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`
`oozz-sssmmm-ostssosooro
`‘nil JOURNAL or Winery mo Ennmmu msmwcs
`Copyright. 0 1996 by The American Society for Pharmacology and Experimental Therapeutics
`JPET 272:619—827. 1996
`
`
`Vol. 272. No. 2
`Printed in USA
`
`Repression of Angiotensin ii and Potentiation of Bradykinin
`Contribute to the Synergistic Effects of Dual Metalloprotease
`inhibition in Heart Failure
`
`NICK C. TRIPPODO. BALKRUSHNA C. PANCHAL and MAXINE FOX
`
`Department a! Phannacologr. The Bristol—Myers Squibb Pharmaceutical Research Institute, Princeton, New Jersey
`Accepted for publication October 20, 1994
`
`
`blockade and NEP-I than after either treatment alone. This
`Neutral endopeptidase inhibition (NEP-l) and angiotansin con-
`vening enzyme inhibition (ACE-I) act synergistically to produce
`indicated that repression of Ang it contributes importantiy to
`acute beneficial hemodynarnic effects in models of heart fail-
`the NEP-i/ACE-l interaction. Bradykinin 32 receptor antago-
`ure. Blockade of the iormation of angiotansin II (Ang It) acting
`nism by Hoe 140 at 100 polka. i.v. significantly blunted the
`together with potentiation oi the natriuretic peptides, bradykinin
`decrease in left ventricular and diastolic pressure but not the
`and other vasoactive peptides may mediate the interaction at
`decrease In left ventricular systolic pressure after duel NEP-l/
`dual enzyme inhibition. In this study. the potential roles of Ang
`ACE-l (SO-28503 and enalaprilat each at 30 umoi/‘itg. l.v.). This
`II repression and bradykinin potentiation were evaluated in
`suggests that bradykinin potantlation contributes to the pre—
`conscious cardiomyopathic hamsters with compensated heart
`load-reducing, but not the silenced-reducing. acute attests of
`failure. The Ang ll AT, receptor antagonist. SR 47436 (EMS-
`NEP—I/ACE-i. Hence, both Ang II repression and bradykinin
`186295), was administered at 30 pmol/kg, i.v. followed by i.v.
`potentietion are factors contributing to the synergistic hemo-
`dynamic etiects oi combined NEP-i and ACE-I In hamsters with
`infusion at 1 pmong/min in combination with NEP-i (SO-28603
`at 30 pmong l.v.). Cardiac preload (left ventricular and dia—
`heart failure. The bradykinin-medlated enhanced effect of com-
`stolic pressure] and atterload (left ventricular systolic pressure)
`bined NEP-l/ACE-l to reduce cardiac preload could improve
`the beneficial eitects oi ACE-l in the treatment of heart failure.
`decreased significantly more after the combination oi Ang II
`
`
`enalaprilat, and the selective NEP inhibitor, SQ-28603, pro-
`duced decreases in cardiac preload and afierload, whereas
`each treatment alone had minimal effects (’I‘rippodo et al.,
`1993). These studies support the concept that the coadmin-
`istration of ACE-1 and NEP-I leads to synergistic effects and
`could have use in the treatment of hypertension and heart
`failure.
`
`ACE-I blocks the formation ofAng II and hence attenuates
`its vasoconstrictor, antinatriuretic and growth enhancement
`properties. NEP-I prevents the enzymatic inactivation of
`ANP and therefore protects or potentiatea its vasodilatory.
`natriuretic and antiproliferative actions. Studies have shown
`that concurrent administration of NEP-I and ACE-I in mod-
`els of hypertension and heart failure result in an interaction
`that leads to cardiovascular effects greater than those caused
`by either treatment given singly. For instance, the antihy-
`pertensive el‘l'ect of ACE-I in conscious spontaneously hyper-
`tensive rats was enhanced by coadministration of selective
`inhibitors of NEP (Seymour at al., 1991; Pham et al., 1993).
`in dogs with pacing~induced heart failure, NEP-l potentiated
`the vaaodilatory effects ofACE-I (Seymour et cl. 1993); and in
`a similar model, subchronic treatment with ACE-1 potenti-
`ated the renal hemodynamic and excretory responses to
`NEP-I (Marguiiea at (11.. 1991). In cardiomyopathic hamsters
`with heart failure, the combination of the ACE inhibitor,
`
`Possible mechanisms by which HEP-I and ACE-l interact
`to produce enhanced cardiovascular effects have been previ-
`ously discussed (Seymour et 01., 1993, Trippodo et al., 1993).
`Attenuation of the formation of Ang II by ACE-I might un-
`mask the vasodilatory efi‘ecta of ANP and other natriuretic
`peptides such as BNP and GNP. This may be particularly
`relevant in heart failure where the biological actions of ANP
`are blunted, perhaps partly because of the counteracting
`effects of Ang II (Rays et 01.. 1989; Margulies et al.. 1991).
`Potentiation of the vasodilatory effects of bradykinin by both
`ACE-1 and HEP-l might also contribute to the synergism.
`Although bradykinin can be potentiated by ACE-I alone,
`Received for publication May 24. 1994.
`greater enhancement of the activity of this peptide may comeM
`ABBREVIATIONS: ACE-l. angiotensln convening enzyme inhibition; Ang II. angiotensln ll; ANP. atrial natriuretlc peptide: BNP. brain natriuretic
`peptide: CNP, C-type natriuretic peptide; HR. heart rate; LVEDP, left ventricular and diastolic pressure: LVSP. left ventricular systolic pressure;
`MAP. mean arterial pressure; NEP-l. neutral andopeptidase inhibition.
`
`BIOCON PHARMA LTD (IPR2020-01263) Ex. 1003, p. 001
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`

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`Trippodo at sl.
`
`Vol. 272
`
`about when two enzymes involved in its degradation, such as
`ACE and NSF. are inhibited. Finally, ACE and NE? have
`substrates in common other than bradykinin. such as sub
`stance P. and hence additional factors may play a role as
`
`Any combination of two or more of these elements may
`interact to produce synergistic cardiovascular effects. How-
`ever. currently there is no direct evidence showing whether
`any of these factors are important in the combined efl'ecta of
`dual metalloprotease inhibition. The purpose of this study
`was to evaluate the potential contributions of Ang II repres-
`sion and bradykinin potentiation on the cardiovascular ef-
`fects of combined NEP-l and ACE—I in cardiomyopathic ham-
`sters with heart failure. These factors were studied not only
`because of the known effects of the inhibitors on their me-
`tabolism. but also because potent specific inhibitors of the
`Ang II AT, receptor and the bradylu'nin B, receptor were
`available. Ang II ATl receptors mediate virtually all of the
`known biological actions of Ang II ('Iimmerrnans et al., 1993).
`Bradykinin B; receptors are likely responsible for many of
`the cardiovascular and renal efl'ects of bradylrinin (Regoli et
`al.. 1990). In this study we used the Ang II AT1 receptor
`antagonist, SR 47436 (EMS-186295) (Cazaubon et al.. 1993)
`and the bradylrinin 3: receptor antagonist, Hoe 140 (Wirth et
`al., 1991). The results from the use of these specific probes
`indicate that both the repression of Ang II and the potentia-
`tion of bradykinin contribute to the acute synergistic effects
`of NEP-l and ACE-l to reduce cardiac preloed'in cardiomyo-
`pathic hamsters with heart failure.
`
`Methods
`
`All procedures involving animals were in accordance with the
`Bristol-Myers Squibb Pharmaceutical Research Institute Animal
`Care and Use Committee.
`
`Anlrnsl preparation
`
`Cardiomyopathic male hamsters of the BIO TO-2 strain (Sale.
`1999) were obtained from Bio Breeders. lnc.. Fitchburg, MA. The
`animals were housed two to five per cage in a light and dark cycle of
`14 h and 10 h. respectively. for a minimum of 2 wk before study. The
`animals were maintained on Purina 5001 pelleted Chow (Purina.
`Richmond. 1N) and tap water ad libitum; they were studied at
`approximately 260 days of age in a stage of nonedcmatous. compen-
`sated heart failure (For at 111.. 1993; Trippodo er al., 1993; Panchal
`and ’l‘rippodo. 1993). All experiments were conducted in conscious.
`unrestrained. cardiomyopathic hamsters 3 h after placement ofcath-
`eters. At the end of each experiment. the animals were euthanized
`with sodium pentobarbital (100 mg/kg. i.v.).
`The hamsters were briefly anesthetized with methohesital sodium
`(70 lug/kg. i.p.. Brevital. Eli Lilly & 00.. Indianapolis. IN). Polyeth-
`ylene catheters (PEIO connected to PE50) were filled with isotonic
`saline (0.9% NeCl) containing heparin (4 Wind). The P210 ends of
`the catheters were used for cannulation. The right jugular vein was
`cannulated and supplemental doses of methohenital (1 mglkg, i.v.)
`were administered as needed during surgery. The right carotid ar-
`tery was cannulated for the measurement of MAP; in some experi-
`ments the catheter was advanced into the left ventricle for the
`measurement of LVSP and LVEDP. The free ends of the catheters
`were passed under the skin and exterior-bed at the back of the neck
`near the scapula. The animals were allowed to recover unrestrained
`for 3 h before initiating measurements. The arterial or ventricular
`catheter was connected to a pressure transducer (Model P23XL.
`Gould Electronics, Valley View. OK) for measurement of MAP or
`LVEDP and LVSP. Cardiovascular pressures and HR were recorded
`
`in the conscious. freemoving hamster on a polygraph (Model 7D.
`Grass Instruments. lnc.. Quincy. MA). MAP was obtained by elec-
`tronically damping the arterial pulse pressure. LV‘EDP and [NSF
`were determined from the left ventricular pulse tracing as described
`previously (Fox at al. 1993; 'l‘rippodo et at. 1993; Pancho! and Trip
`pods. 1993).
`l-ER was obtained through a tachograph preamplifier
`(7P4H. Grass Instruments. lnc.. Quincy. MA) as triggered by the
`arterial or ventricular pulse. The catheter in the jugular vein was
`used for the administration of agents.
`
`Inhlbiflon ol pressor and depressor responses
`Aug 11 pres-or response. Preliminary experiments were con-
`ductedin conscious cardiomyopathic hamsters to determine a dose
`regimen of SR 47436 (EMS-196295) that would nearly completely
`block the pressor response to Aug 11 for at least 2 h The pressor
`responses to two challenges of Ang II (100 ng/lrg. iv. dissolved in
`0.9% NaCl. 1 ml/lrg) were determined. This does of Ang 11 produced
`more than a 30% increase in MAP. Based on the preliminary exper-
`iments. SR 47436 (EMS-186396) was administered to five cardionr
`yopathic hamsters at 30 umol/lrg. i.v. followed by continuous i.v.
`infusion at 1 umolr'lrglmin. Challenges of Ang ll were then repeated
`at 10~min to 30-min intervals up to 150 min alter the bolus iniection
`of SR 47436 (EMS-156295).
`Brndykinin depressor response. Because i.v. bolus iniections
`of bradylrinin caused respiratory distress in the cardiomyopathic
`hamsters, depressor responses to intraarterial administration ofthis
`peptide were studied. There were no changes in the behavior of the
`animals to suggest pain or distress afier the administration of bra-
`dykinin, is. In eight conscious cardiomyopathic hamsters the de-
`pressor response to 0.9% NaCl (1 ml/kg. i.a.) was determined. This
`was followed by two challenges of bradykinin (10 um. i.a., dis-
`solved in 1 ml/kg 0.9% NaCl). This dose of bradykinin and saline
`produced a depressor response that was in the mid to upper range of
`the bradykinin dose-response relationship for depressor efl'ects.
`Based on preliminary experiments. Hoe 140 was administered at 100
`pig/kg. i.v. (dissolved in 0.9% NaCl. 1 mllkg). Challenges of bradyhi-
`nin were then repeated at 5-min to 30-min intervals up to 150 min.
`A second injection of 0.9% NsCl was administered at the end of the
`experiment.
`
`Cardiovascular effects
`
`In this series of experiments. baseline measurements of LV'EDP.
`LVSP and HR were determined in groups of conscious cardiomyo-
`pathic hamsters. Compounds or vehicles were administered at 1
`ml/lrg. i.v.. unless indicated otherwise. and measurements were re-
`peated at 5-min to 30-min intervals up to 90 min afier administra-
`tion of the last agent.
`SR. 47436 (EMS-196295). 3928903 and the combination of
`these agents. SR 47436 (EMS-186295) was administered at 30
`umolflrg. i.v. (0.3 ml) followed by a continuous i.v. infusion at 1
`umol/lrg/min (0.01 mllmin). 511 47436 (3513186295) was prepared in
`0.028 M KOH and diluted to a final concentration of 0.017 M K0“.
`KOH solution (0.017 M) was administered i.v. to the vehicle group at
`0.3 ml followed by a continuous infusion at 0.01 nil/min. SQ-28603
`was dissolved in 0.84% NaHCO, and administered at 30 umol/ltg. i.v.
`This does of SCI-29603 was previously shown to result in a doubling
`of plasma ANP concentration within 90 min in this model ('l‘rippodo.
`et 01.. 1993). The vehicle for 89-25603 was previously shown to have
`only minimal cardiovascular efl’ecta in cardiomyopathic hamsters
`('l‘rippodo el al., 1993); similar minimal effects were observed in this
`study (see below). One group of cardiomyopathic hamsters received
`the combination of 511 47436 (EMS-186295) and SQ—28603. In this
`group, SR 47436 (EMS-186295) was administered according to the
`same dosage regimen described above; 30 min after the bolus iniec-
`h'on of 311 47438 (EMS-186295). SQ-28603 was administered at 30
`umol/kg. i.v.
`
`BIOCON PHARMA LTD (IPR2020-01263) Ex. 1003, p. 002
`
`

`

`Combination of enalaprilat and Bil-28803 and the efl'octs of
`HOE 140 on this combination. Hoe 140 was administered at 100
`pg/kg, i.v. (dissolved in 0.9% NaCl). Enalaprilat and SQ-2BED3 were
`each dissolved in 0.84% Nal-lCO, and were administered at. 30 umol/
`kg, i.v. 30 min apart (enalsprilst first). One group received all three
`of these agents at the doses indicated above in the following se-
`quence: Hoe 140, followed 5 min later by enalaprilat. followed 30 min
`by 39-23803. The vehicle group in this set of experiments received
`0.9% NaCl, followed 5 min later by 0.84% NaHCO,, followed 30 min
`later by a second injection of 0.84% Naflcoa.
`Eflocts o! Hoe 140 on the combination of SR 471,38 (HMS-
`18-0395) and Sta—25303. One group of cardiomyopathic hamsters
`was administered Hoe 140, 6 min later SR 47436 (EMS-186295) and
`30 min later SQ—26803. The vehicles and doses of the compounds
`were the same as indicated above.
`
`Statistical analyses
`Differences in age, body weight and baseline values among groups
`were evaluated by analysis of variance. Difl‘erences in changes from
`baseline among groups were evaluated by analysis of covariance
`with repeated measures and contrasts. The baseline value for each
`variable was used as the covariate. The level of significance was
`taken at P < .05. All data are expressed as means 1 8.3.1“.
`
`Ang II and bradykinin were purchased from Sigma Chemical Co.
`(St. Louis, MO); Hoe 140 was purchased from Peninsula Laborato-
`ries (Belmont, CA); enslaprilat was supplied by Merck Sharp 8:
`Dohme Research Laboratories (West Point. PA); 89-23603 was syn-
`thesized by Bristol-Myers Squibb Pharmaceutical Research Institute
`(Princeton, NJ) and SR 47436 (EMS—186295) was synthesized by
`Sanofi Recherche (Montpellier, France).
`
`‘ Results
`
`inhibition at preasor and depressor responses
`
`Ang I] pressor response. The pressor responses to du-
`plicate injections of Ang I! were 29 1 2 mm Hg and 31 1 3
`mm Hg (fig. 1). After the administration of SR 47436 (EMS-
`186295), the pressor responses to Ang I] at intervals up
`to 150 min were less than 6 mm Hg. These results indicate
`that nearly complete inhibition of the pressor response to
`
`Presser Response to an: it. M. In
`Conscious esroiomyopslhle Hamsters (n I 5)
`
`SR 07436 (EMS-186295)
`30 pmoilkg.
`i.v. o 1 .pmoinrgimin.
`
`i.v.
`
`-2o-1o to 20 30 40 50 50 70 80 so 120150
`Ilinutss
`
`Fig. 1. Changes in MAP In response to Ang ii, 100 ng/kg. i.v. betore
`and after the administration of SR 41436 {EMS-156295) in conscious
`cardiomyopathtc hamsters (age 263 1 5 days: b.wt. 124 1 6 g).
`
`Mechanisms ot NEPIACE Inhibition
`
`321
`
`Ang II was achieved with this dosage regimen of SR 47436
`(EMS-156295).
`Bradylrinin depressor response. The depressor re-
`sponses to intraarterial injection of 0.9% NaCl alone at the
`beginning and end of the experiment were -9 1 1 mm Hg and
`~10 1 1 mm Hg. respectively (fig. 2). The depressor responses
`to duplicate i.a. injections of bradykinin in 0.9% NaCl were
`-21 1 1 mm Hg and -20 1 1 mm Hg. These results indicate
`that the depressor response to bradykinin alone (minus the
`vehicle effect) was approximately 10 mm Hg. Afier the ad-
`ministration of Hoe 140, the depressor responses to i.a. in-
`jections of bradykinin in 0.9% NaCl did not exceed -11 mm
`Hg for intervals up to 180 min. These results indicate that
`alter the administration of Hoe 140, the depressor responses
`to i.a. injections of bradykinin in 0.9% NaCl was attributable
`mostly to the vehicle effect and that the depressor effect of
`bradykinin alone was nearly completely abolished for the
`duration of study.
`
`Cardiovascular attests
`
`8R 47433 (EMS-183295), Sta-23603 and the combina-
`tion of these agents. Age, body weight and baseline values
`were similar (P > .05) among the four groups of cardiomyo-
`pathic hamsters in this series of experiments. These values
`(means 1 S.E.M.) were as follows in the vehicle (n = 10),
`SQ-28603 (n = 6), EMS-18629501 = 9) and EMS-186295 +
`SQ-23603 (n = 7) groups, respectively: age (days), 256 1 2,
`261 1 4, 258 1 1, 261 1 2; b.wt. (g), 116 1 2,118 '1 3, 117 1
`2 and 117 1 3; LVEDP (mm Hg), 19 1 2, 18 1 3, 17 1 2 and
`21 1 2; LVSP (mm Hg),111 1 a, 117 1 5.112 1 2 and 107
`1 3; HR (beats/min). 350 1 10, 378 1 12, 338 1 16 and 354
`1 6. The changes in LVEDP and LVSP afier the administra-
`tion of SR 47436 (EMS-186295) were not significantly differ-
`ent from those in the vehicle (0.017 M KOH) group (fig. 3).
`After the administration of Sta-28603 alone, LVEDP de-
`creased only slightiy (by <5 mm Hg). although the decrease
`at the 30-min time point was significantly greater than the
`change in the vehicle group. LVSP decreased by approxi-
`mately 10 mm Hg afler the administration of SQ-28603
`alone; the decreases at two time points were significantly
`
`Bop-user Flo-porno lo lrsoyldnin or Selina. i.a.
`In Conscious Croiornyopstlde Nameless (n a GI
`Hoe 140. 100'pmh'r.
`
`
`
`our(mini-1;) {7.8
`
`'2°
`.25
`
`some
`
`
`C] Bradyliinin in saline
`
`
`SWSB'SS 15 30 45 £0 90120150100 5
`“Inui-
`
`Fig. 2. Changes in MAP in response to bradyklnin. 10 pig/kg, i.a. or
`saline (0.9% 11860, 1 ml/kg, i.a. before and after the administration 01
`Hoe 140 in oonsclous cardiomyopathlc hamsters (age 259 1 2 days:
`b.wt. 112 1 2 g).
`
`BIOCON PHARMA LTD (IPR2020-01263) Ex. 1003, p. 003
`
`

`

`TH ppodo at el.
`
`~-— Vehicle
`- - -' - -
`50.25503
`
`.°_ 5“ ‘7‘“
`(EMS-180295)
`
` g 5
`
`-I5
`
`0
`
`15
`
`30
`
`45
`
`80
`
`75
`
`90
`
`' P < 0.05 vs Veh
`
`1 P < 0.05 vs so-zseos
`
`Q .
`OO lb0 n
`
`AO
`.15
`o
`15
`so
`45
`so
`75
`so
`“lfltflll
`
`Fig. 3. Cardiovascular changes in conscious cardlomyopsthlc harn-
`sters alter the administration of vehicle (0.017 M KOH), 80-25603. SR
`47435 (EMS-166295) or the combination of 50-23603 and SR 47436
`(EMS-158295). The group receiving the combination treatment is des-
`ignated as 295 + 500. 80-20603 was administered as an iv. bolus at
`30 umol/kg. SR 47436 (EMS-186295) was administered at 30 umol/kg.
`i.v. iollowed by a continuous iv. infusion at 1 pmong/min. in the
`combination treatment group. 50-28603 was administered 30 min after
`the bolus iniection and start 01 iniusion of SR 47436 (EMS-156295).
`
`greater than the changes in the vehicle group. The combina-
`tion of SQ-2B'603 and SR 47436 (EMS-186295) produced de-
`creases in LVEDP and LVSP that were significantly greater
`than the vehicle effects and the changes due to the adminis-
`tration of these compounds alone. For instance, at 90 min
`after the administration of the combination treatment,
`LV'EDP was decreased by 11 t 3 mm Hg from a baseline of
`21 z 2 mm Hg; LVSP was decreased by 18 t 4 mm Hg from
`a baseline of 107 t 3 mm Hg. HER changes were minimal in
`all groups. although significant increases relative to the ve-
`hicle group were observed at two time points after the ad-
`ministration of SR 47436 (EMS-186295).
`Combination of enalaprilat and 3928-803 and the
`efl'ects of Hoe 160 on this combination. Age, body weight
`and baseline values were similar (P > .06) among the four
`groups of cardiomyopathic hamsters in this series of experi-
`ments. These values (means I S.E.M.) were as follows in the
`vehicle (n = 9), Hoe 140 (n = 6), enalaprilat + SQ.28603 (n
`= 6), Hoe 140 + enalaprilat + SQ-23603 (n = 10) groups,
`respectively: age (days), 259 t 2, 262 t 2, 260 1 1 and 258 2
`
`Vol. 272
`
`2; b.wt. (g). 115 1- 2, 115 t 3, 115 1 1 and 116 1' 2; LVEDP
`(mm Hg), 17 i 2, 19 t 3, 24 t 2 and 20 t 2; LVSP (mm Hg).
`108 t 4, 103 1' 4, 105 1' l and 105 t 5', HR (beats/min). 382
`t 14, 359 1' 9, 363 t 11 and 350 t 14. The changes in
`LVEDP and LVSP after the administration of vehicle (0.84%
`NaHCOs) and Hoe 140 alone were minimal (fig. 4). After the
`administration of the combination of enslaprilat and SQ-
`28603, LVEDP decreased by approximately 15 mm Hg for the
`90-min duration of observation; these changes were signifi—
`cantly greater than those in the vehicle group. This combi-
`nation treatment produced similar significant decreases in
`LVSP. The addition of Hoe 140 to the combination of ensla-
`prilat and SQ-23603 resulted in a significantly smaller de-
`crease (approximately 5 mm Hg) in LV'EDP as compared
`with the combination of enalaprilst and SQ—28603. but did
`not blunt the changes in LVSP. The changes in HR in all
`groups were generally small, although significant decreases
`relative to the vehicle or the enalaprilat plus SCI-28603
`groups were observed at some time points in the animals
`receiving Hoe 140 plus the combination of snalaprilat and
`SQ-2B603.
`Eflecta of Hoe 140 on the combination of SR 4743!!
`(EMS-138295) and Bil-23603. Age (253 z 1 days), b.wt.
`
`we»
`—°'— E9603
`---~- 1100140
`—0'— Hoel40+E0003
`
`‘0 HE
`
`003
`
`anew(mung)
`
`45 -30 45
`
`0
`
`15
`
`30
`
`45
`
`00
`
`75
`
`90
`
`to
`
`' P < 0.05 vs Veh
`1P<o.05st+603
`
`‘3 .30 45
`
`0
`
`15
`
`30
`
`45
`
`00
`
`75
`
`90
`
`
`ALUIP(mml’ill
`(nutshell!),2t2osses 45
` Mill
`
`-30 45
`
`0
`
`30
`15
`flinutee
`
`45
`
`60
`
`75
`
`90
`
`Fig. 4. Cardiovascular changes in conscious cardlomyopathic ham-
`sters alter the administration of vehicle (0.34% Nat-i603). Hoe 140 (H.
`100 pmol/kg, l.v.). the combination of enslsprliat (E, 30 umong. l.v.)
`and 80-28603 {603. 30 urnol/kg. ml. or the combination of Hoe 140
`plus enalspn‘ist and 80-20603 (same doses as above). Arrows indicate
`times of administration of compounds.
`
`BIOCON PHARMA LTD (IPR2020-01263) Ex. 1003, p. 004
`
`

`

`(119 i 3 g) and baseline values for LVEDP (26 z 2 mm Hg),
`LVSP (100 t 2 mm Hg) and HR (347 z 8 beats/min) in the
`group of cardiomyopathic hamsters receiving Hoe 140 plus
`the combination of SR 47436 (EMS-156295) and SCI-28603
`were similar (P > .06) to those receiving this combination
`treatment without Hoe 140. The changes in LVEDP. LVSP
`and HR after the administration of Hoe 140 plus the combi-
`nation of SR 47436 (EMS-186295) and SCI-28603 were nearly
`identical to the changes observed after the administration of
`this combination treatment without Hoe 140 (fig. 5).
`Comparison of the combination, enalaprilat + SQ-
`28603 with the combination. SR 47488tBMS-1382951 +
`SQ28803. Because difl'erent vehicles had to be used in the
`two series of experiments using the ACE inhibitor, enalapri-
`lat or the Ang II antagonist, SR 47436 (BMSv186295), and
`because slightly different vehicle effects were observed, di-
`rect comparison of groups from these two series of experi-
`ments would not be valid. Therefore, to eliminate the “vehicle
`efi'ecte" in such a comparison, the average change observed at
`
`Mechanisms of HEP/ROE Inhibl‘tlon
`
`623
`
`each time point during the administration of 0.017 M KOH
`was subtracted from the changes in each animal receiving
`the combination of SR 4743B (BMSJBBZQS) and SQ-23603.
`Similarly, the average change observed after the administra-
`tion of 0.84% NaHCOa was subtracted from the changes in
`each animal receiving the combination of enalaprilat and
`SQ-28603. These values were then compared as shown in
`figure 6. The results indicate that the changes (minus vehicle
`efl‘ects) in LVSP were similar (P > .05 at all time points) in
`the two groups. However, the changes (minus vehicle effects)
`in LVEDP were greater (P < .05 at two time points) in the
`animals receiving enalaprilat plus ESQ-28603 as compared
`with the animals receiving SR 47436 (EMS-136295) plus
`SQ-ZBGOB. A similar comparison between the groups receiv-
`ing Hoe 140 with each of these combination treatments re-
`vealed no significant differences in the changes (minus vehi-
`cle effects) in LVEDP.
`
`Discussion
`
`'—°— 295¢603
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`The findings demonstrated that in hamsters with compen-
`sated heart failure, Ang II receptor blockade acted synergis-
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`Fig. 5. Cardiovascular changes In conscious cardiomyopethic ham-
`sters after the administration of the combinallon 01 50-28603 and SR
`47436 (EMS-138295) or the combination of Hoe 140 plus SR 47436
`(EMS-136295) and 50-28-603. The results from the combination of
`50-28603 and SR 47436 (EMS-156295} are the same as those shown
`In figure 3. 50-28808 {608) was administered as on LV. bolus at 30
`umong. SR 47436 (EMS-186295) (295) was administered at 30 pmol/
`kg. iv. followed by a continuous l.v. Infusion at 1 umol/kg/mln. Hoe 140
`(H) was administered as an iv. bolus at 100 pig/kg. Arrows indicate
`times 0! administration of compounds.
`
`Fip. e. Cardiovascular changes (minus vehicle eflects) in conscious
`csrdlomyopathlc hamsters otter the administration of the combination
`oi enalaprilat (E, 30 ”moi/kg. l.v.) and 30-28603 (603, 30 umol/kg. l.v.)
`or the combination 0! 80-25603 (303. 30 mong. l.v.) and SR 47436
`(EMS-186295) (295. 30 umong.
`l.v. + 1 pmong/mln.
`l.v.). The
`changes were determined trom the results shown in figures 3 and 4.
`The bottom graph shows the changes (minus vehicle etiects) in LVEDP
`in conscious cardiomyopethlc hamsters receiving Hoe 140 in addition
`to the treatments indicated above: these changes were determined
`from the results In figures 4 and 5.
`
`BIOCON PHARMA LTD (IPR2020-01263) Ex. 1003, p. 005
`
`

`

`popodo at at.
`
`Vol. 272
`
`tically with NEP-l to reduce cardiac preloed and afterload.
`This interaction was similar to that previously observed with
`concurrent ACE-l and NEPJ in this model and suggests that
`repression ofAng II is an important factor contributing to the
`acute synergistic effects of dual inhibition of ACE and NEP.
`The results also showed that blockade of bradykinin B2 re-
`ceptors blunted the synergistic effects of NEP-I and ACE-I on
`cardiac preload. but not on afterload, suggesting that, to
`some extent, potentiation of bradykinin is another contribut-
`
`Previous studies in models of heart failure and hyperten-
`sion have shown that the combination of N'EP-I and ACE-I
`elicited cardiovascular effects greater than those caused by
`either treatment alone (Seymour et al., 1991; Margulies er
`al.. 1991; Seymour e! 01., 1993;th et al., 1993; 'l‘rippodo e!
`41., 1993). This interaction of dual metalloproteass inhibition
`might be partly due to the blockade of the formation ofAng 11
`acting together with the enhancement of several vasodilater
`factors, such as the natriuretic peptides (ANP, BNP and
`CN'P), bradykinin and substance P. We explored the poten-
`tial role of Ang II repression in this interaction by using the
`Ang I1 AT, receptor antagonist, SR 47436 (EMS-186295)
`(Cazaubon et al., 1993) in a model of heart failure previously
`characterized in our laboratory (Fox et al., 1993; Trippodo et
`al., 1993; Pancho] and 'l‘n'ppodo, 1993). After i.v. administra-
`tion of SR 47436 (EMS-186295) at 30 pmol/kg followed by
`continuous i.v. infusion at 1 umol/kg/min in conscious car-
`diomyopathic hamsters, the pressor response to Ang II was
`inhibited by more than 80% for more than 2 h. This dosage
`regimen of the Ang II antagonist was used in subsequent
`studies in combination with the NE? inhibitor, SQ-28603 at
`30 amol/kg, i.v., a dose previously shown to have minimal
`acute cardiovascular effects in this model, but found to have
`synergistic effects with the ACE inhibitor, enalaprilat (Trip-
`podo et al., 1993). In the present study. Ang [I antagonism
`alone had minimal acute cardiovascular effects in the con-
`
`scious cardiomyopathic hamsters, which is consistent with
`the minimal acute effects of ACE-I in this model. Also as
`previously reported, NEP-I alone had only small effects.
`However, the combination of Ang 1! blockade and NEP-I
`produced significant reductions in LV'EDP and LVSP that
`were greater than those produced by either treatment alone.
`These synergistic effects were similar to those observed pre-
`viously with the combination of ACE-l and NEP—l. Together.
`the results suggest that attenuation of the formation of Ang
`II is an important factor contributing to the interaction of
`concurrent ACE inhibition and NEP inhibition in cardiom-
`yopathic hamsters.
`The role of bradykinin potentiation in this interaction was
`also investigated, because bradykinin could be protected by
`inhibition of both ACE and NEP (Ura e! 01., 1987; Erdiis,
`1990; Skidgel, 1992). First it was established that a bolus i.v.
`injection of 100 am of the bradykinin B2 receptor antago-
`nist Hoe 140 (Wirth el al., 1991) completely blocked the
`depressor response to bradykinin for at least 3 h in conscious
`cardiomyopathic hamsters. This dose of Hoe 140 was used to
`determine whether bradykinin blockade altered the cardio-
`vascular response to dual metalloprotease inhibition. The
`combination of enalaprilat (30 umol/kg, i.v.) and SQ-28603
`(30 “moi/kg, i.v.) lowered LVEDP by 16 z 2 mm Hg and
`decreased LVSP by 18 z 4 mm Hg within 60 to 90 min in
`cardiomyopathic hamsters. These changes were similar to
`
`those previously reported (Trippodo et al., 1993). However,
`when the ACE and NEP inhibitors were administered after
`Hoe 140, LVEDP decreased by only 6 t 2 mm Hg (AmJ min).
`Blockade of bradykinin receptors had no such blunting effect
`on reduction in afterload by dual metalloprotease inhibition
`(ALVSPN m," = -24 z 3 mm Hg). These results indicate that
`potentiation of bradykinin is an important mediator of the
`acute praised-reducing effect of NEP-I/ACE—I in hamsters
`with heart failure.
`Hoe 140 did not alter the prelosd and attorload responses
`to the combination of 5923603 and SR 47436 (EMS-186295).
`Hence, potentiation of bradykinin did not appear to be a
`factor in the acute synergistic effects of the combination of
`NEP-I and Ang II receptor blockade. This suggests that
`NEP-l alone was not sufficient to potentiate bradykinin in
`this model, at least to the extent that it would interact with
`other vasoactive agents altered by this treatment. These
`results are consistent with findings in rat isolated, perfused
`mesenteric arteries showing that ACE-I potentiated the va-
`sodilator effects of bradykinin, whereas NEP-l did not affect
`the response (Salgsdo et al., 1992). However. although N'EP-I
`may not have potentiated bradykinin enough for it to exert
`an effect during combined treatment with Ang II receptor
`blockade, it is possible that NEP-I could have protected bra-
`dykinin to the extent that an additive or synergistic effect of
`bradykinin activity could have occurred during the combined
`treatment with ACE-I. For instance, although the major bra-
`dykinin-degrading activity observed in intact cultured hu-
`man umbilical vein endothelial cells could be attributed to
`ACE, nearly 20% of the activity was inhibited by phosphor-
`amidon, indicating a significant contribution of NEP (Graf et
`al., 1992). NEP-I also partially blocked the metabolism of
`bredylrinin by a particulate fraction of rabbit endothelial
`cells of venous origin (Llorens-Cortes et al., 1992). Therefore,
`the bradykinin potentiating effects on prelosd reduction ob-
`served with dual N'EP-llACE-I in the cardiomyopathic harn-
`sters could have been due to ACE-I alone or the combined
`effects of ACE-l and HEP-1.
`It is interesting that bradykinin potentiation contributed
`to the decrease in preload but not to the decrease in afierload
`after dual metalloprotease inhibition in the cardiomyopathic
`hamsters. The hemodynamic events that could account for
`the decrease in LVEDP include: pooling of blood away from
`the heart due to peripheral venodilation; decreased blood
`volume via a fluid shifi across the capillaries or via urinary
`fluid loss and altered left ventricular performance due to the
`reduction in afterload, an increase in contractility or change
`in diastolic function.
`Vanodilation. ACE—l was shown to cause venodilation in
`man (Capewell et al., 1989) and animals (Rays ct al., 1939)
`with heart failure; this has several potential mechanisms,
`including decreased Ang 11, decreased sympathetic nerve
`activity and increased kinins. prostaglandins and vasopres-
`sin (Capewell et al., 1939). NEP-I may have a vasorelaxsnt
`effect on ve