<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.

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.e. equal parts of the (S)- and (R)-enantiomers. The present studies focus on the enantiomers and racemates of a number of 5-HT1A receptor agonists and antagonists (pindolol, propranolol, 8-OH-DPAT and other 8-substituted-2-(di-n-propylamino)tetralin derivatives) and BBB transport in vitro and distribution to the brain in vivo. 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 in vitro 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, (S)&gt;(R). Pretreatment with verapamil, an inhibitor of drug efflux via 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. In vitro results with racemic pindolol, propranolol and tetralins showed no differences in BBB transport between the enantiomers. A more rapid apical to basolateral transport (influx) vs. the basolateral to apical (efflux) transport of propranolol (not pindolol) and most tetralins in vitro indicated active transport across the BBB. In conclusion, the combined in vivo and in vitro 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.

The blood-brain barrier is formed by tightly joined capillary cells with transporter proteins and acts as to regulate the brain concentration of nutrients as well as many drugs. When developing central nervous system drugs it is necessary to measure the unbound drug concentration in the brain, i.e. the unbound brain exposure. This is to ensure that the drug reaches the site of action. Furthermore, when designing new drugs it is extremely valuable to be able to predict brain exposure from a tentative drug structure. Established methods to measure total drug concentrations are of limited (if any) utility since the pharmacologically active, unbound, concentration is not obtained. The aim of the conducted research was to develop an efficient methodology to measure unbound drug in the brain and to generate a dataset for developing computational prediction models describing the relationship between drug structure and unbound brain exposure. First it was demonstrated that unbound brain exposure can be efficiently assessed using a combination of total drug concentrations in the brain and separate measurements of drug binding in the brain slices. The in vitro brain slice method was refined and made high-throughput. Improvements were also made to the in vivo measurements of total concentrations by introducing an appropriate correction for drug in residual blood. Modeling of a 43-drug dataset in the rat showed that unbound brain exposure is related to the drug hydrogen bonding potential and not to lipid solubility, which contrasts the common understanding. Further, the drug concentrations in cerebrospinal fluid approximated unbound concentrations in the brain (r2=0.80) and were also correlated with corresponding measurements in humans (r2=0.56). Therefore, rat-derived prediction models can be used when designing drugs for humans. This thesis work has provided drug industry and academia with efficient tools to obtain and to use relevant estimates of drug exposure in the brain for evaluating drugs candidates.

Malignant brain tumor is one of the most lethal forms of cancers. In the United States alone, approximately 20,500 new cases of primary malignant brain and central nervous system tumors are expected to be diagnosed in 2007 with 12,740 deaths estimated. Treatment of malignant brain tumor remains a major challenge despite recent advance in surgery and other adjuvant therapies, such as chemotherapy. The failure of potential effective chemotherapeutics for brain tumor treatment is usually not due to the lack of potency of the drug, but rather can be attributed to lack of therapeutic strategies capable of overcoming blood brain barrier for effective delivery of drug to the brain tumor. In this thesis, we developed a minimally invasive sustained release system for glioma therapy. The present study was initiated in an effort to incorporated Doxorubicin (DOX) loaded PLGA particle into an agarose gel, which can provide a continuous release of DOX locally to the tumor site. DOX, a toposiomearase II inhibitor, is not currently used clinically for brain tumor treatment because when delivered systemically it does not cross BBB. Our hydrogel particle system can overcome this shortcoming of DOX. The results from this study demonstrate that the DOX/PLGA particle gel system can maintain the bioactivity of DOX and sustained release DOX for at least 15 day in vitro. The result of in vivo study showed the DOX/PLGA particle gel treated group had significantly extend the medium survival of 9L glioma bearing rat from 21 days to 29 days. Therefore, the success experience of this local and sustained delivery device might benefit the development of future glioma therapy strategy.

The blood-brain barrier, formed by brain microvessel endothelial cells, is the restrictive barrier between the brain parenchyma and the circulating blood. It was previously demonstrated in our laboratory that knock down of fatty acid transport proteins FATP-1 and CD36 attenuated apical to basolateral monounsaturated fatty acid transport across human brain microvessel endothelial cells (HBMEC). Arachidonic acid (AA; 5,8,11,14 - cis-eicosatetraenoic acid) is a conditionally essential, polyunsaturated fatty acid [20:4(n-6)] and a major constituent of brain lipids. We examined transport of AA across confluent monolayers of HBMEC. Control cells or HBMEC with knock down of FATP-1 or CD36 were cultured on Transwell® plates and incubated apically with [3H]AA and incorporation of [3H]AA into the basolateral medium was determined temporally. [3H]AA was rapidly incorporated into the basolateral medium with time in control cells. Surprisingly, knock down of FATP-1 or CD36 did not alter [3H]AA movement into the basolateral medium. The increased permeability mediated by AA was likely caused by a metabolite of AA produced de novo and was confirmed by an increased movement of fluorescent dextran from apical to basolateral medium. HBMECs expressed PGE2 synthase, cyclooxygenase-1 and -2, PGE2 receptors, tight junction proteins and prostaglandin transporters. The AA-mediated increase in membrane permeability was not attenuated by cyclooxygenase inhibitor drugs (NSAIDs). Incubation of the HBMEC monolayers with exogenous PGE2 resulted in attenuation of the AA-mediated permeability increases. The results indicate that AA increases the permeability of the HBMEC monolayer likely via increased production of metabolites or by-products of the lipoxygenase or epoxygenase pathways. These observations may explain the rapid influx of AA into the brain previously observed upon plasma infusion with AA.

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.

The blood-brain barrier, formed by brain microvessel endothelial cells, is the restrictive barrier between the brain parenchyma and the circulating blood. It was previously demonstrated in our laboratory that knock down of fatty acid transport proteins FATP-1 and CD36 attenuated apical to basolateral monounsaturated fatty acid transport across human brain microvessel endothelial cells (HBMEC). Arachidonic acid (AA; 5,8,11,14 - cis-eicosatetraenoic acid) is a conditionally essential, polyunsaturated fatty acid [20:4(n-6)] and a major constituent of brain lipids. We examined transport of AA across confluent monolayers of HBMEC. Control cells or HBMEC with knock down of FATP-1 or CD36 were cultured on Transwell® plates and incubated apically with [3H]AA and incorporation of [3H]AA into the basolateral medium was determined temporally. [3H]AA was rapidly incorporated into the basolateral medium with time in control cells. Surprisingly, knock down of FATP-1 or CD36 did not alter [3H]AA movement into the basolateral medium. The increased permeability mediated by AA was likely caused by a metabolite of AA produced de novo and was confirmed by an increased movement of fluorescent dextran from apical to basolateral medium. HBMECs expressed PGE2 synthase, cyclooxygenase-1 and -2, PGE2 receptors, tight junction proteins and prostaglandin transporters. The AA-mediated increase in membrane permeability was not attenuated by cyclooxygenase inhibitor drugs (NSAIDs). Incubation of the HBMEC monolayers with exogenous PGE2 resulted in attenuation of the AA-mediated permeability increases. The results indicate that AA increases the permeability of the HBMEC monolayer likely via increased production of metabolites or by-products of the lipoxygenase or epoxygenase pathways. These observations may explain the rapid influx of AA into the brain previously observed upon plasma infusion with AA.

Parkinson's disease (PD) is one of the most common motor neurodegenerative diseases, affecting 1-2% of the world's population over the age of 65. It is characterised by a loss of dopamine neurons within the substantia nigra, which is an integral part of the basal ganglia (BG) where dopamine is the most important modulating neurotransmitter. As the BG is primarily involved with the execution of movement, the lack of dopamine input results in dysfunctional motor control. The current PD treatment, L-DOPA, improves these motor symptoms, however only provides patients 5 to 10 years of improved quality of life before debilitating side effects, often worse than the original symptoms, begin. The neuropeptide substance P (SP) is found in high concentration in the substantia nigra, and BG in general, where it is involved in dopamine release. In the late stages of PD, SP content within the substantia nigra and BG is decreased, thus implicating SP in the pathophysiology of PD. However, SP production has not been examined in the early stages of PD when dopaminergic degeneration is first initiated. This thesis therefore sought to characterise the role of SP in dopaminergic degeneration in an experimental model of early PD, the 6-hydroxydopamine model in rats. In contrast to the prevailing dogma that a decline in SP is associated with neurodegeneration in PD, this thesis demonstrates that SP is actually increased within the striatum in early PD, particular in perivascular tissue and within surviving dopaminergic neurons during the degenerative process. Increasing exposure of the dopaminergic neurons to SP, either by inhibition of substance P breakdown with Captopril or by direct injection with SP, exacerbated the disease progression as indicated by more profound neurogenic inflammation, functional deficits and increased dopaminergic cell death. However, when SP was inhibited by treatment with a SP NK₁ receptor antagonist, dopaminergic neurons were conserved, the inflammatory response was reduced and motor function was returned to near normal levels. We conclude that SP is increased in early PD, and that increased SP plays an important role in the degenerative process, specifically, in the genesis of BBB breakdown and initiation of neurogenic inflammation. Treatment with an NK1 antagonist may thus represent a novel therapeutic approach to early stage Parkinson’s disease. / Thesis (Ph.D.) -- University of Adelaide, School of Medical Sciences, 2009