KR20150135430A - Composition and method for inducing epo-mediated haemoglobin expression and mitochondrial biogenesis in nonhaematopoietic cell - Google Patents

Abstract

상기식에서,
R은 글리코실 그룹이다. The present invention provides a composition for inducing erythropoietin (EPO) -mediated hemoglobin (Hb) expression in non-hematopoietic cells of a subject. The composition comprises a compound of formula I and a pharmaceutically acceptable carrier.
Formula I

In this formula,
R is a glycosyl group.

Description

Technical Field [0001] The present invention relates to a composition and method for inducing EPO-mediated hemoglobin expression and mitochondrial in vivo synthesis in non-hematopoietic cells,

The present invention relates to a composition and a method for inducing hemoglobin expression, mitochondrial in vivo synthesis and autolysis in a subject.

Ischemia leads to oxygen depletion, cellular damage, and related organ dysfunction such as heart failure, stroke, chronic obstructive pulmonary disease, ischemic retinopathy, liver damage and acute renal failure. Because mitochondrial dysfunction is a major factor in organ ischemic injury in the case of oxygen loss, mitochondrial oxidative phosphorylation is abruptly terminated, which leads to a major source of ATP production for energy metabolism.

Erythropoietin (EPO) is essential for red cell mass response in response to tissue oxygenation during hypoxia and anemia. The protective effect of EPO has been demonstrated in an experimental model of various tissues and ischemia-induced damage and has contributed to its effect on non-hematopoietic adaptation, inhibition of apoptosis or angiogenic stimulation. Recently, EPO has been shown to stimulate cardiac mitochondrial proliferation through the activation of mitochondrial in vivo synthesis mediated by peroxisome proliferator-activated receptor-coactivator 1-alpha (PGC-1 alpha), a key regulator of cardiac biophysiology . Clinically, EPO reverses cardiac remodeling by inducing protective effects beyond the correction of anemia, improving cardiac function, and improving exercise durability and patient quality of life. These findings emphasize that EPO-mediated protection may depend on its modulatory effect on intracellular bioenergetics.

Hemoglobin (Hb) is the main oxygen transporter in red blood cells. Its main form, hemoglobin A, is a tetramer of two alpha and beta-polypeptide chains, each of which contains a heme group. Recently, it has been unexpectedly found that Hb is unexpectedly expressed in many non-hematopoietic cells capable of promoting tissue oxygen transport or increasing cellular oxygenation and thus providing a unique protective mechanism against hypoxic / ischemic damage.

Sleep is implicated in plastic cerebral changes based on learning and memory. Both rapid eye movement (REM) and non-REM sleep (NREM) play an important role in memory. Behavioral observations in rats show that the learning period is associated with a subsequent increase in REM sleep, whereas REM sleep deprivation impairs cognitive processing or memory of previously learned absolute types of substance. NREM has been found to be positively correlated with the ability to hold a language pair-association list, a narrative memory. In addition, the transition from short-term to long-term memory by reactivation of sharp wave-ripples in the hippocampus during NREM was important for memory cohesion. In addition, induction of a slow oscillatory potential field by application of an oscillatory potential of 0.75 Hz during early nocturnal NREM has been shown to promote retention of hippocampal dependent narrative memory in healthy individuals.

Patients with dementia, such as Alzheimer's disease (AD), often have nocturnally disturbed sleeping surfaces. In early stage AD patients, REM sleep is relatively unaffected by disease progression and is characterized by a significant loss of REM sleep in later stages of AD. These nighttime sleep interferences increase the severity of the increased dementia. Memory loss is accompanied by accumulation of oxidative damage to lipids, proteins, nucleic acids, and mitochondrial decline, all of which can interfere with neuronal function in aging and disease. Sleep deprivation (SD) also induced memory loss and oxidative stress which impaired mitochondrial activity. One study showed that 36h-SD in young adults induces neurophysiologic outcomes similar to those found in elderly people of approximately 60 years of age. Thus, modulation of mitochondrial function and ROS homeostasis may be useful as therapeutic intervention in oxidative stress-related memory loss.

Moreover, both EPO and EPO receptors are expressed in neurons and astrocytes, and EPO is mainly produced by astrocytes in the brain. EPO has been used extensively to promote erythropoiesis in patients with anemia and has recently been shown to have many noncovalent beneficial effects, including cardioprotection and neuroprotection. Early clinical studies have demonstrated cognitive improvement during EPO treatment among patients with chronic renal failure. Recent studies have shown that high dose EPO treatment improves hippocampal flexibility and cognitive performance in patients with neuropsychiatric disorders. High-dose EPO also enhances hippocampal enhancement by regulating the activity of flexibility, synaptic connectivity and memory-related neural networks and improves the spontaneous conditioning stability of cognitive performance in healthy mice.

It is presumed that EPO can play an important role for pharmacological applications in the treatment of SD-induced damage of hippocampal learning and memory by regulating downstream mitochondrial regulator expression. Due to the fact that EPO has limited clinical use because it can not freely pass the BBB, systemic doses of high doses of recombinant Epo (rEpo) will induce neuroprotective activity.

An autophagy or "self digestion process" is an important physiological process for targeting cytosolic components such as proteins, protein aggregates and cell organelles for degradation in lysosomes. The auto-digestive process is also essential to maintain neural homeostasis and its dysfunction is directly related to an increasing number of diseases. In addition, auto-digestion involves the recycling of intracellular nutrients to maintain cell metabolism during starvation and to remove accumulated cellular organelles and proteins accumulated during stress.

Defective self-digestion is a major contributor to diseases including, but not limited to, neurodegeneration, liver disease and cancer. Many human neurodegenerative diseases are associated with abnormal mutations and / or polyubiquitinated protein accumulation and excessive neuronal cell death.

Polygonum multiflorum Thunb is an oriental medicine used for the treatment of anemia, liver disease and other diseases commonly associated with aging. The present invention provides small molecule compounds isolated and identified from red pepper. These compounds have effects in experimental models of cardiovascular disease, cerebral ischemia, Alzheimer's disease and inflammatory disease and have antioxidant and free radical scavenging properties. In addition, the present invention provides the therapeutic effects and physiological mechanisms of the compounds in animal models.

SUMMARY OF THE INVENTION

The present invention provides a composition for inducing erythropoietin (EPO) -mediated hemoglobin (Hb) expression in non-hematopoietic cells of a subject. The composition comprises a compound of formula I and a pharmaceutically acceptable carrier:

(I)

In this formula,

R is a glycosyl group.

Wherein the glycosyl group is selected from the group consisting of dihydroxyacetone, glucose, galactose, glyceraldehyde, threose, xylose, mannose, ribose, ribulose, tagatose, psicose, fructose, sorbose, rhamnose, erythrose, Lactose, maltose, lactulose, trehalose, cellobos, isomaltotriose, nigerrotriose, maltotose, maltose, maltose, Rios, Melitose, Maltolyoulos, Raffinose, Cestose, and combinations thereof.

According to the present invention, the compounds are useful for inducing Hb-a, Hb-, or dimeric Hb expression in non-hematopoietic cells of a subject, enhancing erythropoietin-erythropoietin receptor binding affinity and also erythropoietin- Binds to a combined erythropoietin receptor complex. In addition, the compounds promote endogenous EPO expression in non-hematopoietic cells and stimulate Hb expression.

The non-hematopoietic cells are selected from the group consisting of kidney cells, hepatocytes, myocardial cells, myoblasts, glial cells, neurons and retinal pigment epithelial cells.

The present invention further provides a method for inducing erythropoietin (EPO) -mediated hemoglobin (Hb) expression in non-hematopoietic cells of a subject, comprising administering to said subject a therapeutically effective amount of a compound of formula I as defined above . According to the present invention, the subject is selected from the group consisting of hypoxia, anemia, kidney ischemia, myocardial ischemia, pulmonary ischemia, neurodegenerative diseases, neuropsychiatric disorders, aging-related macular degeneration (AMD) Disease or syndrome.

The present invention further provides a composition for inducing erythropoietin (EPO) -mediated mitochondrial in vivo synthesis in non-hematopoietic cells of a subject, comprising the above-mentioned compounds of formula I and a pharmaceutically acceptable carrier .

According to the present invention, the compounds induce an increase in the expression of mitochondrial members or PGC-1 alpha to induce EPO-mediated mitochondrial in vivo synthesis, promote erythropoietin-erythropoietin receptor binding affinity, Binds to an ethyne-bound erythropoietin receptor complex. In addition, the compound enhances endogenous EPO expression and stimulates Hb expression in non-hematopoietic cells of the subject. The EPO-mediated mitochondrial in vivo synthesis is PGC-1 alpha-dependent.

The non-hematopoietic cells are selected from the group consisting of kidney cells, hepatocytes, myocardial cells, myoblasts, glial cells, neurons and retinal pigment epithelial cells.

The present invention further provides a method for inducing erythropoietin (EPO) -mediated mitochondrial in vivo synthesis in non-hematopoietic cells of a subject, comprising administering to said subject a therapeutically effective amount of the above-mentioned compound of formula I to provide. The compound induces an increase in expression of mitochondrial members or PGC-l [alpha] to induce EPO-mediated mitochondrial in vivo synthesis.

The subject is a subject suffering from a condition selected from the group consisting of hypoxia, anemia, ischemia-related diseases, neurodegenerative diseases, neuropsychiatric disorders, aging-related macular degeneration (AMD) -related diseases, cardiomyopathy, brain aging, chronic liver disease, multiple sclerosis, Heart failure, obesity, diabetes mellitus, kidney disease, atherosclerosis, aging, metabolic syndrome, and combinations thereof.

The ischemic-related diseases include heart ischemia, ischemic neural degeneration, cerebral ischemia, myocardial ischemia, limb ischemia, cerebral ischemia, liver ischemia, retinal ischemia, stroke, nephritis ischemia, pulmonary ischemia, colonic ischemia, cardiovascular ischemia, ≪ / RTI > The neurodegenerative disease is a disease 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 an auto-digesting action in a subject having an auto-digestive defect comprising administering to the subject a therapeutically effective amount of the above-mentioned compound of formula I, wherein said auto- Thereby enhancing the removal of protein aggregates in the subject.

Wherein said protein aggregate is selected from the group consisting of hungtintin, amyloid beta (A [beta]), alpha-synuclein, tau, superoxide dismutase 1 (SOD1) ≪ / RTI > variants and mutated forms of < RTI ID = 0.0 > and / or < / RTI > The cells of the subject are neurons or glial cells.

The auto-exacerbation defect may be selected from the group consisting of neurodegenerative diseases, retinal diseases, Crohn's disease, aging, myocardial hypertrophy, chronic heart failure, tuberculosis, chronic obstructive pulmonary disease (COPD), cystic fibrosis, liver cirrhosis, Is a disease selected from the group consisting of Paget's disease of bone, obsessive-compulsive disorder, anterior temporal dementia, glomerular disease, metabolic disease, glycogen storage disease type II, inflammatory bowel disease and Pompe disease. The neurodegenerative disease is a disease 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 an auto-digestion effect in a subject having an auto-digestive defect. The composition comprises a compound of formula (I) as mentioned above and a pharmaceutically acceptable carrier.

In addition, the present invention provides a method for preventing amnesia in a subject, comprising administering to the subject a therapeutically effective amount of a compound of formula (I) as defined above. The compounds induce erythropoietin (EPO) to activate autogenous action in the subject.

The self-digestion enhances protein removal in the subject.

The auto-digestive defect is a neurodegenerative disease selected from the group consisting of Huntington's disease, Alzheimer's disease, Parkinson's disease and insomnia.

A patent or a patent application file contains one or more drawings in color. The patent or patent application file will be provided at the office upon the necessary billing and payment.
Figures 1A-1B show characterization of EH-201. (a) HPLC profile of EH-201. A Mightysil RP-18 column (4.6 x 250 mm id, 5 탆) was eluted with a gradient of MeOH / H ₂ O (20/80, v / v) to 100% MeOH for 60 min at a detection wavelength of 280 and 300 nm ml / min. < / RTI &gt; (b) Positive ion mode LC-APCI / MS / MS of EH-201.
Figures 2A-2J show that EH-201 is a potent inducer of EPO expression. (a) Chemical structure of EH-201. (b, c) EH-201-treated kidney slices and hepatocytes were analyzed for EPO expression by Q-PCR and Western blotting. (d, e) primary mouse myocardial cells and (f, g) C2C12 canaliculus cells were treated with EH-201 and the effects on the expression of EPO and EPOR were analyzed by QPCR and Western blotting. (h) The bone marrow cells were incubated with EH-201 for 48 hours and the expression of EPO was detected by Q-PCR. (i) the bone marrow cells were incubated with EH-201 and the colonies counted on day 9 for burst-forming units-erythroid (BFU-E). (j) Quantification of the differentiated erythrocyte streaks was performed using hemoglobin colorimetric method. The control group represents vehicle treatment. The values provide an average + SEM (n = 6 for each). * P &lt; 0.01, * P < 0.05 vs. control, Student's t-test.
Figures 3a-3g show that the induction of mitochondrial in vivo synthesis by EH-201 is mediated by EPO. EH-201-treated kidney slices and primary myocardial cells in the presence or absence of (a, b) neutralized EPO antibody (nEPO-ab, 1 ug / ml) and (c, d) EH- And C2C12 canalicular cells were cultured by QPCR (n = 6) and Western blotting (n = 4), citrate synthase activity (n = 3), and mtDNA copy number (n = 6) and by MitoTracker assay ) < / RTI &gt; (n = 6). The control group represents vehicle treatment. (e, f and g) rhEPO was administered to kidney slices, hepatocytes and C2C12 canaliculus cells. The mitochondrial activity was determined by PGC-1 alpha Q-PCR (n = 6), citrate synthase activity (n = 3), mtDNA copy number (n = 6), and mitochondrial assay (n = 6). The control group represents vehicle treatment. PGC-1α siRNA-transfected C2C12 canaliculus cells were treated with rhEPO (n = 6). The control group represents scrambled siRNA treatment. This value is provided as mean + SEM. ** P <0.01, * P <0.05 vs. untreated control, ns, not significant, Student t-test.
Figures 4A-4I show that the induction of hemoglobin expression by EH-201 in non-hematopoietic cells is mediated by EPO. (a) C2C12 canaliculus cells cultured under oxygen steady state or hypoxic (5% CO ₂ ) for 24 hours were subjected to RT-PCR for expression of hemoglobin-alpha (Hb- alpha) and -beta % Agarose gel electrophoresis. (b) rhEPO-treated C2C12 canaliculus cells were analyzed by Q-PCR (n = 6) for hemoglobin expression in the presence or absence of PGC-1α siRNA transfection. The control group represents scrambled siRNA treatment. (c, d) The rhEPO-treated kidney slices and hepatocytes were analyzed by Q-PCR (n = 6) for hemoglobin expression. The control group represents vehicle treatment. (e, f) EH-201-treated hepatocytes in the presence or absence of EH-201-treated kidney slices and primary mouse myocardial cells and (g, h) neutralized EPO antibody (nEPO-ab, 1 ug / And C2C12 canalicular cells were analyzed by Q-PCR (n = 6) for hemoglobin expression. The values are expressed as means ± SEM. ** P <0.01, * P <0.05 versus untreated control group, ^# P <0.05 vs. control group treated with rhEPO (50 ng / ml). (i) The effect of rhEPO and EH-201 on the proliferation of TF-1 cells was determined by trypan blue dye rejection analysis (top of Fig. 4i, n = 6). The presence or absence of neutralizing antibodies (nEPOR-ab, 0.5 μg / ml; nEPO-ab, 1 μg / ml) for the 48 h proliferation assay or (Fig. 4I, bottom, n = 6). The values are expressed as means ± SEM. ^{## P <0.01, * P <} 0.05 vs control (top of FIG. 4i) or rhEPO alone (lower part of Fig. 4i), ## P <O.O1 for rhEPO + EH-201 25 M, Student's t- test.
Figures 5A-5G show that EH-201 increases tolerance and activation of mitochondrial and hemoglobin expression in mice. (a) Resistance of normal mice was measured by rotarod exercise under oxygen steady state or hypoxia (8% O ₂ ) condition (ND: normal diet). (b) Effect of EH-201 on plasma RBC count and hemoglobin level. (c, d) EPO mRNA expression in mouse kidney and liver was measured by Q-PCR 3 days after EH-201 administration. Serum levels of EPO were measured by ELISA. (e, f) After 3 days of administration of EH-201, isolated myocardial tissue was analyzed by Q-PCR for hemoglobin expression and mitochondrial in vivo synthesis was determined by mtDNA copy number. (g) The effect of EH-201 treatment on vehicle hemoglobin (Hb) expression was quantified by TMBZ staining on SDS-PAGE (left part of FIG. 5g). Quantification of Hb expression (tetramer and dimer, right part of Figure 5g). The values are expressed as means ± SEM (n = 5 for each group). ^# P < 0.01, ^# P < 0.05 vs. ND group; # P <0.01, # P <0.05 vs. 7 days ND group by one-way ANOVA with post-hoc study in Turkey.
Figures 6a-6h show that EH-201 has a therapeutic effect on cardiac dysfunction in doxorubicin (Dox) -induced cardiomyopathy in mice. (a) Survival was analyzed using the Kaplan-Meier method (detailed treatment protocol in Materials and Methods). The normal (N) group represents a saline injection. (b) Effect of EH-201 treatment on mice subjected to hypoxic rotarod immunity test two weeks after Dox injection. (c, d) The effects of EH-201 on cardiac abnormalities and function were characterized by ECG and echocardiography. EF, discharge fraction; FS, fraction reduction; LVIDs / d, left ventricular internal diameter at systolic / diastolic. (e) Representative photomicrographs of the left ventricular section of the mouse heart stained with hematoxylin-eosin and mathon trichrome (left part of FIG. 6E, rod = 10 μm). The blue staining indicated fibrosis and quantification of epilepsy fibrosis (right part of Fig. 6E). (f) Dox 2 weeks post-isolated myocardial tissue was analyzed by Q-PCR for hemoglobin expression and (g) TMBZ staining of each myocardial lysate in the treatment group on SDS-PAGE was performed with quantitative values (h) . The values are expressed as means ± SEM (n = 5-6 animals for each group). ** P <0.01, * P <0.05 versus Dox group by one-way ANOVA with post-hoc study in Turkey.
Figures 7a-7h show that EH-201 accelerates the recovery from anemia and renal function in cisplatin-induced nephropathy in mice. (a) a schematic diagram protocol. (b) Epidemiology of the number of RBCs in peripheral blood over time. (c) Time course of blood urea nitrogen (BUN) value after cisplatin injection. (d) Functional recovery of mouse epidemiology treated with EH-201 on day 28. (e) Hematoxylin-eosin staining of the kidney section after administration of EH-201 on day 28 (rod = 100 μm). (f, g) On day 28, expression of EPO in kidney and liver was determined by Q-PCR. (h) Number of BFU-E colonies in isolated bone marrow cells from mice treated at day 28. [ The values are expressed as means ± SEM (n = 5-6 animals for each group). Normal group with normal group; ^## P &lt;0.01; ** P <0.01, * P <0.05 versus control group by one-way ANOVA with post-test study in Turkey.
Figure 8 shows that EH-201 induces Sirtl expression. Sirtl protein expression in EH-201-treated kidney slices and lysates of hepatocytes was analyzed by Western blotting (n = 4). The control group represents vehicle treatment. The values are expressed as means ± SEM. ** P <0.01, * P <0.05 compared with the control group.
Figure 9 shows a ribbon diagram of the results of computer docking for EH-201 for the EPO / EPOR complex. Docking calculations were performed using a docking server for EPO complexed with the extracellular domain of the EPOR protein model (PDB entry code lcn4). The green skeleton (green) using the ball and the stick indicates the ligand molecule EH-201, and the helix (red, left part of FIG. 9) points to the helix A of EPO and the loop (gray, Points to loop 5 of the EPOR. Predictive interaction residues, including PRO ¹⁴⁴ , GLU ¹⁴⁷ , PRO ¹⁴⁹ , Met ¹⁵⁰ , and THR ¹⁵¹ , are located in loop 5 of the EPOR critical for EPO binding.
Figures 10a-c show that EH-201-induced EPO production does not involve Hif-a activation. (a) The hypoxic response element (HRE) -activated luciferase reporter (Luci) transfected HEK 293 cells were incubated with EH-201 under oxygen steady state or hypoxia (5% O ₂ , as a positive control) for 24 hours Respectively. The plasmid for? -galactosidase (? -Gal) was used as a transfection control and pGL3-v was used as a vector control. Similar results were observed in three additional independent experiments. (b) VEGF expression of EH-201 treated hepatocytes was analyzed by Q-PCR (n = 3). The hypoxic condition was used as a positive control. (c) Hif-2α protein expression levels in nuclear lysates of EH-201 treated turkey slices were analyzed by Western blotting (H: 0% ₂ hypoxia as a positive control). The control group represents vehicle treatment. The values are expressed as means ± SEM. Oxygen steady state, ** P <0.01 compared to Student's t-test, * P <0.05.
Figures 11A and 11B show that EH-201 increases mitochondrial function and in vivo synthesis in liver and skeletal muscle. (a, b) After 14 days of administration of EH-201, isolated liver and skeletal muscle were analyzed by PGC-1? Q-PCR, citrate synthase activity and mtDNA copy number for mitochondrial activity. The values are expressed as means ± SEM (n = 5 animals for each group). ** P < 0.01, * P < 0.05 vs. ND group; ^# P <0.01, ^# P <0.05 vs. 7 days ND group by one-way ANOVA with post-hoc study in Turkey.
Figures 12a and 12b show that EH-201 has a therapeutic effect on cardiac dysfunction in doxorubicin (Dox) -induced cardiomyopathy in mice. (a) Effect of EH-201 on body weight of mice 2 weeks after Dox injection. (b) The effect of EH-201 on cardiac function was characterized by ECG, the heart rate provided in beats per second (bps). The values are expressed as means ± SEM (n = 5-6 animals for each group). ** P <0.01, * P <0.05 versus Dox group by one-way ANOVA with post-hoc study in Turkey.
Figures 13a-13f show that EH-201 stimulates EPO expression in primary astrocytes and PC12 neurons. (a) Structure of EH-201. (b, c) Real-time PCR shows that EH-201 treatment increases EPO mRNA in astrocytes and PC12 neurons for 24 hours. Expression of GAPDH was used as an internal control. (d) Western blotting shows that EH-201 treatment increases EPO protein expression in astrocytes and PC12 neurons for 24 hours. The results are expressed as the relative exponent of at least three independent measures of untreated control group SD. * P <0.05, ** P <0.01 compared to untreated control by one-way ANOVA followed by multiple comparison studies in Turkey. (e) Real-time PCR shows that EPO treatment does not increase Hb-a mRNA in astrocytes and PC12 neurons for 24 hours. (f) Real-time PCR shows that EH-201 treatment does not increase Hb-a mRNA in astrocytes and PC12 neurons for 24 hours. Expression of GAPDH was used as an internal control. The results are expressed as the relative exponents of the untreated control group SD of at least three independent measurements. * P <0.05, ** P <0.01 compared to untreated control by one-way ANOVA followed by multiple comparison studies in Turkey.
14A-14F show that EH-201, a nerve EPO inducer, stimulates the expression of mitochondrial regulators (PGC-1α, Hb-β) and antioxidant gene (HO-1) in primary astrocytes and PC12 neurons . (a) Real-time PCR showed that EPO or EH-201 treatment increased PGC-1α for 24 hours. (b) Hb-β and (c) HO-1 mRNA expression in primary astrocytes. (d) Real-time PCR showed that EPO or EH-201 treatment increased PGC-1α for 24 hours and (e) Hb-β and (f) HO-1 mRNA expression in PC12 neurons. Expression of GAPDH was used as an internal control. The results are expressed as relative exponents of untreated controls &lt; RTI ID = 0.0 &gt; SD < / RTI &gt; from at least three independent measurements. * P <0.05, ** P <0.01 by one-way ANOVA followed by Turkey multiple comparison test.
15A-15H show that EH-201 increases mitochondrial activity in primary astrocytes and PC12 neuronal cells, reduces intracellular ROS and attenuates H ₂ O ₂ -induced cytotoxicity. (a, e) Expression of different forms of Hb (monomer: 16 kD, dimer: 32 kD, tetramer: 64 kD) is identified by Hb-β Ab in primary astrocytes and PC12 neurons treated with EH-201. The results are expressed as relative expression of untreated control < RTI ID = 0.0 &gt; SD < / RTI &gt; from at least three independent measurements. * P < 0.05, ** P < 0.01 by Student's t-test. (b, f) The succinate dehydrogenase activity of astrocytes and PC12 cells treated with EPO or EH-201 at 24 hours was determined using MTT reduction assay (n = 8) and the respective control conditions 24 Without processing at the time). The values are mean SD (n = 8). * P < 0.05, ** P < 0.01 compared to untreated control. (c, g) The astrocytes and PC12 cells treated with EPO or EH-201 for 24 hours are exposed to 100 μM H ₂ O ₂ for 6 hours. Intracellular ROS formation is measured using the DCFH-DA assay. The graph shows the results in relative fluorescence units (RFU). The values are mean SD (n = 8). 0.0 &gt;^# P < 0.01 < / RTI &gt; * P <0.05 compared with H ₂ O ₂ control, ** P <0.01. Scale bar: 50 탆. (d, h) Strontic cells and PC12 cells treated with EPO or EH-201 for 24 hours are exposed to 500 μM H ₂ O ₂ for 6 hours. Cytotoxicity is analyzed using Trypan blue. The values are mean SD (n = 3) as compared to untreated controls. 0.0 &gt;^# P < 0.01 < / RTI &gt; * P < 0.05, ** P < 0.01 compared to H ₂ O ₂ control using Student's t-test.
Figures 16A-16F show that EPO is required for neuroprotective effects of EH-201 in astrocytes and PC12 neurons. Concurrent incubation of EH-201 with (a, d) anti-EPO antibody resulted in EH-201 in succinate dehydrogenase activity as assessed by MTT reduction assay (n = 8) in astrocytes and PC12 cells - Lost induced increase. Compared with the control group, * P <0.05, ** P <0.01. (B, E) The simultaneous incubation of EH-201 with anti-EPO antibody for 24 hours resulted in EH-201 mediated by H ₂ O ₂ as assessed by CDFH-DA assay (n = 8) and loss of the reduced ROS generation, (c, f) trypan blue staining (n = 3) with the reduction of H ₂ O _2, as evaluated by a - compared to the mediated cytotoxicity, H ₂ O ₂ * P <0.05, ** P <0.01, compared to the control group using Student's t- test for ^## P <0.01.
Figures 17a-17g show the effect of EH-201 in a mouse model of sleep deprivation-induced memory loss. (a) The process 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. Statistically significant ** p <0.01 compared with SD group; A statistically significant difference compared to the control group, ^## p <0.01. (d) Real-time PCR analysis of Hbβ, PGC-1α and HO-1 expression levels in mouse hippocampus (n = 6). Statistically significant * p <0.05 compared to the SD group, ** p <0.01, and one-way ANOVA followed by a Turkey's multiple comparison statistically significant ^# p <0.05 compared to the control by the test, p ^## &Lt; 0.01. (e) The MTT assay is used as a marker for mitochondrial activity. The values show mitochondrial function after sleep deprivation of untreated control mice and EH-201-treated mice. * P <0.05, ** p <0.01 compared with SD group statistically significant. One-way ANOVA followed by a comparison with the control group by the Turkey's multiple comparison test statistically significant ^## p <0.01. (f) Obtaining passive avoidance through steps during five consecutive training tests in mice treated with EH-201. EH-201 treatment does not affect learning ability in mice. (g) Acquisition of passive avoidance through steps during three consecutive test attempts in mice treated with EH-201. * P <0.05, ** p <0.01 compared with SD group statistically significant. One-way ANOVA followed by a comparison with the control group by the Turkey's multiple comparison test statistically significant ^## p <0.01.
Figure 18 shows EH-201 induction of cellular EPO expression levels in mouse RPE cells. C57 mouse RPE cells were incubated with 0.4, 2, and 10 μg / ml of EH-201 in DMEM supplemented with 10% FCS. The culture was incubated at 37 [deg.] C for 24 hours. After the incubation period, the whole cell lysate was prepared with solution buffer. Whole cell lysates were prepared and applied to Western blot analysis to detect levels of endogenous EPO. GAPDH was used as a loading control. The bars represent the mean ± SD (n = 3 different experiments; ** p <0.01, *** p <0.001).
Figures 19a-19d show induction of auto-digestion by EH-201. Primary mouse hepatocytes were treated with EH-201, rapamycin (autogenous activation factor, Rm, 50 nM) or 3-methyladenine (3MA, 10 nM) at different doses (0.6, 2.5, 10 and 40 μg / mM) (a and b). Under starvation, primary mouse hepatocyte cultures served as an autogenous activation control (a, b). These treated cells were stained with monocarboxylic catarrhine (MDC), followed by fluorescence microscopy (scale bar: 50 mm), and the fluorescence intensity was measured with a spectrophotometer (b). Western immunoblotting was performed with hepatocyte lysates to study the expression of the self-digesting marker protein LC3 using LC3 antibody (c, d). Study the effect of EH-201 on the induction of self-digestion using kidney slices treated with EH-201 for 18 hours; Analysis of autogenous induction was performed by analyzing western blot for LC3. Quantitation of LC3-II / LC3-I was performed using an immunoreactive band with ImageQuant imaging software (Amersham Biosciences). Data are expressed as means ± SEM. ** P <0.01, * P <0.05 compared with the control group.
Figures 20a and 20b show that activation of EH-201 induced self-extinguishing is through induction of hepatocyte growth factor (HGF). Hepatocytes were treated with EH-201 at different doses (0.6, 2.5, 10 and 40 mg / ml) for 24 hours. rhEPO represents recombinant human EPO; rmHGF represents a recombinant rat hepatocyte growth factor and nEPO-ab represents a neutralizing EPO antibody.

The following specific examples are used to illustrate the invention. Those skilled in the art can readily appreciate other advantages and effects of the present invention. The present invention may also be performed by different specific cases, and the details of the guidance may also be based on different viewpoints and applications in various variations, and the changes do not depart from the purpose of the application.

Erythropoietin is abbreviated herein as EPO in the specification and drawings.

Example 1 Extraction, Isolation and Characterization of EH-201

EH-201, 2,3,5,4'-terahydroxystilbene-2-o-beta-d-glucoside (hereinafter referred to as EH-201) &Lt; / RTI &gt; The dried and ground roots of the red algae were extracted with 40% ethanol and then evaporated to form syrup. To enrich the target components, the extract was diluted twice with 15% ethanol, loaded onto a Diaion HP-20 resin column, and subsequently eluted with 20%, 40%, and 70% ethanol, respectively, in succession. The eluate of 40% ethanol was collected and evaporated. The 40% ethanol eluate was then redissolved in 10% ethanol by sonication and separated into five equal volumes of successive portions of ethyl acetate. The residue of ethyl acetate was then passed through a Sephadex LH-20 column eluting with methanol. A light yellow compound, EH-201, was obtained. The overall yield is about 0.5% with the final compound EH-201 in pure form (99.2%) from the crude dry roasted roots of the red pepper. For future clinical testing purposes, crystallization of the compound was further performed. The 30% aqueous-ethanolic solution of EH-201 is then placed in a -20 ° C refrigerator overnight, followed by a 4 ° C refrigerator. Visible crystals were obtained a few days later.

The chemical entities of EH-201 were characterized by LC / MS / MS, UV, ¹ H-NMR and proton-decoupled ¹³ C-NMR data (FIG. 1 and Table 1) and ¹ H- NMR and Bruker AVIII- It was confirmed by de-coupling the ¹³ C-NMR data set-proton using a measuring instrument. The protons and the carbon chemical shifts of EH-201 are listed in Table 1. The LCMS data of the purified EH-201 was analyzed on a Bruker LC / MS / MS column using APCI (atmospheric pressure chemical ionization) mode with positive ion polarity using a gradient of water and methanol of HPLC grade over 60 min with a reversed phase C18 column. MS spectrometer Esquire 2000 (Fig. 1A). The LCMS data are shown in FIG. 1B showing the correct mass of EH-201 at m / z 407.0. At m / z 407.0, the EH-201 ion was further applied to MS / MS analysis where 407.0 ions were isolated and fragmented. The daughter ion obtained at m / z 245.1 corresponds to EH-201 in which its sugar moiety has been lost. Thus, the compound was identified as 2,3,5,4'-tetrahydroxystilbene-2-o-beta-d-glucoside (TSG or THSG) (Fig.

Example 2 Activation of mitochondrial function and hemoglobin expression in non-hematopoietic cells by the compounds of the present invention

This example describes various assays useful in assessing the activation of mitochondrial function and hemoglobin expression in non-hematopoietic cells by the compounds of the present invention. The compounds of the present invention are prepared according to the method provided in Example 1. The efficacy of the compounds is assessed using a series of activity assays and these assays are described in further detail below.

1. Animals

C57BL / 6J male mice (20-25g) without any specific pathogen at 8-10 weeks of age, purchased from the National Laboratory Animal Center (Taiwan), are housed at 5-10 ° C at a constant temperature of 22 ± 2 ° C Standard laboratory food (PMI, Brentwood, MO, USA) and water were fed free-flowing under a 12 hour dark / light cycle. The experimental protocol was approved by the Institution of the Animal Research Committee of National Yang-Ming University (Guide for Animal Experiments, National Yang-Ming University). All efforts have focused on minimizing the animal suffering to reduce the number of animals used and, if available, to use another alternative to in vivo technology. All studies involving animals were reported in accordance with the ARRIVE guidelines for reporting experiments involving animals.

2. Cell culture and treatment

C2C12 myoblasts, HEK293, and TF-I cells were purchased from the Bioresources Collection and Research Center (BCRC, Hsinchu, Taiwan). The C2C12 myofibroblast differentiated into canaliculus cells and treated with the drug for 24 hours. In vitro 250 쨉 m thick kidney slices were prepared from 8-10 week old C57BL / 6J mice as previously described. The slices were treated with drug in media (15 mM HEPES and 20 mM sodium bicarbonate buffered DMEM / F12) in the atmosphere chamber at 37 ° C with 50% O ₂ : 5% C0 ₂ : 45% N ₂ for 18 hours Respectively. Mouse primary hepatocytes were isolated and purified from 8-10 week old C57BL / 6J mice as previously described and plated onto 1% gelatin-coated microplates in DMEM supplemented with 10% FBS (Gibco, Germany). After the hepatocytes were attached, fresh medium containing the drug was added for 24 hours. Newborn C57BL / 6J mouse myocardial cell cultures were prepared from 1 day old C57BL / 6J mice purchased from the Animal Center at the National Yang-Ming University, and the isolated ventricular cells were cultured in DMEM supplemented with 10% FCS Gt; M199 &lt; / RTI &gt; medium (Gibco, Germany). Myocardial cells were incubated in a humidified atmosphere at 37 [deg.] C with 5% CO ₂ on plates previously coated with 1% gelatin. The subconfluences of spontaneously beating cells were achieved after 48 hours of incubation, after which treatment with various drugs was carried out for 24 hours. Bone marrow stromal cell cultures for colony-forming assays and hemoglobin colorimetric assays were prepared as previously described. In the knockdown experiments, C2C12 root canal cells were transfected with scrambled or PGC-lα-specific siRNA using the lipofectamine 2000 reagent according to the manufacturer's instructions (Invitrogen) (Table 2). These cultured cells were treated with rhEPO (recombinant human erythropoietin, Roche, Germany) or EH-201 or co-incubated with EPO neutralizing antibody (R & D, MN) for the indicated time period. Thereafter, the drug-treated cells and tissue lysates were collected and disrupted to examine their mitochondrial activity as well as specific expression of mRNA and protein.

3. Real-time PCR

Total RNA was extracted using TRIzol reagent (Invitrogen) and reverse transcribed with M-MLV reverse transcriptase (Promega). The expression of the EPO, EPOR, PGC-1 alpha, Hb- alpha, Hb- beta and GAPDH mRNA was quantitatively analyzed by quantitative real-time PCR with an ABI 7500 sequence detector (Applied Biosystem) using SYBR Green Master MixR (AB 1-7500) PCR).

The relative mRNA expression levels were determined using the TTCt method with GAPDH as an endogenous control. The primers used are listed in Table 2.

4. Western blot

Total protein (50 μg) was separated by 12% SDS-PAGE and transferred onto PVDF membranes and analyzed with EPO, PGC-1α, GAPDH, PCNA (Santa Cruz, CA), Sirtl (Millipore, Billerica), or Hif- , &Lt; / RTI &gt; Littleton). After incubation with the appropriate horseradish peroxidase-conjugated secondary antibody, the signal was visualized by ECL detection according to the manufacturer's protocol (Perkin-Elmer).

5. Quantification of mtDNA copy number

Total cell DNA was purified using the conventional phenol-chloroform method and the mtDNA copy number was determined as previously described.

6. Mito Tracker Black

The mitochondrial contents were assessed by MitoTracker microplate assay. The treated cells were loaded with 0.1 μM green fluorescent MitoTracker-Green (MTG, Invitrogen) for 60 min at 37 ° C. The intracellular MTG content was measured by a fluorescence optical meter (Thermo Scientific Inc.). Subsequently, the immobilized cells were labeled with H33342 to assess cell density. The MTG / H33258 fluorescence ratio was calculated.

7. Measurement of citrate synthase activity

The citrate synthase activity was measured in tissue lysates. The change in absorbance at 412 nm is measured and the activity is expressed as μmol / min / mg protein.

8. TF-1 cell proliferation assay

Cells of tEPO-sensitive cell line TF-1 were seeded in 96 well microplates with a cell density of 1 x 10 ⁵ cells / ml in RPMI 1640 medium with 2% PBS and the cells were incubated with EPOR neutralizing antibody (Santa Cruz) Lt; / RTI &gt; and EH-201 in the presence or absence of &lt; RTI ID = 0.0 &gt; The cell number was determined by trypan blue dye rejection assay.

9. Evaluation of rotarod resistance

Prior to splitting into treatment groups, 8- to 10-week-old C57B1 / 6J male mice were trained on a Rotarod device (14 rpm) for up to 10 minutes each for 3 consecutive days of training every 3 days, Were excluded from the experiment. After training, eligible mice were divided into EH-201-treated groups (10, 30 or 90 mg / kg per day, n = 5 for each group) for 7 days. On the day of the test, each mouse was subjected to three tests on a rotor rod at 22 rpm under oxygen steady state or hypoxic (8% O ₂ ) atmosphere. Resistance performance was measured by time course until the mouse was exhausted from tiredness and fell from the rotarod image. The maximum test duration was 60 minutes and there was a 30 minute test rest between each test.

10. EPO ELISA

Serum EPO concentrations were analyzed using ELISA kits specific for mouse EPO (R & D, MN) according to the manufacturer's instructions.

11. Doxorubicin-induced cardiomyopathy

Cardiomyopathy was induced in 8-10 week old C57B1 6J male mice by intraperitoneal (i.p.) injection of 15 mg / kg of doxorubicin-HCl (Sigma-Aldrich) and normal saline was injected (n = 6). Seven days after the injection, the presence of doxorubicin-induced cardiomyopathy was confirmed using an electrocardiogram by observing the long-term S-T interval. An average of 80% of the injected mice were successfully induced (27/34), and ineffective mice were excluded from the EH-201 treatment experiments. The cardiomyopathic mice were randomly divided into four equal groups containing a control (Dox, n = 9) and three EH-201-treated groups (n = 6 for each group) for an additional week. EH-201 was orally administered by mixing it with the feed. Normal diets were fed to the Dox group and normal diets containing different doses of EH-201 (10, 30 or 90 mg / kg per day) were fed to the EH-201-treated group. One week later, the mice were subjected to rotarod immunity test, echocardiography and electrocardiogram. The mice were killed after electrocardiography and the isolated hearts were subjected to histological examination and hemoglobin analysis.

12. Hemoglobin staining

Staining for hemoglobin in isolated myocardial tissue lysates was performed using non-reducing SDS-PAGE followed by tetramethylbenzidine (TMBZ, Sigma-Aldrich). The gel was photographed and scanned using a Typhoon Trio (TM) Imager (GE Healthcare). TMBZ staining was removed from the gel by adding 70 mM sodium sulfite solution. Subsequently, sodium sulfite was replaced with 30% isopropanol and the gel was then stained with coma blue for analysis of the protein loading control.

13. Cardiac ultrasound and electrocardiogram

Mice from all treatment groups were euthanized with isoflurane (0.75-1.5% inhalation) and echocardiography was performed in M mode three times for each mouse using an ATL HDI 5000 ultrasound system (Philips Medical Systems) . Three electrodes were used to evaluate the electrocardiogram (ECG) parameters. From lead I, the ECG tracing was recorded using an electrocardiograph connected to the subcutaneous needle electrode in isoflurane-euthanized mice. All probes were connected to amplifiers and digital converters for signal recording in the 100-mV range using low pass 1kHz and high pass 1kHz filters. ECG signals were recorded and analyzed using a learning data system using Lab VIEW software (National instruments, Inc.).

14. Cisplatin-induced nephropathy

Forty 40 8- to 10-week-old C57B1 / 6J male mice were intraperitoneally injected with 3 doses of cisplatin (Sigma-Aldrich) at 4, 6, and 6 mg / kg body weight intervals n = 6) saline (Fig. 6A). On day 13, the collected serum samples were analyzed for urea nitrogen content (BUN). Mice with BUN values greater than 100 mg / dL were selected for this experiment. An average of 70% of the injected mice successfully induced renal insufficiency (26/40) and ineffective mice were excluded from the EH-201 treatment. Subsequently, the mice were randomly divided into four cohorts that included a control (Ctrl, n = 8) and three EH-201 treated groups (n = 6 for each group) for an additional two weeks . Blood samples from all mice were collected every 5 days. The number of RBCs was determined from complete blood cell counts using a Sysmex Kx-21 hematology analyzer (Sysmex America), and the serum BUN levels were measured using a urease GLDH method using a commercially available kit (Urea FS, DiaSys, Germany) .

15. Bone Marrow Stem Cell Colony-Forming Assay

Bone marrow cell suspensions were isolated and cultured from the femurs of 6-week-old C57BL / 6J male mice (National Laboratory Animal Center, Taiwan) to test rupture-forming unit-red blood cells (BFU-E). All cells were cultured in RPMI 1640 medium supplemented with 15% FBS (Gibco, Germany), 1% bovine serum albumin, 0.8% methylcellulose, 0.1 mM 2-mercaptoethanol (Sigma-Aldrich), 2 U / ml EPO / ml &lt; / RTI &gt; IL-3 (Sigma-Aldrich). The colonies (&gt; 50 cells) were counted on day 9 for BFU-E using an inverted microscope.

16. Hemoglobin colorimetric test

For the detection of differentiated erythrocyte streaks, the isolated bone marrow stromal cells were incubated with 1% bovine serum albumin, 7.5 μM 2-mercaptoethanol, 1.4 mM L-glutamate (Sigma-Aldrich), 5 μM FeC13 Aldrich), and 25 [mU / ml] EPO in the presence of drug treatment. Then, the extracted hemoglobin was mixed with a post-treatment solution of 2,7-diaminofluorene (DAF, Sigma-Aldrich). The change in absorbance at 610 nm was monitored continuously at 25 캜 for 1 minute. The initial reaction rate was measured and the amount of Hb in the sample was determined from the Hb standard curve.

17. Lucifer Lazar Reporter Black

HEK293 cells were transfected with a luciferase reporter plasmid (pGL3, Promega) containing four repeats of the minimal hypoxic response elements (HRE) from the EPO gene. The transfected cells were incubated with EH-201 under oxygen steady state for 24 hours. The cells were maintained under simulated hypoxia (75 mM CoCl ₂ ) or hypoxic conditions (5% O ₂ ) as a positive control for Hif-1α activity. After the treatment, the cell lysate was harvested and luciferase expression was measured by the Bion-Luciferase reporter assay system (Promega).

18. Histological analysis

The heart and kidney tissues were fixed with 10% formalin for paraffin embedding. A 5 mu m thick paraffin section (cross-section for the heart) was prepared for the trichrome staining protocol of H & E and Mason (H & E and Masson's). For the analysis of myocardial fibrosis, six random light microscope photographs were taken from visible myocardium with a magnification of 400x for each animal. In these photomicrographs, the degree of fibrosis was quantified by a blinded observer using the Image J program of NIH origin.

19. Isolated &lt; RTI ID = 0.0 &gt; retinal pigment epithelial &lt; / RTI &gt;

6 to 8-week old C57 / BL6 mice a complete eye quickly removed from (National Laboratory Animal Center, Taiwan ROC ) and 8.0 g / L NaCl, 0.2 g / L KCl, 0.8 g / L KH 2 PO 4, and 1.15 g / L And stored in ice-cold PBS containing NaH ₂ PO ₄ . The 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, 2.5 mM ML-glutamine and 1% Respectively. After washing, eyes were then transferred to fresh PBS for incision. Using a micro incision scissor and a vertical incision microscope, an annular cut along the periphery of each eye was performed. The posterior eye cups containing neural retina and lens were placed in fresh GM medium and incubated at 37 ° C for 20 minutes in a 5% CO ₂ incubator to promote the separation of retinal pigment epithelium (RPE) cell sheets from the neural retina. After removal of the RPE sheet from the neural retina, the complete sheet of RPE cells was stripped and collected in Eppendorf tubes. RPE cells were centrifuged at 1500 rpm for 5 min and resuspended in GM medium. The cell suspension (0.5 ml) was added to a 12 well plate. Cells were cultured at 37 ° C in 5% CO ₂ for 10 days with changes in medium (GM) every other day. After 10 days, the cells were washed with EDTA and then trypsinized for 4 minutes to desorb the cells. The cells were harvested into tubes and centrifuged at 1000 rpm for 5 minutes and resuspended in DMEM, 10% FBS, PEN / strep, 1-glutamine, sodium bicarbonate. The cells were plated on a 6 cm dish until they reached the confluence, the time point at which they were trypsinized and grown in a larger dish.

C57 mouse RPE cells were incubated with 0.4, 2, 10 μg / ml EH-201 in DMEM supplemented with 10% FCS. The culture was incubated at 37 [deg.] C for 24 hours. After the incubation period, whole cell lysates were prepared using lysis buffer. Total cell lysates were prepared and applied to Western blot analysis to detect levels of endogenous EPO. GAPDH was used as a loading control.

20. Statistics

All results are expressed as mean ± SEM. Statistical analysis was performed using Student's t-test. We used a one-way ANOVA to investigate differences across animal study groups. The differences between the averages of the experimental groups were determined through the Turkish test. P <0.05 was considered significant.

20. Results

(1) EH-201 is a powerful EPO inducer

In order to determine whether EH-201 has the ability to induce EPO expression, kidney slices and hepatocytes were treated with EH-201 in vitro. EH-201 was observed to induce EPO mRNA and protein expression in a concentration-dependent manner in kidney slices and hepatocytes (Figs. 2b and 2c). According to the gene expression pattern of EPO in human tissues in a publicly available database written by Sui A, et al (Su, A. I. et al), EPO transcripts express surprisingly high levels in human myocardial cells. Thus, it was also tested whether EH-201 could also induce EPO expression in neonatal mouse cardiac cells and C2Cl2 myocytes. EH-201 was observed to induce the expression of EPO and EPO receptor (EPOR) in primary myocardial cells and C2Cl2 myocytes in a concentration-dependent manner (Figures 2d to 2g). Because bone marrow stromal cells can express EPO to mediate hematopoiesis, bone marrow cells were incubated with EH-201 to investigate its effect on erythropoiesis. The expression of EPO mRNA was increased in bone marrow cells exposed to EH-201 (Fig. 2h). EH-201 significantly increased the number of BFU-E colonies (Fig. 2i) and Hb expression (Fig. 2j) in a concentration dependent manner. Thus, EH-201 is an EPO inducer.

(2) Induction of mitochondrial in vivo synthesis by EH-201 is mediated by EPO

In order to determine whether EH-201 affects mitochondrial in vivo synthesis, a series of experiments were conducted to test the effect of EPO inducing agents in non-hematopoietic cells. In the EH-201-treated kidney slice, the activity and mitochondrial copy number of the mitochondrial marker enzyme citrate synthase increased in a concentration-dependent manner and a rapid increase in expression of PGC-l [alpha] was observed (Fig. The stimulatory effect of EH-201 on mitochondrial in vivo synthesis was also observed with hepatocytes, myocardial cells and C2Cl2 myocytes (Figures 3b-3d). However, neutralization-EPO antibody treatment eliminated the effect of EH-201 induced mitochondrial in vivo synthesis (Figures 3c and 3d), whereas EPO treatment increased PGC-1alpha expression and mitochondrial bio synthesis (Figures 3e-3g) . We then investigated whether these effects were mediated by the PGC-1α-dependent pathway using PGC-1α-specific siRNA-transfected C2C12 myocytes. The PGC-1? SiRNA reduced the concurrent failure of EPO inducing PGC-1? MRNA expression and mitochondrial in vivo synthesis by 44% (Fig. 3g), suggesting that activation of mitochondrial in vivo synthesis by EPO is PGC-1? Dependent He said. In addition, since Sirtl, a mammal, regulates mitochondrial function and in vivo synthesis in skeletal muscle and liver with PGC-1α, Sirtl expression was examined and EH-201 treatment was observed to increase Sirtl expression (Figure 8), indicating that the induction of EH-201 of EPO-mediated mitochondrial activity can occur via the Sirtl / PGC-1 alpha pathway. Thus, the increase in PGC-1 alpha due to EH-201 is dependent on induction of mitochondrial in vivo synthesis in non-hematopoietic cells by increased EPO levels.

(3) Induction of hemoglobin expression in non-hematopoietic cells by EH-201 is mediated by EPO

It was further determined whether the expression of hemoglobin (Hb) was regulated by hypoxia-inducible EPO signaling in non-hematopoietic cells. In vitro experiments were performed by incubating C2C12 cells in the absence and presence of hypoxic conditions. Exposure of C2Cl2 myoblasts with hypoxia induced a significant increase in the expression of Hb-? And Hb-? (Fig. 4a). In EPO-treated C2Cl2 muscle cells, induction of Hb-a expression was more sensitive to treatment than induction of Hb-b (Fig. 4b). In addition, expression of both Hb-? And Hb-? Increased in an EPO-treated kidney slice in a concentration-dependent manner, whereas only Hb-? Expression was sensitive to induction in EPO-treated hepatocytes (Figures 4c and 4d) . Hb subunit expression was significantly increased in EH-201 treated non-hematopoietic cells (Figures 4e-4h) and this increase was eliminated by co-neutralization-EPO antibody treatment (Figures 4g and 4h). Studies were also conducted to determine the role of EPO signaling in inducing Hb expression by PGC-1? SiRNA. It was observed that a decrease in PGC-1 alpha expression in C2Cl2 myocytes decreased expression of both Hb- alpha and Hb-beta mRNA and also reduced the induction effect of EPO on Hb- (Fig. 4b). Thus, modulation of Hb expression in non-hematopoietic cells occurs through both EPO-mediated PGC-1? -Dependent and PGC-1?-Independent pathways. These results demonstrate that EPO-mediated signaling is required for EH-201 induction of hemoglobin expression in non-hematopoietic cells.

(4) EH-201 as an enhancer for the binding of EPO to EPOR instead of containing Hif-a activation

To investigate the mechanism of EH-201 activity, a computer docking method was performed to predict the binding of EH-201 to EPOR. EH-201 was found to bind preferentially to the EPO-EPOR complex (EPO / EPOR) rather than the EPO-free pure EPOR (total molecular interactions energies estimated were -7.48 kcal / mol and -6.30 kcal / mol respectively) . Autodock identified two or more preformed binding sites in the EPO / EPOR complex for EH-201 with negative favorable binding glass energy, and the predicted binding of EPOR (Met ¹⁵⁰ , Thr ¹⁵¹ , Figure 9) The interaction residues included hot-spot residues located in loop 5. Since EPO autoclain activity also plays an important role in EPOR activation and EPO production regulation, it was tested to enhance EPOR activation under the presumption that EH-201 could act as a binding enhancer of EPO to EPOR. TF-1 cells (EPOR positive) proliferation assays were performed to assess EPO biological activity. rhEPO was observed to induce TF-1 cell proliferation in a concentration-dependent manner, whereas in the absence of rhEPO, EH-201 alone could not induce cell proliferation (Fig. 4I). Even in the presence of very low concentrations, for example, 2 ng / ml of rhEPO, EH-201 significantly induced TF-I cell proliferation in a concentration dependent manner. Addition of both neutralized EPO or neutralized EPOR antibodies significantly reduced TF-I cell proliferation (Fig. 4I). These data indicate that EPO is required for the activity of EH-201 and that the EPO / EPOR complex may be the target of EH-201 acting as an enhancer of EPO and EPOR binding. In addition, we examined whether EH-201 induced expression of EPO involves activation of Hif as EPO expression is regulated by a hypoxia inducible factor (Hif). As shown in Fig. 10A, using a luciferase reporter driven by a hypoxic response element to evaluate the activation of Hif-la, EH-201 treatment did not activate the promoter activity. In addition, Hif-1α-targeted vascular endothelial growth factor (VEGF) expression was upregulated during hypoxia, whereas EH-201 did not alter VEGF expression (FIG. 10b). EH-201 treatment also did not stabilize Hif-2 alpha protein levels (FIG. 10c). These findings indicate that the induction of EPO by EH-201 is not due to the activation of Hif-1α or Hif-2α.

(5) Administration of EH-201 enhances the ability of mice to perform resistance

When the EPO-induced effect of EH-201 was shown, it was tested whether EH-201 could enhance tolerance performance in mice exhibiting hypoxic stress. Significantly, administration of EH-201 for 3 days increased the time to discharge (Fig. 5a) under both oxygen steady state and hypoxia in a dose dependent manner and was further enhanced on day 7. However, there was only a slight increase in RBC number and Hb content in peripheral blood (Fig. 5b), which led to an increase in endogenous EPO levels, as confirmed by EH-201 production by induction of production of kidney and liver EPO (Figure 5d). &Lt; / RTI &gt; Expression of Hb- alpha and Hb- beta in the myocardium of EH-201 treated mice was significantly increased as shown by the increase in Hb protein expression observed with TMBZ staining (Figure 5g) (Figure 5f) . High doses of EH-201 also induced cardiac mitochondrial in vivo synthesis (Figure 5e). In addition, EH-201 treatment significantly increased PGC-1α expression and mitochondrial content and activity in liver and skeletal muscle (FIGS. 11a and 11b). These results show that EH-201 treatment dramatically enhances the resistance performance and hypoxic endurance of mice through induction of increased endogenous EPO expression in non-hematopoietic tissues and stimulation of mitochondrial in vivo synthesis and Hb expression.

(6) Therapeutic effect of EH-201 on established doxorubicin-induced cardiomyopathy

To assess the therapeutic potential of EH-201 in myocardial ischemia, a doxorubicin (Dox) -induced cardiomyopathy model was used. One week after Dox injection, cardiomyopathic mice as identified by ECG measurements were initiated for EH-201 treatment for 7 days to investigate the therapeutic effect of EH-201. The survival rate of the EH-201 treated group was improved and the high dose group remained viable until the end of the study period (FIG. 6A). After the hypoxic Rotarod tolerance test, no group recovered from the initial changes in body weight (Fig. 12A), whereas the EH-201 treated group exhibited a strong increase in resistance-activity potency (particularly at 30 and 90 mg / kg dose , And the activity of the Dox group was significantly decreased (FIG. 6B). Myocardial injury was measured by ECG for up to 2 weeks after injection of Dox and these ECG parameters were fairly abnormal, reflecting extensive cardiac damage caused by Dox (Figure 6c). After 7 days of administration of EH-201, these ECG indications were significantly restored in mice treated with EH-201 (30, 90 mg / kg), indicating improvement in cardiac activity (Figures 6c and 12b). Echocardiography performed two weeks after dosing of Dox demonstrated that mice receiving Dox alone had significant cardiac deterioration, as characterized by reduced ejection fraction and fractional reductions. The mice treated with EH-201 (30, 90 mg / kg) had a significantly larger ejection fraction and fraction reduction in comparison (Fig. 6d). However, there was no significant difference in left ventricular diameter in the cardiac systolic and diastolic groups between the groups. These results indicate that treatment with EH-201 significantly alleviates the Dox-induced damage of cardiac function. In addition, the Dox-damaged heart appeared with cytoplasmic vacuolization, myofibrillar disappearance and myocardial fibrosis, which was alleviated by EH-201 treatment (Fig. 6e). The image quantification results show that Dox increases the area of fibrosis in the ventricular myenteric membrane compared to normal mice (normal, 1.71+ 0.18% vs. Dox, 8.31% + 0.94%) (Fig. 6e) Indicating that the mice treated with medium for 201 were almost absent. In addition, Hb expression was upregulated in the isolated myocardium of EH-201 treated mice (Fig. 6f, 30, 90 mg / kg) and increased in Hb dimer form (Fig. 6g and 6h). Taken together, these data show that EH-201 has a therapeutic effect, improving cardiac function and ischemic tolerance in Dox-induced cardiomyopathic mice.

(7) EH-201 improves anemia and renal function in cisplatin-induced nephropathy

Because acute kidney injury may result from renal ischemia induced by the use of a renal toxic agent, established cisplatin-induced nephropathy to investigate the effect of EH-201 induced EPO production on anemia with renal dysfunction Mouse model was adopted (Fig. 7A). From 10 days after the first injection of cisplatin, significant anemia and impaired renal function from day 13 were observed (Figures 7b and 7c). Significantly, administration of EH-201 at 30 and 90 mg / kg for 2 weeks (day 28, FIG. 7b) induced almost complete recovery of anemia. Moreover, the BUN levels of the EH-201 30 and 90 mg / kg treatment groups were also significantly recovered (Figure 7d). The histochemical examination revealed renal tubular epithelial cell necrosis, vacuolization and decolonization starting on day 13; Treatment with EH-201 significantly attenuated such renal damage (Figure 7e). In addition, significant increases in EPO and EH-201 treatment in the kidney of anemic mice were not observed to cause any additional increases (Fig. 7f), suggesting that hypoxic stress on the remaining functional kidney cells and kidneys Lt; RTI ID = 0.0 &gt; EH-201 &lt; / RTI &gt; Treatment with EH-201 30 and 90 mg / kg induced significant recovery of liver EPO expression (Figure 7g). In addition, EH-201 administration also activated red cell lineage cells in bone marrow (FIG. 7h). Collectively, these findings show that EH-201 improves recovery from cisplatin-induced anemia and renal dysfunction by inducing the production of EPO.

(8) EH-201 increases cellular EPO expression levels in mouse RPE cells

Figure 18 shows EH-201 induction of cellular EPO expression levels in mouse RPE cells. C57 mouse RPE cells were incubated with 0.4, 2, 10 μg / ml EH-201 in DMEM supplemented with 10% FCS. The culture was incubated at 37 [deg.] C for 24 hours. After the incubation period, the whole cell lysate was prepared as a lysis buffer. Whole cell lysates were prepared and applied to Western blot to detect levels of endogenous EPO. GAPDH was used as a loading control. The bars represent mean ± SD (n = 3 different experiments; ** p <0.01, *** p <0.001).

Example 3 Activation of mitochondrial function and hemoglobin expression in neurons by the compounds of the present invention

This example describes various assays useful for evaluating mitochondrial function and activation of hemoglobin expression in neurons by the compounds of the present invention. The compounds of the present invention were prepared according to the method provided in Example 1. The efficacy of the compounds is assessed using a series of activity assays, which are further described in detail below.

1. Cell culture

Astrocytic-rich cultures were prepared from 1 day old C57BL / 6J mice purchased from the following institution [the Animal Center at the National Yang Ming University]. Briefly, cortical tissue was digested with trypsin and the resulting dissociated cells were suspended in DMEM containing 10% FBS and incubated in a 100 mm culture dish. After 3 days of incubation, the medium was replaced with fresh 10% FBS / DMEM and the cells were maintained at 37 [deg.] C for an additional 3 days. The cells were dissociated into trypsin and suspended in 10% FBS / DMEM and incubated for 7 to 8 days in a 10 cm dish prior to use. Cells produced by this method consist of approximately 90-95% astrocytes, as determined by immunostaining with antibodies to glial fibrillary acidic protein (GFAP), a specific marker for astrocytes. Rat PC12 neurons were maintained in RPMI 1640.

2. RNA isolation and real-time PCR

RNA was prepared using RNA-BeeTM RNA isolation reagent (Tel-Test, Friendswood, TX). Actin, NGF and PGC (Invitrogen) were incubated with AMV-RT (Promega) in a total volume of 5 μg of RNA using ABI Prism 7700 Sequence Detection System and SYBR Green Master Mix Kit (Applied Biosystem, Foster City, CA) Gt; cDNA &lt; / RTI &gt; for RT-PCR analysis of the expression levels of &lt; RTI ID = 0.0 &gt; Expression levels of mouse beta-actin were used as internal standards. Relative gene expression levels were calculated by the 2-AACT method. The fragments (100-250 bp) were amplified using specific primers for each gene. The following primers were used: EPO (5'-AAT GGA GGT GGA AGA ACA GG-3 'and 5'-ACC CGA AGC AGT GAA GTG A-3'), Hb- TGC TGT CTC TG-3 ') and (5'-GTG CCC TTG AGG CTG TCC AAG TGA-3'), PGC-1α (5'-AGC CGT GAC CAC TGA CAA CGA G- (5'-TGT GTT CCT CTG TCA GCA TCA C-3 ') and GAPDH (5'-CGT GTC TGG TTC TGA GTG CTA AG-3' (5'-TCT TCA CCA CCA TGG AGA-3 'and 5'-ACC AAA GTT GTC ATG GAT GAC-3').

3. Western blot

Cells and brain tissue lysates were prepared using radioimmunoprecipitation assay lysis buffer. Approximately 20 μg of protein was loaded and Western blot analysis was performed using a monoclonal mouse antibody, Hb-β (1: 500; sc-31116, USA) against EPO (1: 500; sc-7956, Santa Cruz, California, USA) GAPDH antibody (1: 10,000; ab9385, Abcam, Cambridge, UK) used as a loading control. A horseradish peroxidase-conjugated anti-IgG secondary antibody was used for enhanced chemiluminescence detection (Amersham, Buckinghamshire, UK).

4. Succinate dihydrogenase assay

Astellular cells or PC12 neurons were plated in 96 well plates at 10 ⁴ cells per well. After 24 hours, the cells were incubated for 48 hours in the presence or absence of EPO or EH-201 containing medium (100 占 퐇 per well). The succinate dehydrogenase activity was determined by MTT reduction assay. The activity was normalized to the cellular protein level (measured using a BioRad protein kit) and the change in absorbance was measured using a microplate reader (PerkinElmer Life Sciences Wallac Victor2). Activity was shown relative to control conditions.

5. Production of reactive oxygen species in cells

The astrocytes and PC12 neurons were treated with EPO or EH-201 for 24 hours. The culture medium was replaced with 100 [mu] M H ₂ O ₂ , and the cells were incubated for 6 hours (astrocytes) or 30 minutes (PC12 cells). Production of intracellular reactive oxygen species (ROS) was then measured using 2 ', 7'-dichlorofluorescein diacetate (DCFH-DA; Molecular Probes, Eugene, OR, USA). DCFH-DA accumulates in cells and is hydrolyzed by cytoplasmic esterases to 2 &apos;, 7 ' -dichlorofluorescein. 2 ', 7'-dichlorofluorescein is oxidized by H ₂ O ₂ to give the fluorescent product, 2', 7'-dichlorofluorescein. Briefly, cultures in 96-well plates were washed with DMEM containing 1% FCS and loaded with 50 μM DCFH-DA for 30 minutes at 37 ° C. The wells were then washed twice with Kreb buffer and the cells were lysed using 0.1 N NaOH in 50% methanol. The wells were vortexed for 10 minutes and 2'-7'-dichlorofluorescein (DCF) fluorescence was observed under a fluorescence microscope or in a microplate reader (PerkinElmer Life Sciences Wallac Victor 2).

6. H ₂ O ₂ -induced cytotoxicity in astrocytes and PC12 neurons

The astrocytes and PC12 neurons were treated with EPO or EH-201 for 24 hours. The astrocyte culture medium was replaced with 500 μM H ₂ O ₂ , and the cells were incubated for 6 hours. The PC12 cell culture medium was replaced with 250 [mu] M H ₂ O ₂ and the cells were incubated for 4 hours. Cell viability was determined by rejection of trypan blue as assessed by light microscopy.

7. Sleep deprivation process

Forty-twelve-week-old C57B1 / 6J adult male mice were purchased from the National Institutional Animal Center (Taipei, Taiwan). The mice were housed at constant temperature and fed laboratory food (PMI, Brentwood, MO, USA) and water in a free manner. The experimental procedure was approved by the Institution of National Yang-Ming University. The animals were deprived of sleep (SD) and kept in their home cages (control group) in the same room. Briefly, C57BL / 6J male mice (7 weeks old) were lighted at 6:00 AM and resident on a 12 hour / 12 hour light / cancer schedule and treated for 7 days. The mice are deprived of sleep or uninterrupted in their home cages for 5 hours (mild sleep deprivation) by mild handling commencing at 6 AM. Mice were fed normal diet or normal diets 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

The passive avoidance experiment was carried out as previously described with minimum strain. A two-way shuttle-box with a guillotine door placed between the module test chambers was used. One chamber was illuminated with a 40 W bulb and the other was kept in a dark room. During the training period, the animals were individually placed in an illuminated chamber that was in contact against the guillotine door. When the animal entered the dark chamber, the door was quietly lowered and a 0.5 mA foot shock was applied through the grating floor for 2 seconds. During the test period, the animal was placed back into the illuminated chamber, but no footshock was applied. The latency to enter is recorded in each period.

9. Statistical analysis

All results are expressed as mean and standard deviation (SD). The significance of the mean difference between two or more groups was determined using the one-way ANOVA of the variables followed by the Turkish post-study study. The Student's t-test was used for statistical comparisons of paired samples. A P value of <0.05 was considered statistically significant.

10. Results

(1) EH-201, an inducer of nerve EPO, elevated the expression of EPO in primary astrocytes and PC12 neurons

EH-201, a nerve EPO inducer, elevated EPO expression in primary astrocytes and PC12 neurons. Because the exogenous EPO can not pass through the blood-brain barrier, its clinical use is limited. Therefore, the effect of EH-201, an endogenous nerve EPO inducer, was tested. The structure of EH-201 is shown in Fig. After EH-201 treatment, astrocytes demonstrated a dose-dependent increase in EPO mRNA expression as measured by real-time PCR analysis (Figure 13b). EH-201 treatment also upregulated EPO mRNA expression in PC12 neurons (Figure 13c). Intracellular EPO protein expression in astrocytes and PC12 cells was upregulated during EH-201 treatment (Figure 13d).

(2) EH-201 elevated the expression of mitochondrial regulators PGC-1α and Hb in primary astrocytes and PC12 neurons

After 24 hours of EPO or EH-201 treatment, cell mRNA was extracted to determine EPO-mediated gene expression. Real-time PCR revealed elevated expression of PGC-1 alpha and Hb-beta mRNA expression; HO-1, a known antioxidant gene up-regulated by EPO, was also induced in both astrocytes (Figs. 14a-14c) and PC12 neurons (Figs. 14d-14f) during EH-201 treatment; However, Hb-a expression was not significantly changed (Figs. 13E to 13F).

(3) EH-201 increased mitochondrial activity and attenuated oxidative stress in primary astrocytes and PC12 neurons

Because PGC-1a and Hb are known as mitochondrial regulators, we analyzed which form of Hb is regulated by EH-201. Figures 15a and 15e show that EH-201 increased all three types of Hb expression in astrocytes and PC12 cells. The mitochondrial activity in the treated cells in the presence or absence of EPO or EH-201 was then measured by MTT assay. Figures 15b and 15f show that EPO or EH-201 induced mitochondrial activity in both astrocytes and PC12 cells. We investigated whether EPO or EH-201 mediated upregulation of these genes attenuated H ₂ O _2- induced oxidative stress in astrocytes and PC12 cells. ROS production was assessed in cells cultured after exposure to H ₂ O ₂ using the oxidative probe CM-H2DCFDA. EPO and EH-201 treatment reduced intracellular ROS in astrocytes (Figure 15c) and PC12 cells (Figure 15g). EPO and EH-201 also reduced cytotoxicity in H ₂ O ₂ exposed cells, indicating that mitochondrial regulation and ROS homeostatic effects of EPO are biologically important (FIGS. 15d and 15h).

(4) EPO is required for attenuated EH-201 mediated increased mitochondrial activity and oxidative stress in primary astrocytes and PC12 neurons

After treatment with asthmatic cells and PC12 cells with EH-201, increased mitochondrial activity and reduction of H ₂ O ₂ -induced ROS production and cytotoxicity were assessed to be dependent on EPO. The observed increased mitochondrial activity using EH-201 treatment was blocked in the presence of anti-EPO antibody as measured by MTT assay as compared to cells treated with EH-201 alone (Fig. 16A). Reduction of H ₂ O ₂ -induced ROS production in astrocytes and PC12 cells treated with EH-201 was eliminated when the cells were co-incubated with anti-EPO antibody (FIG. 16b). The anti-EPO antibody also inhibited EH-201 mediated reduction of H ₂ O ₂ -induced cytotoxicity (FIG. 16c).

(5) Effect of EH-201 on mouse depression-induced memory loss

The neuroprotective effect of EH-201 on memory was evaluated by using the SD model. An overview of the above experimental process is shown in Figure 17A. EPO expression was analyzed in hippocampus from each treated animal. Real-time PCR and Western blotting showed an increase in EPO expression in EH-201 fed animals (Fig. 17b and 17c). Hbp, HO-1 and PGC-1? MRNA expression in the hippocampus was analyzed by real-time PCR (Fig. 17d). MTT assay was used to further assess mitochondrial succinate dehydrogenase activity (Figure 17e). In the passive avoidance test, animals fed EH-201 for 3 days did not show any difference in learning ability (FIG. 17f). However, there was a significant improvement in memory performance in the EH-201 fed mice after SD in the passive avoidance test (Figure 17g). Gene expression changes and increased mitochondrial activity in the hippocampus of animal origin supplied with EH-201 in particular at doses of 100 and 200 mg / kg correlate with passive avoidance testing.

Example 4 Induction of Auto-digestion by the Compounds of the Present Invention

This example describes various assays useful for evaluating the self-extinguishing activity induced by the compounds of the present invention. The compounds of the present invention are prepared according to the method provided in Example 1. The efficacy of the compounds is assessed using a series of activity assays and these assays are further described in detail below.

1. POS Phagocytosis Test

After the indicated treatment, the cells were treated with FITC-OS (1 x 10 ⁷ OS / well) and incubated at 37 ° C for 4 hours. Baseline fluorescence was obtained using untreated cells. The cells were washed 4 times with (EBSS) to remove excess POS. Finally, EBSS was added to each well at 100 [mu] l / well and analysis of the average FITC-OS fluorescence was performed with a fluorescence meter to quantify FITC-OS fluorescence at excitation 485 nm and emission 535 nm. The fluorescence-quenched dye was then added per well at 25 [mu] l / well and the dye was incubated at 37 [deg.] C for 30 minutes; The dye was quantitated by fluorescence spectrometer analysis (excitation, 485 nm: emission, 535 nm).

2. Western blot analysis

After the indicated treatment, the cells were washed twice with ice-cold PBS and lysed in extraction buffer (IM Tris, pH 6.8, 10% SDS, IM DTT). 10-15 μg of total protein was separated by SDS-PAGE and analyzed by immunoblotting using chemiluminescence. The primary antibodies used were either LC3B antibody (Gene Tex, USA, 1: 1000), EPO (GAPDH (Santa Cruz, CA, USA, 1: 1000) or GAPDH (Santa Cruz, CA, USA, 1: 1000). The intensity of the protein bands was quantitated using the image j software and compared to the control group The percentage of specific bands was analyzed.

3. Marking of self-extinguishing vacuole using monodosteryl cadaverine

Monodosylated cadaverine (MDC) is a spontaneous fluorescent dye that can be selectively incorporated into autopagosomes and autolysomes. Cells were incubated with 0.05 mM MDC in PBS for 1 h at 37 ° C. After incubation, the cells were washed twice with PBS and immediately analyzed by fluorescence microscopy (here: 380-420 nm, barrier filter 450 nm).

4. Cell culture and treatment

Rat kidney slices and primary mouse hepatocyte cultures have been described previously. These cultures were incubated for 24 hours with EH-201 at different doses (0.6, 2.5, 10 and 40 mg / ml), the autopegmentation activator rapamycin (Rm, 50 nM) or the autodeposition inhibitor 3-methyladenine , 10 mM). Under hunger (stv), the hepatocyte culture was an autopotential activation control.

5. Results

(1) Figs. 19A to 19D show induction of self-digestion by EH-201.

(2) As shown in Figs. 20A and 20B, activation of EH-201-induced auto-digestion is through induction of hepatocyte growth factor (HGF).

The foregoing description only describes features and functions of the present invention, but is not intended to limit the scope of the present invention. It is obvious to those skilled in the art that all equivalent variations and changes previously described in accordance with the purpose and principles of the present invention should be included in the appended claims.

Claims (40)

A composition for inducing erythropoietin (EPO) -mediated hemoglobin (Hb) expression in non-hematopoietic cells of a subject, comprising a compound of formula I and a pharmaceutically acceptable carrier.
Formula I

In this formula,
R is a glycosyl group. The method of claim 1, wherein the glycosyl group is selected from the group consisting of dihydroxyacetone, glucose, galactose, glyceraldehyde, threose, xylose, mannose, ribose, ribulose, tagatose, psicose, fructose, sorbose, Lactose, Lactose, Lactose, Lactulose, Trehalose, Celobos, Isomaltotriose, Lactose, Lactose, Lactose, Lactose, Wherein the composition is selected from the group consisting of nigerroot, maltotriose, melitose, maltotriulose, raffinose, cestose, and combinations thereof. 2. The composition of claim 1, wherein the compound induces Hb-alpha, Hb-beta, or dimeric Hb expression in non-hematopoietic cells of a subject. 2. The composition of claim 1, wherein the compound enhances erythropoietin-erythropoietin receptor binding affinity. 2. The composition of claim 1, wherein the compound binds to an erythropoietin-coupled erythropoietin receptor complex. 2. The composition of claim 1, wherein the compound enhances endogenous EPO expression and stimulates Hb expression in non-hematopoietic cells. 2. The composition of claim 1, wherein the non-hematopoietic cells are selected from the group consisting of kidney cells, hepatocytes, myocardial cells, myoblasts, glial cells, neurons and retinal pigment epithelial cells. A method for inducing erythropoietin (EPO) -mediated hemoglobin (Hb) expression in non-hematopoietic cells of a subject, comprising administering to the subject a therapeutically effective amount of a compound of formula (I).
Formula I

In this formula,
R is a glycosyl group. The method of claim 8, wherein the glycosyl group is selected from the group consisting of dihydroxyacetone, glucose, galactose, glyceraldehyde, threose, xylose, mannose, ribose, ribulose, tagatose, psicose, fructose, sorbose, Lactose, Lactose, Lactose, Lactulose, Trehalose, Celobos, Isomaltotriose, Lactose, Lactose, Lactose, Lactose, Wherein the group is selected from the group consisting of nigerroot, maltotriose, melitose, maltotriose, raffinose, cestose, and combinations thereof. 9. The method of claim 8, wherein the compound induces Hb-alpha, Hb-beta, or dimeric Hb expression in non-hematopoietic cells of the subject. 9. The method of claim 8, wherein said compound enhances endogenous EPO expression and stimulates Hb expression in non-hematopoietic cells of a subject. 9. The method of claim 8, wherein said subject is selected from the group consisting of hypoxia, anemia, renal ischemia, myocardial ischemia, pulmonary ischemia, neurodegenerative diseases, neuropsychiatric disorders, aging-related macular degeneration (AMD) RTI ID = 0.0 &gt; disease or syndrome. &Lt; / RTI &gt; A composition for inducing erythropoietin (EPO) -mediated mitochondrial in vivo synthesis in non-hematopoietic cells of a subject, comprising a compound of formula I and a pharmaceutically acceptable carrier.
Formula I

In this formula,
R is a glycosyl group. The method of claim 13, wherein the glycosyl group is selected from the group consisting of dihydroxyacetone, glucose, galactose, glyceraldehyde, threose, xylose, mannose, ribose, ribulose, tagatose, psicose, fructose, sorbose, Lactose, Lactose, Lactose, Lactulose, Trehalose, Celobos, Isomaltotriose, Lactose, Lactose, Lactose, Lactose, Wherein the composition is selected from the group consisting of nigerroot, maltotriose, melitose, maltotriulose, raffinose, cestose, and combinations thereof. 14. The composition of claim 13, wherein said compound induces an increase in mitochondrial member or PGC-l [alpha] expression to induce said EPO-mediated mitochondrial in vivo synthesis. 14. The composition of claim 13, wherein the compound enhances erythropoietin-erythropoietin receptor binding affinity. 14. The composition of claim 13, wherein said compound binds to an erythropoietin-coupled erythropoietin receptor complex. 14. The composition of claim 13, wherein said compound enhances endogenous EPO expression and stimulates Hb expression in non-hematopoietic cells of a subject. 14. The composition of claim 13, wherein the EPO-mediated mitochondrial in vivo synthesis is PGC-1 alpha-dependent. 14. The composition of claim 13, wherein said non-hematopoietic cells are selected from the group consisting of kidney cells, hepatocytes, myocardial cells, myoblasts, glial cells, neurons and retinal pigment epithelial cells. A method for inducing erythropoietin (EPO) -mediated mitochondrial in vivo synthesis in non-hematopoietic cells of a subject, comprising administering to the subject a therapeutically effective amount of a compound of formula (I).
Formula I

In this formula,
R is a glycosyl group. The method of claim 21, wherein the glycosyl group is selected from the group consisting of dihydroxyacetone, glucose, galactose, glyceraldehyde, threose, xylose, mannose, ribose, ribulose, tagatose, psicose, fructose, sorbose, Lactose, Lactose, Lactose, Lactulose, Trehalose, Celobos, Isomaltotriose, Lactose, Lactose, Lactose, Lactose, Wherein the group is selected from the group consisting of nigerroot, maltotriose, melitose, maltotriose, raffinose, cestose, and combinations thereof. 22. The method of claim 21, wherein said compound induces an increase in mitochondrial member or PGC-l [alpha] expression to induce said EPO-mediated mitochondrial in vivo synthesis. 22. The method of claim 21, wherein said subject is selected from the group consisting of hypoxia, anemia, ischemia-related diseases, neurodegenerative diseases, neuropsychiatric disorders, AMD-related diseases of aging, cardiomyopathy, brain aging, chronic liver disease, Wherein the disease or syndrome is selected from the group consisting of Pemphigus, hypertension, heart failure, obesity, diabetes mellitus, renal disease, atherosclerosis, aging, metabolic syndrome and combinations thereof. 26. The method of claim 24, wherein said ischemic related disease is selected from the group consisting of cardiac ischemia, ischemic neuron degeneration, cerebral ischemia, myocardial ischemia, limb ischemia, cerebral ischemia, liver ischemia, retinal ischemia, stroke, nephritis ischemia, Renal ischemia, and kidney ischemia. 25. The method of claim 24, wherein the neurodegenerative disease is a disease selected from the group consisting of Alzheimer's disease, Parkinson's disease and Huntington's disease. A method for inducing auto-digestion in a subject having a self-extinguishing defect, comprising administering to the subject a therapeutically effective amount of a compound of formula I, wherein the self-digesting enhances the elimination of protein aggregates in the subject Way
Formula I

In this formula,
R is a glycosyl group. 28. The method of claim 27, wherein the glycosyl group is selected from the group consisting of dihydroxyacetone, glucose, galactose, glyceraldehyde, threose, xylose, mannose, ribose, ribulose, tagatose, psicose, fructose, sorbose, Lactose, Lactose, Lactose, Lactulose, Trehalose, Celobos, Isomaltotriose, Lactose, Lactose, Lactose, Lactose, Wherein the group is selected from the group consisting of nigerroot, maltotriose, melitose, maltotriose, raffinose, cestose, and combinations thereof. 28. The method of claim 27, wherein the auto-digestion defect is in a cell that expresses a protein aggregate in the subject. 28. The method of claim 27, wherein the subject's cells are neurons or glial cells. 28. The method of claim 27, wherein the protein aggregate is selected from the group consisting of hungtintin, amyloid beta (A [beta]), alpha-synuclein, tau, superoxide dismutase 1 (SODl), mutants and mutated forms thereof, &Lt; / RTI &gt; 28. The method of claim 27, wherein the auto-digestive defect is selected from the group consisting of neurodegenerative diseases, retinal diseases, Crohn's disease, aging, myocardial hypertrophy, chronic heart failure, tuberculosis, chronic obstructive pulmonary disease (COPD), cystic fibrosis, Wherein the disease is a disease selected from the group consisting of new type, renal failure, muscular dystrophy, bone poultry disease, inclusion body muscle disease, anterior temporal dementia, glomerular disease, metabolic disease, glycogen storage disease type II, inflammatory bowel disease and Pompe disease . 33. The method of claim 32, wherein the neurodegenerative disease is a disease selected from the group consisting of Huntington's disease, Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis (ALS) and insomnia. A composition for inducing auto-digestion in a subject having an auto-digestive defect, comprising a compound of formula (I) and a pharmaceutically acceptable carrier.
Formula I

In this formula,
R is a glycosyl group. The method of claim 34, wherein the glycosyl group is selected from the group consisting of dihydroxyacetone, glucose, galactose, glyceraldehyde, threose, xylose, mannose, ribose, ribulose, tagatose, psicose, fructose, sorbose, Lactose, Lactose, Lactose, Lactulose, Trehalose, Celobos, Isomaltotriose, Lactose, Lactose, Lactose, Lactose, Wherein the composition is selected from the group consisting of nigerroot, maltotriose, melitose, maltotriulose, raffinose, cestose, and combinations thereof. A method for preventing amnesia in a subject, comprising administering to said subject a therapeutically effective amount of the above-mentioned compound of formula (I), wherein said compound activates auto-digestion in the subject.
Formula I

In this formula,
R is a glycosyl group. The method of claim 36, wherein the glycosyl group is selected from the group consisting of dihydroxyacetone, glucose, galactose, glyceraldehyde, threose, xylose, mannose, ribose, ribulose, tagatose, psicose, fructose, sorbose, Lactose, Lactose, Lactose, Lactulose, Trehalose, Celobos, Isomaltotriose, Lactose, Lactose, Lactose, Lactose, Wherein the group is selected from the group consisting of nigerroot, maltotriose, melitose, maltotriose, raffinose, cestose, and combinations thereof. 37. The method of claim 36, wherein the compound induces erythropoietin (EPO) to activate autogenous action in the subject. 37. The method of claim 36, wherein said autopotenting enhances protein removal in a subject. 37. The method of claim 36, wherein the auto-digestive defect is a neurodegenerative disease selected from the group consisting of Huntington's disease, Alzheimer's disease, Parkinson's disease, and insomnia.

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