Organophosphates (OPs) are used in agriculture via pesticides, and warfare and terrorism via nerve agents. OPs can inhibit acetylcholinesterase (AChE) activity in the nervous system, leading to the buildup of acetylcholine (ACh), and overstimulation of the nervous system and eventual asphyxiation and death. The development of novel blood-brain barrier () penetrating pyridinium oxime reactivators have previously demonstrated efficacy towards treatment of OP poisoning after exposure of rats to a sarin or a VX surrogate, nitrophenyl isopropyl methylphosphonate (NIMP) and nitrophenyl ethyl methylphosphonate (NEMP), respectively. An effective oxime antidote capable of penetrating the and restoring nervous system activity after exposure to a cyclosarin surrogate, nitrophenyl cyclohexyl methylphosphonate (NCMP), has yet to be determined. In Chapter 2, in vitro testing of the efficacy of 17 total novel oxime candidates to utilize against NCMP was conducted with a modified Ellman’s AChE assay. Pools of naïve adult male rat brains were utilized as the AChE source. The first variable investigated was the measurement of AChE activity after inhibition with NCMP and subsequent reactivation with one of the oximes. The second variable investigated restoration of AChE activity after simultaneous oxime and NCMP incubation. The final variable investigated the restoration of AChE activity after simultaneous 2-PAM, oxime and NCMP incubation. A thorough kinetic analysis of our best oximes has yet to be accomplished. In Chapter 3, the best oxime antidotes for NEMP and NIMP were used for kinetic analysis with a modified 96-well plate Ellman’s AChE assay. Protein concentrations were analyzed with a modified Lowry protein tube assay to ensure consistent analytical concentrations. The sources of AChE included pooled rat brain and skeletal muscle, and rat and human erythrocytes and plasma. Butyrylcholinesterase (BChE) activity was also measured in the rat and human plasma samples. The results of these studies strengthen the argument that our oxime antidotes can be used as potential therapeutic drugs for OP poisoning. The kinetic data provided critical information to help propose, for Chapter 4, a dynamic pharmacokinetic based model to predict human AChE or BChE activity after exposure to nerve agent surrogates (NEMP and NIMP) and the oximes (44.08 and 44.25).

Submitted by isabela.moljf@hotmail.com (isabela.moljf@hotmail.com) on 2016-08-08T16:59:46Z No. of bitstreams: 1 raphaeldasilvaalvim.pdf: 4975859 bytes, checksum: 45288f5cf716f2e219a7c41484f3c591 (MD5) / Approved for entry into archive by Adriana Oliveira (adriana.oliveira@ufjf.edu.br) on 2016-08-09T11:55:23Z (GMT) No. of bitstreams: 1 raphaeldasilvaalvim.pdf: 4975859 bytes, checksum: 45288f5cf716f2e219a7c41484f3c591 (MD5) / Made available in DSpace on 2016-08-09T11:55:23Z (GMT). No. of bitstreams: 1 raphaeldasilvaalvim.pdf: 4975859 bytes, checksum: 45288f5cf716f2e219a7c41484f3c591 (MD5) Previous issue date: 2013-02-22 / O agente VX, O-etil metillfosfonotioato de S-2-(diisopropilamino)etila, é um dos principais agentes neurotóxicos e a busca por formas de degrada-lo é consideravelmente importante. Neste trabalho, a hidrólise de um composto organofosforado tipo-VX (metilfosfonotioato de O,S-dimetila, DMPT) pela quimissorção dissociativa na superfície de MgO(001) foi estudada pela teoria do funcional da densidade com condições de contorno periódicas. Um mecanismo de degradação que envolve as reações das moléculas de DMPT e de água foi proposto e investigado em dois tipos de modelos de superfície de MgO(001): terraço e dopada com Al. Conformações, diferenças de energia livre, estados de transição e barreiras de reação foram calculados. Inicialmente, foi verificado que apenas a ligação neurotóxica P-S é quebrada na hidrólise do composto DMPT, que pode ocorrer espontaneamente em todo o intervalo de temperatura analisado (100-600 K). Na quimissorção dissociativa da molécula de DMPT, a formação do intermediário MgO:[PO(CH3)(OCH3)]+[SCH3]− é termodinamicamente menos estável que os produtos de hidrólise a partir da temperatura de aproximadamente 335 K para a superfície dopada com Al, que é muito menor que o mesmo processo calculado no terraço (a partir de 500 K). De acordo com a análise de barreira reacional, a possível reconstituição da ligação P-S não ocorre em ambos modelos de superfície de MgO(001) analisados. Contudo, a barreira de energia eletrônica para a reação de dissociação na superfície dopada com Al é cerca de 49,0 kJ/mol menor do que no terraço. Simultaneamente, o processo de formação dos íons H+ e OH- no terraço de MgO(001) é relevante como a etapa inicial de hidroxilação dessa superfície e faz parte do mecanismo de hidrólise catalisada do composto DMPT. A adsorção de uma, duas e três moléculas de água foram obtidas apenas sobre o terraço de MgO(001), pois sabe-se que as moléculas de água são dissociadas espontaneamente em defeitos pontuais. A variação da energia livre de Gibbs para os processos de adsorção e dissociação foi calculada no intervalo de temperatura de 100-600 K. Os resultados termodinâmicos mostraram que a adsorção de uma única molécula de água não conduz à dissociação. Para o dímero e trímero de moléculas de água, uma molécula se dissocia enquanto que as outras moléculas co-adsorvidas estabilizam as espécies iônicas H+ e OH- sobre a superfície. Nos dois casos, os produtos de dissociação na superfície convergiram para a formação de ligações de hidrogênio entre a hidroxila formada e as moléculas de água. Como consequência dessas interações, a superfície protonada coexiste com os íons hidroxila adsorvidos. As barreiras de energia eletrônica não são grandes o suficiente para desfavorecer a dissociação parcial de duas (23,2 kJ/mol) e três (24,9 kJ/mol) moléculas de água, porque elas seriam facilmente superadas. Portanto, a etapa inicial para a hidrólise no terraço de MgO(001) começa a partir de duas moléculas de água, mas o produto dissociado é mais estável quando existem três moléculas de água quimissorvidas. Em relação à migração dos íons H+ e OH- após a dissociação, as barreiras de energia eletrônica calculadas mostraram que esse processo na superfície de MgO(001) é desfavorável. Assim, os processos de dissociação das moléculas de DMPT e H2O na superfície de MgO(001) devem acontecer em regiões próximas para facilitar a etapa seguinte do mecanismo de reação proposto, que é a recombinação iônica de [PO(CH3)(OCH3)]+, [SCH3]-, HO- e H+ para a subsequente formação dos produtos P1 [HOPO(CH3)(OCH3)] e P2 [HSCH3]. Os produtos P1 e P2 não se acumulam sobre a superfície dopada com Al porque estas moléculas são dessorvidas. Portanto, se comparado com a reação de hidrólise do composto de DMPT, 335 K é uma temperatura ideal a fim de se evitar a acumulação dos produtos sobre os defeitos pontuais analisados, com a consequente dessorção espontânea de P1 e P2 e a reconstituição do MgO na etapa final do processo catalítico. No entanto, os sítios do terraço também podem participar do mecanismo de hidrólise catalisada do composto DMPT a partir de 500 K. Neste trabalho, a superfície de MgO(001) atua como um possível catalisador para a degradação do agente VX, mas com uma maior seletividade dos sítios dopados com Al do que os sítios do terraço. Da mesma forma, estes resultados têm uma variedade de importantes aplicações, bem como uma referência para posteriores estudos da reação do composto VX na superfície de MgO(001) com outros tipos de defeitos ou superfícies. Assim, esses resultados contribuem cientificamente para a área de catálise e superfícies de óxidos na desativação química de agentes neurotóxicos, especialmente os agentes tipo-V. Além disso, o presente trabalho permitirá o desenvolvimento de novas tecnologias para a defesa nacional, a fim de permitir a degradação química desses tipos de compostos sem afetar o meio ambiente. / The VX agent, O-ethyl S-(2-diisopropylethylamino)ethyl methylphosphonothioate, is one of the main neurotoxic agents, thus the search for ways to degrate it is considerably important. In this work, the hydrolysis of a VX-like organophosphorus compound (O,S-dimethyl methylphosphonothioate, DMPT) by the dissociative chemisorption on the MgO(001) surface was studied by density-functional theory using periodic boundary conditions. A degradation mechanism involving the reactions of the DMPT and water molecules was proposed and investigated on two types of MgO(001) surfaces: terrace and Al-doped. Conformations, free energy differences, transition states e reaction barriers were calculated. Firstly, it was verified that only the P-S neurotoxic bond breaks in the hydrolysis of the DMPT compound, which can occur spontaneously throughout the analyzed temperature range (100-600 K). In the dissociative chemisorption of the DMPT molecule, the formation of intermediate MgO:[PO(CH3) (OCH3)]+[SCH3]− is thermodynamically fewer stable than the hydrolysis products from the temperature of about 335 K for the Al-doped surface, which is less than the same process calculated on the surface without defects (from 500 K). According to a reactional barrier analysis, the possible reconstitution of the P-S bond does not occur on both cases of analyzed MgO(001) surface models. However, the electronic energy barrier for the dissociation reaction on the Al-doped sites is about 49.0 kJ/mol less than the one on the terrace. At the same time, the process of the formation of H+ and OH− ions on the MgO(001) terrace is relevant as the hydroxylation initial step of this surface and it is part of the catalyzed hydrolysis mechanism of the DMPT compound. The adsorption of one, two and three water molecules were only obtained on the MgO(001) terrace because it is known that water molecules are dissociated spontaneously on point defects. The variation of the Gibbs free energy for the adsorption and dissociation processes was calculated in the 100-600 K temperature range. The thermodinamic results showed that the adsorption of a single water molecule does not lead to dissociation. For the dimer and trimer of water molecules, one molecule dissociates while the others co-adsorbed stabilize the H+ and HO− ionic species on the surface. In the two cases, the dissociation products on the surface converged for the formation of hydrogen bonds among the formed hydroxyl and water molecules. As a consequence of these interactions, the protonated surface coexists with the adsorbed hydroxyl ions. The electronic energy barriers are not large enough to forbid the partial dissociation of two (23.2 kJ/mol) and three (24.9 kJ/mol) water molecules because they would be easily surmounted. Therefore, the initial step for the hydrolysis on the MgO terrace starts from two water molecules, but the dissociated product is more stable when there are three water molecules III chemisorbed. Regarding the migration of the H+ and HO− ions after the dissociation, the calculated electronic energy barriers showed that this process on the MgO(001) surface is unfavorable. Thus, the dissociation processes of the DMPT and H2O molecules on the MgO(001) surface should happen in close regions to facilitate the next step of the proposed reaction mechanism, which is the ionic recombination of [PO(CH3)(OCH3)]+, [SCH3]-, HO- and H+ for the subsequent formation of the P1 [HOPO(CH3)(OCH3)] and P2 [HSCH3] products. The products P1 and P2 did not accumulate on the Al-doped surface because these molecules are desorbed from 197 K. Therefore, if compared to the hydrolysis reaction, 335 K is an ideal temperature to avoid the accumulation of the products on the analyzed point defects, with the consequent spontaneous desorption of P1 and P2 and the MgO reconstitution in the final step of the catalytic process. However, the sites of the terrace can also participate of the DMPT catalyzed hydrolysis mechanism from 500 K. In this work, the MgO(001) surface works as a catalyst for the degradation of VX agent, but with higher selectivity of the Al-doped sites than that of the terrace sites. In the same way, these results have an important variety of applications, as well as reference for further studies of the VX compound reaction on the MgO(001) surface with other kinds of defects or other surfaces. Thus, these results scientifically contribute to the area of catalysis and oxide surfaces in the chemical deactivation of neurotoxic agents, especially the V-type agents. Furthermore, this work will enable the development of new technologies for national defense in order to enable the chemical degradation of these types of compounds without affecting the environment.

Die Einbrennsilikonisierung wird schon lange verwendet, um spezielle Eigenschaften bei pharmazeutischen Glasbehältnissen zu erzielen. Üblicherweise werden Silikonöle verwendet um Gleitfilme auf den Oberflächen vorgefüllter Glasspritzen zu erzeugen oder die Glaswand zu hydrophobieren damit wäßrige Inhalte sauber ablaufen können. Weiterhin kann durch die hydrophobe Deaktivierung der Glaswände eine Reduktion der Adsorption von Wirk- und Hilfsstoffen sowie eine Erhöhung der hydrolytischen Resistenz erreicht werden. Wegen des parenteralen Verwendungszweckes werden meistens Gläser der Glasart 1 entsprechend dem europäischen Arzneibuch eingesetzt. Die Silikonölfilme (Trimethylsiloxy-endgeblockte Polydimethylsiloxane, PDMS) werden aus verdünnter wäßriger Emulsion auf der inneren Behältnisoberfläche gespreitet und anschließend bei Temperaturen oberhalb von 300°C für ca. 10-30 min hitzebehandelt. Neben der Sterilisation und Entpyrogenisierung wird der Silikonfilm durch das Einbrennen verändert. In der Literatur wurde dieser Prozeß bisher als Hitzefixierung oder -härtung bezeichnet in der Annahme, daß das Silikonöl durch Glasbindung oder Quervernetzung immobilisiert wird. Es wurden weitere Effekte wie Verdampfung oder Zersetzungsreaktionen als Folge der Hitzebehandlung angenommen. Die Dissertation hatte daher zum Ziel diese Vorstellungen analytisch zu belegen. Die Eigenschaften einbrennsilikonisierter Gläser lassen sich durch einfache Experimente beschreiben. Durch organische Extraktion der Gläser (z.B. Toluol, Dichlormethan) läßt sich der überwiegende Silikonanteil (ca. 80%) wiederfinden. Nach erschöpfender Extraktion zeigt sich eine beständige Hydrophobie z.B. durch die Ausbildung hoher Randwinkel von Wasser oder das veränderte Ablaufverhalten von Flüssigkeiten in diesen Behältnissen festgestellt werden. Basierend auf diesen Beobachtungen wurde die Anwesenheit einer gebundenen neben einer löslichen Silikonölfraktion nach dem Einbrennen angenommen. Alle analytischen Studien wurden an dieser zwei Lagen Theorie orientiert und durchgeführt. Dazu wurden einige oberflächensensitive Techniken wie z.B. die kombinierte Röntgenphotoelektronen- und Augerelektronenspektroskopie (XPS/AES) und die Flugzeit-Sekundärionenmassenspektroskopie (TOF-SIMS) eingesetzt. Durch chemische Zustandsdiagramme und massenspektroskopische Strukturanalyse konnte die Ausbildung von Bindungen zwischen Glas und Silikon detektiert werden. Kontaktwinkelmessungen zeigten, daß das gebundene Silikon bis ca. 400°C thermostabil ist und oberhalb von 450°C pyrolysiert wird. Die extrahierbare Silikonfraktion wurde ebenfalls mit einigen analytischen Techniken im Vergleich mit den jeweiligen unbehandelten Ausgangsmaterialien untersucht. Chromatographische Studien mittels Größenausschluß- und Hochtemperatur-Gaschromatographie (SEC,GC) zeigten signifikante Unterschiede in der polymeren Zusammensetzung des PDMS. Die Molekulargewichtsmittel stiegen als Folge von Verlusten von niedermolekularen Siloxanen einer Größe zwischen 0-100 Siloxaneinheiten (SU) an. Diese Defizite können auf Verdampfungs- (0-50 SU) und Zersetzungsreaktionen (50-100 SU) zurückgeführt werden. Die Untersuchung des extrahierbaren eingebrannten Silikonöls mit der Fourier Transform Infrarot Spektroskopie (FTIR) zeigte keine strukturelle Veränderung der PDMS Moleküle, z.B. durch Hydrolyse, Oxidation oder Verzweigung an. Die Emulgatoren werden durch das Einbrennen zerstört. Dies wurde durch die 1H-Kernresonanz Spektroskopie (1H-NMR) nachgewiesen. Die Thermostabilität des PDMS wird durch katalytische Effekte der Emulsionshilfsstoffe und durch die Anwesenheit von Sauerstoff erniedrigt, wie aus thermogravimetrische Analysen (TGA) mit kommerziellen Silikonölen und Ölemulsionen modellhaft abgeleitet wurde. Um die Migration von Silikonölspuren in Arzneimittel in silikonisierten Glasbehältnissen zu bewerten wurde eine Atom Absorptions spektroskopische (AAS) Methode entwickelt und entsprechend den ICH-Richtlinien validiert (Bestimmungsgrenze in Liquida 10 mg/L). Die Schlußfolgerung aus den Analysen ist, daß nach der Einbrennsilikonisierung zwei differenzierbare Silikonfraktionen an der Glasoberfläche anwesend sind. Die kovalent gebundene Dünnschicht ist unlöslich und verliert ihren Ölcharakter, ist aber für die Hydrophobisierung ausreichend. In der löslichen Fraktion, werden die mittleren Molekulargewichte durch den Verlust von niedermolekularen Anteilen erhöht. Der Anteil dieser Schicht hängt von der eingesetzten Ausgangsmenge an Silikonöl ab. Durch die Entfernung der niedermolekularen Anteile und die Zerstörung der Emulgatoren können toxikologische Bedenken zurückgestellt werden. / Heat Curing Siliconization of Pharmaceutical Glass Containers The heat curing siliconization has long since been used to introduce special properties to pharmaceutical glass containers. Silicone oils are usually applied to the containers to form lubricating films on the inner surface of prefilled syringes or to gain hydrophobic container walls for clear draining aqueous solutions, e.g. in injection vials. Furthermore the deactivation of the glass wall reduces adsorption of hydrophilic drug compounds and increases the hydrolytic stability of the glass surface. Due to the parenteral targeting the glass containers are made from glass type 1 according to the European Pharmacopoeia. The silicone oil films (= trimethylsiloxy endcapped polydimethylsiloxane, PDMS) are spread from diluted aqueous silicone oil emulsions on the inner surface and successively heat treated above 300°C for 10-30 minutes. Apart from sterilization and depyrogenation, the silicone layer is changed by the burning-in treatment. In literature the process has previously been designated as heat fixing or heat curing assuming that silicone oil is immobilized by bonding to the glass or branching. Further effects such as vaporization or degradation were suggested along with the heating. Proving these suggestions analytically was therefore the aim of the dissertation. The properties of siliconized glass containers can be characterized by simple experiments. Extraction of the glasses with suitable organic solvents (e.g. toluene, dichloromethane) recovers most of the silicone (approximately 80%) after a typical heat curing process. Still, after exhaustive solvent extraction there are persisting hydrophobic properties of the glasses evidenced by high contact angles of water and changed draining behaviour of fluids in the containers. From this observations it was assumed that a bound and a soluble silicone species is present after the heat curing siliconization. All analytical studies were based on the two-layer hypothesis and thus divided in two parts. Several surface analysis techniques were employed to detect and characterize the fixed silicone layer among them the combined X-ray photoelectron and augerelectron spectroscopy (XPS/AES) and time-of-flight secondary ion mass spectroscopy (TOF-SIMS). The formation of new bonds between silicone and glass was evidenced by chemical state plots and mass spectroscopic structural analysis. Contact angle measurements showed that the bound silicone was thermostable up to 400°C and pyrolyzed at temperatures above 450°C. The extractable silicone was also studied with several analytical techniques in comparison with the respective uncured starting materials. Chromatographic studies using size exclusion (SEC) and high temperature gas chromatography (GC) revealed significant changes in the composition of the silicone polymer. The molecular weight averages increase because low molecular weight siloxanes (LMWS) with 0-100 dimethylsiloxane units are removed through the heat curing process. These deficits can be related to vaporization (molecules from 0-50 units) and degradation (molecules from 0-100 units) effects. A Fourier transform infrared spectroscopic (FTIR) investigation of the molecular structure of the extractable PDMS after heat treatment showed no chemical changes e.g. by hydrolysis, branching or oxidation. The emulsifiers are destroyed in the heating process which was proved by a proton nuclear magnetic resonance (1H-NMR) study. The thermostability of the silicones is decreased by katalytic effects of auxiliary substances in the emulsions and the presence of oxygen. This was concluded from model thermogravimetric studies (TGA) with commercial medical grade silicone oils and emulsions. To evaluate migration of silicone traces to drug formulations in siliconized glass containers a trace analytical method by graphite furnace atomic absorption spectrometry (GF-AAS) was developed and validated according to the ICH guidelines. In liquid formulations levels of 10µg/L can be properly quantified. It was concluded that two main species of silicone can be differentiated on the glass surface after heat curing siliconization. The bonded thin layer is insoluble and sufficient for hydrophobic deactivation. The oil properties are lost. The extractable fraction is altered by removal of LMWS which increases molecular weight and viscosity and determines the lubricating properties. Its amount depends on the used silicone mass. No toxicological concern has to be made about LMWS and emulsifiers in the drug product due to their removal in the curing step.

Cystic fibrosis is caused primarily by deletion of Phe508. An exciting discovery was that CFTR’s sister protein, the P-glycoprotein (P-gp) containing the equivalent mutation (ΔY490), could be repaired by a drug-rescue approach. Drug substrates showed specificity, and their mechanism involves direct binding to the transmembrane domains (TMDs) since arginine suppressor mutations were identified in TMDs that mimicked drug-rescue to promote maturation. We tested the possibility of rescuing CFTR processing mutants with a drug-rescue approach. 1) Arginine mutagenesis was performed on TM6, 8, and 12. 2) Correctors were tested for specificity. 3) Truncation mutants were used to map the VX-809 rescue site. Correctors 5a, 5c, and VX-809 were specific for CFTR. VX-809 appeared to specifically rescue CFTR by stabilizing TMD1. Therefore, the TMDs are potential targets to rescue CFTR. Rescue of P-gp and CFTR appeared to occur by different mechanisms since no arginine suppressor mutations were identified in CFTR.

Cystic fibrosis is caused primarily by deletion of Phe508. An exciting discovery was that CFTR’s sister protein, the P-glycoprotein (P-gp) containing the equivalent mutation (ΔY490), could be repaired by a drug-rescue approach. Drug substrates showed specificity, and their mechanism involves direct binding to the transmembrane domains (TMDs) since arginine suppressor mutations were identified in TMDs that mimicked drug-rescue to promote maturation. We tested the possibility of rescuing CFTR processing mutants with a drug-rescue approach. 1) Arginine mutagenesis was performed on TM6, 8, and 12. 2) Correctors were tested for specificity. 3) Truncation mutants were used to map the VX-809 rescue site. Correctors 5a, 5c, and VX-809 were specific for CFTR. VX-809 appeared to specifically rescue CFTR by stabilizing TMD1. Therefore, the TMDs are potential targets to rescue CFTR. Rescue of P-gp and CFTR appeared to occur by different mechanisms since no arginine suppressor mutations were identified in CFTR.

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No. of bitstreams: 1 raphaeldasilvaalvin.pdf: 3200596 bytes, checksum: 01129a5b7d70b7b3e44dd871c5c77b1d (MD5) Previous issue date: 2009-02-19 / CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Os organofosforados encontram aplicações na indústria, nas áreas de corantes, vernizes, couro artificial, isolantes elétricos, impermeabilizantes, plásticos, aditivos de petróleo e dissolventes; também são usados na medicina no tratamento de doenças, como o glaucoma; no uso doméstico e na agricultura encontram aplicabilidade como inseticidas e pesticidas. Além dessas várias utilidades civis, os organofosforados são utilizados, também, como armas químicas de destruição em massa, e possuem estrutura similar àquelas dos compostos utilizados como inseticidas e pesticidas. No caso do VX, um organofosforado utilizado como arma química, a quebra da ligação P­S é essencial na sua degradação química. Embora muitas reações químicas possam ser empregadas para decompor agentes químicos de guerra, somente algumas podem ser utilizadas na prática em uma neutralização, porque estas reações precisam ser simples e os reagentes empregados devem ser estáveis, baratos e de baixa massa molecular. No entanto, muitas das reações que podem ser úteis para a neutralização do agente neurotóxico VX ainda seguem em discussão em recentes pesquisas, mas na maioria delas se limita alguns sucessos a hidrólise catalisada. Nesta dissertação foram estudados processos de hidrólise catalisada do agente VX por MgO(001) por meio de cálculos ab initio. Foi utilizado o programa PWscf ­ Plane­Waves Self Consistent Field. O PWscf utiliza a Teoria do Funcional da Densidade, a partir de um conjunto de base de autofunções dado por ondas planas e pseudopotenciais. Entre outros atributos, este código é capaz de calcular a energia do estado fundamental dos orbitais de Kohn­Sham para um elétron, além de forças atômicas em diferentes condições de tensão, otimização estrutural e estado de transição. A molécula de VX foi substituída por uma molécula menor, chamada de p­VX, em que foram substituídos alguns radicais do VX por grupos metila. Isto foi feito para diminuir o tamanho da molécula, que reduzirá o custo computacional, mas sem afetar substancialmente a química do problema, o estudo da quebra da ligação P­S. O mecanismo de hidrólise proposto esta relacionado com a quebra heterolítica da ligação P­S, com a conseqüente formação de íons intermediários R­P+ e R'­S­, estes, por sua vez, estabilizados por quimissorção na superfície de MgO(001). Em conjunto com essa reação, acontece a dissociação de moléculas de água para a formação dos íons H+ e HO­, cujos íons também são estabilizados na superfície de MgO(001). O passo final é a recombinação desses íons, para gerar os produtos de hidrólise R­POH e R'­SH, seguida pelo processo de dessorção destas moléculas da superfície do catalisador. Para a reação global de hidrólise da molécula de p­VX, a variação da energia interna foi calculada em ­5,66kcal/mol. Foram determinadas as estruturas dos íons R­P+ e R'­S­ estabilizados sobre a superfície de MgO(001), com uma energia de formação calculada em ­0,20kcal/mol, indicando que os intermediários teriam boa estabilidade sobre a superfície se comparados com a molécula de p­VX original. Na quimissorção dissociativa de moléculas de água sobre a superfície de MgO(001), verificou­se que os íons formados somente ficam estabilizados se estiverem a uma distância mínima de 4,70Å. Qualquer distância abaixo desta levará a formação da molécula de água novamente. No processo envolvendo duas moléculas de água, apenas uma delas se dissocia, enquanto a outra estabiliza os íons formados via ligação de hidrogênio. A molécula não dissociada também interage com um sítio superficial de magnésio. Este resultado foi comprovado pelos cálculos de diferença de densidade de carga eletrônica do sistema, determinação do caminho de reação, onde este obteve uma barreira energética calculada em 5,55kcal/mol para a reação direta e em 7,53kcal/mol para a reação inversa, e pela dissociação parcial utilizando um trímero de moléculas de água, com energia calculada em ­5,40kcal/mol. Os resultados permitem concluir que o mecanismo proposto para a hidrólise catalisada do agente neurotóxico VX pelo MgO é possível. Os modelos construídos podem ser modificados para testes de novos catalisadores com estrutura tipo MgO, via adição de defeitos ou dopantes à superfície da estrutura cristalina, visando a elaboração de catalisadores mais eficientes para a reação de hidrólise com o mesmo mecanismo. / The organophosphates are used in industry, in the fields of dyes, varnishes, artificial leather, electrical insulation, waterproofing, plastics, oil additives and solvents, are also used in medicine to treat diseases such as glaucoma, in the household and in agriculture are applied as insecticides and pesticides. Besides these various civilian facilities, the organophosphates are used as well as chemical weapons of mass destruction, and have similar structure to those of compounds used as insecticides and pesticides. In the case of VX, an organophosphate used as a chemical weapon, the fall in P­S binding is essential in its chemical degradation. Although many chemical reactions can be used to decompose the chemical agents of war, only some can be used in practice in a breakthrough, because these reactions need to be simple and the reagents used should be stable, inexpensive and low molecular weight. However, many of the reactions that may be useful for the neutralization of the neurotoxic VX agent still follow under discussion in recent polls, but most of them are confined to some successes catalyzed hydrolysis. In this dissertation we studied processes of the VX agent catalyzed hydrolysis by MgO (001) by means of ab initio calculations. We used the program PWscf ­ Plane Waves Self­Consistent Field. The PWscf using the Density Functional Theory from a set of basic autofunction given by plane waves and pseudopotentials. Among other attributes, this code is able to calculate the energy of the ground state of the Kohn­Sham orbital for an electron, and atomic force under different conditions of stress, structural optimization and transition state. The VX molecule was replaced by a smaller molecule, called a p­VX, which replaced some of the radicals VX for methyl groups. This was done to reduce the size of the molecule, which reduces the computational cost, but not substantially affect the chemistry of the problem, the study of breaking the link P­S. The proposed mechanism of hydrolysis is related to the breaking of the link heterolytic P­S, with the consequent formation of intermediate ion R­P+ and R'­S­, they, in turn, stabilized by quimissorption the MgO(0010 surface. Together with this reaction, is the dissociation of water molecules to the formation of ions H+ and HO­, whose ions are stabilized on the MgO(001) surface. The final step is the recombination of these ions, to generate products of hydrolysis and R­POH and R'­SH, followed by the process of desorption of molecules from the surface of the catalyst. For the overall reaction of hydrolysis of the molecule p­VX, the variation of internal energy was calculated to be ­5.66 kcal/mol. Were determined the structures of ion R­P+ and R'­S­ stabilized on the MgO(001) surface, with an formation energy calculated at ­0.20 kcal/mol, indicating that the middlemen have good stability on the surface is compared with the p­VX molecule original. In dissociative quimissorption of water molecules on the MgO(001) surface, it was found that the ions formed are stable only if a minimum distance of 4.70 Å. Any distance below this will lead to formation of the water molecule again. In the process involving two water molecules, only one is dissociated, while the other ions stabilizes by the hydrogen bonding formed. The non­dissociated molecule also interacts with a surface site of magnesium. This result was confirmed by the calculations of load density difference of the system, determining the reaction path, where he obtained a energy barrier calculated at 5.55 kcal/mol for the direct reaction and 7.53 kcal/mol for the reverse reaction and by partial decoupling using a trimer of water molecules, with energy calculated at ­5.40 kcal/mol. The results show that the proposed mechanism for the catalyzed hydrolysis of the neurotoxic agent VX by MgO is possible. The models constructed can be modified for testing of new catalysts with MgO type structure, via addition of doping or defects on the surface of the crystal structure, to the development of more efficient catalysts for the hydrolysis reaction with the same mechanism.

Les neurotoxiques organophosphorés sont appelés agents chimiques de guerre car ils sont une menace à la fois pour les militaires et pour les populations civiles. La voie percutanée est l’une des principales voies d’entrée pour ces agents, et plus particulièrement pour le VX, très peu volatil. La décontamination des surfaces exposées est alors cruciale afin d’éviter l’intoxication des victimes. En cas d’attentat terroriste, le cuir chevelu humain pourrait être un site préférentiel d’exposition. Cette partie du corps, riche en follicules pileux, pourrait nécessiter des produits et des processus de décontamination adaptés. Ce travail est divisé en 4 parties : 1) Validation d’un modèle de peau in vitro pour le cuir chevelu humain ; 2) Détermination des stratégies de décontamination ; 3) Formulation de nouveaux systèmes de décontamination ; 4) Évaluation de leur efficacité de décontamination. La peau d’oreille de porc est un modèle pertinent pour l’étude de la pénétration percutanée in vitro du VX à travers le cuir chevelu humain. La peau de dessus de tête de porc représente un bon modèle de cuir chevelu humain pour l’étude de l’affinité du VX avec la tige pilaire. L’étude de distribution du VX selon différents temps d’exposition a montré que la majorité du toxique reste à la surface de la peau jusqu’à 2h d’exposition. Il est donc intéressant de décontaminer la peau même après 2h d’exposition aux agents chimiques de guerre. Les microémulsions comprenant un actif détoxifiant (oxime) sont les systèmes les plus efficaces car ils pénètrent en profondeur afin de venir détruire le toxique in situ dans la peau. Les poudres et les émulsions de Pickering ont une action de surface et permettent d’extraire le toxique présent à la surface de la peau et dans les couches superficielles. Les résultats de nos études in vitro ont montré que ces formulations sont significativement plus efficaces que la terre à foulon pour une décontamination après 45 min d’exposition au VX / Organophosphorous nerve agents are designed as chemical warfare agent because they represent a threat both for the military and the civilians. Due to its low volatility, VX mainly remains in its liquid form and mostly presents a contamination by skin contact. Decontamination of exposed body surface is therefore crucial to prevent victims' poisoning. In case of terrorist acts, civilian human scalp could be a preferential site of exposure. This body region, rich in hair follicles, may require adapted decontamination products and procedures. The aims of this work are: 1) Validation of a relevant in vitro human scalp skin model; 2) Determination of decontamination strategies; 3) Formulation of new decontamination systems; 4) Evaluation of their decontamination efficacy. Pig ear skin is a relevant model when studying the in vitro percutaneous penetration of VX through human scalp. Pig skull roof skin could be used when studying the affinity of VX for hair. This study has shown that most of the nerve agent remains on the skin surface up to 2h of exposure, which means that it is worth decontaminating even if contamination occurred 2h before. Microemulsions loading a detoxifying agent (oxime) are the most efficient systems because they are able to penetrate deeper into the skin to neutralize the agent in situ. Adsorbing powders and Pickering emulsions could interact with the agent present on the skin surface and in the superficial layers. Our results from the in vitro experiments have demonstrated that these formulations are more efficient than Fuller's earth for skin decontamination after 45 min of VX exposure

Der Einsatz basischer substituierter Benzilsäureester mit einer dualistischen anticholinergen und dopaminergen Wirksamkeit als potentielle Antiparkinsonika wird zur Zeit untersucht und diskutiert. Dabei spielen die Kenntnisse der Biotransformation dieser Verbindungen eine große Rolle für deren Beurteilung. Ziel der vorliegenden Arbeit war die Untersuchung der Metabolisierung von ausgewählten Benzilaten in der Ratte. Dazu wurden vier verschiedene Derivate ausgewählt: (R,S)-N-Methyl-4-piperidyl 3,4-dimethoxybenzilat (1), (R,S)-N-Methyl-3-piperidyl 3,4-dimethoxybenzilat (2), N-Methyl-4-piperidyl 3,3'-dimethoxybenzilat (3), (R,S)-N-Methyl-3-piperidyl 3,3'-dimethoxybenzilat (4). Nach Aufarbeitung von Urin und Kot erfolgte die Charakterisierung der Metabolite mittels DC, HPLC und UV. Die wichtigste Methode zur Identifizierung stellte die MS dar, die auch in Kopplung mit der GC Anwendung fand. Durch NMR-spektroskopische Untersuchungen gelang der Nachweis der in p-Stellung erfolgten aromatischen C-Oxigenierung. Im Körper traten eine Reihe von Funktionalisierungsreaktionen auf: Esterspaltung, aromatische C-Oxigenierung, O-Dealkylierung, N-Dealkylierung, Lactamisierung, N-Oxidation, Benzophenonbildung und die Methylierung und Glucuronidierung/Sulfatierung phenolischer Derivate. Alle diese Veränderungen wurden in ihrer Vielfalt das erste Mal bei einzelnen Benzilaten beobachtet. Für die 3,4-Dimethoxyester stellten die C-Oxigenierung und bei 2 zusätzlich die Esterspaltung die Hauptabbauwege dar. Bei den 3,3'-Dimethoxyderivaten traten die O-Dealkylierung und ebenfalls die Esterspaltung in den Vordergrund. Die Entstehung der Lactame bei der Metabolisierung konnte durch Vergleich mit synthetisierter Referenzsubstanz nachgewiesen werden. Bei der Gegenüberstellung des Metabolitenspektrums mit den Ergebnissen der biomimetischen Umsetzungen von 1, 2 und 3 konnte eine Übereinstimmung in der Bildung von Benzilsäure (Esterspaltung), N-Oxid, N-dealkyliertem Ester und Benzophenon gezeigt werden, allerdings konnte dieses chemische in vitro-Testsystem nicht als Modell für C-Oxigenierungsreaktionen, O-Dealkylierung und Lactamisierung dienen. / The suitability of basic substituted benzilic esters with partially available dualistic anticholinergic and dopaminergic properties is examined and discussed for the treatment of Parkinson disease recently. The knowledge of the biotransformation is important for the evaluation of these substances. Purpose of the present thesis was the investigation of the metabolism of some selected benzilates in rats. Therefore four different derivatives were chosen: (R,S)-N-Methyl-4-piperidyl 3,4-dimethoxybenzilate (1), (R,S)-N-Methyl-3-piperidyl 3,4-dimethoxybenzilate (2), N-Methyl-4-piperidyl 3,3'-dimethoxybenzilate (3), (R,S)-N-Methyl-3-piperidyl 3,3'-dimethoxybenzilate (4). After purification of urine and faeces, the characterization of the metabolites was determined by TLC, HPLC and UV. MS, also coupled with GC, represented the most important method for identification. The evidence for aromatic oxygenation in p-position was succeeded by NMR-spectroscopy. Several metabolic processes appeared: ester cleavage, aromatic C-oxygenation, O-dealkylation, N-dealkylation, formation of lactam, N-oxidation, oxidation to benzophenone and the methylation and glucuronidation/sulphatation of phenolic derivatives. All these multiple reactions were observed at single benzilates for the first time. C-Oxygenation represented the main decomposition way for the 3,4-dimethoxy esters and, additionally, ester cleavage for 2. O-Dealkylation and ester cleavage were the important reactions of metabolism of the 3,3'-dimethoxy derivatives. The formation of lactams as metabolites could be verified by comparison to synthesized reference substances. In comparison to the metabolic pathways, the results of the biomimetic examinations of 1, 2 and 3 show agreement in the formation of benzilic acids (ester cleavage), N-oxides, N-dealkylated esters and benzophenones. But this chemical method can't be used as modell for C-oxygenation, O-dealkylation and lactamisation.

No description available.

Gegenstand der vorliegenden Arbeit war die Entwicklung und Validierung geeigneter Methoden zur selektiven qualitativen und quantitativen Bestimmung aller relevanten Zersetzungsprodukte von Natriumthiosulfat-Injektionslösungen. Die Besonderheit liegt in der Analytik der schwefelhaltigen Verbindungen neben einem großen Überschuss an Thiosulfat. Neben der Etablierung analytischer Verfahren konnte Aussagen zu stabilitätsrelevanten Parametern getroffen und die Zersetzungsprodukte unterschiedlich stabilisierter Injektionslösungen charakterisiert werden. Als wichtigste Ergebnisse dieser Arbeit: (i) sind der Nachweis und quantitative Bestimmung von Sulfit, Sulfat und erstmals von Sulfid und Polythionaten (Tetrathionat, Pentathionat, Hexathionat) in Natriumthiosulfat-Injektionslösungen durch Einsatz der DPP, IPC, IC und CZE erfolgt, (ii) sind der Nachweis und erstmals die quantitative Bestimmung von Schwefel nach Flüssig-/Flüssig-Extraktion und mittels RP-HPLC in Natriumthiosulfat-Injektionslösungen vorgenommen worden, (iii) ist erstmals die Trennung von Polysulfiden unterschiedlicher Kettenlänge durch Einsatz der CZE gelungen, (iii) ist der Nachweis der Beteiligung von Sulfid und Polythionaten am Zersetzungsgeschehen von Natriumthiosulfat-Injektionslösungen erbracht worden, (iv) ist eine Einschätzung stabilitätsrelevanter Parameter (pH, Pufferkapazität, Disulfit-Zusatz, EDTA, thermische Belastung) erfolgt (v) ist eine Erklärung des beobachteten pH-Wert-Abfalls aus dem System erbracht worden, (vi) wurden methodische Fortschritte bezüglich Nachweisgrenze und Selektivität erreicht und (vii) ist ein direkten Vergleich aller Methoden bezüglich Nachweisgrenze, Präzision, Richtigkeit, Selektivität, Robustheit und immanenter Anfälligkeit bei großem Überschuss einer Komponente möglich. / The present thesis was focused on development and validation of several analytical methods in order to detect and to determine relevant degradation product of sodium thiosulphate injection solutions with certain selectivity. The exceptional matter is the determination of small amount of sulphur containing compounds beneath a large amount of thiosulphate. In addition parameters which influence the stability of the injection solutions were investigated. Furthermore different degradation products of instable injection solutions were characterised. The main results of these thesis are the following: (i) the detection and quantitative determination of sulphite, sulphate and first of sulphide and polythionates (tetrathionate, pentathionate, hexathionate) in sodium thiosulphate injection solutions by use of DPP, IPC, IC und CZE, (ii) the detection and first the quantitative determination of sulphur after liquid- / liquid-extraction and RP-HPLC analysis in sodium thiosulphate injection solutions, (iii) the first separation of polysulphides with different chain length by use of CZE, (iii) the evidence that sulphide and polythionates are involved in degradation of in sodium thiosulphate injection solutions (iv) an evaluation of the influence of pH, buffer capacity, sodium disulphite, EDTA, sterilisation (v) the explanation of the observed pH-depression , (vi) advantages in detection limits and selectivity (vii) comparison of detection limits, accuracy, precision, selectivity and robustness in presence of sample overload.