1. Introduction
Targeted radionuclide therapy of neuroendocrine tumors (NET) via somatostatin receptor (SST) is proven very effective. The NETTER-1 phase III study showed that treatment with the
177Lu-labeled somatostatin analog DOTA-TATE ([1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid
0, Tyr
3,Thr
8]-octreotate), in combination with single-dose nonradiolabeled octreotide (Sandostatin
® LAR
®), significantly improved objective response, progression-free survival and quality of life versus treatment with double-dose octreotide [
1,
2]. Earlier studies involving large cohorts treated with the
90Y- or
177Lu-labeled analog DOTA-TOC ([DOTA
0, Tyr
3]-octreotide) support these findings [
3,
4]. [
177Lu]Lu-DOTA-TOC is under evaluation in the phase III trial COMPETE (NCT03049189) versus the mTOR inhibitor, everolimus, while [
177Lu]Lu-DOTA-TATE (Lutathera
®) is approved by the U.S. Food and Drug Administration (FDA) and the European Medicines Agency (EMA). As a companion to these analogs for radionuclide therapy, two
68Ga-labeled radio-diagnostics, [
68Ga]Ga-DOTA-TATE (NETSPOT
®) and [
68Ga]Ga-DOTA-TOC (SOMAKIT TOC
®), received FDA approval for positron emission tomography (PET) imaging. The molecular basis of these successful approvals rests on the high affinity of the (radio)metallated DOTA-TATE and DOTA-TOC for the somatostatin receptor subtype 2 (SST2), which is known to be overexpressed by NET cells. However, poorly differentiated neuroendocrine carcinoma (NEC), high-grade NETs, and to a certain extent, well-differentiated NETs, may show low and/or heterogeneous SST2 expression [
5,
6], leading to suboptimal tumor targeting with these analogs. On the other hand, these tumors may express or co-express other SST subtypes among the five known ones (SST1-5). SST5 is the second highly expressed subtype in gastroenteropancreatic neuroendocrine tumors (GEP-NETs) [
7], behind the predominant expression of SST2 (at least in primary tumors) and is concomitantly expressed with SST2 in 70–100% of GEP-NETs, in breast cancer, growth hormone (GH)-secreting pituitary adenomas and in 20–50% of intestinal or bronchial NETs [
5,
8,
9,
10]. NETs from G1/2 to G3 show a downregulation of SST2, while SST5 is constantly present [
5,
11]. SST5 is predominantly expressed, compared to SST2, in other tumors such as glioblastomas [
12], tumor capillaries of pancreatic adenocarcinomas [
13] or in lung cancer [
14]. There are also cases of NETs where SST2 is absent while SST5 is present [
6,
15]. There are other tumors where SST5 is expressed, while SST2 is absent, including ACTH pituitary adenoma, cervix carcinoma and ovarian carcinoma [
16]. Therefore, analogs targeting SST2 and SST5 (but also other subtypes), may potentially target a broader spectrum of various tumors and/or increase the radiation dose in a given tumor.
Most of the known analogs are synthetic cyclic octapeptides with a disulfide six-membered ring and SST2-selectivity. We are interested in developing radiolabeled somatostatin analogs for multireceptor subtype targeting. Replacement of key amino acids on the octreotide motif resulted in the analog [DOTA, 1-Nal
3]-octreotide (DOTA-NOC) with a high affinity for the SST2 and SST5 and lower for the SST3 [
17]. Other designs include, highly constrained bicyclic octreotide analogs, consisting of a head-to-tail cyclization and an inner disulfide six-membered ring [
18], the head-to-tail cyclo-hexapeptide pasireotide (Signifor or SOM230) [
19], cyclic nonapeptides with nondisulfide eight-membered ring [
20], and the 14mer and pseudo-14mer cyclic somatostatin-14 (SS-14) mimics, with ring-size of 12, 9, 8 and 6 amino acids—with the higher number ring favoring multireceptor subtype recognition [
21]. All the above-mentioned analogs showed certain limitations, with [
68Ga]Ga-DOTA-NOC being, so far, the only well-established analog for SST2, SST3 and SST5 targeting [
22]. Clinical data with [
68Ga]Ga-DOTA-NOC indicate that multi-receptor subtype targeting is relevant for improving the diagnostic accuracy and sensitivity of PET imaging of SST-expressing tumors [
23,
24]. Such clinical data on therapy are lacking. In fact, the therapeutic equivalent [
177Lu]Lu-DOTA-NOC has been evaluated in only 69 NET patients, compared to [
177Lu]Lu-DOTA-TATE, showing higher uptake in normal tissues with subsequently higher effective dose [
25]. None of the other analogs has been evaluated for targeted radionuclide therapy.
In this work, we evaluate the
177Lu-labeled somatostatin analog ST8950 ((4-amino-3-iodo)-
d-Phe-c[Cys-(3-iodo)-Tyr-
d-Trp-Lys-Val-Cys]-Thr-NH
2) for potential treatment of SST2- and SST5-expressing tumors. ST8950 (identified as peptide #9 in [
26] and as AP102 in [
27,
28] is a disulfide-bridged bis-iodo-substituted octapeptide that exhibits sub-nanomolar affinity to SST2 and SST5 [
26]. ST8950 is as potent as the natural SS-14 in its ability to inhibit growth hormone and prolactin release [
26], it has intermediate agonistic potency between octreotide and pasireotide at both subtypes [
27], and it acutely reduces growth hormone secretion without causing hyperglycemia (a known undesirable effect of pasireotide) in a healthy rat model [
28]. Our previous work demonstrated that coupling of the chelator DOTA and complexation of Ga
3+ does not alter the affinity to SST2, and while it reduces its affinity to SST5, this still remains in a low nanomolar range [
29]. [
68Ga]Ga-DOTA-ST8950 showed high and specific accumulation in SST2 and SST5-expressing tumors in vivo, comparable to [
68Ga]Ga-DOTA-NOC [
29]. Herein we report the comprehensive evaluation of the therapeutic equivalent [
177Lu]Lu-DOTA-ST8950. The influence of Lu-complexation on the affinity for SST2 and SST5 was assessed, together with the internalization and efflux rate of [
177Lu]Lu-DOTA-ST8950 vs. [
177Lu]Lu-DOTA-NOC on intact cells. A series of in vivo characteristics, including the influence of the injected peptide masses on the biodistribution, the specificity, the role of nephroprotective agents on the kidney uptake, the pharmacokinetics over 168 h, the residence time on the tumors and critical organs and finally the dosimetry of [
177Lu]Lu-DOTA-ST8950 were assessed on a dual SST2- and SST5-expressing xenografted model.
3. Materials and Methods
All chemicals were obtained from commercial sources and used without additional purification. ESI-MS was carried out with ESI Bruker Esquire 3000 plus (Bruker Daltonics, Billerica, MA, USA). RP-HPLC was performed on a Bischoff instrument consisting of a LC-CaDi 22–14 interface, a UV-vis Lambda 1010 detector and a flow-through Berthold LB509 γ-detector (BISCHOFF chromatography, Leonberg, Germany), using a Phenomenex Jupiter Proteo 90 Å C12 (250 × 4.6 mm) column (Phenomenex Inc., California, USA). (Eluents: A = H2O (0.1% TFA), B = Acetonitrile (0.1% TFA), Gradient: 95–50% solvent A in 15 min, Flow rate: 1.5 mL/min). Quantitative gamma counting was performed on a COBRA 5003 γ-system well counter from Packard Instruments (Meriden, CT, USA). SPECT/CT images were acquired using a dedicated nanoSPECT/CT system (Bioscan, Mediso, Budapest, Hungary).
Human Embryonic Kidney (HEK) cells were stably transfected with plasmids encoding the human SST2 and SST5 (HEK-SST2 and HEK-SST5) and cultivated as previously described [
27]. The SST2 and SST5 expression was confirmed by western blot and has been previously reported [
27]. Nontransfected HEK cells were used as negative control.
3.1. Synthesis of the (Radio)Metallated Peptide Conjugates and Stability
DOTA-ST8950 (
Figure 1) was custom-made by PolyPeptide (San Diego, CA, USA). DOTA-NOC (
Figure 1) was synthesized by standard Fmoc-solid-phase peptide synthesis, purified by preparative RP-HPLC and characterized by ESI-MS and analytical RP-HPLC (
Figure 1).
natLu-DOTA-ST8950 and
natLu-DOTA-NOC were synthesized after incubation of the DOTA-conjugates with a 2.5-fold excess of
natLuCl
3 × 6H
2O (Sigma Aldrich, St. Louis, MO, USA) in ammonium acetate buffer (Sigma Aldrich, St. Louis, MO, USA), 0.4 M, pH 5 at 95 °C for 30 min. Free metal ions were eliminated via SepPak C-18 cartridge (Waters), preconditioned with methanol (Merck, Darmstadt, Germany) and water. The reaction mixture was loaded and the free
natLu was eluted with water while the metallated peptides were eluted with ethanol, evaporated to dryness, redissolved in water and lyophilized. The
177Lu-labeled conjugates were synthesized by dissolving 5–10 μg (3–6 nmol) of the DOTA-conjugates in 250 μL of sodium acetate buffer (0.4 M, pH 5.0) followed by incubation with [
177Lu]LuCl
3 (10–200 MBq, depending on the planned experiment) for 30 min at 95 °C. The stability of [
177Lu]Lu-DOTA-ST8950 under two different storage conditions, room temperature (RT) and at 4 °C, was evaluated over 24 h after synthesis.
3.2. Log D Measurements
The determination of log D (pH = 7.4) was performed by the “shake-flask” method. To a pre-saturated mixture of 500 µL 1-octanol and 500 µL of phosphate-buffered saline (PBS) (pH 7.4), 10 µL of 1 µM of the 177Lu-labeled conjugates were added. The solution was vortexed for 1 h to reach the equilibrium and then centrifuged (3000 rpm) for 10 min. From each phase, 100 µL of the aliquot was removed and measured in a γ-counter. Each measurement was repeated three times. Care was taken to avoid cross-contamination between the phases. The partition coefficient was calculated as the average of the logarithmic values (n = 3) of the ratio between the radioactivity in the organic and the PBS phase.
3.3. Affinity Studies
The binding affinities of the
natLu-DOTA-ST8950, in comparison to
natLu-DOTA-NOC, were determined on membranes of HEK-SST2 and HEK-SST5 cells. The natural hormone Somatostatin-14 (SS-14) was used as reference compounds.
125I-labeled SS-14 (
125I-SS-14, 81.4 TBq/mmol, Perkin Elmer, Waltham, MA, USA) was used as a radioligand for the competition binding assays. Binding assays were performed as described previously [
27].
3.4. In Vitro Characterization of [177Lu]Lu-DOTA-ST8950
For all cell experiments, HEK-SST2 and HEK-SST5 were seeded at a density of 1 × 106 cells/well in 6-well plates and incubated overnight with Dulbecco’s modified Eagle’s medium (DMEM) with 1% Fetal Bovine Serum (FBS, Biochrom GmbH, Merck Millipore, Darmstadt, Germany) to obtain a good cell adherence. For plating HEK-SST2 and HEK-SST5, the plates were pre-treated with a solution of 10% poly-lysine to promote the cell attachment.
3.4.1. Internalization Assay
The cells were washed with PBS and were incubated with fresh medium (DMEM with 1% FBS) for 1 h at 37 °C. [177Lu]Lu-DOTA-ST8950 and [177Lu]Lu-DOTA-NOC (100 μL, 2.5 nM) were added to the medium (0.9 mL) and the cells incubated (in triplicates) for 0.5, 1, 2 and 4 h at 37 °C, 5% CO2. The internalization was stopped by removing the medium and washing the cells with ice-cold PBS. The cells were then treated twice for 5 min with ice-cold glycine solution (0.05 mol/L, pH 2.8), to distinguish between cell-surface-bound (acid releasable) and internalized (acid resistant) radio-peptide. Finally, the cells were detached with 1 M NaOH at 37 °C. To determine nonspecific cellular uptake, selected wells were incubated with the radio-peptide in the presence of 1000-fold excess of SS-14. Internalization and bound rate are expressed as a percentage of the applied radioactivity.
3.4.2. Cellular Retention (Efflux) Assay
For the cellular retention studies, HEK-SST2 cells were incubated with both radio-peptides (2.5 nM) for 120 min. The medium was then removed and the wells were washed twice with 1 mL ice-cold PBS. The acid wash with a glycine buffer of pH 2.8 was performed twice (5 min each time) on ice to remove the receptor-bound radio-peptide. Cells were then incubated again at 37 °C with fresh buffer (DMEM with 1% FBS). After preselected time points (10, 20, 30, 60, 120 and 210 min) the external medium was removed for quantification of radioactivity and replaced with fresh 37 °C medium. The cells were solubilized in 1 M NaOH and collected for quantification.
3.5. In Vivo Evaluation of [177Lu]Lu-DOTA-ST8950 in Tumor-Bearing Mice
3.5.1. Tumor Implantation
The Veterinary Office (Department of Health) of the Cantonal Basel-Stadt approved the animal experiments (approval no. 2799) in accordance with the Swiss regulations for animal treatment. Female athymic Nude-Foxn1nu/Foxn1+ mice (Envigo, The Netherlands), 4–6 weeks old, were injected subcutaneously with 107 HEK-SST2 cells in the right shoulder and 107 HEK-SST5 cells in the left shoulder, freshly suspended in 100 μL sterile phosphate-buffered saline. The tumors were allowed to grow for 2–3 weeks.
3.5.2. Investigation of the Influence of the Injected Peptide Mass
Groups of mice bearing dual SST2- and SST5-expressing xenografts were injected with two different peptide doses of [177Lu]Lu-DOTA-ST8950: 10 pmol/100 µL/0.5–0.6 MBq and 100 pmol/100 µL/0.6–1.5 MBq. The biodistribution was evaluated at 1 and 4 h post injection. At the selected time points, the mice were sacrificed and the organs of interest were collected, rinsed, blotted, weighed and counted in a γ-counter. The results are expressed as a percentage of injected activity per gram (%IA/g) obtained by extrapolation from counts of an aliquot taken from the injected solution as a standard.
3.5.3. In Vivo Metabolic Stability Studies
The in vivo stability of [177Lu]Lu-DOTA-ST8950 was evaluated after intravenous injection of (100 pmol/100 µL/9.3 MBq) into the tail vein of non-tumor-bearing mice. Blood samples were collected at 30 and 60 min after injection in polypropylene tubes containing ethylenediaminetetraacetic acid (EDTA). After centrifugation at 4 °C, the plasma was collected and 95% ethanol was added in equal volume (1:1 v/v). The mixture was stirred, centrifuged and the supernatant was separated from the precipitated proteins. The solution was treated with acetonitrile in equal volume (1:1, v/v) to promote further precipitation of proteins. After centrifugation, the supernatant was filtered, diluted with water (1:1, v/v) and analyzed by radio-RP-HPLC for the identification and quantification of the intact peptide and possible metabolites.
3.5.4. Specificity and Kidney Protection
The in vivo SST2- and SST5-mediated uptake of [177Lu]Lu-DOTA-ST8950 was assessed in mice bearing HEK-SST(−) negative xenografts after injection of 100 pmol/100 μL/0.3 MBq. The mice were sacrificed 4 h p.i. and accumulation of the radiotracer in the tumors and in all organs of interest was quantified in a γ-counter.
The basic amino acid lysine was evaluated as a nephroprotective agent, in an attempt to reduce the kidney uptake of [177Lu]Lu-DOTA-ST8950. Dual SST2- and SST5-expressing xenografts were treated intravenously with lysine (20 mg/100 μL in PBS) 5 min before the administration of [177Lu]Lu-DOTA-ST8950 (100 pmol/100 μL/0.3 MBq). Animals were sacrificed 4 h p.i. and accumulation of the radiotracer in the tumors and in all organs of interest was quantified in a γ-counter.
3.5.5. Pharmacokinetics of [177Lu]Lu-DOTA-ST8950
Quantitative biodistribution studies of [177Lu]Lu-DOTA-ST8950 (100 pmol/100 μL/0.6–1.5 MBq) was followed from 1 up to 168 h p.i. At the preselected time points, the mice were sacrificed and the organs of interest were collected, rinsed, blotted, weighed and counted in a γ-counter. The results are expressed as a percentage of injected activity per gram (%IA/g) obtained by extrapolation from counts of an aliquot taken from the injected solution as a standard.
3.5.6. SPECT/CT Imaging
Mice bearing dual SST2- and SST5-expressing xenografts were imaged using a nano SPECT/CT system (Bioscan, Mediso, Budapest, Hungary) 4 h after administration of [177Lu]Lu-DOTA-ST8950 (100 pmol/100 µL/6 MBq). A helical CT scan was acquired with the following parameters: current, 177 mA; voltage, 45 kVp; pitch, A helical SPECT scan was acquired using multipurpose pinhole collimators (APT1), 20% energy window width centered symmetrically over the 208 and 113 keV g-peaks of 177Lu, 24 projections, and 1200 s per projection. CT and SPECT images were reconstructed and filtered using the manufacturer’s algorithm, resulting in a pixel size of 0.3 mm for the SPECT and of 0.2 mm for the CT.
3.5.7. Dosimetry
Mice biodistribution data were used to generate time–activity curves for each radiotracer. Because of the absence of a specific radioactivity accumulation in bones and red marrow, a linear relationship between the blood and the red marrow residence times was assumed for estimating the red marrow radiation dose [
40]. The proportionality factor was the ratio between the red marrow mass and the blood mass in humans. OLINDA/EXM 1.0 (OLINDA/EXM
®, Vanderbilt University, USA) was used to integrate the fitted time–activity curves and to estimate the organ and effective doses using the whole-body adult female model. For all calculations, the assumption was made that the mouse biodistribution, determined as the %IA/organ, was the same as the human biodistribution.
3.6. Statistics
Comparison of data was performed using unpaired two-tailed t-test with GraphPad Prism 7 software (GraphPad Software, Inc., San Diego, CA, USA). p values < 0.05 were considered significant.