Compounds activating AMPK and their use

Abstract

The present invention discloses a compound for activating AMPK, which is adenine and / or a pharmaceutically acceptable salt thereof. The invention also discloses the application of the compound, which is suitable for activating AMPK (AMP-activated protein kinase), and using the compound to prevent or treat physiological conditions or diseases, thereby preventing or treating physiological conditions or diseases that can be improved by AMPK in mammals.

Description

[0001] This invention is a divisional application. The filing date of the original application is September 26, 2013, the application number is 201310444518.X, and the invention title is: Compounds that Activate AMPK and Their Use. Technical Field

[0002] This invention relates to adenine, a compound that is suitable for activating AMPK (AMP-activated protein kinase), and the use of this compound in preventing or treating physiological conditions or diseases. Background Art

[0003] AMPK is clearly a cellular energy sensor and responder to energy demands. AMPK is a heterotrimer composed of a catalytic α subunit, and regulatory β and γ subunits, and all subunits are highly conserved in eukaryotes. AMPK activation is achieved by phosphorylation of the conserved threonine 172 residue of the α subunit by its upstream kinases such as LKB1, calcium / calmodulin-dependent protein kinase (Ca 2+ / Calmodulin-dependent kinase) and TAK1. A high AMP / ATP ratio caused by physiological or pathological stress also activates AMPK. After activation, AMPK promotes catabolic pathways and inhibits anabolism, thereby restoring cellular energy balance by reducing ATP consumption and promoting ATP production.

[0004] As a regulator of energy metabolism balance, AMPK is considered a potential drug target for metabolic syndrome, including type 2 diabetes, cardiovascular diseases, fatty liver, etc. Many metabolic syndromes are related to insulin resistance. Insulin resistance is a pathological state in which cells cannot respond to insulin, so excessive glucose in the blood cannot be removed to skeletal muscle or adipose tissue. In muscle cells, AMPK activation increases the expression level of glucose transporter (GLUT4) in a non-insulin-dependent manner through transcriptional regulation, and induces the translocation of GLUT4 to the cell membrane, resulting in an increased rate of glucose uptake by cells. AMPK activation also inhibits fatty acid and cholesterol synthesis by inhibiting acetyl-CoA carboxylase and HMG-CoA reductase, respectively. In addition, AMPK activation leads to the inhibition of several transcription factors, including SREBP-1c, ChREBP and HNF-4a, and down-regulates the protein expression of enzymes related to fatty acid synthesis and gluconeogenesis. The above-mentioned research findings all support that AMPK is a therapeutic target for metabolic syndrome, especially diabetes.

[0005] In addition to regulating energy metabolism balance, AMPK is also involved in the regulation of several cellular mechanisms, including inflammation, cell growth, apoptosis, autophagy, aging, and differentiation. Many studies have shown that AMPK is an inflammation inhibitor. AMPK activation inhibits inflammation by suppressing the signaling of nuclear transcription factor (NF-κB). The signaling of nuclear transcription factor is the main pathway for activating innate and adaptive immunity. When AMPK is activated, it inhibits the transcriptional activity of nuclear transcription factor by stimulating SIRT1, Forkhead box O (FoxO), or peroxisome proliferator-activated receptor co-activator 1α (PGC1α) to achieve the effect of inhibiting inflammation. In addition, several research groups have also demonstrated that AMPK activation inhibits the protein expression of cyclooxygenase-2 (COX-2). Cyclooxygenase-2 is an inducible enzyme that can be regulated by inflammatory cytokines and growth factors. Its function is to convert arachidonic acid into prostaglandins, which leads to inflammation and pain. Therefore, inhibiting the activity or expression of cyclooxygenase has been proven to have anti-inflammatory effects.

[0006] Several AMPK activators have been proven to have anti-inflammatory functions in in vivo experiments. For example, 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR) has been shown to alleviate acute and recurrent colitis induced by trinitrobenzenesulfonic acid or dextran sulfate sodium in a mouse model. AICAR treatment significantly reduced weight loss in diseased mice and alleviated the inflammatory response. In addition, AICAR has a significant therapeutic effect on the animal model of human multiple sclerosis (EAE) and also reduces the severity of lipopolysaccharide-induced lung injury in mice.

[0007] Dysregulation of the cell signaling pathway may lead to abnormal cell growth and eventually result in cancer. Mammalian target of rapamycin (mTOR) is a serine / threonine kinase that regulates cell growth and autophagy. Dysregulation of the mTOR signaling pathway activity has been found in many different cancers, so mTOR inhibitors are considered potential drugs for cancer treatment. A large number of studies have confirmed that AMPK phosphorylates tuberous sclerosis complex 2 (TSC2) and Raptor to inhibit the mTOR pathway. Various AMPK activators, including AICAR, metformin, and phenformin, have been shown to inhibit the mTOR signaling pathway and suppress cancer cell growth. In addition, AMPK activation induces autophagy by inhibiting mTOR complex-1. Since AMPK inhibits mTOR complex-1, the phosphorylation of serine 757 on Ulk1 decreases, and then serine 317 and 777 are phosphorylated by AMPK. The phosphorylation of Ulk1 by AMPK subsequently initiates autophagy.

[0008] In summary, AMPK is considered a good therapeutic target for many human diseases or pathological conditions, including inflammatory diseases, wound healing, neurodegeneration, cancer, oxidative stress, and cardiovascular diseases. In fact, AMPK activators have been applied in clinical trials for at least 24 categories of diseases, including: bacterial and fungal diseases, behavioral and psychological disorders, blood and lymphatic diseases, cancer, tumors, digestive system diseases, ear, nose, and throat diseases, eye diseases, glandular and hormonal related diseases, cardiovascular diseases, immune system diseases, mouth and dental diseases, muscle, skeletal, and cartilage diseases, nervous system diseases, nutritional and metabolic diseases, respiratory diseases, skin and connective tissue diseases, wound healing, etc. Summary of the Invention

[0009] The present invention provides a novel AMPK activator - adenine and the use of this compound for preventing or treating diseases.

[0010] The present invention provides a compound for activating AMPK, which is adenine and / or its pharmaceutically acceptable salts.

[0011] The present invention provides the use of the above compound as a drug for preparing a medicament for treating diseases or physiological conditions that can be improved by AMPK activators.

[0012] The present invention provides the use of the above compound as a drug for preparing a medicament for treating inflammatory physiological conditions or diseases.

[0013] The present invention provides the use of the above-mentioned compound as a drug for preparing a medicament for preventing or treating one or a combination of prediabetes, type 2 diabetes, and metabolic syndrome, or a physiological condition or disease thereof.

[0014] The present invention provides the use of the above-mentioned compound as a drug for preparing a medicament for preventing or treating one or a combination of prediabetes, type 2 diabetes, and metabolic syndrome, or a physiological condition or disease thereof.

[0015] The present invention provides the use of the above-mentioned compound as a drug for preparing a medicament for preventing or treating Alzheimer's disease.

[0016] The present invention provides the use of the above-mentioned compound as a drug for preparing a medicament for treating a disease or physiological condition that can be improved by autophagy.

[0017] The present invention provides the use of the above-mentioned compound as a drug for preparing a medicament for inhibiting scar formation during the wound healing process.

[0018] The present invention provides the use of the above-mentioned compound as a drug for preparing a medicament for enhancing wound healing.

[0019] The present invention provides the use of the above-mentioned compound as a drug for preparing a medicament for protecting and treating cells damaged by reactive oxygen species in mammals.

[0020] The present invention provides the use of the above-mentioned compound as a drug for preparing a medicament for preventing or treating cancer.

[0021] According to an embodiment of the present invention, a novel AMPK activator - adenine is provided, which can activate AMPK intracellularly, and thus can prevent or treat a physiological condition or disease that can be improved by AMPK in mammals.

[0022] According to an embodiment of the present invention, a method for reducing blood glucose by activating AMPK is provided, thereby preventing or treating diseases including: metabolic syndrome, prediabetes, type 2 diabetes, and insulin resistance, wherein an effective dose of adenine and / or a pharmaceutically acceptable salt is administered to a mammal in need of such treatment.

[0023] According to an embodiment of the present invention, a method for anti - inflammation by activating AMPK is provided, thereby preventing or treating an inflammatory condition or disease, wherein an effective dose of adenine and / or a pharmaceutically acceptable salt is administered to a mammal in need of such treatment.

[0024] According to an embodiment of the present invention, a method for inhibiting fibroblast growth by activating AMPK is provided, thereby preventing the formation of scar tissue during wound healing.

[0025] According to an embodiment of the present invention, a method for enhancing wound healing is provided, wherein an effective dose of adenine and / or a pharmaceutically acceptable salt is administered to a mammal in need of such treatment.

[0026] According to an embodiment of the present invention, a method for inhibiting the generation of reactive oxygen species (ROS) is provided to protect or treat mammalian cells from reactive oxygen species damage, wherein an effective dose of adenine and / or a pharmaceutically acceptable salt is administered to a mammal in need of such treatment.

[0027] According to an embodiment of the present invention, a method for inhibiting the growth of cancer cells is provided to prevent or treat cancer, wherein an effective dose of adenine and / or a pharmaceutically acceptable salt is administered to a mammal in need of such treatment.

[0028] The present invention relates to adenine, which is a compound suitable for activating AMPK, and the use of adenine in preventing or treating physiological conditions or diseases, including: prediabetes, insulin resistance, type 2 diabetes, metabolic syndrome, obesity, inflammation, wound healing, Alzheimer's disease, cancer, oxidative stress, and cardiovascular diseases.

[0029] The present invention discovers that adenine is a novel AMPK activator and has various biological functions. In recent years, AMPK activation has been proven to contribute to the prevention and treatment of diseases, such as prediabetes, insulin resistance, type 2 diabetes, metabolic syndrome, obesity, inflammation, wound healing, Alzheimer's disease, cancer, oxidative stress, cardiovascular diseases, and promoting wound healing. The present invention believes that this effect can be attributed to AMPK activation, which results in but is not limited to reducing the expression level of cyclooxygenase-2, inhibiting the production of reactive oxygen species (ROS), and increasing glucose uptake activity.

[0030] Expected indications

[0031] Based on the research results of the present invention (see Examples), adenine can be used as a therapeutic agent for various physiological conditions or diseases by activating AMPK. The following provides exemplary guidance and evidence for expected indications.

[0032] Treatment of adenine for hyperglycemia, prediabetes, insulin resistance, and type 2 diabetes

[0033] Recently, it has been reported that AMPK activators, including metformin, A769662, and AICAR, reduce plasma glucose concentration in diabetic or obese mouse models. In the present invention, 1 μM to 600 μM of adenine significantly increases glucose uptake in muscle cells C2C12 (Table 2). Furthermore, mice fed a high-fat diet were used as a type 2 diabetes animal model to evaluate the effect of adenine on plasma glucose concentration regulation. Compared with the control group of mice fed a high-fat diet, administration of adenine significantly reduced plasma glucose in high-fat diet-fed mice by more than 30%, reduced plasma triglycerides by more than 35%, and reduced body weight by more than 15% (Example 3). The term "hyperglycemia" as used herein refers to a physiological condition characterized by blood glucose levels higher than 126 mg / dL. The term "prediabetes" as used herein refers to a physiological condition characterized by fasting blood glucose levels higher than 100 mg / dL but lower than 140 mg / dL. The term "insulin resistance" as used herein refers to a physiological condition in which the whole body or tissues, including the liver, skeletal muscle, and adipose tissue, are unable to respond to insulin. The term "type 2 diabetes" as used herein also refers to non-insulin-dependent diabetes or adult-onset diabetes. It refers to insufficient insulin production or insulin resistance caused by metabolic disorders, which is usually characterized by fasting blood glucose levels higher than 140 mg / dL. According to this example, adenine was proven to accelerate glucose uptake and thus can be used as an effective treatment for physiological conditions or diseases related to hyperglycemia.

[0034] Treatment of Adenine in Inflammatory Diseases

[0035] Various AMPK activators have been proven to have anti-inflammatory functions in organisms. For example, 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR) has been proven to alleviate acute and recurrent colitis caused by trinitrobenzenesulfonic acid or dextran sulfate sodium in a mouse model. AICAR treatment significantly reduced the weight loss of diseased mice and alleviated the inflammatory response. In addition, AICAR has obvious therapeutic effects on the animal model of human multiple sclerosis (EAE), and also reduces the severity of lipopolysaccharide-induced lung injury in mice. In the present invention, adenine inhibits lipopolysaccharide-induced inflammatory response in vitro: under lipopolysaccharide stimulation, the secretion levels of inflammatory cytokines in macrophages treated with adenine, including tumor necrosis factor α (TNF-α), interleukin-1β (IL-1β), and interleukin-6 (IL-6), were significantly reduced compared with the control group. Adenine also reduced the expression level of cyclooxygenase-2 expressed by human macrophages induced by lipopolysaccharide (Example 4). In a mouse model of trinitrobenzenesulfonic acid-induced inflammatory bowel disease (IBD), the colonic inflammatory cytokines in the adenine treatment group, including tumor necrosis factor (TNF), interferon γ (INFγ), and interleukin (IL-17), were significantly reduced compared with the control group mice, and the weight loss was rescued (Example 5).

[0036] The term "inflammatory cytokine" used herein refers to cytokines that promote systemic inflammatory responses. The term "inflammatory disease" used herein refers to diseases related to inflammation, including but not limited to ankylosing spondylitis, arthritis (osteoarthritis, rheumatoid arthritis, psoriatic arthritis), asthma, atherosclerosis, Crohn's disease, colitis, dermatitis, diverticulitis, fibromyalgia, hepatitis, irritable bowel syndrome, systemic lupus erythematosus, nephritis, Alzheimer's disease, Parkinson's disease, ulcerative colitis, etc. In recent years, many reports have confirmed that AMPK is an upstream regulator of cyclooxygenase-2 and can inhibit the protein expression of cyclooxygenase-2. Consistent with previous studies, the inventors found a novel AMPK activator, adenine, which can effectively inhibit the protein expression of cyclooxygenase-2. Thus, it can be seen that adenine can inhibit cyclooxygenase-2-mediated inflammation. According to the present invention, adenine is found to inhibit inflammation and can therefore be used for the treatment of physiological conditions or diseases related to inflammation.

[0037] Adenine in Wound Healing and Scar Formation

[0038] AMPK is considered to promote cell motility and enhance wound healing. An AMPK activator, resveratrol, has been shown to enhance the healing of surgical wounds. In addition to wound healing, reducing scar formation during the healing process has always been a primary goal of modern medicine. Neonatal wound healing is different from adult wound healing in that it does not involve scar formation, and the difference lies in the activation of cyclooxygenase-2. During adult wound healing, cyclooxygenase-2 activity is elevated via TGF-beta, leading to an increase in prostaglandin production at the wound site. Prostaglandins have been shown to promote fibroblast proliferation and collagen formation, two factors that can lead to scar formation. Therefore, inhibiting the activity of cyclooxygenase-2 is considered to be effective in preventing scar formation. In the present invention, adenine inhibits fibroblast growth (Example 8) and reduces the protein expression of cyclooxygenase-2. In an animal model, direct application of adenine to the wound not only enhances wound healing but also reduces scar formation (Example 9). Based on the above information, topical application of adenine can effectively enhance wound healing and prevent scar formation.

[0039] Neurodegeneration

[0040] Defects in many cellular mechanisms have been shown to be associated with neurodegenerative diseases, including inflammation, intracellular trafficking, autophagy, etc. The function of autophagy is to remove dysfunctional organelles or protein aggregates within cells and plays an important role in cellular homeostasis. The pathogenesis of many neurodegenerative diseases involves the deposition of intracellular or extracellular protein aggregates, and the removal of these protein aggregates has been shown to improve the progression of such diseases. In addition, impairment of the autophagy pathway or removal of proteins responsible for autophagy has been shown to lead to neurodegeneration. AMPK activation has been shown to promote the autophagy pathway. Therefore, promoting the autophagy pathway by activating AMPK can be an effective strategy for preventing or controlling neurodegenerative diseases. AMPK activators have been shown to reduce amyloid deposition via the autophagy pathway. Daily administration of the AMPK activator - resveratrol can increase the lifespan of Alzheimer's disease mice. Another AMPK activator - curcumin has also been shown to be a potential drug for the treatment of Alzheimer's disease. In the present invention, the inventors found that adenine significantly enhances autophagic activity and reduces A accumulation in Neuro-2A nerve cells, and in addition, adenine improves the cognitive function of Alzheimer's disease mice (Examples 6, 7). Based on the above findings, adenine can be used for the treatment of neurodegenerative diseases.

[0041] The term "neurodegeneration" as used herein refers to the progressive loss of neuronal structure or function. 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 ataxia, spinal muscular atrophy, etc.

[0042] Reactive oxygen species-related diseases

[0043] In biological tissues, reactive oxygen species, including superoxide radicals, hydroxyl radicals, and hydrogen peroxide, are constantly generated, and excessive reactive oxygen species are associated with many diseases, including but not limited to: neurological tissue muscle weakness accompanied by ataxia and pigmentary retinopathy (NARP), MELAS syndrome, myoclonic epilepsy with ragged red fibers (MERRF), Leber hereditary optic neuropathy (LHON), KSS syndrome, Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, Friedreich's ataxia (FA), and aging. Many research reports have confirmed that AMPK activators such as AICAR can reduce the production of reactive oxygen species under conditions induced by high glucose, palmitic acid, or albumin. In the present invention, adenine reduces the production of reactive oxygen species in HUVEC cells (Table 6), so adenine can be used for the treatment of reactive oxygen species-related physiological conditions or diseases.

[0044] Cancer

[0045] AMPK activation inhibits the cyclooxygenase-2 and mammalian target of rapamycin pathways, which are important mechanisms for cancer cell growth. Based on the importance of cyclooxygenase-2 and mammalian target of rapamycin in cancer, activating AMPK to inhibit the cyclooxygenase-2 and mammalian target of rapamycin pathways is considered a reasonable cancer treatment strategy. In fact, many research reports have confirmed that AMPK activators interrupt cancer development. For example, phenformin and metformin have been found to inhibit the development and growth of breast cancer tumors in xenograft cancer mouse models. In the present invention, adenine inhibits the cell growth of human hepatocellular carcinoma cells Hep G2, human breast cancer cells MCF7, and colon cancer cells HT29 (Example 11). The 50% growth inhibitory concentrations of adenine for Hep G2, MCF7, and HT29 are 544.1, 537.5, and 531.9 μM, respectively. In the Hep G2 xenograft mouse model, long-term administration of adenine significantly delays tumor growth. According to the present invention, the treatment method of activating AMPK with adenine can prevent or control the formation or development of cancer. Detailed implementation manners

[0046] The present invention will be further described below in conjunction with specific embodiments, so that those skilled in the art can better understand the present invention and be able to implement it, but the exemplified embodiments are not intended to limit the present invention.

[0047] Example 1

[0048] AMPK activity analysis

[0049] Analysis of the effect of adenine on AMPK phosphorylation was performed on mouse muscle cells C2C12, mouse fibroblasts 3T3, human hepatoma cells Hep G2, human breast cancer cells MCF7, human colon cancer cells HT29, human umbilical vein endothelial cells HUVEC, human acute monocytic leukemia cell line THP1, human macrophages U937, mouse microglial cells BV-2, neuroblastoma cells Neuro2A, and dermal papilla cells. Cells were cultured in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum (FBS), 4 mM L-glutamine, 2 mM sodium pyruvate, and 1% penicillin / streptomycin (Invitrogen GibcoBRL, Carlsbad, CA, USA) at 37 °C and 5% CO 2 environment. 3×10 5 cells were seeded in 6-well plates. After 24 hours, the cells were treated with the designated compound for 30 minutes, and then the cells were lysed and analyzed by Western blotting. 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 soaked in 3% bovine serum albumin dissolved in PBS buffer for 60 minutes, and then anti-phosphorylated AMPK (Thr172) antibody (1:2000, Cell signaling) and anti-AMPK antibody (1:2000, Cell signaling) were added and incubated at 4 °C. After 16 hours, the corresponding secondary antibody was added and reacted at room temperature for 1 hour. The immunoreactive bands were detected with chemiluminescent substrate and the signals were recorded on film. The obtained signals were scanned and analyzed using TotalLabQuant software (TotalLab).

[0050] The effects of adenine on AMPK activation are summarized in Table 1. In all tested cells, adenine significantly activated AMPK.

[0051] Table (1)

[0052]

[0053]

[0054] Example 2

[0055] Glucose uptake - in vitro analysis

[0056] The effect of adenine on glucose uptake was analyzed in muscle cells C2C12 using the fluorescent glucose analog (2-NBDG, Molecular Probes). After C2C12 cells were treated with various concentrations of adenine at 37 °C for 30 minutes, 500 μM of the fluorescent glucose analog was added. After culturing at room temperature for 5 minutes, the cells were washed three times with Kreb-Hepes buffer solution and fixed with 70% ethanol. The fluorescence of the glucose analog in the cells was detected with a fluorometer.

[0057] The effects of adenine on glucose uptake were summarized in Table 2. Adenine significantly promoted glucose uptake in C2C12 cells in a concentration-dependent manner. Data are presented as the mean ± standard deviation of three independent experiments.

[0058] Table (2)

[0059] Reagent Concentration (microM) Glucose uptake (% to control) Adenine 1 117±8.1 10 261±13.4 100 315±11.9 600 338±16.5

[0060] Example 3

[0061] The antidiabetic effect of adenine

[0062] To further evaluate the effect of adenine on the regulation of plasma glucose levels, mice fed a high-fat diet were used as a type 2 diabetes animal model for testing. C57BL / 6J mice were housed at 22 °C on a 12-hour light / dark cycle and fed a high-fat diet (60% kcal% fat) or a normal diet ad libitum. Adenine at 0.1 - 50 mg / kg was administered intraperitoneally to 24-week-old mice, and blood glucose levels were measured 1 and 3 hours after injection. Mice fed a high-fat diet were injected intraperitoneally twice a day for 6 days. One hour after the last dose, plasma was collected and the plasma glucose and triglyceride levels were measured.

[0063] Compared with high-fat diet-fed mice injected with physiological saline, it was found that adenine reduced plasma glucose by more than 30%, reduced triglycerides by more than 35%, and reduced body weight by more than 15%.

[0064] Example 4

[0065] Adenine inhibits lipopolysaccharide-induced inflammatory responses

[0066] The effects of adenine on the inflammatory response were evaluated by detecting the protein levels of cyclooxygenase-2 and the secretion levels of tumor necrosis factor α (TNF-α), interleukin-1β (IL-1β), and interleukin-6 (IL-6) in human macrophages. Human acute monocytic leukemia cell line THP1 was induced to differentiate into macrophages by treatment with 50 nM phorbol 12-myristate 13-acetate (PMA) for 24 hours. THP1 macrophages were further stimulated with 50 ng lipopolysaccharide containing 10 - 600 μM adenine or vehicle for 6 hours, and then the cells were lysed and analyzed by Western blotting. Equal amounts of protein were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and then transferred to a polyvinylidene fluoride membrane. After the transferred polyvinylidene fluoride membrane was soaked in 3% bovine serum albumin dissolved in PBS buffer for 60 minutes, anti-cyclooxygenase-2 antibody (1:1000, Cell signaling) and anti-actin antibody (1:5000, Cell signaling) were added and incubated at 4°C. After 16 hours, the corresponding secondary antibody was added and reacted at room temperature for 1 hour. The immunoreactive bands were detected with chemiluminescent substrate and the signals were recorded on a film. The obtained signals were scanned and analyzed with TotalLab Quant software (TotalLab). The secretion levels of tumor necrosis factor α (TNF-α), interleukin-1β (IL-1β), and interleukin-6 (IL-6) were analyzed by enzyme-linked immunosorbent assay.

[0067] The effects of adenine on the immune response are summarized in Table 3. Compared with the control group, the protein expression levels of cyclooxygenase-2 and the secretion levels of tumor necrosis factor α (TNF-α), interleukin-1β (IL-1β), and interleukin-6 (IL-6) in adenine-treated macrophages were significantly decreased.

[0068] Table (3)

[0069]

[0070] Example 5

[0071] Adenine inhibits trinitrobenzenesulfonic acid-induced inflammatory response in vivo

[0072] Furthermore, a mouse model of trinitrobenzenesulfonic acid-induced inflammatory bowel disease (IBD) was used to evaluate the effect of adenine on the inflammatory response. C57BL / 6J mice were housed at 22 °C with a 12-hour light / dark cycle. Recurrent colitis was induced by administering five escalating doses of trinitrobenzenesulfonic acid: 0.5 mg, 0.75 mg, 1.0 mg, 1.25 mg, and 1.5 mg in 50% ethanol, 0.1 mL per mouse per week. After the third administration of trinitrobenzenesulfonic acid, adenine (0.01, 0.1, 5, or 30 mg / kg body weight) or saline was administered intraperitoneally to the mice daily. The mice were sacrificed two days after the fifth administration of trinitrobenzenesulfonic acid. Inflammatory cytokines in the colon tissue lysate, including tumor necrosis factor (TNF), interferon γ (INFγ), and interleukin (IL-17), were analyzed by enzyme-linked immunosorbent assay.

[0073] Colonic inflammatory cytokines, including tumor necrosis factor (TNF), interferon γ (INFγ), and interleukin (IL-17), in the adenine-treated groups were significantly reduced compared with those in the control group mice, and body weight loss was rescued.

[0074] Example 6

[0075] Amyloid-β peptide and autophagy activity analysis

[0076] Neuroblastoma cells Neuro2A were used to analyze the effect of adenine on amyloid-β peptide.

[0077] Neuro2A cells were cultured in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum (FBS), 4 mM L-glutamine, 2 mM sodium pyruvate, and 1% penicillin / streptomycin (Invitrogen GibcoBRL, Carlsbad, CA, USA) at 37 °C in a 5% CO 2 environment. 3×10 5Cells were seeded in 6-well plates. After 24 hours, the cells were transfected with APP695 and treated with adenine for 24 hours. Then the cells were lysed and analyzed by Western blotting. Equal amounts of protein were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and then transferred to polyvinylidene fluoride membranes. After the transferred polyvinylidene fluoride membranes were soaked in 3% bovine serum albumin dissolved in PBS buffer for 60 minutes, anti-amyloid-β peptide antibody (1:1000, Abcam), anti-LC3 antibody (1:1000, Cell signaling), and anti-actin antibody (1:5000, Cell signaling) were added respectively and reacted at 4°C. After 16 hours, the corresponding secondary antibody was added and reacted at room temperature for 1 hour. The immunoreactive bands were detected with chemiluminescent substrates and the signals were recorded on films. The obtained signals were scanned and analyzed with TotalLab Quant software (TotalLab).

[0078] The effects of adenine on amyloid-β peptide and LC3-II / LC3-I ratio are summarized in Table 4. In Neuro2A cells, adenine significantly decreased the amount of amyloid-β peptide and increased the LC3-II / LC3-I ratio. Since the conversion of LC3-I to LC3-II represents autophagic activity, the higher LC3-II / LC3-I ratio in adenine-treated cells compared to control cells reflects the function of adenine in activating autophagy.

[0079] Table (4)

[0080]

[0081] Example 7

[0082] Adenine rescues amyloid-β peptide-induced neurodegeneration in an Alzheimer's disease experimental mouse model

[0083] Amyloid-β peptide 25-35 was purchased from Sigma-Aldrich (St. Louis, Missouri). The peptide was dissolved in sterile saline and cultured at 37 °C for 7 days before injection. C57BL / 6J mice were housed at 22 °C on a 12-hour light / dark cycle. Adult mice were anesthetized with ketamine (500 mg / kg) and xylazine (100 mg / kg) and placed in a stereotaxic injector. 5 nmol of amyloid-β peptide 25-35 was injected into the lateral ventricle with a 10-μl syringe. The coordinates of the lateral ventricle were -0.5 mm (anteroposterior), ±1 mm (mediolateral), and -2.5 mm (dorsoventral) relative to the bregma. Amyloid-β peptide-injected mice were given adenine or saline by intraperitoneal injection daily. The adenine injection doses were 0.01, 0.1, 5, or 30 mg / kg body weight for 4 consecutive weeks. After 4 weeks, the cognitive function of the mice was analyzed by the Morris water maze method. The water maze was a circular pool with a platform placed under the water surface in the target quadrant for the hidden platform test. During the 5-day hidden platform test, the mice were randomly placed in the pool as the starting point for each test, 6 times a day. One day after the 5-day hidden platform test, an exploratory test was conducted. During the exploratory test, the hidden platform was removed and the opposite quadrant of the target quadrant was used as the starting point. The swimming behavior of the mice in the maze for 60 seconds was recorded by a video camera, and the software was used to analyze the time for the mice to find the platform and the swimming path.

[0084] In the hidden platform test, the time taken for adenine-treated mice to find the platform was significantly reduced compared to that of the control group mice. This experimental result confirmed that adenine could rescue the impaired learning and memory functions of Alzheimer's disease experimental mice. Furthermore, in the exploratory test, adenine-treated mice stayed in the target quadrant for a longer time than the control group mice, demonstrating that adenine enhanced memory retention.

[0085] Example 8

[0086] Adenine inhibits fibroblast growth

[0087] The human fibroblast cell line 3T3 was cultured in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum (FBS), 4 mM L-glutamine, 2 mM sodium pyruvate, and 1% penicillin / streptomycin (Invitrogen GibcoBRL, Carlsbad, CA, USA) at 37 °C in a 5% CO 2 environment. In the cell growth assay, 1 × 10 5Cells were seeded in 6-well plates. After 24 hours, the cells were treated with adenine at the specified concentration for 72 hours, and the number of surviving cells was calculated. The cells were detached with trypsin-EDTA and stained with trypan blue, and the number of surviving cells was counted using a hemocytometer.

[0088] The effects of adenine on the growth of 3T3 cells are summarized in Table 5. As shown in the results of Table 5, adenine significantly inhibited the growth of 3T3 cells in a dose-dependent manner. Data are expressed as the mean ± standard deviation of three independent experiments.

[0089] Table (5)

[0090] Adenine (microM) Cell number (% to 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±11.3

[0091] Example 9

[0092] Adenine enhances wound healing and reduces scar formation

[0093] C57BL / 6J mice were housed at 22 °C on a 12-hour light / dark cycle. Adult mice at 12 weeks of age were anesthetized with ketamine (500 mg / kg) and xylazine (100 mg / kg), and a 6-mm skin punch was used to create a wound on the back of the mice. After the wound was formed, 10 - 1200 μM adenine or saline was applied to the wound. The skin wound was then fixed with a semi-permeable transparent film wound dressing. The mice were sacrificed 14 days after treatment with adenine or physiological saline. Scar formation was analyzed by Masson's trichrome staining (tissues were fixed with 4% paraformaldehyde).

[0094] After 14 days of treatment, the wound healing rate of the adenine-treated wounds was faster than that of the control group, and according to histological staining analysis, the regenerated tissue of the adenine-treated wounds had significantly smaller scars than those of the control group wounds.

[0095] Example 10

[0096] Adenine reduces reactive oxygen species generation

[0097] Human umbilical vein endothelial cells HUVEC were cultured in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum (FBS), 4 mM L-glutamine, 2 mM sodium pyruvate, and 1% penicillin / streptomycin (Invitrogen GibcoBRL, Carlsbad, CA, USA) at 37 °C in a 5% CO 2 environment. 2×10 4Cells were seeded in 96-well black plates. After 24 hours, the medium was replaced with DMEM medium containing 5.6 or 30 mM glucose and adenine at the specified concentration. After 24 hours of treatment, intracellular reactive oxygen species were detected with H2DCF-DA. Cells were washed once with PBS buffer and then incubated with 100 μM DCF at 37 °C for 30 minutes. DCF fluorescence was analyzed with a plate fluorometer (excitation wavelength: 485 nm; emission wavelength: 530 nm).

[0098] The effects of adenine on reactive oxygen species generation are summarized in Table 6. Adenine significantly reduced high-glucose-induced reactive oxygen species generation in a dose-dependent manner.

[0099] Table (6)

[0100]

[0101] Example 11

[0102] Cancer cell growth inhibition assay

[0103] The effects of adenine on cancer cell growth were investigated using human hepatocellular carcinoma cells Hep G2, human breast cancer cells MCF7, and human colon cancer cells HT29. Cells were cultured in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum (FBS), 4 mM L-glutamine, 2 mM sodium pyruvate, and 1% penicillin / streptomycin (Invitrogen GibcoBRL, Carlsbad, CA, USA) at 37 °C in a 5% CO 2 environment. 1×10 5 Cells were seeded in 6-well plates. After 24 hours, the cells were treated with adenine at the specified concentration for 72 hours, and the number of viable cells was counted. Cells were detached with trypsin-EDTA and stained with trypan blue, and the number of viable cells was counted with a hemocytometer.

[0104] The 50% growth inhibitory concentrations of adenine for Hep G2, MCF7, and HT29 were 544.1, 537.5, and 531.9 μM, respectively.

[0105] Example 12

[0106] Tumor growth assay

[0107] Human hepatocellular carcinoma cell line Hep G2 was cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 4 mM L-glutamine, 2 mM sodium pyruvate, and 1% penicillin / streptomycin (Invitrogen GibcoBRL, Carlsbad, CA, USA) at 37 °C in a 5% CO 2 2 environment. 5 × 10 6 cells were injected subcutaneously into 8-week-old NOD-SCID mice. After transplantation, the mice were administered adenine at 5, 20, and 50 mg / kg body weight by intraperitoneal injection daily, and the tumor size was measured every 3 days. Fourteen days after transplantation, compared with the control group of mice, the administration of adenine significantly retarded tumor growth.

[0108] The above-described embodiments are merely preferred embodiments given to fully illustrate the present invention, and the protection scope of the present invention is not limited thereto. Equivalent substitutions or transformations made by those skilled in the art on the basis of the present invention are all within the protection scope of the present invention. The protection scope of the present invention shall be subject to the claims.

Claims

1. A use of adenine and / or its pharmaceutically acceptable salt as a drug for the preparation of a drug for preventing or treating physiological conditions or diseases related to reactive oxygen species, characterized in that: The reactive oxygen species-related physiological condition or disease is Alzheimer's disease, wherein the effective dosage of adenine and / or a pharmaceutically acceptable salt thereof is 0.01 mg / kg body weight to 30 mg / kg body weight.

2. The use according to claim 1, characterized in that: The adenine and / or its pharmaceutically acceptable salt is a single effective ingredient.

3. The use according to claim 1, characterized in that: The effective dose of adenine and / or its pharmaceutically acceptable salt is greater than 10 μM.

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