thickening was improved, and the deposition of collagen fibers and muscle fibers was significantly reduced.

[0039] Figure 12 shows the pathological statistical results of preterm pigs treated with ruxolitinib for prevention and treatment of BPD induced by hyperoxia. A) The number of radial alveoli in the JAKi group was significantly increased compared with the control group (P < 0.0001); B) The Szapiel score of alveolitis in the JAKi group was significantly decreased compared with the control group (P < 0.001); C) The proportion of collagen fiber area in the JAKi group was significantly decreased compared with the control group (P < 0.0001); D) The Ashcroft fibrosis score in the JAKi group was significantly decreased compared with the control group (P < 0.001). *: P < 0.05, **: P < 0.01, ***: P < 0.001, ****: P < 0.0001.

[0040] Figure 13 shows the imaging results of preterm pigs after administration of Zabedosertib for prevention and treatment of BPD induced by hyperoxia. A) Control group: uneven lung inflation, diffuse multiple patchy and linear high-density shadows; B) IRAK4i group: reduced diffuse high-density lesions in both lungs compared with control group.

[0041] Figure 14 shows the H&E staining results of preterm pigs treated with zabedosertib after hyperoxia-induced BPD. Compared with the control group (A), the IRAK4i group (B) showed that the alveolar structure was basically intact, the alveolar septa were relatively normal, and the number of alveoli, alveolar cavities, and alveolar wall inflammatory cell infiltration were significantly reduced compared with the control group.

[0042] Figure 15 shows the Masson staining results of preterm pigs treated with zabedosertib during hyperoxia-induced BPD. A) In the control group, alveolar structure was destroyed, alveolar septa were thickened, and more blue collagen fibers and red muscle fibers were observed. A small portion of the alveolar cavity surface was covered by collagen fibers. B) In the IRAK4i group, alveolar structure was more intact, septal thickening was not obvious, and collagen and muscle fiber deposition was significantly reduced.

[0043] Figure 16 shows the pathological statistical results of preterm pigs treated with zabedosertib during hyperoxia-induced bronchopulmonary dysplasia (BPD). A) The radial alveolar count in the IRAK4i group was significantly increased compared with the control group (P < 0.0001); B) The Szapiel score of alveolitis in the IRAK4i group was significantly decreased compared with the control group (P < 0.001); C) The proportion of collagen fiber area in the IRAK4i group was significantly decreased compared with the control group (P < 0.001); D) The Ashcroft fibrosis score in the IRAK4i group was significantly decreased compared with the control group (P < 0.0001). *: P < 0.05, **: P < 0.01, ***: P < 0.001, ****: P < 0.0001.

[0044] Figure 17 shows the imaging results of preterm pigs after receiving treatment with porcine umbilical cord mesenchymal stem cells (pMSCs) simultaneously with hyperoxia-induced BPD. A) Diffuse patchy high-density shadows in both lungs of the control group; B) The high-density shadows in both lungs of the pMSC group were reduced compared to the control group.

[0045] Figure 18 shows the H&E staining results of preterm pigs after receiving porcine umbilical cord mesenchymal stem cell (pMSC) therapy concurrently with hyperoxia-induced bronchopulmonary dysplasia (BPD). Compared with the control group (A), the pMSC group (B) showed normal alveolar structure, and the number of alveoli, alveolar cavities, and alveolar wall inflammatory cell infiltration were all improved compared with the control group.

[0046] Figure 19 shows the Masson staining results of preterm pigs after receiving treatment with porcine umbilical cord mesenchymal stem cells (pMSCs) simultaneously with hyperoxia-induced bronchopulmonary dysplasia (BPD). A) In the control group, alveolar septa were thickened, with more blue collagen fibers and red muscle fibers observed; B) In the pMSC group, focal alveolar septa were widened, with less collagen and muscle fiber deposition compared to the control group.

[0047] Figure 20 shows the pathological statistical results of preterm pigs treated with porcine umbilical cord mesenchymal stem cells (pMSCs) after hyperoxia-induced bronchopulmonary dysplasia (BPD). A) The number of radial alveoli in the pMSC group was significantly increased compared with the control group (P < 0.01); B) The Szapiel score of alveolitis in the pMSC group was significantly decreased compared with the control group (P < 0.05); C) The proportion of collagen fiber area in the pMSC group was significantly decreased compared with the control group (P < 0.01); D) The Ashcroft fibrosis score in the pMSC group was significantly decreased compared with the control group (P < 0.01). *: P < 0.05, **: P < 0.01, ***: P < 0.001, ****: P < 0.0001.

[0048] Invention Details

[0049] Unless otherwise defined, all technical and scientific terms used in this application have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Furthermore, unless the context otherwise requires, singular terms shall include plural forms, and plural terms shall include singular forms. More specifically, as used in this specification and the appended claims, unless the context otherwise clearly indicates, the singular forms “a,” “an,” and “the / said” include plural referents. Unless otherwise stated, the use of “or” means “and / or.” Furthermore, the use of the term “comprising” and other forms such as “including” and “containing” is not limiting.

[0050] As used in this article, the term "immunosuppressant" refers to a drug that inhibits the body's immune response by suppressing the proliferation and function of cells involved in the immune response (such as T cells and macrophages like B cells) to reduce antibody immune responses and induce immune tolerance.

[0051] As used herein, the term "analog" refers to a compound in which one or more atoms or functional groups in the original compound are replaced by different atoms or functional groups, but which retain a similar basic structure and function.

[0052] As used herein, the term "preterm" refers to a human individual born at a gestational age of less than 37 weeks, including gestational ages of less than 35 weeks, or less than or approximately 30 weeks, less than or approximately 29 weeks, less than or approximately 28 weeks, less than or approximately 27 weeks, less than or approximately 26 weeks, less than or approximately 25 weeks, less than or approximately 24 weeks, less than or approximately 23 weeks, or gestational ages of less than or approximately 22 weeks. The term "newborn" generally refers to a human individual born within 28 days of birth.

[0053] As used in this article, the term "gestational age" refers to the time elapsed from the first day of the mother's last menstrual period to the day of delivery. If pregnancy was achieved using assisted reproductive technology, gestational age is calculated by adding two weeks to the age at conception. As used in this article, the term "PMA" ("postmenstrual age") refers to the baby's gestational age plus the number of weeks and days since birth (i.e., chronological age). For example, a newborn born at 32 weeks gestation and now 28 days old has a PMA of 36 weeks.

[0054] As used herein, the term “prevention” encompasses both “prevention” and “treatment.” The term “prevention” refers to a reduced likelihood of acquiring (or being suspected of acquiring) BPD (i.e., resulting in the absence of at least one clinical symptom in a patient who is at risk of or susceptible to BPD but has not yet experienced or exhibited symptoms of the disease). The biological and physiological parameters used to identify such patients are well-known to clinicians.

[0055] As used herein, the term "treatment" includes the application or administration of the drugs or methods disclosed herein (or the application or administration of the drugs or methods of the present invention to cells or tissues derived from the subject) to a subject to achieve the purpose of delaying, slowing, stabilizing, curing, alleviating, resolving, altering, remedying, reducing the severity of, improving, or affecting the disease or condition of BPD, the symptoms of the disease or condition, or the risk thereof (or suspected BPD). The term "treatment" means any indication of successful treatment or improvement of an lesion, pathology, or condition, including any objective or subjective parameter such as reduction, remission, slowing of the rate of deterioration, reduction of the severity of the disease, stabilization or reduction of symptoms, or making the subject more tolerant of said lesion, pathology, or condition, slowing the rate of degeneration or decline, making the endpoint of degeneration less severe, or improving the subject's physiological condition.

[0056] As used in this article, the term "pharmaceutical acceptable" means a medium that does not produce allergic reactions or similar adverse reactions when given to subjects, such as premature infants.

[0057] The term “therapeutic effective dose” as used in this article refers to the amount that can lead to the disappearance or improvement of any indication of BPD-related damage, pathology, or condition in the treated individual.

[0058] Treatment and / or prevention of BPD with immunosuppressants

[0059] Bipolar disorder (BPD) is a common chronic respiratory disease in premature infants. Essentially, it is a chronic lung disease caused by restricted and damaged lung development in premature infants, and its pathophysiological changes are related to gestational age. Premature infants born before 29 weeks of gestation are in the transition from the pseudoglandular stage to the tubular stage of lung development. Various perinatal stimuli, such as intrauterine growth restriction, inflammatory exposure, oxygen administration, and mechanical ventilation, can lead to the cessation of pulmonary vascular and alveolar development. Pathologically, this manifests as simplified development of distal alveoli, potentially forming large sac-like alveolar structures, and reduced ventilation and gas exchange. The development of the alveolar and surrounding vascular microenvironment ceases, leading to remodeling and ultimately BPD. The direct consequence of this pathophysiological process is that respiratory function cannot meet the body's growth and development needs, clinically manifesting as oxygen dependence and multi-system complications following hypoxia. The development of normal lung microstructures is interrupted; alveoli become fewer and larger, and the interstitial spaces thicken. Furthermore, pulmonary vessels develop abnormally, with fewer and / or abnormally distributed alveolar capillaries; pulmonary resistance may increase, and pulmonary hypertension may develop. Currently, there are no specific and effective treatments. Treatment mainly focuses on the mechanisms of BPD occurrence and development, including prevention of lung injury caused by mechanical ventilation (protective respiratory support strategies, fluid restriction, etc.), anti-inflammatory (antibiotics, glucocorticoids), anti-oxidative stress damage (superoxide dismutase), and nutritional support (vitamin A).

[0060] This invention provides a variety of medications for the treatment and / or prevention of birth defects (especially neonatal BPD). These medications can reduce, decrease, or prevent the development of BPD in newborns, reduce the incidence of neonatal BPD, and improve the survival rate and long-term prognosis of children with BPD. The BPD can be induced by or related to any common clinical factors. The medications are primarily based on the principle of immunosuppression, including but not limited to immunoglobulins, JAK inhibitors, IRAK4 inhibitors, and cellular drugs.

[0061] This invention thus provides a method for treating and / or preventing BPD, comprising administering a therapeutically and / or preventively effective amount of the drug to an individual in need (particularly preterm newborns). The drug has an immunosuppressive effect, reducing lung inflammation and fibrosis by altering the levels of inflammatory factors in airway secretions, thereby reducing the severity of BPD and effectively treating and / or preventing BPD. The individual / subject can be a human or a non-human animal, preferably a human.

[0062] The method of the present invention is particularly applicable to newborns, such as newborns with BPD and newborns at risk of developing BPD. Newborns at risk of developing BPD include preterm newborns, newborns at risk of preterm birth, and any other newborns at risk of developing BPD for any reason.

[0063] Those skilled in the art are well aware of determining whether an individual is at risk of developing BPD or exhibiting early signs or symptoms of BPD. For example, in human individuals, BPD can be diagnosed at approximately 28 days or 36 weeks of postnatal maternal and infantile atrophy (PMA).

[0064] In some cases, the diagnosis of BPD can be made in any preterm newborn with lung disease requiring continuous or frequent oxygen support and with abnormal chest X-ray findings. For example, the diagnosis of BPD can be based on the following criteria: preterm infants with a gestational age <32 weeks have radiographically confirmed persistent parenchymal lung disease and require oxygen therapy support (for more than 3 consecutive days) at 36 weeks of PMA to maintain arterial blood oxygen saturation (SaO2) at 90%–95%. Optionally, other diagnostic criteria for BPD can be used, such as those published by the National Institute of Child Health and Human Development (NICHD) in 2001, which require oxygen support [inhaled oxygen concentration (FiO2) >21%] for more than 28 days cumulatively after preterm birth. BPD is graded based on the degree of oxygen dependence of the fetus at 36 weeks of gestation (PMA) (for fetuses with a gestational age <32 weeks) or 56 days after birth (for fetuses with a gestational age ≥32 weeks): mild BPD does not require oxygen; moderate BPD requires oxygen and has a FiO2 <30%; and severe BPD (sBPD) requires a FiO2 ≥30% and / or continuous positive airway pressure or mechanical ventilation. Generally, a chest X-ray is performed on the newborn at 28 days of age to confirm the diagnosis of BPD. Chest X-rays of newborns with BPD typically show a bubbly or spongy appearance.

[0065] Furthermore, those skilled in the art are familiar with methods for determining the effectiveness of BPD treatment, including determining whether the use of the methods or medications disclosed herein to treat and / or prevent BPD is effective. For example, the effectiveness of treatment can be determined by measuring changes or improvements in any of the BPD symptoms described herein, including but not limited to changes or improvements detected by lung histopathology, such as radial alveolar counts, assessment of alveolar inflammation and fibrosis.

[0066] Immunoglobulin drugs

[0067] In some aspects, the present invention provides a method for treating and / or preventing BPD (especially BPD in preterm newborns) by administering gamma globulin or intravenous immunoglobulin (IVIG).

[0068] Typically, gamma globulin is mainly composed of immunoglobulin G. It is an immunoglobulin product isolated and extracted from the plasma of healthy individuals and treated with virus inactivation, containing various antibodies found in the serum of healthy individuals. Similar to gamma globulin, intravenous immunoglobulin is an immunizing agent mainly containing immunoglobulin G, generally administered via intravenous injection. Methods for preparing IVIG and gamma globulin are well known in the art, such as extraction from plasma or preparation via recombinant genetic technology, and commercially available products of IVIG and gamma globulin, such as intravenous immunoglobulin (pH4), are readily available.

[0069] IVIG is extracted from healthy donor blood, concentrated, and purified, and contains a variety of immunoglobulin molecules. IVIG contains broad-spectrum IgG antibodies against viruses, bacteria, or other pathogens. Furthermore, the unique and anti-unique antibodies against immunoglobulins form a complex immune network, thus providing both immune replacement and immunomodulation therapeutic effects. IVIG has been widely used in many clinical fields, providing antibodies for immunodeficiency conditions such as idiopathic immunoglobulin deficiency (CVID) and X-linked immunodeficiency diseases; it can also treat autoimmune diseases such as inflammatory myopathy, multiple sclerosis, and Guillain-Barré syndrome by inhibiting the formation of autoantibodies; IVIG is also used for infectious diseases, some autoimmune skin diseases, immune thrombocytopenic purpura, and graft rejection.

[0070] In some embodiments, when applied to human subjects, the IVIG is human-derived. In other embodiments, when applied to subjects of other species, IVIG derived from that species or extracted from blood from a donor of that species is used. For example, when applied to a pig model, pig-derived IVIG is used. In some embodiments, the IVIG used is either commercially available or laboratory-prepared.

[0071] The inventors have discovered for the first time that IVIG has unexpected technical effects in the prevention and treatment of neonatal bronchopulmonary dysplasia (BPD), while exhibiting high efficacy, few side effects, and high safety for newborns. IVIG can rapidly increase IgG levels in subjects, enhance the body's immune regulation function, significantly reduce lung lesions caused by BPD, reduce the degree of inflammation, and improve pneumonia, alveoli, and pulmonary fibrosis.

[0072] Small molecule inhibitors

[0073] In some aspects, the present invention provides a method for preventing (i.e., treating and / or preventing) BPD (especially BPD in preterm newborns) by administering small molecule inhibitors. The small molecule inhibitors include JAK inhibitors, IRAK4 inhibitors, etc.

[0074] JAK inhibitors selectively inhibit Janus kinase (JAK), blocking the JAK / STAT pathway. The JAK-STAT signaling pathway is a cytokine-stimulated signal transduction pathway discovered in recent years, involved in many important biological processes such as cell proliferation, differentiation, apoptosis, and immune regulation. Janus kinase is a non-receptor tyrosine protein kinase with four family members: JAK1, JAK2, TYK2, and JAK3. Representative JAK inhibitors include those disclosed in US Patent No. 7,598,257 and WO2013082476A1. For example, the JAK inhibitors that can be used in this invention may be selected from, but are not limited to, ruxolitinib, tofacitinib, upadacitinib, baricitinib, CEP-701 (lentatinib, Cephalon Technology), AZD1480 (Astra Zeneca), LY3009104 / INCB28050 (Eli Lilly, Incyte), Pacritinib / SB1518 (S*BIO), VX-509 (Vertex), GLPG0634 (Galapagos), INC424 (Novartis), R-348 (Rigel), CYT387 (YM Bioscience), TG 10138, AEG 3482 (Axon); and their pharmaceutically acceptable salts and prodrugs.

[0075] In some embodiments, the JAK inhibitor is a heteroaryl-substituted pyrrolo[2,3-b]pyridine and pyrrolo[2,3-b]pyrimidine. In some embodiments, the JAK inhibitor is ruxolitinib or its analogues or derivatives, or pharmaceutically acceptable diastereomers, enantiomers, metabolites, salts (e.g., ruxolitinib phosphate), solvates, or solvates of salts. Ruxolitinib is a JAK1, JAK2 tyrosine kinase inhibitor and was the first approved drug for the treatment of myelofibrosis. It is also currently being used in research on hematological diseases and cancer. Ruxolitinib has the following structure:

[0076] As verified in the examples, the inventors have discovered for the first time that JAK inhibitors, represented by ruxolitinib, also have very good technical effects in the treatment and / or prevention of BPD, significantly delaying the onset of BPD and significantly reducing lung lesions caused by BPD, improving pneumonia, alveoli, and pulmonary fibrosis.

[0077] IRAK4 (interleukin-1 receptor-associated kinase 4) is a member of the intracellular serine-threonine kinase family IRAK. Human IRAK4 plays a crucial role in activating the immune system. IRAK4 is expressed by many cells and mediates signal transduction from the following receptors: Toll-like receptors (TLRs) (except TLR3), and the interleukin (IL)-1β family of receptors consisting of IL-1R (receptor), IL-18R, IL-33R, and IL-36R (see Janeway and Medzhitov, Annu. Rev. Immunol., 2002; Dinarello, Annu. Rev. Immunol., 2009; Flannery and Bowie, Biochemical Pharmacology, 2010). For example, when an interleukin-1 receptor (IL-1R) or a Toll-like receptor (TLR) binds to a ligand, IRAK-4 can mediate signal transduction, activating the expression of downstream inflammatory factors. IRAK4 inhibitors are IL1 receptor kinase inhibitors widely used in research on inflammation (such as rheumatoid arthritis, dermatitis, atopic dermatitis, hidradenitis suppurativa) and various types of cancer. The IRAK4 inhibitors that may be used in this invention include, but are not limited to, isoquinoline-based drugs PF-06650833 (Zimlovisertib), CA-4948 (Emavusertib), Zabedosertib (BAY1834845), BAY1830839, EVO101, R835, and GS-5718 (Edecesertib); other IRAK4 inhibitors in clinical trials include MY004567 and TQH3821; and their pharmaceutically acceptable salts and prodrugs. IRAK4 inhibitors may also include those disclosed in WO2020 / 036830.

[0078] In some embodiments, the IRAK4 inhibitor is Zabedosertib or its analogues or derivatives, or pharmaceutically acceptable diastereomers, enantiomers, metabolites, salts, solvates, or solvates of salts thereof. Zabedosertib has the following structure:

[0079] In some embodiments, the Zabedosertib analogue is BAY1830839, which has the following structure:

[0080] In some embodiments, the Zabedosertib analogues include structures in Zabedosertib where the trifluoromethyl pyridine group is replaced with a methyl, difluoromethyl, 1-hydroxyethyl, or other halogen group, or where fluorine is replaced with another halogen.

[0081] As verified in the examples, the inventors have discovered for the first time that IRAK4 inhibitors also have very good technical effects in the treatment and / or prevention of BPD. They can significantly delay the onset of BPD, and lung pathological examination shows a reduction in the degree of inflammation, and improvement in pneumonia, alveoli, and pulmonary fibrosis.

[0082] As used herein, "pharmaceutically acceptable salt" refers to derivatives of the small molecule inhibitor drugs in which the parent compound is modified by converting the present acid or base moiety into its salt form. Examples of pharmaceutically acceptable salts include, but are not limited to, inorganic or organic acid salts of bases such as amines; and bases or organic salts of acids such as carboxylic acids. Pharmaceutically acceptable salts as described herein include, for example, conventional non-toxic salts or quaternary ammonium salts of parent compounds formed from non-toxic inorganic or organic acids. Pharmaceutically acceptable salts as described herein can be synthesized from parent compounds containing basic or acidic moieties using conventional chemical methods. Typically, such salts are prepared in water, or in organic solvents, or in mixtures of both, by reacting the free acid or base form of these compounds with a stoichiometric amount of a suitable base or acid; non-aqueous solvents such as diethyl ether, ethyl acetate, ethanol, isopropanol, or acetonitrile (MeCN) are generally preferred. A list of suitable salts can be found in Remington's Pharmaceutical Sciences, 17th edition, Mack Publishing Company, Easton, Pa., 1985, p. 1418 and Journal of Pharmaceutical Science, 66, 2 (1977), each incorporated herein by reference in its entirety.

[0083] Small molecule inhibitors disclosed herein (e.g., ruxolitinib or zabedosertib) can be formulated for intravenous or oral administration. Further, the small molecule inhibitors can be formulated into pharmaceutical compositions comprising one or more pharmaceutically acceptable excipients, buffers, or salts. In some embodiments, the small molecule inhibitors may also be administered to the subject in combination with additional treatments, including conventional treatments for BPD such as bronchodilators.

[0084] Cellular drugs

[0085] In some aspects, the present invention provides a method for preventing (i.e. treating and / or preventing) BPD (especially BPD in preterm newborns) by administering cellular immunomodulators, such as mesenchymal stem cells (MSCs) or other immunomodulatory cells (e.g., Treg, MDSCs).

[0086] Mesenchymal stem cells (MSCs) are a type of stem cell with pluripotency and self-renewal capacity, and have broad clinical application prospects. MSCs possess multi-lineage differentiation potential, capable of differentiating into various cell types, and are commonly used for the repair and regeneration of fractures, cartilage injuries, muscle injuries, myocardial infarction, and vascular injuries. Regulatory T cells (Tregs) are a subset of T cells that control autoimmune responses in the body. They have immunosuppressive effects, suppressing the immune responses of other immune cells and acting as major controllers of self-tolerance. As part of CD4+ cells, they limit excessive immune responses by inhibiting pro-inflammatory effects, thereby maintaining homeostasis. Myeloid-derived suppressor cells (MDSCs) are a heterogeneous group of cells derived from bone marrow. They are precursors to dendritic cells (DCs), macrophages, and granulocytes, and possess a significant ability to suppress immune cell responses, making them heterogeneous cells with a negative regulatory capacity for immune responses.

[0087] The MSCs used in this invention can have a variety of sources. For example, the MSCs can be derived from umbilical cord, umbilical cord blood, fat, peripheral blood, and pluripotent stem cell differentiation.

[0088] The inventors have discovered for the first time that mesenchymal stem cells also have very good technical effects in the treatment and / or prevention of BPD. After application, the incidence of BPD in experimental animals was well controlled, lung lesions were reduced, the degree of inflammation was reduced, and pneumonia, alveoli and pulmonary fibrosis were improved.

[0089] Drug administration method and dosage

[0090] Those skilled in the art are well aware of determining the appropriate dosage of the immunotherapeutic drugs described herein, including determining the appropriate dosage based on an individual's age and weight. The immunotherapeutic drugs can be administered via various routes, such as parenteral (e.g., subcutaneous, intraperitoneal, intravenous, including intravenous infusion, intramuscular, or intradermal injection) or non-parenteral (e.g., oral, intranasal, intraocular, sublingual, rectal, or local) routes. Specifically, they can be administered to the subject via spray inhalation, infusion, intravenous infusion, oral administration, or intratracheal injection. Pharmaceutical compositions comprising the immunosuppressants of the present invention can be liquid solutions, suspensions, emulsions, pills, capsules, tablets, sustained-release formulations, or powders, etc. Oral formulations may include standard carriers, such as pharmaceutical-grade mannitol, lactose, starch, magnesium stearate, polyvinylpyrrolidone, sodium saccharin, cellulose, magnesium carbonate, etc.

[0091] In some embodiments, IVIG is administered to an individual via intravenous infusion to treat and / or prevent BPD. In some embodiments, a single dose of IVIG administered via intravenous infusion is from 200 mg / kg body weight to 2000 mg / kg body weight, such as 200 mg / kg body weight, 250 mg / kg body weight, 300 mg / kg body weight, 350 mg / kg body weight, 400 mg / kg body weight, 450 mg / kg body weight, 500 mg / kg body weight, 550 mg / kg body weight, 600 mg / kg body weight, 650 mg / kg body weight, 700 mg / kg body weight, 750 mg / kg body weight, 800 mg / kg body weight, 850 mg / kg body weight, 900 mg / kg body weight, 950 mg / kg body weight, or 1000 mg / kg body weight.

[0092] In some embodiments, JAK inhibitors are administered orally to an individual to treat and / or prevent BPD. In some embodiments, a single dose of the JAK inhibitor is from 1 mg / kg body weight to 5 mg / kg body weight, preferably from 2 mg / kg body weight to 3 mg / kg body weight, such as 2.1 mg / kg body weight, 2.2 mg / kg body weight, 2.3 mg / kg body weight, 2.4 mg / kg body weight, 2.5 mg / kg body weight, 2.6 mg / kg body weight, 2.7 mg / kg body weight, 2.8 mg / kg body weight, 2.9 mg / kg body weight, or 3 mg / kg body weight.

[0093] In some embodiments, IRAK4 inhibitors are administered orally to an individual to treat and / or prevent BPD. In some embodiments, a single dose of the IRAK4 inhibitor is from 1 mg / kg body weight to 5 mg / kg body weight, preferably from 2 mg / kg body weight to 3 mg / kg body weight, such as 2.1 mg / kg body weight, 2.2 mg / kg body weight, 2.3 mg / kg body weight, 2.4 mg / kg body weight, 2.5 mg / kg body weight, 2.6 mg / kg body weight, 2.7 mg / kg body weight, 2.8 mg / kg body weight, 2.9 mg / kg body weight, or 3 mg / kg body weight.

[0094] In some embodiments, mesenchymal stem cells are administered to an individual via intravenous infusion or intratracheal injection to treat and / or prevent BPD. In some embodiments, the dose of mesenchymal stem cells administered is 1 × 10⁻⁶. 6 -3×10 7 Cells / kg body weight, preferably 1×10⁻⁶ 6 -2×10 7 Cells / kg body weight, e.g., 1×10 6 Cells / kg body weight, 2×106 Cells / kg body weight, 3×10 6 Cells / kg body weight, 4×10 6 Cells / kg body weight, 5×10 6 Cells / kg body weight, 6×10 6 Cells / kg body weight, 7×10 6 Cells / kg body weight, 8×10 6 Cells / kg body weight, 9×10 6 Cells / kg body weight, 1×10 7 Cells / kg body weight or 2×10 7 Cells per kg of body weight.

[0095] Generally, the age of the subject treated using the method of the present invention is at least 22 weeks gestational age to about 6 months after birth, preferably at least 24 weeks gestational age to about 6 months after birth. In some embodiments, the treatment may continue until the subject is older than 6 months, for example, the treatment continues until the subject is at least 12 months old, or at least 18 months old or older. Gestational age can be determined by any conventional method. For example, gestational age can be calculated from the first day of the last menstrual period.

[0096] In some embodiments, the newborn treated using the method of the present invention is a preterm newborn with a gestational age of 32 weeks or less. In some embodiments, the newborn is a preterm newborn with a gestational age of 28 weeks or less. In some embodiments, the newborn's birth weight is about 1500g or less. In other embodiments, the newborn's birth weight is about 1000g or less.

[0097] Beneficial effects of the present invention

[0098] This invention proposes methods and applications for the treatment and prevention of BPD based on macromolecular drugs, small molecule drugs, and cell products. It will provide a new solution for the high incidence of bronchopulmonary dysplasia in newborns and is expected to reduce the incidence of neonatal BPD, improve the survival rate and long-term prognosis of children with BPD.

[0099] This invention is the first to propose novel immunosuppressive therapy for the treatment and / or prevention of neonatal bronchopulmonary dysplasia (BPD), which can rapidly improve lung function, reduce the incidence of BPD, and is more effective and has fewer side effects and adverse reactions than glucocorticoid immunosuppressants.

[0100] The present invention will be further illustrated below with reference to the accompanying drawings and specific embodiments. It should be understood that the following specific embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Example

[0101] The following examples illustrate the sources of reagents and raw materials:

[0102] Intravenous injection of porcine immunoglobulin (IVIG): prepared in the laboratory, see Example 1, porcine IVIG preparation section;

[0103] Ruxolitinib: Jakavi (Novartis);

[0104] IRAK4 inhibitor: Zabedosertib (BAY 1834845).

[0105] Example 1: Therapeutic effect of macromolecular drug IVIG in neonatal piglet bPD

[0106] This embodiment constructed a clinically relevant neonatal piglet blastocystitis (BPD) model and verified the therapeutic effect of a representative macromolecular drug, IVIG, in neonatal piglet BPD. In short, porcine IVIG was prepared from plasma collected from healthy pigs, and two treatment courses were administered using the porcine BPD model, with a 7-day interval between the two courses. Seven days after the completion of the second course, the efficacy was evaluated in the treatment group and the untreated hyperoxia group (preterm hyperoxia group, control group).

[0107] 1.1 Preparation of a porcine bronchogenic dysplasia (BPD) model (hyperoxia induction)

[0108] Panamanian pregnant sows with confirmed mating dates were selected. At 105 days of gestation, the animals were anesthetized, and piglets were delivered via cesarean section. After resuscitation and stabilization, the newborn piglets were placed in an incubator and given 80-85% oxygen. Respiratory status was closely monitored. Around 2-3 weeks after birth, piglets exhibited obvious clinical signs such as shortness of breath (approximately 80-120 breaths / minute), nasal cyanosis, respiratory distress, oxygen dependence, and difficulty feeding. Lung imaging was performed, and combined with clinical manifestations, other diseases causing respiratory distress, such as infection, were ruled out, confirming a diagnosis of bronchogenic dysphagia (BPD).

[0109] 1.2 Preparation of porcine IVIG

[0110] Plasma was collected from healthy pigs, and following the traditional low-temperature ethanol precipitation method, octanoic acid was used instead of ethanol as the precipitant. The plasma was then purified through stepwise separation. The specific steps are as follows:

[0111] (1) Preparation of FⅠ+Ⅱ+Ⅲ precipitate: Take healthy pig plasma, melt it at room temperature, and combine them; use physiological saline to adjust the plasma protein content, adjust the pH by adding glacial acetic acid and adjust the ethanol concentration by adding anhydrous ethanol, stir the reaction at -5.0℃ for 4 hours, and centrifuge after the reaction is completed to obtain FⅠ+Ⅱ+Ⅲ precipitate.

[0112] (2) Dissolution of FI+II+III precipitate: FI+II+III precipitate was dissolved in 80mM sodium acetate buffer (pH 5.0) at 4°C for 12 hours by stirring. The supernatant was then separated by centrifugation.

[0113] (3) Octyl acid precipitation: Adjust the pH of the supernatant to 5.0 with 4 mol / L acetic acid or 1 mol / L sodium hydroxide, add octyl acid at a concentration of 100 mmol / L, stir and react at 25°C for 3 hours, and then separate the supernatant by centrifugation;

[0114] (4) Caprylic acid virus inactivation: The supernatant was filtered through a 1.0 μm filter membrane, and the pH of the filtrate was adjusted to 5.0 with 4 mol / L acetic acid or 1 mol / L sodium hydroxide. Water for injection or caprylic acid was added to adjust the caprylic acid concentration of the suspension to 80 mmol / L. The mixture was stirred at 25°C for 2 hours and the supernatant was separated by centrifugation.

[0115] (5) Ethanol precipitation: Adjust the pH of the supernatant to 5.0 with 1 mol / L hydrochloric acid or sodium hydroxide, add anhydrous ethanol at a concentration of 15% to carry out the precipitation reaction, stir the reaction at -4.0℃ for 8 hours, and centrifuge to separate the supernatant.

[0116] (6) Ultrafiltration: The supernatant was filtered through a 0.45 μm filter membrane, and the filtrate was concentrated 15 to 20 times with a 30 KD ultrafiltration membrane. It was then subjected to ultrafiltration dialyzing with 10 times the volume of 50 mmol / L phosphate buffer at pH 7.0. The sample was collected after ultrafiltration, and the protein content was controlled to be 30 to 40 mg / ml.

[0117] (7) Anion exchange chromatography: Use phosphate buffer with pH 7.0 and concentration of 50 mmol / L as equilibration buffer to equilibrate the chromatography column to 8-10 column volumes. Calculate the amount of protein loaded per milliliter of packing material not exceeding 70-80% of the maximum packing material capacity. After loading, collect the permeate. Elution of column-bound proteins is eluted with 50 mmol / L phosphate buffer with pH 7.0 containing 2 mol / L NaCl.

[0118] (8) Virus removal filtration using nanomembrane: The pH of the permeate was adjusted to 5.0 with 1 mol / L hydrochloric acid. After pre-filtration through a 0.1 μm filter membrane, the virus was removed by filtration using a Novasip DV20 nanomembrane, with the filtration pressure controlled to be no greater than 0.25 MPa.

[0119] (9) Ultrafiltration: After virus removal, the filtrate is concentrated to 100 mg / ml using a 30 KD ultrafiltration membrane. After ultrafiltration with water for injection, the sample is collected, and the protein content is controlled to be no less than 100 mg / ml.

[0120] (10) Preparation: Determine the protein content of the stock solution after ultrafiltration, dilute with water for injection, add maltose (final concentration 10%), adjust the pH to 4.0 with 1mol / L hydrochloric acid, so that the protein content of the product is 50mg / ml.

[0121] 1.3 IVIG Treatment

[0122] Resuscitated 105-day-old preterm piglets with bronchogenic dysplasia (BPD) were randomly divided into a control group and a treatment group. BPD induction was performed on both groups of piglets as described above. Respiratory function was closely monitored. Preterm piglets typically had a respiratory rate of 30-50 breaths / minute. Shortness of breath (approximately 60-80 breaths / minute) began to appear around week 1, and significantly worsened around week 2 (approximately 80-120 breaths / minute), accompanied by nasal cyanosis, difficulty feeding, and oxygen dependence. Lung imaging was then performed, and BPD was diagnosed based on the symptoms and chest X-ray.

[0123] Piglets in the treatment group received intravenous infusion of IVIG after diagnosis of BPD, for a total of two courses of treatment, each lasting 5 days, once daily. The IVIG dose was 1 g / kg body weight on days 1-2 and 0.5 g / kg body weight on days 3-5. A second course of IVIG treatment was administered after a 7-day interval. Piglets in the control group received an equal volume of 10% maltose solution infusion after diagnosis of BPD, with the treatment time and cycle consistent with the treatment group. After the start of treatment, the oxygen concentration in the pigsty was gradually reduced in both groups, and respiratory symptoms were observed. Seven days after the end of the second course of treatment, necropsy was performed to collect lung tissue for histopathological analysis.

[0124] 1.4 Pathological analysis of lung tissue

[0125] Lung tissues collected from piglets in both the control and treatment groups were washed with pre-cooled physiological saline and then fixed in neutral formalin for 24 hours. The fixed lung lobes were placed in embedding casks and dehydrated according to a prescribed procedure. After dehydration, they were embedded in paraffin, and the paraffin blocks were serially sectioned using a tissue sectioning machine. Staining was performed according to the procedures of the hematoxylin and eosin (H&E) staining kit and the Masson trichrome staining kit, respectively. The radial alveolar count, alveolar inflammation, degree of fibrosis, and proportion of collagen fibers in each group were compared and analyzed.

[0126] (1) Radial alveolar count: Several independent sections were randomly selected from each piglet's slides. A vertical line was drawn from the center of the respiratory bronchioles to the nearest pleura or fibrous septum. The alveoli on the vertical line are the radial alveoli. Six non-overlapping fields of view were randomly selected from each slide, and the number of radial alveoli in each field of view was counted. The average value was calculated, which is the final radial alveolar count of the piglet to assess the degree of alveolarization.

[0127] (2) Assessment of the severity of alveolitis and fibrosis in lung tissue: Several independent slides were randomly selected from each group, and 6 non-overlapping fields of view were randomly selected from each slide. The Szapiel (Table 1) and Ashcroft scoring systems (Table 2) were used for assessment.

[0128] Table 1 Szapiel rating system

[0129] Table 2 Ashcroft Rating System

[0130] In the Ashcroft score, areas occupying more than half of the visual field are scored for fibrosis, which is recorded as the predominant level of fibrosis in that field. First, it is determined whether the tissue in the field is normal or fibrotic. If normal tissue predominates, the score is 0; if fibrotic tissue predominates, the fibrosis level of that area is scored as one-third of 1, 3, 5, 7, or 8 in Table 2. If there is any difficulty in deciding between scores of 1, 3, 5, and 7, scores of 2, 4, and 6 are given.

[0131] (3) Collagen fiber area ratio statistics: Several independent slices were randomly selected from each group, and 6 non-overlapping fields of view were randomly selected from each slice. ImageJ software was used to measure the area of ​​blue collagen fibers and tissue area respectively, and the proportion of collagen fibers was calculated and statistically analyzed.

[0132] Data analysis was performed using Graphpad Prism software. Data that conformed to a normal distribution and had homogeneous variances were analyzed using one-way ANOVA, with pairwise comparisons using t-tests. Data that conformed to a normal distribution but had unequal variances or did not conform to a normal distribution were analyzed using non-parametric tests. A p-value < 0.05 was considered statistically significant.

[0133] result

[0134] 1. Lung imaging results

[0135] Seven days after the treatment group completed the second course of treatment, chest X-rays were performed on both the treatment group and the control group to compare the lung imaging changes between the two groups after IVIG treatment. The results are shown in Figure 1, where the lung lesions in the treatment group were reduced compared to those in the control group.

[0136] 2. Lung tissue pathological results

[0137] The changes in lung structure between the control and treatment groups after IVIG treatment were compared by H&E staining of lung tissue sections. The results are shown in Figure 2. The lung tissue structure in the treatment group was improved compared to the control group, with normal alveolar structure and reduced inflammatory cell infiltration in the alveolar cavities and alveolar walls. This suggests that IVIG has a significant therapeutic effect on BPD.

[0138] 3. Masson staining

[0139] The changes in pulmonary fibrosis between the control and treatment groups after IVIG treatment were compared using Masson staining of lung tissue sections. The results are shown in Figure 3. The treatment group showed improved alveolar septal thickening and relatively less collagen fiber deposition compared to the control group. This suggests that IVIG can effectively improve the degree of pulmonary fibrosis caused by BPD.

[0140] 4. By statistically analyzing the radial alveolar count, alveolitis score, collagen fiber area ratio, and Ashcroft score of lung tissue pathological sections, the changes in alveolar number, inflammation, and fibrosis between the control and treatment groups after IVIG treatment were compared. The results are shown in Figure 4. The results show that after IVIG treatment, the alveoli of the pigs in the treatment group developed significantly, and the severity of alveolitis and fibrosis in the lung tissue was significantly improved.

[0141] Example 2: The preventive and therapeutic effects of the macromolecular drug IVIG on neonatal piglet bPD

[0142] Given the significant efficacy of IVIG in treating BPD, this embodiment primarily studies the role of IVIG in the prevention and treatment of BPD. Specifically, porcine IVIG was selected as the drug. Premature pigs were fed a high-oxygen diet and received two courses of preventative medication starting on the second day after birth, with a 7-day interval between the two courses. Seven days after completing both courses, the efficacy was evaluated in the premature hyperoxia group (control group) and the premature hyperoxia group + preventative medication group (prevention group).

[0143] Premature piglets with a gestational age of 105 days, after resuscitation, were randomly divided into a control group and a prevention group. Both groups were placed in an incubator after birth and treated with 80%–85% hyperoxia. Piglets in the prevention group received intravenous infusion of IVIG on the second day after birth for two courses, each lasting 5 days, once daily. The IVIG dose was 1 g / kg body weight on days 1-2 and 0.5 g / kg body weight on days 3-5. A second course of IVIG treatment was administered after a 7-day interval. The control group received an equal volume of 10% maltose solution concurrently. Seven days after the completion of the second course of treatment, lung tissue was dissected and collected for histopathological analysis. The methods and procedures for lung tissue histopathological analysis, as well as the statistical methods, were the same as in Example 1.

[0144] result

[0145] 1. Lung imaging results

[0146] The lung imaging changes between the control group and the prevention and treatment group after IVIG treatment were compared by chest X-ray. The results are shown in Figure 5. The diffuse high-density shadow lesions in both lungs of the prevention and treatment group were significantly reduced and alleviated.

[0147] 2. Lung tissue pathological results

[0148] The changes in lung structure between the control and treatment groups after IVIG treatment were compared using H&E staining of lung tissue sections. The results are shown in Figure 6. In the treatment group, normal alveolar structure was observed, alveolar septa were relatively intact, and improvements were seen in alveolar enlargement, increased alveolar number, atelectasis, and the degree of alveolitis. This indicates that IVIG has a good effect on preventing and treating BPD and promoting lung development.

[0149] 3. Masson staining

[0150] Masson staining of lung tissue sections was used to compare lung structural changes between the control and treatment groups after IVIG treatment. The results, shown in Figure 7, indicate that the alveolar septa were thinner and collagen fiber deposition was significantly reduced in the treatment group compared to the control group. This suggests that IVIG can effectively inhibit the progression of pulmonary fibrosis caused by bronchopulmonary dysplasia.

[0151] 4. The changes in alveolar count, inflammation, and fibrosis between the control and treatment groups after IVIG treatment were compared using lung tissue pathological sections, including radial alveolar count, alveolitis score, collagen fiber area ratio, and Ashcroft score. The results are shown in Figure 8. The results indicate that after IVIG treatment, the alveoli in the treatment group showed significant development, and the severity of alveolitis and fibrosis in the lung tissue was significantly controlled. This suggests that IVIG treatment can prevent the occurrence and development of BPD, or reduce the severity of BPD lesions.

[0152] Example 3: Prevention and treatment of BPD in newborn pigs by small molecule drugs

[0153] 3.1: The preventive and therapeutic effects of JAK inhibitors on piglet bPD

[0154] To investigate the preventive and therapeutic effects of immunosuppressive small molecule drugs on piglet bronchiectasis (BPD), ruxolitinib, a Janus-activated kinase (JAK) inhibitor, was selected as a representative drug for testing (hereinafter referred to as the JAKi group). Specifically, preterm piglets were treated with ruxolitinib (JAKi group) starting on day 2 after birth, at a dose of 2.0 mg / kg twice daily, concurrently with BPD induction. The control group was a preterm hyperoxia group. The effects of ruxolitinib on BPD were compared between the control group and the JAKi group.

[0155] Resuscitated preterm piglets with a gestational age of 105 days were randomly divided into a control group and a JAKi group. Both groups were placed in an incubator after birth and treated with 80%–85% hyperoxia. The induction procedure for BPD was the same as in Example 1. The JAKi group received oral ruxolitinib treatment starting on day 2 after birth, 2.0 mg / kg / time (mixed with 2 mL of sterile water for injection), twice daily, until the control group successfully established the BPD model, continuing for approximately 4 weeks. The control group piglets received the same amount of sterile water for injection simultaneously. Blood routine tests were monitored regularly; when the platelet count was <100 x 10⁻⁶, the platelet count was recorded. 9 Consider reducing the dosage if the platelet count is <50 x 10⁹ / L. 9 / L or absolute neutrophil count <0.5x10 9 Discontinue medication at / L. Closely monitor respiratory status. When piglets in the control group exhibit significant shortness of breath (respiratory rate of 80-120 breaths / min), nasal cyanosis, labored breathing, oxygen dependence, and difficulty feeding, perform pulmonary imaging examinations. Combine clinical manifestations to rule out respiratory distress caused by infection or other diseases, and set this as the experimental endpoint. Dissect and collect lung tissue under deep anesthesia for histopathological analysis. The methods and procedures for lung tissue histopathological analysis, as well as the statistical methods, are the same as in Example 1.

[0156] result

[0157] 1. Lung imaging results

[0158] The lung imaging changes between the control group and the JAKi group were compared by chest X-ray. The results are shown in Figure 9. Compared with the control group, the JAKi group had clearer lung markings and significantly reduced diffuse high-density shadows in both lungs.

[0159] 2. Lung tissue pathological results

[0160] The changes in lung structure between the control group and the JAKi group after ruxolitinib treatment were compared by H&E staining of lung tissue pathological sections. The results are shown in Figure 10. Compared with the control group, the JAKi group showed normal alveolar structure, more intact alveolar septa, and significant improvement in the number of alveoli, alveolar cavities, and inflammatory cell infiltration of alveolar walls.

[0161] 3. Masson staining

[0162] The changes in lung structure between the control group and the JAKi group after ruxolitinib treatment were compared by Masson staining of lung tissue pathological sections. The results are shown in Figure 11. In the control group, the alveolar septa were thickened, with more blue collagen fibers and red muscle fibers. Some alveolar cavities were covered by collagen fibers. In contrast, the alveolar septa thickening was improved in the JAKi group, and the deposition of collagen fibers and muscle fibers was significantly reduced.

[0163] 4. By statistically analyzing the radial alveolar count, alveolitis score, collagen fiber area ratio, and Ashcroft score of lung tissue pathological sections, the changes in alveolar number, inflammation, and fibrosis between the control group and the JAKi group after ruxolitinib treatment were compared. The results are shown in Figure 12. Compared with the control group, the JAKi group showed a significant increase in radial alveolar count, a significant decrease in Szapiel score, a significant decrease in collagen fiber area ratio, and a significant decrease in Ashcroft fibrosis score.

[0164] 3.2: The preventive and therapeutic effects of IRAK4 inhibitors on piglet bPD

[0165] To investigate the preventive and therapeutic effects of immunosuppressive small molecule drugs on piglet bronchiectasis (BPD), the interleukin-1 receptor-associated kinase 4 inhibitor (IRAK4i) Zabedosertib was selected as a representative drug for testing (hereinafter referred to as the IRAK4i group). Specifically, preterm piglets were treated with Zabedosertib (IRAK4i group) starting on day 2 after birth, at a dose of 2.5 mg / kg / time, twice daily, until the experimental endpoint. The control group was the preterm hyperoxia group. The preventive and therapeutic effects of BPD in piglets were compared between the control group and the IRAK4i group.

[0166] Resuscitated preterm piglets with a gestational age of 105 days were randomly divided into a control group and an IRAK4i group. Both groups were placed in an incubator after birth and treated with 80%–85% hyperoxia. The induction procedure for BPD was the same as in Example 1. The IRAK4i group received oral Zabedosertib (BAY 1834845) 2.5 mg / kg / dose (dissolved in dimethyl sulfoxide / polyethylene glycol 300 / Tween-80 / physiological saline in a ratio of 10:40:5:45) twice daily, starting on the second day after birth, until the BPD model was successfully established in the control group, which lasted approximately 4 weeks. The control group was simultaneously fed dimethyl sulfoxide / polyethylene glycol 300 / Tween-80 / physiological saline in a ratio of 10:40:5:45. Blood routine tests and liver and kidney function were monitored regularly. Closely observe respiratory status. When control group piglets exhibit obvious shortness of breath (respiratory rate of 80-120 breaths / min), nasal cyanosis, dyspnea, oxygen dependence, and worsening respiratory symptoms after feeding, screaming, or activity, lung imaging examinations are performed. Combined with clinical manifestations, other diseases causing respiratory distress, such as infection, are ruled out, and this is set as the experimental endpoint. Under deep anesthesia, lung tissue is dissected and collected for histopathological analysis. The methods and procedures for lung tissue histopathological analysis, as well as the statistical methods, are the same as in Example 1.

[0167] result

[0168] 1. Lung imaging results

[0169] The lung imaging changes between the control group and the IRAK4i group were compared by chest X-ray. The results are shown in Figure 13. The diffuse high-density lesions in both lungs were reduced in the IRAK4i group compared with those in the control group.

[0170] 2. Lung tissue pathological results

[0171] The changes in lung structure between the control group and the IRAK4i group after Zabedosertib treatment were compared by H&E staining of lung tissue pathological sections. The results are shown in Figure 14. Compared with the control group, the IRAK4i group showed that the alveolar structure was basically intact, the alveolar septa were relatively normal, and the number of alveoli, alveolar cavities and alveolar wall inflammatory cell infiltration were significantly reduced compared with the control group.

[0172] 3. Masson staining

[0173] The lung structure changes between the control group and the IRAK4i group after Zabedosertib treatment were compared by Masson staining of lung tissue pathological sections. The results are shown in Figure 15. In the control group, the alveolar structure was destroyed, the alveolar septa were thickened, and more blue collagen fibers and red muscle fibers were seen. A small part of the alveolar cavity surface was covered by collagen fibers. In contrast, the alveolar structure in the IRAK4i group was more intact, the septal thickening was not obvious, and the deposition of collagen fibers and muscle fibers was significantly reduced.

[0174] 4. By statistically analyzing the radial alveolar count, alveolitis score, collagen fiber area ratio, and Ashcroft score of lung tissue pathological sections, the changes in alveolar number, inflammation, and fibrosis between the control group and the IRAK4i group after Zabedosertib treatment were compared. The results are shown in Figure 16. Compared with the control group, the IRAK4i group showed a significant increase in radial alveolar count, a significant decrease in Szapiel score, a significant decrease in collagen fiber area ratio, and a significant decrease in Ashcroft fibrosis score.

[0175] Example 4: The preventive and therapeutic effects of cell-based drugs on piglet bacillary dysplasia (BPD)

[0176] To investigate the preventive and therapeutic effects of cell-based drugs on piglet bronchiectasis (BPD), mesenchymal stem cells (pMSCs) were selected as a representative drug for testing. Specifically, preterm piglets were infused with pMSCs starting on the second day after birth while undergoing hyperoxia induction, continuing until the experimental endpoint. The control group was the preterm hyperoxia group. The preventive and therapeutic effects of pMSCs on piglets were compared between the control group and the pMSC group.

[0177] Mesenchymal stem cells are derived from pig umbilical cords. The methods for isolating and culturing pig umbilical cord mesenchymal stem cells are as follows:

[0178] 1) Soak the pig umbilical cord tissue in 75% alcohol, shake and wash for 15-30 seconds, then quickly transfer it to PBS. Cut the umbilical cord into 3-5 cm lengths, remove the blood vessels in the umbilical cord, and carefully peel off Wharton's jelly.

[0179] 2) Transfer the peeled Wharton's gum to a centrifuge tube, cut the Wharton's gum into small tissue pieces of 0.3-0.5cm square, add an appropriate amount of collagenase, digest in a 37℃ incubator for 10-15 minutes, centrifuge at 300g for 10 minutes, and wash once with PBS;

[0180] 3) Discard the supernatant, and seed the cell suspension into culture flasks at an appropriate density. Incubate at 37°C in a 5% CO2 incubator. Replace the entire medium after 5-7 days, and then every 3 days thereafter, until the cells expand to 70%-80% confluence. Then, increase the culture rate to 8000-10000 cells / cm³. 2 Passage at inoculation density;

[0181] 4) Take pMSCs passaged to P5, culture them to the logarithmic growth phase, digest them with TrypLE, wash them with physiological saline, centrifuge at 300g for 10 min, count them, and then divide them into 1x10⁻¹ cells. 7 The dosage is calculated per cell / kg, and the cells are resuspended in an appropriate volume of physiological saline for later use.

[0182] 4.2 Mesenchymal stem cell treatment

[0183] Resuscitated preterm piglets with a gestational age of 105 days were randomly divided into a control group and a porcine umbilical cord mesenchymal stem cell (pMSC) group. Both groups of piglets were placed in an incubator after birth and subjected to 80%–85% hyperoxia, as in Example 1. The pMSC group received intravenous infusion of pMSCs starting on the second day after birth, at a dose of 1 x 102. 7 Cells / kg, twice a week, until a BPD model was successfully established in the control group, lasting approximately 4 weeks; the control group received the same volume of physiological saline infusion concurrently. Respiratory status was closely monitored. When piglets in the control group exhibited significant shortness of breath (respiratory rate of 80-120 breaths / min), nasal cyanosis, labored breathing, oxygen dependence, and difficulty feeding, lung imaging was performed. This, combined with clinical manifestations, ruled out respiratory distress caused by infection or other diseases, and was set as the experimental endpoint. Lung tissue was collected under deep anesthesia for histopathological analysis. The methods and procedures for lung tissue histopathological analysis were the same as in Example 1.

[0184] result

[0185] 1. Lung imaging results

[0186] The lung imaging changes between the control group and the pMSC group were compared by chest X-ray. The results are shown in Figure 17. The control group had diffuse patchy high-density shadows in both lungs, while the pMSC group had fewer diffuse high-density shadows in both lungs than the control group.

[0187] 2. Lung tissue pathological results

[0188] The changes in lung structure between the control group and the pMSC group after treatment with porcine umbilical cord mesenchymal stem cells were compared by H&E staining of lung tissue pathological sections. The results are shown in Figure 18. Compared with the control group, the pMSC group had normal alveolar structure, and the number of alveoli, alveolar cavities and alveolar wall inflammatory cell infiltration were all improved.

[0189] 3. Masson staining

[0190] The lung structure changes between the control group and the pMSC group after prophylactic administration of porcine umbilical cord mesenchymal stem cells were compared by Masson staining of lung tissue pathological sections. The results are shown in Figure 19. In the control group, the alveolar septa were thickened, with more blue collagen fibers and red muscle fibers, while the pMSC group showed focal alveolar septal widening and less collagen and muscle fiber deposition than the control group.

[0191] 4. By statistically analyzing the radial alveolar count, alveolitis score, collagen fiber area ratio, and Ashcroft score of lung tissue pathological sections, the changes in alveolar number, inflammation, and fibrosis between the control group and the pMSC group after prophylactic administration of porcine umbilical cord mesenchymal stem cells were compared. The results are shown in Figure 20. Compared with the control group, the pMSC group showed a significant increase in radial alveolar count, a significant decrease in Szapiel score, a significant decrease in collagen fiber area ratio, and a significant decrease in Ashcroft fibrosis score.

[0192] The present application has been described in detail above with reference to specific embodiments and exemplary examples. However, these descriptions should not be construed as limiting the present application. Those skilled in the art will understand that various equivalent substitutions, modifications, or improvements can be made to the technical solutions and implementation methods of the present application without departing from the spirit and scope of the present application, and all such modifications and improvements fall within the scope of the present application.

Claims

1. Use of an immunosuppressant in the preparation of a medicament for the treatment and / or prevention of bronchopulmonary dysplasia (BPD) in a subject, wherein the immunosuppressant is selected from immunoglobulin inhibitors, small molecule immunosuppressants, and cellular immunosuppressants.

2. The use of claim 1, wherein the subject is a newborn (e.g., a premature newborn), an infant, a child, an adolescent, or an adult.

3. The use of claim 2, wherein the premature newborn is born at a gestational age of less than 35 weeks, preferably less than or about 32 weeks.

4. The use according to any one of claims 1-3, wherein the immunoglobulin inhibitor is intravenous immunoglobulin (IVIG) or gamma globulin. Preferably, when the subject is a human subject, the immunoglobulin inhibitor is of human origin.

5. The use of any one of claims 1-3, wherein the small molecule immunosuppressant is a JAK inhibitor, such as ruxolitinib or an analogue, stereoisomer, enantiomer or pharmaceutically acceptable salt thereof.

6. Use according to any one of claims 1-3, wherein the small molecule immunosuppressant is an IRAK4 inhibitor, such as Zabedosertib or an analogue, stereoisomer, enantiomer or pharmaceutically acceptable salt thereof.

7. The use of any one of claims 1-3, wherein the cell-based immunosuppressant is a mesenchymal stem cell (MSC) or an immunomodulatory cell, for example, the MSC is selected from umbilical cord-derived mesenchymal stem cells, umbilical cord blood-derived mesenchymal stem cells, adipose-derived mesenchymal stem cells, peripheral blood-derived mesenchymal stem cells, and mesenchymal stem cells differentiated from pluripotent stem cells.

8. Use according to any one of claims 1-7, wherein the medicament further comprises a pharmaceutically acceptable carrier.

9. Use according to any one of claims 1-8, wherein the drug is formulated for administration to the subject by intravenous infusion, intramuscular injection, oral administration, spraying, or intratracheal injection.

10. The use of any one of claims 1-8, wherein the treatment and / or prevention further comprises assessing the efficacy by detecting respiratory function indicators, lung histopathology, immunohistochemical indicators, and / or lung imaging features of the subject. Optionally, the lung tissue pathological features are selected from one or more of the following: radial alveolar count, alveolitis score, collagen fiber area ratio, and Ashcroft fibrosis score.

11. A method for treating and / or preventing bronchopulmonary dysplasia (BPD) in a subject, comprising administering to the subject an effective amount of an immunoglobulin inhibitor, a small molecule immunosuppressant, or a cellular immunosuppressant; Optionally, the immunoglobulin inhibitor is intravenous immunoglobulin (IVIG) or gamma globulin; preferably, when the subject is a human subject, the immunoglobulin inhibitor is human-derived. Optionally, the small molecule immunosuppressant is a JAK inhibitor, such as ruxolitinib or its analogues, stereoisomers, enantiomers or pharmaceutically acceptable salts. Optionally, the small molecule immunosuppressant is an IRAK4 inhibitor, such as Zabedosertib or its analogues, stereoisomers, enantiomers or pharmaceutically acceptable salts. Optionally, the cell-based immunosuppressant is a mesenchymal stem cell (MSC) or an immunomodulatory cell, for example, the MSC is selected from umbilical cord-derived mesenchymal stem cells, umbilical cord blood-derived mesenchymal stem cells, adipose-derived mesenchymal stem cells, peripheral blood-derived mesenchymal stem cells, and mesenchymal stem cells differentiated from pluripotent stem cells.

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