WO2020063463A1 - 西格列羧及其相关化合物的应用 - Google Patents

西格列羧及其相关化合物的应用 Download PDF

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WO2020063463A1
WO2020063463A1 PCT/CN2019/106893 CN2019106893W WO2020063463A1 WO 2020063463 A1 WO2020063463 A1 WO 2020063463A1 CN 2019106893 W CN2019106893 W CN 2019106893W WO 2020063463 A1 WO2020063463 A1 WO 2020063463A1
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sigitalide
sodium
application
pharmaceutically acceptable
fibrosis
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PCT/CN2019/106893
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English (en)
French (fr)
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鲁先平
宁志强
潘德思
孔一迪
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深圳微芯生物科技股份有限公司
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Priority to EP19865916.1A priority Critical patent/EP3858346A4/en
Priority to RU2021111249A priority patent/RU2769446C1/ru
Priority to BR112021005387-5A priority patent/BR112021005387A2/pt
Priority to AU2019349124A priority patent/AU2019349124B2/en
Priority to KR1020217011725A priority patent/KR102661138B1/ko
Priority to US17/279,107 priority patent/US20210386705A1/en
Priority to JP2021516461A priority patent/JP7132434B6/ja
Priority to MX2021003368A priority patent/MX2021003368A/es
Priority to CA3113427A priority patent/CA3113427A1/en
Publication of WO2020063463A1 publication Critical patent/WO2020063463A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/40—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil
    • A61K31/403—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil condensed with carbocyclic rings, e.g. carbazole
    • A61K31/404—Indoles, e.g. pindolol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/40—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil
    • A61K31/403—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil condensed with carbocyclic rings, e.g. carbazole
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P13/00—Drugs for disorders of the urinary system
    • A61P13/12—Drugs for disorders of the urinary system of the kidneys
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis

Definitions

  • the present invention relates to the technical field of medicines, and particularly to the application of sigitalide and related compounds.
  • Sigitalide is an active product of sigitalide sodium, which is an innovative compound in China with independent intellectual property rights intended to treat type 2 diabetes, which can increase insulin sensitivity, lower blood sugar and improve other metabolic synthesis
  • the pharmacological effects of the disease have been confirmed on a variety of experimental models in vitro and in vivo.
  • the structural formulas of the two are as follows:
  • Sigitalide is currently reported for type 2 diabetes, but has not been reported in fibrotic diseases.
  • Fibrosis is a pathophysiological process that can occur in a variety of tissues and organs in the body. Its pathological characteristics are increased fibrous connective tissue and reduced parenchymal cells in organ tissues. The continuous progress of fibrosis can lead to the destruction of organ structure, functional decline and even failure, which is a pathological type with a lethal threat.
  • the organs of the body are composed of two parts: parenchyma and interstitial.
  • the parenchyma refers to the main structure and functional cells of the organ.
  • the interstitial part consists of interstitial cells and extracellular matrix. It mainly maintains the morphological structure and normal function of tissues and organs.
  • Tissue cell damage caused by exogenous or endogenous factors can induce local inflammatory responses and repair processes.
  • the repair process includes limited proliferation of functional cells and the replenishment of interstitial parts; when the damage is large or the damage factors persist, the repair process will continue, and the extracellular matrix produced by the activation of interstitial cells Will occupy the position of parenchymal functional cells, thereby changing the tissue structure and reducing normal physiological functions.
  • fibrosis is a normal repair response to tissues after injury to protect the relative integrity of tissues and organs. Excessive fibrosis changes the normal shape of tissues and organs and weakens their functions.
  • Fibrotic lesions can occur in almost all major human organs, including the lungs (such as idiopathic interstitial pneumonia, etc.), the heart (hypertrophic cardiomyopathy, etc.), the liver (non-alcoholic fatty liver, cirrhosis, etc.), and the kidneys. (Chronic nephritis, etc.), bone marrow (myelofibrosis, etc.), skin (systemic sclerosis, etc.) are involved.
  • Fibrotic lesions are a multi-factor and multi-path pathological process, including at least tissue parenchymal cell injury, inflammatory response, mesenchymal cell activation, and stroma formation. Exogenous factors of parenchymal cell damage are common, such as drug damage. Endogenous factors include autoimmune damage and metabolic toxicity (such as lipotoxicity). The body's normal inflammatory response process is usually controllable.
  • immune-activating factors exogenous Infection, tissue and cell damage
  • TGF ⁇ or platelet-derived growth factor PDGF ⁇ / ⁇ , etc. stimulates mesenchymal cells to activate, proliferate, and produce extracellular matrix such as collagen.
  • TGF ⁇ or platelet-derived growth factor PDGF ⁇ / ⁇ , etc. stimulates mesenchymal cells to activate, proliferate, and produce extracellular matrix such as collagen.
  • Conventional treatment includes corticosteroids and other anti-inflammatory drugs, chemotherapy drugs and anti-platelet-derived growth factor receptor drugs, which usually only partially improve the symptoms of patients.
  • the main treatment goals are still to delay the disease progression and maintain normal physiological functions.
  • Advanced patients can only accept organ transplants as a rescue method. Therefore, there is a huge clinical need for clinical treatment.
  • the sigitalide and its related compounds in the present invention are sigitalide or its stereoisomers, geometric isomers, tautomers, solvates, metabolites, crystalline forms, amorphous, pharmaceutical Acceptable salt
  • the pharmaceutically acceptable salt is ciglitazone sodium or ciglitazone potassium, and the structural formula of the two is as follows:
  • Fibroblast activation and proliferation is an important pathological process in fibrotic diseases. Inhibition of fibroblast proliferation has the potential to treat fibrotic diseases.
  • primary human hepatic stellate cells and primary human skin fibroblasts are used as test objects to detect the in vitro inhibitory effect of ciglitazone sodium and a reference compound on both.
  • the results show that compared with the reference compound pioglitazone, ciglitazone sodium has inhibitory activity on the proliferation of human skin and liver-derived fibroblasts in vitro, while pioglitazone has no obvious inhibitory activity; ciglitazone specifically inhibits fibroblasts.
  • the in vitro proliferation is significantly different from that of other known PPAR agonists such as pioglitazone. Based on this, the present invention proposes the application of sigitalide and its related compounds in the preparation of fibroblast inhibitors.
  • Transforming growth factor ⁇ can activate fibroblasts and induce the expression of extracellular matrix-related genes, and has an important role in the pathological process of fibrotic diseases.
  • TGF- ⁇ 1 stimulates fibroblast activation and induces matrix expression through interaction with TGF- ⁇ receptors, including ⁇ -smooth muscle actin ( ⁇ -SMA) and connective tissue growth factor (CTGF).
  • ⁇ -SMA ⁇ -smooth muscle actin
  • CGF connective tissue growth factor
  • primary human hepatic stellate cells are cultured in a medium containing TGF- ⁇ 1 and sigitalide sodium or a reference compound, and then the ⁇ -smooth muscle actin ( ⁇ -SMA) and connective tissue growth factor (CTGF) are detected.
  • ⁇ -SMA ⁇ -smooth muscle actin
  • CTGF connective tissue growth factor
  • ciglitazone sodium has a better unexpected inhibitory effect on fibroblast proliferation and fibrosis-related gene expression than other PPAR agonists such as pioglitazone and GFT505.
  • the present invention proposes the application of sigitalide and its related compounds in the preparation of extracellular matrix inhibitors of TGF ⁇ -activated fibroblasts.
  • chemokines released by the injury induce monocytes to reach the injury site and activate into macrophages, which in turn produces various inflammatory factors such as tumor necrosis factor TNF ⁇ and monocytes
  • the chemotactic protein MCP-1, etc. initiates the subsequent inflammatory response.
  • the sustained inflammatory response induces fibroblast activation and the production of extracellular matrix, which is an important pathological process of fibrotic diseases. Inhibiting this process has therapeutic uses for fibrotic diseases.
  • the present invention takes human acute monocyte leukemia monocyte cell line (THP-1) as a test object, is activated by a certain concentration of phorbol (Phorbol-12-myristate-13-acetate, PMA), and differentiates into macrophages.
  • THP-1 human acute monocyte leukemia monocyte cell line
  • PMA phorbol-12-myristate-13-acetate
  • This cell model was used to test the effects of sigitalitab sodium and other compounds on the expression of activated THP-1 related inflammatory factors.
  • the results showed that both siglitazone sodium and the control compound GFT505 significantly inhibited phorbol ester-stimulated monocyte activation and related gene expression, but the effect of the control compound GFT505 was less than that of ciglitazone.
  • the present invention proposes the application of sitaglipide and related compounds in the preparation of inflammatory factor inhibitors.
  • Endogenous or exogenous damage factors cause tissue cell death and induce inflammatory response and tissue repair, which is a typical pathology of fibrotic diseases.
  • Carbon tetrachloride (CCl 4 ) is commonly used as a chemical damage factor to simulate liver tissue fibrosis, and is also a widely used rodent liver fibrosis research model.
  • the present invention uses this model to test the inhibitory effect of ciglitazone sodium on liver fibrosis at different doses. The results showed that in a mouse model, carbon tetrachloride (CCl 4 ) can induce parenchymal cell death, inflammatory infiltration, and tissue fibrosis in the liver, which is the same as the clinical pathological phenomenon.
  • Siglitastat sodium can partially inhibit liver inflammation and fibrosis at high, medium and low doses, and has pharmacological activity for the treatment of fibrotic diseases. Based on this and the effects of the foregoing tests, the present invention proposes the application of siglipide and its related compounds in the preparation of a medicine for treating fibrotic diseases.
  • fibrotic diseases caused by tissue damage caused by chronic inflammation include, but are not limited to, systemic sclerosis, chronic nephritis and renal fibrosis, bone marrow fibrosis, idiopathic pulmonary fibrosis, and non-alcoholic fatty liver.
  • the present invention illustrates that in vitro and animal models of sigitalide and its related compounds inhibit fibroblast activation and matrix production, monocyte activation and chemotactic activity in vitro, and in animal models of liver fibrosis In reducing inflammatory activity and tissue fibrosis area, compared with known similar compounds, ciglitazone sodium shows more significant and different activity characteristics, and has better application potential for treating fibrotic diseases.
  • Figure 1 shows the in vitro growth inhibition curve (EdU method) of ciglitazone sodium on human hepatic stellate cells
  • Figure 2 shows the in vitro growth inhibition curve (MTT method) of ciglitazone sodium on human hepatic stellate cells
  • Figure 3 shows the growth inhibition of human skin fibroblasts in vitro (MTT method) by Siglitazone
  • Figure 4 shows the effects of different concentrations of sigitalitab sodium, and the control compounds GFT505 and pioglitazone on the expression of target genes in TGF ⁇ -stimulated fibroblasts;
  • Figure 5 shows the effect of sigitalitab sodium and the control compound GFT505 on the expression of target genes in phorbol ester-stimulated monocytes; where each group of columns from left to right is the solvent control (dimethyl sulfoxide ), Sigitalita Sodium (3 ⁇ mol / L) and GFT505 (3 ⁇ mol / L);
  • Figure 6 shows the effects of sigitalitab sodium and control compounds on the chemotactic migration of THP-1 cells (crystal violet staining results); where A is the solvent control (dimethyl sulfoxide) and B is the solvent control + MCP -1 (10 ng / ml), C is ciglitastat sodium (3 ⁇ mol / l) + MCP-1 (10 ng / ml), D is GFT505 (3 ⁇ mol / l) + MCP-1 (10 ng / ml), E is pioglitazone (3 micromol / l) + MCP-1 (10 ng / ml), F is rosiglitazone (3 micromol / l) + MCP-1 (10 nano G / ml);
  • Figure 7 shows a microscope observation of HE staining of pathological sections of liver tissue
  • Figure 8 shows the results of inflammation scores obtained from HE staining of liver tissue sections
  • Figure 9 is a microscopic observation of Sirius Red staining of liver tissue pathological sections
  • Figure 10 shows the results of sirius red staining fibrosis scores on liver tissue pathological sections.
  • the invention discloses the application of sigitalide and its related compounds. Those skilled in the art can learn from the content of this article and appropriately improve the process parameters for implementation. In particular, it should be noted that all similar replacements and modifications will be apparent to those skilled in the art, and they are all considered to be included in the present invention.
  • the application of the present invention has been described by the preferred embodiments, and it is obvious that relevant persons can implement or apply the technology of the present invention by modifying or appropriately changing and combining the application described herein without departing from the content, spirit and scope of the present invention. .
  • Example 1 Activation of human PPAR receptors by siglitazone sodium
  • the PPAR reporter gene model includes three subtype expression plasmids, an RXR expression plasmid, a reporter gene plasmid with luciferase, and an GFP expression plasmid as an external reference.
  • an RXR expression plasmid In a 96-well plate inoculated with human liver cell line L-02, the number was 15,000 per well, and the plating rate was about 50%. Incubate at 37 ° C for 24 hours.
  • test drug 48 hours after transfection, different concentrations of test drug were added to the cultured cells. In addition, a DMSO solvent control was set up, and three replicate wells were set up for each treatment group.
  • a fluorescence detector to first detect the fluorescence intensity of green fluorescent protein (GFP) in each well (wavelength 485-527nm) as an internal reference standard, and then add 30 ⁇ L of luciferase substrate (Promega, E151A) to each well, shaking it slightly After mixing, the light absorption value at a wavelength of 562nm is detected.
  • the fluorescence detection signal measured by the test drug is compared with the reference signal of the GFP signal after correction. Compared with the correction signal of the solvent control, a relative reporter gene activation intensity is obtained.
  • the activity is calculated by different concentrations drug test half-activation activity (AC 50) (table 1).
  • Table 1 Siglitazone sodium activation activity on three subtypes of PPAR receptors in a reporter gene model.
  • Example 2 Inhibition of fibroblast proliferation by siglitazone sodium
  • Human hepatic stellate cells (human hepatic cells) were purchased from Sciencell, and the cells were seeded in 96-well plates of complete growth medium (Stellate Cell Medium) for 24 hours, and then in serum-free and growth factor-free cells. The medium was starved overnight. The medium was replaced with a fresh medium containing platelet-derived growth factor (PDGF-BB) and different concentrations of the test compound (siglitastat sodium or reference compound PPAR ⁇ / ⁇ dual agonist pioglitazone, purchased from Selleck), and cultured for 24 hours. hour. EdU (5-ethynyl-2'-deoxyuridine) was added to the cells during the last 17 hours of compound treatment.
  • PDGF-BB platelet-derived growth factor
  • EdU 5-ethynyl-2'-deoxyuridine
  • ciglitazone sodium has inhibitory activity on the proliferation of human skin and liver-derived fibroblasts in vitro, while pioglitazone has no significant inhibitory activity.
  • Siglitazone specifically inhibits the proliferation of fibroblasts in vitro and is significantly different from other known PPAR agonists such as pioglitazone.
  • RNA of the sample was extracted and tested by a conventional PCR amplification kit (FastStart Universal SYBR @ Green Master (ROX), Roche) after reverse transcription.
  • the PCR primer design was designed with reference to the standard mRNA sequence of the target gene in NCBI (Table 2) The PCR reaction and detection were performed on ABI StepOnePlus real-time quantitative PCR sequencer.
  • the relative amount of the transcript was calculated by "Delta-Delta CT method" (Livak et al. 2001), using ACTB as the housekeeping gene for normalization, and using the average data of untreated human hepatic stellate cells as a reference As a control, the relative fold change in gene expression was calculated (Figure 4).
  • Fig. 4 show that, under the stimulation of transforming growth factor TGF ⁇ , the expression of matrix-associated genes in fibroblasts is significantly increased, and ciglitazone significantly inhibits this process.
  • the PPAR ⁇ / ⁇ dual agonist GFT505 has a certain inhibitory effect, and the PPAR ⁇ / ⁇ dual agonist pioglitazone has no effect; while for connective tissue growth factor expression, only ciglitazone sodium has a significant and Dose-dependent inhibition. It has been shown that ciglitazone sodium has a better unexpected inhibitory effect on fibroblast proliferation and fibrosis-related gene expression than other PPAR agonists such as pioglitazone and GFT505.
  • Example 4 Effects of Siglitazone Sodium and Control Compounds on the Expression of Related Inflammatory Factors in Phorbol Ester (PMA) Activated Monocyte Cell Line THP-1
  • THP-1 Human acute monocyte leukemia monocyte cell line (THP-1) is activated by a certain concentration of Phorbol-12-myristate-13-acetate (PMA) and differentiates into macrophages.
  • PMA Phorbol-12-myristate-13-acetate
  • This cell model can be used to test the effects of ciglitazone sodium and other compounds on the expression of activated THP-1 related inflammatory factors.
  • THP-1 was inoculated in a 6-well plate containing complete growth medium for 24 hours.
  • the medium was then freshened with fresh PMA and the test compound (ciglitazone sodium) or the reference compound GFT505 (PPAR ⁇ / ⁇ dual agonist).
  • the medium was replaced and cultured for 6 hours.
  • the total RNA of the sample was extracted and a cDNA template was synthesized by a reverse transcription reaction (Transcriptor First Strand cDNA Synthesis Kit, Roche).
  • a conventional PCR kit FastStart Universal SYBR @ Green Master (ROX), Roche) was used to perform semi-quantitative gene expression detection on the ABI StepOne Plus real-time quantitative PCR sequencer.
  • the target gene primers are shown in Table 2.
  • the relative expression change of the target gene uses the internal reference gene ⁇ -actin (ACTB) as a normalization standard, and the comparison with the untreated cell sample is performed to calculate the relative expression change multiple (Figure 5).
  • ACTB internal reference gene ⁇ -actin
  • the monocyte cell line THP-1 is induced by a certain concentration of chemokines (such as monocyte chemoattractant protein 1, MCP-1) to undergo cell migration activities, thereby simulating in vivo processes.
  • chemokines such as monocyte chemoattractant protein 1, MCP-1
  • Transwell technology can be considered as a polycarbonate membrane filter, a stent with permeability.
  • the polycarbonate membrane divides the culture chamber into two upper and lower chambers.
  • the components in the lower culture medium can affect the growth and differentiation and movement of the upper cells.
  • the ability of cells to migrate can be reflected by staining and counting the number of cells in the lower chamber.
  • the THP-1 cell line was cultured to the logarithmic growth phase, and the cells were resuspended and adjusted to a concentration in a 24-well plate.
  • the medium containing the test compound (siglitastat sodium) or the control compound GFT505, pioglitazone, PPAR ⁇ monoagonist rosiglitazone (all purchased from Selleck) was pretreated for 24 hours.
  • Use Transwell according to the manufacturer's instructions add the medium containing chemokine MCP-1 to the lower chamber (ie, the bottom of the 24-well plate), add cell suspension to the upper chamber and incubate for 4 h, then fix the cells on the "ceiling" side of the polycarbonate membrane.
  • the crystal violet solution was stained ( Figure 6), and the number of cells in the lower chamber was counted (Table 3).
  • Example 6 Siglitazone inhibits liver fibrosis in a mouse model induced by carbon tetrachloride
  • mice were injected intraperitoneally with 25% CCl4 (4 ml / kg body weight), and were injected twice a week, which could cause liver injury and chronic liver fibrosis in 3 weeks.
  • Model animals were given for 6 weeks continuously, and non-model control group was given. The solvent was compared with olive oil at the same time.
  • Model animals were randomly divided into groups from 3 weeks.
  • the control drug group was given a gavage solvent (0.2% sodium methylcellulose) from the fourth week.
  • the high, medium and low doses of ciglitastat sodium were given by gavage at 40, 20 respectively.
  • 10 mg / kg body weight of sigitalitab sodium Table 4).
  • liver tissue specimens were collected after the animals were sacrificed, and the plasma transaminase content and liver tissue weight and liver weight / Weight ratio. After the liver tissue was fixed, hepatic sections were stained for hematoxylin-eosin staining (HE staining) and Sirius red staining. The former was used to evaluate cell damage and inflammation, and the latter was used to evaluate tissue fibrosis.
  • G1 blank control group (olive oil + 0.2% sodium methylcellulose) 10
  • G2 Model control (CCl 4 + 0.2% sodium methylcellulose) 10
  • G3 Low dose of CCl 4 + siglitazone sodium (10 mg / kg body weight) 10
  • G4 Medium dose of CCl 4 + sigitastat sodium (20 mg / kg body weight) 10
  • G5 CCl 4 + Siglitastat sodium high dose (40 mg / kg body weight) 10
  • Hepatic inflammation scores were observed and scored using hematoxylin-eosin staining (HE staining) for pathological sections ( Figure 7), and were single-blindly detected and scored by pathologists. Scores were obtained using the Metavir scoring system for inflammatory activity. It consists of four independent indicators, including inflammation in the manifold area, inflammation in the lobule, detrital necrosis, and bridging necrosis (Figure 8).

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Abstract

提供了西格列羧及其相关化合物的应用，通过体外模型和动物模型阐述了西格列羧及其相关化合物在体外抑制成纤维细胞活化与基质产生、单核细胞活化与趋化活性，在肝纤维化动物模型中降低炎症活动和组织纤维化面积，和已知的类似化合物相比，西格列他钠显示出更为显著和不同的活性特点，具有更好的治疗纤维化疾病的应用潜力。

Description

西格列羧及其相关化合物的应用
本申请要求于2018年9月25日提交中国专利局、申请号为201811114946.5、发明名称为“西格列羧及其相关化合物的应用”的中国专利申请的优先权，其全部内容通过引用结合在本申请中。
技术领域
本发明涉及药物技术领域，具体涉及西格列羧及其相关化合物的应用。
背景技术
西格列羧是西格列他钠的活性产物，西格列他钠是我国有自主知识产权的拟治疗2型糖尿病的创新化合物，其可增加胰岛素的敏感性、降低血糖和改善其他代谢综合症的药理作用已经在离体与整体多种试验模型上得到证实，两者的结构式如下：
Figure PCTCN2019106893-appb-000001
西格列羧目前所报道的适用症为2型糖尿病，但是在纤维化疾病中并无相关报道。
纤维化(fibrosis)是一种病理生理过程，可发生于体内多种组织器官，其病理特征为器官组织内纤维结缔组织增多，实质细胞减少。纤维化持续进展可导致器官结构破坏和功能减退乃至衰竭，属于有致命威胁的病理类型。机体器官由实质和间质两部分构成，实质部分是指器官的主要结构和功能细胞，间质部分由间质细胞和细胞外基质组成，主要维持组织器官形 态结构和正常功能运转。
外源或内源性因素引起的组织细胞损伤，会诱发局部的炎症反应和修复过程。在损伤较小的情况下，修复过程包括有限的功能细胞增生和间质部分的补充；在损伤较大或者损伤因素持续存在时，修复过程也将持续进行，间质细胞活化产生的细胞外基质会侵占实质功能细胞的位置，从而改变组织结构和降低正常生理功能。因此本质上纤维化是组织遭受损伤后的正常修复反应，以保护组织器官的相对完整性。而过度的纤维化则会改变组织器官正常形态并削弱其功能。
几乎所有的人体主要器官都可能发生纤维化病变，包括肺脏(如特发性间质性肺炎等)、心脏(肥厚性心肌病等)、肝脏(非酒精性脂肪肝、肝硬化等)、肾脏(慢性肾炎等)、骨髓(骨髓纤维化等)、皮肤(系统性硬化症等)等组织器官都会累及。
纤维化病变是一个多因素多环节的病理过程，至少包括组织实质细胞损伤、炎症反应、间质细胞活化以及基质形成。实质细胞损伤的外源因素常见的如药物损伤，内源性因素包括自身免疫性损伤和代谢毒性(如脂毒性)；机体正常的炎症反应过程通常是可控的，在免疫活化因素(外源感染、组织细胞损伤)消除后通过负反馈因子终止炎症过程，但是如果刺激因素持续存在(慢性感染、自身免疫性抗原等)或免疫功能缺陷会形成慢性炎症；损伤细胞和免疫细胞产生各种因子(转化生长因子TGFβ或血小板源生长因子PDGFα/β等)刺激间质细胞活化、增殖并产生胶原等细胞外基质，上述刺激的持续存在造成基质累积和组织纤维化。因此，纤维化疾病的干预过程需要多个环节的参与。在目前的临床治疗中，纤维化仍是一个不可逆的疾病进展过程，常规治疗包括皮质激素类和其它抗炎药物、化疗药物和抗血小板源生长因子受体药物，通常只能部分改善患者症状，主要治疗目标仍为延缓疾病进展和维持正常生理功能，晚期患者只能接受器官移植作为挽救手段，因此，临床治疗方面存在巨大的临床需求。
发明内容
有鉴于此，本发明的目的在于提供西格列羧及其相关化合物在纤维化 疾病范畴中的系列应用。
本发明中所述西格列羧及其相关化合物为西格列羧或其立体异构体、几何异构体、互变异构体、溶剂化物、代谢产物、晶型、无定形、药学上可接受的盐；
所述药学上可接受的盐为西格列他钠或西格列他钾，两者的结构式如下：
Figure PCTCN2019106893-appb-000002
成纤维细胞的活化增殖是纤维化疾病过程中重要的病理过程。抑制成纤维细胞增殖具有治疗纤维化疾病的应用潜力。本发明以原代人肝星状细胞和原代人皮肤成纤维细胞为试验对象，检测西格列他钠以及参考化合物对两者的体外抑制作用。结果显示，相比参考化合物吡格列酮，西格列他钠对人皮肤和肝脏来源的成纤维细胞体外增殖均有抑制活性，而吡格列酮没有明显的抑制活性；西格列他钠特异性抑制成纤维细胞的体外增殖，和其它已知的PPAR激动剂如吡格列酮的活性特点有明显区别。基于此，本发明提出了西格列羧及其相关化合物制备成纤维细胞抑制剂中的应用。
转化生长因子β(TGF-β)可以激活成纤维细胞并诱导细胞外基质相关基因的表达，在纤维化疾病病理过程中具有重要作用。TGF-β1通过与TGF-β受体的相互作用刺激成纤维细胞活化并诱导基质表达，包括α-平滑肌肌动蛋白(α-SMA)和结缔组织生长因子(CTGF)等。本发明用含有TGF-β1和西格列他钠或参照化合物的培养基培养原代人肝星状细胞，然后检测α-平滑肌肌动蛋白(α-SMA)和结缔组织生长因子(CTGF)的基因表达情况。结果显示，在转化生长因子TGFβ刺激下，成纤维细胞中基质相关基因表达显著升高，西格列他钠显著抑制这一过程。对于平滑肌肌动 蛋白α表达，PPARα/δ双激动剂GFT505有一定抑制作用，但仍不如西格列他钠，PPARγ/α双激动剂吡格列酮没有作用；而对于结缔组织生长因子表达，仅有西格列他钠具有显著的而且是剂量依赖的抑制作用。显示出西格列他钠相比于其它PPAR激动剂如吡格列酮和GFT505对成纤维细胞的增殖和纤维化相关基因表达具有更好的出乎意料的抑制效果。基于此，本发明提出了西格列羧及其相关化合物在制备TGFβ激活的成纤维细胞的细胞外基质抑制剂中的应用。
细胞损伤后诱导的局部炎症效应由单核细胞参与，损伤释放的趋化因子诱导单核细胞到达损伤部位并活化成为巨噬细胞，继而产生各种炎症因子(如肿瘤坏死因子TNFα和单核细胞趋化蛋白MCP-1等)启动后续炎症反应，持续的炎症反应诱导成纤维细胞活化和产生细胞外基质，这是纤维化疾病的重要病理过程。抑制这一过程对于纤维化疾病具有治疗用途。本发明以人急性单核细胞白血病单核细胞株(THP-1)为试验对象，由一定浓度佛波酯(Phorbol-12-myristate-13-acetate,PMA)活化，分化成为巨噬细胞。通过该细胞模型，检测西格列他钠及其他化合物对活化THP-1相关炎症因子表达的影响。结果显示，西格列他钠和对照化合物GFT505均能显著抑制佛波酯刺激的单核细胞活化和相关基因表达，但是对照化合物GFT505的效果较之西格列他钠不足。基于此，本发明提出了西格列羧及其相关化合物在制备炎症因子抑制剂中的应用。
血液循环中的单核细胞在趋化因子的诱导下发生趋化作用并到达损伤部位从而启动和参与后续炎症反应，这是组织纤维化的重要病理过程之一。本发明通过Transwell技术模拟这一过程，并测试西格列他钠或对照化合物对趋化因子诱导的单核细胞系THP-1趋化作用的影响。结果显示，西格列他钠相比对照化合物，显著抑制单核细胞趋化因子诱导的单核细胞THP-1迁移活性(transwell)，吡格列酮有部分抑制活性，另外2个对照化合物没有明显抑制活性。不同PPAR激动剂显示出不同的活性特点，西格列他钠具有更强的抑制活性。基于此，本发明提出了西格列羧及其相关化合物在制备单核细胞趋化作用抑制剂中的应用。
内源或外源损伤因素(如化学毒物)引起组织细胞死亡并诱导炎症反应和组织修复，是纤维化疾病的典型病理。四氯化碳(CCl 4)通常用来作 为模拟肝组织纤维化的化学损伤因素，也是广泛采用的啮齿类动物肝纤维化研究模型。本发明通过该模型测试西格列他钠不同剂量下对肝纤维化过程的抑制作用。结果显示，在小鼠模型上，四氯化碳(CCl 4)可以诱导肝脏的实质细胞死亡、炎症浸润和组织纤维化，与临床病理现象相同。西格列他钠在高、中、低剂量下均能部分抑制肝组织炎症活动度和纤维化程度，具有纤维化疾病治疗的药效活性。基于此以及前述各项试验效果，本发明提出了西格列羧及其相关化合物在制备治疗纤维化疾病药物中的应用。
综合本发明的一系列试验结果，可以得出西格列羧及其相关化合物在治疗纤维化疾病中的明确效果，特别是慢性炎症导致组织细胞损伤引起的纤维化疾病。其中，慢性炎症导致组织细胞损伤引起的纤维化疾病包括但不限于系统性硬化症、慢性肾炎及肾纤维化、骨髓纤维化、特发性肺纤维化和非酒精性脂肪肝。
由以上技术方案可知，本发明通过体外模型和动物模型阐述了西格列羧及其相关化合物在体外抑制成纤维细胞活化与基质产生、单核细胞活化与趋化活性，在肝纤维化动物模型中降低炎症活动和组织纤维化面积，和已知的类似化合物相比，西格列他钠显示出更为显著和不同的活性特点，具有更好的治疗纤维化疾病的应用潜力。
附图说明
图1所示为西格列他钠对人肝星状细胞的体外生长抑制曲线(EdU法)；
图2所示为西格列他钠对人肝星状细胞的体外生长抑制曲线(MTT法)；
图3所示为西格列他钠对人皮肤成纤维细胞的体外生长抑制(MTT法)；
图4所示为不同浓度西格列他钠以及对照化合物GFT505和吡格列酮对TGFβ刺激的成纤维细胞中目标基因的表达影响；
图5所示为西格列他钠和对照化合物GFT505对佛波酯刺激的单核细胞中目标基因的表达影响；其中，每组柱形从左至右依次为溶剂对照(二 甲基亚砜)、西格列他钠(3微摩尔/升)和GFT505(3微摩尔/升)；
图6所示为西格列他钠和对照化合物对THP-1细胞趋化迁移的影响(结晶紫染色结果)；其中，A为溶剂对照(二甲基亚砜)，B为溶剂对照+MCP-1(10纳克/毫升)，C为西格列他钠(3微摩尔/升)+MCP-1(10纳克/毫升)，D为GFT505(3微摩尔/升)+MCP-1(10纳克/毫升)，E为吡格列酮(3微摩尔/升)+MCP-1(10纳克/毫升)，F为罗格列酮(3微摩尔/升)+MCP-1(10纳克/毫升)；
图7所示为肝组织病理切片HE染色的显微镜观察图；
图8所示为肝组织切片HE染色结果的炎症评分结果；
图9所示为肝组织病理切片天狼星红染色的显微镜观察图；
图10所示为肝组织病理切片天狼星红染色的纤维化评分结果。
具体实施方式
本发明公开了西格列羧及其相关化合物的应用，本领域技术人员可以借鉴本文内容，适当改进工艺参数实现。特别需要指出的是，所有类似的替换和改动对本领域技术人员来说是显而易见的，它们都被视为包括在本发明。本发明所述应用已经通过较佳实施例进行了描述，相关人员明显能在不脱离本发明内容、精神和范围内对本文所述应用进行改动或适当变更与组合，来实现和应用本发明技术。
以下就本发明所提供的西格列羧及其相关化合物的应用做进一步说明。
实施例1：西格列他钠对人PPAR受体的活化
PPAR报告基因模型包括三个亚型的表达质粒，RXR表达质粒、带有荧光素酶的报告基因质粒、和作为外参照的GFP表达质粒。人肝细胞株L-02接种96孔板中，数量为15000个/孔，铺板率约为50％。37℃培养24小时。采用罗氏(Roche)公司的FuGENE 6向每孔细胞中转入相应的表达质粒(pHD荧光报告基因质粒+PPARα表达质粒+GFP表达质粒用于PPARα活性检测；pACOX荧光报告基因质粒+PPARγ表达质粒+RXR表达质粒+GFP表达质粒用于PPARγ活性检测；pGL3-PPRE荧光报告基因质粒 +PPARδ表达质粒+RXR表达质粒+GFP表达质粒用于PPARδ活性检测)。
转染48小时后，向培养细胞中分别加入不同浓度的测试药物，另外设置DMSO溶剂对照，每种处理组设置三个重复孔。
加药24小时后，弃去细胞培养液，将96孔板倒置于吸水纸上将细胞培养液彻底去除，每孔加入80μL细胞裂解液(Promega，E153A)，室温下静置5分钟后用移液器吹打均匀，每孔吸取60μL细胞裂解液转移至检测用白板中(Corning，3693)。
使用荧光检测仪首先检测每孔中的绿色荧光蛋白(GFP)的荧光强度(波长485-527nm)作为内参照标准，然后向每孔中加入30μL荧光素酶底物(Promega，E151A)，轻微晃动混匀后检测波长为562nm的光吸收值，测试药物所测得荧光检测信号以内参照GFP信号校正后与溶剂对照的校正信号相比，得到一个相对的报告基因激活强度，通过不同浓度的活性计算测试药物的半数激活活性(AC 50)(表1)。
表1西格列他钠在报告基因模型上对PPAR受体三个亚型的激活活性。
Figure PCTCN2019106893-appb-000003
结果显示，西格列他钠对PPAR三个亚型均有激活活性。
实施例2：西格列他钠对成纤维细胞的增殖抑制
原代人肝星状细胞(human hepatic stellate cell,HSC)购自Sciencell公司，将细胞接种于完全生长培养基(Stellate Cell Medium)的96孔板中24小时，然后在不含血清及生长因子的培养基中饥饿过夜。将培养基用含有血小板源生长因子(PDGF-BB)和不同浓度待测试化合物(西格列他钠或参照化合物PPARγ/α双激动剂吡格列酮，购自Selleck公司)的新鲜培养基替换，培养24小时。在化合物处理的最后17小时向细胞中加入EdU(5-乙炔基-2'-脱氧尿苷)。最后，除去培养基，将细胞用甲醛固定，并根据制造商的说明书(碧云天)，使用荧光Click-it测定法对细胞的DNA中的EdU进行量 化。结果表示为总细胞数中的EdU阳性细胞的百分比，生物学三重复的平均值。化合物对细胞增殖的抑制通过对比对照组的EdU掺入测定值来确定(图1)。
原代人肝星状细胞或原代人皮肤成纤维细胞(human dermal fibroblast,HDF，均购自Sciencell公司)接种于完全生长培养基(Stellate Cell Medium或Fibroblast Cell Medium)的96孔板中24小时，然后在不含血清及生长因子的培养基中饥饿过夜。将培养基用含有血小板源生长因子(PDGF-BB)和不同浓度待测试化合物的新鲜培养基替换，继续培养72小时，采用常规的MTT检测试剂盒(Promega公司)检测。结果表示为MTT底物的相对吸光度(OD490)，取生物学三重复的平均值。化合物对细胞增殖的抑制通过对比对照组的吸光度测定值来确定(图2，3)。
由图1-3可以看出，相比参考化合物吡格列酮，西格列他钠对人皮肤和肝脏来源的成纤维细胞体外增殖均有抑制活性，而吡格列酮没有明显的抑制活性。西格列他钠特异性抑制成纤维细胞的体外增殖，和其它已知的PPAR激动剂如吡格列酮的活性特点有明显区别。
实施例3：西格列他钠和对照化合物对转化生长因子TGFβ激活的成纤维细胞基因表达影响
将人原代肝星状细胞接种于完全生长培养基(Stellate Cell Medium)的6孔板中24小时，将培养基用含有TGF-β1和待测试化合物(西格列他钠)或参照化合物PPARγ/α双激动剂吡格列酮、PPARα/δ双激动剂GFT505(均购自Selleck公司)的新鲜培养基(不含血清及生长因子)替换，培养24小时。提取样品总RNA，反转录后采用常规PCR扩增试剂盒(FastStart Universal SYBR@Green Master(ROX),Roche)进行检测，PCR引物设计参考NCBI中靶基因的标准mRNA序列进行设计(表2)，PCR反应和检测在ABI StepOnePlus实时定量PCR测序仪上进行。
表2用于测定的目标基因引物(包括内参照基因)
引物名称 引物序列(5'端到3'端)
平滑肌肌动蛋白α(α-SMA)正向引物 CGTGGGTGACGAAGCACAG
平滑肌肌动蛋白α(α-SMA)反向引物 GGTGGGATGCTCTTCAGGG
结缔组织生长因子(CTGF)正向引物 GGAGTGGGTGTGTGACGAG
结缔组织生长因子(CTGF)反向引物 GACCAGGCAGTTGGCTCTAA
β肌动蛋白(ACTB)正向引物 CGGGAAATCGTGCGTGAC
β肌动蛋白(ACTB)反向引物 GGAAGGAAGGCTGGAAGAG
单核细胞趋化蛋白1(MCP-1)正向引物 GATCTCAGTGCAGAGGCTCG
单核细胞趋化蛋白1(MCP-1)反向引物 TTTGCTTGTCCAGGTGGTCC
肿瘤坏死因子α(TNFα)正向引物 TTCTCGAACCCCGAGTGACA
肿瘤坏死因子α(TNFα)反向引物 TCTCTCAGCTCCACGCCATT
通过“Delta-Delta CT method”(Livak等Methods 2001)对转录物的相对量进行计算，使用ACTB作为用于归一化的管家基因，并将未处理的人肝星状细胞的平均数据作为参考对照，计算基因表达的相对变化倍数(图4)。
图4结果说明，在转化生长因子TGFβ刺激下，成纤维细胞中基质相关基因表达显著升高，西格列他钠显著抑制这一过程。对于平滑肌肌动蛋白α表达，PPARα/δ双激动剂GFT505有一定抑制作用，PPARγ/α双激动剂吡格列酮没有作用；而对于结缔组织生长因子表达，仅有西格列他钠具有显著的而且是剂量依赖的抑制作用。显示出西格列他钠相比于其它PPAR激动剂如吡格列酮和GFT505对成纤维细胞的增殖和纤维化相关基因表达具有更好的出乎意料的抑制效果。
实施例4：西格列他钠和对照化合物对佛波酯(PMA)活化的单核细胞系THP-1中相关炎症因子表达的影响
人急性单核细胞白血病单核细胞株(THP-1)由一定浓度佛波酯(Phorbol-12-myristate-13-acetate,PMA)活化，分化成为巨噬细胞。可通过该细胞模型，检测西格列他钠及其他化合物对活化THP-1相关炎症因子表达的影响。
THP-1接种于含完全生长培养基的6孔板中24小时，再将培养基用含有PMA和待测试化合物(西格列他钠)或参照化合物GFT505(PPARα/δ双 激动剂)的新鲜培养基替换，培养6小时。提取样品总RNA并通过反转录反应(Transcriptor First Strand cDNA Synthesis Kit，Roche)合成cDNA模板。采用常规的PCR试剂盒(FastStart Universal SYBR@Green Master(ROX),Roche)，在ABI StepOne Plus实时定量PCR测序仪上进行半定量基因表达检测，目标基因引物见表2。
目标基因的相对表达变化采用内参照基因β-肌动蛋白(ACTB)作为归一化的标准，与未经处理的细胞样品进行对照比较，计算相对表达变化倍数(图5)。
图5结果说明，西格列他钠和对照化合物GFT505均能显著抑制佛波酯刺激的单核细胞活化和相关基因表达，但对照化合物GFT505不如西格列他钠。
实施例5：西格列他钠和对照化合物对趋化因子诱导的单核细胞系THP-1趋化作用的影响
在体外模型中，单核细胞系THP-1在一定浓度趋化因子(如单核细胞趋化蛋白1，MCP-1)的诱导下，会发生细胞迁移活动，从而模拟体内的过程。Transwell技术，根据制造商Corning公司的说明书介绍，可认为Transwell是一种聚碳酸酯膜滤器，一种具有通透性的支架。聚碳酸酯膜将培养室划分为上下两室，下层培养液中的成分可影响上层细胞的生长分化以及运动情况。最终通过染色及计数下室细胞量可反映细胞的迁移能力。
THP-1细胞系培养至对数生长期，重新悬浮细胞并调整浓度铺于24孔板中。用含有待测试化合物(西格列他钠)或对照化合物GFT505、吡格列酮、PPARγ单激动剂罗格列酮(均购自Selleck公司)的培养基预处理24小时。根据制造商说明书使用Transwell,在下室(即24孔板底部)加入含趋化因子MCP-1的培养基，上室加入细胞悬液培养4h后，固定聚碳酸酯膜“天花板”面的细胞用结晶紫溶液进行染色(图6)，并计数下室细胞数目(表3)。
表3 transwell实验的培养孔下室细胞计数
Figure PCTCN2019106893-appb-000004
结合表3和图6可以看出，西格列他钠相比对照化合物，显著抑制单核细胞趋化因子诱导的单核细胞THP-1迁移活性(transwell)，吡格列酮有部分抑制活性，但明显不如西格列他钠，另外2个对照化合物没有明显抑制活性。不同PPAR激动剂显示出不同的活性特点，西格列他钠具有更强的抑制活性。
实施例6：西格列他钠抑制四氯化碳诱导的小鼠模型中的肝纤维化过程
BALB/c小鼠腹腔注射25％CCl4(4毫升/公斤体重)，每周注射两次，3周可以造成肝损伤和慢性肝纤维化病变，模型动物均连续给予6周，非模型对照组给予溶剂对照橄榄油相同时间。模型动物从3周起随机分组，对照药物组从第四周开始给予灌胃溶剂(0.2％甲基纤维素钠)，西格列他钠高、中、低剂量组分别灌胃给予40、20、10毫克/公斤体重的西格列他钠原料药(表4)，至第7周试验结束，实验动物处死后收集血浆和肝组织标本，分别测量血浆转氨酶含量和肝组织重量和肝重/体重比。肝组织固定后进行病理切片，分别进行苏木精-伊红染色(HE染色)和天狼星红染色，前者评估细胞损伤和炎症情况，后者评估组织纤维化情况。
表4试验分组与药物处理情况
组别 动物数
G1:空白对照组(橄榄油+0.2％甲基纤维素钠) 10
G2:模型对照(CCl 4+0.2％甲基纤维素钠) 10
G3：CCl 4+西格列他钠低剂量(10毫克/公斤体重) 10
G4：CCl 4+西格列他钠中剂量(20毫克/公斤体重) 10
G5：CCl 4+西格列他钠高剂量(40毫克/公斤体重) 10
肝组织炎症评分采用苏木精-伊红染色(HE染色)的病理切片进行显微镜观察和评分(图7)，由病理学家单盲检测和评分，评分采用炎症活动度Metavir评分系统得出，包括汇管区炎症、小叶内炎症、碎屑坏死、桥接坏死等四个独立指标组成(图8)。
肝组织切片经天狼星红染色后进行形态学观察(图9)，测量采用半自动数字化图像分析系统和测量软件(OsteoMetrics,Inc.)，由病理学家单盲量片，手动画出整张切片的总面积，之后以直线的方式量出整张切片上的纤维化面积，软件将自动计算测量的总面积，纤维化面积％＝总的纤维化面积÷总肝组织切片面积*100(图10)。
从图7-10的结果可以看出，在小鼠模型上，四氯化碳(CCl 4)可以诱导肝脏的实质细胞死亡、炎症浸润和组织纤维化。西格列他钠在高、中、低剂量下均能部分抑制肝组织炎症活动度和纤维化程度，具有纤维化疾病治疗的药效活性。
以上所述仅是本发明的优选实施方式，应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明原理的前提下，还可以做出若干改进和润饰，这些改进和润饰也应视为本发明的保护范围。

Claims (8)

  1. 西格列羧或其立体异构体、几何异构体、互变异构体、溶剂化物、代谢产物、晶型、无定形、药学上可接受的盐在制备治疗纤维化疾病药物中的应用。
  2. 西格列羧或其立体异构体、几何异构体、互变异构体、溶剂化物、代谢产物、晶型、无定形、药学上可接受的盐在制备成纤维细胞抑制剂中的应用。
  3. 西格列羧或其立体异构体、几何异构体、互变异构体、溶剂化物、代谢产物、晶型、无定形、药学上可接受的盐在制备TGFβ激活的成纤维细胞的细胞外基质抑制剂中的应用。
  4. 西格列羧或其立体异构体、几何异构体、互变异构体、溶剂化物、代谢产物、晶型、无定形、药学上可接受的盐在制备炎症因子抑制剂中的应用。
  5. 西格列羧或其立体异构体、几何异构体、互变异构体、溶剂化物、代谢产物、晶型、无定形、药学上可接受的盐在制备单核细胞趋化作用抑制剂中的应用。
  6. 根据权利要求1-5任意一项所述应用，其特征在于，所述西格列羧药学上可接受的盐为西格列他钠或西格列他钾。
  7. 根据权利要求1-5任意一项所述应用，其特征在于，所述纤维化疾病为慢性炎症导致组织细胞损伤引起的纤维化疾病。
  8. 根据权利要求7所述应用，其特征在于，所述慢性炎症导致组织细胞损伤引起的纤维化疾病包括系统性硬化症、慢性肾炎及肾纤维化、骨髓纤维化、特发性肺纤维化和非酒精性脂肪肝。
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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111437393A (zh) * 2020-04-30 2020-07-24 深圳微芯生物科技股份有限公司 用于糖尿病及其并发症治疗的联合用药及其药物组合物
CN111759838A (zh) * 2020-07-02 2020-10-13 深圳微芯生物科技股份有限公司 西格列他的可药用盐药物组合物及其应用

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114949230A (zh) * 2022-06-13 2022-08-30 厦门大学附属第一医院 一种预防和/或治疗急性髓系白血病的联合用药物组合物及其应用

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2011002012A1 (ja) * 2009-07-01 2011-01-06 大日本住友製薬株式会社 Sglt1阻害薬とインスリン抵抗性改善薬を組み合わせてなる医薬
CN102056606A (zh) * 2008-06-03 2011-05-11 贝林格尔.英格海姆国际有限公司 用于治疗非酒精性脂肪肝疾病的dpp-iv抑制剂
CN102883721A (zh) * 2010-03-24 2013-01-16 东亚制药株式会社 用于预防或治疗非酒精性脂肪性肝病的药物组合物以及使用所述药物组合物预防或治疗非酒精性脂肪性肝病的方法
US20170174718A1 (en) * 2015-12-18 2017-06-22 Ardelyx, Inc. Substituted 4-phenyl pyridine compounds as non-systemic tgr5 agonists

Family Cites Families (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20160331729A9 (en) * 2007-04-11 2016-11-17 Omeros Corporation Compositions and methods for prophylaxis and treatment of addictions
JP5697260B2 (ja) * 2009-11-13 2015-04-08 東レ株式会社 糖尿病の治療または予防薬
JP6246895B2 (ja) * 2013-04-22 2017-12-13 カディラ・ヘルスケア・リミテッド 非アルコール性脂肪性肝疾患（ｎａｆｌｄ）のための新規組成物
TN2016000535A1 (en) * 2014-06-13 2018-04-04 Inventiva Ppar compounds for use in the treatment of fibrotic diseases.
CN104744282A (zh) * 2015-02-17 2015-07-01 南通恒盛精细化工有限公司 一种胰岛素增敏剂的制备工艺
WO2017044551A1 (en) * 2015-09-11 2017-03-16 Mitobridge, Inc. Ppar-alpha agonists for treating mitochondrial diseases
RU2621163C1 (ru) * 2016-02-16 2017-05-31 государственное бюджетное образовательное учреждение высшего профессионального образования "Сибирский государственный медицинский университет" Министерства здравоохранения Российской Федерации (ГБОУ ВПО СибГМУ Минздрава России) Антифибролитическое средство
JP6840853B2 (ja) * 2016-12-09 2021-03-10 カディラ・ヘルスケア・リミテッド 原発性胆汁性胆管炎の治療
CN109316601B (zh) * 2017-07-31 2021-11-09 武汉朗来科技发展有限公司 药物组合物及其用途

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102056606A (zh) * 2008-06-03 2011-05-11 贝林格尔.英格海姆国际有限公司 用于治疗非酒精性脂肪肝疾病的dpp-iv抑制剂
WO2011002012A1 (ja) * 2009-07-01 2011-01-06 大日本住友製薬株式会社 Sglt1阻害薬とインスリン抵抗性改善薬を組み合わせてなる医薬
CN102883721A (zh) * 2010-03-24 2013-01-16 东亚制药株式会社 用于预防或治疗非酒精性脂肪性肝病的药物组合物以及使用所述药物组合物预防或治疗非酒精性脂肪性肝病的方法
US20170174718A1 (en) * 2015-12-18 2017-06-22 Ardelyx, Inc. Substituted 4-phenyl pyridine compounds as non-systemic tgr5 agonists

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
LIVAK ET AL., METHODS, 2001
See also references of EP3858346A4

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111437393A (zh) * 2020-04-30 2020-07-24 深圳微芯生物科技股份有限公司 用于糖尿病及其并发症治疗的联合用药及其药物组合物
CN111759838A (zh) * 2020-07-02 2020-10-13 深圳微芯生物科技股份有限公司 西格列他的可药用盐药物组合物及其应用

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