(1 9)
`
`Q j l )
`
`Europaisches Patentamt
`European Patent Office
`Office euroDeen des brevets
`
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`(11)
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`E P 0 7 2 6 0 7 2 A 2
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`(12)
`
`EUROPEAN PATENT APPLICATION
`
`(43) Date of publication:
`14.08.1996 Bulletin 1996/33
`
`(21) Application number: 96101756.3
`
`(22) Date of filing: 07.02.1996
`
`(84) Designated Contracting States:
`AT BE CH DE DK ES FR GB GR IE IT LI LU MC NL
`PT SE
`
`(30) Priority: 08.02.1995 US 386366
`
`(71 ) Applicant: BRISTOL-MYERS SQUIBB COMPANY
`Princeton, NJ 08543-4000 (US)
`
`(51) |nt. CI.6: A61 K 31/415, A61 K 45/06
`
`(72) Inventors:
`• Powell, James R.
`Washington Crossing, PA 18977 (US)
`• Seymour, Andrea Ann
`Lambertville, NJ 08530 (US)
`• Trippodo, Nick C.
`Newtown PA 18940 (US)
`
`(74) Representative: Josif, Albert, Dr.-lng. et al
`Baaderstrasse 3
`80469 Munchen (DE)
`
`(54)
`
`Composition for the treatment of hypertension and congestive heart failure, containing an
`angiotensin II antagonist and an endopeptidase inhibitor
`
`(57)Hypertension and/or congestive heart failure are
`treated with the combination of the angiotensin II antag-
`onist 2-butyl-6,7,8,9-tetrahydro-3-[[2'-(1 H-tetrazol-5-
`yl)[1 , 1 '-biphenyl]-4-yl]methyl]-1 ,3-diazaspiro[4.4]nonan-
`4-one and a selective neutral endopeptidase inhibitor or
`a dual acting neutral endopeptidase inhibitor.
`
`CM
`<
`CM
`o
`CO
`CM
`
`o
`Q_
`LU
`
`Printed by Rank Xerox (UK) Business Services
`2.13.2/3.4
`
`BIOCON PHARMA LTD (IPR2020-01263) Ex. 1002, p. 001
`
`

`

`Description
`
`EP 0 726 072 A2
`
`5
`
`Darrow et al. in European Patent Application 498,361 disclose treating hypertension or congestive heart failure with
`a combination of an angiotensin II antagonist or a renin inhibitor with a neutral endopeptidase inhibitor.
`Matsumoto et al., JASN, September 1993, disclose that the combined therapy of an angiotensin II blocker,
`DUP753, and a neutral endopeptidase inhibitor, candoxatril, may be useful in the treatment of congestive heart failure
`and renal failure.
`Bernhart et al. in United States Patent 5,270,317 disclose a series of N-substituted heterocyclic derivatives which
`possess angiotensin II antagonist activity. Bernhart et al. disclose that such compounds can be used in the treatment
`10 of various cardiovascular complaints, especially hypertension, heart failure, and venous insufficiency, as well as in the
`treatment of glaucoma, diabetic retinopathy and various complaints of the central nervous system. It is also disclosed
`that such compound can be used in combination with other active agents such as tranquilizers, beta-blocking com-
`pounds, a calcium antagonist, or a diuretic.
`Selective neural endopeptidase inhibitors are taught by Delaney et al. in United States Patents 4,722,810 and
`15 5,223,51 6 and the use of selective neutral endopeptidase inhibitors alone or in combination with angiotensin converting
`enzyme inhibitors to treat hypertension are disclosed by Delaney et al. U.K. Patent Application 2,207,351 and by
`Haslanger et al. in United States Patent 4,749,688. The treatment of congestive heart failure by administration of a com-
`bination of a selective neutral endopeptidase inhibitor and an angiotensin converting enzyme inhibitor is disclosed by
`Seymour in United States Patent 5,225,401 .
`Compounds possessing both neutral endopeptidase and angiotensin converting enzyme inhibition activity are dis-
`closed by Flynn et al. in United States Patent 5,366,973, European Patent Application 481 ,522 and PCT Patent Appli-
`cations WO 93/16103, and WO 94/10193, Warshawsky et al. European Patent Applications 534,363, 534,396 and
`534,492, Fournie-Zaluski European Patent Application 524,553, Karanewsky et al. European Patent Application
`599,444, Karanewsky European Patent Application 595,610, Robl et al., European Patent Application 629,627, Robl
`25 United States Patent 5,362,727 and European Patent Application 657,453.
`This invention is directed to the discovery that the angiotensin II antagonist 2-butyl-6,7,8,9-tetrahydro-3-[[2'-(1H-
`tetrazol-5-yl)[1,1'-biphenyl]-4 -yl]methyl]-1,3-diazaspiro[4.4]nonan-4-one acts synergistically with a selective neutral
`endopeptidase inhibitor or a dual acting neutral endopeptidase inhibitor as defined below to reduce cardiac preload and
`afterload and enhance natriureses. The combination of this angiotensin II antagonist and the selective or dual acting
`30 neutral endopeptidase inhibitor produced significant reductions in left ventricular end diastolic pressure (LVEDP) and
`left ventricular systolic pressure (LVSP) that were greater than those produced by either treatment alone. Thus, the
`combination of this particular angiotensin II antagonist and the selective or dual acting neutral endopeptidase inhibitor
`is useful in treating hypertension and/or congestive heart failure.
`The angiotensin II antagonist employed within this invention is the compound 2-butyl-6,7,8,9-tetrahydro-3-[[2'-(1H-
`tetrazol-5-yl)[1 ,1 '-biphenyl]-4 -yl]methyl]-1 ,3-diazaspiro[4.4]nonan-4-one having the structural formula
`
`20
`
`35
`
`( I )
`
`40
`
`45
`
`50
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`55
`
`2
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`BIOCON PHARMA LTD (IPR2020-01263) Ex. 1002, p. 002
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`

`

`EP 0 726 072 A2
`
`known in the literature as SR47436, BMS 186295, or irbesartan and pharmaceutical^ acceptable salts thereof such as
`the potassium and sodium salts. These angiotensin II antagonists and their method of preparation are disclosed by
`Bernhart et al. in United States Patent 5,270,317.
`The selective neutral endopeptiadase inhibitor for use within this invention are those of the formula
`
`( I D
`
`f
`HS — CH2 — CH
`
`i
`
`f
`
`
`r
`C — NH-CH-(CH2)n — C
`
`Rx
`
`and pharmaceutical^ acceptable salts thereof wherein:
`R2 is alkyl of 1 to 7 carbons, trifluoromethyl, phenyl, substituted phenyl, -(CH2)i t04-phenyl, or -(CH2)i ^-sub-
`stituted phenyl;
`R3 is hydrogen, alkyl of 1 to 7 carbons, phenyl, substituted phenyl, -(CH2)i t0 4-phenyl, or -(CH2)i t0 4-substituted
`phenyl;
`R1 is hydroxy, alkoxy of 1 to 7 carbons, or NH2;
`n is an integer from 1 to 15; and
`the term substituted phenyl refers to a substituent selected from lower alkyl of 1 to 4 carbons, lower alkoxy of 1
`to 4 carbons, lower alkylthio of 1 to 4 carbons, hydroxy, CI, Br, or F.
`Preferred are the selective neutral endopeptidase inhibitors of formula II wherein:
`R2 is benzyl;
`R3 is hydrogen;
`n is an integer from 1 to 9; and
`R1 is hydroxy.
`Most preferred for use in this invention is the selective neutral endopeptidase inhibitor of formula II reported in the
`literature as SQ 28,603 which is the compound of formula II wherein:
`R2 is benzyl;
`R3 is hydrogen;
`n is one; and
`R1 is hydroxy.
`The preparation of the selective neutral endopeptidase inhibitors of formula II wherein R2 is other than trifluorome-
`thyl are disclosed by Delaney et al. in United States Patent 4,722,810. The preparation of the selective neutral
`endopeptidase inhibitors of formula II wherein R2 is trifluoromethyl are disclosed by Delaney et al in United States Pat-
`ent 5,223,516.
`Dual acting neutral endopeptidase inhibitors suitable for use within this invention are compounds which possess
`both neutral endopeptidase inhibiting activity and angiotensin converting enzyme inhibiting activity. Particularly useful
`are the dual acting inhibitors of the formula
`
`and pharmaceutical^ acceptable salts thereof wherein:
`p is one or two;
`X is O or S;
`m is zero or one;
`Y is CH2, S or O provided that Y is S or O only when m is one;
`R4 is hydrogen, alkyl of 1 to 7 carbons, phenyl, substituted phenyl, -(CH2)i t0 4-phenyl, -(CH2)i t0 4-substituted
`phenyl, cycloalkyl of 3 to 7 carbons, -(CH2)i t0 4-cycloalkyl of 3 to 7 carbons, heteroaryl, and -(CH2)i t0 4-heteroaryl;
`R5 is hydrogen, alkyl of 1 to 7 carbons, -(CH2)i to4-phenyl and -(CH2)i t0 4-substituted phenyl;
`the term substituted phenyl refers to a substituent selected from lower alkyl of 1 to 4 carbons, lower alkoxy of 1
`to 4 carbons, lower alkylthio of 1 to 4 carbons, hydroxy, CI, Br, or F; and
`
`3
`
`10
`
`15
`
`20
`
`25
`
`30
`
`35
`
`45
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`50
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`55
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`BIOCON PHARMA LTD (IPR2020-01263) Ex. 1002, p. 003
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`

`

`EP 0 726 072 A2
`
`the term heteroaryl refers to monocyclic rings of 5 or 6 atoms containing one or two O and S atoms and/or one
`to four N atoms provided that the total number of heteroatoms in the ring is 4 or less and bicyclic rings wherein the 5 or
`6 membered heteroaryl ring as defined above is fused to a benzene or pyridyl ring.
`Preferred are the dual acting neutral endopeptidase inhibitors of formula III wherein:
`R4 is benzyl, cyclopropylmethyl, or straight or branched chain alkyl of 3 to 5 carbons;
`p is one or two;
`X is O or S;
`m is zero or one;
`Y is CH2, S, or O provided that Y is S or O when m is one; and
`R5 is hydrogen.
`Most preferred for use in this invention is the dual acting neutral endopeptidase inhibitor of formula III wherein:
`R4 is benzyl;
`p is two;
`YisS;
`m is one;
`Y is CH2 ; and
`R5 is hydrogen.
`The dual acting neutral endopeptidase inhibitors of formula III are disclosed in European Patent Application
`629,627 of Robl etal.
`Also useful as neutral endopeptidase inhibitors for use within this invention are the dual acting inhibitors of the for-
`mula
`
`(IV)
`
`O
`II
`HS — CH-C — A
`I
`R4
`
`and pharmaceutical^ acceptable salts thereof wherein:
`
`BIOCON PHARMA LTD (IPR2020-01263) Ex. 1002, p. 004
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`

`

`is
`
`EP 0 726 072 A2
`
`, o r
`
`I
`H
`
`o
`
`COOR5
`
`R4, R5, and p are as defined above;
`R7 and R8 are both hydrogen, or both alkyl of 1 to 7 carbons, or R7 is hydrogen and R8 is alkyl of 1 to 7
`carbons, phenyl, -(CH2)i t0 4-phenyl and -(CH2)i t0 4-substituted phenyl, or R7 and R8 taken together with the car-
`bon to which they are attached complete a cycloalkyl of 3 to 5 carbons.
`R9 is hydrogen or alkyl of 1 to 7 carbons.
`Z is oxo or two hydrogens.
`
`Preferred are the dual acting neutral endopeptidase inhibitors of formula IV wherein:
`R4 is benzyl;
`R7 and R8 are both methyl;
`R9 is hydrogen or methyl, especially hydrogen;
`
`BIOCON PHARMA LTD (IPR2020-01263) Ex. 1002, p. 005
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`

`

`EP 0 726 072 A2
`
`5
`
`10
`
`p is one or two; and
`Z is oxo.
`The compounds of formula IV and their method of preparation are disclosed in European Patent Application
`599,444 and U.S. Patent Application Serial No. 160,540 filed December 1, 1993.
`The angiotensin II antagonist 2-butyl-6,7,8,9-tetrahydro-3-[[2'-(1H-tetrazol-5-yl)[1,1'-biphenyl]-4-yl]methyl-1,3-dia-
`zaspiro[4.4]nonan-4-one and the selective neutral endopeptidase inhibitor or dual acting neutral endopeptidase inhibi-
`tor may be administered from a single dosage form containing both types of compounds, may be administered in
`separate dosage forms taken at the same time, or may be administered separately on a carefully coordinated schedule.
`If administered separately, the two compounds can be administered from within several minutes of each other up to
`about 4 hours apart.
`The selective or dual acting neutral endopeptidase inhibitor can be administered at a dosage range of from about
`0.03 to about 1000 mg. per kg. of body weight per day with a dosage range of from about 0.3 to about 300 mg. per kg.
`of body weight per day being preferred. The angiotensin II antagonist can be administered at a dosage range of from
`about 0.001 to about 50 mg. per kg. of body weight with a dosage range of from about 0.1 to about 10 mg. per kg. of
`15 body weight being preferred.
`Both compounds can be administered orally, parenterally, or one orally and the other parenterally. Each compound
`may be administered from one to about four times per day depending upon the duration of activity of the compounds
`and the severity of the congestive heart failure and/or hypertension being treated.
`The compounds can be formulated, in the amounts described above, according to accepted pharmaceutical prac-
`tice with a physiologically acceptable vehicle, carrier, excipient, binder, preservative, stabilizer, flavor, etc., in the partic-
`ular type of unit dosage form.
`Illustrative of the adjuvents which may be incorporated in tablets are the following: a binder such as gum traga-
`canth, acacia, corn starch or gelatin; an excipient such as dicalcium phosphate or cellulose; a disintegrating agent such
`as corn starch, potato starch, alginic acid of the like; a lubricant such as stearic acid or magnesium stearate; a sweet-
`ening agent such as sucrose, aspartame, lactose or saccharin; a flavoring agent such as orange, peppermint, oil of win-
`tergreen or cherry. When the dosage unit form is a capsule, it may contain in addition to materials of the above type a
`liquid carrier such as a fatty oil. Various other materials may be present as coatings or to otherwise modify the physical
`form of the dosage unit. For instance, tablets or capsules may be coated with shellac, sugar or both. A syrup of elixir
`may contain the active compound, water, alcohol or the like as the carrier, glycerol as stabilizer, sucrose as sweetening
`agent, methyl and propyl parabens as preservatives, a dye and a flavoring such as cherry or orange.
`In the following examples, BMS 186295 refers to SR47436, i.e. the compound 2-butyl-6,7,8,9-tetrahydro-3-[[2'-(1 H-
`tetrazol-5-yl)[1 ,1 '-biphenyl]-4-yl]-methyl]-1 ,3-diazaspiro[4.4]nonan-4-one, and SQ 28603 refers to the compound (±)-N-
`[2-(mercaptomethyl)-1-oxo-3-phenylpropyl]-p-alanine.
`
`20
`
`25
`
`30
`
`35 Example 1
`
`40
`
`45
`
`The studies described in this experiment were conducted in male hamsters of the BIO TO-2 strain when they were
`approximately 260 days of age and weighed on average 1 15 g. These animals develop a genetic form of cardiomyop-
`athy that progresses uniformly among animals through different stages of heart failure. By 240 - 300 days of age the
`cardiomyopathic hamsters are characterized (as compared with control hamsters) by low mean arterial pressure, a
`40% reduction in cardiac output and a decrease in renal blood flow. They display elevated cardiac filling pressure,
`depressed ventricular function, increased peripheral vascular resistance and have an 8-10-fold increase in plasma
`atrial natriuretic peptide concentration. Since most animals at this age do not have gross peripheral edema or elevated
`plasma renin activity, the cardiomyopathic hamsters were considered to be in compensated heart failure.
`The experiments were conducted in conscious, unrestrained, cardiomyphathic hamsters three hours after place-
`ment of cardivascular catheters using brief anesthesia. The catheters allowed measurement of mean arterial pressure,
`left ventricular end diastolic pressure, left ventricular systolic pressure and heart rate, and provided a means for the
`administration of agents intravenously.
`
`so
`
`a) Inhibition of The Pressor Response To Angiotensin II
`
`Preliminary experiments were conducted in conscious cardiomyopathic hamsters to determine a dose regimen of
`BMS 186295 that would nearly completely block the pressor response to angiotensin II for at least two hours. The pres-
`sor responses to two challenges of angiotensin II (100 ng/kg, i.v. dissolved in 0.9% sodium chloride, 1 ml/kg) were
`55 determined. This dose of angiotensin II produced over a 30% increase in mean arterial pressure. Based on the prelim-
`inary experiments, BMS 186295 was administered to 5 cardiomyopathic hamsters at 30 nmol/kg, i.v. followed by con-
`tinuous i.v. infusion at 1 nmol/kg per min. Challenges of angiotensin II were then repeated at 10- to 30-minute intervals
`up to 150 minutes following the bolus injection of BMS 186295. The results are shown below.
`
`6
`
`BIOCON PHARMA LTD (IPR2020-01263) Ex. 1002, p. 006
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`

`

`EP 0 726 072 A2
`
`Minutes
`-20'
`-10'
`BMS-1 86295,
`10'
`20'
`30'
`40'
`50'
`60'
`70'
`80'
`90"
`120
`150'
`
`Change in mean arterial pressure, mm Hg
`29±2
`31±3
`30 nmol/kg, i.v. followed by 1 nmol/kg/min, i v.
`3±2
`2±1
`3±2
`2±1
`1±1
`4±1
`5±2
`4±1
`3±1
`3±1
`2±1
`
`b) Cardiovascular Effects Of BMS 186295. SQ 28603. And the Combination Of These Agents
`
`In this series of experiments baseline measurements of left ventricular end diastolic pressure, left ventricular systo-
`lic pressure and heart rate were determined in groups of conscious cardiomyopathic hamsters. Compounds or vehicle
`were administered intravenously, and measurements were repeated at 30-minute intervals up to 90 minutes after
`administration of the last agent. BMS 186295 was administered at 30 nmol/kg, i.v. (0.3 ml) followed by a continuous i.v.
`infusion at 1 nmol/kg per min (0.01 ml/min). BMS 186295 was prepared in 0.028 M potassium hydroxide and diluted to
`a final concentration of 0.17 M potassium hydroxide. Potassium hydroxide solution (0.17 M) was administered intrave-
`nously 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%
`sodium bicarbonate and administered at 30 nmol/kg, i.v. This dose of SQ 28603 was previously shown to result in a
`doubling of plasma atrial natriuretic peptide concentration within 90 minutes in this model. One group of cardiomyo-
`pathic hamsters received the combination of BMS 186295 and SQ 28603. In this group BMS 186295 was administered
`according to the same dosage regimen described above; 30 minutes after the bolus injection of BMS 186295, SQ
`28603 was administered at 30 nmol/kg, i.v.
`Differences in age, body weight and baseline values among groups were evaluated by analysis of variance. Differ-
`ences 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 <
`0.05. All data are expressed as mean ± standard error of the mean.
`
`Minutes
`Baseline
`BMS 186295 SQ 28603
`30'
`60'
`90'
`
`Left Ventricular End Diastolic Pressure (mm Hg)
`Vehicle
`SQ 28603 BMS 186295 BMS 186295 & SQ 28603
`17±2
`21 ±2
`19±2
`18±3
`18 ±2
`14 ±3
`18 ±2
`20 ±2
`19 ± 1
`14±2
`12±1
`16±2
`18 ± 1
`11±2
`17±3
`16±2
`16±2
`16±3
`18±3
`10±1
`
`BIOCON PHARMA LTD (IPR2020-01263) Ex. 1002, p. 007
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`

`

`EP 0 726 072 A2
`
`Change From Baseline (mm Hg)
`Minutes after last treatment Vehicle
`SQ 28603 BMS 1 86295 BMS 1 86296 & SQ 28603
`1±1
`4±1*
`-1±1
`-10±2*
`30
`-1±1
`-1±2
`-1±1
`60
`-10±3*|
`-2±1
`1±2
`90
`-3±1
`-11±3*|
`*P <0.05 vs Vehicle
`fP <0.05 vs SQ 28603
`
`Minutes
`Baseline
`BMS 186295 SQ 28603
`30'
`60'
`90'
`
`Left Ventricular Systolic Pressure (mm Hg)
`Vehicle
`SQ 28603 BMS 186295 BMS 1 86295 & SQ 28603
`111 ±3
`1 12 ± 2
`117±5
`107±3
`111 ±3
`111 ±1
`104 ±4
`108 ±5
`111 ±5
`114±2
`109±5
`92±2
`111 ±2
`108±3
`105±4
`88±5
`112 ±5
`89 ±4
`105 ±5
`105 ±5
`
`Change From Baseline (mm Hg)
`Minutes After Last Treatment Vehicle
`SQ 28603 BMS 1 86295 BMS 1 86296 & SQ 28603
`-1 ± 3
`2±2
`-8 ± 1*
`30
`-16±3*|
`-12 ±3*
`-1±2
`60
`-3±1
`-20±4*|
`-6 ±4
`-12 ±2
`-18 ±4*
`90
`1±5
`*P <0.05 vs Vehicle
`fP <0.05 vs SQ 28603
`
`Minutes
`Baseline
`BMS 1 86295 SQ 28603
`30'
`60'
`90'
`
`Heart Rate (beats/min)
`SQ 28603 BMS 186295 BMS 1 86295 & SQ 28603
`378 ±12
`338 ±16
`364 ±6
`380 ± 7
`364 ± 1 4
`366 ± 1 2
`366 ± 1 1
`381 ±9
`363 ±20
`366 ±9
`367 ±18
`353 ±10
`378 ±8
`369 ±28
`351 ±9
`
`Vehicle
`350 ±10
`365 ± 8
`347 ±15
`345 ±15
`354 ±13
`
`8
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`BIOCON PHARMA LTD (IPR2020-01263) Ex. 1002, p. 008
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`

`EP 0 726 072 A2
`
`Change From Baseline (mm Hg)
`Minutes after last treatment Vehicle SQ 28603
`BMS 1 86295 BMS 1 86296 & SQ 28603
`30
`-3 ±15
`3±8
`25 ±9
`1±6
`-13 ±12*
`29 ±7*
`-11 ±9
`60
`-5 ±15
`44 ± 1 1 *
`90
`7±13
`-1±9
`-14±5
`*P <0.05 vs Vehicle
`
`15
`
`Discussion of Results
`
`20
`
`Following the administration of BMS 186295, the pressor responses to angiotensin II were less than 17% of the
`response before the administration of the inhibitor. These results indicate that nearly complete inhibition of the pressor
`response to angiotensin II was achieved following the administered dosage regimen of BMS 186295, and suggests
`effective blockade of the angiotensin II receptors.
`The combination of BMS 1 86295 and SQ 28603 produced cardiovascular effects that were greater than those with
`either treatment alone. Specifically, the combination caused significant decreases in left ventricular end diastolic pres-
`sure and left ventricular systolic pressure with no significant change in heart rate. SQ 28603 produced smaller
`25 decreases, whereas BMS 1 86295 had no significant effects on the measured cardiovascular pressures. Thus, the com-
`bination of BMS 1 86295 and SQ 28603 produced beneficial hemodynamic effects in cardiomyopathic hamsters with
`compensated heart failure.
`
`Example 2
`
`30
`
`35
`
`The studies described in this experiment were conducted in dogs that had been rendered hypertensive by prior uni-
`lateral nephrectomy and constriction of the remaining renal artery. This model is characterized by normal basal levels
`of plasma renin activity and is relatively resistant to the anti -hypertensive activity of angiotensin converting enzyme
`inhibitors and ATi receptor antagonists. Furthermore, the 1 -kidney- 1 -clip (IKIC) hypertensive dogs have normal plasma
`concentrations of atrial natriutetic peptide and fail to develop depressor responses to neutral endopeptidase inhibitors.
`The following experiments were conducted in fasted 1 K1 C hypertensive dogs lightly restrained in standard canine
`slings. An indwelling arterial catheter was accessed via a subcutaneous port for measurement of blood pressure via a
`Gould-Statham pressure transducer. Mean arterial pressure (MAP) was continuously recorded on a Gould chart writer
`and stored electronically using a Po-Ne-Mah data acquisition system. During each study, urine was collected at 20
`40 minute intervals via a Foley bladder catheter for determination of urine volume. The concentrations of urinary sodium
`and potassium were measured using ion-selective electrodes and their rates of urinary excretion (nEq/min) were cal-
`culated. Glomerular filtration rate (GFR) and effective renal plasma flow (ERPF) were determined by the renal clear-
`ances of exogenous creatinine and para-aminohippuric acid (PAH), respectively. The concentrations of creatinine and
`PAH in sequential samples of urine and plasma were determined by spectrophotometric assays and the clearances
`45 were calculated by the standard formula.
`Arterial blood samples were drawn at the end of the control period and at 60 minute intervals thereafter for deter-
`mination of the plasma concentrations of atrial natriuretic peptide (ANP), cyclic GMP and plasma renin activity (PRA)
`by separate radioimmunoassays. The plasma and urine samples were preserved and the assays were conducted
`according to standard radioimmunoassay procedures. Urinary excretion rates of cyclic GMP and ANP were calculated
`and expressed as pmol/min and fmol/min, respectively.
`Four 1 K1 C hypertensive dogs were treated with the combination of 30 nmol/kg iv of BMS 1 86295 and 30 nmol/kg
`iv of SQ 28603. Vehicle (0.84% sodium bicarbonate), 30 nmol/kg iv of SQ 28603 and 30 nmol/kg iv of BMS 186295
`were tested in 3 additional groups of 1K1C hypertensive dogs (n=4 to 5/treatment). In each study, baseline measure-
`ments were obtained during two 20 minute control periods. One of the treatments was then administered and sampling
`continued at 20 minute intervals for three hours.
`To minimize inter-animal variability, each data point was expressed as the change from the average control value
`for that parameter. Significant differences among treatments were identified by analysis of variance for repeated meas-
`ures. Contrasts were calculated to identify significant differences from the effects of vehicle and to compare the combi-
`nation of SQ 28603 and BMS 186295 to the individual treatments. Results are given as mean ± SEM.
`
`so
`
`55
`
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`BIOCON PHARMA LTD (IPR2020-01263) Ex. 1002, p. 009
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`

`Results
`
`EP 0 726 072 A2
`
`Table 1
`Mean Arterial Pressure (mm Hg)
`Time (min) after Treatment Vehicle (n=5)
`SQ 28603 (n=4) BMS 1 86295 (n=4) SQ 28603 + BMS
`186295 (n=4)
`140±3
`
`Control
`
`132±3
`
`157±7
`132±8
`Change from control
`-1±1
`-10±1 *
`-8±2*
`-5±2*
`-4±1*
`-1±2*
`-1±2*
`0±2
`-1±3*
`
`6±1
`9±3*
`8±3*
`8±2
`5±3
`3±4
`4±2
`5±2
`7±3
`
`2±2
`2±1 |§
`0±2|§
`-1±3 *
`1±2|
`2±2
`6±2|
`9±3 1
`6±4|
`
`20
`40
`60
`80
`100
`120
`140
`160
`180
`* p<0.05 compared to vehicle
`t p<0.05 compared to BMS 186295
`§ p<0.05 compared to SQ 28603
`
`3±1
`3±2
`2±1
`6±2
`6±2
`6±2
`7±2
`5±2
`6±3
`
`BMS 186295 signigicantly reduced mean arterial pressure (MAP) (Table 1) in the conscious 1K1C hypertensive
`dogs whereas SQ 28603 initially increased MAP. The effects of the combination BMS 186295 and SQ 28603 were not
`consistently different from those of vehicle.
`
`BIOCON PHARMA LTD (IPR2020-01263) Ex. 1002, p. 010
`
`

`

`EP 0 726 072 A2
`
`TABLE 2
`Sodium Excretion (nEq/min)
`Time (min) after Treatment Vehicle (n=5)
`SQ 28603 (n=4) BMS 1 86295 (n=4) SQ 28603 + BMS
`186295 (n=4)
`18±6
`
`Control
`
`60±14
`
`20
`40
`60
`80
`100
`120
`140
`160
`180
`* p<0.05 compared to vehicle
`*t p<0.05 compared to "vehicle or fBMS1 86295
`*t§ p<0.05 compared to "vehicle, fBMS1 86295 or SQ28603
`
`-17±10
`-16±12
`-12±14
`-11±14
`-12±11
`-16±13
`-10±14
`-4±15
`-3±13
`
`18±2
`40±20
`Change from control
`13±4
`23±9
`21±16*
`41±13*
`14±9*
`36±8*
`25±13*
`27±6*
`24±12*
`22±3*
`35±19*
`30±4*
`39±18*
`28±7*
`44±19*
`30±7*
`44±18*
`30±2*
`
`62±31 *|§
`83±34 *|§
`87±18*|§
`70±16*|§
`54±12*|§
`60±21 *|§
`69±22 *|§
`68±16*|
`74±19*|§
`
`0.32±0.17*
`0.51±0.22 *|§
`0.51±0.11*|§
`0.34±0.08 *|
`0.14±0.08*
`0.20±0.07 *
`0.26±0.09 *|
`0.16±0.03 *|
`0.17±0.03*
`
`TABLE 3
`Urine Volume (ml/min)
`Time (min) after Treatment Vehicle (n=5)
`SQ 28603 (n=4) BMS 1 86295 (n=4) SQ 28603 + BMS
`186295 (n=4)
`0.36±0.13
`
`Control
`
`0.64±0.14
`
`0.43±0.16
`0.38+.0.13
`Change from control
`0.29±0.31 *
`0.01±0.07
`0.15+.0.13*
`0.09±0.14*
`0.18+.0.14*
`0.12±0.07*
`0.05±0.06 *
`0.21±0.15*
`-0.02±0.09 *
`0.07±0.08*
`0.12±0.13*
`-0.02±0.10*
`0.20±0.10*
`-0.07±0.12*
`0.24±0.11*
`-0.07±0.16
`-0.02±0.07 *
`0.17±0.06*
`
`30
`
`55
`
`-0.20±0.12
`-0.27±0.14
`-0.26±0.14
`-0.26±0.15
`-0.23±0.14
`-0.30±0.13
`-0.25±0.14
`-0.23±0.15
`-0.22±0.14
`
`20
`40
`60
`80
`100
`120
`140
`160
`180
`* p<0.05 compared to vehicle
`*t p<0.05 compared to "vehicle or fBMS1 86295
`*t§ p<0.05 compared to "vehicle, fBMS1 86295 or SQ28603
`
`11
`
`BIOCON PHARMA LTD (IPR2020-01263) Ex. 1002, p. 011
`
`

`

`EP 0 726 072 A2
`
`BMS 186295 and SQ 28603 each individually increased sodium excretion (TABLE 2) and urine volume (TABLE 3)
`in the conscious 1 K1 C hypertensive dogs. The natriuretic response to the combination of BMS 1 86295 and SQ 28603
`was greater than the activity of either of the compounds administered singly. The increase in the amount of sodium
`excreted during the 3 hours after simultaneous injections of BMS 186295 and SQ 28603 (12.6±3.4 mEq/3 hr) approxi-
`mated the sum of the natriuretic responses to BMS 186295 (5.4±1.0 mEq/3 hr) and to SQ 28603 (5.2±2.3 mEq/3 hr)
`given individually.
`
`TABLE 4
`Glomerular Filtration Rate (ml/min)
`Time (min) after Treatment Vehicle (n=5)
`SQ 28603 (n=4) BMS 1 86295 (n=4) SQ 28603 + BMS
`186295 (n=4)
`46±4
`
`Control
`
`50±4
`
`45±10
`39±5
`Change from control
`-4±2
`-2±5
`6±0
`5±2
`3±4
`4±3
`0±5
`0±7
`0±4
`
`6±11
`3±2
`6±5
`5±2
`5±4
`5±1
`7±4
`9±5
`10±4
`
`-8±7§
`6±3 1
`11±2*
`7±1 *
`4±4
`12±2*§
`9±1 *
`6±2
`5±5
`
`-2±2
`0±4
`2±3
`0±3
`4±3
`-1±2
`1±2
`5±4
`5±3
`
`20
`40
`60
`80
`100
`120
`140
`160
`180
`* p<0.05 compared to vehicle
`§ p<0.05 compared to SQ 28603
`t p<0.05 compared to BMS 186295
`*§ p<0.05 compared to "vehicle or §SQ28603
`
`BIOCON PHARMA LTD (IPR2020-01263) Ex. 1002, p. 012
`
`

`

`EP 0 726 072 A2
`
`127±41
`123±15
`Change from control
`-68±30 *
`6±38
`-5±15
`-54±35
`3±20
`-35±24
`-20±6
`-13±9
`-25±10
`-1±10
`-8±17*
`-15±6
`-11±12
`8±3*
`-1±12
`-14±12
`-12±11
`-4±9
`
`-84±19*§
`-64±27 *§
`-51±21 §
`-45±21 |
`-41±23 1
`-15±8
`-12±12
`-28±14
`-23±16
`
`TABLE 5
`Effective Renal Plasma Flow (ml/min)
`Time (min) after Treatment Vehicle (n=5)
`SQ 28603 (n=4) BMS 1 86295 (n=3) SQ 28603 + BMS
`186295 (n=4)
`142±30
`
`Control
`
`144±18
`
`-25±10
`-27±17
`-25±16
`30±18
`-26±14
`-41±14
`-32±12
`-21±13
`-21±15
`
`20
`40
`60
`80
`100
`120
`140
`160
`180
`* p<0.05 compared to vehicle
`*§ p<0.05 compared to "vehicle or §SQ28603
`t p<0.05 compared to BMS 186295
`§ p<0.05 compared to SQ 28603
`
`5
`
`10
`
`15
`
`20
`
`25
`
`The combination of BMS 186295 and SQ 28603 significantly increased GFR (TABLE 4) at several times during the
`3 hour test when compared with the effects of vehicle even though effective renal plasma flow (TABLE 5) did not
`increase. The increase in GFR alone did not account for the full natriuretic response, as indicated by a significantly rise
`in fractional sodium excretion from 0.26±0.07% to 1 .28±0.29%.
`
`13
`
`BIO