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-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
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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
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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.
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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
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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
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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
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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
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-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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