WO2018126673A1 - 芍药内酯苷作为吲哚胺2,3-双加氧酶(ido)抑制剂的用途 - Google Patents

芍药内酯苷作为吲哚胺2,3-双加氧酶(ido)抑制剂的用途 Download PDF

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WO2018126673A1
WO2018126673A1 PCT/CN2017/095987 CN2017095987W WO2018126673A1 WO 2018126673 A1 WO2018126673 A1 WO 2018126673A1 CN 2017095987 W CN2017095987 W CN 2017095987W WO 2018126673 A1 WO2018126673 A1 WO 2018126673A1
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disease
paeoniflorin
pharmaceutically acceptable
acceptable salt
ido
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French (fr)
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张作光
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张作光
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Priority to EP17890044.5A priority patent/EP3566707A4/en
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    • 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
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
    • A61K31/7048—Compounds having saccharide radicals and heterocyclic rings having oxygen as a ring hetero atom, e.g. leucoglucosan, hesperidin, erythromycin, nystatin, digitoxin or digoxin
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/125—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives containing carbohydrate syrups; containing sugars; containing sugar alcohols; containing starch hydrolysates
    • 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
    • A61P25/14—Drugs for disorders of the nervous system for treating abnormal movements, e.g. chorea, dyskinesia
    • A61P25/16—Anti-Parkinson drugs
    • 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
    • A61P25/24—Antidepressants
    • 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
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P27/00—Drugs for disorders of the senses
    • A61P27/02—Ophthalmic agents
    • A61P27/12—Ophthalmic agents for cataracts
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Definitions

  • the invention belongs to the field of medicine, relates to the use of a bismuth lactone (Albiflorin), and more particularly to the use of paeoniflorin as a guanamine 2,3-dioxygenase (IDO) inhibitor.
  • a bismuth lactone Albiflorin
  • paeoniflorin paeoniflorin as a guanamine 2,3-dioxygenase (IDO) inhibitor.
  • Indoleamine 2,3-dioxygenase is an enzyme containing heme in the cell. It is the only one outside the liver that can catalyze the metabolism of tryptophan to decompose it along the kynurenine pathway to form quinoline. A rate-limiting enzyme for a range of metabolites, including acids. In recent years, studies have fully proved that IDO overactivation can cause the loss of tryptophan in vivo by degrading tryptophan, which may induce cancer, depression, Alzheimer's disease and other related to tryptophan deficiency. The disease occurs.
  • the increase in IDO activity can inhibit the activation of T cells by degrading tryptophan, thereby inhibiting the proliferation of T cells, thereby mediating tumor cells from escaping the immune system and increasing the probability of cancer in the body.
  • IDO inhibitor 1-methyltryptophan (1-MT) can enhance the sensitivity of tumor cells to immune stimulation of T cells in vitro, and can delay tumor cells in animal models in vivo. Growth enhances the anti-tumor effect of chemotherapeutic drugs and inhibits almost all spontaneous tumors (Friberg M, et al. Int J Cancer, 2002, 101: 151-155.).
  • IDO inhibitors were first used in immunotherapy of tumors. In November 2015, Incyte Pharmaceuticals Inc. disclosed that its combination of the selective IDO inhibitor Epacadostat and Merck's anti-PD-1 monoclonal antibody Keytruda demonstrated good efficacy and safety in an early clinical trial.
  • IDO inhibitors can be used to treat neurological diseases such as Alzheimer's disease, Parkinson's disease, depression, cerebral infarction and the like.
  • IDO inhibitors were developed for the prevention and treatment of IDO overexpression leading to tryptophan deficiency-mediated Alz Neurodegenerative diseases such as Haimo disease have become a new focus of global research and development of AD drugs.
  • IDO is also a new target for anti-depression in the pharmaceutical industry.
  • the pathogenesis of depression is still unknown.
  • Most of the existing antidepressants are developed on the basis of the monoamine hypothesis.
  • the response rate of the drug is only 50% to 60%, and there are different degrees of side effects.
  • IDO chronic hepatitis C
  • HCV chronic hepatitis C
  • PSD depressive disorder
  • TrP tryptophan
  • KYN kynurenine
  • the increased toxic effects of metabolites cause excessive activation of NMDA and the production of free radicals, causing brain neuronal lesions in specific parts of the central nervous system.
  • IDO inhibitors may be more effective than SSRIs such as Baiyoujie for depression caused by inflammation and depression of dysentery associated with somatic disease.
  • Accurate treatment of targeted drug delivery for depression is a direction to improve the efficacy of depression for different causes and different biological targets.
  • Products, food additives or nutritional supplements, and the pathological characteristics of the tryptophan metabolism pathway mediated by IDO include cancer, depression, Alzheimer's disease, Parkinson's disease and the like.
  • the bismuth lactone (Albiflorin) of the present invention is a monoterpenoid compound having a molecular formula of C23H28O11 and a molecular weight of 480.46.
  • the molecular structure is as shown in the formula and is a natural active substance derived from the genus Ranunculaceae Paeonia.
  • the pharmaceutically acceptable salt of the compound of the formula (I) may be selected from the group consisting of citrate, hydrochloride, sulfate, malate, tartrate, citrate And one or more of the phosphates.
  • the present invention discloses that paeoniflorin or a pharmaceutically acceptable salt thereof is an IDO inhibitor; the IDO inhibitor paeoniflorin of the present invention or a pharmaceutically acceptable salt thereof can be used for anti-inflammatory or direct inhibition of IDO.
  • Abnormally mediated neurological diseases such as depression, Alzheimer's disease, Parkinson's disease
  • the IDO inhibitor of the present invention, paeoniflorin or a pharmaceutically acceptable salt thereof can also be used for preventing and treating stroke associated with inflammation Post-stroke depression (PSD) and depression associated with physical diseases such as hepatitis, nephritis, and cancer.
  • PSD Post-stroke depression
  • depression associated with physical diseases such as hepatitis, nephritis, and cancer.
  • the invention provides the use of a paeoniflorin or a pharmaceutically acceptable salt thereof, or an extract comprising a paeoniflorin or a pharmaceutically acceptable salt thereof, as an IDO inhibitor.
  • the present invention also provides a paeoniflorin or a pharmaceutically acceptable salt thereof, or an extract comprising paeoniflorin or a pharmaceutically acceptable salt thereof for preparing a medicament, a food, a health supplement for inhibiting the activity of IDO Use in food additives or nutritional supplements.
  • the present invention also provides a paeoniflorin or a pharmaceutically acceptable salt thereof, or an extract comprising the paeoniflorin or a pharmaceutically acceptable salt thereof for preparing for preventing or treating IDO overactivation-mediated coloration of ammonia Pathological characteristics of the acid metabolism pathway; use in medicines, foods, health products, food additives or nutritional supplements;
  • the disease is selected from one or more of the group consisting of AIDS, cancer, neurodegenerative diseases, depression, cataract, age-related yellowing, and autoimmune diseases;
  • the neurodegenerative disease is selected from one or more of Alzheimer's disease, Huntington's disease, and Parkinson's disease;
  • the medicament is a medicament for immunotherapy for treating cancer.
  • the present invention also provides a paeoniflorin or a pharmaceutically acceptable salt thereof, or an extract comprising paeoniflorin or a pharmaceutically acceptable salt thereof for preparation for preventing or treating inflammatory depression, post-stroke depression Disorder (PSD), depressive disorder coexisting with somatic diseases such as cancer, hepatitis, nephritis, stroke associated with inflammation, Alzheimer's disease associated with inflammation or Parkinson's disease associated with inflammation, food, health products, food additives Or use in nutritional supplements.
  • PSD post-stroke depression Disorder
  • somatic diseases such as cancer, hepatitis, nephritis, stroke associated with inflammation, Alzheimer's disease associated with inflammation or Parkinson's disease associated with inflammation, food, health products, food additives Or use in nutritional supplements.
  • the invention provides a method of inhibiting IDO overactivation, comprising administering a therapeutically effective amount of paeoniflorin or a pharmaceutically acceptable salt thereof, or comprising paeoniflorin or a pharmaceutically acceptable An extract of the accepted salt.
  • the invention also provides a method of preventing or treating a pathologically characterized disease of a tryptophan metabolic pathway mediated by IDO overactivation, the method comprising administering a therapeutically effective amount of a paeoniflorin or a pharmaceutically acceptable thereof a salt, or an extract comprising a paeoniflorin or a pharmaceutically acceptable salt thereof;
  • the disease is selected from the group consisting of AIDS, cancer, neurodegenerative diseases, depression, and white One or more of barriers, age-related yellowing, and autoimmune diseases;
  • the neurodegenerative disease is selected from one or more of Alzheimer's disease, Huntington's disease, and Parkinson's disease;
  • the method is an immunological treatment of cancer.
  • the present invention also provides an Alzheimer's disease for preventing or treating inflammatory depression, post-stroke depression (PSD), comorbidity of diseases such as cancer, hepatitis, nephritis and the like, a stroke associated with inflammation, and associated inflammation.
  • a method of Parkinson's disease associated with inflammation comprising administering a therapeutically effective amount of a paeoniflorin or a pharmaceutically acceptable salt thereof, or an extract comprising a paeoniflorin or a pharmaceutically acceptable salt thereof .
  • the present invention provides a composition for inhibiting IDO, comprising a paeoniflorin or a pharmaceutically acceptable salt thereof, or an extract comprising a paeoniflorin or a pharmaceutically acceptable salt thereof Object
  • the composition is a pharmaceutical, food, health supplement, food additive or nutritional supplement.
  • the present invention also provides a composition for preventing or treating a pathological characteristic disease of an IDO overactivation-mediated tryptophan metabolic pathway, comprising a paeoniflorin or a pharmaceutically acceptable salt thereof, or comprising a peony An extract of a lactone or a pharmaceutically acceptable salt thereof;
  • the composition is a drug, a food, a health supplement, a food additive or a nutritional supplement;
  • the disease is selected from one or more of the group consisting of AIDS, cancer, neurodegenerative diseases, depression, cataract, age-related yellowing, and autoimmune diseases;
  • the neurodegenerative disease is selected from one or more of Alzheimer's disease, Huntington's disease, and Parkinson's disease;
  • composition is for use in immunotherapy for cancer.
  • the invention also provides a depressive disorder for preventing or treating inflammatory depression, post-stroke depression (PSD), comorbidity with physical diseases such as cancer, hepatitis and nephritis, a stroke associated with inflammation, and Alz with associated inflammation.
  • a composition of Parkinson's disease or a inflammatory associated Parkinson's disease comprising a paeoniflorin or a pharmaceutically acceptable salt thereof, or an extract comprising a paeoniflorin or a pharmaceutically acceptable salt thereof.
  • the present invention provides a paeoniflorin or a pharmaceutically acceptable salt thereof for use as an IDO inhibitor, or an extract comprising paeoniflorin or a pharmaceutically acceptable salt thereof.
  • the present invention also provides a paeoniflorin or a pharmaceutically acceptable salt thereof for preventing or treating a pathological characteristic disease of a tryptophan metabolic pathway mediated by IDO overactivation, or comprising a paeoniflorin or a pharmaceutically acceptable thereof An extract of an acceptable salt;
  • the disease is selected from one or more of the group consisting of AIDS, cancer, neurodegenerative diseases, depression, cataract, age-related yellowing, and autoimmune diseases;
  • the neurodegenerative disease is selected from the group consisting of Alzheimer's disease, Huntington's disease, and Parkinson's disease.
  • the diseases One or more of the diseases;
  • the paeoniflorin or a pharmaceutically acceptable salt thereof, or an extract comprising paeoniflorin or a pharmaceutically acceptable salt thereof is used for immunological treatment of cancer.
  • the present invention also provides an Alzheimer's disease for preventing or treating inflammatory depression, post-stroke depression (PSD), comorbidity of diseases such as cancer, hepatitis, nephritis and the like, a stroke associated with inflammation, and associated inflammation. Or a pharmaceutically acceptable salt of Parkinson's disease or a pharmaceutically acceptable salt thereof, or an extract comprising a paeoniflorin or a pharmaceutically acceptable salt thereof.
  • PSD post-stroke depression
  • comorbidity of diseases such as cancer, hepatitis, nephritis and the like
  • a stroke associated with inflammation and associated inflammation.
  • a pharmaceutically acceptable salt of Parkinson's disease or a pharmaceutically acceptable salt thereof, or an extract comprising a paeoniflorin or a pharmaceutically acceptable salt thereof or an extract comprising a paeoniflorin or a pharmaceutically acceptable salt thereof.
  • paeoniflorin and the positive drug fluoxetine are antidepressant, their mechanism of action against depression is different: paeoniflorin is an IDO inhibitor by lowering plasma and hippocampus uric acid/ The ratio of tryptophan (KYN/TrP) inhibits overexpression of IDO to antidepressant; the positive drug fluoxetine is not an IDO inhibitor, not anti-depression by inhibiting IDO and regulating the tryptophan metabolic pathway. Sustained immune response is an important cause of long-term depression and recurrence of depression.
  • the paeoniflorin of the present invention is used as an IDO inhibitor in the treatment of inflammatory depression mediated by IDO overactivation, depression in patients with inflammation associated with depression, post-stroke depression (PSD), hepatitis and nephritis.
  • Depressive disorders of the disease and depressive disorders comorbid with cancer are more effective than SSRIs such as flurazepam.
  • the IDO inhibitor paeoniflorin of the present invention can be safer and more effective for the pathologically characterized diseases of the IDO overactivation-mediated tryptophan metabolism pathway:
  • the IDO inhibitor paeoniflorin of the present invention enhances the immune function of T cells by inhibiting the overactivation of IDO in the body, and prevents tumor cells from escaping the immune system to monitor and kill them, and can be used for preparing anticancer drugs for immunotherapy. .
  • the IDO inhibitor paeoniflorin of the present invention can prevent and alleviate the production and development of Alzheimer's disease by inhibiting the overactivation of IDO in the body to reduce the production of tryptophan and quinolinic acid. Preparation of a new mechanism of action for anti-aging and treatment of neurodegenerative diseases such as Alzheimer's disease.
  • the IDO inhibitor paeoniflorin of the present invention is more suitable for preparing a precise treatment for depressive disorder caused by IDO overactivation-induced depression, post-stroke depression (PSD), and cancer-like nephritis cancer. A generation of antidepressants.
  • the IDO inhibitor paeoniflorin of the present invention is a monomeric natural medicine, although it is chemically inhibited by IDO.
  • the formulation has a similar binding mode, but the difference is that the synthetic chemical inhibitor is the combination of the N atom and the iron ion of the IDO, and the paeoniflorin is the combination of the O atom and the iron ion of the IDO. This different structure determines The IDO natural inhibitor paeoniflorin of the present invention has higher safety.
  • Figure 1 is a graph showing the effects of paeoniflorin on IDO and melatonin receptor (MT1) in hippocampus of chronically stressed rats.
  • Figure 2 is a diagram showing the route of serotonin-melatonin metabolism by guanidine uric acid-quinolinate down-regulated by guanidine uric acid-quinoline in the present invention.
  • A is the immobility time of the mouse forced swimming experiment
  • B is the immobility time of the mouse tail suspension experiment
  • the figure shows the total distance of activity in the open field experiment of mice
  • D is the number of uprights in the open field experiment of mice.
  • Figure 4 is a graph showing the effect of paeoniflorin on the content of inflammatory factors in plasma of mice after LPS modeling:
  • Figure A shows the content of IL-6 in mouse plasma; and
  • Figure B shows the content of TNF- ⁇ in mouse plasma.
  • C is the content of CORT in mouse plasma;
  • D is the content of PGE 2 in mouse plasma.
  • Figure 5 is a graph showing the effects of paeoniflorin on the content of inflammatory factors in hippocampus of mice after LPS modeling:
  • Figure A shows the content of IL-6 in mouse hippocampus; and
  • Figure B shows TNF- ⁇ in mouse hippocampus
  • the content of C is the content of CORT in mouse hippocampus;
  • D is the content of PGE 2 in mouse hippocampus.
  • Figure 6 is a graph showing the effect of paeoniflorin on the ratio of kynuric acid (KYN) and tryptophan (TrP) in hippocampal tissue of LPS modeled mice and its effect on IDO activity.
  • FIG. 7 is a result of docking experiments of the paeoniflorin and IDO1 protein molecules of the present invention using MOE2016 software: the first stage docking result of FIG. A; the binding position of the drug lactone glycoside in IDO1; FIG. The binding mode of heme in IDO1; the binding mode of the drug D-lactone and IDO1 active pocket in Figure D; the calculation result of the binding free energy of the drug and the IDO1 in Figure E; the dynamic energy potential curve in Figure F; It is the binding mode of IDO1 with some synthetic inhibitors.
  • the IDO inhibitor paeoniflorin of the present invention induces IDO in a chronic stress rat model (CUMS) Overexpression and effects of depression-like behavior
  • mice were randomly divided into 6 groups (16 in each group): blank group, model group, positive drug fluoxetine hydrochloride group (10 mg/kg), paeoniflorin low dose group (3.5 mg/kg), medium dose The group (7 mg/kg) and the high dose group (14 mg/kg) were administered by continuous intragastric administration for 7 days.
  • LPS (0.83 mg/kg) was intraperitoneally injected, and after 16 hours of injection, an open field test (OFT) was performed; after 24 hours of injection, forced swimming (FST) and tail suspension test (TST) were performed; The eyeballs were taken from the eyeballs, the plasma was taken by centrifugation, and the hippocampus tissues of the mice were taken after sacrifice. The liquid nitrogen was frozen and stored at -80 °C.
  • the experimental data were expressed as mean ⁇ standard deviation, and the data was statistically analyzed and plotted using Origin Pro 8.0 software. Comparisons between groups were made using the One-Way ANOVA method, with P ⁇ 0.05 and P ⁇ 0.01 as significant differences.
  • mice showed a depression-like state after intraperitoneal injection of LPS.
  • Each administration group can alleviate the depression-like state induced by LPS in mice, and reduce the immobility time in forced swimming and tail suspension test.
  • the forced swimming and tail suspension test in the high dose group of paeoniflorin group had significant difference with the model group, see Figure 3.
  • the experimental results showed that the plasma levels of inflammatory factors IL-6, TNF- ⁇ and glucocorticoid CORT were significantly increased in the model group compared with the blank control group, indicating that the mice developed an inflammatory response after intraperitoneal injection of LPS.
  • the medium and high doses of paeoniflorin can significantly reduce the levels of inflammatory cytokines IL-6 and TNF- ⁇ in mouse plasma, and alleviate the inflammatory response induced by LPS, indicating that paeoniflorin has a good anti-inflammatory effect;
  • paeoniflorin can significantly reduce the concentration of CORT in mouse plasma, with the function of reverse-adjusted HPA axis, see Figure 4.
  • the levels of inflammatory factors IL-6, TNF- ⁇ , PGE2 and CORT in mouse hippocampus were detected by ELISA.
  • the results showed that the levels of inflammatory factors and CORT in the hippocampus of the model group were significantly increased compared with the blank control group, indicating that the concentration of inflammatory factors in the brain tissue of mice increased after intraperitoneal injection of LPS, and the HPA axis was hyperactive and dysfunctional.
  • the activity of hippocampal IDO is expressed by the ratio of KYN and TrP and the ratio of KYN/TrP.
  • LPS induced an inflammatory response in mice, which resulted in a significant increase in the ratio of kynuric acid/tryptophan (KYN/TrP) in the hippocampus of the model group compared with the blank control group, indicating overexpression of IDO in the hippocampus.
  • the positive drug fluoxetine administration group failed to correct the activation of hippocampal IDO induced by LPS, while the high dose of paeoniflorin reduced the ratio of kynuric acid/tryptophan (KYN/TrP).
  • P ⁇ 0.05 the activity of IDO was significantly inhibited, see Figure 6.
  • paeoniflorin is an IDO inhibitor that reduces plasma and hippocampus uric acid/tryptophan The ratio of (KYN/TrP) inhibits overexpression of IDO against depression; the positive drug fluoxetine is not an IDO inhibitor, not anti-depression by inhibiting IDO and regulating the tryptophan metabolic pathway. Sustained immune response is an important cause of long-term depression and recurrence of depression.
  • Paeoniflorin, an IDO inhibitor may be more effective than SSRI-type drugs in the treatment of IDO-mediated depression and depression associated with inflammatory somatic disease, allowing for precise treatment of IDO-mediated depression. .
  • the invention finds and proves that paeoniflorin is an IDO inhibitor through the implementation of case 1 (CUMS experiment), behavioral study of case 2 (LPS experiment), targeted metabolomics research and biological target detection.
  • the IDO inhibitor of the present invention inhibits the up-regulation of IDO in blood and hippocampus induced by stress and inflammation, reduces the production of tryptophan to kynurenic acid and quinolinic acid, and promotes tryptophan through serum.
  • the ubiquitin pathway produces 5-HT and melatonin, which can be prepared as an antidepressant for the prevention or treatment of depression, especially for the treatment of inflammatory depression and with various infectious, non-infectious inflammatory somatic diseases. Depressive disorder of the disease.
  • the IDO inhibitor paeoniflorin of the present invention enhances the immune function of T cells by inhibiting the overactivation of IDO in the body, and prevents tumor cells from escaping the immune system to monitor and kill them, and can be prepared for tumor immunotherapy. Anticancer drugs.
  • the IDO inhibitor paeoniflorin of the present invention prevents and reduces the formation and development of Alzheimer's disease by inhibiting the overactivation of IDO in the body and reducing the production of tryptophan to kynurenic acid and quinolinic acid. It is prepared as a drug for anti-aging and for treating neurodegenerative diseases such as Alzheimer's disease.
  • paeoniflorin or a pharmaceutically acceptable salt thereof, an extract containing paeoniflorin, and a composition can be developed to treat, prevent, modulate and ameliorate mediated by IDO. Tryptophan metabolic pathway dysfunction Pathological characteristics of a disease or sub-health state of drugs, foods, health products, food additives or nutritional supplements.
  • Example 3 Using MOE2016 software to dock the IDO1 inhibitor paeoniflorin and IDO1 protein molecules of the present invention
  • IDO protein there are 20 crystal structures of IDO protein, which are determined by many factors such as biological origin, resolution, and small molecular structure of ligand.
  • the molecular structure of IDO1 protein is determined by the crystal structure of number 4PK5.
  • the original ligand of the protein was used as a template to investigate the fitting mode of paeoniflorin and ligand, and more than 20 fitting results were obtained.
  • fitting results are reviewed one by one, and the fitting results are selected to be optimized to produce the first stage docking result, as shown in Fig. 7A.
  • the MOE software Dock module is used for secondary automatic docking to obtain the second batch of docking results, and then the reasonable results are screened.
  • the secondary results were structurally optimized, and the stability of the docking results was tested using a molecular dynamics simulation method with a simulation time of 6 ns.
  • the binding mode of paeoniflorin to IDO1 was finally determined, and the binding free energy was calculated.
  • Paeoniflorin binds to the core region of IDO1, which is adjacent to the heme structure (consisting of iron ions and protoporphyrin).
  • the receptor active pocket is composed of several alpha helix structures with hemene on the inside and a relatively large inlet towards the environment outside the protein, see Figure 7B.
  • Paeoniflorin forms a chelation system with protoporphyrin and histidine His346, encapsulating the intermediate iron ions. Iron ions are the key factor for the catalytic reaction of IDO1. The combination of paeoniflorin and iron ions successfully occupied the active pocket, resulting in competitive inhibition, see Figure 7C.
  • paeoniflorin In addition to binding to iron ions, the other two parts of paeoniflorin also match the polarity of the receptor pocket. In summary, paeoniflorin satisfies the conditions of the IDO1 inhibitor. The combination mode we obtained is also reasonable, see Figure 7D.
  • IDO1 chemical inhibitors that have been reported so far are combined in the same pattern, see Figure 7G. The difference is that they are all bound to iron ions by N atoms, and the paeoniflorin is bound by oxygen atoms. Oxygen atoms have a lower binding free energy than nitrogen atoms, so the paeoniflorin may have a relatively low binding strength and a weak inhibitory activity compared to these chemical inhibitors.

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Abstract

芍药内酯苷或其药学上可接受的盐作为IDO抑制剂的用途，芍药内酯苷或其药学上可接受的盐作为IDO抑制剂，可用于制备预防和治疗由IDO过活化介导的色氨酸代谢途径的病理学特征疾病的药物、食品、保健品、食品添加剂或营养补充剂，这些疾病选自癌症、抑郁症、阿尔茨海默症和帕金森症中的一种或多种。

Description

芍药内酯苷作为吲哚胺2,3-双加氧酶(IDO)抑制剂的用途 技术领域
本发明属于医药领域，涉及一种芍药内酯苷(Albiflorin)的用途，更具体涉及芍药内酯苷作为吲哚胺2,3-双加氧酶(IDO)抑制剂的用途。
背景技术
吲哚胺2,3-双加氧酶(IDO)是一种细胞内含有亚铁血红素的酶，是肝脏以外唯一可以催化色氨酸代谢使其沿犬尿氨酸途径分解生成包括喹啉酸在内的一系列代谢产物的限速酶。近年来研究已充分证明：IDO过活化通过降解色氨酸造成体内色氨酸缺失的微环境，进而可能会诱导癌症、抑郁症、阿尔茨海默症等多种与色氨酸缺失密切相关的疾病发生。
具体地，IDO活性的提高，可通过降解色氨酸切断T细胞的活化，使T细胞的增殖受到抑制，从而介导肿瘤细胞逃避机体免疫系统的攻击，增加了机体发生癌症的几率。已有研究表明，目前公认的IDO抑制剂1-甲基色氨酸(1-MT)在体外能增强肿瘤细胞对T细胞的免疫刺激的敏感性，在体内的动物模型中能延缓肿瘤细胞的生长，增强化疗药物的抗肿瘤效果，而且对几乎所有的自发性肿瘤起抑制作用(Friberg M，et al.Int J Cancer，2002，101：151-155.)。IDO抑制剂首先被用于肿瘤的免疫治疗。2015年11月，美国Incyte制药公司披露，其研发的选择性IDO抑制剂Epacadostat和默沙东公司抗PD-1单抗Keytruda联合使用在一个早期的临床试验中，显示了良好的疗效和安全性。
IDO的过活化，会影响色氨酸的正常代谢，诱发色氨酸代谢通路犬尿胺酸异常增多，喹啉酸水平的上调，研究证明该通路代谢异常与神经系统炎症和神经退行性疾病的发生密切相关，因此，IDO抑制剂可以用于治疗神经系统疾病，例如阿尔茨海默病、帕金森病症、抑郁症、脑梗塞等。在以Aβ-淀粉样蛋白为靶点研发的治疗AD药物折戟沉沙全面失败的背景下，研发IDO抑制剂，将其用于预防与治疗IDO过表达致使色氨酸匮乏介导的阿尔茨海默症等神经退行性疾病，已成为全球研发AD药物新的关注点。
IDO的过活化，会影响色氨酸的正常代谢，促使血清素的合成代谢途径受到抑制，从而可能诱发抑郁症，因此，IDO也是医药界当下重点关注的抗抑郁新靶点。抑郁症发病机制至今不明，现有的抗抑郁药大多是在单胺假说的基础上研发的，用药的反应率只是50％～60％，且都有不同程度的副作用， 据统计“连续接受6个月抗抑郁剂治疗的病人不到25％，有相当大的比例是因为性与睡眠的副作用而停用”(安德鲁.所罗门《忧郁》P085)由于存在着大量未被满足的医疗需求，人们一直在努力寻找新生物靶点、新作用机制的抗抑郁药物，其中IDO抑制剂是近年来全球医药界关注的重点。
临床慢性炎症反应中IDO的激活与抑郁障碍的发生密切相关，例如用IFN-α治疗的慢性丙型肝炎(HCV)患者，在接受治疗期间抑郁症的发生率可高达25％至33％；伴生炎症的脑卒中患者，卒中后一年内抑郁障碍(PSD)累积发病率为41.8％。关于炎症促使IDO活化导致抑郁障碍的作用机制有两种假说：一是色氨酸(TrP)的衰竭，造成5-HT能神经元功能障碍；二是犬尿胺酸(KYN)的神经元活化代谢物增加的毒性作用，造成NMDA过度激活与自由基的产生，引起中枢特定部位脑神经元病变。对于炎症致使IDO过活化诱导的抑郁症和与躯体病共病抑郁障碍，IDO抑制剂可能比百优解等SSRIs药物更有疗效。针对不同病因和不同的生物靶点，对抑郁症进行靶向给药的精准治疗，是提高抑郁症疗效的一个方向。
发明内容
本发明的目的是提供一种芍药内酯苷(Albiflorin)的新用途，更具体涉及芍药内酯苷作为吲哚胺2,3-双加氧酶(IDO)抑制剂的用途；本发明还提供了芍药内酯苷作为吲哚胺2,3-双加氧酶(IDO)抑制剂，可用于制备预防或治疗由IDO介导的色氨酸代谢途径的病理学特征疾病的药物、食品、保健品、食品添加剂或营养补充剂，而由IDO介导的色氨酸代谢途径的病理学特征疾病包括癌症、抑郁症、阿尔茨海默症、帕金森症等疾病。
本发明所述的芍药内酯苷(Albiflorin)为单萜类化合物，其分子式为C23H28O11，分子量为480.46，分子结构如式所示，是一种天然活性物质，来源于毛莨科植物白芍Paeonia lactiflora Pall或川赤芍Paeonia veitchii Lynch的根、牡丹P.suffrsticosa Andrz的根。
Figure PCTCN2017095987-appb-000001
根据本发明提供的用途，其中，式(I)所示化合物的药学上可接受的盐可以选自枸橼酸盐、盐酸盐、硫酸盐、苹果酸盐、酒石酸盐、柠檬酸盐 和磷酸盐中的一种或多种。
本发明披露了芍药内酯苷或其药学上可接受的盐是IDO抑制剂；本发明IDO抑制剂芍药内酯苷或其药学上可接受的盐，可以通过抗炎或是直接抑制IDO的过表达，预防或治疗由IDO介导的色氨酸代谢途径的病理学特征疾病；这类疾病包括因IDO过活化致使免疫系统抑制而诱导的癌症，包括因IDO过活化诱使色氨酸代谢通路异常介导的抑郁症、阿尔茨海默症、帕金森症等神经系统疾病；本发明IDO抑制剂芍药内酯苷或其药学上可接受的盐，还可用于预防和治疗伴生炎症的脑卒中、卒中后抑郁障碍(PSD)及与肝炎、肾炎、癌症等躯体病共病的抑郁障碍。
一方面，本发明提供了芍药内酯苷或其药学上可接受的盐、或者包含芍药内酯苷或其药学上可接受的盐的提取物作为IDO抑制剂的用途。
本发明还提供了芍药内酯苷或其药学上可接受的盐、或者包含芍药内酯苷或其药学上可接受的盐的提取物在制备用于抑制IDO的活性的药物、食品、保健品、食品添加剂或营养补充剂中的用途。
本发明还提供了芍药内酯苷或其药学上可接受的盐、或者包含芍药内酯苷或其药学上可接受的盐的提取物在制备用于预防或治疗IDO过活化介导的色氨酸代谢途径的病理学特征疾病的药物、食品、保健品、食品添加剂或营养补充剂中的用途；
优选地，所述疾病选自AIDS、癌症、神经退行性疾病、抑郁症、白内障、与年龄相关的黄化以及自身免疫性疾病中的一种或多种；
更优选地，所述神经退行性疾病选自阿尔茨海默病、亨廷顿病和帕金森病中的一种或多种；
进一步优选地，所述药物为免疫法治疗癌症使用的药物。
本发明还提供了芍药内酯苷或其药学上可接受的盐、或者包含芍药内酯苷或其药学上可接受的盐的提取物在制备用于预防或治疗炎性抑郁症，卒中后抑郁障碍(PSD)，与癌症、肝炎、肾炎等躯体病共病的抑郁障碍，伴生炎症的中风，伴生炎症的阿尔茨海默症或伴生炎症的帕金森症的药物、食品、保健品、食品添加剂或营养补充剂中的用途。
另一方面，本发明提供了一种抑制IDO过活化的方法，所述方法包括给予治疗有效量的芍药内酯苷或其药学上可接受的盐、或者包含芍药内酯苷或其药学上可接受的盐的提取物。
本发明还提供了一种预防或治疗由IDO过活化介导的色氨酸代谢途径的病理学特征疾病的方法，所述方法包括给予治疗有效量的芍药内酯苷或其药学上可接受的盐、或者包含芍药内酯苷或其药学上可接受的盐的提取物；
优选地，所述疾病选自AIDS、癌症、神经退行性疾病、抑郁症、白内 障、与年龄相关的黄化以及自身免疫性疾病中的一种或多种；
更优选地，所述神经退行性疾病选自阿尔茨海默病、亨廷顿病和帕金森病中的一种或多种；
进一步优选地，所述方法为免疫法治疗癌症。
本发明还提供了一种预防或治疗炎性抑郁症，卒中后抑郁障碍(PSD)，与癌症、肝炎、肾炎等躯体病共病的抑郁障碍，伴生炎症的中风，伴生炎症的阿尔茨海默症或伴生炎症的帕金森症的方法，所述方法包括予治疗有效量的芍药内酯苷或其药学上可接受的盐、或者包含芍药内酯苷或其药学上可接受的盐的提取物。
再一方面，本发明还提供了一种用于抑制IDO的组合物，其包括芍药内酯苷或其药学上可接受的盐、或者包含芍药内酯苷或其药学上可接受的盐的提取物；
优选地，所述组合物为药物、食品、保健品、食品添加剂或营养补充剂。
本发明还提供了一种用于预防或治疗IDO过活化介导的色氨酸代谢途径的病理学特征疾病的组合物，其包括芍药内酯苷或其药学上可接受的盐、或者包含芍药内酯苷或其药学上可接受的盐的提取物；
优选地，所述组合物为药物、食品、保健品、食品添加剂或营养补充剂；
优选地，所述疾病选自AIDS、癌症、神经退行性疾病、抑郁症、白内障、与年龄相关的黄化以及自身免疫性疾病中的一种或多种；
更优选地，所述神经退行性疾病选自阿尔茨海默病、亨廷顿病和帕金森病中的一种或多种；
进一步优选地，所述组合物用于免疫法治疗癌症。
本发明还提供了一种用于预防或治疗炎性抑郁症，卒中后抑郁障碍(PSD)，与癌症、肝炎、肾炎等躯体病共病的抑郁障碍，伴生炎症的中风，伴生炎症的阿尔茨海默症或伴生炎症的帕金森症的组合物，其包括芍药内酯苷或其药学上可接受的盐、或者包含芍药内酯苷或其药学上可接受的盐的提取物。
再另一方面，本发明还提供了用作IDO抑制剂的芍药内酯苷或其药学上可接受的盐、或者包含芍药内酯苷或其药学上可接受的盐的提取物。
本发明还提供了用于预防或治疗IDO过活化介导的色氨酸代谢途径的病理学特征疾病的芍药内酯苷或其药学上可接受的盐、或者包含芍药内酯苷或其药学上可接受的盐的提取物；
优选地，所述疾病选自AIDS、癌症、神经退行性疾病、抑郁症、白内障、与年龄相关的黄化以及自身免疫性疾病中的一种或多种；
更优选地，所述神经退行性疾病选自阿尔茨海默病、亨廷顿病和帕金森 病中的一种或多种；
进一步优选地，所述芍药内酯苷或其药学上可接受的盐、或者包含芍药内酯苷或其药学上可接受的盐的提取物用于免疫法治疗癌症。
本发明还提供了用于预防或治疗炎性抑郁症，卒中后抑郁障碍(PSD)，与癌症、肝炎、肾炎等躯体病共病的抑郁障碍，伴生炎症的中风，伴生炎症的阿尔茨海默症或伴生炎症的帕金森症的芍药内酯苷或其药学上可接受的盐、或者包含芍药内酯苷或其药学上可接受的盐的提取物。
本发明在研究慢性应激大鼠模型(CUMS)和脂多糖(LPS)诱导的小鼠炎症抑郁样行为中，发现慢性应激和LPS诱发的炎症均可使IDO过表达，而本申请的芍药内酯苷或其药学上可接受的盐可以作为IDO抑制剂，通过抗炎和直接抑制IDO的过表达，进而降低犬尿酸/色氨酸的比率，因此可以作为IDO抑制剂，用于治疗由IDO过活化介导的色氨酸代谢途径的病理学特征疾病。
进一步地，本发明发现，虽然芍药内酯苷和阳性药氟西汀都抗抑郁，但它们抗抑郁的作用机制不同：芍药内酯苷是IDO抑制剂,系通过降低血浆和海马区犬尿酸/色氨酸(KYN/TrP)的比率抑制IDO的过表达抗抑郁；阳性药氟西汀不是IDO抑制剂，不是通过抑制IDO和调节色氨酸代谢通路抗抑郁。持续的免疫性反应是导致抑郁症久治不愈和复发的重要原因。而本发明的芍药内酯苷作为IDO抑制剂，在治疗IDO过活化介导的炎性抑郁症，伴生炎症的脑卒中患者抑郁症，卒中后抑郁障碍(PSD)、肝炎和肾炎等躯体病共病的抑郁障碍及与癌症共病的抑郁障碍，比氟西酊等SSRIs药物疗效更佳。
与现有技术相比，本发明IDO抑制剂芍药内酯苷可以针对IDO过活化介导的色氨酸代谢途径的病理学特征疾病进行更加安全有效的精准治疗：
1、本发明IDO抑制剂芍药内酯苷，通过抑制机体内IDO的过活化，增强T细胞的免疫功能，阻止肿瘤细胞逃逸机体免疫系统对其监视和杀伤，可用其制备免疫疗法的抗癌药物。
2、本发明IDO抑制剂芍药内酯苷，通过抑制机体内IDO的过活化减少色氨酸向犬尿胺酸以及喹啉酸的生成，预防和缓解老年痴呆症的生成与发展，可将其制备为新作用机制的抗衰老和治疗阿尔茨海默病等神经退行性疾病的药物。
3、本发明IDO抑制剂芍药内酯苷，更适宜制备对IDO过活化诱导的抑郁症、卒中后抑郁障碍(PSD)、与癌症肝炎肾炎癌症等躯体病共病的抑郁障碍进行精准治疗的新一代抗抑郁药物。
4、本发明IDO抑制剂芍药内酯苷，是单体天然药物，虽与IDO化学抑 制剂相比结合模式相似，但不同之处在于人工合成的化学抑制剂是N原子与IDO的铁离子结合，而芍药内酯苷是以O原子与IDO的铁离子结合，这种不同结构决定了本发明IDO天然抑制剂芍药内酯苷具有更高的安全性。
附图的简要说明
图1为本发明的芍药内酯苷对慢性应激大鼠海马组织IDO、褪黑素受体(MT1)的影响。
图2为本发明的芍药内酯苷通过抑制IDO下调犬尿酸-喹啉酸上调血清素-褪黑素代谢途径的路线图。
图3为本发明的芍药内酯苷对LPS造模后小鼠行为学实验的影响：A图为小鼠强迫游泳实验的不动时间；B图为小鼠悬尾实验的不动时间；C图为小鼠开放场实验中活动的总距离；D图为小鼠开放场实验中的直立次数。
图4为本发明的芍药内酯苷对LPS造模后小鼠血浆中炎症因子含量的影响：A图为小鼠血浆中IL-6的含量；B图为小鼠血浆中TNF-α的含量；C图为小鼠血浆中CORT的含量；D图为小鼠血浆中PGE2的含量。
图5为本发明的芍药内酯苷对LPS造模后小鼠海马组织炎症因子含量的影响：A图为小鼠海马组织中IL-6的含量；B图为小鼠海马组织中TNF-α的含量；C图为小鼠海马组织中CORT的含量；D图为小鼠海马组织中PGE2的含量。
图6为本发明的芍药内酯苷对LPS造模小鼠海马组织中犬尿酸(KYN)和色氨酸(TrP)含量比率及对IDO活性的影响。
图7为使用MOE2016软件对本发明的芍药内酯苷与IDO1蛋白分子对接实验的结果：图A第一阶段对接结果；图B芍药内酯苷在IDO1中的结合位置；图C芍药内酯苷与IDO1中亚铁血红素的结合模式；图D芍药内酯苷与IDO1活性口袋的结合模式；图E芍药内酯苷与IDO1结合自由能的计算结果；图F动力学模拟势能曲线图；图G为IDO1与一些人工合成抑制剂的结合模式。
实施发明的最佳方式
下面结合具体实施案例，进一步阐释本发明。下述实施案例仅限于说明本发明在治疗抑郁症和提高认知功能方面的具体应用，不是用于限制本发明的应用范围。
实施例一
本发明IDO抑制剂芍药内酯苷对慢性应激大鼠模型(CUMS)诱导的IDO 过表达和抑郁样行为的影响
1、在慢性不可预见性温和应激模型(CUMS)大鼠的海马组织中，IDO的分泌明显升高，褪黑素受体1(MT1)明显下降。芍药内酯苷给药7天后(7mg/天、14mg/天)，大鼠海马中的IDO活性受到明显抑制(P<0.01)，大鼠海马中褪黑素受体1(MT1)明显升高(P<0.01)，结果见下表，用蛋白质印迹法(Western Blot)检测芍药内酯苷对IDO及MTI的影响结果见附图1：
芍药内酯苷对大鼠海马组织吲哚胺-2,3-双加氧酶(IDO)
褪黑素受体1(MT1)的影响(Mean±SE)
Figure PCTCN2017095987-appb-000002
(#表示与模型组比较升高P<0.05，##表示与模型组比较升高P<0.01，**表示与模型组比较降低P<0.01)
2、慢性不可预见性温和应激模型(CUMS)大鼠的海马组织中血清素的合成代谢途径受到抑制，而犬尿胺酸和喹啉酸代谢途径上调(P<0.01)。芍药内酯苷给药7天后(7mg/kg、14mg/kg)，大鼠海马中的犬尿胺酸和喹啉酸的含量明显降低(P<0.01)，表明犬尿酸代谢途径下调；海马中的血清素、褪黑素的含量明显增加(P<0.01)，表明血清素、褪黑素代谢途径上调；犬尿胺酸与色氨酸的比率(KYN/TrP)明显下调，表明IDO活性受到抑制，见附图2。
实施例二
本发明IDO抑制剂芍药内酯苷对LPS诱导的IDO活化、小鼠炎症抑郁样行为及认知功能障碍的影响
将小鼠随机分为6组(每组16只)：空白组、模型组、阳性药盐酸氟西汀组(10mg/kg)、芍药内酯苷低剂量组(3.5mg/kg)、中剂量组(7mg/kg)、高剂量组(14mg/kg)，连续灌胃给药7天。
一、实验及检测内容
1、行为学实验：强迫游泳(FST)、悬尾(TST)和开放场测试(OFT)
第7天给药后，腹腔注射LPS(0.83mg/kg)造模，注射16h后，进行开放场测试(OFT)；注射24h后，进行强迫游泳(FST)和悬尾实验(TST)；之后，摘眼球取血，离心取血浆，处死后取小鼠海马组织，液氮速冻，-80℃保存。
2、测定芍药内酯苷、阳性药氟西汀对小鼠血浆、海马组织炎性因子IL-6、TNF-α、PGE2和糖皮质激素CORT含量浓度的影响。
3、测定芍药内酯苷、氟西汀对小鼠海马组织IDO表达的影响。
二、统计学分析
实验数据以平均值±标准偏差表示，使用Origin Pro8.0软件对数据进行统计分析并做图。使用单因素方差分析(One-Way ANOVA)法进行组间比较，以P<0.05和P<0.01为具有显著性差异。
三、实验结果
1、LPS造模后小鼠行为学实验
行为学实验结果：小鼠腹腔注射LPS造模后出现似抑郁状态，各给药组均能不同程度缓解小鼠由LPS诱导的似抑郁状态，减少强迫游泳和悬尾试验中的不动时间，其中芍药内酯苷高剂量给药组小鼠强迫游泳和悬尾试验不动时间与模型组比较有显著性差异，见附图3。
2、小鼠血浆中炎症因子的含量
实验检测结果显示：模型组小鼠与空白对照组比较，血浆中炎症因子IL-6、TNF-α和糖皮质激素CORT的含量显著增加，说明腹腔注射LPS后小鼠体内产生炎症反应，HPA轴亢进。芍药内酯苷中、高剂量给药组能够显著降低小鼠血浆中炎症因子IL-6和TNF-α的含量，缓解LPS诱导产生的炎症反应，说明芍药内酯苷具有良好的抗炎作用；同时芍药内酯苷能够显著降低小鼠血浆中CORT的浓度，具有反向调节亢进的HPA轴的功能，见附图4。
3、小鼠海马组织炎症因子的含量
用ELISA检测小鼠海马组织中炎症因子IL-6、TNF-α、PGE2以及CORT的含量。结果表明，模型组小鼠海马组织中炎症因子和CORT的含量与空白对照组比较有显著的增加，说明腹腔注射LPS后小鼠脑组织炎症因子浓度增加，同时HPA轴的活动亢进，功能紊乱。芍药内酯苷高剂量组小鼠海马组织中炎症因子IL-6和TNF-α的含量与模型组比较明显降低，提示芍药内酯苷具有抗炎作用；海马组织中CORT含量显著降低，提示芍药内酯苷能够反向调节亢进的HPA轴，见附图5。
4、小鼠海马组织中犬尿酸(KYN)/色氨酸(TrP)的比率
海马IDO的活性通过KYN和TrP含量以及KYN/TrP的比率来表达。LPS诱导小鼠炎症反应，使得模型组小鼠海马组织中犬尿酸/色氨酸(KYN/TrP)的比率较空白对照组显著增加，表明海马中IDO的过表达。与模型组比，阳性药氟西汀给药组，没能校正LPS诱导的海马IDO的活化，而芍药内酯苷高剂量组则降低了犬尿酸/色氨酸(KYN/TrP)的比率(P<0.05)，明显抑制了IDO的活性，见附图6。
本研究通过检测LPS小鼠海马组织中KYN/TrP的比率，发现高剂量芍药内酯苷(14mg/kg)能够降低由LPS诱导炎症反应造成的小鼠海马组织中IDO的活性，而阳性药氟西汀给药组(10mg/kg)未能校正LPS诱导的IDO的过表达。
实验结果提示，虽然芍药内酯苷和阳性药氟西汀都抗抑郁，但它们抗抑郁的作用机制不同：芍药内酯苷是IDO抑制剂，系通过降低血浆和海马区犬尿酸/色氨酸(KYN/TrP)的比率抑制IDO的过表达抗抑郁；阳性药氟西汀不是IDO抑制剂，不是通过抑制IDO和调节色氨酸代谢通路抗抑郁。持续的免疫性反应是导致抑郁症久治不愈和复发的重要原因。芍药内酯苷作为IDO抑制剂，在治疗IDO介导的抑郁症和与炎症躯体病共病的抑郁障碍上，可能比SSRI类型的药物更有疗效，可以对IDO介导的抑郁症进行精准治疗。
对实施案例一和实施例二两个案例的分析
本发明通过实施案例一(CUMS实验)、实施案例二(LPS实验)的行为学研究、靶向代谢组学研究和生物靶点检测，发现并证明芍药内酯苷是IDO抑制剂。
1、本发明IDO抑制剂芍药内酯苷，通过抑制应激和炎症诱导引起的血液、海马中IDO上调，减少色氨酸向犬尿胺酸以及喹啉酸的生成，促进色氨酸经过血清素途径生成5-HT和褪黑素，可将其制备为抗抑郁药物，用于预防或治疗抑郁症，特别是用于治疗炎症抑郁症和与各种感染性、非感染性炎症躯体病共病的抑郁障碍。
2、本发明IDO抑制剂芍药内酯苷，通过抑制机体内IDO的过活化，增强T细胞的免疫功能，阻止肿瘤细胞逃逸机体免疫系统对其监视和杀伤，可将其制备用于肿瘤免疫疗法的抗癌药物。
3、本发明IDO抑制剂芍药内酯苷，通过抑制机体内IDO的过活化和减少色氨酸向犬尿胺酸以及喹啉酸的生成，预防和减轻老年痴呆症的生成与发展，可将其制备为抗衰老和治疗阿尔茨海默病等神经退行性疾病的药物。
4、依据本发明，可将芍药内酯苷或其药学上可接受的盐、含有芍药内酯苷的提取物、组合物，开发成治疗、预防、调节和改善由IDO介导的具有 色氨酸代谢途径功能紊乱病理特征疾病或亚健康状态的药物、食品、保健品、食品添加剂或营养补充剂。
实施例三、使用MOE2016软件对本发明的IDO1抑制剂芍药内酯苷与IDO1蛋白分子对接实验
1.IDO1晶体结构的选择
目前已经报道的IDO蛋白晶体结构有20个，经生物来源、分辨率、配体小分子结构等多因素考量，最终确定以编号4PK5的晶体结构为IDO1蛋白的分子对接结构。
2.小分子结构，芍药内酯苷(Albiflorin)，结构如下：
Figure PCTCN2017095987-appb-000003
3.分子对接实验
使用MOE2016软件包完成对接实验及其他相关计算。
步骤：
准备受体结构，纠正结构中的错误，完成偏电荷计算和质子化过程。选择一个合适的亚基作为受体，删除其他部分。
利用MOE软件Flexible Alignment功能，以蛋白中原有配体为模板，考察芍药内酯苷与配体拟合模式，得到20个以上的拟合结果。
逐个审查拟合结果，选择其中比较合理的拟合结果进行优化，产生第一阶段对接结果，见附图7A。
以第一阶段对接结果为基础，使用MOE软件Dock模块进行二次自动对接，得到第二批对接结果，再从中筛查合理的结果。
对二次的结果进行结构优化，使用分子动力学模拟方法测试对接结果的稳定性，模拟时间6ns。
最终确定芍药内酯苷与IDO1的结合模式，计算结合自由能。
结果与讨论
芍药内酯苷结合于IDO1的核心区域，紧靠着亚铁血红素结构(由铁离子和原卟啉构成)。受体活性口袋由若干alpha螺旋结构构成，内侧为亚铁血红素，有一个比较大的入口，朝向蛋白外面的环境，见附图7B。
芍药内酯苷与原卟啉、组氨酸His346共同形成螯合体系，包裹住中间的铁离子。铁离子是IDO1进行催化反应的关键因素，芍药内酯苷与铁离子结合，成功的占据了活性口袋，从而产生竞争性抑制作用，见附图7C。
除了与铁离子结合以外，芍药内酯苷的另外两个部分也与受体口袋的极性想匹配，综合这三点来看，芍药内酯苷满足IDO1抑制剂的条件。我们获得的结合模式，也是比较合理的，见附图7D。
我们使用MOE对第二阶段对接结果进行初步的自由能计算，结果显示，结合自由能为-132kcal/mol，这一结果在数量级上证明了芍药内酯苷与IDO1具有较高的结合自由能，因而推测具有较高的抑制活性，见附图7E。
我们使用MOE对对接结果活性口袋6埃范围内的残基进行了动力学模拟，结果显示，对接结果稳定，未随时间变化而发生变动，见附图7F。
目前已经报道的几种IDO1化学抑制剂均是以相同模式结合，见附图7G。不同之处在于，它们均是以N原子与铁离子结合，而芍药内酯苷是以氧原子结合。氧原子与氮原子相比，结合自由能较低，因此芍药内酯苷与这些化学抑制剂相比，结合强度可能相对较低，抑制活性较弱。
以上对本发明具体实施方式的描述并不限制本发明，本领域技术人员可以根据本发明作出各种改变或变形，只要不脱离本发明的精神，均应属于本发明所附权利要求的范围。

Claims (10)

  1. 芍药内酯苷或其药学上可接受的盐、或者包含芍药内酯苷或其药学上可接受的盐的提取物作为IDO抑制剂的用途。
  2. 芍药内酯苷或其药学上可接受的盐、或者包含芍药内酯苷或其药学上可接受的盐的提取物在制备用于抑制IDO的活性的药物、食品、保健品、食品添加剂或营养补充剂中的用途。
  3. 芍药内酯苷或其药学上可接受的盐、或者包含芍药内酯苷或其药学上可接受的盐的提取物在制备用于预防或治疗IDO过活化介导的色氨酸代谢途径的病理学特征疾病的药物、食品、保健品、食品添加剂或营养补充剂中的用途；
    优选地，所述疾病选自AIDS、癌症、神经退行性疾病、抑郁症、白内障、与年龄相关的黄化以及自身免疫性疾病中的一种或多种；
    更优选地，所述神经退行性疾病选自阿尔茨海默病、亨廷顿病和帕金森病中的一种或多种；
    进一步优选地，所述药物为免疫法治疗癌症使用的药物。
  4. 芍药内酯苷或其药学上可接受的盐、或者包含芍药内酯苷或其药学上可接受的盐的提取物在制备用于预防或治疗炎性抑郁症，卒中后抑郁障碍(PSD)，与癌症、肝炎、肾炎等躯体病共病的抑郁障碍，伴生炎症的中风，伴生炎症的阿尔茨海默症或伴生炎症的帕金森症的药物、食品、保健品、食品添加剂或营养补充剂中的用途。
  5. 一种抑制IDO过活化的方法，所述方法包括给予治疗有效量的芍药内酯苷或其药学上可接受的盐、或者包含芍药内酯苷或其药学上可接受的盐的提取物。
  6. 一种预防或治疗由IDO过活化介导的色氨酸代谢途径的病理学特征疾病的方法，所述方法包括给予治疗有效量的芍药内酯苷或其药学上可接受的盐、或者包含芍药内酯苷或其药学上可接受的盐的提取物；
    优选地，所述疾病选自AIDS、癌症、神经退行性疾病、抑郁症、白内障、与年龄相关的黄化以及自身免疫性疾病；
    更优选地，所述神经退行性疾病选自阿尔茨海默病、亨廷顿病和帕金森病中的一种或多种；
    进一步优选地，所述方法为免疫法治疗癌症。
  7. 一种预防或治疗炎性抑郁症，卒中后抑郁障碍(PSD)，与癌症、肝炎、肾炎等躯体病共病的抑郁障碍，伴生炎症的中风，伴生炎症的阿尔茨海默症或伴生炎症的帕金森症的方法，所述方法包括予治疗有效量的芍药内酯苷或其药学上可接受的盐、或者包含芍药内酯苷或其药学上可接受的盐的提取物。
  8. 一种用于抑制IDO的组合物，其包括芍药内酯苷或其药学上可接受的盐、或者包含芍药内酯苷或其药学上可接受的盐的提取物；
    优选地，所述组合物为药物、食品、保健品、食品添加剂或营养补充剂。
  9. 一种用于预防或治疗IDO过活化介导的色氨酸代谢途径的病理学特征疾病的组合物，其包括芍药内酯苷或其药学上可接受的盐、或者包含芍药内酯苷或其药学上可接受的盐的提取物；
    优选地，所述组合物为药物、食品、保健品、食品添加剂或营养补充剂；
    优选地，所述疾病选自AIDS、癌症、神经退行性疾病、抑郁症、白内障、与年龄相关的黄化以及自身免疫性疾病中的一种或多种；
    更优选地，所述神经退行性疾病选自阿尔茨海默病、亨廷顿病和帕金森病中的一种或多种；
    进一步优选地，所述组合物用于免疫法治疗癌症。
  10. 一种用于预防或治疗炎性抑郁症，卒中后抑郁障碍(PSD)，与癌症、肝炎、肾炎等躯体病共病的抑郁障碍，伴生炎症的中风，伴生炎症的阿尔茨海默症或伴生炎症的帕金森症的组合物，其包括芍药内酯苷或其药学上可接受的盐、或者包含芍药内酯苷或其药学上可接受的盐的提取物。
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