¹⁷⁷Lu Labeled Cyclic Minigastrin Analogues with Therapeutic Activity in CCK2R Expressing Tumors: Preclinical Evaluation of a Kit Formulation

Minigastrin (MG) analogues specifically target cholecystokinin-2 receptors (CCK2R) expressed in different tumors and enable targeted radiotherapy of advanced and disseminated disease when radiolabeled with a beta emitter such as ¹⁷⁷Lu. Especially truncated MG analogues missing the penta-Glu sequence are associated with low kidney retention and seem therefore most promising for therapeutic use. Based on [d-Glu¹,desGlu^2–6]­MG (MG11) we have designed the two cyclic MG analogues cyclo^1,9[γ-d-Glu¹,desGlu^2–6,d-Lys⁹]­MG (cyclo-MG1) and cyclo^1,9[γ-d-Glu¹,desGlu^2–6,d-Lys⁹,Nle¹¹]­MG (cyclo-MG2). In the present work we have developed and preclinically evaluated a pharmaceutical kit formulation for the labeling with ¹⁷⁷Lu of the two DOTA-conjugated cyclic MG analogues. The stability of the kits during storage as well as the stability of the radiolabeled peptides was investigated. A cell line stably transfected with human CCK2R and a control cell line without receptor expression were used for <i>in vitro</i> and <i>in vivo</i> studies with the radioligands prepared from kit formulations. In terms of stability ¹⁷⁷Lu-DOTA-cyclo-MG2 showed advantages over ¹⁷⁷Lu-DOTA-cyclo-MG1. Still, for both radioligands a high receptor-mediated cell uptake and favorable pharmacokinetic profile combining receptor-specific tumor uptake with low unspecific tissue uptake and low kidney retention were confirmed. Investigating the therapy efficacy and treatment toxicity in xenografted BALB/c nude mice a receptor-specific and comparable therapeutic effect could be demonstrated for both radioligands. A 1.7- to 2.6-fold increase in tumor volume doubling time was observed for receptor-positive tumors in treated versus untreated animals, which was 39–73% higher when compared to receptor-negative tumors. The treatment was connected with transient bone marrow toxicity and minor signs of kidney toxicity. All together the obtained results support further studies for the clinical translation of this new therapeutic approach

Novel Bifunctional Cyclic Chelator for ⁸⁹Zr Labeling–Radiolabeling and Targeting Properties of RGD Conjugates

Within the last years ⁸⁹Zr has attracted considerable attention as long-lived radionuclide for positron emission tomography (PET) applications. So far desferrioxamine B (DFO) has been mainly used as bifunctional chelating system. Fusarinine C (FSC), having complexing properties comparable to DFO, was expected to be an alternative with potentially higher stability due to its cyclic structure. In this study, as proof of principle, various FSC-RGD conjugates targeting α_vß₃ integrins were synthesized using different conjugation strategies and labeled with ⁸⁹Zr. <i>In vitro</i> stability, biodistribution, and microPET/CT imaging were evaluated using [⁸⁹Zr]­FSC-RGD conjugates or [⁸⁹Zr]­triacetylfusarinine C (TAFC). Quantitative ⁸⁹Zr labeling was achieved within 90 min at room temperature. The distribution coefficients of the different radioligands indicate hydrophilic character. Compared to [⁸⁹Zr]­DFO, [⁸⁹Zr]­FSC derivatives showed excellent <i>in vitro</i> stability and resistance against transchelation in phosphate buffered saline (PBS), ethylenediaminetetraacetic acid solution (EDTA), and human serum for up to 7 days. Cell binding studies and biodistribution as well as microPET/CT imaging experiments showed efficient receptor-specific targeting of [⁸⁹Zr]­FSC-RGD conjugates. No bone uptake was observed analyzing PET images indicating high <i>in vivo</i> stability. These findings indicate that FSC is a highly promising chelator for the development of ⁸⁹Zr-based PET imaging agents

Additional file 2: Figure S2. of Influence of a novel, versatile bifunctional chelator on theranostic properties of a minigastrin analogue

HPLC chromatograms at different purification steps. Top: Peptide after cleavage of the rink amid resin; Middle: Fraction of the purification from the preparative HPLC; Bottom: pure peptide with <4 % oxidised content

Distribution coefficient and protein binding of mono- and multimeric conjugates of two different MG analogues (-Met/Nle) radiolabelled with gallium-68.

<p>Distribution coefficient and protein binding of mono- and multimeric conjugates of two different MG analogues (-Met/Nle) radiolabelled with gallium-68.</p

Synthesis of FSC-based mono- and multivalent conjugates.

<p>Schematic overview on the synthetical pathway of mono- and multimeric FSC-based minigastrin bioconjugates (stereochemistry omitted).</p

Receptor affinity studies (IC₅₀) with ^natGa-bound peptides as competitor and human ¹²⁵I-[Leu¹⁵]-Gastrin as radioligand on whole A431-CCK2R cells.

<p>Receptor affinity studies (IC₅₀) with ^natGa-bound peptides as competitor and human ¹²⁵I-[Leu¹⁵]-Gastrin as radioligand on whole A431-CCK2R cells.</p

<i>In vitro</i> metabolite assessment, purification and identification of non-radioactive metabolites formed by artificial enzymatic degradation.

<p>Representative RP-HPLC chromatograms (UV absorption at λ = 220 nm) of enzymatic degradation of Fe-Trimer-Met (A) and Fe-Trimer-Nle (B). Below the arrow chromatograms of purified main metabolites are shown including molecular weight of corresponding mass analysis.</p

<i>In vitro</i> metabolite assessment of ⁶⁸Ga-labelled trimers by artificial enzymatic degradation.

<p>Representative radio-RP-HPLC chromatograms of enzymatic degradation of ⁶⁸Ga-labelled trimeric Met- (A) and Nle (B) conjugates <i>in vitro</i> (major metabolite formation assigned with arrows).</p

<i>In vitro</i> stability assessment of radiopeptides in rat organ homogenates.

<p>⁶⁸Ga-labelled mono- and multimeric peptide conjugates incubated with varying concentrations, 1% (A) and 10% (B) respectively, of rat kidney and liver homogenates. Data are presented as mean (n = 2), error bars omitted.</p

Radiolabelling of FSC-based mono- and multimers with gallium-68.

<p>Representative radio-RP-HPLC chromatograms of ⁶⁸Ga-labelled mono- and multimers with Met-sequence (A) and Nle-sequence (B).</p