PROSTATE SPECIFIC MEMBRANE ANTIGEN (PSMA) LIGANDS WITH IMPROVED RENAL CLEARANCE
20250303005 · 2025-10-02
Assignee
- Deutsches Krebsforschungszentrum Stiftung des Öffentlichen Rechts (Heidelberg, DE)
- Albert-Ludwigs-Universität Freiburg (Freiburg, DE)
Inventors
Cpc classification
A61K51/0497
HUMAN NECESSITIES
International classification
Abstract
The present invention generally relates to the field of radiopharmaceuticals and their use in nuclear medicine as tracers, imaging agents and for the treatment of various disease states of PSMA-expressing cancers, especially prostate cancer, and metastases thereof. In particular, the present invention relates to a PSMA binding ligand or a pharmaceutically acceptable salt or solvate thereof comprising a PSMA binding motif Q and a chelator residue A linked via at least one linker L.sup.AQ, the linker comprising at least one N-alkylated, preferably N-methylated amino acid.
Claims
1-17. (canceled)
18. A PSMA binding ligand or a pharmaceutically acceptable salt or solvate thereof comprising: (a) a PSMA binding motif Q; and (b) a chelator residue A linked via at least one linker L.sup.AQ comprising at least one N-alkylated, preferably N-methylated, amino acid X.sub.1, preferably at least one amino acid having the structure X.sub.1, wherein X.sub.1 is N(CH.sub.3)CH.sub.2C(O), more preferably the linker L.sup.AQ comprises the linking unit (X.sub.1).sub.n.sub.
19. The PSMA binding ligand according to claim 18 or a pharmaceutically acceptable salt or solvate thereof, the PSMA binding ligand having the structure (I):
A-L.sup.AQ-Q(I)
20. The PSMA binding ligand according to claim 18 or a pharmaceutically acceptable salt or solvate thereof, the PSMA binding motif Q having the structure: ##STR00050## wherein R.sup.1 is H or CH.sub.3, preferably H, wherein R.sup.2, R.sup.3 and R.sup.4 are independently of each other, selected from the group consisting of CO.sub.2H, SO.sub.2H, SO.sub.3H, OSO.sub.3H, PO.sub.2H, PO.sub.3H and OPO.sub.3H.sub.2.
21. The PSMA binding ligand according to claim 18 or a pharmaceutically acceptable salt or solvate thereof, wherein A is a chelator residue derived from a chelator selected from the group consisting of 1,4,7,10-tetraazacyclododecane-N,N,N,N-tetraacetic acid (=DOTA), N,N-bis[2-hydroxy-5-(carboxyethyl)benzyl]ethylenediamine-N,N-diacetic acid, 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 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-arninopentyl)(hydroxy)amino]-4-oxobutanoyl}amino)pentyl]-N-hydroxysuccinamide (DFO), Diethylenetriaminepentaacetic acid (DTPA), Trans-cyclohexyl-diethylenetriaminepentaacetic acid (CHX-DTPA), 1-oxa-4,7,10-triazacyclododecane-4,7,10-triacetic acid (oxo-Do3A) p-isothiocyanatobenzyl-DTPA (SCN-Bz-DTPA), 1-(p-isothiocyanatobenzyl)-3-methyl-DTPA (1 B3M), 2-(p-isothiocyanatobenzyl)-4-methyl-DTPA (1 M3B) and 1-(2)-methyl-4-isocyanatobenzyl-DTPA (MX-DTPA).
22. The PSMA binding ligand according claim 18 or a pharmaceutically acceptable salt or solvate thereof, wherein A is a chelator residue having a structure selected from the group consisting of: ##STR00051## wherein A preferably has the structure ##STR00052##
23. The PSMA binding ligand according to claim 18 or a pharmaceutically acceptable salt or solvate thereof, wherein the linker L.sup.AQ comprises at least one amino acid building block AS.sup.a, wherein AS.sup.a has the structure: ##STR00053## wherein Q.sup.1 is selected from the group consisting of alkylaryl, arylalkyl, aryl, alkylheteroaryl, heteroarylalkyl and heteroaryl.
24. The PSMA binding ligand according to claim 18 or a pharmaceutically acceptable salt or solvate thereof, wherein the linker L.sup.AQ comprises at least one amino acid building block AS.sup.b, wherein AS.sup.b has the structure (b): ##STR00054## wherein Q.sup.2 is selected from the group consisting of aryl, alkylaryl, arylalkyl, cycloalkyl, heterocycloalkyl, heteroaryl, heteroarylalkyl and alkylheteroaryl, preferably wherein Q.sup.2 is ##STR00055## more preferably ##STR00056##
25. The PSMA binding ligand according claim 18 or a pharmaceutically acceptable salt or solvate thereof, the PSMA binding ligand having the structure (Ia): ##STR00057## wherein R.sup.1 is H or CH.sub.3, preferably H, wherein R.sup.2, R.sup.3 and R.sup.4 are independently of each other, selected from the group consisting of CO.sub.2H, SO.sub.2H, SO.sub.3H, OSO.sub.3H, PO.sub.2H, PO.sub.3H and OPO.sub.3H.sub.2, Q.sup.1 is selected from the group consisting of alkylaryl, arylalkyl, aryl, alkylheteroaryl, heteroarylalkyl and heteroaryl, Q.sup.2 is selected from the group consisting of aryl, alkylaryl, arylalkyl, cycloalkyl, heterocycloalkyl, heteroaryl, heteroarylalkyl and alkylheteroaryl, and wherein q is an integer of from 0-3.
26. The PSMA binding ligand according to claim 18, the ligand having the structure: ##STR00058## wherein A is a chelator residue having the structure ##STR00059## Q2 is ##STR00060## more preferably ##STR00061## q is 1 Q.sup.1 is ##STR00062## and wherein R.sup.3, R.sup.2 and R.sup.4 are CO.sub.2H and R.sup.1 is H and wherein n1 is preferably in the range of from 1 to 25, preferably 2 to 25, more preferably 3 to 15.
27. A complex comprising: (a) a radionuclide, and (b) the PSMA binding ligand of claim 18 or a pharmaceutically acceptable salt or solvate thereof.
28. The complex of claim 27, wherein, the radionuclide is selected from the group consisting of .sup.89Zr, .sup.44Sc, .sup.111In, .sup.90Y, .sup.66Ga, .sup.67Ga, .sup.68Ga, .sup.177Lu, .sup.99mTc, .sup.60Cu, .sup.61Cu, .sup.62Cu, .sup.64Cu, .sup.66Cu, .sup.67Cu, .sup.149Tb, .sup.152Tb, .sup.155Tb, .sup.153Sm, .sup.161Tb, .sup.153Gd, .sup.155Gd, .sup.157Gd, .sup.213Bi, .sup.225Ac, .sup.230U, .sup.223Ra, .sup.165Er, .sup.52Fe, .sup.59Fe, and radionuclides of Pb (such as .sup.203Pb and .sup.212Pb, .sup.211Pb, .sup.213Pb, .sup.214Pb, .sup.209P, .sup.198Pb, .sup.197Pb), more preferably selected from the group consisting .sup.90Y, .sup.68Ga, .sup.177Lu, .sup.225Ac, and .sup.213Bi, more preferably, the radionuclide is .sup.177Lu or .sup.225Ac.
29. A pharmaceutical composition comprising the PSMA binding ligand of claim 18.
30. A method for treating and/or preventing PSMA expressing cancer, in particular prostate cancer and/or metastases thereof, comprising administering to a subject in need the PSMA binding ligand of claim 18.
31. A method for diagnosing PSMA expressing cancer and/or metastases thereof, preferably of PSMA expressing cancer, in particular of prostate cancer and/or metastases thereof, comprising using a diagnostic method comprising the PSMA binding ligand of claim 18.
32. A method for treating and/or preventing PSMA expressing cancer, in particular prostate cancer and/or metastases thereof, comprising administering to a subject in need the complex of claim 27.
33. A method for diagnosing PSMA expressing cancer and/or metastases thereof, preferably of PSMA expressing cancer, in particular of prostate cancer and/or metastases thereof, comprising using a diagnostic method comprising the complex of claim 27.
34. The method of claim 32, wherein the radionuclide is a -emitter, more preferably .sup.177Lu and wherein preferably the activity dosage of the complex is at least 100 kBq/kg body weight, more preferably at least 500 kBq/kg body weight, most preferably at least 1 MBq/kg body weight.
35. The method of claim 33, wherein the radionuclide is an -emitter, more preferably is .sup.225Ac and wherein preferably the activity dosage of the complex is preferably at least 75 kBq/kg body, more preferably at least 100 kBq/kg body, weight.
36. A pharmaceutical composition comprising the complex of claim 27.
Description
FIGURES
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EXAMPLES
[0194] All commercially available chemicals were of analytical grade and used without further purification. .sup.68Ga (half-life 68 min) was obtained from a .sup.68Ge/.sup.68Ga generator (Galliapharm Ge-68/Ga-68 Generator, Eckert & Ziegler) and .sup.177Lu (half-life 6.6 d) was purchased from ITG. The compounds were purified using semipreparative reversed-phase high performance liquid chromatography (RP-HPLC; Chromolith Semi Prep RP-18e, 10010 mm; Merck, Darmstadt, Germany). Compound analysis was performed using analytical RP-HPLC (RP-HPLC; Chromolith RP-18e, 1004.6 mm; Merck, Darmstadt, Germany). Analytical HPLC runs were performed using a linear gradient (5% A (0.1% aqueous TFA) to 100% B (0.1% TFA in CH.sub.3CN)) in 10 min at 2 mL/min. The system Agilent Technologies 1200 Series was equipped with a variable UV and a gamma detector (Ramona*, Elysia). UV absorbance was measured at 220 and 280 nm, respectively. For mass spectrometry a MALDI-MS (Daltonics Microflex, Bruker Daltonics, Bremen, Germany) was used.
Synthesis of DOTA-Sar.SUB.3.-Chx-2-NaI-Lys-urea-Glu, DOTA-Sar.SUB.5.-Chx-2-NaI-Lys-urea-Glu, DOTA-Sar.SUB.10.-Chx-2-NaI-Lys-urea-Glu and DOTA-Sar.SUB.15.-Chx-2-NaI-Lys-urea-Glu
[0195] The synthesis of the pharmacophore Glu-urea-Lys was performed as described previously (1). Briefly, the synthesis started with the formation of the isocyanate of the glutamyl moiety using triphosgene. A resin-immobilized (2-chloro-trityl resin, Merck, Darmstadt) c-allyloxycarbonyl protected lysine was added and reacted for 16 h with gentle agitation. The resin was filtered off and the allyloxy-protecting group was removed by reacting twice with Pd(PPh.sub.3).sub.4 (0.3 eq.) and morpholine (15 eq.) under ambient conditions (1 h, RT).
[0196] Subsequently, the linker between the PSMA pharmacophore and the chelator was introduced by standard Fmoc solid phase protocol. In a first step Fmoc-2-NaIOH and N-Fmoc-tranexamic acid (4 eq. each) with HATU (4 eq.) and DIPEA (10 eq.) were coupled in DMF. Depending on the amino acid sequence Fmoc-sarcosine was coupled three, five, ten and fifteen-times, respectively, with HATU (4 eq.) and DIPEA (10 eq.) in DMF. Afterwards, bis(tBu)DOTA (bis(tBu)-ester of 1,4,7,10-tetraazacyclododecan-1,4,7,10-tetraacetic acid)(4 eq.) with HATU (4 eq.) and DIPEA (10 eq.) were coupled in DMF. The products were cleaved from the resin for 3 hours at RT using TFA/TIPS/H.sub.2O (95/2.5/2.5, v/v/v) and purified using RP-HPLC using a Chromolith RP-18e column (10010 mm; Merck, Darmstadt, Germany) and identified with mass spectrometry.
TABLE-US-00002 TABLE 1 Analytical data of the final compounds. Mass spectrometry (MALDI-MS) was performed with the metal-free substances. m/z m/z (g/mol, M.sub.calc.) (M.sub.found.) DOTA-Sar.sub.3-Chx-2-NaI-Lys-urea-Glu 1255 1255 DOTA-Sar.sub.5-Chx-2-NaI-Lys-urea-Glu 1397 1396 DOTA-Sar.sub.10-Chx-2-NaI-Lys-urea-Glu 1752 1752 DOTA-Sar.sub.15-Chx-2-NaI-Lys-urea-Glu 2107 2109
.SUP.68.Ga-Labeling
[0197] The precursor peptide [2 nmol in HEPES buffer (1 M, pH 4, 40 L)] was added to 40 L [.sup.68Ga]Ga.sup.3+ eluate (40 MBq). The pH was adjusted to 3.8-4.2 using 30% NaOH. The reaction mixture was incubated at 95 C. for 15 minutes. The radiochemical yield (RCY) was determined by RP-HPLC.
.SUP.177.Lu-Labeling
[0198] The precursor peptide [2 nmol in HEPES buffer (0.1 M, pH 7, 50 L)] was added to 10 L [.sup.177Lu]LuCl.sub.3 (10-30 MBq, 0.04 M HCl). The reaction mixture was incubated at 95 C. for 15 minutes. The radiochemical yield (RCY) was determined by RP-HPLC.
Serum Stability
[0199] .sup.177Lu-labeled compounds (10 L) were incubated in 100 L human (BIOIVT (BRH1548721)) or mouse plasma (100 L, BIOIVT (MSE298415)) at 37 C. At different time points of incubation (t=6 h, 24 h, 48 h, 72 h) aliquots of 10 L were taken and plasma proteins precipitated in 30 L acetonitrile. Samples were centrifuged for 5 min at 13000 rpm and the supernatants analyzed by analytical RP-HPLC.
Cell Culture
[0200] PSMA+LNCaP cells (CRL-1740; ATCC; PSMA-positive) and PC-3 cells (CRL-1435; ATCC; PSMA-negative) were cultured in RPMI medium supplemented with 10% fetal calf serum and 2 mmol/L L-glutamine (all from PAA). Cells were grown at 37 C. in humidified air with 5% CO.sub.2 and were harvested using trypsin-ethylenediaminetetraacetic acid (trypsin-EDTA; 0.25% trypsin, 0.02% EDTA, Invitrogen).
Cell Binding and Internalization
[0201] The competitive cell binding assay and internalization experiments were performed as described previously (2). For competitive cell binding, the cells (10.sup.5 per well) were incubated with a 0.8 nM solution of .sup.68Ga-labeled radioligand [Glu-urea-Lys(Ahx)].sub.2-HBED-CC (PSMA-10, precursor ordered from ABX, Radeberg, Germany) in the presence of 12 different concentrations of DOTA-compound (0-5000 nM, 100 L/well). After incubation, the mixture was removed and the wells were washed 3 times with PBS using a multiscreen vacuum manifold (Millipore, Billerica, MA). Cell-bound radioactivity was measured using a gamma counter (Perkin Elmer 2480, Wizard, Gamma Counter). The 50% inhibitory concentration (IC50) values were calculated by fitting the data using a nonlinear regression algorithm (GraphPad Software) (see Table 2)
[0202] For internalization experiments, 10.sup.5 cells per well were seeded in poly-L-lysine coated 24-well cell culture plates 24 h before incubation. After washing, the cells were incubated with 30 nM of the radiolabeled DOTA-compound (radiolabeled with .sup.68Ga or .sup.177Lu) for 45 min at 37 C. (labeling was performed with 5 nmol of precursor peptide). Specific cell uptake was determined by blockage using 500 M 2-PMPA (2-(phosphonomethyl)pentanedioic acid). Cellular uptake was terminated by washing 3 times with 1 mL of ice-cold PBS. To remove surface-bound radioactivity, cells were incubated twice with 0.5 mL glycine-HCl in PBS (50 mM, pH=2.8) for 5 min. The cells were washed with 1 mL of ice-cold PBS and lysed using 0.3 N NaOH (0.5 mL). The surface-bound and the internalized fractions were measured in a gamma counter. The cell uptake was calculated as percent of the initially added radioactivity bound to 10.sup.5 cells [% ID/10.sup.5 cells] (see Table 2 and Table 2a).
PET/MR Imaging
[0203] For the experimental tumor models 510.sup.6 cells of LNCaP (in 50% Matrigel; Becton Dickinson) were subcutaneously inoculated into the right trunk of 7- to 8-week-old male BALB/c nu/nu mice (Janvier). The tumors were allowed to grow until approximately 1 cm.sup.3 in size. For imaging studies, mice were anesthetized (2% isoflurane) and 0.5 nmol of the .sup.68Ga-labeled compound in 0.9% NaCl (pH 7) were injected into the tail vein. PET imaging was performed with PET/MRI scanner (BioSpec 3T, Bruker) with a dynamic scan for 60 min. The images were iteratively reconstructed (MLEM 0.5 algorithm, 12 iterations) and were converted to SUV images. Quantification was done using a ROI (region of interest) technique and data in expressed in time activity curves as SUV.sub.bodyweight. All animal experiments complied with the current laws of the Federal Republic of Germany.
Organ Distribution
[0204] For the experimental tumor models 510.sup.6 cells of LNCaP (in 50% Matrigel; Becton Dickinson) were subcutaneously inoculated into the right trunk of 7- to 8-week-old male BALB/c nu/nu mice (Janvier). The tumors were allowed to grow until approximately 1 cm.sup.3 in size. For the biodistribution studies, mice were anaesthetized with isoflurane and .sup.177Lu-labeled compounds were injected into a tail vein (1-2 MBq; 60 pmol in 100 l). Mice were sacrificed after 1 h, 2 h, 6 h and 24 h p.i. The organs of the mice were measured with standards in a -gamma counter (2480 Automatic Gamma Counter Wizard, PerkinElmer, Waltham, USA).
[0205] The results are shown in
Statistical Aspects
All experiments were performed at least in triplicate and repeated at least for three times. Quantitative data were expressed as meanSD. If applicable, means were compared using Student's t test. P values <0.05 were considered statistically significant.
Results
In Vitro Characterization
[0206] The final product was identified using reversed-phase HPLC/matrix-assisted laser desorption/ionization mass spectrometry. The .sup.68Ga and .sup.177Lu complexation of the compounds resulted in radiochemical yields higher than 99%. All compounds were found to be stable up to 72 h in human and mouse plasma. Furthermore, all compounds showed a high PSMA-binding affinity in the same nanomolar range as the reference PSMA-617 (Table 2) (3). A PSMA-specific cell surface binding and specific internalization comparable to PSMA-617 was also detected for all .sup.68Ga-labeled compounds.
TABLE-US-00003 TABLE 2 Cell binding and internalization data of .sup.68Ga-labeled compounds*. Affinity to PSMA and internalization properties of the .sup.68Ga-labeled compounds were determined in vitro using PSMA.sup.+-cells (LNCaP). For all compounds PSMA-specific internalization and a not significantly complexation-dependent high binding affinity to PSMA in the low nanomolar range were detected. Specifically cell Specifically surface bound internalized k.sub.i [nM].sup. Compound [% AR/10.sup.5 cells].sup. [% AR/10.sup.5 cells].sup. free ligands DOTA-Sar.sub.3-Chx-2-NaI-Lys-urea-Glu 4.42 2.05 2.20 0.98 29.73 24.15 DOTA-Sar.sub.5-Chx-2-NaI-Lys-urea-Glu 2.79 0.50 1.99 1.15 54.28 33.64 DOTA-Sar.sub.10-Chx-2-NaI-Lys-urea-Glu 3.66 1.31 2.31 0.90 96.85 96.39 DOTA-Sar.sub.15-Chx-2-NaI-Lys-urea-Glu 3.71 1.55 2.03 1.71 74.72 35.96 PSMA-617 3.47 1.53 2.55 1.97 2.3 2.9 *Data are expressed as mean SD (n = 3), .sup.68Ga-labeled compounds; k.sub.i-value for PSMA-617 from Benesova et al. (3). Specific cell uptake was determined by blockage using 500 M 2-PMPA. Values are expressed as % of applied radioactivity (AR) bound to 10.sup.5 cells. .sup.radioligand: .sup.68Ga-PSMA-10 (K.sub.d: 3.8 1.8 nM (1), c.sub.radioligand: 0.8 nM)
TABLE-US-00004 TABLE 2a Cell surface binding and internalization (without or with blocking by 2-PMPA) of the .sup.177Lu labeled compounds (7.5 pmol precursor per 10.sup.5 cells) after 45 min (n = 3). Surface Internalization Surface* Block* Internalization* Block* [% AR/10.sup.5 [% AR/10.sup.5 [% AR/10.sup.5 [% AR/10.sup.5 cells] cells] cells] cells] DOTA-Sar.sub.3-Chx-2-NaI- 0.65 0.15 0.03 0.01 0.13 0.08 0.01 0.01 Lys-urea-Glu DOTA-Sar.sub.5-Chx-2-NaI- 0.47 0.12 0.02 0.01 0.10 0.06 0.00 0.00 Lys-urea-Glu DOTA-Sar.sub.10-Chx-2-NaI- 0.45 0.08 0.03 0.02 0.10 0.06 0.00 0.00 Lys-urea-Glu DOTA-Sar.sub.15-Chx-2-NaI- 0.52 0.03 0.02 0.01 0.11 0.05 0.00 0.00 Lys-urea-Glu *Specific cell uptake was determined by blockage using 500 M 2-PMPA. Values are expressed as % of applied radioactivity (AR) bound to 10.sup.5 cells.
[0207] Results are shown in
In Vivo Characterization
[0208] The pharmacokinetic properties of the novel compounds were analyzed using PET/MR imaging in LNCaP-xenograft bearing mice (