Note: Descriptions are shown in the official language in which they were submitted.
I '7
1 S p e c i f i c a t i o n
This invention relates to a therapeutic preparation
comprising a glycosaminoglycan polysulfate except heparin
and at least one cytostatic drug besides otter common
carriers and companion substances.
The conventional methods known in the treatment of malignant
tumors are substantially restricted to either surgical
and/or radiological treatments as well as to the application
of cytostatic drugs the disadvantages of both the surgical
and the radiological methods of treatment are known
When cytostatic drugs are applied in the treatment of
malignant tumors both the malignant and the healthy tissue
cells are damaged and thus, the narrow bounds ox a therapy
may also jeopardize the success of -the treatment in this case.
It is true that "Journal of Medicinal Chemistry 1974, volt
17, No. 12, page 1~35" teaches to increase the efficacy
of the treatment of tumors with cytostatic drugs by
simultaneously adding heparin. It has been noticed that
patients who did no respond to the treatment with Cytostatic
drums without heparin, responded better whey heparin was
used as an accompanying agent However heparin is not the
substance either, for engendering affection of tumor cells
preferably when there is an increased number of such cells
which would mean, that preferably tumors in the advanced
stage could be treated in a particularly efficacious manner
furthermore, one can proceed from the assumption that
the problem ox serious; unwanted secondary effects is
not solved even if the gl~cosaminogl~can pol~sulfate heparin
is added to common cytostatic drugs.
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1 The tax underlit this invention thus resides in
providing a therapeutic preparation which has a selective
and intensified effect on malignant tumor cells, in
particular if there is already a great number of such cells.
one preparation is meant to directly destroy these cells
to a sigh degree without seriously affecting any healthy
cells or health tissue.
this task is solved by the therapeutic preparation defined
herein before. With the aid of the combination according
lo to the invention it is possible to selectively "open"
the tumor cells Peg by damaging the membrane) and to
concertedly introduce cytotoxically and cytolytically,
respectively, effective substances, so that -these substances
are effective even if the tumor is already in an advanced
stage of growth.
Glycosaminoglycan polysulfates also described as sulfated
polyanions or formerly designated mucopolysaccharide polo-
sulfuric acid esters are known from the therapy ox
various diseases. Substances counted among this family are,
for example, heparins the coagulation characteristics of
which have long been utilized. Other compounds of this
class of substances are applied in the treatment of
h~perlipoidaemiae and h~percholesteraemiae Peg heparins
and hepari~oids~ or they are used as an anti arthritic
Peg extracts of cartilage and chondroitin polysulfate)
furthermore, the substances observed have been known to
distinctively and specifically inhibit enzyme systems
(Schaffrath et at. in: ~oppe-Seylers Z. Fishily. Chum.
I 499 (1976)). Investigations of biocytocultures
revealed that - after incubative - malignant cells accumulate
and store glycosaminoglycan polysulfates in a more intensive
manner than benign cells
Tao
1 Intensified accumulation of glycosaminoglycan polysulfate
results in an inhibition of enzyme systems and thus,
in an inhibition of biosynthetic performances in the cell
metabolism of malignant cells. Hence a synergistic effect
occurs in addition to the effect of the cytostatic drugs
simultaneously applied, thus permitting that the latter can
be used in concentrations which do either not or only
slightly impair benign cells.
Comparative investigations between heparin and other
gl~cosaminoglycan pol~sulfates, in particular chondroitin
polysulfate have shown that the mode of action of glyco-
saminoglycan polysulfates can indeed be different.
It has turned out that gl~cosaminoglycan polysulfates other
than whopper are particularly characterized by an intensified
effect occurring whenever the tumors already have a
great number of cells. though there is no final scientific
explanation for this selective mode Of Bavaria
it is assumed that both kind and degree of sulfation are
responsible for the phenomenon observed
It has been absolutely surprising that glycosaminoglycan
polysulfates other than heparin would show -this particularly
advantageous mode of action
According to the invention the following substances can
be applied as cytostatic drugs: alkylating cytostatic drugs
such as nitrogen mustard derivatives and ethyleneimine
derivatives, a~timetabolites such as methotrexate,
antagonists of Purina and pyrimidine bases, cytostatically
effective antibiotic agents and alkaloids such as
cholchicine or vincristin .
In accordance with the invention preferably chondroitin
polysulfate is used as glycosaminogl~can polysulfate~
Furthermore, the task underlying the invention is solved
by adding the proteolytic enwomb trypsin to the therapeutic
preparation
1 The general biochemical expert knowledge includes that
the effect of both substances is canceled to a certain
degree by the proteol~tic enzyme try pin because of
interaction with glycosa~inoglycan pol~sulfates.
Owing to these experiences the person skilled in the art
has not been able to continue his work within the meaning
of the present invention. Because of reciprocities between
all substances used according to the invention, which
have still not entirely been understood it is not an
inhibiting effect that occurs but - in an absolutely
unexpected miner - a synergistic one, as will be one
by the experiments of the present patent application.
literature describes tr~psin as cytolytic substance.
It selectively attacks the malignant cell which has been
already affected by additional application of a glycosamino-
glycan polysulfate.
Addition of trypsin is important because there are
biologically different resistant species also within one
tumor cell family. such resistant tumor cells are
enz~matically partially digested to permit the cytostatic
drugs to become effective Tr~psin therefore increases
the selective effect of the preparation according to the
invention to malignant cells and thus enables concerted
application of tiny doses of cytostatic drugs which would
affect all cells if solely these drugs were applied in
much larger therapeutic doses
'the tumor cells are selectively "opened" by the glycosamino-
glycan polysulfate, if desired, together with trypsin9
with cytotoxically and cytol~tically, respectively effective
substances being concertedly introduced.
'the preparation according to the invention can parenterall~
be given.
'the therapeutic individual dose of the therapeutic preparation
ranges between 5 and OWE my.
-- 5
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1 the quantity ratio of gl~cosaninoglycan pol~sulfate -to
cytostatic drugs ranges between 10:1 and 1:10 with
regard to parts by weight ox the active substances.
the quantity ox cytostatic drums depends on the kind
of cytostatic drugs used.
the invention is explained by the following experiments.
'the following pharmacological active substances have
been applied in these experiments:
a) glycosaminoglycan pol~sul~ates:
lo 1. chondroitin polysulfate
polysulfated chondroitin of bovine tracheal
cartilage; disaccharide units consisting of
D-glucuronic acid and acutely galactosamine;
Content owe
hexosamine: 10~ Molly my thereof approximately
I ox galactosamine
glucuronic acid: 97~3 pmol/100 my
2. hepa~in-sodium
continuity: OWE%
hexosamine: 152 Molly my ~glucosamin~: 145 ymol/
100 my, galactosamine:
0.02 pmol~100 my)
ironic Acadia Molly my
b) cytostatic drugs:
cyclophosphamide
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~erir~ent 1
72 homosexual rats (pry Doyle) were randomized in
6 groups, the individual groups were treated with the
following active substances:
Group A: sodium heparin c~clophosphamide
Group B: sodium heparin
Group C: chondroitin polysulfate -I cyclophospha~ide
Group D: chondroitin polysulfate
Group I: c~clophosphamide
lo Group I: control
or tumor transmission each animal was inoculated with
20~000 cells of a Yoshida~ascites-sarcoma~
the rats were given the hollowing doses of the active agents
(per kg rat):
cyclophosphamide 25 mg/kg
sodium heparin 10 mg/kg
cho~droitin polysulfate 10 mg/kg
Therapy started 24 hours after the tumor had been communicated
temporal sequence ox the experiment
1. Application of the therapeutically active substances
24 hours after Auschwitz inoculation;
2. application of the therapeutically active substances
28 hours after Auschwitz inoculation;
3. application of the therapeutically active substances
72 hours after Auschwitz inoculation.
l 3 weeks after the last application the results ox the
export were valuated.
A table has been draw up ox the results:
Table 1
group withalive~ree from a dead hazing a
12 animals tumor tumor
. ,...... _
A 11 11 1
Jo 1 1 11 '11
C 11 11 'I
lo D 0 0 12 12
2 2 10 10
0 0 12 12
Experiment 2
12 female rats (Sprague Hawley) were randomized in 2 groups
or tumor transmission each animal was inoculated with
20,000 cells of a Yoshida-ascites-sarcoma.
the rats were given the following doses ox the active
substances (per kg rat):
c~clophosphamide 25 mg/kg
sodium heparin 10 mg/kg
chondroitin pol~sul~ate 10 mg/kg
Start of the therapy: 48 hours after tumor transmission
1 After the first application given 48 hours Atari the
tumor had been communicated six further applications
followed
Group a . of the animals was treated with sodium heparin +
cyclopho sphamide .
Group b. of the animals was treated with chondroitin polyp
sulfate cyclophosphamide~ A table 2 has been drawn up
of the results.
able 2
10 Group with alive tree prom a dead having a
12 animals tumor tumor
-- _ _ . _
a 2 2 10 9
by 8 6
It definitely emerges from the results of both experiments
1 and 2 that chondroitin polysulfate acts on malignant
cells in a surprisingly better anywhere if there is already
an increased number ox such cells. Equally good effect
is achieved by the addition of heparin and chondroitin polyp
sulfate in case the substances are applied very early,
that is, at least 24 hours after the tumor has been
communicated Chondroitin polysulfate obviously causes
cytostatic drugs to be incorporated also when the cells
are in their resting stage. Cytostatic drugs alone and
cytostatic drugs and heparin, respectively, destroy tumor
cells almost only in a ~itotic stage that is in a phase
when the cells are divided. to static drugs and heparin
are only slightly superior to cytostatic drugs alone
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1 It a first clinical application extending over a period
of 18 months the two-substance combination glycosamino-
glycan polysulfate, except heparin, and cytostatic drug
was tested on 125 persons suffering from various tumors
such as, for example, carcinoma of the breast, Castro-
intestinal carcinoma bronchial carcinoma, melanoma and
cancer of the over. In addition to the particular
effectiveness to tumors having a great number ox cells,
it has moreover been observed - and this is a very
10 decisive innovation - that the know considerable secondary
effects of the cytostatic drugs (mitomycin, endoxan,
vincristin~ adriblastin) could be clearly reduce
Above all, younger women did either not or only slightly
suffer from loss of hair. Generally there was no sickness,
no vomiting and no diarrhea. What has to be particularly
emphasized is the fact, that the injuries of the medulla
of a bone caused by cytostatic drums could be remarkably
reduced.
Both subjective and objective negative secondary effects
of the sole chemotherapy of cancer are thus either
reduced or canceled by adding a glycosaminoglycan polysul~ate,
except heparin, to a cytostatic drug, with a decisive
improvement of the effect to malignant tumors being
simultaneously achieved.
All effects of the described combination of active
substances according to the invention which have been
mentioned herein before, constitute significant innovations
hitherto unknown but extraordinarily important for
the treatment of malignant tumors.
-- Jo -
I 7
1 Experiment
36 female rats Sprague Doyle) were divided into 3 groups.
The animals were assigned to the groups according to
the usual randomization.
Kind of tumor: Yoshida-ascites (Homer c)
Number of cells of Yos~;da-ascites inoculation: approximately
20,000
Beginning of the therapy: 48 hours (!) after the tumor has
been inoculated.
Group A: chond~oitin polysulfate trgpsin
lo Group I: chondroitin polysulfate + trypsin cyclophosphamide
Group C: control
Doses of the pharmacological active substances:
chondroitin polysulfate 12 mg/kg
trypsin 6 mg/kg
cyclophosphamide 26 mg/kg
temporal sequence of the experiment
1. Application of the therapeutically active substances
48 hours after Auschwitz inoculation;
2. application of the therapeutically active substances
I 3 days after Auschwitz inoculation;
3, application of the therapeutically active substances
4 days after Auschwitz inoculation.
Jo Y
1 JO application ox the therapelltically active substances
6 days after Auschwitz inoculation;
jesting of the e~ficac~:
I days after the Yoshida-ascites tumor has been inoculated
table 3 has been drawn up of the results.
Group with alive free from ahead having a
12 animals tumor tumor
_ .. . __ _
7 0 5 5
lo 8 8 4 4
C 0 0 12 12
Experiment 3 was partly repeated.
36 female rats (Sprague Hawley) were divided in-to 3 groups.
the animals were assigned to the groups according to the
process of randomization.
find of tumor: Yoshida-ascites (hemorrhagic)
umber of cells upon tumor inoculation: 20,000
Beginning ox the therapy: 2 days after tumor inoculation
20 group A : chondroitin polysulfate12 mug
trypsin mg/kg
cyclophosphamide26 mg/kg
Group : control
Group C : cyclophosphamide26 mg/kg
- 12
1 1. Application of -the -therapeutically active substances
2 days after tutor transmission (20,000 cells of the
Yoshida-ascites-sarcoma);
2. application of the therapeutically active substances
3 days after tumor transmission;
3. application of the therapeutically active substances
5 days after tutor transmission;
4. application of the therapeutically active substances
7 days after tumor transmission
Examination of the test results after 21 days.
A table 4 has been drawn up of the results.
table 4
. . .
Group with alive free from a dead having a
12 animals tumor tumor
_ _ __
A 9 9 3 3
B 0 0 12 12
C 2 2 10 10
Similar to the foregoing experiments 24 female rats
(Sprague Doyle) were randomized.
umber of groups: 2
tumor : Yoshida-ascites (hemorrhagic)
Mummer of cells upon tumor inoculation: approx, 23~000
Group A : chondroitin polysulfate 12 mg/kg
-I trypsin my
c~clophosphamide 25 mg~kg
Group : control
3 -
1 Temporal sequence ox the experiment:
tart ox the therapy (1. application of the therapeutically
active substances 2 days after tumor transmission
2. application 3 days after tumor transmission,
3. application 4 days after tumor transmission,
4 application 5 days after tumor transmission,
5. application 6 days after tumor transmission
6. application 7 days alter tumor transmission).
Examination of the results 21 days after inoculation of
lo the tumor.
table has been drawn up ox the results.
Table
Group with alive tree prom a dead having a
12 animals tumor in this tumor
f~Sroup
_ . _ _
A 11 11 1 O
B O 0 12 12
Jo tumor could be found in the deceased animal of group AD
the cause ox death has not been cleared
the results of the experiments show that the experimental
animals exclusively treated Thea the cytostatic drug cycle-
phosphamide have died of the growth of the tumor in the
course of the experiment similar to the control group
The experimental animals which have been given the cytostatic
drug cyclophosphamide together with a ~lycosaminoglycan
polysul~ate and in addition trypsin have shown a
significantly lengthened lifetime and partly, no growth ox
a tumor. When the group By D and E in experiment 1 as jell
as group in experiment 3 and group C in experiment 4
30 are compared with the groups A and G in experiment 1, group :13
in experiment 3, group in experiment 4 an group A in
experiment 5 the synergistic effect owe the preparation
r7 I
awkward; to the invention dental roulettes,.
histological and cytochemical e2~minatio3ls carried out
parallel to the animal e~eriment~ show that a largely
selective effect to malignaIIt cell con be obtained
5 by the combination OX the substances applied
Experiment 6
60 female rat (Sprague Hawley) were classified into
tree groups (randomized).
wind of tumor: ~oshida-ascit~s (hemorrhagic)
10 Fumier of cells of the ~roshida-ascites inoculation: appear.
2û,~)00
tart of the Thorpe: 24 hours after i~oclllation
Group JO cho~droitin polysulfate c;srclophosphamide
+ to in
15 Group B: chondroitin polysulfate + cyclopho~pha~ide
Group C: control
Doses of the pharmacological active substances:
chondroiti~ pol~sul~ate 12 go per kg
c~5rclopho~phamide (trademark: Endoxan) 24 go per kg
Tropez 6 go / per kg
temporal sequence of the application:
application of the aforementioned combination 24 hours
after tumor inoculation. P~rther applications daily
ox the whole sever applications.
table 6 has been drawn up of the results.
-- 15 --
1';"~7
l Table 6
-
Group with alive free from ahead having a
20 animals tutor tumor
_ . .
A 19 19 1 0
B 12 12 8 7
C 0 0 20 20
this experiment was repeated. table 7 has been drawn up
of the results.
able Z
10 Group with alive free from a dead having a
20 animals tumor tumor
I_ __
A 18 18 2 0
B 11 11 9 7
C 1 0 19 19
As is especially shown by the results of experiment 6
a particular synergistic effect results prom the combination
of the cytostatic drug cyclophosphamide and chondroiti~
polysulfate and trypsinr It seems that a still not entirely
understood reciprocity between the chondroitin polysulfate
and trypsin entails an increased permeability of the
malignant cells to the cytostatic drugs.
The experiments 3 to 6, in which the composition: cho~droitin
polysul~ate + cyclophosphamide + trypsin was respectively
compared with either chondroitin polysulfa~e trypsin or
c~clophosphamide or sully with a control or with chondroitin
I
'7
1 polysulfate + cyclophosphamide definitely prove that
the combination according to the invention is surprisingly
superior Each of -the experiments 3 to 6 individually
shows that the chance of survival of the experimental
animals is increased to a disproportionately high degree
when the active substance combination of chondroitin
polysulfate, trypsin and cyclophosphamide is administered.
In addition to the particular effectiveness to tumors
having a great number of cells, it has been observed as
has already been mentioned, that the aggravating secondary
effects of the cytostatic drugs could be substantially
reduced - and this is an absolutely decisive innovation -
yin particular, younger women did oft or not or only
slightly surfer from loss of hair furthermore, twerp
was generally no sickness, no vomiting and no diarrhea.
What has to be particularly emphasized is the fact
that the injuries of the medulla of a bone caused
by -the cytostatic drugs could be remarkably reduced.
