EP0998674A2 - Conjugate for differentiating between healthy and unhealthy tissue - Google Patents
Conjugate for differentiating between healthy and unhealthy tissueInfo
- Publication number
- EP0998674A2 EP0998674A2 EP98946241A EP98946241A EP0998674A2 EP 0998674 A2 EP0998674 A2 EP 0998674A2 EP 98946241 A EP98946241 A EP 98946241A EP 98946241 A EP98946241 A EP 98946241A EP 0998674 A2 EP0998674 A2 EP 0998674A2
- Authority
- EP
- European Patent Office
- Prior art keywords
- conjugate according
- conjugate
- fluorescence
- acid
- carrier
- 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.)
- Withdrawn
Links
- 150000001875 compounds Chemical class 0.000 claims description 31
- 102000008100 Human Serum Albumin Human genes 0.000 claims description 22
- 108091006905 Human Serum Albumin Proteins 0.000 claims description 22
- 102000004169 proteins and genes Human genes 0.000 claims description 14
- 108090000623 proteins and genes Proteins 0.000 claims description 14
- 239000002253 acid Substances 0.000 claims description 13
- 238000002360 preparation method Methods 0.000 claims description 10
- 230000005284 excitation Effects 0.000 claims description 9
- 230000001575 pathological effect Effects 0.000 claims description 8
- 150000002148 esters Chemical class 0.000 claims description 7
- 229920000570 polyether Polymers 0.000 claims description 6
- 239000000969 carrier Substances 0.000 claims description 4
- 125000002887 hydroxy group Chemical group [H]O* 0.000 claims description 4
- 125000003277 amino group Chemical group 0.000 claims description 3
- 238000000034 method Methods 0.000 claims description 3
- 229920001223 polyethylene glycol Polymers 0.000 claims description 3
- KXTAOXNYQGASTA-UHFFFAOYSA-N 2-benzylidenepropanedioic acid Chemical compound OC(=O)C(C(O)=O)=CC1=CC=CC=C1 KXTAOXNYQGASTA-UHFFFAOYSA-N 0.000 claims description 2
- BZTDTCNHAFUJOG-UHFFFAOYSA-N 6-carboxyfluorescein Chemical compound C12=CC=C(O)C=C2OC2=CC(O)=CC=C2C11OC(=O)C2=CC=C(C(=O)O)C=C21 BZTDTCNHAFUJOG-UHFFFAOYSA-N 0.000 claims description 2
- 125000003172 aldehyde group Chemical group 0.000 claims description 2
- 229930002875 chlorophyll Natural products 0.000 claims description 2
- 235000019804 chlorophyll Nutrition 0.000 claims description 2
- MOFVSTNWEDAEEK-UHFFFAOYSA-M indocyanine green Chemical compound [Na+].[O-]S(=O)(=O)CCCCN1C2=CC=C3C=CC=CC3=C2C(C)(C)C1=CC=CC=CC=CC1=[N+](CCCCS([O-])(=O)=O)C2=CC=C(C=CC=C3)C3=C2C1(C)C MOFVSTNWEDAEEK-UHFFFAOYSA-M 0.000 claims description 2
- 229960004657 indocyanine green Drugs 0.000 claims description 2
- 150000004032 porphyrins Chemical class 0.000 claims description 2
- 239000004721 Polyphenylene oxide Substances 0.000 claims 2
- ZAMOUSCENKQFHK-UHFFFAOYSA-N Chlorine atom Chemical compound [Cl] ZAMOUSCENKQFHK-UHFFFAOYSA-N 0.000 claims 1
- 239000002202 Polyethylene glycol Substances 0.000 claims 1
- BHPNXACHQYJJJS-UHFFFAOYSA-N bacteriochlorin Chemical compound N1C(C=C2N=C(C=C3NC(=C4)C=C3)CC2)=CC=C1C=C1CCC4=N1 BHPNXACHQYJJJS-UHFFFAOYSA-N 0.000 claims 1
- 229910052801 chlorine Inorganic materials 0.000 claims 1
- 239000000460 chlorine Substances 0.000 claims 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 claims 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 claims 1
- YHUVMHKAHWKQBI-UHFFFAOYSA-N quinoline-2,3-dicarboxylic acid Chemical compound C1=CC=C2N=C(C(O)=O)C(C(=O)O)=CC2=C1 YHUVMHKAHWKQBI-UHFFFAOYSA-N 0.000 claims 1
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 16
- QOSSAOTZNIDXMA-UHFFFAOYSA-N Dicylcohexylcarbodiimide Chemical compound C1CCCCC1N=C=NC1CCCCC1 QOSSAOTZNIDXMA-UHFFFAOYSA-N 0.000 description 12
- NQTADLQHYWFPDB-UHFFFAOYSA-N N-Hydroxysuccinimide Chemical compound ON1C(=O)CCC1=O NQTADLQHYWFPDB-UHFFFAOYSA-N 0.000 description 9
- 239000000243 solution Substances 0.000 description 9
- -1 succinimidyl esters Chemical class 0.000 description 7
- LYCAIKOWRPUZTN-UHFFFAOYSA-N Ethylene glycol Chemical compound OCCO LYCAIKOWRPUZTN-UHFFFAOYSA-N 0.000 description 4
- KCDCNGXPPGQERR-UHFFFAOYSA-N coumarin 343 Chemical compound C1CCC2=C(OC(C(C(=O)O)=C3)=O)C3=CC3=C2N1CCC3 KCDCNGXPPGQERR-UHFFFAOYSA-N 0.000 description 4
- ADFXKUOMJKEIND-UHFFFAOYSA-N 1,3-dicyclohexylurea Chemical compound C1CCCCC1NC(=O)NC1CCCCC1 ADFXKUOMJKEIND-UHFFFAOYSA-N 0.000 description 3
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-Dimethylformamide Chemical compound CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 description 3
- 206010028980 Neoplasm Diseases 0.000 description 3
- IYRYQBAAHMBIFT-UHFFFAOYSA-N acridine-9-carboxylic acid Chemical compound C1=CC=C2C(C(=O)O)=C(C=CC=C3)C3=NC2=C1 IYRYQBAAHMBIFT-UHFFFAOYSA-N 0.000 description 3
- 150000001718 carbodiimides Chemical class 0.000 description 3
- 238000006243 chemical reaction Methods 0.000 description 3
- 210000004087 cornea Anatomy 0.000 description 3
- 239000012634 fragment Substances 0.000 description 3
- 238000000108 ultra-filtration Methods 0.000 description 3
- 102000009027 Albumins Human genes 0.000 description 2
- 108010088751 Albumins Proteins 0.000 description 2
- 206010061218 Inflammation Diseases 0.000 description 2
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- WGCNASOHLSPBMP-UHFFFAOYSA-N hydroxyacetaldehyde Natural products OCC=O WGCNASOHLSPBMP-UHFFFAOYSA-N 0.000 description 2
- 230000004054 inflammatory process Effects 0.000 description 2
- 239000012528 membrane Substances 0.000 description 2
- 239000002904 solvent Substances 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 125000004400 (C1-C12) alkyl group Chemical group 0.000 description 1
- ACMLKANOGIVEPB-UHFFFAOYSA-N 2-oxo-2H-chromene-3-carboxylic acid Chemical compound C1=CC=C2OC(=O)C(C(=O)O)=CC2=C1 ACMLKANOGIVEPB-UHFFFAOYSA-N 0.000 description 1
- GDRVFDDBLLKWRI-UHFFFAOYSA-N 4H-quinolizine Chemical compound C1=CC=CN2CC=CC=C21 GDRVFDDBLLKWRI-UHFFFAOYSA-N 0.000 description 1
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 1
- KRKNYBCHXYNGOX-UHFFFAOYSA-K Citrate Chemical compound [O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O KRKNYBCHXYNGOX-UHFFFAOYSA-K 0.000 description 1
- 102000008946 Fibrinogen Human genes 0.000 description 1
- 108010049003 Fibrinogen Proteins 0.000 description 1
- 208000001836 Firesetting Behavior Diseases 0.000 description 1
- 102000003839 Human Proteins Human genes 0.000 description 1
- 108090000144 Human Proteins Proteins 0.000 description 1
- 108060003951 Immunoglobulin Proteins 0.000 description 1
- 102000004895 Lipoproteins Human genes 0.000 description 1
- 108090001030 Lipoproteins Proteins 0.000 description 1
- 206010029113 Neovascularisation Diseases 0.000 description 1
- 239000002262 Schiff base Substances 0.000 description 1
- 150000004753 Schiff bases Chemical class 0.000 description 1
- 108090000901 Transferrin Proteins 0.000 description 1
- 102000004338 Transferrin Human genes 0.000 description 1
- VUFKWDHZAUSXGP-UHFFFAOYSA-N acetic acid;3-hydroxychromen-2-one Chemical compound CC(O)=O.C1=CC=C2OC(=O)C(O)=CC2=C1 VUFKWDHZAUSXGP-UHFFFAOYSA-N 0.000 description 1
- OOGYVVYCCYJADG-UHFFFAOYSA-N acridine-1-carboxylic acid Chemical compound C1=CC=C2C=C3C(C(=O)O)=CC=CC3=NC2=C1 OOGYVVYCCYJADG-UHFFFAOYSA-N 0.000 description 1
- ZQAZWHSZYVXGMP-UHFFFAOYSA-N acridine-9-carboxylic acid;hydrate Chemical compound O.C1=CC=C2C(C(=O)O)=C(C=CC=C3)C3=NC2=C1 ZQAZWHSZYVXGMP-UHFFFAOYSA-N 0.000 description 1
- 150000001408 amides Chemical class 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- 239000012062 aqueous buffer Substances 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- 125000004429 atom Chemical group 0.000 description 1
- 150000004036 bacteriochlorins Chemical class 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 229910052799 carbon Inorganic materials 0.000 description 1
- 235000017168 chlorine Nutrition 0.000 description 1
- 125000001309 chloro group Chemical class Cl* 0.000 description 1
- 239000001752 chlorophylls and chlorophyllins Substances 0.000 description 1
- ZYGHJZDHTFUPRJ-UHFFFAOYSA-N coumarin Chemical compound C1=CC=C2OC(=O)C=CC2=C1 ZYGHJZDHTFUPRJ-UHFFFAOYSA-N 0.000 description 1
- 230000008878 coupling Effects 0.000 description 1
- 238000010168 coupling process Methods 0.000 description 1
- 238000005859 coupling reaction Methods 0.000 description 1
- 238000004132 cross linking Methods 0.000 description 1
- 230000007717 exclusion Effects 0.000 description 1
- 210000003608 fece Anatomy 0.000 description 1
- 229940012952 fibrinogen Drugs 0.000 description 1
- 239000010419 fine particle Substances 0.000 description 1
- 239000007850 fluorescent dye Substances 0.000 description 1
- 244000144980 herd Species 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- 102000018358 immunoglobulin Human genes 0.000 description 1
- 229940072221 immunoglobulins Drugs 0.000 description 1
- 210000004185 liver Anatomy 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 229910021645 metal ion Inorganic materials 0.000 description 1
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 description 1
- 230000035515 penetration Effects 0.000 description 1
- 239000003880 polar aprotic solvent Substances 0.000 description 1
- 239000011541 reaction mixture Substances 0.000 description 1
- 230000035484 reaction time Effects 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 238000003756 stirring Methods 0.000 description 1
- 150000003457 sulfones Chemical class 0.000 description 1
- 238000004809 thin layer chromatography Methods 0.000 description 1
- 239000012581 transferrin Substances 0.000 description 1
- 230000000007 visual effect Effects 0.000 description 1
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K49/00—Preparations for testing in vivo
- A61K49/001—Preparation for luminescence or biological staining
- A61K49/0013—Luminescence
- A61K49/0017—Fluorescence in vivo
- A61K49/0019—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules
- A61K49/0021—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules the fluorescent group being a small organic molecule
- A61K49/0026—Acridine dyes
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K49/00—Preparations for testing in vivo
- A61K49/001—Preparation for luminescence or biological staining
- A61K49/0013—Luminescence
- A61K49/0017—Fluorescence in vivo
- A61K49/0019—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules
- A61K49/0021—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules the fluorescent group being a small organic molecule
- A61K49/0036—Porphyrins
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K49/00—Preparations for testing in vivo
- A61K49/001—Preparation for luminescence or biological staining
- A61K49/0013—Luminescence
- A61K49/0017—Fluorescence in vivo
- A61K49/0019—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules
- A61K49/0021—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules the fluorescent group being a small organic molecule
- A61K49/0039—Coumarin dyes
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K49/00—Preparations for testing in vivo
- A61K49/001—Preparation for luminescence or biological staining
- A61K49/0013—Luminescence
- A61K49/0017—Fluorescence in vivo
- A61K49/005—Fluorescence in vivo characterised by the carrier molecule carrying the fluorescent agent
- A61K49/0056—Peptides, proteins, polyamino acids
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/76—Albumins
- C07K14/765—Serum albumin, e.g. HSA
-
- 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
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54353—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals with ligand attached to the carrier via a chemical coupling agent
-
- 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
- G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
- G01N33/582—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with fluorescent label
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
Definitions
- the invention relates to conjugates for distinguishing diseased and healthy tissue, methods for producing such conjugates and their use
- pathological tissue e.g. B. from tumors
- the removal of the same is often an essential step. To do this, it is necessary for the operating doctor to recognize exactly where pathological tissue ends and healthy tissue begins. However, this is often not possible. Outflow of the pathological tissue is therefore overlooked, which then forms the basis for the re-emergence of the pathological tissue
- the present invention is therefore based on the object of providing a means with which pathological and healthy tissue can be distinguished
- the present invention thus relates to a conjugate comprising a compound capable of fluorescence and a carrier, the compound and the carrier being connected via an acid ester or acid amide bond or enane bridge (Schiff base) and the compound being an excitation wave ge of 630 nm or larger and / or 450 nm or smaller.
- a conjugate comprising a compound capable of fluorescence and a carrier, the compound and the carrier being connected via an acid ester or acid amide bond or enane bridge (Schiff base) and the compound being an excitation wave ge of 630 nm or larger and / or 450 nm or smaller.
- carrier includes compounds of any sort ⁇ dung herd or in superficial smaller vessels, such Neovasku- la ⁇ sationen in the region of the cornea (cornea of the eye) for the enrichment of the conjugate in a particular tissue, for example a tumor, a inflammations suitable are Games of such carriers are proteins and polyethers.
- the carrier may have hydroxyl or amino groups to form the acid ester or acid amide bond with the compound capable of fluorescence.
- the proteins are preferably not considered foreign to the body. They can be in native form. In the native form, the proteins have no inter- and / or intramolecular cross-linking.
- the proteins advantageously have a molecular weight of up to 1 000 000 daltons, in particular 30 000 to 1 000 000 daltons. It is also advantageous if the proteins are human proteins. Examples of the proteins are albumin, fibrinogen, transferrin, immunoglobulins and lipoproteins, with human serum albumin (HSA) being preferred. Fragments of the above proteins can also be used.
- the sequence of the proteins or fragments thereof can have changes in one or more amino acids compared to known sequences of the proteins or fragments thereof.
- polyethers examples are polyethylene glycols, especially those with a molecular weight of 100 to 20,000 daltons.
- the polyethylene glycols are preferably esterified or etherified at the terminal hydroxyl group with a C 1 -C 12 -alkyl group, in particular with a methyl group.
- a conjugate according to the invention can have one or more, in particular 2 to 4, the above carriers. If there are several carriers, they can be the same or different from one another. If there are several polyethers, these are advantageously chosen so that the molecular weight of all polyethers is approximately 20,000 daltons or more.
- the term "for fluorescence-capable compound” includes compounds of any kind that can be excited to fluoresce. These compounds can also be photoactive.
- the compound is attached to the support via an acid ester or acid amide bond or enane bridge.
- the compound capable of fluorescence can contain an acid group, e.g. Legs Have carbon, sulfone, phosphonic or Arson acid group, a hydroxyl group, an amino group or an aldehyde group. There may be several of these groups, which may be the same or different from one another.
- the compound capable of fluorescence is excited at a wavelength of 630 nm or greater, preferably 630 to 850 nm and particularly preferably 650 to 850 nm and / or at a wavelength of 450 nm or less, preferably 320 to 450 nm.
- These wavelengths relate to excitation wavelengths which the compound capable of fluorescence has in the conjugate according to the invention; in free form their excitation wavelength can deviate from this.
- porphyrins such as tetrasulfophenylporphyrin (TSPP; excitation wavelength 650 nm when bound to HSA), chlorines, bacteriochlorins, chlorophylls, phthalocyanines, these compounds being able to have metal ions as the central atom.
- TSPP tetrasulfophenylporphyrin
- acridinecarboxylic acid such as acridine-9-carboxylic acid
- coumaric acid such as coumarin 343, coumarin-3-carboxylic acid and hydroxycoumarin acetic acid (excitation wavelength 365 nm when bound to HSA), and indocyanine green (excitation wavelength 805 nm when bound to HSA), as well as derivatives of the above compounds.
- One or more of the compounds capable of fluorescence can be present in the conjugate according to the invention. If there are several, they can be the same or different from each other.
- FIGS. 1 to 3 Particularly preferred conjugates according to the invention are shown in FIGS. 1 to 3.
- Conjugates of the invention may be prepared by reacting the compound capable fluorescence for connection with the carrier to form a shiftureester ⁇ or acid amide bond is covalently linked. Suitable processes and materials required for this purpose are known to the person skilled in the art.
- the conjugates can be prepared by reacting this compound with carbodiimide and hydroxysuccinimide to form reactive succinimidyl esters and then reacting them with the carrier.
- the succinimidyl esters can be prepared jointly or separately.
- the reaction with the compound capable of fluorescence with carbodiimide and hydroxysuccinimide is carried out in a polar aprotic solvent, preferably dimethylformamide or dimethyl sulfoxide (DMSO).
- a polar aprotic solvent preferably dimethylformamide or dimethyl sulfoxide (DMSO).
- the molar ratio of to the fluorescent compound: carbodiimide: hydroxysuccinimide is about 1: 1, 5-3: 5- 1 0.
- the succinimidyl ester formed is then in an aqueous buffer solution, preferably NaHC0 3 , with the carrier, such as albumin, implemented.
- the carrier concentration is approximately 10 to 70 mg / ml.
- the acid group activated in this way can then react with the formation of acid amide or acid ester bonds with OH and NH groups of the carrier, conjugates according to the invention being obtained.
- the conjugates can be cleaned several times, e.g. B. by ultrafiltration, and finally sterile filtered
- Conjugates according to the invention are distinguished by an increased half-life in the organism. Furthermore, conjugates according to the invention accumulate in pathological tissue, in particular in tumor tissue, in inflammation centers and in superficial smaller vessels, e.g. B. from neovascularizations in the cornea. The compound that is capable of fluorescence is excited by light, whereby pathological tissue can be made visible, whereas healthy tissue in which the conjugates according to the invention do not accumulate is not made visible. Furthermore, there is no interference from the inherent fluorescence of the blood or of tissue, for example the liver, which does not distort the visual impression. Furthermore, conjugates according to the invention, in which the compound capable of fluorescence can be excited at 630 nm or greater, have a high depth of penetration. Brief description of the drawings:
- FIG. 1 shows the preparation of a conjugate from acridine-9-carboxylic acid and human serum albumin
- Figure 2 shows the preparation of a conjugate from coumarin 343 and human serum albumin
- Figure 3 shows the preparation of a conjugate from tetrasulfophenylporphin and human serum albumin.
- acridine-9-carboxylic acid hydrate (A9CS) were dissolved in 2 ml of DMSO and about 1 00 mg of N-hydroxysuccinimide (HSI) in a molar ratio of about 10/1 and 30 mg of N, N'-dicyclohexylcarbodiimide (DCC) in a molar ratio of about 1.5 / 1 added.
- HSU N-hydroxysuccinimide
- DCC N'-dicyclohexylcarbodiimide
- the ester becomes a solution of 2 g of human serum albumin (HSA), which is in 1 0 ml of original solution, 1 0 ml of 0.34 M NaHC0 3 and 1 0 ml of methoxypolyethylene glycol (MPEG) is slowly added.
- HSA human serum albumin
- MPEG methoxypolyethylene glycol
- the separation of the undesired accompanying substances in the finished preparation is carried out by ultrafiltration (exclusion limit 1 0 kD) with at least 4 washes.
- Example 2 Preparation of a conjugate according to the invention from coumarin 343 and human serum albumin
- Example 3 Preparation of a conjugate according to the invention from tetra- (4-sulfophenyD-porphin and human serum albumin
- Tetra- (4-sulfophenyl) porphine was dissolved in DMSO at a concentration of 10 mg / ml. Three times the molar amount of DCC and five times the molar amount of HSI was added to the clear dark green solution. After a reaction time of about 3 to 4 hours, the conversion to the TSPP succinimidyl ester (TSPP-SE) has ended, the di-cyclohexylurea formed being deposited in fine particles.
- the analytical control is carried out by means of thin layer chromatography.
- Human serum albumin (HSA, 4g, ie 2 ampoules of 2 g each in 10 ml) were diluted with 2 x 1 0 ml 0, 1 7 M NaHCO 3 and 20 ml methoxypolyethylene glycol 350 and placed in a 100 ml Erlenmeyer flask.
- the above TSPP-SE solution in DMSO was slowly added to this HSA solution with constant stirring, the initially clear solution becoming cloudy as a result of unreacted DCC which is insoluble in aqueous solution.
- the reaction mixture was stirred at room temperature for 30 minutes to complete the reaction.
- the turbidity was then checked using a sterile filter unit (Millipore, Stericup - GV, 0.22 ⁇ m Low Binding Duropore membrane) and the low molecular weight water-soluble components (DMSO, HSI and unbound TSPP) by ultrafiltration through a membrane with a 30 kD cut-off limit (Amicon YM 30 ) separated. A conjugate of TSPP and HSA was obtained. The coupling yield of TSPP to HSA was 85 to 90%.
- Pillar 1 Zorbax GF 450
- Pillar 2 Zorbax GF 450
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- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- Biomedical Technology (AREA)
- General Health & Medical Sciences (AREA)
- Immunology (AREA)
- Molecular Biology (AREA)
- Epidemiology (AREA)
- Animal Behavior & Ethology (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Urology & Nephrology (AREA)
- Hematology (AREA)
- Medicinal Chemistry (AREA)
- Biochemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Organic Chemistry (AREA)
- Biotechnology (AREA)
- Cell Biology (AREA)
- Analytical Chemistry (AREA)
- Physics & Mathematics (AREA)
- Microbiology (AREA)
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Abstract
Die Erfindung betrifft Konjugate, umfassend eine zur Fluoreszenz-fähige Verbindung und einen Träger, wobei die Verbindung und der Träger über eine Säureester- oder Säureamid-Bindung verbunden sind und die Verbindung eine Anregungswellenlänge von 630 nm oder grösser und/oder 450 nm oder kleiner aufweist. Ferner betrifft die Erfindung die Herstellung solcher Konjugate sowie ihre Verwendung.
Description
Konjugat zur Unterscheidung von krankhaftem und gesundem Gewebe Conjugate to differentiate between diseased and healthy tissue
Die Erfindung betrifft Konjugate zur Unterscheidung von krankhaftem und gesundem Gewebe, Verfahren zur Herstellung solcher Konjugate sowie ihre VerwendungThe invention relates to conjugates for distinguishing diseased and healthy tissue, methods for producing such conjugates and their use
In der Behandlung von krankhaftem Gewebe, z. B. von Tumoren, ist die Entfernung desselben oft ein essentieller Schritt. Hierzu ist es notwendig, daß der operierende Arzt genau erkennt, wo krankhaftes Gewebe endet und gesundes Gewebe beginnt. Dies ist allerdings oft nicht möglich Auslaufer des krankhaften Gewebes werden daher übersehen, die dann die Basis für die erneute Entstehung des krankhaften Gewebes darstelltIn the treatment of pathological tissue, e.g. B. from tumors, the removal of the same is often an essential step. To do this, it is necessary for the operating doctor to recognize exactly where pathological tissue ends and healthy tissue begins. However, this is often not possible. Outflow of the pathological tissue is therefore overlooked, which then forms the basis for the re-emergence of the pathological tissue
Der vorliegenden Erfindung liegt somit die Aufgabe zugrunde, ein Mittel bereitzustellen, mit dem krankhaftes von gesundem Gewebe unterschieden werden kannThe present invention is therefore based on the object of providing a means with which pathological and healthy tissue can be distinguished
Erfindungsgemaß wird dies durch die Gegenstande in den Patentansprüchen erreicht.According to the invention, this is achieved by the subject matter in the claims.
Gegenstand der vorliegenden Erfindung ist somit ein Konjugat, umfassend eine zur Fluoreszenz-fahige Verbindung und einen Trager, wobei die Verbindung und der Trager über eine Saureester- oder Saureamid-Bindung oder Enan-Brucke (Schiffsche Base) verbunden sind und die Verbindung eine Anregungswellenlan- ge von 630 nm oder großer und/oder 450 nm oder kleiner aufweist.The present invention thus relates to a conjugate comprising a compound capable of fluorescence and a carrier, the compound and the carrier being connected via an acid ester or acid amide bond or enane bridge (Schiff base) and the compound being an excitation wave ge of 630 nm or larger and / or 450 nm or smaller.
Der Ausdruck "Trager" umfaßt Verbindungen jeglicher Art, die zur Anreicherung des Konjugats in einem bestimmten Gewebe, z B einem Tumor, einem Entzün¬ dungsherd oder in oberflächlich gelegenen kleineren Gefäßen, wie Neovasku- laπsationen im Bereich der Cornea (Hornhaut des Auges) , geeignet sind Bei-
spiele solcher Träger sind Proteine und Polyether. Zur Ausbildung der Säureesteroder Säureamidbindung mit der zur Fluoreszenz-fähigen Verbindung kann der Träger Hydroxyl- oder Amino-Gruppen aufweisen.The term "carrier" includes compounds of any sort ¬ dung herd or in superficial smaller vessels, such Neovasku- laπsationen in the region of the cornea (cornea of the eye) for the enrichment of the conjugate in a particular tissue, for example a tumor, a inflammations suitable are Games of such carriers are proteins and polyethers. The carrier may have hydroxyl or amino groups to form the acid ester or acid amide bond with the compound capable of fluorescence.
Die Proteine werden vorzugsweise nicht als körperfremd angesehen. Sie können in nativer Form vorliegen. In der nativen Form weisen die Proteine kein inter- und/oder intramolekulares Cross-Linking auf. Günstigerweise besitzen die Proteine ein Molekulargewicht von bis zu 1 00 000 Dalton, insbesondere 30 000 bis 1 00 000 Dalton. Ferner ist es günstig, wenn die Proteine humane Proteine sind. Beispiele der Proteine sind Albumin, Fibrinogen, Transferrin, Immunglobuline und Lipoproteine, wobei humanes Serumalbumin (HSA) bevorzugt ist. Es können auch Fragmente vorstehender Proteine verwendet werden. Ferner kann die Sequenz der Proteine bzw. der Fragmente davon Änderungen von einer oder mehreren Aminosäuren gegenüber bekannten Sequenzen der Proteine bzw. Fragmente davon aufweisen.The proteins are preferably not considered foreign to the body. They can be in native form. In the native form, the proteins have no inter- and / or intramolecular cross-linking. The proteins advantageously have a molecular weight of up to 1 000 000 daltons, in particular 30 000 to 1 000 000 daltons. It is also advantageous if the proteins are human proteins. Examples of the proteins are albumin, fibrinogen, transferrin, immunoglobulins and lipoproteins, with human serum albumin (HSA) being preferred. Fragments of the above proteins can also be used. Furthermore, the sequence of the proteins or fragments thereof can have changes in one or more amino acids compared to known sequences of the proteins or fragments thereof.
Beispiele der Polyether sind Polyethylengiykole, insbesondere solche mit einem Molekulargewicht von 1 00 bis 20 000 Dalton. Vorzugsweise sind die Polyethylengiykole an der endständigen Hydroxylgruppe mit einer C.,-C12-Alkylgruppe, insbesondere mit einer Methylgruppe, verestert oder verethert.Examples of the polyethers are polyethylene glycols, especially those with a molecular weight of 100 to 20,000 daltons. The polyethylene glycols are preferably esterified or etherified at the terminal hydroxyl group with a C 1 -C 12 -alkyl group, in particular with a methyl group.
Ein erfindungsgemäßes Konjugat kann einen oder mehrere, insbesondere 2 bis 4, vorstehender Träger aufweisen. Liegen mehrere Träger vor, so können diese gleich oder verschieden voneinander sein. Liegen mehrere Polyether vor, so werden diese günstigerweise so gewählt, daß das Molekulargewicht aller Polyether ca. 20 000 Dalton oder mehr beträgt.A conjugate according to the invention can have one or more, in particular 2 to 4, the above carriers. If there are several carriers, they can be the same or different from one another. If there are several polyethers, these are advantageously chosen so that the molecular weight of all polyethers is approximately 20,000 daltons or more.
Der Ausdruck "zur Fluoreszenz-fähige Verbindung " umfaßt Verbindungen jeglicher Art, die zur Fluoreszenz angeregt werden können. Diese Verbindungen können auch photoaktiv sein. Die Verbindung ist über eine Säureester- oder Säureamid-Bindung oder Enan-Brücke an den Träger gebunden. Zur Ausbildung dieser kann die zur Fluoreszenz-fähige Verbindung eine Säuregruppe, z. B. eine
Carbon-, Sulfon-, Phosphon- oder Arson-Säuregruppe, eine Hydroxylgruppe, eine Aminogruppe oder eine Aldehydgruppe aufweisen. Es können mehrere dieser Gruppen vorliegen, die gleich oder verschieden voneinander sein können. Die zur Fluoreszenz-fähige Verbindung wird bei einer Wellenlänge von 630 nm oder größer, bevorzugt 630 bis 850 nm und besonders bevorzugt 650 bis 850 nm und/oder bei einer Wellenlänge von 450 nm oder kleiner, bevorzugt 320 bis 450 nm angeregt. Diese Wellenlängen beziehen sich auf Anregungswellenlängen, die die zur Fluoreszenz-fähige Verbindung im erfindungsgemäßen Konjugat aufweist; in freier Form kann ihre Anregungswellenlänge davon abweichen. Vertreter dieser Verbindungen sind Porphyrine, wie Tetrasulfophenylporphyrin (TSPP; Anregungswellenlänge 650 nm, wenn es an HSA gebunden ist), Chlorine, Bakteriochlorine, Chlorophylle, Phtalocyanine, wobei diese Verbindungen Metallionen als Zentralatom aufweisen können. Ferner sind Vertreter der zur Fluoreszenz-fähigen Verbindung Carboxyzimtsäure, Carboxyfluorescein, Acridincar- bonsäure, wie Acridin-9-carbonsäure, Cumarinsäure, wie Cumarin 343, Cumarin- 3-carbonsäure und Hydroxycumarinessigsäure (Anregungswellenlänge 365 nm, wenn es an HSA gebunden ist) , und Indocyaningrün (Anregungswellenlänge 805 nm, wenn es an HSA gebunden ist), sowie Derivate vorstehender Verbindungen.The term "for fluorescence-capable compound" includes compounds of any kind that can be excited to fluoresce. These compounds can also be photoactive. The compound is attached to the support via an acid ester or acid amide bond or enane bridge. To form this, the compound capable of fluorescence can contain an acid group, e.g. Legs Have carbon, sulfone, phosphonic or Arson acid group, a hydroxyl group, an amino group or an aldehyde group. There may be several of these groups, which may be the same or different from one another. The compound capable of fluorescence is excited at a wavelength of 630 nm or greater, preferably 630 to 850 nm and particularly preferably 650 to 850 nm and / or at a wavelength of 450 nm or less, preferably 320 to 450 nm. These wavelengths relate to excitation wavelengths which the compound capable of fluorescence has in the conjugate according to the invention; in free form their excitation wavelength can deviate from this. Representatives of these compounds are porphyrins, such as tetrasulfophenylporphyrin (TSPP; excitation wavelength 650 nm when bound to HSA), chlorines, bacteriochlorins, chlorophylls, phthalocyanines, these compounds being able to have metal ions as the central atom. Representatives of the compound capable of fluorescence, carboxycinnamic acid, carboxyfluorescein, acridinecarboxylic acid, such as acridine-9-carboxylic acid, coumaric acid, such as coumarin 343, coumarin-3-carboxylic acid and hydroxycoumarin acetic acid (excitation wavelength 365 nm when bound to HSA), and indocyanine green (excitation wavelength 805 nm when bound to HSA), as well as derivatives of the above compounds.
Von der zur Fluoreszenz-fähigen Verbindung können im erfindungsgemäßen Konjugat eine oder mehrere vorhanden sein. Liegen mehrere vor, dann können sie gleich oder verschieden voneinander sein.One or more of the compounds capable of fluorescence can be present in the conjugate according to the invention. If there are several, they can be the same or different from each other.
Besonders bevorzugte erfindungsgemäße Konjugate sind in den Figuren 1 bis 3 dargestellt.Particularly preferred conjugates according to the invention are shown in FIGS. 1 to 3.
Erfindungsgemäße Konjugate können hergestellt werden, indem die zur Fluoreszenz-fähige Verbindung mit dem Träger unter Ausbildung einer Säureester¬ oder Säureamid-Bindung kovalent verbunden wird. Hierzu geeignete Verfahren sowie benötigte Materialien sind dem Fachmann bekannt.Conjugates of the invention may be prepared by reacting the compound capable fluorescence for connection with the carrier to form a Säureester ¬ or acid amide bond is covalently linked. Suitable processes and materials required for this purpose are known to the person skilled in the art.
Wenn die zur Fluoreszenz-fähige Verbindung eine Säuregruppe aufweist, dann
können die Konjugate hergestellt werden, indem diese Verbindung mit Carbo- diimid und Hydroxysuccinimid zu reaktiven Succinimidylestern und diese dann mit dem Träger umgesetzt werden. Bei Konjugaten mit mehreren zur Fluoreszenz-fähigen Verbindungen kann die Herstellung der Succinimidylester gemeinsam oder getrennt erfolgen.If the compound capable of fluorescence has an acid group, then the conjugates can be prepared by reacting this compound with carbodiimide and hydroxysuccinimide to form reactive succinimidyl esters and then reacting them with the carrier. In the case of conjugates with several compounds capable of fluorescence, the succinimidyl esters can be prepared jointly or separately.
Die Umsetzung der zur Fluoreszenz-fähigen Verbindung mit Carbodiimid und Hydroxysuccinimid erfolgt in einem polaren aprotischen Lösungsmittel, vorzugsweise Dimethylformamid oder Dimethylsulfoxid (DMSO) . Das Mol-Verhältnis von zur Fluoreszenz-fähigen Verbindung : Carbodiimid : Hydroxysuccinimid beträgt etwa 1 : 1 , 5-3 : 5- 1 0. Der gebildete Succinimidylester wird dann in einer wässrigen Pufferlösung, vorzugsweise NaHC03, mit dem Träger, wie Albumin, umgesetzt. Die Trägerkonzentration beträgt etwa 1 0 bis 70 mg/ml. Die so aktivierte Säuregruppe kann dann unter Ausbildung von Säureamid- oder Säureester-Bindungen mit OH- und NH-Gruppen des Trägers reagieren, wobei erfindungsgemäße Konjugate erhalten werden. Die Konjugate können mehrfach gereinigt werden, z. B. durch Ultrafiltration, und schließlich steril filtriert werden, worauf sie applikationsfertig sind.The reaction with the compound capable of fluorescence with carbodiimide and hydroxysuccinimide is carried out in a polar aprotic solvent, preferably dimethylformamide or dimethyl sulfoxide (DMSO). The molar ratio of to the fluorescent compound: carbodiimide: hydroxysuccinimide is about 1: 1, 5-3: 5- 1 0. The succinimidyl ester formed is then in an aqueous buffer solution, preferably NaHC0 3 , with the carrier, such as albumin, implemented. The carrier concentration is approximately 10 to 70 mg / ml. The acid group activated in this way can then react with the formation of acid amide or acid ester bonds with OH and NH groups of the carrier, conjugates according to the invention being obtained. The conjugates can be cleaned several times, e.g. B. by ultrafiltration, and finally sterile filtered, after which they are ready for application.
Erfindungsgemäße Konjugate zeichnen sich durch eine erhöhte Halbwertszeit im Organismus aus. Desweiteren reichern sich erfindungsgemäße Konjugate in krankhaftem Gewebe, insbesondere in Tumorgewebe, in Entzündungsherden und in oberflächlich gelegenen kleineren Gefäßen, z. B. von Neovaskularisationen im Bereich der Cornea an. Durch Licht wird die zur Fluoreszenz-fähige Verbindung angeregt, wodurch krankhaftes Gewebe sichtbar gemacht werden kann, wohingegen gesundes Gewebe, in dem sich die erfindungsgemäßen Konjugate nicht anreichern, nicht sichtbar gemacht wird. Ferner erfolgt keine Störung durch die Eigenfluoreszenz des Blutes oder von Gewebe, z.B. der Leber, wodurch der optische Eindruck nicht verfälscht wird. Weiterhin weisen erfindungsgemäße Konjugate, in der die zur Fluoreszenz-fähige Verbindung bei 630 nm oder größer angeregt werden kann, ein hohe Eindringtiefe auf.
Kurze Beschreibung der Zeichnungen:Conjugates according to the invention are distinguished by an increased half-life in the organism. Furthermore, conjugates according to the invention accumulate in pathological tissue, in particular in tumor tissue, in inflammation centers and in superficial smaller vessels, e.g. B. from neovascularizations in the cornea. The compound that is capable of fluorescence is excited by light, whereby pathological tissue can be made visible, whereas healthy tissue in which the conjugates according to the invention do not accumulate is not made visible. Furthermore, there is no interference from the inherent fluorescence of the blood or of tissue, for example the liver, which does not distort the visual impression. Furthermore, conjugates according to the invention, in which the compound capable of fluorescence can be excited at 630 nm or greater, have a high depth of penetration. Brief description of the drawings:
Figur 1 : zeigt die Herstellung eines Konjugates aus Acridin-9-carbonsäure und humanem Serumalbumin,FIG. 1: shows the preparation of a conjugate from acridine-9-carboxylic acid and human serum albumin,
Figur 2: zeigt die Herstellung eines Konjugates aus Cumarin 343 und humanem Serumalbumin undFigure 2: shows the preparation of a conjugate from coumarin 343 and human serum albumin and
Figur 3: zeigt die Herstellung eines Konjugates aus Tetrasulfophenylporphin und humanem Serumalbumin.Figure 3: shows the preparation of a conjugate from tetrasulfophenylporphin and human serum albumin.
Die folgenden Beispiele erläutern die Erfindung.The following examples illustrate the invention.
Beispiel 1 Herstellung eines erfindungsgemäßen Konjugates aus Acridin-9- carbonsäure und humanem SerumalbuminExample 1 Preparation of a conjugate according to the invention from acridine-9-carboxylic acid and human serum albumin
Die Struktur und die Herstellung des Konjugats sind in Figur 1 dargestellt.The structure and preparation of the conjugate are shown in Figure 1.
Es wurden 20 mg Acridin-9-carbonsäurehydrat (A9CS) in 2 ml DMSO gelöst und etwa 1 00 mg N-Hydroxysuccinimid (HSI) im Molverhältnis von etwa 1 0/1 sowie 30 mg N,N'-Dicyclohexylcarbodiimid (DCC) im Molverhältnis von etwa 1 ,5/1 zugegeben. Nach etwa 6 Stunden ist die Bildung des Hydroxysuccinimidylesters abgeschlossen. Nach dem Abtrennen des Dicyclohexylharnstoffs (DCHH) über einen lösungsmittelbeständigen Filter (0,2 μm) wird der Ester zu einer Lösung von 2 g humanem Serumalbumin (HSA), das in 1 0 ml Originallösung, 1 0 ml 0,34 M NaHC03 und 1 0 ml Methoxypolyethylenglykol (MPEG) gelöst ist, langsam zugegeben. Die bei der Zugabe entstehende leichte Trübung löst sich nach kurzer Zeit wieder auf. Es entsteht eine leicht gelbliche Lösung eines Konjugats aus A9CS und HSA. Die Abtrennung der im fertigen Präparat unerwünschten Begleitstoffe, wie überschüssiges DCC, HSI, nicht gebundenes A9CS, DMSO
und MPEG, erfolgt durch Ultrafiltration (Auschlußgrenze 1 0 kD) mit mindestens 4 Waschvorgängen.20 mg of acridine-9-carboxylic acid hydrate (A9CS) were dissolved in 2 ml of DMSO and about 1 00 mg of N-hydroxysuccinimide (HSI) in a molar ratio of about 10/1 and 30 mg of N, N'-dicyclohexylcarbodiimide (DCC) in a molar ratio of about 1.5 / 1 added. The formation of the hydroxysuccinimidyl ester is complete after about 6 hours. After the dicyclohexylurea (DCHH) has been separated off via a solvent-resistant filter (0.2 μm), the ester becomes a solution of 2 g of human serum albumin (HSA), which is in 1 0 ml of original solution, 1 0 ml of 0.34 M NaHC0 3 and 1 0 ml of methoxypolyethylene glycol (MPEG) is slowly added. The slight cloudiness that occurs during the addition dissolves again after a short time. A slightly yellowish solution of a conjugate of A9CS and HSA is formed. The separation of the undesired accompanying substances in the finished preparation, such as excess DCC, HSI, unbound A9CS, DMSO and MPEG, is carried out by ultrafiltration (exclusion limit 1 0 kD) with at least 4 washes.
Beispiel 2: Herstellung eines erfindungsgemäßen Konjugates aus Cumarin 343 und humanem SerumalbuminExample 2: Preparation of a conjugate according to the invention from coumarin 343 and human serum albumin
Die Struktur und die Herstellung des Konjugates sind in Fig. 2 dargestellt.The structure and preparation of the conjugate are shown in Fig. 2.
In 2 ml DMSO wurden 20 mg Cumarin 343 (C343 = 1 0-Carboxy-2, 3,6,7- tetrahydro-1 H,5H, 1 1 H-[1 ]benzopyranon[6,7,8,ij]chinolizin-1 1 -on) gelöst. Dazu wurden etwa 1 00 mg HSI im Molverhältnis von 1 0/1 und 30 mg DCC im Molverhältnis von etwa 1 , 5/1 zugegeben. Der Ester wurde wie in Beispiel 1 beschrieben isoliert und mit HSA umgesetzt, wobei eine intensiv gelbe Lösung eines Konjugates aus C343 und HSA erhalten wurde. Die Abtrennung von unerwünschten Begleitstoffen erfolgt wie in Beispiel 1 beschrieben.20 mg of coumarin 343 (C343 = 1 0-carboxy-2, 3,6,7-tetrahydro-1 H, 5H, 1 1 H- [1] benzopyranone [6,7,8, ij] quinolizine) were added to 2 ml of DMSO -1 1 -on) solved. About 100 mg of HSI in a molar ratio of 10/1 and 30 mg of DCC in a molar ratio of about 1.5/1 were added. The ester was isolated as described in Example 1 and reacted with HSA, an intensely yellow solution of a conjugate of C343 and HSA being obtained. Unwanted accompanying substances are separated as described in Example 1.
Beispiel 3: Herstellung eines erfindungsgemäßen Konjugates aus Tetra-(4- sulfophenyD-porphin und humanem SerumalbuminExample 3: Preparation of a conjugate according to the invention from tetra- (4-sulfophenyD-porphin and human serum albumin
Die Struktur des Konjugates und dessen Herstellung sind in Figur 3 dargestellt.The structure of the conjugate and its production are shown in FIG. 3.
Tetra-(4-sulfophenyl)-porphin (TSPP) wurde in einer Konzentration von 1 0 mg/ml in DMSO gelöst. Zu der klaren dunkelgrünen Lösung wurde die dreifache molare Menge an DCC und die fünfache molare Menge an HSI zugegeben. Nach etwa 3 bis 4 Stunden Reaktionszeit ist die Umsetzung zum TSPP-Succinimidylester (TSPP-SE) beendet, wobei der gebildete Di-Cyclohexylharnstoff feinkörnig abgeschieden wird. Die analytische Kontrolle erfolgt mittels Dünnschichtchromatographie.Tetra- (4-sulfophenyl) porphine (TSPP) was dissolved in DMSO at a concentration of 10 mg / ml. Three times the molar amount of DCC and five times the molar amount of HSI was added to the clear dark green solution. After a reaction time of about 3 to 4 hours, the conversion to the TSPP succinimidyl ester (TSPP-SE) has ended, the di-cyclohexylurea formed being deposited in fine particles. The analytical control is carried out by means of thin layer chromatography.
Humanes Serumalbumin (HSA, 4g, d.h. 2 Ampullen zu je 2 g in 1 0 ml) wurden
mit 2 x 1 0 ml 0, 1 7 M NaHCO3 und 20 ml Methoxypolyethylenglykol350 verdünnt und in einem 1 00 ml Erlenmeyer-Kolben vorgelegt. Zu dieser HSA-Lösung wurde langsam unter ständigem Rühren vorstehende TSPP-SE-Lösung in DMSO zugefügt, wobei sich die zunächst klare Lösung durch nicht umgesetztes DCC, das in wässriger Lösung unlöslich ist, eintrübt. Nach Beendigung der Zugabe von TSPP-SE wurde das Reaktionsgemisch zur vollständigen Umsetzung 30 Minuten bei Raumtemperatur gerührt. Anschließend wurde die Trübung über eine Sterilfiltereinheit (Millipore, Stericup - GV, 0,22 μm Low Binding Duropore Membrane) und die niedermolekularen wasserlöslichen Komponenten (DMSO, HSI und nicht gebundenes TSPP) durch Ultrafiltration über eine Membran mit 30 kD Ausschlußgrenze (Amicon YM 30) abgetrennt. Es wurde ein erfindungsgemäßes Konjugat aus TSPP und HSA erhalten. Die Kopplungsausbeute von TSPP an HSA betrug 85 bis 90 % .Human serum albumin (HSA, 4g, ie 2 ampoules of 2 g each in 10 ml) were diluted with 2 x 1 0 ml 0, 1 7 M NaHCO 3 and 20 ml methoxypolyethylene glycol 350 and placed in a 100 ml Erlenmeyer flask. The above TSPP-SE solution in DMSO was slowly added to this HSA solution with constant stirring, the initially clear solution becoming cloudy as a result of unreacted DCC which is insoluble in aqueous solution. After the addition of TSPP-SE was complete, the reaction mixture was stirred at room temperature for 30 minutes to complete the reaction. The turbidity was then checked using a sterile filter unit (Millipore, Stericup - GV, 0.22 μm Low Binding Duropore membrane) and the low molecular weight water-soluble components (DMSO, HSI and unbound TSPP) by ultrafiltration through a membrane with a 30 kD cut-off limit (Amicon YM 30 ) separated. A conjugate of TSPP and HSA was obtained. The coupling yield of TSPP to HSA was 85 to 90%.
Die analytische Reinheitskontrolle erfolgte mittels HPLC unter folgenden Bedingungen:The analytical purity control was carried out by means of HPLC under the following conditions:
Vorsäule: Zorbax Diol (50 x 4 mm)Guard column: Zorbax Diol (50 x 4 mm)
Säule 1 : Zorbax GF 450Pillar 1: Zorbax GF 450
Säule 2: Zorbax GF 450Pillar 2: Zorbax GF 450
Laufmittel: 0,2 M Na-citrat, pH 7, 5Mobile solvent: 0.2 M Na citrate, pH 7.5
Fluß: 1 ml/minFlow: 1 ml / min
Detektor 1 : 280 nm (für das Protein)Detector 1: 280 nm (for the protein)
Detektor 2: 420 nm (für TSPP)
Detector 2: 420 nm (for TSPP)
Claims
1 . Konjugat, umfassend eine zur Fluoreszenz-fähige Verbindung und einen Träger, wobei die Verbindung und der Träger über eine Säureester- oder Säureamid-Bindung eine Enan-Brücke verbunden sind und die Verbindung in dem Konjugat eine Anregungswelleniänge von 630 nm oder größer und/oder 450 nm oder kleiner aufweist.1 . A conjugate comprising a compound capable of fluorescence and a carrier, the compound and the carrier being connected via an enane bridge via an acid ester or acid amide bond and the compound in the conjugate having an excitation wavelength of 630 nm or greater and / or 450 nm or less.
2. Konjugat nach Anspruch 1 , dadurch gekennzeichnet, daß der Träger ein Protein ist.2. Conjugate according to claim 1, characterized in that the carrier is a protein.
3. Konjugat nach Anspruch 2, dadurch gekennzeichnet, daß das Protein ein nicht als körperfremd angesehenes, natives Protein ist.3. Conjugate according to claim 2, characterized in that the protein is a native protein not regarded as foreign to the body.
4. Konjugat nach Anspruch 3, dadurch gekennzeichnet, daß das Protein humanes Serumalbumin ist.4. Conjugate according to claim 3, characterized in that the protein is human serum albumin.
5. Konjugat nach Anspruch 1 , dadurch gekennzeichnet, daß der Träger ein Polyether ist.5. Conjugate according to claim 1, characterized in that the carrier is a polyether.
6. Konjugat nach Anspruch 5, dadurch gekennzeichnet, daß der Polyether ein Polethylenglykol ist.6. Conjugate according to claim 5, characterized in that the polyether is a polyethylene glycol.
7. Konjugat nach einem der Ansprüche 1 bis 6, dadurch gekennzeichnet, daß mehrere Träger vorliegen.7. Conjugate according to one of claims 1 to 6, characterized in that several carriers are present.
8. Konjugat nach einem der Ansprüche 1 bis 7, dadurch gekennzeichnet, daß die zur Fluoreszenz-fähige Verbindung eine Säuregruppe, Hydroxylgruppe, Aminogruppe oder Aldehydgruppe aufweist.
8. Conjugate according to one of claims 1 to 7, characterized in that the compound capable of fluorescence has an acid group, hydroxyl group, amino group or aldehyde group.
. Konjugat nach einem der Ansprüche 1 bis 8, dadurch gekennzeichnet, daß die Anregungswellenlänge 630 bis 850 nm betrtägt.. Conjugate according to one of claims 1 to 8, characterized in that the excitation wavelength is 630 to 850 nm.
0. Konjugat nach einem der Ansprüche 1 bis 9, dadurch gekennzeichnet, daß die Anregungswellenlänge 320 bis 450 nm beträgt.0. Conjugate according to one of claims 1 to 9, characterized in that the excitation wavelength is 320 to 450 nm.
1 . Konjugat nach einem der Ansprüche 1 bis 1 0, dadurch gekennzeichnet, daß die zur Fluoreszenz-fähige Verbindung abgeleitet ist von Porphyrin, Chlorin, Bakteriochlorin, Chlorophyll, Phtalocyanin, Carboxyzimtsäure, Carboxyfluorescein, Acridinsäure, Cumarinsäure oder Indocyaningrün sowie den Derivaten davon.1 . Conjugate according to one of Claims 1 to 1 0, characterized in that the compound capable of fluorescence is derived from porphyrin, chlorine, bacteriochlorin, chlorophyll, phthalocyanine, carboxycinnamic acid, carboxyfluorescein, acridic acid, coumaric acid or indocyanine green and the derivatives thereof.
2. Konjugat nach einem der Ansprüche 1 bis 1 1 , dadurch gekennzeichnet, daß mehrere zur Fluoreszenz-fähige Verbindungen vorliegen.2. Conjugate according to one of claims 1 to 1 1, characterized in that there are several compounds capable of fluorescence.
3. Verfahren zur Herstellung eines Konjugats nach einem der Ansprüche 1 bis 1 2, dadurch gekennzeichnet, daß die zur Fluoreszenz-fähige Verbindung und der Träger unter Ausbildung einer Säureester oder Säureamid- Bindung kovalent verbunden werden.3. A process for the preparation of a conjugate according to any one of claims 1 to 1 2, characterized in that the compound capable of fluorescence and the carrier are covalently linked to form an acid ester or acid amide bond.
4. Verwendung eines Konjugats nach einem der Ansprüche 1 bis 1 2 zur Unterscheidung von krankhaftem und gesundem Gewebe.
4. Use of a conjugate according to one of claims 1 to 1 2 for distinguishing pathological and healthy tissue.
Applications Claiming Priority (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
DE19731741 | 1997-07-23 | ||
DE19731741A DE19731741A1 (en) | 1997-07-23 | 1997-07-23 | Conjugate to differentiate between diseased and healthy tissue |
PCT/DE1998/002102 WO1999005521A2 (en) | 1997-07-23 | 1998-07-22 | Conjugate for differentiating between healthy and unhealthy tissue |
Publications (1)
Publication Number | Publication Date |
---|---|
EP0998674A2 true EP0998674A2 (en) | 2000-05-10 |
Family
ID=7836699
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
EP98946241A Withdrawn EP0998674A2 (en) | 1997-07-23 | 1998-07-22 | Conjugate for differentiating between healthy and unhealthy tissue |
Country Status (5)
Country | Link |
---|---|
US (2) | US20020037254A1 (en) |
EP (1) | EP0998674A2 (en) |
JP (1) | JP2001513583A (en) |
DE (1) | DE19731741A1 (en) |
WO (1) | WO1999005521A2 (en) |
Families Citing this family (8)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
DE19847362A1 (en) * | 1998-10-14 | 2000-04-20 | Deutsches Krebsforsch | New polycyclic aromatic compound-protein conjugates, useful for detection and/or treatment of diseased tissue, e.g. tumors or inflammation sites |
DE29911689U1 (en) * | 1999-07-06 | 2000-04-06 | Sterk Peter | Agents for occluding organic tissue |
DE10006570A1 (en) * | 2000-02-14 | 2001-08-23 | Deutsches Krebsforsch | Hemin-protein conjugate useful as magnetic resonance contrast agent, especially for diagnosis of tumors and inflammatory processes |
ATE298341T1 (en) * | 2001-03-21 | 2005-07-15 | Molteni & C | METAL-SUBSTITUTED NON-CENTROSYMMETRIC PHTHALOCYANINE ANALOGS, THEIR PREPARATION AND USE FOR PHOTODYNAMIC THERAPY AND AS IN VIVO DIAGNOSTICS |
CA2851344C (en) * | 2011-09-05 | 2017-10-03 | Hiroshi Maeda | Polymer-type fluorescent molecule probe |
JP6552236B2 (en) | 2015-03-23 | 2019-07-31 | キヤノン株式会社 | Near-infrared dye-bound transferrin, contrast agent for photoacoustic imaging comprising the near-infrared dye-bound transferrin |
JP2017128532A (en) * | 2016-01-20 | 2017-07-27 | キヤノン株式会社 | Method for producing contrast agent for optical imaging and contrast agent for optical imaging |
JP6752582B2 (en) * | 2016-02-08 | 2020-09-09 | キヤノン株式会社 | Contrast agent for photoacoustic imaging |
Family Cites Families (14)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
DE3321041A1 (en) * | 1983-06-10 | 1984-12-13 | Bayer Ag, 5090 Leverkusen | Chromogenic and fluorogenic esters, process for their preparation and also process and composition for the detection and for the photometric or fluorometric determination of phosphatases or sulphatases |
DE3248043A1 (en) * | 1982-12-24 | 1984-06-28 | Bayer Ag, 5090 Leverkusen | Fluorogenic phosphoric esters, process for their preparation, and process and composition for the detection and fluorometric determination of phosphates |
MC1637A1 (en) * | 1983-11-10 | 1986-01-09 | Genetic Systems Corp | POLYMERIZABLE COMPOUNDS CONTAINING POLYPEPTIDES AS AN INTEGRAL PART AND THEIR USE IN IMMUNOLOGICAL TESTS OF POLYMERIZATION-INDUCED SEPARATION |
US4783529A (en) * | 1985-12-03 | 1988-11-08 | Research Corporation Technologies | Rapid synthesis of radiolabeled porphyrin complexes for medical application |
EP0267038A3 (en) * | 1986-11-06 | 1989-07-12 | The University Of British Columbia | Anhydrous enhanced coupling |
US4923819A (en) * | 1987-03-27 | 1990-05-08 | Chimerix Corporation | Time-resolved fluorescence immunoassay |
US5612016A (en) * | 1988-04-01 | 1997-03-18 | Immunomedics, Inc. | Conjugates of antibodies and bifunctional ligands |
US4990447A (en) * | 1988-06-24 | 1991-02-05 | Gist-Brocades Nv | Process for the purification of serum albumin |
US5231004A (en) * | 1990-05-11 | 1993-07-27 | Eastman Kodak Company | Use of heme-containing proteins as stabilizers for enzyme-labeled immunoreactants |
IL102645A (en) * | 1992-07-26 | 1998-02-22 | Yeda Res & Dev | Chlorophyll and bacteriochlorophyll derivatives, their preparation and pharmaceutical compositions comprising them |
US5447838A (en) * | 1992-08-05 | 1995-09-05 | Hybritech Incorporated | Protein-dye conjugate for confirmation of correct dilution of calibrators |
US5563132A (en) * | 1994-06-21 | 1996-10-08 | Bodaness; Richard S. | Two-step cancer treatment method |
US5856479A (en) * | 1996-05-20 | 1999-01-05 | Nisshinbo Industries, Inc. | Fluorescent group-containing carbodiimide compound |
FR2757162B1 (en) * | 1996-12-12 | 1999-03-26 | Cis Bio Int | NON-AGGREGATED FLUORESCENT CONJUGATES |
-
1997
- 1997-07-23 DE DE19731741A patent/DE19731741A1/en not_active Withdrawn
-
1998
- 1998-07-22 EP EP98946241A patent/EP0998674A2/en not_active Withdrawn
- 1998-07-22 JP JP2000504456A patent/JP2001513583A/en not_active Withdrawn
- 1998-07-22 US US09/463,474 patent/US20020037254A1/en not_active Abandoned
- 1998-07-22 WO PCT/DE1998/002102 patent/WO1999005521A2/en not_active Application Discontinuation
-
2004
- 2004-08-13 US US10/917,907 patent/US20050019263A1/en not_active Abandoned
Non-Patent Citations (1)
Title |
---|
See references of WO9905521A3 * |
Also Published As
Publication number | Publication date |
---|---|
US20050019263A1 (en) | 2005-01-27 |
WO1999005521A2 (en) | 1999-02-04 |
JP2001513583A (en) | 2001-09-04 |
DE19731741A1 (en) | 1999-01-28 |
WO1999005521A3 (en) | 1999-04-08 |
US20020037254A1 (en) | 2002-03-28 |
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