In Vivo Stabilized SB3, an Attractive GRPR Antagonist, for Pre- and Intra-Operative Imaging for Prostate Cancer

SB3 (Fig. 2) was radiolabeled with ¹¹¹In (Covidien) as previously described [31]. Small-volume reactions were performed in conical vials. Sodium acetate was used to control pH, resulting in a final pH value of 4–4.5 and quenchers (ascorbic and gentisic acid 3.5 mM and methionine 10 mM as final concentration) were added to prevent radiolysis. A mixture of 1 nmol SB3 and 125 MBq equivalent [¹¹¹In]InCl₃ and quenchers in a final volume of 140 μl were heated for 15 min at 80 °C. To complex non-incorporated In-111, after cooling down to room temperature, 5 μl of 4 mM DTPA was added. To obtain a molar activity of 12.5 MBq/nmol, after the labeling, 9 nmoles of SB3 was added. The incorporation was determined by instant thin layer chromatography (ITLC) silica gel using sodium citrate (0.1 M, pH 5) as solvent [32] and radiochemical purity (RPC) was measured by a HPLC system (Breeze, Waters), containing a 2690 quaternary pump, and radioactivity was monitored with a NaI detector, digital multichannel analyzer, and dedicated software (MetorX B.V). A Symmetry C₁₈ column (5 μm × 4.6 mm × 250 mm, Waters) was used with a gradient profile of 0–5 min 100 % A, 5–5.01 min 60 % B, 5.01–20 min 80 % B, 20–20.01 min 100 % B, 20.01–25 min 100 % B, and 25.01–30 min (flow 1 ml/min), where A was 0.1 % trifluoroacetic acid (TFA) in H₂O and B was LC-MS grade methanol. No further purification was required.

A molar activity of 12.5 MBq/nmol and 125 MBq/nmol was used for the biodistribution and SPECT/MRI studies, respectively. For the in vivo stability studies, both molar activities were used. To determine the affinity of SB3 for the human GRPR in a competition-binding assay, Tyr⁴-Bombesin (Sigma-Aldrich) was radiolabeled with iodine-125 based on the Iodo Beads® (Pierce) method, adapted with 5-min pre-incubation before addition of the peptide, as previously described [33, 34]. In short, 100 μl of PBS 50 mM Pi pH 7.5, sodium iodide solution (10–20 MBq Na[¹²⁵I]I in 0.01 M NaOH), and pre-rinsed Iodo Beads® were pre-incubated for 5 min, and Iodo Beads® were removed. Then, total volume was added to 70 nmol of peptide in PBS, and the mixture was allowed to react for 5 min at room temperature. To exclude non-labeled iodine, purification was performed by solid phase extraction using a J.T. Baker C₁₈ cartridge followed by HPLC purification [34].

Internalization with [¹¹¹In]SB3 was performed in PC-3 cells, a commonly used human-derived androgen independent GRPR-expressing PC cell line (AACR). The cells were seeded in six-well plates at a density of 0.6 × 10⁶ cells per well and cultured overnight. On the day of the experiment, cells were washed twice with 37 °C PBS. Fresh internalization medium, RPMI + Glutamax with 20 mM HEPES, and 1 % bovine serum albumin fraction V pH 7.4, 10⁻⁹ M [¹¹¹In]SB3 was added. The cells were incubated at 37 °C in triplicates for 60 min, and incubation was stopped by removal of the medium and rinsing with cold PBS twice. Thereafter, the cells were incubated for 10 min with acid wash buffer (50 mM Glycin-HCl/100 mM NaCl, pH 2.8). The supernatant was collected (membrane fraction); the cells were rinsed with acid wash buffer and collected in the same tube. Cells were lysed with 0.1 M NaOH and collected in another tube (internalized cell fraction). The amount of activity in the membrane and cell fraction was measured on an automatic gamma counter.

Healthy or xenografted Balb c nu/nu male mice (Janvier) were used in this study. To obtain xenografted mice, animals were injected subcutaneously in the right shoulder with PC-3, 3 × 10⁶ cells in Matrigel (total volume 150 μl, solution 66 % RPMI, 33 % Matrigel) per animal. Prior to tumor cell inoculation, the PC-3 cells were cultured in RPMI medium supplemented with 10 % fetal bovine serum, 5000 IU/ml penicillin, and 5000 μg/ml streptomycin at 37 °C in a humidified atmosphere containing 5 % CO₂. PC295 is an androgen-dependent, patient-derived cell line with high GRPR expression. Xenografts were generated by subcutaneous implantation of the PC295 human prostate tumor as previously described and were stored at − 80 °C until further use [35]. All culture supplies were purchased at Gibco.

For the competition binding assay, fresh frozen PC295 xenograft tissue slices (10 μm) were incubated in triplicate with 80 μl 5 × 10⁻¹⁰ M [¹²⁵I]Tyr⁴-Bombesin, with tracer only or with an increasing concentration (10⁻¹² to 10⁻⁶ M) of unlabeled SB3 for 1 h and exposed to super resolution phosphor screens (PerkinElmer) for 48 h. The phosphor screens were read using the Cyclone (PerkinElmer) and quantified using OptiQuant software. Binding of unblocked [¹²⁵I]Tyr⁴-bombesin to the tissue slices was set at 100 %, and the percentage of binding relative to the unblocked tissue sample was calculated for the blocked tissue sections. These values were used to determine the IC₅₀ value of unlabeled SB3.

To determine the stability of [¹¹¹In]SB3 −/+ PA in vivo, healthy mice (n = 3 per group) were intravenously injected with 200 pmol/25 MBq or 2000 pmol/25 MBq [¹¹¹In]SB3 (volume 100 μl), co-injected with either 300 μg/100 μl PA (Peptides International Inc.) in 0.5 % ethanol or 100 μl saline solution. Five minutes after injection of [¹¹¹In]SB3 −/+ PA, blood (0.5–1 ml) was collected by cardiac puncture, while animals were under isoflurane/O₂ anesthesia, and transferred to blood-collection tubes containing 90 USP units of lithium heparin (spray coated), HPLC grade ethanol was added in a 1:1 (v/v) ratio, and the tubes were placed on ice. Blood samples were centrifuged 5 min at 1250g, and supernatant was collected. Next, the collected supernatant was centrifuged again for 5 min at 12,500g. The secondary supernatant was collected and diluted with MilliQ down to < 25 % ethanol before injection on HPLC. The samples were analyzed by HPLC (Alliance, Waters) including a Symmetry C₁₈ column (5 μm × 4.6 mm × 250 mm). Elution was performed in a 1-ml/min flow, completed in 30 min, with the gradient 0–5 min 100 % A, 5–5.01 min 40 % A 60 % B, 5.01–20 min 20 % A 80 % B, 20–20.01 min 100 % B, 20.01–25 min 100 % B, and 25.01–30 min 100 % A, where A was 0.1 % TFA in H₂O and B was methanol. In one animal, the sample volume was too low for further analysis; this animal was therefore excluded.

Tumor-bearing mice received an intravenous injection of 200 pmol/2.5 MBq [¹¹¹In]SB3, co-injected with either 300 μg/100 μl PA in 0.5 % ethanol or 100 μl saline solution (n = 4 animals per group for each time point). In previous in vivo studies, 200 pmol appeared to be the optimal peptide amount (regarding uptake in tumor and pancreas; data not shown). To determine specificity of tumor and organ uptake, blocking experiments were performed; an additional group of animals (n = 3) was pre-injected with an excess (20 nmol) of unlabeled SB3 followed by injection of the radiotracer −/+ PA. At three different time points p.i. (1, 4, and 24 h), animals were euthanized and blood, organs, and tumors were collected, weighed, and measured in a γ-counter (1480 WIZARD automatic γ-counter, PerkinElmer). For γ-counter measurements, an isotope specific energy window, a counting time of 60 s, and a counting error ≤ 5 % were used. Results were expressed as the percentage of the injected dose per gram tissue (% ID/g tissue). Animals that had ≥ 7 % ID in the tail were excluded.

For SPECT/MRI, PC-3-xenografted mice were intravenously injected with 25 MBq/200 pmol [¹¹¹In]SB3, co-injected with either 300 μg/100 μl PA in 0.5 % ethanol or 100 μl saline solution. Subsequently, imaging was performed 1, 4, and 24 h p.i. of the radiotracer, while animals were anesthetized using isoflurane/O₂, body temperature was maintained using warm air flow, and respiratory rate was monitored. Whole-body SPECT images were acquired using the novel and state-of-the-art four-head multipinhole system (NanoScan SPECT/MRI, Mediso Medical Imaging) in 30 min (28 projections, 29 s/projection, and 7-cm axial field of view). Images were reconstructed using OSEM with six iterations and four subsets on a 120 × 120 matrix with 0.25 × 0.25 × 0.25-mm isotropic voxels. MR-based attenuation correction was applied to all images. T2 MR images were acquired using a spin echo sequence (TR/TE = 4500/52 ms) with a 35-mm-diameter solenoid coil. Other scan parameters were field of view 70 mm, matrix 256 × 128, and slice thickness 1 mm with 0.1-mm spacing between slices. An ROI of the tumor was drawn by an experienced technician on the T2-weighted MR image to measure the intensity in the SPECT image.

All statistical evaluations were performed with GraphPad Prism software (version 5.01). The IC₅₀ value was calculated using a log (inhibitor) vs response model. To compare in vivo stability −/+ PA, radioactivity uptake in the tumor −/+ PA and radioactivity uptake in the tumor without and with an excess of unlabeled peptide an unpaired t test was used. P values < 0.05 were considered statistically significant. Tumor to organ uptake ratios were calculated per animal and expressed as mean and standard deviation per group.

The radiochemical purity and radiometal incorporation were both > 90 %.

In internalization experiments with [¹¹¹In]SB3 in PC-3 cells, the membrane fraction contained almost four times more radioactivity compared to the cell fraction.

Quantified binding of [¹²⁵I]Tyr⁴-bombesin to PC259 tissue sections without and with increasing concentrations of unlabeled SB3 resulted in an IC₅₀ value of 3.5 nM (95 % CI 1.9 to 6.4 nM) (Fig. 3).

In vivo stability of [¹¹¹In]SB3 −/+ PA was analyzed in blood samples, collected 5 min p.i. In the animals receiving 200 pmol [¹¹¹In]SB3, 37.3 ± 2.5 % of the remaining circulating activity consisted of intact radiotracer, while this value significantly increased (P < 0.0001) to 86.7 ± 1.5 % when [¹¹¹In]SB3 was co-injected with PA (Fig. 4a, b). When a 10× higher peptide amount was administered, these values were 42.0 ± 0.2 % vs 87.9 ± 1.1 % (P < 0.0001) of the radiotracer still being intact in the blood, respectively (Fig. 4c, d), indicating a clear increase of stability even with 2000-pmol peptide.

The results of the biodistribution studies are displayed in Fig. 5. A maximum tumor uptake of 10.2 ± 1.5 % ID/g tissue was reached using 2.5 MBq/200 pmol [¹¹¹In]SB3 1 h p.i. in contrast to 19.7 ± 3.5 % ID/g tissue after injection of [¹¹¹In]SB3 + PA. At all studied time points, there was significantly higher (P < 0.0001) tumor uptake when PA was co-administered with [¹¹¹In]SB3. Next to tumor uptake, high radiotracer uptake was also observed in the GRPR-expressing pancreas and in the kidneys, the latter as a result of renal excretion and partial reabsorption of the radiopeptide. The increased in vivo stability of [¹¹¹In]SB3 + PA also resulted in an increase in pancreatic uptake (4.1 ± 2.3 vs 10.1 ± 4.3 % ID/g tissue 1 h p.i. −/+ PA, respectively), while renal uptake remained similar (3.9 ± 1.4 vs 4.5 ± 1.4 % ID/g tissue 1 h p.i. −/+ PA, respectively). At 4 and 24 h p.i., the same pattern was observed. A similar tumor to pancreas radioactivity ratio was found for [¹¹¹In]SB3 −/+ PA (3.4 ± 2.5 vs 2.2 ± 0.8, 1 h p.i. −/+ PA, respectively). The tumor to kidney ratio was more favorable however when the radiotracer was combined with PA (2.8 ± 1.0 vs 4.8 ± 2.2, 1 h p.i. −/+ PA, respectively). Also, radiotracer uptake in the pancreas and the tumor was receptor-specific as can be concluded from the significantly decreased (P ≤ 0.001) uptake when an excess of unlabeled SB3 was co-administered.

In line with the biodistribution studies, SPECT/MR imaging performed 1, 4, and 24 h p.i. of 25 MBq/200 pmol [¹¹¹In]SB3 −/+ PA resulted in clearly improved tumor visualization when the radiotracer was combined with PA, at all imaging time points (Fig. 6a). ROI analysis showed an increase of 70 % in signal intensity (1424 vs 2434 kBq/ml) 1 h p.i. of [¹¹¹In]SB3 only and [¹¹¹In]SB3 + PA, respectively. Intensity curves and the biodistribution time activity curves were normalized to 100 % at 1 h for the + PA group. The normalized curves obtained from SPECT/MR images and biodistribution studies showed a well-comparable pattern for the tumor radioactivity (Fig. 6b).