0026-895X/88/060843.09$02.OO/O
`© by The American
`for Pharmacology
`Society
`Copyright
`All
`rights
`of
`reproduction
`in any
`form reserved.
`34:843-851
`MOLECULAR
`PHARMACOLOGY,
`
`and Experimental
`
`Therapeutics
`
`Binding Profile of the New Antihypertensive
`The Receptor
`Nebivolol
`and Its Stereoisomers
`Compared With Various
`Adrenergic
`Blockers
`
`Agent
`f3-.
`
`PETRUS
`Department
`Beerse,
`B-2340
`
`Received
`
`J. PAUWELS,
`of Biochemical
`Belgium
`June 2, 1988; Accepted
`
`August
`
`23, 1988
`
`WALTER
`Pharmacology
`
`GOMMEREN,
`(P.J.P., W.G., P.A.J.J.,
`
`GUY
`
`VAN
`J.E.L.)
`
`A.
`PAUL
`LOMMEN,
`and of Organic
`Synthesis
`
`J.
`
`JANSSEN,
`(G.V.L.,
`P.A.J.J.),
`
`JOSEE
`Janssen
`
`LEYSEN
`E.
`Research
`
`Foundation,
`
`+
`
`to (cid:1)
`
`SUMMARY
`[the
`Nebivolol
`10
`the
`mixture],
`(R,S,S,S)-racemic
`(S,R,R,R)-
`investi-
`blockers
`were
`fl-adrenergic
`and
`known
`stereoisomers,
`sites
`and
`f32-adrenergic
`receptor
`for binding
`gated
`in vitro
`binding
`peptide,
`and
`ion channel
`neurotransmitter,
`and
`various
`Selective
`neurotransmitter
`uptake.
`of
`inhibition
`for
`sites
`and
`sites
`and
`rat
`receptor
`in rabbit
`/3(cid:1)(cid:1) and
`fl2-adrenergic
`labeling
`of
`[3H]CGP-1
`with
`21 77 and
`[3H]
`lung,
`respectively,
`was
`obtained
`of an appropriate
`concentration
`dihydroalprenolol
`in the presence
`of
`the selective
`(cid:1)32-adrenergic
`blocker
`lCl 1 1 8-551
`the selective
`f31-adrenergic
`blocker
`CGP
`2071 2-A.
`Nebivolol
`revealed
`high
`affinity
`and selectivityfor
`f31-adrenergic
`receptor
`sites
`in the rabbit
`lung membrane
`preparation
`value
`= 0.9 n(cid:1)
`and
`(K(cid:1)
`ratio
`=
`50). The drug
`dissociated
`slowly
`from these
`receptor
`sites.
`The
`activity
`resided
`in the
`(R 67
`1 38);
`the
`(S,R,R,R)-enantiomer
`(R,S,S,S)-enantiomer
`(A 67 1 45)
`revealed
`175 times
`lower
`adrenergic
`binding
`affinity.
`Within
`the
`series
`of
`stereoisomers,
`
`or
`
`(cid:1)2/f.(cid:1)1
`
`of high
`combination
`best
`the
`1 38 showed
`and R 67
`nebivolol
`only
`compounds,
`reference
`the
`Among
`and
`selectivity.
`affinity
`to S1A
`properties.
`Nebivolol
`bound
`these
`CGP 2071 2-A shared
`require-
`of 20 n(cid:1).
`The stereospecific
`binding
`sites with
`a K, value
`from those
`these
`sites were
`different
`ments
`for
`interaction
`with
`affinity
`was
`for
`the
`f31-adrenergic
`receptor
`site.
`S1A binding
`site
`/3-adrenergic
`also
`observed
`with
`the
`potent
`but
`nonselective
`In the various
`other
`blockers
`carvedilol,
`pindolol,
`and
`propranolol.
`binding
`and
`neurotransmitter
`uptake
`investigated
`radioligand
`assays,
`nebivolol
`and
`its stereolsomers
`showed
`activity
`only
`at
`micromolar
`concentrations
`or were
`inactive.
`Clinical
`studies
`have
`shown
`an interesting
`hemodynamic
`profile
`of nebivolol,
`offsetting
`the
`negative
`effects
`on
`left
`ventricular
`generally
`performance
`observed
`classical
`f3-adrenergic
`blockers.
`Several
`hy-
`are
`potheses
`the mechanism
`action
`nebivolol
`summarized.
`
`of
`
`of
`
`with
`regarding
`
`blockers
`unusual
`apparent
`
`dogs,
`systemic
`and
`stroke
`cardiac
`
`by
`
`Nebivolol
`
`(R 65
`
`824)
`
`(nebivolol-hydrochloride
`
`is R 67
`
`555),
`
`is
`
`asymmetric
`four
`and
`
`(cid:1) 3
`
`blood
`In
`(1-3).
`blood
`in
`administration
`No
`such
`
`effect
`
`after
`
`pseudosym-
`a
`,4-dihydro-2H-1-benzopyran-2-methanol],
`atoms
`(Fig.
`carbon
`metrical
`molecule
`with
`four
`pairs
`and
`enantiomeric
`Ten
`stereoisomers,
`comprising
`Nebivolol,
`the
`isolated.’
`mesoforms,
`were
`synthesized
`(S,R,R,R)-
`(R,S,S,S)-enantiomers,
`and
`cemic
`mixture
`of
`the
`In clinical
`being
`investigated
`as
`a new antihypertensive
`agent.
`studies
`to
`found
`with
`hypertensive
`patients,
`nebivolol
`was
`left
`also
`reduce
`heart
`rate
`and
`pressure
`but
`it
`improved
`studies,
`ventricular
`function
`animal
`pharmacological
`with
`ne-
`immediate
`reduction
`pressure
`was
`observed
`bivolol,
`its
`to
`conscious
`spontaneously
`hypertensive
`rats.
`was
`observed
`with
`known
`
`1).
`two
`ra-
`is
`
`fi-
`
`this work
`supported
`was
`het Wetenschappelijk
`
`a grant
`by
`Onderzoek
`
`the
`from
`in Nijverheid
`
`veer
`Instituut
`en Landbouw
`
`Aanmoe-
`(Brus-
`
`Part
`diging
`sels,
`
`of
`van
`Belgium).
`I Van Lommen
`
`et
`
`aL, manuscript
`
`in
`
`preparation.
`
`(3).
`
`atenolol,
`such
`adrenergic
`A further
`observed
`feature
`negative
`lack
`of
`was
`its
`in
`comparison
`anesthetized
`vascular
`resistance
`reduced
`volume.
`At
`equivalent
`output
`output
`stroke
`volume.
`reduced
`using
`tissues
`have
`investigations
`nebivolol
`isoprenaline-induced
`by
`of
`antagonism
`(cid:1)3,-adrenergic
`receptors
`in
`the
`diated
`the
`compound
`was
`300-fold
`less
`However,
`ing
`/32-adrenergic
`receptor-mediated
`effects
`trachea.
`A selective
`action
`of
`at
`in vivo
`tors
`is
`apparent
`aline-induced
`changes
`by
`cardiac
`f3,-adrenergic
`tion
`in
`diastolic
`blood
`tors)
`in
`dogs
`
`or
`of
`
`in
`
`as
`
`and
`isolated
`
`pindolol.
`propranolol,
`nebivolol
`low
`doses
`at
`effect
`inotropic
`cardiac
`Nebivolol
`with
`propranolol.
`cardiac
`and
`increased
`propranolol
`doses,
`Pharmacological
`potent
`revealed
`a
`me-
`effects
`pig
`atrium.
`antagoniz-
`guinea
`
`guinea
`in
`active
`the
`in
`f3, -adrenergic
`nebivolol
`inhibition
`the
`greater
`from
`contractility
`ventricular
`left
`with
`compared
`receptors,
`as
`pressure
`(vascular
`f32-athenergic
`
`of
`
`of
`
`pig
`recep-
`isopren-
`mediated
`the
`reduc-
`recep-
`
`CGP2O71 2-A,
`ABBREVIATiONS:
`(±H2-hydroxy-5-[2(cid:1)(2(cid:1)ydroxy-3-(4-((1-methyl-4-tnfluoromethyl)1H-imidazole-2-yl)phenoxy)propyl)amino)ethoxyJ-
`benzamide
`monomethane
`sulfonate;
`(±(cid:1)[3H]4(cid:1)3-te(cid:1)iarybutylamino-2-hydroxypropoxy)-benzimidazole-2-on
`[3H]CGP-1
`21 77,
`,e(cid:1)hro-1-(7-methylindan-4-yloxy)-3-isopropylaminobutan-2-ol.
`1 1 8551
`
`hydrochloride;
`
`lCl-
`
`843
`
`LOWER DRUG PRICES FOR CONSUMERS, LLC
`Exhibit 1041-1
`IPR2016-00379
`
`(cid:1)
`

`
`844
`
`Pauwels
`
`et a!.
`
`0,
`
`F
`
`0
`
`Fig.
`(a) of
`
`1. Structure
`the four
`
`ofnebivolol
`asymmetric
`
`with indication
`atoms.
`carbon
`
`this
`
`and
`
`of
`
`for
`This
`lung
`the
`and
`[3H]
`The
`with
`
`neurotransmitter
`binding
`receptor
`the
`study,
`In
`were
`investigated.
`nebivolol
`of
`properties
`inhibition
`uptake
`been
`developed
`models
`have
`binding
`radioligand
`Specific
`fi, - and
`receptor
`sites.
`fl2-adrenergic
`labeling
`selective
`rabbit
`and
`rat
`i.e.,
`tissues,
`(i)
`selective
`was
`achieved
`using
`respectively,
`(ii)
`sites,
`for
`and
`f.(cid:1)2-athenergic
`receptor
`(cid:1),-
`CGP
`20712-A
`(4)
`blocker
`selective
`(cid:1)3,-adrenergic
`receptor
`6),
`and
`(iii)
`(5,
`ICI
`118-551
`blocker
`fl2-athenergic
`receptor
`radioligands.
`as
`CGP-12177
`and
`[‘H}dihydroalprenolol
`interaction
`receptor
`stereoselectivity
`of
`the
`fl-adrenergic
`The
`dissociation
`investigated.
`the
`nebivolol
`stereoisomers
`was
`fi,
`- and
`fl2-adrenergic
`receptors
`from
`rate
`of unlabeled
`drugs
`the
`a previously
`described
`tissue-
`of
`was measured
`by modification
`The
`interaction
`of
`nebivolol
`8).
`adsorbed-to-filter
`method
`neurotransmitter
`receptors,
`stereoisomers
`various
`and
`sites
`was
`investigated.
`channels,
`binding
`these
`of
`inhibit
`monoamine
`tency
`to
`synaptosomes
`brain
`tested.
`fl-adrenergic
`The
`nebivolol
`profile
`of
`and
`and
`its
`stereoisomers
`known
`those
`of
`with
`fl-adrenergic
`blockers.
`of
`nebivolol
`profile
`discussed
`light
`of
`macological
`properties
`and
`findings
`in clinical
`
`The
`
`ion
`po-
`rat
`
`in
`uptake
`selectivity
`compared
`were
`biochemical
`The
`reported
`phar-
`studies.
`
`(7,
`
`with
`peptide
`compounds
`was
`
`is
`
`in
`
`the
`
`Materials
`
`and Methods
`
`Tissue
`and
`
`kg)
`
`(-150
`
`preparation.
`female
`rats
`Tissue
`homogenized
`was
`v/w)
`of
`buffer
`(0.25
`and
`rpm).
`
`25
`
`male
`from
`Lungs
`dissected
`g) were
`10 volumes
`in
`M sucrose,
`
`0.15
`
`pH
`
`7.4)
`was
`
`a
`
`placed
`shaking
`was
`
`vials
`in scintillation
`Instagel
`in 8 ml of
`counted
`in
`a Packard
`
`radioactivity
`and
`(Packard,
`II
`Tri-carb
`
`extracted
`was
`Warrenville).
`The
`4530
`liquid
`scintilla-
`
`binding
`with
`
`rabbit
`
`lung
`in
`sites
`[3HJ
`or
`[3HJCGP-12177
`Nonspe-
`binding
`sites.
`1 (cid:1)cM CGP
`presence
`of
`binding
`in
`rat
`lung
`sites
`with
`[‘H]CGP-12177
`or
`
`membranes,
`
`were
`Filters
`by vigorous
`radioactivity
`tion
`counter.
`$,-adrenergic
`to
`binding
`To measure
`added
`membranes,
`118-551
`10 nM IC!
`was
`occlusion
`of
`fl)-adrenergic
`for
`dihydroalprenolol
`was measured
`in the
`additional
`cific
`binding
`To measure
`binding
`to (cid:1)9)-adrenergic
`20712-A.
`300
`added
`nM CGP
`was
`20712-A
`of(cid:1)9,-adrenergic
`additional
`
`for
`occlusion
`defined
`in
`
`the
`
`was
`
`binding
`presence
`
`sites.
`1
`
`of
`
`Non-
`zM IC!
`
`or
`
`4
`
`[3H]dihydroalprenolol
`specific
`binding
`118-551.
`of binding,
`inhibition
`To measure
`1 nM [3H]
`were
`added
`The
`drugs
`was
`used.
`or
`CGP-12177
`spanning
`six
`concentrations,
`least
`incubation
`to
`the
`[3Hjdihydroal-
`[3H]CGP-12177
`of magnitude.
`orders
`of drug
`as
`the
`percent-
`was
`calculated
`the
`binding
`in
`prenolol
`total
`age
`of
`and
`binding
`[3Hjdihydroalprenolol
`or
`[3H]CGP-12177
`values
`(concen-
`IC(cid:1)
`concentration.
`the
`versus
`the
`log
`of
`plotted
`drug
`were
`derived
`binding)
`[3H]ligand
`specific
`inhibiting
`50% of
`tration
`K, values
`graphically.
`Cheng-Prusoff
`to
`the
`according
`calculated
`were
`and Kd
`K, = ICse/(1
`+ C/Kd) with C being
`equation:
`concentration
`the
`[3Hjligand
`(9).
`the
`equilibrium
`dissociation
`constant
`of
`the
`For
`saturation
`binding
`curves,
`[3H]CGP-12177
`[3Hjdihydroal-
`1 nM. K,, and
`prenolol
`was
`at
`concentrations
`between
`plote.
`regression
`lines
`derived
`from
`by
`the
`method
`squares.
`of
`and
`(cid:1),-
`dissociation
`receptor
`as
`
`for
`
`of drugs
`potencies
`[3H]dihydroalprenolol
`at
`in
`mixtures
`specific
`The
`presence
`
`used
`were
`
`or
`and
`0.05
`Linear
`
`receptor
`unlabeled
`of
`the
`measured
`were
`described
`(see
`
`with
`
`at
`
`10
`
`at
`and
`was
`
`at
`
`to
`
`For
`
`.
`
`Scatchard
`values
`Bmas
`of
`least
`calculated
`were
`Measurement
`(cid:1)2-adrenergic
`rates.
`The
`rates
`in
`vitro
`sites
`i3,-
`and
`fl2-adrenergic
`previously
`method
`absorbed-to-filter
`(7,
`above),
`preparation
`membrane
`cations.
`A tissue
`of 10
`drug
`a
`concentration
`preincubation
`positioned
`glass
`fiber
`filters
`GF/B
`adsorbed
`to Whatman
`tissues,
`adsorbed
`to
`drug-loaded
`tion
`apparatus.
`The
`time
`various
`periods.
`At
`buffer
`for
`warm
`rinsed
`with
`the
`tissue,
`adsorbed
`filter,
`the
`rinsing
`period,
`free
`sample
`of
`[3H]CGP-12177
`to quantify
`receptors.
`drug
`half-time
`of
`dissociation
`of
`the
`unlabeled
`was
`
`during
`
`x
`
`to
`
`was
`
`dissociation
`drugs
`from
`the
`using
`a
`tissue-
`with
`modifi-
`8),
`saturated
`with
`IC(cid:1)
`value,
`was
`the
`filtra-
`on
`filters,
`were
`end
`the
`
`of
`with
`of
`
`a
`
`the
`de-
`
`the
`the
`incubated
`Calculation
`as
`previously
`
`(7).
`scribed
`and
`uptake
`neurotransmitter
`receptor
`Radioligand
`assays.
`Radioligand
`guinea
`or
`using
`performed
`were
`binding
`uptake,
`For
`neurotransmitter
`(10).
`preparations
`pig
`brain
`membrane
`The
`(10).
`brain
`regions
`was
`from rat
`fraction
`crude
`synaptosomal
`D2, dopa-
`S2,
`serotonin
`SIA,
`dopamine
`serotonin
`assay
`conditions
`a2-adrenergic,
`histamine
`H,,
`cholinergic-mus-
`mine
`D,,
`a,-adrenergic,
`(cid:1)t opiate,
`benzodiazepine,
`dihydropyridine,
`biogenic
`amine,
`carinic,
`P and
`neurotensin
`receptor
`binding,
`metabolite
`release,
`substance
`serotonin,
`noradrenaline,
`dopamine
`and
`-y-aminobutyric
`acid
`were
`previously
`described.2
`the
`veratridine
`site
`
`binding
`assays
`
`for
`
`as
`
`Binding
`
`to
`
`rat
`
`used
`
`a
`
`and
`and
`uptake
`of
`
`the
`
`2 Leysen,
`
`J.
`
`E., W.
`
`Gommeren,
`
`A. Eens,
`
`D.
`
`de
`
`Chaffoy
`
`de
`
`Courcelles,
`
`J.
`
`C.
`
`and
`
`Stoof,
`psychotic.
`
`J.
`A.
`P.
`J. Pharrnacol.
`
`Janssen.
`Exp.
`
`Biochemical
`247:661-670
`Ther.
`
`profile
`
`of
`(1988).
`
`risperidone,
`
`a
`
`new
`
`anti-
`
`up
`
`of
`
`of
`
`was
`
`were
`or
`occlusion,
`drug
`for
`incubated
`of
`ice-cold
`GF/B
`glass
`twice
`with
`
`(-2
`rabbits
`Zealand
`New
`0.9%
`in
`transferred
`and
`weight
`wet
`(volumes
`per
`NaCl.
`. H20,
`5 mM
`M NaClO4
`tissue,
`x
`EDTA,
`(3
`10
`Polytron
`mixer
`with
`imidazol,
`mM
`10 mm
`at
`x g for
`830
`centrifuged
`homogenate
`The
`sec,
`1500
`and
`rehomogenized
`was
`The
`pellet
`debris.
`nuclei
`and
`cell
`to
`precipitate
`filtered
`combined,
`and
`were
`supernatants
`The
`centrifuged.
`similarly
`per wet weight
`to 40
`diluted
`further
`and
`cheesecloth,
`over
`volumes
`was
`centrifuged
`suspension
`pH
`50 mM Tris.HC1,
`with
`7.7.
`This
`x g for
`pellet
`membranes.
`The
`the
`cell
`precipitate
`to
`20 mm,
`23,600
`buffer
`centrifuged.
`. HC1
`and
`in Tris
`suspension
`washed
`twice
`by
`was
`with
`a Duall
`homogenizer
`in
`homogenized
`final
`pellet
`was
`The
`pH 8.
`. HC1,
`Tris
`50 mM
`volumes
`During
`the
`entire
`preparation
`was
`kept
`0-4”.
`The
`membrane
`suspension
`the
`tissue
`procedure
`aliquots
`stored
`-70’
`into
`distributed
`preparation
`100 volumes
`preparation
`diluted
`membrane
`the
`assays,
`binding
`pH 8.
`. HC1,
`with
`50 mM Tris
`(v/w)
`fi,-
`and
`$2-adrenergic
`Binding
`assays
`to
`tissue
`bation
`mixtures
`composed
`of
`2 ml
`of
`with
`[H]CGP-12177
`[H]dihydroalprenolol,
`(10%
`solvent
`binding
`site
`and
`0.1 ml
`of
`nonspecific
`or
`inhibition,
`determination
`. The
`and
`were mixed
`for
`37”
`at
`15 mm
`buffer,
`pH
`5 ml
`adding
`Tris
`. HCI
`by
`under
`over Whatman
`fiber
`filters
`rapidly
`rinsed
`5 ml
`of
`ice-cold
`
`receptor
`preparation,
`or
`without
`ethanol),
`binding.
`reactions
`were
`8.0,
`and
`rapid
`vacuum.
`The
`. HC1
`buffer,
`Tris
`
`sites.
`Incu-
`0.1 ml
`of
`drug
`for
`drug
`for
`Samples
`stopped
`filtration
`filters
`were
`8.0.
`
`pH
`
`LOWER DRUG PRICES FOR CONSUMERS, LLC
`Exhibit 1041-2
`IPR2016-00379
`
`

`
`measured
`was
`channel
`Na(cid:1)
`(11).
`viously
`described
`(-)-[3H]CGP-12177
`Materials.
`and
`1[propyl-1,2,3-[3Hjdihydroalprenolol-HC1
`from New England
`Nuclear
`tamed
`stereoisomers
`were
`original
`substances
`(Beerse,
`Belgium).
`Other
`drugs were
`panies
`origin.
`
`with
`
`tetraphenylphosphonium
`
`ions
`
`as
`
`pre-
`
`(34
`
`Ci/mmol)
`
`(Dreieich,
`from
`generously
`
`from Amersham
`was
`Ci/mmol)
`was
`(48
`Germany).
`Nebivolol
`Janssen
`Pharmaceutica
`supplied
`by the
`
`ob-
`and
`
`com-
`
`of
`
`selective
`for
`receptor
`of
`Development
`Inhibition
`$2-adrenergic
`and
`labeling
`of
`$2-adrenergic
`and
`selective
`by
`the
`binding
`[3H]CGP-12177
`ICI
`118-551,
`was
`respectively,
`and
`20712-A
`CGP
`blockers
`inhi-
`preparations;
`membrane
`lung
`rat
`rabbit
`and
`in
`measured
`20712-A
`lung,
`CGP
`In
`rabbit
`in Fig.
`are
`shown
`curves
`bition
`80% of
`inhibited
`and
`curve
`inhibition
`a monophasic
`showed
`ICI
`188-
`of
`curve
`inhibition
`The
`binding.
`[3H]CGP-12177
`total
`bound
`15% oftotal
`less
`than
`that
`it was
`noted
`551 was
`biphasic;
`In
`con-
`range.
`the
`nanomolar
`in
`was
`inhibited
`[3H]CGP-12177
`80%
`nanomolar
`concentrations,
`at
`inhibited,
`118-551
`trast,
`ICI
`membrane
`in
`rat
`lung
`binding
`[3HJCGP12177
`total
`of
`showed
`a bi-
`CGP
`20712-A
`preparation,
`In
`this
`preparation.
`[3H]CGP-12177
`25% of
`total
`curve;
`only
`phasic
`These
`findings
`nanomolar
`range.
`inhibited
`the
`in
`binding
`preparations
`were
`lung
`membrane
`rabbit
`rat
`and
`indicated
`sites,
`respectively.
`receptor
`and
`$2-athenergic
`enriched
`experiments,
`the minor
`population
`of
`and
`In
`subsequent
`receptor
`sites
`rabbit
`rat
`lung
`was
`occluded
`in
`and
`adrenergic
`of 10 nM ICI
`118-551
`and
`by
`addition
`300
`nM CGP
`20712-A
`rat
`lung membrane
`preparations,
`respectively.
`rabbit
`and
`3 and
`4 show
`the
`saturation
`binding
`curves
`of
`[‘H]CGP-12177
`in
`rabbit
`and
`rat
`lung
`membrane
`preparations
`under
`conditions.
`Scatchard
`analysis
`revealed
`a
`single
`population
`sites
`binding
`in
`each
`of
`the
`tissues,
`representing
`-adrenergic
`sites
`receptor
`the
`rabbit
`lung
`and
`(cid:1)92-athenergic
`receptors
`in
`the
`in
`sites
`lung.
`Similar
`findings
`were
`obtained
`with
`Kd and
`Bmax
`dihydroalprenolol.
`values
`for
`[‘HJCGP-12177
`[:;H]dihythoalprenolol
`binding
`are
`summarized
`in Table
`
`Results
`
`binding
`
`models
`receptors.
`fi,-
`
`2.
`
`of
`
`the
`
`the
`
`$,
`
`-
`
`to
`Figs.
`
`such
`of
`
`[‘H]
`and
`[‘H]
`
`1.
`
`(cid:1),
`
`th-
`
`inhibition
`was
`that
`in
`
`$,-
`
`rat
`
`Receptor Binding Profile of Nebivolol
`
`845
`
`affinity
`the
`
`and
`
`$2-adrenergic
`/3,-
`to
`receptor
`being
`f.(cid:1),-athenergic
`higher
`affin-
`with
`bound
`$2-adrenergic
`Its
`sites.
`receptor
`The
`[‘H]CGP-12177.
`of
`to
`that
`mem-
`the
`rabbit
`lung
`in
`sites
`density
`of
`f32-adrenergic
`the
`membrane
`preparation.
`[‘Hjdihydroalprenolol
`
`$2(cid:1)
`
`fi,
`
`high
`with
`bound
`CGP-12177
`for
`affinity
`the
`sites,
`receptor
`[‘H]Dihydroalprenolol
`higher.
`slightly
`-adrenergic
`than
`ity
`to
`was
`similar
`affinity
`receptor
`of
`(cid:1)3,-adrenergic
`receptor
`density
`preparation
`equal
`brane
`sites
`rat
`receptor
`densities
`receptor
`in the
`same
`were
`Interaction
`fl-adrenergic
`tor
`sites.
`enantiomer
`
`in
`
`lung
`with
`
`to
`
`The
`binding
`
`various
`recep-
`its
`
`d-
`145
`
`and
`
`stereoisomers,
`and
`$2-adrenergic
`curves
`of nebivolol,
`its
`1-enantiomer
`/3,-
`and
`to
`membrane
`were
`as
`
`R 67
`fl2-adrenergic
`preparations,
`potent
`as CGP
`binding
`to
`rabbit
`times
`less
`active
`100
`binding
`to
`lung
`rat
`inhibited
`nebivolol
`by
`R 67
`were
`138
`and
`R 67
`145 was
`whereas
`stereoisomers
`remaining
`binding
`affinities
`the
`measured
`[‘H]CGP-
`2.
`summarized
`Table
`in
`fi,-
`highest
`for
`affinity
`selec-
`receptor
`a
`(cid:1)l/fl2
`pronounced
`fl,-adrenergic
`(R,R,R,R),
`with
`R 74
`718
`12-fold
`less
`than
`that
`of R 67
`
`was
`the
`obtained
`range.
`its
`of nebivolol,
`fi,-
`with
`blockers
`inhibition
`the
`5 shows
`Fig.
`(S,R,R,R),
`and
`R 67
`138
`[3H]CGP42177
`binding
`(R,S,S,S)
`in
`rabbit
`rat
`lung
`receptor
`Nebivolol
`R 67
`138
`respectively.
`the
`inhibition
`[3H]CGP12177
`in
`20712-A
`preparation.
`R 67
`145 was
`lung
`membrane
`In
`contrast,
`[3H]CGP-12177
`than
`R 67
`138.
`membrane
`preparation
`only
`weakly
`and
`its
`enantiomers.
`Nebivolol
`times
`less
`potent
`than
`118-551
`IC!
`10
`times
`less
`active.
`eight
`The
`nebivolol
`were
`similarly
`investigated;
`and
`fl2-adrenergic
`receptor
`sites
`1(cid:1)1-
`and
`[3H]dihydroalprenolol
`and
`R 67
`138
`receptors
`and
`to
`50-fold.
`40-
`(70-100-fold)
`fl,-adrenergic
`
`and
`and
`
`of
`
`was
`
`on
`sites
`
`two
`
`of
`
`100
`still
`of
`for
`
`with
`
`are
`the
`revealed
`most
`found
`was
`
`showed
`they
`The
`was
`affinity
`
`and
`affinity
`shown
`is
`bound
`potently
`selectivity.
`lacked
`also
`nonselective.
`and
`for
`/3,-adrenergic
`selective
`was
`a selective
`compound
`
`of
`selectivity
`3.
`in
`Table
`- and
`to
`(cid:1)3,
`Levantolol
`CGP
`re-
`for
`
`less
`and
`IC!
`
`12177
`Nebivolol
`adrenergic
`tivity
`selectivity
`but
`its
`138.
`The
`various
`Carvedilol,
`f32-adrenergic
`and
`labetolol
`20712-A
`was
`ceptor
`sites
`
`receptor
`$-adrenergic
`(cid:1)-athenergic
`known
`pindolol,
`and
`receptor
`were
`potent
`whereas
`
`binding
`blockers
`propranolol
`sites
`and
`potent
`highly
`118-551
`
`receptor
`132-adrenergic
`and
`fl2-adrenergic
`$,-
`ity.
`
`sites.
`receptor
`
`Atenolol
`sites
`
`and
`
`showed
`only
`
`low
`moderate
`
`affinity
`selectiv-
`
`for
`
`of
`
`The
`adrenergic
`sorbed-to-filter
`presented
`levantolol
`and
`the
`R 67
`138
`slowly
`from
`Interaction
`
`the
`rates
`dissociation
`were
`sites
`receptor
`The
`technique.
`Labetolol,
`Table
`4.
`in
`within
`a
`dissociated
`receptor
`fl2-athenergic
`IC!
`118-551,
`(S,R,R,R),
`fl2-adrenergic
`and
`the
`f(cid:1),-
`of nebivolol,
`its
`
`compounds
`measured
`half-times
`pindolol,
`few minutes
`sites.
`By
`
`- and
`the
`tissue-ad-
`the
`dissociation
`propranolol,
`the
`from
`both
`contrast,
`nebivolol,
`carvedilol
`dissociated
`and
`receptor
`site.
`stereoisomers,
`
`(cid:1),
`
`from
`using
`of
`
`$2(cid:1)
`
`are
`and
`
`$,-
`
`and
`
`various
`
`was
`
`blockers
`19-adrenergic
`peptide
`and
`ion
`channels,
`binding
`The
`mitter
`uptake.
`measured
`stereoisomers
`receptor
`neurotransmitter
`binding
`ing
`sites.
`The
`and
`values
`as
`-log
`IC.(cid:1),,
`drugs
`the
`potencies
`of
`serotonin,
`norepinephrine,
`
`with
`
`neurotransmitter
`and
`binding
`sites,
`in vitro
`affinity
`of
`in
`radioligand
`binding
`sites,
`ion
`channels
`and
`affinities
`of
`the
`stereoisomers
`K,
`values
`are
`shown
`in Table
`IC50)
`(-log
`inhibit
`the
`dopamine,
`y-aminobutyric
`
`receptors,
`neurotrans-
`the
`nebivolol
`for
`assays
`bind-
`peptide
`expressed
`5. The
`uptake
`of
`acid
`
`to
`and
`
`RABBIT
`
`LUNG
`
`RAT LUNG
`
`20712A
`
`(cid:1),
`
`I’?
`
`0
`
`10
`
`,
`
`9
`
`,
`
`5
`
`,
`
`7
`
`,(cid:1)-,
`
`‘(cid:1)
`
`0
`
`6
`5
`10
`DRUG CONCENTRATION (-log
`
`9
`
`8
`
`7
`
`6
`
`5
`
`Ml
`
`Inhibition of
`Fig. 2.
`lung
`rat
`and
`to rabbit
`21 77 binding
`[3H]CGP-1
`total
`by COP 20712-A and ICI 1 18-551 . Binding
`membrane
`preparations
`was
`and
`[3H]CGP-1 21 77 as described
`in Materials
`with
`1 n(cid:1)i
`performed
`of
`total
`and nonspecific
`binding
`(in the presence
`Methods.
`In rabbit
`lung,
`1 (cid:1)sM CGP 2071 2-A)
`represent
`16,690
`dpm and 3,71 3 ± 623
`± 2,950
`total and nonspecific
`dpm,
`respectively.
`Rat
`lung,
`binding
`(in the presence
`of 1 MM ICI 1 18-551)
`represent
`17,51 1 ± 2,091 dpm and 2,547
`± 387
`dpm,
`respectively.
`[3H]CGP-121
`77 binding
`is expressed
`as percentage
`of
`total
`binding
`in the absence
`of unlabeled
`drugs.
`f3(cid:1), $1-adrenergic
`Curves were constructed
`site;
`fl(cid:1)-adrenergic
`receptor
`site.
`receptor
`$2,
`standard
`error
`of
`three
`separate
`experiments
`using mean
`values
`±
`performed
`in duplicate.
`
`TP2O;;(cid:1)(cid:1)
`
`I 00
`
`80
`
`60
`
`40
`
`20
`
`(cid:1)-
`a-
`
`0 0
`
`I
`
`LOWER DRUG PRICES FOR CONSUMERS, LLC
`Exhibit 1041-3
`IPR2016-00379
`
`(cid:1)
`(cid:1)
`(cid:1)
`(cid:1)
`

`
`binding; #{149},
`
`and Scatchard
`Fig. 3. Saturation
`(inset)
`curve
`binding
`to fl,-adrenergic
`recep-
`21 77 binding
`of
`[3HJCGP-1
`plot
`preparation.
`Binding
`lung membrane
`in rabbit
`tor
`sites
`in the presence
`of 1 0 nM ICI 118-551
`was
`carried
`out
`sites.
`Nonspecific
`block
`fl2-adrenergic
`receptor
`to
`binding
`was
`defined
`in the
`presence
`of 1 (cid:1)M
`CGP
`2071 2-A (0,
`total
`Specific
`binding).
`Curves
`data
`using mean
`were
`constructed
`values
`of binding
`from four
`experiments.
`separate
`SB, specific
`[3H]CGP-
`total
`bound
`1 2177
`binding,
`[3H]CGP-1
`21 77
`in the
`presence
`of 10 nM ICI 118-551 minus nonspecifically
`[3H]CGP-1 21 77 concentration,
`added
`F,
`bound.
`free
`concentration
`of
`[3H]CGP-1
`21 77 minus
`the total
`con-
`K,, value was given by the reciprocal
`centration
`bound.
`the lines. B,,,(cid:1)a value was given
`value
`of
`the slope
`of
`by the intersection
`point with
`the abscissa
`(in pmol/
`ml). Lines were calculated
`using the method
`of
`least
`squares. Values
`are presented
`in Table 1.
`
`0.0
`
`0.5
`
`[3H]CGP-12177
`
`CnM)
`
`E
`
`0
`
`E0
`
`.0
`
`846
`
`Pauwels
`
`et a!.
`
`Rabbit
`
`Lung
`
`(cid:1)thI
`
`0.6-
`
`0.5(cid:1)
`
`0. L.
`
`LI-
`
`LI)
`
`0.3#{149}
`
`0.2(cid:1)
`
`0.1-
`
`0.00
`
`0.02
`
`Specific
`
`0.04
`
`[(cid:1)H]CGP
`
`0.06
`
`-12177
`
`0.08
`
`binding
`
`(pmol/mt)
`
`Rat
`
`Lung
`
`RABBIT
`
`LUNG
`
`RAT LUNG
`
`TOG
`
`80
`
`60’
`
`40
`
`20
`
`100
`
`Q
`
`80
`
`60
`
`(cid:1)0
`
`-,
`
`20
`
`:-
`a.
`
`0 C
`
`I
`
`(cid:1)l0
`
`(cid:1) (cid:1) (cid:1) (cid:1) (cid:1)
`
`010
`
`U-
`
`(I,
`
`0.OL
`
`0.02
`Specific
`
`[3H]
`
`CGP-
`
`12177
`
`binding
`
`(pmol/mI)
`
`(inset)
`4. Saturation
`Fig.
`curve
`binding
`receptor
`to (cid:1)92-adrenergic
`1 21 77
`binding
`as
`out
`Binding
`was
`carried
`preparation.
`2-A was
`that
`n(cid:1)
`CGP
`2071
`300
`Fig.
`3 except
`1 18-551 ,
`fl1-adrenergic
`receptor
`block
`to
`binding. Derived Kd and B(cid:1)5
`values
`specific
`
`and
`
`[3H}CGP-
`of
`plot
`Scatchard
`lung membrane
`sites
`in rat
`in
`the
`legend
`described
`used instead
`of 10 n(cid:1)
`sites.
`0,
`Total
`binding;
`are presented
`in Table
`
`to
`lCI
`U,
`
`1.
`
`1
`TABLE
`and [3H]dihydroalprenolol
`[3HJCGP-12271
`of
`K,, and B(cid:1),..1 values
`binding
`to $i(cid:1)
`and (cid:1)2-adrenergic
`receptor
`sites
`and rat
`in rabbit
`membrane
`preparation.
`K,, and B(cid:1)
`are
`values
`separate
`experiments.
`
`error
`
`of values
`
`obtained
`
`the
`
`means
`
`±
`
`standard
`
`lung
`
`in four
`
`Rabb(cid:1)
`
`lung /(cid:1),
`
`Rat
`
`lung 02
`
`K5
`
`flu
`
`B(cid:1)
`
`(mo//mg
`
`oftissue
`
`K5
`
`flu
`
`B(cid:1)
`
`tmoI/mg
`
`of
`
`tissue
`
`I3HICGP-12177
`(3HjDihydroalprenolol
`
`0.14
`0.89
`
`± 0.01
`± 0.23
`
`9.3
`5.1
`
`± 0.7
`1.3
`±
`
`0.24
`0.21
`
`± 0.04
`0.03
`±
`
`10.7
`7.7
`
`± 0.7
`± 0.4
`
`CONCENTRATION(-logM)
`DRUG
`and f(cid:1)2-adrenergic
`to (cid:1)
`21 77 binding
`[3H]CGP-1
`of
`Inhibition
`5.
`Fig.
`respectively,
`lung membrane
`preparation,
`and rat
`sites
`in rabbit
`receptor
`(0), R 67 138 (#{149}),R 67 1 45 (x), CGP 2071 2-A (U), and ICI
`by nebivolol
`1 18-551
`(0). Rabbit
`lung binding
`was
`in the presence
`of 10 nM CI 118-
`551 ; total
`binding,
`13874
`± 1 327 dpm;
`and nonspecific
`binding,
`2475
`±
`of
`1 03
`dpm.
`Rat
`lung
`binding
`was
`in the
`presence
`300
`CGP
`20712-A;
`total
`binding,
`13384
`± 2199
`dpm;
`and nonspecific
`binding,
`± 228 dpm.
`[3H]CGP-1 21 77 binding
`is expressed
`as percentage
`1 841
`in the presence
`of 10 n(cid:1)
`ICI 1 18-551 and 300 n(cid:1) CGP
`of
`total
`binding
`2071 2-A for
`rabbit
`were constructed
`and rat
`Curves
`lung,
`respectively.
`using mean
`standard
`values
`of
`three
`separate
`experiments
`error
`±
`duplicate.
`
`n(cid:1)
`
`in
`
`are
`
`Table
`in
`SIA binding
`
`6.
`
`to
`
`716,
`40
`
`829,
`the
`
`shown
`preparations
`synaptosomal
`crude
`in
`bound
`stereoisomers
`the
`nebivolol
`of
`Several
`with
`[3H]8hythoxy2(din..propylammno)tetralin(cid:1)
`labeled
`sites
`R 74
`R 65
`825,
`R 67
`138,
`R 65
`260,
`R 74
`nebivolol,
`142
`showed
`and
`R 67
`K,
`nM.
`In
`values
`between
`20
`and
`neurotrans-
`various
`other
`investigated
`radioligand
`binding
`and
`showed
`mitter
`uptake
`assays,
`nebivolol
`and
`its
`stereoisomers
`inactive.
`only
`activity
`at micromolar
`concentrations
`or were
`In order
`to better
`visualize
`the
`profile
`the
`nebivolol
`stereo-
`isomers,
`charts
`have
`been
`constructed
`for
`nebivolol,
`R 67
`pie
`K,
`138,
`R 67
`145
`using
`the
`reciprocal
`the
`values
`for
`of
`receptor
`binding
`and
`IC50
`values
`for
`of monoamine
`inhibition
`uptake
`(Fig.
`6).
`The
`pie
`chart
`shows
`contribution
`the
`relative
`presented
`of
`each
`activity
`in
`sum
`activities
`drug
`of
`of
`the
`in
`Tables
`2,
`and
`For
`nebivolol,
`$,-adrenergic
`receptor
`6.
`binding
`accounts
`for
`93%,
`f32-adrenergic
`receptor
`binding
`
`and
`
`the
`
`5,
`
`of
`
`for
`
`LOWER DRUG PRICES FOR CONSUMERS, LLC
`Exhibit 1041-4
`IPR2016-00379
`
`(cid:1)
`(cid:1)
`(cid:1)
`(cid:1)
`

`
`TABLE 2
`ligands
`with
`sites measured
`receptor
`and fi2-adrenergic
`for
`stereoisomers
`of nebivolol
`Binding
`affinity
`two
`(nM). Binding was
`number of experiments.
`± standard deviations. Numbers
`in parentheses,
`b, K, values
`a, -log
`performed
`1Gw (M), mean
`value
`of 300 n(cid:1)
`and Methods
`presence
`CI 1 1 8-551 with
`rabbit
`lung membrane
`preparation
`and
`1 0 n(cid:1) CGP 2071 7-A with
`rat
`lung membrane
`in the
`111- and
`02-adrenergic
`receptor
`sites,
`respectively.
`
`as described
`preparation
`
`in Materials
`to measure
`
`Receptor
`
`Binding
`
`Profile
`
`of Nebivolol
`
`847
`
`l(cid:1)HlCGP-12177
`
`binding (1 nM)
`
`[(cid:1)HlDihydroal(cid:1)renod
`
`binding (1 nM)
`
`Rabbe
`
`lung 0,
`
`Rat
`
`lung 02
`
`Rabbit
`
`lung 0(cid:1)
`
`Rat
`
`lung 02
`
`A-number,
`Configuration
`
`+ R,S,S,S
`
`Nebivolol
`S,R,R,R
`R65825
`± R,S,R,R
`S,R,S,S
`R 67 138
`S,R,R,R
`R 67 145
`R,S,S,S
`R65260
`S,R,R,S
`R74716
`R,S,S,R
`R74829
`S,R,S,R
`R74714
`S,R,S,S
`R67142
`R,S,R,R
`R 74
`721
`R,R,S,S
`R 74 723
`S,S,S,S
`R74718
`R,R,R,R
`
`a.
`b.
`a.
`b.
`a.
`b.
`a.
`b.
`a.
`b.
`a.
`b.
`a.
`b.
`a.
`b.
`a.
`b.
`a.
`b.
`a.
`b.
`a.
`b.
`
`8.13±0.05(3)
`0.88
`7.53±0.05(3)
`3.5
`8.17 ± 0.06 (3)
`0.8
`5.93 ± 0.1 1 (3)
`140
`7.65±0.07(2)
`2.7
`6.15±0.07(2)
`84
`6.5(2)
`38
`6.60±0.14(2)
`30
`7.5(2)
`3.8
`5.95 ± 0.07
`133
`5.1 (2)
`945
`7.05±0.07(2)
`11
`
`(2)
`
`6.6(2)
`48
`6.15±0.07(3)
`144
`6.76 ± 0.05 (3)
`34
`5.66 ± 0.05 (2)
`423
`6.70±0.14(2)
`39
`5.65±0.2(2)
`433
`6.20±0.14(2)
`122
`5.95±0.07(2)
`217
`6.10±0.14(2)
`153
`± 0.07
`1370
`5.30 ± 0.14 (2)
`971
`5.2(2)
`1222
`
`5.15
`
`(2)
`
`55
`
`41
`
`42
`
`3
`
`15
`
`5.15
`
`3.2
`
`7.2
`
`40
`
`10
`
`1
`
`111
`
`8.72±0.09(4)
`0.91
`7.92±0.09(4)
`5.7
`8.95 ± 0.1 (4)
`0.54
`6.53 ± 0.05 (3)
`138
`8.2(2)
`3
`6.8(2)
`75
`7.05±0.07(2)
`43
`7.25±0.07(2)
`27
`7.90±0.14(2)
`6
`± 0.07
`193
`5.40 ± 0.14 (2)
`1935
`7.65±0.07(2)
`11
`
`6.40
`
`(2)
`
`5.30
`
`6.57±0.05
`44
`6.13±0.11
`281
`6.96 ± 0.15 (3)
`19
`5.66 ± 0.1 1 (3)
`367
`6.40±0.14(2)
`68
`5.69±0.07(2)
`375
`6.15±0.2(2)
`125
`6.0(2)
`166
`6.0(2)
`166
`± 0.14
`857
`5.19 ± 0.07 (2)
`1187
`5.35±0.07
`744
`
`(4)
`
`(3)
`
`(2)
`
`(2)
`
`48
`
`49
`
`35
`
`2.6
`
`23
`
`5
`
`2.9
`
`6.1
`
`28
`
`4.4
`
`0.6
`
`68
`
`TABLE 3
`of various
`Binding
`affinity
`a,
`-log
`(M), mean
`value
`to Table
`
`lC(cid:1)
`2.
`
`fl-adrenergic
`± standard
`deviation.
`
`blockers
`Numbers
`
`and (cid:1)2-adrenergic
`for
`in parentheses,
`number
`
`receptor
`of experiments.
`
`sites measured
`b. K value (nM).
`
`with
`Binding
`
`two
`was
`
`ligands
`performed
`
`as described
`
`in the legend
`
`[(cid:1)HJC GP-12177
`
`CGP20712-A
`
`Atenolol
`
`Levantolol
`
`Labetolol
`
`Carvedilol
`
`Pindolol
`
`Propranolol
`
`ICI 1 18-551
`
`a.
`b.
`a.
`b.
`a.
`b.
`a.
`b.
`a.
`b.
`a.
`b.
`a.
`b.
`a.
`b.
`
`.
`Rabbd lung 13,
`
`7.86±0.05(3)
`1.6
`5.65±0.07(2)
`396
`6.90±0.07(2)
`15
`6.7(2)
`24
`8.65 ± 0.07
`0.24
`± 0.1 1 (3)
`1.4
`± 0.05
`2.8
`6.60 ± 0.14 (2)
`49
`
`(2)
`
`(3)
`
`8.13
`
`7.83
`
`binding (1 flM)
`
`Rat
`
`lung 132
`
`5.2
`1222
`4.8(2)
`7493
`6.60±0.14(2)
`49
`7.0(2)
`19
`9.0 (2)
`0.19
`8.30 ± 0.1 (3)
`1.0
`8.5 (2)
`0.62
`8.60 ± 0.07 (2)
`0.49
`
`Ratio
`P2/P’
`
`763
`
`19
`
`3.2
`
`0.79
`
`0.79
`
`0.7
`
`0.22
`
`0.01
`
`l3HlDihy droalprenold t(cid:1)nding (1 nM)
`
`.
`Rabbd lung 0(cid:1)
`
`Rat
`
`lung $2
`
`8.35(2)
`2.1
`6.25±0.07(2)
`266
`7.5(2)
`15
`7.40±0.14(2)
`19
`± 0.09
`0.43
`8.66 ± 0.1 1 (3)
`1.0
`8.6 (2)
`1 .2
`7.12 ± 0.09 (4)
`36
`
`9.02
`
`(4)
`
`5.3(2)
`835
`4.75±0.07(2)
`2960
`6.50±0.14(2)
`53
`6.79±0.07
`30
`± 0.02 (3)
`0.25
`± 0.15
`0.8
`8.75
`0.29
`8.43 ± 0.10 (3)
`0.62
`
`(2)
`
`(3)
`
`(2)
`
`8.81
`
`8.33
`
`Rate
`P2
`
`397
`11
`
`3.5
`
`1.58
`
`0.58
`
`0.8
`
`0.24
`
`0.02
`
`binding
`and
`SIA
`activities
`other
`contribution
`However,
`which
`
`1.7%,
`of
`the
`relative
`similar.
`tiomer,
`completely
`accounts
`/32-adrenergic
`channel,
`7 and
`
`for
`
`and
`10%.
`
`for
`
`The
`was
`
`contribution
`The
`4.1%.
`binding
`site
`6 is negligible.
`5 and
`in Tables
`listed
`of R 67 138
`activity
`of each
`(S,R,R,R)
`(R,S,S,S)-enan-
`145,
`the
`of R 67
`chart
`the
`is
`f(cid:1),-adrenergic
`receptors,
`bound
`to
`weakly
`only
`this
`compound,
`binding
`to
`SIA sites
`For
`different.
`fl,-adrenergic
`receptor
`binding
`for
`22%,
`and
`33%,
`receptor
`binding,
`the
`veratridine
`the
`Na(cid:1)
`the
`uptake
`of
`serotonine
`and
`between
`
`The
`Tables
`pranolol
`of
`3,
`labetolol
`tor
`sites
`
`15,
`
`in
`shown
`blockers
`f3-adrenergic
`various
`of
`profile
`pro-
`and
`pindolol,
`that
`carvedilol,
`reveals
`It
`8.
`7 and
`K, values
`sites
`with
`to
`binding
`bind
`potently
`also
`respectively.
`In
`addition,
`carvedilol
`and
`84
`nM,
`and
`inhibited
`[:IHIWB
`401
`binding
`to
`a,-adrenergic
`recep-
`a K, value
`with
`of 3 and
`42
`respectively.
`
`is
`
`SIA
`
`nM,
`
`site
`dopamine
`
`of
`
`Discussion
`
`Specificity
`model.
`
`ing
`
`the
`of
`(cid:1),-
`agreement
`
`In
`
`and
`with
`
`(cid:1)-adrenergic
`previous
`
`receptor
`(see
`
`bind-
`Ref.
`12)
`
`reports
`
`LOWER DRUG PRICES FOR CONSUMERS, LLC
`Exhibit 1041-5
`IPR2016-00379
`
`(cid:1)
`(cid:1)
`(cid:1)
`

`
`848
`
`Pauwels
`
`et a!.
`
`4
`
`TABLE
`Half-time
`fl-adrenergic
`are mean
`Values
`experiments.
`
`±
`
`stereoisomers
`of nebivolol
`of dissociation
`from $(cid:1)-
`blockers
`and fl2-adrenergic
`standard
`deviation.
`Numbers
`in
`
`and
`receptor
`parentheses,
`
`sites
`number
`
`of
`
`T(cid:1),
`
`mm
`
`Rabbit
`
`lung fi,
`
`81 ± 19 (1 0)
`109 ± 35
`(8)
`38 ± 8
`(5)
`54±3
`(4)
`47 ± 18
`(9)
`21 ± 6
`(6)
`1 1 ± 6
`(5)
`1 0 ± 3
`(4)
`8 ± 1
`(6)
`7 ± 1
`(3)
`6 ± 2
`(4)
`
`Rat lung (32
`
`32 ± 5 (11)
`37 ± 7
`(4)
`7 ± 2
`(3)
`47±22(6)
`61 ± 28 (3)
`
`7 ± 3
`
`(2)
`
`1 3 ± 5
`7 ± 1
`6 ± 2
`
`(3)
`(2)
`(2)
`
`Nebivolol
`R 67 138
`R 67 145
`lClll8-551
`Carvedilol
`CGP 20712-A
`Levantolol
`Atenolol
`Propranolol
`Pindolol
`Labetolol
`
`in
`
`rabbit
`that
`found
`we
`and
`sites
`receptor
`selective
`obtained
`these
`tissues,
`sites
`[‘HJCGP-12177
`ligands
`presence
`of
`in the
`$2-adrenergic
`blocker
`blocker
`CGP
`20712-A.
`sis
`ofthe
`binding
`data
`of only
`one
`binding
`4).
`The
`use
`of
`occlusion
`of
`the
`applied
`by Nanoff
`adrenergic
`receptor
`Selectivity
`of
`
`an
`
`in fi, -adrenergic
`enriched
`lung was mainly
`We
`receptor
`sites.
`lung
`in
`f.(cid:1)2-athenergic
`rat
`fl2-adrenergic
`receptor
`labeling
`of
`and
`the
`nonselective
`radio-
`respectively,
`with
`(Table
`and
`[3H]dihydroalprenolol
`selective
`the
`appropriate
`concentration
`/31-adrenergic
`ICI
`118-551
`or
`the
`selective
`Scatchard
`analy-
`Using
`these
`conditions,
`ofthe
`radioligands
`indicated
`the
`presence
`site
`in the
`tissues,
`respectively
`(Figs.
`3 and
`selective
`and
`(cid:1)9,-adrenergic
`blockers
`for
`receptor
`respectively,
`recently
`also
`et al.
`selective
`of
`(cid:1)3,-
`f32-
`sites
`cardiac
`nebivolol
`for
`$(cid:1)-
`
`(cid:1),-
`
`1),
`
`of
`
`(cid:1)-
`
`sites,
`(13)
`for
`in rat
`
`was
`labeling
`microsomes.
`and
`fi2-adrenergic
`
`and
`
`re-
`
`sites
`
`series
`showed
`for
`mixture
`these
`
`had
`can
`by
`was
`
`less
`
`other
`and
`stereoisomers
`of
`f(cid:1),-
`blocker
`was
`a potent
`a
`subnano-
`lung.
`It
`showed
`as
`with
`E:IH]CGp42177
`138 was
`d-enantiomer
`R 67
`R 67
`145 was
`175-fold
`less
`1-enantiomer
`R 74
`723
`(S,S,S,S),
`stereoisomer,
`pindolol,
`propran-
`20712-A,
`nM.
`CGP
`affinity
`the
`same
`f3,-adrenergic
`labetolol,
`and
`aten-
`levantolol,
`than
`nebivolol.
`less
`active
`a pronounced
`/3,-athenergic
`50
`times
`less
`potent
`at
`lung membrane
`preparation.
`R 67
`138
`(S,R,R,R)-enan-
`of
`high
`potency
`Nebivolol,
`which
`(R,S,S,S)-enantiomers,
`(R,S,S,S)-enantiomer
`The
`presence
`of
`
`its
`with
`Nebivolol
`in
`rabbit
`measured
`Its
`
`nM,
`
`in
`
`the
`
`compared
`ceptor
`blockers.
`fi-adrenergic
`receptor
`sites
`adrenergic
`K,
`of
`value
`0.9
`molar
`[:IH]dihythoalprenolol
`as
`well
`whereas
`its
`equipotent
`least
`active
`The
`potent.
`showed
`a K, value
`of 945
`carvedilol
`showed
`olol,
`and
`nebivolol.
`In
`contrast,
`range
`as
`17-,
`27-,
`and
`450-fold
`olol were
`In
`addition,
`nebivolol
`showed
`selectivity
`inasmuch
`it was
`as
`rat
`adrenergic
`receptor
`in the
`sites
`the
`In
`the
`of
`stereoisomers,
`combination
`tiomer
`the
`optimal
`receptors.
`selectivity
`f3,-adrenergic
`(S,R,R,R)
`and
`racemic
`of
`the
`The
`inactive
`shared
`properties.
`on
`the
`activity.
`effect
`apparently
`little
`the
`concentration
`reduce
`compound
`only
`[‘H]dihydroalprenolol,
`Using
`enantiomer
`half.
`of nebivolol
`than
`times
`higher
`two
`indeed
`in K,
`was
`not
`observed
`value
`This
`difference
`potency
`difference
`the
`probably
`because
`12177,
`the
`Within
`presently
`experimental
`variation.
`only
`blockers,
`two
`compounds
`series
`of
`/3-adrenergic
`and
`selectivity
`for
`(cid:1),-athenergic
`combined
`high
`affinity
`and
`CGP
`20712-A.
`The
`tors;
`these
`were
`nebivolol
`was
`still
`10 times
`2-fold
`lower
`affinity
`but
`to
`nebivolol.
`Atenolol,
`generally
`referred
`less
`adrenergic
`blocker,
`was
`300
`400
`times
`to
`times
`selective
`than
`nebivolol.
`Striking
`
`(cid:1)32-
`
`and
`is
`
`a
`
`this
`of
`active
`value
`138.
`
`the
`
`a
`
`4
`
`(cid:1),-
`2 to
`were
`
`K,
`the
`of R 67
`[‘H]CGP-
`within
`investigated
`showed
`recep-
`had
`than
`
`that
`with
`was
`
`latter
`selective
`selective
`and
`
`more
`a
`as
`potent
`differences
`
`5
`
`TABLE
`of nebivolol
`profile
`binding
`Receptor
`a,
`-log
`± standard
`value
`lCw (N), mean
`up to a concentration
`Tested
`and Methods.
`cholinergic
`receptors
`muscarinic
`([3Hjdexetimide,
`sites
`([3H]substance
`substance
`P binding
`P,
`for the biogenic
`value
`of 6.3
`(one
`experiment)
`
`release
`
`stereoisomers
`number
`in parentheses,
`Numbers
`b, K values
`of experiments.
`(nM).
`Experimental
`deviation.
`stereolsomers
`of 10’
`nebivolol
`M,
`D(cid:1)
`receptors
`dopamine
`showed
`no
`interaction
`with
`the
`u-opiate receptors
`benzodiazepine
`receptors
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`rat
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`pig forebrain).
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`In addition,
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`binding
`sites
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`amine
`and metaboli