Patent Application: US-85352897-A

Abstract:
the invention relates to use of steroids or steroid analogues in the treatment of chronic and acute inflammation of the airways , particularly asthmatic conditions . it also relates to compounds and compositions which modulate airway remodelling . in a preferred embodiment , the steroid is 2 - methoxyoestradiol .

Description:
______________________________________ahr airway hyperresponsivenessbfgf basic fibroblast growth factordnp - oa dinitro - phenyol treated ovalbuminegf epidermal growth factorfmlp formyl methiony leucyl phenylalaninepdgf platelet - derived growth factorpma phorbol myristate acetate5ht serotonin______________________________________ 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 were 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 flasks . cells at passage numbers 3 to 15 were used for experiments . cells were subscultured 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 peroxidase ( hrp )- conjugated goat anti - mouse ; ig f ( ab &# 39 ;) 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 - methoxyoestradiol 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 mely synthesized dna , according to our 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 . 1 m 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 - methoxyoestradiol 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 sythesis , 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 pbs containing 1 mm edta , for a period of 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 . differenecs 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 - methoxyoestradiol ( 1 , 3 , 5 10 !- estratriene - 2 , 3 , 17 - triol 2 - methylether lot 83h4065 ); 17β - oestradiol (( 1 , 3 , 5 10 !- estratriene - 3 , 17β - diol , cat no . e8876 ); 2 - methoxyoestriol ( 1 , 3 , 5 10 !- estratriene - 2 , 3 , 16α , 17β - tetrol , lot 26f 4038 ; 2 - methoxyoestrone , ( 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 ( 185gbq / 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 &# 39 ;) 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 - methoxyoestradiol to the colchicine binding site on tubulin raised the possibility that its this compound interferes with leukocyte functions such as phagocytosis and locomotion . functional effects of 2 - methoxyoestradiol 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 tertramethyl - benzidine ( menegazzi et al 1992 ). phagocytosis was determined by radioidination of zymosan particles ( shelton & amp ; hosking , 1975 . guinea - pig peritoneal macrophages were harvested and cultured according to our previous studies ( stewart & amp ; phillips , 1889 ). 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 - methoxyoestradiol ( 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 - pgf1 was measured by radioimmunoassay ( 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 - methoxyoestradiol 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 - methoxyoestradiol ( 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 - oestradiol ( 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 - methoxyoestradiol ( 10 μm ) had no effect in this assay . results are summarised in fig1 . table 1______________________________________tetramethylbenzidine oxidation by humanpolymorphonuclear leukocytes fmlp zymosan control 100 nm 400 μg / ml______________________________________basal - 0 . 001 ± 0 . 057 ± 0 . 26 ± 0 . 002 0 . 021 0 . 0092 - methoxy - - 0 . 004 ± 0 . 028 ± 0 . 013 ± estradiol 3 μm 0 . 001 0 . 03 0 . 012______________________________________ data are expressed as change in absorbance value . assays were carried out using 2 × 10 6 pmn in 0 . 5 ml buffer . in pmn , superoxide anion generation in response to fmlp ( 100 nm ) or zymosan ( 400 μg / ml ) was not reduced by concentrations of 2 - methoxyoestradiol up to 10 μm . in phagocytosis experiments , radioiodination of zymosan particles by pmn was reduced by 2 - methoxyeostradiol with significant effects being observed at both 3 and 10 μm , as shown in table 2 . table 2______________________________________ . sup . 125 i uptake by human polymorphonuclear leukocytes . no phs phs * control zymosan control zymosan______________________________________basal 1 . 80 ± 2 . 83 ± 2 . 13 ± 4 . 33 ± 0 . 09 0 . 09 0 . 24 0 . 512 - methoxy - 1 . 97 ± 1 . 97 ± 1 . 87 ± 3 . 27 ± oestradiol 3 μm 0 . 44 0 . 48 0 . 46 0 . 642 - methoxy - 2 . 05 ± 1 . 80 ± 1 . 75 ± 1 . 65 ± oestradiol 10 μm 0 . 44 0 . 49 0 . 45 0 . 15______________________________________ * phs = pooled human serum data are expressed as percentage of . sup . 125 i incorporation into glass fibrefilterable material . assays were carried out using 1 × 10 . sup . pmn . in adherent monocytes the oxidation of tetramethyl benzidine in respone to phorbol myristate acetate ( 1 μm ) pma or zymosan ( 400 μg / ml ) was unaffected by 10 μm 2 - methoxyoestradiol . 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 - methoxyoestradiol ( 10 μm ) in monocytes from at least some donors . the superoxide anion response of guinea - pig macrophages to zymosan or fmlp was reduced by 2 - methoxy - oestradiol as shown in fig8 . however , the response to pma ( 100 nm ) was unaffected . in addition , fmlp ( 100 nm )- induced increases in 6 - oxo - pgf1 αgeneration were completely blocked by 2 - methoxyoestradiol , whereas the response to pma was reduced by only 50 %, and zymosan did not stimulate an increase in the levels of the prostacyclin metabolite as shown in fig9 . ovalbumin ( dnp - oa ) elicited a concentration - dependent release of 3 h !- 5ht which was reduced by 10 μm 2 - methoxyoestradiol 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 - methoxyoestradiol as shown in fig1 . the inhibitory effects of 2 - methoxyoestradiol 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 superoxide 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 - methoxyoestradiol 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 - methoxyoestradiol used ( 10 μm ), the response to thrombin was reduced to approximately 10 % of the control level . this inhibitory effect of 2 - methoxyoestradiol on dna synthesis was not restricted to the presence of thrombin , as similar concentration - related inhibitory effects of 2 - methoxyoestradiol were observed in cells in which dna synthesis was stimulate with either foetal calf serum ( fcs , 1 % v / v ) or basic fibroblast growth factor ( bfgf , 300 pm ) ( fig2 a and 2b . however , dna synthesis in the presence of either egf ( 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 - methoxyoestradiol , after exposure to mitogens , still inhibited dna synthesis . thrombin ( 0 . 3 u / ml )- stimulated dna synthesis was inhibited when 2 - methoxyoestradiol ( 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 - methoxyoestradiol 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 fig 2d . subsequently , additional time points were examined and these studies indicated that the highest level of activity was observed when 2 - methoxyoestradiol 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 - methoxyoestradiol 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 - methoxyoestradiol 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 . table 3______________________________________effect of 2 - methoxyoestradiol on proteinsynthesis rates in unstimulated smooth musclecells . . sup . 3 h !- leucine incorporation (% 2 - methoxy - control ) oestradiol ( μm ) mean ± sem______________________________________ -- 100 0 . 3 135 ± 3 * 1 . 0 84 ± 4 * 3 . 0 77 ± 3 * 10 . 0 113 ± 9______________________________________ * p & lt ; 0 . 05 paired student &# 39 ; s ttest , compared to 100 % ( no pretreatment ) 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 - methoxyoestradiol decreased the 5ht ( 10 nm )- stimulated increase in protein synthesis in a concentration - dependent manner , as summarized in table 4 . table 4______________________________________effect of 2 - methoxyoestradiol on proteinsynthesis rates in 5ht - stimulated smoothmuscle cells . . sup . 3 h !- leucine incorporation (% 2 - methoxy - control ) oestradiol ( μm ) mean ± sem______________________________________ -- 100 0 . 3 91 ± 5 1 . 0 62 ± 2 * 3 . 0 56 ± 2 * 10 . 0 51 ± 6 * ______________________________________ * p & lt ; 0 . 05 paired student &# 39 ; s ttest , compared to 100 % ( no pretreatment ) morphological 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 - methoxyoestradiol . 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 - methoxyoestradiol , but had no effect on the inhibition of dna synthesis by 2 - methoxyoestradiol . these results are illustrated in fig5 . several compounds related to 2 - methoxyoestradiol were examined for inhibition of fcs ( 1 %, v / v )- stimulated dna synthesis , including the parent compound , 17β - oestradiol , and the immediate precursor , 2 - hydroxyoestradiol . 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 - hydroxyoestradiol ( 10 μm ) was equivalent to 2 - methoxyoestradiol ( 10 μm ). a biophasic effect was observed with analogues including 2 - methoxyoestrone and 2 - methoxyoestriol , 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 - oestradiol and 2 - hydroxyoestradiol on protein synthesis are shown in table 5 . table 5______________________________________effect of 2 - methoxyoestradiol on proteinsynthesis rates in unstimulated smooth muscle cells . . sup . 3 h !- leucine incorporation (% control ) 17β - 2 - oestradiol hydroxyoestradiolconcentration mean ± sem mean ± sem______________________________________ 100 100 0 . 3 103 ± 6 107 ± 3 1 . 0 89 ± 4 94 ± 10 3 . 0 87 ± 6 56 ± 8 * 10 . 0 100 ± 9 51 ± 10 * ______________________________________ * p & lt ; 0 . 05 paired student &# 39 ; s ttest , compared to 100 % ( no pretreatment ) we have shown here that 2 - methoxyoestradiol , a natural metabolite of 17β - oestradiol 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 demonstratable 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 - methoxyoestradiol ( 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 - methoxyoestradiol retains its anti - proliferative effect . 2 - methoxyoestradiol 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 - methoxyoestradiol 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 - methoxyoestradiol . 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 egf and 10 % fcs may be inhibited by 2 - methoxyoestradiol . 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 - methoxyoestradiol suggests that actions other than inhibition of dna synthesis by 2 - methoxyoestradiol also contribute to its anti - proliferative actions . several analogues of 2 - methoxyoestradiol were examined to determine whether they shared this anti - proliferative effect . both the parent compound 17β - oestradiol and the immediate precursor , 2 - hydroxy - oestradiol , 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 - methoxyoestrone and 2 - methoxyoestriol showed a bell - shaped concentration - response curve , with a lesser effect at the higher concentrations . collectively , our observations suggest that 2 - methoxyoestradiol 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 - methoxyoestradiol 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 remodeling 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 - methoxyoestradiol , which 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 - methoxyoestradiol 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 - methoxyoestradiol on smooth muscle and other cell types . a number of other properties of 2 - methoxyoestradiol 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 - methoxyoestradiol inhibits antigen - induced mast cell degranulation . this activity supports the use of 2 - methoxyoestradiol in a wide range of allergic conditions , including allergic rhinitis and atopic skin conditions . inhibition of guinea - pig peritoneal macrophage activation of fmlp suggests that the action of 2 - methoxyoestradiol may extend beyond events associated with the cytoskeleton , since fmlf activates g - protein - linked receptors rather than phagocytosis . in addition , the anti - oxidant activities of 2 - methoxyoestradiol 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 the use of 2 - methoxyoestradiol 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 - methoxyoestradiol and related compounds decrease calcium influx into smooth muscle goyache et al ., 1995 ! which 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 . 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