An important element of metabolic regulation of cardiac and skeletal muscle energetics is the interaction of mitochondria with cytoskeleton. Mitochondria are in charge of supplying the cells with energy, adjusting its functional activity under conditions of stress or other aspects of life. Mitochondria display a tissue-specific distribution. In adult rat cardiomyocytes, mitochondria are arranged regularly in a longitudinal lattice at the level of A band between the myofibrils and located within the limits of the sarcomeres. In interaction with cytoskeleton, sarcomeres and sarcoplasmic reticulum they form the functional complexes, the intracellular energetic units (ICEUs). The ICEUs have specialized pathways of energy transfer and metabolic feedback regulation between mitochondria and ATPases, mediated by CK and AK. The central structure of ICEUs is the mitochondrial interactosome (MI) containing ATP Synthasome, respiratory chain, mitochondrial creatine kinase and VDAC, regulated by tubulins. The main role of MI is the regulation of respiration and the intracellular energy fluxes via phosophotransfer networks. The regulation of ICEUs is associated with structural proteins. The association of mitochondria with several cytoskeletal proteins described by several groups has brought to light the importance of structure-function relationship in the metabolic regulation of adult rat cardiomyocytes. To purvey a better understanding of these findings, the present work investigated the mechanism of energy fluxes control and the role of structure-function relationship in the metabolic regulation of adult rat cardiomyocytes. To show these complex associations in adult cardiac cells several proteins were visualized by confocal microscopy: α-actinin and β-tubulin isotypes. For the first time, it was showed the existence of the specific distribution of β-tubulin isotypes in adult cardiac cells. Respiratory measurements were performed to study the role of tubulins in the regulation of oxygen consumption. These results together confirmed the crucial role of cytoskeletal proteins -i.e. tubulins, α-actinin, plectin, desmin, and others- for the normal shape of cardiac cells as well as mitochondrial arrangement and regulation. In addition, in vivo - in situ mitochondrial dynamics were studied by the transfection of GFP-α-actinin, finding that fusion phenomenon does not occur as often as it is believed in healthy adult cardiac cells. / Un élément important de la régulation du métabolisme énergétique des muscles cardiaque et squelettiques est l'interaction des mitochondries avec le cytosquelette. Les mitochondries sont responsables de l'approvisionnement des cellules en énergie, elles sont capables d'ajuster leur activité fonctionnelle en fonction des conditions de stress ou d'autres aspects de la vie. Les mitochondries ont une distribution spécifique selon les tissus. Dans les cardiomyocytes de rats adultes, les mitochondries sont disposées régulièrement dans un entrelacement longitudinal au niveau des bandes A, entre les myofibrilles et dans les limites des sarcomères. En interaction avec le cytosquelette, le sarcomère et le réticulum sarcoplasmique, elles forment des complexes fonctionnels appelés unités énergétiques intracellulaires (ICEUs). Les ICEUs ont des voies spécialisées de transfert d'énergie et de régulation des feedback métaboliques entre les mitochondries et les ATPases, médiée par la CK et l'AK. La structure centrale des ICEUs est l'interactosome mitochondrial (MI) qui confient l'ATP synthasome, la chaîne respiratoire, la créatine kinase mitochondriale et VDAC, qui pourrait être régulé par les tubulines. Le rôle principal du MI est la régulation de la respiration et des flux d'énergie intracellulaires via les réseaux de phosphotransfert. La régulation des ICEUs est liée aux protéines structurales. L'association des mitochondries avec plusieurs protéines du cytosquelette, décrite par plusieurs groupes, a mis en évidence l'importance de la relation structure-fonction dans la régulation métabolique des cardiomyocytes de rats adultes. Pour fournir une meilleure compréhension de ces résultats, le présent travail étudie le mécanisme de contrôle des flux d'énergie et le rôle des relations structure-fonction dans la régulation métabolique de cardiomyocytes de rats adultes. Pour montrer ces associations complexes dans les cellules cardiaques adultes, plusieurs protéines ont été visualisées par microscopie confocale: l'α-actinine et les isoformes des β-tubulines. Pour la première fois, l'existence d'une distribution spécifique des isoformes de β-tubuline dans les cellules cardiaques adultes a été montré. Des mesures respiratoires ont été réalisées pour étudier le rôle des tubulines dans la régulation de la consommation d'oxygène. Ces résultats ont confirmé le rôle déterminant des protéines du cytosquelette -tubulines, α-actinine, plectine, desmine, et autres- pour le maintien de la forme normale des cellules cardiaques, ainsi que de l'arrangement et de la régulation mitochondrial. En outre, la dynamique mitochondriale a été étudiée in vivo et in situ par la transfection de la GFP-α-actinine, ceci permettant la mise en évidence du fait que le phénomène de fusion ne se produit pas aussi souvent qu'on ne le croit pour des cellules cardiaques adultes en bonne santé.

L'évolution est-elle prédictible? Alors que la réponse habituelle est un non presque unanime, un corpus croissant de connaissances suggère qu'il est temps de revoir cette réponse. Même si les mutations sont toujours considérées comme aléatoires, la détection de patterns génétiques sous-jacents aux événements évolutifs ouvre la porte sur des stratégies potentielles permettant de prévoir les trajectoires évolutives suivies par les organismes lorsqu'ils s'adaptent à des contraintes changeantes. Quand les organismes subissent une spécialisation fonctionnelle (à travers la perte de gènes et de fonctions) pour s'adapter à des signaux environnementaux donnés, les trajectoires évolutives possibles qu'ils peuvent emprunter sont restreintes et devraient donc être plus facilement prévisibles. Dans ce contexte d'évolution réductive par spécialisation, les objectifs de cette thèse sont de mieux comprendre l'interaction entre contraintes environnementales, métabolisme, évolution génétique et adaptations fonctionnelles, et dans un deuxième temps de prédire, pour des contraintes données, les trajectoires évolutives qui seront suivies par les organismes pour s'adapter à ces contraintes. Une première approche met l'accent sur l'importance des interactions biotiques en tant que déterminants des trajectoires évolutives, et comment, en modélisant une hausse bénéfique de dépendance envers un bien commun, il est possible de prédire la dynamique d'une population subissant de tels évènements évolutifs. Une deuxième approche étudie comment les changements observés au niveau métabolique et fonctionnel et engendrés par des modifications de contraintes environnementales pourraient être prévus et testés. Sur la base d'une vision centrée sur le métabolisme, des travaux de modélisation et des travaux d’expérimentions ont été combinés pour étudier l'évolution de la spécialisation aux niveaux génétiques, métaboliques et fonctionnels. Nous montrons que les trajectoires évolutives suivies peuvent-être partiellement prédites en fonction de conditions environnementales spécifiques, mais que ces prédictions sont limitées en raison de la complexité du réseau de l'expression génétique. Ce travail exploratoire et interdisciplinaire augmente les connaissances sur les déterminants évolutifs et les trajectoires suivies par l'organisme au cours d’un phénomène de spécialisation. Il démontre également un grand potentiel de prédiction, notamment grâce à une perception métabolique des systèmes. / Is evolution predictable? While the usual response is an almost unanimous No, a growing corpus of knowledge suggests it is time to seriously revisit this answer. Even though mutations are still assumed to be random, the detection of genetic patterns underlying evolutionary events opens the door on potential strategies to forecast the evolutionary trajectories followed by organisms when they adapt to changing constraints. When organisms undergo functional specialization (through genes and function loss) to adapt to given environmental cues, the possible evolutionary paths they can take are restrained and should thus be more easily predictable. In this context of reductive evolution through specialization, the objectives of this thesis are to understand better the interaction between environmental constraints, metabolism, genetic evolution and functional adaptations, and in a second time to predict, for given environmental constraints, the evolutionary trajectories which will be followed by organisms to adapt to these constraints. A first approach focuses on the importance of biotic interactions as being determinants of evolutionary trajectories, and how by modelling a beneficial rise of dependency on a common good, we could predict the dynamic of a population undergoing such evolutionary events . A second approach investigates how changes entailed in metabolisms and functions by a change in environmental constraints could be forecast and tested. Based on a metabolic-centered view, we combined modelling and experimental work to encompass the evolution of specialization at the genetic, metabolic and functional levels. We show that evolution trajectories can partially be predicted according to specific environmental conditions, but that these predictions are limited due to the intricacy of the genetic expression network. This exploratory and interdisciplinary work increases the knowledge on evolutionary determinants and trajectories followed by organism during specialization. It also demonstrate a great potential for predictions, notably through a metabolic perception of the systems.

Les bivalves côtiers Codakia orbicularis et C. orbiculata, de la famille des Lucinidae, abritent des Gammaprotéobactéries endosymbiotiques sulfo-oxydantes dans leurs branchies. Ces deux bivalves vivent dans les herbiers à Thalassia testudinum et hébergent la même bactérie symbiotique selon les analyses effectuées à partir des séquences d’ADNr 16S. Lors de période de stabulation, la population bactérienne symbiotique décroit alors qu’il n’y a pas, dans le même temps, de relargage des symbiotes observé. Des analyses en cytochimie ont montré une forte activité d’enzymes lysosomales lors de ces épisodes de privation de nourriture et de soufre. Il a ainsi été montré que les symbiotes peuvent servir directement de source de nourriture aux bivalves pour survivre lors de ces périodes de crise. Le transfert de carbone des symbiotes vers l’hôte peut être flexible et pourrait consister en un simple transfert de matière organique ou "milking", dans des conditions normales de nutrition et de digestion des symbiotes et devenir du "farming", dans des conditions de stabulation. Jusqu’à ce jour, le symbiote reste non cultivable. De ce fait, l’utilisation de techniques indépendantes de la culture comme les approches –omics ont été mises en place pour étudier la physiologie de cette bactérie symbiotique. Le génome du symbiote a été analysé par Next Generation Sequencing (NGS) permettant ainsi d’obtenir les bases du protéome et ainsi de pouvoir analyser la physiologie du symbiote. Dans ce travail, le protéome des bactéries a été analysé sous différentes conditions. L’oxydation des sulfures est une des voies métaboliques clés du symbiote de Codakia. Cette voie fait très probablement appel au système Sox périplasmique, ainsi qu’à une sulfite réductase cytoplasmique (DsrAB), une APS réductase (AprAB) et une ATP sulfurylase (SopT). De plus, deux autres enzymes additionnelles d’oxydation des sulfures ont pu être mises en évidence dans l’espace périplasmique du symbiote telle que la quinone réductase (Sqr) et la sulfide déhydrogénase (FccAB). Les gènes des enzymes du cycle de Calvin Benson Bassham (CBB) ne semblent pas être tous présents dans le génome du symbiote. Les protéines de la RuBisCO sont abondamment exprimées. Il semblerait que la régénération du ribulose-1,5-bisphosphate soit effectuée de façon non conventionnelle via une phosphofructokinase PPi-dépendante. Une autre caractéristique du CBB est qu’il y a deux formes différentes de RuBisCO codées dans le génome du symbiote. Les deux formes sont exprimées en même temps, mais la forme I de la RuBisCO est 50 fois plus exprimée que la forme II. En plus de la vie autotrophique, plusieurs gènes nécessaires à une vie hétérotrophique sont présents dans le génome. Dans le protéome, les enzymes de la glycolyse et du cycle TCA sont faiblement exprimées. Les protéines du métabolisme du glycogène ont également été identifiées dans le protéome. De plus, plusieurs types de transporteurs comme ABC, TRAP et PTS sont présents dans le génome et certaines formes d’expression de ces transporteurs ont pu être suspectées, y compris lors de la vie intracellulaire du symbiote. De façon inattendue, un groupe de gènes nif est présent dans le génome permettant la fixation de l’azote atmosphérique par le symbiote. Les protéines codées par les gènes clés, comme la nitrogénase NifH/K/D, ont été abondamment trouvées dans le protéome. De plus, l’analyse du protéome montre une régulation forte de ces protéines dans des conditions de stabulation du bivalve hôte. La rubrerythrine est fortement exprimée et servirait à protéger la nitrogénase de l’oxygène au sein des bacteriocytes. L’endosymbiote bactérien code également pour un système de sécrétion de type 6 (T6SS) pour le transport de molécules bactériennes effectrices à travers les membranes du cytoplasme de la cellule hôte et jouerait un rôle possible de communication directe avec l’hôte. / The shallow water bivalves Codakia orbicularis and Codakia orbiculata, both belonging to the family Lucinidae, harbor endosymbiotic sulfur-oxidizing gamma-Proteobacteria in their gills. The bivalves live in seagrass beds of Thalassia testudinum and harbor the same bacterial symbionts according to 16S rDNA sequence analysis. During starvation, the symbiont population decreases while no release of symbionts were observed. We observed lysosomal enzyme activity during sulfide and food starvation with cytochemical staining methods. We suggest that the host uses symbionts as a nutrient source to survive a hunger crisis. The carbon transfer from the symbionts to the host could be flexible and could consist in transfer of organic matter, "milking", under normal feeding conditions and digestion of the symbionts, "farming", under starved conditions. Until now the symbiont alone is not cultivable. Therefore, cultivation-independent techniques, like -omics approaches were used to analyze the physiology of the symbiont. Next generation sequencing (NGS) was employed to sequence the genomes of symbionts from both hosts, display the backbone for proteomics. The soluble- and membrane-associated symbiont proteomes were analyzed during different conditions. The oxidation of sulfide is one key metabolic pathway of the Codakia symbiont, most probably using the periplasmic Sox-system, a cytoplasmatic sulfite reductase (DsrAB), an APS reductase (AprAB) and an ATP sulfurylase (SopT). Furthermore, indications for two additional putative sulfide oxidation systems in the periplasmic space, the sulfide quinone reductase (Sqr) and the sulfide dehydrogenase (FccAB), could be found. The Calvin Benson Bassham cycle (CBB) of the symbiont is not completely encoded in the genome. The key genes, RuBisCO, are abundantly expressed. It is assumed that the regeneration of the ribulose-1,5-bisphosphate is performed unconventionally via a PPi-dependent phosphofructokinase. Another feature of the CBB is that two different forms of RuBisCO are encoded in the genome. Both are expressed at the same time, but RuBisCO form I is about 50x times more expressed. Additional to the autotrophic lifestyle, all genes for the heterotrophic lifestyle are encoded in the genome. In the proteome, the enzymes related to glycolysis and TCA-cycle were low expressed. Interestingly, proteins for glycogen metabolism were identified in the proteome. Additionally, several types of transporters like ABC, TRAP and PTS are encoded in the genome. In the proteome several indications were found for an expression of these transporters, even in the endosymbiotic lifestyle. Unexpectedly, in the genome a nif gene cluster is encoded for gaseous nitrogen fixation as ammonium source. The key genes, the nitrogenase NifH/K/D, were abundantly identified in proteome. Further, the proteome analyses indicate a strictly down-regulation of these proteins under starvation conditions. Rubrerythrin, a strongly expressed protein and is predicted to protect the nitrogenase against oxygen stress. The bacterial endosymbionts encode a specialized secretion system type 6 (T6SS) for the transport of bacterial effector molecules through the membranes to the host cytoplasm and display one possibility for a direct "communication" with the host. In summary, genomics and proteomics analyses of the Codakia symbiont improved the knowledge about the metabolism of the symbiont in lucinid bivalves.. The genomics and proteomics data generated in this study can be used as a basis for further in-depth analyses of the physiology of the symbionts and interaction with the host.

La contraction cardiaque nécessite un apport énergétique important, dont la source principale est l'oxydation des acides gras dans la mitochondrie. Ces acides gras pénètrent dans la mitochondrie sous la forme d'acyl-carnitine. Lors du couplage excitation-contraction, il y a une communication entre la contraction et le métabolisme assurée par les variations de potentiel et les variations calciques concomitantes. Cette communication permet d'adapter la quantité d'énergie synthétisée en fonction des besoins contractiles. Des observations cliniques et expérimentales indiquent que le métabolisme module également les mécanismes de contraction. En effet de fortes modifications de la balance énergétique observée lors de pathologie comme le syndrome métabolique ou le déficit primaire en carnitine induisent des dysfonctions contractiles et des troubles du rythme cardiaque. L'homéostasie mitochondriale semble tenir un rôle important dans cette communication, mais les mécanismes impliqués restent encore mal connus. Dans ce travail nous nous sommes intéressés à comprendre les comment de fortes variations d'acyl-carnitine influencent l'apparition de troubles du rythme et participent au remodelage ventriculaire. Lors d'une approche intégrative nous avons mis en évidence le rôle central de l'adenine nucleotide transporteur (ANT) dans la genèse des troubles du rythme induit par une forte concentration en palmitoyl-carnitine. De plus des études in vivo sur des animaux déficients en carnitine, ont permis de décrire pour la première fois une relation entre une diminution de l'intervalle QT (QT court) avec un désordre métabolique. / Heart contraction requires a considerable amount of energy. Mitochondrial fatty acid oxidation is the major source of energy production in the heart. Fatty acids diffuse through the mitochondrial membrane in the acyl-carnitine form. During excitation-contraction coupling, variations of membrane potential and calcium concentration allow the communication between contraction and metabolism. This communication allows the adaption of energy production into contractile function. Clinical and experimental observations indicate that metabolism modulates contraction mechanisms. In particular, the energetic imbalance observed in metabolic syndrome or primary carnitine deficiency induces a contractile disturbance and arrhythmias. Mitochondrial homeostasis seems to be an important participant though the mechanisms involved in this phenomenon remain to be completely elucidated. In this study, we examined the influence of acylcarnitine concentration variations on cardiac rhythm and ventricular remodeling. Through an integrative approach, we have demonstrated the pivotal role of the adenine nucleotide transporter (ANT) in the apparition of high acylcarnitine concentration associated arrhythmia. Furthermore, in vivo studies with carnitine deficient mice reveal, for the first time, a relationship between the QT interval duration (short QT) and metabolic disturbance.

Le cisplatine est l'agent chimiothérapeutique le plus largement utilisé pour le traitement de la majorité des tumeurs solides, et la résistance des cellules néoplasiques à ce composé cytotoxique pose un problème majeur en oncologie clinique. Ici, nous avons exploré les vulnérabilités métaboliques potentielles de lignées cellulaires du cancer du poumon non à petites cellules résistantes au cisplatine. Il s’est avéré que les clones résistants au cisplatine (Cis-R) étaient plus sensibles à la mort induite par la privation nutritionnelle in vitro et in vivo en comparaison à leurs contrôles parentaux sensibles au cisplatine (Cis-S). La susceptibilité des cellules Cis-R à la privation nutritionnelle pourrait s'expliquer par une dépendance particulièrement forte vis-à-vis de la glutamine. La déplétion en glutamine était suffisante pour restaurer la sensibilité au cisplatine des clones initialement résistants, et la supplémentation en glutamine a permis le sauvetage des clones Cis-R de la mort induite par la privation nutritionnelle. Les analyses du métabolome par spectrométrie de masse et les interventions spécifiques sur le métabolisme de la glutamine ont révélé que, dans les cellules Cis-R, la glutamine est surtout nécessaire pour la biosynthèse des nucléotides plutôt que pour les réactions anaplérotiques, bioénergétiques ou redox. En conséquence, les cancers Cis-R sont devenus extrêmement sensibles au traitement par des antimétabolites ciblant le métabolisme des nucléosides. / Cisplatin is the most widely used chemotherapeutic agent, and resistance of neoplastic cells against this cytoxicant pose a major problem in clinical oncology. Here, we explored potential metabolic vulnerabilities of cisplatin-resistant non-small cell lung cancer and ovarian cancer cell lines. Cisplatin resistant clones were more sensitive to killing by nutrient deprivation in vitro and in vivo than their parental cisplatin-sensitive controls. The susceptibility of cisplatin-resistant cells to starvation could be explained by a particularly strong dependence on glutamine. Glutamine depletion was sufficient to restore cisplatin responses of initially cisplatin-resistant clones, and glutamine supplementation rescued cisplatin resistant clones from starvation-induced death. Mass spectrometric metabolomics and specific interventions on glutamine metabolism revealed that, in cisplatin-resistant cells, glutamine is mostly required for nucleotide biosynthesis rather than for anaplerotic, bioenergetic or redox reactions. As a result, cisplatin-resistant cancers became exquisitely sensitive to treatment with antimetabolites that target nucleoside metabolism.

Le métabolisme des acides gras (AG) est crucial dans le maintien de l’homéostasie. Son implication dans des processus tels que la signalisation, le stockage énergétique, l’isolation thermique, la régulation du comportement ne révèle qu’une fraction de la complexité et de la variabilité des rôles dans lesquels il peut être associé. En outre, ce métabolisme est dérégulé dans de nombreuses pathologies, diabète, obésité, cancers,... C’est pourquoi les enzymes de ce métabolisme constituent des cibles attractives pour développer de nouveaux traitements. Cependant les conséquences de ces dérégulations sur l’organisme sain sont encore mal connues, surtout à l’échelle de chaque organe.L’objectif de ma thèse était d’évaluer comment le métabolisme des AGs participe à la régulation de l’homéostasie au sein d’un organisme entier. Pour cela, j’ai utilisé les possibilités génétiques du modèle drosophile dont le métabolisme est comparable à celui des mammifères. J’ai ainsi montré que la synthèse d’AGs contribue à neutraliser les effets toxiques du sucre alimentaire. Ce processus se fait en coopération avec la voie de la détoxification du méthylglyoxal qui permet de prévenir la formation de composés issus de la glycation non enzymatique. J’ai aussi contribué à montrer que les précurseurs des hydrocarbures et phéromones ont une origine flexible, qui dépend du maintien de l’homéostasie et qui peut perturber les interactions entre individus. Je suis actuellement en train d’étudier la sensibilité à l’inhibition de la synthèse d’AG de différents modèles de croissance dérégulée. Enfin, dans un travail préliminaire, j’ai montré que le métabolisme des AGs est essentiel dans le tube digestif, possiblement en perturbant l’homéostasie hydrique de la larve.L’ensemble de ces résultats aidera à mieux cerner l’importance du métabolisme des AGs dans le maintien de l’homéostasie d’un organisme sain et dans des processus dérégulés. / Fatty acid (FA) metabolism is crucial in maintaining homeostasis, but also in a numerous of processes including signaling, energy storage, protection to temperature loss, regulation of behavior... In addition, FA metabolism is deregulated in several pathologies including diabetes, obesity, and cancers... Therefore, the enzymes that catalyze the reactions of the FA metabolic pathways constitute attractive targets to develop novel therapies. However the consequences of these deregulations in healthy organism are still poorly known, in particular at the level of each organ.The aim of my PhD was to estimate how FA metabolism participates in the regulation of homeostasis within a whole body organism. To address these issues, I used the genetic possibilities of the Drosophila model, whose metabolism is similar to that of mammals.I showed that FA synthesis contributes to neutralize the toxic effects of dietary sugar. This process operates in cooperation with the methylglyoxal detoxification pathway, which prevents the formation of compounds resulting from the non-enzymatic glycation. I also contributed to a project showing that the precursors of hydrocarbons and pheromones have a flexible origin, which depends on lipid homeostasis and may affect sexual recognition between individuals. Currently, I’m studying the consequences of FA synthesis inhibition in various deregulated growth models. Finally, in a preliminary work, I showed that the FA metabolism is essential in the digestive tract, possibly by disrupting water homeostasis in larvae. Taken together, these results will help to characterize the importance of FA metabolism in healthy organism as well as in deregulated processes.

Malaria is one the world’s most devastating diseases. Several classes of drugs are used to treat malaria. Artemisinin combination therapy is the first line treatment of uncomplicated malaria. The artemisinin derivative, artemisone in conjunction with the Pheroid® drug delivery system, is the focus of this thesis. The impact of the Pheroid® on the bioavailability of artemisone was evaluated in vervet monkeys. The resulting artemisone plasma levels were much lower (Cmax of 47 and 114 ng/mL for reference and Pheroid® test formulations respectively) than expected for the dosages administered (60 mg/kg). The Pheroid® improved the pharmacokinetic profile of artemisone in a clinically significant manner. The metabolism of artemisone was assessed in vitro by using human and monkey liver and intestinal microsomes, and recombinant CYP3A4 enzymes. The Pheroid® inhibits the microsomal metabolism of artemisone. In addition, there is a species difference in artemisone metabolism between man and monkey since the in vitro intrinsic clearance of the reference formulation with monkey liver microsomes is ~8 fold higher in the monkey liver microsomes compared to the human liver microsomes and the estimated in vivo hepatic clearance for the monkey is almost twofold higher than in humans. Artemisone has potent antimalarial activity. Its in vitro efficacy was approximately twofold higher than that of either artesunate or dihydroartemisinin when evaluated against P. falciparum W2, D6, 7G8, TM90-C2B, TM91-C235 and TM93-C1088 parasite strains. The Pheroid® drug delivery system did not improve or inhibit the in vitro efficacy of artemisone or DHA. Artemisone (reference and Pheroid® test formulations) and metabolite M1 abruptly arrested the growth of P. falciparum W2 parasites and induced the formation of dormant ring stages in a manner similar to that of DHA. Interaction of artemisone with the p-glycoprotein (p-gp) efflux transporter was investigated. Artemisone stimulates ATPase activity in a concentration-dependent manner, whereas the Pheroid® inhibited this p-gp ATPase activity. P-gp ATPase activity stimulation was fourfold greater in human than cynomolgus monkey MDR1 expressed insect cell membranes. Artemisone alone and artemisone entrapped in Pheroid® vesicles showed moderate apical to basolateral and high basolateral to apical permeability (Papp) across Caco-2 cells. The Papp efflux ratio of artemisone and artemisone entrapped in Pheroid® vesicles were both >5, and decreased to ~1 when the p-gp inhibitor, verapamil, was added. Therefore, artemisone is a substrate for mammalian p-gp. The cytotoxic properties of Pheroid® on Caco-2 cells were assessed and the pro-Pheroid® seems to be non-toxic at concentrations of 1.25%. Vervet monkey plasma caused antibody-mediated growth inhibition of P. falciparum. Heat inactivated or protein A treatment proved useful in the elimination of the growth-inhibitory activity of the drug-free plasma. Plasma samples containing artemisone could not be analysed by the ex-vivo bioassay method. The dual labelling ROS assay did not prove to be useful in the evaluation of ROS production by artemisone and the Pheroid® delivery system. In conclusion, entrapment of artemisone in the Pheroid® delivery system improves the pharmacokinetic properties of artemisone, but does not improve or inhibit its antimalarial efficacy in vitro. The Pheroid® inhibited both the microsomal metabolism of artemisone and P-gp ATPase activity and was shown to be non-toxic at clinically usable concentrations. / PhD (Pharmaceutics), North-West University, Potchefstroom Campus, 2014

Patients on anti-retroviral (ARV) drug treatment are sometimes simultaneously taking other prescribed drugs and/or over-the-counter drugs and/or herbal remedies. Pharmacokinetic drug-drug or herb-drug interactions can occur in these patients, which might be synergistic or antagonistic in nature leading to increased or decreased bioavailability of the ARV. Consequences of bioavailability changes may either be adverse effects due to increased plasma levels, or lack of pharmacological responses due to decreased plasma levels. The aim of this study is to determine if pharmacokinetic interactions exist between selected commercially available herbal products, namely Linctagon Forte®, Viral Choice® and Canova® and the ARV, indinavir, in terms of transport and metabolism in cell culture models. Bi-directional transport of indinavir was evaluated across Caco-2 cell monolayers in four experimental groups, namely indinavir alone (200 μM, negative control group), indinavir in combination with Linctagon Forte®, indinavir in combination with Viral Choice® and indinavir in combination with Canova® at three different concentrations. Verapamil (100 μM), a known P-gp inhibitor, was combined with indinavir in the positive control group. Samples obtained from the transport studies were analysed by means of a validated high performance liquid chromatography (HPLC) method. The apparent permeability coefficient (Papp) values were calculated from the transport results in both directions and the efflux ratio (ER) values were calculated from these Papp values. The metabolism of indinavir was determined in LS180 cells in the same groups as mentioned for the transport study but with ketoconazole (40 μM), a known CYP3A4 inhibitor, as the positive control group. Indinavir and its predominant metabolite (M6) were analysed in the metabolism samples by means of liquid chromatography linked to mass spectroscopy (LC/MS/MS) to determine the effect of the herbal products on the biotransformation of indinavir. The BL-AP transport of indinavir increased in a concentration dependent way in the presence of Linctagon Forte® and Viral Choice® when compared to that of indinavir alone (control group). Canova® only slightly affected the efflux of indinavir compared to that of the control group. Noticeable increases in the efflux ratio values of indinavir were found for Linctagon Forte® and Viral Choice®, whilst the effect of Canova® on the efflux ratio value was negligible. There was a pronounced inhibition of the metabolism of indinavir in LS180 cells over the entire concentration range for all the herbal products investigated in this study. These in vitro pharmacokinetic interactions indicate the selected herbal products may affect indinavir’s bioavailability, but the clinical significance needs to be confirmed with in vivo studies before final conclusions can be made. / MSc (Pharmaceutics), North-West University, Potchefstroom Campus, 2015

Malaria is one the world’s most devastating diseases. Several classes of drugs are used to treat malaria. Artemisinin combination therapy is the first line treatment of uncomplicated malaria. The artemisinin derivative, artemisone in conjunction with the Pheroid® drug delivery system, is the focus of this thesis. The impact of the Pheroid® on the bioavailability of artemisone was evaluated in vervet monkeys. The resulting artemisone plasma levels were much lower (Cmax of 47 and 114 ng/mL for reference and Pheroid® test formulations respectively) than expected for the dosages administered (60 mg/kg). The Pheroid® improved the pharmacokinetic profile of artemisone in a clinically significant manner. The metabolism of artemisone was assessed in vitro by using human and monkey liver and intestinal microsomes, and recombinant CYP3A4 enzymes. The Pheroid® inhibits the microsomal metabolism of artemisone. In addition, there is a species difference in artemisone metabolism between man and monkey since the in vitro intrinsic clearance of the reference formulation with monkey liver microsomes is ~8 fold higher in the monkey liver microsomes compared to the human liver microsomes and the estimated in vivo hepatic clearance for the monkey is almost twofold higher than in humans. Artemisone has potent antimalarial activity. Its in vitro efficacy was approximately twofold higher than that of either artesunate or dihydroartemisinin when evaluated against P. falciparum W2, D6, 7G8, TM90-C2B, TM91-C235 and TM93-C1088 parasite strains. The Pheroid® drug delivery system did not improve or inhibit the in vitro efficacy of artemisone or DHA. Artemisone (reference and Pheroid® test formulations) and metabolite M1 abruptly arrested the growth of P. falciparum W2 parasites and induced the formation of dormant ring stages in a manner similar to that of DHA. Interaction of artemisone with the p-glycoprotein (p-gp) efflux transporter was investigated. Artemisone stimulates ATPase activity in a concentration-dependent manner, whereas the Pheroid® inhibited this p-gp ATPase activity. P-gp ATPase activity stimulation was fourfold greater in human than cynomolgus monkey MDR1 expressed insect cell membranes. Artemisone alone and artemisone entrapped in Pheroid® vesicles showed moderate apical to basolateral and high basolateral to apical permeability (Papp) across Caco-2 cells. The Papp efflux ratio of artemisone and artemisone entrapped in Pheroid® vesicles were both >5, and decreased to ~1 when the p-gp inhibitor, verapamil, was added. Therefore, artemisone is a substrate for mammalian p-gp. The cytotoxic properties of Pheroid® on Caco-2 cells were assessed and the pro-Pheroid® seems to be non-toxic at concentrations of 1.25%. Vervet monkey plasma caused antibody-mediated growth inhibition of P. falciparum. Heat inactivated or protein A treatment proved useful in the elimination of the growth-inhibitory activity of the drug-free plasma. Plasma samples containing artemisone could not be analysed by the ex-vivo bioassay method. The dual labelling ROS assay did not prove to be useful in the evaluation of ROS production by artemisone and the Pheroid® delivery system. In conclusion, entrapment of artemisone in the Pheroid® delivery system improves the pharmacokinetic properties of artemisone, but does not improve or inhibit its antimalarial efficacy in vitro. The Pheroid® inhibited both the microsomal metabolism of artemisone and P-gp ATPase activity and was shown to be non-toxic at clinically usable concentrations. / PhD (Pharmaceutics), North-West University, Potchefstroom Campus, 2014

Patients on anti-retroviral (ARV) drug treatment are sometimes simultaneously taking other prescribed drugs and/or over-the-counter drugs and/or herbal remedies. Pharmacokinetic drug-drug or herb-drug interactions can occur in these patients, which might be synergistic or antagonistic in nature leading to increased or decreased bioavailability of the ARV. Consequences of bioavailability changes may either be adverse effects due to increased plasma levels, or lack of pharmacological responses due to decreased plasma levels. The aim of this study is to determine if pharmacokinetic interactions exist between selected commercially available herbal products, namely Linctagon Forte®, Viral Choice® and Canova® and the ARV, indinavir, in terms of transport and metabolism in cell culture models. Bi-directional transport of indinavir was evaluated across Caco-2 cell monolayers in four experimental groups, namely indinavir alone (200 μM, negative control group), indinavir in combination with Linctagon Forte®, indinavir in combination with Viral Choice® and indinavir in combination with Canova® at three different concentrations. Verapamil (100 μM), a known P-gp inhibitor, was combined with indinavir in the positive control group. Samples obtained from the transport studies were analysed by means of a validated high performance liquid chromatography (HPLC) method. The apparent permeability coefficient (Papp) values were calculated from the transport results in both directions and the efflux ratio (ER) values were calculated from these Papp values. The metabolism of indinavir was determined in LS180 cells in the same groups as mentioned for the transport study but with ketoconazole (40 μM), a known CYP3A4 inhibitor, as the positive control group. Indinavir and its predominant metabolite (M6) were analysed in the metabolism samples by means of liquid chromatography linked to mass spectroscopy (LC/MS/MS) to determine the effect of the herbal products on the biotransformation of indinavir. The BL-AP transport of indinavir increased in a concentration dependent way in the presence of Linctagon Forte® and Viral Choice® when compared to that of indinavir alone (control group). Canova® only slightly affected the efflux of indinavir compared to that of the control group. Noticeable increases in the efflux ratio values of indinavir were found for Linctagon Forte® and Viral Choice®, whilst the effect of Canova® on the efflux ratio value was negligible. There was a pronounced inhibition of the metabolism of indinavir in LS180 cells over the entire concentration range for all the herbal products investigated in this study. These in vitro pharmacokinetic interactions indicate the selected herbal products may affect indinavir’s bioavailability, but the clinical significance needs to be confirmed with in vivo studies before final conclusions can be made. / MSc (Pharmaceutics), North-West University, Potchefstroom Campus, 2015