Epigenetic Drugs: A Longstanding Story

Epigenetic Drugs: A Longstanding Story Dick de Vos In this chapter, the development of decitabine from its synthesis in 1964 to the submission of a registration file in 2004 is reviewed. The proper application of the unique properties of decitabine took quite some time to elucidate. In addition, the practical handling in the clinic was not easy as the prolonged myelosuppression of decitabine made it difficult to determine the preferred dose and schedule. Laboratory studies on DNA methylation and cell differentiation showed possible applications in solid and hematologic malignancies. However, despite many attempts, results in solid tumors have been disappointing thus far. After thorough investigation, decitabine achieved therapeutic application in myelodysplastic syndrome (MDS), in particular in patients with a poor prognosis. Further indications may include acute myeloid leukemia (AML), chronic myelogenous leukemia (CML), hematopoietic stem cell transplantation, sickle cell anemia, and thalassemia. Whereas most drugs are already at the end of their life cycle after 40 years, decitabine is only at the beginning. Its application will broaden with the increase in knowledge of epigenetic mechanisms and their relationship to drug therapy. Semin Oncol 32:437-442 © 2005 Elsevier Inc. All rights reserved.

T

he story of decitabine coincides with that of new drug development by the European Organization for Research and Treatment of cancer (EORTC). The rise in interest in new anticancer drug investigations in The Netherlands and in Europe is also related to the activities of Dr H.M. Pinedo, professor of clinical oncology at the Free University of Amsterdam. Although resident in his department only since 1979, he was enthusiastically promoting research and development of novel anticancer drugs. Within the framework of a coordinated search for new drugs in the treatment of cancer, Pinedo contacted Pharmachemie BV in 1982 regarding decitabine. Pharmachemie BV was at that time a medium-size company in Haarlem, The Netherlands, transitioning from plain generics to more sophisticated products in oncology. Pharmachemie’s president at that time, Arnold de Vita, a true entrepreneur, opened several doors. The question was whether Pharmachemie BV would participate in the development of decitabine. Did the agent have sufficient antitumor activity? And, did decitabine possess unique properties justifying its development as a drug? A literature search revealed several very interesting aspects of decitabine. First, it proved to be active in several in vitro and in vivo laboratory models. Among other investigators, one appeared to be deeply involved in decitabine research:

Pharmachemie BV, Haarlem, The Netherlands. Address correspondence to Dick de Vos, PhD, RPharmacol, Pharmachemie BV, PO Box 552, 2003 RN Haarlem, The Netherlands. E-mail: [email protected]

0093-7754/05/$-see front matter © 2005 Elsevier Inc. All rights reserved. doi:10.1053/j.seminoncol.2005.07.025

Professor Dr R.L. Momparler of the Ste-Justine Hospital, University of Montreal, Canada. Use was made of his insight into the pharmacology of decitabine.1 Enough documentation of the antitumor activity of decitabine was available, but were there possible unique properties? The literature search identified the work of Professor Dr P.A. Jones of the Department of Biochemistry, University of Southern California, Los Angeles, carried out in cooperation with Dr S.M. Taylor of the Pediatrics Department. Phenotypic conversion was induced by 5-azacytidine and decitabine in mouse embryo cells. The effective concentration of decitabine was one tenth that of 5-azacytidine.2 Experimental evidence was presented showing a relationship between cellular differentiation and DNA methylation.3 Inhibition of DNA methylation by decitabine resulted in reactivation of an inactive X-chromosome locus.4 The differentiation of myeloid leukemia cells seemed a novel subject deserving full attention.5 A phase I study of decitabine in children by Rivard and Momparler6 found a potent antileukemic effect in children with acute leukemia. In addition, differentiation induction therapy was a treatment option, later classified as epigenetic therapy, with promising future prospects.7 The decision was made to develop decitabine on the basis of their interesting evidence suggesting that this was a unique therapy.

Background Decitabine, 5-aza-2=-deoxycytidine or 4-amino-1-(2-deoxy␤-D-erythro-pentofuranosyl)-1,3,5-triazin-2(1H)-one (Fig 1) 437

D. de Vos

438

Figure 1 Chemical structure of decitabine.

is an analog of 2=-deoxycytidine. The only difference is that the 5-carbon is replaced by nitrogen. The crystal structure documents the ␤-D-configuration.8 Decitabine was first synthesized by Pliml and Sorm.9 Its antileukemic properties were reported by Sorm and Vesely.10 For activation to occur, decitabine has to be phosphorylated by the enzyme deoxycytidine kinase to 5-aza-dCMP. This is rapidly converted to 5-aza-dCTP by kinases. 5-Aza-dCTP is a good substrate for DNA polymerase alpha.10,11 Deamination of decitabine or 5-aza-dCMP resulted in a complete loss of activity.12,13 The incorporation of decitabine into DNA produces inhibition of the methylation at position 5. Decitabine may exert a cytotoxic action leading to cell kill, but may also affect cell differentiation by demethylation. In general, inactive genes that are methylated become active on demethylation. This dual activity may be expected for decitabine.

imal nausea and vomiting, and transient fatigue. One partial response in a patient with an undifferentiated carcinoma of the ethmoid sinus was seen. The schedule of this initial phase I study is compared with other schedules in Table 1. The ECTG and the Gynecology Group of the EORTC performed several phase II trials in a range of tumors (see Table 2 for a listing of malignancies). Decitabine appeared not to be active in vivo in solid tumors with the schedules studied. Another phase I study was performed by Professor Dr D.J.T. Wagener of the St Radboud Hospital, Nijmegen, The Netherlands. The recommended schedule was 20 mg/m2/d as a 4-hour infusion given on days 1 to 5 every 4 weeks. This schedule was well tolerated and showed mainly leukocytopenia as the doselimiting toxicity.21 Professor J.L. Abbruzzese of the Department of Gastrointestinal Medicine of the M.D. Anderson Cancer Center would later study a variation of this phase I schedule.22 The decitabine pioneer Momparler had already performed some studies in acute leukemia with infusion schedules of various duration and dose.6,23 In some patients, he also observed inhibition of DNA methylation,23,24 but not cell differentiation. The decisive turn in decitabine research came when contact was established with the Leukemia Unit of the Centro di Riferimento Oncologico in Aviano, Italy (Head: Dr A. Colombatti; Director: Dr S. Monfardini). Dr A. Pinto, oncologist, hematologist, and inspired researcher, had a keen Table 1 Some of the Decitabine Schedules Studied Schedule

1982–1989 After the decision of Pharmachemie BV to pursue decitabine research, the drug had to be synthesized in appreciable amounts and formulated for clinical use. For the synthesis of decitabine several methods were available.14,15 While Pharmachemie BV took care of the acquisition of decitabine for pharmaceutical production, formulation studies were set up with laboratory amounts of decitabine. A formulation was designed by Dr S.J. Meulenbelt-Snijders, and the first batch for a phase I study was subsequently produced by Dr T. J. Schoemaker at the Slotervaart Hospital in Amsterdam.16 Later batches were manufactured by Pharmachemie BV, as the company had completed a process to produce decitabine according to international regulations in US Food and Drug Administration (FDA)-approved facilities. Initial development of decitabine was performed in close cooperation with the New Drug Development Office (NDDO) of the EORTC, incepted officially in 1984. Within the framework of the Early Clinical Trials Group (ECTG) of the EORTC a phase I trial was done in the Department of Medical Oncology of the Academic Hospital of the Free University of Amsterdam by Van Groeningen et al.17 The recommended dose for phase II trials was 75 mg/m2 given as three 1-hour infusions at intervals of 7 hours, every 5 weeks. The dose-limiting toxicity was myelosuppression, with a delayed white blood cell nadir occurring at day 22. Other toxicities included mild, reversible elevation of serum creatinine, min-

References

75 mg/m2 as 3 1-h infusions with 7-h interval, q 5 wks 20 mg/m2/d 4-h infusion d 1–5, q 4wks 0.75–30 mg/kg 12- to 30-h infusion 36–80 mg/kg 36- to 44-h infusion 37–81 mg/kg 36- to 60-h infusion 15–90 mg/m2 4-h infusion 3 times daily for 3 d 300 or 500 mg/m2/p d 24-h infusion for 2–5 d 40, 50, 65, 75 mg/m2/24 h for 3 days 90 mg/m2 4-h infusion d 1–5 ⴙ daunorubicin 50 mg/m2 bolus infusion d 1–3 125 mg/m2 6-h infusion q 12h d 1–6 with or amsacrine 120 mg/m2 1-h infusion d 6 and 7 or idarubicin 12 mg/m2 15-min infusion d 5–7 100 mg/m2 6-h infusion q 12-h d 1–5 100 mg/m2 4-h infusion q 12-h d 1–2 200–600 mg/m2 8-h infusion q 5–6 wks 45, 67, 90, 120 mg/m2 2-h infusion d 1–3, cisplatin 33 mg/m2 after decitabine 20–120 mg/m2/12 h as 12-h infusion twice daily for 3 d 0.15 mg/kg/p d 1–5 for 2 wks, then 2 wks off therapy and 2 wks therapy again Abbreviations: h, hour; wks, weeks; d, day; q, every.

17 21 6 6 23 21 21 33

33

33 33 33 34–36 37

40

51

Epigenetic drugs: a longstanding story

439

Table 2 Circumstances in Which Decitabine Was Studied Malignancy

References

Head and neck carcinoma Colorectal carcinoma Non-small cell lung cancer Malignant melanoma Testicular carcinoma Renal adenocarcinoma Ovarian carcinoma Cervix carcinoma Osteosarcoma Wilms tumor Prostate ALL AML CML MDS Allogenic progenitor cell transplantation Sickle cell anemia

18,21 18,21 18,21,34–37 18,21 21 18,21 19,21 20,39 6 6 38 6,21,23,24 6,21,23,24 21,46,47 21,33,41,42 33,49,50 51

Abbreviations: ALL, acute lymphocytic leukemia; AML, acute myeloid liukemia; CML, chronic myelogenous lukemia; MDS, myelodysplastic syndrome.

insight into the scientific and clinical possibilities of decitabine and had already published some of his views.25-28 The first meeting in Aviano with Pinto and his enthusiastic colleagues, in particular Drs V. Zagonel and V. Attadia, on October 22, 1987 appeared to be a milestone in decitabine development. Future application of decitabine in myelodysplastic syndrome (MDS), chronic myelogenous leukemia (CML), and acute myeloid leukemia (AML), as wells as sickle cell anemia and thalassemia, was envisaged, while epigenetic aspects were clearly identified by Pinto. Pinto initiated a range of studies. Decitabine stimulated dose-dependent irreversible hemoglobinization and morphologic differentiation in the human erythroleukemic cell line K562. 5-Azacytidine appeared to be highly toxic and had a lower induction capability than decitabine. Also in vivo the leukemic cell phenotype was modified by decitabine, as demonstrated by changes in membrane differentiation antigens, growth factor receptors, and other cell surface structures, while c-myc oncogene transcription was also altered. The clinical studies of Pinto et al made use of a schedule of administration with a dose of 15 to 30 mg/m2 in MDS patients and 30, 60, or 90 mg/m2 in AML and CML blastic-phase (bp) patients. The dose was given as a 4-hour infusion three times daily for 3 consecutive days, every 28 to 35 days. Responses were seen in all disease entities. A patient with CML-bp showed a complete remission (CR) with marked thrombocytosis suggestive of the induction of a chronic phase. The CML patient remained in CR for more than 2 years at the time of the last study report. Hematologic toxicity was pronounced as expected. Notably lung toxicity, in particular acute respiratory distress syndrome (ARDS) occurred, probably as a result of rapid cytolysis of leukemic lung infiltrates. Decitabine appeared to induce a clinical type II herpes simplex virus (HSV) reactivation in several patients.21 The Hematology Group of the EORTC also became inter-

ested in decitabine and Dr L. Debusscher of the Institute Jules Bordet, Brussels, Belgium performed a phase I–II trial in adult patients with leukemia. The daily dose was 300 or 500 mg/m2 administered as a 24-hour infusion for 2 to 5 days. In 23 of 26 evaluable courses, a (short) antileukemic effect was seen, in five patients a CR, and in a patient who was probably not resistant to cytosine arabinoside (Ara-C), a CR. Toxicity was acceptable and, apart from the expected hematologic toxicity, consisted of liver function tests abnormalities.21 Professors Dr D.J. Richel and Dr R. Willemze of the Leiden University Medical Center, The Netherlands went on with his study of decitabine in acute leukemia in the laboratory and the clinic. Different high-dose schedules were studied, resulting in the recommendation of decitabine 125 mg/m2 as a 6-hour infusion for 6 days, in combination with amsacrine 120 mg/m2 on days 6 and 7. Patients with AML refractory to Ara-C did not respond to decitabine. Of seven patients in first relapse after Ara-C therapy, six achieved a CR and one a partial remission (PR). At the high dose of 250 mg/m2, two patients developed neurologic problems.21,29-31 Many investigators in the field of decitabine attended a workshop on 5-aza-2=-deoxycytidine on March 11, 1989 in Amsterdam after the Sixth National Cancer Institute (NCI)/EORTC symposium on New Drugs in Cancer Therapy in Amsterdam, The Netherlands from March 7–20, 1989. The results of this meeting on preclinical and clinical aspects of decitabine were published as a monograph on decitabine21 concluding the first developmental phase of the drug. One of the invited speakers, Dr J. Vesely of the Institute of Organic Chemistry and Biochemistry, Prague, Czechoslovakia was unable to attend due to political circumstances. Later, he presented his lecture in the Pharmachemie BV office. This historical context positions the development of decitabine in a rapidly changing world.

1989 –1993 An impression of the progress in decitabine development in this period is obtained from a second workshop where decitabine investigators discussed their results. This workshop on 5-Aza-2=-Deoxycytidine and DNA Methyltransferase Inhibitors in the Treatment of Myeloid Leukemias and MDS: Biological Aspects and Clinical Results was held on November 3, 1991 at the Fifth International Symposium on Therapy of Acute Leukemias in Rome, Italy. Pinto was the chairman of this meeting. After the meeting a monograph was published.32 Preclinical as well as clinical subjects were reported. Taylor and Attadia studied the relationship between DNA methylation and differentiation; decitabine was able to promote differentiation and to activate maturation of cells through hypomethylation. Genes involved in this process are being identified. Momparler presented in vitro results on the combination of decitabine and amsacrine, while Willemze discussed preliminary results on a study comparing decitabine ⫹ amsacrine versus decitabine ⫹ idarubicin. Decitabine was administered at 125 mg/m2 as a 6-hour infusion every 12 hours for 6 days. Using his phase I–II schedule, Pinto initiated a study in MDS and in poor-prognosis AML. The latter

D. de Vos

440 study was carried out in cooperation with Professor F. Mandelli of the University “La Sapienza,” Rome, Italy. Decitabine induced a trilineage response in unfavorable MDS and demonstrated a prominent role in the treatment of AML patients in poor physical condition and/or in advanced age.32

1993–1997 An important event occurred in this period: the involvement of professor H.M. Kantarjian of the M.D. Anderson Cancer Center, Houston, TX in the development of decitabine. Novel ideas, a powerful institute, and regulatory insights gave a new impetus to decitabine research and development. Kantarjian not only designed a range of new studies but also stimulated other investigators to embark on fresh laboratory and clinical investigations. He and Pinto chaired the Workshop on Clinical Results With Decitabine (5-Aza-2=-Deoxyxytidine) in Hematologic Malignancies on May 30, 1996 held in Paris, France after the Second Meeting of the European Haematology Association. Again, a monograph was published after the workshop.33 This third workshop was the last organized by Pharmachemie BV. Other laboratory researchers joined the group of decitabine investigators. Dr J.-P.J. Issa of The John Hopkins Oncology Center, Baltimore, MD, and Dr M. Lübbert, University of Freiburg Medical Center, Freiburg, Germany, presented results on the methylation of DNA and the reactivation of tumor-suppressor gene expression.33 New clinical researchers also entered the field: Dr P W. Wijermans of the Department of Haematology of the Leyenburg Hospital, The Hague, The Netherlands and Dr G. Schwartsmann of The South-American Office for New Drug Development, Hopital de Clinicas de Porto Allegre, Porto Allegre, Brazil.33 Wijermans reported results for 29 patients with progressive MDS. Decitabine was given at a dose of 40, 50, 65, or 75 mg/m2/24 h for 3 days. In 15 patients (54%) a response was observed: eight CRs were obtained. The only major toxicity was myelosuppression, leading to five toxic deaths (17%) in this high-risk group. Decitabine was judged to be an active drug in MDS. The results of the Italian decitabine studies on AML and MDS were presented in Paris by Pinto, but unfortunately not published. Schwartsmann combined decitabine with daunorubicin as first-line therapy in AML. Decitabine was given at a dose of 90 mg/m2 as a 4-hour infusion on days 1 to 5, and daunorubicin at 50 mg/m2 was administered as a bolus infusion days 1 to 3. All six patients achieved a CR. Willemze presented his results on the comparison of decitabine and amsacrine versus decitabine and idarubicin in relapsed AML. Decitabine proved to be a good antileukemic agent with considerable toxicity. Patients with normal cytogenetics had a higher CR rate (51.4%) than those with abnormal cytogenetic findings (15.8%).33 Kantarjian offered the results of the first of several new studies. Decitabine was given as part of a conditioning regimen in allogenic progenitor cell transplantation. The drug was given to three patients at a dose of 100 mg/m2 as a 6-hour infusion every 12 hours for 5 days. It was also combined with busufan and cyclophosphamide in four patients. In the latter

case, decitabine was given at a dose of 100 mg/m2 as a 4-hour infusion every 12 hours on days ⫺8 and ⫺7. Busulfan was given at a dose of 1 mg/kg orally every 6 hours on days ⫺6 to ⫺4. On days ⫺3 and ⫺2, 100 mg/kg of cyclophosphamide was administered with mesna. All patients treated with single-agent decitabine achieved a CR. Two patients had delayed engraftment, the cause of which is not clear and merits further investigation. Two more patients who received the combination treatment achieved CR. These encouraging results warrant further study.33

1997–Present Interest in the treatment of solid tumors with decitabine remained. Momparler studied non-small cell lung cancer (NSCLC) with a dose of 200 to 600 mg/m2 as an 8-hour infusion every 5 to 6 weeks, for one or more cycles, in a phase I–II setting. Fifteen patients entered the study and three of them survived more than 15 months. One patient survived up to 81 months demonstrating that decitabine may have some clinical activity in NSCLC.34-36 Schwartsmann saw only three minor responses with decitabine combined with cisplatin.37 Decitabine had only modest activity in hormone-independent prostate cancer applying the schedule of Van Groeningen.17,38 Decitabine combined with cisplatin was also moderately active in cervical cancer.39 Dr J.S. Weber of Keck/ University of Southern California School of Medicine, Los Angeles, studied the continuous infusion of decitabine in a phase I setting, and investigated its effect on the methylation pattern in tumor biopsies before and after therapy. The schedule employed was 20 to 120 mg/m2 a 12-hour infusion administered twice daily for 3 days. Changes in methylation were observed, but no single gene consistently demonstrated evidence of demethylation.40 Using the low-dose schedule of Pinto,21 Wijermans and Lübbert performed a phase II trial in 66 high-risk MDS patients. The overall response rate was 49% with a 64% response rate in patients with an International Prognostic Scoring System (IPSS) high-risk score. Median progression-free survival was 25 weeks and treatment-related mortality was 7%. Low-dose decitabine was particularly active in patients with the worst prognoses.41 These results were confirmed by the combined results of several studies by the same authors.42 Cytogenetic responses to decitabine were also examined; cytogenetic remission was induced in a substantial number of elderly MDS patients with pre-existing chromosomal abnormalities. A cytogenetic response was associated with improved survival compared with patients with no cytogenetic response and a persistently cytogenetically abnormal clone. The promising low-dose decitabine therapy in MDS fits well into the framework of epigenetic therapy.43 Platelet and nonclonal neutrophil responses to decitabine were also studied in detail.44,45 The energetic involvement of Kantarjian led to a number of publications, of which only some will be mentioned. Results were published on decitabine therapy in 20 CML-bp and 17 accelerated-phase CML patients.46 In the CML-bp patients, the response rate was 25%, and in the accelerated-phase pa-

Epigenetic drugs: a longstanding story tients, 53%. Prolonged myelosuppresion was the most significant side effect. Studies were extended and results in 130 patients were reported.47 The response rate in CML-bp was 28%, in the accelerated phase 55%, and in the chronic phase, 63%. The treatment-related death rate was only 3%. It was again confirmed that prolonged myelosuppresion was the most important toxicity. The conclusion was that decitabine had significant anti-CML activity. A lower dose and a longer exposure to decitabine was suggested in CML. This lower dose and longer exposure was studied in a phase I design.48 AML, MDS, and CML patients were included in the trial. The dose of 15 mg/m2 as a 1-hour daily infusion for 10 days appeared to induce the most responses. The application of decitabine in allogenic blood stem cell transplantion was also studied.49,50 Encouraging results were obtained by Dr M. Koshy of the University of Illinois at Chicago, in sickle cell anemia.51 Eight adult patients were treated with a low-dose schedule of decitabine: 0.15 to 0.30 mg/kg on days 1 to 5 for 2 weeks. Within 4 weeks maximum levels of fetal hemoglobin were reached, demonstrating decitabine to be a promising therapy in patients with sickle cell anemia who fail to respond to hydroxyurea therapy. In 1998 Pharmachemie BV became a part of Teva Pharmaceutical Industries Ltd. At that time decitabine did not fit into the product development lines of Teva. Subsequently decitabine was transferred to SuperGen on September 27, 1999. Supergen announced on October 25, 2004 the submission of a file on decitabine, Dacogen, to the European Agency for the Evaluation of Medicinal Products (EMEA) and on November 1, 2004 to the FDA.

Concluding Remarks In 1982, at the start of decitabine research by Pharmachemie BV, new cancer drug development was just beginning in Europe in an organized way comparable to the NCI in the United States. Pinedo wanted to show that also in Europe a regular process of new cancer drug development could be established. The NDDO was actively involved during the first years, even before its official foundation. When the decitabine research shifted to the field of hematology, the involvement of the NDDO waned. After many years of exclusivity between the NDDO and the EORTC, in 1999 the drug development activities of the NDDO Research Foundation were brought under the umbrella of NDDO Oncology BV. From the start of the project it was clear that decitabine possessed unique properties. Momparler, as a real pioneer, proved to be an indefatigable believer. In solid tumors thus far promising laboratory results could not be substantiated in the clinic. Ways had to be found, however, to exploit the epigenetic aspects of decitabine. The first investigator who played a decisive role in finding clinical applications for decitabine was Pinto. On the one hand there were the unique properties of decitabine in DNA methylation and cell differentiation. On the other hand there was the range of tumors. Pinto made an informed connection between laboratory and clinic and founded the future indications of decitabine. The pleasant

441 side effect of my cooperation with Pinto for many years was that the location of the oncology center in Italy went along well with my interest in classical history.52 The second person of decisive importance in the development of decitabine was Kantarjian. He combined a deep vision into the potential of decitabine in hematological oncology with regulatory insights. Kantarjian specified the known possible applications for decitabine and added new ones, for example, in stem cell transplantion. Working with Kantarjian at the same time brought alive other aspects of my interest in history.53 It took 40 years for decitabine to move from synthesis to submission of the file to various authorities. I had the privilege to witness 16 years of that period, and they appear to have been important years for clarifying the mechanisms of action of decitabine and its possible application in the clinic. It was also a privilege to work with so many gifted researchers, some of whom made decitabine what it is now: a drug approvable for MDS. Decitabine definitely offers more therapeutic possibilities: in AML, as an addition to existing therapy or as treatment of poor prognosis or elderly patients, in CML, stem cell transplant, sickle cell anemia, and thalassemia. Decitabine is now in the skilful hands of SuperGen, who did a great job in developing the registration file. One thing is certain, both in the laboratory and in the clinic decitabine research will continue to lead to novel and exciting results that will hopefully improve cancer treatment.11

Acknowledgment It is impossible to thank all of the people who contributed to the development of decitabine. I have to make an exception for my coworkers for all 16 years, by seniority: Drs Marjolijn Koperdraat, Bas Vrijhof, Mischa Ross, Hellen de Kloet, and Wendy van Overveld.

References 1. Momparler RL: Pharmacology of 5-aza-2=-deoxycytidine. FEBS-Symp 1979. Antimetab Biochem Biol Med 57:33-41, 1979 2. Constantinides PG, Taylor SM, Jones, PA: Phenotypic conversion of cultured mouse embryo cells by aza pyrimidine nucleosides. Dev Biol 66:57-71, 1978 3. Jones PA, Taylor SM: Cellular differentiation, cytidine analogs and DNA methylation. Cell 20:85-93, 1980 4. Jones PA, Taylor SM, Mohandas, T, et al: Cell cycle-specific reactivation of an inactive X-chromosome locus by 5-azadeoxycytidine. Proc Natl Acad Sci U S A 79:1215-1219, 1982 5. Sachs L: The differentiation of myeloid leukaemia cells: New possibilities for therapy. Br J Haem 40:509-517, 1978 6. Rivard, GE, Momparler RL, Demers J, et al: Phase I study on 5-aza-2=deoxycytidine in children with acute leukemia. Leuk Res 5:453-462, 1981 7. Egger G, Liang G, Aparicio A. et al: Epigenetics in human disease and prospects for epigenetic therapy. Nature 429:457-463, 2004 8. Davies MJ, Jenkins PR, Prouse LSJ, et al: Structure of 2=-deoxy-5-azacytidine (decitabine) monohydrate. Acta Cryst C47:1418-1420, 1991 9. Pliml J, Sorm F: Synthesis of 2=-deoxy-D- ribofuranosyl-5-azacytosine. Coll Czech Chem Commun 29:2576-2577, 1964 10. Sorm F, Vesely J: Effect of 5-aza-2=-deoxycytidine against leukemic and hemopoietic tissues in AKR mice. Neoplasma 15:339-343, 1968 11. Bouchard J, Momparler RL: Incorporation of 5-aza-2=-deoxycytidine 5=-triphosphate into DNA. Interactions with mammalian DNA polymerase and DNA methylase. Mol Pharmacol 24:109-114, 1983 12. Chabot G, Bouchard J, Momparler RL: Kinetics of deamination of

D. de Vos

442

13.

14.

15.

16.

17.

18.

19.

20.

21.

22.

23.

24.

25.

26.

27.

28. 29. 30.

31.

32.

33.

5-aza-2=-deoxycytidine and cytosine arabinoside by human liver cytidine deaminase and its inhibition by 3-deazauridine, thymidine, and uracil arabinoside. Biochem Pharmacol 32:1327-1328, 1983 Momparler RL, Rossi M, Bouchard J, et al: Kinetic interaction of 5-aza2-deoxycytidine 5=-monophosphate and its 5=-triphosphate with deoxycytilate deaminase. Mol Pharmacol 25:436-438, 1984 Piskala A, Sorm F: Anomeric 4-amino-1-(2-deoxy-D-erythro-pentofuranosyl))-s-triazin-2(1H)-ones (2=-deoxy-5-azacytidine and its ␣-D anomer). Synthesis of 5-azapyrimidine deoxyribonucleosides via acylglucosyl isocyanates. Nucleic Acid Chem 1:443-449, 1978 Piskala A, Synackova M, Tomankova H, et al: Direct synthesis of 5-azapyrimidine 2=-deoxyribonucleosides. Hydrolysis of 5-aza-2=-deoxycytidine. Nuucleic Acid Res 4:109-113, 1978 Meulenbelt-Snijders SJ, Meulenbelt F, Paalman ACA, et al: Formulering van 5-aza-2=-deoxycytidine, een experimenteel cytostaticum. Pharm Weekbl 119:1186-1189, 1984 van Groeningen CJ, Leyva A, O’Brien AMP, et al: Phase I and pharmacokinetic study of 5-aza-2=-deoxycytidine (NSC 127716) in cancer patients. Cancer Res 46:4831-4836, 1986 Abele R, Clavel M, Dodion P, et al: The EORTC Early Clinical Trials Group experience with 5-aza-2=-deoxycytidine (NSC 127716) in patients with colorectal, head and neck, renal carcinomas and malignant melanomas. Eur J Cancer 23:1921-1924, 1987 Sessa C, Ten Bokkel Huinink W, Stoter G, et al: Phase II study of 5-aza-2=-deoxycytidine in advanced ovarian cancer. Eur J Cancer 26: 137-138, 1990 Vermorken JB, Tumolo S, Roozendaal KJ, et al: 5-Aza-2=-deoxycytidine in advanced or recurrent cancer of the cervix. Eur J Cancer 27:216-217, 1991 Momparler RL, de Vos D (eds): 5-Aza-2=-Deoxycytidine. Preclinical and Clinical Studies. Haarlem, The Netherlands, PCH Publications, 1990 Abbruzzese J, Raber M, Vrijhof W, et al: A phase I trial of laboratory derived synergistic chemotherapeutic (sc) combination: 2= deoxy-5azacytidine (DAC) and cisplatin (CDDP). Proc Am Assoc Cancer Res 32:204, 1991 (abstr 1219) Momparler RL, Rivard GE, Gyger M: Clinical trial on 5-aza-2=-deoxycytidine in patients with acute leukaemia. Pharm Ther 30:277-286, 1985 Momparler RL, Bouchard J, Onetto N, et al: 5-Aza-2=-deoxycytidine therapy in patients with acute leukaemia inhibits DNA methylation. Leuk Res 8:181-185, 1984 Pinto A, Attadia V, Di Fiore PP, et al: 2=-Deoxy-5-azacytidine induces functional and morphological differentiation of a human erythroleukemic cell line (K562), in Rich MA (ed): Leukemia Rev Int 1. New York, NY, Marcel Dekker, 1983, pp 207-208 Pinto A, Attadia V, Fusco A, et al: 5-Aza-2=-deoxycytidine induces terminal differentiation of leukemic blasts from patients with acute myeloid leukemias. Blood 64:922-929, 1984 Pinto A, Maio M, Attadia V, et al: Modulation of HLA-DR antigens expression in human myeloid leukaemic cells by cytarabine and 5-aza2=-deoxycytidine. Lancet 2:867-868, 1984 Pinto A, Attadia V: Leukemia therapy by differentiation inducers: The therapeutic concept of differentiation. Cancer J 1:54, 1986 Richel DJ: Experimental therapy in Ara-C sensitive and resistant leukaemia. PhD thesis, University of Leiden, The Netherlands, 1990 Richel DJ, Colly LP, Kluin-Nelemans JC, et al: The antileukaemic activity of 5-aza-2=-deoxycytidine (Aza-dC) in patients with relapsed acute leukaemia. Br J Cancer 64:144-148, 1991 Willemze R, Stegman APA, Colly LP: The use of 5-aza-2=-deoxycytidine (DAC) in adult patients with acute leukaemia. Haematologica 76:43, 1990 (abstr) Pinto A: 5-Aza-2=-deoxycytidine and DNA methyltransferase inhibitors in the treatment of myeloid leukemias and myelodysplastic syndromes: Biological aspects and clinical results. Leukemia 7:Suppl monograph 1, 1993 Kantarjian HM, Pinto A: Workshop on the clinical results with decit-

34.

35.

36.

37.

38.

39.

40.

41.

42.

43.

44.

45.

46.

47.

48.

49.

50.

51.

52. 53.

abine (5-aza-2=-deoxycytabine) in hematologic malignancies. Leukemia 11:Suppl monograph 1, 1997 Momparler RL, Bouffard DY, Momparler LF, et al: Pilot phase I-II study on 5-aza-2=-deoxycytidine (decitabine) in patients with metastaic lung cancer. Anticancer drugs 8:358-368, 1997 Momparler RL, Eliopoulos N, Ayoub J: Evaluation of an inhibitor of DNA methylation, 5-aza-2=-deoxycytidine for the treatment of lung cancer and the future role of gene therapy. Adv Exp Med Biol 465:433446, 2000 Momparler RL, Ayoub J: Potential of 5-aza-2=-deoxycytidine (decitabine) a potent inhibitor of DNA methylation for therapy of advanced non-small cell lung cancer. Lung Cancer 34:S111-S115, 2001 (suppl 4) Schwartsmann G, Schunemann H, Gorini CN, et al: A phase I trial of cisplatin plus decitabine, a new DNA-hypomethylating agent, in patients with advanced solid tumors and a follow-up early phase II evaluation in patients with inoperable non-small cell lung cancer. Invest New Drugs 18:83-91, 2000 Thibault A, Figg WD, Bergan RC, et al: A phase II study of 5-aza-2=deoxycytidine (decitabine) in hormone independent metastatic (D2) prostate cancer. Tumori 84:87-89, 1998 Pohlmann P, DiLeone LP, Cancella AI, et al: Phase II trial of cisplatin plus decitabine, a new DNA hypomethylating agent, in patients with advanced squamous carcinoma of the cervix. Am J Clin Oncol 25:496501, 2002 Aparicio A, Eads CA, Leong LA, et al: Phase I trial of continuous infusion 5-aza-2=-deoxycytidine. Cancer Chemother Pharmacol 51:231239, 2003 Wijermans P, Lübbert M, Verhoef G, et al: Low-dose 5-aza-2=-deoxycytidine, a DNA hypomethylating agent, for the treatment of high-risk myelodysplastic syndrome: A multicenter phase II study in elderly patients. J Clin Oncol 18:956-962, 2000 Lübbert M, Wijermans P, Kunzmann R, et al: Cytogenetic responses in high-risk myelodysplastic syndrome following low dose treatment with the DNA methylation inhibitor 5-aza-2=-deoxycytidine. Br J Haematol 114:349-357, 2001 Ruter B, Wijermans PW, Lübbert M: DNA methylation as a therapeutic target in hematologic disorders: Recent results in older patients with myodysplasia and acute myeloid leukaemia. Int J Hematol 80:128-135, 2004 Bosch van den J, Lübbert M, Verhoef G: The effects of 5-aza-2=-deoxycytidine (decitabine) on the platelet count in patients with intermediate and high-risk myelodysplastic syndromes. Leuk Res 28:785-790, 2004 Lübbert M, Daskalakis M, Kunzmann R, et al: Nonclonal neutrophil responses after successful treatment of myalodysplasia with low dose 5-aza-2=-deoxycytidine (decitabine). Leuk Res 28:1267-1271, 2004 Kantarjian HM, O’Brien SM, Keating M, et al: Results of decitabine therapy in the accelerated and blastic phases of chronic myelogenous leukaemia. Leukemia 11:1617-1620, 1997 Kantarjian HM, O’Brien S, Cortes J, et al: Results of decitabine (5-aza2=-deoxycytidine) therapy in 130 patients with chronic myeloid leukaemia. Cancer 98:522-528, 2003 Issa JP, Garcia-Manero G, Giles FJ, et al: Phase 1 study of low-dose prolonged exposure schedules of the hypomethylating agent 5-aza-2=deoxycytidine (decitabine) in hematopoetic malignancies. Blood 103: 1635-1640, 2004 Ravandi F, Kantarjian H, Cohen A, et al: Decitabine with allogenic peripheral blood stemcell transplantation in the therapy of leukaemia relapse following a prior transplant: Results of a phase I study. Bone Marrow Transplant 27:1221-1225, 2001 Lima de M, Ravandi F, Shahjahan M, et al: Long-term follow-up of a phase I study of high dose decitabine, busulfan, and cyclophosphamide plus allogenic transplantation for the treatment of patients with leukemias. Cancer 97:1242-1247, 2003 Koshy M, Dom L, Bressler L, et al: 2=-Deoxy-5-azacytidine and fetal haemoglobin induction in sickle cell anemia. Blood 96:2379-2384, 2000 Livius T: Ab urbe condita. Various editions and publishers Werfel F: Die vierzig Tage des Musa Dagh. Vienna, Austria, Zsolnay verlag, 1933