Use of small molecule peptides AWR and / or LQR in the preparation of a medicament for treating myositis-associated interstitial lung disease

Abstract

This invention provides the application of small molecule peptides AWR and / or LQR in the preparation of drugs for treating myositis-associated interstitial lung disease (IIM-ILD), belonging to the field of biomedical technology. By comparing the distribution levels of metabolites in the serum of healthy volunteers and IIM-ILD patients, this invention found that two small molecule peptides, alanine-tryptophan-arginine (AWR) and leucine-glutamine-arginine (LQR), were significantly reduced in IIM-ILD patients. Subsequently, by using AWR and LQR in IIM-ILD model mice, it was determined that AWR and LQR can significantly slow down the loss of body weight and multiple pathological indicators such as lung damage in IIM-ILD mice, and reduce the relative expression levels of various inflammatory factors, making them potential clinical drug candidates for IIM-ILD.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to the application of small molecule peptides AWR and / or LQR in the preparation of drugs for treating myositis-related interstitial lung disease. Background Technology

[0002] Myositis, or idiopathic inflammatory myopathy (IIM), is a heterogeneous group of diseases characterized by proximal muscle involvement and chronic inflammation in the extremities. IIM can affect multiple tissues and organs, including the skin, lungs, heart, and joints, with interstitial lung disease (ILD) being the most common clinical manifestation of IIM involving the lungs. Myositis-associated interstitial lung disease (IIM-ILD) is a significant cause of disability and death in IIM patients. Clinically, the incidence of ILD in IIM patients is very high, ranging from 20% to 78% in adult IIM patients and approximately 8% to 13% in adolescent IIM patients. Currently, treatment for IIM-ILD primarily involves high-dose corticosteroids combined with immunosuppressants (such as azathioprine and cyclophosphamide). However, in clinical practice, the side effects of using hormones and immunosuppressants often impose a significant physiological and psychological burden on patients. Therefore, there is an urgent need to develop potential clinical drugs for IIM-ILD that are safe, effective, and have few side effects. Summary of the Invention

[0003] In view of this, the purpose of the present invention is to provide the application of small molecule peptides AWR and / or LQR in the preparation of drugs for treating myositis-associated interstitial lung disease. The small molecule peptides AWR and / or LQR of the present invention can significantly slow down multiple pathological indicators such as weight loss and lung damage caused by myositis-associated interstitial lung disease, and reduce the relative expression of various inflammatory factors.

[0004] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides the application of small molecule peptides AWR and / or LQR in the preparation of drugs for treating myositis-related interstitial lung disease.

[0005] Preferably, the effective concentration of the small molecule peptide AWR is 12.5~25 mg / kg.

[0006] Preferably, the effective concentration of the small molecule peptide LQR is 12.5~25 mg / kg.

[0007] Preferably, the small molecule peptide AWR and the small molecule peptide LQR are used in combination, and the mass ratio of the small molecule peptide AWR to the small molecule peptide LQR is 12.5~25:12.5~25.

[0008] This invention provides a drug for treating myositis-related interstitial lung disease, comprising an active ingredient and excipients; the active ingredient is a small molecule peptide AWR and / or LQR.

[0009] Preferably, the excipients include one or more of starch, dextrin, cellulose and its derivatives, gelatin, povidone, magnesium stearate, organic acids, glycerol and sorbitol.

[0010] Preferably, the dosage form of the drug for treating myositis-related interstitial lung disease is an oral formulation.

[0011] This invention provides the application of small molecule peptides AWR and / or LQR in the preparation of drugs for treating myositis-associated interstitial lung disease (IIM-ILD). By comparing the distribution levels of metabolites in the serum of healthy volunteers and IIM-ILD patients, it was found that two small molecule peptides, alanine-tryptophan-arginine (AWR) and leucine-glutamine-arginine (LQR), were significantly reduced in IIM-ILD patients. Subsequently, by using AWR and LQR in IIM-ILD model mice, it was determined that AWR and LQR can significantly slow down the loss of body weight and multiple pathological indicators such as lung damage in IIM-ILD mice, and reduce the relative expression levels of various inflammatory factors, making them potential clinical drug candidates for IIM-ILD. Attached Figure Description

[0012] Figure 1 Comparison of serum levels of small peptides AWR and LQR between IIM-ILD patients and healthy volunteers; Figure 2 The changes in body weight of mice in each group during the experiment; Figure 3 Results of H&E staining and Masson staining; Figure 4 Results of qPCR detection of expression levels of inflammatory cytokines in the lungs of mice in each group; Figure 5 The results of qPCR detection of the expression levels of pulmonary fibrosis markers in mice of each group. Detailed Implementation

[0013] This invention provides the application of small molecule peptides AWR and / or LQR in the preparation of drugs for treating myositis-related interstitial lung disease.

[0014] In this invention, the small molecule peptide AWR is alanine-tryptophan-arginine, and the small molecule peptide LQR is leucine-glutamine-arginine. In this invention, the effective concentration of the small molecule peptide AWR is preferably 12.5~25 mg / kg, more preferably 15~20 mg / kg.

[0015] In this invention, the effective concentration of the small molecule peptide LQR is preferably 12.5~25 mg / kg, more preferably 15~20 mg / kg.

[0016] In this invention, the small molecule peptides AWR and LQR are preferably used in combination, and the mass ratio of AWR to LQR is preferably 12.5~25:12.5~25, more preferably 15~20:15~20, and even more preferably 1:1. In this invention, when using both AWR and LQR, the total effective concentration is preferably 12.5~25 mg / kg, more preferably 15~20 mg / kg.

[0017] The present invention does not have any special requirements for the synthesis method of the small molecule peptides AWR and LQR; solid-phase peptide synthesis methods well known in the art can be used.

[0018] This invention provides a drug for treating myositis-related interstitial lung disease, comprising an active ingredient and excipients; the active ingredient is a small molecule peptide AWR and / or LQR.

[0019] In this invention, the excipients preferably include one or more of starch, dextrin, cellulose and its derivatives, gelatin, povidone, magnesium stearate, organic acids, glycerin, and sorbitol. In this invention, the dosage form of the drug for treating myositis-related interstitial lung disease is preferably an oral formulation.

[0020] The following examples illustrate the application of the small molecule peptides AWR and / or LQR provided by the present invention in the preparation of drugs for treating myositis-related interstitial lung disease. However, these examples should not be construed as limiting the scope of protection of the present invention.

[0021] Example 1: Comparison of small molecule peptides AWR and LQR in the serum of IIM-ILD patients and healthy volunteers The study included 25 patients with IIM-ILD and 30 healthy volunteers. Serum samples were collected from all participants, and ultra-high performance liquid chromatography-quantitative-time (UHPLC-Q-TOF-MS) was used to perform metabolomics analysis on serum metabolites. This study was approved by the Ethics Committee of Shantou Central Hospital (Approval No.: 2025Research009). Sample extraction: Take an appropriate amount of sample and add pre-cooled methanol / acetonitrile / water solution (2:2:1, v / v), vortex mix, sonicate at low temperature for 30 min, stand at -20℃ for 10 min, centrifuge at 14000 g at 4℃ for 20 min, take the supernatant and vacuum dry, add 100 μL of acetonitrile water solution (acetonitrile:water = 1:1, v / v) to redissolve before mass spectrometry analysis, vortex, centrifuge at 14000 g at 4℃ for 15 min, take the supernatant for sample analysis.

[0022] Chromatographic conditions: Samples were separated using an Agilent 1290 Infinity LC ultra-high performance liquid chromatography (UHPLC) system with a HILIC column; column temperature 25℃; flow rate 0.5 mL / min; injection volume 2 μL; mobile phase composition A: water + 25 mM ammonium acetate + 25 mM ammonia, B: acetonitrile; gradient elution program as follows: 0–0.5 min, 95% B; 0.5–7 min, B linearly decreasing from 95% to 65%; 7–8 min, B linearly decreasing from 65% to 40%; 8–9 min, B maintained at 40%; 9–9.1 min, B linearly decreasing from 40% to 95%; 9.1–12 min, B maintained at 95%; throughout the analysis, samples were placed in an autosampler at 4℃. To avoid the influence of instrument signal fluctuations, samples were analyzed sequentially in a random order. QC samples were inserted into the sample queue to monitor and evaluate the stability of the system and the reliability of the experimental data.

[0023] Mass spectrometry detection conditions: Primary and secondary spectra of the samples were acquired using an AB Triple TOF 6600 mass spectrometer. After separation using an Agilent 1290 Infinity LC ultra-high performance liquid chromatography (UHPLC) system, mass spectrometry analysis was performed using a Triple TOF 6600 mass spectrometer (AB SCIEX), with detection performed in both positive and negative ion electrospray ionization (ESI) modes. The ESI source settings are as follows: Auxiliary heating gas 1 (Gas1): 60, Auxiliary heating gas 2 (Gas2): 60, Curtain gas (CUR): 30psi, Ion source temperature: 600℃, Spray voltage (ISVF): ±5500 V (positive and negative modes); Primary mass-to-charge ratio detection range: 60-1000 Da, Secondary fragment ion mass-to-charge ratio detection range: 25~1000 Da, Primary mass spectrometry scan cumulative time: 0.20 s / spectra, Secondary mass spectrometry scan cumulative time: 0.05 s / spectra; Secondary mass spectrometry is obtained using data-dependent acquisition mode (IDA) and peak intensity value screening mode, Declustering voltage (DP): ±60 V (positive and negative modes), Collision energy: 35±15 eV, IDA settings are as follows: Dynamic exclusion range of isotopic ions: 4 Da, 10 fragment spectra are acquired per scan.

[0024] Data Analysis and Processing: The raw data was converted to .mzXML format using ProteoWizard, and then peak alignment, retention time correction, and peak area extraction were performed using XCMS software. The data extracted by XCMS underwent metabolite structure identification and data preprocessing (null filtering: removing ion peaks with missing values ​​> 50%; null filling: KNN filling; data filtering: filtering features with RSD > 50%). Finally, GraphPad Prism 8.0 was used for data analysis, and the results are shown below. Figure 1 As shown. Figure 1 This study compared the levels of small peptides AWR and LQR in the serum of IIM-ILD patients and healthy volunteers. The study included 25 IIM-ILD patients and 30 healthy volunteers (NC). , p<0.001.

[0025] Depend on Figure 1 It can be seen that the levels of small molecule peptides AWR and LQR in the serum of IIM-ILD patients are significantly reduced.

[0026] Example 2: Experimental study on the delay of disease progression in IIM-ILD model mice by small molecule peptides AWR and LQR. Animal grouping, modeling and drug administration: A total of 36 BALB / C mice were included in the experiment, including 6 normal control group (Ctrl), 6 model group, 6 low-dose AWR group (AWR-L), 6 high-dose AWR group (AWR-H), 6 low-dose LQR group (LQR-L), and 6 high-dose LQR group (LQR-H).

[0027] Animal modeling and drug administration: One week before modeling, mice were administered AWR-L at 12.5 mg / kg, AWR-H at 25 mg / kg, LQR-L at 12.5 mg / kg, and LQR-H at 25 mg / kg via gavage. The Ctrl group and Model group were given an equal volume of sterile water. One week later, except for the normal control group, mice in the other groups underwent modeling. The modeling process was as follows: skeletal muscle from SD rats was homogenized (30 mg / mL), and 1:1 Freund's complete adjuvant was added. After mixing, 0.2 mL / mouse was injected subcutaneously for 5 consecutive weeks, once a week. Simultaneously, mice in each modeling group were intraperitoneally injected with pertussis toxin, 2 μg / mouse, for 2 consecutive weeks, once a week. During the modeling process, small molecule peptides were still administered via gavage. The weight of the mice was recorded weekly during the experiment. After the experiment, serum was collected from each group of mice and stored at -80℃ for subsequent experiments; at the same time, lung tissue was taken from each mouse, fixed with formalin and used for subsequent experimental research.

[0028] Weekly recording and analysis of mouse body weight; changes in body weight in each group of mice are as follows: Figure 2 As shown. Figure 2 In this study, n=6, and data were recorded weekly. It can be seen that in the second week after modeling, the body weight of the model group mice continued to decrease, but the body weight of mice in the high-dose groups of small molecule peptides AWR and LQR maintained a steady upward trend compared to the normal group. At the experimental endpoint, week 6, the body weight of the model group mice was significantly lower than that of the normal group and the high-dose groups of AWR and LQR. The results indicate that AWR and LQR can slow down the decrease in body weight in mice during IIM-ILD modeling.

[0029] Mouse lungs were fixed in formalin for 24 hours, then dehydrated using xylene-ethanol and prepared into paraffin-embedded specimens. The paraffin-embedded specimens were sectioned using a tissue sectioner, with each section measuring 5 μm. The specimens were rehydrated using a gradient of xylene-ethanol concentrations (from 100% to 50%). Staining was performed using H&E and Masson's staining, followed by permeabilization with a gradient of ethanol concentrations (from 50% to 100%)-xylene, and then the slides were mounted and read.

[0030] The results of H&E staining and Masson staining are as follows: Figure 3 As shown. H&E staining was used to evaluate changes in the physiological structure of mouse lung tissue. The results showed that, compared with the normal group of mice, the physiological structure of the lungs of the model group mice was significantly altered, mainly due to the infiltration of inflammatory cells, edema and widening of alveolar septa, and collapse, which disrupted the physiological structure.

[0031] Masson staining was used to evaluate the degree of fibrosis in the lung tissue of mice. The results showed that compared with the normal group of mice, the collagen fiber staining in the model group was significantly increased, indicating the progression of pulmonary fibrosis in mice. However, after treatment with small molecule peptides, especially the intervention of high doses of AWR and LQR, the fibrosis process in the lungs of the model group mice could be significantly reduced.

[0032] Quantitative PCR (qPCR) was used to detect inflammatory factors (including IL-1β, TNF-α, and TGF-β) in the lungs of mice. The results of qPCR detection of the expression levels of inflammatory cytokines in the lungs of mice in each group are as follows: Figure 4 As shown, Figure 4 middle, p<0.05; p<0.01; p < 0.001. The experimental results showed that various inflammatory markers were significantly increased in the model group mice. However, treatment with small molecule peptides, especially the high-dose AWR and LQR groups, significantly reduced the relative expression levels of various inflammatory factors compared to the model group, suggesting that small molecule peptides have significant anti-inflammatory effects.

[0033] Quantitative PCR (qPCR) was used to detect pulmonary fibrosis markers (including Fnt and TGF-β) in mice. The results of qPCR detection of the expression levels of pulmonary fibrosis markers in each group of mice are as follows: Figure 5 As shown, Figure 5 middle, p<0.05; p<0.01; p < 0.001. The experimental results showed that fibrosis markers were significantly increased in the model group mice. However, treatment with small molecule peptides, especially the high-dose AWR and LQR groups, significantly reduced the relative expression levels of fibrosis markers compared to the model group, suggesting that small molecule peptides have a significant anti-fibrotic effect.

[0034] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. Application of small molecule peptides AWR and / or LQR in the preparation of drugs for treating myositis-related interstitial lung disease.

2. The application according to claim 1, characterized in that, The effective concentration of the small molecule peptide AWR is 12.5~25 mg / kg.

3. The application according to claim 1, characterized in that, The effective concentration of the small molecule peptide LQR is 12.5~25 mg / kg.

4. The application according to claim 1, characterized in that, The small molecule peptides AWR and LQR are used in combination, with the mass ratio of AWR to LQR being 12.5~25:12.5~25.

5. A drug for treating myositis-related interstitial lung disease, characterized in that, It includes active ingredients and excipients; the active ingredients are small molecule peptides AWR and / or LQR.

6. The drug for treating myositis-related interstitial lung disease according to claim 5, characterized in that, The excipients include one or more of starch, dextrin, cellulose and its derivatives, gelatin, povidone, magnesium stearate, organic acids, glycerol and sorbitol.

7. The drug for treating myositis-related interstitial lung disease according to claim 5, characterized in that, The medication for treating myositis-related interstitial lung disease is an oral formulation.

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