Biodistribution and pharmacokinetics of amblyomin-X, a novel antitumour protein drug in healthy mice

Background Amblyomin-X is a recombinant protein under development for cancer treatment owing to its selective cytotoxic activity over several tumour cell lines and tumour regression in mice models. The aim of this study was to examine the distribution and pharmacokinetics of amblyomin-X in healthy female mice. Methods Amblyomin-X was injected intravenously into the healthy animals and at controlled times plasma and organs were removed and analysed for identification and quantification of the protein. Alternatively, the labelled protein was injected into mice and tracked in an in vivo imaging system. Results Amblyomin-X was rapidly eliminated from plasma, probably because of its inability to bind to plasma albumin. After 10 min, the protein was found in the thymus and lungs, and later in the heart, liver and kidneys. In the liver, the protein was found until 24 h after a single injection. The in vivo analysis showed the same kinetics profile, besides the identification of amblyomin-X in the bladder region, indicating its elimination via urine. Only fragments of amblyomin-X were observed in the urine. Conclusions These findings suggest that amblyomin-X is rapidly distributed to the tissues, metabolized by the liver or even kidneys, and eliminated in urine in healthy mice. There is no accumulation in any organ.

Biodistribution and pharmacokinetics of amblyomin-X, a novel antitumour protein drug in healthy mice

Background Amblyomin-X is a recombinant protein under development for cancer treatment owing to its selective cytotoxic activity over several tumour cell lines and tumour regression in mice models. The aim of this study was to examine the distribution and pharmacokinetics of amblyomin-X in healthy female mice. Methods Amblyomin-X was injected intravenously into the healthy animals and at controlled times plasma and organs were removed and analysed for identification and quantification of the protein. Alternatively, the labelled protein was injected into mice and tracked in an in vivo imaging system. Results Amblyomin-X was rapidly eliminated from plasma, probably because of its inability to bind to plasma albumin. After 10 min, the protein was found in the thymus and lungs, and later in the heart, liver and kidneys. In the liver, the protein was found until 24 h after a single injection. The in vivo analysis showed the same kinetics profile, besides the identification of amblyomin-X in the bladder region, indicating its elimination via urine. Only fragments of amblyomin-X were observed in the urine. Conclusions These findings suggest that amblyomin-X is rapidly distributed to the tissues, metabolized by the liver or even kidneys, and eliminated in urine in healthy mice. There is no accumulation in any organ.

Amblyomin-X having a Kunitz-type homologous domain, is a noncompetitive inhibitor of FXa and induces anticoagulation in vitro and in vivo

Background: Cancer has long been associated with thrombosis and many of the standard chemotherapeutics used to treat cancer are pro-thrombotic. Thus, the identification of novel selective anticancer drugs that also have antithrombotic properties is of enormous significance. Amblyomin-X is an anticancer protein derived from the salivary glands of the Amblyomma cajennense tick. Methods: In this work, we determined the inhibition profile of Amblyomin-X and its effect on activated partial thromboplastin time (aPTT) and prothrombin time (PT), using various approaches such as, kinetic analyses, amidolytic assays, SDS-PAGE, and mass spectrometry. Results: Amblyomin-X inhibited factor Xa, prothrombinase and tenase activities. It was hydrolyzed by trypsin and plasmin. MS/MS data of tryptic hydrolysate of Amblyomin-X suggested the presence of Cys(8)-Cys(59) and Cys(19)-Cys(42) but not Cys(34)-Cys(55) disulfide bond. Instead of Cys(34)-Cys(55), two noncanonical Cys(34)-Cys(74) and Cys(55)-Cys(74) disulfide bonds were identified. Furthermore, when Amblyomin-X (1 mg/kg) injected in rabbits, it prolonged aPTT and PT. Conclusion: Amblyomin-X is a noncompetitive inhibitor (K-i = 3.9 mu M) of factor Xa. It is a substrate for plasmin and trypsin, but not for factor Xa and thrombin. The disulfide Cys(34)-Cys(55) bond probably scrambles with inter chain seventh free cysteine residues (Cys(74)) of Amblyomin-X. The prolongation of PT and aPTT is reversible. General Significance. In term of anticoagulant property, this is structural and functional characterization of Amblyomin-X. All together, these results and previous findings suggest that Amblyomin-X has a potential to become an anticancer drug with antithrombotic property. (C) 2016 Elsevier B.V. All rights reserved.

Specific role of cytoplasmic dynein in the mechanism of action of an antitumor molecule, Amblyomin-X

The Kunitz-type recombinant protein, Amblyomin-X, is an antitumor recombinant molecule from a cDNA library prepared from the salivary glands of the tick Amblyomma cajennense. The primary target of this protein appears to be the proteasome. Amblyomin-X increased gene and protein expression of distinct subunits of the molecular motor dynein, which plays a key role in the intracellular transport. Herein, Amblyomin-X was specifically taken up by tumor cells through lipid-raft endocytic pathways, but not by fibroblasts. Moreover, dynein inhibitor, ciliobrevin A, decreased Amblyomin-X uptake by tumor cells. Furthermore, incubation of tumor cells with Amblyomin-X inhibited trypsin-like activity of the proteasome, which was restored upon pretreatment with ciliobrevin A. Only in tumor cells treated with Amblyomin-X, we identified proteins bounds to dynein that are related to aggresome formation, autophagy inhibition, and early and recycling endosome markers. In addition, Amblyomin-X was found to interact with dynein, increased Rab11A protein expression and Rab11A co-localization with the light intermediate chain 2 (LIC2) of dynein. Thereby, the results provide new insights on the antitumor mechanism of Amblyomin-X and reveal an unsuspected role of cytoplasmic dynein in its uptake, intracellular trafficking and pro-apoptotic action. (C) 2016 Published by Elsevier Inc

Ação do Amblyomin-X no processo de coagulação sanguínea / The action ofAmblyomin-X in the blood clotting process

Hemostasis is a biological event, responsible for the smooth blood flow in blood vessels, made up of related elements such as coagulation, fibrinolysis and endothelium. Amblyomin-X is a recombinant protein characterized as an inhibitor of the Kunitz type, expressed in the prokaryotic system (E. coli) and the eukaryote system (P. pastoris) and also capable of inhibiting FXa in blood coagulation. FXa is a protein essential for triggering the clotting mechanism. Once in its active form (FXa) causes a thrombin generation and, finally, the formation of the fibrin clot. In this study, we evaluated the effect of Amblyomin-X in human and rabbit plasmas. Amblyomin-X was injected (1 mg / kg) on the marginal ear vein of rabbits and blood samples were collected at different times (30 min, 2, 4 and 24 ), and were submitted to analysis of activated partial thromboplastin time (aPTT) and prothrombin time (PT). In parallel, human blood from healthy donors treated with Amblyomin-X were subjected to the same tests. The results showed that Amblyomin-X was able to prolong the PT and APTT in rabbit’s plasma, and APTT but not PT in human plasma. Furthermore, the kinetics of enzyme inhibition by Amblyomin-X was also evaluated Amblyomin-X inhibited different enzymes (Plasmin, FXa, and Trypsin) in a non-competitive manner. The Ki valves for plasmin, FXa and trypsin inhibition by Amblyomin-X were 3.8, 2.2 and 3.0 µM respectively. A 55% inhibition on the tenase complex was observed and also a 44% inhibition of the Factor Xa, and in both cases the inhibitor concentration was the same (3.0 µM). These results indicate an anti-coagulant, or anti-thrombotic, activity of Amblyomin-X. Therefore, Amblyomin-X might be a promising novel candidate for hemostatic disorders.

A hemostasia é um evento biológico, responsável pela fluidez do sangue nos vasos sanguíneos, composto por elementos relacionados à coagulação, fibrinólise e endotélio. O Amblyomin-X, é uma proteína recombinante caracterizada como inibidor do tipo Kunitz, expressa em sistema procarioto (E. coli) e em sistema eucarioto (P. pastoris) e é capaz de inibir o FXa da coagulação sanguínea. O FXa é uma proteína essencial para o desencadeamento do mecanismo de coagulação uma vez que a sua forma ativa (FXa) provoca a geração de trombina que é responsável pela formação do coágulo de fibrina. Neste estudo, avaliou-se a ação do Amblyomin-X em plasma de humanos e de coelhos. O Amblyomin-X foi injetado (1 mg/kg) pela veia marginal da orelha dos coelhos e amostras de sangue foram coletadas em diferentes tempos (30 min, 2, 4 e 24 horas), e foram submetidos às análises de tempo de tromboplastina parcial ativada (TTPa) e tempo de protrombina (TP). Em paralelo, sangue humano de doadores sadios foram tratados com Amblyomin-X e submetidos aos mesmos ensaios para plasmas de coelhos. Os resultados mostraram que o Amblyomin-X foi capaz de prolongar o TP e o TTPa, em plasma de coelhos e o TTPa em plasma humano, mas não o TP. Também foi avaliada a cinética enzimática de inibição ocasionada pelo Amblyomin-X, verificou-se que o Amblyomin-X inibiu de forma não competitiva as diferentes enzimas (Plasmina, FXa, Tripsina). O Ki para a Plasmina, FXa e Tripsina foi de 3,8; 2,2 e 3,0 µM respectivamente. Verificou-se também inibição de 55% no complexo tenase e 44%, sobre o FXa, utilizando a mesma concentração de inibidor (3,0 µM). Os resultados sugerem ação anticoagulante ou ainda antitrombótica do Amblyomin-X, que poderá tornar-se um novo candidato para distúrbios hemostáticos.