106-0585$02.00/O
`0090-9556/83/1
`DlsposmoN
`Ai’n
`DRUG
`Mamsouui
`by The American
`Copyright
`1983
`t
`
`Society
`
`for Pharmacology
`
`and Experimental
`
`Therapeutics
`
`Vol.
`Printed
`
`11, No.6
`in U. S. A.
`
`METABOLISM
`In Vitro
`
`KETOTIFEN
`OF
`Characterization
`
`MICROSOMES
`LIVER
`BY HUMAN
`of a Tertiary
`Amine
`Glucuronidation
`
`JEAN
`
`F.
`
`LE
`
`BIGOT,
`
`THIERRY
`
`CRESTEILt,
`
`JEAN
`
`A.
`
`KIECHEL5,
`
`and
`
`PHILIPPE
`
`BEAUNEt
`
`‘Centre
`
`de Recherches
`
`Pharmacocin tiques,
`
`Laboratoires
`
`Sandoz
`
`S.A.R.L.,and
`
`tLaboratoire
`
`do Biochimie,
`
`Chu Necker
`
`(Received
`
`September
`
`7.
`
`1 982;
`
`accepted
`
`July
`
`7.
`
`1983)
`
`ABSTRACT:
`
`using human
`in vitro
`was Investigated
`of ketotifen
`Biotransformation
`observed
`pathways
`the four metabolic
`Three
`of
`liver microsomes.
`incubation,
`under
`the
`conditions
`of
`exhibited
`in vivo
`in man were
`the ab-
`namely
`demethylation,
`N-oxidation,
`and N-glucuronidation,
`sent
`route
`beIng
`the ketoreduction,
`which
`probably
`has a cytosolic
`localization.
`The kinetic
`parameters
`of the N-glucuronidatlon
`(KM for
`ketotifen
`and UDPGA
`and V,,,.,) were
`determined
`with
`native
`and
`detergent-treated
`microsomes.
`Treatment
`by
`Triton
`X-1 00
`in-
`
`No sex difference
`reaction.
`the conjugation
`3-fold
`by about
`creased
`did not seem to be Inhibited
`was observed
`and N-glucuronldation
`Thus,
`human
`liver micro-
`either
`by
`bilirubin
`or
`by 4-nltrophenol.
`somes
`are a useful
`and suitable
`in vitro model
`for studying metabolic
`as In the
`routes,
`specific
`for man,
`case
`of
`ketotifen.
`Obviously,
`the
`results
`obtained
`only
`reflect
`partially
`the multiplicity
`of
`in vivo
`can
`events
`and Interpretation
`has to be complemented
`by Investigations
`with
`other
`models.
`
`Downloaded from
`
`dmd.aspetjournals.org
`
` at ASPET Journals on May 24, 2016
`
`came
`10 ODS
`Sphensorb
`Switzerland).
`(Basel,
`/3-glucuronidase
`UDPGA’
`(sodium
`salt),
`York),
`Co.
`X-100 were
`purchased
`from Sigma
`Chemical
`G-6-P,
`G-6-PDH
`came
`from Boehringer
`(Mann-
`
`from
`obtained
`livers were
`Human
`cincu-
`after
`were
`quickly
`removed
`further
`stored
`-80#{176}C. Microsomes
`at
`as
`previously
`described;
`under
`these
`activities
`have
`been
`shown
`previously
`
`Ltd.
`by Sandoz
`supplied
`Phase
`Sep
`(New
`from
`liver)
`and Triton
`(bovine
`(St. Louis, MO). NADP,
`heim, West Germany).
`Fractions.
`Preparation
`of Microsomal
`Livers
`kidney
`transplantation
`donors.
`latory
`arrest,
`frozen
`in dry
`and
`ice,
`liver
`were
`prepared
`from thawed
`experimental
`conditions,
`enzymatic
`to be well
`preserved
`(12).
`were
`incubations
`stated,
`otherwise
`when
`Except
`Incubation
`Procedure.
`5 mM
`pH 7.4, with
`buffer,
`phosphate
`performed
`in 1 ml ofSO mM sodium
`about
`5 psnol UDPGA,
`and
`MgCI2,
`containing
`100 nmol
`I’4C]ketotifen,
`the
`reaction
`was
`stopped
`30 mm,
`mg
`of
`microsomal
`protein.
`After
`tubes
`were
`vigorously
`shaken.
`of
`addition
`of
`2 ml
`acetonitrile
`and
`the
`Proteins
`were
`pelleted
`by
`a
`centrifugation
`at
`5000g.
`these
`10-mm
`Under
`conditions,
`pellets
`retained
`no
`radioactivity.
`Supernatants
`counted
`were
`for
`total
`radioactivity,
`evaporated
`to dryness
`under
`nitrogen,
`used
`and
`analysis
`of metabolites
`by HPLC
`(see
`below).
`Blanks
`were
`performed
`omitting
`either
`microsomes
`or UDPGA.
`For
`kinetic
`constant
`detenmina-
`and
`the
`tions,
`concentration
`ketotifen
`UDPGA
`varied
`2 to
`1tM and
`from 100 to
` zM,
`5000
`respectively.
`of metabolites
`rate
`formation
`The
`effect
`of protein
`concentration
`on the
`the
`6 mg
`and
`to
`from
`investigated
`with
`concentrations
`varying
`was
`10 and
`60 mm. All
`between
`dependence
`by varying
`incubation
`times
`time
`microsomes
`and
`with
`native
`determinations
`were
`performed
`in duplicate
`with
`microsomes
`treated
`by
`I volume
`of
`0.3% Triton
`X-lOO
`for
`30 mm
`at
`+4#{176}C(the
`fmal
`concentration
`of Triton
`in the
`incubation
`medium
`was
`0.06%).
`adding
`by
`tested
`was
`ketotifen
`toward
`activity
`Monooxygenase
`2.5 mM G-6-P,
`mM NADP,
`of0.l5
`system consisting
`NADPH-generating
`absence
`of UDPGA
`to the
`in the
`in the
`presence
`1 U/ml
`G-6-PDH
`performed
`by
`omitting
`the
`then
`Blanks
`were
`incubation
`medium.
`studies
`were
`performed
`by adding
`system.
`Inhibition
`NADPH-generating
`0.04 mM bilinubin
`or 0.1 mM 4-nitrophenol,
`both
`dissolved
`in 0.015
`either
`to
`M sodium
`hydroxide
`the
`incubations
`containing
`1.5 mg
`protein
`of
`Triton-treated
`microsomes.
`Control
`incubations
`were
`performed
`by addi-
`‘ Abbreviations
`used
`are:
`UDPGA,
`UDP-glucuronic
`acid;
`UDPGT,
`UDP-glucu-
`ronyltransferase;
`G-6-P,
`glucose-6-phosphate;
`G-6-PDH,
`glucose-6-phosphate
`dehydrogenase.
`
`2
`by
`
`for
`by
`
`250
`
`a
`
`of
`
`from
`
`0.5
`
`or
`
`enzyme
`is an
`(bilirubin,
`
`(2),
`(LY
`
`for
`
`only
`(rat,
`
`2.4.1.17)
`(EC
`UDP-glucuronyltransferase
`endogenous
`both
`in metabolic
`pathways
`UDP-glucuronyltransferase
`and
`exogenous
`substrates.
`the
`conjugation
`of UDP-glucuronic
`acid
`to
`groups,
`the most
`significant
`alcohols,
`being
`acids,
`thiols,
`and
`amines.
`Biotransformation
`cyproheptadine
`(1),
`tripelennamine
`methyl
`pyridine-substituted
`dioxane
`route
`fen
`(5)
`revealed
`a novel
`of
`ronidation
`oftertiary
`amines
`giving
`In
`compounds
`(6).
`these
`studies,
`urine
`found
`in
`of
`humans
`rhesus
`species
`dog,
`monkey)
`glucuronide
`in appreciable
`amounts.
`character-
`been
`have
`In
`rats,
`two
`UDP-glucuronyltransferases
`4-nitrophenol
`like
`substrates
`ized:
`one
`of
`them
`was
`active
`on
`other
`conjugating
`the
`was
`inducible
`3-methylcholanthrene,
`by
`9). This
`classification
`bilirubin
`and
`by
`phenobarbital
`induced
`by Wishart
`is
`roughly
`in
`with
`the
`one
`agreement
`et a!.
`“neonatal”
`groups
`(10),
`separating
`in
`“late-foetal”
`activities
`microsomes
`have
`according
`to
`their
`ontogenesis.
`In man,
`with
`veloc-
`been
`shown
`to conjugate
`numerous
`substrates
`in vitro,
`those
`ities
`comparable
`to
`observed
`in
`laboratory
`animals
`(1 1).
`the
`Nevertheless,
`small
`number
`of
`studies
`reported
`did
`not
`allow
`to
`those
`authors
`show
`whether
`UDP-glucuronyltransferase
`activ-
`ities
`were
`supported
`one,
`or more
`enzymes.
`by
`In
`this
`report,
`the metabolism
`of ketotifen
`(Zaditen),
`and
`antianaphylactic
`agent,
`particularly
`the
`kinetics
`of
`gi ucuronidation
`investigated
`human
`in vitro
`somes.
`
`involved
`steroids)
`catalyzes
`functional
`numerous
`carboxylic
`phenols,
`vivo with
`studies
`(3),
`cyclobenzaprine
`ketoti-
`108380)
`(4),
`N-glucu-
`the
`metabolism:
`drug
`ammonium
`to
`quaternary
`rise
`N-quaternary
`glucuronides
`and
`higher
`primates
`did
`not
`excrete
`this
`
`in
`
`and
`
`a
`
`were
`(7). Other
`new
`type
`
`of
`
`and
`
`(8,
`proposed
`and
`liver
`
`two,
`
`was
`
`using
`
`type
`a new
`its
`of
`N-
`liver micro-
`
`Materials.
`labeled
`at
`
`I’4ClKetotifen,
`the C-4
`position
`
`Materials
`specific
`the
`of
`
`and Methods
`acitity,
`107 MCi/mg,
`benzocycloheptathiophen
`
`purity
`
`>98%,
`ring,
`was
`
`Send reprint
`cocin#{233}tiques,
`Rueil-Malmaison,
`
`requests
`Laboratories
`France.
`
`to:
`
`Jean
`Sandoz
`
`Le Bigot
`F.
`S.A.R.L.,
`
`de Recherches
`Centre
`1 4 Boulevard
`Richelieu,
`
`Pharma-
`92506
`
`585
`
`1 of 5
`
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`
`

`
`hydroxide.
`not
`alter
`
`It has
`rate
`
`the
`
`BIGOT
`LE
`been
`verified
`of
`formation
`
`a
`
`586
`M sodium
`of0.0l5
`volume
`equal
`ofan
`tion
`of sodium
`hydroxide
`did
`presence
`the
`that
`glucuronide.
`N-quaternary
`ofthe
`analyzed
`were
`and metabolites
`drug
`Unchanged
`Analytical
`Procedure.
`Altex
`110 A
`consisted
`oftwo
`system
`by reversed
`phase
`HPLC.
`The HPLC
`France)
`and
`a
`(Beckman
`programmer
`pumps
`controlled
`by an Altex
`421
`West
`Ger-
`(Wildbad,
`Berthold
`LB
`503
`continuous
`radioactivity
`detector
`Spherisorb
`packed
`with
`many).
`An
`analytical
`300
`x 4.6 mm i.d.
`column
`were
`performed
`with
`10 ODS
`(particle
`size,
`10 pm) was
`used.
`Elutions
`carbamate
`solution
`programmed
`multistep
`gradient
`of 0.01 M ammonium
`ammonium
`carbamate
`and methanoL
`The
`proportion
`of methanol
`to
`solution
`was
`10% (v/v)
`at
`first
`and
`increased
`to 20,
`47.5,
`and
`100%
`70,
`after
`27, 42, 51, and
`60 mm,
`respectively.
`The
`total
`time
`for
`elution
`was
`70
`miii,
`including
`the
`fmal
`10 mm with
`100% methanoL
`The
`solvent
`flow rate
`was
`kept
`constant
`at 2 mi/mm.
`Dry
`samples
`were
`dissolved
`in 300
` tl of
`H O-methanol,
`50:50
`(v/v) mixture.
`After
`filtration,
`200
`s1 were
`injected.
`The
`radioactivity
`of
`the
`column
`effluent
`was
`detected
`by
`use
`of
`a glass
`of
`scintillator
`cell. The
`efficiency
`the
`radioactivity
`detector
`was
`25% and
`the limit
`ofdetection
`was
`1000 dpm which
`correspond
`to 4.2 ng (10 pmol)
`I’4Clketotifen.
`Retention
`reference
`incubation
`
`times
`samples.
`with
`
`metabolites
`formed
`the
`of
`metabolites
`were
`Polar
`$-glucuronidase.
`Metabolites
`
`compared
`were
`analyzed
`before
`were
`collected
`
`of
`those
`with
`and
`after
`and
`their
`
`1
`TABLE
`of ketot fen
`Metabolism
`in Materials
`for 30 mm as described
`were
`conducted
`incubations
`vitro
`In
`proteins
`and
`0.07 mM (‘4C)ketotifen.
`3 mg microsomal
`with
`and Methods
`as pmol metabolites
`formed
`in 1 mm by 1 mg protein
`expressed
`Results
`are
`for one
`typical
`experiment.
`NADPH
`Generating
`System
`microsomes
`Native
`
`Triton-treated
`
`microsomes
`
`+
`-
`+
`
`+
`-
`+
`
`-
`+
`+
`
`-
`+
`+
`
`<1
`41
`16
`<1
`154
`48
`
`38
`<1
`39
`3
`2
`6
`
`251
`<1
`282
`110
`<1
`105
`
` j D JA
`
`i I
`
`N-Oxide
`
`Nor-ketotifen
`
`and
`=
`
`was
`giving
`the
`same
`in the
`urine
`gl ucuronide
`methods,
`gi ucuronic
`piperilydene
`ronides
`
`obtained
`
`ion
`
`the
`
`was
`
`to
`
`No
`
` sn
`
`7 N
`
`V
`
`K#{149}totlf#{149}n
`
`N-GlUcUrOk.tOtlfsfl
`
`R. jc#{149}d
`
`kstotlfsn
`
`N-oxlds
`
`Fio.
`
`1. Proposed
`
`major metabolic
`
`pathways
`
`ofkeio: fen
`
`in man.
`
`ET
`
`AL.
`
`confirmed
`structures
`a Nermag
`Briefly,
`supplemented
`trometer
`France)
`Malmaison,
`injector
`solid
`glass
`SE
`coated
`with
`taken
`spectra
`were
`70
`was
`eV and
`were
`metabolites
`
`spectrometry
`by mass
`Sidar
`11A gas
`R 10-lOB
`a PDP/8
`computer
`by
`used.
`The
`chromatograph
`was
`a glass
`capillary
`column
`and
`(Chrompack,
`Middleburg,
`52
`in the
`electron
`ionization
`the
`emission
`current
`analyzed
`before
`or after
`
`(13).
`elsewhere
`as described
`spec-
`chromatography-mass
`Rueil-
`system
`(Nermag,
`with
`was
`equipped
`(30 m x
`0.3 mm i.d.)
`The
`Netherlands).
`The
`The
`ionization
`voltage
`isA.
`Parent
`drug
`and
`with
`fl-glucuronidase.
`
`mode.
`250
`was
`incubation
`
`a
`
`Results
`different
`with
`
`metabolites
`human
`liver
`
`ketotifen
`of
`microsomes
`
`are
`
`The
`Pathways.
`Metabolic
`incubation
`after
`formed
`in vitro
`1.
`1 and
`fig.
`displayed
`in table
`of
`a NADPH-generating
`In
`the
`presence
`catalyzed
`the
`formation
`of N-oxidized
`the
`nor-ketotifen.
`Microsomes
`catalyzed
`in the
`when
`ketotifen
`UDPGA
`was
`added
`latter
`a NADPH-generating
`system.
`In the
`was
`of
`the
`glucuronide
`reduced,
`suggesting
`conjugation
`oxidation
`and
`reactions.
`When
`and
`ketotifen
`ried
`out with
`cofactors
`in the
`formed.
`were
`no metabolites
`in HPLC
`time
`retention
`the
`longest
`had
`Nor-ketotifen
`ion
`the molecular
`by
`by GLC/MS
`was
`characterized
`fragment
`at m/z
`a characteristic
`295
`(M . )
`and
`=
`time
`metabolite.
`Its
`retention
`a minor
`N-oxidized
`ketotifen
`was
`analysis
`during
`GLC/MS
`itself
`51 miii
`and
`it decomposed
`unchanged
`drug.
`had
`formed
`in vitro
`N-Glucuroketotifen
`glucuronide
`found
`as
`HPLC
`retention
`time
`(22 mm)
`ofthis
`N-quaternary
`of
`treated
`patients.
`The
`structure
`proved
`by different
`formed
`in vivo has
`been
`previously
`the
`showed
`that
`including
`NMR
`spectroscopy,
`which
`the
`acid
`residue
`coupled
`the
`nitrogen
`of
`atom
`ring
`forming
`a N-quaternary
`derivative
`(5). Glucu-
`from
`both
`in vivo and
`experiments
`formed
`in vitro
`
`Downloaded from
`
`dmd.aspetjournals.org
`
` at ASPET Journals on May 24, 2016
`
`of
`
`of
`
`microsomes
`system,
`mainly
`and
`ketotifen
`of N-glucuro-
`formation
`or
`presence
`absence
`the
`formation
`instance,
`a competition
`between
`incubations
`were
`car-
`absence
`of microsomes,
`(74 mm)
`at m/z
`253.
`
`2 of 5
`
`PENN EX. 2040
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`
`

`
`METABOLISM
`
`OF
`
`KETOTIFEN
`
`BY
`
`HUMAN
`
`LIVER
`
`MICROSOMES
`
`incubation
`after
`61 mm (M.’),
`detergent,
`the
`
`with
`-
`
`for
`with
`
`and
`
`fig.
`
`N-
`
`of
`
`to
`for
`
`of
`
`these
`native
`and
`100
`
`the
`com-
`the
`
`in
`
`both
`
`A
`
`0
`
`1500
`
`Incubations
`(S)
`or Triton
`carried
`out
`
`FIG.
`carried
`were
`X-100-deterged
`for 30 mm with
`
`mm
`2. Effect
`concentration
`andprotein
`ofincubation
`oftime
`0.07 mM ketotifen.
`5 mM UDPGA
`out with
`are
`expressed
`as pmol
`microsomes
`(0).
`Results
`native
`(#{149})or Triton
`X-100-deterged
`microsomes
`
`and
`
`3
`
`1
`N-quaiernary
`glucuronide
`with
`3 mg
`were
`conducted
`conjugated
`by
`I mg microsomal
`are
`expressed
`as pmol
`ketotifen
`
`on ketoqfen
`incubations
`A,
`ketotifen
`(0).
`Results
`
`5
`
`S
`
`4
`formation.
`microsomes
`native
`protein
`incubations
`were
`B,
`protein.
`conjugated/minute.
`
`for
`
`587
`
`the
`As
`
`glucuromdation
`expected,
`the KM from
`also
`when
`ketotifen
`
`treat-
`250
`and
`
`ofUDPGA,
`determined.
`the V
`but
`were
`very
`close
`
`concentrations
`lower
`2). At
`(table
`low to be
`accurately
`was
`too
`rate
`with
`detergent
`increased
`ment
` sM.
`The
`V,,,,,
`observed
`to 440
`varied
`(table
`2).
`UDPGA
`by Microsomes
`Glucuronidatlon
`Results
`are
`shown
`in
`table
`3. No
`served
`between
`sexes.
`Solubilization
`glucuronidation
`rate
`about
`3-fold,
`Inhibition
`of N-GIUCUTOnId*tIOn
`two
`KM observed
`with
`The
`two
`isoenzymes.
`two
`of
`So,
`curonyltransferase,
`namely,
`tested
`for
`their
`to inhibit
`
`Human
`difference
`enhanced
`the
`sex.
`of
`and 4-NItrOpbeDOI.
`suggest
`the
`presence
`ketotifen
`substrates
`of UDP-glu-
`known
`well
`and
`4-nitrophenol,
`were
`bilirubin
`the N-glucuronidation
`ketoti-
`
`from Different
`significant
`by
`Triton
`irrespective
`by BIIITUbIn
`might
`
`Livers.
`was
`ob-
`the
`N-
`
`of
`
`ability
`
`Downloaded from
`
`dmd.aspetjournals.org
`
` at ASPET Journals on May 24, 2016
`
`Kinetic
`
`constants
`
`ofkeiot fen
`
`by human
`
`liver
`
`KM determination
`For
`determination
`of UDPGA,
`carried
`out
`30 mm with
`for
`native
`and
`detergent-treated
`
`of
`
`was
`UDPGA
`was
`0.19 mM.
`1.5 mg
`of protein,
`
`5 mM;
`Incubations
`respectively,
`
`for KM
`were
`for
`
`2
`TABLE
`N-glucuronidation
`microsomes
`ketotifen,
`ketotifen
`2.2 and
`microsomes.
`KM
`
`Vm
`
`Detergent-
`treated
`Native
`microsomcs
`microsomes
`pmi. I
`x n#{252}n’X mf’
`protein
`19
`125
`50
`43
`
`118
`
`Substrate
`
`Ketotifen
`
`UDPGA
`
`Native
`microsomes
`
`Detergent-
`treated
`microsomes
`
` M
`
`12.5
`100
`250
`
`42
`
`440
`
`B
`
`0
`
`fl-glucuronidase
`309
`in GLC/MS).
`m/z
`a dual
`action
`on
`exerted
`in
`of N-oxide
`and
`nor-
`formation
`the N-glucuronidation.
`However,
`a competition
`between
`oxidation
`observed
`again.
`conjugation
`
`reaction,
`
`its
`
`kinetic
`
`ketotifen
`unchanged
`retention
`time
`(HPLC
`=
`X-l0O,
`a nonionic
`Triton
`vitro metabolism.
`It
`inhibited
`and
`strongly
`enhanced
`ketotifen
`in the
`presence
`of Triton
`X-lOO,
`and
`conjugation
`pathways
`was
`further
`characterize
`the
`To
`parameters
`were
`determined.
`Concentration
`of Protein
`Dependence
`on the
`Time
`and Effect
`linear
`up
`reaction
`was
`in fig.
`2,
`the
`As
`shown
`N-Glucuronldatlon.
`both
`to 60 mm with
`microsomes,
`and
`detergent-treated
`and
`native
`and
`detergent-
`native
`up
`to 6 and
`1.5 mg
`protein,
`respectively,
`with
`with microsomal
`treated
`microsomes.
`This
`latter
`result
`verified
`was
`formation
`of
`the
`The
`from
`a different
`liver.
`rate
`of
`preparation
`protein
`concentra-
`quaternary
`glucuronide
`was
`a
`function
`of
`the
`tion
`during
`incubation,
`even
`if
`the
`velocity
`(expressed
`as
`pmol
`1 mm by
`glucuronide
`formed
`1 mg
`protein)
`was
`different.
`Thus,
`30 mm with
`1.7
`all
`further
`determinations
`were
`performed
`2.6 mg
`protein
`for
`native
`microsomes
`1.2 to
`1.8 mg
`detergent-treated
`microsomes.
`the N-Glucuronldation.
`Parameters
`Results
`Kinetic
`of
`3. With
`determinations
`displayed
`in
`table
`2 and
`are
`at
`12.5
`microsomes,
`two KM for
`ketotifen
`were
`observed
`ELM. After
`treatment
`with
`detergent,
`UDP-glucuronyltransferase
`exhibited
`only
`one KM value
`at
`about
`40 sM.
`Simultaneously,
`of detergent-treated
`microsomes
`was
`greatly
`enhanced
`Vm.z
`pared
`to
`the
`two
`V
`untreated
`microsomes.
`By
`varying
`of
`UDPGA
`concentrations
`from
`0.1
`to 5 mM,
`only
`KM and
`one V
`were
`obtained
`with
`native
`and
`detergent-treated
`microsomes
`
`3 of 5
`
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`
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`
`dmd.aspetjournals.org
`
` at ASPET Journals on May 24, 2016
`
`LE
`
`BIGOT
`
`ET
`
`AL.
`
`.
`Native
`0 Detergent
`
`microsomes
`treated-microsomeS
`
`.
`
`0.4
`
`IV
`
`-1
`
`(pmol
`
`588
`
`Incubations
`microsomes
`
`carried
`were
`Velocity
`
`(0).
`
`3. Double
`FIG.
`for 30 min with
`out
`is expressed
`as pmol
`
`reciprocalplo:
`5 mM UDPGA
`ketotifen
`conjugated
`
`by human
`of N-glucuronidation
`of kelotVen
`and
`2.2 mg protein
`for native microsomes
`in 1 min
`by
`I mg protein
`and
`ketotifen
`
`(1iM 1)
`
`1
`k.totifefl
`liver microsomes.
`(#{149})and
`I.5 mg protein
`concentration
`in M.
`
`for detergent-treated
`
`3
`TABLE
`liver microsomes
`by human
`ofketot fen
`rate
`N-Glucuronidation
`protein,
`the mean
`x mg’
`as pmol
`x min’
`Results
`are
`expressed
`or
`individual
`performed
`determinations
`were
`when
`three
`or more
`Incubations
`were
`when
`only
`two
`determinations
`carried
`out.
`ducted
`for 30 min, with
`0.1 mM ketotifen,
`5 mM UDPGA,
`of proteins
`for
`native
`microsomes,
`and
`1.2 to
`1.8 for
`microsomes.
`
`± SD
`values
`con-
`were
`1.7 to 2.6 mg
`detergent-treated
`
`Natives
`
`microsomes
`
`Detergent-treated
`
`microsomes
`
`Fold-up
`
`increase
`
`due
`
`to detergent
`
`33
`
`14
`
`30
`
`Men
`±
`(4)
`98 ±
`(3)
`3.3 ± 0.5
`(3)
`
`7
`
`Women
`36 ±
`(3)
`61-90
`(2)
`2.1-2.4
`(2)
`
`substrates
`two
`fen.
`concentration
`a
`at
`glucuronyltransferase
`human
`bilirubin
`nor
`neither
`ofketotifen:
`values
`tion
`obtained
`respectively,
`the
`presence
`sodium
`
`These
`fmal
`
`were
`of
`
`added
`were
`corresponding
`(14,
`
`to
`to
`
`the
`their
`Under
`15).
`inhibited
`1 16 pmol-
`for
`control
`
`medium
`incubation
`KM for
`respective
`conditions,
`these
`the N-glucuronida-
`min’
`- mg
`protein,
`incubations
`(i.e.
`
`in
`
`4-nitrophenol
`of
`1 15 and
`vs.
`122
`hydroxide).
`
`purpose
`liver
`
`Discussion
`study
`present
`ofthe
`the metabolic
`microsomes,
`the
`N-glucuronidation,
`
`was
`
`to investigate
`pathways
`a novel
`route
`
`in vitro with
`of ketotifen
`and
`of
`biotransfor-
`
`present
`allow
`
`the
`metabolites
`and
`in vivo
`N-glucuroketotifen.
`
`results,
`authors
`
`compared
`to
`draw
`found
`urine,
`Two
`
`2)
`
`were
`in human
`
`vivo
`
`obtained
`those
`with
`in
`conclusions.
`following
`the
`human
`liver micro-
`in vitro with
`namely
`nor-ketotifen,
`N-oxide,
`additional
`metabolites,
`the
`
`re-
`
`in
`1)
`
`The
`human
`particularly
`mation.
`The
`man,
`Common
`somes
`and
`
`not
`This
`reductase,
`in microsomal
`under
`partial
`occur
`investigated
`in the
`not
`only
`formed
`in the
`was
`ofa NADPH-generating
`presence
`biotransformation
`of
`Such
`results
`obtained
`a
`glucuronide
`of
`cyproheptadine,
`and
`ketotifen
`species,
`this
`observed
`(dog,
`was
`baboon)
`(5, 7).
`monkey,
`appropriate
`material
`
`ketotifen
`urine
`
`derivative
`but
`were
`conditions.
`ketone
`of
`absent
`may
`was
`
`forming
`vivo with
`108
`380,
`animal
`not
`
`only
`
`duced
`human
`incubation
`location
`virtually
`duction
`which
`roketotifen
`tive
`ofthe
`that
`a prior
`glucuronidation.
`bility
`of
`shown
`in
`prine,
`LY
`investigated
`reaction
`rhesus
`the
`action.
`of
`determination
`The
`in native
`glucuronidation
`but
`only
`ketotifen,
`ities
`for
`for
`ketotifen
`may
`KM values
`of UDP-glucuronyltransferase.
`by
`the
`data
`of Mahu
`et a!.
`liver,
`UDP-glucuronyltransferase
`likely
`explanation
`is based
`UDP-glucuronyltransferase
`may
`of
`the
`substrate
`enzyme,
`affinity
`of
`the
`reticulum
`endoplasmic
`by
`the
`observation
`ofonly
`treated
`microsomes:
`in
`bedded
`in
`the membrane,
`strate
`and
`thus
`exhibit
`
`and
`
`glucuronide,
`its
`found
`in
`vitro
`be
`due
`could
`a
`cytosolic
`preparations.
`anaerobic
`present
`presence
`
`the
`
`in
`
`is
`re-
`
`the
`
`recovered
`were
`the
`described
`under
`intracellular
`to
`(16)
`which
`enzyme
`In microsomes,
`conditions,
`a protocol
`study.
`3) The
`N-glucu-
`of UDGPA
`irrespec-
`system.
`This
`indicates
`for
`is
`not
`necessary
`ketotifen
`possi-
`confirm
`the
`in vitro
`already
`amine
`as
`tertiary
`a
`cyclobenza-
`tripelennamine,
`among
`(1-5).
`However,
`conjugation
`and
`typical
`unusual
`route
`(rabbit,
`rat)
`or was
`a minor
`So,
`human
`liver
`microsomes
`were
`to
`characterize
`this
`enzymatic
`re-
`
`of
`parameters
`kinetic
`the
`two
`showed
`microsomes
`These
`one
`for UDPGA.
`oftwo
`reflect
`the
`presence
`may
`This
`hypothesis
`indicating
`that
`in human
`is heterogeneous
`accessibility
`the
`ketotifen:
`vary
`and
`greatly
`in relation
`its
`to
`membrane.
`This
`one KM value
`case,
`enzyme
`but
`are
`equally
`only
`one
`apparent
`
`on
`for
`
`ketotifen
`the
`N-
`affm-
`apparent
`different
`two
`isoenzymes
`supported
`be
`as
`in rat
`liver,
`A most
`(15).
`the microsomal
`concentration
`the
`apparent
`location
`in the
`is strengthened
`in detergent-
`are
`not
`to
`the
`constant.
`
`em-
`sub-
`In
`
`of
`local
`affect
`
`the
`thus
`transverse
`hypothesis
`for
`ketotifen
`molecules
`accessible
`affinity
`
`that
`
`4 of 5
`
`PENN EX. 2040
`CFAD V. UPENN
`IPR2015-01835
`
`

`
`METABOLISM
`
`OF
`
`KETOTIFEN
`
`BY
`
`HUMAN
`
`MICROSOMES
`
`589
`
`value
`that
`
`the
`Vmaz
`indicating
`active.
`and
`
`and
`the
`
`to
`
`a
`
`rat
`at
`inhibit
`and
`its KM,
`added
`able
`strengthen
`isoenzyme
`Further
`
`The
`likely
`
`as
`only
`has
`
`of
`
`to
`
`of
`
`fmding
`
`be
`are
`
`the
`
`and
`
`human
`by
`ketotifen
`of
`pathways
`metabolic
`four
`was
`absent
`which
`pathway,
`enzymes.
`cytosolic
`in vitro model
`suitable
`The
`advantage
`of
`the
`predictive
`value
`regarding
`of
`investigation
`unusual
`the
`N-glucuronidation
`incomparable
`advantages.
`shown
`by
`case
`of
`the
`partially
`the multiplicity
`to be
`considered
`carefully
`
`of
`
`the
`of
`in
`
`solu-
`detergent
`by
`increased
`is markedly
`addition,
`enzyme
`molecules
`of
`number
`a
`larger
`bilization,
`liver
`microsomes
`values
`of KM of human
`These
`were
`fully
`reported
`previously
`are
`close
`to
`those
`UDPGA
`for
`ketotifen
`and
`UDPGA
`by Mahu
`et a!., whereas
`the Vm,
`for
`4-nitrophenol
`native
`and
`detergent-treated
`microsomes
`were
`both
`measured
`with
`generally
`reported
`with
`low
`compared
`values
`(1 1,
`15). On
`Vmax
`stimulation
`of
`the Vmaz
`by Triton
`X-lOO
`was
`the
`the
`other
`hand,
`found
`by other
`authors
`(15)
`and
`quite
`similar
`comparable
`to those
`(table
`3). No
`sex
`difference
`for
`the N-glucuron-
`from
`liver
`liver
`to
`was
`observed,
`as
`it has
`already
`been
`reported
`idation
`of
`ketotifen
`for
`some
`other
`drug-metabolizing
`enzymes
`from
`human
`liver
`(12).
`In rat,
`bilirubin
`and
`4-nitrophenol
`are
`the well
`known
`substrates
`two
`UDP-glucuronyltransferase
`isoenzymes,
`induced,
`respec-
`of
`9).
`tively,
`by
`phenobarbital
`3-methylcholanthrene
`(8,
`In man,
`Mahu
`(15)
`drew
`conclusion
`of
`a likely
`heterogeneity
`et a!.
`UDP-glucuronyltransferase
`using
`two
`substrates
`as models.
`these
`In
`the
`present
`investigation,
`none
`compounds
`was
`able
`of
`these
`diminish
`the
`glucuronidation
`rate
`of
`ketotifen
`under
`our
`experi-
`mental
`conditions.
`With
`microsomes,
`use
`a
`similar
`by
`to
`procedure,
`4-nitrophenol
`concentration
`close
`its KM,
`has
`been
`shown
`to partially
`bilirubin
`and methylumbeffi-
`(17)
`ferone
`(18)
`conjugation,
`conversely,
`bilirubin,
`at
`a concentra-
`tion
`roughly
`close
`inhibits
`4-nitrophenol
`conjugation
`to the
`previous
`indicating
`(17).
`These
`observations,
`species
`that man
`is the
`sole
`to form
`N-quaternary
`glucuronide
`in appreciable
`amounts,
`the
`assumption
`that
`a specific
`UDP-glucuronyltransferase
`might
`involved
`in
`the
`in
`progress
`reaction.
`N-glucuronidation
`studies
`now
`assumption.
`to verify
`this
`biotransformation
`the
`In
`conclusion,
`of
`exhibited
`three
`liver
`microsomes
`observed
`in vivo in man.
`reduction
`in the
`in vitro
`study,
`is
`to
`implicate
`Human
`liver microsomes
`are
`a useful
`studying
`drug
`metabolism
`in man.
`for
`which
`is
`of major
`interest
`is
`its
`system
`human
`species.
`In
`particular,
`for
`the
`metabolic
`routes-as
`the
`case
`of
`in
`ketotifen-the
`system
`demonstrates
`the
`However,
`results
`obtained,
`of
`reduction
`ketotifen,
`reflect
`the
`in the
`events
`systems;
`this
`in vivo
`the
`interpretation
`of
`data.
`in vitro
`are
`The
`authors
`Acknowledgments.
`for
`Kreis,
`and
`to France-Transplant
`Julien-Larose
`ples.
`They
`thank
`Miss
`C.
`providing
`analysis,
`Dr.
`R. Voges
`for
`Mrs.
`for
`typing
`the manuscript.
`
`indebted
`providing
`her
`for
`radiolabeled
`
`Jilet
`
`to Dr.
`adult
`help
`
`Dr.
`Frantz,
`sam-
`human
`in GLC-MS
`ketotifen,
`
`and
`
`1. C. C. Porter,
`Vandenheuvel:
`
`B. H. Arison,
`Human
`
`References
`D. C. Titus,
`V. F. Gruber,
`metabolism
`ofcyproheptadine.
`
`J. A.
`and W.
`Drug Metab.
`
`4. A.
`
`5.
`
`7. L.
`
`10. G.
`
`12.
`
`13.
`
`C.
`
`14. M.
`
`15.
`
`17. G.
`
`18.
`
`F.
`
`LIVER
`3, 189-197
`(1975).
`Dispos.
`D.
`R. C. Luders,
`J. Manniello,
`M.
`0. A.
`Servando,
`2. N. K. Chaudhuri,
`Metabolism
`oftripelennamine
`in man.
`K. Chao,
`and M. F. Bartlett:
`(1976).
`Drug Melab.
`Dispos.
`4, 372-378
`S. D. White,
`J. Balletto,
`3. H. B. Hucker,
`S. C. Stauffer,
`A.
`and
`B. H. Anison:
`Physiological
`disposition
`and
`cyclobenzapnine
`in the
`rat,
`dog,
`rhesus
`monkey,
`6, 659-672
`(1978).
`Metab.
`Dispos.
`Crabtree,
`Rubin,
`P. H.
`Dhahir,
`R.
`E.
`disposition
`of
`l-3-[(dimethylamino)-(m-dioxan-5-yl)methylj
`dine
`in man.
`7, 149-154
`Drug.
`Metab.
`Dispos.
`J. R. Kiechel,
`E. Schreier,
`and K. Zehnder:
`ADME
`studies
`in animals
`and man.
`Sandoz,
`6. Z. H.
`Israili,
`P. G. Dayton,
`and
`J. R. Kiechel:
`metabolism.
`A survey.
`Drug
`Metab.
`Dispos.
`J.
`Fischer,
`R.
`L. Thies,
`D. Charkowski,
`Formation
`and
`urinary
`excretion
`of
`cyproheptadine
`monkeys,
`chimpanzees,
`and
`humans.
`Drug.
`Metab.
`424 (1980).
`8. K. W.
`Remmer,
`Fr#{246}hling, H.
`Bock,
`and
`3-methylcholanthrene
`phenobarbital
`rat
`liver microsomal
`UDP-Glucuronyltransferase.
`327, 46-56
`Acta
`(1973).
`and H. Pfeil:
`W. Lilienblum,
`9. K. W. Bock, D.
`Josting,
`UDP-Glucuronyltransferase.
`rat-liver
`microsomal
`inducible
`by
`3-methylcholanthrene
`two
`enzyme
`forms
`98, 19-26 (1979).
`J. Biochem.
`bital.
`Eur.
`J. Wishart,
`M. T. Campbell,
`and
`G.
`geneity
`of UDP-Glucuronyltransferase.
`(A. Aitio,
`in Drug
`Biotransformation”
`Press,
`1979.
`North
`Holland
`Biomedical
`A. M. Batt,
`P. Marli#{234}re,and G. Siest: UDP-
`11. J. A. Boutin,
`A.
`Jacquier,
`activities
`in
`human
`liver
`microsomes
`Glucuronyltransferase
`Bio-
`(1981).
`30, 2507-2510
`chem.
`Pharmacol.
`S. Columelli,
`T. Cresteil,
`J. Dc Graeve,
`P. Kremers,
`P. Beaune,
`Lenoux,
`and
`J. E. Gielen:
`Cytochrome
`P-450 monooxygenase
`ities
`in human
`and
`rat
`liver microsomes.
`J. Biochem.
`Eur.
`606 (1981).
`Lavtne,
`D.
`M. Guerret,
`Julien-Larose,
`its metabolites
`fication
`ketotifen
`and
`of
`chromatography-mass
`spectrometny.
`136-142
`(1983).
`Determination
`Heinwegh:
`P. M.
`K.
`Billing,
`Black,
`B. H.
`in
`needle-biopsy
`activity
`transferase
`bilirubin
`UDP-Glucuronyl
`specimens
`of human
`liver.
`29, 27-35
`(1970).
`Acta
`Clin.
`Chim.
`P. Berthelot:
`Characterize-
`P. Mavier,
`and
`A. M. Preaux,
`J. L. Mahu,
`and
`p-nitrophenol
`uridine
`diphosphate
`tion
`ofmicrosomal
`bilirubin
`in human
`a comparison
`glucuronyltransferase
`activities
`rat
`liver.
`(1981).
`26,
`93-102
`Enzyme
`16. R. L. Felsted,
`and N. R. Bachur:
`Ketone
`Basis
`ofdetoxication”
`(W. B. Jakoby,
`1980.
`Press, New York,
`and
`J. Mulder:
`Biinubin
`diphosphate
`glucuronyltransfenase
`289,
`284-292
`(1972).
`Ada
`Sorgel,
`F.
`E.
`Beyhl,
`and
`phosphate
`glucunonyltransferase
`36,
`861-863
`(1980).
`
`A. G. Zacchei,
`metabolism
`and man.
`
`of
`Drug
`
`and
`
`D.
`
`P. Henry:
`
`The
`pyri-
`
`(1979).
`20-5 11).
`(HC
`Ketotifen
`January
`40
`pp.
`1977,
`of drug
`routes
`Novel
`5, 41 1-415
`(1977).
`J. Donham:
`and
`K.
`glucuronide
`Dispos.
`
`in
`8, 422-
`
`Downloaded from
`
`dmd.aspetjournals.org
`
` at ASPET Journals on May 24, 2016
`
`and
`on
`
`Effects
`B. Rexer:
`specificity
`substrate
`Biochim.
`Biophys.
`
`Purification
`Separation
`or phenobar-
`
`of
`of
`
`of
`of
`
`Functional
`J. Dutton:
`In
`“Conjugation
`ed),
`pp.
`179-187.
`
`hetero-
`Reactions
`Elsevien/
`
`P.
`J.
`activ-
`118, 599-
`
`and
`in
`Biomed.
`
`J. R. Kiechel:
`human
`plasma
`Mass
`Spectrom.
`
`Quanti-
`by
`
`gas
`10,
`
`of
`
`with
`
`liven:
`
`In “Enzymatic
`reductases.
`ed),
`vol.
`1, p. 281. Academic
`
`heterogeneity
`from
`
`of microsomal
`liver.
`Biochim.
`
`rat
`
`uridine
`Biophys.
`
`E. Mutschler:
`caused
`
`Inhibition
`furosemide.
`
`by
`
`of
`
`uridine-di-
`Experientia
`
`W.
`
`and
`
`5 of 5
`
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