University of Nigeria Research Publications

MEZUE, Wilfred Chukwuemeka A

utho

r

Title

Neuromuscular and Central Nervous System Effects of

Piperazine Citrate

/Fac

ulty

Medicine and Dentistry

Dep

artm

ent

Pharmacology and Therapeutics

Dat

e

May, 1983

Sign

atur

e

NEUROMUSCULAR AND CENTRAL NERVOUS SYSTEM EFFECTS OF PIPERAZINE CITRATE

A DISSEIITATION SUBMITTED TO THE D~ARTMENT OF FHARMACOLOGY PLND THERAPNTICS

FACULTY OF MEDICINE MID DENTISTRY UNIVERSITY OF NIGERIA

I N PARTIAL FUT;FILM3NT FOR THE DEGREE OF MASTER OF SCIENCE

NEUROMUSCULAR AND CENTRAL NERVOUS SYSTEM

EFFECTS OF P I P E R A Z I N E CITRATE

W. C. MEZUE, MB.BS.(IBARAN) DEPARTMENT O F PHARMACOLOGY AI?D THERAPEUTICS

PAfiTIGTY O F MEDICINE AND DENTISTRY

UNIVERSITY O F NIGERIA

E W G U CAMmJS

SUPERVISOR:

PROFESSOR G. 0. ONUAGULUCHI, B.Sc. (LOND.), PH.D. (GLASG.); L.s.M.(NIG.); M.R.c . s . (~G. ) F.R.C.P.(GLASG. ~c EDIN.); F.x.c.P. (NIG.);

L.R.C.P (ENG.) . DEPARTMENT OF PHAWCOLOQY m T H E R A P ~ T I C S

FACTJLTY O F MEDICINE AND DENTISTRY

U N I V E R S I m O F N I G E R I A

ENUGU CAMPUS

TABLE O F CONTENTS

T B L E O F O N T E S T S

dedication vq.

INTRODUCTION

H I S T O R I C A L REVIE'? 1

ABSORPTION AND E X R E T I O N 2

MECHANISM O F ACTION 3

CHEMISTRY 6

ANTHELMINTIC ACT :VI TY 7

T O X I C I T Y AND S I C 3; E F F E C T S 9

O B J E C T I V E 1 2

GEXERRL EXPERIP.IENTILL METHODS 13

S E C T I O N A: EXPERIMEXtS ON S H E L E T A l MUSCLES

( i) ISOLRlTED FROG RECTUS A B D O P I N I S PREPAFUTIONI 4

(iii) RAT S C I A T I C ::ERVE-GASTROCT EMUS PREPARATION 19

( iv) CAT S C I A T I C LRTERVE-GASTROCNmUS P R E P , r n T I O N

Cv) NEUR0MUSCULI.R COORDINATION I N YOUNG CHICKS 24

CONTENTS CONTD.

SECTION B: EXPERIMENTS ON TI33 CENTRAL NERVOUS SYSTEM

(i) SLEFJ'ING T I M Z I N MICE

(ii) CONVULSANT ACTIVITY O F LEPTAZOL

( iii) CONWLSANT ACTIVITY O F STEUXNINE

RESULTS

ISOLATED FROG RECTUS ABDQMINIS

ISOLATED RAT PHRENIC h T E R % D U W M

RAfP SCXATIC NERVE GMTROCNEMUS

CAT S C I A T I C SERVE GASWCNEMUS

NECJROMUSCULl@ COOIiDmII\ION I N CHICK3

SLEEPING TIME I N MICE

CONWLSAXT ACTIVITY O F LE3eTAZOL

CONWLSPm ACTIVITY OF STRYCHNINE

DISCUSSION

SUMMARY LND CONCLUSION

REE'ERENC ES

In presenting t h i s work, I wj.& &I admwl&w q

Andebtedness t o Bxsfbmw -, #. O., Head,

Department o f Pharr~amLogy and Thewe- Of

JWiclne, U&rcweity of N i e ; e r i k , wha and

sustained the enthuaiaem, read the s U ~ P ~ S

made o r i t & a l -ions and who had the patience ta

take me through the initial step= r e x w m d ~

thanks also go t o Mr. Oneji nctor of the

-+ a9 P&rmaca.ogg and Theraput ics, who gave

j i t ~ necessary technical assisdance; to M r . Oko of the

U c 8 l Il lustration Unit who typerl the manuecripta;

Mr. YWah also of the Medical Il lustration Unit who

did the graphic works and t o the pther s taf f of the

fBedlOPl IUustrctzbn Unit for the* assistance. I a&W

wish t o thank the laboratory at:,ff of the Phnr-

and Therapeutics Department under the leaderahip o f

Mr. Opara.

Finally I owe it a l l to God who made everything

possible.

May, 19836

D E D I C A T I O N

To the memory of q y father who died in

I N T R O D U C T I O N

HISTORY

Biperaaine i s a drug t h a t has proved k.ughJ.7 effective

a g a i n s t both Ascaris lumbricoides and Enterobius (0-ia)

rmicu3_aris. It was f i r s t used e a r l y i n t h e twent ie th

century f o r t h e treatment of gout, because Piperaz ine urate

5s a so luble substance, However, t h e drug proved t o be

i n e f f e c t i v e as a u r f s s u r i c agent and i~ was de le ted from

o f f i c i a l B r i t i s h Pham4ceut ica l publ icat ion, a l though it

continued t o be used f a i r l y extens ive ly i n o t h e r p a r t s of

Europe. t %

I n 1949, Fayard submitted a t h e s i s t o t h e Universi ty

o f P a r i s i n which he described t h e anthelminthic a c t i o n of

p iperaz ine hydra te w i n s t round s scar is umbricoides), 1 He however made it c l e a r i n the paper t h a t t h e o r i g i n a l

observat ion of t h e e f fec t iveness o f piperazine aga ins t round

worms was made by a Rmen P h a m c i s t , Boismre , who recommen-

ded Fayard t o t r y the drug and provided him with a perpara- '

t i o n o f p iperaz ine hyarate f lavoured w i t h apple syrup f o r

t h e purpose.

The use of p iperaz ine sal ts f o r the treatment of

threadworm ( ~ n t e r o b i u s vermicU.aris) began i n 1951

t ~ o u r i ~ u a n d e t a l ) and i n 1953 White and Standen showed t h a t

p iperaz ine c i t r a t e was more e f f e c t i v e than gen t i an v i o l e t

i n the treatment

Goodwin and

2

of threadworms

Standen ( 1 954;

in children.

1958) ahowed

dose of piperazine s a l t equivalent to 411; of t he hexahydrate

was suf f ic ien t t o render the s tools f ree from mund worm

eggs i n over 80 per cent of African p a t ~ e n t a . P u r e t i o n

nei ther increased nor diminished t h e effectiveness of the

drug. Under out-patient c l i n i c c o n d i t i ~ n s i n Britain, a

single dose of piperazine with o r withcut the simultzuuuua

administration o f a purgative was sufflcient t o remove

evidence of threadworm infeceion f r o m over 90 per cent of

children (white and Scopes, 1960) . ABSORPTION AND - E X C m I O N

Piperazine is readily absorbed i rom the gastro-

i n t e s t i n a l t r a c t and i t is not easy t o understand how i t

reaches the threadworms whfoh are harboured i n the large

bowel. A part of the absorbed drug i s degraded i n the body

but most of it is excreted i n :he Qrine. Rogers (1958) was

able to es tabl ish a wide variation i n the r a t e a t which

piperazine was excreted by different individuals, He also

found tha t ths r a t e o f e x c r e t b n by Africans infected with

ascarids was of the same order a,. the r a t e s i n uninfected

Europeans and t h a t fhere was no significant difference

between the ra tes o f excretign of piperazine c i t r a t e ,

adipate and phospk.te.

3

Pharmacokinetic s tudies i n rats using 4~-piperazine

theophylline ethanoate have shown that b i l l i a r y l eve l s are

400 to 4000 times higher than blood leve ls 2 houra a f t e r

administration, and tha t b i l e represents a fwndamantal route

of excretion (Benedetti e t a l , 1971).

MECHARISPI OF ACTION

Piperazine a c t s upon roundmum as a narcotic. Worms

expelled a f t e r treatment with t h i ~ drug are narcotized,

motionless a.nd relaxed but not k;Uled. Goodwin and Standen

( 1 954) showed tha t tone and movement reappared upon immer

sion i n Ringer solution a t 37'~. This was confirmed by

Poynter (1955). Standen (1955) sfurwsd by using' several

piperazine s a l t s against Ascaris lwnbriooides from the pig,

tha t worms placed i n a 1 :200 -t-o 550 solution o f any of the

piperazine s a l t s were narcotized, but recovered i n l e s s than

3 hours i n a d rug4me medium. Recovery was complete even

a f t e r 48 hours exposure to piperazine.

During narcosis, roundworms i n v i t ro show no prelimi-

nary phase of stimulation (Goodwin, 1958). If a drug causes

stimulation of the worms they may knot together and cause

in te s t ina l obstructj,on, o r may migrate into the b i l e duct

o r appendix o r may cause p e f o r a t i o n of the gut wall. This

disadvantage was noted with drugs such as o i l of chenopodium,

4

hexy l r e so rc ino~ and tetrachloroethylene (Goodwin, 1958) . Furthermore, because the worms are eliminated alive during

o r a f t e r treatment with piperazine, the poss ib i l i t y of

absorption o f d i s i n t ~ t i u n products is uin.bal.

Norton and De Beer ( 1 957) observed t h a t piperazine

blocks the response of &caris m u s c l e Lto m a l l conoentmtW

of acetylcholine a s w i l l small Wses of d-tubocurarine.

They concluded t h a t paralysing action of piperazine was

due t o a curare-like e f f e c t on the neummuscular junctions.

However, s tudies on t h e dc4zriCal a c t i v i t y of the

somatic muscle c e l l s of Ascaris cas* doubts on t h e above

hypothesis ( ~ e Bella e t a l , 1963). The contract ion of

Ascaris muscle is i n i t i a t e d by d y t h m i c spike po ten t ia l s

generated by pacemakers i n the muscle membrane i t s e l f and

not by nerve impulses transmitted &cross neuromuscular

junctions. The c e l l s have an ave- r e s t i ng po ten t ia l of

about -30mV interrupted by rhythmic spikes of myogenic

o r i g i n ( 1-7 spikes/sec) . Acetylcholine depolarisee the

muscle ce l l s , thus increasing the frequency of t h e spike

po ten t i a l s and the degree o f contraction.

Piperazine ( ~ o - ~ w / v ) increases the aver* r e s t i n g

p o t e n t i a l above end -mqPessecs. pa-r

These changes were shown t o be s i m i l a r .to those obtained

temporarily by t h e e l e c t r i c a l s t imula t ions o f inh ib i to ry

nerve f i b r e s . It was a l s o shown t h a t p r e c i s e app l i ca t ion

of p iperaz ine t o d i f f e r e n t a r e a s of t h e muscle cell using

e l e c t r o p h o r e t i c medthods caused hyperpolar i sa t ion of the

membrane only when app l i ed t o a region where both exc i t a to ry

and i n h i b i t o r y neuromuscular synapses were loca ted . It

w a s t he re fo re concluded t h a t piperazine may be regarded

as a pharmacological analogue of a n a t u r a l i n h i b i t o r y

neurohormone (Del Cas te l lo e t a l , 1964).

It is o f i n t e r e s t t h a t , l i k e p iperaz ine gamma

aminobutyric a c i d (G-BA) produced hyperpolar i sa t ion and

cessa t ion of spike a c t i v i t y of Ascaris muscle (Del Cas te l lo

e t a l , 1964) GABA being 100ntimes more potent i n t h i s

r e spec t than piperazine . The maintenance of t h e membrane p o t e n t i a l o f v i s c e r a l

muscle, d i k e t h a t of s k e l e t a l muscle, r e l i e s heavi ly on

t h e cont r ibut ion of ch lo r ide ions ( ~ u e d i n ~ and Kuriyama, 1963).

A similar r e l a t i o n s h i p e x i s t s i n the Ascaris muscle, but

t h e f a c t t h a t the p e r i ? n t e r i c f l u i d which normally ba thes

t h e Ascaris muscle is r e l a t i v e l y low i n chlor ide concentra-

t i o n (only 52nm ch lo r ide ions) suggested t h e p r o b a b i l i t y t h a t

anions o ther than ch lo r ide a r e respons ib le f o r the t r a n s p o r t

of the charge necessary t o maintain and increase the

membrane p o t e n t i a l ( ~ o b s o n e t a l , 1962). The p o s s i b i l i t y o f

organic anions of v o l a t i l e f a t t y a c i d s a c t i ng i n t h i s capa4

c i t y has been m i s e d by t h e works of Beuding ( 1 953) ; and

E l l i son e t a l , ( 1960).

Pa ra ly s i s of Ascaris muscle by piperazine i s associa-

t ed with a lnarked decrease i n t h e formation of succ in ic

a c i d which is a metabolic product of the worm (Bueding e t

a l , 1959) . Succinate production supp l i es t h e energy f o r

t he muscular con t rac t ion of t h e worm and it i s probable

t h a t t h i s energy i s provided in Ascaris muscle mitochondria

by a reduct ion of fwnarate t o succ ina te by reduced

diphosphopyridine nucleot ide me t i c and Bueding, 1961 )

coupled with phosphorylation.

CHEMISTRY

Piperazine i s a he te rocyc l i c organic compound with

the following s t r u c t u r a l formula,

It i s a v a i l a b l e a s the hexahydrate which contains

about 445 of base, E nd i n addi t ion, as various salts such

as c i t r a t e , ad ip s t e phosphate, calcium ede ta te , a d t a r t r a t e .

7

Piperazine c i t r a t e contains a variable amount of water

of c rys ta l l iza t ion , It contains not less than 98.5% of

( c ~ H ~ ~ N ~ ) ZC6H807, calculated with reference t o the

anhydrous substance. It is a white, f ine granular powder,

almost ordorless and t a s t e s acid. It is soluble a t 20 0

i n 1.5 par ts of water, but is insdiahle i n 9% aLcob1

and i n solvent ether.

A 5% w/v solution has a pH of 5 t o 6.

ANTHELMINTIC ACTIVITY

Piperazine is uaed mainly i n the treatment of

helminthiasis i n both human and veterinary pmtices ,

The importance of t h i s is only appreoiated when i t is

recalled t h a t helmirrthiasb and especially ascar iaais

against which pipefazine i s most effective consti tutes a

major world wide h e a t h problem. It is sstiima-kd that

even though a s c a d a s i s has a l o w morbidity, i ts global 6 incidence is of the order of 1000x10 infected persona

(Sturchler, 1 982) Ascariasis and hookwo~ infections are

the commonest h w n parasites i n N b r i a , par tUular ly i n

children. Cowper and Woodward (1 961 ) found ascar is i n 26.5

per cent of 21,70C stools examined r o u t i ~ e l y at the University

College Hospital, Ibadan. Gilles (1 964) discovered an

infect ion r a t e of 70 per cent i n 600 v i l l aae r s in a vfilage

near Ibadsn. Okpala ( 1956) reoorded a simi7&r r&e i n

Lagos. He found an in fec t ion r a t e of 73 per cent in mex-

4,700 Lagos child- and a d o l e s w k Frisk ( 1 939) found

a sca r i s i n 90 out of a series of 120 pwdwmort%ld$ Ln Lagos.

The incidence becomes lower i n the more Northern

par t s of Nigeria. Ramsay ( 1 934) f d t h a t only 6 per cent

o f over 7,000 persons exam&& on the Jm Plat- areas

were infected. Collard ( 1 962) i n a $urol.ey i n Katsina

province which is North of Jos found ova i n only 2.1% o f

536 Habe and 0.4% Fulani examined. I t would thus appear

t h a t i n Nigeria incidence i s highest i n the humid climate

of S o u t h s r n Nigeria, s ign i f ican t ly Zower i n the d r i e r

north and t h a t seasonal incidence i s highest during the

rains (cowper, 1966) . Piperazjne has been shown t o ponsess a reasonably

effect ive antl-aacaris a c t i v i t y a t a concentration of

Qmg/ml (~oodwih 1 958 ; Onuaguluch4 19641, It is very

effect ive again4 t both a sca r i a s i s and t r i chu r i a s i s

producing 100% egg reduction i n ascar ias i s a f t e r 20 days

(~gunmekan, 1973).

The most widely used of the four generally available

piperazine s a l t s is piperazine c i t r a t e . An accepted and

safk schedule f o r adul ts i s a s ing le o r a l dose of 'T1Sm$kg

9

(expressed in terms of the hydrate equivalent; 100mg

piperazine hydrate equivalent t o 125mg c i t r a t e ) with a

maximal individual dose o f 4- g (Gi l l e s 1976). For

Enterobiasis &he usual dose i s O.6g three times da i ly f o r

seven days.

There is a l s o a piperazine theophylline ethanoate

which i s used a s a broncholdilator.

TOXICITY AID SIDE EFFECTS

The fact t h a t piporaslno , h a s neuronusoular t q x k e f f ec t s have

been known f o r a long t i n e , even though very l i t t l e has

been wri t ten on t h i s i n the English Li tera ture . The i n i t i a l

report on t h i s was mde a t the turn o f the century when

piperazine was used f o r the treatmen* of gout (Stewart,

1894). It was then shown t o came clonic spasm, tremor

of the upper extremit ies , muscular wakness, impaifed

co-ordination and i n a b i l i t y t o think c l ea r ly o r hal lucinat ion.

Slaughter ( 1896) reported a toxic c l i n i c a l p ic ture consist ing

of somnolence, narrowcd f ixed pupils, cyanosis bradycardia,

depressed r e sp i r a t i on and f lacc id p a r a l y s i s of the l egs

a f t e r administrzt ion ~f I 1.6g of piperazine.

I n 1953 when the treatment of w o r m i n f e s t a t d n s with

piperazine hexahydra t e was intrsducea (white and Standen,

1953) s i m i l a r symptons M e r e noted during treatment o f s i x

10

ch i ld ren and four teen a d u l t s (White and Standen, 1953b)

and t h e s e were thought t o be due t o a s l i g h t overdosage.

Schuch e t a1 (1966) repor ted many w s e s of neurotoxic

s i d e e f f e c t s , inc luding somnolence o r c o n f u s i o w l s t a t e s ,

d i zz iness , vomit t i n g , hypotonia with severe a t a x i c 3-

impairment of g a i t , inco-ordinat ion, dropping of ob j eo t s

and increase i n t h e number of a t t a c k s of pe t i tma l o r

myoclonic je rks . They a l s o observed electro-sncepbalographic

changes i n c h i l d r e n e x h i b i t i n g neurotoxic s i d e $f f e c t s and

noted t h a t i n those with long s tanding centpal nervoub

system d i seases , t h e electro-encephalograph showgd pronounced

slowing of bas ic a c t i v i t y , i n t e r r u p t e d by atypical

spike-wzve p a t t e r n s a f t e r t h e use of piperazirie hemhydra te .

They suggested t h a t Piperazinc trea.tment in any form should

be avoided i n pa t ien t a with known long s t and in3 d i s e a s e

o f t h e c e n t r a l nervous system e s p e c i a l l y i n those with

s e i z u r e s .

Other s i d e e f fec t s include Pausea, vomf Cling, abdominal

pains and d iar rhoea but these a l s o a r e occasional.

Piperazine de r iva t ives a r e cont ra indica ted i n r e n a l disease,

l i v e r disease and el i l epsy , ( ~ i l l e s , 1976) . Hamlyn e t a1

( 1 976) reported two cases of p ipemzine induced h e p a t i t i s .

11

Cases of haemolysis following piperazine have a l s o been

reported. This may be hypersens i t ive i n nature (Shankar

and Gulati, 1960) o r may be r e l a t e d t o Glucose - 6 - Phosphate Dehydrogenase (G-~-PD) def ic iency (~uchanan, f 971).

03 JECT IVE

The wide use of piperazine has been es tab l i shed

but r e p o r t s on i t s neurotoxic s i d e e f f e c t s demand

e luc ida t ion . These r epo r t s suggest t h a t piperazine may

have some cen t r a l nervous system e f f ec t s . Very l i t t l e

work has been done on the pharmacological ac t ions o f

piperazine,

Preliminary work on t h e e f fec t8 of piperazine on

mammalian s k e l e t a l muscle was carried out by Onuaguluchi

who showed t h a t i n the r a t phrenic heme diaphragm prepa-

r a t i on , piperazine has some inh ib i to ry e f f e c t on ncuromus-

c u l a r funct ions (Onuaguluchi, 1966) , I n continuing along

t h i s i n t e r e s t i n g l i n e , i t was intended t o use laboratory

animals t o explore the neuromuscular e f f e c t s of piperazine

and, thus e luc ida te t h e mechanisms of t h e muscular weakness,

tremor of the ext remi t ies and i n some cases, f l a c c i d

pa ra lys i s of the l egs and depressed r e s p i r a t i o n sometimes

observed i n the pa t i en t s t r e a t e d with piperazine. It was

a l so intended t o provide a pharmacological ba s i s f o r a

poss ib le c e n t r a l nervous system e f f ec t s of piperazine such

as p red i spos i t ion t o se izures .

Frogs weighing between 20 and 30g were used, The

f r o g was stunned by a blow on the head nd then p i t t ed ,

thus des t roying the s p i n a l cord. Thefrog Was then

pinned out on a d i s s ec t i ng board and $5 s k b over the

abdomen r e f l e c t e d by sharp d i s s e c t i o n b exrose the r e c t i .

The r e c t i were separated from t h e i r &achmmts t o the

sternum and t h e pe lv ic g i r d l e , and wee t h e i t r ans fe r red

t o an ae r a t ed d i sh conta in ing frog-Iirfiger m lu t i on at

room temperature. The r e c t i were divided bgi txdinal1.y

i n t o two and threads were at tached t p t h e t o p and bottom

of each piece.

The t i s s u e was at tached by one of the th reads t o a

J-shaped ae r a t i ng g l a s s piece i n a 5-ml organ bath

containing f r o g Ringers s o l u t i o n ( N d 6$, KCL 0.14,

C a C 1 2 0.12, NaH PO 0.01, NaHCO 0.:. Gl*ose 2.0g/~). 2 4 3

The so lu t i on was supplied froma r&rvo i r and the

exwriment conducted a t room temperature The t h r e d from

the upper end of t he t i s s u e was at-tachedto a fron-kil

wr i t ing lever , the l e v e r b e i w fir&adjPIsted so t h a t it

15

was balanced before t h e thread was t i e d , A load of about

Ig was then placed on the lever us ing p las t i c ine t o keep

the muscle on a s t r e t c h ,

After allowing '30 t o 45 minutes fo r t he preparation

t o equ i l i b r a t e , acetylcholine w a s added i n doses o f

2ug/ml, 4ug/ml and 6ug/ml and the contract ions induced

recorded over 1.5 minutes. Piperazine alone was added i n

doses of 1mg/ml t o 6mg/ml and allowed t o a c t f o r 1.5

minutes.

A submaximal dose of acetylcholine was selected from

the above and t h i s was added 2 minutes a f t e r doses of

piperazine ranging from 100ug/ml t o 6mg/m1-. Again records

were taken over 1.5 minutes. Similarly the e f f e c t of

tubocurarine on acetylcholine-induced contract ions were

studied.

The pH of t he various concentpations of the drugs

was monitored using pH ind ica tor p p e r but no narked

di f ference between the high and l o w concentrations of

piperazine was observed.

XXPEIIIWTS USING THE ISOLATED FUT PHRENIC NERVE- $ ~ R A G M SREE)N~AT?ON.

Adult a lb ino rats of e i t h e r sex weighing between

l4O-l6Og were used, A rat w a s k i l l e d by a blow on t h e

Lead, i t s t h r o a t out and i t was l e f t t o bleed as much as

~ o s s i b l e . The rat was then l a i d on i t s back and s trapped

')y t h e l e g s and hand t o t h e s i d e s of a d i s s e c t i n g board.

'he s k i n over the ches t and upper abdomen was d i s sec ted

: r e e and t he ches t opened by cu t t i ng t h o u & the xiphoid

p?ocess and the sternum. The inner aspect o f the thorax

was then examined t o exclude any adherence of t he phrenic

n e v e t o t h e a n t e r i o r thorac ic w a l l . When t h i s was found

t o 3ccur t h e nerve w a s gently dislodged by prodding with

b l w : forceps. The i nc i s ion was then continued l a t e r a l l y

leav-ng t h e diaphragri a t tached t o t h e las t few r i b s . The

a n t e r o r chest w a l l lras then mmoved. The phrenic nerve

w a s i d m t i f i e d as i t en te r s t he diaphragm and followed

up i n t ~e mediastinurn; ca re fu l ly d i s sec ted f r e e , secured

with thr?ad and seci ioned high up thus allowing a reaaon-

ab le leng-h for passing through the electrode. The

a n t e r i o r a7dominal : iuscles were cut along the c o s t a l margin

17

and holding the l a a t r i b with i t a attached diaphragm,

with a p a i r of forceps a t r i angu la r segment of the hemi-

diaphragm was dissected out with its apex in the central

tendon a t l e a s t 2-4mm from the entrance of the nerve.

Another thread w a s a t tached t o the apex of the segment

and t h i s was t i e d t o a l i g h t spr ing loaded l eve r with a

sideways-writing point.

The base was secured by another thread t o a J-sbetped

glaw rod tha t a l s o del ivers oxygen t o the Wml organ b t h .

The muscle and nerve were bathed i n oxygenated krebs solu-

t ions ( N a ~ 1 6.92, KCb 0.4, MgS04.7Hp0 0.29; C a C 1 2 0.28,

K v 0.1 6, NaH CO 2.1 , Glucose 2g/l) even though i n t h e 4 2 3

o r i g h a l descr ip t ion by Bulbring (1946) Tyrode so lu t ion

containtng double dextrose was used. The Krebs so lu t ion

was supplied t o the bath from a r e ~ e r v o i r . The tempera-

ture of *e bath was maintained thermostat ical ly a t

37G. 5 '~. The phrenic nerve waQ led through an electrode

which was connected t o a square m e stimulator.

I n some experiments a d i f f e r en t electrode was a l s o

connec t~d d i rec t ly t o the muscle for d i r e c t s t imulat ion.

Stimulation of the nerve was by square impulses a t a

frequency of 1 2 shocks per minute, with 15-20 v o l t s and

a pulse width of (2.5 msec. I n any given experiment fre-

Q~ency , voltage and pulse width were kept constant.

Pipermine 0.2 t o lOmg/ml and d-tubocurarine 8 to

16ug/ml were used i n the study. Anticholinesterose

a c t i v i t y was s tudied using neostigmine up t o gug/ml.

Drugs i n aqueous solut ion were added d i rec t ly to the tuberculin

bath by means of a I m l ' - syringe. Each drug was

allowed to ac t f o r three minutes a f t e r which the solukian

i n the bath was changed. The preparation was washed

several times i n between addition of drugs. Addition of

drugs were made when the contractions had recovered t~

the or ig ina l s i ze or reached a new but steady level . In

the Br i t i sh Pharmacopoiea 1953 edi t ion, an assay method

f o r (-+_I-tubocurarine chloride was described i n which the

drug was i n contact with the t i s s u e f o r 5 minutes and

doses were given every 10 minutes, but the procedure adopted

i n t h i s experiment were a s described by Chou ( 1 947) where

the contact time was 3 minutes i n wder to x u i x ~ M ~ e the

time t h a t would be required t o wash the drug off the

receptors.

EFFECTS OF PIPEUZINE ON THE CONDUCTION SPEED IN NERVXS USLNG THE RAT S C I A T I C N E R V E G A S T R O C N W S P l W A R A T I O N

This experiment w a s planned t o explore t h e p o s s i b i l i t y

t h a t t h e a c t i o n of p iperaz ine on t h e rat phrenic nerve - diaphragn preparat ion, a t l e a s t i n p a r t , maJr be due t o a

l o c a l a n a e s t h e t i c e f f e c t on the nerve.

R a t s of e i t h e r sex weighing between 1 4 0 - 1 6 0 g were

used. The ra t was anaesthesised using pentobarbitone

sodium 40-60mg/kg in jec ted i n t r a p e r i t o n e a l l y . Surg ica l

anaes thes ia was achieved i n 1 5 - 3 0 minutes and t h e ra t

prepared f o r d i s s e c t i o n by s t r app ing the arms and l e g s

t o thg s i d e s of t h e d i s s e c t i n p board i n a f a c e up pos i t ion .

The s c i a t i c nerve was exposed from t h e medial aspect

o f t h e t h i g h , up t o t h e base of the sp ina l column, The

gastrocnemus muscle was d i s sec ted f r e e from i t s bed and

t h e tendon exposed, divided from i t s i n s e r t i o n and a thread

l e d from t h e end of t h i s tendon over a pul ley t o record on

a smoked su r face , The d i s s e c t i o n w a s e s s e n t i q l l y b lun t

t o avoid haemorrhago and minimize trauma t o t h e t i s s u e s .

The s c i a t i c nerve was p r e p ~ r e d f o r s t imula t ion by

a t t a c h i n g e l e c t r o d e t o the nerve, as near t h e point

of emergence of the nerve from t h e s p i n a l cord as poss ib le ,

Between t h e a t t a c h . e r 9 of t h e e lec t rode and t h e poin t of

en tam of the nerve i n t o t h e gastrocnemua, t h e exposed

20

length of the s c i a t i c nerve was placed i n a s m a l l p l a s t i c

trough i n t o which drugs were placed to bath the nerve. To

maintain constant concentrnt ion around the nerve, cotton

wool was placed ins ide the trough and was completely and

continously soaked with the solution. The cotton wool

used were i n ba l l s of the same weight.

The electrode f r o m the s c i a t i c nerve waa connected

t o the output of a standard s t imulat .Or~ (cF 8048). The

external t r i g g e r points of the stimulator were oonnected

t o a Timer Clock, together with a time marker which was

made to write on the smoked surface d i r ec t ly under the

myograph lever. This ensured tha t the same voltage drive

was dis t r ibuted sirm.iLtan&ously t o the time marker afid the

nerve v i a the stimulator, HoweveY! the voltage drive t o

the nerve was enhanced by increasifig the voltage delivered

from the stimulator. The nerve tJ&s thus stimulated a t a

frequency of 1 2 shodrs per minutes, a t 15-20 vol te and a

pulse width of 0.5 m i l l i seconds, I n any given experiment

the frequency, voltage and pulse width were kept constant.

Recordings on the smoked s u r f a ~ e were made a t a drum

speed of 252mm per second. Bimple muscle twitches with

tkme recordings underneath were recorded with or&y

pwsiological s a l i n e bathing the lerve. The cotton wool

was removed and the recordings repeated with piperaeine

2 1

500ug/ml, ba*hing; the m e m e fm perbds of 1, 5 , .tO arsd

15 minutes. The saine procedure was repeated with 2me/ml

and 4rng/1nl o f piperazine and 100ug/ml o f ligylocaine.

Before changing t o a new drug concentration, the

&%ton wool was removed and the nerve washed many t ims

with physiological saline. 1n-kmal.s of 10 minu-t-3s were

allowed to elapse before the nerve w a s agftin s t ~ m O % d .

Cats weighing between 1 - 5 and 2.2kg were used, 6 of

such c a t s being used on t h e whole. The ca t was weighed

and anaesthesised with pentobarbit one sodium 40-60mg/kg

given i n t r ape r i t onea l l y . Surgical anaesthesia was achieved

i n 15 t o 30 minutes. The ca t was then s t r e tched out on

i ts back on the d i s sec t ing t a b l e , the sk in over the neck

opened and t h a t rachea exposed by se -p ra t ing t h e s t r a p

muscles. The t r achea was cannulated jus t below the

la rynx with a Y-shaped carnula t o a 3 o w suct ioning and

a r t i f i c i a l r e sp i r a t i on should t h e t iecessity a r i s e .

La t e r a l t o the t rachea, the i n t e r n a l jugular ve in was

oxposed arLd cannulated.

One of t h e l e g s was then d issec ted t o expose t he

gastrocnemus muscle, which was l i f t e d from i ts bed and

cleaned up t o i t s or ig in . The i . ~ s e r t i o n having been

exposed, t he tendon was cu t very near t he bone and a

s t rong thread secured t o t h i s end. This thread was l e d

over a system of pul leys t o wri te on a smoked surface .

The s c i a t i c nerve ms then dissected f r e e i n the th igh

and followed d i s t ~ -1y t o i ts ent* i n t o t h e muecle. The

branche s t c ~ t he o the r musble s of t h e l e g were

sectioned and an . l e c t r o d e at tached t~ the nerve. The

electrode was l ed from a standard s t imulator preset to

de l ive r 20 to 25v at a frequency of 1 2 pe r minute arrd a

pulse width of 1.5 t o 2m secs. The voltage, frequency

and pulse width were kept constant f o r each experiment,

Haemostasis was s t r i c t l y securcd throughout the

dissect ion and because the ambient tenperature was hi&

it was necessary on some occasions t o give an intravenow

infbsion of physiological sa l ine . M b i s t cotton was w e d

t o protect the raw surfaces and t h i s was kept mois t by

continous slow dropping of s a l ine f r g m a reservoir .

After es tabl ishing a constant height of contraction

following stimulation, the e f f ec t s o f suxamethonium

producing about 50 per cent inh ib i t ion of contrEsct;iun w a s

chosen. After repeating t h i s dose a nwnber of times to

es tabl ish consistency, the animal was pretreated with

piperazine 10 t o 15 minutes before &*en the suxamethonium.

S h U a r l y the e f f ec t s of piperazine on d-tubocurarine

induced block were studied. The e f f ec t s of piperazine

alone were d s o studied.

Dmgs were given i n aqueous solutions in to the in te r -

m1 jugular vein.

E X P E R I ~ N T S ON THE EWECTS OF PIPERAZINZ ON NXUROMUSCULAR CO-ORDINATION I N YOUNG CHICKS

Young chicks of 4 days old were used, Feathers were

removed from the neck t o expose the external jugular veins.

The chicks were weighed a f t e r which each chick was photo-

graphed s tanding.

Drugs i n aqu& solutions were then injected in to the

external jugular veins. The following drugs were injected

a t the doses s t a t ed below:

Suamethonium - 1 O%/k6

( +) -Tub0 curarine - 1 OOug/kg

Piperazine - 1 00m6/kg

Sl - 40 Omg/kg

9 1 - 800mg/kg

The maximurn volume of solution injected was l e s s -than 0.6ml.s.

4.Oug or& suxamethonium was given t o a 40g chick as 0.4ml.s

of 10ug/011 solution of tho drug i n d i s t i l l e d water. 0.33mls

of lOug/ml solution of tubocurCq.rine was given t o a 33g chick

equimledt t o a t o t a l dose of 3.3ug. Piperazine solution

in d i s t i l l e d water was given t o three chicks wei&ung 27g,

35g and 33g, the chicks receiving doses of 2 . 7 ~ ~ 14% and

2 6 . 4 ~ respectively. These were given as 0,27mls of 10mg/m19

0.2.8mls of 5Omg/ml and 0.53mls of 9ug/ml of piperazine.

The chicks were observed f o r one and one half hours

as regards t h e i r a b i l i t y to stand and t h e i r stance; the

a b i l i t y ro r i gh t themselves when l a id on t h e i r sides;

the presence of twitchings and fascicula t ions and whether

tonic extension of the limbs occured. Ehot ographs o f the

chicks were taken i f s ign i f ican t manifestations occured.

m M E N T S ON THE EFFECTS OF PIPERAZINE ON THE SLEEPING TIMB OF NICE UNDBR Pl3B'TOB,WITONE SODnTM

Male albino mice weighing between 19.5 and 25g were

used a l l o f t h e same generation. The nice were divided

i n t o groups each containing 10 mice. Three control m u -

received pentobarbit one alone, each group receiving e i t h e r

40mg/kg, 50mg/kg o r 6Omg/kg. The other groups were pre-

t rea ted with piperazine 200mg/kg and 1000mg/kg, 30 t o

35 minutes before pentobarbitone w a s injected. Aqueous

solut ions of pentobarbitone and piperazine were used a d

the so lu t ions were administered in t raper i toneal ly . The

volume of the drug solut ion w a s kept under 0.5mls

i r respec t ive o f the dose leve l .

The absence of the r i g h t i n g r e f l ex was used as the

c r i t e r i o n f o r s leep. Thus each mouse was considered t o

have s l ep t when it f a i l s t o r i g h t i t s e l f when placed on

i ts s ide . Sleep was considered t o have terminated when

the mouse (which was made t o s leep on i ts side) first

r igh t s i t s e l f f rom the s leeping posi t ion, and is able t o

again r i g h t i t a e l f within one minute when repositioned on

i ts side.

The time to onset of s l eep and the t o t a l dura t ion of

s leep i n each mouse i n each group was c d c U & d and the

averages with t h e S W were a l so calculated.

In o r d e r t o determine whether piperazine on i t s own

had any effect on the r igh t ing reflex, a group o f ?O mice

were given the higher dose of piperazine (i.e. 1000mg/kg

body weight) and t h e e f f ec t s on t h e righting m f b x waa

observed f o r 2 hours.

BiaZERIPIIENTS ON THE EFFECTS OF PIPBRAZIWl3 OX TEE -"~NT ACTIVITY OF LI@TAZOL

Female albino mice weighing between 18 and 25g

were used. The mice were divided into groups, each

containing 10 mice. Three control groups received

l ep t azo l alone; each group receiving e i t h e r 3Omg/kg

40mg/kg o r 60mg/kg. The other groups were pre t rea ted

with piperazine a t dose Sevels of $Omg/kg and 4OOmg/kg

30 minutes before leptazoa was in jected. Aqueous so lu t ions

were in jec ted intmperitol5sally. The t o t a l volume of

the solut ion used was kept under 0.8ml f o r each mouse,

i r respec t ive of the dose leve l s .

The number of mice conwlsing a f t e r in jec t ing lep-

t azo l was recorded, as was the time iq t e rva l between the

i n j ec t ion of the drug and onset of coklvulsion. The

morta l i ty r a t e from l ep t azo l i n mice pretreated o r

without pretreatment with piperazine ma calculated.

EXPERIMENTS ON THE EFFECTS OF PIPERAZINE ON THE COlTVELSmT ACTIVITY OF STEECWNINB IN MICE:

Male albino mice weighing between 1 8 and 2% were

used. Three control groups each containing 10 mice

received strychnine alone, each group receiving e i t h e r

0.0 5rng/kg, 0.1 mg/kg o r 0.1 5mg/kg. The other groups

of 10 mice each were pre t rea ted with 200mg/kg o r 400*

of piperazine 30 minutes before strychnine was injected.

Aqueous solut ions of strychnine and piperazine were

used, and the solut ions were in jected in t raper i toneal ly .

The t o t a l volume of the so lu t ion of strychnine and

piperazine used i n each mouse was under 0.5ml irrespec-

t i v c of the dose l eve l .

The number of mice ~ m v u l s i n g a f t e r i n j ec t ing

strychnine was recorded aa was t h e time in t e rva l

between the in jec t ion of the drug and onset of

c o n d s i o n . The m o r t a l i t y rateg from strychnine i n

mice pretreated o r without pretre,?.tment wi-l;h piperazine

3 1

FROG RECTUS ABDOMINIS PlUSCLE EXP.ERIbEMTS - Eiperazine a f f ec t ed t h e cont rac ture induced by

ace ty lchol ine i n the f r o g muscle i n an i n t e r e s t i n g

manner.

Piperazine a t 100-1 000ug/ml potent ia ted t he a c t i o n

of ace ty lchol ine on the f r o g r e c t u s muscle i n a dose

dependent manner a able 1 : Figs. 1 ; 2).

Higher doses of piperazine greaCer than lmg/ml

reduced the height o f cont rac t ion; the i nh ib i t i on

being almost complete a t 6mg/ml (Figs. 3 ; 4, Tables

2; 3 . However, up t o 6mg/ml of piperazine did not

on i t s own cause m y not iceable e f f e c t on t h e f rog

muscle (Fig. 5).

1. double rec iprocal p lo t of these d a t a ( f i g u r e 6)

showed t h a t piperazine antagonised t h e a c t i o n of ace ty l -

chol ine i n t h i s t i s s u e i n a non-competitive manner, the

d i s soc i a t i on constant of t h i s antagonism, being

2.6n10-'. On the o-ther hmd d-tubocurarine antagonised

ace ty lchol ine i n a competitive manner with a dissocia-

t i o n constant of 2.0x10-~ (Fig. 7 , Table 5 ) .

RESULTS -- numutx of observations In) = 4

PERCENTAGE;

EFFECT$ OF MEGH DOSKS QF P.i..PEi+AZINE

t P i p ) OM COEITMPeCTI(3NS I N D U C E D BY 6ug/rarl

ACETYLCHOLINE (Ackr) HN T!lE FROG PECTUS

ABDOMXNJS MUSCLE.

RAT PHKENIC NERVB-DIAPHRAGM EXPERIMENTS

P i p e e z i n e reduced the he ight of cont rac t ions of

the r a t diaphragm st imulated i n d i r e c t l y v i a the phrenic

nerve ( ~ i g . 8) . Below I mg /ml piperazine had no s i g n i f i -

cant e f f e c t on t h e height of contract ion. However,

above t h i s dose t h e r e was a dose-depeqdent dimunition

i n t h e height of contract ion. Complete (10%) i n h i b i t i o n

was obtained cons i s t en t ly a t 10mg/ml piperazine a able 6 ) .

Tubocurarine produced q u a l i t a t i v e l y similar e f f e c t s

but was about 500 t imes more potent than p ipemzine

(Fig. 9).

The block induced by piperazine l i k e t h a t induced

by b b o c u r a r i n e was overcome by d i r e c t s t imula t ion of

the muscle (Fig. 10). However, unl ike tubocurar ine

t h e neuromuscular block due to piperazine w a s not e a s i l y

reversed by neostigmine (Pig. 1 1 ) . Also a d d i t i o n of

Neostigmine 4 0 ~ ~ / m l two minutes before adding piperazine

d i d not a f f e c t the speed of onset o r the extent of block

induced by piperazine . The e f f e c t s o ' piperazine u n l i k e those of tubocurar ine

were e a s i l y revers 2d by washing; the block induced by

piperazine reversed rapidly i n under one minute follow-

ing only a s ingle wash (Fig. 12) , but t h a t induced by

tubocurarine w a s reversed a f t e r 5-10 miputes and o n l y

with repeated washings,

The block induced by piperazine g-ted with that

induced by tubocurarine . ~ h u s 4mg/d piperazine alone

produced a 30% reduction i n twitch height, and 2ug/ml

tubocurarine alone produced a 75% block. When these

two were given together a 1007; block was achieved

(Fig, 13) . Neostigmine 40ug/ml w a s again unable t o

reverse the block,

RAT GASTROCNEMUS-SCI ATIC mRVE PREPARATION

Piperaz ine increased t h e l a t e n t period and theref ore

the conduction time a long t h e rat s c i a t i c nerve, t h e

e f f e c t being dose r e l a t e d (Fig. 14).

The degree of i n h i b i t i o n increased as t h e dose

of piperazine was increased from 50Oug/ml t o 4mg/ml.

However, ia a l l cases t h i s e f f e c t was not sus ta ined

a f t e r ten minutes; a f t e r f i f t e e n minutes t h e a f f e c t

of the drug had worn o f f considerably (Table 8).

Figure 15 shows t h a t although l ignocaine increased

the l a t e n t period o r conduction time a long t h e nerve,

i t had about 20 t o 40 times t h e a c t i v i t y of piperazine

i n t h i s regard. Moreover the increase i n l a t e n t per iod

was the same over t h e 1 5 minute per iod of observation.

Height of con t rac t ion recopied a t t h e same time was

not cons i s t en t ly a f fec ted . A tendency t o reduce t h e

height of con t rac t ion was noted but this was not s t a t i s -

t i c a l l y s ign i f i can t (Table 9) .

CAT GASTROCNEMUS-SCI ATIC NERVE PREP~*1RkTION

Piperazine affected the neuznomuscular blocks

induced iq the cat gas t rocnems rmscle by suxamethonium

and d-tuboqumrine i n an oppos i te manner ( ~ i g s . 16 t o 19) . P r e t r e p t m n t with piperazine po ten t i a t ed t h e

block due t o d-tubocurarine . Thus 100ug/kg tubocurar ine

produced a 57 percent i n h i b i t i o n , but when t h e animal

w a s p re t r ea ted with 25mg/kg p iperaz ine t h e r e was

v i r t u a l l y a complete block (98.9%) p able 10) .

On the contrary p iperaz ine antagonised t h e

neuromscu la r block produced by suxametihonium. Pre-

t reatment with 25mg/kg p iperaz ine reduced a 63.6%

i n h i b i t i o n produced by 30ug/kg suxamethanim t o

43.4% able 1 1 1. Figure 20 dompares t h e e f f e c t s of suxamethonium

alone and suxame$honiwn a f t e r pre trea-tment with 25mg/kg

piperazine. To Qchieve the same 50 percent i n h i b i t i o n

(m5-) i n t h e presence of p iperaz ine , t h e dose of

suxamethonium had t o be increased f r o m lOug/kg t o 50ug/kg.

On i ts own p iperaz ine bad no s i g n i f i c a n t e f f e c t

on t!l@ ce t sciatic nerve gastrocnehas preparat ion.

$1 EElL)3IT"PQ# . IT* 70,554 *O L&<S X. AEQIJ.5 Yl_le__.l -

EX3)ERItTmTS ON TIiE EFPCCTS OF PISER:!ZINE ON NEURO- rrnscmm CO-ORDIW~~TION IM YOUNG CHICXS ::

Chicks t r e a t e d with 100ug/kg of suxameehonim died

wi th in one minute from s p a s t i c pa ra lys i s , The lags

were exiiended and the neck r e t r a c t e d (JQ. 22).

F a s c i c u l a t i m s and twi tch ing of muscle preceded t h e

s p a s t i c para&ysis.

Chicks t r e a t e d with 100ug/kg of tubocurarine died

wi th in two &mutes i n f l a c c i d pa ra lys i s (~ig. 23).

No twitching o r f a s c i c u l a t i o n s were observed.

Chicks t r e a t e d with 100mg/lq of p ipemzine remained

r a t h e r qu ie t but no o t h e r abnormality wcls observed.

When the dose of p i p e r a z i m w a s increased t o

400mg/kg, t h e chicks had shaking of t h e head a f t e r about

f i f t e e n minutes of the in jec t ion . The chicks were ab le

t o s t a n d a t e r one hour but on a wide bsse (Fig. 24).

They were s t i l l ab le t o r i g h t themselves when placed

on t h e i r s i d e , Sweating ms noted i n t h e chicks as

we t t ing of f e a t h e r s ,

When the dose of p i p e ~ a z i n e was fur-ther increased

t o 800mg/kgt shaking o f -the head recurred i n five minutes

and t h i r t y m u t e s a f t e r the adminis t ra t ion of p iperaz ine

the chicks began t o s tagger bu t were s t i l l a l e t o

erect although on a base much wider than was observed

with 400mg/kg, 'The c h i c k s were unable t o r i g h t them-

selves when p13ced on t h o i r s i d e , b u t on p l a c i n g upright

were a g a i n able t o mainta in t h e i r foo t ing . Sweating

w a s a l s o no ted (Fig. 25) . Figure 21 show a t y p i c a l yount; chick without any

drugs i n j e c t e d .

EFFECTS 01

COORDINATION YOUNG CHICKS.

& drug Injected. L

Effect of d-tubocurarho 1 . ~Ug/kg, b-

. . 'hn FIGURE 24.-

- D w w EXE'BRIMEN!J!S ON NEUROMUSCUL! COORDIN1*.TION

IN YOUNG CHICKS. L

Effect of P i p o r m i n e 400ng/kg

IN YOUNG WICKS, m

EXPERIMENTS ON THE SLEEPING TIME OF NICE UNDER P ~ T O B L ~ R B I T O N 2

Eiperazine increased -tihe duration of s l e e p but- redu-

ced t h e t ime t o onset of s l eep i n mice under pentobar-

b i tone sodium h y p o s i s and t h i s w a s dose r e l a t e d

( ~ a b l e s 1 2 t o 1 5). 1000mg/kg p iperaz ine alone

administered t o these animals produced an i n i t i a l

e x c i t a t i o n with some of t h e animals shaking - the i r heads

and o t h e r s jumping with minimal st imulus. This per iod

of excitement was followed a f t e r 30 t o 45 minutes by a

period o f reduced a c t i v i t y and weakness but s l eep was

not induced and t h e r i g h t i n g r e f l e x remained i n t a c t .

When t h e animals were p r e t r e a t e d with t h i s dose of

piperazine ( 1 OOOmg/kg) before i n j e c t i n g pentobarbitone,

the re was an increase i n t h e dura t ion of s l e e p t h a t w a s

s i g n i f i c a n t a t p f0 .O5 i n a l l cases (Fig. 26) . 1

200mg/kg piperazine a l s o increased t h e s l eep ing

time but t o a smal l e r ex tent (Fig. 27) . Thus the dura t ion

of s l e e p under 40ng/kg pentobarbi t one was increased by

200mg/kg piparazir e by 1 5.4% while 1 OOOmg/kg of pipera-

z ine increased i t by 69.7% (Tables 1 2 and 14).

However the time t o onset of s leep was reduced

more with the smaller dose of piperazine (Figs, 28;

29) , When the animals were pretreated with tOMhg/kg

piperazine the time t o onset of sleep i n the ,group

receiving 40mg/kg pentobarbitone was reduced by 12.99'0

but pretreatment of the same group with 200mg/kg o f

piperazinc reduced the t i m e t o onset of sleep by 34,&

(Tables 1 3 and 1 5 ) . Figures 28 and 29 show the affect of piperaziuLe

on the time t o onset of sleep.

1; + PIP.

-s- Pentobarb. alone

j lW( mins 1

PFECTS OF P I P E R A Z I N E ON LZPiCAZOL IITDUCED CONVlTLSIONS

Tables 16 and 17 show that piperazine increased the

convulsant e f f e c t and t h e a c u t e mor ta l i ty r a t e of

l e p t a z o l i n mice.

Piperazine produces a marked increase i n t h e con-

vu l san t a c t i v i t y of l ep tazo l . T~.ble 16 shows thzdi t h e r e

was no r e a l d i f f e r e n c e between -the e f f e c t of 200mg sad.

400mg/kg of piperazine on t h e corwnlsarrt; a c t i v i t y of

l e p t a z o l .

The e f f e c t on t h e +hour acute m r t a l i t y r a t e

however showed a s i g n i f i c a n t increase when t h e dose of

piperazine w a s increased from 200mgfkg t o 400mg/kg.

There was no mor ta l i ty from 40mg/kg of l e p t a z o l even

when t h e mice were p re t rea ted with 200mg/lcg. However

pretreatment with 400mg/kg of piperazine caused a

mor ta l i ty of $96 a able 17).

Pretreatment with piperaziiie reduced t h e time

required f o r the onset of c o n v u l s i ~ n i n t h e animals.

Thus 60mg/kg l e p t a z o l a lone p r ~ d u c e d convulsion i n t h e

animals i n 5 minutes but pretreatment with piperazine

reduced t h e t ime of onset t o 3.5 mlnu?;es.

EFFECTS OF PIPERAZINE ON STRYCHNINE INDUCED CONVULSIONS

Piperazine a t 200 and 400rng/kg increased the

convulswt a c t i v i t y and the five-hour acute mor ta l i ty

r a t e of mice t r e a t ed with s t rychnine.

This e f f e c t of piperazine w a s seen t o be dose

r e l a t ed . Thus "ce convulsant a c t i v i t y of O,llng/kg of

s t rychnine w a s increased from 2w0 t o 90% by 400mg/kg

piperazine (Table 18) ,

The e f f e c t of piperazine on the 5-hour acute

mor ta l i ty i s shown i n t ab l e 19. Pre-heatrnent with

piperazine increase acu te mor ta l i ty , the increqse a l so

being dose re la ted . Thus whereas -/kg of piperazine

increased t he mor ta l i ty from 10 t o 205, 400mg/kg

increased i t from 10 t o 505, in t h e group rece iv ing 0.1 rng/kg.

However no d i f ference was noted between the

e f f e c t of 200mg/kg and 400mg/k~ of p ipsmzine on the

mor ta l i ty due t o 0.1 5mg/kg of s t rychnine.

83

DISCUS5ION

P ipe rnz ine c i t r a t e l ong e s t a b l i s h e d t o have a po ten t

p a r a l y s i n g a c t i o n on t h e a s c a r i s smooth muscle ( ~ a ~ a r d ,

1949, Goodwin and Standen, 1954) appears t o have some a c t i -

v i t y on s k e l e t a l muscles,

P ipe raz ine appears t o have a d u a l e f f e c t on t h e

c o n t r n c t u r e s induced by a c e t y l c h o l i n e i n t h e f r o g r e c t u s

abdominis muscle , A t low concen t r a t i o n s ( l e s s than I mg/ml)

i t p o t e n t i a t e d t h e a c t i o n of a c e t y l c h o l i n e . A similar

r e s u l t w a s ob ta ined by Onuaguluchi (1966) who found t h a t

at under 2mg/riiL, p ipe raz ine p o t e n t i a t e d t h e a c t i o n of

a c e t y l c h o l i n e , From t h e p re sen t s t u d y concen t r a t i ons

g r e a t e r than 2mg/ml, p ipe raz ine antagonised t h e a c t i o n of

a c e t y l c h o l i n e . This antagonism by p iperaz ine w a s shown t o

be non-competitive, whereas t h e antsgonism produced by

d- tubocurar ine w a s compet i t ive .

I n t h e rat phren ic nerve-diaphragm p repa ra t ion ,

p i p e r a a i n e produced some r e d u c t i o n i n t h e t w i t c h h e i g h t

f o l l o w i n g i n d i r c c t s t imu la t ion . Compared t o t h e a c t i o n o f

d- tubocurar ine tho potency of p ipe raz inc w a s low and t h e

b ind ing t o i ts recep- o r s poor, hence t h e e f f e c t w a s e a s i l y

terminated by washing. I n this prepamtion tW block duc

t o piperazinc was found t o be r e l a t i v e l y irreversible with

ncostignine.

Pros t iga inc and o t h e r a ~ ~ i & l i n o s t e ~ . ~ pmlczng a

durat ion of the end p l a t e po t en t i a l s (Fcng, 1940; Eccles

e t 31, 1942; Eccles and Macfarlane, 1949) md of end p l a t e

cur ren t s ( Takeuchi and Takeuchi, 1 9 59 ; b g l e b y and Stevens,

1972; Kuba e t a l , 1973). It was argued t h a t t h e f r e e

d i f fu s ion of acetylcholine from the s y m p t i c c l e f t would

be t oo rap id t o account f o r the increased time course o f

end p l a t e p o t e n t i a l s and end p l a t e cur ren t s recorded i n the

prmence of an a n t i c h ~ l i n e s t e r a s e which i n h i b i t s t he hydro-

l y s i s of ace ty lchol ine ( ~ c c l e s and Jaeger , 1958; Kuba and

Tornita, 1971). T h i s has l e d t o the suggestion t h a t t he

prolonging e f f e c t of the a n t i c h o l i m s t o r n m on end p l a t e

po ten t i a l s and end plate cwrronts might arise from an

increase i n the m o m l i f e t imes of conductance channels

ac t iva ted by acotylcholino ( ~ u b a and T o d t a , 1 971 ; %globy

and Stevens, 1972). However, Katz and Miledi (1 973)

reported t h a t prostigmine had l i t t l e or no e f f e c t s on

channel l i fe t ime and thsy suggcstcd t h - ~ t i n t h e presence

of prest ignine t h e ac t ion o f acetylcholjno can be prolonged

by delayed d i f fus ion of acetylcholine from the synaptic

c l e f t r e su l t i ng from repeated bdind iEg of acetylcholine

t o receptors.

Whatever is the mechapisn of prolongation of the ac t ion

of acetylcholine, competitive antagonism is reversed by

ant ichol ines terase , but, t h i s was not t he case with

antagonism produced by piperctzine. The f a c t t h a t t he

block produced by piperazine was not reversed by neostig-

nine, whereas tha t produced by d-tubocurarine was reversed

suggests t h a t piperazine may not a c t en t i r e ly on the neuro-

muscular junction, Pipemsine nay a c t pos t synapt ica l ly on

t h e muscle i t s e l f , possibly by blocking calcium ion move-

iacnts, Direct s t i n u l a t i o n of the ~ u s c l c , however caused a

twitch response. This may suggest t h a t piperazine does

not a c t d i r e c t l y on thc muscle. However, t h e f a c t t h a t the

voltage used i n ths d i r ec t s t imulat ion was high r e l a t i v e

t o t h a t used i n tho ind i r ec t stimulation, does not allow

one t o nake f i n a l conclusion on whether pipcrazinc has a.'

86 '

d i r e c t dcprcssant a c t i o n on thc m s c l e .

Pipernzinc may a l s o a c t prcsynapt ica l ly by reducing

conduction a long the phrenic nerve also k t h e d i n t h e sane

f l u i d , i n which case ncos t iga ine w i l l not be a b l e t o

reverse t h e block. The experiments on t h e r a t gastrocnemus-

s c i z t i c nerve prepara t ion was designed t o explore t h i s

p o s s i b i l i t y . I n t h i s p r e w r a t i o n , pipermint? was shown .to

reduce conduction vcloci-ty along the nerve, even though

the e f f c c t d id not l a s t for nore t h a n t e n minutes. Some

o t h e r recent work i n our l a b o r a t o r i e s showed t h a t pipera-

z inc has a weak l o c a l a x k s t h c t i c a c t i o n , Thus t h e inhi -

b i t i o n of con t rac t ion of the rat diaphragn s t i n u l a t d

i n d i r e c t l y v i a the phrenic nerve by pipcrazine nay be

p a r t l y due t o a l o c a l cznaesthctic e f f e c t on t h e nerve.

In t h e c r ~ t gas trocnemus-sciatic nerve prepara t ion ,

p iperaz ine antagonised the e f f c c t of suxancthoniun while it

enhanced those of d-tubocurarine . These f indings suggest

t h a t p i p e r z i n c has a n e f f e c t on the neurunuscular junc-

t i o n even though t h i s c f f z c t mst bc s l i g h t as the drug

even a t very high dose l e v e l s (up t o 200ng/kg) d i d not

depress the response of the gastrocnenus mascle t o s t inu la -

t i o n of the s c i a t i c nerve. If the ac t im of piperazinc

w a s e n t i r e l y a l o c a l ames the t ic ac t ion , it should enhanco

the block induced by both suxanethoniun a d d-tubocumrine.

Zn young chicks, piperazine produced riuscular weakness

and incoordination espeirially a t higher doses. The muscle

weakness following thc i n j ec t ion of piperazine i s f lacc id

i n nzturo. Young chicks thus a f fec tcd adopted a f l acc id

posturp not unlike the posture adopted by the animals

following in jec t ions o f tubocurarino. Butt le and Z a i m i s (

(1949) showed t h a t i n adu l t fowls o r i n chicks an intrnve-

nous i n j ec t ion of decnmethonim caused a r i g id extension

of thc l i n b s and r e t r a c t i o n of thc head. They were ab le

t o show tha t t h i s response wzs n peripheral e f f ec t and

involved the shortening o f the rluscle - a t rue contracture.

On the o ther hand, d-tubocumrinc causes f l acc id

p a n l y s i s in birds: thc block being mtagonised by

m t i c h o l i n e s t e r a s ~ s and by t c t a m s . It would appear

therefore t h a t the neuromuscul~m block due t o piperazine

i s s imi l a r t o t h a t dui. t o d-tubocurarine, and unlike

s u x a ~ e t h o n i ~ induced neuronuscular block.

Work on the e f f ec t s of piperazine on t h e cen t r a l

nervous systen shows t h a t i n n ice it increases the sleep-

ing time due t o pentobarbitone s o w ( ~ D . 0 2 ) . Surpris-

ingly it a l s o increases the convulsant a c t i v i t y o f l o p t a m 1

and strychnine i n nice.

The poss ib i l i t y t h a t the increase i r i s leeping t i n e was

a. r e s u l t of nuscle para lys i s i n the aniLrnls w a s exclu-

ded by showing t h a t t h e highest dose of piperazine used in

the study d i d not have any e f f ec t on the a b i l i t y of t he

nice t o r i g h t thenselves.

Piperazine lowers the convulsive threshold a s shown

the r e s u l t s obtained f ron t h e study us ing lep tazo l and

strychnine. These two ana lep t ics a c t by d i f f e r e n t

nechanisns. Strychnine i n t e r f e r s only with po~ taynap t i c competitiv

inh ib i t ion i n motoneurones(~cc1es f957) by ac t ing ad

antagonist of t he inh ib i t ing s i t e s i n much tt-e sans manner

a s curare blocks ace ty lchol inc a t the neuronuscular junc-

t i o n ( ~ u n o and Weakly, 1972) . Bbcause typ ica l strychnine

convulsions a l s o occur i n sp ina l <minals , the e f f e c t s of

strychnine are of ten a t t r i bu t ed t o a spinal locus of

ac t ion , and the convulsion frequently t c m d a spinal

convulsion. However, othor por t ions of thc cental ncr-

vms system a ra fully exci ted by doses t h z t produce the

manifestat ions i n a ~ p i n a l animal.

I n cont ras t , l e p t a z o l does not hlock e i t h e r presynaptic

o r postsynaptic inh ib i t ion . There is ~ v i . d m c that the

exc i t a to ry e f f e c t of l e p t a z o l nay be due t o a decrease i n

nouroml recovery t i m e ( ~ e w i n and Esplin, 1961 ) . That

p e m i a b i l i t y changes i n t h e c e n t r a l nervous systerz leading

t o a r i s e i n extraneuronal potnssiup, which would p a r t i a l l y

dcpolar ise neuronal nembmnes and so increase t h e i r

e x c i t a b i l i t y , nay Lamount f o r the convulsive a c t i v i t y o f

l c p t a z o l was suggested by t h e work af G r o s s and Woodbury

( 1 972). human

Sleep i n norn.zl/adults cons i s t of 90 ninu tcs cycles

of synchronous non-rapid eye movcrmnt (NFEM) s l e ep during

which the electroepcephalogram ( EEG) shows k-complexes,

sp indles and d e l t z waves; a l t e r n a t i n g with rapid eye nove-

nent (M) s l eep &iring which the re is rapid eye movement,

a narked decm:xe in muscle tone and an asynchronous EEG.

Slow wave s leep (NREM) i s thought to result f ron

inh ib i t ion of the r e t . . i cu l a r ac t iva t ing system (RAS) by f I

a brain s t e n scrotoncrgic system, which a l so is a t p r i n i x '

nechanism for REM sleep. Drugs which deplete brain sero-

to-?in lcve ls , f o r example paracblorophenyldaninc cause

insomnia i n ca t s (Honykciwicz, r1966) . This can be overcone

by adninis ter ing 5-hydroxytrypyophan vhich res tores the

serotonin content of tho ncurones t o normal.

REM s l c ~ p i s i n i t i a t e d and regulated by areas of t he

pontinc r e t i c u l a r fornation. Thcse include the nucleus

coemxleus, which i n i t i a t e the nusclo inh ib i t ion and

a re f lex ia of REM sleep. Available evidence indicate t h a t

no s ing le synaptic t ransmit ter is responsible for REM sleep.

Cathccola~ine and acetylcholine ncurones i n the brain s t e n

a re thought t o be involved i n the i n i t i a t i o n of t h e phasic

events of REM sleep, the muscle inh ib i t ion and EEG desyn-

chronimtion of R.EM sleep, and the co r t i ca l ,ad behavioural

act ivat ion of the mking s t a t e . Delorne ( 1966) and l a t e r

Jouvet (1967, 1969) proposed the dual theorp of sleep.

Wakefulness ---3 slqw-wave s leep --7 slow

wave s leep (with PC3 spikes) v-) REM sleep.

9 1

5-hydroxytryptanine ( s ero tonin) i n i t i a t e s s l o w wave

slcc p. IvlOnoanine Oxidase &stroya 5-hydroxytryptaninc

to 5-hydroxyindoleacetic acid ( 5-HIM) , 5 - H I M r i s e s i n

concentration and a t a c r i t i c a l l eve l both 5-HT and 5-HIAA

i n i t i a t e the PGO a c t i v i t y and t r i gge r s a group of choliner-

g ic nouroncs tha t interconnect t h e raphe systcn and the

r o s t r d pons. The cholinergic nsuronos s t i n u l a t e the nor-

adrenergic neurones of the locus coeruleus which i n turn

t r i g g c r the s igns of REM Sleep.

Rothballer (1957) noted t h a t 5 H T induced a short

period of EEG arousal followed by a period of hypersynchrony

exhibi t ing high voltage d e l t a waves,

Experinents involving abla t iod of area pos t rem and

t h e r e su l t s of topic21 appl icat ion of serotonin and

serotonin blockers point t o t h i s area a s the receptor s i t e

f o r serotonin ac t ion i n i t s EEG synchronising e f f ec t .

Morcst ( 1960) found t h a t nerve f i b r e s leave the f i b r e plexus

of the a rea postrenn in the 'd i rec t ion of the nucleus of

t rac tus s o l i t a r i u s . ' H e a l so observed i n the area postrena,

dendr i t i c arbor izat ions o f neurons located i n the medial

edge of the nucleu t r ac tus so l i t a r iu s . Bonvallet and

92

Allen ( 1 963) showed t h a t arousal produced by r c t l c u l a r and

nociceptive st imulat ion was norc in tense and nore prolonged

i n ca t s i n which d i sc re t e l es ions were placed in the

cephalic par t of thc nucleus of t he t r a c b s ~ l i t a r i u s .

They suggested the existence o f a (negative) feedback

sys ten operating between t h i s nucleus and t h e r e t i c u l a r

ac t i va t ing sys t en which checks excessive arousal, It

thus appears possible t h a t a f t e r reuoval o f the S H T

s ens i t i ve arca postrena i n a n i m l s with i n t a c t brain s ten ,

t hc arousal produced by r o s t r a l l y ac t ing 5-HT is s t i l l

checked by t h i s ncuronal fccd back sys ten whereas after

t ransect ion of t h e b ra in s t en and disconnection of the

feedback loop 5-HI! induced arousal can f u l l y develop.

Thus t he sane nninas nay bc involved in the mechn-

nisn o f s l e e p an8 the ac t iva t ion of the &IS. This would

explain why drugs which a f f e c t amine a tores i n t h e bra in

have e f f ec t s on both s leep and convulsion thresholds.

Reserpine f o r instance depletes the b r a i n of i t s

dopzmine content (Csrlsson, e t a l , , 1958). It a lso depletes

the brain of i ts 5-hydroxytryptarnine ( 5 9 ~ ~ ) and nor-

adrenaline levclz. Chen e t a1, ( 1 954) showed tha t reserpine

lowers t h e threshol t f o r electroshock o r pentylentetrazol

( leptazol) -induced convulsions. Shaepdryser e t a1 ( 1 962)

93 showed t h a t t h e threshold t o electroshock was r a i s e d 3-fold

by adn in i s t e r i ng dopa and a nonoaninc oxiitase i n h i b i t o r i n

r a b b i t s whose b r a i n had previously been depleted of dopanine.

This suggests t h a t t h e threshold lowcrlng e f f c c t of

reserpine f o r convulsions x ight be czusal ly r e l a t e d t o t h e

deple t ion of b m i n a n h c s . btsur;loto and Jouvet (1964)

showed t h a t reserpine i n doses o f 0,51g/kg l e d t o a reduc-

t i o n i n slow s l eep in c a t s and t o a conplcte e l iminat ion

of the tonic components of paradoxical s leep. If reserpine

was followed by i n j ec t i on of 5-hydrwcytroptophan ( 5-HTP)

t h e prccuasor of 5-HT, t he re was no reduct ion i n slow

s leep , whereas dopa shortened m d lessened t h e suppresion

of paradoxical s leep.

Phenothiazines a l s o havc t h e a b i l i t y t o lower t h e

convulsive threshold. They a l s o havc a var ied a b i l i t y

t o prolong ;tnd enhance the e f fec t of na rco t i c and hypnotic

drugs. -cl, 2.:

Prlenothiaeines a re nore l i k e l y t o criuse drug induced

s e i zu re s i n p t i e ~ t s who have e i t h e r a h i s to ry of se izure

d i so rder o r a conf:i t ion t h a t predisposes towards s e i zu re s . (~oodman and G i l m n, 1975).

94

These drugs a c t by b lock i~ lg the dopamine recep to r s i n

c e r t a i n b r a in areas notably t h e corpus s t r i a t u n .

Kebebian c t a1 ( 1 972) showed tht ant ipsychot ic dmgs

such as chlorprormzine and haloper idol were potent

competitive i nh ib i t o r s of t he s t i n u l r ~ t o r y e f f e c t s of

dopanine on adenylatc cyclasc . The increased conc entra-

t i o n of dopanine ne t abo l i t e s - horaovallin%c ac id i n

caudatc nucleus of c a t and r a b b i t (hnden e t a l , 1964;

Loverty c t 31, 1965) a f t c r phenothiazdnes could be expla i -

ned by assuming t h c t t h e rccop to r blockadc r e s u l t s i n a

conpensatory a c t i v a t i o n of s p e c i f i c neurones l ead ing t o

increased re lease o f dopamine. I n o ther bmim m e n s and

e spec i a l l y i n t h e cortex phenothiazines i n h i b i t t h e se-

uptake of nor-adrenalins <md 5-hydroxytryptmine .

It would thus xppenr t2L'tt i n view of -the r o l e o f t h e

same anines i n t h e nechanisn of convulsion and s leep , a

drug t h a t 3Pfects t he r e loa se of auincs i n the c e n t r a l

nervous system nay prolong s l eep ing t i n e while a t the

sane tirile lowering the convulsive threshold . The e f f i c acy

o f the drug i n producing thess e f f o c t s w i l l depend among

95

o the r things on i t s a b i l i t y t o pass the blood bra in

ba r r i e r . Like phenothinzines and reserpine, piperazine

may a f f e c t the a v a i l a b i l i t y of 5-HT 2nd dopanine i n the

b ra in sten. Studies using the Apormorphine, Tryptaaine,

Nor-adrenaline (ATN) t e s t sys ten (Nienegeers e t a l . , 1972)

a r e cer t :~inly indicated i n order t o deternine t h e e f f e c t

of piperazine on dopaminergic and 5-HT receptors i n the

bmin .

The r e su l t s of t h i s study explain z t l e a s t i n pa r t ,

the nnny neurotoxic e f f e c t s noted with prolonged adnini-

s t r a t i o n o f piperazinc and/or the adrrinistrebtion of high

doses of pipcrazino. Thcs c neuro toxio s ide e f f e c t 3 include

sornnolcnce o r confusion,d s t a t e s , dizziness, vomiting,

hypotonia with severe a t ax i c impnirmnt of g a i t ,

inco-ordination, dropping of objects and incroase i n the

number of a t t acks of p e t i t m l or nyoclonic jerks (~c l iuch

e t a l . , 1966; Slaughker, 1896) . Thus the c f f ec t s of pipc-

razine on se izure threshold bezr out ths c l i n i c a l repor ts

t h a t p i p ~ . r a z i l i e induced se izures are norc l i k e l y when t h c

drug i s given t o p a t i o n t s who have a pas t h i s t o r y o f

scizure d i s o r d e r (Schuch e t al., 1966) . Its effect on

pent obarb i t one-induced s l e e p m y a l s o explain t h e occurence

of t o x i c e f f e c t s such as oonnolcnce o r confusional s t a t e s

( s l m g h t e r , 1896; Schuch c t a l . , 1966) .

3-Ui'WiRY

In t he f rog rcc-hxs 25dorninis prepnration piperazine

produced pot e n t i a t ion of ace tylchaline-in2uced contractures

a t l o w doses while a t high doses i t antzgonised the

acetylcholine contractures . This ncuronusculsr antagonisn

was shown t o be non-conpetitive while d-tubocurarine i n

the sapie p r e p r a t i o n was shown t o produce i t s typ i ca l

cocpe tit ive antagonisn.

This w s s confirxed i n t he rat phrenic nerve-diaphragm

preparat ion, where i t was also lshown t h a t t h e neurorzluscular

block produced by piperazine was of n low potency with

poor a f f i n i t y f o r i t s receptors. The r a t phrenic nerve-

dizphragn experincnts a l s o ra ised the poss ib i l i t y t h a t

piperazine nay a c t , a t l e a s t i n ~ r t , presynaptical ly on

the neurone s . I n the r a t gnstrocneraus-sciatic nerve preparat ion

piperazine c i t r a t c increased the l a t e n t perioc? of response

o f the m s c l e t o s c i a t i c nerve s t i m l a t i o n , thus suggesting

a weak loca l anaes thet ic e f f e c t .

However th3.t p ipenz ine has a d i r ec t neuromuscular

jUction effect i n addit ion W ~ S shown i n the cat gastroc-

nems-scia t ic nerve preparation and t h i s was shown t o be

typical ly non-depolarising and non-conpc t i t ive . In in t ac t m iads pipcrazine was shown t o have

profound ::ffccts on tho cen t ra l nervous systen. I n chicks

it was shown tha t the ncurornuscular wcakness produced by

pipcrazine was non-dcpolarising.

Piperazinc increzscd both thc sleeping t ine under

pentobarbitone sodiun and the convulsive a c t i v i t y , o~f

l cp tazo l 'and strychnine i n nice. The t i n e t o onset 01

slccp was a l so rcduccd.

It is suggostcd t h a t i n thc ccn t ra l nervous system

piperazinc nost probably a c t s by zntagonising the neuro-

act ive aninds and tha t fur ther work i s necessary t o ova-

lua tc t h s e f fcc t c.f piper9,zinc on these mines ,

99

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9