Patent Application: US-32545606-A

Abstract:
the present invention relates to methods of identifying potentially therapeutic compounds for the treatment of hepatic disorders , preferably alcohol - induced hepatitis . further , use of mammals in such methods , therapeutic compounds identified using said methods and the use of a chinese herb , ch - 100 , in the treatment of hepatic disorders , particularly alcohol - induced hepatic disorders , are described .

Description:
the following description outlines the experimental basis for the invention and specifically exemplifies preferred forms of thereof . female wistar rats were obtained from the central animal house , university of newcastle , nsw , australia . animals were housed in individual hanging wire cages in a controlled temperature environment with a 12 hr light - dark cycle and were allowed free access to rat chow and drink throughout the study . animals ( 3 - 6 rats per group ) were fed 40 % ethanol in water ( v / v ), isocaloric sucrose or 2 % sucrose according to the regimen described previously 12 . alcohol consuming rats were initially fed a 5 % ethanol in water ( v / v ) solution for the first week , and were then offered an increasing concentration of ethanol in water to 40 % ethanol during a six to eight week period to allow adaptation to the ethanol . in order to provide the same caloric intake to each rat in the isocaloric group of controls , chow and drinking solution were regulated according to the caloric intake of each rat in the isocaloric group . animals were allowed rat chow and 2 % sucrose solution adlibitum in the second control group . the animal protocol described in this study was approved by the animal research ethics committee of the university of newcastle and performed in accordance with the committee guidelines . chinese herbal medicine , ch - 100 ( cathay herbal laboratories pty ltd , surry hills , australia ), contains 19 different traditional herbs and was used to demonstrate the function of the model . the combination ( 4 tablets / kg body weight / day ) was mixed in 20 - 30 g of rat chow and was fed daily for 8 weeks after rats achieved 40 % ethanol solution while maintaining the rats on 40 % ethanol for the 8 week period . concanavalin a ( con a ) ( pharmacia ab , laboratory separation division , uppsala , sweden ) was administered to each rat via the tail vein in a dose of 20 mg / kg body weight dissolved in 300 μl of phosphate buffered saline ( pbs , bioscience , sydney , australia ). con a was injected after 8 weeks on 40 % alcohol . the rats continued to have access to alcohol following con a injection . animals were weighed and sacrificed 24 hours after con a administration . one group used as a control at time 0 did not receive con a . the liver was perfused and then removed for determination of wet weight , histological examination and cd4 + t cell studies . rats were anaesthetised by means of an intraperitoneal injection of ketamine ( parnell laboratories pty ltd , alexandria , nsw , australia ) and rompun ( bayer australia ltd pyrrible , nsw , australia ) ( 1 : 1 v / v , 0 . 08 - 0 . 12 ml / 100 g body weight ). livers were preperfused in situ through the portal vein with sterile pbs containing 0 . 1 % ethylenediaminetetraacetic acid , edta ( prolabo 12 rue pelee , paris , france ) at a pressure of 10 cm h 2 o giving a flow rate of 10 ml / min to remove intrahepatic blood . they were then , perfused with 300 ml of pbs / edta solution at a pressure height of 40 cm h 2 o ( flow rate of 30 ml / min ). the perfusate ( approx . 300 ml ) collected from the inferior vena cava was centrifuged at 1500 rpm for 15 min . after the supernatant was discarded the cells were resuspended in 3 ml of rpmi 1640 medium containing 5 % fcs ( trace scientific , sydney , nsw , australia ). the cell suspension was then layered onto a ficoll / paque density gradient ( pharmacia , uppsala , sweden ). following centrifugation at 1500 rpm for 20 minutes , the cells were harvested from the interface washed three times with pbs before being resuspended in rpmi / 5 % fcs medium 14 . liver - associated lymphocytes were labelled with mouse anti - rat cd4 + ( w3 / 25 ) ( serotec , oxford , uk ) at 4 c for 45 minutes . the cells were then washed twice in cold pbs by centrifugation at 250 × g for 10 minutes . after centrifugation cells were labelled with an optimal concentration of f ( ab ′) 2 rabbit anti - mouse isotype - specific immunoglobulins conjugated with fitc - conjugated f ( ab ′) 2 ( dako , glostrup , denmark ). after incubation for 20 minutes , the cells were resuspended in 3 ml of rpmi 1640 . finally , liver - associated cd4 + t cells were separated using fascan cell sorter . cell viability was & gt ; 99 % as determined by trypan blue exclusion . liver - associated cd4 + t cells were cultured with con a ( 5 μg / ml ) at 1 × 10 6 cells per ml of rpmi 1640 medium containing 5 % fcs , l - glutarnine ( 2 mm ), and penicillin / streptomycin ( 50 μg / ml ) in a 96 well flat - bottomed plate in an atmosphere of 5 % co 2 in air . after 24 hrs , the supernatants were collected centrifuged and stored at − 70 ° c . until use for tnf assays . tnf - α was measured using commercial elisa kits ( genzyme diagnostics , cambridge , mass ., usa ): the limit of sensitivity was 15 pg / ml . plasma from individual rats was obtained at various time intervals after con a injection . plasma transaminase activity was measured by the standard photometric method using a bichromatic analyzer ( department of biochemists , john hunter hospital , newcastle , australia ). plasma was collected at week 0 when rats entered the experiment , and 4 and 8 weeks after 40 % ethanol consumption . lps levels were analysed using the method of limulus amebocyte lysate ( cape cod inc , woods hole , mass ., usa ). the tests were performed exactly according to the instructions provided by the manufacturer . the livers were fixed in 10 % formalin , embedded in paraffin , sectioned , and stained with haematoxylin and eosin for histological examination . immunostaining for cd4 + and cd8 + t cells was carried out using mouse monoclonal antibodies to cd4 + and cd8 + on cryostat sections . the sections were incubated with horse - radish peroxidase rabbit anti - mouse immunoglobulin , and then developed using an avidin - biotin kit ( zymed , south san francisco , calf ., usa ). all histology and immunostaining sections were reviewed by two of the authors who were unaware of the status of the animal being examined . results are expressed as mean ± se . statistical analysis was performed using the student &# 39 ; s t test p values less than 0 . 05 were considered statistically significant . effect of herbal medicine on body weight gain , liver weight , ratio of liver weight to body weight and lps levels in chronic ethanol consuming rats administered con a data of body weight liver weight and ratio of liver weight to body weight were similar to our previous studies in ethanol - fed rats . although liver weight and ratio of liver weight to body weight tend to decrease in ethanol - fed rats administered con a after the treatment of herbal medicine , there was no significant difference ( data not shown ). serum ethanol levels were consistently greater than 20 mmol / l in alcohol fed rats . serum lps levels were significantly elevated in ethanol - fed rats without herbal treatment compared with those in ethanol - fed rats with herbal treatment ( fig1 ). after injection of con a , a significant increase in plasma transaminase levels ( alt ) was detectable as early as 12 hr , with peak levels occurring at 24 hrs ( fig2 ) in placebo rats . the data show that plasma alt levels in alcohol fed rats were higher than those in control animals ( p & lt ; 0 . 01 ), but there was no significant difference between 2 % sucrose and isocaloric sucrose rats . in contrast , alt levels were significantly lower in herbal treated rats ( fig2 ) ( p & lt ; 0 . 01 ). a sharp increase in the serum levels of tnf - α was observed at 4 hr after con a injection in all con a treated animals without herbal treatment . this gradually declined to baseline levels after 24 hrs ( fig3 ). the data showed that following a single injection of con a , a striking increase in the levels of serum tnf - α was detected earlier than the increase in plasma alt levels . tnf - α in alcohol - fed rats were greater compared with the two sucrose control groups , but there was not significant difference between the two control groups ( fig3 ). interestingly tnf - α levels were significantly lower in herbal treated rats than placebo - treated rats following con a treatment ( fig3 ). tnfα secretion in cultures of liver - associated cd4 + t cells as shown in fig4 , the secretion of tnf - α by con a stimulated liver - associated cd4 + t cells was significantly increased at 24 hr after con a injection in placebo rats . tnf - α production by liver - associated cd4 + t cells was significantly increased at 24 hr after con a injection in placebo rats . tnf - α production by liver - associated cd4 + t cells cultured with con a were significantly higher in ethanol exposed rats receiving con a without herbal treatment . in contrast , tnf - α levels secreted by cd4 + t lymphocytes were significantly lower in herbal treated rats than placebo rats following con a injection . under light microscopy , a score of 0 - 4 was recorded for the following : necrosis , cellular infiltration for zone 1 , 2 , 3 separately , and fatty change , and results are summarised in table 1 . in alcohol - fed rats without herbal treatment following con a administration , changes in liver pathology were more marked with marked necrosis of hepatocytes accompanied by cellular infiltration compared with the two control groups . this consisted mainly of mononuclear cells and neutrophils in the portal areas , around the central vein and in hepatic lobules . focal and lobular necrosis was seen with inflammatory cells found around the necrotic areas . the pathological changes were significantly decreased in herbal treated rats compared with placebo rats . immunostaining with mouse anti cd4 + monoclonal antibodies revealed more cd4 + t cells in the portal area and the central vein areas 24 hrs after con a injection in ethanol fed rats without herbal treatment than both groups of control rats . however , after herbal treatment , less cd4 + t cells were identified in ethanol - fed rats and both groups of control rats . the presence of alcohol in an animal may disturb the effects of certain compounds which would otherwise provide beneficial effects in the treatment of hepatic disorders . since the model described above relies on t cell migration to the liver where damage is induced , it presents an opportunity to test such compounds . a donor rat is treated with alcohol and a t - cell activating agent as indicated above . intrahepatic t cells are removed and injected into a recipient rat as described in cao et al 15 the contents of which is hereby incorporated by reference . the recipient rat may be “ primed ” with the compound of interest prior to injection of the donor cells or may receive the cells and subsequently undergo treatment with the compound . analyses are carried out in the manner described . consequently , the need for alcohol administration to the recipient rats is alleviated and interference between the alcohol and compound of interest is avoided . composition of herbal medicine , ch100 herbal components mg ( extract in 360 mg tablet ) artemisiafrigida ( plant ) 32 saliva miltiorrrhiza ( root ) 32 taraxacun mongolicum ( plant ) 28 astragalua membranaccus 24 loranthusparasiticus ( stem ) 24 paeconia lactiflora ( root ) 24 codonopsispilosula ( root ) 20 glecchoma longituba ( plant ) 20 polygonum cuspidatum ( root ) 20 bupleurumfalcatum ( root ) 16 crataeguspinnatifida ( fruit ) 16 gentiana scabra ( whole fruit ) 16 lycium babarum ( fruit ) 16 zizyphusjujuba ( fruit ) 16 curcuma longa ( fruit ) 16 glycyrrhiza uralensis ( root ) 12 polyporus umbelletus ( root ) 18 poria cocos ( root ) 14 pana - cpseudoginseng ( root ) 4 the invention has been described herein , with reference to specific examples , in order to enable the reader to practice the invention without undue experimentation . however , a person having ordinary skill in the art will readily recognise that many of the components and parameters may be varied or modified to a certain extent without departing from the scope of the invention and that the invention may be embodied in many other forms . furthermore , titles , headings , or the like are provided to enhance the reader &# 39 ; s comprehension of this document , and should not be read as limiting the scope of the present invention . finally , throughout this specification , and the claims which follow , unless the context requires otherwise , the word “ comprise ”, and variations such as “ comprises ” and “ comprising ”, will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps . 1 . spinozzi f , bertotto a , rondoni f , gerli i :˜ scalise f , grignani f : t lymphocyte activation pathways in alcoholic liver disease . immunology 73 : 140 - 146 , 1991 . 2 . roselle g a , mendenhall c l , chedid a , moritz t e , gartside p , the veterans affairs cooperative study groups 119 and 275 : alcohol modulation of immune function : clinical and experimental data . alcoholism : clinical and experimental research 19 ( 3 ): 551 - 554 , 1995 3 . mckeever u , o &# 39 ; mahony c , whelan c a , weir d o , feighery c : helper and suppressor t lymphocyte function in severe alcoholic liver disease . clin exp immunol 60 : 39 - 48 , 1985 . 4 . batey r , cao q , madsen g , pang g , russell a , clancy r . decreased tumor necrosis factor -( x and interleukin - 1 ( x production from intrahepatic mononuclear cells in chronic ethanol consumption and upregulation by endotoxin . alcoholism : clin & amp ; exp res 22 : 150 - 156 , 1998 . 5 . cao q , batey r , pang g , clancy r . altered t - lymphocyte responsiveness to polyclonal cell activators is responsible for liver cell necrosis in alcohol - fed rats . alcoholism : clin & amp ; exp res 22 : 723 - 729 , 1998 . 6 . cao q , batey r , pang g , russell a , clancy r . il - 6 , wn - y and tnf - α production by liver - associated t cells and acute liver injury in rats administered concanavalin a . immunology and cell biology 76 : 542 - 549 , 1998 . 7 . winnock m , barcine m g , lukomska b , huet s , saric j , balabaud c , bioulac - sage p : human liver - associated lymphocytes : a review . j gastroenterology and hepatology 10 : s43 - s46 , 1995 . 8 . wang y , huang d s , giger p t , watson r r : influence of chronic dietary ethanol on cytokine production by murine splenocytes and thymocytes . alcoholism : clin and exp res 18 ( 1 ): 54 - 70 , 1994 . 9 . roselle g a , mendenhall c l : tthan ˜ ol - induced alterations in lymphocyte function in the guinea pig . alcoholism : clinical and experimental research 8 ( 1 ): 62 - 67 , 1984 . 10 . chedid a , mendenhall c l , moritz t e , french s w , chen t s , morgan t r , roselle g a , et . al : cell - mediated hepatic injury in alcoholic liver disease . gastroenterology 105 : 254 - 266 , 1993 . 11 . khorutus a , stahnke l , mcclain c j , logan g , allen f l : circulating tumor necrosis factor , interleukin - 1 and interleukin - 6 concentrations in chronic alcoholic patients . hepatology 13 : 267 - 276 , 1991 . 12 . keegan a , martini r , batey r : ethanol - related liver injury in the rat : a model of steatosis , inflammation and pericentral fibrosis . j hepatology 23 : 591 ˜ 600 , 1995 . 13 . mizueara h , o &# 39 ; neil e , seki n , ogawa t , kusunoki c , otsuka k , satoh s , et . al : t cell activation - associated hepatic injury : mediation by tumor necrosis factors and protection by interleukin - 6 . j exp med 179 : 1529 - 1537 , 1994 . 14 . noble a , macary p a , kemeny d m : wn - y and il - 4 regulate the growth and differentiation of cd8 -′″ t cells into subpopulations with distinct cytokine profiles . j immunology 155 : 2928 - 2937 , 1995 . 15 . cao q , batey r , pang g and clancy r ( 1991 ) etlmol - modified t cell activation mediates liver injury in rats administered con a . 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