Optimization of the Pharmacokinetic Profile of [99mTc]Tc-N4-Bombesin Derivatives by Modification of the Pharmacophoric Gln-Trp Sequence

Current radiolabeled gastrin-releasing peptide receptor (GRPR) ligands usually suffer from high accumulation in GRPR-positive organs (pancreas, stomach), limiting tumor-to-background contrast in the abdomen. In novel N4-bombesin derivatives this was addressed by substitutions at the Gln7-Trp8 site within the MJ9 peptide (H-Pip5-phe6-Gln7-Trp8-Ala9-Val10-Gly11-His12-Sta13-Leu14-NH2) either by homoserine (Hse7), β-(3-benzothienyl) alanine (Bta8) or α-methyl tryptophan (α-Me-Trp8), with the aim of optimizing pharmacokinetics. We prepared and characterized the peptide conjugates 6-carboxy-1,4,8,11-tetraazaundecane (N4)-asp-MJ9, N4-asp-[Bta8]MJ9, N4-[Hse7]MJ9 and N4-[α-Me-Trp8]MJ9, and evaluated these compounds in vitro (GRPR affinity via IC50,inverse; internalization; lipophilicity via logD7.4) and in vivo (biodistribution and μSPECT/CT studies at 1 h post injection (p.i.) in PC-3 tumor-bearing CB17-SCID mice). 99mTc-labeling resulted in radiochemical yields (RCYs) > 95%. All 99mTc-labeled MJ9 analogues showed comparable or higher GRPR affinity than the external reference [99mTc]Tc-Demobesin 4. Receptor-bound fractions were noticeably higher than that of the reference. Despite a slightly enhanced lipophilicity, all novel MJ9 derivatives revealed improved in vivo pharmacokinetics compared to the reference. The Bta8-modified ligand revealed the most favorable tumor-to-abdomen contrast at 1 h p.i. Substitutions at the Gln7-Trp8 site within GRPR ligands hold great potential to modify pharmacokinetics for improved imaging.


Introduction
The field of nuclear medicine continues to grow worldwide, as the importance of non-invasive imaging modalities that bring forth biochemical information on various malignancies is continuously increasing [1,2]. Due to increased life expectancy, the probability of developing cancer will also be higher in the future, which is why the need for personalized medicine will rise as well [3]. Despite growing interest in positron emission tomography (PET) imaging as a consequence of its enhanced sensitivity, single-photon emission computed tomography (SPECT) devices are still more common due to lower acquisition costs, which is especially attractive for smaller nuclear medicine centers, and its widespread application in cardiology [4]. Another advantage of SPECT is the good availability of the generator-produced radionuclide technetium-99m that is perfectly suited for the SPECT technology due to its unique physical properties (half-life of~6 h, E γ~1 42 keV). Not surprisingly, technetium-99m is still the most used radionuclide in nuclear medicine, amounting to up to 80% of all nuclear medicine procedures worldwide [2]. Amongst the various chelators that are available for radiolabeling peptides [5][6][7][8], we selected 6-carboxy- 1,4,8,11-tetraazaundecane (N 4 ) because it stabilizes 99m Tc-complexes that display high stability in vivo, has good hydrophilicity, and has already been applied in preclinical and clinical settings for a variety of target structures [9].

In Vitro Characterization
In vitro data of all 99m Tc-labeled MJ9 derivatives as well as the external reference compound, [ 99m Tc]Tc-Demobesin 4, are summarized in Figure 2 and Table S1. It has to be mentioned that in case of the inverse half maximal inhibitory concentration (IC50,inverse) studies, the respective radiolabeled MJ9 analogue is used at a defined concentration (8.0 nM/well), while the competitor applied (3-I-tyr 6 -MJ9) is used in nonradioactive and in rising concentrations (final concentrations of 10 −11 -10 −5 M/well). Hence, in contrast to conventional IC50 values, higher IC50,inverse values refer to higher (GRPR) affinity, as a higher concentration of 3-I-tyr 6 -MJ9 is required to displace the respective radiolabeled MJ9 derivative from the receptor.

In Vivo Characterization
Biodistribution studies in PC-3 tumor-bearing mice were carried out at 1 h post injection (p.i.) (Table 1, Figure 3).

In Vivo Characterization
Biodistribution studies in PC-3 tumor-bearing mice were carried out at 1 h post injection (p.i.) (Table 1, Figure 3).  All 99m Tc-labeled MJ9 derivatives revealed similarly low activity levels in all organs (<4.5 percent injected dose per gram; %ID/g), except for the GRPR-positive pancreas Data are mean ± SD (n = 3).

Discussion
Due to its unique physical properties and widespread availability, the cost-effective generator-produced radionuclide technetium-99m had and still has a great impact on nuclear medicine. To date, the use of 99m Tc-labeled compounds for single-photon emission computed tomography (SPECT) or SPECT/computed tomography (SPECT/CT) is highly established and used most commonly [2]. For this reason, we decided to design a series of pharmacophore-modified GRPR ligands that allow for easy and rapid 99m Tc-labeling via a 6-carboxy-1,4,8,11-tetraazaundecane (N 4 ) chelator conjugated to the N-terminus, and which show improved tumor-to-abdomen contrast, as bombesin-derived compounds tend to accumulate mainly in the pancreas, but also in the stomach (both GRPR-positive organs) and the intestine [17,22,32,33]. Prior work of our group on 177 Lu-labeled GRPR antagonists with modifications at the Gln 7 -Trp 8 site within the pharmacophore [29] in the biological behavior of bombesin derivatives revealed that the introduction of homoserine (Hse 7 ) and β-(3-benzothienyl) alanine (Bta 8 ) into the MJ9 (H-Pip 5 -phe 6 -Gln 7 -Trp 8 -Ala 9 -Val 10 -Gly 11 -His 12 -Sta 13 -Leu 14 -NH 2 ) peptide might result in increased tumor-to-abdomen ratios. We therefore transferred this concept to [ 99m Tc]Tc-N 4 -conjugated MJ9 derivatives and compared the resulting ligands to the unmodified, as well as the α-methyl-tryptophan (α-Me-Trp 8 )-containing analogue in vitro and in vivo.
Identity of ( t Bu) 4 N 4 was confirmed by 1 H-NMR ( Figure 5, see chapter 4.1). Synthesis via solid-phase peptide synthesis and 99m Tc-labeling of the respective peptides was easily accessible and resulted in RCYs > 95% and molar activities of 33 ± 4 GBq/µmol. In later experiments, we were able to achieve molar activities of up to 60 GBq/µmol by reducing the total volume and adding sodium ascorbate only after labeling. Abiraj

Compound Synthesis and Labeling Procedures
MJ9 derivatives and the non-radioactive competitor 3-I-tyr 6 -MJ9 were prepared via standard Fmoc-based solid phase peptide synthesis (SPPS) using a H-Rink amide ChemMatrix ® resin (35-100 mesh particle size, 0.4-0.6 mmol/g loading, Merck KGaA, Darmstadt, Germany). Purification was accomplished by semi-preparative reversed phase high performance liquid chromatography (RP-HPLC). Compound identity was confirmed via electrospray ionization mass spectrometry (ESI-MS) that was acquired on an expression L CMS mass spectrometer (Advion Ltd., Harlow, UK). 99m Tc-Labeling of N4-conjugated GRPR ligands was carried out by the addition of 1.0 nmol precursor (10 −3 M in Tracepur ® water) to a mixture of 25 µ L of Na2HPO4 (0.05 M in Tracepur ® water, pH = 9.5) and 3.0 µ L of disodium citrate sesquihydrate (0.1 M in Tracepur ® water) in saline. After the addition of a freshly prepared solution of 5.0 µ L of SnCl2 (1 mg/mL ethanol), approximately 40 MBq [ 99m Tc][TcO4] − in saline was added and the labeling solution (final volume of 150-750 µ L) was heated to 90 °C for 10 min. Quality control was performed using radio thin layer chromatography and radio RP-HPLC (radio detection and UV detection at λ = 220 nm). All 99m Tc-labeled MJ9 derivatives revealed high GRPR affinity (IC 50,inverse : 2.4-6.3 nM). We decided to determine inverse IC 50 values, as we wanted to investigate the GRPR affinity of the 99m Tc-labeled compounds rather than the unlabeled precursors. We are convinced that there could be an influence of an unlabeled compared to a labeled chelator moiety, which is why in the case of technetium-99m we prefer inverse IC 50 values. Most groups only determine conventional IC 50 values of the respective unlabeled precursors, a comparison to our IC 50,inverse values is difficult [18,22,32]. Abouzayed et al. reported on equilibrium dissociation constants in the (low) picomolar range, which is also difficult to compare with our IC 50,inverse values [35]. Therefore, we included the well-known GRPR ligand [ 99m Tc]Tc-Demobesin 4 as an external reference compound, which exhibited an IC 50,inverse value of approximately 2.5 nM. Moreover, the non-radioactive competitor (3-I-tyr 6 -MJ9), which revealed an IC 50 value of 1.3 ± 0.4 nM in previous studies [29], showed an IC 50,inverse value of 4.9 ± 0.9 nM for its 125 I-labeled counterpart in this study. We thus concluded that each compound comprised sufficiently high GRPR affinity for further studies in vivo.
Furthermore, we also suggested antagonistic behavior for all novel MJ9 analogues due to their high structural similarity to the well-known GRPR antagonist ([ 68 Ga]Ga-/[ 177 Lu]Lu-) RM2, as well as their low internalization percentage determined in the present study. In contrast, it was reported that the GRPR agonist [ 99m Tc]Tc-Demobesin 4 displays a GRPR-mediated internalization of~75% on PC-3 cells [32], which corroborates with our data.
The substitution of a [ 177 Lu]Lu-DOTA by a [ 99m Tc]Tc-N 4 moiety led to a drastic increase in lipophilicity, as previously developed compounds exhibited logD 7.4 values of ≤−1.8 [36], while the 99m Tc-labeled MJ9 derivatives revealed values in a range of −1.8 to −1.1. By the introduction of a D-aspartate moiety, lipophilicity could be slightly reduced, but it remained elevated compared to [ 177 Lu]Lu-DOTA-conjugated derivatives. However, we considered this lipophilicity sufficient for in vivo studies, as the clinically applied GRPR ligand [ 68 Ga]Ga-/[ 177 Lu]Lu-NeoBOMB1 demonstrated logD 7.4 values of −0.9 and −0.6, respectively [29,37]. We are aware that the introduction of a D-aspartate moiety into the N-terminal linker section could also affect other in vivo parameters, such as cell uptake and metabolic stability, amongst others, as it was observed for a GRPR antagonist that even the change of the radiometal can significantly alter these parameters [38]. , which revealed the second highest GRPR affinity, exhibited the second lowest activity levels in the tumor at 1 h p.i. Moreover, the latter conjugate displayed enhanced activity levels in most non-tumor organs, especially visible in the pancreas. For this reason, the α-Me-Trp-for-Trp 8 substitution within the MJ9 sequence did not lead to improved imaging properties.
A limitation of this study is the absence of competition studies to confirm the tumor (and pancreas) specificity of the novel [ 99m Tc]Tc-labeled MJ9 derivatives. Previous studies revealed GRPR-specific tumor (and pancreas) accumulation for the respective [ 177 Lu]Lu-DOTA-conjugated MJ9 analogues [29,30], as other MJ9 derivatives described in the literature also displayed a GRPR-specific uptake pattern and GRPR affinity was high for all novel [ 99m Tc]Tc-labeled MJ9 derivatives. Moreover, internalization studies revealed that >99% of the applied activity could be blocked by an excess of [ nat Lu]Lu-RM2. For all these reasons, we suggest that there is a high probability that the tumor and pancreas uptake of the novel [ 99m Tc]Tc-N 4 -conjugated MJ9 analogues is GRPR-specific. Nevertheless, this has to be addressed in future studies to confirm.
While all these compounds suffered from increased activity levels in the pancreas, we successfully addressed this issue by substituting the Gln 7 -Trp 8 bond within the pharmacophore of MJ9 analogues either by Hse 7 or Bta 8 to improve tumor-to-abdomen contrast, which is attributed to a more rapid activity clearance from healthy organs. Interestingly, Wang et al. recently reported on a series of bombesin analogues modified at the C-terminus, which resulted in high activity levels in the tumor (~16 %ID/g) and low levels in the pancreas (~2 %ID/g) at 1 h p.i. for one compound ([ 68 Ga]Ga-TacsBOMB5) [26]. The authors suggested that [ 68 Ga]Ga-TacsBOMB5 might be more selective for the human than for the murine GRPR. Such interspecies differences between human and animal GRPR were also observed by Maina et al. [42].
Compared to 99m Tc-labeled PSMA inhibitors such as [ 99m Tc]Tc-MIP-1404, our MJ9 analogues revealed comparable or even higher activity levels in the tumor and noticeably lower levels in the kidneys [10] in tumor-bearing animals at 1 h p.i., which was expected, as the latter organ is PSMA-positive but GRPR-negative. However, in general, off-target accumulation was lower for the PSMA inhibitor than for the GRPR-targeted compounds at 1 h p.i., with the exception of the kidneys. We are aware that the comparison of different tumor models and target structures is difficult. Nevertheless, as particularly [ 99m Tc]Tc-N 4 -asp-[Bta 8 ]MJ9 revealed a favorable biodistribution profile and [ 99m Tc]Tc-MIP-1404 demonstrated high uptake in liver and duodenum in prostate cancer patients, there might be a potential for the GRPR ligand that exhibited improved tumor-to-abdomen contrast preclinically.
In summary, we successfully designed pharmacophore-modified MJ9 analogues for 99m Tc-labeling with comparable or even higher GRPR affinity, low internalization but increased lipophilicity. While biodistribution profiles did not confirm our concerns regarding increased lipophilicity, future compounds with a decreased lipophilicity will show whether the contrast in the abdominal area that was already improved in this study can be optimized even further. We could confirm our hypothesis of improving tumor-to-non-tumor organ ratios in the abdomen at early time points by modifying the pharmacophore of the MJ9 peptide either by Hse 7 or Bta 8 . Due to recent observations in our group for [ 177 Lu]Luconjugated MJ9 analogues with regard to in vivo stability, future studies have to elucidate whether this beneficial clearance effect might also be linked to a decreased metabolic stability of the novel [ 99m Tc]Tc-N 4 -conjugated MJ9 derivatives. Nevertheless, based on the preclinical data determined in this work, [ 99m Tc]Tc-N 4 -asp-[Bta 8 ]MJ9 might be a potential candidate for clinical translation because of its favorable contrast at early imaging time points. In general, bombesin-based tracers could take a complementary role to PSMA inhibitors for prostate cancers and, furthermore, might also be applicable for the imaging of breast cancer and gastrointestinal stromal tumors. It seems plausible to conclude that other GRPR ligands sharing the same pharmacophore (Gln 7 -Trp 8 -Ala 9 -Val 10 -Gly 11 -His 12 ) might also benefit from substitutions at the Gln 7 -Trp 8 site.

Materials and Methods
Further general information and characterization of the non-radioactive competitor (3-I-tyr 6 -MJ9), its radiolabeled analogue (3-[ 125 I]I-tyr 6 -MJ9), all N 4 -conjugated MJ9 analogues and the external reference is provided in the supplemental Material ( Figures S1-S12). Electrospray ionization-mass spectra for characterization of the substances were acquired on an expression L CMS mass spectrometer (Advion Ltd., Harlow, UK). 1 H-NMR-spectra were acquired on an AVHD 400 from Bruker (Billerica, MA, USA) at 300 K. Chemical shifts (δ) are given in parts per million (ppm), spectra are calibrated to the residual 1 H solvent signal of DMSO-d 6 at 2.50 ppm and signal multiplicities are described as: s = singlet, m = multiplet. N-Boc-ethylenediamine (4.0 eq.) was slowly added to a solution of 3-bromo-2-(bromomethyl)propionic acid (1.0 eq.) in THF (25 mL/mmol) and stirred for at least 24 h at room temperature. The solvent was removed under reduced pressure at room temperature and the residue was dissolved in acetone/H 2 O (1/1 (v/v), 25 mL/mmol). After the solution was cooled to 0 • C, triethylamine (3.0 eq.) was added. After 5 min, di-tert-butyl dicarbonate (4.0 eq.) was added and the mixture was stirred for 15 h (0 • C→room temperature). Subsequently, the solvents were removed under reduced pressure and the raw product was purified via reversed phase high performance flash chromatography (35-

Compound Synthesis and Labeling Procedures
MJ9 derivatives and the non-radioactive competitor 3-I-tyr 6 -MJ9 were prepared via standard Fmoc-based solid phase peptide synthesis (SPPS) using a H-Rink amide ChemMatrix ® resin (35-100 mesh particle size, 0.4-0.6 mmol/g loading, Merck KGaA, Darmstadt, Germany). Purification was accomplished by semi-preparative reversed phase high performance liquid chromatography (RP-HPLC). Compound identity was confirmed via electrospray ionization mass spectrometry (ESI-MS) that was acquired on an expression L CMS mass spectrometer (Advion Ltd., Harlow, UK).
99m Tc-Labeling of N 4 -conjugated GRPR ligands was carried out by the addition of 1.0 nmol precursor (10 −3 M in Tracepur ® water) to a mixture of 25 µL of Na 2 HPO 4 (0.05 M in Tracepur ® water, pH = 9.5) and 3.0 µL of disodium citrate sesquihydrate (0.1 M in Tracepur ® water) in saline. After the addition of a freshly prepared solution of 5.0 µL of SnCl 2 (1 mg/mL ethanol), approximately 40 MBq [ 99m Tc][TcO 4 ] − in saline was added and the labeling solution (final volume of 150-750 µL) was heated to 90 • C for 10 min. Quality control was performed using radio thin layer chromatography and radio RP-HPLC (radio detection and UV detection at λ = 220 nm).

In Vitro Experiments
Lipophilicity (logD 7.4 ) and internalization. The determination of both lipophilicity and cell membrane-bound as well as internalized fractions (PC-3 cells, 1.5 × 10 5 cells/mL/well) was carried out with 99m Tc-labeled compounds in analogy to a published procedure [29].
Affinity Determinations (IC 50,inverse ). Competitive binding studies were performed in analogy to a previously reported procedure but with slight modifications [29]. The non-radioactive competitor (3-I-tyr 6 -MJ9) was applied in increasing concentrations (final concentration of 10 −11 -10 −5 M/well), while novel 99m Tc-labeled GRPR ligands were used as radioligand (final concentration of 8.0 nM/well). All experiments were performed in triplicate for each concentration. IC 50,inverse determination for each compound was repeated twice. Data are mean ± standard deviation (SD).

In Vivo Experiments
All animal experiments were conducted in accordance with general animal welfare regulations in Germany (German animal protection act, in the edition of the announcement, dated 18 May 2006, as amended by Article 280 of 19 June 2020, approval no. ROB-55.2-1-2532.Vet_02-18-109 by the General Administration of Upper Bavaria) and the institutional guidelines for the care and use of animals. CB17-SCID mice of both genders and aged 2-10 months (Charles River Laboratories International Inc., Sulzfeld, Germany) were al-lowed to acclimate at the in-house animal facility for at least one week before inoculation was performed. The establishment of PC-3 tumor xenografts was carried out as previously published [29]. Exclusion criteria for animals from an experiment were either weight loss higher than 20%, a tumor size above 1500 mm 3 , an ulceration of the tumor, respiratory distress or a change of behavior. None of these criteria applied to any animal from the experiment. Neither randomization nor blinding was applied in the allocation of the experiments. Health status is SPF according to FELASA.
Imaging studies were carried out according to a recently published protocol [29]. Static images were recorded at t = 1 (anesthesia by 2% isoflurane, n = 1) with an acquisition time of t + (45-60 min) using a high-energy general-purpose rat and mouse collimator via MILabs acquisition software v11.00 and v12.26 from MILabs (Utrecht, the Netherlands).

Conclusions
We demonstrated that our concept of modifying the pharmacophore of 99m Tc-labeled GRPR antagonists at the Gln 7 -Trp 8 results in improved tumor-to-organ ratios in the abdominal area at early time points. In addition, we expect that addressing this dipeptide sequence in bombesin analogues might be a valuable tool to modify pharmacokinetics for both imaging and radioligand therapy.

Patents
A patent application on modified GRPR-targeted ligands with TG and HJW as inventors has been filed (WO2021121734A1).