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Targeted Tumor Therapy with Radiolabeled DNA Intercalator: A Possibility? Preclinical Investigations with 177Lu-Acridine.

ABSTRACT: Objective:A DNA intercalating agent reversibly stacks between the adjacent base pairs of DNA and thus is expected to exhibit preferential localization in the tumorous lesions as tumors are associated with enhanced DNA replication. Therefore, radiolabeled DNA intercalators are supposed to have potential to be used in targeted tumor therapy. Working in this direction, an attempt was made to radiolabel 9-aminoacridine, a DNA intercalator, with 177Lu, one of the most useful therapeutic radionuclides, and study the potential of 177Lu-acridine in targeted tumor therapy. Experiments. 9-Aminoacridine was coupled with p-NCS-benzyl-DOTA to facilitate radiolabeling, and the conjugate was radiolabeled with 177Lu. Different reaction parameters were optimized in order to obtain 177Lu-acridine complex with maximum radiochemical purity. In vitro stability of the radiolabeled complex was studied in normal saline and human blood serum. Biological behavior of the radiolabeled agent was studied both in vitro and in vivo using the Raji cell line and fibrosarcoma tumor-bearing Swiss mice, respectively. Results:177Lu-acridine complex was obtained with ~100% radiochemical purity under the optimized reaction conditions involving incubation of 1.5?mg/mL of ligand with 177Lu (1?mCi, 37?MBq) at 100°C at pH ~5 for 45 minutes. The complex maintained a radiochemical purity of >85% in saline at 6?d and >70% in human serum at 2?d postpreparation. In vitro cellular study showed uptake of the radiotracer (5.3 ± 0.13%) in the Raji cells along with significant cytotoxicity (78.06 ± 2.31% after 6?d). Biodistribution study revealed considerable accumulation of the radiotracer in tumor 9.98 ± 0.13?%ID/g within 1?h postadministration and retention therein till 6?d postadministration 4.00 ± 0.16?%ID/g with encouraging tumor to nontarget organ uptake ratios. Conclusions:The present study, although preliminary, indicates the potential of 177Lu-acridine and thus radiolabeled DNA intercalators in targeted tumor therapy. However, further detailed evaluation is required to explore the actual potential of such agents in targeted tumor therapy.

SUBMITTER: Ghosh S

PROVIDER: S-EPMC7397433 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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Targeted Tumor Therapy with Radiolabeled DNA Intercalator: A Possibility? Preclinical Investigations with <sup>177</sup>Lu-Acridine.

Ghosh Subhajit S Das Tapas T Suman Shishu K SK Kumar Chandan C Sarma Haladhar D HD Dash Ashutosh A

BioMed research international 20200725

<h4>Objective</h4>A DNA intercalating agent reversibly stacks between the adjacent base pairs of DNA and thus is expected to exhibit preferential localization in the tumorous lesions as tumors are associated with enhanced DNA replication. Therefore, radiolabeled DNA intercalators are supposed to have potential to be used in targeted tumor therapy. Working in this direction, an attempt was made to radiolabel 9-aminoacridine, a DNA intercalator, with <sup>177</sup>Lu, one of the most useful therap ...[more]

PMID: 32775454

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Project description:BackgroundRadiohybrid PSMA-targeted ligands (rhPSMA) have been introduced as a novel platform for theranostic applications. Among a variety of rhPSMA-ligands developed for radioligand therapy, two stereoisomers [177Lu]Lu-rhPSMA-10.1 and -10.2 have been synthesized and initially characterized in preclinical experiments with the aim to provide an optimized binding profile to human serum albumin, a reduction of charge, and thus accelerated kidney excretion, and unaffected or even improved tumor uptake. As both isomers showed similar in vitro characteristics and tumor uptake at 24 h post injection in tumor bearing mice and in order to identify the isomer with the most favorable pharmacokinetics for radioligand therapy, we carried out in-depth biodistribution and dosimetry studies in tumor-bearing and healthy mice.ResultsrhPSMA-10.1 and -10.2 were radiolabeled with lutetium-177 according to the established procedures of other DOTA-based PSMA ligands and displayed a high and comparable stability in all buffers and human serum (> 97%, 24 h). Biodistribution studies revealed fast clearance from the blood pool (0.3-0.6%ID/g at 1 h) and other background tissues within 48 h. Distinctive differences were found in the kidneys, where [177Lu]Lu-rhPSMA-10.1 displayed lower initial uptake and faster excretion kinetics compared to [177Lu]Lu-rhPSMA-10.2 expressed by a 1.5-fold and ninefold lower uptake value at 1 h and 24 h in healthy animals, respectively. Tumor uptake was comparable and in the range of 8.6-11.6%ID/g for both isomers over 24 h and was maintained up to 168 h at a level of 2.2 ± 0.8 and 4.1 ± 1.4%ID/g for [177Lu]Lu-rhPSMA-10.1 and [177Lu]Lu-rhPSMA-10.2, respectively.ConclusionOur preclinical data on biodistribution and dosimetry indicate a more favorable profile of [177Lu]Lu-rhPSMA-10.1 compared to [177Lu]Lu-rhPSMA-10.2 for PSMA-targeted radioligand therapy. [177Lu]Lu-rhPSMA-10.1 shows fast kidney clearance kinetics resulting in excellent tumor-to-organ ratios over a therapy relevant time course. Meanwhile, [177Lu]Lu-rhPSMA-10.1 is currently being investigated in clinical phase I/II studies in patients with mCRPC (NCT05413850), in patients with high-risk localized PC (NCT06066437, Nautilus Trial) and after external beam radiotherapy (NCT06105918).

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:BackgroundIndividualized dosimetry is recommended for [177Lu]Lu-PSMA radioligand therapy (RLT) which is resource-intensive and protocols are often not optimized. Therefore, a simulation study was performed focusing on the determination of efficient optimal sampling schedules (OSS) for renal and tumour dosimetry by investigating different numbers of time points (TPs).MethodsSampling schedules with 1-4 TPs were investigated. Time-activity curves of the kidneys and two tumour lesions were generated based on a physiologically based pharmacokinetic (PBPK) model and biokinetic data of 13 patients who have undergone [177Lu]Lu-PSMA I&T therapy. Systematic and stochastic noise of different ratios was considered when modelling time-activity data sets. Time-integrated activity coefficients (TIACs) were estimated by simulating the hybrid planar/SPECT method for schedules comprising at least two TPs. TIACs based on one single SPECT/CT measurement were estimated using an approximation for reducing the number of fitted parameters. For each sampling schedule, the root-mean-squared error (RMSE) of the deviations of the simulated TIACs from the ground truths for 1000 replications was used as a measure for accuracy and precision.ResultsAll determined OSS included a late measurement at 192 h p.i., which was necessary for accurate and precise tumour TIACs. OSS with three TPs were identified to be 3-4, 96-100 and 192 h with an additional SPECT/CT measurement at the penultimate TP. Kidney and tumour RMSE of 6.4 to 7.7% and 6.3 to 7.8% were obtained, respectively. Shortening the total time for dosimetry to e.g. 96 h resulted in kidney and tumour RMSE of 6.8 to 8.3% and 9.1 to 11%, respectively. OSS with four TPs showed similar results as with three TPs. Planar images at 4 and 68 h and a SPECT/CT shortly after the 68 h measurement led to kidney and tumour RMSE of 8.4 to 12% and 12 to 16%, respectively. One single SPECT/CT measurement at 52 h yielded good approximations for the kidney TIACs (RMSE of 7.0%), but led to biased tumour TIACs.ConclusionOSS allow improvements in accuracy and precision of renal and tumour dosimetry for [177Lu]Lu-PSMA therapy with potentially less effort. A late TP is important regarding accurate tumour TIACs.

Dataset Information

Targeted Tumor Therapy with Radiolabeled DNA Intercalator: A Possibility? Preclinical Investigations with 177Lu-Acridine.

Publications

Targeted Tumor Therapy with Radiolabeled DNA Intercalator: A Possibility? Preclinical Investigations with <sup>177</sup>Lu-Acridine.

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