Chemistry and radiochemistry
Chemicals and radionuclides
All chemicals were reagent grade and used without further purification. DOTA-MG11 (DOTA-minigastrin 11, DOTA-DGlu-Ala-Tyr-Gly-Trp-Met-Asp-Phe-NH2) and DG2 (Demogastrin 2, N4-Gly-DGlu-(Glu)5-Ala-Tyr-Gly-Trp-Met-Asp-Phe-NH2) [25] were purchased from PiChem (Graz, Austria). PA (phosphoramidon disodium dehydrate, N-(α-rhamnopyranosyloxyhydroxyphosphinyl)-L-leucyl-L-tryptophan × 2Na × 2H2O) was provided by PeptaNova GmbH (Sandhausen, Germany). TO (DL-thiorphan, DL-3-mercapto-2-benzylpropanoylglycine) was a kind gift of Prof. B. Roques (Université René Descartes, Paris, France). Lis (lisinopril dehydrate, ((S)1-1-[N2-(1-carboxy-3-phenylpropyl)-lysyl-proline dehydrate, MK 521) and Race (racecadotril, (RS)-benzyl N-[3-(acetylthio)-2-benzylpropanoyl]glycinate) were purchased from Sigma-Aldrich (Fig. 1).
Indium-111 used for labeling was purchased in the form of 111InCl3 in a solution of 0.05 M HCl (0.5 mL) from Mallinckrodt Medical B.V. (Petten, the Netherlands).
Preparation of [111In-DOTA]MG11
Lyophilized DOTA-MG11 was dissolved in water to a final concentration of 1 mM, and 50 μL aliquots were stored at −20 °C. Labeling with 111In was conducted in an Eppendorf vial containing 0.1 M sodium acetate buffer pH 4.6 in the presence of excess methionine (Met) to prevent oxidation of Met15 in DOTA-MG11 [26]. Freshly prepared sodium ascorbate buffer (10 mM) was added in the vial, followed by 111InCl3 solution (37–74 MBq), Met (1000 nmol), and DOTA-MG11 (10 nmol). The mixture was left to react at 90 °C for 20 min. Prior to performing quality control by HPLC, EDTA in 0.1 M acetate buffer (pH 4.6) was added to a final concentration of 1 mM to the labeling reaction mixture as a “free” 111In3+ scavenger.
Quality control of [111In-DOTA]MG11
For quality control of the labeled reaction mixture, RP-HPLC was performed using system 1: A Waters Chromatograph (Waters, Vienna, Austria) based on a 600 solvent delivery system coupled to a Waters 996 photodiode array UV detector and a Gabi gamma detector (Raytest, RSM Analytische Instrumente GmbH, Germany) employing a 20 μL injection loop was applied. The Millennium Software (Waters, USA) was used for data processing and chromatographic control, and an XTerra RP-18 (5 μm, 4.6 mm × 150 mm) cartridge column (Waters, Germany) was eluted at 1 mL/min flow rate with a linear gradient starting from 0 % B and advancing to 40 % B within 40 min (solvent A = 0.1 % aqueous trifluoroacetic acid (TFA) and B = MeCN). For metabolism studies, HPLC analysis was performed using system 2: A Waters Chromatograph (Waters, Vienna, Austria) with a 600E multisolvent delivery system coupled to a Gabi gamma detector (Raytest, Germany) employing a 0.5-mL injection loop was applied. Data processing and chromatography run were controlled with Empower Software, and a Symmetry Shield RP-18 (5 μm, 3.9 mm × 20 mm) column (Waters, Germany) was eluted adopting linear gradient starting from 0 % B and advancing to 40 % B within 40 min (solvent A = 0.1 % aqueous TFA and B = MeCN) with a flow rate of 1 mL/min. Radioactivity measurements were conducted in an automated well-type gamma counter (NaI(Tl) crystal, Canberra Packard Auto-Gamma 5000 series instrument) calibrated for 111In.
Biology
Cell lines and cell culture
The rat pancreatic tumor cell line AR42J, endogenously expressing the CCK2R [27], was kindly provided by Prof. C. Decristoforo (University Clinic Innsbruck, Austria). The human epidermoid carcinoma A431 cell line transfected to stably express the CCK2R (A431-CCK2R(+)) or devoid of CCK2R expression (A431-CCK2R(−)) was a gift from Prof. O. Boerman (Department of Nuclear Medicine, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands) and Prof. L. Aloj (Istituto di Biostrutture e Bioimmagini, Consiglio Nazionale delle Ricerche, Naples, Italy) [28].
All culture media were purchased from Gibco BRL, Life Technologies (Grand Island, NY, USA), and supplements were supplied by Biochrom KG Seromed (Berlin, Germany). AR42J cells were cultured in F-12K Nutrient Mixture (Kaighn’s Modification), supplemented with 10 % (v/v) fetal bovine serum, 100 U/mL penicillin, 100 μg/mL streptomycin, and 1 mM L glutamine. A431-CCK2R(+/−) cells were grown in Dulbecco’s Modified Eagle medium with GlutaMAX-I supplemented with 10 % (v/v) fetal bovine serum, 100 U/mL penicillin, 100 μg/mL streptomycin, 4500 mg/L D-glucose, and 250 μg/mL G418. Cells were kept in a controlled humidified air containing 5 % CO2 at 37 °C. Splitting of cells with a ratio of 1:2 to 1:5 was performed when approaching confluency using a trypsin/EDTA solution (0.05/0.02 % w/v) [29].
Metabolism in blood
Animal experiments were carried out in compliance with European and national regulations and were approved by national authorities. For metabolic studies, in-house male Swiss albino mice (30 ± 5 g) were used. A bolus containing [111In-DOTA]MG11 (100 μL, 11–22 MBq, 3 nmol of total peptide, normal saline/EtOH 9/1 v/v) was injected in the tail vein of mice, together with (a) vehicle (100 μL; control), (b) PA (100 μL of vehicle containing 300 μg PA; PA), or (c) (100 μL of vehicle containing 150 μg TO; TO). Additional animals intraperitoneally (ip) received a fine dispersed suspension of Race (3 mg Race dissolved in 0.025 mL DMSO and freshly mixed with 0.375 mL saline) 30–40 min prior to radioligand injection. The animals were kept for 5 min in cages with free access to water. They were sacrificed by cardiac puncture under ether anesthesia, and blood was withdrawn with a syringe and immediately placed in a pre-chilled polypropylene vial on ice containing EDTA and Met. Blood samples were centrifuged at 2000g at 4 °C for 10 min. The plasma was collected, an equal volume of MeCN was added, and the mixture was centrifuged for 10 min at 15,000g at 4 °C. The supernatant was collected and concentrated under a gentle N2-flux at 40 °C to a volume of ≈0.1 mL; the concentrate was diluted with physiological saline (0.4 mL) and passed through a Millex-GV syringe-driven filter unit (0.22 μm/13 mm; Millipore, Milford, USA). Suitable aliquots of the filtrate were analyzed by RP-HPLC [15, 29]. The t
R of the parent radiopeptide in the applied chromatographic conditions (system 2) was established by coinjection of samples with [111In-DOTA]MG11.
Biodistribution in AR42J tumor-bearing SCID mice
In-house male SCID mice (NCSR “Demokritos” Animal House) of 6 weeks of age at the time of arrival (18 ± 2 g body weight) were inoculated subcutaneously (sc) in their flanks with a suspension of freshly harvested AR42J cells (1 × 107 cells in ~150 μL saline). Animals were kept in aseptic conditions for 14 days when well-palpable tumors developed at the inoculation sites (0.31 ± 0.17 g) [29]. At the day of biodistribution, animals received a bolus of [111In-DOTA]MG11 (100 μL, 37–74 kBq, 10 pmol total peptide, in saline/EtOH 9/1 v/v) through the tail vein, coinjected with (a) vehicle (100 μL; control group, n = 10), (b) PA (100 μL of vehicle containing 300 μg PA; PA group, n = 10), (c) TO (100 μL of vehicle containing 150 μg TO; TO group, n = 4), (d) Lis (100 μL of vehicle containing 100 μg Lis; Lis group, n = 4), (e) PA plus Lis (100 μL vehicle containing 300 μg PA and 100 μg Lis; PA+Lis group, n = 5), or (f) 30–40 min after ip injection of Race (3 mg Race dissolved in 0.025 mL DMSO and freshly mixed with 0.375 mL saline; Race group, n = 4). In a separate animal group, the mice were coinjected with 100 μL vehicle containing both 300 μg PA and 100 μg DG2 [25] to assess non-specific tumor uptake during in situ NEP inhibition (in vivo CCK2R blockade; block group, n = 4).
Mice had access to drinking water ad libitum until they were euthanized at 4 h pi. Blood samples, organs of interest, and tumors were collected immediately after dissection, weighted, and measured for radioactivity in the gamma counter; only stomachs were emptied of their contents prior to measurements. Biodistribution data were calculated as percent of injected dose per gram tissue (% ID/g) with the aid of suitable standards of the injected dose, using the GraphPad Prism Software (San Diego, CA).
PA and TO dose dependence study in SCID mice bearing twin A431-CCK2R(+/−) tumors
Inocula of freshly harvested A431-CCK2R(+/−) cells (1.6 × 107/1.4 × 107 cell suspensions in 150 μL saline) were sc-injected in the right and left flanks of the SCID mice (male SCID mice, 6 weeks of age and of 18 ± 2 g body weight on arrival day; NCSR “Demokritos” Animal House). The animals were kept in aseptic conditions for 8 days till well-palpable tumors (0.26 ± 0.08 g) developed at the inoculation sites [29]. At the day of the experiment, the mice received a bolus of [111In-DOTA]MG11 (100 μL, 37–74 kBq, 10 pmol total peptide, in saline/EtOH 9/1 v/v) through the tail vein, coinjected with (a) vehicle (100 μL; control group, n = 5), (b) three different doses of PA (3 μg, n = 5; 30 μg, n = 4; or 300 μg PA, n = 10, dissolved in 100 μL vehicle), and (c) three different doses of TO (1.5, 15, or 150 μg TO dissolved in 100 μL vehicle, all groups of n = 4). Mice had free access to drinking water until they were sacrificed at 4 h pi, and biodistribution was conducted as described above.
Statistical analysis
The in vivo data were statistically analyzed with the Student’s t test (Prism™ 2.01, GraphPad Software, San Diego, CA). Analyses were two-tailed and a P value <0.05 was considered statistically significant.