Silylation of Deoxynucleotide Analog Yields an Orally Available Drug with Antileukemia Effects

Abstract

DNA methyltransferase inhibitors have improved the prognosis of myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). However, because these agents are easily degraded by cytidine deaminase (CDA), they must be administered intravenously or subcutaneously. Recently, two orally bioavailable DNA methyltransferase inhibitors, CC-486 and ASTX727, were approved. In previous work, we developed 5-O-trialkylsilylated decitabines that resist degradation by CDA. However, the effects of silylation of a deoxynucleotide analog and enzymatic cleavage of silylation have not been fully elucidated. Enteric administration of OR21 in a cynomolgus monkey model led to high plasma concentrations and hypomethylation, and in a mouse model, oral administration of enteric-coated OR21 led to high plasma concentrations. The drug became biologically active after release of decitabine (DAC) from OR21 following removal of the 5'-O-trisilylate substituent. Toxicities were tolerable and lower than those of DAC. Transcriptome and methylome analysis of MDS and AML cell lines revealed that OR21 increased expression of genes associated with tumor suppression, cell differentiation, and immune system processes by altering regional promoter methylation, indicating that these pathways play pivotal roles in the action of hypomethylating agents. OR21 induced cell differentiation via upregulation of the late cell differentiation drivers CEBPE and GATA-1 Thus, silylation of a deoxynucleotide analog can confer oral bioavailability without new toxicities. Both in vivo and in vitro, OR21 exerted antileukemia effects, and had a better safety profile than DAC. Together, our findings indicate that OR21 is a promising candidate drug for phase I study as an alternative to azacitidine or decitabine.

Figure 1.

Structure of OR21 and its effects on methylation. Structure of 5′-O-triethylsilyl-2′-deoxy-5-azacytidine (OR2100, OR21) and DAC (A). Agents with a log P value of +1.0 to +5.9 are expected to be absorbed after oral administration. Analysis of protein expression in MDS-L and HL60 cells treated with AZA, DAC, or OR21 (B). LINE-1 methylation analysis in MDS-L and SKM1 cells, as determined by bisulfite pyrosequencing (C). MDS-L and SKM1 cells were strongly hypermethylated (88.9% and 82.7%, respectively). Treatment with 100 nmol/L DAC or OR21 significantly decreased LINE-1 methylation levels in MDS-L [AZA, 88.8% (P = 0.867); DAC, 75.2% (P < 0.01); OR21, 74.2% (P < 0.01)] and SKM1 cells [AZA, 72.1% (P < 0.01); DAC, 58.4% (P < 0.01); OR21, 66.9% (P = 0.01)] (mean ± SD; n = 3). LINE-1 methylation analysis in AML cell lines treated with 500 nmol/L OR21. AML cell lines were also hypermethylated (KG1a, 89.1%; Kasumi-1, 90.7%; HL60, 83.7; THP-1, 69.0%; D). Treatment with OR21 tended to induce hypomethylation [KG1a, 82.4% (P = 0.091); Kasumi-1, 78.6% (P = 0.128); HL60, 76.0% (P = 0.089); THP-1, 64.6% (P = 0.243)] (mean ± SD; n = 3).

Figure 2.

Pharmacokinetics and pharmacodynamics of OR21. Sequential plasma samples were taken from monkeys given the prodrug, and plasma drug concentrations were measured over time. After administration of 6.6 μmol/kg OR21, the AUC was 0.30 (± 0.18) μmol/L/hour (A). After administration of 6.6 μmol/kg DAC, the AUC was 0.01 (± 0.01) μmol/L/hour (B). After administration of 10.9 μmol/kg DAC, the AUC was 0.49 (± 0.17) μmol/L/hour and after administration of 10.9 μmol/kg OR21, the AUC was 0.25 (± 0.14) μmol/L/hour (C). After 5-day intravenous administration of 10.9 μmol/kg DAC and duodenal administration (d.a.) of 10.9 μmol/kg OR21, the γ-globin promoter methylation (average three CpG sites at -53, +6, and +17) levels (Cont, 88.3 ± 1.35%) were significantly reduced at day 8 (DAC, 66.5 ± 2.52%, P = 0.02; OR21, 79.5 ± 4.50%, P = 0.02) (D). *, P < 0.05.

Figure 3.

Genes differentially expressed after OR21 treatment due to alteration of promoter DNA methylation. Methylome analysis revealed that OR21-treated HL60 and MDS-L cells had reduced hypermethylation β-values (A and B). Heatmap comparing log₂ fold changes in gene expression among three cell lines (HL60, SKM1, and MDS-L) following treatment with OR21 (C). Analysis revealed enrichment of genes involved in immune system processes (including cancer testis antigen, e.g., PRAME, TGFβ, and CT45A), tumor suppression (e.g., MT1E, F, TGFβ, and ALOX5AP), and cell differentiation (e.g., EVI2A, B, TGFβI, and TYROBO). D and E, Each of these genes were demethylated after OR21 treatment (D). The β-values of each gene decreased (paired t test, E). **, P < 0.01. The delta β-values (OR21-treated minus vehicle-treated) of HL60 and MDS-L cells were below zero (F).

Figure 4.

OR21 induces cell differentiation in MDS and AML cells. Flow cytometry revealed elevated expression of CD11b (granulocytic differentiation marker) in MDS and SKM1 cell lines (MDS-L) following 96 hours treatment with 100 nmol/L AZA, DAC, or OR21 (mean ± SD; n = 3; A). OR21 induced morphologic changes consistent with granulocytic differentiation (i.e., reduced nuclear–cytoplasmic ratio, larger cell size, and higher numbers of multi-lobed nuclei) in MDS-L and SKM1 cells. May–Giemsa staining of cytospin preparations at 96 hours is shown. B, Levels of CEBPA, CEBPB, PU.1, GATA1, and CEBPE mRNAs were measured by quantitative reverse transcription PCR. Upregulation of CEBPE and GATA-1 was observed in MDS-L and SKM1 cells treated with DAC or OR21 (mean ± SD; n = 3; C). *, P < 0.05; **, P < 0.01.

Figure 5.

SKM1 and HL60 xenograft mouse model experiments. Schedule of SKM1 cell xenograft experiments using BALB/c Rag-2/JAK3 double-deficient (BRJ) mice (A). Day 28: flow cytometry of hCD45-positive cells in mice treated with vehicle or OR21 (B). Kaplan–Meier survival curves showing cumulative survival of mice treated with vehicle or OR21 (n = 3 per cohort; C). Median overall survival time was 29 days (vehicle; P = 0.0246); NR = not reached. Statistical analysis was performed using the log-rank test. Schedule of HL60 cell xenograft experiments using BRJ mice (D). Day 37: flow cytometry of hCD45-positive cells in mice treated with vehicle (n = 5), DAC (n = 4), or OR21 (n = 4; E). Kaplan–Meier survival curves showing cumulative survival of mice treated with vehicle, DAC, or OR21 (F). Median overall survival times were 44 days (vehicle; n = 10), 46.5 days (DAC, n = 8, P = 0.164), and 49 days (OR21, n = 10, P = 0.005). Statistical analysis was performed using the log-rank test. DAC-treated mice were more likely to develop anemia than OR21-treated mice (G). Hemoglobin (Hb) levels were 17.5 g/dL (vehicle, n = 8), 15.6 g/dL (DAC, n = 6, P = 0.092), and 17.1 g/dL (OR21, n = 7, P = 1.0). LINE-1 methylation in bone marrow cells decreased after treatment with DAC or OR21 (n = 6 per cohort; H). Mice treated with a higher dose of DAC (2.0 mg/kg) did not live longer, whereas survival was prolonged in mice treated with OR21 (5.4 mg/kg) [median overall survival; vehicle, 48 days; DAC, 32 days (P = 0.040); OR21, 57 days (P < 0.001)] (I). HL60 xenograft mice exposed to late treatment with OR21 (day +28) had more CD11b-positive cells in PB on days +35 to +40, as determined by flow cytometry (n = 4 per cohort; vehicle, 43.3%; DAC, 75.7%, P = 0.229; OR21, 65.5%; P = 0.038; J).

Figure 6.

In vivo safety profile of OR21. A, Kaplan–Meier survival curves showing cumulative survival of mice treated with vehicle, decitabine (DAC; 2.5 mg/kg), or OR21 (7.5 mg/kg; n = 6 per cohort). B, DAC (2.5 mg/kg) exerted high levels of toxicity, causing severe anemia by days +29 and +57. Mice treated with OR21 (7.5 mg/kg) also developed anemia by day +29, but recovered by day +57 while still on the drug (n = 6 per cohort). C, Among mice exposed to low-dose DAC (1.25 mg/kg) or OR21 (3.75 mg/kg) until day +113, those exposed to DAC were more likely to develop anemia (n = 6 per cohort). WBC, white blood cell count; Hb, hemoglobin; platelet count, PLT.