THE RUSSIAN ACADEMY OF NATURAL SCIENCES (RANS)

RESEARCH INSTITUTE OF IMMUNOPATHOLOGY

I ASSERT
Director of Research Institute of Immunopathology of
RANS,
Doctor of medical sciences, professor,
Corresponding member of RANS
__________________ M.T.Abidov
"___" ______________ 2001.

SUMMARY REPORT
On experimental and clinical researches of
preparations VIUSID and ONCOXIN at oncological diseases

Moscow - 2001

Organizations - co-executors of work:

1. Recearch institute of experimental therapy and diagnostics of tumors of Russian

Blokhin cancer recearch centre of Rassian Academy of Medical Sciences
(Moscow).

2. Tomsk regional clinical hospital (Tomsk).

3. Centre of Modern Medicine of Moscow academy of the market of labour and

information technologies (Moscow).
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Object of research:
The biologically active additives to food Viusid (powder for dissolution 3.2 g, ”Catalysis”,
Madrid - Spain) and Oncoxin (capsules 300 mg, "Catalysis", Madrid - Spain).

1. Experimental researches.
The research problems:
1.1. Study of cytotoxic effect of the biologically active additive to food viusid on human _varian
tumor cells _aOv in vitro.
1.2. Study of antitumor activity of the biologically active additives to food viusid and oncoxin in
experiments in vivo on mouse tumors - lymphocyte leukemia P388, melanoma B16 and
epidermoid Lewis lung carcinoma (LLC).

1.1. Research of cytotoxic effect of viusid

on human _varian tumor cells in vitro.
Materials and methods of research.
Research of cytotoxic effect of viusid was carried out on cells of human ovarian carcinoma
CaOv. _ells were cultured in a monolayer in _ulture medium RPMI1640 supplemented with 10%
fetal _alf serum (FCS) at 37°C.
Cytotoxic activity of oncoxin on tumor cells was not investigated in model test systems in
vitro as the preparation was not dissolved in culture medium, DMSO and several other aquos and
organic solvents.
_ytotoxicity of viusid on CaOv cells determined with the help of the MTT-test. The MTT-
test is based on ensyme-dependent restoration of colourless tetrazole salt in viable and
metabolically active cells with formation of light-blue crystals of phormazane.
Briefly, CaOv cell suspensions were placed in wells of 96-well flat bottom plate (1,7 x 104
cells/well) in _ulture medium RPMI1640 supplemented with 10 % FCS in final volume of 180 _l
and incubated within 24 hours at 37°C and 5% __2. Then researched preparation in final
concentration from 1 _g/ml up to 500 _g/ml (20 _l) was added to plate wells. After 48 hours of
incubation, MTT-reagent (100 _l, final concentration 25 _g/ml) was added to plate wells. After the
next 4 hours of incubation, the formed light-blue phormazane crystals were dissolved in 100 _l
DMSO within 20 minutes. Absorption of the painted DMSO solutions was measured on Titertek
Multiscan MCC/340 at length of a wave 570 nm. The results in test-samples estimated in
comparison with control (samples without researched preparation). Inhibition of tumor cell growth
(ICG) in tests was determined by the formula:
ICG = (1 - test / control) _ 100 %.
Cytotoxic activity of a preparation was estimated with the help of parameter IC50
(concentration causing 50% inhibition of cell growth). Preparation was considered to be active at
IC50 < 100 _g/ml. The mistake of measurements did not exceed 10%.

Results of research.
The results of research of viusid cytotoxicity on CaOv cells are presented in the table 1.
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Table 1.
Cytotoxic effect of viusid on CaOv cells in vitro.
Concentration of a preparation, µg/ml Inhibition of cell growth, %
1 6,25
10 9,65
100 9,65
500 8,52

As it is shown in the table, viusid in a wide range of dozes does not render essential
influence on growth of tumor cells in vitro (IC > 500 _g/ml, that exceeds by 5 folds the given
criterion). It is possible to speak only about the insignificant tendency to an oppression of growth of
cells CaOv (6,25-9,65 %).

1.2. Study of antitumor activity of

the biologically active additives to food viusid and oncoxin in vivo.

Materials and methods of research.

Antitumor action of the biologically active additives to food have been studied on mouse
tumors included in list of obligatory models of tumors, which according to the recommendations of
State Pharmacological Committee of Russian Minestry of Healthcare are used at selection of new
antitumor substances - lymphocyte leukemia P388, melanoma B16 and epidermoid Lewis lung
carcinoma (LLC).
In brief, melanoma B16 and LLC cell suspensions (105 cells) were injected subcutaneously
in axxilary region of BDF1 (DBA/2 x C57BL/6) mice. Suspensions of P388 cells (105 cells/mouse)
were inoculated intraperitoneally to BDF1 (DBA/2 x C57BL/6) mice.
Efficacy of treatment in models of melanoma B16 and LLC was estimated by using of
following criteria: 1) inhibition of tumor growth (%) and 2) increase of life-span (%) of tumor-
bearing animals in comparison with control.
In model of leukemia P388, the main criteria of antitumor activity were 1) increase of life-
span and 2) decrease of ascites volume.
The biologically active additives to food were studied at three doses: therapeutic dose, 10-
multiplely decreased and 10-multiplely increased therapeutic doses. Mouse therapeutic doses
recalculated from human therapeutic doses were 640 mg/kg for viusid and 60 mg/kg for oncoxin.
Treatment began in 48 hours after inoculation of solid tumors and in 24 hours after injection of
leukemia _-388 cells. The biologically active additives were administered per os 3 times daily
within 14 days. Water solutions of the food additives prepared ex tempore. Combined application of
viusid and oncoxin was carried out with an interval not less than 30 minutes. 0.9% NaCl solution
without preparations was administered in the same mode to mice of control groups.
The number of animals in test groups was 8, in control - 14.

Criterion of estimation of antitumor effect

Inhibition of tumor growth (ITG, %).
The measurement of three maximal mutually perpendicular sizes (length, width, height) and
calculation of tumor volume was carried out in each animal, and then average volume of a tumor in
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group was calculated. The measurement of volume of a tumor was carried out by with interval of 3
days starting from 7th day of experiment, when the tumors in the control became measurable.
Formula for calculation of tumor volume:
V (mm3) = L (mm) _ W (mm) _ H (mm).

Inhibition of tumor growth was calculated with the formula:

ITG = (V control – V test) / V control x 100 %,
where
V control - mean volume of tumors in control group,
V test – mean volume of tumors in test group.

Increase of life-span (ILS, %).

After determination of mean life-span (MLS) in test and control groups, ILS was calculated
using the formula:
ILS = (MLS test – MLS control)/ MLS control x 100 %,
where
MLS control - mean life-span of animals in control group,
MLS test - mean life-span of animals in test group.

Minimal criteria of activity – inhibition of tumor growth - 50 %, increase of life-span – 25%.

Toxicity of the preparations was estimated by status of internal organs of animals (spleen,
lungs, presence of metastases in lungs), changes of weight of a body in cases of early death of mice
in comparison with those in cases of their death in the control.

Influence of preparations on growth of lung metastases.

Inhibition of metastases (IM) was estimated by change of lung weight of lost animals in test
groups in comparison with lung weight of animals in the control (%). IM was calculated with the
formula:
IM = (WL test – WL control) / WL control x 100 %,
where
WL control – mean weight of lungs of animals in control group,
WL test – mean weight of lung of animals in test group.

Statistical anlysis was perfomed using the Student’s t-test. The data were expressed as mean
± standard deviation. The distinctions between compared groups were considered to be statistically
sigificant at _ < 0.05.

Results of researches
In model of leukemia _-388, viusid at a doses of 64 and 640 mg/kg and oncoxin at a dose of
6 mg/kg were shown to cause the tendency to increase of life-span of tumor-bearing animals (6 %),
however distinction with the control in these cases was statistically insignificant. Combined
application of both preparations at therapeutic doses did not increase life-span of treated mice
(fig.1).
Administration of viusid at a dose of 6400 mg/kg _ 3 times per day within 14 days reduced
volume of ascites up to 0,8±0,08 ml in comparison with the control - 2,0±0,22 ml, _ < 0,05 (table
2).
In some cases, high doses of the preparations (10-multiplely increased therapeutic dose)
caused the death from toxicity: 1 from 8 mice was lost on the day 3 from the beginning of
administration of viusid, 1 animal from 8 was lost on the day 6 in group of mice treated with
oncoxin.
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Viusid and oncoxin were demonstrated to have a certain tendency to inhibition of growth of
melanoma B16 practically in all terms of research (table 3). The most marked tendency to delay the
growth of tumor was shown on the 7-th day of experiment: viusid at a doze of 64 mg/kg (37 %),
oncoxin - 600 mg/kg (56 %).
However, the combined application of these preparations resulted in stimulation of growth
of tumors (62 %) on the day 7.

Table 2.
Antitumor activity of viusid and oncoxin in model of mouse leukemia _-388.
Death
Dose (mg/kg) _
Preparation Increase of Weith of Weith of Volume from
3 times a day/
life-span, spleen (mg) liver (g) of ascites toxicity
interval (h) _
ILS (%) (ml) (n/n**)
number of
administrations
212,8±17,18 1,9±0,07 2,0±0,22
Control
6 126,4±13,94 1,6±0,09 2,2±0,20 0/8
Viusid 64 _ 3/24 _ 14
6 158,3±18,27 1,8±0,12 1,9±0,29 0/8
Viusid 640 _ 3/24 _ 14
-4*** 125,9±10,29 1,5±0,14 1,8±0,23 1/8
Viusid 6400 _ 3/24 _ 14
6 166,5±21,11 1,9±0,13 1,5±0,18 0/8
Oncoxin 6 _ 3/24 _ 14
2 145,6±9,63 1,9±0,11 1,6±0,27 0/8
Oncoxin 60 _ 3/24 _ 14
3 203,6±28,75 2,0±0,21 0,8±0,08* 1/8
Oncoxin 600 _ 3/24 _ 14
-6 164,9±23,16 1,4±0,12 1,2±0,26 0/8
Viusid + 640 _ 3/24 _ 14

Oncoxin 60 _ 3/24 _ 14*

Notes: * - The distinctions with the control are statistically significant, _ < 0,05
** - n/n, ratio of number of lost animals to common number of animals in the group,
*** - The mark "-" means reduction of life-span in test mice in comparison with control.

Viusid and oncoxin at different doses either did not change essentially or reduced the life-
span of mice with melanoma B16.
The part of animals (2/8) was lost on the 8th-11th days. In model of melanoma B16, viusid
has appeared to be more toxic than oncoxin. When used at a doses of 10-multiplely increased and
10-multiplely reduced therapeutic dose, viusid caused the death of 2 from 8 animals.

Table 3.
Antitumor activity of viusid and oncoxin in model of mouse melanoma B16.
Dose (mg/kg) _ Inhibition of tumor growth (%)*** Death
3 times a day/ from
Prepa- interval (h) _ Day after inoculation of tumor ILS (%) toxicity
rations number of (n/n**)
administrations 7 10 14 17
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Viusid 64 _ 3/24 _ 14 37 +2 3 7 -27**** 2/8

Viusid 640 _ 3/24 _ 14 +4**** 11 28 29 -12 0/8

Viusid 6400 _ 3/24 _ 14 16 24 24 23 -27 2/8

Oncoxin 6 _ 3/24 _ 14 +38 26 17 17 -19 0/8

Oncoxin 60 _ 3/24 _ 14 27 13 12 17 2 0/8

Oncoxin 600 _ 3/24 _ 14 56 +12 17 29 -2 0/8

Viusid + 640 _ 3/24 _ 14 +62 +20 2 26 -31 2/8

Oncoxin 60 _ 3/24 _ 14*

Notes: *- Oncoxin was administered 30 minutes later than viusid.
** - n/n, ratio of ratio of number of lost animals to common number of animals in the group,
*** - Inhibition of tumor growth in all test groups was statictically insignificant, _>0,05
****- The mark “+” means stimulation of tumor growth; the mark "-" means reduction of
life-span in test mice in comparison with control .

Table 4.
Antitumor activity of viusid and oncoxin in model of Lewis carcinoma.
Dose (mg/kg) _ Inhibition of tumor growth (%)*** Death
3 times a day/ from
Prepa- interval (h) _ Day after inoculation of tumor ILS toxicity
rations number of (%) (n/n**)
administrations 7 10 14 17

Viusid 64 _ 3/24 _ 14 40 53 27 +2 13 0/8

Viusid 640 _ 3/24 _ 14 70 67 55 43 18 0/8

Viusid 6400 _ 3/24 _ 14 59 61 36 10 10 0/8

Oncoxin 6 _ 3/24 _ 14 7 26 +1**** +17 14 0/8

Oncoxin 60 _ 3/24 _ 14 67 64 42 27 13 0/8

Oncoxin 600 _ 3/24 _ 14 37 29 21 13 -3**** 0/8

Viusid + 640 _ 3/24 _ 14 58 66 51 34 16 0/8

Oncoxin 60 _ 3/24 _ 14*

Notes: *- Oncoxin was administered 30 minutes later than viusid.
** - n/n, ratio of ratio of number of lost animals to common number of animals in the group,
*** - Inhibition of tumor growth in all test groups was statictically insignificant, _>0,05
****- The mark “+” means stimulation of tumor growth; the mark "-" means reduction of
life-span in test mice in comparison with control .
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At the same time in model of Lewis carcinima, viusid and oncoxin rendered marked
antitumor effect. Viusid showed significant therapeutic effect (ITG = 55-70%) within 6 days, and
oncoxin (ITG = 64-70%) - within 4 days (tab. 4).
Revealed therapeutic effect has appeared to be statistically insifignificant probablly due to
high individual variability of sensitivity of mice to the food additives, as it is presented in figures 2
and 3.
The results of study of individual sensitivity of mice have shown that, when used during 14
days at therapeutic dose of 640 __/kg (3 times a day), viusid rendered marked antitumor effect in
88% of animals (tab. 5). The maximal inhibition of tumor growth was revealed on the 5th day of
therapy (ITG = 71%), however this effect was statistically insignificant (_ > 0,05). Whereas after
finish of viusid treatment, statistically significant ITG (52%, _ < 0,05) was observed.
63% of mice were sensitive to oncoxin treatment in model of Lewis carcinoma. Oncoxin
inhibited the tumor growth by 62-81% in comparison with the control (_ < 0,05). Therapeutic effect
was kept within 7 days, and then was reduced.
In model of LLC, it have been revealed not only therapeutic effects of viusid and oncoxin,
but also stimulating action of the obove preparations on metastatic process in lungs (tab. 6). Viusid
stimulated the growth of lung metastases at a doses of 64 mg/kg and 640 mg/kg. Lung weight of
treated animal by 63-83% exceeded the weight of lungs in the control (_ > 0,05).
Oncoxin stimulated the metastase growth in lungs to a lesser degree - by 30-39%. This
effect was not dependent on a dose.
Moderate antitumor effect (ITG = 51-66%) of combined application viusid+oncoxin at
therapeutic doses in model of LLC was statistically insignificant (_ > 0,05).

Table 5.
Antitumor activity of viusid and oncoxin used at therapeutic doses in model of Lewis
carcinoma.
Dose (mg/kg) _ Inhibition of tumor growth (%) Tumors sensitive
3 times a day/ to treatment (%)
Prepa- interval (h) _ Day after inoculation of tumor
ration number of
administrations 7 10 14 17

Viusid 640 _ 3/24 _ 14 71 62 65 52* 88

Oncoxin 60 _ 3/24 _ 14 81* 81* 62** 46 63

Notes: * - statistically significant ITG, _<0,05;

** - _ ≥ 0,05

Table 6.
The action of viusid _ oncoxin on tumor node and internal organs of mice with LLC.
Change of lung
Dose (mg/kg) _ 3
Praparation Weight of tumor Weight of lungs weight (% as
times a day/
(g) (mg) compared with
interval (h) _
control)**
number of
 8

administrations
10,5±0,76 386,3±51,82
Control
10,7±0,97 629,8±100,94 +63
Viusid 64 _ 3/24 _ 14
9,1±1,35 706,6±99,44 +83
Viusid 640 _ 3/24 _ 14
11,9±0,63 529,3±118,32 +37
Viusid 6400 _ 3/24 _ 14
12,1±1,44 507,4±74,76 +31
Oncoxin 6 _ 3/24 _ 14
10,3±0,31 537,3±77,32 +39
Oncoxin 60 _ 3/24 _ 14
8,7±1,64 501,3±67,88 +30
Oncoxin 600 _ 3/24 _ 14
9,6±0,37 457,9±72,49 +19
Viusid + 640 _ 3/24 _ 14

Oncoxin 60 _ 3/24 _ 14*

Notes: *- Oncoxin was administered 30 minutes later than viusid.
** - The distinction with control is statistically insignificant, _>0,
*** - The mark “+” means stimulation of metastase growth in lungs.

2. Clinical researches.
Introduction.
Malignant tumors represent one of the main medico-social problem in the advanced
countries and, along with cerebro-vascular dissorders, the basic reason of the lethality of population.
Among oncological diseases, a special etention is paid to gastro-intestinal malignant tumors.
In many regions of the world, carcinoma of a stomach occupies the second place in structure of
oncologic diseases (13,3 % -16,5 %) and lethality (19,0 %-19,3 %) (Perevodchikova N.I., 1993,
Dvoirin V.V. et al., 1992). Last decade, tumors of colon occupy the third place among neoplastic
diseases of gastro-intestinal canal and tend to significant growth.
Surgical treatment is a single radical method of therapy of malignant tumors of a stomach
and colon. However, postoperative period is accompanied with a number of complications and high
lethality, and the majority of lost patients perishs from complications of septic character. 5-year's
survival varys from 17% up to 51% (_________ _._. With _____., 1988, ______ _._., 1993 etc.),
despite of wide application of cytostatic therapy after radical removal of a tumor.
The increase of efficacy of treatment of the obove pathology is connected both with
improvement of traditional methods of therapy and with deeper understanding of ethilogy and
pathogenesis of tumor process leading to search of the new approaches in the decision of the given
problem. One of key directions in study of malignant tumors including those of gastro-intestinal
localization is the research of interaction of immune system and tumor. The investigation of status
of cellular and humoral immunity has a prognostic importance in the patients with tumor of
mentioned localization. Great attention is paid to study of lymphocyte infiltration of tumor and
regional lymph nodes (Careful _._., 1994). The results of various methods of immunotherapy (from
application of immunomodifiers of a natural origin to use of expensive and complex methods of
cytokine and cellular adoptive immunotherapy) have been estimated (Careful _._., 1994, Ostanin
 9

_._. et al., 1996, etc). However, estimated methods of immunotherapy have not allowed essentially
to lower lethality of the cancer patients of the given category.
Thus, the search of modern immunomodulators with antitumor activity is very expedient
and actual.
The application of effective immunomodifiers is to reactivate suppressed antitumor
immunity and to potentiate the result of traditional therapy. Combined application of various groups
of preparations including immunomodifiers and anti-oxidants in cancer patients receiving cytostatic
therapy after radical operative treatment is poorly investigated.

2.1. Report on clinical approbation of viusid and oncoxin in the patients with
gastro-intestinal tumors in Tomsk regional clinical hospital.

The purpose of the present research is to study the action of viusid and oncoxin on immune
status, quality of life and tumor process in patients with gastro-intestinal tumors receiving
polychemothapy after radical operative treatment.

Materials and methods

The researches were carried out on base of proctologic and oncologic departments of
Tomsk regional clinical hospital using the method of randomized double blind placebo-controlled
test in the patients with carcinoma of stomach and colon __NxMx. The diagnosis was proved with
the data ezophago-gastro-duodenoscopy (EGDS) in patients with cancer of stomach and
fibrocolonoscopy (FCS) in patients with carcinoma of colon, histologic research, computer
tomography, study of tumor markers.
The patients have given the consent to clinical tests. The research was carried out in two
groups of the patients, which were comparable on age, sex and clinical manifestations of disease.
The patients were randomized into test (basic) and control groups. Each group consisted of 20 men
at the age of 45-65. In each group, 10 patients were with carcinoma of stomach and 10 – carcinoma
of colon.
The patients of test group received the combination of viusid and oncoxin along with basic
therapy (cytostatic and symptomatic preparations). The patients of control group, besides the same
basic therapy, received placebo.
After morphological verification of tumor process, the radical operative intervention
(subtotal resection of a stomach in 20 men, hemicolonectomy in 10 patients, resection of a part of
colon in 10 men) was carried out in all examined patients.
Postoperative complications took place in 8 patients. Among them, ther were 6
complications of septic character and 2 cases of bleeding. 60% of the patients had a syptoms of
anemic syndrom already at preoperative period due to chronic bleeding (the mean Hb -
108,3±4,62).
Initially in both groups, there were the same disorders of the immune status. The basic
abnormalities of the immunologic parameters were the following: suppression of T-cell component
of the immunity, decrease of phagocytic activity of neutrophils, moderate decrease of a serum level
of Jg G, and increase of JgA.
Before operation, the increase of a serum level of CEA up to 10,28± 0,84 ng/ml and __ 72-
4 up to 10,74±0,96 units/ml was shown in the patients with carcinoma of a stomach, and, in patients
with cancer of colon, we observed the increased level of CEA (11,08±0,91 ng/ml) and __ 19-9
(112,6±5,31 units/ml).
The main biochemical parameters of blood serum in the patients at preoperative did not
differ significantly from the parameters in group of the healthy persons.
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Clinical analysis of the blood, study of biochemical parameters of the blood, immunologic
parameters (_D2, _D3, CD4, CD8, _D16, and HLA -DR, _D72, Jg M, G, A; CIC, the N__-test,
phagocyte index, phagocyte number) were investigated before and after courses of cytostatic
treatment. The quality of life of the patients was estimated with the help of the questionnaire
EORTC QLQ CORE 30.
Course of cytostatic therapy consisted of i.v. injection of 600 mg/day of cyclophosphane
and 700 mg/day of 5-fluorouracil within 14 days.
1 capsule of oncoxin was applied 3 times a day 45 minutes prior to meal, and 1 powder of
viusid was admistered 3 times a day through 45 mines after meal for the period of realization
cytostatic therapy and further for all period of observation (3 months). A break between courses of
cytostatic chemotherapy took 6-7 weeks (till restoration of parameters of blood and improvement of
quality of life). We analyse the effect of treatment with combination viusid+oncoxin, which has
been carried out during two courses of cytostatic therapy. Before and in several days after courses
of the cytostatic therapy, the researches of the basic hematological, immunological and biochemical
parameters of the blood (table 7, 8) were carried out.

Results of researches.
In the test group of the patients who have received first course of cytostatic therapy, there
were no marked signes of suppression of hemopoesis except for a lymphocyte level (table 7) in
contrast to control group. It is necessary to note significant increase of B-cells and IgG, decrease of
level of IgA and CIC. Despite the reception of oncoxin and viusid, chemotherapy caused the
suppression of oxigen-dependent metabolism of neutrophils in the patients of the test group. The
level of alkaline phophatase was increased significantly after realization of cytostatic therapy,
however these parameter did not exceed that in the group of healthy persons. During course of
chemotherapy in test group, the nausea was observed in 3 patients, and the short-term pains in field
of heart – in 2 men.
In control group, marked negative dynamics of the basic hematological and immunological
parameters of the blood took place. Biochemical parameters also became worse, however their level
did not fall outside the limits of parameters of the healthy persons. In 5 five patients of control
group, the nausea and vomit were observed on a background of chemotherapy, in 2 patients -
disuria.
After 6-7 weeks after first course of chemotherapy the patients were hospitalized for
realization of the second course of cytostatic therapy. In the test group (patients receiving oncoxin
and viusid), parameters of urine analysis and clinical and biochemical anlyses of blood did not
differ essentially with group of the healthy persons. As for immune status, immunologic parameters
were within the normal limits in 6 patients (30%), and the other patients had some deviations from
norm. The mean values of such parameters, as CD4/CD8 index, _D72, IgG and A, and phagocyte
number did not differ authentically from parameters of the healthy persons, other immunologic
parameters were significantly below than parameters of the healthy donors, except for Ig_, which
level remained increased (table 8).
In group of comparison, the overwhelming majority of hematological and immunological
parameters essentially differed with similar parameters of the healthy persons and patients of the
test group (table 8).
There was a tendency in both groups to increase of metabolic activity of neutrophils in
spontaneous NBT-test and simultaneous decrease of phagocyte reserve.
In the patients of test group, the increase of quality of life (from 3,38±0,2 points up to
4,11±0,18 points, _ < 0,05) was established. The majority of the patients of control group also noted
improvement of quality of life, however this difference was not statistically significant (3,56±0,21
points and 3,72±0,19 points, _> 0,05).
 11

9 patients of control group and 5 patients of the basic group had various sharp respiratory
diseases during the period of observation..
During the second course of cytostatic treatment the sequences of a nausea repeated in 3
patients of the basic group, and the pains in the field of heart were observed in 1 man. In group of
comparison, nausea and vomit were observed in 5 patients, allopecia – 2 patients, disuria - 1,
astenia - 8.
The study of hematological, immunological and biochemical parameters of the blood carried
out after the second course of cytostatic therapy (table 8) has allowed to reveal negative dynamics
of the majority obove parameters in control group: authentic decrease of level of leucocytes, Hb,
_D2, _D3, CD4, CD8 and _D16 cells, spontaneous N__-test, and also increase of activity alkaline
phosphotase and bilirubine level.
In test group, the authentic increase _D16, decrease of a level Ig M and phagocyte index
were observed. The increase of activity alkaline phosphotase did not exceed the parameter limits of
the healthy persons.
Ultrasonic research carried out in dynamics and computer tomography have allowed to
exclude presence of metastases in the patients of both groups.
In test group, the insignificant increase of CEA level up to 4,0 ng/ml and __ 72 - 4 up to 5,4
units/ml was shown in 1 patient with carcinoma of stomach.
In group of comparison, the insignificant increase of tumor markers was observed in 3
patients. Among them there were 2 men with a tumor of stomach (CEA - 5,0 and 3,9 ng/ml, __ 72-4
- 3,4 and 4,1 ng/ml) and 1 patient with carcinoma of colon (CEA - 5,5 ng/ml and __-19-9 - 42,5
units/ml).

Table 7
Changes of hematological, biochemical and immunological parameters in the patients
with carcinoma of stomach and colon during first course of complex therapy (cytostatics +
viusid and oncoxin)
1-st course of complex therapy (M±m)
Parameters Healthy
persons,
n=35 Test group, n=20 Control group, n=20
Before After Before After
treatment treatment treatment treatment
Leucocytes _109/l 6,21±0,14 5,5±0,21 5,6±0,17 5,2±0,17 4,4±0,19*
Erythrocytes _1012/_ 4,32±0,16 3,4±0,12 3,6±0,08 3,5±0,12 3,3±0,17
Hb, g/l 134,8±2,4 112,7±1,9 113,8±1,8 112,1±2,5 105±2,4*
9
Thrombocytes _10 /_ 204,7±4,23 201,1±4,2 198,2±3,9 196,6±3,6 188±8,4*
Lymphocytes, abs. 2,31±0,07 1,52±0,03 1,6±0,02* 1,56±0,02 1,48±0,03*
CD2 1,54±0,08 1,11±0,02 1,12±0,02 1,15±0,03 1±0,03*
CD3 1,41±0,07 0,98±0,02 0,99±0,03 0,93±0,04 0,64±0,01*
CD4 0,86±0,06 0,64±0,01 0,66±0,01 0,64±0,04 0,56±0,02*
CD8 0,42±0,05 0,36±0,01 0,37±0,01 0,36±0,03 0,33±0,01*
CD4/ CD8 2,02±0,13 1,7±0,02 1,8±0,03 1,7±0,2 1,6±0,03*
CD16 0,21±0,02 0,15±0,004 0,14±0,003 0,15±0,005 0,13±0,01*
CD72 0,4±0,04 0,32±0,01 0,36±0,1 0,33±0,01 0,29±0,01*
∝HLA-DR 0,21±0,03 0,15±0,005 0,14±0,004 0,15±0,004 0,13±0,01*
Ig M, g/l 1,37±0,11 1,75±0,8 1,82±0,7 1,68±0,08 1,59±0,07
Ig G, g/l 12,43±1,21 10,3±0,24 11,3±0,26* 10,4±0,18 10,1±0,16
 12

Ig A, g/l 2,31±0,07 3,4±0,21 2,97±0,11* 3,48±0,17 3,33±0,1

CIC, units 72,4±2,8 80,1±5,2 71,6±3,7* 81±6,5 86,4±5,8
NBT-test spont.% 14,8±1,12 11,7±0,23 10,7±0,29* 11,8±0,29 10,7±0,3*
NBT-test stimul. % 28,1±2,8 17,49±0,32 14,9±0,33* 15,5±0,31 14,4±0,25*
Phagocyte number 10,8±1,2 10,24±0,34 8,7±0,35* 8,6±0,37 7,9±0,37*
Phagocyte index 89,7±0,11 64,2±1,4 62,1±1,5 64,4±1,4 58,4±1,1*
Index of complete 1,59±0,05 1,16±0,04 1,2±0,17 1,18±0,04 1,1±0,03*
phagocytosis
CEA (stomach cancer) <3 n_/ml 2,16±0,17 2,13±0,3
CEA (colon cancer) <3 n_/ml 2,25±0,14 2,15±0,2
__ 72-4 <3 units/ml 2,08±0,18 1,99±0,18
__ 19-9 <37 units/ml 24,8±2,5 24,32±2,6
_l__, µ_/l 0,3±0,04 0,37±0,02 0,43±0,03 0,38±0,01 0,55±0,03*
_s__, µ_/l 0,35±0,03 0,44±0,03 0,47±0,02 0,45±0,02 0,53±0,02*
Al. phosphatase, units/l 64,5±8,9 44,5±2,6 62,6±1,9* 57,7±2,2 68,3±1,1*
Protein, g/l 76±1,8 74,2±1,2 72,1±0,9 74,3±1,1 71,5±0,8*
Albumin, % 45,4±3,5 43,5±0,37 41,7±0,62 43,1±0,44 43,2±0,35
Bilirubin, µ_/l 11,8±0,7 14,9±0,38 14,5±0,4 14,8±0,4 15,8±0,34*
The note: * - reliability of distinctions from a reference value (p < 0,05).

Table 8
Changes of hematological, biochemical and immunological parameters in the patients
with carcinoma of stomach and colon during second course of complex therapy (cytostatics +
viusid and oncoxin)
2-nd course of complex therapy (M±m)
Healthy Test group, n=20 Control group, n=20
Parameters persons,
n=35 Before treatment After treatment Before After treatment
treatment
Leucocytes _109/l 6,21±0,14 5,9±0,18** 5,7±0,24 5,1±0,22 4,7±0,2*
12
Erythrocytes _10 /_ 4,32±0,16 3,9±0,07 3,88±0,07 3,7±0,07 3,6±0,09
Hb, g/l 134,8±2,4 126±1,9** 125,7±2,2 118,8±1,8 115,6±2,1*
Thrombocytes _109/_ 204,7±4,23 197,9±4,8 204,6±9,0 218,4±8,6 212,2±7,6
Lymphocytes, abs. 2,31±0,07 1,85±0,1** 1,8±0,09 1,5±0,02 1,49±0,04*
CD2 1,54±0,08 1,29±0,12 1,32±0,09 1,15±0,03 1,05±0,04*
CD3 1,41±0,07 1,23±0,13 1,19±0,08 1,06±0,03 0,95±0,04*
CD4 0,86±0,06 0,63±0,03 0,6±0,01 0,62±0,02 0,56±0,02*
CD8 0,42±0,05 0,31±0,01 0,3±0,01 0,35±0,01 0,33±0,01*
CD4/ CD8 2,02±0,13 2,03±0,05** 1,98±0,05 1,75±0,02 1,72±0,04
CD16 0,21±0,02 0,18±0,006** 0,19±0,066* 0,14±0,01 0,13±0,007*
CD72 0,4±0,04 0,4±0,01 0,39±0,01 0,31±0,01 0,32±0,01
∝HLA-DR 0,21±0,03 0,18±0,009** 0,18±0,01 0,15±0,01 0,15±0,007
Ig M, g/l 1,37±0,11 2,2±0,08** 1,9±0,06* 1,55±0,08 1,56±0,08
Ig G, g/l 12,43±1,21 12,7±0,3** 12,6±0,3 9,7±0,33 9,3±0,4
Ig A, g/l 2,31±0,07 2,3±0,11** 2,2±0,12 2,7±0,15 2,68±0,13
 13

CIC, units 72,4±2,8 62±2,2** 61,9±1,8 83,7±5,5 93,1±5,7

NBT-test spont.% 14,8±1,12 15,3±0,3 17,3±0,38* 16,01±0,9 12,63±0,6*
NBT-test stimul. % 28,1±2,8 22,4±0,8** 22,6±0,5 18,7±0,7 18,1±0,8
Phagocyte number 10,8±1,2 11,6±0,5 12,8±0,4* 8,9±0,37 8,4±0,4
Phagocyte index 89,7±0,11 66,5±1,02** 61,9±1,7* 62,0±1,3 60,7±1,8
Index of complete 1,59±0,05 1,34±0,03 1,32±0,03 1,3±0,03 1,2±0,05
phagocytosis
CEA (stomach cancer) <3 ng/ml 2,09±0,2 2,29±0,3
CEA (colon cancer) <3 ng/ml 2,1±0,2 2,24±0,4
__ 72-4 <3 units/ml 2,04±0,4 2,06±0,3
__ 19-9 <37 units/ml 14,9±2,7 24,12±2,7
_l__, µ_/l 0,3±0,04 0,35±0,03 0,37±0,03 0,37±0,03 0,43±0,03
_s__, µ_/l 0,35±0,03 0,38±0,03 0,36±0,03 0,37±0,03 0,44±0,03
Al. phosphatase, units/l 64,5±8,9 35,3±1,7** 41,8±2,1* 54,4±2,2 59,9±2,7*
Protein, g/l 76±1,8 77,8±1,3 76,2±1,03 73,8±1,6 74±1,3
Albumin, % 45,4±3,5 43,4±0,5 43,5±0,5 43,2±0,4 42,7±0,4
Bilirubin, µ_/l 11,8±0,7 12,3±0,5** 13,2±0,6 15,05±0,5 16,9±0,5*
The notes: *- Reliability of distinctions between values before and after treatment – p < 0.05.
** - Reliability of distinctions between values before treatment in test and control
groups – p < 0.05.

2.2. Report on clinical approbation of viusid and oncoxin in oncologic patients in

the Centre of Modern Medicine (Moscow)

Besides placebo-controlled randomized study of the effects of oncoxin and viusid in

complex with traditional methods of cancer therapy including operative treatment and cytostatic
therapy, we carried out an open study of the effect of separate application of oncoxin+viusid in the
patients with IV stage of cancer, who were not subject to operative and cytostatic treatment.

Materials and methods of research.

26 patients with IV stage of cancer were under observation within 3 months:
• 11 patients with cancer of stomach with metastases in liver (8 men), germination
into pancreas (2 men) and colon (1) at the age of 41-73,
• 10 patients with colon cancer with metastases in liver (4) and retroperitoneal
lymph nodes (3), germination into retroperitoneal cellular tissue (3) at the age of
46-68,
• 2 patients with breast cancer with metastases in lungs (1) and backbone (1), 1
patient with melanoma of an ear bowl with metastases in brain, 1 patient with
cancer of vulva, 1 patient with cancer of brain.

The patients have given the consent to clinical tests.

1 capsule of oncoxin was applied 3 times a day 45 minutes prior to meal, and 1 powder of
viusid was admistered 3 times a day through 45 mines after meal for 3 months.

Dynamic observation over a clinical status of the patients was carried out during all
research.
 14

Clinical, biochemical and immunological analysis of the blood, investigation of tumor

markets were carried out before, and in 1,5 and 3 month of treatment with tested preparations.

Results of researches.
It was observed 6 lethal outcomes within 3 months of observation over the patients
receiving a combination oncoxin+viusid: 2 patientswith colon cancer, 1 patient with stomach
carcinoma, 1 – cancer of vulva, 1 - cancer of brain and 1 patient with melanoma.
The results of dynamic observation over the patients with gastro-intestinal tumors are
presented in the table 9.
Initially in most of the patients, we found out anemia, limphopenia, deficiency of _-cell
component of the immunity, decrease of CD4/CD8 ratio, disorders of neutrophil functions,
increased serum level of markers of tumors of stomach and colon, insignificant increase of
transaminases (mainly _l__), alkaline phosphatase and bilirubin.
The treatment did not change essentially the data of clinical, biochemical and
immunological analyses of the blood. However, it is necessary to note the certain tendency to
increase the reduced lymphocyte number in 1,5 months of treatment, mainly for the number of
CD4+ cells. In result the authentic increase of immunoregulating index (_D4/CD8) was observed.
In 1,5 months of treatment, it were also shown statistically significant decrease of number of
circulating immune complexes (CIC) and tendency to restoration of metabolic and phogocytic
functions of neutrophils.
In 3 months of treatment, these insignificant positive effects smoothed out.
It was not revealed a positive influence on parameters of tumor markers in all terms of
research. It was not revealed an essential changes of the ultrasonic sizes of the primary tumors and
metastases.
It has been prooved that it is impossible to process statistically the data obtained from 5
patients with tumors located not in gastro-intestinal canal due to 3 lethal outcome in this group and
large individual variations in the results of hematological, biochemical, immunological researches.
During application of the preparations in 4 patients, the strengthening of dyspeptic disorders
(nausea, vomit) was observed, however it is not obviously possible to make a final conclusion
whether it was connected with the basic disease or with reception of oncoxin and viusid.

Table 9.
The data of of dynamic study of hematological, biochemical, immunological parameters in
the patients with tumors of stomach and colon of a stage IV during application of a combination
viusid+oncoxin.
Parameters Healthy Before In 1,5 month of In 3 months of
persons, treatment, treatment, treatment,
n=35 n=21 n=20 n=18
9
Leucocytes _10 /l 6,21±0,14 6,1±0,29 6,3±0,42 6,0±0,35
12
Erythrocytes _10 /_ 4,32±0,16 3,6±0,07* 3,7±0,12* 3,5±0,08*
Hb, g/l 134,8±2,4 107±4,8* 112±5,3* 105±3,9*
9
Thrombocytes _10 /_ 204,7±4,23 201,5±6,3 187,5±7,8* 197,5±4,9
Lymphocytes, abs. 2,31±0,07 1,72±0,23* 1,82±0,22* 1,71±0,09*
CD2 1,54±0,08 1,21±0,14* 1,24±0,09* 1,22±0,10*
CD3 1,41±0,07 1,14±0,13* 1,24±0,25 1,13±0,11*
CD4 0,86±0,06 0,52±0,03* 0,63±0,18 0,53±0,05*
CD8 0,42±0,05 0,36±0,04 0,38±0,05 0,37±0,03
CD4/ CD8 2,02±0,13 1,44±0,08* 1,66±0,09*† 1,43±0,07*
 15

CD16 0,21±0,02 0,19±0,03 0,20±0,05 0,20±0,08

CD72 0,4±0,04 0,39±0,03 0,38±0,04 0,37±0,05
∝HLA-DR 0,21±0,03 0,18±0,02 0,19±0,08 0,17±0,04
Ig M, g/l 1,37±0,11 1,52±0,13 1,62±0,26 1,61±0,21
Ig G, g/l 12,43±1,21 12,71±0,42 12,57±0,33 12,34±0,58
Ig A, g/l 2,31±0,07 2,16±0,23 2,23±0,17 2,18±0,19
CIC, units 72,4±2,8 82±3,1* 72,8±2,2† 78,3±2,0*
NBT-test spont.% 14,8±1,12 14,9±0,4 14,9±0,5 14,5±0,3
NBT-test stimul. % 28,1±2,8 20,9±1,2* 24,9±3,8 22,3±1,5*
Phagocyte number 10,8±1,2 10,2±0,8 9,9±0,9 10,0±0,6
Phagocyte index 89,7±0,11 56,1±4,3* 66,1±7,1* 59,1±6,4*
Index of complete 1,59±0,05 1,34±0,24 1,38±0,39 1,33±0,09*
phagocytosis
CEA (stomach cancer) <3 ng/ml 18,3±0,8* 21,3±1,9* 25,3±1,4*†
CEA (colon cancer) <3 ng/ml 23,1±2,8* 25,1±4,2* 24,9±2,9*
__ 72-4 <3 units/ml 28,5±1,3* 28,4±3,3* 27,9±4,6*
__ 19-9 <37 units/ml 57,6±5,2* 61,9±7,0* 73,3±6,0*†
_l__, µ_/l 0,3±0,04 0,52±0,09* 0,55±0,08* 0,56±0,12*
_s__, µ_/l 0,35±0,03 0,41±0,08 0,40±0,09 0,45±0,03*
Al. phosphatase, 64,5±8,9 86,3±7,3* 91,3±6,8* 89,8±8,4*
units/l
Protein, g/l 76±1,8 69,8±6,9 68,9±7,1 70,2±5,7
Albumin, % 45,4±3,5 42,1±3,5 41,2±4,9 43,1±2,4
Bilirubin, µ_/l 11,8±0,7 16,6±1,0* 16,9±1,3* 16,0±0,5*
The notes: *- Reliability of distinctions from values of healthy donors – p < 0.05.
† - Reliability of distinctions from reference values (before treatment) – p < 0.05.

3. CONCLUSIONS
The experimental and clinical researches of the biologically active additives to food viusid and
oncoxin ("Catalysis", Madrid - Spain) in cancer patients have shown:
1. Viusid did not render essential direct cytotoxic effect on tumor CaOv cells (human ovarian
carcinoma).
2. In model of mouse leukemia _-388, viusid at a doses of 64 (10-multiplely reduced therapeutic
dose) and 640 mg/kg (therapeutic dose) and oncoxin at a dose of 6 mg/kg (10-multiplely
reduced therapeutic dose) caused the tendency to increase the life-span of tumor-bearing
animals (6%). The application of viusid at a dose of 6400 mg/kg (10-multiple therapeutic dose)
_ 3 times a day within 14 days reduced statistically significantly the volume of tumor ascites.
3. Viusid and oncoxin showed the tendency to suppress the growth of the primary site of
melanoma B16. Greatest on value the tendency to delay the tumor growth was shown on the day
7 of experiment: viusid at a dose of 64 µg/kg (10-multiplely reduced therapeutic dose) - 37 %,
oncoxin at a dose of 600 µg/kg (10-multiplely increased therapeutic dose) - 56 %. Viusid and
oncoxin at different doses either did not change essentially or reduced life-span of mice with
melanoma.
 16

4. Wen used at therapeutic doses, viusid showed antitumor effect (ITG = 52%) in 88% of animals,
and oncoxin inhibited tumor growth (ITG=81-62%) in 63% of animals in model of Lewis
carcinoma (LLC).
5. Viusid and oncoxin stimulated the metastatic processes in lungs in model of Lewis carcinoma.
6. It was not revealed the direct dependence of therapeutic effect (LLC) and toxicity from a dose
of preparations oncoxin and viusid (_-388, B16).
7. Combined application viusid+oncoxin at therapeutic doses in models of leukemia _-388 and
melanoma B16 did not render therapeutic action. In model of LLC, moderate antitumor effect of
the preparation combination was equal to the action of separate preparations at therapeutic
doses.
8. The clinical tests has allowed to reveal their immunomodulating effect and hemo- and
hepatoprotective actions of viusid and oncoxin in patients with gastro-intestinal tumors
receiving cytostatic (Cyclophosphane + 5-Fluorouracil) therapy in postoperative period, that
was undoubtedly reflected in increase of quality of life of these patients.
9. It is possible to assume the presence of clinical antitumor effect of the combination
oncoxin+viusid, as an insignificant increase of serum level of tumor markers was observed in 3
patients of control group and only in 1 patient of the basic group. However limited time of
observation does not allow to make a final conclusion about antitumor effect of viusid and
oncoxin.
10. The application of combination oncoxin+viusid in the cancer patients with IV stage of disease
caused the short-term tendency to improvement of hematological and immunological
parameters. In particular, the increase of the reduced number of lymphocytes (statistically
insignificant) and CD4/CD8 ratio, decrease of the raised number of circulating immune
complexes, improvement of metabolic and phagocytic activities of neutrophils were observed in
1,5 months of treatment. It was not revealed the essential changes of parameters of tumor
markers. Within 3 months, 6 lethal outcomes from 26 examined patients took place.

4. CONCLUSION

A number of the positive effects has been revealed in experimental clinical researches of
oncoxin and viusid in tumor models and cancer patients, so further study of therapeutic activity of
the obove preparation in field of oncology seems to be expedient.
In particular, it is possible to recommend to carry out an additional experimental research of
antitumor action of the preparations in a combination with cytostatics in model systems in vivo.
Presumably, such application of preparations is to potentiate antitumor effect of cytostatics on the
primary tumor site and to exclude stimulation of metastase growth, observed in the present research
in model of Lewis carcinoma.
The independent application of oncoxin and viusid in cancer patients with IV stage of the
diseases did not give marked therapeutic effect, though the temporary improvements of some
immunolofical and hematological f parameters were observed.
The inclusion of viusid and oncoxin in the complex program of treatment of cancer patients
with gastro-intestinal tumors of __NxMx stage after radical operative removal of a tumor allowed to
receive cytoprotective effect on hemopoetic, immunocompetent and liver cells, that promoted the
better unduring of cytostatic preparations. Due to immunomodulatory and anti-oxidant effects, the
preparations strengthened antitumor effect of basic therapy and increase the quality of life of the
patients.
All obove mentioned points to expediency of the further observation over control and test
groups of the patients for estimation of remote results of complex treatment. Thus, besides clinical,
biochemical and immunological analyses of the blood, the steadfast attention should be given to
 17

such parameters as 2- and, probably, 5-year's viability, development of metastases, dynamics of

serum level of tumor markers.

Responsible for realization of clinical

tests in Tomsk regional clinical hospital,
doctor of medical sciences, professor _.I. Kalyuzhina

Responsible for realization of clinical tests

in the Centre of Modern Medicine,
conducting expert of the Centre, candidate of medical sciences _.V.Nelyubov

Responsible for realization of experimental

researches, sientific director of Reseach Institute of
Immunopathology, assistant professor O.V._alyuzhin

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