Downloaded from
`
`jpet.aspetjournals.org
`
` by guest on July 2, 2014
`
`(Xt22.3.’(cid:1)65/8I/2I82.05(4$O2.00/t)
`(802-3565/81/2182-0504$02.0O/0
`THE JOURNAL OP PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS
`THE
`JOURNAl.
`OF
`PHARMACOLOGY
`AND
`EXPERIMENTAL
`THERAPEUTICS
`Copyright (0 1981 by The American Society for Pharmacology and Experimental Therapeutics
`Copyright
`© 1981
`by The American
`Society
`and Experimental
`TherapeutiCS
`Pharmacology
`for
`
`Vol.
`218, No.2
`Vol. 218, No. 2
`Printed in USA.
`Printed
`in U.S.A.
`
`Antagonism of the Cardiovascular and Respiratory Depressant
`Antagonism
`of
`the Cardiovascular
`and Respiratory
`Depressant
`Effects
`of Morphine
`in the Conscious
`Rabbit
`by Physostigmine1
`Effects of Morphine in the Conscious Rabbit by Physostigminel
`
`MARTA WEINSTOCK, ELI EREZ and DORON ROLL
`MARTA
`WEINSTOCK,
`ELI
`EREZ
`and
`DORON
`ROLL
`
`(M. W. , DR.)
`Department of Pharmacology and Experimental Therapeutics, Hebrew University, Hadassah School of Medicine, Jerusalem, Israel (M. W., D.R.)
`Department
`of Pharmacology
`and
`Experimental
`Therapeutics,
`Hebrew
`University,
`Hadassah
`School
`of Medicine,
`Jerusalem,
`Israel
`and Department of Anesthesia. Hadassah Hospital, Hadassah School of Medicine, Jerusalem, Israel (E.E.)
`and
`Department
`of Anesthesia.
`Hadassah
`Hospital.
`Hadassah
`School
`of Medicine,
`Jerusalem,
`Israel
`(E.E.)
`
`Accepted for publication May 8, 1981
`Accepted
`May
`8,
`1981
`publication
`for
`
`ABSTRACT
`ABSTRACT
`Weinstock, Marta, Eli Erez and Doron Roll: Antagonism of the
`Marta,
`Eli Erez
`and Doron
`Roll: Antagonism
`of
`the
`Weinstock,
`cardiovascular and respiratory depressant effects of morphine
`cardiovascular
`and
`respiratory
`depressant
`effects
`of morphine
`in the conscious rabbit by physostigmine. J. Pharmacol. Exp.
`in the
`conscious
`rabbit
`by
`physostigmine.
`J. Pharmacol.
`Exp.
`Ther. 218: 504-508, 1981.
`218:
`Ther.
`504-508,
`1981.
`
`The influence of physostigmine was studied on the effect of
`The
`influence
`of
`physostigmine
`was
`studied
`on
`the
`effect
`of
`morphine on the cardiovascular and respiratory systems in
`morphine
`on
`the
`cardiovascular
`and
`respiratory
`systems
`in
`conscious rabbits. Morphine (4 mg/kg i.v.) caused analgesia,
`conscious
`rabbits.
`Morphine
`(4 mg/kg
`iv.)
`caused
`analgesia,
`bradycardia, hypotension and respiratory depression, as indi-
`bradycardia,
`hypotension
`and
`respiratory
`depression,
`as mdi-
`cated by a fall in respiratory rate of 50%, a rise in blood Paco,
`cated
`by a fall
`in respiratory
`rate
`of 50%,
`a rise
`in blood
`Pac0,
`from 25.1 to 37.2 mm Hg and a fall in pH from 7.40 to 7.24.
`from
`25.1
`and
`a fall
`in pH from
`7.40
`to 7.24.
`to
`37.2
`mm Hg
`These effects lasted 2 to 3 hr and were completely antagonized
`These
`effects
`lasted
`2 to 3 hr and were
`completely
`antagonized
`by naloxone. Physostigmine (2.5 or 5 µg/kg/min) given by
`by
`naloxone.
`Physostigmine
`(2.5
`or
`5 (cid:1)g/kg/min)
`given
`by
`constant i.v. infusion did not significantly alter blood pressure
`constant
`i.v.
`infusion
`did
`not
`significantly
`alter
`blood
`pressure
`
`or heart rate, but decreased blood Paco, from 25.1 to 19 mm
`or
`heart
`rate,
`but
`decreased
`blood
`Paco2
`from
`25.1
`to
`1 9 mm
`Hg and increased pH from 7.40 to 7.46. Pretreatment of rabbits
`of
`rabbits
`Hg
`7.40
`to 7.46.
`Pretreatment
`and
`increased
`pH from
`with physostigmine (5 jug/kg/min) completely prevented both
`with
`physostigmine
`(5 (cid:1)tg/kg/min)
`completely
`prevented
`both
`the fall in blood pressure and blood pH and the rise in Paco,
`the
`fall
`in blood
`pressure
`and
`blood
`pH and
`the
`rise
`in Paco2
`induced by morphine (4 mg/kg) and also significantly reduced
`induced
`by morphine
`(4 mg/kg)
`and
`also
`significantly
`reduced
`both the intensity and duration of bradycardia. Analgesic activ-
`both
`the
`intensity
`and
`duration
`of bradycardia.
`Analgesic
`activ-
`ity of morphine remained unimpaired by physostigmine. Neo-
`of morphine
`remained
`unimpaired
`by
`physostigmine.
`Neo-
`ity
`stigmine (2.5 µg/kg/min) potentiated the bradycardia induced
`stigmine
`(2.5
`(cid:1)ig/kg/min)
`potentiated
`the
`bradycardia
`induced
`by morphine and did not antagonize its hypotensive and res-
`by morphine
`and
`did
`not
`antagonize
`its
`hypotensive
`and
`res-
`piratory depressant effects. The results support the hypothesis
`depressant
`effects.
`The
`results
`support
`the
`hypothesis
`piratory
`that the respiratory and cardiovascular depressant effects of
`that
`the
`respiratory
`and
`cardiovascular
`depressant
`effects
`of
`morphine, but not the analgesia, result from an inhibition of
`morphine,
`but
`not
`the
`analgesia,
`result
`from an
`inhibition
`of
`acetylcholine release from neurons in the central nervous sys-
`acetylcholine
`release
`from neurons
`in the
`central
`nervous
`sys-
`tem.
`tem.
`
`Injection of acetylcholine and other muscarinic agonists into
`Injection
`of
`acetylcholine
`and
`other
`muscarinic
`agonists
`into
`the lateral ventricle or cisterna magna of dogs and rats results
`lateral
`ventricle
`or
`cisterna
`magna
`of dogs
`and
`rats
`results
`the
`in a rise in blood pressure and tachycardia (Lang and Rush,
`in
`a
`rise
`in
`blood
`pressure
`and
`tachycardia
`(Lang
`and
`Rush,
`1973; Sinha et al., 1967; Krstic and Djurkovic, 1978). Hyperten-
`Sinha
`Krstic
`and
`Djurkovic,
`1978).
`Hyperten-
`et
`at.,
`1973;
`1967;
`sion also results from direct electrical stimulation of the vaso-
`also
`results
`from
`direct
`electrical
`stimulation
`of
`the
`vaso-
`sion
`motor areas in the medulla, and this can be prevented by
`motor
`areas
`in
`the
`medulla,
`and
`this
`can
`be
`prevented
`by
`hemicholinium (Sinha et al., 1967). Application of acetylcholine
`hemicholinium
`(Sinha
`Application
`of acetylcholine
`et at.,
`1967).
`to the floor of the 4th ventricle markedly increased respiration
`to
`the
`floor
`of
`the
`4th
`ventricle
`markedly
`increased
`respiration
`rate in decerebrate cats, pretreated with physostigmine (Miller,
`rate
`in decerebrate
`cats,
`pretreated
`with
`physostigmine
`(Miller,
`1949). These data suggest the presence of cholinergic stimula-
`1949).
`These
`data
`suggest
`the
`presence
`of
`cholinergic
`stimula-
`tory pathways to the vasomotor and respiratory centers.
`pathways
`to
`the
`vasomotor
`and
`respiratory
`centers.
`tory
`Morphine and related opiates can increase acetylcholine
`Morphine
`and
`related
`opiates
`can
`increase
`acetylcholine
`levels in the brain. This does not result from stimulation of
`levels
`in
`the
`brain.
`This
`does
`not
`result
`from
`stimulation
`of
`acetylcholine synthesis or from prevention of acetylcholine
`acetylcholine
`synthesis
`or
`from
`prevention
`of
`acetylcholine
`hydrolysis, but is most likely due to an inhibition of acetylcho-
`hydrolysis,
`but
`is most
`likely
`due
`to
`an
`inhibition
`of
`acetylcho-
`line release (Weinstock, 1971). Such an action has been dem-
`line
`release
`(Weinstock,
`1971).
`Such
`an
`action
`has
`been
`dem-
`onstrated for several opiate drugs in different areas of the
`onstrated
`for
`several
`opiate
`drugs
`in
`different
`areas
`of
`the
`central nervous system (Beleslin and Polak, 1965; Jhamandas
`central
`nervous
`system
`(Beleslin
`and
`Polak,
`1965;
`Jhamandas
`et a/., 1971; Domino and Wilson, 1973; Zsilla et al., 1976).
`Domino
`and
`Wilson,
`1973;
`Zsila
`et
`at.,
`1971;
`1976).
`et
`at.,
`Furthermore, morphine is known to produce effects such as
`Furthermore,
`morphine
`is
`known
`to
`produce
`effects
`such
`as
`respiratory depression, hypotension and bradycardia, which are
`depression,
`hypotension
`and
`bradycardia,
`which
`are
`respiratory
`the opposite to those caused by central cholinergic receptor
`the
`opposite
`to
`those
`caused
`by
`central
`cholinergic
`receptor
`
`Received
`for publication
`November
`Received for publication November 5, 1980.
`5,
`198().
`' This work was supported by U.S. Public Health Service Grant ROl DA
`I Thls
`work
`was
`supported
`b(cid:1)
`U.S.
`Public
`Health
`Service
`Grant
`ROl
`DA
`02150-01 from the National Institute of Drug Abuse.
`02150-01
`from
`the
`National
`Institute
`of Drug
`Abuse.
`
`stimulation. It therefore seemed reasonable to suggest that
`It
`therefore
`seemed
`reasonable
`to
`suggest
`that
`stimulation.
`these effects of morphine result from impairment of acetylcho-
`these
`effects
`of morphine
`result
`from
`impairment
`of
`acetylcho-
`line release.
`line
`release.
`Previous studies which have attempted to show a correlation
`Previous
`studies
`which
`have
`attempted
`to show
`a correlation
`between the cardiovascular or respiratory depressant effects of
`between
`the
`cardiovascular
`or
`respiratory
`depressant
`effects
`of
`morphine and acetylcholine release, have been carried out in
`and
`acetylcholine
`release,
`have
`been
`carried
`out
`in
`morphine
`anesthetized animals (Schaumann, 1958; Laubie et al., 1974).
`anesthetized
`animals
`(Schaumann,
`1958;
`Laubie
`1974).
`et
`al.,
`Anesthetic agents, particularly barbiturates, markedly interfere
`Anesthetic
`agents,
`particularly
`barbiturates,
`markedly
`interfere
`with central cholinergic activity as well as with respiratory
`with
`central
`cholinergic
`activity
`as well
`as with
`respiratory
`control (Bradley and Dray, 1973; Borison, 1971; Weinstock et
`control
`(Bradley
`and
`Dray,
`1973;
`Borison,
`1971; Weinstock
`et
`al., 1979).
`1979).
`at.,
`The present study was therefore carried out in conscious
`The
`present
`study
`was
`therefore
`carried
`out
`in
`conscious
`animals. These were pretreated with physostigmine, which
`animals.
`These
`were
`pretreated
`with
`physostigmine,
`which
`increases the amount of acetylcholine available for interaction
`increases
`the
`amount
`of
`acetylcholine
`available
`for
`interaction
`with its receptors by blocking acetylcholinesterase. If such
`with
`its
`receptors
`by
`blocking
`acetylcholinesteras#{232}.
`If
`such
`pretreatment were found to antagonize the cardiovascular and
`pretreatment
`were
`found
`to
`antagonize
`the
`cardiovascular
`and
`respiratory depressant actions of morphine, it would lend sup-
`respiratory
`depressant
`actions
`of morphine,
`it would
`lend
`sup-
`port to the suggestion that interference with cholinergic trans-
`port
`to
`the
`suggestion
`that
`interference
`with
`cholinergic
`trans-
`mission was involved in these effects of morphine.
`mission
`was
`involved
`in these
`effects
`of morphine.
`
`Methods
`Methods
`
`Measurement of cardiovascular and respiratory parameters.
`Measurement
`of
`cardiovascular
`and
`respiratory
`parameters.
`Male rabbits (mixed strain) weighing 2.5 to 3.5 kg were trained to sit
`Male
`rabbits
`(mixed
`strain)
`weighing
`2.5
`to 3.5
`kg were
`trained
`to
`sit
`quietly in a restraining box. Both ear arteries were cannulated with
`quietly
`in
`a
`restraining
`box.
`Both
`ear
`arteries
`were
`cannulated
`with
`transcutaneous catheters (Quick Cath. No. 20, Travenol Laboratories
`transcutaneous
`catheters
`(Quick
`Cath.
`No.
`20, Travenol
`Laboratories
`Ltd., Castlebar, Ireland) which were filled with sterile saline containing
`Ltd.,
`Castlebar,
`Ireland)
`which
`were
`filled with
`sterile
`saline
`containing
`
`ABBREVIATION:
`ABBREVIATION: ATMN, atropine methyl nitrate.
`ATMN,
`atropine
`methyl
`nitrate.
`
`504
`
`NOVARTIS EXHIBIT 2046
`Noven v. Novartis and LTS Lohmann
`IPR2014-00550
`Page 1 of 5
`
`(cid:9)
`(cid:9)
`

`

`Physostigmine
`Antagonism
`of Morphine
`Physostigmine Antagonism of Morphine (cid:9)
`
`505
`505
`
`injection of 4 and 10 mg/kg of morphine respectively. The peak
`injection
`of 4 and
`10 mg/kg
`of morphine
`respectively.
`The
`peak
`hypotensive response (9.8 ± 2.2 and 12.4 ± 1.8 mm Hg) occurred
`hypotensive
`response
`(9.8 ± 2.2
`and
`12.4 ± 1.8 mm Hg)
`occurred
`20 to 30 min after injection of 2 and 4 mg/kg, respectively. The
`20
`to
`30 mm
`after
`injection
`of 2 and
`4 mg/kg,
`respectively.
`The
`response to 10 mg/kg of morphine was inconsistent, with some
`response’to
`10 mg/kg
`of morphine
`was
`inconsistent,
`with
`some
`rabbits displaying a rise of 5 to 10 mm Hg during the first 10
`rabbits
`displaying
`a
`rise
`of
`5 to
`10 mm
`Hg
`during
`the
`first
`10
`min and others, a small nonsignificant fall. Both the bradycardia
`mm and
`others,
`a small
`nonsignificant
`fall.
`Both
`the
`bradycardia
`and hypotensive response to 4 mg/kg of morphine lasted 2.5 to
`and
`hypotensive
`response
`to
`4 mg/kg
`of morphine
`lasted
`2.5
`to
`3 hr.
`3 hr.
`A dose of 4 mg/kg of morphine was therefore chosen for all
`A dose
`of
`4 mg/kg
`of morphine
`was
`therefore
`chosen
`for
`all
`subsequent experiments since it produced the most extensive
`subsequent
`experiments
`since
`it
`produced
`the
`most
`extensive
`and consistent vasodepression and bradycardia.
`and
`consistent
`vasodepression
`and
`bradycardia.
`Pretreatment with ATMN (0.5 mg/kg) completely prevented
`Pretreatment
`with
`ATMN
`(0.5 mg/kg)
`completely
`prevented
`the bradycardia and reduced the fall in blood pressure.
`bradycardia
`and
`reduced
`the
`in
`blood
`pressure.
`the
`fall
`Morphine (4 mg/kg) caused more than a 50% reduction in
`Morphine
`(4 mg/kg)
`caused
`more
`than
`a
`50% reduction
`in
`respiratory rate, which was associated with a 48% increase in
`respiratory
`rate,
`which
`was
`associated
`with
`a 48% increase
`in
`arterial Paco,. Blood pH was reduced from 7.40 to 7.24 (see fig.
`arterial
`Paco(cid:1).
`Blood
`pH was
`reduced
`from
`7.40
`to
`7.24
`(see
`fig.
`1). Maximum respiratory depression occurred between 30 and
`1). Maximum
`respiratory
`depression
`occurred
`between
`30
`and
`60 min after morphine administration and lasted 3 hr.
`60 mm
`after
`morphine
`administration
`and
`lasted
`3 hr.
`A considerable degree of analgesic activity was also seen at
`A considerable
`degree
`of
`analgesic
`activity
`was
`also
`seen
`at
`this dose level, 30 min after injection of morphine, with most of
`dose
`level,
`30 mm after
`injection
`of morphine,
`with
`most
`of
`this
`the rabbits failing to respond to the highest degree of pressure
`the
`rabbits
`failing
`to
`respond
`to
`the
`highest
`degree
`of pressure
`(table 1).
`(table
`1).
`Naloxone, given by continuous i.v. infusion at a dose of 0.1
`Naloxone,
`given
`by
`continuous
`i.v.
`infusion
`at
`a dose
`of
`0.1
`mg/min completely prevented all the above effects of morphine
`mg/mm
`completely
`prevented
`all
`the
`above
`effects
`of morphine
`(4 mg/kg) in four rabbits.
`(4 mg/kg)
`in
`four
`rabbits.
`Influence of anticholinesterase agents on actions of
`Influence
`of
`anticholinesterase
`agents
`on
`actions
`of
`
`MORPHINE (LmgIk(cid:1))
`0. MORPHINE (cid:9)
`o---..o
`/141
`•
`(2 S.s(cid:1)Ik(cid:1)Imui)
`•
`11111111111( It mg /110 • POYSOSTIS14111( I 2 SA/ kg/ ma I
`MOIPNII(
`(&wqIk(cid:1))
`A-..-.(cid:1).
`#{149}PWYSOS1I(cid:1)MII(
`I 50 jig/k,!
`•—• 141111111111( (tom / kg) • PNYSIST1111111E I SA tog/kg/ mut I
`I kg) #{149}PNYSISTISMIN(
`NORPNIU(
`ltmq
`#{163}-6
`mu)
`ep<O.05
`dep<OO1
`‘“pc000l
`.P4,antSf.
`• p<0.05 (cid:9)
`••• p <0 01 (cid:9)
`"' p<0 001 ; 14eAntS
`
`(cid:1)‘\
`
`120
`120
`110
`110
`100
`100
`90
`90
`▪ 110
`00
`g io
`70
`F• 60
`60
`so
`:(cid:1)
`50
`4,
`40
`40
`.Juo
`30
`40
`40
`
`705
`
`-(cid:1)35
`
`ES
`
`E 30
`
`30
`
`(cid:1)(cid:1)25(cid:1)’
`
`6 25
`
`1981
`1981
`
`25 Wm' of heparin. Blood pressure and heart rate were recorded on a
`25 (cid:1)t/ml
`of heparmn.
`Blood
`pressure
`and
`heart
`rate
`were
`recorded
`on a
`Brush Gould recorder by means of a transducer attached to one arterial
`Brush
`Gould
`recorder
`by means
`of a transducer
`attached
`to one
`arterial
`cannula. Drugs were administered through a butterfly needle (no. 23)
`cannula.
`Drugs
`were
`administered
`through
`a
`butterfly
`needle
`(no.
`23)
`placed in a marginal ear vein. Physostigmine or neostigmine was infused
`placed
`in a marginal
`ear vein.
`Physostigmine
`or neostigmine
`was
`infused
`i.v. in a volume of 0.09 ml/min by means of a Harvard constant infusion
`i,v.
`in a volume
`of 0.09 mI/mis
`by means
`of a Harvard
`constant
`infusion
`pump.
`pump.
`Rectal temperature was monitored on a telethermometer (Yellow
`Rectal
`temperature
`(Yellow
`was
`monitored
`on
`a
`telethermometer
`Springs Instrument Company, Yellow Springs, OH) with the aid of a
`Yellow
`Springs,
`OH) with
`the
`aid
`of
`a
`Springs
`Instrument
`Company,
`thermistor probe inserted into the rectum. Respiration rate was counted
`probe
`inserted
`into
`the
`rectum.
`Respiration
`rate was
`counted
`thermistor
`visually and blood gases and pH were measured on Corning automatic
`visually
`blood
`gases
`pH were measured
`on Corning
`automatic
`and
`and
`blood gas analyzer after adjustment to the appropriate body tempera-
`analyzer
`after
`adjustment
`to the
`appropriate
`body
`tempera-
`gas
`blood
`ture.
`ture.
`Analgesia was assessed from the reaction (squeal or attempt at
`Analgesia
`was
`assessed
`from
`the
`reaction
`(squeal
`or
`attempt
`at
`withdrawal) in response to one of four grades of pressure applied to the
`withdrawal)
`in response
`to one
`of
`four
`grades
`of pressure
`applied
`to the
`tip of the tail with a sponge holder clamp. A score of 1 indicated a
`tip
`of
`the
`tail with
`a
`sponge
`holder
`clamp.
`A score
`of
`1 indicated
`a
`positive reaction to the lowest degree of pressure, whereas 5 denoted
`pressure,
`whereas
`5 denoted
`positive
`reaction
`to
`the
`lowest
`degree
`of
`that no reaction to the highest degree of pressure occurred.
`that
`no reaction
`to the
`highest
`degree
`of pressure
`occurred.
`Rabbits were allowed to rest for at least 1 hr under quiet conditions
`Rabbits
`were
`allowed
`to rest
`for
`at
`least
`1 hr
`under
`quiet
`conditions
`after cannulation, before control readings were taken. Drugs were not
`taken.
`Drugs
`were
`not
`after
`cannulation,
`before
`control
`readings
`were
`administered until two consistent values for blood gases were obtained.
`administered
`until
`two
`consistent
`values
`for blood
`gases
`were
`obtained.
`Mean arterial blood pressure, heart and respiration rates were deter-
`Mean
`arterial
`blood
`pressure,
`heart
`respiration
`rates
`were
`deter-
`and
`mined at 10-min intervals for 1 hr and then at 30-min intervals for 2 hr
`mined
`at
`10-mm
`intervals
`for
`for 2 hr
`1 hr
`and
`then
`at
`30-mm
`intervals
`after injection of morphine. Six rabbits were pretreated with ATMN
`after
`injection
`of morphine.
`rabbits
`were
`pretreated
`with
`ATMN
`Six
`(0.5 mg/kg i.v.) and then were given morphine (4 mg/kg) as above. In
`(0.5 mg/kg
`i.v.)
`then
`were
`given morphine
`(4 mg/kg)
`as
`above.
`In
`and
`other rabbits, physostigmine was infused at a concentration of either
`other
`rabbits,
`physostigmine
`was
`infused
`at
`a concentration
`of
`either
`2.5 (seven animals) or 5µg/kg/min (eight animals). Thirty minutes
`(seven
`animals)
`or
`5 (cid:1)zg/kg/min
`(eight
`animals).
`Thirty
`minutes
`2.5
`after commencement of the infusion, morphine was injected slowly
`after
`commencement
`of
`the
`infusion,
`morphine
`was
`injected
`slowly
`over a period of 2 to 3 min and the infusion of physostigmine continued
`over
`a period
`of 2 to 3 mm and
`the
`infusion
`of physostigmine
`continued
`(0.6 ml)
`for an additional 2 hrs. Blood samples (0.6 ml) for blood gas analysis
`for
`an
`additional
`2 hrs.
`Blood
`samples
`for
`blood
`gas
`analysis
`were taken at least twice before drug administration, 30 min after
`were
`taken
`at
`least
`twice
`before
`administration,
`30 mm after
`drug
`infusion of physostigmine, and at 30, 60 and 90 min after injection of
`infusion
`of physostigmine,
`and
`90 mm after
`injection
`of
`and
`at
`30,
`60
`morphine. The volume of blood taken was replaced each time with an
`morphine.
`The
`volume
`of blood
`taken
`was
`replaced
`each
`time with
`an
`equal volume of sterile saline. In five rabbits, neostigmine (2.5 µg/kg/
`equal
`volume
`of
`sterile
`saline.
`In five
`rabbits,
`neostigmine
`(2.5 (cid:1)sg/kg/
`min) was infused for 30 min and then continued after injection of
`mm)
`was
`infused
`for
`30
`and
`then
`continued
`after
`injection
`of
`mm
`morphine (4 mg/kg). Blood pressure, heart and respiration rates and
`morphine
`(4 mg/kg).
`Blood
`pressure,
`heart
`and
`respiration
`rates
`and
`blood gases were measured as above. In six other rabbits, ATMN (0.5
`blood
`gases
`were measured
`as
`above.
`In six
`other
`rabbits,
`ATMN
`(0.5
`mg/kg), or in four animals, hyoscine (10 mg/kg), was given, 15 min
`mg/kg),
`or
`in
`four
`animals,
`hyoscine
`(10 mg/kg),
`was
`given,
`15 mm
`before the infusion of physostigmine, 5 µg/kg/min.
`before
`the
`infusion
`of physostigmine,
`5 (cid:1)sg/kg/min.
`In four rabbits, naloxone was infused i.v. at a concentration of 0.1
`In
`four
`rabbits,
`naloxone
`infused
`iv.
`at
`a concentration
`was
`of
`0.1
`mg/kg/min for 15 min before and for 90 min after injection of 4 mg/kg
`for
`90 mm after
`injection
`of 4 mg/kg
`15 mm
`before
`and
`mg/kg/mm
`for
`of morphine. Blood pressure, heart and respiration rates were recorded
`pressure,
`heart
`and
`respiration
`rates
`were
`recorded
`of morphine.
`Blood
`as described above.
`as
`described
`above.
`Estimation of plasma cholinesterase. Blood (0.3-0.5 ml) was
`Estimation
`of
`plasma
`cholinesterase.
`Blood
`ml)
`was
`(0.3-0.5
`withdrawn into a heparinized syringe during the predrug control period
`a heparirnzed
`syringe
`during
`the
`predrug
`control
`period
`withdrawn
`into
`and at 30 and 60 min after commencement of physostigmine infusion
`and
`at
`30 and
`60 mm
`after
`commencement
`of physostigmine
`infusion
`(i.e., 30 min after morphine injection). The blood was centrifuged
`30 mm
`after
`morphine
`injection).
`The
`blood
`was
`centrifuged
`(i.e.,
`immediately for 10 min at 1000 x g and cholinesterase activity of the
`immediately
`for
`10 mm at
`and
`cholinesterase
`activity
`the
`x
`g
`of
`1000
`plasma was measured within 10 min by the method of Ellman et al.
`plasma
`was measured
`10 mm by
`the method
`of Eliman
`et at.
`within
`(1961). Drugs used were: ATMN, hyoscine hydrobromide, neostigmine
`(1961).
`Drugs
`used were:
`ATMN,
`hyoscine
`hydrobromide,
`neostigmine
`hydrobromide and physostigmine salicylate (Sigma Chemical Com-
`hydrobromide
`physostigmine
`salicylate
`(Sigma
`Chemical
`Corn-
`and
`pany, St. Louis, MO); morphine hydrochloride (U.S. Vitamins Labo-
`pany,
`St.
`Louis,
`MO);
`morphine
`hydrochloride
`(U.S.
`Vitamins
`Labo-
`ratories Division, Tuckahoe, NY); and naloxone hydrochloride (Endo
`ratories
`Division,
`Tuckahoe,
`NY);
`and
`naloxone
`hydrochloride
`(Endo
`Laboratories, Inc., Garden City, NY). Morphine and physostigmine
`Laboratories,
`Inc.,
`Garden
`City,
`NY).
`Morphine
`physostigmine
`and
`were made up freshly for each experiment in sterile saline which
`were
`made
`up
`freshly
`for
`each
`experiment
`in
`sterile
`saline
`which
`included an equal weight of ascorbic acid to prevent oxidation. All
`included
`an
`equal
`weight
`of
`ascorbic
`acid
`to
`prevent
`oxidation.
`All
`doses are expressed in milligrams per kilogram of body weight of the
`doses
`are
`expressed
`in milligrams
`per
`body
`weight
`of
`the
`kilogram
`of
`appropriate salt.
`appropriate
`salt.
`Statistical analysis. Tests of significance for the difference between
`Statistical
`analysis.
`Tests
`ofsignificance
`for
`the
`difference
`between
`means were performed by a two-tailed Student's t test for paired or
`a two-tailed
`Student’s
`test
`for
`paired
`or
`means
`were
`performed
`by
`t
`unpaired data as indicated in "Results".
`unpaired
`data
`as
`indicated
`in “Results”.
`
`\
`
`‘.\
`
`ii
`
`‘ii
`
`-‘
`
`I’
`
`7.5
`
`74
`
`!
`
`73
`
`J.
`
`30
`
`60
`
`9(cid:1)O
`
`7.2
`0
`
`30 (cid:9)
`30
`
`60
`60
`
`90
`90
`
`‘------
`
`-
`
`------
`
`110
`:(cid:1)u11o(cid:1)___
`
`100
`6 100
`
`.(cid:1)
`
`•
`(cid:1).90
`
`00
`
`‘-(cid:1)(cid:1)-
`
`Of •
`
`I
`
`•
`
`I
`
`60
`60
`
`90
`90
`
`20
`▪ 20
`O
`ic(cid:1)’
`a 15
`
`0 (cid:9)
`0
`
`I
`30 (cid:9)
`30
`
`.(cid:1)
`
`▪ AO
`00
`0
`2
`,(cid:1)
`I
`70 0
`90 (cid:9)
`60 (cid:9)
`30
`90
`60
`0
`30
`Time after Injection of Morphine
`Injection
`Time
`of Morphine
`alter
`Imini
`(mini
`
`‘(cid:1)
`
`1 . The
`effect
`of physostigmine
`Fig. 1. The effect of physostigmine pretreatment on the respiratory
`pretreatment
`Fig.
`the
`respiratory
`on
`depression induced by morphine.
`depression
`induced
`by morphine.
`
`TABLE
`TABLE 1
`1
`The
`effect
`of physostigmine
`on analgesic
`ac tivity
`of morphine
`The effect of physostigmine on analgesic activity of morphine
`
`Analgesic
`Analgesic
`Score ±
`Score
`± S E M
`1.38 ± 0.13
`1 .38
`±
`0.13
`4.55 ± 0.24
`4.55
`±
`0.24
`1.71 ± 0.28
`1.71
`±
`0.28
`
`4.91 ± 0.08
`4.91
`±
`0.08
`
`N
`N
`
`21
`21
`9
`7
`
`9 7
`
`12
`12
`
`Results
`Results
`
`Effect of morphine on blood pressure, heart rate, res-
`Effect
`of morphine
`on
`blood
`pressure,
`heart
`rate,
`res-
`piration and pain threshold. Intravenous injection of mor-
`piration
`and
`pain
`threshold.
`Intravenous
`injection
`of mor-
`phine (2 mg/kg) caused significant bradycardia (reduction of
`phine
`(2 mg/kg)
`caused
`significant
`bradycardia
`(reduction
`of
`72 ± 10 beats/min) within 5 min, whereas 1 mg/kg only reduced
`10 beats/mm)
`within
`5 mm,
`whereas
`1 mg/kg
`only
`reduced
`72
`±
`heart rate by 30 ± 9 beats at 60 min. Reductions in heart rate
`heart
`rate
`by
`30 ± 9 beats
`at
`60 mm.
`Reductions
`in
`heart
`rate
`of 108 ± 12 and 102 ± 10 beats/min occurred 5 to 30 min after
`of
`108 ± 12 and
`102 ± 10 beats/mm
`occurred
`5 to
`30 mm
`after
`
`Treatment
`Treatment
`
`Control
`Control
`Morphine (4 mg/kg)
`Morphine
`(4 mg/kg)
`Physostigmine (5 µg/kg/
`Physostigmine(5(cid:1)tg/kg/
`min, 30 min)
`mm,
`30 mm)
`Physostigmine(5(cid:1)g/kg/
`Physostigmine (5 µg/kg/
`min, 30 min) + morphine
`mm,
`30 mm)
`+ morphine
`(4 mg/kg)
`(4 mg/kg)
`
`NOVARTIS EXHIBIT 2046
`Noven v. Novartis and LTS Lohmann
`IPR2014-00550
`Page 2 of 5
`
`(cid:9)
`(cid:1)
`(cid:1)
`(cid:1)
`(cid:1)
`(cid:1)
`(cid:1)
`(cid:1)
`(cid:1)
`(cid:1)
`

`

`Vol. 218
`Vol. 218
`
`depression by physostigmine (5 pg/kg/min) (see table 3). It
`depression
`by
`physostigmine
`(5
`(cid:1)tg/kg/imn)
`(see
`table
`3).
`It
`only antagonized the salivation, miosis and defecation induced
`only
`antagonized
`the
`salivation,
`miosis
`and
`defecation
`induced
`by physostigmine. On the other hand, hyoscine, 10 mg/kg, both
`by
`physostigmine.
`On
`the
`other
`hand,
`hyoscine,
`10 mg/kg,
`both
`increased the respiratory depressant effect of morphine alone
`increased
`the
`respiratory
`depressant
`effect
`of morphine
`alone
`(Pco, at 30 min after morphine, 40 ± 1.2) and completely
`(Pco.
`at
`30 min
`after
`morphine,
`40
`±
`1.2)
`and
`completely
`antagonized the prevention by physostigmine of the respiratory
`antagonized
`the
`prevention
`by
`physostigmine
`ofthe
`respiratory
`depressant effect of morphine (see table 3).
`table
`3).
`depressant
`effect
`of morphine
`(see
`Neostigmine (2.5 µg/kg/min) inhibited plasma cholinesterase
`Neostigmine
`(2.5
`jig/kg/mm)
`inhibited
`plasma
`cholinesterase
`by 80%, but it did not diminish the respiratory depressant effect
`80(cid:1),
`but
`it did
`not
`diminish
`the
`respiratory
`depressant
`effect
`by
`of morphine, nor did it alter significantly the hypotensive
`of morphine,
`nor
`did
`it
`alter
`significantly
`the
`hypotensive
`response. However, it markedly potentiated the cardiac slowing
`However,
`it markedly
`potentiated
`the
`cardiac
`slowing
`response.
`induced by morphine, reducing heart rate to 90 ± 8.0 beats/
`induced
`by morphine,
`reducing
`heart
`rate
`to
`90
`±
`8.0
`beats/
`min, 5 to 10 min after morphine (4 mg/kg). Neostigmine (5 Itg/
`mm,
`5 to
`10 mm
`after
`morphine
`(4 mg/kg).
`Neostigmine
`(5 (cid:1).tg/
`kg/min) caused marked fasciculations and cardiac arrest in
`kg/mm)
`caused
`marked
`fasciculations
`and
`cardiac
`arrest
`in
`three animals when morphine was injected.
`three
`animals
`when
`morphine
`was
`injected.
`
`Discussion
`Discussion
`
`Intravenous injection of morphine in cats, dogs and man
`Intravenous
`injection
`of morphine
`in
`cats,
`dogs
`and
`man
`produces an initial hypotensive response which is mainly due
`produces
`an
`initial
`hypotensive
`response
`which
`is mainly
`due
`to histamine release (Feldberg and Paton, 1951; Evans et al.,
`to
`histamine
`release
`(Feldberg
`and
`Paton,
`1951;
`Evans
`et
`at.,
`
`a(.ne
`Morphine Ili mg / kg) atone
`Morphine
`(4mqIk(cid:1))
`o----o
`(2.5sqlkglmm)
`+ Morphine
`Pliysostigmlne
`.-• (cid:9)
`Physostigmlne (2.5,11k61min) + Morphine
`A-.-A
`(5.0 sc Ik(cid:1)lw#{225}n)+ Morphine
`Physostigmine
`a--a Physostigmim (5.0 m Iklimin) + Morphine
`#{163}-&
`*ft (cid:9)
`**
`*5
`*0
`
`+0
`
`_4:74*II
`
`-----
`
`•,6
`
`T
`
`sst4 ----
`
`*
`
`oi
`0l
`
`E
`E
`
`-12
`
`0
`
`- 20
`
`-40
`
`- 60
`
`Oi
`
`- 80
`
`-100
`
`CHANGE IN MABP
`
`z’-
`
`U L
`
`u
`
`0(cid:1)
`
`4
`
`z.(cid:1)
`-l(cid:1)
`
`z U
`
`-120
`120
`0 5 10 20 310
`60
`05102030
`60
`TIME
`AFTER
`INJECTION
`OF MORPHINE
`TIME AFTER INJECTION OF MORPHINE
`(m in )
`(mm)
`The
`effect
`of physostigmine
`pretreatment
`on hypotension
`and
`Fig. 2. The effect of physostigmine pretreatment on hypotension and
`mean
`arterial
`blood
`pres-
`bradycardia induced by morphine. MABP, mean arterial blood pres-
`bradycardia
`induced
`by morphine.
`MABP,
`sure.
`sure.
`
`Fig.
`
`2.
`
`506 (cid:9)
`506
`
`Weinstock
`et al.
`Weinstock et al.
`
`30 mmby
`
`morphine. Intravenous injection of physostigmine in doses of
`morphine.
`Intravenous
`injection
`of
`physostigmine
`in
`doses
`of
`0.05 to 0.2 mg/kg resulted in biphasic effects on blood pressure
`0.05
`to
`0.2 mg/kg
`resulted
`in biphasic
`effects
`on
`blood
`pressure
`and heart rate, which lasted 20 to 30 min, depending on the
`and
`heart
`rate,
`which
`lasted
`20
`to
`30 mm,
`depending
`on
`the
`dose. An initial phase of bradycardia and hypotension was
`dose.
`An
`initial
`phase
`of
`bradycardia
`and
`hypotension
`was
`followed by a longer phase of hypertension and return of heart
`followed
`by
`a longer
`phase
`of hypertension
`and
`return
`of heart
`rate to normal values. Since one could not study the cardiovas-
`rate
`to
`normal
`values.
`Since
`one
`could
`not
`study
`the
`cardiovas-
`cular effects of morphine in the presence of continuously chang-
`cular
`effects
`ofmorphine
`in the
`presence
`ofcontinuously
`chang-
`ing and short-lived effects of physostigmine, it was decided to
`and
`short-lived
`effects
`of
`physostigmine,
`it was
`decided
`to
`ing
`give physostigmine as a continuous i.v. infusion. When admin-
`give
`physostigmine
`as
`a
`continuous
`iv.
`infusion.
`When
`admin-
`istered in this way at a concentration of 2.5 or 5 µg/kg/min,
`istered
`in
`this
`way
`at
`a
`concentration
`of
`2.5
`or
`5 (cid:1)tg/kg/min,
`physostigmine caused no significant change in either heart rate
`physostigmine
`caused
`no
`significant
`change
`in either
`heart
`rate
`or blood pressure over a period of 2 hr (table 2). Both doses of
`or
`blood
`pressure
`over
`a period
`of 2 hr
`(table
`2). Both
`doses
`of
`physostigmine caused miosis and some salivation and defeca-
`and
`some
`salivation
`and
`defeca-
`physostigmine
`caused
`miosis
`tion 20 to 40 min after commencement of the infusion. The
`tion
`20
`to
`40 mm
`after
`commencement
`of
`the
`infusion.
`The
`effects occurred earlier and were more pronounced with the
`effects
`occurred
`earlier
`and
`were
`more
`pronounced
`with
`the
`higher dose.
`higher
`dose.
`Both concentrations markedly stimulated respiration, result-
`Both
`concentrations
`markedly
`stimulated
`respiration,
`result-
`ing in an increase in blood pH and a 20 to 25% decrease in
`in
`an
`increase
`in
`blood
`pH
`and
`a
`20
`to
`25% decrease
`in
`ing
`Paco, within 30 min of commencement of the infusion (table 2;
`Paco,
`within
`30 mm
`of
`commencement
`of
`the
`infusion
`(table
`2;
`fig. 1). Plasma cholinesterase was inhibited by 48.4 and 55.3%
`fig.
`1).
`Plasma
`cholinesterase
`was
`inhibited
`by
`48.4
`and
`55.3%
`at 30 min by physostigmine, 2.5 and 5 µg/kg/min, respectively.
`at
`physostigmine,
`2.5
`and
`5 jig/kg/mm,
`respectively.
`There was no significant additional inhibition of plasma cholin-
`There
`was
`no
`significant
`additional
`inhibition
`of plasma
`cholin-
`esterase at either dose of physostigmine at 60 min. Slight muscle
`esterase
`at
`either
`dose
`of physostigmine
`at
`60 mm.
`Slight
`muscle
`fasciculations were only evident 2 hr after continuous infusion
`fasciculations
`were
`only
`evident
`2 hr
`after
`continuous
`infusion
`of the larger dose. In four other rabbits given physostigmine
`of
`the
`larger
`dose.
`In
`four
`other
`rabbits
`given
`physostigmine
`(10 µg/kg/min), considerable salivation and defecation occurred
`(10 (cid:1)ig/kg/min),
`considerable
`salivation
`and
`defecation
`occurred
`within 30 min and muscle fasciculations appeared 40 to 60 min
`within
`30 min
`and
`muscle
`fasciculations
`appeared
`40
`to
`60 mm
`after commencement of the infusion. This dose was therefore
`after
`commencement
`of
`the
`infusion.
`This
`dose
`was
`therefore
`not given together with morphine.
`not
`given
`together
`with
`morphine.
`When physostigmine infusion (2.5 or 5 p.g/kg/min) was given
`When
`physostigmine
`infusion
`(2.5
`or
`5 jig/kg/mm)
`given
`was
`for 30 min and then continued after the injection of morphine
`30 mm
`and
`then
`continued
`after
`the
`injection
`of morphine
`for
`(4 mg/kg), it completely prevented the fall in blood pressure.
`(4 mg/kg),
`it
`completely
`prevented
`the
`fall
`in
`blood
`pressure.
`The bradycardia was not significantly altered by the lower dose
`The
`bradycardia
`was
`not
`significantly
`altered
`by
`the
`lower
`dose
`of physostigmine, but it was reduced both in intensity and
`of
`physostigmine,
`but
`it was
`reduced
`both
`in
`intensity
`and
`duration after administration of 5 µg/kg/min of the anticholin-
`duration
`after
`administration
`of 5 (cid:1)tg/kg/min
`of
`the
`anticholin-
`esterase (see fig. 2).
`esterase
`(see
`fig.
`2).
`Pretreatment with physostigmine (2.5 µg/kg/min) markedly
`Pretreatment
`with
`physostigmine
`(2.5
`(cid:1)tg/kg/min)
`markedly
`diminished the elevation in Paco, which occurred after admin-
`diminished
`the
`elevation
`in Paco,
`which
`occurred
`after
`admin-
`istration of morphine (4 mg/kg). It also reduced significantly
`(4 mg/kg).
`It
`also
`reduced
`significantly
`istration
`of morphine
`the fall in blood pH, respiration rate and Pao, 60 min after
`the
`in
`blood
`pH,
`respiration
`rate
`and
`Pao2
`60 mm
`after
`fall
`morphine (see fig. 1). After pretreatment with physostigmine
`morphine
`(see
`fig.
`1). After
`pretreatment
`with
`physostigmine
`(5 µg/kg/min), morphine no longer caused any significant
`(5
`(cid:1)tg/kg/min),
`morphine
`no
`longer
`caused
`any
`significant
`change in blood Paco, pH or Pao, and the fall in respiratory
`change
`in
`blood
`Pa(-o,,
`pH
`or
`Pao.
`and
`the
`fall
`in
`respiratory
`rate was greatly diminished (see table 3).
`rate
`was
`greatly
`diminished
`(see
`table
`3).
`Pretreatment of rabbits with ATMN (0.5 mg/kg) did not
`Pretreatment
`of
`rabbits
`with
`ATMN
`(0.5
`mg/kg)
`did
`not
`significant