PROTEIN TARGET COMPLEX

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 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 selectively interacts directly with an at least cell constituent, wherein the cell constituent is the at least one 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 non-activated extracellularly applied compound and the first intracellular protein in the cell whose plasma membrane has lost integrity, thereby forming a complex of the first intracellular protein and non-activated extracellularly applied compound, and (d) performing a measurement to determine an amount of targeted complexes formed between the non-activated extracellularly applied compound and 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 a 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. A method according to claim 1, wherein the first intracellular protein in claim 1, step (b) is selected from a group consisting of 40 kDa proteins, 100 kDa proteins, enolase, and lyase.

3. A method according to claim 1, wherein the first compound is selected from one or more of an active compound, selected from the group consisting of (i) a therapeutic compound, (ii) a reporter compound a chemotherapeutic, an MRI contrast agent, a microbubble for ultrasound or opto-acoustic imaging, a nanoparticle, a biological active compound, and a molecule suitable for imaging; and a vehicle for transport of the active compound.

4. A method according to claim 1, wherein the first compound is a reporter compound that is amenable to detection by one or more techniques selected from a group consisting of: optical spectroscopies, optical microscopy, acoustical imaging, acoustic spectroscopies, MRI, PET, SPECT, CT, and combinations thereof.

5. A method according to claim 1, wherein the method further comprises: (e) performing one or more further measurements on the sample of the population of cells to determine an amount of the targeted complexes formed between the compound and first intracellular protein in the cell whose plasma membrane has lost integrity with a first technique suitable for detecting the targeted complexes formed to provide a value or values of the targeting, (f) 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; (g) 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 the targeted complexes formed, as a function of time.

6. A method according to claim 5, wherein a value or values above a threshold value indicate targeting of the compound to the intracellular cell protein.

7. A method according to claim 5, 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.

8. A method of claim 1 further comprising providing an assay for screening drugs for therapy such as cancer therapy, wherein the amount of targeted complexes formed between the non-activated extracellularly applied compound and the first intracellular protein in the cell of step (d) is used to screen the effectiveness of said drugs for therapy.

9. A method of claim 1 further comprising performing a diagnostic method, or to monitor and/or determine the effectiveness of a therapy. wherein the an amount of targeted complexes formed between the non-activated extracellularly applied compound and the first intracellular protein in the cell of step (d) is used to screen the effectiveness of said diagnostic method or to monitor the effect of said therapy.

10. A complex of the non-activated extracellularly applied compound bound to the targeted first intracellular protein obtainable by a method according to claim 1.

11. A complex according to claim 10, having a ratio of an amount of compound contained within a given quantity of cells whose plasma membrane has lost integrity divided by an amount of the non-activated extracellularly applied 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.

12. A complex according to claim 10 for detecting radiation.

13. A complex according to claim 10 applied in the production of a medicament.

14. A complex according to claim 10 applied in treatment of cancer(s) or plaque(s) or regeneration or supporting the immune system.

Description

SUMMARY OF FIGURES

[0054] FIGS. 1a-1c show generic structures of three main sub-families of the present cyanine. FIGS. 1d-1e show examples of group R.sub.9 in FIGS. 1b-c. Cyanine is a non-systematic name of a synthetic dye family belonging to polymethine group. Referring to the central carbon chain in FIG. 1; 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 R.sub.5, R.sub.6, R.sub.7, and R.sub.8, may be present, R.sub.5, R.sub.6, R.sub.7, and R.sub.8, 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 R.sub.1, R.sub.2, and/or substituents, R.sub.1, R.sub.25, 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 R.sub.9, R.sub.9 being selected from H, AA and BB, wherein R.sub.10 is selected from, H, SO.sub.3H, Cl, NCO(CH2).sub.q-Y.sub.3 (q=1-6), (CH2).sub.rY.sub.4 (r=1-6), Y.sub.3 and Y.sub.4 are each independently one of H, COOH, SO.sub.3H, CN, [0055] wherein the nitrogen atoms (N) may comprise further functional N-side groups R.sub.3, R.sub.4, wherein R.sub.3, R.sub.4 are preferably selected each individually from (CH.sub.2).sub.mY, wherein Y is selected each individually from a carboxylic acid having 1-4 carbon atoms, a sulfonate group, CN, CC, and CC, 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, [0056] 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 CC bonds.

[0057] The term cyanine refers to any compound whose core-structure is that of sub-family I, II or III. The integer in names of cyanines such as Cy 3, Cy 5, Cy 7 etc. refers to the number of carbon atoms in the chain L. In an exemplary embodiment, the cyanine belongs to one of these families.

[0058] FIG. 2 shows a generic structure of a Rhodamine; R1 to R12 can be hydrogen or a functional group, examples of suitable functional groups include sulfonic acid groups, carboxylic acid groups, sulfonamides, alcohols, amines, esters, ethers, thiols, thio esters and combinations thereof. The term Rhodamine refers to any compound whose core-structure is that shown in FIG. 2.

[0059] FIG. 3(a)-(j) gives the structures of compounds referred to throughout the application.

[0060] FIG. 4a-4f and 5a-5e give further examples of first compounds suitable for the method of the invention. The structures (a)-(j) of FIG. 3 are preferred examples, HQ4 and HQ5 are particularly suitable for the method of the invention. Specifically: FIG. 4 shows structures of Cy3, Cy5, Cy7, Cy3.5, Cy5.5 and Cy3b (RSO.sub.3.sup., H or alkyl; *=OH); FIG. 5 shows a series of Dy dyes.

[0061] FIGS. 6a-6b show two preferred examples of cyanines. FIGS. 7a-7b, 8a-8d, FIGS. 9-11, FIGS. 12a-12b, FIGS. 13a-13b, FIGS. 14-17, and FIGS. 18a-18c show experimental results.

EXAMPLE

[0062] Selective Binding of Cyanine Dyes to Dead Cells In Vitro and In Vivo.

Introduction

[0063] It has been observed that cyanine dyes of the invention, compounds that are used as controls to parent (bio) molecule coupled dyes to determine potential effects or retention of the dye itself, showed high and selective affinity for dead cells. These carboxylate forms of cyanine dyes, in contrast to their maleimide and NHS ester counterparts do NOT contain reactive groups and cannot be used for labeling (bio) molecules.

[0064] It is important to mention that amine reactive dyes cannot be used in vivo because these will immediately and non-selectively covalently bind to all serumand other proteins in the body that contain a free NH.sub.2 (amine) group.

[0065] Using HQ5 carboxylate as lead compound, binding specificity to dead cells in vitro in our newly developed dry ice assay and by FACS analysis has been determined. Moreover, using confocal microscopy and specific markers of earlyand late stage cell death it has been established at which phase cyanine dyes bind to dead/dying cells. In addition, the mechanism and the intracellular target proteins to which these cyanine carboxylates bind to in dead cells has been identified.

[0066] In vivo, using mice bearing a cryo lesion and matrigel, containing dead cells, it has been shown that HQ5 extravasates through intact vessels and does not merely acts as a blood pool agent. In relation to this it is important to mention the paper of the group of C. Keller where they describe the cyanine dye IR-820 to image injured tissues. However, they show that IR-820 is a blood pool agent that just simply leaks out of blood vessels after disruption of the vessels. (Prajapati S I, Martinez C O, Bahadur A N, Wu I Q, Zheng W, Lechleiter J D, McManus L M, Chisholm G B, Michalek J E, Shireman P K, Keller C. Near-infrared imaging of injured tissue in living subjects using IR-820. Mol Imaging. 2009 January-February; 8(1):45-54.)

[0067] Finally, using mice transplanted with the fast growing 4T1-luc2 mouse breast tumor, we showed that HQ5 specifically labeled necrotic areas in the tumors. These necrotic areas arise spontaneously during tumor development due to insufficient blood supply as a result of the fast growth rate.

Method

Dry Ice Assay

[0068] 4T1-Luc cells were seeded onto individual wells from a 12-well cell culture plate and allowed to grow to confluence in RPMI media supplemented with 10% fetal bovine serum. 4T1-luc2 mouse breast cancer cells were used as the adherence of these cells to the bottom of the culture well remains strong after dry-ice treatment. To initiate cryo-induced cell death, media was discarded and dry-ice was applied to the bottom center of each individual well for 15 sec. Subsequently, fresh medium with different concentrations of the first compound of the invention were added and cells were incubated at 37 C. for 15 min. After incubation, the media were discarded and the cells were washed twice with serum free medium and 0.5 ml fresh RPMI was added to each well. Cells were then visualized using a Licor Odyssey equipped with 700 and 800 nm diode laser. Luciferin (1.25 mg/kg) was also added to each well and fluorescence and bioluminescence images were acquired using an IVIS Spectrum (fluorescence: excitation filter: 710 nm, emission filter: 820 nm, exposure time: 90 s, bin: 8, f/stop: 2, field of view: 12.9 cm) (bioluminescence: open filter, exposure time: 30 s, bin: 8, f/stop: 1, field of view: 12.9 cm). Some culture wells were also stained for 15 min with Trypan Blue (0.2% in culture medium) to confirm cell death.

Results

[0069] With reference to FIGS. 6 to 17.

[0070] FIGS. 6a-6b shows chemical structures of cyanine dyes HQ5 and CW800 which were used in the experiments of the example.

[0071] FIGS. 7a-7b shows binding of cyanine dyes HQ5 and CW800 to dead cells or uptake by living cells using the dry-ice assay.

[0072] The carboxylate cyanine dyes HQ5 and CW800 specifically and dose-dependently bind to dead cells in the center of the wells.

[0073] FIGS. 8a-8d shows FACS analysis of living and dead Jurkat cells labelled with HQ5. FACS analysis of living and Staurosporine treated Jurkat cells incubated with HQQ shows that HQ5 specifically labels dead and not living cells.

[0074] FIG. 9 is a bar graph representation of the FACS data of FIG. 8. Uptake of HQ5 by dead cells is doseand time dependent.

[0075] FIG. 10 shows confocal microscopic pictures of Gambogic acid treated 4T1 cells stained with HQ5 or AnnexinV. Gambogic acid kills cells leading to membrane disruption. It is shown that HQ5 stains the cytoplasm of the cells and not the cell nuclei of late apoptotic or necrotic 4T1 cells. Annexin-V-FITC specifically binds to phosphatidyl serine (PS) expressed on the outside of the cell membrane of apoptotic and necrotic cells.

[0076] FIG. 11 shows confocal microscopic pictures of Gambogic acid treated 4T1 cells stained with HQ5, Annexin V and EtD. Both HQ5 and EtD can not cross membranes of living cells. However, when the cell membrane is disrupted, for instance as in late stage apoptotic or necrotic cells, HQ5 stains the cytoplasm and EtD binds to DNA in the cell nucleus. We show that HQ5 and EtD staining co-localizes, indicating late apoptotic or necrotic cells. Annexin V, however, can next to late apoptotic and necrotic cells also stain cells in an early stage of apoptosis. For this Annexin V stains a larger number of cells than both HQ5 and EtD.

[0077] FIGS. 12a-12b relates to identification of intracellular proteins that bind HQ5. In order to try to identify the protein to which the HQ-dyes bind Lysate is (after pre-treatment) incubated with HQ5 and CW800. Exact protocol and technique not specified. HQ5 and CW800 share some but also have different proteins they bind with. A specific HQ5-binding protein has a molecular weight of approx. 40 kD.

[0078] FIGS. 13a-13b shows homogenates of dead and living cells treated with HQ5 (1 and 5 M) and subsequently run on SDS PAGE. It is demonstrated that HQ5 specifically binds to certain intracellular proteins in dead cells whereas it does not bind to living cells. One of the prominent bands that are stained both by HQ5 and CW800 is one of 40 kD. This band was cut out of the gel and analysed by mass spectrometry. This analysis revealed that this 40 K protein band contains proteins that are most abundant in cells, including actin and tubulin.

[0079] FIG. 14 shows different intracellular 40 proteins and BSA run on SDS-PAGE and stained with HQ5.

[0080] FIG. 15 shows saturation curves of HQ5 to dead 4T1 cells, killed by liquid N.sub.2. It is shown that up to a HQ5 concentration of 200 M, no saturation is reached.

[0081] FIG. 16 demonstrates that HQ5 does not indicate dead tissue by acting as a blood pool agent. Specifically, FIG. 16 is an image of a HQ5 injected mouse containing a brain cryo lesion and subcutaneous injected matrigel and matrigel containing dead 4T1 cells. At the site of brain cryo lesion blood vessels are massively destructed whereas ate the sites of matrigel injection the blood vessels are fully intact. The fact that dead cells in the matrigel stain with HQ5 suggests that this dye can extravasate through intact vessels and does not merely act as a blood pool agent.

[0082] FIG. 17 shows CW800-PLGA nano-particle targeting tumors after photodynamic therapy. Nude mouse bearing two 4T1 breast cancer tumors of which one (upper) was treated with photodynamic therapy. The mouse was injected with nano-particles and imaged 3, 6 and 24 hours after probe injection.

[0083] FIGS. 18a-18c is a histological examination of development of spontaneous necrosis in 4T1 tumors over time. In (a) 4T1 tumor after one week stained with HQ5 and TUNEL is shown. TUNEL staining is specific for dead cells. There is no necrosis. In (b) 4T1 tumor after 2 weeks stained with HQ5 and TUNEL is shown. Some signs of the start of Necrosis. In (c) 4T1 tumor at a later time point, e.g. 24 h, stained with HQ5 and TUNEL is shown. Clear necrosis in the center of the tumor as indicated by both HQ5 staining and TUNEL staining. 4T1 tumors spontaneously develop necrotic cores in the center of the tumor due to uncontrolled rapid growth. Necrosis can be identified by HQ5 which nicely co-localize with TUNEL staining.