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Klaritromicin je predstavnik druge generacije makrolidnih antibiotika, u koju spada i poznatiji azitromicin. Makrolidni antibiotici (makrolidi) se koriste u liječenju infekcija uzrokovanih Gram-pozitivnim i Gram-negativnim bakterijama. Vezanjem za veliku podjedinicu bakterijskog ribosoma makrolidi inhibiraju translaciju proteina čime ubijaju bakterijske stanice. Učestalom primjenom makrolida bakterije su razvile različite mehanizme otpornosti na njihovo djelovanje. Stoga je interes znanosti preusmjeren na razvoj novih makrolidnih antibiotika. Za uspješan dizajn novog makrolida važno je razumjeti njegove interakcije s bakterijskim ribosomom. Stoga je cilj ovog rada okarakterizirati strukturu i interakcije klaritromcina s ribosomom izoliranim iz bakterije Escherichia coli u otopini primjenom jedno- i dvodimenzijskih tehnika NMR. Tehnikama NOESY (engl. nuclear Overhauser effect spectroscopy) i ROESY (engl. rotating frame Overhauser effect spectroscopy) određena je konformacija klaritromicina u slobodnom stanju, a pomoću prijenosnog NOESY određena je konformacija vezanog klaritromicina. Nadalje, metodom STD (engl. saturation transfer difference) proučene su interakcije klaritromicina s ribosomom iz bakterije E. coli i određeni su vezni epitopi. Clarithromycin is a representative of the second generation of macrolide antibiotics, which includes better-known azithromycin. Macrolides are used for treating infections caused by Gram-positive and Gram-negative bacteria. Binding of macrolides to a large subunit of the bacterial ribosome inhibits protein translation and kills bacterial cells. Due to a frequent use of macrolides, bacteria have developed different resistance mechanisms. For this reason, the interest of science has been redirected to the development of new macrolide antibiotics. For a successful design of a new macrolide, it is important to understand its interactions with the bacterial ribosome. Therefore, the purpose of this study is to characterize the structure and interactions of clarithromycin with ribosome isolated from Escherichia coli in solution using one- and two-dimensional NMR techniques. Conformational studies of clarithromycin in the free-state were carried out using NOESY and ROESY techniques, while the bound-state conformation was determined from trNOESY correlations. Furthermore, the interactions of clarithromycin with the E. coli ribosome were studied by STD method, which allowed the assessment of binding epitopes.

Velik delež novoodkritih učinkovin v vodi izkazuje slabo topnost in nizko hitrost raztapljanja, kar se kaže v majhni biološki uporabnosti zdravila. Izboljšanje topnosti in hitrosti raztapljanja učinkovine je zato zelo pomemben raziskovalni vidik farmacevtske industrije. Obetaven pristop za izboljšanje topnosti učinkovine je elektrostatsko razprševanje, pri čemer nastanejo trdne disperzije v obliki polimernih mikro- in nanodelcev z vgrajeno učinkovino. V raziskovalnem delu smo najprej z uporabo različnih matematičnih modelov proučili interakcije med klaritromicinom in polimeri, kar je bistvenega pomena za uspešno pripravo trdnih disperzij. Te smo pripravili z odparevanjem topila in elektrostatskim razprševanjem. Za elektrostatsko razprševanje smo na podlagi proučenih interakcij, literaturnih podatkov in preliminarnih poskusov elektrostatskega raprševanja izbrali polimer PVP K30. Da bi dosegli čim bolj stabilen proces elektrostatskega razprševanja smo preizkusili različna topila in njihove kombinacije. S stabilnostnega in toksikološkega vidika se je najbolje izkazala kombinacija n-butanola in tetrahidrofurana. Z omenjeno kombinacijo topil smo pripravili 8% raztopino klaritromicina in PVP K30 1:1 (m/m), ki smo jo razprševali z večkanalno šobo, pri čemer so nastali delci velikosti 1840 nm. Delce in klasično trdno disperzijo, pripravljeno z odparevanjem topila, smo analizirali z DSC, TGA, FTIR, XRPD in in-vitro testom raztapljanja v deionizirani vodi in fosfatnem pufru (pH = 6,8). Ugotovili smo, da se klaritromicin v delcih nahaja v amorfni obliki, v klasični trdni disperziji pa v kristalni obliki solvata z n-butanolom. Obe formulaciji v vodi in pufru izkazujeta višjo hitrost raztapljanja in topnost od same učinkovine. Delce in trdno disperzijo smo starali 1 mesec pri 25°C in RH=0% ter z zgornjimi analiznimi tehnikami ocenili njuno stabilnost. Lastnosti delcev se med staranjem niso spreminjale, pri trdni disperziji pa je prišlo do manjših sprememb trdnega stanja snovi. Elektrostatsko razprševanje je enostavna in učinkovita metoda za pripravo trdnih disperzij v obliki polimernih mikro- in nanodelcev, ki izboljšajo lastnosti raztapljanja vgrajene učinkovine, poleg tega pa delci v primerjavi s klasičnimi trdnimi disperzijami izkazujejo večjo stabilnost. A large portion of newly discovered drug substances exhibits poor solubility and dissolution in water, which reflects in poor drug bioavailability. Solubility and dissolution enhancement is therefore an important aspect in pharmaceutical industry. Electrospraying is a promising technique to increase drug solubility by preparing solid dispersions in the form of micro- or nanoparticles. In the present study we have used different mathematical models to examine interactions between clarithromycin and different polymers, which is highly important for successful preparation of solid dispersions. The latter were prepared by solvent evaporation or electrospraying method. Based on the results of interaction studies, literature sources and preliminary electrospraying experiments, polymer PVP K30 was chosen for our study. In order to achieve a stable eletrospraying process we examined different solvent mixtures. Combination of n-butanol and tetrahyrofuran proved to be optimal in aspects of process stability and solvent toxicity. We prepared 8% solution of clarithromycin and PVP K30 1:1 (m/m) using previously mentioned solvents. The solution was successfully electrosprayed by a multi-nozzle injector, resulting in 1840 nm particles. The particles and a conventional solvent evaporation solid dispersion were analysed by DSC, TGA, FTIR, XRPD and in-vitro dissolution test in deionised water and phosphate buffer (pH = 6,8) by HPLC. Clarithromycin was found to be present in amorphous form in electrosprayed particles and in n-butanol solvated crystalline form in conventional solvent evaporation solid dispersion. Both formulations exhibit superior solubility and dissolution to pure drug in water and buffer. A one month stability study of particles and solid dispersion was performed at 25°C and RH=0% using above mentioned analytical techniques. Minor solid state changes were noticed in solid dispersion while properties of particles remained the same. Electrospraying is an easy and effective method of enhancing drug dissolution by preparing solid dispersions in the form of drug-loaded polymeric micro- and nanoparticles that exhibit superior stability to conventional solvent evaporation solid dispersions.

Danas, u raznim analitičkim laboratorijama postoji veći broj analitičkih protokola,zasnovanih bilo na izuzetno sofisticiranim ili jednostavnijim tehnikama, koji služe zaodređivanje različitih ciljnih analita od farmakološkog značaja. Među tim grupama ciljnih analita pripadaju i antibiotici koji predstavljaju veliko otkriće u oblasti medicine i zahvaljujući njima spašeno je više od sedam miliona života, ali pored navedenih koristi, antibiotici mogu da izazovu veliki broj neželjenih efekata i žučne kiseline zajedno sa svojim derivatima, koji su fiziološki deterdženti, mogu biti citotoksične za organizam ako se njihova koncentracija ne kontroliše. U ovoj doktorskoj disertaciji prikazan je razvoj analitičkih metoda pre svega voltametrijskihmetoda u kombinaciji sa jednostavnim i savremenim elektrodama/senzorima za određivanje odabranih analita kao što je antraciklični antibiotik doksorubicin (DOX), makrolidni antibioticieritromicin-etilsukcinata (EES), azitromicina (AZI), klaritromicina (CLA) i roksitromicina (ROX) i 3-dehidro-deoksiholne kiseline.Voltametrijska karakterizacija i određivanje gore navedenih antibiotika primenom obnovljive srebro-amalgam film elektrode (Hg(Ag)FE)rađena je direktnom katodnom voltametrijom sa pravougaonim talasima (SWV) i visoko osetljivom adsorptivnom voltametrijom sa pravougaonim talasima (SW-AdSV) u Briton-Robinson puferu, kao pomoćnom elektrolitu, obuhvatajući širok opseg pH vrednosti. Odgovor DOX-a primenom Hg(Ag)FEpraćen je u intervalu potencijala od -0,20 do -0,80 V. Za analizu tragova, optimizacija metode ukazuje da su optimalni parametri za analitički pik na potencijalu (Ep ) -0,57 V u odnosu na zasićenu kalomelovu elektrodu (ZKE): pH 6,0, potencijal akumulacije -0,20 V i vreme akumulacije 140 s. U model rastvoru, DOX je određivan u koncentracionom opsegu 4,99-59,64 ng mL-1. Razvijena SW-AdSV metoda je primenjena za određivanje DOX-a u obogaćenom uzorku humanog urina. Niža koncentracija DOX-a 9,89ng mL-1u voltametrijskoj ćeliji je određivana sa relativnom standardnom devijacijom (RSD) manjom od 6,0%. Što se ispitivanih makrolida tiče oni su pokazali redukcione signale u dalekoj negativnoj oblasti potencijala. Ispitivanja direktnom katodnom SWV rađena su u opsegu potencijala od -0,75 V do -2,00 V u odnosu na ZKE, pri čemu su dobijena jedan ili dva redukciona pika u opsegu potencijala od -1,5 V do -1,9 V. Oblik i intenzitet signala zavisi od primenjene pH vrednosti u širokoj pH oblasti. Za analitičke svrhe, radi razvoja direktne katodne SWV i adsorptivne inverzne/striping SWV metode, pogodnim su se pokazale neutralna i slabo alkalna sredina tj. pH 7,0 sa Ep na -1,67 V u odnosu na ZKE za ROX i EES i pH 7,2 sa Ep na -1,85 V u odnosu na ZKE za AZI i pH 7,4 sa Ep na -1,64 V u odnosu na ZKE za CLA. Na osnovu snimljenih cikličnih voltamograma na optimalnim pH vrednostima, može se predložiti adsorptivno-kontrolisan kinetički proces na elektrodi u slučaju sva četiri ispitivana jedinjenja. Takođe, 1H NMR merenja uz potiskivanje signala vode u pH oblasti između pH 6,0 i 10,5 ukazuju na to da su makrolidni molekuli pri optimalnim analitičkim uslovima predominantno u protonovanoj formi preko tercijerne amino grupe što potpomaže, u sva četiri slučaja, njihovu adsorpciju na odgovarajuće polarizovanoj Hg(Ag)FE. Optimizovane direktne katodne SWV metode pokazuju dobru linearnost u koncentracionom opsegu 4,81-23,3 µg mL-1 , 4,53-29,8 µg mL-1 , 1,96-28,6 µg mL-1 i 1,48-25,9 µg mL-1 za AZI, EES, CLA odnosno ROX. Razvijene SW-AdSV metode rezultiraju u linearnom odgovoru pri nižim koncentracionim intervalima 1,0-2,46 µg mL-1 , 0,69-2,44 µg mL-1, 0,05-0,99 µg mL-1 i 0,10-0,99 µg mL-1 , za AZI, EES, CLA i ROX. RSD za sve razvijene metode nije veća od 1,5% izuzev SWV metode u slučaju AZI-a gde je 4,5%. Direktna katodna SWV metoda je uspešno primenjena za određivanje EES-a u farmaceutskom proizvodu Eritromicin® dok SW-AdSV metoda je primenjena u slučaju određivanja EES-a u obogaćenom uzorku humanog urina iza određivanje ROX-a u farmaceutskom proizvodu Runac® . U svim pomenutim slučajevima, primenjena je metoda standardnog dodatka. Pouzdanost i tačnost elaboriranih procedura u slučaju određivanja EES-a u model sistemu i farmaceutskom proizvodu Eritromicin® su potvrđena poređenjem sa rezultatima dobijenim primenom HPLC-DAD metode.Nakon preliminarnih studija 3-dehidro-deoksiholne kiseline/3-dehidro-deoksiholata primenom elektrode od staklastog ugljenika (GCE), gde je uočeno da ne dolazi do formiranja redukcionog signala u Briton-Robinson puferu između pH 5,0 i 11,8 primenom direktne katodne SWV, bizmut-film je izdvojen ex situ na površini iste elektrode od staklastog ugljenika (BiF-GCE) iz uobičajeno korišćenog rastvora za elektrodepoziciju (0,02 mol L-1 Bi(NO3)3, 1,0 mol L-1 HCl i 0,5 mol L-1 KBr) i tako pripremljena elektroda je primenjena za karakterizaciju i određivanje pomenutog jedinjenja u alkalnoj sredini. Redukcioni signal ispitivanog analita od analitičkog značaja je uočen jedino primenom BiF-GCE u Briton-Robinson puferusa pH vrednostima između 9,5 i 11,8 u režimu adsorptivne inverzne/stripingvoltametrije sa pravougaonim talasima, dok u slučaju direktnih katodnih SWV eksperimentalnih uslova uočen je slab redukcioni pik sa niskom strujom maksimuma pika. Optimizovani eksperimentalni uslovi za određivanje 3-dehidro-deoksiholata obuhvataju odgovarajuće kondicioniranje elektrode uključujući kondicioniranje ex situ pripremljene BiF-GCE u Briton-Robinson pomoćnom elektrolitu pH 11,8 do stabilizacije struje bazne linije elektrohemijskim cikliranjem potencijala radne elektrode u potencijalskom opsegu između -1,00 i -2,00 V u odnosu na ZKE (blizu 15 puta) i primenu dva ključna parametara adsorptivne voltametrije sa pravougaonim talasima: vreme akumulacije od 30 s i potencijal akumulacije -1,00 V u odnosu na ZKE. Zbog relativne asimetričnosti dobijenih redukcionih signala ispitivanog analita sa Ep na -1,35 V u odnosu na ZKE, što je takođe prisutno i u slučaju primene SW-AdSV, određivanje ispitivanog analita je zasnovano na linearnoj zavisnosti između površine pika redukcionog signala spitivanog analita i njegove odgovarajuće koncentracije i postignuta granica detekcije je 1,43 µg mL-1 sa dva linearna opsega kalibracione krive od 4,76 µg mL-1 do 13,0 µg mL-1 i od 13,0 µg mL-1 do 23,1 µg mL-1 za razvoj analitičke metode. RSD metode je 3,22%. Dodatni eksperimenti, elektroliza ispitivanog analita na potencijalu -1,55 V (blizu maksimuma pika ciljnog analita) u odnosu na ZKE su rađeni primenom GCE u obliku ploče (površina 33,52 cm 2 ) modifikovane sa ex situpripremljenim bizmut-filmom. Rastvor od interesa uzorkovan je na početku eksperimenta, nakon 2,5 h i nakon 4,5 h tretmana. Ovakvi uzorci su analizirani primenom 1H NMR merenja uz potiskivanje signala vode u puferskom rastvoru pH 11,8. Može se pretpostaviti da tokom elektrolize 3-dehidro-deoksiholata dolazi do redukcije keto grupe prisutne u strukturi ispitivanog analita.Na osnovu literaturnih podataka da neki od ciljnihmakrolidnih antibiotika kao što je npr. azitromicin pokazuju oksidativno ponašanje na elektrodi od ugljenične paste i elektrodi od zlata deteljna karakterizacija i određivanje četiri makrolidna antibiotika rađena je primenom asičneelektrode od ugljenične paste (CPE) koja se sastoji samo od grafitnog praha i parafinskog ulja sa optimizovanih direktnih anodnih SWV metoda. U slučaju EES-a i AZI-a diferencijalna pulsna voltametrija (DPV) je testirana za iste svrhe. Ključni parametar u slučaju razvoja analitičkih voltametrijskih metoda je odabir pH vrednosti pomoćnog elektrolita gde je oblik/simetričnost i intenzitet oskidacionog pika glavni kriterijum prilikom odabira. Kao odgovarajuće pH vrednosti za voltametrijsko određivanje EES-a primenom SWV metode odabrana je pH 8,0 sa Ep na 0,83V u odnosu na ZKE, dok u slučaju DPV metode pH 12,0 sa Ep na 0,55 V u odnosu na ZKE je bila najpogodnija za analitičke svrhe. Za određivanje AZI-a, u slučaju obe SWV i DPV metode pH 7,0 se pokazala najpogodnijom sa Ep analitičkog signala na 0,85 V odnosno 0,80 V u odnosu na ZKE, dok u slučaju CLA i ROX koji su ispitivani samo primenom SWV metode za analitičke svrhe pH 12,0 je bila najpogodnija sredina sa Ep analitičkog signala na 0,65 V odnosno na 0,63 Vu odnosu na ZKE. Postignute granice detkcije primenom nemodifikovane CPE i direktne anodne SWV su uglavnom u submikrogramskom koncentracionom opsegu 0,17 µg mL-1 , 0,32 µg mL-1 i 0,30 µg mL-1, u slučaju EES-a, AZI-a i ROX-a i u niskom mikrogramskom koncentracionom opsegu 1,43 µg mL-1 za CLA. Razvijena SWV metoda sa jednostavnom CPE pokazala se pogodnom za određivanje ROX-a u komercijalnom proizvodu Runac® tableti. U slučaju optimizovanih DPV metoda postignute granice detekcije za EES i AZI su u niskom mikrogramskom koncentracionom opsegu 1,03 µg mL-1 odnosno 1,53 µg mL-1 . U želji da sepostigne niža granica detekcije za AZI, DPV metoda je testirana u kombinaciji sa CPE radnom elektrodom površinski modifikovanom sa zlatnim nanočesticama prečnika 10 nm (Au-CPE) i postignuta granica detekcije je 0,95 µg mL-1 sa Ep analitičkog signala na 0,80 V u odnosu na ZKE. RSD metode u slučaju Au-CPE je 3,5%, dok je u slučaju nemodifikovane CPE 6,0%. Linearnost analitičke metode zasnovane na primeni Au-CPE je dva puta šira nego u slučaju primene nemodifikovane CPE.Na osnovu dobijenih rezultata može se zaključiti da odgovarajuće kombinacije optimizovanih voltametrijskih tehnika sa ekološki prihvatljivim i lako primenljivim radnim elektrodama, kao što su Hg(Ag)FE, BiF-GCE i CPE zajedno sa Au-CPE, rezultuju razvojem pouzdanih analitičkih metoda, kako u oksidacionim tako i u redukcionim proučavanjima, koje često omogućuju određivanje tragova analita od farmakološkog značaja u jednostavnim, a unekim slučajevima i u složenim sistemima.&nbsp, Nowadays in different analytical laboratories there is the increasing number of analytical protocols, either based on highly sophisticated or simpler measurements techniques, which serving for determination of different target analytes of pharmacological importance. Among such target groups of the analyte belongs the antibiotics which present a great discovery in the field of medicine and thanks to them were saved more than seven million people but beside to the mentioned great benefits, antibiotics can cause a large number of side effects and bile acids together with their derivatives which are physiological detergents but if their concentration is notcontrolled they can be cytotoxic to the body. In the present doctoral dissertation the development of analytical methods, primarily analytical voltammetric methods in combination with simple and contemporary electrodes/sensors, for the determination of selected analytes as antracycline antibiotic doxorubicin (DOX), macrolide antibiotics erythromycin ethylsuccinate (EES), azithromycin (AZI), clarithromycin (CLA) and roxithromycin (ROX) and 3-dehydrodeoxycholic acid were performed.Voltammetric characterization and determination of the above mentioned antibiotics using a renewable silver-amalgam film electrode (Hg(Ag)FE) was performed by direct cathodic square-wave voltammetry (SWV) and by highly sensitive adsorptive square-wave voltammetry (SW-AdSV) in aqueous Britton-Robinson buffer solutions as supporting electrolyte covering the wider pH range. The Hg(Ag)FE response of DOX was monitored in the potential range between -0.20 and -0.80 V. For the trace level analysis the method optimization showed that the optimal conditions for the analytical peak with peak potential (Ep) at -0.57 V vs. SCE were: the pH 6.0, the accumulation potential -0.20 V, and the accumulation time 140 s. In the model solutions, DOX was determined in the concentration range of 4.99-59.64 ng mL-1. The developed SWAdSV method was applied for the determination of DOX in spiked human urine sample. The lowest concentration of DOX of 9.89 ng mL-1 in voltammetric vessel was determined with the relative standard deviation (RSD) less than 6%. As for the investigated macrolides, they showed reduction signals in fairly negative potential range. During direct cathodic SWV investigations conducted over the potential range from -0.75 V to -2.00 V vs. SCE, either one or two reduction peaks were obtained in the potential range from -1.5 to -1.9 V. For analytical purposes concerning the development of direct cathodic SWV and adsorptive stripping SWV methods the neutral and slightly alkaline media were suitable as pH 7.0 with Ep at -1.67 V vs. SCE for ROX and EES and pH 7.2 and pH 7.4 with Ep at -1.85 V and -1.64 V vs. SCE for AZI and CLA, respectively. Based on the cyclic voltammograms recorded at these pH values, adsorptioncontrolled electrode kinetics process can be proposed for all four investigated compounds. The water suppressed 1H NMR measurements in the pH range between 6.0 and 10.5 indicated that the macrolide molecules at the optimal analytical conditions are predominantly in protonated form via their tertiary amino groups which supported in all four cases their adsorption on the appropriately polarized Hg(Ag)FE electrode. The optimized direct cathodic SWV methods showed good linearity in concentration ranges 4.81-23.3 μg mL-1, 4.53-29.8 μg mL-1, 1.96-28.6 μg mL-1, and 1.48-25.9 μg mL-1 for AZI, EES, CLA and ROX, respectively. The SW-AdSV methods resulted in the linear responses at lower concentration ranges as 1.0-2.46 μg mL-1, 0.69- 2.44 μg mL-1, 0.05-0.99 μg mL-1 and 0.10-0.99 μg mL-1, for AZI, EES, CLA and ROX, respectively. The RSD for all developed methods was not higher than 1.5% except the SWV method for AZI with 4.7%. The direct cathodic SWV method was successfully applied for the determination of EES in the pharmaceutical preparation Eritromicin®, while SW-AdSV was tested in the case of the spiked urine sample and for determination of ROX in pharmaceutical preparation Runac®. In all above cases, the standard addition method was used. The reliability and accuracy of the above procedures in the case of EES determination in model system and pharmaceutical preparation Eritromicin® were validated by comparing them with those obtained by means of HPLC-DAD measurements.After initial study of 3-dehydro-deoxycholic acid/3-dehydro-deoxycholate by glassy carbon electrode, where the absence of any reduction peak was observed in the Britton-Robinson buffer solutions between pH 5.0 and 11.8 by direct cathodic SWV, a bismuth-film was electrodeposited ex situ on the same glassy carbon electrode surface (BiF-GCE) from the usually used plating solution (0.02 mol L-1 Bi(NO3)3, 1.0 mol L-1 HCl and 0.5 mol L-1 KBr) and such prepared film-electrode was applied for the characterization and determination of the the target analyte in alkaline media. The reduction signal of analytical importance was observed only by BiF-GCE in Britton-Robinson buffer solutions with pH values between 9.5 and 11.8 in adsorptive stripping square-wave voltammetry working regime, while in the case of the direct cathodic SWV experimental protocol only a very poor reduction peak was obtained. The optimized experimental conditions for the 3-dehydro-deoxycholate determination consist of the optimized electrode conditioning including the electrochemical cycling of the ex situ prepared BiF-GCE potentials in the potential span between -1.0 and -2.0 V vs. SCE (nearly 15 times) in the Britton-Robinson supporting electrolyte pH 11.8 till the stabilization of the baseline current, and the application of two key parameters of the adsorptive square-wave voltammetric protocol: the accumulation time as 30 s and accumulation potential as -1.0 V vs. SCE. Because of the relative asymmetry of the obtained reduction signals of the target analyte with peak Ep at -1.35 V vs. SCE, which is still present in the case of the SW-AdSV, the quantification of the target analyte was based on the linear correlation between peak area of the reduction signal and its appropriate concentrations, and reached limit of detection is 1.43 μg mL-1 and with two linear ranges of calibration curve from 4,76 μg mL-1 to 13.0 μg mL-1 and from 13,0 μg mL-1 to 23,1 μg mL-1 for the development of analytical method. The RSD of the method was 3.22%. Additional experiments were performed applying GCE with rectangular form (area 35.32 cm2) modified with ex situ prepared bismuth-film for the electrolysis of the target analyte which was performed at the potential -1.55 V (nearly the peak maxima of the target analyte) vs. SCE. The solution of interest was sampled at the beginning of the experiment, after 2.5 h and after 4.5 h of treatment. Such samples were analysed by simply water suppressing 1H NMR measurements in the buffered solution at pH 11.8. It can be assumed that during electrolysis of 3-dehydrodeoxycholate the reduction of the keto group present in the structure of the target analyte can be occurred.Driven by earlier literature data about the fact that some of the target macrolide antibiotics as e.g. azithromycin showed oxidation behavior at a carbon paste and gold working electrodes detailed characterization and determination of four target macrolide antibiotics were performed on classical carbon paste electrode (CPE) constituted only from graphite powder and paraffin oil with optimized direct anodic SWV methods. In the cases of EES and AZI differential pulse voltammetric (DPV) methods were tested for the same purpose as well. The key parameter in the case of the development of the analytical voltammetric methods is the selection of the pH value of the supporting electrolyte where the shape/simmetry and intensity of the oxidation peak were the criteria. As the appropriate pH value for determination of EES by SWV method the pH 8.0 was selected with Ep at 0.83 V vs. SCE while in the case of the DPV method the pH 12.0 with Ep at 0.55 V vs. SCE was the most suitable for analytical purpose. As for AZI determination, in the case of both SWV and DPV methods the pH 7.0 was the most appropriate supporting electrolyte with the Ep of analytical signal at 0.85 V and 0.80 V vs. SCE, respectively, while in the case of CLA and ROX which were investigated only with SWV method for the analytical purposes the pH 12.0 was the most suitable with Ep at 0.65 V and at 0.63 V vs. SCE. The obtained detection limits applying the bare CPE and the direct anodic SWV are mainly in submicrogram concentration range as 0.17 μg mL-1; 0.32 μg mL-1 and 0.30 μg mL-1 for EES, AZI, and ROX and in the low microgram concentration range as 1.43 μg mL-1 for the CLA, respectively. The developed method succesfully tested for the determination of ROX in the commercial formulation, Runac® tablet. In the case of the optimized DPV methods the obtained detection limits for EES and AZI are in the low microgram concentration range 1.03 μg mL-1 and 1.53 μg mL-1, respectively. For the improvement of the sensitivity for AZI the DPV method was tested in combination with a CPE working electrode surface modified with gold nanoparticles with diameter of 10 nm (Au-CPE) and reached the limit of detection was 0.95 μg mL-1 at Ep of 0.80 V vs. SCE. The RSD of the method in the case of the Au-CPE is 3.5% while in the case of the native CPE 6.0%. The linearity of the Au-CPE based analytical method is twice wider then it is case with the bare CPE applying protocol.Based on the obtained results it can be conclude that the appropriate combination of the optimized voltammetric pulse techniques and the environmentally friendly and easy to use working electrodes as Hg(Ag)FE, BiF-GCE and CPE together with Au-CPE resulted in the development of reliable analytical method either in the oxidation or reduction studies, often allowing trace level determination of pharmacological importance target analytes in simpler and in some case complexes systems.

Procjenjuje se da je s H. pylori inficirano oko 50% odrasle populacije u razvijenim zemljama i 80-90% u zemljama u razvoju. Zbog ograničenog uspjeha eradikacijske terapije i porasta rezistentnih sojeva H. pylori na antibiotske komponente dosadašnjih terapijskih protokola, traže se novi pristupi u liječenju i novi djelotvorniji lijekovi. Prevalencija H. pylori rezistencije na eradikacijske postupke različita je u različitim dijelovima svijeta. Najveći problem predstavlja rezistencija bakterije na klaritromicin, standardni antibiotik u prvoj liniji liječenja, što dovodi do modifikacija terapijskih shema uvođenjem drugih antibiotika. No, ni to se nije pokazalo dugoročno uspješnim zbog brzog razvoja rezistencije koje se značajno razlikuju prema geografskim područjima. S razvojem neinvazivnih i brzih testova osjetljivosti bakterije na različite antibiotike, prvenstveno na klaritromicin i levofloksacin, bit će lakše odrediti individualno prilagođene terapijske režime od kojih možemo očekivati željene eradikacijske rezultate oko 95%. Može se pretpostaviti da će i probiotici, kada se dodaju eradikacijskoj terapiji za H. pylori, imati povoljan učinak na organizam domaćina promjenom sastava crijevne mikroflore, čime mogu utjecati na sniženje učestalosti nuspojava i omogućiti bolje podnošenje terapije i bolju suradljivost bolesnika. Također, cjepivo protiv H. pylori pokazuje veliki potencijal te bi trebalo biti najučinkovitije ako se primjeni tijekom prvih nekoliko mjeseci nakon rođenja, kada najčešće dolazi do infekcije s H. pylori. Idealno cjepivo ne samo da mora imati dobru sigurnost i učinkovitost, već bi trebalo biti relativno jeftino, dati dugoročnu imunost uz minimalne doze ponavljanja. It is estimated that there are about 50% H. pylori infected adults in developed countries and 80-90% of adults in developing countries. Due to the limited success of eradication therapies and the increasing number of resistant strains of H. pylori to antibiotic components of current treatment protocols, new therapy approaches and new effective drugs are required. The prevalence of H. pylori resistance to eradication procedures is different, in different parts of the world. The biggest problem is the bacterial resistance to clarithromycin, the antibiotic included in standard first-line therapy, which lead to modifications of the therapeutic shemes by adding other antibiotics in therapy. However, that hasn't been successful in the long-term meaning, beacuse of the rapid resistance development which differ significantly according to geographical areas. With the development of non-invasive and rapid sensitivity tests, primarily H. pylori sensitivity to clarithromycin and levofloxacin, it will be easier to determine individually-tailored regimes of which we can expect eradication rates of

The concentrations of inflammatory mediators in nasal secretions could reflect the inflammatory status of the nasal mucosa and evolution of sinus disease. The results of previous investigations suggest that macrolide antibiotics could be effective in the treatment of chronic rhinosinusitis and nasal polyposis. The mean goal of this study was to compare the efects of preoperative clarithromycin administration and surgical treatment with sole surgical treatment in patients with nasal polyposis. Therefore, we assessed the immunomodulatory and clinical efects of long-term low-dose clarithomycin treatment in allergic and non-allergic patients with nasal polyps. Koncentracije medijatora zapaljenske reakcije u nosnom sekretu odražavaju status zapaljenja sluzokože nosa, kao i razvoj zapaljenske bolesti u paranazalnim sinusima. Rezultati prethodnih istraživanja sugerišu da primena makrolidnih antibiotika može biti efikasna u lečenju hroničnog rinosinuzitisa i nosno-sinusne polipoze. Osnovni cilj istraživanja bio je da se uporede efekti preoperativne primene makrolidnog antibiotika klaritromicina i naknadnog hirurškog lečenja sa efektima isključive primene hirurškog lečenja u bolesnika sa nosno-sinusnom polipozom. Osim toga, bilo je potrebno proceniti imunomodulacijske i kliničke efekte dugotrajne, niskodozirane terapije klaritromicinom u alergičnih i nealergičnih pacijenata sa nosnom polipozom.

Aim: Inflammatory processes in nasal mucosa are reflected in various local mediators, found both in mucosal tissue and nasal discharge. In this prospective study, we assessed the effects of long-term low-dose oral administration of clarithromycin (CAM) on Th1 cytokines in nasal secretions and on clinical parameters of severity of nasal polyposis. Methods: A total of forty nasal polyp patients (22 nonallergic and 18 allergic) received 500 mg/day single oral dose of CAM for eight weeks. We measured the levels of proinflammatory Th1 cytokines TNF-α, TNF-β, IL-1β, IL-2, IL-12, and IFN-γ in the nasal fluid samples, before and after treatment by CAM, using flow cytometric method. Before and after therapy, we scored each of the 40 patients according to nasal symptom score and endoscopic score. Results: Following treatment, we found significantly reduced levels of TNF-α (p=0.006) in nasal secretions of nonallergic patients, and of IL-1β (p=0.008) in nasal fluid of allergic patients. Our results suggest an association between the reduction of nasal polyp size and reduction of TNF-α levels in nasal fluid of nonatopic patients and an association between the reduction of nasal polyp size and reduction of IL-12 levels in nasal discharge of atopic patients. Macrolide therapy decreased the size of polyps in 10/22 nonatopic and in 9/18 atopic patients. After macrolide therapy, we found 67.83% nonallergic subjects and 55.55% allergic subjects with improved nasal symptomatology. Conclusion: Long-term low-dose treatment with CAM is effective in the management of nasal polyposis, because of its antiinflammatory and immunomodulatory actions., Cilj: Upalni procesi u nosnoj sluznici očituju se u različitim lokalnim medijatorima, u tkivu sluznice i u nosnom sekretu. U ovoj prospektivnoj studiji procijenili smo učinke dugotrajne, niskodozirane terapije klaritromicinom (CAM) na Th1 citokine u nosnome sekretu, kao i na kliničke parametre očitovanja nosne polipoze. Metode: Četrdesetero (22 nealergičnih i 18 alergičnih) bolesnika s nosnom polipozom dobivalo je po jednu dnevnu dozu od 500 mg CAM-a tijekom osam tjedana. Mjerili smo koncentracije proupalnih Th1 citokina TNF-α, TNF-β, IL-1β, IL-2, IL-12 i IFN-γ u uzorcima nosnoga sekreta, prije i nakon terapije CAM-om, primjenom protočne citometrije. Prije i nakon liječenja klinički smo klasificirali svakoga od četrdesetero bolesnika prema nosnome simptom rezultatu i endoskopskom rezultatu. Rezultati: Nakon liječenja detektirali smo značajno niže koncentracije TNF-α (p = 0,006) u nosnome sekretu nealergičnih bolesnika i IL-1β (p = 0,008) u nosnome sekretu alergičnih bolesnika. Naši rezultati sugeriraju povezanost između smanjenja veličine nosnih polipa i snižavanja koncentracije TNF-α u nosnom sekretu u neatopičnih bolesnika, kao i povezanost između smanjenja veličine nosnih polipa i snižavanja koncentracije IL-12 u nosnome sekretu u atopičnih ispitanika. Terapija makrolidnim antibiotikom smanjila je veličinu polipa u 10/22 nealergičnih i u 9/18 alergičnih bolesnika. Nakon makrolidne terapije našli smo 67,83 % nealergičnih i 55,55 % alergičnih ispitanika s poboljšanim nosnim simptomima. Zaključak: Zbog protuupalnih i imunomodulacijskih djelovanja, dugotrajna niskodozirana primjena CAM-a korisna je u liječenju nosne polipoze.