[CANCER
RESEARCH
26 Part 1, 2292-2296, November
1966]
Effects of Hydroxyurea and Related Compounds
Marrow of Experimental Animals
LEONARD
J. LERNER, ALBERT BIANCHI,
Squibb Institute for Medical Research, New Brunswick,
EURIPIDES YIACAS,
Hydroxyurea prevented the leukocytosis that occurs in mice
inoculated with L1210 leukemia. This activity probably ac
counted for the increase in survival time which was found in
these animals when they were treated with hydroxyurea.
Hydroxyurea also reduced all blood cellular elements in the
immature male rat in direct relationship to the dose employed.
The highest daily dose employed in this study (125 mg/rat) also
caused a reduction in body weight gain, food consumption, and
endocrine organ weights.
Compounds related to hydroxyurea were also effective in
reducing the number of RHC and WBC in the rat. Several of
these decreased the WBC while not affecting the other end points
examined.
Introduction
The antileukemic and antitumor activity of hydroxyurea
(Hydrea—Squibb) in the experimental animal has been reported
by a number of investigators, including ourselves (5, 8, 9, 12, 13).
This compound has also been reported to be an effective agent
in the treatment of human malignancies (2, 6, 7, 14). The effec
tiveness of this compound in the therapy of leukemia is due to
its depression of the bone marrow and the resultant depression
in leukocyte production. However, this property is an unwanted
side effect in nonleukemic cancer chemotherapy. Rosenthal
et al. (11) reported in 1928 that the administration of hydroxyurea
in the dog and rabbit resulted in a sharp reduction in bone
marrow production of RBC and WBC. This present study was
undertaken to correlate peripheral blood cell counts with survival
in L1210 leukemic mice treated with hydroxyurea. Another
purpose of this investigation was to determine the effect of
hydroxyurea, its analogs, and related compounds on the blood,
marrow, various organs, and body weight of normal young rats
to ascertain if toxicity and leukopenia were related or separable.
and
Methods
Leukemia was studied in BDFi mice. Ascitic fluid was aspirated
from the peritoneal cavity of DBA/2 JAX mice inoculated with
L1210 leukemia (tumor line LE02R/23) diluted to 100,000
cells/0.1 ml of inoculum and administered i.p. to the test animals.
Standard procedures1 were followed throughout the study.
1Screening Protocol for Lymphoid Leukemia L1210. Cancer
Chemother. Kept., 1: 45-53, 1959.
Received November 15, 1965; accepted June 1, 1966.
2292
ALECK BORMAN
New Jersey
Summary
Materials
AND
on the Blood and
Hydroxyurea dissolved in water was administered s.c. once
daily starting on Day 6 postinoculation. Control mice were
administered the aqueous vehicle. Each dose group consisted
of 10 mice divided into 2 subgroups of 5 animals. Additional
dose groups of 10 mice each were maintained for hematology.
The mice were bled via the tail vein on Days 7, 9, and 11 of the
study and RBC and WBC were made using a Sanborn-Frommer
cell counter. Deaths were recorded daily.
Immature male rats of the Sprague-Dawley strain weighing
approximately 60-70 gm were employed in the study of the effects
of hydroxyurea and related compounds on blood and selected
organs. In the initial study (Tables 3, 4) hydroxyurea was sus
pended in sesame oil and administered s.c. daily for 10 days to
dose groups of 6 animals. In the other studies the test compounds
were dissolved in water and administered s.c. daily for 10 days
to dose groups of 8 or 12 animals. The rats were sacrificed by
decapitation on the day following the last injection. Prior to
sacrifice blood was taken from the tail vein for hématologie
evaluation. The RBC and WBC were made in the SanbornFrommer cell counter. In addition the concentration of neu
tro phils, lymphocytes, and reticulocytes was determined; the
hematocrit was measured. After sacrificing the animals blood
was collected in heparinized tubes and centrifuged, and the
plasma analyzed for cholesterol by a modification of the DuboffStevenson method (1). Various organs were dissected and weighed
on a torsion balance. Bone marrow was taken from the femur
and the cells differentiated with Giemsa stain. Initial and final
body weights were recorded, and the average daily food intake
was determined.
Results
Mice implanted with L1210 survived an average of 9.0 days
and all of the animals were dead by the 11th day post-infection
(Table 1). Daily administration of hydroxyurea prolonged
survival of the leukemic mice and prevented the sharp increase
in the WBC and the decrease in RBC (Table 2). This compound
prolonged life most effectively when administered at a 5-mg
(approximately 250 nig/kg) daily dose. At this dose the mean
survival time was 14 days. All mice were alive on Day 10, whereas
only 1 out of 10 control animals was still living as of that date
The daily dose of 25 mg (approximately 1250 mg/kg) was less
effective in maintaining life (mean survival time, 12.1 days)
possibly reflecting toxicity of the compound. Survival time was
only slightly advanced with a 1-mg (approximately 50 mg/kg)
dosage; however, the WBC remained at less than 50% of that
of the leukemic control animals when determined on the 9th
CANCER
RESEARCH
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VOL. 26
Effect of Hydroxyurea
on Blood and Marrow
TABLE 1
SURVIVALTIME OF BDF, MICE TRANSPLANTEDWITH L-1210 LEUKEMIA
post-transplantation)010101010879101092610810111071101861208513641441152016117118019
OF MICEALIVE (days
DOSE
SURVIVAL
COMPOUNDControlHydroxyureaHydroxyureaHydroxyureaDAILY
TIME
(days)9.09.714.012.1No.
(mg)1525MEAN
TABLE 2
MICE TRANSPLANTEDWITH L-1210 LEUKEMIA
PERIPHERAL BLOOD COUNTS OP
POST-TRANSPLANTATION7No.
COMPOUNDControlHydroxyurea
ofmice1010
of
mice27
0.4=8.0
±
±0.7
10
10.0 ±0.6
1027RBC-7.3
8.5 ±
0.58.9
Hydroxyurea
HydroxyureaNontumor
¥36.0± 5.
± 6.2a
8.4 ± 0.7d
0.3a14.4
8.4 ±
of
mice2
0.5a5.0
±
I*36.0± 12.
± 7.1a
±0.4"
7.8 ±0.6e
10.0 ± 1.7e
9.4 ±0.6
14.4 ± 1.0
10
7
9.2 ±0.7d11No.6RBC4.4«
6.2 ±0.6dWBC84.0
6.5 ±0.8<<WBC100.010.0 ± 1.0e
9RBC3.8
±0.6WBC635.6 ±0.79No.
con
trol/DAILYDOSE(mg)1525DAYS
" RBC (X 106)/cu mm blood. Figures are mean ±S.E.
6 WBC (X 103)/cu mm blood. Figures are mean ±S.E.
c P < 0.05. Significance
calculated
against
d P < 0.01. Significance
calculated
against nontumor
' Figures
represent
average
from
nontumor
2 survivors
on Day
control.
control.
11.
1 Figures for RBC and WBC represent mean ±S.E. calculated from 27 mice sacrificed at various intervals
during the experiment.
TABLE 3
EFFECT OF HYDROXYUREAON PERIPHERAL BLOOD OF INTACT IMMATUREMALE RATS
TreatmentDaily
(mg)Sesame
dose
oilHydroxyurea
0.3'7.0
±
0.820.2±
2.623.0±
0.414.0±
0.32.3±
± 0.1»
±0.4
±0.9
±0.2
± 1.2
2.1 ±0.3» 22.4 ± 2.3
7.4 ± 0.3
24.8 ± 2.0
13.0 ± 2.1
2.0 ± 1.4» 0.6 ±0.1» 11.5 ± 1.0»
5.1 ±0.2» 12.2 ±0.6»
25
4.5 ±0.2»WBC626.9
12.7 ± 1.2»Reticulocytec14.6
0.0»Neutrophils''4.2
0.0»Lymphocytes*21.9
12.6 ±0.4»
125RBC°7.5
Hydroxyurea
Hydroxyurea
Hydroxyurea15
" RBC (X 106)/cu mm blood.
<>WBC (X 103)/cu mm blood.
' % RBC.
d (X 103)/cu mm blood.
•
(X 103)/cu mm blood.
i Mean ±S.E.
oP < 0.01.
day post-infection. It is interesting that at this low dose level
the cell counts determined on Day 7 or 11 were similar to those
of the nontreated L1210 animals determined on Day 7 and 9,
respectively. A more dramatic maintenance of normal blood cell
counts was obtained by treatment
with the higher doses of
hydroxyurea.
NOVEMBER
1966
The peripheral blood of immature male rats also was affected
by the daily s.c. injection of hydroxyurea suspended in sesame
oil (Table 3). Daily doses of 25 mg and 125 mg of this compound
induced a sharp decrease in the number of WBC and RBC. Lower
doses (1 mg and 5 mg) did not alter these cell counts. The % of
reticulocytes was reduced even more dramatically
with drug
2293
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Leonard J. Lerner, Albert Bianchi, Euripides Yiacas, and Aleck Barman
<N
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treatment. The highest dose (125 mg) practically eliminated all
reticulocytes from the peripheral blood and the 25-mg dose
reduced the % of these cells from 14.6 to 2. All doses of hydroxyurea lowered the numbers of neutrophils. The 2 lower
dosages produced a decrease of approximately 50%, whereas the
2 higher doses almost eliminated this white cell. In contrast, the
2 lower doses did not affect the lymphocyte count although the 2
higher doses reduced the numbers of this white cell by approxi
mately 50%. It is interesting to note that although the 25 mg
dose was much less toxic to the animal, it had a comparable
effect on the WBC. Administration of hydroxyurea at 25 mg or
125 mg daily produced marked alterations in the bone marrow.
There was a depletion of all cellular elements and replacement
with numerous fat vacuoles. This was more intense at the higher
dosage. The toxicity of the higher doses of hydroxyurea is further
illustrated by its effect upon body weight gain and food con
sumption (Table 4). The highest dose reduced food intake by
one-half, allowed only a minimal gain in body weight over the
10-day experimental period and i of the animals succumbed
before the end of the study. The 25-mg and 125-mg daily doses
also reduced the weights of the accessory sex glands, thymus,
thyroid, liver, testes, and spleen; however, the adrenal glands
were not significantly altered. Lower dose levels of hydroxyurea
did not change any organ or body weights.
Hydroxyurea was also administered in an aqueous solution to
immature male rats (Tables 5, 6). A 10-mg daily dose produced
a 23% decrease in the EEC, a 43% in WBC, and lowered the
hematocrit. This dose of hydroxyurea also significantly lowered
body weight gain and testes, ventral prostate, seminal vesicle,
thymus, and liver weights. However, adrenal and thyroid weights
were not altered, and the plasma cholesterol level was normal.
Several compounds related to hydroxyurea were also assayed
at a 10-mg daily dose in the immature male rat (Tables 5, 6).
These compounds, with the exception of JV,0-dicarbamoxyl-hydroxylamine (SQ 10726) significantly decreased the number of
WBC, but only 1-methy 1-1-hydroxyurea (SQ 17589) reduced
the RBC. Compounds SQ 10674, 10726, 17526, 17589, and
17843 reduced body weight gain. The latter 2 compounds also
decreased the weights of the testis, ventral prostate, seminal
vesicle, thymus, and liver. SQ 10726 reduced the weights of the
testis, seminal vesicle, and thymus. None of the compounds
altered plasma cholesterol levels.
The results of this study indicate that several of the compounds
(SQ 10391, 10690, 10708, 16819, and 5184) decrease AVEC with
out decreasing RBC or exhibiting toxicity at the dose employed.
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2294
«
Results of the present study in mice with L1210 leukemia
confirm the life-prolonging activity of hydroxyurea reported
by others (9, 12, 13). It is interesting that the increase in survival
time is accompanied by the prevention of a rise in the WBC
count and a concomitant decline in the RBC.
The effect of hydroxyurea on the peripheral blood of the leukemic mouse is not unique since this compound will also lower
the blood cell counts of normal animals. Administration of
hydroxyurea to the immature rat resulted in a reduction of all
RBC and WBC elements in a dose-related manner. This effect
on the peripheral blood is a direct consequence of the destructive
CANCER RESEARCH VOL. 26
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Effect of Hydroxyurea on Blood and Marrow
TABLE 5
EFFECTOF HYDROXYUREA
ANDRELATEDCOMPOUNDS
ON ORGANSANDBLOODOP IMMATURE
MALE RATS
Compound(Sq
No.)108910391106741069010708107261681917589178435184CompoundWater
ofanimals2812888888888Hematocrit37.6
±0.6"RBC«6.0 ±
0.331.8
1.010.5
±
0.4«37.0
±\.¥
4.6 ±
Q.8112.8
±
1.6'9.8
±
-hydroxyureaW-Carbamoyl-A''-formylhydroxylaminel-Acetyl-3-hydroxyurea3-Methyl-l-hydroxyureaA^O-DicarbamoylhydroxylamineHy
0.833.8
±
0.55.4
±
0.7d37.9
±
1.0a12.3
±
0.56.3
±
1.0a12.4
±
0.835.0
±
0.56.1
±
1.539.5
±
0.65.2
±
l.y14.8±
0.936.3
±
0.56.0
±
1.111.1
±
1.2«6.0
±
acid1
0.727.6
±
0.54.6
±
OAd11.0
±
0.2«5.8
±
ureaJV,0-Dicarbamoyl-jV-ethylhydroxylamineUreaNo.
-Methyl -1-hydroxy
0.&39.6
±
1.0a12.8
±
0.638.6
±
0.16.5
±
±0.9a
±0.8G.2
±0.3WBC*18.3
vehicleHydroxyurea1-Ethyl-l
•
RBC (X 106)/cu mm blood.
6WBC (X 103)/cu mm blood.
c Mean ±S.E.
dP < 0.01.
' P < 0.05.
TABLE 6
EFFECTOF HYDROXYUREA
ANDRELATEDCOMPOUNDS
ON ORGANSANDBLOODOF IMMATURE
MALE RATS
Compound
No.)Water
(Sq
ofanimals2812888888888A
Body
weight
(gm)70.0
(gm)1.33
(mg)88.2
prostate
(mg)56.7
vesicle
2.9»54.0
±
0.021.18
±
vehicle108910391106741069010708107261681917589178435184"
(mg)490.4
3.474.4 ±
3.740.4
±
26.3259.5
±
4.3*63.0
±
0.03"1.26
±
4.9e87.6
±
3.8»51.1
±
37.1"402.5
±
3.260.4
±
0.021.29
±
5.179.4
±
4.146.7
±
34.1261.8
±
3.8e72.9
±
66446.6±35.
0.07.25
±
8.294.1
±
4.655.4
db
4.762.0
±
0.08.26
±
5.779.3
±
5.943.3
±
43.4410.9
±
4.156.0
±
0.04.22
±
5.178.8
±
4.837.5
±
24.5390.0
±
\.5!>63.0
±
0.03«.28
±
3.8C53.3
±
29.1e348.3
±
5.190.6
±
57.0e148.6
±
4.249.6
±
0.03.09
±
6.960.1
±
6.229.7
±
2.6"55.4
±
0.04».19
±
3.6"70.7
±
1.4"39.3
±
14.2"348.1
±
4.7669.6
±
0.03e1.36
±
4.2e90.7
±
3.6e48.0
±
31.3e476.9
±
±6.8Testes
±0.04Ventral
±4.9Seminal
±4.0Thymus
±46.3Liver
(gm)6.8
0.34.7
±
0.7e6.1
±
0.36.4
±
0.36.8
±
0.36.4
±
0.36.0
±
0.16.4
±
0.45.2
±
0.2o5.6
±
0.3e6.4
±
±0.4
S.E.6
Mean ±
0.01.e
P <
P < 0.05.N"o.
activity of this compound on the bone marrow. The mechanism
of action of hydroxyurea is probably due to its inhibition of
DNA synthesis as postulated by several workers (3, 4, 10, 15).
As expected, a compound that inhibits the growth and pro
liferation of cells in the bone marrow is probably also inhibitory
to other tissues. This drug inhibited body growth and the growth
of several endocrine and endocrine-responsive tissues. However,
inhibition of organ and body growth was related to dosage.
Antileukemic drugs must decrease the numbers of WBC;
however, any concomitant reduction in the number of RBC or
general toxicity is undesirable. In the studies reported here, 5
compounds related to hydroxyurea were found to be effective
in reducing peripheral WBC without altering the RBC or induc
ing toxicity. Further investigations of these compounds in
experimental leukemias are warranted.
Acknowledgments
We wish to thank Mrs. E. Dzelzkalns, Mrs. M. Dzelzkalns, Miss
J. Baymiller, and Mr. M. DePhilippo for their assistance in these
studies.
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2295
Leonard J. Lerner, Albert Bianchi, Euripides Yiacas, and Aleck Barman
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CANCER RESEARCH VOL. 26
Downloaded from cancerres.aacrjournals.org on June 15, 2017. © 1966 American Association for Cancer Research.
Effects of Hydroxyurea and Related Compounds on the Blood
and Marrow of Experimental Animals
Leonard J. Lerner, Albert Bianchi, Euripides Yiacas, et al.
Cancer Res 1966;26:2292-2296.
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