Patent Application: US-17657693-A

Abstract:
the present invention relates to 24r - scymnol , including its preparation in substantially pure form and to its use in the treatment of liver dysfunction .

Description:
in accordance with a preferred procedure of the present invention , 24r - scymnol has been prepared by hydrolysis of sodium scymnol sulphate with dilute hydrochloric acid in the presence of barium chloride , to give a crystalline product . the physical and chemical data of the product are set out in table 1 : table 1______________________________________physical and chemical data of 24r - scymnol . ______________________________________mp 183 - 184 ° c . ( 190 ° c . )( colorless plate ), h . sub . 2 o insol , meoh , etoh sol acoet slightly sol , chcl . sub . 3 insol ( α ). sub . p . sup . 25 = 40 . 40 °( 0 . 5c , in meoh )( 34 % 2 °( 0 . 9c , inetoh )) high resolution mass ; calcd for c . sub . 27 h . sub . 44 o . sub . 4 432 . 6493 ( des - 2h . sub . 2 o ) found 432 . 3247ir ν kbr . sub . cm . spsb .-. sub . : 3400 , 2950 , 1480 , 1380 , 1080 , 1040 , 980 , 920max . sup . 1 h - nmr ( in cd . sub . 3 od ); δ ( ppm ): 3 . 95 ( 1h , br ), 3 . 80 - 3 . 50 ( 6h , m ), 3 . 48 ( 1h , m ), 2 . 40 - 2 . 15 ( 2h , m ), 2 . 10 - 1 . 10 ( 23h , m ), 1 . 07 ( 3h , d , j = 6 . 01hz ) 0 . 91 ( 3h , s ), 0 . 71 ( 3h , s ). sup . 13 c - nmr ( incd . sub . 3 od ); δ ( ppm ) 74 . 8 ( d ), 73 . 5 ( d ), 73 . 0 ( d ), 69 . 8 ( d ), 62 . 7 ( t ), 62 . 0 ( t ), 50 . 0 ( t ), 49 . 0 ( d ), 48 . 1 ( s ), 43 . 8 ( d ), 43 . 6 ( d ), 41 . 7 ( d ), 41 . 1 ( t ), 37 . 8 ( d ), 37 . 2 ( t ), 36 . 6 ( s ), 36 . 5 ( t ), 34 . 0 ( t ), 33 . 0 ( t ), 31 . 9 ( t ), 30 . 3 ( t ), 29 . 5 ( t ), 28 . 5 ( d ), 25 . 0 ( t ), 24 . 0 ( q ), 18 . 9 ( q ), 13 . 8 ( q ). ______________________________________ the stereochemical configuration at the 24 - position of the compound was determined as r (+) by its crystallographical analysis and the specific optical rotation . the crystal data and atomic parameters of the compound are set out in table 2 and fig1 respectively . in fig1 fractional atomic coordinates (× 10 4 ) are given , with e . s . d .&# 39 ; s in parentheses , and anisotropic thermal parameters ( å 2 × 10 - 4 ). table 2______________________________________crystal data of 24r - scymnol______________________________________c . sub . 27 h . sub . 48 o . sub . 6 . ch . sub . 3 oh . h . sub . 2 o , m = 518 . 71 , orthorhombic , space groupp 2 . sub . 1 2 . sub . 1 2 , a = 18 . 571 ( 1 ), b = 19 . 927 ( 2 ), c = 7 . 984 ( 1 ) å , v = 2954 . 8å . sup . 3 , z = 4 , f ( 000 )= 1144 , dc = 1 . 19 g / cm . sup . 3 , do = 1 . 22 g / cm . sup . 3 , λ ( cu -- kα )= 1 . 54180 å , μ ( cu -- kα )= 7 . 9cm . sup .- 1 , crystal size0 . 2 × 0 . 2 × 0 . 4 mm . ______________________________________ the activity of 24r - scymnol in the treatment of liver dysfunction has been investigated . in prior international patent application no . pct / au87 / 00281 , two assays have been designated to identify characteristic pharmacological activities of the substance , 24r - scymnol sulphate ( or isolutrol ). the bioassays , designated as ( a ) and ( b ), have been based on the following activities : ( a ) the active principle prevented liver damage in mice caused by carbon tetrachloride ; and ( b ) the active principle increased the respiration rate in mice when a toxic substance , such as nicotine , was administered . these assays are useful in ascertaining the existence of activity , however it has been found that they are sometimes unreliable and not reproducible , indicating they are not reliable assays for measuring the degree of activity . 24r - scymnol has activity in these two bioassays , but it is difficult to make comparison of the degree of activity between isolutrol and 24r - scymnol . accordingly , a new assay has been designed to reproducibly measure the degree of activity . this bioassay , designated as bioassay ( c ), measures tyrosine aminotransferase ( ta ) activity in liver of mice . ta is one of important enzymes in liver . the results of this assay are set out in example 2 below . in addition , to elucidate the possible mechanisms involved in mediating the hepatoprotective effects of 24r - scymnol and 24r - scymnol sulphate various biological features have been assayed . these assays included inhibition of the effects of hepatotoxicity in vivo , in which hepatotoxic responses of animals to carbon tetrachloride , acetaminophen ( paracetamol ) and α - amanitine were assessed , along with the acute effects of alcohol ingestion . the results of these assays are set out in examples 4 to 7 below and demonstrate that 24r - scymnol has surprising , and unexpected , hepatoprotective effects , even when compared with 24r - scymnol sulphate . the present invention also provides a pharmaceutical composition comprising 24r - scymnol , together with a pharmaceutically acceptable carrier or diluent therefor . by way of example , 24r - scymnol can be formulated as stable tablets after being mixed as a powder with a known carrier or bulking agent . such pharmaceutical compositions may be used , for example , for the activation of liver function in the treatment of the diseases of the liver such as hepatitis , nephritis , diabetes , etc . further details of this invention will be apparent from the following examples which illustrate the invention without limiting it in any way . the active principle of shark &# 39 ; s bile ( 500 mg ) was dissolved in 7 ml of 1 % hcl containing 400 mg of bacl 2 and the mixture was heated for 5 h at 100 ° c . after cooling , the resulting solution was extracted three times with 50 ml of acoh - buoh ( 1 : 1 ). the organic layer was washed twice with h 2 o . removal of the solvent gave a yellow oil . the resultant residue was dissolved in meoh and applied to reversed phase hplc . 50 mg of 24r - scymnol was obtained . mice ( 5 weeks old ) were orally administered 24r - scymnol sulphate , the active principle of shark &# 39 ; s bile as disclosed in international patent application no . pct / au87 / 00281 ( md ) ( 10 mg / kg ), 24r - scymnol ( 10 mg / kg ), or water ( 6 : 00 pm on the previous day , 9 : 00 am on the day ). after one hour from the last administration , the mice were forced to swim in a body of water at 35 ° c . after 4 hours swimming , the mice were sacrificed by decapitation and their livers were perfused with 0 . 145m kcl to remove blood . half gram of liver was homogenized in 0 . 145m kcl and centrifuged at 10 , 000 × g for 30 min . and ta activity in the supernatant was measured by the method of diammondstone . the activity is shown as the amount of p - hydroxyphenyl pyruvic acid ( p - hpp ) produced by the enzyme reaction for 10 min . the results of the comparison of the activities of the active principle and 24r - scymnol by the assay ( c ) are as follows : ______________________________________ ta activity ( n - mole p - hpp / mg protein / 10 min . ______________________________________control 1007 ± 49 . 6md 1322 ± 63 . 0 ** 24r - scymnol 1351 ± 29 . 2 *** ______________________________________ **: p & lt ; 0 . 01 , ***: p & lt ; 0 . 001 the above results indicate that 24r - scymnol has almost the same activity as the active principle of the shark &# 39 ; s bile . the following materials and methods were used in examples 4 to 7 hereinafter : ultraviolet ( u . v .) absorption spectra were determined on a hitachi u3200 double beam spectrophotometer . male swiss mice were obtained from monash university animal house and australian animal resources aged at 8 - 10 weeks and weighed between 25 - 30 g . the animals were maintained in a temperature controlled animal house on a 12 hour light dark cycle and allowed food and water ad libitum . test animals received 5 mg / kg 24r - scymnol or 24r - scymnol sulphate intraperitoneally in 0 . 01 ml / g saline daily for seven consecutive days . control animals were housed in the same conditions yet received no treatment . a vehicle control however was used in the first experimentation of carbon tetrachloride . hepatotoxins were administered i . p . on the eighth day , 24 hours after the last drug injection . cardiac puncture was performed on mice anaesthetised with nembutal 0 . 6 mg / kg in saline 24 hours after toxin administration and liver excised for use in histology and microsome preparation . blood was centrifuged for 5 mins at 5 , 000 rpm and serum stored at - 15 ° c . for further use . the liver was rapidly rinsed in ice - cold ( 4 ° c .) phosphate buffer 0 . 1m ph 7 . 4 to remove contaminating blood . liver was homogenised in phosphate buffer ( 25 % liver w / v ) using a ultraturax homogeniser at setting 6 with 3 passes for 10 seconds . the homogenate was centrifuged at 4 ° c . ( 12 , 500 rpm ) for 20 mins and the supernatant retained and centrifuged at 40 , 000 rpm for 60 mins in a beckman ultracentrifuge . the microsomal plug was washed in the phosphate buffer and resuspended in 0 . 1m phosphate buffer ph 7 . 4 containing 20 % glycerin ( 50 % liver w / v ) by agitation in a glass hand homogeniser . suspended microsomes were frozen at - 70 ° c . until required . hepatic tissues were taken from the largest lobe and fixed in 20 volumes of formalin / specimen volume for 3 days . after dehydration and clearing , liver was impregnated in molten paraffin and 7 μm sections cut . sections were stained with mayers acid haematoxylin and putts eosin following a modified formula to that of disbrey , b . d . et al . ( 1970 ). the modified steps were as follows : ______________________________________1 . mayers haematoxylin 1 min2 . rinse in tapwater3 . scotts tapwater substitute 30 sec4 . rinse in tapwater5 . putts eosin 30 sec6 . rinse in tapwater7 . dehydrate , clear in xylene , mount in d . p . x . ______________________________________ elevation of the serum enzymes alanine transaminase ( alt ), sorbitol dehydrogenase ( sdh ) and lactate dehydrogenase ( ldh ) were used as an index of hepatotoxicity . balazs et al . ( 1961 ) recognise that the serum enzymes mentioned increase with increasing hepatocyte necrosis and hence are useful and accurate indexes of hepatocellular injury in rats after hepatotoxin administration . alt was determined by the formation of a brown pyruvate hydrazone , quantitated colorimetrically according to the method of rietman and frankel ( 1957 ). sdh and ldh were determined from the absorbance change at 340 nm occurring after the addition of fructose and pyruvate respectively by the method of gerlach , u . ( 1963 ) and kachmar , j . f . ( 1976 ) respectively . additionally , serum triglycerides measured colorimetrically were also used as a measure of liver damage . the method was based on the selective extraction of triglycerides on the hantzch reaction for formaldehyde according to levy , a . l . ( 1972 ). glucose levels in serum were also tested by the biomerieux kit method to detect any hypoglycemia caused by hepatocyte damage . these were performed by a modified version of the lowry method ( markwell et al ., 1978 ) with bovine serum as a standard . all biological data were expressed here as mean ± s . e . m . and the statistical significance was evaluated as the t - test , difference between two means with a significance of ρ ± 0 . 05 . the toxic effects of carbon tetrachloride on the liver have been investigated over many years with particular attention to the pathology , toxicology and biochemistry of ccl 4 induced by liver injury attributable to : * ccl 4 consistently produces liver injury in many species ( the type and severity of injury to the liver can vary from triacylglycerol accumulation through necrosis to cirrhosis and cancer depending on dosage and method of application ), * ccl 4 is of considerable industrial and environmental importance and is a natural product . a single dose of ccl 4 administered to a rat produces centrilobular necrosis and fatty degeneration of the liver . electron microscopic examination of liver section from ccl 4 poisoned rats reveals early swelling , disorganization and degranulation of the endoplasmic reticulum but no equivalent damage to the mitochondria ( thought to be due to the double membrane ), particularly in the central zone . depression of hepatic protein synthesis by ccl 4 has been confirmed which ultimately results in a reduction in the amount of serum triglycerides , since they leave the liver as lipoproteins . in this study , the protective effects of 24r - scymnol and 24r - scymnol sulphate on several ccl 4 induced biochemical and morphological alterations were assessed . the table below shows the protocol established for ccl 4 toxicity challenge . eight groups of five male swiss mice were divided into control and ccl 4 ( 0 . 01 ml / g 5 % solution in olive oil ) treated groups . a saline control group receiving 0 . 01 ml / g saline was challenged with ccl 4 after a week of injections . an untreated group received ccl 4 on day 8 . all groups had unchallenged controls . cardiac puncture was performed on anaesthetised mice on day 9 ( 24 hours after ccl 4 injection ), blood collected for serum assays and liver excised for use in histology and microsome preparation . elevation of the serum enzymes alt and sdh was used as an index of hepatotoxicity along with protein determination and histological assessment according to the methods in example 3 . fig2 shows that alanine transaminase activity was markedly increased 24 hours after the administration of ccl 4 . ( fig2 shows the effect on serum enzymes after ccl 4 treatment . mice were sacrificed 24 hours after ccl 4 administration ( 0 . 01 ml / g , 5 % ccl 4 ) and treated prior according to the method . each value in fig2 represents the average ÷ s . e . m . from 5 animals .) pretreatment with 24r - scymnol significantly reduced this increase however 24r - scymnol sulphate did not . sorbitol dehydrogenase levels in serum increased 100 - fold above untreated mice 24 hours after ccl 4 administration . both 24r - scymnol and 24r - scymnol sulphate afforded significant protection against the sdh leakage with 24r - scymnol being the greater of the two bile salts . liver histology determined 24 hours after ccl 4 administration showed that all mice sustained centrilobular liver cell necrosis . the magnitude of necrosis was reduced when pretreated with the bile salts , again with the greatest protection afforded by 24r - scymnol . gross morphological analysis of mouse liver complemented the effects seen by microscopy . liver cell protein synthesising ability was markedly reduced upon ccl 4 administration 24 hours after administration . mice pretreated with 24r - scymnol however lost no significant protein synthesising ability measured in the liver microsomes ( table 3 ). no apparent harmful effects on the liver were observed after intraperitoneal injection of either bile salt . liver histology and serum enzyme analysis confirmed healthy livers with no difference to controls . before killing , it was noticed that those animals injected with the bile salts were groomed better than the other groups with shinier , healthier coats and displaying a much less aggressive and agitated behaviour . table 3______________________________________effect on mice of carbon tetrachloridetreatment appearance protein ( mg / ml ) ______________________________________control - 12 . 7 ± 0 . 8ccl . sub . 4 +++ . sup . 9 . 3 ± 0 . 6 . sup . 224r - scymnol sulphate ++ 11 . 3 ± 0 . 424r - scymnol + 11 . 7 ± 0 . 7______________________________________mice were treated with bile salts as per methods sec - tion for 7 days . all groups excluding the untreated wereadministered with 0 . 01 ml / g of a 5 % solution ccl . sub . 4 on day8 and all mice sacrificed on day 9 . protein content was es - timated on microsomal samples according to the methodssection . each value represents the average ± s . e . m . from5 animals . . sup . 2 significantly different from the controlvalue p & lt ; 0 . 05 . hepatic tissue was graded for necrosis . key : - nochange ; + slight change ; ++ moderate change ; +++ sig - nificant change . ______________________________________ mice were treated with bile salts as per methods section for 7 days . all groups excluding the untreated were administered with 0 . 01 ml / g of a 5 % solution ccl 4 on day 8 and all mice sacrificed on day 9 . protein content was estimated on microsomal samples according to the methods section . each value represents the average ± s . e . m . from 5 animals . 2 significantly different from the control value ρ & lt ; 0 . 05 . hepatic tissue was graded for necrosis . key : - no change ; + slight change ; ++ moderate change ; +++ significant change . acetaminophen ( paracetamol ) is a derivative of para - aminophenol and was introduced into clinical medicine as an anti - pyretic agent in the late nineteenth century . when taken in therapeutic doses , paracetamol does not cause any appreciable side effects . however liver injury will develop in all patients who ingest sufficient acetaminophen , becoming evident biochemically within 24 - 48 hours of the time of ingestion ( black , m . 1980 ). studies have shown that acetaminophen - induced liver - cell necrosis is not only mediated by a metabolite of the drug , but that the microsomal mixed - function oxidase system is importantly involved in its formation . liver injury falls upon the centrizonal hepatocytes , in which the greatest lobular concentration of the microsomal mixed - function oxidase system is located ( black , m . 1980 ). although the mixed - function oxidase system is centrally involved in generating the acetaminophen toxic metabolite , this pathway plays a minor role in the overall disposition of the drug when it is ingested in therapeutic doses . thus 85 - 90 % of acetaminophen is normally metabolised by glucoronide or sulphate conjugation ( both are saturable processes ) leaving a relatively small amount to be metabolised via other pathways including the mixed - function oxidase system . extensive studies performed by jollow et al ., ( 1973 ) found that acetaminophen metabolites covalently bound to the liver with increased binding also increasing the degree of liver necrosis . the peak level of binding preceded the development of recognisable necrosis by at least one or two hours with maximum binding to liver occurring two hours after acetaminophen administration . by administering doses of acetaminophen in excess of therapeutic dosage and hence forcing the involvement of the mixed - function oxidase system , in this assay it is determined whether 24r - scymnol and 24r - scymnol sulphate are able to inhibit the consequences of this system . three groups of five male swiss mice were intraperitoneally administered with 350 mg / kg acetaminophen on day 8 . two of the groups received the drug after receiving the usual weekly bile salt inoculation . the third group received the drug after no prior treatment and a fourth group was a control receiving no drug or treatment . in a second series of experiments , 5 male swiss mice were intraperitoneally administered with acetaminophen ( 350 mg / kg ) and one hour later received 24r - scymnol i . p . ( 35 μg / g in saline ). five additional mice received 24r - scymnol i . p . ( 35 μg / g in saline ) four hours post acetaminophen administration and another five mice used as treatment controls . all mice were killed 24 hours after acetaminophen administration and blood and liver taken as before . elevation of the serum enzymes , alt , sdh and ldh and triglyceride levels were used as indices of hepatotoxicity according to the methods mentioned in example 3 along with histopathological assessment . all serum enzymes tested increased significantly 24 hours after acetaminophen administration . 24r - scymnol was capable of significantly reducing hepatic damage when administered prior as indicated by significant reductions in all serum enzyme levels except triglycerides . 24r - scymnol sulphate however only reduced sdh and ldh levels significantly . the alanine transaminase increase in serum levels 24 hours after acetaminophen administration was significantly reduced when 24r - scymnol was administered 1 hour after the drug . protection was afforded also against sdh and ldh increase when 24r - scymnol was administered 1 hour post acetaminophen as to was serum triglyceride decrease . no protection at all was noticed against acetaminophen at this dosage when 24r - scymnol was administered 4 hours post acetaminophen administration . fig3 shows serum enzymes alt and sdh , 24 hours after acetaminophen administration . ( fig3 shows the effect on serum enzymes after acetaminophen administration . all animals received treatments according to the method and were sacrificed 24 hours after acetaminophen ( 350 mg / kg ) administration . each value represents the average ÷ s . e . m . from 5 animals .) fig4 shows the serum enzymes alt and sdh after receiving 24r - scymnol post acetaminophen administration . ( fig4 shows the effect on serum enzyme levels of mice receiving acetaminophen prior to bile salt treatment . mice were treated according to the methods and were sacrificed 24 hours after acetaminophen administration . each value represents the average ÷ s . e . m . from 5 animals .). tables 4 and 5 summarise the effect of acetaminophen on serum triglycerides ldh and microsomal protein content . gross morphology of the liver suggested 24r - scymnol to be hepatoprotective against acetaminophen since necrosis was either non visible or very minute . the acetaminophen group showed levels of necrosis of slight to very damaged with 24r - scymnol sulphate very similar to this . histopathological assessment of liver sections showed centrilobular necrosis in all mice receiving acetaminophen . the magnitude of necrosis was significantly reduced by prior treatment with the bile salts , with 24r - scymnol livers containing only odd necrotic hepatocytes . generally the overall appearance of the mice showed a definite improvement in coat color and behaviour in the 24r - scymnol and 24r - scymnol sulphate treated mice as opposed to the controls . the acetaminophen mice showed evidence of fighting among the group , they were agitated and generally in poor physical condition . table 4______________________________________effect of acetaminophen on mice after prior treatment with24r - scymnol and 24r - scymnol sulphate . ldh triglycerides proteintreatment ( u / l ) ( mg / dl ) ( mg / ml ) ______________________________________control 32 ± 9 138 ± 8 . sup . 12 . 7 ± 0 . 8 . sup . acetaminophen 1997 ± 78 . sup . 2 97 ± 6 . sup . 3 10 . 9 ± 0 . 6 . sup . 224r - scymnol sulphate 1101 ± 83 . sup . b 56 ± 2 . sup . 3 10 . 5 ± 0 . 4 . sup . 224r - scymnol 602 ± 29 . sup . b 117 ± 2 . sup . 3 12 . 2 ± 0 . 3 . sup . mice were treated as per the methods section and admin - istered with 350 mg / kg acetaminophen and sacrificed 24 hourslater . protein estimation was performed on liver microsomes . each value represents the average ± s . e . m . . sup . 2 significantlydifferent from control p & lt ; 0 . 05 . . sup . b although significantly differentfrom control also different from acetaminophen treatment alonep & lt ; 0 . 05 . ______________________________________ table 5______________________________________effect on mice receiving 24r - scymnol post acetaminophenadministration . treatment ldh ( u / l ) triglycerides ( mg / dl ) ______________________________________control 32 ± 9 138 ± 8 . sup . acetaminophen 1023 ± 98 . sup . 2 74 ± 4 . sup . 224r - scymnol 1 hr 550 ± 61 . sup . b 90 ± 5 . sup . b24r - scymnol 4 hr 984 ± 111 . sup . 2 70 ± 4 . sup . 2all mice except the control group received acetamino - phen ( 350 mg / kg ) and sacrificed 24 hours after . one groupreceived 35 mg / kg scymnol 1 hour after acetaminophen , another received the bile salt 4 hours after acetaminophenadministration . each value represents the average ± s . e . m . from 5 animals . . sup . 2 significantly different from controlp & lt ; 0 . 05 . . sup . b through significantly different from control alsosignificantly different from acetaminophen alone . ______________________________________ α - amanitine , a liver toxin ( or amatoxin ) produced by the fungus amanita phalloides ( the &# 34 ; green death cap &# 34 ;) invariably causes death at a dosage of 0 . 2 μg / g mouse , though it holds true that different strains of the same species vary in sensitivity to the poison ( wieland , t . 1966 ). an incubation or latency period occurs after exposure to the toxin , lasting up to 15 hours . a cholera - like period soon follows although there is no sign at this stage of liver toxicity . conversely though at 48 hours after ingestion severe liver failure occurs with increased bilirubin and transaminase elevations and hypoglycemia . in severe cases the clinical deterioration may continue , with symptoms such as hepatic encephalopathy , coma and death ( piqueras , j . 1989 ). researchers have found that α - amanitine causes a poverty of protein metabolism due to the impairment of rna synthesis . the liberation of glucose into the bloodstream is arrested due to damaged hepatocytes , unable to utilise glycogen in the normal manner , whilst lipid accumulation in the liver is due to the arrest of lipoprotein secretion ( choppin , j . 1979 ). 24r - scymnol and 24r - scymnol sulphate significantly showed hepatoprotective effects with the toxins carbon tetrachloride and acetaminophen ( see examples 4 and 5 above ) and as an extension of its ability to confer protection to the liver , experiments were modified to test the possible role in protection with the amatoxins . after the usual week of bile salt injections , five mice per group were intraperitoneally administered with 0 . 5 mg / kg α - amanitine in saline . a further five mice received the toxin after no prior treatment . blood and liver were taken in the usual manner , however 48 hours after toxin administration . elevation of the serum enzymes alt , sdh and ldh , along the serum glucose levels were used as indices of hepatocellular damage , along with histopathological assessment according to the methods in example 3 . at the day of sacrifice one of the control mice had died . since there appeared to be no evidence of fighting with the other mice in the group it was concluded that death was due to α - amanitine . the gross morphological appearance of the remaining control mice liver approximated that of moderate necrosis apart from one which showed very slight necrosis . those mice receiving 24r - scymnol showed no signs of liver cell necrosis whilst those receiving 24r - scymnol sulphate showed varying morphological characteristics ranging from none to moderate necrosis . histopathological assessment of liver sections showed hepatic necrosis in mice receiving α - amanitine along with loss of nucleus integrity , evident due to lack of nuclear staining . prior bile salt treatment significantly reduced this necrosis and maintained the integrity of the hepatocyte nucleus . fig5 shows the serum enzymes alt and sdh , 48 hours after α - amanitine administration . ( fig5 shows the effect on mice serum enzyme levels after α - amanitine treatment . mice were treated according to the method and were sacrificed 45 hours after α - amanitine administration . each value represents the average ÷ s . e . m . from 5 animals .). table 6 outlines the effects α - amanitine has on ldh , serum glucose levels and microsomal protein content . all three liver enzymes ; alt , sdh and ldh were markedly increased 48 hours after α amanitine administration . pretreatment with 24r - scymnol sulphate prior to administration of the poison did not significantly modify the increase in serum enzymes levels . pretreatment with 24r - scymnol only afforded protection against alt increase . the hypoglycemia observed 48 hours after α - amanitine administration was significantly reduced with prior treatment with 24r - scymnol yet not 24r - scymnol sulphate . protein synthesis depletion observed in liver microsomes after α - amanitine treatment was significantly reduced upon pretreatment with both 24r - scymnol and 24r - scymnol sulphate . table 6______________________________________effect on mice of amanitine . glucose proteintreatment ldh ( u / l ) ( mg / dl ) ( mg / ml ) ______________________________________control 21 ± 6 . sup . 183 ± 4 . sup . 13 . 2 ± 0 . 7 . sup . amanitine 3741 ± 245 . sup . 2 138 ± 2 . sup . 2 7 . 6 ± 0 . 5 . sup . 224r - scymnol sulphate 4193 ± 198 . sup . 2 125 ± 1 . sup . 2 11 . 3 ± 0 . 8 . sup . b24r - scymnol 1810 ± 57 . sup . 2 167 ± 2 . sup . b 10 . 7 ± 0 . 3 . sup . bmice were treated according to the method and sacrificed48 hours after α - amanitine administration . protein estimationwas performed on liver microsomes according to the standardmethod . each value represents the average ± s . e . m . from 5animals . . sup . 2 significantly different from control p & lt ; 0 . 05 . . sup . bthoughsignificantly different from control also significantly differentfrom α - amanitine treatment alone p & lt ; 0 . 05 . ______________________________________ ninety to 98 % of the alcohol that enters the body is completely oxidised to carbon dioxide and water ( hawkins et al ., 1966 ). ethanol is converted to acetaldehyde by at least 3 enzyme systems all of which are found extramitochondrially . these are alcohol dehydrogenase ( adh ), catalase ( cat ), and the microsomal ethanol oxidising system ( meos ), which uses nadph in conjunction with molecular oxygen ( valenzuela , a . 1989 ). following acute ethanol ingestion , reduced glutathione ( gsh ) levels in the liver decrease whilst those of oxidised glutathione ( gssg ) increase . this is in conjunction with an enhancement of hepatic lipoperoxidation which only occurs in conditions of maximal gsh reduction . tests were carried out to ascertain whether 24r - scymnol and 24r - scymnol sulphate are capable of reducing the effects of acute ethanol intoxication . in addition , the glutathione status of the liver was assessed , along with the serum enzymes alanine transaminase and sorbitol dehydrogenase since it has been demonstrated that they elevate after acute ethanol administration . male swiss mice received 2 g / kg ethanol administered as a 40 % solution on day 8 after usual bile salt treatment . animals were sacrificed 3 hours after ethanol intoxication and blood taken for routine serum assays and liver taken as before . the protein was precipitated from aliquots of freshly homogenised liver with 25 % hpo 3 . following the removal of precipitate by centrifugation at 100 , 000 g for 30 min the supernatant was assayed for gsh and gssg according to the method of hissin , p and hilf , r . ( 1976 ). standard curves were constructed each time samples were assayed , whilst supernatant was used the day of preparation . malondialdehyde is one of the products formed during microsomal lipid peroxidation which is capable of reacting with thiobartituric acid to give a species absorbing at 532 nm . liver homogenate was assayed according to the method of greenwald , r . g . ( 1985 ). protein was precipitated from fresh blood samples by a mixture of 0 . 45 % znso 4 and 0 . 1n naoh which gave a final ph of 7 . 0 . the mixture was centrifuged at 4 ° c . and the supernatant used for ethanol measurements according to hawkins et al . ( 1966 ). elevations of the serum enzymes , alt and sdh were used as indices of hepatotoxicity according to the methods in example 3 . table 7 illustrates the effect bile salts have on the serum ethanol levels 3 hours after ethanol intoxication . table 8 illustrates the in vivo effect of acute ethanol treatment on mice with and without bile salts . no increase in serum enzyme levels representative of hepatocellular injury were observed in the mice after 3 hours of ethanol intoxication . table 7______________________________________ethanol levels in mice after acute ethanol intoxication . treatment ethanol mmol / ml______________________________________control 0 . 003 ± 0 . 001 . sup . ethanol 0 . 156 ± 0 . 012 . sup . 224r - scymnol sulphate 0 . 097 ± 0 . 007 . sup . b24r - scymnol 0 . 103 ± 0 . 009 . sup . bmice were treated according to the methods sectionand sacrificed 3 hours after ethanol administration . eachvalue represents the average ± s . e . m . from 5 animals .. sup . 2 significantly different from untreated value p & lt ; 0 . 05 .. sup . b significantly different from both untreated and controlvalues p & lt ; 0 . 05 . ______________________________________ table 8______________________________________effects of acute ethanol on mice . mda mg / 100 g nmol / mgtreatment gsh gssg protein / 30 min______________________________________control 683 ± 32 . sup . 156 ± 10 . sup . 0 . 43 ± 0 . 008 . sup . ethanol 504 ± 30 . sup . 2 196 ± 12 . sup . 2 0 . 52 ± 0 . 011 . sup . 224r - scymnol so . sub . 4 600 ± 41 . sup . b 183 ± 15 . sup . 2 0 . 49 ± 0 . 010 . sup . b24r - scymnol 613 ± 27 . sup . b 187 ± 11 . sup . 2 0 . 50 ± 0 . 009 . sup . bmice were treated according to the method section andsacrificed 3 hours after ethanol administration . each value re - presents the average ± s . e . m . from 5 animals . . sup . 2 significantlydifferent from control group p & lt ; 0 . 05 . . sup . b though significantlydifferent from control this data is also significantly differentfrom ethanol treatment alone p & lt ; 0 . 05 . ______________________________________ balazs , t ., murray , t ., mclaughlan , j . and grice , h . 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