WO2010125068A2 - Labeled molecular imaging agents, methods of making and methods of use - Google Patents
Labeled molecular imaging agents, methods of making and methods of use Download PDFInfo
- Publication number
- WO2010125068A2 WO2010125068A2 PCT/EP2010/055629 EP2010055629W WO2010125068A2 WO 2010125068 A2 WO2010125068 A2 WO 2010125068A2 EP 2010055629 W EP2010055629 W EP 2010055629W WO 2010125068 A2 WO2010125068 A2 WO 2010125068A2
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- cystine
- labeled
- cells
- imaging agent
- compound
- Prior art date
Links
- 239000012216 imaging agent Substances 0.000 title claims abstract description 62
- 238000000034 method Methods 0.000 title claims abstract description 39
- 229960003067 cystine Drugs 0.000 claims abstract description 89
- XUJNEKJLAYXESH-UHFFFAOYSA-N cysteine Natural products SCC(N)C(O)=O XUJNEKJLAYXESH-UHFFFAOYSA-N 0.000 claims abstract description 22
- 235000018417 cysteine Nutrition 0.000 claims abstract description 22
- WHUUTDBJXJRKMK-VKHMYHEASA-N L-glutamic acid Chemical compound OC(=O)[C@@H](N)CCC(O)=O WHUUTDBJXJRKMK-VKHMYHEASA-N 0.000 claims abstract description 13
- 229930195712 glutamate Natural products 0.000 claims abstract description 13
- 102000003669 Antiporters Human genes 0.000 claims abstract description 10
- 108090000084 Antiporters Proteins 0.000 claims abstract description 10
- 230000002829 reductive effect Effects 0.000 claims abstract description 10
- LEVWYRKDKASIDU-QWWZWVQMSA-N D-cystine Chemical compound OC(=O)[C@H](N)CSSC[C@@H](N)C(O)=O LEVWYRKDKASIDU-QWWZWVQMSA-N 0.000 claims abstract 3
- 230000001640 apoptogenic effect Effects 0.000 claims description 54
- -1 cystine compound Chemical class 0.000 claims description 38
- 150000001875 compounds Chemical class 0.000 claims description 37
- 239000002243 precursor Substances 0.000 claims description 35
- 102100035300 Cystine/glutamate transporter Human genes 0.000 claims description 26
- 108091006241 SLC7A11 Proteins 0.000 claims description 26
- 230000036542 oxidative stress Effects 0.000 claims description 19
- 239000007850 fluorescent dye Substances 0.000 claims description 13
- 238000003384 imaging method Methods 0.000 claims description 13
- 239000012472 biological sample Substances 0.000 claims description 10
- 239000000126 substance Substances 0.000 claims description 10
- 238000002600 positron emission tomography Methods 0.000 claims description 9
- 238000000799 fluorescence microscopy Methods 0.000 claims description 8
- 239000008194 pharmaceutical composition Substances 0.000 claims description 8
- 238000003786 synthesis reaction Methods 0.000 claims description 8
- 230000015572 biosynthetic process Effects 0.000 claims description 7
- 238000004624 confocal microscopy Methods 0.000 claims description 7
- 238000005388 cross polarization Methods 0.000 claims description 7
- 229910052739 hydrogen Inorganic materials 0.000 claims description 7
- 239000001257 hydrogen Substances 0.000 claims description 7
- 125000004435 hydrogen atom Chemical group [H]* 0.000 claims description 7
- 230000003834 intracellular effect Effects 0.000 claims description 7
- 238000000386 microscopy Methods 0.000 claims description 7
- 238000012634 optical imaging Methods 0.000 claims description 7
- 125000003277 amino group Chemical group 0.000 claims description 5
- 125000006239 protecting group Chemical group 0.000 claims description 5
- 238000003325 tomography Methods 0.000 claims description 5
- WSNMPAVSZJSIMT-UHFFFAOYSA-N COc1c(C)c2COC(=O)c2c(O)c1CC(O)C1(C)CCC(=O)O1 Chemical compound COc1c(C)c2COC(=O)c2c(O)c1CC(O)C1(C)CCC(=O)O1 WSNMPAVSZJSIMT-UHFFFAOYSA-N 0.000 claims description 4
- 238000002603 single-photon emission computed tomography Methods 0.000 claims description 4
- 238000012606 in vitro cell culture Methods 0.000 claims description 3
- 125000003636 chemical group Chemical group 0.000 claims description 2
- 239000000758 substrate Substances 0.000 abstract description 23
- 239000003795 chemical substances by application Substances 0.000 abstract description 16
- 210000004027 cell Anatomy 0.000 description 128
- LEVWYRKDKASIDU-IMJSIDKUSA-N L-cystine Chemical compound [O-]C(=O)[C@@H]([NH3+])CSSC[C@H]([NH3+])C([O-])=O LEVWYRKDKASIDU-IMJSIDKUSA-N 0.000 description 52
- 108010078791 Carrier Proteins Proteins 0.000 description 25
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 16
- 238000010186 staining Methods 0.000 description 16
- 238000006243 chemical reaction Methods 0.000 description 15
- XUJNEKJLAYXESH-REOHCLBHSA-N L-Cysteine Chemical compound SC[C@H](N)C(O)=O XUJNEKJLAYXESH-REOHCLBHSA-N 0.000 description 14
- 239000012154 double-distilled water Substances 0.000 description 13
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 12
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 12
- HKSZLNNOFSGOKW-UHFFFAOYSA-N ent-staurosporine Natural products C12=C3N4C5=CC=CC=C5C3=C3CNC(=O)C3=C2C2=CC=CC=C2N1C1CC(NC)C(OC)C4(C)O1 HKSZLNNOFSGOKW-UHFFFAOYSA-N 0.000 description 12
- 238000002372 labelling Methods 0.000 description 12
- HKSZLNNOFSGOKW-FYTWVXJKSA-N staurosporine Chemical compound C12=C3N4C5=CC=CC=C5C3=C3CNC(=O)C3=C2C2=CC=CC=C2N1[C@H]1C[C@@H](NC)[C@@H](OC)[C@]4(C)O1 HKSZLNNOFSGOKW-FYTWVXJKSA-N 0.000 description 12
- CGPUWJWCVCFERF-UHFFFAOYSA-N staurosporine Natural products C12=C3N4C5=CC=CC=C5C3=C3CNC(=O)C3=C2C2=CC=CC=C2N1C1CC(NC)C(OC)C4(OC)O1 CGPUWJWCVCFERF-UHFFFAOYSA-N 0.000 description 12
- 150000001412 amines Chemical class 0.000 description 11
- 206010028980 Neoplasm Diseases 0.000 description 10
- 239000000975 dye Substances 0.000 description 10
- RWSXRVCMGQZWBV-WDSKDSINSA-N glutathione Chemical compound OC(=O)[C@@H](N)CCC(=O)N[C@@H](CS)C(=O)NCC(O)=O RWSXRVCMGQZWBV-WDSKDSINSA-N 0.000 description 10
- DTQVDTLACAAQTR-UHFFFAOYSA-N Trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-N 0.000 description 9
- 238000002347 injection Methods 0.000 description 9
- 239000007924 injection Substances 0.000 description 9
- 230000006907 apoptotic process Effects 0.000 description 8
- 238000001514 detection method Methods 0.000 description 8
- 238000012544 monitoring process Methods 0.000 description 8
- 239000000047 product Substances 0.000 description 8
- 102100033400 4F2 cell-surface antigen heavy chain Human genes 0.000 description 7
- 108050008874 Annexin Proteins 0.000 description 7
- 102000000412 Annexin Human genes 0.000 description 7
- UETNIIAIRMUTSM-UHFFFAOYSA-N Jacareubin Natural products CC1(C)OC2=CC3Oc4c(O)c(O)ccc4C(=O)C3C(=C2C=C1)O UETNIIAIRMUTSM-UHFFFAOYSA-N 0.000 description 7
- 238000001727 in vivo Methods 0.000 description 7
- NCEXYHBECQHGNR-QZQOTICOSA-N sulfasalazine Chemical compound C1=C(O)C(C(=O)O)=CC(\N=N\C=2C=CC(=CC=2)S(=O)(=O)NC=2N=CC=CC=2)=C1 NCEXYHBECQHGNR-QZQOTICOSA-N 0.000 description 7
- NCEXYHBECQHGNR-UHFFFAOYSA-N sulfasalazine Natural products C1=C(O)C(C(=O)O)=CC(N=NC=2C=CC(=CC=2)S(=O)(=O)NC=2N=CC=CC=2)=C1 NCEXYHBECQHGNR-UHFFFAOYSA-N 0.000 description 7
- 229960001940 sulfasalazine Drugs 0.000 description 7
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 6
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 6
- 238000002512 chemotherapy Methods 0.000 description 6
- 230000000694 effects Effects 0.000 description 6
- 238000004128 high performance liquid chromatography Methods 0.000 description 6
- 230000000670 limiting effect Effects 0.000 description 6
- BWHMMNNQKKPAPP-UHFFFAOYSA-L potassium carbonate Chemical compound [K+].[K+].[O-]C([O-])=O BWHMMNNQKKPAPP-UHFFFAOYSA-L 0.000 description 6
- 238000002360 preparation method Methods 0.000 description 6
- 108010024636 Glutathione Proteins 0.000 description 5
- 241001465754 Metazoa Species 0.000 description 5
- 241000699670 Mus sp. Species 0.000 description 5
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 5
- 230000008901 benefit Effects 0.000 description 5
- 229960003180 glutathione Drugs 0.000 description 5
- 230000002285 radioactive effect Effects 0.000 description 5
- 239000000243 solution Substances 0.000 description 5
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 5
- 108010043121 Green Fluorescent Proteins Proteins 0.000 description 4
- 101000800023 Homo sapiens 4F2 cell-surface antigen heavy chain Proteins 0.000 description 4
- 238000004458 analytical method Methods 0.000 description 4
- 230000008809 cell oxidative stress Effects 0.000 description 4
- 238000010438 heat treatment Methods 0.000 description 4
- 239000000203 mixture Substances 0.000 description 4
- 239000013642 negative control Substances 0.000 description 4
- 230000004987 nonapoptotic effect Effects 0.000 description 4
- XJMOSONTPMZWPB-UHFFFAOYSA-M propidium iodide Chemical compound [I-].[I-].C12=CC(N)=CC=C2C2=CC=C(N)C=C2[N+](CCC[N+](C)(CC)CC)=C1C1=CC=CC=C1 XJMOSONTPMZWPB-UHFFFAOYSA-M 0.000 description 4
- 235000018102 proteins Nutrition 0.000 description 4
- 108090000623 proteins and genes Proteins 0.000 description 4
- 102000004169 proteins and genes Human genes 0.000 description 4
- 238000000746 purification Methods 0.000 description 4
- 239000000700 radioactive tracer Substances 0.000 description 4
- 239000012217 radiopharmaceutical Substances 0.000 description 4
- 229940121896 radiopharmaceutical Drugs 0.000 description 4
- 230000002799 radiopharmaceutical effect Effects 0.000 description 4
- 238000011894 semi-preparative HPLC Methods 0.000 description 4
- 239000011780 sodium chloride Substances 0.000 description 4
- 239000002904 solvent Substances 0.000 description 4
- 239000003643 water by type Substances 0.000 description 4
- 238000004435 EPR spectroscopy Methods 0.000 description 3
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 3
- 102000004144 Green Fluorescent Proteins Human genes 0.000 description 3
- 101000605020 Homo sapiens Large neutral amino acids transporter small subunit 1 Proteins 0.000 description 3
- 108091006313 SLC3A2 Proteins 0.000 description 3
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- 125000003118 aryl group Chemical group 0.000 description 3
- 239000000090 biomarker Substances 0.000 description 3
- 239000008280 blood Substances 0.000 description 3
- 210000004369 blood Anatomy 0.000 description 3
- 239000002872 contrast media Substances 0.000 description 3
- 230000002596 correlated effect Effects 0.000 description 3
- 238000000684 flow cytometry Methods 0.000 description 3
- 239000005090 green fluorescent protein Substances 0.000 description 3
- 238000000338 in vitro Methods 0.000 description 3
- 239000003112 inhibitor Substances 0.000 description 3
- XMBWDFGMSWQBCA-RNFDNDRNSA-M iodine-131(1-) Chemical group [131I-] XMBWDFGMSWQBCA-RNFDNDRNSA-M 0.000 description 3
- 210000003734 kidney Anatomy 0.000 description 3
- 230000007246 mechanism Effects 0.000 description 3
- 230000004048 modification Effects 0.000 description 3
- 238000012986 modification Methods 0.000 description 3
- 230000001338 necrotic effect Effects 0.000 description 3
- 229910052757 nitrogen Inorganic materials 0.000 description 3
- 210000000056 organ Anatomy 0.000 description 3
- 230000001575 pathological effect Effects 0.000 description 3
- 229910000027 potassium carbonate Inorganic materials 0.000 description 3
- 238000004007 reversed phase HPLC Methods 0.000 description 3
- 230000002441 reversible effect Effects 0.000 description 3
- 150000003384 small molecules Chemical class 0.000 description 3
- 230000001225 therapeutic effect Effects 0.000 description 3
- 238000011282 treatment Methods 0.000 description 3
- ZCYVEMRRCGMTRW-UHFFFAOYSA-N 7553-56-2 Chemical compound [I] ZCYVEMRRCGMTRW-UHFFFAOYSA-N 0.000 description 2
- 102000034263 Amino acid transporters Human genes 0.000 description 2
- 108050005273 Amino acid transporters Proteins 0.000 description 2
- 108090000672 Annexin A5 Proteins 0.000 description 2
- 102000004121 Annexin A5 Human genes 0.000 description 2
- CPELXLSAUQHCOX-UHFFFAOYSA-M Bromide Chemical compound [Br-] CPELXLSAUQHCOX-UHFFFAOYSA-M 0.000 description 2
- 229940123450 Cystine/glutamate transporter inhibitor Drugs 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
- 108091052347 Glucose transporter family Proteins 0.000 description 2
- 102000042092 Glucose transporter family Human genes 0.000 description 2
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 2
- 239000004471 Glycine Substances 0.000 description 2
- 239000007818 Grignard reagent Substances 0.000 description 2
- 206010061218 Inflammation Diseases 0.000 description 2
- 241000699666 Mus <mouse, genus> Species 0.000 description 2
- 241000699660 Mus musculus Species 0.000 description 2
- 208000006011 Stroke Diseases 0.000 description 2
- 230000002378 acidificating effect Effects 0.000 description 2
- 235000001014 amino acid Nutrition 0.000 description 2
- 238000005349 anion exchange Methods 0.000 description 2
- 230000003078 antioxidant effect Effects 0.000 description 2
- 239000007864 aqueous solution Substances 0.000 description 2
- 238000003556 assay Methods 0.000 description 2
- 125000004429 atom Chemical group 0.000 description 2
- 229940090047 auto-injector Drugs 0.000 description 2
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 2
- 210000004556 brain Anatomy 0.000 description 2
- 210000004748 cultured cell Anatomy 0.000 description 2
- 230000001419 dependent effect Effects 0.000 description 2
- 201000010099 disease Diseases 0.000 description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 2
- 125000002228 disulfide group Chemical group 0.000 description 2
- 239000003480 eluent Substances 0.000 description 2
- 238000010828 elution Methods 0.000 description 2
- KRHYYFGTRYWZRS-BJUDXGSMSA-M fluorine-18(1-) Chemical compound [18F-] KRHYYFGTRYWZRS-BJUDXGSMSA-M 0.000 description 2
- 125000000524 functional group Chemical group 0.000 description 2
- 239000008103 glucose Substances 0.000 description 2
- 150000004795 grignard reagents Chemical class 0.000 description 2
- 229910052736 halogen Inorganic materials 0.000 description 2
- 150000002367 halogens Chemical class 0.000 description 2
- XMBWDFGMSWQBCA-UHFFFAOYSA-N hydrogen iodide Chemical compound I XMBWDFGMSWQBCA-UHFFFAOYSA-N 0.000 description 2
- 238000011503 in vivo imaging Methods 0.000 description 2
- 230000004054 inflammatory process Effects 0.000 description 2
- 238000001990 intravenous administration Methods 0.000 description 2
- 229910052740 iodine Inorganic materials 0.000 description 2
- 239000011630 iodine Substances 0.000 description 2
- 208000028867 ischemia Diseases 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 210000004185 liver Anatomy 0.000 description 2
- 230000004060 metabolic process Effects 0.000 description 2
- 230000017074 necrotic cell death Effects 0.000 description 2
- 230000009871 nonspecific binding Effects 0.000 description 2
- 239000002773 nucleotide Substances 0.000 description 2
- 125000003729 nucleotide group Chemical group 0.000 description 2
- 238000011580 nude mouse model Methods 0.000 description 2
- 230000003287 optical effect Effects 0.000 description 2
- 239000003960 organic solvent Substances 0.000 description 2
- 125000002524 organometallic group Chemical group 0.000 description 2
- 230000036961 partial effect Effects 0.000 description 2
- 230000007170 pathology Effects 0.000 description 2
- 238000012636 positron electron tomography Methods 0.000 description 2
- 235000015320 potassium carbonate Nutrition 0.000 description 2
- 238000001243 protein synthesis Methods 0.000 description 2
- 239000011541 reaction mixture Substances 0.000 description 2
- 238000011084 recovery Methods 0.000 description 2
- 230000000717 retained effect Effects 0.000 description 2
- 238000012552 review Methods 0.000 description 2
- 125000001424 substituent group Chemical group 0.000 description 2
- 238000002560 therapeutic procedure Methods 0.000 description 2
- 150000003573 thiols Chemical class 0.000 description 2
- 230000000451 tissue damage Effects 0.000 description 2
- 231100000827 tissue damage Toxicity 0.000 description 2
- 231100000419 toxicity Toxicity 0.000 description 2
- 230000001988 toxicity Effects 0.000 description 2
- 230000014616 translation Effects 0.000 description 2
- 230000003827 upregulation Effects 0.000 description 2
- 239000011800 void material Substances 0.000 description 2
- 239000008215 water for injection Substances 0.000 description 2
- UKCGLNQRSQIAON-IKABJLMCSA-N (2R)-2-amino-3-[[(2R)-2-amino-2-carboxyethyl]disulfanyl]-3-aminooxypropanoic acid Chemical compound O(N)C([C@@H](C(=O)O)N)SSC[C@@H](C(=O)O)N UKCGLNQRSQIAON-IKABJLMCSA-N 0.000 description 1
- JYVJSLVUOYOEHM-MZZWAUCGSA-N (2R)-2-amino-3-[[(2R)-2-amino-2-carboxyethyl]disulfanyl]-5-(18F)fluoranylpentanoic acid Chemical compound [18F]CCC([C@@H](C(=O)O)N)SSC[C@@H](C(=O)O)N JYVJSLVUOYOEHM-MZZWAUCGSA-N 0.000 description 1
- MGNAZESEUVNMRR-ZEQRLZLVSA-N (2r)-3-[[(2r)-2-carboxy-2-[[5-(dimethylamino)naphthalen-1-yl]sulfonylamino]ethyl]disulfanyl]-2-[[5-(dimethylamino)naphthalen-1-yl]sulfonylamino]propanoic acid Chemical group C1=CC=C2C(S(=O)(=O)N[C@@H](CSSC[C@H](NS(=O)(=O)C3=C4C=CC=C(C4=CC=C3)N(C)C)C(O)=O)C(O)=O)=CC=CC2=C1N(C)C MGNAZESEUVNMRR-ZEQRLZLVSA-N 0.000 description 1
- KFWHPKSKBQSSBW-UHFFFAOYSA-M (4-formylphenyl)-trimethylazanium;trifluoromethanesulfonate Chemical compound [O-]S(=O)(=O)C(F)(F)F.C[N+](C)(C)C1=CC=C(C=O)C=C1 KFWHPKSKBQSSBW-UHFFFAOYSA-M 0.000 description 1
- YOSZEPWSVKKQOV-UHFFFAOYSA-N 12h-benzo[a]phenoxazine Chemical compound C1=CC=CC2=C3NC4=CC=CC=C4OC3=CC=C21 YOSZEPWSVKKQOV-UHFFFAOYSA-N 0.000 description 1
- ZCXUVYAZINUVJD-AHXZWLDOSA-N 2-deoxy-2-((18)F)fluoro-alpha-D-glucose Chemical compound OC[C@H]1O[C@H](O)[C@H]([18F])[C@@H](O)[C@@H]1O ZCXUVYAZINUVJD-AHXZWLDOSA-N 0.000 description 1
- ZWDVQMVZZYIAHO-COJKEBBMSA-N 2-fluoranylbenzaldehyde Chemical compound [18F]C1=CC=CC=C1C=O ZWDVQMVZZYIAHO-COJKEBBMSA-N 0.000 description 1
- ZWDVQMVZZYIAHO-UHFFFAOYSA-N 2-fluorobenzaldehyde Chemical compound FC1=CC=CC=C1C=O ZWDVQMVZZYIAHO-UHFFFAOYSA-N 0.000 description 1
- AUFVJZSDSXXFOI-UHFFFAOYSA-N 2.2.2-cryptand Chemical compound C1COCCOCCN2CCOCCOCCN1CCOCCOCC2 AUFVJZSDSXXFOI-UHFFFAOYSA-N 0.000 description 1
- UOQXIWFBQSVDPP-UHFFFAOYSA-N 4-fluorobenzaldehyde Chemical compound FC1=CC=C(C=O)C=C1 UOQXIWFBQSVDPP-UHFFFAOYSA-N 0.000 description 1
- NIEBHDXUIJSHSL-VYJFISKDSA-N 4-iodanylbenzaldehyde Chemical compound [123I]C1=CC=C(C=O)C=C1 NIEBHDXUIJSHSL-VYJFISKDSA-N 0.000 description 1
- 239000004215 Carbon black (E152) Substances 0.000 description 1
- YPWSLBHSMIKTPR-UHFFFAOYSA-N Cystathionine Natural products OC(=O)C(N)CCSSCC(N)C(O)=O YPWSLBHSMIKTPR-UHFFFAOYSA-N 0.000 description 1
- 102000012192 Cystatin C Human genes 0.000 description 1
- 108010061642 Cystatin C Proteins 0.000 description 1
- 101710133877 Cystine transporter Proteins 0.000 description 1
- FBPFZTCFMRRESA-FSIIMWSLSA-N D-Glucitol Natural products OC[C@H](O)[C@H](O)[C@@H](O)[C@H](O)CO FBPFZTCFMRRESA-FSIIMWSLSA-N 0.000 description 1
- FBPFZTCFMRRESA-KVTDHHQDSA-N D-Mannitol Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-KVTDHHQDSA-N 0.000 description 1
- ILRYLPWNYFXEMH-UHFFFAOYSA-N D-cystathionine Natural products OC(=O)C(N)CCSCC(N)C(O)=O ILRYLPWNYFXEMH-UHFFFAOYSA-N 0.000 description 1
- FBPFZTCFMRRESA-JGWLITMVSA-N D-glucitol Chemical compound OC[C@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-JGWLITMVSA-N 0.000 description 1
- BWGNESOTFCXPMA-UHFFFAOYSA-N Dihydrogen disulfide Chemical compound SS BWGNESOTFCXPMA-UHFFFAOYSA-N 0.000 description 1
- 229910052692 Dysprosium Inorganic materials 0.000 description 1
- 229910052693 Europium Inorganic materials 0.000 description 1
- ILRYLPWNYFXEMH-WHFBIAKZSA-N L-cystathionine Chemical compound [O-]C(=O)[C@@H]([NH3+])CCSC[C@H]([NH3+])C([O-])=O ILRYLPWNYFXEMH-WHFBIAKZSA-N 0.000 description 1
- 239000004158 L-cystine Substances 0.000 description 1
- 235000019393 L-cystine Nutrition 0.000 description 1
- 229930195725 Mannitol Natural products 0.000 description 1
- AFVFQIVMOAPDHO-UHFFFAOYSA-N Methanesulfonic acid Chemical compound CS(O)(=O)=O AFVFQIVMOAPDHO-UHFFFAOYSA-N 0.000 description 1
- 238000010934 O-alkylation reaction Methods 0.000 description 1
- 238000012879 PET imaging Methods 0.000 description 1
- DNIAPMSPPWPWGF-UHFFFAOYSA-N Propylene glycol Chemical class CC(O)CO DNIAPMSPPWPWGF-UHFFFAOYSA-N 0.000 description 1
- 108010001267 Protein Subunits Proteins 0.000 description 1
- 102000002067 Protein Subunits Human genes 0.000 description 1
- 229910052772 Samarium Inorganic materials 0.000 description 1
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 1
- 229930006000 Sucrose Natural products 0.000 description 1
- 229910052771 Terbium Inorganic materials 0.000 description 1
- 206010052779 Transplant rejections Diseases 0.000 description 1
- 208000030886 Traumatic Brain injury Diseases 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- KRHYYFGTRYWZRS-BJUDXGSMSA-N ac1l2y5h Chemical compound [18FH] KRHYYFGTRYWZRS-BJUDXGSMSA-N 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 150000001349 alkyl fluorides Chemical class 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- 125000002344 aminooxy group Chemical group [H]N([H])O[*] 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 238000010171 animal model Methods 0.000 description 1
- 150000004056 anthraquinones Chemical class 0.000 description 1
- 239000000427 antigen Substances 0.000 description 1
- 108091007433 antigens Proteins 0.000 description 1
- 102000036639 antigens Human genes 0.000 description 1
- 239000003963 antioxidant agent Substances 0.000 description 1
- 235000006708 antioxidants Nutrition 0.000 description 1
- 150000001503 aryl iodides Chemical class 0.000 description 1
- 239000000987 azo dye Substances 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000027455 binding Effects 0.000 description 1
- 239000011230 binding agent Substances 0.000 description 1
- 230000031018 biological processes and functions Effects 0.000 description 1
- 238000005341 cation exchange Methods 0.000 description 1
- 150000001768 cations Chemical class 0.000 description 1
- 230000001413 cellular effect Effects 0.000 description 1
- 230000004637 cellular stress Effects 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- PBAYDYUZOSNJGU-UHFFFAOYSA-N chelidonic acid Natural products OC(=O)C1=CC(=O)C=C(C(O)=O)O1 PBAYDYUZOSNJGU-UHFFFAOYSA-N 0.000 description 1
- 238000001311 chemical methods and process Methods 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 239000007810 chemical reaction solvent Substances 0.000 description 1
- 230000021615 conjugation Effects 0.000 description 1
- 230000000875 corresponding effect Effects 0.000 description 1
- 239000008367 deionised water Substances 0.000 description 1
- 229910021641 deionized water Inorganic materials 0.000 description 1
- 238000003745 diagnosis Methods 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 239000012954 diazonium Substances 0.000 description 1
- 239000000539 dimer Substances 0.000 description 1
- 238000006073 displacement reaction Methods 0.000 description 1
- KBQHZAAAGSGFKK-UHFFFAOYSA-N dysprosium atom Chemical compound [Dy] KBQHZAAAGSGFKK-UHFFFAOYSA-N 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- OGPBJKLSAFTDLK-UHFFFAOYSA-N europium atom Chemical compound [Eu] OGPBJKLSAFTDLK-UHFFFAOYSA-N 0.000 description 1
- 230000029142 excretion Effects 0.000 description 1
- 239000012467 final product Substances 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 238000012632 fluorescent imaging Methods 0.000 description 1
- 108091006047 fluorescent proteins Proteins 0.000 description 1
- 102000034287 fluorescent proteins Human genes 0.000 description 1
- 125000004428 fluoroalkoxy group Chemical group 0.000 description 1
- 125000003709 fluoroalkyl group Chemical group 0.000 description 1
- 238000011010 flushing procedure Methods 0.000 description 1
- 238000009472 formulation Methods 0.000 description 1
- 238000012948 formulation analysis Methods 0.000 description 1
- 150000002334 glycols Chemical class 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 229930195733 hydrocarbon Natural products 0.000 description 1
- 150000002430 hydrocarbons Chemical class 0.000 description 1
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 1
- 230000028993 immune response Effects 0.000 description 1
- 238000000099 in vitro assay Methods 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 208000027866 inflammatory disease Diseases 0.000 description 1
- 208000014674 injury Diseases 0.000 description 1
- 238000010253 intravenous injection Methods 0.000 description 1
- 230000026045 iodination Effects 0.000 description 1
- 238000006192 iodination reaction Methods 0.000 description 1
- INQOMBQAUSQDDS-UHFFFAOYSA-N iodomethane Chemical compound IC INQOMBQAUSQDDS-UHFFFAOYSA-N 0.000 description 1
- 210000000738 kidney tubule Anatomy 0.000 description 1
- 150000002634 lipophilic molecules Chemical class 0.000 description 1
- 230000002934 lysing effect Effects 0.000 description 1
- 229910052749 magnesium Inorganic materials 0.000 description 1
- 239000011777 magnesium Substances 0.000 description 1
- 239000000594 mannitol Substances 0.000 description 1
- 235000010355 mannitol Nutrition 0.000 description 1
- 238000004949 mass spectrometry Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- RRHNGIRRWDWWQQ-UHFFFAOYSA-N n-iodoaniline Chemical compound INC1=CC=CC=C1 RRHNGIRRWDWWQQ-UHFFFAOYSA-N 0.000 description 1
- 239000002159 nanocrystal Substances 0.000 description 1
- 229930014626 natural product Natural products 0.000 description 1
- 239000006225 natural substrate Substances 0.000 description 1
- 239000012299 nitrogen atmosphere Substances 0.000 description 1
- 229910052755 nonmetal Inorganic materials 0.000 description 1
- 150000002843 nonmetals Chemical class 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 230000000269 nucleophilic effect Effects 0.000 description 1
- 238000010534 nucleophilic substitution reaction Methods 0.000 description 1
- 210000000496 pancreas Anatomy 0.000 description 1
- 230000037361 pathway Effects 0.000 description 1
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N phenol group Chemical group C1(=CC=CC=C1)O ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 description 1
- 229920001223 polyethylene glycol Polymers 0.000 description 1
- 229940068917 polyethylene glycols Drugs 0.000 description 1
- 229920005862 polyol Polymers 0.000 description 1
- 150000003077 polyols Chemical class 0.000 description 1
- 229920001184 polypeptide Polymers 0.000 description 1
- 150000004032 porphyrins Chemical class 0.000 description 1
- 235000011181 potassium carbonates Nutrition 0.000 description 1
- 230000003389 potentiating effect Effects 0.000 description 1
- 102000004196 processed proteins & peptides Human genes 0.000 description 1
- 108090000765 processed proteins & peptides Proteins 0.000 description 1
- 235000013772 propylene glycol Nutrition 0.000 description 1
- 239000008213 purified water Substances 0.000 description 1
- 239000002096 quantum dot Substances 0.000 description 1
- 238000000163 radioactive labelling Methods 0.000 description 1
- 229910052761 rare earth metal Inorganic materials 0.000 description 1
- 150000002910 rare earth metals Chemical class 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- KZUNJOHGWZRPMI-UHFFFAOYSA-N samarium atom Chemical compound [Sm] KZUNJOHGWZRPMI-UHFFFAOYSA-N 0.000 description 1
- 239000000523 sample Substances 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 239000000600 sorbitol Substances 0.000 description 1
- 230000035882 stress Effects 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 239000005720 sucrose Substances 0.000 description 1
- 235000000346 sugar Nutrition 0.000 description 1
- 150000005846 sugar alcohols Chemical class 0.000 description 1
- 150000008163 sugars Chemical class 0.000 description 1
- 230000002194 synthesizing effect Effects 0.000 description 1
- GZCRRIHWUXGPOV-UHFFFAOYSA-N terbium atom Chemical compound [Tb] GZCRRIHWUXGPOV-UHFFFAOYSA-N 0.000 description 1
- 150000004905 tetrazines Chemical class 0.000 description 1
- JOXIMZWYDAKGHI-UHFFFAOYSA-N toluene-4-sulfonic acid Chemical compound CC1=CC=C(S(O)(=O)=O)C=C1 JOXIMZWYDAKGHI-UHFFFAOYSA-N 0.000 description 1
- 230000008791 toxic response Effects 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 230000009529 traumatic brain injury Effects 0.000 description 1
- 230000008736 traumatic injury Effects 0.000 description 1
- 238000011269 treatment regimen Methods 0.000 description 1
- 125000004665 trialkylsilyl group Chemical group 0.000 description 1
- 150000004654 triazenes Chemical class 0.000 description 1
- 238000002604 ultrasonography Methods 0.000 description 1
- 210000003932 urinary bladder Anatomy 0.000 description 1
- 210000002700 urine Anatomy 0.000 description 1
- 238000010200 validation analysis Methods 0.000 description 1
- 229910001868 water Inorganic materials 0.000 description 1
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K49/00—Preparations for testing in vivo
- A61K49/001—Preparation for luminescence or biological staining
- A61K49/0013—Luminescence
- A61K49/0017—Fluorescence in vivo
- A61K49/0019—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K49/00—Preparations for testing in vivo
- A61K49/001—Preparation for luminescence or biological staining
- A61K49/0013—Luminescence
- A61K49/0017—Fluorescence in vivo
- A61K49/005—Fluorescence in vivo characterised by the carrier molecule carrying the fluorescent agent
- A61K49/0052—Small organic molecules
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K51/00—Preparations containing radioactive substances for use in therapy or testing in vivo
- A61K51/02—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by the carrier, i.e. characterised by the agent or material covalently linked or complexing the radioactive nucleus
- A61K51/04—Organic compounds
- A61K51/0404—Lipids, e.g. triglycerides; Polycationic carriers
- A61K51/0406—Amines, polyamines, e.g. spermine, spermidine, amino acids, (bis)guanidines
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07B—GENERAL METHODS OF ORGANIC CHEMISTRY; APPARATUS THEREFOR
- C07B59/00—Introduction of isotopes of elements into organic compounds ; Labelled organic compounds per se
- C07B59/001—Acyclic or carbocyclic compounds
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C323/00—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups
- C07C323/50—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups containing thio groups and carboxyl groups bound to the same carbon skeleton
- C07C323/51—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups containing thio groups and carboxyl groups bound to the same carbon skeleton having the sulfur atoms of the thio groups bound to acyclic carbon atoms of the carbon skeleton
- C07C323/57—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups containing thio groups and carboxyl groups bound to the same carbon skeleton having the sulfur atoms of the thio groups bound to acyclic carbon atoms of the carbon skeleton the carbon skeleton being further substituted by nitrogen atoms, not being part of nitro or nitroso groups
- C07C323/58—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups containing thio groups and carboxyl groups bound to the same carbon skeleton having the sulfur atoms of the thio groups bound to acyclic carbon atoms of the carbon skeleton the carbon skeleton being further substituted by nitrogen atoms, not being part of nitro or nitroso groups with amino groups bound to the carbon skeleton
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C323/00—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups
- C07C323/50—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups containing thio groups and carboxyl groups bound to the same carbon skeleton
- C07C323/51—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups containing thio groups and carboxyl groups bound to the same carbon skeleton having the sulfur atoms of the thio groups bound to acyclic carbon atoms of the carbon skeleton
- C07C323/57—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups containing thio groups and carboxyl groups bound to the same carbon skeleton having the sulfur atoms of the thio groups bound to acyclic carbon atoms of the carbon skeleton the carbon skeleton being further substituted by nitrogen atoms, not being part of nitro or nitroso groups
- C07C323/58—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups containing thio groups and carboxyl groups bound to the same carbon skeleton having the sulfur atoms of the thio groups bound to acyclic carbon atoms of the carbon skeleton the carbon skeleton being further substituted by nitrogen atoms, not being part of nitro or nitroso groups with amino groups bound to the carbon skeleton
- C07C323/59—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups containing thio groups and carboxyl groups bound to the same carbon skeleton having the sulfur atoms of the thio groups bound to acyclic carbon atoms of the carbon skeleton the carbon skeleton being further substituted by nitrogen atoms, not being part of nitro or nitroso groups with amino groups bound to the carbon skeleton with acylated amino groups bound to the carbon skeleton
-
- 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
- C07F5/00—Compounds containing elements of Groups 3 or 13 of the Periodic Table
- C07F5/02—Boron compounds
- C07F5/022—Boron compounds without C-boron linkages
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07B—GENERAL METHODS OF ORGANIC CHEMISTRY; APPARATUS THEREFOR
- C07B2200/00—Indexing scheme relating to specific properties of organic compounds
- C07B2200/05—Isotopically modified compounds, e.g. labelled
Definitions
- the invention relates generally to labeled molecular imaging agents and more particularly to imaging agents that are taken up by the cells via the cystine/glutamate transporter.
- the cystine/glutamate transporter is not typically expressed or has extremely low expression in most tissues, but is upregulated in cells exposed to oxidative stress.
- Cystine which comprises two disulfide-linked cysteine amino acids, is a natural substrate for this transporter.
- the cystine/glutamate antiporter (xc- system) is an amino acid transporter (designated SLC7A11) made up of two protein subunits; 4F2hc/CD98, a common subunit for a few classes of transport systems, and xCT, which is specific to the cystine/glutamate exchanger.
- SLC7A11 amino acid transporter
- 4F2hc/CD98 a common subunit for a few classes of transport systems
- xCT which is specific to the cystine/glutamate exchanger.
- the effect of upregulation of the transporter is an increase in cystine uptake; which is then reduced to cysteine inside the cell.
- Intracellular cysteine is the rate limiting substrate for glutathione synthesis. Glutathione is the cells primary anti-oxidant to defend against oxidative stress. Intracellular cysteine is incorporated into one of two pathways, glutathione synthesis or protein synthesis.
- DDC N,N'-Didansyl-L-cystine
- DBC DiBodipy L-cystine
- Invitrogen catalog #B20340
- DBC DiBodipy L-cystine
- cystine labeled with 99m Tc is known.
- Tubis and Endow (1968 Int J Appl Rad Isotop; 19: 835-840) discloses the direct reaction of cysteine with "" 1 TcO 4 " wherein the -SH group of cysteine reduces the 99m TcO 4 " while being simultaneously oxidized to cystine.
- cystine labeled with 35 S is known and has the following structure:
- 35 S has been used for example in animal radiotracer studies (see e.g. Hwang et al 1980 J Neurochem; 35: 417-424).
- 18-F-fluorodeoxyglucose that makes use of the glucose transporter.
- 18 F-FDG is preferentially taken up by cells that have an increased requirement for glucose, and then is trapped inside the cell.
- FDG can be used clinically for the diagnosis, staging and monitoring of many cancers as well as monitoring metabolism in the heart and brain.
- 18 F-FDG is not a substrate for sodium- dependent glucose transporters found in the kidney tubules, which prevents its renal resorption and enhances clearance
- EPR electron paramagnetic resonance
- the imaging agents and methods of the invention take advantage of the cellular amino acid transporter (cystine/glutamate antiporter, xc-), which is activated under conditions of cellular oxidative stress.
- These labeled molecular imaging agents and methods of the invention provide several benefits, including but not limited to, their use in a wide variety of diagnostic and therapeutic monitoring applications; they are small molecules and comprise labeled variants of a natural compound found in the body, and would be administered at tracer levels with physiological concentrations well below those that generate toxic response, therefore toxicity/immune response is expected to be low; and because the imaging agents act as a transporter substrate, the agents benefit from amplification of the signal because the molecular imaging agent is trapped once inside the cell, unlike other molecular binders in which the imaging agent's signal is limited to the stoichiometric binding of a cell surface epitope.
- the labeled substrates of the invention for the cystine/glutamate transporter may also be used to introduce compounds in a target for therapeutic purposes
- these imaging agents may be used for any disease or condition that relates to increased oxidative stress.
- the imaging agents may be used image apoptotic cells in vivo.
- Such noninvasive monitoring of apoptotic cell death is useful, for example without limitation, to monitor chemotherapy effectiveness, tissue damage due to ischemia/stroke, traumatic injury, and transplant rejection.
- Imaging of oxidative stress is also useful for diagnosing and monitoring inflammatory diseases or any pathological indication that includes oxidative stress or tissue damage.
- upregulation of the cystine/glutamate transporter is also associated with chemotherapy resistance in some tumors. Therefore, non-invasive imaging of tumors with basal high cystine uptake could result in identification of tumors likely to be resistant to certain therapies; which could result in efficacious changes in treatment regimens.
- An example of the method of the invention, for detecting oxidative stress in cells generally comprises: introducing an imaging agent comprising a labeled cystine into a cystine/glutamate antiporter of the cells; allowing the intracellular labeled cystine to be reduced into a labeled cysteine; detecting the labeled cysteine in the cell.
- the labeled cystine may be detected in apoptotic cells.
- the labeled cystine used in the method for detecting oxidative stress is selected from one of structures I, II, II and IV as defined more specifically hereunder.
- Another embodiment of the imaging agent of the invention generally comprises, a labeled small molecule substrate of a cystine/glutamate transporter (xc-), such as, but not limited to, Structures I, II, III, IV, V and VI, defined more specifically hereunder.
- xc- cystine/glutamate transporter
- a non-limiting example of a method for imaging of the invention generally comprises: using a cystine compound such as, but not limited to, structures I, II, III and IV as defined more specifically hereunder, wherein the compound is detected using, fluorescence microscopy, laser-confocal microscopy, cross-polarization microscopy, optical imaging, nuclear scintigraphy, positron emission tomography, single photon emission computed tomography.
- FIG 1 is a flow diagram of an embodiment of the imaging agent of the invention being transported into a cell via the cystine/glutamate antiporter.
- FIG 2 A is a forward scatter/side scatter plot showing some late apoptotic Jurkat cells (light gray) with a different granularity than non-apoptotic Jurkat cells (darker gray).
- FIG 2B is a multi-quadrant scatter plot showing AnnexinV and propdium iodide (PI) negative cells in quadrant D3 and represent non-apoptotic cells; showing Annexin V-Cy5 positive and propidium iodide (PI) negative cells in Trotulin (PI) negative cells in Triple-quadrant D4 representing early apoptotic cells; and showing Annexin V positive, cells in Supplementrant D2 representing late apoptotic and necrotic cells
- PI propdium iodide
- FIG 3 A-D shows results from flow cytometric analysis of DBC uptake in Jurkat cells with and without staurosporine (STN) to induce oxidative stress/apoptosis and with and without sulfasalazine (sasz) to inhibit the cystine/glutamate transporter.
- FIG 3A is a graph showing untreated cells demonstrating some uptake or nonspecific binding of the DBC molecule.
- FIG 3B is a graph showing that staurosporine induced cells have three subpopulations of cells, low intensity DBC fluorescence representing normal cells, intermediate intensity representing early apoptotic cells and high intensity representing late apoptotic cells.
- FIG 3C is a graph showing that staurosporine induced cells that are exposed to the cystine/glutamate transporter inhibitor sulfasalazine (sasz) have a different result; a population of cells with intermediate DBC staining represents late apoptotic cells, while Early apoptotic cells are in the low DBC intensity population.
- FIG 4D is a bar chart showing the percentage of cells labeled greater than baseline DBC staining for normal, early apoptotic and late apoptotic cells (as determined by AnnexinV and PI staining).
- FIGs. 4A shows uptake of DBC in Jurkat cells treated with STN for 18 hours.
- FIG 4B shows a comparison of the same cells incubated with DiBodipy(650) Cystine (DBC(650)).
- FIG 5 A-C shows results from flow cytometric analysis of MonoBodipyCystine (MBC) uptake in Jurkat cells with Staurosporine (STTSf) to induce oxidative stress/apoptosis with and without sulfasalazine (sasz) to inhibit the cystine/glutamate transporter.
- MBC MonoBodipyCystine
- STTSf Staurosporine
- sasz sulfasalazine
- FIG 5A is a graph showing that staurosporine induced cells have two subpopulations of cells, no MBC fluorescence representing normal cells and high MBC intensity representing early and late apoptotic cells.
- FIG 5B is a graph showing that staurosporine induced that are exposed to the cystine/glutamate transporter inhibitor sulfasalazine (sasz) have a different result; a population of cells with intermediate MBC staining represents late apoptotic cells, while early apoptotic cells show no MBC fluorescence intensity.
- sasz cystine/glutamate transporter inhibitor sulfasalazine
- FIG 5C is a bar chart showing the percentage of cells labeled greater than baseline MBC staining for normal, early apoptotic and late apoptotic cells (as determined by AnnexinV and PI staining).
- FIG 6 A is a bar chart showing the percentage of normal, early apoptotic and late apoptotic cells (as determined by AnnexinV and PI staining) cells labeled with five different fluorescent agents: DBC, DBC(650), DiBodipyCystathionine (DBCystathionine), MBC and a negative control fluorescent molecule (Bodipy-FL C5).
- FIG 6B is a table showing the mean fold intensity shift for early apoptotic cells that are labeled with each of the five agents shown in FIG 6A.
- FIG 7 is a bar graph showing the uptake of monoAO-[' 8 F]-FB A-Cystine in Jurkat and A549 cells in culture.
- FIG 8 A is a graph showing the biodistribution results in naive Balb-c mice in %ED/organ.
- FIG 8B is a graph showing the %ID/gram of the same data shown in FIG 8A.
- FIG 8C is a graph showing biodistribution results in nude mice with A549 xenograft tumors in % ID/gram.
- Figure 8D shows tumor to blood ratios at each timepoint and is derived from the same data shown in 8C.
- FIG 9 is a graph showing the stability of the mono AO-[' 8 F]-FB A-Cystine molecule over time.
- FIG 10 is an image showing the 18 F-AO-FB-Cystine in a PET image in a na ⁇ ve mouse at 60 minutes post injection.
- the present invention provides an imaging agent comprising a labeled cystine compound having structure I,
- R and R' comprises a label selected from a fluorescent label or a radioisotopic label, and the other of R and R' is hydrogen.
- imaging agent is intended to encompass compounds that may be detected using either in vitro or in vivo imaging techniques.
- fluorescent label includes, but is not limited to, fluorescent imaging agents and fluorophores, that are chemical compounds, which when excited by exposure to a particular wavelength of light, emit light at a different wavelength. Fluorophores may be described in terms of their emission profile, or color, and are the component of a molecule that causes the molecule to be fluorescent. It is typically a functional group that absorbs energy of a specific wavelength or range of wavelengths and re-emit energy at different but equally specific wavelengths or ranges.
- radioisotopic label includes, but is not limited to, radioisotopes that are used in a compound to trace or visualize the compound, or the mechanism of a chemical reaction, in a chemical or biological process, or biological substances, organisms and systems. Such labels are useful, for example, in connection with imaging and detection systems.
- the imaging agents of the invention comprise labeled cystine and any analogs of cystine that maintain the attributes necessary to be a substrate of the cystine/glutamate transporter. As shown in FIG 1, the imaging agents serve as a substrate for the cystine/glutamate transporter. Cystine labeled at a free amine is a substrate for the cystine/glutamate transporter. After transport into the cell, R-cystine is reduced to an R-cysteine and an unlabeled cysteine. R-cysteine is not metabolized and cannot exit via the transporter. Therefore it is retained in a cell that is responding to oxidative stress.
- cystine/glutamate transporter is used interchangeably with, and includes, the terms cystine/glutamate antiporter, cystine/glutamate exchanger, cystine transporter, xc(-), Xc(-), system xc(-), and amino acid transport system Xc(-).
- the transport system comprises dimer of two proteins and includes, but is not limited to: protein xCT and protein CD98 (4F2hc, heavy chain of the 4F2 surface antigen, SLC3A2); protein xCT which is the subunit specific to the xc(-) system; protein CD98 which is a subunit common to a number of transporters with different substrates; and protein xCT that may also dimerize with rBAT, another subunit common to multiple transporters.
- protein xCT and protein CD98 (4F2hc, heavy chain of the 4F2 surface antigen, SLC3A2)
- protein xCT which is the subunit specific to the xc(-) system
- protein CD98 which is a subunit common to a number of transporters with different substrates
- protein xCT that may also dimerize with rBAT, another subunit common to multiple transporters.
- the imaging agent of the invention may be detected by its emitted signal.
- the method of detection of the compounds may include, but are not necessarily limited to, fluorescence microscopy, laser-confocal microscopy, cross-polarization microscopy, optical imaging, nuclear scintigraphy, positron emission tomography, and single photon emission computed tomography.
- the imaging agent of the invention comprises a labeled cystine compound comprising a fluorescent label
- suitable methods of detection include fluorescence microscopy, laser-confocal microscopy, cross-polarization microscopy, and optical imaging.
- the fluorescent label is any moiety capable of detection either directly or indirectly in any of the suitable methods of detection.
- Preferred fluorescent labels include groups having an extensive delocalized electron system, e.g.
- cyanines merocyanines, indocyanines, phthalocyanines, naphthalocyanines, triphenylmethines, porphyrins, pyrilium dyes, thiapyrilium dyes, squarylium dyes, croconium dyes, azulenium dyes, indoanilines, benzophenoxazinium dyes, benzothiaphenothiazinium dyes, anthraquinones, napthoquinones, indathrenes, phthaloylacridones, trisphenoquinones, azo dyes, intramolecular and intermolecular charge-transfer dyes and dye complexes, tropones, tetrazines, ⁇ (dithiolene) complexes, ⁇ zsfbenzene-dithiolate) complexes, iodoaniline dyes, t ⁇ O-dithiolene) complexes.
- Fluorescent proteins such as green fluorescent protein (GFP) and modifications of GFP that have different absorption/emission properties are also useful.
- Complexes of certain rare earth metals e.g., europium, samarium, terbium or dysprosium
- fluorescent nanocrystals Quantum dots.
- One or more of the imaging agents of the invention comprises labeled cystine compounds that comprise fluorescent labels where labeling occurs at the amine groups of the cystine.
- Non-limiting examples include: green fluorescent cystine molecules such as DiBodipy(FL)-Cystine and MonoBodipy(FL)-Cystine; red fluorescent cystine molecules such as DiBodipy(650)-Cystine and MonoBodipy(650)- Cystine.
- Fluorescent labels such as Bodipy fluorescent dyes, may be particularly suited for optical in vivo imaging and in vitro detection of cellular oxidative stress.
- a particular labeled cystine compound comprising a fluorescent label has structure IV:
- MBC MonoBodipyCystine
- Conjugation of a fluorescent label to cystine can be carried out by synthetic chemistry techniques well known to the skilled person.
- imaging agents labeled comprising a fluorescent label suitable for optical imaging and their preparation are reviewed by Licha in "Contrast Agents for Optical Imaging” in Optical, Ultrasound, X-Ray and Radiopharmaceutical Imaing, Springer 2002, Krause Ed.
- the imaging agent of the invention comprises a labeled cystine compound comprising a radioisotopic label
- suitable methods of detection include nuclear scintigraphy, positron emission tomography and single photon emission tomography.
- Preferred radioisotopic labels for positron emission tomography include, 11 C, 13 N, 15 O, 17 F, 18 F, 75 Br, 76 Br or 124 I.
- Most preferred positron-emitting radioactive non-metals are 11 C, 13 N, 18 F and 124 I, especially 11 C and 18 F, most especially 18 F.
- a preferred radioisotopic labels for single photon emission tomography is a gamma-emitting radioactive halogen.
- Preferred gamma-emitting radioactive halogens are 123 I, 131 I and 77 Br, especially 123 I.
- One or more of the imaging agents of the invention comprises prothstetic groups to enable radioisotope labeling where labeling occurs at the amine groups of the cystine.
- Non-limiting examples include: aminoxy(AO) derivatives of cystine that are labeled with F- fluorobenzaldehyde.
- the labeled substrates of the invention for the cystine/glutamate transporter may also be used to introduce labeled compounds, such as, but not limited to, a cystine substrate labeled with 131 I, into a target for therapeutic purposes.
- the imaging agent of the invention comprises a labeled cystine compound having structure II:
- R 1 comprises a radioisotopic label
- R 2 is hydrogen when the dotted bond is a single bond
- R 2 is absent when the dotted bond is a double bond.
- This compound is also referred to herein as monoAO-[ 18 ⁇ F]-FBA-Cystine [0037]
- 18 F-labeled cystine compounds include, but are not limited to: [ 18 F]fluoroethyl-cystine or [ 18 F]FE-cystine having the structure V"
- Imaging agents of the invention wherein the labeled cysteine compound comprises a radioisotopic label are suitably prepared from precursor compounds.
- a "precursor compound” comprises a derivative of the labeled cystine compound, designed so that chemical reaction with a convenient chemical form of the radioisotopic label occurs site-specifically; can be conducted in the minimum number of steps (ideally a single step); and without the need for significant purification (ideally no further purification), to give the desired labeled cysteine compound.
- precursors are synthetic and can conveniently be obtained in good chemical purity.
- the precursor compound may optionally comprise one or more protecting groups for certain functional groups. Protecting groups are well-known to those skilled in the art and are described in detail in 'Protective Groups in Organic Synthesis', Theorodora W.
- the precursor compound is ideally provided in sterile, apyrogenic form.
- the precursor compound can accordingly be used for the preparation of a pharmaceutical composition comprising the imaging agent together with a biocompatible carrier suitable for mammalian administration.
- the precursor compound is also suitable for inclusion as a component in a kit for the preparation of such a pharmaceutical composition.
- the precursor compound is provided in solution and as part of a kit or of a cassette designed for use in an automated synthesis apparatus.
- a particular precursor compound of the invention has structure X:
- R" and R' is a precursor group and the other of R" and R'" is hydrogen, and wherein said precursor compound optionally comprises protecting groups on one or more of the hydroxy, carbonyl and amine functional groups.
- a "precursor group” is a chemical group which reacts with a convenient chemical form of the radioisotopic label to incorporate the radioisotopic label site-specifically Suitable such precursor groups are now described in the context of particular radioisotopic labels.
- the radiofluorine atom may form part of a fluoroalkyl or fluoroalkoxy group, since alkyl fluorides are resistant to in vivo metabolism.
- the radiofluorine atom may be attached via a direct covalent bond to an aromatic ring.
- Radiofluorination may be carried out via direct labelling using the reaction of 18 F-fluoride with a precursor group that comprises a good leaving group, such as bromide, mesylate or tosylate.
- F can also be introduced by O-alkylation of a hydroxyl precursor group with [ 18 F] -fluoroalkyl bromide, [ 18 F]-fluoroalkyl mesylate or [ 18 F]-fluoroalkyl tosylate.
- Nucleophilic displacement from an aryl diazonium salt, aryl nitro compound or an aryl quaternary ammonium salt are suitable routes to aryl- F derivatives.
- F- labelling techniques may be found in "Chemistry of Fluorine-18 Radiopharmaceuticals” by Snyder and Kilbourn in Handbook of Radiopharmaceuticals, Ed. MJ. Welch and CS. Redvanly (2003, John Wiley and Sons).
- the precursor compound preferably comprises a precursor group that is: an aryl iodide or bromide (to permit radioiodine exchange); an activated precursor compound aryl ring (e.g. a phenol group); an organometallic precursor compound (e.g. trialkyltin, trialkylsilyl or organoboron compound); or an organic precursor compound such as triazenes or a good leaving group for nucleophilic substitution such as an iodonium salt.
- a precursor group that is: an aryl iodide or bromide (to permit radioiodine exchange); an activated precursor compound aryl ring (e.g. a phenol group); an organometallic precursor compound (e.g. trialkyltin, trialkylsilyl or organoboron compound); or an organic precursor compound such as triazenes or a good leaving group for nucleophilic substitution such as an iodonium salt.
- Suitable boronate ester organoboron compounds and their preparation are described by Kabalka et al (Nucl. Med. Biol., 2002; 29: 841-843 and 2003; 30: 369-373).
- Suitable organotrifluoroborates and their preparation are described by Kabalka et al (Nucl. Med. Biol., 2004; 31 : 935-938).
- Preferred precursor compounds for radioiodination comprise an organometallic precursor group, most preferably a trialkyltin.
- Radiobromination may be carried out using the precursor compounds described above for radioiodinated compounds. Kabalka and Varma have reviewed various methods for the synthesis of radiohalogenated compounds, including radiobrominated compounds (Tetrahedron 1989; 45(21): 6601-21). [0045] A H C-labeled imaging agent of the invention may be synthesised in a straightforward manner by reacting a precursor compound which is a desmethylated version of the imaging agent with 11 C methyl iodide.
- 1 1 C it is also possible to incorporate 1 1 C by reacting a Grignard reagent of the particular hydrocarbon of the desired imaging agent with [ 11 C]CO 2 to obtain a 11 C reagent that reacts with an amine precursor group in the precursor compound to result in the n C-labelled imaging agent of interest.
- a Grignard reagent comprises a magnesium halide precursor group at the desired site of radiolabelling.
- the disulfide bond of structure I is reduced inside the cell and will no longer be a substrate for the xc- transporter so that the agent cannot leave the cell via this route.
- a fluorescent or radioisotope label (R) will be conjugated to an amine so that the resulting reduced intracellular agent will not be metabolized. The resulting agent therefore enters and is retained in the cells with an activated xc- transporter.
- a single- labeled cysteine compound has an H at the R' position, but an additional label may also be appended to the second amine at the R' position.
- cystine is labeled at one or two of the free amines. When imported into the cell via the transporter, it will be reduced to labeled cysteine, which is no longer a substrate of the transporter and therefore could not be expelled from the cell by the same mechanism of entry. Because the amine is conjugated to the label, the labeled cysteine will not be a substrate for glutathione synthesis or protein synthesis and the label will be trapped in the cells.
- the xc- transporter In a wide variety of human tissues and cells examined, the xc- transporter is predominantly expressed brain, but also in pancreas and in cultured cell lines. The xc- transporter expression is very low in most tissues, but can be upregulated under conditions of oxidative stress and when cells are grown in culture. The xc- transporter is induced under a number of conditions, including apoptotic stimuli, oxidative stress, inflammation, cystine deprivation and chemotherapy resistance.
- the imaging agent of the invention as suitably and preferably defined above is preferably provided as a pharmaceutical composition comprising the imaging agent together with a biocompatible carrier in a form suitable for mammalian administration.
- the pharmaceutical composition forms a separate aspect of the invention.
- the "biocompatible carrier” is a fluid, especially a liquid, in which the imaging agent is suspended or dissolved, such that the pharmaceutical composition is physiologically tolerable, i.e. can be administered to the mammalian body without toxicity or undue discomfort.
- the biocompatible carrier is suitably an injectable carrier liquid such as sterile, pyrogen- free water for injection; an aqueous solution such as saline (which may advantageously be balanced so that the final product for injection is either isotonic or not hypotonic); an aqueous solution of one or more tonicity-adjusting substances (e.g. salts of plasma cations with biocompatible counterions), sugars (e.g. glucose or sucrose), sugar alcohols (e.g.
- the biocompatible carrier may also comprise biocompatible organic solvents such as ethanol. Such organic solvents are useful to solubilise more lipophilic compounds or formulations.
- the biocompatible carrier is pyrogen- free water for injection, isotonic saline or an aqueous ethanol solution.
- the pH of the biocompatible carrier for intravenous injection is suitably in the range 4.0 to 10.5.
- these agents that are taken up into cells via the cystine/glutamate antiporter may be used to image cellular oxidative stress in vivo, including without limitation, the imaging of pathologies or conditions that include cellular oxidative stress. Imaging applications that would benefit from these agents include, but are not limited to, chemotherapy treatment monitoring, ischemia/stroke, inflammation, traumatic brain injury and organ transplant monitoring.
- the present invention relates to a method for imaging a biological sample having a cystine/glutamate transporter, said method comprising:
- imaging agent using fluorescence microscopy, laser-confocal microscopy, cross-polarization microscopy, optical imaging, nuclear scintigraphy, positron emission tomography, or single photon emission computed tomography.
- the biological sample is preferably an in vitro cell culture.
- the step of introducing the imaging agent of the invention is carried out by adding the imaging agent suspended in a suitable buffer to the in vitro cell culture, followed by incubation for a defined period of time, preferably at physiological temperature.
- the detecting step is subsequently carried out using fluorescence microscopy, laser- confocal microscopy, cross-polarization microscopy techniques well known to the skilled person. See for example "Principles of Fluorescence Microscopy" Third Edition 2006, Lakowitz, Ed.
- the biological sample is an intact mammalian subject.
- the imaging agent is preferably administered as the pharmaceutical composition of the invention.
- administration to said subject is carried out parenterally, and most preferably intravenously.
- the intravenous route represents the most efficient way to deliver the imaging agent throughout the body of the subject. Intravenous administration does not represent a substantial physical intervention or a substantial health risk.
- the imaging agent of the invention is preferably administered as the pharmaceutical composition of the invention, as defined herein.
- the detecting step of the method of imaging is preferably carried out using positron emission tomography or single photon emission tomography, preferably wherein said imaging agent comprises a cysteine compound of structure II or III as defined above.
- said imaging agent comprises a labeled cystine compound of structure III as defined above.
- the present invention provides a method for detecting oxidative stress in cells comprising:
- said detecting is carried out in apoptotic cells.
- Human Jurkat cells were cultured with or without IuM staurosporine for 16 hours in an in vitro assay for apoptotic cells and stained with propidium iodide(PI) and Cy5-AnnexinV (Annexin). Typically -30-50% of cells were found to be in some stage of apoptosis or necrosis. Flow cytometry was used to identify population of cells defined as normal (PI and Annexin negative), early apoptotic (PI negative, Annexin positive), and late apoptosis and necrosis (PI and Annexin positive). The results are shown in FIGs. 2A and 2B.
- FIG 2A is a forward scatter/side scatter plot showing some late apoptotic cells (light gray) with a different granularity than non- apoptotic cells (darker gray).
- AnnexinV and PI negative cells are shown in quadrant D3 and represent non-apoptotic cells.
- Cells in quadrant D4 indicate early apoptotic cells that are positive for AnnexinV staining, but negative for PI staining.
- Cells in quadrant D2 indicate late apoptotic and necrotic cells that are positive with both AnnexinV and PI staining.
- Jurkat cells were incubated with (FIG 3B) and without (FIG 3A) 1 ⁇ M staurosprine (STN) for 16-18 hours, stained with Annexin V-Cy5 and propidium iodide, and incubated with DBC for 30 minutes.
- DBC is a commercially available product from Invitrogen (catalog #B20340), which is sold for the purpose of reversible thiol labeling of nucleotides, proteins and cells via a disulfide exchange reaction at acidic conditions. DBC has the structure shown below.
- FIG 3D cells were categorized as normal, early apoptotic, or late apoptotic as in Example 1 , and the percentage of cells with increased DBC staining was recorded for each category.
- Early and late apoptotic cells were labeled with DBC, but this was only inhibited by sasz in the early apoptotic cells.
- DBC does label early apoptotic cells via activity of the cystine-glutamate transporter and acts as a transporter substrate even though it is conjugated to two fluorophores via the amines of cystine. This data shows that the uptake seen in early apoptotic cells with DBC is dependent upon the xc- transporter, rather than some other mechanism.
- the apoptotic Jurkat cell uptake of DBC shows not only that the xc- transporter is activated m cells undergoing oxidative stress (and in this case apoptosis), but also that the xc- transporter is promiscuous enough to allow this substrate (cystine) with amine appended green fluorophores (bodipyFL) into the cell.
- a labeled cystine compound was synthesized with a red fluorophore (bodipy650) to further determine the promiscuity of the transporter.
- DBC-650 did not appear to label apoptotic cells any more than normal cells, as shown in FIGs.
- the bodipy-650 fluorophore and associated linker are larger than that of DBC (FIG 4A), and therefore size represents one limitation of what groups are appended to the amines while retaining the ability to pass through the transporter.
- MBC MonoBodipyCystine
- Jurkat cells were incubated with 1 ⁇ M STN for 16-18 hours, stained with Annexin V-Cy5 and propidium iodide, and incubated with MBC for 30 minutes, without (FIG 5A) and with (FIG 5B) the addition of the cystine/glutamate inhibitor (sasz).
- FIG 5 A MBC labeling is visualized as a subpopulation of cells with a high intensity fluorescence shift, which corresponds well with both early and late apoptotic cells (FIG 5C). No intermediate population was evident, suggesting that MBC labeled early apoptotic cells with greater intensity than DBC.
- FIG 5B the addition of sulfasalazine results in fewer labeled cells and with less of a shift in fluorescence intensity.
- This population correlated well with late apoptotic cells and did not correlate well with early apoptotic cells (FIG 5C).
- MBC is smaller than DBC (with only one label) and more closely resembles the natural transporter substrate cystine; resulting in an agent with improved performance for labeling early apoptotic cells.
- MBC there is less background uptake in normal cells, without sacrificing the magnitude of increase in fluorescence intensity for apoptotic cells, even though there is one less fluorophore per cystine compound.
- DBDCystathionine DiBodipy Cystathionine
- Bodipy-FL C5 which is available commercially from Invitrogen that has the following structure.
- FIG 6A shows a comparison of Bodipy staining of normal, early apoptotic and late apoptotic Jurkat cells with DBC, DBC(650), DBCystathionine, MBC and the negative control (Bodipy-FL C5).
- DBC, DBCystathionine, and MBC stain early apoptotic cells
- DBC(650) only stains late apoptotic cells
- the negative control does not show any shift in fluorescence for apoptotic cells.
- DBC, DBCystathionine, and MBC are all equivalent labels for apoptotic cells.
- FIG 6B the fold shift in fluorescence intensity of the early apoptotic cells is compared to normal cells for each agent. It is clear that MBC provides more intense staining than the other fluorescent agents used.
- labeled cystine compounds serve as more appropriate substrates than larger labeled cystine compounds such as DBC-650. Therefore, labeled cystine compounds were synthesized with a single amine conjugated to [ F]aminoxy- fluorobenzadehyde (monoAO-[ 18 F]-FBA-Cystine), and may be used for PET imaging applications. First the aminoxy-cystine precursor (monoAO-cystine) was synthesized, with the following structure.
- [ 18 F]KF (4OmCLmL-I (1480 MBq.mL-1) in purified water) was obtained from either IBA Molecular (Albany, NY) or PETNET Solutions (Albany, NY) and used as received.
- the [ 18 F]fluoride was first immobilized on a Chromafix 30-PS-HCO3 anion exchange cartridge (ABX, Radeberg, Germany), then eluted into a drydown vessel with a 1 mL, 4:1 mixture of acetonitrile:distilled deionized water (ddH 2 O) containing Kryptofix K222 (376 g.mol-1, 8 mg, 2.13xlO "5 mol) and potassium carbonate (138.2 g.mol-1, 2.1 mg, 1.52xlO "5 mol).
- the solvent was removed under partial vacuum and a flow of nitrogen with gentle heating ( ⁇ 45 0 C) (-15 min).
- the source vial and anion exchange cartridge were then washed with 0.5mL of acetonitrile containing K222 (8 mg) and the reaction mixture again brought to dryness under partial vacuum and gentle heating (- 10 min).
- the reaction vessel was repressurized with nitrogen and the azeotropic drydown repeated twice with an additional 0.5mL of acetonitrile.
- This mixture was subsequently passed through a cation exchange cartridge (Waters SepPak Light Accell Plus CM), diluted to 10 mL with ddH2O, and loaded onto a reverse phase Cl 8 SepPak (Waters SepPak Plus C 18).
- CM Waters SepPak Light Accell Plus CM
- [ 18 F]FB-CyS (0.17mCi, 6.3 MBq) was isolated in a minimal amount of DMSO by first eluting the void volume (approx. 0.5mL) followed by collecting 250 to 300 ⁇ L of eluent in a separate flask. RP-HPLC analysis was performed on the isolated product in order to establish radiochemical and chemical purity. Typically, 10 ⁇ L of a O.l ⁇ Ci/ ⁇ L solution was injected for post formulation analysis. Isolated radiochemical yield was 3.6% (6.6% decay corrected from addition of [ 18 F]FBA) and radiochemical purity of 96.8%.
- Analytical HPLC conditions Analysis performed on an HP Agilent 1100 with a G1311A QuatPump, G1313A autoinjector with lOO ⁇ L syringe and 2.OmL seat capillary, Phenomenex Gemini Cl 8 column (4.6mm ⁇ 150mm), 5 ⁇ , IOOA (S/N 420477-10), G1316A column heater, G1315A DAD and Ramon Star GABI gamma- detector. 95:5 ddH 2 O:CH 3 CN with 0.05% TFA, Solvent B: CH 3 CN with 0.05% TFA. Gradient elution: 0 min. 0%B, 1 min. 15%B, lOmin. 31%B, 10.5 min.
- the monoAO-[ 18 F]-FBA-Cystine was used in an in vitro cell uptake assay, wherein monoAO-[ 18 F]-FBA-Cystine was incubated with cultured cells for 30 minutes and washed twice with saline before lysing the cells with IN NaOH and collection for analysis in a gamma counter.
- FIG 7, shows the uptake of monoAO-
- [ F]-FBA-Cystine in two different cell lines indicating differential basal expression and activity of the cystine/glutamate transporter in Jurkat and A549 cell lines.
- FIGs. 8A and 8B A pharmacokinetic profile of this molecule, which was formulated into 7% ethanol in saline, was established in normal mice and is shown in FIGs. 8A and 8B.
- the na ⁇ ve Balb/c mice were injected -15 uCi with 18 F-cystine and time points were taken at 5, 30, 120 min post injection.
- FIG 8A shows the biodistribution results in na ⁇ ve Balb-c mice, % of injected dose (%ID). Clearance from the body is largely due to renal excretion as shown by the profile of the kidney, bladder and urine.
- FIG 8B shows the %ID/gram of the same data shown in FIG 8A.
- FIG 8C shows a similar biodistribution results in %ID/gram from a study done in nude mice with A549 tumor xenografts. At 120 minute and 240 minute time points the radiotracer has cleared sufficiently to detect more %ID/gram in the tumor than in blood or other tissues (except kidney and bladder). Tumor to blood ratios are shown for each time point in FIG 8D. These results suggest that radiolabeled cystine compounds can be used for the detection of cystine/glutamate transporter activity in vivo.
- FIG 9 shows the stability of the mono AO- [ 18 F] -FB A-Cystine molecule over time in saline. As shown, there was not any change in the gamma-trace profile over a 4-hour period.
- FIG 10 shows the monoAO-[' 8 F]-FB A-Cystme in a PET image in a na ⁇ ve mouse at 60 minutes post injection, showing clearance primarily through the kidneys and bladder.
- the cystine may be labeled with 123 I- iodobenzaldehyde, which similar fluorobenzaldehyde, may be prepared by first adding [ 123 I]4-iodobenzaldehyde ([ 123 I]IBA) to a high recovery vial (2 mL, National Scientific) containing the AO-Cys , 2.5 mg). The reaction commences by dissolving the polypeptide in 0.5 mL of ddH 2 O and adding 8 ⁇ L of trifluoroacetic acid followed by the addition of [ 123 I]IBA in 0.5 mL of methanol.
- the vessel is capped, crimped, placed in a heating block and maintained at 60 0 C for 15 minutes; removing a small aliquot ( ⁇ 5 ⁇ L) for analytical HPLC analysis is done to assess the status of the reaction.
- [ 123 I]IB-Cystine is isolated and purified by semi-preparative HPLC.
- the HPLC fraction containing the product is further diluted (5:1) with ddH 2 O and the product subsequently immobilized on a tC18 Plus Sep Pak (Waters). Flushing the SepPak first with 5 mL of ddH 2 O then 30 mL of air gives the [ 123 I]IB-Cystine in a minimal amount of ethanol by first eluting the void volume (approx. 0.5mL) followed by collecting 250 to 300 ⁇ L of eluent in a separate flask. RP-HPLC analysis is then performed on the isolated product to establish radiochemical and chemical purity.
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Epidemiology (AREA)
- Veterinary Medicine (AREA)
- Public Health (AREA)
- General Health & Medical Sciences (AREA)
- Animal Behavior & Ethology (AREA)
- Biomedical Technology (AREA)
- Engineering & Computer Science (AREA)
- Biophysics (AREA)
- Pharmacology & Pharmacy (AREA)
- Optics & Photonics (AREA)
- Medicinal Chemistry (AREA)
- Physics & Mathematics (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Molecular Biology (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
Abstract
Description
Claims
Priority Applications (9)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
BRPI1015323-3A BRPI1015323A2 (en) | 2009-04-27 | 2010-04-27 | imaging agent, methods for imaging a biological sample, and for detecting oxidative stress in cells, pharmaceutical composition, and precursor compound for the synthesis of an imaging agent |
CN201080019270.7A CN102413844B (en) | 2009-04-27 | 2010-04-27 | Labeled molecular imaging agents, methods of making and methods of use |
JP2012506533A JP5709841B2 (en) | 2009-04-27 | 2010-04-27 | Labeled molecular imaging agent, production method and use method |
MX2011011351A MX336722B (en) | 2009-04-27 | 2010-04-27 | Labeled molecular imaging agents, methods of making and methods of use. |
KR1020117025306A KR101686264B1 (en) | 2009-04-27 | 2010-04-27 | Labeled molecular imaging agents, methods of making and methods of use |
AU2010243678A AU2010243678B2 (en) | 2009-04-27 | 2010-04-27 | Labeled molecular imaging agents, methods of making and methods of use |
EP10718133.1A EP2424573B1 (en) | 2009-04-27 | 2010-04-27 | Labeled molecular imaging agents, methods of making and methods of use |
RU2011140242/15A RU2523411C2 (en) | 2009-04-27 | 2010-04-27 | Tagged molecular visualising agents, methods for preparing and methods for using |
CA2759000A CA2759000C (en) | 2009-04-27 | 2010-04-27 | Labeled molecular imaging agents, methods of making and methods of use |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US12/430,573 | 2009-04-27 | ||
US12/430,573 US8834839B2 (en) | 2009-04-27 | 2009-04-27 | Labeled molecular imaging agents, methods of making and methods of use |
Publications (2)
Publication Number | Publication Date |
---|---|
WO2010125068A2 true WO2010125068A2 (en) | 2010-11-04 |
WO2010125068A3 WO2010125068A3 (en) | 2011-01-20 |
Family
ID=42790740
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/EP2010/055629 WO2010125068A2 (en) | 2009-04-27 | 2010-04-27 | Labeled molecular imaging agents, methods of making and methods of use |
Country Status (11)
Country | Link |
---|---|
US (1) | US8834839B2 (en) |
EP (1) | EP2424573B1 (en) |
JP (1) | JP5709841B2 (en) |
KR (1) | KR101686264B1 (en) |
CN (1) | CN102413844B (en) |
AU (1) | AU2010243678B2 (en) |
BR (1) | BRPI1015323A2 (en) |
CA (1) | CA2759000C (en) |
MX (1) | MX336722B (en) |
RU (1) | RU2523411C2 (en) |
WO (1) | WO2010125068A2 (en) |
Cited By (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP2520556A1 (en) | 2011-05-03 | 2012-11-07 | Bayer Pharma Aktiengesellschaft | Radiolabeled amino acids for diagnostic imaging |
JP2014526473A (en) * | 2011-09-08 | 2014-10-06 | ウェスタン ユニバーシティ オブ ヘルス サイエンス | Targeted liposomes in cancer therapy |
JP2014526505A (en) * | 2011-09-16 | 2014-10-06 | ゼネラル・エレクトリック・カンパニイ | Labeled molecular imaging agent and method of use |
US20150202332A1 (en) * | 2014-01-23 | 2015-07-23 | General Electric Company | Labeled molecular imaging agents and methods of use |
RU2717308C1 (en) * | 2019-06-25 | 2020-03-20 | Федеральное государственное бюджетное научное учреждение "Иркутский научный центр хирургии и травматологии" | Fluorescent sensor for detecting lysosomes in vitro |
Families Citing this family (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2014183110A1 (en) * | 2013-05-10 | 2014-11-13 | The University Of Montana | Methods for the detection of brain injury |
US9468693B2 (en) * | 2014-01-23 | 2016-10-18 | General Electric Company | Labeled molecular imaging agents and methods of use |
CN104629752B (en) * | 2015-01-28 | 2016-05-04 | 东南大学 | A kind of fluorescent molecular probe preparation method who identifies apoptotic cell |
Family Cites Families (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5171563A (en) * | 1988-09-30 | 1992-12-15 | Neorx Corporation | Cleavable linkers for the reduction of non-target organ retention of immunoconjugates |
AU9375698A (en) * | 1997-09-03 | 1999-03-22 | Immunomedics Inc. | Fluorination of proteins and peptides for f-18 positron emission tomography |
IL147812A0 (en) * | 2001-03-16 | 2002-08-14 | N S T Neurosurvival Technologi | Method for targeting chemical compounds to cells and pharmaceutical compositions used therein |
US7211240B2 (en) * | 2002-03-01 | 2007-05-01 | Bracco International B.V. | Multivalent constructs for therapeutic and diagnostic applications |
US7666979B2 (en) | 2002-03-01 | 2010-02-23 | Bracco International B.V. | Methods for preparing multivalent constructs for therapeutic and diagnostic applications and methods of preparing the same |
GB0420344D0 (en) * | 2004-09-14 | 2004-10-13 | Amersham Plc | Diagnostic compounds |
-
2009
- 2009-04-27 US US12/430,573 patent/US8834839B2/en not_active Expired - Fee Related
-
2010
- 2010-04-27 CN CN201080019270.7A patent/CN102413844B/en not_active Expired - Fee Related
- 2010-04-27 WO PCT/EP2010/055629 patent/WO2010125068A2/en active Application Filing
- 2010-04-27 MX MX2011011351A patent/MX336722B/en unknown
- 2010-04-27 KR KR1020117025306A patent/KR101686264B1/en active IP Right Grant
- 2010-04-27 CA CA2759000A patent/CA2759000C/en not_active Expired - Fee Related
- 2010-04-27 AU AU2010243678A patent/AU2010243678B2/en not_active Ceased
- 2010-04-27 BR BRPI1015323-3A patent/BRPI1015323A2/en not_active Application Discontinuation
- 2010-04-27 JP JP2012506533A patent/JP5709841B2/en not_active Expired - Fee Related
- 2010-04-27 RU RU2011140242/15A patent/RU2523411C2/en active
- 2010-04-27 EP EP10718133.1A patent/EP2424573B1/en not_active Not-in-force
Non-Patent Citations (6)
Title |
---|
BOLTON, J. LAB. COMP. RADIOPHARM., vol. 45, 2002, pages 485 - 528 |
HWANG ET AL., J NEUROCHEM, vol. 35, 1980, pages 417 - 424 |
KABALKA ET AL., NUCL. MED. BIOL., vol. 29, 2002, pages 841 - 843 |
KABALKA ET AL., NUCL. MED. BIOL., vol. 31, 2004, pages 935 - 938 |
TETRAHEDRON, vol. 45, no. 21, 1989, pages 6601 - 21 |
TUBIS; ENDOW, INT J APPL RAD ISOTOP, vol. 19, 1968, pages 835 - 840 |
Cited By (8)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP2520556A1 (en) | 2011-05-03 | 2012-11-07 | Bayer Pharma Aktiengesellschaft | Radiolabeled amino acids for diagnostic imaging |
WO2012150220A1 (en) | 2011-05-03 | 2012-11-08 | Bayer Pharma Aktiengesellschaft | Radiolabeled amino acids for diagnostic imaging |
JP2014526473A (en) * | 2011-09-08 | 2014-10-06 | ウェスタン ユニバーシティ オブ ヘルス サイエンス | Targeted liposomes in cancer therapy |
US9808425B2 (en) | 2011-09-08 | 2017-11-07 | Western University Of Health Sciences | Targeted liposomes in cancer therapy |
JP2014526505A (en) * | 2011-09-16 | 2014-10-06 | ゼネラル・エレクトリック・カンパニイ | Labeled molecular imaging agent and method of use |
US20150202332A1 (en) * | 2014-01-23 | 2015-07-23 | General Electric Company | Labeled molecular imaging agents and methods of use |
US9468692B2 (en) * | 2014-01-23 | 2016-10-18 | General Electric Company | Labeled molecular imaging agents and methods of use |
RU2717308C1 (en) * | 2019-06-25 | 2020-03-20 | Федеральное государственное бюджетное научное учреждение "Иркутский научный центр хирургии и травматологии" | Fluorescent sensor for detecting lysosomes in vitro |
Also Published As
Publication number | Publication date |
---|---|
EP2424573A2 (en) | 2012-03-07 |
KR101686264B1 (en) | 2016-12-13 |
JP5709841B2 (en) | 2015-04-30 |
WO2010125068A3 (en) | 2011-01-20 |
CA2759000C (en) | 2017-08-08 |
AU2010243678A1 (en) | 2011-10-27 |
JP2012525328A (en) | 2012-10-22 |
CA2759000A1 (en) | 2010-11-04 |
BRPI1015323A2 (en) | 2020-10-06 |
MX336722B (en) | 2016-01-28 |
KR20120015308A (en) | 2012-02-21 |
US8834839B2 (en) | 2014-09-16 |
US20100272641A1 (en) | 2010-10-28 |
AU2010243678B2 (en) | 2015-11-05 |
CN102413844A (en) | 2012-04-11 |
MX2011011351A (en) | 2011-11-18 |
RU2523411C2 (en) | 2014-07-20 |
EP2424573B1 (en) | 2019-01-16 |
CN102413844B (en) | 2014-12-10 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
CA2759000C (en) | Labeled molecular imaging agents, methods of making and methods of use | |
JP2006524250A (en) | Monitoring the metabolic uptake into the bloodstream and tissues by using radioisotope-labeled alkanoic acids | |
US10259781B2 (en) | Imaging agents | |
Lindner et al. | Radiosynthesis of [18F] SiFA lin-TATE for clinical neuroendocrine tumor positron emission tomography | |
JP2013514326A (en) | Labeled integrin binding agent | |
US9789211B2 (en) | Methods and compositions for positron emission tomography myocardial perfusion imaging | |
US11384106B2 (en) | Metal tricarbonyl complexes comprising substituted iminodiactic acid ligands and uses as radioisotope tracers | |
ES2374743T3 (en) | STEREOISOMERS OF FATTY ACID ANALOGS FOR DIAGNOSTIC IMAGE. | |
CN112105393A (en) | Styrylbenzothiazole derivatives and use in imaging | |
RU2543342C2 (en) | Rhenium-188 composition for treating hepatic cancer in individuals, and method for preparing this composition | |
EP2755694B1 (en) | 18f-labeled agents for imaging cystine/glutamate antiporter and oxidative stress | |
JP2010529173A (en) | Measurement of nerve activity | |
McDonagh et al. | Development of a multi faceted platform containing a tetrazine, fluorophore and chelator: Synthesis, characterization, radiolabeling, and immuno-SPECT imaging | |
Lu et al. | Design, synthesis, and biological evaluation of a 99m Tc-labeled small-molecule tracer for PD-L1 imaging | |
Gower-Fry et al. | Silicon–Fluoride Acceptors (SiFA) for 18F-Radiolabeling: From Bench to Bedside | |
Verbruggen | Technetium-99m radiopharmaceuticals: Current situation and perspectives | |
JP5128293B2 (en) | Bridgehead labeled compounds and methods of use thereof | |
KR20130121830A (en) | Isotopic carbon choline analogs | |
Kulkarni | I-123 labeled radiotracers in cardiovascular nuclear medicine | |
Garg et al. | F-18 based radiopharmaceutical and automation of synthesis: New F-18 radiopharmaceuticals | |
Welling et al. | Optimized delivery of an anti-amyloid VHH-2H heavy chain antibody fragment to the mouse brain |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
WWE | Wipo information: entry into national phase |
Ref document number: 201080019270.7 Country of ref document: CN |
|
121 | Ep: the epo has been informed by wipo that ep was designated in this application |
Ref document number: 10718133 Country of ref document: EP Kind code of ref document: A2 |
|
WWE | Wipo information: entry into national phase |
Ref document number: 7352/DELNP/2011 Country of ref document: IN |
|
WWE | Wipo information: entry into national phase |
Ref document number: 2012506533 Country of ref document: JP Ref document number: 2010718133 Country of ref document: EP |
|
WWE | Wipo information: entry into national phase |
Ref document number: 2759000 Country of ref document: CA |
|
ENP | Entry into the national phase |
Ref document number: 20117025306 Country of ref document: KR Kind code of ref document: A |
|
WWE | Wipo information: entry into national phase |
Ref document number: MX/A/2011/011351 Country of ref document: MX |
|
ENP | Entry into the national phase |
Ref document number: 2010243678 Country of ref document: AU Date of ref document: 20100427 Kind code of ref document: A |
|
NENP | Non-entry into the national phase |
Ref country code: DE |
|
ENP | Entry into the national phase |
Ref document number: 2011140242 Country of ref document: RU Kind code of ref document: A |
|
REG | Reference to national code |
Ref country code: BR Ref legal event code: B01A Ref document number: PI1015323 Country of ref document: BR |
|
ENP | Entry into the national phase |
Ref document number: PI1015323 Country of ref document: BR Kind code of ref document: A2 Effective date: 20111021 |