Cell Death Assay

• US 20150010921A1
• Filed: 02/06/2013
• Published: 01/08/2015
• 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, Lwik, Clemens Waltherus Gerardus, Van Beek, Ermond Reijer
• Granted Patent: US 10,830,770 B2
• Time in Patent Office: Days
• US Class Current: 435/7.8
• 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 detecting cell death using a luminescent compound; to the luminescent compounds for particular uses; to a kit comprising compounds and to a protein. The method is applicable for detecting cell death, essentially regardless of the mechanism through which cell death occurred or is occurring and is therefore not limited e.g. to detecting cell death resulting from only one mechanism selected from apoptosis and necrosis.

Claims

1. A method for determining a value or values of one or more parameters of a biological sample,

said sample comprising a population of cells comprising dead cells, and at least a first compound,

said dead cells having at least a first cell constituent substantially present and/or accessible to the at least first compound only in dead cells,

wherein the first compound is selected from a group consisting of non-toxic cyanine and rhodamine containing compounds, wherein the first compound is capable of selectively interacting chemically/physically/biologically directly with the at least the first cell constituent, wherein the first cell constituent is at least one protein,

wherein the method comprises the steps of:

(i) providing the biological sample;

(ii) performing one or more measurements on the sample with at least a first suitable imaging technique providing a value or values, and;

(iii) analysing said value or values of the measurement to determine a value or values of the one or more parameters, optionally by comparison with a set of reference values.

2. The method according to claim 1, wherein a value or values of the measurement above a threshold value indicate binding of the compound to the first cell constituent.

3. The method according to claim 1, wherein the one or more parameters is the presence and/or concentration and/or location of dead cells in the population of cells.

4. The method according to claim 1, wherein the suitable imaging technique is selected from a group comprising:

emission spectroscopy, MRI, PET, SPECT, CT, optical imaging, such as microscopy, acoustical imaging, and combinations thereof.

5. The method according to claim 1, wherein the cyanine and/or rhodamine is a light emissive cyanine or rhodamine in a wavelength range of 200 nm-2500 nm, preferably an infrared wavelength.

6. The method according to claim 1, wherein said protein is selected from the group comprising fibrous proteins, such as 40 kDa proteins, 100 kDa proteins, such as tubulin, such as α

-tubulin, β

-tubulin, γ

-tubulin, δ

-tubulin and ε

-tubulin, actin, such as G-actin, and F-actin, fibrous structural proteins, such as keratin, such as neutral, basic or acidic keratin, such as keratin 1-keratin 20, metalloenzymes, such as enolase, and lyase, CDC37, preferably tubulin or actin, isomers thereof, complexes thereof, and decay products thereof, optionally being a biomarker for cell death.

7. The method according to claim 1, wherein the cyanine and/or rhodamine is a non-activated or de-activated cyanine and/or rhodamine, preferably a cyanine according to FIG. 1 I, II and III, wherein

n is an integer, such as n∈

[2,10], preferably n∈

[4,8],

the chain L has up to n−

1 double bonds, preferably n/2 double bonds,

wherein sub-families II and III may comprise respectively one and two aromatic ring systems (A,B) signified by the curved line(s) C,

wherein A,B are preferably selected each individually from benzene and naphthalene,

wherein further groups R₅, R₆, R₇, and R₈, may be present, R₅, R₆, R₇, and R₈, are preferably selected each individually from H, and alkyl, such as methyl, ethyl, and propyl, preferably methyl,

wherein the aromatic ring systems may comprise further functional groups R₁, R₂, and/or substituents, R₁, R₂, are preferably selected each individually from H, sulfonate, and sulfonamide,

wherein the chain of alternating single and double bonds L may be interrupted by one or more partly and fully saturated ring structures, such as cyclopentene and cylcohexene, and combinations thereof, such as one or more cyclohexene rings, wherein the saturated ring structure may further comprise functional groups R₉, R₉ being selected from H, AA and BB, wherein R₁₀ is selected from, H, SO₃H, Cl, —

N—

C═

O—

(CH2)_q—

Y₃ (q=1-6), —

(CH2)_r—

Y₄ (r=1-6), Y₃ and Y₄ are each independently one of COOH, SO₃H, CN,

wherein the nitrogen atoms (N) may comprise further functional N-side groups R₃, R₄, wherein R₃, R₄ are preferably selected each individually from —

(CH₂)_mY, wherein Y is selected each individually from a carboxylic acid having 1-4 carbon atoms, a sulfonate group, CN, C═

C, and C═

C, and salts thereof,

wherein said N-side groups comprise m carbon atoms, such as m∈

[1,10], preferably m∈

[2,8], more preferably m∈

[3,7], most preferably m=4,5, and 6,

even more preferably at least one of m=4, 5, and 6, preferably one m=6, and the other m preferably is 4, 5 or 6,

wherein said N-side groups comprise one or more functional groups on an end opposing the N, such as a carboxylic acid having 1-4 carbon atoms, an sulfonic group, and salts thereof, such as sodium and potassium salts, most preferably the functional group on the end comprises one or more double C—

C bonds,

such as one of HQ4, HQ5, HQ6, HQ7, ICG, CW 800, ZW800, L4, L7, L11, CY3, CY3b, CY3.5, CY5, CY5.5, CY7, DY-676, DY-681, DY-731, DY-751, and DY-776, and conjugable derivatives thereof, preferably a carboxylate thereof.

8. The method according to claim 1, wherein the cyanine and/or rhodamine is coupled to one or more of:

(i) a radio-active tracer;

(ii) an MRI contrast agent;

(iii) a microbubble for ultrasound or opto-acoustic imaging;

(iv) a nanoparticle;

(v) a molecule suitable for imaging, and (vi) a biological active compound.

9. The method according to claim 1, wherein the interacting takes place in a time in a range of 1-360 minutes;

more preferably 15-240, most preferably 120-180.

10. The method of claim 1, further comprising using the method in an assay for screening drugs for therapy such as cancer therapy.

11. The method of claim 1, further comprising using the method to monitor and/or determine the effectiveness of a therapy.

12. A compound comprising one or more of a cyanine, a rhodamine, and any other first compound selectively interacting chemically/physically/biologically directly with at least a first cell constituent, for use in detecting cell death, being a non-activated cyanine and/or rhodamine, preferably a cyanine according to FIG. 1 I, II and III, wherein

n is an integer, such as n∈

[2,10], preferably n∈

[4,8],

the chain L has up to n−

1 double bonds, preferably n/2 double bonds,

wherein sub-families II and III may comprise respectively one and two aromatic ring systems (A,B) signified by the curved line(s) C,

wherein A,B are preferably selected each individually from benzene and naphthalene,

wherein further groups R₅, R₆, R₇, and R₃, may be present, R₅, R₆, R₇, and R₈, are preferably selected each individually from H, and alkyl, such as methyl, ethyl, and propyl, preferably methyl,

wherein the aromatic ring systems may comprise further functional groups R₁, R₂, and/or substituents, R₁, R₂, are preferably selected each individually from H, sulfonate, and sulfonamide,

wherein the chain of alternating single and double bonds L may be interrupted by one or more partly and fully saturated ring structures, such as cyclopentene and cylcohexene, and combinations thereof, such as one or more cyclohexene rings, wherein the saturated ring structure may further comprise functional groups R₉, being selected from H, AA and BB, wherein R₁₀ is selected from, H, SO₃H, Cl, —

N—

C═

O—

(CH2)_q—

Y₃ (q=1-6), —

(CH2)_r—

Y₄ (r=1-6), Y₃ and Y₄ are each independently one of H, COOH, SO₃H, and CN,

wherein the nitrogen atoms (N) may comprise further functional N-side groups R₃, R₄, wherein R₃, R₄ are preferably selected each individually from —

(CH₂)_mY, wherein Y is selected each individually from a carboxylic acid having 1-4 carbon atoms, a sulfonate group, CN, C═

C, and C═

C, and salts thereof,

wherein said N-side groups comprise m carbon atoms, such as m∈

[1,10], preferably m∈

[2,8], more preferably m∈

[3,7], most preferably m=4,5, and 6,

even more preferably at least one of m=4, 5, and 6, preferably one m=6, and the other m preferably is 4, 5, or 6,

wherein said N-side groups comprise one or more functional groups on an end opposing the N, such as a carboxylic acid having 1-4 carbon atoms, an sulfonic group, and salts thereof, such as sodium and potassium salts, most preferably the functional group on the end comprises one or more double C—

C bonds,

preferably a carboxylate thereof.

13. The compound according to claim 12, wherein the cyanine or rhodamine is one or more of:

(i) infrared emissive;

(ii) capable of binding to a protein, optionally wherein the protein is a biomarker for cell death;

(iii) one of HQ4, HQ5, HQ6, HQ7, ICG, CW 800, ZW800, L4, L7, L11, CY3, CY3b, CY3.5, CYS, CY5.5, CY7, DY-676, DY-681, DY-731, DY-751, and DY-776, and a conjugable derivative thereof, preferably a carboxylate thereof.

14. The compound according to claim 12 for use in one or more of determining presence or absence of Alzheimer'"'"'s disease, determining effectiveness of chemotherapy, determining cell damage in a tissue, such in a heart after a heart-attack, such as in the brains after a stroke, and cell death in a tissue.

15. An assay kit comprising a fluid, such as a physiological fluid, a cyanine and/or rhodamine, a means for storing, and preferably consevations agents, such as an antibacterial agent, such as an azide, preferably in an amount of 0,1-5 ml and preferably in a concentration of cyanine and/or rhodamine of 0,2-50 μ

M.