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Evaluation of Actinium-225 Labeled Minigastrin Analogue [²²⁵Ac]Ac-DOTA-PP-F11N for Targeted Alpha Particle Therapy.

ABSTRACT: The overexpression of cholecystokinin B receptor (CCKBR) in human cancers led to the development of radiolabeled minigastrin analogues for targeted radionuclide therapy, which aims to deliver cytotoxic radiation specifically to cancer cells. Alpha emitters (e.g., actinium-225) possess high potency in cancer cell-killing and hold promise for the treatment of malignant tumors. In these preclinical studies, we developed and evaluated CCKBR-targeted alpha particle therapy. The cellular uptake and cytotoxic effect of actinium-225 labeled and HPLC-purified minigastrin analogue [²²⁵Ac]Ac-PP-F11N were characterized in the human squamous cancer A431 cells transfected with CCKBR. Nude mice bearing A431/CCKBR tumors were used for biodistribution and therapy studies followed by histological analysis and SPECT/CT imaging. In vitro, [²²⁵Ac]Ac-PP-F11N showed CCKBR-specific and efficient internalization rate and potent cytotoxicity. The biodistribution studies of [²²⁵Ac]Ac-PP-F11N revealed CCKBR-specific uptake in tumors, whereas the therapeutic studies demonstrated dose-dependent inhibition of tumor growth and extended mean survival time, without apparent toxicity. The histological analysis of kidney and stomach indicated no severe adverse effects after [²²⁵Ac]Ac-PP-F11N administration. The post-therapy SPECT-CT images with [¹¹¹In]In-PP-F11N confirmed no CCKBR-positive tumor left in the mice with complete remission. In conclusion, our study demonstrates therapeutic efficacy of [²²⁵Ac]Ac-PP-F11N without acute radiotoxicity in CCKBR-positive cancer model.

SUBMITTER: Qin Y

PROVIDER: S-EPMC7696055 | biostudies-literature | 2020 Nov

REPOSITORIES: biostudies-literature

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Evaluation of Actinium-225 Labeled Minigastrin Analogue [<sup>225</sup>Ac]Ac-DOTA-PP-F11N for Targeted Alpha Particle Therapy.

Qin Yun Y Imobersteg Stefan S Blanc Alain A Frank Stephan S Schibli Roger R Béhé Martin P MP Grzmil Michal M

Pharmaceutics 20201112 11

The overexpression of cholecystokinin B receptor (CCKBR) in human cancers led to the development of radiolabeled minigastrin analogues for targeted radionuclide therapy, which aims to deliver cytotoxic radiation specifically to cancer cells. Alpha emitters (e.g., actinium-225) possess high potency in cancer cell-killing and hold promise for the treatment of malignant tumors. In these preclinical studies, we developed and evaluated CCKBR-targeted alpha particle therapy. The cellular uptake and cy ...[more]

PMID: 33198403

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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.

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:BackgroundThe [177Lu]Lu-DOTA-TATE mediated peptide receptor radionuclide therapy (PRRT) of neuroendocrine tumors (NETs) is sometimes leading to treatment resistance and disease recurrence. An interesting alternative could be the somatostatin antagonist, [177Lu]Lu-DOTA-JR11, that demonstrated better biodistribution profile and higher tumor uptake than [177Lu]Lu-DOTA-TATE. Furthermore, treatment with alpha emitters showed improvement of the therapeutic index of PRRT due to the high LET offered by the alpha particles compared to beta emitters. Therefore, [225Ac]Ac-DOTA-JR11 can be a potential candidate to improve the treatment of NETs (Graphical abstract). DOTA-JR11 was radiolabeled with [225Ac]Ac(NO3)3 and [177Lu]LuCl3. Stability studies were performed in phosphate buffered saline (PBS) and mouse serum. In vitro competitive binding assay has been carried out in U2OS-SSTR2 + cells for natLa-DOTA-JR11, natLu-DOTA-JR11 and DOTA-JR11. Ex vivo biodistribution studies were performed in mice inoculated with H69 cells at 4, 24, 48 and 72 h after injection of [225Ac]Ac-DOTA-JR11. A blocking group was included to verify uptake specificity. Dosimetry of selected organs was determined for [225Ac]Ac-DOTA-JR11 and [177Lu]Lu-DOTA-JR11.Results[225Ac]Ac-DOTA-JR11 has been successfully prepared and obtained in high radiochemical yield (RCY; 95%) and radiochemical purity (RCP; 94%). [225Ac]Ac-DOTA-JR11 showed reasonably good stability in PBS (77% intact radiopeptide at 24 h after incubation) and in mouse serum (~ 81% intact radiopeptide 24 h after incubation). [177Lu]Lu-DOTA-JR11 demonstrated excellent stability in both media (> 93%) up to 24 h post incubation. Competitive binding assay revealed that complexation of DOTA-JR11 with natLa and natLu did not affect its binding affinity to SSTR2. Similar biodistribution profiles were observed for both radiopeptides, however, higher uptake was noticed in the kidneys, liver and bone for [225Ac]Ac-DOTA-JR11 than [177Lu]Lu-DOTA-JR11.Conclusion[225Ac]Ac-DOTA-JR11 showed a higher absorbed dose in the kidneys compared to [177Lu]Lu-DOTA-JR11, which may limit further studies with this radiopeptide. However, several strategies can be explored to reduce nephrotoxicity and offer opportunities for future clinical investigations with [225Ac]Ac-DOTA-JR11.

Dataset Information

Evaluation of Actinium-225 Labeled Minigastrin Analogue [²²⁵Ac]Ac-DOTA-PP-F11N for Targeted Alpha Particle Therapy.

Publications

Evaluation of Actinium-225 Labeled Minigastrin Analogue [<sup>225</sup>Ac]Ac-DOTA-PP-F11N for Targeted Alpha Particle Therapy.

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Dataset Information

Evaluation of Actinium-225 Labeled Minigastrin Analogue [225Ac]Ac-DOTA-PP-F11N for Targeted Alpha Particle Therapy.

Publications

Evaluation of Actinium-225 Labeled Minigastrin Analogue [<sup>225</sup>Ac]Ac-DOTA-PP-F11N for Targeted Alpha Particle Therapy.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets

Evaluation of Actinium-225 Labeled Minigastrin Analogue [²²⁵Ac]Ac-DOTA-PP-F11N for Targeted Alpha Particle Therapy.