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The cardiovascular activity of 2-amino-5-(3,4-dichlorophenoxymethyl)-2-oxazoline (APMO)
TOXICOLOGY
AND
APPLIED
PHARMACOLOGY
26,513-522
(1973)
The Cardiovascular Activity 2-Amino-5-(3,4-dichlorophenoxymethyl)-2-oxazoline
of (APMO)’
A. H. ABDALLAH AND H. D. WHITER Chemical
Biology
Research, Received
The Dow December
Chemical 14,1972;
Company, accepted
April
Midland,
Michigan
48640
12,1973
The Cardiovascular Activity of 2-Amino-5(3,4-dichlorophenoxymethyl)2-oxazoline (APMO). ABDALLAH, A. H. AND WHITE, H. D. (1973). Toxicol. Appl. Pharmacol. 26, 513-522. The cardiovascular activity of APMO was studied in dogs. When compared on a mgfkg basis, its pressor effect was less than that of d-amphetamine. APMO has a diphasic action on blood pressure: a depressor followed by a pressor activity. Upon repeated administration, tachyphylaxis developed to its pressor but not to its depressor effect. The pressor effect of the APMO was antagonized either by reserpine or by phenoxybenzamine pretreatments. This finding suggests that the compound is acting indirectly through the releaseof norepinephrine from peripheral nerve endings. Spinalectomy did not significantly affect its pressor activity. This finding indicates that the brain is not involved in its pressor effect. Recently, we reported on the anorexigenic activity in various animal species of 2-amino-5-(3,4-dichlorophenoxymethyl)2-oxazohne (APMO) (Abdallah, 1972, 1973). Our findings showed that, in approximately equipotent anorexigenic doses, the stimulant effects of APMO on the central nervous and the cardiovascular systems were less than those of d-amphetamine. This paper presents the results of the cardiovascular activity of APMO. METHODS Mongrel dogs of either sex (7-l 8 kg) were used. They were anesthetized with pentobarbital sodium, 30 mg/kg (iv). The right carotid or femoral arteries were cannulated and blood pressure was recorded by means of Gould Brush polygraph through a Statham transducer. Heart rate (HR) and lead II electrocardiogram (ECG) were also continuously monitored. The trachea was intubated, and in certain experiments respiration rate was recorded. Drugs were dissolved in saline and injected via an indwelling catheter in the femoral vein. In some experiments, acute bilateral vagotomy was performed 30 min before the injection of the drugs. To study the effect of APM03 in spinal dogs, the spinal preparation was done under thiopental anesthesia according to the method described by Burn and Rand (1958). 1 Portions of this paper have been presented at the 57th Annual Meeting of The Federation American Societies for Experimental Biology, Atlantic City, New Jersey. z Present address: Marion Laboratories, Inc., Kansas City, Missouri. 3 The Dow Chemical Company, Midland, Michigan. Copyright 0 1973 by Academic Press, Inc. All rights of reproduction in any form reserved. Printed in Great Britain
19
513
of
514
ABDALLAH
AND
WHITE
The drug was injected 60 min after surgical procedure. When depletion of catecholamine was desired, reserpine phosphate4 (3 mg/kg, ip) was given 24 hr prior to use (Maxwell et al., 1959). In certain experiments acute bilateral adrenalectomy was performed 60min before starting drug injection. To study the effects of alpha and beta adrenergic blockers and ganglionic blockade, dogs were pretreated with either phenoxybenzamine HC15 in propylene glycol (10 mg/kg, iv), dichloroisoproterenoF (2 mg/kg, iv), propranolol’ (1 mg/kg, iv) or hexamethonium chloride4 (10 mg/kg, iv) 60, 10, 10 and 10 min, respectively, before iv administration of APMO. To study the interaction of APMO with either norepinephrine,’ acetychoIine9 or histamine,‘O 3 doses of each amine was given to anesthetized dogs before and after the administration of APMO (8 mg/kg, iv). In all experiments blood pressure and heart rate were allowed to return to baseline values between successive doses of APMO and between other drugs. During the study of the development of tachyphylaxis to the repeated administration of APMO (4 mg/kg, iv), the drug was injected every 30 min. Whenever applicable the data were analyzed by using a paired t test. RESULTS
The iv administration of APMO caused a diphasic action on blood pressureYa depressor effect followed by a pressor effect. The 2 mg/kg dose of APMO caused a significant increase, and the 4 mg/kg dose caused both a significant decrease and increase in blood pressure (Table 1). The duration of the depressor and pressor effects were 15-60 set and 60-240 set, respectively. On the other hand, the 0.2 and 0.4 mg/kg doses of d-amphetamine caused a significant increase in blood pressure of 46.3 and 55.0 mm Hg, respectively. At these doses, the effect of both compounds on heart and respiration rates were not marked. In bilaterally vagotomized dogs, the progressive iv administration of 0.5, 1, 2, 4, 8 and 12 mg/kg doses to each dog caused a depressor followed by a pressor effect. The depressor effect became significant at the 4 mg/kg dose, and it increased progressively with the dose until it reached a 71.7 % value (Table 2). On the other hand, the pressor effect became significant after the 1 mg/kg dose but it did not exceed 21%. All doses tested had no significant (p > 0.05) effect on heart and respiration rates. The iv administration of 4 mg/kg of APMO to either adrenalectomized or spinalectomized dogs resulted in a similar depressor followed by a pressor response as seen in normal dogs (Table 3). This dose caused a significant (p < 0.05) increase in heart rate of the spinal dogs. In experiments in which the animals were pretreated with reserpine a significant (p < 0.05) reduction of the pressure effect of both APMO and tyramine was noted. The APMO had no significant effect on the heart rate of both normal and reserpinized dogs. Tyramine caused reflex bradycardia in normal dogs, but not in the reserpinized ones. 4 Nutritional Biochem. Corps., Cleveland, Ohio. 5 Donated by Smith, Kline and French Laboratories, Philadelphia, Pennsylvania. 6 Eli Lilly, Indianapolis, Indiana, 7 Donated by Ayerst Laboratories, Inc., New York, New York. * Chemical Procurement Laboratories, Inc., College Port, New York. 9 Aldrich Chemical Co., Milwaukee, Wisconsin. lo Mann Research, New York.
CARDIOVASCULAR
ACTlVITY
515
OF APMO
TABLE 1 EFFECT OF A SINGLE INTRAVENOUS ADMINISTRATION OF APMO HYDROCHLORIDE AND &HETAMINE SULFATE ON CAROTID BLEND PRESSURE, HEART RATE AND RESPIRATION RATE OF ANESTHETIZED Does
Compound APMO
d-Amphetamine sulfate
Dose No. (mg/kg) Dogs 1
5
2
4
4
3
0.2 0.4
4 3
Mean change in blood pressure mm Hg & SE
Percent change in mean heart rate
Percent change in mean respiration rate
0.0 -4.1
0.0
4.8 zk1.7 -6.3 i 3.8 12.5 i 1.4” -18.3 31.7 46.3 55.0
+ 4.4” It 7.3” * 5.2” zt 5.8”
1.3 -3.0 2.8
4.3
-12.5 0.0 -16.5
a Significantlydifferent (p < 0.05) from saline,paired t- test. The baseline of mean blood pressure and mean heart rate of normal, reserpinized, spinalectomized and adrenalectomized dogs were 114, 75.0, 54.5 and 80 mm Hg and 177,97, 158 and 130 beats/min, respectively. The pretreatment of animals with the alpha blocker phenoxybenzamine (10 mg/kg, iv) caused a significant (p < 0.05) reduction of the pressor effects of APMO and norepinephrine and a reversal of the pressor effect to epinephrine’ (Table 4). Moreover. the reflex bradycardia caused by norepinephrine was significantly antagonized by phenoxybenzamine pretreatment. In the case of epinephrine, phenoxybenzamine reversed its effects on both blood pressure and heart rate. This was due to the effect of epinephrine on the beta receptors which was not counteracted by its effect on the alpha receptors. The beta blocker dichloroisoproterenol (DCI, 2 mg/kg, iv) failed to significantly (p < 0.05) alter the effects of APMO and isoproterenol” on blood pressure (Table 5). On the other hand, the same dose of DC1 significantly altered the effects of the sympathomimetic amines on heart rate. In the case of propranolol (1 mg/kg, iv), it significantly (p < 0.05) blocked the pressor effect of APMO. Moreover, it significantly (p < 0.05) blocked the effect of isoproterenol on both blood pressure and heart rate. The ganglionic blocker hexamethonium (10 mg/kg, iv) which significantly (p < 0.05) blocked the pressor effect of nicotine tartrate failed to block the pressor effect of APMO (Table 6). The iv administration of 8.0 mg/kg of APMO had no significant effect on blood pressure or heart rate alterations induced by norepinephrine, acetylcholine and histamine. However, the depressor effect of acetylcholine was slightly blocked by APMO. This fact indicates that APMO may have a weak anticholinergic activity. Upon repeated administration, tachyphylaxis developed to the pressor effect of APMO. On the other hand, its depressor effect increased in proportion to the number of *I Winthrop Laboratories, New York, New York.
Compound
-18.0f4.6" 27.6 zt 6.9"
5 5 3
4.0
8.0
12.0
f 10.1” f 13.6" f 6.2"
zb4.4"
16.0 f 5.3"
5
2.0
-64.6 28.0 -103.3 17.7
10.3 * 1.9"
6
1.0
5.0 dz1.3
mmHgfSE
6
dogs No’
-71.7 12.3
19.6
-45.2
-14.0 21.0
13.4
8.0
4.2
Percent change
Mean change in blood pressure
0.5
Dose bw/W
(1Significantlydifferent (p < 0.05) from saline, paired t test.
APMO
TABLE 2
7.0
9.2
0.7 0.8 5.2
0.8
Percent change in mean heart rate
z 7m
-1 .o
35.0
16.6
5
5
F
$
-29.5 0.0
15.3
Percent change in mean respiration rate
EFFECT OF PROGRESSIVE INTRAVENOUS ADMINISTRATION OF APMO HYDROCHLORIDE ON CAROI-ID BLOOD PRESSURE, HEART RATE AND RESPIRATION RATE OF ANESTHETIZED AND VAGOTOMIZED DOGS
a Significantly
different
group
APMO
Reserpinized dogs
(p . 0.05),
Tyramine
APMO
Spinal dogs
Tyramine
Drug -~-.-.---APMO
paired
I test.
50 ,wlkg 100 &kg 200 /c&t
4 t-m/kg
4 mz/kg
50 ,a/kg 100 wzlkg 200 /cdk 4 mglk
4 mdkg
Dose _._. ______
5 5 5
5
5
5 5 5 3
8
No. dogs _-~
AND
MEAN
DOGS
OF NORMAL,
0.4 t 3.5 4.0 I!Z2.4” 1.0 h 3.8”
-35.0 i 15.5 5.0 i 5.0
13.6 + 4.3” 8.0 ZII1.8 12.2 i 1.8” 14.8 % 2.7“
7.0 i 11.8 -6.0 i 6.8 -23.0 + 7.7
Mean change in heart rate (beatslmin)
RATE
48.0 i 12.3” 1.6 i 1.0
.___
HEART
-4.0 Ik 1.5 37.8 z!c6.4 ll.Of2.2 30.4 i 8.6 72.5 i 10.5 -3.3 i 3.3 35.0 i 2.9 -8.3 l 1.7 49.0 * 13.1 -6.6 * 1.9
Mean change in femoral blood pressure (mm Hg i SE)
OF APMO HYDKOCHLORIDE ON MEAN BLOOD PRESSURE ADRENALECTOMIZED, SPINALECTOMIZED AND RESERPINIZED
APMO
normal
ADMINISTRATION
Adrenalectomized dogs
Normal dogs
from
OF INTRAVENOUS
Treatment
EFFECT
TABLE 3
-
5 ;i 2 0, G 5
F TI ij 5 CT 5 G
0.5 Ccglk 1.Or&k 2.0 fig/kg
0.5 ,&kg 1.Om/kg 2.0 m/kg
Norepipephrine
Epinephrine
5-8 7.0 f 11.8 -34.8 i 3.7 -39.0 f 5.2 -42.8 zt 4.4 -14.6 f 6.6 -27.0 i 7.4 -44.2 f 10.2
-4.0 f 1.5 37.8 f 6.4 32.0 i 4.1 48.0 f 8.6 64.0 i 8.1 31.8 f 6.0 46.0 f 5.8 68.0 f 7.2
Mean change in HR (beats/min” f SE)
-36.0 it 4.9b -47.0 f 4.0b -49.0 f 3.7b
15.0 f 1.6b 22.2 i 4.1b
-6.7 f 1.7 11.4 f l.Ob 13.0 * 2.Q
Mean change in BP (mm Hg + SE)
17.0 + 6.7b 15.8 i 6.6b 25.0 + 5.1b
-10.8 i 3.gb -7.4 i 2.7b
-2.0 i 2.0 -5.6 zt 1.9’
Mean change in HR (beats/min & SE)
Posttreatment
0 To avoid development of tachyphylaxis, the APMO was given to 2 groups of dogs. One group was pretreated with saline, and the other group was pretreated with phenoxybenmmine. APMO, norepinephrine and epinephrine were administered 60 min after phenoxybenzamine (10 mg/kg, iv). b Significantly different from pretreated group (p c O.Ol), paired f test.
4.0 mg/kg
Dose
APMOe
Compound
Mean change in BP (mm Hg f SE)
Pretreatment
EFFECT OF PHENOXYBENZAMINE PRETREATMENT ON MEAN BLOOD PRESSUREAND MEAN HEART RATE OF ANESTHETIZED-DOGS AFTER INTRAVENOUS ADMINISTRATION OF APMO HYDROCHLORIDE, NOREPINEPHRINE AND EPINEPHRINE
TABLE 4
g m
5
E-
$ E
5
-
Isoproterenol
Epinephrine
APMO”
Isoproterenol
0.5 ,&kg 1 .O .&kg 2.0 ,&kg 0.5 /a/kg 1 .O a/kg 2.0 ,&kg
0.5 &dkg 1.0 &kg 2.0 /a/kg 0.5 &kg 1.O adkis 2.0 i&g 4.0 mg/kg
Epinephrine
mg/kg
4.0
-~
Dose
APMO”
Compound --
5 5 5
5 5
5
5-8
5 5
5
5
5
5
5-8
No. dogs
36.0 s-42.4 49.0
20.0 41.8 72.5
--4.0 37.8
-18.6 -23.1 --38.5
30.0 43.0 62.0
-4.0 37.8
+ 1.5 i 6.4 h 6.3 k 5.6 i 5.1 + 8.8 f 6.4 xk 8.5 $ 1.6 i 6.4 i 5.0 + 5.8 + 6.0 + 7.0 + 5.0 i 5.6 68.6 75.8 72.8
-6.3 -32.8 --16.5
7.0%
39.0 58.4~r 67.5
-22.0 -37.0& -53.0
7.0 10.6 10.2 16.3
11.1
11.8
I20.1 rt22.9 4- 17.8
+ 8.6 z!z16.7
_t
rt
9.7 7.7
i 10.0
f
i
$11.8
% 2.3 zt 5.0*
+ 4.6 i 3.3 * 4.5
f 10.4 i 9.9 i 5.8
i 1.2 + 4.0
5.0 k4.5' -5.0 3: 5.2'
10.0 zt 1.6’
33.8 + 8.0 51 .o jk 6.0 64.4 i 5.6
--9.3 18.6
-8.0 -9.0 -~20.0
47.4 54.4 69.0
-3.0 25.0
Meanchange in BP (mm Hg i SE)
* -i f
3.4' 1.8' 3.2"
2.4 +7.1* + 27.9
f
It 1.2
6.4 8.8 15.0
---3.8 -31.0 --18.8
+ 2.4 f 14.0 i 3.0 i- 2.4' I!z3.46 -k 5.8
2.2 _t 1.4
-2.0 5.0 12.5
-4.0 -7.8 -38.8
-2.0
.~.____ Mean change in HR (beats/min rt SE)
Posttreatment
” To avoid development of tachyphylaxis. the APMO was given to two groups of dogs. One group was pretreated with saline and the other group was pretreated either with dichloroisoproterenol or with propranolol. b Significantly different from pretreatment group (p < 0.05), paired f test. c Significantly different from pretreatment group (p - O.Ol), paired f test.
Propranolol, 1 mg/kg, iv
Dichloroisoproterenol, 2 mg/kg, iv
Beta blocker --__ - .__.__
Pretreatment --~I_-.-. _ Mean change Mean change in BP in HR (mm Hg f SE) (beatsjmin & SE) _______
EFFECTOF BETABLOCKEKS PRETREATMENT ON MEAN FEMORALBLWD PRESSURE AND MEAN HEART RATE OF ANESTHEI.IZEDDons AFXR INTRAVENOUS ADMINISTRATION OF APMO, EPINEPHRINEAND~SOPROTERENOL
TABLE
520
ABDALLAH
AND
WHITE
TABLE 6 EFFECT OF HEXAMETHONKJM HCl(l0 mg/kg, iv) ON MEAN BLOOD PRESSUREOF ANESTHETIZED DOGS AFTER INTRAVENOUS ADMINISTRATION OF APMO AND NICOTINE TARTRATE Mean change in Blood pressure (mm Hg f SE)
Compound APMO
No. dogs
Dose 2 mdkg
25 ,&kg
-6.3 12.5 -25.8 23.8 13.5 -33.3 31.7
2 3
50 m/k
Posttreatment
Pretreatment
4 4 4
4 m&g
Nicotine tartrate
i 3.7 i 1.4 zt 0.8 i 5.5 & 11.5 z!Y3.3 It 14.5
-12.5 30.0 -26.0 29.7 0.0 -5.0 0.0
zt 2.5 i 0.0 f 3.1 f 3.9 * 0.0 f 5.8” zt 0.0”
n Significantly different from pretreatment group, paired t test.
doses. The first, second, third, fourth, and fifth dose (4 mg/kg, iv) of APMO, respectively, caused a pressor effect of 37.8,28.8,20.0, 15.9 and 10.3 mm Hg and a depressor effect of -4.0, -9.0, -17.0, -23.8 and -24.6 mm Hg (Fig. 1). The duration of the depressor and pressor effects were 15-60 set and 60-240 set, respectively. Heart rate was not significantly affected.
-301
’
I 2
I 3
4
' 5
J-30
N&OF DOSES
FIG. 1. Effect of repeated administration of APMO HCl (4 mg/kg, iv) on blood pressure and heart rate of anesthetized dogs. This dose has a diphasic effect on blood pressure: a depressor followed by a pressor effect. 0, decrease in blood pressure; 0, increase in blood pressure; A, increase in heart rates; the abscissa represents the number of doses; the left ordinate represents absolute change in mean blood pressure (mm Hg); the right ordinate represents the absolute change in mean heart rate (beats/ min). Each point represents the mean f SE of 8 observations. **Significantly less than first dose (p < 0.01).
CARDIOVASCULARACTIVITYOFAPMO
521
DISCUSSION The fact that neither hexamethonium chloride, spinalectomy, nor adrenalectomy antagonized the pressor effect of APMO indicates that the compound did not exert its pressor effect by acting on the spinal cord and/or ganglia, on the brain, or through the release of epinephrine from the adrenal medulla. Reserpine pretreatment which depletes catecholamine stores of various tissues (Burn and Rand, 1958; Malmfors and Sachs, 1965) antagonized the pressor effects of both APMO and tyramine. Consequently we may conclude that APMO, like tyramine, acts indirectly through the release of catecholamine (Fleckenstein and Burn, 1953; Carlsson er al., 1957). This conclusion is further supported by the finding that the alpha blocker phenoxybenzamine significantly blocked the pressor effect of APMO. The beta blocker propranolol significantly blocked the pressor effect of APMO. This effect of propranolol may be due to some mechanism other than its beta receptors blockade. This idea is supported by Roba et al., (1972), who stated that the hypotensive potencies of beta blockers CP-240-S Sotalol, propranolol and alprenolol in the rat were not related with their beta receptor blocking activities. In few experiments, the depressor effect of APMO was not significantly blocked either by diphenhydramine or by atropine. This fact indicates that the compound was not acting on the cholinergic or histaminic receptors either directly or indirectly. The mechanism of the depressor activity of APMO will be studied in more detail.
ACKNOWLEDGMENTS The authors are indebted to Smith, Kline and French Laboratories and to Ayerst Laboratories for gifts of phenoxybenzamine HCl and propranolol HCl, respectively. The authors would like to thank Dr. A. Renzi for his constructive critique of this manuscript.
REFERENCES A. H. (1972). Pharmacological evaluation of 2-amino-5-(3,4-dichlorophenoxy)methyl-2-oxazoline: A new anorectic compound. Proc. Int. Congr. Pharmacoi. 5th, p. 1. ABDALLAH, A. H. (1973). Comparative study of the anorexigenic activity of 5-(3,4 dichlorophenoxymethyl)-2-amino-2-oxazoline*HCl and d-amphetamine in different species. ABDALLAH,
Tonicol. Appl. Pharmacol. 25,344-353. BURN, J. H. AND RAND, M. J. (1958). The action of sympathomimetic amines in animals treated with reserpine. J. Physiol. (London) 144, 314-336. CARLSSON, A., ROSENGREN, E., BERTHER, A. AND NILSSON, J.(1957). Effectofreserpine onthe metabolism of catecholamine. In: Psychotropic Drugs (S. Garrattini and V. Ghetti, eds.),
pp. 363-372. Elsevier, Amsterdam. FLECKENSTEIN, A. AND BURN, J. H. (1953). The effect of denervation on the action of sympathomimetic amines on the nictitating membrane. &it. J. Pharmacol. Chemother. 8, 69-78. MALMFORS, J. AND SACHS, C. (1965). Direct studies on the disappearance of the transmitter and changes in the uptake-storage mechanism of degenerating adrenergic nerves. Acta Physiol. &and. &I,21 I-223.
522
ABDALLAH
AND
WHITE
R. A., POVALSKI, H. AND PLUMMER, A. J. (1959). A differential effect of reserpine on pressor amine activity and its relationship to other agents producing this effect. J. Pharmacol. Exp. Ther. 125, 178. ROBA, J., LAMBELIN, G. AND DE-SCHAEPDRYVER, A. F. (1972). Antihypertensive activity of four /I-blocking agents in spontaneously hypertensive rats. Arch. Znt. Pharmacodyn. 200, 182-190. MAXWELL,
AND
APPLIED
PHARMACOLOGY
26,513-522
(1973)
The Cardiovascular Activity 2-Amino-5-(3,4-dichlorophenoxymethyl)-2-oxazoline
of (APMO)’
A. H. ABDALLAH AND H. D. WHITER Chemical
Biology
Research, Received
The Dow December
Chemical 14,1972;
Company, accepted
April
Midland,
Michigan
48640
12,1973
The Cardiovascular Activity of 2-Amino-5(3,4-dichlorophenoxymethyl)2-oxazoline (APMO). ABDALLAH, A. H. AND WHITE, H. D. (1973). Toxicol. Appl. Pharmacol. 26, 513-522. The cardiovascular activity of APMO was studied in dogs. When compared on a mgfkg basis, its pressor effect was less than that of d-amphetamine. APMO has a diphasic action on blood pressure: a depressor followed by a pressor activity. Upon repeated administration, tachyphylaxis developed to its pressor but not to its depressor effect. The pressor effect of the APMO was antagonized either by reserpine or by phenoxybenzamine pretreatments. This finding suggests that the compound is acting indirectly through the releaseof norepinephrine from peripheral nerve endings. Spinalectomy did not significantly affect its pressor activity. This finding indicates that the brain is not involved in its pressor effect. Recently, we reported on the anorexigenic activity in various animal species of 2-amino-5-(3,4-dichlorophenoxymethyl)2-oxazohne (APMO) (Abdallah, 1972, 1973). Our findings showed that, in approximately equipotent anorexigenic doses, the stimulant effects of APMO on the central nervous and the cardiovascular systems were less than those of d-amphetamine. This paper presents the results of the cardiovascular activity of APMO. METHODS Mongrel dogs of either sex (7-l 8 kg) were used. They were anesthetized with pentobarbital sodium, 30 mg/kg (iv). The right carotid or femoral arteries were cannulated and blood pressure was recorded by means of Gould Brush polygraph through a Statham transducer. Heart rate (HR) and lead II electrocardiogram (ECG) were also continuously monitored. The trachea was intubated, and in certain experiments respiration rate was recorded. Drugs were dissolved in saline and injected via an indwelling catheter in the femoral vein. In some experiments, acute bilateral vagotomy was performed 30 min before the injection of the drugs. To study the effect of APM03 in spinal dogs, the spinal preparation was done under thiopental anesthesia according to the method described by Burn and Rand (1958). 1 Portions of this paper have been presented at the 57th Annual Meeting of The Federation American Societies for Experimental Biology, Atlantic City, New Jersey. z Present address: Marion Laboratories, Inc., Kansas City, Missouri. 3 The Dow Chemical Company, Midland, Michigan. Copyright 0 1973 by Academic Press, Inc. All rights of reproduction in any form reserved. Printed in Great Britain
19
513
of
514
ABDALLAH
AND
WHITE
The drug was injected 60 min after surgical procedure. When depletion of catecholamine was desired, reserpine phosphate4 (3 mg/kg, ip) was given 24 hr prior to use (Maxwell et al., 1959). In certain experiments acute bilateral adrenalectomy was performed 60min before starting drug injection. To study the effects of alpha and beta adrenergic blockers and ganglionic blockade, dogs were pretreated with either phenoxybenzamine HC15 in propylene glycol (10 mg/kg, iv), dichloroisoproterenoF (2 mg/kg, iv), propranolol’ (1 mg/kg, iv) or hexamethonium chloride4 (10 mg/kg, iv) 60, 10, 10 and 10 min, respectively, before iv administration of APMO. To study the interaction of APMO with either norepinephrine,’ acetychoIine9 or histamine,‘O 3 doses of each amine was given to anesthetized dogs before and after the administration of APMO (8 mg/kg, iv). In all experiments blood pressure and heart rate were allowed to return to baseline values between successive doses of APMO and between other drugs. During the study of the development of tachyphylaxis to the repeated administration of APMO (4 mg/kg, iv), the drug was injected every 30 min. Whenever applicable the data were analyzed by using a paired t test. RESULTS
The iv administration of APMO caused a diphasic action on blood pressureYa depressor effect followed by a pressor effect. The 2 mg/kg dose of APMO caused a significant increase, and the 4 mg/kg dose caused both a significant decrease and increase in blood pressure (Table 1). The duration of the depressor and pressor effects were 15-60 set and 60-240 set, respectively. On the other hand, the 0.2 and 0.4 mg/kg doses of d-amphetamine caused a significant increase in blood pressure of 46.3 and 55.0 mm Hg, respectively. At these doses, the effect of both compounds on heart and respiration rates were not marked. In bilaterally vagotomized dogs, the progressive iv administration of 0.5, 1, 2, 4, 8 and 12 mg/kg doses to each dog caused a depressor followed by a pressor effect. The depressor effect became significant at the 4 mg/kg dose, and it increased progressively with the dose until it reached a 71.7 % value (Table 2). On the other hand, the pressor effect became significant after the 1 mg/kg dose but it did not exceed 21%. All doses tested had no significant (p > 0.05) effect on heart and respiration rates. The iv administration of 4 mg/kg of APMO to either adrenalectomized or spinalectomized dogs resulted in a similar depressor followed by a pressor response as seen in normal dogs (Table 3). This dose caused a significant (p < 0.05) increase in heart rate of the spinal dogs. In experiments in which the animals were pretreated with reserpine a significant (p < 0.05) reduction of the pressure effect of both APMO and tyramine was noted. The APMO had no significant effect on the heart rate of both normal and reserpinized dogs. Tyramine caused reflex bradycardia in normal dogs, but not in the reserpinized ones. 4 Nutritional Biochem. Corps., Cleveland, Ohio. 5 Donated by Smith, Kline and French Laboratories, Philadelphia, Pennsylvania. 6 Eli Lilly, Indianapolis, Indiana, 7 Donated by Ayerst Laboratories, Inc., New York, New York. * Chemical Procurement Laboratories, Inc., College Port, New York. 9 Aldrich Chemical Co., Milwaukee, Wisconsin. lo Mann Research, New York.
CARDIOVASCULAR
ACTlVITY
515
OF APMO
TABLE 1 EFFECT OF A SINGLE INTRAVENOUS ADMINISTRATION OF APMO HYDROCHLORIDE AND &HETAMINE SULFATE ON CAROTID BLEND PRESSURE, HEART RATE AND RESPIRATION RATE OF ANESTHETIZED Does
Compound APMO
d-Amphetamine sulfate
Dose No. (mg/kg) Dogs 1
5
2
4
4
3
0.2 0.4
4 3
Mean change in blood pressure mm Hg & SE
Percent change in mean heart rate
Percent change in mean respiration rate
0.0 -4.1
0.0
4.8 zk1.7 -6.3 i 3.8 12.5 i 1.4” -18.3 31.7 46.3 55.0
+ 4.4” It 7.3” * 5.2” zt 5.8”
1.3 -3.0 2.8
4.3
-12.5 0.0 -16.5
a Significantlydifferent (p < 0.05) from saline,paired t- test. The baseline of mean blood pressure and mean heart rate of normal, reserpinized, spinalectomized and adrenalectomized dogs were 114, 75.0, 54.5 and 80 mm Hg and 177,97, 158 and 130 beats/min, respectively. The pretreatment of animals with the alpha blocker phenoxybenzamine (10 mg/kg, iv) caused a significant (p < 0.05) reduction of the pressor effects of APMO and norepinephrine and a reversal of the pressor effect to epinephrine’ (Table 4). Moreover. the reflex bradycardia caused by norepinephrine was significantly antagonized by phenoxybenzamine pretreatment. In the case of epinephrine, phenoxybenzamine reversed its effects on both blood pressure and heart rate. This was due to the effect of epinephrine on the beta receptors which was not counteracted by its effect on the alpha receptors. The beta blocker dichloroisoproterenol (DCI, 2 mg/kg, iv) failed to significantly (p < 0.05) alter the effects of APMO and isoproterenol” on blood pressure (Table 5). On the other hand, the same dose of DC1 significantly altered the effects of the sympathomimetic amines on heart rate. In the case of propranolol (1 mg/kg, iv), it significantly (p < 0.05) blocked the pressor effect of APMO. Moreover, it significantly (p < 0.05) blocked the effect of isoproterenol on both blood pressure and heart rate. The ganglionic blocker hexamethonium (10 mg/kg, iv) which significantly (p < 0.05) blocked the pressor effect of nicotine tartrate failed to block the pressor effect of APMO (Table 6). The iv administration of 8.0 mg/kg of APMO had no significant effect on blood pressure or heart rate alterations induced by norepinephrine, acetylcholine and histamine. However, the depressor effect of acetylcholine was slightly blocked by APMO. This fact indicates that APMO may have a weak anticholinergic activity. Upon repeated administration, tachyphylaxis developed to the pressor effect of APMO. On the other hand, its depressor effect increased in proportion to the number of *I Winthrop Laboratories, New York, New York.
Compound
-18.0f4.6" 27.6 zt 6.9"
5 5 3
4.0
8.0
12.0
f 10.1” f 13.6" f 6.2"
zb4.4"
16.0 f 5.3"
5
2.0
-64.6 28.0 -103.3 17.7
10.3 * 1.9"
6
1.0
5.0 dz1.3
mmHgfSE
6
dogs No’
-71.7 12.3
19.6
-45.2
-14.0 21.0
13.4
8.0
4.2
Percent change
Mean change in blood pressure
0.5
Dose bw/W
(1Significantlydifferent (p < 0.05) from saline, paired t test.
APMO
TABLE 2
7.0
9.2
0.7 0.8 5.2
0.8
Percent change in mean heart rate
z 7m
-1 .o
35.0
16.6
5
5
F
$
-29.5 0.0
15.3
Percent change in mean respiration rate
EFFECT OF PROGRESSIVE INTRAVENOUS ADMINISTRATION OF APMO HYDROCHLORIDE ON CAROI-ID BLOOD PRESSURE, HEART RATE AND RESPIRATION RATE OF ANESTHETIZED AND VAGOTOMIZED DOGS
a Significantly
different
group
APMO
Reserpinized dogs
(p . 0.05),
Tyramine
APMO
Spinal dogs
Tyramine
Drug -~-.-.---APMO
paired
I test.
50 ,wlkg 100 &kg 200 /c&t
4 t-m/kg
4 mz/kg
50 ,a/kg 100 wzlkg 200 /cdk 4 mglk
4 mdkg
Dose _._. ______
5 5 5
5
5
5 5 5 3
8
No. dogs _-~
AND
MEAN
DOGS
OF NORMAL,
0.4 t 3.5 4.0 I!Z2.4” 1.0 h 3.8”
-35.0 i 15.5 5.0 i 5.0
13.6 + 4.3” 8.0 ZII1.8 12.2 i 1.8” 14.8 % 2.7“
7.0 i 11.8 -6.0 i 6.8 -23.0 + 7.7
Mean change in heart rate (beatslmin)
RATE
48.0 i 12.3” 1.6 i 1.0
.___
HEART
-4.0 Ik 1.5 37.8 z!c6.4 ll.Of2.2 30.4 i 8.6 72.5 i 10.5 -3.3 i 3.3 35.0 i 2.9 -8.3 l 1.7 49.0 * 13.1 -6.6 * 1.9
Mean change in femoral blood pressure (mm Hg i SE)
OF APMO HYDKOCHLORIDE ON MEAN BLOOD PRESSURE ADRENALECTOMIZED, SPINALECTOMIZED AND RESERPINIZED
APMO
normal
ADMINISTRATION
Adrenalectomized dogs
Normal dogs
from
OF INTRAVENOUS
Treatment
EFFECT
TABLE 3
-
5 ;i 2 0, G 5
F TI ij 5 CT 5 G
0.5 Ccglk 1.Or&k 2.0 fig/kg
0.5 ,&kg 1.Om/kg 2.0 m/kg
Norepipephrine
Epinephrine
5-8 7.0 f 11.8 -34.8 i 3.7 -39.0 f 5.2 -42.8 zt 4.4 -14.6 f 6.6 -27.0 i 7.4 -44.2 f 10.2
-4.0 f 1.5 37.8 f 6.4 32.0 i 4.1 48.0 f 8.6 64.0 i 8.1 31.8 f 6.0 46.0 f 5.8 68.0 f 7.2
Mean change in HR (beats/min” f SE)
-36.0 it 4.9b -47.0 f 4.0b -49.0 f 3.7b
15.0 f 1.6b 22.2 i 4.1b
-6.7 f 1.7 11.4 f l.Ob 13.0 * 2.Q
Mean change in BP (mm Hg + SE)
17.0 + 6.7b 15.8 i 6.6b 25.0 + 5.1b
-10.8 i 3.gb -7.4 i 2.7b
-2.0 i 2.0 -5.6 zt 1.9’
Mean change in HR (beats/min & SE)
Posttreatment
0 To avoid development of tachyphylaxis, the APMO was given to 2 groups of dogs. One group was pretreated with saline, and the other group was pretreated with phenoxybenmmine. APMO, norepinephrine and epinephrine were administered 60 min after phenoxybenzamine (10 mg/kg, iv). b Significantly different from pretreated group (p c O.Ol), paired f test.
4.0 mg/kg
Dose
APMOe
Compound
Mean change in BP (mm Hg f SE)
Pretreatment
EFFECT OF PHENOXYBENZAMINE PRETREATMENT ON MEAN BLOOD PRESSUREAND MEAN HEART RATE OF ANESTHETIZED-DOGS AFTER INTRAVENOUS ADMINISTRATION OF APMO HYDROCHLORIDE, NOREPINEPHRINE AND EPINEPHRINE
TABLE 4
g m
5
E-
$ E
5
-
Isoproterenol
Epinephrine
APMO”
Isoproterenol
0.5 ,&kg 1 .O .&kg 2.0 ,&kg 0.5 /a/kg 1 .O a/kg 2.0 ,&kg
0.5 &dkg 1.0 &kg 2.0 /a/kg 0.5 &kg 1.O adkis 2.0 i&g 4.0 mg/kg
Epinephrine
mg/kg
4.0
-~
Dose
APMO”
Compound --
5 5 5
5 5
5
5-8
5 5
5
5
5
5
5-8
No. dogs
36.0 s-42.4 49.0
20.0 41.8 72.5
--4.0 37.8
-18.6 -23.1 --38.5
30.0 43.0 62.0
-4.0 37.8
+ 1.5 i 6.4 h 6.3 k 5.6 i 5.1 + 8.8 f 6.4 xk 8.5 $ 1.6 i 6.4 i 5.0 + 5.8 + 6.0 + 7.0 + 5.0 i 5.6 68.6 75.8 72.8
-6.3 -32.8 --16.5
7.0%
39.0 58.4~r 67.5
-22.0 -37.0& -53.0
7.0 10.6 10.2 16.3
11.1
11.8
I20.1 rt22.9 4- 17.8
+ 8.6 z!z16.7
_t
rt
9.7 7.7
i 10.0
f
i
$11.8
% 2.3 zt 5.0*
+ 4.6 i 3.3 * 4.5
f 10.4 i 9.9 i 5.8
i 1.2 + 4.0
5.0 k4.5' -5.0 3: 5.2'
10.0 zt 1.6’
33.8 + 8.0 51 .o jk 6.0 64.4 i 5.6
--9.3 18.6
-8.0 -9.0 -~20.0
47.4 54.4 69.0
-3.0 25.0
Meanchange in BP (mm Hg i SE)
* -i f
3.4' 1.8' 3.2"
2.4 +7.1* + 27.9
f
It 1.2
6.4 8.8 15.0
---3.8 -31.0 --18.8
+ 2.4 f 14.0 i 3.0 i- 2.4' I!z3.46 -k 5.8
2.2 _t 1.4
-2.0 5.0 12.5
-4.0 -7.8 -38.8
-2.0
.~.____ Mean change in HR (beats/min rt SE)
Posttreatment
” To avoid development of tachyphylaxis. the APMO was given to two groups of dogs. One group was pretreated with saline and the other group was pretreated either with dichloroisoproterenol or with propranolol. b Significantly different from pretreatment group (p < 0.05), paired f test. c Significantly different from pretreatment group (p - O.Ol), paired f test.
Propranolol, 1 mg/kg, iv
Dichloroisoproterenol, 2 mg/kg, iv
Beta blocker --__ - .__.__
Pretreatment --~I_-.-. _ Mean change Mean change in BP in HR (mm Hg f SE) (beatsjmin & SE) _______
EFFECTOF BETABLOCKEKS PRETREATMENT ON MEAN FEMORALBLWD PRESSURE AND MEAN HEART RATE OF ANESTHEI.IZEDDons AFXR INTRAVENOUS ADMINISTRATION OF APMO, EPINEPHRINEAND~SOPROTERENOL
TABLE
520
ABDALLAH
AND
WHITE
TABLE 6 EFFECT OF HEXAMETHONKJM HCl(l0 mg/kg, iv) ON MEAN BLOOD PRESSUREOF ANESTHETIZED DOGS AFTER INTRAVENOUS ADMINISTRATION OF APMO AND NICOTINE TARTRATE Mean change in Blood pressure (mm Hg f SE)
Compound APMO
No. dogs
Dose 2 mdkg
25 ,&kg
-6.3 12.5 -25.8 23.8 13.5 -33.3 31.7
2 3
50 m/k
Posttreatment
Pretreatment
4 4 4
4 m&g
Nicotine tartrate
i 3.7 i 1.4 zt 0.8 i 5.5 & 11.5 z!Y3.3 It 14.5
-12.5 30.0 -26.0 29.7 0.0 -5.0 0.0
zt 2.5 i 0.0 f 3.1 f 3.9 * 0.0 f 5.8” zt 0.0”
n Significantly different from pretreatment group, paired t test.
doses. The first, second, third, fourth, and fifth dose (4 mg/kg, iv) of APMO, respectively, caused a pressor effect of 37.8,28.8,20.0, 15.9 and 10.3 mm Hg and a depressor effect of -4.0, -9.0, -17.0, -23.8 and -24.6 mm Hg (Fig. 1). The duration of the depressor and pressor effects were 15-60 set and 60-240 set, respectively. Heart rate was not significantly affected.
-301
’
I 2
I 3
4
' 5
J-30
N&OF DOSES
FIG. 1. Effect of repeated administration of APMO HCl (4 mg/kg, iv) on blood pressure and heart rate of anesthetized dogs. This dose has a diphasic effect on blood pressure: a depressor followed by a pressor effect. 0, decrease in blood pressure; 0, increase in blood pressure; A, increase in heart rates; the abscissa represents the number of doses; the left ordinate represents absolute change in mean blood pressure (mm Hg); the right ordinate represents the absolute change in mean heart rate (beats/ min). Each point represents the mean f SE of 8 observations. **Significantly less than first dose (p < 0.01).
CARDIOVASCULARACTIVITYOFAPMO
521
DISCUSSION The fact that neither hexamethonium chloride, spinalectomy, nor adrenalectomy antagonized the pressor effect of APMO indicates that the compound did not exert its pressor effect by acting on the spinal cord and/or ganglia, on the brain, or through the release of epinephrine from the adrenal medulla. Reserpine pretreatment which depletes catecholamine stores of various tissues (Burn and Rand, 1958; Malmfors and Sachs, 1965) antagonized the pressor effects of both APMO and tyramine. Consequently we may conclude that APMO, like tyramine, acts indirectly through the release of catecholamine (Fleckenstein and Burn, 1953; Carlsson er al., 1957). This conclusion is further supported by the finding that the alpha blocker phenoxybenzamine significantly blocked the pressor effect of APMO. The beta blocker propranolol significantly blocked the pressor effect of APMO. This effect of propranolol may be due to some mechanism other than its beta receptors blockade. This idea is supported by Roba et al., (1972), who stated that the hypotensive potencies of beta blockers CP-240-S Sotalol, propranolol and alprenolol in the rat were not related with their beta receptor blocking activities. In few experiments, the depressor effect of APMO was not significantly blocked either by diphenhydramine or by atropine. This fact indicates that the compound was not acting on the cholinergic or histaminic receptors either directly or indirectly. The mechanism of the depressor activity of APMO will be studied in more detail.
ACKNOWLEDGMENTS The authors are indebted to Smith, Kline and French Laboratories and to Ayerst Laboratories for gifts of phenoxybenzamine HCl and propranolol HCl, respectively. The authors would like to thank Dr. A. Renzi for his constructive critique of this manuscript.
REFERENCES A. H. (1972). Pharmacological evaluation of 2-amino-5-(3,4-dichlorophenoxy)methyl-2-oxazoline: A new anorectic compound. Proc. Int. Congr. Pharmacoi. 5th, p. 1. ABDALLAH, A. H. (1973). Comparative study of the anorexigenic activity of 5-(3,4 dichlorophenoxymethyl)-2-amino-2-oxazoline*HCl and d-amphetamine in different species. ABDALLAH,
Tonicol. Appl. Pharmacol. 25,344-353. BURN, J. H. AND RAND, M. J. (1958). The action of sympathomimetic amines in animals treated with reserpine. J. Physiol. (London) 144, 314-336. CARLSSON, A., ROSENGREN, E., BERTHER, A. AND NILSSON, J.(1957). Effectofreserpine onthe metabolism of catecholamine. In: Psychotropic Drugs (S. Garrattini and V. Ghetti, eds.),
pp. 363-372. Elsevier, Amsterdam. FLECKENSTEIN, A. AND BURN, J. H. (1953). The effect of denervation on the action of sympathomimetic amines on the nictitating membrane. &it. J. Pharmacol. Chemother. 8, 69-78. MALMFORS, J. AND SACHS, C. (1965). Direct studies on the disappearance of the transmitter and changes in the uptake-storage mechanism of degenerating adrenergic nerves. Acta Physiol. &and. &I,21 I-223.
522
ABDALLAH
AND
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R. A., POVALSKI, H. AND PLUMMER, A. J. (1959). A differential effect of reserpine on pressor amine activity and its relationship to other agents producing this effect. J. Pharmacol. Exp. Ther. 125, 178. ROBA, J., LAMBELIN, G. AND DE-SCHAEPDRYVER, A. F. (1972). Antihypertensive activity of four /I-blocking agents in spontaneously hypertensive rats. Arch. Znt. Pharmacodyn. 200, 182-190. MAXWELL,