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`
`jpet.aspetjournals.org
`
` at Umd Of New Jersey on March 28, 2013
`
`0022-3565/96/2783-1252$03.OO/O
`Jouic(cid:1)u
`OF PHARMACOLOGY (cid:1)r(cid:1)n
`THE
`by The American
`Copyright
`C 1996
`278:1252-1261,
`JPET
`1996
`
`Exp(cid:1)xmtzwr(cid:1)ii
`THERAPEUTICS
`for Pharmacology
`Society
`
`and
`
`Experimental
`
`Therapeutics
`
`vol.
`Printed
`
`278, No. 3
`in USA.
`
`of
`Characterization
`Effects
`of Olopatadine
`Ocular Allergic
`Diseases
`
`and Antihistaminic
`the Ocular Antiallergic
`(AL-4943A),
`a Novel Drug for Treating
`
`N. A. SHARIF,
`
`S. X. XU, S. T. MILLER,
`A/con
`Laboratories,
`Inc., Fort Worth,
`Texas
`Accepted
`for publication
`May 30, 1996
`
`D. A. GAMACHE
`
`and
`
`J. M. YANNI
`
`acid
`de-
`
`ABSTRACT
`[(Z)-1 1 -[3-(dimethylami-
`KW-4679)
`(AL-4943A;
`Olopatadine
`1 -dihydrodibenz[b,e]oxepine-2-acetic
`no)propyiidene]-6,1
`under
`drug
`antiailergic/antihistaminic
`is an
`hydrochloride]
`the compound
`of
`ocular
`use. The effects
`topical
`for
`velopment
`(histamine,
`tryptase
`of proinflammatory
`mediators
`on
`release
`human
`conjunctival
`and
`prostagiandin
`D2) from monodispersed
`mast
`cells were
`assessed.
`Histamine
`subtype
`binding
`receptor
`affinities
`and
`functional
`potencies
`were
`determined
`ligand
`with
`binding
`and
`phosphoinositide
`turnover
`assays,
`respectively.
`and
`Olopatadine
`inhibited
`the
`release
`of histamine,
`tryptase
`(IC50
`prostaglandin
`D2,
`in a concentration-dependent
`manner
`Evaluation
`of
`the
`interaction
`of olopatadine
`559
`(cid:1)tM).
`with
`=
`the H1
`histamine
`receptors
`revealed
`a relatively
`high
`affinity
`for
`(K1 = 31 .6 nM,
`lower
`receptor
`, n
`but
`pK,
`=
`7.5
`±
`0.1
`=
`7)
`100 MM, p/<1 =
`(K1 =
`n =
`affinities
`for H2 receptors
`4.0
`±
`0.19,
`(K, = 79.4
`7) and H3 receptors
`(cid:1)tM,
`pK1 =
`7). The
`n =
`4.1
`±
`0.16,
`H1
`selectivity
`of
`olopatadine
`was
`superior
`to
`that
`of
`other
`
`such
`
`as ketotifen,
`studied,
`levo-
`antihistamines
`used
`ocularly
`The
`profiling
`pheniramine.
`of
`ol-
`antazoline
`and
`cabastine,
`opatadine
`binding
`assays
`receptor
`revealed
`in 42 nonhistamine
`nonhistamine
`two
`only
`with
`that
`olopatadine
`interacts
`receptor/
`(plC50
`5-6).
`degree
`Olopata-
`uptake
`sites
`to any
`significant
`phosphoinositide
`turnover
`in
`dine
`inhibited
`histamine-induced
`(IC50 =
`10 nM,
`cells
`human
`conjunctival
`epithelial
`plC50
`=
`8.0,
`(IC50 = 1 5.8-31
`n = 4) and in other
`.6 nM,
`cells
`ocular
`human
`apparent
`noncompetitive
`an-
`plC50
`7.5-7.8)
`and
`exhibited
`with
`an estimated
`dissocia-
`tagonist
`properties
`in these
`cells,
`n = 6). This combi-
`tion
`constant
`(Kb) of 1 9.9
`nM (pKb
`7.7,
`inhibition
`and selective
`H1
`nation
`of mast
`cell mediator
`release
`olopatadine
`may
`be partic-
`that
`receptor
`antagonism
`suggests
`ularly
`useful
`in the treatment
`of ocular
`allergic
`diseases.
`Indeed,
`olopatadine
`has
`recently
`shown
`clinical
`efficacy
`in an allergic
`conjunctivitis
`model
`in human
`subjects.
`
`to
`responses
`Schwartz,
`and
`strongly
`suggest
`
`agents
`pharmacological
`et
`at.,
`Irani
`1989;
`experimental
`that
`should
`be
`evalu-
`methods
`have
`target
`cells
`
`Recently,
`in
`
`these
`
`treating
`as
`target
`drug
`
`conjunctivitis
`cells.
`evaluation
`
`Allergic
`characterized
`is
`conjunctivitis
`presence
`edema
`itching,
`of hyperemia,
`1993;
`Schaefer,
`and
`Abelson
`1991;
`at.,
`cell
`resident
`mast
`of
`the
`granulation
`of preformed
`tissue,
`conjunctival
`release
`sized
`proinflammatory
`mediators
`and
`of appropriate
`produce
`the
`receptors
`Because
`histamine
`is the
`predominant
`mediator
`initiating
`the
`symptoms,
`alone
`nists,
`or
`in
`combination
`vasoconstrictors,
`or
`mast
`Schaefer,
`Berdy
`1993;
`et at.,
`and
`control
`to alleviate
`choice
`It
`has
`been
`demonstrated
`species
`and
`from
`different
`differ
`in morphological,
`ties
`(Katz
`, 1985;
`et
`at.
`
`with
`cell
`1991)
`ocular
`that
`mast
`tissues
`within
`and
`cytochemical
`Irarn
`1990).
`et at.,
`
`newly
`
`the
`by
`(Berdy
`et
`De-
`1993).
`in
`human
`synthe-
`activation
`noted
`above.
`mast
`cell
`antago-
`agonist
`and
`of
`
`been
`
`clinically
`tearing
`and
`Li
`et
`at.,
`population
`and
`subsequent
`symptoms
`preformed
`histamine
`topical
`adrenergic
`alpha
`(Abelson
`stabilizers
`drugs
`the
`have
`diseases.
`allergic
`from
`different
`cells
`same
`animal
`the
`functional
`proper-
`Mast
`cell
`popula-
`
`Received
`
`for
`
`publication
`
`March
`
`18,
`
`1996.
`
`tions
`in their
`differ
`also
`(Befus
`at.,
`et
`1987;
`Iram
`observations
`These
`1990).
`drugs
`for
`antiallergic
`HCMC
`using
`ated
`by
`developed
`to
`allow
`been
`(Miller
`1996).
`et at.,
`has
`Histamine
`of proinfiammatory
`ing an allergic
`antihistamiic
`tivity
`response
`rently
`available
`conjunctival
`lems,
`such
`onset
`of
`1991),
`that
`cies
`have
`antagonists.
`
`as
`action
`limit
`prompted
`Although
`
`role
`cell
`
`a
`
`has
`
`a cascade
`in evoking
`a well-established
`dur-
`degranulation
`events
`upon mast
`use
`of
`the
`Therefore,
`response
`(Beavan,
`1982).
`hypersensi-
`immediate
`drugs
`to attenuate
`an
`is well
`of
`the
`However,
`many
`known.
`histamine
`antagonists
`for
`treating
`hypersensitivity
`have
`combination
`low affinity/selectivity
`for H1
`receptors,
`and
`short
`duration
`of
`action
`(Berdy
`their
`clinical
`use
`in humans.
`These
`further
`research
`to
`discover
`this
`strategy
`recently
`
`of
`
`cur-
`ocular
`prob-
`slow
`at.,
`et
`deficien-
`better
`culminated
`
`H1
`
`ABBREVIATIONS:
`
`HCE, human
`
`conjunctival
`
`epithelial;
`
`HCF, human
`
`comeal
`
`fibroblasts;
`
`HCMC,
`
`human
`
`conjunctival
`
`mast
`
`cell(s);
`
`5HT, 5-hy-
`
`roxytryptamine;
`
`HTM3,
`
`human
`
`trabecular
`
`meshwork;
`
`IP,
`
`inositol
`
`phosphate;
`
`PGD2,
`
`prostaglandin
`
`D2; P1, phosphoinositide(s).
`
`1252
`
`APOTEX EX1055
`
`Page 1
`
`(cid:1)
`

`
`Downloaded from
`
`jpet.aspetjournals.org
`
` at Umd Of New Jersey on March 28, 2013
`
`1996
`
`Olopatadlne,
`
`Ocular
`
`Antiallergic
`
`I 253
`
`at.,
`
`emedastine
`
`with
`
`of
`development
`clinical
`and
`discovery
`the
`in
`(Sharif
`antthistamines
`1994),
`Yanni
`, 1994;
`et
`et
`at.
`additional
`still
`sought.
`are
`properties
`therapeutic
`Olopatadine
`{(Z)-11-[13-(dimethyl-
`KW-4679)
`(AL-4943A,
`amino)propylidenel-6,11-dihydrodibenz[b,elloxepine-2-acetic
`acid
`hydrochloride]
`is a new antiallergic/antihistamiic
`drug
`that
`prevents
`immunologically
`stimulated
`HCMC
`histamine
`release
`con-
`and
`potently
`blocks
`histamine-induced
`in
`vitro
`microvascular
`permeability
`et at. , in
`junctival
`in
`vivo
`(Yanni
`of action
`fast
`onset
`press).
`Olopatadine
`exhibited
`a relatively
`topical
`a
`and
`long
`duration
`of
`(up
`24
`hr
`to
`action
`after
`administration)
`in
`pig
`conjunctivitis
`of
`model
`guinea
`has
`(Yanni
`good
`press).
`shown
`also
`Olopatadine
`et
`at.,
`cacy and potency
`conditions
`such
`ofallergic
`models
`in animal
`bronchial
`(Ishii
`and chronic
`rhinitis
`asthma,
`allergic
`urticaria
`1991;
`Ohshima
`of passive
`anaphy-
`Inhibition
`et at.,
`1992).
`at.,
`laxis
`was maintained
`for
`administration
`of ol-
`after
`p.o.
`9 hr
`indicating
`an
`acceptable
`duration
`of action
`this
`opatadine,
`1991;
`Ohshima
`1992).
`drug
`et at.,
`et at.,
`1) to
`follows:
`The
`ofthe
`present
`studies
`as
`were
`4-fold,
`release
`of hista-
`correlate
`effects
`of olopatadine
`the
`on
`mine
`release
`of
`other
`mast
`cell-derived
`mediators
`2)
`(tryptase
`PGD2)
`from
`HCMC;
`to
`compare
`histamine
`and
`receptor
`and
`selectivity
`affinity
`of olopatadine
`subtype
`with
`those
`ocularly
`used
`antihistamines
`relevant,
`other
`and
`pertinent
`reference
`pharma-
`compounds;
`to determine
`the
`an
`antial-
`olopatadine
`and
`ketotifen,
`cological
`specificity
`of
`lergic/antihistaminic
`compound,
`relative
`the
`studying
`systems
`finities
`ofthe
`at
`a range
`ofother
`receptor
`compounds
`to
`unrelated
`to
`and
`4)
`determine
`the
`histamine;
`in
`vitro
`efficacy
`potency,
`possible
`modes
`of action
`of olopatadine
`and
`in antagonizing
`histamine-induced
`P1 hydrolysis
`in cultured
`ocular
`cells
`to correlate
`human
`and
`these
`parameters
`with
`receptor
`binding
`affinities.
`
`the
`
`in
`
`(Ishii
`aims
`
`the
`
`with
`
`of
`
`3)
`
`by
`
`effi-
`as
`et
`
`of
`
`aS-
`
`Materials
`
`and Methods
`
`mediator
`et
`
`of
`
`cells
`press).
`ob-
`
`was
`equili-
`tissue
`200 U
`for
`30
`filter
`This
`strin-
`
`7
`
`a
`in
`by
`
`HCMC
`preparation
`detailing
`Methods
`release.
`mediator
`with
`these
`studies
`release
`and
`HCMC
`monodispersed
`in
`Yanni
`1996;
`(Miller
`described
`have
`been
`al.,
`et
`al.,
`tissues
`donor
`postmortem
`from
`isolated
`Briefly,
`HCMC
`were
`banks
`at various
`eye
`death,
`tamed,
`within
`8 hr
`after
`transported
`and
`tissue
`conjunctival
`medium.
`The
`in Dexol
`corneal
`preservation
`RPMI
`medium
`culture
`and
`1640
`placed
`supplemented
`in
`brated
`incubator.
`After
`37#{176}Cin an
`overnight
`at
`the
`this
`time,
`transferred
`to Tyrode’s
`buffer
`containing
`and
`0.1% gelatin
`was
`ofcollagenase
`200 U ofhyaluronidase
`gram
`oftissue,
`per
`and
`mm
`at
`37#{176}C.The
`incubation
`mixture
`filtered
`over
`Nitex
`was
`cloth
`washed
`with
`equal
`of
`the
`latter
`buffer.
`an
`volume
`and
`digestion
`procedure
`was
`performed
`followed
`by
`a more
`twice,
`and
`digestion
`with
`2000
`U each
`of collagenase
`hyaluronidase
`gent
`from
`combined
`per
`gram
`of
`tissue,
`for
`30 mm at
`37#{176}C.The
`filtrates
`the
`digestion
`procedures
`were
`centrifuged
`825
`x g for
`at
`each
`of
`and the
`mm,
`pelleted
`cells
`were
`resuspended
`in
`calcium/magnesium-
`free Tyrode’s
`over
`buffer
`and
`centrifuged
`(825
`x g
`for
`30 mm)
`Percoll
`(1.058
`glliter)
`cushion.
`The mast
`cells
`that were
`pelleted
`fashion
`were
`resuspended
`in Tyrode’s
`buffer
`and
`washed
`this
`another
`centrifugation
`step.
`Toluidine
`blue
`0
`staining
`and
`trypan
`the
`blue
`exclusion
`by
`cells
`permitted
`the
`counting
`and
`viability
`isolated
`assessment
`the
`mast
`cells.
`release
`To measure
`the
`of endogenous
`5000
`mast
`cells
`were
`of
`suspensions
`15 mm and
`then
`exposed
`pound(s)
`a total
`of 1 ml. The
`reaction
`
`of
`
`for
`volume
`
`mediators
`treated
`to 10 (cid:1)g/ml
`was
`stepped
`
`from these
`the
`test
`with
`anti-human
`by the
`
`cells,
`corn-
`IgE
`in
`addition
`of
`
`Tyrode’s
`
`buffer
`
`at
`centrifugation
`were
`experiments
`tryptase
`histamine,
`are
`kits
`of these
`rnedi-
`preformed
`by
`determined
`corn-
`had
`been
`X. The
`concen-
`1.4
`±
`to
`be
`
`4.6
`
`brain
`
`a
`
`in
`compounds,
`test
`with
`determined
`was
`23#{176}Cfor 40 mm and
`GF/B
`glass
`fiber
`with
`2 X 6 ml
`of
`radioactivity
`bound
`to
`(3-counter,
`with
`four
`
`by
`
`for
`
`affinity
`1 nM,
`at
`
`and
`gelatin-containing
`ice-cold
`these
`for
`7 mm.
`500
`X g
`from
`Supernatants
`collected
`for
`analyzed
`-20#{176}Cuntil
`at
`and
`stored
`The
`sensitivities
`by radioimmunoassays.
`PGD2
`and
`respectively.
`Total
`1 nM,
`2 ng/ml
`and
`3.125
`pg/mi,
`concentrations
`were
`and
`tryptase)
`ator
`(histamine
`assaying
`collected
`cells
`mast
`that
`supernatants
`from
`to 0.1% Triton
`disrupted
`pletely
`30-mm
`exposure
`were
`of
`histamine
`trations
`and
`tryptase
`determined
`respectively.
`13.9
`±
`and
`23.6
`pg/cell
`pg/cell,
`assays.
`receptor
`subtype
`re-
`Histamine
`binding
`Histamine
`below.
`described
`as
`conducted
`were
`binding
`ceptor
`subtype
`assays
`recep-
`H2
`(for H1
`and
`forebrains
`pig
`Briefly,
`frozen-thawed
`guinea
`in 20 ml
`homogenized
`rat
`forebrains
`(for H3 receptors)
`were
`and
`tors)
`a Brinkrnan
`7.4),
`with
`phosphate-buffered
`saline
`(pH
`ice-cold
`of
`were
`centri-
`10 sec),
`and
`for
`tissue
`disrupter
`5-7
`(setting
`Polytron
`were
`40,000
`x
`g
`at
`4#{176}C.The
`15 mm
`at
`fuged
`supernatants
`discarded
`phos-
`and
`tissue
`were
`rehomogenized
`in fresh
`pellets
`the
`final
`and
`as
`described
`above.
`The
`centrifuged
`saline
`phate-buffered
`potassium
`in
`50 mM sodium
`phosphate
`homogenized
`pellets
`were
`at
`-40#{176}Cuntil
`used
`in the
`binding
`assays.
`buffer
`(pH 7.5)
`and
`frozen
`1979),
`H2 (Gajtkowski
`et al.,
`et al.,
`et al.,
`Chang
`The
`(Hill
`1977;
`H1
`1990)
`histamine
`receptor
`subtype
`binding
`1983)
`and H3 (Korte
`et at.,
`assays were
`previously
`described,
`minor
`rnodifi-
`with
`performed
`as
`(Sharif
`et
`at.,
`1994).
`of
`Briefly,
`50
`(cid:1)i.l
`[3H]pyrilamine
`(1-2
`cations
`[3H)tiotidine
`or N-[3H]methylhistamine
`nM)
`riM)
`(1-2
`(4-S
`nM),
`were
`incubated
`10
`to
`20 mg/nil
`with
`frozen-thawed
`homoge-
`unlabeled
`nates
`in
`the
`or
`absence
`of
`presence
`binding
`total
`volume
`(cid:1)il. The
`nonspecific
`of 500
`at
`conducted
`5 mM histamine.
`The
`assays
`were
`over Whatman
`by rapid
`filtration
`then
`terminated
`filters
`(prewetted
`with
`0.3%
`polyethyleneimine)
`ice-cold
`50 mM Tris-HC1
`The
`buffer
`(pH 7.4).
`filters
`was
`determined
`with
`a Wallac
`Big-Spot
`samples
`being
`counted
`simultaneously.
`To determine
`the
`relative
`ofolopatadine
`riM and
`100
`receptors,
`it was
`tested
`at
`NovaScreen
`38
`receptor
`binding
`assays
`four
`additional
`in
`Corp.,
`Hanover,
`and
`MD)
`with
`standard
`performed
`in-house
`assays
`niques.
`broad
`profiling
`the
`For
`comparison,
`ketotifen,
`another
`for
`determined
`in
`parallel
`compound.
`allergic/antihistaminic
`receptor-
`of histamine
`determination
`The
`studies.
`P1 turnover
`was
`hydrolysis
`mediated
`P1
`previously
`described
`as
`performed
`et a!.,
`(Sharif
`modifications
`minor
`with
`1990),
`(Sharif
`and Whiting,
`1994).
`HTM3
`and
`cells
`cells,
`immortalized
`HCE
`primary
`Briefly,
`HCF, which
`(Sharifet
`in
`, 1994,
`H1 receptors
`to express
`a!.
`are
`known
`studies.
`cells
`were
`The
`functional
`for
`these
`were
`press,
`a,b),
`used
`with
`ml myo-
`1 (cid:1)tCi/0.5
`incubated
`plates
`and
`24-well
`in
`cultured
`medium
`(supplemented
`Eagle
`in
`Dulbecco’s
`modified
`[3H]inositol
`and
`10% fetal
`bovine
`gentamicin
`50
`4 mM L-glutamine,
`(cid:1)g/ml
`with
`P1. After
`membrane
`this
`the
`cell
`37#{176}C,to
`for
`24
`hr
`at
`label
`serum)
`cells
`were
`exposed
`to
`hista-
`the
`and
`aspirated
`medium
`was
`time
`modified
`Eagle
`medium
`(con-
`in Dulbecco’s
`tiM to
`1 mM)
`mine
`io mM LiCl)
`for 60 mm
`at 23#{176}C,
`the
`production
`taming
`to
`stimulate
`To
`subsequent
`accumulation
`of
`[3H]IPs
`(Berridge
`1982).
`and
`et a!.,
`to
`drugs
`determine
`the
`potencies
`of
`antagonists,
`the
`were
`added
`the
`30 mm before
`in
`and
`the
`cells
`the
`addition
`of histamine
`were
`kept
`total
`of 90 mm.
`The medium
`was
`aspi-
`a
`contact
`with
`the
`cells
`for
`rated
`the
`end
`ofthis
`incubation,
`and
`the
`assay
`was
`stopped
`by the
`After
`15 mm,
`0.9 ml
`of
`addition
`of
`1 ml
`ofice-cold
`0.1 M formic
`acid.
`to Bio-Rad
`the
`cell
`lysates
`were
`transferred
`Econo-columns
`contain-
`had
`ing
`1 ml
`of AG1X8
`ion-exchange
`resin
`in
`formate
`form,
`which
`been
`previously
`wetted
`with
`of deionized
`water.
`The
`columns
`10 ml
`were
`washed
`with
`10 ml
`deionized
`water
`to
`remove
`the
`free
`of
`myo-[3H]inositol,
`the
`water-soluble
`[3H]IPs
`were
`eluted
`in 0.1
`from
`the
`columns
`4 ml
`of
`1.2 M ammonium
`formate
`
`of
`
`nonhistamine
`for
`(cid:1)M in a battery
`10
`(Oceanix
`Biosciences
`prostanoid
`receptor
`receptor
`binding
`tech-
`at NovaScreen
`also
`ocularly
`anti-
`
`was
`
`utilized
`
`the
`(10
`
`at
`
`and
`with
`
`then
`made
`
`Page 2
`
`

`
`Downloaded from
`
`jpet.aspetjournals.org
`
` at Umd Of New Jersey on March 28, 2013
`
`I 254
`
`Sharif
`
`et al.
`
`Vol. 278
`
`then
`(15 ml) was
`by scintifiation
`counter.
`data
`functional
`curve-fitting
`et
`a!.,
`1991).
`derived
`assays
`produce
`pKb
`represent
`three
`from
`
`were
`corn-
`The
`from
`(K5)
`50%
`
`or
`
`Ci/
`
`and
`data
`
`acid. A water-accepting
`fluid
`M formic
`scintillation
`quantified
`[3H]IPs
`were
`added
`to the
`eluates
`and
`the
`6000
`13-scintillation
`counting
`with
`a Beckman
`LC
`binding
`and
`Data
`analyses.
`Competition
`iterative,
`sigmoidal
`by using
`a nonlinear,
`analyzed
`puter
`prograrn
`(Michel
`and Whiting,
`1984;
`Sharif
`inhibition
`constants
`(drug
`dissociation
`constants)
`drug
`(ICe) and
`the
`receptor
`binding
`assays
`from the
`functional
`represent
`the molar
`drug
`required
`to
`concentrations
`inhibition
`of
`the
`binding/response,
`pK(cid:1)
`whereas
`the
`negative
`logarithms
`of
`All
`parameters.
`more
`experiments
`are
`shown
`as mean
`± S.E.M.
`(87
`[3H]tiotidine
`(24 Ci/mmol),
`Materials.
`[3H)Pyrilamine
`and
`myo-[3H]inositol
`mmol),
`N-[3H]methylhistamine
`(84 Ci/mrnol)
`DuPont-NEN
`(Boston,
`all
`Ci/rnmol)
`were
`purchased
`from
`(15-17
`Co.
`(St.
`MA).
`Percoll
`was
`purchased
`Chemical
`Sigma
`Louis,
`from
`were
`purchased
`from
`Pel-
`Frozen
`guinea
`pig
`and
`rat
`brains
`MO).
`Freez
`(Rogers,
`AR).
`Histaminergic
`agomsts
`and
`antagonists
`were
`purchased
`from Research
`Biochemicals
`International
`(Natick,
`MA).
`Olopatadine
`(AL-4943A,
`KW-4679)
`was
`synthesized
`at
`Kyowa
`Hakko
`Kogyo Co.
`(Shizuoka,
`Japan).
`Anti-human
`IgE was
`purchased
`from
`Cortex
`Biochemicals
`(San
`Leandro,
`CA).
`Radioimrnunoassay
`systems
`for
`proinflammatory
`mediator
`quantitation
`were
`purchased
`from
`the
`following
`companies;
`histamine,
`Immunotech
`Inc.
`(West-
`brook,
`ME);
`tryptase,
`Kabi-Pharmacia
`(Fairfield,
`NJ);
`PGD2,
`Amer-
`sham
`Corp.
`(Arlington
`Heights,
`
`these
`
`IL).
`
`Results
`
`1), when
`(fig.
`Olopatadine
`release.
`mediator
`HCMC
`added to monodispersed
`preparations
`15 mm be-
`of HCMC
`fore
`release
`in a
`inhibited
`histamine
`challenge
`with
`anti-IgE,
`manner
`2).
`Calculated
`IC50
`concentration-dependent
`values
`independent
`experiments
`314
`were
`from
`these
`cell
`cytotoxicity,
`as evi-
`504
`(cid:1).tM and
`(cid:1)tM,
`p.M.
`No mast
`cell mediator
`release,
`was
`denced
`by enhanced
`mast
`observed
`of olopatadine
`10-fold
`higher
`than
`with
`concentrations
`concentrations
`tested.
`effective
`maximally
`In
`separate
`experiments,
`the
`effects
`the mast
`cell
`release
`of
`tryptase
`and
`with
`the
`effect
`upon
`histamine
`release.
`centrations
`ranging
`from
`100
`(cid:1)tM to
`of
`mediator
`release.
`Concentrations
`positively
`culture
`supernatants
`were
`the
`(fig.
`3a) with
`those
`of histamine
`in
`Similarly,
`the
`concentrations
`PGD2
`of
`those
`0.905)
`(fig.
`3b) with
`of histamine
`(r
`obtained
`from
`HCMC
`cultures
`treated
`with
`fore
`the
`immunological
`challenge.
`
`(fig.
`
`three
`859
`
`the
`
`upon
`correlated
`at
`con-
`HCMC
`into
`0.98)
`
`of olopatadine
`PGD2
`were
`Olopatadine
`2 mM inhibited
`released
`tryptase
`correlated
`(r
`=
`same
`supernatants.
`were
`also
`correlated
`in supernatants
`olopatadine
`
`be-
`
`=
`
`0
`
`CO2H
`
`N(CH3)2
`
`Olopatadine
`
`Fig.
`
`I . Chemical
`
`structure
`
`of olopatadine.
`
`high
`
`1).
`
`=
`
`be
`
`of
`
`Mean IC(cid:1) =559pM ± 277
`
`100(cid:1)
`
`80
`
`60
`
`40
`
`0 0I
`
`E a
`
`)
`U)
`a)
`a)
`a)
`
`CEa
`
`)
`Cl)
`
`II
`
`.-
`
`0C0
`
`.(cid:1)
`
`.0
`
`C
`
`-5.0
`
`-4.5
`
`-4.0
`
`-3.5
`
`-3.0
`
`-2.5
`
`Log
`
`[Olopatadine]
`
`(M)
`
`Inhibition
`2.
`Fig.
`Calculated
`lC(cid:1)
`504 (cid:1)M and
`
`by olopatadine.
`from HCMC
`release
`of histamine
`values
`for
`the three
`separate
`experiments
`were
`314
`859
`Data
`shown
`are mean
`± S.D.
`
`(cid:1)M.
`
`and
`specific
`
`Olopatadine
`competed
`for
`N-[3H]methylhistamine
`receptors,
`respectively,
`Well-known
`(figs.
`4-6).
`triprolidine,
`pyrilamine,
`had
`high
`receptor
`compound
`aminopenti-
`H3-selective
`known
`imetit
`and
`affinities
`
`H1
`
`aS-
`
`N-
`(figs.
`
`for H1
`and
`7)
`
`re-
`a
`
`(cid:1)M,
`pK(cid:1)
`
`pK(cid:1)
`
`=
`
`affinity
`
`=
`
`binding.
`subtype
`receptor
`Histamine
`agonists
`and
`antagonists
`histaminergic
`[3H]tiotidine
`and
`[3Hlpyrilamine,
`and
`H3
`histamine
`binding
`to H1, H2
`in a concentration-dependent
`manner
`such
`as
`key
`H1-selective
`ligands,
`tripelennamine
`and
`promethazine,
`finities.
`Likewise,
`the
`H2-selective
`had
`dine
`a high
`affinity.
`In
`addition,
`H2
`compounds
`like
`clobenpropit,
`thioperamide,
`methylhistamine
`exhibited
`H3
`receptor
`4-6;
`table
`affinity
`high
`a relatively
`exhibited
`Olopatadine
`ceptors
`0.10,
`7.5
`pK(cid:1)
`31.6
`nM,
`n
`(IC(cid:1)
`=
`=
`=
`±
`100
`(K1 =
`receptors
`at H2
`lower
`affinity
`significantly
`and H3
`receptors
`0.19,
`= 4.0
`(K(cid:1)
`(cid:1)M,
`79.4
`n
`=
`7)
`±
`0.16,
`and
`H2:H1
`2).
`The
`1 and
`(figs.
`4-6;
`tables
`4.1
`n
`=
`7)
`±
`H1
`indicating
`olopatadine,
`affinity
`ratios
`for
`receptor
`H3:H1
`selectivity
`2500,
`3164
`and
`H2
`and
`H3
`receptors,
`were
`over
`respectively,
`com-
`demonstrating
`that
`this
`is an H1-selective
`was
`2). This
`pound
`(table
`H1-selective
`profile
`of
`olopatadine
`used
`superior
`to
`that
`some
`other
`ocularly
`to
`shown
`evaluated,
`antihistamines
`as
`affinity
`such
`ketotifen
`(H2:H1
`(H2:H1
`500),
`levocabastine
`affinity
`ratio
`ratio
`1032),
`=
`=
`= 1000)
`ratio
`antazoline
`(H2:H1
`affinity
`pheniramine
`and
`affinity
`was
`(table
`(H2:H1
`ratio
`2),
`but
`olopatadine
`406)
`=
`less H1-selective
`than
`chiorpheniramine
`ra-
`(H2:H1
`affinity
`tio
`12,589)
`6270)
`and
`pyrilamine
`(H2:H1
`ratio
`=
`(table
`2).
`To define
`nM to
`10
`pertaining
`prostaglandins,
`nological
`factors,
`and
`in
`another
`The
`ratory.
`
`of olopatadine,
`specificity
`the
`(at
`(cid:1).tM)
`assays,
`in
`38
`nonhistamine
`receptor
`to neurotransmitters,
`peptides,
`regulatory
`and
`uptake
`sites,
`second messengers
`immu-
`at NovaScreen
`(Oceanix
`Biosciences
`Corp.)
`four
`prostanoid
`receptor
`assays
`in
`labo-
`results
`(table
`3; NovaScreen
`task
`order
`0251)
`
`it was
`tested
`binding
`sites,
`
`our
`
`1
`
`Page 3
`
`(cid:1)
`

`
`Downloaded from
`
`1996
`
`Olopatadine,
`
`Ocular
`
`Antiallergic
`
`I 255
`
`(b]
`Olopatadine]
`
`100pM
`178pM
`
`316pM
`
`56OpM
`1mM
`
`3m
`
`(cid:1).
`
`xxi
`
`250
`
`200
`
`150
`
`100
`
`50
`
`.
`
`.
`
`/.
`
`7’.
`
`/1..
`
`#{149}(cid:1)(cid:1)//
`
`.
`
`./
`
`I r =0.905
`
`-I
`E
`
`0 0
`
`)
`0.
`
`C,(cid:1)1a
`
`(9
`
`(a]
`
`Olopatadine]
`
`316pM
`
`56OpM
`
`1mM
`
`3mM
`
`7
`
`6
`
`5
`
`2
`
`0)
`0.
`a)
`U)
`w3
`0.
`
`I-
`
`.
`
`.
`
`I r =0.980
`
`00
`
`02
`
`04
`
`06
`08
`Histamine
`
`10
`
`12
`(pg/cell)
`
`14
`
`16
`
`18
`
`00
`
`02
`
`04
`
`06
`08
`Histamine
`
`10
`
`12
`(pg/cell)
`
`14
`
`16
`
`18
`
`3. a, correlation
`Fig.
`various
`concentrations
`preparations
`upon
`
`released
`and tryptase
`of histamine
`concentrations
`between
`between
`concentrations
`b, correlation
`of olopatadine.
`with olopatadine.
`immunological
`challenge
`after
`treatment
`
`from HCMC
`of histamine
`
`upon
`and
`
`immunological
`PGD2
`released
`
`treatment
`after
`challenge
`from monodispersed
`
`with
`HCMC
`
`jpet.aspetjournals.org
`
` at Umd Of New Jersey on March 28, 2013
`
`AMINOP.
`RANIT.
`IMETIT
`LEVOC.
`
`THIOP.
`
`.
`0
`.
`0
`
`A L(cid:1)
`
`I 00
`
`80
`
`40
`
`0
`
`0)
`C
`V
`
`C C
`
`.)
`
`C.)
`a)
`0.
`Cl,f
`
`x C
`
`o
`(cid:1)20
`
`0)
`C
`(cid:1)0
`C
`co
`C.)
`
`C.)
`a)
`0.
`Cl)
`
`xa
`
`)
`
`-Vii
`
`9
`
`-8
`
`-7
`
`-6
`
`-5
`
`-4
`
`log
`
`[Antagonist]
`
`(M)
`
`-(cid:1)i I
`
`-(cid:1)io
`
`-(cid:1)
`
`-(cid:1)
`
`-(cid:1)7
`
`-6
`
`log
`
`[Antagonist]
`
`-(cid:1)I
`
`-(cid:1)3
`
`-(cid:1)
`
`(M)
`
`binding
`(cid:1)H]pynIamine
`of specific
`Inhibition
`4.
`Fig.
`antagonists.
`of histamine
`olopatadine
`and a variety
`age
`results
`eight
`experiments.
`The
`to
`from
`three
`shown for clarity. #{149},
`I,
`triprolidine;
`0, promethazine;
`El, diphenhydramine;
`A, olopatadine;
`& levocabastine;
`0 ,
`Y,
`thioperamide.
`imetit;
`
`by
`to H1 receptors
`Data
`are the aver-
`error
`bars
`are
`not
`chlorpheniramine;
`+ , cimetidine;
`
`5.
`Fig.
`to H2 receptors
`binding
`[(cid:1)H]tiotidine
`specific
`of
`Inhibition
`Data
`are the
`antagonists.
`a variety
`of histamine
`olopatadine
`and
`age
`results
`error
`bars
`are
`to
`three
`eight
`experiments.
`The
`from
`#{149},aminopotentidine;
`#{149},
`imetit;
`ranitidine;
`shown
`for
`clarity.
`A, olopatadine;
`levocabastine;
`(cid:1),
`
`0,
`thioperamide.
`
`by
`aver-
`not
`LI,
`
`10
`
`for
`
`that
`revealed
`inhibition
`((cid:18)50%
`5)
`(plC50
`fiected
`lower
`7.3). Olopatadine
`38 nonhistamine
`contrast,
`ketotifen
`significant
`((cid:18)50%
`
`had
`olopatadine
`of binding
`at
`5HT2
`receptors
`and
`affinities
`than
`possessed
`receptor
`tested
`inhibition
`
`some
`(cid:1)M)
`(plC50
`its H1
`receptor
`a very
`low affinity
`systems
`examined
`in
`the
`same
`42
`of binding
`at
`
`detectable
`5HT
`6),
`
`affinity
`sites
`re-
`=
`
`the
`
`uptake
`but
`these
`affinity
`(pK(cid:1)
`for
`other
`(table
`3).
`In
`exhibited
`affinities
`
`assays
`10
`(cid:1)tM)
`
`and
`
`for
`atpha-2
`M1
`and
`task
`order
`receptors
`mine
`Functional
`olopatadine,
`mine-induced
`possessing
`types
`centration-dependently
`
`dopamine,
`adrenergic,
`M2 muscarinic
`receptors
`to its
`0251),
`in addition
`4).
`(tables
`1 and
`determine
`studies.
`To
`for
`its
`tested
`it was
`P1
`in
`a
`turnover
`H1
`histamine
`and
`
`D2
`
`5HT2,
`(table
`affinity
`
`H2
`
`histamine
`4; NovaScreen
`for H1 hista-
`
`high
`
`ability
`variety
`receptors.
`potently
`inhibited
`
`the
`
`to
`of
`
`vitro
`in
`antagonize
`ocular
`human
`Olopatadine
`histamine-in-
`
`potency
`
`of
`
`hista-
`cell
`con-
`
`Page 4
`
`(cid:1)
`(cid:1)
`(cid:18)
`(cid:18)
`

`
`Downloaded from
`
`jpet.aspetjournals.org
`
` at Umd Of New Jersey on March 28, 2013
`
`Vol. 278
`
`2
`TABLE
`histaminerglc
`and various
`of olopatadine
`H1 selectivitY
`H2 and H3 receptor
`selectivities
`relative
`to their
`subtypes
`the affinity
`receptor
`ratios
`at
`the
`different
`histamine
`Data
`represent
`1 . The respective
`H2
`based
`on
`the molar
`values
`derived
`from data
`in Table
`250 and
`selectivities
`of
`ranitidine
`and
`cimetidine,
`relative
`to H1 receptors,
`were
`1 6. The respective
`H3 selectivities
`of N-methylhistamine,
`thioperamide
`and his-
`to H1 receptors, were 63,000,
`202,532 and 50,000.
`tamine,
`relative
`
`llgands
`
`p U S
`Corn o nd
`
`H1 Selectivity
`Relative to H2
`
`H1 Selectivity
`Relative to H3
`
`H1 ligands
`Pyrilamine
`Chlorpheniramine
`Tnprolidine
`Olopatadine
`Ketotifen
`Antazoline
`Tripelennamine
`Levocabastine
`Promethazine
`Pheniramine
`Diphenhydramine
`H2 ligands
`Cimetidine
`Ranitidine
`H3 ligands
`Imetit
`N-Methylhistamine
`Thioperamide
`Histamine
`
`12,589
`6,270
`4,750
`3,164
`1 032
`1 000
`731
`500
`251
`406
`126
`
`0.1
`0
`
`0.2
`0.8
`0.3
`0.1
`
`12,589
`2,460
`
`6.6
`
`2,500
`1,953
`800
`6,454
`64
`25,280
`197
`2,000
`
`0.8
`0.4
`
`0
`0
`0
`0
`
`1256
`
`Sharifetal.
`
`I 00
`
`80
`
`60
`
`40
`
`20
`
`0
`
`-12-11-10-9
`
`-8
`-7
`[Antagonist]
`
`-(cid:1)
`
`-#{232}
`-(cid:1)I
`(M)
`
`-(cid:1)3
`
`log
`
`0)
`C
`
`VC
`
`co
`C)
`
`C.)
`a
`Cl,
`
`(cid:1))
`
`6.
`
`A,
`
`of specific
`Inhibition
`Fig.
`N-(cid:1)H]methylhistamine
`by olopatadine
`and a variety
`of histamine
`ceptors
`the average
`results
`from three
`to eight
`experiments.
`#{149},
`imetit;
`0,
`not
`shown
`for
`clarity.
`clobenpropit;
`L(cid:1),
`N-methylhistamine;
`chlorpheniramine;
`, olopatadine.
`dine;
`
`to H3 re-
`binding
`Data are
`antagonists.
`bars
`are
`The error
`I,
`LI,
`thioperamide;
`#{149}
`levocabastine;
`raniti-
`,
`
`P1 turnover
`in
`8.0
`plC50
`=
`±
`7.8
`± 0.04,
`n
`nM,
`plC50
`31.6
`as primary
`table
`5). The
`blocking
`and HTM3
`
`=
`
`duced
`10 nM,
`plC50
`(IC50
`=
`histamine
`8;
`7 and
`histamines
`HCE
`cells,
`comparison.
`In
`additional
`
`for
`HCF
`
`of HCE
`primary
`0.06,
`in HCF
`(IC50
`n
`immortalized
`and
`4)
`=
`9), which
`= 7.5
`n
`±
`do (Pang
`HTM3
`cells
`et at.,
`of
`some
`ocularly
`potencies
`histamine-induced
`P1
`cells
`are
`also
`
`=
`
`cultures
`4),
`in
`0.1,
`
`(IC50
`cells
`15.8
`=
`HTM3
`respond
`1994)
`used
`turnover
`in table
`
`=
`nM,
`cells
`to
`(figs.
`anti-
`in
`5 for
`
`=
`
`shown
`
`mechanistic
`
`studies,
`
`olopatadine
`
`(1-300
`
`nM)
`
`produced
`
`preincubated
`the
`histamine
`sive
`decrease
`(fig. 9). Thus,
`antagonist
`experimental
`(Furchgott,
`(pKb)
`from these
`studies
`nM).
`Similar
`=
`19.9
`
`in
`
`cells
`HTM3
`with
`concentration-response
`in
`the
`histamine-induced
`olopatadine
`exhibited
`properties
`the HTM3
`conditions.
`The
`estimated
`1972;
`Brown
`et
`was
`calculated
`studies
`
`apparent
`cells
`
`shift
`a rightward
`a progres-
`curves,
`with
`response
`maximal
`noncompetitive
`under
`the
`present
`dissociation
`constant
`of olopatadine
`, 1984)
`at.
`to be 7.7
`± 0.2
`6)
`(n
`=
`conducted
`in
`the
`HTM3
`
`of
`
`(Kb
`cells
`
`1
`TABLE
`(AL-4943A)
`for olopatadine
`estimates
`Affinity
`Data are mean (cid:1) S.E.M.
`from three
`to eight
`independent
`
`and other
`experiments.
`
`histaminergic
`The values
`
`subtypes
`receptor
`for histamine
`ligands
`in parentheses
`represent
`the mean Hill coefficients
`shown
`
`of
`
`the inhibition
`
`plots.
`
`Cornpounds
`
`H1 ligands
`Triprolidine
`Pyrilamine
`Ketotifen
`Chlorpheniramine
`Promethazine
`Tnpelennamine
`Diphenhydramine
`Pheniramine
`Antazoline
`Olopatadine
`Levocabastine
`H2 ligands
`Aminopotentidine
`Cimetidine
`Ranitidine
`H3 ligands
`Clobenpropit
`Imetit
`Thioperamide
`N-Methylhistamine
`Histamine
`
`Receptor Affinities
`
`(pK1) and Hill Coefficients
`
`H1 Receptors
`
`H2 Receptors
`
`H3 Receptors
`
`(1.1)
`± 0.06
`9.1
`9.1 ± 0.06
`(0.9)
`(1 .0)
`8.9 ± 0.05
`(0.8)
`8.9 ± 0.10
`(1.0)
`8.9 ± 0.10
`8.5 ± 0.1 0 (1 .0)
`7.9 ± 0.20
`(1.1)
`7.5 ± 0.03
`(0.9)
`7.4 ± 0.05
`(1.0)
`7.5 ± 0.10
`(1 .1)
`7.3 ± 0.08
`(1.0)
`
`(0.6)
`± 0.20
`4.6
`4.3 ± 0.30
`(0.6)
`4.6 ± 0.20 (1.0)
`
`5.6 ± 0.03
`(0.6)
`(0.4)
`4.3 ± 0.10
`(0.8)
`4.2 ± 0.30
`(1 .0)
`4.2 ± 0.34
`3.7 ± 0.08 (0.4)
`
`5.5 ± 0.12
`5.0
`± 0.05
`5.9 ± 0.05
`5.1 ± 0.04
`6.5 ± 0.31
`5.6
`± 0.03
`5.8 ± 0.06
`4.9 ± 0.03
`4.4 ± 0.05
`4.0 ± 0.19
`4.6 ± 0.08
`
`7.4 ± 0.03
`5.8
`± 0.13
`6.8 ± 0.03
`
`(0.6)
`(0.8)
`(0.7)
`(0.8)
`(0.5)
`(0.6)
`(0.6)
`(0.6)
`(0.8)
`(0.7)
`(0.6)
`
`(0.6)
`(0.6)
`(0.8)
`
`(0.7)
`± 0.21
`5.7
`5.1 ± 0.26 (0.7)
`4.3 ± 0.07
`(0.9)
`4.3 ± 0.27
`(0.4)
`4.8 ± 0.09
`(0.7)
`
`5.3 ± 0.10(0.8)
`5.0
`± 0.05
`(0.9)
`5.6 ± 0.04
`(1.0)
`5.5 ± 0.01
`(1.1)
`4.5 ± 0.41
`(0.7)
`(0.7)
`± 0.25
`4.8
`4.6 ± 0.30(0.7)
`5.2 ± 0.22
`(0.9)
`4.5
`± 0.12
`(1.2)
`(0.9)
`4.1
`± 0.16
`± 0.19
`(0.8)
`5.5
`
`7.1 ± 0.20 (0.7)
`(0.7)
`4.7
`± 0.03
`4.8 ± 0.09
`(0.9)
`
`(0.7)
`9.7 ± 0.10
`(0.9)
`9.7 ± 0.03
`9.1 ± 0.09(0.9)
`9.0 ± 0.17(0.9)
`8.4 ± 0.18
`(0.9)
`
`Page 5
`
`(cid:1)
`

`
`Downloaded from
`
`jpet.aspetjournals.org
`
` at Umd Of New Jersey on March 28, 2013
`
`I 257
`
`in our
`
`At10(cid:1)M
`
`16.2
`2.6
`22.7
`3.2
`-4.9
`-7.7
`2.1
`30.4
`3.8
`-0.9
`86.9
`0.4
`36.6
`78.4
`41.5
`25.8
`-5.0
`0.8
`-1.7
`-6.0
`
`-2.7
`
`4.4
`15.3
`2.9
`
`10.2
`2.8
`
`21.9
`8.3
`-10.8
`-3.7
`
`8
`16
`0
`6
`-12.0
`3.6
`10.2
`7.4
`
`1.4
`0.7
`
`-13.4
`51.2
`
`1996
`
`TABLE 3
`In vitro
`binding
`receptor
`Data are averaged
`results
`Minus numbers
`laboratory.
`
`specificity
`profile
`of
`to determine
`of olopatadine
`this drug
`at NovaScreen,
`from two experiments,
`each performed
`in duplicate,
`conducted
`of bInding was considered
`stimulation
`of bIndIng.
`InhibitIon
`signify
`an apparent
`
`Olopatadine,
`
`Ocular
`
`Antlallergic
`
`except
`significant
`
`for
`
`indicated
`those
`when it was a50%.
`Inhibition of Receptor Binding at Different Concentrations
`
`that were
`
`conducted
`
`Receptor/Subtype
`
`Radioligand
`
`AtlnM
`
`At0.1(cid:1)M
`
`%
`
`17.0
`0.0
`PG. prostaglandln;
`2-D-Penicillarnine.
`proprionlc
`binding.
`
`48.9’
`14.7
`6.1
`-4.1
`NE. noreplnephrlne;
`leukotriene;
`LT.
`5-D-Peniclllamine-enkephalin;
`TBOB,
`12.13-dibutyrate.
`acid;
`Phorbol
`PDBU,
`
`IL,
`1-
`
`antihistamines
`other
`with
`for
`pyrilamine
`ifies
`(Kb
`6.3
`(Kb
`levocabastine
`plC6
`(K6
`tine
`1994)
`(figs.
`11).
`
`=
`
`=
`nM,
`0.79
`10 and
`
`=
`
`yielded
`also
`nM,
`pKb
`1.58
`=
`nM,
`plC,,
`8.2,
`9.1
`0.2,
`
`=
`
`±
`
`noncompetitive
`8.8
`0.1,
`=
`±
`n =
`2) and
`4)
`(Shari.f
`=
`
`n
`
`pro-
`4),
`
`n =
`emedas-
`et at.,
`
`Discussion
`
`containing
`cells,
`Mast
`possessing
`tors
`and
`proinfiammatory
`
`a
`mediators,
`
`preformed
`capability
`have
`
`proinfiammatory
`to
`synthesize
`been
`recognized
`
`media-
`additional
`as playing
`
`isoguavacine)
`
`rH]CPX8
`rH]CGS-21680
`rH]Prazosin
`[#{176}HIRX-781094
`[1251]lodocyanopindolol
`[125l]lodocyanopindolol
`rH]SCH-2330
`(cid:1)H]Sulpinde
`[(cid:1)H]GABA
`[(cid:1)H]GABA
`(cid:1)H]Pyrilamine
`rH]Tiotidine
`rHI5HT
`rH]Ketansenne
`rHlPirenzepine
`rHJAFDX384
`rH]NMCI
`rHJCGS-19755
`[(cid:1)H]Kainic
`acid
`rHJAMPA
`
`(+unlabeled
`
`(cid:1)H]Flunitrazepam
`
`rHIDAMGO
`rHIDPDPE
`rH]U69593
`
`rHiBradykinin
`rHjSubstance
`
`P
`
`w-[125l]-Conotoxin
`(cid:1)H]Nitrendipine
`rHITBOB
`125l-Charybdotoxin
`
`rH]PGE2
`rH]PGE2
`(cid:1)H]lloprost
`rHJSQ29548
`rH]LTB4
`rHILTD4
`[(cid:1)H]SQ29548
`rHIPAF
`
`rH]Forskolin
`rHIPDBU
`
`(cid:1)H]Dimethylimipramine
`(cid:1)H]Citalopram
`
`1 1.9
`4.2
`11 .1
`-6.0
`- 1 .8
`-8.5
`1 .9
`5.8
`-5.0
`-0.9
`0.8
`-5.4
`-13.9
`-2.6
`5.1
`10.2
`-5.2
`2.7
`3.3
`-20.4
`
`-5.4
`
`-0.6
`-14.0
`- 1 .0
`
`4.9
`4.9
`
`9.7
`1 0.8
`-5.2
`-9.5
`
`-9
`-5
`
`2
`3
`17.8
`5.3
`16.6
`6.6
`
`-3.7
`9.4
`
`-6.2
`3.0
`
`12.1
`1 .1
`19.6
`2.8
`0.4
`-9.3
`8.9
`5.9
`-2.4
`7.7
`76.8
`5.9
`-6.8
`17.9
`33.9
`23.5
`-4.1
`-7.0
`-0.7
`-14.9
`
`-2.9
`
`6.0
`3.4
`2.6
`
`4.5
`2.6
`
`1 1.8
`1 1 .9
`-7.5
`3.1
`
`1 1
`-2
`
`4
`2
`18.7
`2.5
`12.6
`2.1
`
`-3.2
`0.4
`
`-15.8
`8.6
`
`1
`2
`1
`2
`
`1
`2
`
`P
`
`and autocoids
`
`A(cid:1)
`
`factors
`
`Neurotransmitters
`1
`Adenosine
`2
`Adenosine
`Alpha-i
`-adrenergic
`Alpha-2
`adrenergic
`Beta-i
`adrenergic
`Beta-2
`adrenergic
`Dopamine
`1
`Dopamine
`2
`GABA#{216}
`GABA(cid:1),
`Histamine
`Histamine
`Serotonin
`Serotonin
`Muscarinic
`Muscarinic
`Nicotinic
`NMDA
`Kainate
`Quisqualate
`site
`Regulatory
`Benzodiazepine
`Opioids
`Mu opioid
`Delta
`opioid
`Kappa
`opioid
`Peptides
`Bradykinin
`Substance
`Ion channels
`Calcium (N-type)
`Calcium (1- and L-types)
`Chloride
`Potassium
`Prostanoids
`EP3
`PG(cid:1)’
`EP4 PG
`IP PG”
`TP PGb
`LTB4
`LTD4
`Thromboxane
`PAF
`Second messengers
`Forskolin
`ester
`Phorbol
`Uptake
`sites
`NE uptake
`5HT uptake
`Immunological
`[125l]-IL-la
`IL-i
`[‘25l]-C5A
`C5A
`Complement
`acid; NMDA, N-methyl-o-aspartate;
`GABA,
`y-aminobutyric
`3-dipropyixanthine;
`cyciopentyi-1
`a (cid:1)
`iodide; DAMGO,
`DPDPE,
`N-methylcarbonylcholine
`lnterleukin.
`Tyr-D-Ala-GIy-NMe-Phe-GIy-ol;
`NMCI,
`butylblcycloorthobenzoate;
`PAF, Platelet-activating
`factor
`AMPA, Aipha-amino-3-hydroxy-5-methylisoxazole-4
`b (cid:1)
`in-house.
`performed
`(cid:1)M olopatadine
`C Subsequent
`experiments
`with 1 to 100
`failed to show any significant
`Inhibition
`of r25fl-IL-la
`
`Page 6
`
`

`
`1258
`
`Sharif
`
`et al.
`
`Vol. 278
`
`4
`TABLE
`specificity
`receptor
`of
`to determine
`of ketotifen
`profile
`binding
`In vitro
`results from two experiments.
`each performed
`Data are averaged
`in duplicate,
`conducted
`numbers
`signify an apparent
`of binding.
`Inhibition
`of binding was considered
`stimulation
`
`this
`drug
`for those
`at NovaScreen,
`except
`significant when it was (cid:18)50%.
`
`that were conducted
`laboratory.
`in our
`Abbreviations
`are as in table 3.
`
`Minus
`
`Receptor
`
`Radioligand
`
`Inhibition of
`
`Receptor Binding at Different Concentrations
`
`Downloaded from
`
`jpet.aspetjournals.org
`
` at Umd Of New Jersey on March 28, 2013
`
`Isoguavacine)
`
`rHICPX
`rH]CGS-21680
`rH]Prazosin
`094
`rH]RX-78i
`[125l]lodocyanop
`[125l]lodocyanop..
`rH]SCH-2330
`(cid:1)H]Sulpinde
`rH]GABA
`rH]GABA
`(cid:1)H]Pynlamine
`[(cid:1)H]Tiotidine
`rH]5HT
`rHjKetanserine
`(cid:1)H]Pirenzepine
`rH)AFDX384
`[(cid:1)H]NMCI
`[(cid:1)H]CGS-i9755
`r’(cid:1)’<.(cid:1).inic
`acid
`rH]AMPA
`
`(+unlabeled
`
`rH]Flunitrazepam
`
`rHIDAMGO
`rHIDPDPE
`rH)U69593
`
`(cid:1)H]Bradykinin
`[(cid:1)HJSubstance
`
`P
`
`w(125l]-Conotoxin
`(cid:1)H]Nitrendipine
`rHITBOB
`125l-Charybdotoxin
`
`rH]PGE2
`rH]PGE2
`rH]lloprost
`rH]SQ29548
`rH]LTB4
`rH]LTD4
`rH]SQ29548
`rH]PAF
`
`rH]Forskolin
`rHIPDBU
`
`(cid:1)H]Dimethyl-imipramine
`rH]Cit&opr(cid:1).m
`
`[125I]-lL-i
`[125l1-C5A
`
`a
`
`At 1 nM
`
`-3.3
`6.0
`0.0
`8.8
`1.3
`-11.0
`-4.5
`5.5
`-4.8
`-3.7
`60.3
`8.3
`20.0
`1.7
`9.7
`11.9
`-8.9
`-4.4
`-4.9
`-14.9
`
`7.4
`
`-3.8
`2.1
`-5.0
`
`-6.7
`0.7
`
`7.4
`3.3
`-8.3
`2.9
`
`1.8
`4.7
`2.7
`3.3
`0.6
`-3.7
`-1.7
`-7.4
`
`-11.2
`-2.4
`
`-16.5
`5.6
`
`-12.3
`-5.2
`
`At 0.1 (cid:1)M
`
`%
`
`9.3
`0.4
`8.9
`19.3
`-3.2
`-13.4
`9.2
`15.7
`-2.2
`9.7
`87.9
`25.5
`0.2
`58.4
`48.2
`58.4
`7.9
`-0.4
`2.8
`-7.5
`
`0.9
`
`-5.9
`3.1
`-1.2
`
`19.4
`0.2
`
`17.0
`15.1
`-7.1
`3.9
`
`5.1
`0
`-0.1
`-1.5
`-3.3
`4.4
`15.8
`4.0
`
`10.0
`4.0
`
`-12.9
`6.5
`
`7.8
`-0.9
`
`At 10 (cid:1)M
`
`29.7
`-0.4
`37.8
`91.0
`0.6
`-5.4
`29.8
`84.2
`-3.1
`18.8
`88.2
`83.1
`-0.5
`95.6
`99.0
`96.9
`0.5
`16.5
`-2.6
`-15.0
`
`20.7
`
`22.7
`45.3
`16.5
`
`6.9
`-1.4
`
`25.4
`-3.1
`-6.4
`6.6
`
`-2.2
`1.0
`-0.5
`0.6
`4.6
`3.8
`9.9
`25.4
`
`-1.7
`1.9
`
`8.9
`32.6
`
`33.8
`2.4
`
`1
`2
`
`1
`2
`I
`2
`
`1
`2
`
`P
`
`Neurotransmitters
`1
`Adenosine
`Adenosine
`2
`Alpha-i
`Alpha-2
`Beta-i
`Beta-2
`Dopamine
`Dopamine
`GABA,,
`GABA,,
`Histamine
`Histamine
`Serotonin
`Serotonin
`Muscarinic
`Muscarinic
`Nicotinic
`NMDA
`Kainate
`Quisqualate
`site
`Regulatory
`Benzodiazepine
`Opioids
`Mu opioid
`Delta
`opioid
`opioid
`Kappa
`Peptides
`Bradykinin
`Substance
`Ion channels
`Calcium (N-type)
`Calcium (1- and L-types)
`Chloride
`Pota