in the following examples the invention will be described in more detail . however , the described embodiments mentioned below are only given as examples and should not be limiting to the present invention . other solutions , uses , objectives , and functions within the scope of the invention as claimed in the below described patent claims should be apparent for the person skilled in the art . if not other stated all chemicals and cell culture reagents were purchased from sigma - aldrich ( st louis , mo ., usa ). commercially available human tumor cell lines were purchased from atcc ( atcc / lgc - standards , borás , sweden ). cell lines from urinary bladder ( um - uc - 3 , # crl - 1749 ), colorectal carcinoma ( colo205 , # ccl - 222 and ht29 , # htb - 38 ), prostate carcinoma ( lncap , # crl - 1740 ) and breast carcinoma ( zr - 75 - 1 , # crl - 1500 ) were purchased and stored in liquid nitrogen . cells were cultured in roswell park memorial institute media ( rpmi - 1640 ) or minimum essential medium with earl &# 39 ; s salts ( emem ) supplemented with 10 % foetal calf serum ( fcs , sigma ) and cultured using a 37 ° c . cell culture incubator ( forma scientific , ninolab , kungsbacka , sweden ) with 5 % co 2 humidified atmosphere . tumor cell lines were handled aseptically using a laminar airflow ( laf ) bench ( holten 2448 , ninolab , sweden ) and stored in a liquid nitrogen tank and were initiated in culture as previously described ( falk p . experimental models of the peritoneal environment : effects of tgf - beta and hyaluronan . göteborg : university of gothenburg ; 2008 ). briefly , ampoules of frozen cells were thawed by submerging into a 37 ° c . water bath for 45 s . cells were resuspended in 10 ml of preheated rpmi - 1640 with 10 % fcs and centrifuged in 260 g for 10 min . with supernatant discarded , cell pellets were further resuspended with 5 ml complete medium into 25 cm 2 cell culture flasks ( cell +, sarstedt , nümbrecht , germany ). tumor cells were cultured until sub - confluence and further sub - cultured with trypsin ( 0 . 5 %), edta ( 0 . 2 %) solution ( t4174 , te - 10x , sigma ) in sterile sodium chloride . culture media was changed two - three times a week . culture flasks ( cell +, sarstedt , nümbrecht , germany ) and culture plates ( falcon / bd , franklin lakes , n . j ., usa ) were used for experimental set - ups . cells were regularly monitored using an inverted microscope connected to a digital photo equipment ( axiovert 25 and axiovision , zeiss , carl zeiss ag , germany ) ( fig1 ). described culture techniques and equipment have been used extensively in the laboratory ivermectin ( sigma ) is hydrophobic and was diluted in 95 % alcohol in the first step and then subsequently gently diluted to 1 × 10e - 4 mol / l by adding additional solvent with decreasing amount of alcohol . the following concentrations were used in the experimental set - up ; 1 × 10e - 4 to 1 × 10e - 10 mol / l . abamectin ( sigma ) is hydrophobic and was diluted using methanol and subsequentially gently diluted with decreasing amount of alcohol . concentrations of 10 and 100 μmol / l were used . selamectin ( sigma ) is hydrophobic and according to manufacturing instruction dimethylsulphoxide ( dmso ) was used as solvent . further dilutions were made using gently dilution with decreasing amounts of alcohol . concentrations of 10 and 100 μmol / l were used . since the alcohol concentrations reach above 1 % in assay concentration for the highest avermectin concentrations control experiments were performed using the same alcohol concentration but without active substance . ketoconazole is an antifungal drug used in fungal skin infections and in combination with chemotherapy . ketoconazole is a hydrophobic substance and was dissolved in methanol and further gently diluted using culture media . concentrations of 10 , 20 and 40 μmol / l were used alone and together with ivermectin , selamectin or abamectin . albendazole is a drug indicated for the treatment of a variety of worm infestations . albendazole is a hydrophobic substance and was dissolved in dmso and further gently diluted using alcohol and culture media . concentrations of 20 and 40 μmol / l were used alone and together with ivermectin , selamectin or abamectin . ibuprofen ( sigma ) is a nonsteroidal anti - inflammatory drug ( nsaid ) used for relief symptoms from pain and fever . ibuprofen is hydrophilic and was initially diluted using sterile water and further diluted using culture medium rpmi - 1640 or emem . concentrations of 10 , 20 and 40 μmol / l were used alone or together with ivermectin , selamectin or abamectin . diclofenac sodium ( sigma ) is a nonsteroidal anti - inflammatory drug ( nsaid ) used for relief symptoms from pain . diclofenac sodium is hydrophilic and was initially diluted using sterile water and further diluted using culture medium rpmi - 1640 or emem . concentrations of 10 , 40 and 100 μmol / l were used alone or together with ivermectin , selamectin or abamectin . celecoxib ( sigma ) is a nsaid substance and a specific cox - 2 inhibitor . celecoxib is strongly hydrophobic substance and it was initially dissolved in dimethylsulfoxide ( dmso ) to 32 mmol / l . further dilutions were made due to gently addition of a 50 / 50 ( alcohol 70 %/ rpmi 1640 ) mixture to 3 . 2 mmol / l . additional gentle addition of rpmi 1640 + 1 % fetal calf serum ( fcs ) were made to 1 mmol / l . additional serial dilutions using rpmi 1640 + 1 % fcs to 10 , 20 and 40 μmol / l were used alone or together with ivermectin , selamectin or abamectin . the effect of ivermectin , selamectin , abamectin alone , or in combination with ketoconazole , albendazole , ibuprofen , diclofenac and celecoxib on the morphology of cultured human tumor cells was studied . the effect of ivermectin in the presence of increasing concentration of ivermectin was also investigated . cells were grown into sub - confluence and then sub - cultured into 12 and 24 well plates ( sarstedt , falcon / bd ) for morphology studies , or in 96 well plates ( sarstedt , falcon / bd ) for proliferation studies . viability of urinary bladder cells was studied using the tryphan blue exclusion method ( freshney r . culture of animal cells : a manual of basic techniques . new york : alan r liss inc ; 1987 ). the proliferation rates of the different human tumor cell lines were studied using a method based on the conversion of a sodium salt ( xtt - assay , sigma ) into a stable orange color that could be analyzed in a spectrophotometer . the color change is proportional to the mitochondrial dehydrogenase activity resulting in an effective technique for measuring cell toxicity ( roehm n w , rodgers g h , hatfield s m , glasebrook a l . an improved colorimetric assay for cell proliferation and viability utilizing the tetrazolium salt xtt . j immunol methods 1991 ; 142 ( 2 ): 257 - 65 ) or cell proliferation . the color change was measured using a 96 - well plate reader with software to calculate the absorbance at 450 nm ( v - max / softmax pro , molecular devices , usa ). cells were cultured until approx 50 % confluence and then pre - incubated for 24 h with rpmi - 1640 or emem containing 1 % fcs only . rpmi 1640 and emem without phenol red was used during the experiments to reduce the red color background signal in the xtt - assay . prior to the experimental start all cells were gently washed once with rpmi 1640 and emem with 1 % fcs . increasing concentrations of substance ivermectin was added with an 8 - channel pipette ( titertek , kem - en - tec , taastrup , denmark ) together with sterile pipette tips ( molecular bioproducts , san diego , calif ., usa ). cell cultures including active substances were incubated for 24 hours ( and 96 hours ). untreated wells including culture medium only served as untreated controls . the xtt - assay substrate was added and after gentle agitation using a micro plate shaker , absorbencies at 450 and 650 nm were measured . levels from medium were subtracted and compared with control wells , using culture medium only . the non - parametric kruskal - wallis test was used to detect overall differences and if significant , a mann - whitney u test was performed between individual groups . a p - value & lt ; 0 . 05 were considered statistically significant . results are presented in the figures as mean ± sem . results in tables are presented as mean ± sd . all statistical calculations were performed using the statview 5 . 0 package ( abacus concepts , berkeley , calif ., usa ). calculations of absorbance and concentrations were analyzed with the softmax pro software using both linear and 4 - parameter curve fit ( softmax pro , molecular devices , usa ). cultures were established from all tested tumor cell lines . tumor cells could be cultured , sub - cultured , frozen and refrozen into liquid nitrogen without affecting growth performance . the presence of ivermectin affected all the human cultured tumor cell lines in a negative manner compared to cells treated with the medium control . most negative observations were made in the concentration interval 10e - 4 to 10e - 6 mol / l . most of the tumor cells were affected with damaged cell structure and / or cell loss in anchorage dependent tumor cells . fig2 shows morphological effects of ivermectin on cultured tumor cell lines . it can be seen that concentrations above 10e - 5 mol / l affected morphology and proliferation on most of the cultured tumor cells . the colorectal cell lines ht29 was not morphologically affected even at the highest concentration , while the colo205 line was affected even at 10e - 6 mol / l . both the prostate cell line lncap and the breast cancer cell line 2r - 75 - 1 was affected by the highest concentration 10e - 4 mol / l only ( objective magnification × 20 ). fig3 shows tryphan blue exclusion of human urinary bladder cancer cells . it can be seen that ivermectin affected cells in a dose - dependent manner ( 3 a - b ), compared to untreated medium control ( 3 c ). it is notable that an alcohol concentration above 3 % also affected most of the tumor cells in a negative manner . in the initial experiments with the urinary bladder cells the alcohol concentration in the highest ivermectin concentration were 9 % however , during the later experiments this concentration was reduced to 3 %. this was achieved due to gentle dilution adding small amounts of dilution medium , instead of strictly performed serial dilutions . morphological effects on the cultured tumor cells are illustrated in fig4 . the presence of ivermectin decreased the proliferation rate of all human tumor cells compared to cells treated with medium only ( see table 2 ). proliferation rates are expressed as mean ± standard deviation for each concentration in per cent compared to untreated medium control . at ivermectin 10e - 4 mol / l all cell lines reduced their proliferation rate . for the urinary bladder cell line ( um - uc - 3 ) the addition of ivermectin reduced proliferation significantly ( p & lt ; 0 . 01 ) compared to untreated control , with most pronounced effect at 10e - 4 mol / l ( 34 ± 3 %, p & lt ; 0 . 05 ) and 10e - 5 ( 34 ± 8 %, p & lt ; 0 . 05 ). in the colorectal cell line ( colo205 ) ivermectin reduced the overall proliferation rate ( p & lt ; 0 . 0001 ) compared to control . fig5 shows the proliferation rates in % of untreated control for the colorectal tumor cell line colo205 . this effect was most distinct at 10e - 4 mol / l ( 4 ± 1 %, p & lt ; 0 . 01 ), at 10e - 5 mol / l ( 6 ± 1 %, p & lt ; 0 . 01 ), at 10e - 6 mol / l ( 42 ± 12 %, p & lt ; 0 . 01 ) and at ivermectin 10e - 7 mol / l ( 69 ± 9 %, p & lt ; 0 . 05 ). notable is that the dilution buffer including the same alcohol concentration , but without active substance ivermectin , also affected proliferation ( proliferation in %, mean ± sd compared to control ). in the other colorectal cell line ( ht29 ) the overall proliferation rate was significantly reduced by the addition of ivermectin ( p & lt ; 0 . 0001 ) with most effect seen at 10e - 4 ( 5 ± 1 %, p & lt ; 0 . 01 ) and 10e - 5 ( 85 ± 11 %, p & lt ; 0 . 05 ) mol / l . the prostatic cell line ( lncap ) significantly reduced the proliferation rate ( p & lt ; 0 . 0001 ) compared to untreated control when treated with ivermectin . this effect was most pronounced at 10e - 4 ( 8 ± 1 %, p & lt ; 0 . 01 ) and 10e - 5 ( 753 %, p & lt ; 0 . 01 ) mol / l . finally , also the human breast cancer cell line ( 2r - 75 - 1 ) was affected by ivermectin by reduction in cell proliferation rate ( p & lt ; 0 . 01 ) compared to untreated control . this effect was only seen at 10e - 4 mol / l ( 7 ± 2 %, p & lt ; 0 . 01 ). experimental set ups with the different human cell lines were run in duplicates with 6 - 8 replications in each experiment . results are summarized in table 2 . as earlier described there are a certain effects of alcohol present in the dilution media . this was most pronounced when the colorectal cell line colo205 was investigated . even alcohol concentration below 3 % affected these cell types in a negative manner ( fig4 and 5 ). ivermectin in combination with ketoconazole , ibuprofen , diclofenac and / or celecoxib affect morphology and viability of cultured tumor cell lines in the urinary bladder cell line ( um - uc - 3 ) a synergistic effect were seen when ivermectin were combined with ketoconazole , diclofenac or ibuprofen . cells were damaged and detached from the culture upon stimulation . cell structure was damaged and a lot of cells stimulated by the combination showed a ballooned appearance , compared to untreated medium control . fig6 shows the morphology of urinary bladder cells ( um - uc - 3 ) after 24 hours with ivermectin stimulation . the morphology of the cells was affected when treated with ivermectin ( 100 μm ). however , a synergistic effect was seen with affected morphology when ivermectin were combined with ketoconazole ( 10 , 20 and 40 μm ). a ) control cells , b ) cells affected of ivermectin , 100 μm . c - e ) synergistic effects were seen with ivermectin 100 μm in combination with ketoconazole 10 μm c ), 20 μm , d ) and 40 μm ( e ) compared to ivermectin 100 μm alone ( b ). additional , effects were also seen when ivermectin and diclofenac were combined . fig9 indicates synergistic effect on the morphology when ivermectin 10 μm and diclofenac 100 μm were combined . in the colorectal tumor cell line ( colo205 ) affected cell structure was seen with ivermectin 1001 μm , but no effect were seen at ivermectin 10 μm . a small synergistic effect was seen when ivermectin 100 μm were combined with ketoconazole 10 , 20 and 40 μm , similar to the um - uc - 3 cells . surprisingly , a synergistic effect was also seen at ivermectin 10 μm in combination with ketoconazole 10 , 20 and 40 μm . in these cultures cells showed an increase in cell detachment and ballooned structure , compared to untreated medium control ( fig7 ). similar to the urinary bladder cell line the combination of ivermectin 10 μm and diclofenac 1001 μm generated a synergistic increase in cell death for these cells . the colorectal cell line ht29 was affected when ivermectin 100 μm was used . no synergistic effects were seen in ivermectin 10 or 100 μm , when combined with ketoconazole , ibuprofen and celecoxib , compared to untreated control . a possible synergistic effect was seen when ivermectin 10 μm were combined with diclofenac 40 and 100 μm . moreover , an effect was seen in the morphological appearance when ivermectin were combined with albendazole 20 and 40 μm . fig8 shows that the prostatic cell line lncap was affected after high doses of ivermectin ( 100 μm ), with cell damage and detachment of cells 24 hours after ivermectin stimulation ( c ). no effect was seen at ivermectin 1 ( a ) and 10 μm ( b ). ketoconazole did affect morphology at concentrations 20 and 40 μm with cell death and detachment as expected . at ketoconazole 10 μm ( d ) cells were not microscopically affected , but interestingly the combination with ivermectin 10 μm affected the morphology and caused partly detachment of cells from the surface ( e ), f ) control . a small synergistic effect was seen when ivermectin 10 μm were combined with diclofenac 40 μm and 100 μm . ivermectin in combination with ketoconazole , ibuprofen and celecoxib affects the proliferation rate in the urinary bladder cell line um - uc - 3 ( table 3 ) a synergistic effect in proliferation rate were seen when ivermectin was combined with ketoconazole , ibuprofen or celecoxib . ivermectin only reduced the proliferation rate to 90 ± 1 % ( p & lt ; 0 . 05 , n = 6 ). interestingly , when ivermectin ( 100 μm ) were used together with ketoconazole ( 20 μm ) the proliferation rate decreased to 63 ± 18 % ( p & lt ; 0 . 05 , n = 6 ) and in combination with ketoconazole ( 40 μm ) it decreased to 42 ± 24 % ( p & lt ; 0 . 01 , n = 6 ) compared to untreated medium control ( table 3 ). in the colorectal cell line ( colo2065 ) ivermectin alone affected proliferation rate at the 100 μm level . synergistic effects in proliferation rate were seen when ivermectin were combined with ketoconazole or ibuprofen . this effect was not only seen at ivermectin 100 μm , but also at 10 μm . when ivermectin 10 μm were combined with ketoconazole 10 , 20 or 40 μm the proliferation rate decreased to 83 . 1 ± 16 . 9 % ( p & lt ; 0 . 05 , n = 6 ) with ketoconazole 10 μm , 88 . 1 ± 17 . 6 % ( p & lt ; 0 . 05 , n = 6 ) using ketoconazole 20 μm and at ketoconazole 40 μm the proliferation rate decreased to 57 . 9 ± 24 . 6 % ( p & lt ; 0 . 01 , n = 6 ), compared to medium control ( see table 4 ). the colorectal cell line ht29 was affected in decreased proliferation rate when ivermectin 100 μm was used . no additional effects in proliferation rate were seen combined with ketoconazole , ibuprofen or celecoxib , compared to untreated control . however , when ivermectin 10 μm was combined with albendazole 20 and 40 μm affected morphology were seen with decreased tumor growth . the prostatic cell line ( lncap ) reduced the proliferation rate compared to untreated control when ivermectin at 100 μm was used alone or in combination with ketoconazole , ibuprofen or celecoxib as can be seen in table 5 . ketoconazole alone at 20 and 40 μm decreased the proliferation rate , compared to untreated medium controls . a synergistic effect in proliferation rate was seen when ivermectin 10 μm and ketoconazole 10 μm were combined 86 . 0 ± 0 . 2 % ( p & lt ; 0 . 05 , n = 6 ). this effect was not seen with ivermectin 10 μm ( 106 . 4 ± 2 . 2 ) and ketoconazole 10 μm ( 104 . 6 ± 3 . 0 ) alone , compared to untreated control . the breast cell line ( zr - 75 - 1 ) reduced the proliferation rate compared to untreated control when ivermectin 100 μm was used alone or in combination with ketoconazole , ibuprofen or celecoxib . since ivermectin alone reduced proliferation rate at both 10 and 100 μm no additative effects could be seen when combinations were made . however , surprisingly to the authors , synergistic effects were seen already at ivermectin 1 μm when combined with ibuprofen 20 and 40 μm and celecoxib 40 μm ( see table 6 ). as earlier described there are a certain effects of alcohol present in the dilution media . alcohol concentration above 3 % affected cell proliferation in a negative manner . this was most pronounced when the colorectal cell line colo205 was investigated ; even alcohol concentration below 3 % affected these cell types in a negative manner . in order to evaluate more members from the avermectin family selamectin and abamectin were investigated using similar concentrations as for ivermectin . morphology studies indicated that both selamectin and abamectin at 100 μm have similar negative effect on morphology as for ivermectin 100 μm . this is demonstrated for the colorectal cell line ( colo205 ) as indicated in fig1 . moreover , when albendazole were added at 20 and 40 μm together with ivermectin 10 μm a synergistic effect was seen in the same cell line with decreased growth of cells . this was not seen when ivermectin 10 μm and albendazole 20 and 40 μm were tested alone ( fig1 ). the present studies are based on a case report describing a middle age man suffering from an advanced urothelial cancer with extensive metastasis . due to a tropical infection this patient received the substance ivermectin . surprisingly to the authors , this treated both the tropical infection and the urothelial cancer . the reason for this is not known however a direct effect of the substance ivermectin could not be precluded . the patient case in the present study did not receive any other medication than substance ivermectin . when verifying the effect of ivermectin on a palette of human tumor cells , it could be demonstrated that ivermectin , in a dose - response manner , reduces the proliferation rate in human cultured tumor cells . cells used derived from urinary bladder , colorectal region , prostate and mammary glands . ivermectin reduced the cell proliferation rate in micromolar concentrations in the five different human tumor cell lines , from four different locations . all cell lines reduce the proliferation rate at 100 micromolar and several of them at even lower concentrations . additionally , when selamectin and abamectin , other members in the avermectin family , was used alone or in combination with members from the nsaid family similar effects were seen on several of the different tumor cell lines tested in the experimental models . thus , the observed effect alone or in combination with other substances was not only from the substance ivermectin , but seems also be generated from several members of the avermectin family . however , the above experiments demonstrated that ivermectin and other members from the avermectin family ( i . e in particular abamectin and selamectin ), in combination with ketoconazole , albendazole , ibuprofen , diclofenac and / or celecoxib in a dose - response manner exhibited a synergistic effect on cell morphology and / or reduction in proliferation rate in human cultured tumor cell lines . cells used derived from urinary bladder , colorectal region , breast and prostate tissue . in conclusion , the avermectin compounds ivermectin , abamectin andselamectin reduced cell proliferation in urinary , colorectal , prostate and breast tumor cell lines . when combined with ketoconazole , ibuprofen , diclofenac or celecoxib , the combination provided synergistic effects in affecting the morphology of cultured tumor cells and in the reduction of the proliferation rate . this might introduce several lines of therapeutic substance for all those individuals that suffer from several forms of cancer .
the present invention includes a composition comprising an effective amount of an avermectin compound , and an effective amount of an imidazole antifungal compound , and / or an effective amount of non - steroidal anti - inflammatory drug or suitable pharmaceutically acceptable salts thereof , for use in the inhibition of tumor growth and treating urothelial , colorectal , prostate and / or breast cancers . furthermore , the invention includes a method of inhibiting tumor growth and treating said cancers comprising administering to a subject in need thereof an effective amount of said composition .