US20220354933A1 - Methods of treating myointimal proliferation - Google Patents
Methods of treating myointimal proliferation Download PDFInfo
- Publication number
- US20220354933A1 US20220354933A1 US17/747,379 US202217747379A US2022354933A1 US 20220354933 A1 US20220354933 A1 US 20220354933A1 US 202217747379 A US202217747379 A US 202217747379A US 2022354933 A1 US2022354933 A1 US 2022354933A1
- Authority
- US
- United States
- Prior art keywords
- npp1
- enpp1
- sirna
- fusion protein
- cells
- 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.)
- Pending
Links
- 238000000034 method Methods 0.000 title claims abstract description 53
- 230000035755 proliferation Effects 0.000 title abstract description 61
- 108020001507 fusion proteins Proteins 0.000 claims abstract description 132
- 102000037865 fusion proteins Human genes 0.000 claims abstract description 132
- 108010009413 Pyrophosphatases Proteins 0.000 claims abstract description 11
- 102000009609 Pyrophosphatases Human genes 0.000 claims abstract description 11
- 101150011046 NPP1 gene Proteins 0.000 claims description 144
- 101100080092 Phytophthora capsici NLP1 gene Proteins 0.000 claims description 144
- 108010067341 ectonucleotide pyrophosphatase phosphodiesterase 1 Proteins 0.000 claims description 70
- 230000000694 effects Effects 0.000 claims description 63
- 108090000765 processed proteins & peptides Proteins 0.000 claims description 44
- 238000011282 treatment Methods 0.000 claims description 41
- 102000004196 processed proteins & peptides Human genes 0.000 claims description 35
- 229920001184 polypeptide Polymers 0.000 claims description 32
- 206010020718 hyperplasia Diseases 0.000 claims description 25
- 241001465754 Metazoa Species 0.000 claims description 16
- 230000006378 damage Effects 0.000 claims description 16
- 208000027418 Wounds and injury Diseases 0.000 claims description 15
- 210000001715 carotid artery Anatomy 0.000 claims description 15
- 208000014674 injury Diseases 0.000 claims description 15
- 108060003951 Immunoglobulin Proteins 0.000 claims description 13
- 102000018358 immunoglobulin Human genes 0.000 claims description 13
- 210000005166 vasculature Anatomy 0.000 claims description 9
- 208000033173 Generalized arterial calcification of infancy Diseases 0.000 claims description 8
- 208000004900 arterial calcification of infancy Diseases 0.000 claims description 8
- 230000004044 response Effects 0.000 claims description 6
- 238000010171 animal model Methods 0.000 claims description 5
- 206010068872 Toe walking Diseases 0.000 claims description 3
- 239000012634 fragment Substances 0.000 abstract description 63
- 108090001050 Phosphoric Diester Hydrolases Proteins 0.000 abstract description 8
- 102000004861 Phosphoric Diester Hydrolases Human genes 0.000 abstract description 8
- 210000004027 cell Anatomy 0.000 description 112
- 108020004459 Small interfering RNA Proteins 0.000 description 98
- 210000004509 vascular smooth muscle cell Anatomy 0.000 description 94
- 101000812677 Homo sapiens Nucleotide pyrophosphatase Proteins 0.000 description 93
- 102100039306 Nucleotide pyrophosphatase Human genes 0.000 description 87
- 241000700159 Rattus Species 0.000 description 55
- 108090000623 proteins and genes Proteins 0.000 description 51
- 102000004169 proteins and genes Human genes 0.000 description 50
- 125000003275 alpha amino acid group Chemical group 0.000 description 49
- 235000018102 proteins Nutrition 0.000 description 48
- 230000014509 gene expression Effects 0.000 description 44
- 235000001014 amino acid Nutrition 0.000 description 35
- 150000001413 amino acids Chemical class 0.000 description 34
- 108020004999 messenger RNA Proteins 0.000 description 32
- 239000002609 medium Substances 0.000 description 29
- 241000699670 Mus sp. Species 0.000 description 26
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 26
- 239000012091 fetal bovine serum Substances 0.000 description 23
- 239000013642 negative control Substances 0.000 description 20
- XUJNEKJLAYXESH-UHFFFAOYSA-N cysteine Natural products SCC(N)C(O)=O XUJNEKJLAYXESH-UHFFFAOYSA-N 0.000 description 19
- 102000006602 glyceraldehyde-3-phosphate dehydrogenase Human genes 0.000 description 19
- 108020004445 glyceraldehyde-3-phosphate dehydrogenase Proteins 0.000 description 19
- 235000018417 cysteine Nutrition 0.000 description 18
- 230000030279 gene silencing Effects 0.000 description 18
- 230000001965 increasing effect Effects 0.000 description 18
- 238000002474 experimental method Methods 0.000 description 17
- 230000003197 catalytic effect Effects 0.000 description 16
- 201000010099 disease Diseases 0.000 description 16
- 208000015181 infectious disease Diseases 0.000 description 16
- 230000008685 targeting Effects 0.000 description 16
- 238000001890 transfection Methods 0.000 description 16
- 241000699666 Mus <mouse, genus> Species 0.000 description 15
- 239000002245 particle Substances 0.000 description 15
- 239000000523 sample Substances 0.000 description 15
- 230000002018 overexpression Effects 0.000 description 14
- 210000004899 c-terminal region Anatomy 0.000 description 13
- 239000003981 vehicle Substances 0.000 description 13
- 208000004434 Calcinosis Diseases 0.000 description 12
- IQFYYKKMVGJFEH-XLPZGREQSA-N Thymidine Chemical compound O=C1NC(=O)C(C)=CN1[C@@H]1O[C@H](CO)[C@@H](O)C1 IQFYYKKMVGJFEH-XLPZGREQSA-N 0.000 description 12
- OIRDTQYFTABQOQ-KQYNXXCUSA-N adenosine Chemical compound C1=NC=2C(N)=NC=NC=2N1[C@@H]1O[C@H](CO)[C@@H](O)[C@H]1O OIRDTQYFTABQOQ-KQYNXXCUSA-N 0.000 description 12
- 230000002308 calcification Effects 0.000 description 12
- 235000003642 hunger Nutrition 0.000 description 12
- 230000037351 starvation Effects 0.000 description 12
- 102000004190 Enzymes Human genes 0.000 description 11
- 108090000790 Enzymes Proteins 0.000 description 11
- IQFYYKKMVGJFEH-UHFFFAOYSA-N beta-L-thymidine Natural products O=C1NC(=O)C(C)=CN1C1OC(CO)C(O)C1 IQFYYKKMVGJFEH-UHFFFAOYSA-N 0.000 description 11
- 229940088598 enzyme Drugs 0.000 description 11
- 239000012098 Lipofectamine RNAiMAX Substances 0.000 description 10
- 230000010261 cell growth Effects 0.000 description 10
- 238000012761 co-transfection Methods 0.000 description 10
- 208000035475 disorder Diseases 0.000 description 10
- 239000003814 drug Substances 0.000 description 10
- 230000004927 fusion Effects 0.000 description 10
- 238000003753 real-time PCR Methods 0.000 description 10
- 230000001225 therapeutic effect Effects 0.000 description 10
- 229940122361 Bisphosphonate Drugs 0.000 description 9
- 239000002126 C01EB10 - Adenosine Substances 0.000 description 9
- UDMBCSSLTHHNCD-UHFFFAOYSA-N Coenzym Q(11) Natural products C1=NC=2C(N)=NC=NC=2N1C1OC(COP(O)(O)=O)C(O)C1O UDMBCSSLTHHNCD-UHFFFAOYSA-N 0.000 description 9
- UDMBCSSLTHHNCD-KQYNXXCUSA-N adenosine 5'-monophosphate Chemical compound C1=NC=2C(N)=NC=NC=2N1[C@@H]1O[C@H](COP(O)(O)=O)[C@@H](O)[C@H]1O UDMBCSSLTHHNCD-KQYNXXCUSA-N 0.000 description 9
- 150000004663 bisphosphonates Chemical class 0.000 description 9
- 239000000203 mixture Substances 0.000 description 9
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 8
- 101150017770 ENPP1 gene Proteins 0.000 description 8
- 238000003556 assay Methods 0.000 description 8
- 230000004663 cell proliferation Effects 0.000 description 8
- 239000002299 complementary DNA Substances 0.000 description 8
- 230000005764 inhibitory process Effects 0.000 description 8
- 229940124597 therapeutic agent Drugs 0.000 description 8
- 108010088751 Albumins Proteins 0.000 description 7
- 102000009027 Albumins Human genes 0.000 description 7
- 238000004458 analytical method Methods 0.000 description 7
- 230000000903 blocking effect Effects 0.000 description 7
- 230000037396 body weight Effects 0.000 description 7
- 239000000872 buffer Substances 0.000 description 7
- 230000007812 deficiency Effects 0.000 description 7
- 238000003197 gene knockdown Methods 0.000 description 7
- 238000011534 incubation Methods 0.000 description 7
- 239000002773 nucleotide Substances 0.000 description 7
- 239000000758 substrate Substances 0.000 description 7
- 239000006228 supernatant Substances 0.000 description 7
- WSFSSNUMVMOOMR-UHFFFAOYSA-N Formaldehyde Chemical compound O=C WSFSSNUMVMOOMR-UHFFFAOYSA-N 0.000 description 6
- 210000004263 induced pluripotent stem cell Anatomy 0.000 description 6
- 238000001990 intravenous administration Methods 0.000 description 6
- 238000004519 manufacturing process Methods 0.000 description 6
- 230000003584 silencer Effects 0.000 description 6
- 238000005406 washing Methods 0.000 description 6
- 241000124008 Mammalia Species 0.000 description 5
- 241000283973 Oryctolagus cuniculus Species 0.000 description 5
- 229950006790 adenosine phosphate Drugs 0.000 description 5
- 230000001086 cytosolic effect Effects 0.000 description 5
- 235000011180 diphosphates Nutrition 0.000 description 5
- 230000002255 enzymatic effect Effects 0.000 description 5
- 239000000463 material Substances 0.000 description 5
- 125000003729 nucleotide group Chemical group 0.000 description 5
- -1 nucleotide sugars Chemical class 0.000 description 5
- RWOAVOYBVRQNIZ-BFHYXJOUSA-N p-nitrophenyl thymidine 5'-monophosphate Chemical compound O=C1NC(=O)C(C)=CN1[C@@H]1O[C@H](COP(O)(=O)OC=2C=CC(=CC=2)[N+]([O-])=O)[C@@H](O)C1 RWOAVOYBVRQNIZ-BFHYXJOUSA-N 0.000 description 5
- 238000001262 western blot Methods 0.000 description 5
- 102000008186 Collagen Human genes 0.000 description 4
- 108010035532 Collagen Proteins 0.000 description 4
- CKLJMWTZIZZHCS-REOHCLBHSA-N L-aspartic acid Chemical compound OC(=O)[C@@H](N)CC(O)=O CKLJMWTZIZZHCS-REOHCLBHSA-N 0.000 description 4
- 241001529936 Murinae Species 0.000 description 4
- 229940122907 Phosphatase inhibitor Drugs 0.000 description 4
- 238000011529 RT qPCR Methods 0.000 description 4
- 208000005475 Vascular calcification Diseases 0.000 description 4
- 229960005305 adenosine Drugs 0.000 description 4
- 125000000539 amino acid group Chemical group 0.000 description 4
- 235000003704 aspartic acid Nutrition 0.000 description 4
- 239000007640 basal medium Substances 0.000 description 4
- 230000008901 benefit Effects 0.000 description 4
- 239000013592 cell lysate Substances 0.000 description 4
- 230000001413 cellular effect Effects 0.000 description 4
- 239000003795 chemical substances by application Substances 0.000 description 4
- 229920001436 collagen Polymers 0.000 description 4
- 230000007423 decrease Effects 0.000 description 4
- XPPKVPWEQAFLFU-UHFFFAOYSA-J diphosphate(4-) Chemical compound [O-]P([O-])(=O)OP([O-])([O-])=O XPPKVPWEQAFLFU-UHFFFAOYSA-J 0.000 description 4
- 239000003937 drug carrier Substances 0.000 description 4
- 238000005516 engineering process Methods 0.000 description 4
- 239000000499 gel Substances 0.000 description 4
- 235000013922 glutamic acid Nutrition 0.000 description 4
- 239000004220 glutamic acid Substances 0.000 description 4
- 125000000291 glutamic acid group Chemical group N[C@@H](CCC(O)=O)C(=O)* 0.000 description 4
- 230000012010 growth Effects 0.000 description 4
- 238000001802 infusion Methods 0.000 description 4
- 239000007924 injection Substances 0.000 description 4
- 238000002347 injection Methods 0.000 description 4
- 230000003562 morphometric effect Effects 0.000 description 4
- 238000013425 morphometry Methods 0.000 description 4
- 230000035772 mutation Effects 0.000 description 4
- 238000010606 normalization Methods 0.000 description 4
- 230000036961 partial effect Effects 0.000 description 4
- 230000002829 reductive effect Effects 0.000 description 4
- 238000003757 reverse transcription PCR Methods 0.000 description 4
- 210000000329 smooth muscle myocyte Anatomy 0.000 description 4
- 239000000243 solution Substances 0.000 description 4
- 238000007920 subcutaneous administration Methods 0.000 description 4
- 210000005167 vascular cell Anatomy 0.000 description 4
- XZKIHKMTEMTJQX-UHFFFAOYSA-N 4-Nitrophenyl Phosphate Chemical compound OP(O)(=O)OC1=CC=C([N+]([O-])=O)C=C1 XZKIHKMTEMTJQX-UHFFFAOYSA-N 0.000 description 3
- BTJIUGUIPKRLHP-UHFFFAOYSA-N 4-nitrophenol Chemical compound OC1=CC=C([N+]([O-])=O)C=C1 BTJIUGUIPKRLHP-UHFFFAOYSA-N 0.000 description 3
- 108700039887 Essential Genes Proteins 0.000 description 3
- 102000004160 Phosphoric Monoester Hydrolases Human genes 0.000 description 3
- 108090000608 Phosphoric Monoester Hydrolases Proteins 0.000 description 3
- 239000002202 Polyethylene glycol Substances 0.000 description 3
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 3
- 238000002835 absorbance Methods 0.000 description 3
- 230000002378 acidificating effect Effects 0.000 description 3
- LNQVTSROQXJCDD-UHFFFAOYSA-N adenosine monophosphate Natural products C1=NC=2C(N)=NC=NC=2N1C1OC(CO)C(OP(O)(O)=O)C1O LNQVTSROQXJCDD-UHFFFAOYSA-N 0.000 description 3
- 230000005754 cellular signaling Effects 0.000 description 3
- 239000007795 chemical reaction product Substances 0.000 description 3
- 239000003085 diluting agent Substances 0.000 description 3
- 239000001963 growth medium Substances 0.000 description 3
- 230000036541 health Effects 0.000 description 3
- 230000007062 hydrolysis Effects 0.000 description 3
- 238000006460 hydrolysis reaction Methods 0.000 description 3
- 238000000338 in vitro Methods 0.000 description 3
- 238000001727 in vivo Methods 0.000 description 3
- 238000010348 incorporation Methods 0.000 description 3
- 239000008176 lyophilized powder Substances 0.000 description 3
- 239000012528 membrane Substances 0.000 description 3
- 150000007523 nucleic acids Chemical group 0.000 description 3
- 239000008194 pharmaceutical composition Substances 0.000 description 3
- 229920001223 polyethylene glycol Polymers 0.000 description 3
- 238000002360 preparation method Methods 0.000 description 3
- 239000011535 reaction buffer Substances 0.000 description 3
- 238000011084 recovery Methods 0.000 description 3
- 208000024891 symptom Diseases 0.000 description 3
- RWQNBRDOKXIBIV-UHFFFAOYSA-N thymine Chemical compound CC1=CNC(=O)NC1=O RWQNBRDOKXIBIV-UHFFFAOYSA-N 0.000 description 3
- 238000002604 ultrasonography Methods 0.000 description 3
- FWMNVWWHGCHHJJ-SKKKGAJSSA-N 4-amino-1-[(2r)-6-amino-2-[[(2r)-2-[[(2r)-2-[[(2r)-2-amino-3-phenylpropanoyl]amino]-3-phenylpropanoyl]amino]-4-methylpentanoyl]amino]hexanoyl]piperidine-4-carboxylic acid Chemical compound C([C@H](C(=O)N[C@H](CC(C)C)C(=O)N[C@H](CCCCN)C(=O)N1CCC(N)(CC1)C(O)=O)NC(=O)[C@H](N)CC=1C=CC=CC=1)C1=CC=CC=C1 FWMNVWWHGCHHJJ-SKKKGAJSSA-N 0.000 description 2
- 206010060965 Arterial stenosis Diseases 0.000 description 2
- CIWBSHSKHKDKBQ-JLAZNSOCSA-N Ascorbic acid Chemical compound OC[C@H](O)[C@H]1OC(=O)C(O)=C1O CIWBSHSKHKDKBQ-JLAZNSOCSA-N 0.000 description 2
- BSYNRYMUTXBXSQ-UHFFFAOYSA-N Aspirin Chemical compound CC(=O)OC1=CC=CC=C1C(O)=O BSYNRYMUTXBXSQ-UHFFFAOYSA-N 0.000 description 2
- 241000271566 Aves Species 0.000 description 2
- 239000005552 B01AC04 - Clopidogrel Substances 0.000 description 2
- 241000283707 Capra Species 0.000 description 2
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 2
- 241000588724 Escherichia coli Species 0.000 description 2
- DBVJJBKOTRCVKF-UHFFFAOYSA-N Etidronic acid Chemical compound OP(=O)(O)C(O)(C)P(O)(O)=O DBVJJBKOTRCVKF-UHFFFAOYSA-N 0.000 description 2
- WHUUTDBJXJRKMK-UHFFFAOYSA-N Glutamic acid Natural products OC(=O)C(N)CCC(O)=O WHUUTDBJXJRKMK-UHFFFAOYSA-N 0.000 description 2
- 229940121710 HMGCoA reductase inhibitor Drugs 0.000 description 2
- 108091006905 Human Serum Albumin Proteins 0.000 description 2
- 102000008100 Human Serum Albumin Human genes 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
- TWRXJAOTZQYOKJ-UHFFFAOYSA-L Magnesium chloride Chemical compound [Mg+2].[Cl-].[Cl-] TWRXJAOTZQYOKJ-UHFFFAOYSA-L 0.000 description 2
- PVNIIMVLHYAWGP-UHFFFAOYSA-N Niacin Chemical compound OC(=O)C1=CC=CN=C1 PVNIIMVLHYAWGP-UHFFFAOYSA-N 0.000 description 2
- 239000000020 Nitrocellulose Substances 0.000 description 2
- 229920001213 Polysorbate 20 Polymers 0.000 description 2
- 229940124158 Protease/peptidase inhibitor Drugs 0.000 description 2
- 238000011530 RNeasy Mini Kit Methods 0.000 description 2
- 239000006180 TBST buffer Substances 0.000 description 2
- DRTQHJPVMGBUCF-XVFCMESISA-N Uridine Chemical compound O[C@@H]1[C@H](O)[C@@H](CO)O[C@H]1N1C(=O)NC(=O)C=C1 DRTQHJPVMGBUCF-XVFCMESISA-N 0.000 description 2
- 229960001138 acetylsalicylic acid Drugs 0.000 description 2
- 150000001510 aspartic acids Chemical class 0.000 description 2
- OQFSQFPPLPISGP-UHFFFAOYSA-N beta-carboxyaspartic acid Natural products OC(=O)C(N)C(C(O)=O)C(O)=O OQFSQFPPLPISGP-UHFFFAOYSA-N 0.000 description 2
- 210000004204 blood vessel Anatomy 0.000 description 2
- 239000002775 capsule Substances 0.000 description 2
- 229960004588 cilostazol Drugs 0.000 description 2
- RRGUKTPIGVIEKM-UHFFFAOYSA-N cilostazol Chemical compound C=1C=C2NC(=O)CCC2=CC=1OCCCCC1=NN=NN1C1CCCCC1 RRGUKTPIGVIEKM-UHFFFAOYSA-N 0.000 description 2
- GKTWGGQPFAXNFI-HNNXBMFYSA-N clopidogrel Chemical compound C1([C@H](N2CC=3C=CSC=3CC2)C(=O)OC)=CC=CC=C1Cl GKTWGGQPFAXNFI-HNNXBMFYSA-N 0.000 description 2
- 229960003009 clopidogrel Drugs 0.000 description 2
- 238000011260 co-administration Methods 0.000 description 2
- 150000001875 compounds Chemical class 0.000 description 2
- 238000002591 computed tomography Methods 0.000 description 2
- 238000002607 contrast-enhanced ultrasound Methods 0.000 description 2
- 230000007850 degeneration Effects 0.000 description 2
- 230000000431 effect on proliferation Effects 0.000 description 2
- 230000003511 endothelial effect Effects 0.000 description 2
- 229940009626 etidronate Drugs 0.000 description 2
- 229940125753 fibrate Drugs 0.000 description 2
- 239000007850 fluorescent dye Substances 0.000 description 2
- 230000006870 function Effects 0.000 description 2
- 230000035876 healing Effects 0.000 description 2
- 238000010842 high-capacity cDNA reverse transcription kit Methods 0.000 description 2
- 239000002471 hydroxymethylglutaryl coenzyme A reductase inhibitor Substances 0.000 description 2
- 238000013115 immunohistochemical detection Methods 0.000 description 2
- NOESYZHRGYRDHS-UHFFFAOYSA-N insulin Chemical compound N1C(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(NC(=O)CN)C(C)CC)CSSCC(C(NC(CO)C(=O)NC(CC(C)C)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CCC(N)=O)C(=O)NC(CC(C)C)C(=O)NC(CCC(O)=O)C(=O)NC(CC(N)=O)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CSSCC(NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2C=CC(O)=CC=2)NC(=O)C(CC(C)C)NC(=O)C(C)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2NC=NC=2)NC(=O)C(CO)NC(=O)CNC2=O)C(=O)NCC(=O)NC(CCC(O)=O)C(=O)NC(CCCNC(N)=N)C(=O)NCC(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC(O)=CC=3)C(=O)NC(C(C)O)C(=O)N3C(CCC3)C(=O)NC(CCCCN)C(=O)NC(C)C(O)=O)C(=O)NC(CC(N)=O)C(O)=O)=O)NC(=O)C(C(C)CC)NC(=O)C(CO)NC(=O)C(C(C)O)NC(=O)C1CSSCC2NC(=O)C(CC(C)C)NC(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CC(N)=O)NC(=O)C(NC(=O)C(N)CC=1C=CC=CC=1)C(C)C)CC1=CN=CN1 NOESYZHRGYRDHS-UHFFFAOYSA-N 0.000 description 2
- 230000008069 intimal proliferation Effects 0.000 description 2
- 239000007927 intramuscular injection Substances 0.000 description 2
- 238000010255 intramuscular injection Methods 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 239000012139 lysis buffer Substances 0.000 description 2
- 239000011159 matrix material Substances 0.000 description 2
- 239000000178 monomer Substances 0.000 description 2
- 235000001968 nicotinic acid Nutrition 0.000 description 2
- 229960003512 nicotinic acid Drugs 0.000 description 2
- 239000011664 nicotinic acid Substances 0.000 description 2
- 229920001220 nitrocellulos Polymers 0.000 description 2
- 108020004707 nucleic acids Proteins 0.000 description 2
- 102000039446 nucleic acids Human genes 0.000 description 2
- 230000007170 pathology Effects 0.000 description 2
- 239000000137 peptide hydrolase inhibitor Substances 0.000 description 2
- 229920002401 polyacrylamide Polymers 0.000 description 2
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 description 2
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 description 2
- 230000018883 protein targeting Effects 0.000 description 2
- 230000001105 regulatory effect Effects 0.000 description 2
- 230000002441 reversible effect Effects 0.000 description 2
- 230000006807 siRNA silencing Effects 0.000 description 2
- 230000008477 smooth muscle tissue growth Effects 0.000 description 2
- 239000011780 sodium chloride Substances 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
- 239000007929 subcutaneous injection Substances 0.000 description 2
- 238000010254 subcutaneous injection Methods 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 235000000346 sugar Nutrition 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- 238000002560 therapeutic procedure Methods 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- 230000003442 weekly effect Effects 0.000 description 2
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 1
- 238000010600 3H thymidine incorporation assay Methods 0.000 description 1
- 102100031126 6-phosphogluconolactonase Human genes 0.000 description 1
- 108010029731 6-phosphogluconolactonase Proteins 0.000 description 1
- 241000251468 Actinopterygii Species 0.000 description 1
- 102000007469 Actins Human genes 0.000 description 1
- 108010085238 Actins Proteins 0.000 description 1
- 241000272525 Anas platyrhynchos Species 0.000 description 1
- 241000024188 Andala Species 0.000 description 1
- 200000000007 Arterial disease Diseases 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- DWRXFEITVBNRMK-UHFFFAOYSA-N Beta-D-1-Arabinofuranosylthymine Natural products O=C1NC(=O)C(C)=CN1C1C(O)C(O)C(CO)O1 DWRXFEITVBNRMK-UHFFFAOYSA-N 0.000 description 1
- 102100021961 Bis(5'-adenosyl)-triphosphatase ENPP4 Human genes 0.000 description 1
- 241000283690 Bos taurus Species 0.000 description 1
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 1
- 241000282472 Canis lupus familiaris Species 0.000 description 1
- 241000700198 Cavia Species 0.000 description 1
- 238000003734 CellTiter-Glo Luminescent Cell Viability Assay Methods 0.000 description 1
- 241000282693 Cercopithecidae Species 0.000 description 1
- 102100036093 Ectonucleotide pyrophosphatase/phosphodiesterase family member 7 Human genes 0.000 description 1
- 241000283086 Equidae Species 0.000 description 1
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 1
- 108010037362 Extracellular Matrix Proteins Proteins 0.000 description 1
- 102000010834 Extracellular Matrix Proteins Human genes 0.000 description 1
- 108091006020 Fc-tagged proteins Proteins 0.000 description 1
- 241000282326 Felis catus Species 0.000 description 1
- 241000287828 Gallus gallus Species 0.000 description 1
- 108010018962 Glucosephosphate Dehydrogenase Proteins 0.000 description 1
- 241001272567 Hominoidea Species 0.000 description 1
- 241000282412 Homo Species 0.000 description 1
- 101000897056 Homo sapiens Bis(5'-adenosyl)-triphosphatase ENPP4 Proteins 0.000 description 1
- 101000876377 Homo sapiens Ectonucleotide pyrophosphatase/phosphodiesterase family member 7 Proteins 0.000 description 1
- 101000766306 Homo sapiens Serotransferrin Proteins 0.000 description 1
- 102000008394 Immunoglobulin Fragments Human genes 0.000 description 1
- 108010021625 Immunoglobulin Fragments Proteins 0.000 description 1
- 102000004877 Insulin Human genes 0.000 description 1
- 108090001061 Insulin Proteins 0.000 description 1
- 206010022489 Insulin Resistance Diseases 0.000 description 1
- 206010048858 Ischaemic cardiomyopathy Diseases 0.000 description 1
- 230000004988 N-glycosylation Effects 0.000 description 1
- 101150071357 NPP2 gene Proteins 0.000 description 1
- 108091028043 Nucleic acid sequence Proteins 0.000 description 1
- 108091005461 Nucleic proteins Proteins 0.000 description 1
- 241000282579 Pan Species 0.000 description 1
- 229930040373 Paraformaldehyde Natural products 0.000 description 1
- 208000031481 Pathologic Constriction Diseases 0.000 description 1
- 241001494479 Pecora Species 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
- 241000286209 Phasianidae Species 0.000 description 1
- 102000009569 Phosphoglucomutase Human genes 0.000 description 1
- 101100080097 Phytophthora capsici NLP2 gene Proteins 0.000 description 1
- 108010076504 Protein Sorting Signals Proteins 0.000 description 1
- 238000002123 RNA extraction Methods 0.000 description 1
- 208000035977 Rare disease Diseases 0.000 description 1
- 208000004531 Renal Artery Obstruction Diseases 0.000 description 1
- 206010038378 Renal artery stenosis Diseases 0.000 description 1
- 101000995829 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) Nucleotide pyrophosphatase Proteins 0.000 description 1
- 238000000692 Student's t-test Methods 0.000 description 1
- 241000282898 Sus scrofa Species 0.000 description 1
- 102000004338 Transferrin Human genes 0.000 description 1
- 108090000901 Transferrin Proteins 0.000 description 1
- 102100023935 Transmembrane glycoprotein NMB Human genes 0.000 description 1
- HSCJRCZFDFQWRP-JZMIEXBBSA-N UDP-alpha-D-glucose Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1OP(O)(=O)OP(O)(=O)OC[C@@H]1[C@@H](O)[C@@H](O)[C@H](N2C(NC(=O)C=C2)=O)O1 HSCJRCZFDFQWRP-JZMIEXBBSA-N 0.000 description 1
- HSCJRCZFDFQWRP-UHFFFAOYSA-N Uridindiphosphoglukose Natural products OC1C(O)C(O)C(CO)OC1OP(O)(=O)OP(O)(=O)OCC1C(O)C(O)C(N2C(NC(=O)C=C2)=O)O1 HSCJRCZFDFQWRP-UHFFFAOYSA-N 0.000 description 1
- 238000005411 Van der Waals force Methods 0.000 description 1
- VFRROHXSMXFLSN-VANKVMQKSA-N [(2s,3s,4r,5s)-2,3,4,5-tetrahydroxy-6-oxohexyl] dihydrogen phosphate Chemical compound OP(=O)(O)OC[C@H](O)[C@H](O)[C@@H](O)[C@H](O)C=O VFRROHXSMXFLSN-VANKVMQKSA-N 0.000 description 1
- 230000001594 aberrant effect Effects 0.000 description 1
- 230000002159 abnormal effect Effects 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 239000004480 active ingredient Substances 0.000 description 1
- 230000002776 aggregation Effects 0.000 description 1
- 238000004220 aggregation Methods 0.000 description 1
- RWHOZGRAXYWRNX-VFUOTHLCSA-N alpha-D-glucose 1,6-bisphosphate Chemical compound O[C@H]1[C@H](O)[C@@H](COP(O)(O)=O)O[C@H](OP(O)(O)=O)[C@@H]1O RWHOZGRAXYWRNX-VFUOTHLCSA-N 0.000 description 1
- APKFDSVGJQXUKY-INPOYWNPSA-N amphotericin B Chemical compound O[C@H]1[C@@H](N)[C@H](O)[C@@H](C)O[C@H]1O[C@H]1/C=C/C=C/C=C/C=C/C=C/C=C/C=C/[C@H](C)[C@@H](O)[C@@H](C)[C@H](C)OC(=O)C[C@H](O)C[C@H](O)CC[C@@H](O)[C@H](O)C[C@H](O)C[C@](O)(C[C@H](O)[C@H]2C(O)=O)O[C@H]2C1 APKFDSVGJQXUKY-INPOYWNPSA-N 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 210000001367 artery Anatomy 0.000 description 1
- 235000010323 ascorbic acid Nutrition 0.000 description 1
- 229960005070 ascorbic acid Drugs 0.000 description 1
- 239000011668 ascorbic acid Substances 0.000 description 1
- 239000008228 bacteriostatic water for injection Substances 0.000 description 1
- DRTQHJPVMGBUCF-PSQAKQOGSA-N beta-L-uridine Natural products O[C@H]1[C@@H](O)[C@H](CO)O[C@@H]1N1C(=O)NC(=O)C=C1 DRTQHJPVMGBUCF-PSQAKQOGSA-N 0.000 description 1
- 210000000988 bone and bone Anatomy 0.000 description 1
- 239000000969 carrier Substances 0.000 description 1
- 238000006555 catalytic reaction Methods 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
- 239000006285 cell suspension Substances 0.000 description 1
- 238000012054 celltiter-glo Methods 0.000 description 1
- YRQNKMKHABXEJZ-UVQQGXFZSA-N chembl176323 Chemical compound C1C[C@]2(C)[C@@]3(C)CC(N=C4C[C@]5(C)CCC6[C@]7(C)CC[C@@H]([C@]7(CC[C@]6(C)[C@@]5(C)CC4=N4)C)CCCCCCCC)=C4C[C@]3(C)CCC2[C@]2(C)CC[C@H](CCCCCCCC)[C@]21C YRQNKMKHABXEJZ-UVQQGXFZSA-N 0.000 description 1
- 239000012707 chemical precursor Substances 0.000 description 1
- 235000013330 chicken meat Nutrition 0.000 description 1
- 210000004978 chinese hamster ovary cell Anatomy 0.000 description 1
- 239000002131 composite material Substances 0.000 description 1
- 230000021615 conjugation Effects 0.000 description 1
- 210000002808 connective tissue Anatomy 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 239000012228 culture supernatant Substances 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 230000001419 dependent effect Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 230000029087 digestion Effects 0.000 description 1
- XZTWHWHGBBCSMX-UHFFFAOYSA-J dimagnesium;phosphonato phosphate Chemical compound [Mg+2].[Mg+2].[O-]P([O-])(=O)OP([O-])([O-])=O XZTWHWHGBBCSMX-UHFFFAOYSA-J 0.000 description 1
- 238000006471 dimerization reaction Methods 0.000 description 1
- 230000003467 diminishing effect Effects 0.000 description 1
- 238000009826 distribution Methods 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 210000004177 elastic tissue Anatomy 0.000 description 1
- 238000007824 enzymatic assay Methods 0.000 description 1
- 230000006862 enzymatic digestion Effects 0.000 description 1
- 210000002744 extracellular matrix Anatomy 0.000 description 1
- 239000000835 fiber Substances 0.000 description 1
- 239000011888 foil Substances 0.000 description 1
- 238000004108 freeze drying Methods 0.000 description 1
- 239000003517 fume Substances 0.000 description 1
- 239000007903 gelatin capsule Substances 0.000 description 1
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 1
- 239000010931 gold Substances 0.000 description 1
- 229910052737 gold Inorganic materials 0.000 description 1
- 239000000710 homodimer Substances 0.000 description 1
- 230000002209 hydrophobic effect Effects 0.000 description 1
- 208000011111 hypophosphatemic rickets Diseases 0.000 description 1
- 238000003384 imaging method Methods 0.000 description 1
- 230000005847 immunogenicity Effects 0.000 description 1
- 230000028709 inflammatory response Effects 0.000 description 1
- 230000000977 initiatory effect Effects 0.000 description 1
- 229940125396 insulin Drugs 0.000 description 1
- 239000007928 intraperitoneal injection Substances 0.000 description 1
- 231100000518 lethal Toxicity 0.000 description 1
- 230000001665 lethal effect Effects 0.000 description 1
- 230000000670 limiting effect Effects 0.000 description 1
- 239000002502 liposome Substances 0.000 description 1
- 239000006193 liquid solution Substances 0.000 description 1
- 229940097364 magnesium acetate tetrahydrate Drugs 0.000 description 1
- 229910001629 magnesium chloride Inorganic materials 0.000 description 1
- XKPKPGCRSHFTKM-UHFFFAOYSA-L magnesium;diacetate;tetrahydrate Chemical compound O.O.O.O.[Mg+2].CC([O-])=O.CC([O-])=O XKPKPGCRSHFTKM-UHFFFAOYSA-L 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 230000003340 mental effect Effects 0.000 description 1
- 239000003094 microcapsule Substances 0.000 description 1
- 238000013508 migration Methods 0.000 description 1
- 230000005012 migration Effects 0.000 description 1
- 238000010369 molecular cloning Methods 0.000 description 1
- 238000007491 morphometric analysis Methods 0.000 description 1
- 230000003387 muscular Effects 0.000 description 1
- 230000007935 neutral effect Effects 0.000 description 1
- 231100000252 nontoxic Toxicity 0.000 description 1
- 230000003000 nontoxic effect Effects 0.000 description 1
- 238000012633 nuclear imaging Methods 0.000 description 1
- 230000000414 obstructive effect Effects 0.000 description 1
- 229920002113 octoxynol Polymers 0.000 description 1
- 238000012634 optical imaging Methods 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 208000001040 ossification of the posterior longitudinal ligament of the spine Diseases 0.000 description 1
- 229920002866 paraformaldehyde Polymers 0.000 description 1
- 239000008188 pellet Substances 0.000 description 1
- 229940049954 penicillin Drugs 0.000 description 1
- 230000002093 peripheral effect Effects 0.000 description 1
- 239000000546 pharmaceutical excipient Substances 0.000 description 1
- 230000003285 pharmacodynamic effect Effects 0.000 description 1
- 230000000144 pharmacologic effect Effects 0.000 description 1
- 108091000115 phosphomannomutase Proteins 0.000 description 1
- 230000035790 physiological processes and functions Effects 0.000 description 1
- 229920000642 polymer Polymers 0.000 description 1
- 229920000136 polysorbate Polymers 0.000 description 1
- 238000002600 positron emission tomography Methods 0.000 description 1
- 230000003389 potentiating effect Effects 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 230000002265 prevention Effects 0.000 description 1
- 230000002062 proliferating effect Effects 0.000 description 1
- 230000002035 prolonged effect Effects 0.000 description 1
- 230000000069 prophylactic effect Effects 0.000 description 1
- 230000001681 protective effect Effects 0.000 description 1
- XNSAINXGIQZQOO-SRVKXCTJSA-N protirelin Chemical compound NC(=O)[C@@H]1CCCN1C(=O)[C@@H](NC(=O)[C@H]1NC(=O)CC1)CC1=CN=CN1 XNSAINXGIQZQOO-SRVKXCTJSA-N 0.000 description 1
- 238000007420 radioactive assay Methods 0.000 description 1
- 239000011541 reaction mixture Substances 0.000 description 1
- 238000010188 recombinant method Methods 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 238000010839 reverse transcription Methods 0.000 description 1
- 150000003839 salts Chemical group 0.000 description 1
- 238000002603 single-photon emission computed tomography Methods 0.000 description 1
- 235000020183 skimmed milk Nutrition 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 238000012453 sprague-dawley rat model Methods 0.000 description 1
- 238000010561 standard procedure Methods 0.000 description 1
- 238000007619 statistical method Methods 0.000 description 1
- 230000036262 stenosis Effects 0.000 description 1
- 208000037804 stenosis Diseases 0.000 description 1
- 239000011550 stock solution Substances 0.000 description 1
- 229960005322 streptomycin Drugs 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 239000013589 supplement Substances 0.000 description 1
- 238000001356 surgical procedure Methods 0.000 description 1
- 230000002123 temporal effect Effects 0.000 description 1
- 210000001042 thoracic artery Anatomy 0.000 description 1
- 229940104230 thymidine Drugs 0.000 description 1
- 210000001519 tissue Anatomy 0.000 description 1
- 239000012581 transferrin Substances 0.000 description 1
- 230000009261 transgenic effect Effects 0.000 description 1
- 108091007466 transmembrane glycoproteins Proteins 0.000 description 1
- PIEPQKCYPFFYMG-UHFFFAOYSA-N tris acetate Chemical compound CC(O)=O.OCC(N)(CO)CO PIEPQKCYPFFYMG-UHFFFAOYSA-N 0.000 description 1
- GPRLSGONYQIRFK-MNYXATJNSA-N triton Chemical compound [3H+] GPRLSGONYQIRFK-MNYXATJNSA-N 0.000 description 1
- 208000001072 type 2 diabetes mellitus Diseases 0.000 description 1
- 241000701161 unidentified adenovirus Species 0.000 description 1
- DRTQHJPVMGBUCF-UHFFFAOYSA-N uracil arabinoside Natural products OC1C(O)C(CO)OC1N1C(=O)NC(=O)C=C1 DRTQHJPVMGBUCF-UHFFFAOYSA-N 0.000 description 1
- 229940045145 uridine Drugs 0.000 description 1
- 230000002792 vascular Effects 0.000 description 1
- 208000019553 vascular disease Diseases 0.000 description 1
- 230000035899 viability Effects 0.000 description 1
- 238000003260 vortexing Methods 0.000 description 1
- 230000036642 wellbeing Effects 0.000 description 1
- XRASPMIURGNCCH-UHFFFAOYSA-N zoledronic acid Chemical compound OP(=O)(O)C(P(O)(O)=O)(O)CN1C=CN=C1 XRASPMIURGNCCH-UHFFFAOYSA-N 0.000 description 1
- 229960004276 zoledronic acid Drugs 0.000 description 1
Images
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/43—Enzymes; Proenzymes; Derivatives thereof
- A61K38/46—Hydrolases (3)
- A61K38/465—Hydrolases (3) acting on ester bonds (3.1), e.g. lipases, ribonucleases
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/43—Enzymes; Proenzymes; Derivatives thereof
- A61K38/46—Hydrolases (3)
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
- A01K67/027—New or modified breeds of vertebrates
- A01K67/0271—Chimeric vertebrates, e.g. comprising exogenous cells
-
- 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
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2227/00—Animals characterised by species
- A01K2227/10—Mammal
- A01K2227/105—Murine
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2267/00—Animals characterised by purpose
- A01K2267/03—Animal model, e.g. for test or diseases
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
- A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/30—Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y301/00—Hydrolases acting on ester bonds (3.1)
- C12Y301/04—Phosphoric diester hydrolases (3.1.4)
- C12Y301/04001—Phosphodiesterase I (3.1.4.1)
Definitions
- Myointimal proliferation is an arterial wall smooth muscle cell (SMC) proliferative disorder (Painter, TA, Artif Organs. 1991 February; 15(1):42-55).
- myointimal proliferation involves the migration and proliferation of vascular smooth muscle cells (VSMCs), as well as the involvement of the extracellular matrix in the intima i.e., the innermost coat of a blood vessel consisting of an endothelial layer backed by connective tissue and elastic tissue (see, e.g., Kraiss L W, Clowes A W, In: Sumpio S A N, ed. The Basic Science of Vascular Disease . New York, N.Y.: Futura Publishing; 1997:289-317; and Yang Z, Luscher T F. Eur Heart J. 1993; 14(suppl):193-197).
- VSMCs vascular smooth muscle cells
- Ectonucleotide pyrophosphatase pyrophosphorylase 1 is an ectoenzyme that cleaves ATP to produce extracellular pyrophosphate (PPi).
- Ectonucleotide pyrophosphatase/phosphodiesterase 1 (NPP1/ENPP1/PC-1) deficiency is a rare disease caused by mutations in NPP1, a type II transmembrane glycoprotein.
- NPP1 cleaves a variety of substrates, including phosphodiester bonds of nucleotides and nucleotide sugars and pyrophosphate bonds of nucleotides and nucleotide sugars.
- NPP1 deficiency has been associated with idiopathic infantile arterial calcification (IIAC), insulin resistance, hypophosphatemic rickets, and ossification of the posterior longitudinal ligament of the spine.
- IIAC idiopathic infantile arterial calcification
- insulin resistance a rare autosomal recessive and nearly always fatal disorder
- calcification of the internal elastic lamina of muscular arteries and stenosis due to myointimal proliferation.
- There are more than 160 cases of IIAC that have been reported world-wide. The symptoms of the disease most often appear by early infancy, and the disease is lethal by 6 months of age, generally because of ischemic cardiomyopathy, and other complications of obstructive arteriopathy including renal artery stenosis.
- the present invention relates to uses of isolated recombinant human soluble NPP1 that lacks N-terminal cytosolic and transmembrane domains and fusion proteins thereof for the treatment of NPP1-deficiency and/or myointimal proliferation disorders. Any disorder that is characterized by myointimal proliferation is within the scope of the present invention.
- methods for treating a human patient having detected myointimal proliferation e.g., as assessed by immunohistochemical detection, ultrasound (e.g., intravascular ultrasonography, carotid ultrasound, or contrast-enhanced ultrasound (CEU)), X-ray computed tomography (CT), nuclear imaging (e.g., positron emission tomography (PET) or single-photon emission computed tomography (SPECT)), optical imaging, or contrast enhanced image) are provided, the method comprising administering to the patient one or more doses of a recombinant human soluble ectonucleotide pyrophosphatase phosphodiesterase (hsNPP1), active fragment or fusion protein thereof.
- ultrasound e.g., intravascular ultrasonography, carotid ultrasound, or contrast-enhanced ultrasound (CEU)
- CT X-ray computed tomography
- PET positron emission tomography
- SPECT single-photon emission computed tomography
- the methods of treating myointimal proliferation e.g., as assessed by immunohistochemical detection
- the method comprising: a) identifying a human patient as having myointimal proliferation and b) administering to the identified patient one or more doses of a recombinant human soluble ectonucleotide pyrophosphatase phosphodiesterase (hsNPP1), active fragment or fusion protein thereof.
- hsNPP1 human soluble ectonucleotide pyrophosphatase phosphodiesterase
- a NPP1 fusion protein is administered.
- Preferred fusion proteins comprise and NPP1 component an Fc region of an immunoglobulin and, optionally, a targeting moiety.
- the targeting moiety is Asp 10 (SEQ ID NO: 18).
- the targeting moiety comprises at least eight consecutive aspartic acid or glutamic acid residues (SEQ ID NOS 20 and 21, respectively).
- Particular NPP1 fusion proteins for administration in accordance with the methods disclosed herein have the amino acid sequence set forth in SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11 or SEQ ID NO:12.
- the human patient has an NPP1 deficiency resulting in insufficient production of adenosine or adenosine monophosphate (AMP).
- administration of a recombinant hsNPP1 according to the methods described herein is sufficient to normalize adenosine or adenosine monophosphate (AMP) production in the human patient.
- administration of a recombinant hsNPP1 according to the methods described herein is sufficient to prevent arterial stenosis in the patient.
- any suitable amount of the recombinant hsNPP1 can be administered to the human patient.
- the hsNPP1 is administered in one or more doses containing about 0.5 mg/kg, 1.0 mg/kg, 2.0 mg/kg, 3.0 mg/kg, 4.0 mg/kg, 5.0 mg/kg, 6.0 mg/kg, 7.0 mg/kg, 8.0 mg/kg, 9.0 mg/kg, 10.0 mg/kg, 11.0 mg/kg, 12.0 mg/kg, 13.0 mg/kg, 14.0 mg/kg, 15.0 mg/kg, 16.0 mg/kg, 17.0 mg/kg, 18.0 mg/kg, 19.0 mg/kg, or 20.0 mg/kg.
- the hsNPP1 is administered in one or more doses containing about 1.0 mg/kg to about 5.0 mg/kg NPP1. In another embodiment, the hsNPP1 is administered in one or more doses containing about 1.0 mg/kg to about 10.0 mg/kg NPP1.
- the time period between doses of the hsNPP1 is at least 2 days and can be longer, for example at least 3 days, at least 1 week, 2 weeks or 1 month. In one embodiment, the administration is weekly, bi-weekly, or monthly.
- the recombinant hsNPP1 can be administered in any suitable way, such as intravenously, subcutaneously, or intraperitoneally.
- the recombinant hsNPP1 can be administered in combination with one or more additional therapeutic agents.
- additional therapeutic agents include, but are not limited to Bisphosphonate, Statins, Fibrates, Niacin, Aspirin, Clopidogrel, and speakerarin.
- the recombinant hsNPP1 and additional therapeutic agent are administered separately and are administered concurrently or sequentially.
- the recombinant hsNPP1 is administered prior to administration of the additional therapeutic agent.
- the recombinant hsNPP1 is administered after administration of the additional therapeutic agent.
- the recombinant hsNPP1 and additional therapeutic agent are administered together.
- the patient does not have arterial calcification. In one another, the patient does have arterial calcification.
- an isolated recombinant human sNPP1, fragment or fusion protein thereof uses of an isolated recombinant human sNPP1, fragment or fusion protein thereof.
- the use of an isolated recombinant human sNPP1, fragment or fusion protein thereof for the manufacture of a medicament for treating myointimal proliferation is provided.
- the invention provides the use of an isolated recombinant human sNPP1, fragment or fusion protein thereof for treating myointimal proliferation.
- the myointimal proliferation is not associated with arterial calcification.
- the myointimal proliferation is associated with arterial calcification.
- FIGS. 1A-1C depict mRNA ( FIG. 1A ) and protein ( FIG. 1B ) ENPP1 expression in human primary VSMCs from six different donors, as well as enzyme activity from three of the donors ( FIG. 1C ).
- FIGS. 2A-2B depict inhibition of ENPP1 mRNA expression relative to a negative control. Specifically, FIG. 2A depicts inhibition of ENPP1 mRNA expression relative to a negative control for five siRNA constructs, 48 hours post siRNA transfection. FIG. 2B depicts inhibition of ENPP1 mRNA expression relative to a negative control six and eleven days post siRNA transfection with construct #4.
- FIGS. 3A-3B depict the effect of silencing ENPP1 by siRNA on proliferation of human primary VSMCs from two different donors.
- FIGS. 4A-4B depict the effect of silencing ENPP1 by siRNA on human primary VSMC cell growth on Day 3 ( FIG. 4A ) and Day 4 ( FIG. 4B ).
- FIGS. 5A-5B depict the effect of siRNA silencing on levels of ENPP1 mRNA expression ( FIG. 5A ), cell growth ( FIG. 5B ), and enzyme activity ( FIG. 5C ) in rat VSMCs.
- FIGS. 6A-6B depict the effect of adenosine ( FIG. 6A ), AMP ( FIG. 6A ), and PPi ( FIG. 6B ) on proliferation of rat VSMCs that were knocked down with ENPP1.
- FIG. 7 depicts the effect of bisphosphonate on proliferation of rat VSMCs.
- FIGS. 8A-8B depict the effect of silencing ENPP1 using different siRNA constructs on proliferation in rat VSMCs. Specifically, FIG. 8A depicts the effect of silencing ENPP1 by two siRNA on proliferation of rat VSMCs. FIG. 8B depicts inhibition of ENPP1 mRNA expression relative to a negative control for two siRNA constructs, 48 hours post siRNA transfection.
- FIG. 9A depicts the effect of Ad-mENPP1/Ad-rENPP1 on Mouse/Rat ENPP1 messenger RNA specifically in rat VSMCs.
- FIG. 9B shows the distribution of GFP following co-transfection of siRNA and Ad-GFP.
- FIG. 10 shows that siRNA specific to rat ENPP1 partially affected Ad-mENPP1 on Mouse ENPP1 messenger RNA specifically in co-transfection siRNA with Ad-mRNPP1 in rat VSMCs.
- FIG. 11 depicts the effect of Ad-rENPP1 on ENPP1 protein expression.
- FIGS. 12A-12B depict the effect of mENPP1 overexpression on enzyme activity in rat VSMCs ( FIG. 12A ) and A10 cells ( FIG. 12B ).
- FIGS. 13A-13B shows that silencing ENPP1 increases proliferation on rat VSMCs and overexpression of mouse or rat ENPP1 inhibits proliferation on rat VSMCs that either downregulated ENPP1 expression (si-rENPP1) or normal ENPP1 expression (si-NC).
- FIG. 14 depicts the effect of silencing ENPP1 and overexpression of mouse or rat ENPP1 on rat VSMCs cell growth.
- FIG. 15 shows that FBS has a negative effect on stability of ATP in cultured supernatant.
- CM contains 5% FBS
- Starvation media contains 0.25% FBS.
- ATP function was reduced over 60% in CM after 30 minutes in culture at 37° C.
- FIG. 16 shows that FBS has a negative effect on stability of ATP in cultured supernatant.
- CM contains 5% FBS
- Starvation media contains 0.25% FBS.
- ATP function was reduced over 87% after 2 hours in culture at 37° C.
- FIG. 17 shows that FBS has a negative effect on stability of ATP in cultured supernatant.
- CM contains 5% FBS
- Starvation media contains 0.25% FBS.
- ATP function was almost entirely lost after 24 hours in culture at 37° C.
- FIGS. 18A-18B shows that heat denatured human/mouse ENPP1-Fc protein completely lost their enzymatic activity.
- FIGS. 19A-19C depicts the effect of murine ENPP1-Fc protein ( FIG. 19A ), human ENPP1-Fc ( FIG. 19B ) and human ENPP1-FC-D10 ( FIG. 19C ) on proliferation of rat primary VSMCs.
- FIGS. 20A-20C depict ENPP1 expression by human primary VSMCs and human induced pluripotent stem cell (hiPSC)-derived vascular smooth muscle cells (iVSMCs), as assessed by qRT-PCR ( FIG. 20A ) and Western Blot ( FIGS. 20B-C ).
- hiPSC human induced pluripotent stem cell
- iVSMCs vascular smooth muscle cells
- FIG. 21 depicts the effect of silencing ENPP1 by siRNA on the growth of human induced pluripotent stem cell (hiPSC)-derived vascular smooth muscle cells (iVSMCs).
- hiPSC human induced pluripotent stem cell
- iVSMCs vascular smooth muscle cells
- FIGS. 22A-22C depict the effect of murine ( FIG. 22A ) and human ENPP1-Fc ( FIGS. 22B-22C ) protein on the proliferation of human induced pluripotent stem cell (hiPSC)-derived vascular smooth muscle cells (iVSMCs).
- hiPSC human induced pluripotent stem cell
- iVSMCs vascular smooth muscle cells
- FIG. 23 depicts PPi levels in the supernatant, as assessed by a PPi assay using human iVSMCs.
- FIG. 24 depicts the effect of bisphosphonate on proliferation of human iVSMCs.
- FIG. 25 is a diagram of the carotid artery ligation and sectioning for histological analysis. Five ⁇ m sections were cut spanning 250 ⁇ m from the point of ligation. Every fifth section was analyzed.
- FIG. 26 is a histological analysis (Von Gieson's stain) of sections either 100 (upper) or 200 (lower) ⁇ m from point of ligation from wild-type (WT), ttw/ttw, vehicle-treated ttw/ttw or rhENPP1-treated ttw/ttw/mice from left to right, respectively.
- the internal elastic lamina (IEL), external elastic lamina (EEL) and lumen (L) are indicated by arrows.
- the scale bar represents 100 ⁇ m.
- FIGS. 27A-27C are a morphometric quantitation of medial ( FIG. 27A ) and intimal areas ( FIG. 27B ), as well as the I/M ratio ( FIG. 27C ). *p ⁇ 0.001.
- FIGS. 28A-28B show the effect of therapeutic Enpp1 treatment on intimal hyperplasia in ttw/ttw mouse started 7 days after carotid ligation.
- FIG. 28A shows the degree of intimal hyperplasia 7 days post ligation in WT and ttw/ttw mice.
- FIG. 28B shows the histological analysis (Von Gieson's stain) of sections either 100 (upper) or 200 (lower) ⁇ m from point of ligation from WT, vehicle-treated ttw/ttw or rhENPP1-treated ttw/ttw mice from left to right, respectively.
- the internal elastic lamina (IEL), external elastic lamina (EEL) and lumen (L) are indicated by arrows.
- the scale bar represents 100 ⁇ m.
- FIGS. 29A-29C are a morphometric quantitations of medial ( FIG. 29A ) and intimal areas ( FIG. 29B ), as well as the I/M ratio ( FIG. 29C ) on treatment day 14. *p ⁇ 0.05, **p ⁇ 0.01.
- NPP1 and “ENPP1” refer to the same protein and are used interchangeably herein.
- “About” as used herein when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass variations of ⁇ 20% or ⁇ 10%, more preferably ⁇ 5%, even more preferably ⁇ 1%, and still more preferably ⁇ 0.1% from the specified value, as such variations are appropriate to perform the disclosed methods.
- fragment refers to an active subsequence of the full-length NPP1.
- a “fragment” of a protein or peptide can be at least about 20 amino acids in length; for example, at least about 50 amino acids in length; at least about 100 amino acids in length; at least about 200 amino acids in length; at least about 300 amino acids in length; or at least about 400 amino acids in length (and any integer value in between).
- the fragments may range in size from four amino acid residues to the entire amino acid sequence minus one amino acid.
- a protein “comprising at least a portion of the amino acid sequence of SEQ ID NO: 1” encompasses the full-length NPP1 and fragments thereof.
- an “isolated” or “purified” soluble NPP1 protein or biologically active fragment or fusion protein thereof is substantially free of cellular material or other contaminating proteins from the cell or tissue source from which the NPP1 protein, biologically active fragment or NPP1 fusion protein is derived, or substantially free from chemical precursors or other chemicals when chemically synthesized.
- the language “substantially free of cellular material” includes preparations of NPP1 protein, biologically active fragment, or NPP1 fusion protein in which the protein is separated from cellular components of the cells from which it is isolated or recombinantly produced.
- the language “substantially free of cellular material” includes preparations of NPP1 protein, biologically active fragment or NPP1 fusion protein having less than about 30% (by dry weight) of non-NPP1 protein/fragment/fusion protein (also referred to herein as a “contaminating protein”), more preferably less than about 20% of non-NPP1 protein/fragment/fusion protein, still more preferably less than about 10% of non-NPP1 protein/fragment/fusion protein, and most preferably less than about 5% non-NPP1 protein/fragment/fusion protein.
- non-NPP1 protein/fragment/fusion protein also referred to herein as a “contaminating protein”
- NPP1 protein, fusion protein, or biologically active fragment thereof is recombinantly produced, it is also preferably substantially free of culture medium, i.e., culture medium represents less than about 20%, more preferably less than about 10%, and most preferably less than about 5% of the volume of the protein preparation.
- ranges throughout this disclosure, various aspects of the invention can be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the range.
- the term “subject” encompasses mammals and non-mammals.
- mammals include, but are not limited to, humans, chimpanzees, apes monkeys, cattle, horses, sheep, goats, swine, rabbits, dogs, cats, rats, mice, guinea pigs, and the like.
- non-mammals include, but are not limited to, birds, fish and the like.
- the term “therapeutically effective amount” refers to a nontoxic but sufficient amount of an agent (e.g., hsNPP1 proteins) which, as compared to a corresponding subject who has not received such amount, results in improved treatment, healing, prevention, or amelioration of a disease, disorder, or side effect, or a decrease in the rate of advancement of a disease or disorder.
- an agent e.g., hsNPP1 proteins
- the term also includes within its scope amounts effective to enhance normal physiological function.
- myointimal proliferation refers to abnormal proliferation of the smooth muscle cells of the vascular wall (e.g., the intima of a blood vessel).
- treating includes the application or administration of the NPP1 proteins, active fragments and fusion proteins of the invention to a subject, or application or administration of NPP1 proteins, active fragments and fusion proteins of the invention to a subject who has myointimal proliferation, with the purpose of curing, healing, alleviating, relieving, altering, remedying, ameliorating, preventing, improving, or affecting the disease or disorder.
- treating refers to any indicia of success in the treatment or amelioration of an injury, pathology or condition, including any objective or subjective parameter such as abatement; remission; diminishing of symptoms or making the injury, pathology or condition more tolerable to the subject; slowing in the rate of degeneration or decline; making the final point of degeneration less debilitating; or improving a subject's physical or mental well-being.
- Treatment may be therapeutic or prophylactic.
- the treatment or amelioration of symptoms can be based on objective or subjective parameters; including the results of a physical examination.
- the present invention relates to uses of an isolated recombinant human soluble NPP1 (“sNPP1”) which lacks an N-terminal portion (i.e., lacking cytosolic and transmembrane domains) and fusion proteins thereof for the treatment of NPP1-associated diseases, such as myointimal proliferation.
- sNPP1 isolated recombinant human soluble NPP1
- the subject can be a human patient having deficiencies in NPP1 activity (NPP1 deficiency).
- NPP1 deficiency the patient exhibits low levels of pyrophosphate and/or suffers from a disease or disorder associated with low levels of pyrophosphate.
- the human patient has an NPP1 deficiency resulting in insufficient production of adenosine or adenosine monophosphate (AMP).
- AMP adenosine monophosphate
- the dosage of fusion protein administered to a subject will vary depending upon known factors such as age, health and weight of the recipient, type of concurrent treatment, frequency of treatment, and the like.
- a dosage of active ingredient i.e., fusion protein
- fusion protein can be between about 0.0001 and about 50 milligrams per kilogram of body weight.
- Precise dosage, frequency of administration and time span of treatment can be determined by a physician skilled in the art of administration of therapeutic proteins.
- a preferred embodiment of the present invention involves a method of treating myointimal proliferation, which includes the step of identifying a human patient as having myointimal proliferation and administering to the identified patient a therapeutically effective amount of a recombinant human soluble ectonucleotide pyrophosphatase phosphodiesterase (hsNPP1), active fragment or fusion protein thereof.
- hsNPP1 human soluble ectonucleotide pyrophosphatase phosphodiesterase
- a therapeutically effective amount of protein ranges from about 0.001 to 50 mg/kg body weight.
- an effective dosage ranges from about 0.001 to 50 mg/kg body weight.
- certain factors may influence the dosage required to effectively treat a subject, including but not limited to the severity of the disease, previous treatments, the general health and/or age of the subject, and other diseases present.
- treatment of a subject with a therapeutically effective amount of protein can include a single treatment or, preferably, can include a series of treatments. It will also be appreciated that the effective dosage of protein used for treatment may increase or decrease over the course of a particular treatment.
- a therapeutically effective amount of protein or polypeptide ranges from about 0.001 to 50 mg/kg body weight, preferably about 0.01 to 25 mg/kg body weight, more preferably about 0.1 to 20 mg/kg body weight, and even more preferably about 1 to 10 mg/kg, 2 to 9 mg/kg, 3 to 8 mg/kg, 4 to 7 mg/kg, or 5 to 6 mg/kg body weight.
- the hsNPP1 is administered in one or more doses containing about 0.1 mg/kg, 0.2 mg/kg, 0.3 mg/kg, 0.4 mg/kg, 0.5 mg/kg, 1.0 mg/kg, 2.0 mg/kg, 3.0 mg/kg, 4.0 mg/kg, 5.0 mg/kg, 6.0 mg/kg, 7.0 mg/kg, 8.0 mg/kg, 9.0 mg/kg, 10.0 mg/kg, 11.0 mg/kg, 12.0 mg/kg, 13.0 mg/kg, 14.0 mg/kg, 15.0 mg/kg, 16.0 mg/kg, 17.0 mg/kg, 18.0 mg/kg, 19.0 mg/kg, 20.0 mg/kg, 21.0 mg/kg, 22.0 mg/kg, 23.0 mg/kg, 24.0 mg/kg, 25.0 mg/kg, 26.0 mg/kg, 27.0 mg/kg, 28.0 mg/kg, 29.0 mg/kg, 30.0 mg/kg, 31.0 mg/kg, 32.0 mg/kg, 33.0 mg/kg, 34.0 mg/kg,
- about 0.5 to about 30 mg, about 0.5 to about 20 mg, about 0.5 to about 10 mg, or about 0.5 to about 5 mg are administered to the patient.
- the hsNPP1 is administered in one or more doses containing about 1.0 mg/kg to about 5.0 mg/kg hsNPP1.
- the hsNPP1 is administered in one or more doses containing about 1.0 mg/kg to about 10.0 mg/kg hsNPP1.
- certain factors may influence the dosage required to effectively treat a subject, including, but not limited to, the severity of the disease or disorder, previous treatments, the general health and/or age of the subject, and other diseases present.
- treatment of a subject with a therapeutically effective amount of a protein, polypeptide, or antibody can include a single treatment or, preferably, can include a series of treatments.
- the time period between doses of the hsNPP1 is at least 2 days and can be longer, for example at least 3 days, at least 1 week, 2 weeks or 1 month.
- the therapeutically effective dose of sNPP1, biologically active fragment or fusion protein thereof is administered to a patient between one time every 5 days and one time every 30 days for a period of time determined by a practitioner of skill in the art of medical sciences. In another embodiment, the period of time will be the remainder of the patient's life span.
- the dosing frequency is between one time every 5 days and one time every 25 days. In another embodiment, the dosing frequency is between one time every 5 days and one time every 21 days. In another embodiment, the dosing frequency is between one time every 7 days and one time every 14 days.
- hsNPP1, biologically active fragment or fusion protein thereof can be administered one time every 5 days, one time every 6 days, one time every 7 days, one time every 8 days, one time every 9 days, one time every 10 days, one time every 11 days, one time every 12 days, one time every 13 days, or one time every 14 days.
- hsNPP1, biologically active fragment or fusion protein thereof is administered about weekly.
- sNPP1, biologically active fragment or fusion protein thereof is administered about bi-weekly.
- the dosing frequency is one time about 30 days. It will also be appreciated that the effective dosage of soluble sNPP1 protein, biologically active fragment or fusion protein thereof used for the treatment may increase or decrease over the course of a particular treatment.
- about 1 mg/kg of hsNPP1, biologically active fragment or fusion protein is administered to the patient once a week. In one embodiment, about 2 mg/kg of hsNPP1, biologically active fragment or fusion protein is administered to the patient once a week. In one embodiment, about 3 mg/kg of hsNPP1, biologically active fragment or fusion protein is administered to the patient once a week. In one embodiment, about 4 mg/kg of sNPP1, biologically active fragment or fusion protein is administered to the patient once a week. In one embodiment, about 5 mg/kg of hsNPP1, biologically active fragment or fusion protein is administered to the patient once a week.
- about 6 mg/kg of hsNPP1, biologically active fragment or fusion protein is administered to the patient once a week. In one embodiment, about 7 mg/kg of hsNPP1, biologically active fragment or fusion protein is administered to the patient once a week. In one embodiment, about 8 mg/kg of hsNPP1, biologically active fragment or fusion protein is administered to the patient once a week. In one embodiment, about 9 mg/kg of hsNPP1, biologically active fragment or fusion protein is administered to the patient once a week. In one embodiment, about 10 mg/kg of hsNPP1, biologically active fragment or fusion protein is administered to the patient once a week.
- hsNPP1 biologically active fragment or fusion protein
- biologically active fragment or fusion protein can be administered by, for example, subcutaneous injections, intramuscular injections, and intravenous (IV) infusions or injections.
- IV intravenous
- hsNPP1 biologically active fragment or fusion protein is administered intravenously by IV infusion by any useful method.
- hsNPP1 biologically active fragment or fusion protein can be administered by intravenous infusion through a peripheral line.
- hsNPP1, biologically active fragment or fusion protein can be administered by intravenous infusion through a peripherally inserted central catheter.
- hsNPP1 biologically active fragment or fusion protein is administered intravenously by IV injection.
- hsNPP1 biologically active fragment or fusion protein is administered via intraperitoneal injection.
- hsNPP1 biologically active fragment or fusion protein is administered by subcutaneous injections.
- hsNPP1 biologically active fragment or fusion protein is administered by intramuscular injections.
- hsNPP1 biologically active fragment or fusion protein is administered via a pharmaceutically acceptable capsule of the therapeutic protein.
- the capsule can be an enteric-coated gelatin capsule.
- the method involves administering the soluble NPP1 protein or NPP1 fusion protein of the invention alone, or in combination with other agent(s).
- exemplary therapeutic agents include, but are not limited to bisphosphonate, Statins, Fibrates, Niacin, Aspirin, Clopidogrel, and speakerarin.
- the method involves administering an NPP1 protein or an NPP1 fusion protein of the invention as therapy to compensate for reduced or aberrant NPP1 expression or activity in the subject having an NPP1-deficiency or other associated disease or disorder.
- the isolated sNPP1 proteins, fragments, and fusion proteins can be administered before, after or concurrently with the agent or can be co-administered with other known therapies.
- Co-administration of the isolated sNPP1 proteins, fragments, and fusion proteins of the present invention with other therapeutic agents may provide two agents which operate via different mechanisms which yield an increased therapeutic effect. Such co-administration can solve problems due to development of resistance to drugs.
- this disclosure relates to a method for reducing myointimal proliferation in a subject in need thereof.
- the methods described herein provide a way to reduce myointimal proliferation in a subject (e.g., human patient).
- the human patient has an NPP1 deficiency resulting in insufficient production of adenosine or adenosine monophosphate (AMP).
- administration of a recombinant hsNPP1 according to the methods described herein is sufficient to normalize adenosine or adenosine monophosphate (AMP) production in the human patient.
- administration of a recombinant hsNPP1 according to the methods described herein is sufficient to prevent arterial stenosis in the patient.
- the present invention employs soluble NPP1 (e.g., hsNPP1) that has a biologically active NPP1 domain of NPP1 (i.e., NPP1 components that contain at least one extracellular catalytic domain of naturally occurring NPP1 for the pyrophosphatase and/or phosphodiesterase activity).
- the soluble NPP1 proteins of the invention comprise at least the NPP1 domain essential to carry out the pyrophosphatase and/or phosphodiesterase activity.
- the soluble NPP1, fragment, and fusion proteins thereof can form functional homodimers or monomer.
- a soluble NPP1 protein or NPP1 fusion protein thereof can be assayed for pyrophosphatase activity as well as the ability to increase pyrophosphate levels in vivo.
- SEQ ID NO:5 shows the amino acid sequences of a soluble NPP1 containing amino acids from 107 to 925 of SEQ ID NO:1.
- SEQ ID NO:6 shows the amino acid sequence of a soluble NPP1 containing amino acids from 187 to 925 of SEQ ID NO:1.
- SEQ ID NO:7 shows the amino acid sequence of the Fc region of human IgG1 including the hinge region.
- SEQ ID NO:8 shows the amino acid sequence of the Fc of human IgG1 including a partial hinge region.
- SEQ ID NO:9 shows the amino acid sequence of a NPP1-Fc fusion protein.
- the NPP1 component contains SEQ ID NO:5, and the Fc sequence includes the hinge region.
- SEQ ID NO:10 shows the amino acid sequence of a NPP1-Fc fusion protein.
- the soluble NPP1 contains SEQ ID NO:5, and the Fc sequence includes the partial hinge region.
- SEQ ID NO:1 shows the amino acid sequence of a NPP1-Fc fusion protein.
- the soluble NPP1 contains SEQ ID NO:6, and the Fc sequence includes the hinge region.
- SEQ ID NO:12 shows the amino acid sequence of a NPP1-Fc fusion protein.
- the soluble NPP1 contains SEQ ID NO:6, and the Fc sequence includes the partial hinge region.
- Preferred soluble NPP1 proteins and NPP1 fusion proteins of the invention are enzymatically active in vivo (e.g., human).
- the soluble protein comprises amino acid sequence having at least 60, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% sequence identity to the following sequence:
- SEQ ID NO:2 is the amino acid sequence of a sNPP1 that contains the cysteine-rich region, catalytic region and c-terminal region.
- any desired enzymatically active form of soluble NPP1 can be used in the methods described herein.
- the enzymatically active sNPP1 can increase PPi levels in suitable enzymatic assays, and can be assayed for pyrophosphatase activity, phosphodiesterase activity, or pyrophosphatase and phosphodiesterase activity.
- the sNPP1 contains at least an NPP1 component that lacks the N-terminal cytosolic and transmembrane domains of naturally occurring transmembrane NPP1.
- SEQ ID NO:1 is the amino acid sequence of wild-type NPP1 protein.
- the cytosolic and transmembrane regions are underlined.
- the potential N-glycosylation sites are in bold.
- the amino acid motif “PSCAKE” (SEQ ID NO:17) in bold is the start of a soluble NPP1 which includes the cysteine rich region.
- the NPP1 component contains the cysteine-rich region (amino acids 99-204 of SEQ ID NO:1) and the catalytic region (amino acids 205-591 of SEQ ID NO:1) of naturally occurring human NPP1.
- the NPP1 component also includes the C-terminal region (amino acids 592 to 925 of SEQ ID NO:1), and has the amino acid sequence of SEQ ID NO:2.
- the C-terminal region can be truncated if desired.
- preferred NPP1 components include the cysteine-rich region and catalytic region of human NPP1 (amino acids 99-591 of SEQ ID NO:1) or the cysteine-rich region, the catalytic region and the C-terminal region of human NPP1 (SEQ ID NO:2).
- Other preferred NPP1 components contain only a portion of the cysteine-rich domain and have the sequence of amino acids 107 to 925 of SEQ ID NO:1 or amino acids 187 to 925 of SEQ ID NO:1.
- the cysteine rich region of NPP1 (i.e., amino acids 99 to 204 of SEQ ID NO: 1) can facilitate dimerization of the sNPP1.
- the sNPP1, including fusion proteins, can be in the form of a monomer of functional homodimer.
- the amino acid sequence of the NPP1 component can be a variant of the naturally occurring NPP1 sequence, provided that the NPP1 component is enzymatically active.
- NPP1 variants are enzymatically active and have at least 80%, at least 85%, at least 90%, at least 95% and more preferably at least 96% amino acid sequence identity to the corresponding portion of human NPP1 (e.g., over the length of the cysteine-rich region, the catalytic region, the c-terminal region, the cysteine-rich region plus the catalytic region, the cystein-rich region plus the catalytic region plus the c-terminal region.
- Preferred NPP1 variants have at least 90%, preferably at least 95%, more preferably at least 97% amino acid sequence identity to (i) the amino acid sequence of residues 205-591 of SEQ ID NO: 1, (ii) the amino acid sequence of residues 99-591 of SEQ ID NO:1, (iii) the amino acid sequence of residues 99-925 of SEQ ID NO:1, (iv) the amino acid sequence of residues 107-925 of SEQ ID NO:1, or (v) the amino acid sequence of residues 187-925 of SEQ ID NO:1.
- Suitable positions for amino acid variation are well-known from NPP1 structural studies and analysis of disease-associated mutations in NPP1.
- substitution of the following amino acids occurs in certain disease-associated mutations that reduce NPP1 enzymatic activity, and variations of the amino acids at these positions should be avoided: Ser216, Gly242, Pro250, Gly266, Pro305, Arg349, Tyr371, Arg456, Tyr471, His500, Ser504, Tyr513, Asp538, Tyr570, Lys579, Gly586; Tyr659, Glu668, Cys726, Arg774, His777, Asn792, Asp804, Arg821, Arg888, and Tyr901. (See, e.g., Jansen, S. et al., Structure 20:1948-1959 (2012)).
- the soluble NPP1 protein can be a fusion protein recombinantly fused or chemically bonded (e.g., covalent bond, ionic bond, hydrophobic bond and Van der Waals force) to a fusion partner.
- the fusion protein has at least 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity to SEQ ID NO: 3 or SEQ ID NO:4.
- SEQ ID NO:4 is the amino acid sequence of sNPP1-Fc-D10 (SEQ ID NO: 4). The Fc sequence is underlined.
- the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second amino acid or nucleic acid sequence for optimal alignment and non-homologous sequences can be disregarded for comparison purposes).
- the length of a reference sequence aligned for comparison purposes is at least 30%, preferably at least 40%, more preferably at least 50%, even more preferably at least 60%, and even more preferably at least 70%, 80%, or 90% of the length of the reference sequence (e.g., sNPP1 amino acid sequence of SEQ ID NO:2; amino acids 107-925 of SEQ ID NO:1 or amino acids 187-925 of SEQ ID NO:1).
- amino acid residues or nucleotides at corresponding amino acid positions are then compared.
- a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position (as used herein amino acid is equivalent to amino acid or nucleic acid “homology”).
- amino acid is equivalent to amino acid or nucleic acid “homology”.
- the percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences.
- the comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm.
- the percent identity between two amino acid sequences is determined using the Needleman and Wunsch (J Mol Biol 1970, 48, 444-453) algorithm which has been incorporated into the GAP program in the GCG software package (available at www.gcg.com), using either a Blosum 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6.
- the percent identity between two amino acid is determined using the algorithm of E. Meyers and W. Miller (CABIOS, 1989, 4, 11-17) which has been incorporated into the ALIGN program (version 2.0 or 2.0U), using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4.
- the sNPP1 can consist of or consist essentially of an NPP1 component as described herein.
- the sNPP1 can be in the form of a fusion protein that contains an NPP1 component and one or more other polypeptides, referred to as fusion partners, optionally through a suitable linker in each instance, or in the form of a conjugate between an NPP1 component and another molecule (e.g., PEG).
- fusion partners optionally through a suitable linker in each instance
- another molecule e.g., PEG
- each fusion partner is preferably located c-terminally to the NPP1 component.
- fusion proteins that contain an NPP1 component that contains the cysteine-rich region and catalytic region, and one or more fusion proteins that are located c-terminally to the NPP1 component, are preferred over other configurations of NPP1 fusion proteins because they can be expressed at sufficient levels and are sufficiently stable to be used as therapeutic proteins.
- Any suitable fusion partner can be included in the fusion protein.
- a number of fusion partners are well-known in the art that can provide certain advantages, such as reduced aggregation and immunogenicity, increased the solubility, improved expression and/or stability, and improved pharmacokinetic and/or pharmacodynamics performance. See, e.g., Strohl, W. R. BioDrugs 29:215-239 (2015).
- albumin, albumin fragments or albumin variants e.g., human serum albumin and fragments or variants thereof
- albumin, albumin fragments or albumin variants can be incorporated into fusion proteins and that such fusion proteins can be easily purified, stable and have an improved plasma half-life.
- Suitable albumin, albumin fragments and albumin variants that can be used in the sNPP1 fusion proteins are disclosed, for example in WO 2005/077042A2 and WO 03/076567A2, each of which is incorporated herein by reference in its entirety. Fusions to human transferrin are also known to improve half-life. See, e.g., Kim B J et al., J Pharmacol Expr Ther 334(3):682-692 (2010); and WO 2000/020746. Peptides that bind to albumin or transferrin, such as antibodies or antibody fragments, can also be used. See, e.g., EP 0486525 B1, U.S. Pat. No.
- the fusion protein can also include a CTP sequence (see also, Fares et al., Endocrinol 2010, 151, 4410-4417; Fares et al., Proc Natl Acad Sci 1992, 89, 4304-4308; and Furuhashi et al., Mol Endocrinol 1995, 9, 54-63).
- the fusion partner is the Fc of an immunoglobulin (e.g., Fc or human IgG1).
- the Fc can include CH1, CH2 and CH3 of human IgG1, and optionally the human IgG1 hinge region (EPKSCDKTHTCPPCP (SEQ ID NO:13)) or a portion of the human IgG1 hinge region (e.g., DKTHTCPPCP (SEQ ID NO:14) or PKSCDKTHTCPPCP (SEQ ID NO:15)) if desired.
- the Fc can include CH2 and CH3 of human IgG1, or the Fc of human IgG2 or human IgG4, if desired.
- the sNPP1 fusion protein comprises an NPP1 component and a peptide that increases the half-life of the fusion protein, most preferably the Fc of an immunoglobulin (e.g., Fc or human IgG1).
- a “protein that increases the half-life of the fusion protein” refers to a protein that, when fused to a soluble NPP1 or biologically active fragment, increases the half-life of the soluble NPP1 polypeptide or biologically active fragment as compared to the half-life of the soluble NPP1 polypeptide, alone, or the NPP1 biologically active fragment, alone.
- the half-life of the NPP1 fusion protein is increased 50% as compared to the half-life of the NPP1 polypeptide or biologically active fragment, alone. In another embodiment, the half-life of the NPP1 fusion protein is increased 60% as compared to the half-life of the NPP1 polypeptide or biologically active fragment, alone. In another embodiment, the half-life of the NPP1 fusion protein is increased 70% as compared to the half-life of the NPP1 polypeptide or biologically active fragment, alone. In another embodiment, the half-life of the NPP1 fusion protein is increased 80% as compared to the half-life of the NPP1 polypeptide or biologically active fragment, alone. In another embodiment, the half-life of the NPP1 fusion protein is increased 90% as compared to the half-life of the NPP1 polypeptide or biologically active fragment, alone.
- the half-life of the NPP1 fusion protein is increased 2 fold, 3 fold, 4 fold, 5 fold, 6 fold, 7 fold, 8 fold, 9 fold, or 10 fold as compared to the half-life of the NPP1 polypeptide or biologically active fragment, alone.
- Methods for determining the half-life of a protein or fusion protein are well known in the art. For example, Zhou et al., Determining Protein Half-Lives, Methods in Molecular Biology 2004, 284, 67-77 discloses numerous methods for testing of the half-life of a protein.
- the fusion protein can be conjugated to polymers or other suitable compounds that extend half-life, such as polyethylene glycol (PEG), can be conjugated to the NPP1 fusion proteins.
- PEG polyethylene glycol
- the peptide which increases the half-life of the fusion protein is a CTP sequence (see also, Fares et al., 2010 , Endocrinol., 151(9):4410-4417; Fares et al., 1992 , Proc. Natl. Acad. Sci, 89(10):4304-4308; and Furuhashi et al., 1995 , Molec. Endocrinol., 9(1):54-63).
- the peptide which increases the half-life of the fusion protein is an Fc domain of an Ig.
- Fusion partners may also be selected to target the fusion protein to desired sites of clinical or biological importance (e.g., site of calcification).
- desired sites of clinical or biological importance e.g., site of calcification.
- peptides that have high affinity to the bone are described in U.S. Pat. No. 7,323,542, the entire teachings of which are incorporated herein by reference.
- Peptides that can increase protein targeting to calcification sites can contain a consecutive stretch of at least about 4 acidic amino acids, for example, glutamic acids or aspartic acids.
- the peptide that targets the fusion protein to calcification sites will comprise between 4 and 20 consecutive acidic amino acids, for example 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 consecutive amino acids selected from glutamic acid and aspartic acid.
- the peptide can consist solely of glutamic acid residues, solely of aspartic acid residues, or be a mixture of glutamic acid and aspartic acid residues.
- a particularly preferred moiety for targeting to sights of calcification is Asp 10 (SEQ ID NO:18).
- the NPP1 fusion protein of the invention comprises an NPP1 polypeptide and a moiety that increase protein targeting to calcification sites such as a consecutive stretch of acidic amino acids, for example, glutamic acids or aspartic acids.
- Suitable peptide linkers for use in fusion proteins are well-known and typically adopt a flexible extended conformation and do not interfere with the function of the NPP1 component or the fusion partners.
- Peptide linker sequences may contain Gly, His, Asn and Ser residues in any combination.
- the useful peptide linkers include, without limitation, poly-Gly, poly-His, poly-Asn, or poly-Ser. Other near neutral amino acids, such as Thr and Ala can be also used in the linker sequence.
- linkers include those disclosed in Maratea et al., Gene 1985, 40, 39-46; Murphy et al., Proc Natl Acad Sci USA 1986, 83, 8258-8262; U.S. Pat. Nos. 4,935,233 and 4,751,180.
- Other suitable linkers can be obtained from naturally occurring proteins, such as the hinge region of an immunoglobulin.
- a preferred synthetic linker is (Gly 4 Ser) n , where n is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 (SEQ ID NO:19). Preferably, n is 3 or 4.
- the linker is (Gly 4 Ser) 3 (SEQ ID NO:16) and the fusion protein include a linker with the amino acid sequence GlyGlyGlyGlySerGlyGlyGlyGlySerGlyGlyGlyGlySer (SEQ ID NO:16).
- the linker is from 1 to about 50 amino acid residues in length, or 1 to about 25 amino acids in length. Frequently, the linker is between about 8 and about 20 amino acids in length.
- Preferred NPP1 fusion proteins comprise from N-terminus to C-terminus an NPP1 component, optionally a linker, an Fc region of an immunoglobulin (e.g., human IgG1 Fc optionally including hinge or a portion thereof), optionally a second liner, and optionally a targeting moiety.
- an Fc region of an immunoglobulin e.g., human IgG1 Fc optionally including hinge or a portion thereof
- optionally a targeting moiety when present, are each located C-terminally to the NPP1 component.
- the NPP1 component preferably comprises the cysteine-rich region and the catalytic domain of NPP1, lacks the N-terminal cytosolic and transmembrane domains, and optionally contains the C-terminal region.
- a preferred fusion protein comprises, from N-terminus to C-terminus, an NPP1 component comprising the cysteine-rich domain, the catalytic domain and the C-terminal region of human NPP1; and the Fc region, including hinge, of a human immunoglobulin.
- the Fc region is from human IgG1.
- the fusion protein has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:3.
- SEQ ID NO:3 is the amino acid sequence of sNPP1-Fc fusion protein.
- a preferred fusion protein of this type has the amino acid sequence of SEQ ID NO:3.
- Another preferred fusion protein comprises, from N-terminus to C-terminus, an NPP1 component comprising the cysteine-rich domain, the catalytic domain and the C-terminal region of human NPP1; a linker (e.g., (Gly 4 Ser) 3 (SEQ ID NO:16)); and the Fc region, including hinge, of a human immunoglobulin.
- the Fc region is from human IgG1.
- Another preferred fusion protein comprises, from N-terminus to C-terminus, an NPP1 component comprising the cysteine-rich domain, the catalytic domain and the c-terminal region of human NPP1; the Fc region, including hinge or a portion thereof, of a human immunoglobulin; and a moiety targeting the fusion protein to sites of calcification.
- the Fc region is from human IgG1.
- the moiety targeting the fusion protein to sites of calcification is Asp 10 (SEQ ID NO:18). More preferably, the Fc region is from human IgG1 and the moiety targeting the fusion protein to sites of calcification is Asp 10 (SEQ ID NO:18).
- the fusion protein has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:4.
- a preferred fusion protein of this type has the amino acid sequence of SEQ ID NO:4.
- Another preferred fusion protein comprises, from N-terminus to C-terminus, an NPP1 component comprising the cysteine-rich domain, the catalytic domain and the c-terminal region of human NPP1; a linker (e.g., (Gly 4 Ser) 3 (SEQ ID NO:16)); the Fc region, including hinge or a portion thereof, of a human immunoglobulin; and a moiety targeting the fusion protein to sites of calcification.
- the Fc region is from human IgG1.
- the moiety targeting the fusion protein to sites of calcification is Asp 10 (SEQ ID NO:18). More preferably, the Fc region is from human IgG1 and the moiety targeting the fusion protein to sites of calcification is Asp 10 (SEQ ID NO:18).
- Another preferred fusion protein comprises, from N-terminus to C-terminus, an NPP1 component comprising a portion of the cysteine-rich domain, the catalytic domain and the c-terminal region of human NPP1; optionally a linker (e.g., (Gly 4 Ser) 3 (SEQ ID NO:16)); the Fc region, including hinge or a portion thereof, of a human immunoglobulin.
- the Fc region is from human IgG1.
- the fusion protein has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, or SEQ ID NO:12.
- Preferred fusion protein of this type have the amino acid sequence of SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11 or SEQ ID NO:12.
- a fusion protein of SEQ ID NO:3 is administered in accordance with the methods described herein.
- a fusion protein of SEQ ID NO:4 is administered in accordance with in the methods described herein.
- a fusion protein of SEQ ID NO:9 is administered in accordance with in the methods described herein.
- a fusion protein of SEQ ID NO:10 is administered in accordance with the methods described herein.
- a fusion protein of SEQ ID NO:11 is administered in accordance with the methods described herein.
- a fusion protein of SEQ ID NO:12 is administered in accordance with the methods described herein.
- Fusion proteins of the present invention can be prepared using standard methods, including recombinant techniques or chemical conjugation well known in the art. Techniques useful for isolating and characterizing the nucleic acids and proteins of the present invention are well known to those of skill in the art and standard molecular biology and biochemical manuals can be consulted to select suitable protocols for use without undue experimentation. See, for example, Sambrook et al., 1989, “Molecular Cloning: A Laboratory Manual”, 2 nd ed., Cold Spring Harbor, the content of which is herein incorporated by reference in its entirety.
- the isolated recombinant human sNPP1, fragment, and fusion proteins thereof can be produced in any useful protein expression system including, without limitation, cell culture (e.g., CHO cells, COS cells, HEK203), bacteria such as Escherichia coli ( E. coli ) and transgenic animals, including, but no limited to, mammals and avians (e.g., chickens, quail, duck and turkey).
- cell culture e.g., CHO cells, COS cells, HEK203
- bacteria such as Escherichia coli ( E. coli ) and transgenic animals, including, but no limited to, mammals and avians (e.g., chickens, quail, duck and turkey).
- avians e.g., chickens, quail, duck and turkey.
- a construct that encodes the sNPP1 and includes a suitable signal sequence e.g., from human Ig heavy chain, NPP2, NPP4, NPP7 or human serum albumin, for example
- compositions typically include a pharmaceutically acceptable carrier or excipient.
- compositions comprising such carriers, including composite molecules are formulated by well-known conventional methods (see, for example, Remington's Pharmaceutical Sciences, 14 th ed., Mack Publishing Co., Easton, Pa.), the entire teachings of which are incorporated herein by reference.
- the carrier may comprise a diluent.
- the pharmaceutical carrier can be a liquid and the fusion protein may be in the form of a solution.
- the pharmaceutical carrier can be wax, fat, or alcohol.
- the pharmaceutically acceptable carrier may be a solid in the form of a powder, a lyophilized powder, or a tablet.
- the carrier may comprise a liposome or a microcapsule.
- the pharmaceutical compositions can be in the form of a sterile lyophilized powder for injection upon reconstitution with a diluent.
- the diluent can be water for injection, bacteriostatic water for injection, or sterile saline.
- the lyophilized powder may be produced by freeze drying a solution of the fusion protein to produce the protein in dry form.
- the lyophilized protein generally has increased stability and a longer shelf life than a liquid solution of the protein.
- the present invention is further exemplified by the following examples.
- the examples are for illustrative purpose only and are not intended, nor should they be construed as limiting the invention in any manner.
- VSMCs Human Primary Vascular Smooth Muscle Cells
- VSMCs vascular smooth muscle cells
- baseline ENPP1 gene expression in human VSMCs was assessed via real time polymerase chain reaction (qRT-PCR), western blot analysis, and an assay to detect cell based ENPP1 enzymatic activity, according to the following protocols.
- GAPDH glyceraldehyde-3-phosphate dehydrogenase
- VSMCs were detached, washed in PBS and the cell lysates were prepared in lysis buffer containing 1% each of protease inhibitor, Phosphatase Inhibitor Cocktail 3 (cat #P0044; Sigma) and Phosphatase Inhibitor Cocktail 2 (cat #P5726; Sigma).
- the cell lysate was quantified and denatured, then equal amounts of the protein were loaded on 4-12% Bis polyacrylamide gels.
- the proteins in gels were electrophoretically transferred to nitrocellulose membrane using iBlot® 2 Dry Blotting System.
- Cell based ENPP1 enzyme activity was assayed using the colorimetric substrate, p-nitrophenyl thymidine 5′-monophosphate (Cat #T4510, Sigma). Cells were seeded into the well of 96 well maxisorp plate at 300,000 cells/well and 100,000 cells/well and substrate at a final concentration of 1 mM in reaction buffer (200 mM Tris-HCl pH9, 1M NaCl, 10 mM MgCl2, 0.1% (v/v) Triton-X 100) was added to the plate.
- reaction buffer 200 mM Tris-HCl pH9, 1M NaCl, 10 mM MgCl2, 0.1% (v/v) Triton-X 100
- Enzyme activity is measured the reaction product based on the ability of phosphatases to catalyze the hydrolysis of PNPP to p-nitrophenol with absorbance at 405 nm using a continuous spectrophotometric assay using in a FlexStation® Plate Reader (Molecular Devices) in a kinetic mode with 21 reads at 31 sec intervals. Standard curve was generated using ENPP1-Fc protein ranged from 0 ng/ml to 90 ng/ml. Data was analyzed at 10 minutes respectively.
- FIG. 1 depicts mRNA ( FIG. 1A ) and protein ( FIG. 1B ) ENPP1 expression in human VSMCs from six different donors, as well as enzyme activity from three of the donors ( FIG. 1C ). As shown in FIG. 1 , there was significant natural variability in baseline ENPP1 expression in human primary VSMCs.
- ENPP1 siRNA Human VSMCs (donor 3) were transfected with either ENPP1 siRNA or control siRNA.
- the following siRNA constructs were used: 1 (ENPPA (CDS) Location: 825), 2 (ENPPA (CDS) Location: 813), 3 (ENPPA (CDS) Location: 1272), 4 (ENPPA (CDS) Location: 447), and 5 (ENPPA (CDS) Location: 444).
- siRNA targets [Silencer Select ENPP1 siRNA #1: s10264 (Cat #4390824), siRNA #2 s10265 (Cat #4390824), siRNA #3: s10266 (Cat #4390824), siRNA #4: s224228 (Cat #4392420), Silencer ENPP1 siRNA #5: 144240 (Cat #AM90824); Life Technologies] to human ENPP1 or control using the Lipofectamine RNAiMAX (cat #13778500; ThermoFisher Scientific) following the manufacturer's instructions.
- Cells were seeded in 60 mm dish at density 3500 cells/0.32 cm in complete medium (Vascular Cell Basal medium ATCC #PCS-100-030 supplemented with Vascular Smooth Muscle Cell Growth Kit ATCC #PCS-100-042, ATCC). Cells were treated with either an siRNA that specifically targets human ENPP1 or a negative control siRNA at a concentration of 100 nM in OPTI-MEM (cat #31985; ThermoFisher Scientific) and incubated at 37° C. Cells were harvested 48 hours or 6 days or 11 days after transfection (cells for 6 day and 11 day time points were harvested and reseeded at 48 hours), total RNA was extracted and mRNA levels were assayed by reverse transcription and real-time PCR. Levels of ENPP1 mRNA expression are reported as percentage of inhibition in mRNA expression relative to negative-siRNA after normalization to GAPDH mRNA levels.
- ENPP1 mRNA expression was inhibited by 90% or greater relative to the negative control for all five siRNA constructs, 48 hours post siRNA transfection. Specifically, ENPP1 mRNA expression was inhibited by 91.3% using siRNA construct #1, 93.1% using siRNA construct #2, 94% using siRNA construct #3, 93.6% using siRNA construct #4, and 90.2% using siRNA construct #5, relative to the negative control. Moreover, as shown in FIG. 2B , ENPP1 mRNA expression was inhibited by 84.2% six days post siRNA transfection with construct #4 (relative to the negative control) and 74.6% eleven days post siRNA transfection with construct #4 (relative to the negative control). Accordingly, the data indicates that siRNA sufficiently silences ENPP1 expression for a prolonged period of time.
- Human VSMCs (Donor 1 and Donor 3, passage 4) were seeded in 60 mm dishes at density 0.3*10e6 cells/60 mm dish in Complete Medium (Cat #PCS-100-042, PCS-100-030; ATCC). After overnight recovery, they were transfected with ENPP1 siRNA or control siRNA in OPTI-MEM. After 48 hours, cells were harvested and reseeded at 2500 cells/well into 96 well plate. Cells were cultured in basal medium containing 2% or 5% FBS. Cell proliferation was evaluated by [3H] thymidine uptake. [3H]-thymidine was added in the last 18 hours of culture. Results are expressed as CPM ⁇ SEM. Experiments were triplicated.
- silencing of ENPP1 by siRNA increased proliferation of human primary VSMCs from these two different donors (e.g., by about 1.75 fold or greater) compared to negative control siRNA.
- siRNA ENPP1 (CDS) at 48 hrs.
- Human VSMCs (Donor 1 and Donor 3, passage 4) were seeded in 60 mm dishes at density 0.3*10e6 cells/60 mm dish in Complete Medium (Cat #PCS-100-042, PCS-100-030; ATCC). After overnight recovery, they were transfected with ENPP1 siRNA or control siRNA in OPTI-MEM. After 48 hours, cells were harvested and reseeded at 2500 cells/well into 96 well plate. Cells were cultured in basal medium containing 5% FBS for 3 or 4 days. Cells were detached and stained with AOPI at indicated time point, cell number was measured using auto cell counter Cellometer Auto 2000. Results were expressed as Cell number ⁇ SEM. Experiments were triplicated.
- results As shown in FIGS. 4A (Day 3) and 4 B (Day 4) representing Donor 3, silencing ENPP1 using siRNA increased cell growth in human primary VSMCs. Specifically, cell growth was at least two fold or greater 3-4 days after silencing ENPP1 using two different constructs. These results are consistent in light of results found from independent experiments, independent analysts, different constructs, same donor, and different methods.
- VSMCs Rat Primary Vascular Smooth Muscle Cells
- VSMCs Primary rat vascular smooth muscle cells
- ATCC vascular cell basal media
- collagenase type II 2 mg/ml Cat #17101-015, Gibco
- VSMCs were cultured in vascular cell basal media (ATCC #PCS-100-030) containing 5% fetal bovine serum, and growth supplements (Cat #PCS-100-042, ATCC containing with 5 ng/ml rhFGF, 5 ⁇ g/ml rh Insulin, 50 ⁇ g/ml Ascorbic acid, 10 mM L-gutamine, 5 ng/ml rhEGF, penicillin 10 Units/ml, streptomycin 10 ⁇ g/ml, and Amphotercin B 25 ng/ml).
- VSMCs were subcultured and used between passages 3-4.
- ENPP1 knockdown was achieved by transfection with siRNA. Specifically, transfection of VSMCs with siRNA was performed using Lipofectamine RNAiMAX (cat #13778500; ThermoFisher Scientific) according to the manufacturer's instructions.
- Rat primary VSMCs (passage 3) were seeded into 60 mm dish at density 0.3*10e6/60 mm dish in complete medium and transfected with either one of a siRNA specific targets to rat ENPP1 or negative control siRNA at a concentration of 100 nM in OPTI-MEM (cat #31985; ThermoFisher Scientific) and incubated at 37° C. Cells were harvested at 48 hours and reseeded in the wells of 6 well plate at 37500 cells/well in complete medium. Cells were then harvested at indicated time points after transfection, total RNA was extracted and mRNA levels were assayed by reverse transcription and real-time PCR. Levels of ENPP1 mRNA expression are reported as percentage of inhibition in mRNA expression relative to negative-siRNA after normalization to GAPDH mRNA levels (see FIG. 5A ).
- Rat VSMCs (passage 3) were transfected with either ENPP1 siRNA or control siRNA for 48 hours, then seeded into the wells of 6-well plate at 37500 cells/well (2 wells per condition), the cells were stimulated with Complete Medium (Cat #PCS-100-042, PCS-100-030; ATCC). Cells were detached at indicated time points and stained with AOPI, cell number was measured using auto cell counter, Cellometer Auto 2000. Results are expressed as Cell number ⁇ SEM (see FIG. 5B ). Experiments were triplicated.
- ENPP1 enzyme activity was assayed using the colorimetric substrate, p-nitrophenyl thymidine 5′-monophosphate (Cat #T4510, Sigma). Cells were seeded into the well of 96 well plate at 20000 cells/well and substrate at 1 mM p-nitrophenyl thymidine 5′-monophosphate in reaction buffer was added. Enzyme activity was measured the reaction product based on the ability of phosphatases to catalyze the hydrolysis of PNPP to p-nitrophenol with absorbance at 405 nm using a continuous spectrophotometric assay using in a FlexStation® Plate Reader (Molecular Devices) in a kinetic mode with 21 reads at 31 sec intervals. Standard curve was generated using ENPP1-Fc protein ranged from Ong/ml to 90 ng/ml. Data was analyzed using SoftMax Pro software at 10 minutes. ENPP1 activity in each sample was calculated based on the standards (see FIG. 5C ).
- siRNA silencing of ENPP1 was robust and durable ( FIG. 5A ), increased cell growth ( FIG. 5B ), and decreased enzyme activity ( FIG. 5C ).
- Rat VSMCs (p3) were transfected with either rat ENPP1-siRNA or control siRNA for 48 hrs and then seeded into wells of 96-well plate at 2500 cells/well. Cells were then cultured in complete medium in the presence and absence of Adenosine monophosphate (AMP) (Cat #A1752, Sigma), adenosine (A4036, Sigma) or PPi (Cat #71515, Sigma). Cell proliferation was evaluated on day 3 by [3H] thymidine uptake. Results are expressed as CPM ⁇ SEM. Experiments were triplicated.
- AMP Adenosine monophosphate
- A4036 adenosine
- PPi Cat #71515, Sigma
- adenosine and AMP inhibited proliferation in rat VSMCs that were knocked down with ENPP1 and without regulated ENPP1.
- PPi did not affect proliferation in rat VSMCs ( FIG. 6B ).
- Rat VSMCs (p3) were transfected with either rat ENPP1-siRNA or control siRNA for 48 hrs., then seeded into wells of 96-well plate at 2500 cells/well, cells were cultured in complete medium in the presence and absence of Etidronate (Cat #P5248, Sigma) at indicated concentration. Cell proliferation was evaluated on day 3 by [3H] thymidine uptake. Results are expressed as CPM ⁇ SEM. Experiments were triplicated. Data was reproducible with Zoledronate as well.
- bisphosphonate did not appear to inhibit proliferation in rat VSMCs.
- Rat VSMCs were transfected with siRNA against rat ENPP1 si206 (ENPP1 sequence start position: 462; (Cat #SASI_Rn01_00111206 NM_053535, Sigma)), rat ENPP1 sil53 (ENPP1 sequence start position: 516 (Cat #SASI_RnO2_00266153 NM_053535, Sigma)), or a negative control ( FIG. 8A ).
- cells were seeded into wells of 96-well plate at 1250 cells/well in complete medium. After 4 hours, following treatment conditions were added in complete medium: PDGF (Cat #P8953, Sigma), Cilostazol (Cat #0737, Sigma), PDGF+Cilostazol at indicated concentration.
- [3H] thymidine was added in the last 18 hours of culture.
- Cell proliferation was evaluated by [3H] thymidine uptake.
- [3H]-thymidine was added in the last 18 hours of culture. Results are expressed as CPM ⁇ SEM. Experiments were triplicated.
- silencing ENPP1 by siRNA increased proliferation in rat VSMCs, but was inconsistent between constructs.
- siRNA target rENPP1 (SASI_Rn01_00111206) Cat #PDSIRNA2D, Sigma
- Ad-mENPP1 Vector Biolab (Lot #20150616T #10; Vector Biolabs)
- Ad-rENPP1 Life Tech+Vector Biolab (Lot #20150714T #11; Vector Biolabs)
- Ad-rENPP1 GeneWiz+Vector Biolab (Lot #20150714T #9; Vector Biolabs).
- Ad-mENPP1/Ad-rENPP1 Induces Overexpression of Mouse/Rat ENPP1 mRNA Specifically in Rat VSMC
- Rat VSMCs (passage 3) were seeded in the wells of 6-well plates at 6000 cells/0.32 cm2 in complete medium. After overnight culture, the cells were transfected with siRNA targets to rat ENPP1 using Lipofectamine RNAiMAX. After 4 hours incubation, siRNA was removed and the adenoviral vector Ad-GFP (cat #1060, Vector Biolabs) was added to the cells at multiplicities of infection (MOI) dose of 400. The plates were spun at 37° C. for 1.5 hrs. at 900 g and incubated at 37° C. for next 30 minutes before removing the adenoviral particles and washing with PBS. The siRNA particles were added again and left for overnight infection. The efficacy of adenovirus infectivity was measured 45 hours after infection of Ad-GFP under a fluorescent microscope (DMI8 Leica Microsystems) at 100 ⁇ magnification.
- Co-transfection with Ad-mENPP1 and siRNA-NC The primary Rat Vascular Smooth Muscle Cells (Rat VSMC) (Passage 3) were seeded at 6000 cells/0.32 cm2 in a 6 well dish in complete medium (Vascular Cell Basal medium ATCC #PCS-100-030 supplemented with Vascular Smooth Muscle Cell Growth Kit ATCC #PCS-100-042). After overnight recovery of the cells, the cells were infected with siRNA particles using Lipofectamine RNAiMAX (cat #13778500; ThermoFisher Scientific) diluted in OPTI-MEM (cat #31985; ThermoFisher Scientific) containing 0.25% FBS.
- Lipofectamine RNAiMAX cat #13778500; ThermoFisher Scientific
- OPTI-MEM cat #31985; ThermoFisher Scientific
- Ad-rENPP1 Lot #20150714T #9; Vector Biolabs
- Ad-rENPP1 Lot #20150714T #11; Vector Biolabs
- Ad-mENPP1 Lot #20150616T #10; Vector Biolabs
- the plates were spun at 37° C. for 1.5 hrs at 900 g and incubated at 37° C. for next 30 minutes before removing the adenoviral particles and washing with PBS.
- the siRNA particles were added again and left for overnight infection. Cells were harvested 48 hours after infection with Ad-mENPP1.
- RNA isolation and reverse transcription were performed using the TaqMan® Gene Expression Cells-to-CTTM Kit (Cat #AM1729, Thermofisher Scientific) as per manufacturer's instructions in a 96-well plate format, 96 hours post co-transfection.
- the resulting cDNA is amplified using the TaqMan Universal PCR Master Mix and detected by real-time PCR using QuantStudioTM 7 Flex System in a 384 well-plate.
- TaqMan probes for rat ENPP1 (AJKAK71), Menpp1 (Mm00501088_m1) and housekeeping gene, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) Rn01775763_g1 were obtained from ThermoFisher Scientific.
- Target gene expression level was normalized by GAPDH level in each sample and Relative expression level was calculated using ⁇ Ct method.
- the Rat ENPP1 (AJKAK71) taqman probe was custom designed to detect the rENPP1 in the adenoviral vector cassette while the mouse ENPP1 (Mm00501088_m1) taqman probe was a premade probe from ThermoFisher Scientific.
- Ad-mENPP1/Ad-rENPP1 induced overexpression of Mouse/Rat ENPP1 messenger RNA specifically in rat VSMC. Nearly 100% of VSMCs expressed high level GFP 45 hrs. after co-transfection with siRNA and Ad-GFP ( FIG. 9B ). Moreover, there was specific overexpression of mouse and rat ENPP1 in co-transfected Rat VSMC starting at the 48 hour timepoint, which and persisted through 96 hours.
- Rat VSMCs (passage 3) were seeded in the wells of 6-well plates at 6000 cells/0.32 cm 2 in complete medium. After overnight culture, the cells were transfected with siRNA targets to rat ENPP1 (si206) or siRNA negative control (siNC) using Lipofectamine RNAiMAX. After 4 hours incubation, siRNA was removed and the adenoviral vector contains mouse ENPP1 cDNA sequence (Ad-mENPP1) was added to the cells at multiplicities of infection (MOI) dose of 400. The plates were spun at 37° C. for 1.5 hrs at 900 g and incubated at 37° C.
- MOI multiplicities of infection
- siRNARat enpp1 SASI_Rn01_00111206
- mENPP1 mRNA overexpression persisted in the presence of siRNA targeting rat ENPP1, but with moderate interference of mouse ENPP1.
- Mouse ENPP1 exhibited 92% homology to rat ENPP1 at the gene level. Accordingly, siRNA knock down of rat ENPP1 expression also partially down regulated mouse ENPP1 expression.
- Rat VSMCs (passage 3) were seeded in the wells of 6-well plates at 6000 cells/0.32 cm2 in complete medium. After overnight culture, the cells were transfected with siRNA targets to rat ENPP1 (si206) or siRNA negative control (siNC) using Lipofectamine RNAiMAX. After 4 hours incubation, siRNA was removed and the adenoviral vector contains mouse ENPP1 cDNA sequence (Ad-rENPP1) was added to the cells at multiplicities of infection (MOI) dose of 400. The plates were spun at 37° C. for 1.5 hrs at 900 g and incubated at 37° C.
- MOI multiplicities of infection
- ENPP1 protein expression was successfully rescued by Ad-rENPP1.
- Rat VSMCs (passage 3) (upper) or rat non-differentiated VSMCs A-10 cells (ATCC, CRL-1476) were seeded in the wells of 6-well plates at 6000 cells/0.32 cm2 in complete medium. After overnight culture, the cells were transfected with siRNA targets to rat ENPP1 (si206) or siRNA negative control (siNC) using Lipofectamine RNAiMAX. After 4 hours incubation, siRNA was removed and the adenoviral vector contains mouse ENPP1 cDNA sequence (Ad-rENPP1) was added to the cells at multiplicities of infection (MOI) dose of 400. The plates were spun at 37° C.
- MOI multiplicities of infection
- siNC Silicone Select ENPP1 siRNA s10265 (5 nmol)
- Cat #4390824 ThermoFisher Scientific siRNA Rat enpp1 (SASI_Rn01_00111206)
- Cat #PDSIRNA2D Sigma.
- Rat VSMCs (passage 3) were seeded in the wells of 96-well plate at 6000 cells/well in complete medium. After overnight culture, the cells were transfected with siRNA targets to rat ENPP1 using Lipofectamine RNAiMAX. After 4 hours incubation, siRNA was removed and the adenoviral vector Ad-rENPP1 or Ad-mENPP1 was added to the cells at multiplicities of infection (MOI) dose of 400. The plates were spun at 37° C. for 1.5 hours at 900 g and incubated at 37° C. for next 30 minutes before removing the adenoviral particles and washing with PBS. The siRNA particles were added again and left for overnight infection. Day 1 post transfection, starvation media (0.25% FBS in basal media) was added to the starvation condition, complete media was added to non-starvation condition.
- MOI multiplicities of infection
- Results are expressed as Cell number SEM. Experiments were triplicated.
- silencing ENPP1 increased proliferation in rat VSMCs, whereas overexpression of mouse or rat ENPP1 inhibited proliferation.
- Rat VSMCs were seeded in 35 mm dish at density 75000 cells/9.6 cm 2 in completed medium. After overnight culture, the cells were transfected with siRNA targets to rat ENPP1 or siRNA negative control (siNC) for overnight. The cells were then starved with base medium contains 0.25% FBS. After 24 hrs starvation, the cells were reseeded into the well of 96 well plates at 2500 cells/0.32 cm2 in CM. After 4 hrs, once the cells adhered, ATP treatments (FLAAS, Sigma) were added at 1M in final concentration.
- siRNA targets to rat ENPP1 or siRNA negative control (siNC) for overnight.
- base medium contains 0.25% FBS. After 24 hrs starvation, the cells were reseeded into the well of 96 well plates at 2500 cells/0.32 cm2 in CM. After 4 hrs, once the cells adhered, ATP treatments (FLAAS, Sigma) were added at 1M in final concentration.
- ATP is unstable in complete medium containing 5% FBS in culture at 37° C.
- FIG. 18 heat denatured human/mouse ENPP1-Fc protein completely lost enzymatic activity.
- rat VSMCs were seeded in the well of the 6-well plates in complete medium contains 5% FBS. After overnight culture, the cells were transfected with siRNA targets to rat ENPP1 (SASI_Rn01_00111206 Cat #PDSIRNA2D, Sigma) or siRNA negative control (Silencer Select ENPP1 siRNA s10265 (5 nmol) Cat #4390824, ThermoFisher Scientific) for overnight. The cells were then starved with base medium contains 0.25% FBS.
- siRNA targets SASI_Rn01_00111206 Cat #PDSIRNA2D, Sigma
- siRNA negative control Silencer Select ENPP1 siRNA s10265 (5 nmol) Cat #4390824, ThermoFisher Scientific
- the cells were reseeded into the well of 96 well plates and cultured with completed medium contains 5% FBS in presence with 300 ⁇ M ATP and mENPP1-Fc protein ( FIG. 19A ), hENPP1-Fc ( FIG. 19B ), or hENPP1-Fc-D10 ( FIG. 19C ) protein, at the indicated concentration.
- the cultured medium was replaced daily. Proliferation was measured at day 3 using MicroBeta 3H-Thymidine incorporation. Thymidine was added in the last 18 hours.
- FIGS. 19A-C treatment with any of the ENPP1 proteins (mENPP1-Fc ( FIG. 19A ), hENPP1-Fc ( FIG. 19B ), and hENPP1-Fc-D10 ( FIG. 19C )) inhibited proliferation on rat primary VSMCs.
- Heat denatured ENPP1 had no effect on proliferation of rat VSMCs.
- Example 5 Human Induced Pluripotent Stem Cell (hiPSC)-Derived Vascular Smooth Muscle Cells (iVSMCs)
- hiPSC human induced pluripotent stem cell
- iVSMCs vascular smooth muscle cells
- the resulting cDNA is amplified using the TaqMan Universal PCR Master Mix and detected by real-time PCR using QuantStudioTM 7 Flex System.
- TaqMan probes for human ENPP1, Hs01054038_m1 and housekeeping gene, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) Hs99999905_m1 were obtained from ThermoFisher Scientific.
- Target gene expression level was normalized by GAPDH level in each sample and Relative expression level was calculated using 2- ⁇ Ct method ( FIG. 20A ).
- the membranes were blocked for 1 h in blocking buffer (Licor TBS blocking buffer (P/N 927-50000) and incubated with Rabbit pAb to human ENPP1 (PA527905 by Thermo Fischer Scientific) at 1:500 and GAPDH (14C10) Rabbit anti GAPDH mAB by Cell Signalling Technology (cat #2118L) at 1:1000 in blocking buffer containing 0.2% Tween20 overnight at 4° C., followed by a Donkey-anti-Rabbit Antibody conjugated with the fluorescent dye IRDye® 800CW (cat #926-32213; LICOR) or Donkey-anti-Rabbit Antibody conjugated with the fluorescent dye IRDye® 680RD (cat #926-68073; LICOR) for 1 h at room temperature, respectively.
- blocking buffer Lior TBS blocking buffer (P/N 927-50000) and incubated with Rabbit pAb to human ENPP1 (PA527905 by Thermo Fischer Scientific) at 1:500 and GAPDH (14
- the signals were detected using the Odyssey CLx Imaging System (LI-COR Biosciences). Signals of ENPP1 protein were normalized with level of endogenous protein GAPDH in each sample.
- the relative protein expression for the human primary iVSMC donors and the VCMC donors are depicted in FIGS. 20B and 20C , respectively.
- human primary iVSMCs expressed a high level of ENPP1.
- iPSC differentiated VSMCs p2
- ENPP1 siRNA or negative control siRNAs either consisting of a scrambled nucleotide sequence or directed to actin. After 48 hours transfection, the cells were detached, stained with AOPI and counted in auto cell counter Cellometer 2000.
- silencing ENPP1 by siRNA increased the growth of human iVSMCs as compared to both of the negative controls.
- hiPSC human induced pluripotent stem cell
- iVSMCs vascular smooth muscle cells
- the human iPSC derived VSMCs were seeded at 3500 cells/0.32 cm2 in collagen 1 coated 60 mm dishes in SmGM-2 Smooth Muscle Growth Medium-2 (Cat #CC-3182, Lonza). After overnight culture, the cells were transfected with siRNA targets to human ENPP1 using Lipofectamine RNAiMAX (cat #13778500, ThermoFisher Scientific) for overnight. Following 48 hours starvation with 0.25% FBS, cells were stained with AOPI and counted with auto cell counter, Cellometer 2000. The effect of ENPP1-Fc protein on proliferation in VSMCs was measured using 3H thymine incorporation.
- PPi is produced in an ENPP1 enzyme catalyzed reaction. PPi level was measured in culture supernatant using a radioactive assay. In order to avoid precipitation of magnesium pyrophosphate, assay components were divided into stock solutions. First, a master mix was prepared with 49.6 mM Trizma Acetate (cat #93337; Sigma-Fluka), 4.5 mM Magnesium acetate tetrahydrate (cat #63049; Sigma-Fluka); 3.5 ⁇ M NAPD-Na2 (cat #10128058001; Roche); 16.2 ⁇ M D-Glucose-1,6-diphosphate (cat #G6893; Sigma); 6.6 ⁇ M Uridine-5-Diphosphoglucose (cat #U4625; Sigma); 0.002 U/l Phosphoglucomutase (cat #P3397; Sigma); 0.003 U/l Glucose-6-phosphate dehydrogenase (cat #10165875001;
- reaction mix was filtered and 80 ⁇ l of supernatant was transferred to Iso beta plates (cat #6005040; Perkin Elmer) keeping the same plate position using 960 LTS Wide-orifice tips in 10 racks, presterilized.
- Iso beta plates catalog #6005040; Perkin Elmer
- Add 120 ⁇ l of scintillation liquid Ultima Gold catalog #6013321; Perkin Elmer
- the plated were incubated for 1 hour and then read via a micro beta counter.
- the effect of Bisphosphonate on proliferation in human iVSMCs that silenced ENPP1 was evaluated using 3H thymine incorporation.
- the human iVSMCs were seeded at 3500 cells/0.32 cm2 in collagen 1 coated 60 mm dishes in SmGM-2 Smooth Muscle Growth Medium-2 (Cat #CC-3182, Lonza). After overnight culture, the cells were transfected with siRNA targets to human ENPP1 using Lipofectamine RNAiMAX (cat #13778500, ThermoFisher Scientific) for overnight. Following 48 hours starvation with 0.25% FBS, cells were stained with AOPI and counted with auto cell counter, Cellometer 2000.
- bisphosphonate did not appear to inhibit proliferation in human iVSMCs.
- Carotid artery ligation in the mouse is a common model for investigating the response of the vasculature to mechanical injury. Damage to the vessel induces an inflammatory response and endothelial activation, resulting in smooth muscle cell proliferation and narrowing of the lumen of the vessel. Accordingly, carotid artery ligation in the tip-toe-walking (ttw) mouse, which contains a mutation in Enpp1 and serves as a model of generalized arterial calcification of infancy (GACI), was used to investigate the role of Enpp1 treatment on intimal hyperplasia.
- ttw tip-toe-walking
- FIG. 25 shows the carotid ligation procedure.
- the left carotid artery is exposed through a small incision in the neck and is ligated with a suture approximately 2 mm proximal from the carotid bifurcation.
- the animals are allowed to recover for 14 days, at which time the carotid arteries are harvested and fixed in 4% paraformaldehyde in PBS for sectioning and histological analysis.
- Serial sections of 5 ⁇ m were taken spanning 250 ⁇ m from the ligation. Every fifth section was analyzed with Von Gieson's stain and morphometric analysis was performed using Image J software.
- FIG. 26 Representative stained sections from either 100 ⁇ m (top) or 200 ⁇ m (bottom) from the ligation in WT and vehicle or rhEnpp1-treated ttw/ttw are shown from left to right, respectively.
- Von Gieson's solution stains elastic collagen fibers and distinguishes the internal (IEL) and external elastic lamina (EEL) from the lumen of the vessel (L).
- IEL internal
- EEL external elastic lamina
- the carotid ligation caused intimal hyperplasia resulting in narrowing of the lumen, with more severe narrowing closer to the ligature (100 ⁇ m) and less severe occlusion further away (200 ⁇ m).
- FIGS. 27A-C show morphometric quantitation of the results. Measurement of the circumference of the external and internal elastic lamina and the luminal border allows quantitation of the medial (M) and intimal (I) areas. The medial area, between the external and internal lamina, remained constant ( FIG. 27A ). The intimal area within the lumen showed a statistically-significant increase in ttw/ttw and vehicle-treated ttw/ttw mice relative to WT mice ( FIG. 27B ). The rhENPP1-treated ttw/ttw mice were similar to WT mice in both the intimal area and the I/M ratio, with the results again being statistically significant ( FIG. 27C ). These results support the protective effect of rhENPPP1 when administered prior to carotid ligation.
- FIG. 28A shows the degree of intimal hyperplasia present at 100 and 200 ⁇ m 7 days post carotid ligation, prior to the initiation of ENPP1 treatment. Histological assessment of the therapeutic effect of rhENPP1 when initiated at 7 days post ligation is shown in FIG. 28B , with representative sections at 100 ⁇ m (top) and 200 ⁇ m (bottom) from the ligation in vehicle-treated (left) and rhENPP1-treated (right) ttw/ttw mice presented. Despite the beginning of some intimal hyperplasia in the untreated animals at 7 days post ligation ( FIG. 28A ), treatment with rhENPP1 beginning at this point still showed benefit as the degree of luminal occlusion at both 100 and 200 ⁇ m was less than in the vehicle-treated animals 14 days post ligation.
- FIG. 29 (A-C) shows the morphometric quantitation of the data.
- the I/M ratio of vehicle-treated ttw/ttw mice was increased compared to WT mice, with the results again being statistically significant.
- the I/MV ratio of rhENPP1-treated ttw/ttw mice was between the levels of WT and vehicle-treated ttw/ttw mice, not significantly different compared to WT and vehicle-treated ttw/ttw mice, indicating a decelerating effect of rhENPP1 on already started intimal proliferation.
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Animal Behavior & Ethology (AREA)
- Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Chemical & Material Sciences (AREA)
- Pharmacology & Pharmacy (AREA)
- Veterinary Medicine (AREA)
- Public Health (AREA)
- Medicinal Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Environmental Sciences (AREA)
- Gastroenterology & Hepatology (AREA)
- Immunology (AREA)
- Epidemiology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Animal Husbandry (AREA)
- Zoology (AREA)
- Biodiversity & Conservation Biology (AREA)
- Cell Biology (AREA)
- Cardiology (AREA)
- Organic Chemistry (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Heart & Thoracic Surgery (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Urology & Nephrology (AREA)
- Vascular Medicine (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Peptides Or Proteins (AREA)
Abstract
Description
- This application is a continuation of U.S. patent application Ser. No. 16/309,047, filed on Dec. 11, 2018, which is a 35 U.S.C. 371 national stage filing of International Application No. PCT/US2017/037695, filed on Jun. 15, 2017, which claims the benefit of U.S. Provisional Application No. 62/350,936, filed on Jun. 16, 2016. The contents of any patents, patent applications, and references cited throughout this specification are hereby incorporated by reference in their entireties.
- The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on May 5, 2022, is named INZN_001_C01US_SeqListing.txt and is 89370 bytes in size.
- Myointimal proliferation (also known as myointimal hyperplasia) is an arterial wall smooth muscle cell (SMC) proliferative disorder (Painter, TA, Artif Organs. 1991 February; 15(1):42-55). Specifically, myointimal proliferation involves the migration and proliferation of vascular smooth muscle cells (VSMCs), as well as the involvement of the extracellular matrix in the intima i.e., the innermost coat of a blood vessel consisting of an endothelial layer backed by connective tissue and elastic tissue (see, e.g., Kraiss L W, Clowes A W, In: Sumpio S A N, ed. The Basic Science of Vascular Disease. New York, N.Y.: Futura Publishing; 1997:289-317; and Yang Z, Luscher T F. Eur Heart J. 1993; 14(suppl):193-197).
- Ectonucleotide pyrophosphatase pyrophosphorylase 1 (NPP1) is an ectoenzyme that cleaves ATP to produce extracellular pyrophosphate (PPi). Ectonucleotide pyrophosphatase/phosphodiesterase 1 (NPP1/ENPP1/PC-1) deficiency is a rare disease caused by mutations in NPP1, a type II transmembrane glycoprotein. NPP1 cleaves a variety of substrates, including phosphodiester bonds of nucleotides and nucleotide sugars and pyrophosphate bonds of nucleotides and nucleotide sugars. NPP1 deficiency has been associated with idiopathic infantile arterial calcification (IIAC), insulin resistance, hypophosphatemic rickets, and ossification of the posterior longitudinal ligament of the spine. IIAC, a rare autosomal recessive and nearly always fatal disorder, is characterized by calcification of the internal elastic lamina of muscular arteries and stenosis due to myointimal proliferation. There are more than 160 cases of IIAC that have been reported world-wide. The symptoms of the disease most often appear by early infancy, and the disease is lethal by 6 months of age, generally because of ischemic cardiomyopathy, and other complications of obstructive arteriopathy including renal artery stenosis.
- Although defects in the NPP1 protein have been implicated in such serious disease as IIAC, currently no treatment is available for those who are affected by the disease. Current therapeutic options have limited efficacy and undesirable and/or unacceptable side effects. Braddock, D. et al., (WO 2014/126965A2) discloses compositions and methods for treating pathological calcification and ossification by administering NPP1. Quinn, A. et al., (WO 2012/125182A1) discloses a NPP1 fusion protein to treat conditions including Generalized Arterial Calcification of Infancy (GACI), arterial calcification, insulin resistance, hypophasphatemic rickets, and ossificaiton of the posterior longitudinal ligament of the spine.
- In spite of considerable research in the field, there is a continuing need for new therapies to effectively treat NPP1-deficiencies, including myointimal proliferation disorders
- The present invention relates to uses of isolated recombinant human soluble NPP1 that lacks N-terminal cytosolic and transmembrane domains and fusion proteins thereof for the treatment of NPP1-deficiency and/or myointimal proliferation disorders. Any disorder that is characterized by myointimal proliferation is within the scope of the present invention.
- In one aspect, methods for treating a human patient having detected myointimal proliferation (e.g., as assessed by immunohistochemical detection, ultrasound (e.g., intravascular ultrasonography, carotid ultrasound, or contrast-enhanced ultrasound (CEU)), X-ray computed tomography (CT), nuclear imaging (e.g., positron emission tomography (PET) or single-photon emission computed tomography (SPECT)), optical imaging, or contrast enhanced image) are provided, the method comprising administering to the patient one or more doses of a recombinant human soluble ectonucleotide pyrophosphatase phosphodiesterase (hsNPP1), active fragment or fusion protein thereof.
- In another aspect, the methods of treating myointimal proliferation (e.g., as assessed by immunohistochemical detection) in a human patient, are provided, the method comprising: a) identifying a human patient as having myointimal proliferation and b) administering to the identified patient one or more doses of a recombinant human soluble ectonucleotide pyrophosphatase phosphodiesterase (hsNPP1), active fragment or fusion protein thereof.
- In one embodiment, a NPP1 fusion protein is administered. Preferred fusion proteins comprise and NPP1 component an Fc region of an immunoglobulin and, optionally, a targeting moiety. In one embodiment, the targeting moiety is Asp10 (SEQ ID NO: 18). In another embodiment, the targeting moiety comprises at least eight consecutive aspartic acid or glutamic acid residues (
SEQ ID NOS 20 and 21, respectively). Particular NPP1 fusion proteins for administration in accordance with the methods disclosed herein have the amino acid sequence set forth in SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11 or SEQ ID NO:12. - In another embodiment, the human patient has an NPP1 deficiency resulting in insufficient production of adenosine or adenosine monophosphate (AMP). In another embodiment, administration of a recombinant hsNPP1 according to the methods described herein is sufficient to normalize adenosine or adenosine monophosphate (AMP) production in the human patient. In another embodiment, administration of a recombinant hsNPP1 according to the methods described herein is sufficient to prevent arterial stenosis in the patient.
- Any suitable amount of the recombinant hsNPP1 can be administered to the human patient. In one embodiment, the hsNPP1 is administered in one or more doses containing about 0.5 mg/kg, 1.0 mg/kg, 2.0 mg/kg, 3.0 mg/kg, 4.0 mg/kg, 5.0 mg/kg, 6.0 mg/kg, 7.0 mg/kg, 8.0 mg/kg, 9.0 mg/kg, 10.0 mg/kg, 11.0 mg/kg, 12.0 mg/kg, 13.0 mg/kg, 14.0 mg/kg, 15.0 mg/kg, 16.0 mg/kg, 17.0 mg/kg, 18.0 mg/kg, 19.0 mg/kg, or 20.0 mg/kg. In another embodiment, the hsNPP1 is administered in one or more doses containing about 1.0 mg/kg to about 5.0 mg/kg NPP1. In another embodiment, the hsNPP1 is administered in one or more doses containing about 1.0 mg/kg to about 10.0 mg/kg NPP1.
- The time period between doses of the hsNPP1 is at least 2 days and can be longer, for example at least 3 days, at least 1 week, 2 weeks or 1 month. In one embodiment, the administration is weekly, bi-weekly, or monthly.
- The recombinant hsNPP1 can be administered in any suitable way, such as intravenously, subcutaneously, or intraperitoneally.
- The recombinant hsNPP1 can be administered in combination with one or more additional therapeutic agents. Exemplary therapeutic agents include, but are not limited to Bisphosphonate, Statins, Fibrates, Niacin, Aspirin, Clopidogrel, and varfarin. In one embodiment, the recombinant hsNPP1 and additional therapeutic agent are administered separately and are administered concurrently or sequentially. In one embodiment, the recombinant hsNPP1 is administered prior to administration of the additional therapeutic agent. In another embodiment, the recombinant hsNPP1 is administered after administration of the additional therapeutic agent. In another embodiment, the recombinant hsNPP1 and additional therapeutic agent are administered together.
- In one embodiment, the patient does not have arterial calcification. In one another, the patient does have arterial calcification.
- In another aspect uses of an isolated recombinant human sNPP1, fragment or fusion protein thereof are provided. In one embodiment, the use of an isolated recombinant human sNPP1, fragment or fusion protein thereof for the manufacture of a medicament for treating myointimal proliferation is provided. In another embodiment, the invention provides the use of an isolated recombinant human sNPP1, fragment or fusion protein thereof for treating myointimal proliferation. In one embodiment, the myointimal proliferation is not associated with arterial calcification. In another embodiment, the myointimal proliferation is associated with arterial calcification.
- Other features and advantages of the invention will be apparent from the following detailed description and claims.
-
FIGS. 1A-1C depict mRNA (FIG. 1A ) and protein (FIG. 1B ) ENPP1 expression in human primary VSMCs from six different donors, as well as enzyme activity from three of the donors (FIG. 1C ). -
FIGS. 2A-2B depict inhibition of ENPP1 mRNA expression relative to a negative control. Specifically,FIG. 2A depicts inhibition of ENPP1 mRNA expression relative to a negative control for five siRNA constructs, 48 hours post siRNA transfection.FIG. 2B depicts inhibition of ENPP1 mRNA expression relative to a negative control six and eleven days post siRNA transfection withconstruct # 4. -
FIGS. 3A-3B depict the effect of silencing ENPP1 by siRNA on proliferation of human primary VSMCs from two different donors. -
FIGS. 4A-4B depict the effect of silencing ENPP1 by siRNA on human primary VSMC cell growth on Day 3 (FIG. 4A ) and Day 4 (FIG. 4B ). -
FIGS. 5A-5B depict the effect of siRNA silencing on levels of ENPP1 mRNA expression (FIG. 5A ), cell growth (FIG. 5B ), and enzyme activity (FIG. 5C ) in rat VSMCs. -
FIGS. 6A-6B depict the effect of adenosine (FIG. 6A ), AMP (FIG. 6A ), and PPi (FIG. 6B ) on proliferation of rat VSMCs that were knocked down with ENPP1. -
FIG. 7 depicts the effect of bisphosphonate on proliferation of rat VSMCs. -
FIGS. 8A-8B depict the effect of silencing ENPP1 using different siRNA constructs on proliferation in rat VSMCs. Specifically,FIG. 8A depicts the effect of silencing ENPP1 by two siRNA on proliferation of rat VSMCs.FIG. 8B depicts inhibition of ENPP1 mRNA expression relative to a negative control for two siRNA constructs, 48 hours post siRNA transfection. -
FIG. 9A depicts the effect of Ad-mENPP1/Ad-rENPP1 on Mouse/Rat ENPP1 messenger RNA specifically in rat VSMCs.FIG. 9B shows the distribution of GFP following co-transfection of siRNA and Ad-GFP. -
FIG. 10 shows that siRNA specific to rat ENPP1 partially affected Ad-mENPP1 on Mouse ENPP1 messenger RNA specifically in co-transfection siRNA with Ad-mRNPP1 in rat VSMCs. -
FIG. 11 depicts the effect of Ad-rENPP1 on ENPP1 protein expression. -
FIGS. 12A-12B depict the effect of mENPP1 overexpression on enzyme activity in rat VSMCs (FIG. 12A ) and A10 cells (FIG. 12B ). -
FIGS. 13A-13B shows that silencing ENPP1 increases proliferation on rat VSMCs and overexpression of mouse or rat ENPP1 inhibits proliferation on rat VSMCs that either downregulated ENPP1 expression (si-rENPP1) or normal ENPP1 expression (si-NC). -
FIG. 14 depicts the effect of silencing ENPP1 and overexpression of mouse or rat ENPP1 on rat VSMCs cell growth. -
FIG. 15 shows that FBS has a negative effect on stability of ATP in cultured supernatant. CM contains 5% FBS, Starvation media contains 0.25% FBS. ATP function was reduced over 60% in CM after 30 minutes in culture at 37° C. -
FIG. 16 shows that FBS has a negative effect on stability of ATP in cultured supernatant. CM contains 5% FBS, Starvation media contains 0.25% FBS. ATP function was reduced over 87% after 2 hours in culture at 37° C. -
FIG. 17 shows that FBS has a negative effect on stability of ATP in cultured supernatant. CM contains 5% FBS, Starvation media contains 0.25% FBS. ATP function was almost entirely lost after 24 hours in culture at 37° C. -
FIGS. 18A-18B shows that heat denatured human/mouse ENPP1-Fc protein completely lost their enzymatic activity. -
FIGS. 19A-19C depicts the effect of murine ENPP1-Fc protein (FIG. 19A ), human ENPP1-Fc (FIG. 19B ) and human ENPP1-FC-D10 (FIG. 19C ) on proliferation of rat primary VSMCs. -
FIGS. 20A-20C depict ENPP1 expression by human primary VSMCs and human induced pluripotent stem cell (hiPSC)-derived vascular smooth muscle cells (iVSMCs), as assessed by qRT-PCR (FIG. 20A ) and Western Blot (FIGS. 20B-C ). -
FIG. 21 depicts the effect of silencing ENPP1 by siRNA on the growth of human induced pluripotent stem cell (hiPSC)-derived vascular smooth muscle cells (iVSMCs). -
FIGS. 22A-22C depict the effect of murine (FIG. 22A ) and human ENPP1-Fc (FIGS. 22B-22C ) protein on the proliferation of human induced pluripotent stem cell (hiPSC)-derived vascular smooth muscle cells (iVSMCs). -
FIG. 23 depicts PPi levels in the supernatant, as assessed by a PPi assay using human iVSMCs. -
FIG. 24 depicts the effect of bisphosphonate on proliferation of human iVSMCs. -
FIG. 25 is a diagram of the carotid artery ligation and sectioning for histological analysis. Five μm sections were cut spanning 250 μm from the point of ligation. Every fifth section was analyzed. -
FIG. 26 is a histological analysis (Von Gieson's stain) of sections either 100 (upper) or 200 (lower) μm from point of ligation from wild-type (WT), ttw/ttw, vehicle-treated ttw/ttw or rhENPP1-treated ttw/ttw/mice from left to right, respectively. The internal elastic lamina (IEL), external elastic lamina (EEL) and lumen (L) are indicated by arrows. The scale bar represents 100 μm. -
FIGS. 27A-27C are a morphometric quantitation of medial (FIG. 27A ) and intimal areas (FIG. 27B ), as well as the I/M ratio (FIG. 27C ). *p<0.001. -
FIGS. 28A-28B show the effect of therapeutic Enpp1 treatment on intimal hyperplasia in ttw/ttw mouse started 7 days after carotid ligation.FIG. 28A shows the degree ofintimal hyperplasia 7 days post ligation in WT and ttw/ttw mice.FIG. 28B shows the histological analysis (Von Gieson's stain) of sections either 100 (upper) or 200 (lower) μm from point of ligation from WT, vehicle-treated ttw/ttw or rhENPP1-treated ttw/ttw mice from left to right, respectively. The internal elastic lamina (IEL), external elastic lamina (EEL) and lumen (L) are indicated by arrows. The scale bar represents 100 μm. -
FIGS. 29A-29C are a morphometric quantitations of medial (FIG. 29A ) and intimal areas (FIG. 29B ), as well as the I/M ratio (FIG. 29C ) on treatment day 14. *p<0.05, **p<0.01. - Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in practice or testing of the present invention, the preferred methods and materials are described.
- For clarity, “NPP1” and “ENPP1” refer to the same protein and are used interchangeably herein.
- “About” as used herein when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass variations of ±20% or ±10%, more preferably ±5%, even more preferably ±1%, and still more preferably ±0.1% from the specified value, as such variations are appropriate to perform the disclosed methods.
- As used herein, the term “fragment”, with regard to NPP1 proteins, refers to an active subsequence of the full-length NPP1. A “fragment” of a protein or peptide can be at least about 20 amino acids in length; for example, at least about 50 amino acids in length; at least about 100 amino acids in length; at least about 200 amino acids in length; at least about 300 amino acids in length; or at least about 400 amino acids in length (and any integer value in between). The fragments may range in size from four amino acid residues to the entire amino acid sequence minus one amino acid. Thus, a protein “comprising at least a portion of the amino acid sequence of SEQ ID NO: 1” encompasses the full-length NPP1 and fragments thereof.
- An “isolated” or “purified” soluble NPP1 protein or biologically active fragment or fusion protein thereof is substantially free of cellular material or other contaminating proteins from the cell or tissue source from which the NPP1 protein, biologically active fragment or NPP1 fusion protein is derived, or substantially free from chemical precursors or other chemicals when chemically synthesized. The language “substantially free of cellular material” includes preparations of NPP1 protein, biologically active fragment, or NPP1 fusion protein in which the protein is separated from cellular components of the cells from which it is isolated or recombinantly produced. In one embodiment, the language “substantially free of cellular material” includes preparations of NPP1 protein, biologically active fragment or NPP1 fusion protein having less than about 30% (by dry weight) of non-NPP1 protein/fragment/fusion protein (also referred to herein as a “contaminating protein”), more preferably less than about 20% of non-NPP1 protein/fragment/fusion protein, still more preferably less than about 10% of non-NPP1 protein/fragment/fusion protein, and most preferably less than about 5% non-NPP1 protein/fragment/fusion protein. When the NPP1 protein, fusion protein, or biologically active fragment thereof is recombinantly produced, it is also preferably substantially free of culture medium, i.e., culture medium represents less than about 20%, more preferably less than about 10%, and most preferably less than about 5% of the volume of the protein preparation.
- Ranges: throughout this disclosure, various aspects of the invention can be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the range.
- As used herein, the term “subject” encompasses mammals and non-mammals. Examples of mammals include, but are not limited to, humans, chimpanzees, apes monkeys, cattle, horses, sheep, goats, swine, rabbits, dogs, cats, rats, mice, guinea pigs, and the like. Examples of non-mammals include, but are not limited to, birds, fish and the like.
- As used herein, the term “therapeutically effective amount” refers to a nontoxic but sufficient amount of an agent (e.g., hsNPP1 proteins) which, as compared to a corresponding subject who has not received such amount, results in improved treatment, healing, prevention, or amelioration of a disease, disorder, or side effect, or a decrease in the rate of advancement of a disease or disorder. The term also includes within its scope amounts effective to enhance normal physiological function.
- As used herein, “myointimal proliferation” (also referred to as “myointimal hyperplasia”) refers to abnormal proliferation of the smooth muscle cells of the vascular wall (e.g., the intima of a blood vessel).
- The term “treating” includes the application or administration of the NPP1 proteins, active fragments and fusion proteins of the invention to a subject, or application or administration of NPP1 proteins, active fragments and fusion proteins of the invention to a subject who has myointimal proliferation, with the purpose of curing, healing, alleviating, relieving, altering, remedying, ameliorating, preventing, improving, or affecting the disease or disorder. The term “treating” refers to any indicia of success in the treatment or amelioration of an injury, pathology or condition, including any objective or subjective parameter such as abatement; remission; diminishing of symptoms or making the injury, pathology or condition more tolerable to the subject; slowing in the rate of degeneration or decline; making the final point of degeneration less debilitating; or improving a subject's physical or mental well-being. Treatment may be therapeutic or prophylactic. The treatment or amelioration of symptoms can be based on objective or subjective parameters; including the results of a physical examination.
- The present invention relates to uses of an isolated recombinant human soluble NPP1 (“sNPP1”) which lacks an N-terminal portion (i.e., lacking cytosolic and transmembrane domains) and fusion proteins thereof for the treatment of NPP1-associated diseases, such as myointimal proliferation.
- The subject can be a human patient having deficiencies in NPP1 activity (NPP1 deficiency). In one embodiment, the patient exhibits low levels of pyrophosphate and/or suffers from a disease or disorder associated with low levels of pyrophosphate. In another embodiment, the human patient has an NPP1 deficiency resulting in insufficient production of adenosine or adenosine monophosphate (AMP).
- Generally, the dosage of fusion protein administered to a subject will vary depending upon known factors such as age, health and weight of the recipient, type of concurrent treatment, frequency of treatment, and the like. Usually, a dosage of active ingredient (i.e., fusion protein) can be between about 0.0001 and about 50 milligrams per kilogram of body weight. Precise dosage, frequency of administration and time span of treatment can be determined by a physician skilled in the art of administration of therapeutic proteins.
- A preferred embodiment of the present invention involves a method of treating myointimal proliferation, which includes the step of identifying a human patient as having myointimal proliferation and administering to the identified patient a therapeutically effective amount of a recombinant human soluble ectonucleotide pyrophosphatase phosphodiesterase (hsNPP1), active fragment or fusion protein thereof.
- As defined herein, a therapeutically effective amount of protein (i.e., an effective dosage) ranges from about 0.001 to 50 mg/kg body weight. The skilled artisan will appreciate that certain factors may influence the dosage required to effectively treat a subject, including but not limited to the severity of the disease, previous treatments, the general health and/or age of the subject, and other diseases present. Moreover, treatment of a subject with a therapeutically effective amount of protein can include a single treatment or, preferably, can include a series of treatments. It will also be appreciated that the effective dosage of protein used for treatment may increase or decrease over the course of a particular treatment.
- As defined herein, a therapeutically effective amount of protein or polypeptide (i.e., an effective dosage) ranges from about 0.001 to 50 mg/kg body weight, preferably about 0.01 to 25 mg/kg body weight, more preferably about 0.1 to 20 mg/kg body weight, and even more preferably about 1 to 10 mg/kg, 2 to 9 mg/kg, 3 to 8 mg/kg, 4 to 7 mg/kg, or 5 to 6 mg/kg body weight. In one embodiment, the hsNPP1 is administered in one or more doses containing about 0.1 mg/kg, 0.2 mg/kg, 0.3 mg/kg, 0.4 mg/kg, 0.5 mg/kg, 1.0 mg/kg, 2.0 mg/kg, 3.0 mg/kg, 4.0 mg/kg, 5.0 mg/kg, 6.0 mg/kg, 7.0 mg/kg, 8.0 mg/kg, 9.0 mg/kg, 10.0 mg/kg, 11.0 mg/kg, 12.0 mg/kg, 13.0 mg/kg, 14.0 mg/kg, 15.0 mg/kg, 16.0 mg/kg, 17.0 mg/kg, 18.0 mg/kg, 19.0 mg/kg, 20.0 mg/kg, 21.0 mg/kg, 22.0 mg/kg, 23.0 mg/kg, 24.0 mg/kg, 25.0 mg/kg, 26.0 mg/kg, 27.0 mg/kg, 28.0 mg/kg, 29.0 mg/kg, 30.0 mg/kg, 31.0 mg/kg, 32.0 mg/kg, 33.0 mg/kg, 34.0 mg/kg, 35.0 mg/kg, 36.0 mg/kg, 37.0 mg/kg, 38.0 mg/kg, 39.0 mg/kg, 40.0 mg/kg, 41.0 mg/kg, 42.0 mg/kg, 43.0 mg/kg, 44.0 mg/kg, or 45.0 mg/kg. In another embodiment, about 0.5 to about 30 mg, about 0.5 to about 20 mg, about 0.5 to about 10 mg, or about 0.5 to about 5 mg are administered to the patient. In another embodiment, the hsNPP1 is administered in one or more doses containing about 1.0 mg/kg to about 5.0 mg/kg hsNPP1. In another embodiment, the hsNPP1 is administered in one or more doses containing about 1.0 mg/kg to about 10.0 mg/kg hsNPP1. The skilled artisan will appreciate that certain factors may influence the dosage required to effectively treat a subject, including, but not limited to, the severity of the disease or disorder, previous treatments, the general health and/or age of the subject, and other diseases present.
- Moreover, treatment of a subject with a therapeutically effective amount of a protein, polypeptide, or antibody can include a single treatment or, preferably, can include a series of treatments. In one embodiment, in the range of between about 0.1 to 20 mg/kg body weight, one time per week, twice per week, once in about 10 days, once in about 12 days, once in about 14 days, once in about 17 days, once in about 20 days, once in about about 25 days, or once in about 30 days. In one embodiment, the time period between doses of the hsNPP1 is at least 2 days and can be longer, for example at least 3 days, at least 1 week, 2 weeks or 1 month. In another embodiment, the therapeutically effective dose of sNPP1, biologically active fragment or fusion protein thereof is administered to a patient between one time every 5 days and one time every 30 days for a period of time determined by a practitioner of skill in the art of medical sciences. In another embodiment, the period of time will be the remainder of the patient's life span. In another embodiment, the dosing frequency is between one time every 5 days and one time every 25 days. In another embodiment, the dosing frequency is between one time every 5 days and one time every 21 days. In another embodiment, the dosing frequency is between one time every 7 days and one time every 14 days. hsNPP1, biologically active fragment or fusion protein thereof can be administered one time every 5 days, one time every 6 days, one time every 7 days, one time every 8 days, one time every 9 days, one time every 10 days, one time every 11 days, one time every 12 days, one time every 13 days, or one time every 14 days. In some embodiments, hsNPP1, biologically active fragment or fusion protein thereof is administered about weekly. In other embodiments, sNPP1, biologically active fragment or fusion protein thereof is administered about bi-weekly. In one embodiment, the dosing frequency is one time about 30 days. It will also be appreciated that the effective dosage of soluble sNPP1 protein, biologically active fragment or fusion protein thereof used for the treatment may increase or decrease over the course of a particular treatment.
- In one embodiment, about 1 mg/kg of hsNPP1, biologically active fragment or fusion protein is administered to the patient once a week. In one embodiment, about 2 mg/kg of hsNPP1, biologically active fragment or fusion protein is administered to the patient once a week. In one embodiment, about 3 mg/kg of hsNPP1, biologically active fragment or fusion protein is administered to the patient once a week. In one embodiment, about 4 mg/kg of sNPP1, biologically active fragment or fusion protein is administered to the patient once a week. In one embodiment, about 5 mg/kg of hsNPP1, biologically active fragment or fusion protein is administered to the patient once a week. In one embodiment, about 6 mg/kg of hsNPP1, biologically active fragment or fusion protein is administered to the patient once a week. In one embodiment, about 7 mg/kg of hsNPP1, biologically active fragment or fusion protein is administered to the patient once a week. In one embodiment, about 8 mg/kg of hsNPP1, biologically active fragment or fusion protein is administered to the patient once a week. In one embodiment, about 9 mg/kg of hsNPP1, biologically active fragment or fusion protein is administered to the patient once a week. In one embodiment, about 10 mg/kg of hsNPP1, biologically active fragment or fusion protein is administered to the patient once a week.
- hsNPP1, biologically active fragment or fusion protein can be administered by, for example, subcutaneous injections, intramuscular injections, and intravenous (IV) infusions or injections.
- In one embodiment, hsNPP1, biologically active fragment or fusion protein is administered intravenously by IV infusion by any useful method. In one example, hsNPP1, biologically active fragment or fusion protein can be administered by intravenous infusion through a peripheral line. In another example, hsNPP1, biologically active fragment or fusion protein can be administered by intravenous infusion through a peripherally inserted central catheter.
- In another embodiment, hsNPP1, biologically active fragment or fusion protein is administered intravenously by IV injection.
- In another embodiment, hsNPP1, biologically active fragment or fusion protein is administered via intraperitoneal injection.
- In another embodiment, hsNPP1, biologically active fragment or fusion protein is administered by subcutaneous injections.
- In another embodiment, hsNPP1, biologically active fragment or fusion protein is administered by intramuscular injections.
- In still another embodiment, hsNPP1, biologically active fragment or fusion protein is administered via a pharmaceutically acceptable capsule of the therapeutic protein. For example, the capsule can be an enteric-coated gelatin capsule.
- In one embodiment, the method involves administering the soluble NPP1 protein or NPP1 fusion protein of the invention alone, or in combination with other agent(s). Exemplary therapeutic agents include, but are not limited to bisphosphonate, Statins, Fibrates, Niacin, Aspirin, Clopidogrel, and varfarin. In one embodiment, the method involves administering an NPP1 protein or an NPP1 fusion protein of the invention as therapy to compensate for reduced or aberrant NPP1 expression or activity in the subject having an NPP1-deficiency or other associated disease or disorder. In one embodiment, the isolated sNPP1 proteins, fragments, and fusion proteins can be administered before, after or concurrently with the agent or can be co-administered with other known therapies. Co-administration of the isolated sNPP1 proteins, fragments, and fusion proteins of the present invention with other therapeutic agents may provide two agents which operate via different mechanisms which yield an increased therapeutic effect. Such co-administration can solve problems due to development of resistance to drugs. In particular aspects, this disclosure relates to a method for reducing myointimal proliferation in a subject in need thereof.
- The methods described herein provide a way to reduce myointimal proliferation in a subject (e.g., human patient). In one embodiment, the human patient has an NPP1 deficiency resulting in insufficient production of adenosine or adenosine monophosphate (AMP). In another embodiment, administration of a recombinant hsNPP1 according to the methods described herein is sufficient to normalize adenosine or adenosine monophosphate (AMP) production in the human patient. In another embodiment, administration of a recombinant hsNPP1 according to the methods described herein is sufficient to prevent arterial stenosis in the patient.
- sNPP1
- The present invention employs soluble NPP1 (e.g., hsNPP1) that has a biologically active NPP1 domain of NPP1 (i.e., NPP1 components that contain at least one extracellular catalytic domain of naturally occurring NPP1 for the pyrophosphatase and/or phosphodiesterase activity). The soluble NPP1 proteins of the invention comprise at least the NPP1 domain essential to carry out the pyrophosphatase and/or phosphodiesterase activity.
- In one embodiment, the soluble NPP1, fragment, and fusion proteins thereof can form functional homodimers or monomer. In another embodiment, a soluble NPP1 protein or NPP1 fusion protein thereof can be assayed for pyrophosphatase activity as well as the ability to increase pyrophosphate levels in vivo.
- Described herein are various amino acid sequences of soluble NPP1 compounds, fusion partners and fusion proteins that are suitable for use according to the methods provided herein. SEQ ID NO:5 shows the amino acid sequences of a soluble NPP1 containing amino acids from 107 to 925 of SEQ ID NO:1. SEQ ID NO:6 shows the amino acid sequence of a soluble NPP1 containing amino acids from 187 to 925 of SEQ ID NO:1. SEQ ID NO:7 shows the amino acid sequence of the Fc region of human IgG1 including the hinge region. SEQ ID NO:8 shows the amino acid sequence of the Fc of human IgG1 including a partial hinge region. SEQ ID NO:9 shows the amino acid sequence of a NPP1-Fc fusion protein. The NPP1 component contains SEQ ID NO:5, and the Fc sequence includes the hinge region. SEQ ID NO:10 shows the amino acid sequence of a NPP1-Fc fusion protein. The soluble NPP1 contains SEQ ID NO:5, and the Fc sequence includes the partial hinge region. SEQ ID NO:1 shows the amino acid sequence of a NPP1-Fc fusion protein. The soluble NPP1 contains SEQ ID NO:6, and the Fc sequence includes the hinge region. SEQ ID NO:12 shows the amino acid sequence of a NPP1-Fc fusion protein. The soluble NPP1 contains SEQ ID NO:6, and the Fc sequence includes the partial hinge region.
- Preferred soluble NPP1 proteins and NPP1 fusion proteins of the invention are enzymatically active in vivo (e.g., human). In one embodiment, the soluble protein comprises amino acid sequence having at least 60, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% sequence identity to the following sequence:
-
(SEQ ID NO: 2) PSCAKEVKSCKGRCFERTFGNCRCDAACVELGNCCLDYQETCIEPEHTW TCNKFRCGEKRLTRSLCACSDDCKDKGDCCINYSSVCQGEKSWVEEPCE SINEPQCPAGFETPPTLLFSLDGFRAEYLHTWGGLLPVISKLKKCGTYT KNMRPVYPTKTFPNHYSIVTGLYPESHGIIDNKMYDPKMNASFSLKSKE KFNPEWYKGEPIWVTAKYQGLKSGTFFWPGSDVEINGIFPDIYKMYNGS VPFEERILAVLQWLQLPKDERPHFYTLYLEEPDSSGHSYGPVSSEVIKA LQRVDGMVGMLMDGLKELNLHRCLNLILISDHGMEQGSCKKYIYLNKYL GDVKNIKVIYGPAARLRPSDVPDKYYSFNYEGIARNLSCREPNQHFKPY LKHFLPKRLHFAKSDRIEPLTFYLDPQWQLALNPSERKYCGSGFHGSDN VFSNMQALFVGYGPGFKHGIEADTFENIEVYNLMCDLLNLTPAPNNGTH GSLNHLLKNPVYTPKHPKEVHPLVQCPFTRNPRDNLGCSCNPSILPIED FQTQFNLTVAEEKIIKHETLPYGRPRVLQKENTICLLSQHQFMSGYSQD ILMPLWTSYTVDRNDSFSTEDFSNCLYQDFRIPLSPVHKCSFYKNNTKV SYGFLSPPQLNKNSSGIYSEALLTTNIVPMYQSFQVIWRYFHDTLLRKY AEERNGVNVVSGPVFDFDYDGRCDSLENLRQKRRVIRNQEILIPTHFFI VLTSCKDTSQTPLHCENLDTLAFILPHRTDNSESCVHGKHDSSWVEELL MLHRARITDVEHITGLSFYQQRKEPVSDILKLKTHLPTFSQED - SEQ ID NO:2 is the amino acid sequence of a sNPP1 that contains the cysteine-rich region, catalytic region and c-terminal region.
- Any desired enzymatically active form of soluble NPP1 can be used in the methods described herein. The enzymatically active sNPP1 can increase PPi levels in suitable enzymatic assays, and can be assayed for pyrophosphatase activity, phosphodiesterase activity, or pyrophosphatase and phosphodiesterase activity. Typically, the sNPP1 contains at least an NPP1 component that lacks the N-terminal cytosolic and transmembrane domains of naturally occurring transmembrane NPP1.
- SEQ ID NO:1 is the amino acid sequence of wild-type NPP1 protein. The cytosolic and transmembrane regions are underlined. The potential N-glycosylation sites are in bold. The amino acid motif “PSCAKE” (SEQ ID NO:17) in bold is the start of a soluble NPP1 which includes the cysteine rich region.
- In preferred aspects, the NPP1 component contains the cysteine-rich region (amino acids 99-204 of SEQ ID NO:1) and the catalytic region (amino acids 205-591 of SEQ ID NO:1) of naturally occurring human NPP1. Typically, the NPP1 component also includes the C-terminal region (amino acids 592 to 925 of SEQ ID NO:1), and has the amino acid sequence of SEQ ID NO:2. However, the C-terminal region can be truncated if desired. Accordingly, preferred NPP1 components include the cysteine-rich region and catalytic region of human NPP1 (amino acids 99-591 of SEQ ID NO:1) or the cysteine-rich region, the catalytic region and the C-terminal region of human NPP1 (SEQ ID NO:2). Other preferred NPP1 components contain only a portion of the cysteine-rich domain and have the sequence of amino acids 107 to 925 of SEQ ID NO:1 or amino acids 187 to 925 of SEQ ID NO:1.
- The cysteine rich region of NPP1 (i.e., amino acids 99 to 204 of SEQ ID NO: 1) can facilitate dimerization of the sNPP1. The sNPP1, including fusion proteins, can be in the form of a monomer of functional homodimer.
- The amino acid sequence of the NPP1 component can be a variant of the naturally occurring NPP1 sequence, provided that the NPP1 component is enzymatically active. NPP1 variants are enzymatically active and have at least 80%, at least 85%, at least 90%, at least 95% and more preferably at least 96% amino acid sequence identity to the corresponding portion of human NPP1 (e.g., over the length of the cysteine-rich region, the catalytic region, the c-terminal region, the cysteine-rich region plus the catalytic region, the cystein-rich region plus the catalytic region plus the c-terminal region. Preferred NPP1 variants have at least 90%, preferably at least 95%, more preferably at least 97% amino acid sequence identity to (i) the amino acid sequence of residues 205-591 of SEQ ID NO: 1, (ii) the amino acid sequence of residues 99-591 of SEQ ID NO:1, (iii) the amino acid sequence of residues 99-925 of SEQ ID NO:1, (iv) the amino acid sequence of residues 107-925 of SEQ ID NO:1, or (v) the amino acid sequence of residues 187-925 of SEQ ID NO:1. Suitable positions for amino acid variation are well-known from NPP1 structural studies and analysis of disease-associated mutations in NPP1. For example, substitution of the following amino acids occurs in certain disease-associated mutations that reduce NPP1 enzymatic activity, and variations of the amino acids at these positions should be avoided: Ser216, Gly242, Pro250, Gly266, Pro305, Arg349, Tyr371, Arg456, Tyr471, His500, Ser504, Tyr513, Asp538, Tyr570, Lys579, Gly586; Tyr659, Glu668, Cys726, Arg774, His777, Asn792, Asp804, Arg821, Arg888, and Tyr901. (See, e.g., Jansen, S. et al., Structure 20:1948-1959 (2012)).
- In one embodiment, the soluble NPP1 protein can be a fusion protein recombinantly fused or chemically bonded (e.g., covalent bond, ionic bond, hydrophobic bond and Van der Waals force) to a fusion partner. In another embodiment, the fusion protein has at least 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity to SEQ ID NO: 3 or SEQ ID NO:4. SEQ ID NO:4 is the amino acid sequence of sNPP1-Fc-D10 (SEQ ID NO: 4). The Fc sequence is underlined.
- To determine the percent identity of two amino acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second amino acid or nucleic acid sequence for optimal alignment and non-homologous sequences can be disregarded for comparison purposes). In a preferred embodiment, the length of a reference sequence aligned for comparison purposes is at least 30%, preferably at least 40%, more preferably at least 50%, even more preferably at least 60%, and even more preferably at least 70%, 80%, or 90% of the length of the reference sequence (e.g., sNPP1 amino acid sequence of SEQ ID NO:2; amino acids 107-925 of SEQ ID NO:1 or amino acids 187-925 of SEQ ID NO:1). The amino acid residues or nucleotides at corresponding amino acid positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position (as used herein amino acid is equivalent to amino acid or nucleic acid “homology”). The percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences.
- The comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm. In a preferred embodiment, the percent identity between two amino acid sequences is determined using the Needleman and Wunsch (
J Mol Biol 1970, 48, 444-453) algorithm which has been incorporated into the GAP program in the GCG software package (available at www.gcg.com), using either a Blosum 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6. In another embodiment, the percent identity between two amino acid is determined using the algorithm of E. Meyers and W. Miller (CABIOS, 1989, 4, 11-17) which has been incorporated into the ALIGN program (version 2.0 or 2.0U), using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4. - The sNPP1 can consist of or consist essentially of an NPP1 component as described herein. Alternatively, the sNPP1 can be in the form of a fusion protein that contains an NPP1 component and one or more other polypeptides, referred to as fusion partners, optionally through a suitable linker in each instance, or in the form of a conjugate between an NPP1 component and another molecule (e.g., PEG). When the sNPP1 is in the form of a fusion protein, each fusion partner is preferably located c-terminally to the NPP1 component. Without wishing to be bound by any particular theory, it is believed that fusion proteins that contain an NPP1 component that contains the cysteine-rich region and catalytic region, and one or more fusion proteins that are located c-terminally to the NPP1 component, are preferred over other configurations of NPP1 fusion proteins because they can be expressed at sufficient levels and are sufficiently stable to be used as therapeutic proteins.
- Any suitable fusion partner can be included in the fusion protein. Advantageously, a number of fusion partners are well-known in the art that can provide certain advantages, such as reduced aggregation and immunogenicity, increased the solubility, improved expression and/or stability, and improved pharmacokinetic and/or pharmacodynamics performance. See, e.g., Strohl, W. R. BioDrugs 29:215-239 (2015). For example, it is well-known that albumin, albumin fragments or albumin variants (e.g., human serum albumin and fragments or variants thereof) can be incorporated into fusion proteins and that such fusion proteins can be easily purified, stable and have an improved plasma half-life. Suitable albumin, albumin fragments and albumin variants that can be used in the sNPP1 fusion proteins are disclosed, for example in WO 2005/077042A2 and WO 03/076567A2, each of which is incorporated herein by reference in its entirety. Fusions to human transferrin are also known to improve half-life. See, e.g., Kim B J et al., J Pharmacol Expr Ther 334(3):682-692 (2010); and WO 2000/020746. Peptides that bind to albumin or transferrin, such as antibodies or antibody fragments, can also be used. See, e.g., EP 0486525 B1, U.S. Pat. No. 6,267,964 B1, WO 04/001064A2, WO 02/076489A1, WO 01/45746, WO 2006/004603, and WO 2008/028977. Similarly immunoglobulin Fc fusion proteins are well-known. See, e.g., Czajkowsky D M et al., EMBO Mol Med 4(10):1015-1028 (2012), U.S. Pat. Nos. 7,902,151; and 7,858,297, the entire teachings of which are incorporated herein by reference in their entirety. The fusion protein can also include a CTP sequence (see also, Fares et al.,
Endocrinol 2010, 151, 4410-4417; Fares et al., Proc Natl Acad Sci 1992, 89, 4304-4308; and Furuhashi et al.,Mol Endocrinol 1995, 9, 54-63). Preferably, the fusion partner is the Fc of an immunoglobulin (e.g., Fc or human IgG1). The Fc can include CH1, CH2 and CH3 of human IgG1, and optionally the human IgG1 hinge region (EPKSCDKTHTCPPCP (SEQ ID NO:13)) or a portion of the human IgG1 hinge region (e.g., DKTHTCPPCP (SEQ ID NO:14) or PKSCDKTHTCPPCP (SEQ ID NO:15)) if desired. In some fusion proteins, the Fc can include CH2 and CH3 of human IgG1, or the Fc of human IgG2 or human IgG4, if desired. Preferably, the sNPP1 fusion protein comprises an NPP1 component and a peptide that increases the half-life of the fusion protein, most preferably the Fc of an immunoglobulin (e.g., Fc or human IgG1). As used herein, a “protein that increases the half-life of the fusion protein” refers to a protein that, when fused to a soluble NPP1 or biologically active fragment, increases the half-life of the soluble NPP1 polypeptide or biologically active fragment as compared to the half-life of the soluble NPP1 polypeptide, alone, or the NPP1 biologically active fragment, alone. In one embodiment, the half-life of the NPP1 fusion protein is increased 50% as compared to the half-life of the NPP1 polypeptide or biologically active fragment, alone. In another embodiment, the half-life of the NPP1 fusion protein is increased 60% as compared to the half-life of the NPP1 polypeptide or biologically active fragment, alone. In another embodiment, the half-life of the NPP1 fusion protein is increased 70% as compared to the half-life of the NPP1 polypeptide or biologically active fragment, alone. In another embodiment, the half-life of the NPP1 fusion protein is increased 80% as compared to the half-life of the NPP1 polypeptide or biologically active fragment, alone. In another embodiment, the half-life of the NPP1 fusion protein is increased 90% as compared to the half-life of the NPP1 polypeptide or biologically active fragment, alone. - In another embodiment, the half-life of the NPP1 fusion protein is increased 2 fold, 3 fold, 4 fold, 5 fold, 6 fold, 7 fold, 8 fold, 9 fold, or 10 fold as compared to the half-life of the NPP1 polypeptide or biologically active fragment, alone. Methods for determining the half-life of a protein or fusion protein are well known in the art. For example, Zhou et al., Determining Protein Half-Lives, Methods in Molecular Biology 2004, 284, 67-77 discloses numerous methods for testing of the half-life of a protein. If desired, the fusion protein can be conjugated to polymers or other suitable compounds that extend half-life, such as polyethylene glycol (PEG), can be conjugated to the NPP1 fusion proteins.
- In one embodiment, the peptide which increases the half-life of the fusion protein is a CTP sequence (see also, Fares et al., 2010, Endocrinol., 151(9):4410-4417; Fares et al., 1992, Proc. Natl. Acad. Sci, 89(10):4304-4308; and Furuhashi et al., 1995, Molec. Endocrinol., 9(1):54-63).
- In another embodiment, the peptide which increases the half-life of the fusion protein is an Fc domain of an Ig.
- Fusion partners may also be selected to target the fusion protein to desired sites of clinical or biological importance (e.g., site of calcification). For example, peptides that have high affinity to the bone are described in U.S. Pat. No. 7,323,542, the entire teachings of which are incorporated herein by reference. Peptides that can increase protein targeting to calcification sites can contain a consecutive stretch of at least about 4 acidic amino acids, for example, glutamic acids or aspartic acids. Typically, the peptide that targets the fusion protein to calcification sites will comprise between 4 and 20 consecutive acidic amino acids, for example 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 consecutive amino acids selected from glutamic acid and aspartic acid. The peptide can consist solely of glutamic acid residues, solely of aspartic acid residues, or be a mixture of glutamic acid and aspartic acid residues. A particularly preferred moiety for targeting to sights of calcification is Asp10 (SEQ ID NO:18).
- In one embodiment, the NPP1 fusion protein of the invention comprises an NPP1 polypeptide and a moiety that increase protein targeting to calcification sites such as a consecutive stretch of acidic amino acids, for example, glutamic acids or aspartic acids.
- Suitable peptide linkers for use in fusion proteins are well-known and typically adopt a flexible extended conformation and do not interfere with the function of the NPP1 component or the fusion partners. Peptide linker sequences may contain Gly, His, Asn and Ser residues in any combination. The useful peptide linkers include, without limitation, poly-Gly, poly-His, poly-Asn, or poly-Ser. Other near neutral amino acids, such as Thr and Ala can be also used in the linker sequence. Amino acid sequences which can be usefully employed as linkers include those disclosed in Maratea et al.,
Gene 1985, 40, 39-46; Murphy et al., Proc Natl Acad Sci USA 1986, 83, 8258-8262; U.S. Pat. Nos. 4,935,233 and 4,751,180. Other suitable linkers can be obtained from naturally occurring proteins, such as the hinge region of an immunoglobulin. - A preferred synthetic linker is (Gly4Ser)n, where n is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 (SEQ ID NO:19). Preferably, n is 3 or 4. For example, in some embodiments the linker is (Gly4Ser)3 (SEQ ID NO:16) and the fusion protein include a linker with the amino acid sequence GlyGlyGlyGlySerGlyGlyGlyGlySerGlyGlyGlyGlySer (SEQ ID NO:16). Typically, the linker is from 1 to about 50 amino acid residues in length, or 1 to about 25 amino acids in length. Frequently, the linker is between about 8 and about 20 amino acids in length. Preferred NPP1 fusion proteins comprise from N-terminus to C-terminus an NPP1 component, optionally a linker, an Fc region of an immunoglobulin (e.g., human IgG1 Fc optionally including hinge or a portion thereof), optionally a second liner, and optionally a targeting moiety. Thus, the Fc region and the optional targeting moiety, when present, are each located C-terminally to the NPP1 component. The NPP1 component preferably comprises the cysteine-rich region and the catalytic domain of NPP1, lacks the N-terminal cytosolic and transmembrane domains, and optionally contains the C-terminal region.
- A preferred fusion protein comprises, from N-terminus to C-terminus, an NPP1 component comprising the cysteine-rich domain, the catalytic domain and the C-terminal region of human NPP1; and the Fc region, including hinge, of a human immunoglobulin. Preferably, the Fc region is from human IgG1. In particular embodiments, the fusion protein has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:3. SEQ ID NO:3 is the amino acid sequence of sNPP1-Fc fusion protein.
- A preferred fusion protein of this type has the amino acid sequence of SEQ ID NO:3.
- Another preferred fusion protein comprises, from N-terminus to C-terminus, an NPP1 component comprising the cysteine-rich domain, the catalytic domain and the C-terminal region of human NPP1; a linker (e.g., (Gly4Ser)3 (SEQ ID NO:16)); and the Fc region, including hinge, of a human immunoglobulin. Preferably, the Fc region is from human IgG1.
- Another preferred fusion protein comprises, from N-terminus to C-terminus, an NPP1 component comprising the cysteine-rich domain, the catalytic domain and the c-terminal region of human NPP1; the Fc region, including hinge or a portion thereof, of a human immunoglobulin; and a moiety targeting the fusion protein to sites of calcification. Preferably, the Fc region is from human IgG1. Preferably, the moiety targeting the fusion protein to sites of calcification is Asp10 (SEQ ID NO:18). More preferably, the Fc region is from human IgG1 and the moiety targeting the fusion protein to sites of calcification is Asp10 (SEQ ID NO:18). In particular embodiments, the fusion protein has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:4. A preferred fusion protein of this type has the amino acid sequence of SEQ ID NO:4.
- Another preferred fusion protein comprises, from N-terminus to C-terminus, an NPP1 component comprising the cysteine-rich domain, the catalytic domain and the c-terminal region of human NPP1; a linker (e.g., (Gly4Ser)3 (SEQ ID NO:16)); the Fc region, including hinge or a portion thereof, of a human immunoglobulin; and a moiety targeting the fusion protein to sites of calcification. Preferably, the Fc region is from human IgG1. Preferably, the moiety targeting the fusion protein to sites of calcification is Asp10 (SEQ ID NO:18). More preferably, the Fc region is from human IgG1 and the moiety targeting the fusion protein to sites of calcification is Asp10 (SEQ ID NO:18).
- Another preferred fusion protein comprises, from N-terminus to C-terminus, an NPP1 component comprising a portion of the cysteine-rich domain, the catalytic domain and the c-terminal region of human NPP1; optionally a linker (e.g., (Gly4Ser)3 (SEQ ID NO:16)); the Fc region, including hinge or a portion thereof, of a human immunoglobulin. Preferably, the Fc region is from human IgG1. In particular embodiments, the fusion protein has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, or SEQ ID NO:12. Preferred fusion protein of this type have the amino acid sequence of SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11 or SEQ ID NO:12. In particularly preferred aspects, a fusion protein of SEQ ID NO:3 is administered in accordance with the methods described herein. In other particularly preferred aspect, a fusion protein of SEQ ID NO:4 is administered in accordance with in the methods described herein. In other particularly preferred aspect, a fusion protein of SEQ ID NO:9 is administered in accordance with in the methods described herein. In other particularly preferred aspect, a fusion protein of SEQ ID NO:10 is administered in accordance with the methods described herein. In other particularly preferred aspect, a fusion protein of SEQ ID NO:11 is administered in accordance with the methods described herein. In other particularly preferred aspect, a fusion protein of SEQ ID NO:12 is administered in accordance with the methods described herein.
- Fusion proteins of the present invention can be prepared using standard methods, including recombinant techniques or chemical conjugation well known in the art. Techniques useful for isolating and characterizing the nucleic acids and proteins of the present invention are well known to those of skill in the art and standard molecular biology and biochemical manuals can be consulted to select suitable protocols for use without undue experimentation. See, for example, Sambrook et al., 1989, “Molecular Cloning: A Laboratory Manual”, 2nd ed., Cold Spring Harbor, the content of which is herein incorporated by reference in its entirety.
- The isolated recombinant human sNPP1, fragment, and fusion proteins thereof, can be produced in any useful protein expression system including, without limitation, cell culture (e.g., CHO cells, COS cells, HEK203), bacteria such as Escherichia coli (E. coli) and transgenic animals, including, but no limited to, mammals and avians (e.g., chickens, quail, duck and turkey). For expression, a construct that encodes the sNPP1 and includes a suitable signal sequence (e.g., from human Ig heavy chain, NPP2, NPP4, NPP7 or human serum albumin, for example) in frame with the sequence of the sNPP1 and operably linked to suitable expression control elements.
- The sNPP1, including the fusion proteins, and physiologically acceptable salt forms thereof are typically formulated into a pharmaceutical composition for administration in accordance with the methods described herein. Pharmaceutical compositions typically include a pharmaceutically acceptable carrier or excipient. Compositions comprising such carriers, including composite molecules, are formulated by well-known conventional methods (see, for example, Remington's Pharmaceutical Sciences, 14th ed., Mack Publishing Co., Easton, Pa.), the entire teachings of which are incorporated herein by reference. The carrier may comprise a diluent. In one embodiment, the pharmaceutical carrier can be a liquid and the fusion protein may be in the form of a solution. The pharmaceutical carrier can be wax, fat, or alcohol. In another embodiment, the pharmaceutically acceptable carrier may be a solid in the form of a powder, a lyophilized powder, or a tablet. In one embodiment, the carrier may comprise a liposome or a microcapsule. The pharmaceutical compositions can be in the form of a sterile lyophilized powder for injection upon reconstitution with a diluent. The diluent can be water for injection, bacteriostatic water for injection, or sterile saline. The lyophilized powder may be produced by freeze drying a solution of the fusion protein to produce the protein in dry form. As is known in the art, the lyophilized protein generally has increased stability and a longer shelf life than a liquid solution of the protein.
- Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents of the specific embodiments described herein. Such equivalents are intended to be encompassed by the following claims. Any combination of the embodiments disclosed in the any plurality of the dependent claims or Examples is contemplated to be within the scope of the disclosure.
- The disclosure of each and every U.S. and foreign patent and pending patent application and publication referred to herein is specifically incorporated herein by reference in its entirety, as are the contents of Sequence Listing and Figures.
- The present invention is further exemplified by the following examples. The examples are for illustrative purpose only and are not intended, nor should they be construed as limiting the invention in any manner.
- To assess whether ENPP1 knockdown increases proliferation in human primary vascular smooth muscle cells (VSMCs) the following experiments were conducted using human primary VSMCs obtained from ATCC and ThermoFisher Scientific.
- 1A. ENPP1 Gene Expression
- First, baseline ENPP1 gene expression in human VSMCs was assessed via real time polymerase chain reaction (qRT-PCR), western blot analysis, and an assay to detect cell based ENPP1 enzymatic activity, according to the following protocols.
- Real Time Polymerase Chain Reaction (qRT-PCR): Total RNA was isolated from human VSMCs using a Qiagen Rneasy Mini kit (cat #74106) and QIAshredder (cat #79656, QIAGEN, Valencia, Calif.) as per manufacturer's instructions. The isolated RNA was quantified using a Nanodrop2000 (Thermoscientific) and reverse transcribed to cDNA using High-Capacity cDNA Reverse Transcription Kit (Cat #4368814; ThermoFisher Scientific). The resulting cDNA was amplified using the TaqMan Universal PCR Master Mix and detected by real-time PCR using
QuantStudio™ 7 Flex System. TaqMan probes for human ENPP1, Hs01054038_m1 and housekeeping gene, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) Hs99999905_m1 were obtained from ThermoFisher Scientific. Target gene expression level was normalized by GAPDH level in each sample and Relative expression level was calculated using 2-ΔΔ Ct method. - 1B. Western Blot Analysis
- VSMCs were detached, washed in PBS and the cell lysates were prepared in lysis buffer containing 1% each of protease inhibitor, Phosphatase Inhibitor Cocktail 3 (cat #P0044; Sigma) and Phosphatase Inhibitor Cocktail 2 (cat #P5726; Sigma). The cell lysate was quantified and denatured, then equal amounts of the protein were loaded on 4-12% Bis polyacrylamide gels. The proteins in gels were electrophoretically transferred to nitrocellulose membrane using
iBlot® 2 Dry Blotting System. Following treatment with blocking buffer (5% skimmed milk in 1XTBST (Cat #IBB-180; Boston Bioproducts)), and incubated with Rabbit pAb to human ENPP1 (PA527905 by Thermo Fischer Scientific) at 1:500 or GAPDH (14C10) Rabbit anti GAPDH mAB by Cell Signaling Technology (cat #2118L) at 1:1000 in blocking buffer for overnight at 4° C., followed by a Goat-anti-Rabbit Antibody conjugated with HRP (cat #7074; Cell signaling technology) for 1 hr. at room temperature. The signals were detected using the Protein Simple FluorChem R system (Part #92-15313-00, ProteinSimple). Signals of ENPP1 protein were normalized with level of endogenous protein GAPDH in each sample. - 1C. Cell Based ENPP1 Enzymatic Activity
- Cell based ENPP1 enzyme activity was assayed using the colorimetric substrate, p-
nitrophenyl thymidine 5′-monophosphate (Cat #T4510, Sigma). Cells were seeded into the well of 96 well maxisorp plate at 300,000 cells/well and 100,000 cells/well and substrate at a final concentration of 1 mM in reaction buffer (200 mM Tris-HCl pH9, 1M NaCl, 10 mM MgCl2, 0.1% (v/v) Triton-X 100) was added to the plate. Enzyme activity is measured the reaction product based on the ability of phosphatases to catalyze the hydrolysis of PNPP to p-nitrophenol with absorbance at 405 nm using a continuous spectrophotometric assay using in a FlexStation® Plate Reader (Molecular Devices) in a kinetic mode with 21 reads at 31 sec intervals. Standard curve was generated using ENPP1-Fc protein ranged from 0 ng/ml to 90 ng/ml. Data was analyzed at 10 minutes respectively. - Results:
FIG. 1 depicts mRNA (FIG. 1A ) and protein (FIG. 1B ) ENPP1 expression in human VSMCs from six different donors, as well as enzyme activity from three of the donors (FIG. 1C ). As shown inFIG. 1 , there was significant natural variability in baseline ENPP1 expression in human primary VSMCs. - 1D. ENPP1 Knockdown Using siRNA Targeting Human ENPP1
- Human VSMCs (donor 3) were transfected with either ENPP1 siRNA or control siRNA. The following siRNA constructs were used: 1 (ENPPA (CDS) Location: 825), 2 (ENPPA (CDS) Location: 813), 3 (ENPPA (CDS) Location: 1272), 4 (ENPPA (CDS) Location: 447), and 5 (ENPPA (CDS) Location: 444). Human VSMCs (
donor 3, passage 4) were transfected with either siRNA targets [Silencer Select ENPP1 siRNA #1: s10264 (Cat #4390824),siRNA # 2 s10265 (Cat #4390824), siRNA #3: s10266 (Cat #4390824), siRNA #4: s224228 (Cat #4392420), Silencer ENPP1 siRNA #5: 144240 (Cat #AM90824); Life Technologies] to human ENPP1 or control using the Lipofectamine RNAiMAX (cat #13778500; ThermoFisher Scientific) following the manufacturer's instructions. Cells were seeded in 60 mm dish atdensity 3500 cells/0.32 cm in complete medium (Vascular Cell Basal medium ATCC #PCS-100-030 supplemented with Vascular Smooth Muscle Cell Growth Kit ATCC #PCS-100-042, ATCC). Cells were treated with either an siRNA that specifically targets human ENPP1 or a negative control siRNA at a concentration of 100 nM in OPTI-MEM (cat #31985; ThermoFisher Scientific) and incubated at 37° C. Cells were harvested 48 hours or 6 days or 11 days after transfection (cells for 6 day and 11 day time points were harvested and reseeded at 48 hours), total RNA was extracted and mRNA levels were assayed by reverse transcription and real-time PCR. Levels of ENPP1 mRNA expression are reported as percentage of inhibition in mRNA expression relative to negative-siRNA after normalization to GAPDH mRNA levels. - Results: As shown in
FIG. 2A , ENPP1 mRNA expression was inhibited by 90% or greater relative to the negative control for all five siRNA constructs, 48 hours post siRNA transfection. Specifically, ENPP1 mRNA expression was inhibited by 91.3% usingsiRNA construct # 1, 93.1% usingsiRNA construct # siRNA construct # 3, 93.6% usingsiRNA construct # 4, and 90.2% usingsiRNA construct # 5, relative to the negative control. Moreover, as shown inFIG. 2B , ENPP1 mRNA expression was inhibited by 84.2% six days post siRNA transfection with construct #4 (relative to the negative control) and 74.6% eleven days post siRNA transfection with construct #4 (relative to the negative control). Accordingly, the data indicates that siRNA sufficiently silences ENPP1 expression for a prolonged period of time. - 1E. Effect of ENPP1 Knockdown on Proliferation
- Human VSMCs (
Donor 1 andDonor 3, passage 4) were seeded in 60 mm dishes at density 0.3*10e6 cells/60 mm dish in Complete Medium (Cat #PCS-100-042, PCS-100-030; ATCC). After overnight recovery, they were transfected with ENPP1 siRNA or control siRNA in OPTI-MEM. After 48 hours, cells were harvested and reseeded at 2500 cells/well into 96 well plate. Cells were cultured in basal medium containing 2% or 5% FBS. Cell proliferation was evaluated by [3H] thymidine uptake. [3H]-thymidine was added in the last 18 hours of culture. Results are expressed as CPM±SEM. Experiments were triplicated. - Results: As shown in
FIGS. 3A (Donor 1) and 3B (Donor 3) and Table 1 (Donor 3), silencing of ENPP1 by siRNA increased proliferation of human primary VSMCs from these two different donors (e.g., by about 1.75 fold or greater) compared to negative control siRNA. -
TABLE 1 Inhibition of ENPP1 siRNA ENPP1 (CDS) at 48 hrs. siRNA ENPP1 813 84.7% siRNA_NC N/A 0.0% - 1F. Effect of ENPP1 Knockdown on Cell Proliferation
- Human VSMCs (
Donor 1 andDonor 3, passage 4) were seeded in 60 mm dishes at density 0.3*10e6 cells/60 mm dish in Complete Medium (Cat #PCS-100-042, PCS-100-030; ATCC). After overnight recovery, they were transfected with ENPP1 siRNA or control siRNA in OPTI-MEM. After 48 hours, cells were harvested and reseeded at 2500 cells/well into 96 well plate. Cells were cultured in basal medium containing 5% FBS for 3 or 4 days. Cells were detached and stained with AOPI at indicated time point, cell number was measured using auto cellcounter Cellometer Auto 2000. Results were expressed as Cell number ±SEM. Experiments were triplicated. - Results: As shown in
FIGS. 4A (Day 3) and 4B (Day 4) representingDonor 3, silencing ENPP1 using siRNA increased cell growth in human primary VSMCs. Specifically, cell growth was at least two fold or greater 3-4 days after silencing ENPP1 using two different constructs. These results are consistent in light of results found from independent experiments, independent analysts, different constructs, same donor, and different methods. - To assess whether ENPP1 knockdown increases proliferation, the following experiments were conducted using rat primary VSMCs.
- First, an in vitro primary rat VSMC Model system was established. Primary rat vascular smooth muscle cells (VSMCs) were prepared by using enzymatic digestion of thoracic arteries from 3-week-old Sprague-Dawley rats. Small fragments were minced and digested at 37° C. in vascular cell basal media (ATCC) supplemented collagenase type II 2 mg/ml (Cat #17101-015, Gibco) for 3 hours, mix every 15 minutes and replace digestion solution hourly. Then, the cell suspension was centrifuged at 1000 rpm for 10 min at 4° C., the pellet was resuspended in complete medium and cultured into T75 flasks. Cells were cultured in vascular cell basal media (ATCC #PCS-100-030) containing 5% fetal bovine serum, and growth supplements (Cat #PCS-100-042, ATCC containing with 5 ng/ml rhFGF, 5 μg/ml rh Insulin, 50 μg/ml Ascorbic acid, 10 mM L-gutamine, 5 ng/ml rhEGF,
penicillin 10 Units/ml,streptomycin 10 μg/ml, and Amphotercin B 25 ng/ml). VSMCs were subcultured and used between passages 3-4. ENPP1 knockdown was achieved by transfection with siRNA. Specifically, transfection of VSMCs with siRNA was performed using Lipofectamine RNAiMAX (cat #13778500; ThermoFisher Scientific) according to the manufacturer's instructions. - 2A. Effect of Silencing ENPP1 on Pharmacological Activity
- Rat primary VSMCs (passage 3) were seeded into 60 mm dish at density 0.3*10e6/60 mm dish in complete medium and transfected with either one of a siRNA specific targets to rat ENPP1 or negative control siRNA at a concentration of 100 nM in OPTI-MEM (cat #31985; ThermoFisher Scientific) and incubated at 37° C. Cells were harvested at 48 hours and reseeded in the wells of 6 well plate at 37500 cells/well in complete medium. Cells were then harvested at indicated time points after transfection, total RNA was extracted and mRNA levels were assayed by reverse transcription and real-time PCR. Levels of ENPP1 mRNA expression are reported as percentage of inhibition in mRNA expression relative to negative-siRNA after normalization to GAPDH mRNA levels (see
FIG. 5A ). - Rat VSMCs (passage 3) were transfected with either ENPP1 siRNA or control siRNA for 48 hours, then seeded into the wells of 6-well plate at 37500 cells/well (2 wells per condition), the cells were stimulated with Complete Medium (Cat #PCS-100-042, PCS-100-030; ATCC). Cells were detached at indicated time points and stained with AOPI, cell number was measured using auto cell counter,
Cellometer Auto 2000. Results are expressed as Cell number ±SEM (seeFIG. 5B ). Experiments were triplicated. - ENPP1 enzyme activity was assayed using the colorimetric substrate, p-
nitrophenyl thymidine 5′-monophosphate (Cat #T4510, Sigma). Cells were seeded into the well of 96 well plate at 20000 cells/well and substrate at 1 mM p-nitrophenyl thymidine 5′-monophosphate in reaction buffer was added. Enzyme activity was measured the reaction product based on the ability of phosphatases to catalyze the hydrolysis of PNPP to p-nitrophenol with absorbance at 405 nm using a continuous spectrophotometric assay using in a FlexStation® Plate Reader (Molecular Devices) in a kinetic mode with 21 reads at 31 sec intervals. Standard curve was generated using ENPP1-Fc protein ranged from Ong/ml to 90 ng/ml. Data was analyzed using SoftMax Pro software at 10 minutes. ENPP1 activity in each sample was calculated based on the standards (seeFIG. 5C ). - As shown in
FIGS. 5A-5C , siRNA silencing of ENPP1 was robust and durable (FIG. 5A ), increased cell growth (FIG. 5B ), and decreased enzyme activity (FIG. 5C ). - 2B. Effect of Adenosine, AMP, or PPi on Proliferation of Rat VSMCs
- Rat VSMCs (p3) were transfected with either rat ENPP1-siRNA or control siRNA for 48 hrs and then seeded into wells of 96-well plate at 2500 cells/well. Cells were then cultured in complete medium in the presence and absence of Adenosine monophosphate (AMP) (Cat #A1752, Sigma), adenosine (A4036, Sigma) or PPi (Cat #71515, Sigma). Cell proliferation was evaluated on
day 3 by [3H] thymidine uptake. Results are expressed as CPM±SEM. Experiments were triplicated. - As shown in
FIG. 6A , adenosine and AMP inhibited proliferation in rat VSMCs that were knocked down with ENPP1 and without regulated ENPP1. However, PPi did not affect proliferation in rat VSMCs (FIG. 6B ). - 2C. Effect of Bisphosphonate on Proliferation of Rat VSMCs
- Rat VSMCs (p3) were transfected with either rat ENPP1-siRNA or control siRNA for 48 hrs., then seeded into wells of 96-well plate at 2500 cells/well, cells were cultured in complete medium in the presence and absence of Etidronate (Cat #P5248, Sigma) at indicated concentration. Cell proliferation was evaluated on
day 3 by [3H] thymidine uptake. Results are expressed as CPM±SEM. Experiments were triplicated. Data was reproducible with Zoledronate as well. - As shown in
FIG. 7 , bisphosphonate did not appear to inhibit proliferation in rat VSMCs. - 2D. Effect of Silencing ENPP1 on Proliferation of Rat VSMCs
- Rat VSMCs were transfected with siRNA against rat ENPP1 si206 (ENPP1 sequence start position: 462; (Cat #SASI_Rn01_00111206 NM_053535, Sigma)), rat ENPP1 sil53 (ENPP1 sequence start position: 516 (Cat #SASI_RnO2_00266153 NM_053535, Sigma)), or a negative control (
FIG. 8A ). After 48 hours transfection, cells were seeded into wells of 96-well plate at 1250 cells/well in complete medium. After 4 hours, following treatment conditions were added in complete medium: PDGF (Cat #P8953, Sigma), Cilostazol (Cat #0737, Sigma), PDGF+Cilostazol at indicated concentration. [3H] thymidine was added in the last 18 hours of culture. Cell proliferation was evaluated by [3H] thymidine uptake. [3H]-thymidine was added in the last 18 hours of culture. Results are expressed as CPM±SEM. Experiments were triplicated. - As shown in
FIGS. 8A-8B , silencing ENPP1 by siRNA increased proliferation in rat VSMCs, but was inconsistent between constructs. - Experiments were conducted to assess whether overexpressed ENPP1 rescues proliferation of VSMCs. The following constructs were used: (1) siRNA target rENPP1:(SASI_Rn01_00111206) Cat #PDSIRNA2D, Sigma, (2) Ad-mENPP1: Vector Biolab (Lot
#20150616T # 10; Vector Biolabs), (3) Ad-rENPP1: Life Tech+Vector Biolab (Lot#20150714T # 11; Vector Biolabs), and (4) Ad-rENPP1: GeneWiz+Vector Biolab (Lot#20150714T # 9; Vector Biolabs). - 3A. Ad-mENPP1/Ad-rENPP1 Induces Overexpression of Mouse/Rat ENPP1 mRNA Specifically in Rat VSMC
- Co-transfection with Ad-GFP and siRNA: Rat VSMCs (passage 3) were seeded in the wells of 6-well plates at 6000 cells/0.32 cm2 in complete medium. After overnight culture, the cells were transfected with siRNA targets to rat ENPP1 using Lipofectamine RNAiMAX. After 4 hours incubation, siRNA was removed and the adenoviral vector Ad-GFP (cat #1060, Vector Biolabs) was added to the cells at multiplicities of infection (MOI) dose of 400. The plates were spun at 37° C. for 1.5 hrs. at 900 g and incubated at 37° C. for next 30 minutes before removing the adenoviral particles and washing with PBS. The siRNA particles were added again and left for overnight infection. The efficacy of adenovirus infectivity was measured 45 hours after infection of Ad-GFP under a fluorescent microscope (DMI8 Leica Microsystems) at 100× magnification.
- Co-transfection with Ad-mENPP1 and siRNA-NC: The primary Rat Vascular Smooth Muscle Cells (Rat VSMC) (Passage 3) were seeded at 6000 cells/0.32 cm2 in a 6 well dish in complete medium (Vascular Cell Basal medium ATCC #PCS-100-030 supplemented with Vascular Smooth Muscle Cell Growth Kit ATCC #PCS-100-042). After overnight recovery of the cells, the cells were infected with siRNA particles using Lipofectamine RNAiMAX (cat #13778500; ThermoFisher Scientific) diluted in OPTI-MEM (cat #31985; ThermoFisher Scientific) containing 0.25% FBS. After 4 hours, remove the media and add the adenoviral particles at MOI=400. Three adenoviral particles tested are Ad-rENPP1 (Lot
#20150714T # 9; Vector Biolabs), Ad-rENPP1 (Lot#20150714T # 11; Vector Biolabs) and Ad-mENPP1 (Lot#20150616T # 10; Vector Biolabs). The plates were spun at 37° C. for 1.5 hrs at 900 g and incubated at 37° C. for next 30 minutes before removing the adenoviral particles and washing with PBS. The siRNA particles were added again and left for overnight infection. Cells were harvested 48 hours after infection with Ad-mENPP1. Total RNA was extracted and mRNA levels were assayed by reverse transcription and real-time PCR using primer specific to mouse ENPP1 or rat ENPP1. Levels of mENPP1 mRNA expression are reported as percentage of mRNA expression relative to negative control Ad after normalization to GAPDH mRNA levels. - Real Time Polymerase Chain Reaction (qRT-PCR): RNA isolation and reverse transcription were performed using the TaqMan® Gene Expression Cells-to-CT™ Kit (Cat #AM1729, Thermofisher Scientific) as per manufacturer's instructions in a 96-well plate format, 96 hours post co-transfection. The resulting cDNA is amplified using the TaqMan Universal PCR Master Mix and detected by real-time PCR using
QuantStudio™ 7 Flex System in a 384 well-plate. TaqMan probes for rat ENPP1 (AJKAK71), Menpp1 (Mm00501088_m1) and housekeeping gene, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) Rn01775763_g1 were obtained from ThermoFisher Scientific. Target gene expression level was normalized by GAPDH level in each sample and Relative expression level was calculated using Δ Ct method. - The Rat ENPP1 (AJKAK71) taqman probe was custom designed to detect the rENPP1 in the adenoviral vector cassette while the mouse ENPP1 (Mm00501088_m1) taqman probe was a premade probe from ThermoFisher Scientific.
- As shown in
FIG. 9A , Ad-mENPP1/Ad-rENPP1 induced overexpression of Mouse/Rat ENPP1 messenger RNA specifically in rat VSMC. Nearly 100% of VSMCs expressed high level GFP 45 hrs. after co-transfection with siRNA and Ad-GFP (FIG. 9B ). Moreover, there was specific overexpression of mouse and rat ENPP1 in co-transfected Rat VSMC starting at the 48 hour timepoint, which and persisted through 96 hours. - 3B. mENPP1 mRNA Over-Expression Persists in Presence of siRNA Targeting Rat ENPP1, but with Moderate Interference of Mouse ENPP1
- Co-transfection with Ad-ENPP1 and siRNA: Rat VSMCs (passage 3) were seeded in the wells of 6-well plates at 6000 cells/0.32 cm2 in complete medium. After overnight culture, the cells were transfected with siRNA targets to rat ENPP1 (si206) or siRNA negative control (siNC) using Lipofectamine RNAiMAX. After 4 hours incubation, siRNA was removed and the adenoviral vector contains mouse ENPP1 cDNA sequence (Ad-mENPP1) was added to the cells at multiplicities of infection (MOI) dose of 400. The plates were spun at 37° C. for 1.5 hrs at 900 g and incubated at 37° C. for next 30 minutes before removing the adenoviral particles and washing with PBS. The siRNA particles were added again and left for overnight infection. Cells were harvested 48 hours after infection of Ad-mENPP1. Total RNA was extracted and mRNA levels were assayed by reverse transcription and real-time PCR. Levels of ENPP1 mRNA expression are reported as percentage of mRNA expression relative to negative-siRNA after normalization to GAPDH mRNA levels. Ps: siNC (Silencer Select ENPP1 siRNA s10265 (5 nmol)) Cat #4390824, ThermoFisher Scientific. siRNARat enpp1 (SASI_Rn01_00111206) Cat #PDSIRNA2D, Sigma.
- As shown in
FIG. 10 , mENPP1 mRNA overexpression persisted in the presence of siRNA targeting rat ENPP1, but with moderate interference of mouse ENPP1. Mouse ENPP1 exhibited 92% homology to rat ENPP1 at the gene level. Accordingly, siRNA knock down of rat ENPP1 expression also partially down regulated mouse ENPP1 expression. - 3C. Successful Rescue ENPP1 Protein Expression by Ad-rENPP1
- Co-transfection with Ad-ENPP1 and siRNA: Rat VSMCs (passage 3) were seeded in the wells of 6-well plates at 6000 cells/0.32 cm2 in complete medium. After overnight culture, the cells were transfected with siRNA targets to rat ENPP1 (si206) or siRNA negative control (siNC) using Lipofectamine RNAiMAX. After 4 hours incubation, siRNA was removed and the adenoviral vector contains mouse ENPP1 cDNA sequence (Ad-rENPP1) was added to the cells at multiplicities of infection (MOI) dose of 400. The plates were spun at 37° C. for 1.5 hrs at 900 g and incubated at 37° C. for next 30 minutes before removing the adenoviral particles and washing with PBS. The siRNA particles were added again and left for overnight infection. Cells were harvested 72 hours after infection of Ad-rENPP1. ENPP1 protein levels were measured using in cell western blot assay.
- In cell western: The cells were fixed in 4% Formaldehyde (w/v), Methanol-free (cat #28908, Pierce™) for 20 min at room temperature. The formaldehyde was removed under a fume hood and the cells were washed twice with 200 μl of PBS. The cells were permeabilized in 100p/well (Cat #3603, Costar) of 0.1% Triton x-100 in PBS for 20 min at Room temperature. Then, the cells were blocked using LICOR TBS blocking buffer (P/N 927-50000) for 1 hr. at Room Temperature followed by overnight incubation at 4° C. with 2.5 μg/ml primary antibody Goat anti-ENPP1/PC1 (Cat #OAEB02445, Aviva) in LICOR TBS blocking buffer containing 0.2% Tween20. The wells were washed thrice with 200 μl of 1×TBST and incubated with secondary antibody IRDye® 800CW Donkey-anti-Goat (P/N 926-32214) Antibody at 1:1000 for 1 hr. at Room Temperature covered in foil. The wells were washed thrice with 200 μl of 1×TBST and rinsed with TBS once to get rid of tween. Image the plate in LICOR Odessey Clx.
- As shown in
FIG. 11 , ENPP1 protein expression was successfully rescued by Ad-rENPP1. - 3D. Overexpression of mENPP1 Rescues Enzyme Activity in rVSMCs and A10 Cells
- Co-transfection with Ad-ENPP1 and siRNA: Rat VSMCs (passage 3) (upper) or rat non-differentiated VSMCs A-10 cells (ATCC, CRL-1476) were seeded in the wells of 6-well plates at 6000 cells/0.32 cm2 in complete medium. After overnight culture, the cells were transfected with siRNA targets to rat ENPP1 (si206) or siRNA negative control (siNC) using Lipofectamine RNAiMAX. After 4 hours incubation, siRNA was removed and the adenoviral vector contains mouse ENPP1 cDNA sequence (Ad-rENPP1) was added to the cells at multiplicities of infection (MOI) dose of 400. The plates were spun at 37° C. for 1.5 hrs at 900 g and incubated at 37° C. for next 30 minutes before removing the adenoviral particles and washing with PBS. The siRNA particles were added again and left for overnight infection. Cells were harvested 72 hours after infection of Ad-rENPP1. Cell based ENPP1 enzyme activity was using the colorimetric substrate, p-
nitrophenyl thymidine 5′-monophosphate (Cat #T4510, Sigma). Cells were seeded into the well of 96 well Maxisorp plate at 20000 cells/well and substrate at 1 mM p-nitrophenyl thymidine 5′-monophosphate in reaction buffer was added. Enzyme activity is measured the reaction product based on the ability of phosphatases to catalyze the hydrolysis of PNPP to p-nitrophenol with absorbance at 405 nm using a continuous spectrophotometric assay using in a FlexStation® Plate Reader (Molecular Devices) in a kinetic mode with 21 reads at 31 sec intervals. Standard curve was generated using ENPP1-Fc protein ranged from Ong/ml to 90 ng/ml. Data was analyzed using SoftMax Pro software at 10 minutes. ENPP1 activities in each sample was calculated based on the standards. siNC (Silencer Select ENPP1 siRNA s10265 (5 nmol)) Cat #4390824, ThermoFisher Scientific siRNA Rat enpp1 (SASI_Rn01_00111206) Cat #PDSIRNA2D, Sigma. - As shown in
FIGS. 12A-12B , over-expression of mENPP1 rescued enzyme activity in rVSMCs and A10 cells (nondifferentiated rat VSMCs). Rat ENPP1 activity was not detected in A10 cells, but, low enzyme activity was observed in primary VSMCS transfected with Ad-rENPP1 (GeneWiz). - 3E. Silencing ENPP1 Increases Proliferation in Rat VSMCs, Whereas Over-Expression of Mouse or Rat ENPP1 Inhibits Proliferation
- Co-transfection with Ad-ENPP1 and siRNA: Rat VSMCs (passage 3) were seeded in the wells of 96-well plate at 6000 cells/well in complete medium. After overnight culture, the cells were transfected with siRNA targets to rat ENPP1 using Lipofectamine RNAiMAX. After 4 hours incubation, siRNA was removed and the adenoviral vector Ad-rENPP1 or Ad-mENPP1 was added to the cells at multiplicities of infection (MOI) dose of 400. The plates were spun at 37° C. for 1.5 hours at 900 g and incubated at 37° C. for next 30 minutes before removing the adenoviral particles and washing with PBS. The siRNA particles were added again and left for overnight infection.
Day 1 post transfection, starvation media (0.25% FBS in basal media) was added to the starvation condition, complete media was added to non-starvation condition. - Forty eight hours post transfection, complete media was added to starvation condition cells. Cell proliferation was evaluated at
Day 4 post-transfection by [3H] thymidine uptake. [3H] thymidine was added in the last 18 hours of culture (seeFIGS. 13A-13B ). - In a separate experiment, forty eight hours post transfection, cells were seeded into wells of 24-well plate at 15000 cells/well in complete medium for 4 hours, followed by 48 hours starvation in 0.25% FBS, the cells were cultured in base media contains 5% FBS (lower). Cells were stained with AOPI and counted using auto cell counter 72 hours later (See
FIG. 14 and Table 2) siNC (Silencer Select ENPP1 siRNA s10265 (5 nmol)) Cat #4390824, ThermoFisher Scientific siRNA Rat enpp1 (SASI_Rn01_00111206) Cat #PDSIRNA2D, Sigma. - Results are expressed as Cell number SEM. Experiments were triplicated.
-
TABLE 2 Sample ID Viability (%) siNC + AdNC 89.35 si-rENPP1 + AdNC 85.2 si-rENPP1 + AdmENPP1 89.25 si-rENPP1 + AdrENPP1 85.9 - As shown in
FIGS. 13A-13B andFIG. 14 , silencing ENPP1 increased proliferation in rat VSMCs, whereas overexpression of mouse or rat ENPP1 inhibited proliferation. - In summary, the above experiments demonstrated that silencing ENPP1 increased proliferation and cell growth of VSMCs in all tested systems. Moreover, over expression of mouse or rat ENPP1 using an Ad vector inhibited proliferation and cell growth in rat VSMCs.
- Rat VSMCs were seeded in 35 mm dish at density 75000 cells/9.6 cm2 in completed medium. After overnight culture, the cells were transfected with siRNA targets to rat ENPP1 or siRNA negative control (siNC) for overnight. The cells were then starved with base medium contains 0.25% FBS. After 24 hrs starvation, the cells were reseeded into the well of 96 well plates at 2500 cells/0.32 cm2 in CM. After 4 hrs, once the cells adhered, ATP treatments (FLAAS, Sigma) were added at 1M in final concentration. After 30 minutes, 100 μl of supernatant was collected and tested via CellTiter-Glo® Luminescent Cell Viability Assay (cat #G7572, Promega) in a black/opaque plate. Also, cell titer glo with the seeded cells was performed per manufacturer's instruction. This protocol was repeated 2 hours and 24 hours post ATP treatment.
- As shown in
FIG. 15 (30 minute),FIG. 16 (2 hour), andFIG. 17 (24 hour), ATP is unstable in complete medium containing 5% FBS in culture at 37° C. Moreover, as shown inFIG. 18 heat denatured human/mouse ENPP1-Fc protein completely lost enzymatic activity. - In a separate experiment, rat VSMCs were seeded in the well of the 6-well plates in complete medium contains 5% FBS. After overnight culture, the cells were transfected with siRNA targets to rat ENPP1 (SASI_Rn01_00111206 Cat #PDSIRNA2D, Sigma) or siRNA negative control (Silencer Select ENPP1 siRNA s10265 (5 nmol) Cat #4390824, ThermoFisher Scientific) for overnight. The cells were then starved with base medium contains 0.25% FBS. After 24 hrs starvation, the cells were reseeded into the well of 96 well plates and cultured with completed medium contains 5% FBS in presence with 300 μM ATP and mENPP1-Fc protein (
FIG. 19A ), hENPP1-Fc (FIG. 19B ), or hENPP1-Fc-D10 (FIG. 19C ) protein, at the indicated concentration. The cultured medium was replaced daily. Proliferation was measured atday 3 using MicroBeta 3H-Thymidine incorporation. Thymidine was added in the last 18 hours. - As shown in
FIGS. 19A-C , treatment with any of the ENPP1 proteins (mENPP1-Fc (FIG. 19A ), hENPP1-Fc (FIG. 19B ), and hENPP1-Fc-D10 (FIG. 19C )) inhibited proliferation on rat primary VSMCs. Heat denatured ENPP1 had no effect on proliferation of rat VSMCs. - The following experiments were conducted using human induced pluripotent stem cell (hiPSC)-derived vascular smooth muscle cells (iVSMCs).
- 5A. ENPP1 Expression
- Human iPSC derived VSMCs (donors BJ, BLS) and human primary VSMCs (
donors QuantStudio™ 7 Flex System. TaqMan probes for human ENPP1, Hs01054038_m1 and housekeeping gene, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) Hs99999905_m1 were obtained from ThermoFisher Scientific. Target gene expression level was normalized by GAPDH level in each sample and Relative expression level was calculated using 2-ΔΔ Ct method (FIG. 20A ). - Western Blot Analysis: The VSMCs were detached, washed in PBS and the cell lysates were prepared in lysis buffer containing 1% each of protease inhibitor (p8340; Sigma), Phosphatase Inhibitor Cocktail 3 (cat #P0044; Sigma) and Phosphatase Inhibitor Cocktail 2 (cat #P5726; Sigma). The cell lysate was quantified and denatured, then equal amounts of the protein were loaded on 4-12% Bis polyacrylamide gels. The proteins level was measured once the gel was electrophoretically transferred to nitrocellulose membrane using
iBlot® 2 Dry Blotting System. The membranes were blocked for 1 h in blocking buffer (Licor TBS blocking buffer (P/N 927-50000) and incubated with Rabbit pAb to human ENPP1 (PA527905 by Thermo Fischer Scientific) at 1:500 and GAPDH (14C10) Rabbit anti GAPDH mAB by Cell Signalling Technology (cat #2118L) at 1:1000 in blocking buffer containing 0.2% Tween20 overnight at 4° C., followed by a Donkey-anti-Rabbit Antibody conjugated with the fluorescent dye IRDye® 800CW (cat #926-32213; LICOR) or Donkey-anti-Rabbit Antibody conjugated with the fluorescent dye IRDye® 680RD (cat #926-68073; LICOR) for 1 h at room temperature, respectively. The signals were detected using the Odyssey CLx Imaging System (LI-COR Biosciences). Signals of ENPP1 protein were normalized with level of endogenous protein GAPDH in each sample. The relative protein expression for the human primary iVSMC donors and the VCMC donors are depicted inFIGS. 20B and 20C , respectively. - As shown in
FIGS. 20A-B , human primary iVSMCs expressed a high level of ENPP1. - 5B. Effect of Silencing ENPP1 on Growth of Human iVSMCs.
- Human iPSC differentiated VSMCs (iVSMCs) (p2) were seeded into the wells of 6-well plate and transfected with ENPP1 siRNA or negative control siRNAs, either consisting of a scrambled nucleotide sequence or directed to actin. After 48 hours transfection, the cells were detached, stained with AOPI and counted in auto
cell counter Cellometer 2000. - As shown in
FIG. 21 , silencing ENPP1 by siRNA increased the growth of human iVSMCs as compared to both of the negative controls. - 5C. In Vitro Proof of Concept Human iVSMCs
- Additional experiments were conducted using human induced pluripotent stem cell (hiPSC)-derived vascular smooth muscle cells (iVSMCs) to assess the effect of murine and human ENPP1 protein on proliferation. The following ENPP1 proteins were used: mENPP1-mG1FC: ZLC022, hENPP1-FC: 105-FC, and hENPP1-FC-D10: 105-FC-D10.
- The human iPSC derived VSMCs were seeded at 3500 cells/0.32 cm2 in
collagen 1 coated 60 mm dishes in SmGM-2 Smooth Muscle Growth Medium-2 (Cat #CC-3182, Lonza). After overnight culture, the cells were transfected with siRNA targets to human ENPP1 using Lipofectamine RNAiMAX (cat #13778500, ThermoFisher Scientific) for overnight. Following 48 hours starvation with 0.25% FBS, cells were stained with AOPI and counted with auto cell counter,Cellometer 2000. The effect of ENPP1-Fc protein on proliferation in VSMCs was measured using 3H thymine incorporation. Cells were reseeded in the wells of a 96 well plate that was pre-coated withcollagen 1 in completed medium presence with 300 μM ATP in addition to absence or presence of varied concentration of mENPP1-Fc, hENPP1-Fc, or hENPP1-FC-D10 protein for 3 days. Culture medium was replaced daily. 3H thymine was added in the last 18 hours of culture. Results presents as mean±SEM of four wells (n=4). A similar pattern was confirmed in three independent assays. The statistical analysis was performed by student T test. - As shown in
FIGS. 22A-C , all of the ENPP1 proteins significantly inhibited proliferation in human iVSMCs at all concentrations tested. - 5D. PPi Assay Using Human iVSMCs
- PPi is produced in an ENPP1 enzyme catalyzed reaction. PPi level was measured in culture supernatant using a radioactive assay. In order to avoid precipitation of magnesium pyrophosphate, assay components were divided into stock solutions. First, a master mix was prepared with 49.6 mM Trizma Acetate (cat #93337; Sigma-Fluka), 4.5 mM Magnesium acetate tetrahydrate (cat #63049; Sigma-Fluka); 3.5 μM NAPD-Na2 (cat #10128058001; Roche); 16.2 μM D-Glucose-1,6-diphosphate (cat #G6893; Sigma); 6.6 μM Uridine-5-Diphosphoglucose (cat #U4625; Sigma); 0.002 U/l Phosphoglucomutase (cat #P3397; Sigma); 0.003 U/l Glucose-6-phosphate dehydrogenase (cat #10165875001; Roche) and MilliQ water. Add 0.00118 U/
l Uridine 5′-diphosphoglucose pyrophosphorylase (cat #U8501; Sigma) and 0.0002 μCi/μl Uridine diphospho-D-[6-3H] glucose (cat #NET1163; Perkin Elmer) to the master mix right before adding to the samples, and 25 μl of sample was added into 115 μl master mix. After 30 minutes incubation at 37° C., 200 μl of cold 3% activated charcoal (cat #C5510; Sigma) was added and incubated 30 min at 4° C. with vortexing every 10 min intervals. The contents of the tube was transferred into a 96 well filter plate (cat #8130; Pall Corporation). The reaction mix was filtered and 80 μl of supernatant was transferred to Iso beta plates (cat #6005040; Perkin Elmer) keeping the same plate position using 960 LTS Wide-orifice tips in 10 racks, presterilized. Add 120 μl of scintillation liquid Ultima Gold (cat #6013321; Perkin Elmer) and mixed well. The plated were incubated for 1 hour and then read via a micro beta counter. - As shown in
FIG. 23 , PPi was detected in the supernatant collected from human iVSMCs treated with mENPP1-Fc. Similar results were obtained using both hENPP1-Fc and hENPP1-Fc-D10. - In summary, all three ENPP1 proteins (mENPP1-Fc, hENPP1-Fc, and hENPP1-Fc-D10) significantly inhibited proliferation in human iVSMCs that silenced ENPP1. Heat denatured ENPP1 had no effect on proliferation of iVSMCs. The inhibition was more potent in human iVSMC than it was in rat VSMCs.
- 5E. Effect of Bisphosphonate on Proliferation of Human iVSMCs
- The effect of Bisphosphonate on proliferation in human iVSMCs that silenced ENPP1 was evaluated using 3H thymine incorporation. The human iVSMCs were seeded at 3500 cells/0.32 cm2 in
collagen 1 coated 60 mm dishes in SmGM-2 Smooth Muscle Growth Medium-2 (Cat #CC-3182, Lonza). After overnight culture, the cells were transfected with siRNA targets to human ENPP1 using Lipofectamine RNAiMAX (cat #13778500, ThermoFisher Scientific) for overnight. Following 48 hours starvation with 0.25% FBS, cells were stained with AOPI and counted with auto cell counter,Cellometer 2000. Cells were reseeded in well of 96 well plate at 2500 cells/well and cultured in complete medium in the presence and absence of Etidronate (Cat #P5248, Sigma) at indicated concentration. Cell proliferation was evaluated onday 3 by [3H] thymidine uptake. Results are expressed as CPM±SEM. Experiments were triplicated. - As shown in
FIG. 24 , bisphosphonate did not appear to inhibit proliferation in human iVSMCs. - Carotid artery ligation in the mouse is a common model for investigating the response of the vasculature to mechanical injury. Damage to the vessel induces an inflammatory response and endothelial activation, resulting in smooth muscle cell proliferation and narrowing of the lumen of the vessel. Accordingly, carotid artery ligation in the tip-toe-walking (ttw) mouse, which contains a mutation in Enpp1 and serves as a model of generalized arterial calcification of infancy (GACI), was used to investigate the role of Enpp1 treatment on intimal hyperplasia.
-
FIG. 25 shows the carotid ligation procedure. In anesthetized animals, the left carotid artery is exposed through a small incision in the neck and is ligated with a suture approximately 2 mm proximal from the carotid bifurcation. The animals are allowed to recover for 14 days, at which time the carotid arteries are harvested and fixed in 4% paraformaldehyde in PBS for sectioning and histological analysis. Serial sections of 5 μm were taken spanning 250 μm from the ligation. Every fifth section was analyzed with Von Gieson's stain and morphometric analysis was performed using Image J software. - To determine the effect of Enpp1 on intimal hyperplasia, 6 to 7-week old homozygous ttw/ttw mice were treated with either vehicle or recombinant human Enpp1 (rhEnpp1) at 10 mg/kg by subcutaneous (SC) injection every other day. The mice were treated for 7 days prior to carotid ligation, and treatment continued for 14 days post-surgery when the carotid arteries were harvested for analysis.
- The histological analysis is shown in
FIG. 26 . Representative stained sections from either 100 μm (top) or 200 μm (bottom) from the ligation in WT and vehicle or rhEnpp1-treated ttw/ttw are shown from left to right, respectively. Von Gieson's solution stains elastic collagen fibers and distinguishes the internal (IEL) and external elastic lamina (EEL) from the lumen of the vessel (L). In the WT mice, the carotid ligation caused intimal hyperplasia resulting in narrowing of the lumen, with more severe narrowing closer to the ligature (100 μm) and less severe occlusion further away (200 μm). In contrast, in the ttw/ttw mice the degree of intimal hyperplasia appeared to be increased, as the lumen at 200 μm is almost completely occluded. The ttw/ttw mice treated with rhENPP1 showed much less intimal hyperplasia than those treated with vehicle, approaching the degree seen in WT animals. This suggests that the presence of Enpp1 prior to and after the carotid ligation protected against intimal hyperplasia. -
FIGS. 27A-C show morphometric quantitation of the results. Measurement of the circumference of the external and internal elastic lamina and the luminal border allows quantitation of the medial (M) and intimal (I) areas. The medial area, between the external and internal lamina, remained constant (FIG. 27A ). The intimal area within the lumen showed a statistically-significant increase in ttw/ttw and vehicle-treated ttw/ttw mice relative to WT mice (FIG. 27B ). The rhENPP1-treated ttw/ttw mice were similar to WT mice in both the intimal area and the I/M ratio, with the results again being statistically significant (FIG. 27C ). These results support the protective effect of rhENPPP1 when administered prior to carotid ligation. - In order to determine if ENPP1 could have a therapeutic effect if given after the carotid ligation, 6 to 7-week old ttw/ttw mice were subjected to carotid ligation and allowed to recover. rhEnpp1 treatment (10 mg/kg SC every other day) was initiated 7 days following carotid ligation and continued until the carotid arteries were harvested at 14 days post-ligation.
-
FIG. 28A shows the degree of intimal hyperplasia present at 100 and 200 μm 7 days post carotid ligation, prior to the initiation of ENPP1 treatment. Histological assessment of the therapeutic effect of rhENPP1 when initiated at 7 days post ligation is shown inFIG. 28B , with representative sections at 100 μm (top) and 200 μm (bottom) from the ligation in vehicle-treated (left) and rhENPP1-treated (right) ttw/ttw mice presented. Despite the beginning of some intimal hyperplasia in the untreated animals at 7 days post ligation (FIG. 28A ), treatment with rhENPP1 beginning at this point still showed benefit as the degree of luminal occlusion at both 100 and 200 μm was less than in the vehicle-treated animals 14 days post ligation. -
FIG. 29 (A-C) shows the morphometric quantitation of the data. The medial area, between the external and internal lamina, remained constant. The vehicle-treated ttw/ttw mice and rhENPP1-treated ttw/ttw mice had similar intimal area, both showed significant more proliferated intimal area than WT mice (p<0.01 and p<0.05, respectively). The I/M ratio of vehicle-treated ttw/ttw mice was increased compared to WT mice, with the results again being statistically significant. However, the I/MV ratio of rhENPP1-treated ttw/ttw mice was between the levels of WT and vehicle-treated ttw/ttw mice, not significantly different compared to WT and vehicle-treated ttw/ttw mice, indicating a decelerating effect of rhENPP1 on already started intimal proliferation. - In summary, in response to carotid artery ligation for two weeks, vehicle treated ttw/ttw mice showed accelerated neointimal hyperplasia. In contrast, ENPP1-Fc treated carotid ligated ttw/ttw mice displayed a significant reduction in intimal proliferation, comparable to the proliferation level of ligated WT mice. The results demonstrate that subcutaneous administration of recombinant ENPP1-Fc fusion protein prevents intimal hyperplasia in an animal model of GAC. This finding suggests that ENPP1 enzyme replacement is a potential therapeutic approach for treating intimal hyperplasia in GACI patients.
-
SUMMARY OF SEQUENCE LISTING SEQ ID NO: 1 amino acid sequence of wild-type NPP1 protein MERDGCAGGGSRGGEGGRAPREGPAGNGRDRGRSHAAEAPGDPQAAASLLAPMDVGE EPLEKAARARTAKDPNTYKVLSLVLSVCVLTTILGCIFGLK PSCAKEVKSCKGRCFERTF GNCRCDAACVELGNCCLDYQETCIEPEHIWTCNKFRCGEKRLTRSLCACSDDCKDKGD CCINYSSVCQGEKSWVEEPCESINEPQCPAGFETPPTLLFSLDGFRAEYLHTWGGLLPVI SKLKKCGTYTKNMRPVYPTKTFPNHYSIVTGLYPESHGIIDNKMYDPKMNASFSLKSKE KFNPEWYKGEPIWVTAKYQGLKSGTFFWPGSDVEINGIFPDIYKMYNGSVPFEERILAVL QWLQLPKDERPHFYTLYLEEPDSSGHSYGPVSSEVIKALQRVDGMVGMLMDGLKELNL HRCLNLILISDHGMEQGSCKKYIYLNKYLGDVKNIKVIYGPAARLRPSDVPDKYYSFNYE GIARNLSCREPNQHFKPYLKHFLPKRLHFAKSDRIEPLTFYLDPQWQLALNPSERKYCGS GFHGSDNVFSNMQALFVGYGPGFKHGIEADTFENIEVYNLMCDLLNLTPAPNNGTHGSL NHLLKNPVYTPKHPKEVHPLVQCPFTRNPRDNLGCSCNPSILPIEDFQTQFNLTVAEEKIIK HETLPYGRPRVLQKENTICLLSQHQFMSGYSQDILMPLWTSYTVDRNDSFSTEDFSNCLY QDFRIPLSPVHKCSFYKNNTKVSYGFLSPPQLNKNSSGIYSEALLTTNIVPMYQSFQVIWR YFHDTLLRKYAEERNGVNVVSGPVFDFDYDGRCDSLENLRQKRRVIRNQEILIPTHFFIVL TSCKDTSQTPLHCENLDTLAFILPHRTDNSESCVHGKHDSSWVEELLMLHRARITDVEHI TGLSFYQQRKEPVSDILKLKTHLPTFSQED SEQ ID NO: 2 amino acid sequence of sNPP1 that contains cysteine-rich region, catalytic region and c-terminal region PSCAKEVKSCKGRCFERTFGNCRCDAACVELGNCCLDYQETCIEPEHIWTCNKFRCGEK RLTRSLCACSDDCKDKGDCCINYSSVCQGEKSWVEEPCESINEPQCPAGFETPPTLLFSL DGFRAEYLHTWGGLLPVISKLKKCGTYTKNMRPVYPTKTFPNHYSIVTGLYPESHGIIDN KMYDPKMNASFSLKSKEKFNPEWYKGEPIWVTAKYQGLKSGTFFWPGSDVEINGIFPDI YKMYNGSVPFEERILAVLQWLQLPKDERPHFYTLYLEEPDSSGHSYGPVSSEVIKALQR VDGMVGMLMDGLKELNLHRCLNLILISDHGMEQGSCKKYIYLNKYLGDVKNIKVIYGP AARLRPSDVPDKYYSFNYEGIARNLSCREPNQHFKPYLKHFLPKRLHFAKSDRIEPLTFY LDPQWQLALNPSERKYCGSGFHGSDNVFSNMQALFVGYGPGFKHGIEADTFENIEVYN LMCDLLNLTPAPNNGTHGSLNHLLKNPVYTPKHPKEVHPLVQCPFTRNPRDNLGCSCNP SILPIEDFQTQFNLTVAEEKIIKHETLPYGRPRVLQKENTICLLSQHQFMSGYSQDILMPL WTSYTVDRNDSFSTEDFSNCLYQDFRIPLSPVHKCSFYKNNTKVSYGFLSPPQLNKNSSG IYSEALLTTNIVPMYQSFQVIWRYFHDTLLRKYAEERNGVNVVSGPVFDFDYDGRCDSL ENLRQKRRVIRNQEILIPTHFFIVLTSCKDTSQTPLHCENLDTLAFILPHRTDNSESCVHGK HDSSWVEELLMLHRARITDVEHITGLSFYQQRKEPVSDILKLKTHLPTFSQED SEQ ID NO: 3 amino acid sequence of sNPP1-Fc fusion protein PSCAKEVKSCKGRCFERTFGNCRCDAACVELGNCCLDYQETCIEPEHIWTCNKFRCGEK RLTRSLCACSDDCKDKGDCCINYSSVCQGEKSWVEEPCESINEPQCPAGFETPPTLLFSL DGFRAEYLHTWGGLLPVISKLKKCGTYTKNMRPVYPTKTFPNHYSIVTGLYPESHGIIDN KMYDPKMNASFSLKSKEKFNPEWYKGEPIWVTAKYQGLKSGTFFWPGSDVEINGIFPDI YKMYNGSVPFEERILAVLQWLQLPKDERPHFYTLYLEEPDSSGHSYGPVSSEVIKALQR VDGMVGMLMDGLKELNLHRCLNLILISDHGMEQGSCKKYIYLNKYLGDVKNIKVIYGP AARLRPSDVPDKYYSFNYEGIARNLSCREPNQHFKPYLKHFLPKRLHFAKSDRIEPLTFY LDPQWQLALNPSERKYCGSGFHGSDNVFSNMQALFVGYGPGFKHGIEADTFENIEVYN LMCDLLNLTPAPNNGTHGSLNHLLKNPVYTPKHPKEVHPLVQCPFTRNPRDNLGCSCNP SILPIEDFQTQFNLTVAEEKIIKHETLPYGRPRVLQKENTICLLSQHQFMSGYSQDILMPL WTSYTVDRNDSFSTEDFSNCLYQDFRIPLSPVHKCSFYKNNTKVSYGFLSPPQLNKNSSG IYSEALLTTNIVPMYQSFQVIWRYFHDTLLRKYAEERNGVNVVSGPVFDFDYDGRCDSL ENLRQKRRVIRNQEILIPTHFFIVLTSCKDTSQTPLHCENLDTLAFILPHRTDNSESCVHGK HDSSWVEELLMLHRARITDVEHITGLSFYQQRKEPVSDILKLKTHLPTFSQEDPKSCDKT HTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK GQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 4 amino acid sequence of sNPP1-Fc-D10 PSCAKEVKSCKGRCFERTFGNCRCDAACVELGNCCLDYQETCIEPEHIWTCNKFRCGEK RLTRSLCACSDDCKDKGDCCINYSSVCQGEKSWVEEPCESINEPQCPAGFETPPTLLFSL DGFRAEYLHTWGGLLPVISKLKKCGTYTKNMRPVYPTKTFPNHYSIVTGLYPESHGIIDN KMYDPKMNASFSLKSKEKFNPEWYKGEPIWVTAKYQGLKSGTFFWPGSDVEINGIFPDI YKMYNGSVPFEERILAVLQWLQLPKDERPHFYTLYLEEPDSSGHSYGPVSSEVIKALQR VDGMVGMLMDGLKELNLHRCLNLILISDHGMEQGSCKKYIYLNKYLGDVKNIKVIYGP AARLRPSDVPDKYYSFNYEGIARNLSCREPNQHFKPYLKHFLPKRLHFAKSDRIEPLTFY LDPQWQLALNPSERKYCGSGFHGSDNVFSNMQALFVGYGPGFKHGIEADTFENIEVYN LMCDLLNLTPAPNNGTHGSLNHLLKNPVYTPKHPKEVHPLVQCPFTRNPRDNLGCSCNP SILPIEDFQTQFNLTVAEEKIIKHETLPYGRPRVLQKENTICLLSQHQFMSGYSQDILMPL WTSYTVDRNDSFSTEDFSNCLYQDFRIPLSPVHKCSFYKNNTKVSYGFLSPPQLNKNSSG IYSEALLTTNIVPMYQSFQVIWRYFHDTLLRKYAEERNGVNVVSGPVFDFDYDGRCDSL ENLRQKRRVIRNQEILIPTHFFIVLTSCKDTSQTPLHCENLDTLAFILPHRTDNSESCVHGK HDSSWVEELLMLHRARITDVEHITGLSFYQQRKEPVSDILKLKTHLPTFSQEDPKSCDKT HTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK GQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTOKSLSLSPGK DDDDDDDDDD SEQ ID NO: 5 amino acid sequences of soluble NPP1 containing amino acids from 107 to 925 of SEQ ID NO: 1 SCKGRCFERTFGNCRCDAACVELGNCCLDYQETCIEPEHIWTCNKFRCGEKRLTRSLCA CSDDCKDKGDCCINYSSVCQGEKSWVEEPCESINEPQCPAGFETPPTLLFSLDGFRAEYL HTWGGLLPVISKLKKCGTYTKNMRPVYPTKTFPNHYSIVTGLYPESHGIIDNKMYDPKM NASFSLKSKEKFNPEWYKGEPIWVTAKYQGLKSGTFFWPGSDVEINGIFPDIYKMYNGS VPFEERILAVLQWLQLPKDERPHFYTLYLEEPDSSGHSYGPVSSEVIKALQRVDGMVGML MDGLKELNLHRCLNLILISDHGMEQGSCKKYIYLNKYLGDVKNIKVIYGPAARLRPSDV PDKYYSFNYEGIARNLSCREPNQHFKPYLKHFLPKRLHFAKSDRIEPLTFYLDPQWQLAL NPSERKYCGSGFHGSDNVFSNMQALFVGYGPGFKHGIEADTFENIEVYNLMCDLLNLTP APNNGTHGSLNHLLKNPVYTPKHPKEVHPLVQCPFTRNPRDNLGCSCNPSILPIEDFQTQ FNLTVAEEKIIKHETLPYGRPRVLQKENTICLLSQHQFMSGYSQDILMPLWTSYTVDRND SFSTEDFSNCLYQDFRIPLSPVHKCSFYKNNTKVSYGFLSPPQLNKNSSGIYSEALLTTNIV PMYQSFQVIWRYFHDTLLRKYAEERNGVNVVSGPVFDFDYDGRCDSLENLRQKRRVIR NQEILIPTHFFIVLTSCKDTSQTPLHCENLDTLAFILPHRTDNSESCVHGKHDSSWVEELL MLHRARITDVEHITGLSFYQQRKEPVSDILKLKTHLPTFSQED SEQ ID NO: 6 amino acid sequence of soluble NPP1 containing amino acids from 187 to 925 of SEQ ID NO: 1 EKSWVEEPCESINEPQCPAGFETPPTLLFSLDGFRAEYLHTWGGLLPVISKLKKCGTYTK NMRPVYPTKTFPNHYSIVTGLYPESHGIIDNKMYDPKMNASFSLKSKEKFNPEWYKGEPI WVTAKYQGLKSGTFFWPGSDVEINGIFPDIYKMYNGSVPFEERILAVLQWLQLPKDERP HFYTLYLEEPDSSGHSYGPVSSEVIKALQRVDGMVGMLMDGLKELNLHRCLNLILISDH GMEQGSCKKYIYLNKYLGDVKNIKVIYGPAARLRPSDVPDKYYSFNYEGIARNLSCREP NQHFKPYLKHFLPKRLHFAKSDRIEPLTFYLDPQWQLALNPSERKYCGSGFHGSDNVFS NMQALFVGYGPGFKHGIEADTFENIEVYNLMCDLLNLTPAPNNGTHGSLNHLLKNPVY TPKHPKEVHPLVQCPFTRNPRDNLGCSCNPSILPIEDFQTQFNLTVAEEKIIKHETLPYGRP RVLQKENTICLLSQHQFMSGYSQDILMPLWTSYTVDRNDSFSTEDFSNCLYQDFRIPLSP VHKCSFYKNNTKVSYGFLSPPQLNKNSSGIYSEALLTTNIVPMYQSFQVIWRYFHDTLLR KYAEERNGVNVVSGPVFDFDYDGRCDSLENLRQKRRVIRNQEILIPTHFFIVLTSCKDTS QTPLHCENLDTLAFILPHRTDNSESCVHGKHDSSWVEELLMLHRARITDVEHITGLSFYQ QRKEPVSDILKLKTHLPTFSQED SEQ ID NO: 7 amino acid sequence of Fc region of human IgG1 including hinge region EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKF NWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPI EKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNY KTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 8 amino acid sequence of Fc of human IgG1 including partial hinge region DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV DGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP VLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 9 amino acid sequence of NPP1-Fc fusion protein [(107-925)-Fc] SCKGRCFERTFGNCRCDAACVELGNCCLDYQETCIEPEHIWTCNKFRCGEKRLTRSLCA CSDDCKDKGDCCINYSSVCQGEKSWVEEPCESINEPQCPAGFETPPTLLFSLDGFRAEYL HTWGGLLPVISKLKKCGTYTKNMRPVYPTKTFPNHYSIVTGLYPESHGIIDNKMYDPKM NASFSLKSKEKFNPEWYKGEPIWVTAKYQGLKSGTFFWPGSDVEINGIFPDIYKMYNGS VPFEERILAVLQWLQLPKDERPHFYTLYLEEPDSSGHSYGPVSSEVIKALQRVDGMVGML MDGLKELNLHRCLNLILISDHGMEQGSCKKYIYLNKYLGDVKNIKVIYGPAARLRPSDV PDKYYSFNYEGIARNLSCREPNQHFKPYLKHFLPKRLHFAKSDRIEPLTFYLDPQWQLAL NPSERKYCGSGFHGSDNVFSNMQALFVGYGPGFKHGIEADTFENIEVYNLMCDLLNLTP APNNGTHGSLNHLLKNPVYTPKHPKEVHPLVQCPFTRNPRDNLGCSCNPSILPIEDFQTQ FNLTVAEEKIIKHETLPYGRPRVLQKENTICLLSQHQFMSGYSQDILMPLWTSYTVDRND SFSTEDFSNCLYQDFRIPLSPVHKCSFYKNNTKVSYGFLSPPQLNKNSSGIYSEALLTTNIV PMYQSFQVIWRYFHDTLLRKYAEERNGVNVVSGPVFDFDYDGRCDSLENLRQKRRVIR NQEILIPTHFFIVLTSCKDTSQTPLHCENLDTLAFILPHRTDNSESCVHGKHDSSWVEELL MLHRARITDVEHITGLSFYQQRKEPVSDILKLKTHLPTFSQEDEPKSCDKTHTCPPCPAPE LLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPR EEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTL PPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLT VDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 10 amino acid sequence of NPPl-Fc fusion protein [(107-925)-partial hinge Fc] SCKGRCFERTFGNCRCDAACVELGNCCLDYQETCIEPEHIWTCNKFRCGEKRLTRSLCA CSDDCKDKGDCCINYSSVCQGEKSWVEEPCESINEPQCPAGFETPPTLLFSLDGFRAEYL HTWGGLLPVISKLKKCGTYTKNMRPVYPTKTFPNHYSIVTGLYPESHGIIDNKMYDPKM NASFSLKSKEKFNPEWYKGEPIWVTAKYQGLKSGTFFWPGSDVEINGIFPDIYKMYNGS VPFEERILAVLQWLQLPKDERPHFYTLYLEEPDSSGHSYGPVSSEVIKALQRVDGMVGML MDGLKELNLHRCLNLILISDHGMEQGSCKKYIYLNKYLGDVKNIKVIYGPAARLRPSDV PDKYYSFNYEGIARNLSCREPNQHFKPYLKHFLPKRLHFAKSDRIEPLTFYLDPQWQLAL NPSERKYCGSGFHGSDNVFSNMQALFVGYGPGFKHGIEADTFENIEVYNLMCDLLNLTP APNNGTHGSLNHLLKNPVYTPKHPKEVHPLVQCPFTRNPRDNLGCSCNPSILPIEDFQTQ FNLTVAEEKIIKHETLPYGRPRVLQKENTICLLSQHQFMSGYSQDILMPLWTSYTVDRND SFSTEDFSNCLYQDFRIPLSPVHKCSFYKNNTKVSYGFLSPPQLNKNSSGIYSEALLTTNIV PMYQSFQVIWRYFHDTLLRKYAEERNGVNVVSGPVFDFDYDGRCDSLENLRQKRRVIR NQEILIPTHFFIVLTSCKDTSQTPLHCENLDTLAFILPHRTDNSESCVHGKHDSSWVEELL MLHRARITDVEHITGLSFYQQRKEPVSDILKLKTHLPTFSQEDDKTHTCPPCPAPELLGGP SVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY NSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSR EEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDK SRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 11 amino acid sequence of NPP1-Fc fusion protein [(187-925)-Fc] EKSWVEEPCESINEPQCPAGFETPPTLLFSLDGFRAEYLHTWGGLLPVISKLKKCGTYTK NMRPVYPTKTFPNHYSIVTGLYPESHGIIDNKMYDPKMNASFSLKSKEKFNPEWYKGEPI WVTAKYQGLKSGTFFWPGSDVEINGIFPDIYKMYNGSVPFEERILAVLQWLQLPKDERP HFYTLYLEEPDSSGHSYGPVSSEVIKALQRVDGMVGMLMDGLKELNLHRCLNLILISDH GMEQGSCKKYIYLNKYLGDVKNIKVIYGPAARLRPSDVPDKYYSFNYEGIARNLSCREP NQHFKPYLKHFLPKRLHFAKSDRIEPLTFYLDPQWQLALNPSERKYCGSGFHGSDNVFS NMQALFVGYGPGFKHGIEADTFENIEVYNLMCDLLNLTPAPNNGTHGSLNHLLKNPVY TPKHPKEVHPLVQCPFTRNPRDNLGCSCNPSILPIEDFQTQFNLTVAEEKIIKHETLPYGRP RVLQKENTICLLSQHQFMSGYSQDILMPLWTSYTVDRNDSFSTEDFSNCLYQDFRIPLSP VHKCSFYKNNTKVSYGFLSPPQLNKNSSGIYSEALLTTNIVPMYQSFQVIWRYFHDTLLR KYAEERNGVNVVSGPVFDFDYDGRCDSLENLRQKRRVIRNQEILIPTHFFIVLTSCKDTS QTPLHCENLDTLAFILPHRTDNSESCVHGKHDSSWVEELLMLHRARITDVEHITGLSFYQ QRKEPVSDILKLKTHLPTFSQEDEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMI SRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLH QDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLV KGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSV MHEALHNHYTQKSLSLSPGK SEQ ID NO: 12 amino acid sequence of NPP1-Fc fusion protein [(187-925)-partial hinge Fc] EKSWVEEPCESINEPQCPAGFETPPTLLFSLDGFRAEYLHTWGGLLPVISKLKKCGTYTK NMRPVYPTKTFPNHYSIVTGLYPESHGIIDNKMYDPKMNASFSLKSKEKFNPEWYKGEPI WVTAKYQGLKSGTFFWPGSDVEINGIFPDIYKMYNGSVPFEERILAVLQWLQLPKDERP HFYTLYLEEPDSSGHSYGPVSSEVIKALQRVDGMVGMLMDGLKELNLHRCLNLILISDH GMEQGSCKKYIYLNKYLGDVKNIKVIYGPAARLRPSDVPDKYYSFNYEGIARNLSCREP NQHFKPYLKHFLPKRLHFAKSDRIEPLTFYLDPQWQLALNPSERKYCGSGFHGSDNVFS NMQALFVGYGPGFKHGIEADTFENIEVYNLMCDLLNLTPAPNNGTHGSLNHLLKNPVY TPKHPKEVHPLVQCPFTRNPRDNLGCSCNPSILPIEDFQTQFNLTVAEEKIIKHETLPYGRP RVLQKENTICLLSQHQFMSGYSQDILMPLWTSYTVDRNDSFSTEDFSNCLYQDFRIPLSP VHKCSFYKNNTKVSYGFLSPPQLNKNSSGIYSEALLTTNIVPMYQSFQVIWRYFHDTLLR KYAEERNGVNVVSGPVFDFDYDGRCDSLENLRQKRRVIRNQEILIPTHFFIVLTSCKDTS QTPLHCENLDTLAFILPHRTDNSESCVHGKHDSSWVEELLMLHRARITDVEHITGLSFYQ QRKEPVSDILKLKTHLPTFSQEDDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPE VTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWL NGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFY PSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEA LHNHYTQKSLSLSPGK SEQ ID NO: 13 human IgG1 hinge region EPKSCDKTHTCPPCP SEQ ID NO: 14 portion of human IgG1 hinge region DKTHTCPPCP SEQ ID NO: 15 portion of human IgG1 hinge region PKSCDKTHTCPPCP SEQ ID NO: 16 Linker (Gly4Ser)3 SEQ ID NO: 17 amino acid motif that is start of soluble NPP1 which includes cysteine rich region PSCAKE SEQ ID NO: 18 D10 targeting moiety 19 synthetic linker (Gly4Ser)n,
Claims (13)
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US17/747,379 US20220354933A1 (en) | 2016-06-16 | 2022-05-18 | Methods of treating myointimal proliferation |
Applications Claiming Priority (4)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US201662350936P | 2016-06-16 | 2016-06-16 | |
PCT/US2017/037695 WO2017218786A1 (en) | 2016-06-16 | 2017-06-15 | Methods of treating myointimal proliferation |
US201816309047A | 2018-12-11 | 2018-12-11 | |
US17/747,379 US20220354933A1 (en) | 2016-06-16 | 2022-05-18 | Methods of treating myointimal proliferation |
Related Parent Applications (2)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US16/309,047 Continuation US11364284B2 (en) | 2016-06-16 | 2017-06-15 | Methods of treating myointimal proliferation |
PCT/US2017/037695 Continuation WO2017218786A1 (en) | 2016-06-16 | 2017-06-15 | Methods of treating myointimal proliferation |
Publications (1)
Publication Number | Publication Date |
---|---|
US20220354933A1 true US20220354933A1 (en) | 2022-11-10 |
Family
ID=59258368
Family Applications (2)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US16/309,047 Active 2039-01-18 US11364284B2 (en) | 2016-06-16 | 2017-06-15 | Methods of treating myointimal proliferation |
US17/747,379 Pending US20220354933A1 (en) | 2016-06-16 | 2022-05-18 | Methods of treating myointimal proliferation |
Family Applications Before (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US16/309,047 Active 2039-01-18 US11364284B2 (en) | 2016-06-16 | 2017-06-15 | Methods of treating myointimal proliferation |
Country Status (3)
Country | Link |
---|---|
US (2) | US11364284B2 (en) |
EP (2) | EP3471747A1 (en) |
WO (1) | WO2017218786A1 (en) |
Families Citing this family (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP3471747A1 (en) | 2016-06-16 | 2019-04-24 | Alexion Pharmaceuticals, Inc. | Methods of treating myointimal proliferation |
WO2019067502A1 (en) * | 2017-09-27 | 2019-04-04 | Alexion Pharmaceuticals, Inc. | Methods of improving cardiovascular function and treating cardiovascular disease using a recombinant ectonucleotide pyrophosphatase phosphodiesterase (npp1) |
KR20230047334A (en) * | 2020-05-27 | 2023-04-07 | 이노자임 파마, 인코포레이티드 | Compositions and methods for inhibiting vascular smooth muscle cell proliferation |
MX2022015772A (en) * | 2020-06-09 | 2023-05-19 | Inozyme Pharma Inc | Soluble enpp1 or enpp3 proteins and uses thereof. |
Family Cites Families (25)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US4751180A (en) | 1985-03-28 | 1988-06-14 | Chiron Corporation | Expression using fused genes providing for protein product |
US4935233A (en) | 1985-12-02 | 1990-06-19 | G. D. Searle And Company | Covalently linked polypeptide cell modulators |
SE509359C2 (en) | 1989-08-01 | 1999-01-18 | Cemu Bioteknik Ab | Use of stabilized protein or peptide conjugates for the preparation of a drug |
US6267964B1 (en) | 1989-08-01 | 2001-07-31 | Affibody Technology Sweden Ab | Stabilized protein or peptide conjugates able to bond albumin having extended biological half-lives |
DK1032662T3 (en) | 1997-11-07 | 2006-07-03 | Trillium Therapeutics Inc | Methods and Compositions for Immunomodulation |
US6272859B1 (en) | 1998-10-02 | 2001-08-14 | Caterpillar Inc. | Device for controlling a variable geometry turbocharger |
EP1240337B1 (en) | 1999-12-24 | 2006-08-23 | Genentech, Inc. | Methods and compositions for prolonging elimination half-times of bioactive compounds |
JP4336771B2 (en) | 2001-03-09 | 2009-09-30 | モルフォシス アーゲー | Serum albumin binding moiety |
US7858297B2 (en) | 2001-12-18 | 2010-12-28 | Centre National De La Recherche Scientifique Cnrs | Chemokine-binding protein and methods of use |
AU2003213246A1 (en) | 2002-02-21 | 2003-09-09 | University Of Virginia Patent Foundation | Bone targeting peptides |
EP1572936A2 (en) | 2002-03-05 | 2005-09-14 | Eli Lilly And Company | Heterologous g-csf fusion proteins |
EP1576172A2 (en) | 2002-06-21 | 2005-09-21 | Dyax Corporation | Serum protein-associated target-specific ligands and identification method therefor |
WO2004100987A2 (en) | 2003-05-06 | 2004-11-25 | Regeneron Pharmaceuticals, Inc. | Methods of using il-1 antagonists to treat neointimal hyperplasia |
NZ548612A (en) | 2004-02-09 | 2009-11-27 | Human Genome Sciences Inc | Albumin fusion proteins comprising tandem GLP-1 |
US20050287284A1 (en) | 2004-06-28 | 2005-12-29 | Shukla Triveni P | Processed meats comprising dietary fiber gel |
US20090142347A1 (en) | 2004-09-29 | 2009-06-04 | The Burnham Institute For Medical Research | Tissue-Nonspecific Alkaline Phosphatase (TNAP): a Therapeutic Target for Arterial Calcification |
AU2007293614A1 (en) | 2006-09-08 | 2008-03-13 | Ablynx N.V. | Serum albumin binding proteins with long half-lives |
ES2628841T3 (en) | 2010-03-12 | 2017-08-04 | Alexion Pharmaceuticals, Inc. | Npp1 fusion proteins |
WO2012125182A1 (en) | 2011-03-11 | 2012-09-20 | Synageva Biopharma Corp | Npp1 fusion proteins |
WO2014126965A2 (en) | 2013-02-13 | 2014-08-21 | Yale University | Compositions and methods for treating pathological calcification and ossification |
ES2899895T3 (en) | 2014-12-19 | 2022-03-15 | Inozyme Pharma Inc | Tissue Calcification Treatment Procedures |
EP3298140B1 (en) | 2015-05-19 | 2024-04-24 | Yale University | Compositions for treating pathological calcification conditions, and methods using same |
US20180371434A1 (en) | 2015-11-20 | 2018-12-27 | Yale University | Compositions for Treating Ectopic Calcification Disorders, and Methods Using Same |
EP3471747A1 (en) | 2016-06-16 | 2019-04-24 | Alexion Pharmaceuticals, Inc. | Methods of treating myointimal proliferation |
WO2019067502A1 (en) | 2017-09-27 | 2019-04-04 | Alexion Pharmaceuticals, Inc. | Methods of improving cardiovascular function and treating cardiovascular disease using a recombinant ectonucleotide pyrophosphatase phosphodiesterase (npp1) |
-
2017
- 2017-06-15 EP EP17734573.3A patent/EP3471747A1/en not_active Withdrawn
- 2017-06-15 WO PCT/US2017/037695 patent/WO2017218786A1/en active Search and Examination
- 2017-06-15 US US16/309,047 patent/US11364284B2/en active Active
- 2017-06-15 EP EP20199861.4A patent/EP3827835A1/en active Pending
-
2022
- 2022-05-18 US US17/747,379 patent/US20220354933A1/en active Pending
Also Published As
Publication number | Publication date |
---|---|
US20200306349A1 (en) | 2020-10-01 |
EP3827835A1 (en) | 2021-06-02 |
EP3471747A1 (en) | 2019-04-24 |
WO2017218786A1 (en) | 2017-12-21 |
US11364284B2 (en) | 2022-06-21 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
US20220354933A1 (en) | Methods of treating myointimal proliferation | |
US10577406B2 (en) | FKBP-L polypeptides and uses in angiogenesis-mediated disorders | |
RU2770698C2 (en) | Methods of treating tissue calcification | |
EP2750686B1 (en) | Polypeptide comprising frataxin and a c-terminal mitochondria penetrating peptide for use in the treatment of friedreich's ataxia | |
ES2356286T3 (en) | COMPOSITIONS TO INHIBIT THE C-ALFA PROTEINCINASE FOR THE TREATMENT OF MELLITUS DIABETES. | |
JP2010536852A (en) | Regulation of synapse formation | |
EP3074034B1 (en) | Treatment of inflammatory disease using caveolin linked to an internalisation peptide | |
ES2831031T3 (en) | Fusion proteins for CNS treatment | |
US10550151B2 (en) | Cell-penetrating compositions and methods using same | |
Kim et al. | Newly designed Protein Transduction Domain (PTD)‐mediated BMP‐7 is a potential therapeutic for peritoneal fibrosis | |
WO2019035919A1 (en) | Compositions and methods for inhibition of l-plastin activity in osteoclasts to reduce bone loss | |
Min et al. | Newly synthesized peptide, Ara‐27, exhibits significant improvement in cell‐penetrating ability compared to conventional peptides | |
US20200369726A1 (en) | Peptides and other agents for treating pain and increasing pain sensitivity | |
US9987327B2 (en) | Methods for treating myocardial infarction comprising administering sonic hedgehog | |
WO2015044379A1 (en) | A dyrk1a polypeptide for use in preventing or treating metabolic disorders |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
AS | Assignment |
Owner name: ALEXION PHARMACEUTICALS, INC., CONNECTICUT Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNORS:YAN, YAN;SHAH, ANUMEHA;SAIGAL, ASHMITA;AND OTHERS;SIGNING DATES FROM 20170619 TO 20180209;REEL/FRAME:060650/0966 Owner name: INOZYME PHARMA, INC., MASSACHUSETTS Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNOR:ALEXION PHARMACEUTICALS, INC.;REEL/FRAME:060650/0836 Effective date: 20200831 Owner name: WESTFAELISCHE WILHELMS-UNIVERSITAET MUENSTER, GERMANY Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNORS:RUTSCH, FRANK;NITSCHKE, YVONNE;REEL/FRAME:060650/0826 Effective date: 20181204 |
|
STPP | Information on status: patent application and granting procedure in general |
Free format text: DOCKETED NEW CASE - READY FOR EXAMINATION |