WO2013013490A1 - Utilisation d'un médicament ciblant cyslt1 dans la préparation d'un médicament pour la prévention ou le traitement de maladies auto-immunes - Google Patents

Utilisation d'un médicament ciblant cyslt1 dans la préparation d'un médicament pour la prévention ou le traitement de maladies auto-immunes Download PDF

Info

Publication number
WO2013013490A1
WO2013013490A1 PCT/CN2012/000842 CN2012000842W WO2013013490A1 WO 2013013490 A1 WO2013013490 A1 WO 2013013490A1 CN 2012000842 W CN2012000842 W CN 2012000842W WO 2013013490 A1 WO2013013490 A1 WO 2013013490A1
Authority
WO
WIPO (PCT)
Prior art keywords
cells
eae
montelukast
blood
mice
Prior art date
Application number
PCT/CN2012/000842
Other languages
English (en)
Chinese (zh)
Inventor
谢欣
杜昌升
王烈峰
吕婕
魏巍
Original Assignee
中国科学院上海药物研究所
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 中国科学院上海药物研究所 filed Critical 中国科学院上海药物研究所
Publication of WO2013013490A1 publication Critical patent/WO2013013490A1/fr

Links

Classifications

    • 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
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • 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
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/47—Quinolines; Isoquinolines
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • 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
    • A61P37/00—Drugs for immunological or allergic disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators

Definitions

  • the present invention relates to the field of biomedicine, and in particular to a medicament for targeting a cysteine-leukotriene receptor CysLTl Use in the preparation of a medicament for the prevention or treatment of an autoimmune disease.
  • MS Multiple sclerosis
  • CNS central nervous system
  • the pathological manifestations are demyelinating and degenerative diseases of the central nervous system caused by immune inflammation.
  • Experimental Autoimmune Encephalomyelitis is a CD4 + T cell-mediated demyelinating disease with similar pathological features to MS. It is an ideal animal model for studying the pathogenesis, treatment and prevention of MS.
  • EAE Complete Freund's adjuvant with myelin, Proteolipid Protein, and Myelin Oligodendrocyte Glycoprotein (MOG), immune-related susceptible mice, or self-activated T cells
  • EAE 5-8
  • the integrity of the blood-brain barrier is disrupted, and peripheral CD4 + T cells infiltrate into the CNS, leading to infiltration and aggregation of macrophages and dendritic cells in the CNS, activating microglia, and ultimately leading to nerve detachment.
  • leukotriene A 4 (Leukotriene A 4, LTA 4) by LTA4 hydrolase (leukotriene A4 hydrolase, LTA4H) hydrolysis of leukotriene B 4 (Leukotriene B 4, LTB 4 ), or leukotriene C4 synthase (leukotriene C4 synthase, LTC4S) catalyzed Formation of cysteine - leukotriene C 4 (leukotriene C 4, LTC 4) and until generation of cysteine - leukotrienes D 4 (Leukotriene D 4 , LTD 4 ) and cysteine-leukotriene E4 ( Leukotriene E 4 , LTE 4 ) (11).
  • LTB 4 The biological activity of LTB 4 is achieved by two G-protein coupled receptors (GPCRs), leukotriene B4 receptors 1 and 2 (Leukotriene B4 receptor 1 and 2, BLT1 and BLT2) (12); Cysteinyl leukotriene (CysLTs) is also mediated by two GPCRs, namely cysteine-leukotriene receptors 1 and 2 (CysLTl and CysLT2) (13).
  • GPCRs G-protein coupled receptors
  • Cysteinyl leukotriene receptors 1 and 2 Cysteinyl leukotriene receptors 1 and 2
  • Modulators of the leukotriene signaling pathway include 5-LO inhibitors (eg, zileuton) and CysLTl antagonists (eg, montelukast, zafirlukast, and pulilast) Pranlukast, which ranks among the 3 most widely used drugs for the treatment of asthma, and is the only oral drug (14-17).
  • 5-LO inhibitors eg, zileuton
  • CysLTl antagonists eg, montelukast, zafirlukast, and pulilast
  • Pranlukast which ranks among the 3 most widely used drugs for the treatment of asthma, and is the only oral drug (14-17).
  • autoimmune diseases including multiple sclerosis, rheumatoid arthritis, lupus erythematosus, inflammatory bowel disease, and the like.
  • the drug that targets the cysteine-leukotriene receptor CysLT1 includes a CysLT1 antagonist, and more preferably, the CysLT1 antagonist includes montelukast, zaruz Special (zafirlukast) and pranlukast.
  • the autoimmune diseases include multiple sclerosis, rheumatoid arthritis, erythematosus, and inflammatory bowel disease. It was found that CysLT1 was up-regulated in immune tissues and spinal cord in EAE mice, and CysLTs levels were significantly increased in blood and cerebrospinal fluid. After administration of EAC mice to two antagonists of CysLTl (zarutast and montelukast), the onset time of EAE mice was delayed, the symptoms were alleviated, the incidence was reduced, and CNS inflammatory cell infiltration was reduced.
  • CysLTl signaling does not affect the differentiation of sensitized T cells, most likely by increasing the secretion of IL-17a by MOG-specific T cells, the permeability of the blood-brain barrier, and the induction of T cell chemotaxis leading to the pathogenesis of EAE.
  • a CysLTl antagonist By administering a CysLTl antagonist, it can block the chemotaxis of T cells and reduce the permeability of the blood-brain barrier. That is to say, the anti-asthmatic CysLT1 antagonist can be used for clinical treatment of diseases such as multiple sclerosis, and the present invention not only reveals some mechanisms of the onset of MS, but also provides a new therapeutic target for clinical intervention of the disease.
  • FIG. 1 is a graph showing changes in gene expression of CysLT receptor and synthetase during the pathogenesis of EAE.
  • the mRNAs of spleen, lymph nodes, brain and spinal cord of EAE mice and control mice on the 5th, 9th, 12th, 15th, 18th and 21st day after immunization were extracted and analyzed by real-time quantitative PCR. gene expression. The ratio of specific gene expression to ⁇ -actin ( ⁇ -actin) expression in the same tissue sample was compared to the control.
  • AD CysLTl and CysLT2 gene expression changes in spleen (sputum), lymph node ( ⁇ ), brain (C) and spinal cord (D); EH: in spleen (E), lymph node (F), brain (G) and Changes in 5-LO, LTA4H, and LTC4S gene expression in the spinal cord (H); I: Competitive enzyme immunoassay for detection of CysLTs concentrations in brain, spinal cord, serum, and cerebrospinal fluid (day 10 post immunization). Data are expressed as mean SEM (6 mice per group:) and the results are representative data from two independent experiments, compared to controls, *p ⁇ 0.05, ⁇ 0.01 and ⁇ 0.001, (Student's - test).
  • Figure 2 is a graph showing that the CysLTl receptor antagonist attenuates the condition of EAE.
  • E and F are normal or after immunization H&E staining (E) and fast blue staining (F) pictures of paraffin sections of mouse lumbar spinal tissue treated with saline and montelukast (10 mg/kg, day 3 administration, tissue on day 17).
  • E H&E staining
  • F fast blue staining
  • HJ is a quantitative analysis of the total number of cell infiltration, CD4 + T cell infiltration and demyelination in EG, and the data is expressed by mean SEM.
  • Three mice in each group were taken, and 20 sections of each mouse spinal cord were analyzed for comparison with the normal group, ***;? ⁇ 0.001; compared with the saline control group; ⁇ 0.001 (Student's -test).
  • Figure 3 is a graph showing that montelukast treatment reduces pathogenic T cell infiltration in CNS.
  • EG In the presence of differentiation factor and various concentrations of LTD 4 or montelukast, naive CD4 + T cells derived from the spleen of 8-9 weeks old mice are in the body ⁇ ⁇ 1 ( ⁇ ), ⁇ ⁇ - 17 (F) or T reg (G) differentiation status, data from three independent experiments, expressed as mean SEM.
  • HK Spleen cells from normal and EAE-administered saline or montelukast mice were stunned with MOG 35 _ 55 for 48 hours, and supernatants were collected and IL-17a, IFN- ⁇ , IL-4 and TGF were detected by ELISA. The content of cytokines such as ⁇ .
  • Figure 5 is a graph showing that the CysLTl antagonist blocks the increase in blood-brain barrier permeability caused by LTD 4 .
  • D montelukast inhibits the protein level of ZO-1 in bEnd.3 cells induced by LTD 4 , the data is expressed as mean SEM, the data is from three independent experiments, compared with the control group, ⁇ 0.05 ; and LTD 4 treatment group Compare, # p ⁇ 0.05 (Student's -test).
  • Figure 6 is a graph showing that montelukast inhibits the chemotaxis of LTD 4 to immune cells.
  • A On the 10th day after immunization, the mouse spleen cells were added to the upper chamber of the Transwell transmembrane compartment sandwich culture system, and the upper and lower chambers were separated by a membrane with micropores, and various concentrations of LTD 4 were added to the lower chamber.
  • 1 ⁇ of montelukast was added to the upper and lower chambers, and 100 nM LTD 4 was added to the lower chamber; after 1.5 hours, cells that migrated from the upper chamber to the lower chamber were counted by flow cytometry. .
  • C57BL/6 female rats were purchased from Shanghai Experimental Animal Center (Shanghai, China) and raised in the SPF laboratory of Tongji University Experimental Animal Center. The experiment was started in 8-9 weeks and the light-dark 12-hour cycle was alternated. Food and clean drinking water. All experiments were approved and conducted in accordance with the guidelines of the Animal Health Committee of Tongji University.
  • the Dynal® Mouse CD4 Cell Negative Sorting Kit was purchased from Invitrogen (Carlsbad, CA).
  • ELISA kits for IL-17a, IFN- ⁇ (gamma interferon), TGF- ⁇ (transforming growth factor- ⁇ ) and IL-4 (interleukin-4) were purchased from Daktronics (Shenzhen, China).
  • the scores were as follows: 0 points, no clinical symptoms; 1 point, tail sputum; 2 points, mild sputum ( Unilateral or bilateral hind limb weakness, incomplete paralysis; 3 points, paraplegia (complete bilateral paralysis); 4 points, paraplegia and forelimb weakness or paralysis; 5 points, sudden death or death.
  • mice were anesthetized and fixed by PBS perfusion and 4% paraformaldehyde perfusion.
  • Spinal tissue samples were fixed overnight in 4% paraformaldehyde.
  • inflammatory cell infiltration was analyzed by sputum & sputum staining, and demyelination of the spinal cord was analyzed by fast blue staining.
  • Frozen sections were stained with anti-mouse CD4-resistant and corresponding fluorescent secondary antibodies.
  • RNA from the spinal cord, cerebral cortex, spleen cells and lymph node cells was extracted using TRIzol (Invitrogen) according to the product instructions and reverse transcribed into cDNA using a six-base random primer and M-MLV reverse transcriptase (Progema).
  • Real-time quantitative PCR was performed on a LightCycler quantitative PCR machine (Stratagene) using the SYBR Green JumpStartTM Taq ReadyMixTM kit (Sigma) and each gene primer sequence to detect the expression of each gene.
  • the primer sequences are as follows:
  • CysLTl upstream bow 1 object 5,-CTCCAAGGCACCAAGCAGAC-3, ,
  • CysLTl downstream bow 1 5,-TGCCAAAGAAACCCACAACAG-3, ;
  • CysLT2 upstream primer 5,- CGAAGGCAGAGGCACAGATT-3, ,
  • CysLT2 downstream primer 5,-GAACCAAATCACTGAAGTATGCCT-3, ;
  • 5-LO upstream primer 5,-CACGCATCTGGTGTCTGAGG-3, ,
  • LTA4H downstream primer 5,-CGTAGAGCCAGGTGTTCCAAT-3, ;
  • LTC4S upstream primer 5,-CCTGTGCGGACTGTTCTACCT-3, ,
  • LTC4S downstream bow 1 5,-GCCATCGCCACCAGCA-3, ;
  • ⁇ -actin downstream primer 5, -CC AGTTGGTAAC AATGCCATGTT-3, .
  • Cysteine-leukotriene levels in brain, spinal cord, serum and cerebrospinal fluid were measured using competitive enzyme immunoassay (EIA, Cayman chemical) according to product instructions.
  • EIA competitive enzyme immunoassay
  • the tissue was homogenized in methanol, and the precipitated protein was removed by centrifugation. The supernatant was vacuum dried and used for detection.
  • the blood samples were taken from the eyelids, and after taking blood, they were allowed to stand at 4 ° C for 30 minutes, and after centrifugation at 4500 g for 10 minutes, the supernatant was collected as serum.
  • the capillaries collect cerebrospinal fluid from the large occipital bone.
  • cysteine-leukotriene content is expressed as the number of pg (pg) per milligram (mg) of tissue sample, and then compared to the value of normal control mice; serum and cerebrospinal fluid samples, half
  • the cystine-leukotriene content is expressed as the number of pg (pg) per ml (ml) of liquid and is compared to the value of normal mice.
  • the spinal cord and brain tissue were homogenized on ice with a tissue homogenizer and filtered through a 70 ⁇ m ⁇ cell filter.
  • the cell suspension was centrifuged at 500 g for 10 minutes at 4 ° C, and then resuspended in 8 ml of 37% Percoll reagent.
  • Into 4 ml of 70% Percoll reagent centrifuge at 25 ° C for 750 g for 25 minutes. Cells in the middle layer of 37% to 70% Percoll were collected and flow cytometrically detected.
  • CD4 + T cells in the spleens of 8-9 weeks old C57BL/6 mice were separated by magnetic beads, and the isolated CD4 + T cells were added with anti-CD3 (2 g/ml; 145-2C11; BD Pharmingen) and anti-CD28 ( 2 ⁇ ; 37.51; BD Pharmingen) After induction of antibody, IL-12 (10 ng/ml; Peprotech) and anti-IL-4 (10 g/ml; 11B11; BD Pharmingen) were added to induce differentiation into T H 1 cells.
  • TGF- ⁇ (5 ng/ml; Peprotech), IL-2 (50 U/ml; Peprotech) and anti-IFN- ⁇ (10 ⁇ ; XMG1.2; BD Pharmingen) induced differentiation into T rcg Cells, or added anti-IL-4, anti-IFN- ⁇ and TGF- ⁇ (3 ng/ml), IL-6 (30 ng/ml; eBioscience), tumor necrosis factor (tumor necrosis factor) 10 ng / ml; Peprotech) Wo port IL- ⁇ (10 ng / ml ; Peprotech) of T H -17 "differentiation factor combination" to induce cells to differentiate into T H -17.
  • Various concentrations of LTD4 and montelukast were added simultaneously to assess their effects on T cell differentiation.
  • mice or EAE mice were given saline or montelukast on the 10th day after immunization, the spleen was removed and treated with red blood cell lysate, and then leukocytes were collected by centrifugation at 1000 g for 3 minutes. The leukocytes were resuspended in DMEM medium containing 10% FBS, plated in 96-well plates at 2 X 10 5 /well/100 ⁇ , and stimulated with MOG 35 _ 55 (20 ⁇ 8 / ⁇ 1) for 48 hours. After clearing, according to the specific ELISA kit, according to the product description, the cytokines IL-17a, IFN- ⁇ , IL-4 and TGF- ⁇ were detected.
  • Blood-brain barrier permeability test Blood-brain barrier permeability was measured in vivo using sodium fluorescein as a tracer according to the method described in the literature (21). Briefly, mice were injected intraperitoneally with ⁇ 10% sodium fluorescein, and after 45 minutes, the heart was bled and PBS was perfused. The brain and spinal cord were weighed and then centrifuged at 10,000 g for 10 minutes after homogenization of 1.5 ml of cold 7.5% trichloroacetic acid to remove the soluble precipitate. The supernatant was further diluted 1:10 with 7.5% trichloroacetic acid.
  • the fluorescence value of the ⁇ supernatant sample was measured by a Flexstation multiwell plate reader (Molecular Devices) with an excitation wavelength of 485 nm and an emission wavelength of 530 nm. Fluorescein standard (0.064-10 ( ⁇ g/ml) was used to plot the standard curve to calculate the content of sodium fluorescein in the sample. The fluorescence value of CNS was compared with the fluorescence value of the serum in the same animal. Control mice were compared.
  • the in vitro co-culture of the blood-brain barrier was performed using a 0.4 ⁇ m pore size, 12 mm transmembrane chamber (Transwells, Corning) (22-23).
  • the chamber membrane was coated with laminin (sigma) for 30 minutes, and the mouse brain microcapillary endothelial cells bEnd.3 were plated into each chamber at a density of 6.0 X 10 4 cells/cm 2 .
  • the stromal C6 cells PO X IO 4 were plated in the lower 12-well plates. The medium was changed every two days, and the medium was removed after 4-5 days.
  • the upper chamber was washed with PBS, and then 0.5 ml of buffer containing 1% BSA was added, and LTD 4 (100 nM) or LTD 4 (100 nM) was added respectively.
  • + Montelukast (1 ⁇ ) ⁇ Methanol (0.1%) was a negative solvent control.
  • the liquid in the lower layer is the same as the liquid in the upper chamber.
  • 20 ⁇ 10 mg/ml sodium fluorescein was added to the upper chamber, and the ⁇ liquid was taken out from the lower well at a fixed time point for fluorescence detection and then returned to the well.
  • the fluorescence values of the samples were determined by a Flexstation multiwell plate reader (Molecular Devices) with an excitation wavelength of 485 nm and an emission wavelength of 530 nm.
  • bEnd.3 cells were treated with negative solvent control (0.1% methanol), LTD 4 (100 ⁇ ), or LTD 4 (100 nM) + montelukast (1 ⁇ ), and the cells were washed with PBS. Lysis in lysate (50 mM Tris pH 7.4, 150 mM NaCl, 0.1% Chaps, 1 mM EDTA, 1 mM NaF, 1 mM Na 3 V0 4 and protease inhibitor:), sonicated on ice for 30 seconds, with dioctonine Acid protein quantitation (BCA method) Determine protein concentration. The same amount of protein sample was loaded, separated by SDS-PAGE and electrophoresed to PVDF membrane. Western blot was performed using mouse anti-ZO-1 antibody and related HRP-labeled secondary antibody.
  • the in vitro chemotaxis assay was performed using a 5 ⁇ pore size penetrating chamber (Transwells, Corning).
  • the spleen cells of EAE mice were isolated and preincubated with RPMI-1640 medium containing 1% BSA. After hours, the cells were washed and diluted to a density of 1 X 10 7 cells/ml with RPMI-1640 medium containing 20 mM HEPES and 0.5% BSA. Each concentration of LTD 4 was added to the lower well and 100 ⁇ L of the cell suspension was added to the upper chamber.
  • the present invention is immunized with MOG 35 _ 55 C57BL / 6 mice induced EAE.
  • the present invention detects gene expression of several proteins involved in CysLTs production and signal transduction, including two receptors (CysLTl and CysLT2) and three enzymes (5-LO, LTA4H). Expression levels of LTC4S) on days 5, 9, 12, 15, 18 and 21 after EAE immunization. In the spleen and lymph nodes (Figs. 1A and 1B), CysLTl showed a significant up-regulation from the fifth day after immunization, that is, the preclinical stage of the disease.
  • CysLTl expressed the highest point at the onset of EAE (12 days after immunization) and then slowly decreased (Fig. 1A).
  • CysLTl expression is up-regulated and maintained on a platform.
  • CysLTl expression only showed a slight but significant difference in up-regulation after the onset of EAE (Day 15, Figure 1C).
  • CysLT1 is up-regulated with the onset of EAE and continues to rise throughout the course of the disease (Fig. 1D).
  • CysLT2 had only a slight but significant difference in upregulation on days 5 and 9 of the brain, but no change in expression at other time points and in other tissues (Fig. 1A-D).
  • montelukast significantly reduced the infiltration of leukocytes into the spinal cord compared with the control group.
  • Fast blue staining showed extensive demyelination in the white matter of the spinal cord of EAE mice in the saline control group, while demyelination was significantly reduced after administration of montelukast (Fig. 2F and 21).
  • In situ immunofluorescence staining showed that the dose of CD4 + T cells in the spinal cord of EAE mice was significantly reduced after administration of montelukast.
  • the inventors used flow cytometry to verify the infiltration of CNS leukocytes on the 17th day after immunization.
  • the total number of CNS infiltrates after administration of montelukast (Fig. 3A) and the accumulation of CD4 + T cells in the CNS were reduced (Fig. 3B and 3C).
  • TH- 17 EAE and T H 1 is the major pathogenic CD4 + T cells (24).
  • the absolute number and percentage of T H 1 were significantly decreased (Fig. 3D and 3E).
  • T H -17 in the percentage of CD4 + T cells infiltration has not changed, but the absolute number of T H -17 cells significantly decreased ( Figure 3D and 3F).
  • the administration of CysLT1 antagonists to block the CysLT1 signaling pathway can significantly reduce the inflammatory cell infiltration and demyelination of the CNS, thereby significantly reducing the condition of EAE.
  • CysLTl signaling does not affect T cell differentiation in vivo and in vitro
  • the inventors then tested whether CysLT1 signaling interferes with the differentiation of T cells.
  • montelukast did not significantly alter the ratio of spleen and blood leukocytes (CD45 + cells:), CD4 + T cells, CD8 + T cells, and B cells (Figs. 4A and 4C).
  • EAE mice given montelukast or saline After that, there was no difference in the ratio of T H 1 , T H -17 or T rcg cells in CD4 + cells ( FIGS. 4B and 4D ).
  • the inventors further examined whether CysLTs or montelukast directly affect T H 1, TH-17 or T RCG differentiation.
  • Immune CD4 + T cells were sorted from spleen cells of 8-9 weeks old female C57BL/6 mice by immunomagnetic beads. After activation with anti-CD3 and anti-CD28 antibodies, different differentiation factors and various concentrations of LTD were added. 4 (0.1, 0.3 and 1 ⁇ ) and montelukast (1, 3 and 10 ⁇ ), cells were induced to differentiate into T H 1, TH-17 or T RCG cells. Three days later cells were collected intracellular IFN- Y, IL-17a or staining of Foxp3. Flow analysis revealed that LTD 4 and montelukast did not affect the in vitro differentiation of T H 1 (Fig. 4E), T H- 17 (Fig. 4F) or T reg cells (Fig. 4G).
  • the inventors wanted to test whether montelukast affects the cytokine secretion capacity of splenocytes in MOG re-stimulation.
  • EAE mice given montelukast or saline were sacrificed on the 10th day after immunization, and the splenocytes were isolated and stimulated with MOG 35 _ 55 (20 ⁇ M) at 48 ° C for 48 hours.
  • the supernatant was collected and assayed for IL-17a, IFN- ⁇ , IL-4 and TGF- ⁇ levels by ELISA (25-26).
  • CysLTl signal does not affect or modulate inflammatory T cell proliferation or differentiation of T cells, but it can reduce MOG-specific T H -17 cytokine production.
  • the inventors' experimental results show that montelukast can inhibit leukocyte infiltration, but at the same time, the inventors did not find significant changes in the number of T cells in peripheral immune tissues. Based on the above observations, the inventors speculated that montelukast may affect the infiltration process of leukocytes. . Because changes in the permeability of the blood-brain barrier are associated with the severity of clinical symptoms of EAE (3), and it has been reported that CysLTs can increase microvascular permeability (27-28), the inventors speculate whether CysLTl receptor activation This will lead to an increase in the permeability of the EAE blood-brain barrier. The inventors used sodium fluorescein as a tracer molecule to detect the integrity of the blood-brain barrier (3).
  • the inventors then used the blood-brain barrier model co-cultured with bEnd.3 and C6 in vitro to examine the effect of CysLTs on the blood-brain barrier (22-23).
  • LTD 4 significantly increased the permeability of sodium fluorescein, which increased the permeability of the blood-brain barrier model in vitro.
  • montelukast Fig. 5B.
  • CNS capillary endothelial cells form dense junctions that help maintain low permeability of the blood-brain barrier (29). The integrity of the dense junction directly determines the permeability of the blood-brain barrier (30).
  • LTD 4 was treated on bEnd.3 cells after 24-36 hours of treatment.
  • the level of dense junction protein ZO-1 was significantly reduced (Fig. 5C).
  • the reduced ZO-1 level was almost completely restored (Fig. 5D).
  • this bell-shaped dose response curve is visible in many chemotactic agents.
  • montelukast administration reduced LTD 4- induced spleen cell chemotactic activity (Fig. 6A).
  • surface staining analysis of spleen cells chemoked into the lower plate showed that LTD 4 had stronger chemotactic activity (2.7-fold vs. 1.6-fold) on CD4 + T cells compared with whole spleen cells.
  • Administration of montelukast almost completely inhibited the chemotaxis of LTD 4 to CD4 + T cells.
  • G protein-coupled receptors are the largest receptor superfamily with more than 1,000 members, and they are widely involved in various biological activities and are associated with many human diseases (37). Due to their important physiological roles and localization to the cell surface, GPCRs are currently the most important target for drug action on the market (38).
  • CysLTs are mainly produced by mast cells, macrophages, eosinophils, and basophils. CysLTs play an important role in asthma, allergic rhinitis and other respiratory diseases. A small number of reports have shown that enzymes that inhibit the synthesis of leukotrienes (such as cPLA2a or 5-LO) do not reduce the production of IFN- ⁇ or IL-17a (19), and these studies are consistent with the findings of the inventors. That is, blocking CysLTl can attenuate the condition of EAE, but does not affect the cell differentiation of T H 1 or T H- 17.
  • receptors for BLT1 and LTB 4 may be required for the production of T H 1/T H -17, as BLTT mice show delayed onset of EAE, reduced symptoms of EAE, and decreased levels of T H 1 and T H- 17 cytokines ( 12).
  • Another effect that affects the infiltration process is the permeability of the blood-brain barrier.
  • blood-brain barrier permeability There is a clear association between blood-brain barrier permeability and clinical manifestations of EAE (21). It has been reported that high concentrations of CysLTs increase vascular permeability in the early stages of lactic acid-induced peritonitis (47). Other studies have also shown that CysLTl antagonists, including montelukast, may inhibit tumor metabolism by inhibiting capillary permeability (27). Therefore, the inventors hypothesized that the CysLTl signal may contribute to the destruction of the blood-brain barrier in the pathogenesis of EAE, which may be blocked by montelukast.
  • the inventors found a significant increase in the permeability of the blood-brain barrier in EAE mice, particularly in the spinal cord, which is consistent with previous reports (3), and administration of montelukast inhibits the blood-brain barrier in EAE mice. Increased permeability.
  • LTD 4 can increase the permeability of the blood-brain barrier, and this phenomenon can be blocked by montelukast.
  • CysLTs are involved in respiratory and inflammatory diseases, but their role in autoimmune diseases is rarely mentioned.
  • the inventors have discovered that CysLTs regulate T cell chemotaxis and blood-brain barrier in the pathogenesis of EAE.
  • Permeability and also demonstrated that two CysLT1 antagonists currently used to treat asthma can reduce CNS inflammatory cell infiltration, reduce the clinical symptoms of EAE, and find that montelukast shows significant even in the course of disease progression. Therapeutic effect.
  • the new use of old drugs is an important source of innovative drug discovery. The present invention not only reveals some mechanisms of the onset of MS, but also provides new therapeutic targets and new drugs for clinical intervention of diseases.
  • Tyrosine Kinase 2 plays critical roles in the pathogenic CD4 T cell responses for the development of experimental autoimmune encephalomyelitis. J Immunol 183:7539-7546.
  • TAK-779 decreased Experimental autoimmune encephalomyelitis by reducing inflammatory cell migration into the central nervous system, without affecting T cell function.

Landscapes

  • Health & Medical Sciences (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • General Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Animal Behavior & Ethology (AREA)
  • Medicinal Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • General Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Engineering & Computer Science (AREA)
  • Immunology (AREA)
  • Epidemiology (AREA)
  • Rheumatology (AREA)
  • Pain & Pain Management (AREA)
  • Biomedical Technology (AREA)
  • Neurology (AREA)
  • Neurosurgery (AREA)
  • Orthopedic Medicine & Surgery (AREA)
  • Physical Education & Sports Medicine (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

L'invention concerne une utilisation d'un médicament ciblant un récepteur cystéine-leucotriène CysLT1 dans la préparation d'un médicament pour la prévention ou le traitement de maladies auto-immunes. Les antagonistes de CysLT1 comprennent Montelukast, Zafirlukast et Pranlukast et sont utilisés pour le traitement ou la prévention de la sclérose en plaque, de la polyarthrite rhumatoïde, du lupus érythémateux ou d'une maladie intestinale inflammatoire.
PCT/CN2012/000842 2011-07-28 2012-06-15 Utilisation d'un médicament ciblant cyslt1 dans la préparation d'un médicament pour la prévention ou le traitement de maladies auto-immunes WO2013013490A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN201110213472.1 2011-07-28
CN2011102134721A CN102895661A (zh) 2011-07-28 2011-07-28 靶向CysLT1的药物在制备用于预防或治疗自身免疫性疾病的药物中的用途

Publications (1)

Publication Number Publication Date
WO2013013490A1 true WO2013013490A1 (fr) 2013-01-31

Family

ID=47568346

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2012/000842 WO2013013490A1 (fr) 2011-07-28 2012-06-15 Utilisation d'un médicament ciblant cyslt1 dans la préparation d'un médicament pour la prévention ou le traitement de maladies auto-immunes

Country Status (2)

Country Link
CN (1) CN102895661A (fr)
WO (1) WO2013013490A1 (fr)

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2969010A1 (fr) * 2013-03-13 2016-01-20 Inflammatory Response Research, Inc. Utilisation de levocetirizine et de montelukast dans le traitement des maladies auto-immunes
US9669025B2 (en) 2013-03-13 2017-06-06 Inflammatory Response Research, Inc. Use of levocetirizine and montelukast in the treatment of vasculitis
US9925183B2 (en) 2014-09-15 2018-03-27 Inflammatory Response Research, Inc. Levocetirizine and montelukast in the treatment of inflammation mediated conditions
US11103500B2 (en) 2013-03-13 2021-08-31 IRR, Inc. Use of levocetirizine and montelukast in the treatment of traumatic injury
CN113491696A (zh) * 2017-07-05 2021-10-12 江阴优培尔康药业有限公司 包含孟鲁司特与贻贝粘附蛋白的组合的局部制剂
CN114931578A (zh) * 2022-05-16 2022-08-23 中国医学科学院皮肤病医院(中国医学科学院皮肤病研究所) 孟鲁司特在制备用于治疗系统性红斑狼疮药物中的应用

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9044479B2 (en) 2010-06-16 2015-06-02 Bruce Chandler May Use of levocetirizine and montelukast in the treatment of influenza, common cold and inflammation

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101835749A (zh) * 2007-07-30 2010-09-15 奥斯拜客斯制药有限公司 取代的吲哚

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101835749A (zh) * 2007-07-30 2010-09-15 奥斯拜客斯制药有限公司 取代的吲哚

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
REETTA HOLMA ET AL.: "Acute effects of the cys-leukotriene-1 receptor antagonist, montelukast, on experimental colitis in rats.", EUROPEAN JOURNAL OF PHARMACOLOGY, vol. 429, no. 1-3, 2001, pages 309 - 318 *
SIMON D CROWTHER ET AL.: "Current treatment of asthama-focus on leukotrienes.", EXPERT OPINION ON PHARMACOTHERAPY, vol. 1, no. 5, 2000, pages 1021 - 1040 *

Cited By (15)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11103500B2 (en) 2013-03-13 2021-08-31 IRR, Inc. Use of levocetirizine and montelukast in the treatment of traumatic injury
EP2969010A4 (fr) * 2013-03-13 2016-10-05 Inflammatory Response Res Inc Utilisation de levocetirizine et de montelukast dans le traitement des maladies auto-immunes
US9669025B2 (en) 2013-03-13 2017-06-06 Inflammatory Response Research, Inc. Use of levocetirizine and montelukast in the treatment of vasculitis
US9669026B2 (en) 2013-03-13 2017-06-06 Inflammatory Response Research, Inc. Use of levocetirizine and montelukast in the treatment of autoimmune disorders
US11344545B2 (en) 2013-03-13 2022-05-31 IRR, Inc. Use of levocetirizine and montelukast in the treatment of autoimmune disorders
EP3300734A3 (fr) * 2013-03-13 2018-04-18 Inflammatory Response Research, Inc. Utilisation de lévocétirizine et de montélukast dans le traitement des maladies auto-immunes
EP2969010A1 (fr) * 2013-03-13 2016-01-20 Inflammatory Response Research, Inc. Utilisation de levocetirizine et de montelukast dans le traitement des maladies auto-immunes
US10201537B2 (en) 2013-03-13 2019-02-12 IRR, Inc. Use of levocetirizine and montelukast in the treatment of autoimmune disorders
US10206919B2 (en) 2013-03-13 2019-02-19 IRR, Inc. Use of levocetirizine and montelukast in the treatment of vasculitis
US10195193B2 (en) 2014-09-15 2019-02-05 IRR, Inc. Levocetirizine and montelukast in the treatment of inflammation mediated conditions
US10792281B2 (en) 2014-09-15 2020-10-06 IRR, Inc. Levocetirizine and montelukast in the treatment of inflammation mediated conditions
US9925183B2 (en) 2014-09-15 2018-03-27 Inflammatory Response Research, Inc. Levocetirizine and montelukast in the treatment of inflammation mediated conditions
US11590125B2 (en) 2014-09-15 2023-02-28 IRR, Inc. Levocetirizine and montelukast in the treatment of inflammation mediated conditions
CN113491696A (zh) * 2017-07-05 2021-10-12 江阴优培尔康药业有限公司 包含孟鲁司特与贻贝粘附蛋白的组合的局部制剂
CN114931578A (zh) * 2022-05-16 2022-08-23 中国医学科学院皮肤病医院(中国医学科学院皮肤病研究所) 孟鲁司特在制备用于治疗系统性红斑狼疮药物中的应用

Also Published As

Publication number Publication date
CN102895661A (zh) 2013-01-30

Similar Documents

Publication Publication Date Title
Wang et al. Antiasthmatic drugs targeting the cysteinyl leukotriene receptor 1 alleviate central nervous system inflammatory cell infiltration and pathogenesis of experimental autoimmune encephalomyelitis
WO2013013490A1 (fr) Utilisation d'un médicament ciblant cyslt1 dans la préparation d'un médicament pour la prévention ou le traitement de maladies auto-immunes
Keely et al. Immune dysregulation in the functional gastrointestinal disorders
Napolitani et al. Prostaglandin E2 enhances Th17 responses via modulation of IL‐17 and IFN‐γ production by memory CD4+ T cells
El Annan et al. Characterization of effector T cells in dry eye disease
Sheibanie et al. Prostaglandin E2 exacerbates collagen‐induced arthritis in mice through the inflammatory interleukin‐23/interleukin‐17 axis
Li et al. Enhanced high‐mobility group box 1 (HMGB 1) modulates regulatory T cells (T reg)/T helper 17 (T h17) balance via toll‐like receptor (TLR)‐4‐interleukin (IL)‐6 pathway in patients with chronic hepatitis B
Ferstl et al. Histamine receptor 2 modifies iNKT cell activity within the inflamed lung
Baumann et al. Nasal levels of soluble IL‐33R ST 2 and IL‐16 in allergic rhinitis: inverse correlation trends with disease severity
Niu et al. Matrine regulates Th1/Th2 cytokine responses in rheumatoid arthritis by attenuating the NF‐κB signaling
Ma et al. Anti‐asthmatic effects of baicalin in a mouse model of allergic asthma
Hengel et al. Mosaic theory revised: inflammation and salt play central roles in arterial hypertension
Huilan et al. Role of the subgroups of T, B, natural killer lymphocyte and serum levels of interleukin‐15, interleukin‐21 and immunoglobulin E in the pathogenesis of urticaria
Lalor et al. T h1‐mediated experimental autoimmune encephalomyelitis is CXCR 3 independent
Fischer et al. The potential role of T cell migration and chemotaxis as targets of glucocorticoids in multiple sclerosis and experimental autoimmune encephalomyelitis
Wang et al. γ-aminobutyric acid transporter 1 negatively regulates T cell-mediated immune responses and ameliorates autoimmune inflammation in the CNS
Newman et al. Members of the novel UBASH3/STS/TULA family of cellular regulators suppress T‐cell‐driven inflammatory responses in vivo
Guo et al. Sodium chloride exacerbates dextran sulfate sodium-induced colitis by tuning proinflammatory and antiinflammatory lamina propria mononuclear cells through p38/MAPK pathway in mice
Dixit et al. Leukotriene B4 activates intracellular calcium and augments human osteoclastogenesis
AU2015322125B2 (en) Methods and compositions for modulating TH-GM cell function
Wu et al. Soluble CD83 alleviates experimental allergic rhinitis through modulating antigen-specific Th2 cell property
Mattingly et al. Use of systemic proteasome inhibition as an immune-modulating agent in disease
Kohashi et al. Effective treatment with oral administration of rebamipide in a mouse model of Sjögren's syndrome
Tang et al. Abnormal activation of the Akt-GSK3β signaling pathway in peripheral blood T cells from patients with systemic lupus erythematosus
Wan et al. 5-HT induces regulatory B cells in fighting against inflammation-driven ulcerative colitis

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 12817274

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 12817274

Country of ref document: EP

Kind code of ref document: A1