DIANS ium and Calcium levels in dila. bettc. erience in Managing Sickle-Cell esium, Brit. Med. J. 2, 226.29. in. Path. 13, 99. srUDI.ES ON CENTRAL NERVOUS SYSTEM DEPRESSANTS (IX) 2): Serum magnesium levels in The action of some tranquillizing agents on the coaxically J.l: 1158. stimulated guineapig ileum

in Body /Iuids, J. Bioi. Chem. By P. C. DANDlYA AND K. L. lc\EMNANI 1,1405 . Department of Pharmacology, S. M. S. Medical College, Jaipur

. (1962) : Some constituents of (Received May 21, 1964)

Tranquillizers, namely, benactyzine hydrochloride, pipe thanate hydrochloride, elermination of serum magne. trifluoperazine, rescinnamine, carisoprodol, captodiamine, and emylcamate were investi- 69,39. gated for their mechanism of action on the intestine. All the carbamate derivatives except for the estimation of serum emylcamate failed to cause reduction or abolition of twitch due to electrical stimulation. From our findings it appears that some of the tranquillizers namely reserpine, rescinnamine, chlorpro azine, prochlorperaz ine, azacyclonal, and hydroxyzine act on intestine by com- petitive antagonism of acetylcholine at the receptor groups, whereas drugs like benactyzine, pipethanate, captodiamine and trifluoperazine appear to depress the twitch of intestine by directly acting on the muscle.

Paton (1957) has shown that morphine and morphine-like analgesics depress the twitch and tetanus of coaxicaUy stimulated guinea pig ileum by reducing acetyl- choline (Ach.) released from cholinergic nerve endings. Schaumann (1957) also, though using a different technique, has shown that morphine inhibited the release of Ach. from isolated small intestine of guineapig. Experimental evidence has been presented by this worker which suggests that reduced release of Ach. could not be explained by an inhibition of the synthesis of Ach. nor by stabilization of the bound form of Ach. in the tissue. Apparently morphine reduces the excitability of post- ganglionic structures and thereby the liberation of Ach. from nerve endings during the process of excitation. Kosterlitz and Robinson (1955) found that morphine has an inhibiting effect on the action of 5-hydroxytryptamine (serotonin), whose action on the ileum is now believed to involve nervous pathwa (Robertson, 1953; Rocha E Silva, et al., 1953 and Gaddum and Hameed, 1954). In clinical practice, it has been observed that several tranquillizers also act on the gastrointestinal tract, causing thereby various disturbances of gastrointestinal tract. The effects of some of the ataractic drugs on coaxically stimulated guineapig ileum were reported earlier from this laboratory (Dandiya, 1963). The results of this study warranted similar investigation on some more ataractic drugs in an effort to elucidate their mechanism of action on intestine. The compounds studied were: Benactyzine 2-diethylaminoethyl ester of benzilic acid. Pipethanate - 2-piperid-l-ylethyl benzilate, 162 c. N. S. DEPRESSANTS (IX)

Trifluoperazine 10-[3-( 4-methylpiperazin-l-yl) propyl]-2-trifluoromethylpheno- thiazine. Rescinnamine 3 : 4: 5-trimethoxycinnamate ester of metbylreserpate. Carisoprodol = 2-Carbamyloxymethyl-2-isopropyl-carbamyloxymetbylpeutane. Cap todiam ine - p-butyIthiodiphenylmethyl-2-dimethylaminoethyl sulphide. Emylcamate Carbamate of l-ethyl-l-methyl-l-propanoI.

MATERIALS AND METHODS The technique of Paton (1957) with modifications as reported from this laboratory earlier (Dandiya, 1963) was employed. All experiments were done on pieces of guineapig's small intestine. Guineapig was killed by a blow on the neck. The small intestine was taken out and placed in Kreb's solution. A small piece (nearly Il") of the ileum was taken, emptied of its contents and tied off at both the ends to prevent the mucus in the lumen from oozing into the bath. One end of the gut piece was tied on to a glass tubing and suspended in Kreb's solution bubbled with oxygen, while the other end was clipped to a silver electrode. The capacity of bath was 50 ml. and temperature was controlled between ~o_37° c. Current pulses of 1 m. sec. duration and 4 volts to produce maximal response to a single shock were applied to the electrodes after an interval of every 15 seconds. The contractions of the gut were recorded on a smoked drum by means of a suffi· ciently light lever. The silver wire was attached to the lever for recording the intestinal movements. Appropriate quantity of the drug under test was added to the bath in the form of an aqueous solution. All the drug solutions were made in distilled water excepting that of rescinnamine which was dissolved in a few drops of glacial acetic acid and the volume was made up with water. The volume of each solution added to the bath was less than 1 ml, Appropriate concentrations of Ach. were also added to the bath, so as to cause a muscle contraction almost equivalent to that induced by elect- rical stimulation. The sensitivity of the muscle to Ach. was always tested before and after treatment with the drug. If pretreatment with the drug did not bring any response due to control doses of Ach. then higher doses of Acb. were tried. The doses of various drugs employed are expressed in terms of their salts.

RESULTS Benactyzine was found to be very potent in abolishing the twitch of guineapig ileum due to electrical stimulation and in suppressing the response to Ach. In doses as low as 1 p.g it was able to prevent the twitch due to electrical stimulation, response to very high doses (250 times the control dose) of Ach. was less than that due to the control dose prior to drug treatment (Fig. 1). Pipethanate in doses as low as 1 ug was able to abolish twitch due to electrical stimulation, but response to twice the control dose of Ach. was less than that due to the control dose of Ach. prior to drug treatment (Fig. 2).

,

" yl]-2-trifluoromethylpheno-

of methylreserpate. carbamyloxymetbylpentane. ylaminoethyl sulphide. N ropanol.

ions as reported from this experiments were done on n 1I rII I I Jled by a blow on the neck. t t t l' t '002 ••S,u.g. Kreb's solution. A small .ost·4 . Ach vi T '1 Ach. its contents and tied off O.OQ2~9· 8ClhaC+yz;n~ oozing into the bath. One 1,4.1.9 spended in Kreb's solution a silver electrode. The trolled between ~o_37° C. Fig. 1. The effect of benactyzine hydrochloride on twitches (4/min) and acetylcholine response of guineapig ileum. At 'N' normal twitches are shown. Acetylcholine 0.002 p.g was added ce maximal response to a to the bath while electrical stimulation was temporarily stopped. At 'W' the ileum was rval of every 15 seconds. washed. On treatment with benactyzine hydrochloride (1 p.g) the twitch was reduced drum by means of a suffi- and then abolished. Response to acetylcholine after treatment with the drug could be elicited only with 0.5 p.g. r for recording the intestinal dded to the bath in the form e in distilled water excepting of glacial acetic acid and eh solution added to the Ach. were also added to the ent toJhat induced by elect- was, always tested before the drug did not bring any f Ach. were tried. d in terms of their salts.

hing the twitch of guineapig response to Ach. In doses ctrical stimulation, response as less than that due to the

olish twitch due to electrical Fig. 2. The effect of pipethan ate hydrochloride on twitcbes (4/min) and acetylcboline response of guineapig ileum. At 'N' normal twitches are shown. Acetylcholine 0.1 p.g was added to eh. was less than that due to the bath while electrical stimulation was temporarily stopped. At 'W' the ileum was washed. On treatment with pipethanate hydrochloride (l p.g) the twitch was first reduced and then abolished. Response to acetylcholine after treatment with the drug could be elicited only with 0.2 {Jog.

• 164 C. N. S. DEPRESSANTS (IX)

Captodiamine in moderate doses (30 to 100 JLg) gradually abolished the twit due to electricalstimulation, and high doses'of Ach. failed to elicit response (Fig.3

Fig. 3. The effect of captodiamine hydrochloride on twitches (4/min) and acetylcholine response guineapig ileum. At 'N' normal twitches are shown, Acetylcholine 0.01 p.gwasadded the bath while electrical stimulation was temporarily stopped. At 'W' the ileumVIII washed. On treatment with captodiamine hydrochloride (100 p.g) the twitch reduced and gradually abolished. Response to acetylcholine after treatment with thed", could not be elicited even with 30 pg.

Trifluoperazine also had similar effect on the twitch height. In doses ran~DI between 30 p.g to 100 p.g it was able to gradually abolish the twitch due to eJectri stimulation, and Ach. in high doses (20 times the control dose) could elicit response (Fig. 4).

Rescinnamine when tried in moderate doses (100 p.g) abolished the twitchdue to electrical stimulation. Soon after the treatment with the drug, control dose Ach. was able to produce subnormal response but after a lapse of 1-2 minutes on~ exceptionally high doses of Ach. (2500 times the control dose) could elicit response and that too was less than due to control dose prior to treatment with the drug. But when tried in low doses (10-30 p.g) rescinnamine exhibited depressant effect very gradually. After treatment with the drug for 10 minutes, twitch in responseto electrical stimulation was gradually suppressed, and control dose of Ach. could elicit only subnormal response (Fig. 5). adualJy abolished the twitch to elicit response (Fig. 3).

Fig. 4.- The effect of trifluoperazine on the twitches (4/min) and acetylcholine response of guineapig ileum. At 'N' normal twitches are shown. Acetylcholine 0.01 p.g was added to the bath while electrical stimulation was temporarily stopped. At 'W' the ileum was washed. On treatment with trifluoperazine (30 p.g) the twitch was gradually reduced and then abolished. Response to acetylcholine after treatment with the drug could be elicited only with 0.2 Il-g.

) and acetylcholine response of ylcholine 0.01 p.g was added to ped. At 'W' the ileum was de (100 p.g) the twitch was after treatment with the drug

height. In doses ranging e twitch due to electrical se) could elicit response

abolished the twitch due drug, control dose of se of 1-2 minutes only e) could elicit response atment with the drug. Fig. 5. The effect of rescinnamine on twitches (4/min) and acetylcholine response of guineapig ibited depressant effect ileum. At 'N' normal twitches are shown. Acetylcholine 0.3 p.g was added to the bath es, twitch in response to while electrical stimulation was temporarily stopped. At 'W' the ileum was washed. On treatment with rescinnamine (30 Il-g) no' effect on twitches was observed but after lapse of 01 dose of Ach. could 10 minutes the twitches first became variable and reduced, and then were abolished. When electrical stimulation failed to produce response, it was stopped and acetylcholine 0.3 p.g was added to the bath. The response to acetylcholine obtained was considerably shorter as compared to pretreatment response. 166 C. N. S. DEPRESSANTS (IX)

Carisoprodol in moderate doses (lOO fLg) had no effect on the twitch height due to electrical stimulation. In this property it resembles with another dicarbamate derivative meprobamate as reported earlier.

Emylcamate in doses ranging between 100 p.g to 300 Itg had no effect on the twitch height due to electrical stimulation and on the response due to Ach. Butin doses as high as 1 mg., it gradually abolished the twitch due to electrical stimulation and control doses of Ach. could elicit subnormal response. (Fig. 6).

Fig. 6. The effect of emylcamate on twitches (4/min) and acetylcholine response of guineapig ileum. At 'N' normal twitches are shown. Acetylcholine O.l.1tg was added to thebath while electrical stimulation was temporarily stopped. At 'W' the ileum was washed. On treatment with emylcamate 1 mg. the twitches were gradually abolished. The twitch produced by Acetylcholine 0.1 p.g was considerably shorter as compared to the contraction in untreated muscle.

DISCUSSION Present observations when considered along with the results reported earlier from this laboratory (Dandiya, 1963), throw light on the mechanism of action of these drugs. The combined results are classified in the Table I. Benactyzine and pipethanate have been found to be more potent as compared to hydroxyzine, azacyclonal, and captodiamine. These latter compounds have prevented the response due to electrical stimulation only in high concentrations. . ~ P. C. DANDlYA AND K. L. HEMNANI 167

TABLE I ffect on the twitch height

with another dicarbamate Inhibition of Inhibition of Class Name response to elec- response to Ach. trical stimulation

Ilg had no effect on the nse due to Ach. But in Diphenyl methane to electrical stimulation Derivatives Azacyclonal ++ ++ (Fig. 6). Benactyzine +++++ +..1-+++ Pipethanate +++++ +++ Captodiamine +++ ++++ Hydroxyzine ++ ++

Phenothiazines Chlorpromazine ++ ++ Prochlorperazine +t ++ Trifluoperazine ++++ ++++

Rauwolfia Alkaloids ... Reserpine ++ + Rescinnamine ++ +

Carbamates Meprobamate 0 0 line response of guineapig 1 p.g wasadded to the bath Carisoprodol 0 0 the ileum was washed. On lIy abolished. The twitch Emylcamate ompared to the contraction + + Phenaglycodal 0 0 esults reported earlier Key -Very Strong ++++--r Strong ++++ cbanism of action of Moderate +++ Mild ++ Very Mild + No effect 0 re potent as compared Iter compounds have high concentrations.

\ 168 C. N. S. DEPRESSANTS (IX)

These results are not surprising keeping in view the fact that benactyzine and pipethanate possess potent anticholinergic action. Among phenothiazines, trifluoperazine has been found to be depressantof intestine in doses of 30-100 fJ.g and in this respect it has been found to be almost three times more potent than chlorpromazine and prochlorperazine. On comparing the structure and activity of the above mentioned phenothiazines, it appears that the potent action of trifluoperazine on intestine may be due to 2-trifluoromethyl sub- stitution in the phenothiazine molecule in place of the 2-chloro group. It is well known tlHtt trifluoperazine is many times more potent in its tranquillising action than chlorpromazine, although unlike the latter compound it has not been shownto possess potent anticholinergic activity (Bradley, 1963). This anomaly therefore needs further investigation.

The pattern of action on intestine shown by reserpine and rescinnamine is more or less similar. However in doses of 100 fJ.gthe onset of inhibition demo- nstrated by rescinnamine as compared to that due to reserpine, is slower but more complete.

Carbamate derivatives have hardly exhibited any action worth mentioning o Qept thllt gf ern yloa mu te whioh ha hown mild depressant action. These observe- tions on carbamates confirm the present knowledge that these drugs act centrally and there is very little direct effect of these drugs on the intestine.

In conclusion it can be stated that although like morphine and codeine, the tranquillising agents reserpine, rescinnamine, chlorpromazine, prochlorperazine, azacyclonal, and hydroxyzine were able to abolish twitch due to electrical stimula- tion when employed in appropriate concentrations, unlike the two analgesic agents these also appreciably prevented the response to Ach. Although these drugs do not possess any structural similarity with Ach, but the possibility of a competitive antagonism cannot be altogether ruled out.

On the other hand, some of the drugs namely benactyzine, pipethanate, captodiamine, and trifluoperazine when used in appropriate concentrations, not only abolished the twitch due to electrical stimulation but also the response due to Ach. even when tried in considerably high doses. It appears that there is likelihood that these drugs act directly on the muscle.

The authors are thankful to Shri M. K. Menon for assrstrng in some of the experimental work and are also indebted to the respective pharmaceutical houses for the supply of the following drugs :-

Trifluoperazine Hcl. (Stelazine) Smith, Kline & French Labs. Ca todiamine Hcl. (Suvren) Ayerst, Mckenna & Harrison Ltd., Montreal. Carisoprodol (Soma) Wall ace Labs., New Brunswick. P. C. DANDlYA AND K. L. HEMNANI 169

Reed & Carnrick, New Jersey. e fact that benactyzine and Pipethanate Hc1. (Sycotrol) Benactyzine Hcl (Suavitil) and Eroylcamate Merck Sharp & Dohme Research Lab, West Point, 1-'a. en found to be depressant of has been found to be almost REFERENCES hlorperazine. On comparing Bradley, P. B. (1963) In "Physiological pharmacology" Ed. Root, W. S. and Hofmann, F. G. othiazines, it appears that the Academic Press, New York, I, 464. due to 2-trifluoromethyl sub- Dandiya, P. C. (1963) Arch. into Pharmacodyn., 141, 216. he 2-chloro group. It is well Gaddum, J. H. and Hameed, K. A. (1954) Brit. J. Pharrnacol., 9, 240. n its tranquillising action than Kosterlitz, H. W. and Robinson, J. A. (1955) J. PhysioI. (Lond.), 129,18. d it has not been shown to Paten, W. D. M. (1957) Brit. J. PharmacoI.,12, 119. 3). This anomaly therefore Robertson, P. A. (1953) J. Physiol. (Lond.), 121,54. Rocha E. Silva, M., Valle, J. R. and Picarelli, Z. P. (1953) Brit. J. Pharrnacol., 8,378. serpine and rescinnamine is Scbaumann, W. (1957) Brit. J. Pharmaccl., 12,115. e onset of inhibition demo- reserpine, is slower but more

y action worth mentioning isant action. These observa- these drugs act centrally and stine. e morphine and codeine, the romazine, prochlorperazine, tch due to electrical stimula- ike the two analgesic agents Although these drugs do not possibility of a competitive

y benactyzine, pipethanate, 'ate concentrations, not only o the response due to Ach. that there is likelihood that

ing in some of the experimental s for the supply of the following

e & French Labs. kenna & Harrison Ltd.,

bs., New Brunswick.