the application of antioncological drugs in human patients has enabled great progress to be made in the treatment of common malignant tumors such as breast , colon and lung carcinoma 1 , 2 , 3 , 4 , 5 !. however , results are as yet limited . one of the main drawbacks is the incomplete destruction of micrometastases not removable at primary surgery . it has been shown that when masses of atypical cells undergoing the avascular growth phase reach 150 to 500 μm in diameter 6 !, they present a decrease in the diffusion of nutrient or toxic components towards the center of microspherules . during this phase central cells are still viable and enhance with tritiated thymidine ; in contrast , radiolabelled chemotherapeutic drugs such as methotrexate 7 ! fall to reach the core of the lesion . thus , the diffusion gradient becomes a limiting factor so that greater active drug concentration is required in the intercellular space . it is precisely at this point where the main limitation of chemotherapy appears , as its toxic effects on bone marrow , kidney , digestive mucosa and liver prevent indiscriminate application . progress has been achieved in the application of massive doses with the help of hematopoietic stimulating factors ( g - csf and gm - csf ) which allow bone marrow recovery following high - dose chemotherapy 8 , 9 , 10 !. despite this accomplishment , barely 5 , 000 mg / m 2 of cyclophosphamide can be administered , which is equivalent to 6 or 7 times the usual dose 8 , 9 , 10 !, with some decrease in bone marrow toxicity . in the present study , doses were raised to 10 or 15 times the habitual level of the three associated drugs , by means of a procedure which does not alter the efficacy of the drugs on atypical cells and protects all affected organs . the chemotherapeutic combination chosen for this purpose was cyclophosphamide , methotrexate and 5 - fluoruroacil ( cmf ). on the basis of data gleaned from the literature , neither oligoelements nor phospholipases have been considered to modify significantly the action of chemotherapeutic drugs on atypical cells 11 - 14 !. to illustrate , selenium ( se ) has been reported incapable of producing in vitro protection of atypical fibrosarcoma cells against antineoplasic drugs 13 !. likewise , tobey &# 39 ; s work 15 , 16 ! on zinc ( zn ) and se show that the in vitro protective effect on normal cells is not significant in the case of neoplasic cells . cmf doses of 500 mg / m 2 , 40 mg / m 2 and 600 mg / m 2 , respectively , are regarded as habitual . as already stated , these drugs were chosen for being the most commonly employed association for human breast cancer 1 , 2 , 17 , 18 , 19 !. as protective elements for normal cells , the oligoelements zn , se and mn were used on the basis of plentiful data . for instance , in the case of zn alone , its protective in vitro function against the effect of alkylating agents in cell cultures 15 , 16 ! enabled the application of 10 times the chemotherapeutic dose in pretreated versus non - pretreated cultures , and up to 16 times such dose { when } associated with se and cu . the mechanism of action would appear independent of metallothionein synthesis , a protein capturing heavy metals 16 !, which has been demonstrated for the protection afforded by pre - treatment with zn prior to cis - platin application 11 , 20 !. in the case of alkylating agents , zn acts through an increase in glutathion content and glutathion - s - transferase activity 20 !. it has been observed that weight loss induced by cyclophosphamide in rats may be reversed by addition of cysteine employed to produce glutathion 12 , 20 !. likewise , hemorrhagic cystiris caused by acroleine ( a cyclophosphamide metabolite ) is also inhibited by injection of cysteine and glutathion 21 !. these same components are employed to prevent cyclophosphamide teratogenesis in pregnant rats 22 !. however , the therapeutic efficacy of the drug remains unaltered by such protective effects 12 , 21 !. as regards methotrexate , zn - stimulated glutathion action is associated to transport of the drug within cells 23 !. se also exerts an effect on glutathion metabolism by increasing the enzymatic activity of se - dependent glutathion peroxidase 24 , 25 , 26 !, which prevents the effects of carcinogens and of alkylating agents . interestingly , such enzymatic increase takes place selectively in certain organs ( kidney , colon mucosa and small bowel ), which may explain the prevention of toxicity in certain tissues 27 !, a finding confirmed by the reduction in renal toxicity of cis - platin exerted by se 13 !. besides , it has been shown that less dna chain fractures are induced by carcinogens following se pre - treatment 28 !. both zn and se are able to inhibit carcinogenesis secondary to antineoplasic drugs employed in human patients and to the most common carcinogens used in experimental animals 13 , 16 , 24 , 25 , 26 , 29 - 31 !. for its part , mn acts as a superoxide dismutase catalyst in the prevention of cell damage induced by oxidizing agents , particularly the superoxide ion 32 , 33 !. it has been demonstrated that glutathion depletion due to gamma - glutamyl cysteine synthetase inhibition blocks the production of gamma - interferon secreting cells by roughly 80 % 34 !. the prevention of this process by means of zn and of se may help to improve the immune response in animals under treatment with alkylating agents . the inclusion of phospholipase a 2 in our schedule was due to the fact that it makes up the association originally used with success for the control of experimental rat mammary tumors induced by n - nitroso - methylurea 35 !. studies carried out in our laboratory show that the combination of oligoelements , zn , se and mn , plus phospholipase a 2 ( op ) has a satisfactory effect on sprague - dawley rats with experimentally induced mammary tumors , achieving prolonged survival , improvement in general status , decrease in tumor size and body weight increase 35 !. on the basis of the foregoing background , in vivo tolerance studies were performed in sprague - dawley rats to systemic treatment with the cmf combination , by administering continuous op medication starting one month prior to the application of various cmf dose schedules . sodium selenite , magnesium chloride , manganese chloride , zinc chloride , cobalt powder , cerium powder , molibden powder and silicon powder are drugs of analytical grade and purchased from aldrich chemical co ., usa . phospholipase a2 is a purified fraction from venom of crotalus dussirus terrorificus and from lachesis muta . in a first step , stock solutions of each oligoelement are prepared in destilled water . the concentration of these solutions is 4 × 10 - 3 g / ml . salts are directly dissolved in destilled water ; powders are previously treated as follows : silicon : 50 ml of naoh ( 10 % w / v ) are added to 4 gr . of powder with continuous stirring at 100 ° c ., during 30 minutes . complete dissolution is obtained after 15 to 20 hs at room temperature . the solution is filtered and destilled water is added to complete a final volume of 1 liter . molybdenum : 100 ml of no 3 h ( 10 % v / v ) are added carefully to 4 gr . of powder with continuous stirring at room temperature until complete dissolution . the solution is filtered and destilled water is added to complete a final volume of 1 liter . cobalt / cerium ; 100 ml of clh ( 10 % v / v ) are added to 4 gr . of powder , with continuous stirring until complete dissolution . the solution is filtered and destilled water is added to complete a final volume of 1 liter . the stock solution containing 10 - 4 g / ml of phospholipase a2 is prepared in 50 mm sodium phosphate buffer , ph 7 . this solution is stored at - 20 ° c . the final solution is prepared mixing aliquots from the different stock solutions of the oligoelements and adding destilled water in order to obtain a final concentration of 4 × 10 - 6 g / ml for each component . the phospholipase a 2 is added to a final concentration of 4 × 10 - 9 g / ml . the ph of the solution must be adjusted to 7 . 2 - 7 . 4 with phosphate buffer before adding the phospholipase . 1 ml of the solution is administered daily by sub - cutaneous injection . eighteen batches of sprague - dawley strain rats were employed to determine the ld 50 of the most commonly employed combination for the treatment of human breast tumors , consisting of cyclophosphamide , methotrexate and 5 - fluoruroacil ( cmf ). once mortality curves had been traced , animal batches were given oligoelements ( se , zn and mn ) plus phospholipase a 2 , followed 15 days later by cmf administration . a clearcut significant shift towards the fight was observed in survival curves ( p & lt ; 0 . 001 ) with a cmf ld 50 dose equivalent to roughly 18 , 3 times the dose given to humans . all animals were necropsied at the time of death or when killed two months after cmf administration . anatomopathological studies disclosed renal tubule lesions , hepatic centrolobular steatosis and diverse degrees of bone marrow aplasia . rats not protected with oligoelements and phospholipase a 2 presented more severe lesions such as grade iii bone marrow aplasia , greater incidence of massive sepsis , acute renal tubule necrosis and greater incidence of malignant tumors ( 37 . 5 % of chronic myeloid leukemia ). protected survivors displayed ad integrum recovery from the described anatomopathological lesions . such results afford the possibility of applying this treatment to human patients subjected to high - dose chemotherapy , in order to improve their tolerance . roughly 4 - month - old male sprague - dawley rats , inbred in our laboratory and weighing 380 - 420 g at the start of the experiments , were used . animals were kept in groups of 5 each per cage , with water and food ad libitum , temperature at 22 °- 23 ° c ., humidity at roughly 56 % and 12 hours light cycle . body weight was monitored every other day . throughout , batches of rats protected and unprotected with op medication were available . treatment with chemotherapeutic drugs ( cmf ): for tolerance studies , a combination of cyclophosphamide , methotrexate and 5 - fluoracil ( cmf ), ( the most commonly administered for human mammary carcinoma 1 , 2 , 17 , 18 , 19 ! ), was given as a single dose . the lowest cmf dose ( indicated as 2 - fold ) was calculated by extrapolating the double value of that used in human patients , namely , cyclophosphamide 500 mg / m 2 , methotrexate 40 mg / m 2 and 5 - fluoracil 600 mg / m 2 . the range of doses employed were multiples of the latter and their expression was 4 -, 10 -, 12 -, 14 -, 16 -, 18 - and 20 - fold , respectively . cytostatic multidrug injections were delivered by intraperitoneal route . protection with oligoelements and phospholipase a 2 ( op ): each rat receiving op protection was injected daily , starting 10 days before cmf administration ; with a solution of oligoelements zn , se and mn ( 0 . 15 μg / 100 g body weight and phospholipase a 2 ( 10 ng / 100 g body weight ), supplemented with phosphatidyl - inositol and phosphatidyl - choline in buffered vehicle , as described elsewhere 14 , 15 !. the op combination was administered concentrated in a 0 . 5 - ml volume by subcutaneous route and 0 . 1 ml by oral route , during a maximum of 70 days . protection protocol : all batches consisted of 8 rats each , with a total of 128 animals . batches 1 , 3 , 5 , 7 , 9 , 11 , 13 and 15 received 2 -, 4 -, 10 -, 12 -, 14 -, 16 -, 18 - and 20 - fold cmf , respectively , while batches 2 , 4 , 6 , 8 , 10 , 12 , 14 and 16 received op medication starting 10 days earliest as indicated above , besides the given range ( 2 - to 20 - fold ) of cmf doses . parameters recorded : the following studies were carried out on all batches : a ) body weight monitoring ( every other day ); b ) observation of collateral macroscopic effects including changes in fur coloration , hair loss and nasal and gut hemorrhage ; c ) ld 50 determination ; and d ) necropsy to determine the cause of death in every case and for histopathological study of organs and tissues . all animals were autopsied at the time of spontaneous death or when killed 60 days after cmf administration . viscerae block and the sixth dorsal vertebrae were removed and immediately fixed in 10 % formaldehyde buffer . after careful macroscopic study , specimens of lung , liver , heart , kidney , spleen , genitalia and bone marrow were harvested for histological study . tissues were imbedded in paraffin and stained with hematoxylin - eosin , giemsa , periodic acid - schiff ( pas ) and other special techniques as required . survival curves for control and protected batches of rats , for each dose assayed , were fitted by the kaplan - meier method 36 !, then compared by means of the mantel - cox 37 ! and wilcoxon 38 ! tests , to render compatible results throughout . effective ld 50 doses were estimated for both control and protected batches by the litchfield - wilcoxon method 39 !. anatomopathological lesions found mainly involved bone marrow , kidney and , to a much lesser extent , liver . besides , they differed remarkably according to the time of death . thus , in rats unprotected found dead from 7 to 12 days ( 95 % mortality ) after administering cmf , the severity of bone marrow aplasia found in 100 % ranged from grade ii to iii ( fig8 ) and generalized acute sepsis with bacterial colonies was evident in one or more studied organs ( 80 %) ( fig1 , 11 ), while acute renal tubular necrosis ( 60 %) ( fig1 ) and centrolobular hepatic lesions were relatively slight ( 40 %) ( fig1 ). a second group comprising 5 % of unprotected rats died spontaneously after 25 days / at 13 - 25 days of cmf application . in this group anatomopathological lesions were scarce , without evidence of bone marrow aplasia , but with kidney lesions or sepsis in some cases . protected animals displayed differences in the cause of death versus the control group , which proved more noteworthy with certain critical cmf doses . at the 16 - fold cmf dose , 80 % of controls died of sepsis , whereas barely 20 % of op - protected rats in this group showed signs of sepsis . for the same dose , 80 % of unprotected animals presented bone marrow aplasia , whereas only 10 % of protected rats displayed this lesion at the time of death . in studies performed on survival animals killed at 60 days , practically all controls exhibited myeloid leukemia in bone marrow ( fig1 , 15 ). in contrast , none of the protected rats did so , showing normocellular or slightly hypercellular bone marrows . on the whole , in control groups failing to present aplasia at the time of death , a great percentage displayed chronic myeloid leukemia ( fig1 ), which in one case proved blastic in nature ( fig1 ). in contrast , none of the op - protected rats in any group or for any dose presented leukemia . on the basis of the foregoing studies , it was found that ld 50 for unprotected control rats was 14 . 1 - fold the basic one versus 18 . 3 - fold for op - protected animals , indicating a great shift in tolerance to cytostatic treatment which would enable therapeutical doses to be raised with a satisfactory safety margin . recalling that a 14 - fold dose failed to cause deaths in protected groups in contrast to 50 % mortality in controls , the basal dose might well be increased 2 - or 3 - fold without appreciable danger . furthermore , with the 16 - fold dose mortality was 20 % in op - protected batches , where 80 % of deaths were due to hemorrhage related to bone marrow alterations and associated to acute renal tubular necrosis , collateral effects liable to be controlled in humans by transfusion of platelets and clotting factors . it is worthwhile stressing the two chronological phases of spontaneous death by chemotherapy in rats not protected with op . the first phase was around 10 days and due to sepsis in 80 % of cases , associated almost invariably to acute renal tubular necrosis and bone marrow aplasia : barely 20 % of unprotected animals died of acute renal tubular necrosis or bone marrow aplasia , remaining free of sepsis . a second phase of deaths was discernible at 15 - 25 days , due to causes poorly evidenced morphologically , but most likely of a central circulatory nature ( cardiac , of large vessels or circulatory function ), with a recovered bone marrow on occasion hyperplasic , splenic myelopoiesis and frequent chronic myeloid leukemia in bone marrow . a certain degree of chronic metabolic failure , whether hepatic or renal , cannot be ruled out during this second stage . in op - protected animals , death due to generalized sepsis at 8 - 12 days decreased drastically from 80 to 20 %, and mortality was mainly attributable to acute renal tubular necrosis and bone marrow aplasia during this first phase . such finding strengthens the hypothesis of an immunostimulating action exerted by the oligoelements , already described in the literature 34 , 40 ! and which the present authors have reported for the treatment of experimentally - induced rat tumors 35 !. in protected animals , no deaths were recorded in the second phase thats its 25 days post - cmf . at the time of spontaneous death or when killed , unprotected control rats invariably presented either of two bone marrow pathologies : bone marrow aplasia in the earlier phase or chronic myeloid leukemia , at times biastic in nature , in the later phase . in contrast , there were no cases of myeloid leukemia in treated groups , thus demonstrating the protective effect of op medication against the carcinogenic action of the chemotherapeutic agents employed . such beneficial effect seems mainly attributable to selenium and to zinc 13 , 16 , 24 , 25 , 26 , 29 , 30 , 31 !. likewise , the inhibitory action of preventive op protection has also been shown against chemical carcinogenesis induced by nmu in rats paper in progress !. admittedly , a crucial objection which may be raised concerns the possible inhibition of destructive effects on neoplasic cells caused by protective oligoelements . this issue has been dealt with by numerous authors , to reach the conclusion that the antineoplasic activity of chemotherapy is not significantly affected 11 , 12 , 13 , 14 !. though somewhat paradoxical , this phenomenon is partly explained by the diverse sensitivity towards oligoelements displayed by transformed as compared to normal cells . as already pointed out , selenium is incapable of protecting atypical cells in vitro against antineoplasic drugs 13 ! and zinc exhibits a similar behavior 15 , 16 !. it is also likely that oligoelements may exert a different effect on neoplasic versus normal cells mainly because of subsequent metabolic changes during cell transformation . thus , it has been found that in vitro selenium exerts opposite effects on camp metabolism , in the presence of murine hepatoma cells or normal hepatocytes , with the former undergoing a marked increase in camp 41 , 42 !. such effects leading to growth inhibition of atypical cells 43 ! are probably due to differences in phosphodiesterases present in either cell type . bearing in mind the peculiar blood supply features of metastasic microspheres , it may be conjectured that the poorly irrigated center of the tumoral mass is free of the penetration of chemotherapeutic agents up to quiescent cells 7 , 44 , 45 !. instead , the possibility of trebling the therapeutic dose would allow increasing the concentration gradient at the tumoral border , thus favoring penetration up to the center of microspheres . in the present study we have clearly established the protective effect of treatment with phospholipases and oligoelements on normal rats receiving large doses of chemotherapeutic agents . previously , we had shown the therapeutical effect of this combination on animals with experimentally - induced mammary tumors 43 !, as well as the preventive action on carcinogenesis 44 !. in a further stage , it is therefore essential to carry out a combined study with chemotherapeutic agents on animals with experimental tumors , which will enable the confirmed protective action to be evaluated and determine whether the proposed medication positively modifies the antineoplasic effect of chemotherapy . overall , our results show improved tolerance to the toxic effects of the chemotherapeutic drugs assayed , by means of combined treatment with phospholipase a 2 and oligoelements . such low - cost medication , lacking any deleterious collateral effects and having confirmed immunostimulating and antineoplasic activity , thus represents a useful clinical tool . to sum up , the present studies on tolerance to cytostatic drugs by male sprague - dawley rats allow the following conclusions to be drawn : animals injected intraperitoneally with cmf ( cyclophosphamide , methotrexate , 5 - fluoruracil ), in doses equivalent to those received by human patients with mammary tumor ( cyclophosphamide 500 mg / m 2 , methotrexate 40 mg / m 2 and 5 - fluoruracil 600 mg / m 2 ), showed a drop in body weight as from the day following administration of the combined drugs , and proving more marked as a greater cmf dose was employed . toxic macroscopic effects became evident as the assayed dose was increased , as a multiple of the basic dose indicated above . at doses greater than 10 - fold the basic dose , nasal and gut hemorrhage appeared to a diverse degree starting on the day after cmf administration , correlating closely with the dose . as from the 10 - fold dose there was also an obvious increasingly brownish fur coloration , whose degree likewise correlated with the cmf dose . animals protected with op ( oligoelements plus phospholipase a 2 ) presented similar features to those already described . however , body weight recovery proved statistically faster in op - protected rats than in unprotected controls . cmf proved lethal for unprotected control animals as from the 10 - fold dose . the estimated ld 50 was equivalent to 14 . 1 times the basic dose . such lethal action became evident in rats protected daily with op medication starting from 14 times the basic dose , the estimated ld 50 for treated batches being 18 . 3 times the basic dose . the anatomopathological findings showed that protected animals suffered significantly less generalized sepsis and medullar aplasia . also was stated a protective effect on chronic mielloyd leukemia post - chemotherapy in long term survival animals . these findings imply a better tolerance to the toxic effects of the chemotherapeutic drugs assayed , when supplemented with the combined medication of oligoelements and phospholipase a 2 , since the ld 50 increased significantly in protected rats versus unprotected controls . such correlation provides a valuable therapeutic tool , particularly as the assayed drugs are routinely employed for the treatment of human mammary carcinoma and the doses assayed in the animals are equivalent to those used in oncological medicine . in the present work twenty one patients were studied , who underwent hemicholectomy owing to colonic carcinomas , and who since surgery developed liver metastases confirmed by biopsy or by reliable imaging . diagnostic procedures . metastases could not be treated by surgical methods due to their number , localization , size or poor general condition of the patients . due to different causes , age , general condition , intolerance or refusal to being treated , none of them received complete chemotherapy . based on previous reports ( 1 , 2 , 3 , 4 , 5 , 6 , 7 , 8 , 9 , 10 , 11 , 12 ), different combinations of zinc , selenium and manganese , with the addition of phospholipase a 2 , were assayed with tumor cells cultured in vitro . after wards they were tested in rats bearing chemically induced mammary carcinomas with an excellent therapeutic response . the patients were treated with the best combination determined by experimental assays . because of the good evolution observed , the treatment continued for diverse periods , up to 36 months , and six complete remissions , five partial remissions , stabilization in six cases and progression in four were recorded . in all such cases the common feature was an excellent survival condition . in order to investigate whether oligoelements and phospholipase a 2 may exert a modulatory action on tumor cell growth and proliferation , in vitro studies were performed employing the clonogenic agar technique which allows differential growth of transformed cells . tumors were obtained by intraperitonal ( i . p .) injection of three 50 mg / kg doses of n - nitroso - n - methylurea ( nmu ) to inbred sprague - dawley female rats , aged 50 , 80 and 110 days , as described previously ( 12 , 40 , 41 , 42 ). neoplasms were histopathologically classified as mammary duct carcinomas . tumors for in vitro experiments were surgically obtained during the exponential growth phase having a diameter no greater than 2 cm . tumor specimens were carefully trimmed and cut into 1 - 2 mm pieces , washed twice and digested enzymatically at 37 ° c . for 20 min with pronase - dnase . the supernatant of the digestion was carefully extracted and centrifuged at 1000 rpm for 3 min . the cellular pellet was washed three times with hanks &# 39 ; solution and then resuspended in tissue culture medium to a final concentration of 2 × 10 6 cells / ml . viability was assessed by trypan blue dye exclusion test . a single cell suspension from tumoral tissue was cultured in soft agar under serum free medium conditions . approximately 4 - 5 × 105 cells were plated per well . the bottom layer consisted of mccoys &# 39 ; a medium , adequately enriched with a final agar concentration of 0 . 5 %. the upper layer contained the the cellular suspension and 0 . 3 % of agar . multiwells were incubated at 37 ° c . in an humidified atmosphere of 5 % of co 2 in air . the number of colonies on day 21 after plating was the parameter used to evaluate the mitogenic or inhibitory effect of the different drugs . the three various oligoelements ( mn , se and zn ) were tested adding 20 μl of each dilution per well . dose - response curves were traced with each point run in quadruplicate . results were expressed as percentage of inhibition calculated as the relation between the number of colonies developed for each treatment and the number of colonies in the control well . combination of oligoelements plus phospholipase a 2 at different concentrations . combination of oligoelements at the most effective dilutions plus phospholipase a 2 at different concentrations . patients were selected taking into account their exclusion from other therapeutic schedules , whether surgical due to the multiplicity of liver metastases , chemotherapeutic because of age , general condition , intolerance or rejection of treatment . these criteria were followed in order to ensure the greatest reliability in the analysis of results . twelve of the patients ( 57 %) were males , whose ages ranged from 60 to 84 years with a mean value of 69 . 5 years ( table 1 ). out of the 21 selected patients , only three had previously received a single , incomplete , chemotherapeutic cycle and all discontinued treatment after the first dose because of intolerance or by personal decision . moreover , chemotherapy had been given at least one year before inclusion / admision : on . patients with clinical evidence of disease progression or with an increase in oncological marker serum levels , were not excluded from this study . ten of the patients presented a diagnosis of metastatic disease at baseline ( table 1 ). a further six developed metastases during the first year of illness and the remainder , one year later . eighty percent of the patients were in duke &# 39 ; s c or d stage at the time of diagnosis of the primitive tumor , 15 % were in b stage and 5 % in d stage . in eight patients liver metastases were confirmed by biopsy and in the remainder on the basis of repeated and progressive imaging studies of diverse complexity . all treated patients had histological confirmation of the primitive tumor . most cases corresponded to moderately differentiated adenocarcinomas ( 43 %, table 2 ); 15 % were classified as well differentiated , with only one case scarcely differentiated , while the remainder failed to exhibit recognizable typification . in twelve cases metastasis localization was diffuse , while in the remainder foci were located in the right hepatic lobe ( table 1 ). only three of the patients had undergone surgery one year before starting the treatment , but by the time they entered this trial , they were in relapse and the disease was in progression . metastasis size ranged from 2 cm , in cases of multiple secondary lesions , to a maximum of 15 cm in patients with greater progression ; while the mean value was 5 . 5 cm ( table 1 ) results obtained showed that the combination of oligoelements led to a significant dose - dependent inhibition of cell proliferation . maximal inhibition was produced by dilutions lower than 10 - 3 of the main solution and reached 60 %; ed 50 was 10 - 4 ( fig1 ). independently , phospholipase a 2 presented a dose - dependent antagonic behaviour . at dilutions lower than 10 - 5 , it produced a slight inhibition in the formation of colonies , while at concentrations bigger than to 10 - 6 it showed a stimulating effect on cellular proliferation . combined treatment with oligoelements and phospholipase showed similar inhibitory effects to the one produced by oligoelements alone . a dose dependent inhibition was observed with a maximum of 60 % as from the 10 - 5 dilutions and with ed50 at the 10 - 4 dilution ( fig3 ). the combination of oligoelements in inhibitory concentrations with phospholipase in stimulating dilutions showed growth inhibition slightly greater than the combination of both in equal concentrations ( fig3 ). these results allowed to extrapolate the corresponding dose for the treatment of sprague - dawley rats with mammary nmu - induced carcinomas , obtaining excellent results with a significant rise in survival rate ( 12 ). these doses agreed with the ones applied by other authors in in vitro studies with zn and se to evaluate the protective differential effect on normal cells produced by these elements before treatment with alkaline agents ( 3 )( 8 ). the treatment of sprague - dawley rats with this combination of phospholipase and oligoelements initiated prior to the administration of carcinogen produces a significant decrease ( 80 %) in the development of mammary tumors ( unpublished data ). similar effects on carcinogenesis inhibition have been reported for se ( 2 )( 5 )( 10 ). toxicity studies on spague - dawley rats applying 10 , 100 and 500 times the therapeutic dose for a prolonged time evidenced neither toxicity nor variations in the survival rate of the animals . besides , no morphological lesions were found at necropsy autopsy ( 12 ). other authors have described similar results in toxicity studies with se ( 7 ) and zn ( 45 ). for the treatment of human patients , 1 . 5 μg / kg daily doses of zn , se and mn were given together with 0 . 10 ug / kg of phospholipase a 2 by parenteral and sublingual routes . these doses were obtained by extrapolation of in vitro studies on cellular cultures and in vivo studies on experimental tumors . in all twenty one patients included in this protocol , great improvement in survival was observed with a mean time of 35 ± 19 months from the initiation of treatment with phospholipase and oligoelements . one or more hepatic metastases were already present in 95 % of the cases at the time of the starting treatment . moreover , we recorded a mean survival of 69 . 6 ± 30 months from the date of diagnosis of the primitive tumor . at the time of analysing presented results , all patients were alive , having been admitted to the protocol after the detection of hepatic metastasis in 95 % of cases ( table 3 ). the main criteria on for patient selection was their exclusion from other therapeutic approaches . in 80 % of cases treated patients were classified as dukes c or d ( stage 1 ). the general condition of the patients remarkably improved by the treatment with a normalization of humoral parameters in many cases , while oncological markers became negative , and pain disappeared ( table 3 ). patients failed to present signs of intrahepatic bile duct obstruction or signs of liver failure in spite of hepatic metastasis extension and its multiplicity in some cases . no signs of toxicity were observed due to medication components . neither were there any complications due to intramuscular parenteral administration in spite of the need of daily administration during long periods ( up to 49 months ). to date , all patients are alive . nine of them are in very good general condition ; nine in good condition and the other three in poor condition due to the aggravation of pathologies unrelated to hepatic metastasis . remissions were complete in six cases , partial in five , remaining stable in six and progression was observed in four . in a review of world literature we have not found a single case of colon adenocarcinoma with hepatic metastasis presenting spontaneous regression . scarce cases only occur in clear renal cell carcinomas , neuroblastomas , malignant melanoma , and chorioncarcinoma ( 45 ) ( 47 ) ( 48 ) ( 49 ) ( 50 ). this phenomenon is also observed in congenital leukemia associated to down syndrome type alterations and other chromosomic aberrations ( 51 )( 52 )( 53 ), but has not been described in other ontological entities . due to this , believe that the ten cases presented in this study do not correspond to spontaneous regression but to a response to applied therapeutics . on the other hand , in cases of chemotherapy - treated hepatic metastasis , results are very poor . for instance , with 5 - fluroracyl as a single therapeutic agent , a response of approximately 20 % is achieved without improving survival time ( 54 )( 55 ). by supplementing 5 - fluroracyl with leukovorine , which inhibits thymidilate synthetase allowing a major inhibition of dna synthesis by 5 - fluroracyl ( 56 )( 57 ), improved response rates are obtained but survival rates are not modified significantly ( 58 ). the use of chemotherapic agents in high doses , for example melphalan ( 120 mg / m ), following leukopoiesis stimulants , such as gm - csf for bone marrow rescue , has resulted in a response of 33 % with a duration of only 2 or 3 months ( 59 ). in a series of treated patients , compiled by moertel , mean survival was estimated to be 5 . 5 months ( 60 )( 61 ) from the time of liver involvement diagnosis . these data are consistent with other authors estimating mean survival rate to be roughly 6 months , and in two months in the case of alkaline phosphatase increase ( advanced disease marker ) ( 62 ). the most encouraging studies with 5 - fluroracyl perfusion directly in to the hepatic artery showed that survival could not be extended beyond 10 to 11 months , with 20 to 40 % of morbility due to the method ( 63 ). hepatectomy for colon adenocarcinoma hepatic metastasis is a well - known method . the first intervention of this kind was reported by garre in 1888 ( 64 ). in some selected cases it improves survival remarkably . however it is only indicated in cases where one hepatic lobule presents at most three lesions ( 65 ). in these cases , 36 % survival is achieved after a 5 - year term , especially in patients with a single metastasis ( 66 )( 67 )( 68 ). in large series of thousands of patients it has been shown that only 5 % of the total cases belong to this category . hepatic metastasis resections can only improve survival in roughly 1 or 2 % of cases ( 65 ). in our series of patients only one had been selected for hepatectomy . at the initiation of treatment that patient was in relapse . surgical methods in the above conditions may eradicate the disease and extend survival periods substantially . unfortunately , this method cannot be applied in all cases . when multiple metastasic lesions appear together with bilobular involvement , adverse physical conditions of patients and signs of terminal disease ( hepatomegaly , ascites , high alkaline phosphotase and liver failure , among others ), chemotherapy has not proven to be an adequate substitute to surgery as regards longer survival . therefore , it seems highly encouraging to obtain a mean survival time of 35 month from the start of the treatment with phospholipase and oligoelements in patients excluded from other protocols and free from any other treatment . although we are not proposing our method as an alternative to others , we believe it is indeed useful in the above cases . it is also applicable as a treatment complementary to hepatectomy and conventional chemotherapy since we have already improved the results of this latter procedure ( 35 months versus 11 months of survival ) without toxic or undesirable effects . the mechanisms of action through which our combined phospholipase - oligoelements treatment exerts its therapeutic effect are most likely multiple in nature . the marked response obtained in cellular cultures , in a dose - dependent pattern , indicates the existence of direct inhibition mechanisms of cellular proliferation not mediated by the inmune system . a review of the data accumulated for this purpose on cell differentiation systems and apoptosis has led us to propose independent explanations . phospholipase - oligoelements treatment apparently affects the cellular capacity of synthesis repair and expression of dna chains , probably because oligoelements , whether separately or combined are able to influence some of the enzymes involved in the physiological repair of mutated chains . data supporting this mechanism can be obtained from experimental studies on carcinogenesis . accordingly , nickel ( ni ) in combination with the peptide glycil - glycil - histidine produces the generation of free oxygen radicals from h 2 o 2 , which acting in the proximity of dna chains can induce double linkages in cytocine - cytocine and thymidine - thymidine tandems with mutagenic effects ( 69 )( 70 ). this action may be inhibited or reverted by superoxide dismutase , an enzyme that eliminates the superoxide anion and which is located either in the mitochondrion , where it needs mn for its action , or in the cytosol , requiring cu and zn as catalysts ( 71 )( 72 ). eukariotic cells are protected from h 2 o 2 by the catalase and the glutathion peroxidase present in both cytosol and mitochondrion ( 73 ), the latter having se as catalyst . superoxide dismutase are increases in rats treated with an excess of se in their diet to prevent carcinogenesis induced by diverse agents as nmu and dimethylhydrazine , among others . ( 5 )( 7 )( 9 ). this enzyme is considered responsible at least partially for the benefical effect of se . elimination of o 2 and h 2 o 2 is essential since these components in the presence of fe react releasing hidroxyl radicals ( oh ) against which no cellular mechanism exists . the latter are capable of oxidizing any biological molecule ( membrane lipids , cellular proteins and nucleic acids ). damage to the suppressor genes as p53 ( 17p ) and ddc ( 18q ), which , due to their presence in only one of the allels ( loss of heterocygotia ), or mutated in both allels , or in the remaining allels , or the deleted ones , cause the loss of their regulatory function over cellular proliferation and differentiation ( 74 )( 75 )( 76 )( 77 ). it may be assumed that the favourable action of the oligoelements on the reparair of the damaged gene repair can restitute the atypical cells to the normal circuit , making them return to normal differentiation and apoptosis ( programmed cell death ). this mechanism has been proposed by tobey and by samson and schwartz in relation to the action of zn as an inducer of cellular resistance against alkylating agents and their mutagenicity ( 3 )( 9 )( 78 ). a review of the preventive and reparative mechanisms of the mutagenic effects of o 2 , h 2 o 2 and oh groups , include the three elements employed here , se , zn and mn , as essential catalysts of the critical enzymatic steps of the protective process . their administration in minimal excess and the improvement of their availability through parenteral injection and chemical form are essential facts to explain the remarkable survival differences ( both in treated rats and human patients ). however , there are other possible mechanism of action involving a wide range of molecular and immune path ways . for instance , se has proven to exert an inhibitory effect on the proliferation of atypical cells derived from hepatomas , an effect mediated by the increase in intracellular camp due to inhibition of specific phosphodiesterases ( 30 ), synergic with that of mn which in vitro is a direct activator of the adenylyl cyclase . the final result is a marked increase in the camp pool . the inhibitory effect on cell proliferation produced by camp is widely recognized ( 79 ). there is evidence of the releasing effect exerted by phospholipase a 2 on histamine and serotonine from mastocytes ( 80 )( 81 ). under certain conditions , histamine acts by strongly inhibiting cellular proliferation both in vivo and in vitro ( 40 )( 82 )( 83 ). the presence of histamine receptors in different types of human mammary tumors and melanoma , among others , has been described . ( 84 )( 85 )( 86 ). in such cases , histamine effects are exerted by h1 receptors increasing camp intracellular levels ( 79 ). serotonine acts by the same mechanism , directly and indirectly , through 5ht - 1a and 5ht - 4 receptors activating adenylyl cyclase with a synergic effect as the above mentioned ( 87 )( 88 ). a scarcely studied effect of serotonine receptors is the stimulation of phospholipase c , with the subsequent increase in phosphatidylinositol hydrolisis and intracellular calcium mobilization ( 5ht 1c and 5ht 2 receptors ). this effect is specifically exerted on hippocampal neurons , cortical entorrinal and fronto - cyngulate sectors ( 87 )( 89 )( 90 ). the final result of this stimulation is to increase impulse levels from neurons in those cortical areas ( 91 )( 92 ). some authors attribute this mechanism to 5ht 3 receptors ( 89 ). it is worthwhile to correlate these data with previous work on the action of phospholipase - type venoms on cerebral peptides ( 93 ). both mechanisms involving phospholipases and serotonine explain the positive action on appetite and change of attitude towards disease , which were noted in human patients and rats treated with phospholipases and oligoelements . a remarkable property of 5ht - 1a receptors is their different activity according to cell differentiation degree . it has been demonstrated that these receptors increase camp levels in undifferentiated cells ; but they inhibit adenylyl cyclase in well differentiated ones ( 88 ). this could explain their different mode of action inhibiting atypical cell proliferation but without affecting normal t lymphocytes . ( 87 )( 88 ). by ultrastructural immunolabelling , the presence in the mastocyte granules of prostaglandine e 2 , cyclo - oxygenase and a great pool of phospholipids not arranged in bilayers and forming membrane stuctures , but as an amorphous matrix ( 81 )( 94 ), has been shown . at the time of granule release these phospholipids form microvesicles when coming in contact with water . such microvesicles are linked to the granule &# 39 ; s membrane and simultaneously to the celt &# 39 ; s membrane , thus forming channels that allow exocytosis . besides , granules contain a great quantity of arachidonic acid ( 95 ) which makes them an adequate source to produce eicosanoic acids . the presence of phospholipases is essential and , since arachidonic acid is usually sterificated in sn - 2 phospholipid position , an a 2 phopholipase is needed ( 96 ), which in turn requires a high concentration of calcium for its action , normally found in the granule . in the case of the mastocyte , the enzyme is situated in the matrix of the granule , unlike the macrophage that possesses it in the membrane ( 87 )( 97 )( 98 ). as mentioned above , sht - 2 - 1c and 5ht - 2 serotonine receptors stimulate the increase in intracellular calcium , requirement for phospholipase a 2 activation . therefore , phospholipase a 2 causes a feedback circuit which leads to the secretion of mastocyte granules activating in turn the cascade of arachidonic acid through endogenous phospholipase a 2 and the pool of granule phosphoinositides . such pathway precedes pge2 production by cyclo - oxygenase in the already secreted mastocytes granules ( 81 ). it is well known that the effects of the above mentioned components on the cellular micro - environment herald the initiation of inflammatory processes with the subsequent afflux of immunocompetent cells and the necrosis of abnormal tissue components through a specific or non - specific immunomechanism such as t killer lymphocytes and macrophages , culminating in the necrosis of tumoral tissue . added to growth inhibition of atypical tissues produced by the regulation of intracellular camp levels or its metabolizing enzymes , this finding explains at least in theory the results obtained with the medication under study . a final mechanism suggested for this therapeutic proposal is the necrosis of neoplasic cells mediated by tnf and macrophages . as early as 1978 , mathwes described the existence of a tumoral necrotizing factor in rabbit monocytes ( 99 )( 100 ). previously , carwell in 1975 had reported the appearance of a factor in endotoxine - induced serum producing tumoral necrosis , but without identifying it . it was found that the then so - called tns needed lysosome integrity as well as membrane phospholipase a 2 for its action ( 101 ). later , mathwes established that cellular damage is initially exerted on mitochondria , also suggesting that cytotoxine required the presence of some metal as its action is inhibited by edta , presumably affecting a metaloenzyme cell membrane ( 102 )( 103 )( 104 ). in 1985 richards described tnf inhibition by corticoid due to arachidonic acid blockade . corticoids are supposed to induce a nondialyzable protein called lipomoduline or macrochortine that inhibits phospholipase a 2 . the presence of o2 - free radicalys is also necessary for the action of tnf and the metabolism of arachidonate , an essential stage of the cytolitie process ( 100 ). confirming the necessity of phospholipase a 2 for tnf action , mutch in 1992 established that the resistance of some cell lines derived from gynecological tumors to necrosis by tnf is due to the production by these cells of a protein that prevents the activation of the above enzyme ( 104 ). mechanisms initiated by tnf are not only destructive . some authors have described its effects on the production of lymphokines in lymphocytes and in macrophages ( csf - 1 , mcp ; monocyte chemoatratan protein 1 ) and the expression of several genes such as nfk8 which then stimulate several lymphokines and adhesive molecules that play an active role in tissue defense , inflammation and remodelation processes ( 105 )( 106 )( 107 )( 108 )( 109 ). however , the main action of tnf is still exerted as a result of the stimuli by diverse lymphokines such as i12 , enhancing the production of gamma ifn in t lymphocytes ( 110 ), or i14 , also stimulating gamma ifn and requiring macrophages to exert the suppressor effect on mouse tumors . this inhibition is carried out by means of arnm stimulation for i15 , ifn gamma and tnf t lymphocytes ( 111 )( 112 ). the above effects are important to disclose the origin of chronic inflammatory processes and immune induced inflammation . it may therefore be concluded that the addition of the final effector enzyme of all these mechanisms ( phospholipase a 2 ) by a direct route ( parenteral ) may in some way trigger the above inflammatory processes by offering an effector for the action of alpha tnf . arachidonic acid is produced from membrane phospholipids leading to the release of o 2 - free radicals and the production of prostaglandines by cyclo - oxygenase and other enzymes ( 81 )( 109 ). it is worthwhile stressing that when performing the analysis of these results ( march , 1994 ), all patients were alive . nine of them were in a very good general condition ; nine in good condition and the other three were in poor condition due to aggravation of pathologies unrelated to hepatic metastasis . remissions were complete in six cases and partial in five , while six remained stable and progression was observed in only four cases , with excellent life quality throughout . we could state that while oligoelements protect normal cells from the effects of oxygen free radicals and may perhaps induce the apoptosis of atypical cells , allowing the repair of their genomic damage , phospholipase a 2 promotes the destruction of atypical cells , both in a direct and indirect way . in a direct way , by means of activation of the same mechanisms of cellular destruction inhibited by oligoelements , that is the release of o 2 free radicals . in an indirect way , by means of histamine and serotonine release from mastocyte granules and acting as final effector substrate of the effects mediated by gamma inf , i12 , i14 and tnf already present in the tumoral microenvironment . finally , the afflux of immuncompetent cells and macrophages to the site of development of the destructive processes may produce feedback of the mentioned mechanisms . it might seem contradictory to support the fact that the action of the two groups of compounds utilized is antagonic . nevertheless , it has been reported that oligoelements exert entirely different effects on atypical cells as compared with normal ones . for instance , it has been demonstrated that normal cell culture pretreatment with zn , cu and se makes it 16 times more resistant to high doses of melaphalan than untreated cultures . on the other hand , atypical cell cultures show scarce ( 30 %) or null improvement of resistance to alkylating agents ( 8 ). consequently , we believe that the previously discussed hypothesis , provides a valid explanation for the therapeutic basis of our treatment . since the mechanism of action is non - specific , it may be applied to several neoplasic pathologies and , hence , to a wide range of patients . it is also evident that possible modifications of oncogen and oncoprotein expressions , as well as the capacity to induce apoptosis promoted by the application of phospholipase and oligoelements must be further investigated in cellular cultures . table 1__________________________________________________________________________ primitive hepatic hepatic hepaticpatient tumor metastasis metastasis metastasis metastasis # diagnosis diagnosis size location sugery chemotherapy__________________________________________________________________________1 04 - 14 - 86 05 - 08 - 86 multiple diffused n / a n / a centimet . 2 12 - 15 - 89 07 - 30 - 91 6 . 2 . 5 and right and n / a n / a 2 cm . left lobules3 11 - 14 - 89 09 - 08 - 89 8 cm . then right hepatec - n / a post - surg . 8 cm . lobule tom 05 - 08 - 91 2 cm . 11 - 17 - 89 rec . 5 - 8 - 914 09 - 04 - 90 11 - 05 - 92 3 cm . right n / a n / a lobule5 01 - 20 - 91 01 - 25 - 91 4 cm . segments n / a n / a 7 cm . 4 and 56 11 - 25 - 91 11 - 28 - 91 3 cm . right n / a n / a 4 cm . lobule 7 cm . 7 11 - 07 - 92 01 - 07 - 92 7 cm . multiple n / a n / a 8 cm . diffused8 10 - 16 - 90 10 - 29 - 90 multiple multiple n / a n / a centimet . diffused9 05 - 13 - 91 11 - 03 - 91 8 cm . segment 8 n / a single dose no tolerance10 09 - 14 - 90 07 - 15 - 91 5 cm . right n / a n / a lobule__________________________________________________________________________ table 2__________________________________________________________________________ primite hepatic hepatic hepaticpatient tumor metastasic metastasis metastasic metastasic # diagnosis diagnosis size location surgery chemoterapy__________________________________________________________________________11 2 - 10 - 89 1 - 9 - 91 1 cm . caudal no no lobe12 1 - 4 - 90 2 - 9 - 91 centimetric multiple no no difused13 8 - 11 - 92 8 - 27 - 92 15 cm . hepatic dome no no multiple14 3 - 26 - 92 3 - 26 - 92 3 cm . right lobe no no and 3 . 2 cm . 15 2 - 15 - 92 2 - 15 - 92 centimetric multiple no discontinued after hepatic artery trombosis16 1 - 14 - 92 2 - 14 - 92 3 to 4 cm . multiple no no17 8 - 16 - 91 8 - 16 - 91 centimetric left lobe no no multiple18 11 - 26 - 86 3 - 9 - 91 centrimetric residual heptatecto no right lobe my 3 - 9 - 90 6 - 26 - 9019 12 - 15 - 78 2 - 17 - 90 1 cm . residual left hepatecto no 1 - 25 - 86 lobe my 10 - 15 - 86 2 - 17 - 90 1 - 23 - 8920 5 - 2 - 91 10 - 15 - 91 1 cm . and right lobe no no 0 . 92 cm . 21 1 - 10 - 92 3 - 11 - 92 until 3 cm . multiple no no difused__________________________________________________________________________ table 3______________________________________ age h . m . patient ( in histological dukes diagnosis # sex yrs .) type grade method______________________________________1 female 72 semidiff . c hepatic adenocar . biopsy2 male 65 infiltranting b repeated differen . image adenocar . methods3 male 80 well c biopsy and differentiated hepatectomy adenocar . 4 male 80 moderately b repeated differentiated image adenocar . methods5 male 66 adenocar . c repeated image methods6 male 70 adenocar . c biopsy , hepatic cuneus7 female 60 infiltrating c biopsy , adenocar . hepatic cuneus8 female 67 differentiated c repeated adenocar . image methods9 female 68 differentiated c repeated adencar . image methods10 male 67 well differ . c repeated adenocar . image methods______________________________________ table 4______________________________________pa - age h . m . tient ( in histological dukes diagnosis # sex yrs .) type grade method______________________________________11 female 80 semi - c repeated image differentiated methods adenocarcinoma12 female 67 well - c repeated image differentiated methods adenocarcinoma13 female 61 semi - c intraoperatorial differentiated observatory adenocarcinoma and image methods14 female 73 scarcely - d intraoperatorial differentiated observatory adenocarcinoma and image methods15 male 74 semi - c intraoperatorial differentiated observatory adenocarcinoma and image methods16 male 72 semi - c repeated image differentiated methods adenocarcinoma17 female 64 semi - b introperatorial differentiated observatory adenocarcinoma and image methods18 male 57 semi - c repeated image differentiated methods adenocarcinoma19 male 67 adencarcinoma c repeated image methods20 male 84 well - c repeated image differentiated methods adenocarcinoma21 male 68 semi - c repeated image differentiated methods adenocarcinoma______________________________________ table 5__________________________________________________________________________ survival survival from primitive gradepatient from treatment tumor of general # initiation diagnosis remission status karnofsky__________________________________________________________________________1 94 months 96 months complete excellent 1002 29 months 50 months stable very good 903 27 months 127 months complete very good 1004 37 months 44 months complete poor 40 ( hemiplegia ) 5 33 months 37 months partial poor 50 ( uremia ) 6 26 months 33 months partial good 707 23 months 25 months stable good 708 39 months 46 months complete very good 1009 22 months 33 months progressive fair 6010 39 months 43 months stable very good 80__________________________________________________________________________ table 6__________________________________________________________________________ survival survival from primitive gradepatient from treatment tumor of general # initiation diagnosis remission status karnofsky__________________________________________________________________________11 38 months 56 months complete good 8012 37 months 50 months stable very good 9013 19 months 19 months stable very good 9014 24 months 24 months progression regular 6015 25 months 25 months partial very good 100 remission16 25 months 26 months progression good 8017 31 months 31 months partial very good 90 regerssion18 36 months 45 months stable very good 9019 49 months 90 months complete very good 100 remision20 27 months 36 months partial good 80 remission21 24 months 26 months progression good 80__________________________________________________________________________ the invention has been described with reference to various specific and preferred embodiments and techniques . however , it should be understood that many variations and modifications may be made while remaining within the spirit and scope of the invention .
an immuno - enhancer composition of at least one oligoelement selected from the group consisting of sodium selenite , magnesium chloride , manganese chloride , zinc chloride , cobalt , cerium , molybdenum , and silicon ; and phospholipase a2 . the preparation of the oligoelement and phospholipase a2 composition and its use in immunotherapy , treatment of oncologic diseases and degenerative diseases .