The effect of drug dose and drug exposure time on the binding, internalization, and cytotoxicity of radiolabeled somatostatin analogs

Abstract

Background

Creation of protease-resistant somatostatin analogs has allowed development of these peptides as clinically useful drugs. Widespread diagnostic use of radiolabeled somatostatin analogs has enhanced interest in the binding and intracellular distribution of these peptides. The degree of drug internalization and length of drug retention may be critical for drug-induced cytotoxicity. We hypothesized that the ability of a radiolabeled peptide to bind to a cell, be internalized, and induce cytotoxicity is proportional to both the radioligand concentration and the exposure time.

Materials and methods

To test this hypothesis, somatostatin receptor-expressing cells (IMR-32) were incubated with ¹¹¹In-pentetreotide, a sst 2 preferring somatostatin analogue. Radioligand exposure time and/or concentration were varied.

Results

Prolonged exposure to a fixed concentration of radioligand resulted in progressive increases in whole cell binding and internalization over time. Cells exposed to a relatively fixed number of μCi-Hr yielded constant whole cell binding and internalization. Increasing the μCi-Hr resulted in a proportionate increase in binding. Cytotoxicity was also proportional to the dose of radiation regardless of whether the exposure was internalized radiation (μCi-Hr from ¹¹¹In-pentetreotide) or from external beam radiation (cGy).

Conclusion

Both drug exposure time and drug concentration contribute to cell binding and cytotoxicity in this model and their relative contributions are inversely related.

Introduction

The 14-amino-acid peptide somatostatin is recognized as an inhibitor of peptide release in the pituitary, the central nervous system, the gastrointestinal tract, the immune system, and the pancreas [1]. These effects are mediated by membrane receptors. Five somatostatin receptor subtypes have been identified (sst 1 through sst 5) and cloned 1, 2, 3. The development of protease-resistant somatostatin analogs with increased biological half-lives has enhanced the clinical utility of this class of agents. Somatostatin analogs that are prepared as sustained release formulations are now available and widely used clinically (Sandostatin® LAR, Novartis Pharmaceuticals, Inc., East Hanover, NJ). Somatostatin analogs also have been radiolabeled and are used as diagnostic (OctreoScan® Mallinckrodt Medical, St. Louis, MO) and as therapeutic agents 4, 5. Other analogs with selective affinity for specific receptor subtypes and higher overall binding affinities are being developed for diagnostic and therapeutic applications [5].

Recent evidence from our laboratory and others has shown that somatostatin binding to a membrane receptor with subsequent triggering of G-protein-related signal transduction cascades may influence some, but not all, physiological processes 6, 7, 8, 9, 10, 11. Internalization of the receptor-ligand complex, transport of the peptide to the nucleus, and binding to DNA has been described by Hornick et al. and may account for some of the actions of somatostatin on cell growth/differentiation or regulation of specific gene expression [12]. These effects may be independent of classic signal transduction pathways.

Fresh or frozen membrane preparations have been widely used for competitive binding analysis; however, membrane-based assays cannot assess the internalized fraction of radioligand or examine peptide-induced cytotoxicity. In an effort to overcome the limitations of these membrane assays, we and others have used whole cell binding assays to study somatostatin analogue binding and intracellular transport [12]. Prolonged (24–48 h) exposure of somatostatin receptor-expressing cells to iodine or indium-labeled somatostatin analogs leads to progressive increases in whole cell binding, internalization, and receptor-dependent nuclear transport of the radioligand [12]. Exposure of somatostatin receptor-expressing cells to Auger-emitting radiolabeled somatostatin analogs also induces dose-dependent/receptor-dependent cytotoxicity both in vitro and in vivo 13, 14, 15, 16. These observations have led us to investigate the relative contribution of radioligand concentration and radioligand exposure time on binding, internalization, and cytotoxicity in somatostatin receptor-expressing cells.

Receptor binding is classically defined as both specific and saturable. Each cell population has a receptor density that is relatively stable over short periods of time. Based on these concepts, we hypothesized that the amount of ligand that is specifically bound to a cell membrane and internalized within a cell is a function of receptor density, ligand concentration, and drug exposure time. Of these three parameters, receptor density is the least easily altered. Ligand concentration and drug exposure time may be the critical elements that can alter cell binding, internalization, and cytotoxicity. The experiments outlined below demonstrate that binding and internalization increases with both increasing drug concentration (μCi or pM) and increasing length of exposure (h). Significantly, the same relationships exist for cytotoxicity. Thus, nearly identical total cell binding, internalization, and cytotoxicity can be achieved by exposing receptor-expressing cells to high concentrations of ligand for short periods of time or to low concentrations of ligand for proportionately longer periods of time. We propose that a more accurate description of ligand binding can be achieved by using a dose-time unit (μCi-Hr or pM-Hr) rather than a dose unit (cpm bound or pmol bound) alone.