tio.nof aortic nerve on blood presia. J. Physiol.,109 : 392, 1949

ehyde and methylpentynol on n56.

ight subclavian artery. Am.:}. P",," • Key words: c\omipramine imipramine chlorpromazine neuroleptic activity

]uly-SeptemInd.]. Physiol. P

Method for ~tudyi ng competenceor dcnervatlOn Am ':I p'. . I

• • J. 'O'SIO '.

ERIMENTAL EVALUATION OF THE POSSIBLE NEUROLEPTIC ACTIVITYOF CLOMIPRAMINE*

J. J. BALSA RA AND A. G. CHANDORKAR

output following intravenous in'57-665,1944, Cited in Bioi, Atstr ••

ofsympathetic preganglionic acti

Department of Pharmacology,V. M. Medical College, Solapur-413 003

. J. Physiol.,222 I 1-15 1972. ,.ectncal stimulation of the carotid •

Summary: Clomipramine, a new antidepressant, differs from imipramine by having chlorine inposition 3 of the aromatic ring and in this respect resembles chlorpromazine. Clomipramine was thereforetested for neuroleptic activity. Clomipramine and imipramine were ineffective in inhibiting the tractionresponse and pinna reflex in mice and in inducing catalepsy in rat. Compared to chlorpromazinethey were less potent in blocking conditioned avoidance response and in decreasing spontaneous motoractivity and exploratory behaviour. In contrast to chlorpromazine, clomipramine like imipraminewas found to enhance methamphetamine-induced stereotyped behaviour. Thus clornipramine likeimipramine possesses negligible neuroleptic activity.

INTRODUCTION

Clomipramine hydrochloride (ANAFRAN[L) a newly introduced tricyclic antidepressa.itrug (2) differs from imipramine only by a chlorine substitution in position 3 of the aromatic

ring(11). In the experimental tests commonly used to investigate antidepressant activity clomi-mine was found to be less effective than imipramine in antagonising reserpine or tetrabenazine

mduced ptosis and catalepsy and in potentiating amphetamine induced hyperthermia (11).

~ an~ pH on the electrocardiogrm Btol. Abstr., 30: No. 28728, 19

us adminjstr~tio~ of dextran on car-,1951. CIted ID BioI. Abstr., 26

In promazine molecule, substitution of a chlorine in position 2 increases its neurolepticpotency i. e. potency for depressing motor activity and conditioned avoidance responses inanimals i•••id for altering psychotic behaviour in man (1). As the chemical structure of imipraminebearssom : resemblance to that of promazine, a relevant question would be whether substitutionof ( chlor.ne in position 3 of the aromatic ring of imipramine imparts to it neuroleptic activity(Fig. I). Hence on this basis the present investigation was undertaken to evaluate the neurolepticactivity of do nipramine and characterise it with reference to chlorpromazine a standard neuro-I~?:ic drug.

MATERIALS AND METHODS

Male albino mice and rats, weighing between 20 to 30 g and 120 to 180 g respectively, wereused for the study.

Clomipramine hydrochloride (CJMJ), imipramine hydrochloride (IMl), chlorpromazinehydrochloride (CPZ) and methamphetamine hydrochloride (MAMPH) were dissolved in distilled

·This paper was read at the Decennial Conference of Indian Pharmacological Societyheld at Calcutta in December, 1<)77.

264 Balsara and Chandorkar July-SeptembtrInd. J. Physiol. P

water. Doses quoted refer to the salts. The strength of the solutions was so adjusted that thequisite dose ofadrug was injected intraperitoneally in a constant volume of 0.1 mljlO kg bodyin mice and in a volume of 0.2 mljlOO g body weight in rats. Control animals receiveditoneal injection of distilled water. Groups of 10 animals per dose level of the drug were

Fig. I: Illustrate'S the structural rcsernblence of imipramine to promazine and of clomipramine to chlorprom .

Traction response in mice:

The method followed was that of Courvoisier et al. (5). Mice were suspended froma horizontal wire by their fore paws, and the animals which were able to draw themselves up totouch the wire with one hind paw within 5 sec of being placed on the wire were used for furtherstudy. Both control and drug treated groups were tested for the traction response at 30 and 60,min time interval after the injection. The response was said to be inhibited when the animalwas unable to draw itself up to touch the wire within 5 sec of placement.

Pinna reflex in mice:The pinna reflex was elicited by stimulating the external auditory meatus of each ear

with a fine hair and involves the twitch tremor or laying back of the ear in response to stimubtion of the external auditory meatus (J 3). Both control and drug-treated groups were tested forthe pinna reflex at 30 and 60 min time interval after the injection. the reflex was said to benegative when no response could be elicited from either ear.

5

:5

8 2

9 IO~H2C~2CH2N<CH3PROMAZINE CH3

ClCH2CHtCH2N(CH3

CHLORPROMAZINE CH3

olume 22umber 3

nditioned avoidance response I

Effect on CAR was stusing Cook's pole climbing re

to make 3 consecutive correcttilled water (control group) orThe drug effect on CAR waspole on hearing the buzzer but

The EDso of the drug for inlputed by the method of Mill

Methamphetamine-induced ster

MAMPH was used istereotyped behaviour, charactof the animals. Control groinjection. Drug-treated grou

The rats were kept icm) and were observed visualmined by the method of Laloccurence of continuous sniffi

The intensity of SBThe SE was assessed accordifollows:

Score:

0:

1 :

The rats are hyperaclocomotion and inr"

Continuous sniffingslight responsiveness

Continuous sniffinglicking, very brief pexperimenter.

Continuous gnawinglocomotion. No re

2 :

3

The termination ofphase of' hyperactivity and ~and grooming. The end potthe failure of the rat to resustatistically analysed by the

July-SeptemberInd. J. Physiol. P

ions was so adjusted thatolume of 0.1 mll10 kg body \Vi

atcol animals received inttaose level of the drug were u

Volume 22umber 3 Clomipramine and Neuroleptic Activity 265

Conditionedavoidance response (CAR) in rats:

Cl2CH2CH2N(CH3MAZINE CH3

Effect on CAR was studied in trained rats by the technique of Cook and Weidley (3)usingCook's pole climbing response apparatus. On the day of the experiment, the animal hadto make 3 consecutive correct avoidance responses prior to the intraperitoneal injection of dis-tilledwater (control group) or drug. The animals were tested again 30 min after the injection.Thedrug effect on CAR was expressed as the percentage of animals which failed to climb thepoleon hearing the buzzer but did climb the pole in response to the electric shock.

The EDso of the drug for inhibiting the traction response, pinna reflex and the CAR was com-puted by the method of Miller and Tainter (8).

Methamphetamine-induced stereotyped behaviour (SB) in rats:

MAMPH was used in the dose of 4 mgjkg which in a preliminary trial had inducedstereotyped behaviour, characterised by continuous sniffing and small head movements, in 100 %of the animals. Control group received distilled water followed 30 min later by MAMPHinjection. Drug-treated groups received CIMI, IMI or CPZ followed 30 min later by MAMPH.

The rats were kept in individual cages made of netting (floor area 21 cm x 27 cm, ht 20cm) and were observed visually. The latency of onset and the duration of stereotypy were deter-mined by the method of Lal and Sourkes (7). The onset of stereotypy was determined by theoccurence of continuous sniffing, small head movements with the rat sitting in a crouched posture.

The intensity of SB was assessed by observing the animals for 5 min at 30 min intervals.The SB was assessed according to the scoring system suggested by Costall et al. (4) and is asfollows:

i()r cIomipramine to chlorpron:a,,,jlleScore:

o : The rats are hyperactive as seen by increase in exploratory activity, increase in forwardlocomotion and inr+ease in rearing and responsiveness towards the experimenter.

I : Continuous sniffing and small head movements, periodic exploratory activity, andslight responsiveness towards the experimenter.

2 : Continuous sniffing and small head movements, discontinuous gnawing, biting andlicking, very brief periods of locomotor activity. No responsiveness towards theexperimenter.

3 : Continuous gnawing, biting and licking, no exploratory activity, occasional backwardlocomotion. No responsiveness towards the experimenter.

I. Mice were suspended fromable to draw themselves up to:he wire were Used for furtherraction response at 30 and 60be inhibited when the animaiement.

auditory me?tus of each earle ear in response to stimula-reated groups were tested forI. the reflex was said to be

The termination of MAMPH induced SB was characterised by the animal entering aphase of hyperactivity and by the gradual emergence of normal activity like forward locomotionand grooming. The end point was evaluated by lifting the rat out of the cage and observingthe failure of the rat to resume SB lasting 30 sec within 20 sec of replacement. The results werestatistically analysed by the Student's unpaired t-test.

266 Balsara and Chandorkar July-September II nd, J. Physiol. Pha

Spontaneous motor activity (SMA) in rats:The technique described by Vad et al. (12) was employed for recording SMA. On

one animal was placed in the activity cage at a time. After waiting for 10 min to allowtheinitial excitement to pass away, the up and down i.e. vertical movements of the animal we~recorded by the lever of the Marey's tambour on a moving kymograph for the next 30 min.The animal was then administered drug or distilled water and was kept aside for 20 min. Itwas then placed in the activity cage and after 10 min interval, the record was again taken for30min.

Exploratory behaviour of mice:Effect on exploratory behaviour of mice was studied by the method of Shillite (10). One

control group was always used simultaneously with groups to which various doses of drugshadbeen administered. Mice were placed one at a time on the left hand corner of a wooden boardmeasuring 61 X 61 cm onto which 12 tunnels 7.5 cm long and 4 cm in diameter were fixed arran-ged in a symmetrical pattern. The tunnels were numbered. Drugs were given 30 min beforethe observations, while the control group received distilled water. Each mouse was watchedfor 5 min after it was placed on the board. The number of different tunnels entered in thefirst minute, the total number of tunnels entered as well as the total number of different tunnelsentered during the 5 min observation period, by the control and drug treated groups was noted.The experiments were conducted at the same time each afternoon.

The significance of differences between means was assessed by the use of Student's un-paired t-test.

Induction of catalepsy in rats:The animal was tested for catalepsy 30 min after the drug injection. The animals were

positioned with their front paws resting on a wooden block 12 cm high and scored as catalepticif they maintained this unnatural position for atleast one min (9).

RESULTS

1. Influence on traction response in mice:CIMI and IMI, upto 60 mgjkg body weight, were inactive in inhibiting the traction res·

ponse while the EDso± S.E.M. of CPZ was 5.62 mg±0.31.

2. Influence on pinna reflex in mice:CIMI and IMI, upto 60 mgjkg body weight, were Inactive in inhibiting the pinna reflex

while the EDso ± S.E.M. of CPZ was 3.54 mg ± 0.15.

3. Effect on conditioned avoidance response (CAR) in rats:EDso ± S.E.M. of CIMI and IMI for inhibiting the CAR Were 25.12 mg ± 1.15 and

28.18 mg ± 1.40 respectively while that of CPZ was 4.07 mg ± 0.14. CIMI and IMI though

equipotent (P>O.05) in blockithan CPZ in this respect.

4. Effect on methamphetamine

Pretreatment with eithetime required for the onset ofthe duration and the intensityrats (Table I).

TABLE J; Effect ofmetham

Treatment dose mg/kg

1. MAMPH 4

2. ClMl 10+ MAMPH 4

3. IMI 10+ MAMPH 4

4. CPZ 10+ MAMPH4

*Drug effect for a particular paraNumerals following the drugs in

5. Effect on spontaneousmo

CIMI and IMI in ain dose to 20 mglkg of ClMlto 40 mg/kg reduced the SMmarkedly.

6. Effect on {xploratory be

ClMI and IMI did ndose of 10mgjkg bcdy weighsignificantly decreased theentered during the first mintunnels entered during the 5control group (Table 1I).

7. Induction of catalepsy i

elMI and JMI when

Clomipramine and Neuroleptic Activity 267

ipotent (P>0.05) in blocking the CAR were, on a weight basis, about six times less potentn CPZ in this respect.

Effe~ton methamphetamine-induced SB in rats:

recording SMA.for IQ min to alloents of the animalph for the next 30t aside for 20 min.

d was again taken f

Pretreatment with either ClMI or lMl not only significantly (P<O.OOl) decreased themerequired for the onset of MAMPH induced SB but also significantly (P<O.OOl) prolonged

duration and the intensity of SB. Stereotyped behaviour did not occur in CPZ pretreateds (Table 1).

od of Shillite (I0).ious do es of drurner of a wooden

iameter were fixedere given 30 rnin

h mouse was watct tunnels entered inber of different tunated groups was no

TABLE I: Effect of c1omipramine, imipramine and chlorpromazine onmethamphetamine induced stereotyped behavior in rats.

Onset in min Duration in min Intensity cumulati:e scoreMean :S.E.M. Mean:S.E.M. MfQll±S.E.M.

MAMPH 4 19.3±104 2.11±6.70 1. 7±0.08

elMI 10+ MAMPH 4 12.8±0.87* 260±4.71* 2.6±O.12*

IMI 10+ MAMPH 4 12.1±0.64* 266±4.89* 2.11±0.08*

CPZ 10+ MA IPH 4 0 0e use of Student's

·Urug effect for a partir-ular parameter when compared with the control was statistically significant (P < 0.001).Numerals following the drugs indicate their doses (mg/kg).

n. The animalsnd scored as catafe

Effect on spontaneous motor activity (SMA) in rats:

ClMI and lMl in a dose of to mgjkg did not affect the SMA. However, an increaseindose to 20 mgjkg of ClMI and IMl slightly reduced the SMA and a further increase in doseto 40 mgjkg reduced tbe SMA markedly. CPZ in a dose of 2 and 4 mgjkg reduced the SMAmarkedly.

iting theEffect on exploratory behaviour of mice:

CIMl and IMl did not significantly affect the exploratory behaviour when used in thedose of 10 mgjkg bcdy weight. However, CIM[ (20 mgjkg), INn (20 mgjkg) and CPZ (2 mgjkg)significantly decreased the exploratory behaviour of mice. The number of different tunnelsentered during the first min during the 5 min of 'observation period and the total number oftunnels entered during the 5 min period was significantly lower than that of their respectivecontrol group (Table I I).

.12 mg ± 1.] 5I and JMI tho

Induction of catalepsy in rats:

ClMI and IMI when used in the dose range of to to 40 mgjkg, did not induce catalepsy

J ulv-Scprcmber268 Balsara and Chandorkar Ind . .1. Physiol. P

in rats. Doses beyond 40 mgjkg induced ataxia and motor incoordination and were there!!not tested. CPZ (l0 mg]kg) induced catalepsy in 100% of the animals tested.

TAi<LE Il: Effect of clomipramine, imipramine and chlorpromazine on the exploratory behaviour ofmi

Treatmentdose mglkg

Total number of different Total number of differed Tolal number of tUllnelstunnels entered during the funnels entered during the entered dnrine u.e 5 /I,;n

first min( mean±S.E.M.) 5 minperiod (mean±S.E.M). period (meall±S.E.M)

2.0±0.33 7.0±0.39 l5.6±I.071.9±0.3l 7.4±0.47 IG.l±0.86

2.1±0.44 6.7±0.49 15. !±0.871.8±0.41 G.9±O.43 15.8±0.92

2.1±0.23 8.0±0.39 l8.U±C.B31.1±0.17* 5.0±0.14* 14.2±0.75*

2.0±0.25 7.6±0.33 17.S±0.811.2±O.19* 4.9±0.31* 14.7±0.SI*

2.0±0.21 7.4±0.47 17.6±0.770.7±0.15* 3.9±0.27* 12.4±O.73*

I 1. Control2. CIMl10

I. Control2. IMIIO

I. Control2. CIMI20

I. Control2. IMI20

I. Control2. CPZ 2

IT

III

*Significant in relation to corresponding controls (P <0.05 to 0.01).Numerals following the drugs indicate their doses (mg/kg).

DISCUSSION

The neuroleptic drugs like chlorpromazine are comparatively more potent and selectivein inhibiting the traction response and the pinna reflex, in blocking CAR, in antagonising am-phetamine effects, in decreasing SMA and exploratory behaviour and in inducing catalepsyand hence these test procedures are commonly used to evaluate neuroleptic activity of a drug(6).

Clomipramine and imipramine (upto 60 mgjkg ip) were found to be ineffective in inhi-biting the traction response and pinna reflex. Further upto 40 I'ng/kg dose they did not inducecatalepsy in rats. These results are similar to those of Theobald et al. (J I) who have reportedthat clomipramine (upto 100 mg/kg, se) did not inhibit the traction response and (upto 50mslk«, se) produced only slight cataleptic effect in mice.

Clomipramine and imipramine, though equi-effective in blocking CAR were, on weightbasis, six times less potent than chlorpromazine. Even Theobald et al. (J 1) have reported thatclomipramine (upto 20 mgjkg, ip) did not significantly alter the rate of key pressing in theSidman's conditioned avoidance test.

Clomipramine and imipramine were found, on weight basis, to be about JO times lesspotent than chlorpromazine in decreasing SMA and exploratory behaviour. The orientationmotility in mice is also affected only by high doses (EDso 40 mg/kg, ip) of clomipramine (11).

ume 22ber 3

Clomipramine, like imiprhamphetamine-induced stereo

'ne was found to potentiatdoses (EDso 75 mg/kg, se) it

leptics like chlorpromazine haine toxicity, when used in a I

On the basis of our findissesses negligible neuroleptic

roleptic activity to imipramine.

The authors are gratefulof imipramine hydrochloride antheir generous gift of chlorpromtance and to the Dean, V.M. M

1. Byck, R. Drugs and the tre~unby Goodman, L.S. and A. Gi m

2 Collins, G. H. The: use of.par~. stateS. s-. J. P;ychlal., 122. 189

Cook, L. and E. Weidley. Beha3. 7740-752, }95 .Costall, B., R.J. NaylorandJ. E.

4. rebrai injections. Eur. J. Pharm

5. Courvoisier, S:' R'ID~~protc~~~'la phenothiazme. n syp. 373, 1957.

6 Hill, R. T. and D. H. Tedeschi.. In" An Introduction to Psycho

p.237, 1971.LIS and TH. L. Sourkes. Po

7. a~d o~her agents. Arch. 1nl. Ph

M iller, L. C. and M. L.Tainter.8. in Pharmacology' by Turner, R

Rogers, K. J. and P. Slater. B9. Pharmaf.,23: 135-137,1971.

Shillito, E.E. A method for in10. Phnrmac., 40 : 113-123, 1970.

Theoha1d, W., O. Buch, RA ...11. dimethylaminopropyl)-IO,II-dt

Vad B. G., D.S. Shrotri and.12. Ind. J. Physiol- Phnrmac-, 7: 153

W·tk· L B P Spitalettaand\3. 1 an, . ., . pL_ Exbin the mouse. J. ,,,,.mac. .

J uly-ScplcmbInd . .T. Physiol, P

ation and wereIs te ted.

Total number of tunnelsmtered dl/rin~ Le .'i IlIm

~. period (meull±S.E.M)

J5.6±1.07J6.1±0.86

15. !±0.87

15.8±0.92

18.U±O.83

14.2±0.75*

17.8±0.8114.7±0.81*

J7.6±0.7712'+±0.73'"

re potent and select.R, in antagonising ain inducing catale

ptic activity of a de

be ineffective in insethey did not indu11) who have reportesponseand (upto 5

CAR were, on weigh11) have reported thatf key pressing in th

about 10 ti rnes lesur. The orientationclomiprarnine (I I).

Clomipramine and Neuroleptic Activity 269

Clomipramine, like imipramine, but unlike chlorpromazine was found to enhance thehamphetamine-induced stereotyped behaviour. In the study of Theobald et al. (11) clomi-'ne was found to potentiate amphetamine-induced hyperthermia in rat and only at verydoses (EDso 75 mgjkg, se) it protected grouped mice against amphetamine toxicity. Neu-

Iepticslike chlorpromazine however, are capable of protecting grouped mice against amphe-inetoxicity, when used in a low dose range of 2.5 to 5 mglkg (6).

On the basis of our findings, we therefore, conclude that clomipramine like imipramineessesnegligible neuroleptic activity and the 3 chlorine substitution has not imparted neu-

rolepticactivity to imipramine.

ACKNOWLEDGEMENTS

The authors are grateful to Ciba-Geigy Ltd, Basle, Switzerland, for their generous giftofimipramine hydrochloride and clomipramine hydrocbloride, to May and Baker (India) fortheirgenerous gift of chlorpromazine hydrochloride, to Mr. S.S. Chavan for his technical assis-tanc~and to the Dean, V.M. Medical College, Solapur, for providing facilities.

REFERENCES

Byck, R. Drugs and the treatment of psychiatric disorders. In "The Pharmacological Basis of Therapeutics"by Goodrnan, L.S. and A. Gilman. 5th ed, New-York, Macmillan Publishing Co., Inc., P. 157, 1975.

Collins, G. H. The use of parenteral and oral chlorimiprarnine (Anafranil) in the treatment of depressivestates. Br. J. Psychiai., 122: 189-190, 1973.

Cook, L. and E. Weidley. Behavioral effects of some psychopharmacological agents. Arm. N. r. Acad. Sci., 66 I

740-752, 1957.

Costall, B., R.J. Taylor and]. E. Olley. Stereotypic and anti cataleptic activities of amphetamine after in trace-rebral injections. EI/r.}. Phnrmac., 18 : 83-94, 1972.

Courvoisier, S., R. Ducrot and L. Julou. ouveaux aspects experimentaux de I' activite cen~rals des derives dela phenothiazine. In "Psychotropic Drugs" by Garattini, S. and V. Ghetti, Amsterdam, Elesvier Publishing Co.,p. 373, 1957.

Hill, R. T. and D. H. Tedeschi. Animal testing and screening procedures in evaluating psychotropic drugs.In "An Introduction to Psychopharmacology" by Rech, R. H. and K. E. Moore, New York, Raven Press,p.237, 1971.Lal, S. and TH. L. Sourkes, Potentiation and inhibition of the amphetamine stereotypy in rats by neurolepticsand othcr agents. Arch. Int, Pharmncodyn., 199 : 289-301, 1972.

Miller, L. C. and M. L. Tainter. Proc. Soc. E,.fJ. Biol. Med., 57: 261, 1944. Quoted from 'Screening Methodsin Pharmacology' hy Turner, R. A. London, Academic Press, p. 61, 1965.

Rogers, K. J. and P. Slater. Brain acetylcholine and monoamines during experimental catatonia. J. Pharm.Pharmac.,23 : 135-137, 1971.

Shillito, E.E. A method for investigating the effects of drugs on the exploratory behavior of mice. Br. J.Pharmnc., 40 ; 113-123, 1970.

Theohald, W., O. Buch, H.A. Kunz and Cl. Morpurgo. Pharmacology of the antidepressant 3-chloro-5-(3-dimethylaminopropyl)-IO, l l-dib-, dro-5H-dibenz (b,f) azepirie HCI. Arrneimittel-Forschung., 17 : 561-564, 1967.

Vad, B. G., D.S. Shrotri and .1. H. Balwani. A new technique for recording spontaneous motor activity.Ind. J. Ph;ysiol. Phnrmac., 7; 153-157,1963.

Witkin, L. B., P. Spitaletta and A.J. Plummer. Effects of some central depressants on two simple reflexesin the mouse. J. Phnrmac, Exp. The,., 126 : 330-333, 19.';Q.