Design, synthesis and evaluation of somatostatin analogs for improved imaging and radionuclide therapy

The development of more selective anticancer drugs, with better discrimination between tumor cells and normal cells, is possibly the most important goal of current anticancer research. One avenue towards the development of more selective, better anticancer drugs consists of the targeted delivery of bioactive molecules to the tumor environment by means of binding molecules that are specific for tumor-associated markers. Since the discovery that many tumor types overexpress receptors for peptide hormones there has been an exponential growth in the development of radiolabeled peptides for diagnostic and therapeutic applications in oncology. The first diagnostically studied and also radiotherapeutically employed regulatory peptides were analogs of somatostatin (SS). Despite the explosion of knowledge in recent years in the somatostatin field, a great deal remains to be discovered. In particular, developing the potential of somatostatin analogs for cancer treatment will require a more complete understanding of their intracellular actions and interactions. However, there is no doubt that SS-analog radiotherapy has significantly improved the quality of life of many patients and this is very important for a palliative treatment. Limitations to somatostatin-based peptide receptor radiotherapy are principally due to poor tumor penetration of the radioligand and insufficient accumulation within the neoplastic cell. In addition, low or variable expression of somatostatin receptor subtypes may lead to poor tumor localization of radiolabeled somatostatin analogs. This dissertation presents attempts to overcome these problems associated with targeted therapy and/or imaging with radiolabeled somatostatin analogs. In a first step, we focused on the developing of new radiolabeled somatostatin-based analogs with improved pharmacological profile. Thus, employing parallel solid phase we synthesized a series of analogs of DOTA-octreotide with modifications on the third residue (Phe3). From the small library synthesized two conjugates showed very promising preclinical properties, DOTANOC and DOTA-BOC. Moreover, the Thr8-modified versions of these two compounds (DOTA-NOC-ATE and DOTA-BOC-ATE) also displayed superior preclinical profiles if compared with somatostatin analogs used in the clinic. Subsequently, these four new derivatives are currently in clinical trials and the first promising results are already described herein. Nevertheless, we planned additional structural studies to better understand the results obtained in this series of compounds. In a second step we aimed at the improvement of pharmacokinetics for this type of conjugates. The biggest problem associated with this category of drugs is their excretion pathway. A drawback of the derivatives discussed above is their increased lipophilicity. This is translated in vivo by increased hepatobilliary excretion, hence impairing on the abdominal imaging. Our strategy was the introduction of hydrophilic spacers between the chelator and
the peptide. The derivatives thus obtained partially lose the broad affinity profile of the parent
compounds, although the modifications performed are remote from the pharmacophore.
Nevertheless, they show increased internalization in sstr2-expressing cells and good
biodistribution profile in rats. However, the animal tumor model used did not permit a
genuine evaluation of the hepatobiliary excretion pathway.
While favoring the kidney clearance route, nephrotoxicity can be one of the
consequences, particularly in case of targeted radiotherapy. That is why we proposed the
addition of a PAH unit (recognized by the OAT system of the kidneys) to the DOTAsomatostatin
analogs, expecting a faster excretion, without accumulation in these organs.
Nevertheless, this unit should probably be situated on the chelator or at the peptide Nterminus,
because the conjugate described herein, with PAH at the C-terminus of DOTA-
[Tyr3]-octreotide, loses almost completely the pharmacological properties of the parent
compound (probably due to the proximity to the pharmacophore sequence).
In a third step we proposed a strategy to modify the pharmacodynamics of DOTAsomatostatin
derivatives, by targeting the cell nucleus. Thus, we added an NLS unit to
DOTA-[Tyr3]-octreotide. After testing the best design of such a construct, we selected the
best derivative and performed biological trials. Our hypothesis was confirmed in nuclei
isolation assays, showing increased cell nuclear uptake for the trifunctional NLS-conjugate in
comparison with the parent compound. Moreover, the new molecule has increased uptake and
higher retention in tumoral cells in vitro. These promising results, if confirmed in vivo, could
allow the successful use of Auger-electron emitting radionuclides in targeted radiotherapy.
As mentioned at the beginning of this section, understanding the intracellular
trafficking of somatostatin receptors is part of the work for developing the potential of
somatostatin analogs for therapy. Since phosphorylation is believed to be a critical step in
both receptor internalization and desensitization, an antibody directed against the
phosphorylated receptor may give useful insights into this process. This is the reason for the
phosphorylated peptide synthesis described herein, meant for antibody production. The first
biological assays performed with the obtained antibody are encouraging.
With the advances in proteomics and genomics more targets are being identified and
the opportunities of using combined therapies are increasing. The treatment of any cancer is
unlikely to be optimal with a single drug. There are of course major issues to be addressed,
including the difficulty of selecting the best combination, but a major problem remains the
complete elucidation of intracellular trafficking of such drugs and their targets, particularly in
the field of GPCRs as molecular targets for cancer diagnosis and therapy.