WO2015042821A1 - 活化ampk的化合物及其使用 - Google Patents

活化ampk的化合物及其使用 Download PDF

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Publication number
WO2015042821A1
WO2015042821A1 PCT/CN2013/084294 CN2013084294W WO2015042821A1 WO 2015042821 A1 WO2015042821 A1 WO 2015042821A1 CN 2013084294 W CN2013084294 W CN 2013084294W WO 2015042821 A1 WO2015042821 A1 WO 2015042821A1
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treatment
adenine
disease
preparation
compound
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PCT/CN2013/084294
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English (en)
French (fr)
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邱壬乙
陈翰民
郭正宜
林俊材
黄纯芳
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华安医学股份有限公司
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Priority to CA3112108A priority Critical patent/CA3112108C/en
Priority to CA3112114A priority patent/CA3112114C/en
Priority to JP2016516852A priority patent/JP6254263B2/ja
Priority to ES13894637T priority patent/ES2863173T3/es
Priority to DK13894637.1T priority patent/DK3050567T3/da
Priority to EP13894637.1A priority patent/EP3050567B1/en
Priority to CA2925511A priority patent/CA2925511C/en
Priority to AU2013401767A priority patent/AU2013401767B2/en
Priority to KR1020207010397A priority patent/KR102248806B1/ko
Priority to CA3112089A priority patent/CA3112089C/en
Application filed by 华安医学股份有限公司 filed Critical 华安医学股份有限公司
Priority to PCT/CN2013/084294 priority patent/WO2015042821A1/zh
Priority to KR1020167010883A priority patent/KR102101465B1/ko
Priority to PL13894637T priority patent/PL3050567T3/pl
Priority to KR1020207010398A priority patent/KR102221723B1/ko
Priority to SG11201602268XA priority patent/SG11201602268XA/en
Priority to MYPI2016701058A priority patent/MY182801A/en
Priority to EP20194630.8A priority patent/EP3769766A1/en
Publication of WO2015042821A1 publication Critical patent/WO2015042821A1/zh
Priority to IL244752A priority patent/IL244752B/en
Priority to AU2019203668A priority patent/AU2019203668B2/en
Priority to AU2020244377A priority patent/AU2020244377B2/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
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/519—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with heterocyclic rings
    • A61K31/52—Purines, e.g. adenine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/08—Solutions
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/02—Drugs for dermatological disorders for treating wounds, ulcers, burns, scars, keloids, or the like
    • 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
    • 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
    • A61P3/00—Drugs for disorders of the metabolism
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/04—Anorexiants; Antiobesity agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/06—Antihyperlipidemics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P39/00—General protective or antinoxious agents
    • A61P39/06—Free radical scavengers or antioxidants
    • 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

Definitions

  • the present invention relates to adenine, which is useful for activating AMPK (AMP-activated protein kinase) and using the compound for preventing or treating a physiological condition or disease.
  • AMPK AMP-activated protein kinase
  • AMPK is clearly a sensor of cellular energy and a responder to energy needs.
  • AMPK is a heterotrimer consisting of catalytic alpha subunits, regulatory ⁇ , and gamma subunits, all of which are highly reserving in eukaryotes.
  • AMPK activation is the retention of the 172th threonine residue by its upstream kinase such as LKB1, calcium ion/calcium-dependent protein kinase (Ca 2+ /Calmodulin dependent kinase) and TAK1 phosphorylation of alpha subunits.
  • the sputum/ ⁇ ratio caused by physiological or pathological stress also activates AMPK:.
  • Activation of AMPK promotes catabolic pathways and inhibits anabolism, restoring cellular energy balance by reducing ATP consumption and promoting ATP production.
  • AMPK As an energy metabolism balance regulator, AMPK is considered to be a potential drug target for metabolic syndrome, including type 2 diabetes, cardiovascular disease, and fatty liver. Many metabolic syndromes are associated with insulin resistance. Insulin resistance is a pathological condition in which cells cannot respond to insulin, so excessive glucose in the blood cannot be removed to the skeletal muscle or fat tissue. In muscle cells, AMPK activation increases the expression of glucose transporter (GLUT4) by transcriptional regulation in a non-insulin-dependent manner and induces the transfer of GLUT4 to the cell membrane resulting in an increase in the rate of glucose uptake by the cells. AMPK activation also inhibits fatty acid and cholesterol synthesis by inhibiting acetyl-CoA carboxylase and HMG-CoA reductase, respectively.
  • GLUT4 glucose transporter
  • AMPK activation also inhibits fatty acid and cholesterol synthesis by inhibiting acetyl-CoA carboxylase and HMG-CoA reductase, respectively.
  • AMPK activation leads to inhibition of several transcription factors, including SREBP-lc, ChREBP and HNF-4a, and decreases the protein expression of enzymes involved in fatty acid synthesis and gluconeogenesis.
  • SREBP-lc SREBP-lc
  • ChREBP ChREBP
  • HNF-4a HNF-4a
  • AMPK is also involved in the regulation of several cellular mechanisms, including inflammatory responses, cell growth, cell death, autophagy, aging, and differentiation. Many studies show AMPK is an inhibitor of inflammatory response. AMPK activation inhibits the inflammatory response by inhibiting the signaling of nuclear transcription factor (NF- ⁇ ⁇ ). The signaling of nuclear transcription factors is the main pathway for the activation of innate immunity and acquired immunity. When AMPK is activated, it inhibits nuclear transcription factor by stimulating SIRT1, Forkhead box O (FoxO) or eroxisome proliferator-activated receptor co-activator 1 a (PGC1 a ). Transcriptional activity to achieve an effect of inhibiting the inflammatory response.
  • SIRT1 Forkhead box O
  • PDC1 a eroxisome proliferator-activated receptor co-activator 1 a
  • Cyclooxygenase-2 is an inducible enzyme regulated by inflammatory cytokines and growth factors. Its function is to convert arachidonic acid into prostaglandins, which leads to inflammatory reactions and pain, thus inhibiting cyclooxygenase. Activity or performance has been shown to be anti-inflammatory.
  • AICAR 5-aminoimidazole-4-carboxamide ribonucleoside
  • EAE human multiple sclerosis animal model
  • the mammalian rapamycin target protein is a monoamine/threonine kinase that regulates cell growth and autophagy.
  • the dysregulation of the mammalian target of rapamycin target protein signaling is found in many different cancers, and thus mammalian rapamycin target protein inhibitors are considered to be potential drugs for cancer therapy.
  • Numerous studies have demonstrated that AMPK phosphorylation of tuberous sclerosis complex 2 (TSC2) and Raptor achieves a pathway that inhibits mammalian rapamycin target proteins.
  • AMPK activators include AICAR, metformin, and phenformin have been shown to inhibit the mammalian rapamycin target protein message pathway and inhibit cancer cell growth.
  • AMPK activation induces autophagy by inhibiting the mammalian rapamycin target protein complex-1. Since AMPK inhibits mammalian rapamycin target protein complex-1, phosphorylation of 757 serine on Ulkl is reduced, followed by phosphorylation of AMPK in 317 and 777, and phosphorylation of Ulkl by AMPK. Activate autophagy.
  • AMPK is considered to be a good treatment for many human diseases or pathological conditions, including inflammatory diseases, wound healing, neurodegenerative, cancer, oxidative stress and cardiovascular disease.
  • AMPK activators have been used in clinical trials for at least 24 diseases including: bacterial and fungal diseases, behavioral and psychological disorders, blood and lymphatic diseases, cancer, cancer, digestive diseases, ENT diseases, eye diseases, glands Body and hormone related diseases, cardiovascular diseases, immune system diseases, mouth and dental diseases, muscles, bones, cartilage diseases, nervous system diseases, nutritional and metabolic diseases, respiratory diseases, skin and connective tissue diseases, wounds Healing, etc.
  • AMPK activators have been used in clinical trials for at least 24 diseases including: bacterial and fungal diseases, behavioral and psychological disorders, blood and lymphatic diseases, cancer, cancer, digestive diseases, ENT diseases, eye diseases, glands Body and hormone related diseases, cardiovascular diseases, immune system diseases, mouth and dental diseases, muscles, bones, cartilage diseases, nervous system diseases, nutritional and metabolic diseases, respiratory diseases, skin and connective tissue diseases, wounds Healing, etc
  • the present invention provides a compound for activating AMPK which is adenine and/or a pharmaceutically acceptable salt thereof.
  • the above compounds treat a disease or physiological condition which is ameliorated by an AMPK activator, wherein the adenine and/or a pharmaceutically acceptable salt thereof is administered to a mammal in need of such treatment.
  • the above compound which can reduce the secretion of inflammatory cytokines and the expression of cyclooxygenase-2 in a cell, thereby treating an inflammatory physiological condition or disease, wherein the adenine and/or its pharmaceutically acceptable salt, Give the mammal in need of this treatment.
  • the above compound which increases the uptake of cellular glucose, thereby preventing or treating a physiological condition or disease selected from the group consisting of pre-diabetes, type 2 diabetes, metabolic syndrome, wherein the adenine and/or its medicinal
  • a physiological condition or disease selected from the group consisting of pre-diabetes, type 2 diabetes, metabolic syndrome, wherein the adenine and/or its medicinal
  • the salt received is given to the mammal in need of this treatment.
  • the above compound which reduces plasma triglyceride in a mammal and reduces body weight, thereby preventing or treating obesity, wherein the adenine and/or a pharmaceutically acceptable salt thereof is administered to a mammal in need of such treatment.
  • the above compound which inhibits the accumulation of amylin-like peptide in a cell, thereby preventing or treating Alzheimer's disease, wherein the adenine and/or a pharmaceutically acceptable salt thereof is administered to a mammal in need of such treatment.
  • the above compound which inhibits the growth of fibroblasts, thereby inhibiting scarring during wound healing Forming, wherein the adenine and/or a pharmaceutically acceptable salt thereof, is administered to a mammalian compound that is in need of such treatment, which enhances wound healing, wherein the adenine and/or its pharmaceutically acceptable salt, Give the mammal in need of this treatment.
  • the above compound which inhibits the production of a cell reactive oxygen species, thereby protecting and treating cells damaged by the reactive oxygen species in a mammal, wherein the adenine and/or a pharmaceutically acceptable salt thereof is administered to the subject in need thereof mammal.
  • the present invention provides the use of the above compounds as a medicament for the preparation of a medicament for the treatment of a disease or physiological condition which is ameliorated by an AMPK activator.
  • the present invention provides the use of the above compounds as a medicament for the preparation of a inflammatory physiological condition or disease.
  • the present invention provides the use of the above compound as a medicament for the preparation of a physiological condition or disease for preventing or treating pre-diabetes, type 2 diabetes, metabolic syndrome or a combination thereof.
  • the present invention provides the use of the above compound as a medicament for the preparation of a physiological condition or disease for preventing or treating pre-diabetes, type 2 diabetes, metabolic syndrome or a combination thereof.
  • the present invention provides the use of the above compound as a medicament for the preparation of a medicament for preventing or treating Alzheimer's disease.
  • the present invention provides the use of the above compounds as a medicament for the preparation of a medicament for the treatment of a disease or physiological condition which is ameliorated by autophagy.
  • the present invention provides the use of the above compounds as a medicament for inhibiting scar formation in a wound healing process.
  • the present invention provides the use of the above compounds as a medicament for the preparation of a wound healing enhancement.
  • the present invention provides the use of the above compounds as a medicament for the preparation of a medicament for protecting and treating cells damaged by reactive oxygen species in a mammal.
  • the present invention provides the use of the above compounds as a medicament for the preparation or prevention of cancer.
  • a novel AMPK activator adenine
  • adenine is provided to activate AMPK intracellularly, thereby preventing or treating physiology which can be improved by AMPK in a mammal. Condition or disease.
  • a method of lowering blood glucose by activating AMPK thereby preventing or treating a disease comprising: metabolic syndrome, pre-diabetes, type 2 diabetes, insulin resistance, wherein an effective dose of a gland is administered to a mammal in need of such treatment ⁇ and/or pharmaceutically acceptable according to embodiments of the present invention, providing a method for preventing or treating an inflammatory condition or disease by activating AMPK, wherein an effective dose of adenine is administered to a mammal in need of such treatment. / or pharmaceutically acceptable salts.
  • a method of inhibiting the growth of fibroblasts by activating AMPK is provided to prevent scar tissue formation during wound healing.
  • a method of enhancing wound healing wherein an effective amount of adenine and/or a pharmaceutically acceptable salt is administered to a mammal in need of such treatment.
  • a method of inhibiting reactive oxygen species (ROS) production is provided to protect or treat cells of a mammal from reactive oxygen species injury, wherein an effective dose of adenine is administered to a mammal in need of such treatment / or pharmaceutically acceptable salts.
  • ROS reactive oxygen species
  • a method of inhibiting the growth of cancer cells thereby preventing or treating cancer, wherein an effective dose of adenine and/or a pharmaceutically acceptable salt is administered to a mammal in need of such treatment.
  • the present invention relates to adenine, which is useful for activating AMPK and for using adenine to prevent or treat physiological conditions or diseases, including: pre-diabetes, insulin resistance, type 2 diabetes, metabolic syndrome, obesity, inflammation, wound healing, Alzheimer's disease, cancer, oxidative stress and cardiovascular disease.
  • adenine is a novel AMPK activator and has various biological functions.
  • AMPK activation has been shown to contribute to disease prevention and treatment, such as pre-diabetes, insulin resistance, type 2 diabetes, metabolic syndrome, obesity, inflammation, wound healing, Alzheimer's disease, cancer, oxidative stress, Cardiovascular disease and promote wound healing.
  • the present invention contemplates that this effect can be attributed to AMPK activation resulting from, but not limited to, a decrease in cyclooxygenase-2 expression, inhibition of the production of ROS (ROS), and an increase in glucose uptake activity.
  • ROS ROS
  • adenine can be used as a therapeutic agent for various physiological conditions or diseases by activating AMPK.
  • Exemplary guidance and evidence for the intended indications are provided below.
  • AMPK activators include metformin, A769662, AICAR in diabetic or obese mice mode to reduce plasma glucose concentrations.
  • 1 ⁇ 600 ⁇ of adenine significantly increased the glucose uptake of muscle cells C2C12 (Table 2).
  • mice fed a high fat diet as an animal model of type 2 diabetes were evaluated for the effect of adenine on plasma glucose concentration.
  • adenine administration significantly reduced plasma glucose in high-fat diet-fed mice by more than 30%, and reduced plasma triglyceride by more than 35%, and more than 15% of body weight loss (Examples) 3).
  • hyperglycemia refers to a physiological condition characterized by a blood glucose of more than 126 g/dl.
  • pre-diabetes refers to a physiological condition characterized by a fasting blood glucose above 100 g/dl but below 140 g/dl.
  • insulin resistance refers to a physiological condition in which the system or tissues including the liver, skeletal muscle, and adipose tissue are unable to respond to insulin.
  • second type diabetes as used herein also refers to non-insulin dependent diabetes or adult onset diabetes. It refers to insulin production or insulin resistance caused by metabolic disorders, which is usually characterized by fasting blood glucose above 140 g/dL. According to this embodiment, adenine is confirmed to accelerate glucose uptake and thus can be an effective treatment for physiological conditions or diseases associated with hyperglycemia.
  • AICAR 5-aminoimidazole-4-carboxamide ribonucleoside
  • EAE human multiple sclerosis animal model
  • adenine inhibits lipopolysaccharide-induced inflammatory response in an in vitro assay: inflammatory cytokine secretion by adenine-treated macrophages stimulated by lipopolysaccharide, including tumor necrosis factor a (TNF-a) ), interleukin-1 ⁇ (-1 ⁇ ) and interleukin-6 (IL-6), significantly reduced compared with the control group.
  • TNF-a tumor necrosis factor a
  • IL-6 interleukin-6
  • Adenine also reduces human giants The amount of cyclooxygenase-2 exhibited by phagocytes induced by lipopolysaccharide (Example 4).
  • IBD trinitrobenzene sulfonic acid-induced inflammatory bowel disease
  • colonic inflammatory cytokines including tumor necrosis factor (TNF), interferon Y (INF Y ), and adenine treatment groups were administered.
  • Interleukin (IL-17) was significantly reduced compared to control mice and saved weight loss (Example 5).
  • inflammatory cytokine refers to a cytokine that promotes a systemic inflammatory response.
  • inflammatory disease refers to an inflammation-related disease, including but not limited to ankylosing ridge inflammation, arthritis (osteoarthritis, rheumatoid arthritis, dryness arthritis), asthma, arteries. Atherosclerosis, Crohn's disease, colitis, dermatitis, colonic diverticulitis, fibromyalgia, hepatitis, irritating colon, systemic lupus erythematosus, nephritis, Alzheimer's disease, Parkinson's disease, Ulcerative colitis and the like.
  • AMPK is an upstream regulator of cyclooxygenase-2, which inhibits the protein expression of cyclooxygenase-2.
  • adenine a novel AMPK activator, adenine, is effective in inhibiting the protein expression of cyclooxygenase-2, thereby indicating that adenine inhibits cyclooxygenase-2 mediated inflammation.
  • adenine is found to inhibit inflammation and thus can be used as a treatment for an inflammatory related physiological condition or disease.
  • AMPK is thought to promote cell motility and enhance wound healing.
  • An AMPK activator, resveratrol has been shown to enhance the healing of surgical wounds.
  • resveratrol has been shown to enhance the healing of surgical wounds.
  • reducing the formation of scars during healing has been the preferred target of modern medicine.
  • Newborn wound healing differs from adult wound healing without the formation of scars, the difference being the activation of cyclooxygenase-2.
  • cyclooxygenase-2 activity is elevated via TGF-beta, resulting in increased prostaglandin production at the wound.
  • Prostaglandins have been shown to promote fibroblast proliferation and collagen formation, both of which can lead to scar formation.
  • adenine inhibits fibroblast growth (Example 8) and reduces the protein expression of cyclooxygenase-2.
  • direct administration of adenine at the wound not only enhances wound healing and reduces scar formation (Example 9). Based on the above information, topical application of adenine can effectively enhance wound healing and prevent scar formation.
  • AMPK activation has been shown to promote autophagy pathways. Therefore, activation of AMPK to promote autophagy pathways can be an effective strategy for preventing or controlling neurodegenerative diseases.
  • AMPK activators have been shown to reduce starch-like deposition via autophagy pathways.
  • Daily administration of the AMPK activator, resveratrol increases the lifespan of Alzheimer's mice.
  • Another AMPK activator, curcumin has also been shown to be a potential drug for the treatment of Alzheimer's disease.
  • the inventors have found that adenine significantly enhances autophagy activity and decreases A accumulation in neuron Neuro-2A, and adenine improves cognitive function in mice with Alzheimer's disease (Examples 6, 7). . According to the above findings, adenine can be used as a treatment for neurodegenerative diseases.
  • neural degeneration refers to a condition in which the structure or function of a neuron is gradually lost.
  • Neurodegenerative diseases are the result of neurodegeneration, including but not limited to Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, spinocerebellar atrophy, spinal muscular muscles Atrophy and so on.
  • reactive oxygen species including superoxide radicals, hydroxyl radicals, and hydrogen peroxide are continuously produced, and an excess of reactive oxygen species is associated with many diseases, including but not limited to: nerve tissue muscle weakness Movement disorders and pigmentary retinitis (NARP), MELAS syndrome, muscle-hopping epilepsy with red diaphragmatic fibrosis (MERRF), Leber hereditary optic atrophy (LHON), KSS syndrome, Alzheimer's disease, Parkinson's disease Disease, Huntington's disease, amyotrophic lateral sclerosis, Friedreich's ataxia (FA) and aging.
  • AMPK activators such as AICAR can reduce reactive oxygen species production under high glucose, palmitic acid or albumin induction conditions.
  • adenine reduces the production of reactive oxygen species in HUVEC cells (Table 6), and thus adenine can be used as a treatment for a reactive oxygen-related physiological condition or disease.
  • AMPK activation inhibits cyclooxygenase-2 and mammalian rapamycin target protein pathways, which are important mechanisms for cancer cell growth. Based on cyclooxygenase-2 and mammalian rapamycin targets The importance of proteins for cancer, activation of AMPK to inhibit cyclooxygenase-2 and mammalian rapamycin target protein pathways is considered a reasonable cancer treatment strategy. In fact, many studies have confirmed that AMPK activators disrupt cancer development. For example, phenformin and metformin are found to inhibit breast cancer tumor development and growth in allogeneic cancer mouse models.
  • adenine inhibits cell growth of human liver cancer cells Hep G2, human breast cancer cells MCF7, and colon cancer cells HT29 (Example 11).
  • the 50% growth inhibitory concentrations of adenine on Hep G2, MCF7, and HT29 were 544.1, 537.5, and 531.9 ⁇ , respectively.
  • long-term administration of adenine significantly delayed tumor growth.
  • the formation or development of cancer can be prevented or controlled by the treatment of adenine-activated AMPK.
  • mouse muscle cell C2C12 mouse fibroblast 3T3, human hepatoma cell Hep G2, human breast cancer cell MCF7 and human colon cancer cell HT29 human umbilical vein endothelial cell HUVEC, human acute mononuclear leukocyte cell line THP1, human macrophage Cell U937, mouse microglial BV-2, neuroblastoma cell Neuro2A, and dermal papilla cell Dermal Papilla were used to analyze the effects of adenine on AMPK phosphorylation.
  • the cells were supplemented with 10% fetal bovine serum (FBS), 4 mM L-glutamine, 2 mM sodium pyruvate and 1% penicillin I streptomycin (Invitrogen Gibco BRL, Carlsbad, CA, USA) at 37 in Dulbecco's modified Eagle's medium (DMEM).
  • FBS fetal bovine serum
  • L-glutamine 4 mM L-glutamine
  • 2 mM sodium pyruvate 1% penicillin I streptomycin
  • C 5% C0 2 culture.
  • 3x10 5 cells were seeded in 6-well plates, and cells were treated with the indicated compounds for 30 minutes after 24 hours, followed by lysis of the cells and analysis by Western blotting. Equal amounts of protein were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis followed by transfer to a polyvinylidene fluoride membrane.
  • adenine on glucose uptake was analyzed using a fluorescent glucose analog (2-NBDG, Molecular Probes) in muscle cells C2C12.
  • C2C12 cells were treated with adenine at various concentrations for 30 minutes at 37 ° C, 500 ⁇ fluorescent glucose analog was added, and after incubation for 5 minutes at room temperature, the cells were washed three times with Kreb-Hepes buffer solution and 70% ethanol. fixed. Fluorescence of intracellular glucose analogs was detected by a fluorometer.
  • mice fed a high-fat diet were tested as an animal model of type 2 diabetes.
  • C57BL/6J mice were housed at 22 ° C for 12 hours day/night and fed a high-fat diet (60% kcal% fat) or normal diet on an unrestricted diet.
  • a high-fat diet (60% kcal% fat) or normal diet on an unrestricted diet.
  • 0.1-50 gram/kg of adenine was administered to 24-week-old mice by intraperitoneal injection, and blood glucose levels were measured 1 and 3 hours after injection.
  • Intraperitoneal injection of high fat diet to feed mice twice a day and After 6 days of administration, plasma was collected and plasma glucose and triglyceride levels were measured 1 hour after the last administration.
  • adenine was found to reduce plasma glucose by more than 30%, triglyceride by more than 35%, and body weight by more than 15%.
  • Adenine inhibits inflammatory response induced by lipopolysaccharide
  • TNF-a tumor necrosis factor alpha
  • IL-1 ⁇ interleukin-1 P
  • IL-6 interleukin-6
  • Equal amounts of protein were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and then transferred to a polyvinylidene fluoride membrane. After the transfer, the polyvinylidene fluoride membrane was immersed in 3% bovine serum albumin dissolved in PBS buffer for 60 minutes, and then added anti-cyclase-2 antibody (1:1000, Cell signaling), anti-motor antibody antibody. (1:5000, Cell signaling), at 4. C role. After 16 hours, the corresponding secondary antibody was added to react at room temperature for 1 hour. The immunoreactive zone is detected by cold light and the signal is recorded in the negative film. The resulting signal was scanned and analyzed by TotalLab Quant software (TotalLab). The secretion of tumor necrosis factor a (TNF-a), interleukin-1 ⁇ (IL-1 ⁇ ) and interleukin-6 (IL-6) was analyzed by enzyme-linked immunosorbent assay.
  • TNF-a tumor necrosis factor a
  • IL-1 ⁇ interleukin-1
  • adenine-treated macrophages exhibited cyclooxygenase-2 protein expression and tumor necrosis factor a (TNF-a), interleukin-1 P (IL-1 ⁇ ) and mediators.
  • TNF-a tumor necrosis factor a
  • IL-1 ⁇ interleukin-1 P
  • mediators The secretion of leuko-6 (IL-6) was significantly decreased.
  • Adenine ⁇ TNF a (% IL-1 ⁇ (% to IL-6 (% to COX-2 (%
  • Adenine inhibits trinitrobenzenesulfonic acid-induced inflammatory response in organisms
  • mice were raised at 22. C, 12 hours day/night cycle.
  • Five doses of trinitrobenzene sulfonic acid were gradually increased: 0.5 mg, 0.75 mg, 1.0 mg, 1.25 mg, and 1.5 mg dissolved in 50% ethanol, respectively, and O.lmL was administered to mice every week to induce recurrent colitis.
  • mice were given adenine (0.01, 0.1, 5 or 30 mg/kg body weight) or physiological saline daily by intraperitoneal injection.
  • mice were sacrificed two days after the fifth administration of trinitrobenzenesulfonic acid.
  • Inflammatory Cytokines in Colon Tissue Dissolution Tumor necrosis factor (TNF), interferon Y (INF Y ) and interleukin (IL-17) were analyzed by enzyme-linked immunosorbent assay.
  • the colonic inflammatory cytokines including tumor necrosis factor (TNF), interferon Y (INF ⁇ ) and interleukin (IL-17), were significantly reduced in the adenine treatment group compared with the control group, and the weight loss was saved. .
  • TNF tumor necrosis factor
  • INF ⁇ interferon Y
  • IL-17 interleukin
  • the neuroblastoma cell Neuro2A was used to analyze the effect of adenine on the starch-like peptide.
  • DMEM Dulbecco's modified Eagle's medium
  • FBS Taiwan bovine serum
  • penicillin I streptomycin Invitrogen Gibco BRL, Carlsbad, CA, USA
  • Equal amounts of protein were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and then transferred to a polyvinylidene fluoride membrane.
  • the transferred polyvinylidene fluoride membrane was immersed in 3% bovine serum albumin dissolved in PBS buffer for 60 minutes, and then anti-mycoplasmic ⁇ -peptide antibody (1:1000, Abeam), anti-LC3 antibody (1: 1000, Cell signaling), anti-motor protein antibody (1:5000, Cell signaling), at 4. C role. After 16 hours, the corresponding secondary antibody was added and allowed to react at room temperature for 1 hour. The immunoreactive zone is detected by cold light and the signal is recorded in the negative film. The resulting signal was scanned and analyzed by TotalLab Quant software (TotalLab).
  • adenine significantly reduced the amount of beta-like peptide and increased LC3-II/LC3-I proportion. Since LC3-I conversion to LC3-II represents autophagy activity, the higher LC3-II/LC3-I ratio in adenine-treated cells reflects the function of adenine-activated autophagy.
  • the water maze was carried out in a circular pool, and the platform was placed under the water surface of the target quadrant to hide the platform. Test. During the 5-day hidden platform test, each test mouse was randomized. Placed in the pool as a starting point, 6 tests per day. On the 5th, the exploratory test was conducted 1 day after the hidden platform test. In the exploratory experiment, the hidden platform was removed and the target quadrant was used as the starting point. Recording the mouse in the labyrinth for 60 seconds, using software to analyze the mouse to find the platform time and In the hidden platform trial, mice treated with adenine had a significantly reduced time to find the platform compared to the control group. The results of this trial confirmed that adenine can rescue the impaired learning and memory function of Alzheimer's disease mice. Furthermore, adenine-treated mice in the exploratory trial stayed at the target line time longer than the control group, demonstrating that adenine promotes memory retention.
  • Adenine inhibits fibroblast growth
  • Human fibroblast strain 3T3 contains 10% fetal bovine serum (FBS), 4 mM L-glutamine, 2 mM sodium pyruvate and 1% penicillin I streptomycin (Invitrogen GibcoBRL, Carlsbad, CA, USA) in Dulbecco's modified Eagle's medium (DMEM). 37. C, 5% C0 2 culture. In the cell growth assay, lxlO 5 cells were seeded in 6-well plates.
  • FBS fetal bovine serum
  • L-glutamine 4 mM L-glutamine
  • 2 mM sodium pyruvate 1% penicillin I streptomycin
  • DMEM Dulbecco's modified Eagle's medium
  • cells were treated with a given concentration of adenine for 72 hours, and the number of viable cells was counted.
  • Cells were isolated by trypsin-EDTA and stained with trypan blue, and the number of viable cells was counted using a hemocytometer.
  • Adenine cell number (% to 1)
  • the healing rate of adenine treated wounds was faster than that of the control group, and according to the tissue staining analysis, the adenine-treated wound regeneration tissue showed significantly less scar than the control group.
  • Adenine reduces reactive oxygen species production
  • HUVEC Human umbilical vein endothelial cells HUVEC with Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum (FBS), 4 mM L-glutamine, 2 mM sodium pyruvate and 1% penicillin I streptomycin (Invitrogen GibcoBRL, Carlsbad, CA, USA) 37. C, 5% C0 2 culture. 2xl0 4 cells were seeded in 96-well black dishes. After 24 hours, the medium was replaced with DMEM medium containing 5.6 or 30 mM glucose and the indicated concentrations of adenine were added. After 24 hours of treatment, the intracellular reactive oxygen species were detected by H 2 DCF-DA.
  • DMEM Dulbecco's modified Eagle's medium
  • FBS fetal bovine serum
  • penicillin I streptomycin Invitrogen GibcoBRL, Carlsbad, CA, USA
  • the cells were washed once with PBS buffer and cultured at 100 ⁇ M DCF for 37 minutes at 37 ° C.
  • DCF fluorescence was analyzed by a disc fluorescence analyzer (excitation wavelength: 485 nm; scattering wavelength: 530 nm).
  • Adenine glucose reactive oxygen species generation (% of 5.6 mM
  • adenine The effects of adenine on the growth of cancer cells were observed in human hepatoma cells Hep G2, human breast cancer cells MCF7 and human colon cancer cells HT29.
  • the cells were supplemented with 10% fetal calf serum (FBS), 4 mM L-glutamine, 2 mM sodium pyruvate and 1% penicillin I streptomycin (Invitrogen Gibco BRL, Carlsbad, CA, USA) at 37 in Dulbecco's modified Eagle's medium (DMEM). C, 5% C0 2 culture.
  • l xlO 5 cells were seeded in 6-well plates. After 24 hours, cells were treated with adenine at the indicated concentrations for 72 hours, and the number of viable cells was counted. Cells were separated by trypsin-EDTA and stained with trypan blue, and the number of viable cells was counted using a hemocytometer.
  • the 50% growth inhibitory concentrations of adenine on Hep G2, MCF7, and HT29 were 544.1, 537.5, and 531.9 ⁇ , respectively.
  • Hep G2 contains 10% fetal bovine serum (FBS), 4 mM L-glutamine, 2 mM sodium pyruvate and 1% penicillin I streptomycin (Invitrogen GibcoBRL, Carlsbad, CA, USA) in Dulbecco's modified Eagle's medium (DMEM).
  • FBS fetal bovine serum
  • L-glutamine 4 mM L-glutamine
  • 2 mM sodium pyruvate 1% penicillin I streptomycin
  • DMEM Dulbecco's modified Eagle's medium
  • mice 5% C0 2 culture. 5 ⁇ 10 6 cells were injected subcutaneously into 8-week old NOD-SCID mice. After transplantation, mice were given 5, 20, 50 grams daily by intraperitoneal injection.
  • adenine administration significantly delayed tumor growth compared to control mice.

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Abstract

本发明公开了一种用于活化AMPK的化合物，其为腺嘌呤及/或其医药学上可接受的盐，并公开了该化合物可用于哺乳动物以预防或治疗可被AMPK改善的生理状况或疾病。

Description

活化 AMPK的化合物及其使用
技术领域 本发明关于腺嘌呤， 此化合物适用于活化 AMPK (AMP-活化蛋白激 酶)， 并使用该化合物于预防或治疗生理状况或疾病。 背景技术
AMPK明确为细胞能量的感应器及能量需求的回应者。 AMPK为异三 元体由催化性 α次单元体、 调节性 β 、 γ次单元体组成， 所有次单元体在 真核生物具高度保留性。 AMPK 活化是借由其上游激酶如 LKB1、 钙离子 / 携钙素依赖的蛋白质磷酸激酶 （Ca2+/Calmodulin dependent kinase)及 TAK1 磷酸化 α次单元体具保留性的第 172 苏胺酸残基， 由生理或病理压力造成 高 ΑΜΡ/ΑΤΡ 比例亦活化 AMPK：。 AMPK活化后会促进分解代谢路径进行 并抑制合成代谢， 借由减少 ATP消耗及促进 ATP生成进而恢复细胞能量平 衡。
作为一能量代谢平衡调节者， AMPK被认为对代谢症候群， 包括第二 型糖尿病、 心血管疾病、 脂肪肝等为一具潜力的药物标的。 许多代谢症候 群都与胰岛素抵抗有关。 胰岛素抵抗是为一病理状态， 在此状况细胞无法 对胰岛素进行回应， 因此血液中过多的葡萄糖无法移除至骨胳肌或脂肪组 织。 在肌肉细胞中， AMPK 活化以非胰岛素依赖性方式， 通过转录调控增 加葡萄糖运输蛋白 （GLUT4)表现量， 并诱导 GLUT4转移至细胞膜上导致 细胞摄取葡萄糖速率增加。 AMPK活化亦分别借由抑制乙酰辅酶 A羧化酶 (acetyl-CoA carboxylase) 及羟甲基戊二酸单酰辅酶 A 还原酶 （HMG-CoA reductase) 抑制脂肪酸及胆固醇合成。 此外 AMPK活化导致数个转录因子 的抑制， 包括 SREBP-lc, ChREBP and HNF-4a,并下降调节脂肪酸合成及糖 质新生作用相关酵素的蛋白质表现。 上述提及的研究发现， 均支持 AMPK 是为代谢症候群尤其糖尿病的治疗标的。
除了能量代谢平衡调节外， AMPK亦参与数个细胞机制的调节， 包括 发炎反应、 细胞生长、 细胞雕亡、 自体吞噬、 老化及分化。 许多研究显示 AMPK为发炎反应抑制者。 AMPK 活化通过抑制细胞核转录因子 (NF- κ Β) 的讯息传递抑制发炎反应。 细胞核转录因子的讯息传递为活化先天免疫及 后天免疫主要路径， 当 AMPK活化后会通过刺激 SIRT1、 Forkhead box O (FoxO) 或 eroxisome proliferator-activated receptor co-activator 1 a (PGC1 a ) 抑制细胞核转录因子的转录活性以达到抑制发炎反应的效果。此外数个 研究室亦证明 AMPK活化抑制环氧化酶 -2 (cyclooxygenase-2 , COX-2) 的蛋 白质表现。 环氧化酶 -2 是为一诱导性酵素， 可被发炎性细胞激素及生长因 子调控， 其功能为将花生四烯酸转化成前列腺素因而导致发炎反应及疼痛， 因此抑制环氧化酶的活性或表现已被证实具抗发炎作用。
数个 AMPK活化剂已于生物体内实验被证实具抗发炎功能。 举例而 言, 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR) 于小鼠模式已 被证实可缓解由三硝基苯磺酸或右旋糖酐硫酸酯钠所引起的急性及复发性 结肠炎， AICAR 治疗显著降低疾病小鼠的体重减少并减缓发炎反应。 此外 AICAR 对于人类多发性硬化症动物模式 (EAE)有明显的治疗效果， 亦降低 以脂多醣诱导小鼠肺损伤的严重程度。
细胞讯息传递路径失调可能会导致细胞异常生长， 最终导致癌症。 哺 乳动物雷帕霉素标靶蛋白 （mTOR) 是为一丝胺酸 /苏胺酸激酶，其调控细胞 生长及自体吞噬作用。 哺乳动物雷帕霉素标靶蛋白讯息传递路径的活性失 调在许多不同癌症被发现， 因此哺乳动物雷帕霉素标靶蛋白抑制剂被认为 是癌症治疗的潜力药物。 大量研究证实 AMPK磷酸化 tuberous sclerosis complex 2 (TSC2)及 Raptor达到抑制哺乳动物雷帕霉素标靶蛋白路径。各 种 AMPK活化剂包括 AICAR、 metformin, phenformin 已被证实抑制哺乳 动物雷帕霉素标靶蛋白讯息路径， 并抑制癌细胞生长。 此外 AMPK活化通 过抑制哺乳动物雷帕霉素标靶蛋白复合体 -1 诱导自体吞噬作用。 由于 AMPK抑制哺乳动物雷帕霉素标靶蛋白复合体 -1 , Ulkl上第 757丝胺酸磷 酸化减少 , 其后第 317及 777丝胺酸被 AMPK磷酸化， Ulkl被 AMPK磷 酸化后随的启动自体吞噬作用。
综上所述， AMPK被认为对许多人类疾病或病理状况， 包括发炎性疾 病、 伤口愈合、 神经退化、 癌症、 氧化压力及心血管疾病为一很好的治疗 标的。 事实上， AMPK活化剂已被应用于至少 24类疾病的临床试验，包括: 细菌及真菌疾病、 行为及心理失调、 血液及淋巴疾病、 癌症、 肿瘤、 消化 系统疾病、 耳鼻喉疾病、 眼疾、 腺体及贺尔蒙相关疾病、 心血管疾病、 免 疫系统疾病、 嘴巴及牙齿疾病、 肌肉、 骨胳、 软骨疾病、 神经系统疾病、 营养及代谢性疾病、 呼吸道疾病、 皮肤及结締组织疾病、 伤口愈合等。 发明内容 本发明提供了一种新颖 AMPK 活化剂-腺嘌呤及使用该化合物于预防 或治疗疾病。
本发明提供一种用于活化 AMPK的化合物，其为腺嘌呤及 /或其医药学 上可接受的盐。
上述化合物治疗可被 AMPK活化剂改善的疾病或生理状况，其中所述 腺嘌呤及 /或其医药学上可接受的盐， 给予需此治疗的哺乳动物。
上述化合物， 其可降低细胞中发炎性细胞激素的分泌及环氧化酶 -2 的 表现， 因此治疗发炎性生理状况或疾病， 其中所述腺嘌呤及 /或其医药学上 可接受的盐， 给予需此治疗的哺乳动物。
上述化合物， 其增加细胞葡萄糖的摄取， 因此预防或治疗选自由以下 组成的群的生理状况或疾病： 前期糖尿病、 第二型糖尿病、 代谢症候群， 其中所述腺嘌呤及 /或其医药学上可接受的盐， 给予需此治疗的哺乳动物。
上述化合物， 其减少哺乳动物的血浆三酸甘油酯及减少体重， 因此预 防或治疗肥胖情况， 其中所述腺嘌呤及 /或其医药学上可接受的盐， 给予需 此治疗的哺乳动物。
上述化合物， 其抑制细胞中类淀粉 胜肽的累积， 因此预防或治疗阿 兹海默症， 其中所述腺嘌呤及 /或其医药学上可接受的盐， 给予需此治疗的 哺乳动物。
上述化合物， 其加强细胞自体吞噬活性， 因此治疗可被自体吞噬作用 改善的疾病或生理状况， 其中所述腺嘌呤及 /或其医药学上可接受的盐， 给 予需此治疗的哺乳动物。
上述化合物， 其抑制纤维母细胞生长， 因此于伤口愈合过程抑制疤痕 形成， 其中所述腺嘌呤及 /或其医药学上可接受的盐， 给予需此治疗的哺乳 上化合物， 其加强伤口愈合， 其中所述腺嘌呤及 /或其医药学上可接受 的盐， 给予需此治疗的哺乳动物。
上述化合物， 其抑制细胞活性氧族的生成， 因此于哺乳动物中保护及 治疗受活性氧族伤害的细胞，其中所述腺嘌呤及 /或其医药学上可接受的盐， 给予需此治疗的哺乳动物。
上述化合物， 其抑制癌细胞生长， 因此预防或治疗癌症， 其中所述腺 嘌呤及 /或其医药学上可接受的盐， 给予需此治疗的哺乳动物。
本发明提供上述化合物作为制备治疗可被 AMPK活化剂改善的疾病或 生理状况的药物的应用。
本发明提供上述化合物作为制备治疗发炎性生理状况或疾病的药物的 应用。
本发明提供上述化合物作为制备预防或治疗前期糖尿病、 第二型糖尿 病、 代谢症候群的一种或其组合的生理状况或疾病的药物的应用。
本发明提供上述化合物作为制备预防或治疗前期糖尿病、 第二型糖尿 病、 代谢症候群的一种或其组合的生理状况或疾病的药物的应用。
本发明提供上述化合物作为制备预防或治疗阿兹海默症的药物的应 用。
本发明提供上述化合物作为制备治疗可被自体吞噬作用改善的疾病或 生理状况的药物的应用。
本发明提供上述化合物作为制备伤口愈合过程抑制疤痕形成的药物的 应用。
本发明提供上述化合物作为制备加强伤口愈合的药物的应用。
本发明提供上述化合物作为制备哺乳动物中保护及治疗受活性氧族伤 害的细胞的药物的应用。
本发明提供上述化合物作为制备预防或治疗癌症药物的应用。
综上， 根据本发明的实施例， 提供一新颖 AMPK活化剂-腺嘌呤， 可于 细胞内活化 AMPK , 因此可于哺乳动物预防或治疗可被 AMPK改善的生理 状况或疾病。
根据本发明的实施例， 提供一通过活化 AMPK降低血糖的方法， 从而 预防或治疗疾病包括:代谢症候群、 前期糖尿病、 第二型糖尿病、 胰岛素抵 抗， 其中给予需要此治疗的哺乳动物有效剂量的腺嘌呤及 /或医药学上可接 根据本发明的实施例， 提供一通过活化 AMPK抗发炎的方法， 从而预 防或治疗发炎性状况或疾病， 其中给予需要此治疗的哺乳动物有效剂量的 腺嘌呤及 /或医药学上可接受的盐。
根据本发明的实施例， 提供一通过活化 AMPK抑制纤维母细胞生长 的方法， 从而于伤口愈合期间预防疤痕组织生成。
根据本发明的实施例， 提供一加强伤口愈合的方法， 其中给予需要此 治疗的哺乳动物有效剂量的腺嘌呤及 /或医药学上可接受的盐。
根据本发明的实施例， 提供一抑制反应性氧族 （ROS) 生成的方法， 从 而保护或治疗哺乳动物的细胞远离反应性氧族伤害， 其中给予需要此治疗 的哺乳动物有效剂量的腺嘌呤及 /或医药学上可接受的盐。
根据本发明的实施例， 提供一抑制癌细胞生长的方法， 从而预防或 治疗癌症， 其中给予需要此治疗的哺乳动物有效剂量的腺嘌呤及 /或医药学 上可接受的盐。
本发明是关于腺嘌呤， 此化合物适用于活化 AMPK, 并使用腺嘌呤于 预防或治疗生理状况或疾病， 包括： 前期糖尿病、 胰岛素抵抗、 第二型糖 尿病、 代谢症候群、 肥胖、 发炎、 伤口愈合、 阿兹海默症、 癌症、 氧化压 力及心血管疾病。
本发明发现， 腺嘌呤为一新颖的 AMPK活化剂， 并具有各种生物性功 能。 近年来， AMPK 活化已被证实有助于疾病的预防及治疗， 如前期糖尿 病、 胰岛素抵抗、 第二型糖尿病、 代谢症候群、 肥胖、 发炎、 伤口愈合、 阿兹海默症、 癌症、 氧化压力、 心血管疾病以及促进伤口愈合。 本发明认 为， 此效果可归因于 AMPK活化所导致但不限于降低环氧化酶 -2表现量、 抑制应性氧族（ROS ) 的生产及葡萄糖摄取活性的增加。
预期适应症 基于本发明的研究成果 （见实施例）， 腺嘌呤可通过活化 AMPK作为使 用于各种生理状况或疾病的治疗剂。 以下提供预期适应症的示例性引导及 证据。
腺嘌呤于高血糖、 前期糖尿病、 胰岛素抵抗、 第二型糖尿病的治疗 最近已有报告指出 AMPK活化剂包括 metformin, A769662, AICAR于糖 尿病或肥胖小鼠模式降低血浆葡萄糖浓度。 于本发明中， 1 μΜ〜600 μΜ 的 腺嘌呤显著增加肌肉细胞 C2C12的葡萄糖摄取（表 2)。 更进一步以高脂肪 饲料喂养小鼠作为第二型糖尿病动物模型评估腺嘌呤对血浆葡萄糖浓度调 节效果。 与控制组的高脂肪饲料喂养小鼠比较， 给予腺嘌呤显著减少高脂 肪饲料喂养小鼠的血浆葡萄糖 30%以上，并减少血浆三酸甘油酯 35%以上， 15%以上的体重减少 （实施例 3)。 这里使用的术语「高血糖」是指一生理状 况， 其特征为血糖高于 126亳克 /分升。 这里使用的术语 「前期糖尿病」是 指一生理状况，其特征为空腹血糖高于 100亳克 /分升但低于 140亳克 /分升。 这里使用的术语 「胰岛素抵抗」是指一生理状况， 其中全身或组织包括肝 脏、 骨胳肌、 脂肪组织无法对胰岛素反应。 这里使用的术语 「第二型糖尿 病」亦指非胰岛素依赖型糖尿病或成人发病型糖尿病。 是指代谢失调引起 的胰岛素生产不足或胰岛素抵抗， 其特征通常为空腹血糖高于 140亳克 /分 升。 根据此实施例， 腺嘌呤被证实加速葡萄糖摄取， 因此可作为与高血糖 有关的生理状况或疾病的有效治疗方式。
腺嘌呤于发炎性疾病的治疗
各种 AMPK 活化剂已被证实于生物体内具抗发炎功能。 举例而言， 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR) 于小鼠模式已被证 实可缓解由三硝基苯磺酸或右旋糖酐硫酸酯钠所引起的急性及复发性结肠 炎， AICAR 治疗显著降低疾病小鼠的体重减少并减缓发炎反应。 此外 AICAR 对于人类多发性硬化症动物模式 (EAE)有明显的治疗效果， 亦降低 以脂多醣诱导小鼠肺损伤的严重程度。 于本发明中， 腺嘌呤于体外试验中 抑制脂多醣诱导的发炎反应： 于脂多醣刺激下， 经腺嘌呤处理的巨噬细胞 的发炎性细胞激素分泌量，包括肿瘤坏死因子 a (TNF- a )、介白素 -1 β ( -1 β ) 及介白素 -6 (IL-6), 与控制组相比有显著性减少。 腺嘌呤亦减少人类巨 噬细胞因脂多醣诱导表现的环氧化酶 -2表现量 （实施例 4)。 于三硝基苯磺 酸诱导的发炎性肠病 (IBD)小鼠模式中， 给予腺嘌呤治疗组别的结肠发炎性 细胞激素，包括肿瘤坏死因子 (TNF)、 干扰素 Y (INF Y )及介白素 (IL- 17)均较 控制组小鼠显著减少， 且挽救体重损失 (实施例 5)。
这里使用的术语 「发炎性细胞激素」是指促进系统性发炎反应的细胞 激素。 这里使用的术语 「发炎性疾病」是指与发炎相关的疾病， 包括但不 限制于僵直性脊推炎、 关节炎 （骨关节炎、 风湿性关节炎、 干癣性关节炎)、 气喘、 动脉粥样硬化、 克隆氏症、 结肠炎、 皮肤炎、 大肠憩室炎、 纤维肌 痛症、 肝炎、 激躁性结肠症、 系统性红斑性狼疮、 肾炎、 阿兹海默症、 帕 金森式症、 溃疡性大肠炎等。 近年来， 许多报告已证实 AMPK是环氧化酶 -2上游调控者， 可抑制环氧化酶 -2的蛋白质表现。 与先前研究相符， 发明 人发现一新颖 AMPK活化剂， 腺嘌呤， 可有效抑制环氧化酶 -2的蛋白质表 现， 由此可知腺嘌呤可抑制环氧化酶 -2 介导的发炎。 根据本发明， 腺嘌呤 被发现可抑制发炎， 因此可作为发炎相关的生理状况或疾病的治疗。
腺嘌呤于伤口愈合及疤痕形成
AMPK被认为可促进细胞能动性并加强伤口愈合。 一 AMPK活化剂， 白藜芦醇， 已被证实可加强手术伤口的愈合。 除了伤口愈合外， 在愈合过 程中减少疤痕的形成一直为现代医学的首选目标。 新生儿伤口愈合不同于 成人的伤口愈合， 不会伴随疤痕的形成， 此间差异在于环氧化酶 -2的活化。 于成人的伤口愈合过程， 环氧化酶 -2活性会经由 TGF-beta被提升， 因而导 致伤口处前列腺素生产的增加。 前列腺素已被证实可促进纤维母细胞增生 及胶原蛋白的形成， 此两种因素可导致疤痕形成。 因此， 抑制环氧化酶 -2 的活性被认为可有效预防疤痕形成。 于本发明中， 腺嘌呤抑制纤维母细胞 生长 （实施例 8) 并降低环氧化酶 -2的蛋白质表现。 于动物模式中， 于伤口 处直接施用腺嘌呤不仅可加强伤口愈合并可减少疤痕生成 (实施例 9)。 依据 上述资料， 局部施用腺嘌呤可有效加强伤口愈合并防止疤痕形成。
神经退化
许多细胞机制的缺陷已被证实与神经退化性疾病有关， 包括发炎、 细 胞内运输、 自体吞噬等。 自体吞噬的功能在于移除细胞内失去功能的胞器 或蛋白质团块， 并且对细胞内平衡扮演重要角色。 许多神经退化性疾病的 发病机制涉及细胞内或细胞外蛋白质团块的沉积， 而移除这些蛋白质团块 已显示可改善此类疾病的进展。 此外， 自体吞噬途径受损或移除负责自体 吞噬的蛋白质已被证实导致神经退化。 AMPK 活化已证实可促进自体吞噬 路径。 因此， 通过活化 AMPK以促进自体吞噬路径可作为预防或控制神经 退化性疾病的有效策略。 AMPK 活化剂已被证实可经由自体吞噬路径减少 类淀粉沉积。每日给予 AMPK活化剂-白藜芦醇可增加阿兹海默症小鼠的寿 命。 另一 AMPK 活化剂-姜黄素亦被证实可作为阿兹海默症治疗的潜在药 物。 于本发明中， 发明人发现腺嘌呤于神经细胞 Neuro-2A中显著加强自体 吞噬活性并降低 A累积， 此外腺嘌呤改善阿兹海默症疾病小鼠的认知功能 (实施例 6、 7)。 根据上述发现， 腺嘌呤可作为神经退化性疾病的治疗使用。
这里使用的术语「神经退化」是指神经元结构或功能逐渐丧失的情况。 神经退化性疾病是为神经退化的结果， 包括但不限制于阿兹海默症、 帕金 森氏症、 汉丁顿舞蹈症、 肌萎缩性脊髓侧索硬化症、 脊髓小脑萎缩症、 脊 髓性肌肉萎缩症等。
反应性氧族相关疾病
生物组织中， 反应性氧族包括超氧化物自由基、 羟基自由基及过氧化 氢不断的被产生， 且过量的反应性氧族与许多疾病有关， 包括但不限制于: 神经组织肌肉无力伴随运动失调与色素沉积性视网膜炎 （NARP)、 MELAS 症候群、 肌抽跃癫痫合并红色褴褛肌纤维症 （MERRF)、 Leber遗传性视神 经萎缩症 （LHON)、 KSS 症候群、 阿兹海默症、 帕金森氏症、 汉丁顿舞蹈 症、 肌萎缩性脊髓侧索硬化症、 弗里德赖希氏共济失调 （FA) 及老化。 许多 研究报告证实 AMPK活化剂如 AICAR, 于高葡萄糖、棕梠酸或白蛋白诱导 状况下， 可减少反应性氧族产生。 于本发明中， 腺嘌呤降低 HUVEC 细胞 中反应性氧族的生产 (表 6), 因此腺嘌呤可作为反应性氧族相关的生理状况 或疾病的治疗使用。
癌症
AMPK活化抑制环氧化酶 -2与哺乳动物雷帕霉素标靶蛋白途径， 此二 路径为癌细胞生长的重要机制。 基于环氧化酶 -2 与哺乳动物雷帕霉素标靶 蛋白对癌症的重要性， 活化 AMPK以达抑制环氧化酶 -2与哺乳动物雷帕霉 素标靶蛋白途径被认为是合理的癌症治疗策略。 事实上， 许多研究报告证 实 AMPK活化剂中断癌症发展， 举例而言， phenformin及 metformin于异 体移植的癌症小鼠模式被发现抑制乳癌肿瘤发展及生长。 于本发明中， 腺 嘌呤抑制人类肝癌细胞 Hep G2、人类乳腺癌细胞 MCF7及结肠癌细胞 HT29 的细胞生长 （实施例 11)。 腺嘌呤对 Hep G2、 MCF7、 HT29的 50%生长抑 制浓度分别为 544.1 , 537.5 and 531.9 μ Μ。 于 Hep G2 移植小鼠模式， 长期 给予腺嘌呤显著延迟肿瘤生长。 根据本发明， 以腺嘌呤活化 AMPK的治疗 方式， 可预防或控制癌症的形成或发展。 具体实施方式 下面结合具体实施例对本发明作进一步说明， 以使本领域的技术人员 可以更好的理解本发明并能予以实施， 但所举实施例不作为对本发明的限 定。
实施例 1
AMPK 活性分析
于小鼠肌肉细胞 C2C12、小鼠纤维母细胞 3T3、人类肝癌细胞 Hep G2、 人类乳腺癌细胞 MCF7 及人类结肠癌细胞 HT29 人类脐带静脉内皮细胞 HUVEC、 人类急性单核白血球细胞株 THP1、 人类巨噬细胞 U937、 小鼠微 神经胶细胞 BV-2、神经母细胞瘤细胞 Neuro2A及毛乳突细胞 Dermal Papilla 进行腺嘌呤对 AMPK 磷酸化影响的分析。 细胞以 Dulbecco's modified Eagle's medium (DMEM) 含 10% 胎牛血清 （FBS), 4mM L-glutamine, 2 mM sodium pyruvate及 1% penicillin I streptomycin (Invitrogen GibcoBRL, Carlsbad, CA, USA) 于 37 。 C、 5% C02环境下培养。 3xl05细胞接种于 6-well 盘， 24小时后以指定化合物处理细胞 30分钟，接着溶解细胞并以西 方墨点法进行分析。 等量蛋白质以十二烷基硫酸钠聚丙烯酰胺凝胶电泳进 行分离，接着转印至聚偏氟乙烯膜。转印后的聚偏氟乙烯膜浸泡至溶于 PBS 缓冲液的 3% 牛血清白蛋白 60分钟后， 分别加入抗磷酸化 AMPK (Thrl72) 抗体（1 :2000, Cell signaling)、 抗 AMPK抗体（1 :2000, Cell signaling) , 于 4 。 C 作用。 16小时后加入对应的二抗于室温下反应 1小时。 具免疫反应带 以冷光受质侦测， 并以底片纪录信号。 所得的信号扫描后以 TotalLab Quant 软件 （TotalLab) 进行分析。
腺嘌呤对 AMPK活化的影响统整于表 1。 于所有测试细胞中， 腺嘌呤 均显著活化 AMPK：。 表 (1 )
细胞 腺嘌呤浓度 （microM) AMPK 活化 （fold to
control)
C2C12 1 1.2
10 1.7
100 3.2
200 3.9
600 4.1
3T3 1 1.1
10 1.5
100 2.9
200 4.0
600 4.2
HepG2 1 1.1
10 2.1
100 3.3
200 3.8
600 4.2
MCF7 1 1.2
10 1.6
100 2.5
200 3.4
600 3.7
Figure imgf000012_0001
Papilla
10 1.4
100 2.1
200 2.5
600 2.8
实施例 2
葡萄糖摄取-生物体外分析
使用萤光葡萄糖类似物 (2-NBDG, Molecular Probes)于肌肉细胞 C2C 12 分析腺嘌呤对葡萄糖摄取的影响。 C2C12细胞以各浓度的腺嘌呤于 37°C下 处理 30分钟后， 加入 500 μΜ萤光葡萄糖类似物， 于室温下培养 5分钟后， 细胞以 Kreb-Hepes 缓冲溶液清洗三次， 并以 70%乙醇固定。 细胞内葡萄糖 类似物的萤光以萤光光度计侦测。
腺嘌呤对葡萄糖摄取的影响统整于表 2。 腺嘌呤显著促进 C2C12细胞 的葡萄糖摄取且具浓度依赖性。 数据表示为三个独立实验的平均值 ±标准 差。 表 (2)
Figure imgf000013_0002
Figure imgf000013_0001
腺嘌呤的抗糖尿病效果
为了进一步评估腺嘌呤对血浆葡萄糖水平调节的影响， 以高脂肪饲料 喂养小鼠作为第二型糖尿病动物模型进行试验。 C57BL/6J 小鼠饲养于 22 ° C、 12小时曰 /夜循环并以不限制饮食方式喂养高脂肪饲料 (60% kcal% fat) 或正常饲料。 0.1-50 亳克 /公斤的腺嘌呤以腹腔注射方式给于 24周龄小鼠， 注射后 1 与 3小时测量血糖值。 腹腔注射高脂肪饲料喂养小鼠一天两次并 持续 6天， 最后一次投药 1小时后， 收集血浆并测量血浆葡萄糖及三酸甘 油脂含量。
与施打生理食盐水的高脂肪饲料喂养小鼠相比， 发现腺嘌呤降低血浆 葡萄糖量大于 30%, 降低三酸甘油脂量大于 35%, 并且降低体重 15%以上。
实施例 4
腺嘌呤抑制脂多醣引起的发炎反应
借由检测人类巨噬细胞内环氧化酶 -2 蛋白质量及肿瘤坏死因子 α (TNF- a )、 介白素 -1 P (IL-1 β ) 及介白素 -6 (IL-6)分泌量评估腺嘌呤对发炎 反应的影响。 以 50 ηΜ ΡΜΑ处理 24小时， 诱导人类急性单核白血球细胞 株 THP1 分化至巨噬细胞。 THP1巨噬细胞进一步以 50 ng脂多醣含 10〜600 μ Μ腺嘌呤或载体刺激 6小时， 接着溶解细胞并以西方墨点法进行分析。 等量蛋白质以十二烷基硫酸钠聚丙烯酰胺凝胶电泳进行分离， 接着转印至 聚偏氟乙烯膜。 转印后的聚偏氟乙烯膜浸泡至溶于 PBS缓冲液的 3% 牛血 清白蛋白 60分钟后， 分别加入抗环氧化酶 -2 抗体（1:1000, Cell signaling), 抗机动蛋白抗体 (1:5000, Cell signaling), 于 4 。 C 作用。 16小时后加入对 应的二抗于室温下反应 1 小时。 具免疫反应带以冷光受质侦测， 并以底片 纪录信号。所得的信号扫描后以 TotalLab Quant 软件 （TotalLab) 进行分析。 肿瘤坏死因子 a (TNF- a )、 介白素 -1 β (IL-1 β )及介白素 -6 (IL-6)分泌量以 酵素连结免疫吸附法分析。
腺嘌呤对免疫反应的影响统整于表 3。 与控制组胞比较， 经腺嘌呤处理 的巨噬细胞， 其环氧化酶 -2 蛋白质表现量及肿瘤坏死因子 a (TNF- a )、 介 白素 -1 P (IL-1 β )及介白素 -6(IL-6)分泌量均显著下降。 表 (3)
腺 嘌 呤 TNF a (% IL-1 β (% to IL-6(% to COX-2 (%
(microM) to control) control) control) to control)
0 100+4.7 100+11.3 100+8.5 100+2.9
10 85±9.1 91+8.4 88+6.3 81+4.4
100 41+2.6 29±5.5 21±7.8 59+3.5
600 23+1.8 17±3.7 14+6.2 38+5.3 实施例 5
腺嘌呤于生物体抑制三硝基苯磺酸诱发的发炎反应
进一步以三硝基苯磺酸诱导的发炎性肠病 (IBD)小鼠模式， 评估腺嘌呤 对发炎反应的影响。 C57BL/6J小鼠饲养于 22。 C、 12小时日 /夜循环。 以五 个逐步提升的三硝基苯磺酸剂量： 0.5 mg、 0.75 mg, 1.0 mg, 1.25 mg与 1.5 mg 溶于 50%乙醇分别每周给予小鼠 O.lmL诱发复发性结肠炎。 第三次给 予三硝基苯磺酸后每日以腹腔注射方式给予小鼠腺嘌呤 (0.01, 0.1, 5 或 30 mg/kg体重)或生理食盐水。第五次给予三硝基苯磺酸后两天即将小鼠牺牲。 结肠组织溶解液的发炎性细胞激素： 肿瘤坏死因子 (TNF)、干扰素 Y (INF Y ) 及介白素 (IL-17)以酵素连结免疫吸附法分析。
给予腺嘌呤治疗组别的结肠发炎性细胞激素包括肿瘤坏死因子 (TNF)、 干扰素 Y (INF γ )及介白素 (IL-17), 均较控制组小鼠显著减少， 且挽救体重 损失。
实施例 6
类淀粉 胜肽及自体吞噬活性分析
以神经母细胞瘤细胞 Neuro2A分析腺嘌呤对类淀粉 β胜肽的影响。
Neuro2A细胞以 Dulbecco's modified Eagle's medium (DMEM)含 10% 月台牛血清 (FBS), 4mM L-glutamine, 2 mM sodium pyruvate及 1% penicillin I streptomycin (Invitrogen GibcoBRL, Carlsbad, CA, USA) 于 37 ° C、 5% C02环境下培养。 3xl05细胞接种于 6-well 盘， 24小时后， 细胞以 APP695 进行转染， 并以腺嘌呤处理细胞 24小时， 接着溶解细胞并以西方墨点法进 行分析。 等量蛋白质以十二烷基硫酸钠聚丙烯酰胺凝胶电泳进行分离， 接 着转印至聚偏氟乙烯膜。 转印后的聚偏氟乙烯膜浸泡至溶于 PBS缓冲液的 3% 牛血清白蛋白 60 分钟后， 分别加入抗类淀粉 β胜肽抗体 （1: 1000, Abeam), 抗 LC3抗体 (1 :1000, Cell signaling), 抗机动蛋白抗体 (1 :5000, Cell signaling), 于 4 。 C 作用。 16小时后加入对应的二抗于室温下反应 1 小时。 具免疫反应带以冷光受质侦测， 并以底片纪录信号。 所得的信号扫 描后以 TotalLab Quant 软件 （TotalLab) 进行分析。
腺嘌呤对类淀粉 β胜肽及 LC3-II/LC3-I 比例的影响统整于表 4。 于 Neuro2A细胞中，腺嘌呤显著性减少类淀粉 β胜肽量并且增加 LC3-II/LC3-I 比例。 由于 LC3-I 转换至 LC3-II代表自体吞噬的活性， 于腺嘌呤处理的细 胞中， 较控制组细胞较高的 LC3-II/LC3-I 比例， 反映腺嘌呤活化自体吞噬 作用的功能。
表 (4)
Figure imgf000016_0001
实施例 7
腺嘌呤于阿兹海默症实验小鼠模式解救类淀粉 β胜肽诱导的神经退化 类淀粉 β胜肽 25-35购买自 Sigma-Aldrich (St. Louis, Missouri)„ 胜肽溶 解于无菌生理食盐水并注射前培养于 37 。 C下 7天。 C57BL/6J小鼠饲养 于 22。 C、 12小时日 /夜循环。成鼠以 ketamine (500 亳克 /公斤)及 xyline (100 亳克 /公斤)麻醉， 并放置于立体定位注射仪。 5 nmol 的类淀粉 β胜肽 25-35 以 10 μΐ注射器注射至侧脑室，侧脑是座标为 -0.5 mm (前后向）、 ± 1 mm (内 外侧向）、 -2.5 mm (背腹向)相对于前囱。 每日以腹腔注射方式给予类淀粉 β 胜肽注射小鼠腺嘌呤或生理食盐水， 腺嘌呤注射剂量为 0.01, 0.1, 5 或 30 亳克 /公斤体重， 连续注射 4周。 4周后， 小鼠认知功能以莫里斯水迷宫方 法分析。 水迷宫以圆形水池进行， 平台置于目标象限的水面下以进行隐藏 平台试验。 于 5 天隐藏平台试验期间， 每次试验小鼠均随机放置于水池内 做为起始点， 每日 6次试验。 5日隐藏平台试验后 1 日进行探索性试验。 进 行探索性试验时， 移除隐藏平台并以目标象限对面象限作为起始点。 以摄 录机纪录小鼠于迷宫中泳动 60秒的情形， 以软件分析小鼠寻找平台时间及 于隐藏平台试验中， 经腺嘌呤治疗的小鼠， 其寻找到平台所花费时间 较控制组小鼠显著性减少。 此试验结果证实腺嘌呤可挽救阿兹海默症实验 小鼠受损的学习及记忆功能。 再者， 腺嘌呤治疗小鼠于探索性试验中， 其 停留于目标象线时间较控制组小鼠长， 证明腺嘌呤增进记忆保留。
实施例 8
腺嘌呤抑制纤维母细胞生长
人类纤维母细胞株 3T3以 Dulbecco's modified Eagle's medium (DMEM) 含 10%胎牛血清 （FBS), 4mM L-glutamine, 2 mM sodium pyruvate 及 1% penicillin I streptomycin (Invitrogen GibcoBRL, Carlsbad, CA, USA) 于 37 。 C、5% C02环境下培养。 于细胞生长试验中， lxlO5细胞接种于 6-well盘,
24小时后， 以指定浓度的腺嘌呤处理细胞 72小时， 计算存活的细胞数。 细 胞以 trypsin-EDTA分离并以 trypan blue进行染色， 以血球计数器计算存活 细胞数。
腺嘌呤对 3T3细胞生长的影响统整于表 5。 由表 5结果可知，腺嘌呤显 著抑制 3T3 细胞生长并具剂量依赖性。 数据表示为三个独立实验的平均值 士标准差。 表 (5)
腺嘌呤 细胞数 （％ to
(microM) control)
0 100+4.3
10 91+2.7
50 73±8.1
100 64+5.3
200 48±2.8
500 33+6.4
1000 27+1 1.3 C57BL/6J 小鼠饲养于 22° C、 12 小时日 /夜循环。 12 周龄成鼠以 ketamine (500 亳克 /公斤)及 xyline (100亳克 /公斤)麻醉，于小鼠背部以 6-mm 皮肤取样器制造伤口。 形成伤口后， 于伤口施用 10〜1200 μ Μ腺嘌呤或食 盐水。 皮肤伤口接着以半透性透明片伤口敷料固定。 小鼠以腺嘌呤或生理 食盐水处理 14天后牺牲。疤痕形成以 Masson's trichrome染色分析 （组织以 4% paraformaldehyde固定)。
经 14天处理后， 腺嘌呤处理伤口的愈合速度较控制组快， 且根据组织 染色分析， 以腺嘌呤处理的伤口再生组织， 其疤痕显著较控制组伤口小。
实施例 10
腺嘌呤降低反应性氧族生成
人类脐带静脉内皮细胞 HUVEC 以 Dulbecco's modified Eagle's medium (DMEM) 含 10% 胎牛血清 （FBS), 4mM L-glutamine, 2 mM sodium pyruvate 及 1% penicillin I streptomycin (Invitrogen GibcoBRL, Carlsbad, CA, USA) 于 37 。 C、 5% C02环境下培养。 2xl04细胞接种于 96-well 黑盘， 24小时后，将培养基置换成含 5.6或 30 mM葡萄糖的 DMEM 培养基并加入指定浓度的腺嘌呤。 处理 24 小时后， 细胞内反应性氧族以 H2DCF-DA侦测。 细胞以 PBS缓冲液清洗 1次后， 培养于 100 μ M DCF 37 ° C 30 分钟。 DCF萤光以盘式萤光分析仪分析 （激发波长 :485 nm; 散射波 长： 530 nm)。
腺嘌呤对反应性氧族生成的影响统整于表 6。腺嘌呤显著性减少高葡萄 糖诱导的反应性氧族生成并具剂量依赖性。 表 (6)
腺嘌呤 葡萄糖 反应性氧族生成 （％ of 5.6 mM
(microM) (mM) glucose)
0 30 275± 8.1
10 30 211± 4.3
100 30 1 16+ 1.7
200 30 38.1± 2.9 600 30 21.7士 3.1
1200 30 22.4± 2.5
实施例 11
癌细胞生长抑制试验
以人类肝癌细胞 Hep G2、 人类乳腺癌细胞 MCF7及人类结肠癌细胞 HT29进行腺嘌呤对癌细胞生长的影响。 细胞以 Dulbecco's modified Eagle's medium (DMEM) 含 10% 胎牛血清 （FBS), 4mM L-glutamine, 2 mM sodium pyruvate 及 1% penicillin I streptomycin (Invitrogen GibcoBRL, Carlsbad, CA, USA) 于 37 。 C、 5% C02环境下培养。 l xlO5细胞接种于 6-well 盘， 24小时后， 以指定浓度的腺嘌呤处理细胞 72小时， 计算存活的 细胞数。 细胞以 trypsin-EDTA分离并以 trypan blue进行染色， 以血球计数 器计算存活细胞数。
腺嘌呤对 Hep G2、 MCF7、 HT29 的 50%生长抑制浓度分别为 544.1, 537.5 and 531.9 μ Μ。
实施例 12
肿瘤生长试验
人类肝癌细胞 Hep G2以 Dulbecco's modified Eagle's medium (DMEM) 含 10%胎牛血清 （FBS), 4mM L-glutamine, 2 mM sodium pyruvate 及 1% penicillin I streptomycin (Invitrogen GibcoBRL, Carlsbad, CA, USA) 于 37
。 C、 5% C02环境下培养。 5xl06细胞以皮下注射方式注射至 8 周龄 NOD-SCID 小鼠。 移植后， 每日以腹腔注射方式给予小鼠 5、 20、 50 亳克
/公斤体重的腺嘌呤， 每 3日测量肿瘤大小。 移植后 14天， 相较于控制组小 鼠， 给予腺嘌呤显著延缓肿瘤的生长。
以上所述实施例仅是为充分说明本发明而所举的较佳的实施例， 本发 明的保护范围不限于此。 本技术领域的技术人员在本发明基础上所作的等 同替代或变换， 均在本发明的保护范围之内。 本发明的保护范围以权利要 求书为准。

Claims

权利要求书
1、 一种用于活化 AMPK的化合物， 其特征在于， 其为腺嘌呤及 /或其 医药学上可接受的盐。
2、 如权利要求 1的化合物，其特征在于，其治疗可被 AMPK活化剂改 善的疾病或生理状况， 其中所述腺嘌呤及 /或其医药学上可接受的盐， 给予 需此治疗的哺乳动物。
3、 如权利要求 1的化合物， 其特征在于， 其降低细胞中发炎性细胞激 素的分泌及环氧化酶 -2 的表现， 因此治疗发炎性生理状况或疾病， 其中所 述腺嘌呤及 /或其医药学上可接受的盐， 给予需此治疗的哺乳动物。
4、 如权利要求 1的化合物， 其特征在于， 其增加细胞葡萄糖的摄取， 因此预防或治疗选自由以下组成的群的生理状况或疾病： 前期糖尿病、 第 二型糖尿病、 代谢症候群， 其中所述腺嘌呤及 /或其医药学上可接受的盐， 给予需此治疗的哺乳动物。
5、 如权利要求 1的化合物， 其特征在于， 其减少哺乳动物的血浆三酸 甘油酯及减少体重， 因此预防或治疗肥胖情况， 其中所述腺嘌呤及 /或其医 药学上可接受的盐， 给予需此治疗的哺乳动物。
6、 如权利要求 1的化合物， 其特征在于， 其抑制细胞中类淀粉 胜肽 的累积， 因此预防或治疗阿兹海默症， 其中所述腺嘌呤及 /或其医药学上可 接受的盐， 给予需此治疗的哺乳动物。
7、 如权利要求 1的化合物， 其特征在于， 其加强细胞自体吞噬活性， 因此治疗可被自体吞噬作用改善的疾病或生理状况， 其中所述腺嘌呤及 /或 其医药学上可接受的盐， 给予需此治疗的哺乳动物。
8、 如权利要求 1的化合物， 其特征在于， 其抑制纤维母细胞生长， 因 此于伤口愈合过程抑制疤痕形成， 其中所述腺嘌呤及 /或其医药学上可接受 的盐， 给予需此治疗的哺乳动物。 99、、 如如权权利利要要求求 11的的化化合合物物，， 其其特特征征在在于于，， 其其加加强强伤伤口口愈愈合合，， 其其中中所所述述 腺腺嘌嘌呤呤及及 //或或其其医医药药学学上上可可接接受受的的盐盐，， 给给予予需需此此治治疗疗的的哺哺乳乳动动物物。。
1100、、 如如权权利利要要求求 11 的的化化合合物物，， 其其特特征征在在于于，， 其其抑抑制制细细胞胞活活性性氧氧族族的的生生 成成，， 因因此此于于哺哺乳乳动动物物中中保保护护及及治治疗疗受受活活性性氧氧族族伤伤害害的的细细胞胞，， 其其中中所所述述腺腺嘌嘌 呤呤及及 //或或其其医医药药学学上上可可接接受受的的盐盐，， 给给予予需需此此治治疗疗的的哺哺乳乳动动物物。。
1111、、 如如权权利利要要求求 11 的的化化合合物物，， 其其特特征征在在于于，， 其其抑抑制制癌癌细细胞胞生生长长，， 因因此此 预预防防或或治治疗疗癌癌症症，， 其其中中所所述述腺腺嘌嘌呤呤及及 //或或其其医医药药学学上上可可接接受受的的盐盐，， 给给予予需需此此 治治疗疗的的哺哺乳乳动动物物。。
1122、、 权权利利要要求求 11所所述述化化合合物物作作为为制制备备治治疗疗可可被被 AAMMPPKK活活化化剂剂改改善善的的疾疾 病病或或生生理理状状况况的的药药物物的的应应用用。。
1133、、 权权利利要要求求 11 所所述述化化合合物物作作为为制制备备治治疗疗发发炎炎性性生生理理状状况况或或疾疾病病的的药药 物物的的应应用用。。
1144、、 权权利利要要求求 11 所所述述化化合合物物作作为为制制备备预预防防或或治治疗疗前前期期糖糖尿尿病病、、 第第二二型型 糖糖尿尿病病、、 代代谢谢症症候候群群的的一一种种或或其其组组合合的的生生理理状状况况或或疾疾病病的的药药物物的的应应用用。。
1155、、 权权利利要要求求 11 所所述述化化合合物物作作为为制制备备预预防防或或治治疗疗前前期期糖糖尿尿病病、、 第第二二型型 糖糖尿尿病病、、 代代谢谢症症候候群群的的一一种种或或其其组组合合的的生生理理状状况况或或疾疾病病的的药药物物的的应应用用。。
1166、、 权权利利要要求求 11 所所述述化化合合物物作作为为制制备备预预防防或或治治疗疗阿阿兹兹海海默默症症的的药药物物的的 应应用用。。
1177、、 权权利利要要求求 11 所所述述化化合合物物作作为为制制备备治治疗疗可可被被自自体体吞吞噬噬作作用用改改善善的的疾疾 病病或或生生理理状状况况的的药药物物的的应应用用。。
1188、、 权权利利要要求求 11 所所述述化化合合物物作作为为制制备备伤伤口口愈愈合合过过程程抑抑制制疤疤痕痕形形成成的的药药 物物的的应应用用。。
1199、、 权权利利要要求求 11所所述述化化合合物物作作为为制制备备加加强强伤伤口口愈愈合合的的药药物物的的应应用用。。
2200、、 权权利利要要求求 11 所所述述化化合合物物作作为为制制备备哺哺乳乳动动物物中中保保护护及及治治疗疗受受活活性性氧氧
Figure imgf000021_0001
2211、、 权权利利要要求求 11所所述述化化合合物物作作为为制制备备预预防防或或治治疗疗癌癌症症药药物物的的应应用用。。
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