Lin, Yvonne SBergagnini-Kolev, Mackenzie Camille2020-08-142020-08-142020BergagniniKolev_washington_0250E_21610.pdfhttp://hdl.handle.net/1773/46150Thesis (Ph.D.)--University of Washington, 2020Vitamin D (cholecalciferol or ergocalciferol) is essential for regulating serum calcium and maintaining bone integrity. As vitamin D is not biologically active, it must undergo two sequential enzyme-catalyzed hydroxylations to form the active metabolite, 1α,25-hydroxyvitamin D (1α,25(OH)2D), via the intermediate, 25-hydroxyvitamin D (25(OH)D). The predominant circulating form of 25(OH)D, 25(OH)D3, is metabolized to other metabolites, including 24,25-dihydroxyvitamin D3 (24,25(OH)2D3), 4β,25-dihydroxyvitamin D3 (4β,25(OH)2D3), 25-hydroxyvitamin D-3-O-sulfate (25(OH)D3-S), and 25-hydroxyvitamin D-3-O-glucuronide (25(OH)D3-G). Changes in enzyme expression due to disease, ontogeny, or drug interactions may alter the circulating concentrations of 25(OH)D3 and its metabolites, and contribute to an increased risk of vitamin D deficiency. The aim of this dissertation was to explore mechanisms of altered vitamin D homeostasis in patients with cystic fibrosis (CF) and in pregnant women, and to evaluate 4β,25(OH)2D3 as an endogenous biomarker of CYP3A4 activity. Chapter 2 describes the development and validation of a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the simultaneous quantification of vitamin D2, vitamin D3, 25(OH)D2, 25(OH)D3, 1α,25(OH)2D2, 1α,25(OH)2D3, 24R,25(OH)2D3, 4β,25(OH)2D3, 1β,25(OH)2D3, and 1α,24,25(OH)3D3. We measured the serum concentration of 25(OH)D3 and selected metabolites in a cohort of patients with CF (n = 83) and age-, sex-, and race-matched healthy controls (n=82) (Chapter 3). Patients with CF had 15%, 35%, and 40% lower circulating concentrations of 1α,25(OH)2D3, 4β,25(OH)2D3, and 25(OH)D3-S, respectively. We found no difference in the clearance or half-life of d6-25(OH)D3 in patients with CF compared to healthy controls (n = 5 per group) after IV bolus administration of d6-25(OH)D3. To assess changes in 25(OH)D3 metabolism during pregnancy, we measured 25(OH)D3 and selected metabolites in a longitudinal study of healthy women (n = 15) before, during, and after pregnancy (Chapter 4). The change in the concentration of 25(OH)D3 and its metabolites were evaluated using linear mixed-effects, modeling. The model predicted increase in the concentration of 25(OH)D3 was 45% during pregnancy. Serum concentrations of 1α,25(OH)2D3, 4β,25(OH)2D3, and 25(OH)D3-G increased by 200%, 14%, and 38%, respectively, as estimated by the linear mixed-effects model from pre-pregnancy to 36 weeks of gestation (p < 0.001). Additionally, 25(OH)D3-S concentrations decreased 25% from pre-pregnancy to 36 weeks of gestation (p < 0.001 for all). Protein binding was altered during pregnancy; vitamin D binding protein (VDBP) serum concentrations increased by 190% and albumin concentrations decreased 26% from pre-pregnancy to 36 weeks of gestation, resulting in a 20% decrease in the percent unbound of 25(OH)D3. To evaluate the potential of 4β,25(OH)2D3 as a biomarker of CYP3A4 activity, we developed semi-mechanistic pharmacokinetic-pharmacodynamic models for midazolam, clarithromycin, rifampin, 25(OH)D3, and 4β,25(OH)2D3. The midazolam, clarithromycin, and rifampin models were developed using published clinical data. Model parameters for 25(OH)D3 and 4β,25(OH)2D3 were estimated using clinical data following administration of placebo (control), clarithromycin, rifampin, or clarithromycin and rifampin in combination for 14 days. The sensitivity of 4β,25(OH)2D3 to detect induction or mechanism-based inhibition of CYP3A4 was compared to midazolam, a sensitive probe drug for CYP3A4 activity. For mechanism-based inhibition, we found that a 5.4-fold increase in midazolam AUC corresponded with a 46% decrease in the serum concentration 4β,25(OH)2D3. For induction, we found that an 44% decrease in midazolam AUC corresponded with a 200% increase in 4β,25(OH)2D3 activity. Further clinical studies are necessary to validate 4β,25(OH)2D3 as a biomarker of CYP3A4 activity. Although there are still many unanswered questions, the research presented in this dissertation furthers our understanding of vitamin D disposition in health and disease, and provides more insight into the complexity of vitamin D homeostasis.application/pdfen-USCC BYBiomarkerCYP3A4Cystic FibrosisPregnancyVitamin DPharmaceutical sciencesPharmaceuticsThesis