QUANTITATIVE STUDIES IN CHEMOTHERAPY

I. THE TRYPANOCIDAL ACTION OF ANTIMONY

COMPOUNDS

CARL VOEGTLIN AND HOMER W. SMITH

WITH THE COSPERATION OF

MARIAN M. CRANE, KATHERINE D. WRIGHT AND MABEL A. CONNELL

From the Division of Pharmacology, Hygienic Laboratory, United States Public

Health Service

Received for publication May 12, 1920

INTRODUCTION

The methods used for the elaboration of new drugs with

specific action the parasites
upon of infectious diseases are es-
sentially empirical in nature. They consist in testing hundreds
of chemicals in the hope of finding an effective drug. It is not
surprising that under such circumstances progress is retarded
principally by an almost complete lack of information regarding
the fundamental mechanism by means of which the drug kills
the parasite within the host. The present study was undertaken
in anticipation of elaborating methods which would yield quanti-
tative information as regards the chemotherapeutic value of
certain drugs and which would afford an opportunity of studying
the mechanism involved in the process of sterilization of an
infected animal. For this purpose no new drugs have been
synthetised or studied, but our attention has been confined to
a few drugs which are known to possess specific action. We
shall report in this paper a large number of experiments made
with certain antimony compounds which seemed especially
adapted for our purpose.
Th trypanocidal properties of antimony were discovered by
Plimmer and Thompson (1908), who found that tartar emetic
was very effective in removing trypanosomes from the blood of
453
454 CARL VOEGTLIN AND HOMER W. SMITH

infected rats. Similar results with this drug were obtained by

Mesnil and Brimont (1908). Thompson and Cushny (1909)
tested out other antimony preparations. Rowntree and Abel
(1910) report extensive experiments with antimonythioglycollate,
in which the metal is organically combined. Kolle, Hartoch
and Sch#{252}rmann (1914) obtained good results from the intra-
muscular injection of antimonytrioxide. Certain organic anti-
mony preparations were studied by Uhienhuth, MUlzer and HUgel
(1913). Plimmer, Fry and Ranken (1911) obtained good results
from metaffic antimony in fine suspension. Voegtlin, Lake and
Myers, in some work which will soon be published, found that
antimony in the form of the lactate is a very effective substance.
Various antimony preparations have been successfully used in
the treatrhent of trypanosomiasis in man and the domestic ani-
mals. Great improvement in the patient follows such treatment
and sometimes a cure may be effected.

EXPERIMENTAL

The selection of the disease for this investigation was of con-

siderable importance. In order to simplify the work as much
as possible, albino rats infected with Trypanosoma equiperdum
were chosen for the following reasons. First, the disease is
essentially an infection of the blood and not of the tissues, a
fact which renders possible, by means of an intravenous injection,
the bringing of the drug into immediate contact with the parasites.
Second, the disease is easily propagated in rats and runs a regular
course which leads to death within a few days. Third, the
course of the disease can be accurately followed by counting
the number of parasites in the blood according to the method
of Kolmer (1915).
Methods. A rat showing from 600,000 to 800,000 parasites
per cubic millimeter of blood is bled without special aseptic
precautions into 1.5 to 2 cc. of saline solution containing one
per cent of sodium citrate. This suspension is then injected
intraperitoneally into rats, using 0.25 to 0.5 cc. per animal.
Twenty-four hours later the rats so injected show 50,000 to
 QUANTITATIVE STUDIES IN CHEMOTHERAPY 455

100,000 trypanosomes per cubic millimeter of blood, and die

as a rule within three days. The strain of T. equiperdum used
was obtained from the Department of Agriculture.
The method of counting the parasites in the blood of an in-
fected animal is essentially the one proposed by Kolmer (1915),
who made use of the ordinary blood pipettes for the counting
of red and white cells. He found that counts made upon blood
obtained from the tail gave an accurate index of the number of
trypanosomes in the general circulation, an observation which
we were able to confirm.
The details of the method are as follows: The tip of the tail
is nipped off and the tail is gently whipped against the table
to produce congestion. Slight pressure beginning at the base
of the tail is sufficient to promote free bleeding. The first drop
of blood appearing should be discarded. The blood pipette is
then filled to the 5 mark with blood and made up to the 101
mark with the diluting fluid. The pipette should be shaken for
at least one minute in order to obtain a homogeneous distri-
bution of the parasites. The suspension is then placed in a
Zeiss counting chamber. As the red cells are laked by the dilut-
ing fluid, the parasites are easily distinguished from the leucocytes.
The number of squares which must be counted to secure accurate
counts will of course depend somewhat upon the number of
parasites present. With a large number of parasites fewer
squares have to be counted.
The diluting fluid is so designed that it will lake the red cells
and at the same time stain the trypanosomes. Kolmer used a
fluid of the following composition:
cc.
Formalin (40 per cent) 2.0
Glacial acetic 2.0
Distilled water 96.0
Carbolfuchsin 0.2

We used this fluid for some time with success, until it failed under
peculiar and unexplained circumstances, when it began to cause
a coagulum of unlaked red cells and trypanosomes. After
considerable experimentation, we found that this difficulty could
456 CARL VOEGTLIN AND HOMER W. SMITH

be overcome by using a slightly hypotonic fluid which was pre-

pared as follows: 20 mgm. of crystal violet is dissolved in 200 cc.
of water with slight warming; 700 mgm. of NaCl is added and
the solution is cooled and filtered. A small amount of formalin
(2 cc. in 200 cc.) was used in some experiments and seemed to
fix the trypanosomes and prevent their loss of shape. This
fluid should be made fresh every day.
Toxicity of compounds. Four antimony compounds were
studied, namely, antimonyllactate, antimonyl-potassium-tar-
trate, antimonic-potassium-tartrate, and antimonylthioglycoilate.
The antimonyllactate which contains the antimony in the tri-
valent form was prepared from lactic acid and antimony trioxide.
The substance appears as a thick syrup from which aqueous
solutions are made up . containing the desired metal content.
Solutions of this compound in water are probably dissociated
into antimonyl and lactic acid ions. The antimonyl-potassium-
tartrate (tartar emetic) was “Baker’s analysed.” The anti-
monic potassium tartrate was prepared by oxidizing a solution
of antimonyl-potassium-tartrate with chlorine gas; the solution
was then boiled to drive off the excess chlorine and neutralized
with sodium carbonate just before its use. It should be em-
phasized that this preparation contains the metal in the penta-
valent form. The antimonylthioglycollate was prepared after
the method of Abel and Rowntree from antimonytrioxide and
thioglycoffic acid. In solutions of this compound the antimony
occurs as a complex organic ion.
As a prerequisite for the chemotherapeutic study of the anti-
mony preparations, it was essential to first determine their
toxicity. This was established on albino rats weighing from
80 to 150 grams. An aqueous solution of the drug was injected
into the saphenous vein and the latter was ligated below the
site of injection. The concentration of the solution injected
varied with the toxicity, but the total volume of the injected
fluid never exceeded 1 cc. The rate of injection was kept uni-
formly at 1 cc. in 100 seconds. . The animals were kept under
observation for five days during which time they were fed on
corn, oats and milk. The minimal dose at which the majority
 QUANTITATIVE STUDIES IN CHEMOTHERAPY 457

of a series of animals died was considered the minimal lethal

dose (M. L. D.). The next lower dose at which the majority
of the animals survived will be referred to as the maximum
tolerated dose (M. T. D.). For the determination of the toxicity
of the drug for the host (M. L. D.) and for the parasite-minimum
effective dose (M. E. D.), the doses were graded on the logarithmic
scale as follows: 1, 1.5, 2.25, 3.75, 5, 7.5, 10, etc.; such a gradation
gives a variation of approximately 50 per cent between successive
doses. In confirmation of previous observations on the toxicity
of antimony and arsenic compounds, it was found that the toxicity

TABLE 1

Minimum toxic dose and minimum effective dose of antimony preparations

expressed in cubic centimeter8 of a 1/100 antimony-equivalent solution
per kilo body weight.

M.L .D. M.T.D. M.E.D.

Antimonyllactate 10 7.5 2
Antimonyl-potassium-tartrate 15 10.0 4
Antimonic-potassium-tartrate 400 250.0 Ineffective
Antimonythioglycollate 500 (?) 75

of any one compound varies considerably in different individuals

of the same species. For this reason the figures given are not
to be considered as absolute, and there seems to be no justifi-
cation for splitting the doses any further.
The dosage is always expressed on a molecular or antimony
equivalent basis. The data referring to the toxicity of these
compounds will be found in table 1.
Trypanocidal action. For the determination of the minimum
effective dose it is essential to make trypanosome counts on the
blood of the infected animals before the drug is injected in order
to secure animals of the desired size of infection. It will be
shown later that there exists a definite relation between the size
of the infection (the absolute number of parasites present) and
the amount of the drug necessary to kill all of the parasites.
On account of the practical difficulties of obtaining a large number
of rats with exactly the same size of infection, we have allowed
458 CARL VOEGTLIN AND HOMER W. SMITH

ourselves a certain margin in choosing animals showing 100,000

to 300,000 trypanosomes per cubic millimeter.
The time of disappearance of the parasites from the blood-
stream after the intravenous injection of the drug varies with
the type of drug studied. No standard interval for blood exami-
nations can be established for work of this kind. In order to
keep the results uniform we have made at least two counts on
the day of the injection and the last count at the end of twenty-
four hours. In this time the slowest-acting drug so far studied
by us will have reduced the number of parasites to a minimum,
so that inspection of the blood of the animals will show which
doses have proved effective. The data referring to minimum
effective doses will be found in table 1.
The largest part of the work reported in this paper deals with
antimonyllactate, and this compound was chosen for the study
of the fundamental mechanism by means of which the drug kills
the parasites within the host. This drug seemed especially
suited for this purpose as preliminary experiments had shown
that its intravenous injection is followed by the rapid disappear-
ance of the parasites from the bloodstream, a fact which indicates
that this particular drug is acting directly on the parasite without
first being changed by tissues of the host. An attempt was
made to vary the conditions within certain limits in order to
obtain as large a variety of evidence as possible. Thus both
high and low infections have been studied with each drug and
the dose itself has been varied over a wide range. Each experi-
ment is, however, comparable with the others, as standard
methods of counting and constant technique for the injections
were used throughout this investigation. A count was made
just previous to the injection of the drug which was always
given intravenously, and at intervals of from one to five minutes
after the injection until the reaction between the parasite and
the drug had come to an end.
The results of the experiments are illustrated by figures made
on a logarithmic instead of a linear basis, on account of the wide
range in the values involved.
QUANTITATIVE STUDIES IN CHEMOTHERAPY 459

T,MJ iN Mm UTIS.

FIG. 1. FIG. 2.
460 CARL VOEGTLIN AND HOMER W. SMITH

FIG. 3. Fio. 4.
 QUANTITATIVE STUDIES IN CHEMOTHERAPY 461

ANTI MONYLLAC ATE

‘ri t i
r.7scc t%00 5OWTI *4 PER KILO.

/00.000

100.000
I:

\_
N
2

/0,000

‘I

/,000
I
-0-0- fl,,vrrnoNvL POTAS$SUM T*7*AT(

4.0cc ijoo aoL. -.

-*-- #{228}IW*M0#IYL P07’ASSIVM T.QflTR.41Z

Z 0cc Mt00
i. coo O.PfRNILO.(CCWZiXAANVtN7

FIG. 6.
TIME IN M,NiT($

FIG. 5.
462 CARL VOEGTLIN AND HOMER W. SMITH

Fia. 7.
 QUANTITATIVE STUDIES IN CHEMOTHERAPY 463

DISCUSSION

Inasmuch as this paper deals principally with an investigation

of the fundamental mechanism involved in the sterilization of
the infected animal, the discussion of the pentavalent antimony
compound and of the thioglycollate will be taken up in the paper
dealing with the arsenicals. The evidence regarding the anti-
monials would undoubtedly seem insufficient to support our
conclusions if presented alone, and will be better understood
after a prospective of the entire work.
Fate of parasites. Previous investigators have shown that
trypanosomes are driven from the bloodstream fairly rapidly
by trypanocidal agents, but since most of them used intra-
peritoneal or subcutaneous injections, the actual rate of dis-
appearance was rendered complex by absorption and possibly
also by the destruction of the drug by the host. No particular
attention has been paid, so far as we know, to this rate of dis-
appearance, nor to its bearing upon the fundamental reaction:
drug vs. parasite. Investigation disclosed that it was an excep-
tionally regular process, considering its biological nature, and
that for practical purposes the curves obtained by following the
trypanosome count in the bloodstream after the administration
of the drug could be considered as an accurate index of the re-
action between the drug and the parasites. That the curves
were not influenced wholly or in part as a result of the destruction
of the parasites by the host after they had been killed by the
drug, was established by the injection into normal rats of parasites
killed in vitro. For this purpose the parasites were killed either
by adding a sufficient amount of drug (antimonyllactate) to a
suspension of trypanosomes in saline, or by means of distilled
water after the method of Reynolds and Schoening (1918),
whereby a nearly pure suspension of dead trypanosomes is
secured. Immediately after the intravenous injection of normal
trypanosomes large numbers of the parasites can be found in
the blood, the count soon becoming constant, except for the
gradual increase due to the growth of the infection. When,
however, an examination is made one minute after the injection

THE JOUR. OF PHARM. AND EXPER. THERAP.. VOL. XY, NO. 8

464 CARL VOEGTLIN AND HOMER W. SMITH

of dead trypanosomes, not a single parasite can be found. This

disposal of dead parasites by the host is evidently an exceedingly
rapid process. This destruction is probably very largely due to
cytolysis in the blood stream itself, for the extreme rapidity of
their disappearance both after the injection of enormous numbers
of dead individuals and after the injection of the drug into highly
infected rats, precludes both filtration by the fixed cells of the
body and phagocytosis. Again, if the drug is added to a saline
suspension of normal trypanosomes, they gradually disappear
even at room temperature, and much more rapidly at 37#{176}C.
Moreover, the parasites are never seen to agglutinate in the
bloodstream after the administration of the drug, nor are they
to be found in the leucocytes. The dissolution of the parasites
may be due to the action of autolytic enzymes, or to proteolytic
enzymes in the blood. Whatever the cause, the destructive
action is so rapid that it does not complicate the ultimate result
and the curves obtained in this work may truly be considered
as “death curves” in the sense that they represent the progressive
reaction between the drug and the large number of parasites.
Value of method. The difficulties encountered in most work
on the “fundamental reaction” are eliminated here. Accurate
and direct counts can be readily secured, with sufficient frequency
to follow the reaction closely. This is of particular importance
in the early part of the experiment, and it enables us to follow
the changes closely until 99 per cent of the parasites have been
killed. There may be something of importance to be learned
from the remaining 1 per cent, but it is unlikely.
So far as we are aware, this investigation represents the first
detailed study of the phenomena of sterilization in tivo and for
that reason is especially interesting. Obviously certain compli-
cations render the process more complex, but when it is remem-
bered that the conditions under which the curative action is
called forth are very similar to our experimental conditions,
this fact rather enhances the value of the present report.
Nature of curve. A comparison of our results with those ob-
tained by various observers through in vitro experiments shows
a striking similarity. The principal difficulties encountered in
 QUANTITATIVE STUDIES IN CHEMOTHERAPY 465

in vitro work with bacteria, red blood cells, are diminished here
to a minimum. The uniformity of the individual curves shows
that the experimental error is slight, and considering the rapidity
of the process, the frequency with which counts may be made

TIM IN MINUTES.

FIG. 8

is of considerable advantage. Once the blood is withdrawn from

the rat and diluted, the destruction of the trypanosomes is
checked, and unlike haemolytic experiments, the counts may
be made in a more leisurely fashion.
466 CARL VOEGTLIN AND HOMER W. SMITH

Several outstanding features of the curves may be slightly

emphasized. The process of death with trypanosomes in vivo
is as regular as similar processes in vitro involving bacteria or
red blood cells. This process appears to proceed in each case

/000.000

I00000

10,000

0
IN

I.000

‘0
T/ME ill

FIG. 9. This chart illustrates the close agreement of the curves calculated
from the formula K = dcdt with the actual observations, which were taken from
the preceding experiments.

according to some law which obtains in the mechanism !of

the reaction, drug vs. parasite. Two types of curves are
evident. First, one in which the number of trypanosomes
dying in each interval of time is proportionate to the number
QUANTITATIVE STUDIES IN CHEMOTHERAPY 467

‘-I

#{231}E4
468 CARL VOEGTLIN AND HOMER W. SMITH

TABLE 2

Data relating to curves given in figure 10

‘
CURVE I. DISINFECTION OF S. PYOGENES AUREUS CURVE II. HEMOLYSIS BY ULTRAVIOLET LIGHT

BY PHENOL (CHICK 1910) (BROOKS 1918)

p t. Number Percent- Percent-

T’ ime of bac-
teria (per age
bacteria age bac-
teria sur- Percentage
total time Percentage
cells laked Percentage
cells unlaked
ime drop) dead viving

0 0 1293.0 0 100.0 0 0 100.0

1 6.0 1141.0 12.0 88.0 0.75 1.0 99.0
3 20.0 1044.0 19.0 81.0 1.8 2.0 98.0
4 26.0 952.0 26.2 73.8 2.9 3.0 97.0
5 33.0 708.0 45.3 54.7 5.6 5.0 95.0
6 40.0 543.0 57.2 42.8 8.5 12.0 88.0
7 46.0 401.0 69.0 31.0 13.3 29.0 71.0
8 53.0 243.2 81.2 18.8 22.0 55.0 45.0
9 60.0 202.5 84.4 15.6 31.0 72.0 28.0
10 66.0 156.1 87.9 12.1 44.7 87.0 13.0
12 80.0 45.4 96.5 3.5 100.0 100.0 0
15 100.0 21.8 98.3 1.7

CURVE III. EFFECT OF ANTIMONYLLACTATE ON CURVE IV. EFFECT OF ANTIMONYLLACTATE ON

TRYPANOSOMES TRYPANOSOMES

Thou-
. Thou- Percent- Percent- Percent- Percent-
Percent- san age age Percent- sands age age
tryp&
Time age total trYPa trypa- trypano- Time age total trypa- trypano-
time om nosomes somes Sur- time #{176} nosomes somes sur-
surviv-
dead viving 8urvIV- dead viving

0 0 320.0 0 100.0 0 0 84.0 0 100.0

1 5.9 285.0 11.0 89.0 1 10 70.0 16.5 84.5
2 11.8 255.0 20.0 80.0 2 20 58.0 31.0 69.0
3 17.6 245.0 23.5 76.5 3 30 47.5 43.5 56.5
4 23.5 165.0 49.0 51.5 4 40 36.0 57.0 43.0
5 29.4 110.0 64.0 36.0 5 50 25.0 70.0 30.0
6 35.4 75.0 77.0 23.0 6 60 16.0 81.0 19.0
7 41.2 48.0 85.0 15.0 7 70 11.0 86.9 13.1
8 47.0 35.0 89.0 11.0 8 80 8.0 91.5 8.5
9 53.0 19.0 94.0 6.0 9 90 4.0 95.25 4.75
10 59.0 12.0 96.0 4.0 10 100 2.0 97.60 2.40
11 65.0 11.0 97.0 3.0
12 70.5 8.0 98.0 2.0
13 76.5 5.0 99.0 1.0
14 82.5 4.0 99.0 1.0
16 99.3 1.5 99.5 0.5
17 100.0 0 100.0 0
 QUANTITATIVE STUDIES IN CHEMOTHERAPY 469

left. This is the process of the monomolecular reaction. In

such cases it is evidently necessary to postulate an induction
period, for the process does not immediately acquire its maxi-
mum velocity. An example of this type is curve III, figure 3.
This curve has been plotted again in figure 10 with curves show-
ing the rate of lysis of red cells by ultra-violet light, quoted from
Brooks (1918) and the rate of death of S. pyogenes aureus exposed
to phenol, quoted from Chick (1910). A similar induction period
is evident in both of the other curves cited. In figure 10 all of
the curves have been drawn to the same scale, on a percentage
basis for the sake of comparison. The relative time required
for the destruction of the red cells and the bacteria was much
greater than that required for the destruction of the trypano-
somes. The other type of curve is typical of the majority of
our results (curve IV, fig. 10). From the formula log dcdt =

K, calculated values have been obtained for a few of these curves.

They are given in figure 9 together with a mixed curve, in which
it is supposed that the real process is a mixture of both types,
the early part being of an exponential nature (X in fig. 9), the
latter part of a monomolecular nature (Y in fig. 9). It is clear
that with the exponential type it is not necessary to postulate
any induction period. It seems entirely possible that the true
process is best represented by the exponential curve, and that
it is “slowed up” (curve bent towards the right) in its latter
stages, by a rapidly diminishing concentration of drug in the
blood-stream, or some similar incidental factor.
For the details and the significance of the mathematical ex-
planation of curves of this nature, the reader must be referred
to the investigations of Chick, Arrhenius, Osterhout, Brooks and
others. No attempt can be made to consider here all of the points
of view which have been expressed in the last few years, in an
effort to account for this “process of death.”
Rekztion between size of infection, dose and time. With few
exceptions the process proceeds with regularity throughout, i.e.,
the type of reaction does not change materially in any one ex-
periment. However, different experiments show different rates
of reaction with the same dose of drug. Figure 2 shows several
470 CARL VOEGTLIN AND HOMER W. SMITH

experiments performed with large doses of the drug. It will be

seen that the higher infections are more readily influenced by
the drug, inasmuch as the rate of reaction is greater. It is not
unreasonable to assume that with increasing numbers of the
parasites in the blood stream, toxic metabolism products may
accumulate, or the nutrient value of the medium (blood) may
become impaired, thus rendering the strain as a whole less resist-
ant. In the later stages of the disease, immune bodies may
also have been generated by the host, which might assist the
drug in its action on the parasites and thus complicate the proc-
ess. Previous work has shown that the parasites taken from
highly infected rats are just as virulent as others, but that the
former do not show as great resistance when preserved outside
of the body, as do parasites taken from moderately infected rats.
The old infections die more rapidly; they nevertheless require
just as high a threshold of drug in the bloodstream as do the
younger infections, as will be seen from the analysis of sub-
effective doses (fig. 7).
A priori one might expect that the rate of reaction would bear
some relation to the size of the dose, considering infections of
the same age. But when the dose is gradually decreased it is
found that there is no consistent change in the rate of reaction
until subeffective doses are reached. We are apparently dealing
with a process which, within the limits of minimum effective
dose to minimum toxic dose, proceeds at a practically constant
rate. At sub-effective doses the curve starts downward normally,
turns abruptly, and multiplication of the remaining parasites is
resumed. Under such conditions, such parasites as are left, are
.obviously not injured to an appreciable extent. This recalls the
results obtained with partial hemolysis, where it has been shown
that no partially hemolyzed cells can be found (Handowsky,
1912).
The most significant feature brought out by the experiments
with sub-effective doses is the sharply defined threshold concen-
tration which must be maintained in order to kifi all of the para-
sites. This threshold is dependent to some extent upon the
munber of parasites present. This fact is made evident by glanc-
 QUANTITATIVE STUDIES IN CHEMOTHERAPY 471

ing at the figures for the lower doses. It is the curves of the
high infections which always break first. This relation between
the size of the infection and the size of the dose necessary to prove
effective is open to two explanations. First, it may be that a
certain amount of drug is consumed in killing a certain number
of parasites. It wifi be remembered that a certain amount of
saponin is consumed in hemolysis. However, it seems more
probable that the whole phenomenon produced by sub-effective
doses is to be attributed to absorption of the drug by the tissues
of the host. It is necessary that a certain concentration of the
drug be maintained in the bloodstream throughout the time re-
quired for the process to reach completion, i.e., until all of the
parasites are dead. If absorption by the host has lowered the
amount of drug in the bloodstream below this concentration
before this time, the dose will be sub-effective. Thus various
sub-effective doses of antimonyllactate cease to act not when a
certain number of parasites have been killed, but after a certain
period of time has elasped, during which absorption by the host
has lowered the concentration of the drug in the blood below
the minimum effective threshold (fig. 7). Therefore, the effec-
tiveness of a compound wifi depend to a considerable extent upon
the rapidity with which it is withdrawn from the blood stream
provided that we are dealing with a blood infection and a cura-
tive agent which acts directly upon the parasites.
Minimun effective dose. Because the trypanocidal activity of
a compound is made evident by the reduction of the number of
parasites in the blood and because a fairly well-defined threshold
is necessary to effect this reduction, this method has been used
to standardize various compounds. Although a few parasites
may survive, if the count has been reduced by 99 per cent, it is
obvious that the reactive threshold has been reached. (See figs.
1 to 7.) Therefore, in this and later work, the term “minimum
effective dose” means the minimum dose which given intravenously
will reduce the trypanosomes to none or a very few, regardless of
the time necessary to effect this reduction. We have found it
necessary in establishing the minimum effective dose to confine
ourselves to infections of 100,000 to 300,000 per cubic millimeter
472 CARL VOEGTLIN AND HOMER W. SMITH

of blood. Thus the minimum effective dose of antimonyllactate

is in the neighborhood of 2 cc. M/100 solution per kilo, although
larger doses are required for infections of 800,000 and smaller’
doses are effective for infections of 10,000 to 50,000.
Split doses. In order to gain more definite information as to
the rate of absorption of the drug from the bloodstream by the
tissues of the host, we performed several experiments with split
doses. It will be seen from figure 8 that half of the minimum
effective dose produces a very slight effect. If the other half is
given within about thirty minutes, the blood is freed from para-
sites, showing that the second half supplemented the first half
to make it an effective dose. If we wait longer, however, it
requires more than the minimum effective dose to kill all of the
parasites. This experience supports the hypothesis previously
advanced, that the absorption of the drug by the tissues of the-
host proceeds rather slowly.
These findings cannot of course be generalized as it is necessary
to study this question with each drug. The possibility of the
disposition, of the drug through a chemical change with the for-’
mation of an inactive substance should not be neglected, although
we do not believe that this plays a significant part in the case
of antimonyllactate, a substance which is relatively stable.

SUMMARY

1. The specific action of the antimonyllactate on the try-

panosomes of infected rats can be studied fairly accurately by
following the disappearance.of the parasites from the bloodstream.
2. A sharply defined threshold (minimum effective dose) is
observed, below which the drug has no appreciable effect upon the
parasites. This threshold is in part due to the nature of the
reaction between the drug and the parasites, and in part to ab-
sorption of the drug by the tissues of the host.
3. The process curve is an orderly one, and consistent through-
out individual experiments. However, various experiments in-
dicate two general types of reactions, differing in reaction velocity.
In one type the reaction velocity remains constant throughout-
 QUANTITATIVE STUDIES IN CHEMOTHERAPY 473

the experiment. In the other, the reaction velocity is constantly

increasing.
4. A method for the rapid and accurate determination of the
trypanocidal power of drugs (minimum effective dose) is
recommended.
REFERENCES

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