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`
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`
`0022-3565/90/2541-0176$02.OO/O
`JOURNAL
`OF
`PHARMACOLOGY
`THE
`by The American
`Copyright
`0
`
`1990
`
`AND
`Society
`
`EXPERIMENTAL
`for Pharmacology
`
`THERAPEUTICS
`and
`Experimental
`
`Therapeutics
`
`Vol. 254, No.
`Printed
`in U.S.A.
`
`1
`
`24.1 1 Inhibition in Conscious
`Effect of Endopeptidase
`Cardiomyopathic
`Hamsters
`
`GLENN
`
`J. SMITS,
`
`DEAN
`
`E. McGRAW,
`
`ANGELO
`
`J. TRAPANI
`
`and
`
`EDWARD
`
`B. BLAINE
`
`St. Louis, Missouri
`and Development,
`Searle Research
`Accepted
`for publication
`April 2, 1990
`
`ABSTRACT
`peptide
`natriuretic
`of atrial
`concentration
`plasma
`An
`elevated
`in both
`failure
`(CHF)
`heart
`of congestive
`(ANP)
`is characteristic
`for
`facilitating
`the biological
`One
`strategy
`humans
`and
`animals.
`is to inhibit
`its metabolism.
`actions
`of
`this
`circulating
`hormone
`enzyme
`known
`to
`degrade
`Neutral
`endopeptidase
`24.1 1 , an
`ANP,
`has
`been
`implicated
`in the metabolic
`clearance
`of
`this
`hormone.
`Inhibition
`of endopeptidase
`24.1 1 may
`produce
`in-
`creases
`in the local
`concentrations
`of ANP in organs
`rich
`in the
`enzyme,
`such
`as the
`kidney,
`thereby
`enhancing
`local
`actions,
`natriuresis.
`Exogenous
`administration
`of ANP to CHF
`pa-
`e.g.
`tients
`produces
`limited
`natriuresis,
`perhaps
`because
`of
`the
`as-
`sociated
`fall
`in arterial
`pressure.
`This
`approach
`of blocking
`en-
`dopeptidase
`24.1 1 activity
`in CHF could
`offset
`the general
`effects
`of
`exogenous
`administration
`of ANP
`and
`result
`in enhanced
`natriuresis.
`We therefore
`undertook
`studies
`to determine
`if thior-
`
`circulat-
`increase
`would
`24.1 1 inhibitor,
`an endopeptidase
`phan,
`cardiomy-
`in conscious
`ing ANP levels
`and enhance
`natnuresis
`opathic
`hamsters
`with
`CHF.
`Cardiomyopathic
`hamsters
`had
`significantly
`lower
`(P <
`.05)
`basal mean
`arterial
`pressures
`in
`comparison
`to normal
`hamsters
`(90 ± 2 vs.
`± 3 mm Hg).
`1 35
`Treatment
`with
`thiorphan
`(1 0 mg/kg
`iv.
`bolus
`followed
`by
`10
`mg/kg/hr
`i.v.
`infusion)
`did not
`change
`arterial
`pressure
`in either
`group
`and produced
`a 3-fold
`increase
`in urinary
`sodium excretion
`not
`in the
`cardiomyopathic
`group
`but
`in the
`normal
`hamsters.
`This
`natriuretic
`response
`in the cardiomyopathic
`hamsters
`was
`associated
`with
`a doubling
`of
`the plasma
`concentration
`of ANP.
`These
`results
`indicate
`that
`inhibition
`of endopeptidase
`24.11
`increases
`natnuresis
`and circulating
`ANP concentrations
`in this
`model
`of CHF.
`Thus,
`altering
`the metabolism
`of endogenous
`ANP may
`provide
`a new therapeutic
`approach
`for
`the manage-
`ment
`of CHF.
`
`endocrine
`and
`hemodynamic,
`possesses
`ANP
`Several
`of CHF.
`the
`symptoms
`could
`alleviate
`which
`treating
`agent
`as
`therapeutic
`evaluated
`ANP
`have
`demonstrated
`and
`Crozier
`1986)
`1986;
`(Cody
`et
`at.,
`et
`at.,
`a markedly
`ANP
`exogenous
`natriuretic
`response
`depressed
`in CHF
`for
`responsible
`mechanisms
`which
`The
`patients.
`been
`have
`not
`of ANP-induced
`natriuresis
`this
`attenuation
`the
`may
`contrib-
`depressor
`activity
`of ANP
`identified;
`however,
`to underperfusion
`1986).
`the
`kidneys
`(Crozier
`of
`ute
`at.,
`et
`Rather
`an
`alternative
`exogenous
`ANP,
`than
`administering
`is
`to
`decrease
`a therapeutic
`agent
`as
`approach
`to
`utilizing
`ANP
`indicates
`hormone.
`Evidence
`the
`the metabolism
`of
`endogenous
`inhibitors
`that
`ANP
`is metabolized
`by
`endopeptidase
`24.11
`and
`of
`this
`enzyme
`prevent
`the
`degradation
`of ANP
`(Olins
`et
`1987,
`1989;
`Sonnenberg
`1988;
`Stephenson
`and
`Kenny,
`et
`at.,
`in-
`expected
`to
`Local
`concentrations
`of ANP
`could
`be
`1987).
`crease
`such
`greatly
`during
`endopeptidase
`inhibition
`in
`tissues
`as the kidney
`which
`contain
`(Gee
`of
`large
`amounts
`enzyme
`might
`1985).
`Thus,
`an
`inhibitor
`endopeptidase
`24.11
`of
`et
`at.,
`prove
`to
`be more
`effective
`than
`exogenous
`ANP
`promoting
`
`actions
`studies
`CHF
`
`at.,
`
`in
`
`natriuresis
`because
`cular
`functions,
`can
`counteract
`tively
`raising
`expect
`enhanced
`would
`be maintained.
`natriuretic
`to
`response
`be amplified
`in conditions
`such
`as CHF
`(Burnett
`This
`study
`was
`1986).
`bition
`of
`endopeptidase
`uresis
`and
`increase
`in conscious
`hamsters
`
`the
`
`exogenous
`resulting
`ANP-induced
`ANP
`concentrations
`because
`natriuresis
`Moreover,
`endopeptidase
`of
`elevated
`1986;
`at.,
`et
`undertaken
`would
`24.11
`circulating
`CHF.
`with
`
`broadly
`arterial
`In
`the
`
`one
`
`to
`
`ANP
`decreased
`natriuresis.
`within
`renal
`might
`24.11
`circulating
`Cody
`et
`determine
`elicit
`an
`levels
`of
`
`affects
`pressure
`contrast,
`kidney,
`perfusion
`predict
`inhibition
`levels
`1986;
`whether
`enhanced
`endogenous
`
`cardiovas-
`which
`selec-
`by
`could
`one
`pressure
`that
`the
`would
`of ANP
`Laragh,
`inhi-
`natri-
`ANP
`
`aL,
`
`Methods
`
`Male
`from
`
`CM and NM hamsters,
`protocol.
`and
`Instrumentation
`weighing
`to
`Canadian
`Hybrid
`Farms
`obtained
`g, were
`160
`120
`with methohexital
`sodium
`anesthetized
`While
`Canada).
`(Nova
`Scotia,
`(60 mg/kg
`femoral
`arterial
`venous
`and
`barbiturate,
`a short-acting
`i.p.),
`pressure
`and
`to administer
`inserted
`to monitor
`arterial
`cannulas
`were
`saline
`thiorphan.
`flared
`of a length
`of PE-190
`tubing
`(Clay-
`end
`Adams,
`Parsippany,
`was
`inserted
`into
`the
`dome
`the
`urinary
`
`or
`
`The
`NJ)
`
`of
`
`renal
`
`a
`
`for
`have
`to
`are
`
`the
`
`in
`
`Received
`
`for
`
`publication
`
`July
`
`17,
`
`1989.
`
`ABBREVIATiONS:
`
`ANP,
`
`atrial natriuretic
`
`peptide;
`
`CHF,
`
`congestive
`
`heart
`
`failure; CM, cardiomyopathic;
`
`NM,
`
`normal;
`
`MAP, mean
`
`arterial
`
`pressure;
`
`UV, urine
`
`flow;
`
`r-ANP,
`
`rabbit
`
`ANP;
`
`lgG,
`
`immunoglobulin
`
`G; UNaV,
`
`urinary
`
`sodium excretion.
`
`176
`
`NOVARTIS EXHIBIT 2003
`Biocon v. Novartis, IPR2020-01263
`Page 1 of 4
`
`

`

`Endopeptidase
`
`24.11
`
`InhibItion
`
`In CHF
`
`177
`
`pressure,
`
`UV, UNaV and heart
`
`weights
`
`in CM and NM
`
`1
`TABLE
`arterial
`Basal
`hamsters
`Atnal Wt., atrial weight
`Values are mean ± S.E.M.
`in mg/i
`Ventr. Wt., ventricular weight
`g of body weight.
`in mg/100
`
`00 g of body weight;
`
`Strain
`
`Atri(cid:1) Wt.
`
`Ventr. WI.
`
`MAP
`
`LR4aV
`
`(IV
`
`360±4
`10)
`(n =
`362 ± 20
`(n =
`5)
`
`mm Hg
`135±3
`17)
`(n =
`90 ± 2
`(n =
`16)
`
`(cid:1)Eq/min
`0.10±0.01
`17)
`(n =
`0.26
`± 0.05
`16)
`(n =
`
`(cid:1)d/min
`2.6±0.5
`17)
`(n =
`2.9 ± 0.5
`(n =
`16)
`
`NM
`
`CM
`
`. P <
`
`30±1
`(n = 10)
`100 ± 10’
`(n =
`5)
`.05 vs. NM
`
`hamsters.
`
`Thiorphan
`
`Saline
`
`1.10
`
`1.00
`
`0.90
`
`0.80
`
`0.70
`
`CE 0(cid:1)
`
`1990
`
`bladder
`ened,
`ters.
`
`via
`they
`
`a midline
`were
`placed
`
`abdominal
`into
`Lucite
`
`incision.
`holders
`
`Before
`which
`
`hamsters
`the
`protected
`the
`
`awak-
`cathe-
`
`hours
`NaCl
`
`for
`was
`
`on
`
`in
`
`n
`was
`
`At
`collected
`
`An
`and
`
`with
`
`=
`
`to recover.
`animals
`all
`animals
`started
`equilibration,
`a
`50-mm
`during
`a 30-mm
`control
`to one
`of
`two
`treatments;
`or
`given
`a
`10 mg/kg
`i.v.
`infusion.
`This
`groups;
`CM-Saline
`(CMs
`treated
`n
`on
`saline;
`8). Once
`collected
`the
`
`infusion
`i.v.
`continued
`urine
`period.
`either
`bolus
`i.v.
`randomization
`(CMs
`main-
`thiorphan;
`and
`NM-
`9)
`thiorphan
`the
`was
`MAP
`and
`this
`experi-
`of
`end
`for measurement
`
`was
`The
`they
`of
`
`of
`
`anesthe-
`were
`experiment
`the weights
`
`to
`
`was
`
`Downloaded from
`
`jpet.aspetjournals.org
`
` at ASPET Journals on April 10, 2019
`
`0.60
`
`0.50
`
`0.40
`
`0.30
`
`0.20
`
`0.10
`
`0.00
`
`c:(cid:1)
`
`cardiomyopathic
`
`Normal
`
`LU
`
`.>
`
`Isz
`
`C 5
`
`)
`C)
`
`CC
`
`S
`.C
`C.)
`
`thiorphan
`the 30-mm period during
`change
`1. Absolute
`Fig.
`in UNaV for
`i.v.
`infusion)
`or saline infusion
`iv. bolus and 10 mg/kg/hr
`in
`(1 0 mg/kg
`CM and NM hamsters.
`are mean ± S.E.M.
`Values
`for CM-Saline
`(n = 8),
`8), NM-Saline
`CM-Thiorphan
`(n =
`(n =
`9) and NM-Thiorphan
`(n =
`8)
`change in sodium excretion
`groups.
`The absolute
`was
`calculated
`as the
`value measured
`for
`the 30-mm experimental
`difference
`between
`the
`30-mm control
`period
`that
`determined
`for
`period.
`P <
`.05 vs.
`and
`the
`CM-Saline.
`
`of
`of
`
`the
`
`regardless
`
`(P <
`UV
`the
`
`.05)
`was
`ab-
`of
`
`the CM or NM hamsters,
`in either
`experiment
`the
`or
`saline.
`thiorphan
`with
`treatment
`sodium
`more
`slightly
`excreted
`The
`CM hamsters
`than
`whereas
`period
`control
`during
`NM hamsters
`1 shows
`Figure
`1).
`(table
`strains
`comparable
`between
`solute
`administration
`the
`by
`produced
`in UNaV
`increases
`experimental
`the
`thiorphan
`saline
`during
`or
`Sodium
`period.
`received
`which
`excretion
`did not
`change
`in NM or CM hamsters
`saline
`with
`treated
`throughout
`the
`experiment.
`NM
`hamsters
`thiorphan
`sodium
`in
`did
`exhibit
`a
`significant
`increase
`not
`when
`compared
`saline.
`with
`excretion
`to NM hamsters
`infused
`treatment
`In
`contrast
`CM hamsters
`thiorphan
`responded
`to
`with
`a 3-fold
`significant
`increase
`in UNaV.
`This
`natriuresis
`was
`its magnitude
`varied
`but
`considerably
`between
`individual
`mals
`within
`the
`group.
`Changes
`UV
`paralleled
`sodium
`excretion
`not
`shown).
`(data
`concentration
`of ANP
`The
`plasma
`
`in
`
`ani-
`of
`
`those
`
`was
`
`6-fold
`
`higher
`
`in
`
`the
`
`the
`allowed
`were
`Two
`then
`(cid:1)zl/min)
`(16
`of 0.9%
`After
`experiment.
`the
`throughout
`recorded
`and MAP
`was
`collected
`animals
`randomly
`were
`assigned
`saline
`alone
`were
`maintained
`on
`a 10 mg/kg/hr
`thiorphan
`followed
`by
`procedure
`resulted
`in four
`experimental
`n =
`tamed
`saline;
`8), CM-Thiorphan
`n
`8),
`NM-Saline
`(NMs
`maintained
`=
`Thiorphan
`(NMs
`treated
`with
`thiorphan;
`or
`saline
`infusions
`were
`underway,
`urine
`recorded
`for
`a 30-mm
`experimental
`period.
`blood
`was
`of
`mental
`period,
`2 ml
`arterial
`ANP
`concentration.
`plasma
`randomly)
`chosen
`10 NM,
`hamsters
`(5 CM and
`Some
`the
`conclusion
`of
`at
`the
`tized with
`sodium
`pentobarbital
`the
`viscera
`and
`determine
`of
`examine
`the
`gross morphology
`ventricles.
`of
`dissected
`cardiac
`atria
`and
`biochemical
`volume
`Urine
`Physiological
`and
`analysis.
`calculated
`was
`excretion
`terinined
`gravimetrically
`and
`sodium
`photometry
`flame
`by
`urine
`concentration,
`as measured
`sodium
`MA)
`and
`Lexington,
`1L943,
`Instrumentation
`Laboratories,
`connecting
`Arterial
`pressure
`was monitored
`continuously
`by
`CA)
`transducer
`cannula
`to
`a Statham
`P231D
`(Gould,
`Oxnard,
`2800S
`polygraph
`was
`coupled
`to a Gould
`Pressure
`Processor
`and
`Inc.,
`Cleveland,
`OH). MAP
`was
`MAP
`derived
`electronically.
`pled
`at
`2-mm
`intervals
`quantitate
`the
`average
`arterial
`of
`during
`each
`30-mm
`period
`an
`experiment.
`determined
`was
`plasma
`The
`concentration
`of ANP
`in
`the
`method,
`(Trapani
`previously
`double-antibody
`as
`described
`et al,
`were
`using
`rANP(99-126)
`duplicate
`All
`determinations
`performed
`as standard,
`r-[’251]ANP
`anti-human
`ligand,
`labeled
`as
`the
`goat
`anti-rabbit
`ANP
`at
`Unlabeled
`serum.
`serum
`and
`IgG
`50% displacement
`in
`this
`r(cid:1)[lThIJANP
`produced
`of bound
`interassay
`coefficients
`less
`than
`and
`intra-
`of variation
`were
`Data
`were
`analyzed
`two-way
`Statistics.
`by
`one-way
`and
`Student’s
`test
`for
`and
`variance
`with
`equal
`variance
`data
`t
`for
`comparisons
`test
`rank-sum
`unequal
`variances
`with
`software
`package
`statistical
`Institute
`Inc.,
`Cary,
`(SAS
`considered
`to be different
`were
`when
`P <
`.05.
`Data
`are
`mean
`± S.E.M.
`Drugs.
`Racemic
`phenyl-propyl]-glycine)
`1982;
`cedures
`(Bindra,
`human
`ANP
`serum
`and
`Peninsula
`Laboratories
`from
`Amersham
`Corp.
`
`to
`
`in
`
`rabbit
`
`urine
`the
`
`de-
`from
`(model
`flow.
`arterial
`which
`(Gould
`sam-
`was
`pressure
`
`a
`
`by
`1989).
`
`ANP
`pg/tube
`21
`assay.
`The
`8%.
`analysis
`and Wilcoxon
`using
`the
`SAS
`NC).
`Values
`expressed
`
`of
`
`as
`
`(cid:1)(±)-N-[1-oxo-2-(mercaptomethyl)-3-
`thiorphan
`synthesized
`by
`previously
`was
`ANP(99-126),
`Roques
`et at.,
`1985).
`were
`antirabbit
`IgG serum
`goat
`(Torrance,
`CA).
`r(cid:1)[1nI]ANP
`Heights,
`IL).
`(Arlington
`
`pro-
`published
`anti-
`rabbit
`from
`obtained
`was
`purchased
`
`Results
`
`CM hamster
`The
`atrial
`enlarged
`mass.
`normalized
`weight,
`in the CM hamsters
`was
`no
`difference
`CMs
`were
`visibly
`scribed
`previously
`1969;
`Franch
`et at.,
`edema
`and
`other
`surgery.
`in
`displayed
`Also
`.05)
`in the
`CM animals.
`
`remarkably
`a
`having
`as
`characterized
`is
`the
`atrial
`table
`1, where
`in
`This
`is
`shown
`per
`greater
`weight,
`was
`3 times
`100
`g ofbody
`there
`compared
`to NM hamsters.
`Although
`from the
`in the mass
`of
`the
`ventricles,
`those
`been
`de-
`dilated.
`These
`characteristics
`have
`in animals
`with
`severe
`heart
`(Basjusz
`failure
`marked
`Several
`hamsters
`with
`et at.,
`signs
`of
`disease
`did
`survive
`the
`
`1988).
`advanced
`
`not
`
`table
`
`1, MAP
`MAP
`did
`
`was
`not
`
`significantly
`change
`during
`
`lower
`the
`
`(P <
`course
`
`NOVARTIS EXHIBIT 2003
`Biocon v. Novartis, IPR2020-01263
`Page 2 of 4
`
`

`

`178
`
`Smltsetal.
`
`Vol. 254
`
`CM hamsters
`treated
`similarly
`plasma
`ANP
`not
`alter
`the
`
`the
`in
`than
`saline
`with
`infused
`administration
`2). Thiorphan
`(fig.
`concentrations
`130% in the
`CM animals
`levels
`in the NM animals.
`
`NM
`
`hamsters
`increased
`but
`
`did
`
`Discussion
`
`in
`
`Downloaded from
`
`jpet.aspetjournals.org
`
` at ASPET Journals on April 10, 2019
`
`of
`
`for
`have
`at.,
`et
`it has
`
`to
`the
`
`to
`
`of
`
`secondary
`kidney
`concentration
`of ANP
`to
`the
`natriuresis
`of
`endopeptidase
`24.11
`Endopeptidase
`the metab-
`with
`There
`are
`activity.
`in
`addition
`24.11
`1986;
`Ura
`at.,
`A recent
`report
`24.11
`accounted
`kinins.
`That
`inhibitor
`of endo-
`sodium
`and
`renal
`response
`exhibited
`to
`the
`actions
`and
`intrarenal
`
`to
`at
`
`distal
`ob-
`
`et
`
`of
`
`et
`1986).
`
`(Shima
`tubule
`proximal
`early
`would
`the
`dase
`at.,
`et
`1988)
`by
`excretion
`sodium
`increase
`allowing
`ANP
`intact
`more
`sites
`reach
`in
`of
`portions
`distal
`proximal
`and
`both
`the
`increased
`with
`coupled
`local
`effects,
`Therefore,
`these
`nephron.
`delivery
`in plasma
`levels
`elevation
`of ANP
`the
`kidney
`an
`to
`via
`peptide,
`could
`of
`the
`the
`concentration
`of ANP
`greatly
`increase
`at natriuretic
`sites
`Also,
`because
`arterial
`along
`the
`nephron.
`pressure
`influence
`reduced
`did
`the
`antinatriuretic
`not
`fall,
`a factor.
`be
`renal
`perfusion
`would
`not
`pressure
`compared
`in CM as
`The
`increase
`ANP
`levels
`in
`plasma
`NM hamsters
`aspect
`was
`an interesting
`thiorphan
`treated
`with
`difference
`of
`this
`study.
`The
`most
`probable
`explanation
`for
`this
`is
`of
`the
`existence
`of multiple
`mechanisms
`the
`clearance
`than
`found
`more
`circulating
`ANP.
`Several
`investigators
`one
`binding
`site
`for ANP
`(Leitman
`1986;
`Olins
`et
`at.,
`that
`Takayanagi
`and
`been
`proposed
`et
`at.,
`1988;
`1987)
`of
`one
`these
`sites
`serves
`a storage/clearance
`function
`to regulate
`plasma
`levels
`of
`the
`hormone
`(Maack
`1987).
`Because
`at.,
`et
`de-
`number
`binding
`sites
`of ANP
`there
`evidence
`that
`the
`creases
`under
`conditions
`ANP
`chronically
`plasma
`of
`elevated
`levels
`(Morton
`Schiffrin
`is
`reasonable
`it
`1986),
`et at.,
`et at.,
`1987;
`have
`to
`suggest
`that
`CM hamsters
`may
`number
`a reduced
`capacity
`binding
`sites
`and,
`thus,
`a reduced
`circulating
`to clear
`ele-
`ANP
`receptor-mediated
`mechanisms.
`the
`addition,
`In
`via
`vated ANP
`the
`levels
`may
`saturate
`clearance
`nearly
`remaining
`sites.
`As
`a result,
`the
`of plasma
`in CHF
`regulation
`ANP
`levels
`may
`be more
`dependent
`on degradation
`24.11.
`by endopeptidase
`Under
`such
`conditions,
`endopeptidase
`result
`inhibition
`should
`in increased
`circulating
`levels
`but
`ofANP
`in CHF
`not
`in normal
`animals
`in which
`ANP
`clearance
`mechanisms
`are
`fully
`operative.
`the
`to
`of ANP
`increased
`While
`in ANP
`increases
`plasma
`elevated
`critical
`delivery
`enhanced
`sites
`renal
`explain
`may
`fully
`nephron
`of
`the
`regions
`effects
`CM hamsters,
`other
`in
`the
`served
`cannot
`inhibition
`time.
`be
`excluded
`this
`interfering
`elicit
`natriuresis
`by
`may
`inhibition
`renal
`peptides
`which
`have
`other
`olism
`substrates
`for
`endopeptidase
`known
`several
`to ANP,
`bradykinin
`(Shimamori
`including
`aL,
`and
`substance
`P
`1987)
`(Nau
`et
`at.,
`1987)
`indicated
`endopeptidase
`(Ura
`that
`et aL,
`50 to
`degradation
`for
`75% of
`the
`of
`intrarenal
`showed
`that
`an
`also
`phosphoramidon,
`study
`peptidase
`24.11,
`caused
`in urinary
`an
`increase
`kinin
`excretion.
`Therefore,
`the
`renal
`of
`some
`may
`the
`CM hamsters
`attributable
`be
`by
`bradykinin
`or an interaction
`between
`bradykinin
`ANP.
`inhibition
`summary,
`In
`increase
`an
`produced
`ing
`levels
`of ANP
`previous
`studies
`renal
`exhibit
`a depressed
`present
`the
`The
`results
`of
`inhibitors
`endopeptidase
`peutic
`for
`the
`
`all
`
`delivery
`levels,
`and
`
`at
`
`is
`
`of
`
`of
`approach
`
`regulate
`which
`systems
`humoral
`of many
`is one
`ANP
`and
`we
`determined
`study,
`present
`In
`the
`balance.
`electrolyte
`of
`activity
`be
`ANP
`could
`endogenous
`natriuretic
`whether
`the
`an
`enzyme
`24.11,
`endopeptidase
`by
`inhibiting
`potentiated
`the
`circulation.
`Our
`clearance
`from
`participate
`in its
`may
`which
`endopeptidase
`of
`an
`inhibitor
`indicate
`that
`thiorphan,
`results
`by
`310% in hamsters
`sodium
`excretion
`increased
`24.11,
`levels
`of ANP.
`while
`doubling
`their
`plasma
`CHF
`in
`patients
`When
`ANP
`was
`infused
`i.v.
`natriuretic
`comparison
`response
`was
`blunted
`individuals
`(Cody
`one
`In
`1986).
`1986;
`Crozier
`et at.,
`et
`at.,
`response
`natriuretic
`(Crozier
`the
`attenuated
`at.,
`1986),
`in arterial
`associated
`a significant
`fall
`pressure.
`Our
`with
`profile.
`Treatment
`with
`show
`a
`substantially
`different
`endopeptidase
`inhibitor
`produced
`no
`effect
`on MAP
`and
`with
`with
`enhanced
`natriuresis.
`CHF
`responded
`hamsters
`These
`suggest
`data
`may
`of
`endopeptidase
`24.11
`inhibition
`that
`sodium
`increase
`by mechanisms
`other
`elevating
`excretion
`ANP
`possibly
`by protecting
`ANP
`from
`concentration,
`plasma
`degradation
`within
`By
`inhibiting
`endopeptidase
`at
`kidney.
`the
`the
`glomerulus
`(Shima
`1988),
`thiorphan
`may
`in-
`et
`aL,
`creased
`the
`of ANP
`at
`critical
`renal
`concentration
`local
`thereby
`natriuresis.
`Furthermore,
`unmasking
`enhanced
`site,
`a natriuretic
`action
`ANP
`exerts
`of
`from
`the
`tubular
`side
`nephron,
`is possible
`blocking
`brush-border
`endopepti-
`
`than
`
`have
`
`this
`
`if
`
`the
`
`it
`
`that
`
`Thiorphan
`
`i::i
`
`Saline
`
`1100
`
`1000
`
`900
`
`800
`
`700
`
`600
`
`500
`
`400
`
`300
`
`200
`
`100
`
`E C
`
`)
`
`0.
`
`C.z
`
`0
`
`cardeomyopathic
`Normal
`in CM
`(ANPir)
`concentrations
`of ANP-immunoreactivity
`Fig. 2. Plasma
`treated
`iv. bolus
`and NM hamsters
`with
`saline
`or
`thiorphan
`(1 0 mg/kg
`for CM-Saline
`and 10 mg/kg/hr
`iv.
`infusion).
`Values
`are mean
`± S.E.M.
`8), CM-Thiorphan
`(n =
`(n =
`8), NM-Saline
`(n =
`9) and NM-Thiorphan
`(n
`.05 vs. NM-Saline;
`tP <
`.05 vs. CM-Saline.
`8). P <
`
`=
`
`with
`
`in
`
`et
`
`fluid
`
`with
`
`the
`
`CHF,
`to
`healthy
`study
`was
`data
`the
`the
`
`endopeptidase
`of
`in urinary
`sodium
`in CM hamsters.
`which
`demonstrated
`responsiveness
`study
`24.11
`treatment
`
`suggest
`may
`provide
`of CHF.
`
`24.11
`excretion
`These
`results
`that
`humans
`to
`exogenous
`that
`utilization
`a useful
`
`thiorphan
`by
`circulat-
`and
`differ
`from
`with
`CHF
`ANP.
`
`of
`
`thera-
`
`Acknowledgments
`The
`authors
`this
`study
`
`of
`
`to thank
`wish
`and
`review
`of
`
`Dr. Gillian
`this manuscript.
`
`M. Olins
`We
`
`for
`also
`
`her
`extend
`
`evaluation
`thoughtful
`our
`appreciation
`
`to
`
`NOVARTIS EXHIBIT 2003
`Biocon v. Novartis, IPR2020-01263
`Page 3 of 4
`
`

`

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`
`jpet.aspetjournals.org
`
` at ASPET Journals on April 10, 2019
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`24.11
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`
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`800 N. Lindbergh
`
`H. Blame,
`Boulevard,
`
`Ph.D.,
`R & D, Monsanto
`Searle
`St. Louis, Mo 63167
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`NIXON,
`degeneration
`N.Y. Acad.
`Enzyme
`
`C. W.
`HOMBURGER,
`AND
`congestive
`heart
`and
`156:
`105-129,
`1969.
`Inhibiting
`Cmpounds,United
`
`Sd.
`
`F.:
`failure
`
`NOVARTIS EXHIBIT 2003
`Biocon v. Novartis, IPR2020-01263
`Page 4 of 4
`
`