Design, Radiolabelling, In Vitro And In Vivo Evaluation Of Novel Neurotensin Analogues

Neurotensin (NT) is a tridecapeptide(Glu-Leu-Tyr-Glu-Asn-Lys-Pro-Arg-Arg-Pro-Tyr -Ile-Leu). It is present in both central nervous system (CNS) where it plays the roles of neurotransmitter or neuromodulator, and in some peripheral tissues, mainly in gastrointestinal tract, where it may be considered as a hormone. Some researches revealed that most of the effects of neurotensin were results from the specific interaction with cell surface neurotensin receptors. About 75% tumors of all ductal pancreatic adenocarcinomas were neurotensin receptor positive, whereas normal pancreatic tissues, pancreatitis and endocrine pancreatic cancers did not overexpress neurotensin receptor. Neurotensin analogs can be used in diagnosis of tumors that can overexpress NT receptors. This thesis was focused on the design and evaluation of two novel NT(8-13) analogs. [Objective] Firstly, two NT(8-13) analogs NT1(Arg-Lys-Pro-Tyr-Tle-Leu) and NT2 (Lys-Arg-Pro-Tyr-Tle-Leu) were designed. Then, NT1 and NT2 were labelled with Na¹²⁵I by Chloramine T (Ch-T). In order to explore the possibility of these two analogs used in tumor receptor imaging, in vitro and in vivo properties of these two ¹²⁵I labelled neurotensin analogs were investigated.[Methods] (1)Labelling experiment: NT(8-13) analogs NT1 and NT2 were labelled with Na¹²⁵I by Ch-T, purified by Sep-Pak reversed-phase column and determinded by high performance liquid chromatograph(HPLC). Then, the labelling rate and radiochemical purity were calculated.(2)In vitro binding assay: the number of the cells, the incubated temperature and time were studied in order to determine the optimal conditions for in vitro tests in next steps. The saturable binding experiment between ¹²⁵I-NT2 and HT29 cell receptor was carried out in order to investigate the binding affinity. The competition experiment was done and showed that NT1 displaced ¹²⁵I-NT2 bound to its specific receptor. The data were analyzed by the software (Prism 4.0) to calculate K_d and IC₅₀, respectively. (3)Biodistribution: in vivo biodistribution studies of ¹²⁵I-NT1 and ¹²⁵I-NT2 were performed in ICR normal mice and Balb/c nude mice bearing HT29 xenografts. After intravenous injection of ¹²⁵I-NT1 or ¹²⁵I-NT2, the animals were killed in different interval time. Then, blood, heart, liver, spleen, lung, kidneys, stomach and muscle as well as tumortissue (in the case of nude mice) were collected respectively and weighed, measured by a y counter. Finally, the radioactivity (cpm) was converted into ID%/g tissue and the results were expressed as averaged values with the corresponding standard deviation.[ Results ]?Labelling experiment: the labelling rate of 125 I-NT1 and 125I-NT2 were 68% and 76%, respectively. The radiochemical purity of two neurotensin analogs was both above 98% and kept the level after 24 hours in 4°C refrigerator. (§) in vitro binding assay: the specific activity of 125I-NT2 was 9.71 *1012 Bq/mmol in saturable experiment. The proper experimental condition of 125I-NT2 binding to HT29 cells was as follows: cell number, temperature and time of incubation were 106, 25 °C and lh, respectively. The binding of 5I-NT2 to HT29 cells showed saturability and the concentrations of Kd and Bmax were 0.745±0.418 nmol/L and 0.103±0.060pmol/106cell, respectively. IC50 of NT1 in competition experiment was 1.333nmol/L. (3) Biodistribution.- the biodistribution of ICR mice showed that NT analogs NT1 and NT2 were quickly cleared from blood. The radioactivity was mainly accumulated in stomach, lung and kidneys and excreted by kidney and digestive tract. NT1 and NT2 were accumulated in tumor. They exhibited strong target toward colon carcinoma. The biodistribution of nude mice indicated that the radioactivity in blood and kidney remained in high level and had similar ratio of T/NT for these two peptides.[Conclusion] (Dlodination of NT1 and NT2 by Chloramine T can give high radiochemical purity and high specific activity of 125I-NT1 and 125I-NT2. ? NT2 and NT1 have good specificity and high affinity of binding to HT29 cell receptor.? Although both 125I labelled two novel neurotensin analogs NT1 and NT2, which are in the interconvertible site of lysine and arginine at N-terminal, have similar properties in binding neurotensin receptor, NT2 may be a promising targeting agent in the imaging of colon adenocarcinoma than NT1 because of less organ damage.