Twins in spirit - episode I: comparative preclinical evaluation of [68Ga]DOTATATE and [68Ga]HA-DOTATATE

Background Recently, an intra-patient comparison demonstrated that the somatostatin (sst) ligand [68Ga]HA-DOTATATE ([68Ga]DOTA-3-iodo-Tyr3-octreotate) provides PET images comparable to or superior to those obtained with [68Ga]DOTATATE. To provide a comprehensive basis for nevertheless observed slight differences in tracer biodistribution and dosimetry, the characteristics of [68Ga]HA-DOTATATE were investigated in a detailed preclinical study. Methods Affinities of natGa-HA-DOTATATE and natGa-DOTATATE to sst1–5 were determined using membrane preparations and [125I]SST-28 as radioligand. Internalization into AR42J cells was studied in dual-tracer studies with [125I]TOC as internal reference. Biodistribution was investigated using AR42J tumor-bearing CD1 mice, and specificity of tracer uptake was confirmed in competition studies by coinjection of 0.8 mg TOC/kg. Results Sst2 affinities (IC50) of [natGa]HA-DOTATATE (1.4 ± 0.8 nM, logP: −3.16) and [natGa]DOTATATE (1.2 ± 0.6 nM, logP: −3.69) were nearly identical. Both compounds displayed IC50 > 1 μM for sst1,3,4, while sst5 affinity was markedly increased for natGa-HA-DOTATATE (102 ± 65 nM vs >1 μM for natGa-DOTATATE). [natLu]HA-DOTATATE and [natLu]DOTATATE showed slightly lower, identical sst2 affinities (2.0 ± 1.6 and 2.0 ± 0.8 nM, respectively) and sst3 affinities of 93 ± 1 and 162 ± 16 nM. Internalization of [68Ga]HA-DOTATATE was tenfold higher than that of [125I]TOC but only sixfold higher for [68Ga]DOTATATE and [177Lu]HA-DOTATATE. While [68Ga]HA-DOTATATE and [68Ga]DOTATATE had shown similar target- and non-target uptake in patients, biodistribution studies in mice at 1 h post injection (n = 5) revealed slightly increased non-specific uptake of [68Ga]HA-DOTATATE in the blood, liver, and intestines (0.7 ± 0.3, 1.0 ± 0.2, and 4.0 ± 0.7 %iD/g vs 0.3 ± 0.1, 0.5 ± 0.1, and 2.7 ± 0.8 %iD/g for [68Ga]DOTATATE). However, sst-mediated accumulation of [68Ga]HA-DOTATATE in the pancreas, adrenals, and tumor was significantly enhanced (36.6 ± 4.3, 10.8 ± 3.2, and 33.6 ± 10.9 %iD/g vs 26.1 ± 5.0, 5.1 ± 1.4, and 24.1 ± 4.9 %iD/g, respectively). Consequently, tumor/background ratios for [68Ga]HA-DOTATATE in the AR42J model are comparable or slightly increased compared to [68Ga]DOTATATE. Conclusions The present preclinical data fully confirm the general biodistribution pattern and excellent in vivo sst-targeting characteristics previously observed for [68Ga]HA-DOTATATE in patients. The effect of slightly enhanced lipophilicity on background accumulation and normal organ dose is compensated by the high uptake of [68Ga]HA-DOTATATE in tumor. Thus, [68Ga]HA-DOTATATE represents a fully adequate, freely available substitute for [68Ga]DOTATATE and, given the superb sst-targeting characteristics of [177Lu]HA-DOTATATE in vitro, potential applicability for sst-targeted PRRT.


Background
The excellent suitability of 68 Ga-labeled octreotide analogs for functional imaging of somatostatin (sst) receptoroverexpressing tumors is well documented throughout the literature and has been thoroughly reviewed in recent publications [1,2] [3]. Recently, [ 68 Ga]DOTANOC ([ 68 Ga] DOTA-Nal 3 -octreotide) has also entered the clinical arena [4]. Due to its high affinity to sst 2 , sst 3 and sst 5 , significantly more and smaller lesions are detected using [ 68 Ga]DOTANOC compared to the sst 2 -selective highaffinity ligand [ 68 Ga]DOTATATE, suggesting it to be the preferred agent for the imaging application [5].
However, in the context of sst-targeted endoradiotherapy using the respective 177 Lu-or 90 Y-labeled peptides, [ 177 Lu]DOTATATE compared favorably to [ 177 Lu] DOTANOC [6]. Due to its high sst 2 selectivity, [ 177 Lu]DOTATATE showed significantly lower uptake/ dose delivered to normal tissues, while there were no significant differences concerning tumor kinetics and mean absorbed tumor dose. Thus, in the context of patient selection for sst-targeted endoradiotherapy with [ 177 Lu] DOTATATE, [ 68 Ga]DOTATATE-PET represents the imaging strategy of choice for an accurate assessment of sst expression levels.
Unfortunately, the availability of DOTATATE is governed by patent restrictions. With the intent to provide a suitable alternative with unchallenged in vivo performance, we recently introduced [ 68 Ga]HA-DOTATATE ([ 68 Ga]high-affinity DOTATATE, i.e., [ 68 Ga]DOTA-3iodo-Tyr 3 -octreotate, Figure 1). In the first in vitro studies, Ga-HA-DOTATATE had shown unchangedly high sst 2 affinity compared to Ga-DOTATATE and increased affinity to sst 5 [7]. Given  HA-DOTATATE-PET revealed slightly enhanced uptake of the high-affinity analog in sst-expressing tissues as well as a marginally increased accumulation in the excretion organs. These characteristics lead to a remarkably uniform performance of [ 68 Ga]DOTATATE and [ 68 Ga] HA-DOTATATE in PET imaging of sst-expressing NETs [8]. However, not unexpectedly, the observed slight increase in specific and non-specific tissue accumulation of [ 68 Ga]HA-DOTATATE leads to somewhat higher organ doses compared to [ 68 Ga]DOTATATE [9]. To provide a basis for understanding the underlying reasons for these differences and thus for the adequate interpretation of our observations in patients, we performed a detailed preclinical evaluation of [ 68 Ga]HA-DOTATATE in comparison to [ 68 Ga]DOTATATE. Envisaging its potential use in peptide receptor radiotherapy, [ 177 Lu]HA-DOTATATE was also included in the in vitro evaluation for a first comparative assessment of its binding and internalization characteristics.

D-Phe Cys
The peptides were eluted applying various gradients of 0.1% TFA (trifluoroacetic acid) in H 2 O (solvent A) and 0.1% TFA in acetonitrile (solvent B) over 15 min at a constant flow of 1 mL/min. Preparative RP-HPLC was performed on the same HPLC system using a Multospher 100 RP 18-5 (250 × 10 mm i.d.) column (CS GmbH, Langerwehe, Germany) at a constant flow of 5 mL/min. UV detection was performed at 220 nm using a 206 PHD UV-vis detector (Linear™ Instruments Corporation, Reno, NV, USA). For radioactivity measurement, the outlet of the UV photometer was connected to a NaI(Tl) welltype scintillation counter from EG&G Ortec (München, Germany). Radio-TLC was carried out using a Varian silica impregnated ITLC chromatography paper (Varian Inc., Palo Alto, CA, USA) and a 1:1 (v/v) mixture of 1 M aq. NH 4 OAc and MeOH as mobile phase. TLC strips were analyzed using a miniGita TLC analyzer (Raytest, Straubenhardt, Germany).
Mass spectra were recorded on the LC-MS system LCQ from Finnigan (Bremen, Germany) using a Hewlett Packard series 1100 HPLC system (Agilent Technologies Inc., Santa Clara, CA, USA).

DOTATATE and HA-DOTATATE
DOTATATE and HA-DOTATATE were synthesized according to a previously published protocol [10]. Briefly, the sequences H 2 N-D-Phe-Cys(Trt)-Tyr(tBu)-D-Trp-Lys (Dde)-Thr(tBu)-Cys(Trt)-Thr(tBu)-OH (DOTATATE) and H 2 N-D-Phe-Cys(Trt)-3-iodo-Tyr-D-Trp-Lys(Dde)-Thr(tBu)-Cys(Trt)-Thr(tBu)-OH (HA-DOTATATE) were assembled on 2-CTC resin using a standard Fmoc protocol. After cleavage from the solid support using TFA/ H 2 O/TIBS (triisobutylsilane) (95/2.5/2.5 (v/v/v)), the Lys 5 (Dde)-protected peptides were cyclized using H 2 O 2 in an aqueous THF (tetrahydrofurane) solution at pH 7. DOTA coupling was carried out in aqueous DMF (dimethylformamide) solution using unprotected DOTA and NHS and EDCI as coupling reagents. Subsequent removal of the Lys 5 (Dde) protecting group was carried out using 2% of hydrazine hydrate in DMF. Crude peptides were then purified via preparative HPLC. Ga labeling 68 Ga-labeling of DOTATATE and HA-DOTATATE (5 nmol) was carried out in a fully automated, GMPcompliant procedure using a Scintomics Gallelut + module (Scintomics, Fürstenfeldbruck, Germany) [11,12]. Briefly, a 68 Ge/ 68 Ga generator with SnO 2 matrix (iTHEMBA LABS, South Africa) was eluted with 1.0 M aq. HCl, and in order to minimize the reaction volume, a fraction of 1.25 mL containing approx. 80% of the eluted 68 Ga activity was used for the labeling reaction. HEPES (3.7 M, 800 μL) was used to adjust the pH of the reaction mixture to pH 3. After heating to 95°C for 5 min, the reaction mixture was passed through a SPE cartridge (SepPak® C18, Waters, Eschborn, Germany). The cartridge was washed with water and the 68 Ga-labeled peptides were eluted in a small volume of ethanol. For in vitro studies, the product fraction was diluted with PBS and used as such for further dilutions. For the in vivo biodistribution studies, the ethanol content of the product solution was evaporated in vacuo.
For quality control, radio-TLC was carried out using a Varian silica impregnated glass fiber TLC chromatography paper (Agilent Technologies, Waldbronn, Germany) and a 1:1 (v/v) mixture of 1 M aq. NH 4 OAc and MeOH as mobile phase. TLC strips were analyzed using a BioScan TLC analyzer (BioScan, Tucson, AZ, USA). Lu as calculated from the specific activity at the day of the experiment) was added. The mixture was diluted with NH 4 OAc (0.1 M) to a total volume of 200 μL (pH = 5 in the final reaction mixture) and heated to 95°C for 30 min. Upon cooling to room temperature and determination of the labeling yield using Radio-TLC, the labeling mixture was directly diluted with assay buffer to a final peptide concentration of 20 nM and used as such for the subsequent in vitro studies.
Radioiodination of (Leu 8 ,D-Trp 22 ,Tyr 25 )-somatostatin-28 and Tyr 3 -octreotide Radioiodination of (Leu 8 ,D-Trp 22 ,Tyr 25 )-somatostatin-28 and Tyr 3 -octreotide (TOC) was carried out using the IodoGen® method. Briefly, 200 μg of peptide were dissolved in 0.5 mL TRIS iodination buffer (25 mM Tris-HCl, 0.4 M NaCl, pH 7.5) and transferred to an Eppendorf reaction tube coated with 150 μg of IodoGen®. Upon addition of [ 125 I]NaI (18 to 20 MBq, Hartmann Analytik, Braunschweig, Germany), the reaction vessel was briefly vortexed, and the labeling reaction was allowed to proceed for 15 min at RT. The peptide solution was then removed from the insoluble oxidizing agent. Separation of the radioiodinated peptides from unlabeled precursor was achieved using gradient RP-HPLC (gradient: 22% to 37% solvent B within 20 min, flow: 1 mL/min). For the subsequent in vitro studies, the respective HPLC product fraction was used as such and diluted to the required concentration using assay buffer.
In vitro studies Determination of IC 50 using cell membranes Cell membrane preparations of cells expressing the human sst 1-5 were obtained from Millipore (Schwalbach, Germany) and PerkinElmer (Rodgau, Germany). Competition binding studies were performed in close analogy to the manufacturer's protocol. Briefly, solutions of unlabeled competitor ( nat Ga-DOTATATE, nat Lu-DOTATATE, nat Ga-HA-DOTATATE, nat Lu-HA-DOTATATE; 10 μL, concentration range from 1 × 10 −11 to 1 × 10 −4 M, n = 3 per concentration and peptide) in binding buffer (50 mM HEPES, 5 mM MgCl 2 , 1 mM CaCl 2 , 0.2% BSA, buffered to pH 7.4 using 1 N NaOH) were pipetted into a 96-well plate. To each well, 10 μL of (Leu 8 ,D-Trp 22 ,[ 125 I]Tyr 25 )somatostatin-28 in binding buffer (approx. 100,000 cpm) were added. Then, 5 μg of cell membrane in 80 μL of binding buffer were added per well and incubated for 60 min at RT. The final radioligand concentration in the incubation mixture was 0.42 nM. Membranes were then aspired onto a glass fiber filtermat (Printed Filtermat B, Wallach, Turku, Finland), which had been preconditioned with 0.33% polyethylenimine for 30 min and then washed five times with 300 μL (per well) of wash buffer (50 mM HEPES, 500 mM NaCl, 0.1% BSA, buffered to pH 7.4 using 1 N NaOH), using a Mach II M Harvester 96 (Tomtec CE, Etten-Leur, The Netherlands). Membranes were washed ten times with 150 μL (per well) of wash buffer. The filtermat was then cut into squares containing the membrane from the respective wells, and membrane bound activity was counted in a γ-counter. IC 50 values were calculated from bound activity [cpm] using PRISM 6 software (Graph Pad Software, San Diego, CA, USA).
To avoid the influence of inter-experimental variations in cell count and cell viability on absolute internalization data, all internalization experiments were performed as dual-tracer studies using [ 125 I]TOC as an internal reference. Experiments were carried out as previously described with minor modifications [14]. Briefly, after preconditioning of the cells (approx. 200,000 cells/well) with 200 μL of assay medium for a minimum of 15 min, 25 μL (per well) of either assay medium (total internalization) or 50 μM unlabeled TOC (nonspecific internalization) were added, followed by the addition of 25 μL of assay medium containing a mixture of the respective 68 Ga/ 177 Lu-labeled DOTA-peptide and [ 125 I]TOC, respectively. Final concentrations of 68 Ga/ 177 Lu-peptide and [ 125 I]TOC in the assay medium were 1 nM ( 68 Ga)/2 nM ( 177 Lu) and 0.1 nM, respectively, and were calculated based on the specific activities of the radioligands used. After incubation at 37°C for different time points up to 60 min, the incubation medium was removed, and cells were rinsed with 250 μL of unsupplemented medium. The combined medium fractions represent the amount of free radioligand. Receptor bound (acid releasable) radioactivity was then removed using 2 × 250 μL of ice cold acid wash buffer (0.02 M NaOAc buffered with AcOH to pH = 5). The internalized activity was released by incubation with 250 μL of 1 N NaOH, transferred to vials and combined with 250 μL of PBS used for rinsing the wells. Quantification of the amount of free, acid-releasable, and internalized activity was performed in a γ-counter. Data for total internalized ligand (mean ± SD) were corrected by non-specific internalization (mean ± SD) at the respective time point using Excel considering the laws of error propagation.

AR42J tumor model
To establish tumor growth, AR42J cells were detached from the surface of the culture flasks using 1 mM EDTA in PBS, centrifuged, and resuspended in serum-free culture medium. Concentration of the cell suspension was 2.5 to 5 × 10 6 cells/100 μL. Nude mice (CD-1 nu/nu, female, 6 to 8 weeks, from Charles River WIGA GmbH, Sulzfeld, Germany) were injected with 100 μL of the cell suspension subcutaneously into the flank. Ten days after tumor transplantation, all mice showed solid palpable tumor masses (tumor weight 20 to 90 mg) and were used for the experiments.

Biodistribution studies
The 68 Ga-labeled peptides, 1.1 to 1.7 MBq (depending on the respective specific activity of the radiotracer; the peptide amount per mouse was kept constant at 25 pmol) in 100 μL of PBS (pH 7.4), were injected i.v. into the tail vein of nude mice bearing an AR42J tumor. For competition studies, 20 μg TOC (0.8 mg/kg) were coinjected with the radioligands. The animals (groups of 4 to 5) were sacrificed 1 h post injection and the organs of interest were dissected. The radioactivity was measured in weighted tissue samples using a γ-counter. Data are given in %iD/g and are means ± SD.

Radiolabeling
The synthesis of [ 68 Ga]DOTATATE and [ 68 Ga]HA-DOTATATE was performed according to an optimized protocol, yielding both tracers in >99% radiochemical purity and specific activities (SA) of 62 ± 11 and 68 ± 10 GBq/μmol, respectively. [ 177 Lu]HA-DOTATATE was obtained in the same radiochemical purity but with a lower SA of 38 GBq/μmol due to the comparably low specific activity of the 177 Lu used.
Radioiodination via the IodoGen® method afforded the reference ligands [ 125 I]TOC and [Leu 8 ,D-Trp 22 ,[ 125 I]Tyr 25 ]somatostatin 28 in radiochemical purities >99% and yields of 68 ± 4% and 54 ± 5% after HPLC purification, respectively. Since the HPLC conditions applied allowed very efficient separation of the radioiodinated products from the unlabeled precursors (Δt R ≥ 5 min) and no co-eluting carrier peak was observed in the labeled peptide peak in the quality control UV chromatograms, the specific activity of [ 125 I]TOC and [Leu 8 ,D-Trp 22 ,[ 125 I]Tyr 25 ]-somatostatin 28 was assumed to be that of the radioiodide used for their preparation (≥74 GBq/μmol).

Lipophilicity
The lipophilicities of the respective 68 Ga-and 177 Lucomplexes of DOTATATE and HA-DOTATATE are summarized in Table 1. As expected, iodine-forhydrogen substitution in Tyr 3 leads to an increase in lipophilicity of the [ 68 Ga/ 177 Lu]HA-DOTATATE analogs compared to their respective DOTATATE counterparts. Furthermore, both 177 Lu-labeled compounds display an enhanced lipophilicity compared to their corresponding 68 Ga analogs. This was also anticipated based on the documented different geometries of the 68 Ga-DOTA and the 177 Lu-DOTA chelates. While all carboxylate pendant arms of DOTA (in addition to the DOTA-peptide-amide bond) are involved in the 177 Lu-DOTA complex for complete saturation of the coordination sphere of 177 Lu, one of the carboxylate pendant arms is free in the 68 Ga-DOTA complexes [15].
In vitro studies IC 50 studies using membrane preparations (sst [1][2][3][4][5] Affinities (IC 50 in nM) of the respective nat Ga and nat Lu complexes of DOTATATE and HA-DOTATATE as well as nat Y-HA-DOTATATE for hsst [1][2][3][4][5] are summarized in Table 2. Data were determined using membrane preparations of CHO cells stably transfected with the respective sst subtype. Since in our earlier studies [7,8] only preliminary results (n = 2 experiments) had been published, further experiments were performed to validate these data.
In comparison to the previously published affinities, absolute IC 50 values are slightly increased when averaging Partition coefficients (logP O/PBS ) of the radioligands between PBS (pH 7.4) and n-octanol were determined using a shake flask method (n = 6) [13].
the full data set. However, it was confirmed that nat Ga-DOTATATE and nat Ga-HA-DOTATATE as well as the corresponding nat Lu pair, respectively, have identical and high hsst 2 affinities, with slightly lower affinities of the nat Lu-peptides (as well as nat Y-HA-DOTATATE) compared to the corresponding nat Ga analogs. This observation is in agreement with previous studies by Reubi et al. [16] and Antunes et al. [4] that demonstrated a generally enhanced sst 2 affinity of various sst-targeted nat Ga-DOTA-octapeptides over their respective nat Lu/ nat Y/ nat In analogs. It is important to note, however, that the absolute IC 50 values obtained in the present study using membrane preparations (Table 2) are substantially higher than those obtained by Reubi et al. for nat Ga-DOTATATE and nat Y-DOTATATE using receptor autoradiography (0.20 ± 0.04 and 1.6 ± 0.4 nM, respectively) [16]. The reduced 'sensitivity' of our experimental setup might also explain why nat Ga-DOTATATE and nat Lu-DOTATATE showed virtually no hsst 4 and hsst 5 affinity in this study, while affinities in the range of 200 to 500 nM were observed in the autoradiography assay [16].
In this context, the confirmed substantially enhanced hsst 5 affinities of nat Ga-/ nat Lu-HA-DOTATATE compared to the respective DOTATATE analogs, which represents the rationale for terming these compounds 'high-affinity DOTATATE' (HA-DOTATATE), are even more noteworthy.
Unfortunately, at the time of the second series of experiments, the hsst 3 ChemiSCREEN® membrane (Millipore, Schwalbach, Germany) preparation was not available from the manufacturer anymore, and therefore, the data set for this sst subtype could not be completed under the original experimental conditions. To nevertheless be able to fully evaluate the sst subtype specificities of nat Ga-/ nat Lu-HA-DOTATATE, membrane preparations from CHO-K1 cells transfected with hsst 2-5 were purchased from an alternative supplier (PerkinElmer), and experiments were repeated under experimental conditions identical to the previous experiments in triplicate. Unexpectedly, absolute IC 50 values were found to be generally increased by a factor of 2.5 to 3.5 compared to the values obtained using the ChemiSCREEN® membranes, independently of the hsst subtype assayed. For example, hsst 2A affinities determined for nat Ga-and nat Lu-DOTATATE and nat Ga-, nat Lu-, and nat Y-HA-DOTATATE in this assay were 2.9 ± 0.6, 7.3 ± 2.6, 3.8 ± 1.0, 6.6 ± 1.4, and 6.7 ± 0.1 nM, respectively. Despite the upward shift in absolute IC 50 values, these data very well reflect the relative affinities determined using the ChemiSCREEN® membranes. The same was also observed for the data obtained for hsst [3][4][5] .
Thus, despite the limited numerical compatibility of the two separate data sets, both satisfactorily reflect the important differences in the sst subtype affinity profiles of the different DOTATATE and HA-DOTATATE analogs, namely the substantially enhanced hsst 5 (and hsst 3 ) affinities of the HA-DOTATATE compounds. Therefore, the data in Table 2 were deemed satisfactorily significant to serve as representative values. An improved internalization efficiency of radiolabeled TATE analogs compared to the respective TOC or OC analogs is well documented throughout the literature [14,17,18]. Thus, as expected, all radiometallated TATE analogs in this study showed significantly enhanced internalization compared to the internal standard [ 125 I]TOC. At all time points, internalization efficiency was highest for [ 68 Ga]HA-DOTATATE (1,000 ± 113% of HA-DOTATATE nicely reflects the difference in sst 2 affinity of the compounds (Table 2) and is in accordance with results from the literature [4]. In contrast, the almost doubled internalization of [ 68 Ga]HA-DOTATATE compared to [ 68 Ga]DOTATATE was unexpected, considering their identical sst 2 affinities. However, it has already been demonstrated that internalization efficiency of TATE analogs does not necessarily correlate with sst 2 affinity [14,19]. Furthermore, the fact that the sst 2 affinities were determined in membrane preparations expressing the human receptor, while internalization studies were carried out using rat-sst 2expressing AR42J cells, might also contribute to the discrepancies observed between binding and internalization data ( Figure 2).   tumor-bearing mice fully mirror the observations made in our previously published comparative PET imaging and dosimetry studies in patients [7][8][9]. Due to its enhanced internalization efficiency, [ 68 Ga]HA-DOTATATE shows high uptake in sstexpressing mouse tissues such as the stomach, adrenals, pancreas, and the AR42J tumor xenografts (increased by a factor of 1.4 to 2 compared to [ 68 Ga]DOTATATE) [21]. Sst specificity of tracer uptake in these organs was confirmed in a competition study by coinjection of an excess of unlabeled TOC (Table 3 and Figure 3). Interestingly, sstmediated tracer accumulation was also observed in the lung, spleen, and intestines both for [ 68 Ga]DOTATATE and [ 68 Ga]HA-DOTATATE, underlining the suitability of both radioligands for the sensitive in vivo detection of even low sst expression levels. Of the two compounds investigated, [ 68 Ga]HA-DOTATATE showed a slightly higher proportion of non-blockable tissue uptake in the competition study, reflecting the higher blood activity concentration of this compound under the experimental conditions.

Biodistribution studies
Again, the preclinical mouse data obtained in this study parallel the findings from our human PET studies, where [ 68 Ga]HA-DOTATATE showed somewhat improved  sst-targeting efficiency compared to [ 68 Ga]DOTATATE, reflected by a slight increase in SUV mean for the spleen and pituitary [8]. However, while the mouse biodistribution data in this study reveal fundamental differences between both the general biodistribution and sstmediated tissue accumulation of [ 68 Ga]HA-DOTATATE and [ 68 Ga]DOTATATE, respectively, these opposed effects were not observed to the same extent in the human studies. For example, tracer uptake (SUV mean ) in normal organs such as the liver and kidney as well as in primary tumors and their metastases were found to be nearly identical for both radioligands in patients [8].
Obviously, tissue distribution of [ 68 Ga]HA-DOTATATE and [ 68 Ga]DOTATATE in humans is mainly dominated by the very similar tracer pharmacokinetics of both compounds, whereas in mice, the respective influence of both the increased sst-targeting efficiency and of the slightly enhanced lipophilicity of [ 68 Ga]HA-DOTATATE on tracer biodistribution is much more pronounced. Interestingly, the comparable in vivo performance of [ 68 Ga]HA-DOTATATE and [ 68 Ga]DOTATATE in patients is well reflected by the very similar tumor-tobackground ratios (Figure 4) obtained for the two compounds in the AR42J xenograft model. Here, the high sst-targeting efficiency of [ 68 Ga]HA-DOTATATE is counterbalanced by the enhanced tracer accumulation in non-target organs occasioned by the delayed clearance kinetics of [ 68 Ga]HA-DOTATATE. It is important to note that, given the early time point after tracer injection (1 h p.i.), these data certainly do not display the full sst-targeting potential of [ 68 Ga]HA-DOTATATE. Its increased blood activity levels at 1 h p.i. may lead to improved bioavailability and even higher tumor uptake at later time points, which might prove advantageous for PRRT using [ 177 Lu]HA-DOTATATE. However, longer circulation times also might entail increased hematotoxicity, and therefore, it needs to be carefully evaluated in preclinical therapeutic studies, to what extent the differences between [ 177 Lu]DOTATATE and [ 177 Lu]HA-DOTATATE will have an impact on the therapeutic effects of the compounds.

Conclusions
The previous observations from our patient studies demonstrating nearly identical performance of as suggested by affinity and internalization data, may have potential for targeted radiotherapy using, e.g., 177 Lu. To assess its suitability for this application, however, careful evaluation taking into account the slightly altered pharmacokinetics of HA-DOTATATE compared to DOTATATE and the potential risks associated therewith is warranted.