RADIOLABELED BETA-GALACTOSIDASE SUBSTRATE FOR PET IMAGING OF SENESCENCE

Abstract

The present invention relates to novel compounds useful for visualizing cell senescence in vitro and in vivo, the preparation of said compounds and their use. In particular, the present invention pertains to novel hexose and particularly galactose derivatives which are useful as senescence tracers in vitro and in vivo.

Claims

1. A compound of the formula:
G-S-L, or a salt thereof wherein G is ##STR00018## R is H, substituted or unsubstituted C.sub.1 to C.sub.5 alkyl, substituted or unsubstituted C.sub.1 to C.sub.10 cycloalkyl, or substituted or unsubstituted C.sub.1 to C.sub.10 heterocycloalkyl, * represents the binding site between G and S, S is ##STR00019## wherein X is independently H, halogen, methyl halogen, OH or SH, wherein Y is independently C, S, N, or O, with the proviso that at least 3 C-atoms are present, wherein # represents the binding site between S and L, and wherein L is ##STR00020## wherein R is CH.sub.2, NH, S, or O, wherein n is 0 or 1, wherein R is substituted or unsubstituted C.sub.1 to C.sub.5 alkyl, wherein Z is a radioactive detectable label, a radioactive therapeutic residue, a chelator coordinating a radioactive detectable label or a chelator coordinating a radioactive therapeutic residue.

2. The compound according to claim 1, wherein G is ##STR00021##

3. The compound according to claim 1, wherein R is H or CH.sub.3, X is independently H or methyl halide, Y is independently C or N with the proviso that at least 5 C-atoms are present, R is NH, n is 1, and R is a linear ether having 2 to 5 C-atoms.

4. The compound according to claim 1, wherein R is H, S is ##STR00022## ##STR00023##

5. The compound according to claim 1, wherein the compound is ##STR00024##

6. The compound according to claim 1, wherein the radioactive detectable label is .sup.11C, .sup.40K, .sup.13N, .sup.15O, .sup.18F, .sup.75Br, .sup.76Br, .sup.82Rb, .sup.68Ga, .sup.64Cu, .sup.62Cu, .sup.123I, .sup.124I, .sup.125I, .sup.131I, .sup.210At, .sup.211At and .sup.111In.

7. The compound according to claim 1, wherein the radioactive therapeutic residue is .sup.32P, .sup.60Co, .sup.64Cu, .sup.89Sr, .sup.90Y, .sup.177Lu, .sup.186Re and .sup.153Sm.

8. A method for detecting senescent cells, comprising the steps of contacting cells suspected to comprise senescent cells with the compound of claim 1 or the salt thereof, wherein Z is the radioactive detectable label or the chelator coordinating the radioactive detectable label, thereby enriching said compound in senescent cells, if senescent cells are present among said cells, and detecting the senescent cells having the compound enriched therein.

9. A method for detecting tumor cells, comprising: contacting cells suspected of comprising tumor cells with the compound of claim 1 of the salt thereof, wherein Z is a radioactive detectable label or the chelator coordinating the radioactive detectable label, thereby enriching the compound in tumor cells, if tumor cells are present among said cells, and detecting tumor cells having the compound enriched therein.

10. A method for determining the efficiency of cancer treatment, comprising: contacting cells with the compound of claim 1 or the salt thereof, wherein Z is the radioactive detectable label or the chelator coordinating the radioactive detectable label.

11. A method for determining the efficiency of cancer treatment, the method comprising: (i) contacting cells of a subject undergoing a cancer treatment with the compound of claim 1 or the salt thereof, wherein Z is the radioactive detectable label or the chelator coordinating the radioactive detectable label, thereby enriching said compound in cancer cells, if present in said cells, and thereby determining a first extent to which cancer cells have enriched therein the compound; (ii) repeating step (i) after a certain time period during the cancer treatment to determine a second extent to which cancer cells have enriched therein the compound; and (iii) comparing the first and second extent, thereby determining the efficiency of cancer treatment.

12. A method for treating cancer in a subject in need of a treatment, comprising: administering to the subject a therapeutically effective amount of the compound of claim 1 or the salt thereof, wherein Z is the radioactive therapeutic residue or the chelator coordinating the radioactive therapeutic residue.

13. The method of claim 12, wherein the compound is administered to the subject in a pharmaceutical composition comprising the compound and an inert, non-toxic, pharmaceutically suitable exci

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0054] In the Figures

[0055] FIG. 1 shows a motive explaining functioning of a radio-labelled hexose substrate;

[0056] FIGS. 2A-2C show the tracer 9 as a substrate of commercially available betagalactosidase;

[0057] FIGS. 3A-3B show stability of the tracer 9 under in-vivo conditions;

[0058] FIGS. 4A-4B show in vitro validation of tracer in HCT116 and Hras cells;

[0059] FIG. 5 shows uptake of tracer 9 in senescent versus control HCT116 tumors;

[0060] FIG. 6 shows tracer 9 uptake in senescent versus control Hras tumors; and

[0061] FIGS. 7A-7B show immunohistochemical staining from (a) HCT116 and (b) Hras driven tumor models.

EMBODIMENTS

[0062] According to a preferred embodiment of the present invention G is

##STR00006##

R is as indicated above. Preferably R is H, thereby defining a beta-D-galactose residue, in particular a beta-D-galactopyranoside, which is the best possible sugar moiety for cleavage by beta-galactosidase.

[0063] According to another preferred embodiment of the present invention R is H or CH.sub.3, X is independently selected from H or methyl halide, Y is independently selected from C and N with the proviso that at least 5 C-atoms are present, R is NH, n is 1, and R is a linear ether having 2 to 5 C-atoms.

[0064] According to still another preferred embodiment of the present invention R is H, S is selected from the group consisting of

##STR00007## ##STR00008##

##STR00009##

[0065] In said compounds, L may be as indiacted above.

##STR00010##

[0066] In case L is R is preferably selected from the group consisting of CH.sub.2OCH.sub.2, C.sub.2H.sub.4OCH.sub.2, CH.sub.2OCH.sub.2OCH.sub.2, CH.sub.2OCH.sub.2OCH.sub.2, C.sub.2H.sub.4OCH.sub.2OCH.sub.2, CH.sub.2OC.sub.2H.sub.4OCH.sub.2, C.sub.2H.sub.4OC.sub.2H.sub.4OCH.sub.2, C.sub.2H.sub.4OCH.sub.2OC.sub.2H.sub.4, CH.sub.2OCH.sub.2OCH.sub.2OCH.sub.2, C.sub.2H.sub.4OCH.sub.2OCH.sub.2OCH.sub.2, and CH.sub.2OC.sub.2H.sub.4OCH.sub.2OCH.sub.2. R is CH.sub.2, NH or O, preferably NH. Even more preferably, Z is .sup.18F. In case R is CH.sub.2, it is preferred that n is 0 and R is an unsubstituted Ci to C.sub.5 alkyl. More preferably, Z is .sup.18F.

[0067] In case L is

##STR00011##

Z is preferably .sup.18F.

[0068] Particular preferred compounds of the present invention R is H, S is selected from the group consisting of

##STR00012##

[0069] In said compounds, G is

##STR00013##

wherein R is H.

L is

[0070] ##STR00014##

More preferably, Z is .sup.18F.

[0071] According to a preferred embodiment of the present invention the compound is

##STR00015##

[0072] The second structure employing a spacer molecule is particularly suitably for comparatively large radioactive detectable labels, such as .sup.123I, .sup.124I, .sup.125I, .sup.131I, .sup.210At, .sup.211At and .sup.111In, and large radioactive therapeutic residues, such as .sup.89Sr, .sup.186Re and .sup.153Sm. It is however, preferred that Z is .sup.18F.

[0073] According to another preferred embodiment of the present invention the radioactive detectable label is selected from the group consisting of .sup.11C, .sup.40K, .sup.13N, .sup.15O, .sup.18F, .sup.75Br, .sup.76Br, .sup.82Rb, .sup.68Ga, .sup.64Cu, .sup.62Cu, .sup.123I, .sup.124I, .sup.125I, .sup.131I, .sup.210At, .sup.211At and .sup.111In, preferably .sup.11C, .sup.18F, .sup.68Ga, .sup.64Cu, and .sup.124I, more preferably .sup.18F.

[0074] According to still another preferred embodiment of the present invention the radioactive therapeutic residue is selected from the group consisting of .sup.32P, .sup.60Co, .sup.64Cu, .sup.89Sr, .sup.90Y, .sup.177Lu, .sup.186Re and .sup.153Sm.

[0075] According to a preferred embodiment of the present invention the present compound is for use in surgery. The present compound is preferably employed in combination with at least one inert, non-toxic, pharmaceutically suitable excipient.

[0076] According to a preferred embodiment of the present invention the present compound is for use as a medicament. The present compound is preferably employed in combination with at least one inert, non-toxic, pharmaceutically suitable excipient.

[0077] According to another preferred embodiment of the present invention the present compound for use in a method for detecting cell senescence.

[0078] According to another preferred embodiment of the present invention the present compound is for use in a method of determining the efficiency of cancer treatment.

[0079] The cancer to be diagnosed and/or treated by the present compounds may be selected from prostate carcinoma, colorectal carcinoma, breast cancer, lung tumors, tumors of the male or female genitourinary system, malign melanoma, cervix and throat tumor/cervical tumor, malign lymphoma, neoplasia of the hematopoietic system and musculoskeletal tumors.

[0080] According to still another preferred embodiment of the present invention the method for detecting cell senescence comprises contacting cells with the present compound.

[0081] According to still another preferred embodiment of the present invention the method for determining the efficiency of cancer treatment comprises contacting cells with the present compound.

[0082] According to still another preferred embodiment of the present invention the method is performed in vivo.

[0083] According to still another preferred embodiment of the present invention the method is performed in vitro.

[0084] The above methods can be performed both in vivo, e.g. in a human patient for monitoring the efficiency of cancer treatment, or in vitro, e.g. for screening for new medicaments.

[0085] The present compound will preferably be administered parenterally. For this administration route, the present compounds may be administered in suitable administration forms. Parenteral administration can take place with avoiding of an absorption step (e.g. intravenous, intraarterial, intracardiac, intraspinal or intralumbar) or with inclusion of an absorption step (e.g. intramuscular, subcutaneous, intracutaneous, percutaneous or intraperitoneal). Administration forms suitable for parenteral administration are, inter alia, preparations for injection and infusion in the form of solutions, suspensions, emulsions, lyophilisates or sterile powders.

[0086] The present compounds may be converted into the stated administration forms. This can take place in a manner known per se by mixing with inert, non-toxic, pharmaceutically acceptable excipients. These excipients include inter alia carriers (for example microcrystalline cellulose, lactose, mannitol), solvents (e.g. liquid polyethylene glycols), emulsifiers and dispersants or wetting agents (for example sodium dodecyl sulfate, polyoxysorbitan oleate), binders (for example polyvinylpyrrolidone), synthetic and natural polymers (for example albumin), stabilizers (e.g. antioxidants such as, for example, ascorbic acid), colors (e.g. inorganic pigments such as, for example, iron oxides) and taste and/or odor corrigents.

[0087] The principle underlying the present invention is generally shown in FIG. 1. A radio-labelled glycosidase substrate, i.e. a compound of the present invention, is converted by a glycosidase, in particular beta-galactosidase, to the corresponding sugar and the radio-labelled alcohol. The present compound is shown with residue W indicative of the moieties forming a hexose derivative, such as a galactose derivative, preferably beta-D-galactose. The glycosidase, particularly the beta-galactosidase, is overexpressed and accumulated in senescent cells. The radiolabelled alcohol in turn is accumulated in acidic lysosomes and may be detected by a radiology device, such as a PET/CT device.

[0088] The compounds of the present invention may be generally prepared by the route indicated in reaction scheme I.

##STR00016## ##STR00017##

[0089] In the upper row of reaction scheme I, generation of compound 5 is shown. Compound 5 does not exhibit a radioactive label, i.e. F is .sup.19F. Reaction I is conducted with HBF.sub.4, NaNO.sub.2 at a temperature of 0 C. The yield of compound 2 is 63% w/w. Reaction II is conducted with Ag.sub.2CO.sub.3 and at a temperature of 0 C. The yield of compound 4 is between 40 to 60% w/w. Reaction III is conducted with NaOMe and MeOH at a temperature in the range from 20 to 25 C. followed by reaction IV in presence of amberlite IR.sub.120.

[0090] In the lower row of reaction scheme I, generation of compound 9 (tracer 9), a compound according to the present invention, is shown. Reaction II is conducted with Ag.sub.2CO.sub.3 at a temperature of 0 C. Reaction VI is conducted with Ag.sub.2CO.sub.3 at a temperature of 0 C. Reaction V is conducted with .sup.18F.sup. and DMSO at a temperature of 150 C. for 5 min. Reaction VI is conducted with NaOMe/MeOH, or MeOH, Et.sub.3N, H.sub.2O (10:1:1)

[0091] The compounds of the present invention show a valuable range of pharmalogical effects which could not have been predicted. They are capable of marking senescent cells in vitro and in vivo. Particularly, in vivo marking is made to such an extent that senescent cells may be unambiguously identified in course of a surgical procedure which in turn allows targeted elimination of such cells, particularly cancer cells.

[0092] It is to be understood that the above description is intended to be illustrative only and not restrictive. Many embodiments will be apparent to those skilled in the art upon reviewing the above description. By way of example, the invention has been described preliminarily with reference to synthesis as well as diagnosis of tracer 9. It should be clear that all kinds of suitable detectable labels and therapeutic residues may be synthesized and attached to the present compounds. The scope of the invention should, therefore, be determined not with reference to the above description, but should instead be determined with reference to the appended claims, along with the full scope of equivalents to which such claims are entitled.

[0093] The percentage data in the following tests and examples are, unless indicated otherwise, percentages by weight; parts are parts by weight. Solvent ratios, dilution ratios and concentration data of liquid/liquid solutions are based in each case on volume. The statement w/v means weight/volume. Thus, for example, 10% w/v means: 100 ml of solution or suspension contain 10 g of substance.

EXAMPLES

[0094] General

[0095] Optimized radio synthesis utilizes a TRACERIab FX N Pro (GE). .sup.18F is produced as hydrofluoric acid (HF) using a PETtrace cyclotron (GE). .sup.18F labelling relied on the nucleophilic substitution of an aromatic nitro group. The synthetic route is described in reaction scheme I.

[0096] The following subcutaneous animal models are used for tracer evaluation.

[0097] A xenograft model of colorectal cancer cell line (HCT116) with therapy induced senescence, doxorubicin is used as a chemotherapeutic. Senescence is induced in HCT116 xenograft tumors by intravenous (i.v.) administration of doxorubicin (10 mg/kg). Through tail vain of the mice, 5 days later dynamic PET/MRT scans (1 h) with compound 9 are performed.

[0098] As the second model, transgenic liver progenitor cell line (Hras) was used for subcutaneous allografts in nude mice. The cell line expresses Hras.sup.G12v and p53shRNA under doxycycline (doxy) conditions. After the removal of doxycycline, the p53 mRNA can be restored and translated to p53 protein. The high amount of p53 triggers senescence. The animals bearing Hras tumors received doxy-water (0.2 mg/ml) for tumor development and 14 days, after removal of doxy-water, the PET/MRT scans are performed with compound 9.

Example 1

[0099] Example 1 discloses synthesis of compounds according to reaction scheme I.

[0100] Synthesis of Compound 4

[0101] (2R,3S, 4S, 5R,6S)-2-(acetoxymethyl)-6-((2-fluoropyridin-3-yl)oxy)tetrahydro-2H-pyran-3,4,5-triyltriacetate, 4: A solution comprising 2,3,4,6-TetraOacetyl--D-galactopyranosyl bromide 3 (1.0 g, 2.4 mmol), Ag.sub.2CO.sub.3 (0.74 g, 2.7 mmol), MS-4 (5 g) and 2-fluoro-pyridin-3-ol 5 (0.33 g, 2.9 mmol) in anhydrous DCM (15 mL) is stirred overnight, in the dark, at room temperature, under an argon atmosphere. TLC analysis indicated the complete consumption of the starting bromide and the formation of a new nonpolar product. The solution is filtered through a celite, which was rinsed with DCM (310 mL). After removing the solvent under reduced pressure, the crude residue is purified directly using silica gel column chromatography, using an increasing gradient of EtOAc in PE. The product afforded is a colorless solid (0.51 g, 51%).

[0102] TLC: Rf=0.5 (EtOAc: PE=1:1); .sup.1H NMR (600 MHz, Chloroform-d) 7.94 (d, J=4.6 Hz, 1H, ArH), 7.58 (t, J=7.8 Hz, 1H, ArH), 7.13 (dd, J=7.8, 4.8 Hz, 1H, ArH), 5.50 (dd, J=10.5, 7.9 Hz, 1H), 5.45 (d, J=3.4 Hz, 1H), 5.10 (dd, J=10.5, 3.4 Hz, 1H), 4.96 (d, J=7.9 Hz, 1H), 4.22 (dd, J=11.4, 6.8 Hz, 1H), 4.15 (dd, J=11.4, 6.3 Hz, 1H), 4.01 (td, J=6.8, 1.1 Hz, 1H), 2.19 (s, 3H, OAc), 2.11 (s, 3H, OAc), 2.04 (s, 3H, OAc), 2.02 (s, 3H, OAc); .sup.13C NMR (151 MHz, CDCl.sub.3) 170.26 (CO.sub.quart), 170.13 (CO.sub.quart), 170.04 (CO.sub.quart), 169.44 (CO.sub.quart), 154.75 (d, J=239.9 Hz), 141.59 (d, J=13.4 Hz, ArC), 139.49 (d, J=25.5 Hz, ArC), 130.22 (d, J=3.5 Hz, ArC), 121.85 (d, J=4.3 Hz, ArC), 121.23 (CH), 101.12 (CH), 71.38 (CH), 70.5 (CH), 68.29 (CH), 66.70 (CH), 61.17 (CH.sub.2), 21.03 (OAc), 20.63 (OAc), 20.61 (OAc), 20.55 (OAc).

[0103] Synthesis of Compound 5

[0104] (2S,3R,4S,5R,6R)-2-((2-fluoropyridin-3-yl)oxy)-6-(hydroxyl-methyl)tetrahydro-2H-pyran-3,4,5-triol, 5: Catalytic sodium methoxide is added to a solution of 4 (0.5 g, 1.1 mmol) in dry methanol (5 mL). The solution is stirred at room temperature until TLC analysis revealed that the hydrolysis of the acetylated sugar is complete. Acetic acid (0.2 mL) is added, after which the solvents are removed under reduced pressure. The remaining solid was of high purity, but subsequent recrystallization from methanol afforded 6 as a highly pure crystalline solid (225 mg, 73%).

[0105] TLC: R.sub.f=0.25 (EtOAc: MeOH=9: 1); .sup.1H NMR (600 MHz, Deuterium Oxide) 7.79 (dd, J=8.1, 5.0 Hz, 1H, ArH), 7.72 (t, J=8.9, 8.1 Hz, 1H, ArH), 7.26 (dd, J=8.0, 5.0 Hz, 1H, ArH), 5.06 (d, J=7.8 Hz, 1H), 3.95 (dd, J=3.4, 1.8 Hz, 1H), 3.84-3.78 (m, 2H), 3.75-3.70 (m, 3H), 1.85 (dd, J=2.3, 1.1 Hz, 1H); .sup.130 NMR (151 MHz, Deuterium Oxide) 153.70 (d, J=238.5 Hz, CF), 141.49, 139.72 (d, J=23.9 Hz, ArC), 139.29 (d, J=11.6 Hz, ArC), 128.11 (d, J=3.7 Hz, ArC), 122.75 (d, J=4.2 Hz, ArC), 101.24 (CH), 75.68 (CH), 72.41 (CH), 70.31 (CH), 68.36 (CH), 60.68 (CH.sub.2); HRMS (ESI): [M+Na].sup.+.sub.(theor.)=298,06974, measured=298,06998

[0106] Synthesis of Compound 7

[0107] (2R,3S,4S,5R,6S)-2-(acetoxymethyl)-6-((2-nitropyridin-3-yl)oxy)tetrahydro-2H-pyran-3,4,5-triyltriacetate, 7: A solution comprising 2,3,4,6-TetraOacetyl--D-galactopyranosyl bromide 3 (1.0 g, 2.4 mmol), Ag.sub.2CO.sub.3 (1.3 g, 4.8 mmol), MS-4 (5 g) and 2-nitro-pyridin-3-ol 6 (1.4 g, 9.7 mmol) in anhydrous DCM (10 mL) is stirred over night, in the dark, at room temperature, under an argon atmosphere. TLC analysis indicates the complete consumption of the starting bromide and the formation of a new nonpolar product. The solution is filtered through a celite, which is rinsed with DCM (310 mL). After removing the solvent under reduced pressure, the crude residue is purified directly using silica gel column chromatography, using an increasing gradient of EtOAc in PE. The product 7 afforded is a colorless solid (0.7 g, 63%).

[0108] TLC: Rf=0.65 (EtOAc: PE=4: 1); .sup.1H NMR (600 MHz, Chloroform-0 6 8.26 (dd, J=4.6, 1.4 Hz, 1H, ArH), 7.82 (dd, J=8.4, 1.4 Hz, 1H, ArH), 7.54 (dd, J=8.4, 4.6 Hz, 1H, ArH), 5.51 (dd, J=10.5, 7.9 Hz, 1H), 5.47 (dd, J=3.4, 1.2 Hz, 1H), 5.10 (dd, J=10.5, 3.4 Hz, 1H), 5.06 (d, J=7.9 Hz, 1H), 4.25 (dd, J=11.4, 6.1 Hz, 1H), 4.16 (dd, J=11.4, 6.1 Hz, 1H), 4.06 (ddd, J=6.1, 6.1, 1.3 Hz, 1H), 2.19 (s, 3H, OAc), 2.14 (s, 3H, OAc), 2.06 (s, 3H, OAc), 2.01 (s, 3H, OAc); .sup.13C NMR (151 MHz, CDCl.sub.3) 170.21 (CO.sub.quart), 170.04 (CO.sub.quart), 170.01 (CO.sub.quart), 169.32 (CO.sub.quart), 150.42 (ArC.sub.quart), 144.25 (ArC.sub.quart), 142.91 (ArC), 129.86 (ArC), 128.29 (ArC), 100.94 (CH), 71.65 (CH), 70.31 (CH), 67.64 (CH), 66.58 (CH), 61.26 (CH.sub.2), 20.62 (OAC), 20.60 (OAC), 20.56 (OAC), 20.51 (OAC); HRMS (ESI): [M+Na].sup.+.sub.(theor.)=493,10649 measured=493,10686.

[0109] Synthesis of Compounds 8 and 9

[0110] Radiolabeling of 7 proceeds readily in DMSO at 150 C. Azeotropically dried [K.2.2.2]+.sup.18F.sup. fluoride complex is used, producing the acetylated .sup.18F-labelled intermediate, which is purified using semi-preperative HPLC. The tracer was concentrated by trapping onto a 018 SPE cartridge, deprotected with NaOH (185 gt, 2.0 M) to produce tracer 9 and eluted with 10% ethanol in water (3 mL). It is appropriately formulated with HCl (195 gt, 2.0 M), NaHCO.sub.3 (500 L, 1.0 M) and water (5 mL) to ensure subisotonic sodium concentration (0.1 M) and a final pH (7.5) that is biologically tolerated. The tracer is produced with a radiochemical purity of >98%, the decay corrected yield was 18.6+/2.5% (n=10) with a molar radioactivity of 18.83.5 GBq* mole.sup.1 (n=5).

Example 2

[0111] To assess whether tracer 9 is a substrate of beta-galactosidase, it is incubated at 30 C. for 30 minutes in citrate buffer of pH 5.5 containing commercially available betagalactosidase (Sigma). The reaction is stopped by adding 2 volume equivalents of acetonitrile, followed by 5 minutes of centrifugation. The supernatant is examined directly using radio-HPLC. The reaction produces the radioactive metabolite with the same retention time as the non-radioactive 2-fluoropyridinol. The expected radioactive metabolite is compared with the authentic non-radioactive standard (FIG. 2). In particular, compound 10 is radioactive 2-fluoropyridinol (FIG. 2a), compound 9 is tracer 9 (FIG. 2b), and compounds 1 and 2 are other compounds of reaction scheme I (FIG. 2c). Compound 11 is galactose.

[0112] The tracer 9 is incubated at 37 C. in mouse serum to assess its stability. After 1 hour, 2 volume equivalents of acetonitrile are added and the mixture is centrifuged for 5 minutes. The supernatant is examined directly using radio-HPLC. Serum stability of tracer 9 after 1 h (FIG. 3a) vs tracer 9 (FIG. 3b) is derivable from FIG. 3. The experiment reveals a few minor radioactive metabolites, but tracer's integrity remains largely intact.

[0113] In-vitro evaluation of compound 9 is shown in FIG. 4. HCT116 (FIG. 4a) and Hras cells (FIG. 4b) are treated with 100-200 pCi of tracer and incubated for 50 minutes. The cells are measured in a gamma-counter, with the activity normalized to the number of cells used. In both cases, the senescent cells show significantly higher uptake compared to the controls.

Example 3

[0114] The PET-tracer is tested in two different models in vitro. In HCT116 cells senescence is induced by overnight incubation with doxorubicin, followed by 4 days of culture under normal conditions. Senescence in a HRas driven liver progenitor cell line with a doxycycline regulatable p53-specific shRNA. After induction of senescence, both cell lines are incubated with 3.7-7.4 MBq of tracer for 50 minutes. The cells are then washed, counted and measured in a gamma counter. The activity taken up by the cells is analyzed and normalized to a million cells.

[0115] In vivo tests are performed in nude mice bearing s.c. HCT116 or HRas driven liver progenitor cells. Mice bearing HCT116 tumors are treated with doxorubicin (10 mg/Kg bodyweight) and imaged 4 days after the onset of senescence. Mice bearing Hras driven tumor are supplied with doxycycline in their drinking water, which has been later removed to induce senescence. After induction of senescence, the tracer is administered i.v. and PET scans are performed. The %ID/cc and tumor-to-muscle ratios are determined through quantitative analysis of the PET data, permitting evaluation of the tracers. In vitro senescent HCT116 cells show an uptake of 11 kBq/1 mio cells, while the uptake in non-senescent control cells is only 4 kBq/1 mio cells. In the HRas driven liver progenitor model senescent cells show an uptake of 192 kBq/1 mio cells and is significantly increased in comparison to non-senescent cells (63 kBq/1 mio cells).

[0116] The in vivo tracer uptake in senescent and non-senescent HCT116 tumors (FIG. 5) is 1.7+/0.7 (n=7) vs 1.1+/0.4 (n=5) %ID/cc respectively; the respective TMR values are 1.7 and 1.1. In particular, FIG. 5 indicates that tracer 9 shows higher uptake in senescent vs control HCT116 tumors. Both %ID/cc and TMR are higher in senescent vs control tumors. The excised tumors are subjected to ex vivo analyses. Autoradiography confirmed findings, showing higher tracer uptake in the senescent tumor section vs control. Senescent tumor slices also showed higher X-Gal staining than control sections.

[0117] In the HRas driven model (FIG. 6), the tracer uptake in senescent tumors is significantly increased to that of control tumors (1.5+/0.3 (n=16) vs 0.9+/0.3 (n=11) %ID/cc); the TMR is subject to a similar trend, with respective values of 2.1 and 1.2. FIG. 6 indicates that tracer 9 shows higher uptake in senescent vs control HRas tumors. Both %ID/cc and TMR are higher in senescent vs control tumors. The excised tumors are subjected to ex vivo analyses. Autoradiography confirmed findings, showing higher tracer uptake in the senescent tumor section vs control. Senescent tumor slices also show higher X-Gal staining than control sections. The nonsenescent tumor expresses GFP, which can be visualized with optical imaging.

[0118] Induction of senescence is confirmed by ex vivo beta-gal staining and immunohistology of Ki67, Caspase3, HP1, p53 and p16 (FIG. 7). FIG. 7 shows immunohistochemical staining from HCT116 (FIG. 7a) and HRas (FIG. 7b) driven tumor models. In both cases, immunohistochemical analysis was performed and reveals an increase of the expected senescence markers HP1 and p53. The Hras driven tumors also show high expression of ki67. The low abundance of caspase-3 in both tumor models suggests that apoptosis is not a key contributor to the tumors status and confirms assertion of senescence.