Sylvie Froidevaux

Neuroendocrine tumor targeting: Study of novel gallium-labeled somatostatin radiopeptides in a rat pancreatic tumor model

Somatostatin analogs labeled with radionuclides are of considerable interest in the diagnosis and therapy of SSTR‐expressing tumors, such as gastroenteropancreatic, small cell lung, breast and frequently nervous system tumors. In view of the favorable physical characteristics of the Ga isotopes 67Ga and 68Ga, enabling conventional tumor scintigraphy, PET and possibly internal radiotherapy, we focused on the development of a Ga‐labeled somatostatin analog suitable for targeting SSTR‐expressing tumors. For this purpose, 3 somatostatin analogs, OC, TOC and TATE were conjugated to the metal chelator DOTA and labeled with the radiometals 111In, 90Y and 67Ga. They were then evaluated for their performance in the AR4‐2J pancreatic tumor model by testing SSTR2‐binding affinity, internalization/externalization in isolated cells and biodistribution in tumor‐bearing nude mice. Surprisingly, we found that, compared to 111In or 90Y, labeling with 67Ga considerably improved the biologic performance of the tested somatostatin analogs with respect to SSTR2 affinity and tissue distribution. 67Ga‐labeled DOTA‐somatostatin analogs were rapidly excreted from nontarget tissues, leading to excellent tumor‐to‐nontarget tissue uptake ratios. Of interest for radiotherapeutic application, [67Ga]DOTATOC was strongly internalized by AR4‐2J cells. Furthermore, our results suggest a link between the radioligand charge and its kidney retention. The excellent tumor selectivity of Ga‐DOTA somatostatin analogs together with the different applications of Ga in nuclear oncology suggests that Ga‐DOTA somatostatin analogs will become an important tool in the management of SSTR‐positive tumors.

Preclinical Comparison in AR4–2J Tumor-Bearing Mice of Four Radiolabeled 1,4,7,10-Tetraazacyclododecane-1,4,7,10-Tetraacetic Acid-Somatostatin Analogs for Tumor Diagnosis and Internal Radiotherapy1

Somatostatin analogs labeled with radionuclides are of considerable interest in nuclear oncology as diagnostic or therapeutic tools for somatostatin receptor (SSTR)-expressing tumors. We investigated the suitability of DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid) as a replacement for the widely used diethylenetriaminepentaacetic acid, to enable stable labeling of somatostatin analogs with both therapeutic (90Y) and diagnostic (111In) radionuclides. The three clinically relevant somatostatin agonists, octreotide, vapreotide, and lanreotide, together with the newly designed Tyr3-octreotide (TyrOc), were conjugated to DOTA and labeled with 90Y or 111In. For all DOTA-somatostatin analogs tested, irrespective of the incorporated radionuclide, we observed favorable biodistribution profiles in AR4–2J tumor-bearing mice: 1) a rapid clearance from all SSTR-negative tissues except kidney; 2) a specific uptake in SSTR-positive tissues, including tumor; and 3) an excellent tumor penetration. The ma...

A Combinatorial Peptoid Library for the Identification of Novel MSH and GRP / Bombesin Receptor Ligands

A tripeptoid library was synthesized using 69 different primary amines in initially 69 individual reactions by the mix and split approach. The resulting library consisted of 328,509 (69(3)) single compounds, divided in 69 subpools each containing 4,761 entities. The 69 subpools were tested in two binding assays, one for alpha-MSH (alpha-melanotropin) and one for GRP (gastrin-releasing peptide)/bombesin. The sublibraries with the highest affinity to the MSH receptor (i.e. melanocortin type 1 or MC1 receptor) and, respectively, the GRP-preferring bombesin receptor were identified by an iterative process. Individual tripeptoids with good binding activity were resynthesized, analyzed and their dissociation constants and biological activity determined. The KD of the most potent MC1 receptor ligand was 1.58 mumol/l and that of the GRP-preferring bombesin receptor 3.40 mumol/l. Extension of this latter tripeptoid structure whose KD value increased to 280 nmol/l. A similar increase in activity was not observed with the most potent MSH tripeptoid ligand when extended by one residue, but a compound suitable for radioiodination and lacking the N-terminal amino group had a slightly higher binding activity than the tripeptoids (KD approximately 850 nmol/l). These results demonstrate that testing a peptoid library containing 328,509 single compounds led to the successful identification of new ligands for both the MC1 receptor as well as the GRP-preferring bombesin receptor.

Characterization of Transferrin Receptor in an Immortalized Cell Line of Rat Brain Endothelial Cells, RBE4

The content and distribution of transferrin receptors in an immortalized cell line, RBE4, derived from rat cerebral capillary endothelial cells was investigated using the monoclonal antibody MRC OX-26 (OX-26 mAb) specific for the rat transferrin receptor. An ELISA assay was developed with which the OX-26 mAb can be determined quantiatively. The detection limit of the assay was 10 pg or 0.07 fmol of murine antibody. With this technique accurate measurement of native antibody is now possible without the need for isotope labeling (iodination). Immunostaining of confluent monolayers of RBE4 cells using an antibody directed against the tight junction associated protein ZO-1 was indicative for structural intactness of RBE4 cell monolayers. OX-26 immunostaining demonstrated localization of the transferrin receptor at the plasma membrane and/or in the cytosol. Binding studies showed saturation of OX-26 mAb binding. The antibody binding analysis gave a dissociation constant (KD) of 17.1 +/- 1.2 nmol/l. The total amount of transferrin receptors present per cell was 70,800 +/- 17,000. Our results indicate that receptor binding of OX-26 mAb can be studied using an in vitro cell culture model of rat brain mircrovessel endothelium in conjunction with an ELISA technique for detection of native antibody. This approach will be used to investigate mechanisms of transendothelial transport of OX-26 in vitro.

Receptor-Mediated Tumor Targeting with Radiopeptides. Part 1. General Concepts and Methods: Applications to Somatostatin Receptor-Expressing Tumors

Radiolabeled peptides have become important tools in nuclear oncology, both as diagnostics and more recently also as therapeutics. They represent a distinct sector of the molecular targeting approach, which in many areas of therapy will implement the old “magic bullet” concept by specifically directing the therapeutic agent to the site of action. In this three-part review, we present a comprehensive overview of the literature on receptor-mediated tumor targeting with the different radiopeptides currently studied. Part I summarizes the general concepts and methods of targeting, the selection of radioisotopes, chelators, and the criteria of peptide ligand development. Then, the >400 studies on the application to somatostatin/somatostatin-release inhibiting factor receptor-mediated tumor localization and treatment will be reviewed, demonstrating that peptide radiopharmaceuticals have gained an important position in clinical medicine.

Dimeric DOTA-α-Melanocyte-Stimulating Hormone Analogs: Synthesis and In Vivo Characteristics of Radiopeptides with High In Vitro Activity

Dimeric analogs of α-melanocyte-stimulating hormone (α-MSH) labeled with radiometals are potential candidates for diagnosis and therapy of melanoma by receptor-mediated tumor targeting. Both melanotic and amelanotic melanomas (over-)express the melanocortin-1 receptor (MC1-R), the target for α-MSH. In the past, dimerized MSH analogs have been shown to display increased receptor affinity compared to monomeric MSH, offering the possibility of improving the ratio between specific uptake of radiolabeled α-MSH by melanoma and nonspecific uptake by the kidneys. We have designed three linear dimeric analogs containing a slightly modified MSH hexapeptide core sequence (Nle-Asp-His-d-Phe-Arg-Trp) in parallel or antiparallel orientation, a short spacer, and the DOTA chelator for incorporation of the radiometal. In vitro, all three peptides were more potent ligands of the mouse B16-F1 melanoma cell melanocortin-1 receptor (MC1-R) than DOTA-NAPamide, which served as standard. The binding activity of DOTA-diHexa(NC-NC)-amide was 1.75-fold higher, that of diHexa(NC-NC)-Gly-Lys(DOTA)-amide was 3.37-fold higher, and that of DOTA-diHexa(CN-NC)-amide was 2.34-fold higher. Using human HBL melanoma cells, the binding activity of diHexa(NC-NC)-Gly-Lys(DOTA)-amide was sixfold higher than that of DOTA-NAPamide. Uptake by cultured B16-F1 cells was rapid and almost quantitative. In vivo, however, the data were less promising: tumor-to-kidney ratios 4 hr postinjection were 0.11 for [111In]DOTA-diHexa(NC-NC)-amide, 0.26 for diHexa(NC-NC)-Gly-Lys([111In]DOTA)-amide, and 0.36 for [111In]DOTA-diHexa(CN-NC)-amide, compared to 1.67 for [111In]DOTA-NAPamide. It appears that despite the higher affinity to the MC1-R of the peptide dimers and their excellent internalization in vitro, the uptake by melanoma tumors in vivo was lower, possibly because of reduced tissue penetration. More striking, however, was the marked increase of kidney uptake of the dimers, explaining the unfavorable ratios. In conclusion, although radiolabeled α-MSH dimer peptides display excellent receptor affinity and internalization, they are no alternative to the monomeric DOTA-NAPamide for in vivo application.

HOMOLOGOUS REGULATION OF MELANOCORTIN-1 RECEPTOR (MC1R) EXPRESSION IN MELANOMA TUMOR CELLS IN VIVO

ABSTRACT As G protein-coupled receptors (GPCRs) are the target of numerous signaling molecules, including about half of the therapeutic drugs currently used, it is important to understand the consequences of homologous (ligand-induced) receptor regulation. Continuous exposure of GPCRs to agonist in vitro most frequently results in receptor down-regulation, but receptor up-regulation may occur as well. These phenomena are expected to play a role in the physiological adaptation to endogenous ligands and also in the response to repetitive administration of drugs in the clinic. However, there is little information on homologous regulation of GPCRs in vivo. Here, we report on the regulation of melanocortin-1 receptor (MC1R) expression in melanoma cells implanted into mice. Two melanoma cell lines were investigated, D10 and B16F1, which in vitro had previously been shown to undergo homologous receptor up- and down-regulation, respectively. After implantation into mice and exposure to the natural MC1R agonist α-melanocyte-stimulating hormone (α-MSH), cell-surface MC1R expression was evaluated by competition binding experiments in tumor membrane preparations. In B16F1 cells, a single injection of 50 to 500 µg α-MSH induced a rapid but moderate dose-dependent MC1R down-regulation which could be totally reverted within 16–24 h. By continuous administration of α-MSH via osmotic minipumps, MC1R down-regulation was considerably amplified and reached the level observed in vitro, demonstrating that prolonged receptor interaction was necessary to induce a maximal effect in vivo. Similar results were obtained in vitro, which demonstrates that homologous MC1R regulation in B16F1 cells is essentially independent of the physiological environment. In D10 cells, however, up-regulation could not be reproduced in vivo, suggesting that MC1R up-regulation is more dependent on the physiological environment. These results demonstrate the importance of in vivo receptor regulation studies, in particular in view of the potential use of MC1R as a target for melanoma therapy.

A Microplate Binding Assay for the Somatostatin Type-2 Receptor (SSTR2)

The clinical importance of somatostatin type-2 receptors (SSTR2) and the study of novel analogues of somatostatin such as OctreoScan or [Tyr3]-octreotide containing DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid) as metal chelator led us to develop a methodology to monitor the expression of SSTR2 on tumours of pancreatic origin (e.g. rat AR4-2J cancer cells). Usual binding assay protocols using the commercial [125I][Tyr1]-somatostatin radioligand failed, even in the presence of a cocktail of protease inhibitors with a broad spectrum of activity, possibly due to the high susceptibility of this tracer to proteases expressed in pancreatic cells. We prepared our own radioligand [125I][Tyr2]-octreotide which was shown to be much more resistant to degradation after incubation with AR4-2J plasma membranes. As expected, the increased stability of [125I][Tyr3]-octreotide was associated with good binding to SSTR2. Addition of appropriate protease inhibitors further increased the specific binding of [125I][Tyr3]-octreotide to AR4-2J plasma membranes without affecting the stability of the tracer, suggesting that the protease inhibitors also protect the integrity of SSTR2. Optimal conditions (time, temperature, medium) were developed for a binding assay in 96-well plates using AR4-2J plasma membranes in order to make the assay suitable for high-throughput analysis. This protocol was the basis for studying the in vivo regulation of SSTR2 expression in AR4-2J cells implanted into scid mice after exposure to different compounds.

DOTA α‐Melanocyte‐Stimulating Hormone Analogues for Imaging Metastatic Melanoma Lesions

Abstract: Scintigraphic imaging of metastatic melanoma lesions requires highly tumor‐specific radiopharmaceuticals. Because both melanotic and amelanotic melanomas overexpress melanocortin‐1 receptors (MC1R), radiolabeled analogues of α‐melanocyte‐stimulating hormone (α‐MSH) are potential candidates for melanoma diagnosis. Here, we report the in vivo performance of a newly designed octapeptide analogue, [βAla3, Nle4, Asp5, D‐Phe7, Lys10]‐α‐MSH3–10 (MSHOCT), which was conjugated through its N‐terminal amino group to the metal chelator 1,4,7,10‐tetraazacyclododecane‐1,4,7,10‐tetraacetic acid (DOTA) to enable incorporation of radiometals (e.g., indium‐111) into the peptide. DOTA‐MSHOCT displayed high in vitro MC1R affinity (IC50 9.21 nM). In vivo [111In]DOTA‐MSHOCT exhibited a favorable biodistribution profile after injection in B16‐F1 tumorbearing mice. The radiopeptide was rapidly cleared from blood through the kidneys and, most importantly, accumulated preferentially in the melanoma lesions. Lung and liver melanoma metastases could be clearly imaged on tissue section autoradiographs 4 h after injection of [111In]DOTA‐MSHOCT. A comparative study of [111In]DOTA‐MSHOCT with [111In]DOTA‐[Nle4, D‐Phe7]‐α‐MSH ([111In]‐DOTA‐NDP‐MSH) demonstrated the superiority of the DOTA‐MSHOCT peptide, particularly for the amount of radioactivity taken up by nonmalignant organs, including bone, the most radiosensitive tissue. These results demonstrate that [111In]DOTA‐MSHOCT is a promising melanoma imaging agent.

Melanocortins and Melanoma

Cutaneous melanoma is the cancer with the steepest increase in incidence in the Caucasian population (1) and is currently the most common cancer among young adults (2). Mortality rates are increasing correspondingly, and the disease still leads to death in one of every four to five patients. Ultraviolet light exposure has been identified as the main exogenous risk factor. A highly pigmented skin type protects from the deleterious effects of ultraviolet irradiation and is associated, consequently, with a lower risk. As the melanocortins are well-known stimulators of melanogenesis not only in melanocytes but also in melanoma cells, the question arises as to whether these peptides have a protective function or represent an additional risk factor for melanoma development. Experimental investigations in vivo were initiated by Lee et al. (3) who were the first to demonstrate that daily injections of α-melanocyte-stimulating hormone (α-MSH) into B16 tumor-bearing mice not only induced a marked increase in tyrosinase activity and melanogenesis of the tumors but also had a tendency to retard proliferation of the tumors. This growth retardation was shown to be negatively correlated with the metastatic potential of the cells (4): B16-F1 cells (→low metastatic potential) were more affected by α-MSH than B16-F5 cells (→intermediate metastatic potential). The growth of B16-F10 cells (→high metastatic potential) was not affected by α-MSH, although the number of MSH receptors did not differ significantly between F1 and F10 cells (5). This indicates that the response of melanoma cells to melanocortin peptides is complex and not simply a question of MSH receptor numbers expressed on the cell surface. This chapter reviews the literature of the past ten years by addressing the following topics: the functional effects of MSH peptides on melanogenesis and intracellular signaling in melanoma cells as well as on cell proliferation and metastasis; the regulation of MSH (melanocortin 1 [MC1]) receptor expression on melanoma cells; the role of ectopically produced proopiomelanocortin (POMC) peptides, and finally the potential application of MSH peptides to tumor targeting and therapy. The literature published before 1988 was extensively covered in an earlier review (5).