The two known estrogen receptors, ERa and ERb, are the products of different genes on separate chromosomes. Of these, ERa has been the most extensively studied, and its expression in breast cancer determines the ER+ phenotype. ERb, on the other hand, was discovered only recently and its role in breast cancer pathology remains unclear. ERb inhibits E2-induced proliferation of T47D breast cancer cells in addition to decreasing the expression of cell cycle related genes. Clinical studies have shown a positive correlation between ERâ expression with disease-free survival and overall survival in breast cancer patients. ERb activation with a selective ERb agonist could antagonize the stimulating activity of the ERa in breast cancer cells, and such an ERb agonist could help overcome acquired resistance. Therefore, this work began a search for such agents. A one-pot hydrozirconation-transmetallation-aldimine addition sequence that leads to allylic amides, homoallylic amides and C-cyclopropylalkylamides was significantly accelerated by microwave technology and used for library preparation. The conventional methodology provided a first generation discovery library. A potentially antiestrogenic compound was identified in a transcriptional screening assay from this library, C-cyclopropylalkylamide 26a (O-ethyl-N-{2-[(1S*,2R*)-2-{(R*)[(diphenylphosphinoyl)amino](phenyl)methyl}cyclopropyl]-ethyl}-N-[(4-methylphenyl)sulfonyl]carbamate; a.k.a. CK1-183). Following up on these findings and with the goal to expand the scope of the synthesis methodology, a second generation library of allylic amides and C-cyclopropylalkylamides was prepared. The new library was screened in a fluorescence polarization based homogenous in vitro assay at ERa, and hits were further evaluated in cell-based assays. Three new C-cyclopropylalkylamides, 37c, 37a and 39c, were identified with improved potency over the lead agent 26a against 17b-estradiol (E2) stimulated MCF-7 cells. This second generation library was screened against both ERs. The screening results served to build an SAR model of allylic amides and C-cyclopropylalkylamides at ERa and ERb. A hit from the ERa screen, C-cyclopropylalkylamide 37d (N-(R*)-(((1R*,2R*)-2-butylcyclopropyl)-(4-(phenyl)phenyl)methyl)benzamide), contained a biphenyl core and served as a starting point for the design and synthesis of a third generation of C-cyclopropylalkylamide ER targeting agents. Biphenyl C-cyclopropylalkylamides represent novel structural scaffolds for design and synthesis of ERa and ERb targeting agents and a novel avenue for selective estrogen receptor modulator (SERM) development.

Significant variability in the pharmacokinetics of drugs such as cyclosporine, tacrolimus, sirolimus and mycophenolic acid, is seen in liver transplant patients. These agents are primarily metabolized by CYP3A4 or UGT1A1, and are also substrates for drug transporters such as P-glycoprotein, multidrug resistance protein 2 (MRP2) and bile salt export pump (BSEP). Factors modulating the expression and activity of these enzymes and transporters will lead to changes in the clearance of immunosuppressive agents. Inflammation associated with infection or organ rejection after transplantation can modulate the expression and activity of CYP3A4, UGT1A1 and various drug transporters. HIV-protease inhibitors (HIV-PIs) used for the treatment of HIV infection are shown to modulate the blood levels of immunosuppressive agents. The primary goal of this dissertation research was to evaluate the effect of endogenous chemicals such as cytokines and exogenous compounds such as HIV-protease inhibitors on human hepatic drug metabolizing enzymes and transporters using primary cultures of human hepatocytes. The results from this research indicate that HIV-protease inhibitors generally decrease CYP3A4 activity inspite of increasing CYP3A4 mRNA and protein. The effect of PIs on CYP3A4 recovers over time. PIs also increased UGT1A1 mediated metabolism as well as mRNA expression of transporters. These effects are dependent on the HIV-protease inhibitor used and its concentration. Additionally, HIV-PIs increased the expression and activity of hepatic efflux transporters. These effects of HIV-PIs are found to be potentially mediated through alteration in PXR and CAR expression. Cytokines, released during infection or inflammation process, were observed to downregulate the expression and activity of UGTs and transporters through modulation of PXR and CAR.

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Experiments in this dissertation were conducted to characterize the effects of d-fenfluramine on body temperature and the mechanisms by which d-fenfluramine alter body temperature. The experiments were conducted in conscious male Sprague-Dawley rats. Body temperature was measured in all animals using telemetry. The results of the experiments indicated that d-fenfluramine altered body temperature in animals kept 28, 22, 16 and 4 degrees Centigrade. D-fenfluramine produced hyperthermia in animals kept at 28 degrees Centigrade and varying degrees hypothermia at normal and cooler ambient temperatures. Further experiments were conducted to explore the effects of d-fenfluramine on brown adipose tissue (BAT) thermogenesis, cutaneous vascular tone and whole body oxygen consumption. In animals kept at 22 and 4 degrees Centigrade, we found that d-fenfluramine activated BAT, as indicated by a decrease in BAT norepinephrine content, to the same magnitude. Thus, the hypothermia seen at normal and cooler ambient temperature was not due to lack of BAT activation. Also, activation of BAT by d-fenfluramine was mediated through the sympathetic nervous system and through release of central serotonin, since ganglionic blocker pentolinium and serotonin reuptake inhibitor fluoxetine blocked d-fenfluramine-mediated BAT activation. In animals kept at 16 degrees Centigrade, d-fenfluramine increased tail-skin temperature (Tsk), an index of cutaneous vascular tone, indicating that d-fenfluramine produced cutaneous vasodilation. d-fenfluramine-induced increase in Tsk was mediated through withdrawal of the sympathetic vasoconstrictor tone to the tail, since pentolinium blocks this effect. In animals kept at 28 degrees Centigrade, d-fenfluramine produced a decrease in Tsk, indicating vasoconstriction. The effects of d-fenfluramine on the Tsk were mediated through release of serotonin, since fluoxetine blocked these effects. D-fenfluramine increased whole body oxygen consumption, an index of metabolic activity and the increase was due to BAT activation, since pentolinium prevented the increase. Thus, although d-fenfluramine increased metabolic activity through BAT activation, the increase was insufficient to make up for the heat loss produced by cutaneous vasodilation and thus produces hypothermia. The hyperthermia seen at 28oC is due to activation of BAT and the subsequent inability of the animal to lose the excess heat due to cutaneous vasoconstriction produced by d-fenfluramine at 28 degrees Centigrade.

Organ transplantation is an accepted therapy for diseases that result in chronic irreversible failure of various organs. During transplantation, the transplanted organ is subjected to two inflammatory processes, alloantigen-independent (ischemia/reperfusion injury) and alloantigen-dependent (rejection), both of which involve release of cytokines. The objectives of this dissertation were to evaluate the effect of acute rejection of liver in rats on hepatic and extra-hepatic drug metabolizing capacity for phase I and II enzymes, to evaluate the effect of chronic rejection of liver in humans on hepatic metabolizing capacity of phase I and II enzymes, to evaluate the effect of acute and chronic rejection of liver on hepatic and extra-hepatic protein and mRNA expression of P-glycoprotein and to evaluate the effect of different cytokines on the constitutive and inducible hepatic CYP3A4 activity and protein expression in human hepatocytes. Alloantigen-independent inflammation and altered blood flow caused a reduction in hepatic mRNA of different phase I and II enzymes. However, this reduction did not significantly alter protein levels of all CYP450 enzymes studied. Occurrence of rejection resulted in further reduction in mRNA, protein levels and activity of all CYP450s studied. Syngeneic and allogeneic transplantation caused reduction in the metabolic capacity of extra-hepatic tissues and increased expression of P-gp in the liver. Chronic rejection of the liver in humans selectively altered the activity and protein expression of different phase I and II enzymes and increased P-gp protein expression. In human hepatocyte cultures, IL-1â, IL-6 and TNF-á decreased the activity and protein expression of both constitutive and induced forms of CYP3A4 enzyme. Pre-exposing or co-exposing the hepatocytes to cytokines reduced the ability of rifampicin to induce CYP3A4. In conclusion, acute and chronic rejection of liver significantly altered the expression and activity of several drug metabolizing enzymes and transporters. The magnitude of these alterations was higher in acute rejection. Acute rejection also caused alterations in the metabolizing ability and transporters expression in renal and pulmonary tissues. Cytokines play a major role in modulating the activity of drug metabolizing enzymes and transporters and may contribute to the large inter-individual variation in the pharmacokinetics of drugs in transplant patients.

The primary goal of this dissertation research was to investigate the effect of dehydroepiandrosterone (DHEA) administration on recovery from a GABA-agonist challenge in healthy young and older adults. DHEA and its sulfate metabolite DHEA-S are natural steroid hormones that are produced primarily in the adrenal glands and act as precursors to other hormones (e.g., testosterone and estrogen) in the body. Animal studies have demonstrated antagonistic effects of DHEA and DHEA-S at the GABA receptor complex. When compared to young adults, the elderly have lower concentrations of DHEA and DHEA-S and recover more slowly from the CNS effects of benzodiazepines, the most commonly prescribed class of GABA-agonists. To investigate the role of DHEA and/or DHEA-S as excitatory steroids in the human brain, a GABA-agonist challenge study was proposed. Secondary goals included evaluations of SEM variability over repeated same-day testing and assessing the influence of age and endogenous concentrations of DHEA and DHEA-S on SEMs. Both young (20 to 30 years) and older (60 to 79 years) men and women participated in this four-way crossover of placebo, DHEA, alprazolam plus placebo and alprazolam plus DHEA. For the 12.5 hours after the drug or placebo was administered, responses mediated via the GABA-A receptor complex (saccadic eye movements (SEMs), sedation, memory, and psychomotor performance) were assessed and blood samples were collected for the purpose of determining DHEA, DHEA-S, and alprazolam concentrations. These data demonstrate that DHEA administration enhanced impairment in older men and women and did not alter impairment in young men and women. DHEA administration did not accelerate recovery from GABA-agonist challenge in the older subjects. In fact, the opposite effect was seen with the older men and women who demonstrated saccades with slower velocities and longer durations during the recovery phase of the DHEA/Alp treatment. A surprising outcome of this report is the number of older subjects who experienced impairment at a level where they were unable to complete the SEM tasks in both treatments. The number of older subjects unable to perform the SEM tasks during the DHEA/Alp treatment was significantly greater than during the PL/Alp treatment.

Population pharmacokinetic (PK) and pharmacodynamic (PD) modeling using a mixed effect modeling (MEM) approach has been widely used for various drug classes during development. The MEM approach provides a significant advantage when analyzing large scale clinical trials and special population where only a few samples are available per subject. The aims of this thesis are to explore the applications and advantages of MEM approach in the analysis of target populations (e.g., late-life depression, intensive care unit patients) from various aspects. 1): To characterize the sources of variability and evaluate the impact of patients¡¯ specific characteristics on SSRIs disposition using hyper-sparse concentration data. This study demonstrated that age and weight are significant covariates on citalopram clearance and volume of distribution. The age effect persists across the entire age range (22 to 93 years). Thus elderly subjects may need to receive different dose of citalopram based on their age. The other late-life depression study shows that weight and CYP2D6 polymorphisms significantly impact on maximal velocity (Vm) of paroxetine elimination. Thus, female and male subjects with different CYP2D6 genotypes may receive different dose based on their metabolizer genotype. 2): To optimize a dosing strategy for general medical and intensive care unit (ICU) patients receiving enoxaparin by continuous intravenous infusion. The study suggests that dose should be individualized based on patients¡¯ renal function and weight. It is also found that patients in the ICU tend to have higher exposure, thus should receive lower dose than those in the general medical unit. 3): To evaluate the consistency of exposure using the deviation between model-predicted and observed concentrations (Cpred/Cobs ratio) and assess the stability and robustness of using the ratio in reflecting erratic adherence patterns. The simulations demonstrate that ratio could be used as the indicator of the extreme adherence conditions for both long and short-half life drug. The knowledge gained in the thesis will contribute to the understanding the sources of variability in target population, including subjects specific characteristics, enzyme genetics and adherence, under conditions of highly sparse concentration sampling. This provides a basis whereby the magnitude and consistency of exposure can be examined in conjunction with the maintenance response of subjects in a future study as response data become available.

Cytochrome P450 (CYP) enzymes are an important class of heme-containing proteins that catalyze oxidation reactions leading toward the removal of a wide variety of endogenous and exogenous substrates including prescription drugs. The activities of CYP enzymes are regulated primarily at the transcription level involving the regulatory sequences at the 5-flanking region of the CYP genes. The objective of this dissertation study was to characterize the function of the 5-flanking sequences of selected CYP genes primarily responsible for drug metabolism. Various sequences from the 5-flanking regions of different CYP genes (CYP1A2, CYP2C9, CYP2C18, CYP2D6, CYP2E1, and CYP3A4) were cloned in expression vectors and tested for their activity in driving reporter gene expression in mouse livers and in transfected HepG2, 293, and BL-6 cells under optimized conditions. It was demonstrated that among the tested 5-flanking regions of CYP genes, the CYP2D6 promoter showed the highest activity both in vivo and in vitro. The activities of various 5-flanking regions of CYP genes in sustaining transgene expression were then tested in mouse liver and compared to those of other promoter sequences. As a result, the CYP2D6 promoter showed the highest activity and its activity was comparable to that of many established promoters. The mechanism underlying CYP promoter activities in vivo and in vitro were then studied using the CYP2C9 promoter as a model. Activities of various 5-flanking sequences of CYP2C9 were evaluated by using deletion mutations of plasmid constructs in combination with transfection in mouse livers and in HepG2 cells. Finally, the role of PXR and CAR nuclear receptors in regulating CYP2C9 activation was investigated. The results show that both CAR and PXR are essential for CYP2C9 activation and that the regulatory elements reside in the proximal 1-2 kb region upstream of the CYP2C9 gene.

Etoposide is a widely used topoisomerase II inhibitor particularly useful in the clinic for treatment of disseminated tumors, including childhood leukemia. However, its use is associated with the increased risk of development of secondary acute myelogenous leukemias. The mechanism behind this is still unclear. It was hypothesized that etoposide ortho-quinone, a reactive metabolite previously shown to be generated in vitro by myeloperoxidase, the major oxidative enzyme in the bone marrow cells from which the secondary leukemias arise, might be a contributor to the development of treatment-related secondary leukemias. Experiments showed that the glutathione adduct of etoposide ortho-quinone was formed in myeloperoxidase-expressing human myeloid leukemia HL60 cells treated with etoposide, that its formation was enhanced by addition of the myeloperoxidase substrate hydrogen peroxide, and that the glutathione adduct level was dependent on myeloperoxidase. Both the normoisotopic and a stable isotope-labeled version of the glutathione adduct were synthesized. The latter was used for liquid chromatography-mass spectrometry-based quantitative analyses of the adduct formed by the cells. Discodermolide and dictyostatin are two strucuturally related natural products that possess potent microtubule stabilizing activity. Discodermolide advanced to Phase II clinical trials, but the trials were halted for unannounced reasons. Here, both agents were found to be extensively metabolized by human liver microsomes in vitro. In order to determine the metabolic soft spots in the molecules, the chemical structures of the metabolites of discodermolide and dictyostatin were elucidated by liquid chromatography-mass spectrometry. At least eight metabolites of discodermolide and six metabolites of dictyostatin were formed in human liver microsomes in vitro. The terminal diene groups on discodermolide and dictyostatin were found to be the metabolic soft spots. Results from these studies can be used in future medicinal chemistry design and synthesis work to decrease metabolic rate and improve drug metabolism and pharmacokinetic properties, therefore decreasing the doses needed and perhaps even the toxicity.

Therapeutic hypothermia decreases neurological damage in patients experiencing out-of-hospital cardiac arrest (CA). In addition to hypothermia, critically ill patients are treated with an extensive pharmacotherapeutic regimen. The majority of these medications are hepatically eliminated via the cytochrome P450 (CYP450) system. Changes in drug clearance could limit the putative benefit of hypothermic therapy. With the increased use of therapeutic hypothermia and the fact that critically ill patients receive multiple medications, it is crucial to understand the effects of hypothermia on the disposition and metabolism of drugs used in this population. Thus, it was the overall aim of this research to investigate the effects of therapeutic hypothermia on CYP450-mediated metabolism in an animal model of CA, in human liver microsomes, and in normal healthy subjects. Specifically, hypothermia produced a ~2 fold decrease in the systemic clearance (CLS) of intravenous chlorzoxazone, a specific CYP2E1 probe substrate, in a CA rat model when compared to CA rats treated under normothermic temperatures. The mechanism behind this decrease in CLS was a hypothermia-mediated decrease in the affinity of CYP2E1 for chlorzoxazone. We extended the experimental period to investigate the effects of hypothermia after re-warming, on CYP2E1 and CYP3A2 activity and expression. Our results indicate that rats with CA treated under normothermic temperatures demonstrated a significant decrease in the activities of CYP2E1 and CYP3A2, 24 hrs after injury compared to control. Furthermore, CA significantly decreased the expression of CYP3A2, but not the expression of CYP2E1. CA also produced a ~ 10-fold increase in plasma concentrations of interleukin-6 (IL-6) compared to Control. The CA-mediated reduction in CYP3A2 and CYP2E1 activity, mRNA, and the increase in IL-6 plasma concentrations was attenuated by hypothermia. We also investigated the effects of mild and moderate hypothermia on CYP2E1 and CYP3A4 enzyme kinetics in human liver microsomes. Both mild and moderate hypothermia significantly decreased the Vmax of CYP2E1 and CYP3A4. However, hypothermia increased the Km of CYP2E1 but not CYP3A4. These data demonstrate that mild and moderate hypothermia may produce isoform specific alterations of human CYP450-mediated metabolism. Lastly, in a pilot analysis, we showed that mild hypothermia may potentially alter the ClS and the volume of distribution (Vss) of midazolam in mildly hypothermic normal healthy volunteers. Collectively, this work provides evidence that therapeutic hypothermia alters CYP450-mediated metabolism both during cooling and after re-warming. Based on the magnitude of these changes it is clear that intensivists should be cognizant of these alterations and monitor drug levels and outcomes in their patients when possible. In addition to increased clinical attention, future research efforts are essential to delineate precise dosing guidelines and mechanisms of the effects of hypothermia on drug disposition, metabolism, and response.