The development of treatments for neurological disorders such as Alzheimer’s and Parkinson’s disease begins by understanding what these diseases affect and the consequences of further manifestation. One particular region where these diseases can produce substantial problems is the blood-brain barrier (BBB). The BBB is the selective diffusion barrier between the circulating blood and the brain. The barrier’s main function is to maintain CNS homeostasis and protect the brain from the extracellular environment. The progression of BBB research has advanced to the point where many have modeled the BBB in vitro with aims of further characterizing and testing the barrier. Particularly, the pharmaceutical industry has gained interest in this field of research to improve drug development and obtain novel treatments for patients so the need for an improved model of the BBB is pertinent in their discovery. In the Cal Poly Tissue Engineering lab, an in vitro tissue engineered BBB system has previously been obtained and characterized for the initial investigation of the barrier and its components. However, certain limitations existed with use of the commercial system. Therefore, the focus of this thesis was to improve upon the capabilities and limitations of this commercialized system to allow further expansion of BBB research. The work performed was based on three aims: first to design and develop an in-house bioreactor system that could be used to cultivate the BBB; second, to characterize flow and functional capabilities of the bioreactor; third, to develop protocols for the overall use of the bioreactor, to ultimately allow co-cultures of BAEC and C6 glioma cells, and further the progression toward creating an in vitro model of the BBB. The work of this thesis demonstrates development of an in-house custom bioreactor system that can successfully culture cells. Results showed that the system was reusable, could be sterilized and monitored, was easily used by students trained in the laboratory, and allowed non-destructive scaffold extraction. This thesis also discusses the next set of experiments that will lead to an in vitro model of the BBB.

P2X7 receptors are ion-gated channels activated by ATP. Under pathological conditions, the extensive release of ATP induces sustained P2X7 receptor activation, culminating in induction of proinflammatory pathways with inflammasome assembly and cytokine release. These inflammatory conditions, whether occurring peripherally or in the central nervous system (CNS), increase blood-brain-barrier (BBB) permeability. Besides its well-known involvement in neurodegeneration and neuroinflammation, the P2X7 receptor may induce BBB disruption and chemotaxis of peripheral immune cells to the CNS, resulting in brain parenchyma infiltration. For instance, despite common effects on cytokine release, P2X7 receptor signaling is also associated with metalloproteinase secretion and activation, as well as migration and differentiation of T lymphocytes, monocytes and dendritic cells. Here we highlight that peripheral immune cells mediate the pathogenesis of Multiple Sclerosis and Parkinson’s and Alzheimer’s disease, mainly through T lymphocyte, neutrophil and monocyte infiltration. We propose that P2X7 receptor activation contributes to neurodegenerative disease progression beyond its known effects on the CNS. This review discusses how P2X7 receptor activation mediates responses of peripheral immune cells within the inflamed CNS, as occurring in the aforementioned diseases.

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Blood-brain barrier (BBB) is a unique membranous barrier, which segregates brain from the circulating blood. It works as a physical and metabolic barrier between the central nervous system (CNS) and periphery. In mammals, endothelial cells were shown to be of BBB and are characterized by the tight junctions along with efflux system which are responsible for the restriction of movement of molecules within the cells. Efflux system consists of multidrug resistance proteins such as P-glycoprotein (P-gp). P-gp removes substances out back from the brain to the blood before they reach to the brain. So the barrier is impermeable to many compounds such as amino acids, ions, small peptides and proteins, making it the most challenging factor for the development of new drugs for targeting CNS. Curcumin is a bioactive compound that has a number of health promoting benefits such as anti-inflammatory, anticancer, anti-oxidant agent; as well as a role in neurodegenerative diseases, but low oral bioavailability is the major limiting factor. Low water solubility and rapid metabolism are the two important factors responsible for poor bioavailability of curcumin. Galaxolide is a musk compound and previously known for the bioaccumulation of toxic components in the aquatic animals by interference with the activity of multidrug/multixenobiotic resistance efflux transporters (MDR/MXR). The bioavailability of curcumin can be enhanced when administered with galaxolide. This study was carried out to investigate the effect of galaxolide on the permeation of curcumin through the epithelial cell monolayers. MDCKII-MDR1 cell monolayer is used an in vitro blood-brain barrier model while Caco-2 monolayer is used as an in vitro intestinal model, which also expresses the P-glycoprotein. The curcumin and galaxolide were separately solubilised in the DMSO and used in combination to perform permeation study, to determine the effect of galaxolide on curcumin permeation through epithelial cell monolayers. The galaxolide shows an efflux protein inhibition activity and this activity was used to enhance permeation of curcumin through the Caco-2 monolayer. In summary, galaxolide is a novel permeation enhancer molecule, which can be used for the improvement of drug delivery of other bioactive compounds in future. / Department of Social Welfare, Govt. of Maharashtra (India)

Thesis (MCurr)--Stellenbosch University, 2012. / ENGLISH ABSTRACT: The global epidemic of HIV continues with an estimated 2.2 million children under 15 years of age worldwide living with HIV and 640 000 newly infected in 2004 (WHO, 2009). HIV crosses the blood–brain barrier which may lead to neuronal damage and death. There is controversial evidence within available research on effects of HIV on cognitive and motor development in children because of the limitations imposed by study designs, study populations and study methodological quality. The aims of the review were: - To conduct a systematic review of published research to establish the effects and the prevalence of HIV infection on cognitive and motor development in children. - To critically appraise the methodological quality of published research regarding cognitive and motor development of HIV infected children. The objectives of the review were: - To assess evidence on the cognitive and motor development of HIV-1 infected children - To describe anthropometric outcomes including: weight for age, weight for height, height for age and head circumference in children with a HIV infection. - To assess the methodological quality of studies on the cognitive and motor development of HIV infected children. The following databases were searched for identification of articles; MEDLINE, Google Scholar, AIDSTRIALS, AIDSLINE and CINHAL. The search time frame included published works from inception to July 2011 without language restrictions. Analytical observational trials that assessed at least one outcome (cognitive or motor development or 1 of the anthropometric outcomes) between HIV positive and HIV negative children aged 5 years and below or children with a mean age of less than 5 years were employed. Two review authors independently searched for eligible studies, evaluated methodological quality and extracted the data. Meta-analysis was carried out using Rev Man 5.1 using the risk ratio for categorical data and standard mean difference for continuous data. Fifteen studies with a total of 3 086 participants met the inclusion criteria. HIV infected children were 2.45 times at higher risk of developing cognitive developmental delay than HIV negative children (RR, 95% CI, 1.95, 3.07, P < 0.00001). Infected children scored - 0.54 less than HIV negative children (SMD 95% CI, -0.70, -0.39, 97, p < 0.00001) for cognitive development and -0.68 in motor development (SMD 95% CI, -0.82, -0.55, p< 0.00001). The risk of motor developmental delays was 2.95 times in HIV positive compared with HIV negative children (RR 95% CI, 2.19, 3.99, p < 0.00001). HIV infected children are slower in aspects of cognitive and motor development compared to their HIV negative counterparts. They also showed delays in anthropometric outcomes; weight for age and height for age. Study design influenced results of the studies with children scoring more on cross sectional than cohort studies. There is still need to develop culturally appropriate or standardise neurodevelopment tools as most African studies still rely on international tools. More evidence is needed on the effectiveness of HAART in reducing cognitive and motor delay. / AFRIKAANSE OPSOMMING: Die wêreldwye MIV epidemie duur voort met ongeveer 2.2 miljoen kinders onder 15 jarige ouderdom wat wêreldwyd met MIV leef en 640 000 onlangs in 2004 geïnfekteerd (WHO, 2009). MIV strek oor die bloed-brein grens wat kan lei tot neuronale skade en die dood. Daar is kontroversiële bewys binne beskikbare navorsing oor die effek wat MIV het op kognitiewe en motoriese ontwikkeling in kinders, vanweë die beperkinge wat geplaas word deur studie ontwerpe, studie bevolkings en studie metodologiese kwaliteit. Die doelwitte van die oorsig is om - ‘n sistematiese oorsig van gepubliseerde navorsing te doen om sodoende die effek en voorkoms van MIV infeksie op kognitiewe en motoriese ontwikkeling by kinders vas te stel - ’n kritiese waardering van die metodologiese kwaliteit van gepubliseerde navorsing te doen ten opsigte van die kognitiewe en motoriese ontwikkeling van MIV geïnfekteerde kinders. Die doelwitte van die oorsig is om - assessering te doen van die bewyse van kognitiewe en motoriese ontwikkeling by MIV-1 geïnfekteerde kinders - antropometriese uitkomste te beskryf, insluitend: gewig vir ouderdom, gewig vir hoogte, hoogte vir ouderdom en omtrek van die hoof by kinders met ’n MIV infeksie - die metodologiese kwaliteit te assesseer van studies op die kognitiewe en motoriese ontwikkeling van MIV geïnfekteerde kinders. Die volgende databasisse is nagevors vir die identifisering van artikels: MEDLINE, Google Scholar, AIDSTRIALS, AIDSLINE en CINHAL. Die tydraamwerk vir navorsing het gepubliseerde werk ingesluit vanaf aanvang tot Julie 2011 sonder taalbeperkings. Analitiese waarneembare toetse wat ten minste een uitkoms geassesseer het (kognitiewe of motoriese ontwikkeling of 1 van die antropometriese uitkomste) tussen MIV positiewe en MIV negatiewe kinders van 5 jarige ouderdom en jonger, of kinders met ’n gemiddelde ouderdom van minder as 5 jaar is betrek. Twee oorsig outeurs het onafhanklik vir geskikte studies gesoek, metodologies geëvalueer en data getrek. Meta-analise was uitgevoer deur gebruik te maak van Rev Man 5.1 met behulp van die risiko-ratio vir kategoriese data en die standaard gemiddelde verskil vir aaneenlopende data. Vyftien studies met ’n totaal van 3 086 deelnemers met die insluitingskriteria. MIV geïnfekteerde kinders het 2.45 keer ’n hoër risiko gehad om kognitiewe ontwikkelingsvertraging te ontwikkel as MIV negatiewe kinders (RR, 95% CI, 1.95, 3.07, P< 0.0000). Geïnfekteerde kinders het ’n -0.54 telling behaal, minder as MIV negatiewe kinders (SMD 95% CI, -0.70, -0.39,97 p < 0.00001) vir kognitiewe ontwikkeling en -0.68 vir motoriese ontwikkeling (SMD 95% CI, -0.82, -0.55, p< 0.00001). Die risiko van motoriese ontwikkelingsvertragings was 2.95 keer by MIV positiewe in vergelyking met MIV negatiewe kinders (RR 95% CI, 2.19, 3.99. p < 0.00001). MIV geïnfekteerde kinders is stadiger in aspekte van kognitiewe en motoriese ontwikkeling in vergeyking met hulle MIV negatiewe eweknieë. Hulle het ook vertragings getoon in antropometriese uitkomste; gewig vir ouderdom en hoogte vir ouderdom. Studie ontwerpe het uitslae beïnvloed van die kinders wat ’n hoër telling behaal het met deursnee as in kohort studies. Daar is nog ’n behoefte om kultureel geskikte of gestandaardiseerde neuro-ontwikkelingsinstrumente te ontwikkel, omdat die meeste Afrika-studies nog steeds staat maak op internasionale instrumente. Meer bewyse is nodig aangaande die effektiwiteit van HAART om kognitiewe en motoriese vertraging te verminder.

Metal-based engineered nanomaterials (ENMs) have potential to revolutionize diagnosis, drug delivery and manufactured products, leading to greater human ENM exposure. It is crucial to understand ENM pharmacokinetics and their association with biological barriers such as the blood-brain barrier (BBB). Physicochemical parameters such as size and surface modification of ENMs play an important role in ENM fate, including their brain association. Multifunctional ENMs showed advantages across the highly regulated BBB. There are limited reports on ENM distribution among the blood in the brain vasculature, the BBB, and brain parenchyma. In this study, ceria ENM was used to study the effect of size on its pharmacokinetics. Four sizes of ceria ENMs were studied. Five nm ceria showed a longer half-life in the blood and higher brain association compared with other sizes and 15 and 30 nm ceria had a higher blood cell association than 5 or 55 nm ceria. Because of the long circulation and high brain association of 5 nm ceria compared with other sizes, its distribution between the BBB and brain parenchyma was studied. The in situ brain perfusion technique showed 5 nm ceria (99%) on the luminal surface of the BBB rather than the brain parenchyma. For biomedical applications in the central nervous system (CNS), it is vital to develop stable and biocompatible ENMs and enhance their uptake by taking advantage of their unique properties. Cross-linked nanoassemblies entrapping iron oxide nanoparticles (CNA-IONPs) showed controlled particle size in biological conditions and less toxicity in comparison to Citrate-IONPs. CNA-IONPs considerably enhanced MRI T2 relaxivities and generated heat at mild hyperthermic temperatures (40 ~ 42°C) in the presence of alternating magnetic field (AMF). Numerous researchers showed mild whole body hyperthermia can increase BBB permeability for potential brain therapeutic application. Compared to conventional hyperthermia, AMF-induced hyperthermia increased BBB permeability with a shorter duration of hyperthermia and lower temperature, providing the potential to enhance IONP flux across the BBB with reduced toxicity. Overall, ENMs with optimized physicochemical properties can enhance their flux across the BBB into the brain with desirable pharmacokinetics, which provide great potential for diagnosis and therapy in the CNS.

La délivrance de médicament dans l’organisme vers des organes cibles tout en minimisant les effets secondaires représente un énorme défi scientifique. Les recherches actuelles révèlent qu’il existe de nombreuses embuches pour acheminer des composés thérapeutiques vers le système nerveux central. De nombreuses maladies (l’autisme, la schizophrénie, la maladie d’Alzheimer…) liées au système nerveux central nuisent à la qualité de vie et entrainent des coûts importants pour la société. Ce mémoire repose sur l’amélioration de l’accessibilité de composés thérapeutiques vers le cerveau en passant la barrière hémato-encéphalique, une barrière biologique difficilement franchissable. Pour introduire des médicaments dans le système nerveux central, il faut passer cette barrière, ce qui est très difficile, car elle est remarquablement efficace pour protéger le milieu cérébral. C’est pourquoi nous allons développer une nouvelle stratégie consistant à élaborer un nouveau type de transporteur. Nous proposons d’utiliser des macrolactames ayant la propriété de s’empiler sous forme de tubes supramoléculaires d’une stabilité adéquate. Il sera alors possible d’y greffer des médicaments et aussi des agents d’ouverture de la barrière hémato-encéphalique. Ce mémoire présente l’élaboration de ces nouveaux macrocycles chiraux, les résultats de différentes analyses structurales prouvant la présence de tubes et de systèmes robustes et enfin la fonctionnalisation du macrocycle par un agent médicamenteux (doxorubicine). / Abstract : Delivering drug into the body to target specific organs, while minimizing side effects, is an enormous scientific challenge. Current research reveals that there are many pitfalls for delivering therapeutic compounds to the central nervous system. Many diseases (autism, schizophrenia, Alzheimer's, etc.) linked to the central nervous system affect the quality of life and entail significant costs for society. This thesis is based on the improvement in the accessibility of therapeutic compounds to the brain by passing the blood-brain barrier, a biological barrier difficult to cross. To introduce drugs into the central nervous system, this barrier must be overcome. This is very difficult because it is remarkably effective in protecting the brain. This is why we will develop a new strategy based on a new type of transporter. We propose to use macrolactams having the property of stacking in the form of supramolecular tubes of adequate stability. It will then be possible to graft medicines and also agents capable of opening the blood-brain barrier. This manuscript describes the development of these new chiral macrocycles, the results of various structural analyses proving the presence of robust tubes and systems, and finally the functionalization of the macrocycles by a medicinal agent (doxorubicin).

Le système nerveux central est un organe privilégié et protégé, notamment grâce à l’existence des barrières histologiques entre le sang et les tissus nerveux. La barrière-hémato encéphalique (BHE) et la barrière hémato-rétinienne (BHR) séparent respectivement le parenchyme cérébral et la rétine des composés contenus dans l’espace vasculaire, grâce à l’expression de jonctions serrées et de transporteurs membranaires permettant une régulation spécifique des échanges entre le sang et le parenchyme nerveux. Ce travail a porté sur l’étude d’un nouveau transporteur de cations organiques mis en évidence fonctionnellement à la BHE de la souris. Ce transporteur appartenant très probablement à la superfamille des solute carrier (SLC), fonctionne comme un antiport proton. Actuellement, sa présence ne peut être démontrée que de façon fonctionnelle car son identité moléculaire est encore inconnue. Cet antiport proton constitue un nouvel acteur de la perméabilité cérébrale et ouvre une nouvelle voie d’accès au cerveau. Nous nous sommes tout d’abord attachés à approfondir les connaissances fonctionnelles de ce transporteur en étudiant de nouveaux substrats et tissus d’expression. Le transport cérébral de psychotropes a été étudié in vivo par la technique de perfusion carotidienne in situ chez la souris et in vitro grâce à une lignée de cellules endothéliales cérébrales humaines immortalisées (hCMEC/D3). Nous avons démontré que la haute perméabilité cérébrale de la cocaïne fait intervenir à la fois une diffusion passive et surtout une diffusion médiée par un antiport proton. La vitesse d’entrée des substances d’abus dans le cerveau est associée à un plus fort risque d’addiction et fait de ce transporteur un nouvel acteur critique de la régulation du passage cérébral. En effet, d’autres substances comme la nicotine et certaines amphétamines comme le MDPV et l'ecstasy sont également des substrats de cet antiport. Ce transporteur apparaît comme une cible pharmacologique potentielle dans la prise en charge de toxicomanies. Malgré la diversité chimique et pharmacologique d’interactions des composés avec cet antiport, les concentrations nécessaires pour l’inhiber dépassent celles retrouvées dans le sang. Pour aider l’identification d’inhibiteurs sélectifs et efficaces nous avons développé un modèle pharmacophorique d’inhibiteurs du transporteur à partir de données générées in vitro et de l’approche FLAPpharm. Ce modèle semble prédictif de nouveaux composés pouvant constituer de meilleurs inhibiteurs de ce transporteur. L’étude des échanges in vivo au niveau du tissu nerveux nous a menés à étudier l’impact de transporteurs ABC et de l’antiport-proton au niveau cérébral et rétinien à l’aide de substances spécifiques ou de substrats mixtes comme le vérapamil. L’antiport proton est fonctionnel au niveau de la BHR et transporte notamment la clonidine, le DPH et le vérapamil. Cependant, dans le cas d’un substrat mixte P-gp et SLC (ex : vérapamil), ce transport d’influx n’est visible à la BHE que lorsque la P-gp est neutralisée. Au contraire, à la BHR l’influx lié à cet SLC est visible naturellement. L’impact de la P-gp à la BHR étant 6.3-fois plus faible ce processus est probablement moins masqué. Cette étude illustre la difficulté actuelle de prédire l’impact fonctionnel d’un transporteur pour des substrats multi-spécifiques et l’existence d’une priorisation du transport. Enfin, nous avons essayé d’identifier l’antiport proton au niveau moléculaire par une méthode de photo-activation à l’aide d’un composé adapté. Cette méthode s’est avérée efficace pour fixer une molécule sur le transporteur, permettant par la suite de l’isoler plus facilement. En conclusion, ce travail a permis de mettre en évidence l’importance de l’antiport proton dans la distribution cérébrale de psychotropes et d’ouvrir de nouvelles perspectives dans l’addiction et la compréhension du transport de substrats multi-spécifiques. / The central nervous system is a privilege organ protected by histological barriers between the blood and the nervous tissue. The blood-brain barrier (BBB) and the blood-retinal barrier (BRB) separate cerebral parenchyma and retina from the circulating blood and both express tight junctions and membrane transporters, allowing a precise regulation of the exchanges between the blood and nervous tissues. We studied a new cationic transporter functionally evidenced at the mouse BBB. This molecularly unknown transporter belong to the solute carrier super family (SLC) and is a proton antiporter. It could constitute a new actor in the cerebral permeability and may be a new brain access pathway. First, we worked on the functional identification studying new substrates and new localization. Psychotropic brain transport was studied in vivo by brain in situ perfusion on mouse and in vitro with human immortalized endothelial cells (hCMEC/D3). We showed that cocaine brain entry depends on passive diffusion but also mainly on a proton antiporter. Brain entry rate of drugs of abuse is associated with modulation of addiction liability, making this transporter a new component of brain entry of cocaine, and also nicotine and some amphetamines such as ecstasy and MDPV. This proton antiporter appears to be a new potential target in addiction. Various chemical entities interact with this transporter; however concentrations used to inhibit the transporter are much higher than the one possibly found in the blood. In order to help find or design new selective and potent inhibitors, we developed a pharmacophore model of the proton antiporter inhibitors using in vitro data and the FLAPpharm approach. The model predicts well new possible inhibitors of this transporter. We also studied the impact of the ABC transporters and the proton antiporter at the BBB and the BRB using specific or multi-specific substrates such as verapamil. The proton antiporter is functionally expressed at the BRB and transports clonidine, DPH and verapamil. However, for the multi-specific (P-gp and SLC) compound verapamil, influx transport by the proton antiporter is visible at the BBB only when P-gp efflux is neutralized. On the contrary, at the BRB, the proton antiporter influx is always visible. This is certainly due to the lower impact (by 6.3 fold) of P-gp at the BRB compared to the BBB. These results show the difficulty to predict the functional impact of a transporter for multi-specific compounds and a probable transport prioritization. Finally we worked on the molecular identification of the proton antiporter using a photolabeling method. This work evidenced the importance of the proton antiporter in the brain distribution of psychotropic and drugs of abuse and opened toward new perspectives in addiction and transport comprehension.