experiment was carried out using cells , transformed with genetic construct , expressing oncogene ras , and thus modeling conditions of cells underwent cancerous transformation . ( see model description in levina e m , domnina l v , rovensky y a , vasiliev j m ., ( 1996 ), exp . cell . res ., november 25 , pp 159 - 65 ). mouse ras - transformed fibroblast cells had strongly expressed changed morphology ( see fig1 . ), one close to morphology of metastasizing cancerous cells ( decrease in surface square , cell elongation ). morphological analysis demonstrated cell surface square mean values to be significantly decreased in comparison to control ( see fig2 .). cytoskeleton elements and focal contact proteins — vinculin and paxillin — content was drastically decreased in these cells . cells were poorly adherent and spread - eagled what led to monolayer structure disorder . all these features are peculiar for metastasizing cancerous cells with comparatively increased mobility . however incubation with skq1 led to significant changes in cells morphology ( see fig1 .). mean cell surface square value increased manifold ( see fig2 .) morphological changes correlated with actinic fibrils increase in content , focal contact reinforcement and cell better adherence to the surface . ras - transformed fibroblasts treated with skq1 formed monolayers similar to those of normal fibroblasts . obtained results demonstrate that in model system treatment of metastasizing cancerous cells with skq1 led to normalization of its condition . study of skq1 anti - tumoral activity on a - 431 human skin cancer heteroxenografts transplanted to hiv mice experiment was carried out taking into account russian the federation ministry of health care and international recommendations , published in the following sources : larionov l f , chemotherapy of malignant tumor , moscow ., 1962 ; treshchalina e m and co - authors , methodical recommendations on study of anti - tumoral activity of pharmacological substances in the book : “ guidance on experimental ( preclinical ) study of new pharmacological substances ” edited by corresponding member of russian academy of medical sciences prof . habriev r u , 2005 ; experimental evaluation of antitumor drugs in the usa and ussr and clinical correlations , nci monograph 55 , 1980 ; survey and antitumor toxicity test systems , eortc screening and pharmacology group , 1989 ; anticancer drug development guide : preclinical screening , clinical trials , and approval , edt . teicher b . a ., 1997 . before introduction skq1 was diluted with sterile physiological saline up to two use rates 0 . 5 μm and 5 . 0 μm . preparation was introduced intraperitoneally in single doses 5 . 0 nm / kg and 50 . 0 nm / kg in the volume 0 . 2 ml / mouse . experiment was carried out using a - 431 cell line [ 1 ], lodged in russian collection of collection of cell cultures and possessed the following features : origin : human , epithelial carcinoma j . natl . cancer inst . 1973 . 51 : 14171423 . morphology : epithelial . cultivation method : monolayer cultivation conditions : 10 % bovine embryonal serum dmem culture . procedure of seeding : cell removal using 0 . 25 % trypsin , 0 . 02 % versene ( 3 : 7 ), ratio of seeding 1 : 3 1 : 6 , optimal density 2 . 04 . 0 × 104 cell per sm 2 cryopreservation media , 10 % dmso , 1 . 0 × 106 cells / ml per ampoule viability after cryopreservation : 83 % ( trypan blue staining on zero passage ) contamination control : no bacteria , fungi or microplasma detected genetic identity control : karyological , enzymatic ( ldh , g6pdh ) analysis . caryology : 2n − 46 , chromosome variability limit 5777 , chromosome modal number 6871 tumorigenicity : tumorigenic in hiv mice and in mice treated with antithymocyte serum . other features : large number of receptors to epidermal growth factor . application area : growth factors study , cell biology . collection : atcc crl 1555 ; ecacc 85090402 ; inc ras . a - 431 cells were grown on rpmi - 1640 medium that contained 10 % calf serum and 1 mm of glutamine . cells were transferred to a flask in ratio 1 : 2 or 1 : 3 from full monolayer . medium was changed every 2 - 3 days . cultivated cell suspension then was used to engraft hiv mice . experiment was performed using hiv female mice balb / c nude at the age 8 - 9 . 5 weeks . all mice were raised in gu ronc . mice were managed in specialized virus - free chamber with sterile aeration , fed with sterile briquette food and water under normalized temperature condition and moisture . cells of a - 431 cell line were hypodermically implanted only once in number 1 . 7 - 1 × 10 6 per mouse in 0 . 2 ml of nutrition medium rpmi - 1640 . anti - tumoral effect estimation was carried out using common rate t / c ( treatment / control ), evaluated as a ratio of average tumor volumes in treated and control groups given in percents . rate was taken evaluated using the following formula t / c %=( vo / vk )/( vk )× 100 %, where vo and vk — mean volumes of tumor in treated and control groups thereof . thus in control group t / c = 100 %. mean volume was evaluated using the following formula vm ( mm 3 )= a × b × c . t / c ≦ 42 % was taken as a minimal efficiency criterion [ anticancer drug development guide , edt . by b . a . teicher , humana press , totowa , n . j ., 1997 , p . 101 - 125 ]. for tumor growth speed analysis ( standarcy value ) a ratio of mean tumor value in following estimations to a first one was teken . evaluation started after appearance of palpable tumor ( 9 day after implantation ). after that a multiple tumor volume evaluations were performed until 20 th day after transplantation . in such a way in treated groups first evaluation was performed in process of treatment , and three next evaluations — during 6 days after end of the treatment . tumor evaluations were stopped on 21 st - 24 th day after first mouse died in three experiments . tolerance to the treatment was estimated according to time of death corresponded to treatment with each preparation . mice were mortified by ether anaesthesia overdose , an autopsy was performed , tumors were then extracted and a histological and immunohistochemical assays were carried out . tumors were fixed in 10 % formaldehyde , set into paraffin wax . histological assays were carried out on tumor histological sections , stained with hematoxylin and eosin . study of number of micro - vessels was performed with standard immunological method using antibodies to cd34 antigen . presence of vessel mimicry was , estimated by tumor section staining with schiff &# 39 ; s reagent . at the “ zero ” day all mice were hypodermically engrafted with cells of a - 431 cell line in concentration 1 . 7 × 10 6 per mouse in 0 . 2 ml of nutrition medium rpmi - 1640 . after implantation all mice very divided in groups 7 mice in each . two of the studying groups intraperitoneally received skq1 preparation every day from 1 st to 14 th day after tumor implantation . group 1 ( control ): average weight of the mouse 20 . 3 [ 19 . 6 ÷ 21 . 0 ] g , intraperitoneally received 0 . 2 ml of physiological saline from 1 st to 14 th day after tumor implantation . group 2 : average weight of the mouse 19 . 9 [ 19 . 2 ÷ 20 . 6 ] g , from 1 st to 14 th day after tumor implantation mice were intraperitoneally injected with skq1 preparation in single dose 5 . 0 nm / kg , sum dose 70 . 0 nm / kg . group 3 : average weight of the mouse 19 . 8 [ 19 . 1 ÷ 20 . 5 ] g , from 1 st to 14 th day after tumor implantation mice were intraperitoneally injected with skq1 preparation in single dose 50 . 0 nm / kg , total dose 700 . 0 nm / kg . group 1 ( control ). mean tumor volume values ( vm ) comprised 458 [ 318 ÷ 598 ] mm 3 , 1295 [ 959 ÷ 1631 ] mm 3 , 1996 [ 1319 ÷ 2673 ] mm 3 and 2564 [ 2064 ÷ 3064 ] mm 3 on 9 th , 14 th , 17 th and 20 th day after transplantation thereof . ratio of mean volumes of growing tumors comprised correspondingly 2 . 83 - 4 . 36 - 5 . 6 times . growths factors demonstrate tumor development speed to be normal for mice hypodermic tumors in 20 day period . first mouse died on 22 nd day of tumor . autopsy revealed no visual sings of pathological changes in mouse organs . rest of mice were mortified on 23 rd day after transplantations , again autopsy demonstrated no pathological changes in mice organs . group 2 ( skq1 preparation in single dose 5 . 0 nm / kg ). mean tumor volume values ( vm ) comprised 289 [ 87 ÷ 1491 ] mm 3 , 95 [ 515 ÷ 1395 ] mm 3 , 1349 [ 903 ÷ 1793 ] mm 3 and 1863 [ 919 ÷ 2807 ] mm 3 on 9 th , 14 th , 17 th and 20 th day after transplantation thereof . anti - tumoral effect at these stages comprised t / c = 63 , 74 , 67 , 73 % thereof . no statistical differences with control group were found ( p & gt ; 0 . 05 ). treatment tolerance stated satisfactory . mice started to die on 7 - 8 days after end of the treatment — two mice died on 21 st - 22 nd days after transplantation ; autopsy revealed no visual sings of pathological changes in mice organs . rest of mice were mortified on 23 rd day after transplantations , again autopsy demonstrated no pathological changes in mice organs . group 3 ( skq1 preparation in single dose 50 . 0 nm / kg ). mean tumor volume values ( vm ) comprised 730 [ 430 ÷ 1030 ] mm 3 , 2078 [ 1328 ÷ 2828 ] mm 3 , 2678 [ 1667 ÷ 3689 ] mm 3 and 3557 [ 2042 ÷ 5072 ] mm 3 on 9 th , 14 th , 17 th and 20 th day after transplantation thereof . anti - tumoral effect at these stages comprised t / c = 159 , 160 , 134 , 146 % thereof . treatment tolerance stated satisfactory . all mice were mortified on 23 rd day after transplantations , autopsy demonstrated no pathological changes in mice organs . in group 3 , received treatment with skq1 preparation in single dose 50 . 0 nm / kg , t / c comprised 134 - 160 % what gives evidence of potential stimulation of tumor growth . in group 2 after 8 injections of skq1 preparation in single dose 5 . 0 nm / kg tumor growth was inhibited and t / c comprised 63 %. total dose up to moment of the effect registration comprised 40 . 0 nm / kg . further injections led to no additional effect but no growth acceleration was registered either . a suggestion that the preparation can be efficient when given 8 times in single dose 5 . 0 nm / kg ( total dose 40 . 0 nm / kg ) was made . at the “ zero ” day all mice were hypodermically engrafted with cells of a - 431 cell line in concentration 1 . 0 × 10 6 per mouse in 0 . 2 ml of nutrition medium rpmi . after implantation all mice very divided in 2 groups 10 mice in each . experimental group intraperitoneally received skq1 preparation every day from 1 st to 8 th day after tumor implantation : group 1 ( control ): average weight of the mouse 20 . 8 [ 19 . 8 ÷ 21 . 8 ] g , intraperitoneally received 0 . 2 ml of physiological saline from 1 st to 8 th day after tumor implantation . group 2 : average weight of the mouse 20 . 1 [ 19 . 6 ÷ 20 . 6 ] g , from 1 st to 8 th day after tumor implantation mice were intraperitoneally injected with skq1 preparation in single dose 5 . 0 nm / kg , sum dose 40 . 0 nm / kg . group 1 ( control ). mean tumor volume values ( vm ) comprised 155 [ 117 ÷ 193 ] mm 3 , 1008 [ 828 ÷ 1188 ] mm 3 , 1641 [ 1152 ÷ 2130 ] mm 3 and 2936 [ 2261 ÷ 3611 ] mm 3 on 8 th , 14 th , 17 th and 24 th day after transplantation thereof . ratio of mean volumes of growing tumors comprised correspondingly 6 . 5 - 10 . 5 - 18 . 9 times . growths factors demonstrate a comparatively high speed of tumor development in this experiment . first mouse died on 24 th day of tumor . autopsy revealed no visual sings of pathological changes in mouse organs . rest of mice were mortified on 24 th day after transplantations , again autopsy demonstrated no pathological changes in mice organs . group 2 ( skq1 preparation in single dose 5 . 0 nm / kg ). mean tumor volume values ( vm ) comprised 95 [ 55 ÷ 135 ] mm 3 , 724 [ 441 ÷ 1007 ] mm 3 , 1582 [ 1007 ÷ 2157 ] mm 3 and 2953 [ 1730 ÷ 4176 ] mm 3 on 8 th , 14 th , 17 th and 24 th day after transplantation thereof . anti - tumoral effect at these stages comprised t / c = 61 , 72 , 96 , 101 % thereof . first mouse died on the 16 yh day after treatment end and in the same day with first death in control group . autopsy of died and mortified mice revealed no visual singes of pathological changes in mice organs . obtained results demonstrated , that 8 - day treatment with 5 . 0 nm / kg single dose of skq1 preparation of hiv mice with human skin cancer a - 431 heteroxenografts led to replicable inhibition of tumor growth ( t / c = 61 %). at the “ zero ” day all mice were hypodermically engrafted with cells of a - 431 cell line in concentration 1 . 0 × 10 6 per mouse in 0 . 2 ml of nutrition medium rpmi . after implantation all mice very divided in 2 groups 9 mice in each . studied group intraperitoneally received skq1 preparation every day from 1 st to 14 th day after tumor implantation : group 1 ( control ): average weight of the mouse 18 . 8 [ 18 . 3 ÷ 19 . 3 ] g , intraperitoneally received 0 . 2 ml of physiological saline from 1 st to 8 th day after tumor implantation . group 2 : average weight of the mouse 18 . 8 [ 18 . 3 ÷ 19 . 3 ] g , from 1 st to 14 th day after tumor implantation mice were intraperitoneally injected with skq1 preparation in single dose 5 . 0 nm / kg , sum dose 70 . 0 nm / kg . group 1 ( control ). mean tumor volume values ( vm ) comprised 94 [ 9 ÷ 179 ] mm 3 and 567 [ 274 ÷ 1107 ] mm 3 on 14 th and 21 st days after transplantation thereof . growths factors demonstrate a comparatively low speed of tumor development in this experiment . first mouse died on 21 st day of tumor . autopsy revealed no visual sings of pathological changes in mouse organs . rest of mice were mortified on 23 th day after transplantations , again autopsy demonstrated no pathological changes in mice organs . group 2 ( skq1 preparation in single dose 5 . 0 nm / kg ). mean tumor volume values ( vm ) comprised 39 [ 6 ÷ 72 ] mm 3 and 256 [ 94 ÷ 418 ] mm 3 on 14 th and 21 st day after transplantation thereof . immediately after end of the treatment a significant anti - tumoral effect was registered t / c comprised t / c = 41 %. tolerance to the treatment stated satisfactory . first mouse died on the 7th day after treatment end . autopsy of died and mortified mice revealed no visual singes of pathological changes in mice organs . obtained results demonstrated , that immediately after end of 14 - day treatment with 5 . 0 nm / kg single dose of skq1 preparation of hiv mice with human skin cancer a - 431 heteroxenografts preparation possessed minimal anti - tumoral effect t / c = 42 %. histological and immunohistochemical analyses of tumor sections were performed on material obtained in experiment 1 . histological assay revealed tumor xenografts possess typical structure of non - differentiated tumor of epithermal type . comparison of control samples with those of group 2 demonstrated that skq1 preparation in single dose 5 nm / kg causes partial tumor differentiation : signs of keratinization appear in tumor tissue . number of micro - vessels was counted in so - called hot spots — tumor areas with maximal vascularization . according to our data , number of micro - vessels in tumors ( 23 rd day , 9 th day after end of the treatment ) didn &# 39 ; t statistically differ in all three groups . however , micro - vessels in group 2 were mentioned to be of smaller diameter than in control group . sample staining with pas reagent revealed tumors in control group to possess sings of vasculagenic mimicry , while no such singes were mentioned in tumors in group 2 . experimental study on skq1 treatment effect on hiv mice with human skin cancer a - 431 hypodermic xenografts revealed that long - termed injection of skq1 in single doses 5 . 0 nm / kg didn &# 39 ; t cause tumor growth acceleration , and in some cases led to growth 50 % inhibition . injection of skq1 in single dose 5 . 0 nm / kg causes partial differentiation of a - 431 tumor tissue , decreases sings of vasculagenic mimicry , though does not affect total number of micro - vessels . study on skq1 preparation anti - tumoral effect on mice tumors ( hypodermic introduction of ehrlich &# 39 ; s carcinoma ) experiment was performed according to recommendations listed in description of experimental example 2 . injection of preparation started after 48 hours after transplantation and proceeded during 5 , 10 , 15 and 30 days . for tumor treatment efficiency estimation tumor volumes were evaluated many times after end of short courses or once after end of 30 - day course . efficiency was judged according to standard tumor growth suppression factor ( tgs ) in comparison to control group that didn &# 39 ; t receive a treatment and results were calculated in percents . obtained results were then statistically calculated , using confidence intervals of mean values of compared values , difference stated reliable if p & lt ; 0 . 05 . as was demonstrated , skq1 preparation in single dose 0 . 5 nm / kg possessed short - time inhibition effect on tumors of mice with hypodermic ehrlich &# 39 ; s carcinoma immediately after end of 5 - day course ( tgs = 40 - 50 %). ten - fold dose increase or increase of injection number didn &# 39 ; t lead to prolongation of said effect . thus 5 - day treatment course should be considered as minimal efficient course . two human large intestine carcinoma cell sub - lines that differ by p53 status — wild type cells ( hct116 p53 +/+)) and cells with inactivated p53 ( hct116 p53 −/−)) ( cell line cultivation see in bunz , f ., dutriaux , a ., lengauer , c ., waldman , t ., zhou , s ., brown , j . p ., sedivy , j . m ., kinzler , k . w ., and vogelstein , b . ( 1998 ) science , 282 , pp 1497 - 1500 , see model description in sablina a a , chumakov p m , kopnin b p . ( 2003 ), j . biol . chem ., 278 , pp . 27362 - 27371 ). cells in amount 1 million were hypodermically implanted to thymus - deprived mice . mice in each group were divided to 4 sub - groups 10 mice in each : group received water to drink group received water with skq1 — 0 . 01 nmol of preparation per mouse per day ( 0 . 5 nm / kg / day ) group received water with skq1 — 0 . 1 nmol of preparation per mouse per day ( 5 nm / kg / day ) group received water with skq1 — 1 nmol of preparation per mouse per day ( 50 nm / kg / day ) size of tumors was evaluated every 3 days . after 4 weeks mice were mortified , tumors were fixed for histological assay . according to obtained data , addition of skq1 preparation in drinking water led to tumor growth suppression in comparison to control group , one that received pure water ; changes in growth speed were significantly higher in tumors with inactivated p53 than in tumors with p53 of wild type ( see fig3 ). the most efficient concentration appears to be 0 . 1 nmol per mouse per day ( 5 nm / kg / day ). due to received data addition of skq1 has stronger effect on tumor with inactivated p53 compared to tumors with wild - type p53 . for development of oncological diseases joint therapy that combines skq1 and chemotherapy , a study on skq1 influence on single doxorubicin injection efficiency was performed ( one of mechanisms of the preparation is activation of oxidative processes in normal and cancerous cells ). skq1 was introduced before , simultaneously or after introduction of doxorubicin in maximum dose 500 nm / kg ( in order to block oxidative mechanism of doxorubicin influence ). table 2 demonstrates results of study on efficiency of joint introduction of skq1 introduced in dose 500 nm / kg in 0 . 2 ml / mouse during 5 days in form of alcohol solution and doxorubicin introduced in all variants intraperitoneally in a dose 7 mg / kg on 7 th day after transplantation . schemes of doxorubicin application allow its introduction before , after and during the course of skq1 . skq1 monotherapy was demonstrated to 33 % suppress growth of tumor immediately after end of course . anti - tumoral effect of doxorubicin , applied on 7 th day — when tumor was already fully developed , was less expressed . tgs comprised 26 % and 36 % on 10 th and 14 th day thereof . of all studied schemes of joint application of skq1 and doxorubicin , the most efficient appears to be a scheme with 5 - day - long course of skq1 “ before ” introduction of doxorubicin . maximal tgs was registered on 10 th day ( 61 %) and stayed up to 21 st day . an important thing is that effect of skq1 on 7 th day was the same in groups received skq1 as monotherapy and with aftergoing introduction of doxorubicin — tgs comprises 33 and 39 % thereof . different schemes of joint application of skq1 and doxorubicin proved to be ineffective . in these variants therapeutic effect didn &# 39 ; t exceed effect of doxorubicin and were lower than one of skq1 . due to this , the most efficient scheme appears to be one that comprises introduction of doxorubicin after skq1 course ( 5 - day - long ). addition of skq1 to drinking water appeared to increased hiv mice immunity to infectious diseases , what was registered though estimation of mouse life - span when managed in chamber without sterile aeration . skq1 efficiency demonstrated to depend on p53 status in the tumor . addition of skq1 preparation ( maximal effect — 5 nm of skq1 ) to drinking water inhibits growth of tumors with inactivated skq1 stronger than in tumors of p53 wild type . application of skq1 ( 500 nm , 5 - day - long course ) before introduction of doxorubicin appear to be most efficient ( maximal tgs was registered on 10 th day ( 61 %) and remains the same until 21 st day ). different schemes of joint therapy — application of doxorubicin before or during skq1 course — appear to be ineffective .
the invention relates to pharmacology , medicine and oncology , in particular , to a class of chemical compounds , the structure of which corresponds to formula and which can be used in pharmaceutical compositions for preventing and treating an extended range of oncological diseases , including , in the form of an anti - tumoral preparation and a preparation which is used for combined therapy of oncological diseases .