example 1 : insulin - mimicker activity of sodium tungstate on a model of human iddm ( type 1 diabetes ) the effects of sodium tungstate on the highly hyperglycemic streptozocin - induced diabetic rats ( a good animal model of human iddm ) were determined as follows : streptozotocin and sodium tungstate were from sigma ( st . louis , mo .). enzymes and biochemical reagents were from either boehringer mannheim ( mannheim , germany ) or sigma . all other chemicals were of analytical grade . adult male wistar ( 200 g ) rats were kept under constant 12 - hour light - dark cycle and were allowed to eat and drink ad libitum . when stated , diabetes was induced by a single endovenous injection of streptozocin ( 60 mg / kg body weight ) in 0 . 9 % nacl with 100 mm sodium citrate buffer ( ph 4 . 5 ). diabetes was confirmed by determination of glucosuria and glycemia ( glucose and glycemia strips . boehringer mannheim ). diabetic rats were used 5 - 7 days after streptozocin injection . at the beginning of the experiment , both diabetic and healthy animals were divided into two different groups . in the first group ( untreated ), rats received a solution of 0 . 9 % nacl as drinking water . the other animals ( treated ) were given a solution of 2 mg / ml of sodium tungstate in 0 . 9 % nacl . the treatment was carried out for 15 days . during this period , the fluid and food intake was measured each day between 4 and 6 p . m . at the end of the experiment , rats were anesthetized with diethylether and killed by decapitation between 9 and 11 a . m . immediately , blood was collected to measure serum parameters . meanwhile , livers were quickly sliced and fragments were either used immediately to measure enzyme activities or rapidly frozen in liquid n 2 for later determinations . glycemia was determined by the hexokinase method ( glucoquant . boehringer mannheim ). determinations were adapted to a cobas bio autoanalyzer ( roche biomedica . basel , switzerland ). serum insulin levels were determined by radioimmunoassay ( insik - 5 , soren biomedica . saluggia , italy ). table 1 shows the variations of serum parameters due to tungstate administration . results are expressed as the mean ± ( standard errors ) for the number of animals in parenthesis . it is observed that tungstate slightly increased serum glucose concentration in healthy rats . however , tungstate administration to diabetic rats counteracted the hyperglycemia observed in these animals , from 541 mg / dl ( 30 . 1 mm ) to 195 mg / dl ( 10 . 8 mm ). insulin serum levels were also measured . table 1 shows that tungstate administration did not change serum insulin levels either in healthy or in diabetic rats . table 1______________________________________effect of tungstate in a model of iddm glycemia ( mg / dl ) insulin ( ng / ml ) ______________________________________healthy ratsuntreated ( 11 ) 169 ± 6 6 . 8 ± 1 . 9treated ( 10 ) 190 ± 3 6 . 1 ± 2 . 1diabetic ratsuntreated ( 10 ) 541 ± 33 1 . 3 ± 0 . 3treated ( 10 ) 195 ± 22 2 . 1 ± 0 . 7______________________________________ example 2 : effect of tungstate on a model of human niddm ( type 2 diabetes ) to determine whether the effects of tungstate are of general application , experiments using an animal model which resembles human niddm were performed . this model is the rat treated neonatally with low doses of streptozocin , which develops slight hyperglycemia at 3 - 4 months . it was found that sodium tungstate treatment also normalizes glycemia in these animals , as shown in fig1 for ten animals treated daily with 300 - 350 mg / kg ( in the figure : hu = healthy untreated ; ht = healthy treated ; du = diabetic untreated ; dt = diabetic treated ). all rats responded to the treatment and no macroscopic negative effect was observed . furthermore , all animals survived the fifteen day treatment . it was checked that tungstate is able to mimic insulin in cultured healthy rat hepatocytes . tungstate at concentrations of 1 mm , 100 μm and 10 μm , was able to increase glycogen content to values similar to those values found for insulin . for the in vivo experiments , 200 g body weight male wistar rats have been used . different concentrations of sodium tungstate were administrated intragastrically , in order to calculate de ld 50 value , i . e ., the concentration of sodium tungstate capable of killing the half of the animals , in ten days . tungstate was dissolved with water and adjusted by clorhidric acid to ph 7 . 4 . the single dose was administrated between 10 - 12 a . m . and the rats were allowed to eat to eat and drink ad libitum . the different doses used were the following ones : -- 1000 mg / kg with nine animals only two of them died due to the treatment . -- 2500 mg / kg with five animals and none of them died . -- 4640 mg / kg with ten animals and six died after the administration . -- 6500 mg / kg with five animals and three of them died . -- 8000 mg / kg with five animals and all of them died . the ld 50 was calculated by the method of horn , h . j . ( biometrics 1956 , vol . 12 , pp . 311 - 322 ). the value obtained was 3922 mg / kg of body weight , which is much smaller than the dose used for the treatment of diabetic rats ( 400 mg / kg ), pointing out that the pharmacological effects were achieved at doses far from the toxic ranges . to assess the possible role of tungstate in the in vivo treatment of diabetes , a long - term experiment was performed . sixteen rats were made diabetic by an intraperitoneal injection of streptozocin ( 70 mg / kg body weight ). during two weeks , the animals received as drinking water a solution of 0 . 7 mg / ml of sodium tungstate , to get used to the tungstate taste . after this period , the dose was changed to a therapeutical one of 2 mg / ml . the animals were treated during 8 months , and no toxicological effects were observed in the healthy animals treated with tungstate . in the diabetic animals normalization of glycemia was observed more or less after two weeks of tungstate administration , and this normalization was maintained during all the treatment . treatment prevented the appearance of diabetic complications , i . e . cataract , in the diabetic animals . fig2 shows the evolution of body weight ( w , empty squares ) and glycemia ( g , filled squares ) in one of the treated rats which can be considered representative . the black triangle marks the beggining of the terapeutical treatment . the cytotoxic effects of tungstate were checked in primary cultured rat hepatocytes . the hepatocytes were isolated by collagenase digestion . four rats were used to perform these experiments , after the hepatocytes were allowed to attach to the collagen - coated dishes and differentiate , the cells were incubated for 24 hours with different doses of tungstate ( 0 . 01 , 0 . 1 and 1 mm ). the toxicity of tungstate was evaluated by the release of a cytosolic enzyme to the extracellular medium , since when the cells die their intracellular content is released . the cytosolic enzyme activity measured was lactate dehydrogenase ( ldh ). the activity of this enzyme was determined by spectrophotometric assays with commercially available kits ( boehringher mannheim ) in a cobas - bio autoanalizer . one unit of ldh activity is the amount of enzyme catalyzing the formation of 1 μmol of nadh per minute at 30 ° c . the reaction catalyzed by ldh is : after the incubation with different doses of tungstate , an increase in ldh release was observed at 1 mm dose after 24 hours of incubation . the mitochondrial function was analyzed in rat hepatocytes , this test is based on the reduction of yellow tetrazolium salts to an insoluble blue compound due to the activity of the mitochondrial enzyme succinate dehydrogenase . the test was performed after 24 hours of incubation with sodium tungstate . the cytotoxic indexes , ic 10 and ic 50 , were calculated mathematically form the dose - response curves . these indexes indicate the theoretical concentration of tungstate which induces some toxic effects on the 10 or 50 % of the cells , respectively . for rat hepatocytes , four rats were used and the cells were incubated with different doses of sodium tungstate between 0 . 26 - 2 mm to calculate the ic values , which were : ic 10 = 0 . 6 mm and ic 50 = 1 , 2 mm . human muscle cultures were initiated from satellite cells of muscle biopsies of two patients . aneural - muscle cultures were established in a monolayer according to the explant - preexplantation technique . in primary cultured human muscle cells , the toxicity of tungstate was evaluated by cytosolic enzymes releasing to the extracellular medium , since when the cells died their intracellular content is released . two cytosolic enzymes &# 39 ; activities were measured : lactate dehydrogenase ( ldh ) and creatine kinase ( ck ). creatine kinase and lactate dehydrogenase activities were determined by spectrophotometric assays with commercially available kits ( boehringher mannheim ) in a cobas - bio autoanalizer . one unit of ldh activity is the amount of enzyme catalyzing the formation of 1 μmol of nadh per minute at 30 ° c . the reaction catalyzed by ldh is : one unit of ck activity is the amount of enzyme catalyzing the formation of 1 μmol of atp per minute at 25 ° c . the reaction catalyzed by ck is : the culture medium was supplemented by different concentrations of sodium tungstate ( 0 . 01 , 0 . 1 , 0 . 5 and 1 mm ), and only at the highest dose ( 1 mm ) an increase in ldh and ck release was observed , after two days of incubation . at lower doses no increase in the release of these enzymes was detected , even when the cells were maintained with sodium tungstate for 15 days . therefore , tungstate at doses of 0 . 5 mm or below is not toxic for human muscle cells . the mitochondrial function was also analyzed in human hepatocytes , this test is based on the reduction of yellow tetrazolium salts to an insoluble blue compound due to the activity of the mitochondrial enzyme succinate dehydrogenase . the test was performed after 24 hours of incubation with sodium tungstate . the cytotoxic indexes , ic 10 and ic 50 , were calculated as mentioned before , and two liver biopsies were analyzed , with doses of tungstate between 0 . 26 - 6 mm . the values obtained were higher than in rats : ic 10 = 1 mm and ic 50 = 3 mm ; so in order to produce the same effect in human hepatocytes than in rat hepatocytes a higher concentration of tungstate is needed . therefore tungstate is less toxic in human liver cells than in rat liver cells . while the invention has been illustrated and described as embodied in tungsten ( vi ) compositions for oral treatment of diabetes mellitus , it is not intended to be limited to the details shown , since various modifications and structural changes may be made without departing from the spirit of the present invention . without further analysis , the foregoing will so fully reveal the gist of the present invention that others can , by applying current knowledge , readily adapt it for various applications without omitting features that , from the standpoint of prior art , fairly constitute essential characteristics of the generic or specific aspects of this invention .
the pharmaceutical composition contains an effective amount of a tungsten compound , preferably a tungstate or a isopolytungstate , and more preferably sodium tungstate . in the method of lowering blood sugar in a human suffering from type i or type ii diabetes mellitus , an effective amount of the composition is administered orally . compared with the known vanadium compositions , the invention has the advantage of providing a similar high sugar - lowering activity but with a much lower oral toxicity at effective doses , both short - and long - term . compared with insulin , the invention does not present the adverse effect of inducing hypoglycemia when administered in excess .