Određivanje tkivne farmakokinetike levofloksacina i daidzeina primenom mikrodijalizne metode

Abstract

Doktorska disertacija posvećena je implementaciji mikrodijalizne metode za in vivo uzorkovanje levofloksacina i daidzeina iz intersticijalne tečnosti poprečno-prugastog mišića, i ispitivanju interakcije ove dve supstance u cilju povećanja terapijske efikasnosti levofloksacina. Takođe je ispitana i bezbednost konkomitantne primene levofloksacina i daidzeina. Uticaj daidzeina na prodor levofloksacina pasivnom difuzijom ispitan je PAMPA metodom. Na animalnom modelu, nakon oralne primene levofloksacina i daidzeina pojedinačno i u kombinaciji određene su koncentracije navedenih supstanci u plazmi, intersticijalnoj tečnosti mišića buta primenom mikrodijalizne metode i u homogenatima tkiva. Razvijena je metoda visokosenzitivne tečne hromatografije za detekciju obe supstance u plazmi i uzorcima dijalizata. Ispitan je uticaj daidzeina na farmakokinetiku levofloksacina u plazmi, mišiću, bubregu, jetri, plućima, mozgu, urinu i fecesu. Takođe je ispitan uticaj daidzeina na osetljivost bakterija na levofloksacin, u cilju ispitivanja mogućnosti njegove primenjivosti kao inhibitora efluks pumpi. Interakcija daidzeina i levofloksacina ispitana je na kliničkim izolatima bakterija primenom modifikovane metode šahovske ploče. In silico ispitivanjima određene su interakcije levofloksacina i daidzeina na nivou transportera. Levofloksacin se nakon peroralne primene rapidno distribuiše postižući visoke koncentracije u plazmi i u intersticijumu, sa indeksom distribucije u tkiva od 0,78, ali sa velikim interindividualnim razlikama u vrednostima maksimalnih koncentracija u tkivu. Nakon peroralne primene, uzorkovanja plazme i primene mikrodijalize, pokazano je da se daidzein distribuiše u mišićno tkivo, sa indeksom distribucije u tkiva od 0,35. Daidzein je doveo do povećanja slobodnih koncentracija levofloksacina u mišiću, mozgu, smanjio izlučivanje levofloksacina u urin i doveo do povećanja koncentracija levofloksacina u fecesu. Zajednička primena daidzeina i levofloksacina nije imala negativan uticaj na biohemijske i histološke parametre toksičnosti. Nije zabeležen porast vrednosti biohemijskih parametara funkcije jetre i bubrega niti narušavanje tkivne tkivne arhitektonike ovih organa. Daidzein je povećao osetljivost testiranih bakterija na levofloksacin što ide u prilog daljim ispitivanjima daidzeina u pravcu razvoja efluks inhibitora sa visokim translacionim potencijalom. Kroz uticaj na farmakokinetiku i osetljivost bakterija na levofloksacin, daidzein povećava vrednosti farmakokinetsko/ farmakodinamskih indeksa povezanih sa antibakterijskom aktivnošću fluorohinolona.

This doctoral dissertation is dedicated to the implementation of microdialysis method for the in vivo sampling of levofloxacin and daidzein in striated muscle, and to the study of the interaction between these compounds with the aim of increasing therapeutic efficacy of levofloxacin. The safety of concomitant use of levofloxacin and daidzein was also examined. The effect of daidzein on the penetration of levofloxacin by passive diffusion was investigated by the PAMPA method. In an animal model, after oral administration of levofloxacin and daidzein individually and in combination, the concentrations of both substances were determined in plasma, muscle interstitial fluid and in tissue homogenates. A high-performance liquid chromatography method was developed for the detection of levofloxacin and daidzein in plasma, dialysate and tissue samples. The effect of daidzein on the pharmacokinetics of levofloxacin in plasma, muscle, kidney, liver, lung, brain, urine and feces was examined. The effect of daidzein on bacterial susceptibility to levofloxacin was determined to examine its potential use as an efflux pump inhibitor. The interaction of daidzein and levofloxacin was investigated on clinical isolates of bacteria by the modified checkerboard method. Interactions between levofloxacin and daidzein at the transporter level have been studied using in silico methods. Levofloxacin rapidly distributed after oral administration, achieving high plasma and interstitial muscle fluid concentration, with a tissue distribution index of 0.78, but with large interindividual differences in maximum tissue concentrations. Following oral administration, plasma sampling, and microdialysis, daidzein has been shown to distribute into muscle tissue with a tissue distribution index of 0.35. Daidzein led to an increase in the free concentrations of levofloxacin in muscle, brain, decreased urinary excretion of levofloxacin and an increase in levofloxacin concentration in feces. Concomitant use of daidzein and levofloxacin did not adversely affect biochemical and histological parameters of toxicity. No increase in the values of biochemical parameters of liver and kidney function or disturbance of the tissue architecture of these organs was observed. Daidzein increased the susceptibility of the tested bacteria to levofloxacin, which supports further studies of daidzein as a promising efflux pump inhibitor with high translation potential. Through its effect on the pharmacokinetics and susceptibility of bacteria to levofloxacin, daidzein increases the pharmacokinetic/ pharmacodynamic index values associated with the antibacterial activity of fluoroquinolones.