Cerebral ischemia occurs when blood flow to the brain is insufficient to meet metabolic demand. This can result from cerebral artery occlusion that interrupts blood flow, limits CNS supply of oxygen and glucose, and causes an infarction/ischemic stroke. Ischemia initiates a cascade of molecular events in neurons and cerebrovascular endothelial cells including energy depletion, dissipation of ion gradients, calcium overload, excitotoxicity, oxidative stress, and accumulation of ions and fluid. Blood-brain barrier (BBB) disruption is associated with cerebral ischemia and leads to vasogenic edema, a primary cause of stroke-associated mortality. To date, only a single drug has received US Food and Drug Administration (FDA) approval for treatment of acute ischemia/reperfusion injury, recombinant tissue plasminogen activator (rt-PA). While rt-PA therapy restores perfusion to ischemic brain, considerable tissue damage occurs when cerebral blood flow is re-established. Therefore, there is a critical need for novel therapeutic approaches that can "rescue" salvageable brain tissue and/or protect BBB integrity during cerebral hypoxia and subsequent reoxygenation stress (H/R). One approach that may enable neural tissue rescue following H/R is CNS delivery of drugs with brain protective effects such as HMG-CoA reductase inhibitors (i.e., statins). Our present in vivo data demonstrates that atorvastatin, a commonly prescribed statin, attenuates poly (ADP-ribose) polymerase (PARP) cleavage in the brain following H/R, suggesting neuroprotective efficacy. However, atorvastatin use as a CNS therapeutic is limited by poor blood-brain barrier (BBB) penetration. Therefore, we examined regulation and functional expression of the known statin transporter Oatp1a4 at the BBB under H/R conditions. In rat brain microvessels H/R (6% O₂, 60 min followed by 21% O₂, 10 min) increased Oatp1a4 expression. Brain uptake of taurocholate (i.e., Oap1a4 probe substrate) and atorvastatin were reduced by Oatp inhibitors (i.e., estrone-3-sulfate, fexofenadine), suggesting involvement of Oatp1a4 in brain drug delivery. Pharmacological inhibition of TGF-β/ALK5 signaling with the selective inhibitor SB431542 increased Oatp1a4 functional expression, suggesting a role for TGF-β/ALK5 signaling in Oatp1a4 regulation. Taken together, our novel data show that targeting an endogenous BBB drug uptake transporter (i.e., Oatp1a4) may be a viable approach for optimizing CNS drug delivery for treatment of diseases with an H/R component.

<p>Insects are continually exposed to potentially toxic endogenous compounds and xenobiotics that require rapid elimination from the body. Xenobiotic resistance in insects has evolved predominantly by increasing the activity of detoxification enzymes and/or by increasing toxin excretion via the Malpighian (renal) tubules. The tubules have long been known to transport organic anions at high rates. This thesis examines the mechanisms of excretion and detoxification of the organic anion methotrexate (MTX) by isolated tissues of the fruit fly <em>Drosophila melanogaster</em>. A radioisotope tracer technique and the Ramsay assay were used to measure MTX secretion. Quantitative PCR (qPCR) was used to evaluate the expression of the genes for putative organic anion transporters. My results show that MTX transport across the Malpighian tubule epithelium is active, saturable, Na⁺-independent and inhibited by a wide range of organic anions including MK-571, probenecid and Texas Red. Pharmacological studies and qPCR analyses suggest multiple transporters are involved in the movement of MTX across the Malpighian tubules. Moreover, chronic exposure of larvae to dietary MTX or salicylate dramatically increases the transepithelial transport of MTX by isolated Malpighian tubules, suggesting that excretion of MTX is upregulated by exposure to these organic anions in the diet. In addition, treatments known to increase expression of specific detoxification enzymes, such as the P450 monoxygenases (P450s) and the glutathione-S-transferases (GSTs), also led to an increase in expression levels of multidrug efflux transporter (MET), multidrug resistance like protein 1 (dMRP) as well as to increased secretion of MTX by the tubules. This latter finding suggests a coordinated response to toxin exposure, so that when detoxification pathways are increased, there is a corresponding increase in the capacity for elimination of the products of P450 and GST enzymes. Finally, the last section of this thesis has shown that RNAi knockdown of a single organic anion transporter gene in the principal cells of <em>D. melanogaster</em> Malpighian tubules is associated with reductions in the expression of multiple, functionally-related genes. Importantly, these results indicate that dMRP andMET are not the dominantMTX transporters in the tubules when flies are reared onMTX-enriched diets. However, reductions in the expression of organic anion transporting polypeptide (OATP) are associated with reduced secretion of the organic anionsMTX, fluorescein and Texas Red. Taken together, these results suggest that OATP and at least one additional transporter, as yet unidentified, are required forMTX secretion. In conclusion, the results of my research contribute to our understanding of the mechanisms of organic anion detoxification and excretion in flies exposed to dietary toxins.</p> / Doctor of Science (PhD)

In vivo studies of hepatobiliary disposition are challenging. The hepatobiliary system is complex, as its physiological localization, complex cellular structure with numerous transporters and enzymes, and the interindividual variability in protein expression and biliary flow will all affect the in vivo disposition of a drug under investigation. The research included in this thesis has focused on the involvement of hepatic transport proteins in the hepatobiliary disposition of rosuvastatin. The impact that several transport inhibitors had on the pharmacokinetics of rosuvastatin was investigated in healthy volunteers and in pigs. The effects were considerable, following inhibition of sinusoidal transport proteins by cyclosporine and rifampicin. These inhibitors significantly reduced the hepatic extraction of rosuvastatin by 50 and 35%, respectively, and the plasma exposure increased by factors of 9.1 and 6.3, respectively. Drug-drug interactions (DDI) resulting in markedly higher plasma exposures are important from a drug safety perspective as increased extrahepatic exposure of statins is associated with an increased risk of severe side-effects, such as myopathy which in rare cases could develop into rhabdomyolysis. The DDI caused by cyclosporine and rifampicin can probably be attributed to inhibition of hepatic uptake transporters. In contrast, inhibition of canalicular transporters by imatinib did not significantly affect the pharmacokinetics of rosuvastatin, which suggests that the intracellular concentration of the inhibitor in the hepatocyte was insufficient to affect the transport of rosuvastatin, or that imatinib is not a sufficiently potent inhibitor in vivo. Furthermore, gemfibrozil administered as a single dose into the jejunum in healthy volunteers and pigs did not affect the plasma or biliary pharmacokinetics of rosuvastatin. The previously reported DDI in humans upon repeated dosing with gemfibrozil might be explained by the accumulation of metabolites able to affect the disposition of rosuvastatin. The investigations presented in this thesis conclude that transport proteins are of considerable importance for the hepatobiliary disposition of rosuvastatin in vivo. The Loc-I-Gut catheter can be applied for the investigation of biliary accumulation and to determine bile specific metabolites, however it has limitations when conducting quantitative measurements. In the porcine model, hepatic bile can be collected for up to six hours and enables the determination of the hepatic extraction in vivo.