Protein Target Complex

• US 20150004626A1
• Filed: 02/06/2013
• Published: 01/01/2015
• Est. Priority Date: 02/06/2012
• Status: Active Grant

• Current Assignee: HQ Medical Netherlands BV
• Original Assignee: HQ Medical Netherlands BV
• Inventors: Maring, Markwin Hendrik, Lwik, Clemens Waltherus Gerardus, Van Beek, Ermond Reijer
• Granted Patent: US 10,641,771 B2
• Time in Patent Office: Days
• US Class Current: 435/7.4
• 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 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, wherein the method comprises:

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

(b) providing a first non-activated extracellularly applied first compound, wherein the first compound is substantially unable to cross a healthy cell membrane;

(c) chemically/physically/biologically interacting the first compound and the first protein in a cell whose plasma membrane has lost integrity, and

(d) performing a measurement to determine an amount of interaction between the first compound and the cell whose plasma membrane has lost integrity.

2. The method according to claim 1, wherein the first protein is selected from a 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.

3. The method according to claim 1, wherein the first compound is selected from one or more of

a compound capable of interacting chemically/physically/biologically with the first protein;

a fluorescent compound, such as a cyanine, a rhodamine, and a conjugate thereof, preferably non-activated or de-activated compound, such as a carboxylate thereof;

an active compound, such as;

(i) a therapeutic compound, such as a medicament, (ii) a reporter compound such as a tracer, such as a radio-active tracer, a chemotherapeutic, an MRI contrast agent, a microbubble for ultrasound or opto-acoustic imaging, a nanoparticle such as a quantum dot, a biological active compound, and a molecule suitable for imaging; and

a vehicle for transport of the active compound.

4. The method according to claim 1, wherein the first compound is a reporter compound such as a compound that is amenable to detection by one or more techniques selected from a group consisting:

optical spectroscopies, optical microscopy, acoustical imaging, acoustic spectroscopies, MRI, PET, SPECT, CT, and combinations thereof.

5. The method according to claim 1, wherein the cyanine and/or rhodamine is 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 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 H, 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, preferably a carboxylate thereof, preferably HQ 4 or HQ5.

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

(e) performing one or more further measurements on the sample with a first technique suitable for detecting the first compound to provide a value or values of the first compound;

(f) analysing the value or values to determine one or more values of the one or more parameters of the cells whose plasma membrane has lost integrity of the biological sample, and;

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

7. The method according to claim 6, wherein a value or values above a threshold value indicate binding of the compound to the first cell protein.

8. The method according to claim 6, wherein the one or more parameters is the presence and/or concentration and/or location of cells whose plasma membrane has lost integrity in the population of cells.

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

10. The method of claim 1, further comprising using the method to at least one of diagnose a medical condition, monitor effectiveness of a therapy, and determine the effectiveness of a therapy.

13. A complex of the first compound and the first protein obtainable by a method according to claim 1.

14. The complex of claim 13, characterized by a ratio of an amount of first compound being bound to a given quantity of cells whose plasma membrane has lost integrity divided by an amount of first compound being bound to a same given quantity of living cells ([amount dead cell]/[amount living cell]), wherein the ratio is larger than 10, preferably larger than 10², more preferably larger than 10⁵, even more preferably larger than 107, such as larger than 10⁹.

15. A method of using a complex according to claim 13, the method comprising at least one of:

using the complex to discriminate between live cells and cells whose plasma membrane has lost integrity;

using the complex to detect radiation;

using the complex to treat cancer(s) and/or plaque(s) and/or regeneration and/or support the immune system; and

using the complex to as a medicament.

19. A dead cell comprising the first compound and the first protein obtainable by a method according to claim 1.

11. An assay kit comprising a fluid, such as a physiological fluid, a first nonactivated compound that if brought together with a first intracellular protein is able to interact chemically/physically/biologically therewith, wherein the first non-activated compound is one or more of

(a) an active compound, such as:

(i) a therapeutic compound, such as a medicament, (ii) a reporter compound such as a tracer, such as a radio-active tracer, a chemotherapeutic, an MRI contrast agent, a microbubble for ultrasound or opto-acoustic imaging, a nanoparticle such as a quantum dot, a biological active compound, and a molecule suitable for imaging;

(b) a vehicle for transport of the active compound;

(c) a means for storing, a means for transferring, and any further suitable product.

12. A compound comprising a cyanine and/or rhodamine for discriminating between live cells and cells whose plasma membrane has lost integrity, wherein the cyanine and/or rhodamine is 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 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 H, 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, preferably a carboxylate thereof, preferably HQ 4 or HQ5.

16-18. -18. (canceled)