Oligoproline-Based Mono- and Bivalent Minigastrin and Octreotide Analogues: Synthesis, in Vitro, and in Vivo Characterization

Abstract

Medullary thyroid cancer (MTC) and small cell lung cancer (SCLC) are both neuroendocrine tumours (NETs) with only very limited therapy options. The overexpression of peptide binding G-protein coupled receptors on the surface of cancer cells can be used for selective tumour targeting with radioactively labelled peptides for imaging and therapy. However, peptidic radioligands for the so-called peptide receptor radionuclide therapy (PRRT) often have limitations in terms of stability, specificity, bioavailability, and tumour tissue retention. The heterogeneous expression profile of the target receptors is another important factor that can be limiting for PRRT, since target receptor expression levels can fall below the threshold of detection depending on the state of the disease. With this thesis several strategies to address these limitations were investigated. We explored the advantages of multivalent ligands with more than one recognition motif for the simultaneous targeting of either two receptors of the same type (homodivalent ligands) or two different types of receptors (heterodivalent ligands). The cholecystokinin receptor subtype 2 (CCK2R) and the somatostatin receptor subtype 2 (SSTR2) were chosen as target receptors, since they are both overexpressed on the cells of several types of NETs including MTC and SCLC. Minigastrin analogues to target the CCK2R and octreotide analogues to target the SSTR2 were conjugated over a rigid oligoproline backbone, which ensures well defined distances between the recognition motifs, providing additional information about a distance-dependence of the biological performance. We successfully developed homodivalent ligands to target the CCK2R with higher in vitro cellular uptake compared to the non-conjugated reference compound, demonstrating the benefit of multivalency. We further could show a distance-dependence in the in vitro performance of the homodivalent octreotide based ligands that target the SSTR2. The heterogeneity of NETs was successfully addressed by our heterodivalent ligand with good CCK2R mediated and SSTR2 mediated cellular uptake into tumour cells, which therefore is less dependent on the expression level of one individual target receptor. The search for optimized recognition motifs for multivalent ligands led to the investigation and optimization of novel monovalent minigastrin- and octreotide-type ligands. Minigastrin analogues with different linkers gave important insights into the structure activity relationship between gastrins and the CCK2R (especially regarding the role of the pentaglutamic acid sequence in gastrin), whereas modifications of octreotides led to the development of a highly potent [Tyr3]octreotide analogue. In addition, the attachement of a human serum album binding moiety to monovalent minigastrin analogues for significantly improved pharmacokinetics was investigated.