A New Labeling Method of ^99mTc-PSMA-HBED-CC

Abstract

Objective

⁶⁸Ga-PSMA-HBED-CC (⁶⁸Ga-PSMA-11) was approved by the U.S. Food and Drug Administration as the first prostate-specific membrane antigen (PSMA)-targeted positron emission tomography (PET) imaging drug for patients with prostate cancer. However, the utility of ⁶⁸Ga-PSMA-HBED-CC may be limited due to PET/CT or PET/MR accessibility and ⁶⁸GaCl₃ availability produced from ⁶⁸Ge/⁶⁸Ga generator or cyclotron. Thus, in-house preparation of ^99mTc-PSMA-HBED-CC was developed as an alternative to ⁶⁸Ga-PSMA-HBED-CC to be ubiquitous and affordable in the worldwide population.

Methods

A solution of ^99mTc-pertechnetate was added to PSMA-HBED-CC and 4% SnCl₂·2H₂O in a 10-mL sterile vial. The mixture was heated at 100°C for 15 minutes and then allowed to cool to room temperature. Labeling conditions were optimized to maximize the radiochemical yield of ^99mTc-PSMA-HBED-CC. The chelation completeness was monitored using instant thin layer chromatography, and the stability of ^99mTc-PSMA-HBED-CC was subsequently evaluated.

Results

The radiolabeling of ^99mTc-PSMA-HBED-CC was successful using the appropriate amount of 10 µg PSMA-HBED-CC 3 µg SnCl₂·2H₂O and ^99mTc-pertechnetate 370 MBq at 100°C for 15 minutes, yielded the best result in high radiochemical yield (71.49 ± 2.42%), radiochemical purity (98.29 ± 2.65%), and specific activity of 37.84 ± 1.47 GBq/µmol. ^99mTc-PSMA-HBED-CC is stable with radiochemical purity of more than 95% within 4 hours at room temperature.

Conclusion

A new labeling method of ^99mTc-PSMA-HBED-CC was developed. Quality control parameters of ^99mTc-PSMA-HBED-CC met the criteria in accordance with the European Pharmacopoeia.

 

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Keywords

prostate cancer - PSMA-HBED-CC - PSMA-11 - technetium-99m - ^99mTc-PSMA-HBED-CC

Introduction

Prostate cancer is the most common malignancy found in men and the second leading cause of cancer death worldwide.[1] Over the past two decades, the initial diagnosis and follow-up have been serum prostate-specific antigen levels, digital rectal examination, and some conventional imaging techniques including ultrasound, computed tomography, magnetic resonance imaging, and bone scintigraphy, but none provides highly specific and sensitive detection. Although transrectal ultrasound-guided prostate biopsy is currently accepted as the gold standard to provide the histopathological diagnosis of prostate cancer,[2] it is an invasive procedure that resulted in a risk of side effects and the accuracy of diagnosis. The accurate definition of tumor burden and its staging is particularly important for effective treatment selection. Therefore, molecular imaging, a noninvasive method, has been employed to visualize the tumor in both soft tissue and bone with higher specific and sensitive detection, monitored response to therapy to improve management of prostate cancer, clinical outcome, and patient's quality of life.

The Food and Drug Administration (FDA) approved ⁶⁸Ga-PSMA-HBED-CC (⁶⁸Ga-PSMA-11, previous name: ⁶⁸Ga-DKFZ-PSMA-11, generic name: ⁶⁸Ga-Gazetotide) as the first prostate-specific membrane antigen (PSMA)-targeted positron emission tomography (PET) imaging drug for men with prostate cancer.[3] The development of ⁶⁸Ga-PSMA-HBED-CC, which targets PSMA, has offered new perspectives for prostate cancer diagnosis and evaluation of therapeutic response.[4] PSMA is a cell surface transmembrane protein with 750 amino acids type II glycoprotein that primarily expresses in normal prostate epithelium and is overexpressed in prostate cancer cells including bone metastasis.[5] X-ray crystal structure analysis of PSMA, also known as N-acetylated L-aspartyl-L-glutamate peptidase (NAALADase I), has identified the critical interaction of potent inhibitors within the hydrophobic active site of the enzyme.[6] Consequently, several classes of NAALADase I inhibitors had been exploited for structure-based design platforms, leading to the novel synthesis of PSMA-HBED-CC.[7] [8] [9] To date, ⁶⁸Ga-PSMA-HBED-CC has explicitly demonstrated superior detection of PSMA-positive prostate cancer lesions in recurrent and metastatic sites over conventional imaging methods[10] [11] and two other FDA-approved PET tracers, ¹⁸F-fluciclovine and ¹¹C-choline that are used in patients with suspected cancer recurrence.[12] Recently, the FDA also approved ¹⁸F-piflufolastat as the second PSMA-targeted PET imaging drug with prostate cancer.

Besides ⁶⁸Ga-PSMA-HBED-CC, the preferential use of ^99mTc-labeled urea-based PSMA inhibitor has received interest as an alternative option to widespread the advantages of PSMA imaging due to a number of prostate cancer patients who are scheduled on PSMA imaging. The hybrid modality of single-photon emission CT (SPECT)/CT offers a wide range of workhorses in nuclear medicine with lower financial access, especially the remote medical center in which PET/CT facility is not available. Although the spatial resolution of ^99mTc is not as good as that of ⁶⁸Ga, ^99mTc provides a sufficiently long half-life of 6 hours in both preparation and accumulation in the target site. Moreover, the decay range of ^99mTc is short enough to minimize radiation exposure to patients and medical staff.

While some ^99mTc-labeled PSMA tracers have been previously reported using various PSMA ligands with several forms of complexation, for example, [^99mTc(CO)₃(L)₃]⁺,[13] [14] [15] MAG3-based ^99mTc-PSMA-I&S,[16] ^99mTc-MIP,[17] [18] [19] [20] ^99mTc-HYNIC-PSMA,[21] [22] peptide-chelator-based ^99mTc-DUPA,[23] ^99mTc-PSMA-T4,[24] ^99m Tc-PSMA-tricarbonyl-HBED-CC,[25] and ^99mTc-PSMA-HBED-CC,[26] it challenges to develop a convenient labeling method in a single step without coligand for ^99mTc-complexation. According to our experience in theranostics, we adapted the routine standard labeling procedure of ⁶⁸Ga-PSMA-HBED-CC with the rationale that HBED-CC would serve as a suitable chelator in a mimic manner to diethylenetriamine pentaacetic acid (DTPA) as shown in [Fig. 1]. Our attention focused on optimizing the labeling parameters to improve the radiosynthesis of ^99mTc-PSMA-HBED-CC.

Fig. 1 Chemical structures of (A) diethylenetriamine pentaacetic acid (DTPA), (B) PSMA-HBED-CC, and (C) imposed chelating motif.

 

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Material and Methods

PSMA-HBED-CC was purchased from ABX advanced biochemical compounds (GmbH, Germany). Sodium ^99mTc-pertechnetate was purchased from Global Medical Solution (Thailand). Stannous chloride dihydrate and hydrochloric acid were purchased from Sigma-Aldrich (Germany). The stock solution of 4% stannous chloride was freshly prepared and kept in a refrigerator. All chemicals and solvents were used without further purification unless otherwise noted. The C18 cartridge (Sep-Pak Light, lot no. 045732200A) was purchased from Waters (United States). The TLC scanner Raytest model MiniGita was used.

Preparation of 4% Stannous Chloride Stock Solution

SnCl₂·2H₂O 0.144 mg was added to 37% HCl 0.75 mL, followed by heating at 100°C for 5 minutes. After cooling down to room temperature, 6 N HCl 2.25 mL was added to 4% SnCl₂·2H₂O, which should be freshly prepared before labeling.

 

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Labeling of PSMA-HBED-CC with Tc-99m

To a mixture of 4% SnCl₂·2H₂O solution calculated as an amount of SnCl₂ and an aliquot of PSMA-HBED-CC (10 µg in H₂O 100 µL) in 10 mL sterile vial, ^99mTc-pertechnetate 370 MBq was added. The labeling was performed at 100°C for 15 minutes in a heating block, followed by a 10-minute cool down to reach room temperature. The crude product was passed through a C18 cartridge. ^99mTc-PSMA-HBED-CC was slowly purged from a C18 cartridge using EtOH:H₂O (1:1) 2 mL to the final product vial.

 

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Quality Control

Radiochemical purity (RCP) analyses were performed using instant thin-layer chromatography (iTLC) on silica paper strips as a stationary phase with two different mobile phases. The free form of ^99mTcO₄ ⁻ was determined using 0.9% normal saline, whereas ^99mTc-colloid formation was determined using acetone as the mobile phase. The radioactivity distribution on iTLC strips was also determined on a TLC scanner. Subsequently, radiochemical yield (RCY) was calculated. The pH of the final product was determined using a pH indicator.

 

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Stability Test

The chemical stability of ^99mTc-PSMA-HBED-CC was carried out by incubating the final product radioactivity in samples range 10.22 ± 0.40 mCi at room temperature for 6 hours and monitored by iTLC every hour. The radioconcentration used for the stability test was 2.04 mCi/mL. No stabilizer was added.

 

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Results

Labeling of ^99mTc-PSMA-HBED-CC

The labeling parameters were investigated to achieve the highest possible RCY. The quantity of PSMA-HBED-CC used in each experiment remained constant at 10 µg (0.011 µmol). In accordance with the DTPA cold kit formulation, the appropriate radioactivity of ^99mTc-pertechnetate was determined in direct correlation with approximate 370 MBq (10 mCi), while maintaining the solution's pH at 5.0, as indicated in [Table 1].

Table 1 Labeling parameters in ^99mTc-PSMA-HBED-CC

Iteration	SnCl2·2H2O (µg)	99mTcO4 − (mCi)	Labeling condition	Free form of 99mTcO4 − (%)	99mTc-colloid (%)	RCY (%)
1	0.5	10.02	Room temperature 15 min	99.58	0.17	0.25
2	0.5	10.32	100°C, 15 min	88.89	0.00	11.11
3	1.0	9.97	100°C 15 min	81.82	0.00	18.18
4	2.0	10.16	100°C, 15 min	64.29	10.00	25.71
5	2.5	10.61	100°C, 15 min	20.00	20.00	60.00
6	3.0	11.02	100°C, 15 min	9.09	25.00	65.91
7	3.5	11.64	100°C, 15 min	0.00	41.67	58.33
8	4.0	11.15	100°C, 15 min	0.00	50.00	50.00

Abbreviation: RCY, radiochemical yield.

In the preliminary experiment, 10.02 mCi of ^99mTcO₄ ⁻ was combined with 0.5 µg of SnCl₂ at room temperature for 15 minutes that resulted in a RCY of 0.25%, with 99.58% of ^99mTcO₄ ⁻ remaining unbound. Subsequently, the reaction temperature was increased to 100°C for 15 minutes, in alignment with the standard procedure for labeling ⁶⁸Ga-PSMA-HBED-CC. This adjustment resulted in a RCY of 11.11%, affirming the choice to set the reaction conditions at 100°C for 15 minutes. In iterations 3 to 8, the amount of SnCl₂ was progressively adjusted to enhance the RCY. By increasing the SnCl₂ quantity by 0.5 µg in each iteration, the highest RCY of 65.91% was achieved in the sixth iteration. However, when the amount of SnCl₂ exceeded 3.5 µg, complete chelation occurred between PSMA-HBED-CC and ^99mTcO₄ ⁻, which also led to a significant rise in the formation of hydrolyzed species, ultimately diminishing the RCY. Total radioactivities of the final product recovered after labeling are 10.12 ± 0.58 mCi.

 

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Chemical Stability of ^99mTc-PSMA-HBED-CC

The chemical stability of ^99mTc-PSMA-HBED-CC was evaluated using the optimized labeling method in the 6th iteration that produced the highest RCY. The compound was incubated at room temperature for 6 hours, and RCP was checked hourly through iTLC ([Fig. 2]). The findings are shown in [Fig. 3]. In general, the RCP of Tc-99m radiopharmaceuticals should meet or exceed 95%. This study demonstrated that the RCP of ^99mTc-PSMA-HBED-CC remained above 95% for up to 4 hours after labeling, and stayed above 90% at the 5th and 6th hours.

Fig. 2 Instant thin-layer chromatography (iTLC) chromatogram of ^99mTc-PSMA-HBED-CC; (left) in acetone as mobile phase, (right) in 0.9% normal saline as mobile phase.

Fig. 3 Chemical stability of ^99mTc-PSMA-HBED-CC at room temperature.

 

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Discussion

Since the identification of PSMA as an antigen and the discovery of the specific antibody 7E11-C5 (capromab) for both normal and malignant prostate epithelium, as reported by Horoszewicz et al,[27] PSMA has emerged as a crucial target for prostate cancer cells.[28] [29] [30] In 2012, Eder et al developed the urea-based PET tracer ⁶⁸Ga-PSMA-HBED-CC (formerly ⁶⁸Ga-DKFZ-PSMA-11) to address the limitations of the lead antibody J591.[31] The U.S. FDA approved ⁶⁸Ga-PSMA-HBED-CC as the first PET imaging agent for PSMA-positive lesions in men with prostate cancer in December 2020[3] and ¹⁸F-piflufolastat as the second PET imaging agent for PSMA-positive lesions in men with prostate cancer in May 2021.[3] Inspired by this breakthrough, our study aimed to develop ^99mTc-PSMA-HBED-CC as a SPECT imaging analog to increase accessibility for prostate cancer diagnosis.

PSMA-HBED-CC was chosen for this study due to its widespread clinical use and commercial availability. The dose of PSMA-HBED-CC was standardized at 10 μg. To optimize the manual labeling of ^99mTc-PSMA-HBED-CC, various labeling conditions and amounts of calculated SnCl₂ were assessed. The results, presented in [Table 1], indicate that ^99mTc-PSMA-HBED-CC is a thermodynamically favorable product. Using 2.5 to 4.0 μg of SnCl₂ resulted in a RCY exceeding 50%. When the SnCl₂ amount exceeded 3.5 μg, no uncomplexed Tc-99m was detected, indicating effective reduction of TcO₄ ⁻. However, higher amounts of SnCl₂ also increased colloid formation. Optimal quantitative radiolabelling of 10 μg of PSMA-HBED-CC was achieved with 3.0 μg of SnCl₂. To prevent colloid formation, both SnCl₂ and PSMA-HBED-CC must be present in the reaction mixture before adding TcO₄ ⁻ to form the desired complex.

The chemical stability of ^99mTc-PSMA-HBED-CC was evaluated by incubating it at room temperature. As shown in [Fig. 2], it retained RCP above 95% for up to 4 hours. After this period, free Tc-99m increased. Therefore, it is recommended to use ^99mTc-PSMA-HBED-CC within 4 hours of preparation or store it in a refrigerator to maintain stability.

Vats et al[26] previously reported the preparation of ^99mTc-PSMA-HBED-CC using 50 mg of PSMA-HBED-CC, 40 mg of SnCl₂, and TcO₄ ⁻ 740 MBq at pH 5, yielding a RCY of 60 ± 5%, a RCP greater than 98%, and specific activity of 15 ± 5 GBq/µmol. However, they did not conduct stability tests. Economically, our study used 10 µg of PSMA-HBED-CC, 3 µg of SnCl₂, and 370 MBq of ^99mTc-pertechnetate at 100°C for 15 minutes, achieving a higher RCY (71.49 ± 2.42%), RCP (98.29 ± 2.65%), and specific activity (37.84 ± 1.47 GBq/µmol). This method is more cost-effective and easier to manipulate.

 

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Conclusion

To optimize the labeling of PSMA-HBED-CC with ^99mTc-pertechnetate for prostate cancer imaging, the labeling procedure should be carried out at 100°C for 15 minutes using 3 µg of SnCl₂, minimizing the presence of free ^99mTc-pertechnetate and colloid formation. Purification with a C18 cartridge is required to achieve RCP that complies with the European Pharmacopoeia standards. The stability of ^99mTc-PSMA-HBED-CC remains robust for up to 4 hours at room temperature, with RCP exceeding 95%. It is recommended to use the labeled product within 4 hours of preparation.

 

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Conflict of Interest

None declared.

Authors' Contributions

B.P. and S.S. contributed to the conceptualization, methodology, formulation, formal analysis, and visualization. B.P. performed the kit manufacturing. S.S. managed funding and acquisition. S.S. was a major contributor in writing the manuscript. All authors have read and agreed to the final version.

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Publication History

Article published online:
26 June 2025