-286- THE JOURNAL OF CH1BA MEDICAL SOCIETY 41 1. Vo

CHEMOTHERAPY OF CLONORCHIASIS SINENSIS

1: Therapeutic e旺� ectof 1， 4-bis-trichloromethylbenzol in animals

experimental1y infected with Clonorchis sinensis.

MUNEO YOKOGAWA， HIROTAKA KOYAMA， HIROYUKI YOSHIMURA

and CHAO SIUNG TSAI

Department of Parasitology， School of Medicine， ChibαUniversiか"� Chiba， Japan

(Received for ρublication， Seρtember 18， 1965)

Introduction

Various drugs thus far tried against clonorchiasis sinensis such as emetine hydrochloride，

antimony preparations， chloroquine diphosphate and dithiazanine iodide， have fai!ed to

show promise in c1inical trials.

Lammler (1960 to 1964)(4-7) has recently reported that 1， 4-bis-trichloromethylbenzol

(Hetol) and beta， beta， beta， tris-( 4-chlorophenyl-propionate-4' -methyl-piperazid・hydro-

chloride (Hetolin) are both e旺� ectiveagainst the liver flukes of cattle and sheep， Fasciola

hepatica and Dicrocoelium dendriticum.

1n this study the authors conducted the first use of 1，4-bis-trichloromethylbenzol (Hetol)

for the treatment of animals experimentally infected with Clonorchis sinensis and obtained

highly satisfactory results.

1n the toxicity tests no side effects at all with the usual therapeutic dosage were observed.

Following on the animal experiments， the treatment of human c1onorchiasis sinensis was

also carried out and has turned out to be satisfactory. 1n this paper the authors present

first the results of the animal experiments.

Materials and Method

Metacercariae of Clonorchis sinensis were isolated from the freshwater fish， Pseudorasbora

ρarva， the second intermediate host of Clonorchis sinensis， from a breeding pond at Kita-

kawabe village， Kita-saitama county， Saitama prefecture， by the artificial digestion

technique.

The animals used in the experiments were 3 dogs， 8 rabbits， and 20 rats.

The drug used was 1， 4-bis-trichlorome-

thylbenzol supplied from Hoechst Co. 1ts

l守)-cイcbc1. structural formula Is shown in Fig. 1t is a colorless and odorless powder insoluble in water. The drug has already been placed

on the market by Hoechst Co.， in Germany Fig. 1. Chemical structure of 1， 4-bis-

for the trichloromethylbenzol

"，Hetol“under the trade name

treatment of liver flukes of cattle and sheep， Fasciola hφatica and Dicrocoelium dendri-

ticum.

The dosage in the dog was 150 mg/kg or 200 mg/kg every other day for 5 doses. 1n the

No.3 YOKOGAWA， KOYAMA， YOSHIMURA and TSAI: -287-CHEMOTHERAPY OF CLONORCHIASIS SINENSIS

rabbit， 50mg/kg， 75mg/kg， 100mg/kg， 200mg/kg， or 300mgjkg was given every other

day for 5 doses. The rats were divided in 6 dosages groups 50mgjkg every day for 5

doses， 75mgjkg every day for 5 doses， 75mg/kg every other day for 5 doses， 100mgjkg

every other day for 5 doses， 200mgjkg every other day for 5 doses， and 300mg/kg every

other day for 5 doses. The" administration of the daily dose in "these animals was by

direct oral administration once a day.

The treatment was begun as a rule about 40 days after the infection.

The number of eggs per gram of feces (E. P. G.) was calculated by a modification of

Stoll's egg counting method. If the egg number became too small to be determined by

Stoll's method， all the eggs in 0.5gm of feces were counted by AMS III centrifugation

technique and the E. P. G. was calculated from this.

The modification of Stol1 's method employed was as follows. The smal1 -sized fiask

graduated with 28cc and 30cc was used. Up to the graduation of 28cc， 1/10N NaOH was

first placed， followed by feces up to the graduation of 30cc. Then the coIi tents were well

shaken and mixed with glass baIrs. The number of the eggs in 0.15cc of the mixture

was counted under microscope. The resultant number was multiplied by 100. The value

so obtained was eggs per ml， but， for convenience's sake， it was dealt with as eggs per

gram feces (E. P. G.).

Evaluatiou" of the drug effect

E. P. G. was counted every 3 or 4 days during the treatment and once a week after the

completion of treatment until autopsy. In the autopsy of the animal the contents of the

gall bladder were examined宣� rst. The liver tissue along the bile duct was cut open by

scissors to find worms. Then， the sliced liver tissues were pressed between two slide

glasses and examined for worms under the dissecting microscope.

All the worms found were examined in Tyrode's solution warmed at 370C for motility，

degeneration， atrophy， etc. tinder the dissecting microscope. Subsequently， carmine or

haematoxylin staining was perfonned in the routine manner.

For the toxicity test， 50mg/kg， 100mg/kg， 200mg/kg， 300mg/kg， and 400mgjkg of

Hetol were given -every other day for 5 doses in mice; they were then autopsied for

macroscopic and histo-pathologic examinations of the main internal organs at 7 to 10 days

after the administrations.

Results

1. Toxicity of Hetol in mice.

As shown in Table 1 none of the animals in the groups given 50mg/kg and 100mgjkg，

respectively， of Hetol every other day for 5 doses， showed any pathological changes. In

the group given 200mg/kg a moderate degree of weight loss was observed in 2 of 4 mice.

In one of these two mice a mild fatty degeneration in the liver was observed.

In the groups given 300 mg/kg and 400mg/kg， respectively， a marked fatty degeneration

was observed in the liver. In many of the animals in these groups， weight loss was

significant.

1. Results of the treatment with Heto 2.

(a) Effect in dogs infected with Clonorchis sinensis:

Dog No. 1， female， was treated with Hetol 100mg/kg every other day for n doses from

THE ]OURNAL OF CHIBA MEDICAL SOCIETY

Table 1. Toxicity-test of Hetol in mice

-288 41 1.Vo

Dose Pathological changesBody weight (gm)

ωω-aω

ロ ωロ』

ω#υ

入門

Group mgjkg p.o・� increase or decrease

every other day after treat.

k内・

M

何〉。

ωロ℃万一日

'ωω ω何一担 ロコ

』

ω〉コ

切ロロ-

ω口同一一ヤロ叩ω』

Aωω

明ハザH

I 50 mgjkgx5

II 100mgjkgx5

III 200mgjkgx5

-0.6 -0.5 +0.1

-2.0 -3.5 -2.8

O

-1.9 -1.9 -4.7 -3.8

十�

十 +

+ -+ 十持十十時一�

+ 十

十十

IV 300mgjkgx5 +1.0 + + -3.0 +ト料 一 十 一+

一ー十

持十�

+

-0.7 十 十十

400mgjkgx5 -5.9 件糊+ 十� 件十十+持+十州-9.3

V

-6.4 +十時 一 件

Control -2.3 +0.8 十� +0.9 -2.0 + +0.8

0 +0.5 +1.3

長.+: slight congestion *・持 .十十:� moderate fatty degeneration

持一+

the 43rd day after infection with 100 metacercariae of Clonorchis sinensis.

As shown in Table 2，the E.P. G.，which averaged 450 before treatment，showed a marked

lncrease to 1，800 at the 4th day of treatment. At the 7th day， thg E. P. G. radically

decreased to 600，and at the 14th day to 30. At the 21st day after the :first day of treat-

ment the E. P. G. became zero. At the 28th day，it was found to be 20，but no eggs were

found at the 35th and 42nd day. This dog was autopsied at the 48th day after the :first

day of treatment. Only 6 dead worms，markedly atrophied and deformed，were found in

the gal1 bladder. A marked degeneration and disintegration were noticed in the posterior

half of the dead worms，but a large number of eggs was found within their uteri.

The body weight of the dog before treatment and before autopsy was 8.3 and 7.5kg，

respectively.

Dog No. 2，male，was given Hetol 200 mgjkg every other day for 5 doses from the 40th

day after infection.

The E. P. G. averaged 800 before treatment，increased to 1，100 at the 4th day of treat-

ment and to 1，900 at the 7th day. The E. P. G. thereafter rapidly decreased to 700 at the

14th day and became zero at the 21st day.

Thirty-four dead worms，presumably not long dead，were recovered from the bile ducts

at autopsy. The uteri of these dead worms were also :filled with a large number of eggs.

On the surface of the liver of the dog the several greyish white nodules as granulomas

caused by the disintegrated worms were observed.

No.3 YOKOGA W A， KOYAMA， YOSHIMURA and TSAI: -289-CHEMOTHERAPY OF CLONORCHIASIS SINENSIS

Table 2. Results of treatment with Hetol in dogs experimentally

infected with Clonorchis sinensis.

Results of stool examinations with Stoll'~ method or AMS III centrifugation method.

Eggs Per Gram Feces (EPG) No. of meta- Doses of Hetol Total-0Z

cercariae of mg/kg p. o. doses

-dHωハ� .げ

� -ωω』八宇�

ω』。ぃ志向

Days after the first day of treatment

Clonorchis

切。口

sznenszs glven every other day mg/kg口同 4 7 14 21 28

。35

。42

600 30 0 201 100 5x100 500 450 1，800

2 100 5x200 1，000 1，1� 00800 1，900 700 0

3 900 5x200 1，000 4，700 7，400 2，600 100 0 0 0 0 15持� 1/ 2，300持� 2，400骨� 900持� 200特� 100持� 70持� 85長� 45持

Results of Autopsy

0

No. worms recovered at autopsy living dead

Days after the beginning of treatment Dog No.

6。1 48

2 0 34

f-4

22 3 0 A

VAU

48

e m nr w

2持� ina worm 、、� /tpo

、、，ノーTcyst irL m

ey

qu

ιEw

QMvd

業� Pαrα:gonimusωesteγmαni

The body weight of this dog before treatment and before autopsy was 7.1 and 7.9kg，

respectively.

Dog No. 3， female， was infected with 900 metacercariae of Clonorchis sinensis and 15

metacercariae of Rαragonimus westerl1ωni.

200 mg/kg of Hetol was given every other day for 5 doses. The treatment was begun

at 132 days after the infection with Ctonorchis sinensis and at 143 days after the infection

with Parαgonimus westermani. The E. P. G. of Clonorchis sinensis， averaged 4，700 before

treatment， rose at the 4th day to 7，400， with subsequent decreases to 2，600 at the 7th day

and 100 at the 14th day. Thereafter in the examinations for eggs at the 21st， 28th， 35th，

and 42nd day， no eggs at al1 were discovered. No worms were found at the autopsy at

the 48th day. The body weight which before treatment was 5.3 kg rose considerably to

7.5kg at the autopsy. The E. P. G. of Paragonimus weste1'mani was， as shown in Table 2，

2，300 before treatment， and decreased markedly to 900 at the 7th and down to 200 and

100 at the 14th day and 21st day， respectively. At the autopsy 7 worm cysts of Parago・�

nimus westermani were found 'in the lungs. One cyst at the upper part of the right lung

contained 2 living worms and in the remaining 6 worm cysts no worms were found but

only Rαragonimus eggs and necrotic masses.

(b) E妊� ectin rabbits infected with Clonorchis sinensis:

Eight rabbits weighing 3 kg or so (2 of them were controls) were each infected with

100 metacercariae of Clonorchis sinensis.

Rabbit No. 1 was treated with 50 mg/kg of Hetol every other day for 5 doses from 54

days after infection. The E. P. G. is shown in Table 3. It averaged 360 before treatment，

increased to 950 at the 4th day of treatment， but it decreased to 400 at the 7th day.

Increase was again observed at the 35th day to 360， which was nearly equal to the E.P.G.

before treatment. Thirty living worms with active motility were found in the bile ducts

4

-290- THE JOURNAL OF CHIBA MEDICAL SOCIETY

Table 3. Results of treatment with Hetol in rabbits experimentally

infected with Clonorchis sinensis.

41 1. Vo

Each rabbit infected with. 100 metacercariae of

Clonorchis sinensis.

。. 4Lls

g

aEggs Per Gram Feces (EPG) No. of worms

lV6L

rTLALV

-Ha2

ot-

a

L

H

ふ�

唱白丘� ωロ

�

日�

‘τ

d

m

o

α

g

Sdf/

Kevery other day

『�

Doses of Hetol zzssdu.

1 5x 50 250 360 950 400 530 150 140 360

出�

recovered at FTAo

autopsy・制局

ω』一戸�

ω』。一志向

vdquDa

JU y

a

mgjkg p. o. rr e

一戸口�

74 28 35 living dead

ιιa

ht4

em i

唱�

。。

。

唱�

可

止

内

�

qOi

qtU2 5x 75 375 300 1，600 440 160 40 40 25

3 5x 75 255 390 440 750 60 30 140 200噌� LiA

uハυ

ハ�

5x100 500 270 800 740 45 2

5x200 1，000 340 740 200 140 UA

U

。� 2。5x300 1，500 200 360 340 85 256 Control

。235270 400 3002602602007

303004608 820 600

and one living worm in the gall bladder， thus a total of 31 living worms were detected

at autopsy.

Rabbit No. 2 was given Hetol 75 mgjkg every other day for 5 doses from 54 days after

infection. The E. P. G. averaged 300 before treatment and increased remarkably to 1，600

at the 4th day of treatment. But it decreased gradual1 y to 440 at the 7th day， 160 at the

14th day， ahd 40 at the 21st day. At the 35th day， the E. P. G. was 25. Thirteen worms

exhibiting considerable deformation and atrophy were found in the bile ducts at the

autopsy. Many of the worms showed swollen， degenerated， or necrotic changes in the

posterior half part of the bodies. However， the anterior half of the bodies still showed

some slight motility and the worms were judged to be alive.

Rabbit No. 3 was also given 75 mgjkg of Hetol every other day for 5 doses. The E.P.G.

averaged 390 before treatment， increased to 440 and 750 at the 4th day and 7th day，

respectively. But， it decreased thereafter down to 60 at the 14th day and 30 at the 21st

day. The E. P. G. subsequently rose again to 140 at the 28th day and 200 at the 35th

day. Twenty-one worms were recovered from the bile ducts at autopsy. Nineteen of them

were alive and showed active motility. Only the two remaining worms were found to be

in the course of atrophy and disintegration.

Rabbit No. 4 was given 100 mgjkg of Hetol every other day for 5 doses from the 54th

day after infection. The E. P. G. averaged 270 before treatment， rose to 800 and 740 at

the 4th and 7th day， respectively. The E. P. G. decreased to 45 at the 14th day and it

became zero at the 21st day. Only 2 dead worms were found in the bile ducts at the

autopsy.

Rabbit No. 5 was given 200 mgjkg of Hetol every other day for 5 doses from 54 days

after infection. The E. P. G. averaged 340 before treatment， showed a temporary elevation

to 740 at the 4th day and subsequently decreased to 300 at the 7th day and 140 at the

14th day. The E. P. G. became zero at the 21st day.

Only two dead worms were found in the bile ducts in the liver at the autopsy.

Rabbit No. 6 was given 300 mgjkg of Hetol every other day for 5 doses from the 54th

No.3 YOKOGA W A， KOY AMA， YOSHIMUR.t¥ and TSAI: -291-CHEMOTHERAPY OF CLONORCHIASIS SINENSIS

day after infection. The E. P. G. averaged 200 before treatment， increased a litt1 e to 360

at the 4th day， but it subsequently decreased to 340 at the 7th day and 85 at the 14th

day. The E. P. G. became zero at the 18th day. Only two dead worms， considerably

disintegrated， were found in the gall bladder at autopsy.

Control rabbits No. 7 and No. 8 were examined for E. P. G. in parallel to the treated

rabbits from 30 days after infection. The E. P. G. in the control rabbits did not show

such a remarkable rise or faU as observed in the treated group. At the autopsy 35 living

worms and 2 dead worms were found in rabbit No. 7 and 30 living worms in No. 8.

(c) Effect in rats infected with Clonorchis sinensis:

Each of 20 rats was given 30 metacercariae of Clonorchis sinensis， and excluding two

rats as controls， they were divided in 6 groups as follows. Group 1 consisting of 2 rats

given 50 mg/kg Hetol every day for 5 doses: group II consisting of 2 rats given 75 mg/kg

Hetol every day for 5 doses: gorup III consisting of 2 rats given 75 mg/kg Hetol every

other day for 5 doses: group IV consisting of 4 rats given 100 mg/kg Hetol every other

day for 5 doses: group V consisting of 4 rats given 200 mg/kg Hetol every other day for 5

doses: and group VI consisting of 4 rats given 300 m/kg Hetol every other day for 5 doses.

Treatment was begun 51 to 54 days after infection， and all the animals were autopsied

at from the 22nd to 23rd day after the first day of treatment.

Results of treatment of the 2 rats， No. 1 and No. 2， of the group 1 are shown in Table

4. In rat No. 1 the E. P. G. averaged 1，800 defore treatment and increased to 11，500 at

the 4th day. But， it decreased to 1，200 at the 7th day. No eggs were found in the feces

at the 14th day and thereafter. No worms were found at the autopsy. The E. P. G. in

rat No. 2 was 1，300 before treatment， increased to 2，100 at the 4th day， and subsequently

decreased rapidly to 300 at the 7th day and 70 at the 14th day. The E. P. G. at the 21st

day was 60. Only one living worm， markedly atrophied and degenerated， was recovered

from the bile duct at the autopsy.

In rat No. 3 of the group II， the E. P. G. wa. s 4，050 before treatme'n t， and 3，400， 2，300， and

400 at the 4th， 7th and 14th day， respectively. The E. P. G. became zero at the 21st day.

In rat No. 4 E. P. G.， 2，700 before treatment， markedly increased to 20，000 at the 4th day

and 7，000 at the 7th day. But， it decreased to 500 at the 14th day and became zero at

the 21st day. Only one dead worm was found in the bile duct of No. 3 rat and no worm

was found from No. 4 rat at the autopsy.

E. P. G. of rat No. 5 and rat No. 6 of group III are shown in the same table. The

E. P. G. in rat No. 5 before treatment was 3，600 and following the treatment it showed

temporary increases， i. e. to 7，000 at the 4th day and 5，900 at the 7th day. The E. P. G.

at the 14th day showed decrease to 1，000， but at 21st day it was still 600. In rat No. 5 four

living worms were found in the bile ducts， 2 of them were normal， while the remaining

2 showed marked degeneration and atrophy in the posterior half of the body. In rat No.

6， the E. P. G. before treatment was 2，200 and at the 4th， 7th and 14th day it was 3，4� 00，

1，800， and 2，800， respectively， and it was zero at the 21st day. No worms were found at

autopsy.

In rats No. 7， No. ~， No. 9， and No. 10 of group V， the E. P. G. before treatment was

5，400， 6，500， 2，000， and 2，900， respectively. In rat No. 7， the E. P. G. at the 4th day showed

a remarkable rise to 50，000， but at the 7th day it was 20，400 subsequently with rapid and

marked decrease， down to 900 at the 14th day and to zero at the 21st day. Near1 y the

I

II

III

IV

V

VI

l'.:l c.!) l'.:l

Table 4， Results of treatment with Hetol in rats experimentally infected with 30 metacercariae of Clonorchis sinensis.

。。dead

Eggs Per Gram Feces (EPG) Days from the No. of worms recovered Rat Total doses beginning of at autopsy

inLqoa

唱

吐

Dose mgjkg p. o.Group No. mgjkg Before treatment to 同，出何一『�

同

CCzkrF

livingtreatment 4 7 14 21 autopsy

。。 5x50 daily 250 1，800 11，500 1，200 0 0 22 0

60 1持。。 1，300 2，100 300 2270// //

ハ

ハ�

ハ�

ハ

凸�

1inu

UAU

UAUAUAU

UAUAUAU

UAU

υAU�

5x75 daily 375 4，050 3，400 2，300 400 23

20，000 7，000 23500// // 2，700

5x75 every other day 4持600 0

375 22 22

1，0005，9007，0003，600 。3，4� 00 1，800

可�

5 6

。司わ出回国� 冨開� わ〉円、印。わ� 同，� 円�

KF

U

H

HMW

J

2，200 2，800// //

20，4� 00 AUAUAυnv

5x 100 every other day 500 5，400 50，000

内�

つ�

2222

2222 2222 2222

qhqrM

LqL

qdqd

dqa

900 AUAUかり 6，500 13，000 9，300 0

かか � 2，000 ハリハ

ハ�

ハ�

UAUAUAU

UAυAUAU

噌�

// // 2，900

400nvnuE4 149-QUA

i141

FhuRU

吐�

咽�

み

唱

1-

υ

ハ�

AUAUAUAU

UAUAUAU

5x200 every other day 1，000 5，800 5，000 1，600 1，500

// // 4，100 26，000 5，000 14

// // 1，550

// // 2，800 。1，500 10，700 22，000 1，100 。5，100 24，000 1，100

晶1

5x300 every other day

か りE

A--1

Control 。19 5，700 15，000 26，100 12，500 9，100 23 11 。20 10，600 13，000 19，4� 00 8，000 10，900 23 12

特� 2of 4: degenerated

円

400ム

�

か か 2，900

かか 4，650

〈。� --B

No.3 YOKOGAWA， KOYAMA， YOSH1MURA and TSA1: CHEMOTHERAPY OF CLONORCH1AS1S S1NENS1S 一� 293-

same tendency as the above was observed in rat No. 8. 1n rats No. 9 and No. 10 whose

E. P. G. during treatment was not determined， E. P. G. was zero at the 21st day. No

worms were found in any of them at the autopsy. The only findings at the autopsy were

that the lumina of the bile ducts were markedly enlarged and their walls were much

thickened.

1n rats No. 11， No. 12 No. 13， and No. 140f group V， the E. P. G. before treatment was

5，800，4，100，1，500， and 2，800， respectively. 1n rat No. 11，it was found to decrease gradually，

being 5，000 at the 4th day， 1，600 at the 7th day， and 1，500 at the 14th day; and it became

zero at the 21st day. 1n rat No. 12， the E. P. G. showed a conspicuous rise to 26，000 at

the 4th day， but decreased to 5，000 at the 7th day and 14 at the 14th day. E.P.G. became

zero at the 21st day. 1n rat No， 13 and rat No. 14， the E. P. G. also became zero at the

21st day. No worms were found in any of them at autopsy.

1n rats No. 15， No. 16， No. 17， and No. 18 of group V1， the E. P. G. before treatment

was 10，700 5，100， 2，900， and 4，650， respectively. The E. P. G. in rat No. 15 increased to

22，000 at the 4th day which was about twice as much as the count before treatment. Sub-

sequently， it dropped radicalIy， down 1，100 at the 7th day and zero at the 14th day. 1n

rats No. 16， No. 17 and No. 18 the E. P. G. became zero at the 14th day as in rat No. 15.

No worms were found in any of the rats at autopsy. The only findings were remarkably

enlarged bi1e duct lumen and the thickening of its walls.

E. P. G. of the non-treated control rats of No. 19 and No. 20 were determined at the

same time as done in the treated groups. The fiuctuations in E. P. G. as shown in Table

4 were fairly' notable， but no tendency to decrease was observed in the E. P. G. At the

autopsy 11 and 12 living worms were found from the bile ducts in rats No. 19 and No. 20

respecti vely.

Discussion

Hetol (1，4-bis胴� trichloromethylbenzol)，� one of the therapeutic agents synthesized in the

Laboratory of Parasitology of Hoechst Co.， Germany， against Fasciola he， ρatica has been

fully examined for its e百� ectand toxicity by Behrens (1960)ω， Enigk and Duwel (1960)ω，

Kamel et al. (1960)く� 3)，� and .Lammler (1960)ω.

Other known therapeutic agents against Fasciola heραtica are hexachlorophen， hexachlo-

rethane， bithionol， etc. Lゑ� mmler (1964)ω-ωreported that these agents， other than Hetol，

were entirely ine百� ectiveagainst Dicrocoeliunz dendriticum 'which is also a fiuke parasitizing

the liver of the hosts.

The present authors were interested in the fact that bithionol which was highly e妊� ective

against Fasciola hφαtica and Paragonimus westermani， was ineffective against Clonorchis

sinensis unless a very high dose was used. On the use of Hetol against Clonorchis sinensis，

Lammler (1964)(7) has recently reported that it was found e百� ectivein cats infected with

opisthorchis felineus. 1n the Soviet Union， Plotnikovand Yoldigina (1962)C9) found Hexa四�

chloroparaxylol， which has a similar structure to Hetol， e旺� ectiveto some extent when

administered at doses of 100 to 1，000 mgjkg to cats infected with 0ρisthorchis sp. These

reports， however， are available here only in abstract， no original reports having yet been

received. No detai1s of the aboye studies are known here yet.

1ndependently from the above nientioned studies， the present authors tried Hetol in dogs，

rabbits， and rats experimentally infected with Clonorchis sinensis， and have obtained very

41 1. Vo-294- THE JOURNAL OF CHIBA MEDICAL SOCIETY

promising results as described in this paper.

The dogs， rabbits， and rats in the groups given 100 mgjkg every other day for 5 doses

for a lotal of 500 mgjkg all exhibited complete cure. As for the minimal e百� ectivedosage，

the administration of 50 mgjkg every other day for 5 doses (totaling 250 mgjkg) in the

rabbits was entirely ineffective. In two rabbits given 75 mgjkg every other day for 5 doses

(totaling 375 mgjkg)， a temporary increase in E. P. G. followed by a gradual decrease in

E. P. G. was observed but at the autopsy， 13 living worms and 21 living worms were found

respectively. Most of the worms found from one of the rabbits showed much atrophy and

degeneration， while the majority of 21 worms from the other rabbit were normal. This

:fi nding shows that the dosage of 75 mgjkg every other day for 5 doses is insu伍� cientin the

rabbit.

In contrast， the experiments with the rats showed that， following the administration of

50 mgjkg of Hetol daily for 5 doses (totaling 250 mgjkg)， a complete cure was observed in

one rat and only one living but degenerated worm was deteeted in the other rat at autopsy，

thus indicating the treatment fairly e妊� ective. In the two rats given 75 mgjkg every day

for 5 doses (375 mgjkg totaling) a complete cure was achieved. In the other two rats given

75 mgjkg every other day for 5 doses (totaling 375 mgjkg)， while one of the rats showed

complete cure， the other rat showed the presence of 4 worms consisting of 2 showing

remarkable atrophy and degeneration and 2 nearly normal. These :fi ndings may indicate

that， even when the total of the drug given be equal， there is some difference in e:ffi cacy

between daily administration and administration every other day. Further studies are

needed in this connection.

The mechanism of action of Hetol in Clonorchis sinensis is not fully clari:fi ed yet. In this

experiment it was found that the E. P. G. increased temporarily following the administ-

ration of Hetol but later decreased with eventual disappearance. It should also be remarked

here that in the dead worms found at autopsy， although the degeneration and necrosis of

the. their uteri were almost always posterior half of the bodies were very remarkable，

:fi lled with a large number of eggs.

It is known in the experimental treatment of Paragonimus infection with bithionol by

Yokogawa et al. (1 961)(10) that there is no remarkable temporary increase in E. P. G.

following its administration， that the uterus of Paragonimus worms becomes empty before

they die， and that， thus， the formation of eggs is inhibited :fi rst， before the death of the

worms. This :fi nding with bithionol is signi:fi cantly di妊� erentfrom what we have found

with Hetol in the treatment of Clonorchis infection.

In the rats， the disappearance of the eggs in feces occured relatively ear1 y after treatment

comp訂� edwith those in the rabbits and dogs. This di妊� erencemight be ascribable to the

following di首位� ence. While the rat has no gall bladder， the rabbit and the dog have ‘

a gall bladder， in which the eggs remaining in the gall bladder w il1 be excreted in feces

for a while even after the worms have stopped laying eggs. Besides， when dead fiukes

reach the gall bladder， the necrosis and disintegration of the fiukes there does not preclude

the possibility of eggs continuing to be excreted. The time of the death of the worms

and the time of the disappearance of the eggs do not necessarily coincide. This must be

taken Into consideration when assessing completeness of cure by stool examination.

following the treatment of cats infected with Otisthorchis，reported that∞(1963) 1. Liu et a

with heX: achlorophene， the dead fiukes were excreted in the feces. No study on this fact

No.3 YOKOGAWA， KOYAMA， YOSHIMURA and TSAI: -295-CHEMOTHERAPY OF CLONORCHIASIS SINENSIS

was made in the present studies.

、� Evenin the dog heavi1 y infected with 900 metacercariae of Clonorchis sinensis a complete

cure was observed with the use of 200 mg/kg every other day for 5 doses. However，

further sfudies are desirable on the relationship between therapeutic dosages and the

density of infection. Hetol has been found also fair1 y effective， though in only one case，

against Paragonimus westermani.

Somewhat different results were obtained between dai1 y administration and administ-

ration every other day. It seems to be quite important to clarify the absorption of Hetol

and its excretion in the host.

The acute toxicity in the mouse has been reported to be less than 4，000 mg/kg， in the

rat to be 2，800 mg/kg， and in the rabbit to be 2，200 mg/kg. As for chronic toxicity， 15

doses of 200 mg/kg in the rat have been reported to have produced no toxicity. In our

toxicity studies with the mouse， we observed no abnormal changes at all with 5 x100 mg/

kg. M i1 d hepatic changes were observed in some of the animals given 5 x 200 mg/kg. At

5x300 mg/kg， hepatic fatty degeneration and icterus were pronounced.

The results of the present experimental studies have ir吋� icatedthat， in any of the

animal species used， the use of 100 mg/kg every other day for 5 doses brought about

complete cure. Even a further reduction in the dosage might be feasible without reducing

the above therapeutic e伍� ciency. So， the use of Hetol in the treatment of clonorchiasis

sinensis may be regarded as being possible without concern for its toxicity.

The present studies have demonstrated that the rat， too， may be used as well as other

animals， cats， dogs and rabbits since in the rat about one-third of 30 metacercariae given

become adults， whose detection is very easy. This knowledge is very useful for thera-

peutic experiments.

Summary

Exp・erimentaltreatments were performed with the use of 1，� 4-bis-trichloromethylbenzol

(Hetol) in 3 dogs， 8 rabbits， and 20 rats infected with metacercariae of Clonorchis sinensis.

The results of treatment were evaluated by Stoll's egg counting method and AMS III

centrifuge technique and by autopsy after -treatment.

It has invariably been found in the above studies that， when Hetol was given at daily

doses of 100 mg/kg every other day for 5 d， oses， the E. P. G. temporari1 y increased after

the 2nd or 3rd dose and then it decreased radically， the E. P. G. became zero within 21

days after the first day of treatment， and all these animals were found to be completely

cured at the autopsy.

In rabbits given 75 mg/kg of Hetol every other day for 5 doses， the E. P. G. increased

again after treatment， and a number of living worms was found at the autopsy. In the

rats， on the contrary， the same dose as above given every day for a tota: l of 5 doses did

definitely prove effective.

Doses of 50 mg/kg every other day for 5 doses were ine妊� ectivein' the rabbit， but the

same dose given every day for 5 days produced complete cure in the rat.

Differences in the therapeutic results according to the dosage and method of administ-

ration of Hetol and according to the species of the experimental animal， is discussed in

this paper.

Another thing which has been clarified by the present studies is that 200 mg/kg every

-296ー� THE JOURNAL OF CHIBA MEDICAL SOCIETY Vol. 41

other day for 5 doses produced no appreciable toxicity in the mouse.

The results of the present studies thus far described have indicated the possibility of

the use of Hetol in the treatment of clonorchiasis sinensis in man.

The authors are grateful to Hoechst Co. of Germany for the supply of Hetol for use .in

our present studies and also to Dr. L込� mmlerwho has kindly provided us with literature，

etc. on Hetol. The authors also mention with gratitude the fact that the present studies

were performed under a grant-in-aid from the Education Ministry out of its research .sub-

sidies for the fiscal year 1964.

The research reported in this document has been made possible through the support and

sponsorship of the U S Dept. of Army through its Far East Research Office and also was

supported in part by a Scientific Research Grant from the Ministry of Education in Japan.

References

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467-470.，.Wschr. 67 1. Tierarzt

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