European Jownar of Pharnlacology, 0

1991

ADONIS

EJP

202 ( 199 1I 323-330

Elsevier Science Publishers B.V. All

rights

323

resewed 0014-2YYY/Yl/$O3.50

OOl42YYYYlOO6l8K

52034

e armgiote a Pancras C. Wong, Scott D. Hart, John V. Duncia and Pieter B.M.W.M. l’immermans The Du Pant Merck Pharmaceurical

Cornparty, Wilmingtott, DE, U.S.A.

Received 10 May lYY1, accepted 2 July 1991

DuP 753 (or EXP3174) and PD123177 are nonpeptidc angiotensin (AH&specific ligands. which show high affinities for two AI1 receptor subtypes, i.e. AT, and AT, sites, respectively. In furcsemidc-treated conscious dogs with high renin, DuP 753 and EXP3714, but not PD123177, were as effective as captopril in lowering blood pressure. Both DuP 753 and EXP3174 exhibited seleclive vascular antagonism of AIL In conscious dogs with normal rcnin, DuP 753, but not captopril or EXP3174, caused a dose-dependent but transient decrease in blood pressure. In anesthetized dogs, DuP 753 and captopril caused similar renal vasodilatation and natriuresis. The renal hemodynamic effects of DuP 753 and captopril were more pronounced in dogs with sodium depletion. These results suggest that the AT, receptor mediates the prcssor and renal effects of AI1 in dogs. The acute transient hypotcnsive effect of DuP 753 in normal-rcnin conscious dogs is probably unrelated to AI1 antagonism. Angiotensin

II receptor antagonists;

Angiotcnsion

receptors; Antihypcrtensivc system; (Dog)

1. Introduction DuP 753 (2-n-butyl-Cchloro-5-hydroxymethyl-l-1(2’~lH-tetrazol-5-yl~biphenyl-4-yl~methyl]imidazole, potassium salt) is an orally active nonpeptide angiotensin II (All) receptor antagonist in both animals and humans (Carini and Duncia, 1988; Chiu et al., 1990; Christen et al., 1990; Wong et al., 1990b,c). It is an effective antihypertensive agent in renal hypertensive rats (one-ligate, two-kidney type), a high renin model of experimental hypertension, but not in deoxycorticosterone acetate-salt hypertensive rats, a low renin model (Wang et al., 1990d). It also decreased blood pressure in spontaneously hypertensive rats (Wang et al., 1990e). Unlike the peptide AI1 receptor antagonists and angiotensin converting enzyme inhibitors, DuP 753 does not appear to have agonistic and bradykinin potentiating effects, respectively (Chiu et al., 1990; Wong et al., 1990b,c,d,e). Thus, DuP 753 represents a potential therapeutic agent and a useful physiological probe for studying the renin-angiotensin system (RAS). In the rat, DuP 753 generates an active metabolite,

Corresptindrnce

to: P.C. Wang, The Du Pont Merck Pharmaceutical

Cnmpany. Experimenlal Station. P.O. Box 80400. Wilmington. IZJE 10880-0400.U.S.A. Tel. 1.302.oYS.7lX4. I’UX1.302.6YS.7OS4. * Part XIII in ;I sericb; for pwl XII. set Carini ct al. (IYYI).

agents; Renal functions: Renin-angiotensin

EXP3174 (2-n-butyl-4-chloro-I-[(2’-(IH-tetrazol-5ylYiiphenyl-4-yl)methyl]imidazole-5-carboxylic acid), which is most likely responsible for part of the antihypertensive effect of DuP 753 in rats (Wang et al., 199Of). As the in vivo pharmacology of DuP 753 and EXP3174 has been mainly examined in rats, we characterized in this study the pharmacological profiles of DuP 753 and EXP3174 in dogs to ascertain their effects in another species. Because the RAS plays a minimum role in the control of blood pressure in conscious normotensive animals (Hollenberg, 1979). we studied the blood pressure effects of DuP 753 and EXP3174 in conscious dogs pretreated with furosemide, which is known to activate the RAS (Keeton and Campbell, 1981). Recently, two AII receptor subtypes have been identified in the adrenal gland, brain, uterus and vascular tissues of both rats and humans, and they can be revealed with specific receptor ligands (Chiu et al., 1989; Whitebread et al., 1989; Chang and Lotti, 1990; Dudley et al., 1990). The All receptors that have high binding affinity for DuP 753 are classified as AT, sites and those that have high affinity for PD123177 (1-(4-amino-3-methyl-phenyl)methyl-5-diphenylacetyl4,5,6,7-tetrahydro-I H-imidazo[4,5-clpyridinc-6-carboxylic acid) as AT, sites (Bumpus et al., 1991) (PD123177 is a Warner-Lambert compound, i.e. compound 13

kley et al.. lOS91,previously designated as EXP65S et al.. ~9~9~~.To determine the re!ative involvcments of AT, and AT, in blood pressure control, the effect of PDl23177 on blood pressure in furosemidetreated dogs was studied. Gne of the major functions of AII is the regulation of renal function. which has been widely studied with angiotensin converting enzyme inhibitors (for review, see Hail. 1986). However, the specificity of effects of in converting enzyme inhibitors, which also e degradation of bradykinin (for review, see 19791, is ._uestionable. Thus, the renal effects of the a~giotensin converting enzyme inhibitor captopril and the All receptor antagonist DuP 753 were compared in anesthetized dogs.

2.12.

Gro1ip

2

To assess the efficiency of oral absorption of DuP 753 and EXP3174, the i.v. and oral hypotensive effects of these antagonists were determined in furosemidetreated dogs. Dogs were pretreated with furosemide as described above. In the first series, saline vehicle (0.5 ml/kg), DuP 753 at 1 and 3 mg/kg and EXP3174 at 0.1, 0.3 and 1 mg/kg were given i.v. and their effects on MAP and HR were recorded at 5 to 180 min post-dose. In the second series, water vehicte (2 mi/kg~, DuP 753 at 10 and 30 mg/kg and EXP3174 at 3 and IO mg/kg were given orally by gavage and the experiment was monitored for 8 h. In the third series, the effect of I’D123177 at 10 mg/kg i.v. on MAP was evaluated. 2.1.3. Group 3

Mongrel dogs (g-13 kg) (White Eagle Labs. Doylestown, PA, U.S.A.) of either sex, which had been trained to stand in a sling, were anesthetized with thiopental sodium (20 mg/kg i.v.1, followed by endotracheal intubation. Anesthesia was maintained hy a m~ure of halothane and oxygen. Under aseptic conditions. the right femoral artery was cannulated with a chronic catheter device filled with heparin (I000 l-l/ml). The catheter device is a silastic catheter which has a vascular-access-port (Norfock Medical Products, Inc., Skokie, IL. U.S.A.) attached to it (Garner and Laks, 1987). One day following implantation, animals were trained to stand in a sling for continuous measurement of arterial pressure with a Gould pressure transducer (Gould Inc., Oxnard, CA, U.S.A.). Mean arterial pressure (MAP) and heart rate (HR) were recorded on a Grass polygraph (Grass Instrument Co., Quincy, MA, U.S.A.) and a digital computer (Buxco Electronics, Inc., Sharon, CT, U.S.A.). Dogs with access ports were used for experiments at least 2 days after surgery.

In this series, dogs were not pretreated with furosemide. AI1 at 0.1 pg/kg, phenyiephrine at 30 pg/kg or vasopressin at 0.1 IU/kg were injected i.v. before and subsequently 10 min after each successive cumulative iv. dose of DuP 753 (I, 3 and 10 mg/kg at intervals of 40 min) to ascertain I!“:! ?;Pecit:c.ijr of DuP 753. A similar study was also conducted with EXP3174 at 0.3 to 3 or 1 to 10 mg/kg i.v. 2. I. 4. Group 4 In this series, dogs were not pretreated with furosemide. To ascertain that DuP 753 and EXP3174 were devoid of agonistic activities in dogs, effects of cumulative i.v. doses (I,3 and 10 mg/kg at intervals of 40 min) of these antagonists on MAP and HR were examined. Captoprii given at 1, 3 and 10 mg/kg i.v. cumulatively was also included for comparison. Because DuP 753 at 1 to 10 mg/kg i.v. caused transient hypotension and bradycardia (see the Result Section), we examined whether this was due to potassium generated by DuP 753. Thus, effects of the free base of DuP 753 or KCI at 1 and 2 mg/kg i.v. (an equivalent atnount of potassium as 10 mg/kg of DuP 753) on MAP and HR were also measured. In another group, dogs were first pretreated with captoprii at 10 mg/kg i.v. to block the influence of the RAS, and saline vehicle or DuP 753 & 10 mg/kg i.v. was then given 30 min later.

2.1.1. Groap 1

Dogs were treated with furosemide at 10 mg/kg at 18 (given i.m.1 and at 2 h (given i.vJ before the experiment to elevate their plasma renin activity (PRA). Food and water were withdrawn from these dogs after the first dose of furosemide. Water vehicle (2 ml/kg) and single doses of captopril at 0.3, 1, 3 and 10 mg/kg were given orally by gavage and their effects on MAP and HR were recorded at 5 to 180 min post-dose. In some dogs, an arterial blood sample of 2 ml was taken before and after furosemide treatment for the determination of basal PRA.

2.2. Renal jbnction studies in anesthetizeddogs Mongrel dogs f8- 12 kg) of either sex were studied in six groups. Groups I, II and III were fed a standard dog chow (Canine 2000, Agway Inc., Syracuse, NY, U.S.A.) providing approximately 104 meq of sodium/ day, while those in groups IV, V and VI were maintained for 7 days on a low sodium diet which supplied about 5 meq of sodium/day (Canine h/d diet, Hill’s Pet Products, Inc., Topeka, KS, U.S.A.). In addition, furosemide at IO mg/kg i.m. was given daily to dogs in

325

groups IV, V and VI on day 2 to day 5 of the low-saltdiet regimen. The animals were anesthetized with pentobarbital sodium (30 mg/kg i.v.). Artificial ventilation was provided with a Harvard respirator (Harvard Apparatus, South Natick, MA, U.S.A.). The right and left cephalic veins were cannulated for administration of drugs and continuous infusion of pentobarbital sodium at 6 mg/kg per h. A retroperitoneal flank incision was made to expose the left kidney. The left ureter and gonadal vein were cannulated for collection of urine and renal venous blood samples, respectively. Renal blood flow (RBF) was measured with a calibrated flow probe placed around the left renal artery and the probe was linked to a Carolina electromagnetic flowmeter (Carolina Medical Electronics, Inc., King, NC, U.S.A.). Arterial blood pressure was monitored from a cannulated brachial artery with a Gould transducer. MAP and RBF were recorded continuously on a Grass polygraph. Samples of arterial blood were collected from a cannulated femoral artery. Glomerular filtration rate (GFR) was determined from the renal arteriovenous extraction of ‘2”l[iothalamate] (Hall et al., 1977). A priming dose of 0.5 pCi/kg of iothalamate was administered, followed by a sustaining intravenous infusion of 0.003 &i/kg per min in 0.2 ml/kg per min. Glomerular filtration rate was calculated by the f’ollowing formulas: GFR = (1 - Hct) x FF x RBF and FF = (A - V/)/A, where FF is the filtration fraction and Hct is the arterial hematocrit determined by the microcapiilary method, while A and V are the systemic arterirdl and renal venous ‘2”l-radioactivities, respectively. Ulice sodibm and potassium concentrations were measured by flame photometry (Instrumentation Laboratory, Lexington, MA, U.S.A.). Renal vascular resistance (RVR) was calcuiated by dividing MAP by RBF. Dogs in groups I, 11 and Ill were infused with 0.9% saline by i.v. drip i500 ml) during the surgery period. After a 45-min-stabilization period, two control 15min clearance periods were recorded. Arterial and venous blood samples (1 ml each) were collected at the middle of the clearance period. Saline at 0.5 ml/kg, captopril at 1.5 mg/kg and DuP 753 at 5 mg/kg were given i.v. in groups I, 11 and Ill, respectively, and also in groups IV, V and VI, respectively. Two 15-min clearance periods were then monitored. Values of renal function determined in the second control and treatment clearance periods were used for data analysis. In some dogs, an arterial blood sample in the control period was also collected for PRA determination. 2.3. Analyses and statistics PRA was determined by radioimmunoassay (Haber et al., 1969) using B Du Pont NEN radioimmunoassay kit (Boston, MA, U.S.A.). Statistical analyses used were

analysis of variance, and Duncan’s new multiple-range test for multiple comparison (Cody and Smith, 1987). These analyses were carried out by a computer package, Statistical An+is System (SAS Institute Inc., Cary, NC), in a VAX computer. The level of significance was taken at P < 0.05. All data were expressed as the means + S.E.M. 2.4 Drugs All, phenylephrine and vasopressin were obtained from Sigma Chemical Company (St. Louis, MO, U.S.A.). Furosemide was purchased from HoechstRoussell Pharmaceuticals (Somerville, NJ, U.S.A.). ‘251[iothalamatel (Glofil) was obtained from ISO-TEX Corp. (Friendswood, TX, U.S.A.). DuP 753 (Carini and Duncia, 19881, EXP3174 (Carini et al., 19891, PD123177 (Blankley et al., 1989) and captopril (Ondetti and Cushman, 1978) were synthesized at The Du Pont Merck Pharmaceutical Company (Wilmington, DE, U.S.A.). Solutions of DuP 753 were prepared in water or saline. EXP3174 and ilie free base of DuP 753 were dissolved in a mixture of 5% NaHCO, and 5% dextrose at a pH of 8-9.

3. Results 3.1. Blood pressure effect in conscious dogs PRA in conscious dogs was 1.5 + 0.5 ng angiotensin per h (n = 4). Furosemide treatment in-

I (AI)/ml

Percent of control mean arterial pressure 140 -1

120

i

100

0 Vehicle -3 Capwpril.

0.3 mglkg po

0

1 mglkg po

Captopnl.

0 Ceptopnl.

3 m-a/kg PO

V Capwrit.

10 molto

Furosemide-treated conscious dog

po

I

EO 1

60’

I /0

!_ 20

,40

-7. w---m60 60

100

120

140

160

180

Time (min) Fig. I. Effects of vehicle (n = 7) and captopril at 0.3. I. 3 and IO mg/kg i.v. (n = 3 per dose) on mean arterial pressure (expressed as percentage of control mean arterial pressure) in conscious furosemide-treated dogs. Basal values of mean arterial pressure in the groups treated with the vehicle and captopril at 0.3, I. 3 and 10 mg/kg were 111*6. 104+ll. 108*2. 103+3 and 113+8 mm Hg. respectively. Overall effects of captopril at 0.3 to 10 mg/kg i.v. were significantly different from vehicle (P < 0.05, two-factor experiments with repcabzd measurements on one factor). Value:, represent the means rt S.E.M.

RA to 19.0 & 7.2 ng Al/ml per h (n = 3). ~~~t~~~ri~ at 0.3 to 80 mg/kg p.o. caused a dose-dependent decrease in MAP in furosemide-treated conscious dogs Ifig. 11. Given either i.v. at 1 and 3 mg/kg or p.o. at 10 and 30 mg/kg. DuP 753 also decreased MAP dose dependently in furosemide-treated dogs (fig. 2). Sirn~~ar bypotensiv~ effects were aIso tibscnred with EXP3174 at (3.1 to 1 mg/*kg i.v. or 3 and 10 mg/kg p.o. (fig. 3). HR was not significantly changed by either DuP 733 or EXP3174 (data not shown). On the contrary, PD123i77 at PO mg/kg i.v. (n = 41 did not alter MAP in furosemide-treated dogs (data not shown). In conscious dogs, DuP 7S3 at I, 3 and 10 mg/kg i.v. caused a dose-dependent inhibition of the AlI pressor

craicd

Percent of control mean 140

artenal pressure

Furosemide-treated

conscious

Percent 140

of control

mean arterial pressure -

Vehicle @ EXP3174.0 1 mg/kg IV EXP3174.0 3 m&kg IY

120

EXP3174. t mgfkg IV

100 -

SO-

60-

1 0

/

20

Furosemide-treated

1

40

t

!,

60

80

100

conscious dog

!

120

t

l40

I

160

t

180

Time (min)

Percent of control mean arterial pressure -

dog

140 -/

V&T&@

i

Furosemid~treated

conscious dog

0 Vehicle

120

120

0 EXP3l74. 3 mg/kg po EXP3l74,lO

60 -

-0

f 40

20

60

I

1

120

100

80

f

140

I

160

I I I

m@k@ PO

l;O

Time Imin)

Percent of control mean arterial pressure -

140-1

I 0 “*MC* ! e OuP 753. 10 mg/ke 00

120 i

DuP 763.30 mg/kp PO

100 2

80 -

60

~ro~emide-treater conscious dog

0

1

._.._ _

_~~__,_~~

---- -2

3

4

5

6

7

8

Time (hr) Fin _. 2. Upper pu& Effects of vehicle (n = 4) and DuP 753 at 1 (n = 4) and 3 (n = 7) mg/kg i.v. on mean artrrial pressure (expressed as psrcentage of control mean arterial pressure) in conscious fumsemide-treat2d dogs. Basal values of mean arterial pressure in the groups trrated with the vehicle and DuP 753 at 1 and 3 mg/kg were 99 +S. I16 rt 6 and 10X+4 mm Hp. LOMW pat]& Effects of vehicle (n = 4) and DuP 753 at 10 (n = 121 and 30 (n = 8) mg/kg p.o. on mean arterial pressure in conscious furosemide-treated dogs. Basal values of mean arterial prsssure in groups treated with the vehicle ano DuP 753 at IQ and 30 mg/kg wer2 !03?4, 9h* 7 and 94+4 mm Hs, respectively. Overall effects of DuP 753 given either i.v. or p.o. were significantly different frcm me corresponding vehicle (P < 0.05. two-factnr experiments with repeated measurements on one factor). Values represent the mransIS.E,M.

Fig. 3. Upperpurrrl: Effects of vehicle (n = 4) and EXP3174 at 0.1.0.3 and I mg/kg i.v. (n = 4 per dose) on mean arterial pressure fexpressed as percentage of control mean arterial pressure) in conscious furosemide-treated dogs. Basal values of mean arterial pressure in the groups treated with the vehicle and EXP3174 at 0.1, 0.3 and I mg/kg were 99 t 5, 89 F 5, 88 + 8 and IO5 + 3 mm Hg. Lower panrel: Effects of vehicle Cn= 4) and EXP3174 at 3 and 10 mg/kg p.o. (n = 4 per dose) on mean arterial pressure in conscious furosemidetreated dogs. Basal values of mean arterial pressure in groups treated with the vehicle and EXP3174 at 3 and 10 mg/kg were 103 +4. 90-t 6 and 9h+4 mm Hg, respectively. Overall effects of EXP3174 given either i.v. or p.o. were significantly different from the corresponding vehicle fP < 0.05, two-factor experiments with repeated measurements on one factor). Values represent the means+ S.E.M.

response and did not alter the pressor responses to phenylephrine and vasopressin (fig. 4). A similar selective AI1 antagonism was also observed with EXP3174 (fig. 5). As shown in fig. 6, DuP 753 at 1, 3 and 10 mgfkg i.v. caused a dose-dependent and transient hypotensive effect in conscious dags without furosemide pretreatment. The transient decrease in MAP by DuP 753 was also accompanied with a transient decrease in HR in these dogs (data not shown). On the contrary, neither EXP3174 nor captopril at the same doses decreased MAP (fig. 6) or HR (data not shown). In the

327

._ W

a

I:

60

J

5”

%

sl

9

40

4

40

.a =

30

*C

30

B

20

2 u

10

P

z

zQ

it

r” u

10 0

1

3

10

0

0.3

0

1

1

3

Phenylepbrine

0

I

3

10

0

3

10

70

z0

60

Vasqmssin

10

Fig. 4. Effects of DuP 753 on the pressor responses to angiotensin II (0.1 pg/kg i.v.), phenylephrine (30 pg/kg i.v.) and vasopressin (0.1 lU/kg i.v.) in conscious dogs. Basal values of mean arterial pressure in these 3 groups of dogs were 115$-6, 11X+6 and 121f3 mm Hg, respectively. Values represent the means + S.E.M. * P c 0.05. onefactor experiments with repeated measurements followed by Duncan’s new multiple-range test.

vehicle- (n = 4) and the captopril (10 mg/kg i.vJtreated (n = 4) conscious dogs, DuP 753 at 10 mg/kg i.v. transiently decreased MAP by 69 f 3 and 61 + 3%,

TABLE

* P 5 0.05

0

0

70 60

2

60

Fig. 5. Effects of EXP3174 on the prcssor responses to angiotensin II (0.1 pg/kg i.v.). phenylephrine (30 pg/kg i.v.) and vasopressin (0.1 W/kg i.v.1 in conscious dogs. Basal values of mean arterial pressure in these 3 groups of dogs were 88 f 8. I13 + I1 and 94k 9 mm Hg. respectively. Values represent the meansfS.E.M. * P ~0.05, onefactor experiments with repeated measurements followed by Duncan’s new multiple-range test.

respectively. The free base of DuP 753 at 1, 3 and 10 mg/kg i.v. also reduced MAP by 13 + 3, 51 + 14 and 67 + 4%, respectively (n = 4). However, KCI at 1 and 2 mg/kg i.v. did not alter MAP (data ?ot shown, n = 4).

I

Basal mean arterial pressure (MAP, mm Hg). renal blood flow (RBF, ml/min per g kidney,, renal vascular resistance (RVR. mm Hg/(ml/min per g kidney)), glomerular filtration rate (GFR, ml/min per g kidney), filtration fraction (FF), urinary sodium excretion WNa. ,ueq/min Per g kidney). urinary potassium excretion (UK, peq/min per g kidney) and urine flow WV, ml/min) in groups of sodium-replete and deplete anesthetized dogs treated with vehicle, DuP 753 at 5 mg/kg i.v. and captopril at I.5 mg/kg i.v. Values represent mean f S.E.M. ND, not determined. Sodium-deplete

Sodium-repiete

MAP RBF RVR GFR FF UNa UK uv

Vehicle tn = 8)

DuP 753 (n = 9)

Captopril (n = 8)

Vehicle tn = 4)

DuP 753 tn = 8)

Captopril In= 7)

99 *4 3.6 kO.4 29 +3 0.73 * 0.09 0.32f0.01 3.18f0.51 0.59 f O.bX 0.31 * 0.05

if)9 *4 ?.l +0.2 37 *3 0.62 f 0.05 0.34 >-0.01 2.01 kO.41 0.44 * 0.05 0.27 + 0.06

109 +4 3.6 to.2 31 *3 0.x 1 & 0.09 0.37 f 0.02 2.83 k 0.71 0.48 * 0.09 0.27 f 0.08

109 +5 2.6 kO.4 46 f8 0.40 f 0.09 0.27 + 0.04 ND ND ND

113 +6 2.7 rtO.4 48 +1 0.5 I * 0.09 0.32 f 0.01 ND ND ND

112 +5 3.7 +O.h 36 +l 0.57 f 0.09 0.28 f 0.02 ND ND ND

Percent of control mean arterial pressure 130

3

3

1

-15

/ 0

/ 15

! 30

10

II 45

60

mglkg IV

11 90

75

105

1

changes in MAP and RBF were not significant. In sodium-deplete dogs, the renal hemodynamic effects (i.e. MAP, RBF and RVR) of captopril at 1.5 mg/kg i.v. and DuP 753 at 5 mg/kg i.v were more pronounced. FF was signficantly decreased by DuP 753 and captopril (table 2). As urine flow was very low in sodium-deplete anesthetized dogs (15% of the urine flow in sodium-replete anesthetized dogs), UNa, UK and UV were not recorded in these dogs.

caused similar renal effects except that the

1 120

4. Discussion

Time (inin) Fig. 6. Effects of DuP 753, EXP3174 and captopril tn = 4 per group) on mean arterial pressure (expressed as percentage of control mean arterial pressure) in conscious dogs. Basal values of mean arterial pressure in the groups treated with the DuP 753, EXP3174 and captopril were 118+ 5, 94 + 9 and 111 f 3 mm Hg, respectively. The maximum decrease in mean arterial pressure by DuP 753 was significantly different from the control value at all doses tested (P < 0.05, one-factor experiments with repeated measurements). Values represent the means + S.E.M.

3.2. Renal function studies in anesthetized

dogs

PRA averaged 1.2 + 0.3 and 21.0 5 2.7 ng AI/ml per h in sodium-replete (n = 111 and sodium-deplete anesthetized dogs (n = 91, respectively. Basal MAP, RBF, RVR., GFR, FF, urinary sodium excretion (UNal, urinary potassium excretion (UK) and urine flow (UV) in groups of sodium-replete and deplete anesthetized dogs treated with vehicle, DuP 753 at 5 mg/kg i.v. and captopril at 1.5 mg/kg i.v. are shown in table 1. Captopril at 1.5 mg/kg i.v. decreased MAP and RVR, did not alter GFR and FF, and increased RBF, UNa and UV in sodium-replete dogs but the increase in UK was not significant (table 21. DuP 753 at 5 mg/kg i.v. TABLE

Our study shows that oral administration of captopril, which blocks the formation of AI1 from AI (Ondetti and Cushman, 1978; Hollenberg, 1979), decreased blood pressure significantly in furosemidetreated but not in untreated conscious dogs. This suggests the importance of AI1 in the regulation of blood pressure in furosemide-treated dogs. Given p.o., DuP 753 and EXP3174 were 3 to 10 times less potent than captopril but were as effective as captopril in lowering blood pressure maximally by about 30% in furosemide-treated dogs. Both in terms of potency and duration of action, DuP 753 appears to be less effective in lowering blood pressure in furosemide-treated dogs than in renal artery-ligated hypertensive rats (Wang et al., 1990b,dl. Preliminary evidence from pharmacokinetic studies suggests that DuP 753 generates a lesser amount of the active metabolite EXP3174 in dogs than in rats, which may explain a weaker action of DuP 753 in dogs iD.D. Christ, N.Y. Wong and G.N. Lam, unpublished observations). As DuP 753 has an oral bioavailability of about 27% in dogs (Christ et al., 19901, the total amount of DuP 753 delivered at 10

2

Effects of vehicle, DuP 753 at 5 mg/kg iv. and captopril at 1.5 mg/kg iv. on mean arterial pressure (MAP), renal blood fio;N (RBF), renal vascular resistance (RVR), glomerular filtration rate (GFR), filtration fraction (FF), urinary sodium excretion (UNa), urinary potassium excretion (UK) and urine flow WV) in sodium-replete and deplete anesthetized dogs. Values represent means + S.E.M. ND, not determined. a P < 0.05, compared to the corresponding vehicle-treated group by one-way ananalysis of variance followed by Duncan’s new multiple-range test. % Change From Control Sodium-replete Vehicle (n = 8) MAP RBF RVR GFR FF UNa UK uv

-2*

1

3* -2+ Irt

5 6 5

-8+ 8 -4& 13 9*15 -2* 1.:

Sodium-deplete DuP 753 fn = 8)

Captopril fn = 8)

Vehicle fn = 4)

DuP 753 fn = 8)

Captopril (n = 7)

-Sf 15f -18+ 2*

-11* 4” 16&- 3” -23* 4”

1 -5* 4f 3 -8i 2 -1*13 -6*13 ND ND ND

29 -24* 49*11” -47* 5’

-24f2 = S/1*8’ -so*3 p -14*8 -42kS ’ ND ND ND

1 4 38 4

-9f 3 133+43 u 74+_16” 124*42’

3* 6 -9f 6 112*31” 54* I9 116*49 1’

-19-1: -44+ ND ND ND

9

6’

329

mg/kg p.o. should be equivalent to that at 2.7 my/kg iv. However, the duration of action of DuP 753 at 10 mg/kg p.o. appears to be longer than that at 3 mg/kg i.v. Conceivably, a slower rate of absorption by the oral route, a more intense compensatory reflex activation by the i.v. route due to a more rapid decrease in blood pressure, or more active metabolites generated by the oral route may possibly account for the longer duration of action of DuP 753 given p.o. Since the i.v. and p.o. doses of EXP3714 which decreased blood pressure by about 30% were 0.3 aqd i0 mg/kg, respectively, the oral absorption of EXP3174 appears to be poor in dogs. However, the duration of hypotensive effect of EXP3174, wh!ch was at least equal to that of captopril, was better than that of DuP 753. The AT, specific ligand PD123177 did not alter blood pressure in furosemidc-treated dogs. This is consistent with our previous finding in which DuP 753 but not PD123177 reduced the vasoconstrictor effect of AI1 in rats and lowered blood pressure in renal artery-ligated hypertensive rats with high PRA (Wong et al., 1990a). These results suggest that the AI1 receptor in the vascular smooth muscle of rats and dogs appears to be of the AT, type. DuP 753 and EXP3174 inhibited the pressor response to AI1 but not those to vasopressin and phenylephrine, suggesting selective AI1 antagonism at vascular AI1 receptors. However, we noted that DuP 753 at 1 to 10 mg/kg i.v. caused a dose-dependent and transient decrease in blood pressure in these conscious dogs, which were not pretreated with furosemide and had a normal PRA of about 1.5 ng AI/ml per h. In contrast, EXP3174 and captopril, even up to 10 mg/kg i.v. (at least 30 times the effective dose to block the RAS), did not change blood pressure. Captopril prctreatment also did not affect this hypotensive effect of DuP 753. These results suggest that the hypotensive effect of DuP 753 in normal-renin conscious dogs is probably unrelated to AI1 antagonism. Interestingly, DuP 753 even up to 100 mg/kg i.v. did not change blood pressure in normal-renin conscious normotensive rats (Wong et al., 1990b), suggesting that the hypotensive response to DuP 753 in normal-renin conscious normotensive dogs is species dependent. We noted that this hypotensive effect of DuP 753 was accompanied with a decrease in HR. Since DuP 753 was given iv. as a potassium salt, we examined whether the potassium generated by DuP 753 may have caused bradycardia and hypotension. However, the free base of DuP 753 up to 10 mg/kg i.v. caused a similar short-lasting decrease in blood pressure as its potassium salt. Moreover, KC1 at 1 and 2 mg/kg i.v. (an equivalent amount of potassium as 10 mg/kg of DuP 753) did not affect blood pressure and HR. Thus, the amount of potassium released by DuP 753 is not enough to induce

hypotension

and bradycardia.

As the activity of the RAS varies with sodium intake Weeton and Campbell, 19811, renal effects of DuP 753 and captopril were examined in both the sodium-replete and deplete dog. To minimize the influence of the acute and transient action of DuP 753, the renal effects of DuP 753 was assessed at 15-30 min post-dose. Under this condition, DuP 753 and captopril caused significant renal effects in these anesthetized dogs. Interestingly, in sodium-replete dogs with PRA of 1.2 ng AI/ml per h, captopril and DuP 753 increased urine flow and sodium excretion, suggesting that there is an influence of the RAS on renal function even during sodium repletion. Since GFR was not significantly altered by captopril or DuP 753, the natriuretic actions of these agents may be attributed to the blockade of AILstimulated proximal tubular sodium reabsorption (Hall, 1986). The renal vasodilator effects of DuP 753 and captopril were more pronounced during sodium depletion. GFR was not significantly decreased by DuP 753 and captopril, while FF was signifcantly decreased in sodium-deplete dogs. These results suggest that AI1 may exert a greater vasoconstrictor effect at the renal efferent arterioles especially in sodium-deplete dogs (Hall et al., 1977). The favorable renal action of captopril such as renal vasodilatation and natriuresis has also been shown in essential hypertensive patients and has been imphcated to be beneficial for its overall antihypertensive efficacy (Hollenberg, 1985). As DuP 753 shares many of these renal responses of captopril, it is anticipated that the renal actions of DuP 753 may be of clinical importance during long-term therapy. There was a slight hypotensive effect of captopril observed in the anesthetized but not in conscious sodium-replete dogs, although the values of PRA in both groups of dogs were similar. The reasons for this discrepancy are not clear but factors such as differences in experimental conditions, i.e. anesthetized vs. conscious, cannot be excluded. In conclusion, our studies demonstrate that DuP 753 and EXP3174 are orally active, AI1 receptor antagonists that lower blood pressure in furosemide-treated dogs. Similar to the results obtained in rats (Wang et al., 1990b,c,e), the nonpeptide AI1 receptor antagonists do not have agonistic activities (i.e. no pressor effects) in dogs. In both sodium-replete and sodium-depelete dogs, DuP 753 causes similar renal effects as CaPtOPril. The mechanism accounting for the transient hYWtensive effect of DuP 753 in normal-renin conscious normotensive dogs, which is unrelated to AII receptor blockade, remains to be determined.

Acknowledgments The authors thank Drs. David J. Carini, Philip Ma and Roy T. Uycda for synthesis of EXP3174, PD123177. captopril and the free

h;tse ot Q~P 75.3. Richard Hallowell and Gordon A. Slack are achnowlrged for their excellent technical assistance.

J.C.. J.C. Hodges. J.S. Kiely and S.R. Klutchko, lY8Y. 4.5,6.7-T~trahydr~~-lH-imiduzo[S..S-C]pyridine-h-carb~~~iic acid analogs having antihypertensive activity. U.S. Patent 4.X12.462. issued to Warner-Lamhert Co. (Morris Plains, NJ. U.S.A.). Bumpus. FM., K.J. Catt. A.T. Chiu. M. DeGasparo. T. Goodfriend. A. hsain. M.J. Peach. D.G. Taylor. Jr. and P.B.M.W.M. Timmermans. 1’191, Nomenclature for angiotensin receptors, Hypertension (In press). Carini. D.J. and J.V. Duncia. 1988, Angiotensin II receptor blocking imidazules. European Patent Application 0253310. Carini. D.J.. P.C. Wong and J.V. Duncia. 1989, Angiotensin II receptor blocking imidazoles and combinations thereof with diuretics and NSAIDS. European Patent Application 0324377. C’arini. D.J.. J.V. Duncia. P.E. Aldrich. A.T. Chiu. A.L. Johnson, M.E. Pierce. J.B. Santella. G.J. Wells, R.R. Wexler. PC. Wong and P.B.M.W.M. Timmermans. IYYI. Nonpeptide angiotensin II receptor ;ntagonists: The discovery of a series of n-thiphenylmethyllimidazoles as potent. orally-active antihypertensives. J. Med. Chem. tin press). Chang. R.S.L. and V.J. Lotti. IYYO. Two distinct angiotensin II receptor binding sites in rat adrenal revealed by new selective nonpeptide ligands. Mol. Pharmacol. 29. 347. Chiu. A.T.. W.F. Herhlin. DE. McCall. R.J. Ardecky. D.J. Carini. J.V. Duncia. L.J. Pease. P.C. Wong, R.R. Wexler. A.L. Johnson and P.B.M.W.M. Timmermans, l9XY, Identification of angiotensin II receptor subtypes, Biochem. ?‘ophys. Res. Commun. 165, 196. Chiu. A.T., D.E. McCall. W.A. Price, P.C. Wong, D.J. Carini. J.V. Dunch. A.L. Johnson, R.R. Wexler. S C. Yoo and P.B.M.W.M. Timmermans, 1990, Nonpeptide angiot, nsin II receptor antagonists. VII. Cellular and biochemical pharmarr,ilogy of DuP 753, an orally active antihypertensive agent, J. Pharmacol. Exp. Ther. 253. 71 I. Christ. D.D.. N. Wong, G.N. Lam and C.Y. Quon. 1990, Pharmacokinetics of the novel angiotensin II receptor antagonist DuP 752 in dogs, FASEB J. 4, A462. Christen. Y.. B. Waeber. J. Nussberger. M. Porchet. R. Lee, K. Maggon. P. Timmermans and H.R. Brunner, 1990. Eight-day administration of the orally active angiotensin II antagonist DuP 753 to normal subjects, J. Hypert., 8 (SuppI. 3) Sl6. Cody. R.P. and J.K. Smith. 1987. Applied Statistics and the SAS Programming Language (Elsevier Science Publishing Co., New York) p. 107. BP~~&.

Dudley. D.T., R.L. Panek. T.C. Major, G.H. Lu, R.F. Bruns. B.A. Klinkefus. J.C. Hodges and R.E. Weishaar, 1990. Subclasses of angiotensin II binding sites and their functional significance, Mol. Pharmacol. 3X. 370.

Garner. D. and M.M. Laks, 1985, New implanted chronic catheter device for determining blood pressure and cardiac ouput in conscious dog, Am. J. Physiol. 249, H68l. Haher. E., T. Koerner. L.B. Page, B. Kliman and A. Purnode, 1969. Application of radioimmunoassay for angiotensin I to the physiologic measurements of plasma renin activity in normal subjects, J. Clin. Endocrinol. Metab. 29, 1349. Hall, J.E.. 1986. Control of sodium excretion by angiotensin II: intrarenal mechanisms and blood pressure regulation, Am. J. Physiol. 250, RYhO. Hall. J.E., A.C. Guyton. T.E. Jackson, T.C. Coleman, T.E. Lohmeier and N.C. Trippodo, 1977. Control of glomrrular filtration rate by renin-angiotensin system, Am. J. Physiol. 233, F366. Hollenberg, N.K., 1979, Pharmacologic interruption of the reninangiotensin system, Ann. Rev. Pharmacol. Toxicol. 19, 559. Hollenberg, N.K., 1985, Angiotensin-converting enzyme inhibi:;on: renal aspects, J. Cardiovasc. Pharmacol. 7, S40. Keeton. T.K. and W.B. Campbell, 1981, The pharmacologic alteration of renin release, Pharmacol. Rev. 31, 81. Ondetti. M.A. and D.W. Cushman, 1978, Proline derivatives and related compounds. US Patent 4,105,776. Issued to E.R. Squibb and Sons. Inc. (Princeton, NJ, U.S.A.). Whitebread. S.. M. Mele. B. Kamber and M. de Gasparo. 1989, Preliminary biochemical characterization of two angiotensin I1 receptor subtypes, Biochem. Biophys. Res. Commun. 163, 284. Wong, P.C., SD. Hart, A.M. Zaspel, A.T. Chiu, R.J. Ardecky, R.D. Smith and P.B.M.W.M. Timmermans, 1990a. Functional studies of nonpepttde angiotensin II receptor subtype-specific ligands: DuP 753 (All-l) and PD123177 (All-2). J. Pharmacol. Exp. Ther. 255,584. Wong, P.C., W.A. Price, A.T. Chiu, D.J. Carini. J.V. Duncia, A.L. Johnson, R.R. Wexler and P.B.M.W.M Timmermans, 199Ob, Nonpeptide angiotensin II receptor antagonists: studies with EXP9270 and DuP 753, Hypertension 15, 823. Wong. PC.. W.A. Price, A.T. Chiu, J.V. Duncia, D.J. Carini, A.L. Johnson, R.R. Wexler and P.B.M.W.M. Timrnermans, 199Oc, Nonpeptide angiotensin II receptor antagonists. VIII. Characterization of functional antagonism displayed by DuP 753, an orally active antihypertensive agent, J. Pharmacol. Exp. Ther. 252, 719. Wong, P.C.. W.A. Price, A.T. Chiu, J.V. Duncia, D.J. Carini, A.L. Johnson, R.R. Wexler and P.B.M.W.M. Timmermans, 1990d. Nonpeptide angiotensin II receptor antagonists. IX. Antihypertensive activity in rats of DuP 753, an orally active antihypertensive agent, J. Pharmacol. Exp. Ther. 252, 726. Wong, PC., W.A. Price, A.T. Chiu, J.V. Duncia, D.J. Carini, A.L. Johnson, R.R. Wevler and P.B.M.W.M. Timrnermans? 1990e, Hypotensive action of DuP 753, an angiotensin II antagonist, in spontaneously hypertensive rats. Nonpeptide angiotensin II receptor antagonists: X, Hypertension 15, 459. Wong, P.C., W.A. Price, A.T. Chiu. J.V. Duncia, D.J. Carini, A.L. Johnson, R.R. Wexler and P.B.M.W.M. Timmermans, 199Of, Nonpeptide angiotensin II receptor antagonists. XI. Pharmacology of EXP3174: an active metabolite of DuP 753. an orally active antihypertensive agent, J. Pharmacol. Exp. Ther. 255. 211.