This thesis examines the utility of USPIO-MRI to provide a tool of tracking macrophage recruitment to sites of neuroinflammation within the CNS. Recruited macrophages and microglia resident in CNS tissue play a key role in the pathophysiology of a number of neuroinflammatory diseases such as neuropathic pain and multiple sclerosis. Under activated conditions, microglia and macrophages will phagocytose invading cells and CNS debris. It has been shown that ultrasmall superparamagnetic particles of iron oxide (USPIO), such as Sinerem, injected systemically, are engulfed by macrophages, which in turn migrate to sites of tissue injury. USPIOs can be visualised as a distinct reduction in signal intensity on T2* weighted MR images. However, there are still some issues regarding the distinction between iron-laden recruited macrophages and the entry of free iron across a permeable blood brain barrier (BBB) in disease cases. Hence, it was shown that intravenously injected Sinerem is cleared from the peripheral circulation within 24 hours, indentifying this as a time point as suitable for MCP-1 injection. Data showed that free USPIO can be visualised in the brain and that there is a linear relationship between Sinerem concentration and T2* signal intensity changes. MCP-1 induces macrophage recruitment to the site of microinjection and causes BBB breakdown at between 3 and 4 hours. In particular it was shown that T2* signal intensity changes are seen, in the presence of an intact BBB, as a result of Sinerem laden macrophages. This finding was verified by the co-localisation of ED-1 positive cells and Prussian blue positive regions. It was demonstrated that there is a strong correlation between T2* signal changes and the number of macrophages. This demonstrates that USPIO-MRI can be used to characterise macrophage infiltration in neuroinflammation in the presence of an intact BBB.

A hipertensão arterial cursa com disfunção autonômica e lesão da barreira hematoencefálica (BHE) em áreas de controle autonômico. Demonstramos recentemente que o treinamento aeróbio corrige a lesão da BHE, e a disfunção autonômica, a qual se encontra correlacionada com a integridade da BHE observada nos hipertensos treinados. O objetivo deste trabalho é avaliar a expressão gênica e proteica de componentes da BHE envolvidos na mediação das respostas cardiovasculares à hipertensão e ao treinamento aeróbio (T). Ratos espontaneamente hipertensos (SHR) e seus controles normotensos (WKY) (250-300g) foram submetidos ao protocolo de T em esteira ou mantido sedentários (S) por 4 semanas. Ao final do T os animais dos grupos experimentais foram canulados para aquisição das variáveis hemodinâmicas. A seguir procedeu-se à infusão intra-arterial de 2 corantes (Rodamina-d, 70 KD, e FITC-d,10 KD) e 20 min após os encéfalos foram coletados para realização de ensaios de fluorescência no Núcleo Paraventricular do Hipotálamo (PVN). Outros ratos dos grupos experimentais foram perfundidos com salina via transcardíaca e realizada a microdissecção do PVN. O mRNA foi extraído e sua concentração de foi analisada pela técnica de RT-PCR. Para investigar os efeitos da hipertensão e do T nos componentes da BHE, foram utilizados os seguintes primers: Occludina, Claudina-5, Zônula Ocludens 1 (proteínas da junção oclusiva), Caveolina-1 (indicador de transporte transcelular), Laminina alfa 1 e Colágeno 4 (componentes da membrana basal), PDGFRβ (marcador de pericitos)e Aquaporina-4 (indicador de podócitos de astrócitos), todos eles normalizados para o HPRT endógeno. Os dados de PCR em tempo real foram quantificados pelo método 2Δ ΔCT. Além disso, outros ratos dos mesmos grupos experimentais foram perfundidos com paraformaldeído 4% para a fixação do encéfalo. O tecido foi crioprotegido e seccionado em criostato, 30 um, os cortes foram incubados em anticorpos primários (Reca-1(marcador endotelial), Claudina-5, Caveolina-1, PDGFRβ e Aquaporina-4) e secundários (Alexa Flúor 488 e 594), e sua quantificação no PVN foi realizada através da densidade integrada. Não foi observada diferença na pressão arterial entre os grupos T e S, porém, houve bradicardia de repouso nos animais T (SHR-T:317±3 e WKY-T:308±2) comparados com os animais S (SHR-S: 344±4 e WKY-S: 323±3). A permeabilidade da BHE foi reduzida e normalizada pelo T nos animais hipertensos (SHR-S: 13,6±1,2% e SHR-T: 3,8±0,4%; WKY-S: 3,9±0,2% e WKY-T: 4,1±0,16%), e análise do RT-PCR não mostrou nenhuma diferença para Claudina, PDGFRβ e Aquaporina-4 entre os T e S. A expressão gênica de Caveolina-1 estava aumentada nos SHR comparado aos WKY, e o T foi capaz de reduzir sua expressão (SHR-T: 1,05±0,1). O que foi confirmado pela expressão proteica no PVN: a Caveolina-1 encontrava-se aumentada significativamente nos SHR-S em relação aos WKY, e o T reduziu sua expressão no PVN dos SHR. Conclusão: Nossos dados sugerem que o aumento da permeabilidade da BHE no PVN de hipertensos é devida ao aumento de transcitose, identificada pela expressão de Caveolina-1 e que o treinamento aeróbio reverte esta permeabilidade ao reduzir o transporte transcelular sem alterar o transporte paracelular. / The arterial hypertension courses with autonomic dysfunction and Blood Brain Barrier (BBB) damage in areas of autonomic control. We recently demonstrated that aerobic training corrects the damage to the BBB, and autonomic dysfunction, which is correlated with the integrity of the BBB observed in trained hypertense subjects. The objective of this work is to evaluate the gene and protein expression of BBB components involved in mediating cardiovascular responses to hypertension and aerobic training (T). Spontaneously hypertensive rats (SHR) and their normotensive controls (WKY) (250-300g) were submitted to treadmill protocol or maintained sedentary (S) for 4 weeks. At the end of the T, the animals of the experimental groups were cannulated to acquire the hemodynamic variables. Intra-arterial infusion of two dyes (Rhodamine-d, 70 KD, and FITC-d, 10 KD) and 20 min after brains were collected for fluorescence assays in the Paraventricular Nucleus of hypothalamus (PVN). Other rats from the experimental groups were perfused with transcardiacally with saline and the PVN was microdissected. The mRNA was extracted and its concentration was analyzed by the RT-PCR technique. To investigate the effects of hypertension and T in the BBB components, the following primers were used: Occludin, Claudin-5, Zonula Ocludens 1 (tight junction proteins), Caveolina-1 (indicator of transcellular transport), Laminin α 1 and Collagen-4 (basement membrane components), PDGFRβ (pericyte marker) and Aquaporin-4 (astrocyte podocyte indicator), all standardized for endogenous HPRT. Real-time PCR data were quantified by the method 2ΔΔCT. In addition, other rats from the same experimental groups were perfused with 4% paraformaldehyde for fixation of the brain. The tissue was cryoprotected and cross-sectioned, 30 m, sections were incubated on primary (Reca-1 (endothelial marker), Claudin-5, Caveolin-1, PDGFRβ and Aquaporin-4) and secondary antibodies (Alexa Fluor 488 and 594), and its quantification in thePVN was performed through the integrated density. There was no difference in blood pressure between the T and S groups, but there was resting bradycardia in the T animals (SHR-T: 317 ± 3 and WKY-T: 308 ± 2) compared to S controls (SHR-S: 344 ± 4 and WKY-S: 323 ± 3). The permeability of BBB was reduced and normalized by T in hypertensive animals (SHR-S: 13.6 ± 1.2% and SHR-T: 3.8 ± 0.4%; WKY-S: 3.9 ± 0, 2% and WKY-T: 4.1 ± 0.16%), and RT-PCR analysis showed no difference for Claudin-5, PDGFRβ and Aquaporin-4 between T and S. Caveolin-1 gene expression was increased in SHR compared to WKY, and T was able to reduce its expression (SHR-T: 1.05 ± 0.1). This was confirmed by protein expression in PVN: Caveolin-1 was significantly increased in SHR-S relative to WKY, and T reduced its expression in the PVN of SHR. Conclusion: Our data suggest that increased permeability of BBB in the PVN of hypertense individuals is due to the increase transcytosis as identified by Caveolin-1 expression and that aerobic training reverses this permeability by reducing transcellular transport without altering the paracellular transport.

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.

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.

Brain diseases including neurological disorders and tumors remain undertreated due to the challenge in accessing the brain, and blood-brain barrier (BBB) restricting drug delivery, which also profoundly limits the development of pharmacological treatment. Focused ultrasound (FUS) with acoustic agents including microbubbles and nanodroplets remains as the only method to open the BBB noninvasively, locally, and transiently to assist drug delivery. For an ideal medical system to serve a broad patient population, it requires precise and flexible targeting with simulation to personalize treatment, real-time monitoring to ensure safety and effectiveness, and rapid application, as repetitive pharmacological treatment is often required. Since none of current systems fulfills all the requirements, here we designed a neuronavigation-guided FUS system with protocol assessed in in vivo mice, in vivo non-human primates, and human skulls from in silico preplanning, online FUS treatment and real-time acoustic monitoring and mapping, to post-treatment assessment using MRI. Both sedate and awake non-human primates were evaluated with total treatment time averaging 30 min and 3-mm targeting accuracy in cerebral cortex and subcortical structures. The FUS system developed would enable transcranial FUS in patients with high accuracy and independent of MRI guidance.

La barrière hémato-encéphalique (BHE) est la structure essentielle au système nerveux central (SNC), localisée au sein des capillaires cérébraux. Elle est responsable du maintien de l’homéostasie cérébrale ainsi que de sa protection. En effet, les pathologies affectant le SNC sont souvent associées à une altération au niveau de la BHE. L’un des nombreux facteurs, pouvant altérer l’homéostasie du SNC, est l’hypoxie puisque le cerveau est le premier consommateur d’oxygène. De nombreuses études ont démontré qu’une hypoxie aigüe (modélisation de l’ischémie cérébrale transitoire ou l’AVC), altère la perméabilité de cette barrière. Cependant, certaines pathologies sont liées à une hypoxie intermittente, notamment les troubles respiratoires associés au sommeil, comme le syndrome d’apnées du sommeil. Cette hypoxie intermittente ne semble pas être sans conséquence sur la structure et la fonction du cerveau, car des études menées chez l’Homme ont démontré que ces patients souffraient de troubles cognitifs. L’enjeu dans l’étude de ces pathologies est d’avoir une meilleure compréhension des mécanismes amenant à l’altération de la BHE, afin de développer des stratégies thérapeutiques adéquates. Toutefois, peu d’études à ce jour se sont intéressées à l’impact de l’hypoxie intermittente sur la BHE en raison de la complexité du cerveau. De ce fait, les modèles in vitro de BHE semblent être des outils adéquats à l’étude de la BHE en conditions pathologiques. L’objectif de ce travail de thèse a été de mettre en place un modèle in vitro de BHE permettant l’étude de l’impact de stress hypoxiques répétés sur l’intégrité de cette barrière.Le modèle mis en place est un modèle de co-culture « contact » mettant en jeu des cellules endothéliales cérébrales immortalisées de souris et des astrocytes immortalisés de rat. En effet, de nombreuses études ont montré l’importance des interactions entre les astrocytes et les cellules endothéliales, dans la mise en place du phénotype de barrière de la BHE. Nous avons fait le choix de développer un modèle murin afin d’être en adéquation avec les études menées, chez le petit animal, au laboratoire. Ce modèle a été caractérisé en termes de résistance électrique transendothelial (TEER), de perméabilité membranaire, de jonctions serrées et de transporteurs d’efflux. Ce modèle répond aux critères attendus pour un modèle in vitro de BHE et est rapide à mettre en place.L’impact cellulaire de l’hypoxie intermittente est difficile à étudier in vitro en raison de la rapidité du phénomène. De ce fait, nous avons mis en place une méthode alternative d’induction de stress hypoxique par un agent chimique, l’hydralazine, qui soit reproductible, contrôlable et réversible, afin d’étudier les mécanismes cellulaires et moléculaires induits au niveau de cette BHE. / The blood-brain barrier (BBB) is the essential structure of the central nervous system (CNS), located at the level of brain capillaries. The BBB is responsible for the maintenance of cerebral homeostasis and its protection. Indeed, diseases affecting the CNS are often related to a disorder at the BBB’s site. One of the several factors which can alter the homeostasis of the CNS, is hypoxia because the brain is one of the the first consumer of oxygen of the body. Several studies showed that acute hypoxia (mimic transient brain ischemia or stroke), could alter BBB permeability. However, some disorders are related to intermittent hypoxia, particularly sleep related breathing disorders, such as sleep apnea syndrome. This intermittent hypoxia does not seem to be innocent on the brain structure and function, since studies in humans have demonstrated that these patients suffer from cognitive disorders. The challenge in the study of such pathologies is represented by a better understanding of mechanisms leading to this alteration, in order to develop adequate therapeutic strategies. Nevertheless, few studies have focused, to date, on the impact of intermittent hypoxia on the BBB, due to the complexity of the brain structure in vivo. In this regard, in vitro BBB model appear to be suitable tools for the study of BBB in pathologies conditions. The aim of this thesis was to set up an in vitro BBB model suitable for the study of BBB’s integrity after repeated hypoxic stress exposure.

<p>Delivery of drugs to the brain requires passage across the blood-brain barrier (BBB). Both for drugs already on the market and for new drugs under development, it is important to know to what extent a drug enters the CNS. Many drugs used clinically are racemic mixtures, <i>i.e.</i> equal parts of the (<i>S</i>)- and (<i>R</i>)-enantiomers. </p><p>The present studies focus on the enantiomers and racemates of a number of 5-HT_1A receptor agonists and antagonists (pindolol, propranolol, 8-OH-DPAT and other 8-substituted-2-(di-<i>n</i>-propylamino)tetralin derivatives) and BBB transport <i>in vitro</i> and distribution to the brain <i>in vivo.</i> Assays (HPLC-based) were set up or developed for determination of the racemates and the pure enantiomers (chiral column) of drugs in plasma and brain tissue. BBB transport was assessed <i>in vitro</i> using bovine brain endothelial cells cocultured with rat astrocytes. The physicochemical constants (log P, pKa) and plasma protein binding were determined. Pindolol, propranolol and several tetralines accumulated over time in brain tissue. For pindolol and propranolol, but not for most tetralins, the distribution to the brain was stereoselective, (<i>S</i>)>(<i>R</i>). Pretreatment with verapamil, an inhibitor of drug efflux <i>via</i> P-glycoprotein, differentially decreased the brain/plasma ratios of the enantiomers of pindolol and propranolol, indicating that verapamil may also inhibit an influx transport mechanism. <i>In vitro</i> results with racemic pindolol, propranolol and tetralins showed no differences in BBB transport between the enantiomers. A more rapid apical to basolateral transport (influx) <i>vs</i>. the basolateral to apical (efflux) transport of propranolol (not pindolol) and most tetralins <i>in vitro</i> indicated active transport across the BBB. </p><p>In conclusion, the combined <i>in vivo</i> and <i>in vitro</i> results are consistent with active transport of the studied compounds across the BBB rather than passive diffusion due to their lipophilicity. Some, but not all, chiral drugs are stereoselectively distributed to the brain. Stereoselective plasma protein binding or stereoselective transport across brain endothelial cells does not seem to explain the stereoselective accumulation of pindolol and propranolol. The stereochemical configuration of compounds contributes to their pharmacokinetic as well as their pharmacodynamic uniqueness. The characteristics of the enantiomers of chiral compounds need to be determined empirically rather than based on generalizations from structural or physicochemical information.</p>

<p>This thesis examines the transport of exogenous molecules across the blood-brain barrier (BBB), focusing on active efflux, using positron emission tomography (PET), computer simulation and modelling. P-glycoprotein (P-gp) inhibition was studied using [¹¹C]verapamil and [¹¹C]hydroxyurea was investigated as a new marker for active efflux transport. Simulations were carried out to explore the importance of the efflux transporter location in the BBB. Brain concentrations of [¹¹C]verapamil, [¹¹C]GR205171 and [¹⁸F]altanserin were compared in various laboratory animal species and in humans.</p><p>A central aspect of the studies has been the novel combination of dynamic PET imaging of the brain pharmacokinetics of a labelled drug, administered through an exponential infusion scheme allowing time-resolved consequence analysis of P-gp inhibition, and mathematical modelling of the obtained data. The methods are applicable to drugs under development and can be used not only in rodents but also in higher species, potentially even in humans, to investigate the effects of P-gp or other transporters on drug uptake in the brain.</p><p>The inhibition of P-gp by cyclosporin A (CsA) and the subsequent change in brain concentrations of [¹¹C]verapamil occurred rapidly in the sense that [¹¹C]verapamil uptake increased rapidly after CsA administration but also in the sense that the increased uptake was rapidly reversible. The P-gp inhibition was best described by an inhibitory indirect effect model in which CsA decreased the transport of [¹¹C]verapamil out of the brain. The model indicated that approximately 90% of the transport of [¹¹C]verapamil was P-gp-mediated. The low brain concentrations of [¹¹C]hydroxyurea appeared to be a result of slow transport across the BBB rather than active efflux. This exemplifies why the extent and the rate of brain uptake should be approached as two separate phenomena. The brain-to-plasma concentration ratios for the three studied radiotracers differed about 10-fold be-tween species, with lower concentrations in rodents than in humans, monkeys and pigs. The increase in brain concentrations after P-gp inhibition was somewhat greater in rats than in the other species. </p><p>The findings demonstrate a need to include the dynamics of efflux inhibition in the experimental design and stress the importance of the choice of species in preclinical studies of new drug candidates. </p>

A. A study of Stichodactyla helianthus toxin’s potential to penetrate the buccal mucosa Introduction:  Buccal mucosa is an alternative route for drug administration and has advantages over other conventional routes by avoiding both enzymes in the gastro intestinal system and the hepatic first passage mechanism. Stichodactyla helianthus toxin (ShK) is a peptide toxin that blocks potassium channels in T lymphocytes and could be a future treatment for autoimmune diseases when finding a suitable way of administration. Aim:  The purpose of this part of the study was to develop a robust and reproducible assay for identification and quantification of ShK. The method was then employed for a proof of principle study; determining the concentration of ShK following an in vitro permeability experiment, to evaluate the potential of ShK penetrating the buccal mucosa in porcine tissue.  Materials and Methods:  An HPLC method was developed and validated. A piece of porcine buccal mucosa was used as a membrane because of its similarities with human buccal mucosa, and cinched in between a modified Ussing Chamber consisting of a donor and a receptor chamber. Samples were withdrawn from the receptor chamber to determine the amount of ShK that had penetrated the membrane. Results: The HPLC method developed for quantification of ShK demonstrated high accuracy and precision. No concentrations of ShK were able to be quantified from the receptor chambers. Conclusions:  A robust assay for quantification of ShK was developed but the results from the experiment indicated that ShK could not penetrate the buccal mucosa membrane. B. An investigation of the permeability alterations in the blood-brain barrier associated with Alzheimer’s disease   Introduction:  The blood brain barrier (BBB) protects the brain from potential dangerous substances by different barrier properties such as tight junctions and efflux transporters such as P-glycoprotein. Previous studies have showed that the barrier functions may be altered in Alzheimer’s disease and thereby increase the exposure to substances that are normally excluded from the brain parenchyma. This could be an issue regarding safety and toxicity of medications used among Alzheimer patients. Aim:  The aim of this part of the study was to investigate the difference in brain uptake of verapamil, digoxin, loperamide, propanolol, diazepam and sucrose between 3xTg-AD mice and wild type control mice. Materials and Methods: Female 3xTg-AD mice and control mice of the age 11.5-13.5 months were used. In Situ brain perfusion with radiolabeled substances (n=5-12) was performed and the brain uptake ratio of the substances was compared and statistically analyzed.  Results: No difference in the vascular volume was found when comparing 3xTg-AD with control mice. The ratio of diazepam was observed to be higher in the cortex and propranolol higher in the hippocampus, of 3xTg-AD mice. The uptake ratio of verapamil was higher in both the hippocampus and cortex of 3xTg-AD mice whereas digoxin appeared to be lower in the cortex of 3xTg-AD mice. There was no difference in uptake ratio of loperamide between 3xTg-AD and control mice. Conclusions:  This study in addition to previously executed studies in our laboratory, showed that the membrane thickness is age dependent in 3xTg-AD and that further studies needs to be conducted on the expression of P-glycoprotein in the BBB in 3xTg-AD and control mice.

This thesis examines the transport of exogenous molecules across the blood-brain barrier (BBB), focusing on active efflux, using positron emission tomography (PET), computer simulation and modelling. P-glycoprotein (P-gp) inhibition was studied using [11C]verapamil and [11C]hydroxyurea was investigated as a new marker for active efflux transport. Simulations were carried out to explore the importance of the efflux transporter location in the BBB. Brain concentrations of [11C]verapamil, [11C]GR205171 and [18F]altanserin were compared in various laboratory animal species and in humans. A central aspect of the studies has been the novel combination of dynamic PET imaging of the brain pharmacokinetics of a labelled drug, administered through an exponential infusion scheme allowing time-resolved consequence analysis of P-gp inhibition, and mathematical modelling of the obtained data. The methods are applicable to drugs under development and can be used not only in rodents but also in higher species, potentially even in humans, to investigate the effects of P-gp or other transporters on drug uptake in the brain. The inhibition of P-gp by cyclosporin A (CsA) and the subsequent change in brain concentrations of [11C]verapamil occurred rapidly in the sense that [11C]verapamil uptake increased rapidly after CsA administration but also in the sense that the increased uptake was rapidly reversible. The P-gp inhibition was best described by an inhibitory indirect effect model in which CsA decreased the transport of [11C]verapamil out of the brain. The model indicated that approximately 90% of the transport of [11C]verapamil was P-gp-mediated. The low brain concentrations of [11C]hydroxyurea appeared to be a result of slow transport across the BBB rather than active efflux. This exemplifies why the extent and the rate of brain uptake should be approached as two separate phenomena. The brain-to-plasma concentration ratios for the three studied radiotracers differed about 10-fold be-tween species, with lower concentrations in rodents than in humans, monkeys and pigs. The increase in brain concentrations after P-gp inhibition was somewhat greater in rats than in the other species. The findings demonstrate a need to include the dynamics of efflux inhibition in the experimental design and stress the importance of the choice of species in preclinical studies of new drug candidates.