Journal of Pharmacology and Experimental Therapeutics v 96 (l949)
EXPERIMENTAL CHEMOTHERAPY OF TRYPANOSOMIASIS
I. EFFECT OF P-PHENYLENE DIGUANIDINE AND RELATED COMPOUNDS
AGAINST EXPERIMENTAL INFECTIONS WITH Trypanosoma equiperdum
R. I. HEWITT. A. GUMBLE, S. KUSHNER, S R. SAFIR, L. M. BRANCONE AND Y.
SUBBAROW
Lederle Laboratories Division, American Cyanamid Company, Pearl River, New
York
Received for publication April 4, 1949
INTRODUCTION
A search was begun in our laboratories in 1944 for a compound which might possess
advantages over existing agents for the treatment of trypanosomiasis, and particularly for one
which would be effective when administered orally. Very early in the course of this screening
program it was found that p-phenylene diguanidine possessed curative properties against
Trypanosoma equiperdum in mice when administered either orally or intraperitoneally. It was
not as powerful a trypanoride as Bayer 205 or several other known compounds, but it
possessed several interesting properties which encouraged extensive investigations. The
parasitological data obtained with p-phenylene diguanidine and several related compounds in
experimental animals is presented in this paper, and the chemistry of the group is discussed in
detail elsewhere (1).
The effectiveness of guanidine compounds in experimental trypanosomiasis was first
demonstrated by British investigators. King, Lourie and Yorke (2) showed that a series of
homologues of Synthalin possessed trypanocidal properties. The trypanocidal action rose as
the number of methylene groups in the alkylchain was increased; the decane, dodecane
and tetradecane derivatives were the most active. Activity was then found in alkylene
diamidines in which two guanyl groups were attached to the ends of the methylene chain without
the intervention of other groups. Lourie and Yorke (3) then proceeded to aromatic diamidines
and selected 4,4'-diamidino stilbene (Stilbamidine) as the most active member of the
group. St. A. Heathcote (4) gives a recent summary of the pharmacology and
therapeutic uses of Stilbamidine and related compounds.
The guanidine compounds described in the present report, and more particularly pphenylene diguanidine, are not so strikingly effective in laboratory infections with trypanosomes
as are the aromatic diamidines. p-Phenylene diguanidine, however, has a relatively simple
structure, and it cures T. equiperdum infections in mice in well-tolerated doses when
administered orally.
MATERIAL AND METHODS. Mice infected experimentally with T. equiperdum
were used as laboratory hosts throughout the major part of these investigations. Each
mouse was inoculated intraperitoneally with a saline suspension containing approximately
50,000 to 100,000 trypanosomes, or an average of 2.5 million trypanosomes per kilogram
body weight in 20 gram mice. This parasite produces highly consistent infections in
untreated mice, with death occurring almost invariably within three to five days
after inoculation with the above number of trypanosomes. The strain of T.
779
equiperdum used was obtained in 1945 through the kindness of Dr.A.LTatum,
University of Wisconsin.
Some deviations from the traditional testing methods for trypanocides used by any
investigators have been used in our program. Most screening tests performed with
arsenicals have involved the "single dose technique"; that is, one dose of the drug is
administered 24 hours after inoculation with parasites. Minimum therapeutic doses and
curative doses are then determined according to the number of animals which survive
over a thirty-day period. The single dose which kills one-half of the animals (LD50) divided
by the single dose which cures one-half of the animals (CD50.) is called the
chemotherapeutic index. This is a well recognized standard and is used in a
number of other in vivo biological assays.
The single dose technique appears to provide a satisfactory trypanocidal assay
with compounds which are not excreted rapidly. For screening new compounds about
which nothing is known concerning absorption and excretion, however, the exclusive use
of this method could conceivably bypass many types of compounds which might possess
trypanocidal properties when administered more frequently. In our screening program,
therefore, multiple doses of compounds were administered to infected mice over a period
of at least three days after inoculation with parasites. The variations of the technique used
are described below.
For routine screening, compounds soluble in water were
administered intraperitoneally in most cases, and compounds insoluble in water were
administered orally in a 2.5 per cent suspension of corn starch. This was purely a mutter
of convenience in the initial screening tests, since in our hands the intraperitoneal method
of administration when possible was easier, faster and less liable to produce death by
mechanical injury' in mice than oral administration. If sufficient compound was available,
50 mgm. per kgm. intraperitoneally, or 200 mgm. per kgm. orally was used as the initial
dosage for the first screening test. If death from toxicity occurred at these doses the
quantity was halved and tested again. Active compounds were retested at several
dosage ranges.
Method A. For preliminary assays of all new compounds, dosage was
administered once on the day of inoculation with parasites, and twice daily thereafter with
eight hours between doses, for three days. Untreated controls were always included.
Since T. equiperdum invariably kills mice on the third to fifth days after inoculation,
trypanocidal or suppressive effects could be determined one or two days after treatment
was stopped, without necessitating counting the numbers of trypanosomes present. This
procedure provides a quick method for spotting active substances, and is also valuable for
comparing the action of different derivatives within any group of compounds. In screening
tests mice were not held for extended observation periods after cessation of treatment.
Method B. After preliminary tests had shown that p-phenylene diguanidine
appeared to be the most promising of any of the guanidine derivatives tested, several
series of mice were treated twice or three times daily for from 3½ days to 2 weeks after
inoculation with parasites, in order to determine whether the curative dose could be
lowered by repented dosage. The first groups of mice listed in table 2 were treated once
on the day of inoculation with trypanosomes, 3 times daily for the next 4 days, with 4-hour
780
intervals between dosage, twice on the 6th day, and once on the 7th day. The second
groups were treated similarly for the first week, and were then treated 3 times daily
from the 8th to the 12th days, with 2 doses on the 13th day and 1 dose on the 14th
day. The third groups were treated once on the 1st and 7th days, and twice on the 2nd to
6th days with 8-hour intervals between doses. The fourth group was treated once on the
1st, 7th and 14th days, and twice daily on the 2nd to 6th days and the 8th to 13th days.
These mice were then observed for 30 days after cessation of treatment to determine
curative effects. Mice which lived throughout the period of observation were considered
cured. It was assumed that the mice which died during this period succumbed to their
trypanosomes, even though parasite counts were not made immediately before death.
This procedure has been used by many investigators previously, and has become a more
or less standard practice in the experimental chemotherapy of T. equipped infections. The
comparison between the activity of several guanidines, Stilbamidine, and Bayer 205 given
in table 7 were made on the basis of multiple doses with all compounds listed.
Method C. In order to test the efficacy of p phenylene diguanidine against T.
equiperdum after the parasites had had an opportunity to become well established in the
host. one series of mice was not treated until 54 hours after inoculation with parasites.
These data are given in table 9.
Several different samples of p-phenylene diguanidine were used for bioassay. The
first two batches received were impure bicarbonate salts.
These were
tested as such, and the second batch was then purified. The hydrochloride was
prepared from the second batch, recrystallized from aqueous alcohol. A third batch was
received as the mixed carbonate and bicarbonate salts. Unless otherwise noted, the
results of tests given in the tabular data were obtained with the hydrochloride.
DEFINITION OF TERMS: The following definition of terms is given as they
are used in the present paper:
Suppressive effect. Treated mice lived for varying periods of time after all
controls were dead, but less than 50 per cent survived the 30-day observation period after
cessation of treatment.
Curative effect. Fifty per cent or more treated mice survived for 30 days after
cessation of treatment.
Minimum therapeutic dose (M.Th.D.). The least dose needed to produce a
suppressive effect.
Curative dote (CD50). The least dose needed to produce a curative effect.
Lethal dote (LD50). The least dose needed to kill 50 per cent or more mice within
24 hours after a single dose or on the first or second day of multiple dosages when
multiple dosage was used.
Chemotherapeutic Index (C.L.). The lethal dose divided by the curative dose.
When multiple dosages were
used
the
chemotherapeutic
index
was
781
determined on
this
basis.
RESULTS. p-Phenylene Diguanidine. The oral and intraperitoneal curative doses
of this compound in mice are lifted for 21 series of tests in table 3. Tables 1 and 2
illustrate the method used for determining the curative dose. It will be noted in table 3 that
some variation occurred with different samples of the compound and in different
experiments with the same sample. In general, less variation occurred when doses were
administered for seven days or longer. When treatment was administered for seven
doses ( t wice daily for three and one-half days), using Method B, and the curative
(CD 50 ) intraperitoneal dose of the hydrochloride salt approximated 4 mgm. per kgm,
and the curative (CD50,) oral dose was about 20 mgm. per kgm. When the treatment
period was extended to one or two weeks, the curative (CD50) intraperitoneal dose was 2
mgm. per kgm., and the curative (CD50) oral dose about 15 mgm. per kgm. When
compared with intraperitoneal curative doses of Stilbamidine and Bayer 205 administered
under similar conditions (table 7), p-phenylene diguanidine is much the least effective of
the three compounds. The oral curative doses, however, present a somewhat different
picture. It is five times more effective when given orally than Bayer 205, and one-sixth as
effective as Stilbamidine administered orally. The ratio of intraperitoneal to oral
treatment (table 7) also demonstrates the marked difference between these two routes
of administration with these three compounds. For example, 500 times more Bayer 205 is
required to produce cures by oral treatment than by intraperitoneal treatment, and 120
times more Stilbamidine. Only five times more p-phenylene diguanidine is required to
obtain a curative oral dose than a curative intraperitoneal dose. The mode of action of any
of these trypanocidal substances is thus far unknown
In rabbits infected with T. equiperdum, 10 mgm. per kgm. of p-phenylene
diguanidine administered intraperitonealiy or intravenously, twice daily for fourteen days,
will produce cures, but 5 mgm. per kgm. will not. Rabbits were inoculated intravenously
with a saline suspension of trypanosomes obtained from a heavily infected mouse, and
were held before treatment until symptoms appeared or until trypanosomes were found
in the blood. T.equiperdum in rabbits is notably more resistant to treatment with
arsenicals than the same parasite in mice (6).
782
TABLE
1
Comparison of tingle dote and multiple dose treatment with p-phenylene
diguanidine (T. equiperdum in mice)
DEATHS—AFTER
INOCULATION
NO. OF
DOSE .1.2.
Mgm./kgm
0.2
0.4
0.8
1.6
3.0
6.3
Controls
0.2
0.4
0.8
1.6
3.0
6.3
12.5
25
50
100
Controls
NO. OF
TREATMEN
ANIMALS
T
7*
7*
7*
7*
7*
7*
None
1†
1†
1†
1†
1†
1†
1†
1†
1†
1†
None
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
1-2
days
3-5
days
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
10
10
10
1
1
0
10
10
10
8
10
6
5
1
0
1
0
10
6-15 16-30
days days
—
—
—
9
1
0
—
1
1
—
—
—
—
—
—
—
—
—
1
1
—
__
—
2
—
4
5
9
10
7
3
—
ACTIVITY
None
None
None
Suppressive
Curative
Curative
None
None
None
None
Suppressive
Suppressive
Suppressive
Suppressive
Suppressive
Curative
* One treatment on the first day of inoculation with trypanosomes; two treatments daily
for the next three days, with approximately eight hour intervals between daily doses.
† One dose administered twenty-four hours after inoculation with trypanosomes.
When treatment with p-phenylene diguanidine was delayed in mice for 54 hours
after inoculation with T, equiperdum the oral curative dose (CD50) was about 25 mgm. per
kgm, and the intraperitoneal curative dose (CD50) was about 8 mgm. per kgm. (Table 9).
This demonstrates that when the compound is introduced shortly before the crisis or peak
of parasitemia, somewhat higher doses are needed to clear the blood of trypanosomes
than when treatment is initiated within a few hours after infection.
783
TABLE 2
Effect Of Multiple Treatments With P-Phenylene Diguanidine Against
Trypanosoma, Equiperdum In Mice (Series E-493)
DOSE, MGM/KGM.
ORAL
NO.
MICE
USED
DEATHS
WEEKS AFTER INOCULATION
1
1
2.5
5
7.5
10
15
20
25
T.I.D. 15 doses
T.I.D. 15 doses
T.I.D. 15 doses
T.I.D. 15 doses
T.I.D. 15 doses
T.I.D. 15 doses
T.I.D. 15 doses
T.I.D. 15 doses
10
10
10
10
10
10
10
10
10
10
1
1
2.5
5
7.5
10
15
20
25
T.I.D. 33 doses
T.I.D. 33 doses
T.I.D. 33 doses
T.I.D. 33 doses
T.I.D. 33 doses
T.I.D. 33 doses
T.I.D. 33 doses
T.I.D. 33 doses
10
10
10
10
10
10
10
10
10
10
1
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
37
10
10
7
2
1 B.I.D. 12 doses
2.5 B.I.D. 12 doses
5 B.I.D. 12 doses
7.5 B.I.D. 12 doses
10 B.I.D. 12 doses
15 B.I.D. 12 doses
20 B.I.D. 12 doses
25 B.I.D. 12 doses
1 B.I.D. 25 doses
2.5 B.I.D. 25 doses
5 B.I.D. 25 doses
7.5 B.I.D. 25 doses
10 B.I.D. 25 doses
15 B.I.D. 25 doses
20 B.I.D. 25 doses
25 B.I.D. 25 doses
Controls……………...
2
6
5
1
3
1
3
4
5
1
1
1
2
2
7
7
4
1
1
1
1
1
10
10
9
4
1
3
4
1
3
1
1
3
5
1
1
37
784
3
2
6
TOTAL
DEATH
PERCENT
0 WKS
EFFECT
Per cent
100
100
90
90
2
00
20
0
None
None
Suppressive
Suppressive
Curative
Curative
Curative
Curative
100
100
100
70
50
10
0
0
None
None
Suppressive
Suppressive
Curative
Curative
Curative
Curative
100
100
100
70
10
10
10
10
100
100
100
100
60
60
10
0
100
None
None
None
Suppressive
Curative
Curative
Curative
Curative
None
None
None
Suppressive
Suppressive
Suppressive
Curative
Curative
* Dosage intervals explained in the text.
In one experiment designed to test the efficacy of administering the compound in
the diet, 50 per cent of infected mice survived for 30 days after one week's feeding on
0.125 per cent p-phenylene diguanidine in the; feed. Higher concentration s
of the drug in the feed were not tolerated. The drug was mixed with finely ground food
pellets and placed in small earthenware food cups available to the mice et all hours of the
day and night for one week.
TABLE 3
Estimation of curative dose of p-phenylene diguanidine in 21different test
series. (T. equiperdum in mice)
* Sample No. 1
Sample No. 2
Sample No. 2A
Sample No. 2B
Sample No. 3
= First batch, impure bicarbonate.
= Second batch, impure bicarbonate.
= Purified carbonate from No. 2.
= Purified and recrystullized hydrochloride from No. 2
= Third batch; bicarbonate 42%, carbonate 58%.
Other guanidines and related compounds. Eleven compounds related
structurally to p-phenylene diguanidine are listed in table 4. These produced no
trypanocidal effects in the doses indicated. It should be noted particularly that ortho and
785
meta phenylene diguanidine were completely inactive. In table 5 eleven compounds are
listed which showed varying decrees of effectiveness against T. equiperdum. Four of
these (83-L, 318-L, 331-L and 340L) possess activity which is very near to that of pphenylene diguanidine. With the exception of 331-L (bis-(4-guanidophenyl)-sulfide
carbonate), however, these compounds are less active when administered orally than pphenylene diguanidine. Compound 331-L is as effective as p-phenylene diguanidine, and
possibly slightly more so, when administered either orally or intraperitoneally (table 6). Its
activity, however, is not markedly different from the simpler p-phenylene diguanidine.
Several Fuchsin-guanidine compounds were also made, because of the marked
similarity in dosage and activity between basic Fuchsia and p-phenylene diguanidine. The
trypanocidal activity of fuchsin dyes has been known for nearly fifty years
TABLE 4
Compounds related to p-phtnylene diguanidine which show no
trypanocidal activity in the doses used
 Toxic at higher doses.
Basic Fuchsia produces cures in mice infected with T equiperdum at almost
exactly the same oral and intraperitoneal levels as p-phenylene diguanidine. It is
considerably more toxic, however. Biguanidine and triguanidine substitutions were made
on the fuchsin molecule, and the activity remained at about the same level as either of the
parent structures, providing methyl groups were not added to one of the phenyl rings.
Triphenylmethane-4, 4’, 4"-triguanidine was more active than triphenyuncthane-4, 4'diguandine. Because of the toxicity of these compounds, however, investigations of the
group were stopped.
786
TABLE 5
Compounds related to p-phenylene diguanidine which show
trypanocidal activity
the single dose needed to produce cures. The chemotherapeutic indices given for pphenylene diguanidinc when administered in multiple doses (table 6) are lower than
those which have been reported for Melarsen (5), p-arseno-phenyl-butyric acid (6)
and Stilbamidine (3) when administered in a single dose.
The fact that few guanidines related to p-phenylene diguanidine showed significant
trypanocidal activity was rather surprising. Although all possible deviations in structure
were nut investigated, a sufficient number of related derivatives were tested to
demonstrate that p-phenylene diguanidinc per se probably possesses the maximum
potentialities in this particular group of guanidines.
Chronic toxicity studies will be helpful for evaluating the potentialities of
p-phenylene diguanidine for clinical use in man. Some studies have been undertaken with
787
regard to the distribution of the compound in the central nervous system of laboratory
animals, but the occurrence of other guanidines in normal blood and body fluids
interfered with quantitative determinations.
TABLE 6
Comparison of four guanidines by the multiple dose technique. (Series E-417)
(T. equiperdum in mice)
TABLE 7
Comparison of Stilbamidine, Bayer 205 and four guanidine derivatives. Multiple
dose
technique. (T. equiperdum in mice)
788
TABLE 8
Comparison of three arsenicals by the single dote technique
(T. equiperdum in mice)
* Eagle et al.,1944.
† Weinman, 1946.
No effect has been demonstrated by p-phenylene diguanidine or related
derivatives against Leishmania donovani in hamsters, Schistesoma mansoni in mice,
Litomosoides carinii in cotton rats, Plasmodium lophurae in ducks or Trypanosoma cruzi
in mice.
Effect
of
DOSE
TABLE 9
p-phenylene diguanidine against T. equiperdum when
is started fifty-four hours after inoculation
NO. OF
NO. OF
TREATMENTS
*
MICE
dosage
DEATHS—DAYS AFTER
INOCULATION
1-2
days
3-5
days
6-15
days
16-30
days
ACTIVITY
mgm./kgm.
5.0 oral
13
10
10
None
10.0 oral
13
10
10
None
15.0 oral
13
10
7
1
2
Suppressive
20.0 oral
13
10
6
3
Suppressive
25.0 oral
13
10
1
3
Curative
1.0 Ip.
13
10
10
None
2.0 Ip.
13
10
9
1
None
4.0 Ip.
13
10
2
1
3
Suppressive
8.0 Ip.
13
10
1
1
Curative
None
Controls
10
10
* Treated twice daily for seven days with the exception of the sixth day then a single dose
as administered.
789
SUMMARY
Data are given which demonstrate the trypanocidal activity of p-phenylene
diguanidine and related derivatives against Trypanosoma equiperdum in mice. This
compound is much less effective when administered parenterally than arsenicals and
other known trypanocides such as Bayer 205 or Stilbamidine. It is effective when
administered in multiple doses orally, however, and for this reason is considered to be of
potential interest for trial in man or domestic animals. Of the related guanidines tested
none showed markedly greater activity or were better tolerated than pphenylene diguanidine.
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ORG. CHEM.,13: 924, 1948.
2. KING,H.,LOURLE,E.,AND YORKE, W.: LANCET, 233: 1360, 1937.
3. LOURIE, E.,AND YORKE, W.: ANN. TROP. MED., 33: 289, 1939.
4. ST. A. HEATHCOTE, R.: J. TROP. MED. AND HYG , 49: 1, 33, 1946.
5. WEINMAN, D.: AM. J.TROP. MED., 26 (SUPP.): 95, 1946.
6. EAGLE,
H., HOGAN, R., DOAK,
HEALTH REPORTS, 59: 765, 1944
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STEINMAN,
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