Abstract

Introduction and background

Somatostatin receptors (SSTRs) are G-protein coupled membrane receptors that were first described in rat pituitary tumor cells by Schonbrunn and Tashjian in 1978 [1]. Five different human subtypes have been identified, named SSTR1 to 5 [2]. While the genes for SSTR1, 3, 4 and 5 are intronless, the SSTR2 gene produces two splice variants, SSTR2A and B, differing only in the length of their cytoplasmic tail [3,4]. SSTRs are expressed by a wide variety of normal human tissues, both in various regions of the brain and peripheral organs, such as the spleen, adrenals, pituitary gland, pancreas, liver, gastro-intestinal tract, kidneys and lungs, each exhibiting a characteristic expression pattern of the different SSTR subtypes [5-8]. SSTRs have also been identified in several human tumor types. Neuroendocrine tumors (NETs) represent one of the groups with the highest incidence of SSTR expression [9]. For instance in gastroenteropancreatic (GEP) NETs, SSTRs are present in 80 to 100% of cases, except for insulinomas, which have a lower incidence of 50 to 70% [10]. Other NETs expressing SSTRs include pituitary adenomas, pheochromocytomas, paragangliomas, lung carcinoids, small-cell lung cancers, Merkel cell carcinomas, medullary thyroid carcinomas and neuroblastomas [11]. A wide variability in receptor density and subtype expression has been observed across the different NET types, but also within individual tumor types [9,11]. In the majority of cases, SSTR2 is most abundant, even when other subtypes are present [8,12]. A large variety of other solid and hematological malignancies may also variably express SSTRs. These include meningiomas, gliomas, lymphomas, and breast, lung, renal cell, pancreato-biliary tract, liver cell, colorectal, ovarian and prostatic carcinomas [9,11,12].

The presence of SSTRs on tumor cells may open up an important window of opportunity for the clinical management of those tumors in terms of imaging and therapeutic options. Especially in NETs this opportunity has already been extensively exploited. SSTR overexpression is the foundation on which the use of somatostatin analogs (SSAs) such as octreotide in the pharmacological treatment of NETs is based [13]. According to the most recent European Neuroendocrine Tumor Society (ENETS) consensus guidelines, SSAs are indicated for symptom relief in case of functioning tumors that cause hormone production, as well as for tumor growth inhibition [14].

Furthermore, SSAs can be labeled with radionuclides. These radionuclides either have a heavy nucleus (Z > 83) or possess an imbalance in proton/neutron ratio, or are in a metastable energy state and will undergo radioactive decay. The excess of energy in the nucleus of the unstable element can result in emission of either particles (α, β^+/-) and/or electromagnetic radiation (gamma ray photons (γ)) and as a secondary effect X-rays, conversion electrons and Auger electrons. The specific decay characteristics of the radionuclide attached to the vector molecule determine if the radiopharmaceutical can be used for diagnostic (molecular imaging) or therapeutic (targeted radionuclide therapy) purposes. As such, SSTR imaging now occupies a key position in the clinical management of NETs [15-17]. Moreover, radiolabeling of SSTR targeting agents with therapeutic radionuclides may allow for vectorized radionuclide therapy, also called peptide receptor radionuclide therapy (PRRT). Currently, PRRT by means of radiolabeled SSAs represents an established, evidence-based treatment modality in case of inoperable/metastatic well-differentiated NETs [18] and its role has been enforced by the excellent results obtained in the randomized, controlled NETTER-1 trial [19].

In this review, we will focus on molecular imaging of the SSTR and more specifically on the developments leading to the next generation of clinical PET tracers targeting the SSTR. Peptides can be radiolabeled with radiohalogens (e.g. iodine isotopes and fluorine-18) by standard carbon-halogen bond formation or with radiometals (e.g. indium-111 and gallium-68) using suitable bifunctional chelators that are covalently linked to the biologically active peptide [20]. Therefore, peptide-based radiopharmaceuticals typically consist of the vector moiety (the biologically active peptide) which is linked by means of a chelator, and possibly through an additional linker, to the radionuclide [21]. Figure 1 shows the chemical structure of all relevant chelators discussed in this review with their matching radionuclides, in combination with the different SSAs that have been applied in clinical practice or clinical studies. Crucial for an effective SSTR tracer is its potential to bind the relevant SSTR [22]. It is important to realize that even small changes in the amino acid sequence of the peptide or a different choice of chelator or radionuclide might result in a different affinity profile [22,23] (see also Tables 1 and ​and2).2). Recent advances in molecular imaging of the SSTR, relating to the choice of radionuclide and vectors molecules, will be discussed. Figure 2 gives an overview of the likely future directions in the field of clinical SSTR PET imaging.

Figure 1

Chemical structures of the relevant SSTR tracers that have been applied in clinical practice or clinical studies: radionuclides with their matching chelators or fluorine-18 labeled constructs plus somatostatin receptor agonists or antagonists. (BASS: pNO₂-Phe-c(D-Cys-Tyr-D-Trp-Lys-Thr-Cys)D-TyrNH₂; JR11: Cpa-c[D-Cys-Aph(Hor)D-Aph(Cbm)-Lys-Thr-Cys]-D-Tyr-NH₂).

Figure 2

Overview of the likely future directions in the field of clinical SSTR PET imaging.

From SPECT to PET. SSTR imaging: current status

SSTR imaging was first performed in humans in the late 1980s using ¹²³I-Tyr³-octreotide [24]. However, due to several disadvantages such as a cumbersome radiolabeling procedure, high cost, limited availability of Na¹²³I, and considerable amount of intestinal accumulation of activity complicating interpretation of images, iodine-123 was soon replaced by indium-111, bound to the peptide by means of the chelator diethylenetriaminepentaacetic acid (DTPA) [25,26]. ¹¹¹In-DTPA-octreotide (or ¹¹¹In-pentetreotide) has long been the standard in SSTR imaging [22,27]. Indium-111 is a γ-emitting radionuclide, thus imaging is performed by means of planar scintigraphy or single photon emission computed tomography (SPECT), with or without computed tomography (CT). However, there are some drawbacks to the use of indium-111, such as unfavorable nuclear physical characteristics resulting in suboptimal image quality and relatively high effective doses, limited availability and high costs [27,28]. Successful efforts have been made to label SSAs with technetium-99m instead [27-29]. One of these compounds is ^99mTc-ethylenediamine-N,N’-diacetic acid/hydrazinonicotinamide-Tyr³-octreotide (^99mTc-EDDA/HYNIC-TOC) (see Figure 1), which is registered in Poland (^99mTc-Tektrotyd; Polatom) and used in several mainly Eastern European countries.

A major leap forward was the introduction of SSAs labeled with the positron-emitting radionuclide gallium-68 for positron emission tomography (PET) applications. This was made possible by the development of 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), a macrocyclic chelator capable of forming stable complexes with a multitude of 2⁺ and 3⁺ charged radiometals [30,31] that can be coupled to SSAs (see Figure 1). PET offers several advantages over SPECT, such as a higher sensitivity and spatial resolution and the possibility for straightforward image quantification [22]. The first clinical publication in this field reported on the use of ⁶⁸Ga-DOTA-TOC (⁶⁸Ga-DOTATOC) in patients with meningiomas [32], closely followed by a publication on the application of ⁶⁸Ga-DOTATOC in NET patients [33], both in 2001. Since then, ⁶⁸Ga-labeled SSA PET has rapidly emerged as an established technique for SSTR imaging, especially in NETs where it represents the molecular imaging modality of choice [17,34]. Tracers currently in clinical use are ⁶⁸Ga-DOTATOC, ⁶⁸Ga-DOTA-Tyr³-octreotate (⁶⁸Ga-DOTATATE) and ⁶⁸Ga-DOTA-1-NaI³-octreotide (⁶⁸Ga-DOTANOC), collectively referred to as ⁶⁸Ga-DOTA-peptides (see Figure 1).

Table 1 shows the affinity profiles, determined by means of in vitro binding studies, of most of the SSTR PET ligands discussed in this review as compared to ¹¹¹In-DTPA-octreotide and octreotide. All listed ⁶⁸Ga-DOTA-peptides show higher affinity for SSTR2 than ¹¹¹In-DTPA-octreotide [23,35]. Therefore, in tumors where SSTR2 is the most overexpressed subtype, such as NETs, this offers an additional benefit on top of the physical advantages associated with PET. As such, smaller lesions as well as lesions with low-to-moderate SSTR expression can be detected using ⁶⁸Ga-DOTA-peptide PET imaging versus ¹¹¹In-DTPA-octreotide SPECT [15]. Indeed, several studies comparing both techniques in a head-to-head manner consistently reported a superior performance of ⁶⁸Ga-DOTA-peptide PET in NET patients [36-44] (see Table 3). An example is shown in Figure 3. An additional advantage is the fact that ⁶⁸Ga-DOTATATE and ⁶⁸Ga-DOTATOC are the theranostic twins of ¹⁷⁷Lu-DOTATATE and ⁹⁰Y-DOTATOC, currently the most frequently used radiopharmaceuticals for PRRT, and are as such ideally suited to identify eligible patients [45].

Figure 3

Head-to-head comparison of ¹¹¹In-DTPA-octreotide scintigraphy (A: planar anterior, B: planar posterior, C: transversal SPECT image) and ⁶⁸Ga-DOTATOC PET (D: maximal-intensity projection, E: transversal slice) of a patient with ileal NET and liver, lymph node and peritoneal metastases (patient data from: [41]). More lesions can be visualized on the ⁶⁸Ga-DOTATOC PET images. Dashed lines in (A, B and D) denote the level of transversal slices in (C and E). Scale bar applies to PET images. (SUV = standardized uptake value).

The affinity profiles of the ⁶⁸Ga-DOTA-peptides show some differences (see Table 1), with the most prominent being the fact that ⁶⁸Ga-DOTANOC also has high affinity for SSTR5 and to a lesser extent for SSTR3, while ⁶⁸Ga-DOTATOC shows some affinity towards SSTR5 and ⁶⁸Ga-DOTATATE only binds to SSTR2 [23,35]. On the other hand, the affinity of ⁶⁸Ga-DOTATATE for SSTR2 is an order of magnitude higher than that of the other ⁶⁸Ga-DOTA-peptides. Therefore, some differences in lesion detection rate might be expected. A meta-analysis regarding the diagnostic role of ⁶⁸Ga-DOTATOC and ⁶⁸Ga-DOTATATE reported a high sensitivity (93% and 96%, respectively) and specificity (85% and 100%, respectively) for both tracers [46]. A head-to-head comparison of ⁶⁸Ga-DOTATOC and ⁶⁸Ga-DOTATATE in 40 NET patients showed a comparable diagnostic accuracy, although significantly fewer lesions were detected with the latter (262 vs. 254) [47]. In a similar head-to-head comparison of ⁶⁸Ga-DOTATATE and ⁶⁸Ga-DOTANOC in 20 NET patients, a comparable diagnostic accuracy was found as well with both tracers, with a slight, but not statistically significant difference in the number of detected lesions (130 vs. 116) [48]. Conversely, in another comparison study in 18 GEP NET patients, ⁶⁸Ga-DOTANOC PET detected significantly more lesions than ⁶⁸Ga-DOTATATE (238 vs. 212 out of 248 lesions), but the authors state that the clinical relevance of this observation has to be confirmed in larger trials [49]. Currently, there is no recommendation on which type of ⁶⁸Ga-DOTA-peptide is preferred [17,34,50] and logistic reasons such as availability of the precursor peptide will guide the choice in clinical practice. Moreover, there are some practical obstacles to the implementation of ⁶⁸Ga-DOTA-peptide PET that may differ from country to country. Examples are lack of ⁶⁸Ge/⁶⁸Ga generators registered or cleared for human use, no availability of precursor peptide for human use, no reimbursement or the implementation is economically not viable.

Radionuclide

Gallium-68 has the theoretical advantage that it is available from ⁶⁸Ge/⁶⁸Ga generators and as such cyclotron independent. However, despite the excellent results achieved with ⁶⁸Ga-DOTA-peptides, their use in routine clinical practice is often limited to large nuclear medicine departments. Unlike the ⁹⁹Mo/^99mTc generator, the current generation of ⁶⁸Ge/⁶⁸Ga generators requires a dedicated radiopharmacy staff. Moreover, production and quality control of gallium-68 radiopharmaceuticals is subject to strict regulations imposed by pharmaceutical legislation [51,52]. Nevertheless, due to the significant boost to clinical PET by the introduction of ⁶⁸Ga-PSMA-HBED-CC [53], more and more nuclear medicine departments installed these ⁶⁸Ge/⁶⁸Ga generators and production facilities. Furthermore, the aforementioned issues may be largely solved with the next generation ⁶⁸Ge/⁶⁸Ga generators that have received regulatory approval (e.g. IRE ELiT, Fleurus, Belgium) and the use of kit-based labeling approaches, such as SomaKit TOC^TM (Advanced Accelerator Applications S.A.) and NETSPOT^® (Advanced Accelerator Applications USA), that have received approval by the European Medicines Agency (EMA) and the Food and Drug Administration (FDA), respectively. As the overall activity yield per production batch is low (capacity of two to four patients per production) and half-life of gallium-68 is relatively short (68 minutes), there is only limited potential for centralized production and distribution. However, some possibilities may be opened up in this field by advances in the cyclotron production of gallium-68 [54].

Another disadvantage of gallium-68 is its relatively high positron energy (E_mean = 0.83 MeV) and thus relatively long positron range (R_mean = 3.5 mm), which may compromise spatial resolution [55]. Therefore, the possibilities of using other PET radionuclides for SSTR imaging are currently being explored. The physical characteristics of all radionuclides relevant for PET imaging discussed below are summarized in Table 4.

Copper-64

A frequently used non-standard PET radionuclide is copper-64 [54,56]. Its half-life (12.7 hours) allows for centralized production, while its low positron energy (E_mean = 0.28 MeV) corresponding to a short positron range (R_mean = 0.8 mm) allows for high spatial resolution PET imaging [54,55]. Copper-64 has a relatively low positron branching ratio of 17.5% and its decay is accompanied by emission of β^- particles and Auger electrons adding up to its radiation burden. Therefore, it could be used for therapeutic purposes as well, making it suited for theranostic applications [57], taking into account extensive shielding needed in practice due to the simultaneous high energy gamma and positron emission. Copper-64 can either be produced in a reactor or with a cyclotron [54,57].

As early as 2001, Anderson et al. reported a first clinical evaluation of the dosimetry and pharmacokinetics of ⁶⁴Cu-TETA-octreotide - copper-64 bound to octreotide through the chelator 1,4,8,11-tetraazacyclotetradecane-N,N’,N’’,N’’’-tetraacetic acid (TETA) (see Figure 1) - in eight NET patients and its diagnostic properties were compared to ¹¹¹In-DTPA-octreotide [58]. In two patients, ⁶⁴Cu-TETA-octreotide detected clearly more lesions than ¹¹¹In-DTPA-octreotide. In one patient mild uptake in a lung lesion was observed with ¹¹¹In-DTPA-octreotide, but not picked up by ⁶⁴Cu-TETA-octreotide. However, delayed images which might have shown the lesion were not available for this patient. Pharmacokinetic assessment revealed fast blood clearance with partial urinary excretion. On the other hand, the percentage injected activity in the liver increased with time due to dissociation of the copper-64 isotope, indicating poor in vivo stability [57,58].

The next clinical studies on copper-64 labeled SSAs date from more than 10 years later. Pfeifer et al. prospectively evaluated ⁶⁴Cu-DOTATATE in a first-in-human study in 14 NET patients [59]. PET images with high spatial resolution were obtained. High and stable tumor-to-background ratios were observed on both the early (one hour post injection (p.i.)) and late (three hours p.i.) PET scans, indicating a high tracer internalization rate. Although some dissociation of copper-64 was suggested by increasing hepatic activity, in vivo stability of the tracer was sufficient for imaging purposes. All patients underwent conventional ¹¹¹In-DTPA-octreotide SPECT/CT as well. Additional lesions were detected on ⁶⁴Cu-DOTATATE PET in six patients (43%) and in five of these patients lesions were identified in organs not previously known as disease-involved. No lesions were observed with ¹¹¹In-DTPA-octreotide SPECT that were not revealed by ⁶⁴Cu-DOTATATE PET.

In a subsequent larger prospective study, Pfeifer et al. confirmed the diagnostic superiority of ⁶⁴Cu-DOTATATE PET over ¹¹¹In-DTPA-octreotide SPECT by means of a head-to-head comparison in 112 NET patients [60]. PET images were acquired one hour after injection. The sensitivity and diagnostic accuracy of ⁶⁴Cu-DOTATATE PET (both 97%) were significantly higher than those of conventional ¹¹¹In-DTPA-octreotide SPECT (87% and 88%, respectively). Twice as many lesions were detected using ⁶⁴Cu-DOTATATE PET, and more importantly, in 40 patients (36%) lesions were identified in organs not previously known as disease-involved. In 35 of these 40 patients the true-positive nature of these supplemental involved organs was confirmed by long-term follow-up of 42-60 months.

Of special interest is a recent study published by Johnbeck et al. comparing ⁶⁴Cu-DOTATATE and ⁶⁸Ga-DOTATOC PET in 59 NET patients on a head-to-head basis [61]. On a patient level, ⁶⁴Cu-DOTATATE and ⁶⁸Ga-DOTATOC performed equally well. However, ⁶⁴Cu-DOTATATE detected significantly more additional true-positive lesions, confirmed by at least 30 months of follow-up, than ⁶⁸Ga-DOTATOC (33 versus 7). The authors attributed this difference in lesion detection rate to the shorter positron range of copper-64, with consequent higher spatial resolution and less partial volume effect, rather than to the use of a different peptide. Figure 4 shows an example of a patient where more lesions are seen in the intestinal region with ⁶⁴Cu-DOTATATE than with ⁶⁸Ga-DOTATOC. Tumor-to-background ratios as a measure for image contrast were not significantly different between the two tracers. Although the radiation burden of ⁶⁴Cu-DOTATATE is higher than that of ⁶⁸Ga-DOTATOC (5.7-8.9 mSv vs. 2.8-4.6 mSv), the use of ⁶⁴Cu-DOTATATE offers several advantages for use in routine clinical practices associated to the half-life of copper-64, such as supply to peripheral sites from a central production unit and a more flexible scanning window ranging from one hour to at least three hours after injection. A preclinical evaluation of ⁶⁴Cu-DOTATOC has been published [62], but to our knowledge no subsequent clinical studies have been performed.

Figure 4

PET/CT (left) and PET (right) scans with ⁶⁸Ga-DOTATOC and ⁶⁴Cu-DOTATATE of a patient with intestinal NET and multiple metastases. Additional lesions are seen in the intestinal region with ⁶⁴Cu-DOTATATE. This research was originally published in JNM. Johnbeck CB, Knigge U, Loft A, Berthelsen AK, Mortensen J, Oturai P, Langer SW, Elema DR and Kjaer A. Head-to-head comparison of ⁶⁴Cu-DOTATATE and ⁶⁸Ga-DOTATOC PET/CT: a prospective study of 59 patients with neuroendocrine tumors. J Nucl Med. 2017;58:451-457. © SNMMI.

Copper-64 has also been successfully coupled to TATE by means of a new bifunctional chelator, 5-(8-methyl3,6,10,13,16,19-hexaaza-bicyclo[6.6.6]icosan-1-ylamino)-5-oxopentanoic acid (MeCOSar), forming ⁶⁴Cu-SARTATE [63]. Initial preclinical results are promising showing a high uptake in SSTR2-positive tumors [63]. Further preclinical and clinical studies on this new radiopharmaceutical are ongoing (Australian New Zealand Clinical Trials Registry (ANZCTR) identifier: ACTRN12615000727549) [57].

Vector

Developments in SSTR PET radiopharmaceuticals do not only focus on the choice of radionuclide but also on the characteristics of the vector molecule. The radiopharmaceuticals described above contain the somatostatin receptor agonist octreotide, or an analog of octreotide, as vector molecule, with a predominance of TOC and TATE. Improved tumor targeting may be achieved for instance by using vector molecules having a higher binding affinity for the SSTR or a broader affinity profile for the different receptor subtypes or by using compounds recognizing a higher number of binding sites, such as the SSTR antagonists.

Somatostatin receptor antagonists

All SSTR-targeting radiopharmaceuticals described above are somatostatin agonists. After binding to the SSTR, the ligand-receptor complex is usually internalized, allowing tracer metabolites to accumulate in the target cells [110]. For a long time, it was believed that this process of internalization and subsequent accumulation of the radioligands was essential for high-contrast imaging of SSTR-positive lesions [111]. However, in 2006, Ginj et al. observed in cell cultures expressing human SSTR2 and SSTR3, that antagonists labeled considerably more receptor sites than agonists [110]. This was reflected by the significantly higher tumor uptake seen in mice bearing SSTR2 and SSTR3-expressing tumors after injection with the corresponding antagonist as compared to those injected with the agonist [110]. Especially striking was the fact that counterintuitive to this observation the SSTR2 antagonist, ¹¹¹In-DOTA-pNO₂-Phe-c(D-Cys-Tyr-D-Trp-Lys-Thr-Cys)D-TyrNH₂ (¹¹¹In-DOTA-BASS) (see Figure 1), showed a more than sevenfold lower affinity for the SSTR2 than the SSTR2 agonist ¹¹¹In-DTPA-TATE in this study (50% inhibitory concentration (IC₅₀) of 9.4 ± 0.4 nM vs. 1.3 ± 0.2 nM) [110]. In 2011, Wild et al. published a pilot study in five patients, one metastatic follicular thyroid carcinoma and four NETs, evaluating the biodistribution, tumor uptake and detection of tumor lesions with the SSTR antagonist ¹¹¹In-DOTA-BASS in a head-to-head comparison with ¹¹¹In-DTPA-octreotide [112]. Tumor uptake was up to four times higher with the antagonist, while renal, liver and spleen uptake were lower. ¹¹¹In-DOTA-BASS detected more lesions than ¹¹¹In-DTPA-octreotide (25 vs. 17 out of 28). The three missed bone lesions were negative on the ¹¹¹In-DTPA-octreotide scan as well.

Further advances in the preclinical setting in search of more potent SSTR2 antagonists labeled with PET radioisotopes led to the identification of Cpa-c[D-Cys-Aph(Hor)D-Aph(Cbm)-Lys-Thr-Cys]-D-Tyr-NH₂ (JR11) (see Figure 1) as a promising compound for implementation in the clinical field [113]. Based on the results of previous affinity studies and an in vivo biodistribution study [114], ⁶⁸Ga-NODAGA-JR11 (⁶⁸Ga-OPS202) was selected for a first clinical evaluation by Nicolas et al. [115,116]. 12 patients with GEP NET and a positive ⁶⁸Ga-DOTATOC PET/CT scan in the previous six months were administered two microdoses of ⁶⁸Ga-NODAGA-JR11 with ascending peptide masses at different study visits and underwent subsequent PET/CT scans. ⁶⁸Ga-NODAGA-JR11 showed a fast blood clearance and favorable biodistribution with both peptide doses as compared to ⁶⁸Ga-DOTATOC (lower hepatic, pancreatic, gastro-intestinal and splenic uptake with ⁶⁸Ga-NODAGA-JR11) [115,116]. This was reflected by higher tumor-to-background ratios observed with ⁶⁸Ga-NODAGA-JR11 and further translates to a significantly higher number of detected tumor lesions and higher lesion-based overall sensitivity (94% and 88% for 50 µg and 15 µg ⁶⁸Ga-NODAGA-JR11, respectively, vs. 59% for ⁶⁸Ga-DOTATOC) [116]. An example is shown in Figure 5. The effective dose is in line with ⁶⁸Ga-DOTA-peptides (24 ± 2 µSv/MBq for ⁶⁸Ga-NODAGA-JR11 vs. 21 ± 3 µSv/MBq for ⁶⁸Ga-DOTATATE and ⁶⁸Ga-DOTATOC [117]), although some differences in organ doses are observed due to a slightly different biodistribution [115]. Overall, ⁶⁸Ga-NODAGA-JR11 was well tolerated [115]. However, SSTR antagonists could possibly counteract the effects of SSAs, which may be important in patients with functioning NETs [111]. Therefore, caution is required in anticipation of more safety data [111]. Currently a multicenter clinical trial evaluating the optimal dose and safety of ⁶⁸Ga-NODAGA-JR11 for PET imaging is ongoing (clinicaltrials.gov identifier: NCT03220217).

Figure 5

Maximal-intensity projections (A and C) and PET/CT (B and D) scans with ⁶⁸Ga-NODAGA-JR11 (A and B) and ⁶⁸Ga-DOTATOC of a patient with ileal NET and bilobar liver metastases. Liver magnetic resonance imaging (MRI) was performed four months later, with delayed post-contrast acquisitions (E) and diffusion-weighted images (F), confirming the additional metastases missed or questionable (arrow with question mark) with ⁶⁸Ga-DOTATOC. Note the lower background activity in the liver, intestine and thyroid with ⁶⁸Ga-NODAGA-JR11. Dashed lines in (A and C) denote the level of transversal slices in (B, D, E and F). This research was originally published in JNM. Nicolas GP, Schreiter N, Kaul F, Uiters J, Bouterfa H, Kaufmann J, Erlanger TE, Cathomas R, Christ E, Fani M and Wild D. Sensitivity comparison of ⁶⁸Ga-OPS202 and ⁶⁸Ga-DOTATOC PET/CT in patients with gastroenteropancreatic neuroendocrine tumors: a prospective phase II imaging study. J Nucl Med. 2018;59:915-921. © SNMMI.

The higher tumor uptake achieved with SSTR antagonists may also prove useful for therapeutic purposes. Preclinical studies comparing ¹⁷⁷Lu-DOTA-JR11 (¹⁷⁷Lu-OPS201) with ¹⁷⁷Lu-DOTATATE observed higher tumor uptake and longer residence times, resulting in higher tumor doses delivered by the antagonist as compared to the agonist [118,119]. In a clinical pilot study by Wild et al. in four patients with progressive NETs, more than threefold higher tumor doses and twofold higher tumor-to-kidney and tumor-to-bone marrow dose ratios were observed using a test dose of ¹⁷⁷Lu-DOTA-JR11 as compared to ¹⁷⁷Lu-DOTATATE [120]. All patients were subsequently treated with ¹⁷⁷Lu-DOTA-JR11, resulting in partial remission in two patients, mixed response in one patient and stable disease in the last patient, and as such proving the clinical feasibility of PRRT using radiolabeled SSTR antagonists. Currently, a multicenter clinical trial evaluating the safety and efficacy of ¹⁷⁷Lu-DOTA-JR11 (clinicaltrials.gov identifier: NCT02592707) as well as a study evaluating the theranostic couple ⁶⁸Ga-DOTA-JR11 and ¹⁷⁷Lu-DOTA-JR11 (clinicaltrials.gov identifier: NCT02609737) are ongoing.

Radiolabeled SSTR antagonists might also prove to be especially useful for imaging and therapy of cancer types with a typically lower SSTR expression such as breast cancer [121]. Several preclinical studies observed enhanced tumor targeting in various human SSTR2-expressing tumor samples, including breast carcinoma, by means of in vitro autoradiography using an SSTR2 antagonist in comparison to the SSTR2 agonist [121-123]. This finding was not confirmed in a recent preclinical study by Dude et al. using the human luminal breast cancer model, ZR-75-1, with endogenous SSTR2 expression and negligible expression of other SSTR subtypes, where ⁶⁸Ga-NODAGA-JR11 had a lower tumor uptake than ⁶⁸Ga-DOTATOC and ⁶⁸Ga-DOTATATE [124]. This was tentatively explained by the authors by the fact that they used an endogenously expressing cell line, which may have a lower amount of low-affinity, antagonist-specific binding sites. Interestingly, although ⁶⁸Ga-DOTATATE has a higher affinity for the SSTR2 than ⁶⁸Ga-DOTATOC, the latter was found to have the highest tumor uptake. Additional studies are warranted to further investigate the role of SSTR2 antagonists in breast cancer imaging [124].

Conclusion

Advances in SSTR PET ligands occur on two major fronts: the radionuclide and the peptide vector. Other radionuclides could offer a solution to practical, economical and regulatory barriers to the adoption of ⁶⁸Ga-DOTA-peptide PET, with additional physical advantages such as lower positron range and longer half-life. Developments concerning peptide vectors are mainly driven by the need for improved lesion targeting, especially for tumors with low SSTR expression. Therefore, advances on both fronts are largely complementary. Several promising new PET ligands for clinical SSTR imaging are currently in the pipeline and good results have been demonstrated in phase II trials. Clinical adoption in the near future is a realistic scenario.

Acknowledgements

Abstract

1. Introduction

Colorectal cancer (CRC) is the third most commonly occurring cancer in men and the second in women worldwide, with more than 1.4 million new cancer cases every year [1]. India has a relatively low prevalence of CRC in comparison to other countries, mainly attributed to differences in dietary patterns and lifestyles [2]. It is widely known that curcumin (1,7-bis(4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5-dione), a phyto-polyphenol pigment isolated from the dried rhizomes of Curcuma longa L., has been used for centuries in Asian countries as a dietary spice and traditional drug to treat many different diseases [3]. Besides having antioxidant, anti-inflammatory, anti-bacterial and anti-amyloid properties, curcumin has been shown also to possess anti-cancer activities [4,5]. In particular, the ability to inhibit proliferation of a wide variety of tumour types, including colorectal cancer, has been highlighted by recent studies in vitro and in vivo [6,7,8]. Several studies suggest that curcumin changes the gene expression profiles and signalling pathways such as those involving COX-2 enzymes as well as NF-κB, cyclin D1 and p53 proteins [9,10,11]. Although the mechanism of curcumin’s high accumulation and cytotoxic activity against HeLa, MCF-7, HT29, and HCT-116 cancer cell lines are still unclear [10,11], curcumin itself is a promising tumour-targeting moiety for the development of early diagnosis and therapeutic agents. Nuclear medicine imaging provides information about biological processes occurring at a molecular level in vivo; this occurs by following the fate of radiolabelled compounds. Such radiotracers can be substrates for metabolic pathways overexploited in tumour cells or have particular affinity for receptors overexpressed in tumours. Hence, curcumin derivatives labelled with proper β⁺ emitter radionuclides or curcumin-based radio-metal complexes have the potential to be diagnostic tools for positron emission tomography (PET). Currently, radiolabelled curcuminoids have been mostly investigated for imaging of the central nervous system (CNS) where several curcumin-based probes labelled with fluorine-18 have demonstrated promise in the early detection of Alzheimer’s disease [12,13]. In oncology, curcuminoids as imaging agents are underinvestigated. We have recently reported that curcuminoid complexes labelled with gallium-68 demonstrated preferential uptake in HT29 colorectal cancer and K562 lymphoma cell lines compare to normal human lymphocytes. In that study, the core radio-metal was directly linked to two curcuminoid molecules through the keto-enol moiety [14,15]. The curcuminoid complexes showed good stability in PBS and human serum but were rapidly degraded in whole human blood. These findings, in addition to the low stability and solubility of curcumin structures in physiological media [16], limited the possibility to further study these kind of compounds in vivo. In the present study, we report a new gallium-68-labelled curcumin derivative with curcumin linked to an efficient gallium-68 chelator, 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), and its initial validation as a marker for early detection of CRC. The synthetic pathway, chemical characterization, labelling methods, stability, cell uptake, internalization and efflux studies on HT29 cells and in vivo micro-PET imaging and biodistribution in colorectal tumour bearing mice of this radiotracer are herein reported.

2. Results

2.1. Synthesis and Chemical Characterization

Synthesis of 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic(1,7-bis(4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5-dione (DOTA-C21) was carried out in two steps as shown in Figure 1. The curcumin-based conjugate was identified and completely characterized by ESI-LC/MS (Figure S1) and ¹H-,¹³C-NMR spectroscopy (Figures S2, S3). ESI-LC/MS spectrum exhibits two isomeric peaks (m/z = 798.3; [M + H]⁺) that were attributed to contemporary presence of two main isomeric forms (diketo and keto-enol) as previously reported for curcumin and its derivatives [17,18]. Gallium complexes were obtained by reacting a DOTA-C21 solution with a Ga(NO₃)₃ solution at 95 °C for 30 min in a 1.5:1 metal to ligand molar ratio using a 0.4 M ammonium acetate solution as buffer. Complexes formation was identified by ESI-LC/MS analysis thanks to the typical isotopic pattern of gallium ion (Figures S4, S5). Similar to the free ligand analysis, the spectrum exhibits two compounds with the same m/z (retention times: 2.8 and 3.3 min, respectively) both corresponding to Ga-DOTA-C21 complexes (m/z = 864.2 [M⁶⁹Ga + H]⁺ – 866.2 [M⁷¹Ga + H]⁺). To elucidate chelation mode and binding sites, ¹H-NMR titration of DOTA-C21 with Ga^{3 +} was also performed (Figure S6) and the gallium(III)-complex was characterized by NMR (Figure 2, Figure S7) as well as LC-MS/MS fragmentation experiments (Figure 3, Figure S8).

Figure 1

Synthetic pathway for the preparation of DOTA-C21 and its complexation with Ga(III) by reacting DOTA-C21 with Ga(NO₃)₃ at 1.5:1 metal-to-ligand molar ratio in 0.4 M ammonium acetate solution for 30 min at 95 °C.

Figure 2

Aliphatic region overlap of ¹H-, ¹³C-HSQC-NMR spectra of DOTA-C21 (red (CH₂) and purple (CH/CH₃)) and Ga-DOTA-C21 (green (CH₂) and blue (CH/CH₃)). All the spectra were acquired in MeOD-d₄ at 25 °C ([DOTA-C21] = 0.63 mM). Atom numbering refers to Figures 11 and 12.

Figure 3

LC-MS/MS fragmentation experiments on Ga-DOTA-C21 complexes with m/z 864.5 [M + H]⁺. HPLC-MS chromatogram (A). Fragmentation pathway of the m/z 864.5 [M + H]⁺ ion corresponding to the peak with retention time 5.9 min (B). Fragmentation pathway of the m/z 864.5 [M + H]⁺ ion corresponding to the peak with retention time 6.5 min (C).

¹H- and ¹³C-NMR spectra displayed a strong shift of both protons and carbon with respect to the free ligand when gallium(III) is added. In particular, methylene protons of the chelator ring, briefly r₁ and r₂, are no longer chemical shift equivalent since the interconversion between pseudo-axial (H_a) and pseudo-equatorial (H_e) conformation is prevented by metal complexation. Geminal protons H_a(r₁) and H_e(r₁) appear as broad triplets at 4.00 ppm and 3.38, respectively, due to similar values of geminal (²J_HH) and vicinal (³J_HH) coupling constants (~14Hz). The same situation occurs for H_a(r₂) and H_e(r₂) that fall at 3.47 ppm and 3.41 ppm, respectively. Assignments of ring protons was confirmed by ¹H,¹³C-HSQC spectrum, H_a/H_e(r₁) show cross-peaks with carbon at 54.65 ppm (C(r₁)) and H_a/H_e(r₂) with a carbon at 57.11 ppm (C(r₂)). The methylene groups of acetate arms are not chemical shift equivalent in gallium complex and appear as broad singlets in proton spectrum. The following reasonable assignments can be proposed (for atom numbering refers to Section 4.1.1 and Section 4.1.2): CH₂ in α to amide and nitrogen (atom numbering 15) ¹H- 3.79 ppm and ¹³C- 60.64 ppm; CH₂ in α to carboxylic group involved in gallium coordination (atom numbering 24 and 26) ¹H- 3.83 ppm and ¹³C- 59.41 ppm; CH₂ in α to free carboxylic group (atom numbering 25) ¹H- 3.74 ppm, ¹³C- 61.68 ppm.

Moreover, UV absorption and fluorescent emission spectra upon increasing addition of Ga³⁺ from free ligand were acquired (Figures S9, S10). These analyses showed that DOTA-C21 displays an absorption maximum at 410 nm, typical of curcumin structure in the keto-enol form. Upon addition of gallium (III) solution, an increase in absorbance is observed at 290 nm, while no changes are observed for the absorption maximum. Fluorescence emission spectra of DOTA-C21 (λ_ex 410 nm) do not show any significant change after gallium(III) addition.

2.2. Radiolabelling of the Chelator-Curcuminoid Derivatives with Gallium-68

Radiolabelling was performed using two methods. In the first, postprocessing of the generator elution was performed by cation exchange purification [19] and kinetics of the labelling were studied between 1 and 20 min by TLC. Kinetics were fast and the incorporation yield was 97 ± 2% after 5 min at 95 °C (n = 3). Postprocessing of the generator elution minimizes the metal cation impurities allowing a complete radiolabelling of DOTA-C21 by using only 10 nmol of the precursor. Representative TLC analyses are shown in Figure S11. In the second method, labelling without processing of the elution was attempted with increasing amount of precursor (20, 40, 60, 80 nmol). In this case, a reproducible radiolabelling could only be achieved with 80 nmol of DOTA-C21 after 10 min at 95 °C, with an incorporation yield ranging from 60 to 80%. The crude mixture was further purified by SPE to eliminate unreacted gallium-68 and hydrolysed products. Radiochemical purity was assessed by UHPLC (RCP > 95%, RCY = 55 ± 5%, n = 10). Representative UHPLC chromatograms (radiochemical detector) of a ⁶⁸Ga-DOTA-C21 preparation before and after SPE purification are shown in Figure 4. Similarly, to the complexation reaction with natural gallium, two main radioactive species were obtained with a retention time of 4.7 and 5.1 min, respectively. Based on the data gathered for the non-radioactive preparation, the peaks were assigned to two ⁶⁸Ga-DOTA-C21 isomeric complexes. A further peak at 5.3 min was recorded (ca. 4% of total radioactivity) and was likely due to a gallium-68 complexes with a degraded form of the precursor at labelling conditions. In both methods, the pH was kept constant around 4.

Figure 4

Representative UHPLC chromatograms (radiochemical detector) of a ⁶⁸Ga-DOTA-C21 preparation before (A) and after (B) SPE purification. (1) ⁶⁸Ga-free, (2) ⁶⁸Ga-hydrolyzed products, (3) ⁶⁸Ga-labelled byproducts, (4) and (5) ⁶⁸Ga-DOTA-C21 isomers, (6) ⁶⁸Ga-labelled degradation product.

2.3. In Vitro Stability Studies

In vitro stability of ⁶⁸Ga-DOTA-C21 complexes in PBS, human serum (HS) and whole human blood (HB) were determined according to peak integration of analytical UHPLC (Figure 5). Stability of ⁶⁸Ga-DOTA-C21 in PBS was more than 88 ± 3% after 120 min at 37 °C. In HS, the radiotracer was also stable with 85 ± 3% of intact compound after 120 min of incubation. On the contrary, in whole blood, ⁶⁸Ga-DOTA-C21 was partially degraded to more polar metabolites already after 10 min of incubation. The amount of intact compound ranged from 74 ± 2% after 10 min to 57 ± 3% after 120 min of incubation.

Figure 5

Stability of ⁶⁸Ga-DOTA-C21 complex incubated over time (10, 40, 70, 120 min.) in different media (n = 3, mean ± SD).

2.4. Uptake, Internalization and Efflux in Colorectal Cancer Cell Line

In vitro ⁶⁸Ga-DOTA-C21 uptake studies demonstrated time-dependent cellular accumulation in HT29 colorectal cancer cell line. Total uptake increased continuously in the first 60 min of incubation up to 121 ± 4 KBq of radiotracer per mg of protein (n = 3). At 60 min post incubation, 83% of total radioactivity was internalized in the cells. Conversely, ⁶⁸GaCl₃, used as a negative control, demonstrated significantly lower in vitro cell uptake (20 ± 4 KBq/mg of protein after 60 min of incubation, n = 3) confirming the curcuminoid-mediated uptake by HT29 cells (Figure 6A,B). The cellular uptake of ⁶⁸Ga-DOTA-C21 is comparable to that of the compound obtained by direct labelling of curcumin with gallium-68 (i.e., ⁶⁸Ga(CUR)₂ complexes [15]), however the internalized fraction was more than doubled. To test for receptor-mediated uptake, HT29 cells were preincubated with a 200-fold excess of calcitriol, the natural ligand for vitamin D receptor (VDR), and then ⁶⁸Ga-DOTA-C21 was added. The experiments were performed to assess whether ⁶⁸Ga-DOTA-C21 uptake was mediated by VDR since it has been reported that curcumin has a structure suitable to bind nuclear vitamin D receptor. The difference between uptake with or without calcitriol was not statistically significant (n = 3, Figure 6C) suggesting that the VDR receptor was not involved in the uptake or that the receptor cannot be saturated in these conditions.

Open in a separate window

Figure 6

Uptake and internalization at different time points of ⁶⁸Ga-DOTA-C21 in HT-29 colorectal cancer cell line (n = 3, mean ± SD) (A). Comparison between the uptake of ⁶⁸Ga-DOTA-C21 and ⁶⁸GaCl₃ in HT-29 cells indicating curcumin structure dependent accumulation (n = 3, mean ± SD) (B). Comparison between the uptake of ⁶⁸Ga-DOTA-C21 and ⁶⁸Ga-DOTA-C21 + calcitriol in HT-29 cells (n = 3, mean ± SD) (C).

For efflux studies, cells were incubated with ⁶⁸Ga-DOTA-C21 and the uptake was stopped after 60 min by removing the supernatant. Then, cells were washed, fresh medium was added and they were incubated again for different periods of time. Media were collected and radioactivity released from the cells was measured.⁶⁸Ga-DOTA-C21 showed a slow externalization pattern with around 75% (n = 3) of the radiotracer remaining within the cell after 60 min of incubation (Figure 7).

Figure 7

Externalization of ⁶⁸Ga-DOTA-C21 in HT-29 colorectal cancer cell line at different time points (n = 3, mean ± SD).

2.5. PET Imaging and Biodistribution

HT29 tumour-bearing mice were injected i.p. with ⁶⁸Ga-DOTA-C21 and PET/CT imaging was taken 1 h and 2 h post injection (Figure 8). The following day, the same cohort of mice was injected i.v. with ⁶⁸Ga-DOTA-C21 and dynamic PET/CT imaging was registered for the first 10 min to assess initial distribution of the tracer. The 1 h post injection time point was retaken to evaluate potential differences in the biodistribution pathway related to the type of injection. Mice were sacrificed at 1.5 h post injection, and ex-vivo biodistribution performed. As per PET/CT imaging analysis, upon i.v. injection, ⁶⁸Ga-DOTA-C21 was taken up rapidly in the heart and liver, and began accumulating into the tumour within the first minute of circulation. The tumour was immediately visible over background (muscle). At 1 h post injection, tumour accumulation of the tracer was 2.27 ± 0.85% ID/cc (n = 3) with a tumour to muscle ratio (T/M) of 1.91 ± 0.47 (n = 3). ⁶⁸Ga-DOTA-C21 appeared to have both renal and hepatic clearance. Radioactivity in the heart, which reflected the blood pool, was surprisingly high at 1 h post injection (5.8 ± 0.7% ID/cc, n = 3). Similar biodistribution was observed when the tracer was delivered i.p. At 1 h post injection, the tumour had an uptake of 1.89 ± 0.45% ID/cc (n = 5) and a T/M of 3.59 ± 2.76 (n = 5). At 2 h post injection, tumour uptake increased to 2.60 ± 0.27% ID/cc (n = 5) which was reflected by an increase in the T/M to 5.41 ± 3.62 (n = 5) (Figure 9 and Figure S12A–C). It is worth noting that, when analysing tumour uptake in each single animal, there was no appreciable variation between 1 h and 2 h post injection in animals with an initial low tumour uptake. On the contrary, animals with a tumour uptake greater than 4% ID/cc after one hour, tended to accumulate more radiotracer (Figure S12D). No correlation was found between T/M ratio and tumour size (Figure S12E). Ex-vivo biodistribution confirmed the described accumulation (Figure 9). Tumour uptake was 3.08 ± 0.53% ID/cc (n = 5) with a T/M of 2.29 ± 0.62 (n = 5). As already observed by PET, clearance occurred via both renal and hepatobiliary excretion, bone uptake was barely detectable over background and no significant radioactivity was seen in the intracranial region, suggesting that ⁶⁸Ga-DOTA-C21 is unlikely to cross an intact blood–brain barrier. Blood radioactivity was high, suggesting that a high amount of the radiotracer was in circulation or bound to blood constituents such as serum albumin. Further studies evaluating the clearance mechanism of ⁶⁸Ga-DOTA-C21 are underway to understand the high blood circulation, however one explanation may be that ⁶⁸Ga-DOTA-C21 pharmacokinetics follow a two-compartment model [20,21].

Figure 8

Representative microcoronal-PET/CT scans of HT29 colorectal tumour bearing mouse at 60 min (A) and 120 min (B) post i.p. injection of ca. 37 MBq of ⁶⁸Ga-DOTA-C21.

Open in a separate window

Figure 9

Biodistribution in multiple organs of ⁶⁸Ga-DOTA-C21 in HT-29 colorectal cancer bearing nude mice (n = 5, mean ± SD) at 90 min post i.v. injection. (A). Ratios of the tumour uptake to major organs (n = 5, mean ± SD) for ⁶⁸Ga-DOTA-C21 at 90 min post i.v. injection (B).

3. Discussion

The use of curcumin and its derivatives as potential therapeutics in many medical applications has been largely attested by a long list of scientific literature [22,23,24]. We previously demonstrated that ⁶⁸Ga-labelled curcumin complexes had preferential accumulation in HT29 colorectal cancer cells compared to other cancer cell lines and lymphocyte cultures [15]. However, the stability of these complexes in blood was low as the curcuminoid backbone was directly linked to the radio-metal through the keto-enol moiety, resulting in positively charged 2:1 ligand to metal ratio complexes. The present study is focused on evaluating whether a suitable curcumin derivative that is more stably labeled with gallium-68 provides a radiotracer that preferentially accumulates in CRC lesions, and could be a useful early detection agent which enables nuclear medicine imaging techniques. CRC is globally the third most common type of cancer and is usually diagnosed by invasive techniques like sigmoidoscopy or colonoscopy, during which a colon specimen is collected. These procedures are then followed by medical imaging (usually CT scan) to determine if the disease has spread. While small polyps may be removed during colonoscopy, the cancerous state of large polyps may be assessed by a biopsy [25]. The introduction of a PET radiotracer that specifically accumulates in CRC might be useful for verifying the nature of these lesions without resort to invasive methods. Moreover, treatments used for colorectal cancer may include some combination of surgery, radiation therapy, chemotherapy and targeted therapy and nuclear medicine imaging may be a suitable technique to stage and assess follow-up of the patients.

In the present study, the structure of the first generation of gallium-68 curcumin complexes has been improved by adding DOTA, a strong gallium-68 chelator. DOTA was linked through an (amino)ethyl spacer to one of the phenol groups of the curcumin backbone. With the addition of DOTA, the new bio-conjugate (namely DOTA-C21) has an enhanced water solubility and a higher stability of the complex with gallium-68 is achieved in physiological conditions. Despite its solubility in water, ¹H-NMR spectrum of DOTA-C21 in D₂O (Figure S2A) exhibited extremely broad signals in the aromatic region, suggesting that the hydrophobic backbone of curcumin interacted strongly by intramolecular π−π stacking. The signals could be sharpened by reducing the solvent polarity, for example, switching to methanol-d₄, which reduced aggregation. The ¹H-NMR spectrum in methanol-d₄ (Figure S2B) provided signals in the aromatic region attributable to the asymmetric curcumin moiety of DOTA-C21, while methylene signals of the ethyl spacer and DOTA were found in the aliphatic region (3–4.5 ppm). The structure of curcumin in DOTA-C21 is asymmetric due to the alkylation of one phenol group with the (amino)ethyl spacer, as clearly shown by two slightly shifted spin systems with equivalent spectral pattern (Figures S2, S3). Complete assignment and chemical structure with atom numbering is reported in Section 4.1.1.

As shown in the ESI-LC-MS analysis (Figure S1), DOTA-C21 was present in two isomeric forms that could be assigned to the keto-enol and di-keto tautomer, respectively [17,18]. As the equilibrium was influenced by solvent polarity and pH, the ratio between the two forms could vary over the different analysis conditions. Due to the rapid interconversion between the two isomeric forms also in physiological conditions, it was not expected that the two isomers exhibited different in vivo properties but this assumption should be verified with further studies.

When Ga-DOTA-C21 complexes were synthesized mimicking the radiolabelling experimental conditions (95 °C, 30 min, L:M 1.5:1, 0.4 M NH₄Ac), complex formation was confirmed by mass spectrometry and two compounds with the same m/z, corresponding to gallium-DOTA-C21 complexes at 1:1 metal to ligand molar ratio, were identified by ESI-LC/MS (Figure S4). The isotopic pattern (dashed square, Figures S4C and S5) highlighted the presence of gallium with its characteristic isotopic distribution in both compounds. Further LC/MS fragmentation experiments were carried out in order to exclude the involvement of the keto-enol moiety in the coordination sphere. As reported in Figure 3, the fragmentation pattern of the two isobaric peaks (panels B and C) were completely equivalent and could be attributed to cleavage of the curcumin structure, while gallium was always strongly bound by DOTA (Figure 10). This finding supported the hypothesis that the two isobaric peaks were due to the keto-enol and di-keto tautomers and not to different coordination modes.

Figure 10

Molecular fragments of Ga-DOTA-C21 attributed to the correspondent m/z signals detected in panel B and C of Figure 3.

To elucidate chelation mode and binding sites in depth, ¹H-NMR titration of DOTA-C21 with Ga³⁺ was performed in methanol-d₄ at 25 °C (Figure S6). The signals at 3.2 ppm (methylene groups of tetraazacyclododecane) and at 3.87 ppm (methylene groups in α to carboxylic groups) in the free ligand broadened and shifted at the first Ga^{3 +} addition, suggesting the involvement of the chelator in gallium coordination. A complete overview of 1:1 metal to ligand molar ratio complex formation for both proton and carbon resonances was shown by the overlap of ¹H-,¹³C-HSQC-NMR spectra of the free ligand (DOTA-C21) and its gallium complex (Ga-DOTA-C21) (Figure 2). The metal coordination induces a de-shielding effect on all methylene groups of the chelator ring resulting in an increase of both proton and carbon chemical shifts. Particularly, metal complexation removes chemical shift equivalence of ring methylene protons, as clearly showed by ¹H-,¹H-COSY and ¹H-,¹³C-HSQC-NMR spectra (Figure S7 and Figure 4). Methylene group r₁ shifts from ¹H/¹³C δ (ppm) 3.35/49.77 to 4.00/54.65 and 3.38/54.65, respectively. A lower shift is observed for methylene group r₂ from ¹H/¹³C δ (ppm) 3.26/49.64 to 3.47/57.11 and 3.41/57.11, respectively. These outcomes suggest the assignment of type r₁ to those methylene groups in α position to carboxylic moiety directly involved in metal coordination (atom numbering 17, 18, 21 and 22). All atom numbering herein reported refers to Figure 11 and Figure 12), while r₂ type can be attributed to those methylene groups in α position to non-coordinating carboxylic (amidic) moiety (atom numbering 16, 19, 20 and 23). Methylene groups of the carboxylic arms in α to both nitrogen and carboxylic (amidic) moiety undergo the effect of metal coordination in a different way if the carboxylic group is involved in coordination or not. As shown in Figure 4, methylenes of the carboxylic arms shift from ¹H/¹³C δ (ppm) 3.75–3.87/54.20 to 3.83/59.41, 3.79/60.64 and 3.74/61.68, respectively. Conversely, the chemical shifts of the ethyl spacer (H12 and H13) are unaffected by metal coordination.

Figure 11

Chemical structure of DOTA-C21 with atom numbering.

Figure 12

Chemical structure of Ga-DOTA-C21 with atom numbering.

Curcumin and curcuminoids act as fluorophores in the visible spectrum with strong solvent dependence, and consequently may be employed as fluorescent probes for distribution studies in cells and tissues. The typical main absorbance, due to the keto-enol group, was around 420 nm and the emission was around 500 nm, when excited at 420 nm wavelength [14]. In particular, the absorption of DOTA-C21 showed a maximum at 410 nm in water solution. When a titration with Ga^{3 +} was performed, no variations were observed at λ_max suggesting that the keto-enol moiety was not involved in gallium complexation. On the other hand, an increase in absorbance around 300 nm upon metal addition was detected, supporting that DOTA was the main moiety involved in metal chelation (Figure S9). The free ligand showed weak fluorescence emission upon excitation at λ_max (410 nm) with a maximum at 525 nm (Figure S10). These results differ from previously reported curcumin studies. In fact, while fluorescence quenching occurred due to gallium chelation by the keto-enol moiety [14], herein, emission spectra of DOTA-C21 were only slightly affected by gallium addition. This opposite behaviour furthermore confirmed the involvement of DOTA in gallium chelation rather than the keto-enol group, confirming what was seen by MS and NMR analyses.

Initially, DOTA-C21 was labelled with gallium-68 by preprocessing the generator elution by cation exchange purification [26]. This resulted in efficient labelling with high RCY and RCP after 5 min of incubation of just 10 nmol of precursor. In order to simplify the procedure, labelling was also attempted without preprocessing of the generator elutions. In these conditions, labelling was less effective, further purification of the final product was necessary due to the presence of high amounts of hydrolysed products, and 80 nmol of precursor was needed to drive the reaction to completion. This behavior could be ascribed to the presence of high levels of metal impurities that come from the generator and potentially compete with gallium-68 in the coordination with DOTA [19]. It is worth noting, that DOTA is known to have slow kinetics of complexation and requires harsh conditions for labelling. For these reasons, use of DOTA as a chelator for gallium-68 may enhance hydrolysis of the product and decomposition of the precursor. Use of chelators with smaller cavities such as NOTA and derivatives, or some acyclic chelators recently reported in literature such as HBED-CC or THP, would likely resolve these problems [27].

Similar to other gallium-68 labelled curcuminoid derivatives reported in literature [15] but differently from ⁶⁸GaCl₃, ⁶⁸Ga-DOTA-C21 exhibited a time-dependent uptake in HT29 cells suggesting a relationship between the curcumin-like structure and the ability of the cells to bind and internalize the radiotracers. In particular, the addition of a specific chelator connected to a phenolic moiety appears to be advantageous for the internalization of the compound when compared to the our previously reported tracers where the keto-enol group was exploited to form gallium-68 complexes [15]. Once internalized or bound to the cell membrane, ⁶⁸Ga-DOTA-C21 showed a slow efflux rate and 75% of the compound was still retained by the cells after 60 min of incubation. The mechanism by which curcumin or its derivatives are taken up in colorectal tumours has not been elucidated so far. It has been reported that curcumin has a structure suitable to bind nuclear vitamin D receptor and plays a role in colon cancer chemoprevention thanks to this property [28,29] but no specific experiments have been performed by the authors to confirm this finding. To test the hypothesis of VDR-mediated curcumin uptake, we analysed the VDR expression in HT29 cells and we found that this expression was higher than in other tumour cell lines and human lymphocytes. Then, to assess VDR-specificity, calcitriol, the hormonally active metabolite of vitamin D and VDR natural ligand, was used as a blocking agent. This would clarify the potential involvement of these receptors in ⁶⁸Ga-DOTA-C21 uptake. Unfortunately, uptake of ⁶⁸Ga-DOTA-C21 was not influenced by the presence of the excess of calcitriol (Figure 6C) therefore providing evidence that curcumin and calcitrol are not competitive substrates for the same binding site on VDR. In conclusion, seeing as this is a topic of great interest, further studies are necessary to evaluate the cellular uptake mechanism of curcumin and its radiolabelled derivatives. When the mechanism of accumulation will be elucidated, considerations about the structure-affinity of ⁶⁸Ga-DOTA-C21 in comparison to other compounds will be possible.

The stability tests in whole blood (Figure 7) attested to a certain lability of the curcumin backbone that was probably partially metabolized into more polar products, which then underwent renal excretion in physiological conditions, as proved during the biodistribution study (kidney uptake 11.86 ± 1.1% ID/g at 1.5 h post injection). However, it was shown that 57 ± 3% of ⁶⁸Ga-DOTA-C21 complexes remained intact after 120 min of incubation in human blood, comparing favourably with our other previously reported gallium-68 complexes [14], where the metal, coordinated by the keto-enol moiety, was rapidly released. Stability of ⁶⁸Ga-DOTA-C21 in mouse blood appears comparable, if not superior, to previously reported F-18 labelled curcumin derivatives where the amount of unchanged radiotracer in rat plasma after 10 min post injection comprised only 45% of the total injected radioactivity [30]. ⁶⁸Ga-DOTA-C21 exhibited a slow absorption rate into the main organs and a high amount of activity (12.73 ± 1.9% ID/g) was recorded in blood after 1.5 h post injection suggesting a strong binding interaction with serum albumin as reported for curcumin and their derivatives in general [30,31]. Significant uptake of ⁶⁸Ga-DOTA-C21 occurred in the intestine (13.56 ± 3.3% ID/g), lungs (8.42 ± 0.8% ID/g), liver (5.81 ± 0.5% ID/g) and heart (4.70 ± 0.4% ID/g). ⁶⁸Ga-DOTA-C21 rapidly accumulated in colorectal xenograft tumours with an uptake of 3.57 ± 0.3% ID/g after 1.5 h post injection. Compared to previously reported ¹⁸F-labelled curcumin derivatives, which exhibit fast clearance from the blood to the liver and fast hepatobiliary excretion to the intestine, ⁶⁸Ga-DOTA-C21 blood clearance was slow and it showed both renal and hepatobiliary excretion. Generally, higher kidney and lower intestine uptake with respect to the fluorinated compounds were found. However, the amount of radiotracer accumulated in the intestine might still be a concern since it could mask the colorectal cancer uptake in a potential human examination. Liver uptake was comparable, indicating that ⁶⁸Ga-DOTA-C21 remained partially metabolised, likely via hexahydrocurcumin-glucuroniside conjugation route. Similar to ¹⁸F-radiolabelled compounds, brain uptake was almost negligible [30,32].

4. Materials and Methods

5. Conclusions

The new DOTA derivative of curcumin (DOTA-C21) displayed coordinating property typical of DOTA structure, with the involvement of nitrogen atoms of the rings and two carboxylic arms, as demonstrated by NMR and MS analysis. Gallium-68 labelling conditions are harsh (95 °C, 5′), however they provide good RCY and RCP in spite of the methods used for the reaction (prepurification of the eluate or direct labelling and postpurification). Despite high instability of curcumin in physiological conditions, stability of ⁶⁸Ga-DOTA-C21 in mouse blood appears comparable, if not superior, to previously reported ¹⁸F-labelled curcumin derivatives, and these data are compatible with diagnostic applications. ⁶⁸Ga-DOTA-C21 rapidly accumulated in colorectal xenograft tumours but after 1.5 h post injection, exhibits a slow absorption rate into the main organs and a high amount of activity in blood. Unfortunately, the high uptake in blood, liver and intestines prevents the direct use of this derivatives as an imaging agent for colorectal cancer. However, further studies are needed to understand the mechanism of accumulation of ⁶⁸Ga-DOTA-C21 and to design strategies to increase its selectivity for colorectal tumour since the molecular structure of this radiotracer is an interesting foundation to develop further compounds with improved stability and pharmacokinetics.

Acknowledgments

Abbreviations

Supplementary Materials

The following supplementary materials are available online http://www.mdpi.com/1420-3049/24/3/644/s1: Chemical characterization of ligand and complexes, Quality controls on radioactive preparations, Figure S1: ESI-LC-MS analysis of DOTA-C21,Figure S2: ¹H-NMR spectra of DOTA-C21 in D₂O and in MeOD-d₄ at 25 °C, Figure S3: ¹³C-NMR spectra of DOTA-C21 in MeOD-d₄ at 25 °C, Figure S4: ESI-LC/MS analysis of Ga-DOTA-C21, Figure S5: Detailed ESI-LC/MS of one Ga-DOTA-C21 isomer, Figure S6: ¹H-NMR titration of DOTA-C21 with Ga³⁺, Figure S7: Aliphatic region of ¹H-,¹H-COSY-NMR spectrum of Ga-DOTA-C21 in MeOD-d₄ at 25 °C ([DOTA-C21] = 0.63 mM), Figure S8: LC/MS fragmentation experiments on curcumin, Figure S9: UV–vis absorption spectra of the system Ga³⁺/DOTA-C21, Figure S10: Fluorescence emission spectra of the system Ga^{3 +} /DOTA-C21,Figure S11: TLC analyses of ⁶⁸Ga-DOTA-C21 prepared by using the post processing method, Figure S12: Average biodistribution of ⁶⁸Ga-DOTA-C21 extracted from micro-PET data at various time points post i.p. and i.v. injection. Correlation between T/M ratio and tumour size, Figure S13: In vivo stability of ⁶⁸Ga-DOTA-C21 in murine blood samples.

The information comes from:
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6384893/

Abstract

1. Introduction

Immunoglobulins, in particular monoclonal antibodies (mAbs) are highly attractive targeting structures due to their extraordinary specificity and selectivity and are well established for therapeutic applications, particularly in the field of oncology [1,2]. Because of their favorable targeting abilities and therefore an ever increasing clinical importance, mAbs have regained interest also as imaging agents, in particular for positron emission tomography (PET) applications [3,4,5]. In principle mAbs can be directly radiolabelled either by direct incorporation of radiohalogens or by attaching a chelator to the protein in case of radiometals. Prolonged circulation time and slow distribution within the organism, however, restricts its use to long-lived radionuclides e.g., zirconium-89 (3.26 d) and iodine-124 (4.18 d). This multiday circulation paired with slow radioactive decay provides unfavorable radiation to healthy tissue and adds significantly to the overall radiation burden of patients.

In order to overcome this problem, various pretargeting methodologies have been reported and reviewed recently [6,7,8], enabling a straight forward two-step approach. Thereby the modified antibody is administered, allowed to accumulate at the target site and be eliminated from the bloodstream followed by injection of the radioactive payload to form the radioimmunoconjugate in vivo. This provides certain advantages as it facilitates the use of short-lived radioisotopes for PET applications e.g., gallium-68 (1.13 h), fluorine-18 (1.83 h) and copper-64 (12.7 h) and significantly reduces the radiation dose to healthy tissue, since the radioligand either finds its binding partner to form stable conjugates or is rapidly eliminated due to its small size. Furthermore, it allows to apply radiolabelling at high temperatures using high concentrations of organic solvents if necessary—harsh conditions, where the structural integrity of antibodies would be severely in danger when direct labelling was performed.

Among these pretargeting strategies the inverse electron-demand Diels-Alder reaction (IEDDA) between 1,2,4,5-tetrazines (Tz) and trans-cyclooct-2-enes (TCO) has gained enormous attention mainly due to the exceptionally fast reaction kinetics and high selectivity between the reaction partners even in complex biological systems as encountered in vivo [9,10]. Various preclinical studies demonstrated the feasibility of this approach for molecular imaging using PET-radioisotopes with promising results [11,12,13,14]. The application on humans, however, remains unsuccessful due to the increased blood volume in humans and may therefore lead to insufficient accumulation due to accelerated elimination of the small-sized radioligand.

Related to this, recent study investigated whether an increase of Tz motifs by chelator scaffolding, i.e., multimerization, can improve the binding efficiency and thereby improve imaging contrast. This could contribute in particular to the application of gallium-68 for pretargeted immuno-PET imaging. For this purpose we utilized the macrocyclic chelator Fusarinine C for the design of mono- and multimeric Tz-conjugates as presented in Scheme 1 for a proof-of-concept study, on potentially improved pretargeting for imaging with gallium-68 by applying multimerization. We chose non-internalizing anti-CD20 antibody rituximab (RTX) modified with TCO as targeting vector. Radiolabelling was conducted at room temperature within minutes and the ⁶⁸Ga-labelled conjugates showed reasonable hydrophilicity and excellent stability in human serum. Protein binding, however, remained comparable within the conjugates but was generally high. The ⁶⁸Ga-labelled multimeric conjugates showed a higher binding capacity towards TCO-motif bearing RTX. Furthermore, cell-binding studies revealed highly specific targeting properties and the binding of [⁶⁸Ga]Ga-FSC-Tz multimers to CD20-expressing Raji cells increased with the number of Tz-motifs attached to the chelator. Imaging studies in a simplified pretargeting mouse model proved the trend for improved targeting. We therefore conclude, that multimerization bears a great potential to improve IEDDA related pretargeting when short-lived PET-radioisotopes, particularly gallium-68, are used. Further investigations in established tumor models are warranted to confirm these promising findings.

2. Results

2.2. In Vitro Evaluation

Stability studies of ⁶⁸Ga-labelled conjugates in fresh human serum and PBS as control revealed high stability as no major degradation was observed over a period of 4 h. Corresponding radio-RP-HPLC chromatograms are presented in Figure S5. The results of logD studies and the ability to bind to serum proteins of ⁶⁸Ga-labelled conjugates are summarized in Table 2. They revealed suitable hydrophilicity with minor decrease when increasing the number of Tz residues. All conjugates showed very high protein binding with minor differences between mono- and multimeric [⁶⁸Ga]Ga-Tz-ligands.

Table 2

Distribution coefficient (logD) and protein binding of ⁶⁸Ga-labelled FSC-based Tz-conjugates.

68Ga-Labelled Compound	LogD (pH 7.4)	Protein Binding (%)
		1 h	2 h	4 h
Tz-monomer	−1.64 ± 0.02	61.8 ± 0.2	63.8 ± 2.1	64.0 ± 1.4
Tz-dimer	−1.35 ± 0.01	67.0 ± 2.4	65.9 ± 1.3	68.4 ± 0.3
Tz-trimer	−1.00 ± 0.06	70.5 ± 0.7	69.5 ± 0.4	67.8 ± 0.4

Data are presented as mean ± SD (n = 3)

The non-internalizing anti-CD20 monoclonal antibody rituximab was modified with the TCO motif similar to a previously published procedure [15] and corresponding FACS analysis of CD20-expressing Raji cells incubated with both, TCO-modified and non-modified RTX showed high target specificity (Figure S6), thus demonstrating that the binding ability was not altered by the TCO modification.

The binding capacity of ⁶⁸Ga-labelled mono- and multimeric Tz-ligands was assessed via competitive binding on immobilized RTX-TCO using the non-labelled conjugates as competitor and is presented in Figure 1. The binding of the [⁶⁸Ga]Ga-Tz-monomer was reduced by 50% at a competitor concentration of 486 ± 52 nM when challenged with the non-labelled monomer, whereas the non-labelled dimer (112 ± 6 nM) and trimer (100 ± 10 nM) reduced the binding at significantly lower concentrations. The binding of the [⁶⁸Ga]Ga-Tz-dimer was reduced by half at 95 ± 25 nM in competition with its non-labelled counterpart and at a comparable concentration with the non-labelled trimer (92 ± 15 nM), whereas a decrease to 50% was only achieved at a much higher concentration in competition with non-labelled monomer (865 ± 263 nM). Binding studies of the [⁶⁸Ga]Ga-Tz-trimer showed a comparable trend as the non-labelled trimer reduced the binding by 50% at 147 ± 49 nM and the dimer at 258 ± 60 nM; whereby a significantly higher amount of the monomer (2987 ± 1664 nM) was needed for a 50% binding reduction. Overall in all assays improved binding of di- and trimer over monomer was observed.

Open in a separate window

Figure 1

Competitive binding studies of [⁶⁸Ga]Ga-Tz-monomer (A), [⁶⁸Ga]Ga-Tz-dimer (B) and [⁶⁸Ga]Ga-Tz-trimer (C) on immobilized RTX-TCO using the non-labelled counterparts as competitor.

The results of cell-binding studies on CD20-expressing Raji cells pre-treated with RTX or RTX-TCO prior to incubation with ⁶⁸Ga-labelled Tz-ligands are presented in Figure 2. All ⁶⁸Ga-labelled conjugates showed highly specific targeting properties as the amount of unspecific bound radioligand to RTX pre-treated Raji cells was negligible low (<1%). The binding of ⁶⁸Ga-labelled Tz-ligands on RTX-TCO bound Raji cells increased with the grade of multimerization and was 4.01 ± 0.24% for [⁶⁸Ga]Ga-Tz-monomer, 7.35 ± 0.77% for [⁶⁸Ga]Ga-Tz-dimer and 15.93 ± 0.88 for [⁶⁸Ga]Ga-Tz-trimer, respectively.

Figure 2

Cell-binding studies of ⁶⁸Ga-labelled FSC-based Tz-ligands on CD20-expressing Raji cells pre-treated with anti-CD20 antibody RTX (negative control, black bars) and its TCO modified counterpart (white bars).

2.3. In Vivo Evaluation

Biodistribution studies in non-tumour xenografted BALB/c mice 1 h after administration of the ⁶⁸Ga-labelled Tz-ligands are shown in Figure 3. In general, accumulation in non-targeted tissue was significantly lower for the [⁶⁸Ga]Ga-Tz-monomer compared to the [⁶⁸Ga]Ga-Tz-trimer, whereby the difference between the multimers was less pronounced. In particular, the multimeric Tz-ligands showed slower blood clearance and higher accumulation in renal tissue compared to the monomeric conjugate. The conjugates generally showed low accumulation in non-targeted tissue and low retention in critical organs (e.g., muscle, bone).

Figure 3

Biodistribution studies of ⁶⁸Ga-labelled mono- and multimeric FSC-based pretargeting agents in normal BALB/c mice 1 h p.i. presented as percentage of total injected activity per gram tissue (n = 3).

Imaging studies using BALB/c mice i.m. injected with RTX and RTX-TCO as tumour surrogate 5 h prior to r.o. administration of the radioligand and imaging performed 90 min p.i. are presented in Figure 4. The results mainly confirmed the suitable in vivo distribution profile of FSC-based Tz-ligands radiolabelled with gallium-68 with main activity in kidneys and bladder and some blood pool activity for the ⁶⁸Ga-labelled multimers. Moreover, the accumulation in RTX-TCO positive muscle tissue increased with ascending numbers of Tz motifs and was ~1% for the [⁶⁸Ga]Ga-Tz-monomer, ~2% for the [⁶⁸Ga]Ga-Tz-dimer and ~3% for the [⁶⁸Ga]Ga-Tz-trimer. Surprisingly, the accumulation in TCO negative tissue was also approximately 1% showing negligible differences between the different radioligands.

Figure 4

Static µPET/CT image of i.m. RTX(-TCO) pre-treated normal BALB/c mice 5 h after treatment and 90 min p.i. of the ⁶⁸Ga-labelled Tz-monomer (a), Tz-dimer (b) and Tz-trimer (c). (1 = coronal slice; 2 = 3D volume rendered projections; both prone position).

3. Discussion

IEDDA-based pretargeting has become increasingly popular for molecular imaging as well as radioimmunotherapy over the past few years [16,17,18,19,20,21]. Despite recent advancements towards structural improvement of cyclen- and TACN-based Tz-probes [22,23] or the synthesis of TCO-bearing dendrimers [24], the design of targeting probes bearing multiple Tz-motifs for potentially improved pretargeting has scarcely been investigated. Devaraj and co-workers reported on polymer modified tetrazines (PMT) radiolabelled with fluorine-18 [25] and gallium-68 [26] for PET applications while Zlatni et al. recently demonstrated the targeting applicability for microbubbles bearing multiple Tz-motifs towards prostate specific membrane antigen for ultrasound related diagnostic purposes [15]. However, investigations on the use of small-sized multimeric Tz-ligands have not been reported yet.

FSC is a suitable chelating scaffold for PET radiometals, particularly gallium-68 and zirconium-89 [27,28]. Its unique structural properties enable straight forward mono- and multimeric tracer design [29,30,31,32]. This novel chelator, therefore, represented a very suitable scaffold for the synthesis of mono- and multimeric small-sized Tz-bearing probes for radiolabelling with gallium-68 to evaluate potential benefits for IEDDA-based pretargeting by increasing the number of Tz-residues. The resulting conjugates showed reasonable hydrophilicity and increasing the number of Tz-residues from one to three did not alter the water solubility too much, although being somewhat lower in comparison to linker modified monomeric conjugates [23]. Unexpectedly, all FSC-based Tz-conjugates showed high protein binding with negligible differences between the compounds. This can be related to the introduction of the pegylated Tz residue, since non-modified ⁶⁸Ga-labelled FSC-based precursors showed a protein binding of <3% (Kaepookum et al. unpublished results). This phenomenon has not been reported in the case of monomeric DOTA- and TACN-based derivatives [22,23] and might be disadvantageous by slowing down the distribution of the tracer to the TCO-interaction site. In vivo results, however, did not really reveal a major problem with too slow pharmacokinetics, since the washout from non-targeted tissue was still sufficiently rapid. Competitive binding assessments showed significantly reduced binding of [⁶⁸Ga]Ga-Tz-monomer by a factor of five when challenged with the multimeric conjugates in comparison with the non-labelled monomer. This corresponded with the significantly higher amounts of Tz-monomer needed, 8 to 20 fold respectively, to reduce the binding of the ⁶⁸Ga-labelled multimers by 50%, thus indicating that the binding capacity increased with the number of Tz residues. This was substantiated by the results of the cell binding studies, since the binding to CD20-expressing Raji cells increased by a factor of 1.8 for the [⁶⁸Ga]Ga-Tz-dimer and 3.9 for the [⁶⁸Ga]Ga-Tz-trimer in comparison to the [⁶⁸Ga]Ga-Tz-monomer. Biodistribution studies in non-tumour xenografted healthy BALB/c mice indicated suitable pharmacokinetics and showed a multimerization typical profile exhibiting slower blood clearance and increased kidney retention, similar to prior findings with the FSC-scaffold [31,32]. Imaging studies using a tumour surrogate model confirmed this trend and demonstrated increased binding of the ⁶⁸Ga-labelled multimeric Tz-conjugates. The accumulation in RTX-TCO pretreated muscle tissue doubled for the [⁶⁸Ga]Ga-Tz-dimer and was 3-fold higher for the [⁶⁸Ga]Ga-Tz-trimer compared to the [⁶⁸Ga]Ga-Tz-monomer.

In regard to imaging in living mice there was a clear improvement of target specific accumulation when switching from mono- to multimers. The use of our simplified tumour surrogate animal model, however, requires further investigations in established animal models to address the limitations of this study:

(1) The accumulation in non-targeted tissue, i.e., i.m. injection of RTX resulted to be ~1%. This effect was even more pronounced when choosing a shorter time interval of 2 h between i.m. administration of the mAb and radioligand injection (data not shown) and was only seen at the injection site but generally not in other tissue. We speculate that this is related to the injection of the antibody leading to tissue damage with increased tissue permeability and unspecific accumulation of the radioligand.

(2) Intravenously injected mAb stimulates accumulation at the target interaction site, but a non-negligible amount remains in circulation, thereby increasing the background signal when administering the radioligand. In order to reduce this amount, high molecular weight TCO-scavenger molecules exhibiting low vascular permeability (clearing agents) have been established with great success, significantly improving target-to-background (TTB) ratios [33,34]. The need for clearing agents was completely neglected in this study and we are fully aware that our animal model does not reflect the real situation regarding TTB ratios.

In summary, we have been able to show that FSC is a suitable scaffold for the design of multimeric Tz-conjugates for radiolabelling with gallium-68. Although multimeric Tz-ligands exhibited significant improvements towards IEDDA-based pretargeting additional studies in tumour models are warranted to explore the full potential of this promising concept. Furthermore, a highly interesting therapeutic approach is the release of drugs directly at the interaction site from antibody-drug conjugates (ADCs) mediated via IEDDA reaction between Tz and TCO [35,36,37]. It might be of interest for future perspectives if multimeric Tz-conjugates can boost the release of drugs improving this highly promising “click-to-release” strategy.

4. Materials and Methods

Acknowledgments

Abbreviations

Supplementary Materials

The following are available online at http://www.mdpi.com/1424-8247/11/4/102/s1, Figure S1: ¹H-NMR spectrum of DAFC-PEG₅-Tz (Tz-monomer), Figure S2: ¹H-NMR spectrum of of MAFC-(PEG₅-Tz)₂ (Tz-dimer), Figure S3: ¹H-NMR spectrum of FSC-(PEG₅-Tz)₃ (Tz-trimer), Figure S4: Representative RP-HPLC chromatograms of mono- and multimeric FSC-Tz conjugates (A, UV/vis chromatograms) and their ⁶⁸Ga-labelled counterparts (B, radio-chromatograms), Figure S5: Representative radio-RP-HPLC chromatograms of the stability assessment of ⁶⁸Ga-labelled mono- and multimeric FSC-Tz conjugates incubated with fresh human serum (A) and PBS (B), Figure S6: Fluorescence-activated cell sorting of unstained Raji cells, RTX, RTX-TCO, secondary antibody (APC), RTX + APC and RTX-TCO + APC (order from left to right).

Author Contributions

L.V. carried out the synthesis of FSC-Tz conjugates under guidance of B.M. and both provided the corresponding part of the manuscript; B.M. evaluated the NMR data; D.S. evaluated the protocol for radiolabelling with gallium-68, reviewed the literature, summarized the results and has written the major part of the manuscript; C.R. performed cell culture, animal housing and animal care; S.M. modified the antibody and evaluated the radiolabelled conjugates in vitro as well as in vivo. K.S. provided the Raji cells and carried out FACS analysis of the antibodies. M.P. evaluated the tumour-surrogate model and performed in vivo imaging studies. C.D. was responsible for conceptualization, funding acquisition, project administration and supervision.

Funding

This research was funded by the Austrian Science Foundation (FWF) grant P 25899-B23 and by the Czech Ministry of Education Youth and Sports grant LO1304.

Conflicts of Interest

The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, and in the decision to publish the results.

The information comes from:
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6316846/