Treatment of asthma and airway diseases

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.

This invention relates to a method of treating chronic and acute 
inflammation of the airways, including asthmatic conditions. The invention 
also relates to steroid or steroid analogues used in the treatment, and to 
pharmaceutical compositions comprising these compounds as the active 
agent. In a preferred embodiment, the active component inhibits 
inflammation and smooth muscle cell proliferation in the airway wall. It 
may also have at least one other activity selected from anti-angiogenesis, 
anti-oxidation and the ability to disrupt microtubule formation. 
BACKGROUND OF THE INVENTION 
Two distinct classes of agents are currently used in the treatment of 
asthma. Symptomatic relief is provided by using bronchodilators which 
include the .beta..sub.2 -adrenoceptor agonists such as salbutamol and 
salmeterol. Other agents with bronchodilatory properties include the 
muscarinic-receptor antagonist, ipratropium bromide, and phosphodiesterase 
inhibitors such as theophylline. 
The second class of agents is prophylactic, and includes glucocorticoids 
such as beclomethasone dipropionate. Disodium cromoglycate and nedocromil 
sodium are also used, even though these are less effective than the 
glucocorticoids. 
However, none of these agents completely reverses airway 
hyperresponsiveness or prevents catastrophic life-threatening and fatal 
episodes of asthma in all patients. The fact that these conditions prevail 
and sometimes are the cause of death highlights the fact that the benefits 
from these agents are sub-optimal. 
Asthma is now regarded as a disease of chronic airways inflammation 
characterised by eosinophilic bronchitis Frigas et al., 1991!. In common 
with other chronic inflammatory diseases, the inflammation in asthma 
initiates tissue remodelling, which has been documented in the airways in 
post mortem studies Dunnill et al., 1969! and by bronchial biopsy from 
living donors Brewster et al., 1990; Bai & Pare, 1995!. The remodelling 
involves: epithelial sloughing; marked infiltration of eosinophils into 
the mucosa; activation of mast cells and lymphocytes; enlargement of 
mucous glands; deposition of wound-type collagen immediately below the 
true basement membrane of the epithelium and throughout the mucosa; and an 
increase in the number of myofibroblasts. In addition, there is an 
increase in the volume and number of blood vessels in asthmatic airways, 
indicating that an angiogenesis accompanies the remodelling process 
Kuwano et al., 1993!. The overall volume of the airway wall is increased 
James et al., 1989! in association with an increase in the volume of 
airway smooth muscle Kuwano et al., 1993! which results from both 
hypertrophic and hyperplastic responses Ebina et al., 1993!. 
Airway hyperresponsiveness (AHR) is the excessive bronchoconstrictor 
response of asthmatic subjects to a diverse array of stimuli. The concept 
that the airway wall thickening is central to the development of AHR has 
gained acceptance during the last 10 years. The thickening of the airways 
has been shown by mathemathical modelling studies to amplify the 
consequences of smooth muscle shortening-a given amount of smooth muscle 
shortening is calculated to cause a much greater increase in airways 
resistance in asthmatics compared with healthy subjects (eg 40% shortening 
gives a 15-fold increase in healthy subjects, but a 290-fold increase in 
asthmatics) James et al., 1989!. The airway wall area is increased by 
50-250%, with larger increments being observed in the larger airways 
James et al., 1989!. The muscle increases in volume by 2-3 fold, and the 
extent of the increase is related to the severity of asthma Kuwano et 
al., 1993!. The nature of the change has not been extensively 
investigated, but it comprises both hyperplagia and hypertrophy Ebina et 
al., 1993!. After prolonged allergen avoidance by allergic asthmatics, 
decreases in airways responsiveness to the levels observed in healthy 
subjects have been demonstrated, and are accompanied by a resolution of 
the symptoms Platts-Mills et al., 1987!. Studies such as this are 
consistent with the notion that the structural changes in the asthmatic 
airway are also reversible. 
These long-term changes in the asthmatic airway offer new targets for 
therapeutic intervention Stewart et al., 1993!. Consequently there has 
been considerable interest in identifying the mechanisms for this airway 
wall remodelling response and the influence of existing anti-asthma drugs 
on these processes. A large number of factors have been established as 
mitogens for cultured airway smooth muscle from various species, including 
humans see Stewart et al., 1995a for a review!. as expected, the stimuli 
belonging to the growth factor families, including basic fibroblast growth 
factor (bFGF), platelet-derived growth factor (PDGF.sub.BB) and epidermal 
growth factor (EGF) are the most effective proliferative agents Hirst et 
al., 1992; Stewart et al., 1995a!. Thrombin is also an effective growth 
factor Tomlinson et al., 1994!, whereas bronchoconstrictors such as 
endothelin-1 and the thromboxane A.sub.4 mimetic, U46619, are only weakly 
active, and some other constrictors such as histamine and neurokinins are 
completely inactive Stewart et al., 1995a!. 
In human cultured airway smooth muscle, continuous exposure to 
.beta.-adrenoceptor agonists reduces the proliferative responses to a wide 
range of mitogens, including thrombin, EGF and the thromboxane A.sub.2 
analogue, U46619 Tomlinson et al., 1994; 1995!. Furthermore, 
dexamethasone and other anti-inflammatory steroids also have an 
anti-proliferative effect on cultured airway smooth muscle Stewart et 
al., 1995b!, but the magnitude of the inhibition depends on the mitogen 
that stimulates proliferation in the first instance. It is also important 
to note that long-term treatment with inhaled anti-inflammatory steroids 
produces only a modest reduction in AHR Sotomayor et al., 1984; Lungren 
et al., 1988!, whereas .beta..sub.2 -agonists are reported to have either 
no effect or to increase AHR Wahedna et al., 1993!. Thus, the two most 
commonly used and most effective drug classes for the treatment of asthma 
have sub-optimal effects on AHR, and are therefore unlikely to be 
effective in regulating the structural changes associated with airway 
remodelling that contribute to the progression and development of the 
condition. 
We have been investigating potential ways of arresting or modulating the 
remodelling process and have surprisingly identified a steroid and 
analogues thereof which are suitable for this purpose. 
SUMMARY OF THE INVENTION 
2-methoxyoestradiol is a natural metabolite of 17.beta.-oestradiol, the 
physiological estrogen in humans. 
It is produced in a two-step process, involving hydroxylation of estrogen 
to produce a catecholoestrogen followed by methoxylation to produce the 
corresponding methoxyoetrogen by an inducible cytochrome p450 pathway 
Spink et al., 1994!. Hitherto considered to be biologically inactive 
Rosner et al., 1991!, in cell culture studies it has been established 
that 2-methoxyoestradiol inhibits proliferation of certain transformed 
cell lines Lottering et al., 1992! and of actively proliferating or 
non-quiescent endothelical cells and fibroblasts Fotsis et al., 1994!. 
Fotsis et al also showed that administration of 2-methoxyoestradiol 
inhibited the growth of tumours by suppressing tumour-induced 
angiogenesis, rather than by direct inhibition of tumour cell 
proliferation. It was proposed that the compound reduced basal membrane 
breakdown, thus preventing cell migration into the extracellular matrix 
and rendering it a potential anti-angiogenic agent for the treatment of 
solid tumors or angiogenic diseases. Inhibition of tumour 
neovascularization was also demonstrated in Klauber et al, 1997. 
The anti-proliferative effects of 2-methoxyoestradiol on cultured smooth 
muscle cells from rabbit aorta Nishigaki et al., 1995! also suggested the 
usefulness of this compound in the prevention of progresion of 
atherosclerosis, a disease caused by cellular events that differ from 
those seen in asthma and AHR. 
The mechanism of the anti-proliferative effects ha not yet been 
established. Lottering et al. (1992) suggested that elevation of cyclic 
adenosine monophosphate (cAMP) explains the inhibitory effects of 
2-methoxyoestradiol on DNA synthesis, whereas inhibition of microtubule 
assembly during spindle formation in mitosis is considered to explain the 
inhibitory effects on cell division Fotsis et al., 1994!. The other 
biological effects of 2-methoxyoestradiol are not extensively 
characterized. In pig endometrial cell cultures, 2-methoxyoestradiol 
inhibits the synthesis of PGF.sub.2.alpha. Zhang & Davis, 1992!. 
Non-genomic actions of 2-methoxyoestradiol include microtubule disruption 
via binding at the colchicine site on tubulin D'Amato et al., 1994; 
Aizu-Yokota et al., 1995! and relaxation of vascular smooth muscle 
Goyache et al., 1995!. 
In International patent publication No. WO95/04535, estradiol derivatives 
which exert anti-mitotic effects by inhibiting tubulin polymerisation in 
vitro are disclosed. It is inferred from the in vitro studies that the 
compounds inhibit endothelial cell proliferation. 
The present invention relates to effects of 2-methoxyoestradiol and 
inhibition of inflammatory cell activation. It particularly relates to 
treatment or prevention of airway diseases such as asthma. 
None of the documents referred to above suggest or disclose the invention. 
For example, inhibition of smooth muscle cell proliferation and 
inflammatory cell activation in the airway cannot be predicted by the in 
vitro observations described in WO95/04535 and the mechanism of these 
activities do not appear to be related to actions on microtubule assembly. 
The anti-proliferative effect of 2-methoxyoestradiol on rabbit vascular 
smooth muscle Nishigaki et al, supra! also cannot be extrapolated to 
airway smooth muscle, since there are known differences in responsiveness 
of the cells from these two different sources. 
Some agents which enhance endothelial cell proliferation, eg. heparin, 
actually inhibit proliferation of airway smooth muscle cells. Therefore, 
the anti-proliferative effects of 1-methoxyoestradiol in endothelial cells 
Fotsis et al, WO95/04535, supra! do not suggest that smooth muscle would 
respond in the same way. 
We have found that 2-methoxyoestradiol inhibits the release of 
myeloperoxidase from polymorphonuclear leukocytes obtained from human 
peripheral blood, as well as the phagocytic activity of these cells. The 
reduction of phagocytic activity in these cells provides evidence of its 
anti-inflammatory properties. This is surprising, particularly because 
2methoxyoestradiol is known not to have significant affinity for 
glucocorticoid or for estrogen receptors Merriam et al, 1980!. 
These properties render 2-methoxyoestradiol and related compounds of 
benefit in the treatment of conditions which include but are not limited 
to asthma, chronic obstructive airway diseases and other airway diseases 
characterised by inflammation. Other conditions amenable to treatment by 
the methods of the invention include, for example, emphysema, pneumonia or 
airway diseases characterised by one or both of proliferative and 
inflammatory conditions eg. neutrophil infiltration, or pulmonary 
infectious diseases the symptoms or sequelae or which result from 
activation of resident and inflammatory cells. 
Conditions such as allergic rhinitis may also be treated, since we have 
also found that 2-methoxyoestradiol inhibits degranulation of the mast 
cell-related cell line, RBL2H3. This inhibitory effect of 
2-methoxyoestradiol was selective for antigen-stimulated release since the 
response to the protein kinase C stimulant, PMA, and to the calcium 
ionophore A23187, were unaffected. Thus, the effect on antigen release is 
not likely to result from a non-specific action on microtubule-dependent 
granule extrusion. Our results indicate that 2-methoxyoestradiol and 
related steroids having these activities are useful for treating allergic 
conditions that include but are not limited to rhinitis and atopic skin 
conditions. Without wishing to be bound to any particular mechanism of 
action, these data suggest that specific, signal transduction mechanisms 
involving receptors are involved and contribute to the inhibition of 
inflammatory in the airway. 
We have further found that 2-methoxyoetradiol inhibits DNA synthesis and 
cell division in airway smooth muscle stimulated with a range of growth 
factors, including FCS and bFGF. In addition, serotonin-stimulated 
increases in protein synthesis rates are inhibited by 2-methoxyoestradiol, 
raising the possibility of anti-hypertrophic effects, in addition to 
inhibition of cell proliferation. Our observations, together with the 
anti-angiogenic activity of 2-methoxyoetradiol, indicate that this and 
related compounds may have therapeutic value in the treatment of airway 
diseases characterised by inflammation as described above, in particular 
in the treatment of chronic asthma, with particular impact on the airway 
wall remodelling and hence on airway hyperresponsiveness. Analogues of 
2-methoxyoestradiol were also tested for their ability to inhibit DNA 
synthesis, and the results indicate that these also may have therapeutic 
value. 
In a first aspect, the invention provides a method of treating a disease 
characterised by chronic or acute airway inflammation, comprising the step 
of administering a steroid or steroid analogue having the ability to 
modulate remodelling of the airway to a mammal in need of such treatment. 
Preferably the mammal is a human, cat, horse or bovine, and more 
preferably is human. The steroid 2-methoxyoestradiol is especially 
preferred for use in accordance with the method of the invention. Steroids 
or analogues thereof which do not have effective glucocorticoid activity 
at the dosage level used in accordance with this invention are 
particularly desired. 
In a second aspect, the invention provides a method of treating a disease 
characterised by chronic or acute airway inflammation, comprising the step 
of administering a steroid or steroid analogue to a mammal in need of such 
treatment, wherein said steroid or analogue inhibits phgocytic activity of 
polymorphonuclear leucocytes. Preferably, the release of myeloperoxidase 
from the leucocytes is also inhibited. The activation of macrophages may 
also be inhibited. 
In one embodiment, the method according to the invention further modulates 
remodelling of the airway by inhibiting smooth muscle cell proliferation 
and inflammation. The remodelling may further be modulated by inhibition 
of one or more activities selected from the group consisting of 
angiogenesis, formation of oxidants and microtubule function in the airway 
wall. 
In a particularly preferred embodiment, the method of the invention is used 
in the treatment of a disease selected from the group consisting of 
asthma, airway hyperresponsiveness, brochoconstriction, emphysema, 
pneumonia, atopic disease such as allergic rhinitis and pulmonary 
infection. 
In a third aspect, the invention provides a steroid or steroid analogue 
which modulates airway remodelling by inhibiting inflammation of the 
airway wall. Preferably, the compound also has the ability to inhibit 
proliferation of airway smooth muscle cells, particularly in response to a 
mitogenic stimulus. 
In a fourth aspect, the invention relates to a steroid or steroid analogue 
which modulates airway remodelling by inhibiting phagocytic activity. In a 
preferred embodiment, the phagocytic activity of polymorphonuclear 
leucocytes is inhibited by 2methoxyestradiol. In another embodiment, the 
release of myeloperoxidase from polymorphonuclear leucocytes is also 
suppressed. In a particularly preferred embodiment, the steroid or steroid 
analogue does not exhibit glucocorticoid activity. 
In a fifth aspect, the invention provides a steroid or analogue as 
described above, further having anti-angiogenic activity and/or 
anti-oxidant activity. In a particularly preferred embodiment, the steroid 
or steroid analogue of the invention also has the ability to disrupt 
microtubules in the airway wall. 
In a sixth aspect, the invention provides a composition comprising a 
steroid or steroid analogue as described above, optionally together with 
one or more pharmaceutically acceptable carriers and excipients. Examples 
of such carriers and excipients include but are not limited to dry 
micronised powders together with lactose, or recently developed 
hydrofluoroalkanes. The composition of the invention may be used in 
formulations for administration via any standard route used in treatment 
of airway diseases or asthma, for example, topical, oral, nasal 
administration or by inhalation. These formulations may be in any 
conventional form such as capsules, cachets, tablets, aerosols, powder 
granules, micronised particles or as a solution. Optionally, the steroid 
or steroid analogue may be complexed with cyclodextrin, and may also be in 
the form of an ester formed with a pharmaceutically acceptable acid such 
as sulphate, acetate, benzoate or the like. A person skilled in the art 
will be able by reference to standard texts, such as Remington's 
Pharmaceutical Sciences 17.sup.th edition, to determine how the 
formulations are to be made and how these may be administered. 
The dose of the steroid or steroid analogue to be administered will depend 
on the condition to be treated and the route of administration, and will 
be at the discretion of the attending physician or veterinarian. Such a 
person will readily be able to determine a suitable dose, mode and 
frequency of adminitration. The composition of the invention may be used 
to treat conditions of chronic or acute airway inflammation, including 
asthma, airway hyperresponsiveness (AHR) or bronchoconstriction. 
Administration 
The compositions described above can be provided as physiologically 
acceptable formulations using known techniques, and these formulations can 
be administered by standard routes. In general, the combinations may be 
administered by the topical, oral, rectal or parenteral (e.g., 
intravenous, subcutaneous or intramuscular) route. In addition, the 
combinations may be incorporated into biodegradable polymers allowing for 
sustained release, the polymers being implanted in the vicinity of where 
delivery is desired, for example, at the site of a tumor. The 
biodegradable polymers and their use are described in detail in Brem et 
al., J. Neurosurg. 74:441-446 (1991). 
The dosage of the composition will depend on the condition being treated, 
the particular derivative used, and other clinical factors such as weight 
and condition of the patient and the route of administration of the 
compound. However, for oral administration to humans, a dosage of 0.01 to 
100 mg/kg/day, preferably 0.01-1 mg/kg/day, is generally sufficient. 
The formulations include those suitable for oral, rectal, nasal, topical 
(including buccal and sublingual), vaginal or parenteral (including 
subcutaneous, intramuscular, intravenous, intradermal, intraocular, 
intratracheal, and epidural) administration. The formulations may 
conveniently be presented in unit dosage form and may be prepared by 
conventional pharmaceutical techniques. Such techniques include the step 
of bringing into association the active ingredient and the pharmaceutical 
carrier(s) or excipient(s). In general, the formulations are prepared by 
uniformly and intimately bringing into associate the active ingredient 
with liquid carriers or finely divided solid carriers or both, and then, 
if necessary, shaping the product. 
Formulations of the present invention suitable for oral administration may 
be presented as discrete units such as capsules, cachets or tablets each 
containing a predetermined amount of the active ingredient; as a powder or 
granules; as a solution or a suspension in an aqueous liquid or a 
non-aqueous liquid; or as an oil-in-water liquid emulsion or water-in-oil 
emulsion and as a bolus, etc. 
A tablet may be made by compression or molding, optionally with one or more 
accessory ingredients. Compressed tablets may be prepared by compressing, 
in a suitable machine, the active ingredient in a free-flowing form such 
as a powder or granules, optionally mixed with a binder, lubricant, inert 
diluent, preservative, surface-active or dispersing agent. Molded tables 
may be made by molding, in a suitable machine, a mixture of the powdered 
compound moistened with an inert liquid diluent. The tablets may 
optionally be coated or scored and may be formulated so as to provide a 
slow or controlled release of the active ingredient therein. 
Formulations suitable for topical administration in the mouth include 
lozenges comprising the ingredients in a flavored basis, usually sucrose 
and acacia or tragacanth; pastilles comprising the active ingredient in an 
inert basis such as gelatin and glycerin, or sucrose and acacia; and 
mouthwashes comprising the ingredient to be administered in a suitable 
liquid carrier. 
Formulations suitable for topical administration to the skin be presented 
as ointments, creams, gels and pastes comprising the ingredient to be 
administered in a pharmaceutical acceptable carrier. A preferred topical 
delivery system in a transdermal patch containing the ingredient to be 
administered. 
Formulations for rectal administration may be presented as a suppository 
with a suitable base comprising, for example, coca butter or a salicylate. 
Formulations suitable for nasal administration, wherein the carrier is a 
solid, include a coarse powder having a particle size, for example, in the 
range of 20 to 500 microns which is administered in the manner in which 
snuff is taken, i.e., by rapid inhalation through the nasal passage from a 
container of the powder held close up to the nose. Suitable formulations, 
wherein the carrier is a liquid, for administration, as for example, a 
nasal spray or as nasal drops, include aqueous or oily solutions of the 
active ingredient. 
Formulations suitable for vaginal administration may be presented as 
pessaries, tampons, creams, gels, pastes, foams or spray formulations 
containing in addition to the active ingredient such as carriers as are 
known in the art to be appropriate. 
Formulations suitable for parenteral administration include aqueous and 
non-aqueous sterile injection solutions which may contain anti-oxidants, 
buffers, bacteriostats and solutes which render the formulation isotonic 
with the blood of the intended recipient; and aqueous and non-aqueous 
sterile suspensions which may include suspending agents and thickening 
agents. The formulations may be presented in unit-dose or multi-dose 
containers, for example, sealed ampules and vials, and may be stored in a 
freeze-dried (lyophilized) conditions requiring only the additions of the 
sterile liquid carrier, for example, water for injections, immediately 
prior to use. Extemporaneous injection solutions and suspensions may be 
prepared from sterile powders, granules and tables of the kind previously 
described. 
Preferred unit dosage formulations are those containing a daily dose a 
unit, daily sub-dose, as herein above recited, or an appropriate fraction 
thereof, of the administering ingredient. 
It should be understood that in addition to the ingredients, particularly 
mentioned above, the formulations of this invention may include other 
agents convention in the art having regard to the type of the formulation 
in question, for example, those suitable for oral administration may 
include flavoring agents. 
In a particularly preferred embodiment of the invention, inflammation and 
proliferation of smooth muscle cells in the airway in the wall of an 
asthmatic patient is inhibited by administration of a composition 
comprising 2-methoxyoetradiol. 
Although the invention will be described with particular reference to 
2-methoxyoestradiol, it will be understood that analogues of this compound 
which have the requisite biological activities may also be used in 
accordance with the invention. These include but are not limited to 
2-hydroxyestradiol, 2-methoxyoestradiol-3methyl ether and 
4-methoxyoetradiol. 
A variety of compounds have been identified as oestradiol derivatives 
having anti-proliferative and/or anti-angiogenic activity in other 
tissues. See, for example, WO95/04535 the entire disclosure of which is 
incorporated herein by this reference. 
Such compounds may be suitable candidates for use in accordance with the 
present invention and are within the meaning of steroids, steroid 
analogues or steroid-like compounds for the purpose of the present 
invention. Preferred compounds have a methoxy group at the 2position of 
the steroid backbone. 
In addition, it is contemplated that further compounds not hitherto known 
will have sufficient structural similarity to the 2-methoxy steroids or 
steroid-like compounds of this invention to have biological activities 
within the scope of this invention. For the purposes of this 
specification, the terms "steroid", "steroid analogue" or "steroid-like" 
are to be understood to encompass 2-methoxyoestradiol., 
2-hydroxyoestradiol, 2-methoxyoestradiol-3, methyl ether, 
4-methoxyoestradiol and other compounds based around a steroid nucleus 
that have the relevant biological activities to be used for the purpose of 
the present invention. Other compounds may have sufficient structural 
and/or electronic resemblance (charge distribution) to 2-methoxyoestradiol 
and have biological activities within the scope of this invention without 
strictly having a steroid nucleus, such compounds are to be considered 
steroid analogous for the purposes of the present invention, for example 
compounds of WO95/04535. Compounds with such activities may be readily 
identified by using assays capable of indicating activities of the type 
described elsewhere in this specification. As an example, a compound may 
be tested for its effects on chronic respiratory obstructive disease by 
measuring airway smooth muscle cell proliferation; effects on allergic 
rhinitis and on infectious diseases may be tested by determining the 
inhibition of inflammatory cell activation, eg. mast cells for rhinitis 
and neutrophils for infectious disease. 
A person skilled in the art will be aware of alternative tests and can 
readily screen compounds for use in accordance with the invention.

DETAILED DESCRIPTION OF THE INVENTION 
Abbreviations 
Abbreviations used herein are as follows: 
______________________________________ 
AHR airway hyperresponsiveness 
bFGF basic fibroblast growth factor 
DNP-OA dinitro-phenyol treated ovalbumin 
EGF epidermal growth factor 
fMLP formyl methiony leucyl Phenylalanine 
PDGF platelet-derived growth factor 
PMA phorbol myristate acetate 
5HT serotonin 
______________________________________ 
General Methods 
Cell culture 
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's Modified Eagle's Medium 
(DMEM), supplemented with 2 mM L-glutamine, 100 .mu.g/ml streptomycin, 
100 U/ml penicillin-G, 2 .mu.g/ml amphotericin B, and 0.25% w/v bovine 
serum albumin (BSA)! containing 3 mg/ml collagenase for 30 minutes at 
37.degree. 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.degree. C. Cell suspensions were 
centrifuged (10 min, 100.times.g, 25.degree. 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.sup.2 Falcon 
culture flasks and incubated (37.degree. C., 5% CO.sub.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.sup.2 Falcon culture flasks. Cells at passage numbers 3 
to 15 were used for experiments. 
Immunocytochemistry 
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.degree. 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.degree. 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.degree. C. (horseradish peroxidase (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 .alpha.-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 .alpha.-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 .alpha.-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. 
DNA and protein synthesis 
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.sub.2, in air at 37.degree. 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.degree. C., 5% CO.sub.2) 
before being pulsed with .sup.3 H!-thymidine (1 .mu.Ci/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 .mu.l 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.times.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 .sup.3 H!-leucine replaced .sup.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. 
Cell counting 
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 .mu.l 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 .mu.l of PBS (20% FCS) 
was added to prevent cell lysis by trypsin and cells were counted directly 
in a haemocytometer. 
Statistical analyses 
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. 
Materials 
All chemicals used were of analytical grade or higher. The compounds used 
and their sources were as follows: 2-methoxyoestradiol 
(1,3,510!-estratriene-2,3,17-triol 2-methylether lot 83H4065); 
17.beta.-oestradiol ((1,3,510 !-estratriene-3, 17.beta.-diol, cat no. 
E8876); 2-methoxyoestriol (1,3,510!-estratriene-2,3,16.alpha., 
17.beta.-tetrol, lot 26F 4038; 2-methoxyoestrone, (2,3-dihydroxy 
1,3,510!-estratriene-17-one, lot 110F4003), 2-hydroxyestradiol, 
(1,3,510!-estratriene-2,3,17.beta.-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's Modified Eagle's Medium (DMEM), Flow 
Laboratories, Scotland. 6-.sup.3 H!-thymidine (185GBq/mmol, 5 Ci/mmol), 
Amersham, UK; Microscint .about.O scintillant, Canberra-Packard, 
Australia. The antibodies used for immunocytochemistry were anti-smooth 
muscle .alpha.-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. 
EXAMPLES 1 
Effect of 2-methoxyoestradiol on Leucocyte Activity 
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 & 
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 & Hosking, 
1975. 
Guinea-pig peritoneal macrophages were harvested and cultured according to 
our previous studies (Stewart & Phillips, 1889). Cells were incubated with 
stimuli including the chemotactic tripeptide, formyl methiony leucyl 
Phenylalanine (fMLP, 100 nM), Zymosan (400 .mu.g/ml) or phorbol myristate 
acetate (PMA, 100 nM) for 30 min in the presence or absence of 
2-methoxyoestradiol (10 .mu.M) added 15 min before the stimuli. Superoxide 
anion was determined by superoxide dismutase-sensitive reduction of 
cytochrome c (Stewart & Harris, 1992) and the stable metabolite of 
prostacyclin, 6-oxo-PGF1 was measured by radioimmunoassay (Stewart & 
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 .sup.3 H!-5HT (1 .mu.Ci/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.times.g, 4.degree. C., 5 min) 
and aliquots taken for determination of the amount of .sup.3 H!-5HT 
released. All experiments were carried out in quadruplicate. 
The results showed that 2-methoxyoestradiol (3 .mu.M) reduced oxidation of 
tetramethyl-benzidine in leukocytes stimulated with either zymosan (400 
.mu.g/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 
.mu.M). 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 
.mu.M) had no effect in this assay. Results are summarised in FIG. 1. 
TABLE 1 
______________________________________ 
Tetramethylbenzidine oxidation by Human 
polymorphonuclear leukocytes 
fMLP Zymosan 
Control 100 nM 400 .mu.g/ml 
______________________________________ 
Basal -0.001.+-. 0.057.+-. 
0.26.+-. 
0.002 0.021 0.009 
2-methoxy- -0.004.+-. 0.028.+-. 
0.013.+-. 
estradiol 3 .mu.M 
0.001 0.03 0.012 
______________________________________ 
Data are expressed as change in absorbance value. Assays were carried out 
using 2.times.10.sup.6 PMN in 0.5 ml buffer. 
In PMN, superoxide anion generation in response to fMLP (100 nM) or zymosan 
(400 .mu.g/ml) was not reduced by concentrations of 2-methoxyoestradiol up 
to 10 .mu.M. In phagocytosis experiments, radioiodination of zymosan 
particles by PMN was reduced by 2-methoxyeostradiol with significant 
effects being observed at both 3 and 10 .mu.M, 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 0.51 
2-methoxy- 1.97.+-. 
1.97.+-. 1.87.+-. 
3.27.+-. 
oestradiol 3 .mu.M 
0.44 0.48 0.46 0.64 
2-methoxy- 2.05.+-. 
1.80.+-. 1.75.+-. 
1.65.+-. 
oestradiol 10 .mu.M 
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 .times. 10.sup. 
PMN. 
In adherent monocytes the oxidation of tetramethyl benzidine in respone to 
phorbol myristate acetate (1 .mu.M) PMA or zymosan (400 .mu.g/ml) was 
unaffected by 10 .mu.M 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 .mu.M) 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 FIG. 8. However, the 
response to PMA (100 nM) was unaffected. In addition, fMLP (100 
nM)-induced increases in 6 -oxo-PGF1 .alpha.generation 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 FIG. 9. 
Ovalbumin (DNP-OA) elicited a concentration-dependent release of .sup.3 
H!-5HT which was reduced by 10 .mu.M 2-methoxyoestradiol as can be seen in 
FIG. 10. However, the basal release of .sup.3 H!-5HT and that in response 
to either PMA (100 nM) or the calcium ionophone A23187 (10 .mu.M) were 
unaffected by 2-methoxyoestradiol as shown in FIG. 11. 
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. 
EXAMPLE 2 
Effect of 2-methoxyoestradiol on DNA Synthesis 
Incubation of human cultured airway smooth muscle cells with 0.3-10 .mu.M 
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 .sup.3 
H!-thymidine, as shown in FIG. 2a. At the highest concentration of 
2-methoxyoestradiol used (10 .mu.M), 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) (FIGS. 2a 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 (FIG. 2c). The DNA synthesis in response to 10% FCS 
(27.2.+-.7.8 times more than the unstimulated level of .sup.3 
H!-thymidine incorporation) was significantly greater (p&lt;0.05, paired 
Student'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 .mu.M) 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 (.about.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 (FIG. 2d). 
EXAMPLE 3 
Effect of 2-methoxyoestradiol on protein synthesis and cell numbers 
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 .sup.3 H!-leucine in the presence of thrombin (0.3 
U/ml), FCS (1% v/v) or bFGF (300 pM) was 1 .mu.M, and was similar to the 
results for inhibition of .sup.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 .mu.M. At 10 
.mu.M, there was no significant inhibitory effect in the presence of 
thrombin or bFGF, as shown in FIG. 3. 2-methoxyoestradiol alone caused a 
small stimulation of .sup.3 H!-leucine incorporation at 0.3 .mu.M. Higher 
concentrations (1 and 3 .mu.M) had small inhibitory effects and at 10 
.mu.M 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 .mu.M as shown in FIGS. 4a and 
b. 
TABLE 3 
______________________________________ 
Effect of 2-methoxyoestradiol on protein 
synthesis rates in unstimulated smooth muscle 
cells. 
.sup.3 H!-leucine incorporation (% 
2-methoxy- control) 
oestradiol (.mu.M) 
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's ttest, compared to 100% (no pretreatment) 
EXAMPLE 4 
Serotonin-stimulated .sup.3 H!-leucine incorporationj in Smooth Muscle 
Cells. 
Serotonin (5HT) at concentrations from 0.1 nM up to 10 .mu.M had no effect 
on incorporation of .sup.3 H!-thymidine, but 10 nM 5HT increased 
incorporation of .sup.3 H!-leucine. Preincubation with 0.3-10 .mu.M 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 protein 
synthesis rates in 5HT-stimulated smooth 
muscle cells. 
.sup.3 H!-leucine incorporation (% 
2-methoxy- control) 
oestradiol (.mu.M) 
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's ttest, compared to 100% (no pretreatment) 
EXAMPLE 5 
Morphological effects of 2-methoxyoestradiol 
Morphological changes including the manifestation of a rounded appearance 
of the normally spindle-shaped cells were observed at concentrations of 3 
and 10 .mu.M 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 .mu.M). 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 
FIG. 5. 
EXAMPLE 6 
Effects of analogues of 2-methoxyoestradiol 
Several compounds related to 2-methoxyoestradiol were examined for 
inhibition of FCS (1%, v/v)-stimulated DNA synthesis, including the parent 
compound, 17.beta.-oestradiol, and the immediate precursor, 
2-hydroxyoestradiol. The lower concentrations of each of these compounds 
enhanced FCS (1%)-stimulated DNA synthesis, as shown in FIG. 6. At higher 
concentrations, the enhancement was reversed, and inhibition was observed 
at 10 .mu.M of these compounds. The inhibitory effect of 
2-hydroxyoestradiol (10 .mu.M) was equivalent to 2-methoxyoestradiol (10 
.mu.M). A biophasic effect was observed with analogues including 
2-methoxyoestrone and 2-methoxyoestriol, which enhanced 
thrombin-stimulated DNA synthesis at concentrations up to 3 .mu.M, but the 
level of enhancement declined at 10 .mu.M and is shown in FIG. 7. The 
effects of 17-.beta.-oestradiol and 2-hydroxyoestradiol on protein 
synthesis are shown in Table 5. 
TABLE 5 
______________________________________ 
Effect of 2-methoxyoestradiol on protein 
synthesis rates in unstimulated smooth muscle cells. 
.sup.3 H!-leucine incorporation (% 
control) 
17.beta.- 2- 
oestradiol hydroxyoestradiol 
Concentration 
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's ttest, compared to 100% (no pretreatment) 
We have shown here that 2-methoxyoestradiol, a natural metabolite of 
17.beta.-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 .mu.M) 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 
17.beta.-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 .mu.M. 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, 
17.beta.-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 & 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'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. 
References cited herein are listed on the following pages. 
References 
Aizu-Yokotta, E., Susaki, A. & 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. & Roches, 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. & Cantabrana, B. (1995). 
Non-genomic effects of catecholestrogens in the in vivo rat uterine 
contraction. General Pharmacology, 26, 219. 
D'Amato, R. J., Lin, C. M., Flynn, E., Folkman, J. & 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. (1969). A comparison of 
the quantitative anatomy of the bronchi in normal subjects, in status 
asthmaticus, in chronic bronchitis, and in emphysema. Thorax. 24, 176. 
Ebina, M., Takahashi, T., Chiba, T., Motomiya, M. (1993). Cellular 
hypertrophy and hyperplasia of airway smooth muscle underlying bronchial 
asthma. A 3-D morphometric study. Am. Rev. Resp. Dis. 148,720. 
Fostis, T., Zhang, Y., Pepper, M. S., Adlecrutz. H., Montesano, R., 
Nawroth, P. & Schweiger, L. (1994). The endogenous oestrogen metabolite 
2-methoxyoestradiol inhibits angiogenesis and suppresses tumour growth. 
Nature, 368,237. 
Frigas, E., Motojima, S. & Gleich, G. J. (1991). The eosinophilic injury to 
the mucosa of the airways in the pathogenesis of bronchial asthma. Eur J 
Respir Dis., 13, 123S. 
Hirst, S. J., Barnes, P. J., Twort, C. H. C. (1992). Quantifying 
proliferation of cultured human and rabbit airway smooth muscle cells in 
response to serum and platelet-derived growth factor. Am. J. Resp. Cell. 
Mol. Biol. 7, 574. 
James, A. L., Pare, P. D., Hogg, J. C. (1989). The mechanics of airway 
narrowing in asthma. Am. Rev. Respir. Dis., 139,242, 
Klauber, N., Panangi, S., Flynn, E., Hanel, E. & D'Amato, R. J. (1997) 
Inhibition of angiogenesis and breast cancer in mice by the microtubule 
inhibitors 2-methoxyoestradiol and taxol. Cancer Research 57: 81-86. 
Kuwano, K., Bosken, C. H., Pase, P. D., Bai, T. R., Wiggs, B. R. & Hogg, J. 
C. (1993). Small airways dimensions in asthma and chronic airway 
obstructive pulmonary disease Am. Rev. Resp. Dis., 148,1220. 
Lottering, M. L., Haag, M. & Seegers, J. C. (1992). Effects of 
17-.beta.-oestradiol metabolites on cell cycle events in MCF-7 cells. 
Cancer Research, 52,5926. 
Lungren, R., Soderberg, M., Horstedt, P. and Stenling, R. (1988). 
Morphological studies on bronchial mucosa biopsies from asthmatics before 
and after ten years of treatment with inhaled steroids. Eur. Repir. J., 
1,883. 
Menegazzi, R., Zaborchi, G., Knowles, A., Cramer, C. and Patrarca, P. 
(1992). A new one-step assay on whole cell suspensions for peroxidase 
secretion by human neutrophils and oesinophils. J. Leuk-Biol. 52:612. 
Merriam, G. R., Machusky, N. J., Picard, M. K. & Naftolin, F. (1980) 
Comparative properties of the catecholestrogens, I: methylation by 
catechol-O-methyltransferase and binding to cytosol estrogen receptors. 
Steroids, 36: 1-11. 
Nishigaki, I., Sasguri, Y. & Yagi, K. (1995). Anti-proliferative effect of 
2-methoxyoestradiol on cultured smooth muscle cells from rabbit aorta. 
Atherosclerosis, 113,167. 
Pare, P. & Bai, T. R. (1995). The consequences of chronic allergic 
inflammation. Thorax, 50,328. 
Platts-Mills, T. A. E. & Chapman, M. D. (1987). Dust mites: immunology, 
allergic disease, and environmental control. J. Allergy Clin. Immunol., 
80, 755. 
Rosner, W., Hryb, D. J., Khan, M. S., Nakhla, A. M. & Ronmas, N. A. (1991). 
Sex-hormone binding globulin: anatomy and physiology of a new regulatory 
system. Journal of Steroid Biochemistry & Molecular Biology, 40,813. 
Shelton, M. J and Hosking S. (1975) Neutrophil and Opsonic function in 
children with recurrent infections I. Neurophil iodination. Aust. J. Med. 
Tech. 6:54. 
Sotomayor, H., Badier, M. M Vervloet, D., Orehek, J. (1984). Seasonal 
increase of carbachol airway responsiveness in patients allergic to grass 
pollen. Reversal by corticosteroids. Am. Rev. Resp. Dis., 130,56. 
Spink, D. C., Hayes, C. L., Young, N. R., Christou, M., Sutter, T. R., 
Jefcoate, C. R. & Gierthy, J. F. (1994). The effect of 
2,3,7,8-tetrachlorodibenzo-p-dioxin on estrogen metabolism in MCF-7 breast 
cancer cells: evidence for induction of a novel 17-beta estradiol 
4-hydroxylase. Journal of Steroid Biochemistry & Molecular Biology, 
51,251. 
Stewart A G, and Harris T. (1992). Adenosine inhibits platelet-activating 
factor but not tumour necrosis factor .alpha.-induced priming of human 
neutrophils. Immunology 78:152-158. 
Stewart A G, Tomlinson P R T & Wilson J W. (1995a). Regulation of airway 
wall remodelling: prospects for the development of novel anti-asthma 
drugs. Advances in Pharmacology 33,200. 
Stewart A G, Fernandes D J, & Tomlinson P R T. (1995b). Glucocorticoids 
inhibit mitogenic responses of human cultured airway smooth muscle. 
British Journal of Pharmacology 116,3219. 
Stewart, A. G. & Phillips, W. A. (1989) Intracellular platelet-activating 
factor regulates eicosanoid generation in guinea-pig peritoneal 
macrophages. British Journal of Pharmacology, 98: 141-148. 
Stewart A G, Tomlinson P R, Fernandes D J, Wilson J and Harris T (1995c). 
Tumour necrosis factor .alpha. modulates mitogenic responses of human 
cultured airway smooth muscle. AM. J. Respir. Cell Mol. Biol. 12,110. 
Stewart A G, Tomlinson P R T, & Wilson, J. (1993). Airway wall remodelling 
in asthma: a novel target for the development of anti-asthma drugs. Trends 
in Pharmacological Sciences 14.275. 
Stewart A G, Schachte L & Tomlinson P R. (1997). Regulation of airway 
smooth muscle proliferation by .beta..sub.2 -adrenoceptor agonists. In: 
(Ed. A G Stewart) Airway wall remodelling in the pathogenesis of asthma. 
CRC PRess. Boca Raton, chapter 11 pages 295-335. 
Tomlinson P R T, Wilson J & Stewart A G. (1994). Inhibition by salbutamol 
of the proliferation of human airway smooth muscle cells grown in culture. 
British Journal of Pharmacology 111,641. 
Tomlinson P R T, Wilson J W & Stewart A G. (1995). Salbutamol inhibits the 
proliferation of human airway smooth muscle cells grown in culture: 
relationship to elevated cAMP levels. Biochemical Pharmacology 49,1809. 
Wahedna, I Wong, C. S. Wisniewski, A. F. Z., Pavord, I. D. & Tattersfield, 
A. E. (1993). Asthma control during and after cessation of regular 
.beta.2-agonist treatment. Am Rev Respir. Dis., 148,707. 
Zhang, Z & Davis, D. L. (1992). Cell-type specific responses in 
prostaglandin secretion by glandular and stromal cells from pig 
endometrium treated with catecholestrogens, methoxyestrogens and 
progesterone. Prostaglandins, 44,53-64.