WO2017214579A1 - Compositions and methods for treating osteoporesis - Google Patents
Compositions and methods for treating osteoporesis Download PDFInfo
- Publication number
- WO2017214579A1 WO2017214579A1 PCT/US2017/036876 US2017036876W WO2017214579A1 WO 2017214579 A1 WO2017214579 A1 WO 2017214579A1 US 2017036876 W US2017036876 W US 2017036876W WO 2017214579 A1 WO2017214579 A1 WO 2017214579A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- bone
- cbi
- increasing
- osteoporosis
- cobinamide
- Prior art date
Links
- 239000000203 mixture Substances 0.000 title claims abstract description 67
- 238000000034 method Methods 0.000 title claims abstract description 29
- 210000000988 bone and bone Anatomy 0.000 claims abstract description 107
- 230000001965 increasing effect Effects 0.000 claims abstract description 81
- 210000002997 osteoclast Anatomy 0.000 claims abstract description 76
- ZOOGRGPOEVQQDX-UUOKFMHZSA-N 3',5'-cyclic GMP Chemical compound C([C@H]1O2)OP(O)(=O)O[C@H]1[C@@H](O)[C@@H]2N1C(N=C(NC2=O)N)=C2N=C1 ZOOGRGPOEVQQDX-UUOKFMHZSA-N 0.000 claims abstract description 47
- 230000014509 gene expression Effects 0.000 claims abstract description 40
- 210000000963 osteoblast Anatomy 0.000 claims abstract description 39
- 206010030247 Oestrogen deficiency Diseases 0.000 claims abstract description 38
- 230000004069 differentiation Effects 0.000 claims abstract description 33
- 150000003839 salts Chemical class 0.000 claims abstract description 33
- 208000001132 Osteoporosis Diseases 0.000 claims abstract description 32
- 230000011164 ossification Effects 0.000 claims abstract description 32
- 210000004409 osteocyte Anatomy 0.000 claims abstract description 32
- 230000006907 apoptotic process Effects 0.000 claims abstract description 26
- 229910052500 inorganic mineral Inorganic materials 0.000 claims abstract description 26
- 210000002966 serum Anatomy 0.000 claims abstract description 26
- 239000011707 mineral Substances 0.000 claims abstract description 25
- 206010065687 Bone loss Diseases 0.000 claims abstract description 24
- 239000012453 solvate Substances 0.000 claims abstract description 24
- 230000001582 osteoblastic effect Effects 0.000 claims abstract description 20
- 239000008194 pharmaceutical composition Substances 0.000 claims abstract description 17
- 238000009472 formulation Methods 0.000 claims abstract description 14
- 208000001685 postmenopausal osteoporosis Diseases 0.000 claims abstract description 14
- 230000002401 inhibitory effect Effects 0.000 claims abstract description 13
- 230000001105 regulatory effect Effects 0.000 claims abstract description 10
- 239000003814 drug Substances 0.000 claims description 29
- 239000007788 liquid Substances 0.000 claims description 15
- 239000003826 tablet Substances 0.000 claims description 14
- 239000002502 liposome Substances 0.000 claims description 13
- 239000007924 injection Substances 0.000 claims description 11
- 238000002347 injection Methods 0.000 claims description 11
- 239000007943 implant Substances 0.000 claims description 10
- 238000001802 infusion Methods 0.000 claims description 8
- 229940124597 therapeutic agent Drugs 0.000 claims description 8
- 239000002105 nanoparticle Substances 0.000 claims description 7
- 239000002775 capsule Substances 0.000 claims description 6
- 238000004519 manufacturing process Methods 0.000 claims description 5
- 230000001225 therapeutic effect Effects 0.000 claims description 5
- 238000007912 intraperitoneal administration Methods 0.000 claims description 4
- 229940090046 jet injector Drugs 0.000 claims description 4
- 229940090048 pen injector Drugs 0.000 claims description 4
- 239000000843 powder Substances 0.000 claims description 4
- 239000007921 spray Substances 0.000 claims description 4
- 238000007920 subcutaneous administration Methods 0.000 claims description 4
- 239000000443 aerosol Substances 0.000 claims description 3
- 239000003937 drug carrier Substances 0.000 claims description 3
- 239000008187 granular material Substances 0.000 claims description 3
- 239000002245 particle Substances 0.000 claims description 3
- 239000006187 pill Substances 0.000 claims description 3
- 239000000829 suppository Substances 0.000 claims description 3
- 235000001892 vitamin D2 Nutrition 0.000 claims description 3
- 239000011324 bead Substances 0.000 claims description 2
- 238000007918 intramuscular administration Methods 0.000 claims description 2
- 238000007913 intrathecal administration Methods 0.000 claims description 2
- 238000001990 intravenous administration Methods 0.000 claims description 2
- 239000011859 microparticle Substances 0.000 claims description 2
- 239000000546 pharmaceutical excipient Substances 0.000 claims description 2
- 238000007910 systemic administration Methods 0.000 claims description 2
- 150000001875 compounds Chemical class 0.000 abstract description 25
- 241000699670 Mus sp. Species 0.000 description 107
- MWUXSHHQAYIFBG-UHFFFAOYSA-N Nitric oxide Chemical compound O=[N] MWUXSHHQAYIFBG-UHFFFAOYSA-N 0.000 description 98
- 210000004027 cell Anatomy 0.000 description 58
- 239000003981 vehicle Substances 0.000 description 56
- ZOOGRGPOEVQQDX-UHFFFAOYSA-N cyclic GMP Natural products O1C2COP(O)(=O)OC2C(O)C1N1C=NC2=C1NC(N)=NC2=O ZOOGRGPOEVQQDX-UHFFFAOYSA-N 0.000 description 38
- 230000000694 effects Effects 0.000 description 36
- 238000002474 experimental method Methods 0.000 description 35
- 108010025832 RANK Ligand Proteins 0.000 description 31
- 102000014128 RANK Ligand Human genes 0.000 description 31
- 108020004999 messenger RNA Proteins 0.000 description 31
- 102000004654 Cyclic GMP-Dependent Protein Kinases Human genes 0.000 description 26
- 108010003591 Cyclic GMP-Dependent Protein Kinases Proteins 0.000 description 26
- 230000001054 cortical effect Effects 0.000 description 20
- 239000002609 medium Substances 0.000 description 20
- 229940079593 drug Drugs 0.000 description 19
- 210000002303 tibia Anatomy 0.000 description 19
- 108010032050 Tartrate-Resistant Acid Phosphatase Proteins 0.000 description 17
- 108010035042 Osteoprotegerin Proteins 0.000 description 16
- 102000007591 Tartrate-Resistant Acid Phosphatase Human genes 0.000 description 16
- 102100032236 Tumor necrosis factor receptor superfamily member 11B Human genes 0.000 description 16
- XXUPLYBCNPLTIW-UHFFFAOYSA-N octadec-7-ynoic acid Chemical compound CCCCCCCCCCC#CCCCCCC(O)=O XXUPLYBCNPLTIW-UHFFFAOYSA-N 0.000 description 16
- 239000000243 solution Substances 0.000 description 16
- 102000002260 Alkaline Phosphatase Human genes 0.000 description 15
- 108020004774 Alkaline Phosphatase Proteins 0.000 description 15
- 239000003795 chemical substances by application Substances 0.000 description 15
- 230000035755 proliferation Effects 0.000 description 15
- 102000004067 Osteocalcin Human genes 0.000 description 14
- 108090000573 Osteocalcin Proteins 0.000 description 14
- 108090000623 proteins and genes Proteins 0.000 description 14
- XQRJFEVDQXEIAX-JFYQDRLCSA-M cobinamide Chemical compound [Co]N([C@@H]1[C@H](CC(N)=O)[C@@]2(C)CCC(=O)NC[C@H](O)C)\C2=C(C)/C([C@H](C\2(C)C)CCC(N)=O)=N/C/2=C\C([C@H]([C@@]/2(CC(N)=O)C)CCC(N)=O)=N\C\2=C(C)/C2=N[C@]1(C)[C@@](C)(CC(N)=O)[C@@H]2CCC(N)=O XQRJFEVDQXEIAX-JFYQDRLCSA-M 0.000 description 13
- 108060000903 Beta-catenin Proteins 0.000 description 12
- 102000015735 Beta-catenin Human genes 0.000 description 12
- 238000011529 RT qPCR Methods 0.000 description 12
- 102000013814 Wnt Human genes 0.000 description 12
- 108050003627 Wnt Proteins 0.000 description 12
- 238000002360 preparation method Methods 0.000 description 12
- 108020004463 18S ribosomal RNA Proteins 0.000 description 11
- 239000003623 enhancer Substances 0.000 description 11
- -1 phospho Chemical class 0.000 description 11
- 230000002829 reductive effect Effects 0.000 description 11
- 230000004083 survival effect Effects 0.000 description 11
- CIWBSHSKHKDKBQ-JLAZNSOCSA-N Ascorbic acid Chemical compound OC[C@H](O)[C@H]1OC(=O)C(O)=C1O CIWBSHSKHKDKBQ-JLAZNSOCSA-N 0.000 description 10
- 108010051219 Cre recombinase Proteins 0.000 description 10
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 10
- 241001529936 Murinae Species 0.000 description 10
- 239000000262 estrogen Substances 0.000 description 10
- 238000009806 oophorectomy Methods 0.000 description 10
- 239000007787 solid Substances 0.000 description 10
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 10
- 230000004913 activation Effects 0.000 description 9
- 230000003247 decreasing effect Effects 0.000 description 9
- 229940011871 estrogen Drugs 0.000 description 9
- 230000011664 signaling Effects 0.000 description 9
- 238000011282 treatment Methods 0.000 description 9
- 238000001262 western blot Methods 0.000 description 9
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 8
- 241001465754 Metazoa Species 0.000 description 8
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 8
- 241000700605 Viruses Species 0.000 description 8
- 238000010603 microCT Methods 0.000 description 8
- 150000002823 nitrates Chemical class 0.000 description 8
- 210000004940 nucleus Anatomy 0.000 description 8
- 229940082615 organic nitrates used in cardiac disease Drugs 0.000 description 8
- 230000026731 phosphorylation Effects 0.000 description 8
- 238000006366 phosphorylation reaction Methods 0.000 description 8
- 239000000126 substance Substances 0.000 description 8
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 7
- DBMJMQXJHONAFJ-UHFFFAOYSA-M Sodium laurylsulphate Chemical compound [Na+].CCCCCCCCCCCCOS([O-])(=O)=O DBMJMQXJHONAFJ-UHFFFAOYSA-M 0.000 description 7
- DEGAKNSWVGKMLS-UHFFFAOYSA-N calcein Chemical compound O1C(=O)C2=CC=CC=C2C21C1=CC(CN(CC(O)=O)CC(O)=O)=C(O)C=C1OC1=C2C=C(CN(CC(O)=O)CC(=O)O)C(O)=C1 DEGAKNSWVGKMLS-UHFFFAOYSA-N 0.000 description 7
- 235000010980 cellulose Nutrition 0.000 description 7
- 229920002678 cellulose Polymers 0.000 description 7
- 238000003125 immunofluorescent labeling Methods 0.000 description 7
- 238000000338 in vitro Methods 0.000 description 7
- 229960002378 oftasceine Drugs 0.000 description 7
- 230000036542 oxidative stress Effects 0.000 description 7
- 102000004169 proteins and genes Human genes 0.000 description 7
- 235000019333 sodium laurylsulphate Nutrition 0.000 description 7
- 210000000689 upper leg Anatomy 0.000 description 7
- 108010001789 Calcitonin Receptors Proteins 0.000 description 6
- 102100038520 Calcitonin receptor Human genes 0.000 description 6
- 102000003952 Caspase 3 Human genes 0.000 description 6
- 108090000397 Caspase 3 Proteins 0.000 description 6
- 108090000259 Cyclin D Proteins 0.000 description 6
- 102000001770 Low Density Lipoprotein Receptor-Related Protein-5 Human genes 0.000 description 6
- 108010015167 Low Density Lipoprotein Receptor-Related Protein-5 Proteins 0.000 description 6
- 229910002651 NO3 Inorganic materials 0.000 description 6
- NHNBFGGVMKEFGY-UHFFFAOYSA-N Nitrate Chemical compound [O-][N+]([O-])=O NHNBFGGVMKEFGY-UHFFFAOYSA-N 0.000 description 6
- 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 6
- 102000004243 Tubulin Human genes 0.000 description 6
- 108090000704 Tubulin Proteins 0.000 description 6
- 239000013543 active substance Substances 0.000 description 6
- 238000004458 analytical method Methods 0.000 description 6
- 239000007864 aqueous solution Substances 0.000 description 6
- 230000008901 benefit Effects 0.000 description 6
- 230000002950 deficient Effects 0.000 description 6
- 230000001419 dependent effect Effects 0.000 description 6
- 238000001727 in vivo Methods 0.000 description 6
- 230000003834 intracellular effect Effects 0.000 description 6
- 238000002372 labelling Methods 0.000 description 6
- 150000002632 lipids Chemical class 0.000 description 6
- 238000011068 loading method Methods 0.000 description 6
- 239000000463 material Substances 0.000 description 6
- 238000013042 tunel staining Methods 0.000 description 6
- ZDJHIEHUVPCEDK-IDTAVKCVSA-N 8-(4-chlorophenylthio)-cGMP Chemical compound N1([C@H]2[C@@H]([C@@H]3OP(O)(=O)OC[C@H]3O2)O)C=2NC(N)=NC(=O)C=2N=C1SC1=CC=C(Cl)C=C1 ZDJHIEHUVPCEDK-IDTAVKCVSA-N 0.000 description 5
- 208000006386 Bone Resorption Diseases 0.000 description 5
- 102000003910 Cyclin D Human genes 0.000 description 5
- 108020004414 DNA Proteins 0.000 description 5
- 238000002965 ELISA Methods 0.000 description 5
- 108010046938 Macrophage Colony-Stimulating Factor Proteins 0.000 description 5
- 102100028123 Macrophage colony-stimulating factor 1 Human genes 0.000 description 5
- IOVCWXUNBOPUCH-UHFFFAOYSA-M Nitrite anion Chemical compound [O-]N=O IOVCWXUNBOPUCH-UHFFFAOYSA-M 0.000 description 5
- SNIOPGDIGTZGOP-UHFFFAOYSA-N Nitroglycerin Chemical compound [O-][N+](=O)OCC(O[N+]([O-])=O)CO[N+]([O-])=O SNIOPGDIGTZGOP-UHFFFAOYSA-N 0.000 description 5
- 239000000006 Nitroglycerin Substances 0.000 description 5
- 102000003982 Parathyroid hormone Human genes 0.000 description 5
- 108090000445 Parathyroid hormone Proteins 0.000 description 5
- 229920002472 Starch Polymers 0.000 description 5
- 230000001195 anabolic effect Effects 0.000 description 5
- 230000001640 apoptogenic effect Effects 0.000 description 5
- 235000010323 ascorbic acid Nutrition 0.000 description 5
- 239000011668 ascorbic acid Substances 0.000 description 5
- 210000001185 bone marrow Anatomy 0.000 description 5
- 230000024279 bone resorption Effects 0.000 description 5
- 239000000872 buffer Substances 0.000 description 5
- 239000001913 cellulose Substances 0.000 description 5
- 238000011161 development Methods 0.000 description 5
- 230000018109 developmental process Effects 0.000 description 5
- 239000000839 emulsion Substances 0.000 description 5
- 230000006870 function Effects 0.000 description 5
- 229960003711 glyceryl trinitrate Drugs 0.000 description 5
- 210000004349 growth plate Anatomy 0.000 description 5
- 229920000609 methyl cellulose Polymers 0.000 description 5
- 239000001923 methylcellulose Substances 0.000 description 5
- 235000010981 methylcellulose Nutrition 0.000 description 5
- 230000001089 mineralizing effect Effects 0.000 description 5
- 238000002156 mixing Methods 0.000 description 5
- 239000002840 nitric oxide donor Substances 0.000 description 5
- 230000003647 oxidation Effects 0.000 description 5
- 238000007254 oxidation reaction Methods 0.000 description 5
- 239000000199 parathyroid hormone Substances 0.000 description 5
- 229960001319 parathyroid hormone Drugs 0.000 description 5
- 239000000047 product Substances 0.000 description 5
- 230000004044 response Effects 0.000 description 5
- 235000019698 starch Nutrition 0.000 description 5
- 238000013268 sustained release Methods 0.000 description 5
- 239000012730 sustained-release form Substances 0.000 description 5
- PRDFBSVERLRRMY-UHFFFAOYSA-N 2'-(4-ethoxyphenyl)-5-(4-methylpiperazin-1-yl)-2,5'-bibenzimidazole Chemical compound C1=CC(OCC)=CC=C1C1=NC2=CC=C(C=3NC4=CC(=CC=C4N=3)N3CCN(C)CC3)C=C2N1 PRDFBSVERLRRMY-UHFFFAOYSA-N 0.000 description 4
- 108010085238 Actins Proteins 0.000 description 4
- 102000007469 Actins Human genes 0.000 description 4
- 229920001817 Agar Polymers 0.000 description 4
- 108700028369 Alleles Proteins 0.000 description 4
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 4
- 101000761509 Homo sapiens Cathepsin K Proteins 0.000 description 4
- 102000004264 Osteopontin Human genes 0.000 description 4
- 108010081689 Osteopontin Proteins 0.000 description 4
- 241000700159 Rattus Species 0.000 description 4
- 241000283984 Rodentia Species 0.000 description 4
- 239000008272 agar Substances 0.000 description 4
- 239000003263 anabolic agent Substances 0.000 description 4
- 229940124325 anabolic agent Drugs 0.000 description 4
- 239000011230 binding agent Substances 0.000 description 4
- 239000001768 carboxy methyl cellulose Substances 0.000 description 4
- 239000000969 carrier Substances 0.000 description 4
- FDJOLVPMNUYSCM-WZHZPDAFSA-L cobalt(3+);[(2r,3s,4r,5s)-5-(5,6-dimethylbenzimidazol-1-yl)-4-hydroxy-2-(hydroxymethyl)oxolan-3-yl] [(2r)-1-[3-[(1r,2r,3r,4z,7s,9z,12s,13s,14z,17s,18s,19r)-2,13,18-tris(2-amino-2-oxoethyl)-7,12,17-tris(3-amino-3-oxopropyl)-3,5,8,8,13,15,18,19-octamethyl-2 Chemical compound [Co+3].N#[C-].N([C@@H]([C@]1(C)[N-]\C([C@H]([C@@]1(CC(N)=O)C)CCC(N)=O)=C(\C)/C1=N/C([C@H]([C@@]1(CC(N)=O)C)CCC(N)=O)=C\C1=N\C([C@H](C1(C)C)CCC(N)=O)=C/1C)[C@@H]2CC(N)=O)=C\1[C@]2(C)CCC(=O)NC[C@@H](C)OP([O-])(=O)O[C@H]1[C@@H](O)[C@@H](N2C3=CC(C)=C(C)C=C3N=C2)O[C@@H]1CO FDJOLVPMNUYSCM-WZHZPDAFSA-L 0.000 description 4
- 239000003085 diluting agent Substances 0.000 description 4
- 239000000284 extract Substances 0.000 description 4
- 239000000796 flavoring agent Substances 0.000 description 4
- 239000012737 fresh medium Substances 0.000 description 4
- 239000000499 gel Substances 0.000 description 4
- 230000001976 improved effect Effects 0.000 description 4
- 230000006698 induction Effects 0.000 description 4
- 239000003550 marker Substances 0.000 description 4
- 230000001404 mediated effect Effects 0.000 description 4
- 230000035515 penetration Effects 0.000 description 4
- 238000010186 staining Methods 0.000 description 4
- 230000000638 stimulation Effects 0.000 description 4
- 235000000346 sugar Nutrition 0.000 description 4
- 239000000375 suspending agent Substances 0.000 description 4
- 239000000725 suspension Substances 0.000 description 4
- 241000701161 unidentified adenovirus Species 0.000 description 4
- YNRCBOXEDICOIX-CLFYSBASSA-N (Z)-[bis(2-aminoethyl)amino]-hydroxyimino-oxidoazanium Chemical compound NCCN(CC[NH3+])[N+](\[O-])=N\[O-] YNRCBOXEDICOIX-CLFYSBASSA-N 0.000 description 3
- VOXZDWNPVJITMN-ZBRFXRBCSA-N 17β-estradiol Chemical compound OC1=CC=C2[C@H]3CC[C@](C)([C@H](CC4)O)[C@@H]4[C@@H]3CCC2=C1 VOXZDWNPVJITMN-ZBRFXRBCSA-N 0.000 description 3
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 3
- 230000007730 Akt signaling Effects 0.000 description 3
- 208000010392 Bone Fractures Diseases 0.000 description 3
- 101150011252 CTSK gene Proteins 0.000 description 3
- 229920002134 Carboxymethyl cellulose Polymers 0.000 description 3
- 108090000625 Cathepsin K Proteins 0.000 description 3
- 102000004171 Cathepsin K Human genes 0.000 description 3
- 102100024940 Cathepsin K Human genes 0.000 description 3
- 206010017076 Fracture Diseases 0.000 description 3
- 102000001267 GSK3 Human genes 0.000 description 3
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 3
- 108010014905 Glycogen Synthase Kinase 3 Proteins 0.000 description 3
- 241000282412 Homo Species 0.000 description 3
- 101001128158 Homo sapiens Nanos homolog 2 Proteins 0.000 description 3
- 101001124991 Homo sapiens Nitric oxide synthase, inducible Proteins 0.000 description 3
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 3
- GUBGYTABKSRVRQ-QKKXKWKRSA-N Lactose Natural products OC[C@H]1O[C@@H](O[C@H]2[C@H](O)[C@@H](O)C(O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@H]1O GUBGYTABKSRVRQ-QKKXKWKRSA-N 0.000 description 3
- 235000010643 Leucaena leucocephala Nutrition 0.000 description 3
- 240000007472 Leucaena leucocephala Species 0.000 description 3
- AFVFQIVMOAPDHO-UHFFFAOYSA-N Methanesulfonic acid Chemical compound CS(O)(=O)=O AFVFQIVMOAPDHO-UHFFFAOYSA-N 0.000 description 3
- 102100024193 Mitogen-activated protein kinase 1 Human genes 0.000 description 3
- 241000699666 Mus <mouse, genus> Species 0.000 description 3
- 102100029438 Nitric oxide synthase, inducible Human genes 0.000 description 3
- 102000049398 Vasodilator-stimulated phosphoproteins Human genes 0.000 description 3
- 229930003779 Vitamin B12 Natural products 0.000 description 3
- 235000010443 alginic acid Nutrition 0.000 description 3
- 229920000615 alginic acid Polymers 0.000 description 3
- 229940072107 ascorbate Drugs 0.000 description 3
- 239000003833 bile salt Substances 0.000 description 3
- 230000004071 biological effect Effects 0.000 description 3
- 210000002449 bone cell Anatomy 0.000 description 3
- 210000002798 bone marrow cell Anatomy 0.000 description 3
- 235000010948 carboxy methyl cellulose Nutrition 0.000 description 3
- 239000008112 carboxymethyl-cellulose Substances 0.000 description 3
- 229940105329 carboxymethylcellulose Drugs 0.000 description 3
- WPYWMXNXEZFMAK-UHFFFAOYSA-N cinaciguat Chemical compound C=1C=C(C(O)=O)C=CC=1CN(CCCCC(=O)O)CCC1=CC=CC=C1OCC(C=C1)=CC=C1CCC1=CC=CC=C1 WPYWMXNXEZFMAK-UHFFFAOYSA-N 0.000 description 3
- 229950002128 cinaciguat Drugs 0.000 description 3
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 description 3
- 239000006071 cream Substances 0.000 description 3
- 230000007423 decrease Effects 0.000 description 3
- 239000006196 drop Substances 0.000 description 3
- 229930182833 estradiol Natural products 0.000 description 3
- 229960005309 estradiol Drugs 0.000 description 3
- 235000019634 flavors Nutrition 0.000 description 3
- YWXYYJSYQOXTPL-SLPGGIOYSA-N isosorbide mononitrate Chemical compound [O-][N+](=O)O[C@@H]1CO[C@@H]2[C@@H](O)CO[C@@H]21 YWXYYJSYQOXTPL-SLPGGIOYSA-N 0.000 description 3
- 229960003827 isosorbide mononitrate Drugs 0.000 description 3
- 239000008101 lactose Substances 0.000 description 3
- 239000000865 liniment Substances 0.000 description 3
- 239000006210 lotion Substances 0.000 description 3
- 239000000314 lubricant Substances 0.000 description 3
- HQKMJHAJHXVSDF-UHFFFAOYSA-L magnesium stearate Chemical compound [Mg+2].CCCCCCCCCCCCCCCCCC([O-])=O.CCCCCCCCCCCCCCCCCC([O-])=O HQKMJHAJHXVSDF-UHFFFAOYSA-L 0.000 description 3
- 239000012528 membrane Substances 0.000 description 3
- 239000003921 oil Substances 0.000 description 3
- 235000019198 oils Nutrition 0.000 description 3
- 239000002674 ointment Substances 0.000 description 3
- 230000004072 osteoblast differentiation Effects 0.000 description 3
- 239000006072 paste Substances 0.000 description 3
- 230000037361 pathway Effects 0.000 description 3
- 230000008092 positive effect Effects 0.000 description 3
- 239000003755 preservative agent Substances 0.000 description 3
- 238000011002 quantification Methods 0.000 description 3
- 239000003642 reactive oxygen metabolite Substances 0.000 description 3
- 239000003381 stabilizer Substances 0.000 description 3
- 239000004094 surface-active agent Chemical class 0.000 description 3
- 230000035488 systolic blood pressure Effects 0.000 description 3
- 238000012360 testing method Methods 0.000 description 3
- 210000001519 tissue Anatomy 0.000 description 3
- JOXIMZWYDAKGHI-UHFFFAOYSA-N toluene-4-sulfonic acid Chemical compound CC1=CC=C(S(O)(=O)=O)C=C1 JOXIMZWYDAKGHI-UHFFFAOYSA-N 0.000 description 3
- 238000011200 topical administration Methods 0.000 description 3
- 108010054220 vasodilator-stimulated phosphoprotein Proteins 0.000 description 3
- 235000019163 vitamin B12 Nutrition 0.000 description 3
- 239000011715 vitamin B12 Substances 0.000 description 3
- 239000001993 wax Substances 0.000 description 3
- VBICKXHEKHSIBG-UHFFFAOYSA-N 1-monostearoylglycerol Chemical compound CCCCCCCCCCCCCCCCCC(=O)OCC(O)CO VBICKXHEKHSIBG-UHFFFAOYSA-N 0.000 description 2
- IZHVBANLECCAGF-UHFFFAOYSA-N 2-hydroxy-3-(octadecanoyloxy)propyl octadecanoate Chemical compound CCCCCCCCCCCCCCCCCC(=O)OCC(O)COC(=O)CCCCCCCCCCCCCCCCC IZHVBANLECCAGF-UHFFFAOYSA-N 0.000 description 2
- 238000010600 3H thymidine incorporation assay Methods 0.000 description 2
- IGAZHQIYONOHQN-UHFFFAOYSA-N Alexa Fluor 555 Chemical compound C=12C=CC(=N)C(S(O)(=O)=O)=C2OC2=C(S(O)(=O)=O)C(N)=CC=C2C=1C1=CC=C(C(O)=O)C=C1C(O)=O IGAZHQIYONOHQN-UHFFFAOYSA-N 0.000 description 2
- 102100022524 Alpha-1-antichymotrypsin Human genes 0.000 description 2
- GUBGYTABKSRVRQ-XLOQQCSPSA-N Alpha-Lactose Chemical compound O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@H]1O[C@@H]1[C@@H](CO)O[C@H](O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-XLOQQCSPSA-N 0.000 description 2
- 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 2
- 102100026189 Beta-galactosidase Human genes 0.000 description 2
- 102000003727 Caveolin 1 Human genes 0.000 description 2
- 108090000026 Caveolin 1 Proteins 0.000 description 2
- 241000207199 Citrus Species 0.000 description 2
- 229920002261 Corn starch Polymers 0.000 description 2
- FBPFZTCFMRRESA-KVTDHHQDSA-N D-Mannitol Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-KVTDHHQDSA-N 0.000 description 2
- FEWJPZIEWOKRBE-JCYAYHJZSA-N Dextrotartaric acid Chemical compound OC(=O)[C@H](O)[C@@H](O)C(O)=O FEWJPZIEWOKRBE-JCYAYHJZSA-N 0.000 description 2
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 2
- 206010015548 Euthanasia Diseases 0.000 description 2
- WSFSSNUMVMOOMR-UHFFFAOYSA-N Formaldehyde Chemical compound O=C WSFSSNUMVMOOMR-UHFFFAOYSA-N 0.000 description 2
- 239000005715 Fructose Substances 0.000 description 2
- 229930091371 Fructose Natural products 0.000 description 2
- RFSUNEUAIZKAJO-ARQDHWQXSA-N Fructose Chemical compound OC[C@H]1O[C@](O)(CO)[C@@H](O)[C@@H]1O RFSUNEUAIZKAJO-ARQDHWQXSA-N 0.000 description 2
- 108010010803 Gelatin Proteins 0.000 description 2
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Chemical compound OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 2
- 238000006595 Griess deamination reaction Methods 0.000 description 2
- 229920002907 Guar gum Polymers 0.000 description 2
- WZUVPPKBWHMQCE-UHFFFAOYSA-N Haematoxylin Chemical compound C12=CC(O)=C(O)C=C2CC2(O)C1C1=CC=C(O)C(O)=C1OC2 WZUVPPKBWHMQCE-UHFFFAOYSA-N 0.000 description 2
- 101000678026 Homo sapiens Alpha-1-antichymotrypsin Proteins 0.000 description 2
- 108010001336 Horseradish Peroxidase Proteins 0.000 description 2
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 description 2
- OFOBLEOULBTSOW-UHFFFAOYSA-N Malonic acid Chemical compound OC(=O)CC(O)=O OFOBLEOULBTSOW-UHFFFAOYSA-N 0.000 description 2
- 229930195725 Mannitol Natural products 0.000 description 2
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 2
- 102000007637 Soluble Guanylyl Cyclase Human genes 0.000 description 2
- 108010007205 Soluble Guanylyl Cyclase Proteins 0.000 description 2
- 238000000692 Student's t-test Methods 0.000 description 2
- QAOWNCQODCNURD-UHFFFAOYSA-L Sulfate Chemical compound [O-]S([O-])(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-L 0.000 description 2
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 2
- 240000008042 Zea mays Species 0.000 description 2
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 description 2
- 235000002017 Zea mays subsp mays Nutrition 0.000 description 2
- 239000002253 acid Substances 0.000 description 2
- 230000001464 adherent effect Effects 0.000 description 2
- 239000000783 alginic acid Substances 0.000 description 2
- 229960001126 alginic acid Drugs 0.000 description 2
- 150000004781 alginic acids Chemical class 0.000 description 2
- 230000002424 anti-apoptotic effect Effects 0.000 description 2
- 229960005070 ascorbic acid Drugs 0.000 description 2
- 239000000440 bentonite Substances 0.000 description 2
- 229910000278 bentonite Inorganic materials 0.000 description 2
- SVPXDRXYRYOSEX-UHFFFAOYSA-N bentoquatam Chemical compound O.O=[Si]=O.O=[Al]O[Al]=O SVPXDRXYRYOSEX-UHFFFAOYSA-N 0.000 description 2
- 108010005774 beta-Galactosidase Proteins 0.000 description 2
- IQFYYKKMVGJFEH-UHFFFAOYSA-N beta-L-thymidine Natural products O=C1NC(=O)C(C)=CN1C1OC(CO)C(O)C1 IQFYYKKMVGJFEH-UHFFFAOYSA-N 0.000 description 2
- 230000002146 bilateral effect Effects 0.000 description 2
- 229940093761 bile salts Drugs 0.000 description 2
- 230000036983 biotransformation Effects 0.000 description 2
- 210000004369 blood Anatomy 0.000 description 2
- 239000008280 blood Substances 0.000 description 2
- 239000006172 buffering agent Substances 0.000 description 2
- 239000003729 cation exchange resin Substances 0.000 description 2
- 229940023913 cation exchange resins Drugs 0.000 description 2
- 235000020971 citrus fruits Nutrition 0.000 description 2
- 239000003086 colorant Substances 0.000 description 2
- 235000005822 corn Nutrition 0.000 description 2
- 210000005257 cortical tissue Anatomy 0.000 description 2
- HCAJEUSONLESMK-UHFFFAOYSA-N cyclohexylsulfamic acid Chemical compound OS(=O)(=O)NC1CCCCC1 HCAJEUSONLESMK-UHFFFAOYSA-N 0.000 description 2
- 230000007547 defect Effects 0.000 description 2
- 230000007812 deficiency Effects 0.000 description 2
- 229960003964 deoxycholic acid Drugs 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
- 235000014113 dietary fatty acids Nutrition 0.000 description 2
- 238000010790 dilution Methods 0.000 description 2
- 239000012895 dilution Substances 0.000 description 2
- 229960001760 dimethyl sulfoxide Drugs 0.000 description 2
- 239000002270 dispersing agent Substances 0.000 description 2
- 238000004090 dissolution Methods 0.000 description 2
- 239000002552 dosage form Substances 0.000 description 2
- 230000002526 effect on cardiovascular system Effects 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 230000002708 enhancing effect Effects 0.000 description 2
- CCIVGXIOQKPBKL-UHFFFAOYSA-M ethanesulfonate Chemical compound CCS([O-])(=O)=O CCIVGXIOQKPBKL-UHFFFAOYSA-M 0.000 description 2
- 239000000194 fatty acid Substances 0.000 description 2
- 229930195729 fatty acid Natural products 0.000 description 2
- 150000004665 fatty acids Chemical class 0.000 description 2
- 239000012530 fluid Substances 0.000 description 2
- MHMNJMPURVTYEJ-UHFFFAOYSA-N fluorescein-5-isothiocyanate Chemical compound O1C(=O)C2=CC(N=C=S)=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 MHMNJMPURVTYEJ-UHFFFAOYSA-N 0.000 description 2
- 229910052731 fluorine Inorganic materials 0.000 description 2
- 125000001153 fluoro group Chemical group F* 0.000 description 2
- 239000012634 fragment Substances 0.000 description 2
- 229920000159 gelatin Polymers 0.000 description 2
- 239000008273 gelatin Substances 0.000 description 2
- 235000019322 gelatine Nutrition 0.000 description 2
- 235000011852 gelatine desserts Nutrition 0.000 description 2
- 239000011521 glass Substances 0.000 description 2
- 239000000665 guar gum Substances 0.000 description 2
- 235000010417 guar gum Nutrition 0.000 description 2
- 229960002154 guar gum Drugs 0.000 description 2
- 230000036541 health Effects 0.000 description 2
- 235000003642 hunger Nutrition 0.000 description 2
- 125000004435 hydrogen atom Chemical group [H]* 0.000 description 2
- 230000002209 hydrophobic effect Effects 0.000 description 2
- 238000003384 imaging method Methods 0.000 description 2
- 230000005764 inhibitory process Effects 0.000 description 2
- JVTAAEKCZFNVCJ-UHFFFAOYSA-N lactic acid Chemical compound CC(O)C(O)=O JVTAAEKCZFNVCJ-UHFFFAOYSA-N 0.000 description 2
- 239000000787 lecithin Substances 0.000 description 2
- 235000010445 lecithin Nutrition 0.000 description 2
- 230000000670 limiting effect Effects 0.000 description 2
- 230000007774 longterm Effects 0.000 description 2
- 235000019359 magnesium stearate Nutrition 0.000 description 2
- 239000000594 mannitol Substances 0.000 description 2
- 235000010355 mannitol Nutrition 0.000 description 2
- 239000011159 matrix material Substances 0.000 description 2
- 230000004060 metabolic process Effects 0.000 description 2
- 239000001788 mono and diglycerides of fatty acids Substances 0.000 description 2
- 210000005087 mononuclear cell Anatomy 0.000 description 2
- DNIAPMSPPWPWGF-UHFFFAOYSA-N monopropylene glycol Natural products CC(O)CO DNIAPMSPPWPWGF-UHFFFAOYSA-N 0.000 description 2
- 238000010172 mouse model Methods 0.000 description 2
- 230000007935 neutral effect Effects 0.000 description 2
- 229960003753 nitric oxide Drugs 0.000 description 2
- 229910052757 nitrogen Inorganic materials 0.000 description 2
- 230000005937 nuclear translocation Effects 0.000 description 2
- 239000003960 organic solvent Substances 0.000 description 2
- 230000001599 osteoclastic effect Effects 0.000 description 2
- 239000012188 paraffin wax Substances 0.000 description 2
- 230000035699 permeability Effects 0.000 description 2
- 230000000144 pharmacologic effect Effects 0.000 description 2
- 239000002504 physiological saline solution Substances 0.000 description 2
- 229920001223 polyethylene glycol Polymers 0.000 description 2
- 229920000642 polymer Polymers 0.000 description 2
- 235000013855 polyvinylpyrrolidone Nutrition 0.000 description 2
- 239000001267 polyvinylpyrrolidone Substances 0.000 description 2
- 229920001592 potato starch Polymers 0.000 description 2
- 239000002243 precursor Substances 0.000 description 2
- 238000003753 real-time PCR Methods 0.000 description 2
- 238000003757 reverse transcription PCR Methods 0.000 description 2
- 239000000523 sample Substances 0.000 description 2
- 210000003491 skin Anatomy 0.000 description 2
- 239000002002 slurry Substances 0.000 description 2
- 239000011734 sodium Substances 0.000 description 2
- 239000011780 sodium chloride Substances 0.000 description 2
- FHHPUSMSKHSNKW-SMOYURAASA-M sodium deoxycholate Chemical compound [Na+].C([C@H]1CC2)[C@H](O)CC[C@]1(C)[C@@H]1[C@@H]2[C@@H]2CC[C@H]([C@@H](CCC([O-])=O)C)[C@@]2(C)[C@@H](O)C1 FHHPUSMSKHSNKW-SMOYURAASA-M 0.000 description 2
- PRAKJMSDJKAYCZ-UHFFFAOYSA-N squalane Chemical compound CC(C)CCCC(C)CCCC(C)CCCCC(C)CCCC(C)CCCC(C)C PRAKJMSDJKAYCZ-UHFFFAOYSA-N 0.000 description 2
- 230000000087 stabilizing effect Effects 0.000 description 2
- 239000008107 starch Substances 0.000 description 2
- 230000037351 starvation Effects 0.000 description 2
- 239000008223 sterile water Substances 0.000 description 2
- 125000001424 substituent group Chemical group 0.000 description 2
- 238000001356 surgical procedure Methods 0.000 description 2
- 239000006188 syrup Substances 0.000 description 2
- 235000020357 syrup Nutrition 0.000 description 2
- 239000000454 talc Substances 0.000 description 2
- 229910052623 talc Inorganic materials 0.000 description 2
- 235000012222 talc Nutrition 0.000 description 2
- 230000008685 targeting Effects 0.000 description 2
- 239000002562 thickening agent Substances 0.000 description 2
- 229940104230 thymidine Drugs 0.000 description 2
- 235000015112 vegetable and seed oil Nutrition 0.000 description 2
- 239000008158 vegetable oil Substances 0.000 description 2
- 239000002023 wood Substances 0.000 description 2
- BVKSYBQAXBWINI-LQDRYOBXSA-N (2s)-2-[[(2s)-2-[[(2s)-2-[[(2s)-2-[[(2s)-2-[[(2s)-6-amino-2-[[(2s)-2-amino-5-(diaminomethylideneamino)pentanoyl]amino]hexanoyl]amino]-5-(diaminomethylideneamino)pentanoyl]amino]-3-hydroxypropanoyl]amino]-5-(diaminomethylideneamino)pentanoyl]amino]propanoy Chemical compound OC(=O)CC[C@@H](C(O)=O)NC(=O)[C@H](C)NC(=O)[C@H](CCCN=C(N)N)NC(=O)[C@H](CO)NC(=O)[C@H](CCCN=C(N)N)NC(=O)[C@H](CCCCN)NC(=O)[C@@H](N)CCCN=C(N)N BVKSYBQAXBWINI-LQDRYOBXSA-N 0.000 description 1
- AAWZDTNXLSGCEK-LNVDRNJUSA-N (3r,5r)-1,3,4,5-tetrahydroxycyclohexane-1-carboxylic acid Chemical compound O[C@@H]1CC(O)(C(O)=O)C[C@@H](O)C1O AAWZDTNXLSGCEK-LNVDRNJUSA-N 0.000 description 1
- IXPNQXFRVYWDDI-UHFFFAOYSA-N 1-methyl-2,4-dioxo-1,3-diazinane-5-carboximidamide Chemical compound CN1CC(C(N)=N)C(=O)NC1=O IXPNQXFRVYWDDI-UHFFFAOYSA-N 0.000 description 1
- IIZPXYDJLKNOIY-JXPKJXOSSA-N 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine Chemical compound CCCCCCCCCCCCCCCC(=O)OC[C@H](COP([O-])(=O)OCC[N+](C)(C)C)OC(=O)CCC\C=C/C\C=C/C\C=C/C\C=C/CCCCC IIZPXYDJLKNOIY-JXPKJXOSSA-N 0.000 description 1
- 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
- HSTOKWSFWGCZMH-UHFFFAOYSA-N 3,3'-diaminobenzidine Chemical compound C1=C(N)C(N)=CC=C1C1=CC=C(N)C(N)=C1 HSTOKWSFWGCZMH-UHFFFAOYSA-N 0.000 description 1
- OHXPGWPVLFPUSM-KLRNGDHRSA-N 3,7,12-trioxo-5beta-cholanic acid Chemical compound C1CC(=O)C[C@H]2CC(=O)[C@H]3[C@@H]4CC[C@H]([C@@H](CCC(O)=O)C)[C@@]4(C)C(=O)C[C@@H]3[C@]21C OHXPGWPVLFPUSM-KLRNGDHRSA-N 0.000 description 1
- QTBSBXVTEAMEQO-UHFFFAOYSA-M Acetate Chemical compound CC([O-])=O QTBSBXVTEAMEQO-UHFFFAOYSA-M 0.000 description 1
- OGSPWJRAVKPPFI-UHFFFAOYSA-N Alendronic Acid Chemical compound NCCCC(O)(P(O)(O)=O)P(O)(O)=O OGSPWJRAVKPPFI-UHFFFAOYSA-N 0.000 description 1
- 235000019489 Almond oil Nutrition 0.000 description 1
- 239000005995 Aluminium silicate Substances 0.000 description 1
- 241000416162 Astragalus gummifer Species 0.000 description 1
- 229940122361 Bisphosphonate Drugs 0.000 description 1
- 241000283707 Capra Species 0.000 description 1
- PTHCMJGKKRQCBF-UHFFFAOYSA-N Cellulose, microcrystalline Chemical compound OC1C(O)C(OC)OC(CO)C1OC1C(O)C(O)C(OC)C(CO)O1 PTHCMJGKKRQCBF-UHFFFAOYSA-N 0.000 description 1
- VYZAMTAEIAYCRO-UHFFFAOYSA-N Chromium Chemical compound [Cr] VYZAMTAEIAYCRO-UHFFFAOYSA-N 0.000 description 1
- KRKNYBCHXYNGOX-UHFFFAOYSA-K Citrate Chemical compound [O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O KRKNYBCHXYNGOX-UHFFFAOYSA-K 0.000 description 1
- 102000008186 Collagen Human genes 0.000 description 1
- 108010035532 Collagen Proteins 0.000 description 1
- AAWZDTNXLSGCEK-UHFFFAOYSA-N Cordycepinsaeure Natural products OC1CC(O)(C(O)=O)CC(O)C1O AAWZDTNXLSGCEK-UHFFFAOYSA-N 0.000 description 1
- 206010052895 Coronary artery insufficiency Diseases 0.000 description 1
- 229920001353 Dextrin Polymers 0.000 description 1
- 239000004375 Dextrin Substances 0.000 description 1
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 1
- 206010048554 Endothelial dysfunction Diseases 0.000 description 1
- 239000001856 Ethyl cellulose Substances 0.000 description 1
- ZZSNKZQZMQGXPY-UHFFFAOYSA-N Ethyl cellulose Chemical compound CCOCC1OC(OC)C(OCC)C(OCC)C1OC1C(O)C(O)C(OC)C(CO)O1 ZZSNKZQZMQGXPY-UHFFFAOYSA-N 0.000 description 1
- 108010078321 Guanylate Cyclase Proteins 0.000 description 1
- 102000014469 Guanylate cyclase Human genes 0.000 description 1
- 206010019280 Heart failures Diseases 0.000 description 1
- 101001128156 Homo sapiens Nanos homolog 3 Proteins 0.000 description 1
- 101001124309 Homo sapiens Nitric oxide synthase, endothelial Proteins 0.000 description 1
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical class [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 description 1
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 description 1
- 108060003951 Immunoglobulin Proteins 0.000 description 1
- JVTAAEKCZFNVCJ-UHFFFAOYSA-M Lactate Chemical compound CC(O)C([O-])=O JVTAAEKCZFNVCJ-UHFFFAOYSA-M 0.000 description 1
- 208000003263 MASS syndrome Diseases 0.000 description 1
- FYYHWMGAXLPEAU-UHFFFAOYSA-N Magnesium Chemical compound [Mg] FYYHWMGAXLPEAU-UHFFFAOYSA-N 0.000 description 1
- 208000030136 Marchiafava-Bignami Disease Diseases 0.000 description 1
- 208000029725 Metabolic bone disease Diseases 0.000 description 1
- VVQNEPGJFQJSBK-UHFFFAOYSA-N Methyl methacrylate Chemical compound COC(=O)C(C)=C VVQNEPGJFQJSBK-UHFFFAOYSA-N 0.000 description 1
- 229920000168 Microcrystalline cellulose Polymers 0.000 description 1
- 229920001410 Microfiber Polymers 0.000 description 1
- 102000009645 Mitochondrial Aldehyde Dehydrogenase Human genes 0.000 description 1
- 108010009513 Mitochondrial Aldehyde Dehydrogenase Proteins 0.000 description 1
- 229920000715 Mucilage Polymers 0.000 description 1
- 102000006538 Nitric Oxide Synthase Type I Human genes 0.000 description 1
- 108010008858 Nitric Oxide Synthase Type I Proteins 0.000 description 1
- 102100028452 Nitric oxide synthase, endothelial Human genes 0.000 description 1
- CTQNGGLPUBDAKN-UHFFFAOYSA-N O-Xylene Chemical compound CC1=CC=CC=C1C CTQNGGLPUBDAKN-UHFFFAOYSA-N 0.000 description 1
- 229910019142 PO4 Inorganic materials 0.000 description 1
- 102000003992 Peroxidases Human genes 0.000 description 1
- 102000004160 Phosphoric Monoester Hydrolases Human genes 0.000 description 1
- 108090000608 Phosphoric Monoester Hydrolases Proteins 0.000 description 1
- 239000002202 Polyethylene glycol Substances 0.000 description 1
- 229940124158 Protease/peptidase inhibitor Drugs 0.000 description 1
- AAWZDTNXLSGCEK-ZHQZDSKASA-N Quinic acid Natural products O[C@H]1CC(O)(C(O)=O)C[C@H](O)C1O AAWZDTNXLSGCEK-ZHQZDSKASA-N 0.000 description 1
- 229940124044 RANKL antagonist Drugs 0.000 description 1
- 238000002123 RNA extraction Methods 0.000 description 1
- 108010091086 Recombinases Proteins 0.000 description 1
- 102000018120 Recombinases Human genes 0.000 description 1
- 239000012722 SDS sample buffer Substances 0.000 description 1
- 238000002105 Southern blotting Methods 0.000 description 1
- 235000021355 Stearic acid Nutrition 0.000 description 1
- 229930182558 Sterol Natural products 0.000 description 1
- 229930006000 Sucrose Natural products 0.000 description 1
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 1
- OUUQCZGPVNCOIJ-UHFFFAOYSA-M Superoxide Chemical compound [O-][O] OUUQCZGPVNCOIJ-UHFFFAOYSA-M 0.000 description 1
- FEWJPZIEWOKRBE-UHFFFAOYSA-N Tartaric acid Natural products [H+].[H+].[O-]C(=O)C(O)C(O)C([O-])=O FEWJPZIEWOKRBE-UHFFFAOYSA-N 0.000 description 1
- 229920001615 Tragacanth Polymers 0.000 description 1
- 239000013504 Triton X-100 Substances 0.000 description 1
- 229920004890 Triton X-100 Polymers 0.000 description 1
- 229920001938 Vegetable gum Polymers 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 238000009825 accumulation Methods 0.000 description 1
- 235000011054 acetic acid Nutrition 0.000 description 1
- DPXJVFZANSGRMM-UHFFFAOYSA-N acetic acid;2,3,4,5,6-pentahydroxyhexanal;sodium Chemical compound [Na].CC(O)=O.OCC(O)C(O)C(O)C(O)C=O DPXJVFZANSGRMM-UHFFFAOYSA-N 0.000 description 1
- 150000007513 acids Chemical class 0.000 description 1
- NIXOWILDQLNWCW-UHFFFAOYSA-N acrylic acid group Chemical group C(C=C)(=O)O NIXOWILDQLNWCW-UHFFFAOYSA-N 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 239000004480 active ingredient Substances 0.000 description 1
- 230000003044 adaptive effect Effects 0.000 description 1
- 239000000654 additive Substances 0.000 description 1
- 239000000853 adhesive Substances 0.000 description 1
- 239000000556 agonist Substances 0.000 description 1
- 229940062527 alendronate Drugs 0.000 description 1
- 239000008168 almond oil Substances 0.000 description 1
- 229910052782 aluminium Inorganic materials 0.000 description 1
- XAGFODPZIPBFFR-UHFFFAOYSA-N aluminium Chemical compound [Al] XAGFODPZIPBFFR-UHFFFAOYSA-N 0.000 description 1
- 229910000147 aluminium phosphate Inorganic materials 0.000 description 1
- 235000012211 aluminium silicate Nutrition 0.000 description 1
- 150000003863 ammonium salts Chemical class 0.000 description 1
- 238000010171 animal model Methods 0.000 description 1
- 229940069428 antacid Drugs 0.000 description 1
- 239000003159 antacid agent Substances 0.000 description 1
- 230000000123 anti-resoprtive effect Effects 0.000 description 1
- 239000003146 anticoagulant agent Substances 0.000 description 1
- 229940127219 anticoagulant drug Drugs 0.000 description 1
- 239000000427 antigen Substances 0.000 description 1
- 108091007433 antigens Proteins 0.000 description 1
- 102000036639 antigens Human genes 0.000 description 1
- 239000003963 antioxidant agent Substances 0.000 description 1
- 230000003078 antioxidant effect Effects 0.000 description 1
- 235000006708 antioxidants Nutrition 0.000 description 1
- 239000007900 aqueous suspension Substances 0.000 description 1
- 235000021311 artificial sweeteners Nutrition 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 235000013871 bee wax Nutrition 0.000 description 1
- 239000012166 beeswax Substances 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 229940077388 benzenesulfonate Drugs 0.000 description 1
- SRSXLGNVWSONIS-UHFFFAOYSA-M benzenesulfonate Chemical compound [O-]S(=O)(=O)C1=CC=CC=C1 SRSXLGNVWSONIS-UHFFFAOYSA-M 0.000 description 1
- SRSXLGNVWSONIS-UHFFFAOYSA-N benzenesulfonic acid Chemical compound OS(=O)(=O)C1=CC=CC=C1 SRSXLGNVWSONIS-UHFFFAOYSA-N 0.000 description 1
- 229940092714 benzenesulfonic acid Drugs 0.000 description 1
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 1
- DHCLVCXQIBBOPH-UHFFFAOYSA-N beta-glycerol phosphate Natural products OCC(CO)OP(O)(O)=O DHCLVCXQIBBOPH-UHFFFAOYSA-N 0.000 description 1
- GHRQXJHBXKYCLZ-UHFFFAOYSA-L beta-glycerolphosphate Chemical compound [Na+].[Na+].CC(CO)OOP([O-])([O-])=O GHRQXJHBXKYCLZ-UHFFFAOYSA-L 0.000 description 1
- 230000033558 biomineral tissue development Effects 0.000 description 1
- 150000004663 bisphosphonates Chemical class 0.000 description 1
- 210000002459 blastocyst Anatomy 0.000 description 1
- 230000000903 blocking effect Effects 0.000 description 1
- 230000036772 blood pressure Effects 0.000 description 1
- 230000037182 bone density Effects 0.000 description 1
- 230000008468 bone growth Effects 0.000 description 1
- 210000004271 bone marrow stromal cell Anatomy 0.000 description 1
- 230000004097 bone metabolism Effects 0.000 description 1
- 230000010072 bone remodeling Effects 0.000 description 1
- 230000008416 bone turnover Effects 0.000 description 1
- 210000004556 brain Anatomy 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- 239000007975 buffered saline Substances 0.000 description 1
- 239000001506 calcium phosphate Substances 0.000 description 1
- 229910000389 calcium phosphate Inorganic materials 0.000 description 1
- 235000011010 calcium phosphates Nutrition 0.000 description 1
- CJZGTCYPCWQAJB-UHFFFAOYSA-L calcium stearate Chemical compound [Ca+2].CCCCCCCCCCCCCCCCCC([O-])=O.CCCCCCCCCCCCCCCCCC([O-])=O CJZGTCYPCWQAJB-UHFFFAOYSA-L 0.000 description 1
- 235000013539 calcium stearate Nutrition 0.000 description 1
- 239000008116 calcium stearate Substances 0.000 description 1
- OSGAYBCDTDRGGQ-UHFFFAOYSA-L calcium sulfate Inorganic materials [Ca+2].[O-]S([O-])(=O)=O OSGAYBCDTDRGGQ-UHFFFAOYSA-L 0.000 description 1
- 238000012754 cardiac puncture Methods 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 238000001516 cell proliferation assay Methods 0.000 description 1
- 230000005754 cellular signaling Effects 0.000 description 1
- 229940106189 ceramide Drugs 0.000 description 1
- 150000001783 ceramides Chemical class 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 229940009025 chenodeoxycholate Drugs 0.000 description 1
- RUDATBOHQWOJDD-BSWAIDMHSA-N chenodeoxycholic acid Chemical compound C([C@H]1C[C@H]2O)[C@H](O)CC[C@]1(C)[C@@H]1[C@@H]2[C@@H]2CC[C@H]([C@@H](CCC(O)=O)C)[C@@]2(C)CC1 RUDATBOHQWOJDD-BSWAIDMHSA-N 0.000 description 1
- 239000007910 chewable tablet Substances 0.000 description 1
- 210000000349 chromosome Anatomy 0.000 description 1
- 230000001684 chronic effect Effects 0.000 description 1
- 235000015165 citric acid Nutrition 0.000 description 1
- 229940110456 cocoa butter Drugs 0.000 description 1
- 235000019868 cocoa butter Nutrition 0.000 description 1
- 229920001436 collagen Polymers 0.000 description 1
- 238000007398 colorimetric assay Methods 0.000 description 1
- 238000011284 combination treatment Methods 0.000 description 1
- 239000002299 complementary DNA Substances 0.000 description 1
- 238000013270 controlled release Methods 0.000 description 1
- 230000001276 controlling effect Effects 0.000 description 1
- 229920001577 copolymer Polymers 0.000 description 1
- 208000029078 coronary artery disease Diseases 0.000 description 1
- 230000002596 correlated effect Effects 0.000 description 1
- 125000004122 cyclic group Chemical group 0.000 description 1
- 229940009979 dehydrocholate Drugs 0.000 description 1
- 239000003405 delayed action preparation Substances 0.000 description 1
- 229960001251 denosumab Drugs 0.000 description 1
- 238000000586 desensitisation Methods 0.000 description 1
- 230000001627 detrimental effect Effects 0.000 description 1
- 235000019425 dextrin Nutrition 0.000 description 1
- 210000003275 diaphysis Anatomy 0.000 description 1
- 235000005911 diet Nutrition 0.000 description 1
- 230000037213 diet Effects 0.000 description 1
- 238000007907 direct compression Methods 0.000 description 1
- BFMYDTVEBKDAKJ-UHFFFAOYSA-L disodium;(2',7'-dibromo-3',6'-dioxido-3-oxospiro[2-benzofuran-1,9'-xanthene]-4'-yl)mercury;hydrate Chemical compound O.[Na+].[Na+].O1C(=O)C2=CC=CC=C2C21C1=CC(Br)=C([O-])C([Hg])=C1OC1=C2C=C(Br)C([O-])=C1 BFMYDTVEBKDAKJ-UHFFFAOYSA-L 0.000 description 1
- 231100000673 dose–response relationship Toxicity 0.000 description 1
- 230000003828 downregulation Effects 0.000 description 1
- 238000007908 dry granulation Methods 0.000 description 1
- 229940112141 dry powder inhaler Drugs 0.000 description 1
- 230000009977 dual effect Effects 0.000 description 1
- 239000008157 edible vegetable oil Substances 0.000 description 1
- 230000003028 elevating effect Effects 0.000 description 1
- 239000003995 emulsifying agent Substances 0.000 description 1
- 230000008694 endothelial dysfunction Effects 0.000 description 1
- 230000002255 enzymatic effect Effects 0.000 description 1
- 210000002615 epidermis Anatomy 0.000 description 1
- 230000003628 erosive effect Effects 0.000 description 1
- 150000002148 esters Chemical class 0.000 description 1
- BEFDCLMNVWHSGT-UHFFFAOYSA-N ethenylcyclopentane Chemical compound C=CC1CCCC1 BEFDCLMNVWHSGT-UHFFFAOYSA-N 0.000 description 1
- 235000019325 ethyl cellulose Nutrition 0.000 description 1
- 229920001249 ethyl cellulose Polymers 0.000 description 1
- 239000003925 fat Substances 0.000 description 1
- 235000019197 fats Nutrition 0.000 description 1
- 238000011049 filling Methods 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 235000013355 food flavoring agent Nutrition 0.000 description 1
- 230000037406 food intake Effects 0.000 description 1
- 230000004927 fusion Effects 0.000 description 1
- 210000004602 germ cell Anatomy 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- 229940074045 glyceryl distearate Drugs 0.000 description 1
- 229940075507 glyceryl monostearate Drugs 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 229940126514 guanylate cyclase activator Drugs 0.000 description 1
- 239000003119 guanylate cyclase activator Substances 0.000 description 1
- 230000013632 homeostatic process Effects 0.000 description 1
- 230000006801 homologous recombination Effects 0.000 description 1
- 238000002744 homologous recombination Methods 0.000 description 1
- 229930195733 hydrocarbon Natural products 0.000 description 1
- 150000002430 hydrocarbons Chemical class 0.000 description 1
- 239000008172 hydrogenated vegetable oil Substances 0.000 description 1
- 229910052588 hydroxylapatite Inorganic materials 0.000 description 1
- 238000010191 image analysis Methods 0.000 description 1
- 238000010166 immunofluorescence Methods 0.000 description 1
- 102000018358 immunoglobulin Human genes 0.000 description 1
- 238000011532 immunohistochemical staining Methods 0.000 description 1
- 238000003364 immunohistochemistry Methods 0.000 description 1
- 238000002513 implantation Methods 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 238000011065 in-situ storage Methods 0.000 description 1
- 238000010348 incorporation Methods 0.000 description 1
- 230000001939 inductive effect Effects 0.000 description 1
- 239000004615 ingredient Substances 0.000 description 1
- 239000003112 inhibitor Substances 0.000 description 1
- 229910001867 inorganic solvent Inorganic materials 0.000 description 1
- 239000003049 inorganic solvent Substances 0.000 description 1
- 230000035987 intoxication Effects 0.000 description 1
- 231100000566 intoxication Toxicity 0.000 description 1
- 230000010189 intracellular transport Effects 0.000 description 1
- 239000007927 intramuscular injection Substances 0.000 description 1
- 238000010255 intramuscular injection Methods 0.000 description 1
- 239000007928 intraperitoneal injection Substances 0.000 description 1
- 239000003456 ion exchange resin Substances 0.000 description 1
- 229920003303 ion-exchange polymer Polymers 0.000 description 1
- 230000007794 irritation Effects 0.000 description 1
- NLYAJNPCOHFWQQ-UHFFFAOYSA-N kaolin Chemical compound O.O.O=[Al]O[Si](=O)O[Si](=O)O[Al]=O NLYAJNPCOHFWQQ-UHFFFAOYSA-N 0.000 description 1
- 239000004310 lactic acid Substances 0.000 description 1
- 235000014655 lactic acid Nutrition 0.000 description 1
- 229940067606 lecithin Drugs 0.000 description 1
- 231100001231 less toxic Toxicity 0.000 description 1
- 239000012669 liquid formulation Substances 0.000 description 1
- 230000004777 loss-of-function mutation Effects 0.000 description 1
- 239000007937 lozenge Substances 0.000 description 1
- 210000004705 lumbosacral region Anatomy 0.000 description 1
- 239000006166 lysate Substances 0.000 description 1
- 229910052749 magnesium Inorganic materials 0.000 description 1
- ZLNQQNXFFQJAID-UHFFFAOYSA-L magnesium carbonate Chemical compound [Mg+2].[O-]C([O-])=O ZLNQQNXFFQJAID-UHFFFAOYSA-L 0.000 description 1
- 239000001095 magnesium carbonate Substances 0.000 description 1
- 229910000021 magnesium carbonate Inorganic materials 0.000 description 1
- 235000014380 magnesium carbonate Nutrition 0.000 description 1
- 238000012423 maintenance Methods 0.000 description 1
- 210000004962 mammalian cell Anatomy 0.000 description 1
- 230000035800 maturation Effects 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 150000004667 medium chain fatty acids Chemical class 0.000 description 1
- 238000002844 melting Methods 0.000 description 1
- 230000008018 melting Effects 0.000 description 1
- 230000009245 menopause Effects 0.000 description 1
- 230000002503 metabolic effect Effects 0.000 description 1
- 229910052751 metal Inorganic materials 0.000 description 1
- 239000002184 metal Substances 0.000 description 1
- 230000031864 metaphase Effects 0.000 description 1
- 229940071648 metered dose inhaler Drugs 0.000 description 1
- 229940098779 methanesulfonic acid Drugs 0.000 description 1
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 description 1
- 235000010270 methyl p-hydroxybenzoate Nutrition 0.000 description 1
- 229940102838 methylmethacrylate Drugs 0.000 description 1
- 235000019813 microcrystalline cellulose Nutrition 0.000 description 1
- 239000008108 microcrystalline cellulose Substances 0.000 description 1
- 229940016286 microcrystalline cellulose Drugs 0.000 description 1
- 239000003658 microfiber Substances 0.000 description 1
- 230000003278 mimic effect Effects 0.000 description 1
- 239000002480 mineral oil Substances 0.000 description 1
- 230000002438 mitochondrial effect Effects 0.000 description 1
- 230000009456 molecular mechanism Effects 0.000 description 1
- 239000004570 mortar (masonry) Substances 0.000 description 1
- LNOPIUAQISRISI-UHFFFAOYSA-N n'-hydroxy-2-propan-2-ylsulfonylethanimidamide Chemical compound CC(C)S(=O)(=O)CC(N)=NO LNOPIUAQISRISI-UHFFFAOYSA-N 0.000 description 1
- JXTPJDDICSTXJX-UHFFFAOYSA-N n-Triacontane Natural products CCCCCCCCCCCCCCCCCCCCCCCCCCCCCC JXTPJDDICSTXJX-UHFFFAOYSA-N 0.000 description 1
- 229940097496 nasal spray Drugs 0.000 description 1
- 239000007922 nasal spray Substances 0.000 description 1
- 235000021096 natural sweeteners Nutrition 0.000 description 1
- 239000006199 nebulizer Substances 0.000 description 1
- 230000020395 negative regulation of osteoclast differentiation Effects 0.000 description 1
- 239000002687 nonaqueous vehicle Substances 0.000 description 1
- 231100000956 nontoxicity Toxicity 0.000 description 1
- QIQXTHQIDYTFRH-UHFFFAOYSA-N octadecanoic acid Chemical compound CCCCCCCCCCCCCCCCCC(O)=O QIQXTHQIDYTFRH-UHFFFAOYSA-N 0.000 description 1
- OQCDKBAXFALNLD-UHFFFAOYSA-N octadecanoic acid Natural products CCCCCCCC(C)CCCCCCCCC(O)=O OQCDKBAXFALNLD-UHFFFAOYSA-N 0.000 description 1
- 238000001543 one-way ANOVA Methods 0.000 description 1
- 238000005457 optimization Methods 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 230000002611 ovarian Effects 0.000 description 1
- 239000003002 pH adjusting agent Substances 0.000 description 1
- 229940124641 pain reliever Drugs 0.000 description 1
- 230000036961 partial effect Effects 0.000 description 1
- 230000007170 pathology Effects 0.000 description 1
- 230000007310 pathophysiology Effects 0.000 description 1
- 229920001277 pectin Polymers 0.000 description 1
- 239000001814 pectin Substances 0.000 description 1
- 235000010987 pectin Nutrition 0.000 description 1
- 239000003961 penetration enhancing agent Substances 0.000 description 1
- XYJRXVWERLGGKC-UHFFFAOYSA-D pentacalcium;hydroxide;triphosphate Chemical compound [OH-].[Ca+2].[Ca+2].[Ca+2].[Ca+2].[Ca+2].[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O XYJRXVWERLGGKC-UHFFFAOYSA-D 0.000 description 1
- 239000000137 peptide hydrolase inhibitor Substances 0.000 description 1
- 108040007629 peroxidase activity proteins Proteins 0.000 description 1
- 239000008177 pharmaceutical agent Substances 0.000 description 1
- 239000010452 phosphate Substances 0.000 description 1
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 1
- DCWXELXMIBXGTH-QMMMGPOBSA-N phosphonotyrosine Chemical compound OC(=O)[C@@H](N)CC1=CC=C(OP(O)(O)=O)C=C1 DCWXELXMIBXGTH-QMMMGPOBSA-N 0.000 description 1
- BZQFBWGGLXLEPQ-REOHCLBHSA-N phosphoserine Chemical compound OC(=O)[C@@H](N)COP(O)(O)=O BZQFBWGGLXLEPQ-REOHCLBHSA-N 0.000 description 1
- 230000000704 physical effect Effects 0.000 description 1
- 230000035479 physiological effects, processes and functions Effects 0.000 description 1
- 229940068196 placebo Drugs 0.000 description 1
- 239000000902 placebo Substances 0.000 description 1
- 229920000447 polyanionic polymer Chemical class 0.000 description 1
- 229920000036 polyvinylpyrrolidone Polymers 0.000 description 1
- 231100000683 possible toxicity Toxicity 0.000 description 1
- 230000003244 pro-oxidative effect Effects 0.000 description 1
- 230000003651 pro-proliferative effect Effects 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- ALDITMKAAPLVJK-UHFFFAOYSA-N prop-1-ene;hydrate Chemical group O.CC=C ALDITMKAAPLVJK-UHFFFAOYSA-N 0.000 description 1
- 235000010232 propyl p-hydroxybenzoate Nutrition 0.000 description 1
- QELSKZZBTMNZEB-UHFFFAOYSA-N propylparaben Chemical class CCCOC(=O)C1=CC=C(O)C=C1 QELSKZZBTMNZEB-UHFFFAOYSA-N 0.000 description 1
- 239000003223 protective agent Substances 0.000 description 1
- 230000001681 protective effect Effects 0.000 description 1
- ZHNFLHYOFXQIOW-LPYZJUEESA-N quinine sulfate dihydrate Chemical compound [H+].[H+].O.O.[O-]S([O-])(=O)=O.C([C@H]([C@H](C1)C=C)C2)C[N@@]1[C@@H]2[C@H](O)C1=CC=NC2=CC=C(OC)C=C21.C([C@H]([C@H](C1)C=C)C2)C[N@@]1[C@@H]2[C@H](O)C1=CC=NC2=CC=C(OC)C=C21 ZHNFLHYOFXQIOW-LPYZJUEESA-N 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 239000011347 resin Substances 0.000 description 1
- 229920005989 resin Polymers 0.000 description 1
- 229920006395 saturated elastomer Polymers 0.000 description 1
- 235000003441 saturated fatty acids Nutrition 0.000 description 1
- 150000004671 saturated fatty acids Chemical class 0.000 description 1
- 238000003345 scintillation counting Methods 0.000 description 1
- 239000000565 sealant Substances 0.000 description 1
- 102000034285 signal transducing proteins Human genes 0.000 description 1
- 108091006024 signal transducing proteins Proteins 0.000 description 1
- 230000019491 signal transduction Effects 0.000 description 1
- 231100000161 signs of toxicity Toxicity 0.000 description 1
- 229910052708 sodium Inorganic materials 0.000 description 1
- 235000015424 sodium Nutrition 0.000 description 1
- 235000010413 sodium alginate Nutrition 0.000 description 1
- 239000000661 sodium alginate Substances 0.000 description 1
- 229940005550 sodium alginate Drugs 0.000 description 1
- 235000019812 sodium carboxymethyl cellulose Nutrition 0.000 description 1
- 229920001027 sodium carboxymethylcellulose Polymers 0.000 description 1
- NRHMKIHPTBHXPF-TUJRSCDTSA-M sodium cholate Chemical compound [Na+].C([C@H]1C[C@H]2O)[C@H](O)CC[C@]1(C)[C@@H]1[C@@H]2[C@@H]2CC[C@H]([C@@H](CCC([O-])=O)C)[C@@]2(C)[C@@H](O)C1 NRHMKIHPTBHXPF-TUJRSCDTSA-M 0.000 description 1
- 239000001509 sodium citrate Substances 0.000 description 1
- NLJMYIDDQXHKNR-UHFFFAOYSA-K sodium citrate Chemical compound O.O.[Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O NLJMYIDDQXHKNR-UHFFFAOYSA-K 0.000 description 1
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 1
- OABYVIYXWMZFFJ-ZUHYDKSRSA-M sodium glycocholate Chemical compound [Na+].C([C@H]1C[C@H]2O)[C@H](O)CC[C@]1(C)[C@@H]1[C@@H]2[C@@H]2CC[C@H]([C@@H](CCC(=O)NCC([O-])=O)C)[C@@]2(C)[C@@H](O)C1 OABYVIYXWMZFFJ-ZUHYDKSRSA-M 0.000 description 1
- VMSNAUAEKXEYGP-YEUHZSMFSA-M sodium glycodeoxycholate Chemical compound [Na+].C([C@H]1CC2)[C@H](O)CC[C@]1(C)[C@@H]1[C@@H]2[C@@H]2CC[C@H]([C@@H](CCC(=O)NCC([O-])=O)C)[C@@]2(C)[C@@H](O)C1 VMSNAUAEKXEYGP-YEUHZSMFSA-M 0.000 description 1
- AECTYFQKWPXOSR-DGMAEHPPSA-M sodium;(4r)-4-[(3r,5r,8r,9s,10s,13r,14s,17r)-3-hydroxy-10,13-dimethyl-2,3,4,5,6,7,8,9,11,12,14,15,16,17-tetradecahydro-1h-cyclopenta[a]phenanthren-17-yl]pentanoate Chemical compound [Na+].C([C@H]1CC2)[C@H](O)CC[C@]1(C)[C@@H]1[C@@H]2[C@@H]2CC[C@H]([C@@H](CCC([O-])=O)C)[C@@]2(C)CC1 AECTYFQKWPXOSR-DGMAEHPPSA-M 0.000 description 1
- 239000008247 solid mixture Substances 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 235000010199 sorbic acid Nutrition 0.000 description 1
- 239000004334 sorbic acid Substances 0.000 description 1
- 229940075582 sorbic acid Drugs 0.000 description 1
- 235000010356 sorbitol Nutrition 0.000 description 1
- 239000000600 sorbitol Substances 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 229940032094 squalane Drugs 0.000 description 1
- 238000007619 statistical method Methods 0.000 description 1
- 239000008117 stearic acid Substances 0.000 description 1
- 150000003432 sterols Chemical class 0.000 description 1
- 235000003702 sterols Nutrition 0.000 description 1
- 230000004936 stimulating effect Effects 0.000 description 1
- 210000002784 stomach Anatomy 0.000 description 1
- 230000035882 stress Effects 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 239000005720 sucrose Substances 0.000 description 1
- 150000008163 sugars Chemical class 0.000 description 1
- IIACRCGMVDHOTQ-UHFFFAOYSA-M sulfamate Chemical compound NS([O-])(=O)=O IIACRCGMVDHOTQ-UHFFFAOYSA-M 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 230000002459 sustained effect Effects 0.000 description 1
- 239000007916 tablet composition Substances 0.000 description 1
- 239000011975 tartaric acid Substances 0.000 description 1
- 235000002906 tartaric acid Nutrition 0.000 description 1
- 229940095064 tartrate Drugs 0.000 description 1
- BHTRKEVKTKCXOH-BJLOMENOSA-N taurochenodeoxycholic acid Chemical compound C([C@H]1C[C@H]2O)[C@H](O)CC[C@]1(C)[C@@H]1[C@@H]2[C@@H]2CC[C@H]([C@@H](CCC(=O)NCCS(O)(=O)=O)C)[C@@]2(C)CC1 BHTRKEVKTKCXOH-BJLOMENOSA-N 0.000 description 1
- AWDRATDZQPNJFN-VAYUFCLWSA-N taurodeoxycholic acid Chemical compound C([C@H]1CC2)[C@H](O)CC[C@]1(C)[C@@H]1[C@@H]2[C@@H]2CC[C@H]([C@@H](CCC(=O)NCCS(O)(=O)=O)C)[C@@]2(C)[C@@H](O)C1 AWDRATDZQPNJFN-VAYUFCLWSA-N 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
- 231100000419 toxicity Toxicity 0.000 description 1
- 230000001988 toxicity Effects 0.000 description 1
- 239000003053 toxin Substances 0.000 description 1
- 235000010487 tragacanth Nutrition 0.000 description 1
- 239000000196 tragacanth Substances 0.000 description 1
- 229940116362 tragacanth Drugs 0.000 description 1
- 230000037317 transdermal delivery Effects 0.000 description 1
- 230000032258 transport Effects 0.000 description 1
- 238000011269 treatment regimen Methods 0.000 description 1
- QORWJWZARLRLPR-UHFFFAOYSA-H tricalcium bis(phosphate) Chemical compound [Ca+2].[Ca+2].[Ca+2].[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O QORWJWZARLRLPR-UHFFFAOYSA-H 0.000 description 1
- YNJBWRMUSHSURL-UHFFFAOYSA-N trichloroacetic acid Chemical compound OC(=O)C(Cl)(Cl)Cl YNJBWRMUSHSURL-UHFFFAOYSA-N 0.000 description 1
- 230000007306 turnover Effects 0.000 description 1
- 150000004670 unsaturated fatty acids Chemical class 0.000 description 1
- 235000021122 unsaturated fatty acids Nutrition 0.000 description 1
- 229940014499 ursodeoxycholate Drugs 0.000 description 1
- RUDATBOHQWOJDD-UZVSRGJWSA-N ursodeoxycholic acid Chemical compound C([C@H]1C[C@@H]2O)[C@H](O)CC[C@]1(C)[C@@H]1[C@@H]2[C@@H]2CC[C@H]([C@@H](CCC(O)=O)C)[C@@]2(C)CC1 RUDATBOHQWOJDD-UZVSRGJWSA-N 0.000 description 1
- 239000006200 vaporizer Substances 0.000 description 1
- 230000024883 vasodilation Effects 0.000 description 1
- 239000011345 viscous material Substances 0.000 description 1
- 238000005303 weighing Methods 0.000 description 1
- 230000004584 weight gain Effects 0.000 description 1
- 235000019786 weight gain Nutrition 0.000 description 1
- 238000005550 wet granulation Methods 0.000 description 1
- 239000008096 xylene Substances 0.000 description 1
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/555—Heterocyclic compounds containing heavy metals, e.g. hemin, hematin, melarsoprol
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P19/00—Drugs for skeletal disorders
- A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
Definitions
- This invention generally relates to physiology and bone metabolism.
- nitrosyl- cobinamide for: treating, ameliorating, preventing or reversing osteoporosis; preventing bone loss due to estrogen deficiency; inhibiting osteoclast differentiation; reducing osteoclast numbers; preventing estrogen deficiency-induced osteocyte apoptosis; increasing serum cGMP concentration; increasing bone formation or bone mass, or increasing trabecular bone volume, trabecular number, and trabecular bone mineral density; increasing osteoblast numbers or regulating osteoblastic gene expression, comprising administering a nitrosyl-cobinamide (NO-Cbi), or a bioisostere, stereoisomer, tautomer, hydrate, solvate or pharmaceutically acceptable salt thereof to: an individual; an individual with osteoporosis or at risk of developing osteoporosis.
- NO-Cbi nitrosyl- cobinamide
- Nitric Oxide (NO)/cGMP/protein kinase G (PKG) signaling pathway mediates anabolic effects of estrogens and mechanical stimulation in bone cells by increasing osteoblast proliferation and osteocyte survival.
- nitrates are limited by induction of oxidative stress and development of tolerance, and may increase cardiovascular mortality after long-term use.
- Organic nitrates generate NO in vivo after mitochondrial biotransformation; nitrates are used to treat coronary insufficiency and heart failure, and epidemiological studies suggest their use may reduce fracture risk. 19"21 Based on these data and preclinical studies showing a bone-protective effect of organic nitrates in OVX rats, 22, 23 several clinical trials have examined the skeletal effects of organic nitrates in post-menopausal women, showing an increase in bone formation markers, a decrease in bone resorption markers, and improved bone mineral density in women with estrogen deficiency. 24
- Nitrates are the only NO donors currently FDA-approved for long-term use, but clinical benefits of organic nitrates are limited by development of tolerance and induction of oxidative stress. 27"30 Nitrates require enzymatic activation to release NO, and this reaction generates reactive oxygen species, especially O2 " , which can have detrimental effects in the cardiovascular and skeletal systems. 10 ' 29 ' 31 SUMMARY
- a nitrosyl-cobinamide NO-Cbi
- a bioisostere, stereoisomer, tautomer, hydrate, solvate or pharmaceutically acceptable salt thereof to: an individual; an individual with osteoporosis or at risk of developing osteoporosis,
- the nitrosyl-cobinamide, or the bioisostere, stereoisomer, tautomer, hydrate, solvate or pharmaceutically acceptable salt thereof is formulated as a pharmaceutical composition, or is formulated as a pharmaceutical composition comprising a pharmaceutically acceptable carrier or excipient thereof.
- the nitrosyl-cobinamide, or the bioisostere, stereoisomer, tautomer, hydrate, solvate or pharmaceutically acceptable salt thereof, or the pharmaceutical composition is administered or co-administered with another therapeutic agent, wherein optionally therapeutic agent is administered simultaneously, before or after the nitrosyl-cobinamide, or a bioisostere, stereoisomer, tautomer, hydrate, solvate or pharmaceutically acceptable salt thereof, or the pharmaceutical composition.
- the nitrosyl-cobinamide, or the bioisostere, stereoisomer, tautomer, hydrate, solvate or pharmaceutically acceptable salt thereof, or the pharmaceutical composition is administered in or with an implant or a bone implant.
- the nitrosyl-cobinamide, or the bioisostere, stereoisomer, tautomer, hydrate, solvate or pharmaceutically acceptable salt thereof, or the pharmaceutical composition is administered as or is formulated in a device, an implant, a bone implant, a bead, a tablet, a pill, a capsule, a liquid, a gel, a geltab, a powder, a spray or aerosol, a cachet, a suppository, a dispersible granule, a product of manufacture, a liposome, a particle, a microparticle or a nanoparticle.
- the nitrosyl-cobinamide, or the bioisostere, stereoisomer, tautomer, hydrate, solvate or pharmaceutically acceptable salt thereof, or the pharmaceutical composition is administered as or is formulated in or as a kit, a pump, a device, a subcutaneous infusion device, a continuous subcutaneous infusion device, an infusion pen, a needles, a reservoir, an ampoules, a vial, a syringe, a cartridge, a pen, a disposable pen or jet injector, a prefilled pen or a syringe or a cartridge, a cartridge or a disposable pen or jet injector, a two chambered or multi-chambered pump.
- the therapeutic formulation comprises a nitrosyl-cobinamide (NO-Cbi), or a bioisostere, stereoisomer, tautomer, hydrate, solvate or pharmaceutically acceptable salt thereof.
- NO-Cbi nitrosyl-cobinamide
- bone implants comprising a nitrosyl- cobinamide, or a bioisostere, stereoisomer, tautomer, hydrate, solvate or pharmaceutically acceptable salt thereof.
- FIG. 1A-I illustrate data showing that NO-Cbi enhances cGMP/PKG and Erk/Akt signaling, gene expression, proliferation and survival in POBs, as described in detail in Example 1, below:
- FIG. 1(A) graphically illustrates data from experiments where POBs were incubated in medium with 0.1% FBS (3 x 10 5 cells/ml) for 2 h prior to receiving 10 ⁇ NO-Cbi (NOCbi) for the indicated times, and stable NO oxidation products (nitrite plus nitrate, NOx) were measured in the medium by the Griess reaction;
- FIG. 1(B) and FIG. 1(C) graphically illustrate data from experiments where POBs were treated with vehicle or NO-Cbi at the indicated concentrations for 30 min, and intracellular cGMP concentrations were measured by ELISA (FIG. IB);
- FIG. 1(D) and FIG. 1(E) graphically illustrate data from experiments where serum- deprived POBs were treated with vehicle or 10 ⁇ NO-Cbi for 10 min and ERK and Akt activation were assessed by blotting with phospho-specific antibodies;
- FIG. 1(F) illustrates immunofluorescence images from experiments where POBs were serum-starved for 36 h in medium containing 1% BSA with 10 ⁇ NO-Cbi or vehicle; apoptosis was assessed by immunofluorescence staining with antibodies specific for cleaved caspase-3 and FITC-coupled secondary antibodies (green); nuclei were counterstained with Hoechst 33342 (blue);
- FIG. 1(G) graphically illustrates data from experiments where POBs cultured in medium with 0.1% FBS for 18 h were treated with 10 ⁇ NO-Cbi or vehicle for 1 h, and transferred to fresh medium containing 3 H-thymidine for 24 h;
- FIG. 1(H) and FIG. 1(1) graphically illustrate data from experiments where confluent POBs were differentiated in ascorbate-containing medium for 14 d and some cells received 10 ⁇ NO-Cbi (open bars) or vehicle (filled bars) for the last 24 h.
- FIG. 2A-F illustrate data from experiments showing that NO-Cbi stimulates Wnt signaling and mPOB proliferation via PKG II, as described in detail in Example 1, below:
- FIG. 2A illustrates data from experiments where POBs isolated from mice homozygous for prkg2 alleles flanked by LoxP sites ("floxed" PRKG2 f/f ) were infected with adenovirus expressing ⁇ -galactosidase (LacZ, control) or CRE recombinase (CRE), and forty-eight h later, relative amounts of prkg2 mRNA were determined by qRT-PCR, and knockdown efficiency of PKG II protein was analyzed by Western blotting, with caveolin-1 serving as a loading control;
- FIG. 2B illustrates data from experiments where cells were infected as in FIG. 2A, but 30 h later were transferred to medium containing 0.1% FBS, and 18 h later were treated with 10 ⁇ NO-Cbi or vehicle for 10 min, and Akt and GSK-3P phosphorylation were assessed using antibodies specific for Akt(pSer 473 ) and GSK-3P(pSer 9 ), with total GSK-3P serving as a loading control; densitometric quantitation is shown on the right, with relative amounts of pAkt and pGSK-3 found in vehicle-treated control virus-infected cells assigned a value of 1;
- FIG. 2C illustrates data from experiments where PRKG2 f/f POBs were infected with control or Cre virus and transferred to 0.1% FBS as in FIG. 2B, they were treated with NO-Cbi or vehicle for 6 h, prior to detecting ⁇ -catenin by immunofluorescence staining, and the bottom panel shows nuclei counterstained with Hoechst 33342, and numbers below indicate the percentage of cells showing nuclear ⁇ -catenin;
- FIG. 2D illustrates data from experiments where cells were infected and cultured as in FIG. 2B, and they were treated with NO-Cbi or vehicle for 1 h prior to measuring 3 H- thymidine incorporation into DNA for 24 h;
- FIG. 2E illustrates data from experiments where cells were infected with control or CRE virus as described in FIG. 2 A, and treated with 10 ⁇ NO-Cbi or vehicle for 24 h.
- Expression of Wingless type MMTV-integration site family- la (Wntla), low-density lipoprotein receptor- related protein-5 (Lrp5), ⁇ -catenin (bCat), cyclin D (CycD), alkaline phosphatase (ALP), osteocalcin (OCN), and tubulin (Tubal) mRNAs were measured by qRT-PCR and normalized to 18S rRNA, with relative mRNA levels in untreated cells assigned a value of 1; and
- FIG. 2F illustrates data from experiments where cells POBs cultured in 10 % FBS were treated with 10 ⁇ NO-Cbi for the indicated times, and Lrp5 protein (open symbols) and mRNA (filled symbols) were assessed by Western blotting and qRT-PCR, respectively.
- FIG. 3A-D illustrate data showing that NO-Cbi inhibits osteoclast differentiation, as described in detail in Example 1, below:
- FIG. 3 A and FIG. 3B illustrate data from experiments where murine bone marrow mononuclear cells were cultured in the presence of M-CSF, with RANKL added after 3 d; together with RANKL, cells received vehicle or NO-Cbi at the indicated concentrations;
- FIG. 3C illustrates data from experiments where cells were cultured as in FIG. 3 A, but some cultures received 10 ⁇ NO-Cbi, 5 ⁇ DETA-NONOate (Deta-NO, which releases 2 moles of NO/mol of drug), or 100 ⁇ 8-pCPT-cGMP together with RANKL; and
- FIG. 3D illustrates data from experiments where expression of TRAP, cathepsin K (Ctsk), and calcitonin receptor (CalcR) mRNAs were determined by qRT-PCR and normalized to 18S rRNA, with relative mRNA levels in vehicle-treated cells assigned a value of 1.
- FIG. 4A-G illustrate data showing that NO-Cbi increases serum cGMP
- FIG. 4A illustrates data from experiments where werum cGMP concentrations were measured by ELISA 1 h after the last injection of vehicle or NO-Cbi;
- FIG. 4B illustrates data from experiments where the number of trabecular osteoblasts per bone perimeter (N.Ob/B.Pm) was counted at the proximal tibia;
- FIG. 4C, FIG. 4D and FIG. 4F illustrate data from experiments where trabecular calcein labeling was assessed at the tibia (FIG. 4C), with quantification of mineral apposition rate (MAR, FIG. 4D), mineralizing surface per bone surface (MS/BS, panel E), and bone formation rate (BFR, FIG. 4F); and
- FIG. 4G illustrates data from experiments where RNA was extracted from femurs, and the relative abundance of osteocalcin (OCN), osteopontin (Sppl), alkaline
- ALP phosphatase
- Collal collagen-al
- Lrp5 low-density lipoprotein receptor- related protein-5
- Tubulin Tubulin
- FIG. 5 A-E illustrate data showing that NO-Cbi prevents estrogen deficiency-induced osteocyte apoptosis, where mice were subjected to OVX or sham operation and were treated with vehicle or NO-Cbi as described in Fig. 4, as described in detail in Example 1, below:
- FIG. 5A, FIG. 5B and FIG. 5C illustrate data from experiments where the percentage of apoptotic osteocytes was assessed in trabecular (FIG. 5 A, FIG. 5B) and cortical bone (FIG. 5C) by TUNEL staining (black nuclei) of tibial sections:
- FIG. 5E illustrate data from experiments where Erk activity in cortical (top panel) and trabecular (bottom panel) bone-lining cells was assessed by immunofluorescence staining using a phospho-Erk-specific antibody and horse radish peroxidase-coupled secondary antibody (brown).
- FIG. 6A-C illustrate data showing that NO-Cbi regulates RANKL/OPG and reduces osteoclasts in OVX mice, and mice subjected to OVX or sham-operation were treated with vehicle or NO-Cbi as described in Fig. 4, as described in detail in Example 1, below:
- FIG. 6 A and FIG. 6B illustrate data from experiments where osteoclasts were identified by TRAP staining (red), and the number of trabecular osteoclasts per bone perimeter (N.Oc/B.Pm) was counted at the proximal tibia; and FIG. 6C illustrates data from experiments where RNA was extracted from femurs, and the relative abundance of RANKL, OPG, CTSK, and TRAP mRNA was quantified by qRT-PCR and normalized to 18S rRNA.
- FIG. 7A-D illustrate data showing that NO-Cbi increases trabecular bone mass in OVX mice.
- Mice subjected to OVX or sham operation were treated with vehicle or NO- Cbi as described in Fig. 4, as described in detail in Example 1, below:
- FIG. 7A illustrates data from experiments where tibiae were analyzed by micro-CT imaging, and three-dimensional reconstruction of the trabecular bone at the proximal tibia below the growth plate is shown;
- FIG. 7B, FIG. 7C and FIG. 7D illustrate data from experiments where trabecular bone volume/tissue volume (FIG. 7B), trabecular number (FIG. 7C), and trabecular bone mineral density (FIG. 7D) were quantified at the proximal tibia.
- FIG. 8 illustrates endocortical calcein labeling, where the endocortical calcein labeling, as illustrated in the FIG. 8A images, was assessed at the tibia, with
- FIG. 9 illustrates images of a TUNEL staining of apoptotic osteocytes in cortical tibial bone, where the percentage of apoptotic osteocytes was assessed in cortical bone by TUNEL staining (black nuclei) of tibial sections, as described in Fig. 5C.
- NO-Cbi nitrosyl-cobinamide
- a nitrosyl-cobinamide NO-Cbi
- a bioisostere, stereoisomer, tautomer, hydrate, solvate or pharmaceutically acceptable salt thereof administered to: an individual; an individual with osteoporesis or at risk of developing osteoporesis.
- NO-Cbi nitrosyl-cobinamide
- NO-Cbi nitrosyl-cobinamide
- ovariectomized mice had lower serum cGMP concentrations, which were largely restored to normal by treatment with NO-Cbi, or low-dose estrogen replacement.
- Micro-CT analyses of tibiae showed that all three pharmacological interventions significantly improved trabecular bone architecture in ovariectomized animals, with similar effect sizes.
- NO-Cbi reversed ovariectomy -induced osteocyte apoptosis as efficiently as estradiol, and enhanced bone formation parameters in vivo, consistent with in vitro effects on osteoblast proliferation, differentiation, and survival. Ovariectomy dramatically increased osteoclast numbers, and this effect was completely reversed by estradiol. NO-Cbi significantly decreased the number of osteoclasts in ovariectomized mice, suggesting cGMP-independent effects of NO-Cbi in osteoclasts.
- estrogen deficiency represents a state of relative NO and cGMP deficiency
- NO-dependent or NO-independent guanylate cyclase stimulation is a novel, anabolic treatment strategy for post-menopausal osteoporosis.
- Cbi prevents osteoporosis in ovariectomized mice.
- Most drugs used for treating osteoporosis inhibit osteoclasts (which resorb bone) but do not stimulate osteoblasts (which make new bone).
- NO-Cbi both inhibits osteoclasts and stimulates osteoblasts, so it is an ideal drug for treating osteoporosis, including post-menopausal osteoporosis, and no drug like it is currently available.
- NO-Cbi or nitrosyl cobinamide is a compound having the structure:
- bioisosteres provided herein are compounds comprising one or more substituent and/or group replacements with a substituent and/or group having substantially similar physical or chemical properties which produce substantially similar biological properties to a NO-Cbi, or stereoisomer, racemer or isomer thereof.
- the purpose of exchanging one bioisostere for another is to enhance the desired biological or physical properties of NO-Cbi without making significant changes in chemical structures.
- bioisosteres of compounds as provided herein are made by replacing one or more hydrogen atom(s) with one or more fluorine atom(s), e.g., at a site of metabolic oxidation; this may prevent metabolism (catabolism) from taking place.
- the fluorine atom is similar in size to the hydrogen atom the overall topology of the molecule is not significantly affected, leaving the desired biological activity unaffected. However, with a blocked pathway for metabolism, the molecule may have a longer half-life or be less toxic, and the like.
- NO-Cbi compositions used to practice emboidments described herein are prepared in a wide variety of dosage forms according to any means suitable in the art for preparing a given dosage form.
- Pharmaceutically acceptable carriers can be either solid or liquid.
- solid form preparations include a tablet, a pill, a capsule, a liquid, a gel, a geltab, a powder, a spray or aerosol, a cachet, a suppository, a dispersible granule, and the like.
- a solid carrier can include one or more substances which can also act as diluents, flavoring agents, buffering agents, binders, preservatives, tablet disintegrating agents, or an encapsulating material.
- Suitable solid carriers include magnesium carbonate, magnesium stearate, talc, sugar, lactose, pectin, dextrin, starch, gelatin, acacia, tragacanth, methylcellulose, sodium
- liquid form preparations include solutions, suspensions, syrups, slurries, and emulsions.
- suitable liquid carriers include any suitable organic or inorganic solvent, for example, water, alcohol, saline solution, buffered saline solution, physiological saline solution, dextrose solution, water propylene glycol solutions, and the like, preferably in sterile form.
- compositions used to practice embodiments described herein are formulated and administered to the subject as pharmaceutically acceptable salts.
- pharmaceutically acceptable salts include acid addition salts such as those containing hydrochloride, sulfate, phosphate, sulfamate, acetate, citrate, lactate, tartrate, methanesulfonate, ethanesulfonate, benzenesulfonate, p- toluenesulfonate, cyclohexylsulfamate and quinate.
- Such salts can be derived using acids such as hydrochloric acid, sulfuric acid, phosphoric acid, sulfamic acid, acetic acid, citric acid, lactic acid, tartaric acid, malonic acid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, cyclohexylsulfamic acid, and quinic acid, according to means known and established in the art.
- acids such as hydrochloric acid, sulfuric acid, phosphoric acid, sulfamic acid, acetic acid, citric acid, lactic acid, tartaric acid, malonic acid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, cyclohexylsulfamic acid, and quinic acid, according to means known and established in the art.
- aqueous solutions are prepared by dissolving the active component in water and adding suitable colorants, flavors, stabilizing and thickening agents as desired.
- Aqueous suspensions can also be made by dispersing the finely divided active component in water with viscous material, such as natural or synthetic gums, resins, methylcellulose, sodium carboxymethylcellulose, and other well- known suspending agents.
- solid forms are according to any means suitable in the art.
- capsules are prepared by mixing the composition with a suitable diluent and filling the proper amount of the mixture in capsules.
- Tablets are prepared by direct compression, by wet granulation, or by dry granulation.
- Their formulations usually incorporate diluents, binders, lubricants and disintegrators as well as the compound.
- Non- limiting examples of diluents include various types of starch, cellulose, crystalline cellulose, microcrystalline cellulose, lactose, fructose, sucrose, mannitol, kaolin, calcium phosphate or sulfate, inorganic salts such as sodium chloride and powdered sugar.
- Powdered cellulose derivatives are also useful.
- tablet binders include starches, gelatin and sugars such as lactose, fructose, glucose and the like.
- Natural and synthetic gums are also convenient, including acacia, alginates, methylcellulose, polyvinylpyrrolidone and the like.
- Polyethylene glycol, ethylcellulose and waxes can also serve as binders.
- compositions used to practice embodiments described herein are formulated with lubricants, which can be used in a tablet formulation to prevent the tablet and punches from sticking in the die.
- lubricants can be chosen from such slippery solids as talc, magnesium and calcium stearate, stearic acid and
- Tablet disintegrators are substances which swell when wetted to break up the tablet and release the compound, and include starches such as corn and potato starches, clays, celluloses, aligns, gums, methylcellulose, agar, bentonite, wood cellulose, powdered natural sponge, cation-exchange resins, alginic acid, guar gum, citrus pulp, carboxymethyl cellulose, and sodium lauryl sulfate. Tablets can be coated with sugar as a flavor and sealant, or with film-forming protecting agents to modify the dissolution properties of the tablet.
- the compounds can also be formulated as chewable tablets, by using large amounts of pleasant-tasting substances such as mannitol in the formulation, as is now well-established in the art.
- compositions used to practice embodiments described herein are formulated as liquid formulations or solid form preparations, e.g., which can be intended to be converted, shortly before use, to liquid form preparations.
- liquid forms include solutions, suspensions, syrups, slurries, and emulsions.
- Liquid preparations can be prepared by conventional means with
- compositions can contain, in addition to the active agent, colorants, flavors, stabilizers, buffers, artificial and natural sweeteners, dispersants, thickeners, solubilizing agents, and the like.
- compositions can be in powder form for constitution with a suitable vehicle such as sterile water, saline solution, or alcohol, before use.
- compositions used to practice embodiments described herein are formulated in solution in both the un-ionized and ionized forms. Generally lipid soluble or lipophilic drugs diffuse most readily across mucosal membranes. In alternative embodiments, compositions used to practice embodiments described herein are formulated with buffering agents, pH-adjusting agents, or ionizing agents to adjust the ratio of unionized:ionized forms of the NO-Cbi.
- compositions used to practice embodiments described herein are formulated with permeation enhancers or permeability enhancers, which can significantly enhance the permeability of lipophilic and nonlipophilic drugs, including the NO-Cbi.
- penetration enhancers as described in Cooper et al. (1987) "Penetration Enhancers", in Transdermal Delivery of Drugs, Vol. II, Kyodonieus et al., Eds., CRC Press, Boca Raton, Fla are used.
- Additional forms of chemical enhancers also can be used, such as those enhancing lipophilicity, have been developed to improve transport when physically mixed with certain therapeutic agents and provide more predictable absorption, including for example as described in U.S. Pat. Nos.
- carriers can be coupled to pharmaceutical agents described herein to enhance intracellular transport, as described e.g., by Ames et al. (1973) Proc. Natl. Acad. Sci. USA, 70:456-458 and (1988) Proc. Int. Symp. Cont. Rel. Bioact. Mater., 15: 142.
- permeation enhancers include bile salts such as sodium cholate, sodium glycocholate, sodium glycodeoxycholate, taurodeoxycholate, sodium deoxycholate, sodium lithocholate chenocholate, chenodeoxycholate, ursocholate, ursodeoxycholate, hydrodeoxycholate, dehydrocholate, glycochenocholate, taurochenocholate, and taurochenodeoxycholate.
- bile salts such as sodium cholate, sodium glycocholate, sodium glycodeoxycholate, taurodeoxycholate, sodium deoxycholate, sodium lithocholate chenocholate, chenodeoxycholate, ursocholate, ursodeoxycholate, hydrodeoxycholate, dehydrocholate, glycochenocholate, taurochenocholate, and taurochenodeoxycholate.
- permeation enhancers such as sodium dodecyl sulfate (“SDS”), dimethyl sulfoxide (“DMSO”), sodium lauryl sulfate, salts and other derivatives of saturated and unsaturated fatty acids, surfactants, bile salt analogs, derivatives of bile salts, or such synthetic permeation enhancers as described in U.S. Pat. No. 4,746,508 can be used.
- SDS sodium dodecyl sulfate
- DMSO dimethyl sulfoxide
- sodium lauryl sulfate sodium lauryl sulfate
- salts and other derivatives of saturated and unsaturated fatty acids surfactants, bile salt analogs, derivatives of bile salts, or such synthetic permeation enhancers as described in U.S. Pat. No. 4,746,508 can be used.
- SDS sodium dodecyl sulfate
- DMSO dimethyl sulfoxide
- DMSO, SDS, and medium chain fatty acids (about C-8 to about C-14) their salts, derivatives, and analogs are used.
- the permeation enhancer concentration within the dissolvable matrix material can be varied depending on the potency of the enhancer and rate of dissolution of the dissolvable matrix. Other criteria for determining the enhancer concentration include the potency of the drug and the desired lag time. The upper limit for enhancer concentration is set by toxic effect to or irritation limits of the mucosal membrane.
- compositions used to practice embodiments described herein are formulated with a disintegrating agent.
- Tablet disintegrators are substances which swell when wetted to break up the tablet and release the compound, and include starches such as corn and potato starches, clays, celluloses, aligns, gums, methylcellulose, agar, bentonite, wood cellulose, powdered natural sponge, cation-exchange resins, alginic acid, sodium alginate, guar gum, citrus pulp, carboxymethyl cellulose, polyvinyl- pyrrolidone, and sodium lauryl sulfate.
- Acrylic type polymers can also advantageously be used as disintegrators, including methacrylic copolymers of type C (as disclosed in U.S. Pat. No. 6,696,085).
- compositions can be formulated for use in topical administration.
- Such formulations include, e.g., liquid or gel preparations suitable for penetration through the skin such as creams, liniments, lotions, ointments or pastes, and drops suitable for delivery to the eye, ear or nose.
- compositions used to practice embodiments described herein are formulated as creams, drops, liniments, lotions, ointments and pastes are liquid or semi-solid compositions for external application.
- Such compositions can be prepared by mixing the active ingredient(s) in powdered form, alone or in solution or suspension in an aqueous or non-aqueous fluid with a greasy or non-greasy base.
- the base can comprise complex hydrocarbons such as glycerol, various forms of paraffin, beeswax; a mucilage; a mineral or edible oil or fatty acids; or a macrogel.
- Such compositions can additionally comprise suitable surface active agents such as surfactants, and suspending agents such as agar, vegetable gums, cellulose derivatives, and other ingredients such as preservatives, antioxidants, and the like.
- compositions used to practice embodiments described herein are formulated for injection into the subject or individual.
- the compositions can be formulated in aqueous solutions such as water or alcohol, or in physiologically compatible buffers such as Hanks's solution, Ringer's solution, or physiological saline buffer.
- the solution can contain formulatory agents such as suspending, stabilizing and/or dispersing agents.
- Injection formulations can also be prepared as solid form preparations which are intended to be converted, shortly before use, to liquid form preparations suitable for injection, for example, by constitution with a suitable vehicle, such as sterile water, saline solution, or alcohol, before use.
- compositions used to practice embodiments described herein are formulated in sustained release vehicles or depot preparations. Such long acting formulations can be administered by implantation (for example subcutaneously or intramuscularly) or by intramuscular injection.
- the compositions can be formulated with suitable polymeric or hydrophobic materials (for example, as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt.
- suitable polymeric or hydrophobic materials for example, as an emulsion in an acceptable oil
- ion exchange resins for example, as sparingly soluble derivatives, for example, as a sparingly soluble salt.
- liposomes and emulsions are well-known examples of delivery vehicles suitable for use as carriers for hydrophobic drugs.
- compositions used to practice embodiments described herein are formulated and/or administered by systemic administration, e.g., by oral, intravenous, intraperitoneal and intramuscular delivery.
- compositions used to practice embodiments described herein are formulated and/or administered by infusion or injection (intravenously, intramuscularly, intracutaneously, subcutaneously, intrathecal, intraduodenally, intraperitoneally, and the like).
- the compositions can also be administered intranasally, vaginally, rectally, orally, or transdermally.
- the compositions are administered intravenously. Administration can be at the direction of a physician.
- compositions used to practice embodiments described herein are formulated and/or administered via buccal administration, and the
- compositions can take the form of spray, liquid, tablets, troche or lozenge formulated in conventional manner.
- Compositions for oral or buccal administration can be formulated to give controlled release of the active compound.
- Such formulations can include one or more sustained-release agents known in the art, such as glyceryl mono-stearate, glyceryl distearate and wax.
- compositions used to practice embodiments described herein are formulated for topical administration and/or administered or applied topically. Such administrations include applying the compositions externally to the epidermis, the mouth cavity, eye, ear and nose.
- compositions for use in topical administration include, e.g., liquid or gel preparations suitable for penetration through the skin such as creams, liniments, lotions, ointments or pastes, and drops suitable for delivery to the eye, ear or nose.
- compositions used to practice embodiments described herein are formulated for and/or administered via inhalation.
- Compositions can be inhaled through the nose or mouth.
- inhalation can occur via a nasal spray, dry powder inhaler, metered-dose inhaler, vaporizer, and nebulizer.
- alternative pharmaceutical delivery systems are employed.
- Non-limiting examples of such systems include liposomes and emulsions.
- Certain organic solvents such as dimethyl-sulfoxide can also be employed.
- compounds can be delivered using a sustained-release system, such as semipermeable matrices of solid polymers containing the therapeutic agent.
- sustained-release materials are well known by those skilled in the art.
- Sustained-release capsules can, depending on their chemical nature, release the compounds over a range of several days to several weeks to several months.
- nanoparticles, nanolipoparticles, vesicles and liposomal membranes comprising compounds and compositions are used to practice the methods and embodiments as provided herein.
- multilayered liposomes comprising compounds used to practice embodiments as provided herein, e.g., as described in Park, et al., U.S. Pat. Pub. No. 20070082042.
- the multilayered liposomes can be prepared using a mixture of oil-phase components comprising squalane, sterols, ceramides, neutral lipids or oils, fatty acids and lecithins, to about 200 to 5000 nm in particle size, to entrap a composition used to practice embodiments as provided herein.
- Liposomes can be made using any method, e.g., as described in Park, et al., U.S. Pat. Pub. No. 20070042031, including method of producing a liposome by encapsulating an active agent (e.g., compounds and compositions as provided herein, or a compound used to practice methods as provided herein), the method comprising providing an aqueous solution in a first reservoir; providing an organic lipid solution in a second reservoir, and then mixing the aqueous solution with the organic lipid solution in a first mixing region to produce a liposome solution, where the organic lipid solution mixes with the aqueous solution to substantially instantaneously produce a liposome encapsulating the active agent; and immediately then mixing the liposome solution with a buffer solution to produce a diluted liposome solution.
- an active agent e.g., compounds and compositions as provided herein, or a compound used to practice methods as provided herein
- liposome compositions used to practice embodiments as provided herein comprise a substituted ammonium and/or polyanions, e.g., for targeting delivery of a compound as provided herein, or a compound used to practice methods as provided herein, to a desired cell type or organ, e.g., brain, as described e.g., in U.S. Pat. Pub. No. 20070110798.
- nanoparticles comprising compounds as provided herein, e.g., used to practice methods as provided herein in the form of active agent-containing
- nanoparticles e.g., a secondary nanoparticle
- a fat-soluble active agent used to practice embodiments as provided herein, or a fat-solubilized water- soluble active agent to act with a bivalent or trivalent metal salt.
- solid lipid suspensions can be used to formulate and to deliver compositions used to practice embodiments as provided herein to mammalian cells in vivo, in vitro or ex vivo, as described, e.g., in U.S. Pat. Pub. No. 20050136121.
- the effective amount of a composition to be administered can be dependent on any number of variables, including without limitation, the species, breed, size, height, weight, age, overall health of the subject, the type of formulation, the mode or manner or administration, or the severity of the osteoporesis.
- the appropriate effective amount can be routinely determined by those of skill in the art using routine optimization techniques and the skilled and informed judgment of the practitioner and other factors evident to those skilled in the art.
- a composition to be administered can be dependent on any number of variables, including without limitation, the species, breed, size, height, weight, age, overall health of the subject, the type of formulation, the mode or manner or administration, or the severity of the osteoporesis.
- the appropriate effective amount can be routinely determined by those of skill in the art using routine optimization techniques and the skilled and informed judgment of the practitioner and other factors evident to those skilled in the art.
- a composition to be administered can be dependent on any number of variables, including without limitation, the species, breed, size, height, weight, age, overall health of the subject, the type of formulation
- therapeutically effective dose of the compounds described herein will provide therapeutic benefit without causing substantial toxicity to the subject.
- a concentration of NO-Cbi administered is in a range of about 0.01% to about 90% of the dry matter weight of the composition.
- NO-Cbi comprises up to about 50% of the dry matter weight of the composition.
- NO-Cbi comprises up to about 40% of the dry matter weight of the composition.
- NO-Cbi comprises up to about 30% of the dry matter weight of the composition.
- NO-Cbi comprises up to about 25% of the dry matter weight of the composition.
- NO-Cbi comprises up to about 20%) of the dry matter weight of the composition.
- NO-Cbi comprises up to about 15%) of the dry matter weight of the composition.
- NO-Cbi comprises up to about 10%> of the dry matter weight of the composition.
- individuals or subjects are administered NO-Cbi in a daily dose range of about 0.001 mg/kg to about 10 mg/kg of the weight of the subject.
- the dose administered to the subject can also be measured in terms of total amount of drug administered per day; for example, individuals or subjects are administered NO-Cbi in a dosage range of about 0.01 to about 500 milligrams of NO-Cbi per day: 0.05 milligrams of NO-Cbi per day; about 0.1 milligrams of NO-Cbi per day; about 0.5 milligrams of NO-Cbi per day; about 1 milligrams of NO-Cbi per day; about 5 milligrams of NO-Cbi per day; about 10 milligrams of NO-Cbi per day; about 25 milligrams of NO- Cbi per day; about 50 milligrams of NO-Cbi per day; about 100 milligrams of NO-Cbi per day; about 150 milligrams of NO-Cbi per day; or about
- Treatment can be initiated with smaller dosages that are less than the optimum dose of NO-Cbi, followed by an increase in dosage over the course of the treatment until the optimum effect under the circumstances is reached. If needed, the total daily dosage can be divided and administered in portions throughout the day.
- dosing can occur one to four or more times daily for as long as needed. The dosing can occur less frequently if the
- compositions are formulated in sustained delivery vehicles.
- the dosage schedule can also vary depending on the active drug concentration, which can depend on the needs of the subject.
- compositions used to practice embodiment described herein can be co-administered with other therapeutic agents that are selected for their particular usefulness against the condition that is being treated.
- therapeutic agents can be pain relievers, blood thinners/anticoagulants, clot busters, stomach antacids, compounds which lessen untoward effects of the compositions, other known agents that lower blood pressure.
- the compositions can be co-administered with cobalamin (vitamin B12) to reduce or eliminate potential toxicity of the administered cobinamide.
- the administration of these additional compounds can be simultaneous with the administration of NO-Cbi, or can be administered in tandem, either before or after the administration of NO-Cbi, as necessary.
- Any suitable protocol can be devised whereby the various compounds to be included in the combination treatment are administered within minutes, hours, days, or weeks of each other.
- repeated administrations are in a cyclic protocol.
- Example 1 Nitrosyl-cobinamide (NO-Cbi) is effective for treating osteoporosis
- compositions provided herein including exemplary nitrosyl-cobinamide (NO-Cbi), a direct NO-rel easing agent, is effective for treating osteoporosis in a mouse model of estrogen deficiency-induced osteoporosis.
- NO-Cbi nitrosyl-cobinamide
- NO-Cbi In murine primary osteoblasts, NO-Cbi increased intracellular cGMP, Wnt/ ⁇ - catenin signaling, proliferation, and osteoblastic gene expression, and protected cells from apoptosis.
- NO-Cbi In ovariectomized (OVX) C57B1/6 mice, NO-Cbi increased serum cGMP concentrations, bone formation, and osteoblastic gene expression, and prevented estrogen deficiency-induced osteocyte apoptosis.
- NO-Cbi additionally prevented an ovariectomy-induced increase in osteoclasts, likely due to a reduction in the RANKL/osteoprotegerin gene expression ratio, which regulates osteoclast differentiation, and due to direct inhibition of osteoclast differentiation, observed in vitro in the presence of excess RANKL. While positive NO effects in osteoblasts were mediated by cGMP/PKG, the osteoclast-inhibitory effects appeared to be largely cGMP-independent. Consistent with its effects on osteoblasts and
- NO-Cbi increased trabecular bone mass in OVX mice.
- NO-Cbi represents a novel direct NO-releasing agent that, in contrast to nitrates, does not generate superoxide, and combines anabolic and anti-resorptive effects in bone, making it an effective agent for treating osteoporosis.
- Antibodies against Akt, Akt(pSer 473 ), Erkl(pTyr 204 ), GSK-3p, GSK- 3P(pSer 9 ), and cleaved caspase-3 were from Cell Signaling, and antibodies against Erkl/2, and ⁇ -actin were from Santa Cruz Biotechnology.
- a ⁇ -catenin-specific antibody and fluorophore-labeled secondary antibodies were from InVitrogen.
- DETA-NONOate was from Cayman and the cGMP agonist 8-(4-chlorophenylthio)-cGMP (8-pCPT-cGMP) from BioLog.
- NO-Cbi was generated by reducing dinitro-cobinamide under deoxygenated conditions. Ascorbic acid and dinitro-cobinamide were incubated at a ratio of 5: 1 for 1 h at RT, and then the solution was purged with nitrogen to remove any free NO. NO-Cbi was stable at RT for at least 1 month when protected from light and filter- sterilized.
- mice Ten week-old female C57B1/6 mice were purchased from Jackson Laboratories. They were maintained in accordance with the "Guide for the Care and Use of Laboratory Animals" (2011, 8 th ed., Washington, D.C., Natl. Research Council, Natl. Academys Press), and all experiments were approved by the Institutional Animal Care and Use Committee of the University of California, San Diego. Mice were housed at 3-4 animals per cage in a temperature-controlled environment with a 12 h light/dark cycle; they were fed standard Teklad Rodent Diet with ad lib access to food and water.
- mice weighing 19.5-22.0 g were randomly divided into three groups— Groups 1 and 2 (eight mice each) underwent bilateral ovariectomy, while Group 3 (six mice) underwent a sham operation.
- Group 1 had to be euthanized post-operatively because of suture failure.
- mice received daily i.p. injections six days per week for five weeks, either vehicle (0.1 ml 9.25 mM ascorbic acid, Group 1), or NO-Cbi (10 mg/kg/d given as 0.1 ml of 1.85 mM NO-Cbi, Group 2).
- mice carrying floxed prkg2 alleles (PRKG2 f/f mice).
- PRKG2 f/f mice To generate PRKG2 f/f mice, we PCR-amplified genomic DNA fragments encoding prkg2 exon III with flanking sequences from 129/SvJ ES cells using KOD polymerase (EMD Millipore Corporation).
- the prkg2-f ⁇ oxed construct was assembled in the vector pDL L (gift from Ju Chen of UCSD) and consisted of a 4.2 kb 5' arm, a 0.55 kb fragment including exon III flanked by LoxP sites, a 1.3 kb neo cassette flanked by FRT sites, and a 3kb 3' arm. All PCR products and fusion sites were sequenced.
- the construct was electroporated into Rl ES cells derived from 129/SvJ mice, and G418-resistant clones were isolated and screened by PCR; homologous recombination was confirmed by Southern blot analysis using probes outside of the 5' and 3' arms.
- a recombinant clone with normal chromosome analysis in 20 metaphase spreads was injected into C57B1/6 blastocysts to establish germline chimeric mice.
- Heterozygous PRKG2 f/+ mice were mated with homozygous FLPeR mice containing FLPe recombinase (from Jackson Laboratories) to remove the neo cassette.
- POBs murine primary osteoblasts
- Osteoclasts were generated from murine bone marrow as described. 37 Briefly, bone marrow cells were plated at 10 6 cells/cm 2 in a- Minimal Essential Medium with 10% FBS and 50 ng/ml M-CSF, and non-adherent cells were discarded after 48 h. RANKL (150 ng/ml) was added on day 3, medium was replaced on days 5 and 7; the indicated drugs were added with fresh medium on days 3,5, and 7. On day 8, cultures were fixed and stained for TRAP using a commercially- available kit (Sigma), or were harvested for RNA isolation.
- RANKL 150 ng/ml
- Protein extracts were prepared from mouse bones as described previously. 36 Briefly, -50 mg of pulverized bone was incubated for 15 min on ice in 50 mM Tris-HCl pH 8.0, 150 mM NaCl, 2 mM EDTA, 1% Triton X-100, 0.1% SDS, 0.5% sodium deoxycholate, 2 mM Na 3 V0 4 , 10 mM NaF, and protease inhibitor cocktail. The samples were centrifuged at 13,000 g for 15 min at 4°C. Supernatants were boiled in SDS sample buffer and proteins were resolved by SDS-PAGE and analyzed by Western blotting as described. 13 Films were scanned using Image J software.
- NO production was measured based on nitrite and nitrate accumulation in the medium with a two-step colorimetric assay, as previously described. 13 Serum cGMP concentrations were measured by ELISA using a kit according to the manufacturer's protocol (Biomedical Technologies Inc., MA).
- POBs in 6-well dishes were cultured in 0.1% FBS overnight and treated with NO-Cbi for 1 h; cells were then transferred to fresh medium with 0.1% FBS and 10 ⁇ of [methyl- 3 H]thymidine (20 Ci/mmol, final concentration 0.5 ⁇ ) for 24 h. Cells were extracted in situ in ice-cold 10% trichloroacetic acid, precipitated DNA was collected on glass microfiber filters, and radioactivity on washed filters was measured by scintillation counting.
- Histomorphometric measurements were performed between 0.25 and 2.25 mm distal to the growth plate as described. 38
- Sections (8 ⁇ thick) were de-paraffinized in xylene and rehydrated in graded ethanol and water.
- slides were placed in 10 mM sodium citrate buffer (pH 6.0) at 80-85°C, and allowed to cool to room temperature for 30 min. Endogenous peroxidase activity was quenched in 3% hydrogen peroxide for 10 min.
- Slides were blocked with 5% normal goat serum and incubated overnight at 4°C with anti-phospho- ERK antibody (1 : 100 in blocking buffer), followed by HRP-conjugated secondary antibody for 1 h at room temperature. After development with 3,3-diaminobenzidine substrate (Vector Laboratories, Inc., Burlingame, CA), slides were counterstained with hematoxylin for 2 min.
- Micro-CT Micro-CT analyses were performed on ethanol-fixed tibiae, using a Skyscan 1076TM (Kontich, Belgium) scanner at 9 ⁇ voxel size, and applying an electrical potential of 50 kVp and current of 200 ⁇ , with a 0.5 mm aluminum filter, as described. 38 Mineral density was determined by calibration of images against 2 mm diameter hydroxyapatite rods (0.25 and 0.75 g /cm 3 ). Cortical bone was analyzed by automatic contouring 3.6 mm-4.5 mm distal to the proximal growth plate, using a global threshold to identify cortical bone, and eroding one pixel to eliminate partial volume effects. Trabecular bone was analyzed by automatic contouring the proximal tibial metaphysis 0.36 -2.1 mm distal to the growth plate and using an adaptive threshold to select the trabecular bone.
- mice would provide 90% power to detect the difference in bone volume/tissue volume (BV/TV) between OVX and sham-operated mice, whereas 8 mice per group were required to detect an absolute increase of 0.35% in BV/TV in drug-treated OVX mice with 90% power (a error set at 5%).
- BV/TV bone volume/tissue volume
- NO-Cbi enhances cGMP/PKG and Erk/ Akt signaling, gene expression, proliferation, and survival in POBs.
- cobinamide binds NO with high affinity (K a ⁇ 10 10 M "1 ), yielding NO-Cbi, where one water molecule on cobinamide is replaced by one NO molecule.
- NO-Cbi is stable in aqueous solutions for at least 16 h at room temperature even when exposed to air, but it releases NO rapidly in the presence of NO scavengers or when added to cells in culture medium.
- 32 Murine POBs produced nitrite and nitrate— stable NO oxidation products— yielding a medium concentration of -10 ⁇ over 2 h (Fig. 1A).
- NO-Cbi increased the amount of nitrite and nitrate within 10 min, and by 30 min, the amount of NO oxidation products generated was maximal and accounted for >90% of the NO added to the cells as NO-Cbi (Fig. 1 A).
- NO-Cbi increased the intracellular cGMP concentration in a dose-dependent fashion, resulting in near- maximal phosphorylation of the PKG substrate vasodilator-stimulated phosphoprotein (VASP) at 3-10 ⁇ (Fig. 1B,C).
- VASP vasodilator-stimulated phosphoprotein
- NO-Cbi raised mRNA expression of osteoblast differentiation-related genes, including osteocalcin (OCN), osteopontin (Sppl), collagenl-al (Collal), alkaline phosphatase (ALP), and low-density lipoprotein receptor-related protein-5 (Lrp5), with tubulin (Tubal) expression serving as a control for RNA quality (Fig. 1H).
- OCN osteocalcin
- Sppl osteopontin
- Collal Collal
- ALP alkaline phosphatase
- Lrp5 low-density lipoprotein receptor-related protein-5
- Tubulin (Tubal) expression serving as a control for RNA quality
- NO-Cbi decreased osteoblast expression of RANKL mRNA, whereas it increased expression of the RANKL antagonist OPG, suggesting that NO-Cbi could negatively affect osteoclast differentiation (Fig. II).
- NO-Cbi stimulates Wnt signaling and mPOB proliferation via PKG II.
- the Wnt/p-catenin pathway controls osteoblast differentiation, proliferation, and survival, and is essential for bone mass acquisition and maintenance; increased or decreased gene expression and gain- or loss-of-function mutations of pathway components cause high versus low bone mass phenotypes— for example, expression and activity of the Wnt co-receptor Lrp5 positively correlate with bone mass in humans and mice.
- Stability and nuclear translocation of ⁇ -catenin are negatively controlled by glycogen synthase kinase-3 (GSK-3), which in turn is negatively controlled by phosphorylation on a site targeted by Akt and PKG II.
- NO-Cbi treatment increased Wntla, Lrp5, and ⁇ - catenin mRNA expression in the presence, but not in the absence of PKG II (Fig. 2E).
- Lrp5 protein increased in parallel to mRNA in response to NO-Cbi (Fig. 2F).
- Transcript levels of the Wnt/p-catenin target genes cyclin D (CycD), ALP, and OCN were increased by NO-Cbi in PKG Il-expressing, but not in PKG Il-deficient cells (Fig. 2E).
- NO-Cbi inhibits osteoclast differentiation.
- NO-Cbi dramatically reduced the number of TRAP -positive osteoclasts, with maximal effects observed at 10 ⁇ (Fig. 3A,B). Less pronounced inhibition was observed with the NO donor DETA-NONOate at a concentration calculated to release equivalent amounts of NO (Fig. 3C). In contrast, the cGMP analog 8-pCPT-cGMP, at a concentration that maximally activated PKG I and II in intact cells, had a much smaller, non-significant effect on the number of TRAP-positive cells (Fig. 3C).
- NO-Cbi increases serum cGMP concentration, bone formation, and osteoblastic gene expression in OVX mice.
- NO-Cbi could prevent bone manifestations of estrogen deficiency
- OVX bilateral ovariectomy
- sham operation injected the OVX mice with vehicle or NO-Cbi for five weeks, starting one week postoperatively.
- NO-Cbi dose 10 mg/kg/d
- serum cobinamide concentrations below our limits of detection ( ⁇ 1 ⁇ ).
- 38 OVX mice had lower serum cGMP concentrations compared to sham-operated mice - but NO-Cbi significantly increased serum cGMP at 1 h post administration (Fig. 4A).
- NO-Cbi reversed the decrease in osteoblast numbers found in OVX mice, and significantly increased mineral apposition rate (MAR), mineralizing surfaces (MS/BS), and bone formation rate (BFR) on trabecular bone surfaces (Fig. 4B-F). Similarly, NO-Cbi increased MAR and BFR on endocortical surfaces, although endocortical MS/BS was not affected (Fig. 8).
- NO-Cbi prevents estrogen deficiency-induced osteocyte apoptosis.
- Estrogen deficiency-induced bone loss is, in part, due to osteocyte apoptosis. 11 ' 38 ' 42
- osteocyte protective effects of estrogen require
- NO-Cbi regulates RANKL/OPG expression and reduces osteoclasts in OVX mice.
- Estrogens reduce osteoclast survival, 9, 43 and, as expected, we found an increased number of TRAP-positive osteoclasts on trabecular bone in the OVX mice (Fig. 6 A,B; few osteoclasts were seen on cortical surfaces under all conditions).
- Treating OVX mice with NO-Cbi reduced osteoclast numbers to values found in sham-operated animals (Fig. 6A,B).
- femurs of NO-Cbi-treated OVX mice contained less RANKL and more OPG mRNA, and mRNA expression of the osteoclast marker genes CTSK and TRAP was decreased (Fig. 6C).
- NO-Cbi reduced osteoclast numbers and osteoclastic gene expression in OVX mice, at least, in part, by reducing the RANKL/OPG ratio.
- NO-Cbi increases trabecular bone mass in OVX mice.
- NOS3-deficient mice have reduced bone mass due to defects in osteoblast number and maturation, and they have exaggerated bone loss after ovariectomy, with a blunted response to estrogens 47"49
- NO-generating organic nitrates reduce bone loss from estrogen deficiency in rats (as measured by DXA), whereas NOS inhibitors block estrogen's bone-protective effects and prevent bone formation induced by mechanical stimulation in rodents. 16"18 ' 22 ' 23
- serum concentrations of the NO metabolites nitrite and nitrate correlate with estradiol concentrations, and increase with estrogen administration.
- the NO-independent soluble guanylate cyclase activator cinaciguat increased bone formation and osteoblast marker genes and ameliorates bone loss in OVX mice; however, in contrast to NO-Cbi, multipliguat did not affect osteoclast parameters significantly.
- NOS2 induction by RANKL serves as a feed-back inhibition of RANKL-induced osteoclast differentiation
- NOS2-deficient osteoclasts show increased differentiation in response to RANKL. Adding NO donors to mature osteoclasts inhibits their resorptive activity.
- NO-Cbi treatment of OVX mice decreased osteoclast numbers and reduced osteoclast-specific TRAP and CTSK mRNAs in bone; these results correlated with a decrease in RANKL and increase in OPG mRNAs in bone.
- the cGMP- elevating agent cinaciguat did not influence osteoclast numbers, osteoclast-specific genes, RANKL, or OPG in OVX mice. 38
- NO- Cbi reduced RANKL and increased OPG mRNA in cultured POBs, whereas cinaciguat did not affect these genes. 38
- RANKL and OPG are key regulators of osteoclast differentiation produced primarily by cells of the osteoblastic lineage.
- isosorbide mononitrate decreased a bone resorption marker (N-terminal telopeptide) and increased a bone formation marker (alkaline phosphatase) compared to placebo.
- nitroglycerin increased BMD at the lumbar spine and hip, and increased cortical thickness at the radius and tibia; it also decreased N- terminal telopeptide and increased alkaline phosphatase. 25
- NO-Cbi is derived from the penultimate vitamin B12 precursor cobinamide; cobinamide is found at low concentrations in human serum, and we have found in rodents that cobinamide has no toxicity at >50- fold higher doses than those used in this study and those required for delivery of pharmacological amounts of NO (G.R.Boss et al., unpublished data and 32 ).
- cobinamide After NO release, cobinamide is generated, and cobinamide can bind O2 " and other reactive oxygen species, 35 providing a potential added benefit of protecting cells from oxidative stress.
- Repeated administration of NO-Cbi does not induce tolerance, and NO-Cbi is stable and can be administered by multiple routes, including oral ingestion (G.R. Boss et al., unpublished data and 32 ).
- OVX mice are an accepted model for bone loss due to estrogen deficiency, and show changes in bone architecture and turnover similar to those observed in post-menopausal women, including increased bone resorption and increased osteoblast/osteocyte apoptosis. 9, 42, 44, 46 Some authors have observed increased bone formation markers in parallel with increased resorption in OVX mice, whereas we and others found no change in mineral apposition rate and a trend towards reduced mineralizing surface and bone formation rate after OVX. 66"69 These differences may be attributable to differences in mouse strain and age, the type of bone examined, and the time interval after OVX.
- NO-Cbi regulates bone remodeling by promoting osteoblast proliferation, differentiation, and survival, and by simultaneously inhibiting osteoclast differentiation.
- NO-Cbi improved bone mass in OVX mice, a frequently-used model of post-menopausal osteoporosis. 9 ' 44 ' 70
- a direct NO-releasing agent as a bone-anabolic agent in animals or humans.
- Figure 1 NO-Cbi enhances cGMP/PKG and Erk/Akt signaling, gene expression, proliferation and survival in POBs.
- A POBs were incubated in medium with 0.1% FBS (3 x 10 5 cells/ml) for 2 h prior to receiving 10 ⁇ NO-Cbi (NOCbi) for the indicated times. Stable NO oxidation products (nitrite plus nitrate, NOx) were measured in the medium by the Griess reaction (NOx present in medium without cells was subtracted).
- B,C POBs were treated with vehicle or NO-Cbi at the indicated concentrations for 30 min, and intracellular cGMP concentrations were measured by ELISA (B).
- VASP phosphorylation was analyzed by Western blot using a phospho-Ser 259 -specific antibody, with densitometric quantitation of pVASP normalized to ⁇ -actin shown above.
- D,E Serum-deprived POBs were treated with vehicle or 10 ⁇ NO-Cbi for 10 min and ERK and Akt activation were assessed by blotting with phospho-specific antibodies.
- osteocalcin OCN
- osteopontin Sppl
- collagen 1-Al Cold-dextrin
- ALP alkaline phosphatase
- Lrp5 low-density lipoprotein receptor-related protein-5
- Tubulin Tubulin
- RNKL receptor activator of nuclear factor kappa-B ligand
- OPG osteoprotegerin
- Panels A-I show means ⁇ SEM of at least three independent experiments; *p ⁇ 0.05, **p ⁇ 0.01, ***p ⁇ 0.001, for the comparison between NO-Cbi- and vehicle-treated cells.
- ### p ⁇ 0.01 for the comparison between cells in starvation versus control medium.
- Figure 2 NO-Cbi stimulates Wnt signaling and mPOB proliferation via PKG II.
- (C) PRKG2 f/f POBs were infected with control or Cre virus and transferred to 0.1% FBS as in B; they were treated with NO-Cbi or vehicle for 6 h, prior to detecting ⁇ -catenin by immunofluorescence staining.
- the bottom panel shows nuclei counterstained with Hoechst 33342. Numbers below indicate the percentage of cells showing nuclear ⁇ -catenin.
- D Cells were infected and cultured as in B; they were treated with NO-Cbi or vehicle for 1 h prior to measuring 3 H-thymidine incorporation into DNA for 24 h.
- Wingless type MMTV-integration site family- la Wntla
- low-density lipoprotein receptor- related protein-5 Lrp5
- bCat ⁇ -catenin
- CycD cyclin D
- ALP alkaline phosphatase
- OCN osteocalcin
- Tubulin (Tubal) mRNAs were measured by qRT-PCR and normalized to 18S rRNA, with relative mRNA levels in untreated cells assigned a value of 1.
- F POBs cultured in 10 % FBS were treated with 10 ⁇ NO-Cbi for the indicated times, and Lrp5 protein (open symbols) and mRNA (filled symbols) were assessed by Western blotting and qRT-PCR, respectively.
- Panels A-F show means ⁇ SEM of at least three independent experiments; *p ⁇ 0.05, **p ⁇ 0.01, ***p ⁇ 0.001, for the comparison between NO-Cbi-treated versus vehicle-treated cells infected with control virus, and # p ⁇ 0.05, ## p ⁇ 0.01, ### p ⁇ 0.001 for comparison between NO-Cbi-treated cells infected with control versus CRE virus.
- Figure 3 NO-Cbi inhibits osteoclast differentiation.
- A,B Murine bone marrow mononuclear cells were cultured in the presence of M-CSF, with RANKL added after 3 d; together with RANKL, cells received vehicle or NO-Cbi at the indicated concentrations. Tartrate-resistant acid phosphatase (TRAP)-positive cells (red) were counted on day 8.
- C Cells were cultured as in A, but some cultures received 10 ⁇ NO-Cbi, 5 ⁇ DETA- NONOate (Deta-NO, which releases 2 moles of NO/mol of drug), or 100 ⁇ 8-pCPT- cGMP together with RANKL.
- Panels B-D show means ⁇ SEM of at least three independent experiments; *p ⁇ 0.05, **p ⁇ 0.01, ***p ⁇ 0.001, for the comparison between vehicle-treated versus drug-treated cells.
- FIG. 4 NO-Cbi increases serum cGMP concentration, bone formation, and osteo- blastic gene expression in OVX mice.
- Eleven week-old mice were subjected to ovariectomy (OVX) or sham operation, and 7 d later daily i.p. injections were started with either vehicle (Veh) or NO-Cbi (10 mg/kg/d) for 6 d/week for a total of 5 weeks. Mice additionally received calcein 7 d and 4 d prior to euthanasia.
- Veh ovariectomy
- NO-Cbi 10 mg/kg/d
- Mice additionally received calcein 7 d and 4 d prior to euthanasia.
- Serum cGMP concentrations were measured by ELISA 1 h after the last injection of vehicle or NO-Cbi.
- (B) The number of trabecular osteoblasts per bone perimeter (N.Ob/B.Pm) was counted at the proximal tibia.
- C-F Trabecular calcein labeling was assessed at the tibia (C), with quantification of mineral apposition rate (MAR, panel D), mineralizing surface per bone surface (MS/BS, panel E), and bone formation rate (BFR, panel F).
- Figure 5 NO-Cbi prevents estrogen deficiency-induced osteocyte apoptosis. Mice were subjected to OVX or sham operation and were treated with vehicle or NO-Cbi as described in Fig. 4.
- Figure 6 NO-Cbi regulates RANKL/OPG and reduces osteoclasts in OVX mice.
- Mice subjected to OVX or sham-operation were treated with vehicle or NO-Cbi as described in Fig. 4.
- A,B Osteoclasts were identified by TRAP staining (red), and the number of trabecular osteoclasts per bone perimeter (N.Oc/B.Pm) was counted at the proximal tibia.
- C RNA was extracted from femurs, and the relative abundance of
- RANKL, OPG, CTSK, and TRAP mRNA was quantified by qRT-PCR and normalized to 18S rRNA. Data were calculated according to the AACT method using the mean of the vehicle-treated OVX group. Data represent the mean ⁇ SEM from 6 mice per group. *p ⁇ 0.05, **p ⁇ 0.01, ***p ⁇ 0.001 for the indicated pair-wise comparisons.
- Figure 7 NO-Cbi increases trabecular bone mass in OVX mice. Mice subjected to OVX or sham operation were treated with vehicle or NO-Cbi as described in Fig.4.
- A Tibiae were analyzed by micro-CT imaging, and three-dimensional reconstruction of the trabecular bone at the proximal tibia below the growth plate is shown.
- B-E Trabecular bone volume/tissue volume (B), trabecular number (C), and trabecular bone mineral density (D) were quantified at the proximal tibia as described in Materials and Methods.
- the osteocyte an endocrine cell ... and more.
- Neer RM Arnaud CD
- Zanchetta JR et al Effect of parathyroid hormone (1-34) on fractures and bone mineral density in postmenopausal women with osteoporosis.
- osteoclast attachment requires inositol-l,4,5-trisphosphate receptor-associated cGMP-dependent kinase substrate.
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Medicinal Chemistry (AREA)
- Pharmacology & Pharmacy (AREA)
- Physical Education & Sports Medicine (AREA)
- Life Sciences & Earth Sciences (AREA)
- Animal Behavior & Ethology (AREA)
- General Health & Medical Sciences (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Orthopedic Medicine & Surgery (AREA)
- Epidemiology (AREA)
- Rheumatology (AREA)
- Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Organic Chemistry (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
In alternative embodiments, provided are compounds, including formulations and pharmaceutical compositions, and methods of making and using them, for treating, ameliorating, preventing or reversing osteoporesis, including post-menopausal osteoporosis. In alternative embodiments, provided are methods for, and uses of nitrosyl-cobinamide (NO-Cbi) for: treating, ameliorating, preventing or reversing osteoporosis; preventing bone loss due to estrogen deficiency; inhibiting osteoclast differentiation; reducing osteoclast numbers; preventing estrogen deficiency-induced osteocyte apoptosis; increasing serum cGMP concentration; increasing bone formation or bone mass, or increasing trabecular bone volume, trabecular number, and trabecular bone mineral density; increasing osteoblast numbers or regulating osteoblastic gene expression, comprising administering a nitrosyl-cobinamide (NO-Cbi), or a bioisostere, stereoisomer, tautomer, hydrate, solvate or pharmaceutically acceptable salt thereof to: an individual; an individual with osteoporosis or at risk of developing osteoporosis.
Description
COMPOSITIONS AND METHODS FOR TREATING
OSTEOPORESIS
RELATED APPLICATIONS
This application claims the benefit of priority to U. S. Provisional Patent
Application Serial No. (USSN) 62/348,577 filed June 10, 2016. The aforementioned application is expressly incorporated herein by reference in its entirety and for all purposes.
GOVERNMENT RIGHTS
This invention was made with government support under grants AR051300, R21-
AR065658, U01-NS58030, P01-AG007996, P30-CA023100, P30-NS047101, P30- AR04603 and NS058030, awarded by the National Institutes of Health (NIH), DHHS. The government has certain rights in the invention.
TECHNICAL FIELD
This invention generally relates to physiology and bone metabolism. In particular, in alternative embodiments, provided are compounds, including formulations and pharmaceutical compositions, and methods of making and using them, for treating, ameliorating, preventing or reversing osteoporesis, including post-menopausal osteoporosis. In alternative embodiments, provided are methods for, and uses of nitrosyl- cobinamide (NO-Cbi) for: treating, ameliorating, preventing or reversing osteoporosis; preventing bone loss due to estrogen deficiency; inhibiting osteoclast differentiation; reducing osteoclast numbers; preventing estrogen deficiency-induced osteocyte apoptosis; increasing serum cGMP concentration; increasing bone formation or bone mass, or increasing trabecular bone volume, trabecular number, and trabecular bone mineral density; increasing osteoblast numbers or regulating osteoblastic gene expression, comprising administering a nitrosyl-cobinamide (NO-Cbi), or a bioisostere, stereoisomer, tautomer, hydrate, solvate or pharmaceutically acceptable salt thereof to: an individual; an individual with osteoporosis or at risk of developing osteoporosis.
BACKGROUND
Most FDA-approved treatments for osteoporosis target osteoclastic bone resorption. Only parathyroid hormone (PTH) derivatives improve bone formation, but
they have drawbacks, so new bone-anabolic agents are needed. The Nitric Oxide (NO)/cGMP/protein kinase G (PKG) signaling pathway mediates anabolic effects of estrogens and mechanical stimulation in bone cells by increasing osteoblast proliferation and osteocyte survival. Nitrates, which generate NO, reduce bone loss in estrogen- deficient rats and increase bone mineral density in post-menopausal women. However, nitrates are limited by induction of oxidative stress and development of tolerance, and may increase cardiovascular mortality after long-term use.
Organic nitrates generate NO in vivo after mitochondrial biotransformation; nitrates are used to treat coronary insufficiency and heart failure, and epidemiological studies suggest their use may reduce fracture risk.19"21 Based on these data and preclinical studies showing a bone-protective effect of organic nitrates in OVX rats,22, 23 several clinical trials have examined the skeletal effects of organic nitrates in post-menopausal women, showing an increase in bone formation markers, a decrease in bone resorption markers, and improved bone mineral density in women with estrogen deficiency.24
Organic nitrates are the only NO donors currently FDA-approved for long-term use, but clinical benefits of organic nitrates are limited by development of tolerance and induction of oxidative stress.27"30 Nitrates require enzymatic activation to release NO, and this reaction generates reactive oxygen species, especially O2", which can have detrimental effects in the cardiovascular and skeletal systems.10' 29' 31 SUMMARY
In alternative embodiments, provided are methods for:
- treating, ameliorating, preventing or reversing osteoporosis or postmenopausal osteoporosis;
- preventing bone loss due to estrogen deficiency;
- inhibiting osteoclast differentiation;
- reducing osteoclast numbers;
- preventing estrogen deficiency-induced osteocyte apoptosis;
- increasing serum cGMP concentration;
- increasing bone formation and bone mass, or increasing trabecular bone volume, trabecular number, and trabecular bone mineral density; or
- increasing osteoblast number and regulating osteoblastic gene expression,
comprising
administering a nitrosyl-cobinamide (NO-Cbi), or a bioisostere, stereoisomer, tautomer, hydrate, solvate or pharmaceutically acceptable salt thereof to: an individual; an individual with osteoporosis or at risk of developing osteoporosis,
thereby:
- treating, ameliorating, preventing or reversing osteoporosis or postmenopausal osteoporosis;
- preventing bone loss due to estrogen deficiency;
- inhibiting osteoclast differentiation;
- reducing osteoclast numbers;
- preventing estrogen deficiency-induced osteocyte apoptosis;
- increasing serum cGMP concentration;
- increasing bone formation or bone mass, or increasing trabecular bone volume, trabecular number, and trabecular bone mineral density; or
- increasing osteoblast number and regulating osteoblastic gene expression.
In alternative embodiments, the nitrosyl-cobinamide, or the bioisostere, stereoisomer, tautomer, hydrate, solvate or pharmaceutically acceptable salt thereof, is formulated as a pharmaceutical composition, or is formulated as a pharmaceutical composition comprising a pharmaceutically acceptable carrier or excipient thereof.
In alternative embodiments, the nitrosyl-cobinamide, or the bioisostere, stereoisomer, tautomer, hydrate, solvate or pharmaceutically acceptable salt thereof, or the pharmaceutical composition, is administered or co-administered with another therapeutic agent, wherein optionally therapeutic agent is administered simultaneously, before or after the nitrosyl-cobinamide, or a bioisostere, stereoisomer, tautomer, hydrate, solvate or pharmaceutically acceptable salt thereof, or the pharmaceutical composition.
In alternative embodiments, the nitrosyl-cobinamide, or the bioisostere, stereoisomer, tautomer, hydrate, solvate or pharmaceutically acceptable salt thereof, or the pharmaceutical composition, is administered in or with an implant or a bone implant.
In alternative embodiments, the nitrosyl-cobinamide, or the bioisostere, stereoisomer, tautomer, hydrate, solvate or pharmaceutically acceptable salt thereof, or the pharmaceutical composition, is administered as or is formulated in a device, an implant, a bone implant, a bead, a tablet, a pill, a capsule, a liquid, a gel, a geltab, a
powder, a spray or aerosol, a cachet, a suppository, a dispersible granule, a product of manufacture, a liposome, a particle, a microparticle or a nanoparticle.
In alternative embodiments, the nitrosyl-cobinamide, or the bioisostere, stereoisomer, tautomer, hydrate, solvate or pharmaceutically acceptable salt thereof, or the pharmaceutical composition, is administered as or is formulated in or as a kit, a pump, a device, a subcutaneous infusion device, a continuous subcutaneous infusion device, an infusion pen, a needles, a reservoir, an ampoules, a vial, a syringe, a cartridge, a pen, a disposable pen or jet injector, a prefilled pen or a syringe or a cartridge, a cartridge or a disposable pen or jet injector, a two chambered or multi-chambered pump.
In alternative embodiments, provided are Uses of a nitrosyl-cobinamide (NO-
Cbi), or a bioisostere, stereoisomer, tautomer, hydrate, solvate or pharmaceutically acceptable salt thereof in the manufacture of a medicament for:
- treating, ameliorating, preventing or reversing osteoporosis or postmenopausal osteoporosis;
- preventing bone loss due to estrogen deficiency;
- inhibiting osteoclast differentiation;
- reducing osteoclast numbers;
- preventing estrogen deficiency-induced osteocyte apoptosis;
- increasing serum cGMP concentration;
- increasing bone formation or bone mass, or increasing trabecular bone volume, trabecular number, and trabecular bone mineral density; or
- increasing osteoblast number and regulating osteoblastic gene
expression.
In alternative embodiments, provided are therapeutic formulations for use in:
- treating, ameliorating, preventing or reversing osteoporosis or postmenopausal osteoporosis;
- preventing bone loss due to estrogen deficiency;
- inhibiting osteoclast differentiation;
- reducing osteoclast numbers;
- preventing estrogen deficiency-induced osteocyte apoptosis;
- increasing serum cGMP concentration;
- increasing bone formation or bone mass, or increasing trabecular bone volume, trabecular number, and trabecular bone mineral density; or
- increasing osteoblast number and regulating osteoblastic gene expression,
wherein the therapeutic formulation comprises a nitrosyl-cobinamide (NO-Cbi), or a bioisostere, stereoisomer, tautomer, hydrate, solvate or pharmaceutically acceptable salt thereof.
In alternative embodiments, provided are bone implants comprising a nitrosyl- cobinamide, or a bioisostere, stereoisomer, tautomer, hydrate, solvate or pharmaceutically acceptable salt thereof. The details of one or more embodiments as provided herein are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims.
All publications, patents, patent applications cited herein are hereby expressly incorporated by reference for all purposes.
BRIEF DESCRIPTION OF THE DRAWINGS
The drawings set forth herein are illustrative of embodiments of the invention and are not meant to limit the scope of the invention as encompassed by the claims.
FIG. 1A-I illustrate data showing that NO-Cbi enhances cGMP/PKG and Erk/Akt signaling, gene expression, proliferation and survival in POBs, as described in detail in Example 1, below:
FIG. 1(A) graphically illustrates data from experiments where POBs were incubated in medium with 0.1% FBS (3 x 105 cells/ml) for 2 h prior to receiving 10 μΜ NO-Cbi (NOCbi) for the indicated times, and stable NO oxidation products (nitrite plus nitrate, NOx) were measured in the medium by the Griess reaction;
FIG. 1(B) and FIG. 1(C) graphically illustrate data from experiments where POBs were treated with vehicle or NO-Cbi at the indicated concentrations for 30 min, and intracellular cGMP concentrations were measured by ELISA (FIG. IB);
FIG. 1(D) and FIG. 1(E) graphically illustrate data from experiments where serum- deprived POBs were treated with vehicle or 10 μΜ NO-Cbi for 10 min and ERK and Akt activation were assessed by blotting with phospho-specific antibodies;
FIG. 1(F) illustrates immunofluorescence images from experiments where POBs were serum-starved for 36 h in medium containing 1% BSA with 10 μΜ NO-Cbi or vehicle; apoptosis was assessed by immunofluorescence staining with antibodies specific for cleaved caspase-3 and FITC-coupled secondary antibodies (green); nuclei were counterstained with Hoechst 33342 (blue);
FIG. 1(G) graphically illustrates data from experiments where POBs cultured in medium with 0.1% FBS for 18 h were treated with 10 μΜ NO-Cbi or vehicle for 1 h, and transferred to fresh medium containing 3H-thymidine for 24 h; and
FIG. 1(H) and FIG. 1(1) graphically illustrate data from experiments where confluent POBs were differentiated in ascorbate-containing medium for 14 d and some cells received 10 μΜ NO-Cbi (open bars) or vehicle (filled bars) for the last 24 h.
FIG. 2A-F illustrate data from experiments showing that NO-Cbi stimulates Wnt signaling and mPOB proliferation via PKG II, as described in detail in Example 1, below:
FIG. 2A illustrates data from experiments where POBs isolated from mice homozygous for prkg2 alleles flanked by LoxP sites ("floxed" PRKG2f/f) were infected with adenovirus expressing β-galactosidase (LacZ, control) or CRE recombinase (CRE), and forty-eight h later, relative amounts of prkg2 mRNA were determined by qRT-PCR, and knockdown efficiency of PKG II protein was analyzed by Western blotting, with caveolin-1 serving as a loading control;
FIG. 2B illustrates data from experiments where cells were infected as in FIG. 2A, but 30 h later were transferred to medium containing 0.1% FBS, and 18 h later were treated with 10 μΜ NO-Cbi or vehicle for 10 min, and Akt and GSK-3P phosphorylation were assessed using antibodies specific for Akt(pSer473) and GSK-3P(pSer9 ), with total GSK-3P serving as a loading control; densitometric quantitation is shown on the right, with relative amounts of pAkt and pGSK-3 found in vehicle-treated control virus-infected cells assigned a value of 1;
FIG. 2C illustrates data from experiments where PRKG2f/f POBs were infected with control or Cre virus and transferred to 0.1% FBS as in FIG. 2B, they were treated with NO-Cbi or vehicle for 6 h, prior to detecting β-catenin by immunofluorescence staining, and the bottom panel shows nuclei counterstained with Hoechst 33342, and numbers below indicate the percentage of cells showing nuclear β-catenin;
FIG. 2D illustrates data from experiments where cells were infected and cultured as in FIG. 2B, and they were treated with NO-Cbi or vehicle for 1 h prior to measuring 3H- thymidine incorporation into DNA for 24 h;
FIG. 2E illustrates data from experiments where cells were infected with control or CRE virus as described in FIG. 2 A, and treated with 10 μΜ NO-Cbi or vehicle for 24 h. Expression of Wingless type MMTV-integration site family- la (Wntla), low-density lipoprotein receptor- related protein-5 (Lrp5), β-catenin (bCat), cyclin D (CycD), alkaline phosphatase (ALP), osteocalcin (OCN), and tubulin (Tubal) mRNAs were measured by qRT-PCR and normalized to 18S rRNA, with relative mRNA levels in untreated cells assigned a value of 1; and
FIG. 2F illustrates data from experiments where cells POBs cultured in 10 % FBS were treated with 10 μΜ NO-Cbi for the indicated times, and Lrp5 protein (open symbols) and mRNA (filled symbols) were assessed by Western blotting and qRT-PCR, respectively.
FIG. 3A-D illustrate data showing that NO-Cbi inhibits osteoclast differentiation, as described in detail in Example 1, below:
FIG. 3 A and FIG. 3B illustrate data from experiments where murine bone marrow mononuclear cells were cultured in the presence of M-CSF, with RANKL added after 3 d; together with RANKL, cells received vehicle or NO-Cbi at the indicated concentrations;
FIG. 3C illustrates data from experiments where cells were cultured as in FIG. 3 A, but some cultures received 10 μΜ NO-Cbi, 5 μΜ DETA-NONOate (Deta-NO, which releases 2 moles of NO/mol of drug), or 100 μΜ 8-pCPT-cGMP together with RANKL; and
FIG. 3D illustrates data from experiments where expression of TRAP, cathepsin K (Ctsk), and calcitonin receptor (CalcR) mRNAs were determined by qRT-PCR and normalized to 18S rRNA, with relative mRNA levels in vehicle-treated cells assigned a value of 1.
FIG. 4A-G illustrate data showing that NO-Cbi increases serum cGMP
concentration, bone formation, and osteoblastic gene expression in OVX mice, as described in detail in Example 1, below:
FIG. 4A illustrates data from experiments where werum cGMP concentrations were measured by ELISA 1 h after the last injection of vehicle or NO-Cbi;
FIG. 4B illustrates data from experiments where the number of trabecular osteoblasts per bone perimeter (N.Ob/B.Pm) was counted at the proximal tibia;
FIG. 4C, FIG. 4D and FIG. 4F illustrate data from experiments where trabecular calcein labeling was assessed at the tibia (FIG. 4C), with quantification of mineral apposition rate (MAR, FIG. 4D), mineralizing surface per bone surface (MS/BS, panel E), and bone formation rate (BFR, FIG. 4F); and
FIG. 4G illustrates data from experiments where RNA was extracted from femurs, and the relative abundance of osteocalcin (OCN), osteopontin (Sppl), alkaline
phosphatase (ALP), collagen-al (Collal), low-density lipoprotein receptor- related protein-5 (Lrp5) and tubulin (Tubal) mRNA was quantified by qRT-PCR and normalized to 18S rRNA.
FIG. 5 A-E illustrate data showing that NO-Cbi prevents estrogen deficiency-induced osteocyte apoptosis, where mice were subjected to OVX or sham operation and were treated with vehicle or NO-Cbi as described in Fig. 4, as described in detail in Example 1, below:
FIG. 5A, FIG. 5B and FIG. 5C illustrate data from experiments where the percentage of apoptotic osteocytes was assessed in trabecular (FIG. 5 A, FIG. 5B) and cortical bone (FIG. 5C) by TUNEL staining (black nuclei) of tibial sections:
FIG. 5D illustrates data from experiments where osteoblast and osteocyte apoptosis was assessed by Western blotting of extracts obtained from tibial bone (after removal of bone marrow), using an antibody specific for cleaved caspase-3, with β-actin serving as a loading control (n =2 mice per group); and
FIG. 5E illustrate data from experiments where Erk activity in cortical (top panel) and trabecular (bottom panel) bone-lining cells was assessed by immunofluorescence staining using a phospho-Erk-specific antibody and horse radish peroxidase-coupled secondary antibody (brown).
FIG. 6A-C illustrate data showing that NO-Cbi regulates RANKL/OPG and reduces osteoclasts in OVX mice, and mice subjected to OVX or sham-operation were treated with vehicle or NO-Cbi as described in Fig. 4, as described in detail in Example 1, below:
FIG. 6 A and FIG. 6B illustrate data from experiments where osteoclasts were identified by TRAP staining (red), and the number of trabecular osteoclasts per bone perimeter (N.Oc/B.Pm) was counted at the proximal tibia; and
FIG. 6C illustrates data from experiments where RNA was extracted from femurs, and the relative abundance of RANKL, OPG, CTSK, and TRAP mRNA was quantified by qRT-PCR and normalized to 18S rRNA.
FIG. 7A-D illustrate data showing that NO-Cbi increases trabecular bone mass in OVX mice. Mice subjected to OVX or sham operation were treated with vehicle or NO- Cbi as described in Fig. 4, as described in detail in Example 1, below:
FIG. 7A illustrates data from experiments where tibiae were analyzed by micro-CT imaging, and three-dimensional reconstruction of the trabecular bone at the proximal tibia below the growth plate is shown; and
FIG. 7B, FIG. 7C and FIG. 7D illustrate data from experiments where trabecular bone volume/tissue volume (FIG. 7B), trabecular number (FIG. 7C), and trabecular bone mineral density (FIG. 7D) were quantified at the proximal tibia.
FIG. 8 illustrates endocortical calcein labeling, where the endocortical calcein labeling, as illustrated in the FIG. 8A images, was assessed at the tibia, with
quantification of mineral apposition rate (MAR, FIG. 8B), bone formation rate (BFR, FIG. 8C), and mineralizing surface per bone surface (MS/BS, FIG. 8D); data represent means ± SEM from n= 6 sham-operated mice, n= 7 vehicle-treated OVX mice, and n=8, NO-Cbi-treated OVX mice; *p < 0.05 and **p < 0.01 for the indicated pair-wise comparisons.
FIG. 9 illustrates images of a TUNEL staining of apoptotic osteocytes in cortical tibial bone, where the percentage of apoptotic osteocytes was assessed in cortical bone by TUNEL staining (black nuclei) of tibial sections, as described in Fig. 5C.
FIG. 10 graphically illustrates data from a Micro-CT analysis of tibial cortical bone, where tibiae were analyzed by micro-CT, and cortical (cross-sectional) thickness (FIG. 10A) and cortical tissue mineral density (FIG. 10B) were quantified at the mid- tibial diaphysis, as described in Example 1, below; data represent means ± SEM from n= 6 sham-operated mice, n= 7 vehicle-treated OVX mice, and n=8 NO-Cbi-treated OVX mice.
Like reference symbols in the various drawings indicate like elements.
Reference will now be made in detail to various exemplary embodiments of the invention, examples of which are illustrated in the accompanying drawings. The following detailed description is provided to give the reader a better understanding of
certain details of aspects and embodiments of the invention, and should not be interpreted as a limitation on the scope of the invention.
DETAILED DESCRIPTION
In alternative embodiments, provided are compounds, including formulations and pharmaceutical compositions, and methods of making and using them, for treating, ameliorating, preventing or reversing osteoporesis, In alternative embodiments, provided are methods for, and uses of nitrosyl-cobinamide (NO-Cbi) for: treating, ameliorating, preventing or reversing osteoporosis; preventing bone loss due to estrogen deficiency; inhibiting osteoclast differentiation; reducing osteoclast numbers; preventing estrogen deficiency-induced osteocyte apoptosis; increasing serum cGMP concentration;
increasing bone formation or bone mass, or increasing trabecular bone volume, trabecular number, and trabecular bone mineral density; or increasing osteoblastic gene expression, comprising administering a nitrosyl-cobinamide (NO-Cbi), or a bioisostere, stereoisomer, tautomer, hydrate, solvate or pharmaceutically acceptable salt thereof to: an individual; an individual with osteoporesis or at risk of developing osteoporesis.
As described in Example 1, below, we examined the skeletal effects of a novel NO donor, nitrosyl-cobinamide (NO-Cbi), derived from the vitamin B12 precursor cobinamide; it directly releases NO without biotransformation or generation of reactive oxygen species.32"35 We found that NO-Cbi prevented bone loss in OVX mice by enhancing osteoblast activity and inhibiting osteoclast differentiation.
We tested nitrosyl-cobinamide (NO-Cbi), a novel NO-donor with anti -oxidant properties, in a mouse model of estrogen deficiency-induced osteoporosis (3). Compared with sham-operated mice, ovariectomized mice had lower serum cGMP concentrations, which were largely restored to normal by treatment with NO-Cbi, or low-dose estrogen replacement. Micro-CT analyses of tibiae showed that all three pharmacological interventions significantly improved trabecular bone architecture in ovariectomized animals, with similar effect sizes. NO-Cbi reversed ovariectomy -induced osteocyte apoptosis as efficiently as estradiol, and enhanced bone formation parameters in vivo, consistent with in vitro effects on osteoblast proliferation, differentiation, and survival. Ovariectomy dramatically increased osteoclast numbers, and this effect was completely reversed by estradiol. NO-Cbi significantly decreased the number of osteoclasts in ovariectomized mice, suggesting cGMP-independent effects of NO-Cbi in osteoclasts.
We conclude that estrogen deficiency represents a state of relative NO and cGMP deficiency, and that NO-dependent or NO-independent guanylate cyclase stimulation is a novel, anabolic treatment strategy for post-menopausal osteoporosis. These data confirm an important role of NO/cGMP signaling in bone biology.
We have found that the novel nitric oxide (NO) donor nitrosyl-cobinamide (NO-
Cbi) prevents osteoporosis in ovariectomized mice. Most drugs used for treating osteoporosis inhibit osteoclasts (which resorb bone) but do not stimulate osteoblasts (which make new bone). We found that NO-Cbi both inhibits osteoclasts and stimulates osteoblasts, so it is an ideal drug for treating osteoporosis, including post-menopausal osteoporosis, and no drug like it is currently available.
"NO-Cbi" or nitrosyl cobinamide is a compound having the structure:
Methods of making and formulating and dosaging NO-Cbi are described in Boss et al, USPN 8,222,242.
In alternative embodiments, also provided are bioisosteres of compounds of NO-
Cbi. In alternative embodiments, bioisosteres provided herein are compounds comprising one or more substituent and/or group replacements with a substituent and/or group having substantially similar physical or chemical properties which produce substantially similar biological properties to a NO-Cbi, or stereoisomer, racemer or isomer thereof. In one embodiment, the purpose of exchanging one bioisostere for another is to enhance the desired biological or physical properties of NO-Cbi without making significant changes in chemical structures. For example, in one embodiment, bioisosteres of compounds as provided herein are made by replacing one or more hydrogen atom(s) with one or more
fluorine atom(s), e.g., at a site of metabolic oxidation; this may prevent metabolism (catabolism) from taking place. Because the fluorine atom is similar in size to the hydrogen atom the overall topology of the molecule is not significantly affected, leaving the desired biological activity unaffected. However, with a blocked pathway for metabolism, the molecule may have a longer half-life or be less toxic, and the like.
In alternative embodiments, NO-Cbi compositions used to practice emboidments described herein are prepared in a wide variety of dosage forms according to any means suitable in the art for preparing a given dosage form. Pharmaceutically acceptable carriers can be either solid or liquid. Non-limiting examples of solid form preparations include a tablet, a pill, a capsule, a liquid, a gel, a geltab, a powder, a spray or aerosol, a cachet, a suppository, a dispersible granule, and the like. A solid carrier can include one or more substances which can also act as diluents, flavoring agents, buffering agents, binders, preservatives, tablet disintegrating agents, or an encapsulating material. Suitable solid carriers include magnesium carbonate, magnesium stearate, talc, sugar, lactose, pectin, dextrin, starch, gelatin, acacia, tragacanth, methylcellulose, sodium
carboxymethyl-cellulose, polyethylene glycols, vegetable oils, agar, a low melting wax, cocoa butter, and the like. Non-limiting examples of liquid form preparations include solutions, suspensions, syrups, slurries, and emulsions. Suitable liquid carriers include any suitable organic or inorganic solvent, for example, water, alcohol, saline solution, buffered saline solution, physiological saline solution, dextrose solution, water propylene glycol solutions, and the like, preferably in sterile form.
In alternative embodiments, compositions used to practice embodiments described herein are formulated and administered to the subject as pharmaceutically acceptable salts. Non-limiting examples of pharmaceutically acceptable salts include acid addition salts such as those containing hydrochloride, sulfate, phosphate, sulfamate, acetate, citrate, lactate, tartrate, methanesulfonate, ethanesulfonate, benzenesulfonate, p- toluenesulfonate, cyclohexylsulfamate and quinate. Such salts can be derived using acids such as hydrochloric acid, sulfuric acid, phosphoric acid, sulfamic acid, acetic acid, citric acid, lactic acid, tartaric acid, malonic acid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, cyclohexylsulfamic acid, and quinic acid, according to means known and established in the art.
In alternative embodiments, aqueous solutions are prepared by dissolving the active component in water and adding suitable colorants, flavors, stabilizing and
thickening agents as desired. Aqueous suspensions can also be made by dispersing the finely divided active component in water with viscous material, such as natural or synthetic gums, resins, methylcellulose, sodium carboxymethylcellulose, and other well- known suspending agents.
In alternative embodiments, solid forms are according to any means suitable in the art. For example, capsules are prepared by mixing the composition with a suitable diluent and filling the proper amount of the mixture in capsules. Tablets are prepared by direct compression, by wet granulation, or by dry granulation. Their formulations usually incorporate diluents, binders, lubricants and disintegrators as well as the compound. Non- limiting examples of diluents include various types of starch, cellulose, crystalline cellulose, microcrystalline cellulose, lactose, fructose, sucrose, mannitol, kaolin, calcium phosphate or sulfate, inorganic salts such as sodium chloride and powdered sugar.
Powdered cellulose derivatives are also useful. Non-limiting examples of tablet binders include starches, gelatin and sugars such as lactose, fructose, glucose and the like. Natural and synthetic gums are also convenient, including acacia, alginates, methylcellulose, polyvinylpyrrolidone and the like. Polyethylene glycol, ethylcellulose and waxes can also serve as binders.
In alternative embodiments, compositions used to practice embodiments described herein are formulated with lubricants, which can be used in a tablet formulation to prevent the tablet and punches from sticking in the die. The lubricant can be chosen from such slippery solids as talc, magnesium and calcium stearate, stearic acid and
hydrogenated vegetable oils. Tablet disintegrators are substances which swell when wetted to break up the tablet and release the compound, and include starches such as corn and potato starches, clays, celluloses, aligns, gums, methylcellulose, agar, bentonite, wood cellulose, powdered natural sponge, cation-exchange resins, alginic acid, guar gum, citrus pulp, carboxymethyl cellulose, and sodium lauryl sulfate. Tablets can be coated with sugar as a flavor and sealant, or with film-forming protecting agents to modify the dissolution properties of the tablet. The compounds can also be formulated as chewable tablets, by using large amounts of pleasant-tasting substances such as mannitol in the formulation, as is now well-established in the art.
In alternative embodiments, compositions used to practice embodiments described herein are formulated as liquid formulations or solid form preparations, e.g., which can be intended to be converted, shortly before use, to liquid form preparations. In alternative
embodiments, liquid forms include solutions, suspensions, syrups, slurries, and emulsions. Liquid preparations can be prepared by conventional means with
pharmaceutically acceptable additives such as suspending agents (e.g., sorbitol syrup, cellulose derivatives or hydrogenated edible fats or oils); emulsifying agents (e.g., lecithin or acacia); non-aqueous vehicles (e.g., almond oil, oily esters, or fractionated vegetable oils); and preservatives (e.g., methyl or propyl-p-hydroxybenzoates or sorbic acid). In alternative embodiments, preparations can contain, in addition to the active agent, colorants, flavors, stabilizers, buffers, artificial and natural sweeteners, dispersants, thickeners, solubilizing agents, and the like. In alternative embodiments, compositions can be in powder form for constitution with a suitable vehicle such as sterile water, saline solution, or alcohol, before use.
In alternative embodiments, compositions used to practice embodiments described herein are formulated in solution in both the un-ionized and ionized forms. Generally lipid soluble or lipophilic drugs diffuse most readily across mucosal membranes. In alternative embodiments, compositions used to practice embodiments described herein are formulated with buffering agents, pH-adjusting agents, or ionizing agents to adjust the ratio of unionized:ionized forms of the NO-Cbi.
In alternative embodiments, compositions used to practice embodiments described herein are formulated with permeation enhancers or permeability enhancers, which can significantly enhance the permeability of lipophilic and nonlipophilic drugs, including the NO-Cbi. In alternative embodiment, penetration enhancers as described in Cooper et al. (1987) "Penetration Enhancers", in Transdermal Delivery of Drugs, Vol. II, Kyodonieus et al., Eds., CRC Press, Boca Raton, Fla are used. Additional forms of chemical enhancers also can be used, such as those enhancing lipophilicity, have been developed to improve transport when physically mixed with certain therapeutic agents and provide more predictable absorption, including for example as described in U.S. Pat. Nos.
4,645,502; 4,788,062; 4,816,258; 4,900,555; 3,472,931; 4,006,218; and 5,053,227.
In alternative embodiments, carriers can be coupled to pharmaceutical agents described herein to enhance intracellular transport, as described e.g., by Ames et al. (1973) Proc. Natl. Acad. Sci. USA, 70:456-458 and (1988) Proc. Int. Symp. Cont. Rel. Bioact. Mater., 15: 142. In alternative embodiments, permeation enhancers include bile salts such as sodium cholate, sodium glycocholate, sodium glycodeoxycholate, taurodeoxycholate, sodium deoxycholate, sodium lithocholate chenocholate,
chenodeoxycholate, ursocholate, ursodeoxycholate, hydrodeoxycholate, dehydrocholate, glycochenocholate, taurochenocholate, and taurochenodeoxycholate. Other permeation enhancers such as sodium dodecyl sulfate ("SDS"), dimethyl sulfoxide ("DMSO"), sodium lauryl sulfate, salts and other derivatives of saturated and unsaturated fatty acids, surfactants, bile salt analogs, derivatives of bile salts, or such synthetic permeation enhancers as described in U.S. Pat. No. 4,746,508 can be used. In alternative
embodiments, DMSO, SDS, and medium chain fatty acids (about C-8 to about C-14) their salts, derivatives, and analogs are used. In alternative embodiments, the permeation enhancer concentration within the dissolvable matrix material can be varied depending on the potency of the enhancer and rate of dissolution of the dissolvable matrix. Other criteria for determining the enhancer concentration include the potency of the drug and the desired lag time. The upper limit for enhancer concentration is set by toxic effect to or irritation limits of the mucosal membrane.
In alternative embodiments, compositions used to practice embodiments described herein are formulated with a disintegrating agent. Tablet disintegrators are substances which swell when wetted to break up the tablet and release the compound, and include starches such as corn and potato starches, clays, celluloses, aligns, gums, methylcellulose, agar, bentonite, wood cellulose, powdered natural sponge, cation-exchange resins, alginic acid, sodium alginate, guar gum, citrus pulp, carboxymethyl cellulose, polyvinyl- pyrrolidone, and sodium lauryl sulfate. Acrylic type polymers can also advantageously be used as disintegrators, including methacrylic copolymers of type C (as disclosed in U.S. Pat. No. 6,696,085).
The compositions can be formulated for use in topical administration. Such formulations include, e.g., liquid or gel preparations suitable for penetration through the skin such as creams, liniments, lotions, ointments or pastes, and drops suitable for delivery to the eye, ear or nose.
In alternative embodiments, compositions used to practice embodiments described herein are formulated as creams, drops, liniments, lotions, ointments and pastes are liquid or semi-solid compositions for external application. Such compositions can be prepared by mixing the active ingredient(s) in powdered form, alone or in solution or suspension in an aqueous or non-aqueous fluid with a greasy or non-greasy base. The base can comprise complex hydrocarbons such as glycerol, various forms of paraffin, beeswax; a mucilage; a mineral or edible oil or fatty acids; or a macrogel. Such compositions can additionally
comprise suitable surface active agents such as surfactants, and suspending agents such as agar, vegetable gums, cellulose derivatives, and other ingredients such as preservatives, antioxidants, and the like.
In alternative embodiments, compositions used to practice embodiments described herein are formulated for injection into the subject or individual. For injection, the compositions can be formulated in aqueous solutions such as water or alcohol, or in physiologically compatible buffers such as Hanks's solution, Ringer's solution, or physiological saline buffer. The solution can contain formulatory agents such as suspending, stabilizing and/or dispersing agents. Injection formulations can also be prepared as solid form preparations which are intended to be converted, shortly before use, to liquid form preparations suitable for injection, for example, by constitution with a suitable vehicle, such as sterile water, saline solution, or alcohol, before use.
In alternative embodiments, compositions used to practice embodiments described herein are formulated in sustained release vehicles or depot preparations. Such long acting formulations can be administered by implantation (for example subcutaneously or intramuscularly) or by intramuscular injection. Thus, for example, the compositions can be formulated with suitable polymeric or hydrophobic materials (for example, as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt. Liposomes and emulsions are well-known examples of delivery vehicles suitable for use as carriers for hydrophobic drugs.
In alternative embodiments, compositions used to practice embodiments described herein are formulated and/or administered by systemic administration, e.g., by oral, intravenous, intraperitoneal and intramuscular delivery. In alternative embodiments, compositions used to practice embodiments described herein are formulated and/or administered by infusion or injection (intravenously, intramuscularly, intracutaneously, subcutaneously, intrathecal, intraduodenally, intraperitoneally, and the like). The compositions can also be administered intranasally, vaginally, rectally, orally, or transdermally. In alternative embodiments, the compositions are administered intravenously. Administration can be at the direction of a physician.
In alternative embodiments, compositions used to practice embodiments described herein are formulated and/or administered via buccal administration, and the
compositions can take the form of spray, liquid, tablets, troche or lozenge formulated in conventional manner. Compositions for oral or buccal administration, can be formulated
to give controlled release of the active compound. Such formulations can include one or more sustained-release agents known in the art, such as glyceryl mono-stearate, glyceryl distearate and wax.
In alternative embodiments, compositions used to practice embodiments described herein are formulated for topical administration and/or administered or applied topically. Such administrations include applying the compositions externally to the epidermis, the mouth cavity, eye, ear and nose. Compositions for use in topical administration include, e.g., liquid or gel preparations suitable for penetration through the skin such as creams, liniments, lotions, ointments or pastes, and drops suitable for delivery to the eye, ear or nose.
In alternative embodiments, compositions used to practice embodiments described herein are formulated for and/or administered via inhalation. Compositions can be inhaled through the nose or mouth. In some embodiments, inhalation can occur via a nasal spray, dry powder inhaler, metered-dose inhaler, vaporizer, and nebulizer.
In alternative embodiments, alternative pharmaceutical delivery systems are employed. Non-limiting examples of such systems include liposomes and emulsions. Certain organic solvents such as dimethyl-sulfoxide can also be employed. In alternative embodiments, compounds can be delivered using a sustained-release system, such as semipermeable matrices of solid polymers containing the therapeutic agent. The various sustained-release materials available are well known by those skilled in the art.
Sustained-release capsules can, depending on their chemical nature, release the compounds over a range of several days to several weeks to several months.
In alternative embodiments, nanoparticles, nanolipoparticles, vesicles and liposomal membranes comprising compounds and compositions are used to practice the methods and embodiments as provided herein. Provided are multilayered liposomes comprising compounds used to practice embodiments as provided herein, e.g., as described in Park, et al., U.S. Pat. Pub. No. 20070082042. The multilayered liposomes can be prepared using a mixture of oil-phase components comprising squalane, sterols, ceramides, neutral lipids or oils, fatty acids and lecithins, to about 200 to 5000 nm in particle size, to entrap a composition used to practice embodiments as provided herein.
Liposomes can be made using any method, e.g., as described in Park, et al., U.S. Pat. Pub. No. 20070042031, including method of producing a liposome by encapsulating an active agent (e.g., compounds and compositions as provided herein, or a compound
used to practice methods as provided herein), the method comprising providing an aqueous solution in a first reservoir; providing an organic lipid solution in a second reservoir, and then mixing the aqueous solution with the organic lipid solution in a first mixing region to produce a liposome solution, where the organic lipid solution mixes with the aqueous solution to substantially instantaneously produce a liposome encapsulating the active agent; and immediately then mixing the liposome solution with a buffer solution to produce a diluted liposome solution.
. In one embodiment, liposome compositions used to practice embodiments as provided herein comprise a substituted ammonium and/or polyanions, e.g., for targeting delivery of a compound as provided herein, or a compound used to practice methods as provided herein, to a desired cell type or organ, e.g., brain, as described e.g., in U.S. Pat. Pub. No. 20070110798.
Provided are nanoparticles comprising compounds as provided herein, e.g., used to practice methods as provided herein in the form of active agent-containing
nanoparticles (e.g., a secondary nanoparticle), as described, e.g., in U.S. Pat. Pub. No. 20070077286. In one embodiment, provided are nanoparticles comprising a fat-soluble active agent used to practice embodiments as provided herein, or a fat-solubilized water- soluble active agent to act with a bivalent or trivalent metal salt.
In one embodiment, solid lipid suspensions can be used to formulate and to deliver compositions used to practice embodiments as provided herein to mammalian cells in vivo, in vitro or ex vivo, as described, e.g., in U.S. Pat. Pub. No. 20050136121.
In alternative embodiments, the effective amount of a composition to be administered can be dependent on any number of variables, including without limitation, the species, breed, size, height, weight, age, overall health of the subject, the type of formulation, the mode or manner or administration, or the severity of the osteoporesis. The appropriate effective amount can be routinely determined by those of skill in the art using routine optimization techniques and the skilled and informed judgment of the practitioner and other factors evident to those skilled in the art. Preferably, a
therapeutically effective dose of the compounds described herein will provide therapeutic benefit without causing substantial toxicity to the subject.
In alternative embodiments, a concentration of NO-Cbi administered is in a range of about 0.01% to about 90% of the dry matter weight of the composition. In some aspects, NO-Cbi comprises up to about 50% of the dry matter weight of the composition.
In some aspects, NO-Cbi comprises up to about 40% of the dry matter weight of the composition. In some aspects, NO-Cbi comprises up to about 30% of the dry matter weight of the composition. In some aspects, NO-Cbi comprises up to about 25% of the dry matter weight of the composition. In some aspects, NO-Cbi comprises up to about 20%) of the dry matter weight of the composition. In some aspects, NO-Cbi comprises up to about 15%) of the dry matter weight of the composition. In some aspects, NO-Cbi comprises up to about 10%> of the dry matter weight of the composition.
In alternative embodiments, individuals or subjects are administered NO-Cbi in a daily dose range of about 0.001 mg/kg to about 10 mg/kg of the weight of the subject. The dose administered to the subject can also be measured in terms of total amount of drug administered per day; for example, individuals or subjects are administered NO-Cbi in a dosage range of about 0.01 to about 500 milligrams of NO-Cbi per day: 0.05 milligrams of NO-Cbi per day; about 0.1 milligrams of NO-Cbi per day; about 0.5 milligrams of NO-Cbi per day; about 1 milligrams of NO-Cbi per day; about 5 milligrams of NO-Cbi per day; about 10 milligrams of NO-Cbi per day; about 25 milligrams of NO- Cbi per day; about 50 milligrams of NO-Cbi per day; about 100 milligrams of NO-Cbi per day; about 150 milligrams of NO-Cbi per day; or about 200 milligrams of NO-Cbi per day. Treatment can be initiated with smaller dosages that are less than the optimum dose of NO-Cbi, followed by an increase in dosage over the course of the treatment until the optimum effect under the circumstances is reached. If needed, the total daily dosage can be divided and administered in portions throughout the day.
For effective treatment of osteoporesis, or for stimulation of bone growth, one skilled in the art can recommend a dosage schedule and dosage amount adequate for the subject being treated. In alternative embodiments, dosing can occur one to four or more times daily for as long as needed. The dosing can occur less frequently if the
compositions are formulated in sustained delivery vehicles. The dosage schedule can also vary depending on the active drug concentration, which can depend on the needs of the subject.
In alternative embodiments, compositions used to practice embodiment described herein can be co-administered with other therapeutic agents that are selected for their particular usefulness against the condition that is being treated. For example, such therapeutic agents can be pain relievers, blood thinners/anticoagulants, clot busters, stomach antacids, compounds which lessen untoward effects of the compositions, other
known agents that lower blood pressure. The compositions can be co-administered with cobalamin (vitamin B12) to reduce or eliminate potential toxicity of the administered cobinamide.
In alternative embodiments, the administration of these additional compounds can be simultaneous with the administration of NO-Cbi, or can be administered in tandem, either before or after the administration of NO-Cbi, as necessary. Any suitable protocol can be devised whereby the various compounds to be included in the combination treatment are administered within minutes, hours, days, or weeks of each other. In alternative embodiments, repeated administrations are in a cyclic protocol. The invention will be further described with reference to the examples described herein; however, it is to be understood that the invention is not limited to such examples.
EXAMPLES
Example 1 : Nitrosyl-cobinamide (NO-Cbi) is effective for treating osteoporosis
This example describes data demonstrating that exemplary compositions provided herein, including exemplary nitrosyl-cobinamide (NO-Cbi), a direct NO-rel easing agent, is effective for treating osteoporosis in a mouse model of estrogen deficiency-induced osteoporosis.
In murine primary osteoblasts, NO-Cbi increased intracellular cGMP, Wnt/β- catenin signaling, proliferation, and osteoblastic gene expression, and protected cells from apoptosis. Correspondingly, in ovariectomized (OVX) C57B1/6 mice, NO-Cbi increased serum cGMP concentrations, bone formation, and osteoblastic gene expression, and prevented estrogen deficiency-induced osteocyte apoptosis. NO-Cbi additionally prevented an ovariectomy-induced increase in osteoclasts, likely due to a reduction in the RANKL/osteoprotegerin gene expression ratio, which regulates osteoclast differentiation, and due to direct inhibition of osteoclast differentiation, observed in vitro in the presence of excess RANKL. While positive NO effects in osteoblasts were mediated by cGMP/PKG, the osteoclast-inhibitory effects appeared to be largely cGMP-independent. Consistent with its effects on osteoblasts and
osteoclasts, NO-Cbi increased trabecular bone mass in OVX mice. NO-Cbi represents a novel direct NO-releasing agent that, in contrast to nitrates, does not generate
superoxide, and combines anabolic and anti-resorptive effects in bone, making it an effective agent for treating osteoporosis.
MATERIALS AND METHODS
Materials. Antibodies against Akt, Akt(pSer473), Erkl(pTyr204), GSK-3p, GSK- 3P(pSer9), and cleaved caspase-3 were from Cell Signaling, and antibodies against Erkl/2, and β-actin were from Santa Cruz Biotechnology. A β-catenin-specific antibody and fluorophore-labeled secondary antibodies were from InVitrogen. DETA-NONOate was from Cayman and the cGMP agonist 8-(4-chlorophenylthio)-cGMP (8-pCPT-cGMP) from BioLog.
Preparation of NO-Cbi. NO-Cbi was generated by reducing dinitro-cobinamide under deoxygenated conditions. Ascorbic acid and dinitro-cobinamide were incubated at a ratio of 5: 1 for 1 h at RT, and then the solution was purged with nitrogen to remove any free NO. NO-Cbi was stable at RT for at least 1 month when protected from light and filter- sterilized.
Animal Experiments. Ten week-old female C57B1/6 mice were purchased from Jackson Laboratories. They were maintained in accordance with the "Guide for the Care and Use of Laboratory Animals" (2011, 8th ed., Washington, D.C., Natl. Research Council, Natl. Academies Press), and all experiments were approved by the Institutional Animal Care and Use Committee of the University of California, San Diego. Mice were housed at 3-4 animals per cage in a temperature-controlled environment with a 12 h light/dark cycle; they were fed standard Teklad Rodent Diet with ad lib access to food and water. After one week of acclimatization, mice weighing 19.5-22.0 g were randomly divided into three groups— Groups 1 and 2 (eight mice each) underwent bilateral ovariectomy, while Group 3 (six mice) underwent a sham operation. One mouse in Group 1 had to be euthanized post-operatively because of suture failure. Beginning one week post- surgery, mice received daily i.p. injections six days per week for five weeks, either vehicle (0.1 ml 9.25 mM ascorbic acid, Group 1), or NO-Cbi (10 mg/kg/d given as 0.1 ml of 1.85 mM NO-Cbi, Group 2). This NO-Cbi dose did not significantly reduce systolic blood pressure (< 10 mm Hg), consistent with our previous report,32 and the NO-Cbi- treated mice showed no signs of toxicity, and had similar weight gain during the experiment as vehicle-treated OVX mice. Double calcein labeling was performed by intraperitoneal injection of calcein (25 mg/kg) at seven and three days before euthanasia. Mice were euthanized one hour after the last drug or vehicle injection by CO2 intoxication
and exsaguination; blood samples were collected by cardiac puncture and allowed to clot. Femoral and tibial bones were dissected for quantitative RT-PCR, histology, and micro- CT analyses.
Mice carrying floxed prkg2 alleles (PRKG2f/f mice). To generate PRKG2f/f mice, we PCR-amplified genomic DNA fragments encoding prkg2 exon III with flanking sequences from 129/SvJ ES cells using KOD polymerase (EMD Millipore Corporation). The prkg2-f\oxed construct was assembled in the vector pDL L (gift from Ju Chen of UCSD) and consisted of a 4.2 kb 5' arm, a 0.55 kb fragment including exon III flanked by LoxP sites, a 1.3 kb neo cassette flanked by FRT sites, and a 3kb 3' arm. All PCR products and fusion sites were sequenced. The construct was electroporated into Rl ES cells derived from 129/SvJ mice, and G418-resistant clones were isolated and screened by PCR; homologous recombination was confirmed by Southern blot analysis using probes outside of the 5' and 3' arms. A recombinant clone with normal chromosome analysis in 20 metaphase spreads was injected into C57B1/6 blastocysts to establish germline chimeric mice. Heterozygous PRKG2f/+ mice were mated with homozygous FLPeR mice containing FLPe recombinase (from Jackson Laboratories) to remove the neo cassette.
Culture of murine primary osteoblasts (POBs). POBs were isolated from the femurs and tibiae of 8-12 week-old C57/B16 mice, or from PRKG2f/f mice in a mixed
background, and were grown in DMEM supplemented with 10% FBS, as described.15' 36 In some cases, ascorbate (0.3 mM) and β-glycerolphosphate (10 mM) was added to the medium to induce differentiation. The cells were used at passages 1-5, and were checked for mineralization capacity.36 To delete exon II of PKG II, cells from PRKG2f/f mice were infected with adenovirus encoding CRE recombinase (or control virus expressing LacZ) at an MOI of 50 and used 48 h later.
Culture of murine primary osteoclasts. Osteoclasts were generated from murine bone marrow as described.37 Briefly, bone marrow cells were plated at 106 cells/cm2 in a- Minimal Essential Medium with 10% FBS and 50 ng/ml M-CSF, and non-adherent cells were discarded after 48 h. RANKL (150 ng/ml) was added on day 3, medium was replaced on days 5 and 7; the indicated drugs were added with fresh medium on days 3,5, and 7. On day 8, cultures were fixed and stained for TRAP using a commercially- available kit (Sigma), or were harvested for RNA isolation.
Quantitative RT-PCR assays. Frozen bone shafts (frozen at -80°C after removal of bone marrow cells) were pulverized with a mortar and pestle in liquid nitrogen. RNA
was purified using Trizol-reagent (Molecular Research Center, Inc.) and 1 μg of RNA was reverse-transcribed and quantitative PCR was performed using a MX3005P™ realtime PCR detection system with Brilliant II SYBR® Green QPCR Master Mix™ (Agilent Technologies).15 All primers were tested with serial cDNA dilutions. Relative changes in mRNA expression were analyzed using the 2"ΔΔα method, with 18S rRNA serving as an internal reference; we used mean ACT values (gene of interest minus 18S rRNA) measured in the OVX/vehicle-treated group to calculate ΔΔΟΤ values for the OVX/NO- Cbi group.
Preparation of bone cell extracts and Western blotting. Protein extracts were prepared from mouse bones as described previously.36 Briefly, -50 mg of pulverized bone was incubated for 15 min on ice in 50 mM Tris-HCl pH 8.0, 150 mM NaCl, 2 mM EDTA, 1% Triton X-100, 0.1% SDS, 0.5% sodium deoxycholate, 2 mM Na3V04, 10 mM NaF, and protease inhibitor cocktail. The samples were centrifuged at 13,000 g for 15 min at 4°C. Supernatants were boiled in SDS sample buffer and proteins were resolved by SDS-PAGE and analyzed by Western blotting as described.13 Films were scanned using Image J software.
Quantitation of NOx and cGMP. NO production was measured based on nitrite and nitrate accumulation in the medium with a two-step colorimetric assay, as previously described.13 Serum cGMP concentrations were measured by ELISA using a kit according to the manufacturer's protocol (Biomedical Technologies Inc., MA).
Proliferation Assay. POBs in 6-well dishes were cultured in 0.1% FBS overnight and treated with NO-Cbi for 1 h; cells were then transferred to fresh medium with 0.1% FBS and 10 μθ of [methyl-3H]thymidine (20 Ci/mmol, final concentration 0.5 μΜ) for 24 h. Cells were extracted in situ in ice-cold 10% trichloroacetic acid, precipitated DNA was collected on glass microfiber filters, and radioactivity on washed filters was measured by scintillation counting.
Immunofluorescence Staining. POBs were plated on glass coverslips, transferred to medium containing 0.1% FBS or 0.1% BSA, and incubated in the absence or presence of NO-Cbi for the indicated time. Cells were fixed and permeabilized and incubated with cleaved caspase-3- or β-catenin-specific antibodies (both at 1 : 100 dilution), followed by secondary antibodies conjugated to FITC or AlexaFluor 555, respectively; nuclei were counterstained with Hoechst 33342.36 Images were analyzed with a Keyence BZ-X700™ fluorescence microscope.
Bone histomorphometry, TRAP and TUNEL staining. Tibiae were fixed in 70% ethanol, dehydrated, and embedded in methyl-methacrylate and sectioned at the
University of Alabama, Birmingham, Center for Metabolic Bone Disease. Some sections were stained with Masson's tri chrome, or stained for tartrate-resistant acid phosphatase, while unstained sections were used for assessing fluorochrome labeling.36 TUNEL staining of de-plasticized sections was performed as described.36 Slides were scanned with a Hamamatsu Nanozoomer 2.0 HT Slide Scanning System™, and image analysis was performed using the Nanozoomer Digital Pathology NDP.view2™ software.
Histomorphometric measurements were performed between 0.25 and 2.25 mm distal to the growth plate as described.38
Immunohi stochemi stry . Femurs were fixed overnight in 10% neutral formalin solution, decalcified in 10% EDTA (pH 7.5) for 5 days, and embedded in paraffin.
Sections (8 μπι thick) were de-paraffinized in xylene and rehydrated in graded ethanol and water. For antigen retrieval, slides were placed in 10 mM sodium citrate buffer (pH 6.0) at 80-85°C, and allowed to cool to room temperature for 30 min. Endogenous peroxidase activity was quenched in 3% hydrogen peroxide for 10 min. Slides were blocked with 5% normal goat serum and incubated overnight at 4°C with anti-phospho- ERK antibody (1 : 100 in blocking buffer), followed by HRP-conjugated secondary antibody for 1 h at room temperature. After development with 3,3-diaminobenzidine substrate (Vector Laboratories, Inc., Burlingame, CA), slides were counterstained with hematoxylin for 2 min.
Micro-CT. Micro-CT analyses were performed on ethanol-fixed tibiae, using a Skyscan 1076™ (Kontich, Belgium) scanner at 9 μπι voxel size, and applying an electrical potential of 50 kVp and current of 200 μΑ, with a 0.5 mm aluminum filter, as described.38 Mineral density was determined by calibration of images against 2 mm diameter hydroxyapatite rods (0.25 and 0.75 g /cm3). Cortical bone was analyzed by automatic contouring 3.6 mm-4.5 mm distal to the proximal growth plate, using a global threshold to identify cortical bone, and eroding one pixel to eliminate partial volume effects. Trabecular bone was analyzed by automatic contouring the proximal tibial metaphysis 0.36 -2.1 mm distal to the growth plate and using an adaptive threshold to select the trabecular bone.
Statistical Analyses. Graph Pad Prism 5™ was used for two-tailed Student t-test (to compare two groups) or one-way ANOVA with Bonferroni post-test analysis (to compare
more than two groups); p<0.05 was considered significant. For in vivo experiments, we tested our primary hypothesis, that NO-Cbi affects bone architecture and parameters of bone formation in OVX mice, and our secondary hypothesis that ovariectomy induces bone loss compared to sham-operated animals, using the Student t-test to assess for differences between means of two groups. Based on variability data from our published studies,38 we estimated that a sample size of 6 mice would provide 90% power to detect the difference in bone volume/tissue volume (BV/TV) between OVX and sham-operated mice, whereas 8 mice per group were required to detect an absolute increase of 0.35% in BV/TV in drug-treated OVX mice with 90% power (a error set at 5%).
RESULTS
NO-Cbi enhances cGMP/PKG and Erk/ Akt signaling, gene expression, proliferation, and survival in POBs.
We have shown that cobinamide binds NO with high affinity (Ka ~1010 M"1), yielding NO-Cbi, where one water molecule on cobinamide is replaced by one NO molecule.33 NO-Cbi is stable in aqueous solutions for at least 16 h at room temperature even when exposed to air, but it releases NO rapidly in the presence of NO scavengers or when added to cells in culture medium.32 Murine POBs produced nitrite and nitrate— stable NO oxidation products— yielding a medium concentration of -10 μΜ over 2 h (Fig. 1A). Adding 10 μΜ NO-Cbi to the cells increased the amount of nitrite and nitrate within 10 min, and by 30 min, the amount of NO oxidation products generated was maximal and accounted for >90% of the NO added to the cells as NO-Cbi (Fig. 1 A). NO-Cbi increased the intracellular cGMP concentration in a dose-dependent fashion, resulting in near- maximal phosphorylation of the PKG substrate vasodilator-stimulated phosphoprotein (VASP) at 3-10 μΜ (Fig. 1B,C).
We previously showed that estrogens activate Erk and Akt in a NO/cGMP/PKG- dependent manner in osteocytes and osteoblasts, and that activation of these signaling proteins is essential for estrogen's anti-apoptotic effects in osteocytes/blasts.14 We also showed that the pro-proliferative effects of fluid shear stress on osteoblasts are mediated by NO/cGMP/PKG via Src activation of Erk.15 Consistent with increasing intracellular cGMP, NO-Cbi increased Erk and Akt phosphorylation in murine POBs, protecting cells from serum starvation-induced apoptosis and stimulating proliferation (Fig. 1D-G). In the POBs, NO-Cbi raised mRNA expression of osteoblast differentiation-related genes, including osteocalcin (OCN), osteopontin (Sppl), collagenl-al (Collal), alkaline
phosphatase (ALP), and low-density lipoprotein receptor-related protein-5 (Lrp5), with tubulin (Tubal) expression serving as a control for RNA quality (Fig. 1H). NO-Cbi decreased osteoblast expression of RANKL mRNA, whereas it increased expression of the RANKL antagonist OPG, suggesting that NO-Cbi could negatively affect osteoclast differentiation (Fig. II).
NO-Cbi stimulates Wnt signaling and mPOB proliferation via PKG II.
The Wnt/p-catenin pathway controls osteoblast differentiation, proliferation, and survival, and is essential for bone mass acquisition and maintenance; increased or decreased gene expression and gain- or loss-of-function mutations of pathway components cause high versus low bone mass phenotypes— for example, expression and activity of the Wnt co-receptor Lrp5 positively correlate with bone mass in humans and mice.39 Stability and nuclear translocation of β-catenin are negatively controlled by glycogen synthase kinase-3 (GSK-3), which in turn is negatively controlled by phosphorylation on a site targeted by Akt and PKG II.40' 41 To examine the role of NO and PKG II in osteoblast Wnt signaling, we used POBs isolated from mice which carry floxed PKG II alleles (PRKG2f/f mice) and infected cells with adenovirus encoding CRE recombinase to induce PKG II deficiency (Fig. 2A). In cells infected with control virus encoding LacZ, NO-Cbi induced Akt and GSK-3 phosphorylation and β-catenin nuclear translocation, but these effects were largely lost in CRE virus-infected, PKG Il-deficient cells (Fig. 2B,C). Similarly, NO-Cbi-induced proliferation was prevented by CRE- mediated PKG II knockout (Fig. 2D). NO-Cbi treatment increased Wntla, Lrp5, and β- catenin mRNA expression in the presence, but not in the absence of PKG II (Fig. 2E). Lrp5 protein increased in parallel to mRNA in response to NO-Cbi (Fig. 2F). Transcript levels of the Wnt/p-catenin target genes cyclin D (CycD), ALP, and OCN were increased by NO-Cbi in PKG Il-expressing, but not in PKG Il-deficient cells (Fig. 2E). We conclude that NO-Cbi stimulates Wnt/p-catenin signaling in murine POBs in a PKG II- dependent manner.
NO-Cbi inhibits osteoclast differentiation.
To examine the effect of NO-Cbi on osteoclast differentiation in vitro, we cultured adherent murine bone marrow cells with recombinant M-CSF and RANKL.37 NO-Cbi dramatically reduced the number of TRAP -positive osteoclasts, with maximal effects observed at 10 μΜ (Fig. 3A,B). Less pronounced inhibition was observed with the NO donor DETA-NONOate at a concentration calculated to release equivalent amounts of
NO (Fig. 3C). In contrast, the cGMP analog 8-pCPT-cGMP, at a concentration that maximally activated PKG I and II in intact cells, had a much smaller, non-significant effect on the number of TRAP-positive cells (Fig. 3C). Consistent with the effects of NO-Cbi on osteoclast differentiation, mRNA expression of the osteoclast-specific genes TRAP, cathepsin K (Ctsk), and calcitonin receptor (CalcR) was markedly reduced in NO- Cbi-treated osteoclast cultures (Fig. 3D).
NO-Cbi increases serum cGMP concentration, bone formation, and osteoblastic gene expression in OVX mice.
To test if NO-Cbi could prevent bone manifestations of estrogen deficiency, we subjected mature C57B1/6 mice to bilateral ovariectomy (OVX) or sham operation, and injected the OVX mice with vehicle or NO-Cbi for five weeks, starting one week postoperatively. We used a NO-Cbi dose (10 mg/kg/d) that had no significant effect on systolic blood pressure and resulted in serum cobinamide concentrations below our limits of detection (<1 μΜ). Similar to our previous results,38 OVX mice had lower serum cGMP concentrations compared to sham-operated mice - but NO-Cbi significantly increased serum cGMP at 1 h post administration (Fig. 4A). NO-Cbi reversed the decrease in osteoblast numbers found in OVX mice, and significantly increased mineral apposition rate (MAR), mineralizing surfaces (MS/BS), and bone formation rate (BFR) on trabecular bone surfaces (Fig. 4B-F). Similarly, NO-Cbi increased MAR and BFR on endocortical surfaces, although endocortical MS/BS was not affected (Fig. 8).
Consistent with these results, mRNA expression of osteoblast differentiation-related genes (OCN, Sppl, ALP, Collal, and Lrp5) was higher in femoral shafts of NO-Cbi- treated OVX mice compared to vehicle-treated OVX mice, whereas tubulin-al mRNA was unchanged (Fig. 4G). Together, these results indicate that NO-Cbi increases osteoblastic activity in OVX mice.
NO-Cbi prevents estrogen deficiency-induced osteocyte apoptosis.
Estrogen deficiency-induced bone loss is, in part, due to osteocyte apoptosis.11' 38' 42 We showed previously that the osteocyte protective effects of estrogen require
NO/cGMP/PKG signaling, and are at least partly dependent on Erk activation.14
Consistent with previous reports,14' 38' 42 we saw a significant increase in TUNEL-positive, apoptotic osteocytes in trabecular and cortical bone of OVX mice, with NO-Cbi reducing osteocyte apoptosis to values found in sham-operated mice (Fig. 5A-C, Fig. 9, for cortical bone). Similar results were obtained when we examined the amount of cleaved caspase-3
in bone lysates from NO-Cbi- versus vehicle-treated OVX mice (Fig. 5D).
Immunohistochemical staining of pErk was evident in bone-lining osteoblastic cells of sham-operated mice, but was faint or absent in OVX mice; treating OVX mice with NO- Cbi induced prominent pErk staining in bone-lining cells (Fig. 5E; isotype-matched control immunoglobulin produced no staining, not shown).
NO-Cbi regulates RANKL/OPG expression and reduces osteoclasts in OVX mice.
Estrogens reduce osteoclast survival,9, 43 and, as expected, we found an increased number of TRAP-positive osteoclasts on trabecular bone in the OVX mice (Fig. 6 A,B; few osteoclasts were seen on cortical surfaces under all conditions). Treating OVX mice with NO-Cbi reduced osteoclast numbers to values found in sham-operated animals (Fig. 6A,B). Compared to vehicle-treated OVX mice, femurs of NO-Cbi-treated OVX mice contained less RANKL and more OPG mRNA, and mRNA expression of the osteoclast marker genes CTSK and TRAP was decreased (Fig. 6C). Thus, NO-Cbi reduced osteoclast numbers and osteoclastic gene expression in OVX mice, at least, in part, by reducing the RANKL/OPG ratio.
NO-Cbi increases trabecular bone mass in OVX mice.
To determine the effects of NO-Cbi on estrogen deficiency-induced bone loss, we analyzed tibial microarchitecture by micro-CT. Consistent with previous reports,38' 44 OVX mice had significantly lower trabecular bone volume, trabecular number, and trabecular bone mineral density compared to sham-operated mice; NO-Cbi partly restored all three parameters (Fig. 7A-D). We found no difference in cortical thickness or cortical tissue mineral density between sham-operated and OVX mice, and NO-Cbi did not affect cortical bone parameters (Fig. 10). Estrogen deficiency-induced bone loss is more pronounced in trabecular than cortical bone, and cortical loss may take longer than trabecular loss and varies with age and strain of mice.38' 44-46
Discussion
A better understanding of the molecular mechanisms controlling bone homeostasis has led to development of multiple drugs for osteoporosis that inhibit bone resorption by targeting osteoclast differentiation and/or function.2, 5 However, the only clinically- available agents that stimulate osteoblast activity and bone formation are PTH analogs, but they also stimulate osteoclasts, limiting their effectiveness.2' 5 Therefore, bone- anabolic agents that simultaneously inhibit osteoclast function are needed. We found that the NO donor NO-Cbi improved trabecular bone architecture and increased bone
formation markers in OVX mice, while dramatically decreasing osteoclasts. Our results confirm bone-anabolic effects of NO, explore mechanisms of NO actions in bone cells, and demonstrate in vitro and in vivo effectiveness of a novel, direct NO-releasing agent.
An important role of NO in osteoblast biology is supported by rodent studies: First, NOS3-deficient mice have reduced bone mass due to defects in osteoblast number and maturation, and they have exaggerated bone loss after ovariectomy, with a blunted response to estrogens47"49 And second, NO-generating organic nitrates reduce bone loss from estrogen deficiency in rats (as measured by DXA), whereas NOS inhibitors block estrogen's bone-protective effects and prevent bone formation induced by mechanical stimulation in rodents.16"18' 22' 23 In humans and rats, serum concentrations of the NO metabolites nitrite and nitrate correlate with estradiol concentrations, and increase with estrogen administration.23, 24 Estrogen deficiency reduces NOS expression and activity, leading to a state of relative NO- and, consequently, cGMP-deficiency.38' 50 NO-Cbi restored serum cGMP concentrations in OVX mice, and the drug's positive effects on osteoblast proliferation, differentiation, and survival were likely mediated via increased intracellular cGMP concentrations and PKG activation, because the drug increased Wnt/p-catenin signaling in a PKG II-dependent fashion. We have previously shown that cGMP-elevating agents and cGMP analogs increase osteoblast proliferation, differentiation, and survival, with PKG II activation of Src required for Erk and Akt activation, and PKG I phosphorylation of Bel -2 contributing to anti-apoptotic effects in osteoblasts and osteocytes.14' 15' 38 Consistent with its in vitro effects on POBs, NO-Cbi increased osteoblast number, mineral apposition and bone formation rates; it enhanced osteoblastic gene expression, reduced osteocyte apoptosis, and increased trabecular bone volume and BMD in OVX mice. Like NO-Cbi, the NO-independent soluble guanylate cyclase activator cinaciguat increased bone formation and osteoblast marker genes and ameliorates bone loss in OVX mice; however, in contrast to NO-Cbi, cinciguat did not affect osteoclast parameters significantly.38
NO appears to have dual functions in osteoclast biology: low NO concentrations generated by NOS1 promote, while higher NO concentrations generated by NOS2 inhibit osteoclast differentiation and survival.51' 52 NOS 1 -deficient mice have an increased bone mass with reduced bone turnover due to defects in osteoclast differentiation and function, indicating a positive role of NO in osteoclasts.51, 53 However, NOS2 induction by RANKL serves as a feed-back inhibition of RANKL-induced osteoclast differentiation,
and NOS2-deficient osteoclasts show increased differentiation in response to RANKL. Adding NO donors to mature osteoclasts inhibits their resorptive activity.54"56
NO-Cbi treatment of OVX mice decreased osteoclast numbers and reduced osteoclast-specific TRAP and CTSK mRNAs in bone; these results correlated with a decrease in RANKL and increase in OPG mRNAs in bone. In contrast, the cGMP- elevating agent cinaciguat did not influence osteoclast numbers, osteoclast-specific genes, RANKL, or OPG in OVX mice.38 In agreement with these in vivo results, NO- Cbi reduced RANKL and increased OPG mRNA in cultured POBs, whereas cinaciguat did not affect these genes.38 RANKL and OPG are key regulators of osteoclast differentiation produced primarily by cells of the osteoblastic lineage.2 Consistent with our results in POBs, other workers have shown down-regulation of RANKL by NO, but not by cGMP analogs, in bone marrow stromal cells.57 In addition to reducing the RANKL/OPG ratio in bone and POBs, NO-Cbi directly inhibited osteoclast
differentiation in vitro, in the presence of excess RANKL and M-CSF, whereas a cGMP analog had little effect. cGMP-independent NO effects on osteoclast differentiation have been reported previously.52' 58 Thus, in addition to inducing anabolic responses in osteoblasts, NO-Cbi inhibited osteoclastogenesis directly and indirectly, by reducing the RANKL/OPG ratio.
Epidemiological data link the use of organic nitrates (i.e., nitroglycerin and iso- sorbide mononitrate) to reduced fracture risk,19"21 and four prospective, randomized trials showed a positive effect of these nitrates on bone density, at doses lower than those used for vasodilation.24 Firs^ in young women who underwent ovariectomy, nitroglycerin was as effective as estrogen replacement in preventing bone loss.59 Second, in post-menopausal women with established osteoporosis, subjects randomized to isosorbide mononitrate showed similar improvement in BMD as subjects who received a bisphosphonate.60 Third, in healthy post-menopausal women, isosorbide mononitrate decreased a bone resorption marker (N-terminal telopeptide) and increased a bone formation marker (alkaline phosphatase) compared to placebo.61 And fourth, in osteopenic post-menopausal women, nitroglycerin increased BMD at the lumbar spine and hip, and increased cortical thickness at the radius and tibia; it also decreased N- terminal telopeptide and increased alkaline phosphatase.25 These results indicate positive effects of nitroglycerin on bone formation and possibly on bone strength— with cortical changes superior to those observed with PTH— but the studies were
underpowered to assess fracture risk. ' In contrast, another trial in post-menopausal women failed to show an effect of nitroglycerin on BMD, possibly because treatment adherence was poor.26
A major problem with organic nitrates is that they must be activated by
mitochondrial aldehyde dehydrogenase, which leads to oxidative stress, development of tolerance, and induction of endothelial dysfunction.28, 29 In fact, several large trials of chronic nitrate use in patients with coronary artery diseases have shown increased mortality in the treated patients,63' 64 which may be attributable to increased oxidative stress.27 Since oxidative stress is implicated in the pathophysiology of estrogen deficiency- and age-related osteoporosis,10 and oxidative stress can lead to decreased NO bio-availability and soluble guanylate cyclase desensitization,30 the beneficial effects of nitrates in post-menopausal osteoporosis are likely limited by their pro-oxidant properties.
Considerable effort has been directed towards developing second generation, direct NO-releasing agents, but to date, none have shown efficacy in clinical trials, and some generate toxic metabolites in the process of NO release, making them unsuitable for clinical use.30' 65 We have developed NO-Cbi as a novel, direct NO donor with major advantages over existing, FDA-approved nitrates. NO-Cbi is derived from the penultimate vitamin B12 precursor cobinamide; cobinamide is found at low concentrations in human serum, and we have found in rodents that cobinamide has no toxicity at >50- fold higher doses than those used in this study and those required for delivery of pharmacological amounts of NO (G.R.Boss et al., unpublished data and32). After NO release, cobinamide is generated, and cobinamide can bind O2" and other reactive oxygen species,35 providing a potential added benefit of protecting cells from oxidative stress. Repeated administration of NO-Cbi does not induce tolerance, and NO-Cbi is stable and can be administered by multiple routes, including oral ingestion (G.R. Boss et al., unpublished data and32).
A limitation of our study is that ovariectomy does not mimic the gradual cessation of ovarian function occurring during menopause, however, OVX mice are an accepted model for bone loss due to estrogen deficiency, and show changes in bone architecture and turnover similar to those observed in post-menopausal women, including increased bone resorption and increased osteoblast/osteocyte apoptosis.9, 42, 44, 46 Some authors have observed increased bone formation markers in parallel with increased resorption in
OVX mice, whereas we and others found no change in mineral apposition rate and a trend towards reduced mineralizing surface and bone formation rate after OVX.66"69 These differences may be attributable to differences in mouse strain and age, the type of bone examined, and the time interval after OVX. We did not observe cortical bone loss post OVX, but bone loss in OVX mice also varies greatly among different inbred strains, with age of the mice, the site examined, and the time interval after surgery.38' 44" 46 Another limitation of our study is that we examined a single dose of NO-Cbi; this dose was chosen based on its lack of effect on systolic blood pressure. Further studies are needed to optimize treatment dose and schedule, and to test the effect of oral NO- Cbi administration.
In conclusion, we have shown that NO-Cbi regulates bone remodeling by promoting osteoblast proliferation, differentiation, and survival, and by simultaneously inhibiting osteoclast differentiation. NO-Cbi improved bone mass in OVX mice, a frequently-used model of post-menopausal osteoporosis.9' 44' 70 We are unaware of previous work using a direct NO-releasing agent as a bone-anabolic agent in animals or humans. These studies represent proof of concept for the effectiveness of NO-Cbi as an anabolic agent for treating osteoporosis.
FIGURE LEGENDS
Figure 1 : NO-Cbi enhances cGMP/PKG and Erk/Akt signaling, gene expression, proliferation and survival in POBs. (A) POBs were incubated in medium with 0.1% FBS (3 x 105 cells/ml) for 2 h prior to receiving 10 μΜ NO-Cbi (NOCbi) for the indicated times. Stable NO oxidation products (nitrite plus nitrate, NOx) were measured in the medium by the Griess reaction (NOx present in medium without cells was subtracted). (B,C) POBs were treated with vehicle or NO-Cbi at the indicated concentrations for 30 min, and intracellular cGMP concentrations were measured by ELISA (B). VASP phosphorylation was analyzed by Western blot using a phospho-Ser259-specific antibody, with densitometric quantitation of pVASP normalized to β-actin shown above. (D,E) Serum-deprived POBs were treated with vehicle or 10 μΜ NO-Cbi for 10 min and ERK and Akt activation were assessed by blotting with phospho-specific antibodies.
Densitometric quantitation of pErk and pAkt normalized to total Erk and Akt, respectively, is shown above. (F) POBs were serum-starved for 36 h in medium containing 1% BSA with 10 μΜ NO-Cbi or vehicle; apoptosis was assessed by immunofluorescence staining with antibodies specific for cleaved caspase-3 and FITC-
coupled secondary antibodies (green); nuclei were counterstained with Hoechst 33342 (blue). (G) POBs cultured in medium with 0.1% FBS for 18 h were treated with 10 μΜ NO-Cbi or vehicle for 1 h, and transferred to fresh medium containing 3H-thymidine for 24 h; thymidine incorporation into DNA was measured as described in Experimental Procedures. (H,I) Confluent POBs were differentiated in ascorbate-containing medium for 14 d and some cells received 10 μΜ NO-Cbi (open bars) or vehicle (filled bars) for the last 24 h. Expression of osteocalcin (OCN), osteopontin (Sppl), collagen 1-Al (Collal), alkaline phosphatase (ALP), low-density lipoprotein receptor-related protein-5 (Lrp5), tubulin (Tubal), receptor activator of nuclear factor kappa-B ligand (RANKL), and osteoprotegerin (OPG) mRNAs were determined by qRT-PCR and normalized to 18S rRNA, with relative mRNA levels in vehicle-treated cells assigned a value of 1. Panels A-I show means ± SEM of at least three independent experiments; *p < 0.05, **p < 0.01, ***p < 0.001, for the comparison between NO-Cbi- and vehicle-treated cells. In panel F, ###p <0.01 for the comparison between cells in starvation versus control medium.
Figure 2: NO-Cbi stimulates Wnt signaling and mPOB proliferation via PKG II. (A) POBs isolated from mice homozygous for prkg2 alleles flanked by LoxP sites ("floxed" PRKG2f/f) were infected with adenovirus expressing β-galactosidase (LacZ, control) or CRE recombinase (CRE). Forty-eight h later, relative amounts of prkg2 mRNA were determined by qRT-PCR, and knockdown efficiency of PKG II protein was analyzed by Western blotting, with caveolin-1 serving as a loading control. (B) Cells were infected as in A, but 30 h later were transferred to medium containing 0.1% FBS, and 18 h later were treated with 10 μΜ NO-Cbi or vehicle for 10 min. Akt and GSK-3P phosphorylation were assessed using antibodies specific for Akt(pSer473) and GSK-3P(pSer9 ), with total GSK-3P serving as a loading control; densitometric quantitation is shown on the right, with relative amounts of pAkt and pGSK-3 found in vehicle-treated control virus-infected cells assigned a value of 1. (C) PRKG2f/f POBs were infected with control or Cre virus and transferred to 0.1% FBS as in B; they were treated with NO-Cbi or vehicle for 6 h, prior to detecting β-catenin by immunofluorescence staining. The bottom panel shows nuclei counterstained with Hoechst 33342. Numbers below indicate the percentage of cells showing nuclear β-catenin. (D) Cells were infected and cultured as in B; they were treated with NO-Cbi or vehicle for 1 h prior to measuring 3H-thymidine incorporation into DNA for 24 h. (E) Cells were infected with control or CRE virus as described in A, and treated with 10 μΜ NO-Cbi or vehicle for 24 h. Expression of Wingless type
MMTV-integration site family- la (Wntla), low-density lipoprotein receptor- related protein-5 (Lrp5), β-catenin (bCat), cyclin D (CycD), alkaline phosphatase (ALP), osteocalcin (OCN), and tubulin (Tubal) mRNAs were measured by qRT-PCR and normalized to 18S rRNA, with relative mRNA levels in untreated cells assigned a value of 1. (F) POBs cultured in 10 % FBS were treated with 10 μΜ NO-Cbi for the indicated times, and Lrp5 protein (open symbols) and mRNA (filled symbols) were assessed by Western blotting and qRT-PCR, respectively. Panels A-F show means ± SEM of at least three independent experiments; *p < 0.05, **p < 0.01, ***p < 0.001, for the comparison between NO-Cbi-treated versus vehicle-treated cells infected with control virus, and #p < 0.05, ##p < 0.01, ###p < 0.001 for comparison between NO-Cbi-treated cells infected with control versus CRE virus.
Figure 3 : NO-Cbi inhibits osteoclast differentiation. (A,B) Murine bone marrow mononuclear cells were cultured in the presence of M-CSF, with RANKL added after 3 d; together with RANKL, cells received vehicle or NO-Cbi at the indicated concentrations. Tartrate-resistant acid phosphatase (TRAP)-positive cells (red) were counted on day 8. (C) Cells were cultured as in A, but some cultures received 10 μΜ NO-Cbi, 5 μΜ DETA- NONOate (Deta-NO, which releases 2 moles of NO/mol of drug), or 100 μΜ 8-pCPT- cGMP together with RANKL. (D) Expression of TRAP, cathepsin K (Ctsk), and calcitonin receptor (CalcR) mRNAs were determined by qRT-PCR and normalized to 18S rRNA, with relative mRNA levels in vehicle-treated cells assigned a value of 1.
Panels B-D show means ± SEM of at least three independent experiments; *p < 0.05, **p < 0.01, ***p < 0.001, for the comparison between vehicle-treated versus drug-treated cells.
Figure 4: NO-Cbi increases serum cGMP concentration, bone formation, and osteo- blastic gene expression in OVX mice. Eleven week-old mice were subjected to ovariectomy (OVX) or sham operation, and 7 d later daily i.p. injections were started with either vehicle (Veh) or NO-Cbi (10 mg/kg/d) for 6 d/week for a total of 5 weeks. Mice additionally received calcein 7 d and 4 d prior to euthanasia. (A) Serum cGMP concentrations were measured by ELISA 1 h after the last injection of vehicle or NO-Cbi. (B) The number of trabecular osteoblasts per bone perimeter (N.Ob/B.Pm) was counted at the proximal tibia. (C-F) Trabecular calcein labeling was assessed at the tibia (C), with quantification of mineral apposition rate (MAR, panel D), mineralizing surface per bone surface (MS/BS, panel E), and bone formation rate (BFR, panel F). (G) RNA was
extracted from femurs, and the relative abundance of osteocalcin (OCN), osteopontin (Sppl), alkaline phosphatase (ALP), collagen-al (Collal), low-density lipoprotein receptor- related protein-5 (Lrp5) and tubulin (Tubal) mRNA was quantified by qRT- PCR and normalized to 18S rRNA. Data were calculated according to the AACT method, using the mean of the vehicle-treated OVX group. Data for panels A-F are the means ± SEM from n= 6 sham operated mice, n= 7 vehicle treated OVX mice, and n=8 NO-Cbi- treated OVX mice; data in panel G represent 6 mice per group. *p < 0.05, **p < 0.01, ***p < 0.001 for the indicated pair-wise comparisons.
Figure 5: NO-Cbi prevents estrogen deficiency-induced osteocyte apoptosis. Mice were subjected to OVX or sham operation and were treated with vehicle or NO-Cbi as described in Fig. 4. (A-C) The percentage of apoptotic osteocytes was assessed in trabecular (A,B) and cortical bone (C) by TUNEL staining (black nuclei) of tibial sections. Data in B and C represent means ± SEM from n= 6 mice per group. *p < 0.05, **p < 0.01, for the indicated pair-wise comparisons. (D) Osteoblast and osteocyte apoptosis was assessed by Western blotting of extracts obtained from tibial bone (after removal of bone marrow), using an antibody specific for cleaved caspase-3, with β-actin serving as a loading control (n =2 mice per group). (E) Erk activity in cortical (top) and trabecular (bottom) bone-lining cells was assessed by immunofluorescence staining using a phospho-Erk-specific antibody and horse radish peroxidase-coupled secondary antibody (brown); isotype-matched control IgG produced no signal (not shown).
Figure 6: NO-Cbi regulates RANKL/OPG and reduces osteoclasts in OVX mice. Mice subjected to OVX or sham-operation were treated with vehicle or NO-Cbi as described in Fig. 4. (A,B) Osteoclasts were identified by TRAP staining (red), and the number of trabecular osteoclasts per bone perimeter (N.Oc/B.Pm) was counted at the proximal tibia. (C) RNA was extracted from femurs, and the relative abundance of
RANKL, OPG, CTSK, and TRAP mRNA was quantified by qRT-PCR and normalized to 18S rRNA. Data were calculated according to the AACT method using the mean of the vehicle-treated OVX group. Data represent the mean ± SEM from 6 mice per group. *p < 0.05, **p < 0.01, ***p < 0.001 for the indicated pair-wise comparisons.
Figure 7: NO-Cbi increases trabecular bone mass in OVX mice. Mice subjected to OVX or sham operation were treated with vehicle or NO-Cbi as described in Fig.4. (A) Tibiae were analyzed by micro-CT imaging, and three-dimensional reconstruction of the trabecular bone at the proximal tibia below the growth plate is shown. (B-E) Trabecular
bone volume/tissue volume (B), trabecular number (C), and trabecular bone mineral density (D) were quantified at the proximal tibia as described in Materials and Methods. Data represent means ± SEM from n= 6 sham-operated mice, n= 7 vehicle-treated OVX mice, and n=8 NO-Cbi-treated OVX mice; *p < 0.05, **p < 0.01, ***p < 0.001 for the indicated pair-wise comparisons.
REFERENCES
(1) Dallas SL, Prideaux M, Bonewald LF. The osteocyte: an endocrine cell ... and more.
Endocr Rev 2013;34(5):658-90.
(2) Kawai M, Modder UI, Khosla S, Rosen CJ. Emerging therapeutic opportunities for skeletal restoration. Nat Rev Drug Discov 2011; 10(2): 141-56.
(3) Watts NB, Bilezikian JP, Camacho PM et al. American Association of Clinical
Endocrinologists Medical Guidelines for Clinical Practice for the diagnosis and treatment of postmenopausal osteoporosis. Endocr Pract 2010; 16 Suppl 3 : 1-37. (4) Khosla S, Bellido TM, Drezner MK et al. Forum on aging and skeletal health:
summary of the proceedings of an ASBMR workshop. J Bone Miner Res
2011;26(l l):2565-78.
(5) Reid IR. Short-term and long-term effects of osteoporosis therapies. Nat Rev
Endocrinol 2015 ; 11 (7) : 418-28.
(6) Neer RM, Arnaud CD, Zanchetta JR et al. Effect of parathyroid hormone (1-34) on fractures and bone mineral density in postmenopausal women with osteoporosis. N
£«g/J ¾/ 2001;344(19): 1434-41.
(7) Barrett-Connor E, Grady D, Stefanick ML. The rise and fall of menopausal
hormone therapy. Annu Rev Public Health 2005;26: 115-40.
(8) Barrett-Connor E, Wade SW, Do TP et al. Treatment satisfaction and persistence among postmenopausal women on osteoporosis medications: 12-month results from
POSSIBLE US. Osteoporos Int 2012;23(2):733-41.
(9) Khosla S, Melton LJ, III, Riggs BL. The unitary model for estrogen deficiency and the pathogenesis of osteoporosis: is a revision needed? J Bone Miner Res
2011;26(3):441-51.
(10) Manolagas SC. From estrogen-centric to aging and oxidative stress: a revised
perspective of the pathogenesis of osteoporosis. Endocr Rev 2010;31(3):266-300.
(11) Tomkinson A, Reeve J, Shaw RW, Noble BS. The death of osteocytes via apoptosis accompanies estrogen withdrawal in human bone. J Clin Endocrinol Metab
1997;82(9):3128-35.
(12) Ozcivici E, Luu YK, Adler B et al. Mechanical signals as anabolic agents in bone.
Nat Rev Rheumatol 20\0;6(\):50-9.
(13) Rangaswami H, Marathe N, Zhuang S et al. Type II cGMP-dependent protein
kinase mediates osteoblast mechanotransduction. J Biol Chem 2009;284: 14796-808.
(14) Marathe N, Rangaswami H, Zhuang S, Boss GR, Pilz RB. Pro-survival Effects of 17beta-Estradiol on Osteocytes Are Mediated by Nitric Oxide/cGMP via
Differential Actions of cGMP-dependent Protein Kinases I and II. J Biol Chem
2012;287(2):978-88.
(15) Rangaswami H, Schwappacher R, Marathe N et al. Cyclic GMP and protein kinase G control a Src-containing mechanosome in osteoblasts. Sci Signal
2010;3(153):ra91.
(16) Samuels A, Perry MJ, Gibson RL, Colley S, Tobias JH. Role of endothelial nitric oxide synthase in estrogen-induced osteogenesis. Bone 2001;29(l):24-9.
(17) Turner CH, Owan I, Jacob DS, McClintock R, Peacock M. Effects of nitric oxide synthase inhibitors on bone formation in rats. Bone 1997;21(6):487-90.
(18) Fox SW, Chambers TJ, Chow JW. Nitric oxide is an early mediator of the increase in bone formation by mechanical stimulation. Am J Physiol 1996;270(6 Pt 1):E955-
E960.
(19) Rejnmark L, Vestergaard P, Mosekilde L. Decreased fracture risk in users of
organic nitrates: a nationwide case-control study. J Bone Miner Res
2006;21(l l): 1811-7.
(20) Pouwels S, Lalmohamed A, van ST et al. Use of organic nitrates and the risk of hip fracture: a population-based case-control study. J Clin Endocrinol Metab
2010;95(4): 1924-31.
(21) Jamal SA, Browner WS, Bauer DC, Cummings SR. Intermittent use of nitrates increases bone mineral density: the study of osteoporotic fractures. J Bone Miner Res 1998; 13(l l): 1755-9.
(22) Wimalawansa SJ, De MG, Gangula P, Yallampalli C. Nitric oxide donor alleviates ovariectomy-induced bone loss. Bone 1996; 18(4):301-4.
(23) Hukkanen M, Platts LA, Lawes T et al. Effect of nitric oxide donor nitroglycerin on bone mineral density in a rat model of estrogen deficiency -induced osteopenia. Bone 2003;32(2): 142-9.
(24) Hamilton CJ, Reid LS, Jamal SA. Organic nitrates for osteoporosis: an update.
Bonekey Rep 20\3;2:259.
(27) Thomas GR, DiFabio JM, Gori T, Parker JD. Once daily therapy with isosorbide-5- mononitrate causes endothelial dysfunction in humans: evidence of a free-radical- mediated mechanism. J Am Coll Cardiol 2007;49(12): 1289-95.
(28) Munzel T, Wenzel P, Daiber A. Do we still need organic nitrates? J Am Coll
Cardiol 2007 49(\2): 1296-8.
(29) Parker JD. Nitrate tolerance, oxidative stress, and mitochondrial function: another worrisome chapter on the effects of organic nitrates. J Clin Invest 2004; 113(3):352- 4.
(30) Lundberg JO, Gladwin MT, Weitzberg E. Strategies to increase nitric oxide
signalling in cardiovascular disease. Nat Rev Drug Discov 2015; 14(9):623-41.
(31) Sydow K, Daiber A, Oelze M et al. Central role of mitochondrial aldehyde
dehydrogenase and reactive oxygen species in nitroglycerin tolerance and cross- tolerance. J Clin Invest 2004; 113(3):482-9.
(32) Broderick KE, Alvarez L, Balasubramanian M et al. Nitrosyl-cobinamide, a new and direct NO-releasing drug effective in vivo. Exp Biol Med (Maywood )
2007;232(11): 1432-40.
(33) Sharma VS, Pilz RB, Boss GB, Magde D. Reactions of nitric oxide with vitamin B12 and its precursor, cobinamide. Biochemistry 2003;42:8900-8.
(34) Spitler R, Schwappacher R, Wu T et al. Nitrosyl-cobinamide (NO-Cbi), a new nitric oxide donor, improves wound healing through cGMP/cGMP-dependent protein kinase. Cell Signal 2013;25(12):2374-82.
(35) Jiang J, Chan A, Ali S et al. Hydrogen sulfide-mechanisms of toxicity and
development of an antidote. Scientific Reports 2016;in press.
(36) Kalyanaraman H, Schwappacher R, Joshua J et al. Nongenomic thyroid hormone signaling occurs through a plasma membrane-localized receptor. Sci Signal
2014;7(326):ra48.
(37) Takahashi N, Udagawa N, Tanaka S, Suda T. Generating murine osteoblasts from bone marrow. In: Helfrich MH, Ralston SH, editors. Methods in Molecula
Medicine, Vol. 80: Bone Research Protocols .Totowa, NJ: Humana Press Inc.; 2003. p. 129-44.
(38) Joshua J, Schwaerzer GK, Kalyanaraman H et al. Soluble guanylate cyclase as a novel treatment target for osteoporosis. Endocrinology 2014;en20141343.
(39) Baron R, Kneissel M. WNT signaling in bone homeostasis and disease: from human mutations to treatments. Nat Med 2013; 19(2): 179-92.
(40) Cohen P, Frame S. The renaissance of GSK3. Nature RevMol Cell Biol
2001;2(10):769-76.
(41) Zhao X, Zhuang S, Chen Y, Boss GR, Pilz RB. Cyclic GMP-dependent protein kinase regulates CCAAT enhancer-binding protein beta functions through inhibition of glycogen synthase kinase-3. JBiol Chem 2005;280:32683-92.
(42) Kousteni S, Bellido T, Plotkin LI et al. Nongenotropic, sex-nonspecific signaling through the estrogen or androgen receptors: dissociation from transcriptional activity . Cell 2001; 104(5):719-30.
(43) Harvey CB, Stevens DA, Williams AJ, Jackson DJ, O'Shea P, Williams GR.
Analysis of thyroid hormone responsive gene expression in osteoblastic cells. Mol Cell Endocrinol 2003;213(l):87-97.
(44) Bouxsein ML, Myers KS, Shultz KL, Donahue LR, Rosen CJ, Beamer WG.
Ovariectomy -induced bone loss varies among inbred strains of mice. J Bone Miner Res 2005;20(7): 1085-92.
(45) Bartell SM, Han L, Kim HN et al. Non-nuclear-initiated actions of the estrogen receptor protect cortical bone mass. Mol Endocrinol 2013;27(4):649-56.
(46) Syed FA, Modder UI, Roforth M et al. Effects of chronic estrogen treatment on modulating age-related bone loss in female mice. J Bone Miner Res
2010;25(l l):2438-46.
(47) Aguirre J, Buttery L, O'Shaughnessy M et al. Endothelial nitric oxide synthase gene-deficient mice demonstrate marked retardation in postnatal bone formation, reduced bone volume, and defects in osteoblast maturation and activity. Am J Pathol 2001; 158:247-57.
(48) Armour KE, Armour KJ, Gallagher ME et al. Defective bone formation and
anabolic response to exogenous estrogen in mice with targeted disruption of endothelial nitric oxide synthase. Endocrinology 2001; 142(2):760-6.
(49) Grassi F, Fan X, Rahnert J et al. Bone re/modeling is more dynamic in the endothelial nitric oxide synthase(-/-) mouse. Endocrinology 2006; 147(9):4392-9.
(50) Mendelsohn ME, Karas RH. Rapid progress for non-nuclear estrogen receptor
signaling. J Clin Invest 2010; 120(7):2277-9.
(51) van't Hof RJ, Macphee J, Libouban H, Helfrich MH, Ralston SH. Regulation of bone mass and bone turnover by neuronal nitric oxide synthase. Endocrinology 2004; 145(l l):5068-74.
(52) Zheng H, Yu X, Collin-Osdoby P, Osdoby P. RANKL stimulates inducible nitric- oxide synthase expression and nitric oxide production in developing osteoclasts. An autocrine negative feedback mechanism triggered by RANKL-induced interferon- beta via NF-kappaB that restrains osteoclastogenesis and bone resorption. J Biol Chem 2006;281(23): 15809-20.
(53) Jung JY, Lin AC, Ramos LM, Faddis BT, Chole RA. Nitric oxide synthase I
mediates osteoclast activity in vitro and in vivo. J Cell Biochem 2003;89(3):613-21. (54) Dong SS, Williams JP, Jordan SE, Cornwell T, Blair HC. Nitric oxide regulation of cGMP production in osteoclasts. J Cell Biochem 1999;73(4):478-87.
(55) Fuller K, Kirstein B, Chambers TJ. Murine osteoclast formation and function:
differential regulation by humoral agents. Endocrinology 2006; 147(4): 1979-85.
(56) Yaroslavskiy BB, Turkova I, Wang Y, Robinson LJ, Blair HC. Functional
osteoclast attachment requires inositol-l,4,5-trisphosphate receptor-associated cGMP-dependent kinase substrate. Lab Invest 2010;90: 1533-42.
(57) Fan X, Roy E, Zhu L et al. Nitric oxide regulates receptor activator of nuclear
factor-kappaB ligand and osteoprotegerin expression in bone marrow stromal cells. Endocrinology 2004; 145(2):751-9.
(58) Ralston SH, Grabowski PS. Mechanisms of cytokine induced bone resorption: role of nitric oxide, cyclic guanosine monophosphate, and prostaglandins. Bone
1996;19(l):29-33.
(59) Wimalawansa SJ. Nitroglycerin therapy is as efficacious as standard estrogen
replacement therapy (Premarin) in prevention of oophorectomy-induced bone loss: a human pilot clinical study. J Bone Miner Res 2000; 15(11):2240-4.
(60) Nabhan AF, Rabie NH. Isosorbide mononitrate versus alendronate for
postmenopausal osteoporosis. Int J Gynaecol Obstet 2008; 103(3):213-6.
(61) Jamal SA, Cummings SR, Hawker GA. Isosorbide mononitrate increases bone formation and decreases bone resorption in postmenopausal women: a randomized trial. J Bone Miner Res 2004; 19(9): 1512-7.
(62) Khosla S. Is nitroglycerin a novel and inexpensive treatment for osteoporosis?
JAMA 2011;305(8):826-7.
(63) Nakamura Y, Moss AJ, Brown MW, Kinoshita M, Kawai C. Long-term nitrate use may be deleterious in ischemic heart disease: A study using the databases from two large-scale postinfarction studies. Multicenter Myocardial Ischemia Research Group. Am Heart J 1999; 138(3 Pt l):577-85.
(64) Ishikawa K, Kanamasa K, Ogawa I et al. Long-term nitrate treatment increases cardiac events in patients with healed myocardial infarction. Secondary Prevention
Group. Jpn Circ J 1996;60(10):779-88.
(65) Miller MR, Megson IL. Recent developments in nitric oxide donor drugs. Br J
Pharmacol 2007; 151(3):305-21.
(66) Modder UI, Sanyal A, Kearns AE et al. Effects of loss of steroid receptor
coactivator-1 on the skeletal response to estrogen in mice. Endocrinology
2004; 145(2):913-21.
(67) Hayashi M, Nakashima T, Taniguchi M, Kodama T, Kumanogoh A, Takayanagi H.
Osteoprotection by semaphorin 3A. Nature 2012;485(7396):69-74.
(68) Duque G, Huang DC, Dion N et al. Interferon-gamma plays a role in bone
formation in vivo and rescues osteoporosis in ovariectomized mice. J Bone Miner Res 2011;26(7): 1472-83.
(69) Pierroz DD, Bonnet N, Baldock PA et al. Are osteoclasts needed for the bone
anabolic response to parathyroid hormone? A study of intermittent parathyroid hormone with denosumab or alendronate in knock-in mice expressing humanized
RANKL. JBiol Chem 2010;285(36):28164-73.
(70) Syed FA, Melim T. Rodent models of aging bone: an update. Curr Osteoporos Rep 2011;9(4):219-28.
A number of embodiments of the invention have been described. Nevertheless, it will be understood that various modifications may be made without departing from the spirit and scope of the invention. Accordingly, other embodiments are within the scope of the following claims.
Claims
1. A method for:
- treating, ameliorating, preventing or reversing osteoporosis or postmenopausal osteoporosis;
- preventing bone loss due to estrogen deficiency;
- inhibiting osteoclast differentiation;
- reducing osteoclast numbers;
- preventing estrogen deficiency-induced osteocyte apoptosis;
- increasing serum cGMP concentration;
- increasing bone formation and bone mass, or increasing trabecular bone volume, trabecular number, and trabecular bone mineral density; or
- increasing osteoblast number and regulating osteoblastic gene expression,
comprising
administering a nitrosyl-cobinamide (NO-Cbi), or a bioisostere, stereoisomer, tautomer, hydrate, solvate or pharmaceutically acceptable salt thereof to: an individual; an individual with osteoporosis or at risk of developing osteoporosis,
thereby:
- treating, ameliorating, preventing or reversing osteoporosis or post- menopausal osteoporosis;
- preventing bone loss due to estrogen deficiency;
- inhibiting osteoclast differentiation;
- reducing osteoclast numbers;
- preventing estrogen deficiency-induced osteocyte apoptosis; - increasing serum cGMP concentration;
- increasing bone formation or bone mass, or increasing trabecular bone volume, trabecular number, and trabecular bone mineral density; or
- increasing osteoblast number and regulating osteoblastic gene expression.
2. The method of claim 1, wherein the nitrosyl-cobinamide, or the bioisostere, stereoisomer, tautomer, hydrate, solvate or pharmaceutically acceptable salt thereof, is
formulated as a pharmaceutical composition, or is formulated as a pharmaceutical composition comprising a pharmaceutically acceptable carrier or excipient thereof.
and optionally the the nitrosyl-cobinamide, or the bioisostere, stereoisomer, tautomer, hydrate, solvate or pharmaceutically acceptable salt thereof, is formulated and/or administered by systemic administration, optionally by oral, intravenous, intraperitoneal and intramuscular delivery, or optionally is formulated and/or
administered by infusion or injection, optionally intravenously, intramuscularly, intracutaneously, subcutaneously, intrathecal, intraduodenally, intraperitoneally, and equivalent, or formulated or administered intranasally, vaginally, rectally, orally, or transdermally.
3. The method of claim 1 or claim 2, wherein the nitrosyl-cobinamide, or the bioisostere, stereoisomer, tautomer, hydrate, solvate or pharmaceutically acceptable salt thereof, or the pharmaceutical composition, is administered or co-administered with another therapeutic agent,
wherein optionally therapeutic agent is administered simultaneously, before or after the nitrosyl-cobinamide, or a bioisostere, stereoisomer, tautomer, hydrate, solvate or pharmaceutically acceptable salt thereof, or the pharmaceutical composition.
4. The method of any of the preceding claims, wherein the nitrosyl- cobinamide, or the bioisostere, stereoisomer, tautomer, hydrate, solvate or
pharmaceutically acceptable salt thereof, or the pharmaceutical composition, is administered in or with an implant or a bone implant.
5. The method of any of the preceding claims, wherein the nitrosyl- cobinamide, or the bioisostere, stereoisomer, tautomer, hydrate, solvate or
pharmaceutically acceptable salt thereof, or the pharmaceutical composition, is administered as or is formulated in a device, an implant, a bone implant, a bead, a tablet, a pill, a capsule, a liquid, a gel, a geltab, a powder, a spray or aerosol, a cachet, a suppository, a dispersible granule, a product of manufacture, a liposome, a particle, a microparticle or a nanoparticle.
6. The method of any of the preceding claims, wherein the nitrosyl- cobinamide, or the bioisostere, stereoisomer, tautomer, hydrate, solvate or
pharmaceutically acceptable salt thereof, or the pharmaceutical composition, is administered as or is formulated in or as a kit, a pump, a device, a subcutaneous infusion device, a continuous subcutaneous infusion device, an infusion pen, a needles, a reservoir, an ampoules, a vial, a syringe, a cartridge, a pen, a disposable pen or jet injector, a prefilled pen or a syringe or a cartridge, a cartridge or a disposable pen or jet injector, a two chambered or multi-chambered pump.
7. Use of a nitrosyl-cobinamide (NO-Cbi), or a bioisostere, stereoisomer, tautomer, hydrate, solvate or pharmaceutically acceptable salt thereof in the manufacture of a medicament for:
- treating, ameliorating, preventing or reversing osteoporosis or postmenopausal osteoporosis;
- preventing bone loss due to estrogen deficiency;
- inhibiting osteoclast differentiation;
- reducing osteoclast numbers;
- preventing estrogen deficiency-induced osteocyte apoptosis;
- increasing serum cGMP concentration;
- increasing bone formation or bone mass, or increasing trabecular bone volume, trabecular number, and trabecular bone mineral density; or
- increasing osteoblast number and regulating osteoblastic gene
expression.
8. A therapeutic formulation for use in:
- treating, ameliorating, preventing or reversing osteoporosis or post- menopausal osteoporosis;
- preventing bone loss due to estrogen deficiency;
- inhibiting osteoclast differentiation;
- reducing osteoclast numbers;
- preventing estrogen deficiency-induced osteocyte apoptosis;
- increasing serum cGMP concentration;
- increasing bone formation or bone mass, or increasing trabecular bone volume, trabecular number, and trabecular bone mineral density; or
- increasing osteoblast number and regulating osteoblastic gene expression,
wherein the therapeutic formulation comprises a nitrosyl-cobinamide (NO-Cbi), or a bioisostere, stereoisomer, tautomer, hydrate, solvate or pharmaceutically acceptable salt thereof.
9. A bone implant comprising a nitrosyl-cobinamide, or a bioisostere, stereoisomer, tautomer, hydrate, solvate or pharmaceutically acceptable salt thereof.
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US201662348577P | 2016-06-10 | 2016-06-10 | |
US62/348,577 | 2016-06-10 |
Publications (1)
Publication Number | Publication Date |
---|---|
WO2017214579A1 true WO2017214579A1 (en) | 2017-12-14 |
Family
ID=60578206
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/US2017/036876 WO2017214579A1 (en) | 2016-06-10 | 2017-06-09 | Compositions and methods for treating osteoporesis |
Country Status (1)
Country | Link |
---|---|
WO (1) | WO2017214579A1 (en) |
Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20090010989A1 (en) * | 2005-09-12 | 2009-01-08 | N0Labs Ab | Coating For Implants and Implants With Improved Osteointegration, and Manufacturing Method |
US8222242B2 (en) * | 2007-04-13 | 2012-07-17 | The Regents Of The University Of California | Nitric oxide releasing compounds |
-
2017
- 2017-06-09 WO PCT/US2017/036876 patent/WO2017214579A1/en active Application Filing
Patent Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20090010989A1 (en) * | 2005-09-12 | 2009-01-08 | N0Labs Ab | Coating For Implants and Implants With Improved Osteointegration, and Manufacturing Method |
US8222242B2 (en) * | 2007-04-13 | 2012-07-17 | The Regents Of The University Of California | Nitric oxide releasing compounds |
Non-Patent Citations (2)
Title |
---|
FRIEBE A ET AL.: "From bedside to bench-meeting report of the 7th International Conference on cGMP '' cGMP: generators, effectors and therapeutic implications '' in Trier, Germany , from June 19th to 21st 2015", NAUNYN-SCHMIEDEBERG'S ARCH PHARMACOL, vol. 388, 2015, pages 1237 - 1246, XP035866953 * |
KALYANARAMAN H ET AL.: "NO /GMP as Mediators of Estrogen Effects in Bone", BMC PHARMACOLOGY AND TOXICOLOGY, vol. 16, no. 1, 2015, pages A10, XP021235410 * |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
Kalyanaraman et al. | A novel, direct NO donor regulates osteoblast and osteoclast functions and increases bone mass in ovariectomized mice | |
DE60202727T2 (en) | Inhibition of histone deacetylase for the treatment of cardiac hypertrophy | |
KR101008752B1 (en) | Synergistic combinations comprising a renin inhibitor for cardiovascular diseases | |
MXPA02010091A (en) | Combination of organic compounds. | |
AU2001273938A1 (en) | Novel medical use of aldosterone synthase inhibitors alone or in combination with ati-receptor antagonists | |
Liu et al. | Rivaroxaban suppresses the progression of ischemic cardiomyopathy in a murine model of diet-induced myocardial infarction | |
JP2019529521A (en) | A combination comprising an SGC activator and a mineralocorticoid receptor antagonist | |
TW202112359A (en) | THE USE OF sGC ACTIVATORS FOR THE TREATMENTOF OPHTHALMOLOGIC DISEASES | |
Tian et al. | Hederagenin protects mice against ovariectomy-induced bone loss by inhibiting RANKL-induced osteoclastogenesis and bone resorption | |
BR112021006132A2 (en) | biphenyl sulfonamide compounds for the treatment of type iv collagen diseases | |
WO2005077418A1 (en) | Combination of renin inhibitor and diuretics | |
US20090036432A1 (en) | Combinations of AT1-antagonists, amiloride or triamterine, and a diuretic | |
WO2017214579A1 (en) | Compositions and methods for treating osteoporesis | |
JP2008044871A (en) | Cardiovascular disease-preventing and treating agent | |
CN103429242A (en) | Method and composition for prevention and treatment of cardiac hypertrophy | |
WO2021037212A1 (en) | Composition used for combating metabolic diseases and uses of composition | |
EP4082549A1 (en) | Drug for preventing dialysis shift or renal death | |
US20070123498A1 (en) | Combination of organic compounds | |
CA3104971A1 (en) | Treatment for age- and oxidative stress-associated muscle atrophy and weakness | |
JP2020530031A (en) | New adjuvant therapy in the treatment of prostate cancer | |
WO2017205517A1 (en) | Methods and compositions for the treatment of secretory disorders | |
WO2010025266A1 (en) | Inhibitors of phosphodiesterase type 5a for treating or preventing muscle disease or the symptoms thereof in a patient | |
US10576151B2 (en) | Ciclopirox for use in modulation of glucose homeostasis | |
CN106074421A (en) | A kind of pharmaceutical composition improving stability | |
MISHCHENKO et al. | Antioxidant composition of echinochrome, ascorbic acid and αtocopherol for treating inflammatory processes in lungs |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
121 | Ep: the epo has been informed by wipo that ep was designated in this application |
Ref document number: 17811129 Country of ref document: EP Kind code of ref document: A1 |
|
NENP | Non-entry into the national phase |
Ref country code: DE |
|
122 | Ep: pct application non-entry in european phase |
Ref document number: 17811129 Country of ref document: EP Kind code of ref document: A1 |