Note: Descriptions are shown in the official language in which they were submitted.
HOECHST AXTIENGESEL~SCHAPT HOE 89/P 050 Dr.~H/Le
Description
Poly er-bound ethotre ate, a proee~s for it~ preparation
~nd it~ use ,~
S There has for years been world-w$de research into the
development of pXarmacologically active polymers, includ-
ing, in particular, polymeric antitumor agents (US
4,460,560, US 4,551,502, ~P 0,040,506, WO 875,031, DE
3,515,178, DE 902,344 ~nd ~P 0,111,388).
An essential aim in this eonnection is the preparation of
cytostatics whose side effects have been reduced or
eliminated ~nd whose therapeutic range hss been improved.
The problems with the cytostatics in eurrent clinical use
derive less from insuffieient eytotoxieity and more from
in~dequate selectivity. This me~ns that the wide range of
highly eytotoxie eompounds aet not only in tumor eells
but also in healthy cells in the body and, in many eases,
also more strongly in individual organs.
Hence there is a need for ~n administration form in wh$eh
a level of eytostatie in the blood whieh is within the
therapeutie range, i.e. with whieh tumor eell~ are
greatly da~aged but healthy eells in the body are damaged
only slightly or not at all, ean be reaehed ~nd main-
tained for a prolonged period, in order to ~ehieve a
greater tumor toxieity with, at the same time, diminished
overall toxieity using doses of ehemotherapeutie whieh
are eon~iderably lower in total.
Variou~ developments have been followed up to dates
- Polymers as eytostaties (poly~nions sueh ~8 pyran
eopolymers, polyvinyl sulfonates ete.) whieh ~re
intended to have a tumor-inhibiting aetion per se.
Frequent disadvantages toxic side effeets and narrow
limits in the molecular weight range (nephrotoxicity
> I~W 50 I~D).
- 2 - ~
- Polymers which have a cytostatic action and in which
constituents of the ~polymer backbone~ have a
certain activity after degradation. Prequent disad-
vantages: limited choice of the building block~ of
the polymer, immunogenicity, side effects of the
polymer~ and, ln particular, insolubillty of the
products in water ~J 58/174,409, DE 3,026,448, DE
3,515,178, D~ 3,026,574, DE 902,344 and DE
3,026,575).
10 - Polymer-bound cytostatics on functional amide side-
groups (WO 875,031, EP 0,111,388, D~ 3,539,951, ~O
8,700,056, EP 0,190,464, US 4,551,502, JP
57/143,325, EP 0,040,506 and JP 57/143,326).
Although the liberation of these cytostatics from the
linkage with the polymer can in theory take place on
endocytosis of the complete con~ugate in the acid pH of
the lysosome, it has been shown in practice that endo-
cytosis takQs place to only a small extent even in the
case of polymer con~ugations with target-~pecific modifi-
cations (for example antibody con~ugates). Purthermore,
ma~or problems arise from the introduction of spacers,
the frequently low loading density and difficult syn-
theses. The in vivo results with such con~uqates to date
have therefore been disappointing.
Phase-specific cytostatics, for example methotrexate,
represent a special case because they act only in a
particular phase of the cell cycle (synthesis phase in
the case of the dihydrofolate reductase inhibitor metho-
trexate). Given the relatively short biological half-life
of methotrexate, a single administration frequently has
no antitumor action whatever. A therapeutic effect is
shown only by multiple individual administrations with
very high doses. A ~rescue therapy~ with antagonists is
frequently necessary in order subsequently to avoid, with
a time lag, too much damage in the healthy cells.
~5
-- 3 --
A polymer-bound form of methotrexate i8 of particular
interest because of the~e problems. However, no satisfac-
tory solution has to date been possible by b$nding
methotrexate to poly-L-lysine, poly-lactide-glycolide
(PLG) derivatives etc. (see W0 875,031).
Surprisingly, very good in vitro and in vivo properties
with relevance for human therapy have now been achieved
by binding methotrexate to water-soluble, biocompatible
polymers carrying hydroxyl groups.
Hence the invention relates to~
1. A polymer-bound methotrexate or methotrexate deriva-
tives, having the following features
- an ester linkage of the ~- and/or 7-carboxyl
group of the methotrexate or of the methotrexate
- 15 derivatives with hydroxyl groups of water-soluble
biocompatible polymers;
- bringing about a prolongation of life of more
than 125~ compared with untreated controls in
mice with L1210 leukemia on a ~ingle intraperi-
toneal administration of 60 mg of methotrexate or
methotrexate derivative, which is bound to the
abovementioned polymer, per kg of body weight.
2. A proce~s for the preparation of the polymer-bound
methotrexate with the features specified in 1. or of
the polymer-bound methotrexate derivatives, which
comprises the methotrexate which carries ~- and/or
7-carboxyl groups, or the corresponding derivatives
of methotrexate, being reacted in the presence of
water-abstracting coupling reagents with a water-
soluble biocompatible polymer carrying hydroxyl
groups.
3. The use of the polymer-bound methotrexate with the
features specified in 1., or of the polymer-bound
methotrexate derivatives, as tumor therapeutic.
. . .. . - ,: .
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The invention is described in detail hereinafter, e~pe-
cially in its preferred embodiments. The lnvention 18
furthermore defined by the content~ of the clalm~.
~ethotrexateorN-t4-[[(2,4-diamino-6-pteridinyl)methyl]-
methylamino]benzoyll-glutamic acid is de~cribed a~ com-
pound with its essential characteristics in the article
by A.R. Chamberlain et al. in Analytical Profiles of Drug
Substances, volume 5, edited by ~. ~lorey, Academic
Pre~s, New York (1976), pages 283-306. The preparation of
the compound is described by Seeger et al. tJ. Am. Soc.
11, 1753 (1949) or Rahmann et al., Medic. Res. Rev., 8,
95 (1988)].
The compounds which can be employed as methotrexate
derivatives or analogs are those which have been modified
on the pteridine ring or in the bridge region, a~ well as
those which have been derivatized on the aromatic ring or
in the glutamic acid iety. However, an e~ential
condition is that at least one carboxyl group which is
neces~ary for linkage to the polymer is retained. Analogs
of thi~ type and processes for the prep~ration thereof
are de~cribed in detail in the article by Rahoann et al.
(~ee above). Particularly preferred derivatives which can
be u~ed are methopterin (~erck Index 10 (1983) 5860) and
aminopterin (Merck Index 10 (1983) 477).
The said compounds can be bound via an e~ter linkage to
water-soluble, biocompntible polymers carrying hydroxyl
groups. By biocompatible polymers are eant compounds
which are physiologically tolerated and can be degraded
and/or excreted in the body. Polymers which can prefe-
rably be u~ed are those in which the proportion of
ionizable groups before the loading with the active
substance is below 10 mol-~.
Preferably employed are water-soluble starches with a
mean molecular weight of 1000 to 200,000, preferably 5000
to 50,000, which can al~o be dified. Also used are
- s -
cellulose acetates, especially water-soluble cellulose
acetate from the company (Celanese/WSCA)~ or destrans,
with a mean molecular weight between 1000 and 200,000,
e~pecially with a mean molecular weight of 40,000 to
70,000, or inulin.
It is also possible to use appropriate synthetic poly-
mers. One esample is poly-~,~-(hydroxyethyl)-D,L-
spartamide of the fon~ula I
C - NN~ C -
CH2~ ~ - CH~
r ~ C~
(CH2)2 (CIH2)2
CH2OH ~ CH2OH n
in which the ratio of m to n is in the range 0-1 to 1-0,
preferably 0.7s0.3 to 0.95s0.05. The molecular weight of
compounds.of this type is appro~imately between 2000 and
100,000, preferably between 5000 and 50,000. Appropriate
compounds and procesffe~ for the preparation thereof are
described in detail in German Offenlegungsschr.ift
3,700,128.
Another example of a ~ynthetic polymer is Am~dated
polylysine fumaramide/polylys~ne glutaramide of the
formula II
:: : : ....... .. . ....
- 6 -
O O
Il 11
-(-NH-(CH2)~ - CH - NH-C-X-C-) D- II
C'O
(CH2)~OH
in which X can be -(CH2)3 or -CH-CH-, m can be the numbers
1 to 10, preferably 2, and n can be the number 5 to 2000.
~hese polymers have a molecular weight between 1000 and
300,000. Polymers of this type and processes for the
preparation thereof are described in German Offenlegungs-
schrift 3,707,369.
A further example is 8 copolymer of the formula IIIcomposed of poly-~,~-(2-hydroxyethyl)-D,L-aspartamide
(compound of the formula I) and polysuccinimide
(formul~ I) ~ ~ III
in which the ratio of y to z is in the range from about
O.99sO.Ol to O.OlsO.99, preferably 2.5s7.5 to 7.Ss2.5.
Particularly preferably employed as polymers in the
process according to the invention are the synthetic
compounds depicted in formulae I, II and III.
,
2S For the prepsration o:E the polymer-bound methotrexate or
of the corresponding derivatives, the active substance is
I dissolved, for example in water, dimethyl sulfoxide
; (DMSO), form~mide, N,N-dimethylformamide or methylene
chloride or a mixture of the last three solvents. The
appropriate polymer is added to the same solvent which
has also been u~ed to dissolve the active substance. ~he
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two mixtures are combined and can be incubated in the
presence of a water-abstracting coupling reagent, where
appropriate with exclusion of light, at a pH in the range
from 7 to 9, preferably 8 to 8.S, and at a temperature of
S 0 to 100C, preferably 20 to 30C, for a period of nbout
1 to 29 hours, preferably with stirring.
It iB poBBible to use as water-abstracting coupling
reagent carbodiimides, alkylphosphonic anhydrides,
carbonyldi~mines etc. Carbonyldiimidazole and dicyclo-
hexylcarbodiimide are particularly preferably used.
The resulting crude product can be purified by precipita-
tion with a ~olvent in which the polymer is insoluble. It
is possible to use for this purpose, for example, tetra-
hydrofuran, acetone, dioxane and alcohols. Further
lS purification can take place using methods for molecular
weight partition such as, for example, ultrafiltration,
dialysis and gel permeation.
The process according to the invention results in a
polymer-bound methotrexate product with a loading of
methotrexate or analogs thereof from 1 to 85%, preferably
lS to 7S%, based on the weight of the polymer-bound
product. The product furthermore has an extinction
coefficient between 0.0001 l/mg and 0.05 l/mg, preferably
between 0.005 l/mg and 0.03 l/mg, in aqueous solution at
pH 7 to 8.5 and a wavelength of 302 nm for methotrexate
and methopterin, or an extinction coefficient of 0.0005
l/mg to 0.04 l/mg at the said pH and a wavelength of 282
nm for aminopterin.
The active substance can be slowly released from the
polymer-bound methotrexates according to the invention
into the body by simple hydrolysis, in contrast to the
stronger binding via functional amide groups, from which
the active substance can be liberated only by enzymatic
cleavage.
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With these polymer-methotrexate con~ugates according to
the invention in vivo, for example in mice with L1210
leukemia, prolongations of life of > 125%, preferably
> 150~, compared with untreated controls are achieved
even with a single administration ~intraperitoneal i.p.)
of 60 mg~kg of body weight methotrexate or derivatives
which are linked according to the invention to the water-
soluble, biocompatible polymer carrying hydroxyl groups
(equivalent of methotrexate or derivative). These figures
were determined as described in Example 7. Methotrexate
or the derivatives are not themselves active under the
said conditions. With 330 mg/kg equivalent of metho-
trexate or derivative it was possible to observe complete
remission without relapse up to the termination of the
experiment a~ter 60 days in 2 of 5 animals. This dose is
itself above the LD50 of free methotrexate.
In addition, the polymer-methotrexate con~ugates accord-
ing to the invention have a higher IC50 in vitro than free
methotrexate (for example on L1210, HT 29, A549 cells).
In con~unction with the in vivo results described sbove,
this is to be regarded as evidence of a desired slow
liberation of methotrexate.
The invention is illustrated by means of examples herein-
after. Unless indicated otherwise, percentage data relate
to weight.
Fsample 1: Preparation of a poly-methotrexate-poly-
~,0-(2-hydroxyethyl)-D,L-aspartamide ester
using carbonyldiimidazole
Methotrexate was purchased from Sigma for all the
examples. The polymer poly-~,0-(2-hydroxyethyl)-D,L-
aspartamide is prepared by the method of P. Neri, G.
Antoni, F. aenvenuti, F. Cocola, G. Gazzei, J. Med. Chem.
16, 893 (1973).
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3 g of methotrexate are dissolved in 15 ml of a mixture
of formamide s N,N-dimethylformamide : CH2Cl2
(10 : 9 s 1). To this is added a solution of 300 mg of
pyrrolidinopyridine and 2.44 g of carbonyldiimidazole in
5 ml of the above solvent mixture. The reaction mixture
is stirred at room temperature (RT) for 1.5 h. At the
same time, 3.1 g of poly-~,~-(2-hydroxyethyl)-D,$-aspar-
tamide (PHEA) are dissolved in 9 ml of the solvent
mixture described and stirred at RT. The two solutions
are combined and stirred at RT with exclusion of light
for 20 h. The crude product is precipitated by pouring
into 250 ml of acetone. The yellow precipitate is sepa-
rated off, washed with acetone, dried and then taken up
in 25 ml of aqueous NaHC03 solution (pH 8-9).
The amber-colored aqueous solution is poured onto a
Sephadex gel chromatography column (PD10, Pharmacia) and
separated into a low molecular weight and a high molecu-
lar weight fraction by eluti~n with water. The high
molecular weight fraction is freeze-dried. The metho-
trexate content is determined by UV spectroscopy at~ - 302 nm in aqueous solution. The product is charac-
terized by lH NNR, CHN analysis and thin-layer chroma-
tography. The lH NMR in D20 corresponds to a sum of the
spectra of methotrexate and PHEA, it being possible to
establish the degree of occupancy from the integral
ratios. The degree of occupancy of 22~ by weight metho-
trexate measured by HPLC corre8pond8 to the result of the
W determination. The coupling reagents migrate, whereas
methotrexate and product remain at Rf = 0, in the TLC in
diethyl ether. The presence of free methotrexate could be
ruled out by ultrafiltration with m~mhranes with various
exclusion limits.
I
Yield: 3 g of polymer-bound methotrexate (about 20% of
theory based on methotrexate). Most of the unreacted
methotrexate is recovered by reprecipitation from the low
molecular weight fraction.
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Xxample 2s Preparation of a poly-methotrexate-dextran
(40,000) ester usinq carbonyldiimidazole
Dextran 40,000 was purchased from Fluka, Buchs,
Switzerland. 3 g of methotrexate are dissolved in 10 ml
of a mixture of formamide : N,N-dimethylformamide s CH2Cl2
( 10 S 9 5 1), and 2.39 g of carbonyldiimidazole and 0.29
g of pyrrolidinopyridine are mixed in 5 ml of the ~bove
solvent mixture and stirred at RT for 2 h. Then a solu-
tion of 3.2 g of dextran 40,000 in 20 ml of the same
solvent mixture is added thereto, and the mixture is
stirred at RT with exclusion of light for 20 h. The crude
product i8 precipitated in 350 ml of dry acetone, washed
with acetone and dried.
The solid is taken up in 25 ml of H20 and poured onto a
PD10 gel chromatography column. Elution with H20 yields a
high and a low molecular weight fraction. The high
molecular weight fraction is freeze-dried and analyzed as
described in Example 1. Unreacted methotrexate can be
recovered by reprecipitation.
Yields 3 g of polymer-bound methotrexate
Occupancys 17.3% by weight methotrexate
F~ample 3s Preparation of apoly-methotrexate-dextran
(40,000) ester using dicyclohexylcar-
bodiimide (DCC)
Dextran 40,000 was purchased from Fluka, Buchs,
Switzerland. 3 g of methotrexate are dissolved in 10 ml
of a mixture of formamide s N,N-dimethylformamide : CH2Cl2
(10 s 9 s 1), and a solution of 2.9 g of DCC with 1.8 g
of N,N-dimethylaminoE~yridine (DMAP) in 5 ml of solvent
mixture (as described above) is added thereto. After
stirring briefly, a solution of 3.2 g of dextran 40,000
in 20 ml of the same solvent mixture is added, and the
mixture is stirred at RT with exclusion of light for
20 h. After a precipitate has been filtered off, the
crude product is precipitated by pouring into 350 ml of
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scetone and washed with scetone and then dried.
The solid is taken up in 30 ml of H20 and poured into a
PD10 gel permeation chromatography coluimn and eluted with
H20. The high molecular weight fraction i8 freeze-dried
and analyzed as described in Example 1.
Yield: 3 g of polymer-bound methotrexate
Oeeupaney: 8.5% by weight methotrexate;
methotrexate reeovery by reprecipitation.
B~ample 4s Preparation of a poly-methotrexate-(,~-
(2-hydroxyethyl)-D,L-aspartamide/polysuc-
cinimide) ester
10 g (103 mmol) of polyanhydroaspartic acid (polysuc-
cinimide) are eonverted only partially with 1.83 g
(30 mmol) of 2-aminoethanol into poly-,~-(2-hydroxy-
ethyl)-D,L-aspartamide. The polyanhydroa6partic acid-co-
~,~-(2-hydroxyethyl)-D,L-aspartamide is charaeterized by
NMR speetroseopy and contains about 30% hydroxyethyl
groups and, in contrast to homo-poly-~,~-(2-hydroxy-
ethyl)~D,L-aspartamide which can be clissolved in cold
water, is now soluble only in hot water.
The xeaction is carried out in analogy to Example 1, but
employing the copolymer in place of poly-~,~-(2-hydroxy-
ethyl)-D,L-aspartamide. The amount of polymer employed
depends on the poly-,~-(2-hydroxyethyl)-D,L-aspartamide
proportion in the copolymer. Polymer eorresponding to 1
mole of OH groups is added for each 1 mole of metho-
trexate. The high molecular weight final product from gel
chromatography contains 20% by weight bound methotrexate
(determined by HPLC). Yield about 20% of theory based on
methotrexate employec!l.
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E~ample 5s Preparation of a poly-methotrexate-inulin
ester
The reaction iB carried out in anslogy to Example 1 but
employing inulin (Fluka AG, Buchs, Switzerland) in place
of poly-~,~-(2-hydroxyethyl)-D,L-aspartamide. An ~mount
of inulin corresponding to 1 mole of monomer units iB
added for 1 mole of methotrexate. The high molecular
weight final product from gel chromatography contains 68~
by weight bound methotrexate. Yield about 70% of theory
based on methotrexate employed.
~a~ple 6: Preparation of a poly-methotrexate-poly-
[(2-hydroxyethyl-~mido)-lysine fumaramide]
ester using carbonyldiimidazole
The polymer is prepared in analogy to German Offen-
legungsschrift 3,707,369 ~xample 2, employing (2-hydroxy-
ethyl)amidolysine in place of lysine methyl ester.
3 g of methotrexate are dissolved in 15 ml of the solvent
mixture described in Examples 1 to 3, and a solution of
2.39 g of carbonyldiimidazole and 0.29 g of pyrrolidino-
pyridine in 5 ml of the solvent mixture are added. After
stirring at RT for 2 h, a solution of 3.3 g of the
polymer in 20 ml of the solvent mixture is added, and the
mixture is stirred at RT with exclusion of light for
20 h. Precipitation is then carried out by pouring into
300 ml of acetone, and the residue i8 washed with acetone
and dried. The solid is dissolved in 25 ml of H20 and
separated into high and low molecular weight fractions on
a PD-10 gel chromatography column. The dried high molecu-
lar weight fraction is analyzed as described in ~xample
1.
Yield: 3.1 g of polymer-bound methotrexate
occupancys 19.1~ by weight methotrexate;
methotrexate recovery by reprecipitation.
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- 13 -
~nmple 7s Preparation of apoly-methotrexate-(water-
soluble starch) ester using carbonyl-
diimidazole
Various commercially available water-soluble starches
were employed. 3 g of methotrexate are dissolved in 15 ml
of the solvent mixture described in ~xamples 1 to 3, and
a solution of 2.39 g of carbonyldiimidazole and 0.29 g of
pyrrolidinopyridine in 5 ml of the same mixture is added.
After stirring at RT for 2 h, 3.2 g of water-~oluble
starch fraction in 30 ml of the ~olvent mixture are
added, and the reaction solution iB stirred at RT with
exclusion of light for 20 h. The crude product is
precipitated by pouring into 300 ml of acetone and, after
washing with acetone, dried. ~he residue is taken up in
30 ml of H20 and sub~ected to ultrafiltration. The high
molecular weight phase (retentate, membrane exclusion
limit = 5000) is freeze-dried and analyzed as described
in Example 1.
Yields 3 g of polymer-bound methotrexate
Occupancys 8% by weight methotrexate;
methotrexate recovery by reprecipitation.
~ample 8s In vitro action of methotrexate-dextran
ester on tumor cell lines.
Proliferation test (methotrexate reduction)
.
L1210, A 549 or HT 29 in the exponential phase of growth
are incubated in a cell density of 5 x 103 cells/ml in
"Rosswell Park Memorial Institute" (RPMI) 1640 medium in
a microtiter plate with 96 wells with various concentra-
tions of the test substance at 37~C, 5% CO2 and 95%
relative humidity for 72 hours. Control experiments
receive merely growth medium in place of test substance.
Quadruplicate determinations are set up for each test
substance and for the control. After incubation for 65
hours, 5 ~1 of a methotrexate solution (2.5 mg/ml in
phosphate-buffered saline solution) are added. In the
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- 14 -
presence of live cells, methotrexate is reduced to a dark
red insoluble formazan dyestuff. This reaction is com-
plete after 7 hours (L1210 cells) or after 24 hours (A
549, HT 29 cells), and the supernatant medium i8 careful-
ly aspirated off. The insoluble dyestuff is dissolved byadding 100 ~1 of DMSO, and the extinction of the result-
ing solution is subsequently measured for each well at a
wavelength of 492 nm in a Multiscan Photometer 340 CC
from Flow.
The ratio of the extinctions of treated and untreated
cells yield~ a dose-effect plot from which the concentra-
tion which kills ~ust 50% of the cells (IC50) can be resd
off. The coe~fficient of variation is less than 15% for
repeat experiments.
Table 1
Substance Cell IC50 (ug/ml)
.
L1210 0.01
Methotrexate (MTX) HT 29 0.008
A 549 0.01
L1219 0.13
NTX-dextran ester HT 29 0.41
A 549 0.58
,
~sample 9: In vivo activity on L1210 leukemia in mice
Obtaining tumors:
Ascites fluid is removed under sterile conditions from
DBA2 mice (female, 18 to 20 g) 7 days after tumor implan-
tation. The ascites fluid is washed three times with PBS
(phosphate-buffered saline), counted and subsequently
diluted in PBS to a final concentration of 10~ cells per
0.2 ml.
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lO~ cells in 0.2 ml of PBS are administered intraperi-
toneally to DBA2 mice (female, 18 to 20 g). This transfer
is repeated once a wee~.
Determination of the antitumor effect:
105 cells from the ascites fluid in 0.2 ml of PBS are
administered intraperitoneally to BDF1 mice ~female, 18
to 20 g). 6 animals are employed for each substance
concentration and for the control.
a) The animals are weighed on day l and day 5 after the
tumor cell implantation. A lo88 of weight of more than
20% on day 5 is used as indicator of a toxic effect of
the substance.
b) At the end of the experiment (death of all animals or
day 60 reached), the median survival time of the treated
groups i8 determined as long as--the latter contained 65%
surviving animals on day 5. The median survival time iB
determined in accordance with the formulas
median survival time (MST) = (X + Y)
In this formula, X is the earliest day on which the
number of surviving animals is N/2, and Y is the earliest
day on which the number of surviving animals is (N/2)-l.
In the case where N is an odd number, the median survival
time corresponds to the time X.
The median ~urvival 1ime is determined only for animals
dying during the course of the experiment. Cured animals
(long-time survivors, LTS) are excluded from the deter-
mination of the median survival time and are listed
separately.
The antitumor effect tumor/control (T/C) is determined
from the median survival time of thé treated groups
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- 16 -
(MSTs~or) and control groups (~STCon~rol) in accordance with
the formula
MST~
T/C % x 100
MSTC
T/C values of more than 125~ are regarded as an indicator
of a significant antitumor activity of the test compound.
The doses which bring about the greatest antitumor effect
in each case (optimal dosage) are listed in Tab. 2.
Animals still alive on day 60 are regarded as cured
(LTS).
Resultss See Table 2
Discussions
It is evident from these results that the esterified
methotrexate polymers are, with a single i.p. administra-
tion, superior to pure methotrexate, which indicates a
slow release action of the polymers.
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