The Preparation Of Glucagon Like Peptide-1 Receptor Agonist Exendin-4 Labeled With Radionuclide And Its Experimental Study On Targeting Insulinoma

Background:Receptor imaging is one of the most important methods for molecular imaging.Because the glucagon like peptide-1?GLP-1?receptor is overexpressed on the cell surface of insulinoma,GLP-1 receptor may be the target for insulinoma targeting imaging and therapy.Exendin-4 is a kind of analogue of GLP-1 receptor agonist,which is suitable for clinical study.So in this experimental research the exendin-4 was selected as targeting carrier for insulinoma,modified with fluorescein isothiocyanate?FITC?,radiolabeled with iodine-131(¹³¹I),technetium-99m(^99mTc)and gallium-⁶⁸(⁶⁸Ga)for molecular probes targeting GLP-1 receptor.The biodistribution in normal mice of these molecular probes was evaluated.And SPECT/CT and mirco PET/CT imaging in nude mice bearing insulinoma were performed,and their imaging efficiency was evaluated so as to provide basic data for further the targeting therapies.Objects:1.The Exendin-4 with highly specific binding to the GLP-1 receptor was selected as a target carrier of radionuclides.2.The radiolabeling method of exendin-4 with ¹³¹I was established,and its biological distribution characteristic in normal mice was observed.3.The exendin-4 was modified with FITC,and its specific targeting for insolinoma cells was determined by fluorescence imaging.4.The radiolabeling method of exendin-4 with ^99mTc was established,and the imaging efficiency of ^99mTc-HYNIC-exendin-4 was observed in nude mice bearing insulinoma by means of SPECT/CT.5.The radiolabeling method of exendin-4 with WCGa was established,and the imaging efficiency of ⁶⁸Ga-DOTA-exendin-4 was observed in nude mice bearing insulinoma by means of the mico PET/CT.Materials and Methods:1.Radiolabeling of exendin-4 with ¹³¹I was performed by chloramine-T method.The labeling ratio and radiochemical purity were determined using paper chromatography and TLC.The octanol-water partition coefficient and stability in vitro in saline and normal human serum of labeled polypeptide were also determined.The biodistribution of ¹³¹I-exendin-4 in normal mice were measured at 1,3,6,12,24h after intravenous injection.SPECT imaging was performed in 3 male mice after injection of ¹³¹I-exendin-4 to observe the radioactive distribution in organs.2.The combination of FITC-Lys⁴⁰-exendin-4 with insulinoma cells was studied by means of fluorescence inverted microscope,confocal microscope and flow cytometry.3.Radiolabeling of exendin-4 with ^99mTc was performed by two-step method,the labeling rate and the radiochemical purity was calculated by using HPLC.The stability in vitro and lipid water partition coefficient of ^99mTc-HYNIC-exendin-4 were determinated.The nude mice bearing insulinoma were injected ^99mTc-HYNIC-exendin-4 via the tail vein of nude mice,and then SPECT/CT imaging with multiphase was undertaken for observing the change of radiodistribution and assessing imaging.efficiency for targeting insulinoma.4.The PET molecular probe of ⁶⁸Ga-DOTA-Ahx-Lys⁴⁰-exendin-4 for targeting GLP-1 receptor was prepared and then imaging efficiency for targeting insulinoma in nude mice bearing insulinoma was assessed by means of micro PET/CT.Results:1.The labeling yield of ¹³¹I-exendin-4 was more than 85%with radiochemical purity of more than 97%.The ¹³¹I-exendin-4 had good stability in saline and normal human serum.The octanol-water partiton coefficient lg P=-1.002.The biodistribution in mice showed that ¹³¹I-exendin-4 had rapid blood clearing,and the radioactivity in kidney was obviously higher than that of other organs as indicated by their uptake of?51.54±13.59?%ID/g at 1h and?11.61 ±0.94?%ID/g at 12h,respectively.Organs such as stomach,lung,intestine and bone had transient radioactive uptake,and then after 3h the radioactivity in those organs rapidly decreased.The radioactivity in other organs always kept at a low level.The observation of SPECT multiphase imaging was also consistent with its radiobistribution.2.The fluorescence inverted microscope and confocal experiment showed that the combination of FITC-Lys⁴⁰-exendin-4 with Rin-m5f cells was mainly on the cell membrane.When different concentration gradients?Onmol/100?l,0.01953125nmol/100?l,0.0390625nmol/100?l,0.078125nmol/100?l,0.15625nmol/100?l,0.3125nmol/100?l,0.625nmol/100?l,1.2nmol/100?l,2.5nmol/100?l,5nmol/100?l,10nmol/100?l,20nmol/100?l?of FITC-Lys⁴⁰-exendin-4 were respectively incubated with Rin-m5f cells,Flow cytometiy quantitative analysis results showed FITC-Lys⁴⁰-exendin-4 may combined with Rin-m5f cells,and this combination increased with increasing the concentration of fluorescent peptides,in which when the fluorescence concentration reached 5nmol/100ul,the fluorescence intensity of the cellular uptake of fluorescent peptide reached saturation.3.^99mTc-HYNIC-Ahx-Lys⁴⁰-exendin-4 was successful prepared with labeling rate of more than 95%.HPLC analysis of the radiolabeled peptide showed it had a single peak as the same peak position as unlabeled peptide,and the radiolabeled peptide still maintained good stability in vitro at room temperature.The lipid water partition coefficient was-0.32.In nude mice bearing insulinoma SPECT/CT imaging showed uptake in tumor of radiolabeled peptide with reaching its peak at 4h after tail vein injection of ^99mTc-HYNIC-exendin-4.It also showed that the labeled polypeptide was excreted mainly through the urinary system,which did not affect the observation of the abdominal organs.The competitive inhibition experiment showed that when pre-injection of excessive exendin-4 was performed only a small amount of the radiolabeled exendin-4 was uptaken by tumor in nude mice bearing insulinoma with high uptake in kidneys as well as a low radioactivity uptake in the other parts of the body.4.⁶⁸Ga-DOTA-Ahx-Lys⁴⁰-exendin-4 was successful prepared.HPLC analysis of the radiolabeled peptide showed it had a single peak as the same peak position as unlabeled peptide,and the radiolabeled peptide had good stability in vitro.The lipid water partition coefficient was-2.23.In nude mice bearing insulinoma Micro PET/CT imaging showed uptake in tumor of radiolabeled peptide with reaching its peak at 60min after tail vein injection of ⁶⁸Ga-DOTA-Ahx-Lys⁴⁰-exendin-4.The competitive inhibition experiment showed that when pre-injection of excessive exendin-4 was performed there was no uptake of the radiolabeled exendin-4 by tumor in nude mice bearing insulinoma.Conclusions:1.¹³¹I-exendin-4 can be easily labeled with a high labeling ratio and with stability in vitro.It has good distribution characteristics in vivo,and is cleared mainly by renal excretion due to its hydrosolubility.2.The fluorescence inverted microscope and confocal experiment show that the combination of FITC-Lys⁴⁰-exendin-4 with Rin-m5f cells is mainly on the cell membrane.And this combination increase with increasing the concentration of fluorescent peptides,in which when the fluorescence concentration reaches 5nmol/100ul,the fluorescence intensity of the cellular uptake of fluorescent peptide reaches saturation.3.^99mTc-HYNIC-Ahx-Lys⁴⁰-exendin-4 and ⁶⁸Ga-DOTA-Ahx-Lys⁴⁰-exendin-4 are successful prepared,and it is shown by SPECT/CT and Micro PET/CT imaging that these radiolabeled peptides can be specifically targeting insulinoma in nude mice bearing insulinoma.