Protein target complex

• US 10,641,771 B2
• Filed: 02/06/2013
• Issued: 05/05/2020
• Est. Priority Date: 02/06/2012
• Status: Active Grant

• Current Assignee: HQ Medical Netherlands BV, Publiekrechtelijk Rechtspersoon Academisch Ziekenhuis Leiden H.O.D.N. Lumc
• Original Assignee: HQ Medical Netherlands BV, Publiekrechtelijk Rechtspersoon Academisch Ziekenhuis Leiden H.O.D.N. Lumc
• Inventors: Maring, Markwin Hendrik, Lowik, Clemens Waltherus Gerardus, Van Beek, Ermond Reijer
• Primary Examiner(s): Gabel, Gailene
• Application Number:
  US14/377,146
• Publication Number:
  US 20150004626A1
• Time in Patent Office: 2,645 Days
• CPC Class Codes: A61K 31/352 condensed with carbocyclic rings, e.g. methantheline cannabinoids A61K31/658
• CPC Class Codes: A61K 31/403 condensed with carbocyclic rings, e.g. carbazole
• CPC Class Codes: A61K 31/404 Indoles, e.g. pindolol
• CPC Class Codes: C07D 209/24 with an alkyl or cycloalkyl radical attached to the ring nitrogen atom
• CPC Class Codes: C07D 498/14 Ortho-condensed systems
• CPC Class Codes: C07D 498/22 in which the condensed system contains four or more hetero rings
• CPC Class Codes: C07K 14/435 from animals; from humans
• CPC Class Codes: G01N 2500/10 involving cells
• CPC Class Codes: G01N 2510/00 Detection of programmed cell death, i.e. apoptosis
• CPC Class Codes: G01N 2800/52 Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis
• CPC Class Codes: G01N 33/5008 for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
• CPC Class Codes: G01N 33/5091 for testing the pathological state of an organism
• CPC Class Codes: G01N 33/533 with fluorescent label
• CPC Class Codes: G01N 33/56966 Animal cells
• CPC Class Codes: G01N 33/57496 involving intracellular compounds
• CPC Class Codes: G01N 33/583 with non-fluorescent dye label

Abstract

The present invention relates to a method for labeling or targeting cells whose plasma membrane has lost integrity, such as dead cells, such as for discriminating between live cells and cells whose plasma membrane has lost integrity; to an embodiment of the method for determining one or more values of one or more parameters of cells of a biological sample; to use of the method in an assay for screening drugs for therapy such as cancer therapy; to use of the method to monitor and/or determine the effectiveness of a therapy; to an assay kit; to a complex; to the complex for use as a medicament; to the complex for use in treatment of cancer(s) and/or plaque(s) and/or regeneration and/or supporting the immune system; and, to a dead cell.

Claims

1. A method for targeting proteins in cells whose plasma membrane has lost integrity, for discriminating between live cells and cells whose plasma membrane has lost integrity, wherein the method comprises:

(a) providing a population of cells in a tissue comprising cells whose plasma membrane has lost integrity and live cells, the cells providing a first intracellular protein;

(b) applying a non-activated extracellularly applied compound to the population of cells, wherein the non-activated extracellularly applied compound is substantially unable to cross an intact cell membrane;

wherein the non-activated extracellularly applied compound is selected from a group consisting of negatively charged non-toxic cyanine containing non-activated extracellularly applied compounds, wherein in the non-activated extracellularly applied compound the cyanine selectively interacts directly with at least one cell constituent, wherein the cell constituent is the first intracellular protein, wherein the first intracellular protein is a protein selected from the group consisting of fibrous proteins, metalloenzymes, Hsp90p co-chaperone (CDC37), binding isomers thereof, and binding decay products thereof,

(c) targeting the cyanine of the cyanine containing non-activated extracellularly applied compound to the first intracellular protein in the cell whose plasma membrane has lost integrity, thereby forming a complex of the first intracellular protein and cyanine containing non-activated extracellularly applied compound, and

(d) performing a measurement to determine an amount of targeted complexes formed between the cyanine of the cyanine containing non-activated extracellularly applied compound and the first intracellular protein in the cell whose plasma membrane has lost integrity thereby discriminating between live cells and cells containing the targeted complexes formed whose plasma membrane has lost integrity, wherein the measurement capable of detecting the targeted complexes formed is selected from the group consisting of;

optical spectroscopies, optical microscopy, acoustical imaging, acoustic spectroscopies, Magnetic Resonance Imaging (MRI), Positron Emission Tomography (PET), Single Photon Emission Computed Tomography (SPECT), Computed Tomography (CT), and combinations thereof.

2. The method according to claim 1, wherein the first intracellular protein in claim 1, step (b) comprises 40 kDa proteins or 100 kDa proteins.

3. The method according to claim 1, wherein the cyanine is a non-activated cyanine according to FIG. 1 I, II and III,

4. The method according to claim 1, wherein the method further comprises:

(e) analysing the measured value in claim 1, step (d) to determine one or more parameters of the cells whose plasma membrane has lost integrity in the population of cells, and;

(f) optionally repeating (d) and/or (e) for a given sample at multiple time-points to determine a change in the value of a parameter, or the amount of targeted complexes formed, as a function of time.

5. The method according to claim 4, wherein the one or more parameters is the presence and/or concentration and/or location of cells whose plasma membrane has lost integrity within the population of cells.

6. The method of claim 1, further comprising providing an assay for screening drugs for therapy,

wherein the amount of targeted complexes formed between the cyanine of the cyanine containing non-activated extracellularly applied compound and the first intracellular protein in the cell of claim 1, step (d) is used to screen the effectiveness of said drugs for therapy.

7. The method of claim 1, further comprising performing a diagnostic method, or monitoring a therapy,

wherein the amount of targeted complexes formed between the cyanine of the cyanine containing non-activated extracellularly applied compound and the first intracellular protein in the cell of claim 1, step (d) is used to screen the effectiveness of said diagnostic method or to monitor the effect of said therapy.

8. A complex of the negatively charged non-toxic cyanine in the cyanine containing non-activated extracellularly applied compound and the targeted first intracellular protein obtained by a method according to claim 1.

9. The complex of claim 8, having a ratio of an amount of the cyanine containing non-activated extracellularly applied compound bound to the targeted first intracellular protein contained within a given quantity of cells whose plasma membrane has lost integrity divided by an amount of the compound that is bound to a same given quantity of living cells ([amount dead cell]/[amount living cell]), wherein the ratio is larger than 10.