Use of deaminotyrosine for the preparation of a medicament for the treatment of pulmonary fibrosis

Abstract

This invention provides the application of deaminotyrosine in the preparation of drugs for treating pulmonary fibrosis, relating to the field of pharmaceutical technology. This invention reveals the mechanism by which deaminotyrosine intervenes in the spatiotemporal evolution and cross-sectoral communication of pulmonary fibrosis: in the early stages of macrophage inflammatory shaping, deaminotyrosine strongly blocks p65 nuclear translocation and inhibits the release of microenvironmental inflammatory factors (such as IL-6), thereby preventing the downregulation of P2RY1 receptors on the macrophage surface due to inflammatory toxicity; in the pro-fibrotic phase, the preserved P2RY1 receptors act as a "brake," suppressing STAT6 hypersensitive activation and blocking the secretion of pro-fibrotic factors (FN1 and SPP1). By disrupting paracrine communication from macrophages to fibroblasts, deaminotyrosine effectively inhibits the activation of lung fibroblasts and the deposition of extracellular matrix. This invention provides a novel, safe, and effective candidate drug for the treatment of pulmonary fibrosis, possessing significant clinical development value.

Description

Technical Field

[0001] This invention provides the application of deaminotyrosine in the preparation of drugs for treating pulmonary fibrosis, belonging to the field of pharmaceutical technology. Background Technology

[0002] Pulmonary fibrosis (PF), especially idiopathic pulmonary fibrosis (IPF), is a chronic, progressive, irreversible interstitial lung disease. The pathological mechanism involves excessive proliferation of fibroblasts / myofibroblasts and excessive deposition of extracellular matrix in lung tissue, ultimately destroying the lung parenchyma structure and causing loss of lung function. Patients with pulmonary fibrosis have an extremely poor prognosis, with a median survival of only 3-5 years after diagnosis.

[0003] Currently, the main drugs approved for the clinical treatment of idiopathic pulmonary fibrosis include nintedanib and pirfenidone. Nintedanib is a tyrosine kinase inhibitor, while pirfenidone has both anti-fibrotic and anti-inflammatory effects. However, these drugs can only slow disease progression and cannot reverse pulmonary fibrosis. They also have significant side effects such as diarrhea, hepatotoxicity, and photosensitivity, leading to poor patient tolerance. Therefore, there is an urgent need in the clinical field to develop novel, highly effective, and safe anti-pulmonary fibrosis drugs. Summary of the Invention

[0004] Based on this, the present invention provides the application of deaminotyrosine in the preparation of drugs for treating pulmonary fibrosis. Deaminotyrosine is a decarboxylation product of tyrosine and a naturally occurring biogenic amine in organisms. This invention is the first to discover and demonstrate that deaminotyrosine can effectively inhibit key pathological processes of pulmonary fibrosis through a novel mechanism of blocking pathological communication between macrophages and fibroblasts.

[0005] This invention provides the use of deaminotyrosine or a pharmaceutically acceptable salt, solvate or prodrug thereof in the preparation of medicaments for the prevention and / or treatment of pulmonary fibrosis.

[0006] The pulmonary fibrosis mentioned herein refers to idiopathic pulmonary fibrosis, silicosis, radiation-induced pulmonary fibrosis, or drug-induced pulmonary fibrosis.

[0007] This invention elucidates, for the first time through in vitro and in vivo experiments, a novel mechanism of action of deaminotyrosine against pulmonary fibrosis, mainly manifested in the following stages: (1) Early defense and NF-κB blockade (pretreatment intervention stage): In the early stage of stimulation by microenvironment damage factors (such as LPS), deaminotyrosine is used to intervene in advance, which strongly blocks the translocation of p65 (NF-κB core transcription factor) in macrophages into the cell nucleus, thus inhibiting the outbreak of early inflammatory factors such as IL-6 / TNF-α from the source.

[0008] (2) Mechanism bridging and P2RY1 receptor protection: The early burst of inflammatory factors such as IL-6 in macrophages can lead to paracrine toxicity, which directly causes the degradation of P2RY1 receptors on their surface. Deaminotyrosine successfully protects and maintains the expression of P2RY1 receptors in subsequent pathological processes through its early anti-inflammatory effects.

[0009] (3) Late-stage endogenous braking and transcriptional remodeling: Upon encountering late-stage fibrotic stimulation such as IL-4, the successfully preserved P2RY1 receptor played a crucial "endogenous braking" role. P2RY1 strongly suppressed the phosphorylation hypersensitive burst of STAT6. After STAT6 inhibition, the expression and secretion of fibronectin (FN1) and osteopontin (SPP1) were significantly reduced, completely eliminating paracrine pathogenic communication to fibroblasts. The transformation of lung fibroblasts into myofibroblasts was blocked, collagen deposition was significantly weakened, and the progression of IPF was ultimately successfully halted.

[0010] Compared with the prior art, the present invention has the following beneficial effects: In a bleomycin-induced mouse model of pulmonary fibrosis, deaminotyrosine treatment significantly reduced inflammation and fibrosis. This invention reveals for the first time that deaminotyrosine intervenes in the pathological process of pulmonary fibrosis through a multi-target mechanism of "macrophage-fibroblast crosstalk," which differs from the mechanism of existing drugs that only act on fibroblasts, thus providing a possibility for combination therapy. Furthermore, deaminotyrosine is expected to have good biocompatibility, high safety, and relatively low formulation development difficulty. Attached Figure Description

[0011] Figure 1 HE staining and Masson staining of bleomycin-induced mouse lung tissue sections for deaminotyrosine, and the content of hydroxyproline in lung tissue.

[0012] Figure 2 This study aimed to investigate the intervention of deaminotyrosine on the early inflammatory microenvironment of macrophages and the maintenance of P2RY1 receptor expression.

[0013] Figure 3 To verify the mechanism by which deaminotyrosine (DAT) suppresses STAT6 activation and remodels the fibrinolytic transcriptional program.

[0014] Figure 4 The result of deaminotyrosine (DAT) blocking macrophage-fibroblast paracrine communication and inhibiting fibroblast activation. Detailed Implementation

[0015] To make the objectives, technical solutions, and advantages of this invention clearer, the preferred embodiments of this invention will be described in further detail below with reference to the examples. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without inventive effort are within the scope of protection of this invention.

[0016] Example 1: Therapeutic effect of deaminotyrosine on bleomycin-induced mouse pulmonary fibrosis model Animal model establishment: C57BL / 6 mice were randomly divided into a sham-operated group, a model group (bleomycin administered at 3 mg / kg), a low-dose deaminotyrosine group (50 mM), and a high-dose deaminotyrosine group (100 mM per day). The model was established by intravenous infusion of bleomycin via endotracheal intubation.

[0017] Dosage regimen: Starting from day 1 after model establishment, patients can freely drink water containing DAT daily for 28 days.

[0018] Result evaluation: Pathology: Lung tissue sections were taken and stained on day 28. The model group showed extensive alveolar structural destruction, inflammatory cell infiltration, and blue collagen deposition. In the deaminotyrosine treatment group, especially the high-dose group, the degree of inflammation and fibrosis was significantly reduced.

[0019] Biochemical indicators: The content of hydroxyproline in lung tissue of the deaminotyrosine treatment group was significantly lower than that of the model group (p<0.01).

[0020] Example 2: Intervention of deaminotyrosine on the early inflammatory microenvironment of macrophages and maintenance of P2RY1 receptor expression Cell model establishment: RAW264.7 cells were used to construct an in vitro macrophage inflammation model by administering LPS stimulation.

[0021] Dosage regimen: Macrophages were pretreated with DAT for 12 hours before being stimulated with LPS to induce inflammation.

[0022] Result evaluation: Inflammatory pathway level: Immunofluorescence and protein detection results showed that p65 translocated extensively into the cell nucleus in the model group, and the inflammatory pathway was significantly activated; DAT pretreatment group could significantly block p65 nuclear translocation and effectively inhibit the activation of the inflammatory pathway.

[0023] Pro-inflammatory factor levels: In the model group, a large number of pro-inflammatory factors such as IL-6 and TNF-α were observed in the macrophage microenvironment, resulting in a severe inflammatory response; DAT pretreatment group could significantly inhibit the abnormal increase of pro-inflammatory factors and significantly improve the inflammatory microenvironment of cells.

[0024] Receptor expression level: LPS-induced high levels of inflammatory factors are the main cause of downregulation of P2RY1 receptor expression and receptor deprivation; DAT pretreatment can effectively rescue and stabilize the expression level of macrophage P2RY1 receptor by inhibiting inflammatory outbreaks.

[0025] Example 3: Deaminotyrosine suppression of STAT6 activation and remodeling of fibrinolytic transcription program Cell model establishment: RAW264.7 cells were used to construct an in vitro model of sequential stimulation (LPS-Primed) by combining LPS with IL-4.

[0026] Drug administration regimen: DAT pretreatment was used to maintain stable expression of the P2RY1 receptor in macrophages, followed by sequential stimulation with LPS and then IL-4 to induce macrophage fibrosis activation. A P2RY1 overexpression (P2RY1-OE) positive intervention group was set up to verify target dependence.

[0027] Result evaluation: Endogenous regulatory protein expression: P2RY1 receptor expression was absent in macrophages in the model group, while high levels of P2RY1 receptor expression were maintained in macrophages in the DAT intervention group.

[0028] STAT6 activation level: Sequential stimulation can induce significant phosphorylation and hypersensitive activation of STAT6 in macrophages in the model group, and abnormal activation of the fibrinolytic transcriptional program; DAT intervention can effectively suppress the abnormal phosphorylation and activation of STAT6 and block the activation of the fibrinolytic transcriptional pathway.

[0029] Fibrinolytic factor expression and secretion: The expression and secretion levels of fibronectin (FN1) and osteopontin (SPP1) in macrophages of the model group were significantly increased; DAT intervention can significantly downregulate the expression and secretion of the two types of fibronectin from the source by reshaping the cell transcription program.

[0030] Target validation results: P2RY1 overexpression (P2RY1-OE) cells can reproduce the intervention effect of DAT, and also effectively suppress STAT6 activation and fibrinolytic factor expression, confirming that the role of DAT in remodeling the macrophage fibrinolytic transcriptional program is strictly dependent on the P2RY1 receptor.

[0031] Example 4: Deaminotyrosine (DAT) blocks macrophage-fibroblast paracrine communication and inhibits fibroblast activation Cell model establishment: RAW 264.7 macrophages and NIH-3T3 lung fibroblasts were used to construct an in vitro paracrine communication system between macrophages and fibroblasts. Macrophages were treated with a sequential stimulation protocol of LPS combined with IL-4, and the conditioned medium of macrophages after different treatments was collected for intervention in lung fibroblasts.

[0032] Drug administration regimen: RAW 264.7 macrophages were pretreated with DAT, followed by LPS and then IL-4 sequential stimulation. The supernatant conditioned medium was collected and used to treat NIH-3T3 lung fibroblasts to induce fibroblast activation.

[0033] Result evaluation: Fibroblast activation level: The conditioned medium for sequentially stimulated macrophages without DAT intervention was rich in fibroblast-promoting factors such as FN1 and SPP1, which could significantly induce lung fibroblast activation and greatly upregulate the expression of myofibroblast markers α-SMA and collagen I (COL-1).

[0034] Paracrine communication blocking effect: Macrophages treated with DAT can effectively inhibit the output of their own fibroblasts, and their conditioned medium cannot effectively activate lung fibroblasts. The expression levels of α-SMA and COL-1 in fibroblasts are significantly reduced.

[0035] Cell communication validation results: The results confirm that DAT can effectively block pathogenic paracrine communication between macrophages and lung fibroblasts by inhibiting the secretion of fibroblast-promoting factors by macrophages, thereby inhibiting the abnormal activation of lung fibroblasts.

[0036] 100 mg of deaminotyrosine is thoroughly mixed with 200 mg of microcrystalline cellulose, 50 mg of croscarmellose sodium, and 5 mg of magnesium stearate, and then compressed into tablets for oral administration.

[0037] Alternatively, deaminotyrosine can be dissolved in physiological saline to prepare a sterile injection solution for intravenous or pulmonary administration.

[0038] Figure 1 This study investigated the in vivo therapeutic effect of deaminotyrosine (DAT) on a bleomycin (BLM)-induced mouse model of pulmonary fibrosis. Figure 1 A: Representative images of mouse lung tissue stained with HE (including full lung and local magnified images), showing that DAT significantly reduced BLM-induced alveolar structural damage and inflammatory cell infiltration in a dose-dependent manner. Figure 1 B: Representative images of Masson trichrome staining of mouse lung tissue, showing that the area of ​​blue collagen deposition in the lung interstitium of the DAT treatment group was significantly reduced compared with the BLM model group. Figure 1 C: Statistical chart of Ashcroft fibrosis pathological scores in lung tissue, confirming that DAT significantly reduced the degree of fibrosis (****p<0.0001). Figure 1 D: Statistical graph of relative collagen expression in lung tissue, showing that DAT treatment significantly inhibits collagen overexpression. Figure 1E: The results of hydroxyproline content measurement in lung tissue further confirmed at the biochemical level that DAT can significantly reduce excessive collagen deposition in the lungs (****p<0.0001). (Note: Sham is the sham-operated group, BLM is the model group, and DAT-L and DAT-H are the low- and high-dose deaminotyrosine groups, respectively).

[0039] Figure 2 This study aimed to preemptively intervene in the macrophage inflammatory microenvironment and maintain P2RY1 receptor expression through deaminotyrosine (DAT). Figure 2 The qPCR results of A and B showed that, under LPS (lipopolysaccharide)-induced inflammatory microenvironment stimulation, DAT (10 μM and 100 μM) significantly inhibited the explosive expression of pro-inflammatory factors IL-6 (A) and TNF-α (B) mRNA in macrophages in a dose-dependent manner. Figure 2 The qPCR (C) and Western Blot (D, E) results of C and E show that LPS stimulation leads to a significant decrease in the mRNA and protein expression levels of the P2RY1 receptor in macrophages (receptor deprivation); while the early intervention of DAT can effectively rescue and maintain the expression level of the P2RY1 receptor (***p<0.001, ****p<0.0001).

[0040] Figure 3 Deaminotyrosine (DAT)-dependent P2RY1 receptor suppression of hypersensitive activation of STAT6 in macrophages. From Figure 3 A and Figure 3 Western blotting and quantitative analysis of B showed that, compared with unactivated (M0) and conventionally activated (M2) macrophages, the phosphorylation level of STAT6 (p-Stat6) in LPS-Primed macrophages after inflammatory shaping exhibited a hypersensitive burst.

[0041] from Figure 3 C and Figure 3 The Western blotting and quantitative analysis results of D showed that DAT intervention could strongly suppress the activation of p-Stat6 in LPS-Primed macrophages; and this inhibitory effect was consistent with the effect of direct overexpression of P2RY1 receptor (P2RY1-OE), proving that the transcriptional remodeling effect of DAT depends on the protection of P2RY1 receptor (**p<0.01).

[0042] Figure 4 Deaminotyrosine (DAT) downregulates fibroblast secretion and blocks paracrine communication between macrophages and fibroblasts. From Figure 4Macrophage assays in AD showed that qPCR (A, B) revealed that LPS-Primed macrophages highly expressed and secreted fibrosis-promoting factors fibronectin (FN1) and osteopontin (SPP1); while DAT intervention (and P2RY1 overexpression) significantly blocked the output of FN1 and SPP1 at the transcriptional level (****p<0.0001). Figure 4 The results of the CE fibroblast end (paracrine communication) experiment showed that when lung fibroblasts were treated with the conditioned medium collected from the above macrophages, Western blotting (F) and quantitative analysis (G, H) showed that the macrophage culture medium without DAT intervention greatly promoted the expression of myofibroblast markers α-SMA and collagen I (COL-1) in fibroblasts; while the macrophage culture medium treated with DAT intervention failed to activate fibroblasts due to the loss of fibroblast-promoting factor output (α-SMA and COL-1 expression were significantly decreased, ***p<0.001), confirming that pathogenic cell communication was successfully blocked.

[0043] The embodiments described above are some, but not all, embodiments of the present invention. The detailed description of the embodiments of the present invention is not intended to limit the scope of the claimed invention, but merely to illustrate selected embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort are within the scope of protection of the present invention.

Claims

1. Application of deaminotyrosine in the preparation of drugs for treating pulmonary fibrosis.

2. The application as described in claim 1, characterized in that, The pulmonary fibrosis is idiopathic pulmonary fibrosis, silicosis, radiation-induced pulmonary fibrosis, or drug-induced pulmonary fibrosis.

3. The application as described in claim 1, characterized in that, In the early inflammatory shaping phase of pulmonary fibrosis, the deaminotyrosine can block the nuclear translocation of p65 in the NF-κB pathway in macrophages and inhibit the outbreak of early pro-inflammatory factors, including IL-6 and TNF-α.

4. The application as described in claim 3, characterized in that, The deaminotyrosine residue inhibits the release of inflammatory factors in the early inflammatory microenvironment, preventing the downregulation of P2RY1 receptor expression on the surface of macrophages due to inflammatory damage, thereby maintaining a high level of P2RY1 receptor expression.

5. The application as described in claim 4, characterized in that, When stimulated by fibrinolytic factors, the P2RY1 receptor, whose expression is maintained by deaminotyrosine, acts as an endogenous brake, suppressing the phosphorylation and hypersensitive activation of STAT6 in macrophages.

6. The application as described in claim 5, characterized in that, The deaminotyrosine significantly reduces the secretion of fibronectin (FN1) and osteopontin (SPP1) from macrophages to the microenvironment by inhibiting STAT6 activation and blocking the fibroblast-promoting transcription program. This disrupts paracrine pathogenic communication between macrophages and fibroblasts, thereby inhibiting the phenotypic transformation of lung fibroblasts to myofibroblasts and collagen deposition.

7. The application as described in claim 1, characterized in that, The drug is in the form of tablets, capsules, injections, or inhalers.

8. The application as described in claim 1, characterized in that, The drug includes deaminotyrosine and one or more pharmaceutically acceptable carriers or excipients.

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