D7
`
`0022-3565/95/2722-0619$03.00/0
`‘THE JOURNAL OF PHARMACOLOGY AND EXPERDSENTAL THERAPEUTICS
`Copyright © 1995 by The American Society for Pharmacology and Experimental Therapeutics
`SPET 272:619-627, 1996
`
`
`Vol. 272, No, 2
`Printed in U.S.A.
`
`Repression of Angiotensin Il 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 of Pharmacology, The Bristol-Myers Squibb Pharmaceutical Research Institute, Princeton, New Jersey
`Accepted for publication October 20, 1994
`
`
`Neutral endopeptidase inhibition (NEP-I) and angiotensin con-
`blockade and NEP-I than after either treatment alone. This
`verting enzymeinhibition (ACE-|) act synergistically to produce
`indicated that repression of Ang #l contributes importantly to
`acute beneficial hemodynamic effects In models of heart fail-
`the NEP-I/ACE-I interaction. Bradykinin B, receptor antago-
`ure. Blockade of the formation of angiotensin Il (AngIl) acting
`nism by Hoe 140 at 100 ywa/kg,i.v. significantly blunted the
`together with potentiation of the natriuretic peptides, bradykinin
`decreasein left ventricular end diastolic pressure but not the
`and other vasoactive peptides may mediate the Interaction of
`decrease in left ventricular systolic pressure after dual NEP-I/
`dual enzymeinhibition. In this study, the potential rales of Ang
`ACE-t (SQ-28603 andenalaprilat each at 30 pmol/kg,I.v.). This
`i repression and bradykinin potentiation were evaluated in
`suggests that bradykinin potentiation contributes to the pre-
`conscious cardiomyopathic hamsters with compensated heart
`load-reducing, but not the afterload-reducing, acute effects of
`failure, The Ang tl AT, receptor antagonist, SR 47436 (BMS-
`NEP-VACE-I. Hence, both Ang Il repression and bradykinin
`186295), was administered at 30 nmol/kg,i.v. followed by Lv.
`potentiation are factors contributing to the synergistic hemo-
`Infusion at 1 wzmolV/kg/min in combination with NEP-I (SQ-28603
`dynamic effects of combined NEP-! and ACE-I in hamsters with
`at 30 uwmol/kg I.v.). Cardiac preload (left ventricular end dia-
`heart failure. The bradykinin-mediated enhanced effect of com-
`stolic pressure) and afterload(left ventricular systolic pressure)
`bined NEP-I/ACE-I to reduce cardiac preload could improve
`decreased significantly more after the combination of Ang Il
`the beneficial effects of ACE-I in the treatment of heart failure.
`
`
`ACE-I blocks the formation of Ang II and hence attenuates
`its vasoconstrictor, antinatriuretic and growth enhancement
`properties. NEP-I prevents the enzymatic inactivation of
`ANP andtherefore protects or potentiates its vasodilatory,
`natriuretic and antiproliferative actions. Studies have shown
`that concurrent administration of NEP-I and ACE-I in mod-
`ela 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 effect of ACE-I in conscious spontaneously hyper-
`tensive rats was enhanced by coadministration of selective
`inhibitors of NEP (Seymour etal., 1991; Pham et ai., 1993).
`In dogs with pacing-induced heart failure, NEP-I potentiated
`the vasodilatory effects ofACE-I (Seymouret a/. 1993); and in
`8 similar model, subchronic treatment with ACE-I potenti-
`ated the renal hemodynamic and excretory responses to
`NEP-I (Margulies et al., 1991). In cardiomyopathic hamsters
`with heart failure, the combination of the ACE inhibitor,
`
`enalaprilat, and the selective NEP inhibitor, SQ-28603, pro-
`duced decreases in cardiac preload and afterload, whereas
`each treatment alone had minimal effects (Trippodoet al.,
`1993), These studies support the concept that the coadmin-
`istration of ACE-I and NEP-I leads to synergistic effects and
`could have use in the treatment of hypertension and heart
`failure.
`Possible mechanisms by which NEP-I and ACE-I interact
`to produce enhanced cardiovascular effects have been previ-
`ously discussed (Seymour et al., 1993, Trippodoef al., 1993).
`Attenuation of the formation of Ang II by ACE-I might un-
`mask the vasodilatory effecta of ANP and other natriuretic
`peptides such as BNP and CNP. 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 (Raya e? al., 1989; Margulies et al., 1991).
`Potentiation of the vasodilatory effects of bradykinin by both
`ACE-I and NEP-I might also contribute to the synergism.
`Although bradykinin can be potentiated by ACE-I alone,
`Received for publication May 24, 1994,
`greater enhancementof the activity of this peptide may come
`ieee
`ABBREVIATIONS:ACE-I, angiotensin convarting enzymeinhibition; AngIl, angiotensin Il; ANP, atrial natriuretic peptide; BNP, brain natriuretic
`peptide; CNP, C-type natriuretic peptide; HR, heart rate; LVEDP,left ventricular end diastolle pressure; LVSP,left ventricular systolic pressure;
`MAP, mean arteria) pressure; NEP-I, neutral endopeptidase inhibition.
`
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`Trippodo et al.
`
`Vol. 272
`
`about when two enzymesinvolved in its degradation, such as
`ACE and NEP,are inhibited. Finally, ACE and NEP have
`substrates in common other than bradykinin, such as sub-
`atance 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 combinedeffects of
`dual metalloprotease inhibition. The purpose of this study
`was to evaluate the potential contributions of AngII repres-
`sion and bradykinin potentiation on the cardiovascular ef-
`fects of combined NEP-I and ACE-I in cardiomyopathic ham-
`sters with heart failure. These factora 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 bradykinin B, receptor were
`available. Ang II AT, receptors mediate virtually all of the
`known biological actions of Ang II (Timmermans etal., 1993).
`Bradykinin B, receptors are likely reaponsible for many of
`the cardiovascular and renal effecta of bradykinin (Regoli e¢
`al., 1990). In this study we used the Ang II AT, receptor
`antagonist, SR 47436 (BMS-186295) (Cazaubonet al., 1993)
`and the bradykinin B, receptor antagonist, Hoe 140 (Wirth et
`al., 1991). The resulta 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
`ofNEP-I and ACE-I to reduce cardiac preload in cnndignnye
`pathic hamsters with heart failure.
`
`Methods
`
`All procedures involving animals were in accordance with the
`Bristol-Myers Squibb Pharmaceutica) Research Institute Animal
`Care and Use Committee.
`
`Animal preparation
`Cardiomyopathic male hamaters of the BIO TOQ-2 strain (Sole,
`1986) were obtained from Bio Breeders, Inc., Fitchburg, MA. The
`animals were housed two to five per cage in a light and darkcycle of
`14h and 10h,reapectively, for a minimum of 2 wk before study. The
`animals were maintained on Purina 5001 pelleted Chow (Purina,
`Richmond, IN) and tap water ad libitum; they were studied at
`approximately 260 days of age in a stage of nonedematous, compen-
`sated heart failure (Fox et al., 1993; Trippodo e¢ al., 1993; Panchal
`and Trippodo, 1993). All experiments were conducted in conscious,
`unrestrained, cardiomyopathic hamsters 3 h after placementofcath-
`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 methohexital sodium
`(70 mg/kg, i.p., Brevital, Eli Lilly & Co., Indianapolis, IN). Pelyeth-
`ylene catheters (PE10 connected to PESO) were filled with isatonic
`saline (0.9% NaCl) containing heparin (4 TU/m)). The PE10 ends of
`the catheters were used for cannulation. The right jugular vein was
`cannulated and supplemental doses of methohexital (1 mg/kg,i.v.)
`were administered as needed during surgery. The rightcarotid ar-
`tery wae 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.Thefree ends of the catheters
`were passed under the skin and exteriorized 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 tranaducer (Mode) P23XL,
`Gould Electronics, Valley View, OH) for measurement of MAP or
`LVEDPand LVSP. Cardiovascular pressures and HR were recorded
`
`in the conscious, free-moving hamster on a polygraph (Model 7D,
`Grass Instruments, Inc., Quincy, MA). MAP waa obtained by elec-
`tronically damping the arterial pulse pressure. LVEDP and LVSP
`were determined from theleft ventricular pulse tracing as described
`previously (Fox ef al, 1993; Trippodo et af, 1993; Panchal and Trip-
`podo, 1993), HR was obtained through a tachograph preamplifier
`(7P4H, Grass Instrumenta, Inc., Quincy, MA) as triggered by the
`arterial or ventricular pulse. The catheter in the jugular vein was
`used for the administration of agenta.
`
`inhibition of pressor and depressor responses
`Ang IJ pressor response. Preliminary experiments were con-
`ducted in conscious cardiomyopathic hamsters to determine a dove
`regimen of SR 47436 (BMS-186295) that would nearly completely
`block the pressor response to Ang II for at least 2 h. The pressor
`responses to two challenges of Ang II (100 ng/kg,i.v. dissolved in
`0.9% NaCl, 1 ml/kg) were determined. Thia dose of AngII produced
`more than a 30% increase in MAP. Based on thepreliminary exper-
`iments, SR 47436 (BMS-186295) was administered to five cardiom-
`yopathic hamatere at 30 pzmol/kg, iv. followed by continuous i.v.
`infusion at 1 pmol/kg/min. Challenges of Ang II were then repeated
`at 10-min to 30-minintervals up to 150 min after the bolus injection
`of SR 47436 (BMS-186296).
`Bradykinin depressor response. Becausei.v. bolus injections
`of bradykinin caused respiratory distress in the cardiomyopathic
`hamsters, depressor reaponsesto intraarterial administrationofthis
`peptide were studied. There were no changes in the behaviorof the
`animals to suggest pain or distress after the administration of bra-
`dykinin, i.a. In eight conscious cardiomyopathic hamstera the de-
`pressor response to 0.9% NaC} (1 ml/kg,i.a.) was determined. This
`was followed by two challenges of bradykinin (10 pg/kg,i.s., 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 effects.
`Based on preliminary experiments, Hoe 140 was administered at 100
`pa/kg, i.v, (dissolved in 0.9% NaCl, 1 ml/kg). Challenges of bradyki-
`nin were then repeated at 5-min to 30-min intervals up to 190 min.
`A secondinjection of 0.9% NaCl was administered at the end of the
`experiment.
`
`Cardiovascular effects
`
`In this series of experiments, baseline measurements of LVEDP,
`LVSP and HR were determined in groups of conscious cardiomyo-
`pathic hamsters. Compounds or vehicles were administered at 1
`ml/kg,i.v., unless indicated otherwise, and measurements were re-
`peated at 6-min to 30-min intervals up to 90 min after administra-
`tion of the last agent.
`SR 47436 (BMS-186295), SQ-28603 and the combination of
`these agents. SR 47436 (BMS-186295) was administered at 30
`pmolkg, iv. (0.3 ml) followed by a continuous i.v. infusion at 1
`pmol/kg/min (0.01 ml/min). SR 47436 (BMS- 186295) waa prepared in
`0.028 M KOH anddiluted to a final concentration of 0.017 M KOH.
`KOHeolution (0.017 M) was administeredi.v. to the vehicle group at
`0.3 ml followed by a continuous infusion at 0.01 ml/min. SQ-28603
`was dissolved in 0.84% NaHCO,and administered at 30 pmol/kg,i.v.
`Thia dose of SQ-28603 waa previously shown to result in a doubling
`of plasma ANP concentration within 90 min in this model (Trippodo,
`et al,, 1993). The vehicle for SQ-28603 was previously shown to have
`onjy minimal cardiovascular effects in cardiomyopathic hamaters
`(Trippodoet al., 1993); similar minimal effects were observed in thia
`atudy (see below). One group of cardiomyopathic hamsters received
`the combination of SR 47436 (BMS-186295) and SQ-28603.In this
`group, SR 47436 (BMS-186295) was administered according to the
`same dosage regimen described above; 30 min after the bolus injec-
`tion of SR 47436 (BMS-186295), SQ-28603 was adminietered at 30
`pmolkg,i.v.
`
`BIOCON PHARMA LTD (IPR2020-01263) Ex. 1003, p. 002
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`Combination of enalaprilat and 8Q-28603 and the effects of
`HOE140 on this combination. Hoe 140 was administered at 100
`pg/kg,i.v. (dissolved in 0,9% NaCl). Enalaprilat and SQ-28603 were
`each dissolved in 0.84% NaHCO,and were administered at 30 zmol/
`kg, i.v. 30 min apart (enalaprilatfirst). One group receivedall three
`of these agenta at the doses indicated above in the following se-
`quence: Hoe 140, followed 5 minlater by enalaprilat, followed 30 min
`by SQ-28603. The vehicle group in this aet of experiments received
`0.9% NaCl, followed 5 min later by 0.84% NaHCQ,,followed 30 min
`later by a second injection of 0.84% NaHCQO,.
`Effects of Hoe 140 on the combination of SR 47436 (BMS-
`186295) and SQ-28803, One group of cardiomyopathic hamsters
`was administered Hoe 140, 6 min later SR 47436 (BMS-186295) and
`30 min later SQ-28603. The vehicles and doses of the compounds
`were the sameas indicated above.
`
`Statistical analyses
`Differences in age, body weight and baseline values among groups
`were evaluated by analysis of variance. Differences 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 waa
`taken at P < .05, All data are expressed as means + S.E.M.
`
`AngIJ and bradykinin were purchased from Sigma Chemical Co.
`(St. Louis, MO); Hoe 140 was purchased from Peninsula Laborato-
`ries (Belmont, CA); enalaprilat waa supplied by Merck Sharp &
`Dohme Research Laboratories (West Point, PA); SQ-28603 was syn-
`thesized by Bristol-Myers Squibb Pharmaceutical Research Institute
`(Princeton, NJ) and SR 47436 (BMS-186295) was synthesized by
`Sanofi Recherche (Montpellier, France).
`
`’ Results
`
`Inhibition of pressor and depressor responses
`Ang II pressor response. The pressor responses to du-
`plicate injections of Ang II were 29 + 2 mm Hg and 31 + 3
`mun Hg(fig. 1). After the administration of SR 47436 (BMS-
`186295), the pressor responses to Ang II at intervals up
`to 150 min were less than 6 mm Hg.Theseresults indicate
`that nearly complete inhibition of the pressor response to
`
`Mechanisms of NEP/ACE Inhibition
`
`621
`
`AngII was achieved with this dosage regimen of SR 47436
`(BMS-186295).
`Bradykinin depressor response. The depressor re-
`aponses to intraarterial injection of 0.9% NaCl alone at the
`beginning and endof the experiment were -9 + 1 mm Hg and
`-10 + 1mm Hg,respectively (fig. 2), The depressor responses
`to duplicate i.a. injections of bradykinin in 0.9% NaCl were
`-21 + 1 mm Hgand-20 + 1 mm Hg, These resulte indicate
`that the depressor response to bradykinin alone (minus the
`vehicle effect) was approximately 10 mm Hg.After the ad-
`ministration of Hoe 140, the depressor responsesto i.a. in-
`jections of bradykinin in 0.9% NaCl did not exceed -11 mm
`Hgfor intervals up to 180 min. These results indicate that
`after 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 effects
`
`SR 47436 (BMS-186295), SQ-28603 and the combina-
`tion of these agents. Age, body weight and baseline values
`were similar (P > .05) amongthe four groups of cardiomyo-
`pathic hamsters in this series of experiments. These values
`(means + S.E.M.) were as follows in the vehicle (n = 10),
`SQ-28603 (n = 6), BMS-186295 (n = 9) and BMS-186295 +
`S$Q-28603 (n = 7) groups, respectively: age (days), 256 + 2,
`261 + 4, 258 + 1, 261 + 2; b.wt. (g), 116 + 2,118 + 3,117 +
`2 and 117 + 3; LVEDP (mm Hg), 19 + 2,18 + 3,17 + 2 and
`21 + 2; LVSP (mm Hg), 111 + 3, 117 + 5, 112 + 2 and 107
`+ 3; HR (beats/min), 350 + 10, 378 + 12, 338 + 16 and 364
`+ 6. The changes in LVEDP and LVSPafter the administra-
`tion of SR 47436 (BMS-186295) were not significantly differ-
`ent from those in the vehicle (0.017 M KOH) group(fig. 3).
`After the administration of SQ-28603 alone, LVEDP de-
`creased only slightly (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 after the administration of SQ-28603
`alone; the decreases at two time pointa were significantly
`
`Depressor Response to Bredykinin or Saline,|.e.
`tn Conscious Cardiomyopsthic Hamsters (n = 8)
`
`Hoe 140, 100pov/kg, Lv.
`
`
`Saline
`[LJ Bradykinin in satine
`
`
`S BSBHS §
`
`15 30 45 60 80 120150180 S
`
`0
`
`_ 3s
`
`“10
`
`a
`25
`
`£E
`
`a3
`
`-15g
`
`Minutes
`'
`Fig. 2. Changes in MAPin response to bradykinin, 10 wg/kg,ia. or
`saline (0.9% NaC), 1 mi/kg, |.a. before and after the administration of
`Hoe 140 In conscious cardiomyopathic hamsters (age 259 + 2 days;
`b.wt. 112 + 2 9).
`
`Preasor Response to Ang th, Iv. In
`Conaclous Cardiomyopathic Hamatere (n = 5)
`
`SR 47436 (BMS-186295)
`
`30 pmot/kg, Lv. + 1 pmol/kg/min, iv, AMAP,mmHg
`
`-20-1010 20 30 40 50 60 70 80 90 120150
`Minutes
`
`Fig. 1. Changes in MAP In response to AngII, 100 ng/kg,i.v. before
`and after the administration of SR 47436 (BMS-186295) in conscious
`cardiomyopathic hamsters (age 263 + 5 days; b.wt. 124 + 6 g).
`
`BIOCON PHARMA LTD (IPR2020-01263) Ex. 1003, p. 003
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`Trippodoet al.
`
`————-—=- Vehicle
`5028603
`
`——o— SA 47436
`(BMS-186295)
`
`Vol. 272
`
`603
`
`05
`
`E —
`
`2; b.wt. (g), 115 + 2, 115 + 3, 116 + 1 and 116 + 2; LVEDP
`(mm Hg), 17 + 2,19 + 3, 24 + 2 and 20 + 2; LVSP (mm Hg),
`108 + 4, 103 + 4, 105 + 1 and 106 + 5; HR (beats/min), 382
`+ 14, 359 + 9, 368 + 11 and 350 + 14. The changes in
`LVEDPand LVSPafter the administration of vehicle (0.84%
`NaHCO,) and Hoe 140 alone were minimal (fig. 4). After the
`administration of the combination of enalaprilat and SQ-
`28603, LVEDPdecreased by approximately 16 mm Hgfor 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 enala-
`prilat and SQ-28603 resulted in a significantly smaller de-
`crease (approximately 5 mm Hg) in LVEDP as compared
`with the combination of enalaprilat 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 SQ-28603
`groups were observed at some time points in the animals
`receiving Hoe 140 plus the combination of enalaprilat and
`SQ-28603.
`Effects of Hoe 140 on the combination of SR 47436
`(BMS-186295) and SQ-2860S, Age (258 + 1 days), b.wt.
`
`Vehicle
`—o— +E+603
`~o-we- Hoe 140
`——e— Hoe 140+ E+ 603
`10 HE
`x 5
`E
`5= -10
`ad
`a 618
`-20
`45 -30
`
`—— 295 +609 : 5
`
`o.°8 2
`
`* P < 0.05 vs Veh
`
`+ © < 0.05 vs SO-28603
`
`-15
`
`0
`
`15
`
`30
`
`45
`
`60
`
`75
`
`90
`
`“15
`
`0
`
`390
`15
`Minutes
`
`45
`
`60
`
`75
`
`90
`
`Fig. 3. Cardiovascular changes in conscious cardiomyopathic ham-
`sters after the administration of vehicle (0.017 M KOH), SQ-28603, SA
`47436 (BMS-186295) or the combination of SQ-28603 and SR 47436
`(BMS-186295). The group receiving the combination treatment Is des-
`ignated as 295 + 603. SQ-28603 was administered as an iv. bolus at
`30 pmol/kg. SR 47436 (BMS-186295) was administered at 30 pmol/kg,
`iv. followed by a continuous Lv. infusion at 1 pmol/kg/min. {n the
`combination treatment group, SQ-28603 was administered 30 min after
`the bolus infection and start of infusion of SR 47436 (BMS- 186295).
`
`greater than the changesin the vehicle group. The combina-
`tion of SQ-28603 and SR 47436 (BMS-186295) produced de-
`creases in LVEDP and LVSPthatweresignificantly 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,
`LVEDP was decreased by 11 + 3 mm Hgfrom a baseline of
`21 + 2 mm Hg; LVSP was decreased by 18 + 4 mm Hgfrom
`@ baseline of 107 + 3 mm Hg. HR 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 (BMS-186295).
`Combination of enalaprilat and SQ-28603 and the
`effects of Hoe 140 on thie combination. Age, body weight
`and baseline values were similar (P > .05) amongthe four
`groups of cardiomyopathic hamstersin this series of experi-
`ments. These values (means + S.E.M.) were as follows in the
`vehicle (n = 9), Hoe 140 (n = 6), enalaprilat + SQ-28603 (n
`= 6), Hoe 140 + enalaprilat + SQ-28603 (n = 10) groups,
`respectively: age (days), 259 + 2, 262 + 2,260 + 1 and 258 +
`
`
`8
`
`SLVSP(mmHg)
` SHR(beats/min)SseoR3egs
`
`-15
`
`Oo
`
`15
`
`30
`
`45
`
`60
`
`75
`
`90
`
`-
`
`10
`
`° P< 0.05 vs Veh
`t © < 0,05 vs E + 603
`
`-45
`
`-30
`
`15
`
`20
`
`30
`15
`Minutes
`
`45
`
`60
`
`75
`
`90
`
`Fig. 4. Cardiovascular changes in conscious cardiomyopathic ham-
`sters after the administration of vehicle (0.849% NaHCO,), Hoe 140 (H,
`100 pmot/kg, |.v.), the combination of enalaprilat (E, 30 pmol/kg,|v.)
`and SOQ-28603 (603, 30 zmol/kg,i.v.), or the combination of Hoe 140
`plus enalaprilat and SQ-26603 (same doses as above). Arrowsindicate
`times of administration of compounds.
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`

`(119 + 3 g) and baseline values for LVEDP (26 + 2 mm Hg),
`LVSP (100 + 2 mm Hg) and HR (347 + 8 beats/min) in the
`group of cardiomyopathic hamsters receiving Hoe 140 plus
`the combination of SR 47436 (BMS-186295) and SQ-28603
`were similar (P > .05) to those receiving this combination
`treatment without Hoe 140. The changes in LVEDP, LVSP
`and HRefter the administration of Hoe 140 plus the combi-
`nation of SR 47436 (BMS-186295) and SQ-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 47436 (BMS-186295) +
`SQ-28603. Because different vehicles had to be used in the
`two series of experiments using the ACEinhibitor, enalapri-
`lat or the Ang II antagonist, SR 47436 (BMS-186295), 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
`effects” in such a comparison, the average change observed at
`
`Mechanisms of NEP/ACEInhibition
`
`623
`
`each time point during the administration of 0.017 M KOH
`was subtracted from the changes in each animalreceiving
`the combination of SR 47436 (BMS-186295) and SQ-28603.
`Similarly, the average change observed after the administra-
`tion of 0.84% NaHCO, 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
`effects) in LVSP were similar (P > .05 at all time points) in
`the two groups. However, the changes (minus vehicle effects)
`in LVEDP weregreater (P < .05 at two time points) in the
`animals receiving enalaprilat plus SQ-28603 as compared
`with the animals receiving SR 47436 (BMS-186295) plus
`SQ-28603. 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
`
`—e— 295 +603
`——e-— Hoe 140+ 295 + 603
`
`Thefindings demonstrated that in hamsters with compen-
`sated heart failure, Ang II receptor blockade acted synergis-
`
`—e— £2803
`10
`—eo—+=295-603
`603
`v
`
`OLVSP(STAT-4Veh)
`
`(mmig) 5 30-15
`—e— E+603 ALVEDP
`(aTAT-Veh)
`
`20
`
`0
`
`1§
`
`30
`
`4s
`
`60
`
`+75
`
`90
`
`“30°
`
`15
`
`oO
`
`1§
`
`30
`
`45
`
`60
`
`75
`
`90
`
`-15
`
`0
`
`1§
`
`30
`
`45
`
`—————
`66
`75
`90
`
`-15
`
`0
`
`15
`
`30
`
`45
`
`60
`
`75
`
`90
`
`40 en
`45-30 -1§
`GO
`15
`30
`45
`60
`75
`90
`
`0
`
`——e—~ Hoe 140+E +603
`—e-— Hoo 140 + 295 » 603
`
`:
`
`=
`3
`‘50
`ezEE
`JE -10
`
`aS
`
`2
`
`20
`30
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`
`30
`15
`Minutes
`
`45
`
`60
`
`75
`
`90
`
`o Y
`
`Fig. 5. Cardiovascular changes in conscious cardiomyopathic ham-
`sters after the administration of the combination of SQ-28603 and SR
`47436 (BMS-186295) or the combination of Hoe 140 plus SR 47436
`(BMS-186295) and SQ-28603, The results from the combination of
`SQ-28603 and SR 47436 (BMS-186295) are the same as those shown
`In figure 3. SQ-28603 (603) was administered as an |.v. bolus at 30
`pmol/kg. SR 47436 (BMS-186295) (295) was administered at 30 pmol/
`kg,iv, followed by a continuousI.v. infusion at 1 wmol/kg/min. Hoe 140
`(H) was administered as an |.v. bolus at 100 yu9/kg. Arrowsindicate
`times of administration of compounds.
`
`Fig. 8 Cardiovascular changes (minus vehicle effects) In conscious
`cardiomyopathic hamsters after the administration of the combination
`of enalaprilat (E, 30 pzmol/kg,I.v.) and SQ-28603 (603, 30 zmol/kg, I.v.)
`or the combination of SQ-28603 (603, 30 pmol/kg, I.v.) and SR 47436
`(BMS-186295) (295, 30 pmolkg, Lv. + 1 pmol/kg/min,
`i.v.). The
`changes were determined from the results shown In figures 3 and 4.
`The bottom graph showsthe changes (minus vehicle effects) in LVEDP
`in conscious cardiomyopathic 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
`
`

`

`Trippodo et al.
`
`Val. 272
`
`tically with NEP-I to reduce cardiac preload and afterload.
`This interaction was similar to that previously observed with
`concurrent ACE-I and NEP-I in this model and suggests that
`repression ofAng Iis an important factor contributing to the
`acute synergiatic effects of dual inhibition of ACE and NEP.
`The resulta also showed that blockade of bradykinin B, 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 NEP-I and ACE-I
`elicited cardiovascular effects greater than those caused by
`either treatment alone (Seymour et al., 1991; Margulies et
`al., 1991; Seymour et al., 1993; Pham etal., 1993; Trippodoet
`al., 1993). This interaction of dual metalloprotease inhibition
`might be partly due to the blockadeofthe formation ofAngII
`acting together with the enhancementof several vasodilator
`factors, such as the natriuretic peptides (ANP, BNP and
`CNP), bradykinin and substance P. We explored the poten-
`tial role of Ang II repression in this interaction by using the
`Ang II AT, receptor antagonist, SR 47436 (BMS-186295)
`(Cazaubonet al., 1993) in a model of heart failure previously
`characterized in our laboratory (Foxet al., 1993; Trippodo e¢
`al., 1993; Panchal and Trippodo, 1993). After i.v. administra-
`tion of SR 47436 (BMS-186295) at 30 pmol/kg followed by
`continuous i.v. infusion at 1 »mol/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 IJ antagonist was used in subsequent
`studies in combination with the NEPinhibitor, SQ-28603 at
`30 p»mol/kg,i.v., a dose previously shown to have minimal
`acute cardiovasculareffects in this model, but found to have
`synergistic effects with the ACE inhibitor, enalaprilat (Trip-
`podo ef al., 1993). In the present study, Ang II antagonism
`alone had minimal acute cardiovascular effects in the con-
`scious cardiomyopathic hamsters, which is consistent with
`the minimal acute effecta of ACE-I in this model. Also as
`previously reported, NEP-I alone had only small effects.
`However, the combination of Ang IT blockade and NEP-I
`produced significant reductions in LVEDP and LVSPthat
`were greater than those produced by either treatmentalone.
`These synergistic effects were similar to those observed pre-
`viously with the combination of ACE-I and NEP-I. Together,
`the reaults suggest that attenuation of the formation of Ang
`II is an important factor contributing to the interaction of
`concurrent ACE inhibition and NEPinhibition in cardiom-
`yopathic hamsters.
`Therole of bradykinin potentiation in this interaction waa
`also investigated, because bradykinin could be protected by
`inhibition of both ACE and NEP (Ura et al., 1987; Erdis,
`1990; Skidgel, 1992). Firat it was established that a bolus i.v.
`injection of 100 g/kg of the bradykinin B, receptor antago-
`nist Hoe 140 (Wirth et al., 1991) completely blocked the
`depressor response to bradykinin for at least 3 h in conscious
`cardiomyopathic hamsters. This dose of Hoe 140 was usedto
`determine whether bradykinin blockade altered the cardio-
`vascular response to dual metalloprotease inhibition. The
`combination of enalaprilat (30 zmol/kg, iv.) and SQ-28603
`(30 pmol/kg, i.v.) lowered LVEDP by 16 + 2 mm Hg and
`decreased LVSP by 18 + 4 mm Hgwithin 60 to 90 min in
`cardiomyopathic hamaters. These changes were similar to
`
`those previously reported (Trippodoe¢ al., 1993). However,
`when the ACE and NEPinhibitors were administered after
`Hoe 140, LVEDP decreased by only 6 + 2 mm Hg (Ago min):
`Blockade of bradykinin receptors had oo such blunting effect
`on reduction in afterload by dual metalloprotease inhibition
`(ALVSPoo min = -24 + 3 mm Hg). These results indicate that
`potentiation of bradykinin is an important mediator of the
`acute preload-reducing effect of NEP-I/ACE-I in hamaters
`with heart failure.
`Hoe 140 did not alter the preload and afterload responses
`to the combination of SQ-28603 and SR 47436 (BMS-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-I 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-I did not affect
`the response (Salgadoet al., 1992). However, although NEP-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
`bradykinin by a particulate fraction of rabbit endothelial
`cells of venous origin (Llorens-Cortes et al., 1992). Therefore,
`the bradykinin potentiating effects on preload reduction ob-
`served with dual NEP-V/ACE-I in the cardiomyopathic ham-
`sters could have been due to ACE-I alone or the combined
`effects of ACE-I and NEP-I.
`It is interesting that bradykinin potentiation contributed
`to the decrease in preload but not to the decrease in afterload
`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 shift 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.
`Venodilation. ACE-I was shown to cause venodilation in
`man (Capewell et al., 1989) and animals (Raya et al., 1989)
`with heart failure; this has several potential mechanisms,
`including decreased Ang II, decreased sympathetic nerve
`activity and increased kinins, prostaglandins and vasopres-
`sin (Capewell e¢ al., 1989). NEP-I may have a vasorelaxant
`effect on veins by potentiation of the natriuretic peptides.
`Although ANP alone is unlikely to cause venodilation (Trip-
`podo et al., 1986; Trippodo and Barbee, 1987; Ford et ai.