Differential potency of beclomethasone esters in-vitro on human T-lymphocyte cytokine production and osteoblast activity

Article abstract of DOI:10.1211/0022357001774174

Beclomethasone dipropionate is an inhaled corticosteroid, used for the treatment of asthma. It is metabolised to 17-beclomethasone monopropionate, which has greater affinity for corticosteroid receptors than the parent compound, and to beclomethasone. We investigated the potency of beclomethasone dipropionate, 17-beclomethasone monopropionate and beclomethasone (compared with dexamethasone as a reference steroid) in two different human cell types, peripheral blood mononuclear cells and osteoblasts. We found that beclomethasone dipropionate, 17-beclomethasone monopropionate (EC50 10^-14 M) and beclomethasone (EC50 approx. 10^-12 M) were much more potent than dexamethasone (EC50 10^-8 M) in inhibiting interleukin-5 production by peripheral blood mononuclear cells. In contrast, beclomethasone dipropionate, 17-beclomethasone monopropionate and beclomethasone were equipotent with dexamethasone (EC50 range 0.3-1.2x10^-9 M) in affecting several functional assays of osteoblasts (e.g. alkaline phosphatase activity and osteocalcin synthesis). These results show that the relative bioactivities of corticosteroids vary between different human cell types, and that affinities observed in receptor binding assays are not necessarily predictive of the bioactivity in cell populations, such as peripheral blood mononuclear cells and osteoblasts, which are putatively relevant to efficacy and side effects respectively.

Full text of DOI:10.1211/0022357001774174

J. Pharm. Pharmacol. 2000, 52: 417±423
Received September 16, 1999
Accepted December 6, 1999
# 2000 J. Pharm. Pharmacol.
Differential Potency of Beclomethasone Esters In-vitro
on Human T-lymphocyte Cytokine Production and
Osteoblast Activity
CELINA SEETO*, HEEJA NAMKUNG-MATTHAI{, SHALINI JAYRAM{, KUNCORO FOE*,
KEN F.BROWN*, J. MARGARET HUGHES*, REBECCA S. MASON{, CAROL L. ARMOUR* AND
J. PAUL SEALE{
Departments of *Pharmacy, {Pharmacology, {Physiology and the *Institute of Biomedical Research,
University of Sydney, NSW 2006, Australia
Abstract
Beclomethasone dipropionate is an inhaled corticosteroid, used for the treatment of asthma.
It is metabolised to 17-beclomethasone monopropionate, which has greater af®nity for
corticosteroid receptors than the parent compound, and to beclomethasone. We investi-
gated the potency of beclomethasone dipropionate, 17-beclomethasone monopropionate
and beclomethasone (compared with dexamethasone as a reference steroid) in two different
human cell types, peripheral blood mononuclear cells and osteoblasts.
We found that beclomethasone dipropionate, 17-beclomethasone monopropionate
M) and beclomethasone (EC50 approx. 10
14
12
(EC50 10
M) were much more potent than
8
dexamethasone (EC50 10 M) in inhibiting interleukin-5 production by peripheral blood
mononuclear cells. In contrast, beclomethasone dipropionate, 17-beclomethasone mono-
propionate and beclomethasone were equipotent with dexamethasone (EC50 range 0Á3±
9
1Á2Â10 M) in affecting several functional assays of osteoblasts (e.g. alkaline phosphatase
activity and osteocalcin synthesis).
These results show that the relative bioactivities of corticosteroids vary between dif-
ferent human cell types, and that af®nities observed in receptor binding assays are not
necessarily predictive of the bioactivity in cell populations, such as peripheral blood
mononuclear cells and osteoblasts, which are putatively relevant to ef®cacy and side
effects respectively.
The introduction of the inhaled corticosteroid,
beclomethasone dipropionate, almost 30 years ago
was a major advance in the treatment of asthma
because patients who had previously required long-
term oral prednisone could switch to this new
inhaled corticosteroid without any apparent
unwanted systemic activity, provided that the
inhaled dose was no more than 2000 mg
daily (Wilcox & Avery 1973). Beclomethasone
dipropionate undergoes hydrolysis to the more
polar products, 17-beclomethasone-monopropio-
nate, beclomethasone-21-monopropionateandbeclo-
methasone. It was initially reported that the
degradation of beclomethasone dipropionate was
an inactivation step because 17-beclomethasone-
monopropionate and 21-beclomethasone-mono-
propionate were thought to be less potent than the
parent beclomethasone dipropionate (Martin et al
1974). More recent studies have demonstrated that
17-beclomethasone-monopropionate binds to ster-
oid receptors with a greater af®nity than beclo-
methasone dipropionate so it appears that
beclomethasone dipropionate is a pro-drug which is
hydrolysed to a more active species, 17-beclo-
methasone-monopropionate (WuÈrthwein & Roh-
dewald 1990).
When corticosteroids are administered by the
inhaled route, they are delivered directly to the
target organ without signi®cant unwanted systemic
activity, provided that clinically recommended
doses are not exceeded. With the development of
sensitive assays it has been possible to detect
beclomethasone in the plasma after the inhalation
of doses as low as 400 mg (Seale & Harrison 1998).
Correspondence: J. P. Seale, Department of Pharmacology,
University of Sydney, NSW 2006, Australia.
E-Mail: jpseale@pharmacol.usyd.edu.au

418
CELINA SEETO ET AL
In some patients this systemic absorption of inhaled
corticosteroids can cause unwanted effects such as
easy bruising of the skin (Capewell et al 1990).
Whether or not inhaled corticosteroids affect bone
metabolism and predispose to the development of
osteoporosis, a well recognised side effect of long-
term oral prednisone, is disputed. Changes in
markers of bone formation such as serum osteo-
calcin have been demonstrated in short-term studies
of inhaled corticosteroids. The clinical signi®cance
of these ®ndings is uncertain as the reductions in
serum osteocalcin were paralleled by reductions in
bone mineral density in one study (Hanania et al
1995), but not in another (Boulet et al 1994).
In clinical studies the ef®cacy of an administered
dose of beclomethasone dipropionate will be the
net result of the parent drug and its metabolites on
the in¯ammatory response in the lung. Similarly,
any unwanted systemic activity will be due to the
net activity of the absorbed parent drug and its
active metabolites. In the absence of any data to the
contrary, it has been assumed that the rank order of
potency of corticosteroids at receptors in one cell
type would predict the rank order at receptors in
other cell types. Thus, the most potent steroid in
models of in¯ammation would be the most potent
in terms of target cells for side effects.
without magnesium and calcium (Trace Bios-
ciences, Seven Hills, Australia) underlayered with
Ficoll-Paque and spun at 707 g for 15 min at room
temperature. The layer of peripheral blood mono-
nuclear cells at the gradient interface was carefully
harvested and washed three times with PBS and
resuspended in culture medium (RPMI 1640,
20 mM Hepes, 20 mg mL
1
gentamicin, 2 mM L-
glutamine and 5% v=v heat inactivated foetal
bovine serum) (Trace Biosciences) at a cell density
1
of 2Â106 cells mL =well and plated into 24-
well culture plates. Cells were stimulated with
of the mitogen, phytohaemagglutinin
1
1 mg mL
(PHA) (Murex Biotech Ltd, Dartford, UK) with the
exception of the negative controls to which the
PHA vehicle (PBS) was added. Immediately
afterwards the appropriate concentration of beclo-
methasone dipropionate, metabolite or vehicle
(®nal ethanol concn 0Á00001%) was added. Beclo-
methasone
monopropionate,
propionate and beclomethasone were added to give
dipropionate,
17-beclomethasone
21-beclomethasone
mono-
5
concentrations of 10 14±10
M
and dex-
amethasone was added to give concentrations of
10 14±10 M. All treatments were performed in
triplicate and cells were cultured for 48 h at 37ꢀC in
a humidi®ed 5% CO2 atmosphere.
9
We have investigated whether this assumption is
valid by determining the rank order of potency of
beclomethasone dipropionate and its metabolites,
using two separate human cell types in-vitro. We
chose peripheral blood mononuclear cells, and their
stimulated release of interleukin-5 (IL-5) to re¯ect
an aspect of the anti-in¯ammatory activity of cor-
ticosteroids (Rolfe et al 1992). The second cell type
was human osteoblasts (Namkung-Matthai et al
1998), which were chosen because of the clinical
interest in the possibility of osteoporosis with long-
term use of inhaled corticosteroids (Toogood et al
1994). The activity of beclomethasone dipropionate
and its esters were compared with a reference
corticosteroid, dexamethasone. In addition, we
assessed the conversion of the parent compound,
beclomethasone dipropionate, to its active meta-
bolite under our experimental conditions.
Determination of interleukin-5 content within sam-
ples. Total IL-5 protein content was determined in
each well culture following cell lysis by freeze=
thawing. A sandwich ELISA using a rat IgG1 anti-
human IL-5 capture antibody (TRFK5), a biotiny-
lated rat IgG2a anti-human IL-5 detection antibody
(JES1-5A10), recombinant human IL-5 standard
and protocols were supplied by PharMingen (San
Diego, CA). The lower limit of detection of IL-5
1
using this protocol was 5 pg mL .
1
Data analysis. All values of IL-5 in pg mL were
converted to a percentage of the positive control
(PHA-stimulated cells) to normalise for the varia-
bility between donors. Differences between treat-
ment and control samples were analysed using two-
way analysis of variance and the post-hoc Fischer
test. P < 0Á05 was considered to be signi®cant.
Materials and Methods
Osteoblasts
Isolation. Human osteoblasts were prepared as
previously described (Namkung-Matthai et al
1998). In brief, the whole bone end was minced and
washed extensively using primary growth medium
before being plated out onto 25-cm2 ¯asks in BGJ
Mononuclear cells
Isolation. Human peripheral blood mononuclear
cells were isolated from white cell concentrates
using Ficoll-Paque (Pharmacia Biotech AMRAD,
Uppsala, Sweden) as described previously by Rolfe
et al (1992). Brie¯y, blood was diluted with an
equal volume of phosphate buffered saline (PBS)
medium containing 10% v=v foetal calf serum
1
(FCS) and 44 mg mL
phosphoascorbate, and

DIFFERENTIAL POTENCY OF BECLOMETHASONE ESTERS IN-VITRO
1
419
supplemented with 30 mg mL
penicillin and
extracted on the same day as the incubation was
performed. The concentrations of parent drug and
decomposition products were monitored by HPLC.
1
40 mg mL streptomycin. Cells grown from the
explanted chips reached con¯uence after 3±4
weeks and were subcultured into 24-well plates.
Cells were plated at a concentration of 5±
To each 10-mL sample was added 10 mL of dex-
in ethanol
1
amethasone 21-acetate 40 mg mL
8Â10 4 cells cm and maintained in BGJ without
(internal standard), and then the mixture was sha-
ken with 40 mL dichloromethane for 15 min. The
organic layer was collected and evaporated to
dryness in-vacuo at 30ꢀC. The resulting residue
was reconstituted in a 1-mL mobile phase and
centrifuged (at 15 000 g for 2 min) before injection
onto the HPLC column (injection volume, 100 mL).
2
antibiotics and supplemented with 10% (v=v) FCS
overnight. The cells were maintained in this med-
ium for the duration of the study with medium
changes every 48 h. Serum-containing medium was
used for assays of [3H]-thymidine incorporation
and alkaline phosphatase activity due to poor
development of osteoblasts and the high level of
osteoblast apoptosis in serum-free medium (Nam-
kung-Matthai et al 1998).
The stock solutions of glucocorticoids were pre-
pared in spectroscopic-grade ethanol so that the
®nal concentration of vehicle did not exceed 0Á1%
v=v. All steroid treatment commenced 24 h after
subculture into 24-well plates and continued for
48 h before assay. The 1a,25-dihydroxyvitamin D3
(1,25(OH)2D3) was prepared in the same manner.
HPLC analysis. Liquid chromatography was per-
formed isocratically at ambient temperature (20ꢀC),
using an Alltima C18 (250Â4Á6 mm i.d.) column
(Alltech Associates, Baulkham Hills, NSW, Aus-
tralia), a mobile phase of methanol±water±
acetonitrile±acetic acid (352 : 166 : 50 : 1, v=v)
and a ¯ow rate of 1Á3 mL min 1 with UV detection
at 242 nm. Under these conditions, retention times
were 4Á7 min for beclomethasone, 6Á0 min for inter-
nal standard, 9Á0 min for 17-beclomethasone mono-
propionate and 20Á0 min for beclomethasone
dipropionate. The assay had been validated for
linearity, precision, accuracy and recovery for
beclomethasone dipropionate, 17-beclomethasone
monopropionate and beclomethasone over the con-
Measurement of proliferation. Cell numbers were
counted using a haemocytometer or Coulter counter
and [3H]-thymidine incorporation as previously
described (Namkung-Matthai et al 1998).
Measurement of differentiated functions. Alkaline
phosphatase activity and the secretion of osteocal-
cin were chosen as indices of bone-derived cell
function and assayed as previously described
(Namkung-Matthai et al 1998).
1
centration range 1±50 mg mL .
Data analysis. Relative peak area ratios were cal-
culated using the standard curves. The apparent
decomposition rate constants (kapp) were estimated
from the slope of log-linear phase of declining
concentration versus time plots. The half-lives
Data analysis. Each experiment examining the
effect of the 5 glucocorticoids on one parameter
was carried out in triplicate with cells obtained
from 4 different donors. Data points were expressed
as percentage of (vehicle) control, then combined
for the 4 cell strains to obtain meansÆ s.e.m.
The EC50 (concentration of drug at which 50%
maximal effect is acheived) for each glucocorticoid
in each assay was obtained as previously described
(Namkung-Matthai et al 1998).
1
=
(t 2) were calculated using the following equation:
1
=
t 2 ˆ 0Á693=kapp (Martin 1993). Results were
expressed as the meanÆ s.d.
Materials and chemicals
Beclomethasone dipropionate and its metabolites
17-beclomethasone monopropionate, 21-beclo-
methasone monopropionate and beclomethasone
were supplied by 3M Pharmaceuticals (St Pauls,
2
MN) and were stored in 100% ethanol as 10
M
Conversion of beclomethasone dipropionate
to esters
stock solutions at 20ꢀC. Beclomethasone dipro-
pionate and its metabolites were serially diluted in
culture medium until the desired concentration
range was achieved. Dexamethasone (Decadron,
Merck Sharp and Dohme, Seven Hills, Australia)
Kinetic experiments, which were carried out for
18 h at 37ꢀC, were initiated by the addition of an
ethanolic solution of beclomethasone dipropionate
1
(5 mg mL ) to cell-culture ¯asks, either with or
3
without human osteoblasts added, yielding an
initial concentration of 10 mM in ethanol±culture
medium (0Á1 : 99Á9, v=v). At predetermined time
intervals, 10 mL samples were removed and
was stored in PBS as a 10 M stock solution at
4ꢀC. Dichloromethane (99Á9%, HPLC grade) was
purchased from Sigma (St. Louis, MO). Acetic acid
of analytical reagent grade was obtained from

420
CELINA SEETO ET AL
Ã
Rohne-Poulenc Chemicals (Clayton South, Vic-
toria, Australia). ChromAR HPLC-grade methanol
and acetonitrile, as well as ethanol (analytical
reagent grade), were purchased from Biolab Sci-
enti®c (Clayton South, Victoria, Australia).
beclomethasone monopropionate, were also able to
inhibit IL-5 protein production though not to
the same extent as beclomethasone and 17-
beclomethasone monopropionate (Figure 1).
12
Beclomethasone, at a concentration of 10 M, and
21-beclomethasone monopropionate, at a con-
10
centration of 10 M, both signi®cantly inhibited
Results
IL-5 protein production by 50% (P ˆ 0Á0166 and
P ˆ 0Á0038, respectively).
Mononuclear cells
In contrast to beclomethasone dipropionate and
its metabolites, a concentration of 10 M dexa-
Interleukin-5 protein production was detected in all
donors (n ˆ 6) following stimulation with PHA.
The IL-5 production following PHA stimulation
8
methasone was required to inhibit IL-5 secretion by
50% (P ˆ 0Á0128).
1
was within the range 150±814 pg mL . There was
no detectable IL-5 production in unstimulated cells.
The parent compound, beclomethasone dipro-
pionate, and its major metabolite, 17-beclometha-
sone monopropionate, caused signi®cant inhibition
of IL-5 protein production at concentrations as high
Osteoblasts
The cells which grew from the trabecular ends of
long bones under these culture conditions exhibited
phenotypic characteristics typical of osteoblasts.
These included high alkaline phosphatase activity,
a marker of osteoblastic differentiation, which was
further stimulated by treatment with 1,25-dihy-
droxyvitamin D and secretion of the bone-marker
protein osteocalcin, when treated with 1,25-dihy-
droxyvitamin D. When maintained in culture for
long periods on Thermanox coverslips, the cells
form a relatively regular multilayer which has an
abundant collagen matrix and which mineralises
after about 4 weeks in culture (Slater et al 1994
a, b).
9
14
as 10 M and at concentrations as low as 10
M
14
(P < 0Á0001 for both). Even at 10
M, both
beclomethasone dipropionate and 17-beclometha-
sone monopropionate signi®cantly inhibited IL-5
protein secretion by 85% and 80%, respectively,
relative to the positive control (Figure 1). The other
metabolites, namely beclomethasone and 21-
All glucocorticoids tested stimulated alkaline
phosphatase activity in bone cells, and all inhibited
thymidine incorporation and 1,25-dihydroxy-
vitamin D-stimulated osteocalcin synthesis by these
cells. When subjected to analysis of variance, the
dose±response curves for each of the glucocorti-
coids were not signi®cantly different from dex-
amethasone for any of the parameters tested in
bone cells. All the EC50s were in the nanomolar
range (Table 1).
Figure 1. Activity of beclomethasone dipropionate, its meta-
bolites and dexamethasone on IL-5 protein production in
human peripheral blood mononuclear cells. Results are
expressed as a percentage of the untreated PHA-stimulated
control value in each donor. The mean and standard error
(vertical bars) of 3±6 experiments are shown. u, Beclometha-
sone dipropionate (n ˆ 4); j, 17-beclomethasone monopro-
pionate (n ˆ 4); m, beclomethasone (n ˆ 6); d, 21-beclo-
methasone monopropionate (n ˆ 6); s, dexamethasone
(n ˆ 3).
Conversion of beclomethasone dipropionate
to esters
Following the incubation of beclomethasone
dipropionate as parent drug at 37ꢀC in the cell
culture ¯asks, either with or without the addition of
human bone-derived cells, serial formation of 17-
Table 1. EC50 (nM) values for beclomethasone esters in osteoblasts.
Assay
17-Beclomethasone 21-Beclomethasone Beclomethasone Beclomethasone Dexamethasone
monopropionate
monopropionate
dipropionate
Thymidine incorporation
Alkaline phosphatase
Osteocalcin
0Á6
1Á1
0Á4
4Á0
1Á6
0Á7
0Á5
0Á3
1Á2
1Á7
1Á6
0Á9
3Á1
0Á4
1Á6

DIFFERENTIAL POTENCY OF BECLOMETHASONE ESTERS IN-VITRO
421
beclomethasone monopropionate and beclometha-
sone was observed. In the culture medium alone,
beclomethasone dipropionate was relatively stable
sensitivity between cells from different donors.
This variability is frequently seen when working
with cells from human donors. The EC50 values for
dexamethasone were similar for peripheral blood
mononuclear cells and osteoblasts. However, the
sensitivity to beclomethasone esters differed
markedly between the two cell populations. In
osteoblasts, the EC50 for beclomethasone dipro-
pionate, 17-beclomethasone monopropionate and
beclomethasone were similar to the EC50 for
dexamethasone. In contrast, beclomethasone and its
esters were much more potent than dexamethasone
in inhibiting IL-5 production by peripheral blood
mononuclear cells. Peripheral blood mononuclear
cells were very sensitive to beclomethasone
1
=
(t 2 ˆ 52Á4Æ 2Á7 h, n ˆ 3, Table 2), with less than
23% of beclomethasone dipropionate being bio-
transformed after 18 h incubation. In contrast, rapid
formation of 17-beclomethasone monopropionate
following incubation of beclomethasone dipropio-
nate with human bone-derived cells was observed
1
=
(t 2 of beclomethasone dipropionate ˆ 3Á3Æ 0Á2 h,
n ˆ 3, Table 2). The intermediate metabolite, 17-
beclomethasone monopropionate was subsequently
1
=
hydrolysed at a much slower rate (t 2 ˆ 12Á6Æ 0Á9 h,
n ˆ 3) to form the ultimate product (beclometha-
sone) with human bone-derived cells (Table 2).
The active metabolite, 17-beclomethasone mono-
propionate was approximately 4-fold more stable
than its parent, beclomethasone diproprionate, in
the presence of human bone-derived cells. Thus,
the overall kinetics following incubation of beclo-
methasone dipropionate with human bone-derived
cells were rate-limited by the decomposition of 17-
beclomethasone monopropionate.
dipropionate and 17-beclomethasone mono-
propionate, with concentrations of 10
14
M inhi-
biting IL-5 production by greater than 50%. In
general, T-lymphocytes (which are the cells in the
population that release IL-5) are very sensitive to
the effects of corticosteroids (Robinson et al 1993).
The effects of beclomethasone dipropionate on
human T-lymphocytes have been reported pre-
viously. Crocker et al (1998) isolated populations
of allergen-speci®c T-lymphocytes after long-term
culture (30 days) in the presence of allergens.
Discussion
These cells were then incubated in beclomethasone
M) for 24 h before sti-
12
These experiments were carried out to determine
the effects of beclomethasone and its esters in two
different human cell populations; ®rstly, peripheral
blood mononuclear cells, comprising mainly T-
lymphocytes, as an index of potency in inhibiting
release of the pro-in¯ammatory cytokine, inter-
leukin-5 (IL-5), and secondly, osteoblasts, as an
index of potency in a target cell for unwanted
systemic activity. Dexamethasone was used as a
reference corticosteroid in both cell populations.
Both the peripheral blood mononuclear cells and
osteoblasts responded to each of the corticoster-
oids, although there was considerable variation in
dipropionate (10 7±10
mulation with PHA. Beclomethasone dipropionate
inhibited IL-5 production in a dose-dependent
9
manner, with an IC50 value of 3Á6Â10 M. The
sensitivity of the T-lymphocytes to beclomethasone
dipropionate in our experiments was approximately
10 000 times that reported by Crocker et al (1998).
It is possible that the 30-day period of incubation
with allergens altered the corticosteroid responses
of the T-lymphocytes used by Crocker et al (1998).
Another possible explanation for the discrepant
®ndings may be that the subsets of allergen-speci®c
T-lymphocytes studied by Crocker et al (1998)
Table 2. Concentration±time data of beclomethasone dipropionate and its metabolites, following
the incubation of beclomethasone dipropionate in culture ¯asks, with and without human
osteoblasts at 37ꢀC.
Incubation media
Time (h)
Mean concn (mM, n ˆ 3)
Beclomethasone 17-Beclomethasone Beclomethasone
dipropionate
monopropionate
Culture ¯asks
0
1
6
18
0
1
10Á0
9Á7
0
0
0Á2
0Á6
1Á6
0
2Á1
3Á9
2Á0
0Á1
0Á2
0Á5
0
0Á2
3Á2
7Á3
9Á2
7Á8
Culture ¯asks ‡ osteoblasts
10Á0
7Á7
6
18
2Á8
0Á6

422
CELINA SEETO ET AL
were less sensitive to beclomethasone dipropionate
than the broader population of lymphocytes repre-
sented in the peripheral blood mononuclear cells
used in our experiments.
binding of the ligand induces shape changes in the
activated receptor complex which differ from
ligand to ligand and which are modi®ed by the
particular receptor-associated proteins present in
that cell type. The ®nal effects will also depend on
dimerisation characteristics of the receptor±ligand
complex, which may involve more than one
receptor type, and how that complex interacts with
target sequences in the DNA of that cell, modi®ed
by the DNA-associated proteins present (Haussler
et al 1998). The more complex model predicts that
different compounds acting through the same
receptor may display different activities in different
cells (McDonnell & Norris 1997).
The mono-ester, 21-beclomethasone mono-
propionate, which has very low steroid receptor
binding (WuÈrthwein & Rohdewald 1990) had
demonstrable biological activity, particularly in the
osteoblasts. This may be due to non-steroid recep-
tor binding effects, which have been reported with
other similar agents such as vitamin D (Norman
1998).
The magnitude of the potency ratios for beclo-
methasone dipropionate : dexamethasone and 17-
beclomethasone monopropionate : dexamethasone
in peripheral blood mononuclear cells was greater
than predicted from binding studies reported by
others (WuÈrthwein & Rohdewald 1990). In our
experiments, 17-beclomethasone monopropionate
was approximately 10 000 more potent than dexa-
methasone in inhibiting IL-5 production yet it
has only 10 times the relative receptor af®nity
(WuÈrthwein & Rohdewald 1990). There are many
steps between steroid receptor binding and cellular
response. These ®ndings suggest that, at some or all
of these steps, the effects of beclomethasone are
ampli®ed or distorted with respect to dexa-
methasone to produce a ®nal cellular effect ratio
which is discrepant from that predicted by receptor
binding studies.
In the experiments with peripheral blood mono-
nuclear cells, the effects of beclomethasone dipro-
pionate and 17-beclomethasone monopropionate on
IL-5 release were indistinguishable, which may
have been due to esterase activity fully metabo-
lising beclomethasone dipropionate to its active
metabolite 17-beclomethasone monopropionate
during the incubation period of 48 h. The decom-
position rate of beclomethasone dipropionate was
about 16 times faster in the presence of osteoblasts
than in culture medium alone, indicating relatively
high esterase activity for beclomethasone dipro-
pionate in osteoblasts. It has been reported that
beclomethasone dipropionate is hydrolysed to 17-
beclomethasone monopropionate and beclometha-
sone in the human lung (Martin et al 1974;
Andersson & Ryrfeldt 1984; WuÈrthwein & Roh-
dewald 1990) and gut homogenates (WuÈrthwein &
Rohdewald 1990), so it is likely that peripheral
blood mononuclear cells possess this esterase
activity as well.
In human osteoblasts, this extraordinary differ-
ential activity of dexamethasone and beclometha-
sone dipropionate was not observed. The vast
differences in bioactivity of the beclomethasone
dipropionate compounds in the two cell types add
to evidence that early models of steroid hormone
action, in which a ligand was thought to switch the
receptor from an inactive to an active state and
receptor af®nities were thought to be the prime
determinants of bioactivity, are clearly over-
simpli®cations. Different oestrogens also have a
wide range of activity in different cell types
(McDonnell & Norris 1997). It seems likely that
Beclomethasone and its esters were far more
potent in inhibiting release of the pro-in¯ammatory
cytokine IL-5 from peripheral blood mononuclear
cells than in affecting functional responses of
osteoblasts, such as alkaline phosphatase activity
and osteocalcin release. If one accepts that a
putative anti-in¯ammatory drug should have
greater effect on a response which is part of the
in¯ammatory reaction and less effect on a cell
involved in unwanted side effects (such as osteo-
blasts) then the beclomethasone esters ful®ll this
criterion.
Finally, these results show the importance of
examining effects of corticosteroids on cellular
responses, as potency ratios observed in receptor
binding assays are not necessarily predictive of the
magnitude of the potency ratios in cell populations
possibly relevant to the ef®cacy and side effects.
Acknowledgements
We thank Mathew Traini for technical assistance
and Dr Peter Gray, 3M Pharmaceuticals, for pro-
viding the samples of beclomethasone dipropionate
and beclomethasone monopropionate. Celina Seeto
is the recipient of a PhD scholarship funded by 3M
Pharmaceuticals.
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