[CANCER RESEARCH 30, 1276-1280, May 1970]
The Use of Leucovorin Orally in Normal and Leukemic L1210
Mice to Prevent the Toxicity and Gastrointestinal
Lesions
Caused by High Doses of Methotrexate
Joel S. Sandberg and Abraham Goldin
Cancer Chemotherapy National Service Center, Chemotherapy, National Cancer Institute, Belhexda, Maryland 20014
SUMMARY
Leucovorin p.o. was as effective as parenterally in pre
venting weight loss and toxic deaths in normal mice from
high doses of methotrexate. Histological sections of proxi
mal jejunum demonstrate the protection afforded by de
layed p.o. leucovorin from the marked disruption of villi
and mucosal glands produced by methotrexate. Mice
bearing leukemia L1210 treated with methotrexate plus
leucovorin p.o. were able to tolerate 2- to 4-fold increases
in the dose of methotrexate tolerated by mice treated
with methotrexate alone. This resulted in a greater in
crease in survival time of leukemic mice. The therapeutic
advantage of the regimen of delayed leucovorin given in
conjunction with methotrexate is maintained when leu
covorin is administered p.o.
INTRODUCTION
The ability of leucovorin (Lederle Laboratories, Pearl
River, N. Y.; /V'-formyltetrahydrofolic acid, citrovorum
factor, folinic acid) to reverse the toxicity and antitumor
activity of folate antagonists has been demonstrated (5).
When leucovorin administration is delayed 12 to 24 hr
after antifolate therapy, the antineoplastic effect of the
folate antagonist is retained, while host toxicity is still
reduced (8).
MTX1 remains in wide use in the treatment of choriocarcinoma (15), leukemia (1), and carcinoma (17, 20).
Leucovorin is used as an antidote for MTX overdosage
(9) and to decrease systemic toxicity after local intraarterial infusions of MTX (14). Leucovorin has generally
been administered parenterally, either i.m. (16) or i.v.
(11). Recently, Djerassi (4) and Bertino (personal com
munication) have used the p.o. route to administer leu
covorin.
In the present studies, the efficacy of p.o. administered
leucovorin was examined in normal and leukemic mice.
MATERIALS
AND METHODS
The experimental methods for the evaluation of the
toxicity and the anti-L1210 effects of MTX and leucovorin
have been described (6).
CDFi (BALB/c X DBA/2) F, hybrid male mice weigh
ing from 18 to 26 g were obtained from the production
colonies of the Cancer Chemotherapy National Service
Center.
Stock ascitic L1210 tumor was taken from DBA/2 male
mice and a uniform suspension was made in Hanks' bal
anced salt solution. L1210 cells, 1 X IO6,in 0.2 ml solution
were injected s.c. in the right flank.
In studies with normal or leukemic mice, MTX was dis
solved in 2% sodium bicarbonate and given s.c. in the intrascapular region every 4th day (7) for a total of 5 treat
ments. Leucovorin was dissolved in distilled water and
administered either p.o. or s.c. in the posterior cervical
area every 4th day 24 hr after MTX administration. The
appropriate concentrations of drugs were injected in a
volume of 0.01 ml/g body weight.
For the histological examination of the small intestine,
normal mice were treated with MTX, 160 mg/kg s.c.,
on Days 1 and 5. Leucovorin was administered p.o. or
s.c. 24 hr following MTX therapy (Days 2 and 6). Mice
were sacrificed on Day 8 and the proximal jejunum was
immediately removed. The tissues were fixed in neutral
buffered formalin, dehydrated, embedded in paraffin,
sectioned at 5 to 6 ^, and stained with hematoxylin and
eosin.
RESULTS
Table 1 shows, in terms of the percentage of toxic
deaths and weight loss, the protection afforded by delayed
leucovorin administration against the toxicity of high doses
of MTX in normal mice. Leucovorin was as effective p.o.
as parenterally in preventing toxic deaths and weight loss.
Leukemic mice receiving MTX and delayed leucovorin
also demonstrated
significantly less weight loss than that
resulting from the same dose of MTX alone. This protec
' The abbreviations used are: MTX, methotrexate; ILS. increase in tion from toxicity enabled the mice bearing L1210 to tol
erate 2- to 4-fold increases in the dose of MTX. The
life-span; g.i.. gastrointestinal.
Received August 15. 1969: accepted November 19. 1969.
ability to use higher doses of MTX resulted in greater
1276
CANCER RESEARCH
VOL. 30
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Oral Leiicovorin
and Methotrexate
Table I
The effect of delayed leucovorin administered p.o. or s.c. on the toxicity and anli-Ll2lO
Activity of methotrexate"
MTX alone
A. Normal mice
MTX
dose(mg/kg)320
deaths,6
(%)100
160
80
40MTX
B. L12IO-
dose
(mg/kg)320
100
100
50ILS'
change'
(g)-6.0
-4.5
-3.5
-0.8Toxic
MTX + delayed leucovorin.
200 p.o.
MTX + delayed leucovorin.
200 s.c.
deaths
(%5050
)
deaths
(%)87.5
change
(g)_2
12.5
0Weight
0ILS
(%)6
(%)53
-2. 9
-1. 4
-0.6 7Toxic
50
12.5
change
(g)-3.6-2.7
-0.7
-0.1ILS
(%)55
160
33
8844
6144
80
44
40Toxic
44Weight
44
55Weight
"CDF, male mice (18 to 26 g) were treated with MTX in doses indicated s.c. every 4th day for 5 treatments. Leucovorin, 200 mg/kg, was
given p.o. or s.c. 24 hr after each dose of MTX.
Mice were observed for toxicity for 90 days.
' Weight change is the body weight difference from Day I to Day 7 (after 2 MTX treatments) minus controls.
J Mice inoculated s.c. in the right hind leg with 10HL1210 cells from stock DBA/2 mice bearing ascitic L1210. MTX treatment started 1 day
after tumor implantation.
ILS % is the percentage increase in median survival time compared with controls.
antitumor effectiveness as evidenced by greater increases
in survival time (Table 1). MTX, 160 mg/kg every 4 days,
plus delayed leucovorin, 200 mg/kg p.o., produced an
88% ILS of tumorous mice as compared with controls.
MTX, 40 mg/kg every 4 days, alone yielded an ILS of
55% with all mice having large local subcutaneous tumors
at the time of death. Higher doses of MTX alone were
toxic. Leucovorin alone had no effect on survival time or
weight change as compared with untreated controls. De
layed leucovorin p.o. was as effective as s.c. in protect
ing against MTX toxicity and enabling the use of higher
doses of MTX.
Histológica!Observations on the Use of Oral Leucov
orin to Prevent the Gastrointestinal Lesions of MTX. The
small intestine of mice treated with MTX alone was
boggy, edematous, and distended with a straw yellow
fluid. The small intestines of mice receiving MTX plus
delayed p.o. or s.c. leucovorin appeared normal on gross
examination.
Fig. 1 illustrates the normal jejunal epithelium of un
treated mice. Leucovorin, 200 mg/kg every 4 days, does
not alter the histological appearance of the jejunum.
High parenteral doses of MTX alone produced marked
disruption of both villi and mucosal glands in the jejunum
(Fig. 2). Fig. 3 illustrates the loss of the orderly basilar
arrangement of the nuclei of the jejunal epithelium of
mice treated with high doses of MTX s.c. The cells are
rounded, enlarged, and beginning to slough. There is
cytoplasmic vacuolization, nuclear swelling, and promi
nent nucleoli. The brush border is not present. Lymphocytic infiltration is prominent.
Leucovorin, 200 mg/kg administered p.o. 24 hr after
each dose of MTX, protected the jejunum against the g.i.
toxicity of MTX. There is slight flattening of the villi, but
the villi and mucosal glands are intact (Fig. 4). There is
slight swelling and piling up of nuclei, but the nuclei are
in the basal position and the brush border is present (Fig.
5).
Delayed leucovorin given s.c. also protected against the
jejunal lesions of MTX, with only slight swelling and
piling up of nuclei on histological section (Fig. 6).
DISCUSSION
In the present studies, delayed leucovorin p.o. pro
tected mice from the weight loss and lethal toxicity pro
duced by high doses of MTX in normal mice. MTX and
delayed leucovorin p.o. resulted in greater increases in
life-span of mice bearing leukemia L1210 than did MTX
alone. Leucovorin administered p.o. provided as much
protection from MTX toxicity in both normal and leukemic mice as did leucovorin administered s.c.
Clinically, the g.i. toxicity of MTX is well documented
(3). The morphology of the g.i. lesions produced by antifolates ranges from the appearance of cytoplasmic inclu
sion bodies (22), flattening of the crypt epithelium (23),
and epithelial metaplasia (24) to desquamation of the epi-
MAY 1970
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1277
Joel S. Sandberg and Abraham Goldin
thelial lining (18) and hemorrhagic ulcérations(21). In the
present study the jejuna! sections taken 72 hr after the
last dose of MTX demonstrated marked disruption of
both villi and mucosal glands. The epithelial cells were
increased in size, as described by Jacobson (21) using rat
intestine. The cells were rounded and showed cytoplasmic
vacuolization, nuclear swelling, and prominent nucleoli.
These morphological lesions resulting from high dose
MTX reflect the effect of an antimetabolite on a tissue
with a high rate of cellular proliferation (13). The average
generation time of mouse ileum crypt epithelial cells has
been determined to be about 19 hr, with DNA synthesis
occupying about 7.5 hr (19). MTX blocks DNA synthesis
by inhibiting dihydrofolate reducÃ-aseand preventing the
synthesis of thymidylic acid from deoxyuridylic acid (3).
Delayed leucovorin is postulated to "rescue" normal
cells which have been inhibited in the synthesis of DNA
but are still viable (2). Leucovorin promptly reverses the
inhibition by MTX of the incorporation of tritiated deoxyuridine into DNA by leukemic cells in vitro (10). Morpho
logically, this is apparently reflected in the prevention of
the g.i. lesions caused by MTX with the addition of de
layed leucovorin. The jejunal sections of mice receiving
MTX plus delayed leucovorin showed slight swelling and
piling up of nuclei, but the villi and mucosal glands were
intact. The p.o. route of leucovorin administration was
as effective as the s.c. route in preserving jejunal struc
ture.
It is apparent that the control of toxicity and therapeu
tic advantage of the regimen of delayed leucovorin given
in conjunction with MTX are maintained when leucovorin
is administered p.o.
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CANCER RESEARCH
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Oral Leucovorin and Methotrexate
Fig. 1. Normal mouse jejunum of untreated controls. H & E, X 160.
Fig. 2. Jejunum of mouse treated with MTX, 160 mg/kg s.c., every 4 days (Days 1 and 5). Section was taken on Day 8. There is marked dis
ruption of both villi and mucosal glands with infiltration of chronic inflammatory cells. H & E, X 160.
Fig. 3. Higher power of Fig. 2 showing flattened, markedly disorganized villi of jejunum of mouse treated with MTX. The epithelium has lost
the orderly basilar arrangement of nuclei and brush border. The cells are rounded and beginning to slough. The cells show cytoplasmic vacuoli/ation, nuclear swelling, and prominent nucleoli. Lymphocytic infiltration is apparent. H & E, X 400.
Fig. 4. Jejunum of mouse treated with MTX, 160 mg/kg s.c.. on Days 1 and 5 and leucovorin, 200 mg/kg p.o., on Days 2 and 6 (24 hr after
MTX therapy). The section was taken on Day 8. The delayed leucovorin has protected the intestine from the profound toxic effects of MTX. There
is increased mitotic activity and some flattening of the villi. H & E. X 160.
Fig. 5. Higher power of Fig. 4, showing fairly normal villi of jejunum of mouse treated with MTX and protected by delayed leucovorin. Nuclei
are basal and villous structure is generally intact. There is some swelling and piling up of nuclei. H & E, X 400.
Fig. 6. Slightly tangential section of jejunal villi of mouse treated with MTX, 160 mg/kg, on Days 1 and 5 and leucovorin, 200 mg/kg s.c., on
Days 2 and 6. The section was taken Day 8. There is some swelling and piling up of nuclei, but nuclei are basal and villous structure is generally
intact. H & E, X 400.
MAY 1970
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1279
Joel S. Sandberg and Abraham Goldin
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CANCER
RESEARCH
VOL. 30
Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 1970 American Association for Cancer Research.
The Use of Leucovorin Orally in Normal and Leukemic L1210
Mice to Prevent the Toxicity and Gastrointestinal Lesions
Caused by High Doses of Methotrexate
Joel S. Sandberg and Abraham Goldin
Cancer Res 1970;30:1276-1280.
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