Evolution of Decitabine Development Accomplishments,Ongoing Investigations,and Future Strategies
Elias Jabbour,MD
Jean-Pierre Issa,MD
Guillermo Garcia-Manero,MD
Hagop Kantarjian,MD
Department of Leukemia,The University of Texas M.D.Anderson Cancer Center,Houston,Texas.Decitabine(5-aza-20-deoxycytidine)is a hypomethylating agent with a dual mechanism of action:reactivation of silenced genes and differentiation at low doses,and cytotoxicity at high doses.The original studies in the1980s used deci-tabine as a classical anticancer drug,at its maximum clinically tolerated dose, 1500to2500mg/m2per course.At these doses,decitabine was found to be active in leukemia,but was associated with delayed and prolonged myelosuppression. After a better understanding of epigenetics in cancer and the role of decitabine in epigenetic(hypomethylating)therapy was gained,it was reevaluated at approximately1/20th of the pre
vious doses(ie,at‘optimal biologic’doses that modulate hypomethylation).In these dose schedules of decitabine(100to150mg/ m2per course),the drug was found to be active with manageable side effects in patients with myelodysplastic syndromes(MDS)and other myeloid tumors.Opti-mizing dosing schedules of decitabine to maximize hypomethylation(low dose, high dose intensity,and multiple cycles)have further improved results,suggest-ing that decitabine is an active therapy that alters the natural course of MDS. Combination therapies that augment the epigenetic effect of decitabine will likely improve responses and extend its use for the treatment of other malignancies. Cancer2008;112:2341–51.Ó2008American Cancer Society.
KEYWORDS:decitabine,hypomethylation,myelodysplastic syndromes,response. T he development of new therapeutic strategies for myelodysplas-tic syndrome(MDS)has been the result of extensive under-standing of the pathobiology of the disease.Therapeutics targeting chromatin structure,angiogenesis,and the bone marrow microen-vironment that nurtures the MDS phenotype have demonstrated significant activity and offered an opportunity to alter the natural history of the disease.1Chromatin remodeling is a powerful mecha-nism of regulating gene expression and protein function.2In extreme states,chromatin remodeling can permanently repress expression of a gene,a situation termed‘epigenetic silencing.’Such silencing is exploited by cancers to fully express the mali
gnant phe-notype.3Evidence supporting a role of epigenetic gene silencing in tumorigenesis stems from studies revealing a large number of genes that are silenced by aberrant DNA methylation in different types of cancers,many of which are involved in the control of cell cycle pro-gression,apoptosis,tissue invasion,and genomic stability.
DNA methylation is remarkably altered in most malignancies, with concomitant global hypomethylation and localized hypermeth-ylation.4This increased methylation affects the regulatory region (gene promoter)located in CpG islands and suppresses gene expres-sion permanently,thereby providing cancers with an alternative to mutations or deletions for inactivation of tumor suppressor genes
Address for reprints:Elias Jabbour,MD,Depart-
ment of Leukemia,Unit428,The University of
Texas M.D.Anderson Cancer Center,1515Hol-
combe Blvd.,Houston,TX77030;Fax:(713)
794-4297;E-mail:
Received November13,2007;revision received
December12,2007;accepted December19,
2007.
ª2008American Cancer Society
DOI10.1002/cncr.23463
Published online8April2008in Wiley InterScience(www.interscience.wiley).
2341
and other critical genes.Leukemias and MDS are characterized by the hypermethylation and silencing of multiple genes.5,6Several genes,including the cyclin-dependent kinase inhibitor p15,have aberrant methylation and are associated with resistance to chemotherapy.7–9
The2cytosine analogs azacitidine(5-azacyti-dine)and decitabine(5-aza-20-deoxycytidine)are methyltransferase inhibitors that exhibited encoura-ging in vitro antileukemic activity.5Decitabine is p
hosphorylated to decitabine triphosphate,which incorporates into DNA,depletes DNA methyltransfer-ase,and induces replication-dependent DNA hypo-methylation.10,11At high doses decitabine produces DNA adducts that results in DNA synthesis arrest and cytotoxicity.10,11At low doses it induces gene expression profile changes that favor differentiation, reduced proliferation,and/or increased apoptosis.12–15 This dual antitumor activity has generated significant interest in investigating hypomethylating agents as an-tineoplastic drugs and biologic response modifiers. This review details the role of decitabine in the treat-ment of MDS and other hematologic malignancies.
History of Decitabine
Decitabine(Fig.1)was first synthesized in1964and its potential antileukemic activity was reported in 1968.16Interest in decitabine was enhanced by pre-clinical studies indicating that decitabine is a more potent antileukemic agent in mice than cytosine ara-binoside,11and by the report of Jones and Taylor10 that it could induce terminal differentiation of a mu-rine embryonic cell line.Momparler et al.17and Riv-ard et al.18initiated what to our knowledge were the first clinical trials of decitabine in acute leukemia. The original studies were classic phase1trials that identified the maximum tolerated dose(MTD)as 1500to2250mg/m2per course.17,18The dose-limit-ing toxicity(DLT)was prolonged myelosuppression. Further clinical studies were disappointing in solid tumors but more promising in a
cute myelogenous leukemia(AML),MDS,and chronic myelogenous leukemia(CML).19The regimens tested involved high doses of decitabine given for1to7days per course. In patients with higher-risk MDS,a low-dose schedule (15mg/m2every8hours for3days5135mg/m2 per course)demonstrated encouraging activity.20The University of Texas M.  D.Anderson Cancer Center leukemia group introduced decitabine into leukemia trials in the U.S.in1992.After a better understanding of its hypomethylating effects,and optimization of dose schedules,a pivotal phase3study was designed and initiated in the year2000to compare low-dose decitabine versus supportive care in patients with MDS.21Based on these data,decitabine(Dacogen; SuperGen,Dublin,Calif)received approval from the U.S.Food and Drug Administration for the treatment of MDS and chronic myelomonocytic leukemia (CMML)in May2006.
Preclinical Studies With Decitabine
DNA hypermethylation and gene silencing
Aberrant DNA methylation is present in many malig-nancies,with concomitant global hypomethylation and localized hypermethylation.4Increased methyla-tion affects regulatory regions located in CpG islands and suppresses gene expression.This provides can-cers with an alternative to mutations or d
eletions for inactivation of tumor suppressor genes and other critical genes.Leukemias and MDS are characterized by hypermethylation and silencing of multiple genes.5,6This process can occur early in the disease course and is also associated with disease progres-sion.The cyclin-dependent kinase inhibitor p15INK4b was described as a frequent target of aberrant meth-ylation in MDS,and its inactivation was associated with an increased risk of progression to AML.7Aber-rant methylation of other similar genes was also described and was associated with resistance to ther-apy and tumor progression.8,9,22
Dual modes of action of decitabine
The antineoplastic action of decitabine results from its incorporation into newly synthesized DNA.It
is FIGURE1.Chemical structure of decitabine.
2342CANCER June1,2008/Volume112/Number11
an S-phase-specific agent11and has a dual,dose-de-pendent mechanism of action.At high doses its cyto-toxic activity is due to covalent trapping the enzyme DNA methyltransferase into DNA.10,11At lower doses its antitumor effect is likely due to its ability to in-hibit DNA hypermethylation and to reactivate tumor suppressor genes.10,11At low doses decitabine does not block cell cycle progression of G1phase cells into the S-phase.Low doses of decitabine incorporated into DNA lead to trapping and depletion of the enzyme DNA methyltransferase.In clonogenic as-says,a1-hour exposure to a10m M concentration of decitabine produced loss of clonogenicity in the same range of cells in S-phase(30–50%).A longer ex-posure time of24hours demonstrated a markedly greater antineoplastic activity,with>95%loss of clonogenicity with a decitabine dose of1m M.23The hypomethylation induced by decitabine resulted in reexpression of tumor suppressor genes,induction of cellular differentiation,and suppression of tumor growth.10,24
Hypomethylation and histone acetylation
Decitabine is phosphorylated and incorporated into DNA.It then covalently binds to DNA methyltrans-
ferases and traps the enzyme to DNA,acting as an ir-reversible inhibitor of its enzymatic activity. Consequently,decitabine induces marked DNA hypo-methylation in vitro and in vivo,15and restores silenced gene expression.Its detailed molecular mechanism is still being deciphered.There appears to be a cascade of biochemical events triggered by promoter DNA methylation that involve initial DNA binding proteins,which attract histone deacetylases and histone methylases,and eventually modify his-tones into a silenced chromatin state.25–27A feedback loop is operational between DNA methylation and histone methylation,whereby each of these bio-chemical modifications at a given gene trigger the other,thus creating a self-reinforcing silencing loop.25,27This silencing loop is interrupted by decita-bine.Decitabine induces hypomethylation and re-verses the silenced histone code at tumor suppressor gene loci.17,18,22This dual effect(hypomethylation/ histone changes)may explain the superior effect of decitabine on gene expression compared with his-tone deacetylase inhibitors.28Decitabine also has sig-nificant effects on the expression of genes not silenced by CpG island methylation.
Decitabine induces the expression of p21,a gene that has demonstrated no DNA hypermethylation in cancer.20,29The effects of decitabine on the histone code are not limited to genes showing silencing by promoter-associated methylation.22In bladder cancer cells,decitabine induced rapid and substanti
al remo-deling of the heterochromatic domains of the p14ARF/p16INK4a locus,reducing levels of dimethy-lated H3-K9and increasing levels of dimethylated H3-K4.It also increased acetylation and H3-K4meth-ylation at the unmethylated p14promoter,suggesting it can induce chromatin remodeling independently of its effects on cytosine methylation.27This silen-cing-independent activity of decitabine is not well understood.Other changes may be reactive,due to the stress of exposing cells to a cytotoxic agent. Thus,the ultimate antineoplastic mechanism of deci-tabine may be very pleiotropic.
In vivo molecular effects of decitabine
Global hypomethylation after decitabine therapy in vivo was observed in early trials,30and was studied in more detail in recent studies in leukemia.15,31 Hypomethylation after decitabine was dose-depend-ent,peaked10to15days after the initiation of ther-apy,and recovered to baseline at4to6weeks.In the AML cell line OCI-AML2,decitabine induced the expression of81of22,000genes;96genes were down-regulated(32-fold change in expression).32 Similar results were obtained with primary AML and MDS cells after treatment with decitabine ex vivo and in vivo,respectively.In contrast,significantly fewer changes in gene expression and cytotoxicity were detected in normal peripheral blood mononu-clear and bone marrow cells or transformed epithe-lial c
ells treated with decitabine.32Hypomethylation demonstrated an inconsistent association with response,with a positive correlation in AML,but an inverse correlation in CML.The latter inverse corre-lation may be due to the death of responsive-hypo-methylated cells and a shift to resistant cells that withstand more hypomethylation.
Daskalakis et al.33documented p15demethyla-tion in marrow DNA samples in9of12patients with MDS treated with decitabine,and evidence of p15 gene reactivation in4responding patients with low baseline expression.They also noted gene reactiva-tion in morphologically dysplastic cells in patients not in complete remission at the time of study, demonstrating the in vivo effect of the drug.In a recent study,p15hypomethylation after decitabine was observed,but there was no correlation between baseline p15methylation or after therapy and response.31
Pharmacokinetics
After intravenous decitabine administration,plasma protein binding is negligible(<1%)and has an excel-lent distribution in body fluids.Due to the nucleo-Treatment With Decitabine/Jabbour et al.2343
side transport system there is rapid equilibration of decitabine between extracellular and intracellular compartments.19,34The plasma half-life in humans is approximately35minutes due to rapid deamina
tion by high levels of cytidine deaminase.18Decitabine crosses the blood-brain barrier,achieving27%to 58%of the plasma concentrations after continuous infusion.35The repeated administration of decitabine in patients with advanced MDS as a3-hour infusion of15mg/m2every8hours for3days does not result in systemic accumulation of the drug,and pharma-cokinetic remains unchanged from cycle to cycle.36,37 When decitabine was given as a1-hour infusion daily for10days every28days,the plasma drug concen-tration-time on Days1and10achieved a mean Cmax of93ng/mL and following a2-compartment infusion model.The mean short and long half-lives were2.7minutes and36.9minutes,respectively,with a trend of decreasing the longer half-life on Day10.38 The oral administration of cytidine analogs such as decitabine and azacitidine is not optimal due to the rapid decomposition in acidic conditions and high first-pass metabolism;the reported bioavailability of these agents ranges from9%to41%.35,38Decitabine is initially activated by deoxycytidine kinase from a monophosphate form to the active triphosphate form,which is then incorporated into DNA by DNA polymerase.Decitabine can be inactivated through its major elimination pathway involving deamination by cytidine deaminase found principally in the liver, but also in granulocytes,intestinal epithelium,and plasma.38Urinary clearance of intact drug accounted for29%of plasma clearance in mice.In humans, total body clearance in the range of124Æ19mL/ min/kg exceeds hepatic blood flow and is explained by extensive extrahepatic deamination.39Decita-bine is not a substrate for the cytochrome P450 enzymes.39
Deoxycytidine analogs enter cells rapidly by a nucleoside-specific transport mechanism.19Azaciti-dine and decitabine are activated to triphosphate forms(azacytidine by uridine-cytidine kinase,decita-bine by deoxycytidine kinase)which are subject to degradation by cytidine deaminase.Azacitidine incorporates primarily into RNA(80%–90%),and to a much lesser extent into DNA.40Incorporation into RNA produces disassembly of polyribosomes,altered RNA methylation,a defective acceptor function of transfer RNA,and marked inhibition of protein syn-thesis.In contrast,decitabine incorporates primarily into DNA.Its incorporation into DNA results in cova-lent trapping of DNA methyltransferase causing inhi-bition of DNA methylation at concentrations that do not cause major suppression of DNA synthesis.39The more direct metabolic activation pathway to DNA incorporation of decitabine makes it a more potent compound compared with azacitidine.41,42
Clinical Experience With Decitabine
Early clinical trials
A phase1pharmacokinetic study of decitabine was conducted in21patients with advanced solid tumors.19The drug was given at dose ranges of25to 100mg/m2infused over1hour every8hours33, and the treatment repeated every3to6weeks. For the75mg/m2and100mg/m2doses,mean peak plas
ma concentrations were0.93m M/mL and 2.01m M/mL,respectively.There was rapid disappear-ance of drug from plasma with a T1/2alpha and T1/2 beta of7minutes and35minutes,respectively.Total urinary excretion was<1%of the administered dose, suggesting that decitabine was eliminated rapidly and largely by metabolic processes.19
Initial phase1trials
The first clinical studies of decitabine in hematologic malignancies used1500to2500mg/m2per course.17,18Response rates with decitabine as a sin-gle agent or in combination with other therapies were30%to60%.17,18,20,24,29,43,44In a phase1trial in 30children with refractory or recurrent leukemia, with administered decitabine doses0.75to80mg/kg, significant reductions of circulating blasts were reported.18Using a dose of37mg/kg in a36-hour infusion,1complete remission was noted in a patient with acute lymphocytic leukemia(ALL).17 Momparler et al.17reported similar dose-dependent activity when decitabine was given to27patients at doses ranging from31to81mg/kg over36to 60hours.Nonhematologic toxicity was rare;no MTD was reported.There was detection of70%inhibition of DNA methylation,but no evidence of blast differ-entiation.17
Despite promising activity,high-dose decitabine regimens were not pursued because of delayed and
prolonged myelosuppression.At these doses the dec-itabine effect was likely a cytotoxic one.At lower dose schedules(15mg/m23times a day for3days), decitabine was found to have encouraging activity in MDS.20At an even lower dose(0.15mg/kg daily over 1hour daily for10days)decitabine was reported to have biologic efficacy in reactivating hemoglobin F in patients with sickle cell disease.45These observa-tions,combined with the short half-life of the drug and its absolute requirement for DNA synthesis for activity,led to a novel phase1trial of decitabine in patients with recurrent or refractory leukemia.31This study tested low-dose longer exposure schedules,
2344CANCER June1,2008/Volume112/Number11
with the intent of finding an‘optimal biologic dose’to modulate the molecular target achieving a response at levels lower than the classic MTD.Fifty patients(44with AML/MDS,5with CML,and1with ALL)were treated with increasing doses of decitabine (5mg/m2,10mg/m2,15mg/m2,and20mg/m2) intravenously over1hour daily,5days a week for2 consecutive weeks.The starting dose per course in this study was30times less than the previous estab-lished MTD.The exposure duration was then increased to15days and20days.The treatment was well tolerated and responses were noted at all dose levels.The objective response rate was32%(16of50 patients).In37patients with AML,5(14%)achieved a complete response(CR)and3(8%)achieved a par-tial response(PR).In7patien
ts with MDS,2(29%) achieved a CR and2(29%)achieved a PR.In5 patients with CML,2(40%)achieved a CR and2 (40%)achieved a PR.Responses were slow and grad-ual;the median time to response was45days(range, 16–70days).The dose of15mg/m2daily310was judged to be optimally effective(11responses in17 treated patients;65%);lower response rates were noted when the dose was escalated or prolonged(2 of19or11%).The low response rate at high doses was consistent with earlier studies in which decita-bine at a dose of500to1000mg/m2per course induced CR in only1of17patients for AML recur-rence(unpublished data).In this study,p15gene promoter DNA methylation studies in peripheral blood mononuclear cells were performed in29 patients(including7patients who achieved CRs or PRs),only15(52%)of whom(including2patients who achieved CRs or PRs)had promoter hypermeth-ylation at baseline consistent with gene silencing(ie, >10%methylated).There was no correlation between p15methylation at baseline or after therapy and response to decitabine.Phase2studies in MDS
Two large phase2studies of decitabine in MDS were recently reported(Table1).In the studies of Wijer-mans et al.20,46,47in Europe,169older patients(me-dian age of70years)with intermediate-risk or high-risk MDS received low-dose decitabine(135mg/m2 total dose per course).The overall response rate was 49%,and the induction mortality rate was7%. Response rates were51%with high-risk diseas
e and 46%with intermediate-1disease.Improvement in thrombocytopenia was noted in63%of patients after 2cycles.48Complete remissions were associated with cytogenetic remissions.49Cytogenetic responses by the International Prognostic Scoring System(IPSS) were:low-risk,3of5(60%)patients;intermediate-risk,6of30patients(20%);and high-risk,10of26 patients(38%).Survival was longer among patients achieving a cytogenetic response compared with those who did not(P5.02).
modulateIn a phase2trial of decitabine in patients with MDS,50testing both dose intensity and subcutaneous route of administration,patients received a total dose of100mg/m2per course,and were randomized in a Bayesian design to1of3arms:1)10mg/m2 administered intravenously over1hour daily for 10days;2)20mg/m2administered intravenously over1hour daily for5days;and3)20mg/m2admi-nistered subcutaneously daily for5days(adminis-tered as2subcutaneous doses).Cycles were administered every4weeks and response or lack of response was evaluated only after at least3cycles were given.Ninety-five patients(median age of 67years)were treated,77of whom had MDS,and18 of whom had CMML.Thirty-two percent had secondary MDS and66%had intermediate-2and high-risk disease.The median number of cycles was 71(range,1–18cycles).Overall,32patients(34%) achieved a CR and69(73%)had an objective
TABLE1
Clinical Results of Single-Agent Decitabine in Patients With MDS(Phase2Trials)
MDS indicates myelodysplastic syndromes;IPSS,International Prognostic Scoring System;INT-2,intermediate-2risk;HI,hematologic improvement;OR,overall response;iv,intravenous;sc,subcutaneously;CR, complete response;PR,partial response;NR,not reported.
Treatment With Decitabine/Jabbour et al.2345

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