WO2012116585A1 - 氨基噻唑类MyD88特异性抑制剂的制药用途 - Google Patents

氨基噻唑类MyD88特异性抑制剂的制药用途 Download PDF

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WO2012116585A1
WO2012116585A1 PCT/CN2012/070808 CN2012070808W WO2012116585A1 WO 2012116585 A1 WO2012116585 A1 WO 2012116585A1 CN 2012070808 W CN2012070808 W CN 2012070808W WO 2012116585 A1 WO2012116585 A1 WO 2012116585A1
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myd88
group
treatment
mice
inhibitor
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French (fr)
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周平
姜凤超
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Hangzhou Zhongmei Huadong Pharmaceutical Co Ltd
Tongji Hospital Affiliated to Tongji Medical College of Huazhong University of Science and Technology
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Hangzhou Zhongmei Huadong Pharmaceutical Co Ltd
Tongji Hospital Affiliated to Tongji Medical College of Huazhong University of Science and Technology
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Priority to JP2013555730A priority Critical patent/JP2014506896A/ja
Priority to EP12751830.6A priority patent/EP2682121B1/en
Priority to CA2828796A priority patent/CA2828796A1/en
Publication of WO2012116585A1 publication Critical patent/WO2012116585A1/zh
Priority to US14/016,128 priority patent/US20140073648A1/en
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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
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/496—Non-condensed piperazines containing further heterocyclic rings, e.g. rifampin, thiothixene or sparfloxacin
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D417/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00
    • C07D417/02—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00 containing two hetero rings
    • C07D417/12—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00 containing two hetero rings linked by a chain containing hetero atoms as chain links
    • 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
    • A61P37/02—Immunomodulators
    • 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
    • A61P37/06—Immunosuppressants, e.g. drugs for graft rejection
    • 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
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • 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 a class of aminothiazole-based MyD88-specific inhibitors for use as immunomodulators in medicine and scientific research, especially in anti-transplant rejection, anti-autoimmune diseases, anti-ischemic reperfusion injury and anti-chronic inflammatory response, anti-internal Uses such as toxemia.
  • the body's immune response is divided into natural immunity and acquired immunity. Among them, the latter has been regarded as the main research object and intervention target of transplantation immunity because of its highly specific recognition function and high response effect.
  • the traditional immune response is thought to be the activation of NF-B by the first and second stimulation signals of the acquired immune system. Activation of NF-B enters the nucleus, initiates transcription, synthesizes and secretes various inflammatory cytokines, triggering the subsequent A series of immune responses.
  • Current anti-rejection drugs are all acting on the acquired immune system.
  • TLR Toll-like Receptor
  • MyD88 is a key molecular node of natural immunity, blocking MyD88 blocks the main response of the innate immune system, and thus produces corresponding immunosuppressive effects. If it can be intervened and blocked with a therapeutic drug, it can block the main signal of the TLR pathway and achieve a series of immune regulation. It is foreseeable that this is an excellent solution for achieving immunotherapy. Thousands of studies around the world have validated the importance of this molecule and blocked the therapeutic effects that can be achieved, but none have found a drug that can inhibit it. Other methods of interference with MyD88 molecules, such as gene knockout, cannot be clinically applied.
  • TLR/MyD88 plays an important role in transplantation immunity, and it has been confirmed by blocking MyD88 knockout mice that blocking MyD88 molecules can induce and maintain transplantation immunity. Tolerance, and in the later related research, the designer of the present invention cooperated with the pharmacy team to produce a synthetic and repeated screening to obtain a specific class of MyD88 inhibitors: a class of aminothiazole small molecule compounds (code TJ-M2010 ), such small molecule compounds can specifically bind due to the structural activation site matching the key activation site of MyD88 molecule, so that competitive binding can inhibit the corresponding signal transduction of MyD88.
  • code TJ-M2010 a class of aminothiazole small molecule compounds
  • the object of the present invention is to provide a class of aminothiazole small molecule compounds as specific inhibitors of MyD88, and to inhibit various MyD88 molecules in innate immunity, and to treat various immune-related diseases.
  • the basis of the specific technical scheme for realizing the present invention is that the present invention firstly proposes such aminothiazole MyD88 molecular analogs as anti-graft rejection, anti-autoimmune diseases, anti-ischemic reperfusion injury and anti-chronic inflammatory reaction, anti-endotoxin Applications such as hemolysis use its inhibitory effect on MyD88 molecules in innate immunity.
  • the MyD88 protein of the present invention consists of two domains: a TIR (toll/IL-1 recptor domain) domain and a DD domain (TIR domain), which is the material basis for the homodimerization of MyD88, and further Activates downstream kinases such as IRAKI or IRAK4.
  • TIR domain a class of MyD88-specific inhibitor TJ-M2010 was synthesized. They specifically bind to the TIR domain of MyD88, interfere with the function of the TIR domain of MyD88, prevent MyD88 from forming homodimers, and disable MyD88, thereby blocking MyD88 pathway transduction, and thus failing to activate NF-B.
  • the inflammatory response is broken and thus plays an important role in the treatment of related inflammation and immune diseases.
  • the molecular structure of the aminothiazole-specific MyD88-specific inhibitor of the present invention is as follows:
  • TJ-M2010 3-(4-(4-methoxyphenyl)piperazin-1-yl)-N-(4-phenylthiazol-2-yl)propanamide
  • TJ-M2010 has a very small molecule, It is structurally stable, can penetrate cell membranes, and can be applied both in vitro and in vivo.
  • the specific inhibitor of the aminothiazole type MyD88, TJ-M2010 is used as an NF-KB inhibitor in the preparation of immunomodulatory drugs.
  • the specific inhibitor of the aminothiazole type MyD88, TJ-M2010 is used as an immunomodulator in the treatment of reducing post-transplant rejection and induction and maintenance of transplantation tolerance.
  • the specific inhibitor of the aminothiazole type MyD88 of the present invention is an immunomodulator and is used for treating various chronic inflammatory diseases.
  • chronic inflammatory bowel disease For example, chronic inflammatory bowel disease, asthma, and the like.
  • TJ-M2010 The specific inhibitor of aminothiazoles of the present invention, TJ-M2010, is used as an immunomodulator in the treatment of various autoimmune diseases.
  • various autoimmune diseases For example, type I diabetes, multiple sclerosis, lupus erythematosus, and the like.
  • the specific inhibitor of the aminothiazole type MyD88 of the present invention is used as an immunomodulator in the treatment of diseases of ischemia-reperfusion injury.
  • diseases of ischemia-reperfusion injury For example, prevention and treatment of ischemia-reperfusion injury after myocardial infarction, ischemia-reperfusion injury after replantation of limbs, ischemia-reperfusion injury after graft, preparation of organ preservation solution and cell preservation solution.
  • a specific inhibitor of the aminothiazole type MyD88 of the present invention, TJ-M2010 is used as an immunomodulator in the treatment of sepsis and endotoxemia.
  • the present invention is advantageous in that a novel class of self-synthesized compound TJ-M2010 is used as a specific inhibitor of MyD88 in an experiment, which fully confirms that it is used for anti-rejection and induction of immune tolerance after transplantation, and treats various inflammations.
  • the reaction, prevention and treatment of ischemia-reperfusion injury have obvious effects. It will be an effective immunosuppressive agent, or a transplant tolerance inducing agent, or a transplant tolerance maintenance agent, an anti-inflammatory drug, and an immunomodulator.
  • These novel compounds are effective in inhibiting the expression of CD80 and CD86, thereby preventing the maturation of DC cells.
  • DC cell maturation has been shown to be one of the key steps in the pathogenesis of a variety of autoimmune diseases such as autoimmune cardiomyopathy, experimental autoimmune grapevine, type I diabetes, multiple sclerosis, and lupus erythematosus. Therefore, this type of MyD88 inhibitor TJ-M2010 can be used for the treatment of such diseases.
  • MyD88 pathway blockade can significantly protect against ischemia-reperfusion injury, so the MyD88 inhibitor TJ-M2010 can be used to prevent ischemia-reperfusion injury after myocardial infarction, ischemia-reperfusion injury after replantation of limbs, It has an important role in many aspects such as ischemia-reperfusion injury, organ preservation solution, and cell preservation solution after graft surgery.
  • TJ-M2010 dose-dependent reduction of T cell activation map
  • TJ-M2010 inhibits up-regulation of co-stimulatory molecule CD80 induced by LPS and CpG
  • TJ-M2010 reduces the real-time quantitative PCR analysis of inflammatory factors in the graft (IL- ⁇ , TNF-a, IL-6 relative levels of three inflammatory factors)
  • Balb/c heart transplantation for C57W/6 is a common control group
  • C57W/6 to C57bl/6 is a homologous control chart
  • TJ-M2010 dose-dependently reduced T cell activation
  • LPS in Figure 3 (b), LPS, CPG, myocardial tissue homogenate media stimulated DC, and TJ-M2010 down-regulated CD80 expression.
  • Figure 3 (c) shows that TJ-M2010 dose-related down-regulation of CD 80 expression on DC surface
  • Figure 3 (d) shows that TJ-M2010 dose-related down-regulation of CD80 expression on macrophage surface.
  • the experimental group in Figure 4 MyD88KO NOD mice, MyD88KO/+NOD mice, NOD mice TJ-M2010 medication group.
  • Fig. 5 shows lymphocyte subset analysis in the spleen of different transplant group receptors (syngene, treatment group, control group), showing CD4 + CD25 + Foxp3 + T in transplant recipients treated with TJ-M2010 Significant up-regulation of cell ratio
  • I/R control group I/R CMC is the vehicle control group I/R TJ-M2010 is the experimental group
  • & b are the total number of cells in the bronchoalveolar neutrophils, respectively.
  • a is the activity of myeloperoxidase in lung tissue
  • b is the concentration of interleukin-6 in lung tissue.
  • Figure 12 is a survival curve of endotoxin death test
  • b is the survival test of sepsis death test.
  • TJ-M2010 is used for anti-rejection after transplantation and induction of transplant immune tolerance.
  • TJ-M2010 is used in mouse heart transplantation model
  • the experiment is divided into four groups, which are:
  • the blank control group no treatment, heart transplantation
  • CMC group vehicle control group
  • homologous heart transplantation group theoretically no rejection, long-term survival
  • TJ-M2010 medication group effect detection group
  • Blank control group Balb/c mice were transplanted into the peritoneal cavity of C57W/6 mice, and no special treatment was performed after operation.
  • CMC control group Balb/c mice were transplanted into the peritoneal cavity of C57W/6 mice. From day 0 to day 6 before transplantation, a solution of sodium carboxymethylcellulose (0.5% CMC) containing no TJ-M2010 was administered intraperitoneally, 200 ⁇ l.
  • the same heart transplantation group C57W/6 mice were transplanted into the abdominal cavity of the same C57W/6 mice, and no special treatment was performed after operation.
  • TJ-M2010 medication group Balb/c mice were transplanted into the peritoneal cavity of C57W/6 mice. TJ-M2010, 150 mg/kg dissolved in CMC was intraperitoneally injected from day 0 to day 6 before cardiac transplantation.
  • the experimental results obtained are shown in the survival curve (see Figure 1).
  • the results show that the blank control group and the literature reported that the rejection time was basically the same, about 8 days; there was no difference in the CMC vehicle control group; the heart in the same heart transplantation group survived for a long time.
  • the average heart graft survival time of the TJ-M2010 group was about 20 days, which was significantly longer than that of the control group.
  • TJ-M2010 combined costimulatory molecule inhibitor-anti-CD154 monoclonal antibody (MR1) used in mouse skin transplantation model experiments were divided into 5 groups: allogeneic skin transplantation control group (reported in the literature for 8-10 days), Single use
  • TJ-M2010 group single use MR1 group and combination drug group (TJ-M2010+MR1).
  • Allogeneic skin transplantation CMC control group Balb/c mice were transplanted into the back of C57W/6 mice, and 0.5% CMC200/day was injected intraperitoneally on days 0-3, 5, 7, 9, 11, 13, 15 days after operation.
  • Isogenic skin transplantation group The skin of C57bl/6 mice was transplanted to the back of C57W/6 mice, and no special treatment was performed after operation.
  • TJ-M2010 group alone Balb/c mice were transplanted to the back of C57W/6 mice, and intraperitoneally injected in 0.5% CMC on days 0-3, 5, 7, 9, 11, 13, 15 days after surgery. TJ-M2010, 150mg/kg/d; MR1 group alone: Balb/c mice were transplanted into the back of C57W/6 mice, and intraperitoneal injection of MR1, 200 ⁇ ⁇ / day on the 0th, 1st, 3rd, 7th day after surgery;
  • Combination group Balb/c mice were transplanted into the back of C57W/6 mice, and intraperitoneally injected with 0.5% CMC on the 0-3, 5, 7, 9, 11, 13, 15 days postoperatively. TJ-M2010, 150 mg/kg/d, and intraperitoneal injection of MR1, 200 ⁇ ⁇ / day on days 0-3, 5, 7, 9, 11, 13, 15 days.
  • the experimental results obtained are shown in the survival curve (see Figure 2).
  • the allogeneic skin graft control group had a graft rejection time of about 10 days, which was consistent with the reported time.
  • TJ-M2010 and MR1 alone have no obvious effect on tolerance induction, but the combined application effect is remarkable, and it can make long-term survival of skin grafts which are difficult to induce tolerance.
  • MyD88 inhibitor has a significant effect on anti-rejection and induction of immune tolerance after transplantation. It will be a special immunosuppressive agent, or a transplant tolerance inducer (a short-term treatment can make long-term survival of a skin graft that is extremely difficult to transplant successfully), or a transplant tolerance maintenance agent (such as a rejection induced by a disease-resistant infection). ). Its unique role is unmatched and replaceable with current immunosuppressive agents.
  • Application Example 2 MyD88 inhibitor is used to treat autoimmune diseases.
  • DCs were added to 50 mM TJ-M2010 for 1 hour, and nephroblastic myocardium supernatant was added, LPS (200 ng/ml), Poly I:C (20 mg/ml), CpG (10 mg/ml) for 12 hours.
  • TJ-M2010 inhibited the up-regulation of co-stimulatory molecule CD80 induced by TLR stimulator (LPS, CpG) in RAW264.7 cells, indicating that TJ-M2010 can effectively block the TLR signaling pathway and inhibit the cellular immune response.
  • LPS TLR stimulator
  • CpG TLR stimulator
  • Raw264.7 The number of cells in the 48-well plate is 9*105/well, and the lml medium per well is first pre-incubated with different concentration gradients TJ-M2010 for 2 h, then CPG is added, the final concentration is 40 ug/ml, 37 ° CC02 incubator Incubate overnight (12 h).
  • DC 48-well plate, the number of cells is 1*106/well, 1 ml medium per well, first pre-incubation with different concentration gradient TJ-M2010 for 2 h, then add LPS, final concentration lug/ml, incubate in 37°CC02 incubator Overnight (12h).
  • Flow-through antibody FITC-labeled anti-CD80, CD86, on-machine detection The above two images show that TJ-M2010 has a concentration-dependent inhibition of DC and macrophage surface CD80 expression.
  • TJ-M2010 inhibits up-regulation of co-stimulatory molecule CD80/CD86 by LPS and CpG.
  • MyD88 inhibitor can reduce CD80 expression and prevent DC cell maturation.
  • DC cell maturation has been shown to be one of the key steps in the pathogenesis of a variety of autoimmune diseases such as autoimmune cardiomyopathy, experimental autoimmune glucones, type I diabetes, multiple sclerosis, and lupus erythematosus. Therefore, MyD88 inhibitors can be used in the treatment of such diseases.
  • Drug group 1 day before antigen injection, 0-3, 5, 7, 9, 11 , 13, 15 days were intraperitoneally injected with TJ-M2010 dissolved in 0.5% CMC, 150mg/kg/d
  • Each group was intraperitoneally injected with mycobacterial antigen and continuously monitored its concentration.
  • MyD88KO heterozygous group gradually increased type I diabetes with time, while MyD88KO homozygous group did not develop type I diabetes.
  • the incidence of type I diabetes in TJ-M2010 group was similar to that of MyD88KO homozygous group, indicating MyD88 pathway and I.
  • the occurrence of type 2 diabetes is inextricably linked. Blocking its pathway can reduce the incidence of diabetes. Therefore, the small molecule MyD88 inhibitor TJ-M2010 may become an effective method for prevention and treatment of type I diabetes.
  • MyD88 inhibitor is used for the prevention and treatment of ischemia-reperfusion injury.
  • In vitro Flow cytometry analysis of CD4+CD25+Foxp3+ T cells in recipients receiving antigen (syngeneic, allogeneic) stimulation of spleen and recipients treated with TJ-M2010
  • TJ-M2010 After application of the MyD88 inhibitor TJ-M2010, analysis of lymphocyte subsets in the spleen of transplant recipients and detection of CD4 + CD25 + Foxp3 + T cell ratios revealed that TJ-M2010 was applied by up-regulating CD4 + CD25 + Foxp3 + T cells. The transplant tolerance status of recipient mice was altered.
  • a large number of literatures indicate that regulatory T cells can regulate the development of inflammation through its immunosuppressive effects, the release of inflammatory factors, pro-inflammatory factors and cytokines during ischemia-reperfusion, resulting in damage. Therefore, the application of TJ-M2010 By inhibiting TLR signaling, inhibition of NF-activation, and reduction of expression of inflammatory factors (IFN- ⁇ and IL-17), thereby reducing damage.
  • MyD88 pathway blocks the reduction of renal ischemia-reperfusion injury:
  • Grouping Normal C57W/6 group (Control), CMC vehicle group, MYD88KO group, TJ-M2010 group, 8 groups were treated for ischemia-reperfusion: anesthesia, blood vessel clamp blocked left kidney, thermostat 31° After 80 minutes, open and remove the right kidney and close the abdomen. Blood was taken 24 hours for BUN, Cr detection.
  • TJ-M2010 group and CMC group were intraperitoneally injected with TJ-M2010 dissolved in 0.5% CMC, 150 mg/kg/d 1 day before surgery and on the day of surgery.
  • CMC group 0.5% CMC solution, 200 ⁇ l.
  • mice 3. Observe the survival time of the mice and make a survival curve.
  • the blood specimen is sent to the pathology department for BUN and Cr testing.
  • MyD88 pathway blockade can obviously protect against ischemia-reperfusion injury, so MyD88 inhibitor can be used to prevent ischemia-reperfusion injury after myocardial infarction, ischemia-reperfusion injury after replantation of limbs, It has an important role in many aspects such as ischemia-reperfusion injury, organ preservation solution, and cell preservation solution after graft surgery.
  • Application Example 4 MyD88 inhibitor is used to treat chronic inflammatory diseases.
  • mice The spleen of C57W/6 mice was taken, and the spleen lymphocytes were separated by mouse lymphocyte separation solution and counted.
  • C57W/6 mouse spleen lymphocytes were labeled with CFSE.
  • Balb/c-derived DCs were mixed with C57W/6 mouse lymphocytes for mixed lymphocyte culture. And grouped as follows: Blank group: CPG and TJ-M2010 were not added during the mixed culture.
  • Control group CPG was added during the mixed culture without adding TJ-M2010.
  • Experimental group 1 CPG and TJ-M2010 were added simultaneously during the mixed culture, and the amount of TJ-M2010 was ⁇ .
  • Experimental group 2 CPG and TJ-M2010 were added simultaneously in the process of mixed culture, wherein the amount of TJ-M2010 was experimental group 3: CPG and TJ-M2010 were added simultaneously in the process of mixed culture, wherein the amount of TJ-M2010 was 40 ⁇ .
  • TJ-M2010 can attenuate CpG stimulation T cell proliferation induced by DC activation
  • Real-time quantitative PCR steps 1. TRIzol method from receptors that receive antigen (isogenic, allogeneic) stimulation Extracting total RA
  • Figure 9 is a real-time quantitative PCR analysis of inflammatory factors (IL- ⁇ , TNF-a, IL-6) in grafts.
  • MyD88 pathway blockade reduces tracheal inflammatory response in mice
  • BLM bleomycin nasal spray pneumonia model: 40 ⁇ ketamine xylazine anesthesia, nasal instillation BLM sulfate (300 ⁇ ⁇ or 15 mg/kg)
  • Bronchoalveolar lavage fluid collects cells and cytokines: cut the trachea, insert plastic cannula, lavage at 37 ° C, 0.3 ml PBS, aspirate the lavage fluid (returning more than 95%), repeat 10 Times.
  • the lavage fluid is divided into two parts: one part for cytokine detection (600g centrifugation lOmin collection supernatant storage -80 °C for testing) part of the cell count (with the lower layer 0.4ml resuspended) 4 ° C count
  • Lung MPO activity test The lungs were fully lavaged through the right heart, the lungs were homogenized, the supernatant was centrifuged, and the precipitate was resuspended in lmlPBS (containing 0.5% HTAB, 5 mM EDTA). Centrifugal, 50 ⁇ 1 supernatant added to the test tube
  • Figure 9 shows that MYD88-/- mice recruited concentrated granulocytes and lymphocytes during bronchitis.
  • Figure a shows the total number of cells on days 1, 7, and 11, and there were statistical differences between WT mice and TJ-M2010BLM group.
  • b The bronchial alveolar neutrophils in WT mice peaked at 24 hours for 7 days and recovered for 11 days.
  • the TJ-M2010 group has significantly reduced recruitment
  • FIG 11 shows that the TJ-M2010BLM group attenuates BLM-induced pulmonary inflammation, which is manifested by a decrease in inflammatory cells and inflammatory factors.
  • Figure b shows the reduction of IL-6 in lung tissue at 24h
  • TJ-M2010BLM group significantly attenuated the pulmonary inflammation caused by BLM, thus demonstrating the anti-inflammatory effect of TJ-M2010.
  • MyD88 pathway blockade can reduce the inflammatory response. Therefore, MyD88 inhibitors can be used to treat a variety of chronic inflammatory diseases such as chronic inflammatory bowel disease, asthma and the like.
  • TJ-M2010 control group was given TJ-M2010 (0.5% CMC solvent, 25mg/ml), the dose was 250mg/kg (200 ⁇ each); the solvent control group was given 0.5% CMC, 20 ( ⁇ L each)
  • TJ-M2010 control group was given TJ-M2010 (0.5% CMC solvent, 25mg/ml), the dose was 250mg/kg (200 ⁇ each); the solvent control group was given 0.5% CMC, 20 ( ⁇ L each)
  • the two MyD88 inhibitors used are effective in delaying endotoxin lethal events and reducing endotoxin lethality.

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Description

氨基噻唑类 MyD88特异性抑制剂的制药用途 技术领域
本发明涉及一类氨基噻唑类 MyD88特异性抑制剂作为免疫调节剂医药和科研领域 的应用， 尤其是在抗移植排斥、抗自身免疫疾病、抗缺血再灌注损伤和抗慢性炎症反应、 抗内毒素血症等方面的用途。
背景技术
在本发明提出之前， 本领域的医学专家十分清楚， 对机体免疫系统的抑制性调控是 治疗多种疾病的关键， 如治疗器官移植后排斥反应、 自身免疫性疾病、慢性炎症性疾病、 缺血再灌注损伤等。 而对免疫系统的调控可以从多方面入手的， 而天然免疫反应作为近 期研究的热点， 是一个极佳的达成免疫抑制的方向。
机体免疫反应分为天然免疫和获得性免疫。 其中， 后者因为其特异性极强的识别功 能和高效的反应效果一直以来被当作移植免疫的主要研究对象和干预标靶。 传统免疫反 应被认为是由获得性免疫系统的第一和第二刺激信号激活 NF- B，激活的 NF- B进入 细胞核， 启动转录， 细胞合成和分泌各种炎性细胞因子， 引发随后的一系列免疫反应。 目前的抗排斥药物都是作用在获得性免疫系统上的。 天然免疫一直被认为是机体的天然 保护屏障， 主要是抗击病毒细菌感染、 外来生物入侵等， 但最近几年大量的研究发现天 然免疫系统在移植免疫、 自身免疫性疾病和缺血损伤等方面发挥着极其重要的作用。 天 然免疫系统中又以 Toll样受体 （Tool-Like Receptor, TLR) 发挥最主要的作用而最受关 注， 目前发现 TLR有至少 14个亚型， 主要分布在 APC等免疫细胞上， 这些亚型除了 TLR3以外， 都要通过髓样分化蛋白 (myeloid -differentation protein 88， MyD88)分子传 递信号。 大量的研究显示各种内源性和外源性的危险因子， 刺激天然免疫系统的各个 TLR, 刺激信号经关键分子 MyD88传导， 最后也激活 NF- B， 其后的免疫反应过程与 前述的一样。
因此， 简而言之， MyD88就是天然免疫的关键分子节点， 阻断了 MyD88就阻断了 天然免疫系统的主要反应， 进而产生相应的免疫抑制效果。 如果能够用一种治疗药物对 其进行干预和阻断， 就能够阻断 TLR途径的主要信号从而达成一系列的免疫调控， 可以 预见这是达成免疫治疗的一条极佳的方案。 目前全世界成千上万的研究报告验证了此分子的重要性及阻断其后能达成的治疗效 果， 但都没有找到一种能对其进行抑制的药物。 而其它的对 MyD88分子的干涉途径如 基因敲除等方法不能临床应用。
本发明设计人在 TLR/MyD88方面所作的一系列前期研究， 验证了 TLR在移植免疫 中起着重要作用， 并通过对 MyD88基因敲除小鼠的研究证实阻断 MyD88分子能够诱导 和维持移植免疫耐受， 而在后期的相关研究中，本发明设计人通过与药学专业团队合作， 制造合成并反复筛选获得了一类 MyD88特异性抑制剂： 一类氨基噻唑类小分子化合物 (代号 TJ-M2010), 此类小分子化合物由于结构上的活化位点与 MyD88分子的关键活 化位点相匹配而能特异性结合， 故能够竞争性结合而抑制 MyD88相应的信号传导。 因 此， 将 MyD88特异性抑制剂 TJ-M2010应用于抗移植排斥、 抗自身免疫疾病、 抗缺血再 灌注损伤和抗慢性炎症反应、 抗内毒素血症等， 开创了此类小分子物质的应用先河， 为 多种天然免疫相关性疾病找到了一种全新的药物治疗可能。
发明内容
本发明的目的在于提供一类氨基噻唑类小分子化合物作为 MyD88特异性抑制剂， 并应用其对天然免疫中 MyD88分子的抑制作用， 治疗多种免疫相关性疾病。 实现本发 明的具体技术方案的基础在于，本发明首先提出将此类氨基噻唑类 MyD88分子类似物作 为抗移植排斥、 抗自身免疫疾病、 抗缺血再灌注损伤和抗慢性炎症反应、 抗内毒素血症 等应用， 是利用其对天然免疫中 MyD88分子的抑制作用。
本发明所述的 MyD88蛋白由两个结构域组成： 包括 TIR (toll/IL-1 recptor domain) 域和 DD域（death domain), TIR域是 MyD88发生自身同源二聚化的物质基础， 进而活 化下游的 IRAKI或 IRAK4等激酶。 经过对 MYD88的 TIR域的拓扑结构学分析后， 合 成一类 MyD88特异性抑制剂 TJ-M2010。 它们能特异性的结合在 MyD88的 TIR域， 干 扰 MyD88的 TIR域的功能， 阻止 MyD88形成同源二聚体， 使 MyD88不能活化， 从而 阻断 MyD88通路转导， 进而不能激活 NF- B， 阻断了炎性反应， 因而在相关的炎症及 免疫疾病治疗方面有重要作用。
本发明所述的应用氨基噻唑类 MyD88特异性抑制剂的分子结构如下：
TJ-M2010-1
Figure imgf000004_0001
Figure imgf000004_0002
2-(4-(p-甲苯基)哌嗪 -1-基) - N-(4-苯基噻唑 -2-基)乙酰胺 TJ-M2010-3
Figure imgf000004_0003
2-(4-苯基哌嗪 -1-基) -N-(4-苯基噻唑 -2-基)乙酰胺
Figure imgf000004_0004
3-(4-(4-甲氧苯基)哌嗪 -1-基) -N-(4-苯基噻唑 -2-基)丙酰胺 本发明 MyD88特异性抑制剂 TJ-M2010 分子极小， 结构稳定， 可穿透细胞膜， 并 可同时在体内外应用。
本发明一类氨基噻唑类 MyD88的特异性抑制剂 TJ-M2010, 作为 NF-KB抑制剂，在 制备免疫调节类药物上的应用。 本发明一类氨基噻唑类 MyD88的特异性抑制剂 TJ-M2010, 作为免疫调节剂， 在治 疗减低移植后排斥反应以及移植免疫耐受诱导和维持方面的应用。
本发明一类氨基噻唑类 MyD88的特异性抑制剂 TJ-M2010, 作为免疫调节剂， 在治 疗多种慢性炎症性疾病上的应用。 例如慢性炎症性肠病、 哮喘等。
本发明一类氨基噻唑类的特异性抑制剂 TJ-M2010,作为免疫调节剂，在治疗多种自 身免疫性疾病上的应用。 例如 I型糖尿病、 多发性硬化症、 红斑狼疮等。
本发明一类氨基噻唑类 MyD88的特异性抑制剂 TJ-M2010, 作为免疫调节剂， 在治 疗缺血再灌注损伤类疾病上的应用。 例如预防和治疗心梗后缺血再灌注损伤、 断肢再植 后缺血再灌注损伤、 移植物术后的缺血再灌注损伤以及器官保存液、 细胞保存液的制作 等。 本发明一类氨基噻唑类 MyD88的特异性抑制剂 TJ-M2010, 作为免疫调节剂， 在治 疗脓毒血症和内毒素血症方面的应用。
本发明的优越性在于，将一类新型自主合成的化合物 TJ-M2010作为 MyD88特异性 抑制剂应用在实验中， 充分证实了该在用于移植后抗排斥和诱导免疫耐受、 治疗各种炎 症反应、 防治缺血再灌注损伤等方面具有明显的效果。 将是一种有效的免疫抑制剂、 或 移植耐受诱导剂、 或移植耐受维持剂、 抗炎药物和免疫调节剂。 该类新型化合物能够有 效抑制 CD80、 CD86表达， 从而阻止 DC细胞的成熟。 DC细胞成熟已被证明为多种自 身免疫性疾病如自身免疫性心肌病、 实验自身免疫性葡萄炎、 I型糖尿病、 多发性硬化 症、红斑狼疮等发病的关键步骤之一。故该类 MyD88抑制剂 TJ-M2010可用于此类疾病 的治疗。 MyD88途径阻断可以明显起到对缺血再灌注损伤的保护作用， 故此类 MyD88 抑制剂 TJ-M2010可用于防止如心梗后缺血再灌注损伤、 断肢再植后缺血再灌注损伤、 移植物术后的缺血再灌注损伤、 器官保存液、 细胞保存液等许多方面起到重要作用。 体 外实验结果表明这类 MyD88抑制剂 TJ-M2010通过抑制该通路能有效减少移植物内炎症 因子水平， 说明其与炎症因子的产生密切相关， 故可能成为各类炎症治疗的有效手段。 附图说明
图 1 心脏移植物生存曲线
图 2 皮肤移植物生存曲线图
图 3 TJ-M2010剂量相关地减少 T细胞活化图 TJ-M2010抑制 LPS、 CpG引起的共刺激分子 CD80的上调图
图 4 TJ-M2010减低糖尿病发生率曲线图
图 5 TJ-M2010影响移植受体体内淋巴细胞亚群柱状图
图 6 TJ-M2010改善小鼠肾脏 IRI生存率生存曲线图
图 7 TJ-M2010保护小鼠肾脏 IRI肾功能 （血肌酐、 尿素氮） 图
图 8 TJ-M2010减弱 CpG刺激 DC活化引发的 T细胞增殖流式图
图 9 TJ-M2010减少移植物内炎性因子实时定量 PCR分析图 （IL-Ιβ, TNF-a,IL-6 三种炎性因子相对水平）
图 10 TJ-M2010减少肺炎模型中炎性细胞渗出图
图 11 TJ-M2010减少肺炎模型中炎症因子渗出图
图 12 内毒素和脓毒血症致死实验生存曲线
附图 1中 Balb/c和 C57W/6为两种小鼠品系
分组： Balb/c 心脏移植给 C57W/6为普通对照组， CMC溶媒对照组， C57W/6到 C57bl/6为同系对照图， TJ-M2010实验组
附图 2中异基因皮肤移植对照组 （文献报导排斥时间为 8-10天）、 单用 TJ-M2010 组、 单用 MR1组和联合用药组 （TJ-M2010+MR1 )
附图 3 (a)中， TJ-M2010剂量相关地减少 T细胞活化； 附图 3 (b)中， LPS、 CPG、 心肌组织匀浆溶媒刺激 DC, TJ-M2010下调 CD80的表达。 附图 3 (c) 为 TJ-M2010剂 量相关性下调 DC表面 CD 80表达， 附图 3 (d) 为 TJ-M2010剂量相关性下调巨噬细胞 表面 CD80表达。
附图 4中实验分组： MyD88KO NOD小鼠， MyD88KO/+NOD小鼠， NOD小鼠 TJ-M2010用药组。
在附图 5中， 图 5为不同移植组别受体脾脏内淋巴细胞亚群分析 （同基因组， 治疗 组， 对照组）， 显示 TJ-M2010治疗的移植受者体内 CD4+CD25+Foxp3+T细胞比例明显 上调
附图 6中 分组： MYD88 ΚΟ,Τ J-M2010,CMC,Control对照各组 8只
I/R对照组 I/R CMC为溶媒对照组 I/R TJ-M2010为实验组
附图 10中&、 b分别为支气管肺泡中细胞总数， 中性粒细胞数 附图 11中 a为肺组织中髓过氧化酶活性， b为肺组织中白细胞介素 -6浓度 附图 12中 a为内毒素血症致死实验生存曲线， b为脓毒血症致死实验生存曲线图。 具体实施方法
应用实例 1 : TJ-M2010用于移植后抗排斥反应和诱导移植免疫耐受。
1 ) TJ-M2010用于小鼠心脏移植模型
实验共分为四组， 分别为：
空白对照组（不做任何处理， 做心脏移植）、 CMC组（溶媒对照组）、 同系心脏移植 组 （理论上同系无排斥， 长期存活）和 TJ-M2010用药组 （效果检测组）。 具体处理方法 如下：
空白对照组： 取 Balb/c小鼠心脏移植到 C57W/6小鼠腹腔， 术后不做特殊处理； CMC对照组： 取 Balb/c小鼠心脏移植到 C57W/6小鼠腹腔， 术后心脏移植前第 0天 到第 6天给予腹腔注射不含 TJ-M2010的羧甲基纤维素钠 （0.5%CMC) 溶液， 200μ1。
同系心脏移植组：取 C57W/6小鼠心脏移植到同系 C57W/6小鼠腹腔，术后不做特殊 处理；
TJ-M2010用药组： 取 Balb/c小鼠心脏移植到 C57W/6小鼠腹腔， 术后心脏移植前第 0天到第 6天给予腹腔注射溶于 CMC的 TJ-M2010, 150mg/kg。
所得到实验结果如生存曲线图 （见附图 1 ) 所示， 结果显示空白对照组和文献报道 排斥时间基本一致， 为 8天左右; CMC溶媒对照组无差异； 同系心脏移植组心脏长期存 活， TJ-M2010用药组平均心脏移植物存活时间 20天左右， 较对照组有明显延长。
2) TJ-M2010联合共刺激分子抑制剂-抗 CD154单抗 （MR1) 用于小鼠皮肤移植模型 实验分为 5组： 异基因皮肤移植对照组 （文献报导排斥时间为 8-10天）、 单用
TJ-M2010组、 单用 MR1组和联合用药组 （TJ-M2010+MR1 )。
具体处理方式如下：
异基因皮肤移植 CMC对照组： 取 Balb/c小鼠皮肤移植到 C57W/6小鼠背部， 术后 第 0-3， 5， 7， 9， 11， 13， 15天腹腔注射 0.5%CMC200 /天； 同基因皮肤移植组： 取取 C57bl/6小鼠皮肤移植到 C57W/6小鼠背部， 术后不做特殊处理。
单用 TJ-M2010组： 取 Balb/c小鼠皮肤移植到 C57W/6小鼠背部， 术后第 0-3， 5， 7， 9， 11， 13， 15天分别腹腔注射溶于 0.5%CMC的 TJ-M2010, 150mg/kg/d; 单用 MR1组： 取 Balb/c小鼠皮肤移植到 C57W/6小鼠背部， 术后第 0， 1， 3， 7， 14天给予腹腔注射 MR1， 200μ§ /天；
联合用药组： 取 Balb/c小鼠皮肤移植到 C57W/6小鼠背部， 术后术后第 0-3， 5， 7， 9， 11， 13， 15天分别腹腔注射溶于 0.5%CMC的 TJ-M2010, 150mg/kg/d， 同时第 0-3， 5， 7， 9， 11， 13， 15天给予腹腔注射 MR1， 200μ§/天。
所得到实验结果如生存曲线图 （见附图 2) 所示， 异基因皮肤移植对照组移植物排 斥时间为 10天左右， 和文献报导时间一致
单用 TJ-M2010组和单用 MR1组皮肤移植物排斥时间 10天左右， 无统计学差异， 而联合用药组 （TJ-M2010+MR1 ) 皮肤移植物长期存活达 150天 （文献认为 >100天为移 植物耐受）
可见 TJ-M2010和 MR1单用对耐受诱导无明显效果， 但联合应用效果显著， 能使及 其难以诱导耐受的皮肤移植物长期存活。
以上实验结果直接和间接说明， MyD88抑制剂在用于移植后抗排斥和诱导免疫耐受 具有明显的效果。 将是一种特殊的免疫抑制剂、 或移植耐受诱导剂 （短期用药即可使极 难移植成功的皮肤移植物长期存活）、或移植耐受维持剂（如可对抗病菌感染所诱发的排 斥）。 其独特的作用是目前的免疫抑制剂不可比拟和替代的。
应用实例 2: MyD88抑制剂用于治疗自身免疫性疾病。
体外实验一细胞流式仪结果：证明 MyD88抑制剂可阻止 DC成熟，用于治疗自身 免疫性疾病。
步骤： 1.应用了 TJ-M2010的 BALB/c小鼠来源骨髓细胞， 破红， 2 x lO 6/ML密度 RPMI1640培养基 （加 GM-CSF10ng/ml， IL-4 10ng/ml)
2.48小时去悬浮细胞， 第六天收悬浮及半贴壁细胞
3. DCs加 50mM TJ-M2010培养 1小时，加入坏死心肌上清液， LPS (200ng/ml), Poly I:C ( 20 mg/ml) ,CpG (10 mg/ml)培养 12小时
4.加流式抗体 FITC标记抗 CD80，CD86， 上机检测
TJ-M2010抑制了 RAW264.7细胞对 TLR刺激物 （LPS、 CpG) 引起的共刺激分子 CD80的上调， 说明 TJ-M2010能够有效的阻断 TLR信号通路， 进而抑制了细胞的免疫 反应。 附图 3 (c)、 (d) 实验过程如下：
Raw264.7: 48孔板 细胞数为 9*105/孔， 每孔 lml培养基体系， 首先加入不同浓度 梯度 TJ-M2010预孵育 2h， 然后加入 CPG， 终浓度 40ug/ml， 37°CC02培养箱孵育过夜 ( 12h)。 加流式抗体 FITC标记抗 CD80，CD86， 上机检测；
DC: 48孔板， 细胞数为 1*106/孔， 每孔 lml培养基体系， 首先加入不同浓度梯度 TJ-M2010预孵育 2h， 然后加入 LPS, 终浓度 lug/ml， 37°CC02培养箱孵育过夜（12h)。 加流式抗体 FITC标记抗 CD80，CD86， 上机检测以上二图中可见 TJ-M2010对 DC和巨 噬细胞表面 CD80表达起一浓度相关性抑制。
附图 3: TJ-M2010抑制 LPS、 CpG引起的共刺激分子 CD80/CD86的上调 以上检测结果说明 MyD88抑制剂可降低 CD80表达， 从而阻止 DC细胞的成熟。 DC细胞成熟已被证明为多种自身免疫性疾病如自身免疫性心肌病、 实验自身免疫性葡 萄炎、 I型糖尿病、 多发性硬化症、 红斑狼疮等发病的关键步骤之一。 故 MyD88抑制剂 可用于此类疾病的治疗。
体内试验： 应用 MyD88-/-以及 TJ-M2010对构建 I型糖尿病模型的影响
实验步骤：
1.实验分组： MyD88KO NOD小鼠， MyD88KO/+NOD小鼠， NOD小鼠 TJ-M2010 用药组
2.用药组：抗原注射前 1天，第 0-3， 5, 7, 9, 11 , 13， 15天分别腹腔注射溶于 0.5%CMC 的 TJ-M2010, 150mg/kg/d
3.各组均腹腔注射分支杆菌抗原并持续监测其浓度
4.清洁级伺养观察 30周，取尾静脉血，测非空腹血糖，连续 2次所测血糖≥22 mmol/L 为糖尿病建模标准
所得 I型糖尿病发生率曲线见附图 4:
结果显示: MyD88KO杂合子组随着时间延长 I型糖尿病发生逐渐增加，而 MyD88KO 纯合子组无 I型糖尿病发生， TJ-M2010组 I型糖尿病发生率与 MyD88KO纯合子组相当， 说明 MyD88通路与 I型糖尿病的发生有必然的联系， 阻断其通路能减少糖尿病的发生， 因此小分子 MyD88抑制剂 TJ-M2010可能成为 I型糖尿病的有效防治方法。
应用实例 3: MyD88抑制剂用于预防和治疗缺血再灌注损伤。 体外实验： 对接受抗原 （同基因， 异基因） 刺激的受体脾脏内淋巴细胞亚群分析及 同时接受过 TJ-M2010治疗的受者体内 CD4+CD25+Foxp3+T细胞比例流式检测
实验步骤：
1、 取不同组别 （同基因， 异基因抗原刺激） 受体脾脏， 碾磨分离淋巴细胞
2、加流式抗体 APC标记的 IFN-γ和 APC标记的 IL-17, APC标记的 CD25和 PE标 记的 Foxp3
3、上流式细胞仪分析不同移植组别受体脾脏内淋巴细胞亚群和 TJ-M2010治疗的移 植受者体内 CD4+CD25+Foxp3+T细胞比例
见附图 5，结果显示 TJ-M2010治疗的移植受者体内 CD4+CD25+Foxp3+T细胞比例明 显上调， 而 IFN-γ和 IL-17明显低于 CMC对照组。
通过应用 MyD88抑制剂 TJ-M2010后， 对移植受体脾脏内淋巴细胞亚群分析和 CD4+CD25+Foxp3+T细胞比例的检测发现 TJ-M2010的应用通过上调 CD4+CD25+Foxp3+T 细胞而改变受体小鼠的移植耐受状态。 而大量文献表明调节性 T细胞可以通过其免疫抑 制作用调节炎症的发展，炎症因子，促炎症因子的释放与缺血再灌注时细胞因子的交联， 从而出现损伤， 因此， TJ-M2010的应用通过抑制 TLR信号， 抑制 NF- 活化， 减少 炎症因子 （IFN-γ和 IL-17) 的表达， 从而减轻损伤。
体内实验部分：
MyD88途径阻断减轻肾脏缺血再灌注损伤实验：
实验步骤：
1.分组： 普通 C57W/6组 （Control), CMC溶媒组， MYD88KO组， TJ-M2010组各 组 8只做缺血再灌注处理： 麻醉， 血管夹阻断左侧肾脏， 置温箱 31°， 80min后开放并切 除右侧肾脏， 关腹。 24小时取血用于 BUN, Cr检测。
2. TJ-M2010组和 CMC组， 术前 1天和手术当天分别腹腔注射溶于 0.5%CMC的 TJ-M2010, 150mg/kg/d; CMC组： 0.5%CMC溶液， 200μ1。
3.观察小鼠生存时间， 做生存曲线。 血标本送病理科做 BUN，Cr检测。
4.结果显示 TJ-M2010显著提高小鼠肾脏 IRI后生存率，而且对肾功能有很好的保护 效果。
结果见附图 6和附图 7。 由以上实验可见 MyD88途径阻断可以明显起到对缺血再灌注损伤的保护作用， 故 MyD88抑制剂可用于防止如心梗后缺血再灌注损伤、断肢再植后缺血再灌注损伤、移植 物术后的缺血再灌注损伤、 器官保存液、 细胞保存液等许多方面起到重要作用。
应用实例 4: MyD88抑制剂用于治疗慢性炎症性疾病。
体外实验： TJ-M2010减弱 CpG刺激 DC活化引发的 T细胞增殖及移植物内炎性因 子实时定量 PCR分析
实验步骤：
1、 取 Bal b/c小鼠股骨， 分离骨髓细胞， 加入 GMS-CSF和 IL-4细胞因子， 培养骨 髓源性 DC
2、 培养至第六天， 吹打细胞， 分离未成熟 DC。 离心， 1640培养基重悬。
3、 加入丝裂霉素 （使终浓度达 5(^g/ml)， 37°C水浴， 15min。 1640洗一次， 计数。
4、 取 C57W/6小鼠脾脏， 利用小鼠淋巴细胞分离液分离脾脏淋巴细胞， 并计数。
5、 取 C57W/6小鼠脾脏淋巴细胞标记 CFSE。
6、 Bal b/c来源的 DC与 C57W/6小鼠淋巴细胞做混合淋巴细胞培养。 并分组如下： 空白组： 在混合培养的过程中不加 CPG及 TJ-M2010。
对照组： 在混合培养的过程中加 CPG而不加 TJ-M2010。
实验组 1 : 在混合培养的过程中同时加 CPG及 TJ-M2010, 其中 TJ-M2010的量为 ΙΟμΜο
实验组 2: 在混合培养的过程中同时加 CPG及 TJ-M2010, 其中 TJ-M2010的量为 实验组 3 : 在混合培养的过程中同时加 CPG及 TJ-M2010, 其中 TJ-M2010的量为 40μΜ。
1、 培养至第三天， 收集细胞， 流式检测 C57W/6小鼠淋巴细胞增殖情况。
流式结果见附图 7
结果显示： 随着 TJ-M2010量的增加， Τ细胞增殖 （CD44为其表面标志） 呈下降趋 势
说明 TJ-M2010可减弱 CpG刺激 DC活化引发的 T细胞增殖
实时定量 PCR步骤： 1. 从接受抗原 （同基因， 异基因） 刺激的受体用 TRIzol 法 抽提总 R A
2. 逆转录为 cDNA，两步法 RT-PCR
3. 做标准曲线对比得出 IL-Ιβ, TNF-a，IL-6相对水平
附图九为移植物内炎性因子 （IL-Ιβ, TNF-a,IL-6) 实时定量 PCR分析
结果显示： 心脏移植物内炎症因子水平 TJ-M2010组明显比对照组低， 有显著统计 学差异
体外实验结果表明 MyD88抑制剂 TJ-M2010通过抑制该通路能有效减少移植物内炎 症因子水平 （IL-Ιβ, IL-6较 CMC异基因移植组明显降低）， 说明其与炎症因子的产生 密切相关， 故可能成为各类炎症治疗的有效手段。
体内试验：
MyD88途径阻断降低小鼠气管炎症反应
方法与步骤：
1.动物分组： C57bl/6(B6)NaCl (20(^l滴鼻）组， C57bl/6(B6)BLM组和 TJ-M2010BLM 组（第 0-3， 5， 7， 9， 11， 13， 15天分别腹腔注射溶于 0.5%CMC的 TJ-M2010, 150mg/kg/d)。
2. BLM (博来霉素）滴鼻制作肺炎模型： 40 μΐ氯胺酮甲苯噻嗪气道麻醉， 鼻腔滴入 法 BLM硫酸盐 (300 μ§ or 15 mg/kg)
3.支气管肺泡灌洗液（BAL)收集细胞及细胞因子：切开气管，插入塑料套管， 37°C， 0.3mlPBS灌洗， 抽吸灌洗液（回抽达 95%以上）， 重复 10次。 灌洗液分两部分： 一部分 用于细胞因子检测 （600g离心 lOmin收集上清存放 -80°C用于检测） 一部分用于细胞计 数 （连同下层 0.4ml重悬） 4°C计数
4.肺匀浆检测组织内细胞及因子： BAL后， 取整肺， 搅碎， 离心取上清 -80°C存放 用于 MPO的检测
5.肺 MPO活性检测： 盐水经右心充分灌洗肺脏， 肺匀浆， 离心去上清， lmlPBS (含 0.5%HTAB ， 5mM EDTA ) 重 悬 沉 淀 。 离 心 ， 50μ1 上 清 加 入 试 管
( 20(^lPBS-HTAB-EDTA，2mlHBSS， ΙΟΟμΙ二盐酸邻联茴香胺（1.25 η¾/ηι1)，100μ1 Η202 0.05%) ,15min, 37°C漩涡水箱， 100 μΐ NaN3 1%中止反应， 460nm检测 MPO吸光度值。
6.细胞计数： MG-1L染色 4min， 95%GS-500染色 8min， 涂片计数
7.因子检测: IL-6水平用 ELISA检测 8.统计学分析： U检验分析统计学差异
附图 9: 显示 MYD88-/-小鼠在支气管炎发生过程中募集中性粒和淋巴细胞减少 实验分组： B6NaCL组， B6BLM组和 TJ-M2010BLM组 （n=4)
在 TJ-M2010组支气管肺泡中性粒细胞募集明显减少。
a图显示第 1， 7， 11天的细胞总数， WT小鼠和 TJ-M2010BLM组有统计学差异； b图显示 WT小鼠支气管肺泡中性细胞 24h达峰，持续 7天， 11天恢复，而 TJ-M2010 组明显募集减少
附图 11图显示 TJ-M2010BLM组减轻 BLM诱导的肺炎症反应， 表现为炎症细胞及 炎症因子的减少。
a图肺组织中 MPO因子 （第 7天检测） 的减少
b图分别显示 24h肺组织中 IL-6的减少
从炎症细胞的募集到炎症因子的释放两个方面的实验可以看出 TJ-M2010BLM组明 显减轻 BLM引起的肺炎症反应， 从而证明 TJ-M2010的抗炎效果。
以上实验说明 MyD88途径阻断可降低炎症反应。 因此 MyD88抑制剂可用于治疗多 种慢性炎症性疾病， 如慢性炎症性肠病、 哮喘等。
应用实例 5: MyD88抑制剂用于治疗内毒素血症以及脓毒血症
第一部分:观察 MyD88抑制剂对内毒素血症小鼠死亡率的影响。小鼠随机分为 2组： 溶剂对照组 （Vehicle) 和实验组 （TJ-M2010防治组）， 各 20只。 其中 TJ-M2010防治组 给予 TJ-M2010 ( 0.5%CMC溶剂， 25mg/ml) 灌胃， 剂量为 250mg/kg (200μ 每只）；溶 剂对照组给予 0.5%CMC灌胃， 20(^L每只。 每天一次， 连续灌胃三天。 第三天灌胃后 1 小时后予 LPS腹腔注射， 每十二小时观察存活情况， 观察三天。 观察得到的生存曲线情 况如图 12 (a)
如图所示， 所用的两种 MyD88抑制剂能有效延迟内毒素致死事件， 降低内毒素的 致死率。
第二部分： 观察 MyD88抑制剂对脓毒症小鼠死亡率的影响。 小鼠随机分成:假手术 组、 模型组、 MyD88抑制剂治疗组。 除假手术组只做开腹后缝合以外，对脓毒症模型组 和 MyD88抑制剂治疗组小鼠施行盲肠结扎穿孔术 (CLP)，复制脓毒症小鼠模型。 术后 lh， 分别予以 0.5%CMC20(^L或 TJ-M2010灌胃 250mg/kg 200μ^ ,1次 /12h，连续 4次。术 后每 12h观察各组小鼠生存率一次,连续观察 72h。 所得观察生存率情况如图 12(b)。 如图可见， MyD88抑制剂 TJ-M2010对小鼠脓毒症死亡时间和最终死亡率有比较明 显改善作用。

Claims

权利要求书
1.一类氨基噻唑类 MyD88特异性抑制剂， 作为免疫调节剂， 具有以下分子结构式: TJ-M2010-1
Figure imgf000015_0001
2-(4-(p-甲苯基)哌嗪 -1-基) - N-(4-苯基噻唑 -2-基)乙酰胺 TJ-M2010-3
Figure imgf000015_0002
2-(4-苯基哌嗪 -1-基) -N-(4-苯基噻唑 -2-基)乙酰胺
TJ-M2010-4
Figure imgf000015_0003
3-(4-(4-甲氧苯基)哌嗪 -1-基) -N-(4-苯基噻唑 -2-基)丙酰胺
其特征在于： 一类氨基噻唑类 MyD88特异性抑制剂， 可用于制备免疫抑制剂类药物， 在治疗减低移植后排斥反应以及移植免疫耐受诱导和维持方面的应用， 或用于作为抗炎 药物， 在治疗多种慢性炎症性疾病上的应用， 或用于制备自身免疫治疗药物， 在治疗多 种自身免疫性疾病上的应用， 或用于制备缺血再灌注损伤保护剂， 在治疗缺血再灌注损 伤类疾病上的应用， 或用于制备内毒素血症和脓毒血症防治药物， 用于内毒素血症和脓 毒血症防治上的应用。
PCT/CN2012/070808 2011-03-02 2012-01-31 氨基噻唑类MyD88特异性抑制剂的制药用途 Ceased WO2012116585A1 (zh)

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