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dc.contributor.authorOseghale, Aluyaen
dc.date.accessioned2019-07-03T16:15:23Zen
dc.date.available2019-07-03T16:15:23Zen
dc.date.issued2019-05en
dc.identifier.urihttp://hdl.handle.net/10675.2/622459
dc.descriptionen
dc.description.abstractThe reactivation of fetal hemoglobin (HbF) in sickle cell disease (SCD) ameliorates the clinical severity of the illness and improves patient's survival. Pharmacological induction of HbF has been a major strategy for SCD treatment and several research studies have focused on a wide variety of agents for their potential to induce HbF. However, hydroxyurea (HU) remains the only Food and Drug Administration (FDA)-approved drug proven to elevate HbF in about 50% of adults with SCD. Efficacy of HU has been very limited due to many side effects including bone marrow suppression, susceptibility to infections and long-term infertility. Our group reported potent HbF induction by sodium butyrate in erythroid cells through p38 MAPK activation. Nevertheless, oral administration of a butyrate (BA) derivative to SCD patients was ineffective due to rapid metabolic inactivation by the liver. Therefore, the need for better therapies exists. This project investigated a novel prodrug conjugate of BA and δ-aminolevulinate (ALA) denoted as AN-233. As an ester, AN-233 [1-(butyryloxy) ethyl-5-amino-4-oxopentanoate] undergoes cellular hydrolysis in an esterase dependent manner to yield two active drugs BA and ALA. In prior studies, oral administration of AN-233 to mice increased total hemoglobin but the effect on HbF was unknown. We proposed the hypothesis; that AN-233 upregulates γ-globin gene expression and elevates HbF synthesis via transcriptional and post-transcriptional mechanisms. We investigated AN-233 using in-vitro, in-vivo and ex-vivo model systems including K562 cell lines, sickle progenitors and β-YAC mice. Treatment of K562 cells showed AN-233 significantly increased mRNA levels. Flow cytometry analyses show the prodrug significantly increased HbF protein expression and Western blotting of whole cell lysates confirmed increased synthesis of HbF. Treatment of CD34+ stem cell-derived primary erythroid cells increased early stage (basophilic) erythroblasts by 2.4fold and decreased late stage (orthochromatophilic) erythroblasts by 2.5 fold. In sickle progenitors, AN-233 again elevated F-cell% by 1.5fold and HbF protein by over 2.6fold. Mechanistic studies in K562 cells show AN-233 significantly elevated heme biosynthesis, decreased phosphorylation of HRI and eIF2α thereby promoting the protein synthesis of globin chains. Additionally, AN-233 enhanced histone acetylation at the γ-globin promoter and LCR DNase hypersensitive site 2 (LCR HS2). Treatment of sickle progenitors with AN-233 decreased %sickled cells by up to 50%. The transcription factor BACH1 was reduced while NRF2 was increased in AN-233 treated K562 cells. In vivo, AN-233 increased F-cell% and F-cell MFI of treated β-YAC mice within 4-weeks. Our data support AN-233 as a potent HbF inducer in erythroid progenitors and in mice. The prodrug represents a drug candidate, which can be developed for the treatment of SCD patients.en
dc.publisherAugusta Universityen
dc.subjectBiologyen
dc.subjectPhysiologyen
dc.subjectPharmacologyen
dc.subjectBiologyen
dc.subjectAN-233, beta-YAC, butyrate, delta-aminolevulinate, Fetal hemoglobin, Sickle cell diseaseen
dc.titleTherapeutic induction of fetal hemoglobin in sickle cell disease: development of a novel prodrug AN-233en
dc.typedissertationen
dc.contributor.departmentBiomedical Sciences
dc.language.rfc3066enen
dc.date.updated2019-07-03T16:15:23Zen
dc.description.advisorPace, Betty S.en
dc.description.degreePh.D.en
dc.description.committeeFulton, Daviden
dc.description.committeeLucas, Rudolfen
dc.description.committeeCsanyi, Gaboren
dc.description.committeeKutlar, Abdullahen
refterms.dateFOA2019-08-07T14:17:02Z


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