Pharmacological Studies of p, N-(3, 4-Methylenedioxy Phenyl) Benzoic Acid (RRL-1364) - part-IISharadini A Dahanukar, UK Sheth
Department of Pharmacology, Seth G.S.Medical College, Parel, Bombay 400012, India
Cholinomimetic activity of p, N- (3, 4-methylene dioxy phenyl) benzoic acid was revealed while studying its hypotensive activity in cats. Further evaluation of this activity showed that this compound had both muscarinic and nicotinic components. The cholinomimetic activity was not similar to anticholinesterase like activity. Species variation was present; in rats it failed to show any activity and no activity was exhibited in vitro suggesting some metabolic transformation.
Pharmacological investigations of the new PABA derivative [p, N- (3, 4-Methylenedioxy phenyl) benzoic acid-RRL1364] revealed dose dependent hypotensive action in anaesthetised cats. This action could be blocked by atropinization of cats. Detailed evaluation of the cholinomimetic activity of RRL-1364 was carried out in cats, which is discussed in the present paper.
Effect on smooth muscle: In 3 cats, the effect on smooth muscle of the intestines was studied in vivo by the method described by Jackson. 
In three guinea pigs, the effect of RRL-1364 was studied on smooth muscle of the bronchial tree using Konzett Rossler method.  Response to RRL-1364 alone and its effect on acetyl choline action was studied.
The effect of RRL-1364 was observed on smooth mscles in vitro by using various isolated tissues like guinea pig ileum, rat uterus, guinea pig tracheal chain (Sheth et al  ).
The responses of RRL-1364 in graded doses and its effect on the action of various agonists like acetyl choline, histamine, 5 hydroxytryptamine was noted.
For this purpose, since RRL-1364 was insoluble in water, propylene glycol was used as a solvent and for each experiment control responses with the solvent alone were recorded.
Effect on secretions: The effect of RRL 1364 was noted on the salivary secretions according to the method described by Jackson . 
Evaluation of the nicotinic component: Blood pressure was recorded in 3 cats anaesthetised with ether chioralose. Atropine sulphate 2 mg/kg was injected slowly i.v. and complete blockade of depressor response to acetyl choline (3-5 µg/kg) was confirmed. RRL-1364 was then injected in a large dose (10 mg/kg) i.v. and the response noted.
Effect on superior cervical ganglion: The contractions of the nictitating membrane of cats were elicited in four cats by pre-ganglionic and post-ganglionic stimulation by rectangular pulses. The effect on responses to injected epinephrine (3.5 .g/kg) was also recorded.
When RRL-1364 was given, i.v. a fall in B.P. was produced and therefore, for better interpretation of results of effect on SCG, RRL-1364 was given in small dose (100 µg) in the local circulation of ipsilateral SCG by cannulating lingual artery' , and the effect observed on the contractions of the nictitating membrane.
Effect on neuromuscular junction: Action of RRL-1364 on the neuromuscular junction was studied in situ on tibialis anterior muscle nerve preparation in 3 anaesthetised cats. RRL-1364-200 µg. was injected intra-arterially.
Effect of neostigmine (200 µg. i.a.) on RRL induced contraction was also observed. In addition, effect of the direct muscle stimulation was noted.
Effect on neuromuscular junction was studied on rat phrenic nerve diaphragm preparation as described by Sheth et al.  Contractions produced by nerve stimulation were recorded on E and M physiograph using myograph type B. RRL1364 was added to the bath solution in a concentration ranging from 10-40 µg/ml and was allowed to act for five minutes and the effect of the compound in different doses on muscle contractions elicited by nerve stimulation was studied.
The isolated frog-rectus abdominis muscle was also used to study the effect of RRL-1364 on the skeletal muscle.
Effect on intestinal movements: Intestinal movements in cats under the influence of RRL-1364 (1 mg/kg) were seen to be augmented. The effect started in 15 minutes, was maximum at 90 minutes and continued with increased intensity till the end of four hours [Figure 1].
Konzett Rossler preparation: The compound did not produce any effect perse and it did not affect the effect of acetyl choline on the bronchial resistance.
Effect on isolated smooth muscle preparations: RRL-1364 in doses of 10-40 µg/ml of the bath solution did not produce any effect on the guinea pig ileum, rat uterus and guinea pig tracheal chain.
In addition, the compound in above mentioned doses had no effect on acetyl choline, histamine and barium chloride induced contractions of the guinea pig ileum, 5 Hydroxytryptamine induced contractions of the rat uterus and histamine and acetyl choline induced contractions of the guinea pig tracheal chain.
Effect on salivary Secretion: RRL 1364 in a dose of 1 mg/kg i.v. did not produce any significant effect on the salivary secretions collected from the submaxillary gland.
Nicotinic effect on blood pressure: RRL-1364 when given in a higher dose (10 mg/kg) after prior atropinization, sufficient to completely block the depressor response of acetyl choline (1.3 µg/ kg)-produced a sharp rise in blood pressure (about 40%) and recovered to the basal pre-drug level by ten minutes [Figure 2].
Effect on superior cervical ganglion and nictitating membrane of cat: Though the contractions of the nictitating membrane were diminished after i.v. administration of RRL-1364, locally administered compound did not alter the responses of the nictitating membrane to the stimulation of pre-ganglionic and post-ganglionic nerves.
Rat phrenic nerve diaphragm preparation: RRL-1364 in doses of 10-40 µg/ ml of the bath solution did not produce any effect on contractions of the rat hemidiaphragm elicited by the phrenic nerve stimulation.
Effect on Neuro-muscular junction: Cat tibialis anterior muscle-nerve preparation: RRL-1364 injected in a cumulative dose of 200 µg. through the femoral artery, produced initial transient fasciculations of the contractions of tibialis elicited by nerve stimulation. This was followed by a decrease in the height of contractions in about three minutes and marked blockade was observed within 7 to 10 minutes.
This blockade continued without any recovery till the end of four hours. The blockade was unaffected by neostigmine 200 µg. given intra-arterially. The responses to direct muscle stimulation, however, were unaltered [Figure 3].
RRL-1364 did not produce any effect per se nor did it have any effect on contractions induced by acetyl choline, in frog rectus abdominis muscle and rat phrenic nerve diaphragm preparation.
During evaluation of RRL-1364-a derivative of para-amino-benzoic acidfor hypotensive activity, it was seen that its hypotensive action could be completely blocked by pre-treatment with atropine. When further studies were carried out for its cholinomimetic activity, it was observed that it has got some action on the muscarinic receptors of the smooth muscles of intestines as recorded by Jackson's enterograph. But there was no significant augmentation of the salivary secretions from the sub-maxillary salivary glands collected from Wharton's duct. There was no effect on bronchial smooth muscles.
RRL-1364 exhibited nicotinic action on the blood pressure through stimulation of the sympathetic ganglia and adrenal medulla. But it had no effect on the superior cervical ganglion when it was given in the local circulation.
The diminution in contractions of the nictitating membrane on systemic administration of RRL-1364, could be due to an accompanied fall in the blood pressure and this hypotensive effect is excluded when the compound is given locally, in a small dose.
On the neuromuscular junction the compound had action resembling that of membrane depolarisers, producing initial fasciculations and later blockade which could not be reversed by the use of anticholinesterase.
Thus the compound RRL-1364 appeared to have complex cholinomimetic actions, having both muscarinic as well as nicotinic components. Like acetyl choline, it produced a fall in blood pressure blocked by atropine, it stimulated the intestinal movements and it produced membrane depolarization at the site of the neuromuscular junction. Its nicotinic action on blood pressure could also be demonstrated but unlike acetyl choline, it had long duration of action; it was absorbed when given intraduodenally  and there was no effect on the salivary secretions.
The cholinomimetic activity of the compound does not appear to be due to anticholinesterase activity as the depressor effect of acetyl choline on blood pressure was unaffected.
Another feature of this compound was its inability to show any muscarinic activity on the isolated smooth muscle of guinea pig ileum, guinea pig tracheal chain and isolated rat uterus. Similarly, it failed to show any effect on the isolated rat phrenic nerve diaphragm preparation and frog rectus abdominis muscle. This could be explained on the basis of species variation because it was seen in our earlier work that the compound had marked and prolonged hypotensive activity in cats, less pronounced and shorter lasting hypotensive activity in dogs and no activity in rats.
Another possibility which could be suggested is that the compound as such may not be effective in vitro and it is active only in vivo. Thus the results of the present study reveal cholinomimetic activity of complex nature, and it is premature to conjecture whether this compound will find a place in the treatment of hypertension. Much is yet to be known about it, for example its effect on hypertensive animals, effects on peripheral biogenic amines, etc. before its therapeutic potential can be considered.
We sincerely thank Dr. P. B. Sattur, Regional Research Laboratories, Hyderabad, for his assistance in synthesizing and supplying the compound.
[Figure 1], [Figure 2], [Figure 3]