EP2978417A1 - Composition et procédé pour induire l'expression d'hémoglobine médiée par l'epo et la biogénèse mitochondriale dans une cellule non hématopoïétique - Google Patents

Composition et procédé pour induire l'expression d'hémoglobine médiée par l'epo et la biogénèse mitochondriale dans une cellule non hématopoïétique

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Publication number
EP2978417A1
EP2978417A1 EP13880425.7A EP13880425A EP2978417A1 EP 2978417 A1 EP2978417 A1 EP 2978417A1 EP 13880425 A EP13880425 A EP 13880425A EP 2978417 A1 EP2978417 A1 EP 2978417A1
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Prior art keywords
disease
epo
group
cell
expression
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EP13880425.7A
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German (de)
English (en)
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EP2978417A4 (fr
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Rong-Tsun Wu
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Wu Sophia Shu Fen
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Wu Sophia Shu Fen
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Publication of EP2978417A1 publication Critical patent/EP2978417A1/fr
Publication of EP2978417A4 publication Critical patent/EP2978417A4/fr
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K31/00—Medicinal preparations containing organic active ingredients
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    • A61K31/7028—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages
    • A61K31/7034—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin
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    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
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    • A61P19/00—Drugs for skeletal disorders
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    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/14—Drugs for disorders of the nervous system for treating abnormal movements, e.g. chorea, dyskinesia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/14—Drugs for disorders of the nervous system for treating abnormal movements, e.g. chorea, dyskinesia
    • A61P25/16—Anti-Parkinson drugs
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/20—Hypnotics; Sedatives
    • 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
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    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P27/00—Drugs for disorders of the senses
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    • A—HUMAN NECESSITIES
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    • A61P3/00—Drugs for disorders of the metabolism
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    • 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
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    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P5/00—Drugs for disorders of the endocrine system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/06—Antianaemics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/04—Inotropic agents, i.e. stimulants of cardiac contraction; Drugs for heart failure
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis

Definitions

  • the present invention relates to a composition and a method for inducing haemoglobin expression, mitochondrial biogenesis and autophagy in a subject.
  • Ischemia causes oxygen deprivation, cell injury and related organ dysfunctions, such as heart failure, stroke, chronic obstructive pulmonary disease, ischemic retinopathy, liver injury, and acute renal failure. Because mitochondrial dysfunction is a key factor in organ ischemia injury, upon loss of oxygen, mitochondrial oxidative phosphorylation rapidly stops, with resulting loss of the major source of ATP production for energy metabolism.
  • EPO Erythropoietin
  • PDC-la peroxisome proliferator-activated receptor coactivator 1-a
  • EPO reverses cardiac remodeling, improves cardiac function, and enhances the exercise tolerance and quality of life of patients by inducing protective effects beyond the correction of anaemia.
  • Haemoglobin is the main oxygen transporter in erythrocytes. Its main form, haemoglobin A, is a tetramer consisting of two a- and ⁇ -polypeptide chains, each carrying a heme group. Recently, Hb was unexpectedly found to be expressed in many nonhaematopoietic cells, which may facilitate tissue oxygen transport or increase cellular oxygenation to provide an intrinsic protective mechanism against hypoxic/ischemic injury.
  • AD Alzheimer's disease
  • REM sleep in early-stage AD patients is relatively unaffected by the disease process, later stages of AD are marked by significant losses of REM sleep.
  • Memory loss is accompanied by the accumulation of oxidative damage to lipids, proteins, nucleic acids, and by mitochondrial decay, all of which can disrupt neuronal function in aging and disease.
  • Sleep deprivation (SD) also induced oxidative stress which resulted in memory loss and impaired mitochondrial activity.
  • EPO and the EPO receptor are expressed in neurons and astrocytes, and EPO is produced primarily by astrocytes in the brain.
  • EPO is widely used to enhance erythropoiesis in patients with anemia and recently has been found to have many non-haematopoietic beneficial effects, including cardioprotection and neuroprotection.
  • An early clinical study has demonstrated cognitive improvement during EPO treatment among patients with chronic renal failure.
  • Recently studies have shown that a high-dose EPO treatment improves hippocampal plasticity and cognitive performance in patients suffering from neuropsychiatric diseases.
  • High-dose EPO also enhances hippocampal long term potentiation by modulating plasticity, synaptic connectivity and activity of memory-related neuronal networks and improves operant conditioning stability of cognitive performance in healthy mice.
  • EPO may play a pivotal role for pharmacological applications in the treatment of SD-induced impairment of hippocampal learning and memory by modulating downstream mitochondrial regulator expression. Due to the fact that EPO has limited clinical use because it cannot freely cross the blood-brain barrier (BBB), only systemic dosing of high-dose recombinant Epo (rEpo) would result in neuroprotective activity.
  • BBB blood-brain barrier
  • Autophagy or "self digestion process” is an important physiological process that targets cytosolic components such as proteins, protein aggregates and organelles for degradation in lysosomes.
  • the autophagic process is also essential for maintaining neuronal homeostasis, and its dysfunction has been directly linked to an increasing number of diseases.
  • autophagy is directed to recycling intracellular nutrients in order to sustain cell metabolism during starvation, and eliminating damaged organelles and proteins that have accumulated during stress.
  • Defective autophagy is a major contributor to diseases which may be, but not limited to, neurodegeneration, liver disease, and cancer.
  • diseases which may be, but not limited to, neurodegeneration, liver disease, and cancer.
  • a lot of human neurodegenerative diseases are associated with aberrant mutant and/ or polyubiquitinated protein accumulation and excessive neuronal cell death.
  • Polygonum multiflorum Thunb is a Chinese medicine used for the treatment of anaemia, liver diseases, and other diseases commonly associated with aging.
  • the present invention provides small molecular compounds isolated and identified from Polygonum multiflorum Thunb. These compounds have effects in experimental models of cardiovascular diseases, cerebral ischemia, Alzheimer's disease and inflammation diseases, and have antioxidant and free radical-scavenging properties. In addition, the present invention provides therapeutic effects and physiological mechanisms of such compounds in animal models.
  • the present invention provides a composition for inducing erythropoietin (EPO)-mediated haemoglobin (Hb) expression in a nonhaematopoietic cell of a subject.
  • the composition comprises a compound represented by formula (I): wherein R is a glycosyl group; and a pharmaceutical acceptable carrier.
  • the glycosyl group is one selected from the group consisting of dihydroxyacetone, glucose, galactose, glyceraldehyde, threose, xylose, mannose, ribose, ribulose, tagatose, psicose, fructose, sorbose, rhamnose, erythrose, erthrulose, arabinose, lyxose, allose, altrose, gulose, idose, talose, sucrose, lactose, maltose, lactulose, trehalose, cellobose, isomaltotriose, nigerotriose, maltotriose, melezitose, maltotriulose, raffinose, kestose and a combination thereof.
  • the compound induces Hb-a, Hb- ⁇ , or dimeric Hb expression in the nonhaematopoietic cell of the subject, enhances erythropoietin-erythropoietin receptor binding affinity and also binds to the erythropoietin-bound erythropoietin receptor complex.
  • the compound enhances endogenous EPO expression and stimulates Hb expression in the nonhaematopoietic cell.
  • the nonhaematopoietic cell is selected from the group consisting of a renal cell, a hepatocyte, a cardiomyocyte, a myoblast, a glial cell, a neuronal cell and a retinal pigment epithelium cell.
  • the present invention further provides a method for inducing erythropoietin (EPO)-mediated haemoglobin (Hb) expression in a nonhaematopoietic cell of a subject, comprising administering to the subject a therapeutically effective amount of the aforementioned compound of formula (I).
  • EPO erythropoietin
  • Hb haemoglobin
  • the subject suffers a disease or syndrome selected from the group consisting of hypoxia, anaemia, renal ischemia, myocardial ischemia, lung ischemia, neurodegenerative disease, neuropsychiatric disease, age-related macular degeneration (AMD) -related disease and a combination thereof.
  • the present invention further provides a composition for inducing erythropoietin (EPO)-mediated mitochondrial biogenesis in a nonhaematopoietic cell of a subject, comprising the aforementioned compound of formula (I) and a pharmaceutical acceptable carrier.
  • EPO erythropoietin
  • the compound induces an increase of a mitochondrial number or PGC- ⁇ expression for inducing the EPO-mediated mitochondrial biogenesis, enhances erythropoietin-erythropoietin receptor binding affinity and also binds to the erythropoietin-bound erythropoietin receptor complex.
  • the compound enhances endogenous EPO expression and stimulates Hb expression in the nonhaematopoietic cell of the subject.
  • the EPO-mediated mitochondrial biogenesis is PGC- la-dependent.
  • the nonhaematopoietic cell is selected from the group consisting of a renal cell, a hepatocyte, a cardiomyocyte, a myoblast, a glial cell, a neuronal cell and a retinal pigment epithelium cell.
  • the present invention further provides a method for inducing erythropoietin (EPO)-mediated mitochondrial biogenesis in a nonhaematopoietic cell of a subject, comprising administering to the subject a therapeutically effective amount of the aforementioned compound of formula (I).
  • the compound induces an increase of a mitochondrial number or PGC- la expression for inducing the EPO-mediated mitochondrial biogenesis.
  • the subject suffers a disease or syndrome selected from the group consisting of hypoxia, anaemia, ischemia-related disease, neurodegenerative disease, neuropsychiatric disease, age-related macular degeneration (AMD)-related disease, cardiomyopathy, brain aging, chronic liver disease, multiple sclerosis, Pompe disease, hypertension, cardiac failure, obesity, diabetes mellitus, renal disease, atherosclerosis, aging, metabolic syndrome and a combination thereof.
  • a disease or syndrome selected from the group consisting of hypoxia, anaemia, ischemia-related disease, neurodegenerative disease, neuropsychiatric disease, age-related macular degeneration (AMD)-related disease, cardiomyopathy, brain aging, chronic liver disease, multiple sclerosis, Pompe disease, hypertension, cardiac failure, obesity, diabetes mellitus, renal disease, atherosclerosis, aging, metabolic syndrome and a combination thereof.
  • the ischemia-related disease is one selected from the group consisting of heart ischemia, ischemic neurodegeneration, brain ischemia, myocardial ischemia, limb ischemia, cerebral ischemia, hepatic ischemia, retinal ischemia, stroke, nephritic ischemia, pulmonary ischemia, intestinal ischemia, cardiovascular ischemia, renal ischemia and kidney ischemia.
  • the neurodegenerative disease is one selected from the group consisting of Alzheimer's disease, Parkinson's disease and Huntington's disease.
  • the present invention further provides a method for inducing autophagy in a subject having an autophagy defect, comprising administering to the subject a therapeutically effective amount of the aforementioned compound of formula (I), wherein the autophagy enhances clearance of protein aggregates in the subject.
  • the autophagy defect is in a cell expressing the protein aggregates in the subject, wherein the protein aggregate is an aggregate selected from the group consisting of hungtingtin, amyloid ⁇ ( ⁇ ), ⁇ -synuclein, tau, superoxide dismutase 1 (SOD1), variants and mutated forms thereof, and a combination thereof.
  • the cell of the subject is a neuronal cell or a glial cell.
  • the autophagy defect is one disease selected from the group consisting of neurodegenerative disease, retinal disease, Crohn's disease, aging, cardiac hypertrophy, chronic heart failure, tuberculosis, chronic obstructive pulmonary disease (COPD), cystic fibrosis, hepatic steatosis, polycystic kidney disease, renal failure, muscle atrophy, Paget's disease of bone, inclusion body myopathy, fronto-temporal dementia, glomerular disease, metabolic disease, glycogen storage disease type II, inflammatory bowel disease, and Pompe disease.
  • the neurodegenerative disease is one selected from the group consisting of Huntington's disease, Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis (ALS) and insomnia.
  • the present invention further provides a composition for inducing autophagy in a subject having an autophagy defect.
  • the composition comprises the aforementioned compound of formula (I) and a pharmaceutical acceptable carrier.
  • the invention provides a method for preventing memory loss in a subject, comprising administering to the subject a therapeutically effective amount of the aforementioned compound of formula (I).
  • the compound induces erythropoietin (EPO) to activate the autophagy in the subject.
  • EPO erythropoietin
  • the autophagy enhances protein clearance in the subject.
  • the autophagy defect is a neurodegenerative disease selected from the group consisting of Huntington's disease, Alzheimer's disease, Parkinson's disease and insomnia.
  • FIG. 1A to FIG. IB show EH-201 characterization.
  • A HPLC profile of EH-201.
  • Mightysil RP-18 column (4.6 x 250 mm i.d., 5 ⁇ ) was used at flow rate of 0.8 ml/min with MeOH/H 2 0 (20/80, v/v) gradient to 100% MeOH in 60 minutes in the detection wavelength of 280 and 300 nm.
  • B Positive ion mode LC-APCI/MS/MS of EH-201.
  • FIG. 2 A to FIG. 2J show that EH-201 is a potent inducer of EPO expression.
  • A The chemical structure of EH-201.
  • B, C The EH-201 -treated kidney slices and hepatocytes were analyzed for EPO expression by Q-PCR and Western blotting.
  • D Primary mice cardiomyocytes and
  • F, G C2C12 myotubes were treated with EH-201, and the effects on EPO and EPOR expression were analyzed by QPCR and Western blotting.
  • H The bone marrow cells were incubated with EH-201 for 48 h, and the expression of EPO was detected by Q-PCR.
  • FIG. 3A to FIG. 3G show that the induction of mitochondrial biogenesis by EH-201 is mediated by EPO.
  • A, B EH-201 -treated kidney slices and primary cardiomyocytes and
  • the control represents vehicle treatment.
  • rhEPO was given to kidney slices, hepatocytes and C2C12 myotubes.
  • the control represents vehicle treatment.
  • the control represents the scrambled siRNA treatment.
  • the values are presented as the means+SEM. **P ⁇ 0.01, *P ⁇ 0.05 versus untreated control, n.s., not significant, Student's t-test.
  • FIG. 4A to FIG. 41 show that the induction of haemoglobin expression in nonhaematopoietic cells by EH-201 is mediated by EPO.
  • A Cultured C2C12 myotubes under normoxia or hypoxia (5% 0 2 ) for 24 hours were analyzed for the expression of haemoglobin-alpha (Hb-a) and -beta (Hb- ⁇ ) by RT-PCR, followed by 1.5% agarose gel electrophoresis.
  • the control represents vehicle treatment.
  • (E, F) EH-201 -treated kidney slices and primary mice cardiomyocytes and
  • the values are represented as means+SEM. **P ⁇ 0.01, *P ⁇ 0.05 versus untreated control, # P ⁇ 0.05 versus rhEPO treated control (50 ng/ml).
  • FIG. 5 A to FIG. 5G show that EH-201 increases endurance performance and activation of mitochondrial activity and haemoglobin expression in mice.
  • A The endurance of normal mice was measured with the rotarod exercise under normoxic or hypoxic (8% 0 2 ) conditions (ND: normal diet).
  • B The effect of EH-201 on plasma RBC numbers and haemoglobin levels.
  • C, D EPO mRNA expression in the kidney and liver of mice was measured by Q-PCR after 3 days of EH-201 administration. The serum levels of EPO were determined by ELISA.
  • E, F Isolated myocardium tissues after 3 days of EH-201 administration were analyzed for haemoglobin expression by Q-PCR, and the mitochondrial biogenesis was determined by mtDNA copy number.
  • FIG. 6A to FIG. 6H show that EH-201 has therapeutic effects on cardiac dysfunction in doxorubicin (Dox)-induced cardiomyopathy in mice.
  • A The survival rate was analyzed using the Kaplan-Meier method (detailed treatment protocol in Materials and Methods). The normal (N) group represents saline injection.
  • B The effect of EH-201 treatment on mice performing the hypoxic rotarod endurance test two weeks after Dox injection.
  • C, D The effect of EH-201 on cardiac abnormality and functionality was characterized by ECG and echocardiography.
  • EF ejection fraction
  • FS fractional shortening
  • LVIDs/d left ventricular internal diameter at systole/diastole.
  • F Isolated myocardium tissues after 2 weeks of Dox were analyzed for haemoglobin expression by Q-PCR and
  • FIG. 7 A to FIG. 7H show that EH-201 accelerates the recovery from anaemia and renal function in cisplatin-induced nephropathy in mice.
  • A Schematic diagram protocol.
  • B The time course kinetics of the RBC numbers in the peripheral blood.
  • C The time course kinetics of the blood urea nitrogen (BUN) values after cisplatin injection.
  • D The functional recovery of the kidneys of mice treated with EH-201 on day 28.
  • FIG. 8 shows that EH-201 induces Sirtl expression.
  • the control represents vehicle treatment.
  • the values are represented as the means+SEM. **P ⁇ 0.01, *P ⁇ 0.05 compared with control.
  • FIG. 9 shows ribbon diagrams of the computational docking results for EH-201 on EPO/EPOR complex. Docking calculations were carried out using DockingServer on EPO complexed with extracellular domain of EPOR protein model (PDB entry code lcn4).
  • the predictive interaction residues including PRO 144 , GLU 147 , PRO 149 , Met 150 , and THR 151 are located in loop 5 of EPOR, which is important for EPO binding.
  • FIG. 10A to FIG. IOC show that EH-201 -induced EPO production does not involve Hif- ⁇ activation.
  • HRE hypoxia response element
  • Luci luciferase reporter
  • transfected HEK 293 cells were incubated with EH-201 under normoxia or hypoxia (5% 0 2 , as the positive control) for 24 hours.
  • the plasmid for ⁇ -Galactosidase ( ⁇ -Gal) was used as a transfection control, and the pGL3-v served as a vector control. Similar results were observed in three additional independent experiments.
  • Hypoxia condition served as a positive control.
  • C The Hif-2a protein expression levels in the nuclear lysates of the EH-201 -treated kidney slices were analyzed by Western blotting (H: 5% 0 2 hypoxia as a positive control). The control represents vehicle treatment. The values are represented as the means+SEM. **P ⁇ 0.01, *P ⁇ 0.05 compared with normoxia, Student's t-test.
  • FIG. 11A and FIG. 11B show that EH-201 increases mitochondrial function and biogenesis in the liver and skeletal muscle.
  • FIG. 12A and FIG. 12B show that EH-201 has therapeutic effects on cardiac dysfunction in doxorubicin (Dox)-induced cardiomyopathy in mice.
  • Dox doxorubicin
  • FIG. 12A and FIG. 12B show that EH-201 has therapeutic effects on cardiac dysfunction in doxorubicin (Dox)-induced cardiomyopathy in mice.
  • A The effect of EH-201 on the body weight of mice two weeks after Dox injection.
  • FIG. 13 A to FIG. 13 F show that EH-201 stimulats EPO expression in primary astrocytes and PC12 neuronal cells.
  • A Structure of EH-201.
  • B, C Real time PCR shows that EH-201 treatment for 24 hours increase EPO mRNA in astrocytes and PC 12 neuronal cells. The expression of GAPDH was used as an internal control.
  • D Western blotting shows that EH-201 treatment for 24 hours increase EPO protein expression in astrocytes and PC12 neuronal cells. The results are expressed as the relative index of untreated controls + SD of at least three independent measurements. *P ⁇ 0.05, **P ⁇ 0.01 compared to untreated controls by one-way ANOVA followed by Tukey' s multiple comparison test.
  • E Real time PCR shows that EPO treatment for 24 hours does not increase Hb-a mRNA in astrocytes and PC12 neuronal cells.
  • F Real time PCR shows that EH-201 treatment for 24 hours does not increase Hb-a mRNA in astrocytes and PC 12 neuronal cells.
  • the expression of GAPDH was used as an internal control. The results are expressed as the relative index of untreated controls + SD of at least three independent measurements. * P ⁇ 0.05, ** P ⁇ 0.01 compared to untreated controls by one-way ANOVA followed by Tukey 's multiple comparison test.
  • FIG. 14A to FIG. 14F show that EH-201, a neuronal EPO inducer, stimulates the expression of the mitochondrial regulator (PGC-la, Hb- ⁇ ) and an antioxidant gene (HO-1) in primary astrocytes and PC12 neuronal cells.
  • PGC-la mitochondrial regulator
  • Hb- ⁇ antioxidant gene
  • HO-1 antioxidant gene
  • FIG. 15A to FIG. 15H show that EH-201 increases mitochondrial activity, decreases intracellular ROS and attenuates H 2 0 2 -induced cell toxicity in primary astrocytes and PC 12 neuronal cells.
  • A, E Different forms of Hb (monomer: 16kD, dimer: 32kD, tetramer: 64kD) expression identify by Hb- ⁇ Ab in primary astrocytes and PC12 neuronal cells treated with EH-201. The results are expressed as the relative expression of untreated controls + SD from at least three independent measurements. *P ⁇ 0.05, **P ⁇ 0.01 by Student's t-test.
  • (C, G) Astrocytes and PC 12 cells treated with EPO or EH-201 for 24 hours are exposed to 100 ⁇ H 2 0 2 for 6 hours. Intracellular ROS formation is measured using the DCFH-DA assay. The graph shows results in relative fluorescence units (RFU). The values are the means+SD (n 8).
  • FIG. 16A to FIG. 16F show that EPO is required for the neuroprotective effects of EH-201 in astrocytes and PC12 neuronal cells.
  • FIG. 17 A to FIG. 17 G show that effects of EH-201 in a mouse model of sleep deprivation-induced memory loss.
  • A Procedure of EH-201 treatment in sleep-deprived (SD) mice.
  • B Real time PCR and
  • C western blot analysis of EPO expression in mouse hippocampus from each group. **p ⁇ 0.01 statistically significant compared with the SD group; **p ⁇ 0.01, statistically significant compared with the control groups.
  • FIG. 18 shows EH-201 induction of cellular EPO expression level in mice RPE cells.
  • FIG. 19A to FIG. 19D show induction of autophagy by EH-201.
  • Primary mice hepatocytes were treated with EH-201 at different doses (0.6, 2.5, 10 and 40 ⁇ g/ml), rapamycin (autophagy activator,Rm, 50 nM) or 3-methyladenine (3MA, 10 mM) for 24 hours (A and B).
  • the primary mice hepatocyte cultures under starvation (stv) acted as autophagy activation control (A, B).
  • These treated cells were stained with monodansylcadaverine (MDC) followed by fluorescent microscopy examination (scale bars: 50 mm); and the fluorescent intensity was measured in spectroflurometer (B).
  • MDC monodansylcadaverine
  • B fluorescent microscopy examination
  • FIG. 20 A and FIG. 20B show that EH-201 induced autophagic activation is through hepatocyte growth factor (HGF) induction.
  • Hepatocytes were treated with EH-201 at different doses (0.6, 2.5, 10 and 40 mg/ml) for 24 hours.
  • rhEPO represented recombinent human EPO
  • rmHGF represented recombinant murine heaptocyte growth factor
  • nEPO-ab represented neutralizing EPO antibody.
  • EPO Erythropoietin
  • EH-201 2,3,5,4' -tetrahydroxystilbene-2-o-beta-d-glucoside (hereinafter referred to as EH-201)(FIG. 2A) was extracted and purified to 99.2% purity.
  • the dried and milled roots of Polygonum multiflorum Thunb. was extracted with 40% ethanol and then evaporated to form syrup.
  • the extract was diluted twice with 15% ethanol, loaded on a Diaion HP-20 resin column and then eluted with sequential 20%, 40%, and 70% ethanol, respectively. The effluent of 40% ethanol was collected and evaporated.
  • the 40% ethanol effluent was then redissolved in 10% ethanol by sonication and partitioned with ethyl acetate of equal volume five times successively.
  • the residue of ethyl acetate was then passed through a Sephadex LH-20 column eluting with methanol.
  • a pale yellow compound, EH-201 was obtained.
  • the overall yield is about 0.5 %o from the crude, dried, milled roots of Polygonum multiflorum Thunb. to final compound EH-201 in pure form (99.2%).
  • the crystallization of this compound was further performed.
  • the 30% aqueous-ethanolic solution of EH-201 was then placed into the -20 °C refrigerator overnight then placed into 4°C refrigerator. An acicular crystal was obtained several days later.
  • EH-201 The chemical identity of EH-201 was confirmed by LC/MS/MS, UV, 'H-NMR and proton-decoupled 13 C-NMR data (FIG. 1 and Table 1), and *H-NMR and proton-decoupled 13 C-NMR data sets using a Bruker AVIII-500 NMR spectrometer. The proton and carbon chemical shifts of EH-201 are listed in Table 1.
  • the LCMS data of the purified EH-201 was performed with a Bruker LC/MS/MS spectrometer Esquire 2000 in APCI (Atmospheric Pressure Chemical Ionization) mode with positive ion polarity, using a gradient of HPLC grade water and methanol over 60 minutes with a reverse phase C18 column (FIG. 1A).
  • the LCMS data is exhibited in FIG. IB showing the correct mass of EH-201 at m/z 407.0.
  • the EH-201 ion at m/z 407.0 is further subjected to MS/MS analysis where only the 407.0 ion was isolated and fragmented.
  • the resulting daughter ion at m/z 245.1 is consistent with the EH-201 loses its sugar moiety. Therefore, the compound was identified as 2,3,5,4'-tetrahydroxystilbene-2-o-beta-d-glucoside (TSG or THSG) (FIG. 2A).
  • Example 2 activation of mitochondrial function and haemoglobin expression in nonhaematopoietic cells by the compound of the present invention
  • This example describes various assays that are useful in evaluating the activation of mitochondrial function and haemoglobin expression in nonhaematopoietic cells by the compound of the present invention.
  • the compound of the present invention is prepared according to the methods provided in Example 1. The potency of this compound is evaluated using a series of activity assays and these assays are further described in detail below.
  • mice Eight-to-ten-week-old specific pathogen-free C57BL/6J male mice (20-25 g), obtained from the National Laboratory Animal Centre (Taiwan) were housed 5-6 per cage at a constant temperature of 22+2°C and fed standard laboratory chow (PMI, Brentwood, MO, USA) and water ad libitum under a 12 hour dark/light cycle.
  • the experimental protocol was approved by the Animal Research Committee of National Yang-Ming University (Guide for Animal Experiments, National Yang-Ming University). All efforts were made to minimize animal suffering, to reduce the number of animals used and to utilize alternatives to in vivo techniques, if available. All studies involving animals were reported in accordance with the ARRIVE guidelines for reporting experiments involving animals.
  • the C2C12 myoblast, HEK293, and TF-1 cells were purchased from Bioresources Collection and Research Centre (BCRC, Hsinchu, Taiwan).
  • the C2C12 myoblasts were differentiated to myotubes and were treated with drugs for 24 hours.
  • Ex vivo 250 ⁇ -thick kidney slices were prepared from eight-to-ten-week-old C57BL/6J mice as previously described. The slices were treated with drugs in the gassed media (DMEM/F12 buffered with 15 mM HEPES and 20 mM sodium bicarbonate) in an atmospheric chamber at 37°C with 50% 0 2 : 5% C0 2 : 45% N 2 for 18 hours.
  • gassed media DMEM/F12 buffered with 15 mM HEPES and 20 mM sodium bicarbonate
  • Neonatal C57BL/6J mouse cardiomyocyte cultures were prepared from post-natal one day-old C57BL/6J mice obtained from the Animal Centre at the National Yang-Ming University as described previously, and the isolated ventricular cells were resuspended in 10% FCS-containing M199 medium (Gibco, Germany).
  • the cardiomyocytes were incubated in a humidified atmosphere at 37 °C with 5% C0 2 on plates precoated with 1% gelatin. The subconfluence of spontaneously beating cells was achieved after 48 hours of culture, after which treatments with various drugs were performed for 24 hours.
  • the bone marrow progenitor cell cultures for the colony-forming assay and the haemoglobin colorimetric assay were prepared as previously described.
  • the C2C12 myotubes were transfected with scrambled or PGC- la-specific siRNA (Table 2) using the Lipofectamine 2000 reagent, according to the manufacturer's instructions (Invitrogen).
  • the EPO, EPOR, PGC- ⁇ , Hb-a, Hb- ⁇ , and GAPDH mRNA expression were quantified by quantitative real-time PCR (Q-PCR) with an ABI 7500 sequence detector (Applied Biosystems) using SYBR Green Master MixR (AB 1-7500).
  • the relative mRNA expression levels were determined using the TTCt method, with GAPDH as the endogenous control.
  • the primers used are listed in Table 2.
  • the total protein (50 ⁇ g) was separated by 12% SDS-PAGE, transferred onto
  • PVDF membranes and probed with antibodies against EPO, PGC- ⁇ , GAPDH, PCNA (from Santa Cruz, CA), Sirtl (Millipore, Billerica), or Hif-2a (Novus Biologicals, Littleton). Following incubation with the appropriate horseradish peroxidase-conjugated secondary antibody, the signals were visualized by ECL detection, according to the manufacturer's protocol (Perkin-Elmer).
  • the total cellular DNA was purified using a conventional phenol-chloroform method, and the mtDNA copy number was measured, as previously described. 6.
  • the mitochondrial content was assessed by the MitoTracker microplate assay.
  • the treated cells were loaded with 0.1 ⁇ green fluorescent MitoTracker-Green (MTG, Invitrogen) for 60 minutes at 37°C.
  • the intracellular MTG content was measured by fluorescence photometry (Thermo Scientific Inc.).
  • the fixed cells were labeled with H33342 to assess the cell density.
  • the MTG/H33258 fluorescence ratios were calculated.
  • the citrate synthase activity was measured in tissue lysates. The changes in absorbance at 412 nm were measured, and the activity was expressed as ⁇ / min/ mg protein.
  • Cells of the tEPO- sensitive cell line TF-l were seeded in 96-well microplates at a cell density of 1 x 10 5 cells/ml in RPMI 1640 medium with 2% FBS, and the cells were treated with rhEPO and EH-201 with or without EPOR neutralizing antibody (Santa Cruz) for 48 hours.
  • the cell numbers were determined by a trypan blue dye exclusion assay.
  • mice Before being divided into treatment groups, eight-to-ten-week-old C57B1/6J male mice were trained on a rotarod apparatus (14 rpm) for a maximum of 10 minutes for each of 3 consecutive training sessions per day for 3 days, and the animals that did not master this task were excluded from the experiments.
  • each mouse On the testing day, each mouse was subjected to three trials on the rotarod at 22 rpm under a normoxic or hypoxic (8% 0 2 ) atmosphere. The endurance performance was measured over time until the mice suffered from exhaustion and fell off of the rotarod. The maximum trial length was 60 minutes, and there was a 30-minute rest period between each trial.
  • the serum EPO concentrations were analyzed using an ELISA kit specific for mouse EPO (R&D, MN), according to the manufacturer's instructions. 11. Doxorubicin- induced cardiomyopathy
  • doxorubicin-HCl Sigma-Aldrich
  • EH-201 was administered orally by mixing it into the feed.
  • the Dox group was fed a normal diet and EH-201 -treating groups were fed normal diet containing different doses of EH-201 (10, 30 or 90 mg/kg per day).
  • the mice were subjected to the rotarod endurance test, echocardiography and electrocardiogram. The mice were killed after electrocardiogram, and the isolated hearts were subjected to histological examination and haemoglobin analysis.
  • the staining for haemoglobin in the isolated myocardium tissue lysates was performed with tetramethylbenzidine (TMBZ, Sigma-Aldrich), following nonreducing SDS-PAGE.
  • TMBZ tetramethylbenzidine
  • the photography and scanning of the gels was performed using a Typhoon TrioTM imager (GE Healthcare).
  • the TMBZ stain was removed from the gels by the addition of a 70 mM sodium sulfite solution. Thereafter, 30% isopropanol was used to replace the sodium sulfite, and then the gels were stained with Coomassie blue for analysis of the protein loading control. 13. Echocardiography and Electrocardiogram
  • mice from all treatment groups were anaesthetized with isoflurane (0.75-1.5% inhalation), and echocardiographic measurements were taken in M-mode in triplicates for each mouse using an ATL HDI 5000 ultrasound system (Philips Medical Systems).
  • electrocardiogram electrocardiogram
  • three electrodes were utilized.
  • the ECG tracings from lead I were recorded by means of an electrocardiograph connected to subcutaneous needle electrodes in the isoflurane-anaesthetized mouse. All probes were connected to an amplifier and digital converter for signal recording at the 100-mV range with low-pass 1 kHz and high-pass 1 kHz filters.
  • An acquisition data system with Lab VIEW software National instruments, Inc. was used to record and analyze the ECG signals.
  • cisplatin Sigma-Aldrich
  • the bone marrow cell suspensions were isolated and cultured from the femurs of six-week-old C57BL/6J male mice (National Laboratory Animal Centre, Taiwan) for assaying burst-forming units-erythroid (BFU-E). All cells were cultured in MEM-alpha medium containing 15% FBS (Gibco, Germany), 1% bovine serum albumin, 0.8% methylcellulose, 0.1 mM 2-mercaptoethanol (Sigma- Aldrich), 2 U/ml EPO (Roche, Germany), and 10 ng/ml IL-3 (Sigma-Aldrich). The colonies (> 50 cells) were counted on day 9 for BFU-E using an inverted microscope. 16. Haemoglobin colorimetric assay
  • the isolated bone marrow progenitor cells were cultured in the presence of the drug treatments in MEM-alpha medium containing 1% bovine serum albumin, 7.5 ⁇ 2-mercaptoethanol, 1.4 mM L-glutamate (Sigma-Aldrich), 5 ⁇ FeC13 (Sigma-Aldrich), and 25 [mU/ml] EPO for 96 hours. Thereafter, the extracted haemoglobin was mixed with the 2,7-diaminofluorene (DAF, Sigma-Aldrich) working solution. The change in absorbance at 610 nm was continuously monitored at 25 °C for one minute. The initial rate of the reaction was measured, and the amount of Hb in the samples was determined from the Hb standard curve.
  • DAF 2,7-diaminofluorene
  • HEK293 cells were transfected with a luciferase reporter plasmid (pGL3, Promega) containing four repeats of the minimal hypoxia response elements (HRE) from the EPO gene.
  • the transfected cells were incubated with EH-201 under normoxia for 24 hours.
  • the cells were kept under mimetic hypoxic (75 mM CoCl 2 ) or hypoxic conditions (5% 0 2 ) as a positive control of Hif- ⁇ activity. After the treatments, the cell lysates were harvested, and the luciferase expression was measured by the Dual-Lucif erase Reporter Assay System (Promega). 18. Histological analysis
  • Intact eyes were removed quickly from 6-8 week old C57/BL6 mice (National Laboratory Animal Center, Taiwan R.O.C.) and stored in ice cold PBS, which contained: 8.0 g/L NaCl, 0.2 g/ L KC1, 0.8 g/L KH 2 P() 4 , and 1.15 g/L NaH 2 P0 4 . Eyes were washed twice in growth medium (GM) consisting of Dulbecco's modified eagle's medium (DMEM) containing high glucose, 10% FBS, 1% penicillin/streptomycin, 2.5m ML-Glutamine and 1% non-essential amino acids. After washing, the eyes were then transferred into fresh PBS for dissection.
  • GM growth medium
  • DMEM Dulbecco's modified eagle's medium
  • Cells were cultured at 37°C in 5% C0 2 for 10 days, with a change of medium (GM) every other day. After 10 days the cells were washed with EDTA and then trypsinized for 4 minutes to detach the cells. The cells were collected in a tube, centrifuged at 1000 rpm for 5 minutes and resuspended in DMEM, 10% FBS, PEN/strep, 1-glutamine, sodium bicarbonate. The cells were plated in 6 cm dish until they reached confluence, at which time they were trypsinized and grown in a larger dish.
  • GM medium
  • C57mice RPE cells were incubated with 0.4, 2, 10 ⁇ g/ml EH-201 in DMEM supplemented with 10% FCS. The cultures were incubated at 37 °C for 24 hours. After incubation period, whole cell lysates were prepared with lysis buffer. Total cell lysates were prepared and subjected to western blot analysis to detect the level of endogenous EPO. GAPDH was used as a loading control. 20. Statistics
  • EH-201 is a potent EPO inducer
  • EH-201 has the ability to induce EPO expression
  • kidney slices and hepatocytes were treated with EH-201 ex vivo.
  • EH-201 was observed to dramatically induce EPO mRNA and protein expression in a concentration-dependent manner in the kidney slices and hepatocytes (FIGS. 2B and 2C).
  • the EPO transcript is expressed at a surprisingly high level in human cardiomyocytes. Therefore, whether EH-201 can also induce EPO expression in neonatal mice cardiomyocytes and C2C12 myocytes was also tested.
  • EH-201 concentration-dependently induced the expression of EPO and EPO receptor (EPOR) in the primary cardiomyocytes and C2C12 myocytes (FIGS. 2D to 2G). Because the bone marrow progenitor cells can express EPO to mediate hematopoiesis, bone marrow cells were cultured with EH-201 to examine its effect on erythropoiesis. The expression of EPO mRNA was increased in the bone marrow cells exposed to EH-201 (FIG. 2H). EH-201 significantly increased the number of BFU-E colonies (FIG. 21) and Hb expression in a concentration-dependent manner (FIG. 2J). Accordingly, EH-201 is an EPO inducer. (2) The induction of mitochondrial biogenesis by EH-201 is mediated by EPO
  • EH-201 influences mitochondrial biogenesis.
  • the activity of the mitochondrial marker enzyme citrate synthase increased in a concentration-dependent manner, and a dramatic increase in the mitochondrial copy number and PGC-la expression was also observed (FIG. 3 A).
  • the stimulatory effects of EH-201 on mitochondrial biogenesis were also observed with hepatocytes, cardiomyocytes, and C2C12 myocytes (FIGS. 3B to 3D).
  • neutralizing-EPO antibody treatment abolished the effects of EH-201 -induced mitochondrial biogenesis (FIGS.
  • haemoglobin Hb was regulated by hypoxia inducible EPO signaling in nonhaematopoietic cells.
  • Hb haemoglobin
  • In vitro experiments were performed by incubating C2C12 cells in the absence and presence of hypoxic conditions. The exposure of the C2C12 myoblasts to hypoxia resulted in a noticeable increase in the expression of Hb-a and Hb- ⁇ (FIG. 4A).
  • the induction of Hb-a expression was more susceptible to treatment than that of Hb- ⁇ (FIG. 4B).
  • Hb-a and Hb- ⁇ were increased in a concentration-dependent manner in the EPO-treated kidney slices, whereas only the expression of Hb- ⁇ was susceptible to induction in the EPO-treated hepatocytes (FIGS. 4C and 4D).
  • the expression of Hb subunits was significantly increased in EH-201 -treated nonhaematopoietic cells (FIGS. 4E to 4H), and this increase was abolished by concomitant neutralizing-EPO antibody treatment (FIGS. 4G and 4H). Studies were also conducted to determine the role of EPO signaling in the induction of Hb expression by PGC-la siRNA.
  • EH-201 binds preferentially to the EPO-bound EPOR complex (EPO/EPOR) rather than the EPO-free naive EPOR (estimated total intermolecular energy -7.48 kcal/ mol and -6.30 kcal/mol, respectively).
  • EPO/EPOR EPO-bound EPOR complex
  • EPO-free naive EPOR estimated total intermolecular energy -7.48 kcal/ mol and -6.30 kcal/mol, respectively.
  • Autodock identified more than two preformed binding sites in the EPO/EPOR complex for EH-201 with negative favorable binding free energy, and the predicted interaction residues on EPOR (Met 150 , Thr 151 , FIG. 9) involved the hot-spot residues located in loop 5.
  • EH-201 may act as binding enhancer of EPO to EPOR, thus enhancing the EPOR activation was tested.
  • a TF-1 cell (EPOR positive) proliferation assay was performed to address the EPO biological activity. It was observed that rhEPO induced the proliferation of TF-1 cells concentration-dependently, whereas, in the absence of rhEPO, EH-201 alone was unable to induce cell proliferation (FIG. 41). In the presence of even very low concentrations of rhEPO, e.g., 2 ng/ml, EH-201 significantly induced TF-1 cell proliferation in a concentration dependent manner.
  • EH-201 -induced expression of EPO involves the activation of the hypoxia-inducible factor (Hif), as EPO expression is regulated by Hif. As shown in FIG. 10A, using a hypoxia response element driven luciferase reporter to assess the activation of Hif- la, EH-201 treatment did not activate the promoter activity.
  • Hif hypoxia-inducible factor
  • Hif- la targeted vascular endothelial growth factor (VEGF) expression was upregulated during hypoxia, whereas EH-201 did not alter the VEGF expression (FIG. 10B). EH-201 treatment also did not stabilize the Hif-2a protein levels (FIG. IOC).
  • EH-201 's EPO-inducing effect, whether EH-201 could enhance endurance performance in mice undergoing hypoxic stress was tested.
  • the administration of EH-201 for 3 days increased the run time to exhaustion under both normoxia and hypoxia in a dose-dependent manner (FIG. 5A), with a further enhancement at 7 days.
  • FIG. 5B there was only a slight increase in RBC counts and Hb content in the peripheral blood (FIG. 5B), which indicated that EH-201 increased the RBC numbers by inducing an increase in the endogenous EPO levels (FIG. 5D), as confirmed by the induction of the production of renal and hepatic EPO (FIG. 5C).
  • Hb-a and Hb- ⁇ in the myocardium of the EH-201 -treated mice was significantly increased (FIG. 5F), as confirmed by an increase in Hb protein expression observed with TMBZ staining (FIG. 5G).
  • High doses of EH-201 also induced cardiac mitochondrial biogenesis (FIG. 5E).
  • EH-201 treatment resulted in significantly increased PGC-la expression and mitochondria content and activity in the liver and skeletal muscles (FIGS. 11A and 11B).
  • EH-201 significantly mitigated the Dox-induced impairment of cardiac function.
  • EH-201 ameliorates anaemia and renal function in cisplatin- induced nephropathy Since acute kidney injury may result from renal ischemia caused by the use of nephrotoxic agents, to examine the effect of EH-201 -induced EPO production on the anaemia with renal insufficiency, an established cisplatin-induced nephropathy mouse model was adopted (FIG. 7A). Significant anaemia from day 10 and impaired renal function from day 13 after the first injection of cisplatin was observed (FIG. 7B and 7C). Notably, the administration of 30 and 90 mg/kg of EH-201 for 2 weeks (on day 28, FIG. 7B) led to an almost complete recovery of anaemia.
  • EH-201 30 and 90 mg/kg treatments induced significant recovery of the hepatic EPO expression (FIG. 7G). Furthermore, EH-201 administration also activated the erythroid progenitor cells in the bone marrow (FIG. 7H).
  • EH-201 increases a cellular EPO expression level in mice RPE cells
  • FIG. 18 shows EH-201 induction of cellular EPO expression level in mice RPE cells.
  • Example 3 activating mitochondrial function and haemoglobin expression in neuronal cells by the compound of the present invention
  • This example describes various assays that are useful in evaluating the activation of mitochondrial function and haemoglobin expression in neuronal cells by the compound of the present invention.
  • the compound of the present invention is prepared according to the methods provided in Example 1. The potency of this compound is evaluated using a series of activity assays and these assays are further described in detail below.
  • Astrocyte-enriched cultures were prepared from one-day-old C57BL/6J mice obtained from the Animal Center at the National Yang Ming University as described below. Briefly, cortical tissue was digested with trypsin, and the resultant dissociated cells were suspended in DMEM containing 10% FBS and incubated in 100-mm culture dishes. After 3 days in culture, the media was replaced with fresh 10% FBS/DMEM, and the cells were maintained at 37 °C for an additional 3 days. The cells were dissociated with trypsin, suspended in 10% FBS/DMEM and incubated in a 10-cm dish for 7-8 days prior to use.
  • RNA was prepared using RNA-BeeTM RNA isolation reagent (Tel-test, Friendswood, TX). An aliquot of 5 ⁇ g total RNA was incubated with AMV-RT (Promega) to produce the cDNA for the RT-PCR analysis of the expression levels of ⁇ -actin, NGF and PGC- ⁇ using the ABI Prism 7700 Sequence Detection System and the SYBR Green Master Mix kit (Applied Biosystems, Foster City, CA). The expression level of mouse ⁇ -actin was used as an internal reference. Relative gene expression levels were calculated with the 2-AACT method. Fragments (100-250 bp) were amplified using specific primers for each gene.
  • EPO EPO
  • Hb- ⁇ 5'- TGA TGC TGA GAA GGC TGC TGT CTC TG-3'
  • PGC- ⁇ 5'- AGC CGT GAC CAC TGA CAA CGA G-3'
  • ⁇ -1(5'- CGC CTT CCT GCT CAA CAT T-3') and (5'-TGT GTT CCT CTG TCA GCA TCA C-3') and GAPDH 5'- TCT TCA CCA CCA TGG AGA AG-3' and 5'- ACC AAA GTT GTC ATG GAT GAC-3').
  • Cell and brain tissue lysates were prepared using a radioimmunoprecipitation assay lysis buffer. Approximately 20 ⁇ g of protein was loaded, and western blot analysis was performed using a monoclonal mouse antibody against EPO (1:500; sc-7956, Santa Cruz, California, USA), Hb- ⁇ (1 :500; sc-31116, Santa Cruz, California, USA) and an anti-GAPDH antibody (1:10,000; ab9385, Abeam, Cambridge, UK) that was used as a loading control. A horseradish peroxidase-conjugated anti-IgG secondary antibody was used for enhanced chemiluminescence detection (Amersham, Buckinghamshire, UK).
  • 2',7'-Dichlorofluorescin is oxidized by H2O2 to give a fluorescent product, 2',7'-dichlorofluorescein.
  • cultures in 96-well plates were washed with DMEM containing 1% FCS and loaded with 50 ⁇ DCFH-DA for 30 minutes at 37 °C .
  • Wells were then washed twice with Kreb's buffer, and the cells were solubilized with 0.1 N NaOH in 50% methanol. The wells were vortexed for 10 minutes, and 2'-7'-dichlorofluorescein (DCF) fluorescence was either observed under fluorescence microscopy or measured in a microplate reader (PerkinElmer Life Sciences Wallac Victor2). 6.
  • DCF 2'-7'-dichlorofluorescein
  • Astrocytes and PC 12 neuronal cells were treated with EPO or EH-201 for 24 hours. Astrocyte culture medium was replaced with 500 ⁇ H2O2, and the cells were incubated for 6 hours. PC12 cell culture medium was replaced with 250 ⁇ H2O2, and the cells were incubated for 4 hours. Cell viability was determined by the exclusion of trypan blue as assessed by light microscopy.
  • C57B1/6J adult male mice Forty 12-week-old C57B1/6J adult male mice were obtained from the National Laboratory Animal Center (Taipei, Taiwan). Mice were housed at a constant temperature and supplied with laboratory chow (PMI, Brentwood, MO, USA) and water ad libitum. The experimental procedure was approved by the Animal Research Committee of National Yang-Ming University. The animals were deprived of sleep (SD) or maintained in their home cages (control group) in the same room. Briefly, C57BL/6J male mice (7 weeks of age) were housed on a 12 hours/12 hours light/dark schedule with lights on at AM 6:00 and were handled for 7 days.
  • mice were sleep-deprived in their home cages for 5 hours by gentle handling beginning at AM 6:00 or left undisturbed (non-sleep-deprived mice). Mice were fed with normal diet or normal diet containing different concentrations of EH-201 (50, 100 or 200 mg/kg per day) for 3 days prior to sleep deprivation. 8. Passive avoidance task
  • EH-201 induced neuronal EPO and elevated expression of EPO in primary astrocytes and PC 12 neuronal cells
  • EH-201 a neuronal EPO inducer, elevated the expression of EPO in primary astrocytes and PC 12 neuronal cells. Because exogenous EPO cannot cross the blood-brain barrier, its clinical use is limited. Thus, the effect of an endogenous neuronal EPO inducer, EH-201, was tested.
  • the structure of EH-201 is shown in FIG. 13 A. After EH-201 treatment, astrocytes demonstrated a dose-dependent increase in EPO mRNA expression, as measured by real time PCR analysis (FIG. 13B). EH-201 treatment also up-regulated EPO mRNA expression in PC 12 neuronal cells (FIG. 13C).
  • EH-201 elevated the expression of mitochondrial regulator PGC-la and Hb in primary astrocytes and PC 12 neuronal cells
  • EPO or EH-201 treatment for 24 hours cellular mRNA was extracted to determine EPO-mediated gene expression.
  • Real time PCR revealed elevated expression of PGC-la and Hb- ⁇ mRNA expression; HO-1, a known antioxidant gene up-regulated by EPO, was also induced during EH-201 treatment, both in astrocytes (FIG. 14A to FIG. 14C) and in PC12 neuronal cells (FIG. 14D to FIG. 14F); Hb-a expression, however, was not significantly changed (FIG. 13E to FIG. 13F).
  • EH-201 increased mitochondrial activity and attenuated oxidative stress in primary astrocytes and PC 12 neuronal cells
  • FIG. 15A and E showed that EH-201 increased all three forms of Hb expression in astrocytes and PC 12 cells.
  • mitochondrial activity in cells treated with or without EPO or EH-201 was measured by the MTT assay.
  • FIG. 15B and F showed that EPO or EH-201 induced mitochondrial activity in both astrocytes and PC12 cells. It was examined whether EPO or EH-201 -mediated up-regulation of these genes attenuates oxidative stress induced by H 2 0 2 in astrocytes and PC 12 cells.
  • EPO and EH-201 treatment decreased intracellular ROS in astrocytes (FIG. 15C) and PC12 cells (FIG. 15G). EPO and EH-201 also decreased cell toxicity in cells exposed to H 2 0 2 , indicating that the mitochondrial regulation and ROS homeostasis effect of EPO is biologically important (FIG. 15D and 15H).
  • EPO is required for EH-201 -mediated increased mitochondrial activity and attenuation of oxidative stress in primary astrocytes and PC 12 neuronal cells
  • FIG. 17 A Effects of EH-201 in a mouse model of sleep deprivation- induced memory loss It was evaluated the neuroprotective effect of EH-201 on memory by using a SD model.
  • the experimental procedure is outlined in FIG. 17 A. It was analyzed the EPO expression in the hippocampus from each treated animal. Real time PCR and western blotting showed an increase in EPO expression in animals fed with EH-201 (FIG. 17B and 17C). Hbp, HO-1 and PGC- ⁇ mRNA expression in the hippocampus was analyzed by real time PCR (FIG. 17D). It was further evaluated the mitochondrial succinate dehydrogenase activity using the MTT assay (FIG. 17E).
  • Example 2 describes various assays that are useful in evaluating the inducing autophagy by the compound of the present invention.
  • the compound of the present invention is prepared according to the methods provided in Example 1. The potency of this compound is evaluated using a series of activity assays and these assays are further described in detail below.
  • FITC-OS lxlO 7 OS/well
  • Untreated cells were used to obtain baseline fluorescence.
  • the cells were washed four times with (EBSS) to remove excess POS.
  • EBSS was added to each well, at 100 ⁇ well, and the analysis of mean FITC-OS fluorescence was achieved by a fluorometer, which quantified the FITC-OS fluorescence at excitation 485 nm and emission 535 nm.
  • fluoro-quenching dye was added per well, at and the dye was incubated at 37 °C for 30 minutes; the dye was quantified by fluorometer analysis of fluorescence (excitation, 485 nm: emission, 535 nm).
  • Monodansylcadaverine is a spontaneously fluorescent dye that can be incorporated selectively into autophagosomes and autolysosomes.
  • Cells were incubated with 0.05 mM MDC in PBS at 37°C for 1 hour. After incubation, cells were washed two times with PBS and immediately analyzed by fluorescence microscopy (excitation: 380-420 nm, barrier filter 450 nm). 4.
  • Fluorescence microscopy excitation: 380-420 nm, barrier filter 450 nm. 4.
  • the culture of murine kidney slices and primary mice hepatocytes have described previously. These cultures were treated with EH-201 at different doses (0.6, 2.5, 10 and 40 mg/ml), autophagy activator rapamycin (Rm, 50 nM) or autophagy inhibitor 3-methyladenine (3MA, 10 mM) for 24 hours.
  • EH-201 autophagy activator rapamycin
  • MA autophagy inhibitor 3-methyladenine
  • FIG. 19A to FIG. 19D show induction of autophagy by EH-201.
  • EH-201 induced autophagic activation is through hepatocyte growth factor (HGF) induction.
  • HGF hepatocyte growth factor

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Abstract

L'invention concerne une composition pour induire l'expression d'hémoglobuline (Hb) médiée par l'érythropoïétine (EPO) dans une cellule non hématopoïétique d'un sujet. La composition comprend un composé représenté par la formule (I), dans laquelle R désigne un groupe glycosyle; et un support pharmaceutiquement acceptable.
EP13880425.7A 2013-03-28 2013-03-28 Composition et procédé pour induire l'expression d'hémoglobine médiée par l'epo et la biogénèse mitochondriale dans une cellule non hématopoïétique Withdrawn EP2978417A4 (fr)

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PCT/US2013/034381 WO2014158165A1 (fr) 2013-03-28 2013-03-28 Composition et procédé pour induire l'expression d'hémoglobine médiée par l'epo et la biogénèse mitochondriale dans une cellule non hématopoïétique

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EP2978417A1 true EP2978417A1 (fr) 2016-02-03
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CN106822160A (zh) * 2017-02-16 2017-06-13 中国人民解放军第四军医大学 二苯乙烯苷在制备肥胖性高血压的治疗药物及保健品中的应用
CN107349213A (zh) * 2017-07-10 2017-11-17 苏州神瑞生物科技有限公司 基于二苯乙烯苷的合成制剂在制备治疗神经退行性疾病药物中应用
CN109999047A (zh) * 2019-04-19 2019-07-12 浙江大学 两步法筛选线粒体疾病药物
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JP2016516745A (ja) 2016-06-09
WO2014158165A1 (fr) 2014-10-02
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HK1219871A1 (zh) 2017-04-21
EP2978417A4 (fr) 2017-03-01

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