The development of 177Lu-DOTA-CC-PSMA following a unified “Click Chemistry” protocol of synthesizing metal nuclide-conjugated radiopharmaceuticals

Background Currently, the synthesis pathway of metal nuclide-labeled radiopharmaceuticals is mainly divided into two steps: first, connecting the chelator with the target molecule, and second, labeling the metal nuclide to the chelator. However, the second step of the reaction to label the metal nuclide requires high temperature (90–100 °C), which tends to denature and inactivate the target molecule, leading to loss of biological activities, especially the targeting ability. A feasible solution may be the click chemistry labeling method, which consists of reacting a metal nuclide with a chelating agent to generate an intermediate and then synthesizing a radiopharmaceutical agent via the click chemistry intermediate and the target molecule-alkyne compound. In this study, through the click chemistry of 177Lu-DOTA-N3 with prostate-specific membrane antigen (PSMA)-alkyne compound, 177Lu-labeled PSMA-targeted molecular probe was synthesized and evaluated for its potential to be cleared from the bloodstream and rapidly distributed to tissues and organs, achieving a high target/non-target ratio. 177Lu-PSMA-617 was utilized as an analogue for comparison in terms of synthesizing efficiency and PSMA-targeting ability. Results A novel 177Lu-labeled PSMA radioligand was successfully synthesized through the click chemistry of 177Lu-DOTA-N3 with PSMA-alkyne compound, and abbreviated as 177Lu-DOTA-CC-PSMA, achieving a radiochemical yield of 77.07% ± 0.03% (n = 6) and a radiochemical purity of 97.62% ± 1.49% (n = 6) when purified by SepPak C18 column. Notably, 177Lu-DOTA-CC-PSMA was characterized as a hydrophilic compound that exhibited stability at room temperature and commendable pharmacokinetic properties, such as the superior uptake (19.75 ± 3.02%ID/g at 0.5 h) and retention (9.14 ± 3.16%ID/g at 24 h) within xenografts of 22Rv1 tumor-bearing mice. SPECT/CT imaging indicated that radioactivity in both kidneys and bladder was essentially eliminated after 24 h, while 177Lu-DOTA-CC-PSMA was further enriched and retained in PSMA-expressing tumors, resulting in the high target/non-target ratio. Conclusion This study demonstrated the potential of click chemistry to unify the synthesis of metal radiopharmaceuticals, and 177Lu-DOTA-CC-PSMA was found for rapid clearance and appropriate chemical stability as a PSMA-targeted radioligand. Supplementary Information The online version contains supplementary material available at 10.1186/s41181-024-00287-7.


Background
The main aim of radiopharmaceutical therapy is to form targeted radiopharmaceuticals by linking therapeutic radioisotopes to targeting molecules that precisely identify tumor cells and bind to certain receptors of the tumor cells (Sgouros et al. 2020;Dhoundiyal et al. 2024;Salerno et al. 2023).The radioisotope accumulates and decays at the tumor site, releasing a certain amount of ionizing radiation, which destroys the tumor tissue; meanwhile, the precise positioning allows for targeted therapy with minimal potential impact on surrounding healthy tissues.Prostate-specific membrane antigen (PSMA), a type II transmembrane glycoprotein consisting of 750 amino acids, is commonly overexpressed in almost all prostate cancer cells (Kiess et al. 2015;Haberkorn et al. 2016;Osborne et al. 2013).In contrast, it is expressed at low levels in normal tissues such as kidneys, salivary glands, and small intestine (He et al. 2022); thus, PSMA-targeted radioligand therapy (PRLT) has become a popular therapy for the treatment of prostate cancer (Cimadamore et al. 2018;Wang et al. 2022Wang et al. , 2023)).
The synthesis of metal nuclide-based radiopharmaceuticals is generally divided into two steps: first, connecting the chelating agent with the target molecule to form a precursor (e.g., PSMA-617, PSMA-HYNIC, and FAPI-04, which are common in clinic), and then, labeling the radioisotope on the precursor.The target molecules, especially antibodies and some heat-sensitive peptides with complex spatial structures, are prone to denaturation and inactivation at high temperatures or rigor reaction conditions.However, the second step of the reaction usually requires high temperatures or weak acidic conditions, leading to problems such as loss of targeting.A possible way to overcome these problems is to bring forward the labeling reaction of metal radionuclides as a prereaction of radio-labeled bioconjugate to bioactive molecules.The click chemistry, a successful method for constructing novel pharmacophores through a series of dependable chemical reactions, has played a significant role in the discovery and optimization of drug leads (Kolb et al. 2001).In 2006, researchers have (Marik and Sutcliffe 2006) pioneered the introduction of Cu-catalyzed azide alkyne cycloaddition (CuAAC) reaction into the synthesis and labeling of radiopharmaceuticals, thereby introducing a novel approach for radionuclide labeling.As the development of radiopharmaceuticals progressed, an increasing number of radionuclides have been employed for click chemistry labeling (Zhong et al. 2023), including both radiodiagnostic radionuclides such as 99m Tc, 18 F, and 68 Ga and radiotherapeutic radionuclides such as 188 Re and 177 Lu (Colombo and Bianchi 2010;Mindt et al. 2008;Choy et al. 2017;Evans et al. 2014;Quigley et al. 2022;Wang et al. 2012).In addition, the synthesis of radiotracers targeting PSMA receptors for use as PRLT by click chemistry has been widely used, e.g., Verena i Böhmer synthesised and in vivo evaluated F-18 labeled PSMA-targeted " 18 F-PSMA-MIC" radiotracers by copper(I)-catalyzed azide-alkane cycloaddition and demonstrated that the binding affinity could be improved by copper(I)-catalyzed azide-alkane cycloaddition to promote alkane binding in PSMA (Böhmer et al. 2020); James Kelly developed high-affinity PSMA inhibitors labeled with F-18 by click chemistry, and showed that the radiosynthesis of F-labeled triazoles is simple and highly productive, with high PSMA affinity and specific uptake (Kelly et al. 2017).
In this study, the potential of click chemistry in simplifying and unifying the synthesis of metal radiopharmaceuticals was proposed and explored using an 177 Lu-labeled PSMA-targeting molecular probe.The 177 Lu-labeled PSMA ligand was achieved under mild conditions by incorporating a triazole ring into the molecular structures of DOTA and PSMA using click chemistry, named 177 Lu-DOTA-CC-PSMA.The effect of this triazole ring on the overall drug properties of the complex was preliminarily investigated, and its biological properties were studied in mice with PSMA-positive tumors.

Determination of the partition coefficient
The partition coefficient was determined by mixing the labeled compound 177 Lu-DOTA-CC-PSMA or 177 Lu-PSMA-617 (10 µL) with n-octanol (600 µL) and phosphate buffer (590 µL, 0.1 M, pH 7.4) in an Eppendorf microcentrifuge tube.The mixture was vigorously stirred for 2 min at room temperature and was centrifuged at 8,000 rpm for 3 min.Samples in triplicate from n-octanol and aqueous layer were obtained, and were counted by γ-counter (n = 6).The partition coefficients were calculated using the following equation: logD 7.4 = log (activity concentration in n-octanol)/(activity concentration in aqueous layer).

Tumor model
All experiments were approved by the Committee on the Management and Use of Laboratory Animals of Shanghai Vista Pharmaceutical Technology Co. Ltd. (Institutional Animal Care and Use Committee number Vista-IA-2-1-2306-01), and all methods were carried out in accordance with relevant guidelines and regulations.The reporting in this manuscript adhered to the recommendations in the ARRIVE guidelines.
For in vivo studies, 6-week-old athymic nu/nu CDX (22Rv1) model male mice (subcutaneously inoculating 1 × 10 6 22Rv1 cells at the base of the right forelimb) were obtained from Nanchang Royo Biotech Co. Ltd.The mice were kept in individually ventilated cages under standard conditions with food and water provided ad libitum.Housing conditions were as following: dark/light cycle 12/12 h, ambient temperature around 21-22 °C and humidity between 40 and 70%.Biodistribution and SPECT/CT imaging were performed 3-4 weeks after cell inoculation (when the tumor reached a size of approximately 400 mm 3 ).99m Tc-HYNIC-PSMA SPECT/CT imaging was performed to verify PSMA-avidity of CDX models before the start of the experiment.Details on SPECT/CT acquisition process and PSMA-avidity assessment are provided in Supplementary Data (Fig. S3).

In vivo biodistribution studies
The 22Rv1 tumor-bearing mice were injected in the lateral tail vein with 177 Lu-DOTA-CC-PSMA (n = 4/group, 3.7 MBq in 100 µL per mouse) or 177 Lu-PSMA-617 (n = 4/group, 3.7 MBq in 100 µL per mouse) under isoflurane anesthesia (1-2% isoflurane) and sacrificed by cervical dislocation 0.5, 1, 4, and 24 h later.The mice were kept under persistent anesthesia and warm throughout the injection period.Blood ( 177 Lu-DOTA-CC-PSMA), organs ( 177 Lu-DOTA-CC-PSMA), and tumors ( 177 Lu-DOTA-CC-PSMA and 177 Lu-PSMA-617) were collected and weighed.For the tumor specimens, liquid necrotic areas were discarded, and only solid tumor parts were examined.Activity content was assessed by γ-counter.Tissue counts and injected dose for individual mice were decay-corrected to the time of euthanasia.Tissue uptake was expressed as the percentage injected dose per gram of tissue (% injected dose/gram tissue, %ID/g).Values of tumor uptake were divided by muscle (blood, kidneys) uptake for each animal to calculate the tumor-tomuscle (blood, kidneys) [T/M (B, K)] ratio.The biodistribution data and target/non-target (T/NT) ratios were reported as mean values with the standard deviation (SD).

SPECT/CT imaging
A total 4 mice with subcutaneous 22Rv1 tumors were scanned as exemplary cases for in vivo SPECT/CT imaging.Under isoflurane anesthesia (1-2% isoflurane) 177 Lu-DOTA-CC-PSMA (18.5 MBq in 100 µL per mouse) injection and whole-body imaging was performed 0.5, 1, 2, 4, 6 and 24 h after injection with modular SPECT/CT scanner.The mice were placed in the supine position on the scanning bed post-injection, respectively, and were scanned using modular high-resolution CT (image acquisition parameters: CT, tube voltage: 50 kV; tube current: 30 µA), which was anaesthetised for 5 min using 1% isoflurane.After the CT acquisition, the mice were placed supine on the scanning bed and scanned using modular high-resolution SPECT (image acquisition parameters: SPECT, algorithm: maximum likelihood algorithm; peak: 140 keV; isometric voxel size: 500 μm), and anaesthesia was applied with 1% isoflurane for 28 min.Images were constructed using maximum likelihood algorithm (50 iterations) and the data were corrected for attenuation.Image processing and analysis were performed using VivoQuant software (Invicro) to form hybrid SPECT/CT images in transverse, coronal and sagittal planes.

Data analysis and statistics
All statistical analyses were performed using SPSS 23.0 software (SPSS Inc.), and graphs were plotted using OriginPro 2019b (OriginLab Inc.).Quantitative data are expressed as the mean ± SD.Independent samples of tumor uptake for each organ, as well as for tumor tissue and T/M (T/B, T/K) ratio, tracer uptakes at each time point were compared with Kruskal Wallis H test, and Mann-Whitney-U test was used as post-hoc test in pairwise comparisons of variables with significant results.Two-sided p-values of < 0.05 were considered statistically significant.
Tumor uptake of 177 Lu-DOTA-CC-PSMA peaked at 30 min (19.75 ± 3.02%ID/g).However, 177 Lu-DOTA-CC-PSMA showed the highest tumor-to-muscle (blood) ratio at 24 h (T/M: 801.21 ± 299.73; T/B: 296.30 ± 71.82), compared with the time point of the tumor  uptake peak (T/M: 22.70 ± 6.07; T/B: 6.29 ± 1.46), indicating fast initial uptake followed by slow tumor activity washout paralleled by a stronger washout in the muscle (blood) (Table 2).Still, the T/M (B) ratio was greater than 1 at all of the examined time points (Table 1). 177Lu-DOTA-CC-PSMA was excreted via the renal route.Among normal organs, the kidneys showed the highest uptake (1.58 ± 0.70%ID/g at 24 h after injection), while others showed very low radioactivity accumulation and rapid elimination (Fig. 2A).The blood drug concentration-time curves of the probe 177 Lu-PSMA-617 and 177 Lu-DOTA-CC-PSMA in healthy mice are shown in Fig. 2B.The curves showed that the pharmacokinetics of 177 Lu-DOTA-CC-PSMA and 177 Lu-PSMA-617 were essentially the same (P > 0.05), with rapid clearance from the blood and rapid distribution to all tissues and organs in the body.In addition, we calculated the blood clearance halflife of the radiopharmaceuticals synthesised by the two methods, which was shorter at 15.16 min for 177 Lu-DOTA-CC-PSMA compared to 177 Lu-PSMA-617 (17.18 min).
In addition, we compared the tumor uptake of 177 Lu-DOTA-CC-PSMA (click chemistry method) and that of 177 Lu-PSMA-617 (conventional labeling method) and found that the tumors had significantly higher uptake of the click chemistry-synthesized 177 Lu-DOTA-CC-PSMA (Fig. 2C, P < 0.05 within 24 h).SPECT/CT imaging SPECT/CT images (Fig. 3) showed high tumor uptake in 22Rv1 tumor-bearing mice at all time points, peaking at 2 h, and tumor uptake even after 24 h, indicating that 177 Lu-DOTA-CC-PSMA has a high tumor affinity with good accumulation and retention capacity.The uptake of 177 Lu-DOTA-CC-PSMA was moderate in the kidneys and low in other normal organs, with a gradual decrease in uptake over time, and the radioactivity essentially disappeared from the kidneys and bladder after 24 h, whereas 177 Lu-DOTA-CC-PSMA was further enriched and retained in the PSMA-positive tumors.High T/M ratios and high T/K ratios indicated good non-target organ clearance efficiency and high T/NT ratios.Thus, targeting molecular probes synthesized by click chemistry with good tumor uptake and rapid clearance shows significant potential in simplifying and unifying the synthesis of metal radiopharmaceuticals.

Discussion
This study explored the potential of click chemistry in simplifying and unifying the synthesis of metal radiopharmaceuticals using 177 Lu-labeled PSMA-targeting molecule probe as an example. 177Lu has a long half-life and can release γ-rays, and these γ-rays in SPECT imaging and external dose assessment have the potential for early diagnosis of tumors, recurrence, and metastatic foci (Liu and Chen 2019).As the pairing metal nuclide of 177 Lu, the positronic nuclide 68 Ga has a similar labeling chemistry and can often be labeled with the same drug precursor, and thus it can be prepared for diagnosis and treatment integration of radiopharmaceuticals (Chen et al. 2018).In a similar way, the proposed protocol is suited for 68 Ga-labeling of bioactive molecules.This study achieves a significant reduction in time compared to other click chemistry methods (Choy et al. 2017), which is not significant for Lu-177 labeling, but as a methodological study is significant for the improvement of the radiochemical yield of other short halflife nuclides, e.g.Ga-68.
The synthesis of 177 Lu-DOTA-CC-PSMA is straightforward and expands the scope of PSMA-617 as a labeling precursor, which simplifies the labeling process by avoiding high-temperature reaction conditions compared with conventional 177 Lu labeling.In this study, the click chemistry method was employed to incorporate a triazole ring into both the DOTA and PSMA molecular structure.The effect of this triazole ring on the Fig. 3 In vivo 177 Lu-DOTA-CC-PSMA of a PSMA-positive tumor model.Whole-body scans at 0.5, 1, 2, 4, 6, and 24 h after injection of 177 Lu-DOTA-CC-PSMA (22Rv1 tumor-bearing mouse) are shown, with clearly visible renal accumulation, low uptake in other normal organs, and tumor uptake peaking at 2 h and remaining at 24 h.The tumor is marked with a white arrow overall drug properties of the complex was subsequently investigated, and its biological properties were studied in mice with PSMA-positive tumors.The results showed that 177 Lu-DOTA-N 3 was successfully labeled with PSMA-alkyne compounds after using click chemistry, and the labeling process was simple and efficient, with mild conditions and high RCP.The 177 Lu-DOTA-CC-PSMA in this study showed good in vitro and in vivo stability, which was still maintained at more than 90% within 144 h, which is more consistent with the stability of 177 Lu-PSMA-617 (> 90% within 144 h). 177Lu-DOTA-CC-PSMA could also maintain stability in vivo, indicating that it can be used for in vivo studies.
177 Lu-DOTA-CC-PSMA is a PSMA-targeted nuclear drug with translational therapeutic promise for PRLT. 177Lu-DOTA-CC-PSMA is of good hydrophilic and low lipidsoluble, so it does not easily cross the blood-brain barrier, and the brain tissue is exposed to less radiation.Our pharmacokinetic results showed that 177 Lu-DOTA-CC-PSMA could be cleared from the blood and rapidly distributed to all tissues and organs in the body, providing a lower blood background signal during imaging, which was conducive to the formation of a high T/B ratio and clear SPECT images.The biodistribution and SPECT/CT imaging results in the 22Rv1 tumor-bearing mice, especially the significant renal excretion, suggested that 177 Lu-DOTA-CC-PSMA was efficiently cleared from the blood and other organs, which is an important aspect of in vivo imaging with targeted molecular probes.Meanwhile, the results of the biodistribution and SPECT/CT imaging showed that 177 Lu-DOTA-CC-PSMA exhibited superior uptake and retention within the tumor, good non-target organ clearance efficiency, and high T/NT ratio. 177Lu-DOTA-CC-PSMA is of the advantage of achieving high accumulation within the tumour, with potential therapeutic value.
In conclusion, the labeling of 177 Lu-DOTA-CC-PSMA synthesized by click chemistry is simple and easy, with mild labeling conditions, qualified quality control, and high labeling rate; 177 Lu-DOTA-CC-PSMA has ideal biodistribution, most of which is excreted via the kidneys; and it offers rapid blood clearance and good stability, demonstrating the potential of click chemistry to unify the synthesis of metal radiopharmaceuticals.However, only the popular 177 Lu-labeled PSMA-targeting molecular probe was used as an example in this study, and more studies on metal nuclide-labeled targeting molecules were not carried out.The potential of click chemistry to unify the synthesis of metal radiopharmaceuticals has yet to be verified for more metal nuclides through the click-chemistry-labeled targeting molecular probes.

Conclusion
In this study, we proposed a scheme to avoid the loss of targeting properties during the degeneration and inactivation of targeting molecules in the process of producing metal nuclide-conjugated radiopharmaceuticals and validated it with 177 Lu-labeled PSMAtargeting molecules.A novel 177 Lu-labeled PSMA-targeted molecular probe, designated as 177 Lu-DOTA-CC-PSMA, was successfully synthesized through the click chemistry of 177 Lu-DOTA-N 3 with PSMA-alkyne compound.The stability and tumor uptake of 177 Lu-DOTA-CC-PSMA synthesized by click chemistry were higher and with consistent pharmacokinetics, compared with 177 Lu-PSMA-617 synthesized by conventional methods.Consequently, metal nuclide-coupled radiopharmaceuticals, represented by

Table 2
In vivo evaluation of tumor uptake ( 177 Lu-DOTA-CC-PSMA and 177 Lu-PSMA-617) and biodistribution of 177 Lu-DOTA-CC-PSMA in mice model.(ID%/g, mean ± SD) n = 4, if not indicated otherwise # n = 4, and ratio instead of ID%/g.p-values are given for comparison of each time point for each tissue.Only mice with tumor inoculation have been used in this analysis