Azacitidine: 10 Years Later1
`
`Alice B. Glover, Brian R. Leyland-Jones,• Hoo G. Chun, Barbara Davies, and Daniel F. Hoth2
`
`Azacitidine has been undergoing clinical trials for almost 20 years and is internationally
`considered to have a useful place in the treatment of acute nonlymphocytic leukemia.
`However, its role in the various combinations for induction, intensification, maintenance, or
`relapse regimens has not yet been clearly defined. This review outlines the last 10 years'
`clinical experience with the drug in acute nonlymphocytic leukemia, analyzes what critical
`information has yet to be obtained, and suggests what phase III trials may still be feasible to
`gather that information. [Cancer Treat Rep 71:737-746, 1987)
`
`Azacit idine is a ring analog of the natural pyrimidine
`nucleoside cytidine synthesized in 1964. Clinical trials
`began in Europe in 1967 and in the United States in
`1970. Preclinical information and results of early clini(cid:173)
`cal trials were comprehensively discussed in a previous
`review (1). In that article the authors described a drug
`with "consistent antitumor activity in patients with
`acute myelogenous leukemia resistant to previous treat(cid:173)
`ment" and "an overall r esponse rate of 36% with 20%
`complete remissions ... in ... acute myelogenous leu(cid:173)
`kemia ... of adults and children." Furthermore. the
`median duration of remission with azacitidine was noted
`to be "encouragingly long." In summary, Von Hoff et al
`(1) suggested four potential directions for the develop(cid:173)
`ment of azacitidine as an antileukemic agent, as follows:
`(a) to define the use of azacitidine in the first-line
`treatment of acute nonlymphocytic leukemia (ANLL);
`(b) to explore in a controlled manner the use of infusions
`of the drug versus other modes of administration; (c) to
`examine the use of sc administration, since this would
`be a more desirable route, especially in children; and (d)
`to define the role of azacitidine in consolidation, main(cid:173)
`tenance, and late intensification regimens.
`The review also suggested two other areas of explora(cid:173)
`tion: (a) to develop the early observations of the protec(cid:173)
`tive effect of a zacitidine on mice exposed to lethal x(cid:173)
`irradiation; and (b) to expand animal experiments using
`azacitidine with cytidine as an antidote, to improve the
`therapeutic index of the drug.
`Alter a brief update on preclinical issues, this paper
`will examine the last 10 years' clinical development of
`azacitidine, summarize how these six recommendations
`have been pursued, and finally examine what still needs
`
`to be done to establish the role of azacitidine in the
`treatment of ANLL.
`
`MECHANISM OF ACTION
`
`Several steps in cellular metabolism are affected by
`azacitidine and previous papers (1-5) have dealt with
`these in some detail. The accompanying paper on the
`biochemistry of azacitidine reviews these data at length
`(6) and only the main features are summarized here.
`Azacitidine is transported into the cell and sequen(cid:173)
`tially phosphorylated by the same mechanisms as for
`cytidine and uridine. 5-Azacytidine trip hosphate is in(cid:173)
`corporated into RNA, d isrupting the synthesis and pro(cid:173)
`cessing of both nuclear and cytoplasmic species and
`leading to inhibition of protein synthesis. It is also in(cid:173)
`corporated to a lesser extent into DNA leading to inhi(cid:173)
`bition of DNA synthesis.
`The most interesting development at the biochemical
`level since previous reviews (1-3) has been the increas(cid:173)
`ing use of azacitidine as a "laboratory tool" to examine
`the role played by DNA methylation upon gene expres(cid:173)
`sion and activation. After incorporation into DNA, aza(cid:173)
`citidine noncompetitively inhibits DNA methyltrans(cid:173)
`ferase, causing a block in cytosine methylation in newly
`replicated DNA (7-12). There is now a large body of
`evidence, which has been comprehensively reviewed
`(13-18), indicating that alterations in the pattern of cy(cid:173)
`tosine methylation are causally related to gene expres(cid:173)
`sion and cell differentiation. This hypomethylation of
`DNA is thus probably the mechanism underlying the
`differentiating activity of azacitidine (19) and may be
`related to its tumorigenic effects (20-27).
`
`1 Received Oct 2, 1986; revised Apr 20, 1987; accepted May 5. 1987.
`2 Investigational Drug Branch, Cancer Therapy Evaluation Program.
`Division of Cancer Treatment. National Cancer Institute, National Insti(cid:173)
`tutes of Health, Bethesda. MO.
`
`• Reprint requests to: Brian R. Leyland-Jones, MD, I nvestigational
`Drug Branch, Cancer Therapy Evaluation Program, Division of Cancer
`Treatment, National Cancer Institute, Landow Bldg, Rm 4C09, National
`Institutes of HEalth, BethESda, MD 20892.
`
`Cancer Treatment Reports Vol. 71. No. 7-8, July/August 1987
`
`737
`CELGENE 2034
`APOTEX v. CELGENE
`IPR2023-00512
`
`

`

`PHARMACOKINETICS
`
`The initial study by Troetel et al (28) had shown that
`the plasma half-life of total radioactivity (parent drug
`plus metabolites) after iv administration was about 3½
`hours. Subsequently, Israili et al (29) obtained similar
`results for total radioactivity. In addition, they identified
`at least two metabolites and/or decomposition products
`of azacitidine in plasma. They also showed that levels of
`the parent drug declined much more rapidly than those
`of total radioactivity so that in one patient levels of aza(cid:173)
`citidine were < 2% of total radioactivity after 30 min(cid:173)
`utes. Renal clearance of radioactivity varied from 74 to
`210 ml/minute in the five patients in Israili's study and
`most of the administered radioactivity was excreted via
`the urine.
`From preclinical studies it is known that azacitidine
`is rapidly deaminated by cytidine deaminase which is
`
`present in serum as well as in the liver, granulocytes,
`and gastrointestinal epithelium. This enzyme can be
`inhibited both in human leukemic cells in vitro and in
`the mouse in vivo by tetrahydrouridine (30,31). The
`relative role and clinical significance of deamination in
`the metabolism of azacitidine in humans as compared
`with other enzymatic degradation pathways cannot be
`determined until a specific and sensitive assay for aza(cid:173)
`citidine is developed.
`
`RESULTS OF CLINICAL TRIALS
`This paper will only review those trials of azacitidine
`conducted in ANLL of adults and children. Phase II
`trials in several solid tumors including colorectal, breast,
`lung, melanoma, head and neck, and renal carcinoma
`did not reveal adequate activity to justify further inves(cid:173)
`tigation in these tumor types. A summary of these trials
`is shown in table 1.
`
`TABLE 1.- AZA in solid t umors (phase II trials only)•
`
`No. of-
`
`Schedule
`
`Tumor
`
`Evaluable patients
`(No. entered)
`
`Responses
`
`Ref No.
`
`Colon cancer
`Pancreatic cancer
`Gastric cancer
`
`Breast cancer
`Lung cancer
`Large cell cancer
`Intestinal cancer
`Melanoma
`Hodgkin's disease
`NHL
`Other cancer
`
`Colon cancer
`Renal cancer
`Breast cancer
`NSCLC
`SCLC
`Testicular cancer
`Melanoma
`Other cancer
`Breast cancer
`Renal cancer
`Colon cancer
`Melanoma
`SCLC
`NSCLC
`Hand N
`Other cancer
`
`Other cancer
`
`Osteogenic sarcoma
`Ewi ng's sarcoma
`
`27
`1
`
`27
`
`29
`24
`
`26
`12
`6
`8
`59
`
`15
`17
`14
`12
`14
`4
`13
`85
`6 (8)
`10 (12)
`7 (9)
`10 (10)
`2 (3)
`26 (33)
`14 (14)
`16 (24)
`
`6
`
`7
`7
`
`l PR
`1 < PR
`
`2 PR
`
`6 PR
`1 PR
`
`0
`0
`1 PR
`2 PR
`9 PR
`
`0
`0
`0
`2 PR
`l PR
`2 PR
`
`0
`1 PR
`0
`1 < PR
`0
`l PR; l < PR
`0
`0
`0
`
`0
`0
`
`32
`
`33
`
`34
`
`35
`
`36
`
`37
`
`38
`
`Disease site (group)
`
`Advanced gastrointestinal
`cancer (Mayo)
`
`Breast cancer (RPMI)
`
`Broad phase II (COG)
`
`Dose
`(mg/m2
`
`)
`
`750
`
`60
`100
`
`60
`
`Daily X 5or
`daily X 10
`
`Daily X 10
`then 2 X/wk
`
`Daily X l0t
`
`Broad phase II (SWOG)
`
`225
`
`Daily X 5
`every 3 wks
`
`Broad phase II (SEG)
`
`150
`
`2 X/wk for
`6 wks
`
`Broad phase II (Mt. Sinai)
`
`30-120
`25-200
`
`24-hr CIV
`+
`Pynuofurin
`
`Sarcoma
`
`150-200
`
`Daily X 5
`
`• AZA = azacitidine; Mayo= Mayo Clinic; RPM! = Roswell Park Memorial Institute; COG = Central Oncology Group; SEG = Southeastern Cancer
`Study Group; Mt. Sinai= Mt Sinai Hospital; CIV = continuous iv infusion; N HL = non-Hodgkin's lymphoma; NSCLC = non-sma ll cell lung cancer;
`SCLC = small cell lung cancer: H and N = head and neck cancer; PR= partial response; and < PR = objective response not meeting criteria for PR.
`t : 1.6 mg/ kg.
`
`738
`
`Cancer Treatment Reports
`
`) 11 l",tvG<.)<.)glc
`
`

`

`TABLE 2.- AZA alone in ANLL"
`
`Evaluable pat ients
`
`T
`
`50
`
`RI
`
`50
`
`Rf
`
`T
`
`No. of-
`
`CRs
`
`RI
`
`10
`
`Rf
`
`IOI
`
`8
`
`8
`
`18
`
`6
`
`18
`
`6
`23
`5
`21
`45
`15
`10
`3
`
`321
`
`14
`335
`
`18
`
`14
`
`9
`
`174
`
`3
`
`0
`5
`0
`3
`1
`2
`
`11
`5
`3
`
`0
`
`51
`
`5
`
`4
`
`6
`10
`3
`
`28
`
`14
`56
`
`3
`
`2
`
`3
`
`24
`
`2
`
`3
`
`7
`
`5
`
`T
`
`5
`
`4
`
`2
`4
`0
`7
`
`4
`2
`
`3
`
`0
`
`100
`750
`75
`
`250
`300
`200
`200
`150
`150-600
`120-300
`
`200-250
`
`60
`100
`180
`300
`150-200
`200
`150
`50-90
`
`Schedule
`
`Every 8 hrs X 15
`Single iv
`4-hr infusion every
`6 hrs X 20
`Iv every 4 hrs X 3
`DailyCIV X 5
`Daily CIV X 5
`Daily CIV X 7
`Daily CIV x 10
`Daily iv X 5
`
`Daily iv X 5
`Daily iv X 5
`Every 8 hrs X 15
`Every 8 hrs X 15
`Daily iv X 5
`Daily iv X 5
`Daily CIV X 5
`Daily iv X 5
`
`Daily CIV x 5
`
`3-hr infusion every
`8 hrs X 16
`
`150-200
`
`Daily iv X 5
`
`Group or
`institution
`
`Dose
`(mg/m2 )
`
`SWOGt
`
`SWOGt
`
`MDA
`
`MSKCC
`BCRC
`WCG
`
`SEG
`University of Chicago
`
`MMC
`
`E COG
`
`Subtotal
`
`CCSG
`Total
`
`PRs
`
`RI
`
`5
`
`Rf
`
`Ref No.
`
`39
`
`40
`
`41
`42
`43
`44
`
`45
`46
`47
`48
`
`49
`
`2
`
`0
`3
`
`• MDA = M. D. Anderson Hospital and Tumor Institute; MSKCC = Memorial Sloan-Kettering Cancer Center : BCRC = Baltimore Cancer Research
`Center; WCG = Western Cancer Study Group; MMC = Maine Medical Center; ECOG = Eastern Cooperative Oncology Group; CCSG = Childre·n's
`Cancer Study Group; T = total No. of evaluable patients who were considered by the invest igators to have had an adequate trial of aiacitidine; Rl =
`No. of patients relapsed after induction on standard therapy; Rf= No. of patients refractory to standard induction regimens; and CR = complete r e(cid:173)
`SJ)Qnse.
`t Breakdown by infusion schedule included all leukemia types, so only total for ANLL given here.
`
`Azacltldlne as a Single Agent In ANLL
`Table 2 shows the azacitidine single-agent data from
`trials sponsored by the National Cancer Institute as of
`June 1985. Some of these studies included acute lym(cid:173)
`phocytic leukemia and blast crisis of chronic myelocytic
`leukemia, but the data presented in table 2 are only for
`ANLL. The single-agent data base shown in the earlier
`review (1) has now been expanded from a total of 200
`patients to a total of 335. The overall complete response
`rate has fallen from 20% to 16.7%. As can be seen, the
`pediatric data base has not been expanded in single(cid:173)
`agent use since 1976. Furthermore, the largest st udy
`(40) observed only eight complete responses among 101
`evaluable patients who received an adequate trial of
`azacitidine.
`
`Effect of Schedule on Activity of Azacltldlne
`Azacitidine was active on both daily and intermittent
`schedules against L1210 leukemia. While its predomi(cid:173)
`nant effects were in the S phase, its action was not
`
`limited to the S phase (3). In early clinical trials three
`schedules were used: daily X 5; every 8 hours X 15; and
`daily continuous infusion X 5-7 days. Because of the
`instability of the drug most of the continuous-infusion
`studies actually employed a 3-hour infusion every 8
`hours (40,45,47). (There has been conflicting advice in
`various publications about the stability of azacitidine in
`iv solutions. This issue has recently been reviewed (50)
`and a comparative analysis of decomposition rate in
`several infusion solutions carried out (51). The recom(cid:173)
`mendation from these studies is that the reconstituted
`solution be further diluted within 30 minutes in lactated
`Ringer's injection, USP, which provides optimum pH
`for stability. Under these conditions there will be 90% of
`intact drug remaining after 2 hours and 80% after 6
`hours. Therefore, the dilute solution should be infused
`over $ 2-3 hours.]
`Table 3 summarizes the response rate in acute leuke(cid:173)
`mia aecording to the three schedules used in the initial
`trials. Since data are pooled from all the trials in table
`2, the analysis includes not only patients with ANLL
`
`Vol. 71 . No. 7-8. July/August 1987
`
`739
`
`o 11
`
`'dbyGoogle
`
`

`

`TABLE 3.-Effector schedule on AZA response rate•
`
`Dose
`(mg/m2)
`
`150-500
`50-100
`150-300
`
`Schedule
`
`Daily X 5
`Every 8 hrs X 15
`Daily CJV x 5-7
`
`No. of
`evaluable patients
`
`122
`110
`138
`
`Response
`rate(%)
`
`CR
`
`PR
`
`21
`24
`23
`
`17
`8
`9
`
`• Data are pooled from all the trials in table 2; therefore Nos. include
`not only patients with ANLL but also patients with acute lymphoeytic
`leukemia a nd blast crisis of chronic myeloeytic leukemia.
`
`but also patients with acute lymphocytic leukemia and
`blast crisis of chronic myelocytic leukemia. Using this
`form of analysis to achieve large denominators, the
`complete response rate does not vary between any of
`these schedules. However, the question of schedule de(cid:173)
`pendence has not been addressed in an appropriate
`prospectively randomized trial with statistically defined
`endpoints.
`It is difficult to assess from pooled data whether toxic(cid:173)
`ity is schedule-dependent. The Southwest Oncology
`Group (SWOG) study (40) tested several different sched·
`ules sequentially. Dose and schedule were altered de-
`
`TABLE 4.-AZA: 2-drug combination regimens in remission induction of ANLL•
`
`No.of-
`
`Evaluable
`patients
`
`CRs
`
`PRs
`
`Group or
`institution
`
`Regimen
`
`Dose
`(mg/m2)
`
`Schedule
`
`T
`
`RI
`
`Rf
`
`T
`
`RI
`
`Rf
`
`RI
`
`Rf
`
`Ref No.
`
`T
`
`0
`
`4
`
`5
`
`5
`
`5
`
`5
`
`52
`
`53
`
`54
`
`56
`
`56
`
`44
`
`57
`
`BCRC
`
`BCRC
`
`SEG
`
`SEG
`
`CALGB
`
`SEG
`
`33
`100
`150-250
`7.6-30
`
`150
`25-150
`
`150
`300
`50
`75
`112-200
`75-150
`200
`150
`
`Every 8 hrs X 15
`Daily CIV X 5
`
`Daily X 5
`Daily X l
`Daily CIV X 5
`Single iv
`
`DailyCIV X 5
`Daily X 5
`3-hr infusion every day X 5
`Daily X 3
`Daily CIV X 4}
`Daily X 4
`Daily CIV X 4}
`Daily X 4
`
`16
`
`27
`
`81
`
`29
`
`27
`
`53
`
`Adult
`8
`
`4
`
`4
`
`0
`
`2
`
`8
`
`2
`
`3
`
`7
`
`81
`
`16
`
`16
`
`7
`
`19
`
`27
`
`53
`
`8
`
`3
`
`7
`
`7
`
`AZA
`MGBG
`AZA
`PF
`AZA
`PF
`AZA
`TGdR
`AZA
`Zorubicin
`I AZA
`\ AMSA
`AZA
`{
`AMSA
`AZA
`AMSA
`AZA
`VP-16
`
`AZA
`AMSA
`AZA
`VP-16
`
`AZA
`VP-16
`IAZA
`\ VP-16
`AZA
`{
`VP-16
`AZA
`VP-16
`AZA
`VP-16
`AZA
`DNR
`
`ECOG
`
`BCRC
`
`NWU
`
`DFCC
`
`DFCC
`
`St. Judet
`
`St. Judet
`
`POGt
`
`CCSG
`
`300
`250
`100
`30
`•CALGB = Cancer and Leukemia Group B; NWU = Northwestern University; DFCC = Dana-Farber Cancer Center; St. Jude = St. Jude Children's
`Hospital; POG = Pediatric Oncology Group; MGBG = mitoguazone; PF= pyrazo(urin; TGdR = deoxythioguanosine; AMSA = amsacrine; VP-16 =
`etoposide; and DNR = daunorubicin.
`tOngoing.
`
`150
`150
`50
`75
`150
`150
`300
`200
`
`800
`200
`150
`100
`300
`200
`300
`250
`
`Daily X 5
`Daily X 5
`
`Every 8 hrs X 15
`Daily X 5
`
`Daily X 5
`Daily X 6
`Daily X 2
`Daily X 3
`
`Daily X 2
`Daily X 3
`
`Daily X 2 }
`Daily X 3
`Daily X 2 }
`Daily X 3
`Daily X 2
`Daily X 3
`Daily X 2
`Daily X3
`Daily X 5
`Daily X 3
`
`18
`
`14
`
`16
`
`13
`
`12
`
`4
`
`6
`
`4
`
`12
`
`18
`
`Pedia.tru:
`14
`
`0
`
`14
`
`16
`
`14
`
`6
`
`16
`
`14
`
`6
`
`7
`
`0
`
`0
`
`0
`
`3
`
`6
`
`6
`
`0
`
`0
`
`3
`
`6
`
`6
`
`0
`
`0
`
`2
`
`0
`
`2
`
`58
`
`59
`
`44
`
`44
`
`44
`
`60
`
`61
`
`62
`
`52
`
`34
`
`36
`
`16
`
`34
`
`28
`
`14
`
`17
`
`11
`
`14
`
`3
`
`4
`
`3
`
`0
`
`4
`
`63
`
`44
`
`740
`
`Cancer Treatment Reports
`
`

`

`pending on the toxicity in the preceding group of pa·
`tients. They found that the proportion of severe nausea
`and vomiting was significantly less in patients receiving
`the low-dose infusion schedules (150-200 mg/ m2/day in
`four divided doses) rather than the high once-weekly
`dose (750 mg/m2 in three divided doses). In this trial
`there were several cases of coma. Upon review of each
`case it was concluded that some may be partially at(cid:173)
`tributable to azacitidine. The incidence rate in the
`higher-dose schedules (300 and 750 mg/m2/day in divided
`doses) was 22%, whereas that on the 150-200-mg/m2/day
`regimens was 4.6%. Thus, the lower-dose infusion sched(cid:173)
`ules appear to be associated with less gastrointestinal
`and neurological toxicity. These lower-dose schedules
`have been almost universally adopted for the more re(cid:173)
`cent studies.
`
`Azacltldlne In Two-Drug Combinations In Relapsed or
`Refractory ANLL
`Table 4 shows an analysis of the two-drug combina(cid:173)
`tion regimens separated into refractory and relapsed
`patients and into adult and pediatric populations. The
`total number of evaluable patients with ANLL who
`have received azacitidine in combination with one other
`drug as second-line or later therapy is now > 360. Two
`observations are immediately apparent. First, the pa(cid:173)
`tient denominator on each regimen (with the possible
`exception of azacitidine + deoxythioguanosine) is inade(cid:173)
`quate to demonstrate superiority of any one combination
`over another. Second, the denominators are again in(cid:173)
`adequate to show superiority of any combination over
`the single-agent results. Even after extracting the data
`
`for the most frequently used combination in adults (aza(cid:173)
`citidine and amsacrine) from the three phase II trials,
`there are still only 15 complete responses and five par(cid:173)
`tial responses among 87 patients. In pediatrics, the
`combination of azacitidine with etoposide was the most
`frequently tested. An overall complete response rate of
`50% using these two drugs was seen in the phase II
`evaluation and complete responses were observed in
`both relapsed and refractory patients. None of these
`two-drug combinations has been brought into front-line
`therapy.
`
`Azacilldine: Mullidrug Combinations
`Table 5 presents these data subdivided by adult and
`pediatric trials. Of particular note in the pediatric trials
`is the D-ZAPO (daunorubicin, azacitidine, cytarabine,
`prednisone, and vincristine) combination (65,66) which
`is the only azacitidine combination brought into front(cid:173)
`line therapy in any leukemia trial, either adult or pedi(cid:173)
`atric. The early promise of this combination suggested
`in the 1976 review (12 complete remissions among 24
`patients) has been substantiated by the much greater
`patient denominator now on this trial (117 complete
`remissions among 163 patients). However, the superior(cid:173)
`ity of D-ZAPO over regimens which do not include aza(cid:173)
`citidine remains to be substantiated in a randomized
`trial.
`The MAZE (amsacrine, azacitidine, and etoposide)
`combination (67,68) has been tested in very few patients
`to date; the numbers are inadequate to substantiate
`whether the difference between the Medical Research
`Council (MRC) trial (seven complete responses among
`
`TA BLE 5.-AZA: multidrug combinations in remission induction of ANLL"
`
`Group or
`institution
`
`Prior
`treatment
`
`Regimen
`
`Dose
`(mgfm2)
`
`Schedule
`
`E valuable
`patients
`
`SWOG
`
`Rel and Ref
`
`CCSGl
`
`No
`
`BCRC
`
`Rel and Ref
`
`MRC
`
`AZA
`VCR
`Pred
`DNR
`AZA
`Ara-C
`Pred
`VCR
`
`AZA
`VP-16
`VBL
`
`AZA
`AMSA
`VP-16
`
`150
`1.5
`60
`30
`50
`25
`40
`1.5
`
`50
`50
`6
`
`100
`100
`100
`
`Pediatrie
`8-hr infusion daily X 4
`Single dose
`Daily X4
`Daily xa
`Every 12 hrs X 8
`Every 8 hrs X 12
`Daily X 4
`Daily X l
`
`Adv.U
`Every 8 hrs X 15
`Daily X5
`Days 1 and 6
`
`Daily X 6
`Daily X6
`Daily X 6
`
`56
`
`163
`
`15
`
`10
`
`No. of-
`
`CRs
`
`8
`
`117
`
`2
`
`7
`
`Daily CIV X 5
`AZA
`100
`AMSA
`Daily X 5
`60
`VP-16
`Daily X 5
`30
`• VCR = vincristine; Pred = prednisone; ara•C = C)1.arabine; VBL = vinblast ine; Rel = relapsed; and Ref = refractory.
`tOngoing.
`
`17
`
`3 ( 15 marrow
`hypoplasia)
`
`SWOG
`
`2 (1-5)
`
`Vol. 71, No. 7-8. July/August 1987
`
`PRs
`
`Ref No.
`
`7
`
`64
`
`60, 66
`
`59
`
`67
`
`68
`
`741
`
`

`

`ten patients) and the SWOG trial (three complete re(cid:173)
`sponses among 27 patients) is genuine. The difference in
`response rate may be due to selection of a lower dose of
`amsaerine and etoposide in the SWOG trial. Patient
`selection could also be a factor, since all patients in the
`MRC trial were in first relapse, whereas the median
`number of previous treatment regimens in the SWOG
`trial was two.
`
`Azacllidlne: Consolidation and Maintenance Regimens
`Table 6 lists the trials in which azacitidine has been
`incorporated into front-line consolidation and mainte(cid:173)
`nance regimens. Most of these trials are ongoing. None
`of these trials was designed to address the role of azacit(cid:173)
`idine as an individual agent. The V APA protocol (71, 72)
`used early and late intensification and sequencing of
`different chemotherapy combinations (which included
`azacitidine) after remission induction with a standard
`combination of cytarabine, daunorubicin, vincristine,
`and prednisone. The aim of the intensive sequential
`regimen was to achieve adequate reduction of residual
`leukemic cells and prevent the emergence of drug-resis(cid:173)
`tant cells. When last reported, with a median follow-up
`of 43 months (72), there was a complete remission rate of
`70% and a 3-year actuarial probability remission dura(cid:173)
`tion of 56% for patients< 18 years of age and of 45% for
`patients 18-50 years of age. The D-ZAPO protocol
`(65,66) brought azacitidine into front-line remission in(cid:173)
`duction regimens in pediatric ANLL. It also continued
`its use in both arms of a randomized maintenance pro(cid:173)
`gram, comparing a more intensive schedule with a less
`intensive schedule (daily rather than intermittent 6-
`thioguanine and cyclophosphamide added to the monthly
`pulses of cytarabine and azacitidine and earlier admin(cid:173)
`istration of intrathecal methotrexate). The more inten(cid:173)
`sive regimen showed a significantly improved duration
`of marrow remission, although survival was similar for
`both schedules. The results of the SEG study using aza(cid:173)
`citidine in two of the three arms of a randomized consol(cid:173)
`idation study in adult ANLL are so far only available in
`abstract form (69,70). In this study there was no advan(cid:173)
`tage to adding beta-deoxythioguanosine to the azaciti(cid:173)
`dine arm in terms of percentages of patients remaining
`in complete remission.
`
`DISCUSSION
`The development of azacitidine over the last 10 years
`has been disappointing in its lack of systematic progres(cid:173)
`sion. Of the four goals delineated in the earlier review
`for azacitidine in ANLL, only one has been partially
`achieved, namely, the demonstration that there is no
`major difference in response rate between the three
`schedules of administration. However, even this conclu(cid:173)
`sion is based on pooled data from trials in all forms of
`leukemia. The other three goals have not been achieved:
`(a) except for one series of pediatric trials, azacitidine
`has not been brought into first-line treatment; (b) the sc
`route of administration has not been explored; and (c)
`
`the role of azacitidine in consolidation, maintenance,
`and intensification regimens has not been defined.
`Over 400 patients have been entered in two-drug
`combination trials, hut no attempt has been made to
`demonstrate in a rigorous manner the superiority of any
`one combination over another.
`Promising preclinical leads such as the use of cytidine
`as a rescue agent (73) and enhancement of in vitro cyto(cid:173)
`static activity by thymidine and deoxycytidine (74) have
`not been pursued systematically. The radioprotective
`effect of azacitidine noted in mice (75) has not been
`investigated further except for one study of combined
`teratogenic effects on the mouse central nervous system
`(76) and one study of chromosomal aberrations in the
`broad bean (77). Azacitidine was not demonstrated to be
`protective in either of these studies. However, the origi(cid:173)
`nal study of Vesely et al (75) undoubtedly needs replica(cid:173)
`tion and extension since it has significant clinical impli(cid:173)
`cations if confirmed.
`In summary, the development of azacitidine, since its
`promising biological activity was first described > 20
`years ago (78), has not established a definitive role in
`either front-line or salvage therapy of ANLL. The con(cid:173)
`sequence is that azacitidine has not become generally
`available to physicians as a marketed drug for the
`treatment of leukemia.
`These problems in the development of azacitidine
`exemplify the difficulties inherent in the development of
`an antileukemic agent which has shown "promising"
`activity in phase II trials. As seen from tables 1 and 3,
`considerable expenditure of patients and investigators'
`resources can easily occur w ithouL conclusive result.s
`being obtained.
`We would like to propose a sequence of priorities for
`antileukemic drug development so as to achieve optimal
`use of all clinical and economic resources. (a) Phar(cid:173)
`macokinetic parameters (particularly clearance and
`elimination half-life) need to be established as early as
`technically possible in the drug's development so that
`optimal schedules for phase II and Ill trials can be de(cid:173)
`veloped. In the case of azacitidine the development of a
`specific and sensitive assay for azacitidine had special
`problems due both to its chemical instability in aqueous
`solution and susceptibility to enzyme degradation in
`serum (4). Progress towards a useful assay in biological
`fluids was made 10 years ago (79) but this has not been
`developed further.
`(b) The aim of single-agent trials of an investigational
`antileukemic drug in relapsed or refractory patients is
`to differentiate an active agent from an inactive one and
`to determine within reasonable confidence limits whether
`the response rate warrants further investigation of the
`drug in this disease. Since these trials provide little
`information about the true therapeutic efficacy of the
`drug, it is important that the number of patients be
`kept to the minimum necessary to provide a reliable
`basis for decision making. The standard approach has
`been to enter enough patients to establish a response
`rate for the agent in relapsed or refractory patients. The
`
`742
`
`Cancer Treatment Reports
`
`

`

`TABLE 6.-AZA: use in front-line intensification regimens after remission induction•
`
`Group or institution
`
`Prouicol No.
`
`Ref No.
`
`Induction regimens
`
`Intensive consolidation/maintenance/postremission(cid:173)
`ind uction regiment
`
`69, 70
`
`Adult
`Ara-C and DNR
`
`AZA (X 3)
`or
`AZA and TGdR (X 3}
`or
`6TG. ara-C, and DNR
`
`44
`
`Ara-C and DNR
`
`AZA, then DNR. then ara-C (X 2)
`
`Ara·C and DNR
`
`Ara·C and DNR
`or
`Ara-C and DNR, then AMSA, then AZA. 6TG, and DNR
`or
`AZA. 6TG, and DNR, then allogeneic BMT
`
`Ara•C and DNR
`
`Ara-C, DNR, and 6TG
`
`Ara-C, DNR, and 6TG
`
`DNR, ara·C, 6TG, and i.t. ara•C (X 4), then DNR, AZA, and i.t.
`ara-C (X 4). then ara-C. 6TG. and i.t. ara-C (X 4)
`DNR + ara-C, then VP-16 + AZA
`AMSA. AZA. and ara-C
`or
`Ara-C + AMSA, then AMSA + AZA
`Ara-C, DNR, and 6TG VP-16, AMSA, and AZA
`
`71, 72
`
`Adult and pediatrie
`VAPA twice
`DNR and ara-C (X 4), then DNR and AZA (X 4). then VCR,
`MePred, MP, and MTX (X 4), then ara-C (X 4)
`
`73
`
`D-ZAPO
`
`Pediatric
`
`74
`
`D-ZAPO
`
`6TG, AZA, ara-C. and VCR
`or
`6TG, AZA, ara-C. VCR, a.nd immunotherapy
`6TG, ara-C, and VCR
`or
`6TG. AZA, ara-C, VCR, and CPM
`
`CCG-251
`
`Ara-C and CNR
`
`1
`SEG
`
`ECOG
`SEG
`
`DFCC
`
`Baylor
`
`Hahnemann Med ical
`College
`
`EST P-B477
`
`AML 81-312f
`
`D81-018-690
`
`D81-044-077f
`
`D82-061-724t
`
`Stanford
`
`D82-051·605t
`
`Wei nstein et al
`
`ccsc
`
`CCSG
`
`CCSG
`
`CCSGt
`
`POG
`
`S!Olf
`
`DFCC
`
`T84-0245f
`
`6TG. ara-C, and i.t. MTX (or allogeneic BMT), then 6TG, VCR,
`ara-C, AZA. and CPM (repeated continuous)
`or
`DNR and ara-C. then Pred and VCR. then AZA and DNR. then
`BCNU and CPM, then 6TG and ara-C
`
`Ara•C and Asp, then 6TG, VCR, ara-C, and CPM, then VP-16,
`DNR, ara-C, AZA, and CPM. then VP-16, DNR, ara-C. DXR,
`and 6TG
`
`6TG, ara.C, and AZA, then ara-C and 6TG, then VCR. ara-C,
`CPM, and Pred
`or
`DNR and ara-C, then ara-C and 6TG. then 6TG. ara-C, and AZA,
`then VCR, ara-C, CPM, and Pred
`VP-16, AZA, and i.t. ara-C (X 3), then ara -C and ONR
`
`Ara-C and DNR
`or
`Ara-C, DNR, VP-16,
`6TG, and DXR
`
`Ara-C and DNR
`or
`Ara-C, VCR, and Dex§
`
`HD ara-C, DNR, and
`i.t. ara-C. then
`ara-C, DNR. and i.t.
`ara-C
`Ara-C, DNR, VCR.
`and Pred
`DNR. ara-C. and 6TG
`
`Hawaii Cancer Center
`
`081-0ll-693t
`
`MRC
`
`TS.1-1410
`
`DNR and ara-C. then DNR and AZA, then VCR. MePred. MP,
`and MTX. then ara-C
`DNR. ara-C, 6TG. then CPM. VCR. and ara-C,
`or
`AMSA. AZA , and VP-16. then ara-C and 6TG every 4 wks, then
`CPM, VCR, and ara-C every 4 wks
`*6TG = 6-thioguanosine; BMT = bone marrow transplant; i.t. = intrathecal; McPred = methylprednisone; MO= mercaptopurine; MTX = metho(cid:173)
`trexate; CPM = cyclophosphamide; BCNU = carmustine; DXR = doxorubicin; Asp = asparaginase; Dex = dexamethasone; and HD = high dose.
`tConsolidation has not been separated from maintenance since in many protocols different working defin itions are used , and in recent practice these
`amount to intensive postremission-induction regimens of comparable durations.
`tOngoing study or final rep-Ort not yet published.
`§ Arm was closed once lesser efficacy was established.
`
`Vol. 71, No. 7-8. July I August 1987
`
`743
`
`o 11
`
`'dbyGooglc
`
`

`

`small numbers in such trials (as exemplified for azacit(cid:173)
`idine in table 2) mean that the confidence limits for the
`estimated response rate are very broad. For example, if
`three responses are observed among 25 patients, the
`bounds of the 95% confidence interval are 3% and 30%.
`In other words, a conclusion as to whether the drug is
`active or inactive usually cannot be reached from such a
`result in a phase II trial. A more appropriate des~n to
`achieve the aim of the trial would be a Fleming two(cid:173)
`stage design (80). This design obliges the investigator
`before starting the trial to specify what response rat.e
`would be of sufficient interest to take the drug int.o
`combination phase II and possibly phase III studies as
`part of a front-line combination regimen. The design
`also allows for early termination should the observed
`response rate be below a predetermined minimum level
`of interest.
`(c) In leukemia the new agent will need to be tested as
`part of a combination as soon as its single-agent activity
`has indicated that further development of the drug is
`warranted. At this stage a randomized pilot phase III
`trial using a sequential design may be appropriate (81-
`84). The aims of such a design are to determine the level
`of activity of a combination; to minimize the number of
`patients entered if early results indicate that the test
`combination is unlikely to produce a clinically meaning(cid:173)
`ful improvement over a standard combination; and to
`avoid overinterpretation of unrandomized phase II
`studies in highly selected patients.
`(d) Establishing the specific contribution of the drug
`to front-line combination regimen is extremely difficult
`in a disease like leukemia, with multiple effective agents
`of comparable activity being used in several different
`combination regimens for induction and salvage. It is
`important to establish whether the new agent is merely
`equivalent to existing agents or constitutes a significant
`medical advance. The clearest definition of a drug's con(cid:173)
`tribution is obtained from a randomized trial in which
`the new agent is the only variable between the two
`arms. There are two possible experimental arms. In one
`arm, the new drug substitutes for a standard drug of
`similar mechanism of action and level of activity. In the
`second arm, the new agent is added to the standard
`induction regimen. The control arm in each case would
`be a standard induction regimen. This approach as(cid:173)
`sumes that there will be small differences in effective(cid:173)
`ness between arms; any design should include early
`stopping rules in the event that one arm is substantially
`superior.
`Azacitidine has been brought into the D-ZAPO front(cid:173)
`line combination with a promising remission rate (72%)
`in pediatric ANLL (65,66). However, without a random(cid:173)
`ized comparison of this regimen versus the same group
`of drugs minus azacitidine (ie, daunorubicin, cytara(cid:173)
`bine, prednisone, and vincristine) and no additional dif(cid:173)
`ferences between the two arms in terms of intensifica(cid:173)
`tion or maintenance regimens, it is impossible to say
`whether azacitidine materially adds to the complete
`remission rate or to survival.
`
`The risk of not proceeding in a sequential fashion is
`that new drugs of poorly d