BLOOD
`
`VOL. 48, NO.3
`
`TheJournal of
`The American Society ofHematology
`
`SEPTEMBER 1976
`
`5-Azacytidine (NSC 102816): A New Drug for the
`Treatment of Myeloblastic Leukemia
`
`By William R. Vogler, Donald S. Miller, and James W. Keller
`(Writing Committee for the Southeastern Cancer Study Group)
`
`5-azacytidine
`analog,
`pyrimidine
`The
`(NSC 102816), was administered by con-
`tinuous intravenous infusion in Ringer's
`lactate in increasing doses to sets of pa-
`tients with metastatic cancer to establish
`a dose sufficient to produce mild toxicity.
`Twenty-one patients
`(23 trials) were
`treated with doses of 50-200 mg/sq m/
`day for 5 days every 2-4 wk. Nausea and
`vomiting were moderate and easily pre-
`ventable. Doses of 100-200 mg/sq m for
`5 days every 14 days produced granulo-
`cytopenia, usually after two courses. Less
`toxicity was observed when courses were
`given every 21-28 days. Forty-flve pa-
`
`tients with previously treated and refrac-
`tory acute myeloblastic leukemia were
`treated. The maiority received doses of
`150 mg/sq m for 5 days every 2 wk.
`Eleven (240/0) complete remissions and
`four partial
`remissions were observed.
`The number of courses to achieve remis-
`sion averaged three and required an
`average of 59 days. Nine patients with
`blastic crisis of chronic myeloblastic leu-
`kemia and four with refractory acute
`lymphoblastic leukemia failed to respond.
`5-Azacytidine administered by continuous
`infusion is well tolerated and is an active
`compound in acute myeloblastic leukemia.
`
`T H E PYRIMIDINE ANALOG, 5-azacytidine (NSC 102816), has been
`
`shown to be an active compound in several animal and human neo-
`plasms.'
`Its clinical usefulness has been hampered by severe nausea and
`~
`vomiting and occasional diarrhea accompanying rapid intravenous injection."
`It has been thought that the drug must be given by rapid intravenous infusion
`
`From the Department of Medicine. Division of Hematology and Oncology. Emory University
`School of Medicine. A tlanta, Ga.. and the Department of Medicine. Division of Hematology and
`Oncology. Duke University. Durham. N.C.
`Submitted February 9. 1976: accepted May 4. 1976.
`This paper was prepared on behalf of the Southeastern Cancer Study Group. See the Appendix
`for a list ofparticipants in this study.
`Supported by the following USPHS Research Grants from the National Cancer Institute: CA03227
`and CAl 1263 to Emory University School of Medicine. Atlanta. Ga.; CA03177 to Duke University
`Medical Center. Durham. N.C.: CA03013 and CA 13148 to the University ofAlabama in Birmingham.
`Birmingham. Ala.: CA03376 to Washington University School of Medicine. St. Louis. Mo.: CAI2223
`to the University of Puerto Rico School of Medicine, San Juan. Puerto Rico; and CA07961 to Temple
`University School of Medicine. Philadelphia. Pa.
`reprint requests: W.R. Vogler. M.D .. 718 Woodruff Memorial Building. Emory
`Address for
`University. Atlanta. Ga. 30322.
`© 1976 by Grune & Stratton. Inc.
`
`Blood, Vol. 48, No.3 (September), 1976
`
`331
`
`CELGENE 2127
`APOTEX v. CELGENE
`IPR2023-00512
`
`

`

`332
`
`VOGLER ET Al.
`
`Israili et al." have shown, using thin-
`because of its instability." However,
`layer chromatography and nuclear magnetic resonance studies, that the T! in
`buffered solutions is 60-100 hr at 25°C. Karon et al.' have found that gastro-
`intestinal toxicity is less without reduction of antitumor activity if drug infu-
`sion is extended over 10-15 min or if the dose is divided. Moertel et al.' also
`have found that dividing the dose is associated with less nausea and vomiting.
`In an attempt
`to circumvent
`the severe gastrointestinal
`toxicity,
`the South-
`eastern Cancer Study Group initiated a study of continuous infusions of
`5-azacytidine in patients with metastatic cancer and leukemia.
`
`MATERIALS AND METHODS
`Criteria for Patient Selection
`Patients with advanced metastatic cancer who had recovered from the toxicity of any prior
`chemotherapy and who had a life expectancy of at
`least 8 wk and previously treated patients with
`acute leukemia or blastic transformation of chronic myelocytic leukemia giving informed consent,
`were eligible for study. Acute leukemia included acute lymphoblastic leukemia and acute myelo-
`blastic leukemia (myeloblastic. myelomonocytic, monocytic). Studies were conducted at six institu-
`tions in the Southeastern Cancer Study Group (Emory University, Duke University, University of
`Alabama, University of Puerto Rico. Washington University, and Temple University).
`
`Pretreatment Studies
`Pretreatment studies included history, physical examination, documentation of measurable neo-
`plastic lesions, bone marrow examination, hemogram, alkaline phosphatase, SGOT, serum pro-
`teins, BUN or creatinine clearance, plasma fibrinogen and prothrombin times.
`
`Studies During Treatment
`Blood counts were obtained at least twice weekly for the 6 wk of study and for 2 wk thereafter.
`Renal and liver function tests were repeated every 2 wk. Plasma fibrinogen and prothrombin times
`were repeated about every 4 wk. In the leukemic patients marrow examinations were done prior to
`subsequent courses of chemotherapy if blasts were absent from the peripheral blood.
`
`Drug Administration
`5-Azacytidine was administered by continuous intravenous infusion over a 5-day period. The
`drug was dissolved in 50-100 ml of Ringer's lactate and infused over a 3-12-hr period. Fresh solu-
`tion was prepared every 3-12 hr.
`
`Treatment Plan
`In patients with metastatic cancers it was planned to treat three patients with 50 mg /sq m per day
`for 5 days followed by a 9-day rest period. If hematologic toxicity occurred, therapy was delayed.
`If no toxicity supervened after 6 wk of therapy (three courses), subsequent sets of three patients
`were to be treated with increments of 50 mg/sq m until
`toxicity occurred. Once toxicity occurred,
`six patients were to be treated at that dose before further escalation.
`It was planned to observe
`all patients for a period of 6 wk at a constant dose followed by an additional 2 wk of follow-up.
`Any patients showing improvement were to be continued on treatment.
`In patients with acute leukemia, it was planned to start at 50 rng/sq m in the first set of three
`patients, but if after a 9-day rest period, there was no change or the white count had increased,
`the dose was to be escalated by 50 rng/sq m increments in the second and subsequent courses until
`a hematologic effect was noted. If no effect on the white count occurred at the 50 rng/sq m starting
`dose, the initial dose was to be escalated in subsequent sets of three patients by 50 mg/sq m incre-
`ments.
`
`

`

`5-AZACYTIDINE
`
`333
`
`Evaluation of Toxicity
`Hematologic, hepatic, and renal toxicity were graded as previously reported.f Gastrointestinal
`toxicity was graded as follows: (I) nausea without vomiting while on therapy (mild); (2) nausea
`and vomiting while on therapy controlled by antiemetics (moderate); (3) nausea and vomiting not
`controlled with antiemetics (severe),
`
`Criteria of Response
`In metastatic cancer patients' objective responses were rated as previously reported.f requiring
`at least a 20°/~ reduction in measurable tumors at 12 wk.
`In patients with acute leukemia responses were judged according to the criteria as previously re-
`ported. 12 Those demonstrating a clearing of leukemic cells from the blood, changes in marrow
`status from an M-3 to 2 or less and a reduction in hepatomegaly and splenomegaly when present,
`but not achieving remission status, were considered to have had an antileukemic effect.
`least one
`Patients with metastatic cancer were judged evaluable for toxicity if they received at
`5-day course of treatment and had adequate follow-up information to determine the effect of 5-
`azacytidine on hemogram, liver. and renal function. Patients were judged evaluable for a thera-
`peutic response if they received at least three courses of therapy.
`In leukemia patients all studies were judged evaluable if they received one 5-day course of 5-
`azacytidine according to protocol.
`
`RESULTS
`
`Metastatic Cancer
`Table I summarizes the toxicity observed in 23 trials in 21 patients entered on
`study with metastatic cancers.
`instances
`In most
`The major hematologic toxicity was granulocytopenia.
`patients received two courses of therapy before toxic levels were reached. The
`mean nadir day was day 32 (range 22-45) with recovery (above 1500/cu mm)
`by day 39 (range 25-56). All six patients given infusions of 150 mg/sq m every
`14 days had granulocytopenia. When the infusions were given every 21 days or
`later, two of four experienced granulocytopenia.
`No patient had hepatic or renal toxicity which was thought to be drug re-
`lated. No changes in fibrinogen or prothrombin time were noted although only
`a few tests were done serially.
`Nausea and/or vomiting occurred in 20 patients, and was considered grade 2
`in 6. Symptoms were worse the first day or two of treatment. Nausea was
`usually well controlled with antiemetics. Only one patient refused subsequent
`courses because of nausea and vomiting. An occasional patient experienced
`mild diarrhea.
`
`Dose
`mg/sq m/day
`
`Frequency
`of Course
`(days)
`
`14
`14
`21
`14
`21-28
`
`50
`100
`100
`150
`150
`175
`200
`
`Table 1. Metastatic Cancer Toxicity
`
`Granulocytes
`
`Hematologic
`
`Platelets
`
`< 1500
`
`<750
`
`< 100,000
`
`<50,000
`
`Hemoglobin
`>3 Drop
`
`1
`0
`0
`1
`1
`0
`0
`
`0
`3
`0
`5
`1
`0
`1
`
`0
`0
`0
`0
`1
`0
`1
`
`0
`0
`0
`0
`0
`0
`0
`
`0
`2
`0
`0
`1
`0
`0
`
`Gastrointestinal
`
`2
`
`0
`1
`0
`3
`1
`1
`0
`
`2
`3
`1
`5
`3
`0
`1
`
`>2
`
`0
`0
`0
`0
`0
`0
`0
`
`No. of
`Patients
`
`..
`
`4
`1
`8
`4
`1
`1
`
`

`

`334
`
`VOGLER ET AL.
`
`leukemia *
`
`AML
`
`Blast phase of CML
`
`ALL
`
`Table 2. 5-Azacytidine in Leukemia (Evaluable Patients)
`
`Dose
`(mgjsq mjday)
`
`No. of
`Pati~nts
`
`Dose
`Escalated
`
`50
`100
`150
`200
`Total
`
`100
`150
`200
`Total
`
`150
`200
`Total
`
`1
`7
`32
`5
`45
`
`2
`4
`3
`9
`
`3
`1
`4
`
`1
`5
`7
`0
`13
`
`1
`0
`0
`1
`
`1
`0
`1
`
`GI Toxicity
`
`Responset
`
`2
`
`0
`2
`8
`2
`12
`
`0
`0
`0
`0
`
`0
`0
`0
`
`>2
`
`0
`0
`0
`0
`0
`
`0
`0
`0
`0
`
`0
`0
`0
`
`CR
`
`0
`3
`7
`1
`11
`
`0
`0
`0
`0
`
`0
`0
`0
`
`1
`3
`16
`3
`23
`
`1
`0
`3
`4
`
`1
`0
`1
`
`PR
`
`ALE
`
`0
`0
`4
`0
`4
`
`0
`0
`0
`0
`
`0
`0
`0
`
`1
`3
`7
`0
`11
`
`1
`3
`3
`7
`
`0
`0
`0
`
`*AML, acute myeloblastic leukemia; CML, chronic myeloblastic leukemia; ALL, acute lymphoblastic
`leukemia.
`t CR, complete remission; PR, partial remission; ALE,antileukemic effed.
`
`No significant therapeutic benefit was observed in this group of 21 patients,
`which included 6 with lung carcinoma, 5 with melanoma, 3 with breast car-
`cinoma, 2 with hypernephroma, 2 with leiomyosarcoma, 1 with mesothelioma,
`1 with colon carcinoma, and 1 with fallopian tube carcinoma. One patient with
`melanoma had stabilization of disease but with less than 20% regression in the
`size of measurable lesions.
`
`Results in Leukemia
`A cute myeloblastic leukemia.
`Forty-nine patients with acute myeloblastic
`leukemia were entered on study and 45 were evaluable. Of the four inevaluable
`patients, two were ineligible for the study, in one the protocol was not followed
`and one was lost to follow-up. A summary of these cases is given in Table 2.
`The initial dose of 50 mgjsq m was administered to only one patient and no
`hematologic effect was noted. The dose was escalated in subsequent courses. A
`reduction in leukocyte count was noted, but no remission occurred despite 13
`courses over a year's time.
`Seven patients began at 100 mgjsq m; in five the dose was escalated to 150
`mgjsq m. One of these five and two others obtained a complete remission and
`three had an antileukemic effect. Five experienced mild nausea and vomiting.
`Thirty-two patients started at 150 mgjsq m. In seven, the dose was escalated
`in subsequent courses and in one, it was reduced. The maximum doses given
`were 300 mgjsq mjday. There were seven complete remissions, four partial re-
`missions, and seven patients who had an antileukemic effect. Mild nausea and
`vomiting occurred in 24 patients.
`Granulocytopenia occurred in ten patients and was usually prolonged. One
`patient developed pleuritic chest pain with the last three of her six courses of
`chemotherapy, and this was thought to be drug related. Hyperglycemia oc-
`curred in two patients, and an inappropriate ADH syndrome was observed in
`one patient.
`
`

`

`5-AZACYTIDINE
`
`335
`
`Five patients were started at 200 mgjsq m, and the doses were not escalated.
`One obtained a complete remission. Two experienced moderate nausea and
`vomiting.
`Courses of 5-azacytidine were repeated every 2 wk except in the event of pro-
`longed cytopenia, when treatment was delayed until evidence of recovery oc-
`curred. Bone marrows were monitored at frequent
`intervals and therapy was
`reinstituted whenever
`the percentage of blasts increased even though the
`peripheral blood count remained low. Many patients could tolerate two courses
`with a 9-day interval, but subsequent courses were delayed.
`Thus, of 45 evaluable patients with acute myeloblastic leukemia, complete
`remissions occurred in II (24°~) and partial remissions in four patients. An
`antileukemic effect was observed in II patients. Reduction of white count was
`achieved in almost all patients. Dose escalation in two of the patients with
`partial remissions failed to induce a complete remission. No gastrointestinal
`toxicity greater than two was observed.
`Table 3 summarizes our experience in those II patients who achieved com-
`plete remissions. The number of courses required to achieve remission varied
`from one to seven with an average of three and seemed to be unrelated to dose.
`The beginning of repeated courses varied from 13 to 29 days with an average of
`15 days. Four patients achieved remission without marrow hypoplasia, and the
`remainder demonstrated prolonged marrow hypoplasia after
`two or more
`courses. The time to achieve remission varied from 27 to 92 days with an aver-
`age of 59 days.
`Patients received varying maintenance programs, and assessment of remis-
`sion duration is difficult. The duration of remission ranged from 26 to 600 days
`with a median of 88 days.
`Blast crisis of chronic myelocytic leukemia.
`There were nine patients with
`blastic transformation treated with 5-azacytidine in doses ranging from 100 to
`200 mgjsq m (Table 2). Seven patients had an antileukemic effect after one to
`three courses, but no remissions were obtained. Three patients had prolonged
`granulocytopenia.
`A cute lymphoblastic leukemia.
`
`Four patients with acute lymphoblastic
`
`Table 3. Acute Myeloblastic Leukemia-Complete Remissions
`
`Starting
`Dose
`(mg/sq m/day)
`
`Pt. No.
`
`No. of
`Courses
`
`100
`
`150
`
`200
`
`1
`2
`3
`
`4
`5
`6
`7
`8
`9
`10
`
`11
`
`2
`2
`7
`
`4
`2
`3
`1
`2
`3
`2
`
`2
`
`Interval
`(days)
`
`15
`20
`14
`
`19,29,19
`13
`14
`
`14
`14
`12
`
`16
`
`Aplasia
`(days)
`
`Days to
`Remission
`
`21
`31
`0
`
`0
`0
`21
`0
`29
`22
`41
`
`26
`
`54
`59
`92
`
`67
`27
`61
`38
`64
`75
`50
`
`58
`
`

`

`336
`
`VOGLER ET Al.
`
`leukemia were treated with 150-200 mg/sq m doses, and no responses were ob-
`served (Table 2).
`
`DISCUSSION
`
`Administration of 5-azacytidine by continuous infusion has greatly increased
`patient tolerance. In a phase I study of twice weekly injections given rapidly,
`100% of patients who received a dose of 100 rng/sq m or larger vomited." In
`contrast only about 25°/~ of patients given infusions experienced vomiting.
`Clinically significant hematologic toxicity was primarily confined to granulo-
`cytes. Bone marrow samples taken during the period of neutropenia were
`megaloblastic. Erythropoiesis was impaired, and some patients developed
`anemia. Platelet toxicity was infrequent. The fact that granulocyte toxicity was
`common, and platelet toxicity uncommon would seem to make this drug ideal
`for treatment of myeloblastic leukemia.
`It would appear that the frequency of administration (every 14 days) resulted
`in cumulative granulocyte toxicity, and less toxicity was seen when the fre-
`quency of courses was reduced to every 21 or more days. However, the every 14
`day schedule appeared to be effective in treating leukemia.
`From these studies the recommended starting dose for treating acute leu-
`kemia is 150 mg/sq ru/day.
`The full spectrum of toxicity of 5-azacytidine has not been fully delineated.
`Although we saw no hepatic toxicity which we could establish as due to the
`drug, Bellet et al." described hepatic coma developing in patients given sub-
`cutaneous injections. All had hepatic metastases at the time. Patients should be
`watched for central nervous system toxicity. One of our patients who had been
`treated for central nervous system leukemia developed an inappropriate ADH
`syndrome.
`Although no significant responses were observed in patients with metastatic
`cancer, the number of patients in each disease category was small, and thus the
`data were insufficient to draw conclusions about its efficacy.
`Other studies have been reported concerning the use of 5-azacytidine in treat-
`ing myeloblastic leukemia. McCredie et al." administered the drug by rapid
`intravenous injection (15-30 min) daily in repeated 5-day courses in doses up to
`400 mg/sq m and observed three complete remissions and four partial
`re-
`sponses in 18 patients. All had been previously treated. Because of the severe
`toxic effects (myelosuppression, nausea, vomiting, diarrhea,
`fever, and occa-
`sional hypotension) they suggested that 5-azacytidine might be better tolerated
`if used at a smaller dose, possibly in combination with another agent. Levi and
`Wiernik 13 gave 5-day courses of 200 rng/sq m/day rapidly by intravenous in-
`jection and obtained five complete remissions among 18 patients. In another
`study Levi and Wiernik 14 gave doses of 100 mg/sq m by rapid intravenous in-
`jection in three divided doses combined with methyl-GAG[methyl glyoxal-bis
`(guanyl-hydrazone) (NSC-21946)] to eight patients with refractory nonlympho-
`cytic leukemia and observed two partial remissions. They experienced similar
`toxicity and concluded that the dosage was too low.
`These studies indicate that 5-azacytidine given by continuous infusion is an
`active agent in acute myeloblastic leukemia. The high degree of success in in-
`ducing remissions in patients with refractory myeloblastic leukemia requires
`
`

`

`5-AZACYTIDINE
`
`337
`
`that further studies be done. The use of 5-azacytidine in combination with
`other agents seems warranted.
`
`ACKNOWLEDGMENT
`5-Azacytidine was supplied by the Division of Cancer Treatment, National Cancer Institute,
`Bethesda, Md.
`
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`9. Vogler WR, Arkun S, Velez-Garcia E:
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`5-Azacytidine, a new, highly effective cancer-
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`3. Li LH, Olin EJ, Buskirk HH, Reineke
`10. Pith ova P, Piskala A, Pitha J, Sorrn F:
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`Azacytidine on L 1210 leukemia. Cancer Res
`LXVI. Hydrolysis of 5-azacytidine and its con-
`30:2760-2769, 1974
`nection with
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`4. Weiss AJ, Stambaugh JE, Mastrangelo
`Chern Commun 30:2801--2811, 1965
`MJ, Laucius JF, Bellet RE: Phase I study of
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`Israili ZH, Vogler WR, Mingioli ES,
`5-azacytidine (NSC-I02816). Cancer Chemo-
`Pinkie
`JL, Smithwick RW, Goldstein
`JH:
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`Studies of the disposition of 5-azacytidine _14C
`5. Moertel CG, Schutt AJ, Reitemeier RJ,
`in man. Pharmacologist 16:231, 1974
`Hahn RG: Phase II
`study of 5-azacytidine
`12. Vogler WR, Huguley CM, Rundles RW:
`(NSC-I 02816) in the treatment of advanced gas-
`Comparison of methotrexate with 6-mercapto-
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`56:649-652, 1972
`purine-prednisone in treatment of acute leu-
`kemia in adults. Cancer 20: 1221-1226, 1967
`6. McCredie KB, Bodey GP, Burgess MA,
`13. Levi JA, Wiernik PH: A comparative
`Gutterman JU, Rodriguez V, Sullivan MP,
`clinical trial of 5-azacytidine and guanazole in
`Freireich EJ: Treatment of acute leukemia with
`adults with
`acute
`non-
`previously treated
`5-azacytidine (NSC-I02816). Cancer Chemo-
`lymphocytic leukemia. Cancer, 1976, in press
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`JA, Wiernik
`PH: Combination
`14. Levi
`7. Karon M, Sieger L, Leimbrock S, Finkle-
`therapy with 5-azacytidine (NSC-I02816) and
`stein JZ, Nesbit ME, Swaney JJ: 5-Azacytidine:
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`A new active agent for
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`8. Bellet RD, Mastrangelo MJ, Engstrom
`
`APPENDIX
`The following members of the Southeastern Cancer Study Group paruci-
`pated in this study: John R. Durant" M.D." George Omura" M.D." Richard
`Garns, M.D." John Carpenter" M.D." Marcel Conrad" M.D." University of Ala-
`bama in Birmingham" Birmingham" Ala.: Harold Silberman" M.D." and
`Donald Miller" M.D." Duke University Medical Center" Durham" N.C.:
`Lawrence E. Cooper" M.D." Charles C. Corley, Jr., M.D., L. Thomas Heffner,
`M.D., Julian Jacobs, M.D., James W. Keller, M.D." Melvin Moore, M.D.,
`W. R. Vogler, M.D., E. F. Winton, M.D., Emory University School of Medi-
`cine, Atlanta, Ga.; Antonio Grillo" M.D., and Enrique Velez-Garcia, M.D.,
`University of Puerto Rico School of Medicine, San Juan, Puerto Rico; Rich-
`ard V. Smalley" M.D." Temple University School of Medicine" Philadelphia,
`Pa.: Cary Presant, M.D., and Edward H. Reinhard, M.D., Washington Uni-
`versity School of Medicine, S1. Louis, Mo.
`
`