RADIOLABELING KIT AND METHOD FOR RADIOLABELING DOTA-HYNIC-panPSMA

Abstract

The present invention relates to methods and kits for radiolabeling DOTA-HYNIC-panPSMA, in particular with actinium-225, terbium-161 or gallium-67 or -68, as well as the therapeutic and diagnostic use of such radiolabeled targeting agents.

Description

[0001] RADIOLABELING KIT AND METHOD FOR RADIOLABELING DOTA-HYNIC-panPSMA

[0002] TECHNICAL FIELD

[0003] The present invention is related to the field of nuclear medicine. More particular, the invention relates to radiolabeling of targeting agents, with radionuclides, more particular metal radio nuclides. The obtained radiolabeled targeting agents, may be used in therapeutic applications such as for treating tumours and cancer, such as prostate cancer or in imaging techniques, such as Positron Emission Tomography (PET) or Single-Photon Emission Computed Tomography (SPECT), for example in vivo imaging diagnosis, such as of tumours and cancer, such as prostate cancer.

[0004] BACKGROUND

[0005] Radiotherapy and diagnosis, often called theragnostics (interchangeably indicated “theranostics”) has been rapidly expanding over the last two decades. One particular approach that has reached a lot of attention is the detection and targeting of prostate specific membrane antigen (PSMA) for diagnosis and radiotherapy of a range of cancers or tumours, and especially of course of prostate cancer. Numerous ligands have been developed to target PSMA, including the Lys-Urea-Gly dipeptide PSMA ligand (cf. review by Eiber et al., 2017 - J Nucl Med 2017; 58:67S-76S).

[0006] The best known example is probably PSMA-11 or gozetotide, comprising an HBED-CC chelator conjugated to the Lys-Urea-Gly dipeptide (Eder et al., 2012 - Bioconjugate Chem. 2012, 23, 688- 697 and Eder et al., 2014 - Pharmaceuticals (Basel) 2014 Jun 30;7(7):779-96) and its DOTA conjugated counterpart PSMA-617 having a slightly different linker between the chelator and the PSMA ligand (Afshar-Oromieh et al., 2015 - J Nucl. Med. 2015;56:1697-1705).

[0007] More recently however, new variants have been tested, with a different linker between the chelator and the PSMA ligand.

[0008] One particular interesting example thereof is the radiotherapeutic177Lu radiolabeled DOTA- HYNIC-iPSMA (or DOTA-HYNIC-panPSMA) molecule reported by Luna-Gutierrez and coworkers (Pharmaceutics. Jul 20 2023; 15(7)) and its99mTc radiolabeled diagnostic companion EDDA- HYNIC-iPSMA (Ferro-Flores et al., 2017 - Nucl Med Biol. 2017 May:48:36-44).

[0009] Lutetium-177 however has certain disadvantages such as relatively low linear energy transfer (LET) and can cause damage to surrounding (non-targeted) tissue. Radiolabeling the existing DOTA-HYNIC-panPSMA molecule with for example actinium-225 or terbium-161 would solve at least some of these radiotherapy issues.

[0010] Actinium-225 has a potentially higher therapeutic efficacy than lutetium- 177 due to emitting alpha particles with high linear energy transfer (LET), which can cause more complex and effective DNA double-strand breaks in cancer cells (Ruigrok et al., 2022 - Eur J Nucl Med Mol Imaging 49, 3627- 3638). Actinium-225 also affects fewer surrounding cells (1-2 cells) compared to lutetium-177, which can impact 100-200 cells. This means actinium-225 can deliver more precise and localized treatment, minimizing damage to healthy tissues. Actinium-225 therapy also tends to have less impact on the red bone marrow and blood system compared to lutetium-177. Finally, the Half-Life of actinium-225 is more favourable (9.92 days), which makes it suitable for prolonged therapeutic effects. These advantages make actinium-225 a promising option for targeted radiotherapy, especially in cases where lutetium-177 is less effective (Lin et al., 2021 - Cancer Biother Radiopharm. 2021 Apr;36(3):237-251).

[0011] Alternatively, also terbium-161 has advantages over lutetium-177 in radiotherapy. Terbium-161 has enhanced therapeutic efficacy over lutetium-177 since it emits beta particles, gamma radiation, and a substantial number of conversion and Auger electrons. These short-ranged electrons can effectively eliminate microscopic metastases that might not be visible on imaging. Terbium-161 also shows improved dose distribution. The emission of conversion and Auger electrons allows for a more localized and effective dose distribution, potentially increasing the therapeutic efficacy compared to lutetium-177. The Half-Life of terbium-161 is 6.95 days, which is comparable to that of lutetium-177, making it suitable for similar clinical applications. The radiation safety can also be improved since terbium-161 provides an immediate decrease in dose rate, which can improve general radiation safety and potentially extend treatment room capacity for radioligand therapy. These properties make terbium-161 a promising candidate for targeted radionuclide therapy, especially in treating minimal residual disease and microscopic metastases (Muller et al., 2023 - Eur J Nucl Med Mol Imaging Volume 50, pages 3181-3184).

[0012] For radiodiagnosis, gallium-67 or -68 radiolabeling has been made relatively simple due to the widespread use of new generations of generators that allow for direct elution into a mixture of chelator-linked targeting agent and buffer. Radiolabeling the DOTA-conjugated HYNIC-panPSMA is hence also a goal of the invention since gallium is easier in use than technetium-99m.

[0013] Radiolabeling a molecule designed for lutetium-177 with another radioligand such as actinium- 225, terbium-161 , or gallium-67 or -68 is however not immediately straight-forward and forms the goal of the present invention. In addition, the present invention provides for improved radiolabeling conditions with lutetium-177.

[0014] SUMMARY OF THE INVENTION

[0015] The present invention lies in providing a radiolabeling method and kit for radiolabeling a DOTA functionalized targeting agent with actinium-225, terbium-161 , lutetium-177 or gallium-67 or -68.

[0016] More preferably, said targeting agent comprises a 1 ,4,7,10-tetraazacyclododecano-N,N',N",N""- tetraacetic acid (DOTA) chelator, bound to the molecule hydrazinonicotinamide (HYNIC), which is heterocyclic in nature, generates a rigid chemical structure that minimises the number of conformers and intramolecular hydrogen bonds, thereby resulting in a spatial orientation of the active site (Lys(Nal)-NH-CO-NH-Glu) in the molecule that favours biological recognition of the PSMA protein (i.e. DOTA-HYNIC-Lys(Nal)-Urea-Glu), as reported by Luna-Gutierrez and coworkers (Pharmaceutics. Jul 20 2023; 15(7)). More preferably the molecule is called DOTA- HYNIC-iPSMA or DOTA-HYNIC-panPSMA and has the chemical name: 1 ,4,7,10- tetraazacyclododecane-N,Nz,NZZ,N"'-tetraacetic acid-HYNIC-Lys(Nal)-Urea-Glu. It has a Molecular Weight of 1038g / mol and is defined by chemical formula (I):

[0017] The invention provides for the following aspects:

[0018] Aspect 1. A method for radiolabeling a DOTA functionalized targeting agent with a metal radionuclide (isotope) being terbium-161 , actinium-225, lutetium-177, gallium-67, or gallium-68, comprising the steps of: a) providing a DOTA functionalized targeting agent; b) optionally, providing a buffering agent or buffer solution, allowing to maintain the pH in the range of 3.5 to 7.0, preferably 3.5 to 6.5, preferably 4.0 to 6.0, preferably 4.5 to 5.5, more preferably 4.8 to 5.3; c) providing a suitable amount of radionuclide solution, preferably a solution of terbium-161 actinium-225 or gallium-67 or -68 in aqueous HCI; d) contacting the solution of step c) into a mixture of a) and b) and allowing the radiolabeling reaction to take place, preferably at a temperature of 80 to 95°C, for a period of between 5 and 35 minutes; thereby obtaining a radiolabeled targeting agent; e) optionally, purifying the radiolabeling mixture by eliminating unbound radionuclide from the radiolabeling mixture, thereby obtaining a purified radiolabeled targeting agent.

[0019] Aspect 2. The method according to aspect 1 , wherein the targeting agent is a PSMA ligand and wherein the DOTA chelator is attached to the targeting agent via a HYNIC linker.

[0020] Aspect 3. The method according to aspect 1 or 2, wherein the DOTA functionalized targeting agent is DOTA-HYNIC-panPSMA defined by chemical formula (I)

[0021] In a preferred embodiment, said DOTA-HYNIC-panPSMA is radiolabelled with Lutetium-177 according to formula II:

[0022]

[0023] In a preferred embodiment, said DOTA-HYNIC-panPSMA is radiolabelled with Actinium-225 according to formula III:

[0024] In a preferred embodiment, said DOTA-HYNIC-panPSMA is radiolabelled with Terbium-161 according to formula IV:

[0025]

[0026] In a preferred embodiment, said DOTA-HYNIC-panPSMA is radiolabelled with Gallium-68 according to formula V or radiolabelled with Gallium-67 according to Formula VI: Aspect 4. The method according to aspect 1 , 2 or 3, said buffering agent or buffer solution is a phosphate, nitrate, HEPES, acetate, formate, TRIS, and citrate or a mixture thereof, preferably an acetate or formate buffer, more preferably a sodium ascorbate, sodium acetate, ammonium acetate, or sodium formate buffer.

[0027] Aspect 5. The method according to any one of aspects 1 to 4, wherein the purification step e), when present, is done by means of solid phase extraction or reverse phase extraction, preferably over a silica-based substrate with strong hydrophobicity such as C18-coated silica, C8- coated silica or C4-coated silica, depending on the targeting ligand to be labeled.

[0028] Typically, SPE substrates are made of high-purity silica, coated with C18, which is a hydrophobic and non-polar stationary phase, selectively retaining non-polar and moderately polar compounds such as lipids, steroids, and pesticides while allowing more polar compounds such as sugars and amino acids (or peptides) to pass through.

[0029] Aspect 6. The method according to any one of previous aspects, wherein additionally a metal inhibitor and / or a free (non-bound) radiometal sequestering agent is added to the radiolabeling mixture.

[0030] Said sequestering agent is used to sequester non-bound radionuclides, while said metal inhibitor is a co-chelating agent, capable of inactivating metals (e.g. due to break-through of other metals during elution of a generator) other than radioactive metal without interfering with the chelation between the radioactive metal and the said chelate-functionalized targeting agent, under the conditions of the labeling reaction.

[0031] Aspect 7. The method according to aspect 6, wherein said metal inhibitor is a sugar, preferably a short-chain sugar or oligosaccharide, such as comprising up to 7 monosaccharide units, and / or wherein said sequestering agent is EDTA, DTPA, or combinations thereof.

[0032] In a preferred embodiment, the sequestering agent is used to sequester non-bound radionuclides, and the sugars as defined herein are used to sequester radiometal impurities resulting from generator elution (possible break-through).

[0033] In a preferred embodiment, said radiometal is terbium-161 and the sequestering agent is EDTA or DTPA to sequester non-bound terbium radionuclides, optionally combined with a sugar as defined herein to sequester radiometal impurities resulting from generator elution (break-through). Aspect 8. The method according to any one of aspects 6 to 7, wherein said metal inhibitor is selected from the group comprising: monosaccharides and their derivatives, disaccharides and their derivatives, and cyclodextrins.

[0034] Aspect 9. The method according to any one of aspects 6 to 8, wherein said metal inhibitor is selected from the group comprising: Glucose, D-Fructose, Beta-cyclodextrin, D-Mannose, and beta-cyclodextrin, more preferably D-mannose.

[0035] Aspect 10. The method according to any one of aspects 6 to 9, wherein said metal inhibitor and said functionalised agent are not chemically linked.

[0036] Aspect 11. The method according to any one of aspects 6 to 10, wherein said metal inhibitor and said functionalised agent are chemically linked, through a linker that is unstable in the radiolabeling conditions.

[0037] Aspect 12. The method according to any one of previous aspects, wherein the chelate- functionalized targeting agent comprises a targeting moiety selected from the list comprising a peptide, a urea-based peptidomimetic, a polypeptide, a protein, a vitamin, a saccharide, an antibody, a nucleic acid, an aptamer, an antisense oligonucleotide, or an organic molecule. In a preferred embodiment, said targeting agent is a PSMA-ligand or inhibitor, more preferably the Lys(Nal)-NH-CO-NH-Glu PSMA inhibitor.

[0038] Aspect 13. The method according to any one of aspects 1 to 12, wherein the radionuclide is terbium-161 and wherein the radiolabeling reaction is carried out in an acetate buffer at a pH of between 4.0 and 5.5, more preferably of between 4.5 and 5, or of about 5, or 5.1. Preferably a 0,4M ammonium acetate buffer salt is used. More preferably, ascorbic acid is added as a stabiliser and / or EDTA is added when high activity e.g. more than 300 or 400MBq (or 10mCi) is added in activity. More particularly, the reaction is carried out at 90°C for about 25 to 35 minutes, preferably for about 30 minutes. Generally, no post-purification of the radiolabeled targeting agent is needed.

[0039] Aspect 14. The method according to any one of aspects 1 to 12, wherein the radionuclide is actinium-225, and wherein the radiolabeling reaction is carried out in a 0,04M HCI radiolabeling mixture with ascorbic acid as a stabiliser (0,5M) at a pH of between 4.0 and 5.5, more preferably of between 4.5 and 5, radiolabeled at about 90 to 95°C, for a time of about 10 to 20 minutes, such as 15 min. Said mixture is optionally formulated into a sodium ascorbate and pentetic acid (DTPA) buffer. A C18 post-radiolabeling purification of the radiolabeled targeting agent may be needed.

[0040] Aspect 15. The method according to any one of aspects 1 to 12, wherein the radionuclide is gallium-67 or -68, and wherein the radiolabeling reaction is carried out in an acteate buffer, such as a sodium acetate buffer at pH of between 3.5 and 5.5, more preferably of between 4.0 and 5.0, at a temperature of about 80 to 95°C, such as at about 90°C for about 5 to 30 minutes, such as for about 10 to 15 minutes. Generally, no post-purification of the radiolabeled targeting agent is needed. An activity of up to 2000 MBq (about 50mCi), such as from 100 to 2000MBq can be used in this setting, but typically in a kit form an activity of up to 50MBq would be used, while in a high activity kit setting, up to 2GBq or more could be used.

[0041] Aspect 16. The method according to any one of the previous aspects, wherein the radionuclide is actinium-225 and wherein the activity is from 1 to 20 MBq, such as up to 5 MBq, such as from 5 to 20MBq.

[0042] Aspect 17. The method according to any one of the previous aspects, wherein the radionuclide is lutetium-177 or terbium-161 and wherein the activity is from 1 to 20 GBq, such as up to 400 MBq, such as from 100 to 400MBq, or up to 20Gbq such as from 400MBq to 10GBq.

[0043] Aspect 18. The method according to any one of the previous aspects, wherein the radionuclide is lutetium-177 and wherein the radiolabelling reaction takes place in a radiolabelling mixture comprising an acetate buffer with pH of between 4.0 and 6.5, more preferably of between 4.5 and 6.0, even more preferably of between 5.0 and 5.5, applying about 85°C for 10 to 15 minutes, preferably wherein said radiolabelling mixture additionally comprises 50mg / mL mannitol, and / or 50mg / mL ascorbic acid.

[0044] Aspect 19. The method according to any one of aspects 1 to 18, wherein additionally a stabiliser is added to the radiolabeling mixture that prevents radiolysis (product degradation) of the chelate-functionalized targeting agent, wherein said stabiliser is selected from the group consisting of: ascorbic acid, dehydroascorbic acid, gentisic acid, cysteine, N-acetylcysteine, and methionine, or a salt thereof, preferably as a solution to the radiolabeling mixture prior to radiolabeling. Aspect 20. The method according to aspect 19, wherein the radiolabeling mixture comprises at least 10.0 mg, preferably at least 15.0 mg, preferably at least 20.0 mg, preferably at least 25.0 mg, preferably at least 30.0 mg of stabiliser, more preferably up to 50.0mg of ascorbic acid.

[0045] Aspect 21. The method according to aspect 19, wherein the radiolabeling mixture comprises at most 100.0 mg, preferably at most 90.0 mg, preferably at most 80.0 mg, preferably at most 70.0 mg, preferably at most 60.0 mg, preferably at most 50.0 mg of stabiliser, more preferably of ascorbic acid.

[0046] Aspect 22. The method according to any one of aspects 19 to 21 , wherein the radiolabeling mixture comprises at least 1.0 mg, preferably at least 1.2 mg, preferably at least 1.4 mg, preferably at least 1.6 mg, preferably at least 1.8 mg, preferably at least 2.0 mg, preferably at least 2.2 mg, preferably at least 2.4 mg, preferably at least 2.5 mg of stabiliser per 1850MBq (50 mCi) metal radionuclide.

[0047] Aspect 23. The method according to any one of aspects 19 to 21 , wherein the radiolabeling mixture comprises at most 100.0 mg, preferably at most 80.0 mg, preferably at most 60.0 mg, preferably at most 40.0 mg, preferably at most 20.0 mg, preferably at most 15.0 mg, preferably at most 10.0 mg, preferably at least 7.5 mg, preferably at most 5.0 mg of stabiliser per 1850MBq (50 mCi) metal radionuclide.

[0048] Aspect 24. The method according to any one of aspects 19 to 21 , wherein the amount of stabiliser in the radiolabeling mixture, is such that the weight ratio of the weight of stabiliser over the weight of the DOTA-functionalized targeting agent in the radiolabeling mixture, is at least 40, preferably at least 80, preferably at least 150, preferably at least 300, preferably at least 600, preferably at least 1000, preferably at least 1500, preferably at least 2000.

[0049] Aspect 25. The method according to any one of aspects 19 to 21 , wherein the solution of stabiliser has a concentration of at least 0.14 mg / ml, preferably at least 0.30 mg / ml, preferably at least 0.50 mg / ml, preferably at least 1.00 mg / ml, preferably at least 2.00 mg / ml, preferably at least 4.00 mg / ml, preferably at least 5.00 mg / ml, preferably at least 7.00 mg / ml, preferably at least 9.00 mg / ml, preferably at least 10.00 mg / ml, preferably wherein the stabilizer is ascorbic acid. Aspect 26. The method according to any one of aspects 19 to 25, wherein the stabiliser is ascorbic acid, dehydroascorbic acid or a salt thereof, and optionally wherein said stabiliser is provided together with a bisulfite and or a metabisulfite.

[0050] Aspect 27. A terbium-161 radiolabeled DOTA functionalized targeting agent obtained by the method according to any one of aspects 1 to 13, and 17 to 26.

[0051] Aspect 28. The radiolabeled targeting agent according to aspect 27, which is terbium-161 radiolabeled DOTA-HYNIC-panPSMA.

[0052] Aspect 29. An actinium-225 radiolabeled DOTA functionalized targeting agent obtained by the method according to any one of aspects 1 to 12, 14, and 16 to 26.

[0053] Aspect 30. The radiolabeled targeting agent according to aspect 29, which is actinium-225 radiolabeled DOTA-HYNIC-panPSMA.

[0054] Aspect 31. A gallium-68 radiolabeled DOTA functionalized targeting agent obtained by the method according to any one of aspects 1 to 12, 15, and 18 to 26.

[0055] Aspect 32. The radiolabeled targeting agent according to aspect 31 , which is gallium-68 radiolabeled DOTA-HYNIC-panPSMA.

[0056] Aspect 33. A radiolabeling kit, suitable for producing an actinium-225, a terbium-161 , or a gallium-68 or -67 radiolabeled DOTA functionalized targeting agent comprising:

[0057] - a DOTA functionalized targeting agent, preferably in dried or lyophilized form;

[0058] - a buffering agent or buffer solution, allowing to maintain the pH in the range of 3.5 to 7, such as 3.5 to 6.5 or 4.0 to 5,5, more preferably 4,5 to 5.0, optionally in dried or lyophilized form;

[0059] - optionally a metal inhibitor and / or a free (non-bound) radiometal sequestering agent;

[0060] - optionally a stabiliser selected from the group consisting of: ascorbic acid, sodium ascorbate, dehydroascorbic acid, gentisic acid, cysteine and methionine, or a salt thereof, preferably as a solution, or in a dried or lyophilized form; - optionally, a post-radiolabeling purification means suitable for eliminating unbound metal radionuclide from the radiolabeling mixture after the radiolabeling reaction took place.

[0061] Aspect 34. The kit according to aspect 33, wherein said metal inhibitor, is a co-chelating agent, capable of inactivating metals other than the desired radionuclide metal without interfering with the chelation between the radionuclide metal and the said chelate-functionalized targeting agent, under the conditions of the labeling reaction.

[0062] Aspect 35. The kit according to aspect 33 or 34, wherein said targeting agent is a PSMA- ligand or inhibitor, more preferably the Lys(Nal)-NH-CO-NH-Glu PSMA inhibitor.

[0063] Aspect 36. The kit according to any one of aspects 33 to 35, wherein the targeting agent is a PSMA ligand and wherein the DOTA chelator is attached to the targeting agent via a HYNIC linker.

[0064] Aspect 37. The kit according to any one of aspects 33 to 35, wherein the DOTA functionalized targeting agent is DOTA-HYNIC-panPSMA as defined by general formula (I)

[0065] In a preferred embodiment, said DOTA-HYNIC-panPSMA is radiolabelled with Lutetium-177 according to formula II:

[0066]

[0067] In a preferred embodiment, said DOTA-HYNIC-panPSMA is radiolabelled with Actinium-225 according to formula III:

[0068] In a preferred embodiment, said DOTA-HYNIC-panPSMA is radiolabelled with Terbium-161 according to formula IV:

[0069]

[0070] In a preferred embodiment, said DOTA-HYNIC-panPSMA is radiolabelled with Gallium-68 according to formula V or radiolabelled with Gallium-67 according to formula V: Aspect 38. The kit according to any one of aspects 33 to 37, wherein said buffering agent or buffer solution is a phosphate, nitrate, HEPES, acetate, formate, TRIS, and citrate or a mixture thereof, preferably a sodium ascorbate, sodium acetate, ammonium acetate, or sodium formate buffer.

[0071] Aspect 39. The kit according to any one of aspects 33 to 38, wherein the post-radiolabeling purification means is a solid phase extraction or reverse phase extraction means, preferably a silica-based substrate with strong hydrophobicity such as C18-coated silica, C8-coated silica, C6- coated silica or C4-coated silica, depending on the targeting ligand to be labeled.

[0072] Typically, SPE substrates are made of high-purity silica, coated with C18, which is a hydrophobic and non-polar stationary phase, selectively retaining non-polar and moderately polar compounds such as lipids, steroids, and pesticides while allowing more polar compounds such as sugars and amino acids to pass through.

[0073] Aspect 40. The kit according to any one of aspects 33 to 39, wherein the DOTA-functionalized targeting agent and the buffer are provided in dried or lyophilized form, either in two separate or in one single vial. In one embodiment, also the metal inhibitor and / or stabilising agent are present in lyophilised form, either in a separate vial, or combined with either the buffer or targeting agent.

[0074] Aspect 41. The kit according to any one of aspects 33 to 40, wherein the terbium-161 , is provided as a solution with an activity of at least 5.0 mCi / ml, preferably at least 7.0 mCi / ml, preferably at least 10.0 mCi / ml, preferably at least 15.0 mCi / ml, preferably at least 20.0 mCi / ml, preferably at least 25.0 mCi / ml, preferably at least 30.0 mCi / ml, preferably at least 35.0 mCi / ml, preferably at least 40.0 mCi / ml, preferably at least 45.0 mCi / ml, preferably at least 50.0 mCi / ml, preferably at least 75.0 mCi / ml, preferably at least 100.0 mCi / ml.

[0075] Aspect 42. The kit according to any one of aspects 33 to 40, wherein the actinium-225 is provided as a solution with an activity of at least 5.0 mCi / ml, preferably at least 7.0 mCi / ml, preferably at least 10.0 mCi / ml, preferably at least 15.0 mCi / ml, preferably at least 20.0 mCi / ml, preferably at least 25.0 mCi / ml, preferably at least 30.0 mCi / ml, preferably at least 35.0 mCi / ml, preferably at least 40.0 mCi / ml, preferably at least 45.0 mCi / ml, preferably at least 50.0 mCi / ml, preferably at least 75.0 mCi / ml, preferably at least 100.0 mCi / ml. Aspect 43. The kit according to any one of aspects 33 to 40, wherein the gallium-68 is provided as a solution with an activity of at least 5.0 mCi / ml, preferably at least 7.0 mCi / ml, preferably at least 10.0 mCi / ml, preferably at least 15.0 mCi / ml, preferably at least 20.0 mCi / ml, preferably at least 25.0 mCi / ml, preferably at least 30.0 mCi / ml, preferably at least 35.0 mCi / ml, preferably at least 40.0 mCi / ml, preferably at least 45.0 mCi / ml, preferably at least 50.0 mCi / ml, preferably at least 75.0 mCi / ml, preferably at least 100.0 mCi / ml.

[0076] Aspect 44. The kit according to any one of aspects 33 to 43, comprising at least 10.0 mg, preferably at least 15.0 mg, preferably at least 20.0 mg, preferably at least 25.0 mg, preferably at least 30.0 mg of stabiliser, wherein said stabiliser preferably is ascorbic acid.

[0077] Aspect 45. The kit according to any one of aspects 33 to 44, wherein the kit comprises at most 100.0 mg, preferably at most 90.0 mg, preferably at most 80.0 mg, preferably at most 70.0 mg, preferably at most 60.0 mg, preferably at most 50.0 mg of stabiliser, more preferably of ascorbic acid.

[0078] Aspect 46. The kit according to any one of aspects 33 to 45, wherein the kit comprises at least 1 .0 mg, preferably at least 1.2 mg, preferably at least 1 .4 mg, preferably at least 1 .6 mg, preferably at least 1.8 mg, preferably at least 2.0 mg, preferably at least 2.2 mg, preferably at least 2.4 mg, preferably at least 2.5 mg of stabiliser per 1850 MBq (50 mCi) metal radionuclide.

[0079] Aspect 47. The kit according to any one of aspects 33 to 46, wherein the kit comprises at most 100.0 mg, preferably at most 80.0 mg, preferably at most 60.0 mg, preferably at most 40.0 mg, preferably at most 20.0 mg, preferably at most 15.0 mg, preferably at most 10.0 mg, preferably at least 7.5 mg, preferably at most 5.0 mg of stabiliser per 1850 MBq (50 mCi) metal radionuclide.

[0080] Aspect 48. The kit according to any one of aspects 33 to 47, comprising at least 10.0 pg, preferably at least 15.0 pg, preferably at least 20.0 pg, preferably at least 25.0 pg of said DOTA- functionalized targeting agent.

[0081] Aspect 49. The kit according to any one of aspects 33 to 48, comprising at least 2.0 pg, preferably at least 4.0 pg, preferably at least 6.0 pg, preferably at least 8.0 pg, preferably at least 10.0 pg of said metal inhibitor. Aspect 50. The kit according to any one of aspects 33 to 49, comprising at least 50.0 mg, preferably at least 75.0 mg, preferably at least 100.0 mg, preferably at least 125.0 mg, preferably at least 150.0 mg of said buffering agent.

[0082] Aspect 51. The kit according to any one of aspects 33 to 50, wherein the amount of stabiliser in the kit, is such that the weight ratio of the weight of stabiliser over the weight of the chelate- functionalized targeting agent in the kit, is at least 40, preferably at least 80, preferably at least 150, preferably at least 300, preferably at least 600, preferably at least 1000, preferably at least 1500, preferably at least 2000. In a preferred embodiment, said stabiliser is ascorbic acid.

[0083] Aspect 52. The kit according to any one of aspects 33 to 51 , wherein the solution of stabiliser, has a concentration of at least 0.14 mg / ml, preferably at least 0.30 mg / ml, preferably at least 0.50 mg / ml, preferably at least 1.00 mg / ml, preferably at least 2.00 mg / ml, preferably at least 4.00 mg / ml, preferably at least 5.00 mg / ml, preferably at least 7.00 mg / ml, preferably at least 9.00 mg / ml, preferably at least 10.00 mg / ml. In a preferred embodiment, said stabiliser is ascorbic acid.

[0084] Aspect 53. The kit according to any one of aspects 33 to 52, wherein the stabiliser is provided in the kit together with a bisulfite and or a metabisulfite.

[0085] Aspect 54. The kit according to any one of aspects 34 to 54, wherein said metal inhibitor is a sugar, preferably a short-chain sugar or oligosaccharide, such as comprising up to 7 monosaccharide units.

[0086] Aspect 55. The kit according to any one of aspects 34 to 54, wherein said metal inhibitor is selected from the group comprising: monosaccharides and their derivatives, disaccharides and their derivatives, and polysaccharides and cyclodextrins.

[0087] Aspect 56. The kit according to any one of aspects 34 to 55, wherein said metal inhibitor is selected from the group comprising: Glucose, D-Fructose, Beta-cyclodextrin, and D-Mannose.

[0088] Aspect 57. The kit according to any one of aspects 34 to 56, wherein said metal inhibitor and said functionalised agent are not chemically linked.

[0089] Aspect 58. The kit according to any one of aspects 34 to 57, wherein said metal inhibitor and said functionalised agent are chemically linked, through a linker that is unstable in the radiolabeling conditions. Aspect 59. An imaging method, comprising the steps of:

[0090] 1) radiolabeling an DOTA functionalized targeting agent with actinium-225, terbium-161 or gallium-68 or -67 according to the method of any one of aspects 1 to 25, preferably by using a kit according to any one of claims 33 to 58,

[0091] 2) administering to a subject a diagnostic amount of said actinium-225, terbium-161 , or gallium- 68 or -67 radiolabeled targeting agent; and,

[0092] 3) detecting said actinium-225, terbium-161 , or gallium-68 or -67 radiolabeled targeting agent when bound to its target using PET or SPECT or PET / CT or SPECT / CT imaging methods.

[0093] More preferably, actinium-225 is preferably used for SPECT or SPECT / CT imaging, while terbium-161 and gallium-68 or -67 is preferably used for PET or PET / CT imaging.

[0094] Aspect 60. A method of detecting a prostate tumour or cancer, comprising the steps of:

[0095] 1) radiolabeling DOTA-HYNIC-panPSMA with actinium-225, terbium-161 , or gallium-68 or -67 according to the method of any one of aspects 1 to 32, preferably by using a kit according to any one of claims 33 to 59,

[0096] 2) administering to a subject a diagnostic amount of actinium-225, terbium-161 , or gallium-68 or -67 radiolabeled DOTA-HYNIC-panPSMA; and,

[0097] 3) detecting binding of said actinium-225, terbium-161 , or gallium-68 or -67 radiolabeled DOTA- HYNIC-panPSMA using PET or SPECT or PET / CT or SPECT / CT imaging methods.

[0098] More preferably, actinium-225 is preferably used for SPECT or SPECT / CT imaging, while terbium-161 and gallium-68 or -67 is preferably used for PET or PET / CT imaging.

[0099] Aspect 61. The method according to aspect 60, wherein said detection is used for:

[0100] (i) initial staging of prostate cancer into intermediate, unfavourable, high, or very high-risk prostate cancer,

[0101] (ii) detecting suspected recurrence of prostate cancer and / or detection of metastasis, (iii) selection for radiotherapeutic treatment such as with Lutetium (177Lu) vipivotide tetraxetan (Pluvicto),

[0102] (iv) monitoring prostate cancer for progression into Non-Metastatic or Metastatic Castration- Resistant Prostate Cancer (nmCRPC or mCRPC), or

[0103] (v) determining response to (radio)therapy.

[0104] (vi) real-time imaging or diagnosis in a subject e.g. for use in PET scans for tracking slow biological processes or progression of those over a period of time. In some embodiments, this aspect can be carried out by a computer or processor and hence is directed by a system for in vivo diagnosis or for in vivo imaging in a subject, said system comprising a processing means (processor) configured to carry out the steps of receive real-time imaging data and presenting said imaging date in real-time to the user, thereby enabling in vivo imaging or in vivo diagnosis of said subject.

[0105] (vii) personalised medicine, or determining a personalised medicine treatment strategy.

[0106] Aspect 62. The method according to any one of aspects 59 to 61 , wherein said detection method is used to replace the need for taking a prostate biopsy or is used in SPECT, SPECT / CT, PET or PET / CT scan with MRI in clinically significant or intermediate favourable prostate cancers, or in MRI for active surveillance of prostate cancer.

[0107] Aspect 63. The actinium-225, terbium-161 , or gallium-68 radiolabeled targeting molecule according to any one of aspects 27 to 32, for use in radiodiagnosis, preferably for use in in vivo real time detection of a targeting agent.

[0108] Aspect 64. The actinium-225 or terbium-161 radiolabeled targeting molecule according to any one of aspects 27 to 30, for use in radiotherapy.

[0109] Aspect 65. A method for treatment of cancer, comprising the step of: administering an effective amount of the actinium-225 or terbium-161 radiolabeled DOTA functionalized targeting agent according to any one of aspects 27 to 30 to a patient in need thereof. In some embodiments, said patient can have been diagnosed using the method of aspect 60 to 64. Aspect 66. A method for treatment of cancer, comprising the step of: administering an effective amount of the actinium-225 or terbium-161 radiolabeled DOTA functionalized targeting agent according to any one of aspects 27 to 30 to a patient in need thereof. Aspect 67. The method according to aspect 66, wherein said cancer is over-expressing PSMA.

[0110] Aspect 68. The method according to aspect 66 or 67, wherein said cancer is selected from the group consisting of: prostate cancer, salivary gland cancer, glioblastoma, thyroid cancer, clear cell renal carcinoma, renal cell carcinoma, hepatocellular carcinoma, lung cancer, adenoid cystic carcinoma of the parotid, and breast cancer.

[0111] Aspect 69. The method according to any one of aspects 66 to 68, wherein said targeting agent is a PSMA ligand, preferably, wherein said targeting agent is Glu-Urea-Lys (Glu-NH-CONH-Lys), more preferably Lys(Nal)-NH-CO-NH-Glu, preferably coupled to the DOTA chelator via a HYNIC linker. Most preferably, said targeting agent has the chemical structure of formula (I):

[0112] In a preferred embodiment, said DOTA-HYNIC-panPSMA is radiolabelled with Lutetium-177 according to formula II:

[0113]

[0114] In a preferred embodiment, said DOTA-HYNIC-panPSMA is radiolabelled with Actinium-225 according to formula III:

[0115] In a preferred embodiment, said DOTA-HYNIC-panPSMA is radiolabelled with Terbium-161 according to formula IV:

[0116]

[0117] In a preferred embodiment, said DOTA-HYNIC-panPSMA is radiolabelled with Gallium-68 according to formula V or radiolabelled with Gallium-67 according to formula VI: In any of the above aspects directed to “methods of treatment”, the corresponding “products for use in treating”, “use of product in or for treating”, and “use of product for the manufacture of a medicament for treating” formatting is also included.

[0118] BRIEF DESCRIPTION OF THE DRAWINGS

[0119] Figure 1. Radiolabeling stability of [161Tb]Tb-DOTA-HYNIC-iPSMA targeting agent using 100, 200, or 400MBq of terbium-161 activity.

[0120] Figure 2. Evolution of uncomplexed161Tb when using different activities for radiolabeling.

[0121] Figure 3. Evolution of radiolysis products over time according to the activity used for radiolabeling.

[0122] Figure 4. Evolution of uncomplexed161Tb with and without addition of EDTA.

[0123] Figure 5. Radiolysis product with and without ascorbic acid.

[0124] Figure 6. Radiolabeling of DOTA-HYNIC-panPSMA with161Tb.

[0125] Figure 7. Radiolabeling of DOTA-HYNIC-panPSMA with177Lu.

[0126] Figure 8. [161Tb]Tb-DOTA-HYNIC-panPSMA and [177Lu]Lu-DOTA-HYNIC-panPSMA radiolabeling stability. Circular datapoints:161Tb; square datapoints:177Lu.

[0127] Figure 9. Saturation binding experiment of [161Tb]Tb-DOTA-HYNIC-panPSMA (9A) and [177Lu]Lu- DOTA-HYNIC-panPSMA (9B) on LNCaP cells.

[0128] Figure 10. Counts per minute for specific versus non-specific [161Tb]Tb-DOTA-HYNIC-iPSMA in LNCaP cells. X-axis is indicative for the concentration, with the tested concentrations being 1 , 5, 10, 25, 50 and 100 nM.

[0129] Figure 11. Membrane-bound fraction and internalized fraction of [161Tb]Tb-DOTA-HYNIC- panPSMA assessed on LNCap cells.

[0130] Figure 12. Membrane-bound fraction and internalized fraction of [177Lu]Lu-DOTA-HYNIC- panPSMA assessed on LNCaP cells.

[0131] Figure 13. Efflux of [161Tb]Tb- DOTA-HYNIC-panPSMA (13A) and [177Lu]Lu- DOTA-HYNIC- panPSMA (13B) assessed on LNCaP cells.

[0132] Figure 14. Uptake of [161Tb]Tb-DOTA-HYNIC-panPSMA (14A) and [177Lu]Lu-DOTA-HYNIC- panPSMA (14B) in different cell lines. Left condition of each cell line: 60 min; middle condition of each cell line: 120 min; right condition of each cell line: 240 min. Figure 15. Dose response curve of [161Tb]Tb-DOTA-HYNIC-iPSMA on PC3-pip cells.

[0133] Figure 16. Radio-TLC chromatograms of Ac-225 labeled DOTA-HYNIC-iPSMA. Left: in the crude reaction mixture. Right: at EOS.

[0134] Figure 17. Plot of fractions collected from the HPLC analysis at EOS applied with a gaussian fit to estimate the retention time and RCP of Ac-225-DOTA-HYNIC-panPSMA.

[0135] Figure 18. Radio-TLC chromatograms of Ac-225 labeled DOTA-HYNIC-panPSMA. Left: in the crude reaction mixture. Right: at EOS.

[0136] Figure 19. Plot of fractions collected from the HPLC analysis at EOS and 4 hr after EOS applied with a gaussian fit to estimate the retention time and RCP of Ac-225-DOTA-HYNIC-panPSMA.

[0137] Figure 20. Radio-TLC chromatograms of Ac-225 labeled DOTA-HYNIC-panPSMA. Left: in the crude reaction mixture. Right: at EOS.

[0138] Figure 21. Plot of fractions collected from the HPLC analysis at EOS applied with a gaussian fit to estimate the retention time and RCP of Ac-225-DOTA-HYNIC-panPSMA.

[0139] Figure 22. Plot of fractions collected from the HPLC analysis at EOS applied with a gaussian fit to estimate the retention time and RCP of Ga-68-DOTA-HYNIC-panPSMA. Panel A: using a TELIX kit-based approach. Panel B: using a cassette- based approach (Eckert & Ziegler).

[0140] DETAILED DESCRIPTION

[0141] As used herein, the singular forms “a”, “an”, and “the” include both singular and plural referents unless the context clearly dictates otherwise.

[0142] The terms “comprising”, “comprises” and “comprised of” as used herein are synonymous with “including”, “includes” or “containing”, “contains”, and are inclusive or open-ended and do not exclude additional, non-recited members, elements or method steps. The terms also encompass “consisting of” and “consisting essentially of”.

[0143] The recitation of numerical ranges by endpoints includes all numbers and fractions subsumed within the respective ranges, as well as the recited endpoints. This applies to numerical ranges irrespective of whether they are introduced by the expression “from... to...” or the expression “between... and...” or another expression.

[0144] The term “about” as used herein when referring to a measurable value such as a parameter, an amount, a temporal duration, and the like, is meant to encompass variations of and from the specified value, in particular variations of + / - 10% or less, preferably + / -5% or less, more preferably + / -1% or less, and still more preferably + / -0.1 % or less of and from the specified value, insofar such variations are appropriate to perform in the disclosed invention. It is to be understood that the value to which the modifier “about” refers is itself also specifically, and preferably, disclosed.

[0145] Whereas the term “one or more”, such as one or more members of a group of members, is clear per se, by means of further exemplification, the term encompasses inter alia a reference to any one of said members, or to any two or more of said members, such as, e.g., any33,34,35,36 or37 etc. of said members, and up to all said members. In another example, “one or more” or “at least one” may refer to 1 , 2, 3, 4, 5, 6, 7 or more.

[0146] The discussion of the background to the invention herein is included to explain the context of the invention. This is not to be taken as an admission that any of the material referred to was published, known, or part of the common general knowledge in any country as of the priority date of any of the claims.

[0147] Throughout this disclosure, various publications, patents and published patent specifications are referenced by an identifying citation. All documents cited in the present specification are hereby incorporated by reference in their entirety. In particular, the teachings or sections of such documents herein specifically referred to are incorporated by reference.

[0148] Unless otherwise specified, all terms used in disclosing the invention, including technical and scientific terms, have the meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. By means of further guidance, term definitions may be included to better appreciate the teaching of the present invention. When specific terms are defined in connection with a particular aspect of the invention or a particular embodiment of the invention, such connotation or meaning is meant to apply throughout this specification, i.e., also in the context of other aspects or embodiments of the invention, unless otherwise defined. For example, embodiments directed to products are also applicable to corresponding features of methods and uses.

[0149] In the following passages, different aspects or embodiments of the invention are defined in more detail. Every aspect or embodiment so defined may be combined with each of the other aspects or embodiments unless stated otherwise. In particular, any feature indicated as being preferred or advantageous in one embodiment may be combined with any other embodiment or embodiments indicated as being preferred or advantageous. Reference throughout this specification to “one embodiment”, “an embodiment” means that a particular feature, structure or characteristic described in connection with the embodiment is included in at least one embodiment of the present invention. Thus, appearances of the phrases “in one embodiment” or “in an embodiment” in various places throughout this specification are not necessarily all referring to the same embodiment. Furthermore, the particular features, structures or characteristics may be combined in any suitable manner, as would be apparent to a person skilled in the art from this disclosure, in one or more embodiments. Furthermore, while some embodiments described herein include some but not other features included in other embodiments, combinations of features of different embodiments are meant to be within the scope of the invention, and form different embodiments, as would be understood by those in the art. For example, in the appended claims, alternative combinations of claimed embodiments are encompassed, as would be understood by those in the art.

[0150] The present invention overcomes one or more of the problems identified and observed in the state of the art and allows stabilize radiolabeled chelate-functionalized targeting agents compositions with a high activity.

[0151] The present invention is related to a method for radiolabeling radiopharmaceuticals, preferably a DOTA functionalized targeting agent, with a metal radionuclide being actinium-225, terbium-161 , or gallium-68, comprising the general steps of: a) providing a DOTA functionalized targeting agent as defined herein; b) optionally, providing a buffering agent or buffer solution, allowing to maintain the pH in the range of 3.5 to 7.0, preferably 3.5 to 6.5, preferably 4.0 to 6.0, preferably 4.5 to 5.5, more preferably 4.8 to 5.3; c) providing a suitable amount of radionuclide (isotope) solution, preferably a solution of terbium- 161 , actinium-225 or gallium-68, typically eluted in aqueous HCI; d) contacting the solution of step c) into a mixture of a) and b) and allowing the radiolabeling reaction to take place, preferably at a temperature of 90 to 95°C, for a period of between 5 and 35 minutes; thereby obtaining a radiolabeled targeting agent; e) optionally, purifying the radiolabeling mixture by eliminating unbound radionuclide or radiolabeling impurities from the radiolabeling mixture, thereby obtaining a purified radiolabeled targeting agent.

[0152] The present inventors have found an efficient way to use actinium-225, terbium-161 , or gallium- 68 as a radiolabel for radiopharmaceuticals, especially radiopharmaceuticals comprising a DOTA, chelator group or a derivative thereof. More particular, actinium-225, terbium-161 , or gallium-68- or -67 labeling of a DOTA-functionalized targeting agent, more preferably a PSMA ligand or inhibitor.

[0153] In one particularly preferred embodiment, the PSMA inhibitor or ligand called DOTA-HYNIC- panPSMA, comprising a 1 ,4,7,10-tetraazacyclododecano-N,N',N",N""-tetraacetic acid (DOTA) chelator, bound to the molecule hydrazinonicotinamide (HYNIC), which is heterocyclic in nature, and generates a rigid chemical structure that minimises the number of conformers and intramolecular hydrogen bonds, thereby resulting in a spatial orientation of the active site (Lys(Nal)-NH-CO-NH-Glu).

[0154] DOTA-HYNIC-panPSMA (also referred to herein as ((1-carboxy-5-(3-(naphthalen-2-yl)-2-(6-(2- (2-(4,8,11-tris(carboxymethyl)-1 ,4,8,11-tetraazacyclotetradecan-1- yl)acetyl)hydrazineyl)nicotinamido)propanamido)pentyl)carbamoyl)glutamic acid) is depicted below in Formula (I) and was demonstrated by the inventors to be efficient using certain specific radiolabeling conditions.

[0155] “Prostate-Specific Membrane Antigen”, abbreviated as “PSMA” and also known in the art as “TAH molecule”, “N-acetyl-L-aspartyl-L-glutamate peptidase I (NAALADase I)”, and “NAAG peptidase” refers to a type II transmembrane glycoprotein that is highly expressed on the surface of prostate epithelial cells and is significantly overexpressed in prostate cancer cells, particularly in metastatic and hormone-refractory forms of the disease. PSMA may also be expressed to a lesser extent in certain non-prostatic tissues, including the kidneys, salivary glands, and small intestine. Due to its differential expression profile, PSMA serves as a valuable clinically relevant biomarker and therapeutic target for diagnostic imaging and targeted treatment of prostate cancer. As indicated in the background section of the present specification, the terms “panPSMA” and “iPSMA” are used interchangeably in the art. Both “DOTA-HYNIC-panPSMA” and “DOTA-HYNIC-iPSMA” used herein are therefore indicative for a molecule characterised by Formula (I).

[0156] The aspects and embodiment as described herein advantageously allow to obtain an appropriate chelation yield, particularly above 50%, more particular above 60%, yet more particular above 70%, still yet more particular above 75%, or even above 80%.

[0157] These chelation yields, optionally together with a preliminary or further final purification of the radiolabeled molecule, allow for a high radiochemical purity, e.g. of above 90%, above 95%, or even of and above 98% radiochemical purity.

[0158] As used herein, radioactivities are expressed in Curie [Ci] as unit. However, the conversion of Curie to Becquerel [Bq] is well known in the art, as 1 Ci = 3.7.1010Bq. Hence, 500 mCi is 1.85.1010Bq.

[0159] Actinium-225 has a potentially higher therapeutic efficacy than e.g. lutetium-177, resulting in more complex and effective DNA double-strand breaks in cancer cells, while affecting fewer surrounding cells as compared to lutetium-177. It can hence lead to both more efficient and more specific radiotherapy, delivering more precise and localized treatment, while minimizing damage to healthy tissues and also tends to have less impact on the red bone marrow and blood system compared to lutetium-177. The Half-Life of actinium-225 is also more favourable (9.92 days), which makes it suitable for prolonged therapeutic effects. These advantages make actinium-225 a promising option for targeted radiotherapy, especially in cases where lutetium-177 is less effective. The longer half-life of actinium-225 also reduces the time pressure for radiotherapy as compared to other radionuclides with a shorter half-life, thereby decreasing human error and increasing accuracy of diagnosis / therapy.

[0160] Terbium-161 also has advantages over lutetium-177 in radiotherapy, also having enhanced therapeutic efficacy over lutetium-177 since it emits beta particles, gamma radiation, and a substantial number of conversion and Auger electrons. These Auger - short-ranged electrons can effectively eliminate microscopic metastases that might not be visible on imaging. The emission of conversion and Auger electrons allows for a more localized and effective dose distribution, potentially increasing the therapeutic efficacy compared to lutetium-177. The Half-Life of terbium- 161 is 6.95 days, which is comparable to that of lutetium-177, making it suitable for similar clinical applications. The radiation safety can also be improved since terbium-161 provides an immediate decrease in dose rate, which can improve general radiation safety and potentially extend treatment room capacity for radioligand therapy.

[0161] For radiodiagnosis, gallium-68 radiolabeling has been made relatively simple due to the widespread use of new generations of generators that allow for direct elution into a mixture of chelator-linked targeting agent and buffer. Radiolabeling the DOTA-conjugated HYNIC-panPSMA is hence also a goal of the invention since gallium is easier in use than technetium-99m.

[0162] In some embodiments, all kit components as described herein can be lyophilized altogether or frozen which ensures a longer shelf life.

[0163] Thus, the main advantages of the invention as disclosed herein that differentiate from the state of the art are:

[0164] - due to the longer half-life, the radiolabeling method can be carried out centrally, for example at a specialised radiochemical laboratory or production site, or close to a actinium-225 or terbium-161 -production site, such as a nuclear reactor or a cyclotron;

[0165] - the possibility of a radiolabeling without the need for heating; and / or,

[0166] - the production of highly active radiopharmaceuticals, allowing longer-term therapeutics and / or diagnostics (e.g. multiple acquisitions of images after one single administration or real-time detection).

[0167] - the possibility of radiolabeling without the need for an automated synthesizer, thereby opening up the possibility of using kits providing the necessary chemical, to be used with different sources of actinium-225, or terbium-161.

[0168] Furthermore, a metal inhibitor can be used in the radiolabeling method for neutralizing, at least partially, interfering (radio)metal species and allowing the desired radioactive metal to react with the chelate-functionalized targeting agent. These metal inhibitors may temporarily or permanently remove metals that compete with radioactive metal for the reaction with the chelate-functionalized targeting agent. Said metal inhibitor is thus unable (or has a lower ability) to chelate the radioactive metal in the said conditions of the labeling reaction, but chelates other metals interfering with the chelation of radioactive metal by the chelate-functionalized targeting agent. The presence of metal inhibitors during the radiolabeling reaction provides an advantageous alternative to current approaches for managing the presence of metallic impurities such as increasing the amount of chelate-functionalized targeting agent or the pre-treatment of the eluate of the generator, these additional purification steps consume time (and radioactivity). Hence, in some embodiments, metal inhibitors are used in the present invention, which are selected for their ability to block the competing metals in the radiolabeling reaction without inhibiting the radioactive metal ions in their chelation reaction with the chelate-functionalized targeting agent. Indeed, these metal inhibitors should not interfere negatively on the main radiolabeling reaction or lead to the formation of secondary radiolabeled species. In other words, metal inhibitors should have a limited or no capacity to complex radioactive metal in the conditions used for the radiolabeling reaction. Limited means at least 100 times less than the chelating agent used for the radiolabeling of the chelate- functionalized targeting agent.

[0169] It is interesting to note that the function of metal inhibitors in some embodiments of the present invention is the opposite of the function of the sequestering agents used in the prior art. Indeed, according to known methods, at the end of the labeling reaction, a sequestering agent having a particular affinity for e.g. the radioactive zirconium may be added to chelate the unreacted portion of the isotope, whereas, according to the present invention an agent capable of reducing the competition of metallic impurities other than the radioactive metal is added at the beginning of the reaction.

[0170] As used herein, an "inhibitor of metal" refers to any molecule capable of interacting with, or competing metals, or the chelating moiety of the chelate-functionalized targeting agent or with radioactive metal directly, to inhibit wholly or partially the chelation the chelate-functionalized targeting agent said competing metals and / or promote the chelating of radioactive metal by said targeting agent.

[0171] The illustrative metal inhibitors described herein are not particularly limiting for the invention but are preferably selected from the group of sugars. Sugars used as agents metal inhibitors in the kit of the invention are generally oligosaccharides (up to 6 or 7 monomeric sugar units or monosaccharides) and for example can be monosaccharides or disacharides or derivatives of monosaccharides such as tetracetose, pentacetose, hexacetose, tetrose, pentose, hexose, D- mannose, D-fructose, and derivatives; and I or disaccharides and their derivatives such as maltose and its derivatives; and I or, cyclodextrins and derivatives thereof.

[0172] Preferably, the metal inhibitor is present in the kit as described herein in micromolar amounts, preferably in nanomolar quantities, preferably in an amount of less than 500 nanomolar, still more preferably in an amount less than 100 nanomoles. In a preferred embodiment, said metal inhibitor is present in an amount of from 20 to 40 wt.% or from 25 to 35 wt.% based on the total weight of the chelate-functionalized targeting agent and metal inhibitor.

[0173] When used, the metal inhibitory agent is usually not bound to the chelate-functionalized targeting agent but may also be chemically bound to the chelate-functionalized targeting agent when the chemical bond is a labile (breakable) bond under the conditions of radiolabeling with the chelate- functionalized targeting agent being released in situ in the conditions of radiolabeling. In one embodiment, said metal inhibitory agent is not chemically bound to the chelate-functionalized targeting agent.

[0174] In case of upscaling the amount of activity to be used, adding a certain amount of stabiliser to the radiolabeling solution prior to performing the radiolabeling reaction avoids radiolysis of the chelate-functionalized targeting agent making it possible to use high activity radiolabeling conditions in the clinic.

[0175] As used here, the term “stabilizer” refers to a compound with the ability to decrease or to prevent the radiolysis of the chelate-functionalized targeting agent and / or other compounds of the obtained radiolabeled chelate-functionalized targeting agent composition. Preferably, the stabilizer allows for a radiochemical purity of the radiolabeled chelate-functionalized targeting agent after 4 hours of at least 95%, and that preferably at radioactive concentrations higher than 7.0 mCi / ml. More preferably, the stabilizer allows for a radiochemical purity of the radiolabeled chelate-functionalized targeting agent after 6 hours of at least 95%, and that preferably at radioactive concentrations higher than 7.0 mCi / ml.

[0176] When used, the stabiliser is preferably selected from the group consisting of: ascorbic acid, dehydroascorbic acid, gentisic acid, cysteine and methionine, or a salt thereof. By means of example, a typical salt of ascorbic acid that can be used as stabilizer is sodium ascorbate.

[0177] In certain embodiments a sequestering agent can also be added to sequester non-bound isotope or radiometal after the radiolabeling reaction was finished, to avoid that said unbound isotope would be delivered to the patient and e.g. attached to the bone or cause damage to non-target tissue. Such a sequestering agent can be a chelator for said isotope such as EDTA or DTPA. In contrast to the metal inhibitor defined herein, which is designed to bind metal impurities resulting from break-through of the generator during eluating, the sequestering agent is directed to capture excess isotope. One of the primary methods for producing actinium-225 is a thorium / actinium radionuclide generator.

[0178] One of the primary methods for producing terbium-161 is through nuclear reactors using neutron irradiation of enriched gadolinium-160 targets, or through cyclotrons using gadolinium-160 targets.

[0179] One of the primary methods for producing gallium-68 is through germanium / gallium generators or cyclotrons (liquid or solid target) well known in the field. One particular example is the IRE GalliEo type (TiO2-based) gallium generator.

[0180] The invention also provides for a radiolabeling kit, suitable for producing [Ac225]-labeled DOTA- HYNIC-panPSMA, [Tb161]-labeled DOTA-HYNIC-panPSMA, or [Ga68]-labelled DOTA-HYNIC- panPSMA:

[0181] - DOTA-HYNIC-panPSMA targeting agent, preferably in lyophilized form;

[0182] - a buffering agent or buffer solution, allowing to maintain the pH in the range of 3.5 to 7, such as 3.5 to 6.5 or 4.0 to 5,5, more preferably 4,5 to 5.0, optionally in lyophilized form;

[0183] - optionally, a purification means suitable for eliminating unbound actinium-225 or terbium- 161 from the radiolabeling mixture after the radiolabeling reaction took place, preferably said means is a solid phase extraction (SPE) means as defined herein.

[0184] The kit can additionally comprise:

[0185] - a metal inhibitor and / or sequestering agent as defined herein;

[0186] - a radiolysis stabiliser selected from the group consisting of: ascorbic acid, sodium ascorbate, dehydroascorbic acid, gentisic acid, cysteine and methionine, or a salt thereof, preferably as a solution; and / or

[0187] - instructions for performing the method described herein.

[0188] The buffering agent or buffer solution in said kit can be a phosphate, nitrate, HEPES, acetate, formate, TRIS, ascorbate, and citrate or a mixture thereof, preferably an acetate or formate buffer, more preferably a sodium acetate or sodium formate buffer, most preferably a sodium acetate buffer.

[0189] In order to further increase the radiochemical purity of the end-product (the radiolabeled targeting agent), the methods and kits described herein can further encompass or comprise a step or means for purifying the targeting agent. Exemplary means are solid phase extraction (SPE) or reverse phase extraction means known in the art. Typically, SPE substrates are made of high-purity silica. For small peptides such as the PSMA-11 targeting agent, preferably a silica-based substrate with strong hydrophobicity such as C18-coated silica can be used, which is a hydrophobic and non-polar stationary phase, selectively retaining non-polar and moderately polar compounds such as lipids, steroids, and pesticides while allowing more polar compounds such as sugars and amino acids to pass through. For larger molecules such as polypeptides and antibodies, different hydrophobic interaction resins with C4- , C6-, C8-, phenyl-, or benzyl- coated silica can be used as a substrate depending on the chemical character of the targeting agent.

[0190] In the kit according to the invention, the DOTA-functionalized targeting agent and the buffer can be provided in an lyophilized form, either in two separate vials or in one single vial. In one embodiment, also the metal inhibitor (non-bound radioisotope sequester) and / or stabilising agent (preventing radiolysis of the targeting agent) are present in lyophilised form, either in a separate vial, or combined with either the buffer or targeting agent. Said vials can be siliconized or can be acid-washed prior to use.

[0191] In a specific embodiment, the radiolabeling kit can comprise the following components:

[0192] Sterile Vial 1 comprising the chelate-functionalized targeting agent and the metal inhibitor, preferably in a sterile (e.g.10 mL) vial;

[0193] Sterile Vial 2 that comprises buffering agent, preferably in a sterile (e.g.10 mL) vial;

[0194] Optionally Vial 3 comprising an aqueous solution of radiolysis stabiliser selected from ascorbic acid, dehydroascorbic acid, gentisic acid, cysteine and methionine, sodium ascorbate, or a salt thereof.

[0195] Alternatively, all components can be present in a single vial, preferably in dried or lyophilized form.

[0196] In some embodiments of the kits described herein, the amount of ascorbic acid, dehydroascorbic acid or a salt thereof in the kit, is so that the weight ratio of the weight of stabiliser over the weight of the buffering agent in the kit, is at least 0.006, preferably at least 0.010, preferably at least 0.030, preferably at least 0.050, preferably at least 0.100, preferably at least 0.150, preferably at least 0.200, preferably at least 0.250, preferably at least 0.300. In a preferred embodiment, said stabiliser is ascorbic acid.

[0197] In some embodiments of the kits described herein, the amount of stabiliser in the kit, is such that the weight ratio of the weight of stabiliser over the weight of the metal inhibitor in the kit, is weight of stabiliser over the weight of the metal inhibitor in the mixture obtained in step e), is at least 100, preferably at least 200, preferably at least 500, preferably at least 1000, preferably at least 2500, preferably at least 5000, preferably at least 10000, preferably at least 20000, preferably at least 50000. In a preferred embodiment, said stabiliser is ascorbic acid.

[0198] In some embodiments of the methods described herein, the amount of stabiliser in the mixture obtained in step e), is so that the weight ratio of the weight of stabiliser over the weight of the buffering agent in the mixture obtained in step e), is at least 0.006, preferably at least 0.010, preferably at least 0.030, preferably at least 0.050, preferably at least 0.100, preferably at least 0.150, preferably at least 0.200, preferably at least 0.250, preferably at least 0.300.

[0199] In some embodiments of the methods described herein, the amount of stabiliser in the mixture obtained in step e), is so that the weight ratio of the weight of stabiliser over the weight of the metal inhibitor in the mixture obtained in step e), is at least 100, preferably at least 200, preferably at least 500, preferably at least 1000, preferably at least 2500, preferably at least 5000, preferably at least 10000, preferably at least 20000, preferably at least 50000.

[0200] In some embodiments of the use described herein, the amount of stabiliser in the radiolabeled chelate-functionalized targeting agent composition, is so that the weight ratio of the weight of stabiliser over the weight of the buffering agent in the radiolabeled chelate-functionalized targeting agent composition, is at least 0.006, preferably at least 0.010, preferably at least 0.030, preferably at least 0.050, preferably at least 0.100, preferably at least 0.150, preferably at least 0.200, preferably at least 0.250, preferably at least 0.300. In a preferred embodiment, said stabiliser is ascorbic acid.

[0201] In some embodiments of the use described herein, the amount of stabiliser in the radiolabeled chelate-functionalized targeting agent composition, is so that the weight ratio of the equivalent weight of stabiliser over the weight of the metal inhibitor in the radiolabeled chelate-functionalized targeting agent composition, is at least 100, preferably at least 200, preferably at least 500, preferably at least 1000, preferably at least 2500, preferably at least 5000, preferably at least 10000, preferably at least 20000, preferably at least 50000. In a preferred embodiment, said stabiliser is ascorbic acid.

[0202] After addition of the radioactive metal solution to the mixture of chelate-functionalized targeting agent and buffer, optionally further comprising a stabilizer and / or a metal inhibitor, the solution obtained is left to the radiolabeling reaction for a short period of time, such as between about 10 and 20 minutes, preferably for about 15 minutes at a temperature of between 90 and 95°C. Shorter and longer incubation periods, while not expressly recited herein, are also envisaged. The invention also discloses a radiolabeled targeting agent with radioactive metal, obtained by any one of the methods as described herein.

[0203] Hence, the invention provides in [Ac225]-labeled DOTA-HYNIC-panPSMA, [Tb161]-labeled DOTA-HYNIC-panPSMA, or [Ga68]-labelled DOTA-HYNIC-panPSMA or [Ga67]-labelled DOTA- HYNIC-panPSMA, obtained by any one of the methods or kits as described herein.

[0204] Said gallium-68 or -67, terbium-161 or actinium-225 radiolabeled compounds can be used in radioimaging and detection, terbium-161 or actinium-225 radiolabeled compounds can also be used in longer-term or even real-time imaging. Therefore, the invention further provides an imaging method comprising the steps of:

[0205] 1) radiolabeling a DOTA functionalized targeting agent as described herein with actinium-225, terbium-161 or gallium-68 or -67 preferably according to the method described herein and / or preferably by using a kit described herein,

[0206] 2) administering to a subject a diagnostic amount of said actinium-225, terbium-161 , or gallium- 68 or -67 radiolabeled targeting agent; and,

[0207] 3) detecting said actinium-225, terbium-161 , or gallium-68 or -67 radiolabeled targeting agent when bound to its target using PET or SPECT or PET / CT or SPECT / CT imaging methods.

[0208] A skilled person is aware that the above-mentioned PET (Positron Emission Tomography) and SPECT (Single Photon Emission Computed Tomography) imaging techniques are particularly suited for the uses and methods described herein. A first particularly preferred imaging technique is SPECT / CT. SPECT / CT imaging has been described at several occasions throughout the art. SPECT / CT imaging is a hybrid medical imaging technique that combines two imaging techniques: SPECT and Computed Tomography (CT). By merging these two technologies, SPECT / CT provides both functional (physiological) and anatomical (structural) information in a single imaging session. The SPECT / CT combination offers several advantages such as a better localisation of the signal due to the addition of the CT image, which is beneficial for deducting precise and accurate teachings from the SPECT data. A further particularly preferred imaging technique is PET / CT, which has also been described in detail throughout the art. Similarly to SPECT / CT is PET / CT imaging a hybrid imaging technique that integrates two imaging modalities: Positron Emission Tomography (PET) and Computed Tomography (CT) which provides both functional and anatomical data in one scan. The PET / CT combination allows for enhanced localization of metabolic activity due to the anatomical reference provided by the CT scan. This synergy between PET and CT is particularly enables more precise and accurate interpretation of functional data, improving diagnostic accuracy and therapeutic planning across a range of clinical applications. In preferred embodiments, compounds radiolabeled with actinium-225 are combined with SPECT or SPECT / CT imaging, and compounds radiolabeled with terbium-161 or gallium-68 or gallium- 67 are preferably used for PET or PET / CT imaging.

[0209] Optionally, the imaging method is a method for imaging a cancer characterized by increased PSMA expression. In such preferred embodiments, the cancer is preferably prostate cancer, and the DOTA functionalized targeting agent is DOTA-HYNIC-panPSMA.

[0210] The particular purpose or reason for performing the imaging method is not particularly limiting in the context of the invention and may therefore serve one or more of the following purposes:

[0211] (i) initial staging of a cancer, for example prostate cancer, into intermediate, unfavourable, high, or very high-risk prostate cancer (or an equivalent quantitative classification of the cancer stage),

[0212] (ii) detecting suspected recurrence of cancer and / or detection of metastasis, such as detecting suspected recurrence of prostate cancer and / or detection of metastasis,

[0213] (iii) selection for radiotherapeutic treatment with any radiopharmaceutical used in the treatment of cancer, preferably prostate cancer, such as Lutetium (177Lu) vipivotide tetraxetan (Pluvicto),

[0214] (iv) monitoring prostate cancer for progression into Non-Metastatic or Metastatic Castration- Resistant Prostate Cancer (nmCRPC or mCRPC),

[0215] (v) determining response to (radio)therapy, preferably wherein the radiotherapy relied on the use of a radiolabeled DOTA functionalized targeting agent, optionally wherein the radiotherapy relied on the use of radiolabeled DOTA-HYNIC-panPSMA,

[0216] (vi) real-time imaging or diagnosis in a subject e.g. for use in PET scans for tracking slow biological processes or progression of those over a period of time, or

[0217] (vii) personalised medicine, or determining a personalised medicine treatment strategy. The term “personalised medicine” enjoys widespread use in the field of medicine and refers to a medical treatment that considers a number of subject-specific parameters such as but not limited to genetic background, environment, lifestyle, and medical history to optimize diagnosis and particularly the use and dosages of therapeutic agents and treatment regimens in general, aiming to provide a subject with more precise, effective, and targeted treatments to optimise the likelihood of successful outcomes and minimizing unnecessary side effects.

[0218] A further advantage of using the radiolabeled DOTA conjugated compounds described herein in any detection, imaging, or diagnostic method is that these provide a non-invasive alternative for invasive alternatives used in cancer diagnostics such as but not limited to prostate biopsies. The term "diagnosing" as used herein is indicative for a process of recognizing, deciding on or concluding on a disease, such as cancer, such as prostate cancer, in a subject on the basis of symptoms and signs and / or from results of various diagnostic procedures (such as, for example, from knowing the presence, absence and / or quantity of one or more biomarkers, in the context of the present invention particularly PSMA or of clinical symptoms characteristic for the diagnosed disease or condition). The prognosis of the subject is not limiting for the invention. The subject may therefore be a subject having a good prognosis or a poor prognosis.

[0219] Said terbium-161 or actinium-225 radiolabeled compounds also can be used in targeted radiotherapy, by delivering a therapeutic dose of radiation directly to cancer cells while minimising damage to healthy tissues.

[0220] A further aspect of the invention is therefore directed to curative use of the radiolabelled DOTA- HYNIC-panPSMA compounds. Envisaged is a radiolabeled DOTA-HYNIC-panPSMA as described in any embodiment herein such as [161Tb]Tb- DOTA-HYNIC-panPSMA and / or [225Ac]Ac- DOTA-HYNIC-panPSMA for use in radiotherapy. Alternatively worded, the invention envisages the use of a radiolabeled DOTA-HYNIC-panPSMA as described in any embodiment herein such as [161Tb]Tb-DOTA-HYNIC-panPSMA and / or [225Ac]Ac-DOTA-HYNIC-panPSMA in radiotherapy, and a method of radiotherapy wherein a radiolabeled DOTA-HYNIC-panPSMA as described in any embodiment herein such as [161Tb]Tb-DOTA-HYNIC-panPSMA and / or [225Ac] Ac- DOTA- HYNIC-panPSMA is administered to a subject in need thereof. Also encompassed is the use of a radiolabeled DOTA-HYNIC-panPSMA as described in any embodiment herein such as [161Tb]Tb- DOTA-HYNIC-panPSMA and / or [225Ac]Ac-DOTA-HYNIC-panPSMA in the manufacture of a medicament for radiotherapy. [161Tb]Tb- DOTA-HYNIC-panPSMA and / or [225Ac] Ac- DOTA-HYNIC- panPSMA are particularly preferred in this context in view of the reduced damage to healthy tissue (i.e., healthy, non-malignant cells) imposed by said compounds when compared to other radiolabeled compounds such as [177Lu]Lu-DOTA-HYNIC-panPSMA.

[0221] The concept of radiotherapy is well known to a skilled person. In brief, radiotherapy is indicative for any medical treatment that involves the controlled use of ionizing radiation to destroy or damage pathological tissue, including but not limited to malignant cells, by delivering a prescribed dose of radiation to a targeted area. Envisaged herein is both the use of radiotherapy using one of the compounds described herein as a stand-alone therapy, but combination therapies with other therapies such as but not limited to surgery, chemotherapy, or immunotherapy are also envisaged.

[0222] Optionally, the therapeutic uses and methods described herein are performed on, administrated to, or used in a subject diagnosed with cancer, wherein the diagnostic method performed in a preceding step on the subject involved the use of any one of the radiolabeled DOTA-HYNIC- panPSMA compounds described herein, such as but not limited to the [177Lu]Lu-DOTA-HYNIC- panPSMA, [225Ac]Ac-DOTA-HYNIC-panPSMA, [161Tb]Tb-DOTA-HYNIC-panPSMA, [68Ga]Ga- DOTA-HYNIC-panPSMA, [67Ga]Ga-DOTA-HYNIC-panPSMA compounds described herein.

[0223] Optionally, the medical use for radiotherapy described above is radiotherapy having as goal the treatment of cancer, i.e. killing malignant cells in a subject. Preferred cancers to be treated by the compounds described herein are prostate cancer, salivary gland cancer, glioblastoma, thyroid cancer, clear cell renal carcinoma, renal cell carcinoma, hepatocellular carcinoma, lung cancer, adenoid cystic carcinoma of the parotid, and breast cancer. Optionally, the cancer is characterised by a solid tumour. Optionally, the cancer is metastatic cancer, such as metastatic prostate cancer, such as optionally metastatic castration-resistant prostate cancer

[0224] It is evident to a person of ordinary skill in the art that treatment of a subject with one or more of the radiolabed DOTA-HYNIC-panPSMA compounds implies the administration to said subject of an therapeutic effective amount of said compound(s). The term “effective amount” or “therapeutic effective amount” as used herein refers to an amount of active compound or pharmaceutical agent that elicits the biological or medicinal response in a subject that is being sought by a researcher, veterinarian, medical doctor or other clinician, which may include a reduction or complete removal of the symptoms associated with the disease or condition being treated. Methods to determine pharmaceutically effective amounts are known in the art and are therefore known to a skilled person.

[0225] It is evident that in the context of therapy, “administration” may be used interchangeably with terms such as “treating”. The terms “treat” or “treatment” encompass both the therapeutic treatment of an already clinically apparent disease or condition, such as the therapy of subject having clinical symptoms related to a cancer, as well as more preventive measures, wherein the aim is to prevent or lessen the chances of clinical development and progression of a cancer which at the time of treatment is not characterized by clinical symptoms (for example prostate cancer diagnosed by the detection of increased PSMA levels when compared to the PSMA level of a normal healthy subject). Beneficial or desired clinical results may include, without limitation, alleviation of one or more symptoms or one or more biological markers, diminishment of extent of disease, stabilized (i.e., not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and the like. “Treatment” can also mean prolonging survival as compared to expected survival if not receiving treatment. In addition, the terms "therapeutic treatment" or "therapy" and the like, refer to treatments wherein the object is to bring a subjects body or an element thereof from an undesired physiological change or disorder, to a desired state, such as a less severe or unpleasant state (e.g., amelioration or palliation), or back to its normal, healthy state (e.g., restoring the health, the physical integrity and the physical wellbeing of a subject), to keep it (i.e., not worsening) at said undesired physiological change or disorder (e.g., stabilization), or to prevent or slow down progression to a more severe or worse state compared to said undesired physiological change or disorder.

[0226] In any of the uses and method described herein, a preferred method of administration of the DOTA functionalized targeting agent is intravenous administration. Hence, in certain embodiments described herein the first DOTA functionalized targeting agent is comprised in a solution optionally including further excipients. Preferred excipients include sodium dihydrogen phosphate (anhydrous), disodium monohydrogen phosphate (anhydrous), sorbitol E420, 1 , 1 , 3, 3-propane tetraphosphonic acid, tetrasodium salt (dihydrate), stannous chloride dihydrate, and means to precisely control the pH of the composition that is administered to the subject such as but not limited to sodium hydroxide and / or hydrochloric acid. Further suitable excipients include without limitation sodium chloride, lactose, mannitol, polysorbate 80, propylene glycol, benzyl alcohol, citric acid, sodium citrate, glycine, and any combination thereof. As a skilled person readily appreciates, different excipients serving different or overlapping functions may be combined in one solution for intravenous administration. Non-limiting examples include: buffering agents, solubilizing agents, preservatives, stabilizers, viscosity modifiers, tonicity adjusters, complexing agents, antimicrobial preservatives, antioxidants, chelating agents, suspending agents, surfactants, water for injection, solvents, or any combination thereof.

[0227] It is evident that any one of the methods and uses may be performed by assistance, or by implementation of, computerised means. “Computerised means” as used throughout the present disclosure relates to electronic systems, devices, and technologies configured to perform specific tasks, processes, or operations. Optionally, the computerised means used to quantify one or more relative signal intensities is configured to perform additional tasks and processes which may include without limitation data processing, data management, communication means, data presentation means, transaction automation (i.e. the ability to automatically purchase a radioimmunoconjugate upon detecting a particular relative signal intensity or a combination of particular relative signal intensities), automated appoint scheduling, automated decision-making (such as for example by means of artificial intelligence), electronic record keeping, control systems, monitoring systems, and digital authentication.

[0228] Further envisaged is a computer system configured for performing, or assisting in performing, any of the herein described uses and methods. Yet further envisaged is a computer program product comprising instructions which when the program is executed by a computer, cause the computer to carry out the herein described uses and methods. It is envisaged that any computer system or any computer-implemented method relying on a computer system described herein may further comprise means of machine learning and / or artificial intelligence of said system to enable and / or improve its functionality. Non-limiting examples of machine learning models, i.e. machine learning algorithms include linear regression, logistic regression, decision trees, support vector machines, naive Bayes, k-nearest neighbors (kNN), k-means, random forest, dimensionality reduction algorithms, and gradient boosting algorithms such as gradient boosting machine (GBM), XGBoost, LightGBM, and CatBoost. The use of artificial intelligence for analysis of scintigrams has been reported in the art and is therefore known to a skilled person (e.g., Hajianfar et al., Zeitschrift fur Medizinische Physik, 2024). Software tools for automatization of analysis have also been described in the art (e.g., Yoshida et al., Clinical Imaging, 2021). A skilled person is therefore aware of these tools and technologies and appreciates the benefits they may entail.

[0229] In certain embodiments, the computer readable data is encrypted and requires authentication or authorization credentials from a user or second computer-readable storage system for a computer system to be able to access said data. Hence, the present invention also envisages computer- readable storage media comprising a data storage material encoded with computer readable data wherein said data comprise one or more scintigrams of a subject, or optionally one or more scintigrams of multiple subjects, after being administered at least once with one or more of the radiolabeled DOTA-HYNIC-panPSMA compounds described herein. In certain embodiments, the computer-readable storage medium is a physical storage medium. In alternative embodiments, the computer-readable storage medium is a non-physical storage medium or a storage medium perceived to be a non-physical storage medium (i.e. a cloud based storage medium). Optionally, the computer readable data comprise information relating to the intensities of certain areas of interest of the one or more scintigrams.

[0230] While the invention has been described in conjunction with specific embodiments thereof, it is evident that many alternatives, modifications, and variations will be apparent to those skilled in the art in light of the foregoing description. Accordingly, it is intended to embrace all such alternatives, modifications, and variations as follows in the spirit and broad scope of the appended claims. The herein disclosed aspects and embodiments of the invention are further supported by the following non-limiting examples. The following specific experimental examples are provided in support of the claimed invention but are not to be seen as limiting the scope of the invention.

[0231] EXAMPLES

[0232] Method for preparing DOTA-HYNIC-panPSMA.

[0233] The method was adapted from WO 2019177449 A1. Briefly, the d i-tert- butyl ester of glutamic acid was used initially to synthesise the molecule, said ester was reacted with carbonyldiimidazole (GDI) in the presence of triethylamine (TEA) to form the acylimizazole derivative, which was activated with methyl triflate (MeOTf) to react with (S)-tert-butyl-2-amino-6- (benzyloxycarbonylamino) hexanoate (Cbz-Lys-Ot-Bu), with subsequent deprotection of the Cbz by hydrogenolysis, thereby giving the derivative Glu-Urea-Lys, which was reacted with the amino acid Fmoc-p-naphthyl alanine (HBTU / HOBt) in the solid phase (MBHA resin), followed by 6-Boc- hydrazinopyridin-3-carboxylic acid (Boc-HYNIC) in the presence of diisopropylethylenamine (DI PEA) and dimethylformamide (DMF) followed by addition of TFA.

[0234] This latter addition step was repeated to introduce DOTA-tris (t-Bu ester).

[0235] Finally, the compound was deprotected with TFA, purified by HPLC, and lyophilised.

[0236] The final product was Glu-NH-CO-NH-Lys(P-naphthyl alanine)-HYNIC-DOTA (DOTA-HYNIC- panPSMA). Reverse-phase HPLC analysis of the lyophilised white solid showed a chemical purity of 98.8% for the compound. The DOTA-HYNIC-panPSMA (0.6 mg) was formulated as a lyophilised pharmaceutical form containing 50 mg mannitol and 100 mg ascorbic acid.

[0237] Examples 1-3 :225Ac labeling 600pg DOTA-HYNIC-panPSMA was reconstituted in a 0.5M ascorbic acid buffer.

[0238] A225Ac solution was eluted from a thorium generator by 0.04M HCL and with a specific activity (cf. Table 1), was added to the DOTA-HYNIC-panPSMA solution, resulting in a 1:1 labelling mixture of ascorbic acid buffer and HCL eluate resulting in a pH of the radiolabelling solution of between 4.5 and 5.0. , incubated for 15 minutes at 95°C by stirring.

[0239] Incorporation was determined by radio-thin layer chromatography (TLC). The compound was purified on an Oasis HLB purification column or a C18 SepPak purification column, washed with 0.05 M sodium citrate, and eluted in absolute ethanol. The compound was then formulated to an activity concentration of 370 kBq / mL in formulation buffer (i.e. sodium ascorbate and diethylenetriamine pentaacetate DTPA) and the radiochemical purity (RCP) of the purified and formulated product was determined by radio-TLC and radio high performance liquid chromatography (HPLC). The compound was re-analysed by radio-HPLC 4 hours after the end of synthesis to assess stability.

[0240] Table 1 :225Ac labelling conditions

[0241] Examples 1 to 3 show that DOTA-HYNIC-panPSMA was radiolabeled with225Ac and showed incorporation of up to 89.5% in the crude reaction mixture by radio-TLC. For Example 2, the radiochemical purity (RCP) was 98.6% by radio-TLC and 99.0% in the final product determined by radio-HPLC. No major signs of instability were observed when the compound was re-analysed 4 hours after synthesis, resulting in an RCP of 97.9%. Based upon the radiolabeling data, the quality control release criteria of >95.0% purity and <5.0% of free225Ac by radio-TLC are met.

[0242] Results

[0243] Example 1 :

[0244] The compound was labeled with 650.0 kBq of Ac-225 and an incorporation of 94.5% was obtained determined by radio-TLC. The compound was purified on an HLB purification column, eluted in 250 pL of abs. EtOH and formulated to an activity concentration of 370 MBq / nmol and a compound concentration of 3.9 nmol / mL in formulation buffer.

[0245] A radiochemical purity (RCP) in the final product of 96.7% was obtained determined by radio- TLC. A sample was withdrawn for radio-HPLC analysis and fractions of 30 seconds were collected and analysed on the well counter the day after when secular equilibrium was reached. An RCP of 98.5% was observed in the final formulation determined by radio-HPLC.

[0246] Due to some In-111 contamination of the fraction collected when the sample was re-run on HPLC 4 hr after EOS no data was obtained at this stability timepoint. The results are given in Figure 16 and 17. Example 2:

[0247] The compound was labeled with 1.5 MBq Ac-225 and an incorporation of 89.5% was obtained determined by radio-TLC. The compound was purified on an C18 seppak purification column, eluted in 250 pL of abs. EtOH and formulated to an activity concentration of 370 MBq / nmol and a compound concentration of 4.1 nmol / mL in formulation buffer.

[0248] A radiochemical purity (RCP) in the final product of 98.6% was obtained determined by radio- TLC. A sample was withdrawn for radio-HPLC analysis and fractions of 30 seconds were collected and analysed on the well counter the day after when secular equilibrium was reached. An RCP of 99.0% was observed in the final formulation determined by radio-HPLC.

[0249] The compound was re-analysed by radio-HPLC 4 hr. after EOS and only minor degrees of instability were observed resulting in an RCP of 97.9%. The results are given in Figure 18 and 19.

[0250] Example 3:

[0251] The compound was labeled with 5.0 MBq Ac-225 and an incorporation of 94.0% was obtained determined by radio-TLC. The compound was purified on a C18 seppak purification column, eluted in 300 pL of abs. EtOH and formulated to an activity concentration of 740 MBq / nmol and a compound concentration of 7.9 nmol / mL in formulation buffer.

[0252] A radiochemical purity (RCP) in the final product of 98.2% was obtained determined by radio- TLC. A sample was withdrawn for radio-HPLC analysis and fractions of 30 seconds were collected and analysed on the well counter the day after when secular equilibrium was reached. An RCP of 99.5% was observed in the final formulation determined by radio-HPLC. The results are given in Figures 20 and 21.

[0253] Radiochemistry conditions and results from the 3 test labelings (Examples 1 to 3) are summarized in Table 1.

[0254] Examples 4-7:161Tb labeling

[0255] For each example 600pg DOTA-HYNIC-panPSMA was dissolved (reconstituted) in an ammonium acetate buffer as indicated in Table 2. To this solution,161TbCl3 solutions in an aqueous 0.05M HCI eluates as indicated in Table 2, were added from a Gadolinium-160 enriched target. The obtained reaction mixtures were heated at 90°C for 30 minutes using a heating-block. The reaction mixtures were then let to cool down for 5 min. Examples 4 to 6 were immediately subjected to radio-UV-HPLC for quality control. To Example 7, 10pL of ascorbic acid at 150g / L was added along with 18pL of EDTA 4mM before being subjected to radio-UV-HPLC. Radio-UV-HPLC analysis were performed using a Phenomenex Vydac C18 column (4mL / min, A = 220nm Cis; 150mm x 4,6mm x 5pm). HPLC conditions were: 0-2min: 100% water in 0.1%TFA (A), 2-4min : 100% ACN (B), 4-5min: 100% B ; 5-7min: 100% A. 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. For the evaluation of the radiolabeling stability, radio-UV-HPLC was repeated daily over a period of 4 days. Non-complexed161Tb and radiolysis products, areas at the retention time of DOTA- HYNIC-panPSMA, solvent and all other peaks were integrated.

[0256] Table 2:161Tb labelling conditions

[0257] Results for Examples 4 to 6:

[0258] Radiochemical purities of [161Tb]Tb-DOTA-HYNIC-panPSMA were 97.36% (100MBq - Example 4), 96.14% (200MBq - Example 5) and 95.74% (400MBq - Example 6). No purification was needed. RCP > 98% might be achieved when using161Tb closer to the production date.

[0259] Figures 1 , 2 and 3 show the evolution of the radiolabeling stability, uncomplexed161Tb and radiolysis products up to 4 days after radiolabeling.

[0260] As seen in Figure 1 , radiolabeling was stable up to 200MBq. At higher activity (e.g., 400MBq), radiolabeling was less stable and rapidly decrease.

[0261] Consequently, uncomplexed161Tb increasing more rapidly at higher activity as displayed in Figure 2 below. Addition of complexing agents significantly improves the final result.

[0262] Finally, monitoring of radiolysis products showed that radiolysis occurs at activity as low as 400MBq (Figure 3). Therefore, anti-oxidants significantly improve the final result.

[0263] For Example 7, 300MBq (150|JL) of161Tb in HCI 0.05M were available.161Tb was used 15 days after production. To deal with potential higher metallic contaminants, ammonium acetate buffer 2M, pH 5.5 was used (300|JL). Similar radiolabeling procedure than test 1 was followed. After cooling down, 1OpL of ascorbic acid at 150g / L was added along with 18pL of EDTA 4mM.

[0264] Radiochemical purity was 95.00% suggesting that using161TbCh 15 days after production is the latest limit. Interestingly, no radiolysis products were seen over a period of 7 days revealing that 1 OpL ascorbic acid at 150g / L is effective as quencher. However, uncomplexed161Tb is still seen revealing that 18pL EDTA 4mM is not high enough (cf. Figures 4 and 5). This finding is confirmed by radio-HPLC showing uncomplexed161Tb and 0.04 min later another peak attributed to [161Tb]Tb-EDTA (not shown). Therefore, higher concentration of EDTA may have to be added for effective complexation of unbound161Tb and increase the shell-life.

[0265] 1GBq of161Tb produced 7days before will be used. Radiolabeling will be performed in ammonium acetate buffer 0.4M, pH 5.1 . The raw solution will be heated at 90°C for 30min and let cool down to room temperature for 5 min. 10pL ascorbic acid at 150g / L plus 1 OpL EDTA 50mM will be added next and the solution subjected to radio-HPLC.

[0266] Example 8: In vitro study of [161Tb]Tb-DOTA-HYNIC-panPSMA and [177Lu]Lu-DOTA-HYNIC- panPSMA

[0267] I. Radiolabeling with161Tb and177Lu

[0268] 1.1. Materials and methods

[0269] Radiolabeling of DOTA-HYNIC-panPSMA (600pg, non-GMP) was performed using161TbCh in HCI 0.05M (non-GMP) provided by Terthera or177LuCh in 0.04M HCI (non-GMP) from ITM. Radiolabeling was performed manually in ascorbate buffer with activities up to 500MBq. The raw solution was heated at 90°C for 30 minutes using a heating-block. The solution was then let to cool down for 5 min and was immediately subjected to radio-UV-HPLC for quality control. QC was then repeated over a period of 33 days for [161Tb]Tb-DOTA-HYNIC-panPSMA and 28 days for [177Lu]Lu-DOTA-HYNIC-panPSMA, to follow demetallation and apparition of radiolysis products. Radio-UV-HPLC analysis were performed using a Phenomenex Luna C18 column (0.6mL / min, Cis; 250mm x 4,6mm x 5pm). HPLC conditions were: 0-0.5min: 95% water in 0.1 %TFA (A) 5% acetonitrile (B), 0.5 - 10min : 40% ACN (B), 10-12min: 40% B ; 12-25min: 95% B. The analytical HPLC system used was a Vanquish Core Thermofisher, pump quaternary, multiple wavelength detector CG, with Chromeleon 7.3 software, gabiNova detector. Radiolabeling stability was evaluated in the vehicle at room temperature (23°C).

[0270] 1.2. Results

[0271] Radiochemical purities of [161Tb]Tb-DOTA-HYNIC-panPSMA and [177Lu] Lu- DOTA-HYNIC- panPSMA were 98.26% and 97.52% at the end of radiolabeling (Figures 6 and 7 respectively) and still 95.55% 33 days after radiolabeling for [161Tb]Tb-DOTA-HYNIC-panPSMA and 95.02% for [177Lu]Lu-DOTA-HYNIC-panPSMA, 28 days after radiolabeling. No purification was needed. Figure 8 shows the stability for both products over time. Both [161Tb]Tb-panPSMA and [177Lu]Lu- panPSMA are highly stable overtime

[0272] II. In vitro assays

[0273] For all in vitro assays, LNCaP, 22Rv1 , PC3-wild type and PC3-pip cells were used. They were grown at 37°C, % CO2, in RPMI medium supplemented with 10% FBS, penicillin and steptomycin. Cells were seeded into 24-wells plates at the density of 2 x 105cells per well, the day before the experiment

[0274] 1. Affinity

[0275] Materials and methods

[0276] Plates were stored at 4 °C for 30 min in order to reduce cell processing and then incubated for 2 h with 250 pL of complete medium containing the radiolabeled peptide at increasing concentrations (1 , 5, 10, 25, 50 and 100 nM) with and without the PSMA inhibitor PMPA used at the concentration of 10pM). After incubation, cells were rinsed twice with ice cold DPBS (250 pL, 14190-144, Gibco). Then, cells were lysed with NaOH 1 M (750 pL) to collect the bound fraction. Radioactivity of each fraction was determined in a gamma-counter (Wizard2, PerkinElmer, USA). Affinity (Kd) was determined by nonlinear regression using Prism 10.5 software (GraphPad Software Inc., USA).

[0277] Results

[0278] [161Tb]Tb-DOTA-HYNIC-panPSMA was used at 0.99 MBq / nmol and [177Lu]Lu-DOTA-HYNIC- panPSMA at 0.81 MBq / nmol. For [161Tb]Tb-panPSMA, non-linear regression provides a Kd value of 8.37nM on LNCaP cells (6,4.104receptors per cell), 47.7 nM on 22Rv1 cells (7942 receptors per cell), 28.22nM on PC3-pip cells (3,80.106receptors per cell) and no measurable specific binding on PC3 wild type cells. The specific binding profile in LNCaP cells is depicted in Figure 9A.

[0279] For [177Lu]Lu-DOTA-HYNIC-panPSMA, non-linear regression provides a Kd value of 23.87nM on LNCaP cells, 59.9 nM on 22Rv1 cells, 33.3 nM on PC3-pip cells and no measurable specific binding on PC3 wild type cells. The specific binding profile in LNCaP cells is depicted in Figure 9B.

[0280] Counts per minute for specific versus non-specific [161Tb]Tb-DOTA-HYNIC-iPSMA in LNCaP cells are depicted in Figure 10. 2. Internalization assay

[0281] Materials and methods

[0282] A total of 25 nM of [161Tb]Tb-DOTA-HYNIC-panPSMA in 250 pL of medium with and without 100 pM of the PSMA inhibitor PMPA was added in each well. Plates were incubated for 60, 120, or 240 min at 37 °C, 5% CO2. Three minutes before the selected time point, plates were stored at 4 °C to stop the internalization process. Media were removed, and wells were rinsed three times with ice cold DPBS (250 pL). A total of 250 pL of sodium acetate (20 mM, pH 5) was added in each well twice and then collected in tubes after 5 min incubation. Then, cells were lysed with NaOH 1 M (750 pL) to collect the bound fraction. Radioactivity of each fraction was determined in a gamma-counter. Identical conditions were used for [177Lu]Lu-DOTA-HYNIC-panPSMA Results are expressed as specific binding as a function of total cell associated radioactivity.

[0283] Results

[0284] [161Tb]Tb-DOTA-HYNIC-panPSMA was used at 0.99 MBq / nmol and [177Lu]Lu-DOTA-HYNIC- panPSMA at 0.81MBq / nmol. On LNCaP cells, [161Tb]TbDOTA-HYNIC-panPSMA showed an initial high membrane-bound fraction, common with other radiolabeled PSMA inhibitors, of 34.15 ± 1.33% at 1 h after incubation. This value decrease to 10.93 ± 0.93% at 4h after incubation. Specific internalization was stable over the 4h time of the study around 6% (Figure 11). On 22Rv1 cells, these values were 3 fold lower, in line with the much lower number of PSMA receptor expressed on the cell surface (see affinity paragraph). On PC3-pip cells the membrane bound fraction reaches 55-70% of the total cell associated radioactivity and the internalized fraction reaches 12-20% (2-3 fold more than LNCaP). No specific internalization was depicted on PC3 wild type cells. A faint specific membrane bound fraction of 4.7 ± 1.82% at 2h was seen.

[0285] On LNCaP cells, the behavior of [177Lu]Lu-DOTA-HYNIC-panPSMA was similar to that of [161Tb]Tb-DOTA-HYNIC-panPSMA with an initial membrane bound fraction of 34% at 1 h but this value decreased slower to 27% at 4h. Consequently, the internalization rate was higher at all time points (from 22% at 1h to 17% at 4h) (Figure 12). On PC3-pip cells, specific internalization was increasing overtime from 11 to 17% of the cell associated radioactivity. The membrane bound fraction showed a high initial binding value of 65% at 1 h, slowly decreasing to 55% at 4h. No specific signal was seen on PC3 wild type cells. 3. Efflux studies

[0286] Materials and methods

[0287] [161Tb]Tb- DOTA-HYNIC-panPSMA was used at 0.99 MBq / nmol and [177Lu]Lu- DOTA-HYNIC- panPSMA at 0.81 MBq / nmol. A total of 25nM of [161Tb]Tb-DOTA-HYNIC-panPSMA or [177Lu]Lu- DOTA-HYNIC-panPSMA in 250 pL of medium was added in each well. Plates were incubated 30 min at 37 °C, 5% CO2. Media were removed, and cells were rinsed with ice cold DPBS (250 pL). A total of 250 pL of sodium acetate (20 mM, pH 5) was added in each well and removed after 5 min of incubation. Cells were rinsed again with ice cold DPBS (250 pL). Then, 250 pL of medium were added in each well and plates were incubated for 60, 120 or 240 min at 37 °C, 5% CO2. Media were collected in tubes, and each well was rinsed twice with ice cold DPBS (250 pL). Then, cells were lysed with NaOH 1 M (750 pL) to collect the bound fraction. Radioactivity of each fraction was determined in a gamma-counter. Results are expressed as percentage of total binding.

[0288] Results

[0289] On LNCaP cells and after 30 minutes internalization, [161Tb]Tb-DOTA-HYNIC-panPSMA showed increasing efflux up to 2h. This value remains constant until 4h reaching 72.75 ± 10.35% (Figure 13A). Similar results were obtained on 22Rv1 cells. On PC3-pip cells, efflux reaches 88-96%. Interestingly on PC3 wild type cells, efflux was as high as 80-85% suggesting that the efflux seen is not specific. Similar results were obtained with [177Lu]Lu-DOTA-HYNIC-panPSMA (Figure 13B).

[0290] 4. Uptake study

[0291] Materials and Methods

[0292] A total of 1 nM of [161Tb]Tb-DOTA-HYNIC-panPSMA or [177Lu]Lu-DOTA-HYNIC-panPSMA in 250 pL of medium was added in each well. Plates were incubated for 60, 120, or 240 min at 37 °C, 5% CO2. Three minutes before the selected time point, plates were stored at 4 °C to stop the internalization process. Media were removed, and wells were rinsed three times with ice cold DPBS (250 pL). Then, cells were lysed with NaOH 1 M (750 pL). Radioactivity of each fraction was determined in a gamma-counter. Results are expressed as percentage of applied dose per million cells. Results

[0293] [161Tb]Tb- DOTA-HYNIC-panPSMA was used at 0.99 MBq / nmol and [177Lu]Lu- DOTA-HYNIC- panPSMA at 0.81 MBq / nmol. [161Tb]Tb- DOTA-HYNIC-panPSMA showed a massive uptake in LNCaP (~200%AD / 106cells) and PC3-pip cells (~300%AD / 106cells) while minimum uptake was seen on 22Rv1 and PC3 wild type cells due the low PSMA expression or absence pf PSMA expression respectively (<3%AD / 106cells). Results are depicted in Figure 14A.

[0294] [177Lu]Lu-DOTA-HYNIC-panPSMA showed a similar pattern of uptake on PC3 wild type cells, 22Rv1 cells and LNCaP. Surprisingly, uptake on PC3-pip cells was not different than LNCaP. Results are depicted in Figure 14B.

[0295] 5. Therapy study

[0296] Materials and methods

[0297] 2500 cells per well were seeded the day before the experiment in 96 well plates in RPMI supplemented with 10% FBS and antibiotics. The medium was then removed and increasing activities of [161Tb]Tb- DOTA-HYNIC-panPSMA and [177Lu]Lu- DOTA-HYNIC-panPSMA (0, 0.1 , 0.2, 0.5, 1 , 2, 5, 10 and 20 MBq / mL) was added to the wells in medium without FBS for 4h at 37°C, 5% CO2. At the end of incubation the radiopharmaceutical was removed and cells were recultured for three additional days in RPMI supplemented with 10% FBS and antibiotics. At the end of the three, cell viability was evaluated using Cell Titer gio according to manufacturer’s instructions. Luminescence was recorded using a multiplate reader (BMG Labtech). Results were expressed in percentage of untreated cells. To correct the results to the exact activity added in each well, standards containing the same activity than the wells were gamma counted owing the normalization factor of 23.97 cpm / Bq of our gamma counter (Perkin Elmer, Wizard2). Standards were counted until they fall into the acceptance range of161Tb of our device (1000 - 1.7.106cpm). IC50 was derived by fitting the curve using the « [inhibitor] vs normalized response » equation using GraphPad software v10.2.

[0298] Results

[0299] The results are given in Figure 15. [161Tb]Tb- DOTA-HYNIC-panPSMA is able to inhibit cell viability in a (log)dose-dependent manner. Curve fitting showed that the IC50 is 1595 kBq, 95%CI [1319 - 1937 kBq], Example 9: Optimisation of lutetium-177 radiolabeling of DOTA-HYNIC-panPSMA.

[0300] 600|jg of precursor was mixed with 50mg / mL mannitol, and 50mg / mL ascorbic acid dissolved in

[0301] 1.1 mL acetate buffer (pH=5.0- 5.5). The precursor solution was labelled with up to 10GBq of 177Lu-LuCI3 solution, applying 85°C for 10 minutes. The lutetium-177 radiolabelled DOTA- HYNIC-panPSMA obtained had a radiochemical purity of above 95% measured by HPLC.

[0302] The conditions and results are depicted in the Table below:

[0303] Example 10: Optimisation of gallium-68 radiolabeling of DOTA-HYNIC-panPSMA

[0304] In this experiment the kit-based approach and cassette based approach were compared using the set-up indicated below for each of them. From the results (cf. Figures 22A and 22B) we can derive that both approaches work well but the cassette based approach is more effective.

[0305] Kit protocol:

[0306] Dissolve 50 microgram of DOTA-HYNIC-iPSMA (Telix) in 1 ml acetate buffer (Telix) and elute

[0307] 68GaCh generator (Eckert & Ziegler) into said mixture with HCI, swivel and incubate for 10 minutes at 95°C in a heating block, swivel and let cool (at room temperature) for 10 minutes. Cassette Protocol (Modular lab easy Radiosyntheser - Eckert & Ziegler):

[0308] Dissolve 50 microgram of DOTA-HYNIC-iPSMA (Telix) in 1ml acetate buffer (Telix) 2.3 ml ammonium acetate (Eckert & Ziegler) and 0.6 ml ethanol / water solution (Eckert & Ziegler) in a reaction vial. Eluting the68GaCh generator (Eckert & Ziegler) and trapping the Gallium on an SOX column. Eluting said gallium from the column into said reaction vial with NaCI / HCI solution (Eckert

[0309] & Ziegler. Heta the mixture for 10 minutes at 90°C and purify over a CM ceramic column.

[0310] Optionally it can be sterilized using know means.

Claims

54CLAIMS1. A method for radiolabeling DOTA-HYNIC-panPSMA defined by chemical formula (I)with a metal radionuclide (isotope) selected from the group consisting of: terbium-161, actinium- 225, lutetium-177, gallium-67, or gallium-68, comprising the steps of: a) providing DOTA-HYNIC-panPSMA; b) optionally, providing a buffering agent or buffer solution, allowing to maintain the pH in the range of 3.5 to 7.0, preferably 3.5 to 6.5, preferably 4.0 to 6.0, preferably 4.5 to 5.5, more preferably 4.8 to 5.3; c) providing a suitable amount of radionuclide solution, preferably a solution of terbium-161 actinium-225 or gallium-67 or -68; d) contacting the solution of step c) into a mixture of a) and b) and allowing the radiolabeling reaction to take place, preferably at a temperature of 80 to 95°C, for a period of between 5 and 35 minutes; thereby obtaining a radiolabeled targeting agent; e) optionally, purifying the radiolabeling mixture by eliminating unbound radionuclide from the radiolabeling mixture, thereby obtaining a purified radiolabeled targeting agent.

2. The radiolabelling method according to claim 1, thereby obtaining DOTA-HYNIC- panPSMA which is radiolabelled with Lutetium-177 according to formula II:

3. The radiolabelling method according to claim 1, thereby obtaining DOTA-HYNIC- panPSMA which is radiolabelled with Actinium-225 according to formula III:

4. The radiolabelling method according to claim 1, thereby obtaining DOTA-HYNIC- panPSMA which is radiolabelled with Terbium-161 according to formula IV:

5. The radiolabelling method according to claim 1, thereby obtaining DOTA-HYNIC- panPSMA which is radiolabelled with Gallium-68 according to formula V or radiolabelled with Gallium-67 according to formula VI:(VI).

576. The method according to any one of claims 1 to 5, wherein said buffering agent or buffer solution is a phosphate, nitrate, HEPES, acetate, formate, TRIS, and citrate or a mixture thereof, preferably an acetate or formate buffer, more preferably a sodium ascorbate, sodium acetate, ammonium acetate, or sodium formate buffer.

7. The method according to any one of claims 1 to 6, wherein the purification step e), when present, is done by means of solid phase extraction or reverse phase extraction, preferably over a silica-based substrate with strong hydrophobicity such as C18-coated silica, C8-coated silica or C4-coated silica, depending on the targeting ligand to be labeled.

8. The method according to any one of claims 1 to 7, wherein additionally a metal inhibitor and / or a free (non-bound) radiometal sequestering agent is added to the radiolabeling mixture preferably wherein said sequestering agent is used to sequester non-bound radionuclides, and the metal inhibitor is used to sequester radiometal impurities resulting from isotope generator elution (possible break-through).

9. The method according to claim 8, wherein said metal inhibitor is a sugar, preferably a short-chain sugar or oligosaccharide, such as comprising up to 7 monosaccharide units, and / or wherein said sequestering agent is EDTA, DTPA, or combinations thereof.

10. The method according to claim 9, wherein said radiometal is terbium-161 and wherein said sequestering agent is EDTA or DTPA to sequester non-bound terbium radionuclides, optionally combined with a metal inhibitor as defined herein to sequester radiometal impurities resulting from isotope generator elution (break-through).

11. The method according to any one of claims 8 to 10, wherein said metal inhibitor is selected from the group comprising: monosaccharides and their derivatives, disaccharides and their derivatives, and cyclodextrins.

12. The method according to claim 11 , wherein said metal inhibitor is selected from the group comprising: Glucose, D-Fructose, Beta-cyclodextrin, D-Mannose, and beta-cyclodextrin, more preferably D-mannose.

13. The method according to any one of claims 8 to 12, wherein said metal inhibitor and said functionalised agent are not chemically linked or are chemically linked through an unstable or cleavable bond in the radiolabeling conditions.

14. The method according to any one of claims 1 , 2, 6 to 9 and 11 to 13, wherein the radiolabel is lutetium-177 and wherein the radiolabelling reaction takes place in a radiolabelling mixture comprising an acetate buffer with pH of between 4.0 and 6.5, more preferably of between 4.5 and 6.0, even more preferably of between 5.0 and 5.5, applying about 85°C for 10 to 15 minutes.

15. The method according to claim 14, wherein said radiolabelling mixture additionally comprises 50mg / mL mannitol, and / or 50mg / mL ascorbic acid.

16. The method according to claim 14 or 15, wherein the radiolabelling is done with an activity of from 10OM Bq to up to 10GBq of177Lu.

17. The method according to any one of claims 1 , 3, 6 to 9 and 11 to 13, wherein the radionuclide is actinium-225, and wherein the radiolabeling reaction is carried out in a HCI radiolabeling mixture with ascorbic acid as a stabiliser at a pH of between 4.0 and 5.5, more preferably between 4.5 and 5, radiolabeled at about 90 to 95°C, for a time of about 10 to 20 minutes, such as 15 min.

18. The method according to claim 17, wherein said mixture is optionally formulated into a sodium ascorbate and pentetic acid (DTPA) buffer.

19. The method according to claim 17 or 18, wherein a post-radiolabeling purification of the radiolabeled targeting agent is done on a silica column, preferably on a C18 silica column.

20. The method according to any one of claims 17 to 19, wherein the activity is from 1 to 20 MBq, such as up to 5 MBq, such as from 5 to 20MBq.

21. The method according to any one of claims 1 , 4, 6 to 13, wherein the radionuclide is terbium-161 and wherein the radiolabeling reaction is carried out in an acetate buffer at a pH of between 4.0 and 5.5, more preferably between 4.5 and 5, or of about 5, or about 5.1.

22. The method according to claim 21 , wherein an ammonium acetate buffer salt is used.

23. The method according to claim 21 or 22, wherein additionally ascorbic acid is added as a stabiliser and / or EDTA is added when more than 300 or 400MBq (10mCi) is added in activity.

24. The method according to any one of claims 21 to 23, wherein the radiolabeling reaction is carried out at about 90°C for about 25 to 35 minutes, preferably for about 30 minutes.

25. The method according to any one of claims 21 to 24, wherein no post-purification of the radiolabeled targeting agent is needed.

26. The method according to any one of claims 1 , 5 to 9 and 11 to 13, wherein the radionuclide is gallium-67 or -68, and wherein the radiolabeling reaction is carried out in an acetate buffer, such as a sodium acetate buffer at pH of between 3.5 and 5.5, more preferably of between 4.0 and 5.0, at a temperature of about 80 to 95°C, such as at about 90°C for about 5 to 30 minutes, such as for about 10 to 15 minutes.

27. The method according to claim 26, wherein no post-purification of the radiolabeled targeting agent is done.

28. The method according to claim 26 or 27, wherein said isotope is added in an activity of up to 2GBq (about 50mCi), such as from 100 to 2GBq typically in an activity of up to 50MBq to 2GBq.

29. The method according to any one of the previous claims, wherein when the radiolabel is terbium-161 or lutetium-177 and wherein the activity is from 1 to 20GBq, such as up to 400MBq, such as from 100 to 400MBq, or up to 20Gbq such as from 400MBq to 10GBq.

30. The method according to any one of the previous claims, wherein additionally a stabiliser is added to the radiolabeling mixture that prevents radiolysis (product degradation) of the chelate- functionalized targeting agent, wherein said stabiliser is selected from the group consisting of: ascorbic acid, dehydroascorbic acid, gentisic acid, cysteine, N-acetylcysteine, and methionine, or a salt thereof, preferably as a solution to the radiolabeling mixture prior to radiolabeling.

31. The method according to claim 30, wherein the radiolabeling mixture comprises at least 10.0 mg, preferably at least 15.0 mg, preferably at least 20.0 mg, preferably at least 25.0 mg, preferably at least 30.0 mg of stabiliser, or at most 100.0 mg, preferably at most 90.0 mg, preferably at most 80.0 mg, preferably at most 70.0 mg, preferably at most 60.0 mg, or at most 50.0 mg of stabiliser.

32. The method according to claim 30 or 31 , wherein the stabiliser is ascorbic acid, dehydroascorbic acid or a salt thereof, more preferably of ascorbic acid, and optionally wherein said stabiliser is provided together with a bisulfite and or a metabisulfite.

33. A lutetium-177 radiolabeled DOTA-HYNIC-panPSMA obtained by the method according to any one of claims 1, 2, or 6 to 16, having the chemical structure of formula (II)34. An actinium-225 radiolabeled DOTA-HYNIC-panPSMA obtained by the method according to any one of claims 1, 3, 6 to 13, or 17 to 20, having the chemical structure of formula (III):

35. A terbium-161 radiolabeled DOTA-HYNIC-panPSMA obtained by the method according to any one of claims 1, 4, 6 to 13, and 21 to 25, having the chemical structure of formula (IV):

36. A gallium-68 or gallium-67 radiolabeled DOTA-HYNIC-panPSMA obtained by the method according to any one of aspects 1 , 5 to 13, and 26 to 28, having the chemical structure of respectfully formula (V) or formula (VI):

37. A radiolabeling kit, suitable for producing a radiolabelled DOTA-HYNIC-panPSMA targeting agent comprising: - a DOTA-HYNIC-panPSMA functionalized targeting agent as defined by general formula (I):, preferably in dried or lyophilized form; - a buffering agent or buffer solution, allowing to maintain the pH in the range of 3.5 to 7, such as3.5 to 6.5 or 4.0 to 5,5, more preferably 4,5 to 5.0, optionally in dried or lyophilized form;- optionally a metal inhibitor and / or a free (non-bound) radiometal sequestering agent;- optionally a stabiliser selected from the group consisting of: ascorbic acid, sodium ascorbate, dehydroascorbic acid, gentisic acid, cysteine and methionine, or a salt thereof, preferably as a solution, or in a dried or lyophilized form;- optionally, a post-radiolabeling purification means suitable for eliminating unbound metal radionuclide from the radiolabeling mixture after the radiolabeling reaction took place.

38. The kit according to claim 37, wherein said metal inhibitor, is a co-chelating agent, capable of inactivating metals other than the desired radionuclide metal without interfering with the chelation between the radionuclide metal and the said chelate-functionalized targeting agent, under the conditions of the labeling reaction.

39. The kit according to any one of claims 37 to 38, wherein said DOTA-HYNIC-panPSMA is radiolabelled with Lutetium- 177 according to formula II:

40. The kit according to any one of claims 37 to 38, wherein said DOTA-HYNIC-panPSMA is radiolabelled with Actinium-225 according to formula III:

41. The kit according to any one of claims 37 to 38, wherein said DOTA-HYNIC-panPSMA is radiolabelled with Terbium-161 according to formula IV:

42. The kit according to any one of claims 37 to 38, wherein said DOTA-HYNIC-panPSMA is radiolabelled with Gallium-68 according to formula V or radiolabelled with Gallium-67 according to formula VI:(VI).

43. The kit according to any one of claims 37 to 42, wherein said buffering agent or buffer solution is a phosphate, nitrate, HEPES, acetate, formate, TRIS, and citrate or a mixture thereof, preferably a sodium ascorbate, sodium acetate, ammonium acetate, or sodium formate buffer.

44. The kit according to any one of claims 37 to 43, wherein the post-radiolabeling purification means is a solid phase extraction or reverse phase extraction means, preferably a silica-based substrate with strong hydrophobicity such as C18-coated silica, C8-coated silica, C6-coated silica or C4-coated silica, depending on the targeting ligand to be labeled.

45. The kit according to any one of claims 37 to 44, wherein the DOTA-functionalized targeting agent and the buffer are provided in dried or lyophilized form, either in two separate or in one single vial. In one embodiment, also the metal inhibitor and / or stabilising agent are present in lyophilised form, either in a separate vial, or combined with either the buffer or targeting agent.6646. The kit according to any one of claims 37 to 45, comprising at least 10.0 mg, preferably at least 15.0 mg, preferably at least 20.0 mg, preferably at least 25.0 mg, preferably at least 30.0 mg of stabiliser, or at most 100.0 mg, preferably at most 90.0 mg, preferably at most 80.0 mg, preferably at most 70.0 mg, preferably at most 60.0 mg, preferably at most 50.0 mg of stabiliser, wherein said stabiliser preferably is ascorbic acid.

47. The kit according to any one of claims 37 to 46, comprising at least 10.0 pg, preferably at least 15.0 pg, preferably at least 20.0 pg, preferably at least 25.0 pg of said DOTA-HYNIC- panPSMA targeting agent.

48. The kit according to any one of claims 37 to 47, comprising at least 2.0 pg, preferably at least 4.0 pg, preferably at least 6.0 pg, preferably at least 8.0 pg, preferably at least 10.0 pg of said metal inhibitor.

49. The kit according to any one of claims 37 to 48, comprising at least 50.0 mg, preferably at least 75.0 mg, preferably at least 100.0 mg, preferably at least 125.0 mg, preferably at least 150.0 mg of said buffering agent.

50. The kit according to any one of claims 37 to 49, wherein the stabiliser is provided in the kit together with a bisulfite and or a metabisulfite.

51. The kit according to any one of claims 37 to 50, wherein said metal inhibitor is a sugar, preferably a short-chain sugar or oligosaccharide, such as comprising up to 7 monosaccharide units.

52. The kit according to any one of claims 37 to 51 , wherein said metal inhibitor is selected from the group comprising: monosaccharides and their derivatives, disaccharides and their derivatives, and polysaccharides and cyclodextrins.

53. The kit according to any one of claims 37 to 52, wherein said metal inhibitor is selected from the group comprising: Glucose, D-Fructose, Beta- cyclodextrin, and D-Mannose.

54. The kit according to any one of claims 37 to 53, wherein said metal inhibitor and said functionalised agent are not chemically linked.6755. The kit according to any one of claims 37 to 54, wherein said metal inhibitor and said functionalised agent are chemically linked, through a linker that is unstable in the radiolabeling conditions.

56. An imaging method, comprising the steps of:1) radiolabeling an DOTA-HYNIC-panPSMA targeting agent with lutetium-177, actinium-225, terbium-161, gallium-67 or gallium-68 according to the method of any one of claims 1 to 32, preferably by using a kit according to any one of claims 37 to 55,2) administering to a subject a diagnostic amount of said radiolabeled targeting agent; and,3) detecting said radiolabeled targeting agent when bound to its target using PET or SPECT or PET / CT or SPECT / CT imaging methods.

57. The imaging method according to claim 56, wherein said radiolabel is lutetium-177 and wherein the imaging method used is SPECT or SPECT / CT imaging.

58. The imaging method according to claim 56, wherein said radiolabel is actinium-225 and wherein the imaging method used is SPECT or SPECT / CT imaging.

59. The imaging method according to claim 56, wherein said radiolabel is terbium-161 and wherein the imaging method used is PET, PET / CT, SPECT or SPECT / CT imaging, preferably SPECT or SPECT / CT imaging.

60. The imaging method according to claim 56, wherein said radiolabel is gallium-67 and wherein the imaging method used is SPECT or SPECT / CT imaging.

61. The imaging method according to claim 56, wherein said radiolabel is gallium-68 and wherein the imaging method used is PET or PET / CT imaging.

62. A method of detecting a tumour or cancer, comprising the steps of:1) radiolabeling DOTA-HYNIC-panPSMA with lutetium-177, actinium-225, terbium-161, gallium- 67 or gallium-68, according to the method of any one of claims 1 to 32, preferably by using a kit according to any one of claims 37 to 55,682) administering to a subject a diagnostic amount of said radiolabeled DOTA-HYNIC-panPSMA; and,3) detecting binding of said radiolabeled DOTA-HYNIC-panPSMA using PET or SPECT or PET / CT or SPECT / CT imaging methods.

63. The method according to claim 62, wherein said imaging methods are defined according to any one of claims 57 to 61 .

64. The method according to claim 62 or 63, wherein said detection is used for:(i) initial staging of prostate cancer into intermediate, unfavourable, high, or very high-risk prostate cancer;(ii) detecting suspected recurrence of prostate cancer and / or detection of metastasis;(iii) selection for radiotherapeutic treatment such as with Lutetium (177Lu) vipivotide tetraxetan (Pluvicto);(iv) monitoring prostate cancer for progression into Non-Metastatic or Metastatic Castration- Resistant Prostate Cancer (nmCRPC or mCRPC);(v) determining response to (radio)therapy;(vi) real-time imaging or diagnosis in a subject e.g. for use in PET scans for tracking slow biological processes or progression of those over a period of time. In some embodiments, this aspect can be carried out by a computer or processor and hence is directed by a system for in vivo diagnosis or for in vivo imaging in a subject, said system comprising a processing means (processor) configured to carry out the steps of receive real-time imaging data and presenting said imaging date in real-time to the user, thereby enabling in vivo imaging or in vivo diagnosis of said subject; or(vii) personalised medicine, or determining a personalised medicine treatment strategy.

65. The method according to any one of claims 62 to 64, wherein said detection method is used to replace the need for taking a prostate biopsy or is used in SPECT, SPECT / CT, PET orPET / CT scan with MRI in clinically significant or intermediate favourable prostate cancers, or in MRI for active surveillance of prostate cancer.

66. The lutetium-177, actinium-225, terbium-161, or gallium-67, or -68 radiolabeled targeting molecule according to any one of claims 33 to 36, for use in radiodiagnosis, preferably for use in in vivo real time detection of a targeting agent.

67. The lutetium-177, actinium-225 or terbium-161 radiolabeled targeting molecule according to any one of claims 33 to 36, for use in radiotherapy.

68. A method for treatment of cancer, comprising the step of administering an effective amount of the lutetium-177, actinium-225 or terbium-161 radiolabeled DOTA-HYNIC-panPSMA according to any one of claims 33 to 35 to a patient in need thereof, preferably wherein said cancer has been detected using the method of any one of claims 62 to 65.

69. The method according to any one of claims 62 to 65 or the method according to claim 68, wherein said cancer is over-expressing PSMA.

70. The method according to any one of claims 62 to 65 or the method according to claim 68, wherein said cancer is selected from the group consisting of: prostate cancer, salivary gland cancer, glioblastoma, thyroid cancer, clear cell renal carcinoma, renal cell carcinoma, hepatocellular carcinoma, lung cancer, adenoid cystic carcinoma of the parotid, and breast cancer.

Images