Use of Cm-CATH2 in the preparation of drugs for treating silicosis

Abstract

This invention provides the use of Cm-CATH2 in the preparation of drugs for treating silicosis, belonging to the field of biomedical technology. This invention provides the application of Cm-CATH2 and / or pharmaceutically acceptable salts of Cm-CATH2 in the preparation of drugs for treating silicosis, and verifies the therapeutic effect of Cm-CATH2 on silicosis animal models by constructing such models. The results show that Cm-CATH2 can act on silicosis at multiple targets after the early stage of silicosis in animal models. It reduces oxidative stress levels by downregulating lactate dehydrogenase (LDH), downregulates matrix metalloproteinase 2 (MMP2) and extracellular matrix glycosaminoglycans (GAG), and ultimately reduces collagen III (COL-III) levels, thereby achieving an anti-fibrotic effect. This multi-target approach can effectively alleviate fibrosis progression. Therefore, Cm-CATH2 can be used to treat silicosis, and a dose-response effect exists, with good efficacy at high doses.

Description

Technical Field

[0001] This invention provides a field of biomedical technology, specifically relating to the use of Cm-CATH2 in the preparation of drugs for treating silicosis. Background Technology

[0002] Silicosis is a systemic disease primarily characterized by pulmonary fibrosis, caused by long-term inhalation of large amounts of free silica (SiO2) dust particles in industrial environments. Workers engaged in long-term mining, quarrying, tunneling, and production in quartz powder factories, glass factories, refractory material factories, ceramic factories, and enamel factories are susceptible to this disease. A characteristic feature of silicosis is its slow progression; even after leaving silica dust exposure, the disease continues to develop slowly. Patients often develop symptoms 10-15 years after exposure to silica dust. Early-stage silicosis involves lung function impairment, but due to the lungs' strong compensatory capacity, patients are often asymptomatic. As the disease progresses, especially when complicated by pulmonary tuberculosis and cor pulmonale, varying degrees of respiratory and cardiopulmonary dysfunction gradually appear. The fundamental pathological changes in silicosis are the formation of silicotic nodules in the lung tissue and diffuse interstitial fibrosis.

[0003] Currently, there is no effective treatment for silicosis, and existing treatment methods all have shortcomings. While lung lavage is feasible, it is limited by indications and contraindications, and may worsen the condition in severe cases or when combined with other lung diseases. Drug therapy, as the most convenient, economical, and effective treatment, has limited efficacy. Tetrandrine is a clinically recommended drug, but its effectiveness still needs improvement. Summary of the Invention

[0004] This invention provides the use of Cm-CATH2 in the preparation of drugs for treating silicosis, wherein Cm-CATH2 acts on multiple targets in silicosis and can effectively alleviate the progression of fibrosis.

[0005] This invention provides the potential use of Cm-CATH2 and / or pharmaceutically acceptable salts of Cm-CATH2 in the preparation of medicaments for treating silicosis.

[0006] In a preferred embodiment of the present invention, the pharmaceutically acceptable salts of Cm-CATH2 include sodium salts and phosphates.

[0007] In a preferred embodiment of the present invention, the therapeutic effect of the drug includes at least one of the following: reducing LDH to reduce oxidative damage levels, downregulating MMP2, downregulating GAG, reducing collagen III levels, and alleviating fibrosis progression.

[0008] In a preferred embodiment of the present invention, reducing the level of oxidative damage includes reducing the level of oxidative damage caused by at least one of the following oxidants: H2O2, OH... - and O2 - .

[0009] In a preferred embodiment of the present invention, the dosage form of the medicament for treating silicosis includes an injectable form.

[0010] The present invention also provides a medicament for treating silicosis, the active ingredient comprising Cm-CATH2 and / or a pharmaceutically acceptable salt of Cm-CATH2, and further comprising pharmaceutically acceptable excipients.

[0011] In a preferred embodiment of the present invention, the dosage form of the drug includes an injection.

[0012] In a preferred embodiment of the present invention, the concentration of Cm-CATH2 and / or a pharmaceutically acceptable salt of Cm-CATH2 in the injection solution is not less than 2 mg / mL.

[0013] In a preferred embodiment of the present invention, the solvent of the injection solution includes sterile physiological saline.

[0014] In a preferred embodiment of the present invention, the administration method of the injection solution, based on rats, includes intravenous injection of 400 μg per dose.

[0015] Beneficial effects: This invention constructs a silicosis animal model and treats the silicosis animal model with Cm-CATH2. The results show that after Cm-CATH2 is applied to the silicosis animal model, it can act on silicosis at multiple targets. By significantly reducing oxidative stress levels, downregulating metalloproteinase MMP2 and extracellular matrix GAG, it ultimately reduces collagen III levels, thereby achieving an anti-fibrotic effect. The multi-target effect can effectively alleviate the progression of fibrosis. Therefore, Cm-CATH2 can be used to treat silicosis, and there is a dose-response effect, with high doses showing good results. Attached Figure Description

[0016] Figure 1 These are gross lung images of rats with silicosis under different treatments. In the images, A: Gamble solution control group; B: silicosis group; C: low-dose Cm-CATH2 group; D: high-dose Cm-CATH2 group; E: Cm-CATH2 control group; F: tetrandrine group.

[0017] Figure 2 The images show the HE staining results of lung tissues under different treatments. In the figures, A: Gamble solution control group; B: Silicosis group; C: Low-dose Cm-CATH2 group; D: High-dose Cm-CATH2 group; E: Cm-CATH2 control group; F: Tetrandrine group.

[0018] Figure 3The images show Masson staining results of lung tissue under different treatments. In the figures, A: Gamble solution control group; B: Silicosis group; C: Low-dose Cm-CATH2 group; D: High-dose Cm-CATH2 group; E: Cm-CATH2 control group; and F: Tetrandrine group. Detailed Implementation

[0019] This invention provides the use of Cm-CATH2 and / or pharmaceutically acceptable salts of Cm-CATH2 in the preparation of medicaments for treating silicosis.

[0020] The Cm-CATH2 described in this invention is a cathelicidins family molecule with good antibacterial and anti-inflammatory activities, capable of recruiting immune cells and exerting immunomodulatory functions. The Cm-CATH2 described in this invention is a linear polypeptide containing 33 amino acid residues, with a theoretical isoelectric point of 12.96, a molecular weight of 4089.97 Da, and a net charge of +12. Its source, preparation method, and toxicity can be found in Chinese Patent CN106188265A. The Cm-CATH2 used in the embodiments of this invention is in powder form, with a purity of 99.7% obtained through Jier biochemical synthesis.

[0021] In a preferred embodiment of the present invention, the pharmaceutically acceptable salts of Cm-CATH2 include sodium salts and phosphates.

[0022] In this invention, after silica dust enters the lung tissue, it first interacts with alveolar macrophages, continuously stimulating and activating macrophages and neutrophils to produce reactive oxidative metabolites (H2O2, OH-). - O2 - Dust (such as silicosis) can lead to lung injury. After macrophages in the alveoli engulf dust, a respiratory burst occurs, stimulating the intracellular electron transport system and secreting various active mediators. These mediators ultimately trigger lipid peroxidation. Under normal conditions, LDH exists in the cytoplasm and lysosomes of alveolar macrophages with intact membrane structures, with very low concentrations in the alveolar lumen. However, after severe damage to the alveolar macrophage cell membrane, it appears in large quantities in the alveoli; therefore, increased LDH enzyme activity in bronchoalveolar lavage fluid is an important indicator of lung macrophage damage, lysis, and necrosis. When dust acts on tissue cells, altering their membrane permeability, intracellular LDH leaks out of the cells. Therefore, changes in serum LDH in pneumoconiosis patients are one of the auxiliary indicators for the diagnosis of pneumoconiosis. Silicosis patients have significantly higher LDH levels than the control group.

[0023] In the early stages of pulmonary fibrosis, namely the alveolitis stage, the MMP-2 / TIMP-1 ratio increases, enhancing its degradation effect and causing damage to lung tissue, triggering pulmonary fibrosis. In the later stages, namely the fibrosis stage, the MMP-2 / TIMP-1 ratio decreases, its degradation effect weakens, matrix deposition increases, and stable pulmonary fibrosis is formed. One of the basic components of the extracellular matrix is ​​GAG (glucose glutamate). Studies have found that the total amount of GAG in silicosis tissue increases, and the higher the degree of fibrosis, the denser its distribution, and it coexists with collagen fibers. Collagen III is one of the typical markers of fibrosis. Therefore, in this embodiment of the invention, the above-mentioned markers were detected.

[0024] In one embodiment of this invention, a rat model of silicosis was treated with Cm-CATH2. Regarding gross changes in the lungs, high-dose Cm-CATH2 exhibited effects similar to tetrandrine, showing normal morphology, a light red color, uniform color, soft texture, smooth lung surface, and near disappearance of nodules and hemorrhages. After HE staining of lung tissue, high-dose Cm-CATH2 significantly reduced the degree of pulmonary fibrosis. Verification revealed that Cm-CATH2 can act on silicosis at multiple targets, reducing oxidative stress levels, downregulating metalloproteinases MMP2 and GAG, and ultimately reducing collagen III levels, thereby achieving an anti-fibrotic effect and effectively alleviating fibrosis progression. Therefore, in this invention, the therapeutic effect of the drug includes at least one of the following: downregulating LDH to reduce oxidative damage levels, downregulating MMP2, downregulating extracellular matrix GAG, reducing collagen III (COL-III) levels, and alleviating fibrosis progression. The reduction of oxidative damage levels described in this invention includes reducing oxidative damage levels induced by at least one of the following oxidants: H2O2, OH... - and O2 - .

[0025] The present invention also provides a medicament for treating silicosis, the active ingredient comprising Cm-CATH2 and / or a pharmaceutically acceptable salt of Cm-CATH2, and further comprising pharmaceutically acceptable excipients.

[0026] In a preferred embodiment of the present invention, the dosage form of the drug includes an injection, and the concentration of Cm-CATH2 and / or a pharmaceutically acceptable salt of Cm-CATH2 in the injection solution is not less than 2 mg / mL. The solvent of the injection solution includes sterile physiological saline. The administration route of the injection solution, based on rats, is 400 μg intravenously per dose.

[0027] To further illustrate the present invention, the use of Cm-CATH2 provided by the present invention in the preparation of drugs for treating silicosis is described in detail below with reference to embodiments, but these should not be construed as limiting the scope of protection of the present invention.

[0028] In the embodiments of the present invention, the materials used are conventional materials in the art unless otherwise specified, and the experimental methods used are also conventional methods in the art unless otherwise specified.

[0029] 1. Construction of silicosis animal model:

[0030] Male SD rats were purchased from Changsha Tianqin Biotechnology Co., Ltd., with the license number SCXK(Xiang)2024-0021. They were raised in the SPF-class animal house of Guizhou University of Traditional Chinese Medicine. A total of 48 rats were raised, each weighing 250 g. The quality certificate number was SCXK(Qian)2021-0003; the experimental facility certificate number was SYXK(Qian)2021-0005. After one week of adaptive feeding at a temperature of about 25°C and a humidity of about 45%, animal modeling was carried out. Prepare Gamble solution, filter and sterilize it with a 0.22 μm filter membrane for standby. Weigh 2500 mg of SiO2, sterilize it by high pressure, and dry it for 3 h for standby. Resuspend SiO2 thoroughly with 50 mL of Gamble solution to make its concentration 50 mg / mL, and inject 1 mL into each rat with a sterile 1 mL syringe. Use the perfusion of Gamble solution as a control. Use tetrandrine injection as a positive control (TGS), and use high-dose injection of Cm-CATH2 after perfusion of Gamble solution as a negative control group (Cm-CATH2 control group). At the early stage of silicosis (i.e., 21 days after dust stimulation), drug administration was carried out.

[0031] The composition of Gamble solution: MgCl2·6H2O 0.212 g / L, NaCl 6.415 g / L, Na2HPO4 0.148 g / L, Na2SO4·2H2O 0.179 g / L, CaCl2·4H2O 0.319 g / L, NaHCO3 2.703 g / L, sodium tartrate 0.180 g / L, sodium citrate 0.186 g / L, sodium lactate 0.175 g / L, sodium pyruvate 0.172 g / L, and glycine 0.118 g / L, pH = 7 - 7.4.

[0032] 2. Preparation of Cm-CATH2 injection

[0033] Weigh Cm-CATH2 powder (purity 99.7%, Gil Biochemical), and dissolve it with sterile normal saline. The concentrations are 1 mg / mL (low-dose group) and 2 mg / mL (high-dose group) respectively.

[0034] 3. Intravenous injection for drug administration

[0035] Inject 0.2 mL into the tail vein of each rat, inject once every 3 days, for a total of 5 times.

[0036] 4. Detection of efficacy determination indicators

[0037] After 36 days of treatment, the rats were sacrificed, and their lung tissue was collected for gross lung observation. HE (hematoxylin-eosin) and Masson's trichrome staining were performed. The tissue was then ground with sterile PBS. The mixture was centrifuged at 3000 rpm for 25 min at 4°C, and the supernatant was collected for ELISA detection according to the kit instructions. The detected indicators were LDH, MMP2, GAG, and collagen III.

[0038] Example 1

[0039] 1. Gross changes in rat lungs

[0040] Gross results of rat lungs as follows Figure 1 As shown, the lungs of rats in the Gamble solution control group were bright red, smooth, and soft, with no focal hemorrhages. The Cm-CATH2 control group was similar. The lungs of rats in the silicosis group were swollen, gray, and firm, with consolidation foci. Scattered, raised, millet-sized grayish-white nodules and old hemorrhages were visible on the surface. The lung tissue in the low-dose Cm-CATH2 group was gray, swollen, and rough, with visible surface nodules and focal hemorrhages. The high-dose Cm-CATH2 group had normal morphology, was light red, uniform in color, and soft in texture. The lung lobes were smooth, and nodules and hemorrhages had largely disappeared. The tetrandrine group was similar to the high-dose Cm-CATH2 group.

[0041] 2. HE staining of lung tissue

[0042] Rat lung tissue staining results as follows Figure 2 As shown in the figure, the lung tissue of rats in the silicosis group differed significantly from that in the Gamble control group. Some alveoli showed shrinkage, alveolar septa widened, alveolar structure was severely disordered, and interstitial vasodilation and congestion were observed, along with significant fibrosis. The degree of lung tissue damage in the low-dose Cm-CATH2 group was not significantly different from that in the silicosis group. Compared to the silicosis group, the high-dose Cm-CATH2 group showed reduced lung fibrosis, decreased alveolar collapse, less alveolar septal widening, reduced interstitial fibrosis, and a decreased number of fibroblasts. In contrast, the lung tissue integrity of the Cm-CATH2 control group was similar to that of the Gamble control group, with virtually no fibrosis.

[0043] 3. Masson staining of lung tissue

[0044] Masson staining results are as follows: Figure 3As shown, the lung tissue structure of rats in the Gamble solution control group was clear, the alveolar structure was intact, and there was no obvious blue collagen fiber deposition. The same was true for the Cm-CATH2 control group. In the silicosis group, the blue-stained area of ​​the lung tissue of rats increased, and the alveolar walls were thickened. Collagen fiber deposition was significantly increased, and pulmonary fibrosis was significantly aggravated. Compared with the silicosis group, the low-dose Cm-CATH2 group showed little difference in blue collagen fiber deposition, while the high-dose Cm-CATH2 group and the tetrandrine group showed mild damage to the lung tissue structure and a significant reduction in blue collagen fiber deposition.

[0045] 4. ELISA detection of LDH, GAG, MMP2, and COL-III

[0046] The ELISA results are shown in Tables 1-4. Early treatment of silicosis significantly increased LDH, MMP2, GAG, and COL-III levels in the silicosis group compared to the control group, consistent with the pathogenesis of silicosis. Low-dose Cm-CATH2 was ineffective compared to the silicosis group, while high-dose treatment significantly reduced LDH, MMP2, and COL-III levels. Although TGS significantly reduced LDH and MMP2, COL-III levels were only slightly lower than in the silicosis group and did not significantly affect their levels. Compared to TGS, both low and high-dose Cm-CATH2 significantly reduced LDH, and high-dose Cm-CATH2 significantly reduced COL-III. Simultaneously, both high-dose Cm-CATH2 and TGS significantly reduced MMP2 and GAG, showing comparable effects, but low-dose treatment was ineffective. Therefore, overall, high-dose Cm-CATH2 is more effective than TGS in treating LDH and COL-III, especially in the early stages of silicosis where TGS is not very effective in reducing collagen III production. However, the effects of Cm-CATH2 and TGS are similar, so high-dose Cm-CATH2 is more effective than TGS in treating early-stage silicosis.

[0047] Regarding drug toxicity and side effects, compared with the Gamble solution control group, Cm-CATH2 downregulated the levels of LDH, MMP2, and GAG in the control group, but did not change the collagen III content, indicating that Cm-CATH2 injection can complete lung tissue repair more quickly and therefore has no toxic side effects.

[0048] In Tables 1 to 4, a) the difference was significant compared to the Gamble solution control group (P<0.05); b) the difference was significant compared to the silicosis group (P<0.05); c) the difference was significant compared to the TGS group (P<0.05); and d) the difference was significant compared to the high-dose Cm-CATH2 group (P<0.05).

[0049] Table 1. Effects of Cm-CATH2 on LDH in silicosis-affected rats.

[0050] Group n LDH (pg / mg pro) Gamble solution control group 8 5555.3275±551.25371 Silicosis group 8 <![CDATA[7556.5293±1084.24995 a ]]> Cm-CATH2 low-dose group 8 <![CDATA[4334.3747±277.04159 abc ]]> Cm-CATH2 high-dose group 8 <![CDATA[4333.1505±212.25233 abc <!-- 4 -->]]> Cm-CATH2 control group 8 <![CDATA[4707.4494±191.35429 a ]]> Tetrandrine A group (TGS) 8 <![CDATA[5273.4076±498.53223 b ]]>

[0051] Table 2. Effects of Cm-CATH2 on MMP2 in silicosis-affected rats.

[0052]

[0053]

[0054] Table 3. Effects of Cm-CATH2 on GAG in silicosis-affected rats.

[0055] Group n GAG (μg / g.Pro) Gamble solution control group 8 2.3643±0.29903 Silicosis group 8 <![CDATA[2.8583±0.26026 a ]]> Cm-CATH2 low-dose group 8 <![CDATA[2.5725±0.59739 cd ]]> Cm-CATH2 high-dose group 8 <![CDATA[2.1990±0.28914 b ]]> Cm-CATH2 control group 8 <![CDATA[2.0684±0.10693 a ]]> Tetrandrine A group (TGS) 8 <![CDATA[2.0641±0.21880 b ]]>

[0056] Table 4. Effects of Cm-CATH2 on collagen III in silicosis-affected rats.

[0057]

[0058]

[0059] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. Use of Cm-CATH2 and / or a pharmaceutically acceptable salt of Cm-CATH2 for the manufacture of a medicament for the treatment of silicosis, characterized in that, The therapeutic effect of the drug includes at least one of the following: reducing the level of oxidative damage, down-regulating metalloproteinase MMP2, down-regulating extracellular matrix GAG, reducing the level of collagen III, and effectively relieving the progression of fibrosis. The dosage form of the drug for treating silicosis includes an injection, and the concentration of Cm-CATH2 and / or a pharmaceutically acceptable salt thereof in the injection is not less than 2 mg / mL.

2. Use according to claim 1, characterized in that, The pharmaceutically acceptable salt of Cm-CATH2 includes a sodium salt and a phosphate salt.

3. Use according to claim 1, characterized in that, The reducing the level of oxidative damage to the cell includes reducing the level of oxidative damage induced by at least one of the following oxidants: H2O2, OH - and O2 - .

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