Over 220 million people worldwide are diagnosed with diabetes and rising prevalence is reported in nearly all surveyed populations. Accordingly, the percentage of pregnancies affected by pre-existing type 1 or 2 diabetes or by diabetes that develops during pregnancy, called gestational diabetes mellitus (GDM), is also on the rise. Today, approximately 8% of all pregnancies are complicated by diabetes. Diabetes alters drug disposition mechanisms in non-pregnant subjects but the impact of diabetes on drug disposition in pregnancy has not been properly evaluated. Atypical drug disposition in pregnancy has implications for maternal and fetal health. Because liver tissue from pregnant women is not readily available, this thesis investigated drug disposition mechanisms primarily in a rat model of experimental GDM. This model consisted of administering streptozotocin, a diabetogenic toxin, to pregnant rats on gestational day 6. One key finding was that elevated circulating lipids in GDM rats competed with drugs (e.g., glyburide and saquinavir) for plasma protein binding so as to increase free drug concentrations. Another key finding was that important hepatic drug efflux transporters (e.g., Mdr1a/b) and metabolic enzymes (e.g., Cyp3a2 and Ugt1a1) were upregulated in GDM as a consequence of, most likely, enhanced nuclear receptor activity (e.g., pregnane X receptor upregulation). Upregulation of hepatic drug efflux transporters and metabolic enzymes, coupled with larger unbound drug fractions, would be expected to increase the hepatic clearance of many drugs. Consistent with this, in GDM, maternal and fetal exposure to the Mdr1 and Cyp3a2 substrate lopinavir was substantially lower than controls post-administration and data supporting enhanced lopinavir metabolite formation were obtained. Placental drug efflux transporters were also examined in this lopinavir study. Elevated placental Mdr1b and Bcrp expression was observed in GDM, which was associated with decreased fetal exposure to lopinavir (even after correcting for maternal unbound concentrations). Taken together, this thesis demonstrates that experimental GDM can significantly impact drug disposition by altering key drug disposition mechanisms. If confirmed in humans, this drug-disease interaction would need to be considered when atypical therapeutic outcomes occur in diabetic pregnancies. Data from experiments with human placentas, obtained from pregnancies complicated by insulin-managed diabetes, is included/discussed.

Although the impairing effects produced by alcohol and their direct effect on the driving task have been well defined for decades, similar information on the effects of Δ9-tetrahydrocannabinol (THC) following cannabis use in relation to driving skill is lacking. A combination of experimental and epidemiological studies is presented that examine the effects of THC on driving and collision risk. Preliminary data from a driving simulation study explores how THC impairs driving ability both acutely and residually and consideration is given to the challenges faced when conducting this type of research. Epidemiological data from a population-level survey demonstrate that the self-reported concurrent behaviours of driving under the influence of alcohol (DUIA) and driving under the influence of cannabis (DUIC) impart an increased risk of past-year collision more than 3 times greater than reporting driving after using a single substance, or not driving following substance use.

Although the impairing effects produced by alcohol and their direct effect on the driving task have been well defined for decades, similar information on the effects of Δ9-tetrahydrocannabinol (THC) following cannabis use in relation to driving skill is lacking. A combination of experimental and epidemiological studies is presented that examine the effects of THC on driving and collision risk. Preliminary data from a driving simulation study explores how THC impairs driving ability both acutely and residually and consideration is given to the challenges faced when conducting this type of research. Epidemiological data from a population-level survey demonstrate that the self-reported concurrent behaviours of driving under the influence of alcohol (DUIA) and driving under the influence of cannabis (DUIC) impart an increased risk of past-year collision more than 3 times greater than reporting driving after using a single substance, or not driving following substance use.

This study determined whether phenotype or genotype tests for thiopurine methyltransferase (TPMT) are cost effective interventions for guiding doses of 6-mercaptopurine in children with acute lymphoblastic leukemia (ALL) compared to standard weight-based dosing. A systematic review of the literature was conducted to assess the accuracy of the TPMT technologies, followed by a cost effectiveness analysis which compared genotype, phenotype and weight-based dosing strategies over a three month time horizon. Both TPMT phenotype and genotype technologies were considered accurate though there is no gold standard. Additionally, included studies were of low methodological quality. Neither of the interventions showed a benefit in survival and both were more costly compared to standard weight-based dosing. At this time there is insufficient evidence to recommend the use of phenotype or genotype testing prior to 6-mercaptopurine therapy to guide initial doses in pediatric ALL patients.

This study determined whether phenotype or genotype tests for thiopurine methyltransferase (TPMT) are cost effective interventions for guiding doses of 6-mercaptopurine in children with acute lymphoblastic leukemia (ALL) compared to standard weight-based dosing. A systematic review of the literature was conducted to assess the accuracy of the TPMT technologies, followed by a cost effectiveness analysis which compared genotype, phenotype and weight-based dosing strategies over a three month time horizon. Both TPMT phenotype and genotype technologies were considered accurate though there is no gold standard. Additionally, included studies were of low methodological quality. Neither of the interventions showed a benefit in survival and both were more costly compared to standard weight-based dosing. At this time there is insufficient evidence to recommend the use of phenotype or genotype testing prior to 6-mercaptopurine therapy to guide initial doses in pediatric ALL patients.

Rationale: Methodological quality of systematic reviews (SRs) remains an area of concern with no consensus on the optimal appraisal instrument to assess quality of published research. Hypothesis: The Grading of Recommendations Assessment, Development and Evaluation (GRADE) is a feasible and useful methodology to assess quality of evidence in pediatric pharmacotherapy. Aim: To demonstrate the applicability of the GRADE to selected topics. Methods: To perform two SRs to illustrate the practical use of the GRADE, highlight methodological challenges encountered and compare the GRADE to the alternative appraisal tool. Results: the GRADE was implemented to conduct two SRs. Judgments on imprecision were challenging. Comparison of the GRADE to alternative tool demonstrated that GRADE may generate different conclusions on overall quality assessment. Conclusion: The GRADE is a valuable innovative tool for assessing quality of evidence, applicable to the field of pediatric pharmacotherapy, with a potential to impact inferences drawn after applying alternative instruments.

Rationale: Methodological quality of systematic reviews (SRs) remains an area of concern with no consensus on the optimal appraisal instrument to assess quality of published research. Hypothesis: The Grading of Recommendations Assessment, Development and Evaluation (GRADE) is a feasible and useful methodology to assess quality of evidence in pediatric pharmacotherapy. Aim: To demonstrate the applicability of the GRADE to selected topics. Methods: To perform two SRs to illustrate the practical use of the GRADE, highlight methodological challenges encountered and compare the GRADE to the alternative appraisal tool. Results: the GRADE was implemented to conduct two SRs. Judgments on imprecision were challenging. Comparison of the GRADE to alternative tool demonstrated that GRADE may generate different conclusions on overall quality assessment. Conclusion: The GRADE is a valuable innovative tool for assessing quality of evidence, applicable to the field of pediatric pharmacotherapy, with a potential to impact inferences drawn after applying alternative instruments.

Cytochromes P450 (CYPs) metabolize a vast array of xenobiotics, including many drugs and toxins. Induction or downregulation of the CYPs can have important consequences such as changes in drug efficacy and altered susceptibility to toxicity. Our study investigated the independent and combined effects of ethanol and nicotine on hepatic and/or brain levels of CYP2E1, CYP2B6 and CYP2A6 in African green monkeys. Monkeys were randomized into four groups (N = 10/group): an ethanol-only group, a nicotine-only group, an ethanol + nicotine group and a control (no drug) group. Ethanol (10% ethanol in sucrose solution) was voluntarily self-administered by the monkeys and nicotine was given as subcutaneous injections (0.5 mg/kg bid). Protein levels and/or in vitro activity were assessed in liver and brain tissue. Also, in vivo pharmacokinetics for chlorzoxazone (metabolized selectively by CYP2E1) and nicotine (metabolized primarily by CYP2A6 and to a lesser extent CYP2B6) were assessed. Hepatic CYP2E1 protein levels, in vitro hepatic CYP2E1 activity and in vivo chlorzoxazone metabolism were increased by ethanol and nicotine, alone and in combination. Hepatic CYP2B6 protein levels and in vitro hepatic CYP2B6 activity were increased by ethanol alone or combined ethanol and nicotine exposure, but were unaffected by nicotine alone. Hepatic CYP2A6 protein levels and in vitro hepatic CYP2A6 activity were decreased by nicotine alone or combined ethanol and nicotine exposure, but unaffected by ethanol alone. Chronic nicotine resulted in higher nicotine plasma levels achieved after nicotine administration, consistent with decreased CYP2A6 activity. Ethanol, alone or combined with nicotine, resulted in lower nicotine plasma levels, an effect that was not mediated by changes in CYP activity. Protein levels of CYP2E1 and CYP2B6 were induced in specific regions and cells in the brain as a result of ethanol self-administration, nicotine treatment and the combined exposure to both drugs. In summary, ethanol and nicotine can alter the expression and/or activity of several important CYP2 family enzymes in primate liver and/or brain.

2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-inducible poly(ADP-ribose) polymerase (TiPARP/ARTD14) is a member of the ARTD family and is regulated by the aryl hydrocarbon receptor (AHR); however, little is known about TiPARP function. In this study we examined the catalytic function of TiPARP and determined its role in AHR transactivation. We observed that TiPARP exhibited auto-mono-ADP-ribosyltransferase activity and ribosylated core histones. RNAi-mediated knockdown of TiPARP in T47D breast cancer and HuH7 hepatoma cells increased TCDD-dependent cytochrome P450 1A1 (CYP1A1) and CYP1B1 mRNA expression and recruitment of AHR to both genes. Overexpression of TiPARP reduced AHR-dependent increases in CYP1A1-reporter gene activity, which was restored by overexpression of AHR, but not ARNT. Deletion and mutagenesis studies showed that TiPARP-mediated inhibition of AHR required the zinc finger and catalytic domains. TiPARP and AHR co-localized in the nucleus, directly interacted and both were recruited to CYP1A1 in response to TCDD. Overexpression of TiPARP enhanced whereas RNAi-mediated knockdown of TiPARP reduced TCDD-dependent AHR proteolytic degradation. TCDD-dependent induction of AHR target genes was also enhanced in Tiparp-/- mouse embryonic fibroblasts compared to wildtype controls. Moreover, livers excised from TCDD-treated Tiparp-/- mice displayed significantly greater AHR target gene expression compared with wildtype or heterozygous mice. Comparison of TiPARP to known negative regulator, AHR repressor (AHRR) revealed TiPARP and AHRR some notable similarities and differences between their mechanisms of repression. Similar to TiPARP, the AHRR was recruited to AHR target regulatory regions in response to TCDD and its overexpression repressed reporter gene activity. However unlike TiPARP, knockdown of the AHRR did not affect AHR transactivation or its proteasomal degradation. Despite some mechanistic similarities, our data suggest that TiPARP and AHRR independently repress AHR transactivation. Overall, our findings show that TiPARP is a mono-ADP-ribosyltransferase and a transcriptional repressor of AHR, revealing a novel negative feedback loop controlling AHR transcriptional regulation.

Dopamine receptors are members of the G protein-coupled receptor superfamily and play important roles in neuronal transmission. A D1-D2 receptor heterooligomer generating a G-protein linked PLC-dependent intracellular calcium signal was previously identified. The discovery of this dopamine mediated calcium signal implicated a direct link between dopamine receptors and calcium generation, but its regulation remained to be elucidated. By measuring calcium signaling with Fluo-4 fluorescence or cameleon FRET, rapid desensitization of the calcium signal in heterologous cells and striatal neurons was demonstrated by pre-treatment with SKF 83959, which selectively activates D1-D2 receptor heteromers, or SKF 83822 which only activates D1 receptor homooligomers. Although SKF 83822 was unable to activate D1-D2 receptor heteromers, it still permitted desensitization of the calcium signal. This suggested that occupancy of the D1 receptor binding pocket by SKF 83822 resulted in conformational changes sufficient for desensitization without activation of the heteromer. BRET and co-immunoprecipitation studies indicated an agonist induced interaction between the D1-D2 receptor heteromer and GRK2. Increased expression of GRK2 led to a decrease in the calcium signal and decreased expression of GRK2 led to an increased calcium signal. Expression of the catalytically inactive and RGS mutated GRK2 constructs each led to a partial recovery of the GRK2-attenuated calcium signal. These results indicated that desensitization of the D1-D2 receptor heteromer mediated calcium signal can occur by agonist occupancy even without activation and is regulated by two distinct functions of GRK2. Immunocytochemistry and calcium assays demonstrated that recycling of internalized D1 and D2 receptors and resensitization of the desensitized calcium signal occurred after dopamine pre-treatment but not SKF 83959, suggesting that the trafficking and resensitization response associated with the D1-D2 receptor heteromer is differentially regulated by specific ligands. Overall, these results suggest that D1-D2 receptor heterooligomers are uniquely regulated from their constituent receptors which are not coupled to Gq.