PSMA-binding agents and uses thereof
11629201 · 2023-04-18
Assignee
Inventors
- Martina Benesova (Turgi, CH)
- Cristina Müller (Nussbaumen, CH)
- Christoph Umbricht (Baden, CH)
- Roger Schibli (Baden, CH)
- Konstantin Zhernosekov (Munich, DE)
Cpc classification
A61B6/5235
HUMAN NECESSITIES
A61K51/0474
HUMAN NECESSITIES
A61K51/0491
HUMAN NECESSITIES
C07K16/44
CHEMISTRY; METALLURGY
International classification
C07K16/44
CHEMISTRY; METALLURGY
Abstract
The present invention provides novel compounds that are useful as radiopharmaceuticals, imaging agents and for treatment of cancer.
Claims
1. A radiolabeled complex comprising a radionuclide and a compound according to general Formula (1)(i) or (1)(ii): ##STR00073## wherein Abm is an albumin binding entity, Pbm is a PSMA binding entity, D is a chelator complexed with a radionuclide, X is each independently selected from O, N, S or P, R.sup.1 and R.sup.2 are each independently selected from H, F, Cl, Br, I, branched, unbranched or cyclic C.sub.1-C.sub.12 hydrocarbyl, C.sub.2-C.sub.12 alkenyl, C.sub.2-C.sub.12 alkylnyl, OR.sup.6, OCOR.sup.6, CHO, COR.sup.6, CH.sub.2OR.sup.6, NR.sup.6R.sup.7, CONR.sup.6R.sup.7, COOR.sup.6, CH.sub.2NR.sup.6R.sup.7, SR.sup.6, ═O, ═S or ═NH, or R.sup.1 and R.sup.2 are joined to form a cyclic structure comprising a branched, unbranched or cyclic C.sub.1-C.sub.10 hydrocarbyl group, wherein said hydrocarbyl group is optionally interrupted by up to 2 heteroatoms and optionally substituted by up to 3 groups independently selected from F, Cl, Br, I, OR.sup.6, OCOR.sup.6, COOR.sup.6, CHO, COR.sup.6, CH.sub.2OR.sup.6, NR.sup.6R.sup.7, CH.sub.2NR.sup.6R.sup.7, and SR.sup.7, ═O, ═S and ═NH, Y is selected from a single bond or a linear, branched or cyclic, optionally substituted C.sub.1-C.sub.12 alkyl, optionally interrupted by up to two heteroatoms, OR.sup.6, OCOR.sup.6, CHO, COR.sup.6, CH.sub.2OR.sup.6, NR.sup.6R.sup.7, COOR.sup.6, CH.sub.2NR.sup.6R.sup.7, SR.sup.6, ═O, ═S or ═NH, wherein one or more of the non-adjacent CH.sub.2-groups may independently be replaced by —O—, —CO—, —CO—O—, —O—CO—, —NR.sup.6—, —NR.sup.6—CO—, —CO—NR.sup.6—, —NR.sup.6—COO—, —O—CO—NR.sup.6—, —NR.sup.6—CO—NR.sup.6—, —CH═CH—, —C≡C—, —O—CO—O—, SR.sup.6—, SO.sub.3R.sup.6—, R.sup.6 and R.sup.7 are each independently selected from H or branched, unbranched or cyclic C.sub.1-12 hydrocarbyl, R.sup.3, R.sup.4 and R.sup.5 are each independently selected from —COH, —CO.sub.2H, —SO.sub.2H, —SO.sub.3H, —SO.sub.4H, —PO.sub.2H, —PO.sub.3H, —PO.sub.4H.sub.2, —C(O)—(C.sub.1-C.sub.10)alkyl, —C(O)—O(C.sub.1-C.sub.10)alkyl, —C(O)—NHR.sup.8, or —C(O)—NR.sup.8R.sup.9, wherein R.sup.8 and R.sup.9 are each independently selected from H, bond, (C.sub.1-C.sub.10)alkylene, F, Cl, Br, I, C(O), C(S), —C(S)—NH-benzyl-, —C(O)—NH-benzyl, —C(O)—(C.sub.1-C.sub.10)alkylene, —(CH.sub.2).sub.p—NH, —(CH.sub.2).sub.p—(C.sub.1-C.sub.10)alkylene, —(CH.sub.2).sub.p—NH—C(O)—(CH.sub.2).sub.q, —(CH.sub.rCH.sub.2).sub.t—NH—C(O)—(CH.sub.2).sub.p, —(CH.sub.2).sub.p—CO—COH, —(CH.sub.2).sub.p—CO—CO.sub.2H, —(CH2).sub.p-C(O)NH—C[(CH.sub.2).sub.q—COH].sub.3, —C[(CH.sub.2).sub.p—COH].sub.3, —(CH2).sub.p-C(O)NH—C[(CH.sub.2).sub.q—CO.sub.2H].sub.3, —C[(CH.sub.2).sub.p—CO.sub.2H].sub.3 or —(CH.sub.2).sub.p—(C.sub.5-C.sub.14)heteroaryl, the spacer comprises at least one C—N bond, the linker is characterized by General Formula (6) ##STR00074## and a, b, p, q, r, and t are each independently an integer selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10; or pharmaceutically acceptable salts, esters, or solvates thereof.
2. The radiolabeled complex according to claim 1, wherein said radiolabeled complex is characterized by General Formula (1a): ##STR00075## wherein D is a chelator complexed with a radionuclide, X is each independently selected from O, N, S or P, R.sup.1 and R.sup.2 are each independently selected from H, F, Cl, Br, I, branched, unbranched or cyclic, optionally substituted, C.sub.1-C.sub.12 hydrocarbyl, C.sub.2-C.sub.12 alkenyl, C.sub.2-C.sub.12 alkylnyl, OR.sup.6, OCOR.sup.6, CHO, COR.sup.6, CH.sub.2OR.sup.6, NR.sup.6R.sup.7, CONR.sup.6R.sup.7, COOR.sup.6, CH.sub.2NR.sup.6R.sup.7, SR.sup.6, ═O, ═S or ═NH, or R.sup.1 and R.sup.2 are joined to form a cyclic structure comprising a branched, unbranched or cyclic C.sub.1-C.sub.10 hydrocarbyl group, wherein said hydrocarbyl group is optionally interrupted by up to 2 heteroatoms and optionally substituted by up to 3 groups independently selected from F, Cl, Br, I, OR.sup.6, OCOR.sup.6, COOR.sup.6, CHO, COR.sup.6, CH.sub.2OR.sup.6, NR.sup.6R.sup.7, CH.sub.2NR.sup.6R.sup.7, and SR.sup.7, ═O, ═S and ═NH, Y is selected from a single bond or a linear, branched or cyclic, optionally substituted C.sub.1-C.sub.12 alkyl, optionally interrupted by up to two heteroatoms, OR.sup.6, OCOR.sup.6, CHO, COR.sup.6, CH.sub.2OR.sup.6, NR.sup.6R.sup.7, COOR.sup.6, CH.sub.2NR.sup.6R.sup.7, SR.sup.6, ═O, ═S or ═NH, wherein one or more of the non-adjacent CH.sub.2-groups may independently be replaced by —O—, —CO—, —CO—O—, —O—CO—, —NR.sup.6—, —NR.sup.6—CO—, —CO—NR.sup.6—, —NR.sup.6—COO—, —O—CO—NR.sup.6—, —NR.sup.6—CO—NR.sup.6—, —CH═CH—, —C≡C—, —O—CO—O—, SR.sup.6—, SO.sub.3R.sup.6—, R.sup.6 and R.sup.7 are each independently selected from H or branched, unbranched or cyclic C.sub.1-12 hydrocarbyl, R.sup.3, R.sup.4 and R.sup.5 are each independently selected from —COH, —CO.sub.2H, —SO.sub.2H, —SO.sub.3H, —SO.sub.4H, —PO.sub.2H, —PO.sub.3H, —PO.sub.4H.sub.2, —C(O)—(C.sub.1-C.sub.10)alkyl, —C(O)—O(C.sub.1-C.sub.10)alkyl, —C(O)—NHR.sup.8, or —C(O)—NR.sup.8R.sup.9, wherein R.sup.8 and R.sup.9 are each independently selected from H, bond, (C.sub.1-C.sub.10)alkylene, F, Cl, Br, I, C(O), C(S), —C(S)—NH-benzyl-, —C(O)—NH-benzyl, —C(O)—(C.sub.1-C.sub.10)alkylene, —(CH.sub.2).sub.p—NH, —(CH.sub.2).sub.p—(C.sub.1-C.sub.10)alkylene, —(CH.sub.2).sub.p—NH—C(O)—(CH.sub.2).sub.q, —(CH.sub.rCH.sub.2).sub.t—NH—C(O)—(CH.sub.2).sub.p, —(CH.sub.2).sub.p—CO—COH, —(CH.sub.2).sub.p—CO—CO.sub.2H, —(CH.sub.2).sub.p—C(O)NH—C[(CH.sub.2).sub.q—COH].sub.3, —C[(CH.sub.2).sub.p—COH].sub.3, —(CH.sub.2).sub.p—C(O)NH—C[(CH.sub.2).sub.q—CO.sub.2H].sub.3, —C[(CH.sub.2).sub.p—CO.sub.2H].sub.3 or —(CH.sub.2).sub.p—(C.sub.5-C.sub.14)heteroaryl, the spacer comprises at least one C—N bond, the linker is characterized by the Structural Formula (6): ##STR00076## wherein X is each independently selected from O, N, S or P, Q is selected from substituted or unsubstituted alkyl, alkylaryl and cycloalkyl, W is selected from —(CH.sub.2).sub.c-aryl or —(CH.sub.2).sub.c-heteroaryl, wherein c is an integer selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10, and a, b, p, q, r, t is each independently an integer selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10, or a pharmaceutically acceptable salt, ester, or solvate thereof.
3. The radiolabeled complex according to claim 2, wherein said radiolabeled complex is characterized by any one of the following General Formulas (12.1)-(12.4) or (13.1)-(13.4): ##STR00077## ##STR00078## or pharmaceutically acceptable salts, esters, or solvates thereof, wherein D, spacer, linker, X, R.sup.1-R.sup.5, a, b, m, n are as defined in claim 2, and d is an integer selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, a, b, d, m, n is each independently an integer selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.
4. The radiolabeled complex according to claim 3, wherein D is a chelator complexed with a radionuclide, the chelator selected from 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), N,N″-bis[2-hydroxy-5-(carboxyethyl)-benzyl]ethylenediamine-N,N″-diacetic acid (HBED-CC), 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA), 2-(4,7-bis(carboxymethyl)-1,4,7-triazonan-1-yl)pentanedioic acid (NODAGA), 2-(4,7,10-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecan-1-yl)-pentanedioic acid (DOTAGA), 1,4,7-triazacyclononane phosphinic acid (TRAP), 1,4,7-triazacyclononane-1-[methyl(2-carboxyethyl)-phosphinic acid]-4,7-bis[methyl(2-hydroxymethyl)phosphinic acid] (NOPO), 3,6,9, 15-tetraazabicyclo[9,3,1]pentadeca-1(15),11,13-triene-3,6,9-triacetic acid (PCTA), N′-{5-[Acetyl(hydroxy)amino]pentyl}-N-[5-({4-[(5-aminopentyl)(hydroxy)amino]-4-oxobutanoyl}amino)pentyl]-N-hydroxysuccinamide (DFO), and Diethylenetriaminepentaacetic acid (DTPA), or derivatives thereof.
5. The radiolabeled complex according to claim 3, wherein R.sup.1 and R.sup.2 are each independently selected from H, halogen and C.sub.1-6 alkyl.
6. The radiolabeled complex according to claim 3, wherein R.sup.1 and R.sup.2 are each independently selected from H, iodine, bromine, and C.sub.1-6 alkyl.
7. The radiolabeled complex according to claim 3, wherein R.sup.1 and R.sup.2 are each independently selected from H, iodine, bromine, and C.sub.1-3 alkyl.
8. The radiolabeled complex according to claim 3, wherein R.sup.1 and R.sup.2 are each independently selected from H, iodine, bromine, and methyl.
9. The radiolabeled complex according to claim 3, wherein the linker is characterized by General Formula (6): ##STR00079##
10. The radiolabeled complex according to claim 3, wherein the linker is characterized by General Formula (6a): ##STR00080##
11. The radiolabeled complex according to claim 3, wherein a, b, d, m, and n are each independently an integer selected from: 0, 1, 2, 3, 4, 5 and 6.
12. The radiolabeled complex according to claim 2, wherein Q is selected from C.sub.5-C.sub.7 cycloalkyl.
13. The radiolabeled complex according to claim 12, wherein Q is cyclohexyl.
14. The radiolabeled complex according to claim 2, wherein W is selected from —(CH.sub.2).sub.c-naphthyl, —(CH.sub.2).sub.c-phenyl, —(CH.sub.2).sub.c-biphenyl, —(CH.sub.2).sub.c-indolyl, —(CH.sub.2).sub.c-benzothiazolyl, wherein c is an integer selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.
15. The radiolabeled complex according to claim 14, wherein W is —(CH.sub.2)-naphthyl.
16. The radiolabeled complex of claim 2, wherein the chelator is selected from the group consisting of: 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), N,N″-bis[2-hydroxy-5-(carboxyethyl)benzyl]ethylenediamine-N,N″-diacetic acid (HBED-CC), 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA), 2-(4,7-bis(carboxymethyl)-1,4,7-triazonan-1-yl)pentanedioic acid (NODAGA), 2-(4,7,10-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecan-1-yl)pentanedioic acid (DOTAGA), 1,4,7-triazacyclononane phosphinic acid (TRAP), 1,4,7-triazacyclononane-1-[methyl(2-carboxyethyl)phosphinic acid]-4,7-bis[methyl(2-hydroxymethyl)phosphinic acid] (NOPO), 3,6,9,15-tetraazabicyclo[9,3,1.]pentadeca-1(15),11, 13-triene-3,6,9-triacetic acid (PCTA), N′-{5-[Acetyl(hydroxy)amino]pentyl}-N-[5-({4-[(5-aminopentyl)(hydroxy)amino]-4-oxobutanoyl}amino)pentyl]-N-hydroxysuccinamide (DFO), and Diethylenetriaminepentaacetic acid (DTPA) or derivatives thereof.
17. The radiolabeled complex of claim 2, where Q is selected from the group consisting of: substituted or unsubstituted C.sub.5-C.sub.14 aryl, substituted or unsubstituted C.sub.5-C.sub.14 alkylaryl, and substituted or unsubstituted C.sub.5-C.sub.14 cycloalkyl.
18. The radiolabeled complex according to claim 1, wherein the chelator is selected from DOTA, DOTA, HBED-CC, NOTA, NODAGA, DOTAGA, TRAP, NOPO, PCTA, DFO, DTPA or derivatives thereof.
19. The radiolabeled complex according to claim 1, wherein each X is O.
20. The radiolabeled complex according to claim 6, wherein Y is a linear or branched, optionally substituted, C.sub.1-C.sub.12 hydrocarbyl.
21. The radiolabeled complex according to claim 1, wherein Y is a linear C.sub.1-C.sub.3 hydrocarbyl.
22. The radiolabeled complex according to claim 1, wherein R.sup.1 and R.sup.2 are each independently selected from H and halogen.
23. The radiolabeled complex according to claim 22, wherein in General Formula (1) the group ##STR00081## comprises any one of Structural Formulas (2a), (2b) or (2c): ##STR00082##
24. The radiolabeled complex according to claim 1, wherein R.sup.3, R.sup.4 and R.sup.5 are each independently selected from —COH, —CO.sub.2H, —SO.sub.2H, —SO.sub.3H, —SO.sub.4H, —PO.sub.2H, —PO.sub.3H, —PO.sub.4H.sub.2.
25. The radiolabeled complex according to claim 24, wherein each of R.sup.3, R.sup.4 and R.sup.5 are selected from —CO.sub.2H.
26. The radiolabeled complex according to claim 1, wherein said radiolabeled complex is characterized by any one of General Formulas (11.1)-(11.3): ##STR00083##
27. The radiolabeled complex according to claim 1, wherein the spacer comprises a linear or branched, optionally substituted C.sub.1-C.sub.20 hydrocarbyl, the hydrocarbyl comprising at least one up to 4 heteroatoms.
28. The radiolabeled complex according to claim 27, wherein the spacer comprises —[CHR.sup.10].sub.u—NR11-, wherein R.sup.10 and R.sup.11 are each be independently selected from H and branched, unbranched or cyclic C.sub.1-C.sub.12 hydrocarbyl, and u is an integer selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.
29. The radiolabeled complex according to claim 27, wherein the spacer comprises a linear or branched, optionally substituted, C.sub.1-C.sub.12 hydrocarbyl.
30. The radiolabeled complex according to claim 27, wherein the spacer comprises a linear or branched, optionally substituted, C.sub.2-C.sub.6 hydrocarbyl.
31. The radiolabeled complex according to claim 27, wherein the spacer comprises a linear or branched, optionally substituted, C.sub.2-C.sub.4 hydrocarbyl.
32. The radiolabeled complex according to claim 27, wherein the linear or branched, optionally substituted C.sub.1-C.sub.20 hydrocarbyl comprises 1 to 4 heteroatoms.
33. The radiolabeled complex according to claim 32, wherein the linear or branched, optionally substituted C.sub.1-C.sub.20 hydrocarbyl comprises 1 to 4 nitrogen atoms.
34. The radiolabeled complex according to claim 1, wherein the linker is characterized by Structural Formula (6a): ##STR00084##
35. The radiolabeled complex according to claim 34, ##STR00085## wherein said radiolabeled complex is characterized by General Formula (1c): or pharmaceutically acceptable salts, esters, or solvates thereof.
36. The radiolabeled complex according to claim 35, said radiolabeled complex being characterized by General Formula (7a): ##STR00086## or pharmaceutically acceptable salts, esters, or solvates thereof.
37. The radiolabeled complex according to claim 36, said radiolabeled complex being characterized by Structural Formula (7a)(i), (7a)(ii) or (7a)(iii), and a complexed radionuclide: ##STR00087## or pharmaceutically acceptable salts, esters, or solvates thereof.
38. The radiolabeled complex according to claim 35, wherein General Formula (1c) comprises a spacer of formula 3(a): ##STR00088##
39. The radiolabeled complex according to claim 1, wherein the spacer comprises at least one amino acid residue.
40. The radiolabeled complex according to claim 39, said radiolabeled complex being characterized by General Formula (7b): ##STR00089## wherein A is an amino acid residue, V is selected from a single bond, N, or an optionally substituted C.sub.1-C.sub.12 hydrocarbyl comprising up to 3 heteroatoms, n is an integer selected from 1, 2, 3, 4 or 5, or pharmaceutically acceptable salts, esters, or solvates thereof.
41. The radiolabeled complex according to claim 40, said radiolabeled complex being characterized by Structural Formula (7b)(i), (7b)(ii), (7b)(iii), or (7b)(iv), and a radionuclide: ##STR00090## ##STR00091## or pharmaceutically acceptable salts, esters, or solvates thereof.
42. The radiolabeled complex according to claim 40, wherein said heteroatom is N.
43. The radiolabeled complex according to claim 39, wherein said amino acid residue(s) is/are selected from (D-/L-) aspartate, glutamate or lysine.
44. The radiolabeled complex according to claim 43, wherein said spacer is characterized by Formula (3b) or Formula (3c): ##STR00092## wherein m is an integer selected from 1 or 2, and n is an integer selected from 1, 2, 3, 4 or 5, ##STR00093## wherein o is an integer selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.
45. The radiolabeled complex according to claim 1, wherein the radionuclide is selected from the group consisting of .sup.94Tc, .sup.99mTc, .sup.90In, .sup.111In, .sup.67Ga, .sup.68Ga, .sup.86Y, .sup.90Y, .sup.177Lu, .sup.151Tb, .sup.186Re, .sup.188Re, .sup.64Cu, .sup.67Cu, .sup.55Co, .sup.57Co, .sup.43Sc, .sup.44Sc, .sup.47Sc, .sup.225Ac, .sup.213Bi, .sup.212Bi, .sup.212Pb, .sup.227Th, .sup.153Sm, .sup.166Ho, .sup.152Gd, .sup.153Gd, .sup.157Gd, and .sup.166Dy.
46. A pharmaceutical composition comprising the radiolabeled complex according to claim 1, and a pharmaceutically acceptable carrier, an excipient, or both a pharmaceutically acceptable carrier and an excipient.
47. A kit comprising a radiolabeled complex according to claim 1 or a pharmaceutically acceptable salt, ester, or solvate thereof.
48. A method of medical diagnosis and/or treatment, comprising: (a) administering the radiolabeled complex according to claim 1, to a patient, and (b) obtaining a radiographic image from said patient.
49. A method of detecting the presence of cells and/or tissues expressing prostate-specific membrane antigen (PSMA) comprising: (a) contacting said PSMA-expressing cells and/or tissues with a compound according to claim 1; (b) applying detection means, optionally radiographic imaging, to detect said cells and/or tissues.
50. The method according to claim 49, wherein radiographic imaging comprises positron emission tomography (PET) or single-photon emission computed tomography (SPECT).
51. The method according to claim 49, wherein said one or more cells or tissues comprise prostate cells or tissues, cancerous prostate cells or tissues, spleen cells or tissues, cancerous spleen cells or tissues, kidney cells or tissues, or cancerous kidney cells or tissues.
52. The method according to claim 49, wherein the presence of PSMA-expressing cells or tissues is indicative of a prostate tumor, a metastasized prostate tumor, a renal tumor, a pancreatic tumor, a bladder tumor, and combinations thereof.
53. The radiolabeled complex of claim 1, wherein the compound is a compound according to any one of Structural Formulas (14), (15) or (16): ##STR00094##
54. The radiolabeled complex according to claim 1, wherein the chelator is selected from: DOTA, NODAGA, DO3AP, DO3AP.sup.PrA and DO3AP.sup.ABn.
55. The radiolabeled complex according to claim 1, wherein Y is a linear or branched, optionally substituted, C.sub.1-C.sub.10 hydrocarbyl.
56. The radiolabeled complex according to claim 1, wherein Y is a linear or branched, optionally substituted, C.sub.1-C.sub.6 hydrocarbyl.
57. The radiolabeled complex according to claim 1, wherein R.sup.1 and R.sup.2 are each independently selected from H, iodine, bromine, and C.sub.1-6 alkyl.
58. The radiolabeled complex according to claim 1, wherein R.sup.1 and R.sup.2 are each independently selected from H, iodine, bromine, and C.sub.1-3 alkyl.
59. The radiolabeled complex according to claim 1, wherein R.sup.1 and R.sup.2 are each independently selected from H, iodine, bromine, and methyl.
60. The radiolabeled complex according to claim 44, wherein n is an integer selected from: 1, 2 and 3.
61. The radiolabeled complex according to claim 1, wherein the chelator is selected from: 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), N,N″-bis[2-hydroxy-5-(carboxyethyl)-benzyl]ethylenediamine-N,N″-diacetic acid (HBED-CC), 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA), 2-(4,7-bis(carboxymethyl)-1,4,7-triazonan-1-yl)pentanedioic acid (NODAGA), 2-(4,7,10-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecan-1-yl)-pentanedioic acid (DOTAGA), 1,4,7-triazacyclononane phosphinic acid (TRAP), 1,4,7-triazacyclononane-1-[methyl(2-carboxyethyl)-phosphinic acid]-4,7-bis[methyl(2-hydroxymethyl)phosphinic acid] (NOPO), 3,6,9, 15-tetraazabicyclo[9,3,1]pentadeca-1(15),11,13-triene-3,6,9-triacetic acid (PCTA), N′-{5-[Acetyl(hydroxy)amino]pentyl}-N-[5-({4-[(5-aminopentyl)(hydroxy)amino]-4-oxobutanoyl}amino)pentyl]-N-hydroxysuccinamide (DFO), and Diethylenetriaminepentaacetic acid (DTPA), or derivatives thereof.
Description
DESCRIPTION OF THE FIGURES
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EXAMPLES
Example 1: Design and In Vitro Evaluation of DOTA-Functionalized Albumin-Binding PSMA Ligands
(28) 1.1 Material and Methods
(29) 1.1.1 Novel PSMA Ligands (Overview):
(30) All seven suggested PSMA ligands with a portable albumin-binding moiety were synthesized via a solid-phase platform which was shown to be very useful for the development of above described albumin-affine PSMA ligands.
(31) A multistep synthesis (19 steps for PSMA-ALB-01, 17 steps for PSMA-ALB-03, 20 steps for PSMA-ALB-04 and PSMA-ALB-05, 17 steps for PSMA-ALB-06, 23 steps for PSMA-ALB-07 and PSMA-ALB-08) provided these compounds in isolated overall yields of 26-49%. Crude products were purified by semi-preparative RP-HPLC assuring the final products with purities >98%. The characterization of above described compounds was performed by analytical RP-HPLC and MALDI-MS or ESI-MS, respectively. Analytical data are presented in Table 1.1.
(32) TABLE-US-00001 TABLE 1.1 Analytical Data of PSMA-ALB-01/03/04/05/06/07/08. MW t.sub.r.sup.b Compound Code Chemical Formula [g/mol] m/z.sup.a [min] PSMA-ALB-01 C.sub.69H.sub.95IN.sub.14O.sub.20 1567.50 1568.59 8.15 PSMA-ALB-03 C.sub.65H.sub.92IN.sub.11O.sub.18 1442.41 1443.57 7.57 PSMA-ALB-04 C.sub.79H.sub.116IN.sub.13O.sub.22 1726.77 1727.42 8.17 PSMA-ALB-05 C.sub.73H.sub.102IN.sub.13O.sub.24 1672.59 1673.41 8.09 PSMA-ALB-06 C.sub.66H.sub.95N.sub.11O.sub.18 1330.55 1331.47 7.24 PSMA-ALB-07 C.sub.77H.sub.107IN.sub.14O.sub.27 1787.68 1788.63 7.89 PSMA-ALB-08 C.sub.78H.sub.110N.sub.14O.sub.27 1675.81 1676.79 7.13 • Mass spectrometry of the unlabeled ligand detected as [M + H]; • Retention time of unlabeled ligand on analytical RP-HPLC. Analytical column (100 × 4.6 mm) utilized Chromolith RP-18e stationary phase with mobile phases consisting of 0.1% TFA in water (A) and ACN (B). For analytical runs, a linear gradient of solvent A (90-10% in 10 min) in solvent B at a flow rate of 1 mL/min was used.
(33) The peptidomimetic pharmacophore for PSMA (L-Glu-NH—CO—NH-L-Lys binding entity; step 1-6) was synthesized analogically as described by Eder et al. Bioconjug. Chem. 2012, 23: 688-697. The linker moiety (2-naphthyl-L-Ala-NH—CO-trans-CHX—N3 or 2-naphthyl-L-Ala-NH—CO-trans-CHx-Me-NH2; step 7-10) was prepared according to standard Fmoc (9-fluorenylmethyloxycarbonyl) protocol as previously introduced by Benesovi et al. JNM 2015, 56: 914-920. These two synthetic intermediate stages providing the PSMA ligand precursor were applied analogically for all four compounds (step 1-8). However, the last building block of the linker area for PSMA-ALB-01 [trans-4-azidocyclohexanecarboxylic acid; step 9-10] was replaced for trans-4-(Fmoc-aminomethyl)cyclohexane-carboxylic acid (step 9-10) in case of PSMA-ALB-03/04/05/06/07/08.
(34) PSMA-ALB-01
(35) For synthesis of PSMA-ALB-01, time-efficient “head-to-tail” click coupling of the purified PSMA-precursor with the free azido group and the purified albumin-binding moiety [4-(p-iodophenyl)butyric acid-L-Lys] with propargyl-Gly (step 11-17) was employed. After the efficient coupling of these two precursors via a triazole ring (step 18), an additional purification was performed to remove an excess of CuSO.sub.4.5H.sub.2O. Finally, PSMA-ALB-01 was obtained by the conjugation of the DOTA chelator in a form of its active ester (DOTA-NHS ester; step 19).
(36) The Structural Formula of PSMA-ALB-01 is shown below:
(37) ##STR00039##
(38) PSMA-ALB-03
(39) For the preparation of PSMA-ALB-03, straight one-way synthesis on the resin support was employed. After the Fmoc-L-Lys(Alloc)-OH coupling to PSMA-precursor, Fmoc deprotection, DOTA tris(tBu)-ester conjugation, Alloc deprotection and 4-(p-iodophenyl)butyric acid conjugation followed (step 11-16). Finally, PSMA-ALB-03 was obtained by agitation and subsequent cleavage from the resin with TFA:TIPS:H.sub.2O mixture (step 17).
(40) The Structural Formula of PSMA-ALB-03 is shown below:
(41) ##STR00040##
PSMA-ALB-04
(42) For the synthesis of PSMA-ALB-04, time-efficient “head-to-tail” coupling of the resin-coated PSMA-precursor with the DOTA-conjugated L-Lys and the purified albumin-binding moiety [4-(p-iodophenyl)butyric acid-L-Lys] through direct conjugation of two secondary amines (step 11-18) was employed. After the efficient coupling of these two precursors using suberic acid bis(N-hydroxysuccinimide) ester (step 19), PSMA-ALB-04 was obtained by agitation and subsequent cleavage from the resin with TFA:TIPS:H.sub.2O mixture (step 20).
(43) The Structural formula of PSMA-ALB-04 is shown below:
(44) ##STR00041##
(45) PSMA-ALB-05
(46) For the preparation of PSMA-ALB-05, straight one-way synthesis on the resin support was employed. After the Fmoc-L-Lys(Alloc)-OH coupling to PSMA-precursor, Fmoc deprotection, Fmoc-D-Asp-OtBu conjugation, Fmoc deprotection, second Fmoc-D-Asp-OtBu conjugation, Fmoc deprotection, 4-(p-iodophenyl)butyric acid conjugation, Alloc deprotection and DOTA tris(tBu)-ester conjugation followed (step 11-19). PSMA-ALB-05 was obtained by agitation and subsequent cleavage from the resin with TFA:TIPS:H.sub.2O mixture (step 20).
(47) The Structural Formula of PSMA-ALB-05 is shown below:
(48) ##STR00042##
PSMA-ALB-06
(49) For the synthesis of PSMA-ALB-06, straight one-way synthesis on the resin support was employed. After the Fmoc-L-Lys(Alloc)-OH coupling to PSMA-precursor, Fmoc deprotection, DOTA tris(tBu)-ester conjugation, Alloc deprotection and p-(tolyl)butyric acid conjugation followed (step 11-16). Finally, PSMA-ALB-06 was obtained by agitation and subsequent cleavage from the resin with TFA:TIPS:H.sub.2O mixture (step 17).
(50) The Structural Formula of PSMA-ALB-06 is shown below:
(51) ##STR00043##
PSMA-ALB-07
(52) For the preparation of PSMA-ALB-07, straight one-way synthesis on the resin support was employed. After the Fmoc-L-Lys(Alloc)-OH coupling to PSMA-precursor, Fmoc deprotection, Fmoc-D-Asp-OtBu conjugation, Fmoc deprotection, second Fmoc-D-Asp-OtBu conjugation, Fmoc deprotection, third Fmoc-D-Asp-OtBu conjugation, Fmoc deprotection, 4-(p-iodophenyl)butyric acid conjugation, Alloc deprotection and DOTA tris(tBu)-ester conjugation followed (step 11-22). PSMA-ALB-07 was obtained by agitation and subsequent cleavage from the resin with TFA:TIPS:H.sub.2O mixture (step 23).
(53) The Structural Formula of PSMA-ALB-07 is shown below:
(54) ##STR00044##
PSMA-ALB-08
(55) For the preparation of PSMA-ALB-08 straight one-way synthesis on the resin support was employed. After the Fmoc-L-Lys(Alloc)-OH coupling to PSMA-precursor, Fmoc deprotection, Fmoc-D-Asp-OtBu conjugation, Fmoc deprotection, second Fmoc-D-Asp-OtBu conjugation, Fmoc deprotection, third Fmoc-D-Asp-OtBu conjugation, Fmoc deprotection, p-(tolyl)butyric acid conjugation, Alloc deprotection and DOTA tris(tBu)-ester conjugation followed (step 11-22). PSMA-ALB-08 was obtained by agitation and subsequent cleavage from the resin with TFA:TIPS:H.sub.2O mixture (step 23).
(56) The Structural Formula of PSMA-ALB-08 is shown below:
(57) ##STR00045##
1.1.2 Synthesis of Psma-Alb-03-08 (Details)
a) Synthesis of the Glutamate-Urea-Lysine Binding Entity
(58) 2-Chlorotrityl chloride resin {(2-CT-Resin; Merck; Catalog number 8550170005), 0.30 mmol, substitution capacity 1.63 mmol/g, 100-200 MESH, 1% DVB, total swelling volume in CH.sub.2Cl.sub.2>4.2 mL/g, [184 mg]} in 5 mL syringe with the filter and combi stopper was first agitated in anhydrous dichloromethane (DCM) for 45 min.
(59) The 2-CT-resin was then washed three times with anhydrous DCM and followed by reaction with 1.2 equiv of Alloc (N-allyloxycarbonyl) as well as Fmoc (N-fluorenylmethoxycarbonyl) protected L-lysine {(Fmoc-Lys(Alloc)-OH; Merck; Catalog number 8521240005), 0.36 mmol, 452.50 g/mol, [163 mg], (1)} and 4.8 equiv of N,N-diisopropylethylamine {(DIPEA), 1.44 mmol, 129.24 g/mol, 0.742 g/ml, [251 μL]} in 3 mL of anhydrous DCM. The coupling of the first protected amino acid on the resin (2) proceeded over the course of 16 h with the gentle agitation. The L-lysine-immobilized resin (2) was washed three times with DCM1 and three times with DCM2. Unreacted chlorotrityl groups remaining on the resin were washed five times with the mixture of DCM, methanol (MeOH), and DIPEA in a ratio of 17:2:1 (20 mL).
(60) Subsequently, the resin with Alloc and Fmoc protected L-lysine was washed three times with DCM1, three times with DCM2, three times with N,N-dimethylformamide (DMF1), and, finally, three times with DMF2. Selective removal of Fmoc-protecting group was realized by washing with the mixture of DMF and piperidine in a ratio of 1:1 once for 2 min and then once again for 5 min in order to get product (3). Alloc protected L-lysine was then washed three times with DMF1, three times with DMF2, three times with DCM1, and, finally, three times with DCM2.
(61) In the next step, 10 equiv of tBu protected L-glutamate hydrochloride {(H-Glu(OtBu)-OtBu.HCl; Merck; Catalog number 8540960005), 3.0 mmol, 295.8 g/mol, [887 mg], i} were used for the generation of the isocyanate of the glutamyl moiety iii. An appropriate amount of tBu-protected L-glutamate was dissolved in 150 mL of DCM2 followed by, shortly afterwards, the addition of 3 mL of DIPEA.
(62) This solution was added dropwise over 4 h to a flask with 1 mmol of ice-cooled bis(trichloromethyl)carbonate {(BTC; Sigma; Catalog number 15217-10G), 296.75 g/mol, [297 mg], ii} in 5 mL of dry DCM.
(63) The L-lysine-immobilized resin with one free NH.sub.2-group (3) was added afterwards in one portion to the solution of the isocyanate of the glutamyl moiety iii and stirred for 16 h in order to obtain resin-immobilized bis(tBu)-Glu-urea-Lys(Alloc) (4).
(64) The obtained product (4) coated on the resin was filtered off and washed three times with DCM1 and three times with DCM2. Cleavage of Alloc-protecting group was realized by reaction with 0.15 equiv of TPP Pd {[tetrakis(triphenylphosphine)palladium(0); Sigma; Catalog number 216666-1G], 0.045 mmol, 1155.56 g/mol, [105 mg]} in the presence of 15 equiv of morpholine {4.5 mmol, 87.12 g/mol, 0.999 g/mL, [392 μL]} in 3 mL of anhydrous DCM. The amount of Pd and morpholine was divided into 2 portions and reacted successively by shaking each for 1 h. The reaction was performed in the dark using aluminum foil.
(65) The resin was then washed three times with DCM1, three times with DCM2, three times with DMF1, and, finally, three times with DMF2. To remove residuals of the palladium, the resin was additionally washed ten times with 1% DIPEA in DMF (300 μL DIPEA in 30 mL DMF2) and subsequently washed ten times for 5 min with a solution of cupral {(sodium diethyldithiocarbamate trihydrate; Sigma; Catalog number D3506-100G), 225.31 g/mol} in DMF2 at the concentration of 15 mg/mL (450 mg cupral in 30 mL DMF2).
(66) The resin-immobilized and bis(tBu)-protected Glu-urea-Lys (5) was then washed three times with DMF1, three times with DMF2, three times with DCM1, three times with DCM2, and, finally, three times with diethylether (Et.sub.2O) and dried under vacuum.
(67) Such prepared Prostate-specific Membrane Antigen (PSMA) binding entity (5) was used for the next reaction in order to synthesize all seven compounds (PSMA-ALB-01/03/04/05/06/07/08).
(68) The outline of the whole previous synthesis of the bis(tBu)-protected Glu-urea-Lys pharmacophore is summarized in Scheme 1.1.
(69) ##STR00046##
(70) The resin-immobilized and bis(tBu)-protected binding entity (5) was first agitated in anhydrous DCM for 45 min. Pre-swollen pharmacophore was washed three times with DCM2, three times with DMF1, and three times with DMF2.
(71) b) Synthesis of the Linker Area
(72) Relative to the resin (0.1 mmol), 4 equiv of Fmoc protected 2-naphthyl-L-alanine {(Fmoc-2Nal-OH; Bachem; Catalog number B-2100), 0.40 mmol, 437.50 g/mol, [175.0 mg]} corresponding to the first building block of the linker area were activated with 3.96 equiv of HBTU {(O-(benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate; Sigma; Catalog number 12804-25G-F), 0.39 mmol, 379.24 g/mol, [147.9 mg]} in the presence of 4 equiv of DIPEA {0.40 mmol, 129.24 g/mol, 0.742 g/mL, [71 μL]} in anhydrous DMF.
(73) Two min after the addition of DIPEA, the solution was added to the DMF pre-swollen immobilized bis(tBu)-protected pharmacophore (5) and agitated for 1 h.
(74) Subsequently, the resin with bis(tBu)-protected Glu-urea-Lys and Fmoc protected 2-naphthyl-L-alanine (6) was washed three times with DMF1 and three times with DMF2. Selective removal of the Fmoc-protecting group from compound (6) was realized by washing with the mixture of DMF and piperidine in a ratio of 1:1 once for 2 min and then once again for 5 min in order to obtain products (7).
(75) In the next step, 4 equiv of the second building block which correspond to azidocyclohexanecarboxylic acid {(N3-1,4-trans-CHC—OH; Iris Biotech; Catalog number HAA2235.0001), 0.40 mmol, 169.18 g/mol, [67.7 mg]} for PSMA-ALB-01 or to Fmoc protected tranexamic acid {(trans-4-(Fmoc-aminomethyl)cyclohexane-carboxylic acid; Sigma; Catalog number 58446-5G-F), 0.40 mmol, 379.45 g/mol, [151.8 mg]} for PSMA-ALB-03/04/05/06/07/08 were activated with 3.96 equiv of HBTU {(Sigma; Catalog number 12804-25G-F), 0.39 mmol, 379.24 g/mol, [147.9 mg]} in the presence of 4 equiv of DIPEA {0.40 mmol, 129.24 g/mol, 0.742 g/mL, [71 μL]} in anhydrous DMF. Two min after the addition of DIPEA, the solution was added to the DMF pre-swollen compound (7) and agitated for 1 hour.
(76) Subsequently, the resin with bis(tBu)-protected Glu-urea-Lys-2-naphthyl-L-alanine and azidocyclohexanecarboxylic acid (8A) was washed three times with DMF1, three times with DMF2, three times with DCM1, three times with DCM2, and, finally, three times with Et.sub.2O and dried under vacuum. Final PSMA-precursor (9A) was obtained by the agitation and subsequent cleavage from the resin within 2 h with the mixture consisting of trifluoroacetic acid (TFA), triisopropylsilane (TIPS) and H.sub.2O in a ratio of 95:2.5:2.5. TFA was evaporated, crude product dissolved in acetonitrile (ACN) and water in a ratio of 1:1 and purified via RP-HPLC.
(77) Additionally, the resin with bis(tBu)-protected Glu-urea-Lys-2-naphthyl-L-alanine and Fmoc protected tranexamic acid (8B) was washed three times with DMF1 and three times with DMF2. Selective removal of Fmoc-protecting group from the compound (8B) was realized by washing with the mixture of DMF and piperidine in a ratio of 1:1 once for 2 min and then once again for 5 min in order to obtain products (9B).
(78) The outline of the whole previous synthesis of the linker area is summarized in Scheme 1.2.
(79) ##STR00047## ##STR00048##
c) Synthesis of PSMA-ALB-03
(80) Relative to the lysine-coated PSMA precursor (9B), 4 equiv of Fmoc as well as Alloc protected L-lysine {(Fmoc-Lys(Alloc)-OH; Merck; Catalog number 8521240005), 0.40 mmol, 452.50 g/mol, [181 mg]} was activated with 3.96 equiv of HBTU {(Sigma; Catalog number 12804-25G-F), 0.396 mmol, 379.24 g/mol, [149 mg]} in the presence of 4 equiv of DIPEA {0.40 mmol, 129.24 g/mol, 0.742 g/mL, [70 μL]} in anhydrous DMF. Two min after the addition of DIPEA, the solution was added to the DMF pre-swollen immobilized bis(tBu)-protected PSMA precursor (9B) and agitated for 1 h.
(81) Selective removal of Fmoc-protecting group from the resulting compound (10B) was realized by washing with the mixture of DMF and piperidine in a ratio of 1:1 once for 2 min and then once again for 5 min in order to obtain the product (11B).
(82) The conjugation of the chelator to the resin-immobilized compound (11B) was performed with 2 equiv of DOTA-tris(t-Bu)ester {([2-(4,7,10-tris(2-(t-butoxy)-2-oxoethyl)-1,4,7,10-tetraazacyclododecan-1-yl)acetic acid]; CheMatech; Catalog number 137076-54-1), 0.20 mmol, 572.73 g/mol [115 mg]}. The chelator building block was activated with 1.98 equiv of HBTU {(Sigma; Catalog number 12804-25G-F), 0.198 mmol, 379.24 g/mol, [75 mg]} in the presence of 4 equiv of DIPEA {0.40 mmol, 129.24 g/mol, 0.742 g/mL, [70 μL]} in anhydrous DMF. Two min after the addition of DIPEA, the solution was added to the resin-immobilized and the DMF pre-swollen compound (11B). The coupling of the DOTA chelator proceeded over the course of 2 h with gentle agitation. The resulting compound (12B) was then washed three times with DMF1, three times with DMF2, three times with DCM1, and, finally, three times with DCM2.
(83) Cleavage of Alloc-protecting group from the compound (12B) was realized by reaction with 0.03 equiv of TPP Pd {(Sigma; Catalog number 216666-1G), 0.03 mmol, 1155.56 g/mol, [35 mg]} in the presence of 30 equiv of morpholine {3.0 mmol, 87.12 g/mol, 0.999 g/mL, [262 μL]} in 3 mL of anhydrous DCM. The reaction was performed for 2 hours in the dark using aluminum foil.
(84) The resin was then washed three times with DCM1, three times with DCM2, three times with DMF1, and, finally, three times with DMF2. To remove residuals of the palladium, the resin was additionally washed ten times with 1% DIPEA in DMF (300 μL DIPEA in 30 mL DMF2) and subsequently washed ten times for 5 min with a solution of cupral {(Sigma; Catalog number D3506-100G), 225.31 g/mol} in DMF2 at the concentration of 15 mg/mL (450 mg cupral in 30 mL DMF2). The resulting compound (13B) was then washed three times with DMF1 and three times with DMF2.
(85) Finally, for the coupling of the albumin-binding moiety, 4 equiv of iodophenyl-butyric acid {([4-(p-iodophenyl)butyric acid]; Sigma; I5634-5G), 0.40 mmol, 290.10 g/mol, [116 mg]} was activated with 3.96 equiv of HBTU {(Sigma; Catalog number 12804-25G-F), 0.396 mmol, 379.24 g/mol, [149 mg]} in the presence of 4 equiv of DIPEA {0.40 mmol, 129.24 g/mol, 0.742 g/mL, [70 μL]} in anhydrous DMF. Two min after the addition of DIPEA, the solution was added to the resin-immobilized and DMF pre-swollen product (13B) and agitated for 1 h.
(86) The resulting compound (14B) was then washed three times with DMF1, three times with DMF2, three times with DCM1, three times with DCM2, and, finally, three times with Et.sub.2O and dried under vacuum.
(87) The final compound PSMA-ALB-03 was obtained by agitation and subsequent cleavage from the resin within 2 h with a mixture consisting of TFA, TIPS and H.sub.2O in a ratio of 95:2.5:2.5. TFA was evaporated, crude product dissolved in ACN and water in a ratio of 1:1 and purified via RP-HPLC.
(88) The outline of the above described synthesis is summarized in Scheme 1.3.
(89) ##STR00049## ##STR00050##
d) Synthesis of PSMA-ALB-04
(90) Relative to the lysine-coated PSMA precursor (9B), 4 equiv of Dde as well as Fmoc protected L-lysine {(Dde-Lys(Fmoc)-OH; Merck; Catalog number 8540000001), 0.40 mmol, 532.63 g/mol, [213 mg]} was activated with 3.96 equiv of HBTU {(Sigma; Catalog number 12804-25G-F), 0.396 mmol, 379.24 g/mol, [149 mg]} in the presence of 4 equiv of DIPEA {0.40 mmol, 129.24 g/mol, 0.742 g/mL, [70 μL]} in anhydrous DMF. Two min after the addition of DIPEA, the solution was added to the DMF pre-swollen immobilized bis(tBu)-protected PSMA precursor (9B) and agitated for 1 h.
(91) The resulting compound (10B) was then washed three times with DMF1 and three times with DMF2. Selective removal of Fmoc-protecting group from the resulting compound (10B) was realized by washing with the mixture of DMF and piperidine in a ratio of 1:1 once for 2 min and then once again for 5 min in order to obtain the product (11B).
(92) The conjugation of the chelator to the resin-immobilized compound (11B) was performed with 3 equiv of DOTA-tris(t-Bu)ester {([2-(4,7,10-tris(2-(t-butoxy)-2-oxoethyl)-1,4,7,10-tetraazacyclododecan-1-yl)acetic acid]; CheMatech; Catalog number 137076-54-1), 0.30 mmol, 572.73 g/mol [171 mg]}. The chelator building block was activated with 2.97 equiv of HBTU {(Sigma; Catalog number 12804-25G-F), 0.297 mmol, 379.24 g/mol, [112 mg]} in the presence of 4 equiv of DIPEA {0.40 mmol, 129.24 g/mol, 0.742 g/mL, [70 μL])} in anhydrous DMF. Two min after the addition of DIPEA, the solution was added to the resin-immobilized and the DMF pre-swollen compound (11B). The coupling of the DOTA chelator proceeded over the course of 2 h with gentle agitation.
(93) The resulting compound (12B) was then washed three times with DMF1 and three times with DMF2. Selective removal of Dde-protecting group from the resulting compound (12B) was realized by washing with the mixture of 2% hydrazine in DMF twice for 5 min and then once again for 10 min in order to obtain the product (13B).
(94) Relative to the resin-coated product (13B), 2 equiv of disuccinimidyl suberate {([suberic acid bis(N-hydroxysuccinimide ester)]; Sigma; 68528-80-3), 0.20 mmol, 368.34 g/mol, [74 mg]} was activated with 1.98 equiv of HBTU {(Sigma; Catalog number 12804-25G-F), 0.198 mmol, 379.24 g/mol, [73 mg]} in the presence of 4 equiv of DIPEA {0.40 mmol, 129.24 g/mol, 0.742 g/mL, [70 μL]} in anhydrous DMF. Two min after the addition of DIPEA, the solution was added to the resin-immobilized and DMF pre-swollen product (13B) and agitated for 1 h.
(95) The resulting compound (14B) was then washed three times with DMF1 and three times with DMF2.
(96) The outline of the above described synthesis is summarized in Scheme 1.4.
(97) ##STR00051## ##STR00052## ##STR00053##
(98) The synthesis was accompanied by the parallel preparation of the albumin-binding precursor starting from the 2-chlorotrityl chloride resin {(2-CT-Resin; Merck; Catalog number 8550170005), 0.20 mmol, substitution capacity 1.63 mmol/g, 100-200 MESH, 1% DVB, total swelling volume in CH.sub.2Cl.sub.2>4.2 mL/g, [123 mg]} in 5 mL syringe with the filter and combi stopper which was first agitated in anhydrous dichloromethane (DCM) for 45 min.
(99) The 2-CT resin was then washed three times with anhydrous DCM and followed by reaction with 1.2 equiv of Dde as well as Fmoc protected L-lysine {(Dde-Lys(Fmoc)-OH; Bachem; Catalog number E-3385.0001), 0.24 mmol, 532.64 g/mol, [128 mg] (15B)} and 4.8 equiv of DIPEA {0.96 mmol, 129.24 g/mol, 0.742 g/mL, [167 μL]} in 3 mL of anhydrous DCM.
(100) The coupling of the first protected amino acid on the resin (16B) proceeded over the course of 16 h with gentle agitation.
(101) The L-lysine-immobilized resin (16B) was washed three times with DCM1 and three times with DCM2. Unreacted chlorotrityl groups remaining on the resin were washed five times with the mixture of DCM, MeOH, and DIPEA in a ratio of 17:2:1 (20 mL).
(102) Subsequently, the resin with Dde and Fmoc protected L-lysine was washed three times with DCM1, three times with DCM2, three times with DMF1, and, finally, three times with DMF2. Selective removal of Fmoc-protecting group was realized by washing with the mixture of DMF and piperidine in a ratio of 1:1 once for 2 min and then once again for 5 min in order to get product (17B).
(103) Dde protected L-lysine was then washed three times with DMF1 and three times with DMF2, three times with DCM1, three times with DCM2 and, finally, three times with Et.sub.2O and dried under vacuum.
(104) Such prepared resin-coated Dde protected L-lysine (17B) was split into two portions and one of them was used for the next reaction. This resin-coated product was agitated in anhydrous DCM for 45 min and subsequently washed three times with DMF and three times with DMF2.
(105) Relative to the lysine-coated resin, 4 equiv of iodophenyl-butyric acid {([4-(p-iodophenyl)butyric acid]; Sigma; I5634-5G), 0.40 mmol, 290.10 g/mol, [116 mg]} was activated with 3.96 equiv of HBTU {(Sigma; Catalog number 12804-25G-F), 0.396 mmol, 379.24 g/mol, [149 mg]} in the presence of 4 equiv of DIPEA {0.40 mmol, 129.24 g/mol, 0.742 g/mL, [70 μL]} in anhydrous DMF. Two min after the addition of DIPEA, the solution was added to the resin-immobilized and DMF pre-swollen product (17B) and agitated for 1 h.
(106) The resin with Dde protected L-lysine and iodophenyl-butyric acid (18B) was washed three times with DMF1 and three times with DMF2. Selective removal of Dde-protecting group from the resulting compound (18B) was realized by washing with the mixture of 2% hydrazine in DMF twice for 5 min and then once again for 10 min in order to obtain the product (19B).
(107) The albumin-targeting moiety (20B) was obtained by agitation and subsequent cleavage from the resin within 2 h with a mixture consisting of 5% TFA in DCM. The mixture of solvents from the product was evaporated, crude product dissolved in ACN and water in a ratio of 1:1 and purified via RP-HPLC.
(108) The outline of the above described synthesis is summarized in Scheme 1.5.
(109) ##STR00054##
(110) Finally, the conjugation of 3 equiv of purified albumin-targeting moiety (20B) to the resin immobilized product (14B) was performed. Product (20B) was dissolved in dry DMF and 100 μL of DIPEA was added. Two min after the addition of DIPEA, the solution (20B) was added to the resin-immobilized and DMF pre-swollen product (14B) and agitated for 1 h.
(111) The resulting compound (21B) was then washed three times with DMF1, three times with DMF2, three times with DCM1, three times with DCM2, and, finally, three times with Et.sub.2O and dried under vacuum.
(112) The final compound PSMA-ALB-04 was obtained by agitation and subsequent cleavage from the resin within 2 h with a mixture consisting of TFA, TIPS and H.sub.2O in a ratio of 95:2.5:2.5. TFA was evaporated, crude product dissolved in ACN and water in a ratio of 1:1 and purified via RP-HPLC.
(113) The outline of the above described synthesis is summarized in Scheme 1.6.
(114) ##STR00055## ##STR00056##
e) Synthesis of PSMA-ALB-05
(115) Relative to the lysine-coated PSMA precursor (9B), 4 equiv of Fmoc as well as Alloc protected L-lysine {(Fmoc-Lys(Alloc)-OH; Merck; Catalog number 8521240005), 0.40 mmol, 452.50 g/mol, [181 mg]) was activated with 3.96 equiv of HBTU {(Sigma; Catalog number 12804-25G-F), 0.396 mmol, 379.24 g/mol, [149 mg]} in the presence of 4 equiv of DIPEA (0.40 mmol, 129.24 g/mol, 0.742 g/mL, [70 μL]} in anhydrous DMF. Two min after the addition of DIPEA, the solution was added to the DMF pre-swollen immobilized bis(tBu)-protected PSMA precursor (9B) and agitated for 1 h.
(116) The resulting compound (10B) was then washed three times with DMF1 and three times with DMF2. Selective removal of Fmoc-protecting group from the resulting compound (10B) was realized by washing with the mixture of DMF and piperidine in a ratio of 1:1 once for 2 min and then once again for 5 min in order to obtain the product (11B).
(117) Relative to the lysine-coated PSMA precursor (11B), 3 equiv of Fmoc as well as tBu protected D-aspartate {(Fmoc-D-Asp-OtBu; Merck; Catalog number 8521440001), 0.30 mmol, 411.45 g/mol, [123 mg]} was activated with 2.97 equiv of HBTU {(Sigma; Catalog number 12804-25G-F), 0.297 mmol, 379.24 g/mol, [112 mg]} in the presence of 4 equiv of DIPEA {0.40 mmol, 129.24 g/mol, 0.742 g/mL, [70 μL]} in anhydrous DMF. Two min after the addition of DIPEA, the solution was added to the DMF pre-swollen immobilized bis(tBu)-protected PSMA precursor (11B) and agitated for 1 h.
(118) The resulting compound (12B) was then washed three times with DMF1 and three times with DMF2. Selective removal of Fmoc-protecting group from the resulting compound (12B) was realized by washing with the mixture of DMF and piperidine in a ratio of 1:1 once for 2 min and then once again for 5 min in order to obtain the product (13B).
(119) Relative to the lysine and aspartate-coated PSMA precursor (13B), 3 equiv of Fmoc as well as tBu protected D-aspartate {(Fmoc-D-Asp-OtBu; Merck; Catalog number 8521440001), 0.30 mmol, 411.45 g/mol, [123 mg]} was activated with 2.97 equiv of HBTU {(Sigma; Catalog number 12804-25G-F), 0.297 mmol, 379.24 g/mol, [112 mg]} in the presence of 4 equiv of DIPEA {0.40 mmol, 129.24 g/mol, 0.742 g/mL, [70 μL]} in anhydrous DMF. Two min after the addition of DIPEA, the solution was added to the DMF pre-swollen immobilized bis(tBu)-protected PSMA precursor (13B) and agitated for 1 h.
(120) The resulting compound (14B) was then washed three times with DMF1 and three times with DMF2. Selective removal of Fmoc-protecting group from the resulting compound (148) was realized by washing with the mixture of DMF and piperidine in a ratio of 1:1 once for 2 min and then once again for 5 min in order to obtain the product (15B).
(121) Relative to the resin-coated product (15B), 4 equiv of iodophenyl-butyric acid {([4-(p-iodophenyl)butyric acid]; Sigma; 15634-5G), 0.40 mmol, 290.10 g/mol, [116 mg]} was activated with 3.96 equiv of HBTU {(Sigma; Catalog number 12804-25G-F), 0.396 mmol, 379.24 g/mol, [149 mg]} in the presence of 4 equiv of DIPEA {0.40 mmol, 129.24 g/mol, 0.742 g/mL, [70 μL]} in anhydrous DMF. Two min after the addition of DIPEA, the solution was added to the resin-immobilized and DMF pre-swollen product (15B) and agitated for 1 h.
(122) The resulting compound (16B) was then washed three times with DMF1, three times with DMF2, three times with DCM1, and, finally, three times with DCM2.
(123) Cleavage of Alloc-protecting group from the compound (16B) was realized by reaction with 0.03 equiv of TPP Pd {(Sigma; Catalog number 216666-1G), 0.03 mmol, 1155.56 g/mol, [35 mg]} in the presence of 30 equiv of morpholine {3.0 mmol, 87.12 g/mol, 0.999 g/mL, [262 μL]} in 3 mL of anhydrous DCM. The reaction was performed for 2 hours in the dark using aluminum foil.
(124) The resin was then washed three times with DCM1, three times with DCM2, three times with DMF1, and, finally, three times with DMF2. To remove residuals of the palladium, the resin was additionally washed ten times with 1% DIPEA in DMF (300 μL DIPEA in 30 mL DMF2) and subsequently washed ten times for 5 min with a solution of cupral {(Sigma; Catalog number D3506-100G), 225.31 g/mol} in DMF2 at the concentration of 15 mg/mL (450 mg cupral in 30 mL DMF2). The resulting compound (17B) was then washed three times with DMF1 and three times with DMF2.
(125) The conjugation of the chelator to the resin-immobilized compound (17B) was performed with 3 equiv of DOTA-tris(t-Bu)ester {([2-(4,7,10-tris(2-(t-butoxy)-2-oxoethyl)-1,4,7,10-tetraazacyclododecan-1-yl)acetic acid]; CheMatech; Catalog number 137076-54-1), 0.30 mmol, 572.73 g/mol [171 mg]}. The chelator building block was activated with 2.97 equiv of HBTU {(Sigma; Catalog number 12804-25G-F), 0.297 mmol, 379.24 g/mol, [112 mg]} in the presence of 4 equiv of DIPEA {0.40 mmol, 129.24 g/mol, 0.742 g/mL, [70 μL]} in anhydrous DMF. Two min after the addition of DIPEA, the solution was added to the resin-immobilized and the DMF pre-swollen compound (17B). The coupling of the DOTA chelator proceeded over the course of 2 h with gentle agitation.
(126) Such product (18B) washed three times with DMF1 and three times with DMF2, three times with DCM1, three times with DCM2 and, finally, three times with Et.sub.2O and dried under vacuum.
(127) The final compound PSMA-ALB-05 was obtained by agitation and subsequent cleavage from the resin within 2 h with a mixture consisting of TFA, TIPS and H.sub.2O in a ratio of 95:2.5:2.5. TFA was evaporated, crude product dissolved in ACN and water in a ratio of 1:1 and purified via RP-HPLC.
(128) The outline of the above described synthesis is summarized in two parts of Scheme 1.7.
(129) ##STR00057## ##STR00058## ##STR00059##
(130) Relative to the lysine-coated PSMA precursor (9), 4 equiv of Fmoc as well as Alloc protected L-lysine {(Fmoc-Lys(Alloc)-OH; Merck; Catalog number 8521240005), 0.40 mmol, 452.50 g/mol, [181 mg]} was activated with 3.96 equiv of HBTU {(Sigma; Catalog number 12804-25G-F), 0.396 mmol, 379.24 g/mol, [149 mg]} in the presence of 4 equiv of DIPEA {0.40 mmol, 129.24 g/mol, 0.742 g/mL, [70 μL]} in anhydrous DMF. Two min after the addition of DIPEA, the solution was added to the DMF pre-swollen immobilized bis(tBu)-protected PSMA precursor (9) and agitated for 1 h.
(131) Selective removal of Fmoc-protecting group from the resulting compound (10) was realized by washing with the mixture of DMF and piperidine in a ratio of 1:1 once for 2 min and then once again for 5 min in order to obtain the product (11).
(132) The conjugation of the chelator to the resin-immobilized compound (11) was performed with 2 equiv of DOTA-tris(t-Bu)ester {([2-(4,7,10-tris(2-(t-butoxy)-2-oxoethyl)-1,4,7,10-tetraazacyclododecan-1-yl)acetic acid]; CheMatech; Catalog number 137076-54-1), 0.20 mmol, 572.73 g/mol [115 mg]}. The chelator building block was activated with 1.98 equiv of HBTU {(Sigma; Catalog number 12804-25G-F), 0.198 mmol, 379.24 g/mol, [75 mg]} in the presence of 4 equiv of DIPEA {0.40 mmol, 129.24 g/mol, 0.742 g/mL, [70 μL]} in anhydrous DMF. Two min after the addition of DIPEA, the solution was added to the resin-immobilized and the DMF pre-swollen compound (11). The coupling of the DOTA chelator proceeded over the course of 2 h with gentle agitation. The resulting compound (12) was then washed three times with DMF1, three times with DMF2, three times with DCM1, and, finally, three times with DCM2.
(133) Cleavage of Alloc-protecting group from the compound (12) was realized by reaction with 0.03 equiv of TPP Pd {(Sigma; Catalog number 216666-1G), 0.03 mmol, 1155.56 g/mol, [35 mg]} in the presence of 30 equiv of morpholine {3.0 mmol, 87.12 g/mol, 0.999 g/mL, [262 μL]} in 3 mL of anhydrous DCM. The reaction was performed for 2 hours in the dark using aluminum foil.
(134) The resin was then washed three times with DCM1, three times with DCM2, three times with DMF1, and, finally, three times with DMF2. To remove residuals of the palladium, the resin was additionally washed ten times with 1% DIPEA in DMF (300 μL DIPEA in 30 mL DMF2) and subsequently washed ten times for 5 min with a solution of cupral {(Sigma; Catalog number D3506-100G), 225.31 g/mol} in DMF2 at the concentration of 15 mg/mL (450 mg cupral in 30 mL DMF2). The resulting compound (13) was then washed three times with DMF1 and three times with DMF2.
(135) Finally, for the coupling of the albumin-binding moiety, 4 equiv of tolyl-butyric acid {([4-(p-tolyl)butyric acid]; ABCR; AB1119212), 0.40 mmol, 178.23 g/mol, [71 mg]} was activated with 3.96 equiv of HBTU {(Sigma; Catalog number 12804-25G-F), 0.396 mmol, 379.24 g/mol, [149 mg]} in the presence of 4 equiv of DIPEA {0.40 mmol, 129.24 g/mol, 0.742 g/mL, [70 μL]} in anhydrous DMF. Two min after the addition of DIPEA, the solution was added to the resin-immobilized and DMF pre-swollen product (13) and agitated for 1 h.
(136) The resulting compound (14) was then washed three times with DMF1, three times with DMF2, three times with DCM1, three times with DCM2, and, finally, three times with Et.sub.2O and dried under vacuum.
(137) The final compound PSMA-ALB-06 was obtained by agitation and subsequent cleavage from the resin within 2 h with a mixture consisting of TFA, TIPS and H.sub.2O in a ratio of 95:2.5:2.5. TFA was evaporated, crude product dissolved in ACN and water in a ratio of 1:1 and purified via RP-HPLC.
(138) The outline of the above described synthesis is summarized in Scheme 1.8.
(139) ##STR00060## ##STR00061##
f) Synthesis of PSMA-ALB-07
(140) Relative to the lysine-coated PSMA precursor (9B), 4 equiv of Fmoc as well as Alloc protected L-lysine {(Fmoc-Lys(Alloc)-OH; Merck; Catalog number 8521240005), 0.40 mmol, 452.50 g/mol, [181 mg]} was activated with 3.96 equiv of HBTU {(Sigma; Catalog number 12804-25G-F), 0.396 mmol, 379.24 g/mol, [149 mg]} in the presence of 4 equiv of DIPEA {0.40 mmol, 129.24 g/mol, 0.742 g/mL, [70 μL]} in anhydrous DMF. Two min after the addition of DIPEA, the solution was added to the DMF pre-swollen immobilized bis(tBu)-protected PSMA precursor (9B) and agitated for 1 h.
(141) The resulting compound (10B) was then washed three times with DMF1 and three times with DMF2. Selective removal of Fmoc-protecting group from the resulting compound (10B) was realized by washing with the mixture of DMF and piperidine in a ratio of 1:1 once for 2 min and then once again for 5 min in order to obtain the product (11B).
(142) Relative to the lysine-coated PSMA precursor (11B), 3 equiv of Fmoc as well as tBu protected D-aspartate {(Fmoc-D-Asp-OtBu; Merck; Catalog number 8521440001), 0.30 mmol, 411.45 g/mol, [123 mg]} was activated with 2.97 equiv of HBTU {(Sigma; Catalog number 12804-25G-F), 0.297 mmol, 379.24 g/mol, [112 mg]} in the presence of 4 equiv of DIPEA {0.40 mmol, 129.24 g/mol, 0.742 g/mL, [70 μL]} in anhydrous DMF. Two min after the addition of DIPEA, the solution was added to the DMF pre-swollen immobilized bis(tBu)-protected PSMA precursor (11B) and agitated for 1 h.
(143) The resulting compound (12B) was then washed three times with DMF1 and three times with DMF2. Selective removal of Fmoc-protecting group from the resulting compound (12B) was realized by washing with the mixture of DMF and piperidine in a ratio of 1:1 once for 2 min and then once again for 5 min in order to obtain the product (13B).
(144) Relative to the lysine and aspartate-coated PSMA precursor (13B), 3 equiv of Fmoc as well as tBu protected D-aspartate {(Fmoc-D-Asp-OtBu; Merck; Catalog number 8521440001), 0.30 mmol, 411.45 g/mol, [123 mg]} was activated with 2.97 equiv of HBTU {(Sigma; Catalog number 12804-25G-F), 0.297 mmol, 379.24 g/mol, [112 mg]} in the presence of 4 equiv of DIPEA {0.40 mmol, 129.24 g/mol, 0.742 g/mL, [70 μL]} in anhydrous DMF. Two min after the addition of DIPEA, the solution was added to the DMF pre-swollen immobilized bis(tBu)-protected PSMA precursor (13B) and agitated for 1 h.
(145) The resulting compound (14B) was then washed three times with DMF1 and three times with DMF2. Selective removal of Fmoc-protecting group from the resulting compound (14B) was realized by washing with the mixture of DMF and piperidine in a ratio of 1:1 once for 2 min and then once again for 5 min in order to obtain the product (15B).
(146) Relative to the lysine and two aspartates-coated PSMA precursor (15B), 3 equiv of Fmoc as well as tBu protected D-aspartate {(Fmoc-D-Asp-OtBu; Merck; Catalog number 8521440001), 0.30 mmol, 411.45 g/mol, [123 mg]} was activated with 2.97 equiv of HBTU {(Sigma; Catalog number 12804-25G-F), 0.297 mmol, 379.24 g/mol, [112 mg]} in the presence of 4 equiv of DIPEA {0.40 mmol, 129.24 g/mol, 0.742 g/mL, [70 μL]} in anhydrous DMF. Two min after the addition of DIPEA, the solution was added to the DMF pre-swollen immobilized bis(tBu)-protected PSMA precursor (15B) and agitated for 1 h.
(147) The resulting compound (16B) was then washed three times with DMF1 and three times with DMF2. Selective removal of Fmoc-protecting group from the resulting compound (14B) was realized by washing with the mixture of DMF and piperidine in a ratio of 1:1 once for 2 min and then once again for 5 min in order to obtain the product (17B).
(148) Relative to the resin-coated product (17B), 4 equiv of iodophenyl-butyric acid {([4-(p-iodophenyl)butyric acid]; Sigma; I5634-5G), 0.40 mmol, 290.10 g/mol, [116 mg]} was activated with 3.96 equiv of HBTU {(Sigma; Catalog number 12804-25G-F), 0.396 mmol, 379.24 g/mol, [149 mg]} in the presence of 4 equiv of DIPEA {0.40 mmol, 129.24 g/mol, 0.742 g/mL, [70 μL]} in anhydrous DMF. Two min after the addition of DIPEA, the solution was added to the resin-immobilized and DMF pre-swollen product (17B) and agitated for 1 h.
(149) The resulting compound (18B) was then washed three times with DMF1, three times with DMF2, three times with DCM1, and, finally, three times with DCM2.
(150) Cleavage of Alloc-protecting group from the compound (18B) was realized by reaction with 0.03 equiv of TPP Pd {(Sigma; Catalog number 216666-1G), 0.03 mmol, 1155.56 g/mol, [35 mg]} in the presence of 30 equiv of morpholine {3.0 mmol, 87.12 g/mol, 0.999 g/mL, [262 μL]} in 3 mL of anhydrous DCM. The reaction was performed for 2 hours in the dark using aluminum foil.
(151) The resin was then washed three times with DCM1, three times with DCM2, three times with DMF1, and, finally, three times with DMF2. To remove residuals of the palladium, the resin was additionally washed ten times with 1% DIPEA in DMF (300 μL DIPEA in 30 mL DMF2) and subsequently washed ten times for 5 min with a solution of cupral {(Sigma; Catalog number D3506-100G), 225.31 g/mol} in DMF2 at the concentration of 15 mg/mL (450 mg cupral in 30 mL DMF2). The resulting compound (19B) was then washed three times with DMF1 and three times with DMF2.
(152) The conjugation of the chelator to the resin-immobilized compound (19B) was performed with 3 equiv of DOTA-tris(t-Bu)ester {([2-(4,7,10-tris(2-(t-butoxy)-2-oxoethyl)-1,4,7,10-tetraazacyclododecan-1-yl)acetic acid]; CheMatech; Catalog number 137076-54-1), 0.30 mmol, 572.73 g/mol [171 mg]}. The chelator building block was activated with 2.97 equiv of HBTU {(Sigma; Catalog number 12804-25G-F), 0.297 mmol, 379.24 g/mol, [112 mg]} in the presence of 4 equiv of DIPEA {0.40 mmol, 129.24 g/mol, 0.742 g/mL, [70 μL]} in anhydrous DMF. Two min after the addition of DIPEA, the solution was added to the resin-immobilized and the DMF pre-swollen compound (17B). The coupling of the DOTA chelator proceeded over the course of 2 h with gentle agitation.
(153) Such product (20B) washed three times with DMF1 and three times with DMF2, three times with DCM1, three times with DCM2 and, finally, three times with Et.sub.2O and dried under vacuum.
(154) The final compound PSMA-ALB-07 was obtained by agitation and subsequent cleavage from the resin within 2 h with a mixture consisting of TFA, TIPS and H.sub.2O in a ratio of 95:2.5:2.5. TFA was evaporated, crude product dissolved in ACN and water in a ratio of 1:1 and purified via RP-HPLC. The outline of the above described synthesis is summarized in two parts of Scheme 1.9.
(155) ##STR00062## ##STR00063## ##STR00064##
g) Synthesis of PSMA-ALB-08
(156) Relative to the lysine-coated PSMA precursor (9B), 4 equiv of Fmoc as well as Alloc protected L-lysine {(Fmoc-Lys(Alloc)-OH; Merck; Catalog number 8521240005), 0.40 mmol, 452.50 g/mol, [181 mg]} was activated with 3.96 equiv of HBTU {(Sigma; Catalog number 12804-25G-F), 0.396 mmol, 379.24 g/mol, [149 mg]} in the presence of 4 equiv of DIPEA {0.40 mmol, 129.24 g/mol, 0.742 g/mL, [70 μL]} in anhydrous DMF. Two min after the addition of DIPEA, the solution was added to the DMF pre-swollen immobilized bis(tBu)-protected PSMA precursor (9B) and agitated for 1 h.
(157) The resulting compound (10B) was then washed three times with DMF1 and three times with DMF2. Selective removal of Fmoc-protecting group from the resulting compound (10B) was realized by washing with the mixture of DMF and piperidine in a ratio of 1:1 once for 2 min and then once again for 5 min in order to obtain the product (11B).
(158) Relative to the lysine-coated PSMA precursor (11B), 3 equiv of Fmoc as well as tBu protected D-aspartate {(Fmoc-D-Asp-OtBu; Merck; Catalog number 8521440001), 0.30 mmol, 411.45 g/mol, [123 mg]} was activated with 2.97 equiv of HBTU {(Sigma; Catalog number 12804-25G-F), 0.297 mmol, 379.24 g/mol, [112 mg]} in the presence of 4 equiv of DIPEA {0.40 mmol, 129.24 g/mol, 0.742 g/mL, [70 μL]} in anhydrous DMF. Two min after the addition of DIPEA, the solution was added to the DMF pre-swollen immobilized bis(tBu)-protected PSMA precursor (11B) and agitated for 1 h.
(159) The resulting compound (12B) was then washed three times with DMF1 and three times with DMF2. Selective removal of Fmoc-protecting group from the resulting compound (12B) was realized by washing with the mixture of DMF and piperidine in a ratio of 1:1 once for 2 min and then once again for 5 min in order to obtain the product (13B).
(160) Relative to the lysine and aspartate-coated PSMA precursor (13B), 3 equiv of Fmoc as well as tBu protected D-aspartate {(Fmoc-D-Asp-OtBu; Merck; Catalog number 8521440001), 0.30 mmol, 411.45 g/mol, [123 mg]} was activated with 2.97 equiv of HBTU {(Sigma; Catalog number 12804-25G-F), 0.297 mmol, 379.24 g/mol, [112 mg]} in the presence of 4 equiv of DIPEA {0.40 mmol, 129.24 g/mol, 0.742 g/mL, [70 μL]} in anhydrous DMF. Two min after the addition of DIPEA, the solution was added to the DMF pre-swollen immobilized bis(tBu)-protected PSMA precursor (13B) and agitated for 1 h.
(161) The resulting compound (14B) was then washed three times with DMF1 and three times with DMF2. Selective removal of Fmoc-protecting group from the resulting compound (14B) was realized by washing with the mixture of DMF and piperidine in a ratio of 1:1 once for 2 min and then once again for 5 min in order to obtain the product (15B).
(162) Relative to the lysine and two aspartates-coated PSMA precursor (15B), 3 equiv of Fmoc as well as tBu protected D-aspartate {(Fmoc-D-Asp-OtBu; Merck; Catalog number 8521440001), 0.30 mmol, 411.45 g/mol, [123 mg]} was activated with 2.97 equiv of HBTU {(Sigma; Catalog number 12804-25G-F), 0.297 mmol, 379.24 g/mol, [112 mg]} in the presence of 4 equiv of DIPEA {0.40 mmol, 129.24 g/mol, 0.742 g/mL, [70 μL])} in anhydrous DMF. Two min after the addition of DIPEA, the solution was added to the DMF pre-swollen immobilized bis(tBu)-protected PSMA precursor (15B) and agitated for 1 h.
(163) The resulting compound (16B) was then washed three times with DMF1 and three times with DMF2. Selective removal of Fmoc-protecting group from the resulting compound (14B) was realized by washing with the mixture of DMF and piperidine in a ratio of 1:1 once for 2 min and then once again for 5 min in order to obtain the product (17B).
(164) Relative to the resin-coated product (17B), 4 equiv of tolyl-butyric acid (0.40 mmol} was activated with 3.96 equiv of HBTU {(Sigma; Catalog number 12804-25G-F), 0.396 mmol, 379.24 g/mol, [149 mg]} in the presence of 4 equiv of DIPEA {0.40 mmol, 129.24 g/mol, 0.742 g/mL, [70 μL]} in anhydrous DMF. Two min after the addition of DIPEA, the solution was added to the resin-immobilized and DMF pre-swollen product (17B) and agitated for 1 h.
(165) The resulting compound (18B) was then washed three times with DMF1, three times with DMF2, three times with DCM1, and, finally, three times with DCM2.
(166) Cleavage of Alloc-protecting group from the compound (18B) was realized by reaction with 0.03 equiv of TPP Pd {(Sigma; Catalog number 216666-1G), 0.03 mmol, 1155.56 g/mol, [35 mg]} in the presence of 30 equiv of morpholine {3.0 mmol, 87.12 g/mol, 0.999 g/mL, [262 μL]} in 3 mL of anhydrous DCM. The reaction was performed for 2 hours in the dark using aluminum foil.
(167) The resin was then washed three times with DCM1, three times with DCM2, three times with DMF1, and, finally, three times with DMF2. To remove residuals of the palladium, the resin was additionally washed ten times with 1% DIPEA in DMF (300 μL DIPEA in 30 mL DMF2) and subsequently washed ten times for 5 min with a solution of cupral {(Sigma; Catalog number D3506-100G), 225.31 g/mol} in DMF2 at the concentration of 15 mg/mL (450 mg cupral in 30 mL DMF2). The resulting compound (19B) was then washed three times with DMF1 and three times with DMF2.
(168) The conjugation of the chelator to the resin-immobilized compound (19B) was performed with 3 equiv of DOTA-tris(t-Bu)ester {([2-(4,7,10-tris(2-(t-butoxy)-2-oxoethyl)-1,4,7,10-tetraazacyclododecan-1-yl)acetic acid]; CheMatech; Catalog number 137076-54-1), 0.30 mmol, 572.73 g/mol [171 mg]}. The chelator building block was activated with 2.97 equiv of HBTU {(Sigma; Catalog number 12804-25G-F), 0.297 mmol, 379.24 g/mol, [112 mg]} in the presence of 4 equiv of DIPEA {0.40 mmol, 129.24 g/mol, 0.742 g/mL, [70 μL]} in anhydrous DMF. Two min after the addition of DIPEA, the solution was added to the resin-immobilized and the DMF pre-swollen compound (17B). The coupling of the DOTA chelator proceeded over the course of 2 h with gentle agitation.
(169) Such product (20B) washed three times with DMF1 and three times with DMF2, three times with DCM1, three times with DCM2 and, finally, three times with Et.sub.2O and dried under vacuum.
(170) The final compound PSMA-ALB-07 was obtained by agitation and subsequent cleavage from the resin within 2 h with a mixture consisting of TFA, TIPS and H.sub.2O in a ratio of 95:2.5:2.5. TFA was evaporated, crude product dissolved in ACN and water in a ratio of 1:1 and purified via RP-HPLC.
(171) The outline of the above described synthesis is summarized in two parts of Scheme 1.10.
(172) ##STR00065## ##STR00066## ##STR00067##
1.1.3: .sup.177Lu-Labeling of PSMA Ligands and In Vitro Evaluation
(173) In vitro studies were conducted with .sup.177Lu-PSMA-ALB-01/-03/-04/-05/-06/-07/-08. This included the preliminary evaluation of labeling efficiencies, n-octanol/PBS distribution coefficients and serum protein binding studies. Furthermore, uptake and internalization experiments were performed using the PSMA-transfected PSMApos PC-3 PIP cell line (positive control) and the mock-transfected PSMAneg PC-3 flu cell line (negative control).
(174) a) PSMA-Ligands and Radionuclides
(175) The PSMA-ligands .sup.177Lu-PSMA-ALB-01/-03/-04/-05/-06/-07/-08 were synthesized as described above. The reference compound (PSMA-617) was purchased from Advanced Biochemical Compounds (ABX GmbH, Radeberg, Germany). No-carrier added .sup.177Lu in 0.05 M HCl was provided by Isotope Technologies Garching (ITG GmbH, Germany).
(176) b) Radiolabeling
(177) The stock solution of PSMA-617 was prepared by dilution in MilliQ water to a final concentration of 1 mM. .sup.177Lu-PSMA-ALB-01/-03/-04/-05/-06/-07/-08 were diluted in MilliQ water/DMSO to obtain a final concentration of 1 mM. All compounds were labeled with .sup.177Lu in a 1:5 mixture of sodium acetate (0.5 M, pH 8) and HCl (0.05 M, pH ˜1) at pH 3.5-4.5. The compounds were labeled with .sup.177Lu at specific activities between 5-50 MBq/nmol, depending on the experimental conditions. The reaction mixture was incubated for 15 min at 95° C., followed by a quality control using high-performance liquid chromatography with a C-18 reversed-phase column (Xterra™ MS, C18, 5 μm, 150×4.6 mm; Waters). The mobile phase consisted of MilliQ water containing 0.1% trifluoracetic acid (A) and acetonitrile (B) with a gradient of 95% A and 5% B to 20% A and 80% B over a period of 15 min at a flow rate of 1.0 mL/min. The radioligands were diluted in MilliQ water containing Na-DTPA (50 μM (micromolar)) prior to injection into HPLC.
(178) c) Determination of the n-Octanol/PBS Distribution Coefficient
(179) .sup.177Lu-PSMA-ALB-01/-03/-04/-05/-06/-07/-08 and PSMA-617 were labeled with .sup.177Lu at a specific activity of 50 MBq/nmol. The radioligand (0.5 MBq; 10 pmol, 25 μL) was then added to a reagent tube containing 1475 μL of PBS pH 7.4 and 1500 μL of n-octanol. The vials were vortexed vigorously followed by a centrifugation step for phase separation. Finally, the radioactivity in a defined volume of PBS and n-octanol was measured in a gamma-counter (Perkin Elmer, Wallac Wizard 1480) to calculate the distribution coefficients, expressed as the logarithm of the ratio of counts per minute (cpm) measured in the n-octanol phase to the cpm measure in the PBS phase.
(180) d) Filter Assay
(181) Plasma binding of .sup.177Lu-PSMA-ALB-01/-03/-04/-05/-06/-07/-08 and .sup.177Lu-PSMA-617 was determined using an ultrafiltration assay.
(182) Therefore, the compounds were labeled with .sup.177Lu at a specific activity of 50 MBq/nmol and incubated in human plasma samples or PBS at room temperature. The free and plasma-bound fractions were separated using a centrifree ultrafiltration device (4104 centrifugal filter units [Millipore]; 30000 Da nominal molecular weight limit, methylcellulose micropartition membranes). The incubated solution was loaded to the ultrafiltration device and centrifuged at 2500 rpm for 40 min at 20° C. Samples from the filtrate were taken an analyzed for radioactivity in a gamma-counter. The amount of plasma-bound compound was calculated as the fraction of radioactivity measured in the filtrate relative to the corresponding loading solution (set to 100%).
(183) e) Cell Internalization Assay
(184) Cell uptake and internalization experiments were performed with .sup.177Lu-PSMA-ALB-01/-03/-04/-05/-06/-07/-08 and the reference compound .sup.177Lu-PSMA-617 using the PSMA-transfected PSMA.sup.pos PC-3 PIP and mock-transfected PSMA.sup.neg PC-3 flu cells in order to investigate the specificity of the novel compounds.
(185) Cells were grown in RPMI cell culture medium supplemented with 10% fetal calf serum, L-glutamine, antibiotics and puromycin (2 μg/mL) at 37° C. and 5% CO2 (standard conditions). Routine cell culture was performed twice a week using PBS/EDTA (2 mM) for washing the cells and trypsin for detachment of the cells. The cells were seeded in 12-well plates (˜3×10.sup.5 cells in 2 mL RPMI medium/well) allowing adhesion and growth overnight at standard conditions. The supernatant was removed and the cells washed with PBS pH 7.4 prior to the addition of RPMI medium without supplements (975 μL/well). The compounds were labeled with .sup.177Lu at a specific activity of 5 MBq/nmol and diluted to 1.5 MBq/mL in 0.05% bovine serum albumin (BSA)/0.9% NaCl solution to prevent adherence to plastic vessels. The cells were incubated with 25 μL (˜37.5 kBq)/well radiolabeled PSMA ligands at standard conditions for 2 h and 4 h, respectively. After incubation, the cells were washed three times with ice-cold PBS and the total uptake of the radioligands was determined (PSMA-bound fraction on the surface and internalized fraction). The fraction of internalized radioligand was evaluated in cells washed with ice-cold PBS, followed by a 10 min incubation with stripping buffer (0.05 M glycine stripping buffer in 100 mM NaCl, pH 2.8) and an additional washing step with ice-cold PBS. Cell samples were lysed by addition of NaOH (1 M, 1 mL) to each well. The samples of the cell suspensions were measured in a γ-counter (Perkin Elmer, Wallac Wizard 1480). After homogenization of the cell suspensions, the protein concentration was determined for each sample using a Micro BCA Protein Assay kit (Pierce, Therma Scientific). The results were expressed as percentage of total added radioactivity per 150 μg/mL protein.
(186) 1.2 Results
(187) 1.2.1 Labeling Efficiency
(188) PSMA-ALB-01 and -03 were successfully labeled with .sup.177Lu at specific activities up to 100 MBq/nmol and excellent radiochemical yields of >98%. PSMA-ALB-04, -05, -06, -07 and -08 were labeled with .sup.177Lu in preliminary tests at specific activities up to 50 MBq/nmol and excellent radiochemical yields of >97%. The specific activity used for the experiments (if not otherwise stated) was 50 MBq/nmol. The radiochemical purity of compounds used for in vitro and in vivo studies was always >97% (
(189) 1.2.2 n-Octanol/PBS Distribution Coefficient
(190) .sup.177Lu-PSMA-ALB-01, -03, -04 and -06 showed similar n-octanol/PBS distribution coefficients (Log D value), while the coefficients of .sup.177Lu-PSMA-ALB-05, -07 and -08 indicated slightly more hydrophilic compounds. In general, the data showed that the introduction of an albumin-binding entity reduces the hydrophilicity as compared to the reference compound .sup.177Lu-PSMA-617, however, all compounds are still hydrophilic with log D values >2.7 (
(191) 1.2.3 Albumin-Binding Properties
(192) The ultrafiltration experiments of .sup.177Lu-PSMA-ALB-01, -03, -04, -05, -06 and -07 revealed high serum protein binding capacities as >94% of the compound did not penetrate the filter when incubated in human plasma. The easy possibility of filtrating the compounds was demonstrating when incubating the compound sin PBS where proteins are not present (
(193) 1.2.4 Internalization
(194) Cell uptake and internalization of PSMA ligands .sup.177Lu-PSMA-ALB-01, -03, -04, -05, -06, -07 and -08 were investigated and compared to the reference compound .sup.177Lu-PSMA-617 using PC-3 PIP/flu cells (
Example 2: In Vivo Evaluation of PSMA Ligands in Tumor Mouse Model
(195) .sup.177Lu-PSMA-ALB-01, -03, -04, -05, -06, -07 and -08 were characterized in vivo. Therefore, immunodeficient Balb/c nude mice were inoculated with PSMApos PC-3 PIP and PSMAneg PC-3 flu cells. After intravenous (i.v.) application of the ligands, extensive biodistribution and SPECT/CT studies were performed. Tumor uptake, tumor/blood ratio, tumor/kidney ratio and tumor/liver ratio of .sup.177Lu-PSMA-ALB-01-08 are summarized in
(196) 2.1 Material and Methods
(197) 2.1.1 Tumor Mouse Model
(198) Mice were obtained from Charles River Laboratories, Sulzfeld, Germany, at the age of 5-6 weeks. Female, athymic nude Balb/c mice were subcutaneously inoculated with PC-3 PIP cells (6×10.sup.6 cells in 100 μL Hank's balanced salt solution (HBSS) with Ca.sup.2+/Mg.sup.2+) on the right shoulder and with PC-3 flu cells (5×10.sup.6 cells in 100 μL HBSS Ca.sup.2+/Mg.sup.2+) on the left shoulder. Two weeks later, the tumors reached a size of about 200-300 mm.sup.3 suitable for the performance of the biodistribution and imaging studies.
(199) 2.1.2 Biodistribution Studies
(200) Biodistribution studies were performed using PC-3 PIP/flu tumor-bearing mice, which were inoculated with tumor cells two weeks prior to injection of PSMA ligands. The radioligands were diluted in 0.9% NaCl and i.v. injected in a volume of 100-200 μL. Mice were euthanized at different time points after injection (p.i.) of the radioligands. Selected tissues and organs were collected, weighed and measured using a gamma-counter. The results were decay-corrected and listed as a percentage of the injected activity per gram of tissue mass (% IA/g).
(201) 2.1.3 SPECT/CT Imaging Studies.
(202) SPECT/CT experiments were performed using a dedicated small-animal SPECT/CT camera (NanoSPECT/CT™, Mediso Medical Imaging Systems, Budapest, Hungary). The PSMA ligands were labeled at a specific activity of 25 MBq/nmol and diluted in saline containing 0.05% BSA. Scans were acquired at 4 h, 24 h and 72 h after injection of the radioligands (25 MBq, 1 nmol, 100 L). Data was reconstructed using NanoSPECT/CT™ software and post-processed using VivoQuant (version 3.0, inviCRO Imaging Services and Software, Boston USA). A Gauss post-reconstruction filter (FWHM=1 mm) was applied and the scale of radioactivity was set as indicated on the images (minimum value=0.095 Bq/voxel to maximum value=95 Bq/voxel).
(203) 2.1.4 Therapy in Mouse Model
(204) Five groups of mice (Groups A to E, n=6) with statistically similar body weights and tumor volumes were injected with only the vehicle (saline containing BSA 0.05%; Group A), .sup.177Lu-PSMA-617 (Groups B and C) and .sup.177Lu-PSMA-ALB-06 (Groups D and E), respectively, at Day 0 of the therapy study (Table 2.1). Mice of Groups B and D received 2 MBq of the radioligand (1 nmol/mouse), whereas mice of Groups C and E received 5 MBq of the radioligand (1 nmol/mouse). The mice were monitored by measuring body weights and the tumor size every other day over 12 weeks. Mice were euthanized when pre-defined endpoint criteria were reached, or when the study was terminated at Day 84. The relative body weight (RBW) was defined as [BW.sub.x/BW.sub.0], where BW.sub.x is the body weight in gram at a given day x and BW.sub.0 the body weight in gram at day 0. The tumor dimension was determined by measuring the longest tumor axis (L) and its perpendicular axis (W) with a digital caliper. The tumor volume (V) was calculated according to the equation [V=0.5*(L*W2)]. The relative tumor volume (RTV) was defined as [TV.sub.x/TV.sub.0], where TV.sub.x is the tumor volume in mm3 at a given day x and TV.sub.0 the tumor volume in mm3 at Day 0.
(205) TABLE-US-00002 TABLE 2.1 Design of Therapy Study tumor body injected radioactivity volume.sup.b weight.sup.b [MBq] [mm.sup.3] [g] Treatment measured.sup.a (average ± SD) (average ± SD) group (n = 6) theoretical (average ± SD) Day 0 Day 0 A saline 88 ± 21 16 ± 1.6 B .sup.177Lu- 2 2.2 ± 0.1 103 ± 24 16 ± 1.2 PSMA-617 C .sup.177Lu- 5 5.7 ± 0.4 104 ± 25 17 ± 0.9 PSMA-617 D .sup.177Lu- 2 2.1 ± 0.3 81 ± 25 15 ± 1.3 PSMA-ALB-56 E .sup.177Lu- 5 5.4 ± 0.5 92 ± 34 15 ± 1.3 PSMA-ALB-56 .sup.aRadioactivity in the syringe measured before and after injecting the respective mouse. .sup.bNo significant differences determined between the values measured for each group (p > 0.05).
(206) The efficacy of the radionuclide therapy was expressed as the tumor growth delay (TGD.sub.x), which was calculated as the time required for the tumor volume to increase x-fold over the initial volume at the Day 0. The tumor growth delay index [TGDI.sub.x=TGD.sub.x(T)/TGD.sub.x(C)] was calculated as the TGD.sub.x ratio of treated mice (T) over control mice (C) for a 2-fold (x=2, TGD2) and 5-fold (x=5, TGD5) increase of the initial tumor volume. As a measure to identify undesired side effects, body weights were compared at the day when the first control mouse had to be euthanized. After euthanasia, kidneys, liver and the brain were collected and weighed. The organ ratios (kidney-to-brain and liver-to-brain) were calculated using the organ masses obtained at the day of euthanasia.
(207) The data was analyzed for significance as indicated in the result part using a one-way ANOVA with Tukey's multiple comparison post-test using GraphPad Prism software (version 7). A value of p<0.05 was considered statistically significant. Survival analysis was performed with Kaplan-Meier curves and a log-rank tests (Mantel Cox).
(208) 2.2 Results
(209) 2.2.1 Biodistribution of .sup.177Lu-PSMA-ALB-01, .sup.177Lu-PSMA-ALB-03
(210) The tissue distribution of .sup.177Lu-PSMA-ALB-01 and .sup.177Lu-PSMA-ALB-03 was investigated over a period of eight days. Compounds .sup.177Lu-PSMA-ALB-01 and .sup.177Lu-PSMA-ALB-03 showed highly similar tissue distribution profiles (
(211) High radioactivity levels could be observed in the blood pool already at early time points and were cleared slowly but steadily over time. The uptake of both radioligands in the PSMA.sup.pos PC-3 PIP tumors was increasing until it reached a plateau and did not drop substantially until the end of the study. The uptake in PC-3 flu tumors was clearly below blood levels, indicating highly PSMA-specific binding and uptake in vivo (
(212) TABLE-US-00003 TABLE 2.2 Biodistribution of 177Lu-PSMA-ALB-01 in PC-3 PIP/flu Tumor-Bearing Mice 1 h.p.i. 4 h.p.i. 8 h p.i. 24 h p.i. 48 h p.i. 96 h p.i. Blood 29.7 ± 4.49 25.6 ± 1.53 21.0 ± 2.86 14.2 ± 1.40 12.0 ± 2.18 6.68 ± 0.85 Heart 10.1 ± 1.10 8.71 ± 0.50 7.16 ± 1.23 5.93 ± 0.65 4.42 ± 0.81 2.70 ± 0.37 Lung 16.6 ± 2.78 14.1 ± 0.99 11.6 ± 0.83 8.62 ± 1.47 7.67 ± 0.83 5.07 ± 0.66 Spleen 5.27 ± 1.64 5.34 ± 0.90 4.05 ± 0.69 3.60 ± 0.43 4.62 ± 1.12 3.12 ± .015 Kidneys 19.4 ± 4.82 24.6 ± 0.38 22.6 ± 2.38 22.7 ± 2.18 25.2 ± 4.15 13.0 ± 1.30 Stomach 3.29 ± 1.75 3.30 ± 0.05 2.45 ± 0.43 1.39 ± 0.07 1.49 ± 0.47 0.81 ± 0.04 Intestines 4.15 ± 1.40 4.17 ± 0.70 2.44 ± 0.17 2.12 ± 0.20 1.84 ± 0.54 1.05 ± 0.17 Liver 5.76 ± 1.21 5.92 ± 0.07 5.31 ± 1.23 2.92 ± 0.67 3.03 ± 0.63 1.88 ± 0.36 Salivary glands 5.52 ± 1.08 5.20 ± 0.73 4.45 ± 0.56 3.38 ± 0.32 3.96 ± 0.98 2.22 ± 0.38 Muscle 2.22 ± 0.88 2.06 ± 0.80 1.63 ± 0.27 1.34 ± 0.14 1.35 ± 0.46 0.82 ± 0.12 Bone 3.15 ± 0.47 3.01 ± 0.09 2.54 ± 0.26 1.58 ± 0.06 1.64 ± 0.34 1.07 ± 0.22 PC-3 PIP Tumor 8.98 ± 2.77 20.4 ± 0.39 25.5 ± 2.02 38.2 ± 2.59 65.6 ± 1.84 62.3 ± 3.56 PC-3 flu Tumor 3.64 ± 2.30 5.03 ± 1.61 4.01 ± 0.79 3.95 ± 0.82 4.64 ± 1.84 2.76 ± 0.23 Tumor-to-blood 0.30 ± 0.06 0.80 ± 0.03 1.22 ± 0.08 2.71 ± 0.39 5.54 ± 0.76 9.39 ± 0.65 Tumor-to-liver 1.56 ± 0.30 3.45 ± 0.03 4.97 ± 1.21 13.5 ± 2.79 22.2 ± 4.11 33.7 ± 4.33 Tumor-to-kidney 0.46 ± 0.04 0.83 ± 0.03 1.13 ± 0.07 1.69 ± 0.07 2.64 ± 0.40 4.80 ± 0.20 144 h.p.i. 192 h.p.i. Blood 5.78 ± 0.90 5.21 ± 1.37 Heart 2.23 ± 0.29 2.12 ± 0.68 Lung 4.90 ± 0.58 4.12 ± 0.96 Spleen 4.32 ± 0.57 4.09 ± 1.12 Kidneys 10.2 ± 3.41 7.56 ± 1.44 Stomach 0.84 ± 0.13 0.73 ± 0.12 Intestines 1.07 ± 0.13 1.02 ± 0.25 Liver 1.56 ± 0.16 1.51 ± 0.37 Salivary glands 1.68 ± 0.53 1.78 ± 0.34 Muscle 0.66 ± 0.15 0.64 ± 0.13 Bone 0.99 ± 0.20 0.86 ± 0.17 PC-3 PIP Tumor 78.4 ± 8.57 75.6 ± 22.0 PC-3 flu Tumor 2.82 ± 0.24 2.73 ± 0.84 Tumor-to-blood 13.8 ± 2.38 14.5 ± 1.84 Tumor-to-liver 50.5 ± 6.28 50.2 ± 7.38 Tumor-to-kidney 8.05 ± 1.77 9.87 ± 1.05
(213) TABLE-US-00004 TABLE 2.3 Biodistribution of .sup.177Lu-PSMA-ALB-03 in PC-3 PIP/flu Tumor-Bearing Mice 1 h.p.i. 4 h.p.i. 8 h p.i. 24 h p.i. 48 h p.i. 96 h p.i. Blood 27.4 ± 3.04 24.3 ± 3.60 23.5 ± 0.74 17.3 ± 1.38 12.5 ± 3.78 7.37 ± 0.64 Heart 9.64 ± 1.21 8.54 ± 1.18 8.12 ± 0.46 6.60 ± 1.01 4.40 ± 1.18 3.15 ± 0.28 Lung 16.6 ± 3.29 14.21 ± 3.49 12.21 ± 1.32 9.86 ± 0.57 7.45 ± 2.06 5.56 ± 0.54 Spleen 4.63 ± 0.56 4.76 ± 1.12 4.10 ± 0.14 3.75 ± 0.21 3.79 ± 0.89 3.23 ± 0.53 Kidneys 17.8 ± 2.49 24.5 ± 4.38 28.8 ± 1.49 24.7 ± 1.85 22.6 ± 2.69 16.1 ± 1.69 Stomach 3.19 ± 0.95 2.86 ± 1.03 2.92 ± 0.17 1.39 ± 0.27 1.49 ± 0.48 0.91 ± .010 Intestines 3.70 ± 0.73 3.71 ± 1.09 3.70 ± 0.40 2.19 ± 0.23 1.73 ± 0.50 1.21 ± 0.26 Liver 5.81 ± 2.65 4.56 ± 1.18 4.87 ± 0.42 3.35 ± 0.26 2.53 ± 0.77 1.78 ± 0.03 Salivary glands 5.60 ± 0.70 5.02 ± 1.17 5.49 ± 0.59 4.69 ± 0.33 3.45 ± 1.09 2.19 ± 0.1 Muscle 1.91 ± 0.16 2.04 ± 0.37 2.01 ± 0.10 1.61 ± 0.18 1.32 ± 0.41 0.91 ± 0.15 Bone 2.82 ± 0.41 2.47 ± 0.39 2.71 ± 0.21 2.02 ± 0.31 1.63 ± 0.56 1.07 ± 0.27 PC-3 PIP Tumor 8.49 ± 0.62 19.9 ± 0.79 31.0 ± 5.79 53.8 ± 5.61 72.3 ± 24.7 75.7 ± 2.46 PC-3 flu Tumor 3.84 ± 1.10 5.32 ± 1.06 5.98 ± 0.91 5.47 ± 0.67 5.69 ± 3.65 3.52 ± 0.54 Tumor-to-blood 0.31 ± 0.04 0.83 ± 0.10 1.32 ± 0.29 3.13 ± 0.35 5.94 ± 1.53 10.3 ± 0.57 Tumor-to-liver 1.64 ± 0.61 4.60 ± 1.34 6.38 ± 1.28 16.1 ± 1.75 29.0 ± 5.26 42.6 ± 1.90 Tumor-to-kidney 0.48 ± 0.07 0.83 ± 0.14 1.07 ± 0.18 2.17 ± 0.06 3.17 ± 0.84 4.7 ± 0.38 144 h.p.i. 192 h.p.i. Blood 6.02 ± 0.60 5.29 ± 0.18 Heart 2.55 ± 0.25 2.08 ± 0.14 Lung 4.99 ± 1.00 4.17 ± 0.68 Spleen 2.94 ± 0.39 3.13 ± 0.90 Kidneys 11.2 ± 3.82 7.35 ± 0.92 Stomach 0.73 ± 0.10 0.78 ± 0.07 Intestines 0.88 ± 0.18 0.96 ± 0.03 Liver 1.50 ± 0.14 1.25 ± 0.20 Salivary glands 1.67 ± 0.27 1.55 ± 0.06 Muscle 0.54 ± 0.07 0.62 ± 0.04 Bone 1.14 ± 0.23 0.83 ± 0.22 PC-3 PIP Tumor 68.9 ± 8.80 58.9 ± 12.4 PC-3 flu Tumor 2.77 ± 0.41 2.42 ± 0.23 Tumor-to-blood 11.4 ± 0.47 11.1 ± 1.97 Tumor-to-liver 46.0 ± 2.53 47.1 ± 2.44 Tumor-to-kidney 6.44 ± 1.27 7.97 ± 0.70
2.2.2 Biodistribution of .sup.177Lu-PSMA-ALB-04 and .sup.177Lu-PSMA-ALB-05
(214) The tissue distribution of .sup.177Lu-PSMA-ALB-04 and .sup.177Lu-PSMA-ALB-05 was investigated over a period of eight days (
(215) Blood activity levels in animals injected with .sup.177Lu-PSMA-ALB-04 was very high at early time points and remained by far the highest. A high PSMApos PC-3 PIP tumor accumulation was observed, which slightly decreased towards the end of the study. The accumulated activity in the PSMA.sup.neg PC-3 flu tumor and other non-target organs was clearly below blood levels, indicating highly PSMA-specific binding and uptake in vivo.
(216) The high levels in the blood pool of animals injected with .sup.177Lu-PSMA-ALB-05 were decreasing quickly and remained stable at low levels until the end of the study. Highest uptake of radioactivity could be observed in the PSMA.sup.pos PC-3 PIP tumors of mice injected with .sup.177Lu-PSMA-ALB-05, which was followed by a steady wash-out from the tumor tissue. The uptake in PC-3 flu tumors and other tissues was clearly below blood levels, indicating PSMA-specific binding and uptake in vivo. Biodistribution data for .sup.177Lu-PSMA-ALB-04 and -05 are shown in Table 2.4 and 2.5 below.
(217) TABLE-US-00005 TABLE 2.4 Biodistribution of .sup.177Lu-PSMA-ALB-04 in PC-3 PIP/flu Tumor-Bearing Mice 1 h.p.i. 4 h.p.i. 8 h p.i. 24 h p.i. 48 h p.i. 96 h p.i. Blood 46.7 ± 9.64 n/d n/d 55.3 ± 3.10 38.2 ± 1.48 11.65 ± 1.22 Heart 16.4 ± 5.71 n/d n/d 22.1 ± 3.70 13.6 ± 0.36 4.81 ± 0.29 Lung 25.6 ± 4.59 n/d n/d 40.1 ± 7.77 24.6 ± 2.02 9.96 ± 1.44 Spleen 11.0 ± 3.56 n/d n/d 13.6 ± 1.90 14.0 ± 1.70 6.76 ± 0.59 Kidneys 17.8 ± 4.49 n/d n/d 41.5 ± 1.44 38.2 ± 4.10 15.3 ± 1.49 Stomach 4.01 ± 0.62 n/d n/d 6.18 ± 0.95 5.04 ± 0.35 1.74 ± 0.11 Intestines 6.22 ± 1.11 n/d n/d 8.13 ± 1.31 7.27 ± 0.82 2.36 ± 0.17 Liver 29.3 ± 9.10 n/d n/d 17.6 ± 2.04 13.1 ± 0.67 4.67 ± 0.82 Salivary glands 9.93 ± 2.33 n/d n/d 12.5 ± 0.42 10.6 ± 0.42 4.08 ± 0.44 Muscle 1.96 ± 0.40 n/d n/d 5.82 ± 1.62 4.62 ± 0.38 1.56 ± 0.56 Bone 4.74 ± 1.31 n/d n/d 8.88 ± 0.19 6.80 ± 0.67 2.54 ± 0.31 PC-3 PIP Tumor 9.56 ± 2.71 n/d n/d 82.8 ± 6.84 93.2 ± 12.4 61.4 ± 7.68 PC-3 flu Tumor 3.46 ± 2.66 n/d n/d 14.0 ± 0.60 12.6 ± 0.82 5.63 ± 0.37 Tumor-to-blood 0.20 ± 0.02 n/d n/d 1.50 ± 0.11 2.45 ± 0.41 5.29 ± 0.74 Tumor-to-liver 0.33 ± 0.06 n/d n/d 4.76 ± 0.67 7.16 ± 1.16 13.4 ± 2.88 Tumor-to-kidney 0.54 ± 0.09 n/d n/d 2.00 ± 0.11 2.47 ± 0.50 4.03 ± 0.56 144 h.p.i. 192 h.p.i. Blood n/d 3.75 ± 1.49 Heart n/d 1.73 ± 0.70 Lung n/d 4.10 ± 1.77 Spleen n/d 5.22 ± 3.17 Kidneys n/d 8.82 ± 3.83 Stomach n/d 0.56 ± 0.19 Intestines n/d 0.67 ± 0.15 Liver n/d 2.45 ± 0.81 Salivary glands n/d 1.96 ± 0.73 Muscle n/d 0.71 ± 0.54 Bone n/d 1.17 ± 0.61 PC-3 PIP Tumor n/d 57.6 ± 17.3 PC-3 flu Tumor n/d 3.31 ± 1.43 Tumor-to-blood n/d 15.8 ± 3.55 Tumor-to-liver n/d 23.9 ± 5.12 Tumor-to-kidney n/d 6.91 ± 2.24
(218) TABLE-US-00006 TABLE 2.5 Biodistribution of .sup.177Lu-PSMA-ALB-05 in PC-3 PIP/flu Tumor-Bearing Mice 1 h.p.i. 4 h.p.i. 8 h p.i. 24 h p.i. 48 h p.i. 96 h p.i. Blood 21.3 ± 6.06 10.2 ± 1.98 n/d 1.67 ± 0.29 1.66 ± 0.37 1.79 ± 0.57 Heart 7.56 ± 1.89 3.82 ± 0.63 n/d 0.65 ± 0.11 0.54 ± 0.15 0.70 ± 0.21 Lung 15.0 ± 1.24 7.07 ± 1.44 n/d 1.80 ± 0.78 1.48 ± 0.62 1.36 ± 0.29 Spleen 5.78 ± 1.40 3.29 ± 0.74 n/d 1.13 ± 0.23 0.71 ± 0.23 0.64 ± 0.29 Kidneys 59.3 ± 1.38 52.8 ± 7.17 n/d 23.9 ± 4.02 12.8 ± 2.62 6.89 ± 0.31 Stomach 2.04 ± 0.43 1.15 ± 0.17 n/d 0.28 ± 0.06 0.29 ± 0.08 0.24 ± 0.07 Intestines 2.71 ± 0.40 1.33 ± 0.25 n/d 0.28 ± 0.05 0.28 ± 0.10 0.30 ± 0.11 Liver 5.69 ± 1.59 2.96 ± 0.50 n/d 0.82 ± 0.35 0.56 ± 0.16 0.74 ± 0.14 Salivary glands 6.17 ± 2.12 2.75 ± 0.72 n/d 0.49 ± 0.10 0.45 ± 0.10 0.46 ± 0.10 Muscle 2.36 ± 1.01 1.30 ± 0.23 n/d 0.19 ± 0.06 0.20 ± 0.08 0.15 ± 0.06 Bone 3.03 ± 0.52 1.67 ± 0.27 n/d 0.31 ± 0.08 0.28 ± 0.05 0.28 ± 0.04 PC-3 PIP Tumor 46.9 ± 0.43 75.3 ± 15.3 n/d 79.4 ± 11.1 60.3 ± 10.7 45.0 ± 7.94 PC-3 flu Tumor 3.72 ± 0.83 2.10 ± 0.20 n/d 0.59 ± 0.10 0.57 ± 0.09 0.49 ± 0.11 Tumor-to-blood 2.31 ± 0.58 7.43 ± 1.43 n/d 48.2 ± 7.04 36.7 ± 1.81 27.1 ± 10.0 Tumor-to-liver 8.65 ± 2.21 25.6 ± 4.58 n/d 106 ± 28.6 110 ± 12.2 62.8 ± 18.8 Tumor-to-kidney 0.79 ± 0.02 1.42 ± 0.19 n/d 3.38 ± 0.58 4.72 ± 0.18 6.51 ± 0.98 144 h.p.i. 192 h.p.i. Blood 1.75 ± 0.35 1.48 ± 0.13 Heart 0.65 ± 0.17 0.59 ± 0.05 Lung 1.25 ± 0.18 1.22 ± 0.26 Spleen 0.56 ± 0.08 0.55 ± 0.10 Kidneys 4.28 ± 0.26 2.70 ± 0.36 Stomach 0.23 ± 0.04 0.16 ± 0.04 Intestines 0.27 ± 0.05 0.24 ± 0.04 Liver 0.72 ± 0.13 0.84 ± 0.06 Salivary glands 0.46 ± 0.09 0.37 ± 0.04 Muscle 0.17 ± 0.04 0.14 ± 0.01 Bone 0.25 ± 0.05 0.26 ± 0.04 PC-3 PIP Tumor 33.9 ± 0.80 27.9 ± 3.24 PC-3 flu Tumor 0.52 ± 0.13 0.45 ± 0.06 Tumor-to-blood 19.9 ± 3.88 19.0 ± 2.97 Tumor-to-liver 47.9 ± 8.28 33.4 ± 1.64 Tumor-to-kidney 7.93 ± 0.30 10.4 ± 0.25
2.2.3 Biodistribution of .sup.177Lu-PSMA-ALB-06, .sup.177Lu-PSMA-ALB-07, .sup.177Lu-PSMA-ALB-08
(219) The tissue distribution of .sup.177Lu-PSMA-ALB-06, -07 and -08 was investigated up to three days post injection (
(220) Blood activity levels of all compounds decreased quickly and were comparable throughout the entire study. The highest PSMA.sup.pos PC-3 PIP tumor accumulation was observed for compound .sup.177Lu-PSMA-ALB-06, which slightly decreased towards the end of the study. The accumulated activity in the PSMA.sup.neg PC-3 flu tumor and other non-target organs was below blood levels, indicating PSMA-specific binding and uptake in vivo for all compounds tested. Biodistribution data for .sup.177Lu-PSMA-ALB-06, -07 and -08 are shown in Table 2.6, 2.7 and 2.8 below.
(221) TABLE-US-00007 TABLE 2.6 Biodistribution of .sup.177Lu-PSMA-ALB-06 in PC-3 PIP/flu Tumor-Bearing Mice 1 h.p.i. 4 h.p.i. 8 h p.i. 24 h p.i. 48 h p.i. 72 h p.i. Blood n/d 16.2 ± 1.40 n/d 1.49 ± 0.50 n/d 0.62 ± 0.06 Heart n/d 5.41 ± 0.82 n/d 0.68 ± 0.18 n/d 0.26 ± 0.02 Lung n/d 9.40 ± 1.55 n/d 2.48 ± 2.68 n/d 0.67 ± 0.05 Spleen n/d 3.14 ± 0.29 n/d 0.76 ± 0.18 n/d 0.53 ± 0.02 Kidneys n/d 18.9 ± 0.77 n/d 10.5 ± 2.13 n/d 5.58 ± 0.62 Stomach n/d 1.89 ± 0.19 n/d 0.28 ± 0.07 n/d 0.13 ± 0.02 Intestines n/d 2.64 ± 0.27 n/d 0.30 ± 0.06 n/d 0.15 ± 0.00 Liver n/d 3.45 ± 1.50 n/d 0.50 ± 0.11 n/d 0.28 ± 0.02 Salivary glands n/d 3.26 ± 0.16 n/d 0.52 ± 0.11 n/d 0.24 ± 0.03 Muscle n/d 1.60 ± 0.38 n/d 0.21 ± 0.04 n/d 0.07 ± 0.02 Bone n/d 2.23 ± 0.08 n/d 0.41 ± 0.15 n/d 0.18 ± 0.01 PC-3 PIP Tumor n/d 76.08 ± 7.67 n/d 108 ± 11.6 n/d 77.9 ± 7.52 PC-3 flu Tumor n/d 3.16 ± 0.39 n/d 0.79 ± 0.23 n/d 0.43 ± 0.03 Tumor-to-blood n/d 4.72 ± 0.51 n/d 77.6 ± 21.8 n/d 127 ± 24.9 Tumor-to-liver n/d 24.29 ± 8.27 n/d 222 ± 49.5 n/d 277 ± 19.3 Tumor-to-kidney n/d 4.02 ± 0.25 n/d 10.4 ± 1.16 n/d 14.1 ± 2.02 96 h.p.i. 192 h.p.i. Blood n/d n/d Heart n/d n/d Lung n/d n/d Spleen n/d n/d Kidneys n/d n/d Stomach n/d n/d Intestines n/d n/d Liver n/d n/d Salivary glands n/d n/d Muscle n/d n/d Bone n/d n/d PC-3 PIP Tumor n/d n/d PC-3 flu Tumor n/d n/d Tumor-to-blood n/d n/d Tumor-to-liver n/d n/d Tumor-to-kidney n/d n/d (n/d = not determined)
(222) TABLE-US-00008 TABLE 2.7 Biodistribution of .sup.177Lu-PSMA-ALB-07 in PC-3 PIP/flu Tumor-Bearing Mice 1 h.p.i. 4 h.p.i. 8 h p.i. 24 h p.i. 72 h p.i. 96 h p.i. Blood n/d 6.67 ± 2.04 n/d 0.79 ± 0.08 0.40 ± 0.06 n/d Heart n/d 2.43 ± 0.78 n/d 0.40 ± 0.00 0.21 ± 0.01 n/d Lung n/d 4.67 ± 0.92 n/d 0.73 ± 0.06 0.43 ± 0.02 n/d Spleen n/d 3.41 ± 1.46 n/d 1.14 ± 0.04 0.49 ± 0.03 n/d Kidneys n/d 67.0 ± 9.50 n/d 51.9 ± 6.34 26.0 ± 1.58 n/d Stomach n/d 1.09 ± 0.30 n/d 0.18 ± 0.06 0.10 ± 0.01 n/d Intestines n/d 1.27 ± 0.45 n/d 0.20 ± 0.03 0.10 ± 0.01 n/d Liver n/d 1.94 ± 1.02 n/d 0.52 ± 0.04 0.44 ± 0.08 n/d Salivary glands n/d 2.09 ± 0.50 n/d 0.43 ± 0.04 0.21 ± 0.01 n/d Muscle n/d 0.78 ± 0.22 n/d 0.13 ± 0.01 0.08 ± 0.01 n/d Bone n/d 1.30 ± 0.27 n/d 0.31 ± 0.10 0.31 ± 0.06 n/d PC-3 PIP Tumor n/d 63.5 ± 27.4 n/d 84.6 ± 14.2 62.6 ± 6.35 n/d PC-3 flu Tumor n/d 1.80 ± 0.27 n/d 0.80 ± 0.17 0.43 ± 0.04 n/d Tumor-to-blood n/d 9327 ± 1.75 n/d 107 ± 12.2 160 ± 37.0 n/d Tumor-to-liver n/d 33.6 ± 6.56 n/d 162 ± 17.3 147 ± 38.4 n/d Tumor-to-kidney n/d 0.88 ± 0.28 n/d 1.64 ± 0.29 2.41 ± 0.18 n/d 144 h.p.i. 192 h.p.i. Blood n/d n/d Heart n/d n/d Lung n/d n/d Spleen n/d n/d Kidneys n/d n/d Stomach n/d n/d Intestines n/d n/d Liver n/d n/d Salivary glands n/d n/d Muscle n/d n/d Bone n/d n/d PC-3 PIP Tumor n/d n/d PC-3 flu Tumor n/d n/d Tumor-to-blood n/d n/d Tumor-to-liver n/d n/d Tumor-to-kidney n/d n/d (n/d = not determined)
(223) TABLE-US-00009 TABLE 2.8 Biodistribution of .sup.177Lu-PSMA-ALB-08 in PC-3 PIP/flu Tumor-Bearing Mice 1 h.p.i. 4 h.p.i. 8 h p.i. 24 h p.i. 72 h p.i. 96 h p.i. Blood n/d 0.41 ± 0.18 n/d 0.08 ± 0.01 0.06 ± 0.02 n/d Heart n/d 0.19 ± 0.07 n/d 0.04 ± 0.01 0.03 ± 0.01 n/d Lung n/d 0.48 ± 0.21 n/d 0.09 ± 0.02 1.28 ± 2.02 n/d Spleen n/d 0.53 ± 0.10 n/d 0.10 ± 0.03 0.11 ± 0.05 n/d Kidneys n/d 27.2 ± 5.93 n/d 13.9 ± 2.32 7.98 ± 0.62 n/d Stomach n/d 0.40 ± 0.25 n/d 0.03 ± 0.01 0.02 ± 0.01 n/d Intestines n/d 0.20 ± 0.09 n/d 0.03 ± 0.01 0.02 ± 0.01 n/d Liver n/d 0.27 ± 0.10 n/d 0.11 ± 0.02 0.12 ± 0.01 n/d Salivary glands n/d 0.19 ± 0.06 n/d 0.07 ± 0.05 0.03 ± 0.02 n/d Muscle n/d 0.06 ± 0.03 n/d 0.02 ± 0.01 0.01 ± 0.01 n/d Bone n/d 0.16 ± 0.03 n/d 0.07 ± 0.02 0.09 ± 0.05 n/d PC-3 PIP Tumor n/d 46.9 ± 16.7 n/d 33.0 ± 5.04 24.1 ± 5.37 n/d PC-3 flu Tumor n/d 0.25 ± 0.19 n/d 0.09 ± 0.05 0.09 ± 0.07 n/d Tumor-to-blood n/d 116 ± 9.46 n/d 421 ± 45.7 416 ± 89.5 n/d Tumor-to-liver n/d 177 ± 2.72 n/d 295 ± 40.1 207 ± 47.1 n/d Tumor-to-kidney n/d 1.70 ± 0.22 n/d 2.39 ± 0.24 3.02 ± 0.68 n/d 144 h.p.i. 192 h.p.i. Blood n/d n/d Heart n/d n/d Lung n/d n/d Spleen n/d n/d Kidneys n/d n/d Stomach n/d n/d Intestines n/d n/d Liver n/d n/d Salivary glands n/d n/d Muscle n/d n/d Bone n/d n/d PC-3 PIP Tumor n/d n/d PC-3 flu Tumor n/d n/d Tumor-to-blood n/d n/d Tumor-to-liver n/d n/d Tumor-to-kidney n/d n/d (n/d = not determined)
2.2.4 SPECT/CT Imaging Studies.
(224) SPECT/CT images of PC-3 PIP/flu tumor-bearing mice were performed at different time points after injection of .sup.177Lu-PSMA-ALB-03 and 177Lu-PSMA-ALB-06. The exact injected activity of .sup.177Lu-PSMA-ALB-03 and .sup.177Lu-PSMA-ALB-06 was 25 MBq and 23 MBq, respectively. The favourable in vivo behavior of .sup.177Lu-PSMA-ALB-03 and .sup.177Lu-PSMA-ALB-06 is shown in
(225) 2.2.5 Therapy in Mouse Model
(226) Control mice (Group A) showed constant tumor growth over time, which was comparable to the tumor growth of mice treated with low activity of .sup.177Lu-PSMA-617 (Group B: 2 MBq/mouse). The tumor growth delay indices of mice of Group B (TGDI.sub.2=0.8, TGDI.sub.5=1.4, Table 2.9) were, therefore similar to the values of control animals where the TGDI was defined as 1. The first control mouse reached an endpoint at Day 16, whereas in Group B one mouse had to be euthanized already at Day 12 (Table 2.9). Mice were effectively treated when using a higher activity of .sup.177Lu-PSMA-617 (Group C: 5 MBq/mouse) or low activity of .sup.177Lu-PSMA-ALB-06 (Group D: 2 MBq/mouse). The TGDI.sub.2 and TGDI.sub.5 were similar for mice of both groups (Groups C and D) and consequently, mice had to be euthanized in the same time range (Group C: Day 26 to Day 40; Group D: Day 28 to Day 44; data not shown). In mice treated with higher activity of .sup.177Lu-PSMA-ALB-06 (Group E: 5 MBq/mouse), the tumor growth was effectively inhibited. In four mice of Group E the tumors disappeared entirely and regrowth was not observed until the end of the study at Day 84.
(227) TABLE-US-00010 TABLE 2.9 Tumor Growth Inhibition (TGI) and Tumor Growth Delay Index with x-Fold Increase of Tumor Size (TGDIx) of .sup.177Lu-PSMA-ALB-06 and .sup.177Lu-PSMA-617 first mouse of group median treatment euthanized survival group group [d] [d] TGDI.sub.2 TGDI.sub.5 A saline 16 18 1.0 ± 0.8 1.0 ± 0.1 B .sup.177Lu- 12 19 0.8 ± 0.3 1.4 ± 0.1 PSMA-617 C .sup.177Lu- 26 32 2.1 ± 0.3 2.5 ± 0.3 PSMA-617 D .sup.177LU- 28 36 1.8 ± 0.5 2.3 ± 0.6 PSMA-ALB-56 E .sup.177Lu- 58 n.d..sup.a n.d..sup.a n.d..sup.a PSMA-ALB-56 .sup.an.d. = not defined since mice were still alive at the end of the study.
(228) Mice that received higher activity of .sup.177Lu-PSMA-617 or low activity of .sup.177Lu-PSMA-ALB-06 showed a significantly increased median survival (Group C: Day 32, Group D: Day 36, Table 2.9,
Example 3: Clinical Evaluation of PSMA Ligands
(229) 3.1: Case 1
(230) The compound PSMA-ALB-06, radiolabeled with therapeutic radionuclide Lutetium-177 was used in the scope of an individual curative trial in a patient with mildly differentiated prostate adenocarcinoma with extensive bilobar liver metastases, as well as disseminated osteoblastic metastases (in the pelvic region), and polytopic voluminous lymph node metastases. The evaluation of the biodistribution and in vivo behavior of the radiolabeled compound PSMA-ALB-06 was performed by means of SPECT-CT measurements.
(231) The SPECT-CT visualization was performed at different time points up to 46 hours post injection (p.i.). The radiolabeled compound PSMA-ABL-06 demonstrated a prolonged blood circulation and improved bioavailability (
(232) These first in-human results confirm the pre-clinical findings on improved pharmacokinetic properties of the compound demonstrating it's potential for the treatment of PSMA positive tumors.
(233) 3.2 Case 2:
(234) The compound PSMA-ALB-06, radiolabeled with a positron emitting radionuclide Gallium-68 was used in an individual curative trial in a patient with metastatic castration-resistant prostate cancer as a diagnostic agents for PET-CT. Malignant tissues could be visualized by means of PET with high specificity, whereas the background radioactivity in off-target healthy organs remains moderate (
Example 4: Investigation of PSMA Ligands in Combination with .SUP.44.Sc for PET Imaging
(235) 4.1 Biodistribution Data of .sup.44Sc-PSMA-ALB
(236) .sup.44Sc was produced at the Injector 2 facility at PSI as previously reported..sup.2 Radiolabeling of PSMA-ALB-06 was performed as previously reported by our group using the clinically-established PSMA-617 ligand..sup.5 Biodistribution studies were obtained in female Balb/c nude mice bearing PSMA-positive PC-3 PIP tumor cells (right shoulder) and PSMA-negative PC-3 flu tumors (left shoulder). For this purpose, the mice were inoculated with tumor cells 12-14 days before injection of the radioligand. The mice were euthanized and dissected at 1 h, 4 h and 6 h post injection (p.i.) (
(237) TABLE-US-00011 TABLE 4.1 Biodistribution Data of 44S c-PSMA-ALB-06 in PC-3 PIP/flu Tumor-Bearing Mice. 44Sc-PSMA-ALB-06 1 h p.i. 4 h p.i. 6 h p.i. Blood 26.6 ± 2.82 18.4 ± 1.00 15.6 ± 0.75 Heart 8.91 ± 0.22 6.48 ± 0.50 5.17 ± 0.17 Lung 14.8 ± 2.41 11.17 ± 0.66 9.44 ± 0.86 Spleen 4.86 ± 0.76 4.10 ± 0.54 3.67 ± 0.37 Kidneys 32.9 ± 4.35 28.8 ± 2.46 21.7 ± 0.42 Stomach 2.68 ± 0.34 1.91 ± 0.16 1.98 ± 0.26 Intestines 3.30 ± 0.59 2.50 ± 0.13 2.07 ± 0.34 Liver 5.86 ± 0.67 3.51 ± 0.44 3.67 ± 0.68 Muscle 3.06 ± 0.34 2.24 ± 0.10 1.83 ± 0.07 Bone 3.59 ± 0.91 2.50 ± 0.35 2.54 ± 0.27 Salivary gland 5.83 ± 0.28 4.55 ± 0.41 4.03 ± 0.22 PC-3 PIP Tumor 25.3 ± 5.91 61.7 ± 7.32 72.9 ± 11.1 PC-3 flu Tumor 5.02 ± 1.23 4.37 ± 0.43 3.87 ± 0.45 Tumor-to-blood 0.94 ± 0.11 3.35 ± 0.20 4.69 ± 0.67 Tumor-to-liver 4.28 ± 0.52 17.93 ± 3.32 20.2 ± 3.43 Tumor-to-kidney 0.77 ± 0.12 2.14 ± 0.15 3.35 ± 0.42
4.2 3. PET/CT Imaging of Mice Injected with .sup.44Sc-PSMA-ALB-06
(238) PET/CT experiments were performed using a small-animal PET/CT camera (G8, Perkin Elmer, U.S.) as previously reported by our group..sup.5 The images were taken at 1 h, 4 h and 20 h after injection of 5 MBq .sup.44Sc-PSMA-ALB-06.
(239)
(240) 4.3 Conclusion
(241) Labeling of PSMA-ALB-06 was successfully performed with 44Sc at a specific activity of at least 5 MBq/nmol. The resulting biodistribution study and PET imaging results indicate similar properties of .sup.44Sc-PSMA-ALB-06 as previously determined for .sup.177Lu-PSMA-ALB-06. Due to the high tumor uptake of .sup.44Sc-PSMA-ALB-06, it is believed that this radioligand may be a useful tool for imaging even small lesions at late time points (>4 h p.i.) when background activity is excreted. A clinical translation of this approach appears most promising and should be one of the next steps in order to confirm the potential of the proposed concept.
Example 5: Design and Preclinical Evaluation of an NODAGA-Functionalized Albumin-Binding PSMA Ligands
(242) A long-circulating PSMA-targeting agent suitable for stable complexation of copper was designed, which enables PET imaging of prostate cancer at delayed time points. Therefore, the DOTA-chelator of PSMA-ALB-06 was replaced with a NODAGA-chelator to obtain PSMA-ALB-89. PSMA-ALB-89 and PSMA-ALB-06 were labeled with .sup.64Cu and tested for radiolytic stability, binding to serum albumin and uptake into PSMA-positive PC-3 PIP and PSMA-negative PC-3 flu tumor cells. Biodistribution and PET/CT imaging studies were performed with in PC-3 PIP/flu tumor-bearing mice.
(243) ##STR00068##
5.1 Material and Methods
(244) Solid-Phase Synthesis of the PSMA-Ligand. The NODAGA-functionalized PSMA ligand, referred to as PSMA-ALB-89, was synthesized using a solid phase platform as reported for the PSMA-ALB-06 (cf. Example 1). The only difference was related to the conjugation of the chelator in the last step of the synthesis (Scheme 5.1). The conjugation was performed with 3 equiv NODAGA-tris(t-Bu)ester [4-(4,7-bis(2-(tert-butoxy)-2-oxoethyl)-1,4,7-triazacyclononane-1-yl)-5(tert-butoxy)-5-oxopentanoic acid] activated with 2.97 equiv O-(benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate (HBTU) in the presence of 4 equiv N,N-diisopropylethylamine (DIPEA) in anhydrous N,N-dimethylformamide (DMF). The coupling of the NODAGA chelator proceeded over the course of 3 h with gentle agitation. The final product was cleaved from the resin and subsequently deprotected within 2 h using a mixture consisting of trifluoroacetic acid (TFA), triisopropylsilane (TIPS) and H.sub.2O in a ratio of 95:2.5:2.5 (v/v).
(245) ##STR00069##
(246) Radiolabeling and Stability. .sup.64Cu was produced via the .sup.64Ni(p,n).sup.64Cu nuclear reaction at the research cyclotron Injector 2 facility at PSI..sup.28 PSMA-ALB-89 and PSMA-ALB-06 were dissolved in MilliQ water containing up to 5.5% sodium acetate (0.5 M, pH 8) to prepare 1 mM stock solutions. The PSMA ligands were labeled with .sup.64Cu in a mixture of sodium acetate (0.5 M) and HCl (0.05 M) at pH 5 at specific activities between 5-50 MBq/nmol. The reaction mixture was incubated for 15 min at 95° C. Quality control of the radioligands was performed using RP-HPLC. The radioligands were used for in vitro and in vivo experiments without further purification steps.
(247) Quality control of .sup.64Cu-labeled PSMA ligands (250 MBq in 120 μL; 50 MBq/nmol) was determined immediately after preparation (t=0 h) using RP-HPLC. The reaction mixtures were diluted in saline to an activity concentration of 250 MBq/500 μL and incubated at room temperature. The integrity of the compounds was investigated over one day (t=1 h, 4 h and 24 h, respectively). The amount of intact radioligand was quantified by integration of the product peak of the HPLC chromatograms in relation to the sum of all radioactive peaks of degradation products of unknown structure and traces of released .sup.64Cu which were set to 100%.
(248) Determination of n-Octanol/PBS Distribution Coefficients (Log D Values). The distribution coefficients (log D values) of the .sup.64Cu-labeled radioligands (50 MBq/nmol) were determined by a shake-flask method using liquid-liquid extraction followed by phase separation as previously reported. Three experiments were performed with five replicates for each radioligand. Statistical significance of the data (p<0.05) was evaluated using an unpaired t-test (GraphPad Prism software, version 7).
(249) Determination of Albumin-binding Properties. The binding of the radioligands to human plasma proteins was determined by an ultrafiltration assay. The .sup.64Cu-labeled PSMA-ligands (5-50 MBq, 0.01 nmol) were diluted in different dilutions of human plasma (Stiftung Blutspende SRK Aargau-Solothurn, Switzerland) or PBS as a control experiment as previously reported. Three independent experiments were performed in duplicates with both radioligands and the data was fitted to a semi-logarithmic plot (non-linear regression, one-site, specific binding) to obtain the half maximum binding (B.sub.50) in GraphPad Prism software (version 7).
(250) Cell Uptake and Internalization. Cell uptake (sum of the surface bound and internalized fraction) and internalization of the radioligands (5 MBq/nmol) were determined using PSMA-positive PC-3 PIP and PSMA-negative PC-3 flu cells.
(251) In Vivo Studies. In vivo experiments were approved by the local veterinarian department and conducted in accordance with the Swiss law of animal protection. All mice were obtained from Charles River Laboratories (Sulzfeld, Germany) at the age of 5-6 weeks. Female, athymic BALB/c nude mice were subcutaneously inoculated with PC-3 PIP cells (6×10.sup.6 cells in 100 μL Hank's balanced salt solution (HBSS) with Ca.sup.2+/Mg.sup.2+) on the right shoulder and with PC-3 flu cells (5×10.sup.6 cells in 100 μL HBSS with Ca.sup.2+/Mg.sup.2+) on the left shoulder 12-14 days before the performance of the experiments.
(252) Biodistribution Studies. Mice were injected into a lateral tail vein with the respective radioligand (5 MBq, 1 nmol, 100 μL) diluted in saline containing 0.05% BSA. The mice were sacrificed at 1 h, 4 h and 24 h after injection (p.i.) and selected tissues and organs were collected, weighed and measured using a γ-counter. Groups of 4-6 mice were used for each time point. The results were decay-corrected and listed as percentage of the injected activity per gram of tissue mass (% IA/g). Data presented as the average±standard deviation (SD). The data sets were analyzed for significance using a one-way ANOVA with Bonferroni's multiple comparison post-test using GraphPad Prism software (version 7). A p-value of <0.05 was considered statistically significant.
(253) PET/CT Imaging Studies. PET/CT experiments were performed at 1 h, 4 h and 24 h after injection of the radioligands (5 MBq/l nmol). Mice were injected into a lateral tail vein with the respective radioligand (5 MBq, 1 nmol, 100 μL) diluted in saline containing 0.05% BSA. PET/CT scans were performed using a small-animal PET/CT scanner (G8, Perkin Elmer, Massachusetts, U.S.) as previously reported. The PET scans lasted for 10 min and were followed by a CT scan of 1.5 min. During the in vivo scans, the mice were anesthetized with a mixture of isoflurane and oxygen. Reconstruction of acquired data was performed using the software of the provider of the G8 scanner. All images were prepared using VivoQuant post-processing software (version 3.0, inviCRO Imaging Services and Software, Boston USA). The images were prepared by cutting 2% of the lower scale to make the tumors, liver and kidneys best visible.
(254) 5.2 Results
(255) Synthesis of the PSMA Ligands. PSMA-ALB-89 was synthesized using a solid-phase support in analogy to the synthesis of PSMA-ALB-06 (Example 1). Instead of conjugating a DOTA-chelator, a NODAGA-chelator was used (Scheme 5.1). This multistep synthesis (17 steps) resulted in a highly pure compound (>98%) in an overall yield of 8.7% after semi-preparative HPLC purification.
(256) Radiolabeling, Stability and In Vitro Properties of .sup.64Cu-Labeled PSMA Ligands. PSMA-ALB-89 and PSMA-ALB-06 were labeled with .sup.64Cu at a specific activity up to 50 MBq/nmol. The radioligands showed high radiochemical purity (>98%) and similar retention times (˜11 min). .sup.64Cu-PSMA-ALB-89 and .sup.64Cu-PSMA-ALB-06 were stable (>92%) over a period of at least 4 h. The n-octanol/PBS distribution coefficient (log D values) of .sup.64Cu-PSMA-ALB-89 (−2.3±0.7) was slightly but not significantly (p>0.05) higher than the log D value of .sup.64Cu-PSMA-ALB-06 (−3.1±0.1).
(257) Albumin-binding Properties. .sup.64Cu-PSMA-ALB-89 and .sup.64Cu-PSMA-ALB-06 showed similar binding to plasma proteins (>92%) when incubated in human plasma. The half-maximum binding (B.sub.50) of .sup.64Cu-PSMA-ALB-89 was reached at a [HSA]-to-[radioligand] ratio of 454. This indicates a slightly increased binding when compared to .sup.64Cu-PSMA-ALB-89 which reached half-maximum binding at a [HSA]-to-[radioligand] ratio of 770 (
(258) Cell Uptake and Internalization. The cell uptake of .sup.64Cu-PSMA-ALB-89 into PC-3 PIP cells was ˜46% and the internalized fraction ˜14% after an incubation period of 2 h at 37° C. The cell uptake increased slightly after 4 h incubation time (˜52%), while the internalized fraction remained unchanged (˜14%). Similar values were determined for .sup.64Cu-PSMA-ALB-06 (
(259) Biodistribution Study. The tissue distribution profile of .sup.64Cu-PSMA-ALB-89 was assessed over a period of 24 h in tumor-bearing mice (
(260) Tumor-to-kidney ratios increased over time, yet, the values were rather low after injection of .sup.64Cu-PSMA-ALB-89. The tumor-to-liver ratios of .sup.64Cu-PSMA-ALB-89 were high. Tumor-to-muscle ratios increased over time to 200±38.2 at 24 h p.i.
(261) TABLE-US-00012 TABLE 5.1 Tumor-to-Background Ratios of .sup.64Cu-PSMA-ALB-89 and .sup.64Cu-PSMA-ALB-06 .sup.64Cu-PSMA-ALB-89 1 h p.i. 4 h p.i. 24 h p.i. Tu-to-blood* 0.91 ± 0.02 3.61 ± 0.30 31.3 ± 3.82 Tu-to-muscle 9.0 ± 1.13 36.3 ± 2.25 200 ± 38.2 Tu-to-kidney 0.40 ± 0.02 0.70 ± 0.04 2.68 ± 0.36 Tu-to-liver 3.37 ± 0.31 13.3 ± 1.20 23.6 ± 3.37 *For all Tu-to-organ ratios: Tu = PSMA-positive PC-3 PIP tumor
(262) PECT/CT Imaging Studies. PET/CT scans were performed over a period of 24 h with PC-3 PIP/flu tumor-bearing mice at different time points after injection of the .sup.64Cu-labeled radioligand (
(263) 5.3 Discussion
(264) In this work, a long-circulating PSMA ligand labeled with .sup.64Cu was synthesized to enable PET even one day after radioligand application. PSMA-ALB-89 was synthesized as previously described for PSMA-ALB-06, however, instead of coupling a DOTA-chelator a NODAGA chelator was employed as previously done in our group for other targeting agents.
(265) PSMA-ALB-89 was radiolabeled reproducibly with .sup.64Cu at high specific activities and radiochemical purity (50 MBq/nmol; >95%) suggesting a high quality of the synthesized ligand as well as excellent radiochemical purity of the .sup.64Cu which was produced in-house at PSI. In vitro, .sup.64Cu-PSMA-ALB-89 and .sup.64Cu-PSMA-ALB-06 where both stable after incubation for several hours at room temperature with only limited degradation detectable after 24 h. These results suggest that the NODAGA- and DOTA-chelator are both forming stable complexes with .sup.64Cu in vitro.
(266) The albumin-binding properties of .sup.64Cu-PSMA-ALB-89 were in the same range as for .sup.64Cu-PSMA-ALB-06 when tested in vitro. Binding specificity to PSMA was not affected by the different chelators either as proven by similar cell-bound and internalized fractions observed in vitro for .sup.64Cu-PSMA-ALB-89 and .sup.64Cu-PSMA-ALB-06.
(267) Biodistribution data obtained in a well-established xenograft mouse model using PSMA-positive and PSMA-negative tumors showed that tumor uptake of .sup.64Cu-PSMA-ALB-89 was significantly increased at all investigated time-points, possibly as a result of the longer blood circulation time. The maximum tumor uptake of .sup.64Cu-PSMA-ALB-89 was reached only at the end of the study (24 h p.i.). PET/CT images confirmed the favorable tissue distribution profile of .sup.64Cu-PSMA-ALB-89 with regard to the high tumor uptake and reduced accumulation in the liver. Low liver uptake is important as prostate cancer may result in liver metastases which may be masked by unspecific radioactivity accumulation otherwise.
(268) 5.4 Conclusion
(269) In this example, the DOTA-chelator of PSMA-ALB-06 was replaced by a NODAGA-chelator to enable stable coordination of .sup.64Cu for PET imaging. .sup.64Cu-PSMA-ALB-89 showed increased in vivo stability which was manifest by an increased tumor accumulation and reduced liver retention of .sup.64Cu-PSMA-ALB-89.
Example 6: Design and Evaluation of Further DOTA-Functionalized PSMA-Binding Ligand
(270) 6.1 Material and Methods
(271) Solid-Phase Synthesis of Albumin-Binding PSMA Ligands. PSMA ligands, referred to as PSMA-ALB-02, PSMA-ALB-05 and PSMA-ALB-07, respectively, were designed and synthesized using a solid phase platform. The PSMA-targeting urea-based pharmacophore—
(272) The next steps of the synthesis, comprising the conjugation of the lysine-based building block and the selective cleavage of the Na-Fmoc-protecting group, were performed equally for all three compounds. Relative to the resin-immobilized and bis(t-Bu)-protected precursor (0.3 mmol; compound (1)), 4 equiv of Nα-Fmoc- and Nε-Alloc-protected
(273) PSMA-ALB-02. The synthesis of PSMA-ALB-02 was accomplished by coupling of the albumin-binding moiety to the resin-immobilized precursor (0.1 mmol; compound (2)) while using 4 equiv of 4-(p-iodophenyl)butyric acid activated with 3.96 equiv HBTU in the presence of 4 equiv DIPEA in DMF over the course of 1 h with gentle agitation. Subsequently, the cleavage of the Nε-Alloc-protecting group from the compound (3) was performed with 0.03 equiv of tetrakis(triphenylphosphine)palladium(0) (TPP Pd) in the presence of 30 equiv morpholine in dichlormethane (DCM) within 2 h in the dark. To remove residuals of the palladium, the resin was additionally washed with 1% DIPEA in DMF and afterwards with a solution of sodium diethyldithiocarbamate in DMF (c=15 mg/mL). Finally, the conjugation of the chelator to the resin-immobilized compound was performed with 2 equiv of DOTA-tris(t-Bu)ester [2-(4,7,10-tris(2-(t-butoxy)-2-oxoethyl)-1,4,7,10-tetraazacyclo-dodecan-1-yl)acetic acid] activated with 1.98 equiv HBTU in the presence of 4 equiv DIPEA in DMF. The coupling of the DOTA chelator proceeded over the course of 2 h with gentle agitation. The resulting compound (4) was washed with DMF, DCM and, finally, with Et.sub.2O followed by drying under vacuum. The product was cleaved from the resin and subsequently deprotected within 2 h using a mixture consisting of trifluoroacetic acid (TFA), triisopropylsilane (TIPS) and H.sub.2O in a ratio of 95:2.5:2.5 (v/v). TFA was evaporated, the crude compound dissolved in ACN and H.sub.2O in a ratio of 1:1 (v/v) and purified via reversed-phase high-performance liquid chromatography (RP-HPLC) using semi-preparative column (Supporting Information). The characterization of PSMA-ALB-02 was performed by analytical RP-HPLC (Supporting Information) and matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) or electrospray ionization mass spectrometry (ESI-MS), respectively. The synthesis outlined above is summarized in Scheme 6.1.
(274) ##STR00070##
(275) PSMA-ALB-05 and PSMA-ALB-07. The synthesis of PSMA-ALB-05 was accomplished by coupling of the
(276) The synthesis and purification of PSMA-ALB-07 was performed in analogy to PSMA-ALB-05 with one additional coupling of a third Fmoc-
(277) ##STR00071## ##STR00072##
(278) Radiolabeling and Stability. The new PSMA ligands (PSMA-ALB-02, PSMA-ALB-05 and PSMA-ALB-07, respectively) as well as PSMA-617 (Advanced Biochemical Compounds, ABX GmbH, Radeberg, Germany) were dissolved in MilliQ water containing 10-15% sodium acetate solution (0.5 M, pH 8) to prepare 1 mM stock solutions for radiolabeling. The PSMA ligands were labeled with .sup.177Lu (no-carrier added .sup.177LuCl.sub.3 in 0.04 M HCl provided by Isotope Technologies Garching (ITG GmbH, Germany)) in a mixture of sodium acetate (0.5 M, pH 8) and HCl (0.05 M) at pH 4 at specific activities between 5-50 MBq/nmol. The reaction mixture was incubated for 10 min at 95° C. Quality control of the radioligands was performed using RP-HPLC (Supporting Information). The radioligand solution was used for in vitro and in vivo experiments without further purification steps.
(279) The stability of the radioligands was determined over time using RP-HPLC. The PSMA ligands were radiolabeled with .sup.177Lu (250 MBq) at a specific activity of 50 MBq/nmol without and with the addition of
(280) Determination of n-Octanol/PBS Distribution Coefficients (Log D Values). The distribution coefficients (log D values) of the 177Lu-labeled radioligands were determined by a shake-flask method using liquid-liquid extraction followed by phase separation as previously reported. Briefly, the PSMA ligands were radiolabeled with .sup.177Lu at a specific activity of 50 MBq/nmol. A sample of the radioligands was mixed with phosphate-buffered saline (PBS) and n-octanol followed by vigorous vortexing. After centrifugation for phase separation, the activity concentration in each layer was measured with a γ-counter (Perkin Elmer, Wallac Wizard 1480). Three experiments were performed with five replicates for each compound.
(281) Filter Assay. The binding capacity of the radioligands to mouse and human plasma proteins was determined by an ultrafiltration assay as previously described (Example 1). The .sup.177Lu-labeled PSMA ligands (50 MBq/nmol) were diluted in mouse plasma (Rockland, USA) and human plasma (Stiftung Blutspende SRK Aargau-Solothurn, Switzerland), respectively, and incubated for 15 min at room temperature. In addition, the radioligands were diluted in PBS (buffer solution without proteins) as a control experiment. Aliquots of the solutions were loaded onto an ultrafiltration device and centrifuged. The filtered activity was measured with a γ-counter and used for calculating the plasma protein-bound activity (retained on the filter membrane) as the percentage of total added activity. Three independent experiments were performed in duplicate with each radioligand (.sup.177Lu-PSMA-ALB-02, .sup.177Lu-PSMA-ALB-05 and .sup.177Lu-PSMA-ALB-07, respectively). Two additional experiments were performed in duplicate using .sup.177Lu-PSMA-617. Statistical analysis (one-way ANOVA with Bonferroni's multiple comparison post-test) was performed using GraphPad Prism software, version 7. A p-value of <0.05 was considered statistically significant.
(282) Cell Uptake and Internalization. The sum of the PSMA-bound fraction on the cell surface and the internalized fraction (referred to as cell uptake) and the internalized fraction of the radioligands were determined at a specific activity of 5 MBq/nmol using PSMA-positive PC-3 PIP and PSMA-negative PC-3 flu cells as previously described (Example 1). The radiolabeling solution was diluted in saline containing 0.05% (w/v) bovine serum albumin (BSA) to prevent adherence to laboratory vials and tubes. Further dilution of the radioligand solution with cell culture medium resulted in a final BSA concentration (0.00125%) which was negligible and had no influence on the cell uptake and internalization of the radioligands. In parallel to each experiment with a novel radioligand, control experiments with .sup.177Lu-PSMA-617 were also performed. The experiments were performed in triplicate and repeated three times for each radioligand.
(283) In Vivo Studies. In vivo experiments were performed in female, athymic BALB/c nude mice at the age of 5-6 weeks (Charles River Laboratories, Sulzfeld, Germany) were used for these studies. The mice were subcutaneously inoculated with PC-3 PIP cells (6×10.sup.6 cells in 100 μL Hank's balanced salt solution with Ca.sup.2+/Mg.sup.2+ (HBSS)) on the right shoulder and with PC-3 flu cells (5×10.sup.6 cells in 100 μL HBSS) on the left shoulder about 12-14 days before the performance of the experiments.
(284) Biodistribution Studies. Biodistribution experiments were performed at 1 h, 4 h, 24 h, 48 h, 96 h and 192 h after injection of the radioligands labeled at a specific activity of 5 MBq/nmol. The tumor mass at the time of radioligand injection was 150±40 mg, which corresponds to an average tumor volume of about 150 mm.sup.3. Mice were injected into a lateral tail vein with the respective radioligand (5 MBq, 1 nmol, 100 μL) diluted in saline. BSA (0.05%) was added to the saline in order to prevent adsorption of the radioligand to vials and syringes. The mice were sacrificed at different time points after injection (p.i.) and selected tissues and organs were collected, weighed and measured using a γ-counter. Groups of 3-6 mice were used for each time point. In addition, blocking studies were performed by injection of 2-(phosphonometyl)-pentanedioic acid (2-PMPA, 500 nmol, 100 μL) diluted in saline. The 2-PMPA solution was injected 15 min prior to the application of .sup.177Lu-PSMA-ALB-02 and the mice were sacrificed at 1 h and 4 h p.i., respectively. The results were decay-corrected and listed as percentage of the injected activity per gram of tissue mass (% IA/g). The area under the curve (AUC) was determined for all three albumin-binding PSMA ligands and .sup.177Lu-PSMA-617 from non-decay-corrected data obtained from the biodistribution data of the tumors, kidneys and blood using GraphPad Prism software, version 7.
(285) Statistical analysis was performed to compare the areas under the curve (AUCs) obtained from the biodistribution data sets using a one-way ANOVA with Bonferroni's multiple comparison post-test using GraphPad Prism software (version 7). A p-value of <0.05 was considered statistically significant.
(286) SPECT/CT Imaging Studies. SPECT/CT experiments were performed at 4 h, 24 h and 72 h after injection of the radioligands. Mice were injected into a lateral tail vein with the respective radioligand (25 MBq, 1 nmol, 100 μL) diluted in saline containing 0.05% BSA. In addition, SPECT/CT scans were performed at 1 h, 4 h and 24 h after injection of .sup.177Lu-PSMA-ALB-02 with mice that received 2-PMPA (500 nmol, 100 μL) or non-radiolabeled PSMA-ALB-02 (100 nmol, 100 μL) 15 min prior to the radioligand injection in order to block PSMA. SPECT/CT scans were performed using a small-animal SPECT/CT scanner (NanoSPECT/CT™, Mediso Medical Imaging Systems, Budapest, Hungary). The SPECT scans lasted for 45 min and were followed by a CT scan of 7.5 min. During the in vivo scans, the mice were anesthetized with a mixture of isoflurane and oxygen. Reconstruction of acquired data was performed using the software of the NanoSPECT/CT™. All images were prepared using VivoQuant post-processing software (version 3.0, inviCRO Imaging Services and Software, Boston USA). Gauss post-reconstruction filter (FWHM=1 mm) was applied to the SPECT images and the scale of radioactivity was set as indicated on the images (minimum value=0.95 Bq/voxel to maximum value=95 Bq/voxel).
(287) 6.2 Results
(288) Synthesis of the PSMA Ligands. The PSMA ligands with an albumin-binding moiety were synthesized via a solid-phase platform employing a standard Fmoc (9-fluorenylmethyloxycarbonyl) protocol (
(289) TABLE-US-00013 TABLE 6.1 Analytical Data of PSMA-ALB-02, PSMA-ALB-05, and PSMA-ALB-07 MW t.sub.r.sup.d chemical purity compound code chemical formula [g/mol] m/z.sup.a [min] [%] logD PSMA-ALB-02 C.sub.65H.sub.92IN.sub.11O.sub.16 1442.41 1443.53 6.2 99.5 −2.8 ± 0.09 PSMA-ALB-05 C.sub.73H.sub.102IN.sub.13O.sub.24 1672.59 1673.41 6.1 99.2 −3.5 ± 0.08 PSMA-ALB-07 C.sub.77H.sub.107IN.sub.14O.sub.27 1787.63 1788.63 5.9 98.5 −3.9 ± 0.25 PSMA-617 C.sub.49H.sub.71N.sub.9O.sub.16 1042.15 1043.32 4.8 98.4.sup.c −4.4 ± 0.15 .sup.aMass spectrometry of the unlabeled ligand detected as [M + H].sup.+. .sup.bRetention time of unlabeled ligand on analytical RP-HPLC. Analytical column (1.00 × 4.6 mm) utilized Chromolith RP-18e stationary phase with mobile phases consisting of 0.1% TFA in water (A) and ACN (B). For analytical runs, a linear gradient of solvent A (90-10% in 10 min) in solvent B at a flow rate of 1 mL/min was used. .sup.cThe purity of PSMA-617 was taken from the ABX GmbH certificate of this compound.
(290) Radiolabeling, Stability and In Vitro Properties of .sup.177Lu-PSMA Ligands. PSMA-ALB-02, PSMA-ALB-05 and PSMA-ALB-07 were readily labeled with .sup.177Lu at a specific activity up to 50 MBq/nmol. The radioligands showed high radiochemical purity of >98%. The addition of
(291) In Vitro Testing of Cell Uptake and Binding to Albumin. Uptake for .sup.177Lu-PSMA-ALB-02, .sup.177Lu-PSMA-ALB-05 and .sup.177Lu-PSMA-ALB-07 into PC-3 PIP cells was in the range of 52-57% whereas the internalized fraction was between 18-24% after an incubation period of 2 h at 37° C. (
(292) The results of the ultrafiltration assay indicated significant plasma protein-binding capacity of .sup.177Lu-PSMA-ALB-02, .sup.177Lu-PSMA-ALB-05 and .sup.177Lu-PSMA-ALB-07 when incubated with mouse plasma (87±1.0%, 77±2.1% and 64±2.1%, respectively) and human plasma (95±1.2%, 95±0.6 and 95±0.1%, respectively). These values were significantly higher (p<0.05) than in the case of .sup.177Lu-PSMA-617, which showed only very low binding to mouse plasma proteins (9.3±1.1%) and some binding to human plasma proteins (57±2.3%). Control experiments performed with PBS revealed <5% retention of the radioligands on the filter presumably due to unspecific adsorption to the filter device (data not shown).
(293) Biodistribution Study. The tissue distribution of .sup.177Lu-PSMA-ALB-02, .sup.177Lu-PSMA-ALB-05 and .sup.177Lu-PSMA-ALB-07 was evaluated in mice bearing PC-3 PIP and PC-3 flu tumors on the right and left shoulder, respectively, over a period of 192 h (
(294) Uptake of all PSMA radioligands into the PC-3 PIP tumors showed similar kinetic profiles. .sup.177Lu-PSMA-ALB-02 showed a fast tumor accumulation which reached 78.4 12.8% IA/g already at 4 h p.i. and was retained at this level over 24 h p.i. (76.4±2.49% IA/g). All novel compounds, in particular .sup.177Lu-PSMA-ALB-02, exhibited high blood activity levels (18-21% IA/g), fast clearance of radioactivity from the blood and fast renal clearance. .sup.177Lu-PSMA-617 reached the maximum tumor uptake of ˜56% IA/g already at 4 h p.i which decreased to ˜20% IA/g after 192 h. It was cleared quickly from the blood resulting in <1% IA/g after 1 h and showed a steady wash-out from the kidneys from ˜10% IA/g at 1 h p.i. to <1% IA/g at 24 h p.i. Radioactivity levels in all other tissues were below the blood levels and decreased continuously over time.
(295) Tumor-to-blood, tumor-to-kidney and tumor-to-liver ratios were high for all novel compounds, in particular .sup.177Lu-PSMA-ALB-02. Due to the fast renal clearance, .sup.177Lu-PSMA-617 showed increased tumor-to-background ratios.
(296) TABLE-US-00014 TABLE 6.2 Tumor-to-Background ratios at 24 and 48 h after Injection .sup.177Lu-PSMA-ALB-02 .sup.177Lu-PSMA-ALB-05 .sup.177Lu-PSMA-ALB-07 .sup.177Lu-PSMA-617 24 h p.i. 48 h p.i. 24 h p.i. 48 h p.i. 24 h p.i. 48 h p.i. 24 h p.i. 48 h p.i. tumor-to-blood 176 ± 27 191 ± 37 48 ± 6.4 38 ± 2.5 107 ± 10 154 ± 7 2730 ± 195 3776 ± 585 tumor-to-kidney 7.2 ± 0.3 8.3 ± 0.5 3.4 ± 0.5 5.0 ± 0.7 1.6 ± 0.2 2.3 ± 0.1 .sup. 49 ± 3.7 81 ± 11 tumor-to-liver 164 ± 20 163 ± 32 106 ± 26 100 ± 32 162 ± 14 131 ± 13 528 ± 51 710 ± 97
(297) Additional studies were performed in order to block PSMA by administration of 2-PMPA prior to the injection of .sup.177Lu-PSMA-ALB-02. In PC-3 PIP tumors the uptake was reduced by 64% (17.6±3.24% IA/g) and 41% (46.0±7.29% IA/g) at 1 h and 4 h p.i. respectively, when compared to unblocked uptake at the same time points. The accumulated radioactivity in the kidneys was reduced by 81% and 59% at 1 h and 4 h after radioligand injection, respectively. In all other organs and tissues slight, but not pronounced reduction of radioactivity accumulation was observed (data not shown).
(298) Non-decay-corrected data of the biodistribution study were used to calculate the areas under the curves (AUCs) for the accumulation of the radioligands in the blood pool, tumors, kidneys and the liver (
(299) TABLE-US-00015 TABLE 6.3 Area under the Curve (AUC) Based on Non-Decay-Corrected, Time-Dependent Biodistribution Data of .sup.177Lu-PSMA-ALB-02, -05 and -07 and Ratios of AUCs .sup.177Lu-PSMA- .sup.177Lu-PSMA- .sup.177Lu-PSMA- .sup.177Lu-PSMA- ALB-02 ALB-05 ALB-07 617 AUC [% IA/g .Math. h] PC-3 PIP tumor 6688 ± 485 6741 ± 421 7007 ± 459 3691 ± 156 blood 145 ± 6.3 387 ± 32 180 ± 7.2 52 ± 1.5 kidneys 1130 ± 62 1837 ± 112 3395 ± 201 99 ± 11 liver 57 ± 5.3 131 ± 11 72 ± 3.6 6.2 ± 1.6 Ratios of AUCs AUC.sub.Tu-to-AUC.sub.Bl 46 17 39 71 AUC-.sub.Tu-to-AUC-.sub.Ki 5.9 3.7 2.1 37 AUC.sub.Tu-to-AUC.sub.Li 117 52 97 592
(300) All novel radioligands showed comparable AUCs for the PC-3 PIP tumor uptake which were almost double as high as the AUC (p<0.05) obtained for .sup.177Lu-PSMA-617. All radioligands showed high tumor-to-blood, tumor-to-kidney and tumor-to-liver ratios of AUCs. The high tumor-to-background values of AUCs were obtained for .sup.177Lu-PSMA-617 are due to the fast blood and kidney clearance of this radioligand (Table 6.2).
(301) SPECT/CT Imaging Studies. SPECT/CT scans were performed with PC-3 PIP/flu tumor-bearing mice at 4 h, 24 h and 72 h after injection of the new radioligands as well as .sup.177Lu-PSMA-617 (