Implementing Ac-225 labelled radiopharmaceuticals: practical considerations and (pre-)clinical perspectives.

BACKGROUND: In the past years, there has been a notable increase in interest regarding targeted alpha therapy using Ac-225, driven by the observed promising clinical anti-tumor effects. As the production and technology has advanced, the availability of Ac-225 is expected to increase in the near future, making the treatment available to patients worldwide. MAIN BODY: Ac-225 can be labelled to different biological vectors, whereby the success of developing a radiopharmaceutical depends heavily on the labelling conditions, purity of the radionuclide source, chelator, and type of quenchers used to avoid radiolysis. Multiple (methodological) challenges need to be overcome when working with Ac-225; as alpha-emission detection is time consuming and highly geometry dependent, a gamma co-emission is used, but has to be in equilibrium with the mother-nuclide. Because of the high impact of alpha emitters in vivo it is highly recommended to cross-calibrate the Ac-225 measurements for used quality control (QC) techniques (radio-TLC, HPLC, HP-Ge detector, and gamma counter). More strict health physics regulations apply, as Ac-225 has a high toxicity, thereby limiting practical handling and quantities used for QC analysis. CONCLUSION: This overview focuses specifically on the practical and methodological challenges when working with Ac-225 labelled radiopharmaceuticals, and underlines the required infrastructure and (detection) methods for the (pre-)clinical application.

Evaluation of the tolerability and safety of [225Ac]Ac-PSMA-I&T in patients with metastatic prostate cancer: a phase I dose escalation study.

BACKGROUND: Life expectancy of patients with metastatic castration-resistant prostate cancer (mCRPC) is still limited despite several systemic treatments. Within five years after diagnosis of primary prostate cancer, 10-20% of the patients have mCRPC and curation is not an option. Radionuclide therapy (RNT) targeted against prostate-specific membrane antigen (PSMA) emerged as a new treatment option and showed effective results in patients with mCRPC. Survival benefit after [177Lu]Lu-PSMA RNT has already been demonstrated in several clinical trials. However, [225Ac]Ac-PSMA (225Ac-PSMA) appears to be an even more promising radiopharmaceutical for the treatment of mCRPC. The use of alpha emitting radionuclides offers advantages over beta emitting radionuclides due to the high linear energy transfer effective for killing tumor cells and the limited range to reduce the radiation effects on the healthy tissue. However, these results are based on retrospective data and safety data of 225Ac-PSMA are still limited. Therefore, a prospective trial is needed to determine the optimal amount of activity that can be administered. METHODS: The 225Ac-PSMA-Imaging & Therapy (I&T) trial is an investigator-initiated phase I, single-center, open label, repeated dose-escalation and expansion trial. Patient with PSMA-positive mCRPC after at least one line of chemotherapy and/or one line of nonsteroidal antiandrogen will be treated with 225Ac-PSMA-I&T in increasing amount of activity per cycle. Dose-escalation following an accelerated 3 + 3 design which allows to open the next dose-level cohort in the absence of dose limiting toxicity while the previous one is still ongoing. Up to 4 treatment cohorts will be explored including up to 3 dose-escalation cohorts and one expansion cohort where patients will be administered with the recommended dose. A total of up to 30 patients will be enrolled in this trial. All patients will be evaluated for safety. Additionally, dosimetry was performed for the patients in the dose-escalation cohorts after the first 225Ac-PSMA-I&T administration. DISCUSSION: This trial will assess the safety and tolerability of 225Ac-PSMA-I&T in patients with mCRPC to recommend the optimal dose for the phase II trial. TRIAL REGISTRATION: ClinicalTrials.gov, (NCT05902247). Retrospectively registered 13 June 2023.

The Balance Between the Therapeutic Efficacy and Safety of [177Lu]Lu-NeoB in a Preclinical Prostate Cancer Model.

PURPOSE: Radiolabeled NeoB is a promising gastrin-releasing peptide receptor (GRPR)-targeting radiopharmaceutical for theranostics of GRPR-expressing malignancies, e.g., prostate cancer (PCa). The aim of this study was to evaluate the effect of different doses of [177Lu]Lu-NeoB on the balance between therapeutic efficacy and safety in a preclinical PCa model. PROCEDURES: To determine the efficacy of [177Lu]Lu-NeoB, PC-3 xenografted mice received 3 sham injections (control group) or 3 injections of 30 MBq/300 pmol, 40 MBq/400 pmol, or 60 MBq/600 pmol [177Lu]Lu-NeoB (groups 1, 2, and 3, respectively) 1 week apart. To quantify tumor uptake, single-photon emission computed tomography/computed tomography (SPECT/CT) imaging was performed 4 h after the first, second, and third injection on a separate group of animals. For safety evaluations, pancreatic and renal tissues of non-tumor-bearing mice treated with the abovementioned [177Lu]Lu-NeoB doses were evaluated 12 and 24 weeks post-treatment. RESULTS: Treatment of PC-3 tumors with all three studied [177Lu]Lu-NeoB doses was effective. Median survival times were significantly (p < 0.0001) improved for treatment groups 1, 2, and 3 versus the control group (82 days, 89 days, 99 days versus 19 days, respectively). However, no significant differences were observed between treatment groups. Quantification of SPECT/CT images showed minimal differences in the average absolute radioactivity uptake, especially after the third injection. Histopathological analysis revealed no clear signs of treatment-related pancreatic toxicity. For the kidneys, atrophy and fibrosis were observed for one animal from group 1 and a chronic inflammatory response was observed for both animals from group 3 at 24 weeks post-treatment. CONCLUSIONS: Treatment with [177Lu]Lu-NeoB is effective in a preclinical PCa model. Adjusting the administered dose could positively impact the risk-benefit balance as a higher dose might not lead to an increased therapeutic effect, but it may lead to an increase in toxicological effects in healthy organs such as the kidneys.

Improved postprocessing of dynamic glucose-enhanced CEST MRI for imaging brain metastases at 3 T.

BACKGROUND: Dynamic glucose-enhanced (DGE) chemical exchange saturation transfer (CEST) has the potential to characterize glucose metabolism in brain metastases. Since the effect size of DGE CEST is small at 3 T (< 1%), measurements of signal-to-noise ratios are challenging. To improve DGE detection, we developed an acquisition pipeline and extended image analysis for DGE CEST on a hybrid 3-T positron emission tomography/magnetic resonance imaging system. METHODS: This cross-sectional study was conducted after local ethical approval. Static Z-spectra (from -100 to 100 ppm) were acquired to compare the use of 1.2 versus 2 ppm to calculate static glucose-enhanced (glucoCEST) maps in 10 healthy volunteers before and after glucose infusion. Dynamic CEST images were acquired during glucose infusion. Image analysis was optimized using motion correction, dynamic B0 correction, and principal component analysis (PCA) to improve the detection of DGE CEST in the sagittal sinus, cerebrospinal fluid, and grey and white matter. The developed DGE CEST pipeline was applied to four patients diagnosed with brain metastases. RESULTS: GlucoCEST was strongest in healthy tissues at 2 ppm. Correcting for motion, B0, and use of PCA locally improved DGE maps. A larger contrast between healthy tissues and enhancing regions in brain metastases was found when dynamic B0 correction and PCA denoising were applied. CONCLUSION: We demonstrated the feasibility of DGE CEST with our developed acquisition and analysis pipeline at 3 T in patients with brain metastases. This work enables a direct comparison of DGE CEST to 18F-fluoro-deoxy-D-glucose positron emission tomography of glucose metabolism in patients with brain metastases. RELEVANCE STATEMENT: Contrast between brain metastasis and healthy brain tissue in DGE CEST MR images is improved by including principle component analysis and dynamic magnetic field correction during postprocessing. This approach enables the detection of increased DGE CEST signal in brain metastasis, if present. KEY POINTS: • Despite the low signal-to-noise ratio, dynamic glucose-enhanced CEST MRI is feasible at 3 T. • Principal component analyses and dynamic magnetic field correction improve DGE CEST MRI. • DGE CEST MRI does not consequently show changes in brain metastases compared to healthy brain tissue. • Increased DGE CEST MRI in brain metastases, if present, shows overlap with contrast enhancement on T1-weighted images.

Replacing Lu-177 with Tb-161 in DOTA-TATE and PSMA-617 therapy: potential dosimetric implications for activity selection.

AIM: To explore the dosimetric effect of substituting Lu-177 with Tb-161 in targeted radionuclide therapy (TRT) using the registered tracers DOTA-TATE and PSMA-617. METHODS: Using established kinetic data for [177Lu]Lu-DOTA-TATE and [177Lu]Lu-PSMA-617, radiation absorbed doses to typical tumour lesion as well as non-target tissues ([177Lu]Lu-DOTA-TATE: kidneys, spleen and liver, [177Lu]Lu-PSMA-617: kidneys, liver and salivary glands) were calculated for Lu-177 and Tb-161. RESULTS: For both DOTA-TATE and PSMA-617, the substitution of Lu-177 with Tb-161 results in an increase in the delivered dose per unit of activity to tumour tissue by 40%. If an equivalent non-target delivered dose is strived for in order not to increase toxicity, based on kidney absorbed dose, 7400 MBq Lu-177 per cycle should be substituted with 5400 MBq Tb-161 for DOTA-TATE and 5300 MBq of Tb-161 for PSMA-617. CONCLUSION: When substituting Lu-177 with Tb-161, activity conversion is necessary in order not to exceed non-target dose limits.

[212Pb]Pb-eSOMA-01: A Promising Radioligand for Targeted Alpha Therapy of Neuroendocrine Tumors.

Peptide receptor radionuclide therapy (PRRT) has been applied to the treatment of neuroendocrine tumors (NETs) for over two decades. However, improvement is still needed, and targeted alpha therapy (TAT) with alpha emitters such as lead-212 (212Pb) represents a promising avenue. A series of ligands based on octreotate was developed. Lead-203 was used as an imaging surrogate for the selection of the best candidate for the studies with lead-212. 203/212Pb radiolabeling and in vitro assays were carried out, followed by SPECT/CT imaging and ex vivo biodistribution in NCI-H69 tumor-bearing mice. High radiochemical yields (≥99%) and purity (≥96%) were obtained for all ligands. [203Pb]Pb-eSOMA-01 and [203Pb]Pb-eSOMA-02 showed high stability in PBS and mouse serum up to 24 h, whereas [203Pb]Pb-eSOMA-03 was unstable in those conditions. All compounds exhibited a nanomolar affinity (2.5-3.1 nM) for SSTR2. SPECT/CT images revealed high tumor uptake at 1, 4, and 24 h post-injection of [203Pb]Pb-eSOMA-01/02. Ex vivo biodistribution studies confirmed that the highest uptake in tumors was observed with [212Pb]Pb-eSOMA-01. [212Pb]Pb-eESOMA-01 displayed the highest absorbed dose in the tumor (35.49 Gy/MBq) and the lowest absorbed dose in the kidneys (121.73 Gy/MBq) among the three tested radioligands. [212Pb]Pb-eSOMA-01 is a promising candidate for targeted alpha therapy of NETs. Further investigations are required to confirm its potential.

First preclinical evaluation of [225Ac]Ac-DOTA-JR11 and comparison with [177Lu]Lu-DOTA-JR11, alpha versus beta radionuclide therapy of NETs.

BACKGROUND: The [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.

DNA-PKcs inhibitors sensitize neuroendocrine tumor cells to peptide receptor radionuclide therapy in vitro and in vivo.

Background: Peptide receptor radionuclide therapy (PRRT) increases progression-free survival and quality of life of neuroendocrine tumor (NET) patients, however complete cures are rare and dose-limiting toxicity has been reported. PRRT induces DNA damage of which DNA double strand breaks (DSBs) are the most cytotoxic. DNA-dependent protein kinase catalytic subunit (DNA-PKcs) is a key player in DSB repair and its inhibition therefore is a potential way to enhance PRRT efficacy without increasing the dosage. Methods: We analyzed effects of combining PRRT and DNA-PKcs inhibitor AZD7648 on viability, cell death and clonogenic survival on SSTR2-expressing cell lines BON1-SSTR2, GOT1 and NCI-H69. Therapy-induced DNA damage response was assessed by analyzing DSB foci levels and cell cycle distributions. In vivo efficacy was investigated in BON1-SSTR2 and NCI-H69 xenografted mice and hematologic and renal toxicity were monitored by blood counts, creatinine levels and analyzing renal morphology. Results: Combining PRRT and AZD7648 significantly decreased viability of BON1-SSTR2, GOT1 and NCI-H69 cells and induced cell death in GOT1 and BON1-SSTR2 cells. A strong effect of AZD7648 on PRRT-induced DSB repair was found. In GOT1 cells, this was accompanied by induction of cell cycle blocks. However, BON1-SSTR2 cells were unable to fully arrest their cell cycle and polyploid cells with high DNA damage levels were detected. In vivo, AZD7648 significantly sensitized BON1-SSTR2 and NCI-H69 xenograft models to PRRT. In addition, combination therapy did not induce significant changes in body weight, blood composition, plasma creatinine levels and renal morphology, indicating the absence of severe acute hematologic and renal toxicity. Conclusion: These results highlight that the potentiation of the therapeutic effect of PRRT by DNA-PKcs inhibition is a highly effective and well-tolerated therapeutic strategy. Based on our findings, we recommend initiation of phase I/II studies in patients to find a safe and effective combination regimen.

Synthesis and radiolabelling of PSMA-targeted derivatives containing GYK/MVK cleavable linkers.

Targeted radionuclide therapy (TRT) is a promising strategy to treat different types of cancer. TRT relies on a targeting vector used to deliver a therapeutic radionuclide specifically to the tumour site. Several low molecular weight ligands targeting the prostate-specific membrane antigen (PSMA) have been synthesized, but their pharmacokinetic properties still need to be optimized. Hereby, we describe the synthesis of new conjugates, featuring the cleavable linkers Gly-Tyr-Lys (GYK) and Met-Val-Lys (MVK), to reduce the dose delivered to the kidneys. Compounds were synthesized by solid-phase peptide synthesis (SPPS) and obtained in greater than 95% chemical purity. Radiolabelling was performed with both In-111 and Lu-177 to validate potential use of the compounds as both imaging and therapeutic agents. Radiochemical purity greater than 80% was obtained for both nuclides, but significant radiolysis was observed for the methionine-containing analogue. The results obtained thus far with the GYK-PSMA conjugate could warrant further biological investigations.

In Vivo Efficacy Testing of Peptide Receptor Radionuclide Therapy Radiosensitization Using Olaparib.

Peptide receptor radionuclide therapy (PRRT), a form of internal targeted radiation treatment using [177Lu]Lu [DOTA0-Tyr3]octreotate, is used to treat patients with metastasized neuroendocrine tumors (NETs). Even though PRRT is now the second line of treatment for patients with metastasized NETs, the majority of patients will not be cured by the treatment. PRRT functions by inducing DNA damage upon radioactive decay and inhibition of DNA damage repair proteins could therefore be used as a strategy to potentiate PRRT. Previous work has shown promising results on the combination of PRRT with the PARP inhibitor olaparib in cell lines and mice and we have been taken the next step for further in vivo validation using two different xenografted mouse models. We observed that this combination therapy resulted in increased therapeutic efficacy only in one model and not the other. Overall, our findings indicate a tumor-type dependent anti-tumor response to the combination of PRRT and olaparib. These data emphasize the unmet need for the molecular stratification of tumors to predetermine the potential clinical value of combining PARP inhibition with PRRT.

Radiolabeling and quality control of therapeutic radiopharmaceuticals: optimization, clinical implementation and comparison of radio-TLC/HPLC analysis, demonstrated by [177Lu]Lu-PSMA.

BACKGROUND: Radiopharmaceuticals are considered as regular medicinal products and therefore the same regulations as for non-radioactive medicinal products apply. However, specific aspects should be considered due to the radiochemical properties. Radiopharmaceutical dedicated monographs are developed in the European Pharmacopoeia to address this. Currently, different quality control methods for non-registered radiopharmaceuticals are utilized, often focusing on radio-TLC only, which has its limitations. When the radiochemical yield (RCY) is measured by radio-TLC analysis, degradation products caused by radiolysis are frequently not detected. In contrast, HPLC analysis defines the radiochemical purity (RCP), allowing for detection of peak formation related to radiolysis. During the introduction and optimization phase of therapeutic radiopharmaceuticals, significant percentages of impurities, like radiolysed construct formation, may have consequential impact on patient treatment. Since more hospitals and institutes are offering radiopharmaceutical therapies, such as [177Lu]Lu-PSMA with an in-house production, the demand for adequate quality control is increasing. Here we show the optimization and implementation of a therapeutic radiopharmaceutical, including the comparison of ITLC and HPLC quality control. RESULTS: Downscaled conditions (74 MBq/µg) were in concordance to clinical conditions (18 GBq/250 µg, 5 mL syringe/100 mL flacon); all results were consistent with an > 98% RCY (radio-TLC) and stability of > 95% RCP (HPLC). Radio-TLC did not identify radiolysis peaks, while clear identification was performed by HPLC analysis. Decreasing the RCP with 50%, reduced the cell-binding capacity with 27%. CONCLUSION: This research underlines the importance of the radiolabeling and optimization including clinical implementation and clarifies the need for cross-validation of the RCY and RCP for quality control measurements. Only HPLC analysis is suitable for identification of radiolysis. Here we have proven that radiolysed [177Lu]Lu-PSMA has less binding affinity and thus likely will influence treatment efficacy. HPLC analysis is therefore essential to include in at least the validation phase of radiopharmaceutical implementation to ensure clinical treatment quality.

Advances in 177Lu-PSMA and 225Ac-PSMA Radionuclide Therapy for Metastatic Castration-Resistant Prostate Cancer.

For patients with metastatic castration-resistant prostate cancer (mCRPC), the survival benefit of classic treatment options with chemotherapy and drugs targeting androgen signaling is limited. Therefore, beta and alpha radionuclide therapy (RNT) have emerged as novel treatment options for patients with mCRPC. Radioligands target the prostate-specific membrane antigen (PSMA) epitopes, which are upregulated up to a thousand times more in prostate cancer cells compared to the cells in normal tissues. For this reason, PSMA is an excellent target for both imaging and therapy. Over the past years, many studies have investigated the treatment effects of lutetium-177 labeled PSMA (177Lu-PSMA) and actinium-225 labeled PSMA (225Ac-PSMA) RNT in patients with mCRPC. While promising results have been achieved, this field is still in development. In this review, we have summarized and discussed the clinical data of 177Lu-PSMA and 225Ac-PSMA RNT in patients with mCRPC.

Safety of [177Lu]Lu-NeoB treatment: a preclinical study characterizing absorbed dose and acute, early, and late organ toxicity.

PURPOSE: The radiolabeled gastrin-releasing peptide receptor (GRPR)-targeting antagonist NeoB is a promising radioligand for imaging and therapy of GRPR-expressing malignancies. In the current study, we aimed to discover the target organs of toxicity and the radiotoxic effects to these organs, when repeated dosages of [177Lu]Lu-NeoB are administered to healthy female and male mice. METHODS: Animals received either 3 injections, with a 7-day interval, of vehicle (control group 1), 1200 pmol [175Lu]Lu-NeoB (control group 2) or 40 MBq/400 pmol, 80 MBq/800 pmol, and 120 MBq/1200 pmol [177Lu]Lu-NeoB (treatment groups 1, 2, and 3, respectively). At week 5, 19, and 43 after the first injection acute, early, and late organ toxicity, respectively, was determined. For this, histopathological and blood analyses were performed. To correlate the observed toxicity to absorbed dose, we also performed extensive biodistribution and dosimetry studies. RESULTS: The biodistribution study showed the highest absorbed doses in GRPR-expressing pancreas, the liver, and the kidneys (the main organs of excretion). Both control groups and almost all animals of treatment group 1 did not show any treatment-related toxicological effects. Despite the high absorbed doses, no clear microscopic signs of toxicity were found in the pancreas and the liver. Histological analysis indicated kidney damage in the form of hydronephrosis and nephropathy in treatment groups 2 and 3 that were sacrificed at the early and late time point. In the same groups, increased blood urea nitrogen levels were found. CONCLUSION: In general, repeated administration of [177Lu]Lu-NeoB was tolerated. The most significant radiotoxic effects were found in the kidneys, similar to other clinically applied radioligands. The results of this study underline the potential of [177Lu]Lu-NeoB as a promising option for clinical therapy.

Preclinical Assessment of the Combination of PSMA-Targeting Radionuclide Therapy with PARP Inhibitors for Prostate Cancer Treatment.

Prostate specific membrane antigen targeted radionuclide therapy (PSMA-TRT) is a promising novel treatment for prostate cancer (PCa) patients. However, PSMA-TRT cannot be used for curative intent yet, thus additional research on how to improve the therapeutic efficacy is warranted. A potential way of achieving this, is combining TRT with poly ADP-ribosylation inhibitors (PARPi), which has shown promising results for TRT of neuroendocrine tumor cells. Currently, several clinical trials have been initiated for this combination for PCa, however so far, no evidence of synergism is available for PCa. Therefore, we evaluated the combination of PSMA-TRT with three classes of PARPi in preclinical PCa models. In vitro viability and survival assays were performed using PSMA-expressing PCa cell lines PC3-PIP and LNCaP to assess the effect of increasing concentrations of PARPi veliparib, olaparib or talazoparib in combination with PSMA-TRT compared to single PARPi treatment. Next, DNA damage analyses were performed by quantifying the number of DNA breaks by immunofluorescent stainings. Lastly, the potential of the combination treatments was studied in vivo in mice bearing PC3-PIP xenografts. Our results show that combining PSMA-TRT with PARPi did not synergistically affect the in vitro clonogenic survival or cell viability. DNA-damage analysis revealed only a significant increase in DNA breaks when combining PSMA-TRT with veliparib and not in the other combination treatments. Moreover, PSMA-TRT with PARPi treatment did not improve tumor control compared to PSMA-TRT monotherapy. Overall, the data presented do not support the assumption that combining PSMA-TRT with PARPi leads to a synergistic antitumor effect in PCa. These results underline that extensive preclinical research using various PCa models is imperative to validate the applicability of the combination strategy for PCa, as it is for other cancer types.

In vitro dose effect relationships of actinium-225- and lutetium-177-labeled PSMA-I&T.

PURPOSE: Targeting the prostate-specific membrane antigen (PSMA) using lutetium-177-labeled PSMA-specific tracers has become a very promising novel therapy option for prostate cancer (PCa). The efficacy of this therapy might be further improved by replacing the ß-emitting lutetium-177 with the α-emitting actinium-225. Actinium-225 is thought to have a higher therapeutic efficacy due to the high linear energy transfer (LET) of the emitted α-particles, which can increase the amount and complexity of the therapy induced DNA double strand breaks (DSBs). Here we evaluated the relative biological effectiveness of [225Ac]Ac-PSMA-I&T and [177Lu]Lu-PSMA-I&T by assessing in vitro binding characteristics, dosimetry, and therapeutic efficacy. METHODS AND RESULTS: The PSMA-expressing PCa cell line PC3-PIP was used for all in vitro assays. First, binding and displacement assays were performed, which revealed similar binding characteristics between [225Ac]Ac-PSMA-I&T and [177Lu]Lu-PSMA-I&T. Next, the assessment of the number of 53BP1 foci, a marker for the number of DNA double strand breaks (DSBs), showed that cells treated with [225Ac]Ac-PSMA-I&T had slower DSB repair kinetics compared to cells treated with [177Lu]Lu-PSMA-I&T. Additionally, clonogenic survival assays showed that specific targeting with [225Ac]Ac-PSMA-I&T and [177Lu]Lu-PSMA-I&T caused a dose-dependent decrease in survival. Lastly, after dosimetric assessment, the relative biological effectiveness (RBE) of [225Ac]Ac-PSMA-I&T was found to be 4.2 times higher compared to [177Lu]Lu-PSMA-I&T. CONCLUSION: We found that labeling of PSMA-I&T with lutetium-177 or actinium-225 resulted in similar in vitro binding characteristics, indicating that the distinct biological effects observed in this study are not caused by a difference in uptake of the two tracers. The slower repair kinetics of [225Ac]Ac-PSMA-I&T compared to [177Lu]Lu-PSMA-I&T correlates to the assumption that irradiation with actinium-225 causes more complex, more difficult to repair DSBs compared to lutetium-177 irradiation. Furthermore, the higher RBE of [225Ac]Ac-PSMA-I&T compared to [177Lu]Lu-PSMA-I&T underlines the therapeutic potential for the treatment of PCa.

Improved Multimodal Tumor Necrosis Imaging with IRDye800CW-DOTA Conjugated to an Albumin-Binding Domain.

PURPOSE: To assess our improved NACA for the detection of tumor necrosis. METHODS: We increased the blood circulation time of our NACA by adding an albumin-binding domain to the molecular structure. We tested the necrosis avidity on dead or alive cultured cells and performed SPECT and fluorescence imaging of both spontaneous and treatment-induced necrosis in murine breast cancer models. We simultaneously recorded [18F]FDG-PET and bioluminescence images for complementary detection of tumor viability. RESULTS: We generated two albumin-binding IRDye800CW derivatives which were labeled with indium-111 with high radiochemical purity. Surprisingly, both albumin-binding NACAs had >10x higher in vitro binding towards dead cells. We selected [111In]3 for in vivo experiments which showed higher dead cell binding in vitro and in vivo stability. The doxorubicin-treated tumors showed increased [111In]3-uptake (1.74 ± 0.08%ID/g after saline treatment, 2.25 ± 0.16%ID/g after doxorubicin treatment, p = 0.044) and decreased [18F]FDG-uptake (3.02 ± 0.51%ID/g after saline treatment, 1.79 ± 0.11%ID/g after doxorubicin treatment, p = 0.040), indicating therapy efficacy. Moreover, we detected increased [111In]3-uptake and tumor necrosis in more rapidly growing EMT6 tumors. CONCLUSIONS: Our albumin-binding NACA based on IRDye800CW facilitates tumor-necrosis imaging for assessment of therapy efficacy and aggressiveness in solid tumors using both fluorescence and SPECT imaging.

Development of [225Ac]Ac-PSMA-I&T for Targeted Alpha Therapy According to GMP Guidelines for Treatment of mCRPC.

Recently, promising results of the antitumor effects were observed in patients with metastatic castration-resistant prostate cancer treated with 177Lu-labeled PSMA-ligands. Radionuclide therapy efficacy may even be improved by using the alpha emitter Ac-225. Higher efficacy is claimed due to high linear energy transfer specifically towards PSMA positive cells, causing more double-strand breaks. This study aims to manufacture [225Ac]Ac-PSMA-I&T according to good manufacturing practice guidelines for the translation of [225Ac]Ac-PSMA-I&T into a clinical phase 1 dose escalation study. Quencher addition during labeling was investigated. Quality control of [225Ac]Ac-PSMA-I&T was based on measurement of Fr-221 (218 keV), in equilibrium with Ac-225 in approximately six half-lives of Fr-221 (T½ = 4.8 min). Radio-(i)TLC methods were utilized for identification of the different radiochemical forms, gamma counter for concentration determination, and HPGe-detector for the detection of the radiochemical yield. Radiochemical purity was determined by HPLC. The final patient dose was prepared and diluted with an optimized concentration of quenchers as during labeling, with an activity of 8-12 MBq (±5%), pH > 5.5, 100 ± 20 µg/dose, PSMA-I&T, radiochemical yield >95%, radiochemical purity >90% (up to 3 h), endotoxin levels of <5 EU/mL, osmolarity of 2100 mOsmol, and is produced according to current guidelines. The start of the phase I dose escalation study is planned in the near future.

GRPr Antagonist 68Ga-SB3 PET/CT Imaging of Primary Prostate Cancer in Therapy-Naïve Patients.

The gastrin-releasing peptide receptor (GRPr) is overexpressed in prostate cancer (PCa) cells, making it an excellent tool for targeted imaging. The 68Ga-labeled GRPr antagonist SB3 has shown excellent results in preclinical and clinical studies and was selected for further clinical investigation. The aims of this phase I study were to investigate 68Ga-SB3 PET/CT imaging of primary PCa tumors and assess safety. More aims included an investigation of biodistribution and dosimetry and a comparison with pathology and GRPr expression. Methods: Ten therapy-naïve, biopsy-confirmed PCa patients planned for prostatectomy were included. A 3-h extensive PET/CT imaging protocol was performed within 2 wk before prostatectomy. Prostate tissue was evaluated for tumor localization and Gleason score, and in vitro autoradiography was performed to determine GRPr expression. Available MRI scans performed within 3 mo before the study were matched. For dosimetry, residence times were estimated and effective dose to the body as well as absorbed doses to organs were calculated using the IDAC dose model, version 2.1. Results: Administration of 68Ga-SB3 (187.4 ± 40.0 MBq, 40 ± 5 µg) was well tolerated; no significant changes in vital signs or laboratory results were observed. 68Ga-SB3 PET/CT showed lesions in 8 of 10 patients. Pathologic analysis revealed a total of 16 tumor lesions, of which PET/CT showed 14, resulting in a sensitivity of 88%. 68Ga-SB3 PET/CT imaging showed uptake in 2 large prostatic intraepithelial neoplasia foci, considered a precursor to PCa, resulting in an 88% specificity. Autoradiography of tumor lesions revealed heterogeneous GRPr expression and was negative in 4 patients. Both PET/CT-negative patients had a GRPr-negative tumor. In autoradiography-positive tumors, the level of GRPr expression showed a significant correlation to tracer uptake on PET/CT. Dosimetry calculations estimated the effective dose to be 0.0144 mSv/MBq, similar to other 68Ga-labeled radiopeptides. The highest absorbed dose was detected in the physiologic GRPr-expressing pancreas (0.198 mGy/MBq), followed by the bladder wall and kidneys. Conclusion:68Ga-SB3 PET/CT is a safe imaging method and a promising tool for early PCa imaging.

Autoradiographical assessment of inflammation-targeting radioligands for atherosclerosis imaging: potential for plaque phenotype identification.

PURPOSE: Many radioligands have been developed for the visualization of atherosclerosis by targeting inflammation. However, interpretation of in vivo signals is often limited to plaque identification. We evaluated binding of some promising radioligands in an in vitro approach in atherosclerotic plaques with different phenotypes. METHODS: Tissue sections of carotid endarterectomy tissue were characterized as early plaque, fibro-calcific plaque, or phenotypically vulnerable plaque. In vitro binding assays for the radioligands [111In]In-DOTATATE; [111In]In-DOTA-JR11; [67Ga]Ga-Pentixafor; [111In]In-DANBIRT; and [111In]In-EC0800 were conducted, the expression of the radioligand targets was assessed via immunohistochemistry. Radioligand binding and expression of radioligand targets was investigated and compared. RESULTS: In sections characterized as vulnerable plaque, binding was highest for [111In]In-EC0800; followed by [111In]In-DANBIRT; [67Ga]Ga-Pentixafor; [111In]In-DOTA-JR11; and [111In]In-DOTATATE (0.064 ± 0.036; 0.052 ± 0.029; 0.011 ± 0.003; 0.0066 ± 0.0021; 0.00064 ± 0.00014 %Added activity/mm2, respectively). Binding of [111In]In-DANBIRT and [111In]In-EC0800 was highest across plaque phenotypes, binding of [111In]In-DOTA-JR11 and [67Ga]Ga-Pentixafor differed most between plaque phenotypes. Binding of [111In]In-DOTATATE was the lowest across plaque phenotypes. The areas positive for cells expressing the radioligand's target differed between plaque phenotypes for all targets, with lowest percentage area of expression in early plaque sections and highest in phenotypically vulnerable plaque sections. CONCLUSIONS: Radioligands targeting inflammatory cell markers showed different levels of binding in atherosclerotic plaques and among plaque phenotypes. Different radioligands might be used for plaque detection and discerning early from vulnerable plaque. [111In]In-EC0800 and [111In]In-DANBIRT appear most suitable for plaque detection, while [67Ga]Ga-Pentixafor and [111In]In-DOTA-JR11 might be best suited for differentiation between plaque phenotypes.