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`
`5-Azacytidine
`
`Increases
`
`HbF
`
`Production
`
`and Reduces
`
`Anemia
`
`in Sickle
`
`Cell
`
`Disease:
`
`Dose-Response
`
`Analysis
`
`of Subcutaneous
`
`and Oral
`
`Dosage
`
`Regimens
`
`By G.J. Dover, S. Charache,
`
`S.H. Boyer, G. Vogelsang,
`
`and M. Moyer
`
`to four
`given
`were
`(5-aza)
`of 5-azacytidine
`doses
`Varying
`and 30 days.
`The
`200,
`1 00,
`cell
`individuals
`for
`500,
`sickle
`(HbF)
`containing
`reticulo-
`percentage
`of
`fetal
`hemoglobin
`two-
`to
`five-fold
`within
`cytes
`(F
`reticulocytes)
`increased
`five
`days
`of 5-aza
`therapy
`in all patients,
`with
`a two-
`three-fold
`rapid
`response
`(<48
`hours
`after
`initial
`dose)
`three
`patients.
`Reticulocyte
`suppression
`was
`not observed
`prior
`to,
`during,
`or after
`therapy
`in those
`patients
`who
`responded
`within
`48 hours.
`Subcutaneous
`5-aza was
`given
`in 35-day
`courses
`consisting
`of every
`day,
`every
`other
`day,
`or
`three
`consecutive
`days
`a week.
`No marrow
`toxicity
`was
`observed
`on
`any
`the
`regimens.
`For
`three
`patients,
`the
`
`of
`
`to
`in
`
`was
`
`the
`on
`observed
`level
`F reticulocyte
`average
`highest
`given
`Oral
`5-aza,
`regimen.
`day
`a week
`consecutive
`three
`comparable
`F reticulo-
`produced
`tetrahydrouridine,
`with
`treated
`for more
`than
`two
`patients
`response.
`In the
`cyte
`days, Hb levels
`increased
`to 1 1 to 12 and 9 g/dl,
`MCV
`100
`and MCH
`increased
`by 25%,
`and lysate
`HbF levels
`peaked
`at 1 2% and 20%.
`Fetal
`erythroid
`characteristics
`(i-antigen.
`galactokinase
`activity,
`and G’y/A7
`ratios)
`did
`correlate
`with
`maximal
`HbF
`production.
`The
`frequency
`of
`vaso-
`decrease
`in both
`patients
`to
`occlusive
`crises
`appeared
`followed
`for more
`than
`1 00 days.
`0 1985
`by Grune
`& Stratton,
`Inc.
`
`not
`
`cell
`
`In
`
`study’
`for
`
`were
`
`at
`complex
`individuals
`5-aza
`
`this
`of
`
`DNA
`cell-cycle-specific
`a
`(5-aza),
`5-AZACYTIDINE
`increase
`to
`been
`shown
`has
`agent,’
`hypomethylating
`in
`one
`animals,2’3
`in
`production
`(HbF)
`hemoglobin
`fetal
`with
`individuals
`in several
`and
`thalassemia,4
`individual
`with
`of
`of our
`first
`phase
`the
`anemia.”6
`sickle
`(55)
`I 00
`treated
`55
`patient
`one
`in
`of
`5-aza
`repeated
`doses
`( i ) a rapid
`days,
`we observed:
`after
`hours
`48
`increase
`(within
`con-
`percentage
`of
`reticulocytes
`in
`the
`each
`dose
`of
`5-aza)
`(2)
`increase
`in the
`percentage
`taming
`HbF
`(F reticulocytes),
`levels
`in
`the
`peripheral
`blood
`of
`HbF
`and
`hemoglobin
`toxicity;
`and
`(3)
`no
`apparent
`without
`evidence
`of marrow
`decrease
`in the
`frequency
`of vaso-occlusive
`crisis.
`Alterations
`in
`the
`pattern
`of
`DNA
`methylation
`CpG
`dinucleotide
`sequences
`around
`the
`f3-globin
`gene
`associated
`with
`increased
`HbF
`production
`in
`treated
`with
`5-aza7(cid:1)
`but
`the mechanism
`by which
`increased
`levels
`of HbF
`production
`remained
`uncertain.
`the
`concerning
`In
`article
`expand
`our
`observations
`we
`in
`An
`anemia.
`55
`effect
`5-aza
`therapy
`patients
`with
`that
`defined
`an
`produces
`has
`optimal
`dose
`been
`as
`which
`least
`at
`count
`of
`leads
`but which
`average
`F reticulocyte
`20%,
`and
`gastrointestinal
`20%
`to minimal
`marrow
`toxicity.
`The
`had
`because
`one
`of
`us
`Saudi
`level
`was
`chosen
`shown7
`that
`mild
`with
`clinically
`Arabian
`55
`patients
`disease
`associated
`had
`with
`increased
`production
`of HbF
`F reticulocyte
`levels
`the
`between
`20% and
`50%. We
`varied
`dose
`and
`frequency
`of
`(SC)
`5-aza
`administration
`in order
`to
`subcutaneous
`then
`mine
`an
`optimal
`dose
`regimen,
`and
`determined
`optimal
`oral
`regimen
`5-aza
`administration,
`prompted
`observations
`that
`was
`orally
`effective
`when
`given
`5-aza
`mice8
`or baboons9
`in conjunction
`with
`a cytidine
`deaminase
`inhibitor,
`tetrahydrouridine
`(THU).
`
`of
`
`deter-
`an
`by
`to
`
`MATERIALS
`
`AND
`
`METHODS
`
`SS
`
`45,
`
`homozygous
`adult
`were
`patients
`All
`Patients.
`transfusion
`therapy
`to chronic
`refractory
`had
`become
`A (i.P.)
`was
`described
`Patient
`disease.
`tions
`of
`their
`D (Mi.)
`were
`26,
`and
`Patients
`B (D.P.),
`C (W.T.),
`treated
`for 500 days.
`A was
`old,
`respectively.
`Patient
`dropped
`due
`to noncompliance
`after
`day
`from
`study
`the
`C was
`treated
`for 200 days.
`Patient
`D discontinued
`therapy
`because
`he elected
`to undergo
`bilateral
`hip replacement
`ing aseptic
`necrosis
`of both
`femonal
`heads.
`All patients
`according
`to
`protocols
`approved
`by The
`iohns
`Hopkins
`
`who
`patients
`for complica-
`previously.’
`and
`23 years
`Patient
`B was
`100.
`Patient
`at day 30
`for preexist-
`were
`treated
`University
`
`and
`
`The
`the
`
`two
`
`gave
`and
`during
`when
`of
`
`consent
`cytotoxic
`first
`the
`5-aza
`therapy
`visits
`
`after
`side
`45
`was
`was
`the
`
`to
`
`making
`
`Investigation
`on Clinical
`Committee
`Joint
`carcinogenic,’0
`the
`potential
`of
`informed
`being
`were
`hospitalized
`All
`patients
`of 5-aza.
`effects
`regimens
`of
`therapy
`and
`during
`therapy
`of
`days
`day.
`remainder
`every
`other
`on
`daily
`or
`given
`with
`subjects
`outpatients
`as
`accomplished
`a week.
`three
`times
`to
`hospital
`obtained
`was
`Injectible
`5-aza
`Drugs.
`Institute.
`Cancer
`Treatment,
`National
`Cancer
`i.
`Posada
`by Dns
`were
`and
`THU
`provided
`5-aza
`and
`stoned
`at
`the National
`Cancer
`Institute,
`of 5-aza
`and THU
`was
`performed
`by The
`Pharmacy,
`using
`precautions
`suitable
`for
`Claimed
`exemptions
`for new drugs
`(INDs)
`and
`Drug
`Administration
`for
`all
`phases
`injection,
`100 mg
`of
`injectible
`azacytidine
`for
`water
`injection
`and
`injected
`as a slurry.
`amount
`and the
`The
`percentage
`of F reticulocytes
`HbFlevels.
`anti-human
`rabbit
`HbF/F
`cell were
`determined
`using
`polyclonal
`containing
`HbF
`HbF.”2
`The
`percentage
`of mature
`enythnocytes
`of
`(percentage
`F cells)
`was
`assayed
`using
`a mouse
`monoclonal
`HbF
`anti-human
`antibody
`developed
`in our
`laboratory.
`Lysate
`HbF
`levels were measured
`by alkali
`denaturation”
`and G’y/A-y
`ratios
`were
`kindly
`measured
`by
`Dr
`Blanche
`and
`Dr Walter
`oed”
`RBC indices
`been
`standardized
`phthalate
`ester
`used
`to measure
`(MCHC)
`and
`dL).’6”7
`
`-
`
`of
`
`of
`
`Division
`the
`from
`pyrogen-free
`Purified
`P. Davignon
`and
`#{176}C.Encapsulation
`70
`Hopkins
`Hospital
`Johns
`chemothenapeutic
`agents.
`were
`filed with
`the Food
`of
`the
`study.
`For
`SC
`was
`suspended
`in 4 mL
`of
`
`of
`
`Alter’4
`
`that
`cell counter
`by an electronic
`were measured
`collected
`in K3EDTA.
`Twenty-step
`with
`blood
`were
`supplied
`by Dr C. Noguchi,
`density
`gradients,
`the mean
`corpuscular
`hemoglobin
`concentration
`the
`percentage
`of
`dense
`cells
`(MCI-IC
`37
`>
`
`had
`
`g/
`
`the
`
`Medicine,
`School
`Labonatonyfor
`
`Laboratory
`and
`of Medicine,
`Biochemical
`
`and
`
`of Pediatrics,
`Departments
`From
`Hopkins
`University
`The
`Johns
`Medicine,
`Medical
`Institute,
`Hughes
`the Howard
`Baltimore.
`Human
`Genetics,
`accepted
`13,
`1984;
`Submitted
`Nov
`Institutes
`Supported
`by National
`(G.J.D.),
`(G.J.D.),
`RCDA
`AM00689
`eral
`Clinical
`Research
`Center),
`and
`Research
`Center),
`by
`a grant
`and
`Committeefor
`Sickle
`Cell Anemia.
`Address
`reprint
`requests
`to Dr G.J. Dover,
`Hopkins
`University
`School
`of Medicine,
`Johns
`Baltimore,
`MD 21205.
`e /985
`by Grune
`& Stratton,
`0006-4971/85/6603-0009$03.00/0
`
`27,
`
`Feb
`ofHealth
`KL027996
`RR00722
`from
`
`the
`
`1985.
`grants
`(S.C.),
`(Outpatient
`Central
`
`HL28028
`No.
`RR35
`(Gen-
`Clinical
`Maryland
`
`907.
`Traylor
`720 Rutland
`
`The
`Ave.
`
`Inc.
`
`Blood, Vol 66, No3(September),
`
`1985:
`
`pp 527-532
`
`527
`
`Apotex v. Cellgene - IPR2023-00512
`Petitioner Apotex Exhibit 1031-0001
`
`

`

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`
`528
`
`DOVER
`
`ET AL
`
`Table
`
`1 . Average
`Subcutaneous
`
`Response
`F Reticulocyte
`(SC)
`and Oral
`(P0)
`
`to Various
`5-Azacytidine
`
`Doses
`
`of
`
`A
`
`Patients
`
`B
`
`C
`
`Pre
`I
`
`II
`
`Ill
`
`IV
`V
`
`(O.2)t
`
`4.4
`1.O(cid:1)
`±
`25.0±5.6(1.2)
`(0.9)
`± 2.7
`± 3.5 (1.3)
`
`17.6
`
`26.1
`
`16.8 ± 7.4 (1.0)
`26.3
`10.0(1.0)
`
`±
`
`3.8
`
`± 0.8
`
`(0.2)
`
`14.4
`
`±
`
`1.2 (0.5)
`
`± 7.3 (1.0)
`21.9
`17.8 ± 5.6 (0.9)
`
`31.6
`
`± 8.7 (1.1)
`
`31.9
`
`±
`
`10.0
`
`(1.0)
`
`Mean
`
`±
`
`1 SD percentage
`
`of F reticulocytes.
`
`1O5/(cid:1)L.
`x
`level
`F reticulocyte
`absolute
`tMean
`day
`other
`II, 2 mg/kg/every
`35
`days
`(SC);
`for
`I, 2 mg/kg/d
`for
`three
`consecutive
`days per week
`(SC);
`III. 2 mg/kg/d
`for
`for
`for
`three
`consecutive
`days per week
`(SC);
`IV, 2 mg/kg/d
`for
`day;
`V. 0.2 mg/kg/d
`three
`consecutive
`(SC)
`2 doses
`per
`week
`(P0)
`+ 200 mg THU (P0)
`for
`five weeks.
`
`days
`35
`for
`five weeks
`five weeks
`days
`per
`
`epi-
`an
`
`as
`fever,
`one
`painful
`
`defined
`were
`crises
`Vasoocclusive
`crises.
`Vasoocclusive
`with
`tachycandia,
`associated
`pain
`sodes
`of
`localized
`bilirubin.
`Only
`total
`elevated
`WBC,
`on an
`increase
`in
`with
`such
`findings.
`episode
`in patient
`A was
`not
`associated
`Miscellaneous.
`by Dr
`Activity
`of
`galactokinase
`was
`measured
`and
`B-l2,
`E. Beutlen.’8
`Erythrocyte
`folate,
`serum
`a-fetopnotein,
`Hop-
`cancinoembryonic
`antigen
`levels were measured
`in The
`Johns
`RBC
`kins Hospital
`Clinical
`Laboratory
`using
`standard
`methods.
`tech-
`by
`i-antigen
`titers
`were measured
`a standard
`agglutination
`Cytidine
`provided
`nique’9
`using
`antisera
`kindly
`by Dr M. Crookston.
`deaminase
`levels20 were measured
`in peripheral
`blood mononuclear
`cells
`separated
`on Percoll.2’
`DNA
`polymorphism
`haplotypes
`were
`determined
`by restriction
`enzyme
`analysis
`of peripheral
`blood
`by Dr
`S. Antonanakis.22
`
`RESULTS
`
`that
`1 indicates
`Figure
`response.
`reticulocyte
`ofF
`Onset
`of F reticulocytes
`described
`increase
`in percentage
`rapid
`the
`four
`for
`patient
`A (5)
`occurred
`in three
`of
`patients
`previously
`2 mg/kg/d
`SC
`5-aza
`given
`as
`a single
`dose
`with
`treated
`consecutive
`days.
`In
`the
`three
`patients
`exhibiting
`three
`response,
`reticulocyte
`levels
`remained
`at
`prether-
`rapid
`levels
`during
`this
`period.
`is
`unclear
`patient
`apy
`It
`he
`responded
`more
`slowly.
`Only
`exhibited
`and
`vomit-
`ing
`and
`a decrease
`in reticulocyte
`production
`5.3
`i05/(cid:1)tL
`pretherapy
`to
`I .5 x
`l0’/i(cid:1)L
`ten
`days
`this
`dosage
`level.
`5-aza
`Effect
`various
`summarizes
`I
`tion.
`B,
`responses
`patients
`A,
`5-aza.
`were
`performed
`during
`period
`of
`treatment.
`changed
`times
`when
`F reticulocyte
`pretreatment
`levels.
`Note
`that,
`as
`
`for
`a
`
`D
`
`x
`
`at
`
`why
`nausea
`
`to
`(2.9
`posttherapy)
`
`doses
`
`and
`
`on F reticulocyte
`the
`mean
`F
`C given
`various
`two
`to
`three
`times
`Drug
`regimens
`levels
`had
`returned
`described
`in
`baboons
`
`produc-
`reticulocyte
`of
`doses
`a week
`were
`to
`by
`
`total
`
`of
`Table
`of
`Assays
`each
`at
`
`4
`
`5
`
`6
`
`DAYS
`
`C,)
`
`>.
`
`C.)
`
`zCC
`
`.)
`
`1 .
`
`cell
`sickle
`in four
`response
`F reticulocyte
`The
`Fig
`Patient
`(5-aza).
`(SC)
`5-azacytidine
`subcutaneous
`given
`patients
`patient
`D.
`C,
`patient
`B,
`0;
`patient
`total
`that
`Note
`(cid:1);
`.;
`time
`of
`period
`unchanged
`during
`counts
`were
`reticulocyte
`top
`bar,
`Shaded
`except
`for patient
`D (see Results).
`all patients
`2 mg/kg/d
`of 5-aza.
`refers
`to days
`of administration
`the
`graph.
`(SC)
`for
`three
`consecutive
`days. Data on patients A, B, and C have
`been previously
`described.27
`
`0.
`this
`
`(SS)
`A.
`
`in
`of
`
`On
`
`to
`
`B
`
`in
`
`seemed
`levels
`reticulocyte
`schedules,
`all
`dose
`treatment.
`A and
`patients
`for
`level
`achieved
`respec-
`± 0.8%,
`3.8%
`1.0% and
`C (pretreatment
`that
`in patient
`regimens
`listed
`of
`the
`drug
`by decreased
`WBC,
`(defined
`to pretherapy
`val-
`compared
`for
`30
`consecutive
`of
`5-aza
`A,
`administration
`In subject
`I).
`III)
`week
`(regimen
`days
`each
`to those
`obtained
`comparable
`dose.
`Alternate-day
`adminis-
`daily
`doses
`(regimen
`IV) were
`
`in
`
`ten
`
`the
`observed
`
`SC
`no
`
`seven
`
`two
`
`F
`
`to
`
`baseline
`response
`the
`F reticulocyte
`levels
`always
`
`±
`
`±
`
`DeSimone,23
`influence
`the mean
`4.4%
`(pretreatment
`less
`than
`tively)
`was
`level,
`14.4%
`1.2%).
`None
`marrow
`toxicity
`Table
`I caused
`platelet,
`or
`reticulocyte
`counts
`ues),
`after
`administration
`even
`days
`at
`2 mg/kg/d
`(regimen
`of 5-aza
`for
`three
`consecutive
`resulted
`in F reticulocyte
`levels
`with
`therapy
`at
`same
`daily
`the
`tration
`(regimen
`or divided
`II)
`than
`less
`effective
`regimen
`III.
`V)
`(regimen
`THU
`with
`Oral
`administration
`5-aza
`of
`production,
`F
`reticulocyte
`C
`patient
`A
`and
`increased
`THU
`(200
`or
`(2 mg/kg/d)
`doses
`although
`oral
`of
`5-aza
`(1.5
`to 2
`THU
`not
`shown).
`alone
`mg/d)
`given
`(data
`did
`not
`an
`equal
`and
`before
`5-aza,
`given
`mg/kg)
`was
`hour
`one
`until
`one
`given
`no food was
`second
`dose was
`given
`5-aza;
`with
`was
`5-aza,
`2 mg/kg/d,
`hour
`after
`the
`second
`When
`dose.
`and
`given
`with
`THU
`for
`successive
`days,
`nausea
`three
`and
`vomiting
`were
`prominent
`complaints
`significant
`suppres-
`seen
`sion
`of WBC,
`platelets,
`and
`reticulocytes
`were
`in
`both
`1 5
`days
`patients
`with
`maximum
`depression
`to
`after
`function
`beginning
`treatment.
`Marrow
`recovered
`within
`initial
`three
`weeks
`of
`the
`dose. When
`0.2 mg/kg/d
`5-aza
`of
`was
`given
`orally
`with
`THU
`(regimen
`V, Table
`1) F reticulo-
`cyte
`levels
`comparable
`to those
`achieved
`with
`optimal
`III
`regimen
`(regimen
`of
`2 mg/kg/d)
`were
`with
`evidence
`of
`cytotoxicity.
`1 .5 to 2 mg/kg
`doses,
`in two
`(divided
`of
`An
`THU
`dose
`oral
`5-aza;
`with
`second
`the
`hour
`before,
`given
`one
`first
`the
`of periph-
`in a suppression
`24
`hours
`resulted
`within
`fasting)
`levels
`to 3 1% and
`33% of
`deaminase
`eral
`blood WBC
`cytidine
`C,
`respectively.
`Cytidine
`pretherapy
`levels
`in patients
`A and
`deaminase
`levels
`had
`returned
`to 85% to 100% of pretherapy
`values
`days
`later
`when
`the
`drug
`was
`given
`for
`consecutive
`days
`each
`week.
`When
`given
`three
`consecutive
`days
`each
`week
`(see
`regimen
`V),
`day
`7 cytidine
`deaminase
`
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`

`529
`
`and
`
`±
`
`±
`cell
`
`2
`
`I
`
`a
`of
`a
`
`of
`
`5-AlA
`
`AND
`
`SICKLE
`
`CELL DISEASE
`
`Tab Ic 2. Hematologic
`
`Response
`
`to Various
`
`D oses
`
`of 5-Azacytidine
`
`for Patients
`
`A and C
`
`Hb(g/dL)
`
`Reticulocytes
`
`(%)
`
`HbF (%)
`
`F Cells
`
`(%)
`
`8.5
`11.3
`1 1.9
`
`12.0
`11.7
`11.3
`8.0
`9.2
`
`1.0k
`±
`± 0.8
`± 0.6
`± 0.7
`± 0.3
`± 0.7
`± O.5t
`± 0.3
`
`12.0
`15.3
`16.8
`17.1
`17.1
`12.5
`13.9
`15.3
`
`± 4.0(4.3)
`± 9.1
`(4.5)
`± 6.4
`(5.0)
`± 5.2
`(5.1)
`± 7.6
`(5.6)
`± 4.1
`(3.5)
`± 2.8
`(3.9)
`± 5.5
`(3.4)
`
`1.5
`7.6
`7.1
`8.0
`8.6
`9.6
`2.9
`17.5
`
`± 0.7
`1.5
`±
`± 0.2
`± 0.8
`1.0
`±
`± 0.7
`± 0.1
`± 0.6
`
`9.3
`41.4
`35.2
`32.6
`36.2
`43.0
`14.4
`63.4
`
`1.7
`±
`± 5.7
`± 3.5
`± 2.8
`± 5.8
`± 2.7
`1.2
`±
`± 3.9
`
`of
`Days
`Therapy
`
`125-160
`165-200
`
`203-240
`245-280
`415-464
`
`140-180
`
`Dose
`
`Pre
`
`I I
`
`I
`
`Ill
`
`IV
`V
`Pre
`V
`
`Patient
`
`A
`
`C
`
`Parentheses
`Table
`‘See
`
`the mean
`indicate
`1 for doses
`of 5-aza.
`
`absolute
`
`reticulocyte
`
`level
`
`x
`
`1 05/j.(cid:1)L.
`
`tPatient
`
`A had 46% HbA,
`
`and patient
`
`C had
`
`56% HbA due to previous
`
`transfusions.
`
`were
`
`54%
`
`and
`
`69%
`
`of
`
`pretherapy
`
`levels
`
`for
`
`both
`
`of
`
`5-aza
`
`hematologic
`other
`count,
`percentage
`2 summarizes
`counts,
`on
`
`too
`
`A
`
`F
`
`to
`
`Downloaded from http://ashpublications.org/blood/article-pdf/66/3/527/1639431/527.pdf by guest on 05 October 2021
`
`the mean MCV
`A,
`(Table
`and MCH
`MCV
`In patient
`3).
`regimen
`III
`(1 14
`were
`increased
`MCH
`the most
`during
`(1 16 ± 4 Fl
`and
`40
`40
`I pg)
`Fl
`and
`and
`regimen
`V
`±
`serum
`patient
`pg).
`Both
`A and C had
`normal
`B,2 and
`red
`folate
`levels
`throughout
`therapy.
`The MCHC
`as measured
`the
`by
`an
`electronic
`cell
`counter
`(patients
`A and
`C)
`or
`by
`pthalate
`ester
`technique
`(patient
`C only)
`did
`not
`change
`(Table
`3).
`The
`proportion
`of
`erythrocytes
`with
`a MCHC
`>37
`was
`followed
`weekly
`in
`patient
`C (Fig
`2).
`Dense
`g/dL
`cells
`disappeared
`between
`day
`40
`and
`coincident
`with
`90,
`decrease
`in reticulocytes
`which
`occurred
`during
`the
`period
`marrow
`depression
`gastrointestinal
`toxicity
`following
`and
`toxic
`oral
`of 5-aza
`(2 mg/kg/d)
`given
`with
`THU.
`high
`dose
`Patient
`A (data
`shown)
`showed
`a similar
`suppression
`not
`dense
`cells
`and
`reticulocyte
`counts
`at
`this
`dosage
`schedule.
`After
`adjustment
`of dosage
`(Table
`I , oral
`regimen
`V) when
`F reticulocyte
`levels
`remained
`above
`20% and
`F cell
`levels
`ranged
`from 65% to 83%,
`the
`percentage
`ofdense
`cells
`varied
`between
`4% and
`8%.
`Pretreatment
`levels
`were
`similar
`(4% to
`6%).
`However,
`the
`patient
`had
`transfused
`prior
`to drug
`been
`his
`therapy
`and
`had
`46% normal
`cells
`in
`blood
`at
`the
`red
`beginning
`ofobservation.
`No
`correlation
`between
`the
`propor-
`crisis
`tion
`of dense
`cells
`and
`vasoocclusive
`(as
`noted
`by Fabry
`C. However,
`et al24) was
`observed
`in patient
`only
`two
`assays
`were
`performed
`on
`patient
`C during
`that
`period
`of
`therapy
`when
`crises
`occurred
`(day
`0 through
`20).
`Markers
`of
`‘fetal
`erythropoiesis.
`
`“
`
`Because
`
`all
`
`patients
`
`Table
`
`3. HbF 1ev els and Erythro
`
`cyte
`
`Indice
`
`s on Pa tients
`
`A and C
`
`F Cells
`(%)
`
`Cell
`
`HbF/F
`(pg)
`
`MCV
`(11)
`
`MCH
`(pg)
`
`MCHC
`(g/dL)
`
`8
`
`33
`40
`33
`42
`
`40
`18
`50
`64
`62
`
`62
`
`7.6
`
`7.2
`6.9
`8.0
`8.1
`
`10.4
`5.5
`7.4
`9.9
`11.3
`
`11.2
`
`95
`
`105
`108
`110
`121
`
`125
`98
`101
`124
`126
`
`123
`
`34
`
`37
`38
`38
`40
`
`44
`34
`34
`42
`42
`
`42
`
`35.8
`
`35.2
`35.2
`34.5
`33.7
`
`35.2
`34.7
`33.7
`33.9
`33.3
`
`34.2
`
`HbF
`(%)
`
`1.8
`
`6.5
`7.3
`6.8
`8.6
`
`9.5
`2.9
`10.9
`15.1
`
`16.8
`16.6
`
`of
`Day
`Therapy
`
`0
`101
`200
`301
`410
`
`500
`
`0 5
`
`2
`100
`
`146
`203
`
`Patient
`
`A
`
`C
`
`A were
`on patient
`Values
`Fla) and on patient
`C by phthalate
`
`determined
`ester
`
`by Coulter
`technique.
`
`Counter
`
`(Hialeah,
`
`oral
`
`to return
`before
`these
`from
`hemoglo-
`apparent,
`persis-
`A over
`
`a
`
`in
`cell
`
`levels
`patients.
`mea-
`on
`doses
`various
`Effect
`of
`reticulocyte
`hemoglobin,
`surements:
`aver-
`Table
`of F cells.
`percentage
`HbF
`and
`percent-
`reticulocyte
`age
`hemoglobin
`concentrations,
`patients
`of F cells
`obtained
`and
`age
`of HbF,
`percentage
`data
`those
`of
`5-aza.
`Only
`various
`and
`C on
`regiments
`are
`summa-
`cells
`were
`present
`obtained
`when
`no
`transfused
`changed
`before
`hemoglobin
`con-
`rized.
`Drug
`regimens
`were
`to pretreatment
`levels,
`but
`after
`centrations
`had
`returned
`dropped
`to pretreatment
`levels.
`reticulocyte
`levels
`had
`C
`Hemoglobin
`concentrations
`rose
`in both
`patients
`A and
`2).
`(Table
`while
`they
`on
`5-aza
`Patient
`A’s
`levels
`were
`were
`those
`of Patient
`consistently
`than
`C even
`though
`HbF
`higher
`Although
`levels
`behaved
`conversely.
`patient
`B’s
`hemoglobin
`on
`from
`III
`values
`rose
`regimen
`g/dL
`(pretherapy)
`8.5
`i 1 .5 g/dL
`(day
`he was
`obtain
`84),
`noncompliant
`to
`data
`transfused
`sufficient
`after
`cells
`had
`disappeared.
`D left
`(see Materials
`Patient
`study
`and Methods)
`shortly
`a second
`for
`after
`of 5-aza.
`Except
`transient
`suppression
`reticulocytes
`of
`following
`the
`inception
`of
`high-dose
`5-aza
`therapy
`(2 mg/kg/d
`+ 200 mg THU),
`the
`proportions
`of
`reticulocytes
`in patients
`A and
`C were
`unchanged
`during
`treatment.
`patients
`our
`to allow
`unwilling
`Because
`concentrations
`hemoglobin
`their
`to
`conclude
`cannot
`we
`regimens,
`changing
`in a higher
`results
`drug
`regimen
`data
`whether
`is
`regimen.
`It
`than
`another
`bin
`concentration
`hemoglobin
`concentrations
`remained
`however,
`that
`in
`than
`pretreatment
`levels
`patient
`tently
`higher
`prolonged
`period
`time
`(500
`posttherapy).
`of
`therapy
`During
`the
`300
`days
`of SC
`HbF/F
`cell.
`patient
`A, negligible
`differences
`in the
`amount
`of HbF/F
`were
`seen
`(Table
`even
`though
`F reticulocyte
`production
`3)
`and
`F cell
`levels
`increased
`dramatically
`(Table
`2).
`In
`con-
`trast,
`during
`oral
`5-aza
`therapy,
`the
`amount
`of HbF/F
`cell
`increased
`37% in patient
`A (from
`7.6
`to 10.4
`pg)
`and
`103% in
`patient
`C (from
`5.5
`1 1.2
`pg)
`(Table
`3). No
`changes
`in
`to
`HbF/F
`cell were
`seen
`in the
`short
`time
`patients
`B and D
`were
`followed.
`RBC
`indices
`both
`patients
`
`the
`dose
`
`we were
`pretreatment
`dosage
`one
`
`days
`first
`
`that
`
`apy
`
`and
`
`“dense
`A and
`
`cells.
`C demonstrated
`
`“
`
`After
`
`days
`100
`increases
`
`of
`in
`
`ther-
`their
`
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`
`530
`
`DOVER
`
`ET AL
`
`Painful Crisis in Patients
`of Vaso-occlusive
`Table 4. Frequency
`A and C Treated
`With
`Subcutaneous
`(SC)
`and Oral
`(P0)
`5-Azacytidine
`
`A
`
`C
`
`Day
`
`Crisis’
`
`Day
`
`Crisis
`
`-166-0
`
`O-125t
`
`125-300
`301-500
`
`70
`
`70
`
`10
`15
`
`-200-0
`
`-
`
`0-35
`
`36-200
`
`100
`
`-
`
`20
`
`0
`
`Pretreat-
`ment
`
`Initialtrial
`
`SC5-Aza
`P05-Aza
`
`Days
`
`in crisis
`
`as defined
`
`in Materials
`
`and Methods.
`
`tPrevious
`5-aza.
`
`report6;
`
`monthly
`
`or
`
`every-two-week
`
`pulses
`
`of
`
`IV or SC
`
`DAYS
`
`Alterations
`Fig 2.
`and
`reticulocytes.
`total
`in F reticulocytes,
`C. Note
`the proportion
`of dense cells (MCHC <37 g/dl)
`in patient
`that suppression
`of
`administration
`in total
`reticulocytes
`followed
`on
`three
`oral
`5-azacytidine
`(2 mg/kg/d
`for
`days) with
`THU
`begun
`of F
`day 35. The numbers
`in the top panel
`refer
`to the percentage
`cells present
`on the corresponding
`days. Regimen
`Ill was
`given
`days 0 through
`35, and regimen
`V was given on days 140 through
`200.
`
`been
`
`and
`
`com-
`treatment,
`5-aza
`starting
`before
`transfused
`had
`and
`enzymes
`before
`antigens
`erythrocyte
`of
`parison
`fetal
`Markers
`of
`the
`be meaningless.
`therapy
`would
`during
`maximal
`levels
`of HbF
`examined
`when
`erythropoiesis
`were
`20% in
`patient
`C) were
`(8% to
`10% in
`patient
`A;
`18% to
`attained.
`The
`activity
`of
`galactokinase,
`an
`erythrocyte
`enzyme
`which
`is
`elevated
`prenatally,’8
`within
`adult
`levels.
`total
`of
`fraction
`a
`of G”y globin
`percentage
`The
`C (normal
`patient
`patient
`A and
`was
`53% in
`HbF
`Gilman
`and
`<50%).
`normal
`adult
`level,
`70%;
`newborn
`III-HincII
`two
`have
`recently
`shown
`Huisman2’
`Hind
`the
`around
`haplotypes
`enzyme
`polymorphism
`restriction
`low
`or
`high
`with
`either
`genes
`globin
`are
`associated
`G’y-A’y-f3
`A,
`homozy-
`patient
`According
`to
`their
`analysis,
`G’y
`levels.
`low G’y
`level
`have
`haplotype
`should
`gous
`for
`haplotype
`Patient
`for
`homozygous
`(<48%).
`+
`+
`red
`The
`predicted
`(>56%)
`G’y level.
`to have
`a high
`be
`would
`and
`titer)
`titer
`was
`elevated
`in patient
`A (I
`/64
`i-antigen
`cell
`blood
`cord
`in patient
`C (normal
`adult
`titer
`0)
`undetectable
`(1 / 1 28).
`Schroeder
`Using
`chromatographic
`methods,26
`titer
`1 or Gower
`no embryonic
`hemoglobin
`(Gower
`could
`detect
`would
`detect
`patient
`using
`methodology
`which
`in either
`little
`as 0.03%.
`a-Fetoprotein
`and
`carcinoembryonic
`antigen
`levels
`remained
`normal
`therapy.
`Vasoocciusive
`Vasoocclusive
`crisis.
`decrease
`in number
`in the
`first
`I 30 days
`A (see
`previous
`report’).
`However,
`with
`treatment
`crisis
`frequency
`decreased
`4). During
`oral
`therapy
`number
`
`was
`
`as
`chains
`73% in
`level,
`that
`
`-
`
`-
`
`-
`
`,
`
`a
`
`+
`
`C,
`
`-
`
`+
`
`2)
`as
`
`during
`
`the
`
`not
`did
`crisis
`in patient
`therapy
`of
`parenteral
`continued
`days
`(Table
`10/
`1 75
`crisis
`days
`was
`also
`
`to
`of
`
`C responded
`Patient
`days).
`(1 5 of 200
`decreased
`day
`30 of
`treatment,
`he
`After
`dramatically.
`crisis.
`It
`is
`important
`to
`further
`uncontrolled,
`and
`observations
`receiving
`when
`they
`Furthermore,
`were
`altered.
`psychiatric
`emotional
`and
`these
`personnel.
`For
`concerning
`amelioration
`during
`therapy.
`
`painful
`
`were
`were
`
`5-aza
`
`the
`
`more
`much
`no clearcut
`had
`that
`these
`note
`patients
`knew
`the
`that
`the
`dosages
`when
`and
`treatment
`received
`intense
`patient
`each
`medical
`and
`nursing
`from
`support
`conclusions
`drawn
`reasons,
`no
`of
`clinical
`features
`disease
`
`can
`of
`
`be
`the
`
`DISCUSSION
`
`ofaction.
`
`All
`
`with
`
`are
`other,
`for
`
`two
`
`1)
`
`a
`
`not
`
`and
`
`treated
`patients
`SS
`four
`Mechanism
`production.
`in HbF
`increases
`significant
`5-aza
`demonstrated
`two major
`is unknown,
`but
`the
`increase
`of
`The
`mechanism
`In
`one,
`erythroid
`pre-
`late
`been
`advanced.
`hypotheses
`have
`through
`some
`action
`of 5-aza27;
`cursors
`“reprogrammed”
`precursors,
`with
`inherent
`an
`in
`the
`early
`erythroid
`of HbF,
`are
`“recruited”
`program
`increased
`production
`destruction
`of
`later
`precur-
`concomitant
`with
`the
`cytotoxic
`by
`question
`examining
`the
`the
`sors.3
`Some
`light
`is
`shed
`on
`response
`to
`the
`drug.
`the
`timing
`of
`in
`vivo
`F reticulocyte
`a rapid
`response
`(Fig
`Three
`of our
`four
`patients
`exhibited
`production.
`Only
`patient
`and
`no decline
`in total
`reticulocyte
`immediate
`rise
`in
`F reticulo-
`D failed
`to show
`a
`significant
`of
`the
`F reticulocyte
`response
`cytes.
`Variation
`in the
`rapidity
`with
`patients
`treated
`a
`single
`was
`also
`observed
`in
`six
`SS
`Institutes
`of Health
`by Ley
`course
`of
`5-aza
`the National
`at
`our
`laboratory:
`five
`of
`them
`in
`and
`Nienhuis
`assayed
`and
`not.27
`The
`rapid
`increase
`in F
`did
`responded
`but
`one
`rapidly,
`hours
`of
`treatment
`without
`48
`reticulocytes
`to
`24
`within
`suggests
`that,
`in most
`suppression
`reticulocytes
`of
`total
`erythroid
`precursors
`to pro-
`patients,
`reprograms
`late
`5-aza
`gastrointestinal
`bone
`duce
`Patient
`D exhibited
`HbF.
`treatment
`course
`of
`which
`marrow
`toxicity
`the
`single
`after
`patient
`The
`one
`with
`produced
`the
`response.
`delayed
`delayed
`response
`at
`the National
`Institutes
`of Health
`treated
`that
`behaved
`in
`similar
`fashion.
`It
`is
`conceivable
`the
`late
`response
`in these
`patients
`reflects
`the
`cytotoxic
`“recruit-
`ment”
`phenomenon,
`but
`that
`responses
`to doses
`of 5-aza
`accompanied
`by
`toxicity
`reflect
`the
`alternate
`“reprogram-
`ming”
`effect.
`It
`intriguing
`is
`patients
`were
`. 10)
`(P
`
`our
`levels
`
`>
`
`that
`to note
`not
`significantly
`on any
`of
`the
`
`levels
`reticulocyte
`absolute
`pretherapy
`different
`from
`regimens
`described
`
`drug
`
`in
`
`(see
`
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`
`5-ALA
`
`AND
`
`SICKLE
`
`CELL DISEASE
`
`531
`
`2).
`
`time
`no
`at
`Furthermore,
`reticulocyte
`total
`in
`declines
`levels
`reticulocyte
`in
`F
`that
`regimens
`during
`These
`observations
`was
`associated
`and
`result
`HbF
`
`A and
`
`C did
`in patients
`precede
`production
`reticulocyte
`were
`F reticu-
`higher
`increased
`that
`preceded
`or
`recruitment
`makes
`suppression,
`
`by
`
`F
`a
`
`an
`
`Table
`transient
`nor
`elevations
`produced
`lower
`counts
`suggest
`responses.
`locyte
`with
`not
`production
`cell
`further
`activity
`in erythroid
`decline
`of marrow
`as
`a
`precursors,
`of
`early
`production.
`increased
`cause
`of
`unlikely
`increased
`With
`sustained
`in
`anemia.
`Improvement
`rose,
`production,
`hemoglobin
`concentration
`reticulocyte
`cell.
`MCH,
`and
`the
`amount
`of HbF/F
`did
`the MCV,
`also
`was
`MCHC,
`however,
`remained
`constant.
`There
`decreased,
`suggestion
`that
`clinical
`symptoms
`(painful
`crises)
`effects.
`raising
`questions
`of
`the
`relationships
`between
`these
`a major
`The
`concentration
`of HbS
`within
`the
`red
`cell
`report6
`determinant
`of HbS
`polymerization.28
`A previous
`treat-
`that
`suggested
`improvement
`in
`anemia
`seen
`in
`5-aza
`ment
`is associated
`with
`decreased
`MCHC
`and
`the
`disappear-
`of
`ance
`from
`the
`blood
`very
`cells
`(ie,
`those
`with
`dense
`higher
`MCHC).
`However,
`patients,
`the
`disappear-
`in those
`also
`of
`ance
`dense
`cells
`and
`in MCHC
`were
`change
`the
`our
`In
`associated
`with
`transient
`suppression.6
`reticulocyte
`reticulo-
`patients
`A and
`C,
`dense
`only
`when
`cells
`decreased
`were
`ten
`times
`cytes
`suppressed
`were
`given
`after
`the
`patients
`optimal
`by Nogu-
`the
`oral
`dose
`Recent
`data
`of 5-aza/THU.
`et
`al’7
`young
`chi
`suggest
`that
`cells
`are
`relatively
`these
`dense
`and
`reticulo-
`cells
`therefore
`might
`to disappear
`if
`be
`expected
`levels
`cyte
`fall.
`However,
`during
`optimal
`therapy
`(regimens
`and
`III
`V),
`when
`reticulocyte
`percentages
`were
`not
`not
`decreased,
`Hb
`levels
`remained
`elevated,
`the MCHC
`did
`and
`change,
`dense
`cells
`persisted
`(ie,
`new ones
`were
`formed
`as old
`as
`fast
`ones
`were
`removed).
`It appears,
`therefore,
`that
`the
`partial
`compensation
`for
`the
`anemia
`in these
`patients
`not
`attributable
`to either
`a decreased
`MCHC
`the
`disap-
`pearance
`of dense
`cells.
`decreased
`for
`A second
`possible
`explanation
`production.
`HbF
`patients
`is
`their
`increased
`our
`with
`increased
`associated
`improvement
`in anemia
`was
`cell.
`This
`in HbF/F
`production
`without
`an
`increase
`may
`55
`patients
`in
`that
`a decrease
`anemia
`without
`production
`plished
`by
`increasing
`F cell
`did
`increase
`HbF/F
`cell.
`In
`contrast,
`HbF/F
`cell
`A and C the
`regimens.
`Surprisingly,
`in both
`patients
`was
`greater
`in the MCH
`(10
`pg and
`8 pg.
`respectively)
`the
`increase
`in HbF/F
`cell
`(3.8
`pg
`in A and
`4.7
`pg
`indicating
`HbS/cell
`also
`had
`increased.
`Assuming
`that
`the MCH
`of F cells
`and
`non-F
`cells
`are
`equal,
`the
`percentage
`of
`total
`hemoglobin
`per
`cell
`attributed
`HbF
`did
`to
`increase
`substantially
`in
`patient
`A (22%
`24%,
`day
`to
`500),
`did
`rise
`patient
`C (i6%
`to
`day
`0 to
`but
`27%,
`200).
`Despite
`these
`differences
`in HbF
`production,
`patients
`had
`less
`anemia;
`the
`lower
`HbF
`level
`in patient
`
`F
`as
`The
`
`a
`
`the
`
`is
`
`is
`
`or
`
`in
`
`anemia
`Initially,
`F cell
`indicates
`accom-
`be
`increasing
`on
`oral
`increase
`than
`in C)
`that
`
`not
`0 to
`day
`both
`
`A
`
`the
`
`in
`
`in
`
`hemoglobin
`average
`with
`associated
`being
`alone
`cannot
`production
`increased
`that
`appears
`patients.
`seen
`in our
`in anemia
`changes
`for
`the
`erythro-
`fetal
`Additional
`ofRBCs.
`characteristics
`Fetal
`course
`the
`were
`observed
`during
`cyte
`characteristics
`Erythro-
`patients.
`MCV
`seen
`in both
`was
`therapy.
`Elevated
`were
`increased
`in patient
`A and
`not
`titers
`cyte
`i-antigen
`patient
`C had
`the
`higher
`F cell
`level
`though
`patient
`C even
`patient
`A had
`higher
`G’y/A’y
`ratios
`the
`time
`of
`assay.
`Only
`restriction
`enzyme
`polymorphism
`than
`predicted
`from
`their
`of
`erythrocyte
`enzymes
`were
`haplotypes.2’
`Fetal
`levels
`This
`mixed
`pattern
`fetal
`observed
`in either
`patient.
`is more
`consistent
`with
`adult
`erythrocyte
`markers
`erythropoiesis’9’29.’#{176}
`than
`changes
`seen
`stress
`with
`true
`pattern
`erythropoiesis.
`fetal
`that
`have
`We
`Variability
`response.
`of
`patients.
`varies
`5-aza
`rapidity
`to
`response
`unknown,
`variability
`this
`Although
`for
`reasons
`the
`cytotoxic
`delayed
`with
`associated
`are
`clearly
`effects
`pro-
`F cell
`levels
`baseline
`reticulocyte
`Because
`response.
`since
`and
`determined7
`duction
`are
`genetically
`disease
`in 55
`subse-
`of
`were
`predictive
`pretherapy
`levels
`of F reticulocytes
`it might
`(Table
`I),
`quent
`reticulocyte
`responses
`F reticulocyte
`high
`baseline
`patients
`expected
`that
`55
`with
`patients
`with
`therapy
`than
`less
`intensive
`levels
`need
`may
`(<5%)
`is
`unclear
`it
`However,
`F reticulocyte
`levels.
`can
`reduce
`if any,
`minimal
`percentage
`of F reticulocyte,
`person
`brings
`Each
`clinical
`severity
`sickle
`cell
`disease.
`F cell
`production.
`on
`therapy
`his
`genetic
`constraints
`of drug
`and
`amount
`Such
`constraints
`may
`influence
`the
`clinical
`symptoms.
`magnitude
`of
`response
`needed
`to reduce
`Furthermore,
`it
`is not
`clear
`whether
`improvements
`in any
`hematologic
`parameter
`(percentage
`of HbF,
`F reticulocyte
`level,
`HbF/F
`cell,
`level,
`and
`percentage
`of
`dense
`cells)
`Hb
`predicts
`clinical
`improvement
`defined
`frequency
`
`higher
`HbF
`
`It
`
`levels.
`account
`
`of
`
`in
`at
`
`not
`and
`the
`
`the
`
`a
`
`F
`
`be
`
`low
`what
`the
`to
`
`the
`
`one
`
`of
`
`of
`
`rather
`
`observed
`between
`are
`the
`of
`
`as
`
`by
`
`of
`
`of
`
`F
`
`of
`
`own
`
`vasoocclusive
`makes
`
`crisis.
`assessment
`
`The
`
`of
`
`absence
`therapeutic
`
`a defin-
`such
`manuevers
`
`of
`
`of
`
`as
`
`disease.
`in 55
`5-aza
`patients.
`of 55
`number
`this
`effects
`of
`long-term
`the
`probably
`carcinogenic
`Although
`available
`regarding
`is
`humans.
`In
`the
`in
`cannot
`determine
`described
`patients
`
`of
`severity
`marker
`able
`difficult.
`more
`Overall
`role
`HbF
`in a
`limited
`observed,
`but
`are
`unknown.
`little
`information
`potential
`of
`5-aza
`clinical
`trials,
`we
`HbF
`levels,
`produced
`of
`alter
`the
`clinical
`course
`of
`The
`question
`carcinogenicity
`other
`cell-cycle-specific
`agents
`genie)
`have
`been
`shown
`to increase
`humans.”
`These
`agents
`should
`similar
`that
`outlined
`in
`clinical
`are
`begun.
`
`We
`Little
`form
`
`in
`
`in
`
`increased
`have
`toxicity
`was
`of
`therapy
`in animals,’#{176}
`carcinogenic
`the
`of
`controlled
`absence
`elevations
`whether
`significantly
`will
`here,
`cell
`disease.
`sickle
`with
`trials,
`but
`such
`impedes
`less
`carcino-
`be
`(which
`may
`HbF
`in animals31”2
`and
`evaluated
`in
`a manner
`report
`controlled
`
`to
`trials
`
`be
`
`before
`
`this
`
`Jones
`SM,
`1 . Taylor
`DNA
`methyltransfenase:
`Mol Biol
`162:679,
`1982
`2. DeSimone
`JP,
`Hellen
`
`PA: Mechanisms
`Use
`of 5-azacytidine
`
`of
`
`of
`action
`containing
`
`eukaryotic
`DNA.
`
`J
`
`P,
`
`Hall
`
`L,
`
`Zweins
`
`D:
`
`5-Azacytidine
`
`fetal
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`3. Tonrealba-de
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`
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`1982
`Sci USA 79:4428,
`Ron AT,
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`Stamatoyannopoulos
`G:
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`
`baboons.
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`T, Knapp
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`
`MS.
`the
`
`Fu
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`
`in
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`Apotex v. Cellgene - IPR2023-00512
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`
`

`

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