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Study of lipid membranes with lysolipids as models of atopic dermatitis
Pospíšilová, Markéta ; Vávrová, Kateřina (advisor) ; Zbytovská, Jarmila (referee)
Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of inorganic and organic chemistry Candidate: Pospíšilová, Markéta Supervisor: Assoc. Prof. PharmDr. Kateřina Vávrová, Ph.D. Title of Diploma Thesis: Study of lipid membranes with lysolipids as models of atopic dermatitis We are protected against the harmful influence of the external environment by a skin barrier, which is located in the stratum corneum. The skin barrier also prevents excessive water loss through the skin. Ceramides have the most important role in the barrier function of the skin. The healthy skin's ceramides are synthesized of glucosylceramide and sphingomyelin via enzymes sphingomyelinase and glucocerebrosidase. If there is a lack of ceramide in the stratum corneum, which means, if these mechanisms work insufficiently, it gives rise to atopic dermatitis. In the stratum corneum of a patient who suffers from atopic dermatitis are glucosylceramide and sphingomyelin hydrolyzed by glucosylceramide/sphingomyeline deacylase which leads to free fatty acids and lysolipids (glucosylsphingosine and sphingosinephosphorylcholine) generation. In this work, we prepared model membranes which simulate a composition of lipids of the stratum corneum of a patient with atopic dermatitis, in which ceramides are replaced by...
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Isosters of transkarbam 12 as transdermal penetration enhancers
Šíma, Martin ; Hrabálek, Alexandr (advisor) ; Vávrová, Kateřina (referee)
SUMMARY: Diploma thesis - Isosters of Transkarbam 12 as skin penetration enhancers Transkarbam 12 (5-(dodecyloxycarbonyl)pentylammonium-5-(dodecyloxycarbo- nyl)pentylcarbamate, T12) is a highly effective skin permeation enhancer. In this diploma thesis, T12 analogs with carbamate and carbonate function groups replacing the ester one were synthetised, and evaluated in vitro as skin permeation enhancers on excised porcine skin, using theophiline as a model drug. The results were expressed by an enhancing ratio and compared with the biological activity of T12. The enhancing activity decreases in the following sequence: ester, carbonate, carbamate. The results thus show that it is the ester group in the molecule of T12 which is essential for its extraordinary effect. In addition, homologues with a different length of the ending and connecting carbon chains were evaluated. While the length of the connecting carbon chain does not influence the biological activity significantly, nonyl to decyl ending carbon chains show the highest acceleration ability. It indicates that the length of the carbon chain, and not its lipofility, is important for the enhancing activity.
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Synthesis and study of 1-O-acylceramides
Jon, Vojtěch ; Vávrová, Kateřina (advisor) ; Opálka, Lukáš (referee)
The extracellular matrix of the uppermost layer of the skin, the stratum corneum (SC), consists of ceramides (Cer), cholesterol (Chol), free fatty acids (FFA) and cholesteryl sulfate (CholS). Cer play an important role in the correct barrier function of mammalian epidermis. A new type of sphingolipids, i.e., 1-O-acyl-Cer, have been found in human SC very recently; however, their role in the SC is unknown. These Cer species contain sphingosine (S) that is N- acylated with non-hydroxylated or α- hydroxylated fatty acid, and moreover, hydroxyl group at C1 in sphingosine is esterified by an additional fatty acid (lignoceric acid, C24 or palmitic acid, C16). Because 1-O-acyl-Cer are not commercially available, we aimed to synthesize physiological 1-O-acyl Cer, i.e., Cer-24NS16, Cer-16NS16 and Cer-24AS16. Moreover, we aimed to study their behaviour on permeability and microstructure of model skin lipid membranes. The 1-O-acyl-Cer were synthesized by an acylation of Cer-NS16 or Cer-AS16 with palmitic or lignoceric acid using N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide and 4- dimethylaminopyridine. Cer-AS16 was prepared by α-bromination of palmitic acid, substitution of bromine by hydroxyl and N-acylation of sphingosine by the prepared acid in the presence N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide...
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Synthesis of ceramides with deuterated sphingosine
Havlíčková, Kateřina ; Vávrová, Kateřina (advisor) ; Hrabálek, Alexandr (referee)
Ceramides are a complex group of lipids belonging to sphingolipids. They are the most important component of stratum corneum (SC) and thus participate in a barrier function of the skin. In spite of the ceramides are a subject of intensive research, the exact effect of their structure to the barrier function of the skin is poorly understood. For studying the structure, properties and dynamics of SC lipid membranes, analogues of lipids labelled by deuterium are often used. The methods using deuterated molecules include neutron scattering, fourier transform infrared spectroscopy (FTIR), Raman spectroscopy, deuterium nuclear magnetic resonance (2 H-NMR) and time of flight secondary ion mass spectrometry (ToF-SIMS). This work describes the synthesis of the ceramide with a deuterated sphingosine chain. The acquired ceramides with a different acyl chain length (deuterated as well as non-deuterated) should be used for studying their properties in SC lipid membranes in the future. The synthesis was based on methods commonly used for preparation of non-deuterated analogues of ceramides. The most important step of the synthesis was the incorporation of the deuterated chain into the molecule of sphingosine. The deuterated terminal alkyne was prepared from the perdeuterated bromoalkane. Next the deuterated...
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Synthesis of transdermal permeation enhancers based on piperidine carboxylic acids
Fratričová, Veronika ; Vávrová, Kateřina (advisor) ; Hrabálek, Alexandr (referee)
Transdermal permeation enhancers are chemical compounds, which promote drug absorption through the skin via the barrier resistance decrease. The model structure was molecule of transkarbam 12. I have synthesized these compounds: 2-(decyloxycarbonyl)piperidinium bromide 2-(dodecyloxycarbonyl)piperidinium bromide 1-acetylpiperidine-2-carboxylic acid decyl ester 1-acetylpiperidine-2-carboxylic acid dodecyl ester (R)-N -acetylpiperidin-2-carboxylic acid decyl ester (R)-N -acetylpiperidin-2-carboxylic acid dodecyl ester (R)-N -acetylpiperidin-3-carboxylic acid decyl ester (R)-N -acetylpiperidin-3-carboxylic acid dodecyl ester 2-(decyloxycarbonyl)piperidinium 2-(decyloxycarbonyl)piperidine-1-carbamate 2-(dodecyloxycarbonyl)piperidinium 2-(dodecyloxycarbonyl)piperidine-1-carbamate Transdermal permeation-enhancing activity of the prepared compounds was evaluated on the porcine skin in the Franz diffusion cells using theophylline as the model drug. The enhancement ratio values were compared with the results of structurally related compounds synthesized in the previous diploma theses. The results showed that the least active substances were the hydrobromides, N-acetylderivatives were more active, comparable with model enhancer transkarbam 12. No significant difference between racemates and (R)-izomers of N-...
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Effect of pH on transdermal delivery of adefovir
Lorencová, Kateřina ; Vávrová, Kateřina (advisor) ; Doležal, Pavel (referee)
Adefovir (PMEA) belong to the group of acyclic nucleoside analogues exhibiting various biological (e.g. antiviral, cytostatic, antiparasitic, immunomodu-latory) activities. Its prodrug adefovir dipivoxil (PMEAd) has recently been approved for the treatment of chronic hepatitis B. The aim of this work was to study permeation properties of 2% PMEA in vitro in the broad spectrum of pH using different vehicles with or within presence of permeation enhancers. We also evaluated penetration of PMEA into the porcine skin. Transdermal permeation of 2% PMEA was studied in vitro using the Franz diffusion cell and full-thickness porcine skin. Tris, phosphate buffer (PB) and water (only for pH 3.4) were used as vehicles, pH was in range from 3.4 to 8.8. 1% Dodecyl 6-dimethylaminohexanoat (DDAK) and 1% Transkarbam 12 (5-dodecyloxycarbonyl) pentylammonium-5-(dodecyloxycarbonyl) pentylcarbamate (T12)) were used as permeation enhancers. The flux values were 1.8 ± 0.5, 0.2 ± 0.1, and 0.7 ± 0.4 μg/cm2 /h from aqueous, PB and Tris donor samples at pH 3.4. The respective skin concentrations at 48 h were 294 ± 75, 110 ± 39, and 178 ± 71 μg/g from these vehicles. The flux values of 2% PMEA in PB were from 0.2 ± 0.1 to 2.7 ± 1.32, and from 9.4 ± 4.3 to 26.9 ± 3.4 μg/cm2 /h without and with the presence of 1% DDAK,...
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Synthesis of novel cardioprotective iron chelators
Hrušková, Kateřina ; Vávrová, Kateřina (advisor) ; Roh, Jaroslav (referee)
Oxidative stress participates in patophysiology of many serious cardiovascular diseases. Free intracellular iron occurs as a catalyst of Haber-Weiss and Fenton reaction between superoxides and peroxides increasing the formation of highly toxic hydroxyl radicals, which cause cell damage. Iron chelators diminish the pool of free iron and thus become perspective agents in therapy of various diseases, e.g. anthracycline-induced cardiotoxicity. Aroylhydrazones group, such as salicylaldehyde-isonicotinoylhydrazone (SIH), appear to be highly efficient chelators. Unfortunately, pharmacokinetic studies focused on these compounds revealed their low stability in plasma. Therefore, I synthesised a series of SIH analogs in order to increase their stability together with preserving the ability to chelate free intracellular iron and to define their structure-activity relationships. A basic hypothesis in design of the novel chelators was using substituted ketone instead of aldehyde, leading potentially to an enhanced stability of hydrazone bond. SIH Two different methods were used during the reactions, a conventional heating in an oil bath and heating in a microwave reactor. The latter caused a significant shortening of the reaction time. In vitro studies of novel compounds showed their higher stability in plasma,...
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Synthesis of ceramide analogs with various sphingosine length and evaluation of their effects on the skin barrier
Školová, Barbora ; Vávrová, Kateřina (advisor) ; Hrabálek, Alexandr (referee)
Ceramides are a complex group of lipids, naturally occurring in the uppermost layer of the epidermis, in the stratum corneum (SC). They constitute a major component of extracellular matrix and they are responsible for the skin barrier properties. Diseases such as atopic dermatitis or psoriasis are associated with the decline in content and changes in the composition of ceramides, which lead to the reduction in the protective functions of the skin. Although the importance of ceramides is known, the relationship between their structure and effect on the barrier function is not yet fully elucidated. Earlier studies indicate that the length of ceramide acyl chain affects the skin permeability. It appears that ceramides with short acyl lose the protective properties. First, I have prepared a series of analogues of ceramides with fifteen carbon atoms chain in the sphingosine part and the acyl part of a length of 2, 4 and 6 carbons. Starting substance of the synthesis was N-protected L-serine methyl ester, which was further protected by the formation of a cyclic acetal and subjected to the reduction of the ester to aldehyde. The resulting aldehyde reacted with 1-alcynide in the presence of HMPA. The triple bond was subsequently reduced to a trans-double bond by lithium in ethylamine. After deprotection of...
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Synthesis and study of 6-((7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)amino)hexanoic acid derivatives as transdermal permeation enhancers
Kučera, Ondřej ; Vávrová, Kateřina (advisor) ; Nováková, Veronika (referee)
Ondřej Kučera Charles University in Prague, Faculty of Pharmacy in Hradec Králové Synthesis and study of 6-((7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)amino)hexanoic acid derivatives as transdermal permeation enhancers Transdermal permeation enhancers are used to increase absorption of drugs through skin or, more importantly, through the stratum corneum, which is the uppermost layer of the skin. Mechanism of action of enhancers is not fully understood. In general the most active enhancers consist of hydrophilic and hydrophobic parts. In this work, we prepared and studied fluorescent permeation enhancers. Fluorescent dye NBD (7-nitrobenzo[c][1,2,5]oxadiazol) is fairly hydrophilic, so we used it as a hydrophilic head of potential enhancers based on dodecyl 6- (dimethylamino)hexanoate (DDAK). Such fluorescent enhancers could help us understand more about the mechanism of action. It enables determination of this enhancer and imaging of its penetration pathways in the skin. We synthesized three esters of 6-((7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)amino)-hexanoic acid (NBD-acid) with ester-linked C8-C12 alkyls. 6-Aminocaproic acid reacted with 4-chloro- NBD and then with a series of alcohols to give us different lengths of alkyl chains. We applied these enhancers to human skin in Franz diffusion cells using two...
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