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Improved Tumor-Targeting with Peptidomimetic Analogs of Minigastrin ¹⁷⁷Lu-PP-F11N.

ABSTRACT: The cholecystokinin-2 receptor (CCK2R) is an attractive target in nuclear medicine due to its overexpression by different tumors. Several radiolabeled peptidic ligands targeting the CCK2R have been investigated in the past; however, their low stability against proteases can limit their uptake in tumors and metastases. Substitution of single or multiple amide bonds with metabolically stable 1,4-disubstituted 1,2,3-triazoles as amide bond bioisosteres proved a promising strategy for improving the tumor-targeting properties of a truncated analog of minigastrin. In this study, we applied the previously studied structural modifications to improve the pharmacokinetic and pharmacodynamic properties of PP-F11N, a minigastrin analog currently in clinical trials. Novel minigastrins (NMGs) as analogs of PP-F11N with one or two amide bonds substituted by 1,2,3-triazoles were synthesized, radiolabeled with ¹⁷⁷Lu³⁺, and subjected to full evaluation in vitro (cell internalization, receptor affinity, stability in blood plasma) and in vivo (stability, biodistribution, SPECT/CT imaging). NMGs with triazoles inserted between the amino acids DGlu¹⁰-Ala¹¹ and/or Tyr¹²-Gly¹³ showed a significantly increased cellular uptake and affinity toward the CCK2R in vitro. Resistance against the metabolic degradation of the NMGs was comparable to those of the clinical candidate PP-F11N. Imaging by SPECT/CT and biodistribution studies demonstrated a higher uptake in CCK2R-positive tumors but also in the CCK2R-positive stomach. The peptidomimetic compounds showed a slow tumor washout and high tumor-to-kidney ratios. The structural modifications led to the identification of analogs with promising properties for progression to clinical applications in the diagnosis and therapy of CCK2R-positive neoplasms.

SUBMITTER: Grob NM

PROVIDER: S-EPMC8199036 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Rationale: A high tumor-to-healthy-tissue uptake ratio of radiolabeled ligands is an essential prerequisite for safe and effective peptide receptor radionuclide therapy (PRRT). In the present study, we searched for novel opportunities to increase tumor-specific uptake of the radiolabeled minigastrin analogue [177Lu]Lu-DOTA-(DGlu)6-Ala-Tyr-Gly-Trp-Nle-Asp-Phe-NH2 ([177Lu]Lu-PP-F11N), that targets the cholecystokinin B receptor (CCKBR) in human cancers. Methods: A kinase inhibitor library screen followed by proliferation and internalization assays were employed to identify compounds which can increase uptake of [177Lu]Lu-PP-F11N in CCKBR-transfected human epidermoid carcinoma A431 cells and natural CCKBR-expressing rat pancreatic acinar AR42J cells. Western blot (WB) analysis verified the inhibition of the signaling pathways and the CCKBR level, whereas the cell-based assay analyzed arrestin recruitment. Biodistribution and SPECT imaging of the A431/CCKBR xenograft mouse model as well as histological analysis of the dissected tumors were used for in vivo validation. Results: Our screen identified the inhibitors of mammalian target of rapamycin complex 1 (mTORC1), which increased cell uptake of [177Lu]Lu-PP-F11N. Pharmacological mTORC1 inhibition by RAD001 and metformin increased internalization of [177Lu]Lu-PP-F11N in A431/CCKBR and in AR42J cells. Analysis of protein lysates from RAD001-treated cells revealed increased levels of CCKBR (2.2-fold) and inhibition of S6 phosphorylation. PP-F11N induced recruitment of β-arrestin1/2 and ERK1/2 phosphorylation. In A431/CCKBR-tumor bearing nude mice, 3 or 5 days of RAD001 pretreatment significantly enhanced tumor-specific uptake of [177Lu]Lu-PP-F11N (ratio [RAD001/Control] of 1.56 or 1.79, respectively), whereas metformin treatment did not show a significant difference. Quantification of SPECT/CT images confirmed higher uptake of [177Lu]Lu-PP-F11N in RAD001-treated tumors with ratios [RAD001/Control] of average and maximum concentration reaching 3.11 and 3.17, respectively. HE staining and IHC of RAD001-treated tumors showed a significant increase in necrosis (1.4% control vs.10.6% of necrotic area) and the reduction of proliferative (80% control vs. 61% of Ki67 positive cells) and mitotically active cells (1.08% control vs. 0.75% of mitotic figures). No significant difference in the tumor vascularization was observed after five-day RAD001 or metformin treatment. Conclusions: Our data demonstrates, that increased CCKBR protein level by RAD001 pretreatment has the potential to improve tumor uptake of [177Lu]Lu-PP-F11N and provides proof-of-concept for the development of molecular strategies aimed at enhancing the level of the targeted receptor, to increase the efficacy of PRRT and nuclear imaging.

Project description:Abstract Introduction: Despite the introduction of new molecular targeted therapies, there is still an unmet need for an effective systemic therapy for advanced medullary thyroid carcinoma (MTC). As MTC expresses cholecystokinine-2 (CCK-2) receptors at a high incidence and density, targeting the CCK-2 receptor with radiolabelled gastrin analogues is a potential approach for radionuclide therapy. Unfortunately, kidney and bone marrow toxicity precluded therapeutic applications of CCK-2 receptor specific radiotracers until now. The aim of this prospective study is the feasibility testing of targeting CCK-2 receptors with the novel gastrin analogue [177Lu-DOTA-(DGlu)6-Ala-Tyr-Gly-Trp-Nleu-Asp-PheNH2] (177Lu-PP-F11N) in six patients with advanced MTC (ClinicalTrials.gov: NCT02088645). Subjects and Methods: Six patients received two injections of 1 GBq 177Lu-PP-F11N, one injection with and the other without Physiogel (Gelofusin = plasma expander for nephroprotection) infusion. Planar scintigraphy and SPECT/CT scans were performed at several time points for up to 72 h post injection in order to calculate tumour- and organ doses using 3D voxel- and MIRD based dosimetry (Dosimetry Research Tool v5.2, Siemens Medical Solutions, USA). Blood samples were taken for bone marrow dose calculations. ECG, blood count and blood chemistry were measured up to 12 weeks after the second administration of 177Lu-PP-F11N in order to evaluate adverse events. Results: Adverse reactions (mainly hypotension, flushing and hypokalemia) were self-limiting and not higher than grade 1, according to CTCAE version 4.03. In all patients, 177Lu-PP-F11N accumulation was visible in tumor tissue, in the kidneys, stomach and the colon. Altogether, 14 tumours were eligible for dosimetry. The median (range) radiation dose for tumours, kidneys and bone marrow was 0.88 Gy/GBq (0.69-2.85), 0.090 (0.045-0.115) and 0.010 (0.008-0.016). These resulted in median tumour-to-kidney dose ratios of 12.8 (6.0-52.4) (without Physiogel) and 12.4 (6.4-29.8) (with Physiogel), which was not significantly different. The median tumour-to-bone marrow dose ratio was 87.9 (52.8-316.4). Discussion/Conclusion: The administration of the novel CCK-2 receptor ligand 177Lu-PP-F11N was safe in all six examined patients. Visualization of metastasized/recurrent disease in all patients provides evidence that CCK-2 receptor targeting with 177Lu-PP-F11N is feasible in patients with MTC. The dosimetry results indicate tumour doses that could enable radionuclide therapy. Dosimetry results for kidneys and bone marrow revealed low organ doses, as well as excellent tumour-to-kidney and tumour-to-bone marrow ratios. Further studies will be necessary to evaluate the theranostic potential of 177Lu-PP-F11N in patients with CCK-2 receptor expressing tumours.

Project description:α-particle emitters have recently been explored as valuable therapeutic radionuclides. Yet, toxicity to healthy organs and cancer radioresistance limit the efficacy of targeted α-particle therapy (TAT). Identification of the radiation-activated mechanisms that drive cancer cell survival provides opportunities to develop new points for therapeutic interference to improve the efficacy and safety of TAT. Methods: Quantitative phosphoproteomics and matching proteomics followed by the bioinformatics analysis were used to identify alterations in the signaling networks in response to TAT with the 225Ac-labeled minigastrin analog 225Ac-PP-F11N (DOTA-(dGlu)6-Ala-Tyr-Gly-Trp-Nle-Asp-Phe) in A431 cells, which overexpress cholecystokinin B receptor (CCKBR). Western blot analysis and microscopy verified the activation of the selected signaling pathways. Small-molecule inhibitors were used to validate the potential of the radiosensitizing combinatory treatments both in vitro and in A431/CCKBR tumor-bearing nude mice. Results: TAT-induced alterations were involved in DNA damage response, cell cycle regulation, and signal transduction, as well as RNA transcription and processing, cell morphology, and transport. Western blot analysis and microscopy confirmed increased phosphorylations of the key proteins involved in DNA damage response and carcinogenesis, including p53, p53 binding protein 1 (p53BP1), histone deacetylases (HDACs), and H2AX. Inhibition of HDAC class II, ataxia-telangiectasia mutated (ATM), and p38 kinases by TMP269, AZD1390, and SB202190, respectively, sensitized A431/CCKBR cells to 225Ac-PP-F11N. As compared with the control and monotherapies, the combination of 225Ac-PP-F11N with the HDAC inhibitor vorinostat (suberoylanilide hydroxamic acid, SAHA) significantly reduced the viability and increased the DNA damage of A431/CCKBR cells, led to the most pronounced tumor growth inhibition, and extended the mean survival of A431/CCKBR xenografted nude mice. Conclusion: Our study revealed the cellular responses to TAT and demonstrated the radiosensitizing potential of HDAC inhibitors to 225Ac-PP-F11N in CCKBR-positive tumors. This proof-of-concept study recommends development of novel radiosensitizing strategies by targeting TAT-activated and survival-promoting signaling pathways.

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Improved Tumor-Targeting with Peptidomimetic Analogs of Minigastrin ¹⁷⁷Lu-PP-F11N.

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Improved Tumor-Targeting with Peptidomimetic Analogs of Minigastrin 177Lu-PP-F11N.

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Improved Tumor-Targeting with Peptidomimetic Analogs of Minigastrin ¹⁷⁷Lu-PP-F11N.