Drug Development Research 20:277-290 (1990)

Antihypertensive, Vasodilating, and Sympatholytic Activities of Ofornine@ in Spontaneously Hypertensive Rats Albert DeFelice, Harlan Lape, Patrick Horan, Richard Frering, Armand Brousseau, Bernard O’Connor, John Herrmann, and Denis Bailey

Department of Pharmacology, Sterling Research Group, Rensselaer, New York

ABSTRACT

DeFelice, A., H. Lape, P. Horan, R. Frering, A. Brousseau, B. O’Connor, J. Herrmann, and D. Bailey: Antihypertensive, vasodilating, and sympotholytic activities of ofornine in spontaneously hypertensive rats. Drug Dev. Res. 20:277-290, 1990.

This study of spontaneously hypertensive (SH) rats documents oral antihypertensive ac- tivity of the vasodilator Ofornine@, an anthranilamide whose effect was sustained, at least in part, by suppression of compensatory renal and neurohumoral responses. Doses of 2.5 to 100 mg/kg p.0. reduced systolic blood pressure (BP) by up to 75 mm Hg. Effect lasted 6-24 hr and did not diminish during a 16 day (25 mg/kg/day) regimen. Metoclopramide (1 0.0 mg/kg) and haloperidol (3.0 mg/kg) antagonized the antihypertensive effect. Antihy- pertensive activity was confirmed by direct (arterial cannula) monitoring where 0.001 -1 .O mg/kg i.v. reduced mean BP by 6-47% without tachycardia. Doses of 1-10 p,g/kg i.a. reduced perfusion pressure of the SH rat hindquarters by 35-74 mm Hg. A dose of 5.0 mg/kg p.0. reduced BP and urine norepinephrine levels of SH rats by 58 mm Hg and approximately 50% without raising plasma renin activity or causing sodium retention. How- ever, Ofornine@ (25 mg/kg p.0. daily for 6 days) did not affect reflex bradycardic or tachy- cardic effects of phenylephrine and sodium nitroprusside in SH rats. These and other results indicate that Ofornine@ reduces SH rat blood pressure through vasodilating and presynaptic adrenolytic activities, and that a dopaminergic mechanism may be involved.

Key words: potential antihypertensive, presynaptic adrenolytic, sympatholytic vasodilator, SH rats

Received final version November 20, 1989; accepted December 19, 1989.

Address reprint requests to Patrick Horan, Sterling Research Group, Rensselaer, NY 12144.

0 1990 Wiley-Liss, Inc.

278 DeFelice et al.

INTRODUCTION

The vasodilator Ofornine@ ( 1 -[2-pyridinyl amino) benzoyl] piperidine) has been shown to reduce blood pressure of hypertensive rats and dogs during 16 to 28 day dosing regimens [Lape et al., 1983; DeFelice and Lape, 19831. The aim of this study was to evaluate possible renal and neurohumoral effects in the SH rat that could underlie, in part, the sustained antihypertensive activity. We examined such effects because even slight decreases in arterial pressure provoke homeostatic pressor reflexes mediated by the kidneys and endocrine and autonomic nervous systems. Baroreceptor and chemoreceptor reflexes act via increases in sympathetic traffic to acutely oppose reductions in blood pressure [Guyton and Cowell, 19641, while the reinin-angiotensin-aldosterone vasoconstrictor system and sodium and water reten- tion are powerful longer-term homeostatic mechanisms [Davis et al. 1961; Cowley et al, 19711. Such adjustments can reduce efficacy of antihypertensive agents. For example, auto- nomic reflexes and sodium retention reduce the depressor effect of the vsodilator hydralazine by as much as 75% [Koch-Weser, 19741, and after 2 years of monotherapy complete tolerance has been observed in approximately 90% of patients [Moyer, 19531. Ad described below, Ofornine@, unlike hydralazine, decreased rather than elevated urinary catecholamine and dopamine excretion, and tended to reduce mean plasma renin activity. It did not elicit tachy- cardia or reduce urinary sodium excretion in the SH rat. Unlike clonidine, this agent did not depress blood pressure or heart rate after intracerebro-ventricular administration. This profile, ability to reduce perfusion pressure of SH rat hindquarters, and inhibition of neurogenic cat nictitating membrane contractions suggests that combined vasodilating and peripheral presyn- aptic adrenolytic activities underlie sustained antihypertensive activity in this model.

MATERIALS AND METHODS Acute Oral and Intravenous Effects on Blood Pressure of SH rats

Adult male spontaneously hypertensive rats (250-350 g; Wistar-Kyoto Strain; Charles River Labs, Wilmington, MA) were fasted overnight. Systolic blood pressure (SBP) was then recorded prior to and at 2, 4, 6, and 24 hr after administration of Oforninea (Sterling Drug, Inc., Rensselaer, NY). Ofornine@ suspended in 1% aqueous gum tragacanth was given by gavage at 1.25, 5, and 25 mgikg in a volume of 1 ml/kg of body weight. Hydralazine (1-4 mg/kg), clonidine (0.1-1.0 mgikg), and vehicle alone (1% gum trag.; 1.0 ml/kg) were also given (N = 5-10 ratsigroup). Some of the rats were pretreated with propranolol (10.0 mgikg), chlorpheniramine (1 .O mg/kg), or metoclopramide (10.0 mg/kg) 1 hr prior to receiving the 5.0 mgikg dose of Ofornine@. Blood pressure of conscious rats was measured indirectly using a miniature sphygmomanometer cuff as described and validated elsewhere [Kersten et al., 1947; Ablondi et al., 19471.

For direct recording of intravenous vasodepressor effects, the SH rats were anethetized with sodium pentobarbital (40 mg/kg i.p.), and the 1. common carotid artery and 1. jugular vein were cannulated for recording arterial blood pressure (Statham-Gould P23 1D transducer; Grass model 5 Polygraph) and administering drugs. After a 20 min stabilization period, rats received either Ofornine@ dissolved in 0.004 N lactic acid-isotonic saline, clonidine dissolved in iso- tonic saline, or saline alone.

For direct recording of effects of central administration, SH rats were anesthetized with sodium pentobarbital (40 mgikg i.p.) and cannulae described elsewhere [Weeks and Jones, 1960; Feldberg and Sherwood, 19531 were inserted into the abdominal aorta and the lateral cerebral ventricle. After 3 days recovery, blood pressure was recorded from the aortic cannula before and for 30 min after ventricular administration of Ofornine@ (10 and 25 pg/kg), or vehicle (isotonic saline; 0.015 ml/kg). Clonidine was given at 1 pg/kg to all rats as either a positive control or 30 min after Ofornine@ or saline to confirm responsiveness of each prep- aration.

Antihypertensive Activity of Ofornine@ in SH Rats 279

Subacute Oral Effects on Blood Pressure

Ofornine@ was given in single daily oral doses of 25 mglkg, alone or combined with hydrochlorothiazide (10 mgikg), for 16 days (N = Ygroup) with controls receiving vehicle (1 ml/kg of 1% gum tragacanth). The SBP was monitored daily (except for weekends) as de- scribed above at 2 ,6 , and 24 hr posttreatment and on the third day after treatment was stopped.

In other studies, Ofornine@ (10.0 mg/kg) or vehicle (1 .O ml/kg of 1% gum tragacanth) was administered alone or combined with either propranolol (10.0 mg/kg), captopril (5.0 mg/kg), or hydrochlorothiazide (10.0 mg/kg) for 5 consecutive days. The SBP was monitored daily at 1, 3, 5 , and 24 hr after treatment.

Acute and Sub-Acute Effects on Plasma Renin Activity (PRA)

SH rats were fasted overnight. Baseline SBP was measured indirectly prior to and 4 hr after oral administration of Ofornine@ (5.0 mg/kg), clonidine (0.1 mg/kg), or vehicle (1% gum tragacanth; 1 .O ml/kg). Rats were then decapitated. Plasma was separated from blood and kept at -70°C until assayed. PRA was determined as rate of angiotensin I formation in vitro. Angiotension I levels were measured by radioimmunoassay (New England Nuclear, Boston, MA).

To assess longer term effects on PRA, other SH rats were medicated with Ofornine@ (25 mglkg), alone or combined with hydrochlorothiazide (10 mg/kg), daily for 14 days. Blood pressure of these and vehicle-treated controls were measured at 2, 6, and 24 hr on day 1, 7, and 14. Rats were sacrificed after the last reading and PRA was determined as described above.

Urine Sodium, Volume, and Catecholamine Excretion

SH rats were fasted and baseline SBP was obtained from the foot as described above. Rats were then given 1% gum tragacanth (30 ml/kg p.0.) in which was dissolved approxi- mately equal antihypertensive doses of Ofornine@, reserpine, hydralazine, or clonidine. Con- trols received vehicle alone. The urinary bladders were emptied, the urine was discarded, and the rats were placed in metabolism cages for collection of urine in beakers containing con- centrated HC1. After 5 hr, bladders were again emptied and the urine was combined with the spontaneously voided sample. The SBP was again measured, the rats were sacrificed and the brain and heart were frozen at -70°C. Tissue catecholamines were isolated by adsorption onto alumina using the method of Anton and Sayre 119621. The analysis and quantitation were carried out on a Bioanalytical Systems Liquid Chromatograph using electrochemical detection. The 5-hour urine sample was analyzed for sodium using an Instrumentation Laboratory flame photometer (Watertown, MA). Urine catecholamines were determined according to the method of Kissinger et al. [ 19751 using alumina, acetic acid elution, liquid chromatography, and electrochemical detection.

Effect on Perfusion Pressure of SH Rat Hindquarter

Cannulae were placed in the right jugular vein and carotid artery for intravenous injec- tions and recording blood pressure. The hindquarter was then perfused with blood drawn from the abdominal aorta and delivered at constant flow using a Holter roller pump. The circuit contained connections for intra-arterial injections, recording perfusion pressure (Statham- Could transducer; Grass polygraph), and norepinephrine infusion. Norepinephrine ( 1 pg/ml) was continuously infused to increase vascular tone and to raise perfusion pressure to a sus- tained level of 200 mm Hg. This baseline perfusion pressure was achieved in all experiments by adjusting norepinephrine infusion rate and pump output which remained constant thereafter. Complete hindquarter vascular isolation was confirmed by total loss of perfusion pressure when the pump was stopped.

After an equilibrium period of 10 min, sodium nitroprusside, hydralazine, phentola-

280 DeFelice et al.

mine, Ofornine@, or vehicle (isotonic saline) was injected into the perfusion circuit at a volume of 0.002 ml. Each agent was tested at three dosages given to five rats in random order. Since blood flow was held constant, reduction in perfusion pressure reflected decreased vascular resistance.

Effect on Cardiovascular Responses to Phenylephrine and Nitroprusside

The aorta of SH rats (300-350 g) were cannulated as described above and basal blood pressure and heart rate were recorded after 4 days recovery. Isotonic saline (0.5 ml/kg), 1-phenylephrine (50 pg/kg), and sodium nitroprusside (8 pg/kg) were then administered in- travenously, in succession. The transient effects of each agent were allowed to subside com- pletely before the next agent was injected. Rats were then randomly divided into two groups and treated for 6 days with either Ofornine@ (25 mglkgiday) or vehicle (1% gum tragacanth, 1 ml/kg). Two hr after treatment on day 6, the rats were retested with the alpha agonist, vasodilator, or vehicle and cardiovascular changes compared to those occurring prior to pre- treatment.

Effects on Preganglionic Stimulation of Cat Nictitating Membrane

The method for inducing transient neurogenic contractures of the cat nictitating mem- brane is described elsewhere [Lape and Hoppe, 19561. Briefly, adult mongrel cats were anesthetized (sodium pentobarbital, 35 mg/kg i.p.), blood pressure was monitored from the femoral artery, and the preganglionic vagosympathetic trunks of the superior cervical ganglia were isolated, severed to effect decentralization, and attached to bipolar electrodes. Nerves were stimulated at 0.5, 5 , or 8 Hz for 10 sec and repeated at 10-15 min intervals, using a Grass S-9 stimulator delivering impulses of 6 msec duration and 1-2 V amplitude. Transient tension changes in preloaded membranes ( 2 g) were recorded as mm deflection on a Grass Polygraph. When the transient contractures had become reproducible, Ofornine@ was injected at 1 .O mgikg i.v. during peak contracture. The membrane was stimulated periodically until basal response and systemic pressure had been restored.

Effect of Metoclopromide or Levophed@ on Duration of Intravenous Antihypertensive Activity

The r. common carotid artery and 1. jugular vein of SH rats were cannulated for monitoring mean blood pressure and heart rate and for drug administrations as described above. After 10 min equilibration, Ofornine@ was injected at 0.1 mgikg i.v. Twenty minutes later, metoclopromide (0.1 mg/kg/min), 1-norepinephrine ( 1 pg/kg/min), or isotonic saline were infused intravenously at 0.1 ml/min. for 10 min. The infusion was then stopped and rats were monitored €or an additional 25-30 min.

Statistical Analysis

Student’s t test was used to compare 2 mean values. For comparing multiple pairs of mean values, analysis of variance (ANOVA) for repeated measures or ANOVA followed by Duncan’s multiple range test was used.

RESULTS Acute Antihypertensive Effects

As shown in Figure 1, Ofornine@ significantly lowered the SBP of SH rats by 17 to 67 mm Hg in a dose-related fashion. Effect was well established at 2 hr, maximal at 4, and lasted more than 6 hr at the higher dosages. Hydralazine ( 1 to 4 mg/kg p.0.) and clonidine (0.1 to 1 .O mg/kg p.0.) reduced SBP by a maximum of 39 to 63 and 37 to 74 mm Hg, respectively (Table 1). Onset ( < 2 hr), time to peak effect (4 to 6 hr) and a duration of action exceeding 6 hr was similar to that observed for Ofornine@.

Antihypertensive Activity of Ofornine@ in SH Rats 281

lgo r I8O 170 1

Ly w 3 160 v,

150 -

82 140 -

m g 130 -

-

3 E

2 2 120 -

v, CA >. 110 -

o----.O VEHICLE (1% GUM TRAG.) A- - - -A 1.25 rng/kg

D - - -0 5.0 rng/kg +0 25 rng/kg

I I I \#

0 2 4 6 24

HRS. POST-TREATMENT Fig. 1. vehicle.

Oral antihypertensive activity of Oforninem in conscious SH rats. *, **, P<0.05 or 0.01 vs.

TABLE 1. Maximum Oral Antihypertensive Effect' of Hydralazine and Clonidine in Spontaneously Hypertensive Rats

Systolic blood Dose pressure (mm HE)

Compound (mg/kg p.0.) Baseline

Hydralazine 0 173 * 2 1 171 * 3 2 170 t 3 4 171 t 3

Clonidine 0.1 173 * 4 0.3 173 2 4 1.0 173 k 4

Max A -5 t 1

-39 t 5* -56 * 3* -63 * 8* -37 t 6* -45 k 2* -74 * 4*

'Usually occurred at 2 or 4 hr post-medication; mean t SEM (N = Sigroup). *P <.01 (ANOVA).

When administered intravenously, Ofornine@ (0.01-1 .O mg/kg) reduced SBP by a max- imum of 30 to 50% within 20 to 25 min and without significantly changing heart rate (Table 2). Pressure were still reduced 30 min after injection when the experiments were terminated. Clonidine (2.5, 5 and 20 Fg/kg) exerted a biphasic effect on blood pressure, raising systolic

282 DeFelice et al.

TABLE 2. Intravenous Antihypertensive Activity of Ofornine@ in Anesthetized SH Ratst Systolic B.P. (mm Hg) Diastolic B.P. (mm Hg) Time to Heart rate (bimin)

Dosea At Max. At Max. m a . change At Max. (mg/kg) Basal Change Basal Change (min) Basal Change

391 t 12 O(vehic1e) 181 t 11 171 f 15 144 +- 18 135 t 15 - 0.01 191 t 4 130 t 21 164 * 19 101 f 19 25 F 2 408 F 20 406 f 18 0.10 168 5 14 89 t 10* 146 f 15 74 t 10" 28 f 4 458 t 18 454 +- 12 1 .o 160 t 15 109 t 12* 130+- 13 89 t 11" 20 +- 6 398 * 12 403 f 10

386 2 16

+Values are mean t SEM (N=S/group) aAdministered as a single bolus. *P<O.Ol vs. baseline value.

and diastolic blood pressure a maximum of 12 to 37 mm Hg for 10 to 15 min followed by decreases in systolic and diastolic pressure and heart rate of45 to 85 mm Hg, 37 to 75 mm Hg, and 31 to 61 beatdmin, respectively. Vasodepressor phase lasted longer than 30 min.

Antihypertensive Activity of Repeated Oral Doses

Marked transient (i.e., <24 hr duration) reductions in systolic pressure of the SH rat similar to those seen in the acute studies occurred during days 1 to 6 of the regimen (Fig. 2). The maximum effect occurred 2-6 hr after medication. After 7 days of therapy, however, SBP usually remained significantly depressed 24 hr after medication. Ofornine@ combined with HCTZ also reduced SBP for 24 hr after 7 days of treatment. Maximum effect of combination treatment was not greater than that seen with Ofornine@ monotherapy. No tolerance to the activity of Ofornine@ alone or combined with HCTZ developed during the 16-day regimens. In both test groups SBP returned to premedication levels within 3 days of drug withdrawal. No significant change in SBP occurred in the control group.

When given at the lower dose of 10.0 mg/kg for 5 days, Ofornine@ depressed SBP daily by 25-30 mm Hg (P<O.OOl) within 3-5 hr of administration. The SBP was reduced by 45-60 mm Hg when Ofornine@ was combined with either propranolol, captopril, or hydrochlorothi- azide, and peak effect usually obtained by day 3. At the doses tested, each of the standards alone reduced SBP by approximately 15-30 mm Hg. Accordingly, the depressor effects of low dose Ofornine@ and these standards were additive (P<0.05), not synergistic, in the SH rat.

Effects on Plasma Renin Activity

Despite a mean reduction in SBP of 48 mm Hg 4 hr after Ofornine@ treatment, plasma renin activity was not elevated (Table 3). An equal antihypertensive dose of clonidine pro- voked a significant increase in renin activity of approximately 60%.

Ofornine@ also did not raise PRA of SH rats treated with 25 mgikg p.0. each day for 14 days. This study confirmed the longer-term antihypertensive activity of Ofornine@ observed in the 16-day study (see above). That is, 24-hr reduction of SBP was achieved after 7 days of therapy whether Ofornine@ was given alone or combined with hydrochlorothiazide and toler- ance did not occur during the 2-week regimens (Table 4). However, the combination of hydrochlorothiazide and Ofornine@ did promote PRA by more than 2-fold.

Urine Sodium and Catecholamine Levels

Concurrent effects of antihypertensive doses of several agents on urine sodium and tissue and urinary catecholomine content are shown in Table 5 . At dosages which reduced SBP by approximately 50 mm Hg, clonidine appreciably promoted urinary volume and sodium excretion. Equivalent antihypertensive doses of the other agents did not significantly affect these parameters.

Antihypertensive Activity of Ofornine@ in SH Rats 283

200

190

180

170

160

150

140

130

120

110

M VEHICLE (1% GUM TRAC.) A- - - -A OFORNINE"

25 rng/kg p.o./day/l6 days

j,' T * :

0 1 2 6 7 8 13 14 15 16 19

t DAYS AFTER START OF MEDICATION MEDICATION

STOPPED

Fig. 2. at 2, 6, and 24 hr each day. *, P<0.05 vs. vehicle.

Blood pressure of SH rats treated once daily with Ofornine@ for 16 days. Pressure was recorded

TABLE 3. Acute Effect of Ofornine@ on Systolic Blood Pressure and Plasma Renin Activity of Conscious SH Ratt

Systolic blood pressure (mm Hg) Plasma renin

Treatment 4 hr activity (mgikg P.o.) Basal Post-treatment (ng AUmlihr) Vehicle 172 * 3 167 + 3 12 2 1 (1% gum trag.) Ofornine@ 172 + 2 124 + 3* 9 + 1

Clonidine 112 2 4 129 + 3* 19 * 2* (0.1)

(5.0)

'All values are mean + SEM; N = 16 ratsigroup. *P<O.Ol vs. basal value (SBP) or value for vehicle (PRA)

Both Ofornine@ and clonidine decreased the amount of norepinephrine excreted in 5 hr by approximately 50%. OfornineB also reduced dopamine excretion by 51% and tended to depress epinephrine excretion as well. Hydralazine, however, promoted mean urine catechol- amine levels, effect on epinephrine being significant at the P<0.05 confidence level. The only significant effect of any of these agents on tissue catecholamine content was an appreciable decrease in cardiac norepinephrine levels in reserpine-treated rats. Accordingly, Ofornine@ did not affect tissue catecholamine levels but markedly reduced urinary norepinephrine and dopamine excretion despite a 54 mm Hg mean reduction in SBP.

284 DeFelice et al.

TABLE 4. Effect of Repeated Doses of Ofornine@ on SH Rat Blood Pressure and PRA' Systolic blood pressure (mm Hg)

Treatment base 1 in e Day 15a (mgikgiday 1 Baseline Day I Day 7 Day 14 SBP PRA Vehicle 1 7 6 2 3 - 1 + - 3 2 2 4 - 1 t 4 1 8 2 t 3 1 3 t 4 ( 1 % gum trag.: 1 ml/kg) Ofornine 177 k 5 -53 t 4* -52 * 4* -51 t 5* 148 t 2" 12 t 2 (25) Ofornine (25) + 176 t 4 -52 t 4" -51 2 3* -54 t 4* 154 t 3* 30 t 3* HCTZ (10)

Maximum change from

'SH rats were treated once daily for 14 consecutive days. All values are mean t SEM (n = 5/group). "Systolic blood pressure (SBP) and plasma renin activity (PRA; ng AI/ml/hr) determined 24 hr after last medication. *P<O.Ol vs. vehicle control.

TABLE 5. Effect of Ofornine@ on Tissue Content and Urinary Excretion of Epinephrine (EPI), Norepinephrine (NE), and Dopamine (DA)+

Analysis of 0-5 hr urine sample Brain Heart

Change in Dose in systolic

(nmoleig) (mg/kg) B.P. Vol. Na+ EPI NE DA (nmole'g) Compound (P.O.) (mmHg) (ml) (uEq) (ng) (ng) (ng) NE DA EPI NE DA

(1% G.T.) ml/kg t 7 .3 t 41 t 10 t 38 t 115 2 0.2 2 0.3 2 0.02 t 0.5 t 0.03 Ofornine 5.0 -54 7.6 121 32 149* 370" 2.9 8.2 0.13 4.22 0.11

2 3 t 0.3 t 18 t 4 t 6 t 41 * 0.1 5 0.2 t 0.03 t 0.05 t 0.02 Hydralazine 2.0 -55 8.0 119 80* 429 1000 3.0 8.8 0.14 5.0 0.14

+ 5 +- 0.4 * 17 t 10 -+ 77 -+ 235 t 0.1 k 0.3 t 0.02 -+ 0.06 t 0.02 Reserpine 1.0 -65 6.4 99 60 256 686 2.6 8.3 0.12 1.2** 0.07

t 4 t 0.4 t 19 t 7 t 25 t 62 2 0.2 t 0.4 2 0.1 -+ 0.1 k 0.01 Clonidine 0.1 -47 12.5* 623* 61 149* 833 2.8 8.3 0.16 5.1 0.16

t 7 t 0.01

'After dosing, urine was collected for 5 hr at which time blood pressure and tissues were obtained. All values are mean t SEM; N = 5 . *P<.05. **P<.Ol.

Control 30 -3 7.5 196 55 281 726 2.6 8.1 0.18 5.0 0.13

t 0.8 t 59 t 8 2 29 t 231 t 0.1 * 0.5 t 0.01 2 0.3

Vasodilating Activity in Hind Limb

Vasodepressor effects of standard vasodilators and Ofornine@ when injected intraarte- rially as single bolus doses are indicated in Table 6. Maximum dose-related reductions in perfusion pressure elicited by sodium nitroprusside (0.1-10 pgikg), hydralazine (1-100 pg/ kg), phentolamine (0.001-0.1 pg/kg) and Ofornine@ (0.1-10 pgikg) ranged from 16 to 123, 7 to 63, 6 to 122, and 4 to 74 mm Hg, respectively. Effects peaked within 5 sec and, at the highest doses, lasted approximately 5 to 10 min except for phentolamine whose effect persisted for 29 min. Rank order of potency was phentolamine > sodium nitroprusside > Ofornine@ > hydralazine.

Effect on Cardiovascular Responses to Phenylephrine and Sodium Nitroprusside

The vasopressor effect of phenylephrine, an alpha adrenoceptor agonist, was not inhib- ited in SH rats pretreated with 25 mgikg of Ofornine@ daily for 6 days as it transiently raised

Antihypertensive Activity of Ofornine@ in SH Rats 285

TABLE 6. Effect of Ofornine@, Sodium Nitroprusside, Hydralazine, and Phentolamine on Perfusion Pressure in the Intact Perfused Hindquarter of Anesthetized Spontaneously Hypertensive Rats'

Test Dose perfusion Time to max. recovery AEDSoa compound (kgikg i.a.) pressure (mm Hg) fall (sec) (minutes) (pgikg i.a.) Control 0.002mUkg 0 (Saline) Sodium 0.10 16 +- 3* 5 1 * 0.2 nitroprusside 1 .0 82 * 5* 5 2 +- 0.2 0.35

10.0 123 t 2* 5 5 * 0.5

Max. fall Time to

- - -

Hydralazine 1 .0 7 +- 1* 5 1 +- 0.2 10.0 25 I+_ 5* 5 2 I+_ 0.2 50.0

100.0 63 t 9* 5 9 +- 5.4 Phentolamine 0.001 6 + - 1 5 12 +- 4.5

0.01 36 +- 5* 5 17 +- 6.4 0.01 29 +- 8.0 0.10 122 +- 8* 5

Ofornine 0.10 4 2 1 5 1 +- 0.7 1 .o 35 +- 9* 5 2 t 0.5 2.0

10.0 74 +- 17* 5 4 +- 1.2

'The limb was perfused with norepinephrine to maintain a basal perfusion pressure of 200 mm Hg. "Approximate intraarterial dose calculated to reduce perfusion pressure by 50 mm Hg. *P<0.05.

TABLE 7. Lack of Effect of Ofornine@ Pretreatment on Responses' of Conscious SH Rat to Phenylephrine or Sodium Nitroprusside

Control: gum tragacanth; 1% daily X 6 days

Ofornine: 25 mgikg daily x 6 days

Change in Change in heart Change in Change in Challenge SBP (mm Hg) rate (bimin) SBP (mm Hg) heart rate (bimin)

agent Day 1 Day 1 Day 1 Day 1 (pg/kg i,a): (Baseline) Day 6 (Baseline) Day 6 (Baseline) Day 6 (Baseline) Day 6 Phenylephrine 50 +- 7 -57 +- I 1 -37 +- 8 (50) Sodium nitroprusside -35 ? 4 -30 * 5 50 * 11 12 +- 9 -30 +- 2 -29 +- qNS 43 +- 6 18 +- 9NS (8) Blood pressure Systolic blood Heart rate Systolic blood Heart rate and heart rate pressure (mm Hg) (beatsimin) pressure (mmHg) (beatsimin)

prior to Day 1 Day 6 Day 1 Day 6 Day 1 Day 6 Day 1 Day 6 challenge: 183 I+_ 5 187 +- 9 414 +- 16412 +- 12 181 * 8 156 +- 5* 421 * 11 440 +- 15

76 t 9 63 +- 5 54 +- gNS -80 +- 18 -72 +- 15NS

'Parameters were directly measured from the abdominal aorta of conscious animals. Values are mean +-

SEM; N = 8-l0igroup. NS, Not significantly different from corresponding change in rats pretreated with vehicle alone. *P<0.01 vs. value prior to receiving Ofornine.

blood pressure by approximately 50 mm Hg before or after Ofornine@ (Table 7). Reflex bradycardic and tachycardic effects of phenylephrine and sodium nitroprusside were also not significantly affected by Ofornine@.

Inhibitory Effect of Dopamine Antagonists on Vasodepressor Effect of Ofornine@

As shown in Figure 3, metoclopramide significantly reduced duration of antihyperten- sive activity of a single intravenous bolus of Ofornine@ in anesthetized SH rats. Both meto-

2% DeFeke et al.

r" E E -

ff 0 3 m

-

160 -

140 -

-

120 -

2 s TOO- E

8 0 -

METOCLOPRRMIDE- IN FUSED

SALl N E- INFUSED

I I I t I I

0 5 10 15 20 0 5 10 15 20 25 t I + POST-INFUSION+ t OFORNINE @ METOCLOPRAMIDE

(0.7 mgkg 1.V.) (0.1 rng'kghinil0 min)

SALINE (0.1 rnl/min/lO min) 01

TIME (Minutes)

Fig. 3. Inhibitory effect of metoclopramide on intravenous antihypertensive activity of Ofornine@ in anesthetized SH rats. Post-metoclopramide infusion blood pressure significantly exceeded that in saline- infused rats (P<0.05; repeated measures ANOVA).

clopramide and haloperidol, but not propronolol or chloropheniramine, will antagonize the oral antihypertensive activity of Ofornine@ as well (Table 8). Metoclopramide alone did not significantly affect blood pressure.

Inhibitory Effect of Ofornine@ on Neurogenic Nictitating Membrane Contractions

The amplitude of low (0.5 Hz) frequency-induced contractures was reduced on average of 5 5 % , whereas responses at the higher stimulation frequencies were unaffected (Table 9). Systolic and diastolic blood pressure was also appreciably reduced, and both the adrenolytic and hypotensive activities persisted for approximately 90 min. Inhibitory effect of guanethi- dine, phentolamine and hexamethonium on high-frequency (8 Hz) contractures in this bioassay is reported elsewhere [Lape and Hoppe, 19561.

lntracerebroventricular Effects of Agents

Unlike clonidine, Ofornine@ did not affect blood pressure or heart rate of conscious SH rat when injected into the lateral cerebral ventricle. However clonidine (1 pgikg) significantly depressed both mean blood pressure and heart rate by approximately 20% whether adminis- tered alone or following Ofornine@.

DISCUSSION

Results of these and related studies indicate that the dose-dependent vasodepressor activity of Ofornine@ derives from combined vasodilating and sympatholytic effects. Vasodi-

Antihypertensive Activity of Ofornine@ in SH Rats

TABLE 8. Inhibitory Effed of Dopamine Antagonists an Ckal Anti-Hypertensive Activity of Ofornine@ in SH Ratsi

Maximum reduction in systolic blood pressure (mm HgP

PretreatmenP Vehicle Ofornine@ (m@g p.0.) (1 mlikg) (5.0 mglkg)

Vehicle -14 f 7 -51 f 2' (1% gum trag.) MeFoclopramide -12 t 6 -22 t 2* (10.0.)

(3.0)

(10.0)

el -0)

'MI vdues are mean U SEM; N=5-1O/group. "Rats were pretreated 1 hr prior to administration of Ofornine@ OE whicfe atme. %BP was monitored indirectly at 2, 6, and 24 hr after adminis- tration of Ofornine@ or antagonists per se. "P<0.05 m. vehicle. *P<O.O5 vs. Ofornine@ eEm with no antagoi%st pretreatrrsent.

Haloperidol -21 t 6 -32 t 4*

Propranolol -14 f 3 -46 f 4

Chlorpheniramine -13 t 7 -51 f 3

287

TABLE 9. Elect of Oforae@ on NenFogenie Cat Nktitating Membrane Contractions'

Contractions (mm): (Syst./diast.; mm Hg) Niciibhng membrane B i d pressure

Stirnulation frequency Pexent Percent OIZ) Basat Posttreatment inhibition Basai Posttreatment reductton

0.5 I2 2 3 5 2 I* 55 2 7 178 2 11 138 t 11* -40 5 10

5.0 37 t 1 37 t 3 O f 7 8.0 39 2 4 37 f 4 -5 2 1 198 3~ 16 140 t 11* -58 t 13

154 +- 12 95 t 11* -59 f 10

'Reganglionic nerves of the superior cervical ganglia of anesthetized cats (sodium pentobarbital; 35 mg/kg i.p.) were shocked prior to and after receiving Ofornine (1.0 mg/kg i.v.). All values are mean 2 SEM (n =4/group). *P<0.05 vs. baseline mean value.

120 +- 7 86 5 8* -35 t 8

lation of the SH rat hindlimb is achieved within the range of doses, which, given intrave- nously, effectively reduce systemic blood pressure of SH rats. Furthermore, decreases in total peripheral vascular resistance rather than cardiac output parallel the hypotensive activity in normotensive dogs [DeFelice and Lape, 19831. Evidently Ofornine@ can directly relax the vascular smooth muscle independent of alpha receptors because it appreciably reduces the perfusion pressure of the denervated hindlimb of the dog [DeFelice and Lape, 19831. Since Ofornine@ does not affect pressor activity of phenylephrine in SH rats, alpha adrenoceptor blockade is not considered to mediate the vasorelaxant effect in this species as well. However, anti-hypertensive activity in the conscious intact SH rat is associated with a marked reduction in urinary catecholamine excretion rather than the increase seen with an equal anti-hyperten- sive dose of hydralazine. This indicates that Ofornine@ can also significantly decrease rate or amount of norepinephrine released from noradrenergic neurons. As Ofornine@ does not have

288 DeFelice et al.

beta adrenoceptor blocking activity [DeFelice and Lape, 19831, interference with noradren- ergic neuronal traffic or function might also account, in part, for the absence of tachycardia or hypereninemia in the SH rat. Such responses would usually be provoked by a fall in blood pressure and reflex stimulation of sympathetic nerve activity [Vander, 1967; Winer et al., 19691. Ofornine@ does, in fact, inhibit the reflex pressor response to temporary occlusion of cat carotid arteries by 30% [DeFelice and Lape, 19831. Since Ofornine@ does not interfere with the cardiovascular effects of phenylephrine or isoproterenol, and is inactive after ventricular administration, but does inhibit neurogenic nictitating membrane contractions, it is possible that this agent has peripheral presynaptic adrenolytic activity.

The observation that Ofornine@ inhibits nictitating membrane contractures evoked by low-, but not high-, frequency preganglionic stimulation suggests a mechanism of action different from that of conventional ganglionic or adrenergic neurone blocking agents. For example, it has been previously demonstratedin this laboratory (Lape and Hoppe, 1956), and confirmed elsewhere (Armstrong and Boura, 1973), that guanethidine, at a dose of 2.0 mg/kg i.v., will block peripheral adrenergic neuronal function by 70% at both low and physiologi- cally high (8-10 Hz) stimulation frequencies with hexamethonium also blocking high-fre- quency transmission by at least 50%. Conversely clonidine, which acts on neuronal alpha-2 adrenoceptors to inhibit norepinephrine release [Starke et al, 19721, preferentially inhibits low-frequency adrenergic nerve transmission [Armstrong and Boura, 19731. An analogous frequency-dependent sympathoinhibitory effect of certain dopaminergic agents has also been demosntrated [Long, 19751. There is no evidence in the present study that Ofornine@ acts at peripheral or central alpha receptors to account for the reduced urinary norepinephrine excre- tion in the SH rat. However, metoclopramide, a dopamine DA, receptor antagonist [Kebabian, 19831, does significantly antagonize both the oral and intravenous antihypertensive activity of Ofornine@. Accordingly, Ofornine@ may stimulate DA, receptors as proposed for certain indirect-acting vasorelaxants. Many dopaminergic vasodilators such as apomorphine, ergot derivatives, and pyribedil relax vascular smooth muscle by blocking adrenergic transmission since they are ineffective in phenoxybenzamine or hexamethonium-pretreated animals [Boy- laert et al., 19781. Such agonists, which are structurally more diverse than direct-acting DA, (vascular) dopaminergic agonists, are believed to bind to DA, receptors, depressing norepi- nephrine release [Goldberg and Kohli, 19831. Such binding by Ofornine@ remains to be determined.

Additional studies are required before concluding that a DA, receptor-mediated presyn- aptic inhibition of sympathetic neurotransmission contributes to the anti-hypertensive effect of Ofornine@. It remains to be determined whether metoclopramide also antagonizes the effect of Ofornine@ on norepinephrine excretion and whether S-sulpiride, a more selective DA, receptor antagonist (Kaiser and Jain, 1985), can confirm effects of metoclopramide. It is possible that Ofornine@ interfers with ganglionic transmission as well as presynaptic norepinephrine re- lease. The presence of inhibitory DA, and DA, receptors on ganglia has been demonstrated (Sabouni et al., 1986).

The profile of activity of Ofornine@ may predict several clinical advantages over non- adrenolytic vasodilators as well as potent neurone blockers, such as guanethidine, usually reserved for patients with relatively intractable hypertension. Because reflex stimulation of the sympathetic nervous system and mobilization of several renal pressor responses are apparently blunted, need for adjunctive therapy to improve extent or duration of effect may be reduced. Indeed, co-therapy with hydrochlorothiazide did not promote the sustained activity of maxi- mally effective repeated doses of Ofornine@ (25 mg/kg) in the SH rat. Additive rather than synergistic effects of a suboptimal dose of Ofornine@ (10 mg/kg) combined with propranolol, hydrochlorothiazide, or captopril for 5 days also suggests that Ofornine@ activity is not significantly opposed by important neurohumoral reflexes, at least in the rat model. The latter study, furthermore, demonstrates that Ofornine@ can be effectively used at less than mono- therapy dosages when combined with compatible antihypertensive medication. The preferen-

Antihypertensive Activity of Oforninea in SH Rats 289

tial interference with low frequenceis of adrenergic transmission, as reported for clonidine [Schmitt, 19701, would allow passage of high rates under conditions of cardiovascular stress. For example, Ofornine@ only partially blocked the pressor response to bilateral carotid artery occlusion in the cat. This could predict reduced liability for postural and exertional hypoten- sion compared to guanethidine, as reported for clonidine and bretylium [Armstrong and Boura, 1973; Boura and Green, 19621.

ACKNOWLEDGMENTS

The authors thank Ms. Gail Fowler for typing the manuscript.

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