The Anti-leukemic Action of Combinations
Known Anti-leukemic Agents*
HOWARD
E. SKIPPER,
(From the 0r@c
JUANITA
B. CHAPMAN,
of Certain
AND MARTELIA
BELL
and Biochemistry Division, Southern Research In@iituie,Birmingham, Ala.)
then obtained by simultaneous administration
of
The present study of the anti-leukemic
action
fractions of the LD50 of the various treatments
of combinations
of certain rather diverse agents
(from different syringes). In the instances where
known to prolong survival time of mice with trans
x-ray was employed in combination
with other
planted leukemia was prompted by the observa
tion of an apparent anti-leukemic
synergism be
TABLE 1
tween urethan
and methyl-bis(@-chloroethyl)a
AcuTE
(SINGLE
INJEcTIoN)
TOXICITY DATA
mine (G).
ON
KNOWN
ANTI-LEUKEMIC
AGENTS
Urethan (3, 4, 5, 11), methyl-bis(@-chloroethyl)
ApproximateLDs.AgentVehicle(mg/kg)Aminopterin
amine (HN@) (3, 4, 9), benzene (8, 6), potassium
arsenite
(3, 6), colchicine (3), 4-aminopteroyl
glutamic acid (aminopterin)
(@, 10), and x-radia
Peanut oil8.5
1,150ColchicineSaline2.42,6-Diaminopurine10
tion (6) have all been shown to alter the course of BenzeneSaline
transplanted
mouse leukemia.
All the above
2508
per cent gum
mentioned agents, with the exception of colchicine,
aeacia5Ca.
Azaguanine10
per cent gum
100
have been used clinically with some degree of tem
acacia5Between
1,000HN2Saline5.2KAsO,Saline18.0UrethanSaline1,800X-radiation600
and
porary paffiative effect (7). In addition,
@,6-dia
minopurinc and 8-azaguanine have been recently
shown to increase the life span of mice with certain
roentgen
strains of transmitted
leukemia (1, 8, 10).
aPulverized
andsuspended
in10percentgumacacia.
From a theoretical
standpoint,
it seemed
NoTE: Observation period Wft5uniformly 10 days.
worth-while to carry out certain trials of this na
ture to determine, if possible, which therapeutic
TABLE 2
agents were additive in their anti-leukemic activ
MAXIMUM
DoSE OF CERTAIN ANTI-LEUKEMIC
ity. Such information might assist in determining
AGENTS TOLERATED
BY MICE ON SEVEN SiJc
the homogeneity
of the mechanism of action of
CESSIVE DAILY INTRAPE1IITONEAL
INJECTIONS
this most heterogeneous group of agents.
Maximum
PROCEDURE AND RESULTS
As a first step, the acute LD50 of the nine thera
peutic procedures
was determined
using CFW
strain mice. The compounds were administered by
the intraperitoneal
route, and total body x-radia
tion was employed. The results of these acute
toxicity determinations
are presented in Table 1.
These values are approximations
obtained in most
instances from three points on a log-probability
plot.
An indication of the acute toxicity of certain
dual combinations of these therapeutic agents was
S This
Society,
work
was
supported
upon recommendation
by
the
American
of the Committee
Research
Institute;
and
Mr.
Ben
on Growth
May,
Alabama.
Received
for publication
September
25, 1950.
Mobile,
Vehicle
Aminopterin
Saline
Benzene
Peanut
dose (mg/kg)
0.23
oil
250
Colchicine
Saline
0.63
HNQ
Saline
0.75
KAsO,
Urethan
Saline
Saline
4.5
700
2,6-Diaminopurine
8-Azaguanine
Gum acacia
Gum acacia
100
31
X-ray5
150r
aGiventwicewithina 5-week
intervalwithnodeaths.Thisis
not a maximum tolerated dose.
materials,
administration
ceded the x-radiation
With knowledge
Cancer
of the National Research Council; the Research Fund of the
Southern
tolerated
Agent
of the compounds
pre
by 30-60 minutes.
of the maximum
tolerated
dose
of the individual agents for seven successive daily
injections
(previously
determined
for anti-len
kemic assays, Table
@),it was possible to select
combination
doses that, if not synergistic with
regard to toxicity, might be used in anti-leukemic
109
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TABLE
3
ANTI-LEUKEMICSCREENINGDATA ON CERTAIN COMBINATIONSOF KNOWN ANTI-LEUKEMICAGENTS
Exp.
no.
1:
Dosage
(mg/kg)
Treatment
Controls
Aminopterin
Urethan
11N2
Am+urethan
Am+HN2
(Am)
0.25
700
0.75
0.12+225
0.12+0.5
Urethan+HN2
2:
450+0.38
Controls
Aminopterin (Am)
Coichicine
Am+colchicine
Am+benzene
Am+KAsO,
3:
Am+x-ray@
Controls
4:
Aminopterin (Am)
Am+colchicine
Controls
0.23
0.63
0.12+0.51
0.12+125
0.12+2.5
0.12+150
r(twice)
0.23
0.12+0 .51
Am+x-ray
Benzene+x-ray
KAsO+x-ray
HN2+x-ray
(Am)
Coichicine
Am+colchicine
Controls
6:
Aminopterin
(Am)
Benzene
X-ray
Urethan
HNQ
Coichicine
KA@,
2,6-Disminopurine
Am+benzene
Am+x-ray
Am+urethan
Am+HN2
Am+colchicine
Am+KAsO,
Am+2,6-diaminopurine
Urethan+HN2
7:
Controls
0.23
0.18
Aminopterin
0.12
0.06
Urethan
HN2
700
0.75
125
2,6-Diaminopurine
8-Azaguanine
2. 0 (twice daily)
350+0.38
50+2. 0(twice
Urethan+HN2
2,6-Diaminopurine+8-azagu
anine
8:
cent
(dsys)
in days
untreated controls
12.5
20.4
16.2
15.3
11—16
16—28
14—55
11—28
19.3
16—23
19.5
19.0
11—28
14—28
12.7
11—17
19.1
16.4
19.1
16.5
15—22
11—55
14—35
14—23
18.5
14—22
17.5
11.0
14—28
9—15
17.5
17.7
12—25
12—26
in
survival
1.0
0.48
+24.4
0.37
0.74
0.89
+
+
+
+
+
+
50.4
29.1
50.4
29.9
45.7
37.8
1.0
0.76
0.01
0.18
0.28
0.66
0.62
0.83
0.65
0.80
0.55
+ 60.0
+ 9.0
+ 88.0
1.0
0.16
0.63
+100.0
1.0
0.009
0.89
0.29
0.41
0.12
0.006
0.18
0.28
0.44
0.66
0.68
0.68
0.63
0.65
0.60
+
+
+
+
+
+
+
11—16
9—14
8—14
10—14
0.91
0.76
59.2
44.2
0.8
7.5
15.8
38.8
86.7
49.2
10.9
17.5
52.5
29.5
41.0
11.5
0.5
12.8
+28.2
10—15
1.0
0.69
+
+
+
+
+
+
+
+
+
+
+
+88.5
11—14
1.0
0.58
I .0
+
+
+
+
+
7—10
0.83
+ 59.1
+ 60.9
— 0.9
7—15
9—14
43.6
56.4
67.7
62.8
62.6
66.1
50.0
8.8
7—14
16.8
15.7
13.9
9.8
14—20
14—19
10—17
8—15
11.3
9—15
+ 28.4
11.1
14.7
8.6
10—13
14—18
7—10
+ 67.0
12.7
11—22
+44.8
1.0
+90.9
1.00
+ 78.4
+ 58.0
0.86
0.64
+
0.18
0.31
0.29
0.74
11.4
+26.1
— 2.8
0.49
0.0
0.0
7—11
8.1
increase
+ 65.8
+ 31.7
+ 48.8
+ 58.5
+ 54.5
10—15
17—25
12—SO
9—17
12—15
11—18
14—20
13—26
14—23
9—20
12—22
15—18
9—11
13—22
9—13
1O@—24
7—10
10—23
7—8
8—15
7—15
9—15
7—9
7—10
R.C.I.*
daily)
2.0
A-methopterin
A-methopterin+8-azaguanine
—Per
Per cent increase
survival over
8.8
Controls
S R.C.I.
extremes
0.23
0.75
250
HN2
Benzene
X-ray
KAs@h
Am+benzene
Am+KAsO@
Controls
Aminopterin
Deaths,
survival
12.0
19.1
17.3
12.1
12.9
150 r(twice)
4.5
15.6
0.12+125
16.0
0.12+2.3
16.4
0.12+150 r (twice) 17.9
15.5
250+75 r (twice)
2.5+150
r (twice)
14.1
15.9
0.5+150
r (twice)
10.0
0.23
16.0
0.63
10.9
0. 12+0.51
15.8
7.8
0.23
15.6
250
7.1
10.8
800 r (twice)
700
10.1
0.75
11.0
0.63
8.7
4.5
8.2
100
8.8
0.12+125
10.0
0 .12+150 r (twice) 11.2
0.12+225
12.2
0.12+0.5
12.5
0.12+0.51
12.7
0.12+2.3
11.9
0.12+50
12.1
22@+0.5
11.7
Aminopterin (Am)
5:
Average
2. 0+10
of
a
candidate
compound
or
7—9
20.3
16—37
17.8
12—28
combination/Per
cent
increase
+150.6
+119.8
in
survival
of
1.00
0.79
aminopterin
(or
A-methopterin).
t X-ray was in allinstances administered on the second and the ninth days after inoculation withleukemic spleen brei.
@
Coichicine given at 0.655 mg/kg by mistake 4-0.15 mg/kg of aminopterin
on ninth day. This probably caused early deaths.
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SKIPPER
et al.—Combinciions
screening of combinations of agents. The selected
combination doses were then checked for toxicity
on groups of four mice each. The highest levels
of the two agents causing no deaths on 10 alter
nate day administrations
followed by a 10-day
observation period were selected for anti-leukemic
screening trials. It was not the purpose of the
present effort to determine accurately the acute
and cumulative
toxicity of the combinations
of
agents studied. Rather, it was intended to gain in
formation as to doses of combinations of important
chemotherapeutic
agents which could be used in
screening against transmitted
mouse leukemia.
Briefly, none of the combinations appeared to be
profoundly synergistic with regard to toxicity in
this cumulative toxicity screening.
After
having
obtained
the
necessary
toxicity
data, a number of combinations
of the above
mentioned agents were screened for anti-leukemic
activity, by the use of a procedure that has proved
generally satisfactory
in several laboratories
(3,
8, 9). The inbred mice employed were of the Akm
strain (brother-sister
inbred for genetic control).
The leukemia transplanted
in this study was the
rather acute AK4 strain which kills untreated
mice in about 7—1@
days. In the usual assay, a
large group of Akm mice received intraperitoneal
inoculations of 0.1 cc. of leukemic spleen brei that
contained about one million cells. Two days later,
these animals were divided into an appropriate
number of experimental groups of ten mice each.
One group was retained as an untreated control;
other groups were treated, starting on the second
post-inoculation
day, at the maximum tolerated
level with the agent or combination of agents un
der study.
The results of these assays are reported in
terms of the average per cent of increase in life
span of treated over untreated
groups of mice
(Table 3).
DISCUSSION
As can be seen in Table 3, emphasis in this in
vestigation has been centered around an attempt
to increase theanti-leukemic
action of aminopterin
or A-methopterin
in this rather acute strain of
mouse leukemia. We have observed no indication
of synergism (in Ak4 leukemia) between the fol
lowing pairs of agents:
1. Aminopterin+urethan
@.Aminopterin+nitrogen
mustard
3. Aminoptcrin+colchicine
6. Aminopterin+x-radiation
7. Aminopterin+@,6-diaminopurine
arsenite
Agents
111
8. A-methopterin+8-azaguanine
9. Benzene+x-radiation
10. Potassium arsenite+x-radiation
11. Nitrogen mustard (HN@)+x-radiation
In three experiments where the combination of
urethan plus nitrogen mustard was employed, it
appears that we have confirmed (using a different
leukemic strain) the synergistic anti-leukemic
ef
feet between these two compounds,
which was
previously reported (9) . However, in this rather
acute leukemia (Ak4) the combination of urethan
plus nitrogen mustard is not as effective as are the
anti-folic acid type compounds.
The present data are not sufficient to allow for
deduction with regard to additive effects of the
various
combination
therapy,
except
perhaps
to in
dicate that generally one-half the maximum toler
ated dose of aminopterin plus one-half the maxi
mum tolerated dose of the other agents studied
was not as effective as the maximum tolerated dose
of aminopterin alone, except perhaps in the case
of aminopterin plus colchicine.
SUMMARY
A number
of combinations
of known
anti
leukemic agents has been screened against mouse
leukemia in search of synergistic activity. Only
urethan plus nitrogen mustard exhibited evidence
of synergistic activity, and this combination was
less effective than aminopterin or A-methopterin
for the rather acute Ak4 strain of leukemia used
in these studies.
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The Anti-leukemic Action of Combinations of Certain Known
Anti-leukemic Agents
Howard E. Skipper, Juanita B. Chapman and Martelia Bell
Cancer Res 1951;11:109-112.
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