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The HERG gene encodes for the delayed rectifier K+ channel in human cardiac tissue and contributes to the repolarization phase of the ventricular action potential. Defects in its activity underlies a cardiac disorders linked to a prolongation in the QT interval known as acquired long QT syndrome. The channel has structural properties that lead to its unintentional inhibition by various classes of drugs and is a source of drug induced cardiac toxicity. To date, no assay has been set as a standard due to variability across laboratories and the use of animals providing variable results due to differences in the ion channels involved in repolarisation. This thesis focuses on the development of testing assays for HERG using animal-free methodology. In Chapter 2, a human embryonic kidney (HEK293) cell line was cultured and transfected with the human HERG gene using animal-free methodologies. The success of the transfection was confirmed using PCR, patch clamp electrophysiology and a non-radioactive rubidium assay. Using a non-radioactive rubidium assay, drug inhibition on the transfected cell line was measured. The IC50 values obtained for a range of drugs were compared to those obtained using electrophysiological studies in the literature and there was a high correlation (r2 = 0.76). In Chapter 3, a human neuroblastoma cell line (SH-SY5Y) was tested for its validity for testing the effect of drugs on the endogenously expressed HERG K+ channel. The drug IC50 values obtained using the Rb+ assay were well correlated (r2= 0.82) with patch clamp studies in HERG transfected HEK293 cells in the literature. Clomipramine a clinically used antidepressant causes prolongation in the QT interval, however its mechanism of action on cardiac cells leading to this cardiotoxic effect is unclear. In this study, clomipramine was tested using HERG transfected HEK293 cells and the neuroblastoma cell line (SH-SY5Y) using a rubidium assay and whole cell patch clamp. Clomipramine inhibited HERG with an IC50 value of 8.35 µM and 2.18 µM in HERG transfected HEK293 cells and the neuroblastoma cell line (SH-SY5Y) using the rubidium assay respectively. Clomipramine inhibited HERG currents with an IC50 value of 0.50 µM using the patch clamp technique in HEK293 cells. The results indicate that the prolongation in the QT interval caused by clomipramine may involve HERG inhibition. The HERG K+ channel is regulated by several protein kinases including protein kinase A and protein kinase B. In Chapter 5, the specific PKC activator and phorbol ester PDA was used to study HERG regulation by PKC in HERG transfected HEK293 cells. PDA caused a reduction in HERG currents in HEK293 cells. The PKC pseudo substrate inhibitor PKC [19-36] did not inhibit the effect of PDA on HERG currents. The results of the study suggest that (1) PDA could be acting directly on the channel and inhibiting its function or (2) PDA is activating other proteins which are affecting HERG currents in the HERG transfected HEK293 cells.

Resveratrol (RES) has been associated with numerous pharmacological effects. Yet its pharmacokinetics is not clearly understood. It is known to get extensively metabolized into its sulfated and glucuronidated metabolites and has very low circulating RES concentrations in plasma. Although the concentrations of conjugated metabolites of RES have been reported to be much greater than that of RES, they have not been evaluated. This also becomes important in light of positive biological activities reported for sulfated metabolites of RES. Conjugation is a complex process which can sometimes be a reversible process and needs comprehensive evaluation to better understand RES and its metabolites' disposition. There has been a debate among the researchers regarding the differences in kinetics of preformed versus in vivo formed metabolites in the light of guidelines issued by regulatory bodies regarding metabolites in safety testing (MIST). We have addressed the above questions in this work, in addition to evaluating brain permeability of a potent RES analog, trimethoxy-trans-stilbene (TMS). Chapter 1 presents a detailed introduction, hypothesis and significance of my work. Chapter 2 includes the development and validation of a bioanalytical method for quantitation of RES and its metabolites on LC/MS/MS. We were able to develop and validate a robust bioanalytical method to quantitate RES and its four major metabolites resveratrol-4'-glucuronide (R4'G), resveratrol-3-glucuronide (R3G), resveratrol-4'-sulfate (R4'S) and resveratrol-3-sulfate (R3S). In Chapter 3, lung as a possible metabolizing organ for RES was evaluated. This study was performed in vivo in mouse model using multiple site of administration and single site of sampling approach. In vitro studies were also performed to confirm the in vivo results. Inter species differences in RES pulmonary metabolism were also studied. We observed lungs to be the major metabolizing organs for RES with inter species differences in its metabolism. Chapter 4 provides detailed pharmacokinetics of sulfated metabolites of RES, i.e. resveratrol-3-sulfate (R3S) and resveratrol-4'-sulfate (R4'S) in mouse model by both systemic and oral routes. In vitro studies were also conducted to test the desulfation in liver. Although we did not observe any significant RES in plasma, we observed from our in vitro studies that sulfated metabolites were desulfated in liver. Chapter 5 explains the detailed pharmacokinetics of glucuronidated metabolites of RES i.e. resveratrol-3-glucuornide (R3G) after both systemic and oral route. R3G was observed to undergo enterohepatic circulation. Explanation of R3G disposition in hepatocytes and enterocytes were proposed based on our own and reported results. In Chapter 6 we compared the differences in the kinetics of preformed versus in vivo formed metabolites using modeling and simulation approach. Individual models for disposition of RES, R3S and R3G were developed. These models were combined to give a global model for RES metabolism into R3S and R3G. Simulations were performed under two assumptions; preformed versus in vivo formed metabolite kinetics a) are same and b) they are not same. Our results reported that preformed and in vivo formed metabolite kinetics are not same at least for hydrophilic phase II metabolites. Chapter 7 includes method development and validation for quantitation of TMS in plasma and brain of mouse. Chapter 8 includes a steady state study to characterize the pharmacokinetic parameters of TMS, which was used to evaluate brain permeability of TMS. In summary we developed a robust bioanalytical method for direct quantitation of RES and its metabolites, found the lung to be a major metabolizing organ for RES, delineated complex kinetics of conjugated metabolites of RES, and showed differences in preformed versus in vivo formed metabolite kinetics and better brain permeability of TMS. / Pharmaceutical Sciences

When developing new active safety systems or improving existing systems, conducting performance evaluations is necessary. By performing these evaluations during early development stages, potential problems can be identified and mitigated before the system moves into the production phase. Testing active safety systems can be difficult since the characteristic scenarios may have complex interactions. Using real vehicles for performing these types of scenarios is difficult, expensive, and potentially dangerous. Alternative methods, such as using inflatable targets, scale models, computer simulations or driving simulators, also suffer from drawbacks. Consequently, using virtual reality as an alternative to the traditional methods has been proposed. In this case, a real vehicle is driven while wearing a head-mounted display that presents the scenario to the driver. This research aims to investigate the potential of such technology. Specifically, this work investigates how the chosen technology affects the driver. This investigation has been conducted through a literature review. A test platform was constructed, and two user studies using normal drivers were performed. The first study focused on the effects of visual time delays on driver behavior. This study revealed that lateral behavior changes with added time delays, whereas longitudinal behavior appears unaffected. The second study investigated how driver behavior is affected by different modes of virtuality. This study demonstrated that drivers perceived mixed reality as more difficult than virtual reality. The main contribution of this work is the detailed understanding of how time delays and different modes of virtuality affect drivers. This is important knowledge for selecting which scenarios are suitable for evaluation using virtual reality. / <p>The series name <em>Linköping Studies in Science and Technology Licentiate Thesis</em> is incorrect. The correct series name is <em>Linköping Studies in Science and Technology Thesis</em>.</p>