Study of renal transmembrane transport of somatostatin and gastrin analogues using cellular renal models

M Volkova1, J Mandikova1, A Laznickova0,2, M Laznicek1, F Trejtnar1. 1Charles University in Prague, Faculty of Pharmacy in Hradec Kralove, Department of Pharmacology and Toxicology, 50005 Hradec Kralove, Czech Republic, 2Charles University in Prague, Faculty of Pharmacy in Hradec Kralove, Department of Biophysics and Physical Chemistry, 50005 Hradec Kralove, Czech Republic

Radiolabeled receptor-specific peptides are successfully used for radiodiagnosis and also for radiotherapy of some neuroendocrine tumours. A limiting factor during radiotherapy is nephrotoxicity caused by the accumulation of the radiopeptides in the renal cortex. The main transport mechanism responsible for the renal uptake is still not fully identified, but the preclinical studies point out the role of endocytic receptor megalin. However, transport of somatostatin analogues by influx transporters for organic acids (OATs) can be another mechanism mediating the renal accumulation. This study was aimed at evaluation of the role of renally expressed OAT1 and megalin in cellular accumulation of radiolabeled somatostatin and gastrin derivatives using an in vitro model.

Human cervical cancer cell line; HeLa, was transiently transfected with cDNA encoding human OAT1 by the liposome transfection method according to the manufacture´s protocol (LipofectamineTM2000). To evaluate a possible contribution of active endocytosis by megalin, pig kidney epithelial cell line LLC-PK1 stably expressing megalin endocytic system and albumin as a specific inhibitor of this transport system were used. As controls we used the cells transiently transfected with empty carrier vector-pCMV6. Incubation under low temperature (4° C) served to reveal general role of active transport processes in the cell uptake. Two somatostatin derivatives, [125I]-somatostatin and [111In-DOTA0, 1-Nal3]-octreotide, and two gastrin analogues, [177Lu-DOTA0]-sargastrin and [177Lu-DOTA0]-minigastrin46, were included in the study.

In functional accumulation study, HeLa cells transiently transfected with hOAT1 showed a strong substrate uptake of specific substrate [3H]paraaminohippuric acid, compared with control cells transiently transfected with empty vector. These findings confirmed proper functioning of used cellular model. A significantly higher uptake in HeLa cells transiently transfected with hOAT1 was not detected in any somatostatin or gastrin analogue. Incubation with the specific megalin substrate/inhibitor albumin, resulted in a significant inhibition of the accumulation of both gastrin analogues. [177Lu-DOTA0]-minigastrin46 13% decrease at 120 min and [177Lu-DOTA0]-sargastrin 38% decrease at 120 min. Accumulation in the model cells was significantly inhibited under low incubation temperature in all tested compounds.

Cellular accumulation in the used model cells was at least partly active process as the incubation under lower temperature confirmed. However, transporters for organic acids seem to play no significant role in the transmembrane transport of all studied radiopeptides. As we supposed, the transport is probably mediated at least partly by megalin endocytic system since accumulation of both gastrin analogues was inhibited by specific megalin substrate albumin.

The study was supported by Charles University in Prague (Project SVV 265003), grant GAUK No. 376411/FaF/C-LEK and by the grant Agency of the Czech Republic – grant no. P304/10/1738.