`WORLD HEALTH ORGANIZATION
`
`IARC MONOGRAPHS
`ON THE EVALUATION
`OF CARCINOGENIC
`RISKS TO HUMANS
`
`VOLUME 50
`
`PHARMACEUTICAL DRUGS
`
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`.ZEN
`
`WORLD HEALTH ORGANIZATION
`
`INTERNATIONAL AGENCY FOR RESEARCH ON CANCER
`
`IARC MONOGRAPHS
`ON THE
`|
`EVALUATION OF CARCINOGENIC
`RISKS TO HUMANS
`
`Pharmaceutical Drugs
`
`VOLUME50
`
`This publication represents the views and expert opinions
`of an IARC Working Groupon the
`Evaluation of Carcinogenic Risks to Humans,
`which met in Lyon,
`
`17-24 October 1989
`
`1990
`
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`
`IARC MONOGRAPHS
`
`In 1969, the International Agency for Research on Cancer (IARC)initiated a
`programme on the evaluation of the carcinogenic risk of chemicals to humans
`involving the production of critically evaluated monographs on individual
`chemicals. In 1980 and 1986, the programme was expandedto include the evalua-
`tion of the carcinogenic risks associated with exposures to complex mixtures and
`other agents.
`The objective of the programmeis to elaborate and publish in the form of
`monographscritical reviews of data on carcinogenicity for agents to which humans
`are knownto be exposed, and on specific exposuresituations; to evaluate these data
`in terms of humanrisk with the help of international working groups of experts in
`chemical carcinogenesis and related fields; and to indicate where additional
`research efforts are needed.
`This project is supported by PHS Grant No. 6 UO1 CA33193-06 awarded by the
`US National Cancer Institute, Department of Health and Human Services.
`Additional support has been provided since 1986 by the Commission of the
`European Communities.
`
`©International Agency for Research on Cancer 1990
`
`ISBN 92 832 1250 9
`
`ISSN 0250-9555
`
`Allrights reserved. Application for rights of reproduction ortranslation, in part
`or in toto, should be madeto the International Agency for Research on Cancer.
`
`Distributed for the International Agency for Research on Cancer
`by the Secretariat of the World Health Organization
`
`PRINTED IN THE UK
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`
`AZACITIDINE
`
`This substance was considered by a previous Working Group, in October 1980,
`underthetitle 5-azacytidine (LARC, 1981). Since that time, new data have become
`available, and these have been incorporated into the monograph and taken into
`consideration in the present evaluation.
`
`1. Chemical and Physical Data
`
`1.1
`
`Synonyms
`
`Chem. Abstr. Services Reg. No.: 320-67-2
`Chem. Abstr. Name: 1,3,5-Triazin-2(1H)-one, 4-amino-1-8-ribofuranosyl
`Synonyms: Antibiotic U 18496; 5-azacytidine; ladakamycin; NSC 102816;
`U-18496; WR-183027
`
`1.2
`
`Structural and molecular formulae and molecular weight
`
`NH
`
`A
`NMoaoO"I
`
`
`
`HOH,C oO
`H H
`
`H
`
`H
`
`OH OH
`
`CgHy2N4O5
`
`—
`
`Mol. wt: 244.2
`
`13
`
`Chemical and physical properties of the pure substance
`
`From Winkley and Robins (1970), unless otherwise specified
`(a) Description: White crystalline powder
`
`-47-
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`48
`
`IARC MONOGRAPHS VOLUME50
`
`(6) Melting-point: 235-237°C (decomposes)
`(c) Optical rotation:
`[o]?6 = +26.6°C (c = 1.00; in water)
`(d) Solubility: Soluble in warm water (40 mg/ml), cold water (14 mg/ml), 0.1N
`hydrochloric acid (28 mg/ml) and 0.1 N sodium hydroxide (43 mg/ml);
`soluble in 35% ethanol (14.2-15.0 mg/l), acetone (1 mg/ml), chloroform
`(1 mg/ml), hexane (1 mg/ml) and dimethyl sulfoxide (52.7 mg/ml) (von
`Hoff et al., 1975)
`(e) Spectrosocopy data: Ultraviolet, infrared and nuclear magnetic resonance
`spectra have been reported (Beisler, 1978).
`Stability. Very unstable in aqueous media, rapid degradation to complex
`products occurring within hoursofdissolution in intravenoussolutions at
`room temperature (Reynolds, 1989)
`
`(f)
`
`1.4 Technical products and impurities
`Trade name: Mylosar
`Azacitidineis available as a lyophilized powderin vials containing 100 mg of
`the compound with 100 mg mannitolfor reconstitution as injections of5 mg/ml (von
`Hoffet al., 1975).
`
`2. Production, Occurrence, Use and Analysis
`
`2.1 Production and occurrence
`(a) Production
`Azacitidine, a pyrimidineanalogue ofcytidine with a nitrogen substituted for a
`5-carbon, can be isolated from a culture of the bacterium Streptoverticillium
`ladakanus,but hasalso been preparedby synthetic methods. One reported method
`involved treatmentof the trimethylsilyl derivative of 4-amino-1,3,5-triazin-2-one
`with 2,3,5-tri-O-acetyl-D-ribofuranosyl bromide, followed by deacetylation to give
`azacitidine (Winkley & Robins, 1970).
`Azacitidine is synthesized in the Federal Republic of Germany (Chemical
`Information Services, 1989-90).
`(b) Occurrence
`Azacitidine is produced by the bacterium Streptoverticillium ladakanus
`(Winkley & Robins, 1970).
`
`2.2 Use
`Azacitidineis a cytostatic agent. It has been used mainly in the treatment of
`acute leukaemia, either as intravenous or
`intramuscular injections or as
`
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`AZACITIDINE
`
`49
`
`intravenousinfusionsat a daily level of 40-750 mg/m? (Weiss et al., 1972; Skoda,
`1975; von Hoffet al., 1975, 1976; von Hoff & Slavik, 1977; Wade, 1977; Glover et al.,
`1987; Reynolds, 1989).
`It is used alone, or in combination with vincristine,
`vinblastine, prednisone, cytarabine or amsacrine,at a daily dose of 50-150 mg/m2
`azacitidine.
`It has also been tested for use in the treatmentof a variety of solid
`tumours (Gloveret al., 1987).
`
`2.3 Analysis
`Azacitidine can be quantified in blood by microbiological assay (Pittillo &
`Woolley, 1969) and in plasma by high-performance liquid chromatography with
`ultraviolet detection (Rustum & Hoffman, 1987).
`
`3. Biological Data Relevant to the Evaluation of
`Carcinogenic Risk to Humans
`
`3.1 Carcinogenicity studies in animals
`
`Intraperitoneal injection
`(a)
`Mouse: In a screening assay based on the accelerated induction of leukaemia
`inastrain highly susceptible to developmentofthis neoplasm, 40 AKR female mice,
`two months of age, were given six intraperitonealinjections of azacitidine at 1.5
`mg/kg bw[purity unspecified] over 20 days, and, becauseoftoxicity, six injections of
`azacitidine at 0.8 mg/kg bw overthe following 30 days. Alhtreated micehad died of
`leukaemia by 60 days. A control groupof40 females survivedfree of disease for the
`observation time of 120 days (Vesely & Cihak, 1973).
`In a screening assay basedontheaccelerated induction of lung tumoursin a
`strain highly susceptible to developmentof this neoplasm,three groupsoften male
`and ten female A/He mice, six to eight weeks of age, received intraperitoneal
`injections of azacitidine [purity unspecified], in a vehicle composed of saline,
`polysorbate-80, carboxymethylcellulose and benzyl alcohol, three times a week for
`eight weeks (total doses, 33, 62 and 90 mg/kg bw (which was the maximum tolerated
`dose)). Control groups received 24 intraperitoneal injections of 0.1 mlvehicle or
`were untreated. All animals were killed 24 weeks after the first injection. The
`numbersof mice with lung tumours, calculated onthe basis of survivors of each sex,
`were 6/11 (54%), 5/15 (33%) and 8/19 (42%) in the groups receiving the high, mid
`andlow doses, respectively. The results for untreated and vehicle-treated groups
`were expressed only as per cent tumour incidence; thus, 22% (males) and 17%
`(females) of untreated controls and 26% (males) and 23% (females) of
`
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`IARC MONOGRAPHS VOLUME 50
`
`vehicle-treated controls developed lung tumours. The numberof lung tumours per
`mouse (counted grossly) in animals of each sex treated with the highest dose was
`0.73 + 0.22 (SE), which wassignificantly higher (p < 0.05) than that in untreated
`(males, 0.22 + 0.03; females, 0.17 + 0.02) or vehicle-treated (males, 0.25 + 0.05;
`females, 0.23 + 0.04) control mice. With lower doses, the increase in the numberof
`lung tumours per mouse was notstatistically significant (Stoner ef al., 1973).
`Groups of 35 male and 35 female B6C3F1 mice, 38 days of age, received
`intraperitoneal injections of azacitidine at 2.2 or 4.4 mg/kg bw (>99% pure) in
`buffered saline three times a week for 52 weeks. Groups of 15 male and 15 female
`mice wereuntreated or received the vehicle only. Surviving mice were killed at 81 or
`82 weeks. All high-dose females died before week 62, with nosignificant increase in
`the incidence of any tumour; of the low-dose females, 17/35 survived until
`terminationof the experiment. Amongmales,7/35 ofthe high-dose group and 13/35
`of the low-dose group survived to the end of the study. The overall numbers of
`survivors
`in untreated and vehicle-treated groups were 25/30 and 20/30,
`respectively. In female mice of the low-dose group, lymphocytic and granulocytic
`neoplasmsof the haematopoietic system were observedin 17/29 animals examined
`histologically, at a highly significant incidence (p < 0.001) compared with the
`vehicle-control group (0/14); 10 of the treated animals had granulocytic tumours
`(nine sarcomas, one leukaemia). A malignant lymphocytic lymphomawas observed
`in 1/15 untreated controls. Noincrease in the incidence of tumours was observedin
`male mice (National CancerInstitute, 1978).
`Groupsof50 male and 50 female BALB/c/Cb/Se mice, eight weeks of age, were
`given intraperitoneal injections of azacitidine at 2.0 mg/kg bw in saline (99% pure)
`once a week for 50 weeks. Control groups received injections of saline. After 25
`weeks, survival was reduced in exposed animals of each sex. The incidence of
`lymphoreticular neoplasmswasincreased, occurring in 12/50 (p < 0.01) males and
`36/50 (p < 0.001) females, compared to 3/50 and 6/50 in control males and females,
`respectively. The incidence of lung adenomaswasincreasedin treated males (27/50
`versus 12/50 [p < 0.01]) but not in females. Mammary gland adenocarcinomas and
`adenoacanthomas were foundin 7/50 treated females and in none ofthe controls.
`The incidence of skin tumours was increased in treated animals of each sex,
`occurring in 3/50 treated males comparedto 0/50 controls [p < 0.05] and in 7/50
`treated females comparedto 1/50 controls [p < 0.01, log ranktest] (Cavaliereet al.,
`1987). [The Working Group noted that adenocanthomas are not described as
`mammary tumoursin reference sources; see Turusov (1973, 1976).]
`Rat: Two groupsof 12 or 8 male Fischerrats, weighing 160-180 g, were given
`intraperitonealinjections of azacitidine at 2.5 or 10 mg/kg bw [purity unspecified] in
`saline twice a weekfor nine months. A control groupof 12 male rats was maintained
`without treatment. All rats were killed at 18 months.
`Interstitial-cell testicular
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`AZACITIDINE
`
`51
`
`tumours werefoundin 1/8 high-dose animals and 9/12 low-dose animals compared
`to 0/12 controls. In the high-dose group, two squamous-cell carcinomasof the skin
`and one skin appendage tumourat thesite of injection were found, compared to
`nonein controls (Carr et al., 1984). [The Working Group noted the small numberof
`animals tested, and the absence in controls of testicular tumours, which occurred
`commonly in a second, shorter study by the sameinvestigators (see below).]
`Groupsof 10, 10 or 100 young adult male Fischer rats, weighing 100-160 2,
`received intraperitoneal injections of azacitidine at 0.025, 0.25 or 2.5 mg/kg bw in
`saline [purity unspecified] three times a week for one year. A control group of 50
`rats was injected with saline. At one year, when the study was terminated, 87/100 of
`animals at the high dose and 10/10 in each of the lower-dose groupswerestill alive.
`The highest dose increased the incidence of testicular interstitial-cell tumours to
`56/87, comparedto 10/49 in controls (p < 0.001). No other tumour was observedin
`controls.
`In the highest dose group, other tumours noted were four lymphomas,
`four renal tumours, one lung tumour, three skin tumours, two mesotheliomas and
`two sarcomas (Carref al., 1988). [The Working Group noted the short duration of
`the experiment and the small numbers of animals in some groups.]
`(b) Transplacental administration
`Mouse: Groups of 32-37 pregnant NMRI mice received intraperitoneal
`injectionsof azacitidine at 1 or 2 mg/kg bwinsaline [purity unspecified] on day 12,
`14 or 16 of gestation. A group of 53 control dams wasinjected with saline. The
`numberofstillbirths was increased at the high dose; survival of offspring was
`decreased in all exposed groups.
`In exposed progeny, increased percentages of
`tumour-bearing animals andincreased incidencesof leukaemias and lymphomas,
`lung tumoursandliver tumours were seen in some groups(see Table 1). Some
`increases in the incidence ofsoft-tissue sarcomas were also seen (Schmahletal.,
`1985).
`
`(c) Administration in combination with other compounds
`Rat: In the experiment by Carr et al. (1984), described above, groups of 6-10
`male Fischer rats were given N-nitrosodiethylamine at 50 mg/kg bw 18 h after
`partial hepatectomy, alone or with azacitidine at 2.5 or 10 mg/kg bw by
`intraperitonealinjection. Liver tumours were found in 2/10 and 8/10 animals given
`the low andthe high dose ofazacitidine, respectively, but notin the group given the
`nitroso compoundalone.
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`52
`
`IARC MONOGRAPHS VOLUME 50
`
`Table 1. Incidences of tumours in the progeny of NMRI mice given azaciti-
`dine by intraperitoneal injection?
`
`Treatment
`Sex
`No. of
`Leukaemias
`Lung tumours
`_Liver tumours
`animals
`and lymphomas
`
`mg/kg bw day of
`gestation
`
`
`No.
`
`%
`
`No %
`
`No.
`
`%o
`
`9.1
`15
`18.2
`30
`49.1
`81
`165
`Males
`3.8
`6
`20.9
`33
`50.6
`80
`158
`Females
`9.7
`ll
`19.5
`22
`24.8
`28
`113
`Males
`8.2
`9
`20.0
`22
`23.6
`26
`110
`Females
`2 #867
`16.3
`29
`23.6
`42
`178
`Males
`14
`1
`
`
`
`
`
`
`Females 18.182171 26 15.2 31 11.7
`Males
`97
`9
`93
`46
`2
`14
`47.4
`11
`113
`Females
`101
`14
`13.9
`43
`42.6
`7
`69
`Males
`153
`97
`63.4
`81
`52.9
`14
`9.2
`Females
`160
`98
`613
`99
`619
`8
`5.0
`
`1
`
`2
`
`1
`
`12
`
`12
`
`16
`
`2
`
`11.4
`18
`49.3
`78
`42.4
`67
`158
`Males
`3.3
`5
`54.3
`82
`37.7
`57
`151
`Females
`
`
`
`
`Controls 28.7=57Males 293 84 195 14 48
`
`
`
`
`279 82Females 29.4
`
`
`53
`19.0
`11
`3.9
`
`16
`
`“From Schmahler al. (1985)
`
`3.2 Other relevant data
`
`(a) Experimental systems
`(i) Absorption, distribution, excretion and metabolism
`Bloodlevels of azacitidine, determined by biological activity, in mice peaked
`within 0.5 h after intraperitoneal or oral administration. Maximal concentrationsof
`azacitidine in blood after administration at 50 mg/kg bw were about2 jig/ml after
`oral administration and 43 jig/ml after intraperitoneal injection (Neil et al., 1975).
`In a study using a microbiological assay, maximal concentrations were found
`in blood 15 minafter intraperitoneal injection of 9.5 and 4.75 mg/kg bw (LD49 and
`0.5 LD49) to mice. Elimination wasrapid, and no azacitidine was detected in blood
`1h after injection of the high doseor 30 min after injection of the low dose. No drug
`was detected in liver, lung, brain, spleen or kidneys (Pittillo & Woolley, 1969).
`In a further study, 44C activity in blood diminished rapidly in mice after
`intraperitoneal administration of labelled azacitidine (Raska et al., 1965). The
`half-time for azacitidine and its radioactive metabolites was calculated by von Hoff
`and Slavic (1977) to be 3.8 h; radioactivity was retained in lymphatic organs.
`
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`AZACITIDINE
`
`53
`
`As reported in an abstract, 50% of a dose [amount and route unspecified]
`administered to mice was excreted in the urine within 8 h; of the excreted
`radioactive material, 4% wasassociated with unchangedazacitidine. Six additional
`radioactive metabolites were found (Colesetal, 1975). In beagle dogs, azacitidine,
`5-azacytosine, urea and guanidine were observedafter intravenous administration
`of azacitidine at 0.5 mg/kg bw; 33% of the administered dose was excretedin urine
`by 4 h (Coles et al., 1974).
`In rabbits, most of the radioactivity (25-40%) was
`excreted in the urine after intravenous administration of labelled azacitidine at 15
`mg/kg bw; only small amounts were excreted via the bile (Chanet al., 1977).
`Azacitidine is phosphorylated andinhibits uridine kinase and orotidylic acid
`hydroxylase (von Hoff et al., 1975, 1976).It is readily deaminated in biological
`systems to 5-azauridine, which is degraded further (Cihak, 1974; Neil et al., 1975;
`Glover & Leyland-Jones, 1987).
`(ii) Toxic effects
`Asreported in an abstract, the intraperitoneal LDs0 for azacitidine in mice
`was 116 mg/kg bw and the oral LDsg, 572 mg;five daily doses increasedthe toxicity
`considerably (Palm & Kensler, 1971).
`After phosphorylation, azacitidine is incorporated into DNA and RNAin
`11210 leukaemiacells in vitro (Li et al., 1970); it inhibits DNA synthesis in the liver of
`partially hepatectomized rats.
`Intraperitoneal
`injection of azacitidine at 10
`timol/100 g bw inhibited thymidine kinase and thymidylate kinase in rat liver
`(Cihak & Vesely, 1972).
`Azacitidineis cytotoxic to Friend erythroleukaemiacells (Hickeyet al., 1986),
`L1210 leukaemiacells (Lief al., 1970) and normalrat hepatocytes (Carretal., 1988)
`in vitro; after a dose of 1 x 10-+M, 32% survivalofrat hepatocytes was observed
`within 24 h.
`(iii) Hypomethylation and effects on gene expression
`After incorporation into DNA,azacitidine inhibits DNA methyl transferase
`noncompetitively, blocking cytosine methylationin newly replicated DNA. Since
`hypomethylation patterns in DNAarerelated to gene expression, this may be the
`mechanism by which azacytidine inducesa range of biological effects (Gloveretal.,
`1987). A numberof in-vitro and in-vivo studies have shown that azacitidine
`treatmentaffects both differentiation (Constantinideset al., 1978; Taylor & Jones,
`1979; Tsao et al., 1984; Csordas & Schauenstein, 1986; Liu et al., 1986; Sématef al.,
`1986; Rothrockefal., 1988) and gene expression (Tennantefa/., 1982; Harrison et al.,
`1983; Rothrocketal., 1983; Sugiyamaetal., 1983; del Sennoet al., 1984; Waalkes &
`Poirier, 1985; Castelazzi et al. 1986; Hickey et al., 1986; Hoshino et al. 1987:
`Ishikawaetal., 1987; Price-Haugheyet al., 1987; Carr et al., 1988; Stephanopoulosef
`al., 1988; Wagneretal., 1988).
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`IARC MONOGRAPHS VOLUME50
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`(iv) Effects on reproduction and prenatal toxicity
`Intraperitoneal administration of azacitidine at 1.5-2.5 mg/kg bw to mice for
`various periods during pregnancy induced very high or total resorption of
`conceptuses when treatment wasgiven in the preimplantation period up to day 6;
`after this time,the incidence ofresorptions was onlyslightly greater than the control
`level (Svata et al., 1966; Seifertovaet al., 1968). Other workers have shown that single
`intraperitoneal doses of 1-2 mg/kg to mice during the period of embryogenesis can
`cause a high resorption rate and malformationsin the majority of surviving fetuses,
`including major central nervous system defects, facial clefts and limb defects
`(Schmahlet al., 1984; Takeuchi & Takeuchi, 1985).
`Intraperitoneal injection of azacitidine at 1-4 mg/kg to mice atlater stages of
`pregnancy, especially on day 15, can result in morphological changesin the brain
`(Langman & Shimada, 1971), and behavioural changes canbe detected in offspring
`whentested as adults (Rodieret al., 1973; Langmanet al., 1975; Rodier, 1979).
`The primary mechanism by which azacitidine causes malformationsin rats is
`thoughtto be induction of cell death, but inhibition of somebut notall of the effects
`of azacitidine by administration of caffeine indicates that more than one
`mechanism maybe involved (Kurishita & Ihara, 1987a,b).
`(v) Genetic and related effects
`In Escherichia coli, azacitidine caused DNA damage (Bhagwat & Roberts,
`1987) and prophageinduction (Barbe et al., 1986). It was mutagenic to E.coli (Fucik
`et al., 1965; Lal et al., 1988) and induced base-pair but not frameshift mutations in
`Salmonella typhimurium (Marquardt & Marquardt, 1977; Podger, 1983; Call et al.,
`1986; Levin & Ames, 1986; Schmucket al., 1986).
`Azacitidine induced mitotic recombinations, mitotic gene conversions and
`reverse mutations but not mitotic chromosomeloss in Saccharomyces cerevisiae
`(Zimmermann & Scheel, 1984). It induced mitotic recombinations, deletions and
`gene mutations in the wing spot assay in Drosophila melanogaster (Katz, 1985) and
`chromosomalaberrations in root meristem cells of Vicia faba (Fuciket al., 1970).
`Azacitidine inhibited DNA synthesis in Chinese hamster CHOcells (Tobey,
`1972) and induced DNAstrandbreaks in HeLa cells (Snyder & Lachmann, 1989).
`It induced mutations at the Aprt locus in Chinese hamster V79 cells in one study (at
`5 1M; Marquardt & Marquardt, 1977) but not in another (at 40 4M; Landolph &
`Jones, 1982). It did not induce mutation at the hprt locus in Syrian hamster BHK
`cells (Boucket al., 1984), primary rat tracheal epithelial cells (Walker & Nettesheim,
`1986) or mouse lymphoma LS5178Y cells (at 4 4M; McGregor ef al., 1989).
`Azacitidine induced mutations at the hprt and tk loci in humanfibroblasts (Call et
`al., 1986) and at the tk locus of mouse lymphoma L5178Y cells (Amacher & Turner,
`1987; McGregoref al., 1989).
`It did not induce ouabain-resistant mutations in
`
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`AZACTTIDINE
`
`55
`
`mouse C3H 10T’4, Chinese hamster V79 (Landolph & Jones, 1982), Syrian hamster
`BHK (Boucket al., 1984) or primary rat tracheal epithelial cells (Walker &
`Nettesheim, 1986).
`Azacitidine induced sister chromatid exchange in a cloned hamstercell line
`(Banerjee & Benedict, 1979), in CHO cells (Hori, 1983) and in human peripheral
`lymphocytesin vitro [only one concentration, 8 1M, was tested] (Laviaet al, 1985).
`In anotherstudy, azacitidine did not induce sister chromatid exchange in human
`lymphocytes (up to 9 4M; Ioannidou ef al., 1989).
`It induced chromosomal
`aberrationsin Chinese hamster Doncells (Karon & Benedict, 1972) and in human
`peripheral lymphocytesin vitro [only one concentration, 8 1M, was tested] (Lavia et
`al., 1985) but not in human lymphoblasts (10 4M; Call et al, 1986).
`Azacitidine induced transformation in mouse C3H/10T% (Benedict ef al.,
`1977), Syrian hamster BHK (Boucket al., 1984), mouse BALB/3T3 (Yasutakeef al.,
`1987) and primary rat trachealepithelial cells (Walker & Nettesheim, 1986).
`Azacitidine did not induce dominant lethal mutation in male mice after
`administration at 5 and 10 mg/kg bw intraperitoneally (Epstein et al., 1972).
`(b) Humans
`The toxicity, cytostatic activity and mechanism ofaction ofazacitidine have
`been reviewed (Cihak, 1974; von Hoff & Slavik, 1977; Glover & Leyland-Jones,
`1987).
`
`Pharmacokinetics
`(i)
`the a-phase
`After an intravenous injection of radiolabelled azacitidine,
`half-time of radioactivity was 16-33 min (Israeli et al., 1976), and the B-phase
`half-time was 3.4-6.2 h (Troetelet al., 1972; Israeliet al, 1976). After 30 min,less than
`2%of the plasma radioactivity cochromatographedwith azacitidine; at least two
`different metabolites or decomposition products were detected by thin-layer
`chromatography(Israeli er al., 1976), and 73-98% ofthe injected radioactivity was
`detected in the urine within three days(Israeli et al., 1976). Similhrresuks=were
`obtained by Troetel et al. (1972).
`Less than 1% of radiolabelled azacitidine was bound to human serum albumin
`in vitro (Israeli et al., 1976).
`(ii) Adverse effects
`The major toxic effects of the clinical use of azacitidine have been
`gastrointestinal, haematological and hepatic (von Hoff et al., 1976; von Hoff &
`Slavik, 1977; Reynolds, 1989). Leukopeniais generally the dose-limiting toxicity; in
`a compilation of several studies with a total of 821 patients, the incidence of
`leukopenia (total
`leukocyte count,
`less than 1500/mm3) was 34% and was
`dose-related. Thrombocytopenia has been reportedless frequently (von Hoff et al.,
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`IARC MONOGRAPHS VOLUME 50
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`1976; von Hoff & Slavik, 1977). Fatal hepatic damage was reportedin four patients
`with previous hepatic dysfunction, who had been treated with azacitidine (Bellet et
`al., 1973).
`
`(iii) Effects on reproduction and prenatal toxicity
`No data were available to the Working Group.
`(iv) Genetic and related effects
`No adequate study wasavailable to the Working Group.
`
`3.3. Case reports and epidemiological studies of carcinogenicity to humans
`No data were available to the Working Group.
`
`4. Summary of Data Reported and Evaluation
`
`4.1 Exposure data
`
`Azacitidine is a cytostatic agent that has been used since the 1970s for the
`treatment of acute leukaemia.
`
`4.2 Experimental carcinogenicity data
`
`Azacitidine was tested for carcinogenicity by intraperitoneal injection in four
`studies in mice and in two studies in rats and by transplacental exposure in one
`study in mice. In one study in mice,it accelerated the developmentof leukaemias;in
`the two long-term studies andin the transplacentalstudy, it increased the incidence
`of lymphoid neoplasms.
`In one of the long-term studies, the incidence of lung
`adenomaswas increased in male mice and that of skin tumoursin mice ofeach sex.
`In the transplacental study in mice,it also increased the incidences of lung andliver
`tumours. It accelerated the induction of lung tumoursin mice. In rats, it increased
`the incidence of testicular tumours.
`to rats enhanced the
`Intraperitoneal administration of azacitidine
`developmentofliver tumours induced by N-nitrosodiethylamine.
`
`4.3 Humancarcinogenicity data
`
`No data were available to the Working Group.
`
`4.4 Other relevant data
`
`During the early stages of gestation, azacitidine induces embryomortality in
`mice; during the organogenesis period, multiple, gross structural malformations
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`AZACITIDINE
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`37
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`can be induced; and duringlater stagesof gestation, mainly central nervous system
`defects have been induced in mice.
`Itis
`Azacitidine is readily deaminated to azauridine and further degraded.
`incorporated into DNA and alters gene expression.
`In humans,
`it causes
`leukopenia.
`Azacitidine causes hypomethylation of DNA both in vivo and in vitro.
`In one study, azacitidine did not induce dominantlethal mutations in mice.
`Contradictory results have been reported with respect
`to the induction of
`chromosomalaberrationsandsister chromatid exchange in humancells. In single
`studies, azacitidine induced gene mutations and DNAstrand breaks in human
`cells.
`It induced chromosomal aberrations in Chinese hamster cells, sister
`chromatid exchange in cloned Chinese hamstercells, gene mutations in Chinese
`hamster and mouse lymphomacells and transformation in various cell lines.
`It
`induced mitotic recombination and mutations in Drosophila. Azacitidine induced
`chromosomal aberrations in Vicia faba.
`In Saccharomycescerevisiae, it induced
`gene mutations and mitotic recombination but not chromosomalloss. It induced
`mutations and DNA damagein Salmonella typhimurium and Escherichia coli. (See
`Appendix 1.)
`
`4.5 Evaluation!
`
`the carcinogenicity of azacitidine in
`
`sufficient evidence for
`There is
`experimental animals.
`No data were available from studies in humans on the carcinogenicity of
`azacitidine.
`In making the overall evaluation, the Working Group also took note of the
`following information. Azacitidine is active in a broad spectrum of assays for
`genetic and related effects,
`including those involving mammalian cells.
`Furthermore, azacitidine, a pyrimidine analogue,
`is incorporated into DNA,
`causing hypomethylation.
`
`Overall evaluation
`
`Azacitidine is probably carcinogenic to humans (Group 2A).
`
`‘For description oftheitalicized terms, see Preamble, pp. 26-29.
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`IARC MONOGRAPHS VOLUME 50
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`5. References
`
`Amacher, D.E. & Turner, G.N.(1987) The mutagenicity of 5-azacytidine and otherinhibitors
`of replicative DNA synthesis in the L5178Y mouse lymphoma cell. Mutat. Res., 176,
`123-131
`Banerjee, A. & Benedict, W.F. (1979) Production of sister chromatid exchangesby various
`cancer chemotherapeutic agents. Cancer Res., 39, 797-799
`Barbe,J., Gilbert, I. & Guerrero, R. (1986) 5S-Azacytidine: survival and induction of the SOS
`response in Escherichia coli K12. Mutat. Res., 166, 9-16
`Beisler,J. (1978) Isolation, characterization, and propertiesof a labile hydrolysis product of
`the antitumor nucleoside 5-azacytidine. J med. Chem., 21, 204-208
`Bellet, R.E., Mastrangelo, M.J., Engstrom, PF. & Custer, R.P. (1973) Hepatotoxicity of
`5-azacytidine (NSC-102816)(A clinical and pathologic study). Neoplasma, 20, 303-309
`Benedict, W.F., Banerjee, A., Gardner, A. & Jones, P.A. (1977) Induction of morphological
`transformation in mouse C3H/10T% clone8 cells and chromosomal damagein hamster
`A(T)C1-3 cells by cancer chemotherapeutic agents. Cancer Res., 37, 2202-2208
`Bhagwat, A.S. & Roberts, R.J. (1987) Genetic analysis of the S-azacytidine sensitivity of
`Escherichia coli K-12. J. Bacteriol., 169, 1537-1546
`Bouck, N., Kokinakis, D. & Ostrowsky, J. (1984) Induction of a step in carcinogenesis thatis
`normally associated with mutagenesis by nonmutagenic concentrations of
`5-azacytidine. Mol. Cell Biol., 4, 1231-1237
`Call, K.M., Jensen, J.C., Liber, H.L. & Thilly, W.G. (1986) Studies of mutagenicity and
`clastogenicity of 5-azacytidine in human lymphoblasts and Salmonella typhimurium.
`Mutat. Res., 160, 249-257
`Carr, B.L, Reilly, J.G., Smith, S.S., Winberg, C. & Riggs, A. (1984) The tumorigenicity of
`5-azacytidine in the male Fischer rat. Carcinogenesis, 5, 1583-1590
`Carr, B.L., Rahbar, S., Asmeron, Y., Riggs, A. & Winberg, C.D. (1988) Carcinogenicity and
`haemoglobin synthesis induction by cytidine analogues. Br. J Cancer, 57, 395
`Castellazzi, M., Vielh, P. & Longacre, S. (1986) Azacytidine-induced reactivation of
`adenosine deaminase in a murine cytotoxic T cell line. Eur. J. Immunol., 16, 1081-1086
`Cavaliere, A., Bufalari, A. & Vitali, R. (1987) 5-Azacytidine carcinogenesis in Balb/c mice.
`Cancer Lett., 37, 51-58
`Chan, K.K., Staroscik, J.A. & Sadée, W. (1977) Synthesis of S-azacytidine-6-C and16-14¢, J
`
`med. Chem., 20, 598-600
`Chemical Information Services (1989-90) Directory ofWorld Chemical Producers, Oceanside,
`NY
`Cihak, A. (1974) Biological effects of 5-azacytidine in eukaryotes. A review. Oncology, 30,
`405-422
`Cihak, A. & Vesely, J. (1972) Prolongation of the lag period preceding the enhancementof
`thymidine and thymidylate kinase activity in regenerating rat liver by 5-azacytidine.
`Biochem. Pharmacol., 21, 3257-3265
`
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`AZACITIDINE
`
`59
`
`Coles, E., Thayer, P.S., Reinhold, V. & Gaudio, L. (1974) Pharmacokinetics of excretion of
`5-azacytidine (NSC 102816) and its metabolites (Abstract No. 286). Proc. Am. Assoc.
`Cancer Res., 15, 72
`Coles, E., Wodinsky, I. & Gaudio, L. (1975) The effects of drug combinations on the
`metabolism and excretion of S-azacytidine (5-Aza Sr, NSC 102816) in BDFmice
`(Abstract No. 362). Proc. Am. Assoc. Cancer Res., 16, 91
`Constantinides, P.G., Taylor, S.M. & Jones, P.A. (1978) Phenotypic conversion of cultured
`mouse embryocells by aza pyrimidine nucleosides. Dev. Biol., 66, 57-71
`Csordas, A. & Schauenstein, K. (1986) Thymus involution induced by 5-azacytidine. Biosci.
`Rep., 6, 603-612
`Epstein, S.S., Arnold, E., Andrea, J., Bass, W. & Bishop, Y. (1972) Detection of chemical
`mutagens by the dominantlethal assay in the mouse. Toxicol. appl. Pharmacol., 23,
`288-325
`Fucik, V., Zadrazil, S., Sormova, Z. & Sorm,F. (1965) Mutageniceffects of 5-azacytidine in
`