CN113368106A - Application of Iguratimod in preparation of medicine for preventing and treating idiopathic pulmonary fibrosis - Google Patents

Abstract

The invention discloses application of itomod in preparation of a medicament for preventing and treating idiopathic pulmonary fibrosis, and belongs to the technical field of medicaments. And discloses application of a pharmaceutical composition containing the Iguratimod as an active ingredient in preparing a medicament for preventing and treating idiopathic pulmonary fibrosis, wherein the Iguratimod is a compound shown as a formula (I).

Description

Application of Iguratimod in preparation of medicine for preventing and treating idiopathic pulmonary fibrosis

Technical Field

The invention relates to application of Iguratimod in preparation of a medicament for preventing and treating idiopathic pulmonary fibrosis, and belongs to the technical field of medicaments.

Background

Idiopathic Pulmonary Fibrosis (IPF) is a diffuse lung disease of unknown cause, progresses rapidly, has short median survival, high mortality, and has no specific therapeutic drugs. Chronic inflammation has long been considered as the most basic factor of IPF pathogenesis, and with the deep understanding of IPF pathogenesis, more and more researches in recent years indicate that irreversible damage to lung tissues caused by the damage of the integrity of the alveolar capillary endothelial barrier and the interstitial transformation process of the alveolar epithelial cells which are important constituent cells of the barrier play an extremely important role in the development of IPF pathogenesis and throughout the disease. Therefore, the anti-inflammatory drug is also searched for a drug capable of targeting the protection of the endothelial barrier function of the alveolar capillary, and a brand-new entry point can be provided for the treatment of IPF.

Sphingosine-1-phosphate (S1P) is a bioactive sphingolipid, and plays an important role in cell migration, vascular barrier, immune cell trafficking, etc. by binding to 5 high affinity receptors (S1P 1-5), i.e., S1P1, S1P2, S1P3, S1P4, and S1P 5. The distribution and physiological functions of different subtype receptors are different in different tissues and organs. The receptor subtypes distributed in lung tissues are S1P1, S1P2, S1P3 and S1P4, while the current research focus is mainly on S1P1, S1P2 and S1P 3.

Fingolimod (FTY 720) is the first marketed S1P1 receptor modulator, which also acts on S1P3, S1P4, S1P5 subtypes, and studies show that FTY720 has no therapeutic effect on IPF, but rather promotes vascular endothelial barrier damage in model animals to cause disease exacerbation (Gendron, D.R., et al, FTY720 proteins pulmonary vascular barrier injury, which leads to disease exacerbation) (Gmad. Pharmacol. The 2017.44: p.50-56; Oo, M.L., et al., Engagement of S1 (1) -definitional peptides to therapeutic in this patent, Clinical study of P.2300, J.3. No. 3. 2. J.90. P.2. J.2. et al., research of P.2. J.3. 3. P.3. 3. P.3. No. 3. P.3. 2. J. 2. and P.3. 3. P.3. 3. A. is also includes, a. A. with a. A. includes, and a. also includes, and a. also includes a. for example, and a. are also includes a. for example, and a. for example, and a. are also includes a. The inventors believe that, since S1P1/S1P3 have different regulatory effects on alveolar-capillary barrier function, the barrier disruption caused by FTY720, probably due to its lack of selectivity for S1P1 and S1P3, does not therefore deny the possibility that S1P1 becomes a target for IPF therapy.

Itomod (SYL927, IMMH002) is a new-result small molecule S1P1 regulator (Chinese patent No. ZL201280032727.7) autonomously synthesized by the institute of medicine of Chinese academy of medicine, and simultaneously acts on S1P4 and S1P5 subtypes (Jin Jin, et al, A novel S1P1 modulator IMMH002 excipients in multiple animal models, acta pharmaceutical Sinica B, DOI:10.1016/j. apsb.2019.11.006.).

(Iguratimod, SYL927, IMMH002)

Etomod has better receptor selectivity and safety than the marketed fingolimod, has been approved by the CFDA in 2016 by the national food and drug administration for the treatment of psoriasis (etomod and tablet lot number 2016L09349/2016L09350), and is currently undergoing phase i clinical studies at the first affiliated hospital of the university of beijing.

Our previous studies indicate that both itomod and FTY720, being S1P1 modulators, can also reduce the inflammatory response, but that there is a difference in selectivity for one receptor subtype (S1P3) which may lead to a difference in pharmacological activity for IPF. Based on the above, the inventor utilizes a bleomycin-induced mouse pulmonary fibrosis model to discuss the effect of itomod on IPF, and deeply researches the biological effects of S1P1 and S1P3 in the generation and development of IPF, thereby completing the invention, providing a new treatment selection for IPF, and providing a theoretical basis for treating IPF by taking S1P1 as a target.

Disclosure of Invention

The technical problem to be solved by the invention is to provide the application of the itomod in preparing the medicament for preventing and treating the idiopathic pulmonary fibrosis.

In order to solve the technical problems, the invention provides the following technical scheme:

in a first aspect, the present invention provides the use of itomod, or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament for the prevention and treatment of idiopathic pulmonary fibrosis, wherein said itomod is a compound of formula (I):

in a second aspect, the present invention provides a use of a pharmaceutical composition for preparing a medicament for preventing and treating idiopathic pulmonary fibrosis, the pharmaceutical composition comprising a therapeutically effective amount of itomod or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier, wherein itomod is a compound represented by formula (I).

The itomod of the present invention may be prepared according to methods well known in the art. The itoimod shown in the formula (I) or the pharmaceutically acceptable salt thereof can be combined with one or more pharmaceutically acceptable solid or liquid excipients and/or auxiliary agents to prepare any dosage form suitable for human or animal use. The content of the itomod shown in the formula (I) in the pharmaceutical composition of the invention is usually 0.1-95 wt%.

The itomod shown in the formula (I) or the pharmaceutical composition containing the same can be administered in a unit dosage form, and the administration route can be intestinal tract or parenteral tract, such as oral administration, intravenous injection, intramuscular injection, subcutaneous injection, nasal cavity, oral mucosa, eyes, lungs, respiratory tract, skin, vagina, rectum and the like. The dosage form for administration may be a liquid dosage form, a solid dosage form, or a semi-solid dosage form. The liquid dosage forms can be solution (including true solution and colloidal solution), emulsion (including o/w type, w/o type and multiple emulsion), suspension, injection (including water injection, powder injection and infusion), eye drop, nose drop, lotion, liniment, etc.; the solid dosage form can be tablet (including common tablet, enteric coated tablet, buccal tablet, dispersible tablet, chewable tablet, effervescent tablet, orally disintegrating tablet), capsule (including hard capsule, soft capsule, and enteric coated capsule), granule, powder, pellet, dripping pill, suppository, pellicle, patch, aerosol (powder), spray, etc.; semisolid dosage forms can be ointments, gels, pastes, and the like. The amino propanediol derivative can be prepared into common preparations, sustained release preparations, controlled release preparations, targeting preparations and various particle drug delivery systems.

The dosage of itomod of formula (I) according to the present invention to be administered may vary widely depending on the nature and severity of the disease to be prevented or treated, the individual condition of the patient or animal, the route and dosage form of administration, etc. Generally, a suitable dosage range per day for the present itomod is 0.01-150mg/Kg body weight, preferably 0.1-100mg/Kg body weight, more preferably 0.1-60mg/Kg body weight, most preferably 0.3-60mg/Kg body weight. The above-described dosage may be administered in one dosage unit or divided into several dosage units, depending on the clinical experience of the physician and the dosage regimen including the use of other therapeutic means.

The itoimod shown in the formula (I) or the pharmaceutical composition containing the same can be taken alone or combined with other therapeutic drugs or symptomatic drugs. When the itomod shown in the formula (I) of the invention has synergistic effect with other therapeutic drugs, the dosage of the itomod is adjusted according to actual conditions.

Has the advantages that: the itoimod shown in the formula (I) has obvious curative effect on a bleomycin-induced mouse pulmonary fibrosis model, can obviously prolong the life cycle of a model animal, improve various lung functions, reduce lung inflammation and slow down pathological injury and fibrosis degree, is safe and nontoxic, and provides a new drug choice for clinical treatment of idiopathic pulmonary fibrosis.

Drawings

Figure 1 therapeutic effect of itomod (SYL927) on bleomycin-induced pulmonary fibrosis in mice. A: effect of itomod (SYL927) on survival of model animals; b: effect of itomod (SYL927) on lung index of model animals;

FIG. 2. Effect of Iguratimod (SYL927) on pulmonary function of model animals;

FIG. 3. Effect of Igotimod (SYL927) on pathological changes in model animals (HE staining);

FIG. 4. Effect of Iguratimod (SYL927) on alterations of lung pathology in model animals (Masson staining);

figure 5 effect of itomod (SYL927) on the number of inflammatory cells in bleomycin-induced bronchoalveolar lavage fluid in mouse pulmonary fibrosis model.

Figure 6. effect of itomod (SYL927) on capillary endothelial barrier function. A: effect of itomod (SYL927) on pulmonary claudin ZO-1 expression in animal models of pulmonary fibrosis; b: effect of itomod (SYL927) on intercellular tight junctions of human umbilical vein endothelial cells; c: effect of itomod (SYL927) on mouse peritoneal capillary leakage.

FIG. 7. Effect of Iguratimod (SYL927) on bleomycin-induced interstitial transformation of pulmonary fibrosis mouse alveolar epithelial cells.

Detailed Description

The present invention is further illustrated below by specific examples in order to provide those skilled in the art with a full understanding of the present invention, but it should be understood by those skilled in the art that the examples of the present invention are not to be construed as limiting the present invention in any way.

Experimental example 1: agonistic activity of S1P1 modulators on the S1P1-5 subtype

The experimental method comprises the following steps: the beta-arrestin method is adopted to measure the agonistic activity of S1P1 regulator (S1P: sphingosine-1-phosphate; itomod-P: active phosphate of itomod) on different subtype S1P receptors, and the experimental result shows that the half effective concentration EC is used as the effective concentration₅₀(nM). Wherein the Igotimod-P is a compound shown as a formula (II):

Igotimod-P was formulated in DMSO as a 5mM stock solution, and S1P was formulated in 0.2N NaOH as a 2.5mM stock solution. PathHunter CHO-K1 EDG1 β -Arrestin cells were resuspended in cell plating solution No. 11 and plated at a density of 5000 cells/well in 384 well plates, 20 μ L per plate. 37 ℃ and 5% CO₂After overnight incubation, 5. mu.L of test compound was added at different concentrations, 37 ℃ with 5% CO₂Incubate for 1.5 hours with a standard curve. After the incubation was completed, 12.5. mu.L of the LPAthHunter assay reagent was added to each well and incubated at room temperature for 1 hour. Envision detects the fluorescent signal. Performing 4-parameter nonlinear regression (Log aginst v.response-Variable slope) on the signal value of each well and the Log value of the corresponding compound concentration to obtain EC₅₀Value). Note: S1P (sphingosine-1-phosphate) is a physiological ligand of S1P 1-5; iguratimod belongs to a prodrug S1P1 regulator, and needs to be metabolized into phosphate Iguratimod-P in vivo to play a role.

The experimental results are as follows: as shown in Table 1, etomod-P has strong agonistic activity to S1P1, S1P4 and S1P5, while FTY720 active phosphate, which is a drug on the market, has strong agonistic activity to S1P3, in addition to S1P1, S1P4 and S1P5 according to previous reports (Stephen Handessan, et al, structural and azacyclic analogs of the immunological regulatory agent FTY720 as molecular probes for pathological 1-phosphate excipients, biological & Medicinal Chemistry Let-ers 2007,17(2),491-494, coating indicating induced by FTY 720-phosphorus-mediated FTY 720-ligand-mediated FTY 1, Mueller incorporated by Nature Chemical engineering, S1P1, incorporated by Nature Chemical company, et al, incorporated by reference, et al, 2).

TABLE 1 agonist Activity of S1P1 modulators on the S1P1-5 subtype

Experimental example 2: pharmacodynamic study of S1P1 modulators on IPF

The experimental method comprises the following steps: the pharmacodynamic effects of S1P1 modulators on IPF were evaluated in bleomycin-induced pulmonary fibrosis animal models. An animal model of bleomycin-induced pulmonary fibrosis was established by intratracheal single injection of bleomycin (3mg/kg /) into C57BL/6 mice. On day 10 after molding (disease peak period), the grouped administration was started and divided into Sham group (Sham), Bleomycin group (Bleomycin), Bleomycin + FTY720 group (Bleomycin +1mg/kg FTY720), Bleomycin + eltomomod low dose group (Bleomycin +0.3mg/kg eltomomod), Bleomycin + eltomomod high dose group (Bleomycin +1mg/kg eltomomod), and 15-25 animals per group, wherein the Sham group and Bleomycin group were given an equal volume of solvent control-double distilled water. The experiment was terminated on day 28 after modeling, survival, lung weight index (lung weight/body weight 1000), and lung function were evaluated according to methods known in the art, HE staining and Masson staining were performed to perform pathological evaluation of lung inflammation and fibrosis, and the number of inflammatory cells such as lymphocytes, neutrophils, and eosinophils in bronchoalveolar lavage fluid of animals was determined using a fully automated hematology analyzer.

The experimental results are as follows: when the experiment is finished on the 28 th day after the model is manufactured, the death rate of the animals in the model group reaches 66 percent; and the itomod can obviously prolong the survival time of animals, only one animal in 16 animals in a group of 0.3mg/kg dies, and the survival rate is obviously improved. FTY720, although capable of increasing animal survival to some extent, was not statistically significant compared to the model (see fig. 1A). The lung weight index can reflect the fibrosis degree of the animal lung to a certain extent, and the study shows that the itomod can obviously reduce the lung weight index of the model animal and is almost close to the level of the animal in the pseudo-operation group, while the lung weight index of the animal in the FTY720 group has no improvement compared with the lung weight index of the model group (see figure 1B). The indexes of lung function also show that the itomod almost obviously improves the indexes of lung function, many indexes are restored to normal animal level, and the FTY720 only has certain improvement effect on 3 indexes of 8 indexes, even has the trend of increasing dynamic resistance (see figure 2). Pathological examination further confirmed that itomod significantly reduced inflammatory lesions and the degree of fibrosis in the lungs of model animals (see fig. 3-4). The research results indicate that the targeted regulation of S1P1 can produce clear and remarkable therapeutic effect on pulmonary fibrosis, and S1P1 is very promising to be a target point for treating IPF.

The detection result of animal bronchoalveolar lavage fluid shows that, being the S1P1 regulator, both the itomod and the FTY720 can obviously reduce the content of inflammatory cells such as lymphocytes and eosinophils infiltrated into the lung (see figure 5). According to the previous experimental research of the research team of the inventor, the life cycle of animals cannot be obviously improved by simply applying immunosuppressive drugs and hormones, but the life cycle improving effect of the itomod is very obvious and is equivalent to or better than that of the marketed positive drug pirfenidone applied in the previous research. Although the action mechanism of the existing clinical treatment medicine of pirfenidone as IPF is unknown, researches show that the pirfenidone can simultaneously play roles in resisting inflammation, inhibiting fibroblast proliferation and inhibiting matrix deposition, thereby achieving good curative effect. Therefore, it is suggested that, while the itomod inhibits inflammation, other more important pathways are necessarily regulated to exert the significant curative effect.

Experimental example 3: effect of S1P1 modulators on alveolar-capillary endothelial barrier function

3.1 Effect of S1P1 modulators on pulmonary Claudin ZO-1 expression in animal models of pulmonary fibrosis

The experimental method comprises the following steps: animal models of bleomycin-induced pulmonary fibrosis were constructed as described in example 2 and the same grouping and administration method was used. And detecting the expression of the zonulin ZO-1 in the lung of each group of animals by a Western Blot method. Mice were sacrificed and lung tissue was dissected and approximately 50mg of the same site tissue sample was weighed onto ice into 2ml round bottom Epp tubes. Mu.l of RIPA lysate (containing 1% protease inhibitor cocktail) was added to each sample and placed in a small steel ball and homogenized in a tissue homogenizer at 50Hz for 60 seconds. After this time, the cells were placed on ice for 30min and vortexed every 10 min. 12000rpm, 4 ℃ centrifugal 20min, supernatant for BCA protein quantification and adding the sample buffer boiling degeneration. Protein electrophoresis was performed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and membrane transfer was performed using a wet-transfer method. 5% skim milk was blocked at room temperature for 1h and incubated overnight at 4 ℃ with ZO-1 primary antibody (1: 1000). The corresponding goat anti-rabbit horseradish peroxidase-labeled secondary antibody (1: 2000) was incubated at room temperature for 1h, and protein band imaging was performed using a chemiluminescence imager (Tianneng, Shanghai).

The experimental results are as follows: in vivo animal experiment research shows that the expression of the zonulin ZO-1 in the lung of pulmonary fibrosis animals is greatly reduced, and most model animals have difficulty in detecting the expression of the protein. The Iguratimod can obviously improve the expression quantity of ZO-1 and restore to a normal animal level. Whereas FTY720 had no significant effect on the reduced ZO-1 (see FIG. 6A). The result proves that selective activation of S1P1 can promote vascular endothelial tight junction for the first time.

3.2 Effect of S1P1 modulators on intercellular tight junctions of human umbilical vein endothelial cells

The experimental method comprises the following steps: confocal detects phalloidin-labeled vascular endothelial cytoskeleton. Igotimod-P/FTY 720-P was formulated with DMSO as a 2mM stock solution. Human umbilical vein vascular endothelial cells ea.hy926(4 ten thousand cells/well/200 ul) were seeded onto 8-well cell culture slides precoated with fibronectin coating and incubated overnight in a 5% CO2 cell incubator at 37 ℃. 2mM Igotimod P/FTY720-P stock solution was diluted to 1. mu.M with EA.hy926 cell culture medium. The intracellular medium requiring 3h of drug treatment was discarded, 200. mu.l of the corresponding Igotimod-P/FTY 720-P and a blank containing 0.1% DMSO were added, and the mixture was incubated in a cell incubator for 2 h. And then adding corresponding drugs into the cells needing drug treatment for 1h according to the operation, and continuously incubating for 1h in a cell incubator. The cells were fixed for 10 min with 4% paraformaldehyde as described for rhodamine-phalloidin, followed by punching with 0.1% Triton X-100. Rhodamine-phalloidin was incubated for 15min in the dark and then nuclei were stained with DAPI. Representative fields of view were selected for image acquisition using a confocal microscope (600 ×).

The experimental results are as follows: it was observed in human umbilical vein endothelial cells, ea.hy926, cultured in vitro that the simple administration of itomod significantly increased the intercellular tight junctions, whereas FTY720 had no significant effect on this. At in vitro levels, it was demonstrated that itomod was able to promote tight junctions between vascular endothelial cells (see fig. 6B).

3.3 Effect of S1P1 modulators on mouse peritoneal capillary leakage

The experimental method comprises the following steps: dexamethasone/FTY 720/Igotimod were formulated with 0.5% CMC-Na. Male Kunming mice (22-25g) were randomized on day 0 of the experiment into control, dexamethasone (0.5mg/kg), FTY720(0.3/3mg/kg) and Iguratimod (0.3/3mg/kg) groups and dosing was initiated. 7-9 of them are used in each group. Each group was administered by continuous gavage for 7 days, once daily. The animals were modeled and treated on day seven of dosing. 90 minutes prior to animal treatment, 1% Evans blue was injected tail vein at 0.3ml/20g and immediately followed by 1% acetic acid (0.1ml/10g) i.p.. The animals were then anesthetized and perfused intraperitoneally with 4ml of saline. Peritoneal lavage fluid was collected and absorbance values were read at 590nm wavelength.

The experimental results are as follows: the prophylactic administration of itomod significantly inhibited acetic acid-induced peritoneal capillary leakage in mice, with an effect strength comparable to that of the positive drug dexamethasone, whereas FTY720 had no significant effect on this (fig. 6C).

The results of this example further suggest that targeted modulation of S1P1 may increase vascular endothelial tight junctions at in vivo levels, preventing vascular leakage, while such protection may be counteracted if S1P3 is also activated. Experimental example 4: effect of Iguratimod on alveolar epithelial intercellular transformation

The experimental method comprises the following steps: animal models of bleomycin-induced pulmonary fibrosis were constructed as described in example 2 and the same grouping and administration method was used. The Western Blot method is used for detecting the protein expression of vascular endothelial transmembrane cadherin (E-Ecadrherin), a matrix marker Vimentin (Vimentin) and a transforming growth factor (TGF-beta) which are lung epithelial markers of various groups of animals. The experimental method is described in example 3.

The experimental results are as follows: the Igotimod can remarkably up-regulate the expression of a pulmonary epithelial marker E-Ecadrein of a model animal, down-regulate the expression of an interstitial marker Vimentin and remarkably inhibit the level of TGF-beta (see figure 7). The results of the above studies suggest that targeting S1P1 may improve IPF by reversing the interstitial transformation process of the alveolar epithelium.

In summary, experimental studies show that the itomod shown in formula (I) of the present invention has significant therapeutic effects on IPF, and provides a new drug choice for clinical treatment. Through deep research, the inventor firstly proposes that S1P1 is a potential drug action target for treating IPF, not only widens the treatment field of an S1P1 regulator, but also provides a new strategy and a new thought for IPF treatment, and deeply clarifies that S1P1 promotes endothelial cell tight junction and inhibits epithelial intercellular substance transformation so as to protect the function of a capillary barrier (the capillary barrier is mainly composed of endothelial cells, epithelial cells and respective basement membranes), and provides a scientific and powerful theoretical basis and an experimental basis for later-stage research and clinical application of the IPF.

Claims (2)

1. Use of Iguratimod shown in formula (I) or pharmaceutically acceptable salts thereof in preparing medicaments for preventing and treating idiopathic pulmonary fibrosis and related lung injury,

2. the application of a pharmaceutical composition in preparing a medicament for preventing and treating idiopathic pulmonary fibrosis and related lung injury is characterized in that the pharmaceutical composition contains a therapeutically effective amount of itoimod shown as a formula (I) or pharmaceutically acceptable salts thereof, and a pharmaceutically acceptable carrier,

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