AU5984094A - Immunoreactive reagents employing dihydrofolate reductase - Google Patents
Immunoreactive reagents employing dihydrofolate reductaseInfo
- Publication number
- AU5984094A AU5984094A AU59840/94A AU5984094A AU5984094A AU 5984094 A AU5984094 A AU 5984094A AU 59840/94 A AU59840/94 A AU 59840/94A AU 5984094 A AU5984094 A AU 5984094A AU 5984094 A AU5984094 A AU 5984094A
- Authority
- AU
- Australia
- Prior art keywords
- residue
- group
- reagent
- dhfr
- groups
- 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.)
- Abandoned
Links
- 108010022394 Threonine synthase Proteins 0.000 title claims description 197
- 102000004419 dihydrofolate reductase Human genes 0.000 title claims description 197
- 239000003153 chemical reaction reagent Substances 0.000 title claims description 51
- 108090000623 proteins and genes Proteins 0.000 claims description 96
- 102000004169 proteins and genes Human genes 0.000 claims description 94
- 239000003446 ligand Substances 0.000 claims description 78
- 125000005647 linker group Chemical group 0.000 claims description 77
- 230000027455 binding Effects 0.000 claims description 68
- 238000000034 method Methods 0.000 claims description 61
- 230000002285 radioactive effect Effects 0.000 claims description 51
- 239000002738 chelating agent Substances 0.000 claims description 49
- -1 90 Y Chemical compound 0.000 claims description 46
- 239000000463 material Substances 0.000 claims description 46
- 239000000126 substance Substances 0.000 claims description 32
- IEDVJHCEMCRBQM-UHFFFAOYSA-N trimethoprim Chemical class COC1=C(OC)C(OC)=CC(CC=2C(=NC(N)=NC=2)N)=C1 IEDVJHCEMCRBQM-UHFFFAOYSA-N 0.000 claims description 30
- 230000008685 targeting Effects 0.000 claims description 28
- 102000003781 Inhibitor of growth protein 1 Human genes 0.000 claims description 27
- 108090000191 Inhibitor of growth protein 1 Proteins 0.000 claims description 27
- FBOZXECLQNJBKD-ZDUSSCGKSA-N L-methotrexate Chemical class C=1N=C2N=C(N)N=C(N)C2=NC=1CN(C)C1=CC=C(C(=O)N[C@@H](CCC(O)=O)C(O)=O)C=C1 FBOZXECLQNJBKD-ZDUSSCGKSA-N 0.000 claims description 25
- 238000002059 diagnostic imaging Methods 0.000 claims description 24
- 206010028980 Neoplasm Diseases 0.000 claims description 21
- 239000003431 cross linking reagent Substances 0.000 claims description 19
- 239000002243 precursor Substances 0.000 claims description 18
- 102000008394 Immunoglobulin Fragments Human genes 0.000 claims description 17
- 108010021625 Immunoglobulin Fragments Proteins 0.000 claims description 17
- 150000001875 compounds Chemical class 0.000 claims description 17
- 102000037865 fusion proteins Human genes 0.000 claims description 16
- 108020001507 fusion proteins Proteins 0.000 claims description 16
- 241000124008 Mammalia Species 0.000 claims description 15
- 125000003396 thiol group Chemical group [H]S* 0.000 claims description 14
- 239000002253 acid Substances 0.000 claims description 13
- 125000000524 functional group Chemical group 0.000 claims description 11
- 229910052751 metal Inorganic materials 0.000 claims description 11
- 239000002184 metal Substances 0.000 claims description 11
- 241000588724 Escherichia coli Species 0.000 claims description 10
- 125000003277 amino group Chemical group 0.000 claims description 9
- CNHYKKNIIGEXAY-UHFFFAOYSA-N thiolan-2-imine Chemical compound N=C1CCCS1 CNHYKKNIIGEXAY-UHFFFAOYSA-N 0.000 claims description 7
- 125000002887 hydroxy group Chemical group [H]O* 0.000 claims description 6
- FLCQLSRLQIPNLM-UHFFFAOYSA-N (2,5-dioxopyrrolidin-1-yl) 2-acetylsulfanylacetate Chemical compound CC(=O)SCC(=O)ON1C(=O)CCC1=O FLCQLSRLQIPNLM-UHFFFAOYSA-N 0.000 claims description 5
- VHYRLCJMMJQUBY-UHFFFAOYSA-N 1-[4-[4-(2,5-dioxopyrrol-1-yl)phenyl]butanoyloxy]-2,5-dioxopyrrolidine-3-sulfonic acid Chemical compound O=C1C(S(=O)(=O)O)CC(=O)N1OC(=O)CCCC1=CC=C(N2C(C=CC2=O)=O)C=C1 VHYRLCJMMJQUBY-UHFFFAOYSA-N 0.000 claims description 5
- 125000003729 nucleotide group Chemical group 0.000 claims description 5
- 239000013612 plasmid Substances 0.000 claims description 5
- FPKVOQKZMBDBKP-UHFFFAOYSA-N 1-[4-[(2,5-dioxopyrrol-1-yl)methyl]cyclohexanecarbonyl]oxy-2,5-dioxopyrrolidine-3-sulfonic acid Chemical compound O=C1C(S(=O)(=O)O)CC(=O)N1OC(=O)C1CCC(CN2C(C=CC2=O)=O)CC1 FPKVOQKZMBDBKP-UHFFFAOYSA-N 0.000 claims description 4
- 238000003384 imaging method Methods 0.000 claims description 4
- HHSGWIABCIVPJT-UHFFFAOYSA-M sodium;1-[4-[(2-iodoacetyl)amino]benzoyl]oxy-2,5-dioxopyrrolidine-3-sulfonate Chemical compound [Na+].O=C1C(S(=O)(=O)[O-])CC(=O)N1OC(=O)C1=CC=C(NC(=O)CI)C=C1 HHSGWIABCIVPJT-UHFFFAOYSA-M 0.000 claims description 4
- 239000003937 drug carrier Substances 0.000 claims description 3
- 238000004519 manufacturing process Methods 0.000 claims description 3
- 150000007942 carboxylates Chemical group 0.000 claims description 2
- 239000008194 pharmaceutical composition Substances 0.000 claims 2
- NZNMSOFKMUBTKW-UHFFFAOYSA-N cyclohexanecarboxylic acid Chemical compound OC(=O)C1CCCCC1 NZNMSOFKMUBTKW-UHFFFAOYSA-N 0.000 claims 1
- HJHVQCXHVMGZNC-JCJNLNMISA-M sodium;(2z)-2-[(3r,4s,5s,8s,9s,10s,11r,13r,14s,16s)-16-acetyloxy-3,11-dihydroxy-4,8,10,14-tetramethyl-2,3,4,5,6,7,9,11,12,13,15,16-dodecahydro-1h-cyclopenta[a]phenanthren-17-ylidene]-6-methylhept-5-enoate Chemical compound [Na+].O[C@@H]([C@@H]12)C[C@H]3\C(=C(/CCC=C(C)C)C([O-])=O)[C@@H](OC(C)=O)C[C@]3(C)[C@@]2(C)CC[C@@H]2[C@]1(C)CC[C@@H](O)[C@H]2C HJHVQCXHVMGZNC-JCJNLNMISA-M 0.000 claims 1
- 235000018102 proteins Nutrition 0.000 description 92
- 239000000243 solution Substances 0.000 description 51
- 239000000203 mixture Substances 0.000 description 47
- 102000005962 receptors Human genes 0.000 description 45
- 210000001519 tissue Anatomy 0.000 description 45
- 239000002953 phosphate buffered saline Substances 0.000 description 36
- 210000004027 cell Anatomy 0.000 description 35
- 238000006243 chemical reaction Methods 0.000 description 33
- 229960001082 trimethoprim Drugs 0.000 description 30
- 239000003795 chemical substances by application Substances 0.000 description 27
- 230000000694 effects Effects 0.000 description 27
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 23
- 239000000427 antigen Substances 0.000 description 22
- 108090000765 processed proteins & peptides Proteins 0.000 description 22
- 235000002639 sodium chloride Nutrition 0.000 description 22
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 description 21
- VWQVUPCCIRVNHF-OUBTZVSYSA-N Yttrium-90 Chemical compound [90Y] VWQVUPCCIRVNHF-OUBTZVSYSA-N 0.000 description 21
- 102000036639 antigens Human genes 0.000 description 21
- 108091007433 antigens Proteins 0.000 description 21
- 230000021615 conjugation Effects 0.000 description 21
- 239000000306 component Substances 0.000 description 20
- 239000011541 reaction mixture Substances 0.000 description 18
- 102000004190 Enzymes Human genes 0.000 description 17
- 108090000790 Enzymes Proteins 0.000 description 17
- 108091034117 Oligonucleotide Proteins 0.000 description 17
- 239000000872 buffer Substances 0.000 description 17
- 229940088598 enzyme Drugs 0.000 description 17
- 241000894007 species Species 0.000 description 17
- 239000000047 product Substances 0.000 description 16
- 235000001014 amino acid Nutrition 0.000 description 15
- 229940024606 amino acid Drugs 0.000 description 15
- 150000001413 amino acids Chemical class 0.000 description 15
- 229910021645 metal ion Inorganic materials 0.000 description 15
- 238000002360 preparation method Methods 0.000 description 15
- 239000003814 drug Substances 0.000 description 14
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 14
- 108091026890 Coding region Proteins 0.000 description 13
- 108090000144 Human Proteins Proteins 0.000 description 13
- 102000003839 Human Proteins Human genes 0.000 description 13
- 230000000295 complement effect Effects 0.000 description 13
- 229940079593 drug Drugs 0.000 description 13
- 239000011347 resin Substances 0.000 description 13
- 229920005989 resin Polymers 0.000 description 13
- 239000011780 sodium chloride Substances 0.000 description 13
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 12
- XJLXINKUBYWONI-NNYOXOHSSA-O NADP(+) Chemical compound NC(=O)C1=CC=C[N+]([C@H]2[C@@H]([C@H](O)[C@@H](COP(O)(=O)OP(O)(=O)OC[C@@H]3[C@H]([C@@H](OP(O)(O)=O)[C@@H](O3)N3C4=NC=NC(N)=C4N=C3)O)O2)O)=C1 XJLXINKUBYWONI-NNYOXOHSSA-O 0.000 description 12
- QTBSBXVTEAMEQO-UHFFFAOYSA-N acetic acid Substances CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 12
- 125000000217 alkyl group Chemical group 0.000 description 12
- 125000004432 carbon atom Chemical group C* 0.000 description 12
- 229960000485 methotrexate Drugs 0.000 description 12
- 230000015572 biosynthetic process Effects 0.000 description 11
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 description 11
- 238000002156 mixing Methods 0.000 description 11
- 230000004048 modification Effects 0.000 description 11
- 238000012986 modification Methods 0.000 description 11
- 229910052757 nitrogen Inorganic materials 0.000 description 11
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical group N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 10
- 239000013522 chelant Substances 0.000 description 10
- 239000004052 folic acid antagonist Substances 0.000 description 10
- 238000006722 reduction reaction Methods 0.000 description 10
- 229960002668 sodium chloride Drugs 0.000 description 10
- 239000002904 solvent Substances 0.000 description 10
- 230000001225 therapeutic effect Effects 0.000 description 10
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 9
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 9
- 238000005859 coupling reaction Methods 0.000 description 9
- 238000005755 formation reaction Methods 0.000 description 9
- 230000009467 reduction Effects 0.000 description 9
- 230000002829 reductive effect Effects 0.000 description 9
- 125000001424 substituent group Chemical group 0.000 description 9
- 229910052717 sulfur Inorganic materials 0.000 description 9
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Natural products NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 description 8
- 150000001412 amines Chemical class 0.000 description 8
- 238000003556 assay Methods 0.000 description 8
- 230000001580 bacterial effect Effects 0.000 description 8
- OZRNSSUDZOLUSN-LBPRGKRZSA-N dihydrofolic acid Chemical compound N=1C=2C(=O)NC(N)=NC=2NCC=1CNC1=CC=C(C(=O)N[C@@H](CCC(O)=O)C(O)=O)C=C1 OZRNSSUDZOLUSN-LBPRGKRZSA-N 0.000 description 8
- 125000005842 heteroatom Chemical group 0.000 description 8
- 238000007254 oxidation reaction Methods 0.000 description 8
- MSTNYGQPCMXVAQ-RYUDHWBXSA-N (6S)-5,6,7,8-tetrahydrofolic acid Chemical compound C([C@H]1CNC=2N=C(NC(=O)C=2N1)N)NC1=CC=C(C(=O)N[C@@H](CCC(O)=O)C(O)=O)C=C1 MSTNYGQPCMXVAQ-RYUDHWBXSA-N 0.000 description 7
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 7
- WHUUTDBJXJRKMK-VKHMYHEASA-N L-glutamic acid Chemical compound OC(=O)[C@@H](N)CCC(O)=O WHUUTDBJXJRKMK-VKHMYHEASA-N 0.000 description 7
- 230000003432 anti-folate effect Effects 0.000 description 7
- 229940127074 antifolate Drugs 0.000 description 7
- 125000003118 aryl group Chemical group 0.000 description 7
- 150000005829 chemical entities Chemical class 0.000 description 7
- 238000010828 elution Methods 0.000 description 7
- 230000003993 interaction Effects 0.000 description 7
- 229910052740 iodine Inorganic materials 0.000 description 7
- 150000002500 ions Chemical class 0.000 description 7
- 239000002609 medium Substances 0.000 description 7
- 230000003647 oxidation Effects 0.000 description 7
- 229910052760 oxygen Inorganic materials 0.000 description 7
- 229920001223 polyethylene glycol Polymers 0.000 description 7
- 239000000376 reactant Substances 0.000 description 7
- 150000003839 salts Chemical class 0.000 description 7
- 239000007787 solid Substances 0.000 description 7
- ZCYVEMRRCGMTRW-UHFFFAOYSA-N 7553-56-2 Chemical compound [I] ZCYVEMRRCGMTRW-UHFFFAOYSA-N 0.000 description 6
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 6
- WHUUTDBJXJRKMK-GSVOUGTGSA-N D-glutamic acid Chemical compound OC(=O)[C@H](N)CCC(O)=O WHUUTDBJXJRKMK-GSVOUGTGSA-N 0.000 description 6
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 6
- 101000908713 Homo sapiens Dihydrofolate reductase Proteins 0.000 description 6
- XUJNEKJLAYXESH-REOHCLBHSA-N L-Cysteine Chemical compound SC[C@H](N)C(O)=O XUJNEKJLAYXESH-REOHCLBHSA-N 0.000 description 6
- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 description 6
- 229910019142 PO4 Inorganic materials 0.000 description 6
- DNIAPMSPPWPWGF-UHFFFAOYSA-N Propylene glycol Chemical compound CC(O)CO DNIAPMSPPWPWGF-UHFFFAOYSA-N 0.000 description 6
- VMHLLURERBWHNL-UHFFFAOYSA-M Sodium acetate Chemical compound [Na+].CC([O-])=O VMHLLURERBWHNL-UHFFFAOYSA-M 0.000 description 6
- 125000002947 alkylene group Chemical group 0.000 description 6
- 239000007864 aqueous solution Substances 0.000 description 6
- 239000000969 carrier Substances 0.000 description 6
- 238000005119 centrifugation Methods 0.000 description 6
- 125000003636 chemical group Chemical group 0.000 description 6
- 229940124447 delivery agent Drugs 0.000 description 6
- 235000019441 ethanol Nutrition 0.000 description 6
- 238000010353 genetic engineering Methods 0.000 description 6
- RAXXELZNTBOGNW-UHFFFAOYSA-N imidazole Natural products C1=CNC=N1 RAXXELZNTBOGNW-UHFFFAOYSA-N 0.000 description 6
- 239000011630 iodine Substances 0.000 description 6
- 230000000670 limiting effect Effects 0.000 description 6
- 229930027945 nicotinamide-adenine dinucleotide Natural products 0.000 description 6
- 239000008188 pellet Substances 0.000 description 6
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 6
- 239000010452 phosphate Substances 0.000 description 6
- 239000000843 powder Substances 0.000 description 6
- 102000004196 processed proteins & peptides Human genes 0.000 description 6
- 239000001632 sodium acetate Substances 0.000 description 6
- 235000017281 sodium acetate Nutrition 0.000 description 6
- 239000012064 sodium phosphate buffer Substances 0.000 description 6
- 239000000758 substrate Substances 0.000 description 6
- 241000283690 Bos taurus Species 0.000 description 5
- QOSSAOTZNIDXMA-UHFFFAOYSA-N Dicylcohexylcarbodiimide Chemical compound C1CCCCC1N=C=NC1CCCCC1 QOSSAOTZNIDXMA-UHFFFAOYSA-N 0.000 description 5
- 108060003951 Immunoglobulin Proteins 0.000 description 5
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-Dimethylformamide Chemical compound CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 description 5
- NINIDFKCEFEMDL-UHFFFAOYSA-N Sulfur Chemical group [S] NINIDFKCEFEMDL-UHFFFAOYSA-N 0.000 description 5
- 239000007983 Tris buffer Substances 0.000 description 5
- 230000009471 action Effects 0.000 description 5
- 125000003295 alanine group Chemical group N[C@@H](C)C(=O)* 0.000 description 5
- 125000003368 amide group Chemical group 0.000 description 5
- UMGDCJDMYOKAJW-UHFFFAOYSA-N aminothiocarboxamide Natural products NC(N)=S UMGDCJDMYOKAJW-UHFFFAOYSA-N 0.000 description 5
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical group [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 5
- 150000001720 carbohydrates Chemical class 0.000 description 5
- 239000010949 copper Substances 0.000 description 5
- 235000018417 cysteine Nutrition 0.000 description 5
- XUJNEKJLAYXESH-UHFFFAOYSA-N cysteine Natural products SCC(N)C(O)=O XUJNEKJLAYXESH-UHFFFAOYSA-N 0.000 description 5
- VHJLVAABSRFDPM-ZXZARUISSA-N dithioerythritol Chemical compound SC[C@H](O)[C@H](O)CS VHJLVAABSRFDPM-ZXZARUISSA-N 0.000 description 5
- 238000000684 flow cytometry Methods 0.000 description 5
- OVBPIULPVIDEAO-LBPRGKRZSA-N folic acid Chemical compound C=1N=C2NC(N)=NC(=O)C2=NC=1CNC1=CC=C(C(=O)N[C@@H](CCC(O)=O)C(O)=O)C=C1 OVBPIULPVIDEAO-LBPRGKRZSA-N 0.000 description 5
- 239000011521 glass Substances 0.000 description 5
- 125000000623 heterocyclic group Chemical group 0.000 description 5
- 102000018358 immunoglobulin Human genes 0.000 description 5
- 238000001727 in vivo Methods 0.000 description 5
- 239000003921 oil Substances 0.000 description 5
- 235000019198 oils Nutrition 0.000 description 5
- 239000001301 oxygen Substances 0.000 description 5
- 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 5
- 229940051022 radioimmunoconjugate Drugs 0.000 description 5
- VUFNRPJNRFOTGK-UHFFFAOYSA-M sodium;1-[4-[(2,5-dioxopyrrol-1-yl)methyl]cyclohexanecarbonyl]oxy-2,5-dioxopyrrolidine-3-sulfonate Chemical compound [Na+].O=C1C(S(=O)(=O)[O-])CC(=O)N1OC(=O)C1CCC(CN2C(C=CC2=O)=O)CC1 VUFNRPJNRFOTGK-UHFFFAOYSA-M 0.000 description 5
- 239000011593 sulfur Chemical group 0.000 description 5
- DTQVDTLACAAQTR-UHFFFAOYSA-N trifluoroacetic acid Substances OC(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-N 0.000 description 5
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 5
- 239000011534 wash buffer Substances 0.000 description 5
- 229910001868 water Inorganic materials 0.000 description 5
- YBJHBAHKTGYVGT-ZKWXMUAHSA-N (+)-Biotin Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 description 4
- 208000001333 Colorectal Neoplasms Diseases 0.000 description 4
- ROSDSFDQCJNGOL-UHFFFAOYSA-N Dimethylamine Chemical compound CNC ROSDSFDQCJNGOL-UHFFFAOYSA-N 0.000 description 4
- 108010010803 Gelatin Proteins 0.000 description 4
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 4
- KDXKERNSBIXSRK-YFKPBYRVSA-N L-lysine Chemical compound NCCCC[C@H](N)C(O)=O KDXKERNSBIXSRK-YFKPBYRVSA-N 0.000 description 4
- GUBGYTABKSRVRQ-QKKXKWKRSA-N Lactose Natural products OC[C@H]1O[C@@H](O[C@H]2[C@H](O)[C@@H](O)C(O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@H]1O GUBGYTABKSRVRQ-QKKXKWKRSA-N 0.000 description 4
- 239000004472 Lysine Substances 0.000 description 4
- 239000002671 adjuvant Substances 0.000 description 4
- 235000004279 alanine Nutrition 0.000 description 4
- 125000003710 aryl alkyl group Chemical group 0.000 description 4
- 235000014633 carbohydrates Nutrition 0.000 description 4
- 239000011203 carbon fibre reinforced carbon Substances 0.000 description 4
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 4
- 238000007385 chemical modification Methods 0.000 description 4
- 238000010168 coupling process Methods 0.000 description 4
- 238000004132 cross linking Methods 0.000 description 4
- 239000008367 deionised water Substances 0.000 description 4
- 229910021641 deionized water Inorganic materials 0.000 description 4
- 235000019152 folic acid Nutrition 0.000 description 4
- 239000011724 folic acid Substances 0.000 description 4
- 239000000499 gel Substances 0.000 description 4
- 239000008273 gelatin Substances 0.000 description 4
- 229920000159 gelatin Polymers 0.000 description 4
- 235000019322 gelatine Nutrition 0.000 description 4
- 235000011852 gelatine desserts Nutrition 0.000 description 4
- 239000005556 hormone Substances 0.000 description 4
- 229940088597 hormone Drugs 0.000 description 4
- 230000007062 hydrolysis Effects 0.000 description 4
- 238000006460 hydrolysis reaction Methods 0.000 description 4
- 238000002372 labelling Methods 0.000 description 4
- 125000005439 maleimidyl group Chemical group C1(C=CC(N1*)=O)=O 0.000 description 4
- 150000002739 metals Chemical class 0.000 description 4
- 239000002773 nucleotide Substances 0.000 description 4
- 230000003287 optical effect Effects 0.000 description 4
- 239000000546 pharmaceutical excipient Substances 0.000 description 4
- 239000008363 phosphate buffer Substances 0.000 description 4
- 229920001281 polyalkylene Polymers 0.000 description 4
- 230000000717 retained effect Effects 0.000 description 4
- 229910052702 rhenium Inorganic materials 0.000 description 4
- 238000003998 size exclusion chromatography high performance liquid chromatography Methods 0.000 description 4
- 239000001488 sodium phosphate Substances 0.000 description 4
- 229910000162 sodium phosphate Inorganic materials 0.000 description 4
- 238000010189 synthetic method Methods 0.000 description 4
- RYFMWSXOAZQYPI-UHFFFAOYSA-K trisodium phosphate Chemical compound [Na+].[Na+].[Na+].[O-]P([O-])([O-])=O RYFMWSXOAZQYPI-UHFFFAOYSA-K 0.000 description 4
- 238000005406 washing Methods 0.000 description 4
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 3
- BVKZGUZCCUSVTD-UHFFFAOYSA-L Carbonate Chemical compound [O-]C([O-])=O BVKZGUZCCUSVTD-UHFFFAOYSA-L 0.000 description 3
- 101150074155 DHFR gene Proteins 0.000 description 3
- 229910052692 Dysprosium Inorganic materials 0.000 description 3
- 238000002965 ELISA Methods 0.000 description 3
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 description 3
- LYCAIKOWRPUZTN-UHFFFAOYSA-N Ethylene glycol Chemical compound OCCO LYCAIKOWRPUZTN-UHFFFAOYSA-N 0.000 description 3
- 229910052693 Europium Inorganic materials 0.000 description 3
- 229910052688 Gadolinium Inorganic materials 0.000 description 3
- WHUUTDBJXJRKMK-UHFFFAOYSA-N Glutamic acid Natural products OC(=O)C(N)CCC(O)=O WHUUTDBJXJRKMK-UHFFFAOYSA-N 0.000 description 3
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 description 3
- QNAYBMKLOCPYGJ-REOHCLBHSA-N L-alanine Chemical compound C[C@H](N)C(O)=O QNAYBMKLOCPYGJ-REOHCLBHSA-N 0.000 description 3
- CKLJMWTZIZZHCS-REOHCLBHSA-N L-aspartic acid Chemical compound OC(=O)[C@@H](N)CC(O)=O CKLJMWTZIZZHCS-REOHCLBHSA-N 0.000 description 3
- OUYCCCASQSFEME-QMMMGPOBSA-N L-tyrosine Chemical compound OC(=O)[C@@H](N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-QMMMGPOBSA-N 0.000 description 3
- PEEHTFAAVSWFBL-UHFFFAOYSA-N Maleimide Chemical compound O=C1NC(=O)C=C1 PEEHTFAAVSWFBL-UHFFFAOYSA-N 0.000 description 3
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 3
- QPCDCPDFJACHGM-UHFFFAOYSA-N N,N-bis{2-[bis(carboxymethyl)amino]ethyl}glycine Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(=O)O)CCN(CC(O)=O)CC(O)=O QPCDCPDFJACHGM-UHFFFAOYSA-N 0.000 description 3
- 229920001213 Polysorbate 20 Polymers 0.000 description 3
- JUJWROOIHBZHMG-UHFFFAOYSA-N Pyridine Chemical compound C1=CC=NC=C1 JUJWROOIHBZHMG-UHFFFAOYSA-N 0.000 description 3
- 229910052772 Samarium Inorganic materials 0.000 description 3
- UIIMBOGNXHQVGW-UHFFFAOYSA-M Sodium bicarbonate Chemical class [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 description 3
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- 229910009523 YCl3 Inorganic materials 0.000 description 3
- 238000010521 absorption reaction Methods 0.000 description 3
- 150000001408 amides Chemical class 0.000 description 3
- 125000000539 amino acid group Chemical group 0.000 description 3
- 235000003704 aspartic acid Nutrition 0.000 description 3
- 125000004429 atom Chemical group 0.000 description 3
- 239000011324 bead Substances 0.000 description 3
- OQFSQFPPLPISGP-UHFFFAOYSA-N beta-carboxyaspartic acid Natural products OC(=O)C(N)C(C(O)=O)C(O)=O OQFSQFPPLPISGP-UHFFFAOYSA-N 0.000 description 3
- 239000002775 capsule Substances 0.000 description 3
- 239000004202 carbamide Substances 0.000 description 3
- 229910052799 carbon Inorganic materials 0.000 description 3
- 239000003054 catalyst Substances 0.000 description 3
- 230000009920 chelation Effects 0.000 description 3
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 description 3
- 229910052802 copper Inorganic materials 0.000 description 3
- 230000008878 coupling Effects 0.000 description 3
- 230000006378 damage Effects 0.000 description 3
- 238000010511 deprotection reaction Methods 0.000 description 3
- 201000010099 disease Diseases 0.000 description 3
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 3
- 239000006185 dispersion Substances 0.000 description 3
- VHJLVAABSRFDPM-QWWZWVQMSA-N dithiothreitol Chemical compound SC[C@@H](O)[C@H](O)CS VHJLVAABSRFDPM-QWWZWVQMSA-N 0.000 description 3
- 125000000816 ethylene group Chemical group [H]C([H])([*:1])C([H])([H])[*:2] 0.000 description 3
- 229940014144 folate Drugs 0.000 description 3
- 238000009472 formulation Methods 0.000 description 3
- 235000013922 glutamic acid Nutrition 0.000 description 3
- 239000004220 glutamic acid Substances 0.000 description 3
- 229910052736 halogen Inorganic materials 0.000 description 3
- 238000004128 high performance liquid chromatography Methods 0.000 description 3
- 125000004464 hydroxyphenyl group Chemical group 0.000 description 3
- 238000010348 incorporation Methods 0.000 description 3
- 239000003112 inhibitor Substances 0.000 description 3
- 230000002401 inhibitory effect Effects 0.000 description 3
- 239000007924 injection Substances 0.000 description 3
- 238000002347 injection Methods 0.000 description 3
- 239000007788 liquid Substances 0.000 description 3
- 230000014759 maintenance of location Effects 0.000 description 3
- 108091005573 modified proteins Proteins 0.000 description 3
- 102000035118 modified proteins Human genes 0.000 description 3
- 239000002674 ointment Substances 0.000 description 3
- 230000005298 paramagnetic effect Effects 0.000 description 3
- 239000006187 pill Substances 0.000 description 3
- 229940068917 polyethylene glycols Drugs 0.000 description 3
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 description 3
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 description 3
- 230000008569 process Effects 0.000 description 3
- 230000005855 radiation Effects 0.000 description 3
- 238000000163 radioactive labelling Methods 0.000 description 3
- WUAPFZMCVAUBPE-UHFFFAOYSA-N rhenium atom Chemical compound [Re] WUAPFZMCVAUBPE-UHFFFAOYSA-N 0.000 description 3
- 239000001509 sodium citrate Substances 0.000 description 3
- NLJMYIDDQXHKNR-UHFFFAOYSA-K sodium citrate Chemical compound O.O.[Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O NLJMYIDDQXHKNR-UHFFFAOYSA-K 0.000 description 3
- 239000007909 solid dosage form Substances 0.000 description 3
- 238000006467 substitution reaction Methods 0.000 description 3
- PXQLVRUNWNTZOS-UHFFFAOYSA-N sulfanyl Chemical compound [SH] PXQLVRUNWNTZOS-UHFFFAOYSA-N 0.000 description 3
- 125000004434 sulfur atom Chemical group 0.000 description 3
- 239000000725 suspension Substances 0.000 description 3
- 239000003826 tablet Substances 0.000 description 3
- 239000005460 tetrahydrofolate Substances 0.000 description 3
- 238000011200 topical administration Methods 0.000 description 3
- OUYCCCASQSFEME-UHFFFAOYSA-N tyrosine Natural products OC(=O)C(N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-UHFFFAOYSA-N 0.000 description 3
- 239000003981 vehicle Substances 0.000 description 3
- PUPZLCDOIYMWBV-UHFFFAOYSA-N (+/-)-1,3-Butanediol Chemical compound CC(O)CCO PUPZLCDOIYMWBV-UHFFFAOYSA-N 0.000 description 2
- MTCFGRXMJLQNBG-REOHCLBHSA-N (2S)-2-Amino-3-hydroxypropansäure Chemical compound OC[C@H](N)C(O)=O MTCFGRXMJLQNBG-REOHCLBHSA-N 0.000 description 2
- JNYAEWCLZODPBN-JGWLITMVSA-N (2r,3r,4s)-2-[(1r)-1,2-dihydroxyethyl]oxolane-3,4-diol Chemical compound OC[C@@H](O)[C@H]1OC[C@H](O)[C@H]1O JNYAEWCLZODPBN-JGWLITMVSA-N 0.000 description 2
- AMGNHZVUZWILSB-UHFFFAOYSA-N 1,2-bis(2-chloroethylsulfanyl)ethane Chemical compound ClCCSCCSCCCl AMGNHZVUZWILSB-UHFFFAOYSA-N 0.000 description 2
- FJQZXCPWAGYPSD-UHFFFAOYSA-N 1,3,4,6-tetrachloro-3a,6a-diphenylimidazo[4,5-d]imidazole-2,5-dione Chemical compound ClN1C(=O)N(Cl)C2(C=3C=CC=CC=3)N(Cl)C(=O)N(Cl)C12C1=CC=CC=C1 FJQZXCPWAGYPSD-UHFFFAOYSA-N 0.000 description 2
- VBICKXHEKHSIBG-UHFFFAOYSA-N 1-monostearoylglycerol Chemical compound CCCCCCCCCCCCCCCCCC(=O)OCC(O)CO VBICKXHEKHSIBG-UHFFFAOYSA-N 0.000 description 2
- HZAXFHJVJLSVMW-UHFFFAOYSA-N 2-Aminoethan-1-ol Chemical compound NCCO HZAXFHJVJLSVMW-UHFFFAOYSA-N 0.000 description 2
- AAZRGUSAJLVIES-UHFFFAOYSA-N 2-[4-[(2,4-diaminopyrimidin-5-yl)methyl]-2,6-dimethoxyphenoxy]acetic acid Chemical compound COC1=C(OCC(O)=O)C(OC)=CC(CC=2C(=NC(N)=NC=2)N)=C1 AAZRGUSAJLVIES-UHFFFAOYSA-N 0.000 description 2
- CVOFKRWYWCSDMA-UHFFFAOYSA-N 2-chloro-n-(2,6-diethylphenyl)-n-(methoxymethyl)acetamide;2,6-dinitro-n,n-dipropyl-4-(trifluoromethyl)aniline Chemical compound CCC1=CC=CC(CC)=C1N(COC)C(=O)CCl.CCCN(CCC)C1=C([N+]([O-])=O)C=C(C(F)(F)F)C=C1[N+]([O-])=O CVOFKRWYWCSDMA-UHFFFAOYSA-N 0.000 description 2
- 229920001817 Agar Polymers 0.000 description 2
- GUBGYTABKSRVRQ-XLOQQCSPSA-N Alpha-Lactose Chemical compound O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@H]1O[C@@H]1[C@@H](CO)O[C@H](O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-XLOQQCSPSA-N 0.000 description 2
- XKRFYHLGVUSROY-UHFFFAOYSA-N Argon Chemical compound [Ar] XKRFYHLGVUSROY-UHFFFAOYSA-N 0.000 description 2
- VTYYLEPIZMXCLO-UHFFFAOYSA-L Calcium carbonate Chemical compound [Ca+2].[O-]C([O-])=O VTYYLEPIZMXCLO-UHFFFAOYSA-L 0.000 description 2
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 2
- 108010001857 Cell Surface Receptors Proteins 0.000 description 2
- 229910052684 Cerium Inorganic materials 0.000 description 2
- RGHNJXZEOKUKBD-SQOUGZDYSA-N D-gluconic acid Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@@H](O)C(O)=O RGHNJXZEOKUKBD-SQOUGZDYSA-N 0.000 description 2
- 229910052691 Erbium Inorganic materials 0.000 description 2
- 241000206672 Gelidium Species 0.000 description 2
- 239000004471 Glycine Substances 0.000 description 2
- 229910052689 Holmium Inorganic materials 0.000 description 2
- 101001024703 Homo sapiens Nck-associated protein 5 Proteins 0.000 description 2
- AVXURJPOCDRRFD-UHFFFAOYSA-N Hydroxylamine Chemical compound ON AVXURJPOCDRRFD-UHFFFAOYSA-N 0.000 description 2
- 102000006496 Immunoglobulin Heavy Chains Human genes 0.000 description 2
- 108010019476 Immunoglobulin Heavy Chains Proteins 0.000 description 2
- 102000017727 Immunoglobulin Variable Region Human genes 0.000 description 2
- 108010067060 Immunoglobulin Variable Region Proteins 0.000 description 2
- ONIBWKKTOPOVIA-BYPYZUCNSA-N L-Proline Chemical compound OC(=O)[C@@H]1CCCN1 ONIBWKKTOPOVIA-BYPYZUCNSA-N 0.000 description 2
- ZDXPYRJPNDTMRX-VKHMYHEASA-N L-glutamine Chemical compound OC(=O)[C@@H](N)CCC(N)=O ZDXPYRJPNDTMRX-VKHMYHEASA-N 0.000 description 2
- AGPKZVBTJJNPAG-WHFBIAKZSA-N L-isoleucine Chemical compound CC[C@H](C)[C@H](N)C(O)=O AGPKZVBTJJNPAG-WHFBIAKZSA-N 0.000 description 2
- ROHFNLRQFUQHCH-YFKPBYRVSA-N L-leucine Chemical compound CC(C)C[C@H](N)C(O)=O ROHFNLRQFUQHCH-YFKPBYRVSA-N 0.000 description 2
- COLNVLDHVKWLRT-QMMMGPOBSA-N L-phenylalanine Chemical compound OC(=O)[C@@H](N)CC1=CC=CC=C1 COLNVLDHVKWLRT-QMMMGPOBSA-N 0.000 description 2
- AYFVYJQAPQTCCC-GBXIJSLDSA-N L-threonine Chemical compound C[C@@H](O)[C@H](N)C(O)=O AYFVYJQAPQTCCC-GBXIJSLDSA-N 0.000 description 2
- KZSNJWFQEVHDMF-BYPYZUCNSA-N L-valine Chemical compound CC(C)[C@H](N)C(O)=O KZSNJWFQEVHDMF-BYPYZUCNSA-N 0.000 description 2
- ROHFNLRQFUQHCH-UHFFFAOYSA-N Leucine Natural products CC(C)CC(N)C(O)=O ROHFNLRQFUQHCH-UHFFFAOYSA-N 0.000 description 2
- 229910052765 Lutetium Inorganic materials 0.000 description 2
- NVGBPTNZLWRQSY-UWVGGRQHSA-N Lys-Lys Chemical compound NCCCC[C@H](N)C(=O)N[C@H](C(O)=O)CCCCN NVGBPTNZLWRQSY-UWVGGRQHSA-N 0.000 description 2
- JGFZNNIVVJXRND-UHFFFAOYSA-N N,N-Diisopropylethylamine (DIPEA) Chemical compound CCN(C(C)C)C(C)C JGFZNNIVVJXRND-UHFFFAOYSA-N 0.000 description 2
- 229910017912 NH2OH Inorganic materials 0.000 description 2
- 229910003827 NRaRb Inorganic materials 0.000 description 2
- 102100036946 Nck-associated protein 5 Human genes 0.000 description 2
- 229910052779 Neodymium Inorganic materials 0.000 description 2
- 101710160107 Outer membrane protein A Proteins 0.000 description 2
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N Phenol Chemical compound OC1=CC=CC=C1 ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 description 2
- 239000002202 Polyethylene glycol Substances 0.000 description 2
- 229910052777 Praseodymium Inorganic materials 0.000 description 2
- ONIBWKKTOPOVIA-UHFFFAOYSA-N Proline Natural products OC(=O)C1CCCN1 ONIBWKKTOPOVIA-UHFFFAOYSA-N 0.000 description 2
- 102000007056 Recombinant Fusion Proteins Human genes 0.000 description 2
- 108010008281 Recombinant Fusion Proteins Proteins 0.000 description 2
- 239000012506 Sephacryl® Substances 0.000 description 2
- MTCFGRXMJLQNBG-UHFFFAOYSA-N Serine Natural products OCC(N)C(O)=O MTCFGRXMJLQNBG-UHFFFAOYSA-N 0.000 description 2
- CDBYLPFSWZWCQE-UHFFFAOYSA-L Sodium Carbonate Chemical compound [Na+].[Na+].[O-]C([O-])=O CDBYLPFSWZWCQE-UHFFFAOYSA-L 0.000 description 2
- PXIPVTKHYLBLMZ-UHFFFAOYSA-N Sodium azide Chemical compound [Na+].[N-]=[N+]=[N-] PXIPVTKHYLBLMZ-UHFFFAOYSA-N 0.000 description 2
- DBMJMQXJHONAFJ-UHFFFAOYSA-M Sodium laurylsulphate Chemical compound [Na+].CCCCCCCCCCCCOS([O-])(=O)=O DBMJMQXJHONAFJ-UHFFFAOYSA-M 0.000 description 2
- WQDUMFSSJAZKTM-UHFFFAOYSA-N Sodium methoxide Chemical compound [Na+].[O-]C WQDUMFSSJAZKTM-UHFFFAOYSA-N 0.000 description 2
- 229920002472 Starch Polymers 0.000 description 2
- 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 2
- 229930006000 Sucrose Natural products 0.000 description 2
- 229910052771 Terbium Inorganic materials 0.000 description 2
- DKGAVHZHDRPRBM-UHFFFAOYSA-N Tert-Butanol Chemical compound CC(C)(C)O DKGAVHZHDRPRBM-UHFFFAOYSA-N 0.000 description 2
- AYFVYJQAPQTCCC-UHFFFAOYSA-N Threonine Natural products CC(O)C(N)C(O)=O AYFVYJQAPQTCCC-UHFFFAOYSA-N 0.000 description 2
- 239000004473 Threonine Substances 0.000 description 2
- 229910052775 Thulium Inorganic materials 0.000 description 2
- KZSNJWFQEVHDMF-UHFFFAOYSA-N Valine Natural products CC(C)C(N)C(O)=O KZSNJWFQEVHDMF-UHFFFAOYSA-N 0.000 description 2
- 229910052769 Ytterbium Inorganic materials 0.000 description 2
- JLCPHMBAVCMARE-UHFFFAOYSA-N [3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-hydroxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methyl [5-(6-aminopurin-9-yl)-2-(hydroxymethyl)oxolan-3-yl] hydrogen phosphate Polymers Cc1cn(C2CC(OP(O)(=O)OCC3OC(CC3OP(O)(=O)OCC3OC(CC3O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c3nc(N)[nH]c4=O)C(COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3CO)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cc(C)c(=O)[nH]c3=O)n3cc(C)c(=O)[nH]c3=O)n3ccc(N)nc3=O)n3cc(C)c(=O)[nH]c3=O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)O2)c(=O)[nH]c1=O JLCPHMBAVCMARE-UHFFFAOYSA-N 0.000 description 2
- 238000009825 accumulation Methods 0.000 description 2
- YRKCREAYFQTBPV-UHFFFAOYSA-N acetylacetone Chemical compound CC(=O)CC(C)=O YRKCREAYFQTBPV-UHFFFAOYSA-N 0.000 description 2
- 239000004480 active ingredient Substances 0.000 description 2
- 235000010419 agar Nutrition 0.000 description 2
- 230000002776 aggregation Effects 0.000 description 2
- 238000004220 aggregation Methods 0.000 description 2
- 150000001299 aldehydes Chemical class 0.000 description 2
- 230000003321 amplification Effects 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 239000005557 antagonist Substances 0.000 description 2
- 230000000890 antigenic effect Effects 0.000 description 2
- 239000002585 base Substances 0.000 description 2
- 230000008901 benefit Effects 0.000 description 2
- 239000000440 bentonite Substances 0.000 description 2
- 235000012216 bentonite Nutrition 0.000 description 2
- 229910000278 bentonite Inorganic materials 0.000 description 2
- SVPXDRXYRYOSEX-UHFFFAOYSA-N bentoquatam Chemical compound O.O=[Si]=O.O=[Al]O[Al]=O SVPXDRXYRYOSEX-UHFFFAOYSA-N 0.000 description 2
- SESFRYSPDFLNCH-UHFFFAOYSA-N benzyl benzoate Chemical compound C=1C=CC=CC=1C(=O)OCC1=CC=CC=C1 SESFRYSPDFLNCH-UHFFFAOYSA-N 0.000 description 2
- 125000001797 benzyl group Chemical group [H]C1=C([H])C([H])=C(C([H])=C1[H])C([H])([H])* 0.000 description 2
- 229960002685 biotin Drugs 0.000 description 2
- 235000020958 biotin Nutrition 0.000 description 2
- 239000011616 biotin Substances 0.000 description 2
- 230000037396 body weight Effects 0.000 description 2
- 238000006664 bond formation reaction Methods 0.000 description 2
- 229940098773 bovine serum albumin Drugs 0.000 description 2
- 239000000337 buffer salt Substances 0.000 description 2
- 201000011510 cancer Diseases 0.000 description 2
- 125000004181 carboxyalkyl group Chemical group 0.000 description 2
- 150000001732 carboxylic acid derivatives Chemical class 0.000 description 2
- 125000002843 carboxylic acid group Chemical group 0.000 description 2
- 239000013626 chemical specie Substances 0.000 description 2
- OSASVXMJTNOKOY-UHFFFAOYSA-N chlorobutanol Chemical compound CC(C)(O)C(Cl)(Cl)Cl OSASVXMJTNOKOY-UHFFFAOYSA-N 0.000 description 2
- 238000004587 chromatography analysis Methods 0.000 description 2
- 229910052804 chromium Inorganic materials 0.000 description 2
- 238000003776 cleavage reaction Methods 0.000 description 2
- 238000000576 coating method Methods 0.000 description 2
- 238000003745 diagnosis Methods 0.000 description 2
- 239000002552 dosage form Substances 0.000 description 2
- 239000006196 drop Substances 0.000 description 2
- 239000012149 elution buffer Substances 0.000 description 2
- 239000003995 emulsifying agent Substances 0.000 description 2
- 230000001804 emulsifying effect Effects 0.000 description 2
- 239000000839 emulsion Substances 0.000 description 2
- 150000002148 esters Chemical class 0.000 description 2
- 125000001495 ethyl group Chemical group [H]C([H])([H])C([H])([H])* 0.000 description 2
- 230000007717 exclusion Effects 0.000 description 2
- 230000029142 excretion Effects 0.000 description 2
- 239000000945 filler Substances 0.000 description 2
- 239000000706 filtrate Substances 0.000 description 2
- 239000012467 final product Substances 0.000 description 2
- 125000001207 fluorophenyl group Chemical group 0.000 description 2
- 239000012634 fragment Substances 0.000 description 2
- 210000001035 gastrointestinal tract Anatomy 0.000 description 2
- ZDXPYRJPNDTMRX-UHFFFAOYSA-N glutamine Natural products OC(=O)C(N)CCC(N)=O ZDXPYRJPNDTMRX-UHFFFAOYSA-N 0.000 description 2
- 235000011187 glycerol Nutrition 0.000 description 2
- 239000008187 granular material Substances 0.000 description 2
- 150000002367 halogens Chemical class 0.000 description 2
- 239000001257 hydrogen Substances 0.000 description 2
- 229910052739 hydrogen Inorganic materials 0.000 description 2
- 125000002883 imidazolyl group Chemical group 0.000 description 2
- 238000000338 in vitro Methods 0.000 description 2
- 229910052738 indium Inorganic materials 0.000 description 2
- 239000003701 inert diluent Substances 0.000 description 2
- 238000003780 insertion Methods 0.000 description 2
- 230000037431 insertion Effects 0.000 description 2
- 238000001990 intravenous administration Methods 0.000 description 2
- 238000002955 isolation Methods 0.000 description 2
- AGPKZVBTJJNPAG-UHFFFAOYSA-N isoleucine Natural products CCC(C)C(N)C(O)=O AGPKZVBTJJNPAG-UHFFFAOYSA-N 0.000 description 2
- 229960000310 isoleucine Drugs 0.000 description 2
- 150000002540 isothiocyanates Chemical class 0.000 description 2
- 239000008101 lactose Substances 0.000 description 2
- 229910052746 lanthanum Inorganic materials 0.000 description 2
- 239000008297 liquid dosage form Substances 0.000 description 2
- 125000003588 lysine group Chemical group [H]N([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])(N([H])[H])C(*)=O 0.000 description 2
- 108010054155 lysyllysine Proteins 0.000 description 2
- HQKMJHAJHXVSDF-UHFFFAOYSA-L magnesium stearate Chemical compound [Mg+2].CCCCCCCCCCCCCCCCCC([O-])=O.CCCCCCCCCCCCCCCCCC([O-])=O HQKMJHAJHXVSDF-UHFFFAOYSA-L 0.000 description 2
- 238000002595 magnetic resonance imaging Methods 0.000 description 2
- 229910052748 manganese Inorganic materials 0.000 description 2
- 102000006240 membrane receptors Human genes 0.000 description 2
- SVPRVJIDOLJYQR-UHFFFAOYSA-N n-chlorobenzenesulfonamide;sodium Chemical compound [Na].ClNS(=O)(=O)C1=CC=CC=C1 SVPRVJIDOLJYQR-UHFFFAOYSA-N 0.000 description 2
- 125000001624 naphthyl group Chemical group 0.000 description 2
- 229940097496 nasal spray Drugs 0.000 description 2
- 239000007922 nasal spray Substances 0.000 description 2
- 230000018791 negative regulation of catalytic activity Effects 0.000 description 2
- 125000004433 nitrogen atom Chemical group N* 0.000 description 2
- 231100000252 nontoxic Toxicity 0.000 description 2
- 230000003000 nontoxic effect Effects 0.000 description 2
- 238000003199 nucleic acid amplification method Methods 0.000 description 2
- 239000004006 olive oil Substances 0.000 description 2
- 235000008390 olive oil Nutrition 0.000 description 2
- 125000001997 phenyl group Chemical group [H]C1=C([H])C([H])=C(*)C([H])=C1[H] 0.000 description 2
- COLNVLDHVKWLRT-UHFFFAOYSA-N phenylalanine Natural products OC(=O)C(N)CC1=CC=CC=C1 COLNVLDHVKWLRT-UHFFFAOYSA-N 0.000 description 2
- 229910052698 phosphorus Inorganic materials 0.000 description 2
- 239000002244 precipitate Substances 0.000 description 2
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 2
- 230000002035 prolonged effect Effects 0.000 description 2
- 125000001436 propyl group Chemical group [H]C([*])([H])C([H])([H])C([H])([H])[H] 0.000 description 2
- 235000013772 propylene glycol Nutrition 0.000 description 2
- 229960004063 propylene glycol Drugs 0.000 description 2
- 125000006239 protecting group Chemical group 0.000 description 2
- 238000002731 protein assay Methods 0.000 description 2
- 125000004076 pyridyl group Chemical group 0.000 description 2
- 125000005493 quinolyl group Chemical group 0.000 description 2
- 238000012552 review Methods 0.000 description 2
- SMQUZDBALVYZAC-UHFFFAOYSA-N salicylaldehyde Chemical compound OC1=CC=CC=C1C=O SMQUZDBALVYZAC-UHFFFAOYSA-N 0.000 description 2
- 230000007017 scission Effects 0.000 description 2
- 238000007790 scraping Methods 0.000 description 2
- 208000000587 small cell lung carcinoma Diseases 0.000 description 2
- 239000007974 sodium acetate buffer Substances 0.000 description 2
- 239000012475 sodium chloride buffer Substances 0.000 description 2
- 235000019333 sodium laurylsulphate Nutrition 0.000 description 2
- 235000019698 starch Nutrition 0.000 description 2
- 238000003756 stirring Methods 0.000 description 2
- 239000005720 sucrose Substances 0.000 description 2
- 239000000829 suppository Substances 0.000 description 2
- 239000000375 suspending agent Substances 0.000 description 2
- 238000003786 synthesis reaction Methods 0.000 description 2
- 229910052713 technetium Inorganic materials 0.000 description 2
- 125000000999 tert-butyl group Chemical group [H]C([H])([H])C(*)(C([H])([H])[H])C([H])([H])[H] 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- UAFBBLZEQSKFBK-UHFFFAOYSA-J tetrasodium;2-[[6-[6-[6-[[bis(carboxylatomethyl)amino]methyl]pyridin-2-yl]-4-(3-isothiocyanato-4-methoxyphenyl)pyridin-2-yl]pyridin-2-yl]methyl-(carboxylatomethyl)amino]acetate Chemical compound [Na+].[Na+].[Na+].[Na+].C1=C(N=C=S)C(OC)=CC=C1C1=CC(C=2N=C(CN(CC([O-])=O)CC([O-])=O)C=CC=2)=NC(C=2N=C(CN(CC([O-])=O)CC([O-])=O)C=CC=2)=C1 UAFBBLZEQSKFBK-UHFFFAOYSA-J 0.000 description 2
- 238000002560 therapeutic procedure Methods 0.000 description 2
- 125000001544 thienyl group Chemical group 0.000 description 2
- 238000004809 thin layer chromatography Methods 0.000 description 2
- 150000003568 thioethers Chemical class 0.000 description 2
- CWERGRDVMFNCDR-UHFFFAOYSA-N thioglycolic acid Chemical compound OC(=O)CS CWERGRDVMFNCDR-UHFFFAOYSA-N 0.000 description 2
- 231100000419 toxicity Toxicity 0.000 description 2
- 230000001988 toxicity Effects 0.000 description 2
- 239000004474 valine Substances 0.000 description 2
- 239000003643 water by type Substances 0.000 description 2
- 239000001993 wax Substances 0.000 description 2
- 239000000080 wetting agent Substances 0.000 description 2
- BAERPNBPLZWCES-UHFFFAOYSA-N (2-hydroxy-1-phosphonoethyl)phosphonic acid Chemical compound OCC(P(O)(O)=O)P(O)(O)=O BAERPNBPLZWCES-UHFFFAOYSA-N 0.000 description 1
- RRJNHRZHTMBPEI-UHFFFAOYSA-N (3-hexadecylsulfanylcyclohexyl) 2-(trimethylazaniumyl)ethyl phosphate Chemical compound CCCCCCCCCCCCCCCCSC1CCCC(OP([O-])(=O)OCC[N+](C)(C)C)C1 RRJNHRZHTMBPEI-UHFFFAOYSA-N 0.000 description 1
- 125000003088 (fluoren-9-ylmethoxy)carbonyl group Chemical group 0.000 description 1
- SHXHPUAKLCCLDV-UHFFFAOYSA-N 1,1,1-trifluoropentane-2,4-dione Chemical compound CC(=O)CC(=O)C(F)(F)F SHXHPUAKLCCLDV-UHFFFAOYSA-N 0.000 description 1
- 229940058015 1,3-butylene glycol Drugs 0.000 description 1
- UOFGSWVZMUXXIY-UHFFFAOYSA-N 1,5-Diphenyl-3-thiocarbazone Chemical compound C=1C=CC=CC=1N=NC(=S)NNC1=CC=CC=C1 UOFGSWVZMUXXIY-UHFFFAOYSA-N 0.000 description 1
- CULQNACJHGHAER-UHFFFAOYSA-N 1-[4-[(2-iodoacetyl)amino]benzoyl]oxy-2,5-dioxopyrrolidine-3-sulfonic acid Chemical compound O=C1C(S(=O)(=O)O)CC(=O)N1OC(=O)C1=CC=C(NC(=O)CI)C=C1 CULQNACJHGHAER-UHFFFAOYSA-N 0.000 description 1
- IIZPXYDJLKNOIY-JXPKJXOSSA-N 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine Chemical compound CCCCCCCCCCCCCCCC(=O)OC[C@H](COP([O-])(=O)OCC[N+](C)(C)C)OC(=O)CCC\C=C/C\C=C/C\C=C/C\C=C/CCCCC IIZPXYDJLKNOIY-JXPKJXOSSA-N 0.000 description 1
- GZCWLCBFPRFLKL-UHFFFAOYSA-N 1-prop-2-ynoxypropan-2-ol Chemical compound CC(O)COCC#C GZCWLCBFPRFLKL-UHFFFAOYSA-N 0.000 description 1
- XEZNGIUYQVAUSS-UHFFFAOYSA-N 18-crown-6 Chemical compound C1COCCOCCOCCOCCOCCO1 XEZNGIUYQVAUSS-UHFFFAOYSA-N 0.000 description 1
- VILCJCGEZXAXTO-UHFFFAOYSA-N 2,2,2-tetramine Chemical compound NCCNCCNCCN VILCJCGEZXAXTO-UHFFFAOYSA-N 0.000 description 1
- JFJNVIPVOCESGZ-UHFFFAOYSA-N 2,3-dipyridin-2-ylpyridine Chemical compound N1=CC=CC=C1C1=CC=CN=C1C1=CC=CC=N1 JFJNVIPVOCESGZ-UHFFFAOYSA-N 0.000 description 1
- IOHPVZBSOKLVMN-UHFFFAOYSA-N 2-(2-phenylethyl)benzoic acid Chemical compound OC(=O)C1=CC=CC=C1CCC1=CC=CC=C1 IOHPVZBSOKLVMN-UHFFFAOYSA-N 0.000 description 1
- URDCARMUOSMFFI-UHFFFAOYSA-N 2-[2-[bis(carboxymethyl)amino]ethyl-(2-hydroxyethyl)amino]acetic acid Chemical compound OCCN(CC(O)=O)CCN(CC(O)=O)CC(O)=O URDCARMUOSMFFI-UHFFFAOYSA-N 0.000 description 1
- HHLZCENAOIROSL-UHFFFAOYSA-N 2-[4,7-bis(carboxymethyl)-1,4,7,10-tetrazacyclododec-1-yl]acetic acid Chemical compound OC(=O)CN1CCNCCN(CC(O)=O)CCN(CC(O)=O)CC1 HHLZCENAOIROSL-UHFFFAOYSA-N 0.000 description 1
- CDAWCLOXVUBKRW-UHFFFAOYSA-N 2-aminophenol Chemical class NC1=CC=CC=C1O CDAWCLOXVUBKRW-UHFFFAOYSA-N 0.000 description 1
- OFLNEVYCAMVQJS-UHFFFAOYSA-N 2-n,2-n-diethylethane-1,1,1,2-tetramine Chemical compound CCN(CC)CC(N)(N)N OFLNEVYCAMVQJS-UHFFFAOYSA-N 0.000 description 1
- CJAZCKUGLFWINJ-UHFFFAOYSA-N 3,4-dihydroxybenzene-1,2-disulfonic acid Chemical compound OC1=CC=C(S(O)(=O)=O)C(S(O)(=O)=O)=C1O CJAZCKUGLFWINJ-UHFFFAOYSA-N 0.000 description 1
- HPOCGNHBIFZCAN-UHFFFAOYSA-N 4-[(2,4-diaminopyrimidin-5-yl)methyl]-2,6-dimethoxyphenol Chemical compound COC1=C(O)C(OC)=CC(CC=2C(=NC(N)=NC=2)N)=C1 HPOCGNHBIFZCAN-UHFFFAOYSA-N 0.000 description 1
- HWLAHKWVFFIJLY-UHFFFAOYSA-N 4-[(dimethylamino)methyl]-2,6-dimethoxyphenol;hydrochloride Chemical compound Cl.COC1=CC(CN(C)C)=CC(OC)=C1O HWLAHKWVFFIJLY-UHFFFAOYSA-N 0.000 description 1
- YCPXWRQRBFJBPZ-UHFFFAOYSA-N 5-sulfosalicylic acid Chemical compound OC(=O)C1=CC(S(O)(=O)=O)=CC=C1O YCPXWRQRBFJBPZ-UHFFFAOYSA-N 0.000 description 1
- KNSJCCNMJYXLOL-UHFFFAOYSA-N 6,12-epoxy-6h,12h-dibenzo[b,f][1,5]dioxocin Chemical compound O1C2=CC=CC=C2C2OC1C1=CC=CC=C1O2 KNSJCCNMJYXLOL-UHFFFAOYSA-N 0.000 description 1
- 239000005725 8-Hydroxyquinoline Substances 0.000 description 1
- HBAQYPYDRFILMT-UHFFFAOYSA-N 8-[3-(1-cyclopropylpyrazol-4-yl)-1H-pyrazolo[4,3-d]pyrimidin-5-yl]-3-methyl-3,8-diazabicyclo[3.2.1]octan-2-one Chemical class C1(CC1)N1N=CC(=C1)C1=NNC2=C1N=C(N=C2)N1C2C(N(CC1CC2)C)=O HBAQYPYDRFILMT-UHFFFAOYSA-N 0.000 description 1
- QTBSBXVTEAMEQO-UHFFFAOYSA-M Acetate Chemical compound CC([O-])=O QTBSBXVTEAMEQO-UHFFFAOYSA-M 0.000 description 1
- 108010051457 Acid Phosphatase Proteins 0.000 description 1
- 102000013563 Acid Phosphatase Human genes 0.000 description 1
- 229920000936 Agarose Polymers 0.000 description 1
- 239000005995 Aluminium silicate Substances 0.000 description 1
- 241000416162 Astragalus gummifer Species 0.000 description 1
- 108090001008 Avidin Proteins 0.000 description 1
- 238000000035 BCA protein assay Methods 0.000 description 1
- 108010077805 Bacterial Proteins Proteins 0.000 description 1
- ROFVEXUMMXZLPA-UHFFFAOYSA-N Bipyridyl Chemical group N1=CC=CC=C1C1=CC=CC=N1 ROFVEXUMMXZLPA-UHFFFAOYSA-N 0.000 description 1
- BTBUEUYNUDRHOZ-UHFFFAOYSA-N Borate Chemical compound [O-]B([O-])[O-] BTBUEUYNUDRHOZ-UHFFFAOYSA-N 0.000 description 1
- FRBYJCORNHQJTG-UHFFFAOYSA-N C(CN(C(C1=CC=CC=C1)C(=O)O)O)N(C(C1=CC=CC=C1)C(=O)O)O Chemical compound C(CN(C(C1=CC=CC=C1)C(=O)O)O)N(C(C1=CC=CC=C1)C(=O)O)O FRBYJCORNHQJTG-UHFFFAOYSA-N 0.000 description 1
- 241000282472 Canis lupus familiaris Species 0.000 description 1
- KXDHJXZQYSOELW-UHFFFAOYSA-N Carbamic acid Chemical class NC(O)=O KXDHJXZQYSOELW-UHFFFAOYSA-N 0.000 description 1
- 229920002134 Carboxymethyl cellulose Polymers 0.000 description 1
- 201000009030 Carcinoma Diseases 0.000 description 1
- 241000282693 Cercopithecidae Species 0.000 description 1
- KRKNYBCHXYNGOX-UHFFFAOYSA-K Citrate Chemical compound [O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O KRKNYBCHXYNGOX-UHFFFAOYSA-K 0.000 description 1
- 206010009944 Colon cancer Diseases 0.000 description 1
- RYGMFSIKBFXOCR-UHFFFAOYSA-N Copper Chemical compound [Cu] RYGMFSIKBFXOCR-UHFFFAOYSA-N 0.000 description 1
- 102000004420 Creatine Kinase Human genes 0.000 description 1
- 108010042126 Creatine kinase 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
- 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
- RGHNJXZEOKUKBD-UHFFFAOYSA-N D-gluconic acid Natural products OCC(O)C(O)C(O)C(O)C(O)=O RGHNJXZEOKUKBD-UHFFFAOYSA-N 0.000 description 1
- FEWJPZIEWOKRBE-JCYAYHJZSA-N Dextrotartaric acid Chemical compound OC(=O)[C@H](O)[C@@H](O)C(O)=O FEWJPZIEWOKRBE-JCYAYHJZSA-N 0.000 description 1
- 235000019739 Dicalciumphosphate Nutrition 0.000 description 1
- RPNUMPOLZDHAAY-UHFFFAOYSA-N Diethylenetriamine Chemical compound NCCNCCN RPNUMPOLZDHAAY-UHFFFAOYSA-N 0.000 description 1
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 1
- LVGKNOAMLMIIKO-UHFFFAOYSA-N Elaidinsaeure-aethylester Natural products CCCCCCCCC=CCCCCCCCC(=O)OCC LVGKNOAMLMIIKO-UHFFFAOYSA-N 0.000 description 1
- 239000004593 Epoxy Substances 0.000 description 1
- 241000283086 Equidae Species 0.000 description 1
- 108090000371 Esterases Proteins 0.000 description 1
- JOYRKODLDBILNP-UHFFFAOYSA-N Ethyl urethane Chemical compound CCOC(N)=O JOYRKODLDBILNP-UHFFFAOYSA-N 0.000 description 1
- PIICEJLVQHRZGT-UHFFFAOYSA-N Ethylenediamine Chemical compound NCCN PIICEJLVQHRZGT-UHFFFAOYSA-N 0.000 description 1
- 241000282326 Felis catus Species 0.000 description 1
- 239000004606 Fillers/Extenders Substances 0.000 description 1
- 241000233866 Fungi Species 0.000 description 1
- 108020004206 Gamma-glutamyltransferase Proteins 0.000 description 1
- 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 1
- 108090000288 Glycoproteins Proteins 0.000 description 1
- 102000003886 Glycoproteins Human genes 0.000 description 1
- 241000282412 Homo Species 0.000 description 1
- 101000945318 Homo sapiens Calponin-1 Proteins 0.000 description 1
- 101000652736 Homo sapiens Transgelin Proteins 0.000 description 1
- 108010001336 Horseradish Peroxidase Proteins 0.000 description 1
- 102000001706 Immunoglobulin Fab Fragments Human genes 0.000 description 1
- 108010054477 Immunoglobulin Fab Fragments Proteins 0.000 description 1
- 102000013463 Immunoglobulin Light Chains Human genes 0.000 description 1
- 108010065825 Immunoglobulin Light Chains Proteins 0.000 description 1
- 102000003855 L-lactate dehydrogenase Human genes 0.000 description 1
- 108700023483 L-lactate dehydrogenases Proteins 0.000 description 1
- 102000004856 Lectins Human genes 0.000 description 1
- 108090001090 Lectins Proteins 0.000 description 1
- 240000007472 Leucaena leucocephala Species 0.000 description 1
- 235000010643 Leucaena leucocephala Nutrition 0.000 description 1
- 102000004895 Lipoproteins Human genes 0.000 description 1
- 108090001030 Lipoproteins Proteins 0.000 description 1
- 206010025323 Lymphomas Diseases 0.000 description 1
- 240000003183 Manihot esculenta Species 0.000 description 1
- 235000016735 Manihot esculenta subsp esculenta Nutrition 0.000 description 1
- 229930195725 Mannitol Natural products 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- KWYHDKDOAIKMQN-UHFFFAOYSA-N N,N,N',N'-tetramethylethylenediamine Chemical compound CN(C)CCN(C)C KWYHDKDOAIKMQN-UHFFFAOYSA-N 0.000 description 1
- FSVCELGFZIQNCK-UHFFFAOYSA-N N,N-bis(2-hydroxyethyl)glycine Chemical compound OCCN(CCO)CC(O)=O FSVCELGFZIQNCK-UHFFFAOYSA-N 0.000 description 1
- OVBPIULPVIDEAO-UHFFFAOYSA-N N-Pteroyl-L-glutaminsaeure Natural products C=1N=C2NC(N)=NC(=O)C2=NC=1CNC1=CC=C(C(=O)NC(CCC(O)=O)C(O)=O)C=C1 OVBPIULPVIDEAO-UHFFFAOYSA-N 0.000 description 1
- 108091028043 Nucleic acid sequence Proteins 0.000 description 1
- 241000283973 Oryctolagus cuniculus Species 0.000 description 1
- 108090000526 Papain Proteins 0.000 description 1
- 235000019483 Peanut oil Nutrition 0.000 description 1
- 241001494479 Pecora Species 0.000 description 1
- 102000057297 Pepsin A Human genes 0.000 description 1
- 108090000284 Pepsin A Proteins 0.000 description 1
- 108090000608 Phosphoric Monoester Hydrolases Proteins 0.000 description 1
- 102000004160 Phosphoric Monoester Hydrolases Human genes 0.000 description 1
- 229920003171 Poly (ethylene oxide) Polymers 0.000 description 1
- 229920000805 Polyaspartic acid Polymers 0.000 description 1
- 108010020346 Polyglutamic Acid Proteins 0.000 description 1
- 229920000388 Polyphosphate Polymers 0.000 description 1
- 102100035703 Prostatic acid phosphatase Human genes 0.000 description 1
- 239000004365 Protease Substances 0.000 description 1
- 241000606701 Rickettsia Species 0.000 description 1
- 239000002262 Schiff base Substances 0.000 description 1
- 150000004753 Schiff bases Chemical class 0.000 description 1
- 244000061456 Solanum tuberosum Species 0.000 description 1
- 235000002595 Solanum tuberosum Nutrition 0.000 description 1
- SSZBUIDZHHWXNJ-UHFFFAOYSA-N Stearinsaeure-hexadecylester Natural products CCCCCCCCCCCCCCCCCC(=O)OCCCCCCCCCCCCCCCC SSZBUIDZHHWXNJ-UHFFFAOYSA-N 0.000 description 1
- 241000282887 Suidae Species 0.000 description 1
- FEWJPZIEWOKRBE-UHFFFAOYSA-N Tartaric acid Natural products [H+].[H+].[O-]C(=O)C(O)C(O)C([O-])=O FEWJPZIEWOKRBE-UHFFFAOYSA-N 0.000 description 1
- WDLRUFUQRNWCPK-UHFFFAOYSA-N Tetraxetan Chemical compound OC(=O)CN1CCN(CC(O)=O)CCN(CC(O)=O)CCN(CC(O)=O)CC1 WDLRUFUQRNWCPK-UHFFFAOYSA-N 0.000 description 1
- 229920001615 Tragacanth Polymers 0.000 description 1
- 102100031013 Transgelin Human genes 0.000 description 1
- GSEJCLTVZPLZKY-UHFFFAOYSA-N Triethanolamine Chemical compound OCCN(CCO)CCO GSEJCLTVZPLZKY-UHFFFAOYSA-N 0.000 description 1
- 241000700605 Viruses Species 0.000 description 1
- 239000003875 Wang resin Substances 0.000 description 1
- 240000008042 Zea mays Species 0.000 description 1
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 description 1
- 235000002017 Zea mays subsp mays Nutrition 0.000 description 1
- 239000001089 [(2R)-oxolan-2-yl]methanol Substances 0.000 description 1
- DGEZNRSVGBDHLK-UHFFFAOYSA-N [1,10]phenanthroline Chemical compound C1=CN=C2C3=NC=CC=C3C=CC2=C1 DGEZNRSVGBDHLK-UHFFFAOYSA-N 0.000 description 1
- NERFNHBZJXXFGY-UHFFFAOYSA-N [4-[(4-methylphenyl)methoxy]phenyl]methanol Chemical compound C1=CC(C)=CC=C1COC1=CC=C(CO)C=C1 NERFNHBZJXXFGY-UHFFFAOYSA-N 0.000 description 1
- YDONNITUKPKTIG-UHFFFAOYSA-N [Nitrilotris(methylene)]trisphosphonic acid Chemical compound OP(O)(=O)CN(CP(O)(O)=O)CP(O)(O)=O YDONNITUKPKTIG-UHFFFAOYSA-N 0.000 description 1
- 239000003655 absorption accelerator Substances 0.000 description 1
- 230000002378 acidificating effect Effects 0.000 description 1
- 125000002252 acyl group Chemical group 0.000 description 1
- 208000009956 adenocarcinoma Diseases 0.000 description 1
- 239000003463 adsorbent Substances 0.000 description 1
- 239000000556 agonist Substances 0.000 description 1
- 235000010443 alginic acid Nutrition 0.000 description 1
- 239000000783 alginic acid Substances 0.000 description 1
- 229920000615 alginic acid Polymers 0.000 description 1
- 229960001126 alginic acid Drugs 0.000 description 1
- 150000004781 alginic acids Chemical class 0.000 description 1
- 125000001931 aliphatic group Chemical group 0.000 description 1
- 125000003545 alkoxy group Chemical group 0.000 description 1
- 125000003282 alkyl amino group Chemical group 0.000 description 1
- 125000005276 alkyl hydrazino group Chemical group 0.000 description 1
- 229910052782 aluminium Inorganic materials 0.000 description 1
- XAGFODPZIPBFFR-UHFFFAOYSA-N aluminium Chemical compound [Al] XAGFODPZIPBFFR-UHFFFAOYSA-N 0.000 description 1
- 235000012211 aluminium silicate Nutrition 0.000 description 1
- 238000010976 amide bond formation reaction Methods 0.000 description 1
- 150000001414 amino alcohols Chemical class 0.000 description 1
- LHIJANUOQQMGNT-UHFFFAOYSA-N aminoethylethanolamine Chemical compound NCCNCCO LHIJANUOQQMGNT-UHFFFAOYSA-N 0.000 description 1
- 150000008064 anhydrides Chemical class 0.000 description 1
- 239000003242 anti bacterial agent Substances 0.000 description 1
- 230000000844 anti-bacterial effect Effects 0.000 description 1
- 230000003127 anti-melanomic effect Effects 0.000 description 1
- 239000003429 antifungal agent Substances 0.000 description 1
- 229940121375 antifungal agent Drugs 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 239000012736 aqueous medium Substances 0.000 description 1
- 229910052786 argon Inorganic materials 0.000 description 1
- 125000006615 aromatic heterocyclic group Chemical group 0.000 description 1
- 238000003491 array Methods 0.000 description 1
- 125000001769 aryl amino group Chemical group 0.000 description 1
- 125000000732 arylene group Chemical group 0.000 description 1
- 125000002648 azanetriyl group Chemical group *N(*)* 0.000 description 1
- 229960002903 benzyl benzoate Drugs 0.000 description 1
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 1
- 239000011230 binding agent Substances 0.000 description 1
- 230000031018 biological processes and functions Effects 0.000 description 1
- 230000033228 biological regulation Effects 0.000 description 1
- 229910052797 bismuth Inorganic materials 0.000 description 1
- JCXGWMGPZLAOME-UHFFFAOYSA-N bismuth atom Chemical compound [Bi] JCXGWMGPZLAOME-UHFFFAOYSA-N 0.000 description 1
- 239000012503 blood component Substances 0.000 description 1
- 230000036760 body temperature Effects 0.000 description 1
- 210000001185 bone marrow Anatomy 0.000 description 1
- 229910052794 bromium Inorganic materials 0.000 description 1
- 239000006172 buffering agent Substances 0.000 description 1
- 235000019437 butane-1,3-diol Nutrition 0.000 description 1
- 125000000484 butyl group Chemical group [H]C([*])([H])C([H])([H])C([H])([H])C([H])([H])[H] 0.000 description 1
- 210000004899 c-terminal region Anatomy 0.000 description 1
- 229910000019 calcium carbonate Inorganic materials 0.000 description 1
- 235000010216 calcium carbonate Nutrition 0.000 description 1
- 239000001506 calcium phosphate Substances 0.000 description 1
- CJZGTCYPCWQAJB-UHFFFAOYSA-L calcium stearate Chemical compound [Ca+2].CCCCCCCCCCCCCCCCCC([O-])=O.CCCCCCCCCCCCCCCCCC([O-])=O CJZGTCYPCWQAJB-UHFFFAOYSA-L 0.000 description 1
- 239000008116 calcium stearate Substances 0.000 description 1
- 235000013539 calcium stearate Nutrition 0.000 description 1
- 125000002837 carbocyclic group Chemical group 0.000 description 1
- 150000001718 carbodiimides Chemical class 0.000 description 1
- BVKZGUZCCUSVTD-UHFFFAOYSA-N carbonic acid Chemical class OC(O)=O BVKZGUZCCUSVTD-UHFFFAOYSA-N 0.000 description 1
- 125000002915 carbonyl group Chemical group [*:2]C([*:1])=O 0.000 description 1
- 125000005708 carbonyloxy group Chemical group [*:2]OC([*:1])=O 0.000 description 1
- 239000001768 carboxy methyl cellulose Substances 0.000 description 1
- 235000010948 carboxy methyl cellulose Nutrition 0.000 description 1
- 125000005026 carboxyaryl group Chemical group 0.000 description 1
- 239000008112 carboxymethyl-cellulose Substances 0.000 description 1
- 239000004359 castor oil Substances 0.000 description 1
- 235000019438 castor oil Nutrition 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 239000013592 cell lysate Substances 0.000 description 1
- 239000001913 cellulose Substances 0.000 description 1
- 229920002678 cellulose Polymers 0.000 description 1
- 229960000541 cetyl alcohol Drugs 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 229910052801 chlorine Inorganic materials 0.000 description 1
- 125000002668 chloroacetyl group Chemical group ClCC(=O)* 0.000 description 1
- 229960004926 chlorobutanol Drugs 0.000 description 1
- HLVXFWDLRHCZEI-UHFFFAOYSA-N chromotropic acid Chemical compound OS(=O)(=O)C1=CC(O)=C2C(O)=CC(S(O)(=O)=O)=CC2=C1 HLVXFWDLRHCZEI-UHFFFAOYSA-N 0.000 description 1
- 235000015165 citric acid Nutrition 0.000 description 1
- 239000011248 coating agent Substances 0.000 description 1
- 229940110456 cocoa butter Drugs 0.000 description 1
- 235000019868 cocoa butter Nutrition 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- 235000005822 corn Nutrition 0.000 description 1
- 235000012343 cottonseed oil Nutrition 0.000 description 1
- 239000002385 cottonseed oil Substances 0.000 description 1
- 125000004122 cyclic group Chemical group 0.000 description 1
- 230000000254 damaging effect Effects 0.000 description 1
- 230000003111 delayed effect Effects 0.000 description 1
- 238000012217 deletion Methods 0.000 description 1
- 230000037430 deletion Effects 0.000 description 1
- 238000001212 derivatisation Methods 0.000 description 1
- 238000000502 dialysis Methods 0.000 description 1
- 125000000664 diazo group Chemical group [N-]=[N+]=[*] 0.000 description 1
- NEFBYIFKOOEVPA-UHFFFAOYSA-K dicalcium phosphate Chemical compound [Ca+2].[Ca+2].[O-]P([O-])([O-])=O NEFBYIFKOOEVPA-UHFFFAOYSA-K 0.000 description 1
- 229940038472 dicalcium phosphate Drugs 0.000 description 1
- 229910000390 dicalcium phosphate Inorganic materials 0.000 description 1
- 235000005911 diet Nutrition 0.000 description 1
- 230000037213 diet Effects 0.000 description 1
- 235000014113 dietary fatty acids Nutrition 0.000 description 1
- IRDLUHRVLVEUHA-UHFFFAOYSA-N diethyl dithiophosphate Chemical compound CCOP(S)(=S)OCC IRDLUHRVLVEUHA-UHFFFAOYSA-N 0.000 description 1
- LMBWSYZSUOEYSN-UHFFFAOYSA-N diethyldithiocarbamic acid Chemical compound CCN(CC)C(S)=S LMBWSYZSUOEYSN-UHFFFAOYSA-N 0.000 description 1
- UGMCXQCYOVCMTB-UHFFFAOYSA-K dihydroxy(stearato)aluminium Chemical compound CCCCCCCCCCCCCCCCCC(=O)O[Al](O)O UGMCXQCYOVCMTB-UHFFFAOYSA-K 0.000 description 1
- 238000007865 diluting Methods 0.000 description 1
- 239000003085 diluting agent Substances 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- WQABCVAJNWAXTE-UHFFFAOYSA-N dimercaprol Chemical compound OCC(S)CS WQABCVAJNWAXTE-UHFFFAOYSA-N 0.000 description 1
- JGUQDUKBUKFFRO-CIIODKQPSA-N dimethylglyoxime Chemical compound O/N=C(/C)\C(\C)=N\O JGUQDUKBUKFFRO-CIIODKQPSA-N 0.000 description 1
- 150000002009 diols Chemical group 0.000 description 1
- 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 1
- KAKKHKRHCKCAGH-UHFFFAOYSA-L disodium;(4-nitrophenyl) phosphate;hexahydrate Chemical compound O.O.O.O.O.O.[Na+].[Na+].[O-][N+](=O)C1=CC=C(OP([O-])([O-])=O)C=C1 KAKKHKRHCKCAGH-UHFFFAOYSA-L 0.000 description 1
- 125000004119 disulfanediyl group Chemical group *SS* 0.000 description 1
- 229950004394 ditiocarb Drugs 0.000 description 1
- 239000008298 dragée Substances 0.000 description 1
- NFDRPXJGHKJRLJ-UHFFFAOYSA-N edtmp Chemical compound OP(O)(=O)CN(CP(O)(O)=O)CCN(CP(O)(O)=O)CP(O)(O)=O NFDRPXJGHKJRLJ-UHFFFAOYSA-N 0.000 description 1
- 238000001962 electrophoresis Methods 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 239000002702 enteric coating Substances 0.000 description 1
- 238000009505 enteric coating Methods 0.000 description 1
- 230000002255 enzymatic effect Effects 0.000 description 1
- 125000004185 ester group Chemical group 0.000 description 1
- BEFDCLMNVWHSGT-UHFFFAOYSA-N ethenylcyclopentane Chemical compound C=CC1CCCC1 BEFDCLMNVWHSGT-UHFFFAOYSA-N 0.000 description 1
- 125000001033 ether group Chemical group 0.000 description 1
- 150000002170 ethers Chemical class 0.000 description 1
- AEOCXXJPGCBFJA-UHFFFAOYSA-N ethionamide Chemical compound CCC1=CC(C(N)=S)=CC=N1 AEOCXXJPGCBFJA-UHFFFAOYSA-N 0.000 description 1
- 229940093499 ethyl acetate Drugs 0.000 description 1
- LVGKNOAMLMIIKO-QXMHVHEDSA-N ethyl oleate Chemical compound CCCCCCCC\C=C/CCCCCCCC(=O)OCC LVGKNOAMLMIIKO-QXMHVHEDSA-N 0.000 description 1
- 229940093471 ethyl oleate Drugs 0.000 description 1
- 125000004672 ethylcarbonyl group Chemical group [H]C([H])([H])C([H])([H])C(*)=O 0.000 description 1
- OGPBJKLSAFTDLK-UHFFFAOYSA-N europium atom Chemical compound [Eu] OGPBJKLSAFTDLK-UHFFFAOYSA-N 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 239000003885 eye ointment Substances 0.000 description 1
- 229930195729 fatty acid Natural products 0.000 description 1
- 239000000194 fatty acid Substances 0.000 description 1
- 239000012894 fetal calf serum Substances 0.000 description 1
- 239000000796 flavoring agent Substances 0.000 description 1
- 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 1
- 229910052731 fluorine Inorganic materials 0.000 description 1
- 229960000304 folic acid Drugs 0.000 description 1
- 102000006640 gamma-Glutamyltransferase Human genes 0.000 description 1
- 238000001502 gel electrophoresis Methods 0.000 description 1
- 239000007903 gelatin capsule Substances 0.000 description 1
- 238000002523 gelfiltration Methods 0.000 description 1
- 239000011491 glass wool Substances 0.000 description 1
- 239000000174 gluconic acid Substances 0.000 description 1
- 235000012208 gluconic acid Nutrition 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- 235000001727 glucose Nutrition 0.000 description 1
- YQEMORVAKMFKLG-UHFFFAOYSA-N glycerine monostearate Natural products CCCCCCCCCCCCCCCCCC(=O)OC(CO)CO YQEMORVAKMFKLG-UHFFFAOYSA-N 0.000 description 1
- SVUQHVRAGMNPLW-UHFFFAOYSA-N glycerol monostearate Natural products CCCCCCCCCCCCCCCCC(=O)OCC(O)CO SVUQHVRAGMNPLW-UHFFFAOYSA-N 0.000 description 1
- ZEMPKEQAKRGZGQ-XOQCFJPHSA-N glycerol triricinoleate Natural products CCCCCC[C@@H](O)CC=CCCCCCCCC(=O)OC[C@@H](COC(=O)CCCCCCCC=CC[C@@H](O)CCCCCC)OC(=O)CCCCCCCC=CC[C@H](O)CCCCCC ZEMPKEQAKRGZGQ-XOQCFJPHSA-N 0.000 description 1
- 239000003102 growth factor Substances 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 125000004415 heterocyclylalkyl group Chemical group 0.000 description 1
- BXWNKGSJHAJOGX-UHFFFAOYSA-N hexadecan-1-ol Chemical compound CCCCCCCCCCCCCCCCO BXWNKGSJHAJOGX-UHFFFAOYSA-N 0.000 description 1
- 125000004836 hexamethylene group Chemical group [H]C([H])([*:2])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])[*:1] 0.000 description 1
- 239000012145 high-salt buffer Substances 0.000 description 1
- 239000003906 humectant Substances 0.000 description 1
- 125000000717 hydrazino group Chemical group [H]N([*])N([H])[H] 0.000 description 1
- BHEPBYXIRTUNPN-UHFFFAOYSA-N hydridophosphorus(.) (triplet) Chemical compound [PH] BHEPBYXIRTUNPN-UHFFFAOYSA-N 0.000 description 1
- 230000002209 hydrophobic effect Effects 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-M hydroxide Chemical compound [OH-] XLYOFNOQVPJJNP-UHFFFAOYSA-M 0.000 description 1
- 125000005027 hydroxyaryl group Chemical group 0.000 description 1
- 125000001841 imino group Chemical group [H]N=* 0.000 description 1
- 230000001900 immune effect Effects 0.000 description 1
- 210000000987 immune system Anatomy 0.000 description 1
- 230000005847 immunogenicity Effects 0.000 description 1
- 230000002779 inactivation Effects 0.000 description 1
- APFVFJFRJDLVQX-UHFFFAOYSA-N indium atom Chemical compound [In] APFVFJFRJDLVQX-UHFFFAOYSA-N 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- 125000006303 iodophenyl group Chemical group 0.000 description 1
- 229910052742 iron Inorganic materials 0.000 description 1
- 125000001261 isocyanato group Chemical group *N=C=O 0.000 description 1
- 125000001449 isopropyl group Chemical group [H]C([H])([H])C([H])(*)C([H])([H])[H] 0.000 description 1
- ZBKFYXZXZJPWNQ-UHFFFAOYSA-N isothiocyanate group Chemical group [N-]=C=S ZBKFYXZXZJPWNQ-UHFFFAOYSA-N 0.000 description 1
- 125000001810 isothiocyanato group Chemical group *N=C=S 0.000 description 1
- 239000007951 isotonicity adjuster Substances 0.000 description 1
- NLYAJNPCOHFWQQ-UHFFFAOYSA-N kaolin Chemical compound O.O.O=[Al]O[Si](=O)O[Si](=O)O[Al]=O NLYAJNPCOHFWQQ-UHFFFAOYSA-N 0.000 description 1
- 229910052747 lanthanoid Inorganic materials 0.000 description 1
- 150000002602 lanthanoids Chemical class 0.000 description 1
- 230000002045 lasting effect Effects 0.000 description 1
- 229910052745 lead Inorganic materials 0.000 description 1
- 239000000787 lecithin Substances 0.000 description 1
- 235000010445 lecithin Nutrition 0.000 description 1
- 229940067606 lecithin Drugs 0.000 description 1
- 239000002523 lectin Substances 0.000 description 1
- 208000032839 leukemia Diseases 0.000 description 1
- 239000012035 limiting reagent Substances 0.000 description 1
- 150000002632 lipids Chemical class 0.000 description 1
- 239000000314 lubricant Substances 0.000 description 1
- 235000019359 magnesium stearate Nutrition 0.000 description 1
- 238000012423 maintenance Methods 0.000 description 1
- 239000000594 mannitol Substances 0.000 description 1
- 235000010355 mannitol Nutrition 0.000 description 1
- 238000001819 mass spectrum Methods 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 238000002844 melting Methods 0.000 description 1
- 230000008018 melting Effects 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- HZVOZRGWRWCICA-UHFFFAOYSA-N methanediyl Chemical compound [CH2] HZVOZRGWRWCICA-UHFFFAOYSA-N 0.000 description 1
- GGIPEPBRQYWGNV-UHFFFAOYSA-N methyl 2-[4-[(2,4-diaminopyrimidin-5-yl)methyl]-2,6-dimethoxyphenoxy]acetate Chemical compound C1=C(OC)C(OCC(=O)OC)=C(OC)C=C1CC1=CN=C(N)N=C1N GGIPEPBRQYWGNV-UHFFFAOYSA-N 0.000 description 1
- YDCHPLOFQATIDS-UHFFFAOYSA-N methyl 2-bromoacetate Chemical compound COC(=O)CBr YDCHPLOFQATIDS-UHFFFAOYSA-N 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 238000012544 monitoring process Methods 0.000 description 1
- CQDGTJPVBWZJAZ-UHFFFAOYSA-N monoethyl carbonate Chemical compound CCOC(O)=O CQDGTJPVBWZJAZ-UHFFFAOYSA-N 0.000 description 1
- 239000004570 mortar (masonry) Substances 0.000 description 1
- 230000001338 necrotic effect Effects 0.000 description 1
- 229910052759 nickel Inorganic materials 0.000 description 1
- MGFYIUFZLHCRTH-UHFFFAOYSA-N nitrilotriacetic acid Chemical compound OC(=O)CN(CC(O)=O)CC(O)=O MGFYIUFZLHCRTH-UHFFFAOYSA-N 0.000 description 1
- 231100000344 non-irritating Toxicity 0.000 description 1
- 239000012457 nonaqueous media Substances 0.000 description 1
- 108020004707 nucleic acids Proteins 0.000 description 1
- 102000039446 nucleic acids Human genes 0.000 description 1
- 150000007523 nucleic acids Chemical class 0.000 description 1
- 239000012011 nucleophilic catalyst Substances 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 150000002895 organic esters Chemical class 0.000 description 1
- 150000002923 oximes Chemical class 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-O oxonium Chemical compound [OH3+] XLYOFNOQVPJJNP-UHFFFAOYSA-O 0.000 description 1
- 125000005740 oxycarbonyl group Chemical group [*:1]OC([*:2])=O 0.000 description 1
- 229960003540 oxyquinoline Drugs 0.000 description 1
- 229940055729 papain Drugs 0.000 description 1
- 235000019834 papain Nutrition 0.000 description 1
- 239000012188 paraffin wax Substances 0.000 description 1
- 244000045947 parasite Species 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 239000000312 peanut oil Substances 0.000 description 1
- 229960003330 pentetic acid Drugs 0.000 description 1
- 229940111202 pepsin Drugs 0.000 description 1
- 239000002304 perfume Substances 0.000 description 1
- 229960003742 phenol Drugs 0.000 description 1
- 150000002989 phenols Chemical class 0.000 description 1
- WVDDGKGOMKODPV-ZQBYOMGUSA-N phenyl(114C)methanol Chemical compound O[14CH2]C1=CC=CC=C1 WVDDGKGOMKODPV-ZQBYOMGUSA-N 0.000 description 1
- 125000000286 phenylethyl group Chemical group [H]C1=C([H])C([H])=C(C([H])=C1[H])C([H])([H])C([H])([H])* 0.000 description 1
- 125000002467 phosphate group Chemical group [H]OP(=O)(O[H])O[*] 0.000 description 1
- 150000003904 phospholipids Chemical class 0.000 description 1
- UEZVMMHDMIWARA-UHFFFAOYSA-M phosphonate Chemical compound [O-]P(=O)=O UEZVMMHDMIWARA-UHFFFAOYSA-M 0.000 description 1
- 150000003009 phosphonic acids Chemical class 0.000 description 1
- 150000008300 phosphoramidites Chemical class 0.000 description 1
- IEQIEDJGQAUEQZ-UHFFFAOYSA-N phthalocyanine Chemical compound N1C(N=C2C3=CC=CC=C3C(N=C3C4=CC=CC=C4C(=N4)N3)=N2)=C(C=CC=C2)C2=C1N=C1C2=CC=CC=C2C4=N1 IEQIEDJGQAUEQZ-UHFFFAOYSA-N 0.000 description 1
- 229920000233 poly(alkylene oxides) Polymers 0.000 description 1
- 229920002401 polyacrylamide Polymers 0.000 description 1
- 229920000768 polyamine Polymers 0.000 description 1
- 108010064470 polyaspartate Proteins 0.000 description 1
- 108010077051 polycysteine Proteins 0.000 description 1
- 239000004848 polyfunctional curative Substances 0.000 description 1
- 229920002643 polyglutamic acid Polymers 0.000 description 1
- 229920002704 polyhistidine Polymers 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
- 239000001205 polyphosphate Substances 0.000 description 1
- 235000011176 polyphosphates Nutrition 0.000 description 1
- 229920001282 polysaccharide Polymers 0.000 description 1
- 229920005990 polystyrene resin Polymers 0.000 description 1
- 239000001267 polyvinylpyrrolidone Substances 0.000 description 1
- 229920000036 polyvinylpyrrolidone Polymers 0.000 description 1
- 235000013855 polyvinylpyrrolidone Nutrition 0.000 description 1
- JCBJVAJGLKENNC-UHFFFAOYSA-M potassium ethyl xanthate Chemical compound [K+].CCOC([S-])=S JCBJVAJGLKENNC-UHFFFAOYSA-M 0.000 description 1
- 238000004321 preservation Methods 0.000 description 1
- 239000003755 preservative agent Substances 0.000 description 1
- 230000002265 prevention Effects 0.000 description 1
- 239000003380 propellant Substances 0.000 description 1
- 208000023958 prostate neoplasm Diseases 0.000 description 1
- 108010043671 prostatic acid phosphatase Proteins 0.000 description 1
- 230000009145 protein modification Effects 0.000 description 1
- UMJSCPRVCHMLSP-UHFFFAOYSA-N pyridine Natural products COC1=CC=CN=C1 UMJSCPRVCHMLSP-UHFFFAOYSA-N 0.000 description 1
- 150000003222 pyridines Chemical class 0.000 description 1
- WKSAUQYGYAYLPV-UHFFFAOYSA-N pyrimethamine Chemical compound CCC1=NC(N)=NC(N)=C1C1=CC=C(Cl)C=C1 WKSAUQYGYAYLPV-UHFFFAOYSA-N 0.000 description 1
- 229960000611 pyrimethamine Drugs 0.000 description 1
- YAAWASYJIRZXSZ-UHFFFAOYSA-N pyrimidine-2,4-diamine Chemical compound NC1=CC=NC(N)=N1 YAAWASYJIRZXSZ-UHFFFAOYSA-N 0.000 description 1
- 150000003856 quaternary ammonium compounds Chemical class 0.000 description 1
- MCJGNVYPOGVAJF-UHFFFAOYSA-N quinolin-8-ol Chemical compound C1=CN=C2C(O)=CC=CC2=C1 MCJGNVYPOGVAJF-UHFFFAOYSA-N 0.000 description 1
- 238000007348 radical reaction Methods 0.000 description 1
- 150000003254 radicals Chemical class 0.000 description 1
- 238000011363 radioimmunotherapy Methods 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
- 238000011084 recovery Methods 0.000 description 1
- 210000000664 rectum Anatomy 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 229910052707 ruthenium Inorganic materials 0.000 description 1
- ORIHZIZPTZTNCU-YVMONPNESA-N salicylaldoxime Chemical compound O\N=C/C1=CC=CC=C1O ORIHZIZPTZTNCU-YVMONPNESA-N 0.000 description 1
- 229920006395 saturated elastomer Polymers 0.000 description 1
- 229910052706 scandium Inorganic materials 0.000 description 1
- SIXSYDAISGFNSX-UHFFFAOYSA-N scandium atom Chemical compound [Sc] SIXSYDAISGFNSX-UHFFFAOYSA-N 0.000 description 1
- 125000001439 semicarbazido group Chemical group [H]N([H])C(=O)N([H])N([H])* 0.000 description 1
- 239000008159 sesame oil Substances 0.000 description 1
- 235000011803 sesame oil Nutrition 0.000 description 1
- 150000004760 silicates Chemical class 0.000 description 1
- RMAQACBXLXPBSY-UHFFFAOYSA-N silicic acid Chemical compound O[Si](O)(O)O RMAQACBXLXPBSY-UHFFFAOYSA-N 0.000 description 1
- 235000012239 silicon dioxide Nutrition 0.000 description 1
- 239000002002 slurry Substances 0.000 description 1
- 229910000030 sodium bicarbonate Inorganic materials 0.000 description 1
- 235000017557 sodium bicarbonate Nutrition 0.000 description 1
- 229910000029 sodium carbonate Inorganic materials 0.000 description 1
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 1
- 235000019832 sodium triphosphate Nutrition 0.000 description 1
- 239000008247 solid mixture Substances 0.000 description 1
- 239000007790 solid phase Substances 0.000 description 1
- 239000004334 sorbic acid Substances 0.000 description 1
- 235000010199 sorbic acid Nutrition 0.000 description 1
- 229940075582 sorbic acid Drugs 0.000 description 1
- 239000000600 sorbitol Substances 0.000 description 1
- 230000009870 specific binding Effects 0.000 description 1
- 239000007921 spray Substances 0.000 description 1
- 206010041823 squamous cell carcinoma Diseases 0.000 description 1
- 239000008107 starch Substances 0.000 description 1
- 210000000130 stem cell Anatomy 0.000 description 1
- 150000003431 steroids Chemical class 0.000 description 1
- 239000011550 stock solution Substances 0.000 description 1
- 229910052712 strontium Inorganic materials 0.000 description 1
- 125000003107 substituted aryl group Chemical group 0.000 description 1
- ACTRVOBWPAIOHC-XIXRPRMCSA-N succimer Chemical compound OC(=O)[C@@H](S)[C@@H](S)C(O)=O ACTRVOBWPAIOHC-XIXRPRMCSA-N 0.000 description 1
- 229960005346 succimer Drugs 0.000 description 1
- JJAHTWIKCUJRDK-UHFFFAOYSA-N succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate Chemical compound C1CC(CN2C(C=CC2=O)=O)CCC1C(=O)ON1C(=O)CCC1=O JJAHTWIKCUJRDK-UHFFFAOYSA-N 0.000 description 1
- 235000000346 sugar Nutrition 0.000 description 1
- 150000008163 sugars Chemical class 0.000 description 1
- 125000000446 sulfanediyl group Chemical group *S* 0.000 description 1
- 125000000475 sulfinyl group Chemical group [*:2]S([*:1])=O 0.000 description 1
- BDHFUVZGWQCTTF-UHFFFAOYSA-M sulfonate Chemical compound [O-]S(=O)=O BDHFUVZGWQCTTF-UHFFFAOYSA-M 0.000 description 1
- 150000003457 sulfones Chemical class 0.000 description 1
- 125000000472 sulfonyl group Chemical group *S(*)(=O)=O 0.000 description 1
- 150000003462 sulfoxides Chemical class 0.000 description 1
- 150000003464 sulfur compounds Chemical class 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 239000004094 surface-active agent Substances 0.000 description 1
- 239000003765 sweetening agent Substances 0.000 description 1
- 239000006188 syrup Substances 0.000 description 1
- 235000020357 syrup Nutrition 0.000 description 1
- 239000000454 talc Substances 0.000 description 1
- 229910052623 talc Inorganic materials 0.000 description 1
- 235000012222 talc Nutrition 0.000 description 1
- 239000011975 tartaric acid Substances 0.000 description 1
- 235000002906 tartaric acid Nutrition 0.000 description 1
- GKLVYJBZJHMRIY-UHFFFAOYSA-N technetium atom Chemical compound [Tc] GKLVYJBZJHMRIY-UHFFFAOYSA-N 0.000 description 1
- BSYVTEYKTMYBMK-UHFFFAOYSA-N tetrahydrofurfuryl alcohol Chemical compound OCC1CCCO1 BSYVTEYKTMYBMK-UHFFFAOYSA-N 0.000 description 1
- YNHJECZULSZAQK-UHFFFAOYSA-N tetraphenylporphyrin Chemical compound C1=CC(C(=C2C=CC(N2)=C(C=2C=CC=CC=2)C=2C=CC(N=2)=C(C=2C=CC=CC=2)C2=CC=C3N2)C=2C=CC=CC=2)=NC1=C3C1=CC=CC=C1 YNHJECZULSZAQK-UHFFFAOYSA-N 0.000 description 1
- WSWJIZXMAUYHOE-UHFFFAOYSA-N tetroxoprim Chemical compound C1=C(OC)C(OCCOC)=C(OC)C=C1CC1=CN=C(N)N=C1N WSWJIZXMAUYHOE-UHFFFAOYSA-N 0.000 description 1
- 229960004809 tetroxoprim Drugs 0.000 description 1
- 229910052716 thallium Inorganic materials 0.000 description 1
- TXBBUSUXYMIVOS-UHFFFAOYSA-N thenoyltrifluoroacetone Chemical compound FC(F)(F)C(=O)CC(=O)C1=CC=CS1 TXBBUSUXYMIVOS-UHFFFAOYSA-N 0.000 description 1
- 230000004797 therapeutic response Effects 0.000 description 1
- 150000007970 thio esters Chemical class 0.000 description 1
- 125000002813 thiocarbonyl group Chemical group *C(*)=S 0.000 description 1
- 125000000101 thioether group Chemical group 0.000 description 1
- 229910052718 tin Inorganic materials 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
- 239000003053 toxin Substances 0.000 description 1
- 231100000765 toxin Toxicity 0.000 description 1
- 108700012359 toxins Proteins 0.000 description 1
- 235000010487 tragacanth Nutrition 0.000 description 1
- 239000000196 tragacanth Substances 0.000 description 1
- 229940116362 tragacanth Drugs 0.000 description 1
- 230000007704 transition Effects 0.000 description 1
- 210000004881 tumor cell Anatomy 0.000 description 1
- 229910052721 tungsten Inorganic materials 0.000 description 1
- 229910052720 vanadium Inorganic materials 0.000 description 1
- 235000015112 vegetable and seed oil Nutrition 0.000 description 1
- 239000008158 vegetable oil Substances 0.000 description 1
- 239000011782 vitamin Substances 0.000 description 1
- 229940088594 vitamin Drugs 0.000 description 1
- 235000013343 vitamin Nutrition 0.000 description 1
- 229930003231 vitamin Natural products 0.000 description 1
- 238000009736 wetting Methods 0.000 description 1
- 229910052727 yttrium Inorganic materials 0.000 description 1
- VWQVUPCCIRVNHF-UHFFFAOYSA-N yttrium atom Chemical compound [Y] VWQVUPCCIRVNHF-UHFFFAOYSA-N 0.000 description 1
- 229910052725 zinc Inorganic materials 0.000 description 1
- DGVVWUTYPXICAM-UHFFFAOYSA-N β‐Mercaptoethanol Chemical compound OCCS DGVVWUTYPXICAM-UHFFFAOYSA-N 0.000 description 1
Classifications
-
- 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
- A61K51/08—Peptides, e.g. proteins, carriers being peptides, polyamino acids, proteins
- A61K51/10—Antibodies or immunoglobulins; Fragments thereof, the carrier being an antibody, an immunoglobulin or a fragment thereof, e.g. a camelised human single domain antibody or the Fc fragment of an antibody
- A61K51/1093—Antibodies or immunoglobulins; Fragments thereof, the carrier being an antibody, an immunoglobulin or a fragment thereof, e.g. a camelised human single domain antibody or the Fc fragment of an antibody conjugates with carriers being antibodies
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
- A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
- A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
- A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
- A61K47/6881—Cluster-antibody conjugates, i.e. the modifying agent consists of a plurality of antibodies covalently linked to each other or of different antigen-binding fragments covalently linked to each other
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
- A61K47/6889—Conjugates wherein the antibody being the modifying agent and wherein the linker, binder or spacer confers particular properties to the conjugates, e.g. peptidic enzyme-labile linkers or acid-labile linkers, providing for an acid-labile immuno conjugate wherein the drug may be released from its antibody conjugated part in an acidic, e.g. tumoural or environment
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/531—Production of immunochemical test materials
- G01N33/532—Production of labelled immunochemicals
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/531—Production of immunochemical test materials
- G01N33/532—Production of labelled immunochemicals
- G01N33/535—Production of labelled immunochemicals with enzyme label or co-enzymes, co-factors, enzyme inhibitors or enzyme substrates
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/573—Immunoassay; Biospecific binding assay; Materials therefor for enzymes or isoenzymes
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2121/00—Preparations for use in therapy
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2123/00—Preparations for testing in vivo
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Immunology (AREA)
- Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- Medicinal Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Molecular Biology (AREA)
- Biomedical Technology (AREA)
- Urology & Nephrology (AREA)
- Hematology (AREA)
- Epidemiology (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Animal Behavior & Ethology (AREA)
- Pharmacology & Pharmacy (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Biotechnology (AREA)
- Cell Biology (AREA)
- Pathology (AREA)
- Microbiology (AREA)
- General Physics & Mathematics (AREA)
- Biochemistry (AREA)
- Food Science & Technology (AREA)
- Optics & Photonics (AREA)
- Oncology (AREA)
- Inorganic Chemistry (AREA)
- Hospice & Palliative Care (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
- Peptides Or Proteins (AREA)
Description
IMMUNOREACTIVE REAGENTS EMPLOYING
DIHYDROFOLATE REDUCTASE Field of the Invention
This invention relates to the therapeutic treatment and diagnostic imaging of cancer by means of a tumor targeted sequential delivery system comprised of a primary non-radioactive targeting immunoreagent and a secondary radioactive delivery agent.
Background of the Invention The various, currently available, radiolabeled immunoreactive proteins which are employed in diagnostic imaging and targeted therapeutic applications suffer from certain of the following disadvantages:
1) toxicity;
2) destruction or excretion of the reagent due to rapid catabolism or metabolism;
3) inadequate energy of radioactive emission which results in a low signal to noise ratio;
4) inefficient covalent bonding of the radioactive component with protein in conjugate preparation;
5) long plasma half-lives of currently available radionuclide-containing immunoreactive proteins result in prolonged exposure of normal tissue to damaging effects of radiation that can produce unacceptable toxic effects in otherwise normal and disease free tissues in the body, especially in those tissues and cells most sensitive to radiation damage, e.g., the stem cells of the bone marrow and gastrointestinal tract;
6) slow clearance of radionuclide from the body;
7) increasing the number of sites of incorporation of radionuclide or chelated radionuclide results in a
reduction in the capacity of the radionuclide-containing immunoreactive protein to bind to its target;
8) the number of chelating agents that can be attached to an immunoreactive protein is limited by the need to restrict chemical conjugations to sites removed from the immunoreactive recognition or binding sites of the protein;
9) the number of chelating agents that can be attached to an immunoreactive protein is limited by the number of available groups such as, for example, amino groups suitable for use in attachment of the chelating agents;
15) the number of chelating agents that can be attached to an immunoreactive protein is limited by the potential immunogenicity of the thus modified protein which, being highly derivatized, could be recognized by the immune system as haptenated;
16) the number of ionic radionuclides that can be associated with an immunoreactive protein is restricted by the number of sites of chelation available; and
17) radioimmunotherapy and diagnostic imaging with the various currently available radionuclide containing immunoreactive proteins can be less than optimal because these radiopharmaceuticals may bind to non-target normal tissue, which binding can result in undesirable toxicity to normal tissue during therapeutic applications as well as in high background signals during diagnostic imaging applications.
It is an object of the present invention to
overcome the aforementioned disadvantages of the
currently available radiolabeled immunoreactive
proteins.
Summary of the Invention
The present invention is directed to systems which are useful in the therapeutic treatment and diagnostic imaging of tissue, particularly of cancerous tissue. For a disease such as cancer, such systems comprise a tumor targeted sequential delivery system comprised of a primary non-radioactive targeting immunoreagent and a secondary radioactive delivery agent.
In one embodiment, the present invention is directed to a non-radioactive targeting immunoreagent (sometimes hereinafter referred to as NRTIR) comprised of the residue of a receptor moiety, a linking group, and the residue of an immunoreactive material, which
NRTIR is administered to a tissue of interest and will bind to sites on the surfaces of cells thereof.
In this embodiment, the present invention is also directed to a radioactive delivery agent (sometimes hereinafter referred to as RDA) comprised of the residue of a ligand which has an affinity for non-covalent binding to a receptor moiety, a linking group, and the residue of a radioactive agent. This RDA is
administered to the environs of the tissue which contains said NRTIR bound thereto. In particular, the ligand residue of this RDA will non-covalently bind to the receptor of said NRTIR which is bound to the cells of said tissue of interest. Thus, an effective amount of radioactivity is provided to said tissue. RDA which is unbound to NRTIR can be removed rapidly from the environs of the tissue.
In one aspect of this embodiment (sometimes hereinafter referred to as System A), the present invention comprises an NRTIR comprised of the residue of a receptor moiety which is comprised of the residue of a proteinaceous active site of a dihydrofolate reductase
enzyme (sometimes hereinafter referred to as a DHFR), a linking group, and the residue of an immunoreactive material. In system A, the present invention also comprises an RDA comprised of a ligand which has an affinity for non-covalent binding to a DHFR receptor moiety, a linking group, and the residue of a
radioactive agent.
Specifically, in System A, the present invention is directed to an NRTIR comprised of a residue of the proteinaceous active site of a dihydrofolate reductase enzyme, a linking group, and the residue of an
immunoreactive material such as a tumor targeting antibody together with an RDA comprised of the residue of a ligand which has an affinity for non-covalent binding to said DHFR receptor moiety, a linking group, and the residue of a radioactive agent comprised of the residue of a chelating agent and a radionuclide.
The NRTIR of system A is comprised of (n) DHFR moieties, each of which can non-covalently bind an RDA comprised of the residue of a ligand with an affinity for non-covalent binding to a DHFR and of (m)
radioactive agents where each of n and m is
independently an integer greater than zero. The total number of radioactive agents capable of being bound per antigen is then the product of (n) multiplied by (m). This is in contrast to the binding to cell surface antigen of previously available radioimmunoconjugates comprised of an immunoreactive protein conjugated to (c) radioactive agents wherein the value of (c) is an integer greater than zero and is limited to the number of conjugations that can be performed on said
immunoreactive protein while retaining the
immunoreactivity for said antigen. This limit to the degree of modification of the immunoreactive protein also applies to the NRTIR of System A, and the value of (n) will be approximately the same as the value of (c).
Thus, in this aspect, the non-covalent binding of the RDA to the antigen-bound NRTIR of the present invention will amplify the maximum number of radioactive agents bound per antigen by a factor of approximately (m) over the maximum value (c) available in previously available radioimmunoconjugates.
In another embodiment, the present invention is directed to an NRTIR comprised of the residue of a ligand which exhibits an affinity for non-covalent binding to a receptor moiety, a linking group, and the residue of an immunoreactive material, which NRTIR is administered to a tissue of interest and will bind to sites on the surfaces of cells thereof.
In this embodiment, the present invention is also directed to an RDA comprised of the residue of a receptor moiety for which a ligand has an affinity for non-covalent binding, a linking group, and the residue of a radioactive agent, which RDA is administered to the environs of the tissue which contains the NRTIR of this embodiment bound thereto. In particular, the ligand of the RDA of this embodiment will non-covalently bind to the receptor of the NRTIR which is bound to the surface of the cells of said tissue of interest. Thus, an effective amount of radioactivity is provided to said tissue. RDA which is unbound to NRTIR can be removed rapidly from the environs of the tissue.
In particular, in one aspect of this other
embodiment (sometimes hereinafter referred to as System B), the present invention comprises a NRTIR comprised of the residue of a ligand which has an affinity for non- covalent binding to a DHFR receptor moiety, a linking group, and the residue of an immunoreactive material. In system B the present invention also comprises an RDA comprised of the residue of a DHFR receptor moiety, a linking group, and the residue of a radioactive agent.
Specifically, in System B, the present invention is directed to a NRTIR comprised of the residue of a ligand which has an affinity for non-covalent binding to a DHFR receptor moiety, a linking group, and the residue of an immunoreactive material such as a tumor targeting antibody. In system B the present invention also comprises an RDA comprised of the residue of a DHFR receptor moiety, a linking group, and the residue of a radioactive agent comprised of the residue of a
chelating agent and a radionuclide.
The NRTIR of System B is comprised of (n) residues of ligands that have an affinity for non-covalent binding to a DHFR, each of which can non-covalently bind an RDA comprised of a DHFR and of (m) radioactive agents where each of n and m is independently an integer greater than zero. The total number of radioactive agents capable of being bound per antigen is then the product of (n) multiplied by (m). This is in contrast to the binding to cell surface antigen of previously available radioimmunoconjugates comprised of an
immunoreactive protein conjugated to (c) radioactive agents. In these conjugates the value of (c) is limited to the number of radioactive agents that can be linked or conjugated to the immunoreactive protein while retaining the immunoreactivity for said antigen. This limit to the degree of modification of the
immunoreactive protein also applies to the NRTIR of System B, and the value of (n) will be approximately the same as the value of (c). Thus, in this aspect, the non-covalent binding of the RDA to the antigen-bound
NRTIR of the present invention will amplify the maximum number of radioactive agents bound per antigen by a factor of approximately (m) over the maximum value (c) available in previously available radioimmunoconjugates.
The present invention is also directed to
pharmaceutical and diagnostic compositions comprising an
NRTIR and a pharmaceutically acceptable carrier, and to pharmaceutical and diagnostic compositions comprising an RDA and a pharmaceutically acceptable carrier.
The present invention is further directed to therapeutic methods comprising the administration, in vitro or in vivo, of a therapeutically effective amount of NRTIR to the environs of a tissue of interest of a patient undergoing such therapy, followed after the lapse of an effective period of time by the subsequent administration of a therapeutically effective amount of RDA to said tissue. During the time between
administrations of NRTIR and RDA said NRTIR binds to sites on cells of the target tissue and unbound NRTIR is removed from the environs of said tissue.
The present invention is further directed to diagnostic imaging methods comprising the sequential administration, in vitro or in vivo, of a diagnostic imaging effective amount of an NRTIR to the environs of a tissue of interest of a patient undergoing such diagnostic imaging, followed after a lapse of an
effective period of time by the subsequent
administration of a diagnostic imaging effective amount of RDA to said tissue. During said effective period of time said NRTIR will bind to sites on cells of said tissue of interest and unbound NRTIR will be removed from the environs of the tissue. Subsequently, after an effective time, an image of all or part of said tissue of interest is obtained.
The present invention provides advantages compared to currently available targeting immune reagents. For example:
the total amount of a therapeutically effective amount and of a diagnostic imaging effective amount of radioactive agent delivered to a tissue site can be achieved with specificity and in amplification over that
which can be otherwise achieved with currently available targeting immune reagents;
sequential delivery to target tissue of the NRTIR and the RDA of this invention can reduce the exposure of non-targeted tissues to damage from radiation;
the non-covalent binding of the ligand to the receptor occurs with high affinity and is selective; the NRTIR and the RDA can be used in both
therapeutic and diagnostic imaging applications;
the above-described NRTIR can accumulate at a tumor tissue site in vivo while it is not substantially accumulated at normal tissue sites;
the in vivo residence half life of the above- described NRTIR is long enough to permit its
accumulation at a tumor site;
the in vivo residence half life of the above- described RDA is shorter than the residence half life of the above-described NRTIR;
RDA that does not bind to cell associated NRTIR is rapidly cleared from the patient;
with respect to the same degree of modification of a targeting immunoreagent directly conjugated by a radionuclide or by a chelate containing a radionuclide in currently available radioimmunoconjugates, an
amplification of the number of radionuclides per site of modification per targeting immune reagent can be
obtained using the materials and methods of this
invention;
the NRTIR can comprise a wide variety of
immunoreactive groups, linking groups, and DHFR active site residues in System A, and a wide variety of
immunoreactive groups, linking groups, and ligand residues which have an affinity for non-covalent binding to DHFR active site residues in System B;
the RDA can comprise a wide variety of spacing, linking and chelating groups, radionuclides, and ligand
residues which ligands have an affinity for non-covalent binding to DHFR in System A, and a wide variety of spacing, linking and chelating groups, radionuclides, and DHFR active site residues in System B; and
a wide variety of compositions of matter with a wide variety of sizes and molecular weights and having a specificity for accumulation in tumors can be prepared in accordance with this invention.
Other advantageous features of this invention will become readily apparent upon reference to the following description of the preferred embodiments.
Description of Preferred Embodiments
In preferred embodiments, the above-described non- radioactive targeting immunoreagent (NRTIR) and
radioactive delivery agent (RDA) are comprised of moieties represented in System A (4 systems) and System B (4 systems) below:
SYSTEM A
SYSTEM B
wherein:
Z is the residue of an immunoreactive group;
Rec is the residue of a receptor, preferably a DHFR;
D is the residue of a ligand that has an affinity for non-covalent binding to a receptor, preferably to a DHFR receptor;
DHFR ligand is the residue of a ligand that has an affinity for non-covalent binding to a DHFR active site;
TMP is the residue of a ligand comprised of a
trimethoprim analog;
MTX is the residue of a ligand comprised of a
methotrexate analog;
L1 and L2 are each independently the residue of a linking group that may independently contain a spacing group;
Q is the residue of a chelating group;
M is a radionuclide; and
n and m are each independently an integer greater than zero.
Preferred embodiments of these materials are further described below.
The term "residue" is used herein in context with a chemical entity. Said chemical entity is comprised of, for example, a ligand, or a trimethoprim analog, or a methotrexate analog, or a receptor moiety, or a
proteinaceous active site of a dihydrofolate reductase enzyme, or a DHFR, or a chelating group, or a
radioactive agent, or a linking group, or a protein reactive group, or an immunoreactive group, or an immunoreactive material, or an immunoreactive protein, or an antibody, or an antibody fragment, or a cross- linking agent such as a heterobifunctional cross-linking agent, or a spacing group. The term "residue" is defined as that portion of said chemical entity which exclusively remains when one or more chemical bonds of which said chemical entity is otherwise comprised when considered as an independent chemical entity, is
altered, modified, or replaced to comprise one or more covalent bonds to one or more other chemical entities. Thus, for example, in one aspect in System A and in System B, the residue of a chelating group is comprised of a chelating group which is at least monovalently modified through attachment to the residue of another chemical entity such as, for example, to the residue of a linking group.
In both System A and System B the immunoreactive group, Z, can be selected from a wide variety of
naturally occurring or synthetically prepared materials, including, but not limited to enzymes, amino acids, peptides, polypeptides, proteins, lipoproteins,
glycoproteins, lipids, phospholipids, hormones, growth factors, steroids, vitamins, polysaccarides, viruses, protozoa, fungi, parasites, rickettsia, molds, and components thereof, blood components, tissue and organ components, pharmaceuticals, haptens, lectins, toxins,
nucleic acids (including oligonucleotides), antibodies (monoclonal and polyclonal), anti-antibodies, antibody fragments, antigenic materials (including proteins and carbohydrates), avidin and derivatives thereof, biotin and derivatives thereof, and others known to one skilled in the art. In addition, an immunoreactive group can be any substance which when presented to an immunocompetent host will result in the production of a specific antibody capable of binding with that substance, or the antibody so produced, which participates in an antigen- antibody reaction.
Preferred immunoreactive groups are antibodies and various immunoreactive fragments thereof, as long as they contain at least one reactive site for reaction with the reactive groups on the residue of the receptor moiety in System A or ligand species in System B or with linking groups (L) as described herein. That site can be inherent to the immunoreactive species or it can be introduced through appropriate chemical modification of the immunoreactive species. In addition to antibodies produced by the techniques outlined above, other
antibodies and proteins produced by the techniques of molecular biology are specifically included.
As used herein, the term "antibody fragment" refers to an immunoreactive material which comprises a residue of an antibody, which antibody characteristically exhibits an affinity for binding to an antigen. The term affinity, as used herein, refers to the thermodynamic expression of the strength of interaction or binding between an antibody combining site (or other ligand) and an antigenic determinant (or receptor) and, thus, of the sterochemical compatibility between them; as such it is the expression of the equilibrium or association
constant for the antibody-antigen (or ligand-receptor) interaction. Antibody fragments exhibit at least a percentage of said affinity for binding to said antigen,
said percentage being in the range of 0.001 per cent to 1,000 per cent, preferably 0.01 per cent to 1,000 per cent, more preferably 0.1 per cent to 1,000 per cent, and most preferably 1.0 per cent to 1,000 per cent, of the relative affinity of said antibody for binding to said antigen.
An antibody fragment can be produced from an antibody by a chemical reaction comprising one or more chemical bond cleaving reactions; by a chemical reaction comprising one or more chemical bond forming reactions employing as reactants one or more chemical components selected from a group comprised of amino acids, peptides, carbohydrates, linking groups as defined herein, spacing groups as defined herein, protein reactive groups as defined herein, and antibody fragments such as are produced as described herein; and by a molecular biological process, a bacterial process, or by a process comprised of a genetic engineering of antibody genes.
An antibody fragment can be derived from an antibody by a chemical reaction comprised of one or more of the following reactions:
(a) cleavage of one or more chemical bonds of which an antibody is comprised, said bonds being selected from, for example, carbon-nitrogen bonds, sulfur-sulfur bonds, carbon-carbon bonds, carbon-sulfur bonds, and carbon-oxygen bonds, and wherein the method of said cleavage is selected from:
(i) a catalysed chemical reaction comprising the action of a biochemical catalyst such as an enzyme such as papain or pepsin which to those skilled in the art is known to produce antibody fragments commonly referred to as Fab and Fab'2, respectively;
(ii) a catalysed chemical reaction comprising the action of an electrophilic chemical catalyst such as a
hydronium ion which, for example, favorably occurs at a pH equal to or greater than 7;
(iii) a catalysed chemical reaction comprising the action of a nucleophilic catalyst such as a hydroxide ion which, for example, favorably occurs at a pH equal to or greater than 7; and
(iv) a chemical reaction comprised of a
substitution reaction employing a reagent which is consumed in a stoichiometric manner such as a
substitution reaction at a sulfur atom of a disulfide bond by a reagent comprised of a sulfhydryl group;
(v) a chemical reaction comprised of a reduction reaction such as the reduction of a disulfide bond; and (vi) a chemical reaction comprised of an oxidation reaction such as the oxidation of a carbon-oxygen bond of a hydroxyl group or the oxidation of a carbon-carbon bond of a vicinal diol group such as occurs in a carbohydrate moiety; or
(b) formation of one or more chemical bonds between one or more reactants, such as formation of one or more covalent bonds selected from, for example, carbon- nitrogen bonds (such as, for example, amide bonds, amine bonds, hydrazone bonds, and thiourea bonds), sulfur- sulfur bonds such as disulfide bonds, carbon-carbon bonds, carbon-sulfur bonds, and carbon-oxygen bonds, and employing as reactants in said chemical bond formation one or more reagents comprised of amino acids, peptides, carbohydrates, linking groups as defined herein, spacing groups as defined herein, protein reactive groups as defined herein, and antibody fragments such as are produced as described in (a), above; or
(c) an antibody fragment can be derived by
formation of one or more non-covalent bonds between one or more reactants. Such non-covalent bonds are
comprised of hydrophobic interactions such as occur in an aqueous medium between chemical species that are
independently comprised of mutually accessible regions of low polarity such as regions comprised of aliphatic and carbocyclic groups, and of hydrogen bond
interactions such as occur in the binding of an
oligonucletide with a complementary oligonucletide; or (d) an antibody fragment can be produced as a result of the methods of molecular biology or by genetic engineering of antibody genes, for example, in the genetic engineering of a single chain immunoreactive group or a Fv fragment.
An antibody fragment can be produced as a result a combination of one or more of the above methods.
In certain embodiments, the immunoreactive group can be an enzyme which has a reactive group for
attachment to the receptor moiety in System A or ligand species in System B or to a linking group as described below. Representative enzymes include, but are not limited to, aspartane, aminotransaminase, alanine aminotransaminase, lactate dehydrogenase, creatine phosphokinase, gamma glutamyl transferase, alkaline acid phosphatase, prostatic acid phosphatase, horseradish peroxidase and various esterases.
If desired, the immunoreactive group can be modified or chemically altered to provide reactive groups for attaching to the residues of the receptor moiety in System A or ligand species in System B or to a linking group as described below by techniques known to those skilled in the art. Such techniques include the use of linking moieties and chemical modification such as described in WO-A-89/02931 and WO-A-89/2932, which are directed to modification of oligonucleotides, and U.S. Patent No. 4,719,182.
Two highly preferred uses for the compositions of this invention are for the diagnostic imaging of tumors and the radiological treatment of tumors. Preferred immunological groups therefore include antibodies
(sometimes hereinafter referred to as Ab) to tumor- associated antigens. Specific non-limiting examples include B72.3 and related antibodies (described in U.S. Patent Nos. 4,522,918 and 4,612,282) which recognize colorectal tumors, 9.2.27 and related anti-melanoma antibodies, D612 and related antibodies which recognize colorectal tumors, UJ13A and related antibodies which recognize small cell lung carcinomas, NRLU-10 and related antibodies which recognize small cell lung carcinomas and colorectal tumors (Pan-carcinoma), 7E11C5 and related antibodies which recognize prostate tumors, CC49 and related antibodies which recognize colorectal tumors, TNT and related antibodies which recognize necrotic tissue, PR1A3 and related antibodies which recognize colon carcinoma, ING-1 and related antibodies, which are described in International Patent Publication WO-A-90/02569, B174 and related antibodies which recognize squamous cell carcinomas, B43 and related antibodies which are reactive with certain lymphomas and leukemias, and anti-HLB and related monoclonal
antibodies. An especially preferred antibody is ING-1.
Scheme 1
As used herein, the term "receptor" refers to a chemical group in a molecule which comprises an active site in said molecule, or to an array of chemical groups in a molecule which comprise one or more active sites in said molecule, or to a molecule comprised of one or more chemical groups or one or more arrays of chemical groups, which group or groups or array of groups comprise one or more active sites in said molecule. An "active site" of a receptor has a specific capacity to bind to or has an affinity for binding to a ligand. With respect to use with the term "receptor" or with the term "active site in a receptor", the term "ligand" as used herein refers to a molecule comprised of a specific chemical group or a specific array of chemical groups which molecule, group, or array of groups is
complementary to and has a specific affinity for binding to a receptor, especially to an active site in a
receptor. Examples of receptors include enzymes which catalyze chemical reactions, and cell surface receptors which bind hormones and drugs. The sites of specific
binding of substrates for said enzymes and of hormones and drugs of said cell surface receptors are examples of active sites of said receptors, and the respective substrates, hormones, and drugs are examples of ligands for said receptors.
Preferred receptors (Rec) in System A and System B are comprised of the residue of an active site of an enzyme, and preferred ligands in System A and System B are comprised of the residue of a substrate for said active site of said enzyme.
An especially preferred receptor is comprised of the active site of any protein having dihydrofolate reductase (DHFR) activity. Said DHFR can be isolated, in whole or in part, from any source and used in this invention without further modification, or it can be chemically modified before or after isolation for use in this invention, or it can be modified by well known techniques of molecular biology and isolated for use in this invention, or said molecular biology modified DHFR can be chemically modified before or after isolation for use in this invention as long as the DHFR active site is maintained in such use.
According to the 1992 Sigma Chemical Company catalog on page 350, DHFR activity is defined in terms of units; one said unit of DHFR enzyme activity is defined as the amount of material needed to convert 1.0 micromole of 7,8-dihydrofolate and NADPH to 5,6,7,8- tetrahydrofolate and NADP per minute at pH 6.5 and 25°C. Thus, as used herein, a chemical species comprised of a group or array of groups that catalyze the conversion of
7,8-dihydrofolate and NADPH to 5,6,7,8-tetrahydrofolate and NADP has DHFR activity and such group or array of groups comprises a dihydrofolate reductase active site, i.e., a DHFR. In addition, the modification of a DHFR by known techniques of molecular biology or by chemical modification may reduce the protein's ability to
catalyze the conversion of 7,8-dihydrofolate and NADPH to 5,6,7,8-tetrahydrofolate and NADP yet retain the protein's capacity to bind a ligand.
Examples of ligands that have an affinity for binding to the active site in said DHFR include 7,8- dihydrofolate, derivatives and analogs of 7,8- dihydrofolate, and residues of said derivatives and analogs which may be agonists or antagonists of DHFR activity with respect to 7,8-dihydrofolate. Preferred examples of ligands which exhibit an affinity for binding to a DHFR are comprised of antifolate drugs as described in "Principals of Drug Action, The Basis of Pharmacology", Third Edition, Churchill Livingstone, New York, 1990, Pratt W. B. and Taylor P., Editors, page 623 which are antagonists of DHFR activity with respect to
7,8-dihydrofolate, and of inhibitors of DHFR activity as described in Blaney, J. M., Hansch, C, Silipo, C, and Vittoria, A., Chemical Reviews, (1984), Vol. 84, pp 333- 407. Additional preferred examples are comprised of derivatives and analogs of said antifolate drugs and inhibitors, and residues of said derivatives and said analogs of said antifolate drugs and inhibitors.
Especially preferred ligands which exhibit an affinity for binding to a DHFR are comprised of residues of the antifolate drugs, pyrimethamine, methotrexate,
tetroxoprim, and trimethoprim. Most preferred are comprised of residues of the antifolate drugs,
methotrexate and trimethoprim.
In one aspect in System A and in System B, the DHFR is comprised of a non-human protein, preferably
comprised of a bacterial or protozoal protein, and more preferably of a bacterial protein. Preferably, said DHFR is comprised of protein derived from S. aureus or from E. Coli. More preferably, said DHFR is comprised of protein derived from E. Coli (strain CV634) infected
with the plasmid pCV29 which harbors the E. coli DHFR gene.
In another aspect in System A and in System B, the DHFR is comprised of a human protein. Preferably, said DHFR is comprised of a recombinant human protein. More preferably, said DHFR is comprised of a recombinant human protein which is modified by genetic engineering techniques, which modifications comprise the independent incorporation, substitution, insertion, and deletion of specific amino acids in a peptide sequence of said protein. Yet more preferably, the DHFR comprised of a thus modified recombinant human protein is comprised of an active site which has an affinity for binding to substrates, which affinity is greater than the affinity of natural human protein for said substrates . Even more preferably, the DHFR comprised of a thus modified recombinant human protein is comprised of an active site which has an affinity for binding to residues of the antifolate drugs, methotrexate and trimethoprim, which affinity is greater than the affinity of natural human protein for binding to the residues of said antifolate drugs. Most preferrably, the DHFR comprised of a thus modified recombinant human protein is comprised of an active site which has an affinity for binding to
residues of the antifolate drug, trimethoprim, which affinity is greater than the affinity of natural human protein for binding to the residues of said antifolate drug.
In yet another aspect, the Z-L-X of System A is comprised of a fusion protein. As used herein, the term
"fusion protein" refers to a genetically engineered material comprised of a protein whose coding region is comprised of the coding region of a residue of a first protein fused, in frame, to the coding region of a residue of a second protein. Preferably, said fusion protein is comprised of a protein whose coding region is
comprised of the coding region of a residue of an immunoreactive reagent fused, in frame, to the coding region of one or more residues of a DHFR. Thus, preferably, said fusion protein is comprised of a residue of an immunoreactive reagent fused to one or more residues of a DHFR. In a prefered embodiment, said fusion protein is comprised of residues of DHFR fused to an immunoglobulin heavy chain in the CHi region, such that when combined with an appropriate light chain the said fusion protein comprises an Fab fragment linked to one or more DHFR. In another preferred embodiment, said fusion protein can be comprised of one or more DHFR fused to an immunoglobulin heavy chain in the CH2 or in the CH3 region. In yet another preferred embodiment, said fusion protein, when comprised of an immunoglobulin light chain, can be comprised of a Fab'2 fragment linked to one or more DHFR. In still another preferred embodiment, said fusion protein can be comprised of one or more DHFR fused to the C-terminal end of an
immunoglobulin single-chain construct and thus be comprised of an Fv fragment linked to one or more DHFR.
The above genetically engineered fusion protein comprising Z-L-X of System A can be comprised a protein whose coding region is independently comprised of the coding region of a residue of a human or of a non-human first protein fused, in frame, to the coding region of a residue of a human or non-human second protein.
Preferably, said coding regions are independently human and bacterial or modified by genetic engineering
techniques as above. More preferably, the fusion protein is comprised of a protein whose coding region is comprised of the coding region of a residue of a human immunoreactive reagent fused, in frame, to the coding region of one or more residues of a bacterial or human DHFR or a genetically engineered modified bacterial or human DHFR. Yet more preferably, the fusion protein is
comprised of a thus modified recombinant human DHFR comprised of an active site which has an affinity for binding to a ligand, which affinity is greater than the affinity of natural human protein for said ligand.
Even more preferably, the fusion protein is comprised of a thus modified recombinant human DHFR comprised of an active site which has an affinity for binding to a ligand comprised of a residue of an antifolate drug such as methotrexate or trimethoprim, which affinity is greater than the affinity of natural human protein for binding to the residue of said ligand. Most preferably, the fusion protein is comprised of a thus modified recombinant human DHFR comprised of an active site which has an affinity for binding to a ligand comprised of a residue of trimethoprim, which affinity is greater than the affinity of natural human protein for binding to the residue of said ligand.
The binding of a ligand to a receptor can comprise the formation of a covalent bond or it can comprise a non-covalent interaction. Preferably, in this invention the binding of a ligand to a receptor comprises a non- covalent interaction, sometimes herein referred to as non-covalent binding.
In System A, the DHFR is covalently linked, i.e., conjugated, to an immunoreactive group, preferably to an antibody or to an antibody fragment, most preferably to ING-1, to form the NRTIR [i.e., Z-(L1-Rec)n] of the System.
In System B, in one embodiment, the DHFR as a component of a radioactive delivery agent [i.e., an RDA, Rec-(L2-Q-M)m] is attached to one or more chelating groups, each by means of a linking group, and the chelating group is associated with a radionuclide.
Preferably the chelating group is TMT (described
hereinbelow), the linking group is as described below, and the radionuclide is 90γ,
In another embodiment, the RDA in System B is comprised of a DHFR that contains one or more
radionuclides that are covalently attached, either directly to one or more components of the DHFR or to one or more components that are attached by a linking group as described below to the DHFR. Preferably, said covalently attached radionuclide is a radioisotope of iodine attached to an aromatic ring-containing moiety.
In another embodiment, the RDA in System B is comprised of a DHFR that contains one or more
radionuclides that are covalently attached, either directly to one or more components of the DHFR or to one or more components that are attached by a linking group as described below to the DHFR. Preferably, said covalently attached radionuclide is selected from a radioisotope of technicium and rhenium attached to a group comprised of a sulfur atom.
In System A, chemical conjugation can be achieved, for example, by a technique comprising the use of a linking group (L1) which is introduced through
modification of, for example, a site on an
immunoreactive group. The introduction of activated groups such as activated ethylene groups (e.g.,
maleimide groups) on to amine groups such as lysine epsilon-amines of a protein is represented in Scheme 1. Other techniques include the use of heterobifunctional linking moieties and chemical modifications such as the examples described in U. S. Patent No. 4,719,182.
Additionally, those chemicals such as SMCC which are commonly commercially available, for example, from
Pierce Chemical Company are included as non-limiting examples.
In both System A and System B in one aspect, chemical conjugation is otherwise achieved by using a linking group (L1 and L2, respectively) which is
introduced through mild reduction of the DHFR (or of the
DHFR modified by reagents which contain disulfide bonds) with a reducing reagent such as dithiothreitol to produce sulfhydryl (SH) sites in the reduced DHFR protein moiety. In System A, addition of the thus reduced DHFR protein moiety to the above described maleimide modified antibody (Ab-M) results in an
antibody/receptor conjugate (Ab-M-S-DHFR protein) linked together by one or more thioether bonds. Additionally, those chemicals which are commonly commercially
available, for example, from Pierce Chemical Company and the like which are useful in the covalent attachment of two proteins are included as non-limiting examples in the coupling of DHFR to antibody in System A.
In System B, addition of the thus reduced DHFR protein moiety to a chelating agent which contains a precursor of a linking group comprised of an activated ethylene group such as a maleimide group results in a DHFR/chelating agent conjugate linked together by one or more thioether bonds. Similarly, addition of the thus reduced immunoreactive protein moiety to the residue of a ligand which contains a precursor of a linking group comprised of an activated ethylene group such as a maleimide group results in a immunoreactive protein moiety/ligand conjugate linked together by one or more thioether bonds.
In System A, other groups are useful in the
coupling of the immunoreactive material to the receptor moiety, particularly if the above reagents are utilized. Suitable reactive sites on the immunoreactive material and on the receptor moiety include:
amine sites of lysine;
terminal peptide amines;
carboxylic acid sites, such as are available in
aspartic acid and glutamic acid;
sulfhydryl sites;
carbohydrate sites;
activated carbon-hydrogen and carbon-carbon bonds which can react through insertion via free radical reaction or nitrene or carbene reaction of a so activated residue; sites of oxidation;
sites of reduction;
aromatic sites such as tyrosine; and
hydroxyl sites.
In System A, the ratio of DHFR to immunoreactive group such as an antibody can vary widely from about 0.5 to 10 or more. In bulk, mixtures comprised of
immunoreactive groups which are unmodified and
immunoreactive groups which are modified with DHFR are also suitable. Such mixtures can have a bulk ratio of DHFR to immunoreactive group of from about 0.1 to about 10.
In System A, in preferred embodiments, the mole ratio of DHFR to immunoreacative group is from about 1:1 to about 6:1. It is specifically contemplated that with knowledge of the DNA sequence that encodes DHFR,
especially bacterial and human DHFR, a fusion protein can be made between the antibody and the DHFR, or portions thereof, through the use of genetic engineering techniques. It is specifically contemplated that in all of these compositions of DHFR bound to antibody, DHFR retains a capacity to bind to the ligands described in the invention.
In System B, the ratio of ligand to immunoreactive group such as an antibody can vary widely from about 0.5 to 10 or more. In bulk, mixtures comprised of
immunoreactive groups which are unmodified and
immunoreactive groups which are modified with ligand are also suitable. Such mixtures can have a bulk ratio of ligand to immunoreactive group of from about 0.1 to about 10. In preferred embodiments, the mole ratio of ligand to immunoreactive group is from about 1 : 1 to about 6:1.
In System A, following the linking of the
immunoreactive group, preferably of an antibody or an antibody fragment, to DHFR, the conjugate is purified by passage of the material through a gel permeation column such as Superose 6 using an appropriate elution buffer or by elution from a HPLC column such as a Shodex WS- 803F size exclusion column. Both these methods separate the applied materials by molecular size resulting in the elution of the antibody/DHFR conjugate in a different fraction from any residual non-conjugated DHFR.
In System A, the concentrations of the antibody in the conjugate solutions are determined by the Bradford (BioRad Catalog # 500-0001) method using bovine
immunoglobulin as the protein standard.
In System B, following the linking of the
immunoreactive group, preferably of an antibody or an antibody fragment, to the residue of a ligand, the conjugate is purified by passage of the material through a gel permeation column such as Superose 6 using an appropriate elution buffer or by elution from a HPLC column such as a Shodex WS-803F size exclusion column. Both these methods separate the applied materials by molecular size resulting in the elution of the
antibody/ligand conjugate in a different fraction from any residual non-conjugated ligand.
In System B, the concentrations of the antibody in the conjugate solutions are determined by the BioRad protein assay using bovine immunoglobulin as the protein standard.
In System A, the ability of the antibody to bind to its target antigen following conjugation to DHFR can be assayed by ELISA or flow cytometry. A 30 cm x 7.5 mm TSK-G3000SW size-exclusion HPLC column (Supelco) fitted with a guard column of the same material can be used to determine the amount of aggregation in the final
conjugate.
In System B, the ability of the antibody to bind to its target antigen following conjugation to the residue of a ligand can be assayed by ELISA or flow cytometry. A 30 cm x 7.5 mm TSK-G3000SW size-exclusion HPLC column (Supelco) fitted with a guard column of the same material can be used to determine the amount of
aggregation in the final conjugate.
In System A, the dihydrofolate reductase enzymic activity of the antibody-associated DHFR can be assayed by following the oxidation of pyridine nucleotides during the reduction of folate to tetrahydrofolate by DHFR as described by Mathews and Huennekens ( J. Biol. Chem. 238, 3436-3442 (1963)). This method can also be used to assay the DHFR inhibitory effects of the novel DHFR binding ligands which are modified to include chelating agents as described in this invention.
In System B, the dihydrofolate reductase enzymic activity of the chelating agent-associated DHFR can be assayed by following the oxidation of pyridine
nucleotides during the reduction of folate to
tetrahydrofolate by DHFR as described by Mathews and Huennekens ( J. Biol. Chem. 238, 3436-3442 (1963)).
This method can also be used to assay the DHFR
inhibitory effects of the novel ligands which have an affinity for binding to DHFR and which are linked to immunoreactive groups as described in this invention.
L1 and L2 in System A and System B are
independently a chemical bond or the residue of a linking group. In one aspect, the phrase "residue of a linking group" as used herein refers to a moiety that remains, results, or is derived from the reaction of a protein reactive group with a reactive site on a
protein. The phrase "protein reactive group" as used herein refers to any group which can react with
functional groups typically found on proteins. However, it is specifically contemplated that such protein
reactive groups can also react with functional groups typically found on relevant nonprotein molecules. Thus, in one aspect the linking groups L1 and L2 useful in the practice of this invention derive from those groups which can react with any relevant molecule "Z" or "Rec" as described above containing a reactive group, whether or not such relevant molecule is a protein, to form a linking group. In one aspect, preferred linking groups thus formed include the linking group, L1, between the immunoreactive group, "Z", and the DHFR active site containing species, "Rec", in the NRTIR System A; the linking group, L1, between the immunoreactive group, "Z", and DHFR ligand species (e.g., "TMP" or "MTX") in the NRTIR in System B; and the linking group, L2, between the DHFR active site containing species, "Rec", and the chelating agent, "Q", in the RDA in System Brand between the DHFR ligand species (e.g., "TMP" or "MTX") and the chelating agent, "Q", in the RDA in
System A.
Preferred linking groups are derived from protein reactive groups selected from but not limited to:
(1) a group that will react directly with amine, alcohol, or sulfhydryl groups on the immunoreactive protein or biological molecule containing the reactive group, for example, active halogen containing groups including, for example, chloromethylphenyl groups and chloroacetyl [ClCH2C(=O)-] groups, activated 2- (leaving group substituted)-ethylsulfonyl and ethylcarbonyl groups such as 2-chloroethylsulfonyl and 2- chloroethylcarbonyl; vinylsulfonyl; vinylcarbonyl;
epoxy; isocyanato; isothiocyanato; aldehyde; aziridino; succinimidoxycarbonyl; activated acyl groups such as carboxylic acid halides; mixed anhydrides and the like; and other groups known to be useful in conventional photographic gelatin hardening agents;
(2) a group that can react readily with modified proteins or biological molecules containing the
immunoreactive group, i.e., proteins or biological molecules containing the immunoreactive group modified to contain reactive groups such as those mentioned in (1) above, for example, by oxidation of the protein to an aldehyde or a carboxylic acid, in which case the "linking group" can be derived from protein reactive groups selected from amino, alkylamino, arylamino, hydrazino, alkylhydrazino, arylhydrazino, carbazido, semicarbazido, thiocarbazido, thiosemicarbazido, sulfhydryl, sulfhydrylalkyl, sulfhydrylaryl, hydroxy, carboxy, carboxyalkyl and carboxyaryl. The alkyl portions of said linking groups can contain from 1 to about 20 carbon atoms. The aryl portions of said linking groups can contain from about 6 to about 20 carbon atoms; and
(3) a group that can be linked to the protein or biological molecule containing the immunoreactive group, or to the modified protein as noted in (1) and (2) above by use of a crosslinking agent. The residues of certain useful crosslinking agents, such as, for example, homobifunctional and heterobifunctional gelatin
hardeners, bisepoxides, and bisisocyanates can become a part of, i.e., a linking group in, for example, the protein-(DHFR active site-containing species) conjugate in System A during the crosslinking reaction. Other useful crosslinking agents, however, can facilitate the crosslinking, for example, as consumable catalysts, and are not present in the final conjugate. Examples of such crosslinking agents are carbodiimide and
carbamoylonium crosslinking agents as disclosed in U.S. Patent No. 4,421,847 and the ethers of U.S. Patent No. 4,877,724. With these crosslinking agents, one of the reactants such as the immunoreactive group must have a carboxyl group and the other such as the oligonucleotide
containing species must have a reactive amine, alcohol, or sulfhydryl group. In amide bond formation, the crosslinking agent first reacts selectively with the carboxyl group, then is split out during reaction of the thus "activated" carboxyl group with an amine to form an amide linkage between, for example, the protein and DHFR active site containing species, thus covalently bonding the two moieties. An advantage of this approach is that crosslinking of like molecules, e.g., proteins with proteins or DHFR active site containing species with themselves is avoided, whereas the reaction of, for example, homo-bifunctional crosslinking agents is nonselective and unwanted crosslinked molecules are obtained.
Preferred useful linking groups are derived from various heterobifunctional cross-linking reagents such as those listed in the Pierce Chemical Company
Immunotechnology Catalog - Protein Modification Section, (1991 and 1992). Useful non-limiting examples of such reagents include:
Sulfo-SMCC Sulfosuccinimidyl 4-(N- maleimidomethyl)cyclohexane-1- carboxylate.
Sulfo-SIAB Sulfosuccinimidyl (4- iodoacetyl)aminobenzoate. Sulfo-SMPB Sulfosuccinimidyl 4-(p- maleimidophenyl)butyrate.
2-IT 2-Iminothiolane. SATA N-Succinimidyl S-acetylthioacetate.
In addition to the foregoing description, the linking groups, in whole or in part, can also be comprised of and derived from complementary sequences of nucleotides and residues of nucleotides, both naturally occurring and modified, preferably non-self-associating oligonucleotide sequences. Particularly useful, non- limiting reagents for incorporation of modified
nucleotide moieties containing reactive functional groups, such as amine and sulfhydryl groups, into an oligonucleotide sequence are commercially available from, for example, Clontech Laboratories Inc. (Palo Alto California) and include Uni-Link AminoModifier (Catalog # 5190), Biotin-ON phosphoramidite (Catalog # 5191), N- MNT-C6-AminoModifier (Catalog # 5202), AminoModifier II (Catalog # 5203), DMT-C6-3 'Amine-ON (Catalog # 5222),
C6-ThiolModifier (Catalog # 5211), and the like. In one aspect, linking groups of this invention are derived from the reaction of a reactive functional group such as an amine or sulfhydryl group as are available in the above Clontech reagents, one or more of which has been incorporated into an oligonucleotide sequence, with, for example, one or more of the previously described protein reactive groups such as heterobifunctional protein reactive groups, one or more of which has been incorporated into, for example, an immune reactive agent or DHFR active site containing moiety of this invention.
In the NRTIR of System A, complementary
oligonucleotide sequences are attached to two components of the conjugate, respectively, one sequence to the immune reactive agent and the complementary
oligonucleotide sequence to the DHFR active site
containing moiety. The hybrid formed between the two complementary oligonucleotide sequences then comprises the linking group between the immune reactive agent and the DHFR active site containing moiety.
In the RDA of System B, the complementary
oligonucleotide sequences are attached to two components of the conjugate, one sequence to the residue comprised of one or more chelating agents and the complementary oligonucleotide sequence to the DHFR active site containing moiety. The hybrid formed between the two complementary oligonucleotide sequences then comprises the linking group between the DHFR active site
containing moiety and the component comprised of one or more chelating agents.
In System B, of course, two or more copies of the same oligonucleotide sequence can be linked, for example, in tandem to one DHFR active site containing moiety and a complementary oligonucleotide sequence comprised of multiple chelating agents can be added. The multiple hybrids formed between the two
complementary oligonucleotide sequences then comprises the linking group between the DHFR active site
containing moiety and multiple chelating agents .
Likewise, in System B, the residue of one or more ligands which have an affinity for non-covalent binding to DHFR can be attached to the immunoreactive group using complementary oligonucleotide hybrids as described above.
In System A, analogously, multiple DHFR sequences can be attached to the immunoreactive protein.
Likewise, in System A, one or more ligands which have an affinity for non-covalent binding to DHFR can be
attached to multiple chelating agents using
complementary oligonucleotide hybrids as described above.
Q in System A and in System B represents the residue of a chelating group. The chelating group of this invention can comprise the residue of one or more of a wide variety of chelating agents that can have a radionuclide associated therewith. As is well known, a
chelating agent is a compound containing donor atoms that can combine by coordinate bonding with a metal atom to form a cyclic structure called a chelation complex or chelate. This class of compounds is described in the Kirk-Othmer Encyclopedia of Chemical Technology, Vol. 5, 339-368.
The residues of suitable chelating agents can be independently selected from polyphosphates, such as sodium tripolyphosphate and hexametaphosphoric acid; aminocarboxylic acids, such as
ethylenediaminetetraacetic acid, N-(2- hydroxyethyl) ethylene-diaminetriacetic acid,
nitrilotriacetic acid, N,N-di(2-hydroxyethyl) glycine, ethylenebis (hydroxyphenylglycine) and diethylenetriamine pentacetic acid; 1,3-diketones, such as acetylacetone, trifluoroacetylacetone, and thenoyltrifluoroacetone;
hydroxycarboxylic acids, such as tartaric acid, citric acid, gluconic acid, and 5-sulfosalicylic acid;
polyamines, such as ethylenediamine, diethylenetriamine, triethylenetetramine, and triaminotriethylamine;
aminoalcohols, such as triethanolamine and N-(2- hydroxyethyl)ethylenediamine; aromatic heterocyclic bases, such as 2,2'-dipyridyl, 2,2'-diimidazole,
dipicoline amine and 1,10-phenanthroline; phenols, such as salicylaldehyde, disulfopyrocatechol, and
chromotropic acid; aminophenols, such as 8- hydroxyquinoline and oximesulfonic acid; oximes, such as dimethylglyoxime and salicylaldoxime; peptides
containing proximal chelating functionality such as polycysteine, polyhistidine, polyaspartic acid,
polyglutamic acid, or combinations of such amino acids; Schiff bases, such as disalicylaldehyde 1,2- propylenediimine; tetrapyrroles, such as
tetraphenylporphin and phthalocyanine; sulfur compounds, such as toluenedithiol, meso-2,3-dimercaptosuccinic acid, dimercaptopropanol, thioglycolic acid, potassium
ethyl xanthate, sodium diethyldithiocarbamate,
dithizone, diethyl dithiophosphoric acid, and thiourea; synthetic macrocylic compounds, such as
dibenzo[18]crown-6, (CH3)6- [14]-4,11-diene-N4, and
(2.2.2-cryptate); and phosphonic acids, such as nitrilotrimethylene-phosphonic acid,
ethylenediaminetetra (methylenephosphonic acid), and hydroxyethylidenediphosphonic acid, or combinations of two or more of the above agents.
Preferred residues of chelating agents contain polycarboxylic acid groups and include:
ethylenediamine-N,N N',N'-tetraacetic acid (EDTA);
N,N,N',N",N"-diethylene-triaminepentaacetic acid (DTPA); 1,4,7,10-tetraazacyclododecane-N,N',N",N"'-tetraacetic acid (DOTA); 1,4,7,10-tetraazacyclododecane-N,N',N"- triacetic acid (DO3A); 1-oxa-4,7,10-triazacyclododecane- N,N',N"-triacetic acid (OTTA) ; and trans (1,2)- cyclohexanodiethylenetriamine pentaacetic acid (CDTPA) .
Preferred residues of chelating agents contain polycarboxylic acid groups and include: B4A, P4A, TMT, DCDTPA, PheMT, macroPheMT, and macroTMT;
In one aspect, other suitable residues of chelating agents are comprised of proteins modified for the chelation of metals such as technetium and rhenium as described in U.S. Patent No. 5,078,985, the disclosure of which is hereby incorporated by reference.
In another aspect, suitable residues of chelating agents are derived from N3S and N2S2 containing
compounds, as for example, those disclosed in U.S.
Patent Nos. 4,444,690; 4,670,545; 4,673,562; 4,897,255; 4,965,392; 4,980,147; 4,988,496; 5,021,556 and
5,075,099.
Other suitable residues of chelating agents are described in PCT/US91/08253, the disclosure of which is hereby incorporated by reference. If Q is comprised of the residue of multiple chelating agents, such agents can be linked together by one or more linking groups such as described above.
The residues of the chelating agent Q are
independently linked to the other components of this invention through a chemical bond or a linking group such as L2 as described above. Preferred linking groups also include nitrogen atoms in groups such as amino, imido, nitrilo and imino groups; alkylene, preferably containing from 1 to 18 carbon atoms such as methylene, ethylene, propylene, butylene and hexylene, such alkylene optionally being interrupted by 1 or more heteroatoms such as oxygen, nitrogen and sulfur or heteroatom-containing groups; carbonyl; sulfonyl;
sulfinyl; ether; thioether; ester, i.e., carbonyloxy and oxycarbonyl; thioester, i.e., carbonylthio,
thiocarbonyl, thiocarbonyloxy, and oxythiocarboxy;
amide, i.e., iminocarbonyl and carbonylimino; thioamide, i.e., iminothiocarbonyl and thiocarbonylimino; thio; dithio; phosphate; phosphonate; urelene; thiourelene; urethane, i.e., iminocarbonyloxy,and oxycarbonylimino; an amino acid linkage, i.e., a
group wherein k=1 and X1, X2, X3 independently are H, alkyl, containing from 1 to 18, preferably 1 to 6 carbon atoms, such as methyl, ethyl and propyl, such alkyl optionally being interrupted by 1 or more heteroatoms such as oxygen, nitrogen and sulfur, substituted or unsubstituted aryl, containing from 6 to 18, preferably 6 to 10 carbon atoms such as phenyl, hydroxyiodophenyl,
hydroxyphenyl, fluorophenyl and naphthyl, aralkyl, preferably containing from 7 to 12 carbon atoms, such as benzyl, heterocyclyl, preferably containing from 5 to 7 nuclear carbon and one or more heteroatoms such as S, N, P or O, examples of preferred heterocyclyl groups being pyridyl, quinolyl, imidazolyl and thienyl;
heterocyclylalkyl, the heterocyclyl and alkyl portions of which preferably are described above; or a peptide linkage, i.e., a
group wherein k>1 and each X independently is
represented by a group as described for X1, X2, X3 above. Two or more linking groups can be used, such as, for example, alkyleneimino and iminoalkylene. It is
contemplated that other linking groups may be suitable for use herein, such as linking groups commonly used in protein heterobifunctional and homobifunctional
conjugation and crosslinking chemistry as described for L1 or L2 above. Especially preferred linking groups include amino groups which when linked to the residue of a chelating agent via an isothiocyanate group on the chelating agent form thiourea groups.
The linking groups can contain various substituents which do not interfere with the coupling reaction between the chelating agent Q and the other components of this invention. The linking groups can also contain substituents which can otherwise interfere with such reaction, but which during the coupling reaction, are prevented from so doing with suitable protecting groups commonly known in the art and which substituents are regenerated after the coupling reaction by suitable deprotection. The linking groups can also contain
substituents that are introduced after the coupling reaction. For example, the linking group can be substituted with substituents such as halogen, such as F, Cl, Br or I; an ester group; an amide group; alkyl, preferably containing from 1 to about 18, more
preferably, 1 to 4 carbon atoms such as methyl, ethyl, propyl, i-propyl, butyl, and the like; substituted or unsubstituted aryl, preferably containing from 6 to about 20, more preferably 6 to 10 carbon atoms such as phenyl, naphthyl, hydroxyphenyl, iodophenyl,
hydroxyiodophenyl, fluorophenyl and methoxyphenyl;
substituted or unsubstituted aralkyl, preferably containing from 7 to about 12 carbon atoms, such as benzyl and phenylethyl; alkoxy, the alkyl portion of which preferably contains from 1 to 18 carbon atoms as described for alkyl above; alkoxyaralkyl, such as ethoxybenzyl; substituted or unsubstituted heterocyclyl, preferably containing from 5 to 7 nuclear carbon and heteroatoms such as S, N, P or O, examples of preferred heterocyclyl groups being pyridyl, quinolyl, imidazolyl and thienyl; a carboxyl group; a carboxyalkyl group, the alkyl portion of which preferably contains from 1 to 8 carbon atoms; or the residue of a chelating group.
Residues of DHFR active site binding derivatives, trimethoprim analogs, and methotrexate analogs:
In Systems A and B, specific examples of ligands that have an affinity for non-covalent binding to a DHFR active site useful in this invention include residues of trimethoprim analogs and methotrexate analogs listed in Rahman et al, Methotrexate and Its Analogs; Medicinal Research Reviews, Vol 8, No. 1, 95-157, and Kuyper et al, Carboxysubstituted Trimethoprim Analogs; J. Med. Chem. Vol 28, 303, (1985). Preferred analogs include those defined in structure (i) and in structure (ii), each comprising a reactive group to permit or facilitate binding of one portion of the ligand to the
immunoreactive species, Z, in System B and to the chelating agent, Q, in System A.
Ligands comprised of trimethoprim analogs that are useful in the practice of this invention include derivatives defined in structure (i) above where: R1, R2, and R3 are independently selected from H, alkyl (such as methyl as a preferred group for one or two of R1, R2, and R3), aralkyl, aryl (including substituted aryl), an alkylene carboxylic acid or amide derivative thereof, an alkylene group that contains one or more heteroatoms (such as, but not limited to, for example, oxygen [as an ether or a hydroxyl group] or sulfur [as a thioether group]), an amino acid group and a peptide group, and at least one of -R1, -R2, and -R3 is of the form represented in structure 13:
(X) x— Lv—[[A]a---[B]b]f—W
wherein :
X is an alkylene group from 1 to 12 carbon atoms which may contain one or more heteroatoms such as oxygen, sulfur or nitrogen; L is a linking group as defined above, preferably the residue of an amide group, a chemical bond, an amino acid residue, or an arylene group which may be substituted by one or more hydroxyl groups; A is an alkylene group, a polyalkylene oxidyl group, an amino acid residue, a peptide residue, X, or a group containing pendant substituents which contain heteroatoms (such as, for example, oxygen in the form of one or more hydroxyl groups, carboxylic acid groups or salts thereof, amido groups, ether groups, sulfur in the form of thioether, sulfone, sulfoxide or sulfonate, nitrogen in the form of amino groups, amido groups or a diazo linkage, or phosphorous in the form of phosphate); B is selected from A but modified to contain one or more radionuclides bound thereto by chelating groups, Q, as defined above, such as, for example, but not limited to, TMT groups or DTPA groups, macrocyclic chelating groups, chelate groups that contain sulfur atoms, chelate groups that contain nitrogen atoms, chelate groups that contain pyridine rings, and chelate groups that contain
carboxylate or phosphate groups; W is selected from H, alkyl, aralkyl, alkylene, carboxylic acid groups, amino groups, amido groups, aryl groups, hydroxyaryl groups, a therapeutically effective and diagnostically effective radioisotope of an atom (such as iodine and the like) that can be covalently attached to a component of structure (i) [as distinct from a therapeutically effective or diagnostically effective radioisotope of an ion that can be bound to a component of structure (i) via a chelate group], a chelate group that may contain a therapeutically effective or diagnostically effective radioisotope, M, of a metal ion such as yttrium, indium, rhenium, copper, scandium, bismuth, lead, or leuticum
and the like; x and v are independently zero or one; a is zero or an integer from one to about 100; b, q, and f are independently integers from one to about 100.
In the RDA of System A, a preferred non-limiting example of a linking group includes the residue of a protected (D)-Glu-(Ala)4-Lys-Lys (4) as well as the residue of a protected (L)-Glu-(Ala)4-(Lys)2. Both 4'- carboxymethyloxy-trimethoprim (structure 5) and
methotrexate analog (structure iia) undergo coupling reactions (see SCHEMES 2, 3 and 4) followed by
deprotection to afford trimethoprim-(D)-Glu-(Ala)4-Lys- Lys (structure iii) and methotrexate (structure iv) and the analogous L-Glu containing species, respectively. The synthetic schemes (2,3,4) describe the synthetic methods for trimethoprim-heptapeptide (structure iii) and methotrexate-heptapeptide (structure iv). Thus, the t-butoxycarbonyl (tBoc) blocked Lys-Lys (structure 2) is prepared using a dehydrative coupling method, for example, using DCC (dicyclohexylcarbodiimide) and two protected lysine groups (SCHEME 2). The FMOC (9- Fluorenylmethoxycarbonyl) group of Lys-Lys is then removed via base treatment, and 4 units of alanine are introduced as shown in SCHEME 2 to afford the blocked Ala-Ala-Ala-Ala-Lys-Lys (structure 3). After adding t- butyl (tBu) blocked D-Glu on the N-terminal of the hexapeptide (structure 3), the resulting heptapeptide (structure 4) is coupled with 4'-carboxymethyloxy- trimethoprim (structure 5) to yield trimethoprim- heptapeptide (structure 6). The acidic deblocking of the peptide (structure 6) affords the desired
trimethoprim-D-Glu-(Ala)4-Lys-Lys (structure 7).
trimethoprim-D-Glu-(Ala)4-Lys-Lys (structure 7) upon treatment with TMT-NCS (structure 8) (SCHEME 3) yields a desirable ligand-to-chelating agent conjugate,
trimethoprim-D-Glu-(Ala)4-[Lys-(TMT)]-[Lys-(TMT)],
(structure 9). A solution of trimethoprim-D-Glu-(Ala)4-
[Lys-(TMT)]-[Lys-(TMT)] (structure 9) in deionized water buffered with 0.5 M sodium acetate at a pH of about 6.0 at room temperature is treated with an aqueous HCl solution of 90YCl3 to afford a radionuclide labeled (90Y) trimethoprim-D-Glu-(Ala)4-[Lys-(TMT)]-[Lys-(TMT)].
Employing the similar synthetic methods, but using a blocked L-Glu derivative, the analogous L-Glu- trimethoprim derived materials are obtained.
Employing the similar synthetic methods, a coupling reaction (Scheme 4) of the heptapeptide (structure 4) and the benzoic acid derivative, 4'-carboxy-methotrexate (structure iia), yields the desired blocked
methotrexate-D-Glu-(Ala)4-Lys-Lys (structure 10).
methotrexate-heptapeptide (structure 11), is obtained from acid deblocking of (structure 10), and reacts with TMT-NCS to afford the desirable methotrexate-D-Glu- (Ala)4-[Lys-(TMT)]-[Lys-(TMT)] (structure 12). A solution of methotrexate-D-Glu-(Ala)4-[Lys-(TMT) ]-[Lys- (TMT) ] (structure 12) in deionized water buffered with 0.5 M sodium acetate at a pH of about 6.0 at room temperature is treated with an aqueous HCl solution of 90YCl3 to afford a radionuclide labeled (90Y)
methotrexate-D-Glu-(Ala)4-[Lys-(TMT)]-[Lys-(TMT)].
Employing the similar synthetic methods, but using a blocked L-Glu derivative, the analogous L-Glu- methotrexate derived materials are obtained.
Racemic mixtures of D and L enantiomers of the above described trimethoprim and methotrexate
derivatives are also useful in this invention.
Additional chelating agents and radionuclides bound to chelating agents are incorporated by preparing, for example, analogous peptides comprised of additional Lys- TMT and Lys-TMT-radionuclide groups. Preferably, the number of such Lys-TMT and Lys-TMT-radionuclide residues is from 1 to about 6, and more preferably from 2 to about 6.
In the System B, the NRTIR is comprised of one or more ligands that have an affinity for non-covalent binding to a DHFR active site each with a suitably substituted linking group (L1) conjugated to the immunoreactive group (Z). Preferably, said ligand that has an affinity for non-covalent binding to a DHFR active site is comprised of a residue of (structure i) or (structure ii) linked to Z by a linking group L1 as defined above. The NRTIR preferably contains 2 to about 10 of such groups, more preferably 2 to about 4.
The delivery agent in the System B is comprised of a DHFR active site moiety conjugated to one or more chelating agents via a linking group as described above.
In one embodiment, both in System A and System B, it is desirable that the radionuclide be a metal ion and that said metal ion be easily complexed to the chelating agent, for example, by merely exposing or mixing an aqueous solution of the chelating agent-containing moiety with a metal salt in an aqueous solution
preferably having a pH in the range of about 4 to about 11. The salt can be any salt, but preferably the salt is a water soluble salt of the metal such as a halogen salt, and more preferably such salts are selected so as not to interfere with the binding of the metal ion with the chelating agent. The chelating agent-containing moiety is preferrably in aqueous solution at a pH of between about 5 and about 9, more preferably between pH about 6 to about 8. The chelating agent-containing moiety can be mixed with buffer salts such as citrate, acetate, phosphate and borate to produce the optimum pH.
Preferably, said buffer salts are selected so as not to interfere with the subsequent binding of the metal ion to the chelating agent.
In therapeutic applications, the RDA of this invention preferably contains a ratio of metal
radionuclide ion to chelating agent that is effectivve in
such therapeutic applications. In preferred
embodiments, the mole ratio of metal ion per chelating agent is from about 1:100 to about 1:1.
In diagnostic imaging applications, the RDA of this invention preferably contains a ratio of metal
radionuclide ion to chelating agent that is effective in such diagnostic imaging applications. In preferred embodiments, the mole ratio of metal ion per chelating agent is from about 1:1,000 to about 1:1.
In another embodiment, the RDA of this invention can comprise a non-radioisotope of a metal ion. The metal ions can be selected from, but are not limited to, elements of groups IIA through VIA. Preferred metals include those of atomic number 12, 13, 20, the
transition elements 21 - 33, 38 - 52, 56, 72 - 84 and 88 and those of the lanthanide series (atomic number 57 - 71).
In another embodiment, the RDA of this invention can comprise a radionuclide. The radionuclide can be selected, for example, from radioisotopes of Sc, Fe, Pb, Ga, Y, Bi, Mn, Cu, Cr, Zn, Ge, Mo, Tc, Ru, In, Sn, Sr, Sm, Lu, Sb, W, Re, Po, Ta and Tl. Preferred
radionuclides include 44Sc, 64Cu, 67Cu, 111ln, 212Pb, 68Ga, 90Y, 153sm, 212Bi, 99mTC, 186Re and 188Re. Of these, especially preferred is 90Y. These radioisotopes can be atomic or preferably ionic.
In another embodiment, the RDA of this invention can comprise a fluorescent metal ion. The fluorescent metal ion can be selected from, but is not limited to, metals of atomic number 57 to 71. Ions of the following metals are preferred: La, Ce, Pr, Nd, Pm, Sm, Eu, Gd,
Tb, Dy, Ho, Er, Tm, Yb, and Lu. Eu is especially preferred.
In another embodiment, the RDA of this invention can comprise one or more paramagnetic elements which are suitable for the use in MRI applications. The
paramagnetic element can be selected from elements of atomic number 21 to 29, 43, 44 and 57 to 71. The following elements are preferred: Cr, V, Mn, Fe, Co, Ni, Cu, La, Ce, Pr, Nd, Pm, Sm, Eu, Gd, Tb, Dy, Ho, Er, Tm, Yb and Lu. Mn, Gd, and Dy are especially
preferred.
An example of a structure of an RDA that has utility in this invention is structure (i) above wherein R1, R2, and R3 are as defined above and preferably wherein one of R1, R2, and R3 is represented by structure 14.
where: R' and R" are selected from components of amino acids that comprise, for example, natural amino acids such as glycine, alanine, leucine, serine, lysine, isoleucine, glutamine, aspartic acid, glutamic acid, proline, threonine, valine, phenylalanine, tyrosine, and the like, as well as unnatural amino acids and racemates of natural amino acids, and R' and R" can independently be selected from H, a polyalkylene oxidyl group, and branched peptide groups which may contain additional chelate groups such as TMT;
mi is an integer between 1 and 10, m2, m3, and m4 are independently selected from zero and an integer between 1 and 10 with the proviso that m2 is at least 1, and preferably 2 to about 5; and
W is selected from OH, NH2, a residue of TMT, an O-alkyl group such as an O-methyl group, and NRaRb wherein RaRb are independently selected from alkyl groups such as methyl groups, H, and a polyalkylene oxidyl moiety such as PEG-OH and PEG-O-alkyl (e.g., PEG-O-CH3) with the PEG having an average molecular weight in the range between 45 and 5,000 daltons.
Another example of a structure that has utility in this invention is structure (i) above wherein R1, R2, and R3 are as defined above and preferably wherein one of R1, R2, or R3 is represented by structure 15.
wherein: R' and R" are selected from components of amino acids that comprise, for example, natural amino acids such as glycine, alanine, leucine, serine, lysine, isoleucine, glutamine, aspartic acid, glutamic acid, proline, threonine, valine, phenylalanine, tyrosine, and the like, as well as unnatural amino acids or racemates of natural amino acids, and R' and R" can be
independently selected from H, a polyalkylene oxidyl group, a branched peptide group which may contain one to about 10 additional chelate groups such as TMT;
ins is an integer between 1 and 10, m6, m7, and m8 are independently selected from zero and an integer between
1 and 10 with the proviso that m7 is at least 1, and preferably 2 to about 5;
W is selected from OH, NH2, a TMT moiety, an O-alkyl group such as an O-methyl group, NRaRb wherein RaRb are independently selected from alkyl groups such as methyl, H, a polyalkylene oxide moiety such as PEG-OH and PEG-O- alkyl (e.g., PEG-O-CH3) with the PEG having molecular weight in the range between 45 and 5,000 daltons;
mg is selected from zero and an integer from 1 to about 12; and
R''' is selected from H and the residue of a TMT moiety with or without a radionuclide, said TMT moiety being linked via a thiourea group.
In another embodiment of this invention, an RDA comprised of at least two metal ions in combination with one another in the same formulation is specifically contemplated. For example, the use of a therapeutically effective dose of a radionuclide such as 90γ+3 together with a diagnostic imaging effective dose of a
paramagnetic ion such as Gd+3, the ratio of the molar concentration of the diagnostic imaging effective ion to the molar concentration of the radionuclide ion being typically greater than one, in a pharmaceutically effective formulation of said RDA permits the
simultaneous magnetic resonance imaging of at least a portion of the tissue of a host patient during
therapeutic treatment of said patient.
In another embodiment of this invention, the use of radioisotopes of iodine is specifically contemplated. For example, if the RDA of System A or of System B is comprised of substituents that can be chemically
substituted by iodine in a covalent bond forming
reaction, such as, for example, substituents containing hydroxyphenyl functionality, such substituents can be labeled by methods well known in the art with a
radioisotope of iodine. The thus covalently linked
iodine species can be used in the aforementioned fashion in therapeutic and diagnostic imaging applications.
In a preferred embodiment, an effective dose of an RDA of System A or of System B as described above in a pharmaceutically acceptable medium is prepared by exposing a composition of a precursor of an RDA (said precursor being comprised of a residue of a ligand that has an affinity for non-covalent binding to a DHFR active site, a linking group, and a residue of a
chelating agent in System A and of a residue of a DHFR active site, a linking group, and a residue of a
chelating agent in System B) to a composition containing a radioactive metal ion such that the molar amount of said radionuclide metal ion is less than the molar amount of the chelating groups comprising the RDA in said composition, said duration of exposure lasting an effective time to permit uptake of said metal ion into said RDA.
In a preferred embodiment, an effective dose of a NRTIR of System A or System B as described above in a pharmaceutically acceptable medium is administered to a patient and said NRTIR is allowed to accumulate at the target site such as at a tumor site in said patient.
Subsequently, at an effective time, an effective dose of a RDA as described above in a pharmaceutically
acceptable medium is administered to said patient, and said RDA is allowed to accumulate at the target site, said target site being the said NRTIR accumulated at said tumor site in said patient.
In a preferred embodiment, a therapeutically effective dose of a NRTIR of System A or System B as described above in a pharmaceutically acceptable medium is administered to a patient or to a tissue from a patient and said NRTIR is allowed to accumulate at the target site such as at a tumor site in said patient.
Subsequently, at a therapeutically effective time, a
therapeutically effective dose of a RDA as described above in a pharmaceutically acceptable medium is administered to said patient, and said RDA is allowed to accumulate at the target site, said target site being the said NRTIR accumulated at said tumor site in said patient.
The present invention also comprises one or more NRTIR as described above formulated into compositions together with one or more non-toxic physiologically acceptable carriers, adjuvants or vehicles which are collectively referred to herein as carriers, for parenteral injection for oral administration in solid or liquid form, for rectal or topical administration, or the like. The present invention also comprises one or more RDA as described above formulated into compositions together with one or more non-toxic physiologically acceptable carriers, adjuvants or vehicles which are collectively referred to herein as carriers, for
parenteral injection, for oral administration in solid or liquid form, for rectal, or topical administration, or the like.
The compositions can be administered to humans and animals either orally, rectally, parenterally
(intravenous, by intramuscularly or subcutaneously), intracisternally, intravaginally, intraperitoneally, intravesically, locally (powders, ointments or drops), or as a buccal or nasal spray. It is specifically contemplated that the NRTIR and the RDA can be
administered by the same route such as orally, rectally, parenterally (intravenous, by intramuscularly or
subcutaneously), intracisternally, intravaginally, intraperitoneally, intravesically, locally (powders, ointments or drops), or as a buccal or nasal spray. It is also contemplated that the NRTIR can be administered by a route different from that of the RDA.
Compositions suitable for parenteral injection may comprise physiologically acceptable sterile aqueous or nonaqueous solutions, dispersions, suspensions or emulsions and sterile powders for reconstitution into sterile injectable solutions or dispersions. Examples of suitable aqueous and nonaqueous carriers, diluents, solvents or vehicles include water, ethanol, polyols (propyleneglycol, polyethyleneglycol, glycerol, and the like), suitable mixtures thereof, vegetable oils (such as olive oil) and injectable organic esters such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersions and by the use of surfactants.
These compositions may also contain adjuvants such as preserving, wetting, emulsifying, and dispensing agents. Prevention of the action of microorganisms can be ensured by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, and the like. It may also be desirable to include isotonic agents, for example sugars, sodium chloride and the like. Prolonged absorption of the injectable pharmaceutical form can be brought about by the use of agents delaying absorption, for example, aluminum monostearate and gelatin.
Solid dosage forms for oral administration include capsules, tablets, pills, powders and granules. In such solid dosage forms, the active compound is admixed with at least one inert customary excipient (or carrier) such as sodium citrate or dicalcium phosphate or (a) fillers or extenders, as for example, starches, lactose, sucrose, glucose, mannitol and silicic acid, (b) binders, as for example, carboxymethylcellulose, alignates, gelatin, polyvinylpyrrolidone, sucrose and acacia, (c) humectants, as for example, glylcerol, (d) disintegrating agents, as for example, agar-agar.
calcium carbonate, potato or tapioca starch, alginic acid, certain complex silicates and sodium carbonate, (e) solution retarders, as for example paraffin, (f) absorption accelerators, as for example, quaternary ammonium compounds, (g) wetting agents, as for example, cetyl alcohol and glycerol monostearate, (h) adsorbents, as for example, kaolin and bentonite, and (i)
lubricants, as for example, talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate or mixtures thereof. In the case of capsules, tablets and pills, the dosage forms may also comprise buffering agents.
Solid compositions of a similar type may also be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugar as well as high molecular weight polyethyleneglycols, and the like. Solid dosage forms such as tablets, dragees, capsules, pills and granules can be prepared with coatings and shells, such as enteric coatings and others well known in the art. They may contain
opacifying agents, and can also be of such composition that they release the active compound or compounds in a certain part of the intestinal tract in a delayed manner. Examples of embedding compositions which can be used are polymeric substances and waxes.
The active compounds can also be in micro- encapsulated form, if appropriate, with one or more of the above-mentioned excipients.
Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups and elixirs. In addition to the active compounds, the liquid dosage forms may contain inert diluents commonly used in the art, such as water or other solvents, solubilizing agents and emulsifiers, as for example, ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl
benzoate, propyleneglycol, 1,3-butyleneglycol,
dimethylformamide, oils, in particular, cottonseed oil, groundnut oil, corn germ oil, olive oil, castor oil and sesame oil, glycerol, tetrahydrofurfuryl alcohol, polyethyleneglycols and fatty acid esters of sorbitan or mixtures of these substances, and the like.
Besides such inert diluents, the composition can also include adjuvants, such as wetting agents,
emulsifying and suspending agents, sweetening, flavoring and perfuming agents.
Suspensions, in addition to the active compounds, may contain suspending agents, as for example,
ethoxylated isostearyl alcohols, polyoxyethylene
sorbitol and sorbitan esters, microcrystalline
cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, or mixtures of these substances, and the like.
Compositions for rectal administrations are
preferably suppositories which can be prepared by mixing the compounds of the present invention with suitable non-irritating excipients or carriers such as cocoa butter, polyethyleneglycol or a suppository wax, which are solid at ordinary temperatures but liquid at body temperature and therefore, melt in the rectum or vaginal cavity and release the active component.
Dosage forms for topical administration of a compound of this invention include ointments, powders, sprays and inhalants. The active component is admixed under sterile conditions with a physiologically
acceptable carrier and any preservatives, buffers or propellants as may be required. Opthalmic formulations, eye ointments, powders and solutions are also
contemplated as being within the scope of this
invention.
Actual dosage levels of active ingredients in the compositions of the present invention may be varied so
as to obtain an amount of active ingredient that is effective to obtain a desired therapeutic response for a particular composition and method of administration. The selected dosage level therefore depends upon the desired therapeutic effect, on the route of
administration, on the desired duration of treatment and other factors.
The total daily dose of the compounds of this invention administered to a host in single of divided dose may be in amounts, for example, of from about 1 nanomol to about 5 micromols per kilogram of body weight. Dosage unit compositions may contain such amounts of such submultiples thereof as may be used to make up the daily dose. It will be understood, however, that the specific dose level for any particular patient will depend upon a variety of factors including the body weight, general health, sex, diet, time and route of administration, rates of absorption and excretion, combination with other drugs and the severity of the particular disease being treated.
In another embodiment, the present invention is directed to a method of diagnosis comprising the
administration of a diagnostic imaging effective amount of the compositions of the present invention to a mammal or to a tissue from said mammal in need of such
diagnosis. A method for diagnostic imaging for use in medical procedures in accordance with this invention comprises administering to the body of a test subject in need of a diagnostic image an effective diagnostic image producing amount of the above-described compositions.
In this method, an effective diagnostic image producing amount of a non-radioactive targeting
immunoreagent (NRTIR) as described above in a
pharmaceutically acceptable medium is administered to a patient and said non-radioactive targeting immunoreagent is allowed to accumulate at the target site such as at a
tumor site in said patient. Subsequently, a diagnostic imaging effective dose of a radioactive delivery reagent (RDA) as described above in a pharmaceutically
acceptable medium is administered to said patient, and said radioactive targeting reagent is allowed to accumulate at the target site, said target site being the said non-radioactive targeting immunoreagent accumulated at said tumor site in said patient. The image pattern can then be visualized.
Alternatively, the NRTIR may be reacted with a diagnostic imaging effective amount of a reagent
comprised of a radionuclide prior to administration to the environs of a tissue of interest of a patient undergoing such diagnostic imaging, waiting for an effective period of time during which time said NRTIR will bind to sites on cells of said tissue of interest and during which time unbound NRTIR will be removed from the environs of said tissue and then obtaining an image as a function of time of all or part of said tissue of interest. When the image of all or part of said tissue of interest is optimal, a diagnostic imaging or a therapeutically effective amount of RDA containing the same or a different radionuclide as that employed on the NRTIR is administered to said tissue of interest of said patient.
In addition to human patients, the test subjects can include mammalian species such as rabbits, dogs, cats, monkeys, sheep, pigs, horses, bovine animals and the like.
After administration of the compositions of the present invention, the subject mammal is maintained for a time period sufficient for the administered
compositions to be distributed throughout the subject and enter the tissues of the mammal. A sufficient time period is generally from about 1 hour to about 2 weeks
or more and, preferably from about 2 hours to about 1 week.
The following examples further illustrate the invention and are not to be construed as limiting of the specification and claims in any way. Specific
embodiments of the invention are illustrated in the following examples.
EXAMPLES
Example 1
5-(4'Hydroxy-3',5'-dimethoxybenzyl)-2,4- dimaminopyridine
To a solution of 22 g (0.2 mol) of 2,4- diaminopyrimidine and 49.5 g (0.2 mol) of 2, 6-dimethoxy- 4-[(N,N-dimethylamino)methyl]phenol hydrochloride in 300 ml of ethylene glycol was added 11 g (0.203 mol) of sodium methoxide. The reaction mixture was heated under nitrogen with stirring to 150-160°C, at which point dimethylamine was evolved. After 3 hours, 80% of the theoretical amount of dimethylamine was collected. The solvent was removed in vacuo (at 85°C), the residual oil was washed with water, and then with acetone. The product was obtained as a tan precipitate (yield 25 g).
Example 2 5- (A'-Methoxycarbonylmethyloxy-3',5'-dimethoxybenzyl)- 2,4-diaminopyrimidine
5-(4'-Hydroxy-3',5'-dimethoxybenzyl)-2,4- diaminopyrimidine (5.52 g; 20 mmol) in 80 ml of DMSO wasw allowed to react with t-BuOH (2.469 g; 22 mmol) and methyl bromoacetate (3.366 g; 22 mM) for 14 hours. The
solvent was removed in vacuo, and the residual solid was vigrously shaken with saturated sodium bicarbonate solution. A light brown solid was filtered and dried to yield 1.5 g of the desired ester, m.p. 149-151°C. A second crop (3 g) of less pure material was also isolated. These two were combined for hydrolysis in Example 3.
Example 3
5-(4'-Carboxymethyloxy-3',5'-dimethoxybenzyl)-2,4- diaminopyrimidine, trimethoprim-4'-O-Acetic Acid
5-(4'-Methoxycarbonylmethyloxy-3',5'- dimethoxybenzyl)-2,4-diaminopyrimidine (4.5 g; 15.48 mM) was dissolved in 50 ml of 2 M NaOH. The clear solution was heated at 60°C for 4 hours, cooled, and allowed to stand overnight. A white precipitate formed which was filtered, washed with acetone, then with ether, and then dried. Yield: 20 g; melting point 274°C. H nmr (DMSO- d6) : d 7.5 (S, 1H), 6.53 (S, 2H), 6.08 (S, 2H), 5.69 (S, 2H), 3.9 (S, 2H), 3.7 (S, 6H) and 3.50 ppm (S, 2H).
Mass spectrum (CI) : MH+ = 357 (Na+ salt).
Preparation of H2N- [D-Glu]=Ala-Ala-Ala-Ala-Lys-Lys-OH
The linear peptide H2N-[D-Glu]=Ala-Ala-Ala-Ala-Lys- Lys-OH was synthesized via solid-phase methodology, on an ABI 430A Automated Peptide Synthesizer. The solid support used in the synthesis was a 4-Alkoxybenzyl alcohol polystyrene resin (Wang resin). The N-alpha- Fmoc protecting group was used throughout the synthesis, with t-butyl side chain protection on D-Glu, and t-BOC protection on the side chain of Lys. The peptide chain
was assembled using the ABI FastMoc™ software protocols (0.25 mmole scale, HBTU activated couplings, 4 fold excess of amino acid, 1 hour) for Fmoc-chemistry. Preparation of trimethoprim-4'-O-acetic acid amide of H2N-[D-Glu]-Ala-Ala-Ala-Ala-Lys-Lys-OH
Addition of trimethoprim-4'-O-acetic acid of
Example 3 to the N-terminus of the above peptide-resin was carried out manually by adding in order: 335 mg trimethoprim (1 mmole) in 25 ml of DMSO, 525 uL of diisopropylethylamine (3 mmole) followed by 380 mg of HBTU (1 mmole). The mixture was allowed to react for 2 hours at room temperature, at which time the mixture was filtered, and the peptide resin washed 3 x 50 ml DMF and then by 3 x 50 ml MeOH.
Removal and deprotection of the peptide was
accomplished by adding 15 ml of a 95:5 TFA/H2O solution to the peptide-resin, in a.sealed vessel and shaken at room temperature for 2 hours. At the end of 2 hours, the mixture was filtered by pouring into a scintered glass funnel. The filtrate volume was then reduced to an oil (@ 3 ml) by rotoevaporation. The peptide was then precipitated by dropping the oil into a centrifuge tube containing 50 ml of Et2O. The peptide was spun down and the ether decanted, and the peptide was allowed to air dry.
Example 4
Preparation of radionuclide labeled ( 90Y)-trimethoprim- D-Gl u-(Ala)4-Lys-(TMT)-Lys- (TMT) (Scheme 1 & 2 )
To a mixture of resin containing D-Glu-(COOtBu)- (Ala)4-Lys-tBOC-Lys-tBoc (4; 30mM) and DCC in 150 ml of methylene chloride is added in portions a solution of 5-
(4'-carboxymethyloxy-3',5'-dimethoxybenzyl)-2,4- diaminopyrimidine (i.e., trimethoprim-4'-O-acetic acid) (5; 30mM) in 100 ml of methylene chloride. The
resulting reaction mixture is stirred 2 days, and the solvent and the urea are removed to afford the blocked trimethoprim-4'-O-acetic acid heptapeptide (6), which upon mild hydrolysis with trifluoroacetic acid, yields trimethoprim-4'-O-acetic acid heptapeptide (7).
A solution of trimethoprim-4'-O-acetic acid heptapeptide (7) (20 mM) in 50 ml of a saturated aqueous sodium bicarbonate at about pH 9 is allowed to react with TMT-isothiocyanate (8; 20mM) at room temperature for 12 hours to afford trimethoprim-4'-O-acetic acid-D- Glu-(ALA)4-[Lys-(TMT)]-[Lys-(TMT)] (9).
A solution of the above trimethoprim-4'-O-acetic acid-D-Glu-(ALA)4-[Lys-(TMT)]-[Lys-(TMT)] (9) in
deionized water buffered with 0.5 M sodium acetate at pH 6.0 at room temperature is treated with a solution of 90YCl3 in aqueous hydrochloric acid, uptake of the radiolabel into the chelator is demonstrated by thin layer chromatography. In excess of 97% of the added 90Y is associated with trimethoprim-4'-O-acetic acid-D-Glu- (ALA)4-[Lys-(TMT)]-[Lys-(TMT)] to form the desired 90Y- labelled product.
Example 5
Preparat i on of radi onucl ide l abel ed ( 90Y) -met hotrexat e- D-Gl u- (Ala ) 4-L ys- (TMT) -Lys- (TMT) (Scheme 3)
To a mixture of resin containing D-Glu-(COOtBu)- (Ala)4-(Lys-tBOC)-(Lys-tBoc) (4; 30mM) and DCC in 150 ml of methylene chloride is added in portions a solution of 4'-carboxy-methotrexate (iia; 30mM) in 100 ml of
methylene chloride. The resulting reaction mixture is stirred 2 days, and the solvent and the urea are removed
to afford the blocked methotrexate-heptapeptide (10), which upon mild hydrolysis with 4 N HCl, yields 4- carboxymethotrexate-heptapeptide (11).
To a solution of 4-carboxymethotrexate-heptapeptide (11; 20 mM) in 50 ml of methylene chloride is allowed to react with TMT-isothiocyanate (8; 20mM) at room
temperature for 12 hours to afford 4'- carboxymethotrexate-D-Glu-(ALA)4-[Lys-(TMT)]-[Lys-(TMT)] (12).
A volume of radioactive Yttrium chloride (90Y in
0.04M hydrochloric acid at a specific activity of >500 Ci/mg: Amersham-Mediphysics) is neutralized using two volumes of 0.5 M sodium acetate pH 6.0 and added to a solution of the above 4'-carboxymethotrexate-D-Glu- (ALA)4-[Lys-(TMT)]-[Lys-(TMT)] (12) in deionized water buffered with 0.5 M sodium acetate at pH 6.0 at room temperature. The labeling is allowed to proceed for one hour and then the labeling efficiency is determined by thin layer chromatography on a Gelman ITAL-SG strip developed in 0.1 M sodium citrate, pH 6.0. In excess of 97% of the added 90Y is taken up by the 4'- carboxymethotrexate-D-Glu-(Ala)4-[Lys-(TMT)]-[Lys-(TMT)] to form the desired 90Y-labeled product.
The following examples illustrate the construction of conjugates between an antibody and dihydrofolate reductase (DHFR). In these examples ING-1 (a chimeric IgG1 antibody) is chosen for the methodologies; other antibodies such as those described herein are useful. The DHFR referred to below is of bacterial origin, produced as a recombinant product from the cloned DHFR gene which is overexpresed in Escherchia coli (E. coli), or of human origin, available as a recombinant protein.
Examples of System A
SYSTEM A
wherein:
Z is the residue of an immunoreactive group;
Rec is the residue of a receptor, preferably DHFR;
D is the residue of a ligand that has an affinity for non-covalent binding to the receptor, preferably to a DHFR receptor;
DHFR ligand is the residue of a ligand that has an affinity for non-covalent binding to a DHFR active site;
TMP is the residue of a trimethoprim analog;
MTX is the residue of a methotrexate analog;
L1 and L2 are each independently the residue of a linking group that may independently contain spacing group,
Q is the residue of a chelating group;
M is a radionuclide; and
n and m are each independently an integer greater than zero.
Example 6
( 6a ) P repa rat i on of Ant ibody-Male imide with Su l fo-SMCC
( ING-1 -Ma l ei mi de) [ Z-L1]
A sulfo-SMCC solution (36 nmoles) in PBS was added to a sample of a chimeric antibody (ING-1; 6 nmoles) solution in phosphate buffer (pH7). The resulting mixture was allowed to stand for 30 minutes with occasional mixing at room temperature.The reaction mixture was diluted with phosphate buffered saline, added to a prewashed PD-10 column (Pharmacia), and eluted with PBS to afford ING-1-maleimide. This material was stored on ice until use.
( 6b) Preparation of mercaptoalkyl-antibody [Z-L1]
A sample of of a chimeric antibody (ING-1; 6 nmoles) solution in 0.1 M carbonate buffer (pH 8.8) is mixed with 200 nmoles of an aqueous solution of 2- iminothiolane. The resulting mixture is allowed to stand for 30 minutes with occasional mixing at room
temperature. The reaction mixture is diluted with phosphate buffed saline, added to a prewashed PD-10 column, and eluted with PBS to afford mercaptoalkyl-ING- 1. This material is stored on ice until use.
(6c) Preparation of mercapto-antibody using SATA A solution containing 6 nmoles of ING-1 in PBS is vortexed while 60 nmoles of SATA (in DMSO) are added. After mixing and standing at room temperature for 60 minutes, the reaction mixture is diluted with PBS, and eluted from a PD-10 column with PBS to afford ING-1-CO- CH2-S-CO-CH3. The acetylthioacetylated antibody is deprotected by the addition of 30 uL of a pH 7.5
solution contaning 100 mM sodium phosphate, 25 mM EDTA, 50 mM NH2OH. The reaction proceeds for two hours at room temperature after which the material is again passed down a PD-10 column by elution with PBS. The final product (ING-1-CO-CH2-SH) is used immediately.
( 6d) Radiolabeling of ING-1 wi th 125I
An aliquot of ING-1 (500 ug) is labeled with 125I monochloride (at about 5 mCi/mg) in the presence of iodogen (Sodium N-chloro-benzenesulfonamide) beads in a volume of 500 uL 100mm phosphate buffer (pH 7.2) at room temperature. After 15 minutes the reaction is
terminated by passage of the labeled antibody down a prewashed NAP-5 column (Pharmacia). The radioiodinated protein is eluted with PBS and stored at 4°C until use.
Example 7 (7a) Preparation of mercapto-DHFR using SATA
A solution containing 50 nmoles of DHFR in PBS is vortexed while 500 nmoles of SATA (in DMSO) are added. After mixing and standing at room temperature for 60 minutes, the reaction mixture is diluted with PBS, and eluted from a PD-10 column with PBS to afford DHFR-CO- CH2-S-CO-CH3. The acetylthioacetylated DHFR is
deprotected by the addition of 25 uL of a pH 7.5
solution contaning 100 mM sodium phosphate, 25 mM EDTA, 100mM NH2OH. The reaction proceeds for two hours at room temperature after which the material is again passed down a PD-10 column by elution with PBS. The final product, DHFR-CO-CH2-SH is used immediately.
(7b) Preparation of mercaptoalkyl-DHFR
A sample of DHFR (50 nmoles) is dissolved in 0.1 M carbonate buffer (pH 9) and 2 mmoles of an aqueous solution of 2-iminothiolane are added. The reactants are vortex mixed and kept at room temperature for 120 minutes. The reaction mixture is quenched by the addition of 2 mmoles of ethanolamine, diluted with phosphate buffed saline. The reaction mixture is added to a prewashed PD-10 column, and eluted with PBS to afford DHFR-HC(NH2 +)CH2CH2CH2SH. For use in conjugation to maleimide-deriviatitzed ING-1 (Example 6a), the product is eluted off the column directly into the antibody solution.
(7c) Preparation of reduced DHFR using dithiothreitol
A solution containing 40 nmoles of DHFR in PBS was vortexed and an equal volume of 500 mM dithiothreitol in PBS was added. After mixing and standing on ice for 60 minutes, the reaction mixture was eluted from a
prewashed PD-10 column with PBS to afford DHFR-SH. For use in conjugation to maleimide-deriviatitzed antibody (Example 6a), the product was eluted off the column directly into the antibody solution.
(7d) Preparation of DHFR-Maleimide using Sulfo-SMCC A sulfo-SMCC solution (300 nmoles) in PBS is added to a sample of DHFR (50 nmoles) in phosphate buffer (pH7) . The resulting mixture is allowed to stand for 30 minutes with occasional mixing at room temperature. The reaction is stopped with 60 nmoles basic tris buffer. The reaction mixture is diluted with phosphate buffed saline, added to a prewashed PD-10 column, and eluted
with PBS to afford DHFR-maleimide. This material is stored on ice until use.
(7e) Radiolabeling of DHFR with 125I
An aliquot of DHFR (500 ug) is labeled with 125I monochloride (at about 5 mCi/mg) in the presence of iodogen (Sodium N-chloro-benzenesulfonamide) beads in a volume of 500 uL 100mm phosphate buffer (pH 7.2) at room temperature. After 15 minutes the reaction is
terminated by passage of the labeled protein down a prewashed NAP-5 column. The iodinated DHFR is eluted with PBS and stored at 4°C until use. (7f) Alternative conjugation method that protects
DHFR active site
In order to prevent the interaction of reagents with the active site of the DHFR enzyme during the derivatization process, the active site of the enzyme is blocked to prevent the entry of the reagents.
A methotrexate/agarose (Sigma) column resin slurry (1 mL: protected from light) is washed twice with a large volume of high salt buffer (100 mM KPO4, 1.0 M KCl, 1.0 mM K2EDTA and 0.5 mM dithioerythritol at pH 6.0) to remove any free methotrexate. The final
centrifuged pellet is mixed with DHFR (1 mmole/5 mL) in 5.0 mL of buffer containing (50 mM KPO4, 1.0 mM K2EDTA and 0.5 mM dithioerythritol at pH 6.0) and left for one hour. The resin with the now attached DHFR is
centrifuged to a pellet and washed x3 with washing buffer (50 mM KPO4, 1.0 mM K2EDTA at pH 6.0). The resin pellet is suspended in a sulfo-SMCC solution (6 mmoles) in PBS (pH 7). The resulting mixture is stirred very slowly for 60 minutes at room temperature. The reactio is stopped by dilution with 10 mL of washing buffer and
the resin is again centrifuged to a pellet and washed 2x with the washing buffer. The resin is poured into a narrow glass Pasteur pipette whose exit is obstructed with a plug of glass wool. Maleimide derivatized DHFR is removed from the resin by elution with 30 mL of eluting buffer (100 mM folic acid, 200 mM KBO3, 1.0 M KCl, 1.0 mM K2EDTA at pH 9.0) to afford DHFR-maleimide. This material is pooled, dialysed against dialysis buffer (20 mM Tris, 1.0 mM K2EDTA; pH 7.2) at 4°C overnight and then concentrated in a Centricon-10®
(Amicon) device to a concentration of approximately 1.0 mg/mL protein. This material is then reacted with antibody containing a free sulfhydryl group.
The folate is removed from the active site of the enzyme by passage of the maleimide DHFR (Example 7a) or antibody-DHFR conjugate (see Example 8 below) through a DEAE-SEPHACEL column (Pharmacia). The column (~50 mL resin) is prewashed with DEAE-washing buffer (10 mM Tris, 1.0 mM K2EDTA, 0.2 mM dithioerythritol; pH 7.2) and the protein loaded on in the same buffer. After washing, the protein is removed from the column with a non-linear gradient of 1000mL of DEAE-washing buffer (10 mM Tris, 1.0 mM K2EDTA, 0.2 mM dithioerythritol; pH 7.2) to 1000 mL of 10 mM Tris, 0.5 M KCl, 1.0 mM K2EDTA, 0.2 mM dithioerythritol; pH 7.2. Fractions eluting from the colum are collected and monitored at 280 nm for protein content. Fractions containing protein are pooled, concentrated as before and dialysed overnight against PBS to produce folate-free material.
Example 8 (8a) Conjugation of DHFP to antibody (formation of Z-L1-
Rec)
The methodologies for conjugation are essentially the same, irrespective of whether the maleimide group is on the antibody or on the DHFR, and irrespective of the method chosen to introduce the sulfhydryl group into the protein. The final molar ratio during the conjugation is maintained at close to equimolar antibody:DHFR in order to control over-conjugation of the proteins which could result in inactivation of one or other or both. The following procedure is applicable to the conjugation of materials in Example 6 to the materials in Example 7.
Following reduction (see Example 7a), a sample (50 nmoles) of DHFR(N)-CO-CH2-SH is eluted off a PD-10 column directly into a solution of maleimide- deriviatitzed ING-1 (5 nmoles) prepared according to Example 6a. After a brief mixing the solution is rapidly concentrated by centrifugation in a Centricon-10® device to a concentration of approximately 3.0 mg/mL protein. The reaction then is allowed to proceed for 4 hours at room temperature. The antibody-DHFR conjugate is transfered to a Centricon-30® concentrator and diluted with PBS. After concentrating the protein down to a volume of approximately 500 uL by centrifugation, the material is again diluted with PBS to 3.0 mL and recentrifuged. This procedure, which separates
unconjugated DHFR and other low molecular weight
products from the retained antibody-DHFR and
unconjugated antibody products, is repeated 4 times or until spectrophotometric monitoring of the filtrate at 280 nm shows that no further protein is being filtered. Finally, the material in the Centricon-30® is
concentrated to approximately 1.0 mg, ING-1/DHFR, per
milliliter solution and applied to a 2.6 x 60 cm
Sephacryl S-200 size-exclusion column equilibrated and eluted with 50 mM sodium phosphate buffer at pH 7.2 supplemented with 150 mM sodium chloride. This column separates unconjugated antibody from antibody-DHFR conjugate. Fractions of the eluate containing the conjugate are pooled and then centrifuged in a
Centricon-30 device to a concentration of approximately 1.0 mg, ING-1/DHFR, per milliliter solution. The conjugate is sterile filtered through a 0.22 μ filter and stored at 4°C until use.
Addition of trace amounts of either 125I-labeled DHFR or 125I-labeled ING-1 to the reaction mixtures, allows the ratio of one protein to the other after conjugation to be calculated.
(8b) Conjugation of reduced DHFR to Antibody-malemide (Scheme 1) Following reduction, a sample (50 nmoles) of DHFR-
SH (Example 7c) was eluted off a PD-10 column directly into a solution of maleimide-deriviatitzed ING-1 (5 nmoles) prepared according to Example 6a. After a brief mixing, the reaction was allowed to proceed for 4 hours at room temperature. The antibody-DHFR conjugate was transfered to a Centricon-30® concentrator, diluted with PBS, and concentrated down to a volume of approximately 500 μL by centrifugation. The concentrated protein was then diluted with PBS to a volume of 3.0 mL and
recentrifuged to a volume of 500 uL. This procedure, which separates unconjugated DHFR and other low
molecular weight products from the retained antibody- DHFR and unconjugated antibody products, was repeated three times. Finally, the material in the Centricon-30® was concentrated to approximately 1.0 mg ING-1-DHFR per milliliter solution and applied to a 10 X 300 mm
Superose 12 FPLC size-exclusion column equilibrated and eluted at 1 mL/min with 50 mM sodium phosphate buffer (pH 7.2) supplemented with 150 mM sodium chloride.The optical density of the eluate was continuously monitored at 280 nm and fractions (1.0 mL) containing the
antibody-DHFR conjugate were pooled. The protein concentration of the pooled material was assayed and the conjugate was stored
at 4°C until use.
Examples of System B
SYSTEM B
wherein:
Z is the residue of an immunoreactive group;
Rec is the residue of a receptor, preferably a DHFR receptor;
D is the residue of a ligand that has an affinity for non-covalent binding to the receptor, preferably to a
DHFR receptor;
DHFR ligand is the residue of a ligand that has an affinity for non-covalent binding to a DHFR active site;
TMP is the residue of a trimethoprim analog;
MTX is the residue of a methotrexate analog;
L1 and L2 are each independently the residue of a linking group that may independently contain spacing group;
Q is the residue of a chelating group;
M is a radionuclide; and
n and m are each independently an integer greater than zero.
Example 9
( 9a) Preparat i on of Ant i body-Ma l emi de with Su l fo-SMCC ( INK-1 -Ma l ei mi de
This product is prepared according to the procedure of Example 6a.
(9b) Preparation of trimethoprim-4'-O-acetic acid
cysteine
To a mixture of resin containing L-Cys (COO-trityl) (30 mM: Advanced Chem Tech) and DCC in 150 ml of methylene chloride is added in portions a solution of 5- (4'-O-acetic acid) (30 mM) in 100 ml of methylene chloride. The resulting reaction mixture is stirred 2 days, and the solvent and the urea are removed to afford the blocked trimethoprim-4'-O-acetic acid cysteine, which upon mild hydrolysis with trifluoroacetic acid, yields trimethoprim-4'-O-acetic acid cysteine.
(9c) Conjugation of trimethoprim-4'-O-acetic acid
cysteine to Antibody (formation of Z-L1-REC) Trimethoprim-4'-O-acetic acid cysteine (50 nmoles) prepared according to Example 9b is added directly into a solution of maleimide-derivatized ING-1 (5 nmoles) prepared according to Example 9a. After a brief mixing the solution is then allowed to proceed for 4 hours at room temperature with intermittent stirring. The antibody-trimethoprim conjugate is transferred to a Centricom-30® concentrator and diluted with PBS. After concentrating the protein down to a volume of
approximately 500 μL by centrifugation, the material is again diluted with PBS to 3.0 mL and recentrifuged.
This procedure, which separates unconjugated
trimethoprim and other lower molecular weight products from the retained antibody-trimethoprim and unconjugated antibody products, is repeated 4 times. Finally, the material in the Centricon-30® is concentrated to approximately 10 mg ING-1-trimethoprim per milliliter solution and applied to a 2.6 x 60 cm Sephacryl S-200 size-exclusion column equilibrated and eluted with 50 mM sodium phosphate buffer at pH 7.2 supplemented with 150 mM sodium chloride. This column separates unconjugated antibody from antibody-trimethoprim conjugate.
Fractions of the eluate containing the conjugate are pooled and then centrifuged in a Centricon-30® device to a concentration of approximately 1.0 mg ING-1- trimethoprim per milliliter solution. The conjugate is sterile filtered through a 0.22 filter and stored at 4°C until use.
Example 10
(10a) Conjugation of DHFR to TMT (formation of REC-L2-O)
Terpyridine methylenetetraacetic acid (TMT) or a suitable derivative thereof can be conjugated to a protein molecule (DHFR) to yield a protein-TMT
conjugate. The DHFR referred to below is of either bacterial origin, produced as a recombinant product from the cloned DHFR gene which is overexpressed in E.coli, or of human origin available as a recombinant protein.
DHFR (50 nmoles) is allowed to react with TMT- isothiocyanate (250 nmoles in 1.0 M carbonate, 150 mM sodium chloride buffer, pH 9.3 in an acid washed, conical, glass reaction vial. The solution is stirred briefly to mix the reactants and then left in the dark at room temperature. After 16 hours, the DHFR/TMT conjugate is separated from unconjugated TMT, by
applying the reaction mixture to a PD-10 chromatography column, which has been pre-washed and equilibrated with
50 mM sodium acetate buffer containing 150 mM sodium chloride at pH 5.6. The pure conjugate is eluted off the column with 2.5 mL of that same buffer, and
concentrated on a Centricon-10® concentration device.
(10b) Radiolaheling of DHFR/TMT with 90Y: (formation of Rec-(L2-O-M)m)
A volume of radioactive Yttrium chloride (90Y in 0.04 M hydrochloric acid at a specific activity of >500 Ci/g: Amersham-Mediphysics) is neutralized using two volumes of 0.5 M sodium acetate pH 6.0. The neutralized 90Y (1.0 mCi) is added to 1.0 mL of DHFR/TMT (1 mg/mL) in 50 mM sodium acetate buffer containing 150 mM sodium chloride at pH 5.6. The labeling is allowed to proceed for one hour and then the reaction mixture is loaded on to a PD-10 chromatography column which has been prewashed and equilibrated in a buffer contaiing SO mM sodium phosphate with 150 mM sodiumchloride pH 7.4
(PBS). The sample is eluted from the column with 1.5 mL of PBS. Fractions of radiolabeled DHFR/TMT (0.5 mL) are collected, assayed for radioactivity, and pooled. The labeling efficiency is determined by removing 1.0 uL of the sample and spotting it on to a Gelman ITLC-SG strip. The strip is developed in a glass beaker contining 0.1 M sodium citrate, pH 6.0 for a few minutes until the solvent front has reached three-quarters of the way to the top of the paper. The strip is inserted into a System 200 Imaging Scanner (Bioscan) which has been optimized for 90Y and is controlled by a Compaq 386/20e computer. In this system free 90Y migrates at the solvent front while the DHFR/TMT (90Y) remains at the origin. Using this system more than 98% of the total 90Y radioactivity is found associated with DHFR/TMT at the origin.
Example 11
Assays on the DHFR conjugates prepared from System A or
System B (11a) Protein Concentration
The concentrations of ING-1 and DHFR for use in the conjugate reactions are determined by the BioRad protein assay (BioRad Catalog # 500-0001) using bovine
immunoglobulin as the protein standard. By inclusion of trace amounts of 125I-labeled DHFR or ING-1 in the reaction mixtures, and by knowing the specific activity of the preparations, the ratio of one protein to the other after conjugation is calculated.
As an alternative to radiolabelling, the DHFR can be tagged with other materials (e.g., TMT (for use with 90Y or Europium fluorescence), biotin, fluorescene isothiocyanate (FITC) etc. to detect and quantify the amount of DHFR present in a solution or conjugated to another protein.
(11b) Immunoreactivity assay by Flow Cytometry
Conjugates of antibody-DHFR (e.g.. Example 8) or antibody-trimethoprim (e.g.. Example 10) are examined for their ability to bind to antigens on the surface of a human tumor cell line to which the antibody had been raised. The immunoreactivity of the conjugates is compared by flow cytometry with a standard preparation of the antibody before being subjected to modification. Target HT-29 cells (a human adenocarcinoma cell line obtained from the American Type Tissue Collection: ATTC) are grown to confluency in tissue culture flasks using McCoy's media supplemented with 10% fetal calf serum. The cells are harvested by scraping the flask walls with a cell scraper. Cells from many separate flasks are pooled, centrifuged to a pellet, resuspended at 5x105/mL
in a solution of ice-cold 50 mM sodium phosphate with 150 mM sodium chloride buffer pH 7.4 (PBS) supplemented with 0.1% bovine serum albumin (Sigma) and 0.02% sodium azide (Flow buffer). The cells are washed in this same buffer and then counted. An antibody standard curve is constructed by diluting a stock solution of ING-1 with an irrelevant (non-binding), isotype-matched control antibody (human IgG1), to give a number of samples ranging in ING-1 content from 10% to 100%. The standard curve is made in flow buffer so that each sample contains 1.0 mg protein per mL. Samples from the standard curve and ING-1-DHFR unknowns, or ING-1- trimethoprim unknowns are then incubated with 5x105 HT29 cells at 4°C for 1 hour. After extensive washing to remove unbound antibody, the cells are resuspended in 100 uL flow buffer and incubated at 4°C for 1 hour with goat-anti-human antibody labelled with fluorescene isothiocyanate. After further washing in flow buffer the samples are analyzed by flow cytometry on a Coulter EPICS 753 flow cytometer. Fluorescein isothiocyanate (FITC) and propidium iodide (PI) are excited using the 488 nm emission line of an argon laser. The output is set at 500 mw in light regulation mode. Single cells are identified by 90 degree and forward angle light scatter. Analysis windows are applied to these parameters to separate single cells from aggregates and cell debris. Fluorescence from FITC and propidium are separated with a 550 nm long pass dichroic filter and collected through a 530 nm band pass filter (for FITC), and a 635 nm band pass filter (for PI). Light scatter parameters are collected as integrated pulses and fluorescence is collected as log integrated pulses. Dead cells are excluded from the assay by placing an analysis window on cells negative for PI uptake. The mean fluorescence per sample (weighted average from 2500 cells) is calculated for each histogram. FITC calibration beads are analysed
in each experiment to establish a fluorescence standard curve. The average fluorescence intensity for each sample is then expressed as the average FITC equivalents per cell. Immunoreactivity is calculated by comparing the average fluorescence intensity of the ING1-DHFR or ING-1-trimethoprim sample with values from the standard curve.
(11c) Immunoreact ivity assay by ELISA
The antigen to which the antibody, ING-1, binds is prepared from LS174T or HT-29 cells (available from ATTC) by scraping confluent monolayers of cells from the walls of culture flasks with a cell scraper. The cells from many flasks are combined and a sample is taken and counted to estimate the total number of cells harvested. At all times the cells are kept on ice. Following centrifugation of the cells at 1500 rpm for 10 minutes at 4°C, the cells are washed once in 25 mL ice-cold 50 mM sodium phosphate buffer, pH 7.4 supplemented with 150 mM sodium chloride (PBS), pelleted under the same conditions and transfered in 10 mL PBS to an ice-cold glass mortar. The cells are homogenized at 4°C using a motor-driven pestle and then centrifuged at 3000 x g for 5 minutes. The antigen-rich supernatant is removed from the other cell debris and subjected to further
centrifugation at 100,000 x g for one hour at 4°C. The pellet (antigen fraction) from this final step is suspended in 100 μL of PBS for every million cells harvested. Following an estimate of the protein
concentration (BioRad BCA protein assay using bovine immunoglobulin as the protein standard) the antigen is stored at -20°C until use.
Each well of a 96-well Costar microtiter plates is coated with antigen by adding 100 μL/well of cell lysate
(10 mg/ml) prepared as above. The microtitre plates are
allowed to dry overnight in a 37°C incubator. After washing the plate five times with 0.05% Tween-20
(Sigma), they were blotted dry. The wells of each plate were blocked by adding 125 μL/well of a 1% BSA (bovine serum albumin, Sigma) solution in PBS and incubated for 1 hour at room temperature. The plates were washed five times with 0.05% Tween-20. Samples (50 μL/well in duplicate) of ING-DHFR or ING-trimethoprim conjugates and standard ING-1 antibody solutions are prepared at a range of concentrations in 1% BSA in PBS. Biotinylated ING-1 (1.0 mg/mL in 0.1% BSA) is added to each well (50 μL/well) and the plates are then incubated for 2 hours at room temperature. Following five washes with 0.05% Tween-20, the plates are blotted dry and incubated at room temperature for one hour with dilute (1:2000 in 0.1% BSA) streptavidin-alkaline phosphatase (Tago).
After a further five washes, color is developed in each well upon the addition of 100 μL per well of phosphatase substrate reagent (Sigma). After one hour at room temperature, the color wis read using a 405 nm filter in a Titertek Multiscan microplate reader. (11d) SDS PAGE gel electrophoresis Samples of ING-1-DHFR or ING-1-trimethoprim
conjugates are subjected to electrophoresis on Novex 8%- 16% reduced and native polyacrylamide gels using SDS buffers to estimate their apparent molecular weight and the degree of heterogeneity of the preparation. Using standards of known molecular weight run on the same gel, a standard curve is constructed of the distance
travelled versus the log of the molecular weight. From this standard curve the relative molecular weights of the bands associated with each conjugate preparation are determined.
( 11e) Determi nat i on of aggregate format ion by size-exclus ion HPLC
A 30 cm x 7.5 mm TSK-G3000SW size-exclusion HPLC column (Supelco) fitted with a guard column of the same material is equilibrated with 12 column volumes of 10 mM sodium phosphate buffer pH 6.0 supplemented with 150 mM sodium chloride using a Waters 600E HPLC system with a flow rate of 1.0 mL per minute at 400-600 PSI. A sample (25 uL) of BioRad gel filtration protein standards is injected on to the column. The retention time of each standard is monitored by a Waters 490 UV detector set at 280 nm. Following the recovery of the final standard, the column is washed with a further 10 volumes of 10 mM sodium phosphate buffer pH 6.0 supplemented with 150 mM sodium chloride. Samples (50 μL) of either native ING- 1 antibody or ING-1-DHFR at 200 μg/mL are injected on to the column and their retention times recorded. From the areas of the retained peaks and the retention time, the amount of aggregated material in the ING-1-DHFR or ING- 1-trimethoprim conjugates sampled are calculated.
(11f) Determination of DHFR activity Among other things, the enzymatic activity of DHFR is used to monitor:
i) Preservation of enzyme activity in a manner analogous to measuring the binding of the antibody to it's antigen, the activity of the enzyme before and after conjugation is assayed to ensure that the act of conjugation does not inhibit the enzyme;
ii) inhibitory effect of drugs (e.g., trimethoprim and the trimethoprim analogs described above in Examples
1-5) on both free and antibody-conjugated DHFR;
iii) to assay the effect of the trimethoprim- based TMT [ 90Y] delivery system; and
iv) measurement of the amount of DHFR in a solution.
According to the 1992 Sigma Chemical Company catalog on page 350, DHFR activity is defined in terms of units; one said unit of DHFR enzyme activity is defined as the amount of material needed to convert 1.0 micromole of 7,8-dihyrofolic acid and the reduced form of β-nicotinamide adenine dinucleotide phosphate (NADPH) to 5,6,7,8-tetrahydrofolate and the oxidized form of β- nicotinamide adenine dinucleotide phosphate (NADP) per minute at pH 6.5 and 25°C)
The activity of the enzyme was measured
spectrophotometrically at 340 nm by following the the rate of oxidation of NADPH to NADP during the reduction of dihydrofolate to tetrahydrofolate. An aliquot (362 μL) of a freshly thawed solution of the enzyme substrate (7,8, dihyrofolic acid (5.44 mM)) was treated with 38 μL β-mercaptoethanol and 600 μL of 100mM Imidazole buffer pH 7.0. To measure the enzyme activity of DHFR, the reaction mixture contained 20 μl (3.2 mg/mL) of NADPH, 20 μL of the above 7,8, dihyrofolic acid mixture, and 955 μL 100mM Imidazole buffer pH 7.0. A sample (5 μL; 120 ng of protein) of DHFR was added to the prewarmed (25°C) reaction mixture, mixed rapidly, and the change in optical density at 340 nm was monitored every 5 seconds for a total of 120 seconds in a Shimadzu 160U ultraviolet spectrophotometer. The activity (units/mL) was calculated from the slope of the optical density vs time plot. (11g) Inhibition of DHFR activity
Trimethoprim (TMP) or the trimethoprim-4'-O-acetic acid amide of H2N-[D-Glu]-Ala-Ala-Ala-Ala-Lys-Lys-OH (TMP-peptide: Example 3), in a range of concentrations, was preincubated with samples of DHFR or ING-1-DHFR
conjugates (Example 8b) for 2 minutes. The activity of DHFR was then assayed as before (Example 11f) and the concentration of TMP or TMP-peptide required to produce 50% inhibition of enzyme activity (IC50) was calculated.
(11h) Ouantitation of DHFR in ING-1-DHFR conjugates,
A sample (1.2 μg of protein) of ING-1/DHFR
conjugate (from Example 8b) was used in place of DHFR in the assay for enzyme activity (Example 11f) . The activity of the conjugate (units/mL) was calculated from the slope of the optical density vs time plot as before. From the activity of the conjugate prepared in Example 8b and the activity of a known standard amount of unconjugated DHFR, the average number of molecules of DHFR conjugated to each antibody molecule was calculated and found to be 0.34 moles of DHFR per mole of antibody.
The present invention has been described in detail with particular reference to certain preferred
embodiments thereof, but it will be understood that variations and modifications can be effected within the spirit and scope of the invention.
Claims (54)
1. A non-radioactive targeting immunoreagent that comprises an immunoreactive material, the residue of one or more receptor moieties or ligands which have an affinity for non-covalent binding to a receptor moiety, and one or more linking groups.
2. A radioactive targeting immunoreagent that comprises the residue of a receptor moiety or a ligand which has an affinity for non-covalent binding to a receptor moiety, one or more chelating agents, one or more linking groups and one or more radionuclides.
3. A targeting immune reagent that comprises moieties represented by the structure
Z-(L1-X)n
wherein:
Z comprises the residue of an immunoreactive protein;
L1 is a chemical bond or a linking group that may contain a spacing group;
X is the residue of a receptor moiety or a ligand which has an affinity for non-covalent binding to a receptor moiety; and
n is an integer greater than zero.
4. A radioactive targeting reagent comprised of moieties represented in the structure
D-(L2-Q-M)m
wherein :
D is the residue of a receptor moiety or a ligand which has an affinity for non-covalent binding to a receptoro moiety; L2 is a chemical bond or a linking group that may contain a spacing group;
Q is the residue of a chelating group;
M is a radionuclide; and
m is an integer greater than zero.
5. The reagent of claim 3 wherein Z is the residue of an antibody or antibody fragment.
6. The reagent of claim 5 wherein the antibody is selected from ING-1; B72.3; 9.2.27; D612; UJ13A; NRLU- 10; 7E11C5; CC49; TNT; PR1A3; B174; B43; and anti-HLB antibodies.
7. The reagent of claim 3 wherein X is the residue of dihydrofolate reductase.
8. The reagent of claim 7 wherein the residue of dihydrofolate reductase is derived from Escherichia coli strain CV634 transformed with the plasmid pCV29.
9. The reagent of claim 3 wherein X is selected from the group consisting of residues of trimethoprim analogues and residues of methotrexate analogs.
10. The reagent of claim 3 wherein L1 is comprised of the residue of a heterobifunctional cross-linking reagent.
11. The reagent of claim 10 wherein the
heterobifunctional cross-linking reagent is selected from the group consisting of sulfosuccinimidyl 4-(N- maleimidomethyl)cyclohexane-1-carboxylate,
sulfosuccinimidyl (4-iodoacetyl) aminobenzoate,
sulfosuccinimidyl 4-(p-maleimidophenyl)butyrate, 2-
Iminothiolane, and N-succinimidyl S-acetylthioacetate.
12. The reagent of claim 3 wherein L1 is comprised of the residue of a modified receptor moiety containing a reactive functional group.
13. The reagent of claim 12 wherein the reactive functional group is selected from the group consisting of amino groups and sulfhydryl groups.
14. The reagent of claim 4 wherein D is selected from the group consisting of residues of trimethoprim analogues and residues of methotrexate analogs.
15. The reagent of claim 4 wherein D is the residue of dihydrofolate reductase.
16. The reagent of claim 15 wherein the residue of dihydrofolate reductase is derived from Escherichia coli strain CV634 transformed with the plasmid pCV29.
17. The reagent of claim 4 wherein L2 is the residue of a heterobifunctional cross-linking reagent.
18. The reagent of claim 17 wherein the
heterobifunctional cross-linking reagent is selected from the group consisting of sulfosuccinimidyl 4-(N- maleimidomethyl)cyclohexane-1-carboxylate,
sulfosuccinimidyl (4-iodoacetyl) aminobenzoate,
sulfosuccinimidyl 4-(p-maleimidophenyl)butyrate, 2- Iminothiolane, and N-succinimidyl S-acetylthioacetate.
19. The reagent of claim 4 wherein L2 is the residue of a modified ligand moiety containing a
reactive functional group.
20. The reagent of claim 19 wherein the reactive functional group is selected from the group consisting of amino groups and sulfhydryl groups.
21. The reagent of claim 4 wherein Q contains a polycarboxylic acid group.
22. The reagent of claim 4 wherein Q is selected from the group consisting of B4A, P4A, TMT, DCDTPA, PheMT, macroPheMT, and macroTMT.
23. The reagent of claim 4 wherein M is a
radioactive isotope of a metal.
24. The reagent of claim 23 wherein the
radioactive isotope is selected from 212pb, 212Bi, 90 Y, 177Lu, 186Re, 188Re, 64Cu, 67Cu, 99mTC/ 111In, and 87Y.
25. A method of making a compound of the
structure:
Z-(L1-X)n
wherein:
Z comprises the residue of an immunoreactive protein;
L1 is a chemical bond or a linking group that may contain a spacing group;
X is the residue of a receptor moiety or the residue of a ligand which has an affinity for non- covalent binding to a receptor moiety; and
n is an integer greater than zero;
comprising:
(i) reacting X with a precursor to a residue of L1 under conditions and for a time period sufficient to form a covalent complex which is a precursor to a residue of L1-X; and (ii) reacting Z with the precursor to the residue of L1- X produced in step (i) under conditions and for a time period sufficient to form a covalent complex Z-(L1-X)n.
26. The method of claim 25, wherein step (ii) comprises:
(iia) reacting Z with a precursor to the residue of Li under conditions and for a time period sufficient to form a covalent complex which is a precursor to a residue of Z-(L1)n; and
(iib) reacting the precursor to a residue of Z-(L1)n produced in step (iia) with a precursor to a residue of L1-X under conditions and for a time period sufficient to form a covalent complex Z-(L1-X)n.
27. A method of making a compound of the
structure:
D-(L2-Q-M)m
wherein:
D is the residue of a receptor moiety or the residue of a ligand which has an affinity for non- covalent binding to a receptor moiety;
L2 is a chemical bond or a linking group that may contain a spacing group;
Q is the residue of a chelating group;
M is a radionuclide; and
m is an integer greater than zero;
comprising:
(i) reacting Q with a precursor to a residue of L2 under conditions and for a time period sufficient to form a covalent complex which is a precursor to a residue of L2-Q
(ii) reacting D with the precursor to the residue of L2-
Q produced in step (i) under conditions and for a time period sufficient to form a covalent complex D-(L2-Q) m, and (iii) reacting said covalent complex D-(L2-Q)m with M under conditions and for a time period sufficient to form a complex D-(L2-Q-M)m.
28. The method of claim 27, wherein step (ii) comprises:
(iia) reacting D with a precursor to a residue of L2 under conditions and for a time period sufficient to form a covalent complex which is a precursor to a residue of D-(L2)m; and
(iib) reacting the precursor to the residue of D-(L2)m produced in step (iia) with a precursor to a residue of L2-Q xinder conditions and for a time period sufficient to form a covalent complex D-(L2-Q)m.
29. The method of claim 25 wherein Z is an antibody or antibody fragment.
30. The antibody of claim 29 wherein the antibody is selected from ING-1; B72.3; 9.2.27; D612; UJ13A;
NRLU-10; 7E11C5; CC49; TNT; PR1A3; B174; B43; and anti- HLB antibodies.
31. The method of claim 25 wherein X is selected from the group consisting of residues of trimethoprim analogs and residues of methotrexate analogs.
32. The method of claim 25 wherein L1 is comprised of the residue of a heterobifunctional cross-linking reagent.
33. The method of claim 32 wherein the
heterobifunctional cross-linking reagent is selected from the group consisting of sulfosuccinimidyl 4-(N- maleimidomethyl)cyclohexane-1-carboxylate,
sulfosuccinimidyl (4-iodoacetyl)aminoben oate. sulfosuccinimidyl 4-(p-maleimidophenyl)butyrate, 2- Iminothiolane, and N-succinimidyl S-acetylthioacetate.
34. The method of claim 25 wherein L1 is comprised of the residue of a modified nucleotide moiety
containing a reactive functional group.
35. The method of claim 34 wherein the reactive fxinctional group is selected from the group consisting of amine groups and sulfhydryl groups.
36. The method of claim 25 wherein X is the residue of dihydrofolate reductase.
37. The method of claim 36 wherein the residue of dihydrofolate reductase is derived from Escherichia coli strain CV634 transformed with the plasmid pCV29.
38. The method of claim 28 wherein D is selected from the group consisting of residues of trimethoprim analogs and residues of methotrexate analogs.
39. The method of claim 28 wherein D is the residue of dihydrofolate reductase.
40. The method of claim 39 wherein the residue of dihydrofolate reductase is derived from Escherichia coli strain CV634 transformed with the plasmid pCV29.
41. The method of claim 28 wherein L2 is comprised of the residue of a heterobifunctional cross-linking reagent .
42. The method of claim 41 wherein the
heterobifunctional cross-linking reagent is selected from the group consisting of sulfosuccinimidyl 4-(NN- maleimidomethyl)cyclohexane-1-carboxylate,
sulfosuccinimidyl (4-iodoacetyl)aminobenzoate,
sulfosuccinimidyl 4-(p-maleimidophenyl)butyrate, 2- Iminothiolane, and N-succinimidyl S-acetylthioacetate.
43. The method of claim 41 wherein L2 is a residue of a ligand moiety containing a reactive functional group.
44. The method of claim 43 wherein the reactive functional group is selected from the group consisting of amine groups, carboxylate groups, hydroxyl groups, and sulfhydryl groups.
45. The method of claim 28 wherein Q contains a polycarboxylic acid group.
46. The method of claim 28 wherein Q is selected from the group consisting of B4A, P4A, TMT, DCDTPA, PheMT, macroPheMT, and macroTMT.
47. The method of claim 28 wherein M is a
radioactive isotope of a metal.
48. The method of claim 47 wherein the radioactive isotope is selected from 212Pb, 212Bi, 90Y, 177Lu,
186Re, 188Re, 64Cu, 67Cu, 99mTc, 111In, and 87Y.
49. A pharmaceutical composition comprising a compound of claim 3 dissolved or dispersed in a
pharmaceutically acceptable carrier.
50. A pharmaceutical composition comprising a compound of claim 4 dissolved or dispersed in a
pharmaceutically acceptable medium.
51. A method of treating a tumor in a mammal comprising administering to said mammal an effective dose of a non-radioactive targeting immunoreagent of claim 3 in a pharmaceutically acceptable medium, waiting for a time period sufficient for said non-radioactive targeting immunoreagent to accumulate at the tumor site in said mammal, and subsequently, administering an effective dose of a radioactive targeting reagent of claim 4 in a pharmaceutically acceptable medixim to said mammal, and waiting for a time period sufficient for said radioactive targeting reagent to accumulate at the target site, said target site being the said non- radioactive targeting immunoreagent accumulated at said tumor site in said mammal.
52. A method of diagnostic imaging in a mammal comprising administering to said mammal an imaging effective dose of a non-radioactive targeting
immunoreagent of claim 3 in a pharmaceutically
acceptable medium, waiting for a time period sufficient for said non-radioactive targeting immunoreagent to accumulate at the imaging site in said mammal, and subsequently, administering an effective dose of a radioactive targeting reagent of claim 4 in a
pharmaceutically acceptable medium to said mammal, and waiting for a time period sufficient for said
radioactive targeting reagent to accumulate at the target site, said target site being the said non- radioactive targeting immunoreagent accximulated at said imaging site in said mammal, and generating an image of said mammal.
53. The reagent of claim 3 wherein X is a residue of a receptor moiety and Z and X comprise a fusion protein.
54. The reagent of claim 53 wherein the receptor moiety is dihydrofolate reductase.
Applications Claiming Priority (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US99042392A | 1992-12-15 | 1992-12-15 | |
US990423 | 1992-12-15 | ||
PCT/US1993/011842 WO1994013327A1 (en) | 1992-12-15 | 1993-12-06 | Immunoreactive reagents employing dihydrofolate reductase |
Publications (1)
Publication Number | Publication Date |
---|---|
AU5984094A true AU5984094A (en) | 1994-07-04 |
Family
ID=25536134
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
AU59840/94A Abandoned AU5984094A (en) | 1992-12-15 | 1993-12-06 | Immunoreactive reagents employing dihydrofolate reductase |
Country Status (5)
Country | Link |
---|---|
EP (1) | EP0675737A1 (en) |
JP (1) | JPH08507750A (en) |
AU (1) | AU5984094A (en) |
CA (1) | CA2151614A1 (en) |
WO (1) | WO1994013327A1 (en) |
Families Citing this family (9)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5688488A (en) * | 1989-04-03 | 1997-11-18 | Purdue Research Foundation | Composition and method for tumor imaging |
DE19910102B4 (en) * | 1999-03-08 | 2006-06-01 | Fischer, Markus, Dr.rer.nat. | Protein conjugates, methods, vectors, proteins and DNA for their production, their use, and medicines and vaccines containing the same |
US20070264194A1 (en) * | 2001-08-10 | 2007-11-15 | The Scripps Research Institute | Peptides That Bind To Atherosclerotic Lesions |
AU2005219733C1 (en) * | 2004-03-05 | 2010-12-16 | Denki Kagaku Kogyo Kabushiki Kaisha | Hyaluronic acid-methotrexate conjugate |
US7807675B2 (en) * | 2004-04-02 | 2010-10-05 | Denki Kagaku Kogyo Kabushiki Kaisha | Hyaluronic acid-methotrexate conjugate |
WO2007088051A2 (en) | 2006-01-31 | 2007-08-09 | Bayer Schering Pharma Aktiengesellschaft | Modulation of mdl-1 activity for treatment of inflammatory disease |
EP2005970A1 (en) | 2007-06-22 | 2008-12-24 | Berlin Science Partners GmbH i.V. | Imaging diagnosis by combining contrast agents |
EP2072060A1 (en) | 2007-12-18 | 2009-06-24 | Institut Curie | Methods and compositions for the preparation and use of toxin conjugates. |
CN112649600A (en) * | 2021-01-13 | 2021-04-13 | 青岛农业大学 | DHFR-based multi-residue fluorescence polarization immunoassay method for sulfonamide synergist drugs |
-
1993
- 1993-12-06 CA CA002151614A patent/CA2151614A1/en not_active Abandoned
- 1993-12-06 WO PCT/US1993/011842 patent/WO1994013327A1/en not_active Application Discontinuation
- 1993-12-06 AU AU59840/94A patent/AU5984094A/en not_active Abandoned
- 1993-12-06 EP EP94905922A patent/EP0675737A1/en not_active Withdrawn
- 1993-12-06 JP JP6514321A patent/JPH08507750A/en active Pending
Also Published As
Publication number | Publication date |
---|---|
JPH08507750A (en) | 1996-08-20 |
EP0675737A1 (en) | 1995-10-11 |
WO1994013327A1 (en) | 1994-06-23 |
CA2151614A1 (en) | 1994-06-23 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
AU588832B2 (en) | Antibody-metal ion complexes | |
US5760191A (en) | Macrocyclic complexing agents and targeting immunoreagents useful in therapeutic and diagnostic compositions and methods | |
US4741900A (en) | Antibody-metal ion complexes | |
US5171563A (en) | Cleavable linkers for the reduction of non-target organ retention of immunoconjugates | |
US6248870B1 (en) | Unsymmetrical complexing agents and targeting immunoreagents useful in therapeutic and diagnostic compositions and methods | |
AU619738B2 (en) | Metal-radionuclide-labeled proteins and glycoproteins for diagnosis and therapy | |
CN107382890B (en) | Homogenic and xenogenic multivalent inhibitors of Prostate Specific Membrane Antigen (PSMA) and uses thereof | |
JPH06501703A (en) | Complexing agents and targeting radioimmunoassays useful in therapeutic and diagnostic imaging compositions and methods | |
JPH0749377B2 (en) | Functional specific antibody | |
EA026305B1 (en) | Alpha-emitting complexes | |
CA2150477A1 (en) | Sequential targeting of tumor sites with oligonucleotide conjugates of antibody and complementary oligonucleotide conjugates of chelated radionuclide | |
AU5984094A (en) | Immunoreactive reagents employing dihydrofolate reductase | |
US6429297B1 (en) | Modified avidin and streptavidin and methods of use thereof | |
WO1994020523A1 (en) | Tumor targeting with l-enantiomeric oligonucleotide conjugates of immunoreagents and of chelated radionuclides | |
EP0436664B1 (en) | Cleavable linkers for the reduction of non-target organ retention of immunoconjugates | |
JP3358141B2 (en) | Complex of complexing agent with metal radionuclide ion and diagnostic composition containing the same | |
WO1994029333A1 (en) | Immunoreactive reagents employing monoamine oxidase | |
CN100457192C (en) | Biotin-derivatives and their conjugates with chelating agents | |
AU6128694A (en) | Immunoreactive reagents employing heterodimers | |
Pollack et al. | Hydrazino-type N 2 S 2 chelators |