Use of lithium carbonate in treatment of pulmonary fibrosis

Abstract

Provided is use of lithium carbonate in the treatment of pulmonary fibrosis. In particular, provided is use of lithium carbonate or a pharmaceutical composition containing the same in the preparation of a drug for treating pulmonary fibrosis. Provided is a method for regulating the phenotype of alveolar macrophages in vitro. The method comprises the following steps: 1) acquiring alveolar macrophages; and 2) making the alveolar macrophages in contact with lithium carbonate or a pharmaceutical composition containing the same.

Description

Use of lithium carbonate in the treatment of pulmonary fibrosis Technical Field

[0001] The present application relates to the fields of biomedicine and chronic disease treatment, and more specifically, to the use of lithium carbonate in the treatment of pulmonary fibrosis. Background Art

[0002] Pulmonary fibrosis (PF) is a chronic, progressive interstitial lung disease (ILD) in which lung tissue thickens, stiffens, or forms scar tissue over time. Its pathological characteristics are damage to alveolar epithelial cells, massive proliferation and activation of fibroblasts, and excessive deposition of extracellular matrix (ECM), which ultimately lead to destruction of lung structure and loss of respiratory function. Common causes of PF include long-term smoking, environmental factors, chronic bronchitis, etc. The mortality rate of PF is high, with the median survival time of diagnosed patients being only 2-4 years, and the survival rate within 5 years being less than 30%. Its mortality rate is higher than that of most tumors, and it is called a "tumor-like disease." Currently, there is still a lack of specific treatments for pulmonary fibrosis in clinical practice, so pulmonary fibrosis is called "cancer that is not cancer."

[0003] Pulmonary fibrosis is a highly coordinated process that integrates multiple cell types and signaling mechanisms across organ systems. Distinct triggers, such as burns, infection, autoimmunity, surgical and nonsurgical wounds, foreign bodies, and tumors, converge on similar fibrotic pathways. Initiation of the wound healing process by one of these triggers triggers an inflammatory response, ultimately recruiting fibroblasts and activating a subset of cells (myofibroblasts) to deposit ECM in the form of collagen and other proteins. While wound healing typically resolves with apoptosis of myofibroblasts, profibrotic activators and myofibroblasts persist in fibrotic disease states. PF has long been considered the result of chronic inflammation. Following tissue injury, epithelial and / or endothelial cells release inflammatory mediators, initiating an antifibrinolytic coagulation cascade and leading to thrombus formation. This is followed by an inflammatory and proliferative phase, during which leukocytes are recruited, activated by chemokines and growth factors, and induced to proliferate. Activated leukocytes further secrete and release profibrotic cytokines. Stimulated epithelial cells, endothelial cells, and myofibroblasts also produce matrix metalloproteinases, which damage the basement membrane and produce additional cytokines and chemokines to recruit and activate neutrophils, macrophages, T cells, B cells, and eosinophils, which are essential components of tissue repair. Activated macrophages and neutrophils clear tissue debris, dead cells, and invading organisms. In the early stages of inflammation, myofibroblasts, a key effector cell of PF, produce large amounts of ECM proteins, and endothelial cells form new blood vessels, promoting the development of fibrosis.

[0004] According to statistics, the annual incidence of PF is 0.07%-0.1% per 100,000 people, which estimates that there are over 300,000 PF patients in my country. PF patients also suffer from severe lung function impairment, ultimately leading to respiratory failure, which severely impacts their quality of life and can even be life-threatening. Currently, the main marketed drugs for PF are pirfenidone and nintedanib. Pirfenidone, approved for marketing in Japan in 2008, is a pyridine compound with pleiotropic effects. Its precise mechanism of action is not fully understood, but it primarily exerts anti-inflammatory, anti-fibrotic, and antioxidant properties by downregulating pro-fibrotic growth factors (including PDGF and TGF-β). It can also slow the rate of decline in forced vital capacity (FVC). However, side effects include photosensitivity, fatigue, rash, gastric discomfort, and anorexia. Patients are also at risk of liver dysfunction, requiring regular liver function monitoring. Nintedanib is a multi-targeted tyrosine kinase inhibitor that inhibits platelet-derived growth factor receptor (PDGFR), vascular endothelial growth factor receptor (VEGFR), and fibroblast growth factor receptor (FGFR). It can significantly reduce the absolute decrease in FVC in patients with pulmonary fibrosis (PF), alleviating disease progression to some extent. Common adverse reactions include diarrhea and nausea, which are generally mild. Some patients may experience abnormal liver function, which should be monitored regularly during medication, and the dosage should be reduced or discontinued as appropriate. Furthermore, numerous studies have shown that current treatment strategies that suppress immune inflammatory responses are not effective for all pulmonary fibrosis conditions and may even be harmful to PF patients. With the exception of lung transplantation, current medications can only alleviate PF symptoms but cannot fundamentally reverse or cure the disease. This is primarily due to the limited understanding of the molecular mechanisms underlying pulmonary fibrosis. Therefore, further research into the pathogenesis of PF is needed to provide new targets for its treatment and facilitate the development of new drugs for the treatment of PF.

[0005] Alveolar macrophages (AMs) are currently considered the core driving force behind the pathological changes in pulmonary fibrosis. Macrophages exhibit remarkable plasticity, allowing them to develop into either the M1 or M2 pathway upon activation. M1 macrophages antagonize fibrosis by secreting TNF-α, IL-6, and NO, and releasing matrix metalloproteinases (MMPs) to degrade the extracellular matrix. M2 macrophages, on the other hand, promote fibrosis by releasing IL-10 and TGF-β. In normal human bronchoalveolar lavage fluid, over 90% of immune cells are AMs, all of which express CD206 (an M2 molecular marker), favoring an M2 phenotype. It is now clear that M2 AMs stimulate fibroblast differentiation into myofibroblasts by releasing TGF-β and PDGF, promoting ECM deposition, and inhibiting ECM degradation by producing tissue inhibitors of metalloproteinases (TIMPs). Therefore, drugs targeting AMs for the treatment of PF have been explored in mouse models and clinical trials. However, while AM-targeted treatments for PF have shown some efficacy in mouse models of acute pulmonary fibrosis, they have shown minimal efficacy in models of established pulmonary fibrosis. Compared to AM, myofibroblasts are the ultimate executors of PF, destroying lung structure by producing large amounts of ECM in the alveoli and interstitium. Therefore, targeting myofibroblasts has become a strategy for treating PF. In lung tissue, fibroblasts can differentiate into α-SMA+ myofibroblasts under the action of TGF-β, producing more collagen and distorting the lung parenchyma structure through their contractile movements. In addition, during the normal repair process, myofibroblasts are eliminated through apoptosis. In contrast to the normal regression process, in situ studies of PF patient lung fibrosis tissue and bleomycin-induced mouse lung fibrosis have shown that myofibroblasts are resistant to apoptosis and accumulate in the lung parenchyma, or that myofibroblasts enter a senescent state to persist, which has become a major obstacle to the treatment of PF. Therefore, the urgent need to find new drugs to target myofibroblast cell death is becoming a new therapeutic strategy to reverse established pulmonary fibrosis.

[0006] In this application, the inventors provide evidence that lithium carbonate (LC), a clinically used mood stabilizer, can polarize AMs to the M1 phenotype and, in combination with TNF-α released by AMs, induce myofibroblast death, thereby reversing pulmonary fibrosis.

[0007] Summary of the Invention

[0008] In one aspect, the present application provides the use of lithium carbonate in the treatment of pulmonary fibrosis. Specifically, the present application provides the use of lithium carbonate or a pharmaceutical composition containing the same in the preparation of a medicament for treating pulmonary fibrosis, preferably idiopathic pulmonary fibrosis. In another aspect, the present application provides a method for treating pulmonary fibrosis, comprising administering a therapeutically effective amount of lithium carbonate or a pharmaceutical composition containing the same to a subject in need thereof.

[0009] In another aspect, the present application provides a method for regulating the phenotype of alveolar macrophages in vitro, the method comprising the following steps:

[0010] 1) obtaining alveolar macrophages; and

[0011] 2) contacting the alveolar macrophages with lithium carbonate or a pharmaceutical composition containing lithium carbonate.

[0012] In some embodiments, the drug promotes the conversion of the phenotype of alveolar macrophages from M2 to M1; promotes the release of inflammatory cytokine TNF-α, and induces myofibroblast death in combination with TNF-α.

[0013] In other embodiments, the drug reduces one or more of the following: collagen deposition, type I collagen expression, and hydroxyproline content in the lung.

[0014] In other embodiments, the drug improves one or more of the following: decreased lung volume levels, decreased lung space levels, decreased number of alveoli, decreased blood oxygen saturation levels, increased respiratory rate, and collapse of the lungs compared to normal levels.

[0015] The term "treat" generally refers to eliminating the disease, arresting the progression of the disease, slowing the progression of the disease, reducing the duration of one or more symptoms associated with the disease, improving or reversing at least one measurable parameter associated with the disease, or increasing the survival of subjects suffering from the disease.

[0016] The medicine or pharmaceutical composition of the present application can be conveniently presented in unit dosage form. The medicine or pharmaceutical composition of the present application can be formulated into any suitable dosage form, such as, but not limited to, injection, tablet, capsule, gel, etc. The medicine or pharmaceutical composition of the present application can also be formulated into a suspension in an aqueous, non-aqueous or mixed medium. The medicine or pharmaceutical composition of the present application includes, but is not limited to, solutions, emulsions, foams and liposome-containing formulations. The pharmaceutical composition of the present application may include one or more penetration enhancers, carriers, and excipients.

[0017] In other embodiments, the pharmaceutical composition contains a therapeutically effective amount of lithium carbonate as an active ingredient and one or more pharmaceutically acceptable carriers or excipients.

[0018] The lithium carbonate of the present application can be used in combination with other active ingredients, as long as they do not produce other adverse effects, such as allergic reactions. The lithium carbonate of the present application can be used as the sole active ingredient or in combination with other drugs. Combination therapy is achieved by administering the individual therapeutic components simultaneously, separately or sequentially.

[0019] In another specific embodiment, lithium carbonate or a pharmaceutical composition containing the same is administered to a subject in a therapeutically effective amount.

[0020] The terms "patient," "subject," "individual," and the like are used interchangeably herein and refer to any human or non-human animal or cell thereof, preferably a human or non-human mammal, that can be amenable to the methods described herein. In specific embodiments, the non-human mammal includes, for example, a camel, a donkey, a zebra, a cow, a pig, a horse, a goat, a sheep, a cat, a dog, a rat, a rabbit, a guinea pig, a mouse, a non-human primate. In specific embodiments, the subject is a human. In specific embodiments, the subject is susceptible to, suspected of having, or has pulmonary fibrosis, preferably idiopathic pulmonary fibrosis.

[0021] The term "administering" can refer to providing a predetermined substance to a subject by any appropriate method. The term "therapeutically effective amount" can refer to an amount of an active ingredient or a pharmaceutical composition that induces an animal or human to exhibit a biological or medical response that is considered by the researcher, veterinarian, doctor, or other clinician, and such an amount can include an amount of an active ingredient or a pharmaceutical composition for inducing alleviation of a disease or a disorder to be treated. It will be obvious to those skilled in the art that the therapeutically effective dose and the number of administrations of the effective ingredient of the present application can vary depending on the desired effect. The administration amount or intake amount can be administered in various administration doses and methods, by distributing the composition according to the body weight, age, sex, health status, diet, administration time, administration method, excretion rate, and severity of the disease of the subject, for example, once a day or multiple times a day.

[0022] The drug or pharmaceutical composition of the present application can be administered by any general route as long as it can reach the target tissue. In other embodiments, the drug is formulated into a dosage form suitable for delivery to the lung by intravenous, intra-arterial, subcutaneous, intramuscular, intraperitoneal, oral, inhalation, intranasal, bronchoscopic, thoracotomy route.

[0023] The present inventors have found through a large number of experimental studies that lithium carbonate can convert the phenotype of alveolar macrophages from M2 type to M1 type and up-regulate the expression of inflammatory cytokine TNF-α, and can induce lung fibroblast death in combination with TNF-α, lithium carbonate can reduce the deposition of collagen in the lung, the expression of type I collagen, and the content of hydroxyproline in the bleomycin-induced pulmonary fibrosis model. BRIEF DESCRIPTION OF DRAWINGS

[0024] FIGS. 1A, 1B, and 1C show that lithium carbonate can convert the phenotype of alveolar macrophages in vitro from M2 type to M1 type and up-regulate the expression of inflammatory cytokine TNF-α.

[0025] FIGS. 2A and 2B show that lithium carbonate can induce death of myofibroblasts in vitro in combination with TNF-α.

[0026] Figure 3 shows that lithium carbonate can reduce the deposition of collagen in the lung of a bleomycin-induced lung fibrosis model.

[0027] Figure 4 shows that lithium carbonate can reduce the expression of collagen type I in the lung of a bleomycin-induced lung fibrosis model.

[0028] Figure 5 shows that lithium carbonate can reduce the content of hydroxyproline in the lung of a bleomycin-induced lung fibrosis model. DETAILED DESCRIPTION

[0029] The present application is further described by the following specific examples, which should not be construed as limiting the scope of the present application.

[0030] Various cell lines, drugs and experimental animals used in the examples:

[0031] Cell lines: primary lung fibroblasts and alveolar macrophages were obtained from C57BL / 6J mouse lung tissue;

[0032] Drugs: bleomycin was purchased from Nippon Kayaku, Cat# ECS001668;

[0033] Animals: C57BL / 6J mice were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.

[0034] Example 1: Lithium carbonate can convert the phenotype of alveolar macrophages in vitro from M2 to M1 type and up-regulate the expression of inflammatory cytokine TNF-a.

[0035] 1. Experimental procedure

[0036] Mouse alveolar macrophages were taken and plated at 5 x 10 4 cells per well in a 24-well plate with DMEM medium supplemented with 15% fetal bovine serum (FBS) and penicillin / streptomycin, and incubated in a 37°C incubator with 5% CO2. After 12h of incubation with IL-4 (10 ng / mL), the M2 type AM was obtained, and then 15mM lithium carbonate (M2 or LC) was added to the experimental wells or not to the control wells for 24h.

[0037] RNA sample collection: 1 mL of TRIZOL (Thermo Fisher Scientific, Cat# 15596018) was added to each well to extract RNA, and reverse transcription was performed. The expression levels of M2 phenotype related genes Tgfb, Arg1, Mrc1 and M1 phenotype related genes Nos2, Tnf of macrophages were analyzed by qPCR.

[0038] Supernatant cytokine content detection: The cell supernatant was collected for cytokine content detection. The levels of TNF-a and TGF-b in the cell supernatant were measured using ELISA kits (ABclonal, Cat# RK00027; ABclonal, Cat# RK00057).

[0039] 2. Experimental results

[0040] qPCR results showed that the transcriptional level of M2 phenotype related genes in alveolar macrophages after lithium carbonate treatment decreased, and the transcriptional level of M1 phenotype related genes increased (Figure 1A).

[0041] ELISA results showed that the secretion level of TGF-b in alveolar macrophages after lithium carbonate treatment decreased, and the secretion level of TNF-a increased (Figure 1B, Figure 1C).

[0042] Example 2: Lithium carbonate can induce the death of myofibroblasts in combination with TNF-a.

[0043] 1. Experimental steps

[0044] After the mice were anesthetized and executed by breaking the neck, they were cleaned with an alcohol wet wipe. The mouse chest was opened, and the lung lobes were taken out aseptically and cut into pieces in sterile PBS. After two washes, the lung lobes were cut into 1 mm-3 mm fragments in culture medium. The tissue was centrifuged at 600 g for 10 minutes, and the particles were resuspended in DMEM with 15% FBS added to a 10 cm culture dish. After the adherent cells were collected for subculture after 4-5 days of culture, the cells were grown to the appropriate density, and 15 mM lithium carbonate and / or 20 ng / mL TNF-a (PeproTech, Cat# 315-01A) were added to each well for 24 h of combined culture.

[0045] The cells were digested with trypsin without EDTA, resuspended with PBS containing PI (BioLegend, Cat# 421301), and stained at room temperature for 15 minutes. After neutralization and centrifugation, the cells were resuspended with PBS, and cell death was analyzed using a FACS flow cytometer. PI-stained negative cells were considered to be live cells.

[0046] 2. Experimental results

[0047] After the addition of lithium carbonate or TNF-a alone, there was no significant change in the death rate of myofibroblasts, but after the addition of lithium carbonate and TNF-a at the same time, the death rate of myofibroblasts was significantly increased, indicating that lithium carbonate combined with TNF-a induced the death of myofibroblasts (Figure 2A and Figure 2B).

[0048] Example 3: Lithium carbonate can reduce the deposition of collagen in the lung in a bleomycin-induced pulmonary fibrosis model.

[0049] 1. Experimental procedures

[0050] Bleomycin (BLM) was found to induce pulmonary fibrosis in patients in clinical applications, and is the most commonly used drug to induce pulmonary fibrosis animal models. BLM can be administered by intratracheal, intraperitoneal, subcutaneous, intravenous and inhalation, the most commonly used is tracheal intubation single dose, usually 3-5 mg / kg. After single dose of BLM, acute inflammatory response will last for 7 days, on the 8th day, inflammation will change to fibrosis, and after 28 days, tissue matrix deposition occurs, reaching the peak of fibrotic response.

[0051] We used the most recognized fibrosis modeling method. 8-week-old male C57BL / 6J mice were given 3 mg / kg bleomycin (BLM) or normal saline by tracheal intubation for 14 days to construct a pulmonary fibrosis mouse model and a model control, and then injected intraperitoneally with 75 mg / kg lithium carbonate (LC) or ddH2O (vehicle) for 10 days. The mice in each group were sacrificed after anesthesia by cervical dislocation.

[0052] The lung tissue was collected and fixed in 4% paraformaldehyde (Servicebio, Cat#P1110) at room temperature for 24 h, and the lung tissue was embedded in paraffin and sectioned. Masson was used to stain the lung tissue for lung collagen to evaluate the degree of lung collagen deposition.

[0053] 2. Experimental results

[0054] The Masson staining results showed that after lithium carbonate treatment, the lung collagen deposition of the bleomycin-induced pulmonary fibrosis model was significantly reduced (Figure 3).

[0055] Example 4: Lithium carbonate can reduce the expression of type I collagen in the lung of a bleomycin-induced pulmonary fibrosis model.

[0056] 1. Experimental procedures

[0057] 8-week-old male C57BL / 6J mice were given 3 mg / kg bleomycin (BLM) or normal saline by tracheal intubation for 14 days to construct a pulmonary fibrosis mouse model and a model control, and then injected intraperitoneally with 75 mg / kg lithium carbonate (LC) or ddH2O (vehicle) for 10 days. The mice in each group were sacrificed after anesthesia by cervical dislocation.

[0058] Pulmonary tissues were collected for immunohistochemistry (IHC) staining. Pulmonary tissues were incubated with Col1a1 specific antibody (Abeam, Cat# ab34710) at 4°C overnight. After incubation with secondary antibody (Invitrogen, Cat# A-21206) for 20 minutes, the tissues were stained with horseradish peroxidase (HRP)-conjugated streptavidin for 20 minutes and developed using DAB solution. The tissues were counterstained with hematoxylin for 10 seconds and washed in distilled water for 10 minutes. The tissues were gradually dehydrated by sequential immersion in 80%, 95% and 100% ethanol. The tissues were then cleared by placing them in clearing agent xylene, which dissolves both alcohol and paraffin, and replacing the alcohol in the tissues with xylene. After mounting with resin, the tissues were observed and photographed by upright microscope (Olympus Microsystem).

[0059] 2. Experimental results

[0060] The results of immunohistochemistry staining showed that the pulmonary collagen type I in bleomycin-induced pulmonary fibrosis model was significantly reduced (Figure 4).

[0061] Example 5: Lithium carbonate can reduce the content of hydroxyproline in the lung of bleomycin-induced pulmonary fibrosis model.

[0062] 1. Experimental procedure

[0063] The conventional methods for pulmonary fibrosis evaluation include biochemical quantification of collagen (hydroxyproline content as a surrogate), histological evaluation of fibrosis distribution (Masson staining) and gene expression analysis of fibrosis markers. Hydroxyproline is one of the main components of collagen tissue and is a unique amino acid in collagen, which is an important biomarker of pulmonary fibrosis.

[0064] 8-week-old male C57BL / 6J mice were given 3 mg / kg bleomycin (BLM) or normal saline by tracheal intubation for 14 days to induce pulmonary fibrosis in mice and model controls, and then treated with 75 mg / kg lithium carbonate (LC) or ddH2O (vehicle) by intraperitoneal injection for 10 days. After anesthesia, the mice in each group were sacrificed by cervical dislocation.

[0065] Pulmonary tissues were collected and weighed, and 2 mL of alkaline hydrolysis solution was added. The tissues were boiled at 95°C for 20 minutes with constant mixing. The pH was adjusted to 6.0-6.8 using reagents provided by a commercial hydroxyproline measurement kit (purchased from Nanjing Jiancheng Biological Engineering Institute, Cat# A030-2). After adsorption with activated carbon, about 4 mL of supernatant was collected for measurement. The hydrolysate was centrifuged at 15000 rpm for 20 minutes at 4°C. Then about 1 mL of supernatant was taken according to the kit instructions for measurement of pulmonary hydroxyproline.

[0066] 2. Experimental results

[0067] The detection results of the hydroxyproline measurement kit showed that the content of hydroxyproline in the lung fibrosis model induced by bleomycin was significantly decreased after lithium carbonate treatment (Figure 5).

[0068] In summary, the scheme of the present application has the following effects: the new treatment strategy of using lithium carbonate to treat pulmonary fibrosis, using the effect of lithium carbonate on transforming the phenotype of alveolar macrophages, transforming M2 type alveolar macrophages into M1 type, to prevent the promoting effect of M2 type alveolar macrophages on pulmonary fibrosis, and antagonizing pulmonary fibrosis through M1 type alveolar macrophages. Lithium carbonate promotes the release of TNF-α by alveolar macrophages, thereby mediating the death of myofibroblasts in combination with TNF-α, reversing pulmonary fibrosis, reducing collagen deposition, type I collagen expression and hydroxyproline content in the lung, thereby achieving the treatment of pulmonary fibrosis, and having good clinical application prospect. Lithium carbonate has been approved for marketing in China for a long time, and its safety and reliability have been verified, and can be directly applied to human body.

Claims

1. Use of lithium carbonate or a pharmaceutical composition containing the same in the manufacture of a medicament for the treatment of pulmonary fibrosis.

2. Use according to claim 1, wherein the pulmonary fibrosis is idiopathic pulmonary fibrosis.

3. Use according to claim 1, wherein the medicament promotes a phenotypic conversion of alveolar macrophages from M2 type to Ml type; promotes the release of inflammatory cytokine TNF-α, and induces myofibroblast death in conjunction with TNF-α.

4. Use according to claim 1, wherein the medicament reduces one or more selected from the group consisting of: deposition of collagen in the lung, expression of collagen type I, and hydroxyproline content.

5. Use according to claim 1, wherein the medicament improves one or more selected from the group consisting of: reduced lung volume level compared to normal level, reduced lung space level, reduced alveolar number, reduced blood oxygen saturation level, increased respiratory rate, and atrophy of the lung.

6. Use according to claim 1, wherein the pharmaceutical composition contains a therapeutically effective amount of lithium carbonate as an active ingredient, and one or more pharmaceutically acceptable carriers or excipients.

7. Use according to claim 1, wherein the medicament is formulated in a dosage form suitable for delivery to the lung via intravenous, intra-arterial, subcutaneous, intramuscular, intraperitoneal, oral, inhalation, intranasal, bronchoscopy, and thoracotomy routes.

8. A method of modulating alveolar macrophage phenotype in vitro, the method comprising the steps of: 1) obtaining alveolar macrophages; and 2) contacting the alveolar macrophages with lithium carbonate or a pharmaceutical composition containing the same. ​

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