Pharmaceutical composition and use thereof

Abstract

The present invention relates to the field of fibrosis prevention and / or treatment and particularly to use of reactive oxygen species in the prevention and / or treatment of fibrosis and diseases clearly related to fibrosis. The present invention provides use of reactive oxygen species and a related compound thereof as drugs in the prevention and / or treatment of fibrosis and diseases clearly related to fibrosis in individuals. The applicant creatively found that reactive oxygen species (ROS) can effectively inhibit fibrosis in individuals.

Description

A pharmaceutical composition and uses thereof

[0001] Cross-reference to Related Applications

[0002] This application is based on and claims priority to Chinese Patent Application No. 202411020561.8, filed on July 26, 2024, the entire contents of which are incorporated herein by reference. TECHNICAL FIELD

[0003] The present application relates to the field of prevention and / or treatment of fibrosis, in particular to the use of reactive oxygen species in the prevention and / or treatment of fibrosis and diseases clearly associated with fibrosis. BACKGROUND

[0004] Fibrosis refers to a pathological process of excessive fibrous connective tissue in organs, accumulation of a large amount of extracellular matrix (such as collagen, fibronectin, etc.), and destruction of tissue structure. In the late stage of various diseases such as viral hepatitis, chronic nephritis, scleroderma, and coronary heart disease, organs will undergo fibrosis, and eventually lead to organ failure. Common fibrotic diseases include pulmonary fibrosis, renal fibrosis, hepatic fibrosis, myocardial fibrosis, and cutaneous fibrosis. Myofibroblasts are the main effector cells of this process: when the tissue is damaged, the body releases various pro-fibrotic factors including TGF-β1, which induces the differentiation of resting fibroblasts into myofibroblasts with high expression of alpha-smooth muscle actin (α-SMA) by regulating the TGF-β / SMAD, PI3K / AKT, and other pathways; subsequently, myofibroblasts promote wound healing through synthesis of extracellular matrix, contraction of wounds, secretion of cytokines, and other pathways. Under normal circumstances, myofibroblasts lose their function after the damaged tissue is repaired. However, in fibrotic lesions, myofibroblasts are continuously activated, leading to excessive accumulation of extracellular matrix, causing irreversible tissue damage and scar formation.

[0005] Currently, the treatment of fibrotic diseases mainly includes drug therapy and organ transplantation. As the only final treatment for patients with fibrotic diseases, organ transplantation is limited in its application due to the difficulty in obtaining donors, high surgical risk, and high surgical cost. Existing drugs cannot reverse the fibrotic process, and their clinical efficacy and safety cannot meet the treatment needs. For example, most of the current drugs for pulmonary fibrosis target TGF-β, endothelin, and connective tissue growth factor, aiming to inhibit the abnormal activation of fibroblasts into myofibroblasts. Two FDA-approved drugs for pulmonary fibrosis, pirfenidone and nintedanib, target this activation process to exert anti-fibrotic effects. However, these drugs can only slow down the decline in lung function in patients with mild to moderate pulmonary fibrosis and cannot reverse the progression of the disease. Clinical data show that they have no significant positive effect on the prognosis of pulmonary fibrosis, with a 5-year mortality rate of 70-80%. The limited efficacy of the above treatment options is likely due to the abnormal activation of fibroblasts at the time of diagnosis (i.e., before treatment), and targeting the initial process of pulmonary fibrosis cannot reverse the phenotype of activated myofibroblasts.

[0006] Reactive oxide species (ROS) is a class of highly active compounds containing oxygen, including superoxide (such as superoxide anion, O 2- ), peroxide (such as hydrogen peroxide, H2O2), hydroxyl radical (OH·), singlet oxygen ( 1 O2), and nitric oxide (NO), etc. Studies have shown that a large number of ROS play an important role in the occurrence and development of fibrosis, and may promote the occurrence and development of fibrosis: ROS promotes the progression of fibrosis through various mechanisms such as promoting oxidative stress, inflammatory response, extracellular matrix changes, and apoptosis. Therefore, researchers speculate that clearing ROS with antioxidants or preventing and treating fibrosis is an effective strategy. Based on this, researchers have tried to treat pulmonary fibrosis with antioxidants such as N-acetylcysteine, but relevant clinical evidence shows that N-acetylcysteine cannot improve the survival rate of patients with pulmonary fibrosis, and may even induce cardiovascular adverse events. This result contradicts previous knowledge, suggesting that ROS may play a more complex role in the occurrence and development of pulmonary fibrosis. SUMMARY

[0007] The problem to be solved by the present application is how to effectively treat or prevent fibrosis, such as pulmonary fibrosis, liver fibrosis, kidney fibrosis, skin fibrosis, myocardial fibrosis, and diseases clearly related to fibrosis, such as pancreatic cancer (pancreatic cancer region fibrosis is severe), non-alcoholic fatty liver, systemic sclerosis, etc.

[0008] The applicant has creatively found that reactive oxygen species (ROS), in particular, low-dose reactive oxygen species, can effectively inhibit fibrosis in individuals.

[0009] According to the above needs, in a first aspect, the present application provides the use of reactive oxygen species and related compounds thereof as a medicament for preventing and / or treating fibrosis and diseases clearly related to fibrosis in a subject.

[0010] In the present application, the "reactive oxygen species" refers to the general term of oxygen-containing free radicals and free radical-prone peroxides. The "reactive oxygen species related compounds" refer to compounds that can produce reactive oxygen species.

[0011] In the present application, "prevention and / or treatment" refers to preventing and / or delaying the occurrence of a disease or condition or normalizing it.

[0012] In one embodiment, the level of reactive oxygen species in the cells of the subject is 2-30 times the level of reactive oxygen species in untreated cells.

[0013] Further, in a preferred embodiment, the level of reactive oxygen species in the cells of the subject is 5-20 times the level of reactive oxygen species in untreated cells.

[0014] Further, the reactive oxygen species is generated by physical methods, chemical methods, or physical methods combined with chemical methods.

[0015] In some specific embodiments, the chemical method can be the application of a chemical agent to the subject. Further, the chemical agent can be hydrogen peroxide, a radionuclide. By applying a chemical agent, the level of reactive oxygen species in the cells of the subject is increased to 2-30 times the level of reactive oxygen species in untreated cells.

[0016] In some specific embodiments, the physical method combined with the chemical method can be photodynamic combined with photosensitizer, or sonodynamic combined with sonosensitizer.

[0017] Further, the photodynamic can be an excitation light source, which can be natural light, artificial light, Cerenkov radiation generated by radionuclides, X-rays. Preferably, the radionuclide can be labeled on a ligand targeting myofibroblasts. More preferably, the radionuclide is 68 Ga、 18 F、 64 Cu、 89 Zr、 131 I、 32 P、 177 Lu、 90 Y.

[0018] Further, the sonodynamic can be ultrasound, and further, the conditions of the ultrasound are 1.0-3.0 MHz, 20-80% duty cycle, 0.5-2 W / cm 2 , 2-10 min.

[0019] Further, the photosensitizer can be a new type of aggregation induced emission material (AIE). Further, the new type of aggregation induced emission material can be a material targeting mitochondria or sensitive to mitochondrial membrane potential.

[0020] In a specific embodiment, the new type of aggregation induced emission material can be a photosensitizer DPA-SCP and its variants as shown in the following structure, or tetraphenylethene (TPE) and triphenylamine (TPA).

[0021] Further, the sonosensitizer can be a sonosensitizer material targeting mitochondria or sensitive to mitochondrial membrane potential. Further, the sonosensitizer material can be a new type of AIE material targeting mitochondria or sensitive to mitochondrial membrane potential. In a specific embodiment, the new type of AIE material can be a sonosensitizer DPA-SCP and its variants as shown in the following structure, or TPE and TPA.

[0022] In an embodiment, the individual is a mammal, including but not limited to rat, mouse, non-human primate, human, dog, cat, horse, cow, sheep, pig, goat.

[0023] Preferably, the individual is a human.

[0024] Further, the fibrosis can be pulmonary fibrosis, liver fibrosis, kidney fibrosis, skin fibrosis, myocardial fibrosis, and diseases directly related to fibrosis, such as pancreatic cancer (pancreatic cancer area fibrosis is severe), non-alcoholic fatty liver, systemic sclerosis, atherosclerosis (fibrous cap structure is formed in the plaque area), etc.

[0025] In a second aspect, the present application provides a composition for preventing and / or treating fibrosis and diseases directly related to fibrosis in an individual, comprising:

[0026] (a) a first active ingredient: a targeting agent labeled with a nuclide, wherein the targeting agent targets fibroblast activation protein (FAP);

[0027] (b) a second active ingredient: a photosensitizer.

[0028] In some preferred embodiments, the content of the first active ingredient ranges from 1% to 99%, based on the total weight of the active ingredients of the composition; more preferably, 10% to 90%; more preferably, 30% to 70%.

[0029] In some preferred embodiments, the second active ingredient is present in an amount ranging from 1% to 99%, based on the total weight of the active ingredients of the composition; more preferably, from 10% to 90%; more preferably, from 30% to 70%.

[0030] In some preferred embodiments, the molar ratio of the first active ingredient to the second active ingredient in the composition is 0.1-9.9:9.9-0.1, more preferably, 1-5:5-1, most preferably, 1-3:3-1.

[0031] Preferably, the nuclide can be 68 Ga, 18 F, 64 Cu, 89 Zr, 131 I, 32 P, 177 Lu, 90 Y. More preferably, the nuclide is 68 Ga.

[0032] Further, the targeting agent is a targeting agent targeting myofibroblasts, various tumor cells (such as pancreatic cancer, brain glioma, etc.), fibrous cap of atherosclerotic plaques, etc. In some specific embodiments, the targeting agent is a targeting agent targeting fibroblast activation protein.

[0033] Specifically, the targeting agent includes monoclonal antibodies mAbF19, ESC11, ESC14, etc., polypeptides FAP-2286, FAP-RGD, quinoline small molecule inhibitors FAPI-04, FAPI-46, etc., non-quinoline small molecule inhibitors iFAP, FL-L3, etc.

[0034] Further, the photosensitizer can be a photosensitive material that can target mitochondria or be sensitive to mitochondrial membrane potential.

[0035] Still further, the photosensitive material can be a novel aggregation induced emission (AIE) material that targets mitochondria or is sensitive to mitochondrial membrane potential. In one specific embodiment, the novel aggregation induced emission material can be photosensitizer DPA-SCP and its variants, or TPE and TPA.

[0036] The pharmaceutical compositions of the present application, when administered (dosed) therapeutically, can provide a variety of effects. In general, these substances can be formulated in a non-toxic, inert, and pharmaceutically acceptable aqueous carrier medium, typically having a pH of about 5-8, more preferably a pH of about 6-8, although the pH can vary depending on the nature of the substance being formulated and the condition being treated. The formulated pharmaceutical compositions can be administered by conventional routes, including, but not limited to, inhalation, oral, intramuscular, intraperitoneal, subcutaneous, intradermal, or topical administration.

[0037] In the case of the targeting agent of the present application, it can be combined with a suitable pharmaceutically acceptable solvent or carrier. Such pharmaceutical compositions contain a therapeutically effective amount of the compound and a pharmaceutically acceptable carrier or excipient. Such solvents include, but are not limited to, saline, buffer, dextrose, water, glycerol, ethanol, and combinations thereof. Such carriers include, but are not limited to, hydrogels, microspheres, nanoparticles, liposomes, micelles, vesicles, and combinations thereof. The pharmaceutical preparation should be matched with the administration method. The targeting agent of the present application can be prepared in the form of a needle, for example, by a conventional method using normal saline or an aqueous solution containing dextrose and other adjuvants. Pharmaceutical compositions such as tablets and capsules can be prepared by conventional methods. Pharmaceutical compositions such as needles, solutions, tablets, and capsules are preferably manufactured under sterile conditions. The amount of active ingredient administered is a therapeutically effective amount, for example, about 1 microgram per kilogram of body weight to about 500 milligrams per kilogram of body weight per day.

[0038] In a third aspect, the present application provides use of the above-mentioned composition in the preparation of a medicament for preventing and / or treating fibrosis and diseases explicitly related to fibrosis in an individual.

[0039] In a fourth aspect, the present application provides a kit comprising:

[0040] (a) a first preparation comprising a targeting agent labeled with a nuclide, wherein the targeting agent targets fibroblast activation protein;

[0041] (b) a second preparation comprising a photosensitizer; and

[0042] Optionally, (c) instructions for use.

[0043] In some preferred embodiments, the instructions for use indicate that the first preparation and the second preparation are used in combination to prevent and / or treat fibrosis in an individual.

[0044] In some preferred embodiments, the first preparation and the second preparation are administered simultaneously, separately, or sequentially in preventing and / or treating fibrosis in an individual.

[0045] In some preferred embodiments, the first preparation and the second preparation are independent of each other.

[0046] In some preferred embodiments, the first agent and the second agent are combined.

[0047] In a fifth aspect, the present application provides a kit comprising:

[0048] (a) a first agent comprising a sonosensitizer; and

[0049] (b) a sonodynamic device capable of generating sonodynamic effect.

[0050] Further, the sonodynamic effect is ultrasound, and further, the ultrasound is at a condition of 1.0-3.0 MHz, 20-80% duty cycle, 0.5-2 W / cm 2 , 2-10 min.

[0051] In a sixth aspect, the present application provides a method for preventing and / or treating fibrosis in a subject, comprising:

[0052] applying to the subject a physical method, a chemical method, or a physical method in combination with a chemical method, so that the level of reactive oxygen species in the cells of the subject is increased to 2-30 times the level of reactive oxygen species in untreated cells.

[0053] In a specific embodiment, the present application provides a method for preventing and / or treating fibrosis in a subject, comprising:

[0054] applying to the subject a composition for preventing and / or treating fibrosis in the subject, comprising:

[0055] a radionuclide-labeled targeting agent, wherein the targeting agent targets fibroblast activation protein, and

[0056] a photosensitizer.

[0057] Further, the concentration of the composition is: radionuclide-labeled targeting agent (0.1-10 mCi / mL), photosensitizer (1-500 μM).

[0058] In a specific embodiment, the targeting agent is 68 Ga-FAPI (1 mCi / mL), and the photosensitizer is DPA-SCP (10 μM).

[0059] In another specific embodiment, the present application provides a method for preventing and / or treating fibrosis in a subject, comprising:

[0060] applying to the subject a reactive oxygen species-related compound for preventing and / or treating fibrosis in the subject, and activating the reactive oxygen species by a physical method, comprising:

[0061] a sonosensitizer, and a sonodynamic effect.

[0062] Further, the concentration of the sonosensitizer is 1-500 μM, and the sonodynamic force is ultrasound.

[0063] In a specific embodiment, the condition of ultrasound is 2 MHz, 50% duty cycle, 1 W / cm 2 , 5 min, and the sonosensitizer is DPA-SCP (10 μM).

[0064] In a seventh aspect, the present application provides a kit combination comprising:

[0065] (a) a first kit comprising a photosensitizer or a sonosensitizer; and

[0066] (b) a second kit comprising a substance capable of generating a photodynamic force or a sonodynamic force.

[0067] Further, the first kit can be a photosensitizer as described above in the present application.

[0068] Further, the first kit can be a sonosensitizer as described above in the present application.

[0069] Further, the second kit can be a radionuclide-labeled targeting agent as described above in the present application, and the targeting agent targets fibroblast activation protein.

[0070] Further, the second kit can be a sonodynamic device, which can generate a sonodynamic force, and further, the sonodynamic force is ultrasound. 。 BRIEF DESCRIPTION OF DRAWINGS

[0071] Figure 1: Western blotting results of fibroblast activation-related indicators after treatment with different concentrations of H2O2;

[0072] Figure 2: Western blotting results of fibroblast activation-related indicators after treatment with different concentrations of H2O2;

[0073] Figure 3: A, fluorescence intensity of AIE taken up by two kinds of cells; B, statistical diagram of fluorescence intensity of AIE taken up by two kinds of cells; C, laser confocal microscope detection results;

[0074] Figure 4: A, flow cytometry results of AIE targeted enrichment in the lungs of pulmonary fibrosis mice; B, statistical diagram of AIE targeted enrichment in the lungs of pulmonary fibrosis mice;

[0075] Figure 5: A and B, proportion of Fibronectin-positive cells in mice; C, proportion of AIE-positive cells in Fibronectin-positive cells; D, proportion of AIE-positive cells in Fibronectin-negative cells; E, statistical diagram of the amount of AIE taken up by two groups of cells;

[0076] Figure 6: FAPI combined with AIE, results of ROS production in myofibroblasts;

[0077] Figure 7: A and B, CCK8 detection of the killing effect of AIE+FAPI on myofibroblasts and fibroblasts;

[0078] Figure 8: Western blot detection of fibroblast activation-related indicators;

[0079] Figure 9: A, tissue staining of lung fibrosis inhibited by the composition of the present application; B, western blot detection of lung fibroblast activation-related indicators;

[0080] Figure 10: Tissue staining of liver fibrosis inhibited by the composition of the present application;

[0081] Figure 11: Western blot detection of liver fibrosis-related indicators in each group of mice;

[0082] Figure 12: Survival rate of renal tubular epithelial cells after treatment;

[0083] Figure 13: After TGF-β stimulated renal tubular epithelial cells, different concentrations of H2O2 were given, and the activation indicators of renal tubular epithelial cells were detected by western blot;

[0084] Figure 14: After BLM induced skin fibrosis in mice, different drugs were given, and skin tissue sections were taken for H&E staining;

[0085] Figure 15: Ultrasound activates AIE, which can produce a large amount of ROS in cells;

[0086] Figure 16: After TGF-β stimulated liver fibroblasts, AIE and ultrasound stimulation were given, and the activation indicators of liver fibroblasts were detected by western blot. DETAILED DESCRIPTION

[0087] The advantages and various effects of the present application will be more clearly presented from the following specific embodiments and examples. Those skilled in the art should understand that these specific embodiments and examples are used to illustrate the present application, not to limit the present application.

[0088] Example 1, active oxygen reverses lung tissue myofibroblasts to fibroblasts

[0089] First, the primary fibroblasts derived from lung tissue were activated into myofibroblasts by stimulating with TGF-β for 24 hours. Then, the myofibroblasts were incubated with different concentrations of hydrogen peroxide (L: 150 nM, the intracellular ROS was 4 times of the control group after adding; H: 300 nM, the intracellular ROS was 10 times of the control group after adding) for 24 hours. The proteins of the above-mentioned cells were collected, and the fibroblast activation related indicators were detected by western. The results are shown in FIG. 1A. As can be seen from the figure, hydrogen peroxide does not promote the activation of fibroblasts, but significantly inhibits the expression of collagen and fibronectin in myofibroblasts, that is, it reverses the activation of fibroblasts.

[0090] Again, the fibroblasts were stimulated with TGF-β for 24 hours, and at the same time, different concentrations of hydrogen peroxide (L: 150 nM; H: 300 nM) were given to incubate with the cells. After 24 hours, the proteins of the above-mentioned cells were collected, and the fibroblast activation related indicators were detected by western. The results are shown in FIG. 1B. As can be seen from the figure, hydrogen peroxide does not promote the activation of fibroblasts, but significantly inhibits the expression of collagen and fibronectin in fibroblasts, that is, it inhibits the activation of fibroblasts.

[0091] Then, different concentrations of hydrogen peroxide (0-90 μM) were used to incubate with fibroblasts, and it can be seen from the western detection that the indicators of fibronectin and collagen did not increase, as shown in FIG. 2, which proves that high-dose ROS also cannot induce the activation of fibroblasts into myofibroblasts.

[0092] Example 2, AIE material can be effectively enriched in individual myofibroblasts

[0093] We first activated fibroblast into myofibroblast by stimulating them with TGF-β for 24 hours. Then we added 10 μΜ AIE materials into fibroblast and myofibroblast for 0.5, 2, 4, 8 hours. We found that the AIE uptake of myofibroblast was significantly higher than that of fibroblast after 8 hours of incubation, which was 4 times of that of fibroblast (as shown in Figure 3A-B). We collected the cells after 8 hours of incubation with AIE and detected their localization by laser confocal microscope (red fluorescence for AIE materials, green fluorescence for mitochondria indicated by dye mitotracker). We found that the mitochondria of myofibroblast were significantly increased compared with fibroblast, and a large amount of AIE materials were co-localized with mitochondria, which were enriched in myofibroblast by targeting mitochondria (Figure 3C).

[0094] We induced lung fibrosis in mice by bleomycin (BLM) and injected AIE (100 μΜ, 50 μΐ^) into the airway. After 24 hours, we prepared cell suspension from the lung tissue of mice and detected the proportion of AIE positive cells by flow cytometry. As shown in Figure 4, Blank group was the mice without injection of AIE materials; AIE group was the single marker group, which was used for comparison with blank group, and the AIE positive gate was framed; PBS-AIE group was the normal mice without lung fibrosis, and the proportion of AIE positive cells in the lung tissue was detected after airway injection of AIE; BLM-AIE group was the mouse model of lung fibrosis induced by bleomycin, and the proportion of AIE positive cells in the lung tissue was detected after airway injection of AIE (Figure 4A). By comparing PBS (i.e. normal mice) and BLM (lung fibrosis mice) groups, we confirmed that the uptake ability of lung tissue of BLM group mice (~50% positive rate, Figure 4B) to AIE materials was significantly stronger than that of PBS group (~18% positive rate, Figure 4B).

[0095] To confirm whether myofibroblasts in lung tissue of pulmonary fibrosis mice can effectively uptake AIE, we labeled the cell suspension of lung tissue of BLM and PBS group mice by flow cytometry with Fibronectin antibody (one of the markers of myofibroblasts). As shown in FIG. 5A, B, the proportion of Fibronectin positive cells in lung tissue of BLM group pulmonary fibrosis mice was 3.21%, which was three times of that in normal mice lung tissue (1.06%), proving the success of modeling. We further detected the proportion of AIE positive cells in Fibronectin positive cells of PBS and BLM groups, which were 52.6% and 62.8% (FIG. 5C), respectively, with no significant difference, proving that Fibronectin positive cells in both PBS and BLM groups can effectively uptake AIE materials. Our purpose is to hope that AIE can target myofibroblasts (i.e. Fibronectin positive cells), so the above results are not inconsistent with our goal. To further confirm whether AIE can target myofibroblasts, we detected the proportion of AIE positive cells in Fibronectin negative cells of BLM group mice (FIG. 5A, lower row), and found that there was also AIE signal in Fibronectin negative cells, accounting for about 40% (FIG. 5D), slightly less than the proportion of AIE positive cells in Fibronectin positive cells of BLM group mice (60%, FIG. 5D). We detected the average fluorescence intensity (MFI) of AIE in Fibronectin - AIE + cells and Fibronectin + AIE + cells to characterize the amount of AIE uptake by these two groups of cells, and confirmed that the amount of AIE material taken up by Fibronectin positive cells (MFI, ~ 15, FIG. 5E) was about 30 times of that by Fibronectin negative cells (MFI, ~ 0.5, FIG. 5E). The above in vivo experimental results confirmed that AIE materials can be effectively enriched in myofibroblasts (i.e. Fibronectin positive cells).

[0096] Example 3, use of the composition of the present application for inhibiting fibrosis

[0097] In the foregoing embodiments, it has been confirmed that AIE can be targeted to enrich myofibroblasts. Subsequently, we detected the amount of ROS in each group of cells after stimulating fibroblasts and myofibroblasts by AIE and FAPI combined with DCFH-DA. The group without the addition of TGF-β is fibroblast, and the group with the addition of TGF-β is activated myofibroblast. As shown in FIG. 6, the ROS in myofibroblast (AIE+FAPI, 25 times of the PBS group) is significantly higher than that in fibroblast (AIE+FAPI, 10 times of the PBS group).

[0098] Subsequently, we detected the killing effect of AIE+FAPI on myofibroblast and fibroblast (incubated for 24 hours) by CCK8, and found that AIE+FAPI can kill myofibroblast to a certain extent, but has no killing effect on fibroblast. However, the killing efficiency of myofibroblast is only 25% (FAPI: 100 μCi), which is much lower than our expectation. This experiment was repeated 3 times, and the killing effect was still low even if the concentration of AIE was increased. It is confirmed that the ROS generated by the combination of FAPI and AIE is mild, and the killing efficiency on cells is low. The results are shown in FIGS. 7A-B.

[0099] At the same time, the proteins of the above-mentioned cells were collected, and the fibroblast activation related indicators were detected by western. The results are shown in FIG. 8. As can be seen from FIG. 8, AIE+FAPI not only does not promote the activation of fibroblast, but also significantly inhibits the expression of collagen and fibronectin in myofibroblast, that is, reverses the activation of fibroblast.

[0100] Example 4, the composition of the present application is used for inhibiting lung fibrosis of mice

[0101] Based on the above in vitro experimental results, we carried out animal experiments (n=6) to confirm whether the effect is effective in vivo. BLM (bleomycin) is a drug for inducing lung fibrosis model, the PBS group is the normal mouse group, and the pirfenidone group is the positive control group. As shown in FIG. 9A, H&E, Masson and Sirius red were used to indicate the degree of lung fibrosis in each group of mice. It can be found that the lung fibrosis of the mice administered with AIE+FAPI has basically recovered, and the treatment effect is better than that of the pirfenidone group. The western results further confirm this result, and AIE+FAPI can effectively reverse lung fibrosis, and the treatment effect is better than that of the pirfenidone group (FIG. 9B).

[0102] Example 5, the composition of the present application for inhibiting liver fibrosis in mice

[0103] The effect of FAPI + AIE combination was also verified on the mouse model of liver fibrosis, referring to the method described in Example 4. As shown in Figure 10 below, the combination can effectively alleviate liver fibrosis. Masson (the blue part is collagen) proves that the liver collagen is reduced after administration. Western detection of tissue fibrosis confirms that the combination can effectively treat liver fibrosis (Figure 11).

[0104] Example 6, active oxygen for inhibiting kidney fibrosis in mice

[0105] Renal tubular epithelial cells are the main effector cells of kidney fibrosis. First, different concentrations of hydrogen peroxide (0.3-600 μM) were used to stimulate renal tubular epithelial cells, and cell activity was detected by CCK8 method. It was found that hydrogen peroxide below 300 μM had no obvious effect on the activity of renal tubular epithelial cells (as shown in Figure 12). Then, referring to the method described in Example 1, the renal tubular epithelial cells were activated by stimulating them with TGF-β for 24 hours. Then, different concentrations of hydrogen peroxide (0.3-100 μM) were used to incubate the renal tubular epithelial cells stimulated by TGF-β for 24 hours, and the proteins of the above cells were collected. Western was used to detect the activation-related indicators, and the results are shown in Figure 13. As can be seen from Figure 13, hydrogen peroxide does not promote the activation of renal tubular epithelial cells, but significantly inhibits the expression of collagen and fibronectin in them, i.e. reverses the activation of renal tubular epithelial cells, confirming that ROS can effectively treat kidney fibrosis.

[0106] Example 7, the composition of the present application for inhibiting skin fibrosis in mice

[0107] Abnormal activation of skin fibroblast is the main cause of skin fibrosis (a typical symptom of systemic sclerosis, also known as scleroderma) and scar formation. We induced skin fibrosis in mice by subcutaneously injecting bleomycin into the skin of the mice. After the modeling was successful, AIE, FAPI and AIE+FAPI were subcutaneously injected into the mice, respectively. The skin tissue sections of the mice in each group were taken and subjected to H&E staining, and the results are shown in Fig. 14. The skin tissue structure of the normal group of mice was complete and clear, the fibrous distribution of the dermis layer was uniform, the interstitial space was normal, the blood vessel wall structure was normal, there was no obvious inflammatory cell infiltration, the hair follicles in the dermis layer were visible, and the subcutaneous accessory fat layer and muscle tissue were clear and normal in structure. The skin tissue structure of the model group of mice was disordered, the number of collagen fibers increased significantly, the fibers were arranged closely, and the interstitial space was narrowed. A large number of inflammatory cells were observed in the dermis layer, the hair follicles in the dermis layer were obviously destroyed or disappeared, and the subcutaneous appendages were reduced (sebaceous glands and sweat glands were significantly reduced or even disappeared, and the fat layer was thinned or disappeared). The injection of AIE or FAPI alone had no obvious therapeutic effect on skin fibrosis in mice, and the histopathological characteristics of the sections showed no obvious changes compared with the model group. However, the skin fibrosis in the AIE+FAPI combined administration group of mice was significantly improved: the dermis layer of the skin was thinned, the number of thick collagen fiber bundles was significantly reduced, and the inflammatory cell infiltration was significantly reduced. The above results show that the composition of the present application can effectively inhibit the abnormal activation of skin fibroblasts, treat skin fibrosis (a typical symptom of systemic sclerosis, also known as scleroderma), and prevent and treat scars.

[0108] Example 8, Ultrasound-activated AIE generates ROS to reverse myofibroblasts to fibroblasts

[0109] The AIE material can also act as a photosensitizer, which can be activated by ultrasound to generate ROS. We co-incubated primary liver fibroblasts with AIE (10 μM) material, and then gave the cells ultrasound stimulation (2 MHz, 50% duty cycle, 1 W / cm 2 , 5 min). DCFH detected a large amount of ROS generated in the cells (green fluorescence signal, as shown in Fig. 15). In order to detect whether the ROS generated by the ultrasound-activated AIE material can also reverse the activation of fibroblasts, we detected the activation-related indicators in the cells by western blot. As shown in Fig. 16, the ROS generated by AIE plus ultrasound can effectively inhibit the activation of liver fibroblasts, confirming that this method can effectively treat fibrosis.

Claims

1. Use of reactive oxygen species and its related compounds as a medicament for preventing and / or treating fibrosis and diseases definitely related to fibrosis in a subject.

2. Use according to claim 1, wherein, The level of reactive oxygen species in the cells of the subject is 2-30 times of the level of reactive oxygen species in untreated cells.

3. Use according to claim 1, wherein, The reactive oxygen species are generated by physical methods, chemical methods, or physical methods combined with chemical methods.

4. The use according to claim 1, wherein, The physical methods combined with chemical methods are photodynamic combined with photosensitizer, or sonodynamic combined with sonosensitizer.

5. A composition for preventing and / or treating fibrosis and diseases definitely related to fibrosis in a subject, comprising: (a) a first active ingredient: a targeting agent labeled with a nuclide, wherein the targeting agent targets fibroblast activation protein; (b) a second active ingredient: a photosensitizer.

6. The composition of claim 5, wherein, The targeting agent is monoclonal antibody mAbF19, ESC11, ESC14, polypeptide FAP-2286, FAP-RGD, quinoline small molecule inhibitor FAPI-04, FAPI-46, non-quinoline small molecule inhibitor iFAP, FL-L3.

7. The composition of claim 5, wherein, The photosensitizer can be a photosensitive material that can target mitochondria or be sensitive to mitochondrial membrane potential.

8. Use of the composition of any one of claims 5-7 in the preparation of a medicament for preventing and / or treating fibrosis and diseases definitely related to fibrosis in a subject.

9. A kit for preventing and / or treating fibrosis and diseases definitely related to fibrosis in a subject, comprising: (a) a first preparation comprising a targeting agent labeled with a nuclide, wherein the targeting agent targets fibroblast activation protein; (b) a second preparation comprising a photosensitizer.

10. A kit combination for preventing and / or treating fibrosis and diseases definitely related to fibrosis in a subject, comprising: (a) a first kit comprising a photosensitizer or a sonosensitizer; and (b) a second kit comprising a light source or a sound source capable of generating photodynamic or sonodynamic force.

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