Studies on the mechanism of hypotensive action of a disubstituted piperazine derivative - (Part II)NK Dadkar, Vaishali N Dadkar, UK Sheth
Department of Pharmacology, Seth G. S. Medical College, Parel, Bombay-400012, India
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.
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.
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. 
Effect on SCVR
Effect of spinal compression vasomotor response  (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. 
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.  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.  R-1230 (200 ,ug/kg) was administered through the cannulated vertebral artery as medulla oblongata in cats is supplied by vertebral artery.  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)  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.  Contractions were recorded on E & M physiograph using a myograph type B.
Cat hindquarter's perfusion was carried out with a sigma motor pump.  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.  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. 
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. 
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,
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. 
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
R-1230 did not produce any effect perse nor did it significantly alter acetylcholine induced contractions.
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.  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 ,, 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.  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.  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  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.  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  and Feldberg  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.
[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7], [Figure 8], [Figure 9], [Figure 10], [Figure 11]