The use of nanocarriers is an intriguing approach in the development of efficacious treatment for brain disorders. The aim of the conducted research was to evaluate and quantify the impact of a liposomal nanocarrier formulation on the brain drug delivery. A novel approach for investigating the blood-brain barrier transport of liposomal DAMGO is presented, including in vivo microdialysis in rat, a high quality LC-MS/MS bioanalytical method and pharmacokinetic model analysis of the data. Factors limiting the brain distribution of the free peptide DAMGO were also investigated. Microdialysis, in combination with plasma sampling, made it possible to separate the released drug from the encapsulated and to quantify the active substance in both blood and brain interstitial fluid over time. The opioid peptide DAMGO entered the brain to a limited extent, with a clearance out of the brain 13 times higher than the clearance into the brain. The brain to blood ratio of unbound drug was not affected when the efflux transporter inhibitors cyclosporine A and elacridar were co-administered with DAMGO. Nor was the transport affected in the in vitro Caco-2 assay using the same inhibitors. This indicates that DAMGO is not transported by P-glycoprotein (Pgp) or breast cancer resistant protein (Bcrp). The blood-brain barrier transport was significantly increased for DAMGO when formulated in liposomes, resulting in 2-3 fold higher brain to blood ratio of unbound DAMGO. The increased brain delivery was seen both for glutathione tagged PEGylated liposomes, as well as for PEGyalted liposomes without specific brain targeting. The improvement in brain delivery was observed only when DAMGO was encapsulated into the liposomes, thus excluding any effect of the liposomes themselves on the integrity of the blood-brain barrier. Modeling of the data provided additional mechanistic understanding of the brain uptake, showing that endocytosis or transcytosis of intact liposomes across the endothelial cell membranes were unlikely. A model describing fusion of the liposomes with the luminal membrane described the experimental data the best. In conclusion, the studies presented in this thesis all contribute to an increased understanding of how to evaluate and improve brain delivery of CNS active drugs and contribute with important insights to the nanocarrier field.

Orientador: Sofia Fernanda Gonçalves Zorzella Pezavento / Resumo: A esclerose múltipla (EM) é uma doença inflamatória crônica e desmielinizante do Sistema Nervoso Central (SNC) que desencadeia diferentes graus de incapacidade física e cognitiva. Os tratamentos para a EM são baseados principalmente em drogas imunomoduladoras visando redução da gravidade e da frequência de recidivas, uma vez que não há cura para a doença. Estudos em modelo experimental de EM, denominado encefalomielite autoimune experimental (EAE) têm demonstrado o efeito imunomodulador da vitamina D (Vit D3) tanto na imunidade inata quanto na adaptativa. Neste contexto, nosso objetivo foi avaliar se a intervenção precoce com Vit D3 é capaz de bloquear a neuroinflamação em um modelo experimental de EM. Para isto, camundongos C57BL/6 fêmeas foram imunizados com MOG (glicoproteína da mielina do oligodendrócito) associada ao Adjuvante Completo de Freund e tratadas com Vit D3 por via intraperitoneal. A Vit D3 diminuiu, de forma significativa, a incidência e o escore clínico da doença. Este efeito protetor foi acompanhado da diminuição de entrada de linfócitos no SNC e também de redução no processo de desmielinização e expressão de MHCII em macrófagos e micróglia. A eficácia da Vit D3 também foi associada com controle local do estresse oxidativo, ou seja, nos animais tratados ocorreu normalização dos níveis de peroxidação lipídica e de proteína carbonil e também de enzimas antioxidantes (superóxido dismutase, catalase e glutationa peroxidase). A terapia com Vit D3 também determino... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Multiple sclerosis (MS) is a chronic and demyelinating inflammatory disease of the Central Nervous System (CNS) that triggers different degrees of physical and cognitive disability. Treatments for MS are mainly based on immunomodulatory drugs that reduce severity and frequency of relapses. Studies in an experimental model of MS, called experimental autoimmune encephalomyelitis (EAE), have demonstrated the immunomodulatory effect of vitamin D (Vit D3) in both innate and adaptive immunity. In this context, our objective was to evaluate whether early intervention with Vit D3 was able to block neuroinflammation in EAE. Encephalomyelitis was induced in female C57BL / 6 mice by immunization with MOG (myelin oligodendrocyte glycoprotein) associated with Freund's Complete Adjuvant and two doses of pertussis toxin. Mice were treated with Vit D3 intraperitoneally. Treatment significantly decreased the incidence and clinical score of the disease. This protective effect was associate to decreased lymphocyte infiltration in the CNS and also reduced demyelination and MHCII expression in macrophages and microglia. Vit D3 also determined local control of oxidative stress, that is, normalization of the levels of lipid peroxidation and carbonyl protein and also of antioxidant enzymes (superoxide dismutase, catalase and glutathione peroxidase). Vit D3 therapy reduced NLRP3, caspase-1 and local IL-1β mRNA expression in the CNS. In addition, this precocious Vit D3 therapy normalized the blood-bra... (Complete abstract click electronic access below) / Doutor

A hipertensão cursa com importante déficit autonômico e lesão da barreira hematoencefálica (BHE) enquanto que o treinamento aeróbio (T) de hipertensos reduz acentuadamente a lesão da BHE, mantendo sua integridade no PVN, NTS e RVLM mesmo na persistência de níveis pressóricos elevados. Esta rápida resposta ao T (2 semanas) é condicionada pela redução da disponibilidade de ANGII nas áreas encefálicas, simultâneo aumento da expressão de podócitos dos astrócitos e desativação da microglia, os quais ocorrem simultaneamente à redução do simpático vasomotor (2 semanas) e antes mesmo do aumento da variabilidade da frequência cardíaca, da atividade parassimpática ao coração, da instalação da bradicardia de repouso e queda parcial da pressão arterial, que se instalam a partir da 4ª semana de T. Alterações na permeabilidade da BHE de hipertensos (lesão com prejuízo estrutural/funcional) e treinados (manutenção da integridade estrutural/funcional) são importantes fatores a condicionar respectivamente a disfunção autonômica na hipertensão ou a sua correção pelo treinamento. / The arterial hypertension is accompanied by important autonomic dysfunction and blood-brain barrier (BBB) lesion while aerobic training (T) in hypertension strongly decreases the BBB lesion, maintaining its integrity on the PVN, NTS and RVLM even in the persistence of high blood pressure (BP) levels. This early response to T (2 weeks) is conditioned by the reduction of ANGII availability, increased expression of astrocytic podocytes and deactivation of the microglia in brain areas. These responses occurred simultaneously with the reduction of vasomotor sympathetic activity (2 weeks) and before the increase of both heart rate variability and parasympathetic activity, resting bradycardia and partial BP fall, appearing only at the 4th week. Changes on the BBB permeability in hypertension (lesion with structural/functional damage) and trained (maintenance of the structural/ functional integrity) are important factors to condition the autonomic dysfunction in hypertension or its correction by the training, respectively.

Pour exercer leur effet princeps, les médicaments doivent atteindre des concentrations nécessaires et suffisantes à leur site d'action tout en évitant la survenue d'effets indésirables. Les antibiotiques sont utilisés pour le traitement d'infection cérébroméningées dont la cible bactériologique se situe dans le liquide céphalorachidien (LCR) ou le liquide extracellulaire cérébral (LEC) un site d'action cérébral qui constitue aussi une cible pour des effets secondaires. La distribution de médicament dans le cerveau et le LCR est limitée par la présence des barrières hémato-encéphalique (BHE) et hématoliquidienne (BHL). De plus, des mécanismes d'efflux diminuent les concentrations tissulaires des médicaments. Pour optimiser l'usage des antibiotiques et diminuer les effets indésirables, il est important d'obtenir des informations pharmacocinétiques tissulaires. Le recueil de LEC par microdialyse cérébrale et le prélèvement de LCR permettent chez des patients de réanimation la comparaison des concentrations libres de médicament. Ce travail constitue une étude de la distribution dans le plasma, le LCR et le LEC de deux antibiotiques : le céfotaxime et le métronidazole. Des patients (après un traumatisme crânien ou un accident vasculaire) ont été traités par céfotaxime et métronidazole pour une pneumopathie. Quatre études pharmacocinétiques ont été réalisées à l'équilibre par microdialyse cérébrale (n=11) pour le recueil du LEC ou par prélèvement de LCR (n=9) par une dérivation ventriculaire externe. Les résultats ont montré que le métronidazole diffusait totalement dans les deux milieux alors que la diffusion du céfotaxime était limitée, probablement en raison de phénomènes d'efflux. / To exert their effect while avoiding adverse events, drugs must reach sufficient concentrations in their site of action. Antibiotics are used for treating infection that bacterial target is in the cerebrospinal fluid (CSF) or brain extracellular fluid (ECF) which is also a target for adverse events. Drug distribution in the brain and CSF may be limited by the presence of the blood-brain barrier (BBB) and blood-CSF barrier (BCSFB). In addition, efflux mechanisms may decrease tissue concentrations of drugs.To optimize the use of antibiotics and reduce adverse events, it is important to obtain tissue pharmacokinetics. Collecting ECF samples via brain microdialysis and CSF samples via external ventricular drain in critically ill patients allow comparison of free drug concentrations. This work is a study of the distribution in plasma, CSF and ECF of two antibiotics, cefotaxime and metronidazole.Patients (after a head injury or stroke) were treated with cefotaxime and metronidazole for pneumonia. Four pharmacokinetic studies were performed at equilibrium by brain microdialysis (n = 11) for collecting ECF or CSF samples (n = 9) by an external ventricular drain. The results showed that metronidazole distributes extensively in both ECF and CSF while the diffusion of cefotaxime was limited, probably due to efflux transporters.

abstract: Traumatic brain injury (TBI) is a leading cause of disability worldwide with 1.7 million TBIs reported annually in the United States. Broadly, TBI can be classified into focal injury, associated with cerebral contusion, and diffuse injury, a widespread injury pathology. TBI results in a host of pathological alterations and may lead to a transient blood-brain-barrier (BBB) breakdown. Although the BBB dysfunction after TBI may provide a window for therapeutic delivery, the current drug delivery approaches remains largely inefficient due to rapid clearance, inactivation and degradation. One potential strategy to address the current therapeutic limitations is to employ nanoparticle (NP)-based technology to archive greater efficacy and reduced clearance compared to standard drug administration. However, NP application for TBI is challenging not only due to the transient temporal resolution of the BBB breakdown, but also due to the heterogeneous (focal/diffuse) aspect of the disease itself. Furthermore, recent literature suggests sex of the animal influences neuroinflammation/outcome after TBI; yet, the influence of sex on BBB integrity following TBI and subsequent NP delivery has not been previously investigated. The overarching hypothesis for this thesis is that TBI-induced compromised BBB and leaky vasculature will enable delivery of systemically injected NPs to the injury penumbra. This study specifically explored the feasibility and the temporal accumulation of NPs in preclinical mouse models of focal and diffuse TBI. Key findings from these studies include the following. (1) After focal TBI, NPs ranging from 20-500nm exhibited peak accumulation within the injury penumbra acutely (1h) post-injury. (2) A smaller delayed peak of NP accumulation (40nm) was observed sub-acutely (3d) after focal brain injury. (3) Mild diffuse TBI simulated with a mild closed head injury model did not display any measurable NP accumulation after 1h post-injury. (4) In contrast, a moderate diffuse model (fluid percussion injury) demonstrated peak accumulation at 3h post-injury with up to 500 nm size NPs accumulating in cortical tissue. (5) Robust NP accumulation (40nm) was found in female mice compared to the males at 24h and 3d following focal brain injury. Taken together, these results demonstrate the potential for NP delivery at acute and sub-acute time points after TBI by exploiting the compromised BBB. Results also reveal a potential sex dependent component of BBB disruption leading to altered NP accumulation. The applications of this research are far-reaching ranging from theranostic delivery to personalized NP delivery for effective therapeutic outcome. / Dissertation/Thesis / Doctoral Dissertation Biomedical Engineering 2018

Reactivators of acetylcholinesterase inhibited by organophosphate inhibitors play an important role in organophosphate poisoning therapy. There are a large number of reactivators being tested for their ability of penetration through blood-brain barrier (BBB) nowadays. The main aim of this thesis is to test this ability in four well know reactivators {--} HI-6, K-027, K-074 and TO-032. The 5% lethal dose (LD50) {--} therapeutic dose - of each reactivator was i.m. (intramuscularly) applied to experimental animals (Wistar rats) into right posterior thighs. Then the animals were sacrificed and the blood (plasma) and brain were collected for HPLC analysis. Subsequently, brain and plasma were frozen at -80 °C and stored until the assay. The reactivator concentrations were measured in plasma and four parts of the brain {--} frontal cortex (FC), basal ganglia (BG), ponto-medullar part (PM) and cerebellum (Ce). There were five animals for each group used for the purpose of the experiment. The concentrations of reactivators in plasma and blood samples were measured using HPLC-UV/VIS methodology. HI-6 concentrations CFC were on the average 4.29% of Caverage in plasma, CBG were on the average 1.9% of Caverage in plasma, CPM were on the average 2.3% of Caverage in plasma, and CCe were on the average 1.85% of Caverage in plasma. These rates were counted from all concentrations within the scope of particular observed parts of the brain. All the other tested reactivators were not quantified in brain at all.

Orientador: Marcelo Lancellotti / Dissertação (mestrado) - Universidade Estadual de Campinas, Insituto de Biologia / Made available in DSpace on 2018-08-17T17:20:26Z (GMT). No. of bitstreams: 1 Pereira_RafaellaFabianaCarneiro_M.pdf: 1664553 bytes, checksum: 346b9075da4d999b30fc615fe83733fd (MD5) Previous issue date: 2011 / Resumo: Neisseria meningitdis, ou meningococo, é uma bactéria comensal da nasofaringe humana. Contudo, algumas linhagens meningocócicas ocasionalmente ultrapassam a mucosa respiratória e a barreira hematoencefálica e causam enfermidades como meningite e septicemia. Como a espécie humana é o único hospedeiro natural para esse patógeno, estudos in vitro com modelos celulares são uma ferramenta importante para a análise da interação entre o meningococo e seu hospedeiro. Este trabalho teve por objetivo avaliar a influência de diferentes linhagens de N. meningitidis na adesão celular, morfologia e expressão de quimiocinas inflamatórias em culturas in vitro de células humanas. Tais parâmetros também foram avaliados em um sistema in vitro de co-cultura celular entre células de origem nervosa e endotelial a fim de mimetizar a barrreira hematoencefálica humana. Os resultados obtidos indicam que a adesão de diferentes linhagens bacterianas em células humanas de sítios específicos do processo infeccioso do meningococo, como Hep-2 (laringe), NCIH460 (pulmão), Hec1b (endotelial) e NG97 (neuroglia) foi capaz de mimetizar o processo fisiopatológico deste microrganismo. Em condições in vitro, células de origem nervosa mostraram-se mais suscetíveis à infecção nos parâmetros avaliados como uma elevada expressão de TNF-?, quimiocina característica em infecções meningocócicas. A linhagem B4 destacou-se entre as linhagens meningocócicas estudadas, apresentando elevados percentuais de adesão em células humanas com valor máximo de adesão em NG97. Células HEp-2 apresentaram poucas alterações morfológicas significativas frente à infecção por N. meningitidis. Tais resultados podem estar associados ao fato do trato respiratório superior ser o habitat natural do meningococo, no qual a interação entre este patógeno e as células hospedeiras seja comensal e não-invasiva. O modelo mimético de barreira hematoencefálica, realizado em transwell, indicou comunicação entre as células que o compõem e uma maior expressão dos níveis de quimiocinas inflamatórias quando comparada à infecção desta bactéria por cada uma das células estudadas isoladamente. Tal modelo foi capaz de mimetizar a barreira hematoencefálica, fato o que torna possível sua aplicação em estudos da passagem de fármacos e outros patógenos por essa barreira. / Abstract: Neisseria meningitidis,or meningococci, is a commensal bacterium of the human nasopharynx. However, occasionally some meningococcal strains can cross the respiratory mucosa and the blood-brain barrier and cause life-threatening diseases such as meningitis and septicaemia. Since the human species is the only natural host for this pathogen, in vitro studies with cellular models are an important tool for the analysis of the meningococci and its host. The aim of this present work was to value the influence of several strains of N. meningitidis in cellular adhesion, morphology and inflammatory chemokines expression in human cells cultivated in vitro. These parameters were also evaluated in a co-cultivated cellular system with glial and endothelial cells aiming mimicking the human blood brain barrier. The results indicate that the adhesion of different bacterial strains from specific sites of infectious process of meningococci as Hep-2 (larynges), NCIH460 (lung), Hec1b (endothelial) and NG97 (glial cells) was capable of mimicking, partially, the pathophysiology of this microorganism. In in vitro conditions, cells from nervous origin showed more susceptibility to infection then others in this evaluated parameters such as a high TNF-? expression, a common chemokine in meningococcals diseases. The B4 strain distinguished from the others by presenting high rates of adhesion in human cells with maximum value on NG97. HEp-2 cells showed few expressives morphologic alterations after meningococcal infection. These results may be associated with the fact that the upper human respiratory tract is the meningoccci natural habitat, in which the interaction between this pathogen and host cells are commensal and not-invasive.The mimicking blood-brain barrier model, performed in transweel, has demonstrated some communication between the cells and greater expression of inflammatory chemokines when compared to the infection in isolated cells. This model was capable of mimicing the blood-brain barrier, which makes its application possible in studies of drugs and others pathogens who might crossover though this barrier. / Mestrado / Bioquimica / Mestre em Biologia Funcional e Molecular

Orientador: Maria Alice da Cruz-Hofling / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-14T00:39:53Z (GMT). No. of bitstreams: 1 Carneiro_CatarinaRaposoDias_D.pdf: 35688895 bytes, checksum: dc0574f3ff57450c0d5ff8840699f5e9 (MD5) Previous issue date: 2009 / Resumo: Venenos animais são fontes de substâncias neuroativas, algumas capazes de provocar paralisia e convulsão em mamíferos, com visível ação no sistema nervoso central (SNC). O veneno da aranha Phoneutria nigriventer (PNV) é composto por neurotoxinas que causam, experimentalmente, permeabilização da barreira hematoencefálica (BHE). A BHE é uma entidade tanto física, quanto molecular, composta pelos microvasos sanguíneos cerebrais, pelos pés astrocitários e pericitos adjacentes engajados no controle do tráfego de moléculas na interface sangue-cérebro. A BHE, embora imprescindível à manutenção da homeostase no SNC, pode representar um obstáculo ao acesso de drogas terapêuticas ao microambiente neural. Nossa proposta foi investigar a ação sistêmica do PNV após 15 min, 2 e 5 h da injeção i.v. em ratos Wistar adultos através de: (1) alterações na expressão das proteínas juncionais, de efluxo e transportador de glicose da BHE; (2) alterações na expressão da proteína conexina-43 (constituinte das junções comunicantes) e da proteína fosfatase pPP2A, uma vez que a fosforilação de resíduos de tirosina das proteínas juncionais tem papel no controle da integridade paracelular; (3) ativação de vias neuronais e sua modulação pelo óxido nítrico (NO). (4) Reação inflamatória e gliose reativa de astrócitos in vivo e in vitro e sua possível modulação pelo NO, (5) Purificação e identificação de toxinas do PNV com ação na BHE. A expressão das proteínas juncionais encontrava-se diminuída aos 15 min e 2 h, porém às 5 h pós-PNV a expressão das proteínas investigadas estava total ou parcialmente recuperada, sugerindo ser esse um dos mecanismos de abertura da BHE. Igualmente, a expressão da proteína de efluxo aumentou indicando mecanismo de clearance do agente tóxico. A expressão da conexina-43, e da pPP2A estavam aumentadas aos 15 min e diminuída às 5 h da injeção do PNV, mostrando não só que as comunicações célula-célula e o mecanismo de adesão célulacélula foram afetados, mas também que as alterações podem ser transitórias. Ademais, vias neuronais foram ativadas em áreas motoras e em núcleos do hipotálamo o que explicaria o comprometimento motor (convulsão, paralisia) e os sinais neurovegetativos (sialorréia, hipertensão, estresse respiratório, edema pulmonar, anúria) vistos em animais envenenados. Muitas dessas vias apontam modulação nitrérgica dos sinais tóxicos do envenenamento, uma vez que a inibição da síntese de NO pelo 7- nitroindazol (7-NI) diminuiu a ativação neuronal em algumas áreas e exacerbou em outras. Os astrócitos incubados com PNV, corroborando com estudos in vivo, expressaram citocinas pró-inflamatórias e apresentaram gliose reativa, porém a inibição da síntese do NO atenuou esses efeitos, confirmando que o NO tem um importante papel nos efeitos do PNV. Toxinas F8a-1 e F10a-1, purificadas do PNV, foram identificadas como responsáveis pela permeabilização da BHE, embora não esteja excluída a contribuição de outros componentes. O entendimento da ação do PNV e de suas toxinas no tecido neural e na BHE pode contribuir para o desenvolvimento de ferramentas úteis para uso clínico e em pesquisa. / Abstract: Animal venoms are source of neuroactive substances, some of them able to provoke paralysis and convulsion in mammals, indicating action on the central nervous system (CNS). The Phoneutria nigriventer spider venom (PNV) is composed of neurotoxins that cause, experimentally, blood-brain barrier (BBB) permeabilization. The BBB is both a physical and molecular entity, constituted by the cerebral microvessels and surrounding astrocytic end-feet and pericytes, all involved in the control of the traffic of molecules at the blood-brain interface. Even so the BBB presence is essential for the maintenance of CNS homeostasis; it also represents an obstacle for the therapeutical drugs access into the neural microenvironment. Our proposal was to investigate acute changes (15 min, 2 and 5 h) after PNV i.v. injection in adult Wistar rats through evaluation of the: (1) alterations in the expression of the BBB-junctional proteins, -efflux proteins, and -glucose transporter; (2) alterations in the expression of connexin-43 protein (gap junctions constituent) and protein phosphatase 2A (pPP2A, since phosphorylation of tyrosine residues from junctional proteins plays a role in controlling junctional integrity); (3) activation of neuronal pathways and its modulation by the nitric oxide (NO). (4) In vivo and in vitro astrocytes inflammatory reaction and reactive gliosis after incubation with PNV and its possible modulation by NO. (5) Purification and determination of BBB-acting from toxins PNV. The expression of the junctional proteins was diminished at 15 min and 2 h post-PNV exposure; however at 5 h the expression of most of the proteins investigated was total or partially recovered, suggesting that this might be one of the BBB opening mechanisms. Similarly, the venom increased the efflux protein expression, indicating ongoing clearance of toxic agents from the neural tissue. The expression of both connexin-43 and pPP2A increased after 15 min and diminished after 5 h of PNV injection, showing not only that cell-cell communication and cell-cell adhesion mechanism were affected, but that these alterations were transitory. Activated neuronal pathways have been observed in brain motor areas and hypothalamic nucleus, explaining the motor impairment (convulsion, paralysis) and neurovegetative signs (salivation, hypertension, respiratory stress, pulmonary edema, anuria) observed in the envenomed animals. Many of these neuronal pathways point to a nitrergic modulation of the envenomed toxic signs, since that NO synthesis inhibition by 7-nitroindazol (7-NI) decreased the neuronal activation in some areas and enhanced in others. The astrocytes incubated with PNV, in agreement with in vivo studies, expressed pro-inflammatory cytokines and presented reactive gliosis, however the pretreatment with 7- NI attenuated these effects, confirming that NO have an important role in the PNV effects. The F8a-1 and F10a-1 toxins, fractionated from the crude PNV, were identified as responsible by BBB permeabilization; despite, other venom components contribution can not be discarded. The understanding of the action of the venom of Phoneutria nigriventer and its toxins in the neural tissue and BBB can contribute for the development of useful tools for clinical and research purposes. / Doutorado / Biologia Celular / Doutor em Biologia Celular e Estrutural

La présence de la glycoprotéine P (P-gp) dans la barrière hémato-encéphalique (BHE) conduit à l’échec de nombreuses thérapies ciblant le système nerveux central (SNC). Cependant la P-gp protège aussi le cerveau contre des composés nocifs, essentiellement lipophiles, endogènes et exogènes susceptibles de passer la BHE par diffusion simple. Par conséquent, toute inhibition de la P-gp qui vise à améliorer la distribution des agents pharmacologiques dans le cerveau doit prendre en compte la neurotoxicité potentielle de cette inhibition. Les premiers travaux ont montré que l’elacridar et le tariquidar, deux modulateurs de la P-gp de troisième génération, augmentaient la distribution dans le cerveau de plusieurs de ses substrats. Malheureusement, d’autres études plus récentes, suggèrent l’utilisation de doses élevées de l’elacridar et du tariquidar pour moduler efficacement l’activité de la P-gp dans la BHE. Néanmoins, ces doses élevées en co-administration avec des substrats de la P-gp peuvent être associées à des interactions pharmacocinétiques et à des profils toxiques, limitant ainsi l'utilisation de ces inhibiteurs.Dans ce contexte, l’objectif principal de cette thèse est d’obtenir une modulation transitoire mais efficace de la P-gp dans la BHE par administration intraveineuse de doses faibles mais thérapeutiques de l’elacridar et du tariquidar sous leur forme libre ou co-encapsulé dans les liposomes. Le lopéramide, substrat de la P-gp, a été également administré sous sa forme libre comme une preuve in vivo d’une inhibition efficace de la P-gp dans la BHE.L'administration simultanée de ces deux modulateurs de la P-gp n’a pas modifié leurs concentrations plasmatiques ou celles du lopéramide, mais a entraîné une importante distribution du lopéramide dans le cerveau en raison de leur activité inhibitrice non- compétitive. De plus, la co-encapsulation de l’elacridar et du tariquidar dans des immunoliposomes stabilisées stériquement a amélioré la demi-vie et la distribution dans le cerveau des ceux deux composés. Par conséquent, la distribution dans le cerveau du lopéramide a été considérablement augmentée, sans aucune modification de sa pharmacocinétique ou distribution tissulaire. Par ailleurs, la diminution partielle de l'activité inhibitrice du tariquidar par des liposomes vides suggère l’utilisation de ce nanovecteur comme une approche de bio-détoxification pour le traitement des surdoses de tariquidar. En résumé, cette thèse propose différentes approches pour exploiter pleinement l’elacridar et le tariquidar. Les résultats décrits dans ce manuscrit devraient ouvrir des pistes intéressantes pour atteindre une inhibition efficace de la P-gp dans la BHE et pour réussir des thérapies ciblant le système nerveux central / Although the P-glycoprotein (P-gp) represents an obstacle in several central nervous system (CNS) pharmacotherapies, the P-gp also protects the brain from intoxication by endogenous and exogenous harmful lipophilic compounds that otherwise could penetrate the blood-brain barrier (BBB) by simple diffusion. Therefore, any modulation of the efflux transporter has to consider the potential neurotoxicity of such modulation. Early studies showed that elacridar and tariquidar, two third-generation P-gp modulators, increase the distribution of several P-gp substrates in the brain. Unfortunately, recent studies suggest the use of high doses of elacridar and tariquidar to efficiently modulate the P-gp at the BBB. Nevertheless, when co-administered with P-gp substrates, these high doses may be associated with pharmacokinetic interactions and toxic profiles, thus limiting the use of these compounds.Hence, this thesis aimed to attain a transient but efficient modulation of the P-gp at the BBB using elacridar and tariquidar but avoiding the use of large doses of these compounds. For this purpose we took advantage of the possible in vivo intravenous co-administration of low but therapeutic doses of elacridar and tariquidar, under their free form or co-encapsulated in liposomes. The brain distribution of free loperamide was determined as an in vivo probe of full inhibition of the P-gp activity at the BBB.The concurrent administration of both free P-gp modulators does not modify their plasma concentrations or those of the P-gp substrate but significantly increased the brain uptake of loperamide as a result of their non-competitive modulatory activity. Moreover, the co-encapsulation of elacridar and tariquidar in targeted sterically stabilized immunoliposomes improved the half-lives and brain distribution of both compounds. Consequently, the brain uptake of free loperamide was significantly enhanced without any modification of its pharmacokinetics or tissue distribution. Moreover, the partial impairment of the modulatory activity of tariquidar by empty liposomes, supports the use of this nanocarrier as a bio-detoxifying approach for the treatment of tariquidar overdoses.In summary, this thesis proposes different approaches for full exploitation of elacridar and tariquidar. The findings described in this manuscript should open interesting avenues to achieve an efficient overcoming of the P-gp at the BBB and succeed CNS pharmacotherapies.

Aim: The general aim of this research is development and evaluation of novel methods for predicting active transport over the human blood-brain-barrier (BBB), while this project specifically aims to i) review the literature and select suitable methods and substrates, ii) develop models for determining in vitro kinetic properties of selected compounds, analyze the in vitro data using the developed models and to use Maximum Transport Activity (MTA) approach (Karlgren et al., 2012), iii) perform Physiology Based Pharmacokinetic (PBPK) modelling and compare to in vivo literature data. Background: Drug permeation to the brain through blood circulation is primarily limited by blood-brain barrier (BBB), due to existence of tight junctions in endothelial cells of blood vessels as well as active efflux and influx transporters in the barrier. Toxicity and CNS related side effects can be caused by peripheral targeted drugs crossing BBB. Hence, prediction of BBB permeability and estimation of drug concentration in the brain tissue are challenging in drug discovery. To resolve this, estimating the human BBB permeability using improved in vitro and in silico predictive models can be a facilitator. Methods: In vitro data provided by the Drug Delivery research group was used to develop in vitro predictive models for BBB penetration of Verapamil, Risperidone, and Prazosin using R-studio 1.2.5. The MTA approach was adjusted for extrapolation of BBB in vitro transporter activity to in vivo condition. For PBPK modelling, we took advantage of PK-Sim® to simulate drug disposition and time profile of Risperidone in human and animal species. Results: It was shown that MDR1 is the major transporter for efflux transport of Prazosin and Risperidone in brain while both BCRP and MDR1 have similar impact on transport of Verapamil. Furthermore, it was presented in PBPK models that the predicted brain concentration of Risperidone increases in rat and nonhuman primate (NHP) when MDR1 And BCRP are knocked out while the brain concentration of Risperidone in dog is not affected by expression level of the efflux transporters. Conclusion: Both MDR1 and BCRP are contributing in efflux transport of Verapamil, Risperidone, and Prazosin across the BBB. Additionally, expression of the efflux transporters shown to have an impact on brain exposure of Risperidone in animal PBPK models.