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The study of pharmaceutical preformulation includes the evaluation of pharmacokinetic, pharmacodynamic and physicochemical properties of the drug molecules that aid the formulation. However, it has a limited role in determining drug dosage optimisation in the formulation. The study of drug-Plasma Protein Binding (PPB), and the lipophilicity, solubility, and ionic behaviours of the desired drug molecules addresses the gap and enhances our undertraining related to the behaviour of the drug molecules in the body. The High-Performance Liquid Chromatography (HPLC) technique was used in the current study to assess drug-PPB interaction. Using Michael Abraham’s ‘Linear Free Energy Relationship’ (LFER) method, two major plasma proteins namely, Human Serum Albumin as HSA and α-1-Acid Glycoprotein as AGP, were used and the structural properties governing drug-plasma protein binding was determined. This is the first time that the effect of ionised species on PPB has been quantitatively evaluated. In addition, the molecular interactions also play a key role in the supramolecular complexes of co-crystals. The project also evaluated the co-crystallisation process and its effect on physicochemical properties of the drug. In the current study, amino acids (AAs) have been observed to be a prominent source of coformers. The AAs showed co-crystals formation with carboxylic acids, nonsteroidal anti-inflammatory drug (NSAID) and citric acid which overcome the hygroscopicity problems and improved the physical stability issues during storage. This study has also identified a new formulation which is helpful for improvement in the stability of effervescent tablets at various relative humidity (RH) conditions which will reduce the manufacturing cost associated with the production of effervescent tablets.

Pharmaceuticals with suitable therapeutic properties often found to encounter challenges with dosage form development due to their poor physicochemical properties. Aim of thesis is to evaluate potential of crystal engineering directed cocrystallisation of active ingredients in modulating their physical attributes. The model compounds considered are isoniazid, caffeine, nifedipine, glyburide, chlorpropamide and riboflavin. Co-formers selected are based on the suitability of functional groups for hydrogen bond formation. Co-crystal screening and preparation methods used include neat grinding (NG), liquid assisted grinding (LAG) and solution crystallisation. Antituberculosis drug, isoniazid, upon cocrystallisation with melamine, solubility has reduced as per high performance liquid chromatography assay, however, antimicrobial properties determined using REMA assay confirms that cocrystal anti-mycobacterial activity is not compromised. Next, caffeine-glutaric acid cocrystal polymorphic forms (Forms I and II) subjected to mechanical property evaluations in multiple faces using nanoindentation and correlated relationship between crystal structure and mechanical property. The results suggest that metastable form, Form I, could display suitable tablet properties to that of thermodynamically stable form, Form II. Subsequently, photosensitive drug, nifedipine, cocrystallised with theophylline and caffeine. Notably, photochemical stability along with solubility and drug release of cocrystals is found to be superior to that of nifedipine. Lastly, crystal engineering principles utilised in preparation of multicomponent crystals of antidiabetic model drugs, glyburide and chlorpropamide with various coformers. Interestingly, during the preparation of chlorpropamide-2-nitrobenzyl alcohol, high Z prime crystal (Z’=3) of 2- nitrobenzyl alcohol is serendipitously identified. In conclusion, crystal engineering based cocrystallisation is a viable technology for modulating physicochemical properties of pharma and nutraceuticals.

Modern separation and spectrometric techniques for biological sample lipidom investigation Due recent progress in field of mass spectrometry the lipidomics, part of metabolomics, is increasing its importance for broad fields of biological study. The aim of this study is to test the lipid extraction techniques and to optimize the preseparation and separation of lipids suitable for mass spectrometry detection. The fragmentation patterns of four, the most abundant lipid classes of glycerolipids (PC, PE, TG, DG), were acquired for the proposed system. These patterns were compared with literature. The most appropriate method for extraction was declared technique according Folch based on methanol and chloroform solution. The preseparation due SPE method is very useful tool for lipid determination. The optimized were focused to reach higher recovery especially in polar lipid fraction. Proposed HPLC system is based on methanol with ammonium buffer, water and isopropanol. The testing was done on three columns with different type of sorbents (Gemini, Syncronis and Kinetex). The separation was evaluated according mass spectrometer response, shape and wide of particular analytes peaks. Composition contains 20% of water was determinate as the best and also the best separation was achieved by Kinetex column. The proposed...

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Thèse réalisée en co-tutelle avec l'Université Claude Bernard de Lyon 1, en France. / La gestion de la douleur neuropathique reste un challenge en médecine, malgré le nombre de traitements actuellement disponible. L’expérimentation animale a généré beaucoup d’informations concernant la douleur, mais ces connaissances demeurent insuffisantes pour développer de nouveaux analgésiques plus efficaces tout en restant sécuritaires. La douleur est un symptôme clinique complexe avec de multiples origines, et les mécanismes de douleur centraux et périphériques dépendent de l’évolution de la pathologie. Il est donc essentiel d’investiguer plus profondément les mécanismes moléculaires responsables de l’initiation et du maintien de la douleur, afin de cibler de nouvelles voies de transmission de la nociception plus prometteuses pour soulager la neuropathie et développer de meilleures stratégies thérapeutiques. Ce projet s’est donc intéressé plus particulièrement à la famille des tachykinines issues du gène TAC1 (substance P, ses précurseurs et métabolites, et neurokinine A sont les peptides ciblés pour ce projet de recherche), une famille de neuropeptides qui joue un rôle critique dans la transmission nociceptive. Pour réaliser cette étude, nous avons d’abord développé une stratégie de quantification afin de quantifier les expressions des différents neuropeptides bioactifs cibles, par HPLC-MS/MS. Puisqu’il existe différentes stratégies de quantification des peptides par HPLC-MS/MS, une méthode analytique fiable et robuste était nécessaire pour répondre aux objectifs de recherche. Nous avons développé une méthode utilisant la quantification relative avec un étalon interne stable marqué isotopiquement. En effet, pour quantifier les neuropeptides d’intérêt de l’étude, c’est la stratégie qui s’est avérée la plus reproductible et précise. Suite à la mise au point de la stratégie de quantification, nous avons utilisé des modèles animaux, souvent nécessaires pour faire progresser la recherche scientifique sur la compréhension de la douleur. Le modèle de constriction chronique du nerf sciatique (CCI) est un modèle validé, largement utilisé pour induire et étudier la douleur neuropathique. Afin de s’assurer du développement de la neuropathie, deux tests comportementaux, les filaments de von Frey et le test de Hargreaves, ont été employés avant et après la CCI. Les cerveaux, les élargissements lombaires et les plasmas des animaux ont été prélevés afin d’effectuer les analyses pour quantifier la modulation d’expression des différents neuropeptides bioactifs cibles entre le groupe animal contrôle et le groupe pathologique. Ceci a révélé une augmentation significative des concentrations spinales de β-tachykinine58-71, SP et SP3-11 chez les rats neuropathiques, signifiant donc que l’expression de ces trois peptides est étroitement liée. Au contraire, la concentration spinale de SP5-11 est diminuée de façon significative chez les animaux neuropathiques. De plus, les concentrations cérébrales de β-tachykinine58-71 et SP sont significativement plus élevées chez les rats neuropathiques. Tandis qu’aucune différence significative n’est notée au niveau des concentrations spinales ou cérébrales de NKA, β- tachykinine58-70, SP6-11 et SP1-7. Ceci suggère que la β-tachykinine58-71, SP, SP3-11 et SP5-11 pourraient potentiellement servir de biomarqueurs de la douleur neuropathique. Nous avons aussi utilisé un modèle de souris knock-out, déficientes au niveau du gène du récepteur TRPV1 (TRPV1-/-), afin d’étudier les modulations d’expression de différentes tachykinines en fonction de la présence ou non du récepteur TRPV1, connu pour être étroitement lié aux tachykinines. Le récepteur vanilloïde TRPV1 est impliqué dans la transmission du signal douloureux en étant surexprimé, contribuant à la ensibilisation. Nos résultats ont montré que les concentrations spinales et cérébrales de SP et NKA sont significativement diminuées dans les tissus des souris TRPV1-/-. Ceci démontre la contribution des tachykinines dans la modulation du seuil douloureux, ainsi qu’un lien entre l’expression de SP et NKA et celle du récepteur TRPV1 dans les systèmes nerveux central et périphérique. En effet, les récepteurs vanilloïdes, et plus particulièrement le récepteur TRPV1, jouent un rôle central dans le processus de stimuli nociceptifs. Il est aussi connu que plusieurs ligands du TRPV1, tels que l’eugénol, la vanilline, le [6]-gingérol, ou des agonistes endogènes comme certains endovanilloïdes atténuent la douleur neuropathique en agissant sur le récepteur TRPV1. Suite à ces résultats, nous nous sommes intéressés aux métabolites de SP. En effet, différents mécanismes permettent la libération de SP suite à une lésion et à l’établissement de la douleur neuropathique. Il est bien documenté que le foie est l’organe principal du métabolisme. Par conséquent, au niveau sanguin, ce seront des métabolites de SP qui pourront être exprimés. Nous avons donc choisi d’étudier la stabilité métabolique de SP dans des microsomes de foie de rat, de souris et d’humain. Nos résultats ont montré que SP est rapidement dégradée et que le profil métabolique est différent selon l’espèce étudiée. Par conséquent, plusieurs métabolites de SP semblent intéressants et possèdent des activités pharmacologiques qui leur sont propres, dont SP1-7, SP3-11 et SP5-11. SP, ses métabolites et ses précurseurs, ainsi que NKA s’avérant intéressants en tant que biomarqueurs potentiels de la douleur neuropathique, il semblait aussi important de développer une méthode pour les quantifier dans le plasma. En effet, il est plus aisé de collecter du plasma pour éviter de sacrifier l’animal dans le but de réaliser des études cinétiques à long terme ou de développer des méthodes de dosages applicables à l’humain. Nous avons donc développé une méthode HPLC-MS/MRM afin de pouvoir estimer le niveau endogène des neuropeptides cibles dans le plasma. En revanche, nous n’avons pas eu la chance de pouvoir quantifier ces peptides dans le plasma collecté lors de l’étude animale. Il reste encore de nombreuses études à réaliser avant de pouvoir répondre à toutes les questions concernant la douleur neuropathique, sa transmission et les traitements possibles. Ce projet de doctorat a permis de commencer à éclairer la recherche sur la voie des tachykinines. / The management of neuropathic pain remains a challenge in medicine, despite the availability of numerous drugs. Animal experimentation has generated a tremendous amount of information about pain, but this knowledge is still insufficient for new more efficient and safe analgesics. Pain is a complex clinical symptom with multiple origins, and peripheral and central pain mechanisms depend on the pathology evolution. Thus, it is essential to further investigate the mechanisms responsible for the initiation and maintenance of pain in order to develop better effective therapies. This project is particularly focused on the tachykinin family encoded by TAC1 gene (substance P, its precursors and metabolites, neurokinin A), a family of neuropeptides that plays a critical role in nociceptive transmission. We initially developed a quantification strategy in order to study the targeted bioactive neuropeptide expression modulation by HPLC-MS and HPLC-MS/MS. And it is critical to develop reliable and robust analytical methods to reach the objectives. So, we developed a method using relative quantification with stable isotopic labeled internal standards. In fact, in order to quantify target neuropeptides, this strategy was the most reproducible and accurate. Following the development of the relative quantification strategy, we used validated animal models, fundamental to better knowledges of painful molecular mechanisms. The model of chronic constriction injury of the sciatic nerve (CCI) is a validated model, widely used to induce and study neuropathic pain. To perform complete data, two behavioral tests, von Frey filaments and Hargreaves test, were used before and after the CCI to ensure the neuropathy establishment. The animal brains, lumbar enlargements and plasmas were collected to quantify the expression modulation of different targeted bioactive neuropeptides between the CCI group versus the control group. HPLC-MS/MS analyses revealed that the spinal β-tachykinin58-71, SP and SP3-11 concentrations were significantly up-regulated in neuropathic animals, meaning these peptide expressions are closely related to pain behavior. In contrast, the spinal SP5-11 concentration in neuropathic animals revealed a significant down-regulation compared with normal animals. Moreover, the brain β-tachykinin58-71 and SP concentrations were significantly up-regulated in neuropathic animals. Interestingly, no significant concentration differences were observed in the spinal cord and brain for NKA, β-tachykinine58-70, SP6-11 and SP1-7. This suggests that β-tachykinin58-71, SP, SP3-11 and SP5-11 could potentially serve as drug efficacy markers in early drug discovery. We also used a knock-out mice model, deficient in the TRPV1 receptor gene (TRPV1- /-) to study the expression modulation of different tachykinins according to the presence or absence the TRPV1 receptor. The TRPV1 receptor is known to be closely related to tachykinins. The over-expressed TRPV1 vanilloid receptor is involved in the transmission of painful signal, leading to the sensitization. Our results revealed that SP and NKA spinal and brain concentrations were significantly decreased in TRPV1-/- mice. These results suggest an important tachykinin contribution in the pain threshold modulation, and a close link between SP, NKA and TRPV1 expressions in the central and peripheral nervous systems. Vanilloid receptors, particularly the TRPV1 receptor, play a central role in the nociceptive stimuli transmission. It is also known that several TRPV1 agonists, such as eugenol, vanillin, [6]-gingerol, or endogenous agonists like endovanilloids alleviate pain in neuropathic and osteoarthritis models via their action on the TRPV1 receptor. There are several mechanisms leading to the SP release following an injury and the neuropathy development. Nowadays, it is well documented the liver is the main organ of the metabolism. Thus, SP metabolites could be expressed in the blood level. So we are interested to SP metabolic stability in rat, mouse and human liver microsomes. Our results showed that SP is rapidly degraded and the metabolic profile is different depending on the species studied. Several SP metabolites seem interesting and have pharmacological activities, including SP1-7, SP3-11 and SP5-11. As all these neuropeptides seemed interested targets like potential biomarkers of neuropathic pain, we developed a method to quantify them in the plasma. Additionally, it is easier to collect plasma to avoid sacrificing the animal in order to achieve long-term kinetic studies or develop assay methods applicable to humans. So we have developed a HPLC-MS/MRM analytical method to estimate the targeted neuropeptide endogenous levels in plasmas. However, we did not have the chance to quantify these peptides in collected plasmas during the animal study. A lot of studies are still required to clarify different pathways involved in neuropathic pain in order to develop better and safer therapeutic strategies. This project allowed to better understanding of mecanisms related to tachykinin activities.

Red Lustrous Wheelmade Ware (RLWm ware) transport and storage vessels have been excavated from Late Bronze Age (LBA) sites across the eastern Mediterranean. These distinctive vessels were traded for the valuable commodity they contained so far unidentified. Seventy-three sherds (61 RLWm ware, 12 in local fabrics) and two visible residues were analysed for organic residues using standard lipid extraction techniques. Seven residues from a previous study were re-examined. Gas chromatography-mass spectrometry identified four materials - beeswax, bitumen, fat/oil and resin. Beeswax, found only in vessels from Hittite sites in Turkey, was probably used as a post-firing treatment. Fat/oil, present in some sherds from every site, represents the contents of the vessels and showed many of the characteristics of degraded plant oil. Two examples contained a plant sterol and three yielded ricinoleic acid, a biomarker for castor oil. Gas-chromatography compound-specific isotope ratio mass spectrometry of selected residues excluded dairy products, ruminant animal fats and fish oils as source materials for the fats/oils, while comparison with a small database of modern oils created during this study does not exclude plant oils. Selected samples analysed by high performance liquid chromatography-tandem mass spectrometry did not reveal wine residues. Data on the elemental composition of the fabric collected during another study was re-analysed and compared with data from a further published study, confirming the remarkable consistency of RLWm ware fabric. Volume calculations were also attempted to give an estimate of the capacity of the main vessel forms.

Les flavanols et leurs oligomères, les procyanidines, sont des composés phénoliques biosynthétisés dans les baies de raisin, dont ils sont extraits pendant la vinification. Une meilleure compréhension de leur évolution pendant la maturation du raisin, la vinification et le vieillissement du vin est importante du fait de leur responsabilité dans beaucoup des propriétés organoleptiques du vin (couleur, amertume et astringence). Plusieurs procyanidines ont été obtenues par une méthode de synthèse permettant de contrôler aussi bien la régio- et la stéréosélectivité de la liaison interflavane que le degré d’oligomérisation. Cette méthode a pu être élargie à la synthèse de procyanidines galloylées. L’étape de couplage, qui restait l’étape limitante de cette synthèse, a pu être améliorée par l’utilisation de la catalyse à l’or. Ces composés ont ensuite été utilisés comme standards afin de les identifier et les quantifier dans du raisin et des vins par chromatographie liquide haute performance. L’étude des intéractions supramoléculaires des composés galloylés a été évaluée par RMN DOSY. En parallèle de ces études, un nouveau composé indolique, possédant un motif glucose dans sa structure, a été identifié et caractérisé pour la première fois dans les vins rouges. / Flavanols and their oligomers, procyanidins, are phenolics compounds biosynthetized in grapes, from which they are extrated during winemaking As they contribute so much to the organoleptics properties of the wine (color, bitterness and astringency), a better comprehension of their evolution during grapes maturation, winemaking and ageing of the wine is particulary important. Several procyanidines were obtained by a synthesis method which allows to control as well the interflavan regio- and stereoselectivity as the degree of oligomerization. This method was extended to the synthesis of galloylated procyanidins. The stage of coupling, which was a limiting stage of this synthesis, could be improved by the use of gold III catalysis. These compounds were then used as standards in order to identify and quantify them in grapes and wines by high performance liquid chromatography. The supramolecular properties of galloylated procyanidins were investigated by NMR DOSY. In parallel to these studies, a new indolic compound bearing a glucose moiety, has been identified and characterized for the first time in red wine.

Processos de fotodegradação de compostos orgânicos tóxicos têm sido bastante estudados. Este trabalho trata da aplicação do processo foto-Fenton para a degradação de atrazina em água (composto modelo). O efeito das concentrações dos seguintes compostos foi avaliado: peróxido de hidrogênio (2 a 6 mmol L-1) e ferrioxalato de potássio (0,2 a 1 mmol L-1). Os experimentos foram realizados em um reator com lâmpada UV - 8W (254nm). O processo de fotodegradação foi monitorado por medidas de espectrofotometria de absorção molecular automatizada por injeção seqüencial (SIA) para determinação de peróxido de hidrogênio e por cromatografia a líquido de alta eficiência (CLAE) para determinação de atrazina e metabólitos. Os experimentos demonstram que o processo de foto-Fenton é viável para o tratamento de atrazina em água. / Photo-degradation processes of toxic organic compounds have been widely studied. This work describes the application of the photo-Fenton process for degradation of atrazine in water. Atrazine was used as a model compound. The effects of the concentration of the following substances were evaluated: hydrogen peroxide (1 to 6 mmol L-1) and potassium ferrioxalate (0.2 to 1 mmol L-1). The experiments were accomplished in a reactor with an 8W UV lamp at 254 nm. The photo-degradation was monitored by molecular absorption spectrophotometry automated by sequential injection analysis (SIA) for determination of hydrogen peroxide and by high performance liquid chromatography (HPLC) for determínation of atrazine and its metabolites. Experimental results demonstrated that the photo-Fenton process is feasíble for the treatment of atrazine.

O desenvolvimento de métodos analíticos e o estudo de estabilidade de formulações cosméticas e farmacêuticas fazem parte do processo de garantia de qualidade, o qual tem por objetivo assegurar a eficácia e segurança no uso de tais produtos pelo consumidor. O ácido kójico é um agente despigmentante que pode estar associado ao ácido glicólico, um agente esfoliante, a fim de ter sua ação potencializada. O objetivo do presente trabalho foi o desenvolvimento de um método analítico para determinação do ácido kójico a 1% associado ou não ao ácido glicólico a 5% em formulações tópicas na forma creme e gel, a base de excipientes comumente utilizados em farmácias de manipulação, e a realização de um estudo acelerado para avaliar a estabilidade das mesmas. Para a determinação do ácido kójico isolado empregou-se a espectrofotometria derivada de 1ª ordem no ultravioleta (UVD), usando-se o método \"zero crossing\" a 256,8 nm, onde a interferência dos excipientes da matriz foi anulada. Para a determinação dos dois ativos associados, foi validado um método por CLAE em fase reversa, compareamento iônico, empregando-se coluna Synergi Hidro® C18, fase móvel tampão NH4H2PO4/H3PO4 30 mM pH 3,0 com TBA (brometo de tetrabutilamônio) 2 mM : acetonitrila (95:5), vazão 0,7 mL/min e detector PDA fixado em 220 nm. As amostras foram extraídas sem grande complexidade e os ativos puderam ser determinados em 12 min. As mesmas condições experimentais foram usadas para o desenvolvimento de um método para a determinação do ácido kójico isolado nas formulações creme e gel por CLAE, sendo que a comparação com o método UVD não mostrou diferença estatisticamente significante para p = 95% quanto à precisão e exatidão. As amostras submetidas ao estudo de estabilidade acelerado, com duração de 90 dias, foram armazenadas em estufa a 40±2ºC e luz a 25±2ºC e também acompanhadas em armário fechado à temperatura ambiente, sendo avaliadas quanto aos caracteres organolépticos, pH, doseamento e comportamento reológico. As formulações submetidas ao estudo mostraram-se estáveis fisicamente, mas as amostras apresentaram decaimento do teor de ácido kójico acima de 5% quando armazenadas em estufa a 40±2ºC. Considerada a condição normal de armazenagem as formulações mantiveram-se dentro da faixa de 90% do teor inicial, podendo o prazo de validade de 90 dias ser considerado como referência para formulações semelhantes em composição e embalagem, desde que guardadas em temperatura ambiente e ao abrigo da luz. / The development of analytical methods and the stability study of cosmetics and pharmaceuticals are part of the quality assurance, which has for objective to guarantee the effectiveness and security in the use of such products for the consumer. Kojic acid is a depigmentant agent that can be used in association with glycolic acid, an exfoliant agent, in order to have its action maximized. The aim of this work was the development of an analytical method to assay kojic acid 1% associated or not with 5% glycolic acid in cream and gel form, based on excipients normally used in compounding formulations, and carried out an accelerated study to evaluate its stability. To determine kojic acid in such formulations it was employed an UV first-derivative spectrophotometric method (UVD), with \"zero crossing\" set in 256,8 nm, where the excipients interference could be annulled. To assay both acids in association it was validated a reversed phase HPLC method with ion pairing, employed a Synergi Hidro® C18 column, mobile phase NH4H2PO4/H3PO4 buffer 30 mmol -1 pH 3,0 plus TBA (tetrabutylammonium bromide) 2 mM : acetonitrila (95:5), flow rate of 0,7 mL/min and detector PDA set in 220 nm. The samples were easily extracted and the run time was 12 min. The same experimental conditions were used to the development of a HPLC method in order to determine the kojic acid isolated in cream and gel formulations. The UVD e HPLC methods were not statistically different in terms of accuracy and precision (p = 95%). The samples submitted to the accelerated stability study, for 90 days, were stored at 40±2ºC and light 25±2ºC. All samples were also stored at room temperature protected from light. Appearance, pH, rheology and amount of kojic and glycolic acids (by HPLC) were evaluated. At the end of the study, all the samples showed physical stability, but presented decline in kojic acid above 5% at 40±2ºC. Samples stored at not accelerated conditions preserved 90% of kojic acid concentration. Therefore, a 90 days expiration date may be considered for formulations with similar composition and packing, when stored at room temperature and protected from light.