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Development of a Selective and Stable Reactive Oxygen Species-activated Anti-Acute Myeloid Leukemia Agent and Localizing DNA Aptamer

Earnest, Kaylin G
http://orcid.org/0000-0001-8050-7511
http://rave.ohiolink.edu/etdc/view?acc_num=ucin153546148016143

Abstract Details

2018, PhD, University of Cincinnati, Arts and Sciences: Chemistry.
Anticancer agents that modify DNA are a mainstay of chemotherapy regimens, but development of new classes of these agents has slowed because of the modifications of DNA in non-cancerous cells. This is what gives rise to serious side effects via poor selectivity. The Merino Lab has developed a pro-drug strategy to achieve specificity by translating the finding that levels of reactive oxygen species (ROS) are elevated in cancers, such as Acute Myeloid Leukemia (AML). This pro-drug approach allows cellular ROS to oxidize the pro-drug into its active form to achieve selective cytotoxicity. Our current lead agent (A100) is shown to have 10-fold selectivity between AML cells over normal CD34+ blood cells in vitro and showed some efficacy in the in vivo AML mouse model. This work started with computational analysis to determine what parts of the molecule were a target for metabolic enzymes. The first step taken to improve the molecule was to add polyethylene glycol (PEG) to the free phenol, increasing both its solubility and metabolic stability. To prove metabolic stability, binding assays against CYP1A2 (Cytochrome P450, Isoform 1A2) were done, as CYP1A2 is known to attack alcohols. The synthetic addition of the PEG increased stability against CYP1A2 by almost 50%. To prove stability in a more complex matrix, total stability was measured via half-life in pooled human liver microsomes. The PEGylated compound (A100-PEG) showed a 7-fold increase in the half-life of A100, as compared to A100 alone. A100 alone was not detectable in mouse blood samples after 15 minutes; however, in all three mouse models, A100-PEG was detectable even after 100 minutes and was calculated to have a 6-fold increase in half-life, as compared to A100. Though stable, A100-PEG only showed similar efficacy in the in vivo AML mouse model; however, an IV injection was used instead of the previous IP injection. Having improved its solubility and metabolic stability, the next step was to improve agent delivery. This was done by developing an aptamer to selectively “shuttle” the molecule to the cancer cells. Aptamers are selective binding nucleic acid macromolecules that are evolved in vitro to bind a specific target, in this case, whole AML cells, through a process called SELEX, systematic evolution of ligands by exponential enrichment. Twelve rounds of SELEX, including two counter selections against fibroblast cells, were completed. Aptamer pools were sequenced, and 3 main candidate sequences were identified. These sequences consisted of two 23 bases primers and a 30-base sequence in between. Binding studies were done using flow cytometry, and the lead sequence was found to have a binding constant of 38+/-2.5nM to AML cells, while having no binding to fibroblast and umbilical cord blood cells at 200nM. A truncation study of the lead sequence was done using 9 shortened sequences, which proved that the 5’ primer was not important for binding. The binding of the lead sequence was tested against 7 primary AML patient samples, and 5 of the 7 cell lines showed binding at 200nM.
Edward Merino, Ph.D. (Committee Chair)
Patrick Limbach, Ph.D. (Committee Member)
Laura Sagle, Ph.D. (Committee Member)
92 p.
Pharmaceuticals
reactive oxygen species; aptamer; acute myeloid leukemia

Recommended Citations

Citations

  • Earnest, K. G. (2018). Development of a Selective and Stable Reactive Oxygen Species-activated Anti-Acute Myeloid Leukemia Agent and Localizing DNA Aptamer [Doctoral dissertation, University of Cincinnati]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=ucin153546148016143

    APA Style (7th edition)

  • Earnest, Kaylin. Development of a Selective and Stable Reactive Oxygen Species-activated Anti-Acute Myeloid Leukemia Agent and Localizing DNA Aptamer. 2018. University of Cincinnati, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=ucin153546148016143.

    MLA Style (8th edition)

  • Earnest, Kaylin. "Development of a Selective and Stable Reactive Oxygen Species-activated Anti-Acute Myeloid Leukemia Agent and Localizing DNA Aptamer." Doctoral dissertation, University of Cincinnati, 2018. http://rave.ohiolink.edu/etdc/view?acc_num=ucin153546148016143

    Chicago Manual of Style (17th edition)

ucin153546148016143
246
© 2018, some rights reserved.Creative Commons License Development of a Selective and Stable Reactive Oxygen Species-activated Anti-Acute Myeloid Leukemia Agent and Localizing DNA Aptamer by Kaylin G Earnest is licensed under a Creative Commons Attribution-NonCommercial-NoDerivs 3.0 Unported License. Based on a work at etd.ohiolink.edu.
This open access ETD is published by University of Cincinnati and OhioLINK.