Trenutno ne postoje farmakopejske ili standardne metode i aparature za ispitivanje oslobađanja djelatne tvari iz topikalnih mikročestica. Cilj ovog rada bio je razviti i validirati diskriminatornu in ...vitro metodu za ispitivanje oslobađanja djelatne tvari iz topikalnih mikročestica, uz korištenje imerzijske ćelije. Za potrebe rada pripremljene su i karakterizirane kitozanske i metakrilatne mikročestice s mupirocinom. Za izradu mikročestica korištena je tehnika sušenja raspršivanjem. Ispitana su sljedeća svojstva mikročestica: učinkovitost uklapanja lijeka, morfologija i veličina čestica, kristalno stanje, termička svojstva, spektralna svojstva, zeta potencijal, higroskopnost te sadržaj vode i ostatnih organskih otapala.
Kitozanske i metakrilatne mikročestice s mupirocinom korištene su kao modelni topikalni terapijski sustavi za razvoj in vitro metode za ispitivanje oslobađanja djelatne tvari. Imerzijska ćelija korištena je u kombinaciji s aparaturom s lopaticom. pH i temperatura medija (pH 5,5, 32°C) odabrani su u skladu s fiziološkim uvjetima na koži. Difuzija lijeka odvijala se preko membrane izrađene od smjese celuloznih estera, koja je pokazala nisku adsorpciju lijeka te nizak otpor difuziji lijeka. Nakon
početnog zastojnog vremena količina oslobođenog lijeka postala je proporcionalna korijenu iz vremena. Nagib u linearnom području krivulje oslobađanja lijeka korišten je kao mjera brzine oslobađanja. Varijacije u brzini okretanja lopatica (25 o/min, 50 o/min, 100 o/min), visini lopatica (1 cm, 2,5 cm) i volumenu medija za ispitivanje oslobađanja (100 ml, 200 ml) nisu značajno utjecale na brzinu oslobađanja. Analiza kitozanskih mikročestica izrađenih s kitozanima različitih molekulskih masa pokazala
je da se kod povećane molekulske mase kitozana smanjuje brzina oslobađanja. S druge strane, brzina oslobađanja bila je veća pri većoj koncentraciji lijeka unutar donorskog odjeljka. Na taj je način pokazana diskriminatornost metode prema razlikama u formulaciji, kao i prema razlikama u koncentraciji uzorka unutar donorskog odjeljka ćelije. Metodom je nadalje potvrđena sličnost serija istog sastava proizvedenih istim procesom. Metoda je validirana u skladu s ICH smjernicama. U sklopu validacije metode potvrđena je njena specifičnost, linearnost, točnost, preciznost i robusnost. Metoda je uspješno primijenjena kod kitozanskih i metakrilatnih čestica, čime je pokazan njen potencijal za karakterizaciju raznih tipova
topikalnih čestičnih terapijskih sustava. Razvijena metoda može biti koristan alat u razvoju formulacije kod takvih terapijskih sustava.
Currently there are no compendial or standard methods and apparatuses for in vitro release testing of topical microparticles. The aim of this study was to develop and validate a discriminative in vitro release method for topical microparticles using the immersion cell. For the purpose of this study chitosan-based and methacrylate-based microparticles with mupirocin were prepared by spray drying. The following characteristics of the microparticles were
examined: encapsulation efficiency, particle size and morphology, crystallinity, thermal properties, spectral properties, surface charge, hygroscopicity, residual organic solvents and water content. Chitosan-based and methacrylate-based microparticles with mupirocin were used as model topical delivery systems for in vitro release method development. The immersion cells were used in combination with paddle dissolution apparatus. The pH and temperature of the release medium (pH 5.5, 32°C) were selected to reflect the physiological skin conditions. Diffusion of the drug occured
across a mixed cellulose ester membrane, which demonstrated low drug adsorption and low diffusional resistance. After an initial lag phase the amount of drug released became proportional to the square root of time. The slope in the linear portion of the release curve was used as a measure of release rate. Variations in paddle rotation speed (25 rpm, 50 rpm, 100 rpm), paddle height (1 cm, 2.5 cm) and volume of release medium (100 ml, 200 ml) did not significantly alter the release rates. Appropriate discriminatory power of the method was confirmed as the method was able to detect differences in formulation, as well as differences in drug concentration inside the sample compartment. The analysis of chitosan-based microparticles prepared with chitosans of different molecular weights has shown that the release rate decreases with increasing molecular weight of chitosan. On the other hand, the release rate increased with increasing drug concentration inside the sample compartment. The method was further used to confirm sameness between batches of the same composition prepared by the same process. The method was validated for its specificity, linearity, accuracy, precision and robustness in line with International Conference on Harmonisation (ICH) guidelines. The method was successfully applied both for chitosan-based and methacrylate-based microparticles, which demonstrates its potential application for various types of topical particulate delivery system.
Uvod: Glavni regulator sistemske homeostaze željeza je hepcidin, čiju sintezu reguliraju status željeza, upalni citokini, eritropoetska aktivnost, hipoksija i anemija. Cilj ovoga rada bio je ispitati ...povezanost između serumskog hepcidina i čimbenika koji ga reguliraju u skupini bolesnika s KOPB-om, da bi se ispitalo kako ovi čimbenici utječu na koncentraciju hepcidina i osiguravanje opskrbe željezom neophodnim za eritropoezu. Hipoteze istraživanja: koncentracija hepcidina u serumu bolesnika s KOPB-om promijenjena je u odnosu na kontrolnu skupinu i mijenja se tijekom egzacerbacije u odnosu na stabilnu fazu bolesti; na koncentraciju hepcidina u KOPB-u utječu upala i/ili hipoksija.
Metode i ispitanici: U istraživanje je bilo uključeno 40 bolesnika s KOPB-om i 30 zdravih ispitanika. U KOPB-u skupini parametri su longitudinalno praćeni u tri vremenske točke: u egzacerbaciji, fazi rezolucije i stabilnoj fazi bolesti. Svim ispitanicima određena je koncentracija hepcidina, pokazatelja statusa željeza (serumsko željezo, TIBC, saturacija transferina, feritin); pokazatelja upale (broj leukocita i neutrofilnih granulocita, IL-6 i CRP-a), eritropoetske aktivnosti (broj retikulocita, topivi transferinski receptor i eritropoetin) i koncentracija hemoglobina. Svim bolesnicima s KOPB-om određeni su parcijalni tlak kisika i saturacija hemoglobina kisikom kao pokazatelji hipoksije.
Rezultati: Serumska koncentracija hepcidina bila je povišena u egzacerbaciji i stabilnoj fazi KOPB-a u odnosu na kontrolnu skupinu, te je pozitivno korelirala s IL-6 i CRP-om. Koncentracija hepcidina je bila pozitivno povezana s feritinom i negativno s TIBC-om. Eritropoetska aktivnost, mjerena apsolutnim brojem retikulocita bila je u svim fazama istraživanja niža od kontrolne skupine, a negativna povezanost je pokazana u egzacerbaciji. Koncentracija hepcidina nije bila povezana s parametrima hipoksije. U kontrolnoj skupini hepcidin je korelirao samo s pokazateljima statusa željeza, negativno s TIBC-om i pozitivno s feritinom.
Zaključak: Istraživanje je pokazalo da prisutna sistemska upala i povišena razina IL-6 u egzacerbaciji i stabilnoj fazi KOPB-a mogu biti odgovorne za opaženi porast koncentracije hepcidina. Porast koncentracije hepcidina mogao bi biti povezan s restriktivnom eritropoezom koja se očituje sniženim brojem retikulocita u svim fazama istraživanja, kao i snižavanju koncentracije hemoglobina u stabilnoj fazi bolesti. Dobiveni rezultati pružaju uvid u dinamičke promjene metabolizma željeza i povezanosti s razinom hepcidina. Sistemska upala prisutna kod velikog broja bolesnika s KOPB-om jača kako bolest napreduje, te bi porast razine IL-6 mogao voditi do daljnjeg porasta koncentracije hepcidina.
Background: Hepcidin is the main regulator of systemic iron homeostasis, and its expression is modulated by iron status, hypoxia, erythroid factors and inflammation. The aim of this study was to examine a relationship between level of hepcidin and iron status, erythropoietic activity, hypoxia and inflammation in exacerbations and stable COPD. We hypothesized that hepcidin concentration is changed compared to control group and is changing in acute exacerbation compared to stable COPD; hepcidin concentration is substantially influenced by inflammation and/or hypoxia.
Methods: The study included 40 COPD patients and 30 healthy subjects. In COPD group parameters were longitudinally monitored at three time points: at exacerbation, on resolution and in stable disease. We determined concentration of hepcidin and hemoglobin; parameters of iron status: serum iron, total iron binding capacity (TIBC), ferritin and calculated transferrin saturation. Soluble transferrin receptors, reticulocyte number (Rtc), and regulatory hormone erythropoietin were measured as indicators of erythropoietic activity. Systemic inflammation was assessed by determination of CRP, IL-6, and number of white blood cells and neutrophils. In COPD group partial oxygen pressure and haemoglobin oxygen saturation were determined.
Results: Hepcidin was elevated in exacerbations and in a stable phase compared to the control group and we found positive correlations of hepcidin with inflammatory markers IL-6 and CRP. Hepcidin also correlated positively with ferritin and inversely with TIBC. Erythropoietic activity, measured by absolute Rtc number, was significantly reduced in COPD compared to the control group in all study phases, and negative correlation with hepcidin was established in exacerbation. In exacerbation and stable disease hepcidin correlated with ferritin and TIBC. No correlations were observed with indices of hypoxia. In the control group, positive associations were observed only with indices of iron status, positive with ferritin and negative one with TIBC.
Conclusion: This study shows that elevated values of IL-6 present in exacerbations and stabile COPD might be responsible for the observed increased hepcidin level. These might be associated with restricted erythropoiesis as shown by lower number of reticulocytes and decreased level of haemoglobin at the stable phase. The results obtained might provide new insights into dynamic changes of iron metabolism and its relation to hepcidin level in COPD. Systemic inflammation present in majority of COPD patients increases over time as disease progresses so raising of IL-6 level could lead to further up-regulation of hepcidin.
Učinak sekundarnog metabolita masline (Olea europaea L.) oleuropeina, njegovog razgradnog produkta hidroksitirosola i vodenog ekstrakta maslinova lista na staničnu vijabilnost medicinski značajne ...gljivične vrste Candida albicans ispitan je in vitro testovima. Metodom mikrodilucije određene su minimalne inhibitorne koncentracije (MIK) koje uzrokuju 80 % smanjenja stanične vijabilnosti i koje su iznosile 12,5 mg/ml za oleuropein, 6,25 mg/ml za hidroksitirosol i 25 mg/ml za vodeni ekstrakt maslinova lista. Kvantitativna analiza s fluorescentnim bojanjem upućuje na zaključak da je apoptoza primarni način stanične smrti u uzorcima tretiranim sa sub-MIK koncentracijama ovih tvari. Budući da je patogenost vrste određena virulentnim čimbenicima, ispitani su učinci oleuropeina i hidroksitirosola na najznačajnije virulentne čimbenike vrste C. albicans. Utvrđen je inhibitorni učinak ovih fenolnih tvari na promjenu u hifalni oblik rasta kod vrste C. albicans induciranu u uvjetima staničnog gladovanja. Pri sub-MIK koncentracijama, oleuropein i hidroksitirosol nisu pokazali inhibitorno djelovanje na stvaranje biofilma ove vrste. Pri subinhibitornim koncentracijama nakon 24 sata inkubacije, oleuropein i hidroksitirosol su uzrokovali modulaciju stanične površinske hidrofobnosti koja se dovodi u svezu s adherencijom vrste C. albicans na biomaterijale. Uzgoj vrste C. albicans u prisutnosti oleuropeina i hidroksitirosola je uzrokovao inhibiciju aktivnosti kandidinih hidrolitičkih enzima, aspartil-proteaza (Sap) i α-glukozidaza. Tretiranjem vrste C. albicans s oleuropeinom i hidroksitirosolom utvrđeno je višestruko djelovanje ovih tvari uključujući oštećenje stanične stijenke, stanične membrane i otpuštanje citoplazmatskog sadržaja (DNA i proteina). S obzirom da neki antimikotici djeluju kao inhibitori biosinteze ergosterola, provedeno je ispitivanje učinka oleuropeina i hidroksitirosola na modulaciju ergosterola kojima je utvrđeno smanjenje ovog sterola u ovisnosti o primijenjenoj koncentraciji. Uz testove određivanja djelovanja na gljivičnu vrstu, ispitani su mogući citotoksični i genotoksični učinci na stanice nositelja. Navedene tvari nisu pokazale hemolitičku aktivnost na humanim eritrocitima pri testiranim koncentracijama u rasponu 3-60 μM. Ispitivanje učinka na humanim limfocitima periferne krvi provedeno kometnim testom pokazalo je protektivno djelovanje ovih tvari na oštećenja DNA inducirana s mutagenom H2O2, pri čemu je hidroksitirosol imao bolji protektivni učinak u usporedbi s oleuropeinom nakon 120 min predtretmana s ispitivanim tvarima u koncentracijama 1, 5 i 10 μM. Navedeni genoprotektivni učinci su posljedica snažnog antioksidativnog djelovanja ovih fenolnih spojeva utvrđenih ABTS, FRAP i CUPRAC metodom.
Activity of oleuropein, a phenol secoiridoid present in olive tree products, its derivative hydroxytyrosol and water extract of olive leaf were investigated using in vitro tests against opportunistic fungal pathogen Candida albicans. Antifungal susceptibility testing was used to estimate the drop of viability up to 80 % in comparison to the control (untreated cells). Minimal inhibitory concentration (MIC) against C. albicans for oleuropein was 12,5 mg/ml, for hydroxytyrosol was 6,25 mg/ml and for water extract of olive leaf was 25 mg/ml. Morphological changes in the nuclei after staining with fluorescent DNA-binding dyes revealed that apoptosis was a primary mode of cell death in analyzed samples treated with sub-MIC concentrations of tested compounds. In order to understand mode of action of oleuropein and hydroxytyrosol, their effect on C. albicans virulence factors essential for development of infection in the host have been investigated. Both oleuropein and hydroxytyrosol modulate morphogenetic coversion and inhibit filamentation of C. albicans induced in the conditions of reduced nutrient availability. On the other hand, treatment with sub-MIC concentractions of these phenolic compounds did not affect C. albicans biofilm formation. The hydrophobicity assay showed that both compounds, in sub-MIC values, have decreased the cellular surface hydrophobicity, a factor associated with adhesion to epithelial cells. Cultivation of C. albicans in the presence of oleuropein and hydroxytyrosol also inhibits, in the dose-dependent manner, acitivity of aspartyl-proteases (Sap), hydrolytic enzymes important in adherence, tissue penetration, invasion and destruction of host tissue. Most therapies for fungal infections target the ergosterol biosynthesis pathway or its end product ergosterol which is necessary for growth and normal function of fungal cells. At subinhibitory concentrations tested compounds have altered sterol content and subsequently affected the cell membrane of C. albicans. Both compounds induced dose-dependent loss of intracellular material to outer space (DNA, protein leakage) and caused membrane depolarisation indicating their direct effect on the membrane. In addition, cytotoxicity and genotoxicity assays were performed to elucidate the effect of oleuropein and hydroxytyrosol on the host cells. Oleuropein and hydroxytyrosol exhibited no hemolytic activity on human erythrocytes at tested concentrations (3-60 μM). Antigenotoxic effects of oleuropein and hydroxytyrosol against H2O2-induced damage in human lymphocytes were evaluated in vitro by alkaline comet assay. Pretreatment of human lymphocytes with each of the substance for 120 min produced dose-dependent reduction of primary DNA damage in the tested cell type. At tested concentrations (1, 5, 10 μM), hydroxytyrosol showed better protective effect than oleuropein against H2O2-induced DNA breaks. Our results suggest that genoprotective effects of these phenolic compounds could be attributed to their radical scavenging and metal ions chelating activity which were determined using ABTS, FRAP and CUPRAC assays.
Novi dokazi ukazuju da je antinociceptivno djelovanje botulinum toksina tipa A (BT-A) središnjeg porijekla. U ovom doktorskom radu smo provjerili ovu pretpostavku u ranije nedovoljno istraženim ...oblicima boli, istraživali interakciju sa središnjim neurotransmitorima kao mogući mehanizam djelovanja te pokušali pobliže utvrditi mjesto djelovanja toksina u središnjem živčanom sustavu (SŽS).
Ispitivanja su izvedena na mužjacima Wistar štakora. U upalnoj, neuropatskoj i bilateralnoj mišićnoj boli ispitan je učinak selektivnih i neselektivnih antagonista opioidnih i GABAA receptora, primijenjenih sistemski, spinalno ili supraspinalno, na antinociceptivno djelovanje periferno (supkutano u šapu) primijenjenog BT-A. U tkivu kralješnične moždine ispitana je aktivacija neuronalnih i glija stanica, ekspresija mRNA proupalnih citokina i μ-opioidnih receptora te ekspresija Leu/Met-enkefalina metodama imunofluorescencije i lančane reakcije polimerazom s reverznom transkripcijom. Istraživano je antinociceptivno djelovanje BT-A nakon periferne, spinalne i supraspinalne primjene te je imunofluorescencijom ispitana enzimska aktivnost BT-A u tkivu SŽS-a.
Opioidni i GABAA antagonisti su ovisno o dozi, sistemski i intratekalno, ali ne i supraspinalno, poništili antinociceptivno djelovanje BT-A u svim ispitanim modelima. Učinak antagonista bio je kratkotrajan. BT-A je smanjio neuronalnu aktivaciju u dorzalnom rogu kralješnične moždine, što su antagonisti blokirali. BT-A je smanjio bol u dosad neistraženim modelima visceralne boli (peritonitis, kolitis). Bilateralno antinociceptivno djelovanje BT-A posljedica je prisutnosti toksina samo na ipsilateralnoj strani. BT-A je smanjio bol nakon periferne i intratekalne primjene, dok primijenjen supraspinalno (cisterna magna, moždane komore) nije djelovao, unatoč nalazu njegove enzimske aktivnosti u pojedinim regijama mozga uključenima u nocicepciju.
BT-A ima segmentalno antinociceptivno djelovanje spinalnoj razini, uz neizravnu aktivaciju endogenog opioidnog i GABA-ergičkog sustava. Ovi bi nalazi mogli biti važni za klinička ispitivanja potencijalno korisnih sinergističkih interakcija s konvencionalnim analgeticima i drugim lijekovima te usmjeriti klinička ispitivanja na nove indikacije i nove načine primjene, poput intratekalne.
Novel evidence suggests that the antinociceptive effect of botulinum toxin type A (BT-A) is of central origin. In this doctoral thesis, we verified this assumption in previously insufficiently investigated types of pain, investigated the interaction with central neurotransmitters as the possible mechanism of action and tried to determine the site of the toxin’s action within the central nervous system (CNS).
Male Wistar rats were used in experiments. We examined the effect of selective and nonselective opioid and GABAA antagonists, applied systemically, spinally and supraspinally, on antinociceptive effect of peripherally (subcutaneously into hind paw) applied BT-A in inflammatory, neuropathic, and bilateral pain. Neuronal and glial cells’ activation, proinflammatory cytokines’ and μ-opioid receptors mRNA expression, and Leu/Met-enkephalin protein expression were analyzed using immunofluorescence and quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) in lumbar spinal cord tissue. Further, we investigated the antinociceptive effect of BT-A following peripheral, spinal and supraspinal application. In parallel, enzymatic activity of BT-A in CNS tissue was examined using immunofluorescence.
Opioid and GABAA antagonists, applied systemically and intrathecally, dose-dependently abolished the antinociceptive effect of peripheral BT-A in all tested models, while no effect was observed following their supraspinal application. The effect of antagonists was short-lasting. BT-A reduced neuronal activation in dorsal horn, which was abolished by both, opioid and GABAA antagonist. BT-A diminished pain in, yet uninvestigated, models of visceral pain (peritonitis and colitis). BT-A’s bilateral antinociceptive action occurs after toxin’s presence on ipsilateral side only. BT-A reduced pain after peripheral and intrathecal application, but not after application in cisterna magna or cerebral ventricles, despite of its enzymatic activity in brain regions involved in nociception.
BT-A has segmental antinociceptive effect at the spinal level, which involves an indirect activation of endogenous opioid and GABA-ergic systems. These findings might guide clinical investigations of potentially useful additive or synergistic effects with conventional analgesics and direct clinical trials for novel indications and the routes of application, like intrathecal.
Polimeri su već neko vrijeme dio farmacijskoga oblikovanja lijekova, bilo da se koriste kao pomoćna sredstva ili kao ambalaža za lijekove. Polimeri imaju jedinstvena fizika!no-kemijska svojstva kao ...što su molekulska masa, viskoznost, geliranje ili topljivost, koja uvelike utječu na njihovu primjenu u farmaciji i medicini. Sve je veća primjena polimera u naprednim terapeutskim sustavima kao što su matrični i spremišni sustavi, osmotske pumpe, degradacijski sustavi te sustavi osjetljivi na okolinu. Poznavanje odnosa struktura-svojstvo polimemih tvari te njihova karakterizacija jedan je od preduvjeta za dizajniranje lijekova pomoću polimemih tvari.
