In vitro binding of selected antimicrobial agents to Cytosorb hemoadsorbent

Sepsa je življenjsko ogrožajoča motnja v delovanju organov, ki nastane zaradi neustreznega odziva organizma na okužbo. V sistemski krvni obtok se sproščajo povečane količine mediatorjev vnetja. To vodi do sistemskih sprememb organizma, kar bistveno poveča smrtnost. Poleg protimikrobnega zdravljenja se v zadnjem času pri zdravljenju sepse uporablja hemoadsorbent CytoSorb, ki odstranjuje mediatorje vnetja. Raziskovalci so in vitro ter na živalskih modelih dokazali, da Cytosorb lahko veže tudi nekatere protimikrobne učinkovine. V magistrski nalogi smo želeli ugotovili v kakšni meri se na CytoSorb in vitro adsorbirajo antibiotiki vankomicin, piperacilin in meropenem. In vitro poskus je bil zasnovan v stekleni čaši, v katerih smo kri segrevali na 37 °C in med mešanjem spremljali vezavo izbranih protimikrobnih učinkovin na hemoadsorbent CytoSorb. Pri tem smo simulirali kri septičnega bolnika tako, da smo v goveji krvi s citratnim antikoagulantom uravnali koncentracijo albumina na 20 g/l in delež hematokrita na 30 %. Uporabili smo enostavni metodi in sicer smo spektrofotometrično določali koncentracijo albuminov, medtem ko smo hematokrit določili v kapilarah po centrifugiranju. Z ustreznim odvzemom plazme in dodatkom fosfatnega pufra s pH 7,4 smo prilagodili govejo kri. Med poskusom smo spremljali tudi delež hemolize. Za njen izračun smo potrebovali vrednosti hemoglobina v polni krvi in plazmi, katerega smo določali spektrofotometrično. In vitro poskus smo izvedli v treh delih. V prvem delu smo z merjenjem hemolize določili ustreznost pogojev poskusa (190 obrt/min, 37 °C). V drugem delu smo dokazali, da terapevtske koncentracije izbranih antibiotikov ne vplivajo na hemolizo prilagojene krvi ter določili vpliv pogojev poskusa na koncentracijo antibiotika. V zadnjem sklopu poskusov smo v prilagojeni krvi določili vezavo antibiotikov na hemoadsorbent CytoSorb. Vezavo antibiotikov na CytoSorb smo spremljali 6 ur z 0,1 g in 1 g CytoSorba v 100 ml goveje krvi. Rezultati naloge nakazujejo zelo verjetno vezavo izbranih protimikrobnih učinkovin na hemoadsorbent CytoSorb. Pri tem smo ugotovili, da različna masa CytoSorba vpliva na delež adsorpcije antibiotikov na hemoadsorbent. Razvit in vitro model je dobro orodje za proučevanje vezave učinkovin na hemoadsorbent in bo zato uporaben v nadaljnjih raziskavah.Sepsis is a life-threatening organ dysfunction caused by the body\u27s inadequate response to infection. It leads to an increased production of mediators of inflammation that are released into the systemic bloodstream. This leads to systemic changes in the organism, which significantly increases mortality. In addition to antimicrobial treatment, the haemadsorber CytoSorb, which removes mediators of inflammation, has recently been used in the treatment of sepsis. In addition to removing mediators of inflammation, it also removes some antimicrobial active substances. In this thesis, we wanted to determine in vitro adsorption of antibiotics vancomycin, piperacillin and meropenem on hemoadsorbent CytoSorb. The in vitro experiment was designed in glass beakers in which blood was heated to 37 °C and during mixing the binding of antimicrobial active substances to the CytoSorb was monitored. We simulated the blood of a septic patient by adjusting the albumin concentration in bovine blood with citrate anticoagulant to 20 g/l and the haematocrit ratio to 30%. We used a simple spectrophotometrical method to determine the albumin concentration, while the haematocrit was determined in the capillaries after centrifugation. The bovine blood was adjusted by appropriate plasma collection and the addition of phosphate buffer at pH 7,4. The haemolysis rate was also monitored during the experiment. To calculate it, we needed the haemoglobin values in whole blood and plasma, which were determined spectrophotometrically. The experiment was carried out in three parts. First, we determined the adequacy of the conditions (190 rpm, 37 °C) by measuring haemolysis. Second, we demonstrated that therapeutic concentrations of selected antibiotics do not affect the haemolysis and determined the influence of the experimental conditions on the antibiotic concentration. Third, we determined the binding of the antibiotics to the CytoSorb in the adapted blood. Antibiotic binding to CytoSorb was monitored for 6 hours with 0.1 g and 1 g of CytoSorb in 100 ml of bovine blood. The results of this research indicate highly probable binding of the selected antibiotics to hemoadsorbent CytoSorb. We also found that the different masses of CytoSorb in bovine blood influence the proportion of antibiotics adsorbed to it. The developed in vitro model is a good tool to study the binding of active substances to hemoadsorbent and will be useful in further studies