ANTITUMOR ANTITUMOR AND ANTIMETASTATIC EFFICIENCY OF ANTITUMOR VACCINE AND AMIXIN COMBINED ACTION IN MICE WITH LEWIS LUNG CARCINOMA

lung carcinoma. An effective plan for use of tumor vaccine and amixin, which is administered orally (25 mg/kg) for 3 hours before each of the five injections of the vaccine was developed. Its use in mice with transplanted Lewis lung carcinoma was significantly exceed the results of the introduction of anti-tumor vaccine alone: index of inhibition of tumor growth respectively 54,59 ± 1,97 and 43,79 ± 0,96%, and the average lifespan of mice – 56,2 ± 2,06 and 47,0 ± 1,50 days. In animals with minimal residual tumor (after surgical removal of the primary tumor) in adjuvant mode of antitumor vaccines amixin was effective with the introduction of the latter in the dose 3 hour before the vaccine or after 1 day after each introduction. The frequency of metastasis was decreased to 70%, the average number of metastases and their volume – 8.8 and 34.0% in comparison with those operated control mice, when compared to similar data of mice who received only the vaccine at 2.0 and 2.25 times. The index of inhibition of metastasis using a combined scheme was 92.6%, vaccine; alone vaccine – 83,08%. Further determination the mechanisms of synergistic effect antitumor vaccines and amixin will enable the use of different biopreparations and will help to develop effective schemes of therapy of cancer patients.

According to current data, antitumor vaccines (AV) have high therapeutic efficiency if combined with adjuvants of various origin, and immune response inducing cytokines [1,2].Of particular scientific interest are the effects of interferon (IFN) relating to tumor's increasing immunogenicity and changing sensitivity to T-lymphocyte cytotoxicity.It was shown that IFN directly impacts tumor growth and differentiation, and induces apoptosis [3].Thus it is necessary to study its role as part of combination with other biological agents.Results of experimental and clinical studies substantiate the use of IFN- as an optimization element of complex treatment to reduce risks of metastasis and improve the quality of life for cancer patients [4,5].
Discovery of the unique properties of IFN stimulated the search for substances activating the synthesis of endogenous IFN, so-called IFN inducers [6][7][8].One of these is amixin (tilorone), oral low molecular weight inducer of endogenous IF, the which activity is related to the immunomodulatory properties [9][10][11].Information on significant antitumor activity of amixin has been obtained in model tumor experimental studies, also in clinical trials in patients with melanoma, breast cancer, renal cell carcinoma [12,13].It has been shown that the combined use of antitumor drugs and amixin contributes to strengthening growth inhibition of model tumors, and reduces metastasis [10,14,15].Particular attention is paid to studying effects of IFN combined with vaccinotherapy [4,16,17].
In our previous studies it has been found that the use of IFN or subalin (recombinant strain of probiotic bacteria B. subtillis 2335/105 with embedded gene of human IFN synthesis) significantly increases the efficiency of vaccinotherapy in case of transplanted model Lewis lung carcinoma (LLC) [18,19].
The aim of present work is development of protocols of effective use of AV and inducer of endogenous IFN, amixin.

Materials and Methods
Mice of the line С57Вl (males 2.5 months, 20-22 g, bred in RE Kavetsky Institute of Experimental Pathology, Oncology and Radiobiology of the National Academy of Sciences of Ukraine) were used in experiments according to the International Animal Research Regulations.
To determine the antitumor efficiency of combined action of AV and amixin, mice were transplanted LLC in femoral muscle (4.5•10 5 living tumor cells per mouse).The cellular suspension for transplantation was prepared according to method requiring preliminary tumor trypsinization.AV were prepared following [20] using tumor tissue and filtrate of culture liquid of B. subtilis B-7025.0.3 ml of AV were injected intramuscularly 5 times at the 1 st , 4 th , 8 th , 12 th and 15 th days after tumor grafting.Amixin was introduced orally at 10.0 and 25.0 mg/kg (0.2 and 0.5 mg in 0.2 ml respectively) exclusively (third and fifth groups) or as part of the combined scheme (second and fourth groups) three hours before PV injection.The first group was given only AV.The efficiency of autologous AV and amixin separately and combined was estimated by comparing common characteristics of LLC growth in control (sixth group) and experimental mice.
The tumor growth dynamics, animal survival and average life expectancy (ALE) were determined.Tumor growth inhibition (TGI) index was calculated as: where Vc and Ve are average tumor volume in control and experimental mice respectively.At 7 th, 14th and 21 st day after LLC grafting, functional activity of peritoneal macrophages (Mph) was assessed by HCT-test, and levels of IgG to LLC antigens was determined in blood serum [21].
Antimetastatic action of the combined protocols of AV and amixin was studied in mice with minimum residual tumor disease.LLC was transplanted (2.5•10 5 cells per mouse) into hind-limb cushion, and at the 19 th day after that primary tumors were surgically removed in all mice (anesthetized by sodium thiopental 60 μml subcutaneously) by cutting off the ligature-bound distal part of affected limb (surgical removal, SR).Subsequently the experimental animals were vaccinated (5 times, 0.3 ml subcutaneously).Only the times of the first vaccination were different: mice of first, second and third groups were given AV at 1 st , 4 th , 7 th , 10 th , and 14 th day after SR; mice of the fourth and fifth groups at the 4 th , 7 th , 10 th , 13 th , and 17 th day after SR.Amixin was given per os (25 mg/kg; 0.5 mg per mouse in 0.25 ml), three hours before each AV injection (1 st combined protocol; 2 nd experimental group) or 24 hours after it (2 nd combined protocol; 3 rd and 5 th experimental groups).Control mice were given similar doses of NaCl saline in same time after SR.
Antimetastatic effect was estimated at the 28 th day after SR (47 th day after LLC grafting) by quantity and volume indexes of metastases in lungs: frequency of metastasis (%); average number of metastases per mouse; average volume of a metastasis.Metastases inhibition index (MII) was calculated as follows: А с and А е -number of animals with metastases in control and experimental groups; M e and M c -average number of metastases in animals of control and experimental groups.
Statistical analysis was performed according to common methods of variation statistics.Results are given as М ± m, where М is arithmetic mean, m is standard error.Differences assessed as probable at Р < 0.05.

Results and Discussion
According to analysis of LLC growth dynamics indexes (Fig. 1), the tumor growth is reduced in all animal groups who received AV and/or amixin.The least tumor volume is found in mice receiving AV and amixin in doses of 25 mg/kg (4 th group).Average values of TGI index during 10 th to 38 th days after LLC transplantation in this group is 54.59 ± 1.97% (Table 1), significantly exceeding the results after only vaccinotherapy -43.79 ± 0.96%, (Р < 0.05).As a trend it's better than combined use of 10 mg/kg AV and amixin, 49.24 ± 1.73% (0.1 < Р < 0.05).
It should be noted that the results of implementation of both combined plans (AV and amixin, 10 mg/kg and 25 mg/kg) significantly exceed the effects of separate treatments by AV or similar doses of amixin (Р < 0.05).LLC growth inhibition in experimental animals occurs together with better survival indexes than in control (Fig. 2).The advantages of combined use of AV and amixin in doses of 25 mg/kg are obvious: mortality in animals of this group is observed in 47 th to 70 th days while all control mice died before the 40 th day.
ALE of animals with transplanted LLC who received AV and/ or amixin significantly exceeds that of the control group (32.87 ± 1.89 days, Р < 0.05) (Table 2).The best results are observed for combined use of AV and amixin in doses of 25 mg/kg (56.2 ± 2.06 days) -ALE of these mice is 70.98% higher than that of the control group, and is quite different from ALE of mice given only AV or amixin in that dose (47.0 ± 1.50 and 43.5 ± 1.55 days respectively).The combined plan with amixin in doses of 10 mg/kg was significantly inferior in this regard.
Analysis of functional activity of peritoneal Mph in HCT-test at the 7 th , 14 th , and 21 st day (Table 3) shows no significant difference in mice indexes of this group compared to the tumor growth control (TGC).But evaluating the levels of IgG to LLC antigens in BS in mice given AV and amixin in doses of 25 mg/kg after injections reveals the highest value among all experimental groups: at the 7 th day after transplantation it is 1.732 ± 0.007 optical units, significantly exceeding IgG levels in control mice (0.747 ± 0.011 optical units, Р < 0.05).Later after transplantation (21 st day), IgG levels in BS of animals of all In another experiment, antimetastatic activity of combined AV and amixin (in doses of 25 mg/kg) in C57Bl mice with LLC is studied.The results of analysis of quantitative characteristics of metastasis at the 28 th day after SR show significant difference in the respective values for experimental and control mice.First of all it should be noted that metastasis occurs in lungs of 100% control mice; in 80% mice given only AV regardless of the time of first vaccination (1 st and 4 th groups); 70% mice of both combined plans of AV and amixin if AV is injected in 24 hours after SR (Table 4).Combined plan with vaccination starting 72 hours after SR shows results similar to that of only using AV (80%).
As for the index of average number of metastasis per mouse, the least values are found in both combined plan groups if AV were injected 24 hours after SR (1.60 ± 0.37; 2 nd and 3 rd groups), which is significantly different from vaccination alone (3.20 ± 0.66;  If the vaccination started 24 hours after SR, MII is 92.6% in both cases of combined use of AV and amixin, and this is significantly higher than in case of vaccination only (83.08%).If vaccination started 72 hours after SR, MII in mice given only AV is reduced to 69.33%; in mice given combined treatment (plan 2) to 59.81%.The results mean that the combined use of AV and amixin exhibits better antimetastatic activity if vaccination started earlier (24 hours) after SR, since delaying the treatment by AV and amixin for 96 hours reduces the antimetastatic effect.The observed particularities are confirmed by studying volume characteristics of metastasis (Table 5).Analysis of average metastasis volume shows practically same results in mice of the 1 st -3 rd groups given only AV or in combination with amixin (22.77 ± 5.59; 25.02 ± 6.48; 20.12 ± 6.17 mm 3 ); similar findings are observed for mice of the 4 th group given only AV 96 hours after SR (25.50 ± 3.62 mm 3 ).But all those results significantly differ from these of mice of the 5 th (49.64 ± 6.03 mm 3 ) and control (74.63 ± 4.46 mm 3 ) groups, supporting the importance of timely early beginning of combined treatment by AV and amixin to achieve high antimetastatic efficiency.

Significant reduction of average volume of metastases is recorded in all vaccinated
Thus, pronounced antimetastatic activity of two plans of combined action of autologous AV and amixin given per os in doses of 25.0 mg/kg (0.5 mg per mouse) three hours before AV injection or 24 hours after, is observed in experiment on model LLC with surgical removal of primary tumor.There is no significant difference between results for these two combined plans according to quantitative and volume characteristics of metastasis for considered indexes.
Significant advantages in using combined plans of vaccination and amixin over only vaccination is shown for the average number of metastasis and metastasis inhibition index (84.11%and 92.06%).All considered indexes support the importance of timely introduction of combined plan of autologous AV and amixin, that is early after tumor removal (in 24 hours).The results are the basis for increasing the vaccinotherapy efficiency using the inducer of endogenous IFN.
Thus, efficient plan of combined use of AV and inducer of endogenous IFN, amixin, is developed: per os introduction of amixin in doses of 25 mg/kg three hours before each of the five AV vaccinations.In mice with transplanted LLC the plan is more successful than only AV vaccinotherapy -tumor growth inhibition index is 54.59 ± 1.97 and 43.79 ± 0.96% respectively, and ALE of mice is 56.2 ± 2.06 and 47.0 ± 1.50 days respectively (Р < 0.05).

Fig. 2 .
Fig. 2. Survival in mice (%) with Lewis lung carcinoma after treatment with AV and/or amixin

Table 2 . Average life expectancy in mice with Lewis lung carcinoma after treatment with AV and/or amixin Group № Group characteristics ALE Difference between groups
(*Р < 0.05)

Table 3 . Levels of oxygen-dependent bactericide activity of peritoneal Mph and IgG to LLC antigens in blood serum of mice given AV and/or amixin Group characteristics Activity level of Mph in НСТ-test, optical unit IgG level, optical unit
Note: * -significant difference with respective value in TGC group (Р < 0.05).