Application of fexofenadine hydrochloride in the preparation of anti-fibrosis drugs

Abstract

The present invention relates to the field of biomedicine, and discloses the use of fexofenadine hydrochloride in the preparation of anti-fibrosis drugs. First, the present invention first discovered that 20 drugs including fexofenadine hydrochloride have the effect of significantly promoting the expression of flavin monooxygenase 2 (FMO2); second, the present invention first discovered that 20 drugs including fexofenadine hydrochloride can effectively inhibit organ fibrosis. Moreover, fexofenadine hydrochloride has no obvious toxic side effects within the effective dose range. Therefore, it can provide a new solution for the clinical treatment of fibrotic diseases, which is of great significance to improving the clinical treatment effect of fibrotic diseases and improving the quality of life of patients.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to the application of 20 compounds such as fexofenadine hydrochloride in the preparation of anti-fibrosis drugs. Background Art

[0002] Damage to normal human tissues triggers a series of complex cellular and molecular responses, such as overactivation of interstitial cells and excessive deposition of extracellular matrix, which can lead to fibrosis. Fibrosis plays a role in the progression of chronic diseases of many organs such as the heart, liver, and lungs. Among them, cardiac fibrosis is very common in the course of various cardiovascular diseases such as myocardial infarction and cardiomyopathy, and is the main cause of heart failure. Therefore, how to inhibit cardiac fibrosis is a key scientific issue and clinical need that needs to be solved urgently.

[0003] Although the causes of fibrosis in different tissues and organs are different, there is a persistent abnormal activation of myofibroblasts mediated by multiple signals such as transforming growth factor, platelet-derived growth factor, and fibroblast growth factor. Although the mechanism that drives organ-specific fibrosis has not been fully elucidated, drugs targeting the above abnormal signals have achieved certain efficacy in clinical trials, such as nintedanib and pirfenidone. So far, only these two drugs have been approved for the treatment of patients with idiopathic pulmonary fibrosis. Therefore, there is an urgent need to develop anti-fibrotic drugs targeting targets that affect myofibroblast activation.

[0004] The applicant's previous work found that the content of flavin monooxygenase 2 (FMO2) in cardiac tissue was significantly reduced after myocardial infarction. Downregulation of FMO2 in cardiac fibroblasts can lead to impaired cardiac function, increased collagen deposition, increased number of myofibroblasts, and activation of the TGFβ1 / Smad3 pathway, suggesting that FMO2 can be used as a target for drugs to treat cardiac fibrosis.

[0005] Compared with newly developed drugs, new uses of old drugs have significant advantages: (1) they have good biological activity and pharmacokinetic properties; (2) they can be directly applied and their efficacy can be observed; (3) they ensure the safety of clinical use. Therefore, screening the FDA-approved drug library has positive significance for the development of drugs that promote FMO2 expression for the treatment of fibrosis. Summary of the invention

[0006] In order to solve the above technical problems, the present invention provides an application of 20 compounds such as fexofenadine hydrochloride in the preparation of anti-fibrosis drugs. The present invention first discovered that 20 compounds such as fexofenadine hydrochloride have the effect of significantly promoting the expression of flavin monooxygenase 2 (FMO2) and can effectively inhibit organ fibrosis. Moreover, there are no obvious toxic and side effects within the effective dose range. Therefore, it can provide a new solution for the clinical treatment of fibrotic diseases, which is of great significance to improving the clinical treatment effect of fibrotic diseases and improving the quality of life of patients.

[0007] The specific technical scheme of the present invention is:

[0008] First, the present invention provides the use of 20 drugs including fexofenadine hydrochloride (nitazoxanide, procyclidine hydrochloride, imiquimod, cefpodoxime proxetil, baclofen, indomethacin, clofazimine, cabozantinib malate, naphazoline hydrochloride, olodaterol, prochlorperazine, chloroquine phosphate, sulfapyridine, tamoxifen citrate, metaproterenol sulfate, ensidipine, cilastatin and apremilast) in the preparation of FMO2 agonists.

[0009] Fexofenadine hydrochloride is currently widely used to treat seasonal allergic rhinitis, chronic idiopathic urticaria and other diseases. The present invention team first discovered through experiments that the above drugs, including fexofenadine hydrochloride, have a significant effect of promoting the expression of flavin monooxygenase 2 (FMO2), and therefore are all new FMO2 agonists.

[0010] Preferably, the FMO2 is FMO2 in fibroblasts.

[0011] Preferably, the fibroblasts are cardiac fibroblasts, dermal fibroblasts or embryonic fibroblasts.

[0012] The present invention team discovered through experiments that fexofenadine hydrochloride, nitazoxanide, procyclidine hydrochloride, imiquimod, cefpodoxime proxetil, baclofen, indomethacin, clofazimine, cabozantinib malate, naphazoline hydrochloride, olodaterol, prochlorperazine, chloroquine phosphate, sulfapyridine, tamoxifen citrate, metaprenaline sulfate, ensidipine, cilastatin or apremilast can promote the expression level of FMO2 in fibroblasts from different species and organs, and reverse the downregulation of FMO2 caused by the TGFβ signaling pathway.

[0013] Secondly, the present invention provides the use of fexofenadine hydrochloride, nitazoxanide, procyclidine hydrochloride, imiquimod, cefpodoxime proxetil, baclofen, indomethacin, clofazimine, cabozantinib malate, naphazoline hydrochloride, olodaterol, prochlorperazine, chloroquine phosphate, sulfapyridine, tamoxifen citrate, metaprenaline sulfate, ensidipine, cilastatin or apremilast in the preparation of a medicament for preventing and / or treating fibrosis.

[0014] Based on the applicant's previous research findings (FMO2 can be used as a target for drugs to treat cardiac fibrosis), the present invention team further discovered through experiments that the above drugs, including fexofenadine hydrochloride, can effectively inhibit the expression of fibrosis markers (α-SMA) and extracellular matrix proteins (periostin, fibronectin) in primary fibroblasts induced by TGF-β, and thus can effectively inhibit the classical signaling pathway of organ fibrosis, thereby having the effect of inhibiting organ fibrosis. In addition, the present invention also confirms that fexofenadine hydrochloride has no obvious toxic side effects within the effective dosage range.

[0015] Preferably, the fibrosis is cardiac fibrosis or skin fibrosis.

[0016] As further preferred, the fibrosis is cardiac fibrosis.

[0017] The present invention finds that the above-mentioned drugs including fexofenadine hydrochloride have a particularly good inhibitory effect on cardiac fibrosis, which is specifically manifested in that they can significantly reduce cardiac collagen deposition after myocardial infarction, thereby inhibiting cardiac fibrosis.

[0018] Preferably, the cardiac fibrosis is cardiac fibrosis caused by ischemic heart disease, hypertensive heart disease, cardiomyopathy, arrhythmia or rheumatic heart disease.

[0019] As further preferred, the cardiac fibrosis is cardiac fibrosis caused by ischemic heart disease.

[0020] The present invention has found through experiments that the above-mentioned drugs including fexofenadine hydrochloride have a good inhibitory effect on cardiac fibrosis caused by ischemic heart disease.

[0021] Preferably, the drug comprises: as main active ingredients, fexofenadine hydrochloride, nitazoxanide, procyclidine hydrochloride, imiquimod, cefpodoxime proxetil, baclofen, indomethacin, clofazimine, cabozantinib malate, naphazoline hydrochloride, olodaterol, prochlorperazine, chloroquine phosphate, sulfapyridine, tamoxifen citrate, metaproterenol sulfate, ensidipine, cilastatin or apremilast, and pharmaceutically acceptable excipients.

[0022] Preferably, the drug is in the form of an oral preparation.

[0023] In addition, the present invention also provides a method for inhibiting fibrosis of cardiac fibroblasts in vitro: treating cardiac fibroblasts cultured in vitro with fexofenadine hydrochloride, nitazoxanide, procyclidine hydrochloride, imiquimod, cefpodoxime proxetil, baclofen, indomethacin, clofazimine, cabozantinib malate, naphazoline hydrochloride, olodaterol, prochlorperazine, chloroquine phosphate, sulfapyridine, tamoxifen citrate, metaprenaline sulfate, ensidipine, cilastatin or apremilast to increase the expression level of FMO2 in the cardiac fibroblasts, and reduce the expression of fibrosis marker α-SMA and extracellular matrix proteins periostin and fibronectin, thereby inhibiting fibrosis of cardiac fibroblasts.

[0024] Compared with the prior art, the beneficial effects of the present invention are:

[0025] (1) The present invention first discovered that 20 drugs including fexofenadine hydrochloride have the effect of significantly promoting the expression of flavin monooxygenase 2 (FMO2) and can be used as a new FMO2 agonist.

[0026] (2) The present invention further found that 20 drugs including fexofenadine hydrochloride can effectively inhibit the expression of fibrosis markers (α-SMA) and extracellular matrix proteins (periostin, fibronectin) in fibroblasts induced by TGF-β, thereby effectively inhibiting the classical signaling pathway of organ fibrosis, thereby having the effect of inhibiting organ fibrosis.

[0027] (3) The present invention confirms that fexofenadine hydrochloride has no obvious toxic side effects within the effective dosage range. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 The top 20 drugs that can increase FMO2 expression were screened from the FDA drug library;

[0029] Figure 2 The results are the validation of the effects of different drugs on FMO2 expression in primary cardiac fibroblasts of neonatal rats;

[0030] Figure 3 To investigate the effect of fexofenadine hydrochloride on the expression of FMO2 in fibroblasts from different species and tissues;

[0031] Figure 4 The expression of FMO2, fibrosis marker protein α-SMA, and extracellular matrix proteins Periostin and Fibronectin were detected by Western blot in primary neonatal rat cardiac fibroblasts treated with fexofenadine hydrochloride in vitro;

[0032] Figure 5 The primary neonatal rat cardiac fibroblasts were treated with fexofenadine hydrochloride in vitro, and the expression of fibrosis marker proteins and extracellular matrix proteins α-SMA, Periostin and Fibronectin were detected by immunofluorescence staining;

[0033] Figure 6 The expression of FMO2, fibrosis marker protein α-SMA, and extracellular matrix proteins Periostin and Fibronectin were detected by Western blot in primary human cardiac fibroblasts treated with fexofenadine hydrochloride in vitro;

[0034] Figure 7 This is a picture showing the cardiac fibrosis detected by Sirius red staining in wild-type mice after daily intragastric administration of 10 mg / kg fexofenadine hydrochloride.

[0035] Figure 8 The results of cardiac function testing by cardiac ultrasound were shown in wild-type mice after daily intragastric administration of 10 mg / kg fexofenadine hydrochloride.

[0036] Fig. 9 The morphological changes of liver, kidney and small intestine of wild-type mice after daily intragastric administration of 10 mg / kg fexofenadine hydrochloride.

[0037] Fig.10 The chart shows the changes in serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), blood urea nitrogen (BUN), and creatinine (Cr) in wild-type mice after daily intragastric administration of 10 mg / kg fexofenadine hydrochloride. DETAILED DESCRIPTION

[0038] The present invention will be further described below in conjunction with the embodiments.

[0039] Overall embodiment

[0040] First, the use of fexofenadine hydrochloride, nitazoxanide, procyclidine hydrochloride, imiquimod, cefpodoxime proxetil, baclofen, indomethacin, clofazimine, cabozantinib malate, naphazoline hydrochloride, olodaterol, prochlorperazine, chloroquine phosphate, sulfapyridine, tamoxifen citrate, mepronaline sulfate, ensidipine, cilastatin or apremilast in the preparation of FMO2 agonists.

[0041] In some specific embodiments, the FMO2 is FMO2 in fibroblasts.

[0042] In more specific embodiments, said fibroblasts are cardiac fibroblasts, dermal fibroblasts or embryonic fibroblasts.

[0043] Secondly, the use of fexofenadine hydrochloride, nitazoxanide, procyclidine hydrochloride, imiquimod, cefpodoxime proxetil, baclofen, indomethacin, clofazimine, cabozantinib malate, naphazoline hydrochloride, olodaterol, prochlorperazine, chloroquine phosphate, sulfapyridine, tamoxifen citrate, metaprenaline sulfate, ensidipine, cilastatin or apremilast in the preparation of drugs for preventing and / or treating fibrosis.

[0044] In some specific embodiments, the fibrosis is cardiac fibrosis or skin fibrosis. As further preferred, the fibrosis is cardiac fibrosis.

[0045] In some specific embodiments, the cardiac fibrosis is cardiac fibrosis caused by ischemic heart disease, hypertensive heart disease, cardiomyopathy, arrhythmia or rheumatic heart disease. As further preferred, the cardiac fibrosis is cardiac fibrosis caused by ischemic heart disease.

[0046] In some specific embodiments, the drug includes: as main active ingredients, fexofenadine hydrochloride, nitazoxanide, procyclidine hydrochloride, imiquimod, cefpodoxime proxetil, baclofen, indomethacin, clofazimine, cabozantinib malate, naphazoline hydrochloride, olodaterol, prochlorperazine, chloroquine phosphate, sulfapyridine, tamoxifen citrate, metaproterenol sulfate, ensidipine, cilastatin or apremilast, and pharmaceutically acceptable excipients.

[0047] In some specific embodiments, the drug is in a formulation for oral administration.

[0048] Furthermore, a method for inhibiting fibrosis of cardiac fibroblasts in vitro: treating cardiac fibroblasts cultured in vitro with fexofenadine hydrochloride, nitazoxanide, procyclidine hydrochloride, imiquimod, cefpodoxime proxetil, baclofen, indomethacin, clofazimine, cabozantinib malate, naphazoline hydrochloride, olodaterol, prochlorperazine, chloroquine phosphate, sulfapyridine, tamoxifen citrate, orphenadrine sulfate, ensidipine, cilastatin or apremilast to increase the expression level of FMO2 in the cardiac fibroblasts, and reduce the expression of fibrosis marker α-SMA and extracellular matrix proteins periostin and fibronectin, thereby inhibiting fibrosis of cardiac fibroblasts. Specific embodiments

[0050] Example 1: Screening of FMO2 agonists

[0051] The 20 drugs including fexofenadine hydrochloride that promote FMO2 expression and have anti-fibrosis effects in the present invention are derived from the screening of the FDA drug library. The team of the present invention learned in the early stage of the study that increasing the expression of FMO2 in cardiac fibroblasts can improve cardiac function after myocardial infarction, reduce collagen deposition and the number of myofibroblasts, and inhibit the activation of the TGFβ1 / Smad3 pathway. To this end, the team of the present invention used the HiBiT Lytic Detection System and the FMO2-hibit stable strain, took Cocl2 as the positive compound, and used the FDA-approved drug library (containing 1143 compounds) to screen drugs that can increase FMO2 expression through high-throughput screening experiments. The specific process is as follows:

[0052] A human dermal fibroblast cell line stably expressing FMO2-Hibit was constructed and cultured in a 384-well plate at 3000 cells / 30ul / well at 37°C and 5% carbon dioxide for 24 hours. The next day, drugs with a final concentration of 5μM or 400μM cocl2 were added to the screening system and cultured for another 24 hours. After 24 hours of drug treatment, the cells were lysed and the chemiluminescence value was detected using the HiBiT Lytic Detection System to quantify Hibit expression.

[0053] Figure 1 The screening results of the FDA drug library: the horizontal axis is the Z value, Z value = (average chemiluminescence value of the drug treatment group - average chemiluminescence value of the control group) / chemiluminescence standard of the drug treatment group; the vertical axis is the -log (Q) value, Q = P value * total number of drugs / drug P value in ascending order; we consider Z-score>0.5 and Q value>1.0 to be positive drugs.

[0054] The TOP20 positive drugs selected according to the -log(Q) value are nitazoxanide, ulipristal acetate, procyclidine hydrochloride, imiquimod, cefpodoxime proxetil, baclofen, indomethacin, clofazimine, cabozantinib malate, naphazoline hydrochloride, olodaterol, prochlorperazine, fexofenadine hydrochloride, chloroquine phosphate, sulfapyridine, tamoxifen citrate, metaprenaline sulfate, ensidipine, cilastatin, and apremilast (excluding the antibiotics cefpodoxime proxetil and sulfapyridine and the cardiotoxic drugs baclofen, indomethacin, chloroquine phosphate, and tamoxifen citrate, and subsequent studies will be conducted on the remaining 14 drugs).

[0055] Example 2: Further verification of the TOP20 compounds screened in Example 1

[0056] Primary rat cardiac fibroblasts (NRCF) were isolated and cultured in low-glucose DMEM medium containing 10% fetal bovine serum. After the cells were confluent, they were passaged in a 12-well plate at a ratio of 1:2. When the cells grew to 70% confluent, the medium was replaced with serum-free low-glucose DMEM medium and cultured overnight. Then, fexofenadine hydrochloride (the same operation was performed for the other 13 compounds) was added to the low-glucose medium (the final drug concentration was 5uM). The control group was cultured for 24 hours with only DMSO. The culture supernatant was discarded, washed twice with PBS buffer, and 100ul / well of RIPA cell lysis buffer containing protease phosphatase inhibitors was added. After standing on ice for 5 minutes, the protein was scraped into a 1.5ml EP tube with a cell scraper, and then it was placed on ice for 15 minutes, and then it was placed in a centrifuge and centrifuged at 12,000g for 10 minutes. After centrifugation, 90ul of the supernatant was taken into a new EP tube. Then the protein concentration was determined by BCA method. After determining the protein concentration, dilute the protein to the same concentration with 2.5X loading buffer containing 2-mercaptoethanol. Then boil the protein in a metal bath at 99℃ for 10 minutes. Then perform Western blot protein electrophoresis and transfer. After transfer, block the PVDF membrane with PBST solution containing 5% bovine serum albumin for 1 hour, then prepare the anti-FMO2 primary antibody (antibody catalog number corresponds to NBP1-85952) with the primary antibody diluent at a volume ratio of 1:1000, incubate overnight at 4℃, recover the primary antibody, wash 5 times with PBST, 3 minutes each time. Then add the prepared HRP-conjugated secondary antibody of the corresponding primary antibody source species. Incubate at room temperature for 1 hour. Discard the secondary antibody and wash 5 times with PBST, 3 minutes each time. Then use the developer to develop the bands. The β-actin internal reference was incubated with HRP direct-labeled antibody (1:6000 dilution) at room temperature for 2 hours, the direct-labeled antibody was discarded, and the sample was washed 5 times with PBST, 3 minutes each time, and then the bands were developed and imaged with developer solution. The gray value was calculated using Image J software.

[0057] Figure 2 The results are the validation results of the effects of different drugs on FMO2 expression in primary cardiac fibroblasts of neonatal rats. Figure 2 As shown in the figure, most drugs (12) that can increase FMO2 expression in human dermal fibroblasts can also increase FMO2 in primary cardiac fibroblasts of neonatal rats, among which the expression of FMO2 increased most significantly after treatment with fexofenadine hydrochloride.

[0058] Example 3: Verification of fexofenadine hydrochloride increasing FMO2 expression in fibroblasts

[0059] Human dermal fibroblast cell lines stably expressing FMO2-Hibit, rat primary cardiac fibroblasts (NRCF), mouse embryonic fibroblasts, and isolated human primary cardiac fibroblasts were propagated into 12-well plates. When the cells grew to 70% confluence, the culture medium was replaced with serum-free low-glucose DMEM medium and cultured overnight. The next day, 0 μM, 1.25 μM, 2.5 μM, 5 μM, 10 μM, and 20 μM fexofenadine hydrochloride (T1470, Taoshu Biological) were added and cultured for 24 hours. After 24 hours of drug treatment, the cells were lysed, and the human dermal fibroblast cell line was used to quantify Hibit expression using the HiBiT Lytic Detection System. The remaining cells were subjected to Western blot experiments (as above) after protein extraction to detect the expression level of FMO2.

[0060] Figure 3 The effect of fexofenadine hydrochloride on the expression of FMO2 in human dermal fibroblast cell lines, rat primary cardiac fibroblasts, mouse embryonic fibroblasts, and human primary cardiac fibroblasts; the experimental results showed that 5μM fexofenadine hydrochloride can increase the expression of FMO2 in fibroblasts of different species and different tissue sources.

[0061] Example 4: Western blot evaluation of the effect of fexofenadine hydrochloride on inhibiting the expression of fibrosis markers and extracellular matrix proteins in primary neonatal rat cardiac fibroblasts

[0062] Primary cardiac fibroblasts (NRCF) from neonatal rats were isolated and cultured in high-glucose DMEM medium containing 10% fetal bovine serum. After the cells were confluent, they were passaged in a 12-well plate at a ratio of 1:2. When the cells were 70% confluent, the medium was replaced with serum-free low-glucose DMEM medium and cultured overnight. TGFβ was then added to the low-glucose medium (the final concentration of TGFβ was 20 ng / ml). The control group only added TGFβ solvent. After 24 hours, fexofenadine hydrochloride was added at a final concentration of 5uM. The control group only added DMSO; the two were cultured together for 24 hours. Proteins were extracted for Western blot experiments (same as above). The primary antibody catalog numbers for aSMA, Periostin, Fibronectin, FMO2, and Collagen 1a1 are ab5694, AF2955, ab2413, NBP1-85952, and ab270993, respectively).

[0063] Figure 4In order to treat primary neonatal rat cardiac fibroblasts with fexofenadine hydrochloride in vitro, the expressions of FMO2, fibrosis marker protein α-SMA, and extracellular matrix proteins Periostin and Fibronectin were detected by Western blot. α-SMA, Periostin and Fibronectin were significantly upregulated after TGFβ treatment, and then inhibited after fexofenadine hydrochloride treatment. Meanwhile, the downregulation of FMO2 caused by the activation of TGFβ signaling pathway was also inhibited after fexofenadine hydrochloride treatment, indicating that fexofenadine hydrochloride can increase the expression of FMO2 and inhibit the activation of cardiac fibroblasts.

[0064] Example 5: Immunofluorescence evaluation of the effect of fexofenadine hydrochloride on the expression of fibrosis markers and extracellular matrix proteins in primary neonatal rat cardiac fibroblasts

[0065] Primary cardiac fibroblasts (NRCF) from neonatal rats were isolated and cultured in high-glucose DMEM medium containing 10% fetal bovine serum. After the cells were confluent, they were passaged in a 24-well plate at a ratio of 1:2. When the cells grew to 70% confluent, the medium was replaced with serum-free low-glucose DMEM medium and cultured overnight. Then TGFβ was added to the low-glucose medium (the final concentration of TGFβ was 20ng / ml). The control group only added TGFβ solvent. After 24 hours, fexofenadine hydrochloride with a final concentration of 5uM was added, and the control group only added DMSO; the two were cultured together for 24 hours. The culture supernatant was discarded, washed twice with PBS buffer, fixed with 4% paraformaldehyde at room temperature for 10 minutes, washed three times with PBS buffer, and permeabilized with 0.5% Triton X-100 (prepared with 1xPBS) at room temperature for 20 minutes; after washing three times with PBS, the cells were blocked with goat serum for 30 minutes at room temperature, and the primary antibody was diluted to the target ratio with antibody diluent, and incubated overnight at 4℃. The next day, take out the wet box and rewarm for 30 minutes, then wash with PBS three times, 5 minutes each time; fluorescent secondary antibody: dilute the fluorescent secondary antibody with antibody diluent, incubate at room temperature in the dark for 2 hours, wash with PBS three times, seal the slide with DAPI staining solution containing anti-quenching agent, dry the slide, and observe under a microscope to obtain fluorescent images.

[0066] Figure 5 In this study, fexofenadine hydrochloride was used to treat primary neonatal rat cardiac fibroblasts in vitro. Immunofluorescence staining was used to detect the expression of fibrosis marker protein α-SMA and extracellular matrix proteins Periostin and Fibronectin (green), and blue is DAPI. The results showed that the fluorescence intensity of these proteins was significantly upregulated after TGFβ treatment, and was inhibited after fexofenadine hydrochloride treatment. This indicates that fexofenadine hydrochloride can inhibit the activation of cardiac fibroblasts.

[0067] Example 6: Evaluation of the effect of fexofenadine hydrochloride in inhibiting the expression of fibrosis markers and extracellular matrix proteins in primary human cardiac fibroblasts

[0068] Human primary cardiac fibroblasts (NRCF) were isolated and cultured in DMEM / F12 medium containing 10% fetal bovine serum. After the cells were confluent, they were passaged in a 12-well plate at a ratio of 1:2. When the cells grew to 70% confluent, the medium was replaced with serum-free DMEM / F12 medium and cultured overnight. Then TGFβ was added to the medium (the final concentration of TGFβ was 20ng / ml). The control group only added TGFβ solvent. After 24 hours, fexofenadine hydrochloride with a final concentration of 5uM was added, and the control group only added DMSO; the two were cultured together for 24 hours. Proteins were extracted for Western blot experiments (same as above). The primary antibody catalog numbers for aSMA, Periostin, Fibronectin and FMO2 are ab5694, AF2955, ab2413 and NBP1-85952, respectively).

[0069] Figure 6 In order to treat primary human cardiac fibroblasts with fexofenadine hydrochloride in vitro, the expressions of FMO2, fibrosis marker protein α-SMA, and extracellular matrix proteins Periostin and Fibronectin were detected by Western blot. α-SMA, Periostin and Fibronectin were significantly upregulated after TGFβ treatment, but inhibited after fexofenadine hydrochloride treatment. Meanwhile, the downregulation of FMO2 caused by activation of TGFβ signaling pathway was also inhibited after fexofenadine hydrochloride treatment. Figure 4-6 The in vitro results showed that fexofenadine hydrochloride could increase the expression of FMO2 and inhibit the activation of cardiac fibroblasts, and had anti-fibrotic potential.

[0070] Example 7: Evaluation of cardiac fibrosis and cardiac function in mice with myocardial infarction treated with fexofenadine hydrochloride

[0071] Eight-week-old C57 mice were used for myocardial infarction modeling and then randomly divided into two groups, with 6 mice in each group. Cardiac ultrasound contractile function evaluation was performed 3 days after modeling to ensure that there was no difference in baseline cardiac function between the two groups. The experimental group was given 10 mg / kg of fexofenadine hydrochloride by gavage every day, and the control group was injected with an equal amount of fexofenadine hydrochloride solvent each time. Samples were collected 4 weeks after modeling, and gross images of the mouse heart were taken. The heart was fixed and dehydrated and then embedded. Then 4um thick paraffin sections were made, starting from the apex of the heart, and sections were collected when the ring-shaped tissue structure appeared. One section was collected every 500um, and 5 levels were collected for each heart. Sirius red staining was then performed, and the proportion of infarct fibrosis area was statistically analyzed using Image J software.

[0072] Figure 7 Wild-type mice were given 10 mg / kg fexofenadine hydrochloride by intragastric administration 3 days after myocardial infarction surgery. Samples were collected 28 days after surgery and the cardiac fibrosis was detected by Sirius red staining. Figure 7 The following are representative Sirius red staining images of the heart at each layer of 500um in the control group and the fexofenadine hydrochloride treatment group. The results show that compared with the control group, the area of ​​fibrosis after fexofenadine hydrochloride treatment is reduced, indicating that fexofenadine hydrochloride has the effect of improving fibrosis after myocardial infarction.

[0073] Example 8: Evaluation of cardiac function in mice with myocardial infarction treated with fexofenadine hydrochloride

[0074] Eight-week-old C57 mice were used for myocardial infarction modeling and then randomly divided into two groups, with 6 mice in each group. The cardiac ultrasound contractile function was evaluated 3 days after modeling to ensure that there was no difference in baseline cardiac function between the two groups. The experimental group was given 10 mg / kg of fexofenadine hydrochloride by gavage every day, and the control group was injected with an equal amount of fexofenadine hydrochloride solvent each time. Ultrasound cardiac contractile function was evaluated 14 and 28 days after modeling.

[0075] Figure 8 Wild-type mice were treated with 10 mg / kg fexofenadine hydrochloride by intragastric administration 3 days after myocardial infarction surgery. The left ventricular ejection fraction was measured by cardiac ultrasound before myocardial infarction and 3, 14, and 28 days after myocardial infarction. Figure 8 As shown, 14 and 28 days after myocardial infarction, the left ventricular ejection fraction in the fexofenadine hydrochloride treatment group was higher than that in the control group and the difference was statistically significant, indicating that fexofenadine hydrochloride has the effect of improving cardiac function after myocardial infarction.

[0076] Example 9: HE staining to evaluate the toxic and side effects of fexofenadine hydrochloride on mice

[0077] When the mice were collected after the myocardial infarction model was established for 28 days and the fexofenadine hydrochloride treatment was completed in Example 7-8, the kidneys, livers and small intestines of the mice were taken, fixed and dehydrated, and then embedded, and paraffin sections with a thickness of 4 microns were made. After the sections were dewaxed, HE staining was performed using a fully automatic staining machine, and the sections were sealed with neutral gum and photographed using a Leica upright microscope.

[0078] Fig. 9 Wild-type mice were given 10 mg / kg fexofenadine hydrochloride by gavage every day 3 days after myocardial infarction surgery. Samples were collected 28 days after surgery to observe the morphological changes of the liver, kidney, and small intestine by HE staining to evaluate the safety of the drug. Fig. 9As shown, compared with the control group, the liver, kidney and small intestine of the mice in the fexofenadine hydrochloride treatment group and the control group did not show pathological changes (such as vacuolar degeneration, pigmentation, cell necrosis and inflammatory cell infiltration), indicating that fexofenadine hydrochloride has no obvious toxic side effects on mice at an effective anti-fibrosis dose and has good biosafety.

[0079] Example 10: Evaluation of the toxic and side effects of fexofenadine hydrochloride on mice by blood biochemical detection

[0080] When the mice in Example 7-8 were subjected to myocardial infarction modeling for 4 weeks and completed fexofenadine hydrochloride treatment, blood was collected into 1.5 ml EP tubes, left to stand at room temperature for 2 hours until the blood coagulated, and then centrifuged at 4°C, 3000 rpm for 10 min to obtain the upper serum, and the levels of creatinine (Cr), urea nitrogen (BUN), alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were measured using an automatic biochemical analyzer.

[0081] Fig.10 Wild-type mice were treated with 10 mg / kg fexofenadine hydrochloride by intragastric administration 3 days after myocardial infarction surgery. The samples were collected 28 days after surgery. The blood biochemical indicators including serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), blood urea nitrogen (BUN), and creatinine (Cr) were used to reflect liver and kidney function and evaluate the safety of the drug. Fig.10 As shown in the figure, the values ​​of creatinine (Cr), urea nitrogen (BUN), serum alanine aminotransferase (ALT) and serum aspartate aminotransferase (AST) in the mice treated with fexofenadine hydrochloride were similar to those in the control group, with no significant difference. This indicates that fexofenadine hydrochloride has no effect on the liver and kidney function of mice at an effective anti-fibrosis dose and has good biosafety.

[0082] In summary:

[0083] (1) The present invention uses HiBiT Lytic Detection System and FMO2-hibit stable transfectants, takes Cocl2 as a positive compound, and uses an FDA-approved drug library (containing 1,143 compounds) to conduct a high-throughput screening experiment. The results show for the first time that 20 drugs, including fexofenadine hydrochloride, have the function of increasing FMO2. Specifically, the drug can increase the expression level of FMO2 in fibroblasts from different species and organs by increasing the transcription of FMO2, and reverse the downregulation of FMO2 caused by the TGFβ signaling pathway. It can effectively inhibit the fibrosis of target organs.

[0084] (2) The present invention uses fexofenadine hydrochloride to treat mice after myocardial infarction surgery. The results of Sirius red staining of the mice's hearts show that after WT is treated with fexofenadine hydrochloride, collagen deposition is significantly reduced compared with the control group, and cardiac function is significantly improved. The present invention uses fexofenadine hydrochloride to treat TGFβ-pretreated human cardiac primary fibroblasts. Western blot results show that the expression of α-SMA, periostin, and fibronectin is significantly downregulated after treatment. The results of the present invention prove that fexofenadine hydrochloride can improve the fibrotic state of the classic myocardial infarction model WT mice and human fibrotic cardiac fibroblasts.

[0085] (3) The present invention has experimentally confirmed that fexofenadine hydrochloride can improve cardiac function and fibrosis status in WT mice subjected to a myocardial infarction model (ligation of the left anterior branch), and has no obvious toxic side effects within the effective dose range.

[0086] The raw materials and equipment used in the present invention, unless otherwise specified, are all commonly used raw materials and equipment in the art; the methods used in the present invention, unless otherwise specified, are all conventional methods in the art.

[0087] The above description is only a preferred embodiment of the present invention and does not limit the present invention in any way. Any simple modification, change and equivalent transformation made to the above embodiment based on the technical essence of the present invention still falls within the protection scope of the technical solution of the present invention.

Claims

1. Use of fexofenadine hydrochloride as a FMO2 agonist in the preparation of a drug for preventing cardiac fibrosis, characterized in that: The fexofenadine hydrochloride is used as the only active ingredient.

2. The use according to claim 1, characterized in that: The medicine comprises: fexofenadine hydrochloride as an active ingredient, and pharmaceutically acceptable excipients.

3. The use according to claim 1, characterized in that: The drug is in the form of an oral preparation.

4. The use according to claim 1, characterized in that: The cardiac fibrosis is cardiac fibrosis caused by ischemic heart disease, hypertensive heart disease, cardiomyopathy, arrhythmia or rheumatic heart disease.

5. The use according to claim 4, characterized in that: The cardiac fibrosis is cardiac fibrosis caused by ischemic heart disease.

6. Use of fexofenadine hydrochloride as a FMO2 agonist in the preparation of a medicament for treating cardiac fibrosis, characterized in that: The fexofenadine hydrochloride is used as the only active ingredient.

7. The use according to claim 6, characterized in that: The medicine comprises: fexofenadine hydrochloride as an active ingredient, and pharmaceutically acceptable excipients.

8. The use according to claim 6, characterized in that: The drug is in the form of an oral preparation.

9. The use according to claim 6, characterized in that: The cardiac fibrosis is cardiac fibrosis caused by ischemic heart disease, hypertensive heart disease, cardiomyopathy, arrhythmia or rheumatic heart disease.

10. The use according to claim 9, characterized in that: The cardiac fibrosis is cardiac fibrosis caused by ischemic heart disease.

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