Ce travail porte sur la mise au point des nanocochléates intégrant à la fois l’amphotéricine B (AmB) et la miltéfosine (hexadécylphosphocholine, HePC) dotés de propriétés adéquates pour une administration par voie orale pour le traitement de la leishmaniose viscérale. Notre premier axe de recherche a été l’étude des interactions de l’AmB et de l’HePC avec les monocouches de lipides de DOPS (dioleoyl phosphatidylsérine) et de Cho (cholestérol) dans le développement de nanocochléates chargés avec ces deux principes actifs. Les résultats expérimentaux nous ont permis de déterminer le rapport drogue/lipides optimal pour la formulation des nanocochléates : 9DOPS/1Cho/0,5AmB/0,5HePC et de confirmer que dans la formulation de nanocochléates, l’AmB et l’HePC sont incorporés de façon stable et se situent au sein des bicouches lipidiques entre des lipides, plutôt que dans la phase aqueuse entre les bicouches.Notre second axe de recherche a porté sur la formulation et la caractérisation des nanocochléates chargés en AmB et en HePC. Ces nanocochléates ont été formulés à partir des liposomes unilamellaires encapsulant ces deux principes actifs. Les paramètres expérimentaux majeurs (taille de liposomes, proportion DOPS/1Cho) ont été optimisés. Les propriétés physico-chimiques des nanocochléates, telles que la taille, la charge de surface, la morphologie, le rendement d'encapsulation, l’organisation des principes actifs et la stabilité pendant le stockage au cours de temps ont été étudiées. En vue d'une formulation orale, la libération des principes actifs in vitro ainsi que la stabilité des nanocochléates in vitro dans les milieux gastro-intestinaux ont également été étudiés d’après la Pharmacopée des Etats-Unis. Enfin, une étude in vivo préliminaire chez le rat portant sur la pharmacocinétique plasmatique d'AmB après administration orale de nanocochléates chargés en AmB avec ou sans HePC en comparaison avec l’Ambisome® et la Fungizone® a été entamée. Les résultats préliminaires ont démontré une absorption significative par voie orale de l’AmB.Ce travail a permis de développer des nanocochléates avec des propriétés appropriées pour une formulation contenant à la fois l’AmB et l’HePC administrable par voie orale. Néanmoins, des optimisations restent à réaliser avant d’envisager une application clinique. / The aim of this work was to formulate nanocochleates containing both Amphotericin B (AmB) and miltefosine (hexadecylphosphocholine, HePC) with properties suitable for administration by the oral route for the treatment of visceral leishmaniasis. The first part of this work was a fundamental study of the interactions between AmB and HePC and monolayers of dioleylphosphatidylserine (DOPS) with or without cholesterol (Cho), to provide a basis for the formulation of nanocochleates containing the two active molecules. The results allowed us to define the optimal proportions for the formulation of nanocochleates: 9DOPS/1Cho/0.5AmB/0.5HePC and to predict that AmB and HePC would be incorporated stably into the formulation and would be located between the lipids in the bilayers rather than in the aqueous phase between the bilayers. The second part of the work was the formulation and characterization of nanococheates containing both AmB and HePC. These were derived from unilamellar liposomes containing the two active molecules. The crucial experimental parameters (size of the liposomes, ratio of DOPS to Cho) were optimized. The physico-chemical properties of the nanocochleates, such as the size, surface charge, morphology, encapsulation yield, the organization of the active molecules and the stability during long-term storage were studied. Since the formulation was destined for the oral route, in-vitro drug release and the stability of the nanocochleates in simulated gastro-intestinal media were studied according to the recommendations of the US Pharmacopeia. Finally, a preliminary in-vivo study of the plasma pharmacokinetics of AmB after oral gavage to rats of nanocochleates contained AmB with or without HePC, in comparison with AmBisome and Fungizone, was carried out. A significant oral absorption of AmB was observed. This work has led to the formulation of nanocochleates containing AmB and HePC with appropriate properties for oral administration. However, further optimization is necessary before such particles will be suitable for clinical use.

Les kinétoplastidés sont des protozoaires flagellés responsables de maladies tropicales négligées mortelles telles que la leishmaniose viscérale (L. donovani et L. infantum) ou la trypanosomiase humaine africaine (T. brucei), pour lesquelles les traitements disponibles sont très limités. Depuis quelques années, on observe un regain d'intérêt pour le développement de nitrohétérocycles aromatiques anti-infectieux tels que le delamanide et le féxinidazole. De récentes études indiquent que l'activité anti-kinétoplastidés de ces dérivés repose sur leur bioactivation sélective par des nitroréductases parasitaires, conduisant à la formation de métabolites réduits électrophiles, fortement cytotoxiques. Suite à des études préliminaires réalisées dans notre équipe en série 8-nitroquinoléin-2(1H)-one, ces travaux de thèse portent sur la synthèse et l'étude in vitro de l'activité antiparasitaire de 80 dérivés notamment fonctionnalisés en positions 3 et 6 du pharmacophore par divers motifs, notamment via la mise au point de réactions d'halogénation sélective et de couplages pallado-catalysés. Ainsi, 5 nouvelles molécules hits (4 anti-kinétoplastidés et 1 sélective de T. brucei) ont été identifiées (0,01 µM ≤ CI50 ≤ 7 µM et 13 < IS < 1500), trois d'entre-elles étant des substrats sélectifs des nitroréductases parasitaires de type I. Afin de préciser les relations structure-activité, une étude des potentiels de réduction a également été menée. Des études physico-chimiques (solubilité, test de perméabilité PAMPA) et pharmacocinétiques in vitro (stabilité microsomale et fixation à l'albumine humaine) sont venues compléter ce travail. Enfin, des évaluations de la mutagénicité et de la génotoxicité de ces hits sur des cellules procaryotes et humaines ont été conduites, dans le but de statuer sur leur potentiel pharmaceutique antiparasitaire humain et vétérinaire. / Kinetoplastids are flagellated protozoan parasites responsible for lethal neglected tropical diseases, such as visceral leishmaniasis (L. donovani and L. infantum) or sleeping sickness (T. brucei brucei), for which very few drugs are available. Nowadays, nitroheterocyclic compounds present a renewed interest as anti-infective agents, as illustrated by the development of fexinidazole and delamanid. Some recent studies demonstrated that the antikinetoplastid activity of these derivatives involves their selective bioactivation by parasitic nitroreductases, leading to the formation of electrophilic reduced metabolites, highly cytotoxic. Based on preliminary studies conducted in our team in 8-nitroquinolin-2(1H)-one series, this PhD work is about the synthesis and in vitro antiparasitic study of 80 derivatives mainly functionalized at positions 3 and 6 of the pharmacophore by various substituents, especially via the optimization of selective halogenation and pallado-catalyzed cross coupling reactions. Thereby, 5 new hit compounds (4 antikinetoplastid and 1 selective of T. brucei) were identified (0.01 µM ≤ IC50 ≤ 7 µM and 13 < SI < 1500), three of them being selective substrates of type I parasitic nitroreductases. In order to refine the structure-activity relationship studies, an analysis of reduction potentials was also conducted. In vitro physicochemical (solubility, PAMPA permeability assay) and pharmacokinetic (microsomal stability and human albumin binding) experiments completed this work. Finally, the mutagenicity and genotoxicity evaluations of these new hit compounds toward prokaryotic and human cells were realized, in order to assess their human and veterinary antiparasitic pharmaceutical potential.

Visceral leishmaniasis (VL) or kala-azar is a parasitic infectious disease that is fatal if left untreated. Two types of Leishmania species are causal agents of VL: Leishmania infantum and Leishmania donovani. VL caused by L.infantum is a zoonosis and is endemic in countries around the Mediterranean basin and in Latin-America. VL caused by L. donovani is assumed to be an anthroponosis and is endemic in East-Africa and the Indian subcontinent.<p><p>VL is considered as a major public health problem in the Indian subcontinent and the annual case load of VL in this focus is represents around 80% of the global burden. In Nepal, a quarter of the country’s population is estimated to be at risk of this disease. The disease in the ISC is caused by L. donovani, which is transmitted from man to man by the bite of the sandfly Phlebotomus argentipes. VL occurs predominantly among the poorest of the poor. Since 2005, the governments of Bangladesh, India and Nepal have been engaged in a collaborative effort to eliminate VL from the region. The strategies to control the disease include early diagnosis and treatment, along with vector control measures, effective disease surveillance, social mobilization and partnership building, and clinical and operational research. In recent years, considerable efforts were made within the elimination initiative. Still, important gaps remain in the understanding of the VL epidemiology, and impact as well as on the best approach to case management or vector control. These knowledge gaps may affect the success of the ongoing VL elimination initiative and make it difficult to meet the set target of bringing the incidence down to less than 1 case per 10,000 by 2015. With this background we focused on some of the knowledge gaps; we wanted to generate evidence and offer sound recommendations for policy makers to underpin the ongoing VL elimination initiative in the Indian subcontinent in general and in Nepal in particular. <p><p>We have - for the first time- described the epidemiology of L. donovani infection in high transmission areas in Nepal. The sero-prevalence of L. donovani infection was 9% in these communities, but there was wide variation between endemic villages (5-15%). The seroprevalence rates remain however substantially lower than those observed in a parallel study in the neighbouring districts in Bihar, India. In our study 39% of individuals who live together in a house with at least one recent VL case were serologically (DAT) positive compared to 9% in the overall study population in the same endemic region. This pattern suggests that untreated VL cases are the main source of transmission and sharing the same household is an important risk factor for L. donovani infection. Therefore, the VL elimination campaign recently initiated an active case detection strategy including the search of active cases of VL and post-kala-azar dermal leishmaniasis (PKDL).<p><p>Generally the risk factors for VL are linked to precarious housing conditions and an environment that provides excellent breeding sites for the sandfly vector.VL has thus been largely considered as a disease of the rural poor. However, with occasional cases being reported also from town e.g. Dharan in south-eastern Nepal, questions were raised about possible extension of transmission to urban areas.<p><p>We conducted an outbreak investigation including a case-control study among the residents of Dharan town. We documented several clusters of VL cases in the more peripheral wards of the town. These are wards with new settlements where the poorest migrants install themselves. They are typically a rural-urban interface with most residents dependent on daily wages as agricultural labourers. However, several factors pointed to urban transmission: firstly, we found a strong association between VL and certain housing factors. Secondly, the clustering of VL cases in space and the intra-household clustering makes urban transmission more likely than infection due to migration. Finally, the entomological data also provide further evidence in support of local transmission of VL inside the town. The vector P. argentipes was captured repeatedly inside the town, and some of them were infested with L. donovani.<p><p>We studied the health seeking behavior and documented the households cost of VL care in a miltefosine-based programme after the intensified implementation of VL control efforts in Nepal. We enrolled 168 patients that had been treated for VL within twelve months prior to the survey in five districts in south-eastern Nepal. We observed a median delay of 25 days to present to the appropriate level of the primary healthcare system. Most patients first visited unqualified local practitioners or traditional faith healers for VL care. With a median total cost of US$ 165 per episode of VL treatment, the economic burden of VL across all households was 11% of annual household income or 57% of median annual per capita income. About half of the households exceeded the catastrophic expenditure threshold of 10% of annual household income. Our findings seem to suggest that, compared to previous studies, the economic burden of VL (as a % of household income) has indeed decreased. However, despite the provision of free diagnostics and drugs by the government, households still incurred substantial medical out-of-pocket expenditure, especially at private providers. The government should consider specific policies to reduce VL care costs such as a conditional cash programme for travel and food, and a better health insurance scheme. <p><p>We monitored clinical outcomes of VL treatment with miltefosine up to 12 months after the completion of therapy and explored the potential role of patient compliance, drug resistance, and reinfection. The initial cure rate was 95.8% and cure rate at 6 months after treatment was 82.5%, which further dropped to 73.3% at 12 months after miltefosine treatment. The relapse rate at 6 months was 10.8% and 20.0% at 12 months i.e. relapse is observed in one-fifth of miltefosine treated VL patients in Nepal. The decreased effectiveness of miltefosine observed in our study is an alarming signal for the ongoing VL elimination initiative and implicates the need for reviewing the drug policy in the Indian subcontinent. Relapse was most common among children (<12 years of age) and continued to occur beyond the commonly used 6-month follow-up period. No significant clinical risk factors or predictors of relapse apart from age <12 years were found. Parasite fingerprints of pre-treatment and relapse bone marrow isolates were similar within 8 tested patients, suggesting that clinical relapses were not due to re-infection with a new strain, but due to true recrudescences. MIL blood levels at the end of treatment were similar for cured and relapsed patients.The MIL-susceptibility of 131 VL isolates was also analysed in vitro with a promastigote assay and the mean promastigote MIL-susceptibility (IC50) of isolates from definite cures was similar to that of relapses.<p><p>We also assessed patient adherence to miltefosine treatment for VL given on an unsupervised ambulatory basis, prescribed under routine conditions (i.e. little or no time for treatment counselling) in government primary healthcare facilities. Our findings showed that adherence is a problem and the target of 90% of capsules taken is not reached in 15% of the enrolled patients. The gastrointestinal related side-effects and treatment-negligence after the resolution of clinical symptoms of VL were the main reasons for poor adherence. Effective counselling during the treatment, a short take-home message on the action and side effects of miltefosine, and on the importance of adherence are the best way to prevent poor adherence.<p><p>Post-kala-azar dermal leishmaniasis is more commonly seen in inadequately treated cases which is considered as a reservoir of infection maintaining disease transmission. The occurrence of PKDL in Nepal is relatively low compared to neighbouring countries involved in the elimination initiative. Supervised and adequate treatment of VL seems essential to reduce the risk of PKDL development. Policy makers should include surveillance and case management of PKDL in the VL elimination programme.<p> / Doctorat en Santé Publique / info:eu-repo/semantics/nonPublished

La leishmaniose viscérale est une maladie systémique mortelle en l’absence de traitement. Elle est due aux protozoaires Leishmania donovani et L. infantum, parasites des phagocytes mononucléés, capables d’envahir les organes lymphoïdes et le foie. Le contrôle de l’infection hépatique repose sur la mise en place d’une réponse granulomateuse efficace, promue par une réponse immunitaire Th1, dans un environnement tissulaire Th2. L’objectif de ce travail était l'étude du rôle d’une cytokine Th2 récemment décrite, l’IL-33, dans cette réponse hépatique complexe encore partiellement incomprise. Des dosages d’IL-33 sur des sérums de patients et la détection de cellules IL-33+ dans le foie d’un patient rennais ont placé l’IL-33 comme un biomarqueur possible de la maladie active. L’IL-33 étant exprimée dans les cellules étoilées du foie au cours d’hépatites chroniques, ces cellules ont été exposées à L. donovani. Leur permissivité aux leishmanies sans toxicité apparente ni perturbation de leurs propriétés fonctionnelles, ainsi que la persistance des leishmanies sur une culture de plusieurs semaines, nous ont conduit à proposer les cellules étoilées comme cellules sanctuaires possibles pour les leishmanies viscérotropes, contribuant donc potentiellement au portage asymptomatique. En revanche, elles ne sont pas apparues comme une source majeure d'IL-33 au cours de la leishmaniose viscérale. Chez des souris C57BL/6 et BALB/c infectées par L. donovani, l'IL-33 a été observée dans des cellules ne s'apparentant pas à des cellules étoilées, et principalement localisées dans les granulomes. Des cellules exprimant son récepteur ST2 ayant été également observées dans le foie, un rôle de l’axe IL-33/ST2 a été recherché. Les résultats obtenus chez des souris BALB/c déficientes en ST2 ou traitées par de l'IL-33 recombinante suggèrent que l'IL-33 régule négativement l'expression de cytokines Th1 (IL-12, IFN-γ) et l'infiltrat de neutrophiles et monocytes dans le foie, limitant ainsi le contrôle de la charge parasitaire. Ainsi, l'IL-33 semble être un facteur de susceptibilité pour la leishmaniose viscérale. En parallèle, des travaux entrepris sur des souris C57BL/6 infectées par L. donovani suggèrent de possibles rôles différentiels de l'IL-33 en fonction de l'environnement immunitaire inhérent au fond génétique de l'hôte. / Visceral leishmaniasis is a life-threatening systemic disease caused by Leishmania protozoans, L. donovani and L. infantum, which invade mononuclear phagocytes in the lymphoid organs and the liver. The control of the hepatic parasite burden depends on the granuloma formation, which is favored by a Th1 immune response in a Th2 tissue microenvironment. The aim of this work was to study the role of the recently described Th2 cytokine IL-33 in this complex immune response, which remains partially misunderstood. IL-33 dosages in different patient sera and IL-33+ cells detected in the liver of a patient from Rennes suggested that IL-33 could be a biomarker for active visceral leishmaniasis. As IL-33 was described in hepatic stellate cells during chronic hepatitis, these cells were exposed to L. donovani in primary culture. The cell permissivity to L. donovani and the parasite persistence during a long term culture led us to propose hepatic stellate cells as a new type of sanctuary cells, which could partially explain asymptomatic carriage. However, these cells were apparently not the main source of IL-33 during visceral leishmaniasis. In infected BALB/c and C57BL/6 mice, IL-33 was detected in the liver in non stellate cells preferentially localized in granulomas. The presence of cells expressing its specific receptor ST2 in the liver led us to explore the role of the IL-33/ST2 axis. BALB/c mice deficient in ST2 or treated with recombinant IL-33 and infected with L. donovani revealed that IL-33 downregulates the expression of Th1 key cytokines (IL-12, IFN-γ) and the recruitment of neutrophils and monocytes. Finally, IL-33 acts as a susceptibility factor during visceral leishmaniasis. Besides, the model of L. donovani infected C57BL/6 mice deficient in IL-33 or treated with recombinant IL-33 suggests possible differential roles of IL-33 depending on the immune environment related to the host genetic background.