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    Estudos De Biodisponibilidade E Biodistribuição Do Amblyomin-X Dissertação
    (Universidade Federal de São Paulo (UNIFESP), 2017-01-31) Boufleur, Pamela [UNIFESP]; Tavassi, Ana Marisa Chudzinski [UNIFESP]; Universidade Federal de São Paulo (UNIFESP)
    Objectives: Amblyomin-X is a recombinant protein of 12,3 kDa that inhibits the blood coagulation factor Xa and induces tumor cell apoptosis and regression of melanoma in mice. Pharmacokinetic studies were needed for the identification and location of Amblyomin-X in healthy animais (tumor-free), to identify the speed 'of distribution and the extent to which the protein is absorbed into the systemic circulation until their excretion, and on which organs the protein has affinity with. Methods: Amblyomin-X was intravenously injected onto the retro orbital plexus of healthy female BALB/c mice in different concentrations (1 mg/Kg and 100 mg/Kg), multiple time post injection were evaluated in plasma, organs (thymus, heart, lung, liver and kidney) and urine for analysis. The analysis of organs and plasma was by SDS-PAGE and liquid chromatography-mass spectrometry (ESI-IT-TOF and MALDI-TOF). Urine samples were treated for the proper extraction of proteins and peptides and also analyzed by LC-MS. In parallel experiments were performed using plasma from mice at concentrations of 5, 50 and 200 mg/Kg in single dose and analyzed 24 hours after and the concentrations of 5 and 50 mg/Kg in repeated dosages of·14 and 28 days. Fluorescent-Iabeled Amblyomin-X (Ambly750S) was used for the evaluation of biodistribution kinetics in healthy animais. The treatment was performed in a single dose (1 mg/Kg) at different times and in daily dose (1 mg/Kg) for 14 days intravenously. Results: The Amblyomin-X at a concentration of 100 mg/Kg was found in the plasma immediately after application and remained detectable within 2 hours, with a Cmax of 38 ug/mL Tmax of 30 minutes. The Amblyomin-X was not detected in plasma samples what was analyzed 24 hours post the application of 5, 50 and 200 mg/Kg (single dose) and 5 to 50 mg/Kg injected daily for 14 and 28 days. In organs analyzed by ESI-IT-TOF Amblyomin-X was identified in thymus after 10 minutes to 6 h, heart in 45 minutes up to 12 h, lung 10 minutes up to 6 hours, spleen over 1 hour remaining detectable for 24 hours, in the liver 45 minutes 24 hours and in the kidney in 45 minutes remaining until 24 hours. Peptides were identified corresponding to the degraded Amblyomin-X in urine post 24 hours of application. Ambly750S was detected in the liver at 1 hour post application, and it started to be excreted via the renal route 4 hours after injection. Fluorescent signals were detected 26 hours in the urinary tract organs, especially the bladder. Conclusions: The xii Amblyomin-X has a quick exit from the plasma by the fact of not being bound to albumin. The Amblyomin-X remains in the body of healthy animais for up to 26 hours after application, was found in kidney up to 26 hours, indicating that the protein is not only metabolised by the liver but also by the kidney. After its passage through the kidney Amblyomin-X was found in the urine, indicating its route of elimination.