KIT FOR LABELING A PROSTATE-SPECIFIC MEMBRANE ANTIGEN LIGAND WITH A RADIOACTIVE ISOTOPE

Abstract

A kit for labeling a prostate-specific membrane antigen (PSMA) ligand with a radioactive isotope such as .sup.68Ga, .sup.177Lu, or .sup.90Y. Radiolabeled PSMA ligands prepared by this kit can be used for both imaging and therapy purposes. The kit includes a disposable reaction vial containing predetermined amounts of a sodium-based buffering agent and a PSMA ligand, both in dried form; or two disposable reaction vials, wherein one of the reaction vials contains predetermined amounts of the sodium-based buffering agent in dried form and the other of the reaction vials that contains predetermined amounts of the PSMA ligand in dried form, wherein the reaction vial containing the PSMA ligand and optionally the sodium-based buffering agent further contains protons adhered to the inner walls of the reaction vial.

Claims

1. A kit for labling a PSMA ligand with a radioactive isotope, said kit comprising: (i) a disposable reaction vial containing predetermined amounts of a sodium-based buffering agent and a PSMA ligand, both in dried form; or two disposable reaction vials, wherein one of said reaction vials contains predetermined amounts of said sodium-based buffering agent in dried form and the other of said reaction vials that contains predetermined amounts of said PSMA ligand in dried form, wherein said reaction vial containing said PSMA ligand and optionally said sodium-based buffering agent further contains protons adhered to the inner walls of said reaction vial; and (ii) instructions for optionally adding said sodium-based buffering agent to the reaction vial containing said PSMA ligand, and for labeling said PSMA ligand with a radioactive isotope selected from the group consisting of .sup.68Ga, .sup.117Lu and .sup.90Y, wherein said PSMA ligand is represented by the general formula I:
R.sub.1COR.sub.2LBX I wherein B is a chelating agent capable of coordinating with said radioactive isotope; X is absent or is of the formula LR.sub.2COR.sub.1; L and L each independently is absent or a linker; R.sub.1 and R.sub.1 each independently is an amino acid residue linked via an amino group thereof to the adjacent CO- group; and R.sub.2 and R.sub.2 each independently is an amino acid residue linked via an amino group thereof to the adjacent CO- group.

2. The kit of claim 1, wherein said sodium-based buffering agent is sodium formate, sodium ascorbate, sodium acetate, sodium hydroxide, or sodium citrate.

3. The kit of claim 2, further comprising at least one of: (i) an additional vial containing a predetermined amount of HCl having a molarity in a range of 0.05 to 0.1N for eluting said radioactive isotope from a radioactive isotope generator; and (ii) an additional vial containing a liquid sodium-based buffering agent for pH adjustment.

4. The kit of claim 1, wherein R.sub.1 and R.sub.1, when present, are a glutamic acid residue.

5. The kit of claim 1, wherein R.sub.2 and R.sub.2, when present, are a glutamic acid residue or a lysine residue.

6. The kit of claim 1, wherein L and L each independently is absent or a linker selected from the group consisting of an amino acid residue forming a peptide bond with R.sub.2 or R.sub.2, respectively, a peptide moiety consisting of 2-6 amino acid residues and forming a peptide bond with R.sub.2 or R.sub.2, respectively, (C.sub.1-C.sub.8)alkylene, (C.sub.2-C.sub.8)alkenylene and (C.sub.2-C.sub.8)alkynylene, wherein said (C.sub.1-C.sub.8)alkylene, (C.sub.2-C.sub.8)alkenylene and (C.sub.2-C.sub.8)alkynylene is optionally substituted with one or more groups each independently is selected from the group consisting of halogen, COR.sub.3, COOR.sub.3, OCOOR.sub.3, OCON(R.sub.3).sub.2, CN, NO.sub.2, SR.sub.3, OR.sub.3, N(R.sub.3).sub.2, CON(R.sub.3).sub.2, SO.sub.2R.sub.3, SO.sub.3H, and S(O)R.sub.3, and further optionally interrupted by one or more identical or different heteroatoms selected from the group consisting of S, O and N, and/or at least one group selected from the group consisting of NHCO, CONH-, and N(C.sub.1-C.sub.8alkyl)-, wherein R.sub.3 each independently is selected from the group consisting of hydrogen, and -(C.sub.1-C.sub.8)alkyl.

7. The kit of claim 6, wherein (i) said linker is an amino acid residue, or a peptide moiety consisting of 2-6 amino acid residues, wherein said amino acid each independently is 6-aminohexanoic acid, 8-aminooctanoic acid, 1-naphthylalanine (1Nal), 2-naphthylalanine (2Nal), or 4-(aminomethyl)cyclohexane carboxylic acid (Amc); or (ii) said linker each independently is (C.sub.1-C.sub.8)alkylene, (C.sub.2-C.sub.8)alkenylene or (C.sub.2-C.sub.8)alkynylene, optionally substituted with one or more groups each independently is selected from the group consisting of halogen, COH, COOH, OCOOH, OCONH.sub.2, SH, OH, NH.sub.2, CONH.sub.2, SO.sub.2H, and S(O)H, and further optionally interrupted by one or more identical or different heteroatoms selected from the group consisting of S, O and N, and/or at least one group selected from the group consisting of NHCO, CONH-, and N(C.sub.1-C.sub.8alkyl)-.

8. The kit of claim 7, wherein said linker is 6-aminohexanoic acid or 8-aminooctanoic acid.

9. The kit of claim 7, wherein said linker is a moiety of 1Nal-Amc, 2Nal-Amc, Amc-1Nal or Amc-2Nal.

10. The kit of claim 1, wherein said chelating agent is N,N-bis[2-hydroxy-5-(carboxyethyl)benzyl] ethylenediamine-N,N-diacetic acid (HBED-CC) or 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), forming amide bond with either L, when present, or R.sub.2, and when X is present, forming another amide bond with either L, when present, or R.sub.2.

11. The kit of claim 1, wherein: R.sub.1 and R.sub.1, when present, are a glutamic acid residue; R.sub.2 and R.sub.2, when present, are a glutamic acid residue or a lysine residue; L and L each independently is absent or a linker selected from the group consisting of an amino acid residue forming a peptide bond with R.sub.2 or R.sub.2, respectively, a peptide moiety consisting of 2-6 amino acid residues and forming a peptide bond with R.sub.2 or R.sub.2, respectively, (C.sub.1-C.sub.8)alkylene, (C.sub.2-C.sub.8)alkenylene and (C.sub.2-C.sub.8)alkynylene, wherein said (C.sub.1-C.sub.8)alkylene, C.sub.2-C.sub.8)alkenylene and (C.sub.2-C.sub.8)alkynylene each independently is optionally substituted with one or more groups each independently is selected from the group consisting of halogen, CORS, COORS, OCOOR.sub.3, OCON(R.sub.3).sub.2, CN, NO.sub.2, SR.sub.3, OR.sub.3, N(R.sub.3).sub.2, CON(R.sub.3).sub.2, SO.sub.2R.sub.3, SO.sub.3H, and S(O)R.sub.3, and further optionally interrupted by one or more identical or different heteroatoms selected from the group consisting of S, O and N, and/or at least one group selected from the group consisting of NHCO, CONH-, and N(C.sub.1-C.sub.8alkyl)-, wherein R.sub.3 each independently is selected from the group consisting of hydrogen, and -(C.sub.1-C.sub.8)alkyl; and said chelating agent is HBED-CC or DOTA, forming an amide bond with either L, when present, or R.sub.2, and when X is present, forming another amide bond with either L, when present, or R.sub.2.

12. The kit of claim 11, wherein L and L, when present, each independently is a linker selected from the group consisting of an amino acid residue, a peptide moiety consisting of 2-6 amino acid residues, (C.sub.1-C.sub.8)alkylene, (C.sub.2-C.sub.8)alkenylene and (C2-C8)alkynylene, wherein said amino acid each independently is 6-aminohexanoic acid, 8-aminooctanoic acid, 1Nal, 2Nal, or Amc; and said (C.sub.1-C.sub.8)alkylene, (C.sub.2-C.sub.8)alkenylene and (C.sub.2-C.sub.8)alkynylene is optionally substituted with one or more groups each independently is selected from the group consisting of halogen, COH, COOH, OCOOH, OCONH.sub.2, SH, OH, NH.sub.2, CONH.sub.2, SO.sub.2H, and S(O)H, and further optionally interrupted by one or more identical or different heteroatoms selected from the group consisting of S, O and N, and/or at least one group selected from the group consisting of NHCO, CONH-, and N(C.sub.1-C.sub.8alkyl)-.

13. The kit of claim 12, wherein L and L each independently is a moiety of 1Nal-Amc, 2Nal-Amc, Amc-1Nal or Amc-2Nal, or a residue of 6-aminohexanoic acid or 8-aminooctanoic acid.

14. The kit of claim 13, wherein (i) X is absent; R.sub.2 is a lysine residue linked via its -amino group to the adjacent CO- group; and (a) L is 6-aminohexanoic acid; and B is HBED-CC linked via a carboxylic group thereof to the amino group of the 6-aminohexanoic acid; or (b) L is Amc-2Nal or Amc-1Nal linked via the carboxylic group of the 2Nal or 1Nal, respectively, to the side chain amino group of R.sub.2; and B is DOTA linked via a carboxylic group thereof to the amino group of the Amc; or (ii) X is present; R.sub.2 and R.sub.2 each is a lysine residue linked via its -amino group to the adjacent CO- group; L and L each is 6-aminohexanoic acid; and B is HBED-CC linked via one carboxylic group thereof to the amino group of L and via another carboxylic group thereof to the amino group of L.

15. The kit of claim 14, wherein said radioactive isotope is .sup.68Ga.

Description

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

[0028] The present invention describes new methods for radiolabeling PSMA ligands with suitable radioactive isotopes, such as .sup.68Ga, .sup.90Y and .sup.177Lu, under mild reaction conditions (e.g., room temperature) and for short periods of time (e.g., 3 to 30 minutes), while still obtaining quantitative radiochemical yields, wherein said radiolabeled PSMA isotope is suitable for the diagnosis or therapy of prostate cancer. Accordingly, the present invention provides methods for radiolabeling PSMA, which are both simple and fast, and require neither sophisticated nor expensive equipment as required by the automated synthesis module currently available, such as the hot-cell. Moreover, since the method of the inveiton enables over 95% binding of the radiaoactive isotop to said PSMA ligand, it eliminates the need for a step of removing unbound isotop from the reaction vial, which is an essential step of the presently known labeling methods, involving, e.g., using a designated cartridge of column for said unbound isotop removal.

[0029] In one aspect, the present invention thus provides a method for labeling a PSMA ligand with a radioactive isotope such as .sup.68Ga, .sup.177Lu, or .sup.90Y, said method consisiting essentially of the steps of: (i) providing a reaction vial containing a predetermined amounts of a sodium-based buffering agent and said PSMA ligand, both in dried form, i.e., either lyophilized or spray dried; (ii) adding a solution of said radioactive isotope in a predetermined amount of HCl having a predetermined molarity to said reaction vial, thus obtaining a solution of said PSMA ligand and said radioactive isotope in said HCl; (iii) mixing the solution obtained in (ii) and then incubating it for a sufficient period of time, thus reacting said PSMA ligand with said radioactive isotope to thereby obtain said PSMA ligand labeled with said radioactive isotope, wherein at least 90%, preferably 95%, of said radioactive isotope in said solution is bound to said PSMA ligand; and (iv) optionally adjusting the pH of the solution in said reaction vial to a pH compatible with physiological conditions by adding a sodium-based buffering agent, [0030] wherein said PSMA ligand is of the general formula I:

R.sub.1COR.sub.2LBX I [0031] wherein B is a chelating agent capable of coordinating with said radioactive isotope; X is absent or is of the formula LR.sub.2COR.sub.1; L and L each independently is absent or a linker; R.sub.1 and R.sub.1 each independently is an amino acid residue linked via an amino group thereof to the adjacent CO- group; and R.sub.2 and R.sub.2 each independently is an amino acid residue linked via an amino group thereof to the adjacent CO- group.

[0032] The terms PSMA ligand, PSMA tracer and PSMA inhibitor, used herein interchangeably, refer to a compound of the general formula I defined above, i.e., a compound comprising (a) an urea core formed by linking two amino acid residues via their amino groups to a carbonyl group, and capable of interacting thus inhibiting the PSMA enzyme; and (b) a chelating agent capable of coordinating with a radioactive isotope such as .sup.68Ga, .sup.177Lu, or .sup.90Y, and linked to said urea core optionally via a linker linked to one of said amino acid residues. Particular PSMA ligands described herein are those having an urea core formed by linking glutamic acid and lysine residues, each via its -amino group, to a carbonyl group, and a chelating agent linked to the side chain amino group of the lysine residue via a linker consisting of an amino acid or a dipeptide.

[0033] The term amino acid as used herein refers to an organic compound comprising both amine and carboxylic acid functional groups, which may be either a natural or synthetic, i.e., non-natural, amino acid in its both L- and D-stereoisomers. The twenty two natural amino acids are aspartic acid (Asp), tyrosine (Tyr), leucine (Leu), tryptophan (Trp), arginine (Arg), valine (Val), glutamic acid (Glu), methionine (Met), phenylalanine (Phe), serine (Ser), alanine (Ala), glutamine (Gln), glycine (Gly), proline (Pro), threonine (Thr), asparagine (Asn), lysine (Lys), histidine (His), isoleucine (Ile), cysteine (Cys), selenocysteine (Sec), and pyrrolysine (Pyl). A non-natural amino acid is any amino acid, modified amino acid and/or an analog thereof, that is not one of those natural amino acids, wherein non-limiting examples of non-natural amino acids include diaminopropionic acid (Dap), diaminobutyric acid (Dab), ornithine (Orn), aminoadipic acid, -alanine, -aminohexanoic acid (Ahx), 6-aminohexanoic acid, 8-aminooctanoic acid, 1-naphthylalanine (1Nal), 2-naphthylalanine (2Nal), 3-(1-naphthyl)alanine, 3-(2-naphthyl)alanine, 4-(aminomethyl)cyclohexane carboxylic acid (Amc), -aminobutiric acid (GABA), 3-(aminomethyl) benzoic acid, p-ethynyl-phenylalanine, p-propargly-oxy-phenylalanine, m-ethynyl-phenylalanine, p-bromophenylalanine, p-iodophenylalanine, p-azidophenylalanine, p-acetylphenylalanine, azidonorleucine, 6-ethynyl-tryptophan, 5-ethynyl-tryptophan, 3-(6-chloroindolyl)alanine, 3-(6-bromoindolyl)alanine, 3-(5-bromoindolyl) alanine, azidohomoalanine, p-chlorophenylalanine, -aminocaprylic acid, O-methyl-L-tyrosine, N-acetylgalactosamine--threonine, and N-acetylgalactosamine--serine. The term amino acid residue refers to a residue of an amino acid results from removal of a hydrogen atom(s) from either an amine or carboxylic acid group thereof, or from both amine and carboxylic groups thereof.

[0034] The term peptide as used herein refers to a chain of 2-8 amino acid monomers linked by peptide bonds, i.e., the covalent bond formed when a carboxyl group of one amino acid reacts with an amino group of another. Particular such peptides are those consisting of 2-6 amino acid residues, more particularly 2, 3, or 4 amino acid residues, i.e., dipeptides, tripeptides or tetrapeptides, respectively.

[0035] The term alkylene as used herein refers to a straight or branched divalent hydrocarbon radical having 1-8 carbon atoms and includes, e.g., methylene, ethylene, propylene, butylene, 2-methylpropylene, pentylene, 2-methylbutylene, hexylene, 2-methylpentylene, 3-methylpentylene, 2,3-dimethylbutylene, heptylene, octylene, and the like. Preferred are (C.sub.1-C.sub.6)alkylene, (C.sub.1-C.sub.4)alkylene, and (C.sub.2-C.sub.4)alkylene. The term alkenylene typically means straight or branched divalent hydrocarbon radicals having 2-8 carbon atoms and one or more double bond such as, without limiting, ethenylene, propenylene, 1- and 2-butenylene, 1- and 2-pentenylene, 1-, 2- and 3-hexenylene, heptenylene, octenylene, and the like. The terms alkynylene typically means straight or branched divalent hydrocarbon radicals having 2-8 carbon atoms and one or more triple bond such as, without limiting, ethynylene, propynylene, 1- and 2-butynylene, 1- and 2-pentynylene, 1-, 2- and 3-hexynylene, heptynylene, octynylene, and the like.

[0036] In certain embodiments, the PSMA ligand being labeled according to the method of the present invention has the general formula I as defined above, wherein R.sub.1 and R.sub.1, if present, are a glutamic acid residue linked via the amino group thereof to the adjacent CO-group.

[0037] In certain embodiments, the PSMA ligand being labeled according to the method of the present invention has the general formula I as defined above, wherein R.sub.2 and R.sub.2, if present, are a glutamic acid residue linked via the amino group thereof to the adjacent CO-group, or a lysine residue linked via either the -amino or side chain amino group thereof to the adjacent CO- group. In particular such embodiments, R.sub.2 and R.sub.2, if present, is a lysine residue linked via its -amino group to the adjacent CO- group.

[0038] In certain embodiments, the PSMA ligand being labeled according to the method of the present invention has the general formula I as defined above, wherein L and L each independently is absent or a linker selected from an amino acid residue forming a peptide bond with R.sub.2 or R.sub.2, respectively, a peptide moiety consisting of 2-8, e.g., 2, 3, 4, 5, or 6, amino acid residues and forming a peptide bond with R.sub.2 or R.sub.2, respectively, (C.sub.1-C.sub.8)alkylene, (C.sub.2-C.sub.8)alkenylene or (C.sub.2-C.sub.8)alkynylene, wherein said (C.sub.1-C.sub.8)alkylene, (C.sub.2-C.sub.8)alkenylene and (C.sub.2-C.sub.8)alkynylene is optionally substituted with one or more, e.g., 1, 2, or 3, groups each independently is selected from halogen, CORS, COORS, OCOOR.sub.3, OCON(R.sub.3).sub.2, CN, NO.sub.2, SR.sub.3, OR.sub.3, N(R.sub.3).sub.2, CON(R.sub.3).sub.2, SO.sub.2R.sub.3, SO.sub.3H, or S(O)R.sub.3, and further optionally interrupted by one or more, e.g., 1, 2, or 3, identical or different heteroatoms selected from S, O or N, and/or at least one group, e.g., 1, 2, or more groups, selected from NHCO-, CONH-, N(C.sub.1-C.sub.8alkyl)-, wherein R.sub.3 each independently is selected from hydrogen, or (C.sub.1-C.sub.8)alkyl.

[0039] In certain particular such embodiments, L and L each independently is a linker selected from an amino acid residue, or a peptide moiety consisting of 2-6 amino acid residues, wherein said amino acid each independently is 6-aminohexanoic acid, 8-aminooctanoic acid, 1-naphthylalanine (1Nal), 2-naphthylalanine (2Nal), or 4-(aminomethyl) cyclohexane carboxylic acid (Amc). Preferred such embodiments are those wherein said linker is 6-aminohexanoic acid, 8-aminooctanoic acid, 1Nal-Amc, 2Nal-Amc, Amc-1Nal, or Amc-2Nal.

[0040] In other particular such embodiments, L and L each independently is a linker selected from (C1-C8)alkylene, (C2-C8)alkenylene or (C.sub.2-C.sub.8)alkynylene, optionally substituted with one or more groups each independently is selected from halogen, COH, COOH, OCOOH, OCONH.sub.2, CN, NO.sub.2, SH, OH, NH.sub.2, CONH.sub.2, SO.sub.2H, SO.sub.3H, or S(O)H, and further optionally interrupted by one or more identical or different heteroatoms selected from S, O or N, and/or at least one group selected from NHCO-, CONH-, N(C.sub.1-C.sub.8alkyl)-.

[0041] The chelating agent HBED-CC is a rarely used acyclic complexing agent especially allowing efficient radiolabelling with, e.g., .sup.68Ga even at ambient temperature. Accordingly, in certain embodiments, the PSMA ligand being labeled according to the method of the present invention has the general formula I as defined above, wherein said chelating agent being capable of coordinating with said radioactive isotope is HBED-CC, forming amide bond with either the linker L, if present, or R.sub.2, and when X is present, forming another amide bond with either the linker L, if present, or R.sub.2.

[0042] An alternative chelating agent, widely used for the complexation of various radioactive isotopes such as .sup.68Ga, .sup.177Lu and .sup.90Y, for both diagnostic and therapeutic application, is DOTA. In other embodiments, the PSMA ligand being labeled according to the method of the present invention has the general formula I as defined above, wherein said chelating agent is thus DOTA, forming amide bond with either the linker L, if present, or R.sub.2, and when X is present, forming another amide bond with either the linker L, if present, or R.sub.2.

[0043] In certain embodiments, the PSMA ligand being labeled according to the method of the present invention has the general formula I as defined above, wherein R.sub.1 and R.sub.1, if present, are a glutamic acid residue; R.sub.2 and R.sub.2, if present, are a glutamic acid residue or a lysine residue preferably linked via its -amino group to the adjacent CO- group; L and L each independently is absent or a linker selected from an amino acid residue forming a peptide bond with R.sub.2 or R.sub.2, respectively, a peptide moiety consisting of 2-8 amino acid residues and forming a peptide bond with R.sub.2 or R.sub.2, respectively, (C.sub.1-C.sub.8)alkylene, (C.sub.2-C.sub.8)alkenylene or (C.sub.2-C.sub.8)alkynylene, wherein said (C.sub.1-C.sub.8)alkylene, (C.sub.2-C.sub.8)alkenylene and (C.sub.2-C.sub.8)alkynylene is optionally substituted with one or more groups each independently is selected from halogen, CORS, COORS, OCOOR.sub.3, OCON(R.sub.3).sub.2, CN, NO.sub.2, SR.sub.3, OR.sub.3, N(R.sub.3).sub.2, CON(R.sub.3).sub.2, SO.sub.2R.sub.3, SO.sub.3H, or S(O)R.sub.3, and further optionally interrupted by one or more identical or different heteroatoms selected from S, O or N, and/or at least one group selected from NHCO, CONH, N(C.sub.1-C.sub.8alkyl)-, wherein R.sub.3 each independently is selected from hydrogen, or -(C.sub.1-C.sub.8)alkyl; said radioactive isotope is .sup.177Lu, or .sup.90Y; and said chelating agent is HBED-CC or DOTA, forming amide bond with either L, if present, or R.sub.2, and when X is present, forming another amide bond with either L, if present, or R.sub.2. In particular such embodiments, L and L, if present, each independently is a linker selected from an amino acid residue, a peptide moiety consisting of 2-6 amino acid residues, (C.sub.1-C.sub.8)alkylene, (C.sub.2-C.sub.8)alkenylene or (C.sub.2-C.sub.8)alkynylene, wherein said amino acid each independently is 6-aminohexanoic acid, 8-aminooctanoic acid, 1Nal, 2Nal, or Amc; and said (C.sub.1-C.sub.8)alkylene, (C.sub.2-C.sub.8)alkenylene and (C.sub.2-C.sub.8)alkynylene is optionally substituted with one or more groups each independently is selected from halogen, CORS, COORS, OCOOR.sub.3, OCON(R.sub.3).sub.2, CN, NO.sub.2, SR.sub.3, OR.sub.3, N(R.sub.3).sub.2, CON(R.sub.3).sub.2, SO.sub.2R.sub.3, SO.sub.3H, or S(O)R.sub.3, wherein R.sub.3 is H, and further optionally interrupted by one or more identical or different heteroatoms selected from S, O or N, and/or at least one group selected from NHCO, CONH, N(C.sub.1-C.sub.8alkyl)-. More particular such embodiments are those wherein L and L each independently is 6-aminohexanoic acid, 8-aminooctanoic acid, 1Nal-Amc, 2Nal-Amc, Amc-1Nal, or Amc-2Nal.

[0044] In certain embodiments, the PSMA ligand being labeled according to the method of the present invention is a compound of the general formula I, wherein R.sub.1 is a glutamic acid residue; R.sub.2 is a glutamic acid residue or a lysine residue preferably linked via its -amino group to the adjacent CO- group; L is 6-aminohexanoic acid, 8-aminooctanoic acid, 1Nal-Amc, 2Nal-Amc, Amc-1Nal, or Amc-2Nal; said radioactive isotope is .sup.68Ga, .sup.177Lu, or .sup.90Y; said chelating agent is HBED-CC or DOTA, forming amide bond with L; and X is absent. In one specific such embodiment, the PSMA ligand is a compound of the general formula I, wherein R.sub.2 is a lysine residue linked via its -amino group to the adjacent CO- group; L is 6-aminohexanoic acid; and B is HBED-CC linked via a carboxylic group thereof to the amino group of the 6-aminohexanoic acid, i.e., 4,6,12,19-tetraazadocosane-1,3,7-tricarboxylic acid, 22-[3-[[[2-[[[5 -(2-carboxyethyl)-2-hydroxyphenyl] methyl](carboxy-methyl)amino]ethyl](carboxymethyl)amino]methyl]-4-hydroxyphenyl]-5,13,20- trioxo-(3 S,7 S)-trifluoroacetate salt, herein identified as DKFZ-PSMA-11 (see Appendix). In other specific such embodiments, the PSMA ligand is a compound of the general formula I, wherein R.sub.2 is a lysine residue linked via its -amino group to the adjacent CO- group; L is Amc-2Nal or Amc-1Nal linked via the carboxylic group of the 2Nal or 1Nal, respectively, to the side chain amino group of R.sub.2; and B is DOTA linked via a carboxylic group thereof to the amino group of the Amc, i.e., 2-[3-(1 -carboxy-5-{3-naphthalen-2-yl-2-[(4-{[(4,7, 10-tris(carboxymethyl)-1,4,7,10-tetraaza-cyclododec-1-yl)- acetylamino]-methyl}-cyclohexanecarbonyl)-amino]-propionylamino} -pentyl)-ureido]-pentanedioic acid, herein identified as DKFZ-PSMA-617, or 2[-3-(1-carboxy-5-{3-naphthalen-1-yl-2-[(-4-{[2-(4,7,10-tris(carboxymethyl)-1,4,7,10-tetraaza-cyclododec-1-yl)- acetylamino]-methyl}-cyclohexanecarbonyl)-amino]-propionylamino}-pentyl)-ureido]-pentanedioic acid, herein identified as DKFZ-PSMA-617-1 (see Appendix). According to the method of the invention, these specific PSMA ligands can be labeled with .sup.68Ga, .sup.177Lu, or .sup.90Y.

[0045] In other embodiments, the PSMA ligand being labeled according to the method of the present invention is a compound of the general formula I, wherein X is present; R.sub.1 and R.sub.1 are a glutamic acid residue; R.sub.2 and R.sub.2 are a glutamic acid residue or a lysine residue preferably linked via its -amino group to the adjacent CO- group; L and L each independently is 6-aminohexanoic acid, 8-aminooctanoic acid, 1Nal-Amc, 2Nal-Amc, Amc-1Nal, or Amc-2Nal; said radioactive isotope is .sup.68Ga, .sup.177Lu, or .sup.90Y; and said chelating agent is HBED-CC or DOTA, forming amide bond with L, and another amide bond with L. In one specific such embodiment, the PSMA ligand is a compound of the general formula I, wherein R.sub.2 and R.sub.2 are a lysine residue linked via its -amino group to the adjacent CO- group; L and L each is 6-aminohexanoic acid; and B is HBED-CC linked via one carboxylic group thereof to the amino group of L and via another carboxylic group thereof to the amino group of L, i.e., (3 S,7S)-22-(3-(((carboxymethyl)(2-((carboxymethyl)(2-hydroxy-5-((3R,7S)-1,3,7-tricarboxy-5,13,20-trioxo-4,6,12,19-tetraazadocosan- 22-yl)benzyl)amino)ethyl)amino)methyl)-4-hydroxyphenyl)-5,13,20-trioxo-4,6,12,19-tetraazadocosane-1,3,7-tricarboxylic acid, herein identified as DKFZ-PSMA-10 (see Appendix). According to the method of the invention, these specific PSMA ligands can be labeled with .sup.68Ga, .sup.177Lu, or .sup.90Y.

[0046] In certain embodiments, the present invention provides a method for labeling a PSMA ligand of the general formula I as defined in any one of the embodiments above, wherein the reaction vial provided in step (i) is pretreated with HCl, thereby adhering protons to the vial's wall. Such pretreatment is aimed at significantly reducing adherence of additional protons to the vial's wall upon introducing an eluted isotope's solution, and thus preventing or minimizing possible pH alterations of said solution and facilitating pH conditions favorable for the speedy complexation of the radioactive isotopes with the chelating agent, even at ambient temperature.

[0047] In certain embodiments, the present invention provides a method for labeling a PSMA ligand of the general formula I as defined in any one of the embodiments above, wherein said buffering agent in step (i) is any suitable sodium-based buffering agent such as sodium formate, sodium ascorbate, sodium acetate, sodium hydroxide, or sodium citrate. Specific embodiments are those wherein said buffering agent is sodium formate.

[0048] In certain embodiments, the present invention provides a method for labeling a PSMA ligand of the general formula I as defined in any one of the embodiments above, wherein the solution added in step (ii) to said reaction vial is obtained by eluting said radioactive isotope from a radioactive isotope generator, immediately before step (ii), using said predetermined amount of HCl as an elution solvent. Such embodiments are preferred, e.g., when the radioactive isotope used has a relatively short half-life such in the case of .sup.68Ga, which should thus be eluted from, e.g., a .sup.68Ge/68Ga-generator, using said predetermined amount of HCl having a predetermined molarity, immediately before its addition to the reaction vial and mixing with said PSMA ligand and said sodium-based buffering agent.

[0049] According to the method of the present invention, the HCl solution obtained in step (ii), containing the PSMA ligand and the radioactive isotope, can be mixed using any suitable technique, e.g., by stirring or shaking, and is then incubated for a sufficient period of time and at a suitable temperature so as to react said PSMA ligand with said radioactive isotope, i.e., labeling said PSMA ligand with said radioactive isotope. According to the method of the invention, following this incubation step, at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of said radioactive isotope is bound to said PSMA ligand, i.e., coordinated with said chelating agent B. For example, in certain embodiments, the radioactive isotope is .sup.68Ga, and the solution in step (iii) is incubated for 2 to 10, 2 to 8, or 2 to 6, minutes at room temperature, so as to obtain at least 90% of said .sup.68Ga coordinated with said chelating agent B; or the radioactive isotope is .sup.177Lu or .sup.90Y, and the solution in step (iii) is incubated for about 30 minutes or more, e.g., for about 30, 35, 40, 45, or 50 minutes, at a temperature of about 100 C. or higher, e.g., 100-110 C., 110-120 C., or 120-130 C., so as to obtain at least 95% or at least 99% of said .sup.177Lu or .sup.90Y coordinated with said chelating agent B.

[0050] Depending on the molarity of the HCl solution containing said radioactive isotope, and consequently the pH of the solution added to the reaction vial in step (ii), the pH of the labeled PSMA ligand-containing solution obtained in step (iv) might have to be adjusted to a pH compatible with physiological conditions, i.e., to a pH in a range of 4.0-8.0. The pH adjustment, if required, may be conducted by adding any suitable sodium-based buffering agent such as sodium formate, sodium ascorbate, sodium acetate, sodium hydroxide, or sodium citrate.

[0051] The Example section hereinafter shows preparation of .sup.68Ga-labeled DKFZ-PSMA-11 in a disposable reaction vial containing particular amounts of DKFZ-PSMA-11 and sodium formate, both in lyophilized form, by adding to said reaction vial .sup.68Ga eluted in a particular volume of HCl 0.1N, and incubating the resulting mixture at room temperature for about 5 minutes. The method exemplified is highly efficient, wherein following the short incubation of the PSMA ligand and the radioactive isotope, about 95% of the radioactive isotope is bound to said PSMA ligand, i.e., coordinated with the HBED-CC.

[0052] In one particular such aspect, the invention thus provides a method for labeling a PSMA ligand with .sup.68Ga, said method comprising the steps of: (i) providing a reaction vial containing about 800-1800 mg sodium formate and about 10 g of said PSMA ligand, wherein both the sodium formate and said PSMA ligand are dried; (ii) adding a solution of .sup.68Ga in about 1.8 ml of HCl 0.1N to said reaction vial, thus obtaining a solution of said PSMA ligand and said .sup.68Ga in said HCl, wherein said solution of .sup.68Ga in HCl is obtained from a .sup.68Ga generator, immediately before this step, using said HCl as an elution solvent; (iii) mixing the solution obtained in (ii) and then incubating it for 2 to 10, preferably 2 to 6, minutes at room temperature, thus reacting said PSMA ligand with said .sup.68Ga and obtaining said PSMA ligand labeled with .sup.68Ga; and (iv) optionally adjusting the pH of the solution in said reaction vial to a pH in a range of 4.0 to 8.0, by adding about 0.1 ml sodium hydroxide 1N, wherein said PSMA ligand is of the general formula I:

R.sub.1COR.sub.2LB I

wherein R.sub.1 is a glutamic acid residue linked via its amino group to the adjacent CO- group; R.sub.2 is a lysine residue linked via its -amino group to the adjacent CO- group; and (a) L is 6-aminohexanoic acid linked via the carboxylic group thereof to the to the side chain amino group of R.sub.2; and B is HBED-CC linked via a carboxylic group thereof to the amino group of the 6-aminohexanoic acid; or (b) L is Amc-2Nal or Amc-1Nal linked via the carboxylic group of the 2Nal or 1Nal, respectively, to the side chain amino group of R.sub.2; and B is DOTA linked via a carboxylic group thereof to the amino group of the Amc.

[0053] In another particular such aspect, the invention provides a method for labeling a PSMA ligand with .sup.177Lu or .sup.90Y, said method comprising the steps of: (i) providing a reaction vial containing sodium formate and 10 g of said PSMA ligand, wherein both the sodium formate and said PSMA ligand are dried; (ii) adding a solution of .sup.177Lu or .sup.90Y in about 0.5 ml of HCl 0.1N to said reaction vial, thus obtaining a solution of said PSMA ligand and said .sup.177Lu or .sup.90Y in said HCl; and (iii) mixing the solution obtained in (ii) and then incubating it for about 30 minutes at a temperature of about 100 C. or higher, thus reacting said PSMA ligand with said .sup.177Lu or .sup.90Y and obtaining said PSMA ligand labeled with .sup.177Lu or .sup.90Y, wherein said PSMA ligand is of the general formula I:

R.sub.1COR.sub.2LB I

wherein R.sub.1 is a glutamic acid residue linked via its amino group to the adjacent CO- group; R.sub.2 is a lysine residue linked via its -amino group to the adjacent CO- group; L is Amc-2Nal or Amc-1Nal linked via the carboxylic group of the 2Nal or 1Nal, respectively, to the side chain amino group of R.sub.2; and B is DOTA linked via a carboxylic group thereof to the amino group of the Amc.

[0054] Although in both particular aspects described above, 10 g PSMA ligand were used, it should be noted that the amount of PSMA ligand may be modified, more particularly increased, so as to reduce reaction time. For instance, using 30 g of PSMA ligand instead of 10 g reduces the reaction time by up to 5 minutes when conducted at room temperature (for labeling said PSMA ligand with .sup.68Ga), and by up to 15-30 minutes when carried out at a temperature of about 100 C. (for labeling said PSMA ligand with .sup.177Lu or .sup.90Y).

[0055] The radioactive isotope-labeled PSMA ligand prepared according to the method of the present invention can be administered to a subject, i.e., a mammal in general or a human in particular, for either imaging or treating a prostate cancer or metastases thereof, depending on the particular radioactive isotope used. In certain embodiments, the method of the invention in any one of the embodiments defined above further comprises a step of administering the solution obtained in step (iv) to a subject for imaging or for treatment of prostate cancer or metastases thereof

[0056] In another aspect, the present invention thus provides a PSMA ligand labeled with a radioactive isotope by the method of the invention as defined in any one of the embodiments above, wherein (i) said radioactive isotope is .sup.68Ga, for use in imaging of prostate cancer or metastases thereof; or (ii) said radioactive isotope is .sup.177Lu or .sup.90Y, for use in treatment of prostate cancer or metastases thereof

[0057] In yet another aspect, the present invention relates to a method for imaging of prostate cancer or metastases thereof, the improvement therein comprising using as a PSMA ligand a PSMA ligand labeled with .sup.68Ga by the shake & bake method defined above.

[0058] In still another aspect, the present invention relates to a method for the treatment of prostate cancer or metastases thereof, the improvement therein comprising using as a PSMA ligand a PSMA ligand labeled with .sup.177Lu or .sup.90Y by the shake & bake method defined above.

[0059] In a further aspect, the present invention provides a kit for carrying out the method of the invention, i.e., a kit comprising: (i) a disposable reaction vial containing a predetermined amounts of both a sodium-based buffering agent and a PSMA ligand of the general formula I as defined above, both in dried form, i.e., either lyophilized or spray dried; or two disposable reaction vials, wherein one of said reaction vials contains said sodium-based buffering agent and the other of said reaction vials contains said PSMA ligand, both in dried form; and (ii) instructions for optionally adding said sodium-based buffering agent to the reaction vial containing said PSMA ligand, and for labeling said PSMA ligand with a radioactive isotope. Sodium-based buffering agents that can be used in the kit of the invention include any suitable such buffering agents, e.g., sodium formate, sodium ascorbate, sodium acetate, sodium hydroxide, or sodium citrate.

[0060] In certain embodiments, the disposable reaction vial contained within the kit of the present invention is pretreated with HCl so as to adhere protons to the vial's wall. Such pretreatment is aimed at significantly reducing adherence of additional protons to the vial's wall upon introducing an eluted isotope's solution, and thus preventing or minimizing any pH alterations of said solution and facilitating pH conditions favorable for complexation of the radioactive isotopes with the chelating agent, even at ambient temperature.

[0061] The radioactive isotope for labeling the PSMA ligand provided with the kit of the invention may be provided as ready-for-use product, i.e., for mixing and incubating with the PSMA ligand and sodium-based buffering agent comprised within the kit of the invention, or alternatively may be eluted from a radioactive isotope generator prior to, and shortly before, mixing and incubating with said PSMA ligand and said sodium-based buffering agent, particularly in cases said radioactive isotope has a relatively short half-life such as in the case of .sup.68Ga. In certain embodiments, the kit of the invention according to any one of the embodiments defined above thus further comprises at least one of: (i) a vial containing a predetermined amount of HCl having a molarity in a range of 0.05 to 0.1N for eluting said radioactive isotope from a radioactive isotope generator; and (ii) a vial containing a sodium-based buffering agent for pH adjustment.

[0062] The invention will now be illustrated by the following non-limiting examples.

EXAMPLES

Example 1

Radio-Synthesis of .SUP.68.Ga-Labeled PSMA Tracer According to a Known Technique

[0063] Disposable cassette kits and chemicals including the PSMA ligand DKFZ-PSMA-11 in GMP-compliant grade used for the radio-synthesis were obtained from ABX advanced biochemical compounds (Germany).

[0064] The synthesis of the .sup.68Ga-labelled PSMA ligand as previously described (Eder M. et al., Novel preclinical and radiopharmaceutical aspects of [.sup.68Ga]Ga-PSMA-HBED-CC: A new PET tracer for imaging of prostate cancer, Pharmaceuticals, 2014, 7, 779-796) was reproduced reliably and slightly modified on a fully automated synthesis module in 80%5% decay corrected radiochemical yield within 35 minutes, applying single-use cassette-based kits.

[0065] An audit trial was recorded for each radio-synthesis including all performed steps and courses of radioactivity on three gamma detectors, gas flow and temperature of the heating unit. The steps involved include: (i) conditioning of the purification cartridge; (ii) elution of the .sup.86 G.sub.e/.sup.68 Ga-generator; (iii) purification of the generator elute; (iv) radio-synthesis of the radiolabeled PSMA ligand; and (v) purification by cation exchange column filtration and sterile filtration of the final product.

[0066] The .sup.68Ga-labelled DKFZ-PSMA-11 was obtained in >95% radiochemical purity as two diastereomers with a pH ranging between 6 and 8. Stability of this tracer in the final product solution was tested for up to 6 hours after the end of radio-synthesis and yielded identical results for the radiochemical purity. The only residual solvent found in the final product solution was ethanol in less than 5% v/v taking the density at 20 C. to be 0.79 g/ml. Bacterial endotoxin testing showed <2 IU/ml and all samples tested were sterile. Filter integrity of the sterile filter was tested using the bubble-point test. Gamma spectrometry showed characteristic peaks at 0.511 and 1.077 MeV.

Example 2

Two Modes of Operation for Using the Automated Radio-Synthesis Module

[0067] The automated synthesis module can be carried out using either reusable or disposable reaction vials.

[0068] When reusable vials are used, the mode of operation includes the steps of: (a) adding into the reaction vial a PSMA ligand and sodium formate to reach a working pH of from about 4.0 to about 8.0; (b) automatically eluting the radioactive isotope generator by fractionation with HCl 0.1N (about 0.5 ml for .sup.90Y or .sup.177Lu, or about 1.8 ml for .sup.68Ga); (c) heating the reaction vial at about 95 C. (30 minutes for .sup.90Y or .sup.177Lu, or 5 minutes for .sup.68Ga); (d) purifying the radiolabeled PSMA tracer by passing the crude product through cation exchange column, where the labeled PSMA tracer remains on the column and the free radioactive isotopes are removed and discarded; (e) passing saline through said column to remove residual HCl; (f) eluting the labeled PSMA tracer from the cation exchange column with about 0.5 ml 50% ethanol; (g) passing saline through said column to dilute the final product to a max 5% ethanol; and (h) sterilizing the elute by passing it through a 0.22 micron filter into a collection vial. Finally, a quality control of the labeled PSMA tracer is performed.

[0069] When disposable vials and reagents are used, in which case all the reagents are contained in a disposable vial and disposable separation-column, the mode of operation includes the same steps mentioned above, with the exeption that the vials and the cation exchange column being used are disposable and therefore do not need to be washed after use.

Example 3

Radio-Synthesis of .SUP.68.Ga-Labelled DKFZ-PSMA-11 Using the Shake & Bake Method

[0070] Radio-synthesis of .sup.68Ga-labeled DKFZ-PSMA-11 was carried out using a kit comprising (i) a disposable reaction vial containing the PSMA ligand (10 g) and sodium formate (800-1800 mg), both in lyophilized form; (ii) an elution vial containing 0.1N HC1, for elution of the .sup.68Ge/68Ga generator; and (iii) a buffer vial containing 1N NaOH, for pH adjustment.

[0071] The procedure of radiolabeling the PSMA tracer was performed by: (a) eluting the .sup.68Ge/68Ga generator by fractionation with about 1.8 ml HCl 0.1N directly into the reaction vial; (b) mixing and incubating the reaction vial at room temperature for about 5 minutes; and (c) adjusting the pH in the reaction vial, if lower than 4.0, by adding 0.1 ml 1N NaOH until reaching a pH in the range of 4.0 to 8.0.

[0072] A similar experiment conducted with a disposable reaction vial containing 30 g of the PSMA ligand, reduced the incubation time by about 2 to 3 minutes.

Example 4

Quality Control Procedures

[0073] The resulting .sup.68Ga-PSMA tracers produced in Examples 1 and 3 above were tested for radiochemical purity by using thin layer chromatography (TLC). The resulting acceptable criteria using 0.1M sodium citrate as the runing solvent are: (a) for free .sup.68Ga: Retardation Factor (RF) of 0.8-1.0 and no more than 10%; and (b) for .sup.68Ga-labeled tracer: RF of 0.0-0.3 and no less than 90%. The resulting acceptable criteria using 1M ammonium acetate+methanol (1:1) as the running solvent are (a) for colloid .sup.68Ga: RF of 0.0-0.2 and no more than 10%; and (b) for .sup.68Ga labeled tracer: RF of 0.8-1.0 and no less than 90%.

[0074] The half-life (t.sub.) of the resulting .sup.68Ga-PSMA tracer was from about 65.0 to about 69.6 minutes.

APPENDIX

[0075]

TABLE-US-00001 Specific PSMA ligands described herein DKFZ-PSMA-11 [00001] DKFZ-PSMA-617 [00002] DKFZ-PSMA-617-1 [00003] DKFZ-PSMA-10 [00004]