Patent Application: US-33091899-A

Abstract:
the invention relates to compositions which modulate airway remodelling comprisiing the sterioid 2 - methyoxy - estradiol or analogues thereof , for administration by inhalation .

Description:
the invention will now be described in detail by way of example only , with reference to the following figures in which : fig1 shows the lack of effect of 2 - methoxyestradiol on horseradish peroxidase - mediated oxidation of tetramethyl benzidine . fig2 shows the effect of 2 - methoxyestradiol ( 0 . 3 - 10 μm , 30 min pretreatment ) on mitogen - induced incorporation of [ 3 h ]- thymidine in cultured human airway smooth muscle cells . mitogens tested were 0 . 3 u / ml thrombin , 1 % fcs , 300 pm bfgf ( fig2 a ) and 10 % fcs , 3 nm egf ( fig2 b ). additional experiments of identical design to those depicted in fig2 a & amp ; b were carried out and the combination of this latter data and that in fig2 a & amp ; b is presented in fig2 c . the time - course of the effect of 2 - methoxyestradiol ( 3 μm ) on thrombin ( 0 . 3 u / ml )- induced dna synthesis were also investigated ( 2 d ). data are presented as the means and standard errors of the means of 3 experiments in 3 different cultures , and are expressed as a percentage of the [ 3 h ]- thymidine incorporation in non - pretreated cells . fig3 shows the effect of 2 - methoxyestradiol ( 0 . 3 - 10 μm , 30 min pretreatment ) on mitogen - induced incorporation of [ 3 h ]- leucine . data are presented as the means and standard errors of the means of 3 experiments in 3 different cultures , and are expressed as a percentage of the [ 3 h ]- leucine incorporation in non - pretreated cells . fig4 shows the effect of 2 - methoxyestradiol ( 0 . 3 - 10 μm , 30 min pretreatment ) on cell number in the presence of ( a ) fcs ( 1 %) or ( b ) bfgf ( 300 pm ). data are presented as the means and standard errors of the means of 3 experiments in 3 different cultures , and are expressed as a percentage of the increase in cell number in non - pretreated cells . fig5 shows the effect of the steroid receptor antagonist , ru 486 ( 1 μm ), on 2 - methoxyestradiol ( 3 μm ) inhibition of fcs ( 1 %)- induced dna synthesis . data are presented as the means and standard errors of the means of 3 experiments in 3 different cultures , and are expressed as a percentage of the [ 3 h ]- thymidine incorporation in non - pretreated cells . ru 486 was added 30 min before 2 - methoxyestradiol , which was added 30 min before fcs . fig6 shows the effect of 17 62 - estradiol and 2 - hydroxyestradiol ( 0 . 3 - 10 μm , 30 min pretreatment ) on fcs ( 1 %)- induced incorporation of [ 3 h ]- thymidine . data are presented as the means and standard errors of the means of 3 experiments in 3 different cultures , and are expressed as a percentage of the [ 3 h ]- thymidine incorporation in non - pretreated cells . data relating to 2 - methoxyestradiol are reproduced from fig1 for ease of comparison . fig7 shows the effects of 2 - methoxyestrone and 2 - methoxyestradiol ( 0 . 3 - 10 μm , 30 min pretreatment ) on thrombin ( 0 . 3 u / ml )- induced incorporation of [ 3 h ]- thymidine . data are presented as the means and standard errors of the means of 3 experiments in 3 different cultures , and are expressed as a percentage of the [ 3 h ]- thymidine incorporation in non - pretreated cells . data relating to 2 - methoxyestradiol are reproduced from fig2 a for ease of comparison . fig8 shows the effect of 2 - methoxyestradiol on superoxide anion release in guinea - pig peritoneal macrophages . this compound completely blocked superoxide anion response of the macrophages to zymosan and reduced those to fmlp . fig9 shows the effect of 2 - methoxyestradiol on prostacylin release in guinea - pig peritoneal macrophages . the results demonstrate that the estradiol completely blocked the macrophage response to fmlp whilst the response to pma was inhibited by 50 %. fig1 is a plot showing the effects of various concentrations of ovalbumin ( dnp - oa ) on mast cell ( rbl2h3 ) degranulation , and the inhibition by 2 - methoxyestradiol . fig1 shows that 2 - methoxyestradiol reduced ovalbumin ( dnp - oa )- stimulated release of [ 3 h ]- 5ht from guinea - pig peritoneal macrophages , but did not affect release in response to pma . human bronchial airway smooth muscle was obtained from macroscopically normal lung resection specimens from lung transplant donors or recipients provided by the alfred hospital ( melbourne ). cultures were prepared as previously described in detail ( tomlinson et al ., 1994 ). briefly , the tissue was partially digested in dulbecco &# 39 ; s modified eagle &# 39 ; s medium ( dmem ), [ supplemented with 2 mm l - glutamine , 100 μg / ml streptomycin , 100 u / ml penicillin - g , 2 μg / ml amphotericin b , and 0 . 25 % w / v bovine serum albumin ( bsa )] containing 3 mg / ml collagenase for 30 minutes at 37 ° c . and approximately 0 . 5 g smooth muscle was further digested by a 2 hour incubation in 0 . 5 mg / ml elastase , followed by an 18 hour incubation in collagenase ( 3 mg / ml ) at 37 ° c . cell suspensions ere centrifuged ( 10 min , 100 × g , 25 ° c . ), washed three times in supplemented dmem , resuspended in 25 ml dmem containing 10 % ( v / v ) heat - inactivated foetal calf serum ( fcs ), seeded into 25 cm 2 falcon culture flasks and incubated ( 37 ° c ., 5 % co 2 ) for 7 to 10 days until monolayer confluence was reached . cells were then harvested weekly by 10 min exposure to 0 . 5 % trypsin , 1 mm edta and passaged at a 1 : 3 split ratio into 75 cm 2 falcon culture flaskes . cells at passage numbers 3 to 15 were used for experiments . cells were subcultured into 8 - well glass tissue culture chamber slides ( labtek ), and grown to 100 % confluency in dmem ( 10 % fcs ). slides were washed three times in pbs , before fixation for 20 seconds in ice - cold acetone and stored for up to four weeks at 4 ° c . before staining . following rehydration in pbs / bsa ( 0 . 25 %) for twenty minutes , the cells were permeabilized by incubation in 0 . 5 % triton x - 100 ( in pbs ) and incubated with primary antibody for at least 60 minutes at 22 ° c . the primary antibody was removed by washing 3 times with 0 . 25 % bsa in pbs , and then the cells were exposed to the secondary antibody for at least 60 minutes at 22 ° c . ( horseradish peroxidate ( hrp )- conjugated goat anti - mouse ; ig f ( ab ′) 2 fragment or goat anti - rabbit igg ). controls were provided by substituting the primary antibody for pbs / bsa ( 0 . 25 %). the staining of the fixed cells was analysed by light microscopy ( olympus bh2 attached to a videopro 32 image analysis system , faulding imaging , clayton , victoria ). the characteristics of the antibodies used to identify the smooth muscle in culture were established on native airway wall specimens . the antibodies used were raised against α - actin , myosin , calponin ( all specific to smooth muscle ), cytokeratin ( epithelial cells ) and pecam - 1 ( cd31 , which is a marker of endothelial cells ). the expression of smooth muscle α - actin , myosin and calponin was observed in all cultures used in this study . these cultures did not express detectable pecam - 1 staining , and less than 5 % of the cells were positive for staining with the monoclonal antibody against cytokeratin . paraffin - embedded sections of the airway adjacent to that used for generation of cultures stained positively for smooth muscle α - actin and myosin in bundles of airway smooth muscle and blood vessels only . the antibody against pecam - 1 stained vascular endothelium , whereas that against cytokeratin stained only the epithelium , confirming the specificity of these antibodies for the target antigens . cells were subcultured into 24 - well plates at a 1 : 3 ratio and allowed to grow to monolayer confluency over a 72 - 96 hour period in an atmosphere of 5 % co 2 in air at 37 ° c . the serum - containing medium was replaced with serum - free dmem for a 24 hour period to produce growth arrest . in some experiments , the cells were pretreated with 2 - methoxyestradiol 30 min before the addition of mitogen . the stimulant ( mitogen ) was added to the appropriate wells together with a supplement containing insulin , transferrin , and selenium ( monomed a , 1 % v / v ). monomed a was added to provide progression factors which are essential for the mitogenic activity of growth factors such as thrombin , epidermal growth factor ( egf ) and basic fibroblast growth factor ( bfgf ) ( stewart et al ., 1995a ). mitogens and inhibitors were left in contact with cells from the time of addition until the end of the experiment , unless indicated otherwise . cells were incubated for 24 hours ( 37 ° c ., 5 % co 2 ) before being pulsed with [ 3 h ]- thymidine ( 1 μci / ml for four hours ) to measure incorporation of radiolabel into newly synthesized dna , according to out previous study ( stewart et al ., 1995 ). incorporation of radioactivity was determined by filtration at the end of the pulse - labelling period . the medium containing the radioactivity was aspirated and the cells were lysed by addition of 200 μl of 0 . 1m naoh . the dna was immobilised by filtration in a binding harvester ( packard filtermate 196 ) on glass fibre filters ( packard , standard ), which were then washed with 3 × 3 ml volumes of distilled water and a single 1 ml volume of 100 % ethanol . the dried filters were counted in a packard topcount liquid scintillation counter . protein synthesis rates were determined in experiments of analogous design to those described above , but [ 3 h ]- leucine replaced [ 3 h ]- thymidine in the pulsing incubation of 4 hours . furthermore , in experiments to determine the effects of mitogens and 2 - methoxyestradiol on the rate of protein synthesis , incubations with mitogen were carried out for a period of 48 hours . the longer duration of these experiments was required to allow sufficient time for cell division to occur . the progression of airway smooth muscle cells through the cell - cycle to mitosis was determined by measuring changes in cell number in experiments of analogous design to those used for dna synthesis , except that the incubations with mitogen were continued for 48 hours . cells were removed from each of the wells of 6 - well culture plates used in these experiments by exposure to 200 μl of 0 . 5 % trypsin in psb containing 1 mm edta , for a period 30 - 45 min to ensure that the cells were completely dissociated from each other and from the culture plate to enable an accurate count to be made . at the end of this period , a further 200 μl of pbs ( 20 % fcs ) was added to prevent cell lysis by trypsin and cells were counted directly in a haemocytometer . each treatment in an individual experiment was carried out in quadruplicate for dna and protein synthesis experiments . each experiment was performed in at least three different cultures obtained from three different individuals . for cell counting , single incubations were carried out in three cultures . results are presented as grouped data from multiple cultures and are expressed as mean ± s . e . of n cultures . the degree of increment was calculated by dividing the response of treated wells by that of the control wells on the same 24 - well plate . the grouped data was analysed by paired t - test after normalisation by log transformation . the bonferroni adjustment for multiple comparisons was used when necessary . differences were considered to be significant when p & lt ; 0 . 05 . all chemicals used were of analytical grade or higher . the compounds used and their sources were as follows : 2 - methoxyestradiol ( 1 , 3 , 5 [ 10 ]- estratriene - 2 , 3 , 17 - triol 2 - methylether lot 83h4065 ); 17β - estradiol (( 1 , 3 , 5 [ 10 ]- estratriene - 3 , 17β - diol , cat no . e8876 ); 2 - methoxyestriol ( 1 , 3 , 5 [ 10 ]- estratriene - 2 , 3 , 16α , 17β - tetrol , lot 26f 4038 ; 2 - methoxyestrone , ( 2 , 3 - dihydroxy 1 , 3 , 5 [ 10 ]- estratriene - 17 - one , lot 110f4003 ); 2 - hydroxyestradiol , ( 1 , 3 , 5 [ 10 ]- estratriene - 2 , 3 , 17β - triol lot 75h0853 ); l - glutamine , essentially fatty acid free bovine serum albumin fraction v ( bsa ), thrombin ( bovine plasma ), sigma , usa ; amphotericin b ( fungizone ), human recombinant basic fgf ( bfgf ), promega , usa ; collagenase type cls 1 , elastase , worthington biochemical , usa ; dulbecco ‘ a ’ phosphate buffer saline ( rbs ), oxoid , england ; trypsin , versene , penicillin - g , streptomycin , monomed a , csl , australia , foetal calf serum ( fcs ), flow laboratories , australia ; dulbecco &# 39 ; s modified eagle &# 39 ; s medium ( dmem ), flow laboratories , scotland . [ 6 - 3 h ]- thymidine ( 185 gbq / mmol , 5 ci / mmol ), amersham , uk ; microscint - o scintillant , canberra - packard , australia . the antibodies used for immunocytochemistry were anti - smooth muscle α - actin ( mouse monoclonal ) ( dako m851 ), monoclonal mouse anti - pecam - 1 ( dako - cd31 , jc / 70a ) ( dako m823 ), dako corporation , usa ; anti - cytokeratin ( mouse monoclonal cy90 , sigma , usa ) anti - mouse ig f ( ab ′) 2 fragment fitc - conjugate ( host sheep ), sheep anti - rabbit ig hrp - conjugate ( silenus ddaf ), silenus , australia , and anti - smooth muscle myosin ( rabbit polyclonal ), provided by professor m sparrow , perth , wash . leucocyte activation is a feature of the pathology of asthma . the binding of 2 - methoxyestradiol to the colchicine binding site on tubulin raised the possibility that this compound interferes with leukocyte functions such as phagocytosis and locomotion . functional effects of 2 - methoxyestradiol were examined on polymorphonuclear leukocytes ( pmn ) and adherent monocytes obtained from human peripheral blood . superoxide anion generation was determined by superoxide dismutase — sensitive reduction of cytochrome c ( stewart & amp ; harris , 1992 ). the release of myeloperoxidase was determined by oxidation of tetramethyl - benzidine ( menegazzi et al 1992 ). phagocytosis was determined by radioiordination of zymosan particles ( shelton & amp ; hosking , 1975 ). guinea - pig peritoneal macrophages were harvested and cultured according to out previous studies ( stewart & amp ; phillips , 1989 ). cells were incubated with stimuli including the chemotactic tripeptide , formyl methiony leucyl phenylalanine ( fmlp , 100 nm ), zymosan ( 400 μg / ml ) or phorbol myristate acetate ( pma , 100 nm ) for 30 min in the presence or absence of 2 - methoxyestradiol ( 10 μm ) added 15 min before the stimuli . superoxide anion was determined by superoxide dismutase - sensitive reduction of cytochrome c ( stewart & amp ; harris , 1992 ) and the stable metabolite of prostacyclin , 6 - oxo - pgf1was measured by radioimmunoassy ( stewart & amp ; phillips , 1989 ). all individual incubations were carried out in duplicate and experiments were carried out in macrophages from 5 guinea - pigs . rbl2h3 cells were cultured in rpmi 1640 containing 10 % fcs and were passaged into 24 well plates for experiments . the cells were sensitised by a 48 hour incubation with 50 % ( v / v ) conditioned medium from a lymphoid cell line secreting anti - dnp ovalbumin antibody . during the last 24 hours of this incubation [ 3 h ]- 5ht ( 1 μci / ml ) was added to each of the wells to label granular amine stores . at the end of the incubation period , the medium was aspirated , the cells were washed twice in rpmi 1640 and incubated in rpmi 1640 ( 0 . 25 % bsa ) in the absence or presence of 2 - methyoxyoestradiol for 15 mins prior to stimulation with dnp - treated ovalbumin , a23187 or pma for 30 mins at which time the supernatants were harvested , subjected to centrifugation ( 1000 × g , 4 ° c ., 5 min ) and aliquots taken for determination of the amount of [ 3 h ]- 5ht released . all experiments were carried out in quadruplicate . the results showed that 2 - methoxyestradiol ( 3 μm reduced oxidation of tetramethyl - benzidine in leukocytes stimulated with either zymosan ( 400 μg / ml ) or fmlp , as shown in table 1 . cell - free supernatants from fmlp stimulated leukocytes also contained myeloperoxidase activity as determined by tetramethyl - benzidine oxidation , but this activity was reduced only by the highest concentration of 2 - methoxy - estradiol ( 10 μm ). in addition , experiments were carried out to examine whether there was a direct effect of 2 - methoxyoestradiol on oxidation of tetramethyl benzidine by purified horseradish peroxidase . 2 - methoxyestradiol ( 10 μm had no effect in this assay . results are summarised in fig1 . in pmn , superoxide anion generation in response to fmlp ( 100 nm ) or zymosan ( 400 μg / ml ) was not reduced by concentrations of 2 - methoxyestradiol up to 10 μm . in phagocytosis experiments , radioiodination of zymosan particles by pmn was reduced by 2 - methoxyestradiol with significant effects being observed at both 3 and 10 μm , as shown in table 2 . in adherent monocytes the oxidation of tetramethyl benzidine in response to phorbol myristate acetate ( 1 μm ) pma or zymosan ( 400 μg / ml ) was unaffected by 10 μm 2 - methoxyestradiol . furthermore , superoxide anion generation in response to pma was also unaffected in this cell type . however , zymosan - stimulated superoxide anion generation appeared to be markedly inhibited by 2 - methoxyestradiol ( 10 μm ) in monocytes from at least some donors . the superoxide anion response to guinea - pig macrophages to zymosan or fmlp was reduced by 2 - methoxyestradiol as shown in fig8 . however , the response to pma ( 100 nm ) was unaffected . in addition , fmlp ( 100 nm )- induced increases in 6 - oxo - pgf1 α generation were completely blocked by 2 - methoxyestradiol , whereas the response to pma was reduced by only 50 %, and zymosan did not stimulate an increase in the levels of the prostacyclin metabolite a shown in fig9 . ovalbumin ( dnp - oa ) elicited a concentration - dependent release of [ 3 h ]- 5ht which was reduced by 10 μm 2 - methoxyestradiol as can be seen in fig1 . however , the basal release of [ 3 h ]- 5ht and that in response to either pma ( 100 nm ) or the calcium ionophone a23187 ( 10 μm ) were unaffected by 2 - methoxyestradiol as shown in fig1 . the inhibitory effects of 2 - methoxyestradiol on pmn myeloperoxidase release and activity , together with the reduction in phagocytosis , indicate that the compound will have an anti - inflammatory effect in vivo . the selective inhibition of zymosan - stimulated superomide anion generation suggests a specific effect on this phagocytic stimulus . these observations and our experiments showing inhibitory effects on macrophage function provide clear evidence of anti - inflammatory properties of benefit in asthma and other chronic obstructive airways diseases , particularly those with demonstrable pmn involvement . incubation of human cultured airway smooth muscle cells with 0 . 3 - 10 μm of 2 - methoxyestradiol for 30 min before mitogen addition , and throughout the remaining 28 hours of the experiment , caused a concentration - dependent reduction in thrombin ( 0 . 3 u / ml )- stimulated incorporation of [ 3 h ]- thymidine , as shown in fig2 a . at the highest concentration of 2 - methoxyestradiol used ( 10 μm ), the response to thrombin was reduced to approximately 10 % of the control level . this inhibitory effect of 2 - methoxyestradiol on dna synthesis was not restricted to the presence of thrombin , as similar concentration - related inhibitory effects of 2 - methoxyestradiol were observed in cells in which dna synthesis was stimulated with either foetal calf serum ( fcs , 1 % v / v ) or basic fibroblast growth factor ( bfgf , 300 pm ) ( fig2 a and 2 b ). however , dna synthesis in the presence of either efg ( 3 nm ) or 10 % fcs was inhibited to a significantly lesser extent than responses to thrombin , bfgf or lower concentrations of fcs ( fig2 c ). the dna synthesis in response to 10 % fcs ( 27 . 2 ± 7 . 8 times more than the unstimulated level of [ 3 h ]- thymidine incorporation ) was significantly greater ( p & lt ; 0 . 05 , paired student &# 39 ; s t - test ) than the response to 0 . 3 u / ml thrombin ( 8 . 4 ± 3 . 1 fold ), 3 nm egf ( 4 . 5 ± 0 . 7 ) or 1 % fcs ( 12 . 7 ± 0 . 4 ), but not significantly different from the response to 300 pm bfgf ( 22 . 5 ± 5 . 3 ). time - course studies were also carried out to determine whether addition of 2 - methoxyestradiol , after exposure to mitogens , still inhibited dna synthesis . thrombin ( 0 . 3 u / ml )- stimulated dna synthesis was inhibited when 2 - methoxyestradiol ( 3 μm ) was added up to 4 hours after the thrombin , with maximum inhibition being observed at 2 hours after thrombin addition . addition of 2 - methoxyestradiol between 4 and 14 hours after the thrombin resulted in a small inhibition (˜ 20 %), whereas addition at 18 hours or later had no effect on the dna synthesis in the presence of this mitogen as shown in fig2 d . subsequently , additional time points were examined and these studies indicated that the highest level of activity was observed when 2 - methoxyestradiol was added either simultaneously or 1 hour after thrombin , but significant inhibition persisted up to 6 hours after thrombin addition ( fig2 d ). in order to determine whether inhibition of dna synthesis also resulted in arrest of cell - cycle progression and inhibition of mitosis , measurements of both protein synthesis and cell numbers after 48 hours of incubation with mitogens were made . the threshold concentration for inhibition of incorporation of [ 3 h ]- leucine in the presence of thrombin ( 0 . 3 u / ml ), fcs ( 1 % v / v ) or bfgf ( 300 pm ) was 1 μm , and was similar to the results for inhibition of [ 3 h ]- thymidine incorporation . the maximum percentage reduction of the response of approximately 30 % was less than the value observed with dna synthesis , and occurred at 3 μm . at 10 μm , there was no significant inhibitory effect in the presence of thrombin or bfgf , as shown in fig3 . 2 - methoxyestradiol alone caused a small stimulation of [ 3 h ]- leucine incorporation at 0 . 3 μm . higher concentrations ( 1 and 3 μm ) had small inhibitory effects and at 10 μm there was no effect . these results are summarised in table 3 . in contrast , the increases in cell number in response to either fcs ( 1 %, v / v ) or bfgf ( 300 pm ) were more sensitive to inhibition by 2 - methoxyestradiol than either protein or dna synthesis , with complete inhibition of the proliferation responses being observed at 3 μm as shown in fig4 a and b . serotonin ( 5ht ) at concentrations from 0 . 1 nm up to 10 μm had no effect on incorporation of [ 3 h ]- thymidine , but 10 nm 5ht increased incorporation of [ 3 h ]- leucine . preincubation with 0 . 3 - 10 μm of 2 - methoxyestradiol decreased the 5ht ( 10 nm )- stimulated increase in protein synthesis in a concentration - dependent manner , as summarized in table 4 . morpholiogical changes including the manifestation of a rounded appearance of the normally spindle - shaped cells were observed at concentrations of 3 and 10 μm of 2 - methoxyestradiol . the shape changes were relatively rapid in onset , being observed within 6 hours , and were maintained for the duration of the incubation . these shape changes were similar to those elicited by incubation of cells with the microtubule disaggregating agent , colchicine ( 0 . 1 - 10 μm ). the steroid receptor antagonist , ru 486 [ stewart et al ., 1995b ] reduced the shape changes in response to either colchicine or 2 - methoxyestradiol , but had no effect on the inhibition of dna synthesis by 2 - methoxyestradiol . these results are illustrated in fig5 . several compounds related to 2 - methoxyestradiol were examined for inhibition of fcs ( 1 %, v / v )- stimulated dna synthesis , including the parent compound , 17β - estradiol , and the immediate precursor , 2 - methoxyestradiol . the lower concentrations of each of these compounds enhanced fcs ( 1 %)- stimulated dna synthesis , as shown in fig6 . at higher concentrations , the enhancement was reversed , and inhibition was observed at 10 μm of these compounds . the inhibitory effect of 2 - methoxyestradiol ( 10 μm ) was equivalent to 2 - methoxyestradiol ( 10 μm ). a biphasic effect was observed with analogues including 2 - methoxyestrone and 2 - methoxyestradiol , which enhanced thrombin - stimulated dna synthesis at concentrations up to 3 μm , but the level of enhancement declined at 10 μm and is shown in fig7 . the effects of 17 - β - estradiol and 2 - hydroxyestradiol on protein synthesis are shown in table 5 . we have shown here that 2 - methoxyestradiol , a natural metabolite of 17β - estradiol which was previously thought to be inactive , has anti - inflammatory activities and inhibits the dna synthesis and subsequent division of airway smooth muscle cells cultured from human bronchi . the anti - inflammatory property renders the compound and its analogues useful in the treatment of inflammatory diseases , e . g ., treatment of asthma and other chronic obstructive airway diseases , particularly those with demonstrable pmn involvement . the inhibitory effect on dna synthesis is not a result of cytotoxicity , since protein synthesis rates were not altered by incubation of cells with the highest concentrations of 2 - methoxyestradiol ( 10 μm ) and no cell detachment from the culture plates was observed at this concentration . without wishing to be bound by any proposed mechanism for the observed advantages , it is possible that the steroid inhibits the cells early in the g1 phase of the cell - cycle ( 2 . 0 hours post - mitogen ), causing maximal inhibition of dna synthesis . it remains to be established whether post - mitogen addition of 2 - methoxyestradiol retains its anti - proliferative effect . 2 - methoxyestradiol inhibited responses to bfgf , thrombin and fcs ( 1 %) with similar potencies , indicating that the effect was not specific to any one mitogen . this observation suggests that 2 - methoxyestradiol acts at early intracellular signalling step ( s ) used by each of these mitogens . nevertheless , the inhibitory effect on dna synthesis was surmountable , with higher concentrations of fcs ( 10 %) being significantly less inhibited by preincubation with 2 - methoxyestradiol . this resistance could be explained by the greater response to the higher concentration of fcs , but a similar argument cannot be made for the resistance to inhibition when the mitogen is egf , which elicited smaller responses than those elicited by thrombin , fcs 1 % or bfgf . however , the proliferative effects of efg and 10 % fcs may be inhibited by 2 - methoxyestradiol . we do not yet have any evidence linking the inhibition of dna synthesis to inhibition of cell proliferation . however , the fact that the latter effect is observed at lower concentrations of 2 - methoxyestradiol suggests that actions other than inhibition of dna synthesis by 2 - methoxyestradiol also contribute to its anti - proliferative actions . several analogues of 2 - methoxyestradiol were examined to determine whether they shared this anti - proliferative effect . both the parent compound 17β - estradiol and the immediate precursor , 2 - hydroxyestradiol , at lower concentrations increased dna synthesis in response to fcs ( 1 %) and inhibited dna synthesis at 3 and 10 μm . it was not established whether these changes in dna synthesis resulted in corresponding changes in cell proliferation . the enhancement of thrombin - stimulated dna synthesis by 2 - methoxyestrone and 2 - methoxyestradiol showed a bell - shaped concentration - response curve , with a lesser effect at the higher concentrations . collectively , our observations suggest that 2 - methoxyestradiol is the most potent of the analogues examined , consistent with earlier observations on the proliferative responses of endothelial cells [ fotsis et al ., 1994 ]. it may also be possible to administer the parent compound , 17β - estradiol , together with agents which induce metabolism to the active compound . for example , inducers of p450 cytochrome and of catecholamine methyl transferase may be used . inhibitors of aryl sulphatase may also be considered . the anti - proliferative effect of 2 - methoxyestradiol and its ability to reduce 5ht - induced increases in protein synthesis indicate both anti - hyperplastic and anti - hypertrophic effects . there is compelling evidence for hyperplasia and hypertrophy in asthmatic airways [ ebina et al ., 1993 ], which account for a large part of the phenomenon of ahr [ james et al ., 1989 ]. reductions in ahr are associated with complete resolution of symptoms in some asthmatics [ platts - mills et al ., 1987 ]. moreover , of all the structural changes documented in the airway wall remodelling response in asthma , an increase in the airway smooth muscle is considered to be of greatest importance [ pare & amp ; bai , 1995 )]. thus a compound such as 2 - methoxyestradiol , whcih prevents the growth response of airway smooth muscle , would reduce ahr and therefore reduce the symptoms of asthma . in addition , the anti - angiogenic activity of 2 - methoxyestradiol [ fotsis et al ., 1994 ] is likely to limit the remodelling response , since it has been established that there is an angiogenic component to the remodelling [ kuwano et al ., 1993 ]. it seems likely that this angiogenesis is required to support the metabolic needs of the increased tissue mass . therefore , prevention of the angiogenesis may arrest the remodelling response independently of any direct inhibitory effects of 2 - methoxyestradiol on smooth muscle and other cell types . a number of other properties of 2 - methoxyestradiol are likely to be of therapeutic benefit in the treatment of asthma , including its established ability to disrupt microtubule formation [ d &# 39 ; amato et al ., 1994 ], which may reduce the exocytotic release of inflammatory mediators from mast cells , macrophages and eosinophils . our data indicate that 2 - methoxyestradiol inhibits antigen - induced mast cell degranulation . this activity supports the use of 2 - methoxyestradiol in a wide range of allergic conditions , including allergic rhinitis and atopic skin conditions . inhibition of guinea - pig peritoneal macrophage activation by fmlp suggests that the action of 2 - methoxyestradiol may extend beyond events associated with the cytoskeleton , since fmlp activates g - protein - linked receptors rather than phagocytosis . in addition , the anti - oxidant activities of 2 - methoxyestradiol may also be of benefit , since the three key inflammatory cell types involved in airway inflammation each have the capacity to generate large amounts of oxygen radicals , and together with nitric oxide may cause significant oxidant damage . these activities also support he use of 2 - methoxyestradiol in the treatment of chronic obstructive airways disease , in which an important role for oxy radicals is well established and there is evidence of airway wall remodelling [ kuwano et al ., 1993 ]. finally , several studies indicate that 2 - methoxyestradiol and related compounds decrease calcium influx into smooth muscle [ goyache et al ., 1995 ] whcih would , if also demonstrated for airways smooth muscle , counteract bronchospasm in asthma . although the examples have been described in some detail for the purpose of clarity and understanding , they represent guidelines only . the person skilled in the art will recognise that various modifications and alterations to the embodiments described herein may be made without departing from the scope of the invention . aizu - yokotta , e ., susaki , a . & amp ; sato , y . ( 1995 ). natural estrogens induce modulation of microtubules in chinese hamster v79 cells in culture . cancer research , 55 , 1863 . brewster , c . e . p , howarth , p . h ., djukanovic , r ., wilson , j ., holgate , s . t . & amp ; roche , w . r . ( 1990 ). myofibroblasts and subepithelial fibrosis in bronchial asthma . am . j . respir . cell mol . biol . 3 , 507 . goyache , f . m ., gutierrez , m ., hidalgo , a . & amp ; cantabrana , b . ( 1995 ). non - genomic effects of catecholestrogens in the in vivo rat uterine contraction . general pharmacology , 26 , 219 . d &# 39 ; amato , r . j ., lin , c . m ., flynn , e ., folkman , j . & amp ; hamel , e . ( 1994 ). 2 - methoxyoestradiol , an endogenous mammalian metabolite , inhibits tubulin polymerization by interacting at the colchicine site . proceedings of the national academy of sciences ( usa ). 91 , 3964 . dunhill , m . s ., massarella , g . r ., anderson , j . a . 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