EP0823894A1 - Azocompounds for the antemortem diagnosis of alzheimer's disease and in vivo imaging and prevention of amyloid deposition - Google Patents
Azocompounds for the antemortem diagnosis of alzheimer's disease and in vivo imaging and prevention of amyloid depositionInfo
- Publication number
- EP0823894A1 EP0823894A1 EP96913856A EP96913856A EP0823894A1 EP 0823894 A1 EP0823894 A1 EP 0823894A1 EP 96913856 A EP96913856 A EP 96913856A EP 96913856 A EP96913856 A EP 96913856A EP 0823894 A1 EP0823894 A1 EP 0823894A1
- Authority
- EP
- European Patent Office
- Prior art keywords
- lower alkyl
- alkyl group
- chrysamine
- tetrazole
- oxadiazole
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Ceased
Links
- 208000024827 Alzheimer disease Diseases 0.000 title claims description 26
- 238000011503 in vivo imaging Methods 0.000 title claims description 15
- 230000002265 prevention Effects 0.000 title claims description 7
- 230000003941 amyloidogenesis Effects 0.000 title description 20
- 238000003745 diagnosis Methods 0.000 title description 11
- XFFSCOOTVXBLCK-QAVVBOBSSA-N OC(=O)c1cc(ccc1O)\N=N\c1ccc(cc1)-c1ccc(cc1)\N=N\c1ccc(O)c(c1)C(O)=O Chemical class OC(=O)c1cc(ccc1O)\N=N\c1ccc(cc1)-c1ccc(cc1)\N=N\c1ccc(O)c(c1)C(O)=O XFFSCOOTVXBLCK-QAVVBOBSSA-N 0.000 claims abstract description 315
- 210000004556 brain Anatomy 0.000 claims abstract description 186
- 238000000034 method Methods 0.000 claims abstract description 148
- 238000009739 binding Methods 0.000 claims abstract description 145
- 230000027455 binding Effects 0.000 claims abstract description 144
- 150000001875 compounds Chemical class 0.000 claims abstract description 83
- 208000037259 Amyloid Plaque Diseases 0.000 claims abstract description 60
- 238000001727 in vivo Methods 0.000 claims abstract description 33
- 230000001988 toxicity Effects 0.000 claims abstract description 25
- 231100000419 toxicity Toxicity 0.000 claims abstract description 25
- 239000008194 pharmaceutical composition Substances 0.000 claims abstract description 24
- 206010002022 amyloidosis Diseases 0.000 claims abstract description 18
- 230000007850 degeneration Effects 0.000 claims abstract description 18
- 238000001574 biopsy Methods 0.000 claims abstract description 17
- 201000010374 Down Syndrome Diseases 0.000 claims abstract description 15
- 206010044688 Trisomy 21 Diseases 0.000 claims abstract description 14
- 125000000217 alkyl group Chemical group 0.000 claims description 112
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 71
- 230000015572 biosynthetic process Effects 0.000 claims description 62
- WCPAKWJPBJAGKN-UHFFFAOYSA-N oxadiazole Chemical compound C1=CON=N1 WCPAKWJPBJAGKN-UHFFFAOYSA-N 0.000 claims description 52
- 229910052740 iodine Inorganic materials 0.000 claims description 44
- 229910052731 fluorine Inorganic materials 0.000 claims description 41
- 150000003536 tetrazoles Chemical class 0.000 claims description 39
- AYNNSCRYTDRFCP-UHFFFAOYSA-N triazene Chemical compound NN=N AYNNSCRYTDRFCP-UHFFFAOYSA-N 0.000 claims description 39
- 229910052739 hydrogen Inorganic materials 0.000 claims description 38
- 229910052794 bromium Inorganic materials 0.000 claims description 37
- 229910052801 chlorine Inorganic materials 0.000 claims description 37
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 claims description 36
- 125000002023 trifluoromethyl group Chemical group FC(F)(F)* 0.000 claims description 36
- 108090000765 processed proteins & peptides Proteins 0.000 claims description 34
- 210000001638 cerebellum Anatomy 0.000 claims description 33
- 125000003784 fluoroethyl group Chemical group [H]C([H])(F)C([H])([H])* 0.000 claims description 33
- -1 12SI Chemical compound 0.000 claims description 29
- 238000003384 imaging method Methods 0.000 claims description 28
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 23
- ATJFFYVFTNAWJD-UHFFFAOYSA-N Tin Chemical compound [Sn] ATJFFYVFTNAWJD-UHFFFAOYSA-N 0.000 claims description 19
- 239000000243 solution Substances 0.000 claims description 18
- 125000005907 alkyl ester group Chemical group 0.000 claims description 17
- JXBKZAYVMSNKHA-UHFFFAOYSA-N 1h-tetrazol-1-ium-5-olate Chemical group OC=1N=NNN=1 JXBKZAYVMSNKHA-UHFFFAOYSA-N 0.000 claims description 16
- 125000002887 hydroxy group Chemical group [H]O* 0.000 claims description 16
- 231100000252 nontoxic Toxicity 0.000 claims description 16
- 230000003000 nontoxic effect Effects 0.000 claims description 16
- 238000002603 single-photon emission computed tomography Methods 0.000 claims description 14
- 125000001424 substituent group Chemical group 0.000 claims description 14
- 229910052799 carbon Inorganic materials 0.000 claims description 13
- 238000002595 magnetic resonance imaging Methods 0.000 claims description 13
- 150000003839 salts Chemical class 0.000 claims description 13
- 125000000623 heterocyclic group Chemical group 0.000 claims description 12
- 241000699670 Mus sp. Species 0.000 claims description 11
- 201000010099 disease Diseases 0.000 claims description 11
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 claims description 11
- 238000010494 dissociation reaction Methods 0.000 claims description 10
- 230000005593 dissociations Effects 0.000 claims description 10
- 239000003937 drug carrier Substances 0.000 claims description 9
- 108700028369 Alleles Proteins 0.000 claims description 8
- 108010060159 Apolipoprotein E4 Proteins 0.000 claims description 8
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims description 7
- 229910052760 oxygen Inorganic materials 0.000 claims description 7
- 208000001072 type 2 diabetes mellitus Diseases 0.000 claims description 7
- 238000004611 spectroscopical analysis Methods 0.000 claims description 6
- 102000036770 Islet Amyloid Polypeptide Human genes 0.000 claims description 5
- 108010041872 Islet Amyloid Polypeptide Proteins 0.000 claims description 5
- 210000005013 brain tissue Anatomy 0.000 claims description 5
- 150000001732 carboxylic acid derivatives Chemical class 0.000 claims description 5
- 229910052757 nitrogen Inorganic materials 0.000 claims description 5
- 206010016207 Familial Mediterranean fever Diseases 0.000 claims description 4
- 230000001746 atrial effect Effects 0.000 claims description 4
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 claims description 4
- 208000011580 syndromic disease Diseases 0.000 claims description 4
- 208000005145 Cerebral amyloid angiopathy Diseases 0.000 claims description 3
- 206010035226 Plasma cell myeloma Diseases 0.000 claims description 3
- 239000003795 chemical substances by application Substances 0.000 claims description 3
- 230000002140 halogenating effect Effects 0.000 claims description 3
- 238000010253 intravenous injection Methods 0.000 claims description 3
- 230000009885 systemic effect Effects 0.000 claims description 3
- 208000023769 AA amyloidosis Diseases 0.000 claims description 2
- 208000018282 ACys amyloidosis Diseases 0.000 claims description 2
- 206010002025 Amyloidosis senile Diseases 0.000 claims description 2
- 206010007509 Cardiac amyloidosis Diseases 0.000 claims description 2
- 206010008111 Cerebral haemorrhage Diseases 0.000 claims description 2
- 208000010859 Creutzfeldt-Jakob disease Diseases 0.000 claims description 2
- 102000012192 Cystatin C Human genes 0.000 claims description 2
- 108010061642 Cystatin C Proteins 0.000 claims description 2
- 206010011878 Deafness Diseases 0.000 claims description 2
- 208000034846 Familial Amyloid Neuropathies Diseases 0.000 claims description 2
- 206010016202 Familial Amyloidosis Diseases 0.000 claims description 2
- 208000007487 Familial Cerebral Amyloid Angiopathy Diseases 0.000 claims description 2
- 108010049003 Fibrinogen Proteins 0.000 claims description 2
- 102000008946 Fibrinogen Human genes 0.000 claims description 2
- 102000004878 Gelsolin Human genes 0.000 claims description 2
- 108090001064 Gelsolin Proteins 0.000 claims description 2
- 208000032838 Hereditary amyloidosis with primary renal involvement Diseases 0.000 claims description 2
- 208000032849 Hereditary cerebral hemorrhage with amyloidosis Diseases 0.000 claims description 2
- 206010019889 Hereditary neuropathic amyloidosis Diseases 0.000 claims description 2
- 101000772194 Homo sapiens Transthyretin Proteins 0.000 claims description 2
- 201000002795 Muckle-Wells syndrome Diseases 0.000 claims description 2
- 206010036105 Polyneuropathy Diseases 0.000 claims description 2
- 108010048233 Procalcitonin Proteins 0.000 claims description 2
- 206010039811 Secondary amyloidosis Diseases 0.000 claims description 2
- 102000054727 Serum Amyloid A Human genes 0.000 claims description 2
- 108700028909 Serum Amyloid A Proteins 0.000 claims description 2
- 102100029290 Transthyretin Human genes 0.000 claims description 2
- 208000024780 Urticaria Diseases 0.000 claims description 2
- 208000033559 Waldenström macroglobulinemia Diseases 0.000 claims description 2
- 208000005881 bovine spongiform encephalopathy Diseases 0.000 claims description 2
- 230000000747 cardiac effect Effects 0.000 claims description 2
- 230000001684 chronic effect Effects 0.000 claims description 2
- 238000005388 cross polarization Methods 0.000 claims description 2
- 208000022993 cryopyrin-associated periodic syndrome Diseases 0.000 claims description 2
- 231100000895 deafness Toxicity 0.000 claims description 2
- 201000007891 familial visceral amyloidosis Diseases 0.000 claims description 2
- 229940012952 fibrinogen Drugs 0.000 claims description 2
- 238000001631 haemodialysis Methods 0.000 claims description 2
- 208000016354 hearing loss disease Diseases 0.000 claims description 2
- 230000000322 hemodialysis Effects 0.000 claims description 2
- 230000002401 inhibitory effect Effects 0.000 claims description 2
- 206010022498 insulinoma Diseases 0.000 claims description 2
- 201000000564 macroglobulinemia Diseases 0.000 claims description 2
- 208000023356 medullary thyroid gland carcinoma Diseases 0.000 claims description 2
- 238000000386 microscopy Methods 0.000 claims description 2
- 201000000050 myeloid neoplasm Diseases 0.000 claims description 2
- 208000021255 pancreatic insulinoma Diseases 0.000 claims description 2
- 230000007824 polyneuropathy Effects 0.000 claims description 2
- CWCXERYKLSEGEZ-KDKHKZEGSA-N procalcitonin Chemical compound C([C@@H](C(=O)N1CCC[C@H]1C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@H](C(=O)N[C@@H](C)C(=O)N[C@@H]([C@@H](C)CC)C(=O)NCC(=O)N[C@@H](C(C)C)C(=O)NCC(=O)N[C@@H](C)C(=O)N1[C@@H](CCC1)C(=O)NCC(O)=O)[C@@H](C)O)NC(=O)[C@@H](NC(=O)[C@H](CC=1NC=NC=1)NC(=O)[C@H](CC=1C=CC=CC=1)NC(=O)[C@H](CCCCN)NC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CC=1C=CC=CC=1)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@@H](NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)[C@@H](NC(=O)CNC(=O)[C@H](CC(C)C)NC(=O)[C@H](CCSC)NC(=O)[C@H]1NC(=O)[C@H]([C@@H](C)O)NC(=O)[C@H](CO)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CC(N)=O)NC(=O)CNC(=O)[C@@H](N)CSSC1)[C@@H](C)O)[C@@H](C)O)[C@@H](C)O)C1=CC=CC=C1 CWCXERYKLSEGEZ-KDKHKZEGSA-N 0.000 claims description 2
- 229920006395 saturated elastomer Polymers 0.000 claims description 2
- 208000008864 scrapie Diseases 0.000 claims description 2
- 230000009758 senescence Effects 0.000 claims description 2
- 230000002194 synthesizing effect Effects 0.000 claims description 2
- 201000007905 transthyretin amyloidosis Diseases 0.000 claims description 2
- JCXJVPUVTGWSNB-UHFFFAOYSA-N Nitrogen dioxide Chemical compound O=[N]=O JCXJVPUVTGWSNB-UHFFFAOYSA-N 0.000 claims 39
- 208000025688 early-onset autosomal dominant Alzheimer disease Diseases 0.000 claims 1
- 208000015756 familial Alzheimer disease Diseases 0.000 claims 1
- 239000000203 mixture Substances 0.000 abstract description 21
- 230000001575 pathological effect Effects 0.000 abstract description 5
- IQFVPQOLBLOTPF-HKXUKFGYSA-L congo red Chemical compound [Na+].[Na+].C1=CC=CC2=C(N)C(/N=N/C3=CC=C(C=C3)C3=CC=C(C=C3)/N=N/C3=C(C4=CC=CC=C4C(=C3)S([O-])(=O)=O)N)=CC(S([O-])(=O)=O)=C21 IQFVPQOLBLOTPF-HKXUKFGYSA-L 0.000 description 54
- HFACYLZERDEVSX-UHFFFAOYSA-N benzidine Chemical compound C1=CC(N)=CC=C1C1=CC=C(N)C=C1 HFACYLZERDEVSX-UHFFFAOYSA-N 0.000 description 41
- 210000001519 tissue Anatomy 0.000 description 40
- 238000003786 synthesis reaction Methods 0.000 description 39
- 210000004027 cell Anatomy 0.000 description 31
- 230000000694 effects Effects 0.000 description 31
- YGSDEFSMJLZEOE-UHFFFAOYSA-N salicylic acid Chemical compound OC(=O)C1=CC=CC=C1O YGSDEFSMJLZEOE-UHFFFAOYSA-N 0.000 description 27
- 230000009467 reduction Effects 0.000 description 24
- 239000000523 sample Substances 0.000 description 24
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N Phenol Chemical compound OC1=CC=CC=C1 ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 description 21
- 239000000126 substance Substances 0.000 description 21
- 238000006243 chemical reaction Methods 0.000 description 20
- 239000002253 acid Substances 0.000 description 19
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 18
- OSWPMRLSEDHDFF-UHFFFAOYSA-N methyl salicylate Chemical compound COC(=O)C1=CC=CC=C1O OSWPMRLSEDHDFF-UHFFFAOYSA-N 0.000 description 18
- 108090000623 proteins and genes Proteins 0.000 description 18
- 230000008499 blood brain barrier function Effects 0.000 description 17
- 210000001218 blood-brain barrier Anatomy 0.000 description 17
- XFFSCOOTVXBLCK-UHFFFAOYSA-N chembl300172 Chemical compound C1=C(O)C(C(=O)O)=CC(N=NC=2C=CC(=CC=2)C=2C=CC(=CC=2)N=NC=2C=C(C(O)=CC=2)C(O)=O)=C1 XFFSCOOTVXBLCK-UHFFFAOYSA-N 0.000 description 15
- CPRRHERYRRXBRZ-SRVKXCTJSA-N methyl n-[(2s)-1-[[(2s)-1-hydroxy-3-[(3s)-2-oxopyrrolidin-3-yl]propan-2-yl]amino]-4-methyl-1-oxopentan-2-yl]carbamate Chemical compound COC(=O)N[C@@H](CC(C)C)C(=O)N[C@H](CO)C[C@@H]1CCNC1=O CPRRHERYRRXBRZ-SRVKXCTJSA-N 0.000 description 15
- 238000002600 positron emission tomography Methods 0.000 description 15
- 102000004169 proteins and genes Human genes 0.000 description 15
- 206010012289 Dementia Diseases 0.000 description 14
- FJKROLUGYXJWQN-UHFFFAOYSA-N papa-hydroxy-benzoic acid Natural products OC(=O)C1=CC=C(O)C=C1 FJKROLUGYXJWQN-UHFFFAOYSA-N 0.000 description 14
- 229960004889 salicylic acid Drugs 0.000 description 14
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 13
- 230000001681 protective effect Effects 0.000 description 13
- 230000002285 radioactive effect Effects 0.000 description 13
- KBPLFHHGFOOTCA-UHFFFAOYSA-N 1-Octanol Chemical compound CCCCCCCCO KBPLFHHGFOOTCA-UHFFFAOYSA-N 0.000 description 12
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 description 12
- VREFGVBLTWBCJP-UHFFFAOYSA-N alprazolam Chemical compound C12=CC(Cl)=CC=C2N2C(C)=NN=C2CN=C1C1=CC=CC=C1 VREFGVBLTWBCJP-UHFFFAOYSA-N 0.000 description 11
- 239000011734 sodium Substances 0.000 description 11
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 10
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 10
- 210000004369 blood Anatomy 0.000 description 10
- 239000008280 blood Substances 0.000 description 10
- 210000002682 neurofibrillary tangle Anatomy 0.000 description 10
- 150000002989 phenols Chemical class 0.000 description 10
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 9
- 206010059245 Angiopathy Diseases 0.000 description 9
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 9
- 241001465754 Metazoa Species 0.000 description 9
- ZMANZCXQSJIPKH-UHFFFAOYSA-N Triethylamine Chemical compound CCN(CC)CC ZMANZCXQSJIPKH-UHFFFAOYSA-N 0.000 description 9
- 230000002490 cerebral effect Effects 0.000 description 9
- 238000001514 detection method Methods 0.000 description 9
- 210000004185 liver Anatomy 0.000 description 9
- 229960001047 methyl salicylate Drugs 0.000 description 9
- 239000002243 precursor Substances 0.000 description 9
- 230000001225 therapeutic effect Effects 0.000 description 9
- 150000001448 anilines Chemical class 0.000 description 8
- 239000000047 product Substances 0.000 description 8
- 238000000159 protein binding assay Methods 0.000 description 8
- 239000002904 solvent Substances 0.000 description 8
- 230000002123 temporal effect Effects 0.000 description 8
- 238000011282 treatment Methods 0.000 description 8
- 230000002776 aggregation Effects 0.000 description 7
- 238000004220 aggregation Methods 0.000 description 7
- 230000000875 corresponding effect Effects 0.000 description 7
- 150000008049 diazo compounds Chemical class 0.000 description 7
- 239000000975 dye Substances 0.000 description 7
- 238000005192 partition Methods 0.000 description 7
- 102000004196 processed proteins & peptides Human genes 0.000 description 7
- 239000011780 sodium chloride Substances 0.000 description 7
- HRDVSBWJVVGOAT-UHFFFAOYSA-N 4-(4-aminophenyl)-2-bromoaniline Chemical group C1=CC(N)=CC=C1C1=CC=C(N)C(Br)=C1 HRDVSBWJVVGOAT-UHFFFAOYSA-N 0.000 description 6
- 241000013355 Mycteroperca interstitialis Species 0.000 description 6
- KDLHZDBZIXYQEI-UHFFFAOYSA-N Palladium Chemical compound [Pd] KDLHZDBZIXYQEI-UHFFFAOYSA-N 0.000 description 6
- DNIAPMSPPWPWGF-UHFFFAOYSA-N Propylene glycol Chemical compound CC(O)CO DNIAPMSPPWPWGF-UHFFFAOYSA-N 0.000 description 6
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 6
- 230000032683 aging Effects 0.000 description 6
- 230000007423 decrease Effects 0.000 description 6
- 230000008021 deposition Effects 0.000 description 6
- 239000003814 drug Substances 0.000 description 6
- 238000002474 experimental method Methods 0.000 description 6
- 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 6
- 230000005764 inhibitory process Effects 0.000 description 6
- 239000010410 layer Substances 0.000 description 6
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 description 6
- VLKZOEOYAKHREP-UHFFFAOYSA-N n-Hexane Chemical compound CCCCCC VLKZOEOYAKHREP-UHFFFAOYSA-N 0.000 description 6
- 238000002360 preparation method Methods 0.000 description 6
- 229940058287 salicylic acid derivative anticestodals Drugs 0.000 description 6
- 150000003872 salicylic acid derivatives Chemical class 0.000 description 6
- 238000011830 transgenic mouse model Methods 0.000 description 6
- UAIUNKRWKOVEES-UHFFFAOYSA-N 3,3',5,5'-tetramethylbenzidine Chemical class CC1=C(N)C(C)=CC(C=2C=C(C)C(N)=C(C)C=2)=C1 UAIUNKRWKOVEES-UHFFFAOYSA-N 0.000 description 5
- 206010029350 Neurotoxicity Diseases 0.000 description 5
- 206010044221 Toxic encephalopathy Diseases 0.000 description 5
- 125000000304 alkynyl group Chemical group 0.000 description 5
- 150000001412 amines Chemical class 0.000 description 5
- 239000007864 aqueous solution Substances 0.000 description 5
- 125000003118 aryl group Chemical group 0.000 description 5
- 238000003556 assay Methods 0.000 description 5
- 230000001413 cellular effect Effects 0.000 description 5
- 230000008878 coupling Effects 0.000 description 5
- 238000010168 coupling process Methods 0.000 description 5
- 238000005859 coupling reaction Methods 0.000 description 5
- ZUOUZKKEUPVFJK-UHFFFAOYSA-N diphenyl Chemical group C1=CC=CC=C1C1=CC=CC=C1 ZUOUZKKEUPVFJK-UHFFFAOYSA-N 0.000 description 5
- 150000002148 esters Chemical class 0.000 description 5
- NIHNNTQXNPWCJQ-UHFFFAOYSA-N fluorene Chemical compound C1=CC=C2CC3=CC=CC=C3C2=C1 NIHNNTQXNPWCJQ-UHFFFAOYSA-N 0.000 description 5
- 210000005153 frontal cortex Anatomy 0.000 description 5
- 238000011534 incubation Methods 0.000 description 5
- 230000003993 interaction Effects 0.000 description 5
- 238000006192 iodination reaction Methods 0.000 description 5
- 125000002346 iodo group Chemical group I* 0.000 description 5
- 238000005259 measurement Methods 0.000 description 5
- 239000000693 micelle Substances 0.000 description 5
- 231100000228 neurotoxicity Toxicity 0.000 description 5
- 230000007135 neurotoxicity Effects 0.000 description 5
- 238000013391 scatchard analysis Methods 0.000 description 5
- 230000009870 specific binding Effects 0.000 description 5
- 238000010186 staining Methods 0.000 description 5
- QLDQYRDCPNBPII-UHFFFAOYSA-N 1,2-benzoxazol-3-one Chemical class C1=CC=C2C(O)=NOC2=C1 QLDQYRDCPNBPII-UHFFFAOYSA-N 0.000 description 4
- IQUPABOKLQSFBK-UHFFFAOYSA-N 2-nitrophenol Chemical class OC1=CC=CC=C1[N+]([O-])=O IQUPABOKLQSFBK-UHFFFAOYSA-N 0.000 description 4
- PVFOHMXILQEIHX-UHFFFAOYSA-N 8-[(6-bromo-1,3-benzodioxol-5-yl)sulfanyl]-9-[2-(2-bromophenyl)ethyl]purin-6-amine Chemical compound C=1C=2OCOC=2C=C(Br)C=1SC1=NC=2C(N)=NC=NC=2N1CCC1=CC=CC=C1Br PVFOHMXILQEIHX-UHFFFAOYSA-N 0.000 description 4
- 102000001049 Amyloid Human genes 0.000 description 4
- 108010094108 Amyloid Proteins 0.000 description 4
- 241000557626 Corvus corax Species 0.000 description 4
- OAKJQQAXSVQMHS-UHFFFAOYSA-N Hydrazine Chemical compound NN OAKJQQAXSVQMHS-UHFFFAOYSA-N 0.000 description 4
- 241000699666 Mus <mouse, genus> Species 0.000 description 4
- 238000005481 NMR spectroscopy Methods 0.000 description 4
- HCHKCACWOHOZIP-UHFFFAOYSA-N Zinc Chemical compound [Zn] HCHKCACWOHOZIP-UHFFFAOYSA-N 0.000 description 4
- 229960000583 acetic acid Drugs 0.000 description 4
- 230000002378 acidificating effect Effects 0.000 description 4
- 238000010171 animal model Methods 0.000 description 4
- 238000000376 autoradiography Methods 0.000 description 4
- 239000004305 biphenyl Substances 0.000 description 4
- 125000006267 biphenyl group Chemical group 0.000 description 4
- 230000000711 cancerogenic effect Effects 0.000 description 4
- 210000000349 chromosome Anatomy 0.000 description 4
- 239000010432 diamond Substances 0.000 description 4
- USIUVYZYUHIAEV-UHFFFAOYSA-N diphenyl ether Chemical compound C=1C=CC=CC=1OC1=CC=CC=C1 USIUVYZYUHIAEV-UHFFFAOYSA-N 0.000 description 4
- 239000012530 fluid Substances 0.000 description 4
- 125000004428 fluoroalkoxy group Chemical class 0.000 description 4
- 230000002209 hydrophobic effect Effects 0.000 description 4
- 238000002347 injection Methods 0.000 description 4
- 239000007924 injection Substances 0.000 description 4
- 210000003734 kidney Anatomy 0.000 description 4
- 229920002521 macromolecule Polymers 0.000 description 4
- 230000007246 mechanism Effects 0.000 description 4
- 230000004060 metabolic process Effects 0.000 description 4
- OJURWUUOVGOHJZ-UHFFFAOYSA-N methyl 2-[(2-acetyloxyphenyl)methyl-[2-[(2-acetyloxyphenyl)methyl-(2-methoxy-2-oxoethyl)amino]ethyl]amino]acetate Chemical class C=1C=CC=C(OC(C)=O)C=1CN(CC(=O)OC)CCN(CC(=O)OC)CC1=CC=CC=C1OC(C)=O OJURWUUOVGOHJZ-UHFFFAOYSA-N 0.000 description 4
- 238000007069 methylation reaction Methods 0.000 description 4
- 230000035772 mutation Effects 0.000 description 4
- 230000007171 neuropathology Effects 0.000 description 4
- 210000000056 organ Anatomy 0.000 description 4
- 230000000144 pharmacologic effect Effects 0.000 description 4
- 208000028591 pheochromocytoma Diseases 0.000 description 4
- XKJCHHZQLQNZHY-UHFFFAOYSA-N phthalimide Chemical compound C1=CC=C2C(=O)NC(=O)C2=C1 XKJCHHZQLQNZHY-UHFFFAOYSA-N 0.000 description 4
- 239000003755 preservative agent Substances 0.000 description 4
- 238000003756 stirring Methods 0.000 description 4
- 238000006467 substitution reaction Methods 0.000 description 4
- 239000000725 suspension Substances 0.000 description 4
- 239000003981 vehicle Substances 0.000 description 4
- KTZQTRPPVKQPFO-UHFFFAOYSA-N 1,2-benzoxazole Chemical compound C1=CC=C2C=NOC2=C1 KTZQTRPPVKQPFO-UHFFFAOYSA-N 0.000 description 3
- KGLPWQKSKUVKMJ-UHFFFAOYSA-N 2,3-dihydrophthalazine-1,4-dione Chemical compound C1=CC=C2C(=O)NNC(=O)C2=C1 KGLPWQKSKUVKMJ-UHFFFAOYSA-N 0.000 description 3
- NEAQRZUHTPSBBM-UHFFFAOYSA-N 2-hydroxy-3,3-dimethyl-7-nitro-4h-isoquinolin-1-one Chemical compound C1=C([N+]([O-])=O)C=C2C(=O)N(O)C(C)(C)CC2=C1 NEAQRZUHTPSBBM-UHFFFAOYSA-N 0.000 description 3
- BRARRAHGNDUELT-UHFFFAOYSA-N 3-hydroxypicolinic acid Chemical compound OC(=O)C1=NC=CC=C1O BRARRAHGNDUELT-UHFFFAOYSA-N 0.000 description 3
- CCTOEAMRIIXGDJ-UHFFFAOYSA-N 4-hydroxy-2-benzofuran-1,3-dione Chemical compound OC1=CC=CC2=C1C(=O)OC2=O CCTOEAMRIIXGDJ-UHFFFAOYSA-N 0.000 description 3
- RUNIWHUHDQHEBB-UHFFFAOYSA-N 9-methylcarbazole-2,7-diamine Chemical compound C1=C(N)C=C2N(C)C3=CC(N)=CC=C3C2=C1 RUNIWHUHDQHEBB-UHFFFAOYSA-N 0.000 description 3
- UJOBWOGCFQCDNV-UHFFFAOYSA-N 9H-carbazole Chemical compound C1=CC=C2C3=CC=CC=C3NC2=C1 UJOBWOGCFQCDNV-UHFFFAOYSA-N 0.000 description 3
- WFDIJRYMOXRFFG-UHFFFAOYSA-N Acetic anhydride Chemical compound CC(=O)OC(C)=O WFDIJRYMOXRFFG-UHFFFAOYSA-N 0.000 description 3
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 3
- 241000894006 Bacteria Species 0.000 description 3
- 208000024806 Brain atrophy Diseases 0.000 description 3
- LCGLNKUTAGEVQW-UHFFFAOYSA-N Dimethyl ether Chemical compound COC LCGLNKUTAGEVQW-UHFFFAOYSA-N 0.000 description 3
- 241000282412 Homo Species 0.000 description 3
- AVXURJPOCDRRFD-UHFFFAOYSA-N Hydroxylamine Chemical compound ON AVXURJPOCDRRFD-UHFFFAOYSA-N 0.000 description 3
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 3
- 238000011887 Necropsy Methods 0.000 description 3
- 238000006350 Schiemann fluorination reaction Methods 0.000 description 3
- 230000005856 abnormality Effects 0.000 description 3
- 238000002835 absorbance Methods 0.000 description 3
- 230000006933 amyloid-beta aggregation Effects 0.000 description 3
- 238000004458 analytical method Methods 0.000 description 3
- 150000001504 aryl thiols Chemical class 0.000 description 3
- 238000011888 autopsy Methods 0.000 description 3
- 239000000987 azo dye Substances 0.000 description 3
- 230000008901 benefit Effects 0.000 description 3
- 235000010290 biphenyl Nutrition 0.000 description 3
- 239000005388 borosilicate glass Substances 0.000 description 3
- 231100000357 carcinogen Toxicity 0.000 description 3
- 239000003183 carcinogenic agent Substances 0.000 description 3
- 238000012512 characterization method Methods 0.000 description 3
- 238000012937 correction Methods 0.000 description 3
- 238000000354 decomposition reaction Methods 0.000 description 3
- 125000000664 diazo group Chemical group [N-]=[N+]=[*] 0.000 description 3
- 238000009826 distribution Methods 0.000 description 3
- 231100000673 dose–response relationship Toxicity 0.000 description 3
- 238000011156 evaluation Methods 0.000 description 3
- 238000001914 filtration Methods 0.000 description 3
- 229910052736 halogen Inorganic materials 0.000 description 3
- 150000002367 halogens Chemical class 0.000 description 3
- XMBWDFGMSWQBCA-UHFFFAOYSA-N hydrogen iodide Chemical compound I XMBWDFGMSWQBCA-UHFFFAOYSA-N 0.000 description 3
- 238000000338 in vitro Methods 0.000 description 3
- 230000000968 intestinal effect Effects 0.000 description 3
- 150000002496 iodine Chemical class 0.000 description 3
- INQOMBQAUSQDDS-UHFFFAOYSA-N iodomethane Chemical compound IC INQOMBQAUSQDDS-UHFFFAOYSA-N 0.000 description 3
- 210000004153 islets of langerhan Anatomy 0.000 description 3
- 231100000053 low toxicity Toxicity 0.000 description 3
- 239000003550 marker Substances 0.000 description 3
- 230000004048 modification Effects 0.000 description 3
- 238000012986 modification Methods 0.000 description 3
- 238000000302 molecular modelling Methods 0.000 description 3
- ARGDYOIRHYLIMT-UHFFFAOYSA-N n,n-dichloro-4-methylbenzenesulfonamide Chemical compound CC1=CC=C(S(=O)(=O)N(Cl)Cl)C=C1 ARGDYOIRHYLIMT-UHFFFAOYSA-N 0.000 description 3
- 210000002241 neurite Anatomy 0.000 description 3
- 238000002610 neuroimaging Methods 0.000 description 3
- 230000002887 neurotoxic effect Effects 0.000 description 3
- 231100001223 noncarcinogenic Toxicity 0.000 description 3
- 230000000269 nucleophilic effect Effects 0.000 description 3
- 125000001997 phenyl group Chemical group [H]C1=C([H])C([H])=C(*)C([H])=C1[H] 0.000 description 3
- 229920003023 plastic Polymers 0.000 description 3
- 239000004033 plastic Substances 0.000 description 3
- 230000003389 potentiating effect Effects 0.000 description 3
- 230000008569 process Effects 0.000 description 3
- 238000000163 radioactive labelling Methods 0.000 description 3
- 239000011541 reaction mixture Substances 0.000 description 3
- 229910000029 sodium carbonate Inorganic materials 0.000 description 3
- 238000010561 standard procedure Methods 0.000 description 3
- 230000007470 synaptic degeneration Effects 0.000 description 3
- 210000003478 temporal lobe Anatomy 0.000 description 3
- 238000012360 testing method Methods 0.000 description 3
- 238000003828 vacuum filtration Methods 0.000 description 3
- 239000011701 zinc Substances 0.000 description 3
- UXTZUUVTGMDXNG-UHFFFAOYSA-N 1,2-benzoxazine-3,4-dione Chemical compound C1=CC=C2C(=O)C(=O)NOC2=C1 UXTZUUVTGMDXNG-UHFFFAOYSA-N 0.000 description 2
- BZQYQHBLAIWZAC-UHFFFAOYSA-N 1h-2,3-benzoxazine Chemical compound C1=CC=C2CON=CC2=C1 BZQYQHBLAIWZAC-UHFFFAOYSA-N 0.000 description 2
- OYFRNYNHAZOYNF-UHFFFAOYSA-N 2,5-dihydroxyterephthalic acid Chemical compound OC(=O)C1=CC(O)=C(C(O)=O)C=C1O OYFRNYNHAZOYNF-UHFFFAOYSA-N 0.000 description 2
- YAUZDUXTENTYLR-UHFFFAOYSA-N 2,7-diaminofluoren-1-one Chemical compound NC1=CC=C2C3=CC=C(N)C(=O)C3=CC2=C1 YAUZDUXTENTYLR-UHFFFAOYSA-N 0.000 description 2
- XGAYQDWZIPRBPF-UHFFFAOYSA-N 2-hydroxy-3-propan-2-ylbenzoic acid Chemical class CC(C)C1=CC=CC(C(O)=O)=C1O XGAYQDWZIPRBPF-UHFFFAOYSA-N 0.000 description 2
- CHZCERSEMVWNHL-UHFFFAOYSA-N 2-hydroxybenzonitrile Chemical compound OC1=CC=CC=C1C#N CHZCERSEMVWNHL-UHFFFAOYSA-N 0.000 description 2
- HRGQNKFLJJXNHV-UHFFFAOYSA-N 3,6-dimethyl-5h-benzo[c]cinnoline-2,7-diamine Chemical compound C1=CC(N)=C2N(C)NC3=CC(C)=C(N)C=C3C2=C1 HRGQNKFLJJXNHV-UHFFFAOYSA-N 0.000 description 2
- AZKSAVLVSZKNRD-UHFFFAOYSA-M 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide Chemical compound [Br-].S1C(C)=C(C)N=C1[N+]1=NC(C=2C=CC=CC=2)=NN1C1=CC=CC=C1 AZKSAVLVSZKNRD-UHFFFAOYSA-M 0.000 description 2
- WHKZBVQIMVUGIH-UHFFFAOYSA-N 3-hydroxyquinoline-2-carboxylic acid Chemical compound C1=CC=C2C=C(O)C(C(=O)O)=NC2=C1 WHKZBVQIMVUGIH-UHFFFAOYSA-N 0.000 description 2
- FDCDAEYGQTVYOH-UHFFFAOYSA-N 4-(4-amino-5-methyl-2-nitrophenyl)-2-methyl-5-nitroaniline Chemical compound C1=C(N)C(C)=CC(C=2C(=CC(N)=C(C)C=2)[N+]([O-])=O)=C1[N+]([O-])=O FDCDAEYGQTVYOH-UHFFFAOYSA-N 0.000 description 2
- SFESZKFPZVUJJG-UHFFFAOYSA-N 4-(4-aminophenyl)-2-iodoaniline Chemical compound C1=CC(N)=CC=C1C1=CC=C(N)C(I)=C1 SFESZKFPZVUJJG-UHFFFAOYSA-N 0.000 description 2
- ZIPLRYYWAXPVMG-UHFFFAOYSA-N 4-oxo-1h-cinnoline-3-carboxylic acid Chemical class C1=CC=C2C(=O)C(C(=O)O)=NNC2=C1 ZIPLRYYWAXPVMG-UHFFFAOYSA-N 0.000 description 2
- IABMSFZLDSRRQZ-UHFFFAOYSA-N 4-oxo-1h-pyridazine-3-carboxylic acid Chemical class OC(=O)C1=NN=CC=C1O IABMSFZLDSRRQZ-UHFFFAOYSA-N 0.000 description 2
- JWPRICQKUNODPZ-UHFFFAOYSA-N 5-fluoro-2-hydroxybenzoic acid Chemical class OC(=O)C1=CC(F)=CC=C1O JWPRICQKUNODPZ-UHFFFAOYSA-N 0.000 description 2
- SNCJAJRILVFXAE-UHFFFAOYSA-N 9h-fluorene-2,7-diamine Chemical compound NC1=CC=C2C3=CC=C(N)C=C3CC2=C1 SNCJAJRILVFXAE-UHFFFAOYSA-N 0.000 description 2
- 102000009091 Amyloidogenic Proteins Human genes 0.000 description 2
- 108010048112 Amyloidogenic Proteins Proteins 0.000 description 2
- PAYRUJLWNCNPSJ-UHFFFAOYSA-N Aniline Chemical compound NC1=CC=CC=C1 PAYRUJLWNCNPSJ-UHFFFAOYSA-N 0.000 description 2
- XKRFYHLGVUSROY-UHFFFAOYSA-N Argon Chemical compound [Ar] XKRFYHLGVUSROY-UHFFFAOYSA-N 0.000 description 2
- 241000282472 Canis lupus familiaris Species 0.000 description 2
- IAZDPXIOMUYVGZ-WFGJKAKNSA-N Dimethyl sulfoxide Chemical compound [2H]C([2H])([2H])S(=O)C([2H])([2H])[2H] IAZDPXIOMUYVGZ-WFGJKAKNSA-N 0.000 description 2
- LVGKNOAMLMIIKO-UHFFFAOYSA-N Elaidinsaeure-aethylester Natural products CCCCCCCCC=CCCCCCCCC(=O)OCC LVGKNOAMLMIIKO-UHFFFAOYSA-N 0.000 description 2
- 206010018341 Gliosis Diseases 0.000 description 2
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 2
- 101000896985 Homo sapiens Carbonyl reductase [NADPH] 1 Proteins 0.000 description 2
- WTDHULULXKLSOZ-UHFFFAOYSA-N Hydroxylamine hydrochloride Chemical compound Cl.ON WTDHULULXKLSOZ-UHFFFAOYSA-N 0.000 description 2
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 description 2
- 241000124008 Mammalia Species 0.000 description 2
- LGDSHSYDSCRFAB-UHFFFAOYSA-N Methyl isothiocyanate Chemical compound CN=C=S LGDSHSYDSCRFAB-UHFFFAOYSA-N 0.000 description 2
- 241000699660 Mus musculus Species 0.000 description 2
- 102100022365 NAD(P)H dehydrogenase [quinone] 1 Human genes 0.000 description 2
- UFWIBTONFRDIAS-UHFFFAOYSA-N Naphthalene Chemical compound C1=CC=CC2=CC=CC=C21 UFWIBTONFRDIAS-UHFFFAOYSA-N 0.000 description 2
- 238000012879 PET imaging Methods 0.000 description 2
- 239000002202 Polyethylene glycol Substances 0.000 description 2
- JUJWROOIHBZHMG-UHFFFAOYSA-N Pyridine Chemical compound C1=CC=NC=C1 JUJWROOIHBZHMG-UHFFFAOYSA-N 0.000 description 2
- SMWDFEZZVXVKRB-UHFFFAOYSA-N Quinoline Chemical compound N1=CC=CC2=CC=CC=C21 SMWDFEZZVXVKRB-UHFFFAOYSA-N 0.000 description 2
- PXIPVTKHYLBLMZ-UHFFFAOYSA-N Sodium azide Chemical compound [Na+].[N-]=[N+]=[N-] PXIPVTKHYLBLMZ-UHFFFAOYSA-N 0.000 description 2
- NINIDFKCEFEMDL-UHFFFAOYSA-N Sulfur Chemical compound [S] NINIDFKCEFEMDL-UHFFFAOYSA-N 0.000 description 2
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 2
- 230000001476 alcoholic effect Effects 0.000 description 2
- 125000003342 alkenyl group Chemical group 0.000 description 2
- 230000029936 alkylation Effects 0.000 description 2
- 238000005804 alkylation reaction Methods 0.000 description 2
- 150000001413 amino acids Chemical group 0.000 description 2
- 239000003963 antioxidant agent Substances 0.000 description 2
- 235000006708 antioxidants Nutrition 0.000 description 2
- 239000008365 aqueous carrier Substances 0.000 description 2
- 239000003125 aqueous solvent Substances 0.000 description 2
- 150000001501 aryl fluorides Chemical class 0.000 description 2
- 150000001502 aryl halides Chemical class 0.000 description 2
- 108010066657 azoreductase Proteins 0.000 description 2
- 230000001580 bacterial effect Effects 0.000 description 2
- 230000003542 behavioural effect Effects 0.000 description 2
- XJHABGPPCLHLLV-UHFFFAOYSA-N benzo[de]isoquinoline-1,3-dione Chemical class C1=CC(C(=O)NC2=O)=C3C2=CC=CC3=C1 XJHABGPPCLHLLV-UHFFFAOYSA-N 0.000 description 2
- 239000000872 buffer Substances 0.000 description 2
- 244000309464 bull Species 0.000 description 2
- 150000004657 carbamic acid derivatives Chemical class 0.000 description 2
- 150000007942 carboxylates Chemical group 0.000 description 2
- 125000002843 carboxylic acid group Chemical group 0.000 description 2
- 231100000315 carcinogenic Toxicity 0.000 description 2
- OAYRYNVEFFWSHK-UHFFFAOYSA-N carsalam Chemical class C1=CC=C2OC(=O)NC(=O)C2=C1 OAYRYNVEFFWSHK-UHFFFAOYSA-N 0.000 description 2
- 230000007541 cellular toxicity Effects 0.000 description 2
- 210000001175 cerebrospinal fluid Anatomy 0.000 description 2
- 239000002738 chelating agent Substances 0.000 description 2
- 235000019878 cocoa butter replacer Nutrition 0.000 description 2
- 238000007796 conventional method Methods 0.000 description 2
- 230000002596 correlated effect Effects 0.000 description 2
- 230000003013 cytotoxicity Effects 0.000 description 2
- 231100000135 cytotoxicity Toxicity 0.000 description 2
- 230000003247 decreasing effect Effects 0.000 description 2
- 230000001419 dependent effect Effects 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 230000018109 developmental process Effects 0.000 description 2
- 239000008121 dextrose Substances 0.000 description 2
- 239000012954 diazonium Substances 0.000 description 2
- 150000001989 diazonium salts Chemical class 0.000 description 2
- IJGRMHOSHXDMSA-UHFFFAOYSA-O diazynium Chemical compound [NH+]#N IJGRMHOSHXDMSA-UHFFFAOYSA-O 0.000 description 2
- TXCDCPKCNAJMEE-UHFFFAOYSA-N dibenzofuran Chemical compound C1=CC=C2C3=CC=CC=C3OC2=C1 TXCDCPKCNAJMEE-UHFFFAOYSA-N 0.000 description 2
- IYYZUPMFVPLQIF-UHFFFAOYSA-N dibenzothiophene Chemical compound C1=CC=C2C3=CC=CC=C3SC2=C1 IYYZUPMFVPLQIF-UHFFFAOYSA-N 0.000 description 2
- JRCLMBQKVGYQAG-UHFFFAOYSA-N dibenzothiophene-2,7-diamine Chemical compound C1=C(N)C=C2C3=CC=C(N)C=C3SC2=C1 JRCLMBQKVGYQAG-UHFFFAOYSA-N 0.000 description 2
- LOKCTEFSRHRXRJ-UHFFFAOYSA-I dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride Chemical compound P(=O)(O)(O)[O-].[K+].P(=O)(O)([O-])[O-].[Na+].[Na+].[Cl-].[K+].[Cl-].[Na+] LOKCTEFSRHRXRJ-UHFFFAOYSA-I 0.000 description 2
- 238000006073 displacement reaction Methods 0.000 description 2
- 238000004090 dissolution Methods 0.000 description 2
- 238000010828 elution Methods 0.000 description 2
- 210000003743 erythrocyte Anatomy 0.000 description 2
- LVGKNOAMLMIIKO-QXMHVHEDSA-N ethyl oleate Chemical compound CCCCCCCC\C=C/CCCCCCCC(=O)OCC LVGKNOAMLMIIKO-QXMHVHEDSA-N 0.000 description 2
- 229940093471 ethyl oleate Drugs 0.000 description 2
- 125000003709 fluoroalkyl group Chemical group 0.000 description 2
- 239000012362 glacial acetic acid Substances 0.000 description 2
- 230000007387 gliosis Effects 0.000 description 2
- 238000010438 heat treatment Methods 0.000 description 2
- 239000001257 hydrogen Substances 0.000 description 2
- 238000003018 immunoassay Methods 0.000 description 2
- 238000005462 in vivo assay Methods 0.000 description 2
- 239000011261 inert gas Substances 0.000 description 2
- 239000000543 intermediate Substances 0.000 description 2
- 238000001990 intravenous administration Methods 0.000 description 2
- 230000026045 iodination Effects 0.000 description 2
- HCZHHEIFKROPDY-UHFFFAOYSA-N kynurenic acid Chemical compound C1=CC=C2NC(C(=O)O)=CC(=O)C2=C1 HCZHHEIFKROPDY-UHFFFAOYSA-N 0.000 description 2
- 229910052751 metal Inorganic materials 0.000 description 2
- 239000002184 metal Substances 0.000 description 2
- BDAGIHXWWSANSR-UHFFFAOYSA-N methanoic acid Natural products OC=O BDAGIHXWWSANSR-UHFFFAOYSA-N 0.000 description 2
- 125000000956 methoxy group Chemical class [H]C([H])([H])O* 0.000 description 2
- ZGKOISWXZKBKQB-UHFFFAOYSA-N methyl 2-hydroxy-5-[[4-[4-[(4-hydroxy-3-methoxycarbonylphenyl)diazenyl]phenyl]phenyl]diazenyl]benzoate Chemical group C1=C(O)C(C(=O)OC)=CC(N=NC=2C=CC(=CC=2)C=2C=CC(=CC=2)N=NC=2C=C(C(O)=CC=2)C(=O)OC)=C1 ZGKOISWXZKBKQB-UHFFFAOYSA-N 0.000 description 2
- 150000004702 methyl esters Chemical class 0.000 description 2
- 230000001035 methylating effect Effects 0.000 description 2
- 238000012544 monitoring process Methods 0.000 description 2
- 230000000877 morphologic effect Effects 0.000 description 2
- 230000004770 neurodegeneration Effects 0.000 description 2
- 231100000189 neurotoxic Toxicity 0.000 description 2
- 150000002832 nitroso derivatives Chemical class 0.000 description 2
- 230000002352 nonmutagenic effect Effects 0.000 description 2
- 239000012038 nucleophile Substances 0.000 description 2
- 235000015097 nutrients Nutrition 0.000 description 2
- 150000002895 organic esters Chemical class 0.000 description 2
- 239000012044 organic layer Substances 0.000 description 2
- 150000004866 oxadiazoles Chemical class 0.000 description 2
- 229910052763 palladium Inorganic materials 0.000 description 2
- 230000001936 parietal effect Effects 0.000 description 2
- 230000007310 pathophysiology Effects 0.000 description 2
- 239000000546 pharmaceutical excipient Substances 0.000 description 2
- 239000008363 phosphate buffer Substances 0.000 description 2
- 239000002953 phosphate buffered saline Substances 0.000 description 2
- 229920001223 polyethylene glycol Polymers 0.000 description 2
- 231100000175 potential carcinogenicity Toxicity 0.000 description 2
- 238000011002 quantification Methods 0.000 description 2
- 230000005855 radiation Effects 0.000 description 2
- 102000005962 receptors Human genes 0.000 description 2
- 238000001953 recrystallisation Methods 0.000 description 2
- 230000002829 reductive effect Effects 0.000 description 2
- 238000004007 reversed phase HPLC Methods 0.000 description 2
- 238000012552 review Methods 0.000 description 2
- 231100000161 signs of toxicity Toxicity 0.000 description 2
- 239000000741 silica gel Substances 0.000 description 2
- 229910002027 silica gel Inorganic materials 0.000 description 2
- 239000000377 silicon dioxide Substances 0.000 description 2
- LPXPTNMVRIOKMN-UHFFFAOYSA-M sodium nitrite Chemical compound [Na+].[O-]N=O LPXPTNMVRIOKMN-UHFFFAOYSA-M 0.000 description 2
- 239000012064 sodium phosphate buffer Substances 0.000 description 2
- CRWJEUDFKNYSBX-UHFFFAOYSA-N sodium;hypobromite Chemical compound [Na+].Br[O-] CRWJEUDFKNYSBX-UHFFFAOYSA-N 0.000 description 2
- 239000000758 substrate Substances 0.000 description 2
- 125000000542 sulfonic acid group Chemical group 0.000 description 2
- 229910052717 sulfur Inorganic materials 0.000 description 2
- 239000011593 sulfur Substances 0.000 description 2
- 238000002560 therapeutic procedure Methods 0.000 description 2
- 125000002088 tosyl group Chemical group [H]C1=C([H])C(=C([H])C([H])=C1C([H])([H])[H])S(*)(=O)=O 0.000 description 2
- 231100000331 toxic Toxicity 0.000 description 2
- 230000002588 toxic effect Effects 0.000 description 2
- ITMCEJHCFYSIIV-UHFFFAOYSA-M triflate Chemical group [O-]S(=O)(=O)C(F)(F)F ITMCEJHCFYSIIV-UHFFFAOYSA-M 0.000 description 2
- GETQZCLCWQTVFV-UHFFFAOYSA-N trimethylamine Chemical compound CN(C)C GETQZCLCWQTVFV-UHFFFAOYSA-N 0.000 description 2
- LWIHDJKSTIGBAC-UHFFFAOYSA-K tripotassium phosphate Chemical compound [K+].[K+].[K+].[O-]P([O-])([O-])=O LWIHDJKSTIGBAC-UHFFFAOYSA-K 0.000 description 2
- 238000002371 ultraviolet--visible spectrum Methods 0.000 description 2
- 235000015112 vegetable and seed oil Nutrition 0.000 description 2
- 239000008158 vegetable oil Substances 0.000 description 2
- 210000003462 vein Anatomy 0.000 description 2
- 125000000391 vinyl group Chemical group [H]C([*])=C([H])[H] 0.000 description 2
- 238000005406 washing Methods 0.000 description 2
- 229910052725 zinc Inorganic materials 0.000 description 2
- ULVUDWVWPYAUPI-UHFFFAOYSA-N (2-amino-5-nitrophenyl)-(3-nitrophenyl)methanone Chemical compound NC1=CC=C([N+]([O-])=O)C=C1C(=O)C1=CC=CC([N+]([O-])=O)=C1 ULVUDWVWPYAUPI-UHFFFAOYSA-N 0.000 description 1
- ULTMNUZMEYTTCU-UHFFFAOYSA-N (ethoxycarbonylamino) 2-(3-hydroxyphenyl)acetate Chemical class CCOC(=O)NOC(=O)CC1=CC=CC(O)=C1 ULTMNUZMEYTTCU-UHFFFAOYSA-N 0.000 description 1
- RYHBNJHYFVUHQT-UHFFFAOYSA-N 1,4-Dioxane Chemical compound C1COCCO1 RYHBNJHYFVUHQT-UHFFFAOYSA-N 0.000 description 1
- PIEXCQIOSMOEOU-UHFFFAOYSA-N 1-bromo-3-chloro-5,5-dimethylimidazolidine-2,4-dione Chemical class CC1(C)N(Br)C(=O)N(Cl)C1=O PIEXCQIOSMOEOU-UHFFFAOYSA-N 0.000 description 1
- HQJQYILBCQPYBI-UHFFFAOYSA-N 1-bromo-4-(4-bromophenyl)benzene Chemical group C1=CC(Br)=CC=C1C1=CC=C(Br)C=C1 HQJQYILBCQPYBI-UHFFFAOYSA-N 0.000 description 1
- SJJCQDRGABAVBB-UHFFFAOYSA-N 1-hydroxy-2-naphthoic acid Chemical compound C1=CC=CC2=C(O)C(C(=O)O)=CC=C21 SJJCQDRGABAVBB-UHFFFAOYSA-N 0.000 description 1
- KJUGUADJHNHALS-UHFFFAOYSA-N 1H-tetrazole Substances C=1N=NNN=1 KJUGUADJHNHALS-UHFFFAOYSA-N 0.000 description 1
- GLDQAMYCGOIJDV-UHFFFAOYSA-N 2,3-dihydroxybenzoic acid Chemical class OC(=O)C1=CC=CC(O)=C1O GLDQAMYCGOIJDV-UHFFFAOYSA-N 0.000 description 1
- AKEUNCKRJATALU-UHFFFAOYSA-N 2,6-dihydroxybenzoic acid Chemical class OC(=O)C1=C(O)C=CC=C1O AKEUNCKRJATALU-UHFFFAOYSA-N 0.000 description 1
- LJRSHKJYQVMZTL-UHFFFAOYSA-N 2,7-dinitro-9h-carbazole Chemical compound [O-][N+](=O)C1=CC=C2C3=CC=C([N+](=O)[O-])C=C3NC2=C1 LJRSHKJYQVMZTL-UHFFFAOYSA-N 0.000 description 1
- HDVGAFBXTXDYIB-UHFFFAOYSA-N 2,7-dinitrofluoren-9-one Chemical compound C1=C([N+]([O-])=O)C=C2C(=O)C3=CC([N+](=O)[O-])=CC=C3C2=C1 HDVGAFBXTXDYIB-UHFFFAOYSA-N 0.000 description 1
- FBFNMTGUOBUGFQ-UHFFFAOYSA-M 2-(2,5-diphenyltetrazol-1-ium-1-yl)-4,5-dimethyl-1,3-thiazole;bromide Chemical compound [Br-].S1C(C)=C(C)N=C1[N+]1=C(C=2C=CC=CC=2)N=NN1C1=CC=CC=C1 FBFNMTGUOBUGFQ-UHFFFAOYSA-M 0.000 description 1
- UEYQJQVBUVAELZ-UHFFFAOYSA-N 2-Hydroxynicotinic acid Chemical compound OC(=O)C1=CC=CN=C1O UEYQJQVBUVAELZ-UHFFFAOYSA-N 0.000 description 1
- IIQLVLWFQUUZII-UHFFFAOYSA-N 2-amino-5-(4-amino-3-carboxyphenyl)benzoic acid Chemical compound C1=C(C(O)=O)C(N)=CC=C1C1=CC=C(N)C(C(O)=O)=C1 IIQLVLWFQUUZII-UHFFFAOYSA-N 0.000 description 1
- DYIFXNDAYPEXGI-UHFFFAOYSA-N 2-hydroxy-3,4-diiodobenzoic acid Chemical class OC(=O)C1=CC=C(I)C(I)=C1O DYIFXNDAYPEXGI-UHFFFAOYSA-N 0.000 description 1
- PJLHEOAOXSHXLR-UHFFFAOYSA-N 2-hydroxy-3-iodobenzoic acid Chemical class OC(=O)C1=CC=CC(I)=C1O PJLHEOAOXSHXLR-UHFFFAOYSA-N 0.000 description 1
- AUJMZPGZDYABQC-UHFFFAOYSA-N 2-hydroxy-5-[[4-(4-hydroxyphenyl)phenyl]diazenyl]benzoic acid Chemical group C1=C(O)C(C(=O)O)=CC(N=NC=2C=CC(=CC=2)C=2C=CC(O)=CC=2)=C1 AUJMZPGZDYABQC-UHFFFAOYSA-N 0.000 description 1
- SWDNKOFGNPGRPI-UHFFFAOYSA-N 2-hydroxy-5-iodobenzoic acid Chemical compound OC(=O)C1=CC(I)=CC=C1O SWDNKOFGNPGRPI-UHFFFAOYSA-N 0.000 description 1
- YGSDEFSMJLZEOE-WGGUOBTBSA-N 2-hydroxybenzoic acid Chemical compound OC1=CC=CC=C1[14C](O)=O YGSDEFSMJLZEOE-WGGUOBTBSA-N 0.000 description 1
- UPHOPMSGKZNELG-UHFFFAOYSA-N 2-hydroxynaphthalene-1-carboxylic acid Chemical class C1=CC=C2C(C(=O)O)=C(O)C=CC2=C1 UPHOPMSGKZNELG-UHFFFAOYSA-N 0.000 description 1
- HUWXDEQWWKGHRV-UHFFFAOYSA-N 3,3'-Dichlorobenzidine Chemical compound C1=C(Cl)C(N)=CC=C1C1=CC=C(N)C(Cl)=C1 HUWXDEQWWKGHRV-UHFFFAOYSA-N 0.000 description 1
- JRBJSXQPQWSCCF-UHFFFAOYSA-N 3,3'-Dimethoxybenzidine Chemical compound C1=C(N)C(OC)=CC(C=2C=C(OC)C(N)=CC=2)=C1 JRBJSXQPQWSCCF-UHFFFAOYSA-N 0.000 description 1
- NUIURNJTPRWVAP-UHFFFAOYSA-N 3,3'-Dimethylbenzidine Chemical compound C1=C(N)C(C)=CC(C=2C=C(C)C(N)=CC=2)=C1 NUIURNJTPRWVAP-UHFFFAOYSA-N 0.000 description 1
- QXGJCWSBOZXWOV-UHFFFAOYSA-N 3,4-dihydroxyphthalic acid Chemical class OC(=O)C1=CC=C(O)C(O)=C1C(O)=O QXGJCWSBOZXWOV-UHFFFAOYSA-N 0.000 description 1
- UDKMUSGWGWZJBZ-UHFFFAOYSA-N 3,6-dihydroxyphthalic acid Chemical compound OC(=O)C1=C(O)C=CC(O)=C1C(O)=O UDKMUSGWGWZJBZ-UHFFFAOYSA-N 0.000 description 1
- YMUHHTGNCDBGHB-UHFFFAOYSA-N 3,7-dinitrodibenzofuran Chemical compound [O-][N+](=O)C1=CC=C2C3=CC=C([N+](=O)[O-])C=C3OC2=C1 YMUHHTGNCDBGHB-UHFFFAOYSA-N 0.000 description 1
- VPQKTIGQQGJDGI-UHFFFAOYSA-N 3-[[4-[4-[(3-carboxy-5-fluoro-2-hydroxyphenyl)diazenyl]phenyl]phenyl]diazenyl]-5-fluoro-2-hydroxybenzoic acid Chemical group OC(=O)C1=CC(F)=CC(N=NC=2C=CC(=CC=2)C=2C=CC(=CC=2)N=NC=2C(=C(C(O)=O)C=C(F)C=2)O)=C1O VPQKTIGQQGJDGI-UHFFFAOYSA-N 0.000 description 1
- WCXJWELXGZYSRK-UHFFFAOYSA-N 3-[[4-[4-[(3-carboxyphenyl)diazenyl]phenyl]phenyl]diazenyl]benzoic acid Chemical group OC(=O)C1=CC=CC(N=NC=2C=CC(=CC=2)C=2C=CC(=CC=2)N=NC=2C=C(C=CC=2)C(O)=O)=C1 WCXJWELXGZYSRK-UHFFFAOYSA-N 0.000 description 1
- HFFADIIGXFRHGG-UHFFFAOYSA-N 3-[[7-[(3-carboxyphenyl)diazenyl]-9h-fluoren-2-yl]diazenyl]benzoic acid Chemical compound OC(=O)C1=CC=CC(N=NC=2C=C3C(C4=CC=C(C=C4C3)N=NC=3C=C(C=CC=3)C(O)=O)=CC=2)=C1 HFFADIIGXFRHGG-UHFFFAOYSA-N 0.000 description 1
- QBMSOLXNIBQCKE-UHFFFAOYSA-N 3-hydroxybenzene-1,2-dicarbohydrazide Chemical compound NNC(=O)C1=CC=CC(O)=C1C(=O)NN QBMSOLXNIBQCKE-UHFFFAOYSA-N 0.000 description 1
- FVMDYYGIDFPZAX-UHFFFAOYSA-N 3-hydroxyphenylacetic acid Chemical compound OC(=O)CC1=CC=CC(O)=C1 FVMDYYGIDFPZAX-UHFFFAOYSA-N 0.000 description 1
- AFPHTEQTJZKQAQ-UHFFFAOYSA-N 3-nitrobenzoic acid Chemical compound OC(=O)C1=CC=CC([N+]([O-])=O)=C1 AFPHTEQTJZKQAQ-UHFFFAOYSA-N 0.000 description 1
- XGVGBXVWAPHQAY-UHFFFAOYSA-N 3-nitrosobenzoic acid Chemical compound OC(=O)C1=CC=CC(N=O)=C1 XGVGBXVWAPHQAY-UHFFFAOYSA-N 0.000 description 1
- MZGVIIXFGJCRDR-UHFFFAOYSA-N 4,6-dihydroxybenzene-1,3-dicarboxylic acid Chemical compound OC(=O)C1=CC(C(O)=O)=C(O)C=C1O MZGVIIXFGJCRDR-UHFFFAOYSA-N 0.000 description 1
- OSWFIVFLDKOXQC-UHFFFAOYSA-N 4-(3-methoxyphenyl)aniline Chemical compound COC1=CC=CC(C=2C=CC(N)=CC=2)=C1 OSWFIVFLDKOXQC-UHFFFAOYSA-N 0.000 description 1
- FWOLORXQTIGHFX-UHFFFAOYSA-N 4-(4-amino-2,3,5,6-tetrafluorophenyl)-2,3,5,6-tetrafluoroaniline Chemical group FC1=C(F)C(N)=C(F)C(F)=C1C1=C(F)C(F)=C(N)C(F)=C1F FWOLORXQTIGHFX-UHFFFAOYSA-N 0.000 description 1
- QWUSZGIKGARVEC-UHFFFAOYSA-N 4-(4-amino-3-bromophenyl)-2-bromoaniline Chemical compound C1=C(Br)C(N)=CC=C1C1=CC=C(N)C(Br)=C1 QWUSZGIKGARVEC-UHFFFAOYSA-N 0.000 description 1
- IKWZVCLCVMQAMF-UHFFFAOYSA-N 4-(4-amino-3-iodophenyl)-2-iodoaniline Chemical compound C1=C(I)C(N)=CC=C1C1=CC=C(N)C(I)=C1 IKWZVCLCVMQAMF-UHFFFAOYSA-N 0.000 description 1
- OCEINMLGYDSKFW-UHFFFAOYSA-N 4-(4-amino-3-nitrophenyl)-2-nitroaniline Chemical compound C1=C([N+]([O-])=O)C(N)=CC=C1C1=CC=C(N)C([N+]([O-])=O)=C1 OCEINMLGYDSKFW-UHFFFAOYSA-N 0.000 description 1
- NMPKGVLDQYRYMC-UHFFFAOYSA-N 4-[[4-[4-[(3-carboxy-4-hydroxynaphthalen-1-yl)diazenyl]-3-iodophenyl]phenyl]diazenyl]-1-hydroxynaphthalene-2-carboxylic acid Chemical group C1=CC=CC2=C(O)C(C(=O)O)=CC(N=NC=3C=CC(=CC=3)C=3C=C(I)C(N=NC=4C5=CC=CC=C5C(O)=C(C(O)=O)C=4)=CC=3)=C21 NMPKGVLDQYRYMC-UHFFFAOYSA-N 0.000 description 1
- SDNFLSIPGYWAGP-UHFFFAOYSA-N 4-[[4-[4-[(4-hydroxy-3-nitrophenyl)diazenyl]-3-iodophenyl]phenyl]diazenyl]-2-nitrophenol Chemical group C1=C([N+]([O-])=O)C(O)=CC=C1N=NC1=CC=C(C=2C=C(I)C(N=NC=3C=C(C(O)=CC=3)[N+]([O-])=O)=CC=2)C=C1 SDNFLSIPGYWAGP-UHFFFAOYSA-N 0.000 description 1
- XXYNZSATHOXXBJ-UHFFFAOYSA-N 4-hydroxyisoindole-1,3-dione Chemical compound OC1=CC=CC2=C1C(=O)NC2=O XXYNZSATHOXXBJ-UHFFFAOYSA-N 0.000 description 1
- RZQPSPDOJRHMNF-PLNGDYQASA-N 4-nitro-2-[(z)-2-(3-nitrophenyl)ethenyl]aniline Chemical compound NC1=CC=C([N+]([O-])=O)C=C1\C=C/C1=CC=CC([N+]([O-])=O)=C1 RZQPSPDOJRHMNF-PLNGDYQASA-N 0.000 description 1
- QYGHXDAYBIFGKI-UHFFFAOYSA-N 4-oxo-1h-pyrimidine-6-carboxylic acid Chemical compound OC(=O)C1=CC(=O)N=CN1 QYGHXDAYBIFGKI-UHFFFAOYSA-N 0.000 description 1
- SONKTIFDCXIZKZ-UHFFFAOYSA-N 5-[2-[4-[4-[2-(3-carboxy-4-methoxyphenyl)ethynyl]phenyl]phenyl]ethynyl]-2-methoxybenzoic acid Chemical group C1=C(C(O)=O)C(OC)=CC=C1C#CC1=CC=C(C=2C=CC(=CC=2)C#CC=2C=C(C(OC)=CC=2)C(O)=O)C=C1 SONKTIFDCXIZKZ-UHFFFAOYSA-N 0.000 description 1
- ICNLTGTXJKJXGX-UHFFFAOYSA-N 5-[[4-[4-[(3-carboxy-4-hydroxy-5-iodophenyl)diazenyl]phenyl]phenyl]diazenyl]-2-hydroxy-3-iodobenzoic acid Chemical group IC1=C(O)C(C(=O)O)=CC(N=NC=2C=CC(=CC=2)C=2C=CC(=CC=2)N=NC=2C=C(C(O)=C(I)C=2)C(O)=O)=C1 ICNLTGTXJKJXGX-UHFFFAOYSA-N 0.000 description 1
- GUXMZPMAIGGGJT-UHFFFAOYSA-N 5-[[4-[4-[(3-carboxy-4-hydroxy-5-propan-2-ylphenyl)diazenyl]-3-iodophenyl]phenyl]diazenyl]-2-hydroxy-3-propan-2-ylbenzoic acid Chemical group OC(=O)C1=C(O)C(C(C)C)=CC(N=NC=2C=CC(=CC=2)C=2C=C(I)C(N=NC=3C=C(C(O)=C(C(C)C)C=3)C(O)=O)=CC=2)=C1 GUXMZPMAIGGGJT-UHFFFAOYSA-N 0.000 description 1
- AEDPLXWALMHGRC-UHFFFAOYSA-N 5-[[4-[4-[(3-carboxy-4-hydroxyphenyl)diazenyl]-3,5-dimethylphenyl]-2,6-dimethylphenyl]diazenyl]-2-hydroxybenzoic acid Chemical group CC1=CC(C=2C=C(C)C(N=NC=3C=C(C(O)=CC=3)C(O)=O)=C(C)C=2)=CC(C)=C1N=NC1=CC=C(O)C(C(O)=O)=C1 AEDPLXWALMHGRC-UHFFFAOYSA-N 0.000 description 1
- RPYTULQHYJIBBG-UHFFFAOYSA-N 5-[[4-[4-[(5-carboxy-6-oxo-1H-pyridin-3-yl)diazenyl]-3-iodophenyl]phenyl]diazenyl]-2-oxo-1H-pyridine-3-carboxylic acid Chemical group N1=C(O)C(C(=O)O)=CC(N=NC=2C=CC(=CC=2)C=2C=C(I)C(N=NC=3C=C(C(O)=NC=3)C(O)=O)=CC=2)=C1 RPYTULQHYJIBBG-UHFFFAOYSA-N 0.000 description 1
- MBJAPGAZEWPEFB-UHFFFAOYSA-N 5-amino-2-(4-amino-2-sulfophenyl)benzenesulfonic acid Chemical group OS(=O)(=O)C1=CC(N)=CC=C1C1=CC=C(N)C=C1S(O)(=O)=O MBJAPGAZEWPEFB-UHFFFAOYSA-N 0.000 description 1
- MYWRHUAHJSRQDG-UHFFFAOYSA-N 5-bromo-6-hydroxy-5-propan-2-ylcyclohexa-1,3-diene-1-carboxylic acid Chemical class CC(C)C1(Br)C=CC=C(C(O)=O)C1O MYWRHUAHJSRQDG-UHFFFAOYSA-N 0.000 description 1
- CVIZZIRBZYUMJV-UHFFFAOYSA-N 5-hydroxyquinoxaline-6-carboxylic acid Chemical compound N1=CC=NC2=C(O)C(C(=O)O)=CC=C21 CVIZZIRBZYUMJV-UHFFFAOYSA-N 0.000 description 1
- BLHCMGRVFXRYRN-UHFFFAOYSA-N 6-hydroxynicotinic acid Chemical compound OC(=O)C1=CC=C(O)N=C1 BLHCMGRVFXRYRN-UHFFFAOYSA-N 0.000 description 1
- ZCYVEMRRCGMTRW-UHFFFAOYSA-N 7553-56-2 Chemical compound [I] ZCYVEMRRCGMTRW-UHFFFAOYSA-N 0.000 description 1
- SVERGYVLDUAOMN-UHFFFAOYSA-N 9-methyl-2,7-dinitrocarbazole Chemical compound C1=C([N+]([O-])=O)C=C2N(C)C3=CC([N+]([O-])=O)=CC=C3C2=C1 SVERGYVLDUAOMN-UHFFFAOYSA-N 0.000 description 1
- 208000000044 Amnesia Diseases 0.000 description 1
- 102000013455 Amyloid beta-Peptides Human genes 0.000 description 1
- 108010090849 Amyloid beta-Peptides Proteins 0.000 description 1
- 101710095339 Apolipoprotein E Proteins 0.000 description 1
- 102100029470 Apolipoprotein E Human genes 0.000 description 1
- 230000007324 Aβ metabolism Effects 0.000 description 1
- 241000212384 Bifora Species 0.000 description 1
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 1
- 241000282461 Canis lupus Species 0.000 description 1
- KXDHJXZQYSOELW-UHFFFAOYSA-M Carbamate Chemical compound NC([O-])=O KXDHJXZQYSOELW-UHFFFAOYSA-M 0.000 description 1
- 208000005623 Carcinogenesis Diseases 0.000 description 1
- 241000700198 Cavia Species 0.000 description 1
- 102000000844 Cell Surface Receptors Human genes 0.000 description 1
- 108010001857 Cell Surface Receptors Proteins 0.000 description 1
- 241000282693 Cercopithecidae Species 0.000 description 1
- 238000005821 Claisen rearrangement reaction Methods 0.000 description 1
- 102000000989 Complement System Proteins Human genes 0.000 description 1
- 108010069112 Complement System Proteins Proteins 0.000 description 1
- 229910021589 Copper(I) bromide Inorganic materials 0.000 description 1
- 229910021591 Copper(I) chloride Inorganic materials 0.000 description 1
- QOSSAOTZNIDXMA-UHFFFAOYSA-N Dicylcohexylcarbodiimide Chemical compound C1CCCCC1N=C=NC1CCCCC1 QOSSAOTZNIDXMA-UHFFFAOYSA-N 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- QUSNBJAOOMFDIB-UHFFFAOYSA-N Ethylamine Chemical compound CCN QUSNBJAOOMFDIB-UHFFFAOYSA-N 0.000 description 1
- 229910004039 HBF4 Inorganic materials 0.000 description 1
- 108091006905 Human Serum Albumin Proteins 0.000 description 1
- 102000008100 Human Serum Albumin Human genes 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
- COLNVLDHVKWLRT-QMMMGPOBSA-N L-phenylalanine Chemical compound OC(=O)[C@@H](N)CC1=CC=CC=C1 COLNVLDHVKWLRT-QMMMGPOBSA-N 0.000 description 1
- 229910013354 LiB4 Inorganic materials 0.000 description 1
- 102000018697 Membrane Proteins Human genes 0.000 description 1
- 108010052285 Membrane Proteins Proteins 0.000 description 1
- 208000026139 Memory disease Diseases 0.000 description 1
- 208000001089 Multiple system atrophy Diseases 0.000 description 1
- 229910017971 NH4BF4 Inorganic materials 0.000 description 1
- 208000012902 Nervous system disease Diseases 0.000 description 1
- DTOVRIGQWTUCKY-UHFFFAOYSA-N OC(=O)C1=C(O)C=CC=C1O.COC(=O)C1=C(O)C=CC=C1O Chemical compound OC(=O)C1=C(O)C=CC=C1O.COC(=O)C1=C(O)C=CC=C1O DTOVRIGQWTUCKY-UHFFFAOYSA-N 0.000 description 1
- 102000012288 Phosphopyruvate Hydratase Human genes 0.000 description 1
- 108010022181 Phosphopyruvate Hydratase Proteins 0.000 description 1
- 239000012980 RPMI-1640 medium Substances 0.000 description 1
- 238000000297 Sandmeyer reaction Methods 0.000 description 1
- 108010071390 Serum Albumin Proteins 0.000 description 1
- 102000007562 Serum Albumin Human genes 0.000 description 1
- BQCADISMDOOEFD-UHFFFAOYSA-N Silver Chemical compound [Ag] BQCADISMDOOEFD-UHFFFAOYSA-N 0.000 description 1
- GKLVYJBZJHMRIY-OUBTZVSYSA-N Technetium-99 Chemical compound [99Tc] GKLVYJBZJHMRIY-OUBTZVSYSA-N 0.000 description 1
- 229920004890 Triton X-100 Polymers 0.000 description 1
- 239000013504 Triton X-100 Substances 0.000 description 1
- XUGUHTGSMPZQIW-UHFFFAOYSA-N [[4-(4-diazonioiminocyclohexa-2,5-dien-1-ylidene)cyclohexa-2,5-dien-1-ylidene]hydrazinylidene]azanide Chemical class C1=CC(N=[N+]=[N-])=CC=C1C1=CC=C(N=[N+]=[N-])C=C1 XUGUHTGSMPZQIW-UHFFFAOYSA-N 0.000 description 1
- 230000002159 abnormal effect Effects 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 238000009825 accumulation Methods 0.000 description 1
- 150000008065 acid anhydrides Chemical class 0.000 description 1
- 150000007513 acids Chemical class 0.000 description 1
- 230000007059 acute toxicity Effects 0.000 description 1
- 231100000403 acute toxicity Toxicity 0.000 description 1
- 150000001345 alkine derivatives Chemical class 0.000 description 1
- 230000004075 alteration Effects 0.000 description 1
- YDPLDMLRERBXAV-UHFFFAOYSA-N aluminum;triazide Chemical compound [Al+3].[N-]=[N+]=[N-].[N-]=[N+]=[N-].[N-]=[N+]=[N-] YDPLDMLRERBXAV-UHFFFAOYSA-N 0.000 description 1
- 150000001408 amides Chemical class 0.000 description 1
- 125000000539 amino acid group Chemical group 0.000 description 1
- 230000003466 anti-cipated effect Effects 0.000 description 1
- 230000000845 anti-microbial effect Effects 0.000 description 1
- 239000004599 antimicrobial Substances 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 229910052786 argon Inorganic materials 0.000 description 1
- 239000012300 argon atmosphere Substances 0.000 description 1
- 150000004982 aromatic amines Chemical class 0.000 description 1
- 150000001499 aryl bromides Chemical class 0.000 description 1
- 150000001503 aryl iodides Chemical class 0.000 description 1
- 210000001130 astrocyte Anatomy 0.000 description 1
- 125000004429 atom Chemical group 0.000 description 1
- 238000006149 azo coupling reaction Methods 0.000 description 1
- 230000004888 barrier function Effects 0.000 description 1
- XVJWBXACABRAEC-UHFFFAOYSA-N benzene;1,1'-biphenyl Chemical group C1=CC=CC=C1.C1=CC=CC=C1C1=CC=CC=C1 XVJWBXACABRAEC-UHFFFAOYSA-N 0.000 description 1
- 150000008316 benzisoxazoles Chemical class 0.000 description 1
- SWJXWSAKHXBQSY-UHFFFAOYSA-N benzo(c)cinnoline Chemical compound C1=CC=C2C3=CC=CC=C3N=NC2=C1 SWJXWSAKHXBQSY-UHFFFAOYSA-N 0.000 description 1
- 150000005130 benzoxazines Chemical class 0.000 description 1
- 238000009835 boiling Methods 0.000 description 1
- 238000005893 bromination reaction Methods 0.000 description 1
- 150000001649 bromium compounds Chemical class 0.000 description 1
- 210000004899 c-terminal region Anatomy 0.000 description 1
- 230000036952 cancer formation Effects 0.000 description 1
- 150000001716 carbazoles Chemical class 0.000 description 1
- 125000004432 carbon atom Chemical group C* 0.000 description 1
- 230000021523 carboxylation Effects 0.000 description 1
- 238000006473 carboxylation reaction Methods 0.000 description 1
- 231100000504 carcinogenesis Toxicity 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
- 210000003710 cerebral cortex Anatomy 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 238000001311 chemical methods and process Methods 0.000 description 1
- 150000001805 chlorine compounds Chemical class 0.000 description 1
- 239000002026 chloroform extract Substances 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- OQENBJBTQPIZKA-GTFORLLLSA-N chrysen-1-amine Chemical compound [14C]1(=CC=CC=2C3=CC=C4C=CC=CC4=C3C=CC1=2)N OQENBJBTQPIZKA-GTFORLLLSA-N 0.000 description 1
- WCZVZNOTHYJIEI-UHFFFAOYSA-N cinnoline Chemical class N1=NC=CC2=CC=CC=C21 WCZVZNOTHYJIEI-UHFFFAOYSA-N 0.000 description 1
- 238000003776 cleavage reaction Methods 0.000 description 1
- 230000007278 cognition impairment Effects 0.000 description 1
- 230000024203 complement activation Effects 0.000 description 1
- 238000010668 complexation reaction Methods 0.000 description 1
- 230000001010 compromised effect Effects 0.000 description 1
- 238000011970 concomitant therapy Methods 0.000 description 1
- OXBLHERUFWYNTN-UHFFFAOYSA-M copper(I) chloride Chemical compound [Cu]Cl OXBLHERUFWYNTN-UHFFFAOYSA-M 0.000 description 1
- 230000001054 cortical effect Effects 0.000 description 1
- 230000003412 degenerative effect Effects 0.000 description 1
- 230000003111 delayed effect Effects 0.000 description 1
- 230000002939 deleterious effect Effects 0.000 description 1
- 238000001212 derivatisation Methods 0.000 description 1
- 206010012601 diabetes mellitus Diseases 0.000 description 1
- 150000004891 diazines Chemical class 0.000 description 1
- QQSBWCNELOEITJ-UHFFFAOYSA-N dibenzofuran-3,7-diamine Chemical compound NC1=CC=C2C3=CC=C(N)C=C3OC2=C1 QQSBWCNELOEITJ-UHFFFAOYSA-N 0.000 description 1
- 150000004826 dibenzofurans Chemical class 0.000 description 1
- 150000005690 diesters Chemical class 0.000 description 1
- 125000000118 dimethyl group Chemical group [H]C([H])([H])* 0.000 description 1
- RBOUZBUAZMPIJV-UHFFFAOYSA-L disodium 5-[[4-[4-[(1-amino-4-sulfonatonaphthalen-2-yl)diazenyl]phenyl]phenyl]diazenyl]-2-hydroxybenzoate Chemical compound [Na+].[Na+].Nc1c(cc(c2ccccc12)S([O-])(=O)=O)N=Nc1ccc(cc1)-c1ccc(cc1)N=Nc1ccc(O)c(c1)C([O-])=O RBOUZBUAZMPIJV-UHFFFAOYSA-L 0.000 description 1
- 238000002224 dissection Methods 0.000 description 1
- 238000011833 dog model Methods 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 230000009977 dual effect Effects 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 238000011067 equilibration Methods 0.000 description 1
- HFTNNOZFRQLFQB-UHFFFAOYSA-N ethenoxy(trimethyl)silane Chemical compound C[Si](C)(C)OC=C HFTNNOZFRQLFQB-UHFFFAOYSA-N 0.000 description 1
- 239000002024 ethyl acetate extract Substances 0.000 description 1
- RIFGWPKJUGCATF-UHFFFAOYSA-N ethyl chloroformate Chemical compound CCOC(Cl)=O RIFGWPKJUGCATF-UHFFFAOYSA-N 0.000 description 1
- VMVZGGPZNHFGKS-UHFFFAOYSA-N ethyl n-(oxomethylidene)carbamate Chemical compound CCOC(=O)N=C=O VMVZGGPZNHFGKS-UHFFFAOYSA-N 0.000 description 1
- 238000001704 evaporation Methods 0.000 description 1
- 230000008020 evaporation Effects 0.000 description 1
- 230000001747 exhibiting effect Effects 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 239000011536 extraction buffer Substances 0.000 description 1
- 239000012091 fetal bovine serum Substances 0.000 description 1
- 239000000706 filtrate Substances 0.000 description 1
- YLQWCDOCJODRMT-UHFFFAOYSA-N fluoren-9-one Chemical compound C1=CC=C2C(=O)C3=CC=CC=C3C2=C1 YLQWCDOCJODRMT-UHFFFAOYSA-N 0.000 description 1
- IRXSLJNXXZKURP-UHFFFAOYSA-N fluorenylmethyloxycarbonyl chloride Chemical compound C1=CC=C2C(COC(=O)Cl)C3=CC=CC=C3C2=C1 IRXSLJNXXZKURP-UHFFFAOYSA-N 0.000 description 1
- KRHYYFGTRYWZRS-BJUDXGSMSA-M fluorine-18(1-) Chemical group [18F-] KRHYYFGTRYWZRS-BJUDXGSMSA-M 0.000 description 1
- 125000005816 fluoropropyl group Chemical group [H]C([H])(F)C([H])([H])C([H])([H])* 0.000 description 1
- 235000019253 formic acid Nutrition 0.000 description 1
- 238000004108 freeze drying Methods 0.000 description 1
- 125000000524 functional group Chemical group 0.000 description 1
- 239000007789 gas Substances 0.000 description 1
- 238000012252 genetic analysis Methods 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- 239000005337 ground glass Substances 0.000 description 1
- 125000005843 halogen group Chemical group 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- 230000002962 histologic effect Effects 0.000 description 1
- 230000007062 hydrolysis Effects 0.000 description 1
- 238000006460 hydrolysis reaction Methods 0.000 description 1
- 238000005470 impregnation Methods 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 239000012535 impurity Substances 0.000 description 1
- 239000003112 inhibitor Substances 0.000 description 1
- 239000007972 injectable composition Substances 0.000 description 1
- 210000000936 intestine Anatomy 0.000 description 1
- 238000007917 intracranial administration Methods 0.000 description 1
- 238000011835 investigation Methods 0.000 description 1
- 239000011630 iodine Substances 0.000 description 1
- 238000010983 kinetics study Methods 0.000 description 1
- 238000002372 labelling Methods 0.000 description 1
- 150000002605 large molecules Chemical class 0.000 description 1
- 230000003902 lesion Effects 0.000 description 1
- 239000003446 ligand Substances 0.000 description 1
- 150000002632 lipids Chemical class 0.000 description 1
- 150000002634 lipophilic molecules Chemical class 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- DLEDOFVPSDKWEF-UHFFFAOYSA-N lithium butane Chemical compound [Li+].CCC[CH2-] DLEDOFVPSDKWEF-UHFFFAOYSA-N 0.000 description 1
- 229910001496 lithium tetrafluoroborate Inorganic materials 0.000 description 1
- 230000004807 localization Effects 0.000 description 1
- 238000009593 lumbar puncture Methods 0.000 description 1
- 229910052943 magnesium sulfate Inorganic materials 0.000 description 1
- 235000019341 magnesium sulphate Nutrition 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 238000004949 mass spectrometry Methods 0.000 description 1
- 230000001404 mediated effect Effects 0.000 description 1
- 230000006984 memory degeneration Effects 0.000 description 1
- 208000023060 memory loss Diseases 0.000 description 1
- 239000002207 metabolite Substances 0.000 description 1
- 229910021645 metal ion Inorganic materials 0.000 description 1
- TZJOTDHMGVBGRS-UHFFFAOYSA-N methyl 2-hydroxy-3-propan-2-ylbenzoate Chemical compound COC(=O)C1=CC=CC(C(C)C)=C1O TZJOTDHMGVBGRS-UHFFFAOYSA-N 0.000 description 1
- SYOCHGYFOGQYNQ-UHFFFAOYSA-N methyl 2-hydroxy-5-[[4-[4-[(4-hydroxy-3-methoxycarbonylphenyl)diazenyl]-3-iodophenyl]phenyl]diazenyl]benzoate Chemical group C1=C(O)C(C(=O)OC)=CC(N=NC=2C=CC(=CC=2)C=2C=C(I)C(N=NC=3C=C(C(O)=CC=3)C(=O)OC)=CC=2)=C1 SYOCHGYFOGQYNQ-UHFFFAOYSA-N 0.000 description 1
- XVXCLDNUWBUICD-UHFFFAOYSA-N methyl 5-bromo-2-methoxybenzoate Chemical compound COC(=O)C1=CC(Br)=CC=C1OC XVXCLDNUWBUICD-UHFFFAOYSA-N 0.000 description 1
- KCLYALWNJLDDLR-UHFFFAOYSA-N methyl 5-ethynyl-2-methoxybenzoate Chemical compound COC(=O)C1=CC(C#C)=CC=C1OC KCLYALWNJLDDLR-UHFFFAOYSA-N 0.000 description 1
- 230000011987 methylation Effects 0.000 description 1
- PQIOSYKVBBWRRI-UHFFFAOYSA-N methylphosphonyl difluoride Chemical group CP(F)(F)=O PQIOSYKVBBWRRI-UHFFFAOYSA-N 0.000 description 1
- 210000000274 microglia Anatomy 0.000 description 1
- 238000010172 mouse model Methods 0.000 description 1
- 238000010995 multi-dimensional NMR spectroscopy Methods 0.000 description 1
- XBECFEJUQZXMFE-UHFFFAOYSA-N n-(4-aminobutyl)acetamide;hydrochloride Chemical compound Cl.CC(=O)NCCCCN XBECFEJUQZXMFE-UHFFFAOYSA-N 0.000 description 1
- MZRVEZGGRBJDDB-UHFFFAOYSA-N n-Butyllithium Substances [Li]CCCC MZRVEZGGRBJDDB-UHFFFAOYSA-N 0.000 description 1
- 230000010807 negative regulation of binding Effects 0.000 description 1
- 210000000478 neocortex Anatomy 0.000 description 1
- 230000001722 neurochemical effect Effects 0.000 description 1
- 230000000626 neurodegenerative effect Effects 0.000 description 1
- 230000002981 neuropathic effect Effects 0.000 description 1
- 230000009871 nonspecific binding Effects 0.000 description 1
- HGASFNYMVGEKTF-UHFFFAOYSA-N octan-1-ol;hydrate Chemical compound O.CCCCCCCCO HGASFNYMVGEKTF-UHFFFAOYSA-N 0.000 description 1
- TVMXDCGIABBOFY-UHFFFAOYSA-N octane Chemical compound CCCCCCCC TVMXDCGIABBOFY-UHFFFAOYSA-N 0.000 description 1
- 208000031237 olivopontocerebellar atrophy Diseases 0.000 description 1
- 125000002524 organometallic group Chemical group 0.000 description 1
- 230000003647 oxidation Effects 0.000 description 1
- 238000007254 oxidation reaction Methods 0.000 description 1
- 239000001301 oxygen Substances 0.000 description 1
- 210000000496 pancreas Anatomy 0.000 description 1
- 239000012188 paraffin wax Substances 0.000 description 1
- 238000010647 peptide synthesis reaction Methods 0.000 description 1
- YNPNZTXNASCQKK-UHFFFAOYSA-N phenanthrene Chemical compound C1=CC=C2C3=CC=CC=C3C=CC2=C1 YNPNZTXNASCQKK-UHFFFAOYSA-N 0.000 description 1
- OPZHGGBXSHYNHS-UHFFFAOYSA-N phenyl n-ethoxycarbonylcarbamate Chemical compound CCOC(=O)NC(=O)OC1=CC=CC=C1 OPZHGGBXSHYNHS-UHFFFAOYSA-N 0.000 description 1
- COLNVLDHVKWLRT-UHFFFAOYSA-N phenylalanine Natural products OC(=O)C(N)CC1=CC=CC=C1 COLNVLDHVKWLRT-UHFFFAOYSA-N 0.000 description 1
- 230000010287 polarization Effects 0.000 description 1
- 125000003367 polycyclic group Chemical group 0.000 description 1
- 229910000160 potassium phosphate Inorganic materials 0.000 description 1
- 235000011009 potassium phosphates Nutrition 0.000 description 1
- 208000022256 primary systemic amyloidosis Diseases 0.000 description 1
- 238000000425 proton nuclear magnetic resonance spectrum Methods 0.000 description 1
- UMJSCPRVCHMLSP-UHFFFAOYSA-N pyridine Natural products COC1=CC=CN=C1 UMJSCPRVCHMLSP-UHFFFAOYSA-N 0.000 description 1
- 239000012857 radioactive material Substances 0.000 description 1
- 239000012217 radiopharmaceutical Substances 0.000 description 1
- 229940121896 radiopharmaceutical Drugs 0.000 description 1
- 230000002799 radiopharmaceutical effect Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 230000004044 response Effects 0.000 description 1
- 230000000717 retained effect Effects 0.000 description 1
- 230000002441 reversible effect Effects 0.000 description 1
- 239000012047 saturated solution Substances 0.000 description 1
- 230000007017 scission Effects 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 238000012031 short term test Methods 0.000 description 1
- 229910052709 silver Inorganic materials 0.000 description 1
- 239000004332 silver Substances 0.000 description 1
- 238000001542 size-exclusion chromatography Methods 0.000 description 1
- 229910052708 sodium Inorganic materials 0.000 description 1
- 235000010288 sodium nitrite Nutrition 0.000 description 1
- 229910001495 sodium tetrafluoroborate Inorganic materials 0.000 description 1
- PUZPDOWCWNUUKD-ULWFUOSBSA-M sodium;fluorine-18(1-) Chemical compound [18F-].[Na+] PUZPDOWCWNUUKD-ULWFUOSBSA-M 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 238000000638 solvent extraction Methods 0.000 description 1
- 210000000952 spleen Anatomy 0.000 description 1
- 238000007447 staining method Methods 0.000 description 1
- 125000001273 sulfonato group Chemical group [O-]S(*)(=O)=O 0.000 description 1
- 208000024891 symptom Diseases 0.000 description 1
- 102000013498 tau Proteins Human genes 0.000 description 1
- 108010026424 tau Proteins Proteins 0.000 description 1
- 229910052713 technetium Inorganic materials 0.000 description 1
- 229940056501 technetium 99m Drugs 0.000 description 1
- 125000003698 tetramethyl group Chemical group [H]C([H])([H])* 0.000 description 1
- HPGGPRDJHPYFRM-UHFFFAOYSA-J tin(iv) chloride Chemical compound Cl[Sn](Cl)(Cl)Cl HPGGPRDJHPYFRM-UHFFFAOYSA-J 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 230000009466 transformation Effects 0.000 description 1
- WMSCYGTVHFMKNS-UHFFFAOYSA-N tributyl(methyl)stannane Chemical group CCCC[Sn](C)(CCCC)CCCC WMSCYGTVHFMKNS-UHFFFAOYSA-N 0.000 description 1
- REDSKZBUUUQMSK-UHFFFAOYSA-N tributyltin Chemical compound CCCC[Sn](CCCC)CCCC.CCCC[Sn](CCCC)CCCC REDSKZBUUUQMSK-UHFFFAOYSA-N 0.000 description 1
- ILWRPSCZWQJDMK-UHFFFAOYSA-N triethylazanium;chloride Chemical compound Cl.CCN(CC)CC ILWRPSCZWQJDMK-UHFFFAOYSA-N 0.000 description 1
- 125000000026 trimethylsilyl group Chemical group [H]C([H])([H])[Si]([*])(C([H])([H])[H])C([H])([H])[H] 0.000 description 1
- CWMFRHBXRUITQE-UHFFFAOYSA-N trimethylsilylacetylene Chemical group C[Si](C)(C)C#C CWMFRHBXRUITQE-UHFFFAOYSA-N 0.000 description 1
- LYRCQNDYYRPFMF-UHFFFAOYSA-N trimethyltin Chemical class C[Sn](C)C LYRCQNDYYRPFMF-UHFFFAOYSA-N 0.000 description 1
- CCRMAATUKBYMPA-UHFFFAOYSA-N trimethyltin Chemical compound C[Sn](C)C.C[Sn](C)C CCRMAATUKBYMPA-UHFFFAOYSA-N 0.000 description 1
- 230000035899 viability Effects 0.000 description 1
- 229920002554 vinyl polymer Polymers 0.000 description 1
- 230000000007 visual effect Effects 0.000 description 1
- 238000012800 visualization Methods 0.000 description 1
- 238000003260 vortexing Methods 0.000 description 1
- 239000003643 water by type Substances 0.000 description 1
- 238000002424 x-ray crystallography Methods 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D209/00—Heterocyclic compounds containing five-membered rings, condensed with other rings, with one nitrogen atom as the only ring hetero atom
- C07D209/02—Heterocyclic compounds containing five-membered rings, condensed with other rings, with one nitrogen atom as the only ring hetero atom condensed with one carbocyclic ring
- C07D209/44—Iso-indoles; Hydrogenated iso-indoles
- C07D209/48—Iso-indoles; Hydrogenated iso-indoles with oxygen atoms in positions 1 and 3, e.g. phthalimide
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C245/00—Compounds containing chains of at least two nitrogen atoms with at least one nitrogen-to-nitrogen multiple bond
- C07C245/02—Azo compounds, i.e. compounds having the free valencies of —N=N— groups attached to different atoms, e.g. diazohydroxides
- C07C245/06—Azo compounds, i.e. compounds having the free valencies of —N=N— groups attached to different atoms, e.g. diazohydroxides with nitrogen atoms of azo groups bound to carbon atoms of six-membered aromatic rings
- C07C245/08—Azo compounds, i.e. compounds having the free valencies of —N=N— groups attached to different atoms, e.g. diazohydroxides with nitrogen atoms of azo groups bound to carbon atoms of six-membered aromatic rings with the two nitrogen atoms of azo groups bound to carbon atoms of six-membered aromatic rings, e.g. azobenzene
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C245/00—Compounds containing chains of at least two nitrogen atoms with at least one nitrogen-to-nitrogen multiple bond
- C07C245/02—Azo compounds, i.e. compounds having the free valencies of —N=N— groups attached to different atoms, e.g. diazohydroxides
- C07C245/06—Azo compounds, i.e. compounds having the free valencies of —N=N— groups attached to different atoms, e.g. diazohydroxides with nitrogen atoms of azo groups bound to carbon atoms of six-membered aromatic rings
- C07C245/10—Azo compounds, i.e. compounds having the free valencies of —N=N— groups attached to different atoms, e.g. diazohydroxides with nitrogen atoms of azo groups bound to carbon atoms of six-membered aromatic rings with nitrogen atoms of azo groups bound to carbon atoms of six-membered aromatic rings being part of condensed ring systems
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C59/00—Compounds having carboxyl groups bound to acyclic carbon atoms and containing any of the groups OH, O—metal, —CHO, keto, ether, groups, groups, or groups
- C07C59/40—Unsaturated compounds
- C07C59/58—Unsaturated compounds containing ether groups, groups, groups, or groups
- C07C59/64—Unsaturated compounds containing ether groups, groups, groups, or groups containing six-membered aromatic rings
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D213/00—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members
- C07D213/02—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members
- C07D213/04—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom
- C07D213/60—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
- C07D213/78—Carbon atoms having three bonds to hetero atoms, with at the most one bond to halogen, e.g. ester or nitrile radicals
- C07D213/79—Acids; Esters
- C07D213/80—Acids; Esters in position 3
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D217/00—Heterocyclic compounds containing isoquinoline or hydrogenated isoquinoline ring systems
- C07D217/22—Heterocyclic compounds containing isoquinoline or hydrogenated isoquinoline ring systems with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to carbon atoms of the nitrogen-containing ring
- C07D217/24—Oxygen atoms
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D221/00—Heterocyclic compounds containing six-membered rings having one nitrogen atom as the only ring hetero atom, not provided for by groups C07D211/00 - C07D219/00
- C07D221/02—Heterocyclic compounds containing six-membered rings having one nitrogen atom as the only ring hetero atom, not provided for by groups C07D211/00 - C07D219/00 condensed with carbocyclic rings or ring systems
- C07D221/04—Ortho- or peri-condensed ring systems
- C07D221/06—Ring systems of three rings
- C07D221/14—Aza-phenalenes, e.g. 1,8-naphthalimide
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D237/00—Heterocyclic compounds containing 1,2-diazine or hydrogenated 1,2-diazine rings
- C07D237/26—Heterocyclic compounds containing 1,2-diazine or hydrogenated 1,2-diazine rings condensed with carbocyclic rings or ring systems
- C07D237/28—Cinnolines
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D237/00—Heterocyclic compounds containing 1,2-diazine or hydrogenated 1,2-diazine rings
- C07D237/26—Heterocyclic compounds containing 1,2-diazine or hydrogenated 1,2-diazine rings condensed with carbocyclic rings or ring systems
- C07D237/30—Phthalazines
- C07D237/32—Phthalazines with oxygen atoms directly attached to carbon atoms of the nitrogen-containing ring
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D257/00—Heterocyclic compounds containing rings having four nitrogen atoms as the only ring hetero atoms
- C07D257/02—Heterocyclic compounds containing rings having four nitrogen atoms as the only ring hetero atoms not condensed with other rings
- C07D257/04—Five-membered rings
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D261/00—Heterocyclic compounds containing 1,2-oxazole or hydrogenated 1,2-oxazole rings
- C07D261/20—Heterocyclic compounds containing 1,2-oxazole or hydrogenated 1,2-oxazole rings condensed with carbocyclic rings or ring systems
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D265/00—Heterocyclic compounds containing six-membered rings having one nitrogen atom and one oxygen atom as the only ring hetero atoms
- C07D265/02—1,2-Oxazines; Hydrogenated 1,2-oxazines
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D265/00—Heterocyclic compounds containing six-membered rings having one nitrogen atom and one oxygen atom as the only ring hetero atoms
- C07D265/04—1,3-Oxazines; Hydrogenated 1,3-oxazines
- C07D265/12—1,3-Oxazines; Hydrogenated 1,3-oxazines condensed with carbocyclic rings or ring systems
- C07D265/14—1,3-Oxazines; Hydrogenated 1,3-oxazines condensed with carbocyclic rings or ring systems condensed with one six-membered ring
- C07D265/24—1,3-Oxazines; Hydrogenated 1,3-oxazines condensed with carbocyclic rings or ring systems condensed with one six-membered ring with hetero atoms directly attached in positions 2 and 4
- C07D265/26—Two oxygen atoms, e.g. isatoic anhydride
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D271/00—Heterocyclic compounds containing five-membered rings having two nitrogen atoms and one oxygen atom as the only ring hetero atoms
- C07D271/02—Heterocyclic compounds containing five-membered rings having two nitrogen atoms and one oxygen atom as the only ring hetero atoms not condensed with other rings
- C07D271/10—1,3,4-Oxadiazoles; Hydrogenated 1,3,4-oxadiazoles
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
- C07F7/00—Compounds containing elements of Groups 4 or 14 of the Periodic Table
- C07F7/22—Tin compounds
- C07F7/2208—Compounds having tin linked only to carbon, hydrogen and/or halogen
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C2603/00—Systems containing at least three condensed rings
- C07C2603/02—Ortho- or ortho- and peri-condensed systems
- C07C2603/04—Ortho- or ortho- and peri-condensed systems containing three rings
- C07C2603/06—Ortho- or ortho- and peri-condensed systems containing three rings containing at least one ring with less than six ring members
- C07C2603/10—Ortho- or ortho- and peri-condensed systems containing three rings containing at least one ring with less than six ring members containing five-membered rings
- C07C2603/12—Ortho- or ortho- and peri-condensed systems containing three rings containing at least one ring with less than six ring members containing five-membered rings only one five-membered ring
- C07C2603/18—Fluorenes; Hydrogenated fluorenes
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C2603/00—Systems containing at least three condensed rings
- C07C2603/02—Ortho- or ortho- and peri-condensed systems
- C07C2603/04—Ortho- or ortho- and peri-condensed systems containing three rings
- C07C2603/22—Ortho- or ortho- and peri-condensed systems containing three rings containing only six-membered rings
- C07C2603/26—Phenanthrenes; Hydrogenated phenanthrenes
Definitions
- the present invention relates to a method of imaging amyloid deposits in brain in vivo to allow antemortem diagnosis of Alzheimer's Disease.
- present invention also relates to therapeutic uses for such compounds.
- AD Alzheimer's Disease
- AD Alzheimer's disease
- AD is definitively diagnosed through examination of brain tissue, usually at autopsy.
- Khachaturian Arch. Neurol . 42: 1097 (1985) ; McKhann et al . , Neurology 34: 939 (1984) .
- ⁇ europathologically this disease is characterized by the presence of neuritic plaques ( ⁇ P) , neurofibrillary tangles ( ⁇ FT) , and neuronal loss, along with a variety of other findings.
- ⁇ P neuritic plaques
- ⁇ FT neurofibrillary tangles
- neuronal loss along with a variety of other findings.
- Mann, Mech . Ageing Dev. 31: 213 (1985) Post-mortem slices of brain tissue of victims of Alzheimer's disease exhibit the presence of amyloid in the form of proteinaceous extracellular cores of the neuritic plaques that are characteristic of AD.
- amyloid cores of these neuritic plaques are composed of a protein called the ⁇ -amyloid (A ⁇ ) that is arranged in a predominately beta-pleated sheet configuration.
- a ⁇ ⁇ -amyloid
- Mori et al . Journal of Biological Chemistry 261 : 17082 (1992) ; Kirschner et al. , PNAS 83: 503 (1986) .
- Neuritic plaques are an early and invariant aspect of the disease. Mann et al . , J. Neurol . Sci . 89: 169; Mann, Mech . Ageing Dev. 31: 213 (1985) ; Terry et al . , J. Neuropathol . Exp . Neurol 46: 262 (1987) .
- a ⁇ probably occurs long before clinical symptoms are noticeable.
- the currently recommended "minimum microscopic criteria" for the diagnosis of AD is based on the number of neuritic plaques found in brain. Khachaturian, Arch . Neurol . , supra (1985) . Unfortunately, assessment of neuritic plaque counts must be delayed until after death. Amyloid-containing neuritic plaques are a prominent feature of selective areas of the brain in AD as well as Downs Syndrome and in persons homozygous for the apolipoprotein E4 allele who are very likely to develop AD. Corder et al . , Science 261: 921 (1993) ; Divry, P., J. Neurol . Psych .
- Congo red stained amyloid is characterized by a dichroic appearance, exhibiting a yellow-green polarization color.
- the dichroic binding is the result of the beta-pleated sheet structure of the amyloid proteins.
- AD Alzheimer's disease
- research efforts to develop methods for diagnosing Alzheimer's disease in vivo include (1) genetic testing, (2) immunoassay methods and (3) imaging techniques.
- AD Alzheimer's disease
- AD is genetically heterogeneous. St. George- Hyslop et al . , Nature 347: 194 (1990) . Linkage to chromosome 21 markers is shown in only some families with early-onset AD and in no families with late-onset AD. More recently a gene on chromosome 14 whose product is predicted to contain multiple transmembrane domains and resembles an integral membrane protein has been identified by Sherrington et al . , Nature 375: 754-760 (1995) . This gene may account for up to 70% of early- onset autosomal dominant AD. Preliminary data suggests that this chromosome 14 mutation causes an increase in the production of A ⁇ .
- AD Alzheimer's disease Using antibodies for in vivo diagnosis of AD would require marked abnormalities in the blood-brain barrier in order to gain access into the brain. There is no convincing functional evidence that abnormalities in the blood-brain barrier reliably exist in AD. Kalaria, Cerebrovascular & Brain Metabolism Reviews 4: 226 (1992) .
- a ⁇ antibodies are also disadvantageous for use in AD diagnostics because they typically stain diffuse deposits of A ⁇ in addition to the neuritic plaques - and the diffuse plaques often predominate. Yamaguchi et al., Acta Neuropathol . , 77: 314 (1989). Diffuse plaques may be a separate type of lesion, not necessarily involved in the dementing process of AD.
- Congo red may be used for diagnosing amyloidosis in vivo in non-brain parenchymal tissues.
- Congo red is probably not suitable for in vivo diagnosis of A ⁇ deposits in brain because only 0.03% of an injected dose of iodinated Congo red can enter the brain parenchyma.
- Radioiodinated bisdiazobenzidine compounds related to Congo red such as Benzo Orange R and Direct Blue 4, have been proposed to be useful in vi tro and in vivo to detect the presence and location of amyloid deposits in an organ of a patient. Quay et al . , U.S. Patent Nos.
- amyloid binding compounds which are similar to Congo red but which enter the brain (Congo Red does not) .
- Such compounds could be used in preventing cell degeneration associated with fibril formation and thereby treat pathological conditions in amyloid associated diseases, such as AD and Downs Syndrome and in treating pancreatic islet cell toxicity in type 2 diabetes mellitus.
- amyloid binding compounds that are non-toxic and bioavailable and, consequently, can be used in therapeutics.
- an amyloid binding compound of Formula I or a water soluble, non-toxic salt thereof is provided, in accordance with one aspect of the present invention, an amyloid binding compound of Formula I or a water soluble, non-toxic salt thereof:
- R' is H or a lower alkyl group
- each Q is independently selected from one of the following structures, each of which contain a carboxylic acid or an acid-like functionality: IA, IB, IC, ID, IE, IF and IG, wherein IA has the following structure:
- each of R 3 , R 4 , R 5 , R 6 , or R 7 independently is H, F,
- N(R') 2 , N0 2 , (C 0)N(R') 2 0(C0)R', OR', C00R' , a tri- alkyl tin, a tetrazole or oxadiazole of the form:
- R 3 , R 4 , R 5 , R 6 , or R 7 is a hydroxy, tetrazole, oxadiazole, N0 2 , or carboxy in both Q's;
- R 1# R 2 , R 3; R 4 , or R 7 is not H when R 5 is hydroxyl and R 6 is carboxy or a lower alkyl ester thereof; at least one of R lf R 2 , R 3 or R 4 is not H when R 5 is amino, R 6 is carboxy or a lower alkyl ester thereof, and R 7 is hydroxyl; at least one of R l t R 3 , R 4 , or R 7 is not H when R 2 is CH 3 , R 5 is hydroxyl and R 6 is carboxy or a lower alkyl ester thereof; at least one of Ri or R 2 , is not H or CH 3 when R 3 , R 4 , or R 7 is CH 3 , R 5 is hydroxyl and R 6 is carboxy or a lower alkyl ester thereof;
- IB has the following structure (IB)
- R 8 and R 9 are lower alkyl
- R 10 ⁇ R u , R 12 , R 13 , R 14 , R 15 or R 16 is a hydroxy, tetrazole, oxadiazole, N0 2 or carboxy in both Q' s;
- IC has the following structure
- ID has the following structure:
- IE has the following structure:
- IF has the following structure:
- R 28 , R 29 , R 30 , R 31 or R 32 is a hydroxy, tetrazole, oxadiazole, N0 2 or carboxy in both Q's;
- IG has the following structure: (IG)
- R 33 , R 34 , R 35 , R 36 , R 37 , R 38 , or R 39 is the r, link defined for Formula I above and each
- R' is H or a lower alkyl group
- R 9 are lower alkyl
- R 33 , R 34 , R 35 , R 36 , R 37 , R 38 , or R 39 is a hydroxy, tetrazole, oxadiazole, N0 2 or carboxy in both Q's.
- K D dissociation constant
- Still another object of the present invention is to provide a method for synthesizing an amyloid binding compound of Formula I, as defined above, or a water soluble, non-toxic salt thereof, wherein at least one of the substituents R- L -R*-* and R 10 -R 39 is selected from the group consisting of 131 I, 123 I, 76 Br, 75 Br, 18 F and 19 F, comprising the step of reacting an amyloid binding compound of Formula I, as defined above, or a water soluble, non-toxic salt thereof wherein at least one of the substituents R ⁇ -R 7 and R 10 -R 39 is a tri-alkyl tin, with a halogenating agent containing 131 I, 123 I, 76 Br, 75 Br, 18 F or 19 F.
- An additional object of the present invention is a pharmaceutical composition for in vivo imaging of amyloid deposits, comprising (a) an amyloid binding compound of Formula I, as defined above, or a water soluble, non-toxic salt thereof, wherein at least one of the substituents R** . -R 7 and R 10 -R 39 is selected from the group consisting of 131 I, 123 I, 76 Br, 75 Br, 18 F, 19 F and a carbon-containing substituent as specified in Formula I wherein at least one carbon is 11 C or 13 C, and (b) a pharmaceutically acceptable carrier.
- Yet another object of the present invention is an in vivo method for detecting amyloid deposits in a subject, comprising the steps of: (a) administering a detectable quantity of the above pharmaceutical composition, and (b) detecting the binding of the compound to amyloid deposit in said subject. It is also an object of the present invention to provide an in vivo method for detecting amyloid deposits in a subject wherein the amyloid deposit is located in the brain of a subject. This method of the invention may be used in a subject who is suspected of having an amyloidosis associated disease or syndrome selected from the group consisting of Alzheimer's Disease, Down's Syndrome, and homozygotes for the apolipoprotein E4 allele.
- Another object of the invention relates to pharmaceutical compositions and methods of preventing cell degeneration and toxicity associated with fibril formation in amyloidosis associated conditions such as AD and Type 2 diabetes mellitus.
- Such pharmaceutical compositions comprise Chrysamine G or the above described derivatives thereof and a pharmaceutically acceptable carrier.
- Such compounds would be non-toxic.
- Another object of the invention relates to the use of the probes as a stain for the visualization and detection of amyloid deposits in biopsy or post-mortem tissue specimens.
- Another object of this invention relates to the use of radiolabeled probes for the quantitation of amyloid deposits in biopsy or postmortem tissue specimens. Another object relates to a method of distinguishing Alzheimer's disease brain from normal brain.
- Fi ⁇ ure IA illustrates the chemical structures of Chrysamine G and several Chrysamine G analogues or derivatives which have been synthesized and tested, including the 3-iodo derivative (3-ICG) , the 3-iodo dimethoxy derivative (3-ICG(OMe) 2 ) , the dimethyl ester derivative (CG(COOMe) 2 ) , the phenol derivative, salicylic acid (SA) , the aniline derivative (1/2CG) , Congo red, the 3,3'-diiodo derivative (3,3'-I 2 CG), the 3,3'-dibromo derivative (3,3' -Br 2 CG) , the 3,3' -dichloro derivative (3,3' -C1 2 CG) , the 3-bromo derivative (3- BrCG) , and the 5-fluorosalicylic acid derivative ( (5- FSA)CG) .
- the numbers in the figure refer to each co pound's ⁇ (in ⁇ M) for inhibition
- Figure IB illustrates the chemical structures of several Chrysamine G analogues or derivatives which have been synthesized and tested, including the 3,3'- dicarboxylic acid derivative (3, 3 ' - (COOH) 2 CG) , the 2, 2 ' -disulfonic acid derivative of Chrysamine G (2,2'- (S0 3 ) 2 CG), the 3-bromo, 3-isopropylsalicylic acid derivative (3-Br- (3-iPrSA) CG) , the 3-isopropylsalicylic acid derivative ( (3-iPrSA) CG) , the 2,4-diphenol derivative (2,4-Diphenol) , the ⁇ -resorcylic acid derivative ( (6-OHSA) CG) , the 3,3 ' ,5,5' - tetramethylbenzidine derivative (3, 3 ' , 5, 5 ' - (CH 3 ) 4 CG) , the 3 , 3 ' -dimethyl
- FIGS 2A-2K illustrate the chemical structures of Chrysamine G and tri-alkyl tin derivatives of analogues of Chrysamine G, in particular heterocyclic analogues. Note that these structures represent one- half of a molecule which is symmetric around the wavy bond shown in the upper right, except that the tri- alkyl tin moiety may only be on one side of the biphenyl group.
- the tri-alkyl tin derivatives are stable intermediate and immediate precursors for the preparation of high specific activity halogenated radioactive derivatives.
- the heterocyclic analogues represent alternative means of placing weakly acidic moieties in the same structural position as the moderately acidic carboxylic acid group of Chrysamine G. These tri-alkyl tin precursor compounds are shown in their protonated form, yet those of skill in the art recognize that their deprotonated forms and tautomers also are embraced by these drawings.
- Figure 3 Displacement curves of [ 14 C] Chrysamine G binding to A ⁇ (10-43) by several structural analogues of Chrysamine G. Abbreviations refer to those used in Figure 1.
- Figure 3A Chrysamine G (open triangles) ; (5-FSA)CG (filled diamonds) ; 3, 3 ' - (COOH) 2 CG (filled squares) ; 2, 2 ' - (S0 3 ) 2 CG (filled circles) .
- Figure 3B Chrysamine G (open triangles) ; Congo red (open circles) ; aniline derivative (open inverted triangles) ; phenol derivative (open squares) ; salicylic acid (X's) . Curves which show increased binding at higher concentrations do so because of the formation of micelles. Bedaux, F. et al., Phar . Weekblad 98: 189 (1963) .
- Figure 4A is a Scatchard plot of Chrysamine G binding to A (10-43) .
- the curved line represents a nonlinear least-squares fit to a two, independent binding site model.
- the straight lines represent the individual components.
- Figure 4B is a Scatchard analysis of [ 14 C] CG binding to typical control (diamonds) and AD brain samples (squares) .
- the dashed line has the same slope as the AD line and is meant to aid in the comparison with the control slope.
- This AD brain sample had 48 NP/x200 magnification, a K D of 0.35 ⁇ M, and a B max of 790 fmol/ ⁇ g protein.
- the control had a K D of 0.48 ⁇ M, and a B ⁇ of 614 fmol/ ⁇ g protein.
- Figure 5 is a graph illustrating the linearity of the binding assay with respect to peptide concentration. Approximately 0.9 ⁇ g of A ⁇ (10-43) was used in the typical assay.
- Figure 6A is a graph illustrating the time course of association of Chrysamine G and A ⁇ (10-43) .
- Figure 6B is the graphic illustration of the determination of the association rate constant (k .
- Figure 6C is a graph of the time course of dissociation of Chrysamine G from A ⁇ (10-43) .
- Figure 7 is a graphic representation of a molecular model of the interaction between Chrysamine G and A ⁇ .
- Figure 8A is a graph illustrating the correlation between the amount of [ 14 C] Chrysamine G bound and the number of neuritic plaques (NP) in AD brain samples.
- Figure 8B is a graph illustrating the correlation between the amount of [ 1 C] Chrysamine G bound and the number of neurofibrillary tangles (NFT) in AD brain samples.
- the filled symbols and heavy lines indicate brains without amyloid angiopathy, the open symbols and dashed lines indicate brains with amyloid angiopathy.
- the y-axis represents total, absolute [ 1 C] Chrysamine G binding (fmol/ ⁇ g protein) in homogenates of brain samples adjacent to those used for staining. Approximately 75 ⁇ g protein and 150 nM [ 14 C] Chrysamine G were used.
- FIGs 9A. 9B and 9C The binding of Chrysamine G to various brain areas in samples of AD brain having more than 20 NPs/x200 magnification, referred to as "High Plaque AD Brains", is shown in figure 9A.
- the binding of Chrysamine G to brain areas in samples of AD brain having less than 20 NPs/x200 magnification, referred to as “Low Plaque AD Brains” is shown in Figure 9B.
- the data points represent the ratio of [ 1 C] Chrysamine G binding in the designated brain area to [ 14 C] Chrysamine G binding in the cerebellum (CB) of the same brain.
- Horizontal bars represent the mean and error bars represent the standard error for control (circles) , and AD brain (diamonds in 9A and 9B) .
- Brain areas include the frontal pole (FP) , head of caudate (CAU) , superior/ middle frontal (SMF) , superior temporal (ST) , inferior parietal (IP) , and occipital (OC) cortex.
- FP frontal pole
- CAU head of caudate
- SMF superior/ middle frontal
- ST superior temporal
- IP inferior parietal
- OC occipital cortex
- Asterisks indicate significant differences compared to control (*p ⁇ 0.05; **p ⁇ 0.001) .
- Two Down' s syndrome brain samples are indicated in Figure 9C.
- the diamonds in 9C represent a brain from a 23 year old Down's syndrome patient not yet symptomatic with AD.
- the triangles in 9C represent a 51 year old Down's syndrome patient who had developed AD as do the vast
- Figure 10 is a graph illustrating the tissue levels of Chrysamine G in mice injected with [ 14 C] Chrysamine G in the lateral tail vein and sacrificed at the times indicated.
- the open symbols and thin lines represent absolute radioactivity in units of cpm/g tissue (left axis) .
- the closed symbols and solid lines represent the ratio of brain radioactivity to that in kidney (top) or blood (middle) . The ratios are plotted on the right axis.
- FIG. 11 Section from the inferior temporal lobe of AD brain stained with Chrysamine G by the method of Stokes and Trickey, J " . Clin . Pathol . 26: 241-242 (1973) . Two neuritic plaques are clearly visible.
- BOTTOM Adjacent sections from the temporal lobe of an AD patient with amyloid angiopathy. Left: Brain section was stained with the Congo red method of Puchtler. Puchtler et al . , J. Histochem. Cytochem. 10: 35 (1962) . The bar represents 20 microns. Right: Adjacent brain section stained by substituting Chrysamine G for Congo red in the Puchtler method. Both sections readily demonstrate the same amyloid- laden vessel . A small amount of background autofluorescence from erythrocytes and lipofuscin also is visible. All photomicrographs were obtained using fluorescein isothiocyanate (FITC) filters.
- FITC fluorescein isothiocyanate
- FIG. 12 A bar graph showing the effect of increasing concentrations of A ⁇ (25-35) in the presence and absence of Chrysamine G on the cellular redox activity of rat pheochromocytoma (PC-12) cells as measured by 3- [4, 5-dimethylthiazol-2-yl] -2, 5- diphenyltetrazolium bromide, MTT, reduction.
- the reduction product of MTT absorbs at 560 nm which is plotted on the vertical axis.
- the effect of A ⁇ (25-35) alone is shown in the filled bars and shows a dose dependent decrease in MTT reduction.
- Significant differences from control no A ⁇ (25-35) , no Chrysamine G
- the protective effect of 20 ⁇ M Chrysamine G is shown in the open bars.
- Significant differences between MTT reduction in the presence and absence of Chrysamine G are shown in black inside the open bars .
- Figure 13 A bar graph showing the protective effect of increasing concentrations of Chrysamine G against the A ⁇ (25-35) -induced reduction of cellular redox activity of rat pheochromocytoma (PC-12) cells as measured by 3- [4, 5-dimethylthiazol-2-yl] -2, 5- diphenyltetrazolium bromide, MTT, reduction.
- the reduction product of MTT absorbs at 560 nm which is plotted on the vertical axis.
- the effect of Chrysamine G in the absence of A ⁇ (25-35) is shown in the filled bars.
- Chrysamine G is a Congo red derivative with the key structural difference being that the sulfonic acid moieties found in Congo red are replaced by carboxylic acid groups in Chrysamine G ( Figure 1) .
- This structural alteration allows Chrysamine G to enter the brain better than Congo red and large macromolecules such as antibodies.
- Chrysamine G may be a more specific marker for AD pathophysiology than antibodies, which would also label diffuse plaques, due to the apparent specificity of Chrysamine G for the neuritic plaque and NFTs.
- the Chrysamine G derivatives of the present invention have each of the following characteristics: (1) specific binding to synthetic A ⁇ in vi tro, (2) binding to neuritic but not diffuse plaques in brain sections (3) ability to cross a non-compromised blood brain barrier in vivo .
- the method of this invention determines the presence and location of amyloid deposits in an organ or body area, preferably brain, of a patient.
- the present method comprises administration of a detectable quantity of a pharmaceutical composition containing a compound of Formula I, as defined above, called a “detectable compound, " or a pharmaceutically acceptable water-soluble salt thereof, to a patient.
- a “detectable quantity” means that the amount of the detectable compound that is administered is sufficient to enable detection of binding of the compound to amyloid.
- An “imaging effective quantity” means that the amount of the detectable compound that is administered is sufficient to enable imaging of binding of the compound to amyloid.
- the invention employs amyloid probes which, in conjunction with non-invasive neuroimaging techniques such as magnetic resonance spectroscopy (MRS) or imaging (MRI) , or gamma imaging such as positron emission tomography (PET) or single-photon emission computed tomography (SPECT) , are used to quantify amyloid deposition in vivo .
- non-invasive neuroimaging techniques such as magnetic resonance spectroscopy (MRS) or imaging (MRI)
- gamma imaging such as positron emission tomography (PET) or single-photon emission computed tomography (SPECT)
- PET positron emission tomography
- SPECT single-photon emission computed tomography
- the radiation emitted from the organ or area being examined is measured and expressed either as total binding or as a ratio in which total binding in one tissue is normalized to (for example, divided by) the total binding in another tissue of the same subject during the same in vivo imaging procedure.
- Total binding in vivo is defined as the entire signal detected in a tissue by an in vivo imaging technique without the need for correction by a second injection of an identical quantity of labeled compound along with a large excess of unlabeled, but otherwise chemically identical compound.
- a "subject” is a mammal, preferably a human, and most preferably a human suspected of having dementia.
- the type of detection instrument available is a major factor in selecting a given label.
- radioactive isotopes and 19 F are particularly suitable for in vivo imaging in the methods of the present invention.
- the type of instrument used will guide the selection of the radionuclide or stable isotope.
- the radionuclide chosen must have a type of decay detectable by a given type of instrument .
- Another consideration relates to the half-life of the radionuclide. The half-life should be long enough so that it is still detectable at the time of maximum uptake by the target, but short enough so that the host does not sustain deleterious radiation.
- the radiolabeled compounds of the invention can be detected using gamma imaging wherein emitted gamma irradiation of the appropriate wavelength is detected.
- Methods of gamma imaging include, but are not limited to, SPECT and PET.
- the chosen radiolabel will lack a particulate emission, but will produce a large number of photons in a 140-200 keV range.
- the radiolabel will be a positron-emitting radionuclide such as 19 F which will annihilate to form two 511 keV gamma rays which will be detected by the PET camera.
- amyloid binding compounds/probes are made which are useful for in vivo imaging and quantification of amyloid deposition.
- the Chrysamine G derivatives may be labeled with 19 F or 13 C for MRS/MRI by general organic chemistry techniques known to the art. See, e . g. , March, J. "ADVANCED ORGANIC CHEMISTRY: REACTIONS, MECHANISMS, AND STRUCTURE (3rd Edition, 1985) , the contents of which are hereby incorporated by reference.
- the Chrysamine G derivatives also may be radiolabeled with 18 F, 11 C, 75 Br, or 76 Br for PET by techniques well known in the art and are described by Fowler, J. and Wolf, A. in POSITRON EMISSION TOMOGRAPHY AND AUTORADIOGRAPHY (Phelps, M. , Mazziota, J., and Schelbert, H. eds . ) 391-450 (Raven Press, NY 1986) the contents of which are hereby incorporated by reference.
- the Chrysamine G derivatives also may be radiolabeled with 123 I for SPECT by any of several techniques known to the art. See, e . g. , Kulkarni, Int. J " . Rad.
- Chrysamine G derivatives may be labeled with any suitable radioactive iodine isotope, such as, but not limited to 131 I, 125 I, or 123 I, by iodination of a diazotized amino derivative directly via a diazonium iodide, see Greenbaum, F. Am . J. Pharm.
- a stable triazene or tri-alkyl tin derivative of Chrysamine G or its analogues is reacted with a halogenating agent containing 131 I, 125 I, 123 I, 76 Br, 75 Br, i8 F or i9 F-
- a halogenating agent containing 131 I, 125 I, 123 I, 76 Br, 75 Br, i8 F or i9 F-
- the stable tri-alkyl tin derivatives of Chrysamine G and its analogues are novel precursors useful for the synthesis of many of the radiolabeled compounds within the present invention.
- these tri-alkyl tin derivatives are one embodiment of this invention.
- the Chrysamine G derivatives also may be radiolabeled with known metal radiolabels, such as Technetium-99m ( 99m Tc) . Modification of the substituents to introduce ligands that bind such metal ions can be effected without undue experimentation by one of ordinary skill in the radiolabeling art.
- the metal radiolabeled Chrysamine G derivative can then be used to detect amyloid deposits.
- the methods of the present invention may use isotopes detectable by nuclear magnetic resonance spectroscopy for purposes of in vivo imaging and spectroscopy.
- Elements particularly useful in magnetic resonance spectroscopy include 19 F and 13 C.
- Suitable radioisotopes for purposes of this invention include beta-emitters, gamma-emitters, positron-emitters, and x-ray emitters. These radioisotopes include 131 I, 123 I, 18 F, "C, 75 Br, and 76 Br.
- Suitable stable isotopes for use in Magnetic Resonance Imaging (MRI) or Spectroscopy (MRS) include 19 F and 13 C.
- Suitable radioisotopes for in vi tro quantification of amyloid in homogenates of biopsy or post-mortem tissue include 125 I, 14 C, and 3 H.
- the preferred radiolabels are 18 F for use in PET in vivo imaging, 123 I for use in SPECT imaging, 19 F for MRS/MRI, and 3 H or 1 C for in vi tro studies.
- any conventional method for visualizing diagnostic probes can be utilized in accordance with this invention.
- the method could be used to diagnose AD in mild or clinically confusing cases. This technique would also allow longitudinal studies of amyloid deposition in human populations at high risk for amyloid deposition such as Down's syndrome, familial AD, and homozygotes for the apolipoprotein E4 allele. Corder et al . , Science 261: 921 (1993) .
- a method that allows the temporal sequence of amyloid deposition to be followed can determine if deposition occurs long before dementia begins or if deposition is unrelated to dementia. This method can be used to monitor the effectiveness of therapies targeted at preventing amyloid deposition.
- the dosage of the detectably labeled Chrysamine G derivative will vary depending on considerations such as age, condition, sex, and extent of disease in the patient, contraindications, if any, concomitant therapies and other variables, to be adjusted by a physician skilled in the art. Dosage can vary from 0.001 mg/kg to 1000 mg/kg, preferably 0.1 mg/kg to 100 mg/kg. Administration to the subject may be local or systemic and accomplished intravenously, intraarterially, intrathecally (via the spinal fluid) or the like. Administration may also be intradermal or intracavitary, depending upon the body site under examination.
- the area of the subject under investigation is examined by routine imaging techniques such as MRS/MRI, SPECT, planar scintillation imaging, PET, and emerging imaging techniques, as well.
- routine imaging techniques such as MRS/MRI, SPECT, planar scintillation imaging, PET, and emerging imaging techniques, as well.
- the exact protocol will necessarily vary depending upon factors specific to the patient, as noted above, and depending upon the body site under examination, method of administration and type of label used; the determination of specific procedures would be routine to the skilled artisan.
- the amount (total or specific binding) of the bound radioactively labelled Chrysamine G or Chrysamine G derivative or analogue is measured and compared (as a ratio) with the amount of labelled Chrysamine G or Chrysamine G derivative bound to the cerebellum of the patient. This ratio is then compared to the same ratio in age-matched normal brain.
- the pharmaceutical compositions of the present invention are advantageously administered in the form of injectable compositions.
- a typical composition for such purpose comprises a pharmaceutically acceptable carrier.
- the composition may contain about 10 mg of human serum albumin and from about 0.5 to 500 micrograms of the labeled Chrysamine G or Chrysamine G derivative per milliliter of phosphate buffer containing NaCl .
- compositions include aqueous solutions, non- toxic excipients, including salts, preservatives, buffers and the like, as described, for instance, in REMINGTON'S PHARMACEUTICAL SCIENCES, 15th Ed. Easton: Mack Publishing Co. pp. 1405-1412 and 1461-1487 (1975) and THE NATIONAL FORMULARY XIV., 14th Ed. Washington: American Pharmaceutical Association (1975) , the contents of which are hereby incorporated by reference.
- non-aqueous solvents examples include propylene glycol, polyethylene glycol, vegetable oil and injectable organic esters such as ethyl oleate.
- Aqueous carriers include water, alcoholic/aqueous solutions, saline solutions, parenteral vehicles such as sodium chloride, Ringer's dextrose, etc.
- Intravenous vehicles include fluid and nutrient replenishers .
- Preservatives include antimicrobials, anti-oxidants, chelating agents and inert gases. The pH and exact concentration of the various components of the pharmaceutical composition are adjusted according to routine skills in the art. See, Goodman and
- compositions of the present invention are those that, in addition to specifically binding amyloid in vivo and capable of crossing the blood brain barrier, are also non-toxic at appropriate dosage levels and have a satisfactory duration of effect.
- a ⁇ (10-43) Chrysamine G derivative binding is first analyzed using synthetic A ⁇ peptide called A ⁇ (10-43).
- the 10-43 peptide was chosen because it has been shown that this peptide provides a model system containing all of the characteristic structural features of A ⁇ peptides. Hilbich et al . , J. Mol . Biol . 218: 149 (1991) .
- the 10- 43 amino acid fragment of A ⁇ was synthesized with 9- fluorenylmethyl chloroformate (FMOC) chemistry by the Peptide Synthesis Facility of the University of Pittsburgh.
- the peptide was characterized by mass spectrometry and the major component had an M R of 3600 g/mole (calc. 3598) .
- the peptide was further purified by the method of Hilbich et al . which, in brief, consisted of sequential size-exclusion chromatography on a Biogel P10 column (2 x 180 cm, 200-400 mesh, Biorad, Richmond, CA) in 70% formic acid followed by a second elution through a Biogel P4 column (2 x 180 cm, 200-400 mesh) in 1M acetic acid.
- Hilbich et al . J. Mol . Biol . 218: 149 (1991).
- the peptide was lyophilized and stored at -80°C until used in the binding assays.
- A_Bi_ ⁇ o acid sequence for A (10 -43 ) is as follows :
- Binding assay to synthetic A ⁇ (10-43) Binding assays were performed in 12 x 75 mm borosilicate glass tubes. Various concentrations of nonradioactive Chrysamine G derivatives were added in 10% ethanol/water. Ethanol was necessary to prevent the micelle formation which occurs with these diazo dye derivatives, since the micelles are trapped by the filter even in the absence of peptide. To the above solution, 25 ⁇ l of a 0.36 mg/ml suspension of A ⁇ (10-43) in H 2 0 was added and 10% ethanol was added to bring the volume to 950 ⁇ l.
- the mixture was rapidly vortexed and the binding reaction was stopped by vacuum filtration through Whatman GF/B filters using a Brandel M-24R Cell Harvester (Gaithersburg, MD) followed by two 3-ml washes with 10% ethanol at room temperature at times of 5, 10, 20, 30, 45, 60, 75, 135, 240, and 300 sec; bound radioactivity was determined as above.
- 25 ⁇ l of 0.36 mg/ml A ⁇ (10-43) were placed in 450 ⁇ l of 10% ethanol followed by 25 ⁇ l of 2.5 ⁇ M [ 14 C] Chrysamine G in 40% ethanol . This mixture was vortexed and incubated for 30 min at room temperature.
- the mixture was diluted with 4.5 ml of 10 ⁇ M nonradioactive Chrysamine G in 10% ethanol at time zero, the mixture was rapidly vortexed, and the dissociation was stopped by filtration as above at times of 0.5, 1.5, 3, 5, and 15 min, and bound radioactivity was determined as above.
- tissue homogenizer PT 10/35, Brinkman Instruments Inc.
- mice Female Swiss-Webster mice were injected in the lateral tail vein with approximately 0.03 ⁇ Ci/g of [ 14 C] Chrysamine G in a 0.9% NaCl solution. Mice were sacrificed by cervical dislocation at intervals of 15 min, 35 min, 1 hr, 4 hr, and 24 hr after injection. The carotid blood, brain, liver, and kidneys were rapidly obtained, weighed, and homogenized in distilled/deionized H 2 0 using a ground glass homogenizer.
- the invention relates to a pharmaceutical composition and method for preventing cell degeneration and toxicity associated with fibril formation in certain "amyloidosis associated" conditions such as Alzheimer's Disease, Down's Syndrome and Type 2 diabetes mellitus, hereditary cerebral hemorrhage amyloidosis (Dutch) , amyloid A (reactive) , secondary amyloidosis, familial mediterranean fever, familial amyloid nephropathy with urticaria and deafness (Muckle-wells Syndrome) , amyloid lambda L-chain or amyloid kappa L-chain (idiopathic, myeloma or macroglobulinemia-associated) A beta 2M (chronic hemodialysis) , ATTR (familial amyloid polyneuropathy (Portuguese, Japanese, Swedish) , familial amyloid cardiomyopathy (Danish) , isolated cardiac amyloid, (systemic senile amyloid
- This method involves administering a pharmaceutical composition comprising Chrysamine G, or one of the above described derivatives thereof, to a subject suspected of having or at high risk of developing such amyloidosis associated condition.
- the therapeutic compounds of the present invention include only non-toxic, non-carcinogenic compounds. That is, the present invention addresses the problems with potential carcinogenicity by using only diazo compounds based on 3, 3 ' , 5, 5 ' - tetramethylbenzidine. 3, 3 ' , 5, 5 ' -Tetramethylbenzidine is a well-studied non-mutagenic, non-carcinogenic substitute for benzidine. Holland et al . .
- alkenyl and alkynyl derivatives of the azo compounds are not substrates for reduction by bacterial or mammalian azo reductases.
- compounds of the present invention intended for therapeutic use are advantageous over existing compounds because they contain either a non- mutagenic, non-carcinogenic benzidine derivative or an alkenyl or alkynyl linkage which is not a substrate for bacterial azo-reductases in the intestines.
- a ⁇ neurotoxicity requires fibril formation and is inhibited by Congo red.
- Congo red the amyloid fibril-binding dye Congo red inhibits fibrillar A ⁇ neurotoxicity by inhibiting fibril formation or by binding preformed fibrils.
- Congo red also has been shown to inhibit pancreatic islet cell toxicity of diabetes-associated amylin, another type of amyloid fibril.
- Lorenzo et al . , supra . See also, Burgevin et al . NeuroReport 5: 2429 (1994) ; Pollack et al . , J. Neurosci . Letters 184: 113-116 (1995) ; Pollack et al . Neuroscience Letters 197: 211 (1995) .
- amyloid-binding compounds such as Chrysamine G and its derivatives, which are similar to Congo red but which, unlike Congo red, enter the brain well, would be effective in preventing cell degeneration and toxicity associated with fibril formation in amyloidosis associated conditions.
- Example 8 and Figures 12 and 13 it is shown that Chrysamine G has effects very similar to those previously reported for Congo red in having a dose- dependent, protective effect in rat pheochromocytoma. Therefore, these in vi tro assays provide a means for selecting compounds for use in pharmaceutical compositions for the prevention of cell degeneration and toxicity associated with fibril formation.
- amyloid-associated neurodegeneration in addition to following amyloid formation serially, the presence of amyloid- associated neurodegeneration, as measured by the formation of dystrophic neurites, synapse loss, neurofibrillary tangle formation and gliosis, also is assessed in serial necropsies in animals treated and untreated with the compounds of the present invention.
- a pharmaceutical composition comprising Chrysamine G or derivatives thereof, is administered to subjects in whom amyloid or amyloid fibril formation, cell degeneration and toxicity are anticipated.
- such subject is a human and includes, for instance, those who are at risk of developing cerebral amyloid, including the elderly,- nondemented population and patients having amyloidosis associated diseases and Type 2 diabetes mellitus.
- the term "preventing” is intended to include the amelioration of cell degeneration and toxicity associated with fibril formation.
- amelioration is meant the prevention of more severe forms of cell degeneration and toxicity in patients already manifesting signs of toxicity, such as dementia.
- the pharmaceutical composition for purposes of preventing cell degeneration and toxicity associated with fibril formation in amyloidosis associated diseases comprises Chrysamine G or a derivative thereof described above and a pharmaceutically acceptable carrier.
- such pharmaceutical composition comprises serum albumin, Chrysamine G or Chrysamine G derivative and a phosphate buffer containing NaCl .
- Other pharmaceutically acceptable carriers include aqueous solutions, non-toxic excipients, including salts, preservatives, buffers and the like, as described, for instance, in REMINGTON'S PHARMACEUTICAL SCIENCES, 15th Ed., Easton: Mack Publishing Co., pp. 1405-1412 and 1461-1487 (1975) and THE NATIONAL FORMULARY XIV., 14th Ed. Washington:
- non-aqueous solvents examples include propylene glycol, polyethylene glycol, vegetable oil and injectable organic esters such as ethyl oleate.
- Aqueous carriers include water, alcoholic/aqueous solutions, saline solutions, parenteral vehicles such as sodium chloride, Ringer's dextrose, etc.
- Intravenous vehicles include fluid and nutrient replenishers.
- Preservatives include antimicrobial, anti-oxidants, chelating agents and inert gases. The pH and exact concentration of the various components the pharmaceutical composition are adjusted according to routine skills in the art. See, Goodman and Gilman's THE PHARMACOLOGICAL BASIS FOR THERAPEUTICS (7th Ed. ) .
- such pharmaceutical composition could be administered orally, in the form of a liquid or solid, or injected intravenously or intramuscularly, in the form of a suspension or solution.
- pharmaceutically effective amount is meant an amount that prevents cell degeneration and toxicity associated with fibril formation. Such amount would necessarily vary depending upon the age, weight and condition of the patient and would be adjusted by those of ordinary skill in the art according to well-known protocols.
- a dosage would be between 0.1 and 100 mg/kg per day, or divided into smaller dosages to be administered two to four times per day. Such a regimen would be continued on a daily basis for the life of the patient.
- the pharmaceutical composition could be administered intramuscularly in doses of .1 to 100 mg/kg every one to six weeks.
- the invention relates to a method of detecting amyloid deposits in biopsy or post-mortem tissue.
- the method involves incubating formalin-fixed tissue with a solution of a compound of Formula I, described above.
- the solution is 25-100% ethanol, (with the remainder being water) saturated with the compound of Formula I .
- the compound stains or labels the amyloid deposit in the tissue, and the stained or labelled deposit can be detected or visualized by any standard method.
- detection means include microscopic techniques such as bright-field, fluorescence, laser- confocal and cross-polarization microscopy.
- the invention relates to a method of quantifying the amount of amyloid in biopsy or post-mortem tissue.
- This method involves incubating a labelled derivative of Chrysamine G, preferably the compounds of Formula I, or a water- soluble, non-toxic salt thereof, with homogenate of biopsy or post-mortem tissue.
- the tissue is obtained and homogenized by methods well known in the art .
- the preferred label is a radiolabel, although other labels such as enzymes, chemiluminescent and immunofluorescent compounds are well known to skilled artisans.
- the preferred radiolabel is 125 I, 14 C or 3 H, the preferred label substituent of Formula I is at least one of R-.- R 7 , R 10 -R 27 . Tissue containing amyloid deposits will bind to the labeled derivatives of Chrysamine G.
- the bound tissue is then separated from the unbound tissue by any mechanism known to the skilled artisan, such as filtering.
- the bound tissue can then be quantified through any means known to the skilled artisan. See Example 3.
- the units of tissue-bound radiolabeled Chrysamine G derivative are then converted to units of micrograms of amyloid per 100 mg of tissue by comparison to a standard curve generated by incubating known amounts of amyloid with the radiolabeled Chrysamine G derivative.
- the invention relates to a method of distinguishing an Alzheimer's diseased brain from a normal brain involving obtaining tissue from (i) the cerebellum and (ii) another area of the same brain, other than the cerebellum, from normal subjects and from subjects suspected of having Alzheimer's disease. See Example 3. Such tissues are made into separate homogenates using methods well known to the skilled artisan, and then are incubated with a radiolabeled Chrysamine G derivative. The amount of tissue which binds to the radiolabeled Chrysamine G derivative is then calculated for each tissue type (e.g.
- cerebellum, non-cerebellum, normal, abnormal cerebellum, non-cerebellum, normal, abnormal
- the ratio for the binding of non-cerebellum to cerebellum tissue is calculated for tissue from normal and for tissue from patients suspected of having Alzheimer's disease. These ratios are then compared. If the ratio from the brain suspected of having Alzheimer's disease is above 90% of the ratios obtained from normal brains, the diagnosis of Alzheimer's disease is made.
- Chrysamine G i.e., 4,4 '-bis (3- carboxy-4-hydroxyphenylazo) -biphenyl
- reaction steps will be referred to as the "Chrysamine G Synthesis" general procedure.
- Benzidine 2HCl (28.9 mg, 0.11 mmole, Sigma Chemical Company, St. Louis, MO) was added to 1.5 ml of 1 :1 DMSO:distilled/deionized H 2 0 in a 50cc round bottom flask.
- Each of the reaction steps were carried out at 0°C unless otherwise specified. Twenty-nine ⁇ l of concentrated HC1 were added, resulting in a clear solution after stirring.
- the UV/visible spectrum in 40% ethanol showed a ⁇ - nax at 389 nm.
- the molar absorptivity of Chrysamine G was determined by calculating the concentration of Chrysamine G through comparison of peak areas to an internal standard by NMR and then immediately running the UV/vis spectrum of an aliquot of the NMR sample diluted in 40% ethanol.
- the molar absorptivity in 40% ethanol at 389 nm was 5.5 x 10 4 AU/(cm «M) .
- Chrysamine G was synthesized by a modification of the above procedure. The tetra-azotization of benzidine was performed as described above except in
- 4,4' -bis (3-carboxy-4-hydroxy-5- isopropylphenylazo) -3-iodobiphenyl is synthesized by substituting 3-bromobenzidine (see below) for benzidine and methyl 3-isopropylsalicylate for methyl salicylate in the Chrysamine G Synthesis described above.
- the bromo derivative thus obtained is converted to the tri- alkyl tin and then to the iodo derivative as described in detail below.
- Chrysamine G are synthesized by substituting 3- bromobenzidine (see below) for benzidine and 1-hydroxy- 2-naphthoic acid (or, for related derivatives, other hydroxynaphthoic acid isomers, hydroxyquinolinecarboxylic acids such as kynurenic acid, or hydroxybenzodiazinecarboxylic acids such as 5- hydroxyquinoxaline-6-carboxylic acid) for methyl salicylate in the Chrysamine G Synthesis described above.
- the bromo derivative thus obtained is converted to the tri-alkyl tin and then to the iodo derivative as described in detail below.
- nitroso compounds are coupled to amines in glacial acetic acid by the Mills reaction.
- Nitroso compounds are made by the method of Coleman et al. , Organic Synthesis Collective Vol. Ill: p668.
- 3-nitrobenzoic acid Aldrich Chem. Co., Milwaukee, WI
- Zinc dust (372 mg) is added in small portions over 5 minutes.
- the temperature begins to rise, but is kept between 50 and 55°C by the use of an ice bath.
- the mixture is stirred at that temperature for 20 minutes and then the zinc residues are filtered and washed with boiling water.
- the combined filtrate and washings are cooled to 0°C in a beaker by the addition of ice.
- a cold solution of sulfuric acid 750 ⁇ l of concentrated acid plus enough ice to bring the temperature to -5°C
- An ice-cold solution of 170 mg of sodium dichromate dihydrate in 750 ⁇ l of water is added all at once with stirring.
- Methoxy derivatives of all phenol compounds are synthesized by the following procedure. To one equivalent of the phenolic Chrysamine G ester derivative dissolved in acetone at an approximate concentration of 5 mg/ml, is added 10 equivalents of methyl iodide (Aldrich Chemical Company, Milwaukee, WI) and 10 equivalents of K 2 C0 3 . The suspension is refluxed for 6-24 hrs, taken to dryness on a rotary evaporator, and extracted with chloroform. The ester is hydrolysed by addition of 1:1 THF/0.1 N NaOH to a concentration of approximately 1 mg/ml and stirring at room temperature from 24-72 hours. Unhydrolysed methoxy-ester is removed by extraction with chloroform, the pH was adjusted to ⁇ 2, and the methoxy-acid was extracted into ethyl acetate.
- substituted benzidines including, but not limited to, those listed below are synthesized by the referenced methods and also can be used in place of unsubstituted benzidine: 3,3'- dibromobenzidine and 3,3 ' -diiodobenzidine (Snyder, H. et al . , J. Am. Chem. Soc . 71: 289-291, 1949) ; 3- bromobenzidine and 3-iodobenzidine (Badger, G. et al . , Aust . J. Chem. 17: 1036-1049, 1964; Holland, V. et al . , Tetrahedron 30: 3299-3302, 1974) .
- Chrysamine G are made by substituting 2,7-diamino- fluorene (Aldrich Chemical Company, Milwaukee, WI) for benzidine in the tetra-azotozation procedures and then coupled by the same procedures used for tetra-azotized benzidine.
- 2, 7-diaminophenanthracene, 2,7- diamino-3, 6-dimethylbenzo [c] cinnoline, 2,7- diaminofluorenone, 9-methyl-2, 7-diaminocarbazole, 3,7- diaminodibenzofuran (unlike the remainder of these polycyclic compounds, the conventional numbering system puts the oxygen bridge atom of dibenzofurans at the 5- position instead of the 9-position, therefore the amino substituents are in the 3 , 7-positions rather than the 2, 7-positions as in the other compounds even though, spatially, the positions are equivalent)
- 2,7- diaminodibenzothiophene, and 2,7- diaminodibenzothiophene-9, 9-dioxide ("benzidine sulphone") are substituted for benzidine in the standard tetra-azotization and coupling procedures.
- 9- Methyl-2, 7-diaminocarbazole compounds may be N- demethylated after coupling if the unsubstituted carbazole is desired.
- 2, 7-Diaminophenanthracene is synthesized by reduction of 2, 7-dinitrophenanthracene made from 2-amino-3 ' , 5-dinitro-cis-stilbene by the method of Ullmann and Mallet Ber. 31: 1694-1696, 1898 and Nunn, A. et al . , J. Chem. Soc . 1952: 2797-2803.
- 2, 7-Diamino-3, 6-dimethylbenzo [c] cinnoline is prepared by NaOBr oxidation of the corresponding hydrazo compound formed via the reduction of 3 ,3 ' -dimethyl- 6,6 ' -dinitrobenzidine (Aldrich Chemical Co., Milwaukee, WI) with zinc in aqueous NaOH by the method of Snyder et al . , J. Amer. Chem . Soc . 71: 289 (1949) .
- 3 , 3 ' -dimethyl-6, 6 ' -dinitrobenzidine can be first tetra-azotized and coupled in place of benzidine, followed by treatment with Zn/NaOH and NaOBr as above.
- 2,7-Diaminofluorenone is synthesized by reduction of 2, 7-dinitrofluorenone made from 2-amino- 3 ' , 5-dinitrobenzophenone by the method of Ullmann and Mallet Ber. 31: 1694-1696, 1898 and Nunn, A. et al . , J. Chem. Soc . 1952: 2797-2803.
- 9-Methyl-2, 7- diaminocarbazole is synthesized by reduction of 9- methyl-2, 7-dinitrocarbazole made by N-methylation of 2, 7-dinitrocarbazole prepared from 2-amino-3 ' , 5- dinitrodiphenylamine as described in Saunders, K.H. and Allen, R.L.M. AROMATIC DIAZO COMPOUNDS 625-640
- the 5-fluoro derivative, 4,4 ' -bis (2-hydroxy-3- carboxy-5-fluorophenylazo) -biphenyl) is synthesized by substituting 5-fluorosalicylic acid (Aldrich Chemical Company, Milwaukee, WI) for salicylic acid.
- Aromatic fluoroalkyl derivatives are synthesized employing the method of Bishop et al . , J. Med. Chem. 34: 1612 (1991) in which Claisen rearrangement of the appropriate O-allyl ethers forms an aromatic allyl derivative which can be further functionalized to yield the fluoroethyl or fluoropropyl derivatives.
- an aromatic iodide can be readily converted to an aromatic alkyne consisting of two to five carbon atoms in length using the palladium- assisted coupling methodology of Sonogashira et al . , Tetrahedron Letters 4467-4470 (1975) . Subsequent derivatization of the alkyne yields the fluoroalkyl derivative.
- Fluoroalkoxy derivatives may be prepared by the method of Chumpradit et al . , J. Med. Chem . 36: 21 (1993) in which alkylation of the appropriate phenol with the appropriate 1-bromo (or iodo or sulfonyloxy) - omega-fluoroalkane yields the corresponding fluoroalkoxy derivative. Radiofluorination of Aromatic Alkylsulfonyloxy and Alkoxysulfonyloxy Derivatives
- Tri-Alkvl Tin Derivatives of Chrysamine G The general structure of the tri-alkyl tin derivative of Chrysamine G is shown in Figure 2B.
- one tri-alkyl tin group will be substituted at the 3-position on one side of the biphenyl moiety, but other positions, including the salicylic acid or heterocyclic moiety are also potential targets.
- These tri-alkyl tin derivatives are stable immediate precursors for preparation of the radioiodinated and radiobrominated compounds to be used in humans . More specifically, these tri-alkyl tin derivatives are used to prepare the halogenated radioactive compounds applicable for use in in vivo imaging of amyloid.
- Tri-alkyl tin derivatives are prepared from the appropriate arylhalides, [ (C 6 H 5 ) 3 P] 3 Pd(0) , and hexaalkylditin by previously published procedures including Kosugi, M. , et al., Chem. Lett . 1981: 829;
- 3-Bromo or 3-iodo-4,4 ' -bis (3-methoxycarbonyl-4- hydroxyphenylazo) -biphenyl or its dimethyl ether are prepared by synthesis of 3-bromo- or 3-iodobenzidine (see above) , tetra-azotization and coupling to methyl salicylate as for the synthesis of Chrysamine G, and methylation of the phenol as described above when the methoxy compound is desired.
- the tributyl or trimethyl tin derivatives are radio-iodinated with Na[ 125 I] or Na[ 123 l] or radio- brominated with Na [ 75 Br] or Na [ 76 Br] by published procedures such as Mathis et al . , J. Labell . Co p . and
- 3- or 4-amino derivatives of salicylic acid, or the corresponding derivatives of the heterocyclic analogues of salicylic acid shown in Figure 2 are converted to the corresponding diazo compounds with sodium nitrite and HCl or H 2 S0 4 .
- the iodine derivatives are directly prepared by forming the diazonium iodide which is then converted into the aryl iodide, or by way of the triazene intermediates. See, e . g. , Greenbaum, F. Am. J. Pharm. 108: 17 (1936), Satyamurthy, N. and Barrio, J., J. Org. Chem . 48: 4394
- Aryl bromides and chlorides are prepared from the diazo compounds by treatment with CuCl or CuBr according to the Sandmeyer reaction or via the triazene as for the iodine derivatives.
- Aryl fluorides are prepared by treating the diazonium compounds with NaBF 4 , HBF 4 , or NH 4 BF 4 according to the Schiemann reaction or via triazene decomposition similar to the iodine derivatives.
- Aryl thiols are prepared from the diazonium compounds by treatment with sulfur-containing nucleophiles such as HS " , EtO-CSS " , and S 2 2 ".
- aryl thiols can be prepared by replacement of aryl halides with sulfur containing nucleophiles. These reactions are described in March, J., ADVANCED ORGANIC CHEMISTRY: REACTIONS, MECHANISMS, AND STRUCTURE (3rd Edition, 1985) .
- 99m Tc derivatives are prepared by complexation with the aryl thiols. Radiolabeling with 1X C can be readily done via N-methylation, O-methylation as described above substituting ["C]methyl iodide, [ U C] alkylation, or [ X1 C] carboxylation of suitable Chrysamine G analogues.
- Radiobromination using 75 Br and 76 Br can be accomplished using either electrophilic (Br + ) or nucleophilic (Br") substitution techniques analogue to radioiodination techniques, see Coenen, H. , supra .
- 2, 6-Dihydroxybenzoic acid ( ⁇ -resorcylic acid) methyl ester (TCI America, Portland, OR) is converted to the hydroxamic acid by the use of hydroxylamine hydrochloride according to the method of B ⁇ shagen (Chem. Ber 100: 954-960; 1967) .
- the hydroxamic acid is converted to the corresponding 3-hydroxy-1,2- benzisoxazole with the use of S0C1 2 and then triethylamine, also by the method of B ⁇ shagen (Chem.
- methyl salicylate is coupled to benzidine as usual and the resulting 4,4 '-bis (3- methoxycarbonyl-4-hydroxyphenylazo) -biphenyl (or the dimethyl ester of Chrysamine G) is converted to the hydroxamic acid and then the benzisoxazole by the method of B ⁇ shagen as described above.
- a third type of 3-hydroxy-1,2-benzisoxazole is synthesized from the dimethyl esters of several isomeric dihydroxy benzenedicarboxylic acids including 4, 6-dihydroxy-1, 3- benzenedicarboxylic acid, 3, 6-dihydroxyphthalic acid, and 2, 5-dihydroxyterephthalic acid (Aldrich Chem. Co., Milwaukee, WI) .
- the dihydroxy/diesters is converted to dihydroxy/dihydroxamic acids by reaction with hydroxylamine by the method of B ⁇ shagen described above.
- Conversion to the double benzisoxazole is effected by treatment with S0C1 2 and triethylamine, again, by the method of B ⁇ shagen described above.
- 3-Hydroxyphthalimide made from 3-hydroxyphthalic anhydride (Aldrich Chemical Company, Milwaukee, WI) is coupled with benzidine or its derivative via the same procedure used for salicylic acid derivatives and then converted to the tri-alkyl tin and iodo-derivatives as described above.
- Synthesis of the phthalhydrazide or 2,3- benzodiazine-1,4 (2H,3H) -dione derivative see Figure 2E
- 3-Hydroxyphthalhydrazide made from the reaction of 3-hydroxyphthalic anhydride (Aldrich Chemical Company,
- 4-Amino-l, 8-naphthalimide is coupled with benzidine or its derivative via standard procedures for diazo coupling of aryl amines, as described in
- the oxadiazoles are synthesized by treatment of the tetrazoles prepared as above with an acid anhydride (such as acetic anhydride) .
- An alternate method is that of Bamford et al . , J. Med. Chem. 38: 3502 (1995) .
- hydrazide derivatives of Chrysamine G or salicylic acid obtained by treatment of the respective esters with hydrazine
- methyl isothiocyanate in the presence of dicyclohexylcarbodiimide.
- the aniline derivative is synthesized by substituting two equivalents of aniline (Fisher Chemical Co., Fair Lawn, NJ) for each equivalent of benzidine.
- the 2, 2 ' -disulfonic acid derivative is synthesized by substituting benzidine-2, 2 ' -disulfonic acid (Pfaltz & Bauer, Inc., Waterbury, CT) for benzidine.
- the phenol derivative is synthesized by substituting one equivalent of phenol for each equivalent of salicylic acid. Congo red (Aldrich certified grade) is obtained commercially.
- Chrysamine G binds well to synthetic A ⁇ (10-43) peptide in vi tro .
- Figure 4A shows a Scatchard analysis of the binding of Chrysamine G to A ⁇ (10-43) .
- the higher affinity component has a K D of 0.257 ⁇ M and a B max of 3.18 nmoles Chrysamine G/mg A ⁇ (10-43) .
- the lower affinity component is less well defined by these data, but appears to have a K D of 4.01 ⁇ M and a B ⁇ of 18.7 nmoles Chrysamine G/mg A ⁇ (10-43) .
- the low affinity component represents the binding of Chrysamine G at high concentrations to a distinct, low-affinity site, not the binding to an impurity in the preparation.
- the amount of Chrysamine G injected in vivo is so low that there is not any binding to the- low-affinity component. At very low concentrations, the ratio of high-to-low affinity binding is very large.
- Chrysamine G binding is linear with peptide concentration over the range employed, as shown in Figure 5.
- A A e 1 t o
- t is the time in min
- Chrysamine G Derivatives Can Inhibit the Binding of Chrysamine G to A ⁇
- K values for the inhibition of [ 14 C] Chrysamine G binding to A ⁇ (10-43) by the Chrysamine G analogues are shown under the chemical structures in Figure 1 and several displacement curves are shown in Figure 3.
- K x is defined as IC 50 / (1+ [L] /K D ) , where [L] is the concentration of [ 14 C] Chrysamine G in the assay (.100- .125 ⁇ M) and K D is 0.26 ⁇ M, the K D of Chrysamine G determined by the Scatchard analysis above.
- Chrysamine G itself gives a K. of 0.37+ 0.04 ⁇ M, a value very consistent with those obtained from the Scatchard and kinetic analyses.
- the activity of the difluoro Chrysamine G derivative suggests that an 18 F difluoro Chrysamine G derivative works for PET imaging and an 19 F difluoro Chrysamine G derivative works for MRS/MRI imaging of brain.
- the 3-ICG is slightly more potent than Chrysamine G.
- the activity of the 3-ICG derivative suggests that an 123 I difluoro Chrysamine G derivative works for SPECT imaging. Methylating the phenol of 3-ICG decreases the affinity by a factor of 10 in 3-IGC(OMe) 2 . Methylating the carboxylate group effected an even greater (about
- ⁇ G is the energy of the binding reaction
- R is the molar gas constant [8.31441 J/ (mole»°K) ]
- T is temperature in °K
- K eq is the equilibrium constant for the reaction:
- the expected energy of binding would be about 19 KJ/mole. From the K A of 73 ⁇ M for the aniline derivative, the energy of binding is 23 KJ/mole which is in acceptable agreement with the predicted value.
- Figure 7 shows a schematic of this model, generated using MacroModel 2.5, in which Chrysamine G spans 5 peptide chains in an anti-parallel beta-sheet conformation.
- the peptides are used without further structural refinement.
- the peptides are aligned so that alternate chains were spaced 4.76 A apart, characteristic of beta-sheet fibrils.
- Alternate peptide chains are drawn in black and white.
- Chrysamine G (black) is energy minimized and aligned with the fibril model to maximize contact with lysine- 16 (light grey ovals in top figure) and the hydrophobic phenylalanine 19/20 region (bottom) .
- the two views are of the same model at approximately 90° from one another.
- the white arrows indicate the direction taken to obtain the alternate view.
- AD brain Under the conditions employed, control and AD brain showed a single binding component.
- the useful Chrysamine G derivatives or analogues provided herein have binding affinities that are at least in the range of 0.01 to 10.0 ⁇ M K D , as measured by binding to either synthetic A ⁇ peptide or Alzheimer's Disease brain tissue; higher affinity compounds having binding affinities in the range of 0.0001 to 0.01 ⁇ M are also useful in the method of the present invention.
- Chrysamine G binding may not be specific for A ⁇ . Instead, Chrysamine G binding may reflect the total amyloid "load" of the brain, comprised of aggregated deposits of A ⁇ in neuritic plaques and cerebrovascular amyloid. Deposits of phosphorylated tau protein in NFTs may contribute to Chrysamine G binding as well. Goedert, M. et al . , PNAS 85: 4051 (1988) . NFTs also are composed of anti- parallel beta-sheet fibrils similar in quaternary - structure to fibrils of A ⁇ . Kirschner et al . , Proc . Na tl . Acad . Sci . U. S . A . 83: 503 (1986) .
- vi tro binding assays such as those described above and below are widely used in the art as models to screen compounds for in vivo binding in brain and to predict success in subsequent in vivo imaging studies. See, Young, A. et al . , Receptor Assays : In Vi tro and In Vivo . in POSITRON EMISSION TOMOGRAPHY AND AUTORADIOGRAPHY (Phelps, M. , Mazziota, J. , and Schelbert, H. eds. ) pp. 73-111 (1986) .
- the labeled Chrysamine G and Chrysamine G derivatives of the invention also may be used in the in vi tro binding assays described above and below to quantitate amyloid deposits in biopsy or post-mortem specimens.
- the average amount of [ 14 C] Chrysamine G bound to control cerebellum is nearly identical to the amount bound to AD cerebellum (Table 2) , supporting the use of cerebellum as an internal control. Therefore, the cerebellar ratio (CBR) accurately reflects the absolute quantity of [ 14 C] Chrysamine G bound and offers the advantage of providing an internal control for each brain. Binding is greater in AD brain whether expressed in absolute terms of fmol/ ⁇ g protein (Table 2) or as a ratio to the binding in the cerebellum of the same brain (Table 3) . The CBR is the more sensitive measure and shows less variability between brains . The use of total binding and CBRs greatly facilitates extension of these ex vivo results to in vivo studies. Accordingly, the results below are expressed in these terms whenever appropriate. Table 2. Comparison of total binding in AD and control brain*.
- the CBR for each sample is obtained by dividing the absolute value of [ 1 C] Chrysamine G binding in that sample by the absolute value of [ 14 C] Chrysamine G binding in the cerebellar sample from that same brain.
- the values in the table are the average CBRs from each brain area ( ⁇ SEM) . High- and low-plaque AD brains combined.
- Figure 9A and 9B shows the binding of
- FIG. 9C shows the data from two patients who had Down's syndrome. Down's syndrome patients all develop deposits of A ⁇ by their fourth decade and many develop AD. Wisniewski et al. , Neurology 35: 957 (1985) ; Schapiro et al . , Neurobiol . Aging 13, 723 (1992) . Both of these patients showed [ 14 C] Chrysamine G binding above the control range. Since the younger patient (23 years old) had amyloid deposits but was not yet clinically demented, Figure 9C suggests that Chrysamine G can detect differences from control in non-demented patients destined to develop AD long before the dementia is clinically evident.
- the compounds and method of the invention provide two useful measurements for differentiating AD brain from normal brain; either (1) total Chrysamine G binding (Table 2) or (2) the ratio of Chrysamine G binding in a given brain area to binding in the cerebellum of the same brain (Table 3) .
- These measurements furnish two great advantages for in vivo quantitation of AD neuritic plaques.
- the instant invention can quantify A ⁇ deposition. without having to expose the subject to a second injection of radioactive material in order to measure non-specific binding. Because of this, the data are expressed as total binding only. In all of the experiments presented, specific binding data yields even greater differences between AD and control brain.
- MRI volume measurements performed in conjunction with the method* of the invention are analogous to those routinely employed in the art. See, Pearlson, G. and Marsh, L. MAGNETIC RESONANCE IMAGING IN PSYCHIATRY in Annual Review of Psychiatry (Vol. 12) Oldham, J. et al . , eds. p. 347-381 (1993). Therefore a method for determining the total radioactivity per volume of brain area would use the following equation:
- S/V CB where S/V ⁇ is the signal/volume in the cerebellum of the same subject during the same imaging study.
- This ratio from any brain area other than cerebellum from a patient suspected of having AD or other pathological condition characterized by the deposition of amyloid could then be compared to the normal range of the analogous ratio from the same brain area of a group of age-matched normal control subjects.
- the ratio of the binding to brain areas with high deposits of neuritic plaques to the cerebellum can be used as the parameter to distinguish Alzheimer from control subjects.
- the octanol-water partition coefficient is a measure of the relative lipophilicity of a compound. The more lipophilic a compound, the more likely it is to cross the blood-brain barrier. See, Goodman and Gilman's THE PHARMACOLOGICAL BASIS FOR THERAPEUTICS
- the octanol/water partition coefficient of Chrysamine G is 60.22 ⁇ 3.97 and that of Congo red is 0.665 ⁇ 0.037 (p ⁇ 0.001). This suggests that Chrysamine G is approximately 90 times more lipophilic than Congo red and therefore is theoretically more likely to cross the mammalian blood-brain barrier.
- the octanol/water partition coefficients for the 3-iodo and 3,3'-diiodo derivatives of Chrysamine G ( Figure 1) are 72.53 ⁇ .74 and 112.9 ⁇ 7.3, respectively.
- CHRYSAMINE G DERIVATIVES TO CROSS THE BLOOD-BRAIN BARRIER AND METABOLISM OF CHRYSAMINE G
- amyloid probes to diagnose AD in vivo requires them to be able to cross the blood-brain barrier and gain access to parenchymal amyloid deposits.
- Chrysamine G The ability of Chrysamine G to cross the blood- brain barrier was studied in Swiss-Webster mice. After i.v. injection, the brain/blood ratio measured at 15 min was over 10:1 and approached 20:1 by 35 min ( Figure 10) . The radioactivity in brain stayed nearly constant over this period, but decreased in the blood and increased in the liver. The brain/kidney ratio was highest at 15 min (over the time points sampled) and approached 0.5. When brain and liver were extracted 60 min after i.v.
- In vivo animal testing provides yet a further basis for determining dosage ranges, efficacy of transfer through the blood barrier and binding ability.
- Particularly preferred for this purpose are the transgenic mouse model of Games et al . , (Nature 373: 523 (1995)) and the "senile animal" model for cerebral amyloidosis; i . e . , animals such as the transgenic mice or aged dogs or monkeys, which are known to develop variable numbers of Alzheimer-type cerebral neuritic plaques, see Wisniewski et al . , J. Neuropathol . & Exp . Neurol . 32: 566 (1973), Selkoe et al . , Science 235: 873 (1987) , are tested for binding and detection efficacy.
- This in vivo assay requires control-biopsy or necropsy monitoring to confirm and quantify the presence of amyloid deposits.
- a dose of approximately 10 mCi of an appropriately radiolabeled derivative of Chrysamine G with a specific activity of approximately 500 Ci/mole or higher is injected intravenously into normal subjects and patients suspected of having AD and monitored by SPECT or PET imaging to analyze the detectability of the derivative in brain relative to other organs and to define the time course of detectability in the brain.
- a dose which can be reliably detected is defined as a
- An imaging-effective dose of an appropriately, radioactively labeled derivative of Chrysamine G is injected into a subject suspected of having brain amyloid deposition due to pathological conditions such as AD. After a period of 15 minutes to 24 hours, the radioactive signal from brain is detected by SPECT or PET. Radioactivity is simultaneously detected in all brain areas included in the field of view of the detector. This field of view will be set up so as to include large portions of the cerebellum, superior • Ill -
- Example 3 temporal cortex, superior/middle frontal cortex, and intervening brain regions. An MRI scan will be performed prior to the study so that corrections can be made for brain atrophy in the areas of interest by methods discussed in Example 3.
- the S/V A , S/V ⁇ , and Ratio A variables discussed in Example 3 will be calculated and compared to analogous normative ratios obtained previously from age-matched normal control subjects.
- the top frame of Figure 11 demonstrates two neuritic plaques stained by Chrysamine G.
- the staining method was that of Stokes and Trickey, J. Clin . Pathol . 26: 241-242 (1973) with Chrysamine G substituted for
- Chrysamine G administered intraperitoneally no notable behavioral effects or toxicity were observed in mice for periods up to 72 hrs.
- the doses of [ 14 C] Chrysamine G administered were on the order of 1 mg/kg. Chrysamine G appeared to show little acute toxicity based on attempts to establish an LD 50 . Even when the maximum volume that can be injected into a mouse without harming it just from fluid volume effects (approx. 0.025 ml/g) of a saturated solution of Chrysamine G was injected into mice (100 mg/kg) , there were no behavioral changes noted for at least 72 hrs, the longest period tested. Doses required for detection of radiolabeled derivatives by SPECT or PET would be orders of magnitude below this dose.
- Congo red has been safely injected into humans in quantities much greater than would be used for the radioactive Chrysamine G derivatives.
- the LD 50 for Congo red has been shown to be 190 mg/kg mouse (Tubis et al . , J. Amer. Pharm . Assoc . 49: 422 (I960)) , which is similar to the >100 mg/kg LD 50 shown for Chrysamine G.
- these two chemically similar compounds cause similar low toxicities in mice.
- Chrysamine G derivatives can similarly be tested for toxicity in mice and other higher mammals by injecting a wide range of concentrations and studying the animals for various signs of toxicity by methods well known in the art. See, Goodman and Gilman's THE PHARMACOLOGICAL BASIS FOR THERAPEUTICS (7th Ed.) .
- Rat pheochromocytoma cells (PC-12) were grown in RPMI 1640 media with 10% fetal bovine serum. Approximately 5,000 exponentially growing cells were plated in 96-well plates in a volume of 100 ⁇ l of media and allowed to incubate at 37°C overnight. The A ⁇ (25- 35) , which had been pre-aggregated at 37°C for 7 days, was pre-incubated with Chrysamine G (CG) or related compounds in aqueous solution prior to addition of 20 ⁇ l to achieve the final concentrations given (0.01 to 10 ⁇ M A ⁇ (25-35) and 0.03 to 20 ⁇ M CG) .
- CG Chrysamine G
- the cells were incubated for 24 hrs prior to the addition of 13.3 ⁇ l of 5 mg/ml MTT (3 , (4, 5-dimethylthiazol-2-yl) 2, 5- diphenyltetrazolium bromide) in sterile phosphate buffered saline. After 4.5 hrs at 37°C, 100 ⁇ l of extraction buffer (20% w/v SDS in 50% DMF/water; pH adjusted to 4.7 with 2.5% of 80% acetic acid and 2.5% IN HC1) was added and the plates were incubated overnight. Hansen et al . , J. Immunol . Methods 119: 203 (1989) . Color development was then measured at 560 nm.
- Chrysamine G are shown in white numbers inside the filled bars.
- the protective effect of 20 ⁇ M Chrysamine G is shown in the open bars.
- Significant differences between MTT reduction in the presence and absence of Chrysamine G are shown in black numbers inside the -open bars .
- Figure 13 demonstrates the protective effect of increasing concentrations of Chrysamine G against the A ⁇ (25-35) -induced reduction of cellular redox activity of PC12 cells.
- the effect of Chrysamine G in the absence of A ⁇ (25-35) is shown in the filled bars. There was no significant difference between control (no A ⁇ , no Chrysamine G) and any of the concentrations of Chrysamine G in the absence of A ⁇ (25-35) .
- MTT reduction in the presence of 1 ⁇ M A ⁇ (25-35) and increasing concentrations of Chrysamine G is shown in the open bars .
- Significant differences in MTT reduction between the presence and absence of A ⁇ (25-35) at each concentration of Chrysamine G are shown in white numbers inside the filled bars.
- Significant differences in MTT reduction between the A ⁇ (25-35) control (no Chrysamine G) and A ⁇ (25-35) plus increasing concentrations of Chrysamine G are shown in black numbers inside the open bars .
- Chrysamine G appears to be more potent than Congo red, showing effects in the range of 0.1 to 1.0 ⁇ M. This is consistent with the Ki values for binding to synthetic A ⁇ of 0.37 ⁇ M for Chrysamine G and 2.8 ⁇ M for Congo red ( Figure 1) .
- Chrysamine G could interfere with the aggregation of A ⁇ .
- Chrysamine G could interfere with the effects (direct or indirect) of A ⁇ on the target cells.
- Congo red does inhibit aggregation of A ⁇ as well as protect against the toxic effects of aggregated A ⁇ . Lorenzo and Yankner, Proc . Natl . Acad. Sci . 91: 12243 (1994) . Interference with aggregation is unlikely in the above experiment since the A ⁇ was pre-aggregated prior to incubation with Chrysamine G.
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
- Heterocyclic Carbon Compounds Containing A Hetero Ring Having Nitrogen And Oxygen As The Only Ring Hetero Atoms (AREA)
Abstract
Amyloid binding compounds which are derivatives of Chrysamine G, pharmaceutical compositions containing, and methods using such compounds to identify Alzheimer's brain in vivo and to diagnose other pathological conditions characterized by amyloidosis, such as Down's Syndrome are described. Pharmaceutical compositions containing Chrysamine G and derivatives thereof and methods using such compositions to prevent cell degeneration and amyloid-induced toxicity in amyloidosis associated conditions are also described. Methods using Chrysamine G derivatives to stain or detect amyloid deposits in biopsy or post-mortem tissue are also described. Methods using Chrysamine G derivatives to quantify amyloid deposits in homogenates of biopsy and post-mortem tissue are also described.
Description
AZO COMPOUNDS FOR THE ANTEMORTEM DIAGNOSIS OF ALZHEIMER'S DISEASE AND IN VIVO IMAGING AND PREVENTION OF AMYLOID DEPOSITION
The present invention was made utilizing funds from the National Institute of Ageing, grant numbers AG-05443, AG-05133 and AG-08974. This is a continuation-in-part application of U.S. patent 5 application Serial No. 08/432,019, filed May 1, 1995 which is a continuation-in-part of U.S. patent application Serial No. 08/282,289, filed July 29, 1994.
Background of the Invention The present invention relates to the
10 identification of compounds that are suitable for imaging amyloid deposits in living patients. More specifically, the present invention relates to a method of imaging amyloid deposits in brain in vivo to allow antemortem diagnosis of Alzheimer's Disease. The
15 present invention also relates to therapeutic uses for such compounds.
Alzheimer's Disease ("AD") is a neurodegenerative illness characterized by memory loss and other cognitive deficits. McKhann et al . , Neurology 34: 939
20 (1984) . It is the most common cause of dementia in the United States. AD can strike persons as young as 40-50 years of age, yet, because the presence of the disease is difficult to determine without dangerous brain biopsy, the time of onset is unknown. The prevalence
25 of AD increases with age, with estimates of the
affected population reaching as high as 40-50% by ages 85-90. Evans et al . , JAMA 262: 2551 (1989) ; Katzman, Neurology 43: 13 (1993) .
By definition, AD is definitively diagnosed through examination of brain tissue, usually at autopsy. Khachaturian, Arch. Neurol . 42: 1097 (1985) ; McKhann et al . , Neurology 34: 939 (1984) . Νeuropathologically, this disease is characterized by the presence of neuritic plaques (ΝP) , neurofibrillary tangles (ΝFT) , and neuronal loss, along with a variety of other findings. Mann, Mech . Ageing Dev. 31: 213 (1985) . Post-mortem slices of brain tissue of victims of Alzheimer's disease exhibit the presence of amyloid in the form of proteinaceous extracellular cores of the neuritic plaques that are characteristic of AD.
The amyloid cores of these neuritic plaques are composed of a protein called the β-amyloid (Aβ) that is arranged in a predominately beta-pleated sheet configuration. Mori et al . , Journal of Biological Chemistry 261 : 17082 (1992) ; Kirschner et al. , PNAS 83: 503 (1986) . Neuritic plaques are an early and invariant aspect of the disease. Mann et al . , J. Neurol . Sci . 89: 169; Mann, Mech . Ageing Dev. 31: 213 (1985) ; Terry et al . , J. Neuropathol . Exp . Neurol 46: 262 (1987) .
The initial deposition of Aβ probably occurs long before clinical symptoms are noticeable. The currently recommended "minimum microscopic criteria" for the
diagnosis of AD is based on the number of neuritic plaques found in brain. Khachaturian, Arch . Neurol . , supra (1985) . Unfortunately, assessment of neuritic plaque counts must be delayed until after death. Amyloid-containing neuritic plaques are a prominent feature of selective areas of the brain in AD as well as Downs Syndrome and in persons homozygous for the apolipoprotein E4 allele who are very likely to develop AD. Corder et al . , Science 261: 921 (1993) ; Divry, P., J. Neurol . Psych . 27: 643-657 (1927) ; isniewski eϋ al . , in Zimmerman, H.M. (ed.) : PROGRESS IN NEUROPATHOLOGY, (Grune and Stratton, N.Y. 1973) pp. 1-26. Brain amyloid is readily demonstrated by staining brain sections with thioflavin S or Congo red. Puchtler et al . , J. Histochem . Cytochem . 10: 35
(1962) . Congo red stained amyloid is characterized by a dichroic appearance, exhibiting a yellow-green polarization color. The dichroic binding is the result of the beta-pleated sheet structure of the amyloid proteins. Glenner, G. N. Eng. J. Med. 302: 1283
(1980) . A detailed discussion of the biochemistry and histochemistry of amyloid can be found in Glenner, N. Eng. J. Med . , 302: 1333 (1980) .
Thus far, diagnosis of AD has been achieved mostly through clinical criteria evaluation, brain biopsies and post mortem tissue studies. Research efforts to develop methods for diagnosing Alzheimer's disease in
vivo include (1) genetic testing, (2) immunoassay methods and (3) imaging techniques.
Evidence that abnormalities in Aβ metabolism are necessary and sufficient for the development of AD is based on the discovery of point mutations in the Aβ precursor protein in several rare families with an autosomal dominant form of AD. Hardy, Nature Genetics 1: 233 (1992) ; Hardy et al . , Science 256: 184 (1992) . These mutations occur near the N- and C-terminal cleavage points necessary for the generation of Aβ from its precursor protein. St. George-Hyslop et al . , Science 235: 885 (1987) ; Kang et al . , Nature 325: 733 (1987) ; Potter WO 92/17152. Genetic analysis of a large number of AD families has demonstrated, however, that AD is genetically heterogeneous. St. George- Hyslop et al . , Nature 347: 194 (1990) . Linkage to chromosome 21 markers is shown in only some families with early-onset AD and in no families with late-onset AD. More recently a gene on chromosome 14 whose product is predicted to contain multiple transmembrane domains and resembles an integral membrane protein has been identified by Sherrington et al . , Nature 375: 754-760 (1995) . This gene may account for up to 70% of early- onset autosomal dominant AD. Preliminary data suggests that this chromosome 14 mutation causes an increase in the production of Aβ. Scheuner et al . , Soc. Neurosci . Abstr. 21: 1500 (1995) . A mutation on a very similar gene has been identified on chromosome 1 in Volga
German kindreds with early-onset AD. Levy-Lahad et al . , Science 269 : 973-977 (1995) .
Screening for apolipoprotein E genotype has been suggested as an aid in the diagnosis of AD. Scott, Nature 366: 502 (1993) ; Roses, Ann. Neurol . 38: 6-14 (1995) . Difficulties arise with this technology, however, because the apolipoprotein E4 allele is only a risk factor for AD, not a disease marker. It is absent in many AD patients and present in many non-demented elderly people. Bird, Ann. Neurol . 38: 2-4 (1995) . Immunoassay methods have been developed for detecting the presence of neurochemical markers in AD patients and to detect an AD related amyloid protein in cerebral spinal fluid. Warner, Anal. Chem . 59: 1203A (1987) ; World Patent No. 92/17152 by Potter; Glenner et al . , U.S. Patent No. 4,666,829. These methods for diagnosing AD have not been proven to detect AD in all patients, particularly at early stages of the disease and are relatively invasive, requiring a spinal tap. Also, attempts have been made to develop monoclonal antibodies as probes for imaging of Aβ. Majocha et al . , J. Nucl . Med . , 33: 2184 (1992) ; Majocha et al . , WO 89/06242 and Majocha et al . , U.S. Patent 5,231,000. The major disadvantage of antibody probes is the difficulty in getting these large molecules across the blood-brain barrier. Using antibodies for in vivo diagnosis of AD would require marked abnormalities in the blood-brain barrier in order to gain access into
the brain. There is no convincing functional evidence that abnormalities in the blood-brain barrier reliably exist in AD. Kalaria, Cerebrovascular & Brain Metabolism Reviews 4: 226 (1992) . Aβ antibodies are also disadvantageous for use in AD diagnostics because they typically stain diffuse deposits of Aβ in addition to the neuritic plaques - and the diffuse plaques often predominate. Yamaguchi et al., Acta Neuropathol . , 77: 314 (1989). Diffuse plaques may be a separate type of lesion, not necessarily involved in the dementing process of AD. The latter is suggested by findings of diffuse plaques in cognitively normal controls and aged dogs. Moran et al . , Medicina Clinica 98: 19 (1992) ; Shimada et al . , Journal of Veterinary Medical Science 54: 137 (1992) ; Ishihara et al . , Brain Res . 548: 196 (1991); Giaccone et al . , Neurosci . Lett . 114: 178 (1990) . Even if diffuse plaques are forerunners of neuritic plaques, the key pathological event in AD may be the process that turns the apparently benign diffuse plaque into the neuritic plaque with its associated halo of degeneration. Therefore, a probe is needed that is specific for the neuritic plaque and NFTs as a more specific marker for AD pathophysiology than antibodies that would also label diffuse plaques.
Recently, radiolabeled Aβ peptide has been used to label diffuse, compact and neuritic type plaques in sections of AD brain. Maggio et al. , WO 93/04194.
However, these peptides share all of the disadvantages of antibodies. Specifically, peptides do not normally cross the blood-brain barrier in amounts necessary for imaging and because these probes react with diffuse plaques, they may not be specific for AD.
Congo red may be used for diagnosing amyloidosis in vivo in non-brain parenchymal tissues. However, Congo red is probably not suitable for in vivo diagnosis of Aβ deposits in brain because only 0.03% of an injected dose of iodinated Congo red can enter the brain parenchyma. Tubis et al . , J. Amer. Pharm . Assn . 49: 422 (1960) . Radioiodinated bisdiazobenzidine compounds related to Congo red, such as Benzo Orange R and Direct Blue 4, have been proposed to be useful in vi tro and in vivo to detect the presence and location of amyloid deposits in an organ of a patient. Quay et al . , U.S. Patent Nos. 5,039,511 and 4,933,156. However, like Congo red, all of the compounds proposed by Quay contain strongly acidic sulfonic acid groups which severely limit entry of these compounds into the brain making it extremely difficult to attain an imaging effective quantity or detectable quantity in the brain parenchyma.
The inability to assess amyloid deposition in AD until after death impedes the study of this devastating illness. A method of quantifying amyloid deposition before death is needed both as a diagnostic tool in mild or clinically confusing cases as well as in
monitoring the effectiveness of therapies targeted at preventing Aβ deposition. Therefore, it remains of utmost importance to develop a safe and specific method for diagnosing AD before death by imaging amyloid in brain parenchyma in vivo . Even though various attempts have been made to diagnose AD in vivo, currently, there are no antemortem probes for brain amyloid. No method has utilized a high affinity probe for amyloid that has low toxicity, can cross the blood-brain barrier, and binds more effectively to AD brain than to normal brain in order to identify AD amyloid deposits in brain before a patient's death. Thus, no in vivo method for AD diagnosis has been demonstrated to meet these criteria. Very recent data suggest that amyloid-binding compounds will have therapeutic potential in AD and type 2 diabetes mellitus. As mentioned above, there are two broad categories of plaques in AD brain, diffuse and neuritic (classical) . Diffuse plaques do not appear to induce morphological reactions such as the reactive astrocytes, dystrophic neurites, microglia cells, synapse loss, and full complement activation found in neuritic plaques. Joachim et al . , Am. J. Pathol . 135: 309 (1989) ; Masliah et al . , loc. ci t . 137: 1293 (1990) ; Lue and Rogers, Dementia 3: 308 (1992) . These morphological reactions all signify that neurotoxic and cell degenerative processes are occurring in the areas adjacent to the compact Aβ
deposits of neuritic plaques. Aβ-induced neurotoxicity and cell degeneration has been reported in a number of cell types in vitro. Yankner et al., Science 250: 279 (1990) ; Roher et al . , BBRC 174: 572 (1991) ; Frautschy et al., Proc. Natl . Acad. Sci. 88: 83362 (1991) ;
Shearman et al . , loc. cit. 91: 1470 (1994) . It has been shown that aggregation of the Aβ peptide is necessary for in vitro neurotoxicity. Yankner, Neurobiol. Aging 13: 615 (1992) . Differences in the state of aggregation of Aβ in diffuse and neuritic plaques may explain the lack of neurotoxic response surrounding the diffuse plaque. Lorenzo and Yankner, Proc. Natl. Acad. Sci., 91: 12243 (1994) . Recently, three laboratories have reported results which suggest that Congo red inhibits Aβ-induced neurotoxicity and cell degeneration in vitro. Burgevin et al . , NeuroReport 5: 2429 (1994); Lorenzo and Yankner, Proc. Natl. Acad. Sci. 91: 12243 (1994) ; Pollack et al . , Neuroscience Letters 184: 113 (1995) ; Pollack et al . , Neuroscience Letters 197: 211 (1995) . The mechanism appears to involve both inhibition of fibril formation and prevention of the neurotoxic properties of formed fibrils. Lorenzo and Yankner, Proc. Natl. Acad. Sci. 91: 12243 (1994) . Congo red also has been shown to protect pancreatic islet cells from the toxicity caused by amylin. Lorenzo and Yankner, Proc. Natl. Acad. Sci. 91: 12243 (1994) . Amylin is a fibrillar peptide
similar to Aβ which accumulates in the pancreas in type 2 diabetes mellitus.
It is known in the art that certain azo dyes may be carcinogenic. Morgan et al . Environmental Heal th Perspectives, 102(supp.) 2: 63-78, (1994) . This potential carcinogenicity appears to be based largely on the fact that azo dyes are extensively .metabolized to the free parent amine by intestinal bacteria. Cerniglia et al . , Biochem. Biophys . Res . Com. , 107: 1224-1229, (1982) . In the case of benzidine dyes (and many other substituted benzidines) , it is the free amine which is the carcinogen. These facts have little implications for amyloid imaging studies in which an extremely minute amount of the high specific activity radiolabelled dye would be directly injected into the blood stream. In this case, the amount administered would be negligible and the dye would by-pass the intestinal bacteria.
In the case of therapeutic usage, these facts have critical importance. Release of a known carcinogen from a therapeutic compound is unacceptable. A second problem with diazo dye metabolism is that much of the administered drug is metabolized by intestinal bacteria prior to absorption. This lowered bioavailability remains a disadvantage even if the metabolites released are innocuous.
Thus, a need exists for amyloid binding compounds which are similar to Congo red but which enter the
brain (Congo Red does not) . Such compounds could be used in preventing cell degeneration associated with fibril formation and thereby treat pathological conditions in amyloid associated diseases, such as AD and Downs Syndrome and in treating pancreatic islet cell toxicity in type 2 diabetes mellitus.
A further need exists for amyloid binding compounds that are non-toxic and bioavailable and, consequently, can be used in therapeutics.
Summary of the Invention
It therefore is an object of the present invention to provide a safe, specific method for diagnosing AD before death by in vivo imaging of amyloid in brain parenchyma. It is another object of the present invention to provide an approach for identifying AD amyloid deposits in brain before a patient's death, using a high-affinity probe for amyloid which has low toxicity, can cross the blood-brain barrier, and can distinguish AD brain from normal brain. It is another object to provide a treatment for AD which will prevent the deposition or toxicity of Aβ. It is another object to provide a technique for staining and detecting amyloid deposits in biopsy or post-mortem tissue specimens. It is another object to provide a method for quantifying amyloid deposition in homogenates of biopsy or post-mortem tissue specimens.
In accomplishing these and other objects, there has been provided, in accordance with one aspect of the present invention, an amyloid binding compound of Formula I or a water soluble, non-toxic salt thereof:
wherein :
- 7 is either N=N, c__≡C, or CR'--CR' (where R'
represents H or a lower alkyl group) ;
X is C(R")2
(wherein R" is each independently is H, F, Cl, Br, I, a lower alkyl group, CF3, CH2-CH2F, 0-CH2-CH2F, CH2-CH2-CH2F, 0-CH2-CH2-CH2F, CN, (C=0) -R' , N(R')2, N02, (C=0)N(R')2 0(CO)R', OR', COOR' , a tri-alkyl tin, a tetrazole or oxadiazole of the form:
wherein R' is H or a lower alkyl group)
or X is CH=CH, N=N, C=0, 0, NR' (where R' represents H or a lower alkyl group) , S, or S02;
each R and R2 independently is H, F, Cl, Br, I, a lower alkyl group, CF3, CH2-CH2F, 0-CH2-CH2F, CH2-CH2- CH2F, 0-CH2-CH2-CH2F, CN, (C=0) -R' , N(R')2, N02, (C=0)N(R')2 0(CO)R', OR', COOR' , a tri-alkyl tin, a tetrazole or oxadiazole of the form:
(wherein R' is H or a lower alkyl group) , or a triazene of the form:
R«
(wherein Rβ and R9 are lower alkyl
_^N^ _N
N
groups) or
each Q is independently selected from one of the following structures, each of which contain a carboxylic acid or an acid-like functionality: IA, IB, IC, ID, IE, IF and IG, wherein IA has the following structure:
wherein: each of R3, R4, R5, R6, or R7 independently is H, F,
Cl, Br, I, a lower alkyl group, CF3, CH2-CH2F, 0- CH2-CH2F, CH2-CH2-CH2F, 0-CH2-CH2-CH2F, CN, (C=0) -R' ,
N(R')2, N02, (C=0)N(R')2 0(C0)R', OR', C00R' , a tri- alkyl tin, a tetrazole or oxadiazole of the form:
(wherein R' is H or a lower alkyl group) , or a triazene of the form:
R
8
I (wherein R8 and R9 are lower alkyl
N R9
groups) or
wherein at least one of R3, R4, R5, R6, or R7 is a hydroxy, tetrazole, oxadiazole, N02, or carboxy in both Q's;
and at least one of R1# R2, R3; R4, or R7 is not H when R5 is hydroxyl and R6 is carboxy or a lower alkyl ester thereof; at least one of Rlf R2, R3 or R4 is not H when R5 is amino, R6 is carboxy or a lower alkyl ester thereof, and R7 is hydroxyl; at least one of Rl t R3, R4, or R7 is not H when R2 is CH3, R5 is hydroxyl and R6 is carboxy or a lower alkyl ester thereof; at least one of Ri or R2, is not H or CH3 when R3, R4, or R7 is CH3, R5 is hydroxyl and R6 is carboxy or a lower alkyl ester thereof;
IB has the following structure (IB)
wherein: each of R_.o. Rn Ri2» _3. Rι . R15 or R16 independently is H, F, Cl, Br, I, a lower alkyl group, CF3, CH2-CH2F, 0-CH2-CH2F, CH2-CH2-CH2F, 0-CH2- CH2-CH2F, CN, (C=0)-R', N(R')2 N02, (C=0)N(R')2 0(CO)R', OR', COOR' , a tri-alkyl tin, a tetrazole or oxadiazole of the form:
(wherein R' is H or a lower alkyl group) , or a triazene of the form:
rein R8 and R9 are lower alkyl
^N^ ,N, (whe
N
groups) or
and wherein at least one of R10ι Ru, R12, R13, R14, R15 or R16 is a hydroxy, tetrazole, oxadiazole, N02 or carboxy in both Q' s;
IC has the following structure
w ere n: at least one of R17, R18, R19, R20/ or R21 is H, F, Cl, Br, I, a lower alkyl group, CF3, CH2-CH2F, 0- CH2-CH2F, CH2-CH2-CH2F, 0-CH2-CH2-CH2F, CN, (C=0) -R' , N(R')2, N02, (C=0)N(R')2 0(C0)R' , OR', COOR' , a tri- alkyl tin, a tetrazole or oxadiazole of the form:
(wherein R' is H or a lower alkyl group) , or a triazene of the form:
(wherein R8 and R9 are lower alkyl
-/N -N-R.
groups) or
ID has the following structure:
wherein: each of R22, R23, or R24 independently is H, F, Cl, Br, I, a lower alkyl group, CF3, CH2-CH2F, 0-CH2- CH2F, CH2-CH2-CH2F, 0-CH2-CH2-CH2F, CN, (C=0) -R' , N(R')2/ N02, (C=0)N(R')2 0(C0)R', OR', COOR' , a tri- alkyl tin, a tetrazole or oxadiazole of the form:
(wherein R' is H or a lower alkyl group) , or a triazene of the form:
(wherein R8 and R9 are lower alkyl
groups) or
represents a heterocyclic ring of one of the six following formulas:
IE has the following structure:
wherein :
each of R25, R26, or R27 independently is H, F, Cl, Br, I, a lower alkyl group, CF3, CH2-CH2F, 0-CH2- CH2F, CH2-CH2-CH2F, 0-CH2-CH2-CH2F, CN, (C=0) -R' , N(R')2, N02, (C=0)N(R')2 0(C0)R' , OR', COOR' , a tri- alkyl tin, a tetrazole or oxadiazole of the form:
(wherein R' is H or a lower alkyl group) , or a triazene of the form:
(wherein R8 and R9 are lower alkyl
groups) or
represents a heterocyclic ring of one of the six following formulas :
IF has the following structure:
(IF
wherein: exactly one of R28, R29, R30, R31 or R32 is the z %
link defined for Formula I above and each other R28/ R29' R3o R3i or R32 independently is H, F, Cl, Br, I, a lower alkyl group, CF3, CH2-CH2F, 0-CH2- CH2F, CH2-CH2-CH2F, 0-CH2-CH2-CH2F, CN, (C=0) -R' , N(R')2, N02, (C=0)N(R')2 0(CO)R', OR', COOR' , a tri- alkyl tin, a tetrazole or oxadiazole of the form:
(wherein R' is H or a lower alkyl group) , or a triazene of the form:
(wherein R8 and R9 are lower alkyl N Rq
groups) or
wherein at least one of R28, R29, R30, R31 or R32 is a hydroxy, tetrazole, oxadiazole, N02 or carboxy in both Q's;
IG has the following structure:
(IG)
wherein: exactly one of R33, R34, R35, R36, R37, R38, or R39 is the r, link defined for Formula I above and each
other R33, R34, R35, R36, R3 , R38, or R39 independently is H, F, Cl, Br, I, a lower alkyl group, CF3, CH2- CH2F, 0-CH2-CH2F, CH2-CH2-CH2F, 0-CH2-CH2-CH2F, CN, (C=0)-R', N(R')2, N02, (C=0)N(R' )2 0(CO)R' , OR' , COOR' , a tri-alkyl tin, a tetrazole or oxadiazole of the form:
(wherein R' is H or a lower alkyl group) , or a triazene of the form:
(wherein R8 and R9 are lower alkyl
groups) or
and wherein at least one of R33, R34, R35, R36, R37, R38, or R39 is a hydroxy, tetrazole, oxadiazole, N02 or carboxy in both Q's.
It is another object of the present invention to provide an amyloid binding compound of Formula I, as defined above, or a water soluble, non-toxic salt thereof, wherein at least one of the substituents R-.-R7 and R10-R39 is selected from the group consisting of
131 I / 123 I # 76 Br # 7 5βr j 1BF ( 19p ( 125 -,^ H2 - CH2 - 18F , O - CH2 - CH2 -
18F, CH2-CH2-CH2-18F, 0-CH2-CH2-CH2-18F and a carbon- containing substituent as specified in Formula I wherein at least one carbon is "C or 13C.
It is a further object of the invention to provide an amyloid binding compound of Formula I, as defined above, or a water-soluble, non-toxic salt thereof, wherein the compound binds to Aβ with a dissociation constant (KD) between 0.0001 and 10.0 μM when measured by binding to synthetic Aβ peptide or Alzheimer's Disease brain tissue.
Still another object of the present invention is to provide a method for synthesizing an amyloid binding compound of Formula I, as defined above, or a water soluble, non-toxic salt thereof, wherein at least one of the substituents R-L-R*-* and R10-R39 is selected from the group consisting of 131I, 123I, 76Br, 75Br, 18F and 19F, comprising the step of reacting an amyloid binding compound of Formula I, as defined above, or a water soluble, non-toxic salt thereof wherein at least one of the substituents Rι-R7 and R10-R39 is a tri-alkyl tin, with a halogenating agent containing 131I, 123I, 76Br, 75Br, 18F or 19F.
An additional object of the present invention is a pharmaceutical composition for in vivo imaging of amyloid deposits, comprising (a) an amyloid binding compound of Formula I, as defined above, or a water soluble, non-toxic salt thereof, wherein at least one of the substituents R**.-R7 and R10-R39 is selected from the group consisting of 131I, 123I, 76Br, 75Br, 18F, 19F and a carbon-containing substituent as specified in Formula I wherein at least one carbon is 11C or 13C, and (b) a pharmaceutically acceptable carrier.
Yet another object of the present invention is an in vivo method for detecting amyloid deposits in a subject, comprising the steps of: (a) administering a detectable quantity of the above pharmaceutical composition, and (b) detecting the binding of the
compound to amyloid deposit in said subject. It is also an object of the present invention to provide an in vivo method for detecting amyloid deposits in a subject wherein the amyloid deposit is located in the brain of a subject. This method of the invention may be used in a subject who is suspected of having an amyloidosis associated disease or syndrome selected from the group consisting of Alzheimer's Disease, Down's Syndrome, and homozygotes for the apolipoprotein E4 allele.
Another object of the invention relates to pharmaceutical compositions and methods of preventing cell degeneration and toxicity associated with fibril formation in amyloidosis associated conditions such as AD and Type 2 diabetes mellitus. Such pharmaceutical compositions comprise Chrysamine G or the above described derivatives thereof and a pharmaceutically acceptable carrier. Such compounds would be non-toxic. Another object of the invention relates to the use of the probes as a stain for the visualization and detection of amyloid deposits in biopsy or post-mortem tissue specimens.
Another object of this invention relates to the use of radiolabeled probes for the quantitation of amyloid deposits in biopsy or postmortem tissue specimens.
Another object relates to a method of distinguishing Alzheimer's disease brain from normal brain.
Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.
Brief Description of the Drawings
Fiσure IA illustrates the chemical structures of Chrysamine G and several Chrysamine G analogues or derivatives which have been synthesized and tested, including the 3-iodo derivative (3-ICG) , the 3-iodo dimethoxy derivative (3-ICG(OMe)2) , the dimethyl ester derivative (CG(COOMe)2) , the phenol derivative, salicylic acid (SA) , the aniline derivative (1/2CG) , Congo red, the 3,3'-diiodo derivative (3,3'-I2CG), the 3,3'-dibromo derivative (3,3' -Br2CG) , the 3,3' -dichloro derivative (3,3' -C12CG) , the 3-bromo derivative (3- BrCG) , and the 5-fluorosalicylic acid derivative ( (5- FSA)CG) . The numbers in the figure refer to each
co pound's ^ (in μM) for inhibition of [14C] Chrysamine G binding to the synthetic peptide, Aβ (10-43) .
Figure IB illustrates the chemical structures of several Chrysamine G analogues or derivatives which have been synthesized and tested, including the 3,3'- dicarboxylic acid derivative (3, 3 ' - (COOH)2CG) , the 2, 2 ' -disulfonic acid derivative of Chrysamine G (2,2'- (S03)2CG), the 3-bromo, 3-isopropylsalicylic acid derivative (3-Br- (3-iPrSA) CG) , the 3-isopropylsalicylic acid derivative ( (3-iPrSA) CG) , the 2,4-diphenol derivative (2,4-Diphenol) , the γ-resorcylic acid derivative ( (6-OHSA) CG) , the 3,3 ' ,5,5' - tetramethylbenzidine derivative (3, 3 ' , 5, 5 ' - (CH3)4CG) , the 3 , 3 ' -dimethyl derivative (3,3 ' - (CH3)2CG) , the the 2,2 ' -dimethyl derivative (2, 2 '- (CH3) 2CG) , the benzisoxazole derivative (CG Benzisoxazole) , and the 3- carboxy alkyne derivative (3- (COOH) -C3C) . The numbers in the figure refer to each compound's Ki (in μM) for inhibition of [14C] Chrysamine G binding to the synthetic peptide, Aβ (10-43) .
Figures 2A-2K illustrate the chemical structures of Chrysamine G and tri-alkyl tin derivatives of analogues of Chrysamine G, in particular heterocyclic analogues. Note that these structures represent one- half of a molecule which is symmetric around the wavy
bond shown in the upper right, except that the tri- alkyl tin moiety may only be on one side of the biphenyl group. The tri-alkyl tin derivatives are stable intermediate and immediate precursors for the preparation of high specific activity halogenated radioactive derivatives. The heterocyclic analogues represent alternative means of placing weakly acidic moieties in the same structural position as the moderately acidic carboxylic acid group of Chrysamine G. These tri-alkyl tin precursor compounds are shown in their protonated form, yet those of skill in the art recognize that their deprotonated forms and tautomers also are embraced by these drawings.
2A) Chrysamine G. 2B) Tri-alkyl tin derivative of Chrysamine G.
2C) Tri-alkyl tin derivative of the 3-Hydroxy-1,2- benzisoxazole analogue.
2D) Tri-alkyl tin derivative of the phthalimide or isoindole-1,3 (2H) -dione analogue. 2E) Tri-alkyl tin derivative of the phthalhydrazide or
2,3-benzodiazine-l,4 (2H,3H) -dione analogue.
2F) Tri-alkyl tin derivative of the 2,3-benzoxazine-
1,4 (3H) -dione analogue.
2G) Tri-alkyl tin derivative of the (2H)1,3- benzoxazine-2,4 (3H) -dione analogue.
2H) Tri-alkyl tin derivative of the (3H)2-benzazine-
1,3 (2H) -dione analogue.
21) Tri-alkyl tin derivative of the 1, 8-Naphthalimide analogue.
2J) Tri-alkyl tin derivative of the tetrazole analogue. 2K) Tri-alkyl tin derivative of the oxadiazole analogue.
Figure 3 Displacement curves of [14C] Chrysamine G binding to Aβ (10-43) by several structural analogues of Chrysamine G. Abbreviations refer to those used in Figure 1. Figure 3A) Chrysamine G (open triangles) ; (5-FSA)CG (filled diamonds) ; 3, 3 ' - (COOH)2CG (filled squares) ; 2, 2 ' - (S03)2CG (filled circles) . Figure 3B) Chrysamine G (open triangles) ; Congo red (open circles) ; aniline derivative (open inverted triangles) ; phenol derivative (open squares) ; salicylic acid (X's) . Curves which show increased binding at higher concentrations do so because of the formation of micelles. Bedaux, F. et al., Phar . Weekblad 98: 189 (1963) .
Figure 4A is a Scatchard plot of Chrysamine G binding to A (10-43) . The curved line represents a nonlinear least-squares fit to a two, independent binding site model. The straight lines represent the individual components.
Figure 4B is a Scatchard analysis of [14C] CG binding to typical control (diamonds) and AD brain
samples (squares) . The dashed line has the same slope as the AD line and is meant to aid in the comparison with the control slope. This AD brain sample had 48 NP/x200 magnification, a KD of 0.35 μM, and a Bmax of 790 fmol/μg protein. The control had a KD of 0.48 μM, and a B^ of 614 fmol/μg protein.
Figure 5 is a graph illustrating the linearity of the binding assay with respect to peptide concentration. Approximately 0.9 μg of Aβ (10-43) was used in the typical assay.
Figure 6A is a graph illustrating the time course of association of Chrysamine G and Aβ (10-43) .
Figure 6B is the graphic illustration of the determination of the association rate constant (k .
Figure 6C is a graph of the time course of dissociation of Chrysamine G from Aβ (10-43) .
Figure 7 is a graphic representation of a molecular model of the interaction between Chrysamine G and Aβ.
Figure 8A is a graph illustrating the correlation between the amount of [14C] Chrysamine G bound and the number of neuritic plaques (NP) in AD brain samples.
Figure 8B is a graph illustrating the correlation between the amount of [1 C] Chrysamine G bound and the number of neurofibrillary tangles (NFT) in AD brain samples. In both Figure 8A and 8B, the x-axis represents the average NP or NFT count per field at x200 magnification in Bielschowsky stained sections of either the superior/middle frontal (n=10) or superior temporal cortex (n=10) . The filled symbols and heavy lines indicate brains without amyloid angiopathy, the open symbols and dashed lines indicate brains with amyloid angiopathy. The y-axis represents total, absolute [1 C] Chrysamine G binding (fmol/μg protein) in homogenates of brain samples adjacent to those used for staining. Approximately 75 μg protein and 150 nM [14C] Chrysamine G were used.
Figures 9A. 9B and 9C. The binding of Chrysamine G to various brain areas in samples of AD brain having more than 20 NPs/x200 magnification, referred to as "High Plaque AD Brains", is shown in figure 9A. The binding of Chrysamine G to brain areas in samples of AD brain having less than 20 NPs/x200 magnification, referred to as "Low Plaque AD Brains", is shown in Figure 9B. The data points represent the ratio of [1 C] Chrysamine G binding in the designated brain area to [14C] Chrysamine G binding in the cerebellum (CB) of the same brain. Horizontal bars represent the mean and error bars represent the standard error for control
(circles) , and AD brain (diamonds in 9A and 9B) . Brain areas include the frontal pole (FP) , head of caudate (CAU) , superior/ middle frontal (SMF) , superior temporal (ST) , inferior parietal (IP) , and occipital (OC) cortex. Asterisks indicate significant differences compared to control (*p<0.05; **p<0.001) . Two Down' s syndrome brain samples are indicated in Figure 9C. The diamonds in 9C represent a brain from a 23 year old Down's syndrome patient not yet symptomatic with AD. The triangles in 9C represent a 51 year old Down's syndrome patient who had developed AD as do the vast majority of Down's syndrome patients by their 40's.
Figure 10 is a graph illustrating the tissue levels of Chrysamine G in mice injected with [14C] Chrysamine G in the lateral tail vein and sacrificed at the times indicated. The open symbols and thin lines represent absolute radioactivity in units of cpm/g tissue (left axis) . The closed symbols and solid lines represent the ratio of brain radioactivity to that in kidney (top) or blood (middle) . The ratios are plotted on the right axis.
Figure 11. TOP: Section from the inferior temporal lobe of AD brain stained with Chrysamine G by the method of Stokes and Trickey, J". Clin . Pathol . 26: 241-242 (1973) . Two neuritic plaques are clearly
visible. BOTTOM: Adjacent sections from the temporal lobe of an AD patient with amyloid angiopathy. Left: Brain section was stained with the Congo red method of Puchtler. Puchtler et al . , J. Histochem. Cytochem. 10: 35 (1962) . The bar represents 20 microns. Right: Adjacent brain section stained by substituting Chrysamine G for Congo red in the Puchtler method. Both sections readily demonstrate the same amyloid- laden vessel . A small amount of background autofluorescence from erythrocytes and lipofuscin also is visible. All photomicrographs were obtained using fluorescein isothiocyanate (FITC) filters.
Figure 12. A bar graph showing the effect of increasing concentrations of Aβ (25-35) in the presence and absence of Chrysamine G on the cellular redox activity of rat pheochromocytoma (PC-12) cells as measured by 3- [4, 5-dimethylthiazol-2-yl] -2, 5- diphenyltetrazolium bromide, MTT, reduction. The reduction product of MTT absorbs at 560 nm which is plotted on the vertical axis. The effect of Aβ (25-35) alone is shown in the filled bars and shows a dose dependent decrease in MTT reduction. Significant differences from control (no Aβ (25-35) , no Chrysamine G) are shown in white inside the filled bars. The protective effect of 20 μM Chrysamine G is shown in the open bars. Significant differences between MTT
reduction in the presence and absence of Chrysamine G are shown in black inside the open bars .
Figure 13. A bar graph showing the protective effect of increasing concentrations of Chrysamine G against the Aβ (25-35) -induced reduction of cellular redox activity of rat pheochromocytoma (PC-12) cells as measured by 3- [4, 5-dimethylthiazol-2-yl] -2, 5- diphenyltetrazolium bromide, MTT, reduction. The reduction product of MTT absorbs at 560 nm which is plotted on the vertical axis. The effect of Chrysamine G in the absence of Aβ (25-35) is shown in the filled bars. There was no significant difference between control (no Aβ (25-35) , no Chrysamine G) and any of the concentrations of Chrysamine G in the absence of Aβ(25- 35) . MTT reduction in the presence of 1 μM Aβ (25-35) and increasing concentrations of Chrysamine G is shown in the open bars . Significant differences in MTT reduction between the presence and absence of Aβ (25-35) at each concentration of Chrysamine G are shown in white inside the filled bars. Significant differences in MTT reduction between the Aβ (25-35) control (no Chrysamine G) and Aβ (25-35) plus increasing concentrations of Chrysamine G are shown in black inside the open bars .
Figure 14. Comparison of the effects of
Chrysamine-G and the inactive phenol derivative on the
toxicity induced by Aβ (25-35) . 1 μM Aβ (25-35) was present in all experiments except control. Chrysamine- G showed protective effects at 0.1 and 1 μM, but the phenol derivative showed no protective effects, and perhaps enhanced the toxicity of Aβ.
Detailed Description of the Preferred Embodiments The present invention exploits the ability of Chrysamine G and radiolabeled derivatives thereof to cross the blood brain barrier in vivo and bind to Aβ deposited in neuritic (but not diffuse) plaques, to Aβ deposited in cerebrovascular amyloid, and to the amyloid consisting of the protein deposited in NFT. Chrysamine G is a Congo red derivative with the key structural difference being that the sulfonic acid moieties found in Congo red are replaced by carboxylic acid groups in Chrysamine G (Figure 1) . This structural alteration allows Chrysamine G to enter the brain better than Congo red and large macromolecules such as antibodies. Tubis et al . , J. Am. Pharmaceut . Assn. 49: 422 (1960) . Also, Chrysamine G may be a more specific marker for AD pathophysiology than antibodies, which would also label diffuse plaques, due to the apparent specificity of Chrysamine G for the neuritic plaque and NFTs. The Chrysamine G derivatives of the present invention have each of the following characteristics: (1) specific binding to synthetic Aβ in vi tro, (2)
binding to neuritic but not diffuse plaques in brain sections (3) ability to cross a non-compromised blood brain barrier in vivo .
The method of this invention determines the presence and location of amyloid deposits in an organ or body area, preferably brain, of a patient. The present method comprises administration of a detectable quantity of a pharmaceutical composition containing a compound of Formula I, as defined above, called a "detectable compound, " or a pharmaceutically acceptable water-soluble salt thereof, to a patient. A "detectable quantity" means that the amount of the detectable compound that is administered is sufficient to enable detection of binding of the compound to amyloid. An "imaging effective quantity" means that the amount of the detectable compound that is administered is sufficient to enable imaging of binding of the compound to amyloid.
The invention employs amyloid probes which, in conjunction with non-invasive neuroimaging techniques such as magnetic resonance spectroscopy (MRS) or imaging (MRI) , or gamma imaging such as positron emission tomography (PET) or single-photon emission computed tomography (SPECT) , are used to quantify amyloid deposition in vivo . The term " in vivo imaging" refers to any method which permits the detection of a labeled Chrysamine G, labeled Chrysamine G derivative or labeled compound of Formula I, as
described above, of the present invention. For gamma imaging, the radiation emitted from the organ or area being examined is measured and expressed either as total binding or as a ratio in which total binding in one tissue is normalized to (for example, divided by) the total binding in another tissue of the same subject during the same in vivo imaging procedure. Total binding in vivo is defined as the entire signal detected in a tissue by an in vivo imaging technique without the need for correction by a second injection of an identical quantity of labeled compound along with a large excess of unlabeled, but otherwise chemically identical compound. A "subject" is a mammal, preferably a human, and most preferably a human suspected of having dementia.
For purposes of in vivo imaging, the type of detection instrument available is a major factor in selecting a given label. For instance, radioactive isotopes and 19F are particularly suitable for in vivo imaging in the methods of the present invention. The type of instrument used will guide the selection of the radionuclide or stable isotope. For instance, the radionuclide chosen must have a type of decay detectable by a given type of instrument . Another consideration relates to the half-life of the radionuclide. The half-life should be long enough so that it is still detectable at the time of maximum uptake by the target, but short enough so that the host
does not sustain deleterious radiation. The radiolabeled compounds of the invention can be detected using gamma imaging wherein emitted gamma irradiation of the appropriate wavelength is detected. Methods of gamma imaging include, but are not limited to, SPECT and PET. Preferably, for SPECT detection, the chosen radiolabel will lack a particulate emission, but will produce a large number of photons in a 140-200 keV range. For PET detection, the radiolabel will be a positron-emitting radionuclide such as 19F which will annihilate to form two 511 keV gamma rays which will be detected by the PET camera.
In the present invention, amyloid binding compounds/probes are made which are useful for in vivo imaging and quantification of amyloid deposition.
These compounds are to be used in conjunction with non- invasive neuroimaging techniques such as magnetic resonance spectroscopy (MRS) or imaging (MRI) , positron emission tomography (PET) , and single-photon emission computed tomography (SPECT) . In accordance with this invention, the Chrysamine G derivatives may be labeled with 19F or 13C for MRS/MRI by general organic chemistry techniques known to the art. See, e . g. , March, J. "ADVANCED ORGANIC CHEMISTRY: REACTIONS, MECHANISMS, AND STRUCTURE (3rd Edition, 1985) , the contents of which are hereby incorporated by reference. The Chrysamine G derivatives also may be radiolabeled with 18F, 11C, 75Br, or 76Br for PET by techniques well known in the art and
are described by Fowler, J. and Wolf, A. in POSITRON EMISSION TOMOGRAPHY AND AUTORADIOGRAPHY (Phelps, M. , Mazziota, J., and Schelbert, H. eds . ) 391-450 (Raven Press, NY 1986) the contents of which are hereby incorporated by reference. The Chrysamine G derivatives also may be radiolabeled with 123I for SPECT by any of several techniques known to the art. See, e . g. , Kulkarni, Int. J". Rad. Appl . & Inst . (Part B) 18: 647 (1991) , the contents of which are hereby incorporated by reference. In addition, the Chrysamine G derivatives may be labeled with any suitable radioactive iodine isotope, such as, but not limited to 131I, 125I, or 123I, by iodination of a diazotized amino derivative directly via a diazonium iodide, see Greenbaum, F. Am . J. Pharm. 108: 17 (1936) , or by conversion of the unstable diazotized amine to the stable triazene, or by conversion of a non-radioactive halogenated precursor to a stable tri-alkyl tin derivative which then can be converted to the iodo compound by several methods well known to the art. See, Satyamurthy and Barrio J. Org . Chem . 48: 4394 (1983) , Goodman et al . , J. Org. Chem . 49: 2322 (1984) , and Mathis et al . , J. Labell . Comp . and Radiopharm . 1994: 905; Chumpradit et al . , J. Med. Chem. 34: 877 (1991) ; Zhuang et al . , J. Med. Chem . 37: 1406 (1994) ; Chumpradit et al . , J. Med. Chem. 37: 4245 (1994) . For example, a stable triazene or tri-alkyl tin derivative of Chrysamine G or its analogues is reacted with a
halogenating agent containing 131I, 125I, 123I, 76Br, 75Br, i8 F or i9 F- Thus, the stable tri-alkyl tin derivatives of Chrysamine G and its analogues are novel precursors useful for the synthesis of many of the radiolabeled compounds within the present invention. As such, these tri-alkyl tin derivatives are one embodiment of this invention.
The Chrysamine G derivatives also may be radiolabeled with known metal radiolabels, such as Technetium-99m (99mTc) . Modification of the substituents to introduce ligands that bind such metal ions can be effected without undue experimentation by one of ordinary skill in the radiolabeling art. The metal radiolabeled Chrysamine G derivative can then be used to detect amyloid deposits.
The methods of the present invention may use isotopes detectable by nuclear magnetic resonance spectroscopy for purposes of in vivo imaging and spectroscopy. Elements particularly useful in magnetic resonance spectroscopy include 19F and 13C.
Suitable radioisotopes for purposes of this invention include beta-emitters, gamma-emitters, positron-emitters, and x-ray emitters. These radioisotopes include 131I, 123I, 18F, "C, 75Br, and 76Br. Suitable stable isotopes for use in Magnetic Resonance Imaging (MRI) or Spectroscopy (MRS) , according to this invention, include 19F and 13C. Suitable radioisotopes for in vi tro quantification of amyloid in homogenates
of biopsy or post-mortem tissue include 125I, 14C, and 3H. The preferred radiolabels are 18F for use in PET in vivo imaging, 123I for use in SPECT imaging, 19F for MRS/MRI, and 3H or 1 C for in vi tro studies. However, any conventional method for visualizing diagnostic probes can be utilized in accordance with this invention.
The method could be used to diagnose AD in mild or clinically confusing cases. This technique would also allow longitudinal studies of amyloid deposition in human populations at high risk for amyloid deposition such as Down's syndrome, familial AD, and homozygotes for the apolipoprotein E4 allele. Corder et al . , Science 261: 921 (1993) . A method that allows the temporal sequence of amyloid deposition to be followed can determine if deposition occurs long before dementia begins or if deposition is unrelated to dementia. This method can be used to monitor the effectiveness of therapies targeted at preventing amyloid deposition.
Generally, the dosage of the detectably labeled Chrysamine G derivative will vary depending on considerations such as age, condition, sex, and extent of disease in the patient, contraindications, if any, concomitant therapies and other variables, to be adjusted by a physician skilled in the art. Dosage can vary from 0.001 mg/kg to 1000 mg/kg, preferably 0.1 mg/kg to 100 mg/kg.
Administration to the subject may be local or systemic and accomplished intravenously, intraarterially, intrathecally (via the spinal fluid) or the like. Administration may also be intradermal or intracavitary, depending upon the body site under examination. After a sufficient time has elapsed for the compound to bind with the amyloid, for example 30 minutes to 48 hours, the area of the subject under investigation is examined by routine imaging techniques such as MRS/MRI, SPECT, planar scintillation imaging, PET, and emerging imaging techniques, as well. The exact protocol will necessarily vary depending upon factors specific to the patient, as noted above, and depending upon the body site under examination, method of administration and type of label used; the determination of specific procedures would be routine to the skilled artisan. For brain imaging, preferably, the amount (total or specific binding) of the bound radioactively labelled Chrysamine G or Chrysamine G derivative or analogue is measured and compared (as a ratio) with the amount of labelled Chrysamine G or Chrysamine G derivative bound to the cerebellum of the patient. This ratio is then compared to the same ratio in age-matched normal brain. The pharmaceutical compositions of the present invention are advantageously administered in the form of injectable compositions. A typical composition for such purpose comprises a pharmaceutically acceptable
carrier. For instance, the composition may contain about 10 mg of human serum albumin and from about 0.5 to 500 micrograms of the labeled Chrysamine G or Chrysamine G derivative per milliliter of phosphate buffer containing NaCl . Other pharmaceutically acceptable carriers include aqueous solutions, non- toxic excipients, including salts, preservatives, buffers and the like, as described, for instance, in REMINGTON'S PHARMACEUTICAL SCIENCES, 15th Ed. Easton: Mack Publishing Co. pp. 1405-1412 and 1461-1487 (1975) and THE NATIONAL FORMULARY XIV., 14th Ed. Washington: American Pharmaceutical Association (1975) , the contents of which are hereby incorporated by reference.
Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oil and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic/aqueous solutions, saline solutions, parenteral vehicles such as sodium chloride, Ringer's dextrose, etc. Intravenous vehicles include fluid and nutrient replenishers . Preservatives include antimicrobials, anti-oxidants, chelating agents and inert gases. The pH and exact concentration of the various components of the pharmaceutical composition are adjusted according to routine skills in the art. See, Goodman and
Gilman's THE PHARMACOLOGICAL BASIS FOR THERAPEUTICS (7th Ed.) .
Particularly preferred pharmaceutical compositions of the present invention are those that, in addition to specifically binding amyloid in vivo and capable of crossing the blood brain barrier, are also non-toxic at appropriate dosage levels and have a satisfactory duration of effect.
Molecular Modeling
Molecular modeling was done on an Evans and Sutherland PS-330 computer graphics system, running the computer modeling program MacroModel (Version 2.5 available from C. Still at Columbia University) to generate the Aβ peptide chains in the anti-parallel beta-sheet conformation. Kirschner et al . , Proc . Natl . Acad. Sci . U. S.A . 83: 503 (1986) . The amyloid peptides were used without further structural refinement . The Aβ peptides were aligned so that alternate chains were spaced 4.76 A apart, characteristic of beta-sheet fibrils. Kirschner, supra . Chrysamine G was energy minimized and aligned with the fibril model to maximize contact with lysine-16 of Aβ (10-43) and the hydrophobic phenylalanine-19 and -20 region.
Characterization of Specific Binding to Aβ Synthetic Peptide: Affinity, Kinetics, Maximum Binding The characteristics of Chrysamine G and
Chrysamine G derivative binding is first analyzed using
synthetic Aβ peptide called Aβ(10-43). The 10-43 peptide was chosen because it has been shown that this peptide provides a model system containing all of the characteristic structural features of Aβ peptides. Hilbich et al . , J. Mol . Biol . 218: 149 (1991) . The 10- 43 amino acid fragment of Aβ was synthesized with 9- fluorenylmethyl chloroformate (FMOC) chemistry by the Peptide Synthesis Facility of the University of Pittsburgh. The peptide was characterized by mass spectrometry and the major component had an MR of 3600 g/mole (calc. 3598) . The peptide was further purified by the method of Hilbich et al . which, in brief, consisted of sequential size-exclusion chromatography on a Biogel P10 column (2 x 180 cm, 200-400 mesh, Biorad, Richmond, CA) in 70% formic acid followed by a second elution through a Biogel P4 column (2 x 180 cm, 200-400 mesh) in 1M acetic acid. Hilbich et al . , J. Mol . Biol . 218: 149 (1991). The peptide was lyophilized and stored at -80°C until used in the binding assays.
A_Bi_αo acid sequence for A (10 -43 ) is as follows :
Binding assay to synthetic Aβ(10-43) Binding assays were performed in 12 x 75 mm borosilicate glass tubes. Various concentrations of nonradioactive Chrysamine G derivatives were added in 10% ethanol/water. Ethanol was necessary to prevent the micelle formation which occurs with these diazo dye derivatives, since the micelles are trapped by the filter even in the absence of peptide. To the above solution, 25 μl of a 0.36 mg/ml suspension of Aβ(10-43) in H20 was added and 10% ethanol was added to bring the volume to 950 μl. After incubating for 10 min at room temperature, 50 μl of [14C] Chrysamine G in 40% ethanol was added, resulting in a final concentration of [14C] Chrysamine G of 100-125 nM depending on the preparation of [14C] Chrysamine G used. The binding mixture was incubated for 30 min at room temperature. Bound and free radioactivity were separated by vacuum filtration through Whatman GF/B filters using a Brandel M-24R Cell Harvester (Gaithersburg, MD) followed by two 3-ml washes with 10% ethanol at room temperature. Filters were equilibrated overnight in 4 ml Cytoscint®- ES scintillant (ICN Biomedicals, Inc., Irvine, CA) in
7.0 ml plastic scintillation vials before counting. In this and all binding assays, incubations were done at least in triplicate and the results expressed as mean ± standard deviation.
Kinetic studies
Kinetics studies of [14C] Chrysamine G binding to Aβ (10-43) were performed in 13 x 100 mm borosilicate glass tubes by the filtration assay described above. For the kinetics of association, 25 μl of 0.36 mg/ml Aβ (10-43) were placed in 475 μl of 10% ethanol and 4.5 ml of 125 nM [14C] Chrysamine G was added to the solution at time zero. The mixture was rapidly vortexed and the binding reaction was stopped by vacuum filtration through Whatman GF/B filters using a Brandel M-24R Cell Harvester (Gaithersburg, MD) followed by two 3-ml washes with 10% ethanol at room temperature at times of 5, 10, 20, 30, 45, 60, 75, 135, 240, and 300 sec; bound radioactivity was determined as above. For the kinetics of dissociation, 25 μl of 0.36 mg/ml Aβ (10-43) were placed in 450 μl of 10% ethanol followed by 25 μl of 2.5 μM [14C] Chrysamine G in 40% ethanol . This mixture was vortexed and incubated for 30 min at room temperature. The mixture was diluted with 4.5 ml of 10 μM nonradioactive Chrysamine G in 10% ethanol at time zero, the mixture was rapidly vortexed, and the dissociation was stopped by filtration as above
at times of 0.5, 1.5, 3, 5, and 15 min, and bound radioactivity was determined as above.
Characterization of Specific Binding to Alzheimer's Disease Brain
Binding of Chrysamine G to AD and control brain homogenates
Autopsy brain samples were obtained from the Neuropathology Core of the Alzheimer's Disease Research Center of the University of Pittsburgh. Controls were defined as not meeting neuropathological criteria for AD (sufficient number of NPs or NFTs) according to the standards specified in a published NIA conference report. Khachaturian, Arch . Neurol . 42: 1097 (1985) . Brain samples from eight control (ages 58-75) , eleven AD (ages 61-84) , and two Down syndrome brains (ages 23 and 51) were studied. There were six high-plaque (>20 NPs/x200 magnification) and five low-plaque (<20 NPs/x200 magnification) AD brains. Two controls were clinically demented but had no NPs or NFTs and received the diagnosis of "Dementia Lacking Distinctive
Histology". Knopman Dementia 4: 132 (1993) . Another control had dementia and olivopontocerebellar atrophy. The other controls had no clinical or histological evidence of neurologic disease. Autopsy samples were immediately frozen at -70 °C and stored at that temperature until homogenized. The numbers of NPs and NFTs were counted in sections of five separate but
adjacent fields (x200 magnification) between cortical layers 2 and 4 in the cortex at the junction of superior and middle frontal gyri and superior temporal isocortex of all brains studied. A qualitative assessment of the presence of amyloid angiopathy in the superior/middle frontal cortex was made. The Bielschowsky silver impregnation method was used to identify NPs and NFTs and Congo red staining was used to identify cerebral amyloid angiopathy. Details of this procedure have been previously published. Moossy et al . , Arch . Neurol . 45: 251 (1988) . Samples used for CG binding to the superior/middle frontal or superior temporal cortex were adjacent on gross dissection to those used for NP and NFT counts. Approximately 100 mg of tissue from the junction of the superior and middle frontal cortex, superior temporal cortex, frontal pole, head of the caudate, inferior parietal cortex, occipital cortex, or cerebellum were homogenized with a Polytron® tissue homogenizer (PT 10/35, Brinkman Instruments Inc.,
Westbury, NY) for 30 sec at setting 6 in 10% ethanol at a concentration of 10-20 mg brain/ml. Not all areas were available from each brain. Aliquots of 25-150 μl tissue (about 25-300 μg of protein by the method of Lowry et al . , J. Biol , Chem. 193: 265 (1951)) were incubated in 12 x 75 mm borosilicate glass tubes at room temperature with 10-750 nM [1 C] CG (26.8 Ci/mole) in a final volume of 1.0 ml of 10% ethanol for 30 min
at room temperature. The standard conditions employed about 150 μg of protein and 75 nM [1 C] CG for the - cerebellar ratio studies and about 75 μg of protein and 150 nM [14C] CG for the correlative studies with NPs, NFTs, and amyloid angiopathy. Ethanol was necessary to prevent the micelle formation which occurs with diazo dye derivatives, since the micelles are trapped by the filter even in the absence of tissue. Bound and free radioactivity were separated by vacuum filtration through Whatman GF/B filters using a Brandel M-24R Cell Harvester (Gaithersburg, MD) followed by two 3-ml washes with 10% ethanol at room temperature. Filters were equilibrated overnight in 4 ml Cytoscint®-ES scintillant (ICN Biomedicals, Inc., Irvine, CA) in 7.0 ml plastic scintillation vials before counting.
Saturable (specific) binding was defined as total binding minus residual (non-saturable) binding in the presence of 20 μM unlabelled CG. In all binding assays, incubations were done at least in triplicate and the results expressed as mean ± standard error unless otherwise specified. Results were expressed either in absolute terms of fmol [1 C] CG bound/μg protein in a given brain area, or as a ratio of the fmol/μg protein in that brain area to the fmol/μg protein in the cerebellum of the same brain.
Octanol/Water Partitioning
Approximately 75 μM solutions of Chrysamine G or its analogues were prepared in 5.0 ml 1-octanol. Five ml of phosphate buffered saline (0.15 M NaCl, 5 mM potassium phosphate, pH 7.4) were added and the layers mixed by rapid vortexing. The mixture was then centrifuged at 1,000 g to facilitate the formation of two clear phases. The layers were separated using a separatory funnel and 600 μl of each layer was diluted with 400 μl of ethanol and the absorbance measured at 389 nm for Chrysamine G or the λmax for each analogue. Concentrations were determined after correction for the molar absorptivity differences in the two solvents and the partition coefficient expressed as the concentration in the octanol layer divided by the concentration in the aqueous layer. Experiments were done in triplicate.
Imaging the Binding of Chrysamine G to Amyloid Deposits in Alzheimer's Disease Brain For visual demonstration of CG binding to tissue, adjacent 8 micron paraffin sections of an AD brain with heavy deposits of cerebrovascular amyloid were stained with both CG and Congo red by the alkaline Congo red method of Puchtler et al . , J. Histochem. Cytochem. 10: 355 (1962) or the method of Stokes and Trickey, J".
Clin . Pathol . 26: 241-242 (1973) . In the CG staining procedure, CG was substituted for Congo red, but the
procedure was otherwise identical. Stained slides were examined using a fluorescein isothiocyanate (FITC) filter.
Determining Compound's Ability to Cross the Blood Brain Barrier
Mouse studies Female Swiss-Webster mice were injected in the lateral tail vein with approximately 0.03 μCi/g of [14C] Chrysamine G in a 0.9% NaCl solution. Mice were sacrificed by cervical dislocation at intervals of 15 min, 35 min, 1 hr, 4 hr, and 24 hr after injection. The carotid blood, brain, liver, and kidneys were rapidly obtained, weighed, and homogenized in distilled/deionized H20 using a ground glass homogenizer. An aliquot was weighed into an 18.0 ml plastic scintillation vial (Beckman Poly-Q-Vial) and counted after addition of 10.0 ml of scintillation cocktail (Cytoscint®-ES (ICN) ) and overnight equilibration. The [1C] Chrysamine G content of the tissues was expressed as cpm/mg tissue. Experiments in which radioactivity was extracted from tissues were performed as above except 0.05 μCi/g of [14C] Chrysamine G was injected and the mice were sacrificed at 60 min. Brain and liver were then removed and extracted with a Folch procedure. Folch et al . , J. Biol . Chem. 226: 447 (1957) . In both tissues, over 95% of the extracted radioactivity was contained in the organic layer. The organic layer was evaporated
to dryness, resuspended in a minimal amount of 10% methanol/90% ACN, and injected onto a silica column (Prep Nova Pak HR Silica, 7.8 x 300 mm, Waters, Milford, MA) and eluted isocratically with the same solvent. Under these conditions, 99% of the radioactivity is eluted in the solvent front, but most lipids are retained longer, making the fraction eluting in the solvent front suitable for injection onto the reverse-phase C4 column system described above. The entire solvent front was collected, dried, and resuspended in 10% ACN/90% sodium phosphate buffer (5mM, pH 6) and injected, along with authentic non- radioactive Chrysamine G, onto the C4 column. One minute fractions were collected and counted after addition of 10 ml of Cytoscint®-ES.
In yet another embodiment, the invention relates to a pharmaceutical composition and method for preventing cell degeneration and toxicity associated with fibril formation in certain "amyloidosis associated" conditions such as Alzheimer's Disease, Down's Syndrome and Type 2 diabetes mellitus, hereditary cerebral hemorrhage amyloidosis (Dutch) , amyloid A (reactive) , secondary amyloidosis, familial mediterranean fever, familial amyloid nephropathy with urticaria and deafness (Muckle-wells Syndrome) , amyloid lambda L-chain or amyloid kappa L-chain (idiopathic, myeloma or macroglobulinemia-associated) A beta 2M (chronic hemodialysis) , ATTR (familial amyloid
polyneuropathy (Portuguese, Japanese, Swedish) , familial amyloid cardiomyopathy (Danish) , isolated cardiac amyloid, (systemic senile amyloidosises) , AIAPP or amylin insulinoma, atrial naturetic factor (isolated atrial amyloid) , procalcitonin (medullary carcinoma of the thyroid) , gelsolin (familial amyloidosis (Finnish) ) , cystatin C (hereditary cerebral hemorrhage with amyloidosis (Icelandic) ) , AApo-A-I (familial amyloidotic polyneuropathy - Iowa) , AApo-A-II (accelerated senescence in mice) , fibrinogen-associated amyloid; and Asor or Pr P-27 (scrapie, Creutzfeld Jacob disease, Gertsmann-Straussler-Scheinker syndrome, bovine spongiform encephalitis) or in cases of persons who are homozygous for the apolipoprotein E4 allele. This method involves administering a pharmaceutical composition comprising Chrysamine G, or one of the above described derivatives thereof, to a subject suspected of having or at high risk of developing such amyloidosis associated condition. Because certain diazo compounds could be carcinogenic, the therapeutic compounds of the present invention include only non-toxic, non-carcinogenic compounds. That is, the present invention addresses the problems with potential carcinogenicity by using only diazo compounds based on 3, 3 ' , 5, 5 ' - tetramethylbenzidine. 3, 3 ' , 5, 5 ' -Tetramethylbenzidine is a well-studied non-mutagenic, non-carcinogenic substitute for benzidine. Holland et al . . Tetrahedron,
30: 3299-3302, (1974) . De Serres and Ashby (eds.) Evaluation of Short -Term Tests for Carcinogens, Elsevier North-Holland, 1981. 3, 3', 5,5'- Tetramethylbenzidine has been used as a substitute for benzidine in the synthesis of azo dyes. Josephy and
Weerasooriya, Chem . -Biol . Interactions, 1984: 375-382, (1984) . Ashby et al . , Carcinogenesis 3: 1277-1282, (1982) . Inventors have synthesized the tetramethyl analog of Chrysamine G (4,4 ' -bis (3-carboxy-4- hydroxyphenylazo) -3 , 3 ' , 5, 5 ' -tetramethylbiphenyl) and have shown it to bind to synthetic Aβ (10-43) with a Ki of 0.75 ± .09 μM, a value about twice that of Chrysamine G.
Any potential problems with lower bioavailability is avoided by the use of alkenyl and alkynyl derivatives of the azo compounds. These compounds are not substrates for reduction by bacterial or mammalian azo reductases.
Indeed, compounds of the present invention intended for therapeutic use are advantageous over existing compounds because they contain either a non- mutagenic, non-carcinogenic benzidine derivative or an alkenyl or alkynyl linkage which is not a substrate for bacterial azo-reductases in the intestines. In vi tro studies have shown that Aβ neurotoxicity requires fibril formation and is inhibited by Congo red. Specifically, it has been shown that the amyloid
fibril-binding dye Congo red inhibits fibrillar Aβ neurotoxicity by inhibiting fibril formation or by binding preformed fibrils. Lorenzo et al . , Proc . Natl . Acad. Sci . USA 91: 12243-12247 (1994) . Congo red also has been shown to inhibit pancreatic islet cell toxicity of diabetes-associated amylin, another type of amyloid fibril. Lorenzo et al . , supra . See also, Burgevin et al . NeuroReport 5: 2429 (1994) ; Pollack et al . , J. Neurosci . Letters 184: 113-116 (1995) ; Pollack et al . Neuroscience Letters 197: 211 (1995) . These data indicate that amyloid-binding compounds such as Chrysamine G and its derivatives, which are similar to Congo red but which, unlike Congo red, enter the brain well, would be effective in preventing cell degeneration and toxicity associated with fibril formation in amyloidosis associated conditions.
In Example 8 and Figures 12 and 13 , it is shown that Chrysamine G has effects very similar to those previously reported for Congo red in having a dose- dependent, protective effect in rat pheochromocytoma. Therefore, these in vi tro assays provide a means for selecting compounds for use in pharmaceutical compositions for the prevention of cell degeneration and toxicity associated with fibril formation. Compounds such a Chrysamine G and the above described derivatives thereof, are tested pursuant to the present invention, for in vivo efficacy in preventing amyloid fibril formation or associated
cellular degeneration, as measured by the formation of dystrophic neurites, synapse loss, neurofibrillary tangle formation and gliosis, in an animal model, such as the "senile animal" model for cerebral amyloidosis, Wisniewski et al . , J. Neuropathol . & Exp . Neurol . 32: 566 (1973) , the mouse model of familial Mediterranean fever (Neurochem. , Inc. Kingston, Ontario, Canada) and the transgenic mouse model of Alzheimer-type neuropathology, Games et al . , Nature 373: 523-527 (1995) . In the familial Mediterranean fever model, the animals develop systemic amyloidosis. In an in vivo assay according to this invention, serial necropsies in animals treated and untreated with the compounds of the present invention to evaluate the inhibition of amyloid formation are compared. In the animal models for cerebral amyloid formation, in addition to following amyloid formation serially, the presence of amyloid- associated neurodegeneration, as measured by the formation of dystrophic neurites, synapse loss, neurofibrillary tangle formation and gliosis, also is assessed in serial necropsies in animals treated and untreated with the compounds of the present invention.
According to the present invention, a pharmaceutical composition comprising Chrysamine G or derivatives thereof, is administered to subjects in whom amyloid or amyloid fibril formation, cell degeneration and toxicity are anticipated. In the preferred embodiment, such subject is a human and
includes, for instance, those who are at risk of developing cerebral amyloid, including the elderly,- nondemented population and patients having amyloidosis associated diseases and Type 2 diabetes mellitus. The term "preventing" is intended to include the amelioration of cell degeneration and toxicity associated with fibril formation. By "amelioration" is meant the prevention of more severe forms of cell degeneration and toxicity in patients already manifesting signs of toxicity, such as dementia.
The pharmaceutical composition for purposes of preventing cell degeneration and toxicity associated with fibril formation in amyloidosis associated diseases comprises Chrysamine G or a derivative thereof described above and a pharmaceutically acceptable carrier. In one embodiment, such pharmaceutical composition comprises serum albumin, Chrysamine G or Chrysamine G derivative and a phosphate buffer containing NaCl . Other pharmaceutically acceptable carriers include aqueous solutions, non-toxic excipients, including salts, preservatives, buffers and the like, as described, for instance, in REMINGTON'S PHARMACEUTICAL SCIENCES, 15th Ed., Easton: Mack Publishing Co., pp. 1405-1412 and 1461-1487 (1975) and THE NATIONAL FORMULARY XIV., 14th Ed. Washington:
American Pharmaceutical Association (1975) , and the UNITED STATES PHARMACOPEIA XVIII. 18th Ed. Washington: American Pharmaceutical Association (1995) , the
contents of which are hereby incorporated by reference.
Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oil and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic/aqueous solutions, saline solutions, parenteral vehicles such as sodium chloride, Ringer's dextrose, etc. Intravenous vehicles include fluid and nutrient replenishers. Preservatives include antimicrobial, anti-oxidants, chelating agents and inert gases. The pH and exact concentration of the various components the pharmaceutical composition are adjusted according to routine skills in the art. See, Goodman and Gilman's THE PHARMACOLOGICAL BASIS FOR THERAPEUTICS (7th Ed. ) .
According to the invention, such pharmaceutical composition could be administered orally, in the form of a liquid or solid, or injected intravenously or intramuscularly, in the form of a suspension or solution. By the term "pharmaceutically effective amount" is meant an amount that prevents cell degeneration and toxicity associated with fibril formation. Such amount would necessarily vary depending upon the age, weight and condition of the patient and would be adjusted by those of ordinary skill in the art according to well-known protocols. In one embodiment, a dosage would be between 0.1 and 100
mg/kg per day, or divided into smaller dosages to be administered two to four times per day. Such a regimen would be continued on a daily basis for the life of the patient. Alternatively, the pharmaceutical composition could be administered intramuscularly in doses of .1 to 100 mg/kg every one to six weeks.
In yet another embodiment, the invention relates to a method of detecting amyloid deposits in biopsy or post-mortem tissue. The method involves incubating formalin-fixed tissue with a solution of a compound of Formula I, described above. Preferably, the solution is 25-100% ethanol, (with the remainder being water) saturated with the compound of Formula I . Upon incubation, the compound stains or labels the amyloid deposit in the tissue, and the stained or labelled deposit can be detected or visualized by any standard method. Such detection means include microscopic techniques such as bright-field, fluorescence, laser- confocal and cross-polarization microscopy. In yet another embodiment, the invention relates to a method of quantifying the amount of amyloid in biopsy or post-mortem tissue. This method involves incubating a labelled derivative of Chrysamine G, preferably the compounds of Formula I, or a water- soluble, non-toxic salt thereof, with homogenate of biopsy or post-mortem tissue. The tissue is obtained and homogenized by methods well known in the art . The preferred label is a radiolabel, although other labels
such as enzymes, chemiluminescent and immunofluorescent compounds are well known to skilled artisans. The preferred radiolabel is 125I, 14C or 3H, the preferred label substituent of Formula I is at least one of R-.- R7, R10-R27. Tissue containing amyloid deposits will bind to the labeled derivatives of Chrysamine G. The bound tissue is then separated from the unbound tissue by any mechanism known to the skilled artisan, such as filtering. The bound tissue can then be quantified through any means known to the skilled artisan. See Example 3. The units of tissue-bound radiolabeled Chrysamine G derivative are then converted to units of micrograms of amyloid per 100 mg of tissue by comparison to a standard curve generated by incubating known amounts of amyloid with the radiolabeled Chrysamine G derivative.
In yet another embodiment, the invention relates to a method of distinguishing an Alzheimer's diseased brain from a normal brain involving obtaining tissue from (i) the cerebellum and (ii) another area of the same brain, other than the cerebellum, from normal subjects and from subjects suspected of having Alzheimer's disease. See Example 3. Such tissues are made into separate homogenates using methods well known to the skilled artisan, and then are incubated with a radiolabeled Chrysamine G derivative. The amount of tissue which binds to the radiolabeled Chrysamine G derivative is then calculated for each tissue type
(e.g. cerebellum, non-cerebellum, normal, abnormal) and the ratio for the binding of non-cerebellum to cerebellum tissue is calculated for tissue from normal and for tissue from patients suspected of having Alzheimer's disease. These ratios are then compared. If the ratio from the brain suspected of having Alzheimer's disease is above 90% of the ratios obtained from normal brains, the diagnosis of Alzheimer's disease is made.
EXAMPLE 1. THE SYNTHESIS PF CHRYSAMINE G AND
DERIVATIVES THEREOF
Synthesis of Chrysamine G
The synthesis of Chrysamine G (i.e., 4,4 '-bis (3- carboxy-4-hydroxyphenylazo) -biphenyl) requires the following reaction steps. These reaction steps will be referred to as the "Chrysamine G Synthesis" general procedure. Benzidine»2HCl (28.9 mg, 0.11 mmole, Sigma Chemical Company, St. Louis, MO) was added to 1.5 ml of 1:1 DMSO:distilled/deionized H20 in a 50cc round bottom flask. Each of the reaction steps were carried out at 0°C unless otherwise specified. Twenty-nine μl of concentrated HC1 were added, resulting in a clear solution after stirring. To the benzidine solution, a solution of 15.5 mg (0.22 mmole) of NaN02 in 300 μl of 1:1 DMSO/H20 was added drop-wise, resulting in a pH of about 2-3. The reaction mixture was stirred for 45 min, and then to this tetra-azotized benzidine mixture was
added drop-wise over a 10 min period to 24.8 mg (0.18 mmole) of methyl salicylate (Aldrich) dissolved in 2.0 ml of 100% DMSO containing 250 mg/ml Na2C03 in suspension, keeping the pH about 10.5. The resulting mixture was stirred for 1 hr at 0°C, and then overnight at room temperature.
After this time, the pH was adjusted to about 7 and the mixture was extracted with three 50 ml portions of chloroform. The combined chloroform extracts were washed with three 50 ml portions of H20, and then taken to dryness yielding the dimethyl ester of Chrysamine G (i.e., 4,4' -bis (3-methoxycarbonyl-4-hydroxyphenylazo) - biphenyl) , which was further purified by recrystallization from chloroform/hexane. The ester was then hydrolysed by dissolution in about 100 ml of
1:1 ethanol:H20 containing four equivalents of NaOH and refluxed for three hours. Evaporation of the ethanol followed by lyophilization of the H20 yielded the tetra-sodium salt of Chrysamine G. The free acid of Chrysamine G was formed by dissolving the tetra-sodium salt in H20, washing once with chloroform to remove any unhydrolysed dimethyl ester, lowering the pH to about 2 and extracting with three 50 ml portions of ethyl acetate. The combined ethyl acetate extracts were washed with three 50 ml portions of H20 and taken to dryness.
Under these conditions, there was no remaining methyl salicylate, salicylic acid, or benzidine, and
only trace amounts of the mono-substituted product, 4- hydroxy-4 ' - (3-carboxy-4-hydroxyphenylazo) -biphenyl by reverse-phase HPLC using a C4 column (Vydac 214-TP510) using a solvent system of sodium phosphate buffer (5 mM, pH 6) :acetonitrile (ACN) 90:10, isocratically, for 10 min and then increased to 50% ACN over the next 20 min at a flow rate of 3.5 ml/min. The column eluant was monitored at 290 and 365 nm with a dual wavelength, diode array detector (Perkin Elmer 235C) . Under these conditions, Chrysamine G eluted at 17.6 min.
The structure of Chrysamine G and derivatives was confirmed by proton NMR at 500 MHz in DMSO-d6 with TMS as the internal standard. The peak assignments for the tetra-sodium salt of Chrysamine G were as .follows with SA referring to protons at the specified ring position on the salicylic acid moiety and BZ referring to protons on the benzidine moiety: SA-3, doublet J=8.73 Hz at 6.75 parts per million (ppm) ; SA-4, doublet of doublets J=8.73 and 2.72 Hz at 7.82 ppm; BZ-2/6, doublet J=8.44 Hz at 7.91 ppm; BZ-3/5, doublet J-8.44 Hz at 7.95 ppm; and SA-6, doublet J-2.72 Hz at 8.28 ppm. The UV/visible spectrum in 40% ethanol showed a λ-nax at 389 nm. The molar absorptivity of Chrysamine G was determined by calculating the concentration of Chrysamine G through comparison of peak areas to an internal standard by NMR and then immediately running the UV/vis spectrum of an aliquot of the NMR sample
diluted in 40% ethanol. The molar absorptivity in 40% ethanol at 389 nm was 5.5 x 104 AU/(cm«M) .
[14C] Chrysamine G was synthesized by a modification of the above procedure. The tetra-azotization of benzidine was performed as described above except in
100% H20. Fifty μl of 2.5 M Na2C03 in H20 were added to 50 μCi of crystalline salicylic acid-carboxy-14C (Sigma) in a 0.5 ml conical glass vial. Sixty μl of the tetra-azotized benzidine mixture was added to the conical vial, vortexed and kept at 0°C for 1 hr. To prevent formation of the mono-substituted benzidine by¬ product, 12.5 μl of 250 mM non-radioactive salicylic acid (Sigma) in 2.5 M Na2C03 was added to the reaction mixture and maintained for 1 hr at 0°C. The vial was kept overnight at room temperature. The entire mixture was dissolved in a minimal amount of 35% ACN and injected onto the C4 column as described above. The peak corresponding to the Chrysamine G standard was collected and lyσphilized. A specific activity of 26.8 Ci/mole was calculated by determining the absorbance at 389 nm and then counting the radioactivity in an aliquot of the same sample. The [1 C] Chrysamine G was stored in 40% ethanol. When the purified t14C] Chrysamine G was re-injected onto the C4 column and eluted isocratically with 21% ACN at 3.5 ml/min, > 98% of the radioactivity co-eluted with authentic Chrysamine G at 10.4 min. Many of the Chrysamine G derivatives were synthesized using this "Chrysamine G
Synthesis" general procedure, with the exceptions noted below. Structures of the derivatives were verified by
NMR. Figure 1 shows the chemical structure of
Chrysamine G and several derivatives. Synthesis of the 3-Isopropylsalicylic Acid Derivative of Chrysamine G
4,4' -bis (3-carboxy-4-hydroxy-5- isopropylphenylazo) -3-iodobiphenyl is synthesized by substituting 3-bromobenzidine (see below) for benzidine and methyl 3-isopropylsalicylate for methyl salicylate in the Chrysamine G Synthesis described above. The bromo derivative thus obtained is converted to the tri- alkyl tin and then to the iodo derivative as described in detail below.
Synthesis of Pyridine and Diazine Derivatives of Chrysamine G
4,4' -bis (5-carboxy-6-hydroxy-3-pyridylazo) -3- iodobiphenyl and related hydroxypyridinecarboxylic or hydroxydiazinecarboxylic acid derivatives of Chrysamine G are synthesized by substituting 3-bromobenzidine (see below) for benzidine and 2-hydroxynicotinic acid (or, for related derivatives, 6-hydroxynicotinic acid, 3- hydroxypicolinic acid, or hydroxydiazinecarboxylic acids such as 4-hydroxypyrimidine-6-carboxylic acid) for methyl salicylate in the Chrysamine G Synthesis described above. The bromo derivative thus obtained is
converted to the tri-alkyl tin and then to the iodo derivative as described in detail below.
Synthesis of Naphthalene, Quinoline, and Benzodiazine Derivatives of Chrysamine G 4,4' -bis (3-carboxy-4-hydroxy-naphthylazo) -3- iodobiphenyl and related hydroxynaphthoic, hydroxyquinolinecarboxylic, or hydroxybenzodiazinecarboxylic acid derivatives of
Chrysamine G are synthesized by substituting 3- bromobenzidine (see below) for benzidine and 1-hydroxy- 2-naphthoic acid (or, for related derivatives, other hydroxynaphthoic acid isomers, hydroxyquinolinecarboxylic acids such as kynurenic acid, or hydroxybenzodiazinecarboxylic acids such as 5- hydroxyquinoxaline-6-carboxylic acid) for methyl salicylate in the Chrysamine G Synthesis described above. The bromo derivative thus obtained is converted to the tri-alkyl tin and then to the iodo derivative as described in detail below.
Synthesis of Nitrophenol Derivatives of Chrysamine G
4,4' -bis (3-nitro-4-hydroxy-phenylazo) -3- iodobiphenyl and related nitrophenol derivatives of Chrysamine G are synthesized by substituting 3- bromobenzidine (see below) for benzidine and 2- nitrophenol (or other nitrophenol isomers for related
derivatives) for methyl salicylate in the Chrysamine G Synthesis described above. The bromo derivative thus obtained is converted to the tri-alkyl tin and then to the iodo derivative as described in detail below.
Alternative Method for the Synthesis of Diazo Compounds
As an alternative method of forming azo compounds, nitroso compounds are coupled to amines in glacial acetic acid by the Mills reaction. Badger, G. et al . , Aust . J. Chem . 17: 1036 (1964) . Nitroso compounds are made by the method of Coleman et al. , Organic Synthesis Collective Vol. Ill: p668. For example, 3-nitrobenzoic acid (Aldrich Chem. Co., Milwaukee, WI) (2.44 mmoles) and a solution of 150 mg of NH„C1 in 5 ml of water are combined in a 50 ml round-bottomed flask and stirred vigorously. Zinc dust (372 mg) is added in small portions over 5 minutes. After the addition of zinc is complete the temperature begins to rise, but is kept between 50 and 55°C by the use of an ice bath. The mixture is stirred at that temperature for 20 minutes and then the zinc residues are filtered and washed with boiling water. The combined filtrate and washings are cooled to 0°C in a beaker by the addition of ice. To this cold mixture of the hydroxylamine, a cold solution of sulfuric acid (750 μl of concentrated acid plus enough ice to bring the temperature to -5°C) is added with stirring. An ice-cold solution of 170 mg of
sodium dichromate dihydrate in 750 μl of water is added all at once with stirring. The resulting 3- nitrosobenzoic acid thus obtained is combined with one- half of an equivalent of benzidine, 2, 7-diaminofluorene or another benzidine derivative in glacial acetic acid and warmed to 70-80°C for 7 hrs and then allowed to stand at room temperature overnight. The mixture is diluted with water and extracted with ethyl acetate giving 4,4 ' -bis (3-carboxy-phenylazo) -biphenyl) , 2,7- bis (3-carboxy-phenylazo) -fluorene, or the corresponding bis (3-carboxy-phenylazo) derivative of the particular benzidine derivative employed.
Synthesis of Methoxy Derivatives
Methoxy derivatives of all phenol compounds are synthesized by the following procedure. To one equivalent of the phenolic Chrysamine G ester derivative dissolved in acetone at an approximate concentration of 5 mg/ml, is added 10 equivalents of methyl iodide (Aldrich Chemical Company, Milwaukee, WI) and 10 equivalents of K2C03. The suspension is refluxed for 6-24 hrs, taken to dryness on a rotary evaporator, and extracted with chloroform. The ester is hydrolysed by addition of 1:1 THF/0.1 N NaOH to a concentration of approximately 1 mg/ml and stirring at room temperature from 24-72 hours. Unhydrolysed methoxy-ester is removed by extraction with chloroform,
the pH was adjusted to ~2, and the methoxy-acid was extracted into ethyl acetate.
Synthesis of Substituted Benzidine Derivatives
For the substituted benzidine compounds, the above Chrysamine G synthesis general procedure is followed except 3, 3 ' -dichlorobenzidine (Pfaltz & Bauer, Waterbury, CT) , 4,4 ' -diaminooctafluorobiphenyl, 3 , 3 ', 5, 5 ' -tetramethylbenzidine (Aldrich Chemical Company, Milwaukee, WI) , 3, 3 ' -dimethylbenzidine (Aldrich Chemical Company, Milwaukee, WI) , 3,3'- dimethoxybenzidine (Aldrich Chemical Company, Milwaukee, WI) , benzidine-3,3 ' -dicarboxylic acid (Pfaltz & Bauer, Waterbury, CT) or lower alkyl esters thereof, or 3 , 3 ' -dinitrobenzidine (Fluka Chemical Corp., Ronkonkoma, NY) are used in place of unsubstituted benzidine. Other substituted benzidines including, but not limited to, those listed below are synthesized by the referenced methods and also can be used in place of unsubstituted benzidine: 3,3'- dibromobenzidine and 3,3 ' -diiodobenzidine (Snyder, H. et al . , J. Am. Chem. Soc . 71: 289-291, 1949) ; 3- bromobenzidine and 3-iodobenzidine (Badger, G. et al . , Aust . J. Chem. 17: 1036-1049, 1964; Holland, V. et al . , Tetrahedron 30: 3299-3302, 1974) .
Synthesis of Phenanthracene, Benzo(c)cinnoline, Fluorene, Fluorenone, Carbazole, Dibenzofuran, Dibenzothiophene and Dibenzothiophene-9, 9-dioxide Derivatives Fluorene (2, 2 ' -methylenebiphenyl) derivatives of
Chrysamine G are made by substituting 2,7-diamino- fluorene (Aldrich Chemical Company, Milwaukee, WI) for benzidine in the tetra-azotozation procedures and then coupled by the same procedures used for tetra-azotized benzidine. Likewise, 2, 7-diaminophenanthracene, 2,7- diamino-3, 6-dimethylbenzo [c] cinnoline, 2,7- diaminofluorenone, 9-methyl-2, 7-diaminocarbazole, 3,7- diaminodibenzofuran (unlike the remainder of these polycyclic compounds, the conventional numbering system puts the oxygen bridge atom of dibenzofurans at the 5- position instead of the 9-position, therefore the amino substituents are in the 3 , 7-positions rather than the 2, 7-positions as in the other compounds even though, spatially, the positions are equivalent) , 2,7- diaminodibenzothiophene, and 2,7- diaminodibenzothiophene-9, 9-dioxide ("benzidine sulphone") are substituted for benzidine in the standard tetra-azotization and coupling procedures. 9- Methyl-2, 7-diaminocarbazole compounds may be N- demethylated after coupling if the unsubstituted carbazole is desired. 2, 7-Diaminophenanthracene is synthesized by reduction of 2, 7-dinitrophenanthracene made from 2-amino-3 ' , 5-dinitro-cis-stilbene by the method of Ullmann and Mallet Ber. 31: 1694-1696, 1898
and Nunn, A. et al . , J. Chem. Soc . 1952: 2797-2803. 2, 7-Diamino-3, 6-dimethylbenzo [c] cinnoline is prepared by NaOBr oxidation of the corresponding hydrazo compound formed via the reduction of 3 ,3 ' -dimethyl- 6,6 ' -dinitrobenzidine (Aldrich Chemical Co., Milwaukee, WI) with zinc in aqueous NaOH by the method of Snyder et al . , J. Amer. Chem . Soc . 71: 289 (1949) . Alternatively, 3 , 3 ' -dimethyl-6, 6 ' -dinitrobenzidine can be first tetra-azotized and coupled in place of benzidine, followed by treatment with Zn/NaOH and NaOBr as above. 2,7-Diaminofluorenone is synthesized by reduction of 2, 7-dinitrofluorenone made from 2-amino- 3 ' , 5-dinitrobenzophenone by the method of Ullmann and Mallet Ber. 31: 1694-1696, 1898 and Nunn, A. et al . , J. Chem. Soc . 1952: 2797-2803. 9-Methyl-2, 7- diaminocarbazole is synthesized by reduction of 9- methyl-2, 7-dinitrocarbazole made by N-methylation of 2, 7-dinitrocarbazole prepared from 2-amino-3 ' , 5- dinitrodiphenylamine as described in Saunders, K.H. and Allen, R.L.M. AROMATIC DIAZO COMPOUNDS 625-640
(Edward Arnold, London, 1985) , the entire contents of which are hereby incorporated by reference. 3,7- Dia inodibenzofuran is synthesized by reduction of 3,7- dinitrodibenzofuran made from 2-amino-3 ' , 5- dinitrodiphenylether as described in Saunders, K.H. and Allen, R.L.M. AROMATIC DIAZO COMPOUNDS 625-640 (Edward Arnold, London, 1985) , the entire contents of which are hereby incorporated by reference. 2,7-
Diaminodibenzothiophene, and 2,7- diaminodibenzothiophene-9, 9-dioxide are prepared by the methods of Courtot and Evain Bull. Soc . Chi . 49 (iv) : 528, 1931 and Cullinane and Dav'ies Rec . Trav. Chim. 55: 881-886 (1936) .
Synthesis of Alkynyl (C≡C) and Vinyl (CH--CH) Derivatives of Chrysamine G
5-Bromosalicylic acid (Aldrich Chemical Company,
Milwaukee, WI) is converted to the methyl ester/methyl ether by reaction with methyl iodide in the presence of K2C03 as described above. The 2-methoxy-5-bromobenzoic acid methyl ester thus obtained is reacted with (trimethylsilyl) acetylene (Aldrich Chemical Company, Milwaukee, WI) in the presence of palladium. The trimethylsilyl group is removed and two equivalents of the resultant 2-methoxy-5-acetylenylbenzoic acid methyl ester is reacted with 4,4 ' -dibromobiphenyl (Aldrich Chemical Company, Milwaukee, WI) in the presence of palladium as above. The resultant alkynyl analogue of Chrysamine G, 4,4 ' -bis (3-carboxy-4- methoxyphenylacetylenyl) -biphenyl is prepared by hydrolysis of the ester as described above. This alkynyl analogue is reduced by conventional methods to form the vinyl analogue of Chrysamine G.
Synthesis of Diiodosalicylic Acid Derivative of Chrysamine G
The 3-iodosalicylic acid derivative of Chrysamine
G, 4,4' -bis (3-carboxy-4-hydroxy-5-iodophenylazo) - biphenyl) , is synthesized by iodination of 5- iodosalicylic acid (Aldrich Chemical Company,
Milwaukee, WI) at the 3-position followed by selective de-iodination of the 5-position. After formation of the methyl ester and recrystallization, the light brown, waxy 3-iodo derivative is substituted for methyl salicylate in the above procedure for the synthesis of Chrysamine G. The desired product is separated by elution from a silica gel column with 75% ChCl3/25% hexane. The ester is then hydrolysed by dissolution in 1:1 ethanol :H20 containing four equivalents of NaOH and refluxed for three hours.
Synthesis of Di-fluoro Chrysamine G
The 5-fluoro derivative, 4,4 ' -bis (2-hydroxy-3- carboxy-5-fluorophenylazo) -biphenyl) , is synthesized by substituting 5-fluorosalicylic acid (Aldrich Chemical Company, Milwaukee, WI) for salicylic acid. [18F] aryl fluorides derivatives of Chrysamine G can be prepared by substituting 18F-labeled precursors such as [18F]LiBF4, in the Schiemann reaction, via triazene decomposition with Cs [18F] , or via nucleophilic 18F- for-X substitution, where X = tosyl, triflate, N02, +N(CH3)3, or halogen. See Fowler, J. and Wolf, A. in
POSITRON EMISSION TOMOGRAPHY AND AUTORADIOGRAPHY (Phelps, M., Mazziota, J., and Schelbert, H. eds . ) 391- 450 (Raven Press, NY, 1986) and Kilbourn, M. Fluorine- 18 labeling of radiopharmaceuticals. (Natl. Acad. Press, Washington, D.C.) (1990) .
Synthesis of Aromatic Fluoroalkyl and Fluoroalkoxy Derivatives
Aromatic fluoroalkyl derivatives are synthesized employing the method of Bishop et al . , J. Med. Chem. 34: 1612 (1991) in which Claisen rearrangement of the appropriate O-allyl ethers forms an aromatic allyl derivative which can be further functionalized to yield the fluoroethyl or fluoropropyl derivatives. Alternatively, an aromatic iodide can be readily converted to an aromatic alkyne consisting of two to five carbon atoms in length using the palladium- assisted coupling methodology of Sonogashira et al . , Tetrahedron Letters 4467-4470 (1975) . Subsequent derivatization of the alkyne yields the fluoroalkyl derivative. Fluoroalkoxy derivatives may be prepared by the method of Chumpradit et al . , J. Med. Chem . 36: 21 (1993) in which alkylation of the appropriate phenol with the appropriate 1-bromo (or iodo or sulfonyloxy) - omega-fluoroalkane yields the corresponding fluoroalkoxy derivative.
Radiofluorination of Aromatic Alkylsulfonyloxy and Alkoxysulfonyloxy Derivatives
Radiofluorination to yield the aromatic
[18F] fluoroalkyl and [18F] fluoroalkoxy derivatives is performed by the method of Mathis et al . , Nucl . Med.
Biol . 19: 571 (1992) in which aromatic alkyl- or alkoxysulfonyloxy (e.g. alkoxytosylate) derivatives are substituted with [18F] fluoride to yield aromatic
[18F] fluoralkyl and [18F] fluoralkoxy compounds.
Radio-Iodination and Radio-Bro ination of
Chrysamine G Derivatives by the Tri-Alkyl Tin Route
Synthesis of Tri-Alkvl Tin Derivatives of Chrysamine G The general structure of the tri-alkyl tin derivative of Chrysamine G is shown in Figure 2B. In general, one tri-alkyl tin group will be substituted at the 3-position on one side of the biphenyl moiety, but other positions, including the salicylic acid or heterocyclic moiety are also potential targets. These tri-alkyl tin derivatives are stable immediate precursors for preparation of the radioiodinated and radiobrominated compounds to be used in humans . More specifically, these tri-alkyl tin derivatives are used to prepare the halogenated radioactive compounds applicable for use in in vivo imaging of amyloid.
General Procedures for the Synthesis of Tri-Alkyl Tin Derivatives
Tri-alkyl tin derivatives are prepared from the appropriate arylhalides, [ (C6H5) 3P] 3Pd(0) , and hexaalkylditin by previously published procedures including Kosugi, M. , et al., Chem. Lett . 1981: 829;
Heck, R. Pure and Appl . Chem. 1978: 691; Echavarren, A. and Stille, J. J. Am. Chem. Soc. 1987: 5478; Mitchell,
T. J. Organometallic Chem. 1986: 1; and Stille, J. Pure and Applied Chem . 1985: 1771. These derivatives also can be obtained by the use of n-BuLi and trialkyl tin chloride by the procedure of Mathis et al . , J. Labell .
Comp . and Radiopharm. 1994: 905.
Synthesis of the 3-trialkyl tin derivative of 4,4 ' -bis (3-methoxycarbonyl-4-hydroxyphenylazo) - biphenyl
3-Bromo or 3-iodo-4,4 ' -bis (3-methoxycarbonyl-4- hydroxyphenylazo) -biphenyl or its dimethyl ether are prepared by synthesis of 3-bromo- or 3-iodobenzidine (see above) , tetra-azotization and coupling to methyl salicylate as for the synthesis of Chrysamine G, and methylation of the phenol as described above when the methoxy compound is desired. Under an argon atmosphere, 1 mmol of the phenolic ester or the methoxy ester, [ (C6H5) 3P] 3Pd(0) (0.1 to 0.2 mmol) , hexabutylditin or hexamethyl ditin (1.25 mmol) , and dioxane (25 ml) is heated at 70°C for 16 hrs. The reaction mixture is cooled and the solvent is
evaporated. Tri-alkyl tin halide is removed with aqueous KF. The organics are extracted with ethyl acetate, dried over magnesium sulfate, filtered, and the solvent is evaporated under reduced pressure. The residue is purified on silica gel to obtain 3- trialkyltin-4,4' -bis(3-methoxycarbonyl-4- hydoxyphenylazo) -biphenyl.
Radio-Iodination or Radio-Bromination of Tri-AIκyl Tin Derivatives
The tributyl or trimethyl tin derivatives are radio-iodinated with Na[125I] or Na[123l] or radio- brominated with Na [75Br] or Na [76Br] by published procedures such as Mathis et al . , J. Labell . Co p . and
Radiopharm . 1994: 905; Chumpradit et al . , J. Med . Chem .
34: 877 (1991) ; Zhuang et al . , J. Med. Chem . 37: 1406
(1994) ; Chumpradit et al . , J. Med. Chem. 37: 4245 (1994) . In general, 0.5 mg of tri-alkyl tin compound, 0.2 ml of anhydrous acetonitrile, 10 μl of 2M H3P04, 2- 100 μl of a solution of high specific activity (>2000 Ci/mmol) Na[125I] or Na[123I] (or Na [75Br] or Na [76Br] ) in pH 9-12 NaOH, and dichloramine-T (DCT) (20 μl of 2.5 mg/ml DCT in acetonitrile) are placed in a 1 ml Reacti- Vial . The vial is capped and the mixture is stirred at room temperature in the dark. The reaction is monitored by HPLC and after 30 min is quenched with 50 μl of 2 M Na2S203. The product is purified by standard chromatographic techniques. Mathis et al . , J. Labell . Comp . and Radiopharm. 1994: 905. Similarly, low specific activity 18F derivatives are prepared by analogous procedures.
General Procedures for the Preparation of Non- Radioactive I, Br, Cl, F and -SH Derivatives
In general, 3- or 4-amino derivatives of salicylic acid, or the corresponding derivatives of the heterocyclic analogues of salicylic acid shown in Figure 2, are converted to the corresponding diazo compounds with sodium nitrite and HCl or H2S04. The iodine derivatives are directly prepared by forming the diazonium iodide which is then converted into the aryl iodide, or by way of the triazene intermediates. See, e . g. , Greenbaum, F. Am. J. Pharm. 108: 17 (1936), Satyamurthy, N. and Barrio, J., J. Org. Chem . 48: 4394
(1983) and Goodman, M. et al . , J. Org. Chem. 49: 2322
(1984) . Aryl bromides and chlorides are prepared from the diazo compounds by treatment with CuCl or CuBr according to the Sandmeyer reaction or via the triazene as for the iodine derivatives. Aryl fluorides are prepared by treating the diazonium compounds with NaBF4, HBF4, or NH4BF4 according to the Schiemann reaction or via triazene decomposition similar to the iodine derivatives. Aryl thiols are prepared from the diazonium compounds by treatment with sulfur-containing nucleophiles such as HS", EtO-CSS", and S2 2". Alternatively, aryl thiols can be prepared by replacement of aryl halides with sulfur containing nucleophiles. These reactions are described in March, J., ADVANCED ORGANIC CHEMISTRY: REACTIONS, MECHANISMS, AND STRUCTURE (3rd Edition, 1985) .
General Procedures for the Preparation of Radioactive C, F and Tc Derivatives
In addition to the above procedures, high specific activity radiolabeling with 99mTc for SPECT or with the positron-emitting radionuclides 11C, 18F, 75Br and 76Br is accomplished according to literature-based methods well known in the art . Some of the potential specific methods are described below, but there are other well- known methods which will be apparent to those skilled in the art and are described in Fowler, J. and Wolf, A. Posi tron emi tter- labeled compounds in POSITRON EMISSION TOMOGRAPHY AND AUTORADIOGRAPHY (Phelps, M., Mazziota, J., and Schelbert, H. eds. ) p 391-450 (Raven Press, NY) (1986) , Coenen, H. et al . , Radiochimica Acta 34: 47 (1983), and Kulkarni, Int. J. Rad. Appl . £ Inst . (Part B) 18: 647 (1991) , the contents of which are hereby incorporated by reference.
99m Tc derivatives are prepared by complexation with the aryl thiols. Radiolabeling with 1XC can be readily done via N-methylation, O-methylation as described above substituting ["C]methyl iodide, [UC] alkylation, or [X1C] carboxylation of suitable Chrysamine G analogues. [18F] aryl fluorides derivatives can be prepared by substituting 18F-labeled precursors such as [18F]LiB4 in the Schiemann reaction described above, via triazene decomposition with Cs [18F] , or via nucleophilic 18F-for-X substitution, where X = tosyl,
triflate, N02, +N(CH3)3, or halogen. Radiobromination using 75Br and 76Br can be accomplished using either electrophilic (Br+) or nucleophilic (Br") substitution techniques analogue to radioiodination techniques, see Coenen, H. , supra .
Synthesis of the 3-Hydroxy-1,2-benzisoxazole derivative and related derivatives (see Figure 2C)
2, 6-Dihydroxybenzoic acid (γ-resorcylic acid) methyl ester (TCI America, Portland, OR) is converted to the hydroxamic acid by the use of hydroxylamine hydrochloride according to the method of Bόshagen (Chem. Ber 100: 954-960; 1967) . The hydroxamic acid is converted to the corresponding 3-hydroxy-1,2- benzisoxazole with the use of S0C12 and then triethylamine, also by the method of Bόshagen (Chem.
Ber 100: 954-960; 1967) . This compound is then coupled with benzidine, 3-bromobenzidine, or other benzidine derivatives via the same procedure used for salicylic acid derivatives. The bromo derivatives can then converted to the tri-alkyl tin and iodo-derivatives as described above.
Alternatively, methyl salicylate is coupled to benzidine as usual and the resulting 4,4 '-bis (3- methoxycarbonyl-4-hydroxyphenylazo) -biphenyl (or the dimethyl ester of Chrysamine G) is converted to the hydroxamic acid and then the benzisoxazole by the method of Bδshagen as described above. A third type of
3-hydroxy-1,2-benzisoxazole is synthesized from the dimethyl esters of several isomeric dihydroxy benzenedicarboxylic acids including 4, 6-dihydroxy-1, 3- benzenedicarboxylic acid, 3, 6-dihydroxyphthalic acid, and 2, 5-dihydroxyterephthalic acid (Aldrich Chem. Co., Milwaukee, WI) . After coupling to benzidine or its derivatives by standard procedures, the dihydroxy/diesters is converted to dihydroxy/dihydroxamic acids by reaction with hydroxylamine by the method of Bδshagen described above. Conversion to the double benzisoxazole is effected by treatment with S0C12 and triethylamine, again, by the method of Bόshagen described above.
Synthesis of the phthalimide or isoindole-1, 3 (2H) - dione derivative (see Figure 2D)
3-Hydroxyphthalimide made from 3-hydroxyphthalic anhydride (Aldrich Chemical Company, Milwaukee, WI) is coupled with benzidine or its derivative via the same procedure used for salicylic acid derivatives and then converted to the tri-alkyl tin and iodo-derivatives as described above.
Synthesis of the phthalhydrazide or 2,3- benzodiazine-1,4 (2H,3H) -dione derivative (see Figure 2E)
3-Hydroxyphthalhydrazide made from the reaction of 3-hydroxyphthalic anhydride (Aldrich Chemical Company,
Milwaukee, WI) with hydrazine is coupled with benzidine or its derivative via the same procedure used for salicylic acid derivatives and then converted to the tri-alkyl tin and iodo-derivatives as described above.
Synthesis of the 2,3-benzoxazine-l,4 (3H) -dione derivative (see Figure 2F)
3-Hydroxyphthalic anhydride (Aldrich Chemical
Company, Milwaukee, WI) is converted to the 2,3- benzoxazine with the use of hydroxylamine. The benzoxazine derivative is then coupled with benzidine or its derivative via the same procedure used for salicylic acid derivatives and then converted to the tri-alkyl tin and iodo-derivatives as described above.
Synthesis of the (2H)1,3-benzoxazine-2,4 (3H) -dione derivative (see Figure 2G)
This compound is synthesized by the method of
Effenberger et al . , { Chem . Ber. 105: 1926-1942; 1972) .
Briefly, phenol is coupled with benzidine or its derivative via the same procedure used for salicylic acid derivatives. This adduct is then converted to the carbamate by reaction with ethoxycarbonylisocyanate
(0=C=N-C0-0-Et) in the presence of triethylamine. This
substituted carbamate (or N-ethoxycarbonyl-carbamic acid-phenyl ester) is converted to the benzoxazinedione by heating in diphenyl ether. The benzoxazinedione is then converted to the tri-alkyl tin and iodo- derivatives, as described above.
Synthesis of the (3H)2-benzazine-l,3 (2H) -dione derivative (see Figure 2H)
3-Hydroxyphenylacetic acid (Aldrich Chemical
Company, Milwaukee, WI) is converted to the amide and then coupled with benzidine or its derivative via the same procedure used for salicylic acid derivatives. This adduct is then converted to the N-(3- hydroxyphenylacetoxy) -carbamic acid ethyl ester derivative by reaction with ethyl chloroformate. This substituted carbamate is converted to the benzazinedione by heating in diphenyl ether. The benzazinedione is then converted to the tri-alkyl tin and iodo-derivatives, as described above.
Synthesis of the 1, 8-Naphthalimide derivative (See Figure 21)
4-Amino-l, 8-naphthalimide is coupled with benzidine or its derivative via standard procedures for diazo coupling of aryl amines, as described in
Saunders, K.H. and Allen, R.L.M. AROMATIC DIAZO COMPOUNDS (Edward Arnold, London, 1985) , the entire contents of which are hereby incorporated by reference.
The diazo naphthalimide is then converted to the tri¬ alkyl tin and iodo-derivatives, as described above.
Synthesis of Tetrazole and Oxadiazole Derivatives (See Figure 2J and 2K) 2-Cyanophenol (Aldrich Chemical Company,
Milwaukee, WI) is converted to the tetrazole by reaction with sodium or aluminum azide according to the method of Holland and Pereira J". Med. Chem. 10: 149
(1967) and Holland U.S. Patent No. 3,448,107. Briefly, 2-cyanophenol or cyanophenol derivatives of Chrysamine G (1 mmol) in 40 ml DMF is treated with sodium azide (10 mmol) and triethylamine hydrochloride (10 mmol) under argon. The mixture is stirred at 120°C for 2 hrs after which the mixture is cooled and worked up in a manner analogous to that described above for Chrysamine G.
The oxadiazoles are synthesized by treatment of the tetrazoles prepared as above with an acid anhydride (such as acetic anhydride) . An alternate method is that of Bamford et al . , J. Med. Chem. 38: 3502 (1995) . In this procedure, hydrazide derivatives of Chrysamine G or salicylic acid (obtained by treatment of the respective esters with hydrazine) are treated with methyl isothiocyanate in the presence of dicyclohexylcarbodiimide.
Synthesis of Chrysamine G Derivatives for Use as Controls
The aniline derivative is synthesized by substituting two equivalents of aniline (Fisher Chemical Co., Fair Lawn, NJ) for each equivalent of benzidine. The 2, 2 ' -disulfonic acid derivative is synthesized by substituting benzidine-2, 2 ' -disulfonic acid (Pfaltz & Bauer, Inc., Waterbury, CT) for benzidine. The phenol derivative is synthesized by substituting one equivalent of phenol for each equivalent of salicylic acid. Congo red (Aldrich certified grade) is obtained commercially.
EXAMPLE 2. CHRYSAMINE G AND CHRYSAMINE G
DERIVATIVES BIND SPECIFICALLY TO Aβ Binding to synthetic Aβ (10-43)
Chrysamine G binds well to synthetic Aβ (10-43) peptide in vi tro . Figure 4A shows a Scatchard analysis of the binding of Chrysamine G to Aβ (10-43) . The higher affinity component has a KD of 0.257 μM and a Bmax of 3.18 nmoles Chrysamine G/mg Aβ (10-43) . The lower affinity component is less well defined by these data, but appears to have a KD of 4.01 μM and a B^ of 18.7 nmoles Chrysamine G/mg Aβ(10-43) . The low affinity component represents the binding of Chrysamine G at high concentrations to a distinct, low-affinity site, not the binding to an impurity in the
preparation. The amount of Chrysamine G injected in vivo is so low that there is not any binding to the- low-affinity component. At very low concentrations, the ratio of high-to-low affinity binding is very large.
The amount of Chrysamine G binding is linear with peptide concentration over the range employed, as shown in Figure 5.
Kinetics of Binding Kinetic studies showed a fairly rapid association (Figure 6A) , essentially complete by 1 min, at a [Chrysamine G] = 112μM with a t of 8.9 ± 1.8 sec and a somewhat less rapid dissociation (Figure 6.C) , tκ = 55 ± 9.4 sec [dissociation rate constant (k-x) = 1.26 x 10"2 sec"1] . Figure 6B shows a transformation of the association kinetic data according to the method of Bennett and Yamamura. Bennett, J.P. and Yamamura, H.I. in NEUROTRANS ITTER RECEPTOR BINDING (N.Y. : Raven Press 1985) pp. 61-89. The linear portion of the association curve in Figure 6A is transformed into the line of Figure 6B, in which In [Beq/ (Beq-Bt) ] is plotted versus time, where Beq is the amount of Chrysamine G bound at equilibrium (4 min) and Bt is the amount bound at time=t. The slope of this line equals kobserved and (kobserved - k_i) / [Chrysamine G] , where k_x is the dissociation rate constant determined from the data in
Figure 6C. The curve in Figure 6C follows the equation:
A =A e 1 t o where At is the amount of Chrysamine G remaining bound at time=t, A0 is the amount of Chrysamine G bound at time=0, t is the time in min, and k.-*. is the dissociation rate constant. From this analysis, the association rate constant (ki) is calculated to be 3.75 x 104 M"1 sec -1 giving a KD = k^/k-L = 0.34 μM, in good agreement with the Scatchard analysis.
Chrysamine G Derivatives Can Inhibit the Binding of Chrysamine G to Aβ
K values for the inhibition of [14C] Chrysamine G binding to Aβ (10-43) by the Chrysamine G analogues are shown under the chemical structures in Figure 1 and several displacement curves are shown in Figure 3. Kx is defined as IC50/ (1+ [L] /KD) , where [L] is the concentration of [14C] Chrysamine G in the assay (.100- .125 μM) and KD is 0.26 μM, the KD of Chrysamine G determined by the Scatchard analysis above. Chrysamine G itself gives a K. of 0.37+ 0.04 μM, a value very consistent with those obtained from the Scatchard and kinetic analyses. Congo red gives a Kx of 2.82±.84 μM. The difluoro derivative of Chrysamine G, (5-FSA)CG, (Figure 1) is one-third as potent as Chrysamine G itself (K.. = 1.16±0.19 μM) . The activity of the
difluoro Chrysamine G derivative suggests that an 18F difluoro Chrysamine G derivative works for PET imaging and an 19F difluoro Chrysamine G derivative works for MRS/MRI imaging of brain. The 3-ICG is slightly more potent than Chrysamine G. The activity of the 3-ICG derivative suggests that an 123I difluoro Chrysamine G derivative works for SPECT imaging. Methylating the phenol of 3-ICG decreases the affinity by a factor of 10 in 3-IGC(OMe)2. Methylating the carboxylate group effected an even greater (about
200-fold) decrease in affinity in CG(COOMe)2. Removing the acid moiety entirely, as in the phenol derivative, completely destroyed binding affinity.
These results suggest that the acid moiety of Chrysamine G analogues plays the major role in binding to Aβ and that the phenol moiety plays an facilitating role. The effect of the phenol could occur through hydrogen bonding to the acid which could serve to stabilize the structural orientation of the acid moiety. The presence of a phenol in the ortho position could also alter the charge distribution of the acid either through hydrogen bonding or through changes in the charge distribution of the aromatic system as a whole. Alternatively, the phenol could directly participate in binding to the amyloid via a bi-dentate attachment of both the phenol and the acid to the amyloid binding site. Adding a second phenol ortho to the carboxylate as in the resorcylic acid derivative,
(6-0HSA)CG, produces the highest affinity compound in this series having a Ki of 0.094 ± .02 μM.
Increasing the lipophilicity of the biphenyl backbone appears to increase the affinity somewhat . The di-halo derivatives, 3,3'-I2CG, 3,3'-Br2CG, and
3,3'-Cl2CG, all have very similar Ki values which are about half that of Chrysamine G.
Distorting the dihedral angle between the phenyl rings of the biphenyl group by substitution at the 2- position markedly diminishes affinity. This is demonstrated by the inactivity of the 2,2 ' -di-sulfonic acid derivative of Chrysamine G, 2, 2 ' - (S03) 2CG. Since the 3,3' di-carboxylic derivative, 3, 3 ' - (COOH) 2CG, shows only a 7-fold loss of activity from Chrysamine G, it is unlikely that the additional acidic moieties are the sole cause for the loss of activity in the 2,2'- disulfonic acid. This 2,2'- derivative is unique in that the bulky sulfonate groups in the 2-position force the biphenyl group out of planarity. Molecular modelling studies showed that the dihedral angle between the two biphenyl benzene rings in the 2,2'- disulfonic acid derivative is 83°. This angle is approximately 35-40° in Chrysamine G and all of the other active derivatives. In an attempt to explore the importance of the bidentate nature of the functional groups of Chrysamine G, the binding of an aniline derivative which represents one-half of a Chrysamine G molecule (Figure
1) was studied. An approximation of the energy of binding can be calculated from the equation:
ΔG = -R T In K eq
where ΔG is the energy of the binding reaction, R is the molar gas constant [8.31441 J/ (mole»°K) ] , T is temperature in °K and Keq is the equilibrium constant for the reaction:
[probe] + [peptide] ** [probe»peptide] and
Using the value of 0.26μM for the KD of Chrysamine G, the energy of binding is roughly 38
KJ/mole. If the aniline derivative binds with one-half of this energy, the expected energy of binding would be about 19 KJ/mole. From the KA of 73 μM for the aniline derivative, the energy of binding is 23 KJ/mole which is in acceptable agreement with the predicted value.
The importance of the hydrophobic region of Chrysamine G and the aniline derivative is demonstrated by the total lack of binding activity of salicylic acid itself. The affinity of Chrysamine G for Aβ appears to be several fold greater than the affinity of Congo red for this peptide. The binding is reversible with a dissociation constant of approximately 250-400 nM, whether measured by Scatchard analysis, kinetic methods, or inhibition of binding. Owing to the non- crystalline, poorly soluble nature of amyloid fibrils, the structure of Congo red or Chrysamine G complexes
with amyloid has never been defined by precise structural techniques such as x-ray crystallography or multi-dimensional NMR. Models of Congo red interactions with amyloid have been proposed. Cooper, Lab. Invest. 31: 232 (1974) ; Romhanyi, Virchows Arch . 354: 209 (1971) . This work suggests that Congo red does not bind to a single amyloid peptide molecule, but binds across several Aβ molecules oriented by virtue of the beta-sheet fibril. Klunk et al . , J. Histochem. Cytochem . 37: 1273 (1989) .
Figure 7 shows a schematic of this model, generated using MacroModel 2.5, in which Chrysamine G spans 5 peptide chains in an anti-parallel beta-sheet conformation. The peptides are used without further structural refinement. The peptides are aligned so that alternate chains were spaced 4.76 A apart, characteristic of beta-sheet fibrils. Alternate peptide chains are drawn in black and white. Chrysamine G (black) is energy minimized and aligned with the fibril model to maximize contact with lysine- 16 (light grey ovals in top figure) and the hydrophobic phenylalanine 19/20 region (bottom) . The two views are of the same model at approximately 90° from one another. The white arrows indicate the direction taken to obtain the alternate view.
The 19.1 A spacing between the carboxylic acid moieties of Chrysamine G matches well with the distance of 19.0 A across the 5 chains (4 x 4.76 A between
adjacent chains shown by Kirschner et al . , Proc . Natl . Acad. Sci . U. S.A . 83: 503 (1986)) . If the native - structure of Aβ involves a hairpin loop structure as Hilbich et al . , suggest (Hilbich et al . , J. Mol . Biol . 218: 149 (1991)), then chains 1 and 2, 3 and 4, 5 and 6, etc . , would be folded halves of the same molecule, but the model would otherwise be the same. Also important to note is the necessity for positively charged amino acid residues in this model, such as lysine-16 in Aβ. Previous work has shown that Congo red binding correlates well with the number of positively charged amino acids in a sample of amyloid fibrils. Klunk et al . , J. Histochem. Cytochem. 37: 1273 (1989) . The bidentate nature of the model in Figure 7 and the importance of hydrophobic interactions is supported by the decrease in affinity of the mono- dentate aniline analogue of Chrysamine G and the inactivity of salicylic acid as well as the increased potency of the more lipophilic compounds having two halogens on the benzidine moiety (see Figure 1) . The importance of the nearly planar biphenyl group is suggested by the inactivity of the 2,2' -disulfonic acid derivative.
EXAMPLE 3. CHRYSAMINE G DISTINGUISHES ALZHEIMER'S DISEASE BRAIN FROM NORMAL BRATTJ
Characterization of the Binding of Chrysamine G to AD Brain Scatchard analyses of the binding of Chrysamine G and Chrysamine G derivatives to AD brain samples were performed in an effort to understand the increased binding of Chrysamine G to AD brain. (Figure 4B, Table
1) . Under the conditions employed, control and AD brain showed a single binding component. The KD in AD brain was 16% lower than control but the difference was not significant (p=.29) . The Bm3LX in AD brain was 36% higher than the Bmax in control brain, but, again, the difference did not reach significance (p=.09) . Therefore, the increased binding in AD brain appears to be mainly due to the presence of more of the same binding component which exists in control brain, rather than the presence of a unique component.
Table 1. Comparison of binding Parameters in AD and control brain
The binding of CG to AD brain significantly correlated with numbers of NPs in the association cortices of the brain. Figure 8A shows the correlation of [1 C] CG binding with numbers of NPs in the superior/middle frontal and superior temporal cortex of AD brain. The correlation with NPs was significant whether controls were included (r=0.69; p=0.001) or if the AD brains were considered alone (r=0.59; p=0.007) . Figure 8B shows a similar correlation with NFT counts . As with NPs, the correlation with NFTs was significant whether controls were included (r=0.60; p=0.001) or if the AD brains were considered alone (r=0.50; p=0.026) . The correlation with NFTs is not surprising since CG is a derivative of Congo red, which stains NFTs. The number of NPs was significantly correlated with the number of NFTs (r=0.82; p=.0001) .
Only qualitative data on the presence or absence of amyloid angiopathy was available for the brains used in this study, so similar correlations could not be performed between CG binding and cerebrovascular amyloid levels . The presence of amyloid angiopathy does appear to be a confounding variable in the correlation of CG binding with NP counts . Figure 8A shows the improved correlation of CG binding to NP counts in brains without amyloid angiopathy (r=0.79; p=0.01) compared to those brains with cerebrovascular amyloid deposits (r=0.49; p=0.15) . A similar
improvement was not found in the correlation to NFT counts.
The KD for [14C] Chrysamine G binding to AD brain is similar to that found for [1 C] Chrysamine G binding to synthetic Aβ in vi tro, suggesting that binding in brain homogenates also may represent interaction with Aβ. The correlation of Chrysamine G binding to NFTs may indicate that Chrysamine G binds to these structures in brain homogenates as well . Alternatively, since the number of NFTs correlates closely with the number of NPs, the correlation of [1 C] Chrysamine G binding to NFTs may just be an epiphenomenon of Chrysamine G binding to NPs.
The useful Chrysamine G derivatives or analogues provided herein have binding affinities that are at least in the range of 0.01 to 10.0 μM KD, as measured by binding to either synthetic Aβ peptide or Alzheimer's Disease brain tissue; higher affinity compounds having binding affinities in the range of 0.0001 to 0.01 μM are also useful in the method of the present invention.
Considering the above, Chrysamine G binding may not be specific for Aβ. Instead, Chrysamine G binding may reflect the total amyloid "load" of the brain, comprised of aggregated deposits of Aβ in neuritic plaques and cerebrovascular amyloid. Deposits of phosphorylated tau protein in NFTs may contribute to Chrysamine G binding as well. Goedert, M. et al . , PNAS
85: 4051 (1988) . NFTs also are composed of anti- parallel beta-sheet fibrils similar in quaternary - structure to fibrils of Aβ. Kirschner et al . , Proc . Na tl . Acad . Sci . U. S . A . 83: 503 (1986) .
Total and Relative Chrysamine G Binding Distinguishes AD From Normal Brain
In vi tro binding assays such as those described above and below are widely used in the art as models to screen compounds for in vivo binding in brain and to predict success in subsequent in vivo imaging studies. See, Young, A. et al . , Receptor Assays : In Vi tro and In Vivo . in POSITRON EMISSION TOMOGRAPHY AND AUTORADIOGRAPHY (Phelps, M. , Mazziota, J. , and Schelbert, H. eds. ) pp. 73-111 (1986) . The labeled Chrysamine G and Chrysamine G derivatives of the invention also may be used in the in vi tro binding assays described above and below to quantitate amyloid deposits in biopsy or post-mortem specimens.
Saturable (specific) binding of [14C] Chrysamine G was observed both in AD brain and control brain homogenates and constituted 60-80% of total binding in AD brain. Non-saturable binding was very similar in AD and control brain. Both saturable and total binding were greater in AD brain than in control . Despite the lower sensitivity obtained when using total binding, this parameter is more predictive of success in in vivo studies which are the ultimate goal of this invention.
Also for the purpose of extension to in vivo studies, it is advantageous if Chrysamine G binding in cortical areas is normalized to a brain area in which Chrysamine G binding is very similar in both AD and control brain. This obviates the need to calculate the absolute quantity bound which is difficult to do in vivo . We examined binding in the cerebellum as a potential control area because classical NPs are exceedingly rare in this brain area (Joachim et al . , Am. J. Pathol . 135: 309 (1989) ) .
The average amount of [14C] Chrysamine G bound to control cerebellum is nearly identical to the amount bound to AD cerebellum (Table 2) , supporting the use of cerebellum as an internal control. Therefore, the cerebellar ratio (CBR) accurately reflects the absolute quantity of [14C] Chrysamine G bound and offers the advantage of providing an internal control for each brain. Binding is greater in AD brain whether expressed in absolute terms of fmol/μg protein (Table 2) or as a ratio to the binding in the cerebellum of the same brain (Table 3) . The CBR is the more sensitive measure and shows less variability between brains . The use of total binding and CBRs greatly facilitates extension of these ex vivo results to in vivo studies. Accordingly, the results below are expressed in these terms whenever appropriate.
Table 2. Comparison of total binding in AD and control brain*.
♦High- and low-plaque AD brains combined.
Table 3. Comparison of total binding in AD and control brain as a ratio to cerebellum* .
*The CBR for each sample is obtained by dividing the absolute value of [1 C] Chrysamine G binding in that sample by the absolute value of [14C] Chrysamine G binding in the cerebellar sample from that same brain. The values in the table are the average CBRs from each brain area (± SEM) . High- and low-plaque AD brains combined.
Figure 9A and 9B shows the binding of
[1 C] Chrysamine G to six brain areas normalized to the cerebellum of the same brain. The binding of Chrysamine G to AD brain areas in AD brains having more than 20 NPs/x200 magnification, "High Plaque AD
Brains", is shown in Figure 9A. The binding of Chrysamine G to AD brain areas in AD brains having -less than 20 NPs/x200 magnification, "Low Plaque AD Brains", is shown in Figure 9B. In all brain areas, the binding to AD brain is significantly greater than the binding to control (see Table 3) . In superior/middle frontal cortex, there is no overlap between control and any of the AD samples . In all brain areas except the occipital cortex, there is no overlap between control and the AD samples having >20 NPs/x200 magnification. In brain areas with the least deposition of classical NPs, such as the occipital cortex (and cerebellum) , the greatest overlap between AD and control was observed. Figure 9C shows the data from two patients who had Down's syndrome. Down's syndrome patients all develop deposits of Aβ by their fourth decade and many develop AD. Wisniewski et al. , Neurology 35: 957 (1985) ; Schapiro et al . , Neurobiol . Aging 13, 723 (1992) . Both of these patients showed [14C] Chrysamine G binding above the control range. Since the younger patient (23 years old) had amyloid deposits but was not yet clinically demented, Figure 9C suggests that Chrysamine G can detect differences from control in non-demented patients destined to develop AD long before the dementia is clinically evident.
The compounds and method of the invention provide two useful measurements for differentiating AD brain from normal brain; either (1) total Chrysamine G
binding (Table 2) or (2) the ratio of Chrysamine G binding in a given brain area to binding in the cerebellum of the same brain (Table 3) . These measurements furnish two great advantages for in vivo quantitation of AD neuritic plaques. First, by providing a means to measure total Aβ binding, rather than specific Aβ binding, the instant invention can quantify Aβ deposition. without having to expose the subject to a second injection of radioactive material in order to measure non-specific binding. Because of this, the data are expressed as total binding only. In all of the experiments presented, specific binding data yields even greater differences between AD and control brain. Second, variations in brain uptake of Chrysamine G derivatives will affect the absolute concentration of Chrysamine G in brain. Some mechanism will be necessary, therefore, to account for these variations between subjects. Each patient can serve as his/her own control by finding a brain area that shows little Aβ deposition (i.e., an experimental "blank") . Since classical NPs are exceedingly rare in the cerebellum (Joachim et a . , Am. J. Pathol . 135: 309 (1989)), Chrysamine G binding to the cerebellum was used as a control for each brain studied. The results were expressed in terms of the ratio of Chrysamine G binding in a given brain area to binding in the cerebellum of the same brain (Figure 9 and Table 3) .
For the purposes of in vivo quantitation of amyloid in AD, the effect of brain atrophy should be considered. Therefore, when using the Chrysamine G and Chrysamine G derivative probes in vivo to quantitate amyloid, brain atrophy can be corrected based on MRI volume measurements. MRI volume measurements performed in conjunction with the method* of the invention are analogous to those routinely employed in the art. See, Pearlson, G. and Marsh, L. MAGNETIC RESONANCE IMAGING IN PSYCHIATRY in Annual Review of Psychiatry (Vol. 12) Oldham, J. et al . , eds. p. 347-381 (1993). Therefore a method for determining the total radioactivity per volume of brain area would use the following equation:
total SPECT or PET signal from brain area "A" MRI determined brain volume
(excluding CSF) in brain area "A"
Designating this measurement as the signal/volume for brain area "A" or S/VA means that the cerebellar ratio would be expressed as: _ . S/VA
Ratio. =
S/VCB where S/V^ is the signal/volume in the cerebellum of the same subject during the same imaging study. This ratio from any brain area other than cerebellum from a patient suspected of having AD or other pathological
condition characterized by the deposition of amyloid could then be compared to the normal range of the analogous ratio from the same brain area of a group of age-matched normal control subjects. The ratio of the binding to brain areas with high deposits of neuritic plaques to the cerebellum can be used as the parameter to distinguish Alzheimer from control subjects.
EXAMPLE 4 THE OCTANO -WATER PARTITION COEFFICIENTS OF CHRYSAMINE G, CHRYSAMINE G DERIVATIVES. AND CONGO RED
The octanol-water partition coefficient is a measure of the relative lipophilicity of a compound. The more lipophilic a compound, the more likely it is to cross the blood-brain barrier. See, Goodman and Gilman's THE PHARMACOLOGICAL BASIS FOR THERAPEUTICS
(7th Ed.). The octanol/water partition coefficient of Chrysamine G is 60.22±3.97 and that of Congo red is 0.665±0.037 (p<0.001). This suggests that Chrysamine G is approximately 90 times more lipophilic than Congo red and therefore is theoretically more likely to cross the mammalian blood-brain barrier. The octanol/water partition coefficients for the 3-iodo and 3,3'-diiodo derivatives of Chrysamine G (Figure 1) are 72.53±.74 and 112.9±7.3, respectively. These octanol/water partition coefficients show that these derivatives, which are non-radioactive analogues of some of the
radiolabeled Chrysamine G derivatives to be used for in vivo studies, are up to 170 times more lipophilic than Congo red and up to twice as lipophilic as Chrysamine G. This suggests they will enter the brain much better than either Congo red or Chrysamine G.
EXAMPLE 5 THE ABILITY OF CHRYSAMINE G AND
CHRYSAMINE G DERIVATIVES TO CROSS THE BLOOD-BRAIN BARRIER AND METABOLISM OF CHRYSAMINE G
Use of the amyloid probes to diagnose AD in vivo requires them to be able to cross the blood-brain barrier and gain access to parenchymal amyloid deposits. The ability of Chrysamine G to cross the blood- brain barrier was studied in Swiss-Webster mice. After i.v. injection, the brain/blood ratio measured at 15 min was over 10:1 and approached 20:1 by 35 min (Figure 10) . The radioactivity in brain stayed nearly constant over this period, but decreased in the blood and increased in the liver. The brain/kidney ratio was highest at 15 min (over the time points sampled) and approached 0.5. When brain and liver were extracted 60 min after i.v. injection of [14C]Chrysamine G, >95% of the recovered radioactivity co-eluted with authentic Chrysamine G on reverse phase HPLC, indicating no significant metabolism of Chrysamine G over this period of time.
Chrysamine G does get into normal mouse brain, and the brain/blood ratio is high. The radioactivity in brain remained relatively constant over the first 30 min while decreasing in blood and increasing in liver. This suggests that the high brain/blood ratio is more a result of efficient removal of Chrysamine G from the blood by the liver than to further accumulation in the brain. At 60 min, essentially all of the radioactivity found in the brain and liver proved to be unchanged Chrysamine G. Congo red does not cross the blood-brain barrier well. Tubis et al . , J. Amer. Phar . Assn . 49: 422 (1960) . Most of the Congo red is cleared by the liver and spleen and the brain/kidney ratio achieved in guinea pigs is approximately 0.07. Tubis et al . , supra . Chrysamine G also is cleared by the liver, but has greater entry into the brain.
In vivo animal testing provides yet a further basis for determining dosage ranges, efficacy of transfer through the blood barrier and binding ability. Particularly preferred for this purpose are the transgenic mouse model of Games et al . , (Nature 373: 523 (1995)) and the "senile animal" model for cerebral amyloidosis; i . e . , animals such as the transgenic mice or aged dogs or monkeys, which are known to develop variable numbers of Alzheimer-type cerebral neuritic plaques, see Wisniewski et al . , J. Neuropathol . & Exp . Neurol . 32: 566 (1973), Selkoe et al . , Science 235: 873 (1987) , are tested for binding and detection efficacy.
This in vivo assay requires control-biopsy or necropsy monitoring to confirm and quantify the presence of amyloid deposits.
Other suitable animal models for use in testing the compositions and methods of the present invention are produced transgenically. For instance, Quon et al . , Nature, 352: 239-241 (1991) used rat neural- specific enolase promoter inhibitor domain to prepare transgenic mice. See also, Wirak et al . , Science, 253: 323-325 (1991) . Still other models have been produced by Intracranial administration of the β/A4 peptide directly to animals (Tate et al . , Bull . Clin . Neurosci . , 56: 131-139 (1991).
It is noted that none of the in vivo animal models may turn out to be extremely good models for AD neuropathology. Instead, they may more closely model the amyloid deposition of normal aging. This is particularly true of the aged-mammal models. All of these models show a preponderance of diffuse plaques as discussed above for the aged dog model. While there is some cerebrovascular amyloid, there are few neuritic plaques, except in the Games et al . , transgenic mouse model. The other transgenic mouse models often show only diffuse plaques. Therefore, while these models may be useful for studying distribution of the probes in the brain, there is a fairly low probability that these models would show the same quantitative differences that would be expected to be seen in AD
brain based on the in vi tro studies of Chrysamine G binding to AD brain described above.
Evaluating the Ability of Chrysamine G Derivatives to Cross the Human Blood-Brain Barrier A dose of approximately 10 mCi of an appropriately radiolabeled derivative of Chrysamine G with a specific activity of approximately 500 Ci/mole or higher is injected intravenously into normal subjects and patients suspected of having AD and monitored by SPECT or PET imaging to analyze the detectability of the derivative in brain relative to other organs and to define the time course of detectability in the brain.
A dose which can be reliably detected is defined as a
"imaging effective dose."
Evaluation of Chrysamine G and Chrysamine G
Derivatives to Distinguish AD from Age-Matched Controls in Humans
An imaging-effective dose of an appropriately, radioactively labeled derivative of Chrysamine G is injected into a subject suspected of having brain amyloid deposition due to pathological conditions such as AD. After a period of 15 minutes to 24 hours, the radioactive signal from brain is detected by SPECT or PET. Radioactivity is simultaneously detected in all brain areas included in the field of view of the detector. This field of view will be set up so as to include large portions of the cerebellum, superior
• Ill -
temporal cortex, superior/middle frontal cortex, and intervening brain regions. An MRI scan will be performed prior to the study so that corrections can be made for brain atrophy in the areas of interest by methods discussed in Example 3. The S/VA, S/V∞, and RatioA variables discussed in Example 3 will be calculated and compared to analogous normative ratios obtained previously from age-matched normal control subjects.
EXAMPLE 6 HISTOLOGIC LOCALIZATION OF CHRYSAMINE G
BINDING TO CEREBROVASCULAR AMYLOID
The top frame of Figure 11 demonstrates two neuritic plaques stained by Chrysamine G. The staining method was that of Stokes and Trickey, J. Clin . Pathol . 26: 241-242 (1973) with Chrysamine G substituted for
Congo red. Except for somewhat lower intensity, these deposits are identical to those stained with Congo red (not shown) . The bottom two frames of the photomicrograph in Figure 11 show adjacent sections from the temporal lobe of an AD patient with amyloid angiopathy. The section in the lower left was stained with the Congo red using the method of Puchtler. Puchtler et al . , J. Histochem. Cytochem. 10: 35 (1962) . The section in the lower right of Figure 11 was stained by substituting Chrysamine G for Congo red in the Puchtler method. Both sections readily demonstrate
the same amyloid-laden vessel. A small amount of background auto-fluorescence from erythrocytes and lipofuscin also is visible. Both photomicrographs were obtained with a laser confocal microscope using fluorescein isothiocyanate (FITC) filters. The bar represents 20 microns.
EXAMPLE 7 ASSESSING THE TOXICITY OF
CHRYSAMINE G AND CHRYSAMINE G
DERIVATIVES
At doses of 10 and 100 mg/kg non-radioactive
Chrysamine G administered intraperitoneally, no notable behavioral effects or toxicity were observed in mice for periods up to 72 hrs. The doses of [14C] Chrysamine G administered were on the order of 1 mg/kg. Chrysamine G appeared to show little acute toxicity based on attempts to establish an LD50. Even when the maximum volume that can be injected into a mouse without harming it just from fluid volume effects (approx. 0.025 ml/g) of a saturated solution of Chrysamine G was injected into mice (100 mg/kg) , there were no behavioral changes noted for at least 72 hrs, the longest period tested. Doses required for detection of radiolabeled derivatives by SPECT or PET would be orders of magnitude below this dose. Congo red has been safely injected into humans in quantities much greater than would be used for the
radioactive Chrysamine G derivatives. The LD50 for Congo red has been shown to be 190 mg/kg mouse (Tubis et al . , J. Amer. Pharm . Assoc . 49: 422 (I960)) , which is similar to the >100 mg/kg LD50 shown for Chrysamine G. Thus, these two chemically similar compounds cause similar low toxicities in mice.
Other Chrysamine G derivatives can similarly be tested for toxicity in mice and other higher mammals by injecting a wide range of concentrations and studying the animals for various signs of toxicity by methods well known in the art. See, Goodman and Gilman's THE PHARMACOLOGICAL BASIS FOR THERAPEUTICS (7th Ed.) .
EXAMPLE 8 ASSESSING THE ABILITY OF CHRYSAMINE G TO PROTECT AGAINST Aβ (25-35) - DUCED TOXICITY
Protection from Aβ (25-35) -induced Toxicity in PC- 12 cells
Rat pheochromocytoma cells (PC-12) were grown in RPMI 1640 media with 10% fetal bovine serum. Approximately 5,000 exponentially growing cells were plated in 96-well plates in a volume of 100 μl of media and allowed to incubate at 37°C overnight. The Aβ (25- 35) , which had been pre-aggregated at 37°C for 7 days, was pre-incubated with Chrysamine G (CG) or related compounds in aqueous solution prior to addition of 20 μl to achieve the final concentrations given (0.01 to 10 μM Aβ(25-35) and 0.03 to 20 μM CG) . The cells were
incubated for 24 hrs prior to the addition of 13.3 μl of 5 mg/ml MTT (3 , (4, 5-dimethylthiazol-2-yl) 2, 5- diphenyltetrazolium bromide) in sterile phosphate buffered saline. After 4.5 hrs at 37°C, 100 μl of extraction buffer (20% w/v SDS in 50% DMF/water; pH adjusted to 4.7 with 2.5% of 80% acetic acid and 2.5% IN HC1) was added and the plates were incubated overnight. Hansen et al . , J. Immunol . Methods 119: 203 (1989) . Color development was then measured at 560 nm. Maximum viability was defined as the absorbance of control wells to which only the 20 μl of distilled, deionized H20 was added. Maximum toxicity was defined by wells in which the cells were lysed by addition of 0.1% (final concentration) of Triton X-100. Incubation of PC12 cells with Aβ (25-35) results in a concentration-dependent decrease in the ability of these cells to reduce MTT (Figure 12) . Figure 12 shows the effect of increasing concentrations of Aβ (25-35) in the presence and absence of Chrysamine G on the cellular redox activity of PC12 cells as measured by
MTT reduction. The reduction product of MTT absorbs at 560 nm which is plotted on the vertical axis. The effect of Aβ (25-35) alone is shown in the filled bars and shows a dose dependent decrease in MTT reduction. Significant differences from control (no Aβ, no
Chrysamine G) are shown in white numbers inside the filled bars. The protective effect of 20 μM Chrysamine G is shown in the open bars. Significant differences
between MTT reduction in the presence and absence of Chrysamine G are shown in black numbers inside the -open bars .
Figure 13 demonstrates the protective effect of increasing concentrations of Chrysamine G against the Aβ (25-35) -induced reduction of cellular redox activity of PC12 cells. The effect of Chrysamine G in the absence of Aβ (25-35) is shown in the filled bars. There was no significant difference between control (no Aβ, no Chrysamine G) and any of the concentrations of Chrysamine G in the absence of Aβ (25-35) . MTT reduction in the presence of 1 μM Aβ (25-35) and increasing concentrations of Chrysamine G is shown in the open bars . Significant differences in MTT reduction between the presence and absence of Aβ (25-35) at each concentration of Chrysamine G are shown in white numbers inside the filled bars. Significant differences in MTT reduction between the Aβ (25-35) control (no Chrysamine G) and Aβ (25-35) plus increasing concentrations of Chrysamine G are shown in black numbers inside the open bars .
As has previously been reported, Congo red protects against the Aβ-induced toxicity at concentrations over 2 μM, achieving complete protection by 20 μM. Burgevin et al . NeuroReport 5: 2429 (1994) ; Lorenzo and Yankner, Proc . Natl . Acad. Sci . 91: 12243 (1994) ; Pollack et al . , Neuroscience Letters 184: 113 (1995) ; Pollack et al . Neuroscience Letters 197: 211
(1995) . Chrysamine G shows a protective effect which is dependent on both the concentration of Aβ (25-35) (Figure 12) as well as the concentration of Chrysamine G (Figure 13) . The protective effect of Chrysamine G is evident at 0.2 μM, a concentration very close to the Ki of Chrysamine G for binding to synthetic Aβ, 0.37 μM (Figure 1) . Chrysamine G appears to be more potent than Congo red, showing effects in the range of 0.1 to 1.0 μM. This is consistent with the Ki values for binding to synthetic Aβ of 0.37 μM for Chrysamine G and 2.8 μM for Congo red (Figure 1) .
In another experiment (Figure 14) , the effect of Chrysamine G and the phenol derivative (see Figure 1) , which does not bind Aβ, was examined in cells incubated with 1 μM Aβ (25-35) . Chrysamine G showed protective effects at 0.1 and 1 μM, but the phenol derivative showed no protective effects, and perhaps enhanced the toxicity of Aβ.
These results suggest that the lipophilic derivative of Congo red, Chrysamine G, prevents Aβ- induced cytotoxicity in cell culture at concentrations very similar to those at which it binds Aβ. This protection shows structural specificity since the phenol derivative which does not bind to synthetic Aβ also does not prevent Aβ-induced cytotoxicity. Since Chrysamine G partitions into the brain well, these results provide evidence that Chrysamine G and Aβ-
binding derivatives of Chrysamine G have therapeutic potential in the treatment of AD.
The mechanism of the protective effect of Chrysamine G is unknown at present . Two broad possibilities exist. First, Chrysamine G could interfere with the aggregation of Aβ. Second, Chrysamine G could interfere with the effects (direct or indirect) of Aβ on the target cells. Congo red does inhibit aggregation of Aβ as well as protect against the toxic effects of aggregated Aβ. Lorenzo and Yankner, Proc . Natl . Acad. Sci . 91: 12243 (1994) . Interference with aggregation is unlikely in the above experiment since the Aβ was pre-aggregated prior to incubation with Chrysamine G. Thus, inhibition of aggregation could prove to be an important therapeutic effect of Chrysamine G but is not a likely explanation for the protective effects of Chrysamine G against pre- aggregated A . The model of Chrysamine G binding to Aβ described in Figure 7, displays how Chrysamine G could "coat" the surface of Aβ. This may change how the fibrillar deposits are recognized by cell-surface receptors or other macromolecules such as complement proteins and interfere with the toxic effects of Aβ which may be mediated by these macromolecules . It is likely that Chrysamine G and Congo red exert multiple effects, both before and after the aggregation of Aβ. This is advantageous from a therapeutic point of view since patients are likely to present at a time when
there are pre-existing Aβ aggregates as well as ongoing amyloid deposition.
Claims
1. An amyloid binding compound of Formula I or a water soluble, non-toxic salt thereof:
wherein:
is either N=N, or (where R'
represents H or a lower alkyl group);
X is C(R")2
(wherein R" is each independently is H, F, Cl, Br, I, a lower alkyl group, CF3, CH2-CH2F, O-CH2-CH2F, CH2-CH2-CH2F, O-CH2-CH2-CH2F, CN, (C=O) -R', N(R')2, NO2, (C=O)N(R')2 O(CO)R', OR', COOR', a tri-alkyl tin, a tetrazole or oxadiazole of the form:
wherein R' is H or a lower alkyl group) or X is CH=CH, N=N, C=O, O, NR' (where R' represents H or a lower alkyl group), S, or SO2; each R1 and R2 independently is H, F, Cl, Br, I, a lower alkyl group, CF3, CH2-CH2F, O-CH2-CH2F, CH2-CH2- CH2F, O-CH2-CH2-CH2F, CN, (C=O) -R', N(R')2, NO2,
(C=O)N(R')2 O(CO)R', OR', COOR', a tri-alkyl tin, a tetrazole or oxadiazole of the form:
(wherein R' is H or a lower alkyl group), or a triazene of the form:
(wherein R8 and R9 are lower alkyl groups) or each Q is independently selected from one of the following structures, each of which contain a
carboxylic acid or an acid-like functionality:
IA, IB, IC, ID, IE, IF and IG, wherein
IA has the following structure:
(
wherein:
each of R3, R4, R5, R6, or R7 independently is H, F, Cl, Br, I, a lower alkyl group, CF3, CH2-CH2F, O- CH2-CH2F, CH2-CH2-CH2F, O-CH2-CH2-CH2F, CN, (C=O) -R', N(R')2, NO2, (C=O)N(R')2 O(CO)R', OR', COOR', a trialkyl tin, a tetrazole or oxadiazole of the form:
(wherein R' is H or a lower alkyl group), or a triazene of the form:
(wherein R8 and R9 are lower alkyl groups) or
wherein at least one of R3, R4, R5, R6, or R7 is a hydroxy, tetrazole, oxadiazole, NO2, or carboxy in both Q's; and at least one of R1 , R2, R3, R4, or R7 is not H when R5 is hydroxyl and R6 is carboxy or a lower alkyl ester thereof; at least one of R1, R2, R3, or R4 is not H when R5 is amino, R6 is carboxy or a lower alkyl ester thereof, and R7 is hydroxyl; at least one of R1, R3, R4, or R7 is not H when R2 is CH3, R5 is hydroxyl and R6 is carboxy or a lower alkyl ester thereof; at least one of R1 or R2, is not H or CH3 when R3, R4, or R7 is CH3, R5 is hydroxyl and R6 is carboxy or a lower alkyl ester thereof;
IB has the following structure:
wherein:
each of R10, R11; R12, R13, R14, R15, or R16
independently is H, F, Cl, Br, I, a lower alkyl group, CF3, CH2-CH2F, O-CH2-CH2F, CH2-CH2-CH2F, O-CH2- CH2-CH2F, CN, (C=O)-R', N(R')2, NO2, (C=O)N(R')2 O(CO)R', OR', COOR', a tri-alkyl tin, a tetrazole or oxadiazole of the form:
(wherein R' is H or a lower alkyl group), or a triazene of the form:
(wherein R8 and R9 are lower alkyl
groups) or and wherein at least one of R10, R11, R12, R13, R14, R15 or R16 is a hydroxy, tetrazole, oxadiazole, NO2 or carboxy in both Q' s;
IC has the following structure:
wherein:
at least one of R17, R18, R19, R20, or R21 is H, F, Cl, Br, I, a lower alkyl group, CF3, CH2-CH2F, O- CH2-CH2F, CH2-CH2-CH2F, O-CH2-CH2-CH2F, CN, (C=O) -R' , N(R')2, NO2, (C=O)N(R')2 O(CO)R', OR', COOR' , a trialkyl tin, a tetrazole or oxadiazole of the form:
(wherein R' is H or a lower alkyl group), or a triazene of the form:
(wherein R8 and R9 are lower alkyl
groups) or ID has the following structure :
wherein :
each of R22, R23, or R24 independently is H, F, Cl, Br, I, a lower alkyl group, CF3, CH2-CH2F, O-CH2- CH2F, CH2-CH2-CH2F, O-CH2-CH2-CH2F, CN, (C=O) -R', N(R')2, NO2, (C=O)N(R')2 O(CO)R', OR', COOR', a trialkyl tin, a tetrazole or oxadiazole of the form:
(wherein R' is H or a lower alkyl group), or a triazene of the form:
(wherein R8 and R9 are lower alkyl groups) or
represents a heterocyclic ring of one of the six following formulas:
IE has the following structure
wherein: each of R25, R26, or R27 independently is H, F, Cl, Br, I, a lower alkyl group, CF3, CH2-CH2F, O-CH2- CH2F, CH2-CH2-CH2F, O-CH2-CH2-CH2F, CN, (C=O) -R', N(R')2, NO2, (C=O)N(R')2 O(CO)R', OR', COOR', a trialkyl tin, a tetrazole or oxadiazole of the form:
(wherein R' is H or a lower alkyl group), or a triazene of the form:
(wherein R8 and R9 are lower alkyl groups) or
represents a heterocyclic ring of one of the six following formulas:
IF has the following structure:
wherein:
exactly one of R28, R29, R30, R31 or R32 is the
link defined for Formula I above and each other R28, R29, R30, R31 or R32 independently is H, F, Cl, Br, I, a lower alkyl group, CF3, CH2-CH2F, O-CH2- CH2F, CH2-CH2-CH2F, O-CH2-CH2-CH2F, CN, (C=O) -R', N(R')2, NO2, (C=O)N(R')2 O(CO)R', OR', COOR', a trialkyl tin, a tetrazole or oxadiazole of the form:
(wherein R' is H or a lower alkyl group), or a triazene of the form:
(wherein R8 and R9 are lower alkyl groups) or
wherein at least one of R28, R29, R30, R31 or R32 is a hydroxy, tetrazole, oxadiazole, NO2 or carboxy in both Q's;
IG has the following structure
wherein:
exactly one of R33, R34, R35, R36, R37, R38, or R39 is the link defined for Formula I above and each
other R33, R34, R35, R36, R37, R38, or R39 independently is H, F, Cl, Br, I, a lower alkyl group, CF3, CH2- CH2F, O-CH2-CH2F, CH2-CH2-CH2F, O-CH2-CH2-CH2F, CN, (C=O) -R' , N(R')2, NO2, (C=O)N(R')2 O(CO)R', OR', COOR', a tri-alkyl tin, a tetrazole or oxadiazole of the form:
(wherein R' is H or a lower alkyl group), or a triazene of the form: (wherein R8 and R9 are lower alkyl groups) or and wherein at least one of R33, R34, R35, R36, R37, R38, or R39 is a hydroxy, tetrazole, oxadiazole, NO2 or carboxy in both Q's.
2. The compound of claim 1 , wherein at least one of the substituents R1-R7 and R10-R39 is selected from the group consisting of 131I, 123I, 76Br, 75Br, 18F, CH2- CH2-18F, O-CH2-CH2-18F, CH2-CH2-CH2-18F, O-CH2-CH2-CH2-18F, 19F, 125I, and a carbon-containing substituent as specified in Formula I wherein at least one carbon is 11C or 13C.
3. The compound of claim 1, wherein said compound binds to Aβ with a dissociation constant (KD) between
0.0001 and 10.0 μM when measured by binding to
synthetic Aβ peptide or Alzheimer's Disease brain tissue.
4. A method for synthesizing a compound of claim 1, wherein at least one of the substituents R1-R7 and R10-R39 is selected from the group consisting of 131I, 12SI, 123I, 76Br, 75Br, 18F and 19F, comprising the step of reacting a compound of claim 1, wherein at least one of the substituents R1-R7 and R10-R39 is a tri-alkyl tin, with a halogenating agent containing 131I, 125I, 123I, 76Br, 75Br, 18F or 19F.
5. A pharmaceutical composition for in vivo imaging of amyloid deposits, comprising (a) a compound of claim 2, and (b) a pharmaceutically acceptable carrier.
6. An in vivo method for detecting amyloid deposits in a subject, comprising the steps of:
(a) administering a detectable quantity of the pharmaceutical composition of claim 5, and
(b) detecting the binding of said compound to amyloid deposit in said subject.
7. The method of claim 6, wherein said amyloid deposit is located in the brain of a subject.
8. The method of claim 6, wherein said subject is suspected of having a disease or syndrome selected from the group consisting of Alzheimer's Disease, familial Alzheimer's Disease, Down's Syndrome and homozygotes for the apolipoprotein E4 allele.
9. The method of claim 6, wherein said detecting is selected from the group consisting of gamma imaging, magnetic resonance imaging and magnetic resonance spectroscopy.
10. The method of claim 9, wherein said gamma imaging is either PET or SPECT.
11. The method of claim 6, wherein said
pharmaceutical composition is administered by
intravenous injection.
12. The method of claim 7, wherein the ratio of (i) binding of said compound to a brain area other than the cerebellum to (ii) binding of said compound to the cerebellum, in said subject, is compared to said ratio in normal subjects.
13. A method of inhibiting cell degeneration and toxicity associated with fibril formation in an amyloidosis-associated condition, said method
comprising the step of administering to a subject having or suspected of having such condition, a pharmaceutically effective amount of Chrysamine G or a derivative thereof.
14. The method of claim 13, wherein said
derivative of Chrysamine G is an amyloid binding compound of Formula I or a water soluble, non-toxic salt thereof:
wherein:
is eitner N=N' or CR'=CR' (where R'
represents H or a lower alkyl group);
X is C(R")2
(wherein R" is each independently is H, F, Cl, Br, I, a lower alkyl group, CF3, CH2-CH2F, O-CH2-CH2F, CH2-CH2-CH2F, O-CH2-CH2-CH2F, CN, (C=O) -R', N(R')2, NO2, (C=O)N(R')2 O(CO)R', OR', COOR', a tri-alkyl tin, a tetrazole or oxadiazole of the form: wherein R' is H or a lower alkyl group) or X is CH=CH, N=N, C=O, O, NR' (where R' represents H or a lower alkyl group), S, or SO2; each R1 and R2 independently is H, F, Cl, Br, I, a lower alkyl group, CF3, CH2-CH2F, O-CH2-CH2F, CH2-CH2- CH2F, O-CH2-CH2-CH2F, CN, (C=O) -R', N(R')2, NO2,
(C=O)N(R')2 O(CO)R', OR', COOR' , a tri-alkyl tin, a tetrazole or oxadiazole of the form:
(wherein R' is H or a lower alkyl group), or a triazene of the form:
(wherein R8 and R9 are lower alkyl groups) or
each Q is independently selected from one of the following structures, each of which contain a
carboxylic acid or an acid-like functionality:
IA, IB, IC, ID, IE, IF and IG, wherein
IA has the following structure:
wherein:
each of R3, R4, R5, R6, or R7 independently is H, F, Cl, Br, I, a lower alkyl group, CF3, CH2-CH2F, O- CH2-CH2F, CH2-CH2-CH2F, O-CH2-CH2-CH2F, CN, (C=O) -R', N(R')2, NO2, (C=O)N(R')2 O(CO)R', OR', COOR', a trialkyl tin, a tetrazole or oxadiazole of the form:
(wherein R' is H or a lower alkyl group), or a triazene of the form: (wherein R8 and R9 are lower alkyl
groups ) or wherein at least one of R3, R4, R5, R6, or R7 is a hydroxy, tetrazole, oxadiazole, NO2, or carboxy in both Q'S; and at least one of R1, R2, R3, R4, or R7 is not H when R5 is hydroxyl and R6 is carboxy or a lower alkyl ester thereof; at least one of R1, R2, R3, or R4 is not H when R5 is amino, R6 is carboxy or a lower alkyl ester thereof, and R7 is hydroxyl; at least one of R1, R3, R4, or R7 is not H when R2 is CH3, R5 is hydroxyl and R6 is carboxy or a lower alkyl ester thereof; at least one of R1 or R2, is not H or CH3 when R3, R4, or R7 is CH3, R5 is hydroxyl and R6 is carboxy or a lower alkyl ester thereof;
IB has the following structure:
wherein:
each of R10, R11, R12, R13, R14, R15, or R16
independently is H, F, Cl, Br, I, a lower alkyl group, CF3, CH2-CH2F, O-CH2-CH2F, CH2-CH2-CH2F, O-CH2- CH2-CH2F, CN, (C=O)-R', N(R')2, NO2, (C=O)N(R')2 O(CO)R', OR', COOR', a tri-alkyl tin, a tetrazole or oxadiazole of the form:
(wherein R' is H or a lower alkyl group), or a triazene of the form:
(wherein R8 and R9 are lower alkyl groups) or
and wherein at least one of R10, R11, R12, R13, R14, R15 or R16 is a hydroxy, tetrazole, oxadiazole, NO2 or carboxy in both Q' s;
IC has the following structure:
wherein:
at least one of R17, R18, R19, R20, or R21 is H, F, Cl, Br, I, a lower alkyl group, CF3, CH2-CH2F, O- CH2-CH2F, CH2-CH2-CH2F, O-CH2-CH2-CH2F, CN, (C=O) -R', N(R')2, NO2, (C=O)N(R')2 O(CO)R', OR', COOR', a trialkyl tin, a tetrazole or oxadiazole of the form:
(wherein R' is H or a lower alkyl group), or a triazene of the form: (wherein R8 and R9 are lower alkyl
groups) or
ID has the following structure:
wherein:
each of R22, R23, or R24 independently is H, F, Cl, Br, I, a lower alkyl group, CF3, CH2-CH2F, O-CH2- CH2F, CH2-CH2-CH2F, O-CH2-CH2-CH2F, CN, (C=O) -R', N(R')2, NO2, (C=O)N(R')2 O(CO)R', OR', COOR', a trialkyl tin, a tetrazole or oxadiazole of the form:
(wherein R' is H or a lower alkyl group), or a triazene of the form:
(wherein R8 and R9 are lower alkyl
groups) or
represents a heterocyclic ring of one of the six following formulas:
IE has the following structure:
wherein:
each of R25, R26, or R27 independently is H, F, Cl, Br, I, a lower alkyl group, CF3, CH2-CH2F, O-CH2- CH2F, CH2-CH2-CH2F, O-CH2-CH2-CH2F, CN, (C=O) -R', N(R')2, NO2, (C=O)N(R')2 O(CO)R', OR', COOR', a trialkyl tin, a tetrazole or oxadiazole of the form:
(wherein R' is H or a lower alkyl group) , or a triazene of the form:
(wherein R8 and R9 are lower alkyl
groups) or represents a heterocyclic ring of one of the six following formulas:
IF has the following structure:
wherein: exactly one of R28 , R29 , R30 , R31 or R32 is the
link defined for Formula I above and each other R28, R29, R30, R31 or R32 independently is H, F, Cl, Br, I, a lower alkyl group, CF3, CH2-CH2F, O-CH2- CH2F, CH2-CH2-CH2F, O-CH2-CH2-CH2F, CN, (C=O) -R', N(R')2, NO2, (C=O)N(R')2 O(CO)R', OR', COOR', a trialkyl tin, a tetrazole or oxadiazole of the form:
(wherein R' is H or a lower alkyl group), or a triazene of the form:
(wherein R8 and R9 are lower alkyl groups) or
wherein at least one of R28, R29, R30, R31 or R32 is a hydroxy, tetrazole, oxadiazole, NO2 or carboxy in both Q's; IG has the following structure:
wherein:
exactly one of R33, R34, R35, R36, R37, R38, or R39 is the link defined for Formula I above and each
other R33, R34, R35, R36, R37, R38, or R39 independently is H, F, Cl, Br, I, a lower alkyl group, CF3, CH2- CH2F, O-CH2-CH2F, CH2-CH2-CH2F, O-CH2-CH2-CH2F, CN, (C=O)-R', N(R')2, NO2, (C=O)N(R')2 O(CO)R', OR', COOR', a tri-alkyl tin, a tetrazole or oxadiazole of the form:
(wherein R' is H or a lower alkyl group), or a triazene of the form:
(wherein R8 and R9 are lower alkyl groups) or and wherein at least one of R33, R34, R35, R36, R37, R38, or R39 is a hydroxy, tetrazole, oxadiazole, NO2 or carboxy in both Q's.
15. The method of claim 14 wherein each R2 is CH3 when Z=Z is N=N.
16. The method of claim 13, wherein said
amyloidosis-associated condition is selected from the group consisting of Alzheimer's Disease, Down's
Syndrome, Type 2 diabetes mellitus, hereditary cerebral hemorrhage amyloidosis (Dutch), amyloid A (reactive), secondary amyloidosis, familial mediterranean fever, familial amyloid nephropathy with urticaria and
deafness (Muckle-wells Syndrome), amyloid lambda
L-chain or amyloid kappa L-chain (idiopathic, myeloma or macroglobulinemia-associated) A beta 2M (chronic hemodialysis), ATTR (familial amyloid polyneuropathy (Portuguese, Japanese, Swedish), familial amyloid cardiomyopathy (Danish), isolated cardiac amyloid, (systemic senile amyloidosises), AIAPP or amylin insulinoma, atrial naturetic factor (isolated atrial amyloid), procalcitonin (medullary carcinoma of the thyroid), gelsolin (familial amyloidosis (Finnish)), cystatin C (hereditary cerebral hemorrhage with
amyloidosis (Icelandic)), AApo-A-I (familial
amyloidotic polyneuropathy - Iowa), AApo-A-II
(accelerated senescence in mice), fibrinogen-associated amyloid; and Asor or Pr P-27 (scrapie, Creutzfeld Jacob disease, Gertsmann-Straussler-Scheinker syndrome, bovine spongiform encephalitis) or in cases of persons who are homozygous for the apolipoprotein E4 allele, and the condition associated with homozygosity for the apolipoprotein E4 allele.
17. A pharmaceutical composition for the
prevention of cell degeneration and toxicity associated with fibril formation in amyloidosis associated
conditions comprising Chrysamine G and a
pharmaceutically acceptable carrier.
18. A pharmaceutical composition for the
prevention of cell degeneration and toxicity associated with fibril formation in amyloidosis-associated conditions comprising a Chrysamine G derivative of claim 1 and a pharmaceutically acceptable carrier.
19. A method of detecting amyloid deposits in biopsy or post-mortem tissue comprising the steps of:
(a) incubating formalin-fixed tissue with a solution of a compound of claim 1 to form a labelled deposit and then,
(b) detecting the labelled deposits.
20. The method of claim 19 wherein said solution is composed of 25-100% ethanol (the remainder being water) saturated with a compound of claim 1.
21. The method of claim 19 wherein said detecting is effected by microscopic techniques selected from the group consisting of bright-field, fluorescence, laser- confocal, and cross-polarization microscopy.
22. A method of quantifying the amount of amyloid in biopsy or post-mortem tissue comprising the steps of:
a) incubating a radiolabeled derivative of
Chrysamine G with a homogenate of biopsy or postmortem tissue,
b) separating the tissue-bound from the tissue- unbound radiolabeled Chrysamine G derivative, c) quantifying the tissue-bound radiolabeled Chrysamine G derivative, and
d) converting the units of tissue-bound
radiolabeled Chrysamine G derivative to units of micrograms of amyloid per 100 mg of tissue by comparison with a standard.
23. The method of claim 22, wherein said
radiolabeled derivative of Chrysamine G is an amyloid binding compound of Formula I or a water soluble, non- toxic salt thereof:
wherein : is either N=N, or CR'=CR' (where R'
represents H or a lower alkyl group);
X is C(R")2
(wherein R" is each independently is H, F, Cl, Br,
I, a lower alkyl group, CF3, CH2-CH2F, O-CH2-CH2F,
CH2-CH2-CH2F, O-CH2-CH2-CH2F, CN, (C=O) -R', N(R')2,
NO2, (C=O)N(R')2 O(CO)R', OR', COOR', a tri-alkyl tin, a tetrazole or oxadiazole of the form:
wherein R' is H or a lower alkyl group) or X is CH=CH, N=N, C=O, O, NR' (where R' represents H or a lower alkyl group), S, or SO2; each R1 and R2 independently is H, F, Cl, Br, I, a lower alkyl group, CF3, CH2-CH2F, O-CH2-CH2F, CH2-CH2- CH2F, O-CH2-CH2-CH2F, CN, (C=O) -R', N(R')2, NO2,
(C=O)N(R')2 O(CO)R', OR', COOR', a tri-alkyl tin, a tetrazole or oxadiazole of the form:
(wherein R' is H or a lower alkyl group), or a triazene of the form: > (wherein R8 and R9 are lower alkyl groups) or each Q is independently selected from one of the following structures, each of which contain a
carboxylic acid or an acid-like functionality:
IA, IB, IC, ID, IE, IF and IG, wherein
IA has the following structure:
wherein:
each of R3, R4, R5, R6, or R7 independently is H, F, Cl, Br, I, a lower alkyl group, CF3, CH2-CH2F, O- CH2-CH2F, CH2-CH2-CH2F, O-CH2-CH2-CH2F, CN, (C=O) -R', N(R')2, NO2, (C=O)N(R')2 O(CO)R', OR', COOR', a trialkyl tin, a tetrazole or oxadiazole of the form:
(wherein R' is H or a lower alkyl group), or a triazene of the form:
(wherein R8 and R9 are lower alkyl
groups) or wherein at least one of R3, R4, R5, R6, or R7 is a hydroxy, tetrazole, oxadiazole, NO2, or carboxy in both Q's; and at least one of R1, R2, R3, R4, or R7 is not H when R5 is hydroxyl and R6 is carboxy or a lower alkyl ester thereof; at least one of R1, R2, R3, or R4 is not H when R5 is amino, R6 is carboxy or a lower alkyl ester thereof, and R7 is hydroxyl; at least one of R1, R3, R4, or R7 is not H when R2 is CH3, R5 is hydroxyl and R6 is carboxy or a lower alkyl ester thereof; at least one of R1 or R2, is not H or CH3 when R3, R4, or R7 is CH3, R5 is hydroxyl and R6 is carboxy or a lower alkyl ester thereof;
wherein:
each of R10, R11, R12, R13, R14, R15, or R16
independently is H, F, Cl, Br, I, a lower alkyl group, CF3, CH2-CH2F, O-CH2-CH2F, CH2-CH2-CH2F, O-CH2- CH2-CH2F, CN, (C=O)-R', N(R')2, NO2, (C=O)N(R')2 O(CO)R', OR', COOR', a tri-alkyl tin, a tetrazole or oxadiazole of the form:
(wherein R' is H or a lower alkyl group), or a triazene of the form:
(wherein R8 and R9 are lower alkyl
groups) or and wherein at least one of R10, R11, R12, R13, R14, R15 or R16 is a hydroxy, tetrazole, oxadiazole, NO2 or carboxy in both Q's;
IC has the following structure:
wherein:
at least one of R17, R18, R19, R20, or R21 is H, F, Cl, Br, I, a lower alkyl group, CF3, CH2-CH2F, O- CH2-CH2F, CH2-CH2-CH2F, O-CH2-CH2-CH2F, CN, (C=O) -R', N(R')2, NO2, (C=O)N(R')2 O(CO)R', OR', COOR', a trialkyl tin, a tetrazole or oxadiazole of the form:
(wherein R' is H or a lower alkyl group), or a triazene of the form:
(wherein R8 and R9 are lower alkyl groups) or
ID has the following structure:
wherein: each of R22, R23, or R24 independently is H, F, Cl, Br, I, a lower alkyl group, CF3, CH2-CH2F, O-CH2- CH2F, CH2-CH2-CH2F, O-CH2-CH2-CH2F, CN, (C=O) -R', N(R')2, NO2, (C=O)N(R')2 O(CO)R', OR', COOR', a trialkyl tin, a tetrazole or oxadiazole of the form:
(wherein R' is H or a lower alkyl group), or a triazene of the form:
(wherein R8 and R9 are lower alkyl
groups) or
represents a heterocyclic ring of one of the six following formulas: IE has the following structure:
wherein:
each of R25, R26, or R27 independently is H, F, Cl, Br, I, a lower alkyl group, CF3, CH2-CH2F, O-CH2- CH2F, CH2-CH2-CH2F, O-CH2-CH2-CH2F, CN, (C=O) -R', N(R')a. NO2, (C=O)N(R')2 O(CO)R', OR', COOR', a trialkyl tin, a tetrazole or oxadiazole of the form:
(wherein R' is H or a lower alkyl group), or a triazene of the form:
(wherein R8 and R9 are lower alkyl groups) or
represents a heterocyclic ring of one of the six following formulas:
IF has the following structure:
wherein:
exactly one of R28, R29, R30, R31 or R32 is the
link defined for Formula I above and each other R28, R29, R30, R31 or R32 independently is H, F, Cl, Br, I, a lower alkyl group, CF3, CH2-CH2F, O-CH2- CH2F, CH2-CH2-CH2F, O-CH2-CH2-CH2F, CN, (C=O) -R', N(R')2, NO2, (C=O)N(R')2 O(CO)R', OR', COOR', a trialkyl tin, a tetrazole or oxadiazole of the form:
(wherein R' is H or a lower alkyl group), or a triazene of the form: (wherein R8 and R9 are lower alkyl
groups ) or
wherein at least one of R28, R29, R30, R31 or R32 is a hydroxy, tetrazole, oxadiazole, NO2 or carboxy in both Q's;
exactly one of R33, R34, R35, R36, R37, R38, or R39 is the link defined for Formula I above and each
other R33, R34, R35, R36, R37, R38, or R39 independently is H, F, Cl, Br, I, a lower alkyl group, CF3, CH2- CH2F, O-CH2-CH2F, CH2-CH2-CH2F, O-CH2-CH2-CH2F, CN, (C=O)-R', N(R')2, NO2, (C=O)N(R')2 O(CO)R', OR', COOR', a tri-alkyl tin, a tetrazole or oxadiazole of the form:
(wherein R' is H or a lower alkyl group), or a triazene of the form:
(wherein R8 and R9 are lower alkyl groups) or and wherein at least one of R33, R34, R35, R36, R37, R38, or R39 is a hydroxy, tetrazole, oxadiazole, NO2 or carboxy in both Q's.
24. The method of claim 23 wherein at least one of the substituents R1-R7 and R10-R39 is labeled with a radiolabel selected from the group consisting of 125I, 3H, and a carbon-containing substituent as specified in Formula I, wherein at least one carbon is 14C.
25. A method of distinguishing an Alzheimer's disease brain from a normal brain comprising the steps of:
a) obtaining tissue from (i) the cerebellum and (ii) another area of the same brain other than the cerebellum, from normal subjects and from subjects suspected of having Alzheimer's disease;
b) incubating said tissues with a radiolabeled Chrysamine G derivative so that amyloid in said tissue binds with said radiolabeled Chrysamine G derivative;
c) quantifying the amount of amyloid bound to said radiolabeled Chrysamine G derivative, according to the method of claim 21;
d) calculating the ratio of the amount of amyloid in the area of the brain other than the
cerebellum to the amount of amyloid in the
cerebellum;
e) comparing said ratio for amount of amyloid in said tissue from normal subjects with ratio for amount of amyloid in tissue from subjects
suspected of having Alzheimer's disease; and f) determining the presence of Alzheimer's disease if said ratio from the brain of a subject
suspected of having Alzheimer's disease is above 90% of the ratios obtained from the brains of normal subjects.
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
EP01103705A EP1110945A3 (en) | 1995-05-01 | 1996-05-01 | Azocompounds for the antemortem diagnosis of Alzheimer's disease and in vivo imaging and prevention of amyloid deposition |
Applications Claiming Priority (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US43201995A | 1995-05-01 | 1995-05-01 | |
US432019 | 1995-05-01 | ||
PCT/US1996/005918 WO1996034853A1 (en) | 1995-05-01 | 1996-05-01 | Azocompounds for the antemortem diagnosis of alzheimer's disease and in vivo imaging and prevention of amyloid deposition |
Related Child Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
EP01103705A Division EP1110945A3 (en) | 1995-05-01 | 1996-05-01 | Azocompounds for the antemortem diagnosis of Alzheimer's disease and in vivo imaging and prevention of amyloid deposition |
Publications (1)
Publication Number | Publication Date |
---|---|
EP0823894A1 true EP0823894A1 (en) | 1998-02-18 |
Family
ID=23714410
Family Applications (2)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
EP96913856A Ceased EP0823894A1 (en) | 1995-05-01 | 1996-05-01 | Azocompounds for the antemortem diagnosis of alzheimer's disease and in vivo imaging and prevention of amyloid deposition |
EP01103705A Withdrawn EP1110945A3 (en) | 1995-05-01 | 1996-05-01 | Azocompounds for the antemortem diagnosis of Alzheimer's disease and in vivo imaging and prevention of amyloid deposition |
Family Applications After (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
EP01103705A Withdrawn EP1110945A3 (en) | 1995-05-01 | 1996-05-01 | Azocompounds for the antemortem diagnosis of Alzheimer's disease and in vivo imaging and prevention of amyloid deposition |
Country Status (10)
Country | Link |
---|---|
EP (2) | EP0823894A1 (en) |
JP (1) | JPH11505526A (en) |
KR (1) | KR19990008328A (en) |
AU (1) | AU712552B2 (en) |
CA (1) | CA2219880A1 (en) |
HU (1) | HUP9900426A3 (en) |
MX (1) | MX9708425A (en) |
NO (1) | NO975002L (en) |
NZ (1) | NZ307369A (en) |
WO (1) | WO1996034853A1 (en) |
Families Citing this family (16)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US6168776B1 (en) * | 1994-07-19 | 2001-01-02 | University Of Pittsburgh | Alkyl, alkenyl and alkynyl Chrysamine G derivatives for the antemortem diagnosis of Alzheimer's disease and in vivo imaging and prevention of amyloid deposition |
US6417178B1 (en) | 1994-07-19 | 2002-07-09 | University Of Pittsburgh | Amyloid binding nitrogen-linked compounds for the antemortem diagnosis of alzheimer's disease, in vivo imaging and prevention of amyloid deposits |
WO1997041856A1 (en) * | 1996-05-08 | 1997-11-13 | Massachusetts Institute Of Technology | ORGANOMETALLIC LIGANDS FOR THE LOCALIZATION AND QUANTIFICATION OF AMYLOID IN VIVO AND $i(IN VITRO) |
WO1998048846A1 (en) * | 1997-04-29 | 1998-11-05 | Nycomed Imaging As | Light imaging contrast agents |
ZA989365B (en) * | 1997-10-15 | 1999-04-15 | Boehringer Mannheim Pharm Corp | Preparation for treating alzheimer's disease |
EP1334091B1 (en) | 2000-08-24 | 2012-09-19 | University of Pittsburgh - Of the Commonwealth System of Higher Education | Thioflavin derivatives and their use in diagnosis and therapy of alzheimer's disease |
US6589504B1 (en) | 2000-09-22 | 2003-07-08 | Pharmacia & Upjohn Company | Compounds and methods for diagnosing and treating amyloid-related conditions |
AU2002211517A1 (en) * | 2000-10-04 | 2002-04-15 | California Institute Of Technology | Magnetic resonance imaging agents for in vivo labeling and detection of amyloid deposits |
US6479528B1 (en) * | 2001-07-31 | 2002-11-12 | Neuronautics, Inc. | Methods for inhibiting or reversing tau filament formation polymerization |
AU2003303666A1 (en) * | 2002-01-29 | 2004-08-13 | Protemix Discovery Limited | Suppression of cytotoxic protein conformers |
GB0307855D0 (en) * | 2003-04-04 | 2003-05-14 | Novartis Ag | Organic compounds |
EP1814876A2 (en) * | 2004-07-15 | 2007-08-08 | The General Hospital Corporation | Heterocyclic dye compounds for in vivo imaging and diagnosis of alzheimer's disease |
JP2007084526A (en) * | 2004-11-26 | 2007-04-05 | Nagasaki Univ | Composition for amyloid-associated disease diagnosis |
WO2006116736A2 (en) * | 2005-04-27 | 2006-11-02 | Siemens Medical Solutions Usa, Inc. | A method for the preparation of imaging probes using click chemistry |
US7910579B2 (en) | 2006-12-25 | 2011-03-22 | Tohoku University | Benzoxazole derivatives |
AR079687A1 (en) * | 2009-12-23 | 2012-02-15 | Bayer Schering Pharma Ag | ADEQUATE FORMULATIONS FOR DIAGNOSIS FOR IMAGES WITH PET |
Family Cites Families (9)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JPS58174459A (en) * | 1982-04-06 | 1983-10-13 | Canon Inc | Recording solution |
JPS59100450A (en) * | 1982-11-30 | 1984-06-09 | Mitsubishi Paper Mills Ltd | Photosensitive body for electrophotography |
JPS60205454A (en) * | 1984-03-29 | 1985-10-17 | Mitsubishi Paper Mills Ltd | Electrophotographic sensitive body |
EP0287909B1 (en) * | 1987-04-08 | 1992-09-02 | Salutar, Inc. | Amyloidosis and alzheimer's disease diagnostic assay and reagents therefor |
US4933156A (en) * | 1987-04-08 | 1990-06-12 | Salutar, Inc. | Amyloidosis and Alzheimer's disease diagnostic assay and reagents therefor |
US5039511A (en) * | 1987-04-08 | 1991-08-13 | Salutar, Inc. | Amyloidosis and alzheimer's disease diagnostic assay and reagents therefor |
JPS63301047A (en) * | 1987-05-30 | 1988-12-08 | Toshiba Corp | Positively chargeable laminate type electrophotographic sensitive body |
JPS63301045A (en) * | 1987-05-30 | 1988-12-08 | Toshiba Corp | Positively chargeable electrophotographic sensitive body |
US5276059A (en) * | 1992-07-10 | 1994-01-04 | The United States Of America As Represented By The Department Of Health And Human Services | Inhibition of diseases associated with amyloid formation |
-
1996
- 1996-05-01 AU AU56692/96A patent/AU712552B2/en not_active Ceased
- 1996-05-01 EP EP96913856A patent/EP0823894A1/en not_active Ceased
- 1996-05-01 NZ NZ307369A patent/NZ307369A/en unknown
- 1996-05-01 CA CA002219880A patent/CA2219880A1/en not_active Abandoned
- 1996-05-01 HU HU9900426A patent/HUP9900426A3/en unknown
- 1996-05-01 WO PCT/US1996/005918 patent/WO1996034853A1/en not_active Application Discontinuation
- 1996-05-01 JP JP8533383A patent/JPH11505526A/en active Pending
- 1996-05-01 EP EP01103705A patent/EP1110945A3/en not_active Withdrawn
- 1996-05-01 KR KR1019970707854A patent/KR19990008328A/en not_active Application Discontinuation
-
1997
- 1997-10-30 NO NO975002A patent/NO975002L/en not_active Application Discontinuation
- 1997-10-31 MX MX9708425A patent/MX9708425A/en unknown
Non-Patent Citations (1)
Title |
---|
See references of WO9634853A1 * |
Also Published As
Publication number | Publication date |
---|---|
WO1996034853A1 (en) | 1996-11-07 |
EP1110945A2 (en) | 2001-06-27 |
KR19990008328A (en) | 1999-01-25 |
HUP9900426A2 (en) | 1999-05-28 |
EP1110945A3 (en) | 2003-05-07 |
MX9708425A (en) | 1998-08-30 |
NZ307369A (en) | 1999-11-29 |
JPH11505526A (en) | 1999-05-21 |
CA2219880A1 (en) | 1996-11-07 |
AU5669296A (en) | 1996-11-21 |
AU712552B2 (en) | 1999-11-11 |
NO975002D0 (en) | 1997-10-30 |
NO975002L (en) | 1998-01-02 |
HUP9900426A3 (en) | 2001-12-28 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
US6417178B1 (en) | Amyloid binding nitrogen-linked compounds for the antemortem diagnosis of alzheimer's disease, in vivo imaging and prevention of amyloid deposits | |
US6133259A (en) | Alkyl, alkenyl and alkynyl chrysamine G derivatives for inhibition of cell degeneration and toxicity associated with amyloid deposition | |
US9833458B2 (en) | Thioflavin derivatives for use in the antemortem diagnosis of Alzheimer's disease and in vivo imaging and prevention of amyloid deposition | |
AU2001286702A1 (en) | Thioflavin derivatives and their use in diagnosis and theraphy of alzheimer's disease | |
AU712552B2 (en) | Azo compounds for the antemortem diagnosis of alzheimer's disease and in vivo imaging and prevention of amyloid deposition | |
MXPA00004371A (en) | Compounds for the antemortem diagnosis of alzheimer's disease and i(in vivo) imaging and prevention of amyloid deposition | |
MXPA99009557A (en) | Alkyl, alkenyl and alkynyl chrysamine g derivatives for the antemortem diagnosis of alzheimer's disease and (in vivo) imaging and prevention | |
CZ20001685A3 (en) | Compounds for diagnosis Alzheimer's disease and in vivo representation and prevention of amyloid deposits | |
CZ370099A3 (en) | Alkyl, alkenyl and alkynyl derivatives of chrysamine G for diagnosis of Alzheimer's disease and for representation in vivo and prevention of amyloid deposits |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
PUAI | Public reference made under article 153(3) epc to a published international application that has entered the european phase |
Free format text: ORIGINAL CODE: 0009012 |
|
17P | Request for examination filed |
Effective date: 19971111 |
|
AK | Designated contracting states |
Kind code of ref document: A1 Designated state(s): AT BE CH DE DK ES FI FR GB GR IE IT LI LU MC NL PT SE |
|
17Q | First examination report despatched |
Effective date: 19990629 |
|
GRAG | Despatch of communication of intention to grant |
Free format text: ORIGINAL CODE: EPIDOS AGRA |
|
STAA | Information on the status of an ep patent application or granted ep patent |
Free format text: STATUS: THE APPLICATION HAS BEEN REFUSED |
|
18R | Application refused |
Effective date: 20020526 |