Reagents and Instrumentation
All reagents and solvents were of the best grade available and were obtained from Sigma-Aldrich, Merck, Fluka, AlfaAesar, VWR, AcrosOrganics and Fisher Scientific and used without further purification. They were provided with a septum. All culture reagents were from Gibco BRL, Life Technologies (Grand Island, NY). N.c.a. 177LuCl3 and 111InCl3 were obtained from Isotope Technologies Garching GmbH (ITG) (Munich, Germany) and b.e.Imaging GmbH (Baden-Baden, Germany), respectively. The GalliaPharm® Ge-68/Ga-68 Generator was available from Eckert & Ziegler (Berlin, Germany).
Deuterated solvents for NMR spectra were commercially available by Deutero. Thin layer chromatography plates from Merck, silica gel 60 F254 coated aluminium plates, were used for the analysis. Silica gel 60 (core size 0.063 / 0.200 mm) from Macherey-Nagel was used for purification by column chromatography.
The purification of the peptide was performed by semipreparative RP-HPLC on a 120-5 C18 Nucleosil column (250 x 21 mm) applying a linear gradient of 15-90% solvent B in 25 min at a flow rate of 12 mL / min (solvent A, 0.1% TFA / H2O; solvent B, 0.1%TFA / Acetonitrile).
The quality control of the peptides as well as the radiolabeled compounds was performed by analytical RP-HPLC from Knauer advanced scientific instrument equipped with a Knauer Smartline Manager 5000, a Smartline Pump 1000 and a Smartline UV Detector 2600. The RP-HPLC runs were performed on an analytical 120-5 C18 Nucleosil column (250 x 4.5 mm) applying a linear gradient of 15-90% solvent B in 25 min at a flow rate of 1 mL / min. (solvent A, 0.1% TFA / H2O; solvent B, 0.1% TFA / Acetonitrile). Ultraviolet detection was performed using a Knauer detector at 240 nm. For radioactivity measurement, a Na(TI) well-type scintillation Gina star was used. The radiotracer solutions were prepared by dilution with 0.9% NaCl.
The human prostate adenocarcinoma cell line PC3 was obtained from CLS Cell Lines Service GmbH, (Eppelheim, Germany). Cell culture media Dulbecco's Modified Eagle Medium (DMEM) with GlutaMax-I Supplement, F-12 Nutrient Mixture with GlutaMax-I Supplement, Dubecco's Phosphate Buffered Saline (DPBS), Fetal Bovine Serum (FBS), Trypsin-EDTA and antibiotic solution Penicillin-Streptomycin were from Gibco BRL, Life Technologies (Grand Island, NY) and purchased from ThermoFisher (Switzerland).
ESI-MS mass spectra were acquired on a Bruker Daltonics Esquire 3000 plus device.
The 1H-, 13C-NMR measurements were performed on a Bruker Avance III HD 400 (400 MHz) or Avance III 600 (600 MHz). LC / MS spectra were measured on an Agilent Technologies 1220 Infinity LC system coupled to an Agilent Technologies 6130B Single Quadrupole LC / MS system.
TLC scans were acquired on an Elysia Raytest TLC scanner using the Gina Star TLC software.
Quantitative γ-counting was performed with a Cobra II Gamma Counter from Packard Instrument (USA).
All experiments were carried out twice in triplicates.
Synthesis of the prochelator AAZTA5-(tBu)4
The precursor AAZTA5-(tBu)4 was successfully synthesized over 4-steps following the protocol described by Sinnes et al. (Sinnes et al. 2019b). Briefly, the synthesis steps are described below and shown in Figure 2:
1,4-Dibenzyl-6-methylpentanoate-6-nitroperhydro-1,4-diazepane (1)
2-nitrocyclohexanone (2.00 g; 13.9 mmol) and Amberlyst A21 (1.05 g) were mixed with dry methanol (35 mL). The solution was heated to 60 °C and stirred under reflux for 1 h. N,N'-dibenzylethylene-diamine (3.36 g; 13.9 mmol) and paraformaldehyde (1.67 g; 55.5 mmol) were added to the solution. The suspension was heated to 80°C and stirred overnight. After completion of the reaction, the suspension was filtered and the filtrate concentrated under vacuum. After column chromatographic purification (CH / EA; 9:1; Rf= 0.27) a yellow oil was obtained as product 1 (5.20 g; 11.8 mmol; 85%).
1,4-Di(tert-butylacetate)-6-methylpentanoate-6-amino-di(tert-butylacetate)-perhydro-1,4-diazepine (3)
1 (1.05 g, 2.39 mmol) was added to a solution containing Pd(OH)2 / C (0.62 g, 10 wt%) and abs. ethanol (20 mL). Acetic acid (411 µL; 7.18 mmol) was added, the solution was saturated with hydrogen and stirred overnight at room temperature. After completion of the reaction, Pd(OH)2 / C was filtered over Celite and the filtrate was concentrated under vacuum. Crude product 2, a white-yellowish solid, was used for further reaction without further purification.
2 (1.05 g; 2.39 mmol) and K2CO3 (1.32 g; 9.57 mmol) were dissolved in dry acetonitrile (30 mL). tert-butyl bromoacetate (1.41 mL; 9.57 mmol) and potassium iodide (0.80 g) were added and stirred overnight at 40 °C. The solution was concentrated under vacuum and purified by column chromatography (CH / EA, 7:1; Rf= 0.15). Compound 3 was obtained as yellow oil (0.89 g; 1.30 mmol; 54%).
N,N’1,4-Di(tert-butylacetate)-6-pentanoicacid-6-(amino-di(tert-butylacetate))-perhydro-1,4-diazepine (4)
3 (293 mg, 0.43 mmol) was dissolved in 1,4-dioxane / water (2:1; 6 mL). 1 M LiOH solution (641 µL; 0.64 mmol) was added and stirred overnight at room temperature. After completion of the reaction, the solution was concentrated, the residue concentrated with 1 M NaHCO3 solution and extracted several times with chloroform. The organic phase was extracted with water, dried with Mg2SO4 and concentrated under vacuum. Compound 4 was obtained a yellowish oil (224 mg; 0.33 mmol; 76%).
Synthesis of the Chelator-Peptide Conjugate
The peptide-chelator conjugate was synthesized manually using standard Fmoc chemistry and Rink amide 4-methylbenzhydrylamine resin. The spacer (Pip) and the prochelator (AAZTA5-(tBu)4) were consecutively coupled to the peptide using 1-[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxid hexafluorophosphate (HATU) as an activating agent. The cleavage of the peptide and the simultaneous deprotection of the side chain-protecting groups was performed using trifluoroacetic acid / triisopropylsilane / H2O (95 / 2.5 / 2.5). The crude conjugate was further purified by semipreparative RP-HPLC as described in the “Reagents and Instrumentation” section.
Radiochemistry
68Ga-LF1: The 68Ga-labeled radiotracer was prepared within 5 min, using the Modular-Lab PharmTracer module by Eckert & Ziegler (Berlin, Germany). The radiolabeling performance of LF1 was assessed at pH 4.0 (0.2 M sodium acetate buffer), at RT using a conjugate amount of 5 - 20 μg (approximately 3 - 12 nmol). Briefly, the 68Ge / 68Ga-generator was eluted with 5 mL HCl 0.1 N and the eluate (~ 400 MBq) was loaded onto a cation exchange column (Strata-XC, Phenomenex). 68Ga was eluted with 700 μL of a mixture of 5.5 M NaCl / 0.1 M HCl directly in a vial containing 400 μL of 1.8 M sodium acetate buffer, 2 mL H2O, 200 μL of EtOH, 5 mg ascorbic acid / 20 μL H2O and the tested amount of the conjugate, followed by SepPak C-18 purification to remove uncomplexed radiometal.
177Lu-LF1 / 111In-LF1 and 177Lu-RM2: The 177Lu- and 111In-labeled radiotracers were prepared by dissolving 5-10 μg (approximately 3 - 6 nmol) of LF1 in 250 μL ammonium acetate buffer (0.5 M, pH 5.4), followed by incubation with 177LuCl3 (30 - 100 MBq) or 111InCl3 (approximately 35 - 40 MBq) for 10 min at RT and were used without any further purification step.
After the labeling with 68Ga, 177Lu or 111In and the quality control of the generated radiotracers, one equivalent of either natGa(NO3)3 or natLuCl3 x 6H2O or natInCl3 x 2H2O were added to the relevant radiolabelling solutions. The final solutions were incubated at RT for 10 min to obtain structurally characterized homogeneous ligands which were used for the saturation binding studies.
Quality control of the radiotracers / Stability
Chemical and radiochemical purity of the tested solutions were determined using an analytical Nucleosil 100-5 C18 column applying the conditions described in the “Reagents and Instrumentation” section.
The presence of free 68Ga3+ and 68Ga3+-colloid in the 68Ga-LF1 preparation was quantified by radio thin layer chromatography (Radio-TLC) using Silica gel 60-plates and two different mobile phase systems: a) 0.1 M Na-citrate; b) methanol / 1 M ammonium acetate (1 / 1, v / v). Using the first Radio-TLC system, the radiopeptide product and 68Ga-colloid remain immobilized at the starting point, whereas free 68Ga3+ ions move with the mobile phase. When the second system is used, only the labelled peptide moves with the mobile phase / solvent front.
The stability of the newly prepared radiotracers was tested via RP-HPLC for a period of 4 h post labeling.
Lipophilicity
The lipophilicity (LogDoctanol/PBS, pH 7.4) was estimated by the “shake-flask” method: The labeled conjugates (100 pmol; 1.08 MBq, 1.16 MBq and 0.50 MBq for 68Ga-LF1, 177Lu-LF1 and 111In-LF1 respectively) were added to a solution of 1-octanol (500 μL) and of PBS (500 μL, pH 7.4). The mixture was vortexed for 1 h to reach the equilibrium and then centrifuged (3000 rpm) for 10 min. From each phase, an aliquot (50 to 100 μL) was pipetted out and measured in a gamma-counter. Each measurement was repeated five times. Care was taken to avoid cross-contamination between the phases. The partition coefficient was calculated as the average log ratio of the radioactivity in the organic fraction and the PBS fraction.
Protein binding studies in human plasma
68Ga-LF1, 177Lu-LF1 and 111In-LF1 (100 pmol; 1.08 MBq, 1.16 MBq and 0.50 MBq for 68Ga-LF1, 177Lu-LF1 and 111In-LF1 respectively) were incubated with human plasma (0.5 mL) at 37 °C for 30 min and 60 min respectively. When the incubating period was completed, proteins were precipitated with a solution of 1 mL of MeOH / ACN (1:1). Centrifugation (10 min, 9660g) for the separation of proteins was then performed. After careful separation of the two phases, the respective activities were measured in a gamma-counter, followed by determination of the percentage of each radiotracer which does not bind to the plasma proteins.
Cell culture
The human prostate epithelial adenocarcinoma cell line PC3, which is known to overexpress GRPr, was cultured at 37 °C and 5% CO2 in Dulbecco's Modified Eagle Medium (DMEM) with GlutaMAX-I supplement and F-12 Nutrient Mix with GlutaMAX-I supplement in a ratio of 1:1. The medium was supplemented with 10% fetal bovine serum (FBS), penicillin (100 U / mL) and streptomycin (100 µg / mL).
Saturation Binding Studies
For receptor saturation analysis, PC3 cells were seeded at a density of 0.8 - 1 million cells per well in 6-well plates and incubated overnight with medium (DMEM : F12 (1:1) containing 1% FBS, 100 U/mL penicillin and 100 μg/mL streptomycin). The next day, the medium was removed, the cells washed and incubated for 1 h at 37 °C with fresh medium. Afterwards, the plates were placed on ice for 30 min followed by incubation with increasing concentrations of either 68/natGa-LF1 or 177/natLu-LF1 or 111/natIn-LF1 or 177/natLu-RM2 (1-100 nM) in phosphate-buffered saline binding buffer pH 7.4. After the addition of the radioligands, the cells were incubated for 120 min at 4 °C. Non-specific binding was determined in the presence of Tyr4-Bombesin at a final concentration of 1 μM. Then the cells were washed twice with ice-cold PBS, followed by solubilization with 1 N NaOH and the cell-associated radioactivity was measured using a gamma-counter. Specific binding was plotted against the total molar concentration of the added radiotracer. The Kd values and the concentration of the radiotracer required to saturate the receptors (Bmax) were determined by nonlinear regression using GraphPad (Prism 8 Graph Pad Software, San Diego, CA). For all the cell studies the values are normalized for 1x106 cells per well and all data are from two independent experiments with triplicates in each experiment.
Internalization Studies
For internalization experiments, PC3 cells were seeded into 6-well plates and treated as described above. Approximately 0.25 pmol of the respective radiopeptides were added to the medium and the cells were incubated (in triplicates) for 0.5, 1, 2, 4 and 6 h at 37 °C, 5% CO2 for 177Lu-LF1 and 111In-LF1 and for 15, 30, 60, 90, 120, 180 and 240 min at 37 °C, 5% CO2 for 68Ga-LF1. To determine nonspecific membrane binding and internalization, excess of Tyr4-Bombesin (final concentration 1 μΜ) was added to selected wells. At each time point, the internalization was stopped by removing the medium and washing the cells twice with ice-cold PBS. To remove the receptor-bound radioligand, an acid wash was carried out twice with a 0.1 M glycine buffer pH 2.8 for 5 min on ice. Finally, cells were solubilized with 1 N NaOH. The radioactivity of the culture medium, the receptor-bound, and the internalized fractions were measured in a γ-counter.
In vitro therapy studies with 177Lu-LF1 in the presence or without rapamycin
For the in vitro therapy studies, PC3 cells were seeded at a density of 50000-100000 cells (depending on the investigating time point: 100000, 75000 and 50000 cells for 24, 48, 72 h respectively) in 12-well plates and incubated overnight with cultivation medium at 37 °C and 5% CO2. The next day, the medium was removed, the cells washed, and culture medium containing either rapamycin (10 nM) or DMSO (control medium) was added. The plates were incubated at 37 °C and 5% CO2 for a period of 6 h. Afterwards, the medium containing rapamycin or DMSO was removed and fresh medium was added in each well. Furthermore, 1.85 MBq of 177Lu-LF1 was added to preselected wells. The monotherapy (either with rapamycin or 177Lu-LF1 alone) and combination therapy study (177Lu-LF1 in presence of rapamycin) were performed in parallel with untreated cells which served as reference. The viability of the cells was accessed by the trypan blue exclusion assay after incubation at 37 °C and 5% CO2 for 24, 48 and 72 h. The experiment was performed twice in triplicate.
Statistical Analysis
All data are expressed as the mean of values ± standard deviation (mean ± SD). Prism 8 Software (GraphPad Software) was used to determine statistical significance at the 95% confidence level, with a P value of less than 0.05 being considered significant.