the description in this application is in particular directed to trichostatin a as a non - limiting example and is never intended to limit the scope of the invention . trichostatin a or derivatives thereof are disclosed to be useful as an antifibrotic agent for the treatment fibrosis . pharmaceutical formulations and use of compounds of trichostatin a are also disclosed . trichostatin a is an antifungal agent originally isolated from streptomyces hygroscopicus by tsuji et al . ( j . antibiot 29 : 1 - 6 , 1976 ). trichostatin a is also useful as an anticancer ( cancer res 47 : 3688 - 3691 , 1987 ) and an antiprotozoal agent ( j . antibiot 41 : 461 - 468 , 1988 ). in the course of experiments we discovered that trichostatin a has a strong antifibrotic effect on hepatic stellate cells which are the major connective tissue producing cells in the liver . trichostatin a can be brought in the form of pharmaceutically acceptable salts . as such pharmaceutically acceptable salts may be used so long as they do not adversely affect the desired pharmacological effects of the compounds . the selection and the production can be made by those skilled in the art . for instance , as a pharmaceutically acceptable salt , and alkali metal salt such as sodium salt or a potassium salt , an alkaline earth metal salt such as calcium salt or a magnesium salt , a salt with an organic base such as an ammonium salt , or a salt with an organic base such as a triethylamine salt or an ethanolamine salt , may be used . subjects to be treated by the present invention include both humans and animals . the antifibrotic agent of the present invention may be administered orally or non - orally . in the case of oral administration , it may be administered in the form of soft and hard capsules , tablets , granules , powders , solutions , suspensions or the like . in the case of non - oral administration , they may be administered in the form of ointments or injection solution , drip infusion formulations , suppositories whereby continuous membrane absorption can be maintained in the form of solid , viscous liquid , or suspension . the selection of the method for preparation of these formulations and the vehicles , disintegrators or suspending agents , can be readily made by those skilled in the art . the antifibrotic agent of the present invention may contain a further substance having antifibrotic activities , in addition to trichostatin a or its pharmaceutically acceptable salts . the amount of the active ingredients in the composition of the present invention may vary depending on the formulation , but is usually from 0 . 1 to 50 % by weight irrespective of the manner of administration . the dose is determined taking into consideration the age , sex , and symptom of the disease of the subject , the desired therapeutic effect , the period for administration , etc . however , preferably a daily dose of the active ingredient is from 0 . 05 to 100 mg for an adult . the following examples are provided to illustrate the present invention , and should not be construed as limiting thereof . trichostatin a 7 -[ 4 -( dimethylamino ) phenyl ]- n - hydroxy - 4 , 6 - dimethyl - 7 - oxo - 2 , 4 - heptadienamide was prepared from the culture broth of streptmyces platensis no . 145 . sodium butyrate was purchased from sigma ( st . louis , mo ., usa ). stock solutions of trichostatin a were prepared in ethanol ( 2 mg / ml ), stored at - 20 ° c ., and diluted as required for each experiment . the final concentration of ethanol in the medium was 0 . 0016 %. stock solutions of sodium butyrate ( 100 mmol / l ) were prepared in distilled water , and diluted as required . effect of trichostatin a and sodium butyrate on the synthesis of collagens type i and iii , and smooth muscle α - actin by hepatic stellate cells the antifibrotic activity of trichostatin a and sodium butyrate were tested using cultures of hepatic stellate cells . stellate cells were isolated from adult wistar rats ( 400 - 550 g ) by enzymatic digestion of the liver with collagenase / pronase / dnaase followed by density gradient centrifugation on nicodenz ( nycomed , oslo , norway ). after isolation cells were suspended in dulbecco &# 39 ; s modified eagle &# 39 ; s medium supplemented with 10 % fetal calf serum , 100 u / ml penicillin , and 100 μg / ml streptomycin , and cultured at 37 ° c . in a humidified atmosphere with 5 % co 2 and 95 % air . at day 3 cells were exposed to trichostatin a ( 1 - 100 nmol / l ) for 24 h . during the subsequent 24 h cells were metabolically labeled with 25 μci / ml of trans 35s - label ( specific activity of 35 s - methionine & gt ; 1 , 000 ci / mmol , icn biomedicals , costa mesa calif .) while exposure to the compounds was continued . after labelling medium was collected , and subjected to immunoprecipitation using antibodies against collagens type i and iii and smooth muscle α - actin . the precipitates were fractionated by sds - page and radioactivity of specific bands were measured by the phosphorimaging technology . for the effect at mrna level , cells were exposed to 100 nmol / l trichostatin a for 24 h . rna was then extracted and analyzed by northern hybridization analysis . table 1 and 2 show the results which were expressed as percentage value relative to the control culture , respectively for trichostatin a and sodium butyrate . note the dose - dependent suppression of collagens type i and iii synthesis by trichostatin a . also , note the strong suppression of smooth muscle α - actin , an activation marker of stellate cells . table 1______________________________________results for trichostatin a 100 nm 10 nm 1 nm______________________________________collagen i 37 . 9 ± 5 . 6 68 . 9 ± 4 . 7 91 . 7 ± 9 . 5collagen iii 30 . 1 ± 9 . 6 73 . 2 ± 20 . 9 71 . 9 ± 21 . 0sm α - actin 15 . 5 ± 7 . 4 54 . 4 ± 5 . 3 87 . 6 ± 0 . 3______________________________________ sodium butyrate suppressed smooth muscle α - actin less effectively , with 50 % reduction at a concentration of 1 mmol / l . inhibition of collagen type iii and smooth muscle α - actin synthesis indicated that trichostatin a was more potent than butyrate by 5 orders of magnitude . table 2______________________________________results for sodium butyrate 10 . sup .- 3 m 10 . sup .- 4 m 10 . sup .- 5 m______________________________________collagen i 107 . 8 ± 9 . 8 127 . 9 ± 16 . 2 92 . 8 ± 11 . 7collagen iii 67 . 9 ± 19 . 1 90 . 6 ± 42 . 2 109 . 4 ± 31 . 1sm α - actin 50 . 0 ± 19 . 9 91 . 6 ± 23 . 4 97 . 9 ± 24 . 4______________________________________ effect of trichostatin a on the gene expression of collagens type i and iii , and smooth muscle α - actin by hepatic stellate cells hepatic stellate cells were isolated and cultured as described in example 1 . cells were exposed to 100 nmol / l trichostatin a for 24 h . rna was then extracted and analyzed by northern hybridization analysis . at day 3 cells were exposed to 100 nmol / l trichostatin a or 1 mmol / l sodium butyrate for 24 h . total rna was extracted by the method of chomczynski and sacchi . northern hybridization was performed using p - labeled cdna probes for rat procollagen α 1 ( i ) ( 1 . 6 kb pst i fragment ), rat procollagen α 1 ( iii ) ( 0 . 5 kb hind iii / ecori fragment ), and glyceraldehyde - 3 - phosphate dehydrogenase ( gapdh ) ( 0 . 5 kb xbai / hindiii fragment ). for smooth muscle α - actin , a crna probe corresponding to the 5 &# 39 ;- untranslated region of mouse smooth muscle α - actin mrna was used as described previously . the results were quantitated by phosphor - imaging and corrected for gapdh . the results at the mrna level are shown in table 3 . collagen type iii and smooth muscle α - actin mrna levels were suppressed to a similar extent as at the protein level . on the other hand , only modest tendency for suppression was observed for collagen type i , suggesting that the suppression of collagen type i was mainly post - translational . table 3______________________________________ 100 nm______________________________________collagen i 79 . 3 ± 13 . 5collagen iii 39 . 0 ± 13 . 5sm α - actin 20 . 6 ± 15 . 4______________________________________ effect of trichostatin a and sodium butyrate on the cell proliferation of hepatic stellate cells finally , the applicant examined the effects of trichostatin a and sodium butyrate on proliferation of stellate cells , since high proliferative activity is one of the major features of myofibroblastic differentiation . cells were cultured in triplicate or quadruplicate in 24 well plates ( costar ). cells at day 2 were exposed to 0 . 01 - 1 mmol / l sodium butyrate or 1 - 100 nmol / l trichostatin a for the 4 subsequent days . culture medium and test compounds were replaced every day . at day 6 cells were trypsinized and counted in a hemocytometer . trichostatin a showed at 100 nmol / l a strong suppressive effect on proliferation . table 4 summarizes these cell - count results . table 4______________________________________cell - count results______________________________________ 10 . sup .- 7 m 10 . sup .- 8 m 10 . sup .- 9 mcontrol trichostatin a trichostatin a trichostatin a______________________________________23 . 7 ± 1 . 9 16 . 2 ± 1 . 0 22 . 0 ± 3 . 0 22 . 9 ± 1 . 0______________________________________ 10 . sup .- 3 m 10 . sup .- 4 m 10 . sup .- 5 mcontrol butyrate butyrate butyrate______________________________________23 . 9 ± 2 . 0 20 . 4 ± 0 . 2 22 . 2 ± 0 . 7 21 . 1 ± 2 . 0______________________________________ finally cells were cultured in triplicate or quadruplicate in 24 well plates . at day 4 cells were exposed tot 0 . 01 - 1 mmol / l sodium butyrate or 1 - 100 nmol / l trichostatin a for 24 h . subsequently , medium was changed and cells were further incubated for 20 h with the same concentrations of sodium butyrate or trichostatin a in the presence of 10 μci / ml [ 3 h ]- thymidine ( specific activity 25 ci / mmol , 10 μci / ml ). radioactivity incorporated into the 2 % perchloric acid / 95 % ethanol / insoluble fraction was measured by scintillation counting . parallel cultures incubated with [ 3 h ]- thymidine in the presence of 10 mmol / l hydroxyurea provided the baseline value , which was subtracted from each measurement . final data were normalized for cell number which was determined by trypsinization of parallel wells . table 5 shows the results expressed in cpm / cell . table 5______________________________________ [. sup . 3 h ]- thymidine incorporation______________________________________ 10 . sup .- 7 m 10 . sup .- 8 m 10 . sup .- 9 mcontrol trichostatin a trichostatin a trichostatin a______________________________________10 . 0 ± 0 . 8 1 . 3 ± 0 9 . 9 ± 0 . 4 9 . 6 ± 0 . 4______________________________________ 10 . sup .- 3 m 10 . sup .- 4 m 10 . sup .- 5 mcontrol butyrate butyrate butyrate______________________________________9 . 9 ± 0 . 4 6 . 2 ± 0 . 2 9 . 5 ± 0 . 3 9 . 5 ± 0 . 2______________________________________ skin fibroblasts were obtained from male wistar rats ( 300 - 400 g ) by the explant tehnique as described previously . all rats were fed ad libitum , and received humane care in compliance with the institution &# 39 ; s guidelines for the care and use of laboratory animals in research . cells were grown in dulbecco &# 39 ; s modified eagle &# 39 ; s medium with 10 % fetal calf serum , and cultured at 37 ° c . in a humidified atmosphere with 5 % co 2 and 95 % air . when the culture became confluent , cells were trypsinized and replated into 75 cm 2 culture flasks in a split ratio of 1 : 4 . experiments were performed using confluent cells between passage 5 and 9 . preliminary experiments have shown that under these conditions , skin fibroblasts had acquired the myofibroblast phenotype at the time of experiments , as evidenced by their large size , prominent stress fibers , and expression of smooth muscle α - actin . confluent cultures of skin fibroblasts were exposed to 1 , 10 and 100 nmol / l trichostatin a for 24 h . for the tgf - β 1 experiment , cells were exposed to 5 ng / ml of human recombinant tgf - β 1 ( calbiochem ) and / or 100 nmol / l trichostatin a for 24 h . after the initial 24 h exposure of these compounds , cells were metabolically labeled for 24 h with 50 μci / ml of trans 35 s - label ( specific activity of 35 s - methionine & gt ; 1 , 000 ci / mmol , icn biomedicals , costa mesa , calif .) in the presence of vitamin c ( 50 μg / ml ) ( merck ) and β - aminopropionitrile ( 64 μg / ml ) ( sigma ), while exposure to trichostatin a and / or tgf - β 1 was continued . labeled media or cell layers were separately harvested and stored at - 70 ° c . protein synthesis was measured by trichloroacetic acid ( tca ) precipitation . equal counts ( 10 6 cpm ) of labeled media or cell lysates were subjected to immunoprecipitation . immunoprecipitation was performed using antibodies against collagen type i ( southern biotechnology , birmingham , ala . ), type iii ( gift by prof . dr . d . schuffan , freie univ . berlin , deutschland ) or smooth muscle α - actin ( clone 1a4 , sigma ). after immunoprecipitation , proteins were separated by sds - page , gels were immersed in amplify ( amersham , little charfort , uk ) and dried , exposed to preflashed autoradiography film ( hyperfilm - mp , amersham ) or quantitatively analyzed by phosphor - imaging ( molecular imager , gs - 525 , biorad , usa ). confluent cultures of fibroblasts were exposed to 100 nmol / l trichostatin a and / or 5 ng / ml tgf - β 1 for 24 h . total rna was extracted by the method of chomczynski and sacchi . northern hybridization was performed as described , using 32 p - labeled cdna probes for rat procollagen α 1 ( i ) ( 1 . 6 kb pst i fragment ), rat procollagen α 1 ( iii ) ( 0 . 5 kb hind iii / ecori fragment ), and gapdh ( glyceraldehyde - 3 - phosphate dehydrogenase ) ( 0 . 5 kb xbai / hindiii fragment ). for smooth muscle α - actin mrna was used as described previously . the results were quantitated by phosphor - imaging and corrected for gapdh . the ratios of protein or mrna levels of treated vs . controls were calculated for each experimental condition and expressed as means ± standard deviation . for the protein study , correction was made for the difference , if any , in the trichloroacetic acid - precipitable counts . the number of experiments used to calculate a mean value was at least 3 . the effect was considered statistically significant , when 1 . 0 did not belong to the 95 % confidence interval of the treated / control ratio . then we metabolically labeled endogenously produced protein with 35 s - methionine , and immunoprecipitated collagen type i and iii using specific antibodies . the immunoprecipitated proteins were separated on sds - page and quantified by phosphor - imaging . we found that at 100 nmol / l , 10 nmol / l , and 1 nmol / l trichostatin a inhibited synthesis of collagen type i by 30 %, 44 %, and 17 %, respectively ( table 6 , row 2 ). synthesis of collagen type iii was inhibited by 41 %, 49 %, and 10 % at the same concentrations of trichostatin a ( table 6 , row 3 ). thus , both collagen type i and iii were inhibited by trichostatin a ( p & lt ; 0 . 05 for 100 nmol / l and p & lt ; 0 . 01 for 10 nmol / l ), with the maximal effect obtained at 10 nmol / l . synthesis of smooth muscle α - actin , a marker for myofibroblast differentiation , was also inhibited by 37 ± 18 %, 36 ± 8 %, respectively , following exposure to 100 nmol / l and 10 nmol / l trichostatin a ( table 6 , row 4 ). these inhibitory effects of trichostatin a was selective , since over all protein synthesis as measured by tca - precipitable counts was not affected . next we explored whether trichostatin a affected the fibrogenic actions of tgf - β 1 in skin fibroblasts . for this purpose , cells were exposed to tfg - β 1 ( 5 ng / ml ) alone , trichostatin a ( 100 ng / ml ) alone , or combination of tfg - β 1 ( 5 ng / ml ) and trichostatin a ( 100 nmol / l ). collagen type i and iii , and smooth muscle α - actin were again immunoprecipitated , separated on sds - page and quantified by phosphor - imaging . as shown in table 7 , exposure to tgf - β 1 stimulated synthesis of collagen type i and iii 3 . 4 and 4 . 7 fold , respectively . although stimulation occurred in every preparation of skin fibroblasts , the magnitude of stimulation varied from culture to culture ranging from 1 . 8 to 5 . 9 fold for collagen type i and from 2 . 5 to 8 . 0 fold for collagen type iii . tgf - β 1 , had a modest stimulating effect ( 1 . 8 fold increase ) on the synthesis of smooth muscle α - actin . strikingly , the stimulating effect of tgfβ 1 was largely abolished , when tgf - β 1 and trichostatin 1 were added simultaneously . finally , we explored at which level trichostatin a exerted its inhibitory effect on the collagen synthesis by skin fibroblasts . for this purpose we again exposed skin fibroblasts to trichostatin a and / or tgf - β1 . after 24 hour exposure , total rna was extracted and subjected to northern hybridization analysis . the radioactivity was measured , corrected for gapdh , and expressed as a ratio to the value of the control culture . as shown in table 8 , tgf - β 1 ( 5ng / ml ) increased gene expression of collagens type i , and iii , and smooth muscle α - actin 2 . 3 fold , 2 . 5 fold and 1 . 7 fold , respectively . trichostatin a alone ( 100 nmol / l ) had a modest suppressive effect on the mrna levels of collagens type i and iii and smooth muscle α - actin . these data suggest that suppressive effect of trichostatin a occurs both at the transcriptional and posttranslational level . the results of the above mentioned examples are summurized in table 6 , 7 and 8 . table 6______________________________________the effect of different concentrations of tsa at the proteinlevel . results are expressed as percentage of control value 100 nm 10 nm 1 nm______________________________________collagen i 70 +/- 13 % 56 +/- 11 % 83 +/- 11 % collagen iii 59 +/- 18 % 51 +/- 11 % 90 +/- 14 % α - sma 63 +/- 8 % 64 +/- 8 % 86 +/- 23 % ______________________________________ table 7______________________________________the effect of tsa on inducing effect of tgf - β atthe protein level tgf - β tgf - β + tsa tsa______________________________________collagen i 347 ± 185 % 121 ± 39 % 70 ± 13 % collagen iii 476 ± 273 % 86 ± 35 % 59 ± 18 % sma 175 ± 196 % 67 ± 6 % 63 ± 8 % ______________________________________ table 8______________________________________the effect of tsa on the inducing effect of tgf - βat the mrna level tgf - β tgf - β + tsa tsa______________________________________collagen i 231 ± 89 % 132 ± 49 % 84 ± 14 % collagen iii 253 ± 94 % 180 ± 51 % 74 ± 10 % sma 171 ± 61 % 114 ± 24 % 85 ± 16 % ______________________________________ the results in this application indicate that histone deacetylase inhibitors provide a novel therapeutic potential in the treatment of fibro - proliferative diseases . in conclusion , we have demonstrated that two unrelated histone deacetylase inhibitors are active antifibronics compounds as two well known experimental models of fibrosis , i . e . hepatic fibrosis and skin fibrosis . the invention further relates to a method for the treatment of humans or animals afflicted with fibrosis , comprising administering to said subject an effective amount of a histone deacetylase inhibitor in particular trichostatin a or a pharmaceutically acceptable salt thereof and optionally a suitable excipient .
compounds of submicromolar concentrations of a histone deacetylase inhibitor , useful as anti - fibrotic agents . the compounds strongly inhibit three main features of myofibroblastic differentiation of cultured hepatic stellate cells and of skin fibroblasts : synthesis of collagens type i and iii , the predominant collagens in fibrosis of liver and other organs ; cellular proliferation ; and expression of smooth muscle α - actin , a marker for activated myofibroblasts . preferred compounds include trichostatin a or a pharmaceutically acceptable salt thereof , and sodium butyrate .