The material on this page was copied from the collection of the National Library of Medicine by a third party and mClY be protected by U S Copyright lal,'I
`
`NOVARTIS EXHIBIT 2036
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`

`\
`
`.1
`
`i
`
`I I f(
`
`ALZHEIMER'S AND
`PARKINSON'S
`DISEASES
`
`Strategies for Research and Development
`
`-0
`C m
`
`Edited by
`Abraham Fisher
`
`Israel Institute for Biological Research
`Ness-Ziona, Israel
`
`Israel Hanin
`
`Loyola University Stritch School of Medicine
`Chicago, illinois
`and-,
`Chaim Lachman
`
`Israel Institute for Biological Research
`Ness-Ziona, Israel
`
`PLENUM PRESS • NEW YORK AND LONDON
`
`NOVARTIS EXHIBIT 2036
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`

`[
`
`Library of Congress Cataloging in Publication Data
`
`OHOLO Conference on Basic and Therapeutic Strategies in Alzheimer's and Other
`Related Neuropsychiatric Disorders (1985: Eilat, Israel)
`Alzheimer's and Parkinson's diseases.
`(Advances in behavioral biology; v. 29)
`
`OHO
`
`"Proceedings of the thirtieth OHOLO Conference on Basic and Therapeutic
`Strategies in Alzheimer's and Other Related Neuropsychiatric Disorders, held in March
`24-27, 1985, in Eilat, Israel"~ T.p. verso.
`Includes bibliographies and index.
`1. Parkinsonism-Congresses. 2. Alzheimer's disease-Congresses; I. Fisher,
`Abraham. II. Hanin, Israel. III. Lachman, Chaim. IV. Title. V. Series. [DNLM: 1.
`Alzheimer's Disease-congresses. 2. Parkinson Disease-congresses. WM 220 038
`1985a)
`RC382.047 1985
`ISBN 0-306-42232-8
`
`85-32056
`
`616.8'3
`
`\'\1 3
`AD~/j
`:J q
`1/.
`} C; t; (0
`
`/Co J
`
`Proceedings of the thirtieth OHOLO COnferElnCe on Basic and
`Therapeutic Strategies in Alzheimer's and Other Related
`Neuropsychiatric Disorders, held March 24-27, 1985, in
`Eilat, Israel
`
`. © 1986 Plenum Press, New York
`A Division of Plenum Publishing Corporation
`233 Spring Street, New York, N.Y. 10013
`
`All rights reserved
`
`No part of this book may be reproduced, stored in a retrieval system, or transmitted
`in any form or by any means, electronic, mechanical, photocopying, microfilming,
`recording, or otherwise, without written permission from the Publisher
`
`Printed in the United States of America
`
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`NOVARTIS EXHIBIT 2036
`Noven v. Novartis and LTS Lohmann
`IPR2014-00550
`Page 3 of 14
`
`

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`
`PHARMACOLOGICAL ACTIVITY OF NOVEL ANTICHOLINESTERASE AGENTS OF POTENTIAL
`
`USE IN THE TREATMENT OF ALZHEIMER'S DISEASE
`
`~1arta Weinstock 1 , Michal Razin 1 , Michael Chorev2 & Zeev
`Tashma2
`Departments of Pharmacology1 and Medicinal Chcmistry2
`School of Pharmacy, Hebrew University, Ein Kerem, Jerusalem
`Israel
`
`INTRODUCTION
`
`In dementia of the Alzheimer type there is a selective loss in the
`cerebral cortex of choline acetyl transferase
`(CAT),
`the enzyme
`that
`synthesizes acetylcholine
`(ACh) 1,2. The degree of dementia and memory
`impairment that occurs in this condition is well correlated with the
`decrement in cortical cholinergic transmission3 • Horeover, scopolamine,
`a cholinergic antagonist, can cause memory impairment in normal individ(cid:173)
`uals similar to that in aging4• These findings suggest that impaired
`cortical cholinergic transmission may be at least in part responsible
`for
`the
`symptomatology of Alzheimer disease.
`In support of
`this
`suggestion it was
`found
`that physostigmine, which prevents
`the
`destruction of ACh,can cause memory improvement in Alzheimer patients5 •
`The extent of improvement of the symptomatology was closely related to
`the degree of inhibition of acetylcholinesterase (AChE)
`in the spinal
`fluid, and thgs to the amount of physostigmine reaching the central
`nervous system •
`
`As potential therapy for dementia, physostigmine has a number of
`disadvantages, the most serious of which is its low therapeutic ratio.
`In most studies in which any improvement in s'ymptomatology was report.ed~
`the dose range in which this occurred was very narrow (1-2. 5mg orally
`or 0.25-0 •. 5mg, i. v. 7), with higher doses causing a decrement in perform(cid:173)
`ance or distressing side effects due to peripheral cholinergic over(cid:173)
`activity. Another disadvantage is its low chemical stability8 and short
`duration of action, which necessitate frequent dosing. Its oral bio(cid:173)
`availability is also unpredictable, and it only appears to produce
`improvement in Alzheimer symptomatology by this route if it is given
`with lecithin9.
`
`The purpose of the present study was to synthesize anticholin(cid:173)
`esterase agents which readily reach the CNS after parenteral and oral
`administration; which have a higher therapeutic ratio than that of phy(cid:173)
`sostigmine, greater chemical stability, and a longer duration of action.
`These advantages should make them more sui table than physostigmine for
`the
`long term treatment of conditions associated with a deficit in
`cholinergic transmission in the central nervous system.
`
`Apart from physostigmine, all of the carbamate anticholinesterases
`which are used medicinally, have a quaternary N-function and thus do not
`
`539
`
`I 1
`
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`

`penetrate the eNS to any significant extent10 . Almost all the synthetic
`carbamates with a tertiary N were designed as insecticides, and have a
`monomethyl substituent on
`the N of
`the carbamate. They are
`thus
`relatively unstable at physiological pH and of short duration 10. One
`such carbamate, miotine, has only been used clinically as a miotic 11
`The dimethyl analogue, has only been used as an insecticide 12
`The
`effect of other mono or dialkyl substitution on the N of the carbamate
`of this structure on AChE activity in vitro or in vivo does not appear
`to have been studied. Accordingly we prepared and tested a series of
`mono and alkyl derivatives of miotine, the activities of some of which
`are described.
`(A patent has been applied for the novel structures).
`Particular emphasis is placed on their abilities to inhibit brain AChE
`and on their relative toxicities.
`
`METHODS
`
`•
`Preparation of mono- and di-substituted phenyl carbamates
`
`The N-monoalkyl and N,N-dialkyl substituted phenyl carbamate s were
`synthesized from d. -m-hydroxyphenylethyl-dimethylamine (I), which was
`itself ~repared according to the procedure described by Stedman and
`Stedman 1 with minor modifications, as shown in the scheme be low:
`" ....
`
`o
`II
`
`..... R 2
`
`&-~ '::R,NCOCI
`~CH-NMe2
`
`I
`Dialkyl Me
`derivatives
`
`Method B
`
`OH & R,NCO
`
`~cH-NMe2
`Me
`I
`
`Method A
`
`o
`1\
`O-C-NH-R 2
`
`·~H-NMe,
`
`Jt'~oalkYl
`derivatives
`
`For the synthesis of the monoalkylphenyl carbamates, a 2-3 fold
`molar excess of the alkyl isocyanate was reacted with phenol I
`in dry
`benzene at room temperature overnight (see Scheme 1 method A). For the
`synthesis of the N ,N-dialkyl-substituted phenyl carbamates, 1.5-2 fold
`molar excess of the corresponding carbamoyl chloride was allowed to
`I
`react with phenol
`in dry acetonitrile in the presence of a similar
`excess of sodium hydride (see Scheme 1 method B). The weak a.cidi ty of
`phenol I
`required the use of a strong ba.se such as sodium hydride to
`produce the phenolate which acts as the nucleophile.
`
`All carbamates were obtained as hydrochloride salts by saturating
`their etheral solutions with HCl(g). These salts were purified by re(cid:173)
`crystallization from ethanol-ether. Purity was assessed by t.l.c. on
`precoated silica ~el plates, reversed-phase HPLC, elemental microchem(cid:173)
`ical analysis and H-n.m.r.
`
`Heasurement of antiAChE activity in vitro
`
`(Sabra strain) weighing 30-40g were sacrificed by
`Hale mice
`cervical dislocation and
`the whole brain minus cerebellum rapidly
`removed and weighed. The brains from 10 mice were homogenized in 1ml/
`100g wet weight phosphate buffer 0.1M pH 8.0, centrifuged at 12,000 rpm
`and the supernatant, discarded. The pellet was mixed with a similar
`volume as above of buffer 0.1H pH 8.0 containing 1% Triton using a
`Vortex Genie at maximum speed for 1 min. The mixture was centrifuged and
`the supernatant which contained most of the solubilized AChE~ was used
`for subsequent determinations of anticholinesterase activity.
`
`540
`
`inh
`ace
`inc
`bef
`mea
`dat
`of
`was
`
`Hea
`
`(s.
`ran
`abs
`(tr
`ing
`giv
`as
`fro
`tha
`
`Ass
`
`dru
`The
`det
`tha
`
`Ta
`
`Dr,
`
`RA
`RA<
`RA
`RA
`RA
`RA
`RA
`RA'
`RA
`
`*
`
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`

`

`The effect of at least three different concentrations of each
`inhibitor was measured on the rate of hydrolysis of 20)11 of 0.075M
`acetyl thiocholine iodide by 25 pI of solubilized AChE. The enzyme was
`incubated with the inhibitor for periods ranging from 2-180 mins at 37 0 C
`before
`the addition of
`the substrate. The rate of hydrolysis was
`measured by the spectrophotometric method of Ellman et 81.13. From these
`data the molar concentration of each agent that inhibited the activity
`of the enzyme by 50% (IC50) at the Hme of peak acti vi ty (:'50-120 min)
`was calculated.
`
`Measurement of antiAChE activity in vivo
`
`At least three doses of each drug were administered subcutaneously
`(s.c.) or orally to mice. Animals were sacrificed at different times
`ranging from 0.25 to 7 hours after drug administration. The presence or
`absence of side effects
`reminiscent of cholinergic hyperacti vi ty
`(tremors, salivation, defecation, fasciculations, difficulty in breath(cid:173)
`ing) were noted for each drug. The brain was rapidly removed at the
`given times stated above and the enzyme AChE extracted and solubilized
`as described in the previous section. The activity of the enzyme removed
`from drug treated mice was measured as described above and compared with
`that of mice given saline (control).
`
`Assessment of acute toxicity
`
`Male mice were given one of at least three different doses of each
`drug orally or s.c., a minimum of 10 mice being alotted to each dose.
`The number of animals that died
`in each group wi thin 3 hours was
`determined, and from these data the LD50 (dose in ~moles/kg which was le(cid:173)
`thal to 50% of the mice) was computed.
`
`Table
`
`Drug
`
`structure,
`relative
`chemical
`between
`1. Relationship
`hydrophobicity and molar refractivity of phenyl carbamates
`
`R1
`
`R2
`
`Capacity factor
`(k' )*
`
`Mol. Refractivity
`
`5.65
`Me
`RA2 (miotine)
`0.5
`H
`10.30
`0.83
`Et
`RA6
`H
`14.96
`n-Pr
`1.48
`RA15
`H
`14.96
`1.37
`i-Pr
`RA13
`H
`Allyl
`14.49
`1.33
`RA14
`H
`26.69
`6.17
`c-Hexyl
`RA12
`H
`11.30
`Me
`RA10
`Me
`1.33
`15.95
`Et
`RA7
`Me
`2.33
`20.60
`Et
`RA8
`Et
`4.33
`* Capacity factor defined as ratio of difference between retention time
`of the compound and that of the unretained solute to that of the
`unretained solute on a reversed phase (C18) HPLC column (SOlvent; 70%
`of 0.1% aqueous TFA soln. + 30% methanol). This factor is a measure
`of the relative hydrophobicity of the compound.
`
`541
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`NOVARTIS EXHIBIT 2036
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`

`

`This experiment was repeated in animals which had been pretreated
`15 mins previous ly with either a tropine methy)ni tra te
`(ATMN 5mg/kg)
`which block only peripheral muscarinic receptors14 or atropine sulphate,
`(5mg/kg) which blocks both central and peripheral muscarinic receptors,
`and the anticholinesterase agents were injected s.c.
`
`Measurement of antiAChE activity in different areas of rat brain
`
`Male and female Sabra r a ts weighing 150-350g were injected s.c.
`with either saline, physostigmine 0.15mg/kg, RA6 1.0mg/kg. RA7 O.5mg/kg
`or RA15 0.5mg/kg (six animals were used for each treatment group). The
`cerebral cortex, hippocampus, corpus striatum and medulla oblongata vTere
`rapidly dissected on ice, weighed individually, homogenized in phosphate
`buffer and extracted and solubilized as described above for mouse brain.
`The activity of the enzyme
`from treated and control rats was also
`measured as described above.
`
`The percent inhibition of AChE by each drug was computed for the
`different brain areas by comparison with the pooled mean of the control
`values (n=12) for each area.
`
`Statistical analyses. Data from the. experiment on the effects of
`drugs on AChE in different areas of rat' brain were analysed by 2-way
`annlysis of variance, followed by Neuman Keul's post hoc comparisons.
`
`RESULTS
`
`The relationship between the N alkyl substituents, relative hydro(cid:173)
`phobicity and molar refractivity is shown in Table 1. In general both
`the latter parameters increased as the size of the mono or disubstit.ut.ed
`alkyl groups became larger.
`
`Table 2. The effect of the novel compounds on AChE acti vi ty in mouse
`brain in vitro and in vivo
`
`Drug
`
`I
`I
`I
`I
`I
`I
`Physostigmine: 0.011
`I 0.013
`RA2
`I
`I 0·40
`RA6
`I
`RA15
`: 0.11
`:12.10
`RAn
`I 0·43
`RA14
`I
`I 0.093
`RA12
`I
`I 0.027
`RA10
`I
`I 3.00
`RA7
`I
`RAS
`:35.0
`
`100
`85
`3
`10
`0.1
`3
`12
`41
`0.4
`0.03
`
`ED50
`IC50 Relative Potency:
`JlM
`to Physostigmine: )lMoles/kg
`I
`I
`I
`I
`I
`I
`I
`I
`I
`I
`I
`I
`I
`I
`I
`I
`I
`I
`I
`I
`I
`I
`
`Relative Potency
`to Physostigmine
`
`100
`100
`11
`3:'5
`2
`15
`13
`81
`22
`2
`
`0.92
`0.92
`8.47
`2.80
`40.0
`6.01
`7.24
`1.14
`4.20
`56.0
`
`542
`
`Ant:
`
`on ;
`sum!
`(mi(
`in
`rel1
`stil
`(RAE
`but
`c-h<
`but
`reSI
`nov<
`2-2(
`iso
`
`cor:
`man,
`e.g
`<n-:
`i-p:
`
`tha)
`ana.
`pot<
`
`caw
`grol
`of
`tha)
`into
`red',
`
`Dru,
`
`Phy
`RA2
`RA6
`RA1 .
`RA1
`RA1
`RA1
`RA7
`RA8
`
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`

`

`AntiAChE activity in mouse brain
`
`The inhibitory activities of the novel carbamates and physostigmine
`on a solubilized preparation of ACQB of mouse whole brain in vitro are
`summarized
`in Table 2. The monomethyl substituted derivative, RA2,
`(miotine), was found to be the most potent inhibitor of brain AChE, both
`in vitro and in vivo. It has a rapid onset of action which is of a
`relatively short duration (90-120 min in vivo)
`like that of physo(cid:173)
`stigmine (Table 3). Increase in the size of the alkyl radical to ethyl
`(RA6) , resulted in a large reduction (>30 fold) in in vitro a c tivity,
`but only a 6-fold decrease, in vivo. Larger substituents, n-propyl, and
`c-hexyl proved to be more potent inhibitors than N-ethyl, or N-allyl,
`but less so,
`than N-methyl, while
`introduction of an i-propyl group
`resulted in a 1000-fold decrease in AChE activity. In general, all the
`novel monosubstituted carbamates were more active in vivo by f ac tors of
`2-20 times,
`than one would have expected from the activities on
`the
`isolated enzyme when compared to physostigmine or miotine. [Table 21.
`
`Comparison of the data in Tables 1 and 2, reveals that there is no
`correlation between in vitro anticholinesterase activity (IC50) of the
`monosubstituted carbamates and any of the physical parameters examined,
`e.g. chain length in extended conformation, methyl (RA2), <ethyl (RA6),
`<n-propyl
`(RA15); molar refractivity, cf. c-hexyl
`(RA12), ethyl and
`i-propyl (RA13); hydrophobicity, cf n-propyl and i-propyl.
`
`The disubsti tuted carbamates were generally less active in vitro
`the corresponding monosubstituted derivatives. Among
`the
`three
`than
`analogues there appeared to be a negative correlation between inhibitory
`potency, and both hydrophobicity and molar refractivity volume .
`
`Introduction of a second methyl group on the N of the carbamate
`caused only a small reduction in inhibitory activity. However, vrhen one
`group was substituted by ethyl, (RA7) in vitro activity fell by 2 orders
`of magnitude. Surprisingly, this compound was considerably more potent
`than one would have expected from the in vitro data when it was injected
`into the whole animal
`Under these conditions its acti vi ty was only
`reduced to 1/3rd of that of the dimethyl derivative.
`
`Table 3. Duration of action of carbamates on brain AChE in mice
`
`Drug
`
`Physostigmine
`RA2
`RA6
`RA15
`RA14
`RA12
`RA10
`RA7
`RA8
`
`Time of peak
`inhibition
`(min. )
`
`% inhibition ~ s.e.
`by ED50 at 3 hrs
`
`ED50 oral
`
`ED50 s.c.
`
`15
`15
`30-120
`15-30
`30
`30-60
`15
`60-120
`30-120
`
`0
`0
`47+1
`26+5
`41+3
`36~3
`0
`33+3
`31~6
`
`4.3
`1.3
`2.6
`4.0
`3.8
`3.0
`3.4
`1.5
`1.4
`
`543
`
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`
`

`

`UJ
`.c.
`u
`
`-< -o
`
`c
`o
`:.0
`E c
`
`80
`
`60
`
`40
`
`20
`
`""1"",
`f" " '1'" -, "
`
`o
`
`.2
`
`A Physo O. 2Smg/kg
`2.5 mgl kg
`
`o RAS
`
`• RA7
`
`2.0mg/kg
`
`' "
`
`3
`
`7
`
`Time in hours after s .c. injection
`
`Fig. 1: Duration of inhibition of brain AChE Rfter s.c. injection of
`physostigmine, RA6 and RA7 in mice
`
`,s
`
`a weak
`to be
`proved
`The diethyl substituted compound, RA8,
`inhibitor, with an IC50 of only 35 pM. All the compounds having a sub(cid:173)
`stitutent larger than methyl, had a slower onset of action, both on the
`isolated solubilized enzyme and
`in
`the whole animal, and a
`longer
`duration of action in vivo, than methyl derivatives and physostigmine
`(Table 3). The latter drugs ceased to inhibit brain AChE 2-3 hours after
`injection, while all the novel compounds with alkyl substituents larger
`than methyl caused significant inhibition for 3-7 hours [Fig. 11.
`
`The maximum inhibition of the brain AChE after oral administration
`of any dose of physostigmine, did not exceed 50%. This was achieved at
`about a 4 times larger dose than the ED50 after s.c. injection [Table
`3]. Higher doses, caused marked respiratory distress, fasciculations and
`tremors. With
`the possible exception of RA10,
`a greater
`than 70%
`inhibition of brain AChE was obtained after oral administration of all
`the other compounds. The
`incidence of untoward
`symptoms due
`to
`cholinergic overactivity was also much lower with these compounds.
`
`Acute toxicity
`
`The acute toxicity of the anticholinesterase agents is shown in
`Table 4, when these were given alone or after pretreatment with ATMN or
`atropine. The therapeutic ratios, defined as the LD50/ED50, of all the
`compounds except RA2 were about 3
`times greater than that of physos(cid:173)
`tigmine, which was only 3.3. Blockade of peripheral muscarinic receptors
`by ATMN, caused a similar increase in LD50 (1.5-2.2 fold.) in all the
`compounds. When muscarinic receptors in the CNS were also blocked by
`
`544
`
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`

`

`atropine, the LD50 of physostigmine and the majority of the compounds
`rose by 2.2-3.5 fold. The disubstitued compounds, RA10 and RA7, however,
`showed a 6-11 fold increase in LD50.
`),
`AntiAChE activity in different areas of rat brain
`
`The AChE activity of different areas of rat brain is shown in Table
`5. While
`the
`cerebral
`cortex,
`and medulla
`showed
`hippocampus
`approximately similar amounts of enzyme activity, that in the striatum
`was about 10-fold higher.
`
`Fig. 2 shows the effect of physostigmine and three novel carbamates
`on AChE activity in 4 areas of rat brain. The doses of the 4 drugs were
`chosen which gave the same degree of inhibition of AChE in the cerebral
`cortex. At these doses, RA6, RA7 and RA15 caused significantly less
`inhibition in the medulla (P<O.05) and RA7 caused a lower effect in the
`striatum, than in the cortex. RA6 and RA7 also produced significantly
`less inhibition in the medulla than did physostigmine. The effect of
`RA15 in the hippocampus was significantly greater than that of all the
`other drugs when given at a dose that inhibited the enzyme in the cortex
`to a similar extent.
`
`DISCUSSION
`
`In the present series of carbamate derivatives in vitro inhibition
`(IC50) of brain AChE varied 3000-fold from the most to least potent
`drug.
`In
`the mono-alkylated derivatives, no correlation was
`found
`between
`the
`IC50 values and hydrophobicity, molar refractivity, or
`length of the most extended conformation of the carbamate moiety. Thus,
`the largest substituent, c-hexyl, showed a much smaller decrease in
`inhi bi tory potency
`compared
`to miotine,
`than did
`the monoethyl
`derivative. On the other hand, introduction of an i-propyl resulted in a
`1000-fold decrease in activity, while n-propyl, which has the same molar
`refractivity and hydrophobicity, was only 10 times less potent than
`miotine.
`
`Table 4. Acute toxicity of carbamates in mice
`
`LD50
`J.1IIloles/kg
`s.c,
`
`Therapeutic
`ratio
`(LD50/ED50)
`
`Degree of protection** afforded
`by pretreatment with
`ATMN*
`Atropine*
`
`3.0
`4.50
`95.7
`30.5
`64.S
`41.5
`12.4
`46.0
`>568
`
`3.3
`4.9
`11.3
`10.9
`10.S
`9.S
`10.9
`11 .0
`>10.0
`
`1.S
`1.8
`1 .5
`1.5
`1.S
`1.2
`1.6
`2.2
`
`3.0
`2.4
`2.7
`3.0
`2.2
`3.5
`5.S
`10.9
`
`Drug
`
`Physo.
`RA2
`RA6
`RA15
`RA14
`RA12
`RA10
`RA7
`RAS
`
`injected 15 min. after atropine methyl nitrate 5 mg/kg or
`* Drug
`atropine sulphate 5 mg/kg
`** LD50 after ATMN or atropine pretreatment
`LD50 of drug alone
`
`545
`
`>.
`..D
`Q)
`c:
`'0
`'6
`Q)
`::2!
`'0
`c
`~
`..0
`::.:::i
`cu
`c: o
`~
`Z
`Q)
`
`:5
`'+-o
`c:
`o
`13
`Q)
`o
`<.)
`Q)
`
`:5
`E
`o
`.!=
`"0
`Q)
`'0..
`o
`<.)
`
`CJ) cu
`3:
`Q)
`0 )
`cu
`0..
`CJ)
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`:5
`c: o
`cu
`.~
`co
`E
`Q)
`J::
`I-
`
`NOVARTIS EXHIBIT 2036
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`IPR2014-00550
`Page 10 of 14
`
`

`

`Table 5. AChE activity in different areas of rat brain
`
`Brain Area
`
`Cerebral cortex
`Hippocampus
`Medulla
`Corpus striatum
`
`(11 )
`( 12)
`( 12)
`(11 )
`
`pM of substrate hydrolysed
`per min per mg. tissue + s.e.
`
`2.73+0.09
`3. 43.:!:.0.09
`5.55+0.27
`27.10+1.10
`
`Furthermore, no clear correlation could be demonstrated between
`anti AChE activity of the carbamates on the isolated enzyme taken from
`mouse brain and that obtained et vivo after injection of the drug into
`mice. All the novel carbamates were relatively much more active in vivo
`in relation to physostigmine or miotine, than in vitro. This discrepancy
`was especially evident in the disubsti tuted analogues, RA 7 and RA8.
`These compounds were 50-60 times more effective in vivo than one would
`have predicted from the data on the isolated enzyme •
`. ,"
`The relatively greater activity of the larger monoalkyl and dialkyl
`substi tuted drugs in the whole animal may be due to a greater chemical
`stability. It has previously been shown that monomethyl carbamates are
`much less stable that dimethyl derivatives at physiolgical pH10 The
`relatively long duration of enzyme inhibition (>7 hours) of all the
`larger alkyl deri vati ves in vivo,
`(compared with about 2 hours for
`physostigmine) suggests that they are chemically more stable at body pH
`and are more slowly metabolized.
`
`Another reason for the greater in vivo activity of the RA compounds
`may be their higher lipid solubility, \'1hich should enable a greater
`proportion of
`the drug
`to
`reach
`the central nervous system. This
`property could also explain
`the more efficient absorption
`from
`the
`gastro-intestinal tract of several of these carbamates, particularly RA7
`and RA8.
`
`Comparison of the acute toxicity of the RA compounds with that of
`physostigmine in mice, showed the former to have considerably higher
`therapeutic ratios, 10-12, compared with 3.3 for physostigmine and 4.5
`for miotine. Furthermore, signs of cholinergic overactivity, fasciculat(cid:173)
`ions,
`tremors, salivation and defecation were seen at the ED50 dose
`(which caused 50% inhibition of the whole brain enzyme) of physostigmine
`but not of the other carbamates. The greater therapeutic ratios of the
`RA compounds appears at first sight.to be surprising since the mortality
`is a direct result of AChE inhibition, and is due to the J2resence of
`excess AChE in the medulla, which causes respiratory arrest1? This was
`demonstrated in
`the present study by pretreating
`the animals with
`atropine which prevents the centrally induced respiratory depression14 ,
`and which raises the LD50 of all the monosubstituted carbamates by a
`factor of about 3. In the presence of such muscarinic blockade, death
`from overdose then results from respiratory muscle paralysis due to
`excess ACh at the neuromuscular junction. At this stage, no antidotes
`are effective and only artificial ventilation can prevent loss of life.
`The fact that the LD50 of RA7 can be increased 11-fold by muscarinic
`receptor blockade, demonstrates a relative lack of effect of this drug
`on somatic muscle. This
`is a distinct advantage
`in
`terms of its
`therapeutic potential.
`
`546
`
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`
`

`

`CERE BRAL
`CORTEX
`
`HIPPOCAMPUS
`
`•
`
`CORPUS STRIA TUM
`
`MEDULLA
`OBLONGATA
`
`w
`..c:
`u
`c:J:
`'I-
`0
`
`t:
`......
`0
`~ ......
`..0
`......
`..c:
`t:
`......
`~
`
`40
`
`20
`
`0
`
`40
`
`20
`
`o
`
`o Physo 0.15mg/kg
`
`II RAG 1.0 mgl kg
`
`0 . 5 mg/kg
`
`• P<0.05 cf Physo.
`
`~ R A15 0 5 mg I kg
`+ P< 0.05 cf Cortex
`
`rAt
`in different areas of
`Inhibition of AChE
`Fig. 2.
`physostigmine and 3 novel carbamates
`* Significantly different from physostigmine in s ame
`P<0.05
`+ Significantly different from value
`P<0.05
`
`in cortex for
`
`brain by
`
`brain area
`
`same drug
`
`lower toxicity of the RA compounds an
`In order to explain the
`attempt was made to determine whether they have a selective effect in
`different brain areas. It was found that physostigmine inhibited AChE to
`the same extent in four areas in the rat brain in spite of the fact that
`these areas contain different amounts of enzyme. In contrast, RA6, RA7
`and RA15 given in doses which blocked AChE in the cerebral cortex by
`35-40%, caused significantly less inhi bi tion in the medulla. The most
`striking difference was seen with RA7 which only reduced AChE in the
`medulla by 10%. Since the ED50 was determined in whole brain, of which
`the cerebral cortex contributes a major portion compared to the medulla,
`this differential effect of the drugs serves to explain their higher
`therapeutic ratio.
`
`547
`
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`
`

`

`the
`in
`a difference
`from
`result
`selective effect may
`The
`distribution of the drugs to these brain areas. Alternatively, it may be
`due
`to
`the presence of AChE
`isoenzymes, which could have different
`affinities for
`the
`inhibitors. Such a differential sensitivitd of
`multiple forms of AChE has been demonstrated for organophosphates1 • It
`remains to be determined whether multiple forms of AChE are present in
`rat brain, and whether they are selectively inhibited by RA compounds.
`
`The data from this study show that larger monoalkyl or dialkyl
`deri va ti ves of miotine, possess several advantages over physostigmine
`for potential therapeutic application in conditions invol ving reduced
`cholinergic
`transmission in the cerebral cortex. If the therapeutic
`effect of these agents results from inhibition of AChE in this brain
`area, compounds RA6, RA 15, RA 14, RA 12, RA 10, RA7 and RA8 all have
`considerably higher therapeutic ratios than physostigmine and show fewer
`side effects at ED50 doses. This may be due to a selective inhibition in
`cortical areas sparing the medulla. RA7 and RA10 have an additional
`advantage in the fact that the lethal effects of drug overdose can be
`prevented by atropine. While
`the duration of significant
`enzyme
`inhibition after physostigmine is less than 2 hours, all the above drugs
`(except RA10) act for periods of 7 hours or more after a
`single
`injection. The longer duration is a distinct advantage in the treatment ·
`of chronic conditions such as Alzheimer's disease. Furthermore, RA6, RA7
`and RA8 show a significantly more efficient oral absorption since their
`potencies when given by
`this
`route closely
`resemble
`those after
`parenteral administration.
`
`Acknowledgement. This research was supported by a grant from the
`Israeli National Council for Research and Development No. 2248.
`
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`Noven v. Novartis and LTS Lohmann
`IPR2014-00550
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`< 0...
`i' 8
`~. ~
`
`, >-.
`, .0
`
`(l)
`
`-0
`(l)
`U
`-0
`0..
`(l)
`.0
`>-.
`ro
`E
`-0
`C ro
`~
`'-ro
`'" 0...
`""2
`:5
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`the
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`mt
`of
`It
`in
`
`cyl
`lne
`~ed
`~ic
`lin
`lye
`.er
`in
`lal
`be
`rme
`19s
`~le
`mt '
`lA7
`dr
`;er
`
`;he
`
`~ic
`
`md
`lry
`
`>n,
`"th
`~d.
`
`~ic
`
`Iry
`
`'al
`;e,
`
`in
`le,
`
`:al
`in
`~O :
`
`lin
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`.I.
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`'ic
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`i
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`NOVARTIS EXHIBIT 2036
`Noven v. Novartis and LTS Lohmann
`IPR2014-00550
`Page 14 of 14
`
`