EP0554331A1 - Agents de diagnostic fluorescents auto-rassemblants - Google Patents

Agents de diagnostic fluorescents auto-rassemblants

Info

Publication number
EP0554331A1
EP0554331A1 EP91919114A EP91919114A EP0554331A1 EP 0554331 A1 EP0554331 A1 EP 0554331A1 EP 91919114 A EP91919114 A EP 91919114A EP 91919114 A EP91919114 A EP 91919114A EP 0554331 A1 EP0554331 A1 EP 0554331A1
Authority
EP
European Patent Office
Prior art keywords
components
aldehyde
hydrazine
target
cells
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Withdrawn
Application number
EP91919114A
Other languages
German (de)
English (en)
Inventor
Darryl C. Rideout
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Scripps Research Institute
Original Assignee
Scripps Research Institute
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Application filed by Scripps Research Institute filed Critical Scripps Research Institute
Publication of EP0554331A1 publication Critical patent/EP0554331A1/fr
Withdrawn legal-status Critical Current

Links

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
    • 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
    • A61K49/0058—Antibodies
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/531—Production of immunochemical test materials
    • G01N33/532—Production of labelled immunochemicals
    • G01N33/533—Production of labelled immunochemicals with fluorescent label
    • 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

Definitions

  • the invention relates to the detection of cells and tissues which are affected with a condition which renders their detection desirable. More specifically, the invention concerns the in situ formation of fluorescent conjugates from separately administered components to detect these targets.
  • U.S. Patent 4,812,449 issued to the applicant herein and incorporated herein by reference discloses the general approach of providing active materials in the form of component precursors so as to permit self assembly in a target microenviron ent.
  • a target refers to an organism, tissue, cell, or other biologically responsive material which it is desired to modify.
  • the target will occur in a general environment which may be an j_n vitro or an n vivo environment, and supplies its own “microenvironment”.
  • the patent discloses a large number of reaction types which may be employed to form a conjugate jLn situ, including hydrazone formation.
  • the self-assembly of a conjugate which is intended to interact with the target in situ has a number of advantages, including selectivity provided by the individual components, delay of the effect until the assembly is completed in the microenvironment in which it is intended to take place, enhancement of rate constants and selectivity, and a resulting dosage enhancement as compared to administration of the preformed conjugate.
  • the invention herein represents a particularly advantageous specific embodiment of this general approach—namely, use of a self-assembling conjugate to generate fluorescence for detection of target conditions either jj vivo or ij vitro.
  • a self-assembling conjugate to generate fluorescence for detection of target conditions either jj vivo or ij vitro.
  • it is possible to enhance the selectivity for target by conjugating one or both of the components of the self-assembling conjugate to a target-specific ligand.
  • the invention is directed to self-assembling conjugates which emit fluorescence and permit the detection of target organisms, cells or tissues in an in vivo or an jLn vitro environment.
  • the precursors of a highly fluorescent material which themselves are not fluorescent, or which emit substantially less fluorescence than does the conjugate and/or which limit fluorescence at different wavelengths from conjugate, are supplied to the environment in which the target is contained. Due either to intrinsic factors characteristic. of the components or to modifications thereof which enhance selectivity, the components are selectively combined in situ in the desired target to permit detection through emission of fluorescence.
  • the conjugates may be cytotoxic.
  • the invention is directed to a method to detect a target organism, cell or tissue in an in vivo or an in vitro environment which method co prises administering to the environment containing the target, components of a conjugate which will self- assemble in the microenvironment of the target to form a fluorescent conjugate, and detecting the fluorescence emitted.
  • the invention is directed to compositions, especially pharmaceutical compositions which contain these components.
  • Figure 1 shows the structures of illustrative aldehyde K, hydrazine L and conjugate M, which conjugate is highly fluorescent.
  • Figure 2 shows the ability of the conjugate M preformed or formed jln situ to label MCF-7 cells .in vitro.
  • Figure 3 shows an isobologram for inhibition of MCF-7 human breast carcinoma cells using combinations of compounds K and L.
  • Figure 4 shows the effect of compounds K, M, and L on fluorescence emitted by MIA PaCa cells in . vitro.
  • Figure 5 shows an isobologram for inhibition of MIA PaCa cells by various combinations of components K and L.
  • the invention is directed to methods to detect target organisms, cells or tissue by emitted fluorescence using fluorescent conjugates which have been formed in situ by two precursor components.
  • the components are preferably an aldehyde and a hydrazine, neither of which is highly fluorescent, but which, when condensed to form the hydrazone, result in a highly fluorescent conjugate.
  • Preferred aldehydes are polyaromatic aldehydes with a high level of conjugation; a similar description would ' apply to preferred embodiments of the hydrazines.
  • Particularly preferred are the aldehyde and hydrazine shown as structures K and L in Figure 1.
  • the invention method relies in part on the ability of the components of the conjugates to migrate preferentially to the target organism, cell or tissue.
  • suitable targets include those moieties whose detection is of medical interest, such as tumor cells, infectious organisms, diseased tissue, and the like.
  • the homing specificity of the components can be enhanced by coupling the component to a targeting ligand using general coupling methods known in the art, including direct coupling, but preferentially utilizing linker molecules which permit more controlled coupling reactions.
  • Commercially available linkers such as those from Pierce Chemical Company, Rockford, IL, may be used, for example.
  • the targeting ligand can be, for example, an antibody or an immunoreactive fragment thereof, such as an Fab or Fab' fragment, a receptor ligand wherein the receptor is characteristic of the target cell, organism or tissue, or a substrate for active molecules present in high concentration in the microenvironment of the target.
  • the targeting ligand can be coupled to either the aldehyde or the hydrazine component or to both. Coupling to both greatly enhances the selectivity of the conjugation.
  • the invention method comprises supplying the environment of the target with the two components either simultaneously or, according to the nature of the target, one or the other of the components may be supplied initially followed by the other.
  • the fluorescence emitted is detected using standard techniques.
  • the application of the present method permits the use of fluorescence analysis jLn vivo rather than histological staining of fixed tissues.
  • the conjugate may also be toxic to the target, and both treatment and monitoring thereof can be simultaneously effected.
  • Example 1 Fluorescent Staining of MCF-7 Cells In Vitro MCF-7 human breast carcinoma cells were cultured under standard conditions and treated with the components K and L shown in Figure 1 at various concentrations of each.
  • K is P-(triphenyl phosphonium methyl) benzaldehyde
  • L is hydrazinostilbazole; they react to obtain the corresponding hydrazone.
  • hydrazone of structure M shown in Figure 1 can be formed iri situ by separate provision of K and L precursors either simultaneously or using prior administration K before administration of L.
  • Figure 2A and B show the phase contrast photograph and fluorescent micrograph of MCF-7 cells treated with a saline control. No fluorescence is seen.
  • C, E and G show the phase contrast photographs and D, F and H show fluorescence micrographs of MCF-7 cells which have been treated with reagents L, K or M, respectively, for 24 hours.
  • panels D and F neither 11.2 ⁇ g/ml L or 16.8 ⁇ g/ml of K provided any fluorescence labeling for these cells.
  • only 5 2.6 ⁇ g/ml of M provided a high contrast micrograph.
  • panels A 1 and B 1 are the . corresponding results for MCF-7 cells incubated simultaneously for 24 hours with 5.6 ⁇ g/ml L and 8.5 ⁇ g/ml K.
  • Panel B shows that a fluorescent signal is
  • panels C and D' show that if L is administered before , even in higher amount, no fluorescence is obtained. Administration of 11.2 ⁇ g/ml L for 24 hours followed by washing and treatment with 16.8 ⁇ g/ml K for 24 hours did not result
  • the cytotoxicity of this conjugate is shown in Figure 5 with respect to MIA PaCa 2 cells. As shown, either 20 ⁇ M L or 55 ⁇ M K were able, taken alone, to effect 50% growth inhibition in MIA PaCa cells over a 48 hour period. However, combinations of these components at less than 10 ⁇ M concentration of each were able to exhibit a comparable cytotoxic Effect.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Immunology (AREA)
  • Engineering & Computer Science (AREA)
  • Biomedical Technology (AREA)
  • Hematology (AREA)
  • Chemical & Material Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Urology & Nephrology (AREA)
  • Molecular Biology (AREA)
  • Animal Behavior & Ethology (AREA)
  • Physics & Mathematics (AREA)
  • Biotechnology (AREA)
  • Microbiology (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • Epidemiology (AREA)
  • Food Science & Technology (AREA)
  • Medicinal Chemistry (AREA)
  • Cell Biology (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
  • Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)

Abstract

On alimente séparément un milieu en composés de conjugués fluorescents qui se lient en présence d'une cellule ou d'un tissu d'organisme cible, de sorte que l'on obtient un conjugué fluorescent pouvant servir à détecter ladite cible.
EP91919114A 1990-10-04 1991-10-03 Agents de diagnostic fluorescents auto-rassemblants Withdrawn EP0554331A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US59247790A 1990-10-04 1990-10-04
US592477 1996-01-26

Publications (1)

Publication Number Publication Date
EP0554331A1 true EP0554331A1 (fr) 1993-08-11

Family

ID=24370806

Family Applications (1)

Application Number Title Priority Date Filing Date
EP91919114A Withdrawn EP0554331A1 (fr) 1990-10-04 1991-10-03 Agents de diagnostic fluorescents auto-rassemblants

Country Status (3)

Country Link
EP (1) EP0554331A1 (fr)
JP (1) JPH06504365A (fr)
WO (1) WO1992006378A1 (fr)

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH0579985A (ja) * 1991-09-24 1993-03-30 Kyoto Daiichi Kagaku:Kk 化学発光増強剤及び増強方法
US8580516B2 (en) * 2008-09-05 2013-11-12 University Of Chicago Methods and compositions for direct detection of DNA damage

Family Cites Families (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4671958A (en) * 1982-03-09 1987-06-09 Cytogen Corporation Antibody conjugates for the delivery of compounds to target sites
US4868103A (en) * 1986-02-19 1989-09-19 Enzo Biochem, Inc. Analyte detection by means of energy transfer
US4812449A (en) * 1986-07-03 1989-03-14 Scripps Clinic And Research Foundation In situ active compound assembly
US4937183A (en) * 1988-02-03 1990-06-26 Cytogen Corporation Method for the preparation of antibody-fragment conjugates
US5047227A (en) * 1988-02-08 1991-09-10 Cytogen Corporation Novel and improved antibodies for site specific attachment of compounds

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO9206378A1 *

Also Published As

Publication number Publication date
WO1992006378A1 (fr) 1992-04-16
JPH06504365A (ja) 1994-05-19

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