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 ::  Abstract
 ::  Introduction
 ::  Material and Methods
 ::  Results
 ::  Discussion
 ::  References
 ::  Article Figures

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ARTICLE
Year : 1976  |  Volume : 22  |  Issue : 4  |  Page : 161-170

Studies on the mechanism of hypotensive action of a disubstituted piperazine derivative - (Part II)


Department of Pharmacology, Seth G. S. Medical College, Parel, Bombay-400012, India

Correspondence Address:
N K Dadkar
Hoechst Research Centre, Mulund, Bombay-400080.
India
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Source of Support: None, Conflict of Interest: None


PMID: 1032831

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 :: Abstract 

The mechanism of hypotensive action of R-1230, a new disub­stituted piperazine derivative was studied in anaesthetised normo­tensive cats. Rapid intravenous injection of R-1230 produced an immediate transient fall in blood pressure attributable to a direct peripheral vasodilator action as concluded from cat's hindquarters' perfusion experiment. The prolonged secondary hypotensive re­sponse appeared to be of complex nature. Inhibition of centrally mediated pressor reflexes on intracerebroventricular administra­tion, accompanied by peripheral adrenergic blockade, points to the dual mechanism of drug induced hypotension. Results in decere­brate and spinal preparations indicated the lower brainstem region lo be the principal site of central hypotensive action.



How to cite this article:
Dadkar N K, Dadkar VN, Sheth U K. Studies on the mechanism of hypotensive action of a disubstituted piperazine derivative - (Part II). J Postgrad Med 1976;22:161-70

How to cite this URL:
Dadkar N K, Dadkar VN, Sheth U K. Studies on the mechanism of hypotensive action of a disubstituted piperazine derivative - (Part II). J Postgrad Med [serial online] 1976 [cited 2020 Sep 24];22:161-70. Available from: http://www.jpgmonline.com/text.asp?1976/22/4/161/42816



 :: Introduction Top


A series of disubstituted piperazine derivatives were evaluated for their struc­ture activity relationship and many of them were found to possess CNS depres­sant, hypotensive and adrenolytic activity. One of these compounds, N 1 -[ (β-3, 4, 5 trimethoxybenzoyl) ethyl] N 4 - (O-metho­xyphenyl) Piperazine (R-1230) revealed a dose dependent hypotensive effect in anaesthetised dogs and cats. The present paper deals with detailed investigations of the hypotensive action of this com­pound in experimental animals.


 :: Material and Methods Top


Effect on Blood Pressure and Heart Rate

Cats of either sex weighing 2.8-4.5 kg were anaesthetised with Pentobarbital sodium (40 mg/kg i.p.). The animals were maintained on positive pressure artificial respiration. Carotid blood pres­sure was recorded using a mercury mano­meter. R-1230 was injected as a soluble maleate salt in doses of 2.5, 5.0 and 10.0 mg/kg through cannulated femoral vein. Blood pressure and ECG (lead II) were recorded before and for a period of 4 hours after the administration of R-1230. Heart rate was calculated from ECG. In order to study the absorption from gastro­intestinal tract, R-1230 (10 mg/kg) was injected intraduodenally. In some animals, blood pressure responses were studied in debuffer preparation where vagus and carotid sinus nerves from both sides were cut. The effects of various blocking agents given i.v. on the depressor responses of R-1230 were studied. The doses employ­ed were as follows:

Atropine (2 mg/kg), propranolol (400µg/ kg in two divided doses), mepyramine (10 mg/kg) and phenoxybenzamine (5 mg/kg).

Effect on spiral and decerebrate cats

In some experiments, blood pressure effects were studied in spinal and decere­brate preparations. [6]

Effect on SCVR

Effect of spinal compression vasomotor response [5] (SCVR) was studied in 3 cats. R-1230 was injected intrathecally in a dose of 1.0 mg. The effect of 1.0 mg of R-1230 injected i.v, was also studied in the same animal.

In anaesthetised dogs, effects of R-1230 (5 mg/kg i.v.) were studied by eliciting the pressor response to bilateral carotid occlusion for 30 sec., electrical stimulation of central and peripheral cut end of vagus and intravenously administered epine­phrine (3-5 µg/ kg) , norepinephrine (2-4 µg/kg) , acetylcholine (5 µg/kg) and histamine (5 µg/kg).

Effect on centrally mediated vasopresser reflexes

In three pentobarbitone anaesthetised dogs, R-1230 (200 µg/kg) was injected through the cannulated vertebral artery. [13]

Responses to carotid occlusion for 30-45 sec. and electrical stimulation of central cut end of vagus nerve were tested before and at various time intervals upto 2 hours after administration of R-1230. In addi­tion, the effect on the pressor responses to peripherally injected epinephrine (2-4 ug/kg) was studied to rule out the leak­age of compounds into the peripheral cir­culation.

Effect of intracerebroventricula.r (ICV) administration of R-1230

To study the central component of drug action, R-1230 (0.5 mg in 0.1 ml) was administered locally to CNS by ICV route. [11] Effect on arterial pressure, pres­sor responses to carotid occlusion and epi­nephrine (3-4 µg/kg) were studied. The effect of same dose administered intra­venously was studied in same animal after blood pressure returned to control levels. Placement of cannula was con­firmed by injection of Indian Ink at the end of experiment.

Effect on direct medullary stimulation

In three chloralose anaesthetised (80 mg/kg i.v.) cats, pressor responses were obtained by electrical stimulation of medullary vasomotor centre by means of Horsley-Clarke Stereotaxic technique. The electrode placement was aided by the parameters described by Wang and Ran­son. [18] R-1230 (200 ,ug/kg) was administer­ed through the cannulated vertebral artery as medulla oblongata in cats is supplied by vertebral artery. [15] Peripheral leakage was excluded by testing the effect of R-1230 on pressor response to injected norepinephrine (2-4 µg kg i.v.).

Effect on linguomandibular reflex

Effect of R-1230 administered through the cannullated vertebral artery on poly-synaptic linguomandibular reflex was studied in chloralose anaesthetised cats) [16] Recording was done on E & M physiograph using myograph B.

Effect on superior cervical ganglion

Three chloralose anaesthetised cats were used for this study. Effect of 1 mg/kg i.v. R.-1230 was studied on superior cervi­cal ganglion by recording the nictitating membrane contractions induced by elec­trical stimulation of pre and post gang­lionic sympathetic nerve trunk using supramaximal rectangular pulses. [14] Con­tractions were recorded on E & M phy­siograph using a myograph type B.

Hindquarter's perfusion

Cat hindquarter's perfusion was car­ried out with a sigma motor pump. [4] The effect of local arterial administration of R-1230 was studied on perfusion pressure to extremities and carotid blood pressure.

Rabbit's aortic strip

Spirally cut strips of descending thoracic aorta were used. [12] Dose/response curves for norepinephrine (5 x 10- 8 -2 x 10 -7 g/ ml of bath) were obtained by cumulative administration of increasing concentra­tions of norepinephrine before and 15 minutes after R-1230 (1 x 10 -8 M). The nature of antagonism was studied by the method of Arunlakshana and Schild. [2]

Isolated Guineapig ileum

Dose response curves for Acetylcholine (5 x 10 -9 -8 x 10- 8 g/ml) and histamine (5 x 10 -8 x 10 -8 g/ml of bath) were obtained by cumulative administration of increasing concentration of agonists, before and 15 minutes after R-1230 (1.6 x 1.0 -7 -3.2 x 10- 7 M), Log dose response curves were plotted and pD 2 value was determined. [17]

Rabbit Heart and Atria

Effects of R-1230 (0.1 mg-1.0 mg) on heart rate and coronary flow were studied in Langendorff's preparation. Effect of 5-100 µg/ml of R-1230 was studied on rabbit atria. Effect of atropine (1 µg/ml) was studied on drug induced depression. Similarly effect of R-1230 was studied on responses to epinephrine and acetylcho­line by administering them before and 5 minutes after R-1230.

Rectus abdominis of frog

Effects of R-1230 (5-50 µg/ml of bath) were studied on acetylcholine induced contractions,


 :: Results Top


Structure of R-1230 is described in [Figure 1].

Effect oil, Brood pressure is shown in [Figure 2].

Administration of R-1230, produced a dose related fall in blood pressure (35­-55% fall) with duration of 140-240 mins. Only ECG change observed was tachy­cardia. Debuffer preparation failed to affect these hypotensive effects. Intraduo­denal administration caused essentially the similar effects except for a gradual onset (5-15 minutes) and sustained effect lasting for 3-4 hours. The hypotensive effect was not altered by various blocking agents studied.

Effect on normotensive, spinal and dece­rebrate cats [Figure 3]

In normotensive animals R-1230 (5 mg/kg i.v.) produced a sharp, sustained fall in blood pressure (45-50% fall) with gradual recovery to basal level in 240 mins. Spinalisation resulted in abolition of this sustained hypotensive effect though an immediate transient fall with immediate recovery was evident. How­ever, in decerebrate preparation, both these components were seen. Thus, there was an initial transient fall with partial recovery in 1-2 mins, and then a secon­dary, gradual fall (50-55% fall) without complete recovery to predrug level upto 240 minutes.

Intrathecal administration of R-1230 had no effect on pressor response to SCVR and intravenous injection of norepinephrine. Same dose injected intravenously could inhibit both SCVR and norepinephrine response. This effect was evident within 10-15 minutes and disappeared after 90 minutes [Figure 4].

R-1230, markedly diminished the pres­sor response to bilateral carotid occlusion and pressor response to electrical stimulation of central cut end of vagus. There was a reversal of pressor response to epi­nephrine. Pressor response to norepine­phrine was markedly inhibited. No alterations in the depressor responses to injections of acetylcholine, histamine and to electrical stimulation of peripheral cut end of vagus were observed [Figure 5].

Effect on centrally Mediated vasopressor reflex [Figure 6]

R-1230, administered through cannulated vertebral artery produced a gradual and sustained fall in blood pressure reach­ing maximum at 10-15 minutes. The pres­sor responses to carotid occlusion and central vagal stimulation were completely ­ inhibited. The pressor response to inject­ed epinephrine was partially inhibited (30% inhibition). Recovery of all these responses was obtained after 90 minutes.

Effect of intracerebroventricular (ICV) administration of R-1230 [Figure 7]

There was a gradual fall in blood pres­sure (15-25% fall) lasting for 45-60 minutes. This was accompanied by inhi­bition of pressor response to carotid oc­clusion without affecting pressor response to intravenous epinephrine. Administration of the same dose intravenously in the same animal did not produce any of the above mentioned effects.

Effect on pressor response to direct medullary stimulation [Figure 8]

There was a significant inhibition (50­60%) of vasopressor responses evoked by direct medullary stimulation. Effect be­came evident in 10 minutes, lasted for 60-90 minutes and disappeared after 120 minutes.

Effect on linguomandibular reflex

R-1230
(200 µg/kg) in the vertebral artery did not produce any change in linguomandibular reflex though a gradual fall in blood pressure was evident.

Effect on superior cervical ganglion [Figure 9]

R-1230 (1 mg/kg i.v.) produced 50% inhibition of the responses of nictitating membrane to pre and postganglionie stimulation of cervical sympathetic nerve. The preganglionic nerve stimulation for a period of 60 sec. was also 50% inhibited and the inhibition was sustained through­out the period of stimulation. Contractile response of nictitating membrane to in­jected epinephrine was totally inhibited. Recovery was seen after 120-180 minutes.

Effect on hindquarters' perfusion [Figure 10]

R-1230 (1-8 µg) produced a transient fall in the mean perfusion pressure to the lower extremities of cats without any alteration in the systemic blood pressure. The dose response relationship was ob­served over a small dose range only.

Study of antagonisms between norepine­phrine and R-1230 [Figure 11]

In three experiments (spinal prepara­tion was used to avoid fall in blood pres­sure due to administration of R-1230),dose response curves for norepinephrine were established alone and following different doses of R-1230. When 1/v versus 1/s was plotted [Figure 11] where v is the pressor response in mm-Hg and s is the dose of norepinephrine in µg/kg it was observed that the resultant lines had different slopes but common inter­cept lying in the line corresponding to infinite dose, This was suggestive of com­petitive antagonism between norepine­phrine and R-1230. [8]

Effect on rabbit's aortic strip

R-1230 could effectively antagonise norepinephrine induced contractions. There was a parallel shift to the right of the dose response lines for norepinephrine and maximum response could be attained with higher doses of norepinephrine in presence of antagonist, The pA 2 , value for R-1230 was 7.83 whereas pA 2 ,-pA 10 was 0.93. The mean log k 2 value was 7.77.

Effect on Guineapig's ileum

There was a dose dependent antagonis­tic effect of R-1230 on acetylcholine and histamine. The cumulative dose response curve for acetylcholine and histamine re­corded in the presence of various concentrations of R-1230 showed a non-compe­titive antagonism. Average pD 2 values for acetylcholine and histamine were found to be 6.55 and 6.79.

Effect on rabbit's heart and atria

R-1230 decreased the rate and ampli­tude of contractions of rabbit heart. Re­covery was seen in 10-15 minutes. No change in coronary flow was evident.

In a dose range of 10-100 µg/ml of organ bath, R-1230 produced a dose dependent diminution in the amplitude and rate of contractions of rabbit atria. Recovery followed after washing out the drug. The depression of atria induced by R-1230 was unaffected by prior atropine treat­ment (1µg/ml of bath). No alterations in responses of atria to epinephrine (0.05-0.1 µg/ml of bath) or acetylcholine (0.1-0.2 µg/ml) were seen on addition of R-1230 in doses of 1-5 pg /ml of bath.

Effect on rectus abdominis of frog

R-1230 did not produce any effect perse nor did it significantly alter acetylcho­line induced contractions.


 :: Discussion Top


Study of blood pressure activity of R­1230 revealed that there was a dose de­pendent reduction in mean arterial pres­sure in anaesthetised cats and dogs. Since hypotensive effect was immediate in onset, possibility of a metabolite being active could be ruled out. The rapid and sustained effect also ruled out the possibility of effect mediated through the release of a vaso depressor substance like histamine.

Hypotensive action was evident even in debuffer preparation which signified that the action was not mediated via an afferent of the parasympathetic nervous system nor through carotid or aortic sinus. In addition, R-1230 in large doses (5 mg/ kg) failed to inhibit the depressor re­sponse of peripheral vagal stimulation. At a dose of 1 mg/kg i.v. it inhibited the effects of both preganglionic and post­ganglionic sympathetic stimulation over 15 sec., and when preganglionic stimula­tion was carried out over 60 sec., inhibi­tion remained sustained throughout the period of stimulation. This excluded the possibility of its ganglion blocking acti­vity. [14] However, nictitating membrane response to exogenously administered adrenaline was also abolished. It could also inhibit pressor responses to exoge­nous epinephrine and norepinephrine and these findings were suggestive of its blocking effects on receptors in peripheral adrenergic effector organs. Stimulant action on β-receptors could also be ruled out as propranolol pretreatment failed to affect its hypotensive response. Inhibition of vasomotor reflexes elicited by carotid occlusion and stimulation of central cut end of vagus suggested the possibility of additional effect on central site of vaso­motor integration.

In the doses studied, this compound did not possess significant action on the myo­cardium. ECG did not reveal any abnor­mality except for reflex tachycardia which was abolished in debuffer preparation.

In vitro studies using rabbit heart and rabbit atria revealed a depressant effect on the rate and force of contraction only at very high dose ranges (0.25 to 1.0 mg for heart and 10-1000 µg/ml for atria).

Since cat's hindquarter perfusion stu­dies revealed a transient fall in perfusion pressure in doses not affecting systemic blood pressure, it appeared that R-1230 has some direct action on vascular smooth muscles. However, this could not account for the prolonged secondary hypotension seen with R-1230. Spinalisation of animals abolished this prolonged secondary hypo­tension, hence the site of action appeared to be in the central nervous system rostral to transection. The absence of hypotensive action in spinal animals could not be explained fully because of initial low blood pressure in such preparations, though it is known that peripherally act­ing vasodilators like nitrites can cause fall in blood pressure even in spinal pre­paration.

Since hypotensive action of R-1230 was not altered by decerebration, at midcol­licular plane, its site of action might be localised to an area lying between the midcollicular and second cervical segment. The reticular formation of the brain stem has areas within it responsible for main­tenance of blood pressure. Neurophysio­logical studies [1],[3],[18] point to the existence of an integrative site in the region of the calamus scriptorius, referred to most com­monly as the vasomotor centre for con­trol of blood pressure.

The possibility exists that R-1230 might have produced hypotension by acting at these sites. Reversal of epinephrine re­sponse with marked inhibition of norepinephrine response, was noted with R­1230. The epinephrine reversal could be abolished by propranolol. But R-1230, when administered after propranolol could inhibit the pressor response to epi­nephrine and norepinephrine. Thus the reversal of epinephrine response was due to alpha adrenergic blocking activity and not due to potentiation of beta receptor activation. Since all the dose response curves of norepinephrine in the absence and presence of various closes of R-1230 were found to meet at a point on the y axis, by the Line-Weaver-Burk Plot ana­lysis, nature of antagonism appeared to be competitive. [8] This was further confirm­ed by the 'in vitro' studies with rabbit aortic strips. There was a parallel shift of the to dose response curves in pre­sence of different concentrations of R-1230 without any alteration in the slopes of the curve. [2] The pA 2 ,-pA 10 value for R­1230, was 0.93, which is in good agree­ment with the theoretical value of 0.95 for competitive antagonism. Further, the mean log k 2 value was also found to be the same as its pA 2 value.

Chai and Wang [7] have shown that re­flexes of afferent nerve stimulation are integrated in the medulla oblongata in the vasomotor centre. Any agent affecting central integrative sites can block the responses. Thus depression of carotid occlusion reflex and inhibition of pressor response to central vagal stimulation by R-1230, raised the possibility that in addi­tion to its peripheral adrenergic blocking activity, it could depress CNS sites re­sponsible for blood pressure regulation. Further studies by the intracerebroven­tricular and intravertebral artery administration of R-1230 confirmed the hypotensive effect of the compound. Centrally mediated reflexes and the pres­sor response to direct medullary stimula­tion were also inhibited by R-1230. In this relation peripheral action was ruled out as the compound administered by these routes did not affect the responses to epinephrine and norepinephrine given intravenously. All these findings led to the conclusion that hypotensive action of these drugs was in part due to the de­pression of the medullary and mesenche­phalic integrative mechanisms involved in blood pressure regulation. It is known that as a result of generalised CNS de­pressant activity, various autonomic and somatic reflexes can be inhibited. Since R-1230 produced hypotensive effect without altering linguomandibular reflex, it was concluded that its hypotensive action was due to selective depression of autonomic centres and not as a result of generalized depression of CNS.

R-1230 administered intrathecally failed to alter pressor response resulting from the compression of spinal cord. R-1230 1 mg/kg given i.v. could abolish SCVR. This was suggestive of peripheral site of action in addition to the effect exerted on brain stem structures mentioned above. Similar actions have been describ­ed for chlorpromazine, hydrallazine, and tolazoline. [5] It is clear that the compound did not act on spinal neurones possibly as a result of failure of drug to penetrate the site because it was given intrathecally. This could be explained in the light of findings of Feldberg and Fleishhauri [10] and Feldberg [9] that response to a drug varies with its site of application whether out­side or inside the brain.

Thus the results of the present study suggest that R-1.230 induced hypotension is of a complex nature. A direct transient vasodilator action at the periphery appears to cause immediate sharp fall in blood pressure. The prolonged hypotensive re­sponse may be attributed to its central vasomotor depression as well as peri­pheral alpha adrenergic blockade caused by the drug.

 
 :: References Top

1.Alexander, R. S.: Tonic and reflex func­tion of medullary sympathetic cardio­vascular centres. J. Neurophysiol., 9: 205-217, 1946.  Back to cited text no. 1    
2.Arunalakshana, O. and Schild, H. O.: Some quantitative uses of drug antagonists, Brit. J. Pharmacol., 14: 48-58, 1959.  Back to cited text no. 2    
3.Back, L. M. N.: Relationships between bulbar, respiratory, vasomotor and somatic facilitatory and inhibitory areas. Am. J. Physiol., 171: 417-435, 1952.  Back to cited text no. 3    
4.Beck, L.: Active reflex dilatation in the innervated perfused hind leg of the dog. Am. J. Physiol., 201: 23-28, 1981.  Back to cited text no. 4    
5.Bhargava, K. P. and Kulsreshtha, J. K.: The spinal compression vasomotor response as a pharmacological tool. Arch, int. Pharmacodyn., 120: 85-93, 1959.  Back to cited text no. 5    
6.Burn, J. H : Practical Pharmacology Blackwell Scientific Publication, Oxford. pp. 22-24, 25-29, 35-36, 37-39, 1952.  Back to cited text no. 6    
7.Chai, C. Y. and Wang, S. C.: Integration of sympathetic cardiovascular mechanism in medulla oblongata of the cat. Am. J. Physiol., 215: 1310-1315, 1968.  Back to cited text no. 7    
8.Chen, G. and Russel, D. A.: A quantita­tive study of blood pressure response to cardiovascular drugs and their antagonists. J. Pharmac. Expt. Ther., 99: 401-408, 1950.  Back to cited text no. 8    
9.Feldberg, W. A.: Pharmacological ap­proach to the brain from its inner and outer surface, Edward Arnold (publishers) Ltd. pp. 9-29, 1963.  Back to cited text no. 9    
10.10.Feldberg, W. and Fleischhauer, K.: Pene­tration of bromophenol blue from the per­fused cerebral ventricles into the brain tissue. J. Physiol., 150: 451-462, 1960.  Back to cited text no. 10    
11.Feldberg, W. and Sherwood, S. L.: In­jections of the drugs into the lateral ven­tricles of cats. J. Physiol., 123: 46-67, 1954.  Back to cited text no. 11    
12.Furchgott, R. F. and Bhadrakom, S.: Re­actions of strips of rabbit aorta to epine­phrine, isopropylarterenol, sodium nitrite and other drugs. J. Pharmac. Expt. Ther., 108: 129-143, 1953.  Back to cited text no. 12    
13.Gaitonde, B. B., McCarthy, L. E. and Borison, H. L.: Central emetic action and toxic effects of digitalis in cats. J. Phar­mac. Expt. Ther., 147: 409-415, 1965.  Back to cited text no. 13    
14.Green, A. F. and Boura. A. L- A.: Eva­luation of drug activities: In, "Pharma­cometrics" Editors-D. R. Laurence and A. L. Bacharach, Academic Press, N.Y. pp. 432-456, 1964.  Back to cited text no. 14    
15.Holms, R. L., Newmann, P. P, and Wol­storcroft, C.: The distribution of carotid and vertebral blood in the brain of the cat. J. Physiol., 140: 236-246, 1958.  Back to cited text no. 15    
16.King, E. E. and Unna, K. R.: The action of mephenesin and other interneuron depressants on the brain stem. J. Phar­mac. Expt. Ther., 11: 293-301, 1954.  Back to cited text no. 16    
17.Van Rossum, J. M.: Cumulative dose re­sponse curve II. Technique for the making of dose response curves in isolated organs and the evaluation of drug parameter. Arch. int. Pharmacodyn., 143: 451-481, 1954.  Back to cited text no. 17    
18.Wang, S. C. and Ranson, S. W.: Auto­nomic responses to electrical stimulation of the lower brain stein. J. Comp. Neu­rol., 71: 437-472, 1939.  Back to cited text no. 18    


    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7], [Figure 8], [Figure 9], [Figure 10], [Figure 11]



 

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