Patent Application: US-94338597-A

Abstract:
previously unknown anti - tumor , anti - infectious , immune system modulatory and anti - depressive properties of a family of barbituric acid derivatives are provided herein . preferred derivatives are 4 , 6 - pyrimidinedione , 1 , 3 - dibutyldihydro - 2thioxo ; and 4 , 5 , 6 - pyrimidinetrione , 1 , 3 - dibutyldihydro - 2thioxo . these compounds are effective for killing tumor cells , especially carcinoma , lymphoma and leukemia cells ; for killing pathogenic organisms , especially viruses ; for stimulating t cell formation and for alleviating symptoms of depression and are readily tolerated by the animal being treated .

Description:
the present invention provides novel methods for use of a family of barbituric acid analogs that are capable of killing , or inhibiting the growth of benign or malignant tumor cells or other pathogenic biological contaminants infecting body tissues , treating conditions of the immune system , or improving symptoms of depression . as demonstrated in the following examples , compounds c1 and c2 are powerful anti - tumor agents , particularly against carcinoma , such as prostate cancer or breast cancer , sarcoma , leukemia and lymphoma . the toxicity of compound c1 is provided in example 7 . studies were also carried out to establish anti - viral , immunomodulatory and anti - depression properties of compound c1 . the following materials and methods are not to be construed as limiting . synthesis of compound c1 : 4 , 6 ( 1h , 5h )- pyrimidinedione , 1 , 3 - dibutyldihydro - 2 - thioxo was synthesized , freeze dried and stored at room temperature for use in the following examples . the synthesis was carried out as described in brown ( 1962 ) as follows : a 3 - necked 2 liter flask fitted with a water condenser , gas inlet , and suba seal was purged with dry n 2 . a freshly prepared solution of sodium ethyrate , prepared by slowly adding 2 . 5 g of na to 50 ml etoh at room temperature , was added to the flask . diethyl malonate ( 17 g , 110 mmol ) was then added with vigorous stirring , followed by the addition of n , n &# 39 ;- dibutyl - 2 - thiourea ( 10 g , 53 mmol ). the mixture was stirred vigorously under n 2 for 3 days . the mixture was cooled to room temperature , 50 ml of water was added carefully and ethanol was removed under reduced pressure . the remaining residue was poured into water ( 200 ml ) and cooled in an ice / water bath . the solution was filtered to remove unreacted starting materials and acidified with dilute hcl . the resulting precipitate was collected by suction filtration , washed with water , and dried thoroughly to give a white solid . the yield was about 75 %. compound c1 was determined to be approximately 98 % pure by tlc and nmr . synthesis of 4 , 5 , 6 ( 1h )- pyrimidinetrione , 1 , 3 - dibutyldihydro - 2 - thioxo , compound c2 : a sample of n , n &# 39 ;- dibutyl - thiobarbituric acid ( 10 g ) was dissolved in toluene ( 100 ml ) and dried seo 2 ( 10 g ) was added . a steady stream of air was passed through the solution , and the solution was heated to reflux for 1 h . after cooling overnight , the solvent was removed under reduced pressure and the residue was chromatographed on activated alumina using chloroform as eluant . the desired compound was isolated after evaporation of the solvent and examined for purity by tlc and nmr . yield -- 6 g . two further analogs of c1 were synthesized and tested for toxicity for lymphoma cells . analog c4 has structure a where r 1 and r 2 are hydrogen , r 3 is s and r 4 is h . analog c5 has structure a where r 1 and r 2 are methyl , r 3 is s and r 4 is h . cell lines for the following examples were obtained from american type culture collection ( rockville , md . ), and were maintained in recommended medium at 37 ° c . in a humidified atmosphere of 5 % co 2 in air . in the following examples , tumor cells were treated with varying concentrations of compounds c1 , c2 , c4 or c5 dissolved in absolute ethanol . to limit any non - specific toxicity , the final concentration of ethanol was not allowed to exceed 0 . 5 %. stock concentrations of 5 mg / ml or 20 mg / ml were used . viability of cells was determined in some studies by the trypan blue dye exclusion method . in other studies , cell viability / proliferation activity was determined by mtt assay by using a kit obtained from promega , ( promega corp ., madison , wis . 53711 ). for this assay , a 100 μl cell suspension containing 5 - 10 3 monolayer cells or 1 × 10 5 suspension cells was placed in the wells of a 96 - well microtiter plate . after overnight incubation , cells were treated with different concentrations of the test compound . control wells received vehicle only . after 24 , 48 , 72 or 96 h of incubation , 50 μl of mtt dye solution was added . after 4 h of incubation at 37 ° c ., 100 μl of solubilization / stop solution was added . after another h of incubation , cells were mixed using a multi - channel pipet and the absorption of the dye color was recorded at 570 nm using a plate reading absorption spectrophotometer . methods of in vitro assay , such as those disclosed in selected methods in cellular immunology , eds . b . b . mishell and s . m . shiigi , w . h . freeman and company , san francisco , calif ., 1980 , and plumb et al ., cancer res ., 49 : 4435 , 1980 were used . these references are incorporated by reference herein . even though the invention has been described with a certain degree of particularity , it is evident that many alternatives , modifications , and variations will be apparent to those skilled in the art in light of the foregoing disclosure . accordingly , it is intended that all such alternatives , modifications , and variations which fall within the spirit and the scope of the invention be embraced by the defined claims . the following examples are included to demonstrate preferred embodiments of the invention . it should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function well in the practice of the invention , and thus can be considered to constitute preferred modes for its practice . however , those of skill in the art should , in light of the present disclosure , appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention . human prostate cancer ( pc - 3 and du - 145 cell lines ) and human breast cancer ( mcf - 7 and bt - 20 cell lines ) were treated with compound c1 or c2 for 24 , 48 , 72 or 96 h at 37 ° c . after the incubation period , viability of the cells was determined by mtt assay ( promega corp ., madison , wis . 53711 ). the resulting data are presented in table table 1______________________________________effect of compound c1 and c2 on prostate and breast cancer cells . percent cell killcell type 24 hr 48 hr 72 hr 96 hr______________________________________ cmpd c1 dose ( μg / ml ) pc - 3 human 50 61 . 7 ± 10 . 0 91 . 0 ± 3 . 6 100 . 0 100 . 0prostate 100 88 . 7 ± 5 . 5 92 . 7 ± 1 . 2 100 . 0 100 . 0cancer 150 90 . 5 ± 7 . 1 94 . 9 ± 5 . 3 100 . 0 100 . 0du - 145 50 0 0 23 . 5 ± 4 . 5 26 . 2 ± 5 . 5human pro - 100 27 . 5 ± 6 . 4 52 . 2 ± 3 . 4 58 . 7 ± 2 . 1 64 . 0 ± 4 . 2state cancer 150 43 . 0 ± 2 . 9 71 . 0 ± 7 . 3 78 . 7 ± 3 . 4 82 . 5 ± 3 . 7mcf - 7 50 39 . 5 ± 10 . 5 67 . 5 ± 5 . 3 85 . 8 ± 3 . 8 97 . 8 ± 1 . 7human breast 100 57 . 3 ± 1 . 5 92 . 8 ± 4 . 9 88 . 0 ± 5 . 4 97 . 8 ± 1 . 7cancer 150 87 . 8 ± 8 . 8 92 . 3 ± 5 . 3 84 . 5 ± 5 . 1 96 . 5 ± 2 . 5bt - 20 50 43 . 3 ± 4 . 1 65 . 5 ± 2 . 4 73 . 3 ± 2 . 9 100 . 0human breast 100 62 . 0 ± 7 . 9 91 . 3 ± 2 . 5 86 . 8 ± 3 . 6 100 . 0cancer 150 94 . 0 ± 4 . 2 96 . 5 ± 2 . 4 86 . 0 ± 3 . 6 100 . 0______________________________________ cmpd c2 dose ( μg / ml ) pc - 3 human 100 32 . 7 ± 6 . 2 46 . 2 ± 4 . 1 51 . 5 ± 6 . 5 75 . 0 ± 1 . 0prostate 150 47 . 0 ± 6 . 7 70 . 7 ± 6 . 0 77 . 7 ± 4 . 6 88 . 7 ± 3 . 2cancer 200 57 . 5 ± 3 . 5 81 . 5 ± 3 . 1 88 . 5 ± 5 . 2 95 . 0 ± 3 . 6du - 145 100 0 . 0 0 . 0 0 . 0 10 . 0 ± 6 . 1human 150 0 . 0 4 . 2 ± 3 . 9 9 . 2 ± 5 . 3 25 . 2 ± 7 . 4prostate 200 0 . 0 20 . 0 ± 3 . 0 36 . 7 ± 2 . 1 43 . 7 ± 4 . 3cancermcf - 7 100 9 . 0 ± 7 . 0 16 . 3 ± 4 . 8 33 . 3 ± 5 . 4 87 . 7 ± 0 . 6human breast 150 26 . 3 ± 7 . 9 43 . 3 ± 7 . 3 61 . 0 ± 5 . 6 97 . 0 ± 0 . 8cancer 200 40 . 0 ± 10 . 6 70 . 3 ± 7 . 8 81 . 3 ± 3 . 2 94 . 3 ± 2 . 1bt - 20 100 29 . 8 ± 6 . 8 51 . 8 ± 1 . 9 57 . 5 ± 2 . 6 99 . 9human breast 150 30 . 0 ± 4 . 4 50 . 0 ± 1 . 7 60 . 8 ± 2 . 8 99 . 9cancer 200 30 . 3 ± 2 . 9 55 . 5 ± 8 . 9 68 . 5 ± 3 . 4 99 . 9______________________________________ these data show that human prostate and breast cancer cells were susceptible to the cytotoxic action of both compounds c1 and c2 . compound c1 killed over 96 % of pc - 3 prostate and both breast cancer cell lines , and over 82 % of du 145 prostate cancer cells after a 96 h treatment at 150 μg / ml . compound c2 killed over 94 % of pc - 3 prostate and both breast cancer cell lines , and over 43 % du 145 prostate cancer cells after a 96 h treatment at 200 μg / ml . cell kill in untreated controls ( vehicle only ) ranged from 5 % to 15 % for all experiments including those in subsequent examples . the daudi human lymphoma cell line and the l1210 mouse leukemia cell line were treated with compound c1 c2 for 24 or 48 h at 37 ° c . the viability of the cells was table 2______________________________________effect of compounds c1 or c2 on leukemia and lymphoma cells % cytotoxicitycell type 24 hr . 48 hr . ______________________________________ c1 dose ( μg / ml ) daudi human 50 91 . 3 ± 4 . 6 99 . 9lymphoma 100 93 . 3 ± 4 . 9 99 . 9 150 94 . 0 ± 4 . 9 99 . 9l1210 mouse 50 81 . 2 ± 4 . 3 99 . 0leukemia 110 93 . 5 ± 3 . 1 99 . 9 150 89 . 2 ± 3 . 2 99 . 9______________________________________ c2 dose ( μg / ml ) daudi human 100 57 . 5 ± 3 . 0 41 . 5lymphoma 150 81 . 3 ± 4 . 0 92 . 0 200 93 . 2 ± 3 . 3 88 . 0l1210 mouse 100 33 . 2 ± 2 . 0 29 . 0 ± 3 . 5leukemia 150 50 . 0 ± 4 . 1 53 . 7 ± 2 . 5 200 58 . 7 ± 2 . 6 72 . 5 ± 2 . 4______________________________________ these results show killing of human lymphoma daudi cells and mouse leukemia l1210 cells with compounds c1 and c2 . exposure to compound c1 resulted in over 99 % killing of these cells after a 48 h incubation period at all concentrations tested . exposure to compound c2 resulted in over 90 % and 58 % killing of these cells , respectively , after a 24 h incubation period at 200 μg / ml . compounds c4 and c5 were also tested for toxicity against human lymphoma daudi cells . incubation at 150 μg / ml produced a cell kill of less than 20 %, indicating that these derivatives are less effective than compounds c1 or c2 . from the data presented herein , it is apparent that the methods of the present invention include near total destruction of various tumor cells . the effect of compound c1 on clonogenic human breast tumor cells was investigated . to assay clonogenic tumor cells , human breast cancer mcf - 7 adenocarcinoma cells ( 1 × 10 3 cells per flask for control and 5 × 10 3 cells for treatment groups ) were plated and , after 24 h of incubation at 37 ° c . in a humidified atmosphere of 5 % co 2 and air , these cells were treated with 20 μg / ml , 40 μg / ml or 80 μg / ml of compound c1 . after additional incubation periods of 24 h and 48 h , cells were washed to remove the drug and were allowed to grow at 37 ° c . in a co 2 incubator . after 10 days , colonies consisting of 50 or more cells were counted . the resulting data are provided in table 3 . table 3______________________________________effect of c1 on the clonogenic growth of human breast cancer mcf - 7cellsc1 dose no . of colonies ( mean ± s . d .) percent ( μg / ml ) 24 hr 48 hr reduction______________________________________0 158 . 5 ± 12 . 2 158 . 5 ± 12 . 2 020 24 . 5 ± 7 . 8 24 . 5 ± 6 . 1 84 . 540 13 . 5 ± 5 . 1 11 . 7 ± 1 . 2 92 . 680 3 . 0 ± 2 . 2 0 ± 0 99 . 99______________________________________ compound c1 caused a virtually complete elimination of tumor cell clonogenic growth under the conditions employed . the activity of compound c1 in vivo against human du - 145 prostate tumors transplanted into athymic mice is demonstrated in the present example . male athymic balb / c nude - nu mice ( 6 - 8 weeks old , 18 - 22 g each ) were purchased from harlan sprague - dawley , inc . ( indianapolis , ind .) and maintained in a germ - free environment . the mice had free access to sterile food and water . human prostate du - 145 cells ( 1 × 10 7 ) were transplanted subcutaneously into athymic mice . solid tumors appearing after 30 days were cut into small pieces ( 2 - 2 mm ) and transplanted into native mice . treatment of three prostate tumor bearing animals was initiated the next day by oral feeding of 400 mg / kg of compound c1 suspended in polyethylene glycol . the treatment schedule consisted of a single dose on alternate days . the control group of animals ( 3 mice ) received vehicle only . after 18 doses of treatment , palpable prostate tumors in the treated group could not be detected , whereas tumors grew to an approximate tumor volume of 20 cm 3 in the control group . at this point , the treatment was terminated and animals were kept under observation for the regrowth of prostate tumors . as of 5 months posttreatment , there were no signs of tumor regrowth while only one control animal bearing prostate tumor was alive at that time point . during the course of this study , side effects such as weight loss , diarrhea or any signs of discomfort in the treated group of animals were not observed , indicating that compound c1 is readily tolerated . the results after an 18 dose treatment are presented in table 4 . table 4______________________________________effect of compound c1 on the growth of du - 145 human prostatetumorstreatment animal tumor area tumor volume % group dose weight ( g ) ( cm . sup . 2 ) ( cm . sup . 3 ) tgi______________________________________control vehicle 28 . 5 19 . 90 ± 7 . 1 20 . 64 ± 7 . 01 onlyc1 400 mg / kg 29 . 3 0 . 0 0 . 0 99 . 0______________________________________ % tgi indicates percent tumor growth inhibition , calculated by using the formula % tgi = 100 ( 1v . sub . t / v . sub . c ), where v . sub . t and v . sub . c are the mean volumes of the treated and control tumors , respectively . these results show that even after an 18 dose treatment , palpable tumors were not detectable in treated animals . in addition , regrowth of treated tumors did not occur for a period of 5 months . no observable side effects , such as weight loss or lethargy , were observed in treated animals . in contrast , tumors grew rapidly to an average tumor volume of 20 . 64 cm 3 in untreated animals . these results clearly show that compound c1 is an effective antitumor agent in vivo and that it is readily tolerated . similar experiments were carried out where implanted human prostate tumors ( 2 × 2 mm ) were allowed to become established . eight days later , the tumor size was ≈ 5 × 5 mm . treatment was initiated which consisted of intramuscular injections of c1 ( 100 mg / kg ) given on alternate days . after 5 weeks of treatment , tumors were not detectable by palpation in 2 of 3 animals . in the third animal , further tumor growth essentially did not occur since the tumor was 6 × 5 . 5 mm in size . untreated control mice ( n = 3 ), had tumors at 5 weeks of average size of 10 × 9mm . human fibrosarcoma cells were cultured in minimum essential medium ( eagle ) with non - essential amino acids and earls balanced salt solution supplemented with 10 % heat - inactivated fetal bovine serum . cells ( 5 × 10 3 in 0 . 5 ml of growth medium ) were plated in 16 well plates and incubated at 37 ° c . in a humidified atmosphere of 5 % co 2 in air for 24 h . cells were then treated with different doses of c1 . control wells received vehicle only (& lt ; 0 . 5 % ethanol in saline ). after further incubation periods of 24 and 48 h , the viability of cells was determined by trypan blue dye exclusion method . the results are provided in table 5 . table 5______________________________________effect of c1 on human fibrosarcoma ht - 1080 cell . sup . atime percent survival ( h ) control 25 μg / ml 50 μg / ml 100 μg / ml 150 μg / ml______________________________________24 92 . 3 ± 3 . 1 0 . 03 ± 0 . 03 0 . 03 ± 0 . 03 0 048 92 . 4 ± 3 . 1 0 0 0 0______________________________________ . sup . a control cells were treated with vehicle ( 0 . 5 % ethanol in saline ) only . virtually all fibrosarcoma cells were killed at 24 h at doses of 100 and 150 μg / ml . at a dose of 50 μg / ml , over 92 % of the cells were killed . at 48 h , virtually all fibrosarcoma cells were killed even at the lowest dose of 25 μg / ml tested . in control wells , approximately 8 % cells were killed at the highest dilution of solvent control . these results show that human fibrosarcoma cells are very susceptible to the cytotoxic action of c1 . through the initial work of hamburger and salmon ( 1977 a , b ), it is possible to culture human tumors using a two - layer soft agar system . this culture system , called the human tumor cloning system , was initially utilized to select the most appropriate anticancer agent for an individual patient &# 39 ; s tumor . later , it was demonstrated that if a patient &# 39 ; s tumor was sensitive to a drug in vitro , the chance of that patient responding clinically to the drug was 81 %, while if the patent tumor was resistant to the drug in vitro , the chance of the patient not responding to the drug was 93 % ( salmon , 1978 ). in an in vitro - in vivo correlation study involving 800 patients retrospectively , it was determined that the true positive rate for this assay was 70 %, while the true negative rate for the assay was 98 % ( von hoff et al ., 1981 ). in a more recent prospective clinical trial of the cloning system involving 604 patients , the percent true positive for the assay was 64 %, while the percent true negative for the assay was 86 % ( von hoff et al ., 1983 ). these studies have demonstrated that in vitro predictive assays can improve the response rate of patients . this system was used to study the response of compound c1 for breast cancer and non - small cell lung carcinoma . collection and preparation of tumor cells : after obtaining informed consent in accordance with federal and institutional guidelines , malignant effusions , ascites and bone marrow aspirates containing tumor cells as well as solid tumor specimens were collected from patients undergoing procedure carried out as a part of their diagnostic workup or as part of treatment for their disease . solid tumors or lymph nodes were minced into 2 to 5 mm fragments in the operating room and immediately placed in mccoy &# 39 ; s medium 5a ( gibco , grand island , n . y .) containing 10 % heat - inactivated newborn calf serum plus 1 % penicillin / streptomycin . within four h , these solid tumors were mechanically disassociated with scissors , forced through no . 100 stainless steel mesh , through 25 gauge needles , and then washed with mccoy &# 39 ; s medium as previously described ( hamburger & amp ; salmon , 1977 , a , b ; salmon , 1978 , von hoff et al ., 1981 , 1983 , 1990 ). ascites , pleural , pericardial fluids and bone marrow were obtained by standard techniques . the fluid or marrow was placed in sterile containers containing 10 units of preservative - free heparin per ml of malignant fluid or marrow . after centrifugation at 150 × g for 10 minutes , the cells were harvested and washed with mccoy &# 39 ; s medium plus 10 % heat inactivated fetal calf serum . the viability of cell suspensions thus obtained was determined with trypan blue . tumor cells to be cloned were suspended in 0 . 3 % agar in enriched cmrl 1066 medium ( gibco , grand island , n . y .) supplemented with 15 % heat - inactivated horse serum , penicillin ( 100 units / ml ), streptomycin ( 2 mg / ml ) glutamine ( 2 mm ), insulin ( 3 units / ml ), asparagine ( 0 . 6 mg / ml ), and hepes buffer ( 2 mm ). different concentrations of compound c1 were added to the above mixture and cells were plated in 35 mm petri dishes in a top layer of agar over an underlayer of agar to prevent growth of fibroblasts . three plates were prepared for each data point and incubated at 37 ° c . after an incubation period of 14 days , plates were removed and the number of colonies were counted in each plate . the number of colonies ( defined as & gt ; 50 cells ) formed were compared in drug treated and untreated ( control ) plates and the percent colonies surviving at each drug dose tested was then calculated as a percentage of control . table 6______________________________________activity of c1 in a human tumor cloning system # responsive tumors / total # evaluatedtumor type 75 μg / ml 100 μg / ml 125 μg / ml______________________________________breast 3 / 8 4 / 8 6 / 8lung carcinoma 0 / 1 1 / 1 1 / 1 ( non - small cell ) ______________________________________ in the above table , a responsive tumor is defined as one where at least 50 % inhibition of tumor colony formation occurred . the data indicate a very high probability of successful inhibition of breast tumor and lung carcinoma with compound c1 . in order to determine the in vivo toxicity of compound c1 , balb / c mice were orally administered a single escalating dose of c1 . a single escalating dose of the test drag is a procedure where a dose is administered to a group of mice ( in this case n = 3 ). if toxicity is not elicited , the next higher dose is given to a fresh group of mice and so on until toxicity is observed . in preliminary repeat dose studies , a suspension of c1 in polyethylene glycol ( c1 is mixed with a 2 . 5 % solution of polyethylene glycol and homogenized in a sterile dounce homogenizer until it flows freely through a 22 × 11 / 2 &# 34 ; with 11 / 4 mm ball feeding needle ) was administered orally to a group of three balb / c mice . a daily dose of 400 mg / kg given for 10 days was easily tolerated and all animals survived the treatment . at the dose of 800 mg / kg , one animal died on day three and another died on day 10 . at the dose of 1000 mg / kg , one animal died on day 2 , another on day 5 and a third animal died on day 10 . at the dose of 1200 mg / kg , all animals died on day 3 . a single dose of up to 1200 mg / kg was easily tolerated . however , a single dose of 2000 mg / kg was lethal . a parenteral dose may be from about 1 μg / kg to about 500 mg / kg . the anti - viral activity of compound c1 was determined as follows ; a stock of herpes simplex virus ( hsv ) was mixed with different concentrations of compound c1 and incubated for 24 h at 37 ° c . the control sample received vehicle only . after the incubation period , samples were serially diluted and 0 . 1 ml of each dilution and the undiluted samples were inoculated into monolayers of vero cells ( african green monkey kidney cells ). overlay medium was added and the monolayers were incubated at 37 ° c . after 3 days of incubation , the overlay medium was removed , and the monolayer cells were fixed with methanol and treated with giemsa stain . plaques were counted and results are shown in table 7 . table 7______________________________________effect of compound c1 on the inactivation of herpes simplex virusdose of c1 * pfu / ml % inactivation log reduction______________________________________0 7 . 1 × 10 . sup . 5 0 . 0 0 . 030 μg / ml 2 . 1 × 10 . sup . 5 70 . 4 0 . 5360 μg / ml l . 5 × 10 . sup . 5 78 . 9 0 . 68120 μg / ml 6 . 5 × 10 . sup . 5 99 . l 2 . 04150 μg / ml 7 . 5 × 10 . sup . 5 99 . 9 3 . 00______________________________________ * pfu = plaque forming units . these results show that a near complete inactivation ( 99 . 9 %) of herpes simplex was obtained in a dose - dependent manner . in a further study , cell free human immunodeficiency virus ( hiv - 1 ) was treated with a range of concentrations of compound c1 . after 2 h at 37 ° c ., treated and control hiv - 1 samples were added to the indicator mt - 4 cells ( cultured human t cells ) in the presence of 2 μg / ml polybrene ™ for 2 h . after washing , mt - 4 cells were cultured in the presence of drug - treated or control ( untreated ) hiv - 1 . cell viability of mt - 4 cells was determined by the trypan blue dye exclusion method . the results are presented in table 8 . table 8______________________________________effect of compound c1 on the inactivation of cell free humanimmunodeficiency virus dose % inactivationcell type ( μg / ml ) day 7______________________________________mt - 4 cells 0 0 ( uninfected ) mt - 4 + hiv - 1 0 0mt - 4 + hiv - 1 75 7 . 7mt - 4 + hiv - 1 100 34 . 1mt - 4 + hiv - 1 125 62 . 6______________________________________ these results show that even a 2 h period of treatment at 125 μg / ml provided a 62 % inactivation of the virus on day 7 as compared to essentially complete killing of cells by the untreated hiv - 1 in the control culture . under these conditions , the drug doses were not toxic to the mt - 4 cells . the effect of compound c1 on the modulation of t cells was studied . after obtaining informed consent , three human hiv - 1 positive volunteer subjects received compound c1 at a dose of 4 . 3 mg / kg for a period of 15 days . blood samples were drawn before and after on day 15 or 30 from the initiation of treatment for regular cbc profile . data from one subject showed that the t cell count increased from 425 to 720 . the normal range of t cells is 650 - 1330 . these subjects also reported an alleviation from lethargy , depression and an increased appetite , feeling of being happy , and desire to live . the effect of compound c1 on growth of human tumor cell lines maintained by the national cancer institute ( bethesda , md .) as standard cell lines for screening compounds for cytotoxicity is provided in the present example . the national cancer institute carried out the studies at the direction of the present inventor . in the tests , the national cancer institute used the srb ( sulforhodamine b ) assay as described by skehan et al ., ( journal of the national cancer institute , 82 : 13 , jul . 4 , 1990 , 1107 - 1112 ). briefly , the assay is as follows . tca - fixed cells were stained for 30 minutes with 0 . 4 % ( wt / vol ) srb dissolved in 1 % acetic acid . at the end of the staining period , srb was removed and cultures were quickly rinsed four times with 1 % acetic acid to remove unbound dye . the acetic acid was poured directly into the culture wells from a beaker . this procedure permitted rinsing to be performed quickly so that desorption of protein - bound dye did not occur . residual wash solution was removed by sharply flicking plates over a sink , which ensured the complete removal of rinsing solution . because of the strong capillary action in 96 - well plates , draining by gravity alone often failed to remove the rinse solution when plates were simply inverted . after being rinsed , the cultures were air dried until no standing moisture was visible . bound dye was solubilized with 10 mm unbuffered tris base ( ph 10 . 5 ) for 5 minutes on a gyratory shaker . optical density ( od ) was read in either a uvmax microtiter plate reader ( molecular devices , menlo park , calif .) or a beckman du - 70 spectrophotometer . for maximum sensitivity , od was measured at 564 nm . because readings were linear with dye concentrations only below 1 . 8 od units , however , suboptimal wavelengths were generally used , so that all samples in an experiment remained within the linear od range . with most cell lines , wavelengths of approximately 490 - 530 nm worked well for this purpose . nine different non - small cell lung carcinoma cell lines , six different colon carcinoma cell lines , six different cns ( central nervous system ) carcinoma cell lines , seven different melanoma cell lines , six different ovarian carcinoma cell lines , and eight different renal carcinoma cell lines were tested . the results are provided in table 9 . table 9______________________________________effect of compound c1 on the inhibition of growth of nci tumor celllines . percent growthcarcinoma 10 . sup . x molar compound c1cell line x = - 7 . 0 x = - 6 . 0 x = - 5 . 0 x = - 4 . 0 x = - 3 . 0______________________________________non - smallcell lungcancera549 / atcc 98 95 95 67 - 98ekvx 98 106 104 94 - 69hop - 62 98 99 96 93 - 100hop - 92 104 110 104 51 - 70nci - h226 96 100 106 90 - 39nci - h23 97 103 98 63 - 62nci - h322m 145 144 136 127 - 100nci - h460 99 100 97 82 - 54nci - h522 100 102 104 47 - 82coloncancerhcc - 2998 113 80 109 76 - 82hct - 116 109 111 118 48 - 100hct - 15 99 102 102 72ht29 102 103 90 76 - 96km12 101 101 102 76 - 90sw - 620 110 100 99 85 - 39cns cancersf - 268 98 100 93 52 - 58sf - 295 104 100 100 47 - 95sf - 539 95 104 101 72 - 100snb - 19 105 99 109 90 - 54snb - 75 106 113 120 83 - 65u251 104 111 102 54 - 55melanomalox imvi 101 106 102 65 - 100malme - 101 101 101 81 - 833mm14 106 100 105 94 - 100sk - mel - 2 110 106 100 87 - 55sk - mel - 28 110 111 108 69 - 75uacc - 257 102 102 96 75 - 77uacc - 62 94 91 88 53 - 79ovariancancerigrov1 105 107 103 88 - 85ovcar - 3 110 114 110 89 - 93ovcar - 4 101 104 105 66 - 75ovcar - 5 102 98 99 97 - 97ovcar - 8 102 106 108 81 - 49sk - ov - 3 95 90 92 85 - 100renalcancer786 - 0 82 115 87 - 100a498 99 105 96 62 - 81achn 99 105 107 89 - 3caki - 1 103 85 73 92 - 100rxf 393 93 93 86 43 - 85sn12c 90 58 89 62 - 42pk - 10 94 94 98 74 - 19uo - 31 92 99 92 63 - 75______________________________________ data of table 9 demonstrate that compound c1 causes cell kill to all listed cell lines at concentrations of greater than about 10 - 4 m . the present data on cytotoxicity of compound c1 extend the in vitro and in vivo data of examples 1 , 3 , 4 , and 6 to five further types of carcinoma cell lines in a total of 33 further tests used by the national cancer institute as standard lines for screening compounds for cytotoxicity . all of the compositions and methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure . while the compositions and methods of this invention have been described in terms of preferred embodiments , it will be apparent to those of skill in the art that variations may be applied to the composition , methods and in the steps or in the sequence of steps of the method described herein without departing from the concept , spirit and scope of the invention . more specifically , it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved . all such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit , scope and concept of the invention as defined by the appended claims . the following references , to the extent that they provide exemplary procedural or other details supplementary to those set forth herein , are specifically incorporated herein by reference . altman , r . f . a . and l . g . spoladore , archiv fur geschwulstforschung 36 : 3 , 207 - 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( india ), 21 : 227 , 1962 . saratikov et al ., izv . sib . otd . nauk ssr biol . nauk ., 2 , 147 , 1973 . tabern , d . l . and volwiler , e . h ., j . amer . chem . soc ., 57 : 1961 , 1935 . toth et al ., ( alkaloida vegyeszeti gyar ), hung , teljes hu 19767 ( cl . c07d239 / 62 ), apr . 28 , 1981 . von hoff , d . d . et al ., j . natl . cancer inst . 82 : 110 - 116 , 1990 .