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Impact of alzheimer’s disease on drug transport across the blood-brain barrier
thesisposted on 27.02.2017, 23:51 by Mehta, Dharmini
Alzheimer’s disease (AD) is a neurodegenerative disorder, characterized by β-amyloid plaques and hyperphosphorylated tau tangles in the brain. Along with these pathological lesions, there have been multiple reports of physical and biochemical alterations of the blood-brain barrier (BBB) in AD. However, less is known about the impact of such BBB changes on the ability of therapeutic agents to traverse the BBB, and their disposition into the central nervous system. Therefore, the aim of the research project was to systematically assess the brain uptake of a series of small probe compounds (with varying mechanisms of transport across the BBB) and the anti-AD drug, MEM, in a relevant animal model of AD. An in situ transcardiac perfusion method was validated in Swiss Outbred mice with marker compounds to ensure the physical integrity and functionality of the BBB, prior to applying this method in a relevant mouse model of AD. This method of transcardiac perfusion was able to discriminate between high penetrating ([3H] diazepam and [3H] propranolol) and low penetrating ([3H] digoxin) marker compounds and depicted the functional activity of selected efflux (P-glycoprotein, P-gp) and influx (glucose transporter, GLUT-1) transporters at the BBB. The cortical and hippocampal uptake of small drug-like, radiolabelled markers of paracellular diffusion, passive transcellular diffusion and the P-gp efflux mechanism were then measured in triple transgenic (3×TG) AD mice and their corresponding wild-type (WT) control, at a young (12-14 months) and old (18-20 months) age point. The brain uptake was measured in cortical and hippocampal regions as these are the two prominent AD-affected regions of the brain. The brain uptake of the paracellular marker, [14C] sucrose, did not differ between WT and 3×TG mice at both age points, suggesting that BBB paracellular integrity was maintained in 3×TG AD mice. The brain uptake of the passively diffusing markers, [3H] diazepam and [3H] propranolol, was unchanged in young 3×TG mice, compared with WT mice. However, the brain uptake of these transcellular marker compounds decreased 54-60% in aged 3×TG mice, when compared to WT mice. This suggested an altered transcellular transport mechanism in 3×TG mice at this older age, where AD pathology is likely to be more pronounced. To clarify the potential mechanism responsible for the reduced transcellular mechanism of transport in 3×TG AD mice, the thickness of the cerebrovascular basement membrane was measured by collagen-IV immunohistochemistry in cortical slices obtained from aged WT and 3×TG mice. A significant 33.5% increase in the thickness of the cerebrovascular basement membrane was observed in 3×TG mice relative to WT mice, suggesting that the reduced uptake of the transcellular markers may have been due to a thickened cerebrovascular basement membrane and an increased path length through which these passively-diffusing molecules had to diffuse. Though P-gp expression is reported to be reduced in AD, surprisingly the brain uptake of the investigated P-gp substrates ([3H] digoxin, [3H] loperamide and [3H] verapamil) was not different between genotypes at both age groups, except for [3H] verapamil whose brain uptake was enhanced in young AD mice. To elucidate the unexpected lack of difference in the brain uptake of P-gp substrates in 3×TG mice, it was essential to measure cerebral microvascular P-gp expression in the 3×TG AD mouse model, as it had not been reported that P-gp expression was actually reduced in this particular mouse model of AD. The methods for isolating cerebral capillaries from mouse brain and the subsequent Western blotting for quantification of P-gp were established and characterized. Using these optimized techniques, it was found that P-gp expression was decreased in two sub-populations of 3×TG mice by 17-42.4% (with 6-7 mice/population); however, in one sub-population of 3×TG mice this reduction was not evident, hinting towards a variable P-gp down-regulation in AD mice. Although P-gp expression was generally reduced in 3×TG mice, the brain uptake of P-gp substrates remained unchanged in AD mice. This may be due to the decreased P-gp expression being counteracted by a thickening in the basement membrane observed, leading to a net effect of no apparent differences in the brain uptake of P-gp substrates between WT and 3×TG mice. After assessing the brain uptake of marker compounds in AD mice, it was considered important to assess the brain uptake of a therapeutic compound used in the treatment of AD, and hence MEM was selected as a model anti-AD drug. Sensitive and robust LC-MS assays were developed and validated for quantifying MEM concentrations in mouse brain homogenate and perfusate. Similar to previous studies, MEM brain uptake was assessed in both young and aged WT and 3×TG mice using transcardiac perfusion and the concentrations measured with the validated LC-MS assay. While the brain uptake of MEM was not significantly different between WT and 3×TG mice at 12-14 months, MEM brain uptake was significantly decreased by 43% in 18-20 month old 3×TG mice relative to WT mice, a similar effect to that observed with regards to [3H] diazepam and [3H] propranolol uptake. It may be presumed that MEM diffuses across the BBB, at least partly, by passive transcellular diffusion (due to its lipophilicity and small molecular weight). Therefore, a potential reason for the reduced brain uptake of MEM in AD mice could be the thickening of the cerebral microvascular membrane, as was postulated for the reduced brain uptake of [3H] diazepam and [3H] propranolol. However, given MEM has been reported to interact with organic cation transporter 2 (OCT2) in human embryonic kidney cells, an active transport phenomenon was also considered to contribute to the brain disposition of MEM. Whether such an active transport process for MEM exists at the BBB is not yet known, and therefore additional studies were carried out to assess the brain disposition of MEM in healthy Swiss Outbred mice using transcardiac perfusion. These studies assessed the concentration dependency of the brain uptake of MEM, determined the impact of known inhibitors of the OCT family on MEM brain uptake, and determined the impact of different perfusate ionic compositions and pH conditions on MEM brain uptake. In summary, the results of these brain uptake studies demonstrated that MEM transport across the BBB was saturable, pH-dependent, independent of transmembrane potential and Na+ ion concentration, and H+-coupled, suggesting the involvement of an organic cation transporter regulated by proton antiport mechanisms, which is most likely to be the organic cation/carnitine transporter, OCTN1. Thus, the observed reduction in the brain uptake of MEM in 3×TG mice could be due to two reasons, 1) cerebrovascular basement membrane thickening that may impede the passive diffusion of MEM and 2) decreased expression of the cationic transporter (most likely OCTN1) at the BBB in 3×TG AD mice, decreasing the active brain uptake of MEM, though further studies are required to confirm this second hypothesis. Overall, the findings from this research project imply that the transcellular mechanism of transport across the BBB is decreased in 3×TG AD mice, and suggest that the anti-AD drug MEM is likely to access the brain via active transport mechanisms and passive diffusion processes. The research described in this thesis is significant, and highlights that the CNS exposure of drugs is likely to differ between AD patients and healthy individuals, warranting further clinical investigations and the consideration of altered regimen design in patients with this neurodegenerative disorder.