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Optimization of the skin barrier model with isolated ceramides of human stratum corneum
Dulanská, Lucia ; Pullmannová, Petra (advisor) ; Zbytovská, Jarmila (referee)
Charles University, Faculty of Pharmacy in Hradec Králové Department of Biophysics and Physical Chemistry Author: Lucia Dulanská Supervisor: Mgr. Petra Pullmannová, Ph.D Title of thesis: Optimization of the skin barrier model with isolated ceramides of human Stratum corneum Stratum corneum (SC), the uppermost layer of the skin, regulates transcutaneous water loss and protects against outer conditions and harmful substances. It consists of cornified cells - corneocytes and extracellular lipid matrix, which is responsible for the barrier functions. Corneocytes are covered with covalently bound lipids creating the corneocyte lipid envelope (CLE). CLE is considered to interconnect the extracellular lipids with corneocytes and to have a templating effect. We aimed to optimize a skin lipid model simulating also the presence of CLE. The lipidic part of the model was prepared from an equimolar mixture of isolated human skin ceramides (hCer), cholesterol and free fatty acids (FFA, either protonated or deuterated) with 5 weight % of cholesteryl sulfate. hCer were extracted from the isolated human SC and purified by the column chromatography. The composition of hCer was determined by the high- performance thin-layer chromatography. The reverse-phase and normal phase silica gel particles served as the CLE...
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In vitro models of skin barrier
Šimek, Matěj ; Zbytovská, Jarmila (advisor) ; Pullmannová, Petra (referee)
Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmaceutical Technology Candidate: Matěj Šimek Consultant: doc. Dr. rer. nat. Mgr. Jarmila Zbytovská Title of thesis: In vitro models of skin barrier The aim of this work is to summarize information about various types of skin models which are used for testing of permeability, toxicity, irritability and other aspects of drugs, through professional, verified and reviewed literature. These characteristics are necessary to know in order to grant optimal safety, effectiveness nad quality of transdermally administered drugs. Transdermal administration of drugs has got lots of benefits in contrast with classic peroral administration. An administration of drugs through this way is quite simple and it can be interrupted quite easily. We can also easily change a place of administration in which a drug is released and the risk of overdosing is very low. Furthermore, transdermal administration makes possible to maintain constant plasmatic concentration of drug in a blood stream and also to prolong the duration of effect of drugs with small halftime thanks to constant releasing of drug. And primarily, transdermally administered drug normally avoids the "first-pass" effect of liver, so the dose of drug can be lowered. The risk of drug...
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Study of transdermal and dermal absorption of 2,6-diaminopurine acyclic nucleoside phosphonates
Diblíková, Denisa ; Vávrová, Kateřina (advisor) ; Zbytovská, Jarmila (referee)
Acyclic nucleoside phosphonates (ANP) are broad-spectrum antivirals highly effective against herpes-, retro- and hepadnaviruses. They also exhibit cytostatic, antiparasitic, immunomodulatory activities. Their transdermal delivery offers an attractive and advantageous route of administration, but is limited due to the polar character of their phosphonate moiety. The aim of this work was to study the possibility of both transdermal and dermal application of a series of 2,6-diaminopurine derivatives including (R)-PMPDAP and (S)-PMPDAP, (S)-HPMPDAP, (S)-8-azaHPMPDAP, cyclic (S)-HPMPDAP and lysolipid prodrugs, i.e., hexadecyloxypropyl (HDP) esters of (R)-HDP-PMPDAP and (S)-HDP- HPMPDAP. Ability of ANP to penetrate trough the skin by themselves is generally very low. For this reason the influence of permeation enhancer dodecylester of 6- (dimethylamino)hexanoic acid (DDAK) through and into human skin was investigated. The evaluation was performed in vitro by using Franz diffusion cells and human skin. The results of this work confirm that ANP (60 mM in 60 % propylene glycol) delivery through the skin is very low (flux 0.53-1.40 nmol/cm2 /h), except for the lysolipid prodrugs (R)-HDP-PMPDAP and (S)-HDP-HPMPDAP), which were not detected in the acceptor phase at all. 1 % DDAK enhanced transdermal flux of...
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Influence of cholesterol concentration on permeability and microstructure of model skin barrier lipid membranes
Červená, Martina ; Vávrová, Kateřina (advisor) ; Zbytovská, Jarmila (referee)
Cholesterol is a substance of a steroidal nature that has a number of functions in the human body. One of them is also an irreplaceable role in the proper functioning of the skin barrier. Cholesterol is an integral part of the lipid matrix, together with ceramides and free fatty acids in an equimolar ratio (1: 1: 1), and 5% cholesterol sulfate, which fills the intercellular space between stratum corneum cells and is responsible for the barrier properties of the skin. Cholesterol is therefore necessary for epidermal homeostasis, but its role in SC permeability is unknown. The aim of this work was to study the influence of cholesterol concentration on the permeability and microstructure of model lipid membranes of the skin barrier. Eight sets of model membranes with decreasing cholesterol concentrations (100%, 80%, 70%, 60%, 40%, 20%, 0%) and cholesterol alone were studied for which permeability and microstructure were monitored.The study of permeability of membranes was carried out in the Franz diffusion cells by measuring four permeation parameters: water loss, electrical impedance measurement, and the cumulative amount of two model drugs (theophylline and indomethacin). The microstructure of these model membranes was verified by X-ray powder diffraction. X-ray powder diffraction measurements...
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Nanocarriers for dermal and transdermal drug administration
Girgel, Josef ; Zbytovská, Jarmila (advisor) ; Holas, Ondřej (referee)
Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmaceutical Technology Candidate: Josef Girgel Consultant: Doc. Dr. rer. Nat. Mgr. Jarmila Zbytovská Title of thesis: Nanocarriers for dermal and transdermal drug delivery The aim of this work was to map the literature and the most logically divide it and to elucidate the mechanisms of the carrier's interaction with the skin, the degree of their ability to penetrate the skin and to evaluate their potential to the next years. Skin administration appears to be more advantageous way than others, mainly due to higher patient's compliance, avoiding the first pass effect of the liver, ensuring a stable blood concentration during transdermal administration and reducing adverse effect of the drug. However, the skin serves as an effective barrier to the external environment, especially its outermost layer called stratum corneum. In a recent year, the interest of many research groups has focused on nanocarriers which are expected to deliver more efficient transport to and through the skin barrier due to their size ranging from tens to hundreds of nanometers. It has been confirmed that some of these carriers are able to permeate the skin. For metallic nanoparticles, their permeation is primary associated with toxicity and they are...
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The permeability and microstructure of model stratum corneum lipid membranes: comparison of non-hydroxylated and (R)- and (S)-α-hydroxylated ceramides
Šilarová, Michaela ; Vávrová, Kateřina (advisor) ; Zbytovská, Jarmila (referee)
Ceramides are lipid components in the uppermost layer of the skin, stratum corneum and they are essential for correct function of a skin barrier. In the stratum corneum, ceramides with cholesterol and free fatty acids are in equimolar ratio. Ceramides contain sphingoid bases, which are amino alcohols sphingosine, phytosphingosine, dihydrosphingosine or 6- hydroxysphingosine. These sphingoid bases are N-acylated by non-hydroxylated, α- hydroxylated or ω-hydroxylated fatty acid, mostly by lignoceric (C24) acid. The aim of this work was to study the permeability and microstructure of the model membranes containing non-hydroxylated ceramides. Moreover, we aimed to study the effect of additional α-hydroxyl group in ceramides including the effect of stereochemistry in position 2. We prepared model membranes based on Cer, free fatty acids (C16-24), cholesterol and a small amount of cholesteryl sulfate (5 wt%). We investigated four permeability markers: electrical impedance, water loss through the membrane, flux of theophylline and flux of indomethacin. The microstructure and miscibility of ceramides with other lipids were studied by infrared spectroscopy and X-ray powder diffraction. The results from experiments showed some differences between ceramides. For example the effect of the type of sphingoid...
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Characterization of liposomal formulations for brain-targeting.
Labajová, Andrea ; Šklubalová, Zdeňka (advisor) ; Zbytovská, Jarmila (referee)
Charles University in Prague, Faculty of Pharmacy in Hradec Králové Department of: Department of Pharmaceutical Technology Consultant: Assoc. Prof. Zdeňka Šklubalová, Ph.D. Robin Tremmel, Ph.D. student Student: Andrea Labajová Title of Thesis: Characterization of liposomal formulations for brain- targeting This paper deals with characterization of liposomal formulations with encapsulated triethylenetetramine (TETA), which is selective CuII - chelator used in the treatment of Wilson's disease for decades. Liposomal formulations were prepared by a film hydration method with subsequent dual asymmetric centrifugation with the addition of 2.5 mol/l TETA solution dissolved at pH3 or/and pH7, respectively. Size exclusion chromatography (SEC) was performed to separate free-TETA from the encapsulated-one at day 1, 2, 3, 4, 5 and 8. Two methods of liposome purification, the one column method and the two columns method, were used. The size and the size distribution of prepared liposomes were measured by photon correlation spectroscopy (PCS) at each day of storage. The concentration of encapsulated TETA, as well as the concentrations of cholesterol before the SEC and after the SEC were determined by HPLC in order to express the encapsulation efficiency. No influence of pH or the method of purification on the...
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The permeability and microstructure of model stratum corneum lipid membranes: comparison of non-hydroxylated and (R)- and (S)-α-hydroxylated ceramides
Šilarová, Michaela ; Vávrová, Kateřina (advisor) ; Zbytovská, Jarmila (referee)
Ceramides are lipid components in the uppermost layer of the skin, stratum corneum and they are essential for correct function of a skin barrier. In the stratum corneum, ceramides with cholesterol and free fatty acids are in equimolar ratio. Ceramides contain sphingoid bases, which are amino alcohols sphingosine, phytosphingosine, dihydrosphingosine or 6- hydroxysphingosine. These sphingoid bases are N-acylated by non-hydroxylated, α- hydroxylated or ω-hydroxylated fatty acid, mostly by lignoceric (C24) acid. The aim of this work was to study the permeability and microstructure of the model membranes containing non-hydroxylated ceramides. Moreover, we aimed to study the effect of additional α-hydroxyl group in ceramides including the effect of stereochemistry in position 2. We prepared model membranes based on Cer, free fatty acids (C16-24), cholesterol and a small amount of cholesteryl sulfate (5 wt%). We investigated four permeability markers: electrical impedance, water loss through the membrane, flux of theophylline and flux of indomethacin. The microstructure and miscibility of ceramides with other lipids were studied by infrared spectroscopy and X-ray powder diffraction. The results from experiments showed some differences between ceramides. For example the effect of the type of sphingoid...
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Study on phase behaviour of selected lipid mixtures
Černochová, Klára ; Zbytovská, Jarmila (advisor) ; Matějka, Pavel (referee)
Charles University in Prague, Faculty of Pharmacy in Hradec Králové Department of Pharmaceutical Technology Consultant: Mgr. Jarmila Zbytovská, Dr. rer. nat. Student: Klára Černochová Title of Thesis: Study on phase behaviour of selected lipid mixtures Ceramides, cholesterol and fatty acids are the main components of the stratum corneum (SC) intercellular lipid matrix. These lipids play the major role in maintaining the skin barrier function. The goal of this work is to study the SC lipid mixtures on the molecular level, characterization of their miscibility and their temperature phase behavior. For this purpose, lipid model systems imitating natural structure of the SC intercellular lipid matrix were prepared. The membranes contained one or more ceramides (CerAP, CerNP, CerNS), cholesterol, stearic acid and cholesterol sulfate. The mixtures were studied by infrared spectroscopy using a heated ATR-crystal. From the measured data, IR spectra and temperature dependency of the position of the particular IR-modes were evaluated. The results showed the presence of hydrogen bindings within the ceramides. These bonds were also observable in the equimolar lipid mixture based on CerNP. Otherwise, lipid mixtures containing CerAP showed very good miscibility of the individual components. The two- and three-...
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Study of lipid membranes with ceramide precursors as models of atopic dermatitis
Staňková, Klára ; Vávrová, Kateřina (advisor) ; Zbytovská, Jarmila (referee)
Charles University in Prague, Faculty of pharmacy in Hradec Králové Department of inorganic and organic chemistry Candidate: Klára Staňková Supervisor: doc. PharmDr. Kateřina Vávrová, Ph.D. Title of diploma thesis: Study of lipids membranes containing ceramide precursors as models of atopic dermatitis Ceramides (Cer) are important determinants for both water-retention function and permeability-barrier function in the stratum corneum (SC). They are synthesized from their precursors, i.e. sphingomyelin (SM) and glycosylceramide (GC) by the enzymes sphingomyelinase (SMase) and glucocerebrosidase (GCase). Their reduced levels have been found in the skin diseases e.g. in atopic dermatitis, and are also a causative factor for the dry and barrier-disrupted skin. The aim of this study was to prepare model skin lipid membranes simulating the defect of SMase and GCase and study their permeability. Models of SC lipid membranes were prepared as an equimolar mixture of Cer or their precursors (either SM or GC) in different ratios, cholesterol, fatty acid mixture and 5 % of cholesterol sulfate. The permeability of these model membranes was evaluated using permeation of theophylline and indometacin, transepidermal water loss and electrical impedance in Franz diffusion cells. The amount of model drug permeated...
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