616.937 : 615.778

THE CHEMOTHERAPEUTIC ACTION OF SYN- THALIN IN EXPERIMENTAL INFECTIONS WITH T. BRUCE1 AND T. CONGOLENSE.*

PAUL BROWNING. From the Departmerit of Bacteriology and Pathology of the University

ar, d Western InJirmary, Glasgow.

THE therapeutic propeyty of the guanidine derivative, synthalin, in try- panosome infections was described by N. and H. von Jancs6 (1935) for T. brucei in mice and independently by Schern and Artagaveytia-Allende (1936) for T . equinum in rats. The former were led to investigate this substance by certain theoretical considerations. From enquiry into the mode of action of germanin (Bayer-205), in which there is a long interval before the lethal effect on the trypanosomes follows the injection of the drug, they concluded that this trypanocidal agent acts on the parasites by affecting their sugar metabolism (N. and H. von Jancs6, 1934 a and b, 1934-35). Schern (192.5) had shown that the vitality of trypanosomes, as manifested by their motility, depended very greatly on the glucose content of the medium in which they were suspended. Also, it had been found by von Issekutz (1933) that when infected animals were treated with germanin and their blood withdrawn a few hours later, the contained trypanosomes showed diminished glycolysis in vitro. The von Jancs6s (1934-35) observed that trypanosomes which had been subjected to deprivation of glucose in vitro, like those which had bee.? acted on by germanin, on introduction subsequently into the body were rapidly phagocytosed by cells of the reticulo-endothelial system in the liver, spleen and bone marrow, whereas such phagocytosis did not occur with untreated virulent trypanosomes. They ascertained further that in infected animals treated with germanin certain degenerative changes occurred in the morphology of the parasites, characterised especially by signs of interference with the processes of division. These appearances were seen readily only with animals in which the phagocytic mechanism of the reticulo-endotheli a1 system had been interfered with recently by the combined procedures of splenectomy and intravenous injection of a preparation of colloidal copper-so-called reticulo-endothelial blockade. In intact infected animals treated with the drug such degenerative changes were not seen because .he trypanosomes a t an early stage of degeneration underwent phagocytosi;. Accordingly they tested the chemotherapeutic action of certain guanidine derivatives which are known to produce hypo- glycEmia. Among thme, synthalin (decamethylene diguanidine hydro- chloride) and synthalin B (the corresponding dodecamethylene compound) are the most effective <and have been suggested as substitutes for insulin, although their action is stated to depend on a different mechanism, being associated with toxic effects on the liver (Bodo and Marks, 1928 ; and others).

~

* The expenses of this work were defrayed by a grant from the Rankine Fund. 323

324 P. BROWNING

After treatment with synthalin the same phenomena of phagocytosis and degeneration were observed as after germanin. Therefore it was concluded that these guanidine derivatives also act by affecting the sugar metabolism of the parasites. I n discussing the means by which this might be brought about, the von Jancs6s inclined to the view that synthalin probably does not act directly on the trypanosomes, but leads to trypanocidal effects indirectly through producing a chronic hypoglyczmia in the host and thus causing a " sugar blockade " of the metabolism of the trypanosomes. I n the event of this being the mechanism of action, they considered that, in contrast to what holds with other trypanocidal agents, resistance to the action of synthalin might not follow repeated administration. It is specially noteworthy that these guanidine compounds are the only purely organic trypanocidal drugs which do not possess a complex ring structure. In view of the great theoretical importance of the observations and conclusiom, further examination of the subject seemed desirable. Since the present work was concluded, observations of Lourie and Porke (1937) have been published, codrming the chemotherapeutic action of synthalin in trypano- some-infected animals and also showing that in witro it exhibits a trypanocidaJ activity comparable with that of the aromatic trivalent arsenicals both on normal strains and on strains which had been rendered resistant to atoxyl or germanin.

Methods. It is unnecessary to describe the procedures in detail since they are

those which have been employed throughout in this laboratory (Browning and Gulbransen, 1934). The trypanosomes used were T. brucei (strain Paris 111) and a strain, derived by the writer from the latter, which had been rendered fast to arsacetin by treatment with that drug but was not a relapse strain. Also two strains of T . congolense were used-one being that previously mentioned (Browning, Cappell and Gulbransen, 1934), the sccond a strain which when once it appears in the blood of inoculated animals always persists, although perhaps with some variations in numbers, till the death of the animal one to several weeks later. All the strains were propagated continuously in mice.

Treatment was given in the form of subcutaneous injections of aqueous solutions of the substances, the dosage being a t the rate of 1 C.C. of the dilution shown for a mouse weighing 20 grammes; the animals ranged in weight from 18 to 24 grammes. The guanidine derivative used was synthalin. It was found that usually a single dose of 1 : 10,000 was tolerated but this amount could not be repeated safely; often subsequent doses of 1 : 13,000 or 1 : 15,000 were tolerated when repeated on 4-9 consecutive days. When death or severe illness from larger doscs resulted, convulsions seldom occurred beforehand. I n some control experiments guanidine carbonate was used, the maximum tolerated dose being 1 : 200, which could be repeated, while a single dose of 1 : 100 often produced convulsions and death. The blood sugar was estimated by the Hagedorn-Jensen method, the animals having been quickly anzsthetised with chloroform and the blood taken from the beating left ventricle. Duplicate estimations were always made and close correspondence obtained, 0.1 C.C. of blood being used for each.

Trypanocidal action of synthalin in vivo. T. brucei infection. Infections with this strain were curable

by styryl-314 in a single dose of 1 : 10,000 and in a proportion of animals with 1 : 20,000, or by arsacetin 1 : 100, given in each

S Y N T H A L I N I N TRYPANOSOME INFECTIONS 325

3.

s 1 : 10,000

S 1 : 15,000 + S 1 : 15,000

-

case 24 hours after inoculation, when scanty parasites were present in the blood ; it may thus be regarded as having a fairly normal sensitiveness to drugs. The arsacetin-resistant strain, which under similar conditions as regards treatment was not affected by a dose of 1 : 40 to 1 : 50 of arsacetin, was approximately as readily influenced by synthalin as was the non-resistant strain. The von Jancsds, howevor, obtained the best therapeutic results with a strain of the former type.

The results, which are exemplified in table I, confirmed the curative action of siynthalin. With the arsacetin-resistant strain as well as the normal, the sterilising action occurred slowly and it was observed that a preliminary multiplication of parasites took place in the blood whether the first dose of the drug was given five hours before or a,fter inoculation.

4. 5. 6. 7-120. - - ~

- - - -

S 1 : 15,000 - + - -

- - - -

TABLE I. Effect of synthalin on. infection with T. brucei.

Strain.

Normal

Arsacetin- fast

Arsacetin- fast

1.

S 1 : 15,000 followed by inoculatioii 4 hrs. later

S 1 : 15,000 followed by inoculation 5 hrs. later

Inoculation followed about 5 hrs. later by S 1 : 15,000

2.

S 1 : 15,000 + S 1 : 15,000 + S 1 : 15,000 +

Days.

In this and the following tables, S = synthalin; +, ++, +++ = trypano- somes present in the blood in increasing numbers on the days indicated ; - = try- panosomes absent as judged by microscopic examination.

No effect on thla course of the infection was produced by guanidine carbonate when given alone in a dose of 1 : 200 five hours before inoculation with the normal strain and repeated on the three following days, or together with a dose of arsacetin 1 : 200 48 hours aftei, inoculation.

T. congolense infection. A distinct therapeutic effect was observed with both strains. This fact is all the more striking in view of the insusceptibility of these trypanosomes toward most chemotherapeutic agents effective against T. brucei. Illustrative details are given in ?;able I1 of a mouse inoculated with strain 11, which does not disappear spontaneously from the blood after having once appeared. After the parasites had reappeared on the 13th day the same dose of synthalin (1 : 15,000) was repeated on the 14th, 15th, 17th, 18th, 20th and 21st days, but without

326 P . BROWNING

1. 2-5. G. 7. a. -__

Inoculated - + +++ +++ S 1 : 15,000 S 1 : 15,000

causing the parasites to disappear from the blood again ; also after ceasing treatment they remained abundant. In other animals the parasites after disappearing in response to treatment with the

TABLE 11.

Effect of synthalin on infection with T . congolense.

10-12 13.

+++ S 1 : 15,000 1 -

+

I-

1. 4. 5. G. 7. ' 8. 9-14. ! 15. - - - ~ - -

each day - I + Inoculated + +++ +++ ++ -

S 1 : 13,000 S 1 : 13,000 S 1 : 13,000 S 1 : 13,000 S 1 : 13,000

Days. !

16. +++ Similar observations were made with the less virulent strain I,

a relapse developing for example on the day following the last of nine consecutive daily doses of synthalin 1 : 13,000 and after the blood had been free from trypanosomes for seven consecutive days. These results indicate clearly that, contrary to the von Jancsb' expectation, resistance of the infection to synthalin becomes established, although it remains undecided as to whether this occurs owing to alterations in the parasites or the host. Schern and Artagaveytia-Allende, however, state that trypanosomes may become synthalin-fast.

Guanidine carbonate in a dose of 1 : 200 given on each of three consecutive days had no effect on the established infection with strain 11.

The blood sugar of mice in relation to the trypunocidul action of synthalin.

The von Jancsds have suggested that possibly a slight but persistent hypoglycEmia produced in the animals by synthalin may cause disturbances in the metabolism of the parasites which

SYNTHALIiV I N TR YPANOSOME INFECTIONS 327

lead to progressive degeneration. They do not, however, give any figures. In ordw to examine this point the blood sugar of seven normal animals which had been fed within a few hours previously and which were similar in weight to those used in the therapeutic experiments was estimated and the percentage of glucose found to be 0.13, 0.134, 0-16, 0.161, 0.178, 0.19 and 0.2 (average = 0.165). These figures are on the whole slightly higher than those of Kalabukhov and Rodionov (1934) obtained by the same method with mice aged 40-120 days, but they differ consider- ably from the figure, 0.245, of Scott (1922). In seven mice whose blood was swarming with T . congolense strain I, but which appeared in good health, the blood sugar percentages were 0.085, 0.09, 0.108 (2 mice), 0.135, 0.143, 0.15 (average = 0.117), that is to say, distinctly lower than in the normal animals. In general contrast t o these, five untreated animals with abundant T . brucei in their blood and within a t most a few hours of death showed the follow- ing values :-0, 0.03 (2 mice), 0.064, and 0.1 per cent. The latter results agree with the general finding that marked hypoglycaemia is an agonal phenomenon in trypanosomiasis (see Lourie and Yorke for a full review of the literature). Synthalin and guanidine carbonate were administered to uninfected mice under conditions shown in tables I V and V. The results indicate that with guanidine, even in doses less than that producing convulsions, a slight hypo- glyczmia can be produced, and a similar result follows with much smaller amounts of the more highly toxic synthalin. But it should be noted that of the two substances the latter alone has trypanocidal action, Further, the fall in blood sugar produced by these compounds is not notably greater than that in chronic infections with the relatively non-pathogenic strain of T. congolense (vide supra). Schern also found that insulin was without significant curative action in tIypanosomiasis and a few experiments in the present work were in agreement with this. Accordingly there is no

TABLE IV. Effect Gf synthalin on blood sugar in mice.

No. of doses each of 1 : 13,000.

l-1- ntervals in hours between doses and examination

of blood.

5

23, 4 23, 4

60, 37, 13 60, 37, 13

48, 29, 24, 4 48, 29, 24, 4

Blood sugar per cent

0.071

0.07 0.08

0.106 0.115

0.1 0.108

328 P . BROWNING

4 51, 28, 4

79, 56, 28, 4 273,49,1 t

729, 499, 20, 1 t '724, 499, 20, 1

TABLE V. Effect of guanidine on blood sugar in mice.

0.09 0.09 0.14 0.1 0.08 0.06

I No. of doses."

1 : 200) 1 : 200x3) 1 : 200x4) 1 : 200 x 2 ; 1 : 100) 1 : 2 0 0 ~ 3 ; 1:50) 1 : 2 0 0 X 3 ; 1 : 5 0 )

Intervals in hours between doses and examination

of blood. Blood sugar

per cent.

* The amounts of thc doses are given in brackets. 't Convulsions followed the last dose.

clear evidence that the hypoglycaemia produced by synthalin is in itself responsible for its chemotherapeutic action in trypanosomiasis.

Summary.

1. The observation that an aliphatic compound, decamethylene diguanidine hydrochloride (synthalin), has chemotherapeutic action in mice infected with T . brucei has been confirmed. The therapeutic result was as marked with an arsacetin-resistant strain as with the same strain before being rendered resistant to the drug.

2 . Infections due to two different strains of T . congolense in mice wwe also influenced by the administration of synthalin. When repeated doses of the drug were given a t daily intervals, however, relapses occurred eventually in the course of the treatment. Relapses were also found to resist further treatment with the drug.

3. Slight hypoglycaemia occurs in untreated mice chronically infected with T . congolense but the infection persists in spite of this. The degree of hypoglyczmia is similar to that in uninfected mice treated respectively with synthalin and with guanidine, but only the former of these drugs has chemotherapeutic action. The above facts do not support the view that the chemotherapeutic action of synthalin is due exclusively or chiefly to its effect on the blood sugar of the host. This conclusion is in conformity with Lourie and Yorlce's observation that synthalin is highly trypanocidal in vitro.

Thanks are due to Mr H. E. Hornby, Tanganyika Territory, for the second strain of T . congolense used in these experiments, and to Messrs Schering-Kahlbaum for placing a supply of synthalin at our disposal.

REFERENCES.

BODO, R., AND MARKS, H. P. . BROWNING, C. H., CAPPELL, 1934. this Journal, xxxix. 65.

D. F., AND GULBRANSEN, R. BROWNING, C. H., AND GUL- 1934. this Journal, xxxix. 75.

1928. J . Physiol., Ixv. 83.

BRANSEN, R.

SYNTHALIN I N TRYPANOSOME INFECTIONS 329

VON ISSEKUTZ, B. . . . .

VON JANCS~, N., ANL~ VON

JANCS~, H. 3 3 3 7

1, 3 9 ,, 3, 3 , 3 ,

KALABUKHOV, N., AND RODI- ONOV, v.

LOURIE, E. M., AND YOllKE, w. SCHERN, K. . . . , . . SCHERN, K., AND ARTAGAVEY-

TIA-ALLENDE, R. SCOTT, E. L. . . . . .

1933. Arch. exp. Path. Pharmak., clxxiii.

1934a. Zbl. Bakt., Abt. I, Orig., cxxxii. 257.

1934b. Ann. Trop. Med. Parasitol., xxviii. 419.

1934-35. Z. Imrnunitatsforsch., lxxxiv. 471. 1935. Ibid., Ixxxvi. 1. 1934. Fol. Hczmatol., lii. 145.

1937. Ann. Trop. Med. Parasitol., xxxi.

1925. Zbl. Bakt., Abt. I, Orig., xcvi. 356,

1936. 2. Immunitditsforsch., lxxxix. 21.

1922. Amer. J . Physiol., lix. 481.

479.

435.

360, 362, 440, 444, 451.