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Integration of nanobiomedicine and TCM in the treatment of cancer and infection diseases of different etiology Ivanenko S.I., Tereshina E.V., Laskaviy V.N., Yakunchikov A.N. WWMA Ag, Switzerland © 2012 WWMA AG © 2012 Sergey Ivanenko, Elena Tereshina, Vladislav Laskaviy, Artem Yakunchikov Copyright by owner © 2012 WWMA AG. All rights reserved. No part of this book may be reproduced in any manner and form, by print, photo print, microfilm or any other means without permission from the owner WWMA AG.

The treatment of cancer faces certain obstacles because of lack of early diagnosis and low effectiveness of surgery methods, chemo- and radiation therapy and hormonal therapy. Both chemo- and radiation therapy enhance immunosuppressive action of tumor. Nowadays some expectations arise with the development of immune therapy. This therapy may be effective at the early stages of tumor progression and soon after surgery intervention. Immune therapy has antirelapse and antimetastasis effect. The investigators of WWMA Ag, Zug, Switzerland are working in the field of antitumor vaccination. They have overcome difficulties of tumor antigen extraction and the way of its delivery in organism by elaboration of alternative methods of activation of immune system and direct action on tumor cells that not only stops proliferation but induces apoptosis of proliferating cells. The principles of immune defense of animals and humans are built upon active immunization or interference against virulent species of infection agents. These principles are based on antibody production against corpuscular and soluble components of microorganisms. Immune defense which develops after disease or vaccination is due to the interaction of phagocytes with the infection agents (nonspecific incomplete phagocytosis). Further on, the antigen presentation by Tlymphocytes and B- lymphocytes activation ends by the antibody production. These mechanisms of immune defense can be fulfilled with true antigens having certain molecular weight. Moreover, proper immune reaction in humans and animals is possible only if at certain age in postnatal period there exists a mature and unsuppressed immune system. There are periods of lifetime when the

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organism cannot produce an adequate immune answer. Nonspecific incomplete phagocytosis in these periods is basic factor if defense. The aim of the investigation was development of immune defence through the triggering of specific complete phagocytosis. Endogeneous and secreted components of cancer cells were used to achieve the aim. The means of targeted delivery and anchoring of these components in immunocompetent organs have been elaborated. Introduction to an organism of extracted components by certain methods secure specific complete phagocytosis of bacteria and fungi and activates antiviral immune defense. The results obtained at the first stage of investigation on tuberculosis, brucellosis, salmonellosis, actinomicosis, candidosis, transmissible gastroenteritis, reproductive-respiratory syndrome in swine demonstrated effective immune defense that insure lifelong resistance to infection. Another attitude is the cell metabolism modulation with the switch from anaerobic to aerobic way of energy production which allows to stop proliferation and in induce apoptosis in tumor cells. In 1939 by of Otto Warburg made the discovery that cancer cells use only anaerobic glucose oxidation. Oxygen penetrating into the cell is used for burning of acetyl in mitochondrion as well as for red-ox reactions in cytoplasm. Oxygen is an electron and proton acceptor in the production of water (mitochondrion) and hydrogen peroxide (mitochondrion and cytoplasm). Hydrogen peroxide regulates the functioning of the proton pump and electron-transport chain in mitochondrion By modulation of the hydrogen peroxide concentrations it is possible to switch from glycolysis to respiration. Hydrogen peroxide may also influence the cell proliferation. We have developed a theoretical scheme of conjugation of proton and electron flows which include methyl group, oxygen reduction and nitric oxide synthesis pathways. In such conjugated system a formic acid aldehyde demonstrate the highest red-ox potential. We have made preparations on the bases of formic acid aldehyde (preparations of the natural metabolite – PNM). PNM efficacy was tested in cell cultures, animals and volunteers.

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Experiments on PNM cytotoxicity were held on human lung carcinoma from ATCC HTB-53 and human small lung carcinoma from ATCC CRL-2066 with means of MTT assay PNM. MTT assay is a standard colorimetric assay for the determination of metabolic dysfunction, leading to reduction of MTT to formazan. This reduction takes place only when reductase enzymes are active, and therefore conversion is often used as a measure of viable (living) cells. When the amount of purple formazan produced by cells treated with an agent is compared with the amount of formazan produced by untreated control cells, the effectiveness of the agent in causing death, or changing metabolism of cells, can be deduced through the production of a dose-response curve. High cytotoxic efficacy of PNM in the certain doses was shown. The incucyte pictures taken during the whole experiment showed that PNM stops the cells proliferation. The switch from aerobic to anaerobic types of the cellular metabolism was shown as well. The medicine has been tested in vitro on 27 human cell cultures including rectal carcinoma (CXF 280, CXF 1130), gastric carcinoma (GXF 1172), liver carcinoma (LIXF 575), bladder carcinoma (BXF 1218), lung carcinoma (LXFA 289 and LXFA 526), small cell carcinoma of lung (LXFS 615 and LXFS 650), large cell carcinoma of lung (LXFL 1647 and LXFL 529), head and neck epithelial squamous cell carcinoma (HNXF 536), mammary adenocarcinoma (MAXF 401, MAXF 1322, MAXF 1384), ovary carcinoma and ovary serous papillary adenocarcinoma (OVXF 550 and OVXF 899 ), pancreatic adenocarcinoma (PAXF 736), prostate carcinoma (PRXF 736), melanoma carcinoma (MEXF 1539, MEXF 514, MEXF 989), pleural mesothelioma carcinoma (PXF 1118), kidney renal adenocarcinoma (RXF 631, RXF 944 LX and RXF 423), rhabdomyosarcoma (SXF 627), Acute lymphoblastic leukemia (ALL), Chronic lymphocytic leukemia (CLL), Acute myelogenous leukemia (AML), Chronic myelogenous leukemia (CML), Hairy cell leukemia (HCL), T-cell prolymphocytic leukemia (T-PLL), Large granular lymphocytic leukemia. The cancer cells of mammal gland MAXF 401 NL occurred to be the most sensitive to PNM, there IC 50 was 0,127 % v/v.

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Table 1. Effect of different PNM concentrations on the effectiveness of growth inhibition of neoplastic cells colonies. A number of neoplastic cell lines where the effect Drug

concentration

of growth inhibition of colonies was registered Absolute value

Relative value(%)

3,6 • 10-5 — 3,6 • 10-3

18

66,6

3,6 • 10-4 — 3,6 • 10-3

4

14,8

3,6 • 10-6 — 3,6 • 10-3

3

11,2

3,6 • 10-7 — 3,6 • 10-3

1

3,7

3,6 • 10-8 — 3,6 • 10-3

1

3,7

27

100

TOTAL:

As the research shows (Table 1), all used 27 lines of neoplastic human cells exposed to PNM in maximum concentration (3,6·10-3) caused the cells death, while the concentration in the range from 3,6·10-5 to 3,6·10-8 caused enhanced inhibition of neoplastic cells. A test of drug's mechanism of action on the breast adenocarcinoma MAXF 401NL cell line showed growth decrease by 25% after 3 days of treatment and the following passage of cells. During the second passage it was registered that the growth of neoplastic cells was reduced by 30% compared to the control. Investigation of PNM antitumor efficacy was held on rats that were devided into groups. In the first group Pliss lymphosarcoma, in the second group sarcoma 45 and in the third group Walker 256 carcinosarcoma were transplantated subcutaneously with 0,5 ml 10% tumor cells suspension in the Hanks solution. PNM was injected intramuscular in dose 0,75 ml per rat in the first twenty-four hours after tumor transplantation and later with 2-3 days interval. The total amount of injected PNM was 6 ml per each rat (8 injections). Antitumor effect was

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evaluated by tumor growth dynamic, percent of tumor growth braking, average lifespan of died animals and percent of cure. Animals with Pliss lymphosarcoma revealed significant tumor growth braking that reached 52% up to 14 days after transplantation, 10% recovered. In animals with sarcoma 45 had 98%. tumor growth braking, 40% recovered. In animals with rapidly growing experimental model of Walker 256 carcinosarcoma tumor growth braking was 75,6% up to 14 days after transplantation up to 14 days after transplantation, 20% recovered. It is worth noticing that a number of animals with Walker 256 carcinosarcoma after 4-5 PNM injections there was tumor degradation followed by fibrous tissue substitution. The life span of the experimental animals didn’t differ from control. PNM not only directly acts on tumor cells but has generalized effect activating immune system. This activation was revealed in experiments when PNM was injected simultaneously with tuberculin and brucellin. Tuberculin and brucellin are compounds secreted by causative agents of tuberculosis and brucellosis. They are gaptens and have weakly expressed or completely absent stage of antigen processing in phagocytes. PNM stimulates the development of respiratory explosion in phagocytic cells thus producing conditions for the completed phagocytosis. PNM injection to rats in doses 1 ml/kg, 10 ml/kg and 25 ml/kg resulted in the enhancement of gulp down activity of phagocytes to Е. соli, and in increase of the phagocytes number. The maximum was achieved when PNM dose was 10 ml/kg: for a phagocytic indicator 20% (р < 0,00 1) and for a phagocytic number 26% (р < 0,001). PNM posses pronounced immunostimulating effect that is expressed by the accumulation of antibody producing cells in the spleen and increase of hemagglutinin in the serum of the immunized animals. In experiments with volunteers the influence of PEM on immune status and general physical and psychical health was investigated. Patient А, 48 aged female with uterus mioma. After immunocorrection course ( two 5 ml PNM injections with interval 14 days) there was normalization of Т- lymphocytes, Т-activines, Т-suppressors, Т-helpers, JgA immunoglobulines.

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Patient T, 56 aged female with lung carcinoma. After immunocorrection course (one 5 ml PNM injection) the total number of Т-lymphocytes, Т-activines, Тhelpers, В- lymphocytes, IgG, IgA, IgM immunoglobulines was increased. The health condition significantly recovered, ability to work restored. Patient C, 38 aged female with mammal gland cancer. After immunocorrection course (three 5 ml PNM injection with the interval between injections 11 and 14 days) there was normalization of Т- lymphocytes, Т-activines, Т-suppressors, Т-helpers, Вlymphocytes IgA, IgG immunoglobulines level. Ability to work restored, lust for life appeared. The continuation of the therapy course during 12 months led to the full recovery. Courses of PNM treatment of 125 patients with mammal gland, intestine and ovary cancer, as well as with melanoma revealed 44-50% recovery (Fig. 1).

Fig. 1. Results of courses of PNM treatment of volunteers aged from 34 to 78 years old. Research project of PNM treatment of patient with cancer revealed its high efficacy as antitumor remedy with immunomodulating properties (Fig. 2). PNM mobilizes and activates macrophages; causes the production of alpha- and betainterferon’s; restricts the production of cytokines of inflammation (interleukins I,6,8, factor of tumor necrosis); stimulates the production of antibodies to different

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antigens of infectious origin; suppresses virus replication; improves organism resistance towards infections caused by viruses, bacteria or fungi.

Fig. 2. Spectrum of PNM therapeutic action.

During the last 20 years in the European countries and USA treatment of patients with cancer includes preparations based on natural products. The integration between western and traditional eastern medicine seems to be the most perspective. The attention is attracted to fungi per se or their extracts. Fungi are the unique ancient organisms that in evolution have occupied the middle place between plants and animals. They have proteins specific for animals and substances produced mainly by plants. Among fungi there two with well known antitumor effect - Linzhi (Ganoderma lucidum) and Cordiceps. The composition of bioactive substances in Linzhi consists of ganoderic acids (triterpenoids), polysaccharides and organic germanium. There more than 50 types of various polysaccharides and their content in Ganoderma lucidum more

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than 40% higher than in other fungi species. Polysaccharides possess immunomodulating properties activating T-lymphocytes. Some polysaccharides demonstrate significant antitumor effect (direct cytotoxic action towards tumor cells). Triterpenoids stipulate detocxication, hepatoprotective, immunomodulating, antiallergic and health strengthening character of theurapeutical efficacy of Ganoderma lucidum preparations. Organic germanium displays antioxidative properties, can reduce immune system functions and brake down the tumor growth. Linzhi preparations have systemic influence on the organism. They normalize hormone production, improve hematopoiesis, defend from oxidative stress and support immunity/ In China today Ganoderma lucidum, that is used as in dry so in liquid forms is admitted a strategic drug and food resource. More thah 1 billion of Chinese patient with cancer use the Linzhi preparation as a remedy. In our experiments on the antitumor efficacy of Linzhi liquid form (LFL) we used Kunmiing and Shanghai mice with transplantated liver cancer. In the control group wiyhout treatment the fast tumor growth was registered while in the experimental group the tumor braking was observed.

А)

Control group

B) Experimental group

Ingibitory action of LFL on the tumor growth was 73%.

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А). Control group

В) Experimental group

In the experiments antiinflamatory effect of LFL was detected (Table 2). In mice treated by LFL the amount of leucocytes was lower than in control group up to 9,8%, 10,8% and 4,8% for LFL (1). Table 2. Groups

Dose

Mice

leucocytes

(g/kg)

шт.

(109/L, X±S)

Control

0.0

10

8.98±1.13

endoksan

0.1

10

3.59±1.83

LFL 1

1.0

10

8.17±1.85*

LFL 2

0.5

10

8.10±1.76*

LFL 3

0.25

10

8.59±1.58*

Preparations

*Comparison with endoksan, P