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Comprehensive investigation of penetration enhancers with complementary analytical techniques.
Vidlářová, Lucie ; Doležal, Pavel (advisor) ; Musilová, Marie (referee)
Transdermal drug delivery system is in the centre of attention in recent years. For efficient dermal drug delivery the drug has to overcome the barrier of the outermost layer of the skin, the stratum corneum. For facilitating dermal drug transport, the barrier properties of the stratum corneum can be varied by applying chemical penetration enhancers. The aim of this work was to characterize various penetration enhancers and investigate their mechanism of action. We combined well established techniques like differential scanning calorimetry (DSC) and infrared spectroscopy (IR) with confocal Raman microscopy (CRM) as an upcoming technique in skin research. CRM offers the possibility of label-free and non- destructive, chemically selective analysis of stratum corneum lipids and proteins. We used isolated human stratum corneum for incubation with the penetration enhancers. As a novel approach, the samples of treated stratum corneum were freeze dried to avoid any discrepancies which might come up with differences in the hydratation state of stratum corneum (SC). Furthermore, the structure of lipids and proteins in the stratum corneum was analyzed. In our study, stratum corneum was treated with dimethyle sulphoxide, propylene glycol, ethylene glycol, ethylene glycol-d4 and oleic acid. We observed that...
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Cleaning Validation of Capsule Filling Machine
Hynková, Aneta ; Doležal, Pavel (advisor) ; Dittrich, Milan (referee)
Charles University in Prague, Faculty of Pharmacy in Hradec Králové Department of Department of Pharmaceutical Technology Candidate Mgr. Bc. Aneta Hynková Consultant Doc. RNDr. Pavel Doležal, CSc. Title of Thesis CLEANING VALIDATION OF CAPSULE FILLING MACHINE Validation refers to the documented verification that the production and control processes associated with the production of a product meet predetermined parameters. The aim of cleaning validation in pharmaceutical industry is to obtain documents confirming that the cleaning procedure of the evaluated production equipment ensures safe processing of medicinal products in terms of the absence of impurities or contaminant. The theoretical part of the thesis provides an overview of the legislative basis for binding guidelines for the validation of cleaning based on risk assessment for the prevention of cross-contamination for production equipments that produce multiple products and an overview of all essential concepts developed and implemented for activities in this area. The practical part is focused on the validation of the cleaning process of the Bosch capsule filling machine, model GKF 2500 ASB 100% as a new device to be put into operation in the Good Manufacturing Practice mode. The equimpent has 100% weight control and a maximum filling...
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Biopharmaceutical aspects of macromolecular carriers for systemic sublingual drug delivery
Berka, Pavel ; Doležal, Pavel (advisor) ; Kubová, Kateřina (referee) ; Kvítek, Libor (referee)
Charles University, Faculty of Pharmacy in Hradec Králové Department of Pharmaceutical Technology Candidate Mgr. Pavel Berka Supervisor Doc. RNDr. Pavel Doležal, CSc. Title of Doctoral Thesis Biopharmaceutical aspects of macromolecular carriers for systemic sublingual drug delivery The primary aim of this work was to experimentally verify the hypothesis on sublingual permeation of macromolecules as drug carriers. Literature data about the sublingual route of drug administration and methods of sublingual and buccal membrane processing for in vitro experiments were reviewed for this purpose. The task to design and verify a new methodology of processing and preservation of sublingual membranes suitable for permeation experiments resulted from this literature review. The new methodology of the "flash freezing" membranes uses the prepared membranes with complete epithelial composition, including the lamina propria and part of the fibrous substrate. Rapid membrane freezing with liquid nitrogen (-180 řC) was performed without cryoprotectants to avoid affecting membrane permeability, with only preliminary fixation of protein structures with sodium azide. For comparison, membranes were also frozen by conventional slow freezing to -20 řC and declared as "frozen" membranes. Neither "fresh" nor "flash frozen"...
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HPMC-based liposomal mucoadhesive films with model peptide as target API
Belcáková, Hedviga ; Nobilis, Milan (advisor) ; Doležal, Pavel (referee)
Charles University, Faculty of Pharmacy in Hradec Kralove Department of Pharmaceutical Chemistry and Pharmaceutical Analysis Candidate: Hedviga Belcáková Supervisor: Prof. Milan Nobilis, CSc., Veronika Fidelj (Heidelberg University, Insitute of Pharmacy and Molecular Biotechnology) Diploma thesis title: HPMC-based liposomal mucoadhesive films with model peptide as target API This thesis describes the preformulation stage of mucoadhesive films intended for liposomal peptide delivery via buccal membrane. The evaluation consisted of thickness, maximum tensile strength, strain, moisture content, in vitro swelling and liposome integrity measurements. The chosen polymer (hypromellose, HPMC) was found to perform optimally in concentrations of 10 % with PEG 400 (5 %) acting as plasticizer and liposome concentration of 2 %. The developed preparation method showed good reproducibilty with room for improvement in the homogenization area. The choice of medium (H2O vs. PBS) showed strong influence on formulation's mechanical properties resulting in significant loss of elasticity and mucoadhesive strength. The addition of liposomes in the third stage had been carried out successfully with only occassional effect on their integrity after dissolution.
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Evolution of mitochondrial protein import
Dohnálek, Vít ; Doležal, Pavel (advisor) ; Novotný, Marian (referee)
1 Abstract Even though mitochondria possess their own genome and ribosomes, majority of mitochondrial proteins is encoded in the nucleus and translated by cytosolic ribosomes. Hence it was necessary to establish transport complexes allowing the import of proteins from the cytosol. These complexes are best described in yeast. However, we are encountering organisms lacking many of the subunits of these complexes with increasing frequency. Therefore, we are presenting the overview of the distribution of the subunits within eukaryotic organisms. We specifically take a closer look at parasitic protist Giardia intestinalis that is well known for its extreme reductions of the import complexes. There have been only few subunits identified so far. Porin Tom40, that is responsible for translocating all the incoming proteins across the outer mitochondrial membrane, has been identified despite the high divergence, while homolog of Sam50 hasn't been successfully identified yet. Sam50 is however believed to be necessary for insertion of Tom40 into the membrane. Vast part of this thesis is dedicated to this phenomenon that is highly uncommon and maybe unique among the eukaryotic organisms.
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Multi-element Systems of Biomaterials Based on Magnesium and Zinc
Hasoňová, Michaela ; Nový, František (referee) ; Vojtěch, Dalibor (referee) ; Doležal, Pavel (advisor)
Dissertation thesis deals with basic research in the field of materials from pure Zn powders and Mg, Zn, and Ca binary mixtures prepared by powder metallurgy. General powder metallurgy principles and methods, a brief description of Mg, Zn, and Ca structure and properties, and the latest research in the field of bulk materials preparation from these elements via powder metallurgy are summarized in the theoretical part of the thesis. The experimental part focuses on the preparation of materials from finer and coarser Zn powder particles by hot pressing at 300 and 400 °C using the pressure of 100, 200, 300, 400, and 500 MPa. Binary mixtures based on Mg with the addition of Zn or Ca were prepared by hot pressing in the solid-state (300 °C) and hot pressing in the semi-solid state (400 °C, 450 °C in the case of Mg-Ca system) using the pressure of 500 MPa. Binary mixtures based on Zn with the addition of Mg or Ca were prepared by hot pressing in the semi-solid state (400 °C) using the pressure of 500 MPa. The prepared materials were evaluated in terms of microstructure, elemental and phase composition, microhardness, flexural strength, and fractography. The results showed that in the case of processed from pure Zn powders, a better combination of the flexural strength and displacement was achieved in the case of the finer Zn powder, namely in the material prepared at a temperature of 400 °C and a pressure of 500 MPa. In the case of mixtures, the best connection between the powder particles was achieved in the case of a material based on finer Zn powder with 0.5 wt.% of Mg, which had a significant effect on the achieved values of flexural strength and displacement. The amount of minor powder in the mixture had a significant effect on the prepared material structure and phase composition, while the processing conditions influenced the reached strength characteristics and fracture mechanism.
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Nanofiber drug carriers 1
Vodičková, Jana ; Doležal, Pavel (advisor) ; Šnejdrová, Eva (referee)
CHARLES UNIVERSITY Faculty of Pharmacy in Hradec Kralove Department of Pharmaceutical Technology Name: Jana Vodičková Title of diploma thesis: Nanofiber drug carriers 1 Supervisor: doc. RNDr. Pavel Doležal, CSc In the theoretical part is mentioned structurally hierarchical division of nanomaterials, characteristics of nonwoven nanotextiles and technology of Nanospider® for production of nonwoven nanofiber mats by electrospining method. A brief description of properties and use of polyurethane including nanofibers is described and contact angle measurement is characterized. The experiment deals with measurement of wettability of composite chitosan-polyurethane nanomembrane. It shows difficult water wettability and low capillarity of tested nanofibrous products. The next part is devoted to measuring the release properties of nanofibrous composite membranes in terms of caffeine release as a model drug. The last part deals with the dissolution of folic acid from non-crosslinked and cross-linked PVA nanofiber membranes. All membranes were provided by Elmarco s.r.o. Liberec in order to measure the liberation profiles of drugs in the screening of pilot plant parameters of electrospining for nanofibering by combinations of different polymers and drugs Key words: Nanospider®, electrospining, polyurethane,...
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Permeation of nanoparticles through sublingual membrane 3
Dvořáková, Pavlína ; Doležal, Pavel (advisor) ; Kováčik, Andrej (referee)
Charles University in Prague, Faculty of Pharmacy in Hradec Králové Department of pharmaceutical technology Candidate: Mgr. Dvořáková Pavlína Consultant: doc. RNDr. Pavel Doležal, CSc. Title of Thesis: Permeation of nanoparticles through sublingual membrane 3. In the theoretical part, attention was paid to the entry of drugs into the body by the sublingual route, description of the oral cavity (mucosa, epithelium, mucus, saliva, MCGs), OCCAT model, and basic concepts of bioimpedance. In the experimental part, the hydrodynamic size of the PD-Chromeon 470 nanoparticles was verified by the DLS method. In the environment of water, even two buffers pH 6.8 and 7.4 according to Sörensen, practically identical results were obtained at the level of 40 nm. Buffers were used as donor, resp. acceptor phase in vitro permeation experiments. The experiments included measurement of impedances of differently processed and stored sublingual membranes from pig tongue. To test the penetration of nanoparticles through these model mucosal barriers, nanoparticles were applied in a concentrated (500 µg / ml), diluted (1: 4) dispersion. The lowest impedance had the sodium azide-preserved membranes, which were frozen at -18 ř C for a long time. These also showed the highest in vitro permeation values of nanoparticles. Rapid...
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Synthesis and evaluation of transdermal drug permeation enhancers
Kopečná, Monika ; Vávrová, Kateřina (advisor) ; Doležal, Pavel (referee) ; Hampl, František (referee)
Penetration enhancers are compounds that facilitate transdermal drug delivery, which is an advantageous pathway of drug administration (compared to conventional methods). However, skin acts as formidable barrier enabling terrestrial life by protecting the inner body from the environmental conditions, microbes and substances. Thus, for drug administration via this pathway, some approach to temporary increase the skin permeability (e.g. permeation enhancer) should be employed. Despite the existence of many enhancer classes, there is still a need for new compounds enhancing broader spectrum of drugs, with reversible mode of action and better safety profile. One of the promising approaches is to explore natural compounds with low toxicity and irritation potential. In this thesis, I first studied various glucose and galactose derivatives bearing structural fragments that were responsible for enhancing activity of previously studied enhancers (e.g. amino acid derivatives, fatty acids and alcohols, modified ceramides). A detailed examination of these sugar- based compounds revealed potent glucoside and galactoside enhancers (12Glc6N and A15) comparable to or better than Span 20 with reversible mode of action and acceptable toxicity on keratinocyte and fibroblast cell lines. Another part of my work builds...
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Bacterial proteins in the biogenesis of mitochondria of unicellular eukaryotes.
Petrů, Markéta ; Doležal, Pavel (advisor) ; Embley, Martin (referee) ; Hashimi, Hassan (referee)
in English Formation of mitochondria by the conversion of a bacterial endosymbiont is the fundamental moment in the evolution of eukaryotes. An integral part of the organelle genesis was the displacement of the endosymbiont genes to host nucleus and simultaneous creation of new pathways for delivery of proteins synthesized now in the host cytoplasm. Resulting protein translocases are complexes combining original bacterial components and eukaryote-specific proteins. In addition to these novel protein import machines, some components of the original bacterial secretory pathways have remained in the organelle. While the function of a widely distributed mitochondrial homolog of YidC, Oxa1, is well understood, the role of infrequent components of Sec or Tat translocases has not yet been elucidated. So far, more attention has been paid to their abundant plastid homologs, which assemble photosynthetic complexes in the thylakoid membrane. In the thesis, the structure and function of prokaryotic YidC, Sec and Tat machineries and their eukaryotic homologs are described. By comparing both organelles of the endosymbiotic origin, the hypothesis is drawn on why these translocases have been more "evolutionary successful" in plastids than in mitochondria.
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