EUROPEAN JOURNAL OF PHARMACOLOGY 10 (1970) 83-86.

NORTH-HOLLAND PUBLISHING COMPANY

A SIMPLE SCREENING METHOD FOR ANTIPARKINSONIAN D R U G S IN M I C E

Takashi NOSE and Michio KOJ1MA

Department o f Pharmacology, Research and Development Division,
Tanabe Seiyaku Co., Ltd., Osaka, Japan

Received 17 October 1969 Accepted 9 January 1970

T. NOSE and M. KOJIMA, A simple screening method for antiparkinsonian drugs in mice, European J. Pharmacol.l 0
(1970) 83-86.
A number of antiparkinsonian and miscellaneous drugs were examined for their ability to protect mice from
physostigmine induced death and tremorine, oxotremorine and nicotine induced tremors. Typical antiparkinsonian
drugs, such as centrally acting anticholinergics (hyoscine, atropine, benztropine, trihexyphenidyi, procyclidine,
biperiden and ethopropazine) and antihistamines (promethazine and diphenhydramine) were effective against
physostigmine induced death, whereas other drugs, ineffective in parkinsonian therapy, were ineffective. The use of
the physostigmine induced death method in preliminary screening for antiparkinsonian drugs in mice was more
specific than either the tremorine or oxotremorine tremor method.

Antiparkinsonian drugs Centrally acting anticholinergics

Physostigmine induced death Tremorine tremor
Oxotremorine tremor Nicotine tremor
Screening method

1. INTRODUCTION It therefore seemed reasonable to expect that

Screening tests for antiparkinsonian drugs general-
ly involve observations on one of the following :
tremor induced by tremorogenic agents, catalepsy
induced by neuroleptics, electroencephalogram arous-
al response induced by cholinergic drugs or electrical
stimulation in the midbrain reticular formation and
parkinson-like postural tremor and rigidity induced
by experimental lesion or electrical stimulation in the
midbrain reticular formation (Friedman and Everett,
1964).
Tremorine has been widely used for the evaluation
of antiparkinsonian drugs since its introduction by
Everett in 1956. A positive correlation between the
antiacetylcholine and antitremorine potencies of anti-
parkinsonian drugs was reported by Ahmed and
Marshall (1962). There is direct and mutual antagon
ism between physostigmine and centrally active anti-
cholinergic drugs on the clinical manifestations of
parkinsonism (Duvoisin, 1967).
antiparkinsonian drugs would be specific central anta-
gonists of physostigmine. In the present experiments,
the ability of drugs to protect mice from physostig-
mine induced death was examined as a method for
screening antiparkinsonian drugs. The specificity of
the method for antiparkinsonian drugs was compared
with that of other methods utilizing tremorine, oxo-
tremorine or nicotine induced tremor.
2. METHODS
All experiments were performed on male albino
mice weighing from 18 to 22 g. Graded doses of test
drugs were administered intraperitoneally to groups
of 5 mice 30 min before an intraperitoneal injection
of physostigmine or tremorogenic agents, with the
exception of nialamide and tetrabenazine (60 min
before), and ct-methyldopa (240 min before). In gen-
eral the largest dose of each drug injected was more
84 T.Nose, M.Kojima, Screening method/or antiparkinsonian drugs

than half of the acute LDso. The dose which protect-
ed 50% of the mice from physostigmine induced
death or tremorogenic agent induced tremor was
calculated by the method of Weil (1952).
2.1. Antagonism of physostigmine induced death
A dose of 2 mg/kg of physostigmine caused tremor
with salivation and chronic convulsions, and death for
all mice within 20 min after administration. The
antiphysostigmine activity of each drug expressed as
an EDso was determined from the number of mice
which survived more than 60 rain.
2.2. Antagonism of tremorine, oxotremorine and
nicotine induced tremors
Antitremor potency was determined from the
protection given by the drug from tremor induced by
tremorine (15 mg/kg), oxotremorine (0.5 mg/kg) or
nicotine bitartrate (10 mg/kg); at these doses the
drugs induced tremor without death in all mice. The
tremor induced by tremorine or oxotremorine in the
head and limbs was accompanied by parasympathetic
stimulation such as salivation, miosis and diarrhoea
which lasted for more than 60 min. The time of onset
and peak effect of oxotremorine occurred earlier than
that of tremorine. The tremor induced by nicotine in
the head and limbs occurred within 3 to 5 min and
was followed by convulsion. The duration of nicotine
induced tremor was shorter than that of tremorine or
oxotremorine. EDso values of drugs were calculated
from numbers of mice which did not show any
tremor within 60 min after administration of tremor-
ogenic agents.
2.3. Drugs
The following test drugs were used: Atropine
sulfate, hyoscine hydrobromide, benztropine mesyl-
ate, trihexyphenidyl hydrochloride, procyclidine
hydrochloride, biperiden hydrochloride, ethopropa-
zine hydrochloride, promethazine hydrochloride, di-
phenhydramine hydrochloride, N-butyl hyoscine
bromide, pentobarbitone sodium, chlorpromazine
hydrochloride, imipramine hydrochloride, caffeine
hydrochloride, methamphetamine hydrochloride,
nialamide, dl-dopa, 5-hydroxytryptophan, hexa-
methonium chloride, d-tubocurarine chloride, diben-
amine hydrochloride, dichloroisoproterenol and
a-methyldopa (dissolved in saline); phenobarbitone,
tetrabenazine, meprobemate and diphenylhydantoin
(suspended in 0.5 percent carboxymethylcellulose
solution).
3. RESULTS
3.1. Antagonism of physostigmine induced death
EDso values of antiparkinsonian and miscellan-
eous drugs against physostigmine induced death are
given in table 1.
Low doses of centrally acting anticholinergics and
antihistamines protected mice from physostigmine
induced death. Benztropine and trihexyphenidyl were

Potency in Physosflgmine Method Drug Potency in Tremorine Method

[ Atropine
[ Hyoscine ]
I Benztropine J
I Trihexyphenidyl I
I Procyclidine
[ Biperiden
Ethopropozine ]
Promethozine J
Diphenhydromine i
Chlorpromozine ]
Imipromine --1
Nialamide J
I 0.1 0.01 log. 0.01 0.1 I
Fig. 1. Effectiveness of drugs in antagonizing physostigmine induced death and tremorine induced tremor in mice. Each bar
indicates the value calculated from 1/ED5o.
 T.Nose, M.Ko]ima, Screening method for antiparkinsonian drugs 85

Table 1
The activity o f various drugs in protecting mice from physostigmine induced death
and tremorine, oxotremorine and nicotine induced tremors.

EDso b of b
Pretreated antilethal ED5o o f antitremor activity
Drug a before activity
(min) against
vs. Tremorine vs. Oxotremorine vs. Nicotine
physostigmine

Atropine sulfate 30 1.9 mg/kg 1.4 mg/kg 1.3 m g / k g - (up to 160) c mg/kg
Hyoscine HBr 30 0.6 0.5 0.2 - (up to 100) c
Benztropine mesylate 30 3.5 1.5 2.7 - (up to 32) c
Trihexyphenidyl HC1 30 5.5 1.5 10.7 60% (80) d
Procyclidine HC1 30 9.3 2.7 28.2 20% (80) d
Biperiden HC1 30 8.9 2.7 13.0 20% (50) d
Ethopropazine HCI 30 24.6 4.7 21.4 26.0
Promethazine HCI 30 24.6 5.4 14.2 - (up to 50) c
Diphenhydramine HCI 30 40.7 10.7 - (up to 50) c 40% (50) d
N-butyl hyoscine bromide 30 - (up to 50) c 40% (80) d - (up to 50) c - (up to 50) c
Pentobarbitone-Na 30 - (up to 40) c - (up to 40) c - (up to 40) c 21.6
Phenobarbitone 30 - (up to 100) c 40% (100) d - (up to 100) c 19.2
Tetrabenazine 60 - (up to 40) c 7.7 - (up to 40) c - (up to 40) c
Chlorpromazine HC1 30 - (up to 100) c 2.3 20% (64) d 6.9
Imipramine HC1 30 - (up to 100) c 49.3 20% (80) d 20.8
Meprobamate 30 - (up to 320) c _ (up to 400) c _ (up to 500) c 30.8
Diphenylhydantoin 30 - (up to 400) c _ (up to 800) c _ (up to 400) c - (up to 200) c
Caffeine 30 - (up to 160) c _ (up to 160) c _ (up to 160) c - (up to 160) c
MethamphetamineHCl 30 -(uptol0) C -(upto40) c -(uptol0) C -(uptol0) C
Nialamide 60 - (up to 320) c 32.5 - (up to 300) c - (up to 300) c
d/-Dopa 30 - (up to 200) c _ (up to 200) c _ (up to 200) c - (up to 200) c
5-Hydroxytryptophan 30 - (up to 40) c - (up to 40) c - (up to 40) c - (up to 40) c
H e x a m e t h o n i u m chloride 30 20% (40) d - (up to 40) c - (up to 40) c 2.2
d-Tubocurarine chloride 30 - (up to 0.5) c - (up to 0.5) c - (up to 0.5) c 20% (0.5) d
Dibenamine HCI 30 - (up to 200) c _ (up to 200) c _ (up to 200) c - (up to 200) c
Dichloroisoproterenol 30 - (up to 80) c 60% (100) d - (up to 100) c 40% (100) d
t~-Methyldopa 240 - (up to 400) c _ (up to 400) c _ (up to 400) c 50% (500) d

a Drug was injected intraperitoneally.

b Dose giving protection in 50% o f mice (mg/kg). After pretreated with drug, mice received intraperitoneally 2 m g / k g o f physos-
tigmine, 15 mg/kg o f tremorine, 0.5 mg/kg of oxotremorine or 10 mg/kg o f nicotine.
c No effect with m a x i m u m dose shown in parentheses.
d Percentage of mice protected against tremorogenic agents by dose shown in parentheses.

the most effective of the synthetic antiparkinsonian 3.2. Antagonism o f tremorine, oxotremorine and
d r u g s a l t h o u g h t h e s e w e r e less e f f e c t i v e t h a n h y o s c i n e nicotine induced tremors
or atropine. Diphenhydramine and ethopropazine, EDso values of antiparkinsonian and miscellan-
u s e f u l in p a r k i n s o n i a n t h e r a p y , w e r e also e f f e c t i v e . eous drugs against tremorine, oxotremorine and
High doses of hexamethonium afforded some protec- n i c o t i n e i n d u c e d t r e m o r s a r e a l s o g i v e n in t a b l e 1.
tion but high doses of the other drugs, including Low doses of centrally acting anticholinergics and
N-butyl hyoscine were inactive. antihistamines antagonized tremor induced by trem-
86 T.Nose, M.Kofima, Screening method ]or antiparkinsonian drugs
orine or oxotremorine. Drugs which antagonized oxo-
tremorine induced tremor also antagonized tremor
induced by tremorine. Tetrabenazine, chlorproma-
zine, imipramine and nialamide also showed some
activity against tremorine or oxotremorine induced
tremor. However, tremor induced by nicotine was not
antagonized by the typical antiparkinsonian drugs,
but was antagonized by miscellaneous drugs such as
phenobarbitone, chlorpromazine, imipramine, etc.
The results obtained for the physostigmine in-
duced death method were similar to those obtained
for the tremorine and oxotremorine induced tremor
methods, except for tetrabenazine, chlorpromazine,
imipramine and nialamide. Fig. 1 shows a histogram
o f the potency o f several drugs in the physostigmine
induced death and tremorine induced tremor meth-
ods. There was a good correlation between the anti-
physostigmine and antitremorine potencies of anti-
parkinsonian drugs.
4. DISCUSSION
There have been previous reports on the use of
physostigmine in screening for antiparkinsonian
drugs: these include the ability of drugs to prevent
physostigmine induced parkinson-like tremor in the
dog (Foucon et al., 1965), physostigmine induced
shivering in the rat (Dandiya and Bhargava, 1968) and
sinistrotorsion in the guinea-pig after an intracarotid
injection of physostigmine (De Jonge and Funcke,
1962). These methods are unsuitable for preliminary
screening tests since they are either too costly or
demand too much time. In the present experiments,
physostigmine induced death was the parameter
measured since, in mice, the tremor was not distinct.
The present results showed that centrally acting
anticholinergics and antihistamines which are useful in
parkinsonian therapy were potent in protecting mice
from physostigmine induced death. However the
tremorine and oxotremorine induced tremor methods
were less specific since some neuroleptics and thymo-
leptics such as tetrabenazine, chlorpromazine, imi-
pramine and nialamide were also active. These results
have been comfirmed previously (Ahmed and Mar-
shall, 1962 ; Everett, 1964 ; Dandiya and Bhargava,
1968 ; Leslie, 1969).
The activities of the drugs in antagonizing physos-
tigmine and tremorine or oxotremorine were similar
and in order of decreasing potency they are : hyos-
cine, atropine, benztropine, trihexyphenidyl, bi-
periden, procyclidine, ethopropazine, promethazine
and diphenhydramine. This order appears to be corre-
lated with the maximum human therapeutic dosage
of antiparkinsonian drugs quoted by Ahmed and
Marshall (1962). The present results also confirmed a
previous report (Cahen and Lynes, 1951) that the
nicotine induced tremor method is not suitable for
the screening of antiparkinsonian drugs because of a
lack of specificity.
In conclusion, tire present physostigmine induced
death method was found to be a convenient method
with high specificity for the preliminary screening of
antiparkinsonian drugs in mice.
ACKNOWLEDGEMENTS
We gratefully acknowledge the technical assistance of Mr.
M. Yamamura. We are also very grateful to Dr. Y. Kowa and
Dr. G. Hayashi for their constant encouragement and valua-
ble suggestions throughout this work.
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