The aryl hydrocarbon receptor (AhR) is a PAS domain transcriptional factor also known as the ¡§dioxin receptor¡¨ or ¡§xenobiotic receptor.¡¨ My thesis work has uncovered an endobiotic role for AhR in hepatic steatosis and other metabolic functions. Activation of AhR induced spontaneous hepatic steatosis, which is characterized by the accumulation of triglycerides. The steatotic effect of AhR was likely due to the combined upregulation of fatty acid translocase CD36/FAT, suppression of fatty acid oxidation, inhibition of hepatic export of triglycerides, and an increase in the mobilization of peripheral fat. Promoter analysis established CD36 as a novel transcriptional target of AhR. Moreover, the steatotic effect of an AhR agonist was inhibited in mice deficient of CD36. Results from this study may help to establish AhR and its target fatty acid translocase CD36 as attractive targets for intervention in fatty liver disease. The liver X receptors (LXRs), both the Ñ and Ò isoforms, are nuclear receptors identified as sterol sensors that modulate cholesterol and lipid metabolism and homeostasis. In the second part of my thesis research, I report a novel LXR-mediated mechanism of androgen deprivation. Genetic or pharmacological activation of the liver X receptor (LXR) in vivo lowered androgenic activity by inducing the hydroxysteroid sulfotransferase 2A1 (SULT2A1), an enzyme essential for the metabolic deactivation of androgens. Activation of LXR also inhibited the expression of steroid sulfatase (STS) in the prostate, which may have helped to prevent the local conversion of sulfonated androgens back to active metabolites. At the physiological level, activation of LXR in mice inhibited androgen-dependent prostate regeneration in castrated mice. Treatment with LXR agonists inhibited androgen-dependent proliferation of prostate cancer cells in LXR- and SULT2A1-dependent manner. The ability of LXRs to regulate androgen metabolism makes them novel therapeutic targets for the treatment and prevention of hormone-dependent prostate cancer. Taken together, my work has revealed novel functions of AhR in lipid metabolism and LXR in androgen deprivation. It is hoped that understandings of the endobiotic functions of AhR and LXR may establish these two receptors as therapeutic targets for the management of metabolic disease in humans.

Breast cancer incidence in women in the United States is 1 in 8 (about 13%). In the U.S., breast cancer death rates are higher than any other cancer besides lung cancer, and more than 25% cancers are classified as breast cancer. In 2008, an estimated 182,460 new cases of invasive, along with 67,770 of non-invasive (in situ), breast cancers were diagnosed in women in the U.S. About 40,480 women in the U.S. were projected to die in 2008 from breast cancer. Paclitaxel (Taxol), a microtubule (MT) stabilizing agent, was originally noted to be useful against breast cancers. Yet, like with many other cancer therapeutic agents, resistance to paclitaxel remains a significant problem in treating malignancies. One potential mechanism for the resistance observed is alterations in microtubule dynamics and altered binding of paclitaxel to its cellular target, the microtubule. Stathmin is a highly conserved, 17kDa protein that functions as an important regulator of microtubule dynamics. Several studies have shown potential correlations between stathmin levels and resistance to paclitaxel. The latest results from our collaborator Prof. Mary Ann Jordan at the University of California-Santa Barbara clearly show that reduction of the level of stathmin in BT549 cells increases their sensitivity to paclitaxel (vide infra). This reduction must obviously result in some changes in the affected cells' proteome, which delivers a signal of regulatory importance to the MT system; The goal of the project was to detect and characterize the earliest proteomic changes, using 2-D DiGE and MALDI-TOF-MS, of BT549 breast cancer cell lines engineered with constitutively lowered stathmin levels. Two proteins, Protein Kinase C epsilon and Microtubule-Associate Protein 6, were identified to be expressed at lower levels with statistical significance, and potential mechanisms exit for those two proteins to interact with stathmin and/or microtubules are discussed. Based on this information, it is proposed that stathmin may play a role in certain integrating as well as diverse intracellular regulatory pathways. It is expected that this more detailed understanding of protein profile changes in these cells will allow for more rational decision-making in further research of the mechanisms leading to paclitaxel resistance.

Preterm birth (PTB), birth prior to 37 weeks of gestational age, is a major cause of early childhood mortality and morbidity in the United States. 17alpha-hydroxyprogesterone caproate (17-OHPC) has recently been documented to reduce the incidence of preterm birth. A complete understanding of the pharmacokinetics of 17-OHPC will help in improving clinical outcome. The goal of this dissertation research was to evaluate the preclinical and clinical pharmacology of 17-OHPC in order to optimize the use of this drug in preventing preterm birth. Studies in human liver microsomes and human hepatocytes indicate that 17-OHPC is metabolized by CYP3A. Significant transplacental transfer of 17-OHPC (cord blood to maternal plasma ratio of 0.3) has been observed in pregnant subjects. Studies were performed in fetal hepatocytes to evaluate the metabolism of 17-OHPC. Fetal hepatocytes demonstrate the ability of human fetal liver to metabolize 17-OHPC to fetal specific metabolites, with oxidation being the major metabolic pathway. Further, 17-OHPC and/or its metabolites inhibit bile salt transport in both adult and fetal hepatocytes. To understand the clinical pharmacology of 17-OHPC, pregnant women who received 17-OHPC for clinical reasons were studied and blood samples collected periodically. Wide-interindividual variation was observed in the pharmacokinetics of 17-OHPC in pregnant subjects. The half life of 17-OHPC was 9 days and plasma concentrations of 17-OHPC did not achieve steady state. The race and body mass index of the pregnant subjects affect the plasma levels of 17-OHPC. In conclusion, since CYP3A is involved in the oxidative metabolism of numerous commonly used drugs; 17-OHPC may be involved in clinically relevant metabolic drug interactions with co-administered CYP3A inhibitors or inducers. Since 17-OHPC crosses the placental barrier and reaches the fetus, use of higher doses of 17-OHPC should be approached with caution. The clinical effectiveness of 17-OHPC in preventing preterm birth has been observed in only 33% of the patients. Given the wide interindividual variability, modification of the starting dose based on BMI and race alongwith monitoring of plasma levels and adjustment of subsequent doses accordingly may be needed to improve therapeutic outcomes in the treatment of preterm birth with 17-OHPC.

Orthotopic liver transplantation (OLT) is the only curative therapy for end-stage liver diseases. To overcome organ shortage, organs from extended criteria donors, which would ordinarily be discarded, are used sometimes. These organs provide additional grafts; however, they are more susceptible to ischemia-reperfusion (I/R) injury. I/R injury, an unavoidable process during OLT, is a major cause of liver graft non-function and failure, requiring urgent re-transplantation, which further depletes the scare organ pool. To date, no therapy is available to reduce or prevent I/R injury. Prostaglandins (PG) have well characterized vasodilatory and anti-platelet aggregatory actions. Many PG analogues, including prostacyclin (PGI2), have been evaluated for their ability to reduce hepatic I/R injury after OLT. Poor stability, intolerable side effects, and the inability to show a significant difference in primary endpoint have limited their clinical application so far. Treprostinil, a relatively new FDA-approved PGI2 analogue, has a higher stability, potency, and longer elimination half-life than other PGI2 analogues available. The objectives of this dissertation were to examine the efficacy of treprostinil in protecting the liver graft against I/R injury during OLT. Proof of concept of treprostinil minimizing hepatic I/R injury was demonstrated in a rat OLT model. Further analysis showed that I/R injury significantly down-regulated CYP2E1, CYP2C11, and CYP3A mRNA, protein expression, and activity, as well as the expression of several hepatic transporters in liver graft post-OLT. Treprostinil improved hepatic expression and activity of CYP450 enzymes and transporters. In particular, Bsep mRNA expression was restored to normal and Mrp2 and P-gp protein expression were up-regulated. In vitro studies confirmed that treprostinil does not inhibit or induce the metabolism of immunosuppressive medications. These findings support co-administration of treprostinil with cyclosporine A, tacrolimus, sirolimus, or mycophenolic acid to adult OLT patients without concern for any drug-drug interaction. This is the first study to examine the efficacy of treprostinil for protection of liver grafts against I/R injury during OLT. A clinical study has been initiated to examine the safety and efficacy of perioperative treprostinil administration to adult OLT patients. Collectively, this work makes significant contributions to the field of liver transplantation and, potentially, solid organ transplantation.

The aim of the dissertation was to identify the systematic contributors that modify the estimated population parameters and that explain sources of variability in drug exposure (Chapter 2-4), response, and clinical outcome (Chapter 5-7). The source of measurable variability evaluated in the thesis include patient characteristics in chapter 2-3, patient behavior in chapter 4 (e.g. dosing history), biological system in chapter 5-7, and inferior clinical practice in chapter 5-7. The dissertation was predominantly non-linear mixed effect modeling and Monte Carlo simulation methods in NONMEM® and R. Our results in chapter 2-4 showed that incorporating covariate information into population PK models identified substantial systematic contributors to the variability in drug exposure for both perphenazine and escitalopram. Race and smoking status in the past week were identified as two significant covariates affecting clearance of perphenazine. CYP 2C19 genotype, age, and weight strongly influenced the CL/F of escitalopram. The measurement error associated with an incorrect or incomplete dosing history affected the population PK parameter estimation of escitalopram in the non-linear mixed effect modeling process. Furthermore, our simulation results in chapter 5-7 showed that three intervention approaches may lead to lower cardiovascular risk compared to current clinical practice strategy: 1) BP can be calibrated with respect to clinic visit times with consideration of PK/PD/dosing regimen. 2) BP-misclassification in current clinical practice is around 20~45% depends on clinic visit time. Optimal clinic visit time exists. In general, patients should avoid early morning and late afternoon visit which lead to the highest BP misclassification. 3) It is important to decrease patients BP in a timely fashion. Initiating antihypertensive treatment with the higher tolerable dose as well as setting a lower goal BP of 120 mm Hg resulted in a significantly lower cardiovascular risk. In conclusion, the dissertation identified three potential interventions to be considered in the clinical practice or antihypertensive drug labeling for better BP management: BP calibration based on clinic visit time; patients should generally have post treatment clinic visit times between 11:00 AM ~ 3:00 PM; a high dose strategy for antihypertensive drug therapy.

The N-end rule pathway relates the in vivo half-life of a protein to the identity of its N-terminal residue. The conjugation of arginine (Arg) from Arg-tRNAArg to N-terminal Asp, Glu, or Cys is a universal eukaryotic protein modification that can lead to ubiquitylation and proteasomal degradation of the resulting Arg-conjugated proteins through the N-end rule pathway. The mammalian ATE1 gene encodes Arg-transferase that mediates all known N-terminal arginylation reactions. ATE1-/- embryos die owing to various cardiovascular defects including ventricular hypoplasia, ventricular septal defect, and late angiogenesis. The genomewide functional proteomics previously identified a set of RGS proteins (RGS4, RGS5, and RGS16) as in vivo substrates of ATE1. These RGS proteins are important negative regulators of Gáq-activated signaling for myocardial growth and vascular maturation/integrity. In my first project, I attempted to determine the role of ATE1-dependent posttranslational arginylation in Gáq-dependent cardiac signaling. I constructed and characterized ATE1-/-GáqTg compound mutant mice, where Gáq is exclusively overexpressed in the heart from áMHC promoter. I found that while Gáq overexpression in the heart rescues significantly cardiac defects in ATE1-/- embryonic hearts, it does not cause a noticeable change in vascular defects. These results together suggest that ATE1 controls cardiac development and signaling in part through Gq-activated signaling pathways. In the second project, I generated RGS5 transgenic mouse (TG) strains overexpressing either MC-RGS5 (wild-type, short-lived) or MV-RGS5 (mutant, long-lived) from vascular smooth muscle-specific SM22á promoter to determine the physiological importance of RGS5 proteolysis in Gq signaling of VSMC. Both MC-RGS5 and MV-RGS5 mice were viable and fertile without any visible defects. However, MC-RGS5 female v mice demonstrated impaired delivery in that newborn pups were often found dead associated with an absence of milk in their stomachs. In contrast, MV-RGS5 mice did not show this phenotype. The mis-regulated RGS5 proteolysis in MC-RGS5 mice may result in the failure in oxytocin-induced uterine and mammary gland smooth muscle contraction. In summary, my research provides an insight into the role of N-end rule pathway in cardiovascular Gq signaling.

Transplant patients at high risk of invasive mold infections receive voriconazole for prophylaxis. Large variability in voriconazole exposure with a fixed dosing regimen was observed. Low exposure of voriconazole predisposes patients for infection. High concentrations are associated with toxicity. The objectives are to characterize the pharmacokinetics of voriconazole in transplant patients, to identify factors associated with the variability in the pharmacokinetics, and to develop adequate dosing guidelines for transplant patients. Liver, lung and pediatric bone marrow transplant (BMT) patients were enrolled. Multiple blood samples were collected within one dosing interval (totally 75 full pharmacokinetic profiles). Voriconazole plasma concentrations were measured using HPLC. Non-compartmental analysis was performed using WinNonlin. Population pharmacokinetic models were developed using NONMEM. Covariate models were built using a forward addition and reverse removal approach. Precision of parameter estimation was evaluated by bootstrapping. Adequate dosing regimens were developed using Monte Carlo simulations. There was a good correlation between AUCo-∞ and trough voriconazole plasma concentrations. Bioavailability of voriconazole is substantially reduced in lung transplant and BMT patients during the early post-transplant period. Pharmacokinetics of voriconazole is significantly associated with postoperative time, liver function and CYP2C19 genotype in liver transplant patients. Pharmacokinetics of voriconazole is significantly associated with postoperative time and cystic fibrosis in lung transplant patients. Pharmacokinetics of voriconazole is significantly associated with liver function in BMT patients. Patients with cystic fibrosis (CF) exhibited a significantly lower bioavailability than non-CF patients. Donor characteristics had no significant correlation with pharmacokinetics of voriconazole in liver transplant patients. Bioavailability of voriconazole is similar between lung transplant and BMT patients. Compared to liver and lung transplant patients, BMT patients had significantly higher clearance and significantly lower volume of distribution. In conclusion, weight-adjusted or fixed dosing regimens resulted in highly variable exposure of voriconazole in liver transplant, lung transplant and BMT patients. Given that trough voriconazole concentration is a good measure of drug exposure (AUC), voriconazole dose can be individualized based on trough concentrations. Population analysis demonstrated inadequacy of oral administration of voriconazole and adequacy of intravenous administration during the first few post-operative days, followed by oral doses for optimal drug exposure.

Hydrodynamic gene delivery (HGD) has emerged as an effective and safe method for transfecting liver hepatocytes in vivo, and has potential for gene therapy of liver fibrosis. The objective of this study was to evaluate the effectiveness and safety of HGD using CCl4 induced fibrotic liver in rats as a model. I demonstrated that there is a progressive reduction of efficiency of HGD in rats with increasing severity of liver fibrosis. Using a reporter plasmid containing luciferase gene, we showed over 2,000-fold decrease in luciferase activity in the liver with advanced fibrosis compared to that of control animals. Reduction in reporter gene expression in fibrotic liver was correlated to lower copy number of plasmid DNA and less amount of luciferase mRNA in the liver. Microscopy analysis revealed significant accumulation of collagen fibers in the boundary of liver lobules and thickened hepatic sinusoidal endothelium. The morphological changes in fibrotic liver are associated with restriction of flow-through across the liver of DNA solution hydrodynamically injected and are responsible for the reduced gene delivery efficiency of the hydrodynamic procedure. Results from electrocardiogram and serum biochemistry show no difference between the control and fibrotic animals undergone HGD. These results suggest that the HGD is a safe method for gene transfer in animals with liver fibrosis but the effectiveness of gene delivery decreases with increase of severity of fibrosis. Future work should focus on adjustment of injection parameters (DNA dose, injection volume, injection speed) for optimal gene delivery to fibrotic liver.

Thesis (Ph. D.)--University OF MICHIGAN.

Thesis (Ph. D.)--University OF MICHIGAN.