PSMA BINDING DUAL MODE RADIOTRACER AND THERAPEUTIC
20220096668 · 2022-03-31
Inventors
- Alexander Josef Wurzer (München, DE)
- Hans-Jürgen Wester (Ilmmünster, DE)
- Matthias Johannes Eiber (Vaterstetten, DE)
Cpc classification
C07B2200/05
CHEMISTRY; METALLURGY
A61K51/0497
HUMAN NECESSITIES
A61K51/0406
HUMAN NECESSITIES
International classification
A61P35/00
HUMAN NECESSITIES
Abstract
The present invention relates to a compound according to formula (V): or a pharmaceutically acceptable salt thereof, optionally containing a chelated radioactive cation and wherein F is optionally .sup.18F.
##STR00001##
Claims
1. The compound according to formula (V): ##STR00023## or a pharmaceutically acceptable salt thereof, containing either a chelated radioactive cation or wherein F is optionally .sup.18F.
2. The compound of claim 1 according to formula (Va): ##STR00024## or a pharmaceutically acceptable salt thereof, wherein; each X is independently OH or O.sup.−; M is a chelated radioactive cation or is absent; and F is optionally .sup.18F
3. The compound of claim 1 or claim 2, wherein the chelated radioactive cation is selected from the cations of Sc, Cu, Ga, Y, In, Tb, Ho, Lu, Re, Pb, Bi, Ac, Er and Th.
4. The compound of claim 3, wherein the chelated radioactive cation is Ga.
5. The compound according to claim 3, wherein the chelated radioactive cation is Lu-177, Y-90, or Ac-225.
6. A method of producing the compound of claim 1, comprising the steps of: a) reacting a compound of formula (I): ##STR00025## with a compound of formula (II): ##STR00026## to form a compound of formula (III): ##STR00027## wherein PG.sub.1 is tBu, PG.sub.2 is Fmoc and PG.sub.3 is Dde; and the reaction conditions involve the use of a base, wherein the base is 2,4,6-collidine or 2,6-dimethylpyridine; b) reacting the compound of formula (III) under conditions suitable for forming a compound of formula (IV): ##STR00028## and c) reacting the compound of formula (IV) under conditions suitable for forming compound (V): ##STR00029##
7. The method according to claim 6, wherein compound (II) is preactivated by reaction with 2-(1H-Benzotriazole-1-yl)-1,1,3,3-tetramethylaminium tetrafluoroborate (TBTU), 1-Hydroxy-7-azabenzotriazole (HOAt) and 2,4,6-collidine prior to reaction with compound (I).
8. The method of claim 7, wherein the preactivation takes place for 5 minutes or less.
9. The compound according to claim 1 or claim 2 of formula (VI) ##STR00030## or a pharmaceutically acceptable salt thereof.
10. The compound according to claim 1 or claim 2 of formula (VII) ##STR00031## or a pharmaceutically acceptable salt thereof.
11. A pharmaceutical or diagnostic composition comprising the compound according to any one preceding claim.
12. A conjugate, compound or composition according to any one preceding claim for use as a cancer diagnostic or imaging agent.
13. A method of imaging and/or diagnosing cancer comprising administering a conjugate, compound or composition according to any one preceding claim to a patient in need thereof.
14. A conjugate, compound or composition according to any one preceding claim for use in the treatment of cancer.
15. A conjugate, compound or composition according to any one preceding claim for the diagnosis, imaging or prevention of neoangiogenesis/angiogenesis.
16. A conjugate, compound or composition according to any one preceding claim for use as a cancer diagnostic or imaging agent or for use in the treatment of cancer wherein the cancer is prostate, breast, lung, colorectal or renal cell carcinoma.
Description
[0093] The Figures illustrate:
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[0122] Right: Radio-HPLC analysis of ‘cold’ urine spiked with the respective [F-18]rhPSAM-7.x tracer for a period of 1 h (7.1., 7.2.), 0.5 h (7.3.) and 2 h (7.4.). HPLC-conditions: Solvent A: H2O+0.1% TFA; Solvent B: MeCN+0.1% TFA; Gradient: 5% isocratic 0-3 min, 25-35% B 3-43 min, 95-95% B 43-48 min; flow: 1 mL/min, column: Nucleosil 100-5 C18, 125×4.6 mm.
[0123]
[0124] Top: Radio-HPLC analysis of urine 30 min p.i. of [F-18]rhPSMA7.3 in mice showing a small proportion at 1.6 min and intact tracer at ca. 34.5 min. [0125] Bottom (left): urine of mice, 30 min p.i. of [F-18]rhPSMA7.3, was diluted and subjected to STRATA-X cartridge fixation. The cartridge was washed and eluted with MeCN/water (60/40 v/v+1% TFA); only intact tracer was detected. [0126] Bottom (right): both the breakthrough from the cartridge fixation (non-retained components) and the fraction finally eluted MeCN/water from the cartridge were analysed by TLC (bottom, right). Whereas 96.1% [F-18]rhPSMA7.3 and only 3.9% [F-18]fluoride were found in the eluate of the cartridge, the reverse ratio was found in the breakthrough of the cartridge (3.4% [F-18]rhPSMA7.3 and only 96.6% [F-18]fluoride).
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[0134] The Examples illustrate the invention.
Example 1: Material and Methods
[0135] The Fmoc-(9-fluorenylmethoxycarbonyl-) and all other protected amino acid analogs were purchased from Bachem (Bubendorf, Switzerland) or Iris Biotech (Marktredwitz, Germany). The tritylchloride polystyrene (TCP) resin was obtained from PepChem (Tübingen, Germany). Chematech (Dijon, France) delivered the chelators DOTAGA-anhydride, (R)-DOTA-GA(tBu).sub.4 and (S)-DOTA-GA(tBu).sub.4. All necessary solvents and other organic reagents were purchased from either, Alfa Aesar (Karlsruhe, Germany), Sigma-Aldrich (Munich, Germany) or VWR (Darmstadt, Germany). Solid phase synthesis of the peptides was carried out by manual operation using an Intelli-Mixer syringe shaker (Neolab, Heidelberg, Germany). Analytical and preparative reversed-phase high pressure chromatography (RP-HPLC) were performed using Shimadzu gradient systems (Shimadzu Deutschland GmbH, Neufahrn, Germany), each equipped with a SPD-20A UV/Vis detector (220 nm, 254 nm). A Nucleosil 100 C18 (125×4.6 mm, 5 μm particle size) column (CS GmbH, Langerwehe, Germany) was used for analytical measurements at a flow rate of 1 mL/min. Both specific gradients and the corresponding retention times t.sub.R are cited in the text. Preparative HPLC purification was done with a Multospher 100 RP 18 (250×10 mm, 5 μm particle size) column (CS GmbH, Langerwehe, Germany) at a constant flow rate of 5 mL/min. Analytical and preparative radio RP-HPLC was performed using a Nucleosil 100 C18 (5 μm, 125×4.0 mm) column (CS GmbH, Langerwehe, Germany). Eluents for all HPLC operations were water (solvent A) and acetonitrile (solvent B), both containing 0.1% trifluoroacetic acid. Electrospray ionization-mass spectra for characterization of the substances were acquired on an expression.sup.L CMS mass spectrometer (Advion Ltd., Harlow, UK). NMR spectra were recorded on Bruker AVHD-300 or AVHD-400 spectrometers at 300 K. pH values were measured with a Seven Easy pH-meter (Mettler Toledo, Gießen, Germany).
[0136] Synthesis Protocols
[0137] 1) Solid-Phase Peptide Synthesis Following the Fmoc-Strategy
[0138] TCP-Resin Loading (GP1)
[0139] Loading of the tritylchloride polystyrene (TCP) resin with a Fmoc-protected amino acid (AA) was carried out by stirring a solution of the TCP-resin (1.95 mmol/g) and Fmoc-AA-OH (1.5 eq.) in anhydrous DCM with DIPEA (4.5 eq.) at room temperature for 2 h. Remaining tritylchloride was capped by the addition of methanol (2 mL/g resin) for 15 min. Subsequently the resin was filtered and washed with DCM (2×5 mL/g resin), DMF (2×5 mL/g resin), methanol (5 mL/g resin) and dried in vacuo. Final loading/of Fmoc-AA-OH was determined by the following equation:
[0140] On-Resin Amide Bond Formation (GP2)
[0141] For conjugation of a building block to the resin bound peptide, a mixture of TBTU and HOBT is used for pre-activation with DIPEA or 2,4,6-trimethylpyridine as a base in DMF (10 mL/g resin) for 5 min. The exact stoichiometry and reaction time for each conjugation step is given in the synthesis protocol. After reaction, the resin was washed with DMF (6×5 mL/g resin).
[0142] On-Resin Fmoc-Deprotection (GP3)
[0143] The resin-bound Fmoc-peptide was treated with 20% piperidine in DMF (v/v, 8 mL/g resin) for 5 min and subsequently for 15 min. Afterwards, the resin was washed thoroughly with DMF (8×5 mL/g resin).
[0144] On-Resin Dde-Deprotection (GP4) The Dde-protected peptide (1.0 eq.) was dissolved in a solution of 2% hydrazine monohydrate in DMF (v/v, 5 mL/g resin) and shaken for 20 min (GP4a). In the case of present Fmoc-groups, Dde-deprotection was performed by adding a solution of imidazole (0.92 g/g resin), hydroxylamine hydrochloride (1.26 g/g resin) in NMP (5.0 mL) and DMF (1.0 mL) for 3 h at room temperature (GP4b). After deprotection the resin was washed with DMF (8×5 mL/g resin).
[0145] Peptide Cleavage from the Resin with Simultaneous Deprotection of Acid Labile Protecting Groups (GP 5)
[0146] The fully protected resin-bound peptide was dissolved in a mixture of TFA/TIPS/water (v/v/v; 95/2.5/2.5) and shaken for 30 min. The solution was filtered off and the resin was treated in the same way for another 30 min. Both filtrates were combined, stirred for additional 5 h and concentrated under a stream of nitrogen. After dissolving the residue in a mixture of tert-butanol and water and subsequent lyophilisation the crude peptide was obtained.
[0147] .sup.natGa-Complexation (GP6)
[0148] For .sup.natGa-complexation, the peptide (1.0 eq.) was dissolved in a 3:1 (v/v) mixture of tBuOH in H.sub.2O and an aqueous solution of Ga(NO.sub.3).sub.3 (3.5 eq.) was added. After heating the resulting mixture for 30 min at 75° C. the peptide was purified by RP-HPLC.
[0149] 2) Synthesis of the PSMA Binding Motif
[0150] Glu-urea-Glu ((tBuO)EuE(OtBu)2)
##STR00014##
[0151] The tBu-protected Glu-urea-Glu binding motif (EuE) was synthesized according to a previously published procedure (scheme 1) for tBu-protected Glu-urea-Lys (EuK).
[0152] Di-tert-butyl (1H-imidazole-1-carbonyl)-L-glutamate (i)
[0153] A solution of DCM containing 2.0 g (7.71 mmol, 1.0 eq.) l-di-tert-butyl-L-glutamate.HCl was cooled on ice for 30 min and afterwards treated with 2.69 mL TEA (19.28 mmol, 2.5 eq.) and 3.3 mg (0.3 mmol, 0.04 eq.) DMAP. After additional stirring for 5 min, 1.38 g (8.84 mmol, 1.1 eq.) of 1,1′-carbonyldiimidazole (CDI) dissolved in DCM were slowly added over a period of 30 min. The reaction mixture was further stirred overnight and enabled to warm to RT. The reaction was stopped using 8 mL saturated NaHCO.sub.3 with concomitant washing steps of water (2×) and brine (2×) and dried over Na.sub.2SO.sub.4. The remaining solvent was removed in vacuo and the crude product (S)-Di-tert-butyl 2-(1H-imidazole-1-carboxamido)pentanedioate (i) was used without further purification.
5-benzyl 1-(tert-butyl) (((S)-1,5-di-tert-butoxy-1,5-dioxopentan-2-yl)carbamoyl)-L-glutamate (ii)
[0154] 2.72 g (7.71 mmol, 1.0 eq.) of the crude product (S)-Di-tert-butyl-2-(1H-imidazole-1-carboxamido) pentanedioate (i) were dissolved in 1,2-dichloroethane (DCE) and cooled on ice for 30 min. To this solution were added 2.15 mL (15.42 mmol, 2.0 eq.) TEA and 2.54 g (7.71 mmol, 1.0 eq.) H-L-Glu(OBzl)-OtBu.HCl and the solution was stirred overnight at 40° C. The remaining solvent was evaporated and the crude product purified using silica gel flash-chromatography with an eluent mixture containing ethyl acetate/hexane/TEA (500:500:0.8; v/v/v). After removal of the solvent, 5-benzyl-1-(tert-butyl)-WS)-1,5-di-tert-butoxy-1,5-dioxopentan-2-yl)carbamoyl)-L-glutamate (ii) was obtained as a colorless oil.
[0155] (tBuO)EuE(OtBu).sub.2 (iii)
[0156] To synthesize (tBuO)EuE(OtBu).sub.2, 3.17 g (5.47 mmol, 1.0 eq.) of 5-benzyl-1-(tert-butyl)-(((S)-1,5-di-tert-butoxy-1,5-dioxopentan-2-yl)carbamoyl)-L-glutamate (ii) were dissolved in 75 mL EtOH and 0.34 g (0.57 mmol, 0.1 eq.) palladium on activated charcoal (10%) were given to this solution. The flask containing the reaction mixture was initially purged with H.sub.2 and the solution was stirred over night at room temperature under light H.sub.2-pressure (balloon). The crude product was purified through celite and the solvent evaporated in vacuo. The product (iii) was obtained as a hygroscopic solid (84%). HPLC
[0157] (10% to 90% B in 15 min): t.sub.R=11.3 min. Calculated monoisotopic mass (C.sub.23H.sub.49N.sub.2O.sub.9): 488.3; found: m/z=489.4 [M+H].sup.+, 516.4 [M+Na].sup.+.
##STR00015##
[0158] 3) Synthesis of the Silicon-Fluoride Acceptor
[0159] 4-(Di-tert-butylfluorosilyl)benzoic acid (SiFA-BA)
##STR00016##
[0160] SiFA-BA was synthesized according to a previously published procedure (scheme 2). All reactions were carried out in dried reaction vessels under argon using a vacuum gas manifold.
[0161] ((4-bromobenzyl)oxy)(tert-butyl)dimethylsilane (i)
[0162] To a stirred solution of 4-bromobenzylalcohol (4.68 g, 25.0 mmol, 1.0 eq.) in anhydrous DMF (70 mL) imidazole (2.04 g, 30.0 mmol, 1.2 eq.) and TBDMSCI (4.52 g, 30.0 mmol, 1.2 eq.) were added and the resulting mixture was stirred at room temperature for 16 h. The mixture was then poured into ice-cold H.sub.2O (250 mL) and extracted with Et.sub.2O (5×50 mL). The combined organic fractions were washed with sat. aq. NaHCO.sub.3 (2×100 mL) and brine (100 mL), dried, filtered and concentrated in vacuo to give the crude product which was purified by flash column chromatography (silica, 5% EtOAc/petrol) to give i as a colourless oil (7.18 g, 95%). .sup.1H NMR (400 MHz, CDCl.sub.3): δ [ppm]=0.10 (6H, s, SiMe.sub.2t-Bu), 0.95 (9H, s, SiMe.sub.2tBu), 4.69 (2H, s, CH.sub.2OSi), 7.21 (2H, d), 7.46 (2H, d). HPLC (50 to 100% B in 15 min): t.sub.R=15 min.
[0163] Di-tert-butyl{4-[(tert-butyldimethylsilyloxy)methyl]phenyl}fluorosilane (ii)
[0164] At −78° C. under magnetic stirring, a solution of tBuLi in pentane (7.29 mL, 1.7 mol/L, 12.4 mmol 2.4 eq.) was added to a solution of ((4-bromobenzyl)oxy)(tert-butyl)dimethylsilane (i) (1.56 g, 5.18 mmol, 1.0 eq.) in dry THF (15 mL). After the reaction mixture had been stirred for 30 min at −78° C., the suspension obtained was added dropwise over a period of 30 min to a cooled (−78° C.) solution of di-tert-butyldifluorosilane (1.12 g, 6.23 mmol, 1.2 eq.) in dry THF (10 mL). The reaction mixture was allowed to warm to room temperature over a period of 12 h and then hydrolyzed with saturated aqueous NaCl solution (100 mL). The organic layer was separated and the aqueous layer was extracted with diethyl ether (3×50 mL). The combined organic layers were dried over magnesium sulfate and filtered. The filtrate was concentrated in vacuo to afford ii as a yellowish oil (1.88 g, 95%). It was used for subsequent reactions without further purification. NMR spectra were in accordance with the data reported in the literature.sup.[2]. HPLC (50 to 100% B in 20 min): t.sub.R=19 min.
4-(Di-tert-butylfluorosilanyl)benzyl Alcohol (iii)
[0165] A catalytic amount of concentrated aqueous HCl (0.5 mL) was added to a suspension of ii (1.88 g, 4.92 mmol, 1.0 eq.) in methanol (50 mL). The reaction mixture was stirred for 18 h at room temperature and then the solvent and the volatiles were removed under reduced pressure. The residue was redissolved in diethyl ether (40 mL) and the solution was washed with saturated aqueous NaHCO.sub.3 solution. The aqueous layer was extracted with diethyl ether (3×50 mL). The combined organic layers were dried over magnesium sulfate and filtered. The filtrate was concentrated in vacuo to afford iii as a yellowish oil (1.29 g, 98%) that solidified. The product was used without further purification. NMR spectra were in accordance with the data reported in the literature.sup.[2]. HPLC (50 to 100% B in 15 min): t.sub.R=8.2 min.
4-(Di-tert-butylfluorosilyl)benzaldehyde (iv)
[0166] A solution of the alcohol iii (1.37 g, 5.10 mmol, 1.0 eq.) in dry dichloromethane (20 mL) was added dropwise to a stirred ice-cooled suspension of pyridinium chlorochromate (2.75 g, 12.8 mmol, 2.5 eq.) in dry dichloromethane (60 mL). After the reaction mixture had been stirred for 30 min at 0° C. and for 2.5 h at room temperature, anhydrous diethyl ether (40 mL) was added and the supernatant solution was decanted from the black gum-like material. The insoluble material was washed thoroughly with diethyl ether and the combined organic phases were passed through a short pad of silica gel (10 cm per g crude product) for filtration. The solvents were removed in vacuo to yield aldehyde iv as a yellowish oil (1.31 g, 96%). NMR spectra were in accordance with the data reported in the literature.sup.[2]. HPLC (50 to 100% B in 15 min): t.sub.R=10.5 min.
4-(Di-tert-butylfluorosilyl)benzoic Acid (v)
[0167] At room temperature, 1 M aqueous KMnO.sub.4 (30 mL) was added to a mixture of iv (1.31 g, 4.92 mmol, 1.0 eq.), tert-butanol (30 mL), dichloromethane (3.3 mL), and 1.25 M NaH.sub.2PO.sub.4.H.sub.2O buffer (20 mL) at pH 4.0-4.5. After the mixture had been stirred for 25 min, it was cooled to 5° C., whereupon excess KMnO.sub.4 (0.78 g, 4.92 mmol, 1.0 eq.) was added. The reaction was then quenched by the addition of saturated aqueous Na.sub.2SO.sub.3 solution (50 mL). Upon addition of 2 M aqueous HCl, all of the MnO.sub.2 dissolved. The resulting solution was extracted with diethyl ether (3×100 mL). The combined organic layers were washed with saturated aqueous NaHCO.sub.3 solution, dried over MgSO.sub.4, filtered, and concentrated under reduced pressure to provide a white solid, which was purified by recrystallization from Et.sub.2O/n-hexane (1:3, for 12 h) to give v (0.84 g, 60%). NMR spectra were in accordance with the data reported in the literature.sup.[2]. HPLC (50 to 100% B in 15 min): t.sub.R=8.5 min.
##STR00017##
[0168] 4) Synthesis of rhPSMA-7.1-7.4
[0169] The first synthetic steps for preparation of the four different isomers of rhPSMA-7 are identical and carried out together, applying the standard Fmoc-SPPS protocol described above, starting from resin bound Fmoc-D-Orn(Dde)-OH. After cleavage of the Fmoc group with 20% piperidine in DMF (GP3), (tBuO)EuE(OtBu).sub.2 (2.0 eq.) was conjugated with HOAt (2.0 eq.), TBTU (2.0 eq.) and DIPEA (6.0 eq.) in DMF for 4.5 h. After cleavage of the Dde-group with a mixture of 2% hydrazine in DMF (GP4a), a solution of succinic anhydride (7.0 eq.) and DIPEA (7.0 eq.) in DMF was added and left to react for 2.5 h. Conjugation of Fmoc-D-Lys(OtBu).HCl (2.0 eq.) was achieved by adding a mixture of HOAt (2.0 eq.), TBTU (2.0 eq.) and DIPEA (6.0 eq.) in DMF to the resin. After pre-activation for 5 min, Fmoc-D-Lys(OtBu).HCl (2.0 eq.) dissolved in DMF was added and left to react for 2.5 h (GP2). Subsequent cleavage of the Fmoc-group was performed, by adding a mixture of 20% piperidine in DMF (GP3). Finally, the resin was split in order to synthesize rhPSMA-7.1-7.4 (scheme 3).
[0170] rhPSMA-7.1 (D-Dap-(R)-DOTA-GA):
##STR00018##
[0171] Fmoc-D-Dap(Dde)-OH (2.0 eq.) was pre-activated in a mixture of HOAt (2.0 eq.), TBTU (2.0 eq.) and 2,4,6-trimethylpyridine (6.7 eq.) in DMF and added to the resin-bound peptide for 2.5 h. Following orthogonal Dde-deprotection was done using imidazole and hydroxylamine hydrochloride dissolved in a mixture of NMP and DMF for 3 h. SiFA-BA (1.5 eq.) was reacted with the free amine of the side chain with HOAt (1.5 eq.), TBTU (1.5 eq.) and DIPEA (4.5 eq.), as activation reagents in DMF for 2 h. After Fmoc-deprotection with piperidine (GP3), (R)-DOTA-GA(tBu).sub.4 (2.0 eq.) was conjugated with HOAT (2.0 eq.), TBTU (2.0 eq.) and 2,4,6-trimethylpyridine (6.7 eq.) in DMF for 2.5 h. Cleavage from the resin with simultaneous deprotection of acid labile protecting groups was performed in TFA, according to GPS. .sup.natGa-complexation of the peptide was carried out, as described in GP6.
[0172] rhPSMA-7.2 (L-Dap-(R)-DOTA-GA):
##STR00019##
[0173] Fmoc-L-Dap(Dde)-OH (2.0 eq.) was pre-activated in a mixture of HOAt (2.0 eq.), TBTU (2.0 eq.) and 2,4,6-trimethylpyridine (6.7 eq.) in DMF for 2.5 h. Following orthogonal Dde-deprotection, conjugation of SiFA-BA and Fmoc-cleavage was carried out as described for rhPSMA-7.1. (R)-DOTA-GA(tBu).sub.4 (2.0 eq.) was conjugated with HOAT (2.0 eq.), TBTU (2.0 eq.) and 2,4,6-trimethylpyridine (6.7 eq.) in DMF for 2.5 h. Cleavage from the resin with simultaneous deprotection of acid labile protecting groups was performed in TFA according to GPS. .sup.natGa-complexation of the peptide was carried out, as described in GP6.
[0174] rhPSMA-7.3 (D-Dap-(S)-DOTA-GA):
##STR00020##
[0175] Fmoc-D-Dap(Dde)-OH (2.0 eq.) was pre-activated in a mixture of HOAt (2.0 eq.), TBTU (2.0 eq.) and 2,4,6-trimethylpyridine (6.7 eq.) in DMF for 2.5 h. Following orthogonal Dde-deprotection, conjugation of SiFA-BA and Fmoc-cleavage was carried out as described for rhPSMA-7.1. (S)-DOTA-GA(tBu).sub.4 (2.0 eq.) was conjugated with HOAT (2.0 eq.), TBTU (2.0 eq.) and 2,4,6-trimethylpyridine (6.7 eq.) in DMF for 2.5 h. Cleavage from the resin with simultaneous deprotection of acid labile protecting groups was performed in TFA according to GP5. .sup.natGa-complexation of the peptide was carried out, as described in GP6.
[0176] rhPSMA-7.4 (L-Dap-(S)-DOTA-GA):
##STR00021##
[0177] Fmoc-L-Dap(Dde)-OH (2.0 eq.) was pre-activated in a mixture of HOAt (2.0 eq.), TBTU (2.0 eq.) and 2,4,6-trimethylpyridine (6.7 eq.) in DMF for 2.5 h. Following orthogonal Dde-deprotection, conjugation of SiFA-BA and Fmoc-cleavage was carried out as described for rhPSMA-7.1. (S)-DOTA-GA(tBu).sub.4 (2.0 eq.) was conjugated with HOAT (2.0 eq.), TBTU (2.0 eq.) and 2,4,6-trimethylpyridine (6.7 eq.) in DMF for 2.5 h. Cleavage from the resin with simultaneous deprotection of acid labile protecting groups was performed in TFA according to GPS. .sup.natGa-complexation of the peptide was carried out, as described in GP6.
[0178] rhPSMA-7.1:
[0179] HPLC (10 to 70% B in 15 min): t.sub.R=10.5 min.
[0180] HPLC (25 to 35% B in 40 min): t.sub.R=31.4 min.
[0181] rhPSMA-7.2:
[0182] HPLC (10 to 70% B in 15 min): t.sub.R=10.4 min.
[0183] HPLC (25 to 35% B in 40 min): t.sub.R=27.9 min.
[0184] rhPSMA-7.3:
[0185] HPLC (10 to 70% B in 15 min): t.sub.R=10.4 min.
[0186] HPLC (25 to 35% B in 40 min): t.sub.R=28.1 min.
[0187] rhPSMA-7.4:
[0188] HPLC (10 to 70% B in 15 min): t.sub.R=10.5 min.
[0189] HPLC (25 to 35% B in 40 min): t.sub.R=29.1 min.
[0190] rhPSMA-7.1-7.4:
[0191] Calculated monoisotopic mass (C63H96FGaN12O25Si): 1536.6 found: m/z=1539.4 [M+H]+, 770.3 [M+2H]2.sup.+.
##STR00022##
[0192] 5).sup.18F-Labelling
[0193] For .sup.18F-labelling a previously published procedure was applied, which was slightly modified. Briefly, aqueous .sup.18F was passed through a SAX cartridge (Sep-Pak Accell Plus QMA Carbonate light), which was preconditioned with 10 mL of water. After drying with 10 mL of air, water was removed, by rinsing the cartridge with 10 mL of anhydrous acetonitrile followed by 20 mL of air. .sup.18F was eluted with 100 μmol of [K.sup.+⊂2.2.2]OH.sup.− dissolved in 500 μl of anhydrous acetonitrile. Before labelling, 30 μmol of oxalic acid in anhydrous acetonitrile (1 M, 30 μL) were added. This mixture was used as a whole or aliquot for fluorination of 10-25 nmol of PSMA-SiFA (1 mM in anhydrous DMSO). The resulting reaction mixture was incubated for 5 minutes at room temperature. For purification of the tracer, a Sep-Pak C18 light cartridge, preconditioned with 10 mL EtOH, followed by 10 mL of H.sub.2O was used. The labelling mixture was diluted with 9 mL PBS (pH 3) and passed through the cartridge followed by 10 mL of H.sub.2O. The peptide was eluted with 500 μL of a 4:1 mixture (v/v) of EtOH in water. Radiochemical purity of the labelled compound was determined by radio RP-HPLC and radio-TLC (Silica gel 60 RP-18 F.sub.254S, mobile phase: 3:2 mixture (v/v) of MeCN in H.sub.2O supplemented with 10% of 2 M aqueous NaOAc and 1% of TFA).
[0194] 6).sup.125I-Labelling
[0195] The reference ligand for in vitro studies ([.sup.125I]I-BA)KuE was prepared according to a previously published procedure. Briefly, 0.1 mg of the stannylated precursor (SnBu.sub.3-BA)(OtBu)KuE(OtBu).sub.2 was dissolved in a solution containing 20 μL peracetic acid, 5.0 μL (21 MBq) [.sup.125I]NaI (74 TBq/mmol, 3.1 GBq/mL, 40 mM NaOH, Hartmann Analytic, Braunschweig, Germany), 20 μL MeCN and 10 μL acetic acid. The reaction solution was incubated for 10 min at RT, loaded on a cartridge and rinsed with 10 mL water (C18 Sep Pak Plus cartridge, preconditioned with 10 mL MeOH and 10 mL water). After elution with 2.0 mL of a 1:1 mix (v/v) of EtOH/MeCN, the radioactive solution was evaporated to dryness under a gentle nitrogen stream and treated with 200 μL TFA for 30 min with subsequent evaporation of TFA. The crude product of ([.sup.125I]I-BA)KuE was purified by RP-HPLC (20% to 40% B in 20 min): t.sub.R=13.0 min.
[0196] In vitro experiments
[0197] 1) Determination of IC.sub.50
[0198] The PSMA-positive LNCaP cells were grown in Dublecco modified Eagle medium/Nutrition Mixture F-12 with Glutamax-I (1:1) (Invitrigon), supplemented with 10% fetal calf serum and maintained at 37° C. in a humidified 5% CO.sub.2 atmosphere. For determination of the PSMA affinity (IC.sub.50), cells were harvested 24±2 hours before the experiment and seeded in 24-well plates (1.5×10.sup.5 cells in 1 mL/well). After removal of the culture medium, the cells were treated once with 500 μL of HBSS (Hank's balanced salt solution, Biochrom, Berlin, Germany, with addition of 1% bovine serum albumin (BSA)) and left 15 min on ice for equilibration in 200 μL HBSS (1% BSA). Next, 25 μL per well of solutions, containing either HBSS (1% BSA, control) or the respective ligand in increasing concentration (10.sup.−10-10.sup.−4 M in HBSS, were added with subsequent addition of 25 μL of ([.sup.125I]I-BA)KuE (2.0 nM) in HBSS (1% BSA). All experiments were performed at least three times for each concentration. After 60 min incubation on ice, the experiment was terminated by removal of the medium and consecutive rinsing with 200 μL of HBSS. The media of both steps were combined in one fraction and represent the amount of free radioligand. Afterwards, the cells were lysed with 250 μL of 1 M NaOH and united with the 200 μL HBSS of the following wash step. Quantification of bound and free radioligand was accomplished in a γ-counter.
[0199] 2) Internalization
[0200] For internalization studies, LNCaP cells were harvested 24±2 hours before the experiment and seeded in 24-well plates (1.25×10.sup.5 cells in 1 mL/well). Subsequent to the removal of the culture medium, the cells were washed once with 500 μL DMEM-F12 (5% BSA) and left to equilibrate for at least 15 min at 37° C. in 200 μL DMEM-F12 (5% BSA). Each well was treated with either 25 μL of either DMEM-F12 (5% BSA) or a 100 μM PMPA solution for blockade. Next, 25 μL of the .sup.68Ga/.sup.18F-labeled PSMA inhibitor (5.0 nM) was added and the cells incubated at 37° C. for 60 min. The experiment was terminated by placing the 24-well plate on ice for 3 min and consecutive removal of the medium. Each well was rinsed with 250 μL HBSS and the fractions from these first two steps combined, representing the amount of free radioligand. Removal of surface bound activity was accomplished by incubation of the cells with 250 μL of ice-cold PMPA (10 μM in PBS) solution for 5 min and rinsed again with another 250 μL of ice-cold PBS. The internalized activity was determined by incubation of the cells in 250 μL 1 M NaOH and the combination with the fraction of a subsequent wash step with 250 μL 1.0 M NaOH. Each experiment (control and blockade) was performed in triplicate. Free, surface bound and internalized activity was quantified in a γ-counter. All internalization studies were accompanied by reference studies using ([.sup.125I]I-BA)KuE (c=0.2 nM), which were performed analogously. Data were corrected for non-specific internalization and normalized to the specific-internalization observed for the radioiodinated reference compound.
[0201] 3) Octanol-Water Partition Coefficient
[0202] Approximately 1 MBq of the labeled tracer was dissolved in 1 mL of a 1:1 mixture (by volumes) of phosphate buffered saline (PBS, pH 7.4) and n-octanol in an Eppendorf tube. After vigorous mixing of the suspension for 3 minutes at room temperature, the vial was centrifuged at 15000 g for 3 minutes (Biofuge 15, Heraus Sepatech, Osterode, Germany) and 100 μL aliquots of both layers were measured in a gamma counter. The experiment was repeated at least six times.
[0203] 4) HSA Binding
[0204] For the determination of HSA binding, a Chiralpak HSA column (50×3 mm, 5 μm, H13H-2433) was used at a constant flow rate of 0.5 mL/min. The mobile phase (A: NH.sub.4OAc, 50 mM in water, pH 7 and B: isopropanol) was freshly prepared for each experiment and only used for one day. The column was kept at room temperature and each run was stopped after detection of the signal to reduce the acquisition time. All substances were dissolved in a 0.5 mg/ml concentration in 50% 2-propanol and 50% 50 mM pH 6.9 ammonium acetate buffer. The chosen reference substances display a range of HSA binding from 13% to 99% since a broad variety of albumin binding regarding the peptides was assumed. All nine reference substances were injected consecutively to establish a non-linear regression with Origin Pro 2016G.
TABLE-US-00001 TABLE 1 Reference substances used for the calibration of the HSA-column. Reference t.sub.R Log t.sub.R Lit. HSA % Log K HSA p-benzylalcohol 2.40 0.38 13.15 −0.82 Aniline 2.72 0.43 14.06 −0.79 Phenol 3.28 0.52 20.69 −0.59 Benzoic Acid 4.08 0.61 34.27 −0.29 Carbamazepine 4.15 0.62 75.00 0.46 p-nitrophenol 5.62 0.75 77.65 0.52 Estradiol 8.15 0.91 94.81 1.19 Probenecid 8.84 0.95 95.00 1.20 Glibenclamide 29.18 1.47 99.00 1.69 The retention time is shown exemplary for a conducted experiment; t.sub.R retention time; Lit. HSA literature value of human serum albumin binding in [%]; Log K HAS logarithmic K of human serum albumin binding.
[0205] In Vivo Experiments
[0206] All animal experiments were conducted in accordance with general animal welfare regulations in Germany and the institutional guidelines for the care and use of animals. To establish tumor xenografts, LNCaP cells (10.sup.7 cells/200 μL) were suspended in a 1:1 mixture (v/v) of Dulbecco modified Eagle medium/Nutrition Mixture F-12 with Glutamax-I (1:1) and Matrigel (BD Biosciences, Germany), and inoculated subcutaneously onto the right shoulder of 6-8 weeks old CB17-SCID mice (Charles River, Sulzfeld, Germany). Mice were used for when tumors had grown to a diameter of 5-8 mm (3-4 weeks after inoculation).
[0207] 1) Biodistribution
[0208] Approximately 1-2 MBq (<0.2 nmol) of the .sup.18F-labeled PSMA inhibitor was injected into the tail vein of LNCaP tumor-bearing male CB-17 SCID mice and sacrificed after 1 h post injection (n=4-5).
[0209] Selected organs were removed, weighted and measured in a γ-counter
[0210] 2) Metabolism Studies
[0211] a) Analytical Set-Up
[0212] Analytical reversed-phase high pressure chromatography (RP-HPLC) were performed using Shimadzu gradient systems (Shimadzu Deutschland GmbH, Neufahrn, Germany), equipped with a SPD-20A UV/Vis detector (220 nm, 254 nm). A Multospher 100 RP18 (125×4.6 mm, 5 μm particle size) column (CS GmbH, Langerwehe, Germany) was used for analytical measurements at a flow rate of 1 mL/min. Eluents for all HPLC operations were water (solvent A) and acetonitrile (solvent B), both containing 0.1% trifluoroacetic acid. Radioactivity was detected through connection of the outlet of the UV-photometer to a HERM LB 500 detector (Berthold Technologies GmbH, Bad Wildbad, Germany). The gradient for all HPLC operations was: 5% B isocratic 0-3 min, 25-35% B 3-43 min, 95-95% B 43-48 min.
[0213] For radio-thin-layer chromatography, aluminum sheets coated with silica gel 60 RP-18 F.sub.254s were used with a mobile phase consisting of a 3:2 mixture (v/v) of MeCN in H.sub.2O supplemented with 10% of 2 M aqueous NaOAc and 1% of TFA. Analysis was performed using either a Scan-RAM radio-TLC detector (LabLogic Systems Ltd., Sheffield, United Kingdom) or a CR 35 BIO phosphorimager (Duerr Medical GmbH, Bietigheim-Bissingen, Germany).
[0214] b) Determination of Metabolic Stability of rhPSMA-7.1-7.4
[0215] For in vivo μmetabolism studies, 8-12 MBq (<0.6 nmol) of the respective .sup.18F-labeled ligand (rhPSMA-7.1-7.4) was injected into the tail vein of female healthy CB17-SCID mice (n=4). Mice were left under anesthesia for 30 min and the urine was collected using a bladder catheter. Urine samples were pooled and centrifuged for 5 min at 9000 rpm to remove suspended solids. The supernatant was directly used for radio-HPLC analysis with the above mentioned conditions. In order to demonstrate that isotopic exchange of .sup.19F with peptide-bound .sup.18F is taking place in urine, each compound was incubated for certain time intervals with urine samples of female healthy CB-17-SCID mice, which where analysed by radio-HPLC and/or radio-TLC. Additionally, this experiment was carried out with the addition of excess Na.sup.19F (0.5 μmop and incubation for 2 h with .sup.18F-labeled rhPSMA-7.3.
[0216] c) Determination of In Vivo Distribution of rhPSMA-7.1-7.4
[0217] In order to quantify the relative uptake of each isomer (rhPSMA-7.1-7.4), a tumor-bearing male CB-17-SCID mouse was injected with the racemic mixture of rhPSMA-7 (180-280 MBq, S.sub.A=247-349 GBq/μmol, produced at the Klinikum rechts der Isar in a fully automated procedure). The animal was left under anesthesia for 30 min and sacrificed. Urine, blood, liver, kidneys and tumor were collected and processed to the hereafter described procedures. The urine sample was centrifuged for 5 min at 9000 rpm to yield a clear solution and directly subjected to radio-HPLC analysis. Blood was diluted to 1 mL with H.sub.2O and centrifuged twice at 13000 g for 5 min. The supernatant was collected and loaded on a Strata X cartridge (33 μm Polymeric Reversed Phase 500 mg, pre-conditioned with 5 mL MeOH, followed by 5 mL H.sub.2O). After washing with 5 mL H.sub.2O, the cartridge was eluted with a 6:4 mixture (v/v) of MeCN in H.sub.2O, supplemented with 1% TFA. The eluate was diluted with water and analysed by radio-HPLC. Tumour, kidneys and liver were homogenised using either a Potter-Elvehjem tissue grinder (Kontes Glass Co, Vineland, USA) or a MM-400 ball mill (Retsch GmbH, Haan, Germany).
[0218] I) Potter-Elvehjem Tissue Grinder
[0219] Tumour and kidneys were separately homogenised in the tissue homogeniser with 1 mL of extraction buffer (850 μL 1 M HEPES pH7.4, 100 μL 20 mM PMPA and 100 μL 1M NaCl) for 30 min. The resulting homogenate was collected and centrifuged at 13000 g for 5 min. Subsequently the supernatant was collected, centrifuged again (13000 g, 5 min) and loaded on a Strata X cartridge (33 μm Polymeric Reversed Phase 500 mg, pre-conditioned with 5 mL MeOH, followed by 5 mL H.sub.2O). After washing with 5 mL H.sub.2O, the cartridge was eluted with a 6:4 mixture (v/v) of MeCN in H.sub.2O, supplemented with 1% TFA. The eluate of each organ was diluted with water and analysed by radio-HPLC.
[0220] II) MM-400 Ball Mill
[0221] The organs (tumour, kidney, liver) were separately homogenised in a 2 mL tube together with 3 grinding balls (3 mm diameter) and 1 mL of extraction buffer (850 μL 1 M HEPES pH7.4, 100 μL 20 mM PMPA and 100 μL 1M NaCl) for 10 min at 30 Hz. The homogenate was centrifuged at 13000 g for 5 min and the supernatant was collected. Subsequently, the pellet was suspended in 1 mL of extraction buffer and homogenized again with the ball mill for 10 min at 30 Hz. After centrifugation (13000 g, 5 min), both supernatants were combined and loaded on a Strata X cartridge (33 μm Polymeric Reversed Phase 500 mg, pre-conditioned with 5 mL MeOH, followed by 5 mL H.sub.2O). After washing with 5 mL H.sub.2O, the cartridge was eluted with a 6:4 mixture (v/v) of MeCN in H.sub.2O, supplemented with 1% TFA. The eluate of each organ was diluted with water and analysed by radio-HPLC. In order to demonstrate that the breakthrough during cartridge loading, is not a result of unbound F-18, the supernatant was also examined by radio-TLC after centrifugation.
[0222] Finally the ratios of the individual isomers were determined from the HPLC profiles of the extracted samples and compared to the ratios of the isomers from the quality control of the racemic mixture of rhPSMA-7. The decay corrected extraction- and cartridge loading-efficiency, as well as the overall extracted activity of the examined samples are given in table 2. The cartridge elution-efficiency was >99% for all experiments.
Example 2: Results
[0223] Chromatographic Peak Assignment
[0224] The chromatographic peak assignment was carried out by comparison of the UV profiles of
[0225] a) the rhPSMA7-rac mixture with
[0226] b) the rhPSMA7-rac mixture coinjected with each enantiopure rhPSMA7 compound.
[0227] The following names are used for the different isomers:
[0228] rhPSMA-rac: [.sup.19F][.sup.natGa]D/L-Dap-R/S-DOTAGA-rhPSMA7
[0229] rhPSMA-7-1: [.sup.19F][.sup.natGa]D-Dap-R-DOTAGA-rhPSMA7
[0230] rhPSMA-7-2: [.sup.19F][.sup.natGa]L-Dap-R-DOTAGA-rhPSMA7
[0231] rhPSMA-7-3: [.sup.19F][.sup.natGa]D-Dap-S-DOTAGA-rhPSMA7
[0232] rhPSMA-7-4: [.sup.19F][.sup.natGa]L-Dap-S-DOTAGA-rhPSMA7
TABLE-US-00002 TABLE 2 Assignment of the different isomers, names, typical retention times (HPLC conditions are given in FIG. 2a-4b, and percentage of each isomer on a typical rhPSAM7-rac mixture. The exact amount can vary for each isomer. t.sub.R typical percentage ligand Name [min] of whole mixture [.sup.19F][.sup.natGa]D-Dap-R-DOTAGA-rhPSMA7 rhPSMA-7-1 31.6 21 [.sup.19F][.sup.natGa]L-Dap-R-DOTAGA-rhPSMA7 rhPSMA-7-2 28.3 22 [.sup.19F][.sup.natGa]D-Dap-S-DOTAGA-rhPSMA7 rhPSMA-7-3 28.9 37 [.sup.19F][.sup.natGa]L-Dap-S-DOTAGA-rhPSMA7 rhPSMA-7-4 30.1 20
[0233] Binding Affinities
[0234] The first set of values (rhPSMA-7.1 and rhPSMA-7.2;
TABLE-US-00003 TABLE 3 Depiction of the individual IC.sub.50 [nM] measurements (as shown in FIG. 6a and 6b). Conditions as described in the legend to FIG. 6a. No rhPSMA7.1 rhPSMA7.2 rhPSMA7.3 rhPSMA7.4 1 8.74 3.17 nd nd 2 6.91 2.97 nd nd 3 7.27 3.36 nd nd 4 5.04 2.64 3.17 3.4 5 2.76 2.91 3.56 6 7.11 3.94 5.35 2.79 7 8.31 5.8 5.74 4.57 8 4.97 4.31 4.59 3.78 9 6.44 4.32 4.45 nd Mean 6.85 3.70 4.37 3.62 SD 1.36 1.01 1.14 0.65 * Value no 5 of the rhPSMA7.1 series was deleted (statistical outlier).
TABLE-US-00004 TABLE 4 Binding affinities (IC.sub.50 [nM]) of other selected PSMA inhibitors (*). No Inhibitor IC.sub.50 [nM] 1 (I-BA)KuE 7.1 ± 2.4 nM 2 DCFPyL 12.3 ± 1.2 nM 3 DKFZ1007 4.2 ± 0.5 nM * carried out in our lab using the identical binding assay (Robu et al. EJNMMI Research 2018; 8: 30).
[0235] Internalization Studies
TABLE-US-00005 TABLE 5 Depiction of the individual internalization rates [% of [.sup.125I]IB-KuE]. rhPSMA7.1 rhPSMA7.2 rhPSMA7.3 rhPSMA7.4 1 61.8 188.7 156.6 209.6 1 70.6 182.7 156.0 202.6 1 68.0 169.5 171.7 209.8 1 67.9 205.3 — — 1 71.5 212.6 — — 1 77.5 192.3 — — Mean 69.55 191.83 161.41 207.33 SD 5.19 15.54 8.88 4.06
TABLE-US-00006 TABLE 6 Internalization values [% of [.sup.125I]IB-KuE] of other selected PSMA inhibitors (*). No Inhibitor internalization [%] 1 PSMA-1007 118 ± 4 2 DCFPyL 118 ± 5 * carried out in our lab using the identical binding assay (Robu et al. EJNMMI Research 2018; 8: 30).
[0236] Lipophilicities (Octanol-Water Partition Coefficient)
[0237] Determianation of the IogP values was carried out in phosphate buffered saline (PBS, pH 7.4) and n-octanol (=logP.sub.oct/PBS).
TABLE-US-00007 TABLE 7 Individual log P measurements for rhPSMA7-isomers 7.1-7.4 isomers, determined in octanol/PBS.sub.7.4 mixtures. rhPSMA7.1 rhPSMA7.2 rhPSMA7.3 rhPSMA7.4 rhPSMA7-rac 1 −2.79 −3.00 −3.03 −3.09 −3.23 2 −2.82 −3.01 −3.08 −3.12 −3.15 3 −2.80 −3.00 −3.07 −3.06 −3.17 4 −2.84 −3.00 −3.02 −3.07 −3.10 5 −2.88 −3.03 −3.06 −3.10 −3.17 6 −2.90 −2.96 −3.07 −3.04 −3.24 7 −2.85 −2.94 −3.06 −2.99 −3.80 8 −2.86 −2.89 −2.86 −2.97 −3.60 9 −3.14 −3.02 −3.39 −3.37 −3.87 10 −3.29 −3.02 −3.33 −3.43 −3.61 11 −3.26 −3.06 −3.34 −3.29 −3.76 12 −3.02 −3.02 −3.34 −3.48 −3.65 13 −3.15 −2.99 −3.20 −3.52 −3.67 14 −3.57 −3.02 −3.39 −3.50 — 15 −3.40 −3.06 −3.40 −3.44 — 16 −3.32 −3.14 −3.41 −3.41 — 17 −3.64 −3.40 −3.48 −3.56 — 18 −3.92 −3.50 −3.49 −3.61 — 19 — −3.45 −3.32 −3.58 — 20 — −3.45 −3.45 −3.54 — 21 — −3.53 −3.53 −3.42 — 22 — −3.48 −3.43 −3.57 — 23 — — −3.56 −3.67 — Mean −3.14 −3.13 −3.26 −3.33 −3.46 SD 0.34 0.22 0.19 0.22 0.29
TABLE-US-00008 TABLE 8 log P values of PSMA-1007, DCFPYL, rhPSMA7-rac and rhPSAM7.1-7.4 isomers; (n = 6), octanol/PBS.sub.7.4. Inhibitor log P PSMA-1007 −1.6 DCFPyL −3.4 .sup.natGa-.sup.18F-rhPSMA7-rac, −3.46 ± 0.29 .sup.68Ga-.sup.natF-rhPSMA7-rac .sup.natGa-.sup.18F-rhPSMA7.1 −3.14 ± 0.34 .sup.natGa-.sup.18F-rhPSMA7.2 −3.13 ± 0.22 .sup.natGa-.sup.18F-rhPSMA7.3 −3.26 ± 0-19 .sup.natGa-.sup.18F-rhPSMA7.4 −3.33 ± 0.22
[0238] Binding of PSMA Inhibitors to Human Plasma Protein
TABLE-US-00009 TABLE 9 HSA binding of of PSMA-1007, DCFPYL, rhPSMA7-rac and rhPSAM7.1-7.4 isomers; (n = 6). Determined on a Chiralpak HSA column (50 × 3 mm, 5 μm, H13H-2433). Inhibitor HSA Binding [%] PSMA-1007 97.8 DCFPyL 14.3 .sup.68Ga-.sup.natF-rhPSMA7-rac 96.7 .sup.natGa-.sup.18F-rhPSMA7.1 97.7 .sup.natGa-.sup.18F-rhPSMA7.2 97.8 .sup.natGa-.sup.18F-rhPSMA.3 96.9 .sup.natGa-.sup.18F-rhPSMA7.4 96.6
[0239] Biodistribution of [.sup.18F][.sup.natGa]rhPSMA7.1-7.4 at 1 h pi
TABLE-US-00010 TABLE 10 Biodistribution (in % ID/g) of .sup.18F-rhPSMAs at 1 h p.i in LNCaP tumor-bearing SCID mice. Data are expressed as mean ± SD (n = 4 for rhPSMA7.1, n = 5 for 7.2, n = 4 for 7.3, n = 5 for 7.4 and n = 3 for 7-rac). [.sup.18F][.sup.natGa]- [.sup.18F][.sup.natGa]- [.sup.18F][.sup.natGa]- [.sup.18F][.sup.natGa]- [.sup.18F][.sup.natGa]- rhPSMA-7-1 rhPSMA-7-2 rhPSMA-7-3 rhPSMA-7-4 rhPSMA-rac blood 0.53 ± 0.13 0.56 ± 0.20 0.96 ± 0.24 1.15 ± 0.30 1.1 ± 0.03 heart 0.53 ± 0.03 0.32 ± 0.13 0.87 ± 0.17 0.71 ± 0.26 0.69 ± 0.07 lung 1.1 ± 0.21 0.89 ± 0.38 2.2 ± 0.35 1.59 ± 0.61 1.4 ± 0.17 liver 0.75 ± 0.62 0.35 ± 0.08 0.69 ± 0.13 0.69 ± 0.20 0.67 ± 0.07 spleen 20.0 ± 4.2 10.1 ± 6.3 16.6 ± 2.6 18.4 ± 9.77 11.1 ± 2.3 pancreas 0.45 ± 0.12 0.21 ± 0.08 0.63 ± 0.44 0.50 ± 0.30 0.60 ± 0.10 stomach 0.28 ± 0.17 0.19 ± 0.08 0.44 ± 0.23 0.25 ± 0.06 0.49 ± 0.07 intestine 0.30 ± 0.16 0.18 ± 0.07 0.35 ± 0.07 0.37 ± 0.09 0.60 ± 0.27 kidneys 220 ± 24.8 87.6 ± 28.8 292 ± 45.1 153 ± 80.3 71.3 ± 13.3 adrenals 2.0 ± 0.25 1.3 ± 0.8 2.2 ± 0.83 3.57 ± 2.38 3.0 ± 0.45 muscle 0.32 ± 0.30 0.13 ± 0.07 0.33 ± 0.15 0.31 ± 0.08 0.36 ± 0.06 bone 0.50 ± 0.31 0.31 ± 0.24 0.38 ± 0.32 0.62 ± 0.30 0.91 ± 0.11 tumor 14.1 ± 4.1 6.5 ± 2.3 18.3 ± 7.2 18.9 ± 3.27 10.4 ± 0.67
[0240] Biodistribution of [.sup.18F][.sup.natGa]rhPSMA7.1-7.4 at 1 h pi with Competition
TABLE-US-00011 TABLE 11 Biodistribution [% ID/g] of .sup.18F-labeled rhPSMA tracers co-injected with PMPA (8 mg/kg) at 1 h p.i in LNCaP tumor- bearing SCID mice. Data are expressed as mean ± SD (n = 3). [.sup.18F][.sup.natGa]- [.sup.18F][.sup.natGa]- [.sup.18F][.sup.natGa]- [.sup.18F][.sup.natGa]- rhPSMA-7-1 rhPSMA-7-2 rhPSMA-7-3 rhPSMA-7-4 blood 0.86 ± 0.40 1.1 ± 0.31 0.55 ± 0.14 0.82 ± 0.17 heart 0.37 ± 0.16 0.47 ± 0.09 0.26 ± 0.04 0.37 ± 0.05 lung 0.85 ± 0.29 1.1 ± 0.32 0.69 ± 0.10 0.74 ± 0.14 liver 0.43 ± 0.07 0.46 ± 0.07 0.46 ± 0.14 0.48 ± 0.14 spleen 0.21 ± 0.08 0.26 ± 0.07 0.35 ± 0.02 0.28 ± 0.15 pancreas 0.16 ± 0.10 0.12 ± 0.05 0.11 ± 0.02 0.18 ± 0.09 stomach 0.97 ± 0.81 0.21 ± 0.06 0.76 ± 0.74 0.20 ± 0.07 intestine 0.66 ± 0.32 0.33 ± 0.10 0.94 ± 0.97 0.36 ± 0.08 kidneys 10.9 ± 2.5 10.9 ± 1.0 15.5 ± 2.2 7.2 ± 2.4 adrenals 0.003 ± 0.004 0.07 ± 0.10 0.07 ± 0.09 0.03 ± 0.04 muscle 0.17 ± 0.15 0.09 ± 0.03 0.09 ± 0.02 0.20 ± 0.05 bone 0.33 ± 0.24 0.57 ± 0.39 0.34 ± 0.22 1.0 ± 0.8 tumor 0.94 ± 0.22 1.0 ± 0.13 1.5 ± 0.4 0.99 ± 0.19
[0241] Quantification of Relative Changes of the Amount of Each rhPSMA7.x Isomer in Blood, Kindey, Liver, Urine and Tumor after Application of [.sup.18F]rhPSMA7-Rac
[0242] With the aim to quantify the relative changes of each rhPSMA7 isomer in blood, liver, kidney, urine and tumor 30 min after injection of [.sup.18F]rhPSMA7-rac into a LNCaP tumor bearing mouse, two different homogenization methods (a potter and a ball mill) were used to extract the tracer from kidney, liver and tumor tissue (see Materials and Methods).
[0243] Table 12 summarizes the observed efficiencies for both homogenization methods and the efficancy of the subsequent solid phase extraction procedure (to separate the tracer from the protein fraction).
TABLE-US-00012 TABLE 12 Determination of the decay corrected extracted activities from the examined tissue samples via the Potter-Elvehjem tissue grinder (n = 1) and the MM-400 ball mill (n = 3). EFFICIENCY [%] Sample SPE cartridge- extraction loading overall Potter-Elvehjem tissue grinder (n = 1): blood 93 93 86 kidney 91 66 60 tumor 90 59 53 MM-400 ball mill (n = 3): blood 98 ± 2 94 ± 2 92 ± 3 liver 97 89 ± 2 86 ± 2 kidney 63 ± 5 68 ± 8 43 ± 8 tumor 64 ± 18 65 ± 3 42 ± 14
[0244] Whereas the extraction of activity from the samples using the potter was quite efficient, the use of the ball mill was disappointing. Nevertheless, even with the ball mill >60% extraction efficiency was reached.
[0245] Taking into account the possible species that could be formed by metabolic cleavage of amide bonds of rhPSMA7, only a) species with significantly increase lipophilicity of b) F18-fluoride seem probable. Thus in principle it seem possible that “iL” species depicted in
[0246] For quantification of each isomer in the racemic mixture and especially for the poorly separated first and second peak (rhPSMA 7.2 and rhPSMA7.3) a graphical approximation was initially used. This approach was based on the assumption that a) each isomer is eluted from the HPLC column with an identical peak shape and b) the different peak heights can be used as first approximation to calculate by means of linear factors less separated peaks (i.e. rhPSMA 7.2 and rhPSMA7.3).
[0247] Based on these assumptions, the first analysis was performed by using one LNCaP tumor bearing mice coinfected with [.sup.18F][.sup.natGa]rhPSMA7-rac. With the aim to validate these experiments by means of three additional experiments and to improve the graphical analyses by a more valid procedure, the Systat software package ‘PeakFit’ was used. PeakFit allows for automated nonlinear separation, analysis and quantification of HPLC elution profiles by deconvolution procedures that uses a Gaussian response function with a Fourier deconvolution/filtering algorithm (https://systatsoftware.com/products/peakfit/).
[0248] A comparison of the graphical analysis of the first experiments revealed that the graphical analysis overestimated the second peak (rhPSMA7.3), whereas the first peak was underestimated. Consequently, all data sets were reanalyzed and quantified by means of PeakFit.
[0249] HPLC-Analyses of 4 Independent Experiments in Tumor Bearing Mice 30 Min p.i.
[0250] 1. Evaluation of Peak 3 and 4 (rhPSMA7.4 and rhPSMA 7.1) by Radio-HPLC
[0251] It was first examined, whether the deconvolution technique shows similar data for the last two peaks (rhPSMA7.4 and 7.1) that have a good separation (although they are not baseline separated).
[0252] 2. Evaluation of all Peaks (rhPSMA7.1, 7.2, 7.3 and 7.4) by Radio-HPLC
[0253]
[0254] 3. Discussion of the HPLC Data
[0255] The radio-HPLC analyses of the radioactivity extracted from the homogenized (kidney, liver, tumor) or diluted (blood) tissues and subsequently immobilized on and eluted from the solid phase extraction cartridge did show no signs of metabolic instability. Thus, no lipophilic metabolic fragments were observed. It should be noted that F-18-fluoride cannot be accurately detected by HPLC under the conditions used for sample preparation (see TLC analysis).
[0256] Although there is a clear trend towards the D-Dap-derivative rhPSMA7.1 and 7.3, the overall changes are low (max 15%). It is also important to stress in this context, that
[0257] Although rhPSMA7.1 has the weakest affinity and internalization of all rhPSMA7 compounds, it shows the largest positive percentage change in blood liver, kidney and tumor.
[0258] Although the reason for this result is unclear, one can speculate that homogenization of the tissue samples, even with the ball mill, did not resulted in a quantitative cell disruption. Thus, the rhPSMA7 tracers with the highest internalization (rhPSMA7.2: 191.83%±15.54%, rhPSMA7.4: 207.33±4.06% and rhPSMA7.3: 161.41%±8.88%) might have been extracted in a less efficient manner, whereas rhPSMA7.1 with its low internalization of only 69.55%±5.29% was efficiently extracted and is consequently overestimated in the HPLC analysis.
[0259] In addition, it seems that the rhPSMA compounds 7.2 and 7.4 are somewhat more rapidly excreted (see values for urine). These compounds show generally negative changes in solid tissues and blood, although both compounds exhibit higher affinities and internalization rates when compared with rhPSMA7.1. Whether this might be caused by metabolic degradation of 7.2 and 7.4 (both are L-Dap derivative) is unclear, since no metabolites, i.e. lyophilic metabolites have been detected. It might however be possible, that such metabolites (see
[0260] TLC-Analysis in Tumor Bearing Mice 30 Min p.i.
[0261] Radio-TLC Analysis was carried out a) on urine samples by directly subjecting a small volume onto a TLC strip, b) by analysis of a small volume of the non-immobilized activity during the SPE process (the ‘breakthrough fraction’), and c) by analysis of a small volume of the cartridge eluates.
TABLE-US-00013 TABLE 13 TLC analysis of blood, organ and urine samples TLC Scanner Phosphoimager .sup.18F- .sup.18F- Comment * Intact tracer Fluoride Intact Fluoride TLC signal intensity [cts] Date Sample [%] [%] tracer [%] [%] (overall very low-low) 30.07.2018 QK Methodological problems Blood
Liver
Kidney 94.04 5.96 369 Urine 82.51 17.49 726 Tumor
01.08.2018 QK 94.27 5.73 96.02 3.98 384 Blood
Liver
93.92.sup.# 6.08.sup.#
Kidney 94.58 5.42 572 Urine 96.2 3.80 98.55 1.55 395 Tumor
02.08.2016 QK Activity level 97.43 2.57 Blood too low for TLC 96.80 3.20 Liver 74.15.sup.# 25.85.sup.# Kidney 96.48 3.52 Urine 95.85 4.15 Tumor 96.47 4.15 * due to the low activity level, the TLC measurements with signal intensity <200 cts have been deleted.
[0262] Discussion of the TLC Data
[0263] Since it is very difficult to detect n.c.a. .sup.18F-fluoride by means of RP-18 chromatography (due to free Si—OH groups of the matrix that interact with nca fluoride), thin layer chromatography was performed to investigate to quantify F-18-Fluoride in the extracted solutions.
[0264] Since none of the reagents and salts normally used for protein precipitation are tested for cold fluoride and to avoid possible liberation of F-18-fluoride from the tracer by isotopic exchange, protein precipitation was not implemented in the sample preparation process—although such protein load often result in limited peak separation, peak tailing and activity that sticks at the start line. The solutions obtained after tissue extraction (or blood centrifugation) were directly used for TLC analysis.
[0265] Although the activity available for analysis was quite low in all samples, the TLC results reveal that the overall content of F-18-fluride was below approx. 6% in the tissue investigated, except: [0266] the urine sample obtained on Jul. 30, 2018 (17.49% free fluoride), [0267] the liver sample obtained on Aug. 2, 2018 (25.85% free fluoride).
[0268] Whereas the analysis of the urine by TLC is regarded as valid result (see Profile in the
[0269] It need to be noted that neither the biodistribution studies, nor the clinical PET scans in humans (status July 2018: approx 1400 scans with [F-18]rhPSMA7-rac) resulted in any suspicious or identifiable F-18-accumulation in bone by liberated F-18-fluoride. To further investigate the liberation of F-18 fluoride from [F-18]rhPSMA7-rac (as observed in one urine sample) we investigated the occurrence of F-18-Fluoride in further urine samples (normal mice) by means of RP-18 HPLC (new RP-18 end-capped column) and TLC analyses.
[0270] Radio-TLC-Analysis of the Formation of F-18-Fluoride in Normal Mice 30 Min p.i.
[0271] For this purpose normal mice were used. Urine samples were collected by means of a catheter over a period of 30 min. The urine was centrifuged and directly subjected to HPLC and TLC.
[0272] As shown in
[0273] Due to the fact that such high amounts of F-18 fluoride were not detected in the HPLC analyses of blood or organs, such as kidneys, tumor, liver etc., that no elevated activity uptake in bone was observed in the biodistribution studies in mice and no elevated activity uptake in bone was observed during the clinical PET scans with the [F-18]rhPSMA7-rac compound since [F-18]rhPSMA7-rac has been established for clinical scanning end of 2017 at the TUM (status end July, 2018: approx. 1400 PET scans in patients with prostate cancer) we concluded that [F-18]fluoride might be formed downstream from glomerular filtration of the tracer, resulting in the formation and subsequent excretion of [F-18]fluoride WITHOUT detectable uptake of F-18-fluoride in blood, organs or bones.
[0274] This assumption is supported by the literature on the toxicology of fluoride that describes relevant amounts of fluoride in KIDNEYS AND URINE. Normal urinary fluoride levels of 0.3 ppm were observed in mice (Bouaziz H et al., Fluoride 2005; 38(1):23-31). In another publication, the average fluoride concentration in the urine of normal mice was determined to be 0.13-0.14 μg/mL (Poesina N D et al. Rom J Morphol Embryol 2014, 55(2):343-349), and Inkielewicz I. et al. found that the fluoride content in the serum of rats is about 5% of the concentration of fluoride in the kidneys (serum: 0.051 μg/mL, kidneys: 0.942 μg/mL) (Fluoride; 36 (4); 263-266). Taken into account that most of the tracer is specifically taken up into and also physiologically cleared by the kidneys, an elevated fluoride level in the kidney, combined with a body temperature of 36.6° C., might result in a continuous elimination of F-18-fluoride from the rhPSMA-compounds in kidneys.
[0275] Consequently, fresh and nonradioactive urine samples collected from normal mice were incubated with [F-18]rhPSMA7.x for various time periods (see legend to
[0276] To further support the hypothesis, 500 nmol cold F-19-fluoride was added to fresh and nonradioactive urine of mice, followed by the addition of [F-18]rhPSMA7.3 and incubation for 2 h. According to the hypothesis, the high concentration of [F-19]fluoride should result in the formation of a significant amount of [F-18]fluoride.
[0277] Since isotopic exchange rates are depending on the concentration of the four relevant species in the equilibrium ([F-18]Fluoride, [F-19]fluoride, [F-18]rhPSMA7.3 and [F-19]rhPSMA7.3), it was also investigated, whether the addition of [F-18]fluoride to fresh and radioactive urine (20,6% [F-18]Fluoride, 79,4% [F-18]rhpsma7.3) followed by the addition of cold [F-19]rhPSMA7.3 tracer also result in the labelling of the radiopharmaceutical [F-18]rhPSMA7-3. Unexpectedly, even a small amount of 5 nmol [F-19]rhPSMA7-3 to the urine above resulted in an increase of [F-18]rhPSMA7.3 from 79.4% to 85.8% (F-18] Fluoride decreased from 20.6% to 14.2%) at room temperature.
[0278] The results obtained by isotopic exchange in urine are considered representative for all tracers conjugated with the 4-(di-tert-butyl[(18)F]fluorosilyl)-benzyl)oxy moiety and thus for all rhPSAM7 isomers.
[0279] Preclinical Dosimetry, Human Biodistribution and Uptake in Tumor Lesions
[0280] Please note that in the following 18F-rhPSMA-7 refers to .sup.natGa-.sup.18F-rhPSMA7-rac and 18F-rhPSMA-7.3 to .sup.natGa-.sup.18F-rhPSMA7.3
[0281] A) Preclinical Dosimetry of 18F-rhPSMA-7 and 18F-rhPSMA-7.3 in Mice
[0282] Aim was to assess the distribution and excretion of .sup.18F-rhPSMA-7 and .sup.18F-rhPSMA-7.3 at different time-points up to 300 minutes following a single intravenous administration in mice and to perform calculations for internal dosimetry.
[0283] Methods
[0284] 3-5 mice were injected per timepoint with a mean 25.6±3.6 MBq of 18F-rhPSMA-7 and 28.5±4.8 MBq of 18F-rhPSMA-7.3, respectively. Mice, severe combined immunodeficiency (SCID) were used for the experiments. All animal experiments were conducted in accordance with general animal welfare regulations in Germany and the institutional guidelines for the care and use of animals.
[0285] Mice were sacrificed at the following timepoints:
[0286] .sup.18F-rhPSMA-7: 10, 20, 40, 60, 120 and 180 minutes after administration.
[0287] .sup.18F-rhPSMA-7.3: 10, 60, 120, 180 and 300 minutes after administration.
[0288] Please note that based on initial experiments exhibiting prolonged renal kidney uptake for .sup.18F-rhPSMA-7.3 a late timepoint (300 min) was used for the final experiments.
[0289] The following tissues/fluids were harvested:
[0290] Urine, blood, heart, lung, spleen, pancreas, liver, stomach (emptied), small intestine (emptied), large intestine (emptied), kidneys, bladder, testis, fat, muscle (partial, femoral), femur, tail and brain. Urine was collected with a pipette in the CO.sub.2 gas chamber. In case of missing urination in the chamber the bladder was aspirated with an insulin syringe. Blood was withdrawn instantly after sacrifice with an insulin syringe from the heart. All other tissues and organs were dissected and transferred directly in plastic containers.
[0291] The weights of the samples in the plastic containers were measured using an electronic balance. The weights of the empty and pre-labeled plastic containers for the dedicated samples were measured beforehand. The tare weight of the plastic containers was subtracted from the weight of the measurement sample with the plastic container. The thus-calculated weight was designated as the weight of the measurement sample.
[0292] The plastic containers containing the measurement samples were placed in specific racks of an automatic gamma counter (PerkinElmer-Wallac, Waltham, USA) for measuring the counting rate over 60 seconds (counts per minute=cpm). In addition, a 1% (v/v) standard (n=5) with a known amount of radioactivity was measured together with the samples to convert the counting rate of the organ samples into activity.
[0293] Data Analysis
[0294] The counting rates of measurement samples were automatically corrected for decay. The radioactivity distribution ratios (unit: percentage of the injected dose (% ID) in the measurement samples were determined using the equation below. The sum of the counting rates from all measurement samples obtained from one mouse was designated as the counting rate for administrated radioactivity.
[0295] The radioactivity distribution ratio per unit weight of the measurement sample (unit: % ID/g) excluding urine and feces samples was determined by using the equation below. The weight of the measurement sample was determined by subtraction of the empty measurement container from the container including the sample.
[0296] Dosimetry Analysis
[0297] For consistency of statistical calculations for each radiotracer the same number of time-points for .sup.18F-rhPSMA-7 and .sup.18F-rhPSMA-7.3 was used. Therefore, for .sup.18F-rhPSMA-7 the 10 min and 20 min time points were combined creating a 15 min endpoint.
[0298] The time-integral of activity for the accumulation in the significant source organs (AUCs) were generated both with numerical integration and physical decay according to J Juan et al., Journal of Pharmaceutical Sciences, 1993, 82:762-763.
[0299] Kirshner et al. established a method that uses linear scaling of the percent injected dose in the animal by the ratio of the organ weights and total body weights of phantoms in both species. [0300] Kirschner A S, Ice R D, Beierwaltes W H. Radiation Dosimetry of 131I-19-Iodocholesterol. J Nucl Med. 1973 Sep. 1; 14(9):713-7. [0301] Kirschner A, Ice R, Beierwaltes W. Letters to the editor. J Nucl Med. (1975):248-9.
[0302] In brief, to calculate a human dosimetry from the biodistribution in the mice, an extrapolation was necessary to account for the differences between the animals and humans. Normal-organ radiation doses were estimated for the 70-kg Standard Adult anatomic model using time-depending organ activity concentrations (in percent of the injected dose per gram, % ID/g) and total-body activities measured in the biodistribution studies in mice.
[0303] Tissue activity concentrations in mice were converted to tissue fractional activities in the 70-kg Standard Adult using the relative fractional organ masses in the Standard Adult and the “standard” 25-gramm mouse. Time-dependent total-body activity was fit to an exponential function and the difference between the injected activity and the total-body activity was assumed to be excreted to the urine because activity concentrations in the liver and GI tracer were low at all time points studied.
[0304] Organ residence time was calculated by numerical integration using the trapezoidal rule and the rest-of-body .sup.18F residence times was calculated as the difference between the total-body residence time and the sum of the organ and urine residence times. The bladder contents residence time was estimated using the dynamic voiding model in the OLINDA/EXM 1.0 dosimetry software. Finally, the Standard Adult mean organ dose equivalents (in mSv/MBq) and effective dose (also in mSv/MBq) were then calculated using OLIN DA/EXM 1.0.
[0305] Final calculation of radiation absorbed dose and dosimetry from biodistribution in mice: The tissues or organs in which a significant accumulation of radioactivity occurs (i.e., source organ) were kidney, spleen, lung, liver and heart. With respect to activity accumulation and clearance, a rapid clearance from blood and clearance to urine but relatively slow build-up in kidney was found.
[0306] Results
TABLE-US-00014 TABLE 14 Dosimetry results for .sup.18F-rhPSMA-7 using a 3.5 h bladder voiding interval. Target Organ Alpha Beta Photon Total EDE Cont. ED Cont. Adrenals 0.00E000 1.95E−03 5.85E−03 7.80E−03 0.00E000 3.90E−05 Brain 0.00E000 1.95E−03 2.54E−03 4.49E−03 0.00E000 2.24E−05 Breasts 0.00E000 1.95E−03 2.29E−03 4.24E−03 6.36E−04 2.12E−04 Gallbladder Wall 0.00E000 1.95E−03 5.54E−03 7.49E−03 0.00E000 0.00E000 LLI Wall 0.00E000 1.95E−03 1.41E−02 1.61E−02 9.66E−04 1.93E−03 Small Intestine 0.00E000 1.95E−03 8.40E−03 1.04E−02 0.00E000 5.18E−05 Stomach Wall 0.00E000 1.95E−03 5.05E−03 7.00E−03 0.00E000 8.40E−04 ULI Wall 0.00E000 1.95E−03 7.31E−03 9.26E−03 0.00E000 4.63E−05 Heart Wall 0.00E000 8.82E−04 3.54E−03 4.42E−03 0.00E000 0.00E000 Kidneys 0.00E000 4.70E−02 1.80E−02 6.51E−02 3.91E−03 3.25E−04 Liver 0.00E000 6.35E−04 3.63E−03 4.27E−03 0.00E000 2.13E−04 Lungs 0.00E000 1.25E−03 3.04E−03 4.30E−03 5.16E−04 5.16E−04 Muscle 0.00E000 1.95E−03 5.73E−03 7.68E−03 0.00E000 3.84E−05 Ovaries 0.00E000 1.95E−03 1.35E−02 1.55E−02 3.87E−03 3.09E−03 Pancreas 0.00E000 1.95E−03 6.13E−03 8.08E−03 0.00E000 4.04E−05 Red Marrow 0.00E000 1.39E−03 5.41E−03 6.80E−03 8.16E−04 8.16E−04 Osteogenic Cells 0.00E000 4.18E−03 4.75E−03 8.93E−03 2.68E−04 8.93E−05 Skin 0.00E000 1.95E−03 2.98E−03 4.93E−03 0.00E000 4.93E−05 Spleen 0.00E000 1.59E−02 1.01E−02 2.61E−02 1.56E−03 1.30E−04 Testes 0.00E000 1.95E−03 9.69E−03 1.16E−02 0.00E000 0.00E000 Thymus 0.00E000 1.95E−03 3.24E−03 5.18E−03 0.00E000 2.59E−05 Thyroid 0.00E000 1.95E−03 3.23E−03 5.18E−03 1.55E−04 2.59E−04 Urinary Bladder Wall 0.00E000 2.45E−01 1.08E−01 3.54E−01 2.12E−02 1.77E−02 Uterus 0.00E000 1.95E−03 2.61E−02 2.80E−02 1.68E−03 1.40E−04 Total Body 0.00E000 2.37E−03 5.39E−03 7.77E−03 0.00E000 0.00E000 Effective Dose Equivalent (mSv/MBq) 3.56E−02 Effective Dose (mSv/MBq) 2.66E−02
TABLE-US-00015 TABLE 15 Dosimetry results for .sup.18F-rhPSMA-7 using a 1.0 h bladder voiding interval. Target Organ Alpha Beta Photon Total EDE Cont. ED Cont. Adrenals 0.00E000 1.95E−03 5.64E−03 7.59E−03 0.00E000 3.79E−05 Brain 0.00E000 1.95E−03 2.54E−03 4.49E−03 0.00E000 2.24E−05 Breasts 0.00E000 1.95E−03 2.25E−03 4.20E−03 6.30E−04 2.10E−04 Gallbladder Wall 0.00E000 1.95E−03 4.96E−03 6.91E−03 0.00E000 0.00E000 LLI Wall 0.00E000 1.95E−03 7.25E−03 9.20E−03 5.52E−04 1.10E−03 Small Intestine 0.00E000 1.95E−03 5.79E−03 7.73E−03 0.00E000 3.87E−05 Stomach Wall 0.00E000 1.95E−03 4.70E−03 6.65E−03 0.00E000 7.98E−04 ULI Wall 0.00E000 1.95E−03 5.33E−03 7.27E−03 0.00E000 3.64E−05 Heart Wall 0.00E000 8.82E−04 3.48E−03 4.36E−03 0.00E000 0.00E000 Kidneys 0.00E000 4.70E−02 1.76E−02 6.47E−02 3.88E−03 3.23E−04 Liver 0.00E000 6.35E−04 3.39E−03 4.02E−03 0.00E000 2.01E−04 Lungs 0.00E000 1.25E−03 3.01E−03 4.26E−03 5.11E−04 5.11E−04 Muscle 0.00E000 1.95E−03 4.01E−03 5.96E−03 0.00E000 2.98E−05 Ovaries 0.00E000 1.95E−03 7.21E−03 9.16E−03 2.29E−03 1.83E−03 Pancreas 0.00E000 1.95E−03 5.86E−03 7.80E−03 0.00E000 3.90E−05 Red Marrow 0.00E000 1.39E−03 4.29E−03 5.68E−03 6.82E−04 6.82E−04 Osteogenic Cells 0.00E000 4.18E−03 4.09E−03 8.27E−03 2.48E−04 8.27E−05 Skin 0.00E000 1.95E−03 2.38E−03 4.32E−03 0.00E000 4.32E−05 Spleen 0.00E000 1.59E−02 9.90E−03 2.58E−02 1.55E−03 1.29E−04 Testes 0.00E000 1.95E−03 5.09E−03 7.03E−03 0.00E000 0.00E000 Thymus 0.00E000 1.95E−03 3.20E−03 5.15E−03 0.00E000 2.57E−05 Thyroid 0.00E000 1.95E−03 3.23E−03 5.17E−03 1.55E−04 2.59E−04 Urinary Bladder Wall 0.00E000 7.87E−02 3.66E−02 1.15 E−01 6.92E−03 5.76E−03 Uterus 0.00E000 1.95E−03 1.12E−02 1.31E−02 7.87E−04 6.56E−05 Total Body 0.00E000 2.27E−03 3.93E−03 6.19E−03 0.00E000 0.00E000 Effective Dose Equivalent (mSv/MBq) 1.82E−02 Effective Dose (mSv/MBq) 1.22E−02
TABLE-US-00016 TABLE 16 Dosimetry results for .sup.18F-rhPSMA-7.3 using a 3.5 h bladder voiding interval. Target Organ Alpha Beta Photon Total EDE Cont. ED Cont. Adrenals 0.00E000 3.12E−03 7.93E−03 1.10E−02 0.00E000 5.52E−05 Brain 0.00E000 3.12E−03 4.07E−03 7.19E−03 0.00E000 3.59E−05 Breasts 0.00E000 3.12E−03 3.55E−03 6.67E−03 1.00E−03 3.34E−04 Gallbladder Wall 0.00E000 3.12E−03 7.46E−03 1.06E−02 0.00E000 0.00E000 LLI Wall 0.00E000 3.12E−03 1.28E−02 1.59E−02 9.53E−04 1.91E−03 Small Intestine 0.00E000 3.12E−03 9.42E−03 1.25E−02 0.00E000 6.27E−05 Stomach Wall 0.00E000 3.12E−03 7.02E−03 1.01E−02 0.00E000 1.22E−03 ULI Wall 0.00E000 3.12E−03 8.57E−03 1.17E−02 0.00E000 5.85E−05 Heart Wall 0.00E000 1.32E−03 5.39E−03 6.71E−03 0.00E000 0.00E000 Kidneys 0.00E000 5.11E−02 2.07E−02 7.18E−02 4.31E−03 3.59E−04 Liver 0.00E000 9.70E−04 5.02E−03 5.99E−03 0.00E000 3.00E−04 Lungs 0.00E000 1.95E−03 4.66E−03 6.61E−03 7.93E−04 7.93E−04 Muscle 0.00E000 3.12E−03 6.55E−03 9.67E−03 0.00E000 4.83E−05 Ovaries 0.00E000 3.12E−03 1.26E−02 1.57E−02 3.92E−03 3.14E−03 Pancreas 0.00E000 3.12E−03 8.29E−03 1.14E−02 0.00E000 5.70E−05 Red Marrow 0.00E000 2.22E−03 6.79E−03 9.01E−03 1.08E−03 1.08E−03 Osteogenic Cells 0.00E000 6.70E−03 6.52E−03 1.32E−02 3.97E−04 1.32E−04 Skin 0.00E000 3.12E−03 3.83E−03 6.95E−03 0.00E000 6.95E−05 Spleen 0.00E000 1.52E−02 1.15E−02 2.67E−02 1.60E−03 1.34E−04 Testes 0.00E000 3.12E−03 8.96E−03 1.21E−02 0.00E000 0.00E000 Thymus 0.00E000 3.12E−03 5.08E−03 8.20E−03 0.00E000 4.10E−05 Thyroid 0.00E000 3.12E−03 5.15E−03 8.27E−03 2.48E−04 4.14E−04 Urinary Bladder Wall 0.00E000 1.56E−01 7.14E−02 2.27E−01 1.36E−02 1.14E−02 Uterus 0.00E000 3.12E−03 2.04E−02 2.36E−02 1.41E−03 1.18E−04 Total Body 0.00E000 3.52E−03 6.33E−03 9.86E−03 0.00E000 0.00E000 Effective Dose Equivalent (mSv/MBq) 2.94E−02 Effective Dose (mSv/MBq) 2.17E−02
TABLE-US-00017 TABLE 17 Dosimetry results for .sup.18F-rhPSMA-7.3 using a 1.0 h bladder voiding interval. Target Organ Alpha Beta Photon Total EDE Cont. ED Cont. Adrenals 0.00E000 3.12E−03 7.79E−03 1.09E−02 0.00E000 5.46E−05 Brain 0.00E000 3.12E−03 4.06E−03 7.18E−03 0.00E000 3.59E−05 Breasts 0.00E000 3.12E−03 3.53E−03 6.65E−03 9.97E−04 3.32E−04 Gallbladder Wall 0.00E000 3.12E−03 7.11E−03 1.02E−02 0.00E000 0.00E000 LLI Wall 0.00E000 3.12E−03 8.45E−03 1.16E−02 6.94E−04 1.39E−03 Small Intestine 0.00E000 3.12E−03 7.78E−03 1.09E−02 0.00E000 5.45E−05 Stomach Wall 0.00E000 3.12E−03 6.80E−03 9.92E−03 0.00E000 1.19E−03 ULI Wall 0.00E000 3.12E−03 7.33E−03 1.05E−02 0.00E000 5.23E−05 Heart Wall 0.00E000 1.32E−03 5.36E−03 6.68E−03 0.00E000 0.00E000 Kidneys 0.00E000 5.11E−02 2.04E−02 7.16E−02 4.29E−03 3.58E−04 Liver 0.00E000 9.70E−04 4.87E−03 5.84E−03 0.00E000 2.92E−04 Lungs 0.00E000 1.95E−03 4.63E−03 6.58E−03 7.90E−04 7.90E−04 Muscle 0.00E000 3.12E−03 5.47E−03 8.59E−03 0.00E000 4.30E−05 Ovaries 0.00E000 3.12E−03 8.61E−03 1.17E−02 2.93E−03 2.35E−03 Pancreas 0.00E000 3.12E−03 8.12E−03 1.12E−02 0.00E000 5.62E−05 Red Marrow 0.00E000 2.22E−03 6.09E−03 8.31E−03 9.97E−04 9.97E−04 Osteogenic Cells 0.00E000 6.70E−03 6.11E−03 1.28E−02 3.84E−04 1.28E−04 Skin 0.00E000 3.12E−03 3.45E−03 6.57E−03 0.00E000 6.57E−05 Spleen 0.00E000 1.52E−02 1.14E−02 2.66E−02 1.59E−03 1.33E−04 Testes 0.00E000 3.12E−03 6.08E−03 9.20E−03 0.00E000 0.00E000 Thymus 0.00E000 3.12E−03 5.06E−03 8.18E−03 0.00E000 4.09E−05 Thyroid 0.00E000 3.12E−03 5.15E−03 8.27E−03 2.48E−04 4.13E−04 Urinary Bladder Wall 0.00E000 5.18E−02 2.66E−02 7.84E−02 4.70E−03 3.92E−03 Uterus 0.00E000 3.12E−03 1.11E−02 1.43E−02 8.55E−04 7.13E−05 Total Body 0.00E000 3.45E−03 5.42E−03 8.87E−03 0.00E000 0.00E000 Effective Dose Equivalent (mSv/MBq) 1.85E−02 Effective Dose (mSv/MBq) 1.28E−02
CONCLUSION
[0307] The radioactivity distribution ratios were highest in kidneys after administration of both .sup.18F-rhPSMA-7 and .sup.18F-rhPSMA-7.3 at all examined time points in mice. Moreover, it was high in the spleen and in the bladder for both radiotracers compared to all other assessed tissues, where activity ratios were lower than 8% ID/g.
[0308] Since the majority of .sup.18F-rhPSMA-7/.sup.18F-rhPSMA-7.3 activity augment in the kidneys and the excretion via bladder reveal high activities, the main excretion route is defined via kidneys and the urinary system.
[0309] Using a 3.5 h and 1.0 h bladder voiding interval the extrapolated total effective doses were 2.66E-02 and 1.22E-02 mSv/MBq for .sup.18F-rhPSMA-7 and 2.17E-02 and 1.28E-02 mSv/MBq for .sup.18F-rhPSMA-7.3, respectively. An injection of up to 370 MBq (10 mCi) for a clinical scan would result in a favorable radiation exposure of less than 5 mSv for both agents assuming a 1 h voiding interval.
[0310] Differences worth to mention between both radiotracers are only evident regarding kidney uptake as .sup.18F-rhPSMA-7.3 tends to accumulate more gradual with longer retention. Yet radiation exposure is comparable between both agents.
[0311] B) Human Biodistribution and Uptake in Tumor Lesions of 18F-rhPSMA-7 and 18F-rhPSMA-7.3
[0312] The following sections describe biodistribution of 18F-rhPSMA-7 and 18F-rhPSMA-7.3. Proof-of-concept evaluation was conducted under compassionate use. The agent was applied in compliance with The German Medicinal Products Act, AMG § 13 2b, and in accordance with the responsible regulatory body (Government of Oberbayern).
[0313] All subjects were examined on a Biograph mCT scanner (Siemens Medical Solutions, Erlangen, Germany). All PET scans were acquired in 3D-mode with an acquisition time of 2-4 min per bed position. Emission data were corrected for randoms, dead time, scatter, and attenuation and were reconstructed iteratively by an ordered-subsets expectation maximization algorithm (four iterations, eight subsets) followed by a postreconstruction smoothing Gaussian filter (5-mm full width at one-half maximum).
[0314] Methods
[0315] Human biodistribution was estimated by analysing clinical .sup.18F-rhPSMA-7- and .sup.18F-rhPSMA-7.3-PET/CT exams in 47 and 32 patients, respectively. Mean injected activities were 324 (range 236-424) MBq vs. 345 (range 235-420) MBq and uptake times were 84 (range 42-166) min and vs. 76 (range 59-122) min for .sup.18F-rhPSMA-7 vs. .sup.18F-rhPSMA-7.3, respectively.
[0316] The mean and maximum standardized uptake values (SUVmean/SUVmax) were determined for background (gluteal muscle), normal organs (salivary glands, blood pool, lung, liver, spleen, pancreas, duodenum, kidney, bladder, bone) and three representative tumor lesions. Tumor uptake was analyzed in 89 lesions (26 primary tumors/local recurrences, 23 bone, 38 lymph node and 2 visceral metastases) and 63 lesions (14 primary tumors/local recurrences, 30 bone, 18 lymph node and 1 visceral metastases) for .sup.18F-rhPSMA-7 and .sup.18F-rhPSMA-7.3, respectively.
[0317] For calculation of the SUV, circular regions of interest were drawn around areas with focally increased uptake in transaxial slices and automatically adapted to a three-dimensional volume of interest (V01) at a 50% isocontour. Organ-background and Tumor-background ratios were calculated.
[0318] Results
[0319] Human biodistribution of .sup.18F-rhPSMA-7 and .sup.18F-rhPSMA-7.3 showed the typical pattern known from other PSMA-ligands. Uptake parameters for .sup.18F-rhPSMA-7 and .sup.18F-rhPSMA-7.3 were very similar with a lower activity retention in the bladder and higher uptake in tumor lesions for .sup.18F-rhPSMA-7.3: SUVmean for .sup.18F-rhPSMA-7 vs. .sup.18F-rhPSMA-7.3 were 16.9 vs. 16.0 (parotid gland), 19.6 vs. 19.6 (submandibular gland), 2.0 vs. 1.9 (blood pool), 0.7 vs. 0.7 (lungs), 7.0 vs. 7.3 (liver), 9.1 vs. 8.5 (spleen), 32.4 vs. 35.5 (kidney), 2.5 vs. 2.8 (pancreas), 10.9 vs. 11.0 (duodenum), 1.1 vs. 1.3 (non-diseased bone) and 10.2 vs. 2.0 (bladder) for .sup.18F-rhPSMA-7 vs. .sup.18F-rhPSMA-7.3, respectively. In particular, uptake values of .sup.18F-rhPSMA-7.3 vs. .sup.18F-rhPSMA-7 were significantly lower for retention in the bladder (2.0±0.8 vs. 6.3±21.2, p<0.05) and significantly higher for tumor lesions (32.5±42.7 vs. 20.0±20.2, p<0.05).
TABLE-US-00018 TABLE 18 SUVmax and SUVmean of normal organs and tumor lesions using .sup.18F-rhPSMA-7. Data are shown as mean, minimum and maximum. SUVmax SUVmean mean min max mean min max background 1.0 0.6 1.8 0.6 0.4 1.2 parotic gland 23.8 8.2 42.3 16.9 5.5 32.7 submandibular gland 27.0 10.1 43.8 19.6 7.0 29.7 bloodpool 2.4 1.6 3.9 2.0 1.1 17.0 lungs 1.1 0.5 3.1 0.7 0.3 2.0 liver 9.5 4.5 25.2 7.0 3.2 17.7 spleen 11.8 4.7 21.0 9.1 3.4 17.1 kidney 44.8 19.1 75.2 32.4 13.2 54.7 pancreas 3.7 1.8 7.9 2.5 1.3 5.5 duodenum 14.8 2.8 32.7 10.9 1.9 23.9 bone 1.7 0.8 3.1 1.1 0.6 2.1 bladder 8.5 0.5 112.0 6.3 0.3 85.7 tumor 27.6 3.1 167.2 20.0 2.1 115.7
TABLE-US-00019 TABLE 19 SUVmax and SUVmean of normal organs and tumor lesions using .sup.18F-rhPSMA- 7.3. Data are shown as mean, minimum and maximum. SUVmax SUVmean mean min max mean min max background 1.0 0.6 1.7 0.7 0.4 1.1 parotic gland 24.6 11.2 38.3 16.0 8.2 25.0 submandibular gland 28.4 14.6 47.4 19.6 10.4 33.4 bloodpool 2.8 1.9 3.9 1.8 1.3 2.5 lungs 1.1 0.7 1.9 0.7 0.4 1.1 liver 9.7 4.6 15.4 7.3 3.2 12.3 spleen 11.4 5.0 22.5 8.5 3.7 17.9 kidney 51.9 30.9 99.9 35.5 20.7 70.6 pancreas 4.2 2.4 7.8 2.8 1.6 5.2 duodenum 16.4 6.1 32.2 11.0 3.0 23.0 bone 2.1 1.1 3.4 1.3 0.7 2.2 bladder 3.1 1.1 6.0 2.0 0.7 4.1 tumor 44.0 2.4 316.0 32.5 1.6 224.1
TABLE-US-00020 TABLE 20 Ratio SUVmax and SUVmean to background of normal organs and tumor lesions using .sup.18F-rhPSMA-7. Data are shown as mean, minimum and maximum. ratio SUVmax ratio SUVmean mean min max mean min max parotid gland 25.2 8.2 45.3 28.3 9.2 54.5 submandibular 28.7 10.1 54.7 33.3 11.7 61.8 gland bloodpool 2.5 1.3 4.8 3.2 1.6 21.3 lungs 1.1 0.4 3.3 1.1 0.4 4.0 liver 10.4 4.7 42.0 11.9 4.6 44.3 spleen 12.5 4.7 35.0 15.1 5.7 39.5 kidney 48.1 18.2 98.7 55.2 19.8 109.3 pancreas 3.9 1.5 11.3 4.3 1.9 10.8 duodenum 15.7 2.8 31.3 18.4 3.2 35.3 bone 1.7 0.9 2.9 1.8 1.0 3.2 bladder 8.7 0.6 112.0 10.2 0.5 142.8 tumor 32.0 3.1 278.6 36.0 3.5 289.3
TABLE-US-00021 TABLE 21 Ratio SUVmax and SUVmean to background of normal organs and tumor lesions using .sup.18F- rhPSMA-7.3. Data are shown as mean, minimum and maximum. ratio SUVmax ratio SUVmean mean min max mean min max parotid gland 24.7 11.9 46.2 25.2 12.4 44.6 submandibular 28.2 14.0 62.1 30.6 15.7 62.3 gland bloodpool 2.8 1.5 5.2 2.9 1.7 4.9 lungs 1.0 0.6 1.8 1.0 0.6 1.8 liver 9.7 4.0 19.0 11.4 4.1 20.7 spleen 11.4 3.6 22.4 13.3 3.9 28.6 kidney 51.8 25.7 93.0 55.6 27.6 95.5 pancreas 4.1 2.2 6.9 4.4 2.3 7.8 duodenum 16.2 6.9 34.3 17.1 4.7 39.4 bone 2.0 1.1 3.2 2.1 1.0 3.6 bladder 3.1 0.9 5.5 3.1 0.9 6.8 tumor 43.6 1.7 321.2 50.8 1.8 356.4
CONCLUSION
[0320] Human biodistribution is similar between .sup.18F-rhPSMA-7 and .sup.18F-rhPSMA-7.3 for most normal organs. However, tracer retention in the bladder is significantly lower and uptake in tumor lesions significantly higher for .sup.18F-rhPSMA-7.3 posing a clear advantage for clinical imaging. Imaging examples with favorable human biodistribution and high uptake of tumor lesions of .sup.18F-rhPSMA-7.3 are shown in