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以功能性生物標記平台開發 老化生技產品 蘇益仁 教授 國家衛生研究院 特聘研究員 成功大學 講座教授 南臺科技大學 講座教授 21世紀疾病與失智症有共通的起源－飲食失調 Major Diseases in 21st Century: Metabolic syndrome (obesity), Cancers, and Ageing Infection ER Stress (內質網壓力） Genetics Stress Nutrition Inflammation (發炎) Metabolic Syndrome (代謝症候群) Oxidative Stress (氧化壓力) Genomic instability DNA damage Ageing (氧化DNA傷害) (基因體不穩定) Repair (修護DNA) WHO Recommendation: Diet & Exercise Cancer 癌 Alzeimer’s disease from Non-Alcoholic Fatty Liver to Cancer Development (1) Non-alcoholic fatty liver to HCC; (2) HCV HCC (3) HBV HCC Cancer Science 332(24), p1519, 2011 Revolution Concept on the Role of Foods in Health 食物在健康所扮演的革命性新觀念 Revolution Concept on the Role of Foods in Health 食物在健康所扮演的革命性新觀念 Health Food is classically considered to be: Anti-oxidants, vitamins, trace metals, fiber, essential fatty acids--etc. ? Are there functional factors which play a role like drugs or have proven therapeutic effects ? How can we step forward ? Natural Products and Healthy Ageing 天然食材與養生長壽 PPAR ligands: L: ligands B: binding proteins Co-act: Co-activator 白藜蘆醇 (Resveratrol) 薑黃 (Curcumin) 不飽和脂肪酸 (Omega 3) 類胡蘿蔔素(Carotenoids) 類黃酮(Flavonoids) 三萜類(Tripternoids) B PGC-1A L B L L L PPARs RXRa Bexarotene Retinoids Co-Act PPRE PPAR a/g ↑ NFkB ↓ Apo E ↑ mTOR ↓ 代謝 抗發炎 抗癌 抗發炎 抗癌 抗老化 防癌、抗癌 8 The key regulator of metabolic syndrome, inflammation, and cancers: PPARs ( peroxisome proliferator activated receptors ): 三種異構物 3 isoforms ( alpha, beta, gamma) Effects of PPAR agonists: lipid regulation, Anti-inflammation, and anti-cell proliferation Drugs with PPAR agonistic activities PPARγ活化劑已開發成為糖尿病用藥 Takeda pioglitazone Glaxosmithkline Rosiglitazone PPARs platform technology to develop functional product from herbs PPARs promoter plasmids Extracts from natural products HepaG2 cell Extracts of Natural products Screening for products with high PPAR agonistic activities 從數百種天然食材中檢測 PPARs的活性大小 PPARγ activation fold compared to troglitazone(0.25ug/ml) Combination of the L,B,C,S showed the best synergistic activation of PPARα and PPARγ Efficacy tests of Metamin 3D—animal and human trials Metamin 3D Clinical Trails in VGH, Taichung, for metabolic syndrome 委託台中榮總進行雙盲試驗─ 人體健康3D改善代謝症候群 Randomized, placebo-controlled in 2 groups 採盲式隨機分成兩組，試驗組給予健康3D，對照組給予安慰 劑。 Oral, tid, for 3 months 一天一次，一次三錠 給予試驗產品12週(3個月) Publication and Patent under application Natural products for anti-oxidant, ageing, anti-inflammation, and chemoprevention Resveratrol: Red Grape Magic • Red wine 5mg/bottle Resveratraol Science 1997: Resveratrol is effective for tumor control at the stages of tumor initiation, promotion, and prevention Synergistic effect of PPAR agonists ( omega 3 ) and resveratrol in PPARγ activation Omega 3 resveratrol PPARγ activation by Metamin 3D and resveratrol 12 10 luciferae fold 8 6 4 2 0 Co ntr o 3D l 20 ul 3D 20 ul 3D +R es 5u l 20 ul 3D +R es 10 ul 20 ul +R e s2 0u l Fig 2. PPAR gamma activation fold of six selected natural products . pioglitazone 0.1uM were used as positive control. Luminescence Values are means ± SEM, n = 3, and are folds of the control (DMSO) mean. resveratrol 2.00 AU 1.50 resveratrol 1.00 0.50 1.50 Standard resveratrol 0.5 mg/ml resveratrol 0.40 AU 0.30 0.20 AU 0.00 0.00 10.00 20.00 30.00 40.00 50.00 60.00 Minutes 1.00 0.50 0.00 0.00 10.00 20.00 30.00 40.00 50.00 60.00 Minutes 綠加 3D+R plus HPLC 圖譜 0.10 0.00 0.00 10.00 20.00 30.00 40.00 50.00 60.00 Minutes Standard resveratrol 0.1 mg/ml Resveratrol 含量經比對標準品後， 為8.421896 mg / 1g ， 大於規格的 6mg/ g Curcumin has antioxidant, anti-inflammatory, and anti-amyloid activity and is a promising compound for the development of AD therapies Curcuma longa (薑黃) In vitro studies with curcumin -- Antioxidant effect -- Anti-inflammatory effect -- Inhibition of b-secretase -- Inhibition of Ab aggregation In vivo studies with curcumin -- Inhibition of Ab deposition -- Inhibition of Ab oligomerization (薑黃素) (CNS Neuroscience&Therapeutics 16, 285-297, 2010) Question The mechanism by which curcumin inhibits Ab deposition remains to be clarified Curcumin Clinical Trials Cancer • Colorectal cancer • Pancreatic cancer • Breast cancer • Prostate cancer • Multiple myeloma • Lung cancer • Cancer lesions • Head and neck cancer •Vitiligo •Psoriasis Inflammatory diseases •Crohn disease •Ulcerative proctitis • Ulcerative colitis •Inflammatory bowel disease • Irritable bowel syndrome •Rheumatoid arthritis • Osteoarthritis • Chronic anterior uveitis •Recurrent anterior uveitis •Post operative Inflammation • Gastric ulcer • Peptic ulcer •H. pylori infection •Idiopathic orbital inflammatory Pseudotumor Skin diseases Neurodegenerative diseases •Dejerine-Sottas disease • Alzheimer's disease O O Cardiovascular diseases OCH3 H3CO HO OH Curcumin •Acute coronary syndrome • Atherosclerosis Metabolic diseases • Diabetes •Diabetic nephropathy •Diabetic microangiopathy • Lupus nephritis Renal diseases •Renal transplantation Viral diseases OTHERS b-Thalassemia •Biliary dyskinesia •Gallbladder contraction •Recurrent respiratory tract infections • Cholecystitis •Hepatoprotection •Chronic arsenic exposure • Alcohol intoxication •Chronic bacterial prostatitis •Acquired immunodeficiency syndrome Gupta, Patchva and Aggarwal:, AAPS J. (in press) 阿茲海默症的學理基礎: 異常構造的β類澱粉蛋白(β -amyloid)無法清除，而累積在神 經細胞中形成細纖維團(fibrillary tangle)或細胞外的斑塊(plague) ，引起神經細胞傷害或死亡，導致傳導功能、發炎等病變。 Two Key Issues for Curcumin in Drug Development No drug has been ever developed 1. No patent: a. Clarify the signal pathway and establish a reporter assay to screen the drug candidates b. Modification of curcumin as a new drug 2. Poor absorption and difficult to reach PK value for drug use: use new delivery system such as lipoid carrier, or to modify curcumin to be water soluble Curcumin showed PPARg activity in HepG2 cells ** Curcumin-Indena The effects of natural compounds on the anti-oxidant activity by assay of NrF2 in Huh7 cells. Method: Luciferase Reporter Assay Huh7-24hr Relative luciferase activity NrF2 activity 5 4 3 2 1 0 Control Curcumin 15μ g/ml(Indena) Resveratrol 25μ M Preliminary data Curcumin could significantly increase ApoE protein expression in HuH-7 cells HuH-7 24h Western blot assay RXRα ApoE β-actin Rapamycin PPARα DMSO Untreated p-mTOR Sigma Curcumin Indena Curcumin 5 5 10 20 10 20 μg/mL Apo E and Alzheimer's disease (AD) Apolipoprotein E (Apo E) is important in plasma lipid metabolism. Apo E polymorphism or genotype is closely related to total plasma cholesterol levels and LDL levels: E2 allele is associated with lower , and E4 with higher levels, compared with common E3 allele. (Hagberg J.et al : Physiol. Genomics 2000;4:101-108) The risk of developing AD: Apo E4 ˃ E3 ˃ E2 (84yrs, 75yrs,68yrs) Mariani SM, Medscape General Medicine 2004;6:47 Cramer PE., et. al. Science 2012, 335: 1503-1506. The flux of Aβ/cholesterol/ApoE through the cell and the potential regulation by PPAR/RXR/LXR PPAR (Wolozin B, Neuron 41, 7-10, 2004) 膽固醇(cholesterol)代謝與 AD的致病機制息息相關 1. Total cholesterol及LDL在AD病人顯著上升 ，且與Aβ42的形成相關(Kuo,1998). 2. 降低Cholesterol的藥物Statins可降低A β及 App片斷(β -CTFs). 3. ApoE可運送cholesterol及結合LDL受體，與 Aβ的沉積相關(Striffmater,1993). 4. Ceramide及cholesterol代謝異常可引發 oxidative stress並引起大腦aging及AD. Hypothesis Curcumin may induce Ab clearance through RXR/LXR-mediated apoE upregulation, ameliorating AD pathophysiology Curcumin ApoE Ab clearance Key Biomarkers for Health: PPAR/RXR and LXR/RXR Regulated by PPAR agonist, Resveratrol and Curcumin L Oxysterol, Curcumin LXR L Glucose regulation Lipid metabolism Anti-cancer Anti-inflammation Alzeimer’s disease L RXRa LXR Glucose regulation Cholesterol metabolism ApoE The effects of Green Cross natural compounds on the activity of PPARγ in Huh7 cells. Method: Luciferase Reporter Assay Huh7-24hr Relative luciferase activity PPARg activity 9 8 7 6 5 4 3 2 1 0 3D, 3DR, new product 均為一天之劑量 Control 50%EtOH Troglitazone 0.125g/mL 3D 3DR New product 3DRC 1顆 3DRC 2顆 3DRC 3顆 3DRC 4顆 The effects of natural compounds on the activity of PPARγ in Huh7 cells: A comparison between 3DRC and Lipoguard ( Viva ) Method: Luciferase Reporter Assay Huh7-24hr Relative luciferase activity PPARg activity 8 7 6 5 4 3 2 1 0 Control 50%EtOH Troglitazone 0.125μ g/mL 3D 3DRC 1X 3DRC 4X 1040210 LipoGuard(上 ) LipoGuard(下 ) The effects of natural compounds on the activity of PPARγ in Huh7 cells. Method: Luciferase Reporter Assay Huh7-24hr Relative luciferase activity PPARg activity 8 7 6 5 4 3 2 1 0 Control 50%EtOH Troglitazone 0.125μ g/mL 3D 3DRC 1X 3DRC 4X 1040210 LipoGuard(上 ) LipoGuard(下 ) The effects of Green Cross natural compounds on the activity of ApoE in Huh7 cells. Method: Luciferase Reporter Assay Huh7-16hr Relative luciferase activity ApoE activity 7 6 5 4 3 2 1 0 3D, 3DR, new product 均為一天之劑量 Control 50%EtOH Curcumin 15g/mL 3D 3DR New product 3DRC 1顆 3DRC 2顆 3DRC 3顆 3DRC 4顆 服用3D+R plus Curcumin 就如每天喝一瓶紅酒、三餐吃咖哩、 喝綠茶、豆漿、綠藻精及芝麻 融合 地中海、日本、印度等國家長壽 的祕密。 「君、臣、佐、使」複方概念是二十一世紀生技 產品開發的主流。 Nature 2011 One for all and all in one