The effects of novel macrocyclic chelates on the targeting properties of the ⁶⁸Ga-labeled Gastrin releasing peptide receptor antagonist RM2

The overexpression of peptide receptors in various types of tumors is becoming more evident, leading to a growing interest in their potential application in cancer diagnosis or targeted therapy [1]. One prominent example is the gastrin-releasing peptide receptor (GRPr), which is predominantly expressed mainly in human tumors such as prostate [2, 3], breast [4, 5] and gastrointestinal stromal tumors [6] and small-cell lung cancer [7]. Due to the benefits of targeting tumors with good vascular permeability and rapid access, the use of peptide ligands for radionuclide targeting of GRPr has been extensively studied for both imaging and therapy purposes [8].

The GRPr, also referred as bombesin receptor subtype 2 (BB2), is part of the G protein-coupled family of bombesin receptor, which includes neuromedin B receptor (NMBR/BB1), and bombesin receptor subtypes 3/4 (BB3/BB4). Of the GRPr-targeting peptide ligands, bombesin and its derivatives have gained considerable attention due to their high affinity. As a result, radiolabeled analogs of these BN-like peptides could be used on the imaging and treatment of GRPr-expressing tumors [9]. BN-based antagonists have demonstrated superior in vivo performance compared to agonists, with improved biodistribution, targeting capabilities, and other attributes [10‐14]. A small peptide called RM2, with the chelator DOTA coupled to D-Phe-Gln-Trp-Ala-Val-Gly-His-Sta-Leu-NH2 via the cationic spacer 4-amino-1-carboxymethyl-piperidine (referred to as DOTA-RM2 in this study), has shown to be one of the potent antagonists with excellent pharmacokinetic properties, indicating that gallium-68 labeled DOTA-RM2 may serve as a good candidate for positron emission tomography (PET) imaging [15]. Currently, GRPr imaging appears to be complementary to prostate-specific membrane antigen (PSMA) imaging in preclinical studies and preliminary human studies in patients with primary limited prostate cancer [16, 17]. Several studies have shown that in recurrent prostate cancer, RM2-PET imaging may be more beneficial in patients with negative findings on conventional imaging, compared to PSMA-PET [18‐20]. In addition, RM2-like drugs expected to be used for treatment have also been extensively studied and reported in recent years [21, 22].

It is well known that the pharmacokinetics and targeting potency of small peptides can be altered by structural modifications. Chelating agents for linking radionuclides are essential for targeting-based polypeptide conjugates. However, the use of different macrocyclic chelators had a profound influence on the biodistribution profile of the radiolabeled conjugates [23, 24]. This is because chelating agents may affect specific interactions between drug molecules and receptors as well as off-target activity. It might be argued that the chelator changes the overall charges and even affects the local charges, lipophilicity, and preferred conformation of the radiolabeled peptide [25, 26]. Therefore, it is not surprising that biodistribution and targeting properties may be significantly affected, as shown for many short peptides [27, 28].

Herein, we investigated in vivo imaging performance based on the targeting polypeptide moiety of the GRPr antagonist RM2 and a new type of chelating group (AAZTA⁵, DATA^5m) of the resulting radioligand. In general, acyclic chelators are considered to have the characteristics of fast radiolabeling kinetics. The downside is the poor stability of the formed complexes [29]. In contrast, macrocyclic chelator-metal complexes are particularly stable, but the formation of complexes often requires high temperatures and consumes a longer period. The hybrid chelator AAZTA and DATA, as hexadentate tribasic ligands, derived from perhydro-1,4-diazepine, combine the advantages of cyclic and non-cyclic chelators [30, 31]. The endocyclic and exocyclic amines in this hybrid structures provide three ligand units and can introduce three or four further donor units through the alkylation of these amines with carboxylic acids [32]. Studies have shown that such hybrid chelators have excellent properties for nucleophiles such as gallium, lutetium, scandium, and copper, and can complex rapidly and stably with the corresponding metals at room temperature [33‐36]. Their highly efficient and robust labelling characteristics of AAZTA and DATA render them promising candidates for utilization in ⁶⁸Ga-PET and the development of kit-type labelling. In recent years, some small molecules have been further developed as radioligands with fast labeling properties, such as TOC [37], PSMA [36] and FAP inhibitors [34, 35]. To the best of our knowledge, AAZTA⁵ and DATA^5m have not been applied to the GRPr-targeted peptide RM2, and investigation into the biological properties of its derivatives are even more limited. Therefore, evaluating these new radiotracers with AAZTA⁵ and DATA^5m as ligands will be informative and valuable.

To investigate the impact of macrocyclic chelators on RM2 peptide, ⁶⁸Ga-AAZTA⁵-RM2, ⁶⁸Ga-DATA^5m-RM2, and reference radioligand ⁶⁸Ga-DOTA-RM2 were synthesized. We hypothesized that these chelators might display different profiles in vitro and in vivo; thus, a radiotracer with optimized properties in imaging or biodistribution will likely be screened. The physicochemical properties and the binding specificity to PC-3 cells of these ⁶⁸Ga-labeled conjugates were evaluated. In addition, the distribution and PET/CT imaging of ⁶⁸Ga-X-RM2 (X = DOTA or DATA^5m or AAZTA⁵) in PC-3 transplanted BALB/c nu/nu mice were also compared.

An important role of chelators is to stably complex radionuclides and thus provide a means for labelling the targeting vector to allow the labelled ligands to interact with the targeted molecules. Many structure-related variables determine radiopharmaceuticals’ general behavior. In general, due to the relatively small size of the peptides, any structural modification of a substrate likely affects the affinity and selectivity to its targeted receptor. For example, the pharmacokinetics of peptides can be tuned by altering the chelators with variable lipophilicity, charge, size, and coordination symmetry [24]. In addition, the introduction of chelators may affect not only the binding but also the function of the targeted peptide, for example, by changing the agonistic effect to an antagonistic one [38]. Recently, the effects of organ distribution, target uptake and image contrast of RM2 derivatives with different chelating agents were investigated. The results show that the chelating agent largely affects the affinity of the molecule, which may be related to its total charge, with an improved affinity when the N-terminal carries an overall positive charge [39]. Thus, modification of chelating groups is one of the simple but effective ways to change the pharmacokinetic properties and targeting characteristics of peptides. This study aims to synthesize and compare RM2 analogs conjugated with homologous macrocyclic chelates and investigate the effect of these building blocks on the imaging performance.

Bifunctional AAZTA derivatives were recently reported and successfully applied to prepare ⁶⁸Ga conjugates for PET imaging [30]. AAZTA⁵ is a suitable derivative that introduces a C5-alkyl spacer with a terminal carboxylic acid functional group on the AAZTA backbone. Incorporating the alkane chain may increase the lipophilicity of the molecule and on the other hand enrich its steric structure, such as chiral isomerism. The influence of the chirality and lipophilicity of a chelator on the biodistribution was estimated but have not been extensively investigated [26, 40]. Similarly, DATA^5m has the same backbone structure as AAZTA⁵, except that one of the carboxyl groups on the primary amine group is replaced by a methyl group. It may be facilitates the formation of stable coordination complexes with small metal ions and azacarboxylates based on 1,4,7-triazacyclononanes [41, 42]. Because the geometry of octahedra is favorable for the coordination of metal ions such as Zn²⁺, Mn²⁺, Fe³⁺ and Ga³⁺, and the presence of primary amine groups can easily lead to the nitrogen atom-functionalization of heptathlete ligands. In addition, the chelating portion in DATA^5m is hybrid because the scaffold contains both cyclic and acyclic structural features, where the flexibility of the acyclic portion facilitates fast complexation while the cyclic portion minimizes the energy barrier to complexation and inhibits the decomplexation processes [32]. This particular chemical property leads to a unique coordination mode with ⁶⁸Ga [37].

The study replaced the chelator of DOTA-RM2 using AAZTA⁵ and DATA^5m. Once labelled with ⁶⁸Ga³⁺, the biological properties were investigated in vitro and in vivo. To ensure the comparability of the research, we performed a head-to-head comparative study along with DOTA-RM2. It turned out that lower temperatures, shorter times, and less quantities of precursors are required when labelling AAZTA⁵-RM2 and DATA^5m-RM2 with ⁶⁸Ga³⁺ (Fig. 2). The results showed that the radiolabeling of DATA^5m-RM2 and AAZTA⁵-RM2 was achieved within 3 min at room temperature. This simplified the reaction conditions is advantageous over DOTA-RM2, which needs to be carried out at a higher temperature condition. At the same time, the amount of precursors required for the complexation of DATA^5m and AAZTA⁵ with ⁶⁸Ga³⁺ was far less than that of DOTA, which was about 1/5–1/2 of that. In this context, a higher specific activity may be obtained for the same amount of DATA^5m-RM2 or AAZTA⁵-RM2. Additionally, the simple and efficient labelling process might further facilitate the rapid kit preparation of ⁶⁸Ga-radiopharmaceuticals, compared to previous kit-based protocols. The superior chemical properties of AAZTA⁵ and DATA^5m may enhance drug preparation efficiency, particularly for hospitals or institutions with inadequate supporting facilities.

Cellular uptake and internalization studies were conducted in PC3 cells to examine the impact of variations in chelating groups on receptor binding capacity. The results showed that all three compounds displayed highly specific binding to GRPr-expressing cells. While their maximal cellular uptake was similar, there was a slight downward trend in the uptake of ⁶⁸Ga-DATA^5m-RM2 after 2 h (Fig. 4a). In addition, it was observed that all three compounds were characterized by a low level of internalization, but AAZTA appeared to be slightly higher than the other ligands. (Fig. 4b). Although the reason was unclear, some studies had reported that the number of carboxylates of chelators may be an essential factor in determining the internalization rate [25]. In vitro and in vivo binding specificity assays showed that AAZTA⁵-RM2 and DATA^5m-RM2 retained GRPr-binding capacity after ⁶⁸Ga-labeling and confirmed that binding of both compounds to cells was receptor-mediated (Figs. 4c, 7). Thus, the introduction of these two chelating groups does not affect the specificity and targeting ability of the new ligand to bind to GRPr to a large extent. Indeed, DATA^5m-RM2 and AAZTA⁵-RM2 are superior in radiochemistry properties, meanwhile, antagonistic property towards the receptor is remained.

Biodistribution studies were performed in PC-3 tumor-bearing mice to further explore the effect of chelators on targeting and other biological properties (Fig. 5; Additional file 1: Table S1). Consistent with the results of in vitro studies, ⁶⁸Ga-DATA^5m-RM2 and ⁶⁸Ga-AAZTA⁵-RM2 showed similar biodistribution compared to ⁶⁸Ga-DOTA-RM2. This was reflected in the similar drug accumulation in tumor and non-target organs for all three compounds. However, it was worth noting that the radioactivity accumulation in the pancreas (known as receptor-positive organs) was significantly lower for ⁶⁸Ga-DATA^5m-RM2 and ⁶⁸Ga-AAZTA⁵-RM2 (Fig. 5). The pancreatic uptake of ⁶⁸Ga-DATA^5m-RM2 and ⁶⁸Ga-AAZTA⁵-RM2 was only 1/3 or even lower than that of ⁶⁸Ga-DOTA-RM2. The biodistribution of drugs can be influenced by multiple factors, with pharmacokinetics playing a significant role in determining drug accumulation in non-target organs. Our hypothesis is that the incorporation of a hybrid chelator into RM2 impacts its pharmacokinetic properties, as it may weaken the interactions within the microenvironment. This could result in a more rapid and reversible dissociation of RM2 from GRP receptors, thereby reducing its accumulation in the pancreas. In addition, tumor uptake was comparable for all tested analogs, but ⁶⁸Ga-DATA^5m-RM2 produced better tumor-to-organ ratios compared to ⁶⁸Ga-DOTA-RM2, especially tumor-to-muscle/bone ratio (Table 2). Although the maximum tumor uptake of the ⁶⁸Ga-AAZTA⁵-RM2 tended to decrease after 30 min, its tumor-to-organ ratios were still excellent. Taken together, compared with ⁶⁸Ga-DOTA-RM2 and ⁶⁸Ga-AAZTA⁵-RM2, ⁶⁸Ga-DATA^5m-RM2 showed better targeting effects and pharmacokinetic properties, which are favorable for imaging quality.

Herein, we present the synthesis and in vitro and in vivo study of novel GRPr-targeted radiotracers, ⁶⁸Ga-DATA^5m-RM2 and ⁶⁸Ga-AAZTA⁵-RM2. We further evaluated their affinity and specificity to GRPr-positive tumors by comparing the pharmacokinetics and PET imaging abilities with ⁶⁸Ga-DOTA-RM2. ⁶⁸Ga-DATA^5m-RM2 and ⁶⁸Ga-AAZTA⁵-RM2 displays favorable pharmacokinetics based on good tumor to non-target organs ratio. The excellent uptake and specificity in GRPr-positive tumors indicates that DATA^5m-RM2 has no less targeting ability than DOTA-RM2. In addition, the DATA^5m chelators show improving radiolabeling characteristics, making them ideal candidates for developing a new generation of ⁶⁸Ga-PET imaging agents that can be labelled in a kit-type manner. Therefore, the ⁶⁸Ga-DATA^5m-RM2 may be the preferable candidate for visualizing GRPr-expressing tumors using PET.

This manuscript presents the in vitro and in vivo study of two new RM2 derivatives by introducing new chelators, DATA^5m and AAZTA⁵, into GRPr antagonist RM2. It shows that the chelator component affects the targeting properties and pharmacokinetics of the ⁶⁸Ga-labeled BN antagonist RM2 to some extent. The results indicate that ⁶⁸Ga-DATA^5m-RM2 and ⁶⁸Ga-DOTA-RM2 had comparable radioactive uptake in GRPr-positive tumors, but lower uptake in normal organs. Thus, ⁶⁸Ga-DATA^5m-RM2 offers a superior tumor-to-organ ratios, together with its much milder radiolabeling procedure, it might be a more viable candidate for further development as a PET agent for visualizing GRPr-positive tumors.

Reagents and solvents, which were purchased from commercial sources, were of analytical or HPLC grade. DATA^5m-RM2, AAZTA⁵-RM2 and DOTA-RM2 were purchased from Nanchang Tanzhen Biological Technology Co., Ltd. (China). ⁶⁸GaCl₃ was eluted from a ⁶⁸Ge–⁶⁸Ga generator (ITM, Germany). Mass spectrometry (MS) was performed using an LC–MS system model LC-2030C (SHIMADZU, Japan). Chemical and radiochemical purity was determined by analytical high performance liquid chromatography (HPLC, SHIMADZU) equipped with a 4.6 mm × 250 mm C18 reversed-phase column (Agilent). All compounds were purified by preparative HPLC (Agilent) equipped with a 10 mm × 250 mm C18 reversed-phase column (Agilent). Radiocounting was performed using a CAPRAC-t γ-counter (Edmonton, Canada). Micro-PET/CT imaging using a small animal PET/SPECT/CT scanner, model InLiView-3000B (NOVEL MEDICAL, China). PC-3 cells were purchased from the Chinese Infrastructure of Cell Line Resource. BALB/c nude mice were purchased from Beijing HFK BIOSCIENCE Co., Ltd., China.