⁶⁸Ga-THP-PSMA: a PET imaging agent for prostate cancer offering rapid, room temperature, one-step kit-based radiolabeling

Abstract

The clinical impact and accessibility of ⁶⁸Ga tracers for the prostate-specific membrane antigen (PSMA) and other targets would be greatly enhanced by the availability of a simple, one-step kit-based labeling process. Radiopharmacy staff are accustomed to such procedures in the daily preparation of ^99mTc radiopharmaceuticals. Currently, chelating agents used in ⁶⁸Ga radiopharmaceuticals do not meet this ideal.

Aim

To develop and evaluate preclinically a ⁶⁸Ga radiotracer for imaging PSMA expression that could be radiolabeled simply by addition of ⁶⁸Ga generator eluate to a cold kit.

Methods

A conjugate of a tris(hydroxypyridinone) (THP) chelator with the established urea-based PSMA inhibitor was synthesized and radiolabeled with ⁶⁸Ga by adding generator eluate directly to a vial containing the cold precursors THP-PSMA and sodium bicarbonate, with no further manipulation. It was analyzed after 5 min by instant thin layer chromatography and HPLC. The product was subjected to in vitro cell-binding studies to determine PSMA affinity using PSMA-expressing DU145-PSMA cells, with their non-expressing analog DU145 as a control. In vivo PET imaging and ex vivo biodistribution studies were carried out in mice bearing xenografts of the same cell lines, with ⁶⁸Ga-HBED-CC-PSMA as a comparator.

Results

Radiolabeling was complete (>95%) within 5 min at room temperature, showing a single radioactive species by HPLC that was stable in human serum for >6 hours and showed specific binding to PSMA-expressing cells with an IC₅₀ of 361 ± 60 nM. In vivo PET imaging showed specific uptake in PSMA-expressing tumors, reaching 5.6 ± 1.2 % ID/cm³ at 40-60 min and rapid clearance from blood to kidney and bladder. The tumor uptake, biodistribution and pharmacokinetics were not significantly different to those of ⁶⁸Ga-HBED-CC-PSMA except for reduced uptake in the spleen.

Conclusion

Conjugation of THP to the PSMA pharmacophore produces a ⁶⁸Ga tracer with equivalent imaging properties but greatly simplified radiolabeling compared to other ⁶⁸Ga-PSMA conjugates. THP offers the prospect of rapid, simple, one-step, room temperature “syringe-and-vial” radiolabeling of ⁶⁸Ga radiopharmaceuticals.

Introduction

Infrastructure in radiopharmacies has been developed around ^99mTc labeling protocols, where a generator is eluted multiple times per day to produce a diverse range of tracers by reconstituting commercially available GMP “cold kits” (1), typically in a high-throughput environment where speed, simplicity, volume and reproducibility of radiolabeling are paramount. The growth of ¹⁸F and ¹¹C PET tracers, with which a kit model is not compatible due to the need for an on-site cyclotron and more complex synthetic chemistry, has spawned more diverse, complex and costly infrastructure to support PET. Nevertheless the generator and kit approach retains its appeal and in principle, GMP ⁶⁸Ga generators now available are amenable to kit production if a simple, mild chelation step can be achieved (2,3). This would make ⁶⁸Ga tracers widely available without the complex and costly infrastructure associated with ¹⁸F and ¹¹C tracer production.

An ideal one-step radiolabeling procedure for ⁶⁸Ga radiolabeling, matching the simplicity typical of long-established ^99mTc-labeling procedures requiring only the addition of generator eluate to the freeze-dried kit vial, has not yet been achieved. Towards this end, the chelating agent must meet several criteria: its labeling should reach completion (>95%) quickly (<5 min) at room temperature and be unaffected by common trace metals, without additional steps to concentrate, buffer or purify. Its complex should resist in vivo transchelation by endogenous proteins such as transferrin, and conjugation and radiolabeling should not induce mixtures of diastereomers, enantiomers or geometric isomers, nor adverse pharmacokinetics e.g. delayed renal clearance or non-specific binding. The current generation of ⁶⁸Ga chelators do not meet these criteria. For example, the widely adopted macrocycle DOTA (4), while complexing Ga³⁺ with extraordinarily high kinetic stability, has very slow complexing kinetics, necessitating heat (e.g. 90 °C, followed by a suitable cooling period), a large amount of the biomolecule, and low pH. Low yields (<95%) necessitate a purification step. These factors add process complexity, limit specific activity and may damage the biomolecule. Conversely, the fast chelation kinetics of HBED-CC makes radiolabeling of ⁶⁸Ga-HBED-CC-PSMA possible at room temperature, but produces an undesirable mixture of cis/trans geometric isomers distinguishable by HPLC (5,6,7). Thus, a heating step is still required, to reduce the number of isomers and increase the yield of one to ~ 90 % (6). Clinical radiosynthesis of tracers based on these chelators is currently performed on cartridge-based synthesis modules, taking 35 min and typically affording 80% ± 5 % decay corrected radiochemical yield (6).

Recently, several groups have introduced new experimental ⁶⁸Ga³⁺ chelators that address these issues but none eliminate all of them. NOTA, TRAP and DEDPA are promising but, like DOTA, require acidic conditions, and are vulnerable to competition from contaminating trace metals. The DATA series of chelators show rapid, room temperature, labeling at pH 5 and the DATA^PPh variant can be labeled in 15 min at pH 7 but require preprocessed eluate (8). A class of chelator that promises to meet the requirements for kit-based labeling is the tris(hydroxypyridinone) (THP) system; it can complex ⁶⁸Ga rapidly at room temperature and close to neutral pH, with high yield and purity. Its performance has previously been evaluated against a range of common chelators (9), including HBED, and it was shown to have superior radiolabeling properties under milder conditions. THP has also been functionalized for conjugation to peptides and proteins and the conjugates retain the required mild radiolabeling and in vivo targeting properties (9–13).

Here we evaluate a THP bioconjugate targeting the prostate-specific membrane antigen (PSMA, over-expressed in prostate cancer), incorporating a small urea-linked dipeptide pharmacophore (4,5,14) (Fig.1). A ⁶⁸Ga-labeled conjugate of this targeting moiety with HBED-CC has shown outstanding clinical promise in several trials in patients with prostate cancer (15,16), but for reasons outlined above is not amenable to a simple, one-step kit-based synthesis. The aims of this work were to determine the potential of ⁶⁸Ga-THP-PSMA to achieve one-step kit-based labeling of a radiopharmaceutical intended for PSMA imaging, and to evaluate preclinically the resulting tracer.

Figure 1.

Structure of (A) DOTA-PSMA (PSMA-617) (4); (B) HBED-CC-PSMA (DKFZ-PSMA-11) (5,6); (C) THP-PSMA

Materials and methods

Synthesis of THP-PSMA

THP-PSMA was synthesized via an orthogonal solid phase strategy described in the Supplemental data.

Preparation of lyophilized kits

Kits for one-step radiolabeling were prepared by lyophilizing an aqueous solution (5.25 mL) containing sodium bicarbonate (44 mg), sodium phosphate monobasic (8.6 mg), sodium phosphate dibasic heptahydrate (8.9 mg) and THP-PSMA (40 µg, 26 nmol) in a plastic vial to afford a white powder.

⁶⁸Ga-THP-PSMA radiolabeling

Radiolabeling and radio-HPLC and -iTLC procedures for ^67/68Ga-DOTA-PSMA (PSMA-617) (4), ^67/68/natGa-HBED-CC-PSMA (DKFZ-PSMA-11) (5, 6), and ^67/natGa-THP-PSMA are described in Supplemental data.

Initial ⁶⁸Ga radiolabeling optimization experiments were performed with an Eckert and Ziegler (E&Z Radiopharma GmbH) ⁶⁸Ge/⁶⁸Ga generator producing 120 – 400 MBq ⁶⁸Ga. The eluate (0.1 M HCl, 5 mL, Sigma Aldrich HPCE grade) was fractionated (10 × 0.5 mL) but not preconditioned to concentrate ⁶⁸Ga or remove trace metal contaminants. Typically, ⁶⁸Ga³⁺ (5-75 MBq, 250 µL of the hottest fraction) was added to a mixture containing THP-PSMA in a range of concentrations (5-0.01 µg in 3 µL) in sodium bicarbonate solution (1 M, 26 µL) producing a solution with a pH of 6.5-7.5. Radiochemical yield was evaluated after 5 min at room temperature using reverse phase HPLC and iTLC. HPLC: ⁶⁸Ga-THP-PSMA R_t = 10.9 min; unbound ⁶⁸Ga R_t = 2.32 min. iTLC: ⁶⁸Ga-THP-PSMA R_f = 0.8-1; unbound ⁶⁸Ga R_f = 0. For in vivo studies THP-PSMA (2 µg) was labeled with ⁶⁸Ga as described above; >95% radiochemical purity was consistently achieved.

To evaluate labeling using the entire eluate without fractionation, radiolabeling of lyophilized kits was performed by addition of E&Z ⁶⁸Ge/⁶⁸Ga generator eluate (5 mL 0.1M HCl, 122-202 MBq) or Galli E_O (IRE ELiT) ⁶⁸Ge/⁶⁸Ga generator eluate (1.1 ml eluate diluted to 5 mL with 0.1 M HCl, 600-660 MBq) directly into a vented freeze-dried kit vial. After mixing, carbon dioxide evolution visibly ceased after 15 s, providing a transparent, colorless solution at pH 6-7 with a final concentration of 5.25 µM THP-PSMA. iTLC was performed 5 min and HPLC 10 min after reconstitution. Decay-corrected radiochemical yield determined by each method was >95% (n = 3 per generator).

Log P_OCT/PBS

⁶⁷Ga labeled radiotracer (50 µL, 10 μM, specific activity 1.5 MBq/nmol) in PBS (prepared as described in Supplemental data) was added to a pre-equilibrated mixture of 500 µL octanol and 450 µL PBS. The mixture was vortex-mixed for 30 min, and the phases separated by centrifugation (10,000 rpm, 10 min). Aliquots from each phase were gamma-counted.

Serum Stability

⁶⁸Ga-THP-PSMA labeled at 5 MBq/nmol as described above. 20 µL was added to 180 µL human serum giving a final THP-PSMA concentration of 2.5 µM. Samples were incubated at 37°C and monitored over 6 h by size exclusion HPLC (details in Supplemental data). ⁶⁸Ga-THP-PSMA without serum and unchelated ⁶⁸Ga³⁺ incubated with serum were also analyzed.

Cell uptake and binding affinity assays

To determine the cellular uptake of each tracer at 37°C and 4°C over time, and whether a plateau (equilibrium) state is reached, PSMA-expressing cells DU145-PSMA and non-PSMA-expressing cells DU145 (17) were seeded in a 24-well plate (0.25 x 10⁶ cells/well), one day before the assay. Two minutes before incubation the medium was replaced with 245 µL fresh medium at 37°C or 4°C, then 5 µL of one of the ⁶⁷Ga PSMA tracers (specific activity 0.75-2.2 MBq/nmol) was added giving a final concentration of 1 nM. The cells were incubated at 37°C or 4°C. At each time point supernatant was removed and the cells were washed with 3 × 0.25 mL PBS to determine the unbound fraction, followed by an acid wash (0.5 M glycine, pH 2.5, at 4°C, 5 mins) to determine cell surface-bound activity. Cells were lysed with 1 M NaOH to determine activity internalized by the cells. Fractions were gamma counted.

To determine the IC₅₀, competitive binding studies were performed with DU145-PSMA cells with 1 nM ⁶⁸Ga-DOTA-PSMA as the probe, blocking with ^natGa-THP-PSMA or ^natGa-HBED-CC-PSMA over a range of concentrations. Full details are described in Supplemental data.

Due to poor solubility of ^natGa-THP-PSMA above 0.25 mM, an alternative measure of affinity was developed, allowing the relative affinity of two gallium PSMA tracers to be determined simultaneously without the ^natGa complex. This method mitigates solubility difficulties and minimizes variance in measurements across samples arising from different cell numbers or radiotracer batches. Two different PSMA tracers, one labeled with ⁶⁷Ga and the other with ⁶⁸Ga, were simultaneously incubated with DU145-PSMA cells in a single well at 4°C for 2 h (the non-internalizing conditions selected to best represent the equilibrium state from the cell uptake studies). The tracers compared were: ^67/68Ga-DOTA-PSMA, ^67/68Ga-HBED-CC-PSMA, and ^67/68Ga-THP-PSMA. Each ⁶⁷Ga tracer was compared to each ⁶⁸Ga tracer and affinity ratios were obtained by measuring both total binding and non-specific binding for each tracer (gamma counting) and calculating the ratio of their specific binding. Full details are described in the Supplemental data.

Mouse model of prostate cancer

Animal studies complied with UK Research Councils’ and Medical Research Charities’ guideline on responsibility in the use of animals in bioscience research, under UK Home Office project and personal licenses. Subcutaneous prostate cancer xenografts were produced in SCID/beige mice (male, 5-12 weeks) by injecting 4 × 10⁶ DU145-PSMA or DU145 cells in the right flank. Imaging was undertaken once the tumor had reached 5-10 mm in diameter (1-4 weeks after inoculation).

PET scanning

PET imaging was performed on four groups of mice (n = 3 in each) under isoflurane anesthesia with a BioScan nanoPET-CT PLUS (Mediso, Hungary). Mice were CT imaged before radiotracer administration and dynamic PET data were collected for the first hour PI. Mice were then immediately culled and their organs harvested, weighed and gamma counted. Mice bearing DU145-PSMA tumors were administered either ⁶⁸Ga-THP-PSMA (5-15 MBq, 50-140 μL, 0.4-0.9 µg, group 1) or ⁶⁸Ga-HBED-CC-PSMA (5-15 MBq, 50-140 μL, 0.6-1.3 µg, group 2) by tail vein injection. A third group (group 3) were co-administered ⁶⁸Ga-THP-PSMA and the PSMA-inhibitor 2-(phosphonomethyl)pentane-1,5-dioic acid (PMPA) (50 µg, Enzo Life Sciences) (18,19). Mice bearing DU145 tumors were administered ⁶⁸Ga-THP-PSMA (group 4). The imaging protocol and image analysis methods are described in the Supplemental data.

Statistical Analysis

Data were analyzed in GraphPad Prism 5 (version 5.04) and are expressed as mean ± SD. Student t-tests were used to determine statistical significance with P < 0.05 considered significant.

Results

Synthesis of THP-PSMA

The resin-bound PSMA inhibitor (7, Fig. S1) was prepared by in situ formation of a bis(tert-butyl) glutamate isocyanate (5, Fig. S1) followed by coupling with resin-bound lys(Dde) (3, Fig. S1). Subsequent deprotection of Dde followed by coupling with glutaric anhydride provided an intermediate bearing a pendant carboxylate (8, Fig. S1). This was activated using HATU/DIPEA chemistry to form an amide bond with the free amine of a THP derivative (20). Cleavage from the solid support with simultaneous glutamate deprotection using TFA formed THP-PSMA (10, Fig. S1). After purification by semi-preparative HPLC, 10 was isolated as a TFA salt. Yield of THP-PSMA(CF₃COO)₃: (C₅₄H₇₇N₁₁O₁₉.(CF₃COO)₃) MW 1526.33, 4 mg, 2.6 µmol, 5.2% yield from resin loading. HPLC: R_t = 13.4 min, >98% purity. Full details of LC-ESI-MS, ¹H and ¹³C NMR characterization are shown in Supplemental data.

Radiolabeling

The THP chelator enabled radiolabeling with unmodified generator eluate, in a single step. ⁶⁸Ga³⁺ (5-75 MBq) in aqueous HCl (0.1 M, 250 µL) was added to pre-prepared THP-PSMA (5-0.01 µg in 3 µL H₂O) in sodium bicarbonate solution (1 M, 27 µL). Five minutes after addition of ⁶⁸Ga³⁺, pH was 6.5-7.5. Radiochemical purity, determined by HPLC and iTLC as a function of THP-PSMA concentration is shown in Fig. 2. Specific activities between 15 and 45 MBq/nmol were consistently achieved with the labeling conditions used for in vivo work (2 µg, 1.3 nmol THP-PSMA, 250 µL 20-60 MBq ⁶⁸Ga eluate, 95% radiochemical purity).

Figure 2.

(A) Dependence of radiochemical purity of ⁶⁸Ga-THP-PSMA on mass of THP-PSMA in 280 µL, as measured by HPLC (red) and iTLC (black) after 5 min; n = 3, mean ± SD. (B) HPLC (λ = 220 nm) of THP-PSMA (blue, R_T = 13.38 min) and ^natGa-THP-PSMA (black, R_T = 10.92 min, excess Ga(NO₃)₃ is present with R_T = 1.47 min), and radio-HPLC of ⁶⁸Ga-THP-PSMA (red, R_T =11.05 min) labeled using the one-step kit and analyzed 10 min post-reconstitution. (C) iTLC of ⁶⁸Ga-THP-PSMA labeled using one-step kit, analyzed 5 min post-reconstitution (Rf unchelated ⁶⁸Ga = 0, Rf ⁶⁸Ga-THP-PSMA = 0.8-1).

One-step kit for radiolabeling

Using kit vials containing lyophilized THP-PSMA (40 μg, 26 nmol), sodium bicarbonate (44 mg) and sodium phosphate buffer (17.5 mg), radiosynthesis of ⁶⁸Ga-THP-PSMA was achieved in one step by direct addition of 5 mL of generator eluate (0.1 M HCl, 122-202 MBq from the E&Z generator or 600-660 MBq from the IRE generator). Five minutes post addition, the pH was 6-7 and iTLC confirmed radiochemical purity above 95% with specific activities of up to 22 MBq/nmol when using the IRE generator (Fig. 2 and S3). iTLC analysis up to 3 h post reconstitution showed no evidence of instability or autoradiolysis (Fig. S3).

Lipophilicity and serum stability

The log P_OCT/PBS values of the three ⁶⁸Ga-PSMA complexes at pH 7.4 were very similar: -5.35 ± 0.1 for ⁶⁷Ga-THP-PSMA (n = 6), -5.40 ± 0.2 for ⁶⁷Ga-HBED-CC-PSMA (n = 6) and -5.40 ± 0.1 for ⁶⁷Ga-DOTA-PSMA (n = 5), indicating that all tracers are hydrophilic and lipophilicity is unlikely to underlie differences in in vivo performance. Serum stability studies of ⁶⁸Ga-THP-PSMA (Figure S4) showed minimal transchelation (<2%) to serum proteins after 6 h incubation.

In vitro cell uptake and binding affinity assays

Uptake of ⁶⁷Ga-DOTA-PSMA, ⁶⁷Ga-HBED-CC-PSMA and ⁶⁷Ga-THP-PSMA in DU145-PSMA and DU145 cells at 37°C and 4°C is shown in Figures 3A and S5. All three tracers showed time-dependent accumulation in PSMA-expressing cells, but very low uptake in non-PSMA-expressing cells, confirming uptake is PSMA-mediated. At 37°C uptake continued to increase with time, preventing measurement of equilibrium binding parameters such as K_d, but when temperature was reduced to 4°C, a plateau was reached for all tracers after approximately 2 h. Relative affinity was therefore measured after 2 h at 4°C. Results showed the K_d of ^67/68Ga-THP-PSMA was 10.4 ± 3.4 times higher than that of ^67/68Ga-HBED-CC-PSMA and 8.6 ± 3.4 times that of ^67/68Ga-DOTA-PSMA. This was confirmed by the directly measured IC₅₀ values of Ga-THP-PSMA (361 ± 60 nM) and Ga-HBED-CC-PSMA (34.3 ± 4.1 nM) (Fig. 3B and S6).

Figure 3.

(A) ⁶⁷Ga-THP-PSMA uptake over time at 4°C and 37°C, 1 × 10⁶ cells/mL with DU145-PSMA or DU145 cells. (B) Representative IC₅₀ experiment for ^natGa-THP-PSMA with 1 nM ⁶⁸Ga-DOTA-PSMA as the probe; (n = 4) for each concentration. IC₅₀ values in main text are the mean of at least 3 experiments (C) Ratio of K_d of two tracers incubated at 1 nM with DU145-PSMA cells at 4°C for 2 h. The ratios were calculated from the specific binding obtained by incubating the ⁶⁷Ga version of a tracer in the same well as the ⁶⁸Ga version of its comparator, and vice versa. Total wells (n = 34) for comparison of ^67/68Ga-THP-PSMA with ^67/68Ga-HBED-CC-PSMA or ^67/68Ga-DOTA-PSMA and (n = 18) for comparison of ^67/68Ga-HBED-CC-PSMA with ^67/68Ga-DOTA-PSMA; mean ± SD.

PET imaging and biodistribution

PET/CT scanning of ⁶⁸Ga-THP-PSMA in SCID/beige mice bearing PSMA-positive xenografts (DU145-PSMA, group 1) showed that excretion of ⁶⁸Ga-THP-PSMA was rapid and exclusively renal, with the majority of activity associated with the bladder and kidneys by the end of the scan (60 min) (Fig. 4 and Fig. S7). Blood clearance, represented by blood pool in the left ventricle, was rapid, decreasing to 1% ID/cm³ within 30 min and 0.5% ID/cm³ by 60 min (Fig. 5A). With images scaled between 0 and 25% ID/cm³, tumors were clearly delineated (Fig. 4). Analysis of the 40 – 60 min post-injection (PI) images gave a tumor uptake value of 5.6 ± 1.2 % ID/cm³ (Fig. 5A). Specificity of tumor uptake was confirmed both by mice bearing a PSMA negative xenograft, but otherwise identical tumor (DU145, group 4) with uptake of 1.5 ± 1.2 % ID/cm³ (p < 0.05 compared to group 1), and by blocking experiments in mice bearing a DU145-PSMA tumor where PMPA was co-administered with ⁶⁸Ga-THP-PSMA (group 3) giving a tumor uptake of 1.0 ± 0.4 % ID/cm³ (p < 0.05 compared to group 1).

Figure 4.

Representative PET/CT images of mice bearing xenografts at 40-60 min PI with PET images scaled from 0-25 % ID/cm³. (A) ⁶⁸Ga-THP-PSMA in DU145-PSMA tumor (group 1); (B) ⁶⁸Ga-THP-PSMA in DU145 tumor (group 4); (C) ⁶⁸Ga-THP-PSMA in DU145-PSMA tumor blocked with PMPA (group 3); (D) ⁶⁸Ga-HBED-CC-PSMA in DU145-PSMA tumor (group 2).

Figure 5.

(A) PET-derived time-activity curves of mice bearing DU145-PSMA tumors, imaged with ⁶⁸Ga-THP-PSMA for 1 h PI: blood pool (left ventricle) (red), tumor (blue) and leg muscle (black); (n = 3, mean ± SD). (B) PET/CT–derived % ID/cm³ in tumor 40-60 min PI. ⁶⁸Ga-THP-PSMA in DU145-PSMA tumor (black), ⁶⁸Ga-THP-PSMA in DU145-PSMA tumor blocked with PMPA (light gray), ⁶⁸Ga-THP-PSMA in DU145 tumor (dark gray) or ⁶⁸Ga-HBED-CC-PSMA in DU145-PSMA tumor (white); (n = 3, mean ± SD); (C) tumor/blood and tumor/muscle ratio of PET-derived % ID/cm³.

For comparison with an established PET tracer, mice bearing DU145-PSMA tumors were imaged with ⁶⁸Ga-HBED-CC-PSMA (group 2) (5,6,15). Time activity curves for ⁶⁸Ga-HBED-CC-PSMA (group 2) and ⁶⁸Ga-THP-PSMA (group 1) showed very similar blood clearance, tumor uptake and renal excretion (Fig. 5A and S7). Image analysis of 40-60 min PET images revealed that tumor uptake of ⁶⁸Ga-THP-PSMA (5.3 ± 0.1 % ID/cm³) was not significantly different to that of ⁶⁸Ga-HBED-CC-PSMA (5.6 ± 1.2 % ID/cm³).

Ex vivo biodistribution data are summarized in table S1 and were consistent with PET image analysis, confirming renal excretion and excellent specificity of ⁶⁸Ga-THP-PSMA for PSMA expressing tumors. ⁶⁸Ga-THP-PSMA showed very similar ex vivo biodistribution to ⁶⁸Ga-HBED-CC-PSMA, apart from markedly lower spleen uptake (3.7 ± 1.3 and 17.6 ± 6.1 % ID/g respectively). Importantly, anesthesia appeared to severely affect tracer uptake in the kidney and excretion to bladder, with large variation in kidney uptake across all groups, so comparison of kidney and bladder activity should be interpreted with caution.

Discussion

The aim of this study was to develop a ⁶⁸Ga radiotracer that targets PSMA with simple and high-yielding radiolabeling procedures suitable for development into a single step kit-formulated GMP radiopharmaceutical. The latter requirement was met by incorporating THP into the design. A THP-PSMA conjugate has been synthesized and characterized, and could be readily radiolabeled with ⁶⁸Ga (and ⁶⁷Ga). Radiolabeling yields of >95% and specific activity 15 - 45 MBq/nmol with unprocessed generator-produced ⁶⁸Ga were achieved in one step at pH 7 and ambient temperature within 5 min without further purification. Based on this we developed a lyophilized kit, which can be labeled/reconstituted simply by adding 5 mL of raw generator eluate. One-step kits can only be used with generators with <0.001% ⁶⁸Ge breakthrough; of these, the E&Z has the largest elution volume (5 mL, 0.1 M HCl) and so the kit was designed accordingly. Within the range used, specific activity was limited only by the activity available from the generator; the highest observed was 22 MBq/nmol with an elution of 660 MBq in 5 mL. Higher specific activities have been obtained in a clinical setting and will be reported alongside first-in-man studies. These radiolabeling properties demonstrate that kit-based radiolabeling of ⁶⁸Ga tracers, analogous to the simple manipulations to which radiopharmacy staff producing ^99mTc radiopharmaceuticals are accustomed, and requiring equipment no more complex than a shielded syringe and vial, is entirely feasible with an appropriate chelator.

As well as rapid and simple radiolabeling, the coordination properties of THP endow its conjugates with other properties well-suited to radiopharmaceutical application. It is highly selective for tripositive metal ions with ionic radius similar to that of Fe³⁺ and Ga³⁺ (21), potentially reducing the need to remove contaminating metal ions in raw generator eluate. Unlike HBED-CC, the tripodal design of THP restricts the number of geometric isomers that form upon Ga³⁺ coordination: upon reverse-phased HPLC, ⁶⁸Ga-THP-PSMA elutes as a single radioactive species (Fig. 2B) shown by LCMS to be a 1:1 complex with gallium (Fig. S2). Any isomerism (such as the Δ/Λ isomerism possible with tripodal complexes) is subject to equilibration that is rapid compared to the timescale of HPLC and in vivo processes, and is hence biologically irrelevant. The lipophilicity is low and comparable to that of established ⁶⁸Ga-PSMA ligands.

Although THP-PSMA was rationally designed based on previous literature (5,19,22,23), we have not yet undertaken structure–activity studies to optimize the specific target affinity of THP-PSMA bioconjugates. In vitro experiments demonstrate that ⁶⁸Ga-THP-PSMA bids specifically to PSMA, although with weaker affinity than ⁶⁸Ga-HBED-CC-PSMA and ⁶⁸Ga-DOTA-PSMA. The simplicity of radiolabeling, excellent serum stability and in vitro PSMA-binding results justified further evaluation of the new tracer in vivo. The in vivo properties of ⁶⁸Ga-THP-PSMA show that incorporation of THP into bioconjugates confers no THP-specific adverse pharmacokinetics. The rapid renal excretion, low non-specific uptake of ⁶⁸Ga-THP-PSMA in non-target tissues and high, specific radioactivity concentration in PSMA-expressing tumors are clinically desirable pharmacokinetic features. In the pre-clinical model used here, tumor uptake and other biodistribution and pharmacokinetic properties of ⁶⁸Ga-THP-PSMA and ⁶⁸Ga-HBED-CC-PSMA are indistinguishable except that spleen uptake of ⁶⁸Ga-THP-PSMA is lower by almost a factor of 5. Although PSMA-targeting radiopharmaceuticals generally appear to share high uptake in spleen in mice, murine spleen is not believed to express PSMA (24). It seems likely therefore that some other target capable of binding the PSMA tracers is present in spleen, and that ⁶⁸Ga-THP-PSMA has enhanced ability to distinguish PSMA from this alternative target.

Concluding remarks

Use of THP as the ⁶⁸Ga³⁺ chelator facilitates rapid chelation under mild conditions and produces a PSMA-targeted bioconjugate that can be labeled in one step by reconstitution of a kit with unprocessed generator eluate. Labeling with such kits requires only a generator, a cold-kit vial, a syringe, QC facilities and shielding. Kit-based labeling can be performed in a few minutes, using the full volume of unprocessed generator eluate (5 mL), without post-synthesis purification, achieving >95 % radiochemical purity. In vivo, ⁶⁸Ga THP-PSMA accumulates in PSMA-expressing tumors, with good tumor to background ratios and ability to delineate PSMA-positive tumor lesions similar to that of ⁶⁸Ga-HBED-CC-PSMA. Kit-based radiolabeling of ⁶⁸Ga radiopharmaceuticals is feasible and would facilitate wider and more economical use of ⁶⁸Ga in hospitals, hence benefitting more patients.