Development and evaluation of albumin binder-conjugated heterodimeric radiopharmaceuticals targeting integrin αvß3 and CD13 for cancer therapy.

PURPOSE: The advancement of heterodimeric tracers, renowned for their high sensitivity, marks a significant trend in the development of radiotracers for cancer diagnosis. Our prior work on [68Ga]Ga-HX01, a heterodimeric tracer targeting CD13 and integrin αvß3, led to its approval for phase I clinical trials by the China National Medical Production Administration (NMPA). However, its fast clearance and limited tumor retention pose challenges for broader clinical application in cancer treatment. This study aims to develop a new radiopharmaceutical with increased tumor uptake and prolonged retention, rendering it a potential therapeutic candidate. METHODS: New albumin binder-conjugated compounds were synthesized based on the structure of HX01. In vitro and in vivo evaluation of these new compounds were performed after labelling with 68Ga. Small-animal PET/CT imaging were conducted at different time points at 0.5-6 h post injection (p.i.) using BxPC-3 xenograft mice models. The one with the best imaging performance was further radiolabeled with 177Lu for small-animal SPECT/CT and ex vivo biodistribution investigation. RESULTS: We have synthesized novel albumin binder-conjugated compounds, building upon the structure of HX01. When radiolabeled with 68Ga, all compounds demonstrated improved pharmacokinetics (PK). Small-animal PET/CT studies revealed that these new albumin binder-conjugated compounds, particularly [68Ga]Ga-L6, exhibited significantly enhanced tumor accumulation and retention compared with [68Ga]Ga-L0 without an albumin binder. [68Ga]Ga-L6 outperformed [68Ga]Ga-L7, a compound developed using a previously reported albumin binder. Furthermore, [177Lu]Lu-L6 demonstrated rapid clearance from normal tissues, high tumor uptake, and prolonged retention in small-animal SPECT/CT and biodistribution studies, positioning it as an ideal candidate for radiotherapeutic applications. CONCLUSION: A new integrin αvß3 and CD13 targeting compound was screened out. This compound bears a novel albumin binder and exhibits increased tumor uptake and prolonged tumor retention in BxPC-3 tumors and low background in normal organs, making it a perfect candidate for radiotherapy when radiolabeled with 177Lu.

Development and Evaluation of DOTA-FAPI-Maleimide as a Novel Radiotracer for Tumor Theranostic with Extended Circulation.

This study aimed to evaluate a novel albumin-binding strategy for addressing the challenge of insufficient tumor retention of fibroblast activation protein inhibitors (FAPIs). Maleimide, a molecule capable of covalent binding to free thiol groups, was modified to conjugate with FAPI-04 in order to enhance its binding to endogenous albumin, resulting in an extended blood circulation half-life and increased tumor uptake. DOTA-FAPI-maleimide was prepared and radiolabeled with Ga-68 and Lu-177, followed by cellular assays, pharmacokinetic analysis, PET/CT, and SPECT/CT imaging to assess the probe distribution in various tumor-bearing models. Radiolabeling of the modified probe was successfully achieved with a radiochemical yield of over 99% and remained stable for 144 h. Cellular assays showed that the ligand concentration required for 50% inhibition of the probe was 1.20 ± 0.31 nM, and the Kd was 0.70 ± 0.07 nM with a Bmax of 7.94 ± 0.16 fmol/cell, indicative of higher specificity and affinity of DOTA-FAPI-maleimide compared to other FAPI-04 variants. In addition, DOTA-FAPI-maleimide exhibited a persistent blood clearance half-life of 7.11 ± 0.34 h. PET/CT images showed a tumor uptake of 2.20 ± 0.44%ID/g at 0.5 h p.i., with a tumor/muscle ratio of 5.64 in HT-1080-FAP tumor-bearing models. SPECT/CT images demonstrated long-lasting tumor retention. At 24 h p.i., the tumor uptake of [177Lu]Lu-DOTA-FAPI-maleimide reached 5.04 ± 1.67%ID/g, with stable tumor retention of 3.40 ± 1.95%ID/g after 4 days p.i. In conclusion, we developed and evaluated the thiol group-attaching strategy, which significantly extended the circulation and tumor retention of the adapted FAPI tracer. We envision its potential application for clinical cancer theranostics.

Head-to-head comparison of different classes of FAP radioligands designed to increase tumor residence time: monomer, dimer, albumin binders, and small molecules vs peptides.

PURPOSE: Fibroblast activation protein-α (FAP)-targeting radioligands have recently demonstrated high diagnostic potential. However, their therapeutic value is impaired by the short tumor residence time. Several strategies have been tested to overcome this limitation, but a head-to-head comparison has never been done. With the aim to identify strengths and limitations of the suggested strategies, we compared the monomer FAPI-46 versus (a) its dimer (FAPI-46-F1D), (b) two albumin binders conjugates (FAPI-46-Ibu (ibuprofen) and FAPI-46-EB (Evans Blue)), and (c) cyclic peptide FAP-2286. METHODS: 177Lu-labeled ligands were evaluated in vitro in cell lines with low (HT-1080.hFAP) and high (HEK-293.hFAP) humanFAP expression. SPECT/CT imaging and biodistribution studies were conducted in HT-1080.hFAP and HEK-293.hFAP xenografts. The areas under the curve (AUC) of the tumor uptake and tumor-to-critical-organs ratios and the absorbed doses were estimated. RESULTS: Radioligands showed IC50 in the picomolar range. Striking differences were observed in vivo regarding tumor uptake, residence, specificity, and total body distribution. All [177Lu]Lu-FAPI-46-based radioligands showed similar uptake between the two tumor models. [177Lu]Lu-FAP-2286 showed higher uptake in HEK-293.hFAP and the least background. The AUC of the tumor uptake and absorbed dose was higher for [177Lu]Lu-FAPI-46-F1D and the two albumin binder conjugates, [177Lu]Lu-FAPI-46-Ibu and [177Lu]Lu-FAPI-46-EB, in HT1080.hFAP xenografts and for [177Lu]Lu-FAPI-46-EB and [177Lu]Lu-FAP-2286 in HEK293.hFAP xenografts. The tumor-to-critical-organs AUC values and the absorbed doses were in favor of [177Lu]Lu-FAP-2286, but tumor-to-kidneys. CONCLUSION: The study indicated dimerization and cyclic peptide structures as promising strategies for prolonging tumor residence time, sparing healthy tissues. Albumin binding strategy outcome depended on the albumin binding moiety. The peptide showed advantages in terms of tumor-to-background ratios, besides tumor-to-kidneys, but its tumor uptake was FAP expression-dependent.

Fatty acid-conjugated radiopharmaceuticals for fibroblast activation protein-targeted radiotherapy.

INTRODUCTION: Radiopharmaceuticals that target cancer-associated fibroblasts (CAFs) have become an increasingly attractive strategy for cancer theranostics. Recently, a series of fibroblast activation protein inhibitor (FAPI)-based radiopharmaceuticals have been successfully applied to the diagnosis of a variety of cancers and exhibited excellent tumor selectivity. Nevertheless, CAF-targeted radionuclide therapy encounters difficulties in cancer treatment, as the tumor uptake and retention of FAPIs are insufficient. To meet this challenge, we tried to conjugate albumin-binding moiety to FAPI molecule for prolonged circulation that may increase the accumulation and retention of radiopharmaceuticals in tumor. METHODS: Two fatty acids, lauric acid (C12) and palmitic acid (C16), were conjugated to FAPI-04 to give two albumin-binding FAPI radiopharmaceuticals, denoted as FAPI-C12 and FAPI-C16, respectively. They had been radiolabeled with gallium-68, yttrium-86, and lutecium-177 for stability study, binding affinity assay, PET and SPECT imaging, biodistribution, and radionuclide therapy study to systematically evaluate their potential for CAF-targeted radionuclide therapy. RESULTS: FAPI-C12 and FAPI-C16 showed high binding affinity to FAP with the IC50 of 6.80 ± 0.58 nM and 5.06 ± 0.69 nM, respectively. They were stable in both saline and plasma. The tumor uptake of [68Ga]Ga-FAPI-04 decreased by 56.9% until 30 h after treated with FAPI-C16 before, and the uptakes of [86Y]Y-FAPI-C12 and [86Y]Y-FAPI-C16 in HT-1080-FAP tumor were both much higher than that of HT-1080-Vehicle tumor which identified the high FAP specific of these two radiopharmaceuticals. Both FAPI-C12 and FAPI-C16 showed notably longer circulation and significantly enhanced tumor uptake than those of FAPI-04. [177Lu]Lu-FAPI-C16 had the higher tumor uptake at both 24 h (11.22 ± 1.18%IA/g) and 72 h (6.50 ± 1.19%IA/g) than that of [177Lu]Lu-FAPI-C12 (24 h, 7.54 ± 0.97%IA/g; 72 h, 2.62 ± 0.65%IA/g); both of them were much higher than [177Lu]Lu-FAPI-04 with the value of 1.24 ± 0.54%IA/g at 24 h after injection. Significant tumor volume inhibition of [177Lu]Lu-FAPI-C16 at the high activity of 29.6 MBq was observed, and the median survival was 28 days which was much longer than that of the [177Lu]Lu-FAPI-04 treated group of which the median survival was only 10 days. CONCLUSION: This proof-of-concept study validates the hypothesis that conjugation of albumin binders may shift the pharmacokinetics and enhance the tumor uptake of FAPI-based radiopharmaceuticals. This could be a general strategy to transform the diagnostic FAP-targeted radiopharmaceuticals into their therapeutic pairs.

Preclinical investigations using [177Lu]Lu-Ibu-DAB-PSMA toward its clinical translation for radioligand therapy of prostate cancer.

[177Lu]Lu-Ibu-DAB-PSMA was previously characterized with moderate albumin-binding properties enabling high tumor accumulation but reasonably low retention in the blood. The aim of this study was to investigate [177Lu]Lu-Ibu-DAB-PSMA in preclinical in vivo experiments and compare its therapeutic efficacy and potential undesired side effects with those of [177Lu]Lu-PSMA-617 and the previously developed [177Lu]Lu-PSMA-ALB-56. BALB/c nude mice without tumors were investigated on Day 10 and 28 after injection of 10 MBq radioligand. It was revealed that most plasma parameters were in the same range for all groups of mice and histopathological examinations of healthy tissue did not show any alternations in treated mice as compared to untreated controls. Based on these results, a therapy study over twelve weeks was conducted with PC-3 PIP tumor-bearing mice for comparison of the radioligands's therapeutic efficacy up to an activity of 10 MBq (1 nmol) per mouse. In agreement with the increased mean absorbed tumor dose, [177Lu]Lu-Ibu-DAB-PSMA (~ 6.6 Gy/MBq) was more effective to inhibit tumor growth than [177Lu]Lu-PSMA-617 (~ 4.5 Gy/MBq) and only moderately less potent than [177Lu]Lu-PSMA-ALB-56 (~ 8.1 Gy/MBq). As a result, the survival of mice treated with 2 MBq of an albumin-binding radioligand was significantly increased (p < 0.05) compared to that of mice injected with [177Lu]Lu-PSMA-617 or untreated controls. The majority of mice treated with 5 MBq or 10 MBq [177Lu]Lu-Ibu-DAB-PSMA or [177Lu]Lu-PSMA-ALB-56 were still alive at study end. Hemograms of immunocompetent mice injected with 30 MBq [177Lu]Lu-Ibu-DAB-PSMA or 30 MBq [177Lu]Lu-PSMA-617 showed values in the same range as untreated controls. This was, however, not the case for mice treated with [177Lu]Lu-PSMA-ALB-56 which revealed a drop in lymphocytes and hemoglobin at Day 10 and Day 28 after injection. The data of this study demonstrated a significant therapeutic advantage of [177Lu]Lu-Ibu-DAB-PSMA over [177Lu]Lu-PSMA-617 and a more favorable safety profile as compared to that of [177Lu]Lu-PSMA-ALB-56. Based on these results, [177Lu]Lu-Ibu-DAB-PSMA may has the potential for a clinical translation.

Impact of the mouse model and molar amount of injected ligand on the tissue distribution profile of PSMA radioligands.

PURPOSE: Various preclinical study designs are described in the literature for the evaluation of PSMA radioligands. In this study, [177Lu]Lu-Ibu-DAB-PSMA, an albumin-binding radioligand, and [177Lu]Lu-PSMA-617 were investigated and compared under variable experimental conditions. METHODS: In vitro cell uptake studies were performed with PC-3 PIP and LNCaP tumor cells using a range of molar concentrations (0.75-500 nM) of both radioligands. Biodistribution and SPECT/CT imaging studies were carried out with the respective tumor mouse models using 0.05 nmol and 1.0 nmol injected ligand per mouse. RESULTS: In both tumor cell lines, the uptake of the radioligands was increased when using low molar concentrations of the respective ligand. The observed saturation effect at high ligand concentrations was more pronounced for LNCaP cells that express PSMA at lower levels than for PC-3 PIP cells. At all investigated timepoints, the in vivo uptake of both radioligands was higher in PC-3 PIP tumors than in LNCaP tumors. A low molar amount of injected ligand increased the PC-3 PIP tumor uptake mainly for [177Lu]Lu-Ibu-DAB-PSMA; however, the molar amount of ligand was relevant for both radioligands when using LNCaP tumors. Renal retention of both radioligands was, however, up to fourfold higher during the first hours after application of a low ligand amount compared to the high ligand amount. CONCLUSION: The results of this preclinical study underline the relevance of the tumor model and applied ligand amount for the characterization of PSMA radioligands. The application of equal preclinical study designs is crucial to allow the comparison of novel radioligands with existing ones and, thus, predict potential advantages of new radioligands in view of a clinical application.

Synthesis and Evaluation of Novel 111In-Labeled Picolinic Acid-Based Radioligands Containing an Albumin Binder for Development of a Radiotheranostic Platform.

Picolinic acid-based metallic chelators, e.g., neunpa and octapa, have attracted much attention as promising scaffolds for radiotheranostic agents, particularly those containing larger α-emitting radiometals. Furthermore, albumin binder (ALB) moieties, which noncovalently bind to albumin, have been utilized to improve the pharmacokinetics of radioligands targeting various biomolecules. In this study, we designed and synthesized novel neunpa and octapa derivatives (Neunpa-2 and Octapa-2, respectively), which contained a prostate-specific membrane antigen (PSMA)-binding moiety (model targeting vector) and an ALB moiety. We evaluated the fundamental properties of these derivatives as radiotheranostic agents using 111In. In a cell-binding assay using LNCaP (PSMA-positive) cells, [111In]In-Neunpa-2 and [111In]In-Octapa-2 specifically bound to the LNCaP cells. In addition, a human serum albumin (HSA)-binding assay revealed that [111In]In-Neunpa-2 and [111In]In-Octapa-2 exhibited greater binding to HSA than their non-ALB-conjugated counterparts ([111In]In-Neunpa-1 and [111In]In-Octapa-1, respectively). A biodistribution assay conducted in LNCaP tumor-bearing mice showed that the introduction of the ALB moiety into the 111In-labeled neunpa and octapa derivatives resulted in markedly enhanced tumor uptake and retention of the radioligands. Furthermore, single-photon emission computed tomography imaging of LNCaP tumor-bearing mice with [111In]In-Octapa-2 produced tumor images. These results indicate that [111In]In-Octapa-2 may be a useful PSMA imaging probe and that picolinic acid-based ALB-conjugated radiometallic complexes may be promising candidates as radiotheranostic agents.

Preclinical Investigations to Explore the Difference between the Diastereomers [177Lu]Lu-SibuDAB and [177Lu]Lu-RibuDAB toward Prostate Cancer Therapy.

[177Lu]Lu-Ibu-DAB-PSMA, a radioligand modified with ibuprofen as the albumin binder, showed higher accumulation in PSMA-positive tumors of mice than the clinically used [177Lu]Lu-PSMA-617 but lower retention in non-targeted tissues than previously developed albumin-binding PSMA radioligands. The aim of this study was to investigate whether the stereochemistry of the incorporated ibuprofen affects the radioligand's in vitro and in vivo properties and to select the more favorable radioligand for further development. For this purpose, SibuDAB and RibuDAB containing (S)- and (R)-ibuprofen, respectively, were synthesized and labeled with lutetium-177. In vitro, the two isomers had similar properties; however, [177Lu]Lu-SibuDAB showed increased binding to mouse and human plasma proteins (91 ± 1 and 88 ± 2%, respectively) compared to [177Lu]Lu-RibuDAB (75 ± 2 and 79 ± 2%, respectively). In vivo, [177Lu]Lu-SibuDAB was metabolically more stable than [177Lu]Lu-RibuDAB with ∼90 vs ∼67% intact radioligand detected in the blood at 4 h post injection (p.i.). In line with the lower albumin-binding affinity, the blood clearance of [177Lu]Lu-RibuDAB in mice was considerably faster [27% of injected activity (% IA), 1 h p.i.] than for [177Lu]Lu-SibuDAB (50% IA, 1 h p.i.). Time-dependent biodistribution studies performed in tumor-bearing athymic nude mice showed high PSMA-specific tumor uptake for both isomers. A twofold increased area under the curve (AUC0→8d) of the blood retention was determined for [177Lu]Lu-SibuDAB as compared to [177Lu]Lu-RibuDAB, whereas the kidney AUC0→8d value of [177Lu]Lu-SibuDAB was only half as high as for [177Lu]Lu-RibuDAB. As a result, a more favorable tumor-to-kidney AUC0→8d ratio was obtained for [177Lu]Lu-SibuDAB, which was also visualized on SPECT/CT images. Based on its improved kidney clearance and higher metabolic stability, [177Lu]Lu-SibuDAB was selected as the more favorable radioligand. Therapy studies performed with [177Lu]Lu-SibuDAB (5 MBq/mouse) demonstrated the anticipated therapeutic superiority over the current gold-standard [177Lu]Lu-PSMA-617 (5 MBq/mouse). The significantly increased survival time of mice treated with [177Lu]Lu-SibuDAB as compared to those injected with [177Lu]Lu-PSMA-617 justifies further development of this novel radioligand toward clinical application.

Structure-Activity Relationships and Pharmacokinetics of 111In-Labeled Glucagon-like Peptide-1 Receptor-Targeting Exendin-4 Derivatives Conjugated with Albumin Binder Moieties.

Insulinomas are neuroendocrine tumors that are derived from pancreatic ß-cells, and they often overexpress the glucagon-like peptide-1 receptor (GLP-1R). Radiolabeled exendin-4 derivatives have been used to noninvasively detect the GLP-1R during the diagnosis and preoperative localization of insulinomas; however, their marked renal accumulation can hinder the imaging of pancreatic tail lesions. In this study, we designed and synthesized 111In-labeled exendin-4 derivatives that possessed 4-(4-substituted phenyl)-moieties as albumin binder (ALB) moieties ([111In]In-E4DA2-4), and studied their structure-activity relationships and pharmacokinetics (as well as those of [111In]In-E4DA1, which we previously reported) to determine their usefulness as radioligands for GLP-1R imaging. 111In-labeling was performed by reacting maleimide precursors with [111In]InCl3 in 2-(N-morpholino)ethanesulfonic acid buffer, and then, the products were conjugated with exendin-4-Cys40. A saturation binding assay using GLP-1R-expressing INS-1 cells was carried out to evaluate the in vitro affinity of the radioligands for the cells. In addition, the affinity of the 111In-labeled derivatives for human serum albumin (HSA) was evaluated in an HSA-binding assay. Furthermore, an in vivo biodistribution study and single-photon emission computed tomography (SPECT) imaging were performed using INS-1 tumor-bearing mice. [111In]In-E4DA1-4 were prepared at radiochemical yields of 6-17%. In the saturation binding assay, [111In]In-E4DA1-4 showed a similar affinity for the INS-1 cells, indicating that the kind of ALB moiety used had no effect on the affinity of the exendin-4 derivatives for the cells. In the HSA-binding assay, [111In]In-E4DA1-4 all bound to HSA. In the biodistribution assay, [111In]In-E4DA1-4 exhibited marked tumor accumulation and retention. In addition, they showed lower renal accumulation than previously reported exendin-4-based radioligands without ALB moieties. The pharmacokinetics of the 111In-labeled exendin-4 derivatives varied markedly according to the kind of ALB moiety used. In particular, [111In]In-E4DA2, which contained a 4-(4-bromophenyl)butyric acid derivative as an ALB moiety, showed the highest tumor accumulation. SPECT imaging with [111In]In-E4DA2 clearly visualized INS-1 tumors with no marked accumulation in normal organs. These results provide important information that will aid the design of novel exendin-4-based radioligands targeting the GLP-1R.

Development of an 111In-Labeled Glucagon-Like Peptide-1 Receptor-Targeting Exendin-4 Derivative that Exhibits Reduced Renal Uptake.

Insulinomas are neuroendocrine tumors that are mainly found in the pancreas. Surgical resection is currently the first-line treatment for insulinomas; thus, it is vital to preoperatively determine their locations. The marked expression of the glucagon-like peptide-1 receptor (GLP-1R) is seen in pancreatic ß-cells and almost all insulinomas. Radiolabeled derivatives of exendin-4, a GLP-1R agonist, have been used with nuclear medicine imaging techniques for the in vivo detection of the GLP-1R; however, their marked renal accumulation can hinder the imaging of pancreatic tail lesions. To develop a GLP-1R imaging probe that exhibits reduced renal accumulation, we designed and synthesized a straight-chain GLP-1R-targeting radioligand, [111In]In-E4DA1, which consisted of exendin-4, DOTADG (a chelator), and an (iodophenyl)butyric acid derivative (an albumin binder [ALB]). We performed preclinical evaluations of [111In]In-E4DA1 to investigate its utility as a GLP-1R imaging probe. [111In]In-E4DA1 and [111In]In-E4D (a control compound lacking the ALB moiety) were prepared by reacting the corresponding precursors with [111In]InCl3 in buffer. Cell-binding and human serum albumin (HSA)-binding assays were performed to assess the in vitro affinity of the molecules for INS-1 (GLP-1R-positive) cells and albumin, respectively. A biodistribution assay and single-photon emission computed tomography imaging were carried out using INS-1 tumor-bearing mice. In the cell-binding assay, [111In]In-E4DA1 and [111In]In-E4D exhibited in vitro binding to INS-1 cells. In the HSA-binding assay, [111In]In-E4DA1 bound to HSA, while [111In]In-E4D showed little HSA binding. The in vivo experiments involving INS-1 tumor-bearing mice revealed that the introduction of an ALB moiety into the DOTADG-based exendin-4 derivative markedly increased the molecule's tumor accumulation while decreasing its renal accumulation. In addition, [111In]In-E4DA1 exhibited greater tumor accumulation than renal accumulation, whereas previously reported radiolabeled exendin-4 derivatives demonstrated much higher accumulation in the kidneys than in tumors. These results indicate that [111In]In-E4DA1 may be a useful GLP-1R imaging probe, as it demonstrates only slight renal accumulation.

Design and Evaluation of Novel Albumin-Binding Folate Radioconjugates: Systematic Approach of Varying the Linker Entities.

Tumor targeting using folate radioconjugates is a promising strategy for theragnostics of folate receptor-positive tumors. The aim of this study was to investigate the impact of structural modifications of folate radioconjugates on their pharmacokinetic properties. Four novel folate radioconjugates ([177Lu]Lu-OxFol-2, [177Lu]Lu-OxFol-3, [177Lu]Lu-OxFol-4, and [177Lu]Lu-OxFol-5), modified with a lipophilic or hydrophilic linker entity in close proximity to the albumin-binding 4-(p-iodophenyl)butanoate entity or the DOTA chelator, respectively, were designed and evaluated for comparison with the previously developed [177Lu]Lu-OxFol-1. A hydrophobic 4-(aminomethyl)benzoic acid linker, incorporated in close proximity to the 4-(p-iodophenyl)butanoate entity, enhanced the albumin-binding properties (relative affinity 7.3) of [177Lu]Lu-OxFol-3 as compared to those of [177Lu]Lu-OxFol-1 (relative affinity set as 1.0). On the other hand, a hydrophilic d-glutamic acid (d-Glu) linker entity used in [177Lu]Lu-OxFol-2 compromised the albumin-binding properties. [177Lu]Lu-OxFol-4 and [177Lu]Lu-OxFol-5, in which the respective linker entities were incorporated adjacent to the DOTA chelator, showed similar albumin-binding properties (0.6 and 1.0, respectively) as [177Lu]Lu-OxFol-1. Biodistribution studies in KB tumor-bearing nude mice revealed twofold higher tumor-to-kidney ratios at 4 h and 24 h after injection of [177Lu]Lu-OxFol-3 (∼1.2) than after injection of [177Lu]Lu-OxFol-1 (∼0.6). The tumor-to-kidney ratios of [177Lu]Lu-OxFol-2 were, however, much lower (∼0.2) due to the high kidney retention of this radioconjugate. The tumor-to-kidney ratios of [177Lu]Lu-OxFol-5 were only slightly increased (∼0.9), and the ratios for [177Lu]Lu-OxFol-4 (∼0.7) were in the same range as for [177Lu]Lu-OxFol-1. SPECT/CT imaging studies demonstrated similar tumor uptake of all radioconjugates but a clearly improved tumor-to-kidney ratio for [177Lu]Lu-OxFol-3 as compared to that for [177Lu]Lu-OxFol-1. Based on these data, it can be concluded that the linker entity in close proximity to the 4-(p-iodophenyl)butanoate entity affects the radioconjugate's pharmacokinetic profile considerably due to the altered affinity to albumin. Changes in the linker entity, which connects the DOTA chelator with the folate molecule, do not have a major impact on the radioconjugate's tissue distribution profile, however. As a result of these findings, [177Lu]Lu-OxFol-3 had a comparable therapeutic effect to that of [177Lu]Lu-OxFol-1 but appeared advantageous in preventing kidney damage. Provided that the kidneys will present the dose-limiting organs in patients, [177Lu]Lu-OxFol-3 would be the preferred candidate for a clinical translation.

Modulation of the Pharmacokinetics of a Radioligand Targeting Carbonic Anhydrase-IX with Albumin-Binding Moieties.

The expression of carbonic anhydrase-IX (CA-IX) in tumors can lead to a poor prognosis; thus, CA-IX has attracted much attention as a target molecule for cancer diagnosis and treatment. An 111In-labeled imidazothiadiazole sulfonamide (IS) derivative, [111In]In-DO3A-IS1, exhibited marked tumor accumulation but also marked renal accumulation, raising concerns about it producing a low signal/background ratio and a high radiation burden on the kidneys. In this study, four 111In-labeled IS derivatives, IS-[111In]In-DO2A-ALB1-4, which contained four different kinds of albumin binder (ALB) moieties, were designed and synthesized with the aim of improving the pharmacokinetics of [111In]In-DO3A-IS1. Their utility for imaging tumors that strongly express CA-IX was evaluated in mice. An in vitro binding assay of cells that strongly expressed CA-IX (HT-29 cells) was performed using acetazolamide as a competitor against CA-IX, and IS-[111In]In-DO2A-ALB1-4 did not exhibit reduced binding to HT-29 cells compared with [111In]In-DO3A-IS1. In contrast, IS-[111In]In-DO2A-ALB1-4 showed a greater ability to bind to human serum albumin than [111In]In-DO3A-IS1 in vitro. In an in vivo biodistribution study, the introduction of an ALB moiety into the 111In-labeled IS derivative markedly decreased renal accumulation and increased HT-29 tumor accumulation and blood retention. The pharmacokinetics of the IS derivatives varied depending on the substituted group within the ALB moiety. Single-photon emission computed tomography imaging with IS-[111In]In-DO2A-ALB1, which showed the highest tumor/kidney ratio in the biodistribution study, facilitated clear HT-29 tumor imaging, and no strong signals were observed in the normal organs. These results indicate that IS-[111In]In-DO2A-ALB1 may be an effective CA-IX imaging probe and that the introduction of ALB moieties may improve the pharmacokinetics of CA-IX ligands.

Feasibility of using a 99mTc-hydroxamamide complex containing an albumin binder moiety for in vivo albumin labeling-based tumor imaging.

Human serum albumin (HSA), which is distributed throughout the blood, is used as a carrier for transporting drugs to tumors based on the enhanced permeability and retention (EPR) effect. To develop an agent for the in vivo radiolabeling of endogenous albumin, we designed and synthesized novel hydroxamamide (Ham)-based technetium-99m (99mTc) complexes, which contained a monovalent or bivalent 4-(4-iodophenyl)butyric acid (IA) derivative as an albumin binder (ALB) moiety ([99mTc]AB2 and [99mTc]ALB2, respectively), and evaluated their utility for in vivo tumor imaging. In an in vitro HSA-binding assay, [99mTc]AB2 and [99mTc]ALB2 showed greater binding to HSA than [99mTc]BHam, a 99mTc-Ham complex without an ALB moiety. In an in vivo biodistribution assay, [99mTc]ALB2 showed marked blood and tumor retention (25.13 and 4.61% injected dose (ID)/g, respectively, at 1 h postinjection), suggesting that the EPR effect had been induced. However, [99mTc]AB2 showed no marked blood or tumor retention (4.16 and 0.75% ID/g, respectively, at 1 h postinjection), probably because the affinity of the monovalent IA derivative for albumin was insufficient to induce the EPR effect. These findings indicated that the multivalent interactions of [99mTc]ALB2 had enhanced its affinity for albumin. 99mTc-complexes containing multivalent ALB moieties may be useful for tumor imaging.

Combined Application of Albumin-Binding [177Lu]Lu-PSMA-ALB-56 and Fast-Cleared PSMA Inhibitors: Optimization of the Pharmacokinetics.

The strategy of using radioligands for targeting the prostate-specific membrane antigen (PSMA) revealed to be promising for the treatment of metastatic castration-resistant prostate cancer (mCRPC). Recently developed albumin-binding PSMA radioligands showed a remarkably increased tumor uptake because of the enhanced blood circulation, but higher accumulation of activity was also observed in off-target organs and tissues. The aim of this study was to investigate the option of using fast-cleared, small-molecular-weight PSMA inhibitors (PSMA-11, 2-PMPA, and ZJ-43) to reduce the kidney uptake of [177Lu]Lu-PSMA-ALB-56, a previously developed albumin-binding PSMA radioligand. Dual-isotope SPECT/CT imaging was performed with tumor-bearing mice coinjected with [177Lu]Lu-PSMA-ALB-56 and a 2.5-fold molar excess of [67Ga]Ga-PSMA-11. At early timepoints after injection, the high renal uptake of [67Ga]Ga-PSMA-11 reduced the accumulation of [177Lu]Lu-PSMA-ALB-56 in the kidneys substantially, whereas the tumor uptake of [177Lu]Lu-PSMA-ALB-56 was only slightly affected. These findings were confirmed in biodistribution studies, which revealed reduced uptake of [177Lu]Lu-PSMA-ALB-56 in the kidneys due to coadministered unlabeled PSMA-11 (9.1 ± 0.8% IA/g vs 46 ± 11% IA/g; 1 h p.i.). The tumor uptake of [177Lu]Lu-PSMA-ALB-56 was almost the same at 1 h p.i., irrespective of whether or not PSMA-11 was coinjected (24 ± 6% IA/g vs 27 ± 7% IA/g). The application of [177Lu]Lu-PSMA-ALB-56 with 2-PMPA or ZJ-43, respectively, showed similar results in biodistribution studies. Among all three tested PSMA inhibitors, 2-PMPA, applied at a 2.5-fold molar excess relative to [177Lu]Lu-PSMA-ALB-56, was most effective to improve the tumor-to-kidney ratios over the first hours after injection of [177Lu]Lu-PSMA-ALB-56. The concept of using a PSMA inhibitor together with [177Lu]Lu-PSMA-ALB-56 appears promising in view of a clinical translation of this and possibly other long-circulating PSMA radioligands.

Albumin-Binding PSMA Radioligands: Impact of Minimal Structural Changes on the Tissue Distribution Profile.

The concept of using ibuprofen as an albumin-binding entity was recently demonstrated by the development of [177Lu]Lu-Ibu-PSMA-01. In the present study, we designed a novel ibuprofen-containing radioligand (Ibu-PSMA-02) with subtle structural changes regarding the linker entity in order to investigate a potential impact on the in vitro and in vivo properties. Ibu-PSMA-02 was prepared using solid-phase synthesis techniques and labeled with lutetium-177. [177Lu]Lu-Ibu-PSMA-02 was evaluated in vitro with regard to its plasma protein-binding properties, PSMA affinity and uptake into PSMA-expressing PC-3 PIP tumor cells. The tissue distribution profile of [177Lu]Lu-Ibu-PSMA-02 was assessed in tumor-bearing mice and dose estimations were performed. The in vitro characteristics of [177Lu]Lu-Ibu-PSMA-02 were similar to those previously obtained for [177Lu]Lu-Ibu-PSMA-01 with respect to plasma protein-binding, PSMA affinity and tumor cell uptake. The in vivo studies revealed, however, an unprecedentedly high uptake of [177Lu]Lu-Ibu-PSMA-02 in PC-3 PIP tumors, resulting in an increased absorbed tumor dose of 7.7 Gy/MBq as compared to 5.1 Gy/MBq calculated for [177Lu]Lu-Ibu-PSMA-01. As a consequence of the high tumor accumulation, [177Lu]Lu-Ibu-PSMA-02 showed higher tumor-to-background ratios than [177Lu]Lu-Ibu-PSMA-01. This study exemplified that smallest structural changes in the linker entity of PSMA radioligands may have a significant impact on their pharmacokinetic profiles and, thus, may be applied as a means for ligand design optimization.

Radioiodinated Portable Albumin Binder as a Versatile Agent for in Vivo Imaging with Single-Photon Emission Computed Tomography.

In this study, radioiodinated 4-( p-iodophenyl)butyric acid ([131I]IBA) was synthesized and evaluated as a portable albumin-binder for potential applications in single photon emission computed tomography imaging of blood pool, tumor, and lymph node with significantly improved pharmacokinetic properties. The [131I]IBA was prepared under the catalyst of Cu2O/1,10-phenanthroline. After that, the albumin-binding capability of [131I]IBA was tested in vitro, ex vivo, and in vivo, respectively. [131I]IBA was obtained with very high radiolabeling yield (>99%) and good radiochemical purity (>98%) within 10 min. It binds to albumin effectively with high affinity (IC50= 46.5 µM) and has good stability. The results of biodistribution indicated that the [131I]IBA was mainly accumulated in blood with good retention (10.51 ± 2.58%ID/g at 30 min p.i. and 4.63 ± 0.17%ID/g at 4 h p.i.). In the SPECT imaging of mice models with [131I]IBA, blood pool, lymph node, and tumors could be imaged clearly with high target-to-background ratio. Overall, the radioiodinated albumin binder of [131I]IBA with long blood half-life and excellent stability could be used to decorate diversified albumin-binding radioligands and developed as a versatile theranostic agent.

Exendin-4 Derivatives with an Albumin-Binding Moiety Show Decreased Renal Retention and Improved GLP-1 Receptor Targeting.

The glucagon-like peptide-1 receptor (GLP-1R) is highly and specifically expressed on the pancreatic ß-cells. It plays an important role in glucose metabolism as well as in ß-cell-derived diseases like diabetes, insulinoma, or congenital and adult hyperinsulinemic hypoglycemia. Radiolabeled exendin-4, a ligand of GLP-1R, has routinely been used in clinics to image insulinomas. However, its major drawback is the high kidney accumulation. Here, we show that the addition of an albumin-binding moiety (ABM) to radiolabeled exendin-4 results in a significant reduction of kidney uptake while retaining its high affinity and specificity to GLP-1R. The four tested peptides were shown to have high affinity to the GLP-1 receptor (IC50 of 3.7 ± 0.6 to 15.1 ± 0.8 nM). The radiolabeled derivatives were taken up into cells efficiently, internalizing between 39 ± 2 and 56 ± 2% after 2 h. Thus, the derivatives with ABM outperformed the reference peptide with its IC50 of 22.5 ± 2.9 nM and internalization of 41 ± 4%. Stability in human blood plasma was slightly enhanced by the addition of the albumin binder. In biodistribution studies, the radioligands exhibited an improved target-to-kidney ratio in comparison to the reference peptide of up to seven-fold. This was confirmed qualitatively in single-photon-emission computed tomography (SPECT)/CT imaging. This study demonstrated in vitro and in vivo that the addition of an ABM to radiolabeled exendin-4 strongly decreased kidney accumulation while retaining affinity to GLP-1R. Thus, exendin-4 derivatives with an albumin-binding moiety could present a viable class of diagnostic tracers for the detection of insulinomas and other GLP-1R-positive tissue in clinical application.

Enhancing Treatment Efficacy of 177Lu-PSMA-617 with the Conjugation of an Albumin-Binding Motif: Preclinical Dosimetry and Endoradiotherapy Studies.

We designed and evaluated a novel albumin-binder-conjugated 177Lu-PSMA-617 derivative, 177Lu-HTK01169, with an extended blood retention time to maximize the radiation dose delivered to prostate tumors expressing prostate-specific membrane antigen (PSMA). PSMA-617 and HTK01169 that contained N-[4-( p-iodophenyl)butanoyl]-Glu as an albumin-binding motif were synthesized using the solid-phase approach. Binding affinity to PSMA was determined by in vitro competition-binding assay. 177Lu labeling was performed in acetate buffer (pH 4.5) at 90 °C for 15 min. SPECT/CT imaging, biodistribution, and endoradiotherapy studies were conducted in mice bearing PSMA-expressing LNCaP tumor xenografts. Radiation dosimetry was calculated using OLINDA software. Lu-PSMA-617 and Lu-HTK01169-bound PSMA with high affinity ( Ki values = 0.24 and 0.04 nM, respectively). SPECT imaging and biodistribution studies showed that 177Lu-PSMA-617 and 177Lu-HTK01169 were excreted mainly via the renal pathway. With fast blood clearance (0.68%ID/g at 1 h postinjection), the tumor uptake of 177Lu-PSMA-617 peaked at 1 h postinjection (15.1%ID/g) and gradually decreased to 7.91%ID/g at 120 h postinjection. With extended blood retention (16.6 and 2.10%ID/g at 1 and 24 h, respectively), the tumor uptake of 177Lu-HTK01169 peaked at 24 h postinjection (55.9%ID/g) and remained at the same level by the end of the study (120 h). Based on dosimetry calculations, 177Lu-HTK01169 delivered an 8.3-fold higher radiation dose than 177Lu-PSMA-617 to LNCaP tumor xenografts. For the endoradiotherapy study, the mice treated with 177Lu-PSMA-617 (18.5 MBq) all reached humane end point (tumor volume >1000 mm3) by Day 73 with a median survival of 58 days. Mice treated with 18.5, 9.3, 4.6, or 2.3 MBq of 177Lu-HTK01169 had a median survival of >120, 103, 61, and 28 days, respectively. With greatly enhanced tumor uptake and treatment efficacy compared to 177Lu-PSMA-617 in preclinical studies, 177Lu-HTK01169 warrants further investigation for endoradiotherapy of prostate cancer.

Design and Preclinical Evaluation of an Albumin-Binding PSMA Ligand for 64Cu-Based PET Imaging.

Recently, we developed an albumin-binding radioligand (177Lu-PSMA-ALB-56), which showed higher PSMA-specific tumor uptake in mice than the previously developed 177Lu-PSMA-617 under the same experimental conditions. Such a radioligand may be of interest also for PET imaging, possibly enabling better visualization of even small metastases at late time-points after injection. The aim of this study was, therefore, to modify PSMA-ALB-56 by exchanging the DOTA chelator with a NODAGA chelator for stable coordination of 64Cu ( T1/2 = 12.7 h; Eß+av = 278 keV). The resulting NODAGA-functionalized PSMA-ALB-89 ligand, and the previously establish DOTA-functionalized PSMA-ALB-56 ligand were labeled with 64Cu and evaluated in vitro and in vivo. Both radioligands showed plasma protein-binding properties in vitro and PSMA-specific uptake in PC-3 PIP cells. Biodistribution studies, performed in tumor-bearing mice, revealed high accumulation of 64Cu-PSMA-ALB-89 in PSMA-positive PC-3 PIP tumor xenografts (25.9 ± 3.41% IA/g at 1 h p.i.), which was further increased at later time-points (65.1 ± 7.82% IA/g at 4 h p.i. and 97.1 ± 7.01% IA/g at 24 h p.i.). High uptake of 64Cu-PSMA-ALB-89 was also seen in the kidneys, however, 64Cu-PSMA-ALB-89 was efficiently excreted over time. Mice injected with 64Cu-PSMA-ALB-56 showed increased accumulation of radioactivity in the liver (25.3 ± 4.20% IA/g) when compared to the liver uptake of 64Cu-PSMA-ALB-89 (4.88 ± 0.21% IA/g, at 4 h p.i.). This was most probably due to in vivo instability of the 64Cu-DOTA complex, which was also the reason for lower tumor uptake (49.7 ± 16.1% IA/g at 4 h p.i. and 28.3 ± 3.59% IA/g at 24 h p.i.). PET/CT imaging studies confirmed these findings and enabled excellent visualization of the PSMA-positive tumor xenografts in vivo after injection of 64Cu-PSMA-ALB-89. These data indicate that 64Cu-PSMA-ALB-89 is favorable over 64Cu-PSMA-ALB-56 with regard to the in vivo stability and tissue distribution profile. Moreover, 64Cu-PSMA-ALB-89 outperformed previously developed 64Cu-labeled PSMA ligands. Further optimization of long-circulating PSMA-targeting PET radioligands will be necessary before translating this concept to the clinics.

Preclinical Development of Novel PSMA-Targeting Radioligands: Modulation of Albumin-Binding Properties To Improve Prostate Cancer Therapy.

The treatment of metastatic castration-resistant prostate cancer (mCRPC) remains challenging with current treatment options. The development of more effective therapies is, therefore, urgently needed. Targeted radionuclide therapy with prostate-specific membrane antigen (PSMA)-targeting ligands has revealed promising clinical results. In an effort to optimize this concept, it was the aim of this study to design and investigate PSMA ligands comprising different types of albumin binders. PSMA-ALB-53 and PSMA-ALB-56 were designed by combining the glutamate-urea-based PSMA-binding entity, a DOTA chelator and an albumin binder based on the 4-( p-iodophenyl)-moiety or p-(tolyl)-moiety. The compounds were labeled with 177Lu (50 MBq/nmol) resulting in radioligands of high radiochemical purity (≥98%). Both radioligands were stable (≥98%) over 24 h in the presence of l-ascorbic acid. The uptake into PSMA-positive PC-3 PIP tumor cells in vitro was in the same range (54-58%) for both radioligands; however, 177Lu-PSMA-ALB-53 showed a 15-fold enhanced binding to human plasma proteins. Biodistribution studies performed in PC-3 PIP/flu tumor-bearing mice revealed high tumor uptake of 177Lu-PSMA-ALB-53 and 177Lu-PSMA-ALB-56, respectively, demonstrated by equal areas under the curves (AUCs) for both radioligands. The increased retention of 177Lu-PSMA-ALB-53 in the blood resulted in almost 5-fold lower tumor-to-blood AUC ratios when compared to 177Lu-PSMA-ALB-56. Kidney clearance of 177Lu-PSMA-ALB-56 was faster, and hence, the tumor-to-kidney AUC ratio was 3-fold higher than in the case of 177Lu-PSMA-ALB-53. Due to the more favorable tissue distribution profile, 177Lu-PSMA-ALB-56 was selected for a preclinical therapy study in PC-3 PIP tumor-bearing mice. The tumor growth delay after application of 177Lu-PSMA-ALB-56 and 177Lu-PSMA-617 applied at the same activities (2 or 5 MBq per mouse) revealed better antitumor effects in the case of 177Lu-PSMA-ALB-56. As a consequence, the survival of mice treated with 177Lu-PSMA-ALB-56 was prolonged when compared to the mice, which received the same activity of 177Lu-PSMA-617. Our results demonstrated the superiority of 177Lu-PSMA-ALB-56 over 177Lu-PSMA-ALB-53 indicating that the p-(tolyl)-moiety was more suited as an albumin binder to optimize the tissue distribution profile. 177Lu-PSMA-ALB-56 was more effective to treat tumors than 177Lu-PSMA-617 resulting in complete tumor remission in four out of six mice. This promising results warrant further investigations to assess the potential for clinical application of 177Lu-PSMA-ALB-56.