Browsing by Subject "Triazoles"

Now showing 1 - 6 of 6
  • Thumbnail Image
    Item
    Aerobic Uptake of Cholesterol by Ergosterol Auxotrophic Strains in Candida glabrata & Random and Site-Directed Mutagenesis of ERG25 in Saccharomyces cerevisiae
    (2012-09-27) Whybrew, Jennafer Marie; Bard, Martin; Lees, N. Douglas; Blacklock, Brenda
    Candida albicans and Candida glabrata are opportunistic human pathogens that are the leading cause of fungal infections, which are increasingly becoming the leading cause of sepsis in immunosuppressed individuals. C. glabrata in particular has become a significant concern due to the increase in clinical isolates that demonstrate resistance to triazole antifungal drugs, the most prevalent treatment for such infections. Triazole drugs target the ERG11 gene product and prevent C-14 demethylation of the first sterol intermediate, lanosterol, preventing the production of the pathways end product ergosterol. Ergosterol is required by yeast for cell membrane fluidity and cell signaling. Furthermore, C. glabrata, and not C. albicans, has been reported to utilize cholesterol as a supplement for growth. Although drug resistance is known to be caused by an increase in expression of drug efflux pumps, we hypothesize a second mechanism: that the overuse of triazole drugs has lead to the increase of resistance by C. glabrata through a 2-step process: 1) the accumulation of ergosterol auxotrophic mutations and 2) mutants able to take up exogenous cholesterol anaerobically in the body acquire a second mutation allowing uptake of cholesterol aerobically. Two groups of sterol auxotrophic C. glabrata clinical isolates have been reported to take up sterol aerobically but do not produce a sterol precursor. Sterol auxotrophs have been created in C. glabrata by disrupting different essential genes (ERG1, ERG7, ERG11, ERG25, and ERG27) in the ergosterol pathway to assess which ergosterol mutants will take up sterols aerobically. Random and site-directed mutagenesis was also completed in ERG25 of Saccharmoyces cerevisiae. The ERG25 gene encodes a sterol C-4 methyloxidase essential for sterol biosynthesis in plants, animals, and yeast. This gene functions in turn with ERG26, a sterol C-3 dehydrogenase, and ERG27, a sterol C-3 keto reductase, to remove two methyl groups at the C-4 position on the sterol A ring. In S. cerevisiae, ERG25 has four putative histidine clusters, which bind non-heme iron and a C-terminal KKXX motif, which is a Golgi to ER retrieval motif. We have conducted site-directed and random mutagenesis in the S. cerevisiae wild-type strain SCY876. Site-Directed mutagenesis focused on the four histidine clusters, the KKXX C-terminal motif and other conserved amino acids among various plant, animal, and fungal species. Random mutagenesis was completed with a procedure known as gap repair and was used in an effort to find novel changes in enzyme function outside of the parameters utilized for site-directed mutagenesis. The four putative histidine clusters are expected to be essential for gene function by acting as non-heme iron binding ligands bringing in the oxygen required for the oxidation-reduction in the C-4 demethylation reaction.
  • Thumbnail Image
    Item
    Changes in breast density and circulating estrogens in postmenopausal women receiving adjuvant anastrozole
    (AACR, 2011-12) Prowell, Tatiana M.; Blackford, Amanda L.; Byrne, Celia; Khouri, Nagi F.; Dowsett, Mitchell; Folkerd, Elizabeth; Tarpinian, Karineh S.; Powers, Pendleton P.; Wright, Laurie A.; Donehower, Michele G.; Jeter, Stacie C.; Armstrong, Deborah K.; Emens, Leisha A.; Fetting, John H.; Wolff, Antonio C.; Garrett-Mayer, Elizabeth; Skaar, Todd C.; Davidson, Nancy E.; Stearns, Vered
    Factors associated with an increased risk of breast cancer include prior breast cancer, high circulating estrogens, and increased breast density. Adjuvant aromatase inhibitors are associated with a reduction in incidence of contralateral breast cancer. We conducted a prospective, single-arm, single-institution study to determine whether use of anastrozole is associated with changes in contralateral breast density and circulating estrogens. Eligible patients included postmenopausal women with hormone receptor-positive early-stage breast cancer who had completed local therapy, had an intact contralateral breast, and were recommended an aromatase inhibitor as their only systemic therapy. Participants received anastrozole 1 mg daily for 12 months on study. We assessed contralateral breast density and serum estrogens at baseline, 6, and 12 months. The primary endpoint was change in contralateral percent breast density from baseline to 12 months. Secondary endpoints included change in serum estrone sulfate from baseline to 12 months. Fifty-four patients were accrued. At 12 months, compared with baseline, there was a nonstatistically significant reduction in breast density (mean change: -16%, 95% CI: -30 to 2, P = 0.08) and a significant reduction in estrone sulfate (mean change: -93%, 95% CI: -94 to -91, P < 0.001). Eighteen women achieved 20% or greater relative reduction in contralateral percent density at 12 months compared with baseline; however, no measured patient or disease characteristics distinguished these women from the overall population. Large trials are required to provide additional data on the relationship between aromatase inhibitors and breast density and, more importantly, whether observed changes in breast density correlate with meaningful disease-specific outcomes.
  • Thumbnail Image
    Item
    Estrogens and their precursors in postmenopausal women with early breast cancer receiving anastrozole
    (Elsevier, 2015-07) Ingle, James N.; Kalari, K. R.; Buzdar, Aman U.; Robson, Mark E.; Goetz, Matthew P.; Desta, Zeruesenay; Barman, Poulami; Dudenkov, Tanda T.; Northfelt, Donald W.; Perez, Edith A.; Flockhart, David A.; Williard, Clark V.; Wang, Liewei; Weinshilboum, Richard M.; Department of Medicine, IU School of Medicine
    PURPOSE: We determined hormone concentrations (estradiol [E2], estrone [E1], estrone conjugates [E1-C], androstenedione [A], testosterone [T]) before and on anastrozole therapy where we also determined plasma concentrations of anastrozole and its metabolites. EXPERIMENTAL: Postmenopausal women who were to receive adjuvant anastrozole for resected early breast cancer were studied. Pretreatment, blood samples were obtained for the acquisition of DNA and for plasma hormone measurements (E2, E1, E1-C, A, and T). A second blood draw was obtained at least 4 weeks after starting anastrozole for hormone, anastrozole and metabolite measurements. For hormone assays, a validated bioanalytical method using gas chromatography negative ionization tandem mass spectrometry was used. Anastrozole and metabolite assays involved extraction of plasma followed by LC/MS/MS assays. RESULTS: 649 patients were evaluable. Pretreatment and during anastrozole, there was large inter-individual variability in E2, E1, and E1-C as well as anastrozole and anastrozole metabolite concentrations. E2 and E1 concentrations were below the lower limits of quantitation in 79% and 70%, respectively, of patients on anastrozole therapy, but those with reliable concentrations had a broad range (0.627-234.0 pg/mL, 1.562-183.2 pg/mL, respectively). Considering E2, 8.9% had the same or higher concentration relative to baseline while on anastrozole, documented by the presence of drug. CONCLUSIONS: We demonstrated large inter-individual variability in anastrozole and anastrozole metabolite concentrations as well as E1, E2, E1-C, A, and T concentrations before and while on anastrozole. These findings suggest that the standard 1mg daily dose of anastrozole is not optimal for a substantial proportion of women with breast cancer.
  • Thumbnail Image
    Item
    Evidence for a Long-Lasting Compulsive Alcohol Seeking Phenotype in Rats
    (American College of Neuropsychopharmacology, 2018-03) Giuliano, Chiara; Peña-Oliver, Yolanda; Goodlett, Charles R.; Cardinal, Rudolf N.; Robbins, Trevor W.; Bullmore, Edward T.; Belin, David; Everitt, Barry J.; Psychology, School of Science
    Excessive drinking to intoxication is the major behavioral characteristic of those addicted to alcohol but it is not the only one. Indeed, individuals addicted to alcohol also crave alcoholic beverages and spend time and put much effort into compulsively seeking alcohol, before eventually drinking large amounts. Unlike this excessive drinking, for which treatments exist, compulsive alcohol seeking is therefore another key feature of the persistence of alcohol addiction since it leads to relapse and for which there are few effective treatments. Here we provide novel evidence for the existence in rats of an individual vulnerability to switch from controlled to compulsive, punishment-resistant alcohol seeking. Alcohol-preferring rats given access to alcohol under an intermittent 2-bottle choice procedure to establish their alcohol-preferring phenotype were subsequently trained instrumentally to seek and take alcohol on a chained schedule of reinforcement. When stable seeking-taking performance had been established, completion of cycles of seeking responses resulted unpredictably either in punishment (0.45 mA foot-shock) or the opportunity to make a taking response for access to alcohol. Compulsive alcohol seeking, maintained in the face of the risk of punishment, emerged in only a subset of rats with a predisposition to prefer and drink alcohol, and was maintained for almost a year. We show further that a selective and potent μ-opioid receptor antagonist (GSK1521498) reduced both alcohol seeking and alcohol intake in compulsive and non-compulsive rats, indicating its therapeutic potential to promote abstinence and prevent relapse in individuals addicted to alcohol.
  • Thumbnail Image
    Item
    The HIF-PHI BAY 85–3934 (Molidustat) Improves Anemia and Is Associated With Reduced Levels of Circulating FGF23 in a CKD Mouse Model
    (Wiley, 2021-06) Noonan, Megan L.; Ni, Pu; Agoro, Rafiou; Sacks, Spencer A.; Swallow, Elizabeth A.; Wheeler, Jonathan A.; Clinkenbeard, Erica L.; Capitano, Maegan L.; Prideaux, Matthew; Atkins, Gerald J.; Thompson, William R.; Allen, Matthew R.; Broxmeyer, Hal E.; White, Kenneth E.; Medical and Molecular Genetics, School of Medicine
    Fibroblast growth factor-23 (FGF23) is a critical factor in chronic kidney disease (CKD), with elevated levels causing alterations in mineral metabolism and increased odds for mortality. Patients with CKD develop anemia as the kidneys progressively lose the ability to produce erythropoietin (EPO). Anemia is a potent driver of FGF23 secretion; therefore, a hypoxia-inducible factor prolyl hydroxylase inhibitor (HIF-PHI) currently in clinical trials to elevate endogenous EPO to resolve anemia was tested for effects on iron utilization and FGF23-related parameters in a CKD mouse model. Mice were fed either a casein control diet or an adenine-containing diet to induce CKD. The CKD mice had markedly elevated iFGF23 and blood urea nitrogen (BUN), hyperphosphatemia, and anemia. Cohorts of mice were then treated with a patient-equivalent dose of BAY 85-3934 (BAY; Molidustat), which elevated EPO and completely resolved aberrant complete blood counts (CBCs) in the CKD mice. iFGF23 was elevated in vehicle-treated CKD mice (120-fold), whereas circulating iFGF23 was significantly attenuated (>60%) in the BAY-treated CKD mice. The BAY-treated mice with CKD also had reduced BUN, but there was no effect on renal vitamin D metabolic enzyme expression. Consistent with increased EPO, bone marrow Erfe, Transferrin receptor (Tfrc), and EpoR mRNAs were increased in BAY-treated CKD mice, and in vitro hypoxic marrow cultures increased FGF23 with direct EPO treatment. Liver Bmp-6 and hepcidin expression were downregulated in all BAY-treated groups. Femur trabecular parameters and cortical porosity were not worsened with BAY administration. In vitro, differentiated osteocyte-like cells exposed to an iron chelator to simulate iron depletion/hypoxia increased FGF23; repletion with holo-transferrin completely suppressed FGF23 and normalized Tfrc1. Collectively, these results support that resolving anemia using a HIF-PHI during CKD was associated with lower BUN and reduced FGF23, potentially through direct restoration of iron utilization, thus providing modifiable outcomes beyond improving anemia for this patient population. © 2021 American Society for Bone and Mineral Research (ASBMR).
  • Thumbnail Image
    Item
    Modeling Sitagliptin Effect on Dipeptidyl Peptidase 4 (DPP4) Activity in Adults with Hematological Malignancies After Umbilical Cord Blood (UCB) Hematopoietic Cell Transplant (HCT)
    (Springer International Publishing, 2014-03) Vélez de Mendizábal, Nieves; Strother, Robert M.; Farag, Sherif S.; Broxmeyer, Hal E.; Messina-Graham, Steven; Chitnis, Shripad D.; Bies, Robert R.; Department of Medicine, IU School of Medicine
    Background and Objectives— Dipeptidyl peptidase-4 (DPP4) inhibition is a potential strategy to increase the engraftment rate of hematopoietic stem/progenitor cells. A recent clinical trial using sitagliptin, a DPP4 inhibitor approved for type 2 diabetes mellitus, has shown to be a promising approach in adults with hematological malignancies after umbilical cord blood (UCB) hematopoietic cell transplant (HCT). Based on data from this clinical trial, a semi-mechanistic model was developed to simultaneously describe DPP4 activity after multiple doses of sitagliptin in subjects with hematological malignancies after a single-unit UCB HCT. Methods— The clinical study included 24 patients that received myeloablative conditioning followed by 4 oral sitagliptin 600mg with single-unit UCB HCT. Using a nonlinear mixed effects approach, a semi-mechanistic pharmacokinetic/pharmacodynamic model was developed to describe DPP4 activity from this trial data using NONMEM 7.2. The model was used to drive Monte-Carlo simulations to probe various dosage schedules and the attendant DPP4 response. Results— The disposition of sitagliptin in plasma was best described by a 2-compartment model. The relationship between sitagliptin concentration and DPP4 activity was best described by an indirect response model with a negative feedback loop. Simulations showed that twice a day or three times a day dosage schedules were superior to once daily schedule for maximal DPP4 inhibition at the lowest sitagliptin exposure. Conclusion— This study provides the first pharmacokinetic/pharmacodynamic model of sitagliptin in the context of HCT, and provides a valuable tool for exploration of optimal dosing regimens, critical for improving time to engraftment in patients after UCB HCT.