EP2978417A1 - Composition and method for inducing epo-mediated haemoglobin expression and mitochondrial biogenesis in nonhaematopoietic cell - Google Patents
Composition and method for inducing epo-mediated haemoglobin expression and mitochondrial biogenesis in nonhaematopoietic cellInfo
- 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
- Authority
- EP
- European Patent Office
- Prior art keywords
- disease
- epo
- group
- cell
- expression
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Withdrawn
Links
- 230000014509 gene expression Effects 0.000 title claims abstract description 124
- 230000001404 mediated effect Effects 0.000 title claims abstract description 32
- 230000001939 inductive effect Effects 0.000 title claims abstract description 29
- 239000000203 mixture Substances 0.000 title claims abstract description 28
- 238000000034 method Methods 0.000 title claims description 45
- 230000008437 mitochondrial biogenesis Effects 0.000 title claims description 28
- 101150002621 EPO gene Proteins 0.000 title description 3
- 229940105423 erythropoietin Drugs 0.000 claims abstract description 175
- 102000003951 Erythropoietin Human genes 0.000 claims abstract description 173
- 108090000394 Erythropoietin Proteins 0.000 claims abstract description 173
- OXCMYAYHXIHQOA-UHFFFAOYSA-N potassium;[2-butyl-5-chloro-3-[[4-[2-(1,2,4-triaza-3-azanidacyclopenta-1,4-dien-5-yl)phenyl]phenyl]methyl]imidazol-4-yl]methanol Chemical compound [K+].CCCCC1=NC(Cl)=C(CO)N1CC1=CC=C(C=2C(=CC=CC=2)C2=N[N-]N=N2)C=C1 OXCMYAYHXIHQOA-UHFFFAOYSA-N 0.000 claims abstract description 169
- 150000001875 compounds Chemical class 0.000 claims abstract description 52
- 125000003147 glycosyl group Chemical group 0.000 claims abstract description 17
- 210000004027 cell Anatomy 0.000 claims description 119
- 230000004900 autophagic degradation Effects 0.000 claims description 36
- 108090000623 proteins and genes Proteins 0.000 claims description 33
- 210000002569 neuron Anatomy 0.000 claims description 31
- 230000002438 mitochondrial effect Effects 0.000 claims description 30
- 206010021143 Hypoxia Diseases 0.000 claims description 28
- 102000004169 proteins and genes Human genes 0.000 claims description 28
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 claims description 25
- 201000010099 disease Diseases 0.000 claims description 24
- 210000003494 hepatocyte Anatomy 0.000 claims description 23
- 210000003734 kidney Anatomy 0.000 claims description 22
- 208000028867 ischemia Diseases 0.000 claims description 19
- -1 erthrulose Chemical compound 0.000 claims description 18
- DBTMGCOVALSLOR-UHFFFAOYSA-N 32-alpha-galactosyl-3-alpha-galactosyl-galactose Natural products OC1C(O)C(O)C(CO)OC1OC1C(O)C(OC2C(C(CO)OC(O)C2O)O)OC(CO)C1O DBTMGCOVALSLOR-UHFFFAOYSA-N 0.000 claims description 16
- SRBFZHDQGSBBOR-IOVATXLUSA-N D-xylopyranose Chemical compound O[C@@H]1COC(O)[C@H](O)[C@H]1O SRBFZHDQGSBBOR-IOVATXLUSA-N 0.000 claims description 16
- PYMYPHUHKUWMLA-UHFFFAOYSA-N arabinose Natural products OCC(O)C(O)C(O)C=O PYMYPHUHKUWMLA-UHFFFAOYSA-N 0.000 claims description 16
- SRBFZHDQGSBBOR-UHFFFAOYSA-N beta-D-Pyranose-Lyxose Natural products OC1COC(O)C(O)C1O SRBFZHDQGSBBOR-UHFFFAOYSA-N 0.000 claims description 16
- RXKJFZQQPQGTFL-UHFFFAOYSA-N dihydroxyacetone Chemical compound OCC(=O)CO RXKJFZQQPQGTFL-UHFFFAOYSA-N 0.000 claims description 16
- 230000007954 hypoxia Effects 0.000 claims description 16
- 230000004770 neurodegeneration Effects 0.000 claims description 16
- 208000007502 anemia Diseases 0.000 claims description 15
- 210000004413 cardiac myocyte Anatomy 0.000 claims description 13
- 230000001419 dependent effect Effects 0.000 claims description 13
- 208000015122 neurodegenerative disease Diseases 0.000 claims description 13
- 208000024827 Alzheimer disease Diseases 0.000 claims description 11
- 230000007547 defect Effects 0.000 claims description 10
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims description 9
- 239000008103 glucose Substances 0.000 claims description 9
- HDTRYLNUVZCQOY-UHFFFAOYSA-N α-D-glucopyranosyl-α-D-glucopyranoside Natural products OC1C(O)C(O)C(CO)OC1OC1C(O)C(O)C(O)C(CO)O1 HDTRYLNUVZCQOY-UHFFFAOYSA-N 0.000 claims description 8
- OWEGMIWEEQEYGQ-UHFFFAOYSA-N 100676-05-9 Natural products OC1C(O)C(O)C(CO)OC1OCC1C(O)C(O)C(O)C(OC2C(OC(O)C(O)C2O)CO)O1 OWEGMIWEEQEYGQ-UHFFFAOYSA-N 0.000 claims description 8
- ODEHMIGXGLNAKK-OESPXIITSA-N 6-kestotriose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)OC[C@@H]1[C@@H](O)[C@H](O)[C@](CO)(O[C@@H]2[C@@H]([C@@H](O)[C@H](O)[C@@H](CO)O2)O)O1 ODEHMIGXGLNAKK-OESPXIITSA-N 0.000 claims description 8
- GUBGYTABKSRVRQ-XLOQQCSPSA-N Alpha-Lactose Chemical compound O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@H]1O[C@@H]1[C@@H](CO)O[C@H](O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-XLOQQCSPSA-N 0.000 claims description 8
- YTBSYETUWUMLBZ-UHFFFAOYSA-N D-Erythrose Natural products OCC(O)C(O)C=O YTBSYETUWUMLBZ-UHFFFAOYSA-N 0.000 claims description 8
- WQZGKKKJIJFFOK-CBPJZXOFSA-N D-Gulose Chemical compound OC[C@H]1OC(O)[C@H](O)[C@H](O)[C@H]1O WQZGKKKJIJFFOK-CBPJZXOFSA-N 0.000 claims description 8
- WQZGKKKJIJFFOK-WHZQZERISA-N D-aldose Chemical compound OC[C@H]1OC(O)[C@@H](O)[C@@H](O)[C@H]1O WQZGKKKJIJFFOK-WHZQZERISA-N 0.000 claims description 8
- WQZGKKKJIJFFOK-IVMDWMLBSA-N D-allopyranose Chemical compound OC[C@H]1OC(O)[C@H](O)[C@H](O)[C@@H]1O WQZGKKKJIJFFOK-IVMDWMLBSA-N 0.000 claims description 8
- LKDRXBCSQODPBY-JDJSBBGDSA-N D-allulose Chemical compound OCC1(O)OC[C@@H](O)[C@@H](O)[C@H]1O LKDRXBCSQODPBY-JDJSBBGDSA-N 0.000 claims description 8
- YTBSYETUWUMLBZ-IUYQGCFVSA-N D-erythrose Chemical compound OC[C@@H](O)[C@@H](O)C=O YTBSYETUWUMLBZ-IUYQGCFVSA-N 0.000 claims description 8
- MNQZXJOMYWMBOU-VKHMYHEASA-N D-glyceraldehyde Chemical compound OC[C@@H](O)C=O MNQZXJOMYWMBOU-VKHMYHEASA-N 0.000 claims description 8
- RXVWSYJTUUKTEA-UHFFFAOYSA-N D-maltotriose Natural products OC1C(O)C(OC(C(O)CO)C(O)C(O)C=O)OC(CO)C1OC1C(O)C(O)C(O)C(CO)O1 RXVWSYJTUUKTEA-UHFFFAOYSA-N 0.000 claims description 8
- SHZGCJCMOBCMKK-UHFFFAOYSA-N D-mannomethylose Natural products CC1OC(O)C(O)C(O)C1O SHZGCJCMOBCMKK-UHFFFAOYSA-N 0.000 claims description 8
- WQZGKKKJIJFFOK-QTVWNMPRSA-N D-mannopyranose Chemical compound OC[C@H]1OC(O)[C@@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-QTVWNMPRSA-N 0.000 claims description 8
- QWIZNVHXZXRPDR-UHFFFAOYSA-N D-melezitose Natural products O1C(CO)C(O)C(O)C(O)C1OC1C(O)C(CO)OC1(CO)OC1OC(CO)C(O)C(O)C1O QWIZNVHXZXRPDR-UHFFFAOYSA-N 0.000 claims description 8
- HMFHBZSHGGEWLO-SOOFDHNKSA-N D-ribofuranose Chemical compound OC[C@H]1OC(O)[C@H](O)[C@@H]1O HMFHBZSHGGEWLO-SOOFDHNKSA-N 0.000 claims description 8
- ZAQJHHRNXZUBTE-NQXXGFSBSA-N D-ribulose Chemical compound OC[C@@H](O)[C@@H](O)C(=O)CO ZAQJHHRNXZUBTE-NQXXGFSBSA-N 0.000 claims description 8
- ZAQJHHRNXZUBTE-UHFFFAOYSA-N D-threo-2-Pentulose Natural products OCC(O)C(O)C(=O)CO ZAQJHHRNXZUBTE-UHFFFAOYSA-N 0.000 claims description 8
- YTBSYETUWUMLBZ-QWWZWVQMSA-N D-threose Chemical compound OC[C@@H](O)[C@H](O)C=O YTBSYETUWUMLBZ-QWWZWVQMSA-N 0.000 claims description 8
- 206010056474 Erythrosis Diseases 0.000 claims description 8
- 229930091371 Fructose Natural products 0.000 claims description 8
- 239000005715 Fructose Substances 0.000 claims description 8
- RFSUNEUAIZKAJO-ARQDHWQXSA-N Fructose Chemical compound OC[C@H]1O[C@](O)(CO)[C@@H](O)[C@@H]1O RFSUNEUAIZKAJO-ARQDHWQXSA-N 0.000 claims description 8
- LKDRXBCSQODPBY-AMVSKUEXSA-N L-(-)-Sorbose Chemical compound OCC1(O)OC[C@H](O)[C@@H](O)[C@@H]1O LKDRXBCSQODPBY-AMVSKUEXSA-N 0.000 claims description 8
- WQZGKKKJIJFFOK-VSOAQEOCSA-N L-altropyranose Chemical compound OC[C@@H]1OC(O)[C@H](O)[C@@H](O)[C@H]1O WQZGKKKJIJFFOK-VSOAQEOCSA-N 0.000 claims description 8
- SHZGCJCMOBCMKK-JFNONXLTSA-N L-rhamnopyranose Chemical compound C[C@@H]1OC(O)[C@H](O)[C@H](O)[C@H]1O SHZGCJCMOBCMKK-JFNONXLTSA-N 0.000 claims description 8
- PNNNRSAQSRJVSB-UHFFFAOYSA-N L-rhamnose Natural products CC(O)C(O)C(O)C(O)C=O PNNNRSAQSRJVSB-UHFFFAOYSA-N 0.000 claims description 8
- GUBGYTABKSRVRQ-QKKXKWKRSA-N Lactose Natural products OC[C@H]1O[C@@H](O[C@H]2[C@H](O)[C@@H](O)C(O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@H]1O GUBGYTABKSRVRQ-QKKXKWKRSA-N 0.000 claims description 8
- GUBGYTABKSRVRQ-PICCSMPSSA-N Maltose Natural products O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@@H](CO)OC(O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-PICCSMPSSA-N 0.000 claims description 8
- MUPFEKGTMRGPLJ-RMMQSMQOSA-N Raffinose Natural products O(C[C@H]1[C@@H](O)[C@H](O)[C@@H](O)[C@@H](O[C@@]2(CO)[C@H](O)[C@@H](O)[C@@H](CO)O2)O1)[C@@H]1[C@H](O)[C@@H](O)[C@@H](O)[C@@H](CO)O1 MUPFEKGTMRGPLJ-RMMQSMQOSA-N 0.000 claims description 8
- PYMYPHUHKUWMLA-LMVFSUKVSA-N Ribose Natural products OC[C@@H](O)[C@@H](O)[C@@H](O)C=O PYMYPHUHKUWMLA-LMVFSUKVSA-N 0.000 claims description 8
- 229930006000 Sucrose Natural products 0.000 claims description 8
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 claims description 8
- HDTRYLNUVZCQOY-WSWWMNSNSA-N Trehalose Natural products O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@@H](O)[C@@H](CO)O1 HDTRYLNUVZCQOY-WSWWMNSNSA-N 0.000 claims description 8
- MUPFEKGTMRGPLJ-UHFFFAOYSA-N UNPD196149 Natural products OC1C(O)C(CO)OC1(CO)OC1C(O)C(O)C(O)C(COC2C(C(O)C(O)C(CO)O2)O)O1 MUPFEKGTMRGPLJ-UHFFFAOYSA-N 0.000 claims description 8
- 206010064930 age-related macular degeneration Diseases 0.000 claims description 8
- 230000032683 aging Effects 0.000 claims description 8
- HDTRYLNUVZCQOY-LIZSDCNHSA-N alpha,alpha-trehalose Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 HDTRYLNUVZCQOY-LIZSDCNHSA-N 0.000 claims description 8
- HMFHBZSHGGEWLO-UHFFFAOYSA-N alpha-D-Furanose-Ribose Natural products OCC1OC(O)C(O)C1O HMFHBZSHGGEWLO-UHFFFAOYSA-N 0.000 claims description 8
- DBTMGCOVALSLOR-VXXRBQRTSA-N alpha-D-Glcp-(1->3)-alpha-D-Glcp-(1->3)-D-Glcp Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@@H](O)[C@@H](O[C@H]2[C@@H]([C@@H](CO)OC(O)[C@@H]2O)O)O[C@H](CO)[C@H]1O DBTMGCOVALSLOR-VXXRBQRTSA-N 0.000 claims description 8
- WQZGKKKJIJFFOK-PHYPRBDBSA-N alpha-D-galactose Chemical compound OC[C@H]1O[C@H](O)[C@H](O)[C@@H](O)[C@H]1O WQZGKKKJIJFFOK-PHYPRBDBSA-N 0.000 claims description 8
- SRBFZHDQGSBBOR-STGXQOJASA-N alpha-D-lyxopyranose Chemical compound O[C@@H]1CO[C@H](O)[C@@H](O)[C@H]1O SRBFZHDQGSBBOR-STGXQOJASA-N 0.000 claims description 8
- PYMYPHUHKUWMLA-WDCZJNDASA-N arabinose Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)C=O PYMYPHUHKUWMLA-WDCZJNDASA-N 0.000 claims description 8
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 claims description 8
- GUBGYTABKSRVRQ-QUYVBRFLSA-N beta-maltose Chemical compound OC[C@H]1O[C@H](O[C@H]2[C@H](O)[C@@H](O)[C@H](O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@@H]1O GUBGYTABKSRVRQ-QUYVBRFLSA-N 0.000 claims description 8
- 229940120503 dihydroxyacetone Drugs 0.000 claims description 8
- 229930182830 galactose Natural products 0.000 claims description 8
- DBTMGCOVALSLOR-AXAHEAMVSA-N galactotriose Natural products OC[C@@H]1O[C@@H](O[C@@H]2[C@@H](O)[C@H](CO)O[C@@H](O[C@H]3[C@@H](O)[C@H](O)O[C@@H](CO)[C@@H]3O)[C@@H]2O)[C@H](O)[C@H](O)[C@H]1O DBTMGCOVALSLOR-AXAHEAMVSA-N 0.000 claims description 8
- 150000002453 idose derivatives Chemical class 0.000 claims description 8
- FBJQEBRMDXPWNX-FYHZSNTMSA-N isomaltotriose Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1OC[C@@H]1[C@@H](O)[C@H](O)[C@@H](O)[C@@H](OC[C@@H]2[C@H]([C@H](O)[C@@H](O)C(O)O2)O)O1 FBJQEBRMDXPWNX-FYHZSNTMSA-N 0.000 claims description 8
- BJHIKXHVCXFQLS-PQLUHFTBSA-N keto-D-tagatose Chemical compound OC[C@@H](O)[C@H](O)[C@H](O)C(=O)CO BJHIKXHVCXFQLS-PQLUHFTBSA-N 0.000 claims description 8
- 239000008101 lactose Substances 0.000 claims description 8
- JCQLYHFGKNRPGE-FCVZTGTOSA-N lactulose Chemical compound OC[C@H]1O[C@](O)(CO)[C@@H](O)[C@@H]1O[C@H]1[C@H](O)[C@@H](O)[C@@H](O)[C@@H](CO)O1 JCQLYHFGKNRPGE-FCVZTGTOSA-N 0.000 claims description 8
- 229960000511 lactulose Drugs 0.000 claims description 8
- PFCRQPBOOFTZGQ-UHFFFAOYSA-N lactulose keto form Natural products OCC(=O)C(O)C(C(O)CO)OC1OC(CO)C(O)C(O)C1O PFCRQPBOOFTZGQ-UHFFFAOYSA-N 0.000 claims description 8
- 208000002780 macular degeneration Diseases 0.000 claims description 8
- FGPATWVHNYVVEE-SKPZHCOCSA-N maltotriulose Chemical compound OC[C@H]1OC(O)(CO)[C@@H](O)[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@H](O[C@@H]2[C@@H]([C@@H](O)[C@H](O)[C@@H](CO)O2)O)[C@@H](CO)O1 FGPATWVHNYVVEE-SKPZHCOCSA-N 0.000 claims description 8
- FYGDTMLNYKFZSV-UHFFFAOYSA-N mannotriose Natural products OC1C(O)C(O)C(CO)OC1OC1C(CO)OC(OC2C(OC(O)C(O)C2O)CO)C(O)C1O FYGDTMLNYKFZSV-UHFFFAOYSA-N 0.000 claims description 8
- QWIZNVHXZXRPDR-WSCXOGSTSA-N melezitose Chemical compound O([C@@]1(O[C@@H]([C@H]([C@@H]1O[C@@H]1[C@@H]([C@@H](O)[C@H](O)[C@@H](CO)O1)O)O)CO)CO)[C@H]1O[C@H](CO)[C@@H](O)[C@H](O)[C@H]1O QWIZNVHXZXRPDR-WSCXOGSTSA-N 0.000 claims description 8
- MUPFEKGTMRGPLJ-ZQSKZDJDSA-N raffinose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO[C@@H]2[C@@H]([C@@H](O)[C@@H](O)[C@@H](CO)O2)O)O1 MUPFEKGTMRGPLJ-ZQSKZDJDSA-N 0.000 claims description 8
- 239000005720 sucrose Substances 0.000 claims description 8
- FYGDTMLNYKFZSV-BYLHFPJWSA-N β-1,4-galactotrioside Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]1O[C@@H]1[C@H](CO)O[C@@H](O[C@@H]2[C@@H](O[C@@H](O)[C@H](O)[C@H]2O)CO)[C@H](O)[C@H]1O FYGDTMLNYKFZSV-BYLHFPJWSA-N 0.000 claims description 8
- 208000000044 Amnesia Diseases 0.000 claims description 7
- 208000026139 Memory disease Diseases 0.000 claims description 7
- 230000006984 memory degeneration Effects 0.000 claims description 7
- 208000023060 memory loss Diseases 0.000 claims description 7
- 210000003098 myoblast Anatomy 0.000 claims description 7
- 208000031229 Cardiomyopathies Diseases 0.000 claims description 6
- 208000032007 Glycogen storage disease due to acid maltase deficiency Diseases 0.000 claims description 6
- 206010053185 Glycogen storage disease type II Diseases 0.000 claims description 6
- 208000023105 Huntington disease Diseases 0.000 claims description 6
- 208000018737 Parkinson disease Diseases 0.000 claims description 6
- 201000004502 glycogen storage disease II Diseases 0.000 claims description 6
- 208000017169 kidney disease Diseases 0.000 claims description 6
- 210000004498 neuroglial cell Anatomy 0.000 claims description 6
- 108020003175 receptors Proteins 0.000 claims description 6
- 102000005962 receptors Human genes 0.000 claims description 6
- 201000006474 Brain Ischemia Diseases 0.000 claims description 5
- 206010063897 Renal ischaemia Diseases 0.000 claims description 5
- 208000031225 myocardial ischemia Diseases 0.000 claims description 5
- 108010075944 Erythropoietin Receptors Proteins 0.000 claims description 4
- 102100036509 Erythropoietin receptor Human genes 0.000 claims description 4
- 102100033448 Lysosomal alpha-glucosidase Human genes 0.000 claims description 4
- 208000013738 Sleep Initiation and Maintenance disease Diseases 0.000 claims description 4
- 108010021188 Superoxide Dismutase-1 Proteins 0.000 claims description 4
- 206010002026 amyotrophic lateral sclerosis Diseases 0.000 claims description 4
- 206010022437 insomnia Diseases 0.000 claims description 4
- 230000000302 ischemic effect Effects 0.000 claims description 4
- 208000019423 liver disease Diseases 0.000 claims description 4
- 210000003583 retinal pigment epithelium Anatomy 0.000 claims description 4
- 208000011580 syndromic disease Diseases 0.000 claims description 4
- 206010008120 Cerebral ischaemia Diseases 0.000 claims description 3
- 208000006545 Chronic Obstructive Pulmonary Disease Diseases 0.000 claims description 3
- 206010019280 Heart failures Diseases 0.000 claims description 3
- 208000001647 Renal Insufficiency Diseases 0.000 claims description 3
- 208000017442 Retinal disease Diseases 0.000 claims description 3
- 208000006011 Stroke Diseases 0.000 claims description 3
- 210000004556 brain Anatomy 0.000 claims description 3
- 206010008118 cerebral infarction Diseases 0.000 claims description 3
- 201000006370 kidney failure Diseases 0.000 claims description 3
- 102100026882 Alpha-synuclein Human genes 0.000 claims description 2
- 102000013455 Amyloid beta-Peptides Human genes 0.000 claims description 2
- 108010090849 Amyloid beta-Peptides Proteins 0.000 claims description 2
- 201000001320 Atherosclerosis Diseases 0.000 claims description 2
- 206010007558 Cardiac failure chronic Diseases 0.000 claims description 2
- 206010007572 Cardiac hypertrophy Diseases 0.000 claims description 2
- 208000006029 Cardiomegaly Diseases 0.000 claims description 2
- 208000011231 Crohn disease Diseases 0.000 claims description 2
- 201000003883 Cystic fibrosis Diseases 0.000 claims description 2
- 201000011240 Frontotemporal dementia Diseases 0.000 claims description 2
- 208000022461 Glomerular disease Diseases 0.000 claims description 2
- 206010056328 Hepatic ischaemia Diseases 0.000 claims description 2
- 206010019708 Hepatic steatosis Diseases 0.000 claims description 2
- 206010020772 Hypertension Diseases 0.000 claims description 2
- 208000022559 Inflammatory bowel disease Diseases 0.000 claims description 2
- 206010022680 Intestinal ischaemia Diseases 0.000 claims description 2
- 208000001145 Metabolic Syndrome Diseases 0.000 claims description 2
- 206010028289 Muscle atrophy Diseases 0.000 claims description 2
- 208000021642 Muscular disease Diseases 0.000 claims description 2
- 201000009623 Myopathy Diseases 0.000 claims description 2
- 208000008589 Obesity Diseases 0.000 claims description 2
- 208000010191 Osteitis Deformans Diseases 0.000 claims description 2
- 208000027067 Paget disease of bone Diseases 0.000 claims description 2
- 201000000690 abdominal obesity-metabolic syndrome Diseases 0.000 claims description 2
- 108090000185 alpha-Synuclein Proteins 0.000 claims description 2
- 208000016738 bone Paget disease Diseases 0.000 claims description 2
- 206010012601 diabetes mellitus Diseases 0.000 claims description 2
- 208000016097 disease of metabolism Diseases 0.000 claims description 2
- 230000002526 effect on cardiovascular system Effects 0.000 claims description 2
- 231100000852 glomerular disease Toxicity 0.000 claims description 2
- 210000003000 inclusion body Anatomy 0.000 claims description 2
- 201000002818 limb ischemia Diseases 0.000 claims description 2
- 210000004072 lung Anatomy 0.000 claims description 2
- 208000030159 metabolic disease Diseases 0.000 claims description 2
- 201000006417 multiple sclerosis Diseases 0.000 claims description 2
- 230000020763 muscle atrophy Effects 0.000 claims description 2
- 201000000585 muscular atrophy Diseases 0.000 claims description 2
- 235000020824 obesity Nutrition 0.000 claims description 2
- 208000030761 polycystic kidney disease Diseases 0.000 claims description 2
- 230000002685 pulmonary effect Effects 0.000 claims description 2
- 208000032253 retinal ischemia Diseases 0.000 claims description 2
- 102000013498 tau Proteins Human genes 0.000 claims description 2
- 108010026424 tau Proteins Proteins 0.000 claims description 2
- 201000008827 tuberculosis Diseases 0.000 claims description 2
- 102000008221 Superoxide Dismutase-1 Human genes 0.000 claims 2
- JAYVHSBYKLLDJC-DSNJPTTOSA-N (E)-2,3,5,4'-tetrahydroxystilbene-2-O-beta-D-glucoside Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]1OC1=C(O)C=C(O)C=C1\C=C\C1=CC=C(O)C=C1 JAYVHSBYKLLDJC-DSNJPTTOSA-N 0.000 description 206
- 241000699670 Mus sp. Species 0.000 description 66
- 238000011282 treatment Methods 0.000 description 59
- 230000000694 effects Effects 0.000 description 46
- 210000001130 astrocyte Anatomy 0.000 description 39
- 241001465754 Metazoa Species 0.000 description 27
- 208000010340 Sleep Deprivation Diseases 0.000 description 27
- 230000001965 increasing effect Effects 0.000 description 24
- 238000003556 assay Methods 0.000 description 21
- 230000006698 induction Effects 0.000 description 21
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 18
- 108020004999 messenger RNA Proteins 0.000 description 18
- 230000004913 activation Effects 0.000 description 17
- 238000001262 western blot Methods 0.000 description 17
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 15
- 238000011529 RT qPCR Methods 0.000 description 14
- 238000002474 experimental method Methods 0.000 description 14
- 238000010162 Tukey test Methods 0.000 description 13
- 238000001543 one-way ANOVA Methods 0.000 description 13
- 238000003753 real-time PCR Methods 0.000 description 13
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 12
- 102100031181 Glyceraldehyde-3-phosphate dehydrogenase Human genes 0.000 description 12
- 108020004445 glyceraldehyde-3-phosphate dehydrogenase Proteins 0.000 description 12
- 230000001146 hypoxic effect Effects 0.000 description 11
- 239000003642 reactive oxygen metabolite Substances 0.000 description 11
- 238000012360 testing method Methods 0.000 description 11
- AOJJSUZBOXZQNB-TZSSRYMLSA-N Doxorubicin Chemical compound O([C@H]1C[C@@](O)(CC=2C(O)=C3C(=O)C=4C=CC=C(C=4C(=O)C3=C(O)C=21)OC)C(=O)CO)[C@H]1C[C@H](N)[C@H](O)[C@H](C)O1 AOJJSUZBOXZQNB-TZSSRYMLSA-N 0.000 description 10
- 238000004458 analytical method Methods 0.000 description 10
- 210000001519 tissue Anatomy 0.000 description 10
- 230000000747 cardiac effect Effects 0.000 description 9
- 241000699666 Mus <mouse, genus> Species 0.000 description 8
- 108020004459 Small interfering RNA Proteins 0.000 description 8
- 238000000692 Student's t-test Methods 0.000 description 8
- 229940090044 injection Drugs 0.000 description 8
- 238000002347 injection Methods 0.000 description 8
- 239000007924 injection Substances 0.000 description 8
- 230000003834 intracellular effect Effects 0.000 description 8
- 230000015654 memory Effects 0.000 description 8
- 230000004898 mitochondrial function Effects 0.000 description 8
- 210000004165 myocardium Anatomy 0.000 description 8
- 230000003472 neutralizing effect Effects 0.000 description 8
- 238000010186 staining Methods 0.000 description 8
- 230000001225 therapeutic effect Effects 0.000 description 8
- 239000003814 drug Substances 0.000 description 7
- 210000003743 erythrocyte Anatomy 0.000 description 7
- 239000001963 growth medium Substances 0.000 description 7
- 210000001320 hippocampus Anatomy 0.000 description 7
- 238000011534 incubation Methods 0.000 description 7
- 239000000411 inducer Substances 0.000 description 7
- 239000006166 lysate Substances 0.000 description 7
- 238000005259 measurement Methods 0.000 description 7
- 239000002609 medium Substances 0.000 description 7
- 108010030844 2-methylcitrate synthase Proteins 0.000 description 6
- FSASIHFSFGAIJM-UHFFFAOYSA-N 3-methyladenine Chemical compound CN1C=NC(N)=C2N=CN=C12 FSASIHFSFGAIJM-UHFFFAOYSA-N 0.000 description 6
- 108010071536 Citrate (Si)-synthase Proteins 0.000 description 6
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 description 6
- 108020005196 Mitochondrial DNA Proteins 0.000 description 6
- 210000002798 bone marrow cell Anatomy 0.000 description 6
- 230000001413 cellular effect Effects 0.000 description 6
- 229940079593 drug Drugs 0.000 description 6
- 210000004185 liver Anatomy 0.000 description 6
- 238000004519 manufacturing process Methods 0.000 description 6
- MLEBFEHOJICQQS-UHFFFAOYSA-N monodansylcadaverine Chemical compound C1=CC=C2C(N(C)C)=CC=CC2=C1S(=O)(=O)NCCCCCN MLEBFEHOJICQQS-UHFFFAOYSA-N 0.000 description 6
- 210000000107 myocyte Anatomy 0.000 description 6
- 238000011002 quantification Methods 0.000 description 6
- 230000001105 regulatory effect Effects 0.000 description 6
- 230000002861 ventricular Effects 0.000 description 6
- UAIUNKRWKOVEES-UHFFFAOYSA-N 3,3',5,5'-tetramethylbenzidine Chemical compound CC1=C(N)C(C)=CC(C=2C=C(C)C(N)=C(C)C=2)=C1 UAIUNKRWKOVEES-UHFFFAOYSA-N 0.000 description 5
- 238000011746 C57BL/6J (JAX™ mouse strain) Methods 0.000 description 5
- 102000006732 Citrate synthase Human genes 0.000 description 5
- 206010016654 Fibrosis Diseases 0.000 description 5
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 5
- 102000019259 Succinate Dehydrogenase Human genes 0.000 description 5
- 108010012901 Succinate Dehydrogenase Proteins 0.000 description 5
- 230000033228 biological regulation Effects 0.000 description 5
- 210000001185 bone marrow Anatomy 0.000 description 5
- 238000004113 cell culture Methods 0.000 description 5
- DQLATGHUWYMOKM-UHFFFAOYSA-L cisplatin Chemical compound N[Pt](N)(Cl)Cl DQLATGHUWYMOKM-UHFFFAOYSA-L 0.000 description 5
- 229960004316 cisplatin Drugs 0.000 description 5
- 231100000135 cytotoxicity Toxicity 0.000 description 5
- 230000003013 cytotoxicity Effects 0.000 description 5
- 238000001514 detection method Methods 0.000 description 5
- 230000004761 fibrosis Effects 0.000 description 5
- 238000011068 loading method Methods 0.000 description 5
- 230000001537 neural effect Effects 0.000 description 5
- 230000008452 non REM sleep Effects 0.000 description 5
- 235000021590 normal diet Nutrition 0.000 description 5
- 230000007959 normoxia Effects 0.000 description 5
- 230000036542 oxidative stress Effects 0.000 description 5
- 238000011084 recovery Methods 0.000 description 5
- 230000009467 reduction Effects 0.000 description 5
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 5
- 241001289529 Fallopia multiflora Species 0.000 description 4
- 102000003745 Hepatocyte Growth Factor Human genes 0.000 description 4
- 108090000100 Hepatocyte Growth Factor Proteins 0.000 description 4
- 108060001084 Luciferase Proteins 0.000 description 4
- 239000005089 Luciferase Substances 0.000 description 4
- 238000000134 MTT assay Methods 0.000 description 4
- 231100000002 MTT assay Toxicity 0.000 description 4
- UIIMBOGNXHQVGW-UHFFFAOYSA-M Sodium bicarbonate Chemical compound [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 description 4
- 108010073929 Vascular Endothelial Growth Factor A Proteins 0.000 description 4
- 102000005789 Vascular Endothelial Growth Factors Human genes 0.000 description 4
- 108010019530 Vascular Endothelial Growth Factors Proteins 0.000 description 4
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 4
- 230000002886 autophagic effect Effects 0.000 description 4
- 230000006378 damage Effects 0.000 description 4
- 229960004679 doxorubicin Drugs 0.000 description 4
- 238000002592 echocardiography Methods 0.000 description 4
- 230000004217 heart function Effects 0.000 description 4
- 150000002500 ions Chemical class 0.000 description 4
- 230000013016 learning Effects 0.000 description 4
- 239000003550 marker Substances 0.000 description 4
- 229910052760 oxygen Inorganic materials 0.000 description 4
- 239000001301 oxygen Substances 0.000 description 4
- 239000013641 positive control Substances 0.000 description 4
- 230000036385 rapid eye movement (rem) sleep Effects 0.000 description 4
- 230000002829 reductive effect Effects 0.000 description 4
- 210000002966 serum Anatomy 0.000 description 4
- 210000002027 skeletal muscle Anatomy 0.000 description 4
- 230000007958 sleep Effects 0.000 description 4
- 239000011780 sodium chloride Substances 0.000 description 4
- GEHJYWRUCIMESM-UHFFFAOYSA-L sodium sulfite Chemical compound [Na+].[Na+].[O-]S([O-])=O GEHJYWRUCIMESM-UHFFFAOYSA-L 0.000 description 4
- 210000000130 stem cell Anatomy 0.000 description 4
- 238000012549 training Methods 0.000 description 4
- DGVVWUTYPXICAM-UHFFFAOYSA-N β‐Mercaptoethanol Chemical compound OCCS DGVVWUTYPXICAM-UHFFFAOYSA-N 0.000 description 4
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 3
- XDFNWJDGWJVGGN-UHFFFAOYSA-N 2-(2,7-dichloro-3,6-dihydroxy-9h-xanthen-9-yl)benzoic acid Chemical compound OC(=O)C1=CC=CC=C1C1C2=CC(Cl)=C(O)C=C2OC2=CC(O)=C(Cl)C=C21 XDFNWJDGWJVGGN-UHFFFAOYSA-N 0.000 description 3
- 238000003222 MTT reduction assay Methods 0.000 description 3
- 108091027981 Response element Proteins 0.000 description 3
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- GLNADSQYFUSGOU-GPTZEZBUSA-J Trypan blue Chemical compound [Na+].[Na+].[Na+].[Na+].C1=C(S([O-])(=O)=O)C=C2C=C(S([O-])(=O)=O)C(/N=N/C3=CC=C(C=C3C)C=3C=C(C(=CC=3)\N=N\C=3C(=CC4=CC(=CC(N)=C4C=3O)S([O-])(=O)=O)S([O-])(=O)=O)C)=C(O)C2=C1N GLNADSQYFUSGOU-GPTZEZBUSA-J 0.000 description 3
- 208000027418 Wounds and injury Diseases 0.000 description 3
- 238000002835 absorbance Methods 0.000 description 3
- 238000010171 animal model Methods 0.000 description 3
- 239000003963 antioxidant agent Substances 0.000 description 3
- 230000003078 antioxidant effect Effects 0.000 description 3
- 230000008499 blood brain barrier function Effects 0.000 description 3
- 210000001218 blood-brain barrier Anatomy 0.000 description 3
- 230000037396 body weight Effects 0.000 description 3
- 230000001756 cardiomyopathic effect Effects 0.000 description 3
- 239000013592 cell lysate Substances 0.000 description 3
- 230000004663 cell proliferation Effects 0.000 description 3
- 238000001516 cell proliferation assay Methods 0.000 description 3
- 238000007398 colorimetric assay Methods 0.000 description 3
- 230000001086 cytosolic effect Effects 0.000 description 3
- 230000007423 decrease Effects 0.000 description 3
- 230000003247 decreasing effect Effects 0.000 description 3
- 239000000539 dimer Substances 0.000 description 3
- 208000022675 doxorubicin induced cardiomyopathy Diseases 0.000 description 3
- 239000000975 dye Substances 0.000 description 3
- 230000004064 dysfunction Effects 0.000 description 3
- 239000003623 enhancer Substances 0.000 description 3
- 230000005284 excitation Effects 0.000 description 3
- 239000000499 gel Substances 0.000 description 3
- 230000000971 hippocampal effect Effects 0.000 description 3
- 235000003642 hunger Nutrition 0.000 description 3
- 208000014674 injury Diseases 0.000 description 3
- 238000007912 intraperitoneal administration Methods 0.000 description 3
- 230000003907 kidney function Effects 0.000 description 3
- 239000012139 lysis buffer Substances 0.000 description 3
- 230000004048 modification Effects 0.000 description 3
- 238000012986 modification Methods 0.000 description 3
- 238000003032 molecular docking Methods 0.000 description 3
- 238000010172 mouse model Methods 0.000 description 3
- 210000005157 neural retina Anatomy 0.000 description 3
- 230000000324 neuroprotective effect Effects 0.000 description 3
- 238000011302 passive avoidance test Methods 0.000 description 3
- 230000037361 pathway Effects 0.000 description 3
- 230000035755 proliferation Effects 0.000 description 3
- 238000011160 research Methods 0.000 description 3
- 238000004904 shortening Methods 0.000 description 3
- 230000011664 signaling Effects 0.000 description 3
- 230000037351 starvation Effects 0.000 description 3
- 230000035882 stress Effects 0.000 description 3
- 239000000126 substance Substances 0.000 description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 3
- 239000012130 whole-cell lysate Substances 0.000 description 3
- 238000001644 13C nuclear magnetic resonance spectroscopy Methods 0.000 description 2
- 238000005160 1H NMR spectroscopy Methods 0.000 description 2
- 208000009304 Acute Kidney Injury Diseases 0.000 description 2
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 2
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical group [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 2
- 206010012289 Dementia Diseases 0.000 description 2
- 239000012594 Earle’s Balanced Salt Solution Substances 0.000 description 2
- WSFSSNUMVMOOMR-UHFFFAOYSA-N Formaldehyde Chemical compound O=C WSFSSNUMVMOOMR-UHFFFAOYSA-N 0.000 description 2
- 108010010803 Gelatin Proteins 0.000 description 2
- 102000053171 Glial Fibrillary Acidic Human genes 0.000 description 2
- 101710193519 Glial fibrillary acidic protein Proteins 0.000 description 2
- 101000987586 Homo sapiens Eosinophil peroxidase Proteins 0.000 description 2
- 108010001336 Horseradish Peroxidase Proteins 0.000 description 2
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 description 2
- WHUUTDBJXJRKMK-VKHMYHEASA-N L-glutamic acid Chemical compound OC(=O)[C@@H](N)CCC(O)=O WHUUTDBJXJRKMK-VKHMYHEASA-N 0.000 description 2
- 241001529936 Murinae Species 0.000 description 2
- 206010028594 Myocardial fibrosis Diseases 0.000 description 2
- 239000012980 RPMI-1640 medium Substances 0.000 description 2
- 208000033626 Renal failure acute Diseases 0.000 description 2
- 102100038836 Superoxide dismutase [Cu-Zn] Human genes 0.000 description 2
- 102000004142 Trypsin Human genes 0.000 description 2
- 108090000631 Trypsin Proteins 0.000 description 2
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 description 2
- PNNCWTXUWKENPE-UHFFFAOYSA-N [N].NC(N)=O Chemical compound [N].NC(N)=O PNNCWTXUWKENPE-UHFFFAOYSA-N 0.000 description 2
- 238000009825 accumulation Methods 0.000 description 2
- 239000012190 activator Substances 0.000 description 2
- 201000011040 acute kidney failure Diseases 0.000 description 2
- 239000012298 atmosphere Substances 0.000 description 2
- 238000000065 atmospheric pressure chemical ionisation Methods 0.000 description 2
- 230000002238 attenuated effect Effects 0.000 description 2
- 108010005774 beta-Galactosidase Proteins 0.000 description 2
- 210000004369 blood Anatomy 0.000 description 2
- 239000008280 blood Substances 0.000 description 2
- 229940098773 bovine serum albumin Drugs 0.000 description 2
- 229910052799 carbon Inorganic materials 0.000 description 2
- 239000006285 cell suspension Substances 0.000 description 2
- 230000007541 cellular toxicity Effects 0.000 description 2
- 230000008859 change Effects 0.000 description 2
- 238000012512 characterization method Methods 0.000 description 2
- 230000003931 cognitive performance Effects 0.000 description 2
- 230000001332 colony forming effect Effects 0.000 description 2
- 230000002596 correlated effect Effects 0.000 description 2
- 210000004748 cultured cell Anatomy 0.000 description 2
- 230000006735 deficit Effects 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 238000002224 dissection Methods 0.000 description 2
- 231100000673 dose–response relationship Toxicity 0.000 description 2
- 238000001378 electrochemiluminescence detection Methods 0.000 description 2
- 208000002854 epidermolysis bullosa simplex superficialis Diseases 0.000 description 2
- 230000000925 erythroid effect Effects 0.000 description 2
- 230000010437 erythropoiesis Effects 0.000 description 2
- 238000000799 fluorescence microscopy Methods 0.000 description 2
- 239000008273 gelatin Substances 0.000 description 2
- 229920000159 gelatin Polymers 0.000 description 2
- 235000019322 gelatine Nutrition 0.000 description 2
- 235000011852 gelatine desserts Nutrition 0.000 description 2
- 210000005046 glial fibrillary acidic protein Anatomy 0.000 description 2
- 230000002440 hepatic effect Effects 0.000 description 2
- 238000004128 high performance liquid chromatography Methods 0.000 description 2
- 230000013632 homeostatic process Effects 0.000 description 2
- 102000044890 human EPO Human genes 0.000 description 2
- 230000006872 improvement Effects 0.000 description 2
- 230000003993 interaction Effects 0.000 description 2
- 238000002955 isolation Methods 0.000 description 2
- 238000004895 liquid chromatography mass spectrometry Methods 0.000 description 2
- 238000001294 liquid chromatography-tandem mass spectrometry Methods 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 238000000386 microscopy Methods 0.000 description 2
- 210000003463 organelle Anatomy 0.000 description 2
- 239000012188 paraffin wax Substances 0.000 description 2
- 210000005259 peripheral blood Anatomy 0.000 description 2
- 239000011886 peripheral blood Substances 0.000 description 2
- 108040007629 peroxidase activity proteins Proteins 0.000 description 2
- 102000013415 peroxidase activity proteins Human genes 0.000 description 2
- 239000013612 plasmid Substances 0.000 description 2
- 230000003389 potentiating effect Effects 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 230000001681 protective effect Effects 0.000 description 2
- ZAHRKKWIAAJSAO-UHFFFAOYSA-N rapamycin Natural products COCC(O)C(=C/C(C)C(=O)CC(OC(=O)C1CCCCN1C(=O)C(=O)C2(O)OC(CC(OC)C(=CC=CC=CC(C)CC(C)C(=O)C)C)CCC2C)C(C)CC3CCC(O)C(C3)OC)C ZAHRKKWIAAJSAO-UHFFFAOYSA-N 0.000 description 2
- 230000008085 renal dysfunction Effects 0.000 description 2
- 230000002441 reversible effect Effects 0.000 description 2
- 239000000523 sample Substances 0.000 description 2
- 230000035939 shock Effects 0.000 description 2
- 229960002930 sirolimus Drugs 0.000 description 2
- QFJCIRLUMZQUOT-HPLJOQBZSA-N sirolimus Chemical compound C1C[C@@H](O)[C@H](OC)C[C@@H]1C[C@@H](C)[C@H]1OC(=O)[C@@H]2CCCCN2C(=O)C(=O)[C@](O)(O2)[C@H](C)CC[C@H]2C[C@H](OC)/C(C)=C/C=C/C=C/[C@@H](C)C[C@@H](C)C(=O)[C@H](OC)[C@H](O)/C(C)=C/[C@@H](C)C(=O)C1 QFJCIRLUMZQUOT-HPLJOQBZSA-N 0.000 description 2
- 235000017557 sodium bicarbonate Nutrition 0.000 description 2
- 229910000030 sodium bicarbonate Inorganic materials 0.000 description 2
- 235000010265 sodium sulphite Nutrition 0.000 description 2
- 239000000243 solution Substances 0.000 description 2
- 238000007619 statistical method Methods 0.000 description 2
- UCSJYZPVAKXKNQ-HZYVHMACSA-N streptomycin Chemical compound CN[C@H]1[C@H](O)[C@@H](O)[C@H](CO)O[C@H]1O[C@@H]1[C@](C=O)(O)[C@H](C)O[C@H]1O[C@@H]1[C@@H](NC(N)=N)[C@H](O)[C@@H](NC(N)=N)[C@H](O)[C@H]1O UCSJYZPVAKXKNQ-HZYVHMACSA-N 0.000 description 2
- 230000004083 survival effect Effects 0.000 description 2
- 238000004885 tandem mass spectrometry Methods 0.000 description 2
- 238000001890 transfection Methods 0.000 description 2
- 238000003211 trypan blue cell staining Methods 0.000 description 2
- 239000012588 trypsin Substances 0.000 description 2
- VFNKZQNIXUFLBC-UHFFFAOYSA-N 2',7'-dichlorofluorescein Chemical compound O1C(=O)C2=CC=CC=C2C21C1=CC(Cl)=C(O)C=C1OC1=C2C=C(Cl)C(O)=C1 VFNKZQNIXUFLBC-UHFFFAOYSA-N 0.000 description 1
- RUVJFMSQTCEAAB-UHFFFAOYSA-M 2-[3-[5,6-dichloro-1,3-bis[[4-(chloromethyl)phenyl]methyl]benzimidazol-2-ylidene]prop-1-enyl]-3-methyl-1,3-benzoxazol-3-ium;chloride Chemical compound [Cl-].O1C2=CC=CC=C2[N+](C)=C1C=CC=C(N(C1=CC(Cl)=C(Cl)C=C11)CC=2C=CC(CCl)=CC=2)N1CC1=CC=C(CCl)C=C1 RUVJFMSQTCEAAB-UHFFFAOYSA-M 0.000 description 1
- JKMHFZQWWAIEOD-UHFFFAOYSA-N 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid Chemical compound OCC[NH+]1CCN(CCS([O-])(=O)=O)CC1 JKMHFZQWWAIEOD-UHFFFAOYSA-N 0.000 description 1
- QFVHZQCOUORWEI-UHFFFAOYSA-N 4-[(4-anilino-5-sulfonaphthalen-1-yl)diazenyl]-5-hydroxynaphthalene-2,7-disulfonic acid Chemical compound C=12C(O)=CC(S(O)(=O)=O)=CC2=CC(S(O)(=O)=O)=CC=1N=NC(C1=CC=CC(=C11)S(O)(=O)=O)=CC=C1NC1=CC=CC=C1 QFVHZQCOUORWEI-UHFFFAOYSA-N 0.000 description 1
- YRNWIFYIFSBPAU-UHFFFAOYSA-N 4-[4-(dimethylamino)phenyl]-n,n-dimethylaniline Chemical compound C1=CC(N(C)C)=CC=C1C1=CC=C(N(C)C)C=C1 YRNWIFYIFSBPAU-UHFFFAOYSA-N 0.000 description 1
- SNCJAJRILVFXAE-UHFFFAOYSA-N 9h-fluorene-2,7-diamine Chemical compound NC1=CC=C2C3=CC=C(N)C=C3CC2=C1 SNCJAJRILVFXAE-UHFFFAOYSA-N 0.000 description 1
- 230000002407 ATP formation Effects 0.000 description 1
- 102100022900 Actin, cytoplasmic 1 Human genes 0.000 description 1
- 108010085238 Actins Proteins 0.000 description 1
- 101100297347 Caenorhabditis elegans pgl-3 gene Proteins 0.000 description 1
- 208000024172 Cardiovascular disease Diseases 0.000 description 1
- 108010078791 Carrier Proteins Proteins 0.000 description 1
- 229910021580 Cobalt(II) chloride Inorganic materials 0.000 description 1
- 108050006400 Cyclin Proteins 0.000 description 1
- 238000003718 Dual-Luciferase Reporter Assay System Methods 0.000 description 1
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 1
- 238000002965 ELISA Methods 0.000 description 1
- 238000008157 ELISA kit Methods 0.000 description 1
- 108090000371 Esterases Proteins 0.000 description 1
- 229910015400 FeC13 Inorganic materials 0.000 description 1
- 108010043121 Green Fluorescent Proteins Proteins 0.000 description 1
- 239000007995 HEPES buffer Substances 0.000 description 1
- 241000282412 Homo Species 0.000 description 1
- 101000920686 Homo sapiens Erythropoietin Proteins 0.000 description 1
- 238000012404 In vitro experiment Methods 0.000 description 1
- 206010061218 Inflammation Diseases 0.000 description 1
- 102100034343 Integrase Human genes 0.000 description 1
- 108010002386 Interleukin-3 Proteins 0.000 description 1
- PIWKPBJCKXDKJR-UHFFFAOYSA-N Isoflurane Chemical compound FC(F)OC(Cl)C(F)(F)F PIWKPBJCKXDKJR-UHFFFAOYSA-N 0.000 description 1
- 229930195714 L-glutamate Natural products 0.000 description 1
- 239000012097 Lipofectamine 2000 Substances 0.000 description 1
- 206010067125 Liver injury Diseases 0.000 description 1
- 101000756628 Mus musculus Actin, cytoplasmic 1 Proteins 0.000 description 1
- 101000987583 Mus musculus Eosinophil peroxidase Proteins 0.000 description 1
- 238000005481 NMR spectroscopy Methods 0.000 description 1
- 206010028851 Necrosis Diseases 0.000 description 1
- 239000002033 PVDF binder Substances 0.000 description 1
- 229930182555 Penicillin Natural products 0.000 description 1
- JGSARLDLIJGVTE-MBNYWOFBSA-N Penicillin G Chemical compound N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)CC1=CC=CC=C1 JGSARLDLIJGVTE-MBNYWOFBSA-N 0.000 description 1
- 102000003728 Peroxisome Proliferator-Activated Receptors Human genes 0.000 description 1
- 108090000029 Peroxisome Proliferator-Activated Receptors Proteins 0.000 description 1
- 206010057249 Phagocytosis Diseases 0.000 description 1
- 102000009339 Proliferating Cell Nuclear Antigen Human genes 0.000 description 1
- 239000012083 RIPA buffer Substances 0.000 description 1
- 238000002123 RNA extraction Methods 0.000 description 1
- 239000012162 RNA isolation reagent Substances 0.000 description 1
- 108010092799 RNA-directed DNA polymerase Proteins 0.000 description 1
- 238000010240 RT-PCR analysis Methods 0.000 description 1
- 241000700159 Rattus Species 0.000 description 1
- 101000595041 Rattus norvegicus Podocalyxin Proteins 0.000 description 1
- 206010062237 Renal impairment Diseases 0.000 description 1
- 206010038923 Retinopathy Diseases 0.000 description 1
- 101000833181 Schizosaccharomyces pombe (strain 972 / ATCC 24843) Glycerol dehydrogenase 1 Proteins 0.000 description 1
- 108050002485 Sirtuin Proteins 0.000 description 1
- 102000011990 Sirtuin Human genes 0.000 description 1
- 206010040844 Skin exfoliation Diseases 0.000 description 1
- 241000278713 Theora Species 0.000 description 1
- 239000007983 Tris buffer Substances 0.000 description 1
- 108010046334 Urease Proteins 0.000 description 1
- 230000001594 aberrant effect Effects 0.000 description 1
- 230000002159 abnormal effect Effects 0.000 description 1
- 230000005856 abnormality Effects 0.000 description 1
- 230000003213 activating effect Effects 0.000 description 1
- 208000012998 acute renal failure Diseases 0.000 description 1
- 230000006978 adaptation Effects 0.000 description 1
- 238000000246 agarose gel electrophoresis Methods 0.000 description 1
- 230000001668 ameliorated effect Effects 0.000 description 1
- 238000000540 analysis of variance Methods 0.000 description 1
- 230000003305 autocrine Effects 0.000 description 1
- 210000004961 autolysosome Anatomy 0.000 description 1
- 210000004957 autophagosome Anatomy 0.000 description 1
- 239000012822 autophagy inhibitor Substances 0.000 description 1
- 230000004888 barrier function Effects 0.000 description 1
- 238000010009 beating Methods 0.000 description 1
- 230000003542 behavioural effect Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- WQZGKKKJIJFFOK-FPRJBGLDSA-N beta-D-galactose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@H]1O WQZGKKKJIJFFOK-FPRJBGLDSA-N 0.000 description 1
- 102000005936 beta-Galactosidase Human genes 0.000 description 1
- 230000002715 bioenergetic effect Effects 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- 229960000074 biopharmaceutical Drugs 0.000 description 1
- 238000004820 blood count Methods 0.000 description 1
- 210000005013 brain tissue Anatomy 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 238000004364 calculation method Methods 0.000 description 1
- 239000004202 carbamide Substances 0.000 description 1
- 230000003683 cardiac damage Effects 0.000 description 1
- 230000005961 cardioprotection Effects 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 239000006143 cell culture medium Substances 0.000 description 1
- 230000005779 cell damage Effects 0.000 description 1
- 208000037887 cell injury Diseases 0.000 description 1
- 230000003833 cell viability Effects 0.000 description 1
- 108091092356 cellular DNA Proteins 0.000 description 1
- 230000019522 cellular metabolic process Effects 0.000 description 1
- 230000002490 cerebral effect Effects 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 208000020832 chronic kidney disease Diseases 0.000 description 1
- 208000022831 chronic renal failure syndrome Diseases 0.000 description 1
- 229940105442 cisplatin injection Drugs 0.000 description 1
- 230000003081 coactivator Effects 0.000 description 1
- 230000001149 cognitive effect Effects 0.000 description 1
- 230000007370 cognitive improvement Effects 0.000 description 1
- 230000001447 compensatory effect Effects 0.000 description 1
- 239000002299 complementary DNA Substances 0.000 description 1
- LBJNMUFDOHXDFG-UHFFFAOYSA-N copper;hydrate Chemical compound O.[Cu].[Cu] LBJNMUFDOHXDFG-UHFFFAOYSA-N 0.000 description 1
- 238000012937 correction Methods 0.000 description 1
- 210000005257 cortical tissue Anatomy 0.000 description 1
- 239000013078 crystal Substances 0.000 description 1
- 238000002425 crystallisation Methods 0.000 description 1
- 230000008025 crystallization Effects 0.000 description 1
- 230000002950 deficient Effects 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- 230000035618 desquamation Effects 0.000 description 1
- 230000006866 deterioration Effects 0.000 description 1
- 230000029087 digestion Effects 0.000 description 1
- 230000037149 energy metabolism Effects 0.000 description 1
- 230000002708 enhancing effect Effects 0.000 description 1
- 210000003013 erythroid precursor cell Anatomy 0.000 description 1
- 239000003797 essential amino acid Substances 0.000 description 1
- 235000020776 essential amino acid Nutrition 0.000 description 1
- 230000007717 exclusion Effects 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 239000011536 extraction buffer Substances 0.000 description 1
- 230000002349 favourable effect Effects 0.000 description 1
- 239000007850 fluorescent dye Substances 0.000 description 1
- 239000012634 fragment Substances 0.000 description 1
- 230000007760 free radical scavenging Effects 0.000 description 1
- 239000012737 fresh medium Substances 0.000 description 1
- 230000006870 function Effects 0.000 description 1
- 239000003102 growth factor Substances 0.000 description 1
- 150000003278 haem Chemical group 0.000 description 1
- 230000003394 haemopoietic effect Effects 0.000 description 1
- 210000005003 heart tissue Anatomy 0.000 description 1
- 230000011132 hemopoiesis Effects 0.000 description 1
- 231100000753 hepatic injury Toxicity 0.000 description 1
- 230000001744 histochemical effect Effects 0.000 description 1
- 238000010562 histological examination Methods 0.000 description 1
- 229910052739 hydrogen Inorganic materials 0.000 description 1
- 238000003119 immunoblot Methods 0.000 description 1
- 230000000984 immunochemical effect Effects 0.000 description 1
- 230000001771 impaired effect Effects 0.000 description 1
- 230000001976 improved effect Effects 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 230000004054 inflammatory process Effects 0.000 description 1
- 208000037906 ischaemic injury Diseases 0.000 description 1
- 229960002725 isoflurane Drugs 0.000 description 1
- 210000003292 kidney cell Anatomy 0.000 description 1
- 238000002372 labelling Methods 0.000 description 1
- 239000003446 ligand Substances 0.000 description 1
- 150000002632 lipids Chemical class 0.000 description 1
- 201000007270 liver cancer Diseases 0.000 description 1
- 208000014018 liver neoplasm Diseases 0.000 description 1
- 230000007787 long-term memory Effects 0.000 description 1
- 230000027928 long-term synaptic potentiation Effects 0.000 description 1
- 238000003468 luciferase reporter gene assay Methods 0.000 description 1
- 210000003712 lysosome Anatomy 0.000 description 1
- 230000001868 lysosomic effect Effects 0.000 description 1
- 230000014759 maintenance of location Effects 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 230000005056 memory consolidation Effects 0.000 description 1
- 206010027175 memory impairment Diseases 0.000 description 1
- 230000007334 memory performance Effects 0.000 description 1
- 230000002503 metabolic effect Effects 0.000 description 1
- XELZGAJCZANUQH-UHFFFAOYSA-N methyl 1-acetylthieno[3,2-c]pyrazole-5-carboxylate Chemical compound CC(=O)N1N=CC2=C1C=C(C(=O)OC)S2 XELZGAJCZANUQH-UHFFFAOYSA-N 0.000 description 1
- 229920000609 methyl cellulose Polymers 0.000 description 1
- 239000001923 methylcellulose Substances 0.000 description 1
- 238000001531 micro-dissection Methods 0.000 description 1
- 210000003470 mitochondria Anatomy 0.000 description 1
- 230000004065 mitochondrial dysfunction Effects 0.000 description 1
- 230000006705 mitochondrial oxidative phosphorylation Effects 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 230000000897 modulatory effect Effects 0.000 description 1
- 239000003068 molecular probe Substances 0.000 description 1
- 239000000178 monomer Substances 0.000 description 1
- 230000017074 necrotic cell death Effects 0.000 description 1
- 230000003589 nefrotoxic effect Effects 0.000 description 1
- 230000006654 negative regulation of apoptotic process Effects 0.000 description 1
- 231100000381 nephrotoxic Toxicity 0.000 description 1
- 230000016273 neuron death Effects 0.000 description 1
- 230000003955 neuronal function Effects 0.000 description 1
- 230000006776 neuronal homeostasis Effects 0.000 description 1
- 230000004112 neuroprotection Effects 0.000 description 1
- 230000003557 neuropsychological effect Effects 0.000 description 1
- 230000000422 nocturnal effect Effects 0.000 description 1
- 108020004707 nucleic acids Proteins 0.000 description 1
- 102000039446 nucleic acids Human genes 0.000 description 1
- 150000007523 nucleic acids Chemical class 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- 230000031868 operant conditioning Effects 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 230000004768 organ dysfunction Effects 0.000 description 1
- 230000004792 oxidative damage Effects 0.000 description 1
- 230000001590 oxidative effect Effects 0.000 description 1
- 238000006213 oxygenation reaction Methods 0.000 description 1
- 229940049954 penicillin Drugs 0.000 description 1
- 230000008782 phagocytosis Effects 0.000 description 1
- 230000000144 pharmacologic effect Effects 0.000 description 1
- 238000002205 phenol-chloroform extraction Methods 0.000 description 1
- 238000005375 photometry Methods 0.000 description 1
- 230000008288 physiological mechanism Effects 0.000 description 1
- 230000035790 physiological processes and functions Effects 0.000 description 1
- 229920001184 polypeptide Polymers 0.000 description 1
- 229920002981 polyvinylidene fluoride Polymers 0.000 description 1
- 231100000857 poor renal function Toxicity 0.000 description 1
- 230000026341 positive regulation of angiogenesis Effects 0.000 description 1
- 230000002035 prolonged effect Effects 0.000 description 1
- 230000004224 protection Effects 0.000 description 1
- 230000009979 protective mechanism Effects 0.000 description 1
- 238000010791 quenching Methods 0.000 description 1
- 230000007420 reactivation Effects 0.000 description 1
- 230000006950 reactive oxygen species formation Effects 0.000 description 1
- 238000004064 recycling Methods 0.000 description 1
- 238000007634 remodeling Methods 0.000 description 1
- 210000005084 renal tissue Anatomy 0.000 description 1
- 239000011347 resin Substances 0.000 description 1
- 229920005989 resin Polymers 0.000 description 1
- 230000004044 response Effects 0.000 description 1
- 239000000790 retinal pigment Substances 0.000 description 1
- 210000000844 retinal pigment epithelial cell Anatomy 0.000 description 1
- 238000003757 reverse transcription PCR Methods 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 229910000162 sodium phosphate Inorganic materials 0.000 description 1
- 238000000527 sonication Methods 0.000 description 1
- 230000004936 stimulating effect Effects 0.000 description 1
- 230000000638 stimulation Effects 0.000 description 1
- 229960005322 streptomycin Drugs 0.000 description 1
- 238000007920 subcutaneous administration Methods 0.000 description 1
- 230000000946 synaptic effect Effects 0.000 description 1
- 239000006188 syrup Substances 0.000 description 1
- 235000020357 syrup Nutrition 0.000 description 1
- 230000009885 systemic effect Effects 0.000 description 1
- 230000000287 tissue oxygenation Effects 0.000 description 1
- 230000007704 transition Effects 0.000 description 1
- 230000032258 transport Effects 0.000 description 1
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 1
- 238000002604 ultrasonography Methods 0.000 description 1
- 210000000689 upper leg Anatomy 0.000 description 1
- 230000003827 upregulation Effects 0.000 description 1
- 210000003934 vacuole Anatomy 0.000 description 1
- 230000002477 vacuolizing effect Effects 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
- 239000012224 working solution Substances 0.000 description 1
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- 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
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
- A61P1/04—Drugs for disorders of the alimentary tract or the digestive system for ulcers, gastritis or reflux esophagitis, e.g. antacids, inhibitors of acid secretion, mucosal protectants
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P11/00—Drugs for disorders of the respiratory system
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P13/00—Drugs for disorders of the urinary system
- A61P13/12—Drugs for disorders of the urinary system of the kidneys
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P19/00—Drugs for skeletal disorders
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P21/00—Drugs for disorders of the muscular or neuromuscular system
-
- 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
-
- 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
-
- 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
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P27/00—Drugs for disorders of the senses
- A61P27/02—Ophthalmic agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P3/00—Drugs for disorders of the metabolism
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P3/00—Drugs for disorders of the metabolism
- A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
- A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- 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
Abstract
Description
Claims
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
PCT/US2013/034381 WO2014158165A1 (en) | 2013-03-28 | 2013-03-28 | Composition and method for inducing epo-mediated haemoglobin expression and mitochondrial biogenesis in nonhaematopoietic cell |
Publications (2)
Publication Number | Publication Date |
---|---|
EP2978417A1 true EP2978417A1 (en) | 2016-02-03 |
EP2978417A4 EP2978417A4 (en) | 2017-03-01 |
Family
ID=51624947
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
EP13880425.7A Withdrawn EP2978417A4 (en) | 2013-03-28 | 2013-03-28 | Composition and method for inducing epo-mediated haemoglobin expression and mitochondrial biogenesis in nonhaematopoietic cell |
Country Status (8)
Country | Link |
---|---|
EP (1) | EP2978417A4 (en) |
JP (1) | JP2016516745A (en) |
KR (1) | KR20150135430A (en) |
CN (1) | CN105188688A (en) |
AU (1) | AU2013384234A1 (en) |
CA (1) | CA2907965C (en) |
HK (1) | HK1219871A1 (en) |
WO (1) | WO2014158165A1 (en) |
Families Citing this family (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN107921053B (en) * | 2015-06-16 | 2020-09-01 | 吴荣灿 | Method for treating or preventing dry eye |
CN107922453B (en) * | 2015-07-23 | 2021-05-25 | 吴荣灿 | Novel compounds as positive allosteric modulators of erythropoietin and erythropoietin receptors for the treatment of erythropoietin-deficient diseases |
CN106822160A (en) * | 2017-02-16 | 2017-06-13 | 中国人民解放军第四军医大学 | Application of the Stibene-glucoside in the medicine and health products for preparing obesity-related hypertension |
CN107349213A (en) * | 2017-07-10 | 2017-11-17 | 苏州神瑞生物科技有限公司 | Applied based on the synthesising preparation of Stibene-glucoside in treatment nerve degenerative diseases medicine is prepared |
CN109999047A (en) * | 2019-04-19 | 2019-07-12 | 浙江大学 | Two-step method screens mitochondrial disease drug |
CN113599357B (en) * | 2021-07-30 | 2023-03-24 | 中国医学科学院生物医学工程研究所 | Application of ROS (reactive oxygen species) -responsive nanoparticles coated with hematopoietic growth factors in preparation of drugs for treating hematopoietic injuries |
Family Cites Families (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2000044370A1 (en) * | 1999-01-29 | 2000-08-03 | Sunstar Inc. | Drugs, foods and oral compositions containing stilbene-type compounds |
US20050042314A1 (en) * | 2003-08-22 | 2005-02-24 | National Yang-Ming University | Extracts of Polygonum multiflorum Thunb., and preparation process and uses of the same |
US7824714B2 (en) * | 2008-06-13 | 2010-11-02 | Development Center For Biotechnology | Chinese herb extract for treating dementia and preparation method thereof |
US20100160243A1 (en) * | 2008-12-24 | 2010-06-24 | National Yang-Ming University | Compounds and methods for enhancing erythropoiesis |
-
2013
- 2013-03-28 JP JP2016505443A patent/JP2016516745A/en active Pending
- 2013-03-28 KR KR1020157030448A patent/KR20150135430A/en not_active IP Right Cessation
- 2013-03-28 EP EP13880425.7A patent/EP2978417A4/en not_active Withdrawn
- 2013-03-28 AU AU2013384234A patent/AU2013384234A1/en not_active Abandoned
- 2013-03-28 CA CA2907965A patent/CA2907965C/en active Active
- 2013-03-28 WO PCT/US2013/034381 patent/WO2014158165A1/en active Application Filing
- 2013-03-28 CN CN201380075192.6A patent/CN105188688A/en active Pending
-
2016
- 2016-07-05 HK HK16107786.5A patent/HK1219871A1/en unknown
Also Published As
Publication number | Publication date |
---|---|
CA2907965A1 (en) | 2014-10-02 |
HK1219871A1 (en) | 2017-04-21 |
WO2014158165A1 (en) | 2014-10-02 |
JP2016516745A (en) | 2016-06-09 |
KR20150135430A (en) | 2015-12-02 |
CN105188688A (en) | 2015-12-23 |
CA2907965C (en) | 2021-12-07 |
EP2978417A4 (en) | 2017-03-01 |
AU2013384234A1 (en) | 2015-10-15 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
JP6879980B2 (en) | Enhancement of autophagy or prolongation of lifespan by administration of urolithin or its precursor | |
Yang et al. | Resveratrol regulates microglia M1/M2 polarization via PGC-1α in conditions of neuroinflammatory injury | |
CA2907965C (en) | Composition and use for treating cardiac failure | |
Liu et al. | Diosmin protects against cerebral ischemia/reperfusion injury through activating JAK2/STAT3 signal pathway in mice | |
Briyal et al. | Neuroprotective and anti-apoptotic effects of liraglutide in the rat brain following focal cerebral ischemia | |
Yao et al. | Toll-like receptor 4 mediates microglial activation and production of inflammatory mediators in neonatal rat brain following hypoxia: role of TLR4 in hypoxic microglia | |
JP6707549B2 (en) | Anti-aging compounds and their use | |
Elsherbiny et al. | ABT-702, an adenosine kinase inhibitor, attenuates inflammation in diabetic retinopathy | |
Sun et al. | Role of p-MKK7 in myricetin-induced protection against intestinal ischemia/reperfusion injury | |
Wang et al. | Pro-survival and anti-inflammatory roles of NF-κB c-Rel in the Parkinson's disease models | |
Shi et al. | GCN2 suppression attenuates cerebral ischemia in mice by reducing apoptosis and endoplasmic reticulum (ER) stress through the blockage of FoxO3a-regulated ROS production | |
Wu et al. | Recombinant adiponectin peptide promotes neuronal survival after intracerebral haemorrhage by suppressing mitochondrial and ATF4‐CHOP apoptosis pathways in diabetic mice via Smad3 signalling inhibition | |
Zhang et al. | The dual neuroprotective-neurotoxic effects of sevoflurane after hemorrhagic shock injury | |
US20160008386A1 (en) | Composition and method for inducing epo-mediated haemoglobin expression and mitochondrial biogenesis in nonhaematopoietic cell | |
Ding et al. | Fibroblast growth factor 21 attenuates ventilator-induced lung injury by inhibiting the NLRP3/caspase-1/GSDMD pyroptotic pathway | |
Amato et al. | The potential of lisosan G as a possible treatment for glaucoma | |
Cai et al. | Up-regulation of Thioredoxin 1 by aerobic exercise training attenuates endoplasmic reticulum stress and cardiomyocyte apoptosis following myocardial infarction | |
Feng et al. | AHNAK-modified microbubbles for the intracranial delivery of triptolide: In-vitro and in-vivo investigations | |
TWI641594B (en) | Iridoid compound and pharmaceutical composition thereof for treating stroke and uses thereof | |
EP3979989A1 (en) | Production and use of extracellular vesicle-contained enampt | |
CN111989103A (en) | Pharmaceutical compositions, methods of treatment and uses thereof | |
US20230277491A1 (en) | Pharmaceutical combination comprising glycolic acid and l-alanine | |
Chen et al. | Uncoupling protein 2 facilitates insulin-elicited protection against lipopolysaccharide-induced myocardial dysfunction | |
US20230054551A1 (en) | Oral pharmaceutical composition and method for delivering nitric oxide to a patient's circulatory system or brain | |
WO2023173359A1 (en) | Metabolic disease therapeutic agent or preventive agent |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
PUAI | Public reference made under article 153(3) epc to a published international application that has entered the european phase |
Free format text: ORIGINAL CODE: 0009012 |
|
17P | Request for examination filed |
Effective date: 20150922 |
|
AK | Designated contracting states |
Kind code of ref document: A1 Designated state(s): AL AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HR HU IE IS IT LI LT LU LV MC MK MT NL NO PL PT RO RS SE SI SK SM TR |
|
AX | Request for extension of the european patent |
Extension state: BA ME |
|
DAX | Request for extension of the european patent (deleted) | ||
RIC1 | Information provided on ipc code assigned before grant |
Ipc: A61P 25/28 20060101ALI20161013BHEP Ipc: A61K 31/05 20060101AFI20161013BHEP Ipc: A61K 31/045 20060101ALI20161013BHEP Ipc: A61K 31/7034 20060101ALI20161013BHEP Ipc: A61P 25/16 20060101ALI20161013BHEP Ipc: A61K 31/70 20060101ALI20161013BHEP |
|
A4 | Supplementary search report drawn up and despatched |
Effective date: 20170131 |
|
RIC1 | Information provided on ipc code assigned before grant |
Ipc: A61K 31/045 20060101ALI20170125BHEP Ipc: A61P 25/16 20060101ALI20170125BHEP Ipc: A61K 31/7034 20060101ALI20170125BHEP Ipc: A61K 31/70 20060101ALI20170125BHEP Ipc: A61K 31/05 20060101AFI20170125BHEP Ipc: A61P 25/28 20060101ALI20170125BHEP |
|
REG | Reference to a national code |
Ref country code: HK Ref legal event code: DE Ref document number: 1219871 Country of ref document: HK |
|
STAA | Information on the status of an ep patent application or granted ep patent |
Free format text: STATUS: REQUEST FOR EXAMINATION WAS MADE |
|
STAA | Information on the status of an ep patent application or granted ep patent |
Free format text: STATUS: THE APPLICATION IS DEEMED TO BE WITHDRAWN |
|
18D | Application deemed to be withdrawn |
Effective date: 20170829 |
|
REG | Reference to a national code |
Ref country code: HK Ref legal event code: WD Ref document number: 1219871 Country of ref document: HK |