Radiolabeled somatostatin analogues are important tools for targeted therapy of tumors of neuroendocrine origin. The retention of the radionuclide in tumor cells is a key factor in maximizing thfe radiation dose ratio of tumor to normal cells. We have evaluated a new series of peptides developed by Drs. Whetstone and Meares (University of California at Davis, USA). The peptides include an additional five amino acid sequence that is cleavable by cathepsin, for the purpose of improving the release and trapping of labeled catabolites within the cell. These were directly compared with radioiodinated glycated octreotate (Gluc-TOCA), which was shown to have the best internalization properties of the 4 peptides previously evaluated. The different peptides analyzed in the current study were: DOTA-Ahx-Oct (OCT), DOTA-Ahx-Ser-Val-Glu-Phe-Ala-Ahx-Oct (P3) and DOTA-Ahx-Gly-Ser-Val-Glu-Phe-Ahx-Oct (P4), where Ahx=epsilon amino hexyl. A comparison of their properties under optimized conditions including binding capacity, internalization, externalization and stability was done in order to determine the best criteria for selection.
Labeling was performed with 131I and 177Lu because of their radiochemical properties and the widespread availability of these radionuclides at a reasonable cost, and with 125I as a reference. The labeling with 177Lu was optimized by varying temperature and reaction time, pH, molar ratio (peptide/Lu) and evaluating labeling efficacy, radiochemical purity and stability. Quality control procedures included reversed phase HPLC (C18 column) and chromatography on various solvents/supports. Determination of specific binding to biological materials was analyzed with D341 Med human medulloblastoma cells, which express high levels of somatostatin receptors. Internalization and externalization were evaluated as a function of time. Paired assays with each of the peptides labeled with 177Lu and radioiodinated Gluc-TOCA as reference were done.
The labeling of OCT, P3 and P4 was successfully achieved using 177LuCl3 (24Ci/mg; 2.6Ci/mL); ammonium acetate buffer and gentisic acid were used to provide optimal pH conditions and protection against radiolysis. Radiochemical purity was higher than 98%. The effect of temperature (40-70ºC) and incubation time (10-60min) was studied for 177Lu-P3; yields >99% could be obtained with 10 min at 70ºC and 30 min at 55ºC while incubation times up to 60 min gave only 80% yield at 40ºC. The first condition was selected for further labelings and similar results were obtained for the labeling of P4 and OCT. Labeling Gluc-TOCA with 125I and 131I gave yields higher than 95% and 93%, respectively. The specific binding, internalization and externalization studies done for all the peptides using the D341 cells showed that although total binding and internalization was higher for 125I-Gluc-TOCA (4.5±0.3% and 34±1% at 2h), the percentage of activity remaining inside the cell (>80%) was higher for the new peptides 177Lu-P3 and 177Lu-P4, which bear the amino acid sequence cleavable by cathepsin inside the cell. These preliminary results are indicative that these new strategies for the design of somatostatin analog peptides could have an important role in targeted radiotherapy.
Acknowledgements: Work supported by a UICC fellowship to HB and the National Institutes of Health
