Influence of a novel, versatile bifunctional chelator on theranostic properties of a minigastrin analogue

Background 6-[Bis(carboxymethyl)amino]-1,4-bis(carboxymethyl)-6-methyl-1,4-diazepane (AAZTA ) is a promising chelator with potential advantages over 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) for radiopharmaceutical applications. Its mesocyclic structure enables fast radiolabelling under mild conditions with trivalent metals including not only 68Ga for positron emission tomography (PET) but also 177Lu and 111In for single-photon emission computed tomography (SPECT) and radionuclide therapy. Here, we describe the evaluation of a bifunctional AAZTA derivative conjugated to a model minigastrin derivative as a potential theranostic agent. Methods An AAZTA derivative with an aliphatic C9 chain as linker was coupled to a minigastrin, namely [AAZTA0, D-Glu1, desGlu2–6]-minigastrin (AAZTA-MG), and labelled with 68Ga, 177Lu and 111In. The characterisation in vitro included stability studies in different media and determination of logD (octanol/PBS). Affinity determination (IC50) and cell uptake studies were performed in A431-CCK2R cells expressing the human CCK2 receptor. μPET/CT and ex vivo biodistribution studies were performed in CCK2 tumour xenograft-bearing nude mice and normal mice. Results AAZTA-MG showed high radiochemical yields for 68Ga (>95 %), 177Lu (>98 %) and 111In (>98 %). The logD value of −3.7 for both [68Ga]- and [177Lu]-AAZTA-MG indicates a highly hydrophilic character. Stability tests showed overall high stability in solution with some degradation in human plasma for [68Ga]- and transchelation towards DTPA for and [177Lu]-AAZTA-MG. An IC50 value of 10.0 nM was determined, which indicates a high affinity for the CCK2 receptor. Specific cell uptake after 60 min was >7.5 % for [68Ga]-AAZTA-MG and >9.5 % for [177Lu]-AAZTA-MG, comparable to other DOTA-MG-analogues. μPET/CT studies in CCK2 receptor tumour xenografted mice not only revealed high selective accumulation in A431-CCK2R positive tumours of 68Ga-labelled AAZTA-MG (1.5 % ID/g in 1 h post injection) but also higher blood levels as corresponding DOTA-analogues. The 111In-labelled peptide had a tumour uptake of 1.7 % ID/g. Biodistribution in normal mice with the [177Lu]-AAZTA-MG showed a considerable uptake in intestine (7.3 % ID/g) and liver (1.5 % ID/g). Conclusion Overall, AAZTA showed interesting properties as bifunctional chelator for peptides providing mild radiolabelling conditions for both 68Ga and trivalent metals having advantages over the currently used chelator DOTA. Studies are ongoing to further investigate in vivo targeting properties and stability issues and the influence of spacer length on biodistribution of AAZTA. Electronic supplementary material The online version of this article (doi:10.1186/s13550-015-0154-7) contains supplementary material, which is available to authorized users.


Background
With the increasing number of applications for peptidebased radiopharmaceuticals, new radiolabelling strategies are of high interest in particular for "theranostic" agents [1], which combine the modalities of therapy and diagnostic imaging [2]. The theranostic principle is unique for radiopharmeuticals by exchanging, for example 68 Ga for diagnostic purposes by 177 Lu as a therapeutic counterpart for the same targeting vector, such as a peptide, thereby translating the diagnostic molecular characterisation into a treatment opportunity for the patient. One challenge in this case is varying labelling properties for different radionuclides. Current chelators like 1,4,7,10tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) have certain limitations [3], in particular providing different coordinations for different metals and requiring high temperatures for radiolabelling. Other chelators such as 1,4,7-triazacyclononane, 1-glutaric acid-4,7 acetic acid (NODAGA) [4] have shown the potential for convenient preparation of 68 Ga-radiopharmaceuticals at room temperature; however, they cannot be used for other trivalent metals, in particular, those for radionuclide therapy applications. A novel and versatile alternative bifunctional chelator for radiopharmaceutical applications is 6-[bis(carboxymethyl)]amino]-6-(9-carboxynonyl)-1,4-diazepane-1,4-diacetic acid, tetra-t-butyl ester (AAZTA) [5]. AAZTA is a heptadentate ligand, based on the perhydro-1,4-diazepine scaffold enabling rapid metal coordination under mild conditions. It was first developed for magnetic resonance imaging (MRI) applications as a chelator for gadolinium [6], but showed significant affinity for several other metal ions [7]. Recently AAZTA has been proposed as potential chelator for 68 Ga and PET applications [8] and has been used for the development of an RGD peptide conjugate as a combined PET/MRI probe radiolabelled with 68 Ga [9].
Advantages of the AAZTA chelator are based on its mesocyclic structure which enables labelling of different radioisotopes at room temperature (RT). This allows application also for heat or oxidation sensitive peptides or other biomolecules.
We were not only interested in the application of AAZTA as bifunctional chelator for potential theranostic applications, allowing radiolabelling with 68 Ga for positron emission tomography (PET), but also other trivalent radiometals such as 111 In for single-photon emission computed tomography (SPECT) and 177 Lu for therapeutic applications. We chose the minigastrin11 (MG11) analogue ([D-Glu 1 , desGlu [2][3][4][5][6] ] − minigastrin, minigastrin = Glu-Glu-Glu-Glu-Glu-Glu-Ala-Tyr-Gly-Trp-Met-Asp-Phe-NH 2 ) as a model targeting sequence which has been widely characterised with other chelators including HYNIC and DOTA [10,11]. Especially, DOTA shows sensitivity towards harsh radiolabelling conditions, in particular, heat due to the easily oxidised Met-residue in the peptide sequence. Minigastrin analogues are also very interesting because of their targeting properties to the cholecystokinin 2 receptor, which is overexpressed in various tumour tissue-like medullary thyroid carcinoma (MTC) and gastrointestinal stromal tumours (GIST). We here report on major characteristics of an AAZTA-MG conjugate, namely [AAZTA 0 , D-Glu 1 , desGlu 2-6 ]-minigastrin (AAZTA-MG), with respect to radiolabelling and in vitro and in vivo properties.

Analytical methods
Reversed-phase high-performance liquid chromatography (RP-HPLC) analysis was performed with an Ul-tiMate 3000 RS UHPLC pump, UltiMate 3000 RS Column Compartment (column oven temperature was set at 25°C), UltiMate 3000 Variable Wavelength Detector (Dionex, Germering, Germany) and a radio detector (GabiStar, Raytest; Straubenhardt, Germany). An ACE 3 μm C 18 150 × 3.0 mm (ACE, Aberdeen, Scotland) with a flow rate of 0.6 mL/min, and UV detection at 220 nm was used. The single runs were performed with the following acetonitrile (ACN)/H 2 O/ 0. Thin layer chromatography (TLC) analysis was performed with silica gel impregnated glass fibre sheets (ITLC-SG stripes; Varian, Lake Forest, CA, USA) as stationary phase and sodium citrate monohydrate (0.21 g/ mL, pH 5) as mobile phase and analysed using TLC scanner (Scan-RAM™, LabLogistic, Sheffield, UK).
The radioactivity of all samples was measured in a 2480 Automatic Gamma counter Wizard 2 3" (Perkin Elmer, Waltham, MA, USA).

Peptide analysis by mass spectrometry
For mass spectrometry a CESI 8000 (Sciex, Brea, CA, USA) equipped with a polyethyleneimine-coated capillary (total length; 100 cm, i.d.; 30 μm, o.d.; 150 μm) was coupled via an ESI module to an LTQ Orbitrap XL ETD (Thermo Scientific, Vienna, Austria) [12]. Peptides were diluted in 0.1 % formic acid and analysed by direct infusion mass spectrometry using a flow rate of 200 nL/min. Spectra were acquired in the Orbitrap at a resolution of R = 60,000; the AGC target value was 1,000,000; maximum ionization time was set to 100 ms.
Amino acid (fivefold excess) coupling was performed by HATU/HOAt activation in NMP at pH 8 to 9 and a reaction time of 1 to 4 h. For the amino acids Asp and Glu, an OtBu side chain protection was applied, whereas for Trp BOC and for Tyr, tBu protection groups were used.
After removing the last Fmoc protection group from the final amino acid, the AAZTA-C 9 chelator CN-AAZTA(tBu) 4 was coupled using a threefold excess of the chelator activated by HATU/HOAt in 10 mL NMP for 10 min at pH >10. The solution was then shaken for 18 h overnight and washed as a last step with NMP.
To cleave the peptide from the resin, a solution of TFA/Triisopropylsilane/H 2 O (v/v/v; 95/2.5/2.5) was added and left to react for 1 h, followed by ether precipitation and a drying step. The resulting peptide was dissolved in 35 % ethanol, purified by RP-HPLC and characterised by ESI-MS (see also Additional file 1).

Radiolabelling
All radiolabelling experiments were performed at RT with reaction times of about 10 min. The radiolabelling yield was determined by radio-HPLC or radio-ITLC.

Stability assay
To verify the stability of radiolabelled AAZTA-MG, tests were performed in the following four different media: ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid (DTPA) solutions in 1000-fold excess over the peptide, human plasma and PBS as control. The labelling solution was diluted to 1 mL and 50 μL of that solution incubated with 150 μL of the challenging solution. After 60, 120 and 240 min, 5 μL of the sample were analysed by TLC with citrate buffer (0.21 g/mL, pH = 5) as mobile phase. The strips were cut into three evenly divided pieces and measured in a gamma counter to determine the free radioligand (solvent front) to labelled peptide (origin) ratio.

Protein binding
For this test radiolabelled peptide solution was diluted to 1 mL with PBS. From this solution, 150 μL were incubated in 850 μL fresh human plasma and in 850 μL of PBS as control. After 30, 60 and 120 min, 25 μL of the solution were analysed by size-exclusion chromatography using MicroSpin G-50 Columns (Sephadex G-50, GE Healthcare, Vienna, Austria). First, the MicroSpin columns were centrifuged at 2000 crf for 1 min to remove the storage buffer, and then, the 25 μL were added and centrifuged again for 1 min with 2000 crf. Both, column (containing the "free" radiolabelled peptide) and eluate (containing the protein-bound fraction), were measured in the gamma counter.
In vitro binding affinities of AAZTA-MG were determined by using CCK2+ cell membranes and 177 Lu-labelled CP04 (a minigastrin analogue with high affinity to the CCK2 receptor) as radioligand incubated with increasing concentrations of the competitor. As competitor AAZTA-MG and [ nat Lu]-and [ nat Ga]-AAZTA-MG were used. The latter were prepared by incubating 10 μg AAZTA-MG with a tenfold molar excess of Lu-chloride in ascorbic acid buffer and 100 μl Ga-bromide in sodium acetate buffer for 10 min, respectively.
The 96-well plates (MulitScreen Filter Plates, HTS) were washed with 250 μL 10 mM TRIS buffered saline (pH 7.3). CCK2 cells were diluted in 20 mM HEPES buffer containing 1 M MgCl 2 -in this hypoosmotic solution the cells were cracked releasing free membranes. Then, 150 μL of cell solution (ca. 8 × 10 5 cells/mL was added into each well and incubated with 50 μL 177 Lu-labelled CP04 (0.97 nM) for 10 min at RT. Hereafter, 50 μL of AAZTA-MG was added at increasing concentrations (0.01-1000 nM), and the plate was incubated for 1 h at 37°C. Unbound radioligand was removed by two washes with 250 μL TRIS buffered saline, and the remaining radioactivity on the filter was measured in the gamma counter. IC 50 values were determined by fitting the percent inhibition data using non-linear curve fitting (Origin 6.1, Northampton, MA, USA).

Internalisation assay
For the determination of the receptor-mediated radioligand uptake, CCK2+ and CCK2− cells were counted to a density of 8 × 10 5 cells, seeded into 6-well plates (Greiner Bio-One) and left to grow overnight at 37°C. On the day of the experiment to each 6-well either 150 μL of PBS/ 0.5 % bovine serum albumin (BSA) solution (total series) or 150 μL blocking solution (1 nM pentagastrin in PBS/ 0.5 % BSA solution, non-specific series) for control were added. Hereafter, 150 μL of the radiolabelled peptide was added (1.3 nM AATZA-MG) into each well and incubated for 1 h at 37°C. To stop the internalisation process medium was removed and the supernatant collected in plastic vials. The wells were washed two times with 1 mL DMEM medium containing 1 % fetal bovine serum, and again, the supernatant was collected in plastic vials (wash fraction).
The cells were rinsed twice with 1 mL ice-cold Glycine-buffer and incubated for 5 min at RT to remove membrane-bound radiolabelled peptide (membranebound fraction). Subsequently, the cells were lysed by addition of two times 1 mL of 2 M NaOH (internalised fraction). All collected solutions were measured in the gamma counter. . Organs (heart, stomach spleen, liver, pancreas, kidneys and intestine), blood and muscle tissue were removed and weighted. Activity of the samples was measured simultaneously in the gamma counter ( 177 Lu as well as 68 Ga) using the respective gamma-energy windows, and the results expressed as percentage of injected dose per gram tissue (% ID/g).
For biodistribution studies in tumour xenograftbearing mice, female athymic BALB/c nude mice (Charles River Laboratories, Sulzfeld, Germany) were used (n = 4 per group). For the induction of tumour xenografts, mice were injected subcutaneously near the front shoulder with 2 × 10 6 cells (CCK2+) and the same amount of CCK2− cells as a control subcutaneously in 100 μL DMEM + 100 μL matrigel. The tumours were allowed to grow until they had reached a volume of 0.3 to 0.6 cm 3 . 1 MBq (~0.3 nmol,~0.4 μg peptide) of either 68 Ga or 111 In-labelled AAZTA-MG (n = 4) was administered retroorbitally. After 60 min, mice were sacrificed, and accumulated radioactivity was determined in organ and tumour tissues and the results expressed as percentage of injected dose per gram tissue (% ID/g).

μPET/CT
The positron emission tomography (PET) and computed tomography (CT) images were acquired with an Albira PET/SPECT/CT small animal imaging system (Bruker Biospin Corporation, Woodbridge, CT, USA).

In vitro characterisation
Distribution coefficient (logD) for [ 68 Ga]AAZTA-MG was −3.6 (±0.18 SD; n = 6) and for [ 177 Lu]AAZTA-MG −3.73 (±0.17 SD; n = 2), respectively. The difference between these two was negligible, which indicates that the peptide labelled with both radiometals is a highly hydrophilic compound. The chelator AAZTA did not dramatically reduce hydrophilicity. Stability tests of [ 68 Ga]AAZTA-MG (n = 3) showed an instability in human plasma and a slight transchelation with EDTA and DTPA. This indicates some release of 68 Ga, which also was observed in the biodistribution study, where blood levels with the 68 Ga-compound were increased. In comparison, [ 177 Lu]AAZTA-MG (n = 3) showed a high stability in PBS, EDTA and human plasma but some transchelation with DTPA (Table 1).
Protein binding in human plasma was significantly higher (p = 0.01 t test) for [ 68 Ga]AAZTA-MG than for [ 177 Lu]AAZTA-MG. The protein-bound fraction increased over time for the 68 Ga-labelled compound from 9.5 to 15.8 %, indicating a slow release of 68 Ga. In contrast to that, the 177 Lu-labelled compound showed 4.5 % protein binding with little variation over time.

μPET-CT imaging
The μPET scans with [ 68 Ga]AAZTA-MG showed good targeting properties for CCK2− receptor-expressing tissue corresponding with the data from biodistribution studies. Radioactivity in kidneys and bladder could be explained by the main renal excretion way. Also, specific uptake in the CCK2+ tumours was observed with no uptake in the CCK2− tumours. Images obtained at three different time points (30, 45, and 60 min p.i.) are shown in Fig. 4. The not only the kidneys and bladder shown were clearly visualized but also the CCK2+ tumour. In contrast, the CCK2− control tumour did not show any signal above background. Over time, washout from kidneys and some hepatobiliary excretion which was predicted from the biodistribution studies can be seen in the images. A dynamic scan over 90 min but only in one slice of PET scan without CT is shown in Fig. 5. High radioactivity accumulation in the gall bladder and the tumour was observed. Kidneys are not shown in this slice, but a distribution in the gastrointestinal tract was seen over time, which also confirms the biodistribution results.

Discussion
Choosing appropriate chelators gives radiopharmaceutical science the opportunity to utilize different radionuclides for diagnosis and to move established radiopharmaceuticals towards therapeutic applications, enabling a theranostic approach. Particularly, the chelator AAZTA shows a novel versatile way of binding trivalent radionuclides, providing mild radiolabelling conditions combined with high-specific activities. So far only few data are available on the properties of AAZTA conjugated biomolecules or peptides. We chose the minigastrin analogue MG11 as a model peptide, since gastrin is an interesting peptide for both diagnostic and therapeutic applications in rare malignant diseases (MTC, GIST) [17][18][19], and this particular peptide was already used in combination with a number of alternative chelating systems. Minigastrin represents a peptide which is sensitive to extensive heating steps, as methionine in its peptide sequence is prone to oxidation leading to loss of receptor binding. In case of the chelator AAZTA, no heating for radiolabelling is required, and the problem of Met-oxidation can be almost eliminated. In our study, all radiolabelling experiments could be carried out at RT whereas for examples for DOTA radiolabelling temperatures of more than 80°C are usually needed.
Coupling of the AAZTA derivative to MG11 was successful and proven by ESI-MS analysis. In this preliminary study, no optimization of the synthesis was performed, which is why relatively low yields were achieved; however, these low yields were sufficient for the initial characterisation of the AAZTA-MG bioconjugate.
Radiolabelling results were overall very good and showed quantitative radiolabelling at high-specific activities for 177 Lu, 68 Ga and 111 In, respectively.
The logD values for [ 68 Ga]AAZTA-MG and [ 177 Lu]-AAZTA-MG indicate a highly hydrophilic character of "Internalised" equates to internalisation ability of AAZTA-MG into the cells, and "Membrane bound" shows the adsorption to the external membrane (membrane-bound fraction); Data are shown as means ± SD the complexes. This high hydrophilicity would typically result in renal excretion of these peptides. Biodistribution and μPET-CT studies, however, revealed partly excretion via the hepatobiliary route. This may be a consequence of the rather lipophilic linker (aliphatic C9 chain) used in the applied AAZTA derivatives or potentially a result of hepatobiliary excretion of certain metabolites, considering the limited metabolic stability of MG11-based peptide conjugates. Overall high stability in different solutions was found for both radiometal labels, with some decomposition in human plasma for [ 68 Ga]AAZTA-MG. This corresponds to the high blood levels seen in the biodistribution study resulting in lower tumour-to-background ratios in the μPET scans. This was also confirmed by the higher protein-binding values of [ 68 Ga]AAZTA-MG as compared to its 177 Lu counterpart. The amount of 68 Ga bound to serum proteins was increasing over time which can be explained by transchelation to transferrin due to a low complex stability. This is in contrast to the findings of Baranjay et al. [20] who reported excellent stability of AAZTA complexes with stability constants (logK) of 22.18 for Ga 3+ and 29.58 for In 3+ , which compares well with the stability constant of 68 Ga-DOTA with 21.3 and 23.9, respectively. Waldron et al. [8] also found excellent stability of [ 68 Ga]-AAZTA complexes labelled at pH 4 and above. However, they also describe unstable "kinetically trapped" side products with low stability towards challenging solutions when labelled at pH 3.3 or lower. We were not able to detect such side products by HPLC with our [ 68 Ga]-AAZTA-MG, radiolabelled at pH 4.5, but cannot fully exclude their presence. Nevertheless, in a recent comparison of different 68 Ga-chelators, AAZTA also showed some trend to release the radiometal in transchelation experiments [21]. Despite the high thermodynamic stability of AAZTA-metal chelates, in comparison to DOTA, our studies indicate limitations in kinetic stability in particular of [Ga]AAZTA complexes.
Therefore, dedicated applications with 68 Ga chelators like DOTA or NOTA may be preferable [22,23]. In contrast to these findings for [ 177 Lu]AAZTA-MG, only limited transchelation towards DTPA was observed, but no indication for in vivo instability was found; also, protein binding was neglectable.
The low IC 50 values of AAZTA-MG showed high affinity for the CCK2 receptor, comparable to other results of MG11 conjugated to DOTA [24]. Also, binding studies with the corresponding nat Ga-and nat Lu-labelled compounds revealed no difference to the "metal-free" peptide. Specific cell uptake after 60-min incubation resulted in high-specific internalisation for both 68 Ga-and 177 Lu-labelled AAZTA-MG conjugates with no significant difference between the two radiometals.
Biodistribution in normal BALB/c mice for 177 Lu-labelled AAZTA-MG showed comparable low retention in the kidneys but higher hepatobiliary excretion to DOTA-MG11 [16]. Biodistribution of 68 Ga-and 111 In-labelled peptide was performed in BALB/c mice and CCK2 tumour xenografted nude mice including μPET studies. They revealed a significant ( 68 Ga p = 0.007; 111 In p = 0.01 t test) difference in accumulation in CCK2+ vs. CCK2− tumours ( Table 2). Accumulation of peptide in the CCK2+ tumour was well above background and allowed to visualize the size of the tumour whereas almost no radioactivity was detected in the CCK2− control tumour. These findings are overall comparable to other μPET scans with DOTA-MG analogues with the exception of a higher uptake in the intestinal tract [22]. However, tumour uptake with 1.74 % ID/g was somewhat lower as for [ 111 In]DOTA-MG11 with 3.04 % ID/g in the same tumour model [16]. This cannot be explained by receptor binding or internalisation behaviour which was comparable for both compounds, therefore probably related to the differences in pharmacokinetics by introducing another bifunctional chelator.
The tumour-to-kidney ratio of 0.9 for 111 In and 0.6 for 68 Ga is also comparable to DOTA conjugated to MG11 [25]. For [ 68 Ga]AAZTA-MG, higher blood levels were observed. This indicates an in vivo instability of the [ 68 Ga]AAZTA complex and correlates with our in vitro tests, in particular, the high protein binding and instability in human blood serum. It also resulted in a tumour-toblood ratio of about 4 with 1.5 % ID/g in CCK2+ tumours as compared to a blood level of 0.4 % ID/g. This is in contrast to Tornesello et al. [25] who reported higher contrast for the [ 68 Ga]DOTA-MG11 over the [ 111 In]DOTA-MG11. For [ 111 In]AAZTA-MG, the tumour-to-blood ratio was considerably higher with a value of 12, which seems slightly lower compared to [ 111 In]DOTA-MG11 in the same tumour model [16]. Again [ 111 In]AAZTA-MG also showed considerable higher excretion via the intestinal tract, which could be explained by the aliphatic linker used for AAZTA conjugation. This could be addressed by modifying the structure of the AAZTA conjugate, e.g., by truncating the spacer length between peptide and chelator to eliminate intestinal activity. Also, conjugation to other