WO1991017999A1 - METHOD FOR LABELLING SUBSTANCES CONTAINING AMINO GROUPS, INCLUDING CELLS SUCH AS BLOOD CELLS, WITH 99mTc AND KIT FOR CARRYING OUT THIS METHOD - Google Patents
METHOD FOR LABELLING SUBSTANCES CONTAINING AMINO GROUPS, INCLUDING CELLS SUCH AS BLOOD CELLS, WITH 99mTc AND KIT FOR CARRYING OUT THIS METHOD Download PDFInfo
- Publication number
- WO1991017999A1 WO1991017999A1 PCT/NL1991/000080 NL9100080W WO9117999A1 WO 1991017999 A1 WO1991017999 A1 WO 1991017999A1 NL 9100080 W NL9100080 W NL 9100080W WO 9117999 A1 WO9117999 A1 WO 9117999A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- labelling
- borohydride
- labelled
- ions
- substance
- Prior art date
Links
- 238000000034 method Methods 0.000 title claims abstract description 38
- 238000002372 labelling Methods 0.000 title claims abstract description 32
- 239000000126 substance Substances 0.000 title claims abstract description 24
- 210000004027 cell Anatomy 0.000 title claims abstract description 9
- 210000000601 blood cell Anatomy 0.000 title claims abstract description 6
- -1 AMINO GROUPS Chemical group 0.000 title claims description 10
- 238000006243 chemical reaction Methods 0.000 claims abstract description 13
- IUTCEZPPWBHGIX-UHFFFAOYSA-N tin(2+) Chemical class [Sn+2] IUTCEZPPWBHGIX-UHFFFAOYSA-N 0.000 claims abstract description 10
- 239000011363 dried mixture Substances 0.000 claims abstract description 8
- 150000002500 ions Chemical class 0.000 claims abstract description 8
- 125000003277 amino group Chemical group 0.000 claims abstract description 6
- 102000004169 proteins and genes Human genes 0.000 claims description 24
- 108090000623 proteins and genes Proteins 0.000 claims description 24
- 229910020889 NaBH3 Inorganic materials 0.000 claims description 2
- 239000012736 aqueous medium Substances 0.000 claims description 2
- 235000018102 proteins Nutrition 0.000 description 23
- 239000000243 solution Substances 0.000 description 10
- 108060003951 Immunoglobulin Proteins 0.000 description 7
- 102000018358 immunoglobulin Human genes 0.000 description 7
- 102000007330 LDL Lipoproteins Human genes 0.000 description 6
- 108010007622 LDL Lipoproteins Proteins 0.000 description 6
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 6
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 6
- 238000009206 nuclear medicine Methods 0.000 description 5
- 102000008946 Fibrinogen Human genes 0.000 description 4
- 108010049003 Fibrinogen Proteins 0.000 description 4
- ATJFFYVFTNAWJD-UHFFFAOYSA-N Tin Chemical compound [Sn] ATJFFYVFTNAWJD-UHFFFAOYSA-N 0.000 description 4
- 229940012952 fibrinogen Drugs 0.000 description 4
- RWSXRVCMGQZWBV-WDSKDSINSA-N glutathione Chemical class OC(=O)[C@@H](N)CCC(=O)N[C@@H](CS)C(=O)NCC(O)=O RWSXRVCMGQZWBV-WDSKDSINSA-N 0.000 description 4
- 229910052713 technetium Inorganic materials 0.000 description 4
- 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
- ZLMJMSJWJFRBEC-UHFFFAOYSA-N Potassium Chemical compound [K] ZLMJMSJWJFRBEC-UHFFFAOYSA-N 0.000 description 3
- 230000004071 biological effect Effects 0.000 description 3
- 239000002738 chelating agent Substances 0.000 description 3
- 239000000539 dimer Substances 0.000 description 3
- 238000002360 preparation method Methods 0.000 description 3
- 239000011780 sodium chloride Substances 0.000 description 3
- GKLVYJBZJHMRIY-UHFFFAOYSA-N technetium atom Chemical compound [Tc] GKLVYJBZJHMRIY-UHFFFAOYSA-N 0.000 description 3
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- 108010024636 Glutathione Proteins 0.000 description 2
- 108091006905 Human Serum Albumin Proteins 0.000 description 2
- 102000008100 Human Serum Albumin Human genes 0.000 description 2
- 241000283973 Oryctolagus cuniculus Species 0.000 description 2
- 229920005654 Sephadex Polymers 0.000 description 2
- 239000012507 Sephadex™ Substances 0.000 description 2
- 102100033571 Tissue-type plasminogen activator Human genes 0.000 description 2
- VEJXYBLYLRPHPK-UHFFFAOYSA-N [Mo].[Tc] Chemical compound [Mo].[Tc] VEJXYBLYLRPHPK-UHFFFAOYSA-N 0.000 description 2
- 239000003795 chemical substances by application Substances 0.000 description 2
- 150000001875 compounds Chemical class 0.000 description 2
- 238000003745 diagnosis Methods 0.000 description 2
- 229940044627 gamma-interferon Drugs 0.000 description 2
- 229960003180 glutathione Drugs 0.000 description 2
- 150000004678 hydrides Chemical class 0.000 description 2
- 238000001727 in vivo Methods 0.000 description 2
- NOESYZHRGYRDHS-UHFFFAOYSA-N insulin Chemical compound N1C(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(NC(=O)CN)C(C)CC)CSSCC(C(NC(CO)C(=O)NC(CC(C)C)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CCC(N)=O)C(=O)NC(CC(C)C)C(=O)NC(CCC(O)=O)C(=O)NC(CC(N)=O)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CSSCC(NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2C=CC(O)=CC=2)NC(=O)C(CC(C)C)NC(=O)C(C)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2NC=NC=2)NC(=O)C(CO)NC(=O)CNC2=O)C(=O)NCC(=O)NC(CCC(O)=O)C(=O)NC(CCCNC(N)=N)C(=O)NCC(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC(O)=CC=3)C(=O)NC(C(C)O)C(=O)N3C(CCC3)C(=O)NC(CCCCN)C(=O)NC(C)C(O)=O)C(=O)NC(CC(N)=O)C(O)=O)=O)NC(=O)C(C(C)CC)NC(=O)C(CO)NC(=O)C(C(C)O)NC(=O)C1CSSCC2NC(=O)C(CC(C)C)NC(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CC(N)=O)NC(=O)C(NC(=O)C(N)CC=1C=CC=CC=1)C(C)C)CC1=CN=CN1 NOESYZHRGYRDHS-UHFFFAOYSA-N 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 231100000252 nontoxic Toxicity 0.000 description 2
- 230000003000 nontoxic effect Effects 0.000 description 2
- 229910052700 potassium Inorganic materials 0.000 description 2
- 239000011591 potassium Substances 0.000 description 2
- IWZKICVEHNUQTL-UHFFFAOYSA-M potassium hydrogen phthalate Chemical compound [K+].OC(=O)C1=CC=CC=C1C([O-])=O IWZKICVEHNUQTL-UHFFFAOYSA-M 0.000 description 2
- 239000003755 preservative agent Substances 0.000 description 2
- 230000005855 radiation Effects 0.000 description 2
- 230000002285 radioactive effect Effects 0.000 description 2
- 239000011541 reaction mixture Substances 0.000 description 2
- 230000035484 reaction time Effects 0.000 description 2
- 210000002966 serum Anatomy 0.000 description 2
- 229910052708 sodium Inorganic materials 0.000 description 2
- 239000011734 sodium Substances 0.000 description 2
- DEQANNDTNATYII-OULOTJBUSA-N (4r,7s,10s,13r,16s,19r)-10-(4-aminobutyl)-19-[[(2r)-2-amino-3-phenylpropanoyl]amino]-16-benzyl-n-[(2r,3r)-1,3-dihydroxybutan-2-yl]-7-[(1r)-1-hydroxyethyl]-13-(1h-indol-3-ylmethyl)-6,9,12,15,18-pentaoxo-1,2-dithia-5,8,11,14,17-pentazacycloicosane-4-carboxa Chemical compound C([C@@H](N)C(=O)N[C@H]1CSSC[C@H](NC(=O)[C@H]([C@@H](C)O)NC(=O)[C@H](CCCCN)NC(=O)[C@@H](CC=2C3=CC=CC=C3NC=2)NC(=O)[C@H](CC=2C=CC=CC=2)NC1=O)C(=O)N[C@H](CO)[C@H](O)C)C1=CC=CC=C1 DEQANNDTNATYII-OULOTJBUSA-N 0.000 description 1
- WXTMDXOMEHJXQO-UHFFFAOYSA-N 2,5-dihydroxybenzoic acid Chemical compound OC(=O)C1=CC(O)=CC=C1O WXTMDXOMEHJXQO-UHFFFAOYSA-N 0.000 description 1
- 102000004506 Blood Proteins Human genes 0.000 description 1
- 108010017384 Blood Proteins Proteins 0.000 description 1
- ZOXJGFHDIHLPTG-UHFFFAOYSA-N Boron Chemical group [B] ZOXJGFHDIHLPTG-UHFFFAOYSA-N 0.000 description 1
- 241000283690 Bos taurus Species 0.000 description 1
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 1
- UJKPHYRXOLRVJJ-MLSVHJFASA-N CC(O)C1=C(C)/C2=C/C3=N/C(=C\C4=C(CCC(O)=O)C(C)=C(N4)/C=C4\N=C(\C=C\1/N\2)C(C)=C4C(C)O)/C(CCC(O)=O)=C3C Chemical compound CC(O)C1=C(C)/C2=C/C3=N/C(=C\C4=C(CCC(O)=O)C(C)=C(N4)/C=C4\N=C(\C=C\1/N\2)C(C)=C4C(C)O)/C(CCC(O)=O)=C3C UJKPHYRXOLRVJJ-MLSVHJFASA-N 0.000 description 1
- 241000557626 Corvus corax Species 0.000 description 1
- XFXPMWWXUTWYJX-UHFFFAOYSA-N Cyanide Chemical compound N#[C-] XFXPMWWXUTWYJX-UHFFFAOYSA-N 0.000 description 1
- 229920002307 Dextran Polymers 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
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 206010073306 Exposure to radiation Diseases 0.000 description 1
- 108010053070 Glutathione Disulfide Proteins 0.000 description 1
- 102000004877 Insulin Human genes 0.000 description 1
- 108090001061 Insulin Proteins 0.000 description 1
- 102000008070 Interferon-gamma Human genes 0.000 description 1
- 108010074328 Interferon-gamma Proteins 0.000 description 1
- LEVWYRKDKASIDU-IMJSIDKUSA-N L-cystine Chemical compound [O-]C(=O)[C@@H]([NH3+])CSSC[C@H]([NH3+])C([O-])=O LEVWYRKDKASIDU-IMJSIDKUSA-N 0.000 description 1
- 108010016076 Octreotide Proteins 0.000 description 1
- 241000212342 Sium Species 0.000 description 1
- 102000005157 Somatostatin Human genes 0.000 description 1
- 108010056088 Somatostatin Proteins 0.000 description 1
- GKLVYJBZJHMRIY-OUBTZVSYSA-N Technetium-99 Chemical compound [99Tc] GKLVYJBZJHMRIY-OUBTZVSYSA-N 0.000 description 1
- 229910021626 Tin(II) chloride Inorganic materials 0.000 description 1
- 108090000373 Tissue Plasminogen Activator Proteins 0.000 description 1
- 108050006955 Tissue-type plasminogen activator Proteins 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 230000002411 adverse Effects 0.000 description 1
- 150000001408 amides Chemical class 0.000 description 1
- 150000001412 amines Chemical class 0.000 description 1
- 235000001014 amino acid Nutrition 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- 230000003127 anti-melanomic effect Effects 0.000 description 1
- 239000000427 antigen Substances 0.000 description 1
- 102000036639 antigens Human genes 0.000 description 1
- 108091007433 antigens Proteins 0.000 description 1
- 238000000376 autoradiography Methods 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
- 239000008366 buffered solution Substances 0.000 description 1
- 229930002875 chlorophyll Natural products 0.000 description 1
- 235000019804 chlorophyll Nutrition 0.000 description 1
- ATNHDLDRLWWWCB-AENOIHSZSA-M chlorophyll a Chemical compound C1([C@@H](C(=O)OC)C(=O)C2=C3C)=C2N2C3=CC(C(CC)=C3C)=[N+]4C3=CC3=C(C=C)C(C)=C5N3[Mg-2]42[N+]2=C1[C@@H](CCC(=O)OC\C=C(/C)CCC[C@H](C)CCC[C@H](C)CCCC(C)C)[C@H](C)C2=C5 ATNHDLDRLWWWCB-AENOIHSZSA-M 0.000 description 1
- XUJNEKJLAYXESH-UHFFFAOYSA-N cysteine Natural products SCC(N)C(O)=O XUJNEKJLAYXESH-UHFFFAOYSA-N 0.000 description 1
- 235000018417 cysteine Nutrition 0.000 description 1
- 229960003067 cystine Drugs 0.000 description 1
- 238000010494 dissociation reaction Methods 0.000 description 1
- 230000005593 dissociations Effects 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 238000001962 electrophoresis Methods 0.000 description 1
- 239000003480 eluent Substances 0.000 description 1
- 230000002255 enzymatic effect Effects 0.000 description 1
- 210000003743 erythrocyte Anatomy 0.000 description 1
- 238000002523 gelfiltration Methods 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- YPZRWBKMTBYPTK-BJDJZHNGSA-N glutathione disulfide Chemical compound OC(=O)[C@@H](N)CCC(=O)N[C@H](C(=O)NCC(O)=O)CSSC[C@@H](C(=O)NCC(O)=O)NC(=O)CC[C@H](N)C(O)=O YPZRWBKMTBYPTK-BJDJZHNGSA-N 0.000 description 1
- 210000003714 granulocyte Anatomy 0.000 description 1
- 229960003569 hematoporphyrin Drugs 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- 230000003054 hormonal effect Effects 0.000 description 1
- 229940088597 hormone Drugs 0.000 description 1
- 239000005556 hormone Substances 0.000 description 1
- 229940106780 human fibrinogen Drugs 0.000 description 1
- 230000001900 immune effect Effects 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 238000010348 incorporation Methods 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 229940125396 insulin Drugs 0.000 description 1
- 238000004255 ion exchange chromatography Methods 0.000 description 1
- 230000014759 maintenance of location Effects 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 239000002609 medium Substances 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 239000002773 nucleotide Substances 0.000 description 1
- 125000003729 nucleotide group Chemical group 0.000 description 1
- QYSGYZVSCZSLHT-UHFFFAOYSA-N octafluoropropane Chemical compound FC(F)(F)C(F)(F)C(F)(F)F QYSGYZVSCZSLHT-UHFFFAOYSA-N 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 229960003330 pentetic acid Drugs 0.000 description 1
- XNGIFLGASWRNHJ-UHFFFAOYSA-L phthalate(2-) Chemical compound [O-]C(=O)C1=CC=CC=C1C([O-])=O XNGIFLGASWRNHJ-UHFFFAOYSA-L 0.000 description 1
- 230000037081 physical activity Effects 0.000 description 1
- 229920000642 polymer Polymers 0.000 description 1
- LJCNRYVRMXRIQR-OLXYHTOASA-L potassium sodium L-tartrate Chemical compound [Na+].[K+].[O-]C(=O)[C@H](O)[C@@H](O)C([O-])=O LJCNRYVRMXRIQR-OLXYHTOASA-L 0.000 description 1
- 229940074439 potassium sodium tartrate Drugs 0.000 description 1
- 239000002244 precipitate Substances 0.000 description 1
- 230000001376 precipitating effect Effects 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 230000002335 preservative effect Effects 0.000 description 1
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 1
- 239000000700 radioactive tracer Substances 0.000 description 1
- 239000012217 radiopharmaceutical Substances 0.000 description 1
- 229940121896 radiopharmaceutical Drugs 0.000 description 1
- 230000002799 radiopharmaceutical effect Effects 0.000 description 1
- 239000012048 reactive intermediate Substances 0.000 description 1
- 229940072272 sandostatin Drugs 0.000 description 1
- HELHAJAZNSDZJO-OLXYHTOASA-L sodium L-tartrate Chemical compound [Na+].[Na+].[O-]C(=O)[C@H](O)[C@@H](O)C([O-])=O HELHAJAZNSDZJO-OLXYHTOASA-L 0.000 description 1
- 229910000033 sodium borohydride Inorganic materials 0.000 description 1
- 239000012279 sodium borohydride Substances 0.000 description 1
- 235000011006 sodium potassium tartrate Nutrition 0.000 description 1
- 239000001433 sodium tartrate Substances 0.000 description 1
- 229960002167 sodium tartrate Drugs 0.000 description 1
- 235000011004 sodium tartrates Nutrition 0.000 description 1
- NHXLMOGPVYXJNR-ATOGVRKGSA-N somatostatin Chemical compound C([C@H]1C(=O)N[C@H](C(N[C@@H](CO)C(=O)N[C@@H](CSSC[C@@H](C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CC=2C=CC=CC=2)C(=O)N[C@@H](CC=2C=CC=CC=2)C(=O)N[C@@H](CC=2C3=CC=CC=C3NC=2)C(=O)N[C@@H](CCCCN)C(=O)N[C@H](C(=O)N1)[C@@H](C)O)NC(=O)CNC(=O)[C@H](C)N)C(O)=O)=O)[C@H](O)C)C1=CC=CC=C1 NHXLMOGPVYXJNR-ATOGVRKGSA-N 0.000 description 1
- 229960000553 somatostatin Drugs 0.000 description 1
- 235000011150 stannous chloride Nutrition 0.000 description 1
- 229940095064 tartrate Drugs 0.000 description 1
- 229940056501 technetium 99m Drugs 0.000 description 1
- 230000001225 therapeutic effect Effects 0.000 description 1
- 230000036962 time dependent Effects 0.000 description 1
- 229910001432 tin ion Inorganic materials 0.000 description 1
- AXZWODMDQAVCJE-UHFFFAOYSA-L tin(II) chloride (anhydrous) Chemical compound [Cl-].[Cl-].[Sn+2] AXZWODMDQAVCJE-UHFFFAOYSA-L 0.000 description 1
- YNJBWRMUSHSURL-UHFFFAOYSA-N trichloroacetic acid Chemical compound OC(=O)C(Cl)(Cl)Cl YNJBWRMUSHSURL-UHFFFAOYSA-N 0.000 description 1
- 239000002753 trypsin inhibitor Substances 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
- A61K51/12—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by a special physical form, e.g. emulsion, microcapsules, liposomes, characterized by a special physical form, e.g. emulsions, dispersions, microcapsules
- A61K51/1203—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by a special physical form, e.g. emulsion, microcapsules, liposomes, characterized by a special physical form, e.g. emulsions, dispersions, microcapsules in a form not provided for by groups A61K51/1206 - A61K51/1296, e.g. cells, cell fragments, viruses, virus capsides, ghosts, red blood cells, viral vectors
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07B—GENERAL METHODS OF ORGANIC CHEMISTRY; APPARATUS THEREFOR
- C07B59/00—Introduction of isotopes of elements into organic compounds ; Labelled organic compounds per se
- C07B59/004—Acyclic, carbocyclic or heterocyclic compounds containing elements other than carbon, hydrogen, halogen, oxygen, nitrogen, sulfur, selenium or tellurium
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
- C07F13/00—Compounds containing elements of Groups 7 or 17 of the Periodic Table
- C07F13/005—Compounds without a metal-carbon linkage
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2123/00—Preparations for testing in vivo
Definitions
- the invention relates to a method for labelling substances
- * * including cells such as blood cells, which contain one or more amino * groups, with a radioisotope, the substance to be labelled being combined in the presence of a reductor with "TcO ⁇ ions.
- radioisotope is understood to be a radio-
- m Tc Labelling with " m Tc is used in biomedical science for, for example, proteins such as monoclonal antibodies, which are particularly useful for many diagnostic and therapeutic applications
- the labelling method must be compatible with the biological and physical activity of the protein; the chemicals used must be compatible with
- the first reaction is carried out at a relatively high temperature, for example 1_0°C, and for a relatively long time, for example . hours.
- a relatively high temperature for example 1_0°C
- a relatively long time for example . hours.
- the reductor used is at least one borohydride.
- borohydrides which are suitable for use in the method of the invention are potassium borohydride and sodium boro ⁇ hydride (NaBH ⁇ ) and potassium borohydride cyanide and sodium boro ⁇ hydride cyanide (NaBH 3 CN) .
- the first-mentioned substances are preferred because particularly high values for the labelling effi- ciency can be obtained with these. In principle, however, all sub ⁇ stances which yield borohydride ions can be used.
- the method according to the invention is particularly efficient if Sn 2+ ions (which, for example, originate from tin(II) chloride) are also present as reductor. If Sn 2+ ions are used, the efficiency of the labelling can be improved appreciably. This effi ⁇ ciency is, for example, better by a factor of 10-100 than without the use of Sn 2+ ions.
- the total amount of tin in the sample (which contains at least 0.1 mg of the substance to be labelled) is in general 1-2000 ng. If a so-called freeze-dried kit is used for carrying out the method according to the invention, the amount of tin can be relatively low and can be, for example, 5 ⁇ 50 ng.
- Tin ions have the tendency to form a colloidal precipitate. In the method according to the invention, this can be counteracted by the use of chelating agents. Chelating agents of this type are known. Examples of chelating agents which are very satisfactory are tartrate/phthalate buffer and diethylenetriaminepentaacetic acid
- preservative is also present in the solution because it has been found that in the absence of such an agent the stability of the tin solution declines. All conventional preservatives can be used, for example
- the temperature in the labelling reaction according to the invention is not critical. Preferably, the temperature of the working environment is then also maintained. In general, this temperature will be 1 ⁇ 25 C.
- An embodiment of the method according to the invention which is preferred is that in which per 0.1 mg of the substance to be labelled at least an amount of 10-500 ng of Sn 2+ ions and 0.01 - 1 ⁇ g of borohydride is used.
- the pH of the chosen solutions can also vary within a wide range. In general, however, it is preferred that the substance to be labelled is present in an aqueous medium having a pH in the range of 3 ⁇ H.
- the " ra Tc0 ⁇ ions to be used are present in a solution having a pH in the range of _.5 ⁇ 7-5.
- the Sn 2+ ions which are optionally to be used are present in a solution having a pH in the range of 0-7, and - the final solution of the labelled substance has a pH in the range of 5-10.
- the labelling reaction will in general be carried out in a buffered solution.
- proteins can be labelled with retention of functional properties.
- functional properties for example, in the case of antibodies the immuno-diagnostic properties, in the case of enzymes the enzymatic properties and in the case of hormones the hormonal properties are preserved.
- proteins can, for example, be labelled using the method according to the invention (the labelling efficiency being given in percent) : aggregated IgG (immunoglobulin) 97_ hematoporphyrin 60% IgG, fractions purified via protein A 88» cystine/cysteine GSSG (glutathione dimer) 9 %
- Hb dimer (haemoglobin) 98% dextran 88% anti- ⁇ -interferon 98%
- HSA dimer human serum albumin
- HSA polymer 75% anti-leucoprotease 93%
- granulocytes PMN
- erythro- cytes erythro- cytes/"mixed" leuco's.
- the labelled proteins can be prepared non-toxic, pyrogen-free and sterile. This is important, especially when the labelled protein has to be used in vivo. In this case the labelling must, of course, have no adverse influence on the biological properties of the protein.
- sodium borohydride is particularly preferred in the labelling method according to the invention which is carried out in one step.
- a high incorporation (for example 95%) of the label is achievable.
- the method according to the invention is especially important for the labelling of biologically active proteins in nuclear medicine, in which radiopharmaceuticals are administered to patients for diagnostic purposes.
- a diagnosis can be made or a diagnosis can be confirmed on the basis of a (sometimes) time-dependent distribution pattern.
- the distribution pattern in the body is recorded using instruments which are suitable for this purpose and which show images or scintigrams.
- the " m Tc label used has the significant
- the method according to the invention can also be used for labelling substances other than proteins, provided these substances
- amino groups examples are amino acids, nucleotides and medicaments containing amino groups, which can be useful in nuclear medicine.
- Cells such as blood cells can also be labelled using the method according to the invention.
- use can be made of 10 substances which combine with the cells, for example troponolate.
- Substances of this type entrain the radioactive technetium.
- the method according to the invention makes efficiencies of between 50 and 90% possible.
- the invention also relates to a kit for carrying out the 15 method according to the invention, which kit at least comprises: a container, which contains a freeze-dried mixture of borohydride, a tin(II) salt and a buffer.
- the invention also relates to a freeze-dried mixture of boro ⁇ hydride, a tin(II) salt and a buffer.
- the labelling can be carried 20 out in a simple and effective manner with the aid of this mixture or the abovementioned kit.
- the 99m Tc ⁇ 4 ion is always “carrier-free” or present in “tracer” amounts and is not an important reaction parameter.
- the Sn 2* concentration in the buffer is preferably 1 mg/ml.
- the labelling efficiency of the method followed was determined by means of gel filtration on a 10 cm long column of Sephadex G50 using 0.15 M NaCl as eluent (the labelled protein elutes on the dead volume) ; via precipitation with trichloroacetic acid, the labelled protein precipitating; via autoradiography following electrophoresis, in which case an initial overall impression is obtained; via HPLC analysis and (especially for human immunoglobulin) via ion exchange chromatography on DEAE-Sephadex A25.
- the following table gives the labelling efficiency which was obtained for various protein preparations under conditions which had been found to be optimum for a human immunoglobulin preparation.
- the LDL label was also found not to exchange with other 2 serum proteins when labelled LDL was incubated in serum.
- SUBSTITUTE SHEET 0.5-1 ⁇ g of NaBH ⁇ in the form of a 1 mg/ml solution in 0.05 N NaOH.
- 0.1-1 mg of protein in 0.1-0.5 ml of 0.15 M NaCl and 100 ⁇ l of "TcOz, from a molybdenum-technetium generator are added to the freeze-dried mixture.
- the freeze-dried mixture of Sn 2+ , NaBH ⁇ and buffer is found to be stable for a few months at 37°C, provided the ampoules are sealed airtight under nitrogen.
- the stability is apparent from the fact that when the labelling is carried out with freeze-dried material which has been stored for a few months at 37 ° C, the labelling efficiency is equally as high as with freshly prepared reaction mixture.
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Abstract
The invention relates to a method for labelling substances, including cells such as blood cells, which contain one or more amino groups, with a radioisotope, the substance to be labelled being combined in the presence of a reductor with 99mTcO4 ions and the reductor used being at least one borohydride, to a kit for carrying out said method, which kit comprises at least a container, which contains a freeze-dried mixture of a borohydride, a tin(II) salt and a buffer, and to a freeze-dried mixture at least consisting of borohydride, tin(II) salt and buffer for use in labelling reactions.
Description
Method for labelling substances containing amino groups, including cells such as blood cells, with ""Tc and kit for carrying out this method.
5 The invention relates to a method for labelling substances,
* * including cells such as blood cells, which contain one or more amino * groups, with a radioisotope, the substance to be labelled being combined in the presence of a reductor with "TcOή ions.
In this context "radioisotope" is understood to be a radio-
10 active particle which originates from the radioactive pertechnetate used.
The labelling of substances with the radionuclide technetium "m, which is used in nuclear medicine, is disclosed, for example, in The Journal of Nuclear Medicine, No. 11 (1970), pp. 1_7, ibid No. 23
15 (1982), pp. 1011-1019 and the book "Technetium in Chemistry and Nuclear Medicine", edited by M. Nicolini et al., published by Raven Press, N.Y., I98O. Technetium-99m is relatively inexpensive, can be simply obtained and has a short half-life of 6 hours. The photo- energy of the gamma radiation is 1.0 keV and no β-radiation is
20 emitted. Consequently, the exposure of the patient to radiation is restricted to a minimum.
Labelling with "mTc is used in biomedical science for, for example, proteins such as monoclonal antibodies, which are particularly useful for many diagnostic and therapeutic applications
25 because of their special biological activity and their specific affinity for certain antigen determinants which are encountered in tissues and in organs in specific cells.
The labelling method must be compatible with the biological and physical activity of the protein; the chemicals used must
30 preferably be non-toxic and the method must preferably be simple to carry out and preferably be suitable for a presentation in the form of an "instant kit". In the International Patent Application W0 86/03010 a method
<#' is described for labelling compounds containing amino groups with
35 "mTc, in which method """TcO/, ions are reacted in an acid medium with an amine or amide, a reactive intermediate being formed which is then allowed to react with the compound to be labelled. With this
SUBSTITUTE SHEET
method the first reaction is carried out at a relatively high temperature, for example 1_0°C, and for a relatively long time, for example . hours. Although the labelling efficiency is satisfactory, the relatively high temperature and the relatively long reaction time constitute a serious drawback.
According to the invention it has been found that this draw¬ back can be overcome by a method as specified in the preamble, which method is characterised in that the reductor used is at least one borohydride. Examples of borohydrides which are suitable for use in the method of the invention are potassium borohydride and sodium boro¬ hydride (NaBHή) and potassium borohydride cyanide and sodium boro¬ hydride cyanide (NaBH3CN) . The first-mentioned substances are preferred because particularly high values for the labelling effi- ciency can be obtained with these. In principle, however, all sub¬ stances which yield borohydride ions can be used.
The method according to the invention is particularly efficient if Sn2+ ions (which, for example, originate from tin(II) chloride) are also present as reductor. If Sn2+ ions are used, the efficiency of the labelling can be improved appreciably. This effi¬ ciency is, for example, better by a factor of 10-100 than without the use of Sn2+ ions. The total amount of tin in the sample (which contains at least 0.1 mg of the substance to be labelled) is in general 1-2000 ng. If a so-called freeze-dried kit is used for carrying out the method according to the invention, the amount of tin can be relatively low and can be, for example, 5~50 ng.
Tin ions have the tendency to form a colloidal precipitate. In the method according to the invention, this can be counteracted by the use of chelating agents. Chelating agents of this type are known. Examples of chelating agents which are very satisfactory are tartrate/phthalate buffer and diethylenetriaminepentaacetic acid
(DTPA) . The last-mentioned agent in particular is very satisfactory.
When the method according to the invention is applied using a freeze-dried kit and, for example, the system tin/DTPA, preservative is also present in the solution because it has been found that in the absence of such an agent the stability of the tin solution declines. All conventional preservatives can be used, for example
SUBSTITUTE SHEET
dihydroxybenzoic acid.
The temperature in the labelling reaction according to the invention is not critical. Preferably, the temperature of the working environment is then also maintained. In general, this temperature will be 1 ~25 C.
An embodiment of the method according to the invention which is preferred is that in which per 0.1 mg of the substance to be labelled at least an amount of 10-500 ng of Sn2+ ions and 0.01 - 1 μg of borohydride is used.
The pH of the chosen solutions can also vary within a wide range. In general, however, it is preferred that the substance to be labelled is present in an aqueous medium having a pH in the range of 3~H. the "raTc0^ ions to be used are present in a solution having a pH in the range of _.5~7-5. the Sn2+ ions which are optionally to be used are present in a solution having a pH in the range of 0-7, and - the final solution of the labelled substance has a pH in the range of 5-10.
The labelling reaction will in general be carried out in a buffered solution.
Using the method according to the invention, proteins can be labelled with retention of functional properties. Thus, for example, in the case of antibodies the immuno-diagnostic properties, in the case of enzymes the enzymatic properties and in the case of hormones the hormonal properties are preserved.
Apart from the proteins named in the examples below, the following proteins can, for example, be labelled using the method according to the invention (the labelling efficiency being given in percent) : aggregated IgG (immunoglobulin) 97_ hematoporphyrin 60% IgG, fractions purified via protein A 88» cystine/cysteine GSSG (glutathione dimer) 9 %
SUBSTITUTE SHEET
glutathione 95. bacterial chlorophyll 11% insulin 83_ chimera (diverse) 80 sandostatin 60% somatostatin 60
92% serum 96% bovine albumin 85%
Hb dimer (haemoglobin) 98% dextran 88% anti-γ-interferon 98%
(MDH) immunoglobulin derivative 98% tPA I 1 F(ab)2)Y22 construct 80%
HSA dimer (human serum albumin) 70% HSA polymer 75% anti-leucoprotease 93%
Cells which may be mentioned are: granulocytes (PMN)/erythro- cytes/"mixed" leuco's.
The labelled proteins can be prepared non-toxic, pyrogen-free and sterile. This is important, especially when the labelled protein has to be used in vivo. In this case the labelling must, of course, have no adverse influence on the biological properties of the protein.
As already mentioned, sodium borohydride is particularly preferred in the labelling method according to the invention which is carried out in one step. A high incorporation (for example 95%) of the label is achievable.
The method according to the invention is especially important for the labelling of biologically active proteins in nuclear medicine, in which radiopharmaceuticals are administered to patients for diagnostic purposes. A diagnosis can be made or a diagnosis can be confirmed on the basis of a (sometimes) time-dependent distribution pattern.
The distribution pattern in the body is recorded using instruments which are suitable for this purpose and which show images or scintigrams. The "mTc label used has the significant
SUBSTITUTE SHEET
advantage that the half-life is relatively short, which prevents unnecessary exposure to radiation.
The method according to the invention can also be used for labelling substances other than proteins, provided these substances
5 contain amino groups. Examples are amino acids, nucleotides and medicaments containing amino groups, which can be useful in nuclear medicine.
Cells such as blood cells can also be labelled using the method according to the invention. In this case, use can be made of 10 substances which combine with the cells, for example troponolate. Substances of this type entrain the radioactive technetium. The method according to the invention makes efficiencies of between 50 and 90% possible.
The invention also relates to a kit for carrying out the 15 method according to the invention, which kit at least comprises: a container, which contains a freeze-dried mixture of borohydride, a tin(II) salt and a buffer.
The invention also relates to a freeze-dried mixture of boro¬ hydride, a tin(II) salt and a buffer. The labelling can be carried 20 out in a simple and effective manner with the aid of this mixture or the abovementioned kit.
The method according to the invention is illustrated in more detail in the following examples, which imply no restriction on the scope of the invention. 25
Example I
The reaction of "mTc with the protein takes place as follows and under the indicated reaction conditions.
30 protein + "mTc0ή + borohydride + Sn2* • ■> protein labelled with "mTc room temperature for 15 minutes
Ϋ _~ The reaction is carried out at room temperature (15- °C) .
35 The following variables were investigated and, insofar as applicable, optimised for, in particular, a human immunoglobulin preparation:
SUBSTITUTE SHEET
1. the degree of acidity at which the reaction is carried out,
2. the concentrations of the borohydride and Sn2+,
3. the protein concentration, _. the reaction time, and 5« the reaction temperature.
The 99mTcθ4 ion is always "carrier-free" or present in "tracer" amounts and is not an important reaction parameter.
Good results, where more than 95% of the 99mTc added is recovered as label bonded to the protein, can be obtained if the reaction mixture consists of
0.1 - 1 mg of protein in 0.1-0.5 ml of 0.15 M NaCl, 0.2-1 μg of Sn2+ added in the form of a solution in a buffer, consisting of potassium hydrogen phthalate ( _0 mM) and potas¬ sium sodium tartrate (10 mM) , having a pH of 5-6. The Sn2* concentration in the buffer is preferably 1 mg/ml. Preferably
SnCl2.2H20 is used,
0.5-1 μg of NaBH/,, added in the form of a 1 mg/ml solution in 0.05 N NaOH, and
100 μl of "TcOz, solution, as obtained from a molybdenum- technetium generator (a so-called "cow").
The labelling efficiency of the method followed was determined by means of gel filtration on a 10 cm long column of Sephadex G50 using 0.15 M NaCl as eluent (the labelled protein elutes on the dead volume) ; via precipitation with trichloroacetic acid, the labelled protein precipitating; via autoradiography following electrophoresis, in which case an initial overall impression is obtained; via HPLC analysis and (especially for human immunoglobulin) via ion exchange chromatography on DEAE-Sephadex A25. The following table gives the labelling efficiency which was obtained for various protein preparations under conditions which had been found to be optimum for a human immunoglobulin preparation.
SUBSTITUTE SHEET
protein labelling efficiency
polyclonal human immunoglobulin 98
5 polyclonal rabbit immunoglobulin 80 monoclonal antibody Y22 (antifibrin) 90 monoclonal antibody Y22, F(ab)2 84 fibrinogen (human) 94 fibrinogen (rat) 83
10 human albumin 67 low density lipoprotein (human) (LDL) 81 low density lipoprotein (rabbit) (LDL) 43 anti-melanoma monoclonal antibody (Nori- _,IgM) 88 gamma-interferon 65
15 α-L-antitrypsin 60 tissue-type plasminogen activator (t-PA) 73
N-terminal "disulphide knot" of fibrinogen 84
Both rat and human fibrinogen were found to have the same
20 "radio-clottability" before and after 4 hours incubation in plasma.
This was >90% in all cases, which indicates that no dissociation of the label occurs and that labelling does not have a negative influence on the clottability of fibrinogen.
The LDL label was also found not to exchange with other 2 serum proteins when labelled LDL was incubated in serum.
By means of standard in vivo and in vitro techniques it was possible to demonstrate that the immunological and biochemical properties of the proteins used were not affected.
30 Example II
The following mixture of substances, involved in the labelling method, is freeze-dried in each of a series of small glass
? ampoules:
<_ - 0.2-1 μg Sn : SnCl2.2H20 dissolved to give a concentration of 1
35 mg of Sn2+ per ml of buffer, consisting of potassium hydrogen phthalate (40 mM) and potassium sodium tartrate (10 mM) , of' pH 5-6, and
SUBSTITUTE SHEET
0.5-1 μg of NaBH^ in the form of a 1 mg/ml solution in 0.05 N NaOH. 0.1-1 mg of protein in 0.1-0.5 ml of 0.15 M NaCl and 100 μl of "TcOz, from a molybdenum-technetium generator are added to the freeze-dried mixture.
After reacting for 15 minutes at room temperature, the labelling is complete and the results are fully comparable with those from example I.
The freeze-dried mixture of Sn2+, NaBHή and buffer is found to be stable for a few months at 37°C, provided the ampoules are sealed airtight under nitrogen. The stability is apparent from the fact that when the labelling is carried out with freeze-dried material which has been stored for a few months at 37°C, the labelling efficiency is equally as high as with freshly prepared reaction mixture.
SUBSTITUTESHEET
Claims
1. Method for labelling substances, including cells such as blood cells, which contain one or more amino groups, with a radio¬ isotope, the substance to be labelled being combined in the presence of a reductor with """TcO^ ions, characterised in that the reductor used is at least one borohydride.
2. Method according to Claim 1, in which the borohydride is NaBHή or NaBH3CN.
3- Method according to one of the preceding claims, in which Sn2+ ions are also present as reductor.
4. Method according to one of the preceding claims, in which the temperature during the labelling reaction is 1 -25°C.
5. Method according to one of the preceding claims, in which per 0.1 mg of the substance to be labelled at least an amount of
1 - 2000, preferably 10-500 ng of Sn2+ ions and 0.01 - 1 μg of borohydride is used.
6. Method according to one of the preceding claims, in which - the substance to be labelled is present in an aqueous medium having a pH in the range of 3~H. the 99mTc0, ions to be used are present in a solution having a pH in the range of 4.5_7-5, the Sn2+ ions which are optionally to be used are present in a solution having a pH in the range of 0-7, and the final solution of the labelled substance has a pH in the range of 5~10.
7. Method according to one of the preceding claims, in which the labelling reaction is carried out for 5~20 minutes.
8. Method according to one of the preceding claims, in which the substance to be labelled is a protein such as a (monoclonal) antibody or a part thereof.
9. Kit for carrying out the method according to one of the preceding claims, at least comprising a container, which contains a freeze-dried mixture of a borohydride,- a tin(II) salt and a buffer.
10. Freeze-dried mixture at least consisting of borohydride, tin(II) salt and buffer for use in labelling reactions.
SUBSTITUTE SHEET
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| NL9001137 | 1990-05-15 | ||
| NL9001137A NL9001137A (en) | 1990-05-15 | 1990-05-15 | METHOD FOR LABELING AMINO GROUPS CONTAINING SUBSTANCES WITH 99MTC AND EQUIPMENT FOR CARRYING OUT THIS METHOD |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| WO1991017999A1 true WO1991017999A1 (en) | 1991-11-28 |
Family
ID=19857100
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| PCT/NL1991/000080 WO1991017999A1 (en) | 1990-05-15 | 1991-05-15 | METHOD FOR LABELLING SUBSTANCES CONTAINING AMINO GROUPS, INCLUDING CELLS SUCH AS BLOOD CELLS, WITH 99mTc AND KIT FOR CARRYING OUT THIS METHOD |
Country Status (3)
| Country | Link |
|---|---|
| AU (1) | AU7990191A (en) |
| NL (1) | NL9001137A (en) |
| WO (1) | WO1991017999A1 (en) |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4424200A (en) * | 1979-05-14 | 1984-01-03 | Nuc Med Inc. | Method for radiolabeling proteins with technetium-99m |
| US4917878A (en) * | 1988-05-02 | 1990-04-17 | Thomas Jefferson University | Novel use of a radiolabelled antibody against stage specific embryonic antigen for the detection of occult abscesses in mammals |
-
1990
- 1990-05-15 NL NL9001137A patent/NL9001137A/en not_active Application Discontinuation
-
1991
- 1991-05-15 AU AU79901/91A patent/AU7990191A/en not_active Abandoned
- 1991-05-15 WO PCT/NL1991/000080 patent/WO1991017999A1/en unknown
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4424200A (en) * | 1979-05-14 | 1984-01-03 | Nuc Med Inc. | Method for radiolabeling proteins with technetium-99m |
| US4917878A (en) * | 1988-05-02 | 1990-04-17 | Thomas Jefferson University | Novel use of a radiolabelled antibody against stage specific embryonic antigen for the detection of occult abscesses in mammals |
Non-Patent Citations (3)
| Title |
|---|
| Chemical Abstracts, vol. 89, 1978, (Columbus, Ohio, US), H. Spies et al.: "Labeling of 6-mercaptopurine with technetium-99m", see page 248, abstract 159552r, & Zentralinst. Kernforsch., Rossendorf Dresden. (Ber.) 1977, ZfK-340, Jahresbericht 56-7 * |
| Journal of Nuclear Medicine, vol. 18, no. 6, June 1977; A.R. Fritzberg et al.: "Evaluation of formamidine sulfinic acid and other reducing agents for use in the preparation of Tc-99m labeled radiopharmaceuticals", pages 553-557 * |
| Journal of Nuclear Medicine, vol. 18, no. 6, June 1977; A.R. Fritzberg et al.: "Evaluation of formamidine sulfinic acid and other reducing agents for use in the preparation of Tc-99m labeled radiopharmaceuticals", pages 553-557, see the whole article * |
Also Published As
| Publication number | Publication date |
|---|---|
| NL9001137A (en) | 1991-12-02 |
| AU7990191A (en) | 1991-12-10 |
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