CONJUGATED AND LABELLED APELIN, PREPARATION AND USES THEREOF

Abstract

The invention relates to the field of imaging, diagnostic, internal vectorized radiotherapy and nuclear medicine. Inventors herein describe new products for use for labelling, detecting and/or imaging angiogenesis, vasculogenesis or a tissue or organ expressing the APJ receptor; for use for detecting, measuring, diagnosing, staging and/or monitoring angiogenesis, vasculogenesis, an angiogenesis- and/or vasculogenesis-related disease or disorder, and/or a disease or disorder inducing or modulating the expression of a APJ receptor in a tissue or organ; for use for preventing or treating angiogenesis, vasculogenesis, an angiogenesis- and/or vasculogenesis-related disease or disorder, and/or a disease or disorder inducing or modulating the expression of a APJ receptor in a tissue or organ; or for use for evaluating or monitoring the therapeutic effect of an angiogenic or antiangiogenic treatment or of an APJ receptor-targeted treatment. Compositions and kits comprising such products are also herein described as well as uses thereof.

Claims

1-17. (canceled)

18. Apelin conjugated to a chelator and labeled with a radioactive element wherein the Apelin amino acid sequence: a) comprises SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4; or b) is selected from the group consisting of SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12 and SEQ ID NO: 13.

19. The conjugated and labeled Apelin according to claim 18, wherein the chelator is selected from 6-amino-6 methylperhydro-1,4-diazepinetetraacetic acid (AAZTA), 1,4,7-triazacyclononane-1,4-diacetic acid (NODA), 1,4,7-triazacyclononane,1-glutaric acid-4,7 acetic acid (NODAGA), 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), 2,2′,2″-(10-(2,6-dioxotetrahydro-2H-pyran-3-yl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid (DOTAGA), 1,4,7-triazacyclononane-triacetic acid (NOTA), N,N′-Bis(2-hydroxybenzyl)-1-(4-bromoacetamidobenzyl)-1,2-ethylenediamine-N,N′-diacetic acid (HBED), N1-hydroxy-N1-(5-(4-(hydroxy(5-(3-(4-isothiocyanatophenyl)thioureido)pentyl)amino)-4-oxobutanamido)pentyl)-N4-(5-(N-hydroxyacetamido)pentyl)succinamide (DFO), triazacyclononane-phosphinate (TRAP), pentetic acid or diethylenetriaminepentaacetic acid (DTPA), bromoacetamidobenzyl(TETA),1,4,7-triazacyclononane-1,4-bis[methylene(hydroxymethyl)phosphinicacid]-7-[methylene(2-carboxyethyl)phosphinicacid])(NOPO), HBED-CC(DKFZ), 2-(4-isothiocyanotobenzyl)-1, 4, 7, 10-tetraaza-1, 4, 7, 10-tetra-(2-carbamonyl methyl)-cyclododecane (TCMC), N—[(R)-2-amino-3-(p-aminophenyl)propyl]-trans-(S,S)-cyclohexane-1,2-diamine-N,N,N′,N″,N″-pentaacetic acid (CHX-A″-DTPA) and a functional derivative thereof.

20. The conjugated and labeled Apelin according to claim 18, wherein the radioactive element is a radionuclide selected from the group consisting of gallium-68 (.sup.68Ga), gallium-67 (.sup.67Ga), lutetium-177 (.sup.177Lu), fluorine-18 (F.sup.18), yttrium-90 (.sup.90Y), bismuth-213 (.sup.213Bi), actinium-225 (.sup.225Ac), lead-212 (.sup.212Pb), indium-111 (.sup.111In), zirconium-89 (.sup.89Zr), terbium-149 (.sup.149Tb), terbium-152 (.sup.152Tb), terbium-155 (.sup.155Tb), terbium-161 (.sup.161Tb) and copper-64 (.sup.64Cu).

21. The conjugated and labeled Apelin according to claim 18 which is selected from the group consisting of [.sup.68Ga]Ga-NODAGA-Apelin, [.sup.68Ga]Ga-DOTA-Apelin, [.sup.68Ga]Ga-DOTAGA-Apelin, [.sup.68Ga]Ga-NOTA-Apelin, [.sup.68Ga]Ga-HBED-Apelin, [.sup.68Ga]Ga-DFO-Apelin, [.sup.68Ga]Ga-AAZTA-Apelin, [.sup.67Ga]Ga-NODAGA-Apelin, [.sup.67Ga]Ga-DOTA-Apelin, [.sup.67Ga]Ga-DOTAGA-Apelin, [.sup.67Ga]Ga-NOTA-Apelin, [.sup.67Ga]Ga-HBED-Apelin, [.sup.67Ga]Ga-DFO-Apelin, [.sup.67Ga]Ga-AAZTA-Apelin, Al[.sup.18F]F-NOTA-Apelin, Al[.sup.18F]F-NODA-Apelin, Al[.sup.18F]F-DOTAGA-Apelin, [.sup.64Cu]Cu-DOTA-Apelin, [.sup.64Cu]Cu-DOTAGA-Apelin, [.sup.89Zr]Zr-DOTA-Apelin, [.sup.89Zr]Zr-DOTAGA-Apelin, [.sup.177Lu]Lu-DOTA-Apelin, [.sup.177Lu]Lu-DOTAGA-Apelin, [.sup.177Lu]Lu-DKFZ-Apelin, [.sup.177Lu]Lu-AAZTA-Apelin, [.sup.225Ac]Ac-DOTA-Apelin, [Pb.sup.212]Pb-TCMC-Apelin, [.sup.213Bi]Bi-DTPA-Apelin, [.sup.90Y]Y-DTPA-Apelin, [.sup.90Y]Y-CHX-A″-DTPA-Apelin and [.sup.111In]In-DTPA-Apelin, [.sup.149Tb]Tb-DOTA-Apelin, [.sup.149Tb]Tb-DOTAGA-Apelin, [.sup.152Tb]Tb-DOTA-Apelin, [.sup.152Tb]Tb-DOTAGA-Apelin, [.sup.155Tb]Tb-DOTA-Apelin, [.sup.155Tb]Tb-DOTAGA-Apelin, [.sup.116Tb]Tb-DOTA-Apelin and [.sup.116Tb]Tb-DOTAGA-Apelin.

22. The conjugated and labeled Apelin according to claim 18 which is selected from the group consisting of [.sup.68Ga]Ga-NODAGA-Apelin, [.sup.68Ga]Ga-DOTA-Apelin, [.sup.68Ga]Ga-DOTAGA-Apelin, [.sup.68Ga]Ga-NOTA-Apelin, [.sup.68Ga]Ga-HBED-Apelin, [.sup.68Ga]Ga-DFO-Apelin, [.sup.68Ga]Ga-AAZTA-Apelin, [.sup.67Ga]Ga-NODAGA-Apelin, [.sup.67Ga]Ga-DOTA-Apelin, [.sup.67Ga]Ga-DOTAGA-Apelin, [.sup.67Ga]Ga-NOTA-Apelin, [.sup.67Ga]Ga-HBED-Apelin, [.sup.67Ga]Ga-DFO-Apelin, [.sup.67Ga]Ga-AAZTA-Apelin, Al[.sup.18F]F-NOTA-Apelin, Al[.sup.18F]F-NODA-Apelin, [.sup.111In]In-DTPA-Apelin, [Cu.sup.64]Cu-DOTA-Apelin, [.sup.64Cu]Cu-DOTAGA-Apelin, [.sup.89Zr]Zr-DOTA-Apelin, [.sup.89Zr]Zr-DOTAGA-Apelin, [.sup.152Tb]Tb-DOTA-Apelin, [.sup.152Tb]Tb-DOTAGA-Apelin, [.sup.155Tb]Tb-DOTA-Apelin and [.sup.55Tb]Tb-DOTAGA-Apelin.

23. The conjugated and labeled Apelin according to claim 18 which is selected from the group consisting of [.sup.177Lu]Lu-DOTA-Apelin, [.sup.177Lu]Lu-DOTAGA-Apelin, [.sup.177Lu]Lu-DKFZ-Apelin, [.sup.177Lu]Lu-AAZTA-Apelin, [.sup.225Ac]Ac-DOTA-Apelin, [Pb.sup.212]Pb-TCMC-Apelin, [.sup.213Bi]Bi-DTPA-Apelin, [.sup.90Y]Y-DTPA-Apelin, [.sup.90Y]Y-CHX-A″-DTPA-Apelin, [.sup.149Tb]Tb-DOTA-Apelin, [.sup.149Tb]Tb-DOTAGA-Apelin, [.sup.161Tb]Tb-DOTA-Apelin, and [.sup.161Tb]Tb-DOTAGA-Apelin.

24. A method of labelling or imaging angiogenesis or vasculogenesis in a subject, or of labelling or imaging in vivo or ex vivo a tissue or organ expressing the APJ receptor, comprising administering the subject with, or exposing the tissue or organ to a conjugated and labeled Apelin according to claim 22 used as a Single Photon Emission computed Tomography (SPECT-CT) radiotracer or as a Positron Emission Tomography-Computed Tomography (PET-CT) radiotracer.

25. The method according to claim 24, wherein the subject is a human being.

26. A method for treating angiogenesis, vasculogenesis or a disease or disorder inducing or modulating the expression of an APJ receptor in a tissue or organ in a subject in need thereof, comprising administering the subject with a conjugated and labeled Apelin according to claim 23.

27. The method according to claim 26, wherein the disease or disorder inducing or modulating the expression of an APJ receptor in a tissue or organ is a solid cancer wherein the cancerous tumor and/or cancerous tumor vasculature expresses an APJ receptor, and the cancer is typically selected from lung cancer, cholangiocarcinoma, liver cancer, gastric cancer, prostate cancer, ovarian cancer, breast cancer, renal cancer, squamous cell carcinoma, multiple myeloma, glioblastoma, colon cancer, obesity-related colon cancer, endometrial cancer and obesity-related endometrial cancer.

28. The method according to claim 26, wherein the subject is a human being.

29. A composition comprising an Apelin conjugated to a chelator and labeled with a radioactive element according to claim 18 and a pharmaceutically acceptable diluent, excipient, carrier or support.

30. A method of detecting, measuring, diagnosing, staging or monitoring angiogenesis or vasculogenesis, or a disease or disorder inducing or modulating the expression of an APJ receptor in a tissue or organ in a subject, comprising administering the subject with a conjugated and labeled Apelin selected from [.sup.68Ga]Ga-NODAGA-Apelin, [.sup.68Ga]Ga-DOTA-Apelin, [.sup.68Ga]Ga-DOTAGA-Apelin, [.sup.68Ga]Ga-NOTA-Apelin, [.sup.68Ga]Ga-HBED-Apelin, [.sup.68Ga]Ga-DFO-Apelin, [.sup.68Ga]Ga-AAZTA-Apelin, [.sup.67Ga]Ga-NODAGA-Apelin, [.sup.67Ga]Ga-DOTA-Apelin, [.sup.67Ga]Ga-DOTAGA-Apelin, [.sup.67Ga]Ga-NOTA-Apelin, [.sup.67Ga]Ga-HBED-Apelin, [.sup.67Ga]Ga-DFO-Apelin, [.sup.67Ga]Ga-AAZTA-Apelin, Al[.sup.18F]F-NOTA-Apelin, Al[.sup.18F]F-NODA-Apelin, [.sup.111In]In-DTPA-Apelin, [Cu.sup.64]Cu-DOTA-Apelin, [.sup.64Cu]Cu-DOTAGA-Apelin, [.sup.89Zr]Zr-DOTA-Apelin, [.sup.89Zr]Zr-DOTAGA-Apelin, [.sup.152Tb]Tb-DOTA-Apelin, [.sup.152Tb]Tb-DOTAGA-Apelin, [.sup.115Tb]Tb-DOTA-Apelin and [.sup.155Tb]Tb-DOTAGA-Apelin, or with a composition comprising an Apelin conjugated to a chelator and labeled with a radioactive element according to claim 18, wherein the Apelin amino acid sequence comprises SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4, and a pharmaceutically acceptable diluent, excipient, carrier or support.

31. The method according to claim 30, wherein the disease or disorder is selected from ischemia, an ischemia-associated disease or disorder, myocardial infarction, a solid cancer, atherosclerosis, an endothelial dysfunction-related disease, a cardiovascular disease, a metabolic disease, diabetes mellitus and obesity.

32. The method according to claim 30, wherein the subject is a human being.

33. A method of evaluating or monitoring the therapeutic effect of an angiogenic or anti-angiogenic treatment, or of an APJ receptor-targeted treatment, in a subject, comprising administering the subject with a conjugated and labeled Apelin selected from [.sup.68Ga]Ga-NODAGA-Apelin, [.sup.68Ga]Ga-DOTA-Apelin, [.sup.68Ga]Ga-DOTAGA-Apelin, [.sup.68Ga]Ga-NOTA-Apelin, [.sup.68Ga]Ga-HBED-Apelin, [.sup.68Ga]Ga-DFO-Apelin, [.sup.68Ga]Ga-AAZTA-Apelin, [.sup.67Ga]Ga-NODAGA-Apelin, [.sup.67Ga]Ga-DOTA-Apelin, [.sup.67Ga]Ga-DOTAGA-Apelin, [.sup.67Ga]Ga-NOTA-Apelin, [.sup.67Ga]Ga-HBED-Apelin, [.sup.67Ga]Ga-DFO-Apelin, [.sup.67Ga]Ga-AAZTA-Apelin, Al[.sup.18F]F-NOTA-Apelin, Al[.sup.18F]F-NODA-Apelin, [.sup.111In]In-DTPA-Apelin, [Cu.sup.64]Cu-DOTA-Apelin, [.sup.64Cu]Cu-DOTAGA-Apelin, [.sup.89Zr]Zr-DOTA-Apelin, [.sup.89Zr]Zr-DOTAGA-Apelin, [.sup.152Tb]Tb-DOTA-Apelin, [.sup.152Tb]Tb-DOTAGA-Apelin, [.sup.155Tb]Tb-DOTA-Apelin and [.sup.155Tb]Tb-DOTAGA-Apelin, or a composition comprising an Apelin conjugated to a chelator and labeled with a radioactive element according to claim 18, wherein the Apelin amino acid sequence comprises SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4, and a pharmaceutically acceptable diluent, excipient, carrier or support.

34. The method according to claim 33, wherein the disease or disorder is selected from ischemia, an ischemia-associated disease or disorder, myocardial infarction, a solid cancer, atherosclerosis, an endothelial dysfunction-related disease, a cardiovascular disease, a metabolic disease, diabetes mellitus and obesity.

35. The method according to claim 33, wherein the subject is a human being.

36. A kit comprising an Apelin comprising SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4, a chelator selected from 6-amino-6 methylperhydro-1,4-diazepinetetraacetic acid (AAZTA), 1,4,7-triazacyclononane-1,4-diacetic acid (NODA), 1,4,7-triazacyclononane,1-glutaric acid-4,7 acetic acid (NODAGA), 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), 2,2′,2″-(10-(2,6-dioxotetrahydro-2H-pyran-3-yl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid (DOTAGA), 1,4,7-triazacyclononane-triacetic acid (NOTA), N,N′-Bis(2-hydroxybenzyl)-1-(4-bromoacetamidobenzyl)-1,2-ethylenediamine-N,N′-diacetic acid (HBED), N1-hydroxy-N1-(5-(4-(hydroxy(5-(3-(4-isothiocyanatophenyl)thioureido)pentyl)amino)-4-oxobutanamido)pentyl)-N4-(5-(N-hydroxyacetamido)pentyl)succinamide (DFO), triazacyclononane-phosphinate (TRAP), pentetic acid or diethylenetriaminepentaacetic acid (DTPA), bromoacetamidobenzyl(TETA),1,4,7-triazacyclononane-1,4-bis[methylene(hydroxymethyl)phosphinicacid]-7-[methylene(2-carboxyethyl)phosphinicacid])(NOPO), HBED-CC(DKFZ), 2-(4-isothiocyanotobenzyl)-1, 4, 7, 10-tetraaza-1, 4, 7, 10-tetra-(2-carbamonyl methyl)-cyclododecane (TCMC), N—[(R)-2-amino-3-(p-aminophenyl)propyl]-trans-(S,S)-cyclohexane-1,2-diamine-N,N,N′,N″,N″-pentaacetic acid (CHX-A″-DTPA) and a functional derivative thereof, and a radioactive element which is a radionuclide selected from gallium-68 (.sup.68Ga), gallium-67 (.sup.67Ga), lutetium-177 (.sup.177Lu), fluorine-18 (F.sup.18), yttrium-90 (.sup.90Y), bismuth-213 (.sup.213Bi), actinium-225 (.sup.225Ac), lead-212 (.sup.212Pb), indium-111 (.sup.111In), zirconium-89 (.sup.89Zr), terbium-149 (.sup.149Tb), terbium-152 (.sup.152Tb), terbium-155 (.sup.55Tb), terbium-161 (.sup.161Tb) and copper-64 (.sup.64Cu), in three distinct containers; or an Apelin-chelator conjugate wherein the Apelin amino acid sequence comprises SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4, in a single container, and a radioactive element which is a radionuclide selected from gallium-68 (.sup.68Ga), gallium-67 (.sup.67Ga), lutetium-177 (.sup.177Lu), fluorine-18 (F.sup.18), yttrium-90 (.sup.90Y), bismuth-213 (.sup.213Bi), actinium-225 (.sup.225Ac), lead-212 (.sup.212Pb), indium-111 (.sup.111In), zirconium-89 (.sup.89Zr), terbium-149 (.sup.149Tb) terbium-152 (.sup.152Tb), terbium-155 (.sup.155Tb) terbium-161 (.sup.161Tb) and copper-64 (.sup.64Cu), in a distinct container.

37. A method for producing an Apelin conjugated to a chelator and labeled with a radioactive element wherein the Apelin amino acid sequence comprises SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4, with the kit of claim 36.

Description

FIGURES

[0160] FIG. 1. Radiochemical purity in reactional medium and in human serum for two hours.

[0161] FIG. 2. APJ expression appreciated by Western-Blot corrected by GAPDH expression in different cell lines.

[0162] FIG. 3. T84 autoradiography in baseline or blocking conditions.

[0163] FIG. 4. Organs biodistribution of [.sup.68Ga]Ga-NODAGA-Apelin13(F13A) in murine model (n=3).

[0164] FIG. 5. PET images of [.sup.68Ga]Ga-NODAGA-Apelin13(F13A) biodistribution in healthy mice for two hours.

[0165] FIG. 6. Tracers [.sup.68Ga]Ga-NODAGA-Apelin13(F13A) and [.sup.68Ga]Ga-NODAGA-RGD.sub.2 accumulation in Matrigel.

[0166] FIG. 7. [.sup.68Ga]Ga-NODAGA-Apelin13(F13A) PET images in Matrigel over 21 Days.

[0167] FIG. 8. Ischemic limb doppler signal and non-ischemic limb doppler signal ratio over time.

[0168] FIG. 9. [.sup.68Ga]Ga-NODAGA-Apelin13(F13A) and [.sup.68Ga]Ga-NODAGA-RGD.sub.2 targeting in ischemic limb (corrected by non-ischemic limb signal) over time.

[0169] FIG. 10. Representative [.sup.68Ga]Ga-NODAGA-Apelin13(F13A) PET images over 21 days in mouse model of hindlimb ischemia.

[0170] FIG. 11. Correlation of [.sup.68Ga]Ga-NODAGA-Apelin13(F13A) PET signal ratio on day 7 and LASER-Doppler signal at the day of surgery (left); Correlation of [.sup.68Ga]Ga-NODAGA-Apelin13(F13A) PET signal ratio on day 7 and reperfusion on day 21 (right).

[0171] FIG. 12. [.sup.68Ga]Ga-NODAGA-Apelin13(F13A) PET signal on day 7 and day-input function (LASER Doppler Day7/Day0) uncorrelation.

[0172] FIG. 13. Tumor to muscle [.sup.68Ga]Ga-NODAGA-Apelin13(F13A) PET signal ratio in baseline or in blocking conditions in ectopic mice model of human colon adenocarcinoma.

[0173] FIG. 14. [.sup.68Ga]Ga-NODAGA-Apelin13(F13A) PET images in baseline or in blocking conditions in ectopic mice model of human colon adenocarcinoma.

[0174] FIG. 15. Tumor to background [68Ga]Ga-NODAGA-Apelin13(F13A) PET signal ratio and [.sup.68Ga]Ga-NODAGA-RGD.sub.2 PET signal ratio in ectopic mice model of human colon adenocarcinoma.

[0175] FIG. 16. Representative radioHPLC chromatogram of [.sup.68Ga]Ga-AP747.

[0176] FIG. 17. Radio-TLC chromatogram of [.sup.67Ga]Ga-AP747.

[0177] FIG. 18. Saturation binding curve of [.sup.67Ga]Ga-AP747 towards APJ receptor on T84 cells.

[0178] FIG. 19. APJ receptor specific internalized and membrane bound fraction of [.sup.67Ga]Ga-AP747 in T84 cells.

[0179] FIG. 20. Cellular efflux of [.sup.67Ga]Ga-AP747 on T84 cells.

[0180] FIG. 21. Radiochromatogram of [.sup.68Ga]Ga-DOTA-Apelin-F13A (Rf0) showing excellent radiochemical purity ≥99% after purification with neglectable [.sup.68Ga]Ga.sup.3+ at the front (Rf1). RadioTLC: ITLC/sg paper in 0.1M sodium citrate solution pH=5.

[0181] FIG. 22. (A): MicroPET signal quantification of [.sup.68Ga]Ga-AP747 and [.sup.68Ga]Ga-RGD.sub.2 expressed as an ipsi—to contralateral signal ratio. (B): Representative images of [.sup.68Ga]Ga-AP747 and [.sup.68Ga]Ga-RGD.sub.2 microPET/CT in the same animal. Dotted orange lines represent both hemispheres.

[0182] FIG. 23. (A) Representative image of [.sup.68Ga]Ga-AP747 microPET/CT 3 days after MCAO in rat. (B) Quantification of [.sup.68Ga]Ga-AP747 microPET/CT signal quantification in ipsi- and contralateral hemisphere before MCAO, on the day of MCAO, and every day for 7 days after MCAO in rat.

EXAMPLES

Example 1

Materials and Methods

NODAGA Conjugation

[0183] Apelin-13(F13A), purchased from Sigma-Aldrich (Merck Millipore) was solubilized in 0.2M bicarbonate buffer (1 mg/ml) and added to 10 equivalents of NODA-GA-NHS ester (CheMatech) in 0.2M bicarbonate buffer. The mixture was left at room temperature (RT) for 2 h. The conjugate was then transferred to a tC18 Cartridge (Sep-Pak) washed 2 times with water to eliminate unreacted small molecules then eluted with EtOH. Solvent was evaporated at RT, PBS was added and conjugate in PBS stored at −20° C.

Radiochemistry

[0184] Gallium was obtained in .sup.68GaCl.sub.3 form using a commercial TiO.sub.2-based .sup.68Ge/.sup.68Ga generator (Galliapharm, Eckert&Ziegler). .sup.68GaCl.sub.3 (200.69±40.97 MBq/0.5 mL) was eluted from a .sup.68Ge/.sup.68Ga generator using 0.1 N HCl, after which 4M ammonium acetate buffer (pH 7.4) was added. This solution was then added to NODAGA-Apelin-13(F13A) (1 μg/μL); final pH of the mixture was 6.0. The reaction mixture was incubated at RT for 5 min.

[0185] Determination of radiochemical purity was done by radio-thin-layer chromatography (ITLC-SG) and was performed using a Ray-test miniGITA radio-TLC scanner detector (Straubenhardt, Ge) (eluents, 1:1 [v/v] mixture of 1M aqueous ammonium acetate solution and methanol and also in Trisodium Citrate 0,1M). Evaluation of .sup.68Ga-NODAGA-Apelin13(F13A) stability was performed in human serum at 60 and 120 min after radiosynthesis.

[0186] NODAGA-RGD was purchased from ABX and radiolabelled as recommended with .sup.68Ga by the manufacturer.

In Vitro Experiments

Western Blot

[0187] APJ expression was evaluated by Western-Blot with cell lysates. Cell lysates were loaded on polyacrylamide gel (NuPAGE, Invitrogen, 4%-12%). After migration (80V, 30 minutes), proteins were transferred to nitrocellulose membrane (checked by Rouge-Ponceau). Membrane was saturated (TBST-3%; BSA, Tris-buffered saline Tween 20%; Bovine Serum Albumine 3%) and then Anti-APJ Apelin Receptor Antibody: 5H5L9 (rabbit monoclonal Invitrogen, 1 μg/mL) was added overnight, under agitation. After TBST wash, secondary antibody: Goat Anti-Rabbit HRP-tagged (Thermofisher) was added for one hour. Chemiluminescent revelation was made thanks to ECL kit (Thermofischer). Membrane images acquisition were performed by Gbox (Syngene). Finally, a stripping was performed to determine GADPH expression.

Autoradiography

[0188] A blocking strategy was performed on cells expressing the highest level of APJ. This strategy consists in adding a large excess (100-fold) of unconjugated peptide (Apelin13(F13A)) before adding inventors' product of interest. Unspecific tracer was eliminated by several washes. The remaining activity, considered as specifically bound to the target, was evaluated by autoradiography.

Human Umbilical Vein Endothelial Cells

[0189] HUVEC cell lines (Laboratoire de Thérapie cellulaire, CHU La Conception AP-HM/C2VN Aix-Marseille Université) were cultivated in EGM-2 medium complemented with 10% fetal bovine serum decomplemented and 1% antimycotic-antibiotic mix. Cell lines were maintained in a humidified 5% CO.sub.2 incubator at 37° C. HUVEC's activation was performed by incubation with TNF-alpha (10 ng/ml) overnight.

Human Colon Adenocarcinoma Cell Line

[0190] T84 cell line (EuroBioDev) was cultivated in DMEM-F12/Glutamax medium complemented with 10% fetal bovine serum decomplemented and 1% antimycotic-antibiotic mix. Cell lines were maintained in a humidified 5% CO.sub.2 incubator at 37° C.

Human Glioblastoma Cell Line

[0191] U87 cell line was cultivated in Dulbecco's modified Eagle's medium complemented with 10% fetal bovine serum, 1% antimycotic-antibiotic mix, and 1% non-essential amino-acid. Cell lines were maintained in a humidified 5% CO.sub.2 incubator at 37° C.

Human Pancreatic Adenocarcinoma Cell Line

[0192] SOJ6 cell line (CRCM, Aix-Marseille Université) was cultivated in DMEM-F12/Glutamax/Pyruvate complemented with 10% fetal bovine serum decomplemented and 1% antimycotic-antibiotic mix. Cell lines were maintained in a humidified 5% CO.sub.2 incubator at 37° C.

Animal Experiments

[0193] All procedures using animals were approved by the Institution's Animal Care and Use Committee (CE71, Aix-Marseille Université) and were conducted according to the 2010/63/EU European Union Directive. Swiss and Swiss Nude mice were housed in enriched cages placed in a temperature- and hygrometry-controlled room with daily monitoring and fed with water and commercial diet ad libitum.

Mouse Model of Hindlimb Ischemia and Matrigel

[0194] Unilateral hindlimb ischemia was performed on 9-week-old male Swiss mice (Janvier Labs) after femoral artery excision under 2% isoflurane anesthesia. LASER Doppler perfusion imaging (Perimed, Craponne, France) was used to assess revascularization from day 0 to day 21 after surgery. Perfusion results are expressed as a ratio of ischemic to non-ischemic limb blood flow. Hindlimb ischemic damage was quantified on Days 1, 3, 7, 10, 13 and 21.

[0195] These mice were also subcutaneously implanted with Matrigel (Dutscher) supplemented with 10% fetal bovine serum under 2% isoflurane anesthesia.

Ectopic Mouse Model of Human Colon Adenocarcinoma

[0196] Human colon adenocarcinoma xenografts were established by subcutaneous injections of 1×10.sup.6 T84 cells into 6-week-old male Swiss nude mice (Charles River) under 2% isoflurane anesthesia.

MicroPET Imaging

[0197] On hindlimb ischemia mouse model (n=8) and Matrigel mouse model (n=7) on day 1, 3, 7, 10, 13 and 21 post-surgery, mice were IV injected with 5-10 MBq of .sup.68Ga-NODAGA-Apelin13(F13A) under 2% isoflurane anesthesia.

[0198] PET images were acquired 60 min after IV injection on a Mediso Nanoscan PET/CT under 2% isoflurane anesthesia. On hindlimb ischemia mouse model and on day 1, 3, 7, 10, 13 and 21 post-surgery, mice were IV injected with 5-10 MBq of [.sup.68Ga]Ga-NODAGA-RGD.sub.2 injection under 2% isoflurane anesthesia. PET images were acquired 60 min after IV injection on a Mediso Nanoscan PET/CT under 2% isoflurane anesthesia.

[0199] For blocking experiments, a 50-fold excess of unconjugated peptide: Apelin-13(F13A) was IV injected 30 min previous .sup.68Ga-NODAGA-Apelin13(F13A), and PET images were acquired 1 hour 30 min after the first IV injection on a Mediso Nanoscan PET/CT under 2% isoflurane anesthesia.

[0200] For biodistribution study in healthy mice (n=3), images were continuously acquired just after .sup.68Ga-NODAGA-Apelin13(F13A) IV injection with 5-6 MBq and recorded up to 2 h post injection on a Mediso NanoPET/CT under 2% isoflurane anesthesia.

[0201] On colon adenocarcinoma mice model (n=3), mice were IV injected with 5-10 MBq of [.sup.68Ga]Ga-NODAGA-RGD.sub.2 or .sup.68Ga-NODAGA-Apelin13(F13A) respectively under anesthesia.

[0202] PET images were acquired 1 h after IV injection on a Mediso NanoPET/CT under 2% isoflurane anesthesia.

[0203] Quantitative region-of-interest (ROI) analysis of the PET images was performed on attenuation- and decay-corrected PET images using InVicro—VivoQuant software and tissue uptake values are presented as an ischemic muscle to contralateral muscle ratio and as a percentage of the injected dose per gram of tissue (% ID/g) which was determined by decay correction for each sample normalized to a standard of known weight, which was representative of the injected dose.

Statistical Analysis

[0204] Biodistribution data were analysed using the Graphpad Prism software (San Diego, Calif.). Data are presented as mean values±SD. Ischemic to contralateral muscle ratio were analyzed using the two-way analysis of variance (ANOVA) and no parametric t-test (Mann Whitney test). Differences were considered statistically significant when p<0.05.

Results

Radiochemistry

[0205] Incubation in human serum didn't lead to significant tampering of [.sup.68Ga]-NODAGA-Apelin13(F13A) radiochemical purity until two hours post-incubation (n=3). Radiolabeling remained stable over time in molecular imaging conditions (<2 h) (FIG. 1).

In Vitro Experiments

Western Blot

[0206] In order to evaluate tissue APJ expression in different cell lines Western Blot was performed (FIG. 2) on U87, SOJ6, T84, HUVECs and TNFα-activated HUVECs. T84 cell line seems to be the line with the higher expression of APJ. Moreover, activated HUVECs express a higher level of APJ than HUVECs in baseline conditions. APJ expression evaluation was corrected by the expression of GADPH (charge indicator).

Autoradiography

[0207] Because T84 has the higher expression level of APJ, a blocking strategy using autoradiography (FIG. 3) was performed on these cells. The remaining activity recorded by autoradiography in blocking condition was significantly reduced compared to classic conditions (P-value=0.0003). AP747 corresponds to NODAGA-Apelin13(F13A).

Animal Experiments

Biodistribution in Healthy Mice

[0208] In healthy mice, PET signal quantification in organs (FIG. 4) highlights a suitable pharmacokinetic of [.sup.68Ga]Ga-NODAGA-Apelin13(F13A) and a fast and mainly urinary excretion without noticeable accumulation in liver, lungs or brain. FIG. 5 shows representative PET biodistribution images of one mouse for two hours.

Mouse Model of Hindlimb Ischemia and Matrigel

[0209] [.sup.68Ga]Ga-NODAGA-Apelin13(F13A) accumulates in Matrigel plug and this accumulation is significantly higher than [.sup.68Ga]Ga-NODAGA-RGD.sub.2 on day 10 (P-value=0.0362), day 13 (P-value=0.0064) and day 21 (P-value=0.0016). [.sup.68Ga]Ga-NODAGA-Apelin13(F13A) PET signal is all the more important over time (P-value=0.0051). Globally, over experiment period [.sup.68Ga]Ga-NODAGA-Apelin13(F13A) PET signal is significantly superior to [.sup.68Ga]Ga-NODAGA-RGD.sub.2 PET signal (p=0.0000017) (FIG. 6). Corresponding PET images are shown on FIG. 7.

[0210] Ischemia-reperfusion monitoring of ischemic limb by LASER-Doppler (FIG. 8) was compared with non-ischemic limb and with perfusion after surgery (Day 1). In ischemic limb [.sup.68Ga]Ga-NODAGA-Apelin13(F13A) PET signal (peak at Day 7) is notably better (P-value=0.0497), than [68Ga]Ga-NODAGA-RGD.sub.2 PET signal (peak at Day 10) (FIG. 9). Corresponding PET images are represented on FIG. 10.

[0211] [.sup.68Ga]Ga-NODAGA-Apelin13(F13A) PET signal in ischemic limb (corrected with non-ischemic limb) is significantly negatively correlated to LASER-Doppler signal at day of surgery (P two-tailed=0.0188). This means that [.sup.68Ga]Ga-NODAGA-Apelin13(F13A) PET signal in ischemic limb is more important when ischemia injury is severe and so the hindlimb perfusion is low (FIG. 11).

[0212] Another correlation was established. Indeed, [.sup.68Ga]Ga-NODAGA-Apelin13(F13A) PET signal in ischemic limb (corrected with non-ischemic limb) is more important when reperfusion is longer (LASER-Doppler Day 21/Day 0) (P two-tailed=0.0196) (FIG. 11).

[0213] [.sup.68Ga]Ga-NODAGA-Apelin13(F13A) PET signal in ischemic limb (corrected with non-ischemic limb) is not linked to day-input function (P two-tailed=0.4236) (FIG. 12).

Ectopic Mouse Model of Human Colon Adenocarcinoma

[0214] PET signal during in vivo blocking experiment on ectopic mouse model of colon adenocarcinoma was quantified and compared to classic conditions. Indeed, in classic conditions [.sup.68Ga]Ga-NODAGA-Apelin13(F13A) PET signal level is significantly higher (793.3%±217.2) than [.sup.68Ga]Ga-NODAGA-Apelin13(F13A) PET signal in blocking conditions (102.6%±31.37) (P-value=0.0235) (FIG. 13). Corresponding PET images are represented on FIG. 14.

[0215] [.sup.68Ga]Ga-NODAGA-Apelin13(F13A) PET signal was compared to [.sup.68Ga]Ga-NODAGA-RGD.sub.2 PET signal (FIG. 15). PET signal quantification was corrected by background signal. [.sup.68Ga]Ga-NODAGA-Apelin13(F13A) PET signal (23.33%21.09) was significantly (P-value=0.0480) higher than that of [.sup.68Ga]Ga-NODAGA-RGD.sub.2 (3.496%±3.841).

CONCLUSIONS

[0216] Inventors herein describe the first radiotracer for PET imaging of APJ (herein identified as “AP747”). They first developed AP747 for detecting, imaging, measuring and/or monitoring APJ expressing-tissue. The radiomarker is usable as a companion tool for modulating therapeutic strategy, and as a tool to evaluate tissue angiogenesis.

[0217] After compound synthesis and its radiolabelling, AP747 targeting against APJ was validated thanks to blocking strategies monitored by autoradiography on cells expressing APJ. Once stability of the tracer had been validated, in vivo evaluation in PET imaging was performed.

[0218] Pharmacokinetic profile in rodent revealed a fast-urinary excretion without hepatic accumulation; an ideal profile for PET imaging agents and theragnostic approaches. Biodistribution evaluation on two well-described and characterized angiogenesis models: Matrigel (simple hypoxic model) and ischemic model (Hindlimb ischemia). In both models, the results showed that the PET signal of [68Ga]Ga-NODAGA-Apelin13(F13A) in angiogenesis outbreaks superior than [.sup.68Ga]Ga-NODAGA-RGD.sub.2 PET signal: gold standard in angiogenesis molecular imaging (Azizi Y et al., 2013).

[0219] Specificity of AP747 PET imaging signal was demonstrated through in vivo blocking experiments (pre-incubation to saturate binding sites of the tracer and appreciate the specificity of [.sup.68Ga]Ga-NODAGA-Apelin13(F13A) with Apelin13(F13A)) on angiogenic and tumoral models, overexpressing APJ. Partial blocking (about 50%) can be explained by the small amount of cold Apelin13(F13A) used because of bad tolerance due to cardiovascular effects of Apelin as previously reported in literature (Benton G et al., 2014).

[0220] Targeting should be reflective of APJ overexpression in hypoxic conditions reported in literature and observed by Western-Blot during HUVEC TNF-stimulation. During angiogenesis sprouting, endothelial cell sprouts growing is known to be VEGF-guided, but other signals (repellent or attractive) can be useful in vessel formation moves. Massive secretion of apelin by endothelial tip cells promotes APJ expression by stalk cells, as well as their proliferation. Lumen formation in stalk cells involves vacuoles fusion and other mechanisms not fully explained, but APJ/Apelin system plays a major role during sprouting, as observed in Apelin-KO animals. Adhesive or repellent interactions between tip cells regulate sprouts and vessels fusion. Activated HUVEC fixation intensity appreciated by autoradiography was correlated to in vivo observations. APJ expression of gastrocnemius muscles of ischemic animals quantified by histology permits to evaluate links between PET signal intensity and APJ tissue expression. PET signal kinetic profile allows to assess APJ expression kinetic in hypoxic conditions (Matrigel and ischemic models). This expression seems to be intense and extended. Profiles observed on Matrigel and ischemic models are different: [0221] In Matrigel model, signal is increasing, probably related to avascular and acellular contents of Matrigel, a minimum of 10 days is required to observe new vessels formation, and some supplementary days for obtain functional vessels (Chapman et al., 2014); [0222] In ischemic model, a peak is observed at day 7 followed by an intensity diminution, probably linked to vascular development from popliteal anastomosis (Gronman, M et al., 2017);

[0223] Moreover, on ischemic model, LASER-Doppler signal at day 7 (peak) is correlated to late reperfusion index (Day 21/Day 1). In this way, [.sup.68Ga]Ga-NODAGA-Apelin13(F13A) seems to be an early predictive factor of tissue perfusion, further argument supporting [.sup.68Ga]Ga-NODAGA-Apelin13(F13A) as a tracer for evaluating tissue angiogenesis intensity.

[0224] In comparison with [.sup.68Ga]Ga-NODAGA-RGD.sub.2, [.sup.68Ga]Ga-NODAGA-Apelin13(F13A) imaging appeared pertinent at an earlier stage, with stronger signal and that lingers longer. Information potency of [.sup.68Ga]Ga-NODAGA-Apelin13(F13A) appears to be much more powerful than [.sup.68Ga]Ga-NODAGA-RGD.sub.2, actually in clinical development. Especially, regarding therapeutic and protector properties of Apelin, alone or as adjuvant, in ischemic pathologies comprising hind limb ischemia (Hasan, J et al., 2004), PET imaging that could evaluate in vivo expression or overexpression of APJ is usable as a tool to determine therapeutic eligibility, to monitor therapeutic efficiency, as prognostic or diagnostic index, like other theragnostic couples in clinical development or trials (Jakobsson, L et al., 2010).

[0225] Results observed in colon adenocarcinoma murine model with high level of [.sup.68Ga]Ga-NODAGA-Apelin13(F13A) fixation, whose specificity had been checked by blocking strategy, are very relevant.

Example 2

Materials and Methods

.SUP.67.Ga-Radiolabelling

[0226] [.sup.67Ga]Ga-citrate (200 MBq, CURIUM) was converted in [.sup.67Ga]GaCl.sub.3 using two Light silica Sep-pac (Waters, refWAT023537). Briefly, [.sup.67Ga]Ga-citrate was loaded on the cartridge and then eluted using 1 mL HCl 0.1M (Rottem, KT720P) in form of [.sup.67Ga]GaCl.sub.3 and subsequently used for radiolabelling as described for [.sup.68Ga]GaCl.sub.3. The final product was formulated in 3 mL PBS.

Radiochemical Purity Control

[0227] Radio-UV-HPLC analyses were performed using a Phenomenex Luna C18 column (4 mL/min, λ=220 nm C18; 150 mm×4.6 mm×5 μm). HPLC conditions were: 0-2 min: 90% ACN (A), 10% water in 0.1% TFA (B), 2-10 min: 90%.fwdarw.10% A; 10%.fwdarw.90% B, 10-12 min: 10% A; 90% B, 12-14 min: 10%.fwdarw.90% A; 90%.fwdarw.10% B. The analytical HPLC system used was a JASCO system with ChromNAV software, a PU-2089 Plus quaternary gradient pump, a MD-2018 Plus photodiode array detector and Raytest Gabi Star detector. TLC analysis were also carried out (miniGITA plate reader, acquisition time of 1 min, Rf impurities≥0.8, Rf .sup.68Ga-bioconjugates ≤0.1 using citrate buffer pH5 as mobile phase and ITLC-SG as stationary phase.

Hydrophilicity

[0228] Hydrophilicity of [.sup.67Ga]Ga-AP747 was assessed by the water-octanol partition/distribution coefficient method. In a centrifuge tube, 500 μL of 1-octanol was added to 500 μL of phosphate-buffered saline (pH 7.4) containing the radiolabeled peptide (50 kBq). After equilibrium, the solution was vigorously stirred for 5 min at room temperature and subsequently centrifuged (4000 rpm, 5 min) to yield two immiscible layers. Aliquots of 100 μL were taken from each layer and the radioactivity in the samples was determined by a gamma counter (Perkin Elmer, Waltham, Mass., USA).

Saturation Binding Assay.

[0229] The affinity of [.sup.67Ga]Ga-AP747 was studied on T84 cells seeded at a density of 250.10.sup.3 cells per well in 24-well plates (Corning®) and incubated overnight with complete medium. Well plates were first set on ice 30 minutes before the beginning of the experiment. [.sup.67Ga]Ga-AP747 was then added to the medium at concentration of (0.1, 1, 10, 100, 250 nM) and cells were incubated (in quadruplicates) for 2 hours at 4° C. Incubation was stopped by removing medium and washing cells twice with ice-cold PBS. Finally, cells were treated with NaOH (1M) and radioactivity was measured in a gamma counter. In order to assess for non-specific affinity, excess non-radioactive apelin-13 (final concentration 1 M), was added to selected wells.

In Vitro Internalization and Efflux Studied Studies.

[0230] T84 cells were cultured as described in saturation binding experiments above.

[0231] For internalization studies, 50 kBq of [.sup.67Ga]Ga-AP747 were added to the medium the day of the experiments and the cells were incubated (in quadruplicates) during 10, 30 or 60 minutes at 37° C. Three minutes before the end of the incubation time, internalization was stopped on ice and the supernatant was removed. Internalization was then stopped by eliminating the supernatant and each well was washed with 3×250 μL of ice-cold PBS. The membrane-bound fraction was retrieved in 2×250 μL sodium acetate buffer (20 mM, pH 5) for 5 min. Finally, cells were treated with 500 μL of NaOH (1 M). The radioactivity of the membrane-bound fraction and the internalized fraction was measured in a gamma counter. The experiment was performed twice. To also verify receptor specificity, blocking experiments were performed by using 1 μM of apelin-13.

[0232] For efflux experiments, 10 kBq of [.sup.67Ga]Ga-AP747 were added to the medium the day of the experiments and the cells were incubated (in octoplicates) for 30 minutes at 37° C. Three minutes before the end of the incubation time, internalization was stopped on ice and the supernatant was removed. Each well was washed with 1 mL of ice-cold PBS. The membrane-bound fraction was retrieved in 2 mL sodium acetate buffer (20 mM, pH 5) for 2 min, each well was rinsed a second time with 1 mL ice-cold PBS and fresh culture medium was added. At each time point (10, 30, 60 and 120 minutes), the efflux was stopped by collecting the medium and washing cells twice with ice-cold PBS. Finally, cells were treated with NaOH (1 M). The radioactivity of the collected culture medium supernatant, the PBS wash fractions, and the total internalized fraction was measured in a gamma counter. The experiment was performed twice.

Radiosynthesis of Al[.SUP.18.F]F-NODA-Apelin-F13A

[0233] Aluminium chloride (AlCl.sub.3.Math.6H.sub.2O), sodium acetate (NaOAc), potassium hydrogenocarbonate (KHCO.sub.3), glacial acetic acid (AcOH), water for HPLC, acetonitrile for HPLC, trifluoroacetic acid and pH paper were purchased from Sigma (France). The analytic reverse phase HPLC column (Luna C18 150×4.6 mm 5 μm) was purchased from Phenomenex (France). Solid-phase extraction cartridge (Sep-Pak QMA light) was purchased from Waters (France). No carrier-added [.sup.18F] fluoride was trapped on the anion-exchange resin. The cartridge was washed with 5 mL of HPLC water. The cartridge carrying the .sup.18F-anions was eluted with 600 μL of a 0.4 M solution of KHCO.sub.3. A pH of 4.5 required for .sup.18F-chelation by addition of glacial acetic acid was obtained. The pH value was determined using pH-paper. 50 μL of the .sup.18F-solution were incubated 10 minutes at room temperature with 3 μL of a 2 mM solution of AlCl.sub.3.Math.6H.sub.2O. Then, 9 μL of NODA-Apelin (2 mM in 0.5 M of NaOAc) were added to the previous reaction mixture. The solution was incubated at 100° C. for 10 minutes. 20 μL of the reaction mixture was injected through HPLC as described upper.

Radiosynthesis of [.SUP.68.Ga]Ga-DOTA-Apelin-F13A

[0234] 70 μL of 1 mol.Math.L.sup.−1 sodium acetate trihydrate solution were added to 4 μg/10 μL of DOTA-Apelin-F13A. 500p of freshly eluted [.sup.68Ga]GaCl.sub.3 were added to the reactor. The mixture was heated at 110° C. for 10 min, then allowed to cool at room temperature for 5 min. A tC18-light cartridge was preconditioned with 1 mL of 90% ethanol, then 2 mL of HPLC water, and loaded with the reaction product. The cartridge was washed with 2 mL of HPLC water. Finally, [.sup.68Ga]Ga-DOTA-Apelin-F13A was eluted from the tC18 cartridge with 0.5 mL of 50% ethanol solution in 0.9% NaCl. Radiochemical purity was checked before and after purification by radio-thin layer chromatography (radioTLC) on ITLC/sg paper with 0.1M sodium citrate solution pH=5 ([.sup.68Ga]Ga-DOTA-Apelin-F13A at Rf0; [.sup.68Ga]Ga.sup.3+ at the front, Rf1).

Orthotopic Mouse Model of Human Glioblastoma

[0235] Human glioblastoma xenografts were achieved by orthotopic injections 5×10.sup.5 U87 cells (3 μL, PBS+/+) into 8-week-old female athymic nude mice (Charles River) under 2% isoflurane anesthesia. Stereotaxic injections using a Hamilton microsyringe were realized in left striatum (coordinates: −2 mm dorsal/ventral, +1 mm lateral, and +1 mm anterior/posterior from bregma). Mice were allowed for resting for 4 weeks.

[.sup.68Ga]Ga-AP747 microPET CT of Orthotopic Mouse Model of Human Glioblastoma

[0236] Mice bearing orthotopic human glioblastoma (n=3) were IV injected with [.sup.68Ga]Ga-RGD.sub.2 (1.99±0.25 MBq) or with [.sup.68Ga]Ga-AP747 (2.95±0.15 MBq) under anesthesia. PET images were acquired 1 h after IV injection on a NanoPET/CT (Mediso) under 2% isoflurane anesthesia. Quantitative region-of-interest (ROI) analysis of the PET images was performed on attenuation- and decay-corrected PET images using VivoQuant software (InVicro) and tissue uptake values were presented as a left-to-right hemisphere activity ratio. Left-to-right hemisphere ratios were compared using the paired t-test.

Middle Cerebral Artery Occlusion (MCAO) Followed with Reperfusion in Rats

[0237] 6-8-month-old female rats were intubated, and mechanically ventilated with 3.0 vol % sevoflurane in a gas mixture of 30% oxygen and 70% nitrogen. Focal cerebral ischemia was induced by occluding the right middle cerebral artery with a monofilament coated with silicone (diameter adjusted to the weight of the animal). After a 60-min ischemia, the filament was withdrawn allowing reperfusion. Rats were allowed for resting for 2 days.

[.sup.68Ga]Ga-AP747 microPET CT of MCAO Rats

[0238] MCAO rats (n=3) were IV injected with [.sup.68Ga]Ga-AP747 (12.05±0.75 MBq) under anesthesia the day before MCAO, right after MCAO, and everyday up to day 7 post-MCAO. MicroPET images were acquired for 20 min, 2 h after injection, on a NanoPET/CT (Mediso) under 2% isoflurane anesthesia. Quantitative region-of-interest (ROI) analysis of the PET images was performed on attenuation- and decay-corrected PET images using VivoQuant software (InVicro) and tissue uptake values were presented as the quantified ipsi- to contralateral microPET signal ratio and compared to the day before MCAO using a one-way ANOVA.

Results

[0239] [.sup.67Ga]Ga-AP747 and [.sup.68Ga]Ga-AP747 were obtained with high radiochemical purity (>95%), high apparent molar activity >10 MBq/μg and high volumic activity >30 MBq/mL.

[0240] Representative radio-HPLC chromatogram of [.sup.68Ga]Ga-AP747 is displayed in FIG. 16. Representative radio-TLC chromatogram of [.sup.67Ga]Ga-AP747 is also displayed in FIG. 17.

Hydrophilicity

[0241] [.sup.67Ga]Ga-AP747 was found to be hydrophilic with a log D.sub.7.4 value of −3.03±0.02.

Saturation Binding Assay

[0242] The specific receptor binding of [.sup.67Ga]Ga-AP747 for APJ receptor was investigated on T84 cells. Saturation binding curves revealed nanomolar affinity was with a K.sub.d value of 11.85±2.8 nM (FIG. 18).

In Vitro Internalization and Efflux Studied Studies.

[0243] The APJ receptor-mediated internalization and the APJ receptor membrane-bound fraction of [.sup.67Ga]Ga-AP747 into T84 cells were analyzed. Specific and time dependent internalization into T84 cells was observed with a maximum of 79.7±7.3% of the cell associated radioactivity being internalized at 60 min. The receptor specific and time dependent membrane-bound fraction of [.sup.67Ga]Ga-AP747 was low (<5%) at any time point (FIG. 19)

[0244] [.sup.67Ga]Ga-AP747 was further evaluated regarding cellular efflux on T84 cells. A high and fast efflux of internalized radioactivity was found for [.sup.67Ga]Ga-AP747. Already 10 minutes post-internalization, 66.7±1.7% of the total binding was externalized. Efflux increased over time to reach 81.2±2.9% at 2 h (FIG. 20).

Radiosynthesis of Al[.SUP.18.F]F-NODA-Apelin-F13A

[0245] NODA-Apelin-F13A was rapidly and successfully radiolabeled with [.sup.18F]F in 10 minutes at 100° C. by chelation via Al-bound .sup.18F. The quality control assessed by HPLC, before purification, showed a labeling efficiency of 47%. Further purification of the Al[.sup.18F]F-NODA-Apelin was achieved through a C18 cartridge.

Radiosynthesis of [.SUP.68.Ga]Ga-DOTA-Apelin-F13A

[0246] DOTA-Apelin-F13A was rapidly and successfully radiolabeled with [.sup.68Ga]Ga. The quality control assessed by TLC, before purification, showed a labeling efficiency of 76%. Further purification of the [.sup.68Ga]Ga-DOTA-Apelin-F13A was achieved through a C18 cartridge reaching a ≥99% radiochemical purity (FIG. 21).

[.sup.68Ga]Ga-AP747 microPET CT of Orthotopic Mouse Model of Human Glioblastoma

[0247] Ipsi-to-contralateral [.sup.68Ga]Ga-AP747 microPET signal quantification ratio was significantly higher than that of [.sup.68Ga]Ga-RGD.sub.2 in the same glioblastoma mice (1.45±0.22 and 0.76±0.25 respectively, *P=0.0346, n=3, FIG. 22).

[.sup.68Ga]Ga-AP747 microPET CT of MCAO Rats

[0248] After no significant modification on the day of MCAO (1.26±0.03, n=3) and the day after (1.74±0.31, n=3), ipsi-to-contralateral [.sup.68Ga]Ga-AP747 microPET signal quantification ratio significantly peaked on day 2 (5.65±0.77, ****P<0.0001, n=3) and day 3 (7.05±0.78, ****P<0.0001, n=3), then slightly decreased on day 4 (3.75±1.62, **P=0.0029, n=3), day 5 (3.34±0.52, *P=0.0121, n=3) and day 6 (3.21±1.27, *P=0.0185, n=3) until back to baseline on day 7 (1.65±0.17, n=3, FIG. 23).

CONCLUSIONS

[0249] Altogether, inventors herein demonstrate that:

[0250] [.sup.68Ga]Ga-AP747 (i.e. [.sup.68Ga]Ga-NODAGA-Apelin) is a powerful radiotracer useful for labelling and/or imaging glioblastoma in vivo as shown in micro PET/CT of orthotopic mouse model of human glioblastoma (FIG. 22). In comparison with [.sup.68Ga]Ga-NODAGA-RGD.sub.2, [.sup.68Ga]Ga-AP747 imaging appeared with stronger signal. Information potency of [.sup.68Ga]Ga-AP747 appears to be much more powerful than [.sup.68Ga]Ga-NODAGA-RGD.sub.2.

[0251] The present results demonstrate that [.sup.68Ga]Ga-AP747 is suitable for use as a Positron Emission Tomography (PET-CT) radiotracer and is suitable for use for labelling and/or imaging in vivo or ex vivo a tissue or organ expressing the APJ receptor or for use in vivo for detecting, measuring, diagnosing, staging and/or monitoring a cancer.

[0252] [.sup.68Ga]Ga-AP747 is also a powerful radiotracer for quantifying APJ receptor expression kinetics following ischemia as shown on a rat model of middle cerebral artery occlusion (MCAO) followed with reperfusion (FIG. 23).

[0253] Inventors successfully generated the tracers [.sup.68Ga]Ga-DOTA-Apelin and Al.sup.18F-NODA-Apelin with a an excellent radiochemical purity greater than 99%.

[0254] They successfully developed the tracer [.sup.67Ga]Ga-NODAGA-Apelin with high radiochemical purity (>95%) for detecting, imaging, measuring and/or monitoring APJ expressing-tissue. After compound synthesis and its radiolabelling, [.sup.67Ga]Ga-NODAGA-Apelin binding on carcinoma cells (T84) was studied and revealed a great (nanomolar) affinity of [.sup.67Ga]Ga-NODAGA-Apelin for APJ receptor on T84 cells. Likewise, inventors showed the APJ receptor-mediated internationalization as well as the high and fast efflux for [.sup.67Ga]Ga-NODAGA-Apelin. As switching from [.sup.68Ga]Ga to [.sup.67Ga]Ga has no influence on the chemical structure of the radiotracer, these in vitro results obtained with [.sup.67Ga]Ga-AP747 can be extrapolated to [.sup.68Ga]Ga-AP747. Similarly, the in vivo results obtained with [68Ga]Ga-AP747 can be extrapolated to [.sup.67Ga]Ga-AP747. Altogether, the results show that [.sup.68Ga]Ga-AP747 and [.sup.67Ga]Ga-AP747 are suitable for use as PET-CT radiotracers.

REFERENCES

[0255] Activation of Catalase by Apelin Prevents Oxidative Stress-linked Cardiac Hypertrophy—Foussal—2010—FEBS Letters—Wiley Online Library.” n.d. Accessed Aug. 19, 2019. [0256] Azizi Y, Faghihi M, Imani A, Roghani M, Nazari A. Post-infarct treatment with [Pyr1]-apelin-13 reduces myocardial damage through reduction of oxidative injury and nitric oxide enhancement in the rat model of myocardial infarction. Peptides. 2013; 46:76-82. [0257] Benton, G., Arnaoutova, I., George, J., Kleinman, H. K. & Koblinski, J. Matrigel: From discovery and ECM mimicry to assays and models for cancer research. Advanced Drug Delivery Reviews. 2014; 79-80, 3-18. [0258] Berta J, Kenessey I, Dobos J, et al. Apelin Expression in Human Non-small Cell Lung Cancer: Role in Angiogenesis and Prognosis. Journal of Thoracic Oncology. 2010; 5(8):1120-1129. [0259] Brame A L, Maguire J J, Yang P, et al. Design, Characterization, and First-In-Human Study of the Vascular Actions of a Novel Biased Apelin Receptor Agonist. Hypertension. 2015; 65(4):834-840. [0260] Carroll A O, Lolait S J, Harris L E, Pope G R. The apelin receptor APJ: journey from an orphan to a multifaceted regulator of homeostasis. 1998. [0261] Cesirio J M S, Brito R B O, Malta C S, et al. A simple method to induce hypoxia-induced vascular endothelial growth factor-A (VEGF-A) expression in T24 human bladder cancer cells. In Vitro CellDevBiol-Animal. 2017; 53(3):272-276. [0262] Chapman, N. A., Dupré, D. J. & Rainey, J. K. The apelin receptor: Physiology, pathology, cell signalling, and ligand modulation of a peptide-activated class A GPCR. Biochem Cell Biol 92. 2014; 431-440. [0263] Chen T, Liu N, Xu G-M, Liu T-J, Liu Y, Zhou Y, Huo S-B, Zhang K (2017) Apelin13/APJ promotes proliferation of colon carcinoma by activating Notch3 signaling pathway. Oncotarget 8:101697-101706. [0264] Dowda B F O, Heiberb M, Chanb A, et al. A human gene that shows identity with the gene encoding the angiotensin receptor is located on chromosome 11. 1993; 136:355-360. [0265] Dray C, Knauf C, Valet P, Masri B. Apelin receptors: From signaling to antidiabetic strategy. Eur J Pharmacol. 2015:1-11. [0266] Eyries Melanie, Siegfried Géraldine, Ciumas Mariana, et al. Hypoxia-Induced Apelin Expression Regulates Endothelial Cell Proliferation and Regenerative Angiogenesis. Circulation Research. 2008; 103(4):432-440. [0267] Foldes G, Horkay F, Szokodi I, et al. Circulating and cardiac levels of apelin, the novel ligand of the orphan receptor APJ, in patients with heart failure. Biochemical and Biophysical Research Communications. 2003; 308(3):480-485. [0268] Gerbier R, Leroux V, Couvineau P, Alvear-perez R. New structural insights into the apelin receptor: identification of key residues for apelin binding. 2019. [0269] Gronman, M. et al. Imaging of αvβ3 integrin expression in experimental myocardial ischemia with [68Ga]NODAGA-RGD positron emission tomography. Journal of Translational Medicine. 2017; 15, 144. [0270] Hall C, Ehrlich L, Venter J, O'Brien A, White T, Zhou T, et al. Inhibition of the apelin/apelin receptor axis decreases cholangiocarcinoma growth. Cancer Lett. 1 févr 2017; 386:179-88. [0271] Harford-Wright E, Andre-Gregoire G, Jacobs K A, et al. Pharmacological targeting of apelin impairs glioblastoma growth. Brain. 2017; 140(11):2939-2954. [0272] Hasan, J. et al. Quantitative Angiogenesis Assays in vivo—A Review. Angiogenesis. 2004; 7, 1-16. [0273] Heo K, Kim Y H, Sung H J, et al. Hypoxia-induced up-regulation of apelin is associated with a poor prognosis in oral squamous cell carcinoma patients. Oral Oncology. 2012; 48(6):500-506. [0274] Hou J, Zhong T, Guo T, et al. Apelin promotes mesenchymal stem cells survival and vascularization under hypoxic-ischemic condition in vitro involving the upregulation of vascular endothelial growth factor. Experimental and Molecular Pathology. 2017; 102(2):203-209. [0275] Iturrioz X, Gerbier R, Leroux V, Alvear-Perez R, Maigret B, Llorens-Cortes C. By Interacting with the C-terminal Phe of Apelin, Phe255 and Trp259 in Helix VI of the Apelin Receptor Are Critical for Internalization. J Biol Chem. 2010; 285(42):32627-32637. [0276] Jakobsson, L. et al. Endothelial cells dynamically compete for the tip cell position during angiogenic sprouting. Nature Cell Biology. 2010; 12, 943-953. [0277] Japp A G, Cruden N L, Barnes G, et al. Acute Cardiovascular Effects of Apelin in Humans: Potential Role in Patients With Chronic Heart Failure. Circulation. 2010; 121(16):1818-1827. [0278] Kawamata Y, Habata Y, Fukusumi S, et al. Molecular properties of apelin: tissue distribution and receptor binding. 2001; 1538:162-171. [0279] Kasai A, Ishimaru Y, Kinjo T, et al. Apelin Is a Crucial Factor for Hypoxia-Induced Retinal Angiogenesis. ATVB. 2010; 30(11):2182-2187. [0280] Kasai A, Ishimaru Y, Higashino K, et al. Inhibition of apelin expression switches endothelial cells from proliferative to mature state in pathological retinal angiogenesis. Angiogenesis. 2013; 16(3):723-734. [0281] Khan P, Maloney P R, Hedrick M, et al. Functional Agonists of the Apelin (APJ) Receptor. In: Probe Reports from the NIH Molecular Libraries Program. Bethesda (Md.): National Center for Biotechnology Information (US); 2010. [0282] Kleinz M J, Davenport A P. Immunocytochemical localization of the endogenous vasoactive peptide apelin to human vascular and endocardial endothelial cells. 2004; 118:119-125. [0283] Langelaan D N, Reddy T, Banks A W, Dellaire G, Dupré DJ, Rainey J K. Biochimica et Biophysica Acta Structural features of the apelin receptor N-terminal tail and first transmembrane segment implicated in ligand binding and receptor trafficking. BBA—Biomembr. 2013; 1828:1471-1483. [0284] Le Gonidec S, Chaves-Almagro C, Bai Y, et al. Protamine is an antagonist of apelin receptor, and its activity is reversed by heparin. The FASEB Journal. 2017; 31(6):2507-2519. [0285] Li F Y, Li L, Qin X, et al. Apelin-induced vascular smooth muscle cell proliferation: the regulation of cyclin D1. Frontiers in bioscience: a journal and virtual library. 2008; 13:3786-3792. [0286] Li X, Zhang X, Li F, et al. 14-3-3 mediates apelin-13-induced enhancement of adhesion of monocytes to human umbilical vein endothelial cells. Acta Biochim Biophys Sin (Shanghai). 2010; 42(6):403-409. [0287] Macaluso N J M, Pitkin S L, Maguire J J, Davenport A P, Glen R C. Discovery of a Competitive Apelin Receptor (APJ) Antagonist. ChemMedChem. 2011; 6(6):1017-1023. [0288] Maguire J J, Kleinz M J, Pitkin S L, Davenport A P. Isoform in the Human Heart Vasoactive Mechanisms and Inotropic Action in Disease. 2009:598-604. [0289] Maloney P R, Khan P, Hedrick M, et al. Discovery of 4-oxo-6-((pyrimidin-2-ylthio)methyl)-4H-pyran-3-yl 4-nitrobenzoate (ML221) as a functional antagonist of the apelin (APJ) receptor. Bioorg Med Chem Lett. 2012; 22(21):6656-6660. [0290] Mota N De, Goazigo A R, Messari S El, et al. Apelin, a potent diuretic neuropeptide counteracting vasopressin actions through inhibition of vasopressin neuron activity and vasopressin release. 2004; 101. [0291] Muto J, Shirabe K, Yoshizumi T, et al. The Apelin-APJ System Induces Tumor Arteriogenesis in Hepatocellular Carcinoma. Anticancer Res. 2014; 34(10):5313-5320. [0292] Picault F-X, Chaves-Almagro C, Projetti F, Prats H, Masri B, Audigier Y. Tumour co-expression of apelin and its receptor is the basis of an autocrine loop involved in the growth of colon adenocarcinomas. Eur J Cancer. 1 févr 2014; 50(3):663-74. [0293] Schreiber C A, Holditch S J, Ikeda A G and Y. Sustained ELABELA Gene Therapy in High-salt Diet-induced Hypertensive Rats [Internet]. Vol. 16, Current Gene Therapy. 2016 [cité 5 mai 2020]. p. 349-60. Disponible sur: http://www.eurekaselect.com/147524/article [0294] Tatemoto K, Hosoya M, Habata Y, et al. Isolation and Characterization of a Novel Endogenous Peptide Ligand for the Human APJ Receptor. 1998; 476:471-476. [0295] Trifonov L, Afri M, Palczewski K, Gruzman* EEK and A. An Expedient Synthesis of CMF-019: (S)-5-Methyl-3-{1-(pentan-3-yl)-2-(thiophen-2-ylmethyl)-1H-benzo[d]imidazole-5-carboxamido}hexanoic Acid, a Potent Apelin Receptor (APJ) Agonist. Medicinal Chemistry. Published Oct. 31, 2018. [0296] Liu M, Li H, Zhou Q, et al. ROS-Autophagy pathway mediates monocytes-human umbilical vein endothelial cells adhesion induced by apelin-13. Journal of Cellular Physiology. 2018; 233(10):6839-6850. [0297] Li Y, Bai Y, Jiang Y, et al. Apelin-13 Is an Early Promoter of Cytoskeleton and Tight Junction in Diabetic Macular Edema via PI-3K/Akt and MAPK/Erk Signaling Pathways. BioMed Research International. 2018. [0298] Picault F-X, Chaves-Almagro C, Projetti F, Prats H, Masri B, Audigier Y. Tumour co-expression of apelin and its receptor is the basis of an autocrine loop involved in the growth of colon adenocarcinomas. Eur J Cancer. 1 févr 2014; 50(3):663-74. [0299] Podgórska M, Diakowska D, Pietraszek-Gremplewicz K, Nienartowicz M, Nowak D. Evaluation of Apelin and Apelin Receptor Level in the Primary Tumor and Serum of Colorectal Cancer Patients. J Clin Med [Internet]. 20 sept 2019 [cité 29 Jan. 2020]; 8(10). [0300] Wang M, Gupta R C, Rastogi S, et al. Effects of acute intravenous infusion of apelin on left ventricular function in dogs with advanced heart failure. J Card Fail. 2013; 19(7):509-516. [0301] Wan Y, Zeng Z, Xi M, et al. Dysregulated microRNA-224/apelin axis associated with aggressive progression and poor prognosis in patients with prostate cancer. Human Pathology. 2015; 46(2):295-303. [0302] Wysocka M B, Pietraszek-gremplewicz K, Nowak D. The Role of Apelin in Cardiovascular Diseases, Obesity and Cancer. 2018; 9:1-15. [0303] Wysocka M B, Pietraszek-Gremplewicz K, Nowak D. The Role of Apelin in Cardiovascular Diseases, Obesity and Cancer. Front Physiol. 2018; 9. [0304] Xin Q, Cheng B, Pan Y, Liu H, yang C, Chen J, et al. Neuroprotective effects of apelin-13 on experimental ischemic stroke through suppression of inflammation. Peptides. 1 Jan. 2015; 63:55-62. [0305] Yeganeh-Hajahmadi M, Najafipour H, Rostamzadeh F. The differential effects of low and high doses of apelin through opioid receptors on the blood pressure of rats with renovascular hypertension. Hypertension Research. 2017; 40(8):732-737. [0306] Zhou N, Fan X, Mukhtar M, et al. Cell—cell fusion and internalization of the CNS-based, 2003; 307:22-36.