10.1177/0091270004271947ARTICLE
`
`MARCUCCI ET ALBIOAVAILABILITY OF AZACITIDINE SUBCUTANEOUS VERSUS INTRAVENOUSCLINICAL STUDIES
`
`
`
`
`
`CLINICAL STUDIES
`
`Bioavailability of Azacitidine Subcutaneous
`Versus Intravenous in Patients With the
`Myelodysplastic Syndromes
`
`Guido Marcucci, MD, Lewis Silverman, MD, Mark Eller, PhD,
`Linda Lintz, BA, and C. L. Beach, PharmD
`
`The primary objectives of this study were to characterize the
`absolute bioavailability of azacitidine after subcutaneous
`(SC) administration and to compare the single-dose
`pharmacokinetics of azacitidine following SC and intrave-
`nous (IV) administration. Six patients with myelodysplastic
`syndromes were randomly assigned according to a crossover
`design to treatment A, consisting of azacitidine administered
`as a single 75-mg/m2 SC dose, or treatment B, consisting of
`azacitidine administered as a single 75-mg/m2 IV infusion
`dose over 10 minutes. A minimum of 7 days and a maximum
`of 28 days were permitted between treatments. The study
`demonstrated good bioavailability of a SC azacitidine dose
`
`compared to an IV infusion treatment. The exposure profiles
`following SC drug administration illustrate measurable
`azacitidine levels with bioavailability (AUC) values within
`89% of those measured following IV administration (range,
`70%-112%). The median IV half-life was 0.36 ± 0.02 hours
`compared to 0.69 ± 0.14 hours for SC administration. Regard-
`less of the route of administration, a single dose of
`azacitidine, 75 mg/m2, was generally well tolerated.
`
`Keywords: Azacitidine; bioavailability; pharmacokinetics;
`myelodysplastic syndromes; epigenetics
`Journal of Clinical Pharmacology, 2005;45:597-602
`©2005 the American College of Clinical Pharmacology
`
`Azacitidine, a ring analog of the pyrimidine
`
`nucleoside cytidine, has effects on cell differentia-
`tion, gene expression, and deoxyribonucleic acid
`(DNA) synthesis and metabolism and, through inhibi-
`tion of DNA methyltransferase, induces reexpression
`of silenced genes in cancer cells.1 Since the early 1970s,
`azacitidine has been investigated in the United States
`for the treatment of acute leukemia in both adults and
`children, and initial clinical trials in adults demon-
`strated the activity of this drug primarily in chemo-
`therapy refractory patients with acute myelogenous
`leukemia (AML).2 Subsequently, azacitidine was also
`evaluated in other types of neoplastic disease and be-
`
`From Ohio State University, Columbus, Ohio (Dr Marcucci); The Mount
`Sinai Medical Center, New York (Dr Silverman); Quintiles Inc, Kansas City,
`Missouri (Dr Eller); and Pharmion Corporation, Overland Park, Kansas
`(Ms Lintz, Dr Beach). Submitted for publication June 30, 2004; revised ver-
`sion accepted October 7, 2004. Address for reprints: Guido Marcucci,
`MD, Ohio State University Medical Center, 4th Floor Starling Loving Hall,
`320 West 10th Avenue, Columbus, OH 43210.
`DOI: 10.1177/0091270004271947
`
`nign hematologic disorders, including solid tumors,
`myelodysplastic syndromes (MDS), and hemoglobin-
`opathies (eg, thalassemia and sickle cell anemia).2
`Treatment options for MDS have been largely inef-
`fective because allogeneic stem cell transplantation is
`the only curative therapy and a viable option for only
`5% of patients. Most patients receive supportive care
`alone for palliation, which does not alter the natural
`course of the disease. MDS ultimately progresses to
`death, either due to bone marrow failure or transforma-
`tion to AML. The rationale for using azacitidine to treat
`MDS is based on the ability of azacitidine to reverse
`epigenetic gene silencing and its effects as a hypo-
`methylating agent, which induces cell differentiation
`in vitro. Two phase II studies demonstrated that
`azacitidine could improve hematopoiesis, which man-
`ifests as increases in peripheral blood counts, de-
`creases in transfusion requirements, and risk of hemor-
`rhage and infection.3 These effects on hematopoiesis
`were confirmed in a phase III randomized study (Can-
`cer and Leukemia Group B [CALGB 9221]), in which
`azacitidine administered subcutaneously (SC), 75 mg/
`m2, daily for 7 days every 28 days was shown to be sig-
`
`J Clin Pharmacol 2005;45:597-602
`
`597
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`MARCUCCI ET AL
`
`nificantly superior to best supportive care and ap-
`peared to alter the natural course of MDS.4,5 The risk of
`transformation to acute leukemia was also markedly
`reduced in this study.
`The maximum tolerated dose (MTD) of azacitidine
`following SC administration has not been determined,
`although SC doses as high as 100 mg/m2 were adminis-
`tered in the CALGB MDS studies. In contrast, the MTD
`for intravenous (IV) administration of azacitidine was
`reported to be in the range of 150 to 200 mg/m2/d when
`given daily for 5 days every 14 to 21 days in children
`aged 2 to 17 with AML.6 The MTD IV dose was reported
`to be as high as 500 mg/m2 when given once weekly or
`150 mg/m2 when given twice weekly to patients with
`solid tumors.7,8 Pharmacokinetic data were not col-
`lected in these studies, likely due to a lack of reliable
`analytical methods.
`The pharmacokinetics and drug disposition of
`azacitidine in humans were initially evaluated using a
`radiolabeled compound, but the azacitidine and drug
`metabolites could not be distinguished.9,10 More re-
`cently, however, a sensitive and selective high-
`performance liquid chromatography with mass spec-
`trometric detection (LC-MS/MS) method has been
`successfully developed and validated to quantify
`plasma concentration of azacitidine.11
`The aim of the present study was to characterize the
`absolute bioavailability of azacitidine after subcutane-
`ous administration and to compare the single-dose
`pharmacokinetics of azacitidine given SC with IV ad-
`ministration. Using LC-MS/MS, we analyzed plasma
`samples collected from MDS patients treated with a sin-
`gle dose of azacitidine 75 mg/m2 given SC or 75 mg/m2
`given IV over a period of 10 minutes. Both the SC
`and IV doses used in this study were based on previ-
`ously published literature. The SC dose of azacitidine
`(75 mg/m2) was the same as that used in CALGB studies
`89213 and 9221.4 The IV dose was based on a study by
`Israili et al,10 in which azacitidine was administered at
`doses of 150 to 250 mg/m2 as an 8- to 10-minute intra-
`venous bolus to 5 subjects.
`
`METHODS
`
`Study Design
`
`This study was a multicenter, randomized, open-label,
`2-treatment, 2-period, complete crossover design. Up
`to 10 male or female patients with MDS (any of the 5
`French-American-British [FAB] classification sub-
`types) were to be enrolled to achieve the target of 6
`completed patients. Patients ≥40 years, with a life ex-
`
`598 • J Clin Pharmacol 2005;45:597-602
`
`pectancy >3 months, an Eastern Cooperative Oncology
`Group (ECOG) performance status of 0-2, and normal
`hepatic and renal function who signed a study-specific
`informed consent were eligible. Patients who were
`pregnant, had a history of severe cardiac or pulmonary
`disease, or received radiation therapy, chemotherapy,
`or other investigational drugs within the previous 30
`days were excluded from the study. Concomitant med-
`ications considered strong microsomal enzyme induc-
`ers (eg, omeprazole, phenobarbital, rifampin, dexa-
`methasone) or inhibitors (eg, selective serotonin
`reuptake inhibitors, macrolide antibiotics, fluoro-
`quinolones, codeine) were to be avoided. Following
`screening procedures and baseline assessments (per-
`formed within 22 days prior to administration of study
`drug), the patients were randomly assigned to treat-
`ment A, consisting of azacitidine administered as a sin-
`gle 75-mg/m2 SC dose, or treatment B, consisting of a
`single-75 mg/m2 IV infusion dose administered over 10
`minutes. The protocol allowed a minimum of 7 days
`and maximum of 28 days between treatments.
`All patients were enrolled at 2 US investigational
`sites, Ohio State University and Mount Sinai Medical
`Center. The Western Investigational Review Board and
`Mount Sinai Medical Center Investigational Review
`Board reviewed and approved the study protocol for
`the 2 investigative sites.
`
`Drug Administration
`
`The study drug, prepared and packaged by Ash Steven
`(Riverview, Mich), was supplied as 100-mg azacitidine
`powder vials. To obtain azacitidine suspension for SC
`administration, the powder vials were reconstituted
`with 4 mL of sterile water for injection, to yield a final
`concentration of 25 mg/mL. Subcutaneous doses up to
`100 mg (4 mL) were administered in a single injection.
`For doses greater than 100 mg, the dose was equally di-
`vided between 2 syringes for SC injection administered
`at the same time in 2 different body regions. The SC in-
`jection was to be administered within 45 minutes fol-
`lowing preparation of the azacitidine suspension. To
`prepare the IV solution, 4 mL of this azacitidine sus-
`pension was diluted up to 50 mL with Lactated
`Ringer’s solution. The azacitidine dose was infused at a
`rate of 5 mL/min, followed by a 10-mL saline infusion.
`The IV infusion was to be administered over 10
`minutes.
`All patients received single doses of azacitidine 75
`mg/m2 as a SC injection (treatment A) or IV infusion
`(treatment B) no later than 10 AM on day 1 of each study
`period.
`
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`BIOAVAILABILITY OF AZACITIDINE SUBCUTANEOUS VERSUS INTRAVENOUS
`
`Blood Sampling
`
`Plasma concentrations of azacitidine were measured in
`blood samples collected through either an indwelling
`catheter or by peripheral venipuncture in tubes con-
`taining potassium EDTA. To minimize aqueous degra-
`dation of azacitidine during sampling, the collected
`blood samples were immediately processed or stored
`in an ice bath following the blood draw and processed
`within 30 minutes. Azacitidine is stable up to 4 days
`when extracted from plasma directly into acetonitrile
`that contains zinc sulfate. Stability is further enhanced
`by evaporating the acetonitrile and storing azacitidine
`at –70°C as a dried residue.
`For SC dosing, samples were collected at 30 minutes
`and 1, 2, 4, 8, 12, 24, and 48 hours following the SC
`dose. For IV dosing, samples were collected at 5, 11,
`and 30 minutes and 1, 2, 4, 8, 12, 24, and 48 hours fol-
`lowing the beginning of the IV infusion. Because sam-
`ples collected after 12 hours of dosing were below the
`quantifiable limit (BQL) in the first 2 patients, the pro-
`tocol was then amended to exclude samples drawn at
`12, 24, or 48 hours postdosing in subsequent patients.
`Once collected, the samples were vortexed, centri-
`fuged, and stored at –70°C until shipment (within 2
`days).
`
`Analytical Method
`
`A validated LC-MS/MS method was used for the deter-
`mination of azacitidine in human plasma. The instabil-
`ity of azacitidine dictated that a portion of the sample
`processing occur at the sites. Prelabeled, capped poly-
`propylene tubes were sent to each site in preparation
`for sample collection. Each tube contained 2 mL of
`acetonitrile and approximately 200 mg of zinc sulfate
`to stabilize azacitidine in plasma. Following vortexing
`and centrifugation of each blood collection tube
`(Vacutainer), a 100-mL aliquot of plasma was trans-
`ferred from each blood collection tube to the appropri-
`ate prelabeled sample collection tube. All tubes were
`vortexed, centrifuged, and stored at –70°C until ship-
`ment (within 2 days). Upon arrival at the bioanalyti-
`cal laboratory, internal standard (uracil beta-D-
`arabinofuranoside [Ara U]) was added to each tube,
`which was subsequently vortexed and centrifuged.
`The organic layer (acetonitrile), containing the ex-
`tracted azacitidine, was transferred to a second appro-
`priately labeled polypropylene test tube and evapo-
`rated to dryness. Sample tubes were stored at –70°C
`until analysis by LC-MS/MS with electrospray ioniza-
`tion. This assay was validated over a range of 10.0 to
`
`600 ng/mL. The in-process accuracy acceptance crite-
`ria for quality control samples were set to 20% of the
`theoretical value. Accuracy, as expressed by mean per-
`cent recovery relative to theoretical concentrations of
`calibration standards, was 95% to 105%, and preci-
`sion, expressed as percent coefficient of variation, was
`4% to 7%.
`
`Pharmacokinetic Analysis
`
`Pharmacokinetic (PK) parameters were calculated us-
`ing noncompartmental techniques with WinNonlin
`Professional Version 4.01 (Pharsight Corporation,
`Mountain View, Calif). Graphics were prepared with
`SAS Version 8.2 or SigmaPlot 7.101 (SPSS, Chicago,
`Ill).
`Pharmacokinetic parameters calculated from
`plasma concentrations included Cmax, tmax, AUC0-∞,
`AUC0-z, AUCext, λz, t1/2, and absolute bioavailability (F).
`Apparent plasma clearance (CLsc) was calculated fol-
`lowing the SC treatment only; systemic plasma clear-
`ance (CL) and volume of distribution (Vd) were calcu-
`lated following the IV treatment only.
`
`Statistical Analysis
`
`Descriptive statistics were presented by treatment for
`the concentration data at each scheduled sample col-
`lection time point and for the derived pharmacokinetic
`parameters. To assess the absolute bioavailability of
`azacitidine, the 75-mg/m2 SC dose (treatment A) was
`compared to the 75-mg/m2 IV infusion dose (treatment
`B), with treatment B as reference. The comparisons
`were evaluated with an analysis of the natural log-
`transformed data. An analysis of variance (ANOVA)
`with terms for sequence, period, and treatment as fixed
`effects and subject nested within sequence as a random
`effect was performed for AUC0-z, AUC0-∞, and Cmax, from
`which a 90% confidence interval for the ratio of
`treatment means was obtained.
`
`RESULTS
`
`Patients
`
`Of a total of 8 patients who were enrolled in this study,
`6 met entry criteria and were randomized to receive ei-
`ther treatment A or B, followed by a crossover to the
`other treatment not previously received. The 6 patients
`(3 men and 3 postmenopausal or surgically sterile
`women) were Caucasian and nonsmokers, 57 to 83
`years of age, and had a mean body weight of 76.8 ± 14.4
`
`CLINICAL STUDIES
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`MARCUCCI ET AL
`
`Table I Summary of Arithmetic Mean ± SD Pharmacokinetic Parameters Following Subcutaneous (SC)
`and Intravenous (IV) Dose Administration (n = 6)
`
`Route
`
`SC
`IV
`
`t1/2, h
`AUC0-z, ng(cid:127)h/mL
`AUC0-∞, ng•h/mL
`Cmax, ng/mL
`0.69b ± 0.14 167.48 ± 48.69
`923.88 ± 473.61
`960.53 ± 458.06
`750.0a ± 403.3
`76076.45 ± 25515.25
`0.36c ± 0.02 146.70 ± 46.91
`1025.11 ± 298.06
`1044.26 ± 285.67
`2750.0 ± 1069.0
`Apparent clearance (CLsc) after SC dosing and systemic clearance (CL) following IV infusion; Vd following IV infusion only; AUC0-8, area under the plasma concentra-
`tion time 0 to infinity, calculated by log-linear trapezoidal summation and extrapolated to infinity by addition of the last quantifiable plasma concentration divided by the elimination rate
`constant λz; AUC0-z, area under the plasma concentration time curve from time 0 to the last measurable time point, calculated by the log-linear trapezoidal
`summation.
`a. Approximately 3 µM.
`b. Approximately 41 minutes.
`c. Approximately 22 minutes.
`
`CL, L/h
`
`Vd, mL
`
`mean plasma azacitidine concentrations with time are
`shown in Figure 1, and the PK results are summarized
`in Table I. Geometric mean peak plasma azacitidine
`concentration following SC administration was 687.30
`ng/mL (approximately 3 µM). Mean IV half-life was
`0.36 ± 0.02 hours (approximately 22 ± 1 minutes),
`whereas SC half-life was 0.69 ± 0.14 hours (approxi-
`mately 41 ± 8 minutes). Apparent mean SC clearance
`(167.48 ± 48.69 L/h) was slightly higher than systemic
`(IV) mean clearance (146.70 ± 46.91 L/h). Volume of
`distribution following IV dosing was 76 ± 26 L. Sys-
`temic clearance exceeded the glomerular filtration rate
`and total renal blood flow, indicating that nonrenal
`elimination (eg, metabolism/hydrolysis/degradation)
`played a role in the elimination of parent drug.
`Subcutaneous administration of azacitidine showed
`good bioavailability compared to IV administration.
`The average of the individual bioavailability estimates,
`based on ratios of AUC0-∞ and AUC0-z arithmetic means,
`was 92% and 90%, respectively, with individual val-
`ues ranging from 60% to 128% for AUC0-∞ and from
`52% to 128% for AUC0-z. Based on the SC/IV ratio of
`the geometric least squares (LS) means, the SC bio-
`availability was 89% and 86% using the pharmaco-
`kinetic parameters AUC0-∞ and AUC0-z, respectively.
`The geometric LS mean ratio for Cmax was 27%. The dif-
`ferences in Cmax are consistent with higher maxi-
`mum exposure expected following IV versus extra vas-
`cular drug administration. Absolute bioavailability of
`89% was calculated based on the ratio of the AUC0-∞
`SC geometric LS mean to the AUC0-∞ IV geometric LS
`mean with a 90% confidence interval of 70% to 112%
`(Table II).
`
`Adverse Events
`
`Overall, 5 of the 6 patients participating in the study ex-
`perienced at least 1 adverse event following SC or IV
`administration. Adverse events were experienced in 5
`patients following SC administration and 4 patients
`
`IV/(B) 75 mg/m2 (N = 6)
`SC/(A) 75 mg/m2 (N = 6)
`
`1
`
`2
`
`3
`
`4
`
`5
`
`Time (h)
`
`4000
`
`3000
`
`2000
`
`1000
`
`Plasma Concentration (ng/mL)
`
`0
`
`0
`
`Figure 1. Mean azacitidine concentration-time profile for subcuta-
`neous (SC) and intravenous (IV) treatments.
`
`kg with a mean body surface area of 1.9 ± 0.2 m2 (range,
`1.68-2.20 m2). According to the FAB classification at
`screening, 2 patients had refractory anemia (RA), and 4
`patients had refractory anemia with excess of blasts
`(RAEB).
`
`Pharmacokinetics
`
`Following SC administration of azacitidine, maximum
`plasma concentrations were observed at 0.5 hours in
`all 6 patients. Following IV infusion, Cmax was observed
`at the end of saline flush (11 minutes) in 4 of the 6 pa-
`tients and 5 minutes into the infusion in 2 of the 6 pa-
`tients. Mean maximum azacitidine plasma concentra-
`tions following IV infusion were approximately 4-fold
`higher than those observed following SC drug admin-
`istration. AUC0-∞ values were well characterized by
`AUC0-z, with an average of 2.3% and 4.8% of the AUC0-∞
`extrapolated following IV and SC drug administration,
`respectively. For each route of administration, the
`
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`BIOAVAILABILITY OF AZACITIDINE SUBCUTANEOUS VERSUS INTRAVENOUS
`
`Table II Comparison of Subcutaneous (SC) and
`Intravenous (IV) Azacitidine Dosing by
`Analysis of Variance
`
`Parameter
`(Unit)
`
`AUC0-∞,
`ng(cid:127)h/mL
`
`AUC0-z,
`ng(cid:127)h/mL
`
`Cmax,
`ng/mL
`
`Geometric
`Least
`Squares
`Mean
`
`Ratio of
`Least
`Squares
`Means, %
`
`90%
`Confidence
`Intervals,
`%
`
`Treatment
`
`SC
`IV
`
`SC
`IV
`
`SC
`IV
`
`896.37
`1011.22
`
`852.76
`987.28
`
`687.30
`2580.32
`
`88.6
`
`70.2-111.9
`
`86.4
`
`64.1-116.4
`
`26.6
`
`19.2-36.9
`
`following IV administration. The most frequently re-
`ported adverse events were nausea (3/6) and vomiting
`(2/6), both observed with IV and SC administration; in-
`jection site bruising (2/6), SC administration only; and
`arthralgia (2/6), IV administration only. Most adverse
`events reported were grade 1 or 2 in intensity; grade 3
`adverse events of hypertension (assessed as possibly
`related by the investigator) and worsening of low he-
`moglobin, which required transfusion (assessed as not
`related to study drug by the investigator), were re-
`ported in 1 patient each. There were no deaths, serious
`adverse events, or withdrawals from the study due to
`adverse events.
`In general, there were no trends or clinically mean-
`ingful changes in laboratory parameters or vital sign
`values from baseline to endpoint. There were no clini-
`cally relevant changes in electrocardiogram findings or
`in physical examination findings.
`
`DISCUSSION
`
`The primary objectives of this study were to charac-
`terize the absolute bioavailability of azacitidine after
`subcutaneous administration and to compare the phar-
`macokinetics of single-dose azacitidine given subcu-
`taneously with those of single-dose azacitidine given
`intravenously. Azacitidine appears to be a high-
`clearance, short half-life drug with good absolute bio-
`availability after SC administration (86%-89%, based
`on geometric LS mean ratios of AUC). Individual arith-
`metic bioavailability (F) values varied from patient to
`patient, ranging from 60% to 128% for AUC0-∞ and from
`
`52% to 128% for AUC0-z. These findings may be due to
`the inherent variability in the disposition of azaciti-
`dine, the bioanalytical measures, or the study design.
`The instability of azacitidine has been well docu-
`mented.12 Therefore, substantial care was given with
`regard to sample handling and analysis. The instability
`of azacitidine necessitated that sample processing was
`performed in part at the sites. Personnel at each site
`were trained and required to pass a sample processing
`quality control test prior to processing samples. Never-
`theless, it cannot be excluded that some variability in
`the data might have been introduced by personnel-
`dependent variations in sample processing. At least 3
`quantifiable samples were obtained for each patient
`following SC dosing, and at least 4 quantifiable sam-
`ples were obtained for each patient following IV dos-
`ing. For both treatments, distribution and elimination
`phases were defined by at least 2 data points. Despite
`variability of the pharmacokinetic parameter and
`bioavailability data, our study demonstrated measur-
`able azacitidine levels following SC dosing, with bio-
`availability (AUC) values within approximately 90%
`of those measured following IV administration (range,
`52%-128%).
`Geometric mean SC and IV Cmax concentrations were
`687 ng/mL and 2580 ng/mL, respectively, which corre-
`sponds to 3 µM (SC) and 11 µM (IV). Half-life differ-
`ences were noted between the 2 routes of adminis-
`tration. Subcutaneous half-lives (0.69 hours) were
`approximately 2-fold greater than IV half-lives (0.36
`hours). Increased SC half-life of azacitidine might be
`due to the lack of adequately defining the distribution
`and elimination phases for both formulations. How-
`ever, it is possible that following SC dosing, additional
`transient time was required for azacitidine to move
`from the SC compartment into the circulatory system.
`Another explanation is that the azacitidine remained
`both stable and bioavailable at the SC depot site until
`entering the plasma compartment.
`The apparent (SC) clearance (167.48 L/h or 2791
`mL/min) and systemic (IV) clearance (146.70 L/h) of
`azacitidine far exceeded the glomerular filtration rate
`(approximately 125 mL/min) and total renal blood
`flow (1200 mL/min) in healthy subjects.13 This suggests
`that nonrenal elimination (eg, metabolism, hydrolysis,
`and/or degradation) plays a role in the elimination of
`parent azacitidine. The role of metabolism in the dis-
`position of azacitidine is potentially quite significant,
`as can be derived by comparing our results with those
`obtained in prior bioavailability studies measuring 14C.
`The half-life calculated based on 14C radioactivity
`appeared longer (3.4-6.2 hours) than that calculated for
`
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`

`MARCUCCI ET AL
`
`both IV and SC administration in our study.7,8 Because
`14C radioactivity, rather than parent drug concentra-
`tion, was measured in the previous studies, the longer
`half-life following 14C azacitidine administration is
`likely due to the presence of circulating metabolites.
`This can also be inferred from a comparison of 14C Cmax
`and 14C AUC values from previous studies with those
`obtained in our study from a specific assay for parent
`drug. After correcting for differences in dose, peak 14C
`plasma concentrations and AUC values after SC ad-
`ministration were approximately 20- and 150-fold
`higher, respectively, than the corresponding values for
`azacitidine determined in our study.
`These pharmacokinetic results, which used more re-
`liable analytic methodologies, raise interesting ques-
`tions when correlated with in vitro and in vivo obser-
`vations. Azacitidine induces cell differentiation in
`vitro at concentrations in the range of 2 to 4 µm, which
`are comparable to the in vivo concentrations achieved
`with SC dosing in this study and are clinically effective
`in MDS. Azacitidine, after incorporation into DNA as a
`substituted cytidine residue, irreversibly combines
`with DNA methyltransferase, leading to its inactiva-
`tion. This forms newly synthesized DNA, which is
`hemimethylated compared to parental DNA, and sub-
`sequently becomes fully hypomethylated after 1 addi-
`tional cycle of DNA synthesis. A once-a-day single-
`bolus injection of azacitidine daily for 7 days every 28
`days is likely to affect only a small population of cells
`in S phase. It would be difficult to explain kinetically
`how this short exposure would be sufficient to produce
`the hypomethylating effect. Repetitive exposures,
`which appear to be clinically important, may explain
`part of this effect. Further studies, correlating pharma-
`cokinetic analyses with the corresponding changes in
`DNA methylation, will be needed to further define the
`mechanism of action. This will be critical, now that
`azacitidine has been approved for use in patients with
`MDS in the United States, to help select an optimized
`dose and schedule both with respect to modulation of
`epigenetic gene silencing and clinical response.
`
`CONCLUSION
`
`This study demonstrated good bioavailability of an SC
`azacitidine dose compared to an IV infusion treatment.
`The exposure profiles following SC drug adminis-
`
`tration illustrate measurable azacitidine levels, with
`bioavailability (AUC) values within 89% of those mea-
`sured following IV administration. A single dose of
`azacitidine, 75 mg/m2, was generally well tolerated, re-
`gardless of the route of administration.
`
`This study was supported by a research grant from Pharmion Cor-
`poration. Dr Marcucci and Dr Silverman received grant funding at the
`time of this study. Ms Lintz and Dr Beach are employees of Pharmion
`Corporation.
`
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`602 • J Clin Pharmacol 2005;45:597-602
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`Apotex v. Cellgene - IPR2023-00512
`Petitioner Apotex Exhibit 1017-0006
`
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