The
effect of rectally administered proteinases in animal models of
malignancies
Wald M., Pouèková P., Zadinová M., Olejár T.
The effect of rectally administered proteinases in animal models of
malignancies. International symposium of pediatric oncology, April
23 - 24, 1998, Prague, Czech Republic, abst. 33, pp. 50. 598 KA
(19-03-3)
Department of Surgery, 2nd Medical School, Charles University,
Prague, Institute of Biophysics, 1st Medical School, Charles
University, Prague.
Abstract
Long-term rectal administration of enzyme mixture containing papain,
trypsin and chymotrypsin in the same ratio as the preparation
Wobe-Mugos E (Mucos Pharma, Germany) was evaluated for its antitumor
effects in three animal models of malignancies. In experimental
studies with B16 melanoma and Lewis‘ lung carcinoma in mice treated
with proteinases prolonged survival and reducing of metastases was
demonstrated. In spontaneous T-lymphoblastic leukemia of rat species
SD/Ipcv, prolonged survival, decreased terminal blast counts and
minimal weight loss was observed in the treated animals.
In C57Bl6 inbred mice inoculated with B16 melanoma cells 30% of the
test group (TG) animals have been cured of cancer. In the rest of TG
(70%) the survival time was prolonged by 58.3% as compared to the
control group (CG) (average survival time - 24 days CG, 38 days TG).
Based on histological and immunohistochemical evaluation a faster
process of metastasizing was found in CG than in the TG.
C57Bl6 mice with the Lewis lung carcinoma was
inoculated subcutaneously and primary tumors were removed on the day
12. The metastatic process (MP) was investigated. In the control
group (CG), 90% of animals died of MP by the day 18 after primary
tumor extirpation. In group A (enzyme therapy /ET/ started from the
day of primary tumor extirpation, lasted till the end of the
experiment) - 30% of mice died of MP (by the day 25). In group B (ET
started 6 days before primary tumor extirpation and lasted till the
end of the experiment) only 10% of animals died of MP (by day the
15). Group C (ET began 24 hours after intracutaneous tumor
inoculation and lasted till the end of experiment) - no MP was
found. None of the CG survived till the end of experiment at 100
days. In groups A, B and C - 60%, 90% and 100% of animals,
respectively, survived till the end of experiment.
In the experiment with rat species SD/Ipcv spontaneous lymphoblastic
leukemia TG presented a significantly lower rate of blast cells in
the peripheral blood during disease development. The average
survival time in TG was 150 ± 45,62 days, in CG 43,83 ± 24,52 days
since the appearance of the first blast cells. One half of TG
survived until the end of experiment (366 days) and was finally
sacrified. Two of surviving animals were completely free of blast
cells at the end of experiment. ET also had a positive effect on the
stable body weight gain in TG in contrast to the body weight loss of
the CG in the terminal stage of the disease.
Preparations consisting of enzyme mixtures (Wobenzym, Phlogenzym,
Wobe-Mugos) are used in various medicine fields for many years.
These drugs are composed of hydrolytic enzymes (proteinases) of
plant (bromelain, papain) and animal (trypsin, chymotrypsin) origin.
Antiedematous, anti-inflammatory, thrombolytic, analgetic and
immunomodulatory properties of these drugs are estimated.
In cancer patients enzyme preparations e.g. can help to reduce side
effects of chemotherapy and improve the tolerance of radiation.
Their ability to block growth of cancer cells and metastasizing is
found questionable and this topic is discussed very often.
Nevertheless, previously performed animal experiments confirmed this
ability. To search after this problem we decided to arrange new
experiments.
Proteinase mixture containing enzymes in the same ratio as the
preparation Wobe-Mugos E [MUCOS Pharma, Germany – one tablet
contains papain 100 mg (=270 F.I.P.E.), trypsin 40 mg (=29 mkat),
chymotrypsin 40 mg (=200 mkat), corresponding to the total
proteolytic activity from 1740 F.I.P.E] were chosen for these
experiments.
-
-
1) Preparation Wobe-Mugos E inhibits the growth of melanoma B16 and development of metastases in experiment on mice
Material and methods:
melanoma Bl6 cells were multiplied in the acsitic fluid
20 inbred C 57Bl6 mice were intradermally inoculated with 2x106 tumor cells
growing primary tumors were surgically removed on the day 10 after the inoculation
previous metastatic activity melanoma B16 studies showed: 100% of mice perished because of metastases within 3 weeks after the 10th day removal of primary tumor on the day of primary tumor removal 2 groups were created:
|
A |
B |
|
(enzyme) |
(control) |
|
10 mice |
10 mice |
|
Wobe Mugos E |
saline |
|
2x45 mg/kg of body weight/day |
2x daily |
|
per rectum |
per rectum |
daily application until the perishing of mice
on the day of perishing an autopsy was performed and organs affected
by the metastatic process were taken for histological and
immunohistochemical examination
Results:
The graph shows that all mice in the control group perished within
30 days of experiment, whereas in the test group 30% of mice
survived for 100 days and were sacrificed after the experiment
termination. Autopsy showed no metastases.
The mean survival time in mice perished of
metastases was in control group 24 days, whereas in Wobe-Mugos E
test group 38 days.
Histological and immunohistochemical evaluation proved a faster and
more developed metastatic process in control group in comparison to
the test one.
|
a |
b |
|
|
|
Histological findings of B16 melanoma: (a) Test group - lungs:
disseminated larger and smaller irregular tumor foci, predominantly
in vessel areas. Polymorphic cells with vesiculiform nucleus and
nucleolus. Sporadic occurrence of polymorphic cells. (b) Control
group - lungs: regressive changes (hemorrhages and necroses) can be
observed in tumors. Tumor cells proliferate on the edge.
|
a |
b |
|
|
|
-
Immunohistochemical evaluation: (a) Test group - lungs:
proliferating cells in lung tumor foci are actively labeled by
peroxidase. (b) Control group - lungs: proliferating cells on
the edge of necrosing tumor foci in lungs are labeled by
peroxidase.
Conclusion:
The results described here demonstrate that the polyenzyme preparation Wobe-Mugos E is capable to inhibit B16 melanoma tumor growth and metastasizing in mice.
-
-
2) The effect of proteinases on survival and hematological parameters in SD/Ipcv rat spontaneous lymphoblastic leukemia.
Material and methods:
in average in 37% of SD/Ipcv rats the spontaneous lymphoblastic leukemia occurs during the first year of life
52 animals (28 males, 24 females) were investigated, peripheral blood count was screened every two weks between the day 44 and 100 of age and then every week till the end of experiment (366 days)
12 rats with lymphoblasts appearing in the peripheral blood were divided into two groups. There were no statistically significant differences from the time of first blast occurrence in the enzyme and control group.
|
A |
B |
|
(enzyme) |
(control) |
|
6 rats |
6 rats |
|
Wobe-Mugos E |
saline |
|
2x45 mg/kg of body weight/day |
0.1 ml 2x daily |
|
per rectum |
per rectum |
Results:
|
The mean survival time: (since the appearance of first
blasts)
|
|
|
Enzyme group |
150 ± 45.62 days. |
|
Control group |
43, 83 ± 24.52 days |
(p=0.004).
Body weight development of included animals was observed and
compared in the period of last six weeks before the perishing or
sacrifice. Rats of enzyme group showed a stable body weight or slow
body weight gain (on average 10,83 g in last 6 weeks). In opposite,
significant weight loss (on average 88,66 g in the last 6 weeks)
were observed in control group.
There was a perceptible difference in the mean
blast count in the peripheral blood of both groups before exitus.
Common blast counts in control animals were from 7x109/L
to 42x109/L (30-72%). In just one animal from the test
group the periphery at the time of exitus was massively flooded with
blasts to the value 11x109/l (37%) (p=0.017):
Maximum total white blood cell counts at the time of exitus reached
in the test group ranged from 9.9x109/L to 29,8 x109/L
and in control group values varied from 21.6x109/L to
58x109/L.
Lymphocyte and neutrophil counts see table.
|
|
A (enzyme group) |
B(control group) |
|
mean survival time |
150 ± 45,62 |
43,83 ± 24,62 |
|
max. total WBC count* |
9,9 – 29,8 x 109/L |
21,6 – 58,0 x 109/L |
|
blast count* |
0,0 – 11,0 x 109/L |
7,0 – 42,0 x 109/L |
|
lymphocyte count* |
9,9 – 29,8 x 109/L |
3,5 – 13,1 x 109/L |
|
neutrophil count* |
3,6 – 6,3 x 109/L |
5,6 – 25,2 x 109/L |
* the last values before exitus
Conclusion:
From the obtained data it could be concluded that the rectal
administration of proteolytic enzyme mixture (Wobe-Mugos E)
significantly prolonged survival time in SD/Ipcv leukemic rats and
is able to alleviate the severity of disease course – mainly
occurrence of blasts in peripheral blood is decreased and treated
animals show better condition without weight loss.
3)
Proteinases (Wobe-Mugos E) reduce metastasizing and prolong survival
time in C57B16 mice with Lewis lung carcinoma
Material and methods:
murine Lewis lung carcinoma tumor cells were multiplied in the
ascitic fluid
40 C57Bl6 mice were intradermally inoculated with 4x106 tumor cells
on the day 12 after the inoculation growing primary tumors were
surgically removed and 4 groups were created:
|
K |
A |
B |
C |
|
10 mice |
10 mice |
10 mice |
10 mice |
|
saline |
Wobe-Mugos E |
Wobe-Mugos E |
Wobe-Mugos E |
|
0.1 ml- 2x daily |
2x45 mg/kg of body |
the same dosage |
the same dosage |
|
per rectum |
per rectum |
per rectum |
per rectum |
|
from the day |
from the day |
start 6 days before removal |
start on the day |
administration was
performed daily until the perishing of mice
observation was performed for 100 days after the primary tumor
removal
after this period all surviving animals were sacrificed.
in perished and sacrificed animals a pathology-anatomical autopsy
including histological examination was performed
lungs, liver, primary tumor relapses, and metastases to axillary
lymphatic nodes were examined
Results:
Group K (control)
All mice perished with the mean survival time of 14,3 days. In 9
mice (90%) a generalization of cancer was a cause of death. In one
case (10%) heart failure was a cause of death.
Group A (test) - drug administration started after removal of
primary tumor. 6 mice (60%) survived until the end of experiment
(day 100) and were sacrificed. No metastases were found. 4 mice
(40%) perished with the mean survival time of 68,7 days and in 3
mice (30%) a generalization of cancer was a cause of death.
Group B (test) - drug administration started 6 days before
removal of primary tumor. 9 mice (90%) survived until the end of
experiment (day 100) and were sacrificed. No metastases were found.
Just 1 mouse (10%) perished on the day 15.
Group C (test) - drug administration started on the day of
primary tumor inoculation. All mice (100%) survived till the end of
experiment and were sacrificed on the day 100. No metastases were
found.
Conclusion:
Study results showed unambiguously that the administration of
Wobe-Mugos E to the mice with Lewis lung carcinoma has a fundamental
influence on the metastasizing process.While none of the mice in
control group survived till the end of experiment -100 days - and a
cause of death was in 90% tumor generalization, in the test groups
60%, 90%, and 100% of mice, resp. survived till the end of
experiment and the animals were free of primary tumor generalization
signs.
Results of al three experiments show longer survival of enzyme treated animal groups and imply that proteinase mixture rectal administration reduces metastatic potential of solid tumors and alleviate severity of spontaneous lymphatic leukemia course.
Direct molecular mechanism of these enzyme effects has not been fully clarified yet.