Preparation And Identification Of Radioiodinated Glycosylated Somatostatin

Background:Natural somatostatin was described in 1973 as a hypothalamic peptide that inhibits the secretion of immunoreactive pituitary growth hormone. SMS acts as an endogenous inhibitory regulator of the secretary and preliferatory response of its target cells. The effects are mediated when SMS binds to its membrane receptor (SSR), which have five subtypes, SSR1-5. The five SSRs bind natural SMS, SMS-14 and SMS-28 with low nanomolar affinity. Natural SMS has a short half-life in vivo due to enzymatic degradation, so more long-acting SMS analogs have been developed during passed 20 years. Octreotide is the best known and most clinically established. However, octreotide and other short synthetic peptide analogs have affinity only to two SSR subtypes, SSR2 and SSR5.SSR are expressed in a majority of human tumors, including most tumors of neuro-endocrine origin, breast tumors, certain brain tumors, renal cell tumors, lymphomas, and prostate cancer. Octreotide is frequently used in the treatment of patients with secreting neuro-endocrine tumors. The treatment effect is palliation of hormonal symptoms. There is an increasing interest in using SMS in cancer treatment due to its potentially anti-proliferate and even apoptotic actions. However, little is known about the effect dose and dose frequency, partly because oflimitations of existing analogs regarding their SSR subtype affinity and in vivo short half-life.Glycosylated SMS is natural SMS-14 that has been conjugated to dextran of strictly defined molecular weight. By using different sizes of the carbohydrate backbone, blood half-life and biodistribution can be modulated. A larger dextran yields a prolonged half-life, allowing conjugation of several SMS and probably increasing the avidity toward SSRs. The kidney excretion threshold for dextran is approx 50 kDa, and by selecting dextran above or below the threshold, the biodistribution and blood half-life are modulated. In the present study, SMS was conjugated to dextran with a mean molecular weight of 10 kDa. The synthesis, radiolabeling, SSR binding affinity, biodistribution, and blood half-life were investigated.Materials and MethodsIn this study, dextran 10 was mildly oxidized with sodium periodate and sodium acetate buffer. The oxidized dextran was incubated with somatostatin, tyramine and cyanoborohydride, and then the mixture was purified on a PD-10 column. A sample of the eluted conjugate was measured in a spectrophotometer at 280 nm. The peptide concentration of the conjugate was determined.Radiolabeling was performed with Iodogen method. The phosphate buffer, Tyramine-DxlO-SMS and NaI25I solution were added to the bottom of tube covered with Iodogen. Stop the reaction at room temperature; The part of elution contained Tyramine-DxlO-SMS was purified by ridding of the free Na125I. The elution was measured in a gamma-counter and the radiolabeling rate was calculated.SD rat cortex membranes were prepared in lab, the membranes protein concentration were measured in a spectrophotometer. Testing receptor-binding characteristic of Tyramine-DxlO-SMS by means of multi-point saturation method and competitive displacement method, especially the latter. Briefly, rat cortex membranes were diluted with binding buffer. Binding assay consisted of radioligand (' 5I-Tyr3-Octreotide) and buffer or increasing concentration of Tyramine-DxlO-SMS and membranes suspension. The membranes were incubated in the waterand were filtrated rapidly over glass-fiber filters and subsequently washed. Specific radioligand binding was defined as total binding minus nonspecific binding. Triplicates were performed for each data point, averaged, and the data was analyzed with the software GraphPad Prism 4.0. Finally, calculated the value IC50. In addition, multi-point saturation method was used in the binding of 125I-Tyramine-Dxl0-SMS and rat cortex membranes.The post-injected rat (three at each time) was scribed by cervical dislocation after different time. Samples of blood and organs were collected. They were carefully rinsed and weighed, and the radioactivity was finally measured in an automatic gamma-counter. The percentage of the injected dose per gram tissue (%ID/g) was calculated.ResultsThe results show that glycosylated somatostatin-14 keep high affinity to somatostatin receptor subtype-2; the value IC50 (IC50 ~ 8.45 nM) was in the same low-nM range as the reference ligand Octreotide(ICso = 1.49 nM). Radioiodinated glycosylated somatostatin-14 targeted to SSR2-positive tissue. The multi-point saturation experiment shows the close result to the former study, the specific binding was 9-61% of the total binding, a complete plateau was not reached after 5 hours. The blood half-life of 125I- Tyramine-DxlO-SMS was 6.7 h post injection. The digestion and excretion is mainly through the hepato-biliary and kidney system. Increased uptake was seen in the adrenals, which are somatostatin receptor positive organs.ConclusionGlycosylated somatostatin-14 has been shown to have ideal targeting properties with excellent tumor/blood ratios demonstrated in endocrine tumors. Clinicians involved with nuclear medicine await SMS derivatives that can be used in targeting radiotherapy. The model described in this paper may fulfill these requirements.