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Abstract |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Functional implications of the recently described fatty acid conjugation of budesonide (BUD) (Tunek, A.,
K. Sjödin, and G. Hallström, Drug Metabol. Dispos. 1997;25:1311-1317; Miller-Larson, A., E. Hjertberg,
H. Mattsson, M. Dahlbäck, A. Tunek, and R. Brattsand, Am. J. Respir. Crit. Care Med. 1997;155:A353
[Abstr.]) were studied in a rat cell line, Rat1, transfected with the activation protein-1 (AP-1)-controlled
regulatory element (TRE) driving the reporter gene 
-galactosidase. TRE is downregulated by glucocorticosteroids (GCS) through interaction with the AP-1 complex. BUD was compared to fluticasone propionate (FP), a potent glucocorticosteroid that does not form fatty acid conjugates. The kinetics and metabolism of the GCS were studied after incubation of either 3H-BUD or 3H-FP with transfected Rat1 cells. Up
to 20% of added BUD was taken up into the cells over 24 h. The great majority of the intracellular radioactivity (80-90%) consisted of lipophilic BUD conjugates. After removing extracellular 3H-GCS, the outflow of radioactivity was studied. Only free BUD and not fatty acid conjugates was detected extracellularly, suggesting that hydrolysis of the conjugates was required to release BUD from the cell. During 165 min, less BUD (about 65% of totally incorporated) was released than FP (more than 90%). In the functional studies, FP was about six times more potent than BUD in downregulating TRE after 24 h continuous
exposure. However, after a 6-h pulse of GCS, the effect of BUD persisted unchanged 18 h later, whereas FP had almost lost its efficacy (P < 0.05 between the drugs). In addition, the reversible conjugation process of BUD resulted in transferable GCS effects. Medium containing released BUD from previously
loaded cells mediated nearly the same downregulatory effect after addition to naive cells as did continuous
treatment. No such transferable effect was seen for FP. In conclusion, the reversible fatty acid conjugation
of BUD resulted in prolonged cellular retention and anti-inflammatory activity after pulse exposure in this
in vitro system. This fatty acid conjugation mechanism appears to add to the beneficial pharmacologic profile of BUD.
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Introduction |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Glucocorticosteroids (GCS) developed for inhalation, such as budesonide (BUD) and fluticasone propionate (FP), exhibit better airway selectivity than earlier generations of steroids developed for systemic use, such as hydrocortisone (HC), prednisolone, and dexamethasone (1). A key chemical feature mediating this selectivity is lipophilicity. Optimal lipophilicity obtained by appropriate substitutions will enhance both receptor affinity (2) and hepatic first-pass inactivation (1). In addition, optimal lipophilicity maximizes dwell time within tissue, which seems to be another key component for the desired airway selectivity of inhaled GCS.
The relationship between lipophilicity and airway potency has hitherto been evaluated in terms of the intact steroids (2, 3) because there has been little evidence of local metabolism. The recently discovered, reversible fatty acid conjugation of BUD adds novel concepts to this discussion. GCS esterified at the C-21 position, such as in the fatty acid conjugates of BUD (4), have been shown to have very low or no affinity to the GCS receptor (1, 2). Thus, these conjugates are considered inactive. BUD can be conjugated to fatty acids within many cell types and in that way can be stored in an inactive but recoverable form. The conjugated BUD can be hydrolyzed by intracellular lipases, releasing free and pharmacologically active BUD (4, 5). Of four GCS (BUD, beclomethasone propionate [BDP], FP, and HC) studied in human lung tissue in vitro, only BUD undergoes a substantial fatty acid conjugation (4, 5). In a rat tracheal model, a more protracted tissue binding has been reported for BUD than for the other three GCS, suggesting that conjugated BUD might also serve as a depot in the tissue (5, 6) in vivo.
Conjugation of steroids to nonpolar compounds in vivo and in vitro has been described previously (7, 8). The lipophilic compounds have been characterized as fatty acid esters (8) and shown to occur for a number of different steroids, including endogenous GCS and estrogens (9, 10). To our knowledge, however, formation of fatty acid esters of a synthetic GCS has not been reported previously.
The aim of the present study was to investigate the functional implications of the fatty acid conjugation of BUD in a well-defined cellular system in vitro in which concentration and exposure time could be adequately controlled. In this system, pulse exposure versus continuous exposure to the GCS was compared. This has clinical relevance because GCS are inhaled one to four times daily and a changed activity profile after pulse exposure may affect both therapeutic and adverse effects. BUD was compared with FP, which has higher receptor affinity (2, 11) than BUD but does not undergo fatty acid conjugation.
As a readout for functional GCS activity, Rat1 cells
transfected with the regulatory element TRE (AP-1 controlled regulatory element) coupled to the reporter gene
-galactosidase was used. Steroids inhibit TRE by binding
of the GCS-receptor (GR) complex to the transcription
factor AP-1 (activation protein-1), which otherwise activates transcription via TRE (12). Activation of AP-1 and
its subsequent binding to regulatory elements such as TRE
is important in mediating the pro-inflammatory effects of many cytokines, growth factors, and proteases (12). Inhibiting AP-1/TRE activation is one important mechanism
behind the therapeutic efficacy of GCS in asthma (13).
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Materials and Methods |
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Reagents
O-nitrophenol--D-galactopyranoside (ONPG) was obtained from Sigma (St. Louis, MO). Cell culture media and
fetal calf serum (FCS) were purchased from GIBCO BRL
(Paisley, UK). BUD, FP, and BUD-oleate were obtained
from Astra Draco AB (Lund, Sweden). All other chemicals were of analytical grade.
Radiochemicals
3H-BUD and 3H-FP (radiochemical purity > 97%) were
obtained from Radiochemical Center (Amersham, Little
Chalfont, UK) or Astra Draco AB. The radiochemical
concentration was 1.1-3.8 mCi/ml for BUD (specific radioactivity, 17-37 Ci/mmol), and 1.3-2.0 mCi/ml for FP
(specific radioactivity, 5.4-33 Ci/mmol). The 3H-GCS were
stored in stock solutions in ethanol at 
20°C at a concentration of 10
4 to 105 mol/liter.
Cell Culture Materials
All cell culture plastics were obtained from Costar (Badhoevedorp, The Netherlands), except 50-ml sterile tubes from Falcon (Labora, Stockholm, Sweden) and microtiter plates from Nunc (Roskilde, Denmark).
Cell Culture
The rat fibroblast cell line, Rat1 (14), was initially obtained from Dr. Å. Oldberg, University of Lund (Lund, Sweden). The Astra Draco-derived Rat1 TRElacZ transfectant was grown in Dulbecco's modified Eagle's medium supplemented with 10% FCS, glutamine (4 mmol/liter), penicillin (50 U/ml), streptomycin (50 IU/ml), and 1% nonessential amino acids (GIBCO). Cells were cultured at 37°C and 5% CO2, and passaged once weekly; experiments were performed on cells within 15 passages. Transrepression (12) mediated by GR/AP-1 interactions with TRE was studied after GCS treatment.
Reporter Gene Assay
The activity of TRE in the transfected Rat1 cells was determined by assaying the reporter gene lac Z (-galactosidase). Enzyme activity was determined by a spectrophotometric assay performed in microtiter plates. Cells were lysed
with 50 µl 0.5% (or 0.1% when protein was determined simultaneously) Triton X-100 followed by the addition of
40 µl of the reaction mixture (1 µl of a solution containing
0.1 mol/liter MgCl2, 4.5 mol/liter -mercaptoethanol, 22 µl
ONPG [4 mg/ml in 0.1 mol/liter sodium phosphate, pH 7.5], and 17 µl 0.1 mol/liter sodium phosphate [pH 7.5]) to each
well. After 60 min at 37°C, 100 µl stop buffer (300 mmol/liter glycine, 15 mmol/liter ethylenediamine tetraacetic acid,
pH 11.3) was added and absorbance read at 420 nm in a plate
reader (Multiscan MCC/340; Labsystems, Stockholm, Sweden). Downregulation of TRE by GCS was expressed as
the percentage of activity present in cells not treated with
GCS.
Pharmacokinetic Studies
Uptake and time course of formation of lipophilic conjugates.
Transfected Rat1 cells were seeded in 10-cm petri
dishes and allowed to grow to about 90% confluency. Medium containing 3H-BUD at a concentration of 3 × 108
mol/liter (diluted from the ethanol stock solution, taking
care not to exceed 0.1% ethanol in the final solution) was
added. After incubation for 30 min, 6 h, or 24 h at 37°C in
5% CO2, cells were rapidly washed five times with medium
and scraped from the dish in a small volume of medium.
The cell suspension was then centrifuged at 1,000 rpm and
4°C for 10 min and the medium carefully removed. Results
are given as percentage of total amount of radioactivity added to the culture, mean of three dishes (standard deviation, SD). In a similar experiment, pellets were extracted
in 2 ml of ethanol (99.9%) overnight at room temperature
and the ethanol phase subjected to high-pressure liquid
chromatography (HPLC) in liquid chromatography-system
1 as described by Tunek and associates (4).
Temporal study of the reversibility of conjugation.
Transfected Rat1 cells were incubated with medium containing
3 × 108 mol/liter 3H-BUD or 3H-FP for 2 h. After rapid
washing three times, GCS-free medium was added to the
cells. During further incubation for 165 min, 2 × 100 µl
was taken for analysis of radioactivity at different time
points. The samples were replaced with fresh medium. At
the end of the experiment, cells were washed and harvested as above for analysis of radioactivity in the ethanol
extracts. Some medium samples were fractionated by HPLC
in LC-system 3 (4) to study the balance between free and
conjugated GCS.
Estimation of GCS concentrations in cells and medium
after pulse administration.
Experiments were performed
as described below (duration or conditioned-medium experiments), except that 3H-BUD and 3H-FP at 107 mol/liter
were used. When mimicking the pulse-duration studies, the
cells were moved to new flasks instead of microplates to
facilitate recovery of medium and cells. Samples (0.5 ml)
from each incubation step were taken and added to scintillation vials. After removal of the last medium from the cells,
they were scraped from the flasks and centrifuged, and radioactivity was determined in the pellet using Ultima Gold
as scintillation fluid. Results were calculated as percent radioactivity recovered from the initial amount added.
Pharmacodynamic Studies
Continuous exposure: dose-response studies.
Transfected
Rat1 cells were grown in microplates to about 90% confluency. Medium was changed to medium containing GCS
(either BUD, BUD-oleate, or FP) at concentrations ranging from 1012 to 106 mol/liter. Cultures were further incubated for 24 h, followed by determination of -galactosidase activity in the cells.
Pulse exposure: comparison of activity of BUD and FP
after a 6-h pulse of GCS.
Transfected Rat1 cells were cultured in flasks with BUD (107 mol/liter) or FP (107 or 2 × 108 mol/liter) for 6 h. Cells were thereafter washed repeatedly by incubating them five times for 5 min with fresh
medium. This washing procedure was included in order to
remove as much free and rapidly extractable (presumably
nonconjugated) GCS as possible. Thus, when washed five
times, 85 to 90% of BUD (compared with rapid washing)
was retained in the cells but only 10 to 30% of FP (see also
Table 1, In cells after wash).
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-galactosidase to the
amount of protein.
Conditioned medium: release of transferable activity into
conditioned medium.
Transfected Rat1 cells were incubated with GCS-containing medium (107 mol/liter BUD
and 107 or 2 × 108 mol/liter FP) for 3 h before changing
to GCS-free medium and culture for an additional hour.
Thereafter, cells were washed intensively five times with 5 min incubation, and the last wash medium was collected.
After the washings (performed to remove free, nonconjugated GCS), cells were transferred to new flasks by gentle
trypsinization as described above. No feeder layer was used.
GCS-free medium was again added and the cultures continued for an additional 3 h to allow release of reversibly
conjugated GCS. Thereafter, the medium conditioned with
the released GCS was collected. The last wash and this 3-h
conditioned medium (CM) were centrifuged at 4°C and 1,000 rpm for 10 min to remove loose cells, and added to new cultures of transfected Rat1 cells grown in microplates. The
plates were incubated at 37°C overnight before determination of the downregulation of TRE mediated by released GCS in the CM. Control cells were treated in the same way
except that all washes and subsequent media contained the
GCS at adequate concentration.
Statistics
For the dose-response studies, parallel sigmoidal curves were fitted to data using nonlinear regression. The relative dose potency was estimated from the horizontal distance of the curves and 95% confidence limits were constructed. In the BUD-FP duration and CM studies, data were analyzed using analysis of variance followed by sequential pairwise comparisons. In the duration studies, this was performed in two ways. In Method 1, comparison between the downregulation of BUD and FP after the 6-h pulse was performed. In Method 2, comparison between the downregulation after the 6-h pulse and the downregulation after continuous incubation (control) was made for each GCS.
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Results |
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Pharmacokinetic Studies
Uptake of BUD and time course of formation of lipophilic conjugates. Incorporation of 3H-BUD into transfected Rat1 cells increased during the investigated period; from a mean of 3.3% (SD 0.24) of totally added radioactivity at 30 min to 15.6% (SD 1.4) at 6 h to 21.3% (SD 1.5) at 24 h. Thus, after 24 h about 20% of the initially added radioactivity had been taken up by the cells. The proportion of the total amount of intracellular radioactivity present as lipophilic conjugates remained at 80 to 90% during the whole time period studied (Figure 1). The main conjugate peak contains BUD-oleate and the smaller peak contains BUD-palmitoleate, but both peaks are probably mixtures of more than one fatty acid ester.
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Release of GCS-derived radioactivity from preloaded Rat1 cells. In another experiment, cells were loaded with 3H-GCS for 2 h. About 20% of total added radioactivity from either BUD or FP was taken up. After washing to remove nonconjugated GCS, 13.3% of BUD and 5.8% of FP remained in the cells. The release of remaining intracellular GCS was followed during 165 min (Figure 2). For FP, a steady state in the medium was reached after only 25 min, whereas the release of BUD was markedly slower, with total extracellular levels constantly increasing. Presumably, the rate-limiting step for release of BUD was hydrolysis of the fatty acid conjugates. Almost 25 times less FP (0.2%) than BUD (5.0%) remained in the cells at the end of the experiment. More than 90% of the FP-related radioactivity in the cells at the beginning of the release period was released over 165 min compared with about 65% of BUD-related radioactivity. HPLC analysis of the BUD medium at 15, 55, and 165 min in a similar experiment revealed that all of the extracellular radioactivity represented intact BUD; no lipophilic conjugates were found in the medium (Figure 3). Note that in HPLC system 3, the conjugates have longer retention time than in system 1 (4) (and are thus not recorded on this part of the tracing). In a separate experiment, the HPLC profiles of cells treated with 3H-BUD or 3H-FP confirmed that no lipophilic conjugates were formed with FP in the transfected Rat1 cells (data not shown).
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Disposition of the GCS during pulse-duration and conditioned-medium experiments. The disposition of total radioactivity during experiments comparing the duration of pharmacologic activity of BUD and FP is shown in Table 1 (Duration Experiments section). After the 6-h pulse incubation and washes, approximately 13% of totally added 3H-BUD radioactivity was estimated to be retained in the cells. The subsequent movements and further incubation over 18 h reduced that proportion to approximately 5% (i.e., more than one-third of the amount after the last wash was still retained within the cells). For FP, the amount in the cells at the end of the 6-h pulse period (after the last wash) was estimated to be approximately 0.7%, presumably due to larger losses during washes. Furthermore, only 0.1% remained in the cells at the end of the 18-h incubation. Thus, at the end, the amount of FP in the cells was 50 times lower than that of BUD (free and conjugated).
A similar kinetic difference between the substances was seen in the CM experiments (Table 1, Conditioned-Medium Experiments section). The generally lower level of GCS in medium and cells reflects the shorter GCS pulse (3 h). The proportion of radioactivity recovered in the last wash was similar for both GCS. However, during production of the CM over the next 3 h, more than 10-fold greater radioactivity was released from the BUD-treated cells (4.1%) than from the FP-treated cells (0.4%). While for BUD this release represented a more than 10-fold increment over what was found in the last wash, there was no difference for the FP-treated cells. In these experiments, as in the pulse experiments, 50 times more BUD than FP was retained intracellularly at the end of the experiment.Pharmacodynamic Studies
Continuous exposure: dose-response.
The relative potencies in downregulating TRE in transfected Rat1 cells
are shown in Table 2 and exemplified in Figure 4. By using
a sigmoidal model to describe the dose-response curves, FP was estimated to be about six times (95% confidence
limits: 3.4-11.8, n = 8) more potent than BUD. On the
basis of that ratio, in the subsequent studies BUD was
compared with two concentrations of FP, where the lower
concentration of 2 × 108 mol/liter was considered roughly
equipotent with 107 mol/liter BUD (as determined during continuous exposure). In addition, BUD was estimated
to be about 150 times more potent (confidence limits:
37-671, n = 3) compared with extracellularly added BUD-oleate.
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Pulse exposure: effect after a 6-h GCS pulse and subsequent
move of the cells to new microplates.
Cells were treated with
a 6-h pulse of BUD or FP, washed intensively, and moved
to new microplates containing a feeder layer of nontransfected cells (to facilitate adherence). The cells had to be
moved to avoid the strong plastic adsorption of FP mentioned previously, which strongly interfered with the experimental outcome. The remaining efficacy 18 h after the
GCS pulse is shown in Figure 5 (note that effects are related to the amount of protein in the cultures to account
for potential differences in adhesion of the moved cells).
The mean level of downregulation in the control cultures
with 24-h continuous exposure was around 60%. After a 6-h pulse, BUD (107 mol/liter) caused 54% downregulation, whereas FP (107 and 2 × 108 mol/liter) downregulated TRE by only 28 and 11%, respectively (P < 0.05 and P < 0.001, compared with BUD). The BUD response to a 6-h pulse did not differ significantly from the corresponding values with continuous incubation, whereas this
was clearly the case for the equipotent FP concentration
(2 × 108 mol/liter) and even for the higher FP concentration, 107 mol/liter. Thus, with pulse exposure FP downregulated TRE significantly less than in the continuously
exposed controls (P < 0.001 for both concentrations).
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Production of conditioned medium containing GCS activity.
Cells were pulsed with BUD and FP for 3 h, intensively washed, and subsequently moved to new flasks, where
released GCS was collected in fresh medium (defined as
CM after the 3 h incubation) over 3 h. The effect of CM
from cells pretreated with BUD (107 mol/liter) or FP
(107 mol/liter or 2 × 108 mol/liter) on naive cells is shown
in Figure 6. At t = 0 (from "last wash"), no downregulatory
activity was mediated by BUD-treated cells. However, the
"last wash" from FP-treated cells mediated a substantial
downregulatory activity (around 23%). This effect was
shown to be the result of FP strongly adhering to the plastics of the initial wells, indicating the necessity of moving
the cells to new flasks. In fact, a similar effect of CM was
obtained from incubations with FP-containing medium
(107 mol/liter) in flasks with or without transfected Rat1
cells. Moving the cells to new flasks prevented this interference (data not shown).
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Discussion |
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Introduction
Materials and Methods
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Discussion
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Asthma is a chronic inflammatory disease of the airways, and inhaled GCS are known to be the first choice of therapy for its long-term treatment. Key properties of these GCS are a combination of high receptor affinity, a high hepatic first-pass inactivation, and a prolonged binding to target tissue (1, 11). These desired properties are improved by a high lipophilicity (2, 16). However, the optimal lipophilicity is not known, and none of the current GCS have been designed for and primarily selected in airway models. Moreover, the models that have been used to describe the properties of inhaled GCS, such as receptor affinity (2, 3, 11, 17) and functional potency in target or non-target cells (18), have been in vitro models. In these models, FP and BDP (for the latter compound partly through its primary hydrolysis product beclomethasone monopropionate) exhibit the highest potency, and they are also the most lipophilic steroids. These in vitro studies were based on a continuous incubation of test GCS, whereas, in the clinical situation, inhalation therapy is administered as local pulses, followed by GCS drainage from target via blood circulation. The importance of a varying extent of prolonged tissue binding will presumably be poorly detected in experiments based on continuous GCS incubation in vitro.
The aim of this study was to compare coupled kinetics
and dynamics (especially regarding duration) of the two
potent GCS, BUD and FP. The latter has higher lipophilicity, twice the receptor affinity, and sometimes an even
higher functional potency compared with BUD in assays
in vitro (2, 11, 19), but lacks the free hydroxyl in the 17
side-chain required for formation of fatty acid conjugates.
Moreover, no fatty acid conjugate formation by FP has
been seen in human or rat tissues as well as in the transfected Rat1 cell line. In inhalation therapy in clinical asthma, the potency difference between BUD and FP has
been reported to be 2-fold (21; based on meta-analysis) or
lacking (22). Furthermore, in a series of controlled trials
BUD demonstrated efficacy even on a once-daily regimen
(23, 24). This suggests that the earlier in vitro tests may not
adequately mimic the clinical efficacy. Thus, one aim was
to design in vitro models where especially duration comparisons could be performed better and, for BUD, to relate results after both continuous and pulse exposure to
the fatty acid conjugation. Transfected Rat1 cells were
shown to conjugate and deconjugate BUD in a similar
manner to the reversible conjugation in airways recently
described (4, 5) and were thus selected as a suitable cell
system.
Kinetics of Fatty Acid Conjugation in Rat1 Cells
The uptake studies revealed that the intracellular formation of fatty acid conjugates of BUD is a rapid process. After only 30 min of incubation, the great majority of intracellular BUD existed in the conjugated form. In vitro studies in microsomal systems show that acetyl coenzyme A and adenosine triphosphate are required for BUD conjugation (4) and that the conjugation is partly blocked by inhibitors of acyl coenzyme A:cholesterol acyltransferase inhibitors (data not shown). Together, these data suggest that BUD conjugation might resemble the metabolic routes of cholesterol or endogenous steroids, such as estrogens (25). However, the exact enzymatic pathway of the conjugation process remains to be elucidated.
The conjugation was reversible; that is, when the medium was replaced with medium without BUD, the nonconjugated steroid but not the lipophilic conjugates was released from the cells. The reversibility of the fatty acid conjugation of BUD seems to be dependent on the action of lipases as reported recently (4). In analogy with already described fatty acid conjugates of estrogens (26), a potential lipase candidate could be hormone-sensitive lipase (27). Further studies of these mechanisms are ongoing in our laboratory.
Presumably due to the reversible conjugation reaction, BUD was more efficiently retained intracellularly than FP, which cannot be conjugated. Thus, about 30 to 50 times more BUD than FP was retained in the cells after prolonged incubation in GCS-free medium. FP was also more easily removed from cells by extensive washings, in spite of its higher lipophilicity (FP being eight times more lipophilic than BUD [5]). However, the lipophilicity of BUD-oleate was shown to be 500 times higher than that of FP (5; data in the files of Astra Draco). A concordant retention of BUD was also seen in vivo and in studies employing rat trachea ex vivo (5, 6), showing that pulse exposure in cell cultures can mimic in vivo kinetics with its continuous blood drainage.
Aspects of Fatty Acid Conjugation Related to Functional GCS Activity
The uptake studies also show that the proportion of conjugate formation seems to be constant over time (i.e., with
various intracellular BUD concentrations). However, prolonged functional steroid activity is best attained when the
cells are exposed to high concentrations of BUD so that
substantial amounts of conjugates are formed. Thus, pulse
incubation of transfected Rat1 cells with 107 mol/liter BUD
resulted in more prolonged activity than 109 mol/liter
BUD (data not shown). This concept is also supported by
the BUD kinetics in situ in the rat trachea (5). Here the most marked retention of BUD was obtained at the large
airway level, where the BUD deposition is most prominent. BUD concentration can be estimated to be about
107 mol/liter at that level, whereas in plasma the concentration is suggested to be around 109 mol/liter (1). Presumably BUD, having a lower lipophilicity and thus higher
water solubility than FP, can rapidly be taken up into the
target tissue and there converted to the much more lipophilic fatty acid conjugates. Thus, the high BUD concentration at target can, by the reversible fatty acid conjugation mechanism, result in a local, marked prolongation of
activity.
BUD-oleate was about 150 times less potent in downregulating TRE than BUD in this study. The minute GCS activity of BUD-oleate might even depend on a small contamination with BUD. Obviously, at extracellular addition to transfected Rat1 cells, the preformed conjugate did not have access to the intracellular enzymes, presumably lipases, that have been shown to hydrolyze the conjugate to active BUD (4, 27). Its very limited water solubility might also contribute to the low activity in this in vitro study. Other studies have shown low receptor affinity of fatty acid conjugates of endogenous steroids (estrogens, androgens, or corticoids [28, 29]) and synthetic GCS (data in files of Astra Draco and 2) coupled to low functional activity in vitro (29).
The potency relationship between BUD and FP varied strongly whether the GCS were administered continuously or as a pulse. Under constant incubation FP was six times more potent, in agreement with earlier in vitro studies (19, 20). On the other hand, after the pulse followed by washing, BUD was clearly more effective. The probable explanation for this shift is that FP is more easily and rapidly extracted from the cells than is the largely conjugated BUD. Thus, the extensive washing produced an approximately 50 times lower intracellular concentration of FP, which correlated to its much lower activity.
To investigate the deconjugation process further, CM
experiments were performed. The CM produced by cells
pretreated with 107 mol/liter BUD mediated a greater
downregulation than did CM from cells pretreated with
2 × 108 or 107 mol/liter FP. This functional difference
was explained by the GCS concentrations released into
the CM. The estimated BUD concentration in CM was 10 times higher than for FP and sufficient to mediate a substantial downregulation of TRE. Of the total cellular
amount of BUD before the CM production (comprising
mainly conjugates), two-thirds were released, suggesting a
marked deconjugation process over the 3-h period. Thus, a
large enough intracellular depot of fatty acid esters of
BUD can release active BUD to other cells as well as prolong GCS activity in the cell where it was first deposited.
Supporting this concept, fatty acid esters of endogenous estrogens are known to prolong duration of activity in vivo
(30, 31), despite lacking receptor affinity (29, 32).
In conclusion, this in vitro study shows that BUD is efficiently taken up and retained in transfected Rat1 cells, mainly in the form of fatty acid conjugates. This and related studies (4) demonstrated the reversibility of the conjugation process, releasing active BUD intra- and extracellularly. Pulse administration of BUD results in a more prolonged duration of GCS activity than after FP. This was demonstrated in cells treated directly and (using naive cells treated with CM) indirectly. The retention and activity of GCS in transfected Rat1 cells have been studied after pulse exposure plus extensive washing, intended to partly mimic the drainage occurring in the tissue by the blood circulation. It is pointed out that BUD and FP are inhaled in asthma as "pulses," normally once to twice a day, suggesting that, for duration studies, pulse administration is a more relevant administration mode in vitro than constant exposure. The concordance of our results in vitro with findings in the rat trachea in vivo (5, 6, 32) supports the use of such investigations in vitro for mechanistic aspects of the conjugation/deconjugation process.
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Footnotes |
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Address correspondence to: Elisabet Wieslander, Astra Draco AB, Preclinical Research and Development, Dept. of Pharmacology, P.O. Box 34, S-221 00 Lund, Sweden. E-mail: elisabet.wieslander{at}draco.se.astra.com
(Received in original form September 29, 1997 and in revised form December 1, 1997).
Acknowledgments: The authors acknowledge Liv Severinsson and Marianne Juhlin at the Department of Cell and Molecular Biology, Astra Draco AB, Lund, Sweden, for providing the transfected Rat1 cell line. The authors are also grateful to Anna Miller-Larsson, Department of Pharmacology, Astra Draco, for helpful discussions; Per Larsson at Biostatistics and Data Management, Astra Draco AB, for statistical expertise; and Nina Åsvatne for typing the manuscript.
Abbreviations AP-1, activation protein-1; BUD, budesonide; CM, conditioned medium; FP, fluticasone propionate; GCS, glucocorticosteroid; HPLC, high-pressure liquid chromatography; SD, standard deviation; TRE, AP-1 controlled regulatory element.
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References |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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1. Brattsand, R., and B. Axelsson. 1997. Basis of airway selectivity of inhaled glucocorticoids. In Inhaled Glucocorticoids in Asthma, Mechanisms and Clinical Actions (Vol. 97, Lung Biology in Health and Disease Series). R. P. Schleimer, W. W. Busse, and P. M. O'Byrne, editors. Marcel Dekker, Inc., New York. 351-379.
2. Würthwein, G., S. Rehder, and P. Rodhewald. 1992. Lipophilicity and receptor affinity of glucocorticoids. Pharm. Ztg. Wiss. 137: 161-167 .
3. Högger, P., and P. Rodhewald. 1994. Binding kinetics of fluticasone propionate to the human glucocorticoid receptor. Steroids 59: 597-602 [Medline].
4.
Tunek, A.,
K. Sjödin, and
G. Hallström.
1997.
Reversible formation of fatty
acid esters of budesonide, an anti-asthma glucocorticoid, in human lung
and liver microsomes.
Drug Metabol. Dispos.
25:
1311-1317
5. Miller-Larsson, A., H. Mattsson, E. Hjertberg, A. Tunek, and R. Brattsand. 1998. Reversible fatty acid conjugation of budesonide: a novel mechanism for prolonged retention of topical steroid in asthmatic tissue. Drug Metab. Dispos. Biol. Fate Chem. (In press)
6. Miller-Larsson, A., H. Mattsson, S. Ohlsson, E. Hjertberg, A. Tunek, M. Dahlbäck, and R. Brattsand. 1994. Prolonged release from the airway tissue of glucocorticoids budesonide and fluticasone propionate as compared to beclomethasone dipropionate and hydrocortisone. Am. J. Respir. Crit. Care Med. 149: A466 . (Abstr.) .
7. Hochberg, R. B., S. L. Pahuja, J. E. Zielinski, and J. M. Larner. 1991. Steroidal fatty acid esters. J. Steroid Biochem. Mol. Biol. 40: 577-585 [Medline].
8.
Mellon-Nussbaum, S.,
L. Ponticorvo, and
S. Lieberman.
1979.
Characterization of the lipoidal derivatives of pregnenolone prepared by incubation of
the steroid with adrenal mitochondria.
J. Biol. Chem.
254:
12500-12505
9. Poulin, R., D. Poirier, C. Theriault, J. Couture, A. Belanger, and F. Labrie. 1990. Wide spectrum of steroids serving as substrates for the formation of lipoidal derivatives in ZR-75-1 human breast cancer cells. J. Steroid Biochem. Mol. Biol. 35: 237-247 .
10.
Hampel, M. R., and
Peng Li-Hia, M. R. J. Pearlman, and W. H. Pearlman.
1978.
Acylation of [3H]corticosterone by acini from mammary gland of lactating
rats.
J. Biol. Chem
253:
8545-8553
11. Brattsand, R., and B. Axelsson. 1992. New inhaled glucocorticosteroids. In New Drugs for Asthma, Vol. 2. P. J. Barnes, editor. IBC Technical Services, London. 193-208.
12. Jonat, C., H. J. Rahmsdorf, K. K. Park, A. C. Cato, S. Gebel, H. Ponta, and P. Herrlich. 1990. Antitumor promotion and antiinflammation: down-modulation of AP-1 (Fos/Jun) activity by glucocorticoid hormone. Cell 62: 1189-1204 [Medline].
13. Barnes, P., and S. Pedersen. 1993. Efficacy and safety of inhaled corticosteroids in asthma. Am. Rev. Respir. Dis. 148(Suppl.): S1-S26 .
14. Topp, W. C.. 1981. Normal rat cell lines deficient in nuclear thymidine kinase. Virology 113: 408-411 [Medline].
15. Bradford, M.. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72: 248-254 [Medline].
16. Brattsand, R.. 1997. What factors determine anti-inflammatory activity and selectivity of inhaled steroids. Eur. Respir. Rev. 7: 356-361 .
17. Phillips, G. H. 1990. Structure-activity relationship of topically active steroids: the selection of fluticasone propionate. Respir. Med. 84(Suppl. A): 19-23.
18. Ek, A., B. M. Larsson, L. Palmberg, and K. Larsson. 1996. Glucocorticoids inhibit the release of cytokines from epithelial cells. Eur. Respir. J. 9(Suppl. 23):126S.
19. Corrigan, C. J., J. K. Bungre, B. Assoufi, A. E. Cooper, H. Seddon, and A. B. Kay. 1996. Glucocorticoid resistant asthma: T-lymphocyte steroid metabolism and sensitivity to glucocorticoids and immunosuppressive agents. Eur. Respir. J. 9: 2077-2086 [Abstract].
20. Roca-Ferrer, J., J. Mullol, E. Lopez, A. Xaubet, L. Pujols, J. C. Fernandez, and C. Picado. 1997. Effect of topical anti-inflammatory drugs on epithelial cell-induced eosinophil survival and GM-CSF secretion. Eur. Respir. J. 10: 1489-1495 [Abstract].
21. Barnes, N., C. Hallett, and T. A. J. Harris. 1996. An overview of morning peak expiratory flow rate (PEFR) for clinical trials comparing fluticasone propionate with budesonide. Eur. Respir. J. 9(Suppl. 23):52S.
22. Agertoft, L., and S. Pedersen. 1997. A randomized double blind dose reduction study to compare the minimal effective dose of budesonide Turbuhaler and fluticasone propionate Diskhaler. J. Allergy Clin. Immunol. 99: 773-780 [Medline].
23. McFadden, E. R., Jr. 1997. Budesonide Turbuhaler once daily administration for patients with mild to moderate chronic asthma. Am. J. Respir. Crit. Care Med. 155(Suppl. 4, Pt. 2):A352. (Abstr.)
24. Venables, T. L., M. B. Addlerstone, A. J. Smithers, M. D. Blagden, D. Weston, T. Gooding, E. P. Carr, and R. M. Follows. 1996. A comparison of the efficacy and patient acceptability of once daily budesonide via Turbuhaler and twice daily fluticasone propionate via disc-haler at an equal dose of 400 mcg in adult asthmatics. Br. J. Clin. Res. 7: 15-32 .
25. Billheimer, J. T., and P. J. Gillies. 1990. Intracellular cholesterol esterification. In Advances in Cholesterol Research. M. Esfahani and J. B. Swaney, editors. The Telford Press, Caldwell, NJ. 7-145.
26. Pahuja, S. L., and R. B. Hochberg. 1995. A comparison of the esterification of steroids by rat lecithin:cholesterol acyltransferase and acyl coenzyme A:cholesterol acyltransferase. Endocrinology 136: 180-186 [Abstract].
27. Tsujita, T., K. Shirai, Y. Saito, and H. Okuda. 1990. Relationship between lipase and esterase. In Isozymes: Structure, Function, and Use in Biology and Medicine. Wiley-Liss, Inc. New York, NY. 915-933.
28. Petrazzuoli, M., S. L. Pahuja, J. M. Larner, and R. B. Hochberg. 1990. Biological activity of the fatty acid ester metabolites of corticoids. Endocrinology 127: 555-559 [Abstract].
29. Zielinski, J. E., S. L. Pahuja, J. M. Larner, and R. M. Hochberg. 1991. Estrogenic action of estriol fatty acid esters. J. Steroid Biochem. Mol. Biol. 38: 399-405 [Medline].
30. MacLusky, N. J., J. M. Larner, and R. Hochberg. 1989. Actions of estradiol-17-fatty acid ester in estrogen target tissue of the rat: comparison with other C-17 metabolites and a pharmacological C-17 ester. Endocrinology 124: 318-324 [Abstract].
31. Larner, J. M., N. J. MacLusky, and R. B. Hochberg. 1985. The naturally occuring C-17 fatty acid esters of estradiol are long-acting estrogens. J. Steroid Biochem. Mol. Biol. 22: 407-413 .
32. Miller-Larsson, A., P. Jansson, A. Runström, A.-B. Jeppsson, and R. Brattsand. 1997. Reversible fatty acid conjugation of budesonide results in a prolonged topical anti-inflammatory activity in airways as compared to fluticasone propionate. Am. J. Respir. Crit. Care Med. 155: A353 . (Abstr.) .
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