68Ga-DOTA-D-Alanine-BoroPro Radiotracer for Imaging of the Fibroblast Activation Protein in Malignant and Non-Malignant Diseases.

Recently, we reported a new fibroblast activation protein (FAP) inhibitor radiopharmaceutical based on the 99mTc-((R)-1-((6-hydrazinylnicotinoyl)-D-alanyl) pyrrolidin-2-yl) boronic acid (99mTc-HYNIC-D-Alanine-BoroPro)(99mTc-HYNIC-iFAP) structure for tumor microenvironment SPECT imaging. This research aimed to synthesize 68Ga-[2,2',2″,2‴-(2-(4-(2-(5-(((S)-1-((S)-2-boronopyrrolidin-1-yl)-1-oxopropan-2-yl)carbamoyl)pyridin-2-yl)hydrazine-1-carbothioamido)benzyl)-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetrayl)tetraacetic acid] (68Ga-DOTA-D-Alanine-BoroPro)(68Ga-iFAP) as a novel radiotracer for PET imaging and evaluate its usefulness for FAP expression in malignant and non-malignant tissues. The coupling of p-SCN-benzene DOTA with HYNIC-iFAP was used for the chemical synthesis and further labeling with 68Ga. Radiochemical purity was verified by radio-HPLC. The specificity of 68Ga-iFAP was evaluated in HCT116 cells, in which FAP expression was verified by immunofluorescence and Western blot. Biodistribution and biokinetic studies were performed in murine models. 68Ga-iFAP uptake at the myocardial level was assessed in mice with induced infarction. First-in-human images of 68Ga-iFAP in healthy subjects and patients with myocardial infarction, glioblastoma, prostate cancer, and breast cancer were also obtained. DOTA-D-Alanine BoroPro was prepared with a chemical purity of 98% and was characterized by UPLC mass spectroscopy, FT-IR, and UV-vis. The 68Ga-iFAP was obtained with a radiochemical purity of >95%. In vitro and in vivo studies demonstrated 68Ga-iFAP-specific recognition for FAP, rapid renal elimination, and adequate visualization of the glioblastoma, breast tumor, prostate cancer, and myocardial infarction sites. The results of this research justify further dosimetry and clinical trials to establish the specificity and sensitivity of 68Ga-iFAP PET for FAP expression imaging.

225Ac-iPSMA-RGD for Alpha-Therapy Dual Targeting of Stromal/Tumor Cell PSMA and Integrins.

Prostate-specific membrane antigens (PSMAs) are frequently overexpressed in both tumor stromal endothelial cells and malignant cells (stromal/tumor cells) of various cancers. The RGD (Arg-Gly-Asp) peptide sequence can specifically detect integrins involved in tumor angiogenesis. This study aimed to preclinically evaluate the cytotoxicity, biokinetics, dosimetry, and therapeutic efficacy of 225Ac-iPSMA-RGD to determine its potential as an improved radiopharmaceutical for alpha therapy compared with the 225Ac-iPSMA and 225Ac-RGD monomers. HEHA-HYNIC-iPSMA-RGD (iPSMA-RGD) was synthesized and characterized by FT-IR, UV-vis, and UPLC mass spectroscopy. The cytotoxicity of 225Ac-iPSMA-RGD was assessed in HCT116 colorectal cancer cells. Biodistribution, biokinetics, and therapeutic efficacy were evaluated in nude mice with induced HCT116 tumors. In vitro results showed increased DNA double-strand breaks through ROS generation, cell apoptosis, and death in HCT116 cells treated with 225Ac-iPSMA-RGD. The results also demonstrated in vivo cytotoxicity in cancer cells after treatment with 225Ac-iPSMA-RGD and biokinetic and dosimetric properties suitable for alpha therapy, delivering ablative radiation doses up to 237 Gy/3.7 kBq to HCT116 tumors in mice. Given the phenotype of HCT116 cancer cells, the results of this study warrant further dosimetric and clinical studies to determine the potential of 225Ac-iPSMA-RGD in the treatment of colorectal cancer.

99mTc-Labeled Cyclic Peptide Targeting PD-L1 as a Novel Nuclear Imaging Probe.

Recent cancer therapies have focused on reducing immune suppression in the tumor microenvironment to prevent cancer progression and metastasis. PD-1 is a checkpoint protein that stops the immune response and is expressed on immune T cells. Cancer cells express a PD-1 ligand (PD-L1) to bind to the T-cell surface and activate immunosuppressive pathways. This study aimed to design, synthesize, and evaluate a 99mTc-labeled PD-L1-targeting cyclic peptide inhibitor (99mTc-iPD-L1) as a novel SPECT radiopharmaceutical for PD-L1 expression imaging. AutoDock software (version 1.5) was used to perform molecular docking for affinity calculations. The chemical synthesis was based on the coupling reaction of 6-hydrazinylpyridine-3-carboxylic acid with a 14-amino-acid cyclic peptide. iPD-L1 was prepared for 99mTc labeling. Radio-HPLC was used to verify radiochemical purity. The stability of the radiopeptide in human serum was evaluated by HPLC. iPD-L1 specificity was assessed by SDS-PAGE. [99mTc]Tc-iPD-L1 cellular uptake in PD-L1-positive cancer cells (HCC827 and HCT116) and biodistribution in mice with induced tumors were also performed. One patient with advanced plantar malignant melanoma received [99mTc]Tc-iPD-L1. The iPD-L1 ligand (AutoDock affinity: -6.7 kcal/mol), characterized by UPLC mass, FT-IR, and UV-Vis spectroscopy, was obtained with a chemical purity of 97%. The [99mTc]Tc-iPD-L1 was prepared with a radiochemical purity of >90%. In vitro and in vivo analyses demonstrated [99mTc]Tc-iPD-L1 stability (>90% at 24 h) in human serum, specific recognition for PD-L1, high uptake by the tumor (6.98 ± 0.89% ID/g at 1 h), and rapid hepatobiliary and kidney elimination. [99mTc]Tc-iPD-L1 successfully detected PD-L1-positive lesions in a patient with plantar malignant melanoma. The results obtained in this study warrant further dosimetric and clinical studies to determine the sensitivity and specificity of [99mTc]Tc-iPD-L1/SPECT for PD-L1 expression imaging.

Synthesis and Evaluation of 177Lu-DOTA-PD-L1-i and 225Ac-HEHA-PD-L1-i as Potential Radiopharmaceuticals for Tumor Microenvironment-Targeted Radiotherapy.

Current cancer therapies focus on reducing immunosuppression and remodeling the tumor microenvironment to inhibit metastasis, cancer progression, and therapeutic resistance. Programmed death receptor 1 (PD-1) is expressed on immune T cells and is one of the so-called checkpoint proteins that can suppress or stop the immune response. To evade the immune system, cancer cells overexpress a PD-1 inhibitor protein (PD-L1), which binds to the surface of T cells to activate signaling pathways that induce immune suppression. This research aimed to synthesize PD-L1 inhibitory peptides (PD-L1-i) labeled with lutetium-177 (177Lu-DOTA-PD-L1-i) and actinium-225 (225Ac-HEHA-PD-L1-i) and to preclinically evaluate their potential as radiopharmaceuticals for targeted radiotherapy at the tumor microenvironment level. Using PD-L1-i peptide as starting material, conjugation with HEHA-benzene-SCN and DOTA-benzene-SCN was performed to yield DOTA-PD-L1-i and HEHA-PD-L1-I, which were characterized by FT-IR, UV-vis spectroscopy, and HPLC. After labeling the conjugates with 225Ac and 177Lu, cellular uptake in HCC827 cancer cells (PD-L1 positive), conjugate specificity evaluation by immunofluorescence, radiotracer effect on cell viability, biodistribution, biokinetics, and assessment of radiation absorbed dose in mice with in duced lung micrometastases were performed. 225Ac-HEHA-PD-L1-i and 177Lu-DOTA-PD-L1-i, obtained with radiochemical purities of 95 ± 3% and 98.5 ± 0.5%, respectively, showed in vitro and in vivo specific recognition for the PD-L1 protein in lung cancer cells and high uptake in HCC287 lung micrometastases (>30% ID). The biokinetic profiles of 177Lu-DOTA-PD-L1-i and 225Ac-DOTA-PD-L1-i showed rapid blood clearance with renal and hepatobiliary elimination and no accumulation in normal tissues. 225Ac-DOTA-PD-L1-i produced a radiation dose of 5.15 mGy/MBq to lung micrometastases. In the case of 177Lu-DOTA-PD-L1-i, the radiation dose delivered to the lung micrometastases was ten times (43 mGy/MBq) that delivered to the kidneys (4.20 mGy/MBq) and fifty times that delivered to the liver (0.85 mGy/MBq). Therefore, the radiotherapeutic PD-L1-i ligands of 225Ac and 177Lu developed in this research could be combined with immunotherapy to enhance the therapeutic effect in various types of cancer.

Improving Overall Survival and Quality of Life in Patients with Prostate Cancer and Neuroendocrine Tumors Using 177Lu-iPSMA and 177Lu-DOTATOC: Experience after 905 Treatment Doses.

177Lu-iPSMA is a novel radioligand developed at ININ-Mexico with a high affinity for the PSMA protein heavily expressed in cancer cells of approximately 95% of patients with metastatic castration-resistant prostate cancer (mCRPC). 177Lu-DOTATOC is a patent-free radioligand, molecularly recognized by somatostatin receptors (SSTR-2) overexpressed in cancer cells of about 80% of patients with metastatic gastroenteropancreatic neuroendocrine tumors (GEP-NET). This translational research aimed to determine the efficacy and safety of 177Lu-iPSMA and 177Lu-DOTATOC developed as GMP pharmaceutical formulations for treating progressive and advanced mCRPC and NET. One hundred and forty-five patients with mCRPC and one hundred and eighty-seven subjects with progressive NET (83% GEP-NET and 17% other NET), treated with 177Lu-iPSMA and 177Lu-DOTATOC, respectively, were evaluated. Patients received a mean dose of 7.4 GBq per administration of 177Lu-iPSMA (range 1-5 administrations; 394 treatment doses) or 177Lu-DOTATOC (range 2-8 administrations; 511 treatment doses) at intervals of 1.5-2.5 months. Efficacy was assessed by SPECT/CT or PET/CT. Results were stratified by primary tumor origin and number of doses administered. Patients with mCRPC showed overall survival (OS) of 21.7 months with decreased radiotracer tumor uptake (SUV) and PSA level in 80% and 73% of patients, respectively. In addition, a significant reduction in pain (numerical scale from 10-7 to 3-1) was observed in 88% of patients with bone metastases between one and two weeks after the second injection. In the GEP-NET population, the median progression-free survival was 34.7 months, with an OS of >44.2 months. The treatments were well tolerated. Only ten patients experienced grade ≥ 3 myelosuppression (3% of all patients). The observed safety profiles and favorable therapeutic responses demonstrated the potential of 177Lu-iPSMA and 177Lu-DOTATOC to improve overall survival and quality of life in patients with progressive and advanced mCRPC and NET.

Chemo-radiotherapy with 177Lu-PLGA(RGF)-CXCR4L for the targeted treatment of colorectal cancer.

Introduction: More than 1.9 million new cases of colorectal cancer and 935,000 deaths were estimated to have occurred worldwide in 2020. Therapies for metastatic colorectal cancer include cytotoxic chemotherapy and targeted therapies in multiple lines of treatment. Nevertheless, the optimal use of these agents has not yet been resolved. Regorafenib (RGF) is an Food and Drug Administration (FDA)-authorized multikinase inhibitor indicated for patients with metastatic colorectal cancer, non-responding to priority lines of chemotherapy and immunotherapy. Nanoparticles have been used in specific applications, such as site-specific drug delivery systems, cancer therapy, and clinical bioanalytical diagnostics. C-X-C Chemokine receptor type 4 (CXCR4) is the most widely-expressed chemokine receptor in more than 23 human cancer types, including colorectal cancer. This research aimed to synthesize and preclinically evaluate a targeted nanosystem for colorectal cancer chemo-radiotherapy using RGF encapsulated in Poly(D,L-lactic-co-glycolic acid) (PLGA) nanoparticles coated with a CXCR4 ligand (CXCR4L) and 177Lu as a therapeutic ß-emitter. Methods: Empty PLGA and PLGA(RGF) nanoparticles were prepared using the microfluidic method, followed by the DOTA and CXCR4L functionalization and nanoparticle radiolabeling with 177Lu. The final nanosystem gave a particle size of 280 nm with a polydispersity index of 0.347. In vitro and in vivo toxicity effects were assessed using the HCT116 colorectal cancer cell line. Results: 177Lu-PLGA(RGF)-CXCR4L nanoparticles decreased cell viability and proliferation by inhibiting Erk and Akt phosphorylation and promoting apoptosis. Moreover, in vivo administration of 177Lu-PLGA(RGF)-CXCR4L significantly reduced tumor growth in an HCT116 colorectal cancer xenograft model. The biokinetic profile showed hepatic and renal elimination. Discussion: Data obtained in this research justify additional preclinical safety trials and the clinical evaluation of 177Lu-PLGA(RGF)-CXCR4L as a potential combined treatment of colorectal cancer.

Toxicity Assessment of [177Lu]Lu-iFAP/iPSMA Nanoparticles Prepared under GMP-Compliant Radiopharmaceutical Processes.

The fibroblast activation protein (FAP) is heavily expressed in fibroblasts associated with the tumor microenvironment, while the prostate-specific membrane antigen (PSMA) is expressed in the neovasculature of malignant angiogenic processes. Previously, we reported that [177Lu]lutetium sesquioxide-iFAP/iPSMA nanoparticles ([177Lu]Lu-iFAP/iPSMA) inhibit HCT116 tumor progression in mice. Understanding the toxicity of [177Lu]Lu-iFAP/iPSMA in healthy tissues, as well as at the tissue and cellular level in pathological settings, is essential to demonstrate the nanosystem safety for treating patients. It is equally important to demonstrate that [177Lu]Lu-iFAP/iPSMA can be prepared under good manufacturing practices (GMP) with reproducible pharmaceutical-grade quality characteristics. This research aimed to prepare [177Lu]Lu-iFAP/iPSMA under GMP-compliant radiopharmaceutical processes and evaluate its toxicity in cell cultures and murine biological systems under pathological environments. [177Lu]Lu2O3 nanoparticles were formulated as radiocolloidal solutions with FAP and PSMA inhibitor ligands (iFAP and iPSMA), sodium citrate, and gelatin, followed by heating at 121 °C (103-kPa pressure) for 15 min. Three consecutive batches were manufactured. The final product was analyzed according to conventional pharmacopeial methods. The Lu content in the formulations was determined by X-ray fluorescence. [177Lu]Lu-iFAP/iPSMA performance in cancer cells was evaluated in vitro by immunofluorescence. Histopathological toxicity in healthy and tumor tissues was assessed in HCT116 tumor-bearing mice. Immunohistochemical assays were performed to corroborate FAP and PSMA tumor expression. Acute genotoxicity was evaluated using the micronuclei assay. The results showed that the batches manufactured under GMP conditions were reproducible. Radiocolloidal solutions were sterile and free of bacterial endotoxins, with radionuclidic and radiochemical purity greater than 99%. The lutetium content was 0.10 ± 0.02 mg/mL (0.9 GBq/mg). Significant inhibition of cell proliferation in vitro and in tumors was observed due to the accumulation of nanoparticles in the fibroblasts (FAP+) and neovasculature (PSMA+) of the tumor microenvironment. No histopathological damage was detected in healthy tissues. The data obtained in this research provide new evidence on the selective toxicity to malignant tumors and the absence of histological changes in healthy tissues after intravenous injection of [177Lu]Lu-iFAP/iPSMA in mammalian hosts. The easy preparation under GMP conditions and the toxicity features provide the added value needed for [177Lu]Lu-iFAP/iPSMA clinical translation.

An evaluation of the present status of quality assurance program implementation in digital mammography facilities in a developing country.

Quality assurance program (QAP) implementation in mammography is a key aspect to guarantee the acquisition of -quality images and, thus, an opportune diagnosis of breast cancer. A QAP involves planned and systematic actions, such as quality control (QC) tests that will produce consistently high-quality images, while considering low radiation exposure for stakeholders. Currently, there is poor evidence of the current QAP implementation status in developing countries, particularly in Latin America. In this work, 47 Mexican digital mammography facilities were evaluated in terms of their QAP implementation's current status during the period October 2021 to April 2022. A survey of three key indicators of compliance/non-compliance with QAPs was carried out. Also, 17 QC test parameters were assessed in all mammography units according to an international framework. The Kruskal-Wallis test, McNemar test, and Spearman correlation were used to identify the significant correlation between the QC tests and QAP indicators. Statistical analysis showed a significant positive correlation between the QAP indicators and QC parameters that were evaluated, especially associated with an increase in image quality. Almost 90% of the facilities evaluated failed in some QAP indicators, and only 44.68% of the facilities had an updated and current QAP implemented. Successful QAP implementation directly impacted on the QC mammography test results, particularly in terms of image quality, thus conditioning a successful mammography acquisition chain. The results obtained in this work suggest that better regulations need to be implemented to guarantee QAP surveillance, which could mean an increase in opportune diagnosis and management of breast cancer, with improved radiological safety for patients and occupational exposure personnel.

Engineered rHDL Nanoparticles as a Suitable Platform for Theranostic Applications.

Reconstituted high-density lipoproteins (rHDLs) can transport and specifically release drugs and imaging agents, mediated by the Scavenger Receptor Type B1 (SR-B1) present in a wide variety of tumor cells, providing convenient platforms for developing theranostic systems. Usually, phospholipids or Apo-A1 lipoproteins on the particle surfaces are the motifs used to conjugate molecules for the multifunctional purposes of the rHDL nanoparticles. Cholesterol has been less addressed as a region to bind molecules or functional groups to the rHDL surface. To maximize the efficacy and improve the radiolabeling of rHDL theranostic systems, we synthesized compounds with bifunctional agents covalently linked to cholesterol. This strategy means that the radionuclide was bound to the surface, while the therapeutic agent was encapsulated in the lipophilic core. In this research, HYNIC-S-(CH2)3-S-Cholesterol and DOTA-benzene-p-SC-NH-(CH2)2-NH-Cholesterol derivatives were synthesized to prepare nanoparticles (NPs) of HYNIC-rHDL and DOTA-rHDL, which can subsequently be linked to radionuclides for SPECT/PET imaging or targeted radiotherapy. HYNIC is used to complexing 99mTc and DOTA for labeling molecules with 111, 113mIn, 67, 68Ga, 177Lu, 161Tb, 225Ac, and 64Cu, among others. In vitro studies showed that the NPs of HYNIC-rHDL and DOTA-rHDL maintain specific recognition by SR-B1 and the ability to internalize and release, in the cytosol of cancer cells, the molecules carried in their core. The biodistribution in mice showed a similar behavior between rHDL (without surface modification) and HYNIC-rHDL, while DOTA-rHDL exhibited a different biodistribution pattern due to the significant reduction in the lipophilicity of the modified cholesterol molecule. Both systems demonstrated characteristics for the development of suitable theranostic platforms for personalized cancer treatment.

[99mTc]Tc-iFAP/SPECT Tumor Stroma Imaging: Acquisition and Analysis of Clinical Images in Six Different Cancer Entities.

Fibroblast activation protein (FAP) is highly expressed on the cancer-associated fibroblasts (CAF) of the tumor stroma. Recently, we reported the preclinical evaluation and clinical biokinetics of a novel 99mTc-labeled FAP inhibitor radioligand ([99mTc]Tc-iFAP). This research aimed to evaluate [99mTc]Tc-iFAP for the tumor stroma imaging of six different cancerous entities and analyze them from the perspective of stromal heterogeneity. [99mTc]Tc-iFAP was prepared from freeze-dried kits with a radiochemical purity of 98 ± 1%. The study included thirty-two patients diagnosed with glioma (n = 5); adrenal cortex neuroendocrine tumor (n = 1); and breast (n = 21), lung (n = 2), colorectal (n = 1) and cervical (n = 3) cancer. Patients with glioma had been evaluated with a previous cranial MRI scan and the rest of the patients had been involved in a [18F]FDG PET/CT study. All oncological diagnoses were corroborated histopathologically. The patients underwent SPECT/CT brain imaging (glioma) or thoracoabdominal imaging 1 h after [99mTc]Tc-iFAP administration (i.v., 735 ± 63 MBq). The total lesions (n = 111) were divided into three categories: primary tumors (PT), lymph node metastases (LNm), and distant metastases (Dm). [99mTc]Tc-iFAP brain imaging was positive in four high-grade WHO III-IV gliomas and negative in one treatment-naive low-grade glioma. Both [99mTc]Tc-iFAP and [18F]FDG detected 26 (100%) PT, although the number of positive LNm and Dm was significantly higher with [18F]FDG [82 (96%)], in comparison to [99mTc]Tc-iFAP imaging (35 (41%)). Peritoneal carcinomatosis lesions in a patient with recurrent colorectal cancer were only visualized with [99mTc]Tc-iFAP. In patients with breast cancer, a significant positive correlation was demonstrated among [99mTc]Tc-iFAP uptake values (Bq/cm3) of PT and the molecular subtype, being higher for subtypes HER2+ and Luminal B HER2-enriched. Four different CAF subpopulations have previously been described for LNm of breast cancer (from CAF-S1 to CAF-S4). The only subpopulation that expresses FAP is CAF-S1, which is preferentially detected in aggressive subtypes (HER2 and triple-negative), confirming that FAP+ is a marker for poor disease prognosis. The results of this pilot clinical research show that [99mTc]Tc-iFAP SPECT imaging is a promising tool in the prognostic assessment of some solid tumors, particularly breast cancer.

[99mTc]Tc-iFAP Radioligand for SPECT/CT Imaging of the Tumor Microenvironment: Kinetics, Radiation Dosimetry, and Imaging in Patients.

Tumor microenvironment fibroblasts overexpress the fibroblast activation protein (FAP). We recently reported the preclinical evaluation of [99mTc]Tc-iFAP as a new SPECT radioligand capable of detecting FAP. This research aimed to evaluate the kinetic and dosimetric profile of [99mTc]Tc-iFAP in healthy volunteers, and to assess the radioligand uptake by different solid tumors in three cancer patients. [99mTc]Tc-iFAP was obtained from lyophilized formulations prepared under GMP conditions with >98% radiochemical purity. Whole-body scans of six healthy subjects were obtained at 0.5, 2, 4, and 24 h after [99mTc]Tc-iFAP (740 MBq) administration. A 2D-planar/3D-SPECT hybrid activity quantitation method was used to fit the biokinetic models of the source organs (volume of interest: VOI) as exponential functions (A(t)VOI). The total nuclear transformations (N) that occurred in the source organs were calculated from the mathematical integration (0,∞) of A(t)VOI. The OLINDA code was used to estimate the radiation doses. Three treatment-naive patients (breast, lung, and cervical cancer) with a prior [18F]FDG PET/CT scan underwent whole-body, chest, and abdominal SPECT/CT scanning after [99mTc]Tc-iFAP (740 MBq) administration. Both imaging methods were compared visually and quantitatively. Oncological diagnoses were performed histopathologically. The results showed favorable [99mTc]Tc-iFAP biodistribution and kinetics due to rapid blood activity removal (t1/2α = 2.22 min and t1/2ß = 90 min) and mainly renal clearance. The mean radiation equivalent doses were 5.2 ± 0.8 mSv for the kidney and 1.7 ± 0.3 mSv for the liver after administration of 740 MBq. The effective dose was 2.3 ± 0.4 mSv/740 MBq. [99mTc]Tc-iFAP demonstrated high and reliable uptake in the primary tumor lesions and lymph node metastases in patients with breast, cervical, and lung cancer, which correlated with that detected by [18F]FDG PET/CT. The tumor microenvironment molecular imaging from cancer patients obtained in this research validates the performance of additional clinical studies to determine the utility of [99mTc]Tc-iFAP in the diagnosis and prognosis of different types of solid tumors.

Targeted Endoradiotherapy with Lu2O3-iPSMA/-iFAP Nanoparticles Activated by Neutron Irradiation: Preclinical Evaluation and First Patient Image.

Prostate-specific membrane antigen (PSMA) is expressed in a variety of cancer cells, while the fibroblast activation protein (FAP) is expressed in the microenvironment of tumors. Previously, we reported the ability of iPSMA and iFAP ligands to specifically target PSMA and FAP proteins, as well as the preparation of stable 177Lu2O3 nanoparticles (<100 nm) functionalized with target-specific peptides. This research aimed to evaluate the dosimetry and therapeutic response of Lu2O3-iPSMA and Lu2O3-iFAP nanoparticles activated by neutron irradiation to demonstrate their potential for theranostic applications in nuclear medicine. The biokinetic behavior, radiation absorbed dose, and metabolic activity ([18F]FDG/micro-PET, SUV) in preclinical tumor tissues (athymic mice), following treatment with 177Lu2O3-iPSMA, 177Lu2O3-iFAP or 177Lu2O3 nanoparticles, were assessed. One patient with multiple colorectal liver metastases (PSMA-positive) received 177Lu2O3-iPSMA under a "compassionate use" protocol. Results indicated no significant difference (p < 0.05) between 177Lu2O3-iPSMA and 177Lu2O3-iFAP, regarding tumor radiation absorbed doses (105 ± 14 Gy, 99 ± 12 Gy and 58 ± 7 Gy for 177Lu2O3-iPSMA, 177Lu2O3-iFAP, and 177Lu2O3, respectively) and tumor metabolic activity (SUV of 0.421 ± 0.092, 0.375 ± 0.104 and 1.821 ± 0.891 for 177Lu2O3-iPSMA, 177Lu2O3-iFAP, and 177Lu2O3, respectively) in mice after treatment, which correlated with the observed therapeutic response. 177Lu2O3-iPSMA and 177Lu2O3-iFAP significantly inhibited tumor progression, due to the prolonged tumor retention and a combination of 177Lu radiotherapy and iPSMA or iFAP molecular recognition. There were negligible uptake values in non-target tissues and no evidence of liver and renal toxicity. The doses received by the patient's liver metastases (42−210 Gy) demonstrated the potential of 177Lu2O3-iPSMA for treating colorectal liver metastases.

225Ac-rHDL Nanoparticles: A Potential Agent for Targeted Alpha-Particle Therapy of Tumors Overexpressing SR-BI Proteins.

Actinium-225 and other alpha-particle-emitting radionuclides have shown high potential for cancer treatment. Reconstituted high-density lipoproteins (rHDL) specifically recognize the scavenger receptor B type I (SR-BI) overexpressed in several types of cancer cells. Furthermore, after rHDL-SR-BI recognition, the rHDL content is injected into the cell cytoplasm. This research aimed to prepare a targeted 225Ac-delivering nanosystem by encapsulating the radionuclide into rHDL nanoparticles. The synthesis of rHDL was performed in two steps using the microfluidic synthesis method for the subsequent encapsulation of 225Ac, previously complexed to a lipophilic molecule (225Ac-DOTA-benzene-p-SCN, CLog P = 3.42). The nanosystem (13 nm particle size) showed a radiochemical purity higher than 99% and stability in human serum. In vitro studies in HEP-G2 and PC-3 cancer cells (SR-BI positive) demonstrated that 225Ac was successfully internalized into the cytoplasm of cells, delivering high radiation doses to cell nuclei (107 Gy to PC-3 and 161 Gy to HEP-G2 nuclei at 24 h), resulting in a significant decrease in cell viability down to 3.22 ± 0.72% for the PC-3 and to 1.79 ± 0.23% for HEP-G2 at 192 h after 225Ac-rHDL treatment. After intratumoral 225Ac-rHDL administration in mice bearing HEP-G2 tumors, the biokinetic profile showed significant retention of radioactivity in the tumor masses (90.16 ± 2.52% of the injected activity), which generated ablative radiation doses (649 Gy/MBq). The results demonstrated adequate properties of rHDL as a stable carrier for selective deposition of 225Ac within cancer cells overexpressing SR-BI. The results obtained in this research justify further preclinical studies, designed to evaluate the therapeutic efficacy of the 225Ac-rHDL system for targeted alpha-particle therapy of tumors that overexpress the SR-BI receptor.

Design, Synthesis and Preclinical Assessment of 99mTc-iFAP for In Vivo Fibroblast Activation Protein (FAP) Imaging.

Fibroblast activation protein (FAP) is expressed in the microenvironment of most human epithelial tumors. 68Ga-labeled FAP inhibitors based on the cyanopyrrolidine structure (FAPI) are currently used for the detection of the tumor microenvironment by PET imaging. This research aimed to design, synthesize and preclinically evaluate a new FAP inhibitor radiopharmaceutical based on the 99mTc-((R)-1-((6-hydrazinylnicotinoyl)-D-alanyl) pyrrolidin-2-yl) boronic acid (99mTc-iFAP) structure for SPECT imaging. Molecular docking for affinity calculations was performed using the AutoDock software. The chemical synthesis was based on a series of coupling reactions of 6-hidrazinylnicotinic acid (HYNIC) and D-alanine to a boronic acid derivative. The iFAP was prepared as a lyophilized formulation based on EDDA/SnCl2 for labeling with 99mTc. The radiochemical purity (R.P.) was verified via ITLC-SG and reversed-phase radio-HPLC. The stability in human serum was evaluated by size-exclusion HPLC. In vitro cell uptake was assessed using N30 stromal endometrial cells (FAP positive) and human fibroblasts (FAP negative). Biodistribution and tumor uptake were determined in Hep-G2 tumor-bearing nude mice, from which images were acquired using a micro-SPECT/CT. The iFAP ligand (Ki = 0.536 nm, AutoDock affinity), characterized by UV-Vis, FT-IR, 1H-NMR and UPLC-mass spectroscopies, was synthesized with a chemical purity of 92%. The 99mTc-iFAP was obtained with a R.P. >98%. In vitro and in vivo studies indicated high radiotracer stability in human serum (>95% at 24 h), specific recognition for FAP, high tumor uptake (7.05 ± 1.13% ID/g at 30 min) and fast kidney elimination. The results found in this research justify additional dosimetric and clinical studies to establish the sensitivity and specificity of the 99mTc-iFAP.

Targeted photodynamic therapy using reconstituted high-density lipoproteins as rhodamine transporters.

Reconstituted high-density lipoprotein (rHDL) nanoparticles are excellent transporters of molecules and very useful for targeted therapy as they specifically recognize the scavenger receptor, class B1 (SR-B1) that is present on the surface of a wide range of tumor cells. However, they have rarely been employed to transport photosensitizers (PS) for photodynamic therapy (PDT). Rhodamine (R) compounds have been dismissed as useful PSs for PDT due to their low 1O2 production, excitation wavelengths with little tissue penetration, and poor selectivity for tumor cells. It was recently demonstrated that when irradiating at 532 nm or with Cerenkov radiation (CR) from a ß-emitting radionuclide, R123, R6G, and RB undergo electron transfer reactions (type I reaction) with folic acid. R6G also produces type I reactions with O2. In this work, the photodynamic effects of the rHDL-R system were evaluated in vitro. rHDL nanoparticles loaded with R123, R6G, and RB were synthesized, and the PS was internalized into T47D tumor cells. When cells were irradiated with a 532-nm laser in the presence of an rHDL-R systems, a cytotoxic photodynamic effect was obtained in the order R6G > R123 > RB. In the presence of CR from a 177Lu source, cytotoxicity showed the order R6G > RB > R123. The higher cytotoxicity induced by R6G in both cases corresponds to higher cellular internalization and larger production of type I and II reactions. Thus, in this work, it is proposed that rHDL-R/177Lu system can be applied in theragnostics as a multimodal radiotherapy-PDT-imaging system (imaging by SPECT or Cerenkov) and in hypoxic solid tumors in which external radiation is not effective and 177Lu-CR acts as light source.

A Multimodal Theranostic System Prepared from High-Density Lipoprotein Carrier of Doxorubicin and 177Lu.

Recently, it was demonstrated that doxorubicin (Dox.HCl), a chemotherapeutic agent, could be photoactivated by Cerenkov radiation (CR). The objective of the present work was to develop a multimodal chemotherapy-radiotherapy-photodynamic therapeutic system based on reconstituted high-density lipoprotein (rHDL) loaded with Dox.HCl and 177Lu-DOTA. 177Lu acts as a therapeutic radionuclide and CR source. The system can be visualized by nuclear imaging. Fluorescence microscopy showed that rHDL-Dox specifically recognized cancer cells (T47D) that are positive for SR-B1 receptors. Encapsulated Dox.HCl was released into the cells and produced reactive oxygen species when irradiated with a 450-nm laser (photodynamic effect). The same effect occurred when Dox.HCl was irradiated by 177Lu CR. Through in vitro experiments, it was confirmed that the addition of 177Lu-DOTA to the rHDL-Dox nanosystem did not affect the specific recognition of SR-B1 receptors expressed in cells, or the cellular internalization of 177Lu-DOTA. The toxicity induced by the rHDL-Dox/177Lu nanosystem in cell lines with high (T47D and PC3), poor (H9C2) and almost-zero (human fibroblasts (FB)) expression of SR-B1 was evaluated in vitro and confirmed the synergy of the combined chemotherapy-radiotherapy-photodynamic therapeutic effect; this induced toxicity was proportional to the expression of the SR-B1 receptor on the surface of the cells used. The HDL-Dox/177Lu nanosystem experienced uptake by tumor cells and the liver-both tissues with high expression of SR-B1 receptors-but not by the heart. 177Lu CR offered the possibility of imparting photodynamic therapy where laser light could not reach.

Antitumoral effects of dovitinib in triple-negative breast cancer are synergized by calcitriol in vivo and in vitro.

Chemotherapy is a standard therapeutic option for triple-negative breast cancer (TNBC); however, its effectiveness is often compromised by drug-related toxicity and resistance development. Herein, we aimed to evaluate whether an improved antineoplastic effect could be achieved in vitro and in vivo in TNBC by combining dovitinib, a multi-kinase inhibitor, with calcitriol, a natural anticancer hormone. In vitro, cell proliferation and cell-cycle distribution were studied by sulforhodamine B-assays and flow cytometry. In vivo, dovitinib/calcitriol effects on tumor growth, angiogenesis, and endothelium activation were evaluated in xenografted mice by caliper measures, Itgb3/VEGFR2-immunohistochemistry and 99mTc-Ethylenediamine-N,N-diacetic acid/hydrazinonicotinamyl-Glu[cyclo(Arg-Gly-Asp-D-Phe-Lys)]2 (99mTc-RGD2)-tumor uptake. The drug combination elicited a synergistically improved antiproliferative effect in TNBC-derived cells, which allowed a 7-fold and a 3.3-fold dovitinib dose-reduction in MBCDF-Tum and HCC-1806 cells, respectively. Mechanistically, the co-treatment induced a cell cycle profile suggestive of cell death and DNA damage (accumulation of cells in SubG1, S, and G2/M phases), increased the number of multinucleated cells and inhibited tumor growth to a greater extent than each compound alone. Tumor uptake of 99mTc-RGD2 was reduced by dovitinib, suggesting angiogenesis inhibition, which was corroborated by decreased endothelial cell growth, tumor-vessel density and VEGFR2 expression. In summary, calcitriol synergized dovitinib anticancer effects in vitro and in vivo, allowing for a significant dose-reduction of dovitinib while maintaining its antiproliferative potency. Our results suggest the beneficial convergence of independent antitumor mechanisms of dovitinib and calcitriol to inhibit TNBC-tumor growth.

Preclinical dosimetric studies of 177 Lu-scFvD2B and comparison with 177 Lu-PSMA-617 and 177 Lu-iPSMA endoradiotherapeutic agents.

PURPOSE: Internal dosimetry has become a very important tool to evaluate the risks and benefits of new endoradiotherapeutic agents. Nowadays, some of the most successful targeted radionuclide therapy (TRT) agents are 177 Lu-DOTA conjugates based on low molecular weight (LMW) Glu-ureido PSMA inhibitors. It has, however, been demonstrated that the DOTA chelating moiety reduces the internalization of the LMW-PSMA agent and its radiation dose to the tumor. Previously, we reported that 177 Lu-scFvD2B, an antibody-based construct, demonstrated statistically significant higher cell uptake and internalization in LNCaP prostate cancer (PCa) cells (PSMA-positive) when compared to the LMW-PSMA agents, 177 Lu-PSMA-617 and 177 Lu-iPSMA, two of the endoradiotherapeutic agents which currently are the most used in PCa therapy. The aim of this study is to estimate the preclinical 177 Lu-scFvD2B organ and tumor-absorbed doses, and to compare the values with those of 177 Lu-PSMA-617 and 177 Lu-iPSMA. METHODS: 177 Lu-scFvD2B, 177 Lu-PSMA-617, and 177 Lu-iPSMA were prepared and their radiochemical purity determined. Biodistribution studies of each radiopharmaceutical were then carried out in healthy mice to define the main source organs (SO) and to calculate the number of disintegrations in each source organs per unit of administered activity (NSO ). Absorbed dose in the main organs were then calculated for each 177 Lu-conjugate by means of OLINDA/EXM 2.1.1 software, using the calculated NSO for both the adult male and the mouse phantoms as program inputs. Images of mice bearing micropulmonary tumors injected with 177 Lu-conjugates were also obtained. Tumor standardized uptake values (SUV) for the different conjugates, obtained from the 3D SPECT image reconstruction of these mice, were used as the number of disintegrations in a tumor site per unit of administered activity (NT ). The tumor-absorbed dose was calculated using the published electron dose S-values for sphere models with diameters ranging from 10 µm to 10 mm and considering a uniform activity distribution and tumor density equivalent to water density. RESULTS: All 177 Lu-labeled agents were obtained in high yield (98%). Dosimetric studies carried out using mouse phantoms demonstrated that organ absorbed doses of 177 Lu-scFvD2B were from 1.4 to 2.3 times higher than those for 177 Lu-iPSMA and from 1.5 to 2.6 times higher than those for 177 Lu-PSMA-617. However, the 177 Lu-scFvD2B values of tumor-absorbed doses for all investigated tumor sizes were from 2.8 to 3.0 times greater than those calculated for 177 Lu-iPSMA and 177 Lu-PSMA-617, respectively. Moreover, 177 Lu-scFvD2B showed the highest tumor/kidney ratio when compared to those reported for 177 Lu-albumin conjugates. CONCLUSIONS: In this preclinical study, we demonstrated the potential of 177 Lu-scFvD2B as a therapeutic agent for PSMA-expressing tumors, due to its higher tumor-absorbed dose when compared with 177 Lu-LMW agents.

Preparation and Dosimetry Assessment of 166Dy2O3/166Ho2O3-iPSMA Nanoparticles for Targeted Hepatocarcinoma Radiotherapy.

This research aimed to prepare 166Dy2O3-iPSMA/166Ho2O3-iPSMA nanoparticles (166Dy2O3/166Ho2O3-iPSMA NPs) and assess the radiation absorbed dose produced by the nanosystem to hepatic cancer cells by using experimental in vitro and in vivo biokinetic data. Dy2O3NPs were synthesized and functionalized with the prostate-specific membrane antigen inhibitor peptide (iPSMA). Fourier transform infrared (FTIR) spectroscopy, transmission electron microscope (TEM), dynamic light scattering (DSL) and zeta potential analyses indicated the formation of Dy2O3-iPSMA NPs (46.11 ± 13.24 nm). After neutron activation, a stable 166Dy2O3/166Ho2O3- iPSMA nanosystem was obtained, which showed adequate affinity to the PSMA receptor in HepG2 cancer cells (Kd = 9.87 ± 2.27 nM). in vitro studies indicated high 166Dy2O3/166Ho2O3-iPSMA internalization in cancer cells, with high radiation doses to cell nuclei (107 Gy) and cytotoxic effects, resulting in a significant reduction in HepG2 cell viability (decreasing to 2.12 ± 0.31%). After intratumoral administration in mice, the nanosystem biokinetic profile indicated significant retention into the tumoral mass, producing ablative radiation doses (>70 Gy).

[68Ga]Ga-iPSMA-Lys3-Bombesin: Biokinetics, dosimetry and first patient PET/CT imaging.

BACKGROUND: The prostate-specific membrane antigen (PSMA) and the gastrin-releasing peptide receptor (GRPR) are overexpressed in prostate cancer (PCa). In preclinical studies, the iPSMA-Lys3-Bombesin (iPSMA-BN) heterodimeric ligand has shown a suitable affinity for PSMA and GRPR. This research aimed to assess the biokinetics and radiation dosimetry of [68Ga]Ga-iPSMA-BN in four healthy volunteers based on biodistribution data obtained from whole-body PET/CT studies, as well as to visualize the [68Ga]Ga-iPSMA-BN tumor uptake in a patient with PCa. METHODS: PET/CT images acquired at 5 min, 0.5, 1, and 2 h after radiotracer administration (124.5 ± 2.1 MBq) were corrected for attenuation, scattering, dead-time, and decay. The activity in the segmented volumes of interest (VOIs) in each source organ at different times was adjusted to mono- and bi-exponential biokinetic models (A(t)VOI), from which the total disintegrations (N) were calculated to assess the internal radiation doses by using the OLINDA V1.1 code. RESULTS: Images from the patient showed an evident uptake by the metastasis (SUVmax of 4.7) and by the organs expressing GRPR (pancreas) and PSMA (salivary glands). The average effective dose was 2.70 ± 0.05 mSv, which was like those known for most of the 68Ga studies, making [68Ga]Ga-iPSMA-BN a promising dual-target PET imaging radiotracer for PCa. CONCLUSIONS: [68Ga]Ga-iPSMA-BN, capable of detecting both PSMA and GRPR with suitable biokinetics and dosimetric patterns, could be a potential complementary diagnostic tool for the improvement of prostate cancer PET imaging.