Novel Therapeutic Agents for the Treatment
`of Myelodysplastic Syndromes
`
`Bruce D. Cheson, James A. Zwiebel, Janet Dancey, and Anthony Murgo
`
`activity
`agents have demonstrated
`Few chemotherapy
`in patients with myelodysplastic syndromes (MOS) and
`supportive management
`remains
`the standard
`of care.
`An increasing number of new drugs in development are
`being directed
`at specific molecular
`or biological
`tar-
`gets of these diseases. Topctecan, a topoisomerase I
`inhibitor,
`has shown single-agent
`activity and is now
`being
`combined
`with
`other
`agents,
`including
`cy~
`tarablne. The aminothiol
`amifostine
`induces
`responses
`in about 30% of patients; however,
`its role is still being
`clarified. Agents
`that
`inhibit histone
`deacetylase
`and
`target DNA hypermethylation,
`thus permitting
`dere-
`pression of normal genes,
`include 5-azacytidine,
`decita-
`bine, phenyl butyrate.
`and depsipeptide. Arsenic triox-
`ide has demonstrated
`impressive
`activity
`in acute
`promyelocytic
`leukemia
`and preclinical
`data
`suggest
`the potential
`for activity in MOS. UCN~OI
`is a novel
`agent
`that
`inhibits protein kinase C and other protein
`kinases important
`for progression
`through
`the G. and
`G2 phases of the cell cycle. Dolastatfn-In has extremely
`potent
`in vitro activity against a variety of tumor
`cell
`lines. Since its dose-limiting toxicities
`include myelosup-
`pression,
`it
`is being studied
`in acute myelogenous
`leukemia (AML) and MOS. Rat may playa role in MOS.
`and activation
`of this gene and its signaling pathways
`may require farnesylation.
`Several
`farnesyl
`transferase
`inhibitors
`are now available for study in patients with
`MOS. An increasing
`body of data suggests
`a possible
`role for angiogenesis
`in MOS. and several antlanglcgen-
`esis agents are in clinical
`trials,
`including thalidomide.
`SU5-416. and anti-vascular
`endothelial
`growth
`factor
`(VEGF) antibodies. Development
`of new drugs
`and
`regimens will be facilitated by recently developed stan-
`dardized response
`criteria. Future clinical
`trials should
`focus on rational
`combinations
`of these
`agents
`and
`others with the goal of curing patients with MOS.
`Semin Oneal 27:560-577. This is a US government work.
`There are no restrictions on its use.
`
`THE MYELOOYSPLASTlC syndromes (MOS)
`
`are a heterogeneous group of hematopoietic
`disorders characterized by pancytopenia,
`generally
`in the setting of a hypercellular bone marrow. MDS
`
`From the Cancer Therapy Evaluation Progrcm (CTEP), Divi~
`sion of Cancer Treatment and Diagnosis, National Cancer
`Institute,
`Bethesda, MD.
`to Bruce D. Cheson, MD, National
`requests
`Address
`reprint
`Cancer Institute, Executive Plaza North-Room 741, Bethesda, MD
`20892.
`This is a US government work. There are no restrictions on its
`""
`
`have historically been referred to as oligo blastic
`leukemia, refractory anemia, smoldering acute leu-
`kemia, or preleukemia.
`In 1982,
`the French-
`American-British
`(FAB) group presented a classifi-
`cation, modified in 1985, which currently is the
`most widely used.t- The FAB group separated
`MOS into five categories: refractory anemia (RA),
`RA with ringed sideroblasts
`(RARS), RA with
`excess blasts (RAEB), and RAEB in transforma-
`tion (RAEB- T). The distinction between RAEB- T
`and acute myelogenous
`leukemia (AML)
`is based
`on histopathology, not clinical features, As a result,
`patients with MDS may exhibit a clinical picture
`consistent with AML with rapidly increasing num-
`bers of blasts. but without
`the requisite number
`to
`fulfill
`the
`criteria
`for
`the diagnosis of AMLJ
`Recently, a World Health Organization
`(WHO)
`steering committee proposed changes to the MDS
`subtypes with the major modifications
`including
`reclassifying chronic myelomonocytic
`leukemia
`(CMML)
`as a myeloproliferative
`disorder
`and
`decreasing the threshold for diagnosing AML from
`30% blasts to 20%4 This system may eventually
`replace the FAB.
`The likelihood of transformation to AML varies by
`FAB subtypes-s:approximately 10% to 20% for RA or
`RARS, 20% to 30% for CMML, 40% to 50% for
`RAEB, and 60% to 75% for RAEB-T. Nevertheless,
`the MDS are uniformly fatal, even without progression
`to AML, because of infection and bleeding.'·!O
`Over the years, a number of scoring and prognos-
`tic systems have been published to facilitate com-
`parisons among reports of various treatments
`for
`MOS_ Recently,
`the International Prognostic Scor-
`ing System (lPSS) has been widely adopted.'!
`Factors
`taken into consideration
`included bone
`marrow blasts, cytogenetics, and cvropenias. Groups
`were identified with relative risks for transforma-
`tion to AML and overall survival. Patients
`in the
`good cytogenetics group were those with a normal
`karyotype; poor risk included patients with com-
`plex abnormalities
`or with an involved chromo-
`some 7; intermediate-risk
`patients consisted of all
`others (Table 1)-
`There are no curative therapies other than stem
`cell transplantation, which is an option for only a
`subset of patients. Therefore, numerous
`therapies
`
`560
`
`Seminars in Oncology, Vol 27, No 5 (October),
`
`2000: pp 560·577
`
`DR. REDDY’S LABS., INC. EX. 1029 PAGE 1
`
`

`

`NEW THERAPEUTIC
`
`AGENTS FOR. MDS
`
`561
`
`Table
`
`I.
`
`International
`
`Prognostic
`
`Scoring
`
`System for
`
`the Myelodysplastic
`
`Syndromes
`
`Prognostic Variable
`
`Bone marrow blasts (%)
`Karyotype
`Cyropanlas
`
`(no. of lineages)
`
`0
`
`<5
`Good
`0/1
`
`0.5
`
`5-10
`Intermediate
`2/3
`
`Score
`
`1.0
`
`Poor
`
`1.5
`
`11-20
`
`2.0
`
`21·30
`
`NOTE. The total of the values for each prognostic
`variable is used to place patients
`high. These risk groups have significantly different outcomes.
`
`in , of 4 risk groups:
`
`low, intermediate-]
`
`, ineermedraee-z.
`
`and
`
`to improve
`have been and are being investigated
`the outlook for these patients. Drugs selected for
`study in MOS have
`typically been those with
`significant activity in AML Thus, cytarabine has
`been most widely evaluated,
`dating
`back more
`than 30 years when Ellison et a[1' first reported
`complete
`remissions with doses of cytarabine
`as
`low as 10 mg/m'/d. Response
`rates were clearly
`dose-dependent, which encouraged
`the develop-
`ment of higher dose regimens. Subsequently, anec-
`dotal
`reports and small series were published in
`which cytarabine
`at 10% to 20% of the standard
`dose administered
`either
`subcutaneously
`or by
`continuous
`intravenous
`infusion appeared to be
`effective in the treatment of AML and MOS."·"
`Additional
`studies and a randomized phase III trial
`failed to support a major role for this therapy.z3.z5
`Anthracyclines
`and related compounds
`have
`had been srudied as single agents only to a limited
`In a study in which hydroxyurea
`and
`extent.26,Z7
`etoposide were compared in patients with CMML,
`response rates and survival were not
`impressive
`with either agent, but favored the former.28 Other
`drugs that have been evaluated include S-tluogua-
`nine and homoharringtonine,
`but both showed
`limited activity.29,JO
`
`NEW AGENTS
`Several agents with unique mechanisms of activ-
`ity are currently
`or will soon be evaluated
`in
`clinical
`trials for patients with MOS.
`Topoisomerase IInhibitors
`Topotecan is a topoisornerase I inhibitor whose
`activity in acute
`leukemia
`led to its testing in
`MOS. The initial report
`included 47 patients with
`RAEB, RAEB- T, or CMML.JI They were a poor-
`risk group, as demonstrated
`by the fact
`that
`the
`median age was 66 years, 70% exhibited cytoge-
`netic
`abnormalities,
`and more
`than
`half were
`
`before topotecan therapy. Topo-
`thrombocytopenic
`tecan was delivered at a dose of 2 mg/m2 as a
`continuous 24~hour infusion for 5 days. Treatment
`resulted in 28% complete remissions and an addi-
`tional13% of patients who experienced significant
`hematologic
`improvement. All eight patients with
`cytogenetic
`abnormalities
`before treatment
`and
`who achieved a complete remission became cvtoge-
`netically normal once in complete remission. The
`median remission duration was 7.5 months with
`38% of patients
`still alive 1 year following treat-
`ment. Whether
`chronic oral topotecan is effective
`is undergoing evaluation.
`The same investigators have shown that cornbi-
`nation of topotecan
`and cvtarabine
`is extremely
`active in patients with MDS. Beran et aP2 reported
`on 86 patients with MOS and CMML, most of
`whom (66%) were previously untreated, but who
`were considered high risk based on age or cvroge-
`netic abnormalities. Topotecan was administered
`ar a dose of 1.25 mg/m' by continuous
`infusion
`daily for 5 days, and cytarabine
`at 1 glm' by a
`2-hour
`infusion
`daily for 5 days. A complete
`remission was attained in 56% of patients, with 7%
`treatment-related
`deaths and a median survival of
`60 weeks."
`Preliminary
`results have been pub-
`lished of aggressive combination
`of
`topotecan,
`fludarabine,
`cytarabine,
`and granulocyte
`colony-
`stimulating factor (G-CSF);
`there were 50% com-
`plete remissions and 40% partial
`remissions, and
`the regimen appeared to be well rolcrarcd.P
`
`Amifostine
`(Ethyol; Alza Pharmaceuricals, Palo
`Amifostine
`is a phosphorylated
`aminothiol
`that
`Alto, CAl
`protects bone marrow progenitors
`and other nor-
`mal
`tissues
`from the
`toxicities
`associated with
`chemotherapy
`or radiation therapy.
`It was devel-
`oped by the Walter Reed Army Medical
`Institute
`
`DR. REDDY’S LABS., INC. EX. 1029 PAGE 2
`
`

`

`S62
`
`during
`the military code name WR-2721)
`(thus,
`the Cold Wat as part of a classified research project
`to identify an agent that would protect military
`personnel
`from radiation in the event of nuclear
`war. Arnifostine was found to afford greater protec-
`tion against radiation than more than 4,000 other
`compounds
`screened. Nevertheless,
`the Army ter-
`minated development
`of this compound in 1988
`because of its poor oral bioavailability
`and the
`prohibitive nausea, vomiting, diarrhea, and abdomi-
`nal cramps with the oral formulation.
`Further research was encouraged by the observa-
`tion that amifostine stimulates hematopoiesis in
`both animal models and in vitro studies, and that
`it
`enhances
`the formation of hematopoietic
`progeni-
`tors from MDS bone marrow. In the initial phase
`1/11studv," the drug was administered at doses of
`100,200, or 400 mg/m' three times per week or 740
`investigators
`mg/m2 weekly for 3 weeks. These
`treated 18 patients at a median age of 73 years.
`FAB types included RA (seven patients), RARS
`(n = 5), RAEB (n = 4), and RAEB-T (n = 2).
`Seventeen patients were anemic, 15 of whom were
`transfusion-dependent,
`12 had an absolute neutro-
`phil count less than 1,000{pL and 14 were thrombo-
`cytopenic. Hematologic
`improvement was ob-
`served
`in 83% with
`the
`three-times-a-week
`schedules,
`including either an increase in neutro-
`phils or a reduction in red blood cell transfusion
`requirements. More than 40% of patients had a rise
`in their platelet counts. However,
`there was accel-
`eration to AML in several patients with RAEB~T.
`Although 61% of patients had clonal cyrogenetic
`abnormalities
`before therapy,
`the
`abnormalities
`persisted even in patients with a hematologic
`response. No data regarding duration of response
`were provided, although responses were reported to
`persist during continuation therapy.
`List et ap5 reported the results of a subsequent
`multicenter
`trial of arnlfostine in 117 patients, 104
`of whom were evaluable at the time of presenta-
`tion. A neutrophil
`response occurred in 10 (33%)
`of 30 patients, and was considered major
`in nine
`and minor in the other. A red blood cell response
`was evaluable in 66 patients, and a major response
`occurred in seven, with three experiencing a minor
`response. A major improvement
`in platelet count
`was seen in seven of 27 patients, with a minor
`response in three others, and 21% of patients had
`an increase in the reticulocyte count. A decrease in
`myeloblasts and sideroblasts occurred in 28% and
`
`CHESON ET AL
`
`response rate was
`respectively. The overall
`31%,
`30%, which is significantly
`lower
`than
`in the
`previous trial. Adverse events that were moderate
`or severe included fatigue
`(14%, 18%), nausea
`09%,
`36%),
`and vomiting
`04%,
`27%).
`In a
`smaller series."
`a single or multilineage
`response
`was noted
`in five of 12 patients
`(58%). The
`absolute neutrophil
`count
`Increased in 25% (by
`102 to 1,560{pL), platelets
`in 50% (by 24,000 to
`49,000{pL), reticulocytes
`in 25% (1.9% to 20%),
`and hemoglobin in 16% (5.3 to 5.6 g{dL).
`In other
`reports,
`results with this agent were
`disappointing.37,38 Hofmann
`et ap8 described 32
`patients with RA/RARS
`(n = 26) and RAEB{
`RAEB-T (n ~ 15) treated at a dose of 200 mg{m'
`three times per week followed by a Zvweek interval,
`for four courses. Limited benefit was observed even
`in patients with low- or intermediate-risk
`disease
`by the IPSS.
`in MDS is still being
`The role of amifostine
`clarified. Nevertheless,
`combinations of amifosrine
`with other agents such as 'i-azacytidine
`are being
`evaluated.
`
`Agents That Target Transcription
`the rno-
`Recent developments
`in understanding
`lecular basis
`for
`transcriptional
`repression
`and
`activation
`have presented
`new possibilities
`for
`cancer therapy. Two mechanisms of gene silencing,
`promoter hvpermethvlauon
`and histone deacetvla-
`tion,
`appear
`to be interrelated.
`The
`utility of
`targeting DNA hypermethylation
`and histone
`deacetylation
`is being explored clinically. Agents
`shown to inhibit
`histone
`deacetylase
`in vitro
`include sodium phenylbutyrate,
`depsipeptide, hy-
`brid polar compounds.'?
`and MS-27-275.40 Hypo-
`methylating agents include 5~azacytidine and 'i-aza-
`Zcdeoxycyudine. The exploration
`of these agents
`in the clinic, either alone or in combination with
`retinoids, demethylation
`agents, and chemothera-
`is a novel and promising area of
`peutic agents I
`cancer therapeutics.
`agents.
`Hypomethylating
`and
`5-Azacytidine
`fi-aaa-Z." deoxycytidine are pyrimidine analogs that
`have been extensively evaluated in patients with
`MDS. These compounds are metabolized intracel-
`lularly to triphosphates
`and subsequently incorpo-
`rated into newly synthesized DNA, where they
`directly inhibit DNA synthesis and inhibit
`the
`activity of DNA rnethvlrransferase,
`the enzyme
`required for 5/ -cytosine methylation
`of cytosine-
`
`DR. REDDY’S LABS., INC. EX. 1029 PAGE 3
`
`

`

`NEW THERAPEUTIC AGENTS FOR MDS
`
`56l
`
`As a result,
`(CpG) dmuclcondes.tl"
`guanosine
`cytosine methylation
`is blocked in newly repli-
`cated DNA, but not
`in the DNA of resting at
`nondividing
`cells.
`Inhibition
`of methylation
`by
`5~azacytidille and decitabine
`is associated with
`transcription of genes previously silenced by meth-
`ylation of promoter region CpG;rich islands, and
`with cellular phenotypic changes; these effects can
`occur at concentrations
`that are too low to inhibit
`DNA synthesis directly or to cause substantial
`cytotoxicity.41,43-45The potential application of
`'i-azacyrtdine and decitabine as inhibitors of DNA
`methylation and inducers of cell differentiation of
`normal and neoplastic hematopoietic progenitor
`cells is an area of active investigation.43,45-48
`5~Azacytidine initially demonstrated activity in
`AML,49-53but with considerable toxicity at doses
`required for response. Since the drug also induces
`in vitro cellular differentiation in association with
`hypomethvlation
`of DNA,
`it was of interest
`for
`study in MDS. Chitambar
`et a)54used a relatively
`low dose (10 to 35 mg/m'/d for 14 days) to treat 13
`patients,
`three of whom achieved a partial rei
`sponse. Cancer and Leukemia Group B (CALGB)
`conducted a phase II trial of S-aaa-
`mvestigarors'?
`cytidine at 75 mgfm'/d by continuous
`infusion for 7
`days every 28 days in 48 patients with MDS and
`noted 11% complete remissions and 25% partial
`remissions. Major toxicities
`included nausea and
`vomiting: one patient died of neutropenic sepsis.
`Subcutaneous administration resulted in slightly
`lower response, rates-7% complete
`remissions,
`17% partial remissions, and 14% with trilineage
`improvement, but less than a partial response.S
`These findings are similar to those achieved with
`low-dose cytarabine.
`The CALGB recently reported the pteliminary
`results of a phase III randomized trial of 5;azacyti;
`dine versusobservation in 191 patients with MDS.57
`The patients were stratified by FAB subtype (19%
`RA, 4% RARS, 42% RAEB, 21% RAEB-T, 6%
`CMML);
`patients with RA or RARS had,
`in
`addition, symptomatic cvropenias.
`'i-Azacyridine
`was administered subcutaneously at a dose of 75
`mg/m'/d fat 7 days every 4 weeks for four cycles.
`Patients on the observation arm could receive
`'i-azacytidine upon progression. Hematologic
`rei
`sponses were significantly higher in patients ran;
`domized to receive 5;azacytidine compared with
`(P < .0001): 63% (6% complete
`observation
`re-
`sponse, 10% partial response, and 47% improve-
`
`merit) versus 7% (all improvement, no complete or
`partial responses). The median time to leukemic
`transformation or death was 22 months for patients
`on the treatment arm, compared with 12 months
`for
`the patients
`randomized
`to observation
`(P = .0034). The 12- and 24-month
`overall sur-
`vival rate was higher in patients randomized to
`receive azacytidine (70% and 4 I % versus 62% and
`25%, respectively), as was the median survival
`time (I8 versus 14 months),
`but
`the differences
`were not yet significant. Treatment with 5~azacyti-
`dine was associated with subjective improvement
`in quality of life as measured by fatigue, dyspnea,
`physical functioning, positive affect, and psycho-
`logic distress.V Whether 5;azacytidine
`improves
`overall survival or reduces transformation to leuke-
`mia will require additional follow-up evaluation.
`5-Aza-2' deoxycytidine
`(decitabine)
`is another
`hypomethylating agent with potent in vitro activ-
`icy.In earlier studies, decitabine administered as an
`intermittent
`intravenous infusion achieved brief
`responses in a small series of patients with MDSj
`however, the majority experienced life-threatening
`neutropenia
`and/or thrornbocvroperua.t? Wijer;
`mans et al60 reviewed the experience with this
`agent
`in MDS and found a 54% tesponse rate of29
`elderly patients,
`although
`there were 17% toxic
`deaths. This drug is under developmenr
`for MDS
`both in Europe and the United States.",60
`Histone deacetylation and DNA hypermethylation.
`Retmoids, other hormone receptors, and the Myel
`Mad/Max network of growth regulators exert their
`effects on gene expression by interacting with
`nuclear corepressor complexes that are present on
`the DNA of promoter regions.61,61 Gene silencing
`occurs with the recruitment ofhistone deacetylases
`and the formation of a nuclear corepressor-histone
`deacetylase complex (NCHDC). Histone deacetv-
`lase catalyzes the removal of acetyl groups from
`histone proteins,
`inducing a conformation change
`that results in an environment unfavorable to gene
`transcription. A NCHDC has been found to play
`an important role in acute promyelocytic leukemia
`(APL), where the NCHDC is recruited by both the
`PML-RARa
`and PLZF-RARa
`fusion
`proteins,
`which form as a consequence
`of chromosomal
`trans locations
`t(l5; 17)
`and
`t(l I; I 7),
`respec-
`tively6J.67 A NCHDC is also recruited by ETO, a
`component of the fusion product resulting from
`the t(8;21) chromosomal
`translocation in AML.68,69
`Moreover,
`inhibitors of histone deacetylase have
`
`DR. REDDY’S LABS., INC. EX. 1029 PAGE 4
`
`

`

`564
`
`CHESON ET AL
`
`repression
`been found to overcome transcriptional
`and to potentiate
`retinoid-induced
`differentiation
`of APL and AML cells.63•66•69 A clinical
`test of this
`observation was performed in a patient with APL
`who had become tefractory to both chemotherapy
`and all-tram retinoic
`acid (ATRA). Administra-
`rion of both ATRA and a histone
`deacetylase
`inhibitor,
`sodium phenylbutyrate
`(see below),
`re-
`sulted in a complete remission. The clinical re-
`sponse was associated with acetylation of histone
`proteins in the leukemic cells."?
`in gene
`While methylation
`of CpG islands
`promoter
`regions has
`long been known
`to be
`associated with gene silencing,
`it was not known
`how such DNA hvpermerhvlauon
`exerts its effect
`on gene transcription. Recent studies have shed
`light on both the role of DNA hypermethylation
`in the inactivation
`of tumor suppressor genes, as
`well as the mechanism of transcriptional
`repres-
`sion. Examples of genes associated with CpG
`hypermerhvlation
`include, among others, RB in
`retinoblastoma, VHL (the von Hippel-Lindau gene)
`p16lNK4A
`and p15lNK4A
`in renal carcinoma,
`(cvclin-dependenr
`kinase inhibitors)
`in solid tu-
`mors and in hematologic malignancies, and hMLHl
`(a DNA mismatch repair gene) in colon cancer."
`The mechanism of gene silencing by DNA hyper-
`methylation
`now appears to involve the recruit-
`ment of a NCHDC by the methyl-Cpff-hinding
`protein, MeCP2.72,73 In fact, the combined admin-
`istration of a demethylaring
`agent and a histone
`deacetvlase inhibitor has been shown to svnergiae
`in reactivating genes that were silenced in cancer
`cells.74 This finding nor only links the processes of
`DNA hypermethylation
`and histone
`deaceryla-
`tion, but
`also presents
`therapeutic
`targets
`for
`agents
`that
`are relatively nontoxic,
`or used at
`nontoxic doses.
`
`Fhenylbutyrate
`(FB) is a low-molecular-weight
`Phenylbutyrate
`phenyl-farty acid that been used clinically to treat
`hyperammonemia
`in children with inborn errors of
`urea synthesis.75 It also been shown to enhance
`fetal hemoglobin production in some patients with
`hemoglobmopathies.ts A number of mechanisms
`have been proposed for the antitumor effect of PB,
`including (1) elimination
`of glutamine necessary
`for nucleic acid and protein synthesis in rapidly
`growing normal and tumor cells77,78;(2) inhibition
`of the mevalonate pathway of cholesterol synthesis
`
`process-
`leading to interference of post-translational
`ing of proteins, modification of lipid metabolism,
`inhibition
`of protein isoprenylation, and regula-
`tion of gene expression through DNA hvpomethyl-
`ation 79,80;(3) activation of a peroxisome prolifera-
`tor-activated
`receptor
`by PB, a transcriptional
`factor regulating lipid metabolism and cell growrh'":
`and (4)
`regulation
`of gene expression
`through
`histone hyperacetylation
`via inhibition of nuclear
`histone deacetylases.82-84
`PB has been shown to induce differentiation,
`tumor cytostasis. and reversion of malignant pheno-
`type in several
`in vitro models.80,85-89 PB, as a
`histone deacetylase inhibitor, may have synergistic
`activity with ATRA in the treatment of APL.65,67,90
`The PML-RAR fusion protein was shown to re-
`cruit a transcriptional
`corepressor
`complex that
`includes a histone deacetvlase. ATRA alone could
`partially
`dissociate
`the
`complex,
`allowing
`in-
`creased transcription, but butyrate (or other inhibi-
`tors of histone deacetylases)
`in combination with
`ATRA was able to completely abrogate the inhibi-
`tion of transcription.
`In light of these observations,
`an APL patient who experienced multiple relapses
`after ATRA treatment was treated with PB in
`combination with ATRA under
`compassionate
`release, and achieved a complete remission.i''
`
`Depsipeptide
`is a bicyclic pep-
`(NSC 630176)
`Depsipepride
`tide originally isolated from Chromobacterium viola~
`cetcn, strain 968, by Fujisawa Pharmaceutical Co
`(Osaka, Japan). In the original observations, depsi-
`peptide selectively decreased the mRNA expres-
`sion of the c-myc oncogene
`and inhibited
`the
`growth of the Ha-ras-transfortned NIH3T3 clonal
`cell
`line, Ras-I. but had no effect on Ha-res
`mRNA expression.vt It did not affect DNA synthe-
`sis, but caused cell cycle arrest at GO/Gl. Recently,
`it has been shown to be a histone
`deacerylase
`mhibiror"
`of
`incubation
`that
`Byrd et al demonstrated
`chronic lymphocytic leukemia cells with depsipep-
`tide resulted in an alteration
`in apoptosis-assoct-
`ated proteins: an increase in Bax with no change in
`Bcl-Z, and a decrease in p27 expression."
`In collaboration with Fujisawa Pharmaceutical
`Co,
`the National Cancer
`Institute
`(NCI)
`is cur-
`rently sponsoring two phase I trials of depsipeptide
`administered as a -l-hour intravenous
`infusion.
`In
`one trial, a once-weekly infusion schedule (days 1,
`
`DR. REDDY’S LABS., INC. EX. 1029 PAGE 5
`
`

`

`NEW THERAPEUTIC AGENTS FOR MDS
`
`565
`
`8, and 15 every 28 days) is used, while the other
`trial evaluates a twice-weekly (days 1 and 5 every
`21 days) schedule.
`
`MS-27-275
`that was
`MS-27-275 is a benzamide derivative
`(Tokyo,
`synthesized
`by Mitsui Pharmaceuticals
`Japan) in a search for novel antitumor agenrs.w
`The compound was found to have histone deacery-
`lase activity in vitro at micromolar concentrations.
`In addition, when administered orally, MS-27-275
`inhibited the growth of a number of tumor xeno-
`grafts. The NCI,
`in collaboration with Mitsui
`Pharmaceuticals, plans to sponsor phase I trials of
`this agent in the near future.
`
`Hylrrid Polar Compounds
`(HMBA) was the
`Hexamethylene
`bisacetamide
`first of the class of hybrid polar compounds
`to be
`evaluated as an antitumor agent
`in MDS and
`AML. The limited clinical activity that was ob-
`served was attributed to the inability to achieve
`the plasma concentrations
`that were required to
`induce differentiation in cells
`in vitro and to
`Subsequently,
`dose-limiting
`rhrombocytopenta.P'
`Richon et aP9 described compounds structurally
`related
`to HMBA,
`but which
`exhibited
`3-log
`greater potency in inducing terminal differentia-
`In
`tion and apoptosis in transformed cell
`lines.
`addition, these compounds possess histone deacety-
`lase inhibitory activity at micromolar concentra-
`tions. Recently, one such compound, Mvcarboxv-
`cinnamic acid bishydroxamide (CBHA), was found
`to induce apoptosis in human neuroblastoma, and
`the effect was associated with CD95jCD95
`ligand
`expression by the tumor cells." These agents
`should be entering into early clinical trials in the
`near future.
`
`Arsenic Trioxide
`Arsenic was used as a medicinal 2,400 years ago
`in the time of the ancient Greeks and Romans.
`Paul Ehrlich used organic arsenicals for the treat-
`ment of syphilis. Arsenicals
`are still
`included as
`ingredients
`in folk remedies of some cultures,
`particularly
`in China
`and other parts of Asia.
`Arsenic was widely used to treat syphilis before the
`advent of penicillin,
`and the organic arsenical
`melarsoprol
`is a recognized treatment
`for the
`meningoencephalitic
`stage of African trypanoso-
`mtasis." Fowler's solution (1% arsenic trioxide in
`
`in the 18th
`formulated
`potassium bicarbonate),
`century to treat a variety of infectious and neoplas-
`tic disorders, was reported by US physicians in the
`1930s to be useful
`in the treatment
`of chronic
`myelogenous
`leukemia (CML), and more recently
`by hematologists in China to treat various forms of
`leukemia,
`including CML.97
`Recent
`interest in the development of arsenic
`trioxide as an anticancer agent emanates
`from
`reports by Chinese investigators98,99of its efficacy
`in the treatment of APL. These favorable results in
`APL were confirmed
`in the United
`States by
`investigators at Memorial Sloan-Kettering Cancer
`Center
`(MSKCC).IOO
`Preclinical
`studies have shown that human APL
`cells are very sensitive to the growth-inhibitorv
`and cytotoxic effects of arsenic trioxide.101,102 Sen-
`sitivity to arsenic trioxide in vitro has also been
`demonstrated against a variety of other tumor
`types, including those derived from myeloid leuke-
`mias other
`than PML,101 myelorna.l'"
`lymphoid
`leukemia and lymphoma,I04-107prostate cancer,lOS
`and a various other solid rumors.P?
`The mechanism of antitumor activity of arsenic
`trioxide is not understood, but it appears to depend
`to some degree on dose and tumor type. The
`principal
`target
`in APL cells is the promvelocvric
`leukemia gene-retinoic
`acid receptor-alpha
`fusion
`transcript
`(PML/RAR_lY)102,110 Arsenic
`trioxide
`causes the degradation
`of the PML/RAR-lY and
`wild-type PML proteins,
`thus inhibiting their af-
`feet on growth and differentiation.lOZ,111,llZ Unlike
`ATRA, arsenic trioxide does not downregulate
`wild-type RAR_lY.102 In NB4 cells, a human APL
`cell line with t05;17)
`and the PML/RAR-o: fusion
`protein,
`the effects of arsenic trioxide treatment
`are dose-dependentl'":
`induction of partial (nonter-
`mtnal) differentiation at relatively low concentra-
`tions
`(0.1 to 0.5 pmol/L) with predominantly
`apoprosis at higher concentrations
`(0.5 to 2 pmol!
`L). However,
`induction of apoptosis by arsenic
`trioxide involves mechanisms other than modula-
`tion of PML or PML/RAR-lY.lol
`For example,
`growth inhibition and apoptosis induced by arsenic
`trioxide has been associated with downregulation
`of bel-2 expression in APL and other myeloid cell
`lines.101,102 Arsenic trioxide is known to react with
`sulfhydryl
`(SH) groups and thus alter many differ-
`ent enzyme systems,
`including those that affect
`protein tyrosine phosphorylation.Uv-U" The antitu-
`mor properties of arsenic trioxide have been attrib-
`
`DR. REDDY’S LABS., INC. EX. 1029 PAGE 6
`
`

`

`566
`
`CHESON ET AL
`
`to effects on mitochon-
`in part,
`least
`uted, at
`dria.105,115,116 Studies with isolated systems suggest
`that arsenic trioxide induces apoptosls by directly
`influencing the mitochondrial permeability transi-
`tion pore, which can be inhibited
`by Bci-Z, an
`endogenous antagonist of permeability transition
`pore funcnoo.!" Mitochondrial
`transmembrane
`potential collapse has been demonstrated in malig-
`nant
`lymphocytic cells undergoing arsenic trioxide-
`induced apoptosis.t'v Cells become more sensitive
`to arsenic trioxide when combined with inhibitors
`of glutathione
`(GSH) synthesis,
`including ascorbic
`acid.105,1l7 Additional data suggest that
`induction
`of mitotic arrest and apoptosis by arsenic trioxide is
`to
`related to the binding of this trivalent arsenical
`two cysteine residues in tubulin,
`thus blocking the
`guanosine
`triphosphate
`(GTP) binding site and
`leading to disruption
`of microtubule
`formation
`during mitosis. us
`Most clinical experience with arsenic trioxide
`comes from trials conducted
`in China
`and the
`United States
`in patients with refractory or re-
`lapsed APL,98.]OO,1I9.120In a study by Shen et al,99
`arsenic trioxide was administered intravenously at
`a fixed dose of 10 mg/d, either
`alone or
`in
`combination with other chemotherapy. Nine of 10
`patients treated with arsenic alone and all five of
`the remaining patients treated with a combination
`regimen achieved
`complete
`hematologic
`remis-
`sions. Niu et aP20 recently reported the results of
`arsenic trioxide
`treatment
`in 58 patients with
`APL. Clinical complete remission was obtained in
`eight of 11 patients (72.7%) with newly diagnosed
`(85.1%) with relapsed
`APL and 40 of 47 patients
`disease, In a pilot study conducted at MSKCC in
`the United Stares.F? arsenic trioxide was adminis-
`tered intravenously
`over 2 to 4 hours at doses
`ranging from 0,06 to 0.2 mg/kg/d; the initial course
`of treatment was continued until
`the bone marrow
`was morphologically clear of leukemia. The me-
`dian duration of induction
`therapy was 33 days
`(range, 12 to 39 days), with one to five courses
`administered to responding patients. A complete
`hematologic
`remission was achieved in 11 of the
`12 patients. The median duration of remission was
`(range, 1 to >9 months),
`5 months
`In eight
`patients,
`the bone marrow tested negative
`by
`reverse-transcription
`polymerase
`chain
`reaction
`(RT PCR)
`for PML/RAR-Cl after
`two courses of
`therapy. The three patients whose bone marrow
`remained RT-PCR-positive
`relapsed during the
`
`second course of treatment. None of the patients
`whose bone marrows were RT~PCR-negative
`re-
`lapsed at the time of the report. The results of this
`single-institution
`study are being confirmed in a
`multicenter
`trial. 1Zl A dose-finding phase I study of
`arsenic trioxide is being conducted by investigators
`at MSKCC to determine the recommended
`phase
`II dose in hematologic malignancies
`other
`than
`APL,12l
`to arsenic trioxide
`attributed
`toxicities
`Major
`involve a variety of organ systems. Skin changes
`are common,
`including
`dryness, hyperkeratosis,
`pruritus,
`rash, erythema,
`and hyperpigmentanon.
`Gastrointestinal
`toxicity includes nausea, vomit-
`ing, abdominal
`pain,
`anorexia,
`and stomatitis.
`Renal and hepatic toxicity and hyperglycemia are
`also observed. Patients can experience a variety of
`constitutional
`symptoms
`and muscular
`skeletal
`complaints,
`such as lassitude, fatigue, weight gain,
`arthralgia,
`bone pain, myalgia,
`toothache,
`and
`headache. Cardiovascular
`and neurologic complt-
`cations may be a particular
`concern in patients
`who receive more than a few courses of therapv.!'?
`Neurotoxicity includes peripheral motor or sensory
`neuropathy and seizures. Some patients experience
`lightheadedness. dizziness, and hypotension during
`the 1~ or 2-hour
`infusion, but
`this
`is usually
`alleviated by extending
`the infusion to 4 hours.
`Other
`cardiac
`effects
`that have been reported
`include second-degree heart block, prolongation of
`the QT interval, and torsades des pointes; patients
`exposed to amphotericin B, or those with hvpoka-
`lemia or hypomagnesemia for any reason, may be at
`higher risk for these cardiac abnonualities.Uv
`Of interest
`is that patients with APL treated
`with arsenic trioxide can develop fluid retention
`and a syndrome
`similar
`to the
`"retinoic
`acid
`syndrome/'122,123with fever, fluid retention, weight
`gain, dyspnea, pneumonitis, and leukocytosis. Signs
`or symptoms of the syndrome are usually mani-
`fested within 1 to