Effect of digitalis upon the resistance of the mouse to diphtheria toxin.

(1)

Effect of digitalis upon the resistance of the mouse to diphtheria toxin. 21

(From the Department of Pharmacology, Medical College, Nagasaki, Japan—

Director : Prof. ]61. Terasaka.)

Effect of digitalis upon the resistance of the mouse to

diphtheria toxin.

By

Dr. Mikio YUNOKI.

(Received 24 June 1939).

It is well known that a large proportion of deaths from diphtheria are due to circulatory failure. In such a condition, naturally the interest from a therapeutic standpoint has become to centre upon the heart and circulation. Now attention is therefore directing to the study of the best means of preventing of a toxaemic condition such as diphtheria. At present time there are only a few reports of studies upon the effect of digitalis to diphtheria toxin.

Haskell (1) studied the effect of injection of digitalis and the lethal dose of diphtheria toxin upon guinea pigs. He found that moderately large single doses of digitalis had no favourable influence on the dulation of life. In a later investigation upon the same problem he suggested that if the heart is severely injured by diphtheria toxin, digitalis has a deleterious action. Bush (2) showed that poisoning by diphtheria toxin increased the susceptibility of frogs and dogs to digitalis. Elmunds and Cooper(3) found that digitalis, given to dogs poisoned with diphtheria toxin, acted as cardiac stimulant, but that the animals were more susceptible to digitalis than normal dogs. In 1932, reports of studies upon the protective treatment of diphtheria toxin with digitalis were made by Myers (4).

He stated that the administration of digitalis to guinea pigs which have received a lethal dose of diphtheria toxin is to a very considerable degree life saving.

Following the appearance of Myers' paper, Gold and Klumpp (5), Edmunds and Sn2-th(6) showed that no conclusive evidence could be found that the previous

• administration of digitalis would protect guinea pigs from the diphtheria toxin.

A persual of the literature makes it obvious that no general agreement exists as to the value of digitalis in case of diphtheria intoxication. The present study was undertaken, therefore, to determine the degree of protective action of digitalis against diphtheria toxin.

Experimental methods

As experimental animals white mice of approximately 15gr were used, the standardized diphtheria toxin (1 : 300 M.L.D. 250 grams guinea pig) was kindly supplied by the Department of Bacteriology of this University.*

I with to thank here Prof. T. Naito of the Department of Bacteriology of this University, for the valuable assistance given in obtaining the necessary material.

(2)

22 ^Yunok‑i .

As a control, a preliminary determination of the minimum lethal dose of the diphtheria toxin to be used was carried out an;'‑ was found to be for lO grams mouse, 0,25 cubic centimetre. As digitalis, digitoxin (Merck) was used in all experiments. Digitoxin was daily injected subcutaneously in an amount of 0,0002mg (1 : 20 M.L.D.), 0,0004mg (1 : 10 M.L.D.), and 0.00lmg (1 : 4 M.L.D.) per gram respectively and these daily subcutaneous injections of digitoxin were given to each group for five, ten, thirteen, fifteen and twenty days. When the members of each group had received the requisite nurnber of injections, each of them was injected subcutaneously with a M.L.D. of diphtheria toxin (0,25cc. per 10g). The mortality and survival time of the untreated controls which had received only toxin were compared with the animals which had received toxin after the previous treatment by digitoxin.

Table L Table 2.

The resistance of mice against diptheria toxin. The resistance of mice against diphtheria toxin of 0,25cc. per lograms. (controls)

*f !1' 'r eJ) e,c

k _:e r¥J

16 16 15 15 lO 13 12 l]

ll ll

20 17 15 13 20

ll 17 16 lO

12.

15 15

cD v, o

:

H

CSH

O bJO

He)

O

O, 25 ll ll ll ll ll ll ll ll ll' ll ll ll ll ll ll

ll ll ll ll ll

O

S

cs̲

l

LH G'

.

4 7 5 5 6 6 4

i3 14 6

Il

8 iO 8

I 2

7 6

days

ll ll ll ll ll ll ll ll ll ll ll ll ll ll ll ll

Survived

ll ll ll ll

,J _C rC ' bfJ

' h , _c:

:,

n,

J)

J

13 12 13 14 9

ll 9 8 7 11 ll 12 8

ll

8 12

(3)

Effect of digitalis upon the resistance of the mouse to diphtheria toxin. 23

Results

l. The resistance ot mice against diphtheria toxin.

At the beginning of each experiments, in order to determine the minimal lethal dose of diphtheria toxin, varying amounts of diphtheria toxin werb injected subcutaneously in the back of animals and observations were made for a period of sixty days after the toxin injection. In the series of experiments (table 1), no death occurred among the animals which had received a toxin injection of 0,1cc. per 10g, while the animals which had received a toxin injectlon of 0,5cc.

per 10g all dled within 2‑7 days after injection and by a toxin injection of lcc.

per 10g all animals died within 2 days. The injection of 0,25cc. per lOg killed 77,30/0 of the animals within 14 days after injection. The experiments given in table I show that the mortality and survival time after toxin injection was almost directly proportional to the dose of diphtheria toxin injected and the minimum lethal dose of diphtheria toxin to be used was found to be for 10 grams of mouse 0,25 cubic centimetre. (Table 1. Table 2)

2. The resistance of mice having received repeated subcutaneous injections of digitoxin against diphtheria toxin.

iR. The resistance ot mice digitalised for five days against diphtheria toxin.

After having been left for a few days to become accustorned to their surrounding, daily subcutaneous injections of digitoxin (0,002"/o) were given for five days. For the purpose of this experiment 30 mice were divided into two groups. 15 mice in one group were injected 0,0002mg per gram every day and 15 in the other group were injected 0,0004010mg of digitoxin per gram every day. After the animals of these two groups had received the injection of digitoxin for five days, each animal was injected with a M.L.D. of diphtheria toxin (0,25cc. per 10g). .

The results are shown in table 3. In the series of 15 mice which received subcutaneous injections of digitoxin 0,0002mg per gram, 3 in this group survived and 12 died. In most cases except a few, the mice seemed to die before or after one week after the toxin injections. In the series of 15 mice which received subcutaneous injections of digitoxin 0,0004mg per gram, 2 in this group survived and 13 died. In most cases except a few, the mice seemed to die before or after one week after the toxin injections. The details of these experimental results indicate clearly that the mortality and survival time of mice which had received digitoxin injections of 0,0002mg or 0,0004mg per gram every day are almost identical with those of the control animals. In the animals of one‑half of each group, the digitoxin injections were given up immediately after the injection of diphtheria toxin, but in the other 1lalf, the digitoxin injections were continued

(4)

24 Yunoki．

till death occurred． Nevertheless，no difference could be found between the one and the other．

From the assembled results of my experiments，it was found that the previous treatment by digitoxin for five days was without any favourable influencc，

Table3・

The resistance of m三ce digita】ised for five d級ys ag＆inst diphtheτia toxin。

③ 鼠

2 診』巽

9コ三客．曽ゆ 3

一_ご

』1のの

＄q 噂bぬ宣

．葺目籍ぎδbの

・黎昌。

．嵩のパのbのq

∩。二

｝8 0宕て1咽．90

お易

角9

り

巻9

粥℃o 宅8 ち℃

←

唱図ト司

山9．90の

．巽いごお8勧

ρ 胃oo 胤8一_属℃

の

㌧一

｝o qo

一_ぢ

葺 ρ

護ご

㊤

℃ 一_鳥一り

bβ

ぶ5

qo o㊤

q q o区

．》

o

．黛

x qo．9

｝ごご．昌む。9

邸

い（_O bρ 目3）

℃．霞の

一蓉三昌o．曽トで

1 ¹¹ ^0．0002 5days 0ン01 0．25 2 8 Givenup 0．01

2 ¹² ^〃 ^〃 ^OPOU ^〃 5 ⁹ ^〃 ^0フ011

3 ¹³ ^〃 ^〃 ^〃 ^〃 7 ^〃 ^〃 ^〃

4 ¹¹ ^〃 ^〃 ^0．01 ^〃 6 7 ^〃 ^0．01

5 ¹⁶ ^〃 ^〃 ^0，014 ^〃 7 ⁹ ^〃 0ρ14

6 H ^〃 ^〃 ^0，011 ^〃 ¹⁰ 8 ^〃 0ρ11

7 ^！3 ^〃 ^〃 ^〃 ^〃 ¹⁵ 7 ^〃 ^0，011

8 ¹² ^〃 ^〃 ^0，0！3 ^〃 7 ⁹ ^〃 ^0，013

9 ¹³ ^〃 ^〃 ^0，014 ^〃 ^Survived ^Continued 0ρ32

10 9 ^〃〃 07008 〃 6 1 ⁷ ^〃 ^0ρ14

11 12 〃〃 0，009 〃 SUrvived 〃 0，016

12 13 〃〃 0，012 〃 r

Survived 〃 0，026

13 12 〃〃 0，011 〃 20 8 ^〃 ^0ン026

14 ¹¹ ^〃〃 0．0096 〃 5 8 ^〃 ^0，019

15 13 〃〃 0，0！2 〃 19 8 ^〃 ^0）022

！6 13 0．0004 5（1ays 0，022 0．25 6 8 Givenup ^0，022

17 Il 〃〃 0ρ2 ^〃 6 8 ^〃 0．02

18 14 〃〃 Q，023 ^〃 ^雪0 9 ^〃 ^0，023

19 14 〃〃 0，023 〃 9 8 ^〃 ^0，023

20 14 〃〃 0，025 〃 9 8 ^〃 ^0，025

21 12 ク〃 0，018 ^〃 43 7 ^〃 ^0，018

22 ¹³ ^〃 ^〃 0，021 ^〃 15 7 ^〃 ^0，021

23 15 〃〃 0，023 ^〃 Survived 〃 0，023

24 ¹⁴ ^〃〃 0，026 ^〃 6 ^】0 ^Continued ^0，049

25 ¹³ 〃〃 0ρ26 ^〃 6 ¹⁰ ^〃 ^0，049

26 ¹⁰ ^〃 ^〃 0，017 ^〃 4 7 ^〃 0．03

27 ¹³ ^〃〃 0，022 ^〃 5 ⁸ ^〃 ^0．04

28 ¹³ ^〃 ^〃 0，022 ^〃 5 8 ^〃 ^0，036

29 12 〃〃 0，023 〃 16 8 ^ク ^0，042

30 ¹⁵ ^〃 ^〃 0ρ25 ^〃 ^SUrvived ^〃 ^0．Q4

(5)

Erfect f digitalis upon th*. r',si , tance or th". mouse t<, dll*hthe"ia to*in. 25

not only with regard to the mortality as shown in table 3, but also with respect to the dulation of life trfter toxin injection. Ilere, any possible beneficial action, of the digitoxin appears extremely doubtful.

B. The resistance of mice digitalised for ten days against diphthcria toxin.

In this series of experiments (table 4), 29 mice were divided into two groups. 15 in one group were given the injection of digitoxin 0,0002mg per gram for ten days and 14 in the other group were given the injection of 0,0004mg per gram every day. After previous treatment by digitoxin for ten days, all animals of both groups were injected with a M.L.D. of diphtheria In one‑b̲alf of all the animals, the digitoxin injections were given up toxin.

immmediately after the toxin injection, but in the other half the digitoxin injections were continued unto death. The results are shown in table 4. In the series of 15 mice which received subcutaneous injections of digitoxin 0,0002mg per gram, 2 in this group survived but 13 died. In the series of 14 mice which received subcutaneous injections of digitoxin 0,0004mg per gram, all animals died.

In most cases except a few, the mice seemed to die before or after one week after the toxin injection. Now the results of these experiments (table 4) were compared with the controls as shown in table 2. The results show decisively that digitalised mice werc not far from the controls" as indicated by mortality, survival time. From the results of these experiments therefore, it was founcl that the previous injection of digitoxin for ten days produced no beneflcial action to diphtheria toxin.

C. The reslstance of mrce digrtallsed for flfteen days agamst d]phthena tcxm In this series of experiments (table 5), 30 mice were divided into two groups.

The animals of the two groups were given injections of the same doses of digitoxin as already described above. The results are shown in table 5. In the series of 15 mice which received subcutaneous injection of digitoxin 0,0002mg per gram, 2 in this group survived but 13 died. In the series of 15 mice which received subcutaneous injection of digitoxin 0,004mg per gram, 3 in this group survived but 12 died. The results embodied in table 5 indicate that the mortality of the digitalised animals shows very little change, as cornpared with the numbers of control animals which had received no digitoxin. On the other hand, the survival time was only slightly longer except for a few animals with

¥; rhom no difference could be found as compared with the control anirnals.

D. The resist･*nce of mice digitalised fcr twenty days against diphtheria tcxin.

In this series. of experiments (table 6), 29 mice were divided into t¥vo

(6)

26 Yunoki．

groups and the animals of the two groups were given injections dose of digitoxin as already described above・ At the end of the for twenty days diphtheria toxin O，25cc．per10grams was injected．

of these experiments are shown in table6． In the series of15

Table4。

The resistance of mice dlgitalised for ten days ag翫illst diphtheria toxin．

一㊤

葺

客多ぎ8 ＆登 ξ℃ 8唱圏謬爵超

of the same

digita1量sation The details mice which

一】 o山○℃bめの o

駈D− q

（^bβ

臼弓ご

1

2 3 4 5 6 7 8 9

10 11 12 13 14 15

16 17 18 19 20 21 22 23 24 25 26 27 28 29

12 12 13 12 12 14 13 10 12 12 12 14 13 13 14

10 11 13 10

」4

13 14 14 14 12 12 12 12 14

00002

〃〃〃〃〃〃〃〃〃〃〃〃〃〃

0，0004 〃〃

〃

〃〃

〃〃〃

〃

10d鼠ys 〃〃〃〃〃〃〃〃〃〃〃〃〃〃

10days

〃〃〃〃〃〃〃〃〃〃〃〃〃

0声022 0，025 07023 0，022 0ラ026 0，028 0，026 0，02 0，022 0，025 0，02 0）021 0，024 0》025 0，024

0ρ3 0，036 0ン04 0シ032 0，055 0ρ5 0フ049 0，047 0，036 0，041 0，045 0，05 0，045 0，047

025

〃〃〃ケ

〃

〃〃〃〃

〃

0フ25

〃

5 8 5 8 8

10 SUrvived

り

SUrvived 10

3 7 5 8

14

1置

9 9 9 7 9 8

『4

10

5 8 9 8

10

璽0

10

8

10

9

10

7

9 8 8 9 8 9

6 6 8 8

10

8

10

7 8 7 8 9 9 8

Given np

〃〃〃〃〃、〃

Continued

〃〃〃〃ウ〃

Given up

〃〃〃〃〃〃〃

Continued

〃〃〃〃〃

0，022 0，025 0ラ023

Gρ22 0ρ26

0，028 0）026 0，02

0ρ37 0ρ37

0，029 0，029

φ，038

0，04 0，041

0703 07036 0204 0，C32 0，055 0，05 0タ049 0，047 0，048 0，063 0ρ8 0，077

0，08！

0，078

(7)

Efflect of digitalis upoll the resistallce of the！nouse to diphtheria toxin・ 2？

received subcutaneous injection of digitoxin O，0002mg Per gram，3in this group survived but12 died． In the series of14mice which received subcutaneous injection of digitoxin O，0004mg per gram，1圭n this group survived but13died。

From the observation of the results embodied in table6，no difference could be

Taめ1e5。

The resistance of mice digitalised for fifteen days against diphtheria toxin。

8ロ^…ii う

客

1

2 3

4

5

6 7 8 9

10 11 12 13 14 15

16 17 18 19 20 21 22 23 24 25 26 27 28 29 30

公9

讐

浬三

．聾

3つ

13 14 12 14 13 13

！3

11 13 14 14 14 13 12 12

14 14 11 12 12 11 15 13 12 11 11 15 14 10 15

の弱一のの8似

2螺

じぽレ

図6bβo り

．眉 q

．曽oo

0，0002 〃〃〃〃〃〃〃〃〃〃〃

〃

0，0004 〃

〃

〃〃〃〃〃〃レ

〃〃〃〃

〃

∴q．聾○

℃薗二

Q｝ oO r

自も咽

．g q む区二【2

15days

〃〃〃〃〃〃〃〃〃〃〃〃〃〃

：5days

〃〃〃〃〃〃〃〃〃〃〃〃〃〃

．葺

，8bO〉く（

．鮒

℃ o 需8 ぢ唱

←

0ρ36

0，037

0ρ26

0，041 0．038 0，036 0，034 0｝029 0，033

0ρ43

0．038 0，039 0，032 0，032 0，032

0ρ79

0ナ074 0，053 0，071 0，056 0，051 0，088 0，06 0，071 0，067 0，063 0ラ071 0，066 0，054 0，08

．臼o

×一〇一一〇の

．雪儀昌

台9bPむ6響

邑3 蒼ε

0，25

〃

．

〃

0，25

〃

〃〃

〃

〃ク

ε

｝o

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ご宕

∩

一

℃o

一_c6 一の_bめ

3。6

12 19

4 5

SUrvived

ノ

9Urvived

12 10

8

26

5

14 15

6

19

6

5

18

4

6

13

7

SUrvived

『

Survived 35

7

5

Survived l6

8 8 8 8

9 7 6 9 9 9 8 8 9 9 9 8 8 8 8

10

8

7 8 8 9

自o

容 9 ￠o り一

・〔区 q g o o ▼」一口 Q

．一一〇区 o 。F「

一口_{o 』同．一}

，嵩粥

．曽

ρ

Given up

〃〃〃〃〃〃〃

C611tinued

〃〃〃〃〃〃

Given u1）

〃〃〃〃〃〃〃

Continued

〃〃〃〃〃〃

｝QO 臭 3）

ε，縄

一蓉 5・属o，聾臼℃

0，036 0フ037 0，026 0，041 0，038 0，036 0ラ034 0，029 0，04 0，054 0》042 0，045 0，043 0，04 0，04

0ン079

0ρ74

07053

0，b7】

0，056 0，051 0，088 0フ06 0，092 0，087 0フ084 0，096 0，084 0，086

0，！07

(8)

28 Vunoki．

found between the mortality of the anima聴which received digitoxin for twenty days and that of the control animals which had received no digitoxin・

The dulation of life after toxin injection，however，was slig耳tly longeぎin comparison with the regular control group・

TabIe6．

The resistance of m1ce（1igit＆1ised for twenty days against diphtlleria toxin・

一￠』 0 qo戸4図（

b幻

qOo一一

o 旨_〇

董 9 ^0．0002 20 days 0ρ29 ^0．25 6 ⁶ ^Gi▽enup

2 ¹² ^〃 ^〃 ^0，039 ^〃 9 ⁸ ^〃

3 ^】3 ^〃 ^〃 ^0，045 ^〃 ¹² ⁷ ^〃

4 ← ¹⁴ ^〃 ^〃 ^0，066 ^〃 ⁱ⁴ ¹² ^〃

5 ¹⁴ ^〃 ^〃 ^0，048 ^〃 ¹⁷ ¹² ^〃

6 ¹⁴ ^〃 ^〃 ^0，048 ^〃 ^SUrvived ^〃

7 ¹¹ ^〃 ^〃 ^0，039 ^〃 9 1 3 ^〃

8 ^！5 ^〃 ^〃 ^0，049 ^． ^Survived ^〃

9 ¹³ ^〃 ^〃 ^0，058 ^〃 ^urvived ^〃

10 15 _〃〃 03051 〃 9 8 ^〃

11 ^】3 〃〃 0，046 〃 ^望7 10 〃

12 ¹¹ 〃〃 0，036 ^〃 ^聖2 7 ^〃

！3 12 〃〃 0，042 〃 6 9 ^〃

14 14 〃〃 0ρ49 ^〃 ¹⁰ 8 ^〃

15 14 _〃〃 0ラ053 ^〃 8 ⁷ ^〃

16 ^】2 0．0004 20 days 0．11 0．25 ¹⁶ ¹⁰ Givellup

17 13 _〃〃 0．08 ^〃 6 ^〃

18 9 〃〃 0，065 〃 Survived ⁹

19 14 〃〃 0．11 〃 12 8 ^〃

20 ¹³ 〃〃 0，096 〃 13 8 ^〃

21 ^！4 〃〃 0，096 ^〃 ^9、 8 ^〃

22 ¹³ 〃〃 0，085 〃 ^璽4 9 ^〃

23 ¹³ 〃〃 0．1 〃 13 9 ^〃

24 9 ^〃 ^〃 ^0，076 ^〃 9 ^ρ7 ^〃

25 13 _〃〃〃 12 10 〃

26 ¹⁵ ^〃 ^〃 ^0ラ1 ^〃 ¹⁰ 9 ^〃

27 ¹⁵ 〃〃 0．11 ^〃 ^Il 9 ^〃

28 ¹⁵ 〃〃 0，H ^〃 ^lI ¹¹ 〃

29 13 〃〃 0，085 〃 9 ⁹ ^〃

嘩

(9)

Errect of digitaus upon the resistance of the mouse to diphtheria toxin. ?9

E. The resistance of mice which received digitoxin 0,00lmg per gram for thirteen days against diphtheria toxin.

With the results of my experiments' as above described, from a practical standpoint, no conclusive evidence could be fouhd that the previous adminis‑

tration of digitoxin would protect mice from the effects of a mimum lethal dose of diphtheria toxin. Myers obtained a protective action against at least two and one‑half lethal ･ doses of toxin by 8 or 13 previous daily injections of 0,05cc. of the tincture of digitalis̲ Accordingly one quarter of the lethal do e of digitoxin (,0.00lmg per grath) was chosen for daily injection, since 'approximately "such mounts yielded positive results . in . /74r,,ers' experiments. In this series of experi‑

ments (Table 7), 10 mice were given an ' subcutaneous injection of digitoxin (0,0040/0 ) per gram 0,00lmg every day. At the end of the digitalisation for thirteen days, diphtheria toxin 0,25cc. per 10 grarns was injected. The results of these experiments are shown in table 7, where it is found that the most cases among 9 mice died before pr after 6 days after toxin injection, but only one slJlrvived.

From the results of the experiments, it is shown that a beheficial dffect of digitalisation on diphtheria toxin could not be fouhd either iri preventing death or in prolonging life. It seeriled to me thaf if 0,00lmg per gram‑.of digitoxin were given daily, the death of the mouse was likely to be hastened. ,

' able 7*

' The resistance of mice which̲ received digitoxin o,oolmg per glram for thirteen days against diphtheria toxin.'

(10)

30 ^Yunoki .

Summary and Conclusions.

1. An attempt was made to determine the influence of digitoxin upon the resistance of mice to diphtheria toxin. The minimum lethal dose of the diph‑

theria toxin (1 : 300 M.L.D. 250g guinea pig) to be used was found to be for 10g of mouse, 0,25 cubic centimetre.

2. The mortality and survlval time of mice ¥ rhich received subcutaneous injection of digitoxin 0,0002mg or 0,0004mg per gram for five days were almost identical in comparison with the regular control animals which had received no digitoxin. Although the period of the digitalisation was prolonged for ten days, the results were without any favourable influence, not only with regard to the mortality, but also with respect to the dulation of life after toxin injection.

3. If the mice were digitalised daily for fifteen or twenty days, the results of this experiment indicated that the mortality of the digitalised animals shows very little change as compared with the control animals, but the survival time for the animals which . had received digitoxin was slightly longer except for a few animals with whom no difference could be found as compared with the

control animals.

4. Digitalising the mice daily with an amount of 0,00lmg of digitoxin per gram for thirteen days, a beneflcial effect of the digitallsation on diphtheria toxin could not be found either in preventing death or in prolonging life, but the injurious effect of it could be seen.

5. With the results of my experiments, from the practical standpoint, no conclusive evidence could be found that the previous administration of digitoxin would protect mice from the effects of the minimum lethal dose of diphtheria toxm.

I wish to express my gratitude to Professor Dr. M. Terasaka for his valuable advice during the course of this work and for his very helpful assistance in the writing of this paper.

I) Haskell : J, am**r. Pharm.

B sh : J. Pbarmacol. (Am.) Assoc. 85 (1925) : 1798. cit.

(Am.) 44 (1932) : 191. S) Pharmacol. (Am.) 61 (1937) :

Ref erences

16 (1927) : 639. Cit.. from J. Pharmacol. (Am. ) 44 (1932) : 191. 2) 3) Edmunds, C. W. and Cooper, R. O.: J. amer med, 13 (1919) : 55.

from J. Pharmacol. (Am.) 61 (1937) : 37. 4) Myers : J. Pharmacol.

Oold and Kltlmpp : Amer. J. med. Sci. 185 (1933) : 509. Cit. from J.

37. 6) Edm nds and Smith : J. Pllarmacol. (Am.) 61 ( 1937) : 37.