Identification of New and Diverse Inducers of Fetal Hemoglobin with High Throughput Screening (HTS)

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 4277-4277 ◽  
Author(s):  
Jose I Sangerman ◽  
Michael S Boosalis ◽  
Ling Shen ◽  
Sarah Haigh ◽  
Ada Kane ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Globin Gene ◽  
Clinical Stage ◽  
Gene Promoter ◽  
Fetal Hemoglobin ◽  
Luciferase Reporter ◽  
Significant Activity ◽  
Globin Mrna ◽  
Globin Promoter

Abstract Abstract 4277 Pharmacologic augmentation of fetal hemoglobin (HbF, γ-globin) production, to replace diminished β-globin chains in the β-thalassemias and to inhibit HbS polymerization in sickle cell disease, is a definitive therapeutic modality. Despite long-term efforts, regulatory approval has been obtained for only one chemotherapeutic agent. Pharmacologic reactivation of high-level HbF expression with non-cytotoxic, tolerable therapeutics is still an unmet medical need for this global health burden. To investigate potential therapeutic libraries for unrecognized HbF inducers, we developed a high-throughput screening (HTS) program to interrogate diverse chemical libraries, including a library of FDA-approved and clinical stage drugs. This program has identified unexpected new and highly potent HbF-inducing drugs, some of which are already in clinical use for other medical indications and have established safety profiles. A human cell-based assay which was previously used in low throughput assays, utilizing a 1.4-kilobase (kb) KpnI-BglII fragment of the HS2 of the locus control region (LCR) linked to the γ-globin gene promoter and the enhanced green fluorescent protein (EGFP) reporter gene, was adapted for high throughput screening and employed as the primary screen. Cytotoxic activity was assayed in a simultaneous counter screen. A number of hits were identified as being more potent than positive controls (such as butyrate). Several hits were immediately eliminated from further development as potential hemoglobinopathy therapeutics because of cytotoxicity (e.g., Idarubicin) or undesirable off-target effects, but nonetheless validated the HTS itself and were validated in secondary confirmatory assays as highly-potent HbF-inducers. The HTS assay identified 8 FDA-approved drugs as potent inducers of γ-globin gene expression, with activity at 1–2 logs lower concentrations (1000-fold higher potency) than prior generation therapeutic candidates. The γ-globin-specificity of hits was determined in a secondary assay employing a stably-transfected dual-luciferase reporter construct containing the LCR and the β-globin promoter linked to renilla luciferase and the Aγ-globin promoter linked to firefly luciferase (μLCRβprRlucAγprFluc cassette). Clinical-stage or clinically-approved agents, including Ambroxol at 1 μM, Desloratadine at 1 μM, Resveratrol at 10 μM, Benserazide at 5 μM, the HDAC inhibitor MS-275 at 5 μM, and an established bioactive, NSC-95397, at 1 μM were all significantly more active in this assay than Butyrate at 2000 μM, with MS-275 and Resveratrol being the most active. These drugs were then assayed for their ability to induce γ-globin mRNA expression in cultured primary human erythroid progenitors, at concentrations which are pharmacologically achievable in humans. Drugs significantly more active in γ -globin mRNA induction than the positive control (2-fold induction) in this system included Ambroxol (3-fold), Desloratadine (up to 6-fold), Resveratrol (up to 3-fold), Benserazide (up to 5-fold), and MS-275 (up to 3.7-fold). Two agents were subsequently studied in anemic baboons, and demonstrated in vivo induction of γ-globin mRNA, HbF, and F-reticulocytes. Unexpectedly, rises in total hemoglobin (>1 gm/dL) also occurred with 2 agents. Thus, a panel of structurally- and functionally-unrelated compounds demonstrate greater HbF-inducing activity, with up to 1000-fold higher potency, than current HbF-inducers which have significant activity in clinical trials. Some of the drugs identified by HTS have entirely benign safety profiles. These candidates could be clinically evaluated rapidly and at significantly less cost than new chemical entities, which require extensive toxicology, manufacturing, and clinical evaluation. These findings demonstrate the utility of a high-throughput screening program based on γ-globin gene promoter induction. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Substitution of the Human β-Spectrin Promoter for the Human Aγ-Globin Promoter Prevents Silencing of a Linked Human β-Globin Gene in Transgenic Mice

1998 ◽  
Vol 18 (11) ◽  
pp. 6634-6640 ◽  
Author(s):  
Denise E. Sabatino ◽  
Amanda P. Cline ◽  
Patrick G. Gallagher ◽  
Lisa J. Garrett ◽  
George Stamatoyannopoulos ◽  
...  
Keyword(s):  
Transgenic Mice ◽  
Fetal Liver ◽  
Globin Gene ◽  
Gene Promoter ◽  
Yolk Sac ◽  
Globin Genes ◽  
Erythroid Cells ◽  
Globin Mrna ◽  
Globin Promoter ◽  
In Erythroid Cells

ABSTRACT During development, changes occur in both the sites of erythropoiesis and the globin genes expressed at each developmental stage. Previous work has shown that high-level expression of human β-like globin genes in transgenic mice requires the presence of the locus control region (LCR). Models of hemoglobin switching propose that the LCR and/or stage-specific elements interact with globin gene sequences to activate specific genes in erythroid cells. To test these models, we generated transgenic mice which contain the human Aγ-globin gene linked to a 576-bp fragment containing the human β-spectrin promoter. In these mice, the β-spectrin Aγ-globin (βsp/Aγ) transgene was expressed at high levels in erythroid cells throughout development. Transgenic mice containing a 40-kb cosmid construct with the micro-LCR, βsp/Aγ-, ψβ-, δ-, and β-globin genes showed no developmental switching and expressed both human γ- and β-globin mRNAs in erythroid cells throughout development. Mice containing control cosmids with the Aγ-globin gene promoter showed developmental switching and expressed Aγ-globin mRNA in yolk sac and fetal liver erythroid cells and β-globin mRNA in fetal liver and adult erythroid cells. Our results suggest that replacement of the γ-globin promoter with the β-spectrin promoter allows the expression of the β-globin gene. We conclude that the γ-globin promoter is necessary and sufficient to suppress the expression of the β-globin gene in yolk sac erythroid cells.

Methylation of α-type embryonic globin gene απrepresses transcription in primary erythroid cells

Blood ◽  
2002 ◽  
Vol 100 (12) ◽  
pp. 4217-4222 ◽  
Author(s):  
Rakesh Singal ◽  
Jane M. vanWert ◽  
Larry Ferdinand
Keyword(s):  
Globin Gene ◽  
Gene Promoter ◽  
Luciferase Reporter ◽  
Globin Genes ◽  
Erythroid Cells ◽  
Histone H4 ◽  
Luciferase Reporter Gene ◽  
Cpg Dinucleotides ◽  
Expression Construct ◽  
Globin Promoter

The inverse relationship between expression and methylation of β-type globin genes is well established. However, little is known about the relationship between expression and methylation of avian α-type globin genes. The embryonicαπ-globin promoter was unmethylated, andαπ-globin RNA was easily detected in 5-day chicken erythroid cells. A progressive methylation of the CpG dinucleotides in the απ promoter associated with loss of expression of απ-globin gene was seen during development in primary erythroid cells. A 315-bpαπ-globin promoter region was cloned in an expression construct (απpGL3E) containing a luciferase reporter gene and SV40 enhancer. The απpGL3E construct was transfected into primary erythroid cells derived from 5-day-old chicken embryos. Methylation of απpGL3E plasmid andαπ-globin promoter alone resulted in a 20-fold and 7-fold inhibition of expression, respectively. The fully methylated but not the unmethylated 315-bpαπ-globin gene promoter fragment formed amethyl cytosine-binding proteincomplex (MeCPC). Chromatin immunoprecipitation assays were combined with quantitative real-time polymerase chain reaction to assess histone acetylation associated with theαπ-globin gene promoter. Slight hyperacetylation of histone H3 but a marked hyperacetylation of histone H4 was seen in 5-day when compared with 14-day erythroid cells. These results demonstrate that methylation can silence transcription of an avian α-type embryonic globin gene in homologous primary erythroid cells, possibly by interacting with an MeCPC and histone deacetylase complex.

Next Generation Clinical-Stage HbF Inducers Exhibit Multiple Molecular Actions In Vitro and In Vivo and Offer Personalized Therapeutic Regimens

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 560-560
Author(s):  
Betty S. Pace ◽  
Jose I Sangerman ◽  
Michael S Boosalis ◽  
Roman Wolf ◽  
Yan Dai ◽  
...  
Keyword(s):  
Globin Gene ◽  
Clinical Stage ◽  
Hdac Inhibitors ◽  
Gene Promoter ◽  
Erythroid Progenitors ◽  
Globin Mrna ◽  
F Cells ◽  
High Level

Abstract High-level expression of fetal (Υ) globin reduces clinical complications in sickle cell disease and this is achieved with hydroxyurea (HU) in young children. However, non-cytotoxic high-potency therapeutics, particularly which can be utilized in combination with HU, are needed for most adolescent and adult patients who have continued serious clinical events. We have identified additional pharmaceutical candidates which induce HbF without cytotoxicity, using a Υ-globin gene promoter linked to GFP for robotic high-throughput screening, and screening five diverse chemical libraries. From a library of US and EU drugs which are approved for treatment of other medical conditions, a small panel of approved therapeutics were found to induce Υ globin expression, and have benign safety profiles, are orally active, and are suitable for long-term use. Three orally active candidates were evaluated in anemic baboons, and two, DLT and PB-04, induced Υ-globin mRNA by 15- to 33-fold over baseline levels. In 3/3 beta-globin locus YAC transgenic mice, one candidate (PB-04; 20 mg/kg) given by intra-peritoneal (IP) injections (for experimental feasibility) 3 times/ week for 5 wks significantly increased F-cells from 0.1 to 9%, 0.4 to 18%, and 0.13 to 12% respectively; and mean fluorescence intensity (MFI) increased by 10- to 33-fold. Responses were observed within one week. In hydroxyurea treated mice (100 mg/kg; IP, 5 days/ wk) F-cells increased from 0.3 to 2.3% on average (p<0.05), and MFI increased by 6- to 7-fold, while water vehicle did not increase F-cells significantly. PB-04 has been used clinically for decades as an excipient solely to prolong the half-life of another pharmaceutical, and is suitable for repurposing. In ChIP assays in K562 cells, PB-04 treatment demonstrated dual actions of displacing HDAC3 by 20-fold and LSD-1 by 3-fold from the Υ globin gene promoter. To investigate potential effects of genetic modifiers of HbF on responses to different HbF inducer classes, erythroid progenitors from 40 sickle and beta thalassemic subjects were sub-genotyped for 3 major quantitative trait loci (QTL) (Bcl-11A, HMIP, Xmn-I) and cultured +/- 7 HbF inducers which are in clinical trials or approved. Most HbF inducers, including decitabine and butyrate used as positive controls, are active in 50-70% of progenitors, with differential Υ-globin mRNA responses observed. Only 10% of progenitors did not respond to any inducing agent. Most progenitors with the Xmn-1 variant responded with higher Υ globin transcription to all inducers. Sodium dimethylbutyrate (HQK-1001) and decitabine, produced 6-fold overall mean induction. PB-04 produced 9-fold mean induction above untreated control levels from the same subject. HDAC inhibitors (Butyrate, MS-275) which suppress Bcl-11A expression, demonstrate higher activity in progenitors from subjects without an underlying SNP in Bcl-11A. Another HDAC inhibitor, SB939, produced responses in 80% of progenitors. 25-30% of subjects’ progenitors exhibit high induction, 12-to 40-fold above untreated controls, to dimethylbutyrate, PB-04, decitabine, and an HDAC1/2 inhibitor 14F, suggesting a “high responder genotype” of which only half had a recognized favorable QTL. Taken together, these in vitro and in vivo studies identify a mini-pipeline of clinical-stage HbF-inducing therapeutics, with both epigenetic and targeted molecular actions, which can be investigated clinically to develop tailored therapeutics and therapeutic combinations for high-level induction of HbF in subgenotyped hemoglobinopathy patients. Seven new and established inducers have been evaluated in erythroid progenitors cultured from sub-genotyped hemoglobinopathy patients, 3 new drugs (PB-04, DLT, RSV) and two HDAC inhibitors (MS-275 and SB939), induced 3- to 40-fold higher Υ-globin mRNA above untreated control levels. Disclosures: Faller: Phoenicia BioSciences, Inc.: Employment, Equity Ownership, Membership on an entity’s Board of Directors or advisory committees, Research Funding. Perrine:Phoenicia BioSciences, Inc.: Employment, Equity Ownership, Membership on an entity’s Board of Directors or advisory committees, Research Funding.

scholarly journals Trienone analogs of curcuminoids induce fetal hemoglobin synthesis via demethylation at Gγ-globin gene promoter

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Khanita Nuamsee ◽  
Thipphawan Chuprajob ◽  
Wachirachai Pabuprapap ◽  
Pornrutsami Jintaridth ◽  
Thongperm Munkongdee ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Promoter Region ◽  
Globin Gene ◽  
Gene Promoter ◽  
Fetal Hemoglobin ◽  
K562 Cells ◽  
Erythroid Progenitor ◽  
Erythroid Progenitor Cells ◽  
Hbf Induction ◽  
Globin Promoter

AbstractThe reactivation of γ-globin chain synthesis to combine with excess free α-globin chains and form fetal hemoglobin (HbF) is an important alternative treatment for β-thalassemia. We had reported HbF induction property of natural curcuminoids, curcumin (Cur), demethoxycurcumin (DMC) and bis-demethoxycurcumin (BDMC), in erythroid progenitors. Herein, the HbF induction property of trienone analogs of the three curcuminoids in erythroleukemic K562 cell lines and primary human erythroid progenitor cells from β-thalassemia/HbE patients was examined. All three trienone analogs could induce HbF synthesis. The most potent HbF inducer in K562 cells was trienone analog of BDMC (T-BDMC) with 2.4 ± 0.2 fold increase. In addition, DNA methylation at CpG − 53, − 50 and + 6 of Gγ-globin gene promoter in K562 cells treated with the compounds including T-BDMC (9.3 ± 1.7%, 7.3 ± 1.7% and 5.3 ± 0.5%, respectively) was significantly lower than those obtained from the control cells (30.7 ± 3.8%, 25.0 ± 2.9% and 7.7 ± 0.9%, respectively P < 0.05). The trienone compounds also significantly induced HbF synthesis in β-thalassemia/HbE erythroid progenitor cells with significantly reduction in DNA methylation at CpG + 6 of Gγ-globin gene promoter. These results suggested that the curcuminoids and their three trienone analogs induced HbF synthesis by decreased DNA methylation at Gγ-globin promoter region, without effect on Aγ-globin promoter region.

MicroRNA-96 Inhibits Fetal Hemoglobin Expression During Erythropoiesis

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 3220-3220
Author(s):  
Imane Azzouzi ◽  
Jeannine Winkler ◽  
Jean-Claude Fauchère ◽  
André P Gerber ◽  
Markus Stoffel ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cord Blood ◽  
Protein Level ◽  
Globin Gene ◽  
Direct Interaction ◽  
Fetal Hemoglobin ◽  
Luciferase Reporter ◽  
Globin Mrna ◽  
Hel Cells

Abstract Abstract 3220 Background: Fetal hemoglobin, HbF (α2γ2), is the main hemoglobin synthesized until birth; it subsequently declines and switches to adult hemoglobin, HbA (α2β2). Certain mutations within the globin gene locus can cause persistent HbF synthesis after birth, ameliorating symptoms in hemoglobinopathies. Analysis from e.g. Hb Corfu and δβ-thalassemia indicates that γ-globin gene expression is post-transcriptionally regulated (Efremov DG., Br J Haematol, Oct 1994; Chakalova L., Blood, 2005). The post-transcriptional regulation of HbF might involve regulatory non-coding RNAs. MicroRNAs (miRNAs) are a small non-coding family of 21 nucleotide RNAs that regulate gene expression post-transcriptionally by targeting mRNAs. After transcription and maturation the miRNA is incorporated into the miRNA-induced silencing complex (miRISC). The miRISC, which contains argonaute proteins (AGO), binds and silences the target mRNAs. Previous studies have reported a role of miRNAs in the developmental switch from HbF to HbA synthesis (Noh SJ, J Transl Med, 2009; Bianchi N., BMB Rep, 2009), however so far no direct interaction between miRNAs and the γ-globin mRNA has been reported. Moreover a function of miRISC in mature red blood cells (RBC) has not been shown yet. Therefore, we aimed (I) to characterize the miRISC composition in RBCs, (II) to identify miRNAs that are directly involved in the regulation of HbF and (III) to study their function during erythropoiesis. Methods: To characterize miRISC in RBCs, we performed co-immunoprecipitations of the miRISC using antibodies directed against human AGO. The immunoprecipitated complexes were analyzed by Western-blot, mass spectrometry and qPCR. To identify miRNAs potentially involved in the regulation of HbF miRNA expression patterns in RBCs from adult and umbilical cord blood were analyzed by qPCR. To confirm the role of selected miRNAs, γ-globin protein levels were measured by ELISA in HEL cell cultures transfected with miRNA precursors or inhibitors as well as in primary erythroid cultures overexpressing miRNAs after lentiviral transduction. The direct interaction between miRNAs and the γ-globin mRNA was analyzed by luciferase reporter assay. Results: AGO1, 2, 3 and 4 and Importin 8 were detected in RBCs both from adult and cord blood. In adult RBC samples (n=3), expressing <1% HbF of total Hb, γ-globin mRNA was bound to AGO2 but not to AGO1 containing miRISC. However in cord blood (n=3), containing 90 % HbF and 30 times more γ-globin mRNA molecules than adult RBCs, the amount of γ-globin mRNA bound to AGO2 containing miRISC was 200 times less (p<0.05). With these indications, we started to analyze miRNA patterns in adult and cord blood RBCs. miR-96, miR-146a, miR-330-3p, let-7a and miR-888 were significantly downregulated in cord blood RBCs compared to adult RBCs. However, luciferase reporter assays demonstrated that only miR-96 was able to bind to the predicted target site within the γ-globin mRNA. Transfection of HEL cells with miR-96 inhibitor showed a twofold induction (p<0.01; n=3) of the γ-globin protein level compared to control cells transfected with scramble RNA. Consistently, transfection with miR-96 precursor in HEL cells showed a 15% reduction (p<0.05, n=3) of the γ-globin protein level. To study the miR-96 mediated inhibition of HbF during erythropoiesis, cord blood derived primary erythroid cultures were transduced with miR-96 lentiviruses. A 40% decrease (p<0.05, n=3) of γ-globin protein expression was observed in erythroid cells overexpressing miR-96 compared to cells transduced with the negative control virus. This γ-globin reduction was observed on 10 and 14 days old cultures, which contained 80% and 65% CD235+ CD71+ erythroblasts, respectively. Conclusions: This study identifies miR-96 as direct inhibitor of the HbF expression during erythropoiesis. Specifically we observed that in cells with low HbF content, γ-globin mRNAs are bound to miRISC and are therefore directly inhibited by miRNAs. We demonstrated that miR-96 is binding to a seedless target site within the γ-globin open reading frame. Finally we showed that miR-96 overexpression reduces the expression of HbF in erythroleukemia cells and during late stages of cord blood derived erythropoiesis. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Activation of delta-globin gene expression by erythroid Krupple-like factor: a potential approach for gene therapy of sickle cell disease

Blood ◽  
1996 ◽  
Vol 88 (10) ◽  
pp. 4051-4057 ◽  
Author(s):  
D Donze ◽  
PH Jeancake ◽  
TM Townes
Keyword(s):  
Gene Expression ◽  
Gene Therapy ◽  
Sickle Cell ◽  
Globin Gene ◽  
Luciferase Reporter ◽  
Beta Globin ◽  
Globin Mrna ◽  
Globin Promoter ◽  
Murine Erythroleukemia ◽  
Globin Gene Expression

Hemoglobin A2 (HbA2; alpha 2 delta 2) is a powerful inhibitor of HbS (alpha 2 beta 2(3)) polymerization. However, HbA2 levels are normally low in sickle cell patients. We show that a major reason for low delta- globin gene expression is the defective CACCC box at -90 in the delta-globin promoter. When the CACCC box defect in delta is corrected, expression of an HS2 delta /Luciferase reporter is equivalent to HS2 beta/Luciferase. Erythroid Krupple-like factor (EKLF), which binds to the CACCC box of the beta-globin gene and activates high-level expression, does not bind to the normal delta-globin promoter. Our goal is to design a modified EKLF that binds to the defective delta-globin promoter and enhances delta-globin gene expression. To test the feasibility of this strategy, we inserted the beta-globin CACCC box at - 90 of the delta-globin gene promoter to produce an HS2 delta CAC-beta construct and quantitated human delta-and beta-globin mRNA in stably transformed murine erythroleukemia (MEL) cells. delta-Globin mRNA in these cells was 22.0% +/- 9.0% of total human globin mRNA (delta/delta + beta) as compared with 3.0% +/- 1.3% in the HS2 delta-beta control. In a second set of experiments a GAL4 DNA-binding site was inserted at - 90 of the delta-globin gene to produce an HS2 delta GAL4-beta construct. This construct and a GAL4(1–147)/EKLF expression vector were stably transfected into MEL cells. delta-Globin mRNA in these cells was 27.8% +/- 7.1% of total human globin mRNA as compared with 9.9% +/- 2.5% in the HS2 delta GAL4-beta plus GAL4(1–147) control. These results show that delta-globin gene expression can be significantly increased by a modified EKLF. Based on these results, we suggest that modified EKLFs, which contain zinc fingers designed to bind specifically to the defective delta-globin CACCC box, may be useful in gene therapy approaches to increase HbA2 levels and inhibit HbS polymerization.

scholarly journals Affordable Luciferase Reporter Assay for Cell-Based High-Throughput Screening

2012 ◽  
Vol 18 (4) ◽  
pp. 453-461 ◽  
Author(s):  
Ellen Siebring-van Olst ◽  
Christie Vermeulen ◽  
Renee X. de Menezes ◽  
Michael Howell ◽  
Egbert F. Smit ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Firefly Luciferase ◽  
Luciferase Assay ◽  
Luciferase Reporter ◽  
Chemical Components ◽  
Simple Liquid ◽  
Luciferase Reporter Construct ◽  
Reagent Cost ◽  
Firefly Luciferase Gene

The firefly luciferase gene is commonly used in cell-based reporter assays. Convenient luciferase assay reagents for use in high-throughput screening (HTS) are commercially available. However, the high cost of these reagents is not within the means of some academic laboratories. Therefore, we set out to develop an affordable luciferase assay reagent applicable in an HTS format using simple liquid-handling steps. The reagent was homemade from individual chemical components and optimized for luminescence intensity and stability. We determined the minimal concentrations of the most expensive components, dithiothreitol (DTT) and D-luciferin, resulting in a total assay reagent cost of less than 1 cent per sample. Signal stability was maximized by omission of coenzyme A and reduction of DTT concentration. The assay was validated in a high-throughput setting using two cancer cell lines carrying a p53-dependent luciferase reporter construct and siRNAs modulating p53 transcriptional activity. Induction of p53 activity by silencing PPM1D or SYVN1 and reduction of p53 activity by silencing p53 remained constant over a 2-h measurement period, with good assay quality (Z′ factors mostly above 0.5). Hence, the luciferase assay described herein can be used for affordable reporter readout in cell-based HTS.

scholarly journals Effects of tetrahydrouridine on pharmacokinetics and pharmacodynamics of oral decitabine

Blood ◽  
2012 ◽  
Vol 119 (5) ◽  
pp. 1240-1247 ◽  
Author(s):  
Donald Lavelle ◽  
Kestis Vaitkus ◽  
Yonghua Ling ◽  
Maria A. Ruiz ◽  
Reda Mahfouz ◽  
...  
Keyword(s):  
Targeted Therapy ◽  
Exposure Time ◽  
Nonhuman Primates ◽  
Cytidine Deaminase ◽  
Globin Gene ◽  
Gene Promoter ◽  
Fetal Hemoglobin ◽  
Competitive Inhibitor ◽  
Time Profile ◽  
Pharmacokinetics And Pharmacodynamics

Abstract The deoxycytidine analog decitabine (DAC) can deplete DNA methyl-transferase 1 (DNMT1) and thereby modify cellular epigenetics, gene expression, and differentiation. However, a barrier to efficacious and accessible DNMT1-targeted therapy is cytidine deaminase, an enzyme highly expressed in the intestine and liver that rapidly metabolizes DAC into inactive uridine counterparts, severely limiting exposure time and oral bioavailability. In the present study, the effects of tetrahydrouridine (THU), a competitive inhibitor of cytidine deaminase, on the pharmacokinetics and pharmacodynamics of oral DAC were evaluated in mice and nonhuman primates. Oral administration of THU before oral DAC extended DAC absorption time and widened the concentration-time profile, increasing the exposure time for S-phase–specific depletion of DNMT1 without the high peak DAC levels that can cause DNA damage and cytotoxicity. THU also decreased interindividual variability in pharmacokinetics seen with DAC alone. One potential clinical application of DNMT1-targeted therapy is to increase fetal hemoglobin and treat hemoglobinopathy. Oral THU-DAC at a dose that would produce peak DAC concentrations of less than 0.2μM administered 2×/wk for 8 weeks to nonhuman primates was not myelotoxic, hypomethylated DNA in the γ-globin gene promoter, and produced large cumulative increases in fetal hemoglobin. Combining oral THU with oral DAC changes DAC pharmacology in a manner that may facilitate accessible noncytotoxic DNMT1-targeted therapy.

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