Rapid kit-based 68Ga-labelling and PET imaging with THP-Tyr3-octreotate: a preliminary comparison with DOTA-Tyr3-octreotate

Background Ge/68Ga generators provide an inexpensive source of a PET isotope to hospitals without cyclotron facilities. The development of new 68Ga-based molecular imaging agents and subsequent clinical translation would be greatly facilitated by simplification of radiochemical syntheses. We report the properties of a tris(hydroxypyridinone) conjugate of the SSTR2-targeted peptide, Tyr3-octreotate (TATE), and compare the 68Ga-labelling and biodistribution of [68Ga(THP-TATE)] with the clinical radiopharmaceutical [68Ga(DOTATATE)]. Methods A tris(hydroxypyridinone) with a pendant isothiocyanate group was conjugated to the primary amine terminus of H2N-PEG2-Lys(iv-Dde)5-TATE, and the resulting conjugate was deprotected to provide THP-TATE. THP-TATE was radiolabelled with 68Ga3+ from a 68Ge/68Ga generator. In vitro uptake was assessed in SSTR2-positive 427-7 cells and SSTR2-negative 427 (parental) cells. Biodistribution of [68Ga(THP-TATE)] was compared with that of [68Ga(DOTATATE)] in Balb/c nude mice bearing SSTR2-positive AR42J tumours. PET scans were obtained 1 h post-injection, after which animals were euthanised and tissues/organs harvested and counted. Results [68Ga(THP-TATE)] was radiolabelled and formulated rapidly in <2 min, in ≥95 % radiochemical yield at pH 5–6.5 and specific activities of 60–80 MBq nmol−1 at ambient temperature. [68Ga(THP-TATE)] was rapidly internalised into SSTR2-positive cells, but not SSTR2-negative cells, and receptor binding and internalisation were specific. Animals administered [68Ga(THP-TATE)] demonstrated comparable SSTR2-positive tumour activity (11.5 ± 0.6 %ID g−1) compared to animals administered [68Ga(DOTATATE)] (14.4 ± 0.8 %ID g−1). Co-administration of unconjugated Tyr3-octreotate effectively blocked tumour accumulation of [68Ga(THP-TATE)] (2.7 ± 0.6 %ID g−1). Blood clearance of [68Ga(THP-TATE)] was rapid and excretion was predominantly renal, although compared to [68Ga(DOTATATE)], [68Ga(THP-TATE)] exhibited comparatively longer kidney retention. Conclusions Radiochemical synthesis of [68Ga(THP-TATE)] is significantly faster, proceeds under milder conditions, and requires less manipulation than that of [68Ga(DOTATATE)]. A 68Ga-labelled tris(hydroxypyridinone) conjugate of Tyr3-octreotate demonstrates specificity and targeting affinity for SSTR2 receptors, with comparable in vivo targeting affinity to the clinical PET tracer, [68Ga(DOTATATE)]. Thus, peptide conjugates based on tris(hydroxypyridinones) are conducive to translation to kit-based preparation of PET tracers, enabling the expansion and adoption of 68Ga PET in hospitals and imaging centres without the need for costly automated synthesis modules.

The SSTR2-targeting radiopharmaceutical [ 68 Ga(DOTA-TATE)] has demonstrated superior clinical resolution and  sensitivity compared to the 111 In-labelled SPECT tracer,  [ 111 In(DTPA-octreotide)], in identifying tumours expressing SSTR2 in neuroendocrine cancer patients [3]. Despite the multistep radiochemistry required, [ 68 Ga(DOTA-TATE)] is used routinely in PET clinics, and in conjunction with 18 F-FDG [5], is important in determining therapeutic regimes of patients presenting with neuroendocrine tumours [1,2,4,9,10]. It is instructive to compare DOTATATE with THP-TATE (Chart 1), both in terms of (i) radiosynthesis in a hospital radiopharmacy and (ii) preclinical biodistribution, in order to evaluate the advantages and disadvantages of this new class of tris(hydroxypyridinone) chelators.

Materials and instrumentation
Mass spectra were recorded on an Agilent 6510 Q-TOF LC/MS mass spectrometer (Agilent, Palo Alto, CA). Instant thin-layer chromatography strips (ITLC-SG) were obtained from Varian Medical Systems UK, Ltd. (Crawley, UK), and ITLC strips were visualised using a Raytest Rita-Star TLC scanner. Semi-preparative reverse-phase HPLC was conducted using an Agilent Eclipse XDB-C18 column (9.4 × 250 mm, 5 μm) coupled to an Agilent 1200 LC system, with a 3 mL min −1 flow rate and UV spectroscopic detection at 220 nm. Mobile phase A contained water with 0.2 % TFA, and mobile phase B contained acetonitrile with 0.2 % TFA. The gradient started with 100 % A at 0 min, and the concentration of B increased at a rate of 1 % min −1 .
Analytical reverse-phase HPLC and radio-HPLC traces were acquired using two different instruments: (1) an Agilent 1200 LC system with an Agilent Zorbax Eclipse XDB-C18 column (4.6 × 150 mm, 5 μm) and UV spectroscopic detection at 220 nm. The radio-HPLC was coupled to a LabLogic Flow-Count detector with a sodium iodide probe (B-FC-3200). Mobile phase A comprised water with 0.1 % TFA, and mobile phase B comprised acetonitrile with 0.1 % TFA. For method 1, the concentration of B increased at a rate of 1.67 % min −1 , with 100 % A at 0 min and 50 % B at 30 min with a flow rate of 1 mL min −1 ; (2) an Agilent Zorbax Eclipse XDB-C18 column (4.6 × 150 mm, 5 μm) with a 1 mL min −1 flow rate and UV spectroscopic detection at 220 nm coupled to a Shimadzu HPLC. This was coupled to a radiation detector consisting of an Ortec model 276 Photomultiplier Base with Preamplifier, Amplifier, BIAS supply and SCA and a Bicron 1M 11.2 Photomultiplier Tube. For method 2, the concentration of B increased at a rate of 6.67 % min −1 , with 100 % A at 0 min and 80 % B at 12 min.
Analytical size-exclusion radio-HPLC traces were acquired using an Agilent 1200 Series HPLC system and a Phenomenex Biosep 2000 (300 × 7.8 mm) size-exclusion column with a phosphate-buffered saline mobile phase.

Radiolabelling
Initial radiolabelling experiments utilised an Eckert and Ziegler 68 Ge/ 68 Ga generator. Aqueous HCl solution (0.1 M, 5 mL) was passed through the generator, and the eluate was fractionated (5 × 1 mL). The second fraction (1 mL, containing 90-100 MBq 68 Ga) was added directly to an ethanol/water solution (50 %/50 %, 50 μL) of THP-TATE (25 μg) and immediately followed by a solution of ammonium acetate (2 M, 200 μL) to obtain a solution of pH 5-6. This solution was immediately applied to an analytical reverse-phase C18 HPLC column. For in vivo and in vitro studies, generator-produced 68 Ga 3+ (800-1000 MBq, iThemba Labs generator) was concentrated on an AG 50WX4 (400 mesh) cation exchange cartridge and eluted with 200 μL 0.9 M HCl in ethanol/water (90 %/10 %) [16]. This volume was diluted in deionised water (800 μL) and directly added to THP-TATE (25 μg) at ambient temperature, followed immediately by addition of aqueous ammonium acetate (2 M, 400 μL) to obtain solutions of pH~6.5, resulting in [ 68 Ga(THP-TATE)]. These solutions were further diluted by addition of saline solution (0.9 % NaCl w/v, 1.1 mL). Within 2-5 min of addition of 68 Ga 3+ to the conjugates, the solutions were subjected to analytical reverse-phase HPLC and ITLC analysis. Synthesis of [ 68 Ga(DOTATATE)] was undertaken using methods previously reported [17]. Briefly, an iThemba Labs generator at approximately 3 months post-calibration was eluted with aqueous HCl (0.4 M, 5 mL). The eluate was passed through an AG 50WX8 (400 mesh) cation exchange resin, and the 68 Ga 3+ was retained on the resin. The resin was washed with a solution of 80 % acetone/0.15 N HCl (1 mL) to remove residual 68 Ge breakthrough, followed by elution of 68 Ga 3+ (using a solution of 97.6 % acetone/0.05 N HCl, 400 μL) into a pre-heated reaction vial containing DOTATATE (42 μg), ascorbic acid and gentisic acid in sterile Milli-Q water (5 mL). After 10 min at 105°C, the reaction mixture was passed through a reverse-phase solid-phase extraction cartridge (Strata-X, 30 mg, Phenomenex). The Strata-X cartridge was rinsed with sterile Milli-Q water, and [ 68 Ga(DOTATATE)] was subsequently recovered with ethanol (500 μL). The ethanol solution containing [ 68 Ga(DOTATATE)] was transferred into a vial containing saline for injection (9 mL), and the resultant mixture passed through a low protein-binding filter. Radiochemical yields ranged from 50 to 70 %, and radiochemical purity was greater than 95 %.

Log P OCT/PBS determination
A solution containing [ 68 Ga(THP-TATE)] (10 μL, synthesised using eluate from an Eckert and Ziegler generator as described above) was added to 500 μL of octanol and 490 μL of aqueous phosphate-buffered saline solution. The mixture was agitated using a vortex for 3-4 min, and the phases separated by centrifugation (4000 rpm, 5 min). Aliquots from each phase (50 μL) were counted for radioactivity in a gamma counter. The experiment was repeated six times.

Serum stability
A solution containing [ 68 Ga(THP-TATE)] (150 μL, synthesised using eluate from an Eckert and Ziegler generator as described above) was added to 1.5 mL of fresh human female O + serum, incubated at 37°C for 5 h, and the reaction mixture was analysed using size-exclusion HPLC chromatography. Concurrently, a solution of 68 Ga 3+ in ammonium acetate (0.33 M, 8 MBq, 300 μL) was added to 1.5 mL of serum and incubated at 37°C for 4 h, followed by analysis using size-exclusion HPLC.

In vitro uptake
The A427 human non-small cell lung carcinoma cell line was obtained from American Type Culture Collection (catalogue number: HTB-53). The SSTR2 overexpressing cell line A427-7 was a gift from Prof. Buck Rogers [40]. A427-7 and parental A427 cells were plated in Minimum Essential Medium (MEM) containing 10 % FBS at 5 × 10 5 cells per well in poly-D-lysine-coated 12well cell culture dishes for 24 h. On the day of the binding assay, cells were washed in PBS and equilibrated in MEM containing 1 % FCS. Cells were then treated with [ 68 Ga(THP-TATE)] (1.5 MBq, 5 μL, 4 μM THP-TATE), with or without blocking TATE peptide (5 μL, 800 μM, 200-fold excess) for 5, 15, 30 and 60 min (A427-7 cells) and 60 min (A427 parental cells) in triplicate. Uptake was terminated by placing the cells on ice. Unbound free tracer was collected, with the supernatant and cold PBS washes combined for this fraction. The surface-bound tracer fraction was collected through two 10-min acid washes (0.1 M glycine in saline, pH 2.3). Finally, the internalised fraction was collected through incubation in 1 M NaOH for 10 min. The activity of these fractions was determined using a gamma counter (Biomedex). Protein concentration in each well was determined using the Pierce BCA Protein Assay Kit (Amersham) on the internalised fractions collected. Results were calculated as a percentage of added radioactivity and normalised to protein concentration. The experiment was repeated three times.

PET scanning and biodistribution
All animal experiments were performed with approval from the Peter MacCallum animal ethics committee. Sixto eight-week-old Balb/c nude mice (Animal Resources Centre, Western Australia) were implanted subcutaneously on the right flank with three million AR42J cells (sourced from ATCC). Once the tumours reached a volume >150 mm 3 , the animals (n = 3) were injected intravenously with 23-28 MBq [ 68 Ga(THP-TATE)] (containing 1 μg of THP-TATE). For blocking studies, animals (n = 3) were coinjected with Tyr 3 -octreotate peptide (400 μg). For [ 68 Ga(DOTATATE)], the animals (n = 3) were injected with 8 MBq of the tracer (containing 1 μg of DOTA-TATE). At 1 h, the animals were anaesthetised and imaged on a Philips MOSAIC small animal PET scanner. The images were reconstructed using a 3D RAMLA algorithm and tracer uptake determined as described previously [41]. On completion of the scan, animals were euthanised and tissues harvested, weighed and radioactivity counted using a gamma counter (Biomedex). Quantitation of PET images was performed using in-house software (MARVn 3.31). Regions of interest were drawn around tissues of interest and uptake ratio calculated as the maximum pixel intensity in the tumour divided by the average uptake in a mediastinal background region, liver or kidneys, as appropriate.

Synthesis and radiolabelling of THP-TATE
Reaction of the bifunctional chelator THP-NCS (Chart 1) with H 2 N-PEG 2 -Lys(iv-Dde) 5 -TATE under microwave conditions resulted in the facile formation of THP-PEG 2 -Lys(iv-Dde) 5 -TATE. Removal of the iv-Dde group from the Lys 5 side-chain resulted in the formation of THP-TATE (Chart 1).
The new THP-TATE peptide conjugate could be radiolabelled with generator-produced eluate that was added directly from the generator or eluate that was preconditioned to concentrate activity and remove any contaminating 68 Ge [16]. In both cases, 68 Ga 3+ in 1 mL HCl solution was added to THP-TATE (10 nmol) at ambient temperature, followed immediately by addition of aqueous ammonium acetate and saline to obtain solutions of pH 5-7, which were then immediately subjected to ITLC and HPLC analysis. This synthetic protocol reproducibly provided the labelled conjugate [ 68 Ga(THP-TATE)] in >95 % radiochemical yield (with <5 % attributable to unchelated 68 Ga 3+ ) and in the case where a generator eluting 750-1000 MBq was utilised, specific activities of 60-80 MBq nmol −1 . Using lower quantities of THP-TATE (5 nmol) resulted in radiochemical yields of 80-90 %, indicating that for every 1 mL solution containing 68 Ga 3+ , at least 25 μg of THP-TATE is required to reliably achieve radiochemical yields >95 %. Without addition of ammonium acetate solution, radiolabelling of THP-TATE was not observed: complex formation did not occur in highly acidic solutions (such as in the final solution isolated after preconditioning the eluate (0.9 M HCl) or that used to elute the generator (0.1 M HCl)).
HPLC and LCMS analyses of the analogous nonradioactive [ nat Ga(THP-TATE)] compound were undertaken to verify the identity of the radiolabelled product. Only a single product was observed in the total ion chromatogram of the LCMS of [ nat Ga(THP-TATE)]. Only two signals were observed in the resulting mass spectrum, corresponding to the dipositive and tripositive ions of [ nat Ga(THP-TATE)] (Fig. 1, inset). Under the HPLC conditions employed, [ 68 Ga(THP-TATE)] possessed a retention time (RT) of 20.23 min (sodium iodide scintillation detection) (Fig. 1, red trace). Nonradioactive [ nat Ga(THP-TATE)] possessed a RT of 20.17 min (UV detection at 220 nm) (Fig. 1, blue trace), with the difference in retention times a result of the configuration of the detectors in series. The co-elution of the non-radioactive and radioactive Ga 3+ -labelled peptides was indicative of the formation of a single radiolabelled product (>95 % radiochemical purity) where the Ga 3+ :THP-TATE stoichiometry = 1:1.

Lipophilicity and serum stability studies
The log P OCT/PBS of [ 68 Ga(THP-TATE) measured −3.20 ± 0.09 (n = 6), almost 0.5 units higher than that of [ 68 Ga(DO-TATATE)] which possesses a log P OCT/PBS of −3.69 [42], indicating that the Ga 3+ -coordinated THP complex is significantly more lipophilic than the DOTA complex.
Serum stability studies were undertaken to determine whether [ 68 Ga(THP-TATE)] releases 68 Ga 3+ to endogenous serum proteins. Addition of generator-produced 68 Ga 3+ to a solution of human serum resulted in 68 Gabound protein adducts that possessed distinct retention times of 6.6, 10.4 and 13.8 min when applied to the sizeexclusion HPLC column utilised in this study (Fig. 2a). Radiolabelled [ 68 Ga(THP-TATE)] possessed a retention time of 31.8 min (Fig. 2b). After incubation of [ 68 Ga(THP-TATE)] in fresh human serum at 37°C for 5 h, the sizeexclusion chromatogram exhibited a strong signal at the same retention time of [ 68 Ga(THP-TATE)] (>98 % integration), as well as small signals between 5 and 15 min (<2 % integration), indicating that less than 2 % of 68 Ga 3+ bound to THP-TATE underwent transchelation to serum proteins (Fig. 2c) during 5 h.

In vitro cell binding and internalisation of [ 68 Ga(THP-TATE)]
To assess the internalisation of [ 68  internalised radioactivity was quantified (Fig. 3). After a 60-min incubation, <4 % of added radioactivity/mg of protein (%AR mg −1 ) was bound to the cell surface, but over 40 %AR mg −1 was internalised. Indeed, at all time points, surface-bound activity measured <10 %AR mg −1 whilst internalised activity increased over the course of the 60-min experiment. A427-7 cells were also co-incubated with [ 68 Ga(THP-TATE)] and an excess of unconjugated Tyr 3 -octreotate (TATE, 200-fold excess compared to THP-TATE) peptide to determine SSTR2-specific uptake [41]. At all time points, internalised and surface-bound activity measured <1 %AR mg −1 (Fig. 3). Lastly, [ 68 Ga(THP-  In PET scans of animals administered [ 68 Ga(THP-TATE)] (Fig. 4a), the tumour of each animal could be clearly delineated, as well as the kidneys. The tumour to background (mediastinum), liver and kidney ratios are listed in Table 1. Excretion was largely renal, with significant amounts of activity in the bladders of all animals at 1 h PI. In contrast, tumours in animals co-administered TATE peptide could not be delineated. Animals administered [ 68 Ga(DOTATATE)] exhibited higher tumour to kidney, tumour to liver, and tumour to background ratios than those of [ 68 Ga(THP-TATE)] (Table 1).

Discussion
The work described here demonstrates that with suitable design of chelators-in this case, the tripodal hexadentate THP chelator-to facilitate extremely fast chelation under mild conditions and low ligand concentration, rapid kit-based synthesis of 68 Ga radiopharmaceuticals is  readily achievable and can be performed in a few minutes using a generator, a kit vial, a syringe and appropriate shielding. This has the potential to greatly increase the availability of 68 Ga radiopharmaceuticals for the benefit of more hospitals and patients. Several methods for radiosynthesis of [ 68 Ga(DOTA-TATE)] have been reported, and although radiochemical yields of between >99 and 95 % can be obtained (obviating a post-synthetic purification step), all require 5-10min reaction time at 80-100°C [11][12][13][14][15][16][17][18] or microwave heating for 1 min at 90°C [11] with pH 3-5 ( Table 2). Radiochemical syntheses typically require between 7 and 30 nmol of DOTATATE (or DOTATOC), although in the case of microwave heating, 0.5-1 nmol of conjugate is sufficient for quantitative radiolabelling [11][12][13][14][15][16][17][18].
In contrast, radiosynthesis to produce [ 68 Ga(THP-TATE)] in specific activities sufficient for in vivo administration could be undertaken in <2 min, at room temperature and formulated to pH 6-7 at the same time the reaction occurs ( Table 2). Under the conditions employed here, it is possible that the rate of reaction of 68 Ga 3+ with THP-TATE is limited only by the rate of diffusion of components in the reaction mixture. Provided 25 μg (equivalent to 10 nmol) of THP-TATE is utilised, radiochemical yields >95 % are routinely achievable. The specific activities achieved (60-80 MBq nmol −1 ) are comparable to specific activities achieved in the clinical production of [ 68 Ga(DOTATATE)].
Several other chelators are capable of achieving nearquantitative radiochemical labelling at room temperature. NOTA/NODAGA conjugates can be radiolabelled at room temperature in radiochemical yields in excess of 95 % at pH 3.5-4 within 10 min [19]. The DEDPA chelator can similarly be radiolabelled in excess of 97 % yield at pH 4.5 in 10 min at nmol levels [31,32]. Whilst TRAP and its derivatives have typically been labelled at elevated temperatures in order to achieve extraordinarily high specific activities, at pH 3.3, near quantitative-radiolabelling (~95 %) can be achieved at μM concentrations in 10 min at room temperature [24]. The advent of bifunctional tris(hydroxypyridinone) chelators increases the pH range at which biomolecules can be radiolabelled at room temperature, permitting labelling at neutral pH and hence 68 Ga PET imaging of fusion proteins, antibody fragments and other proteins that are sensitive to extremes of heat and pH.
Whilst THP-TATE could be labelled using unprocessed, fractionated eluate directly from the generator, a post-processing method to remove any 68 Ge (as required in radiopharmaceutical preparations from some 68 Ge/ 68 Ge generators) that ultimately provided an ethanolic solution (18 % ethanol in aqueous HCl solution) of 68 Ga 3+ was utilised in preparations of [ 68 Ga(THP-TATE)] for in vivo experiments [16]. Radiochemical yields of [ 68 Ga(THP-TATE)] prepared using such solutions were high, heating and post-purification were not required, and the final formulation was suitable for injection into mice ( Table 2).
The bifunctional chelator THP-NCS provides a facile synthetic route to peptide conjugates bearing tris(hydroxypyridinone) chelators [35]. The THP chelator is significantly larger than DOTA, and previous work has suggested that increasing the distance between the THP chelator and the targeting peptide leads to increased receptor affinity [35]. A PEG linker was included in the THP-TATE conjugate to circumvent potential deleterious effects the close proximity of the THP group might exert upon the conjugate affinity for SSTR2 receptors. Synthesis from the Lys(iv-Dde) 5 derivative, PEG 2 -Lys(Dde) 5 -TATE, ensured selective attachment of the isothiocyanate, THP-NCS, to the N-terminus of the peptide.
Less than 2 % of 68 Ga(DOTATATE)] is comparable (11.5 ± 0.6 vs 14.4 ± 0.8 %ID g −1 ) but [ 68 Ga(THP-TATE)] has a longer residence time in the kidney (22.3 ± 4.2 vs 5.6 ± 0.5 %ID g −1 ), higher uptake in the liver (1.4 ± 0.1 vs 0.4 ± 0.04 %ID g −1 ) and higher blood  [35]. Lastly, although we did not detect colloids at the point of HPLC and ITLC analysis, the possibility that unchelated 68 Ga 3+ (<5 %), present in the formulation, resulted in some colloid formation between the point of analysis and the point of in vivo administration, in turn contributing to a small proportion of liver activity in animals administered [ 68 Ga(THP-TATE)], cannot be completely eliminated.
PET scanning experiments and ex vivo biodistribution in animals co-administered TATE peptide (blockade group) demonstrated that TATE peptide effectively blocks SSTR2 receptor binding by [ 68 Ga(THP-TATE)], indicating in vivo specificity of [ 68 Ga(THP-TATE)] for SSTR2. Significantly higher blood and kidney activity in the blockade group was also observed, contrasting most [42,45,46] but not all [47] previous reports that compare preclinical biodistribution of SSTR2 radiotracers in blockade and non-blockade groups of SSTR2-positive tumour-bearing mice. It is possible that the significantly higher blood activity observed in the blockade group compared to the non-blockade group (4.7 ± 0.7 vs 0.6 ± 0.1 %ID g −1 , respectively) is in part a consequence of persistent presence of the radiotracer in circulation in the absence of receptors available for binding, rather than high non-specific organ uptake. In this scenario, higher blood and kidney activity in the blocked group compared to that of the [ 68 Ga(THP-TATE)] group is a result of blocked SSTR2 sites that are no longer able to function as a "sink" for [ 68 Ga(THP-TATE)] [48]. It is also possible that the observed higher blood and kidney activity in the blockade group is a result of slower clearance of [ 68 Ga(THP-TATE)] from circulation via a renal route in the presence of excess TATE peptide.

Conclusions
Simplicity of labelling with minimal need for complex equipment and radiochemical expertise, which is likely to be a key to the wider availability of 68 Ga PET, is afforded by appropriate design of the 68 Ga chelator. The tris(hydroxypyridinone) bifunctional chelator, THP-NCS, provides facile access to the peptide conjugate THP-TATE, which can be radiolabelled with generatorproduced 68 Ga 3+ in high radiochemical yield (>95 %) and specific activities of 60-80 MBq nmol −1 . Radiosynthesis and formulation is rapid (<2 min), proceeds at ambient temperature and simply requires addition of 68 Ga 3+ solution to the conjugate and neutralisation with acetate solution. The resulting tracer, [ 68 Ga(THP-TATE)], specifically binds to SSTR2 and, similar to other agonists of SSTR2, is rapidly internalised. In vivo, [ 68 Ga(THP-TATE)] clears rapidly from circulation, accumulates specifically at SSTR2-positive tumours and is cleared predominantly via a renal pathway. In comparison with [ 68 Ga