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Vol. 280, Issue 3, 1366-1373, 1997
Immunosciences Research Area, Abbott Laboratories, Abbott Park, Illinois
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Abstract |
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 Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Our primary goal has been to discover leukotriene biosynthesis inhibitors with characteristics that are appropriate for use as clinical agents. The success of the use of zileuton in the treatment of asthma led us to explore further the use of the N-hydroxyurea class of 5-lipoxygenase inhibitors as longer-acting compounds with good lung penetration. A variety of in vitro and in vivo methods were used to evaluate a large number of compounds, from which ABT-761 [(R)-N-(3-(5-(4-fluorophenylmethyl)thien-2-yl)-1-methyl-2-propynyl)-N-hydroxyurea] was selected for study. ABT-761 exhibited potent and selective inhibition of leukotriene formation both in vitro and in vivo. More importantly, the compound potently inhibited antigen-induced bronchospasm in guinea pigs when given either prophylactically or therapeutically. In addition, ABT-761 was a potent inhibitor of eosinophil influx into the lungs of Brown Norway rats. These data provide added support for the role of leukotrienes in both bronchospasm and eosinophilic inflammation and characterize ABT-761 as a particularly potent inhibitor of leukotrienes formed in pulmonary tissues. These data combined with the excellent pharmacokinetic characteristics of the compound indicate its potential use in the treatment of leukotriene-dependent human disease.
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Introduction |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Considerable evidence suggests
that leukotrienes contribute to human asthma (Henderson, 1994
; Israel,
1994; Wenzel et al., 1995). Although much of this evidence
was initially derived from research with isolated cells and tissues and
with animal models, more recent clinical results with specific
LTD4 antagonists (Spector et al., 1994) and the
specific 5-lipoxygenase inhibitor zileuton (Israel et al.,
1993) have substantiated the role of leukotrienes in this common
disease. Clinical findings with zileuton indicate that effective
modulation of leukotriene formation in asthmatics requires not only
that an agent be a potent inhibitor but also that tissue concentrations
of the agent be maintained throughout the treatment period. Thus,
optimal therapeutics will require long-acting agents with excellent
tissue distribution. We previously reported a testing strategy that
successfully predicted potency and duration of action for
N-hydroxyurea containing 5-lipoxygenase inhibitors (Bell
et al., 1995). This approach was used in the discovery of
ABT-761
[(R)-N-(3-(5-(4-fluorophenylmethyl)thien-2-yl)-1-methyl-2-propynyl)-N-hydroxyurea] (Brooks et al., 1995). In the present study, we describe the
characteristics of that compound; its biochemical profile, duration of
action and pharmacokinetics in rodents; and its activity in reducing bronchoconstriction and pulmonary inflammation in rodent models of
airway disease.
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Materials and Methods |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Bicinchonic acid protein assay reagents were purchased from Pierce Chemical (Rockford, IL). HPLC columns were purchased from Regis Chemical Co. (Morton Grove, IL). Ficoll-Hypaque Mono-Poly Resolving medium was from Flow Laboratories (McLean, VA). EIA reagents were purchased from Cayman Chemical Co. (Ann Arbor, MI) or from PerSeptive Diagnostics ( Cambridge, MA). Radioimmunoassay kits were from PerSeptive. ABT-761, zileuton, Bay X 1005, MK-476 and ZD-2138 were synthesized in our laboratories.
RBL cell lysate 5-lipoxygenase inhibitor potency.
The
activity of agents in inhibiting the 5-lipoxygenase RBL-1 was performed
according to the method of Jakschik et al. (1980) as
modified in our laboratories (Carter et al., 1991).
Reversibility of human PMNL 5-lipoxygenase inhibition.
The
inhibition of ionophore A23187-induced leukotriene formation in human
neutrophils and the reversibility of inhibition by ABT-761 was
performed as described by Bell et al. (1995).
Human whole blood eicosanoid formation.
Whole blood
eicosanoid formation was measured as described by Bell et
al. (1995).
Biosynthesis of leukotrienes by chopped lung. Cynomolgus monkey lung was obtained from these laboratories. The lung tissue was cut into 1- to 2-mm cubes and continuously oxygenated at 4°C in modified Krebs-Henseleit buffer composed of 118 mM NaCl, 1.1 mM MgSO4·7H2O, 1.8 mM CaCl2, 24.9 mM NaHCO3, 1.0 mM KH2PO4 and 11.1 mM glucose; washed; and resuspended in Krebs' buffer at a concentration of 100 mg/ml. Tissues were preincubated with vehicle (0.5% v/v DMSO) or test compound for 10 min. Calcium ionophore A23187 (20 µM) in DMSO or DMSO alone was added, and tissues were further incubated for 30 min. The tissue samples were transferred to an ice bath and centrifuged at 500 × g for 15 min at 4°C, and supernatants were removed and analyzed for LTB4 by EIA (Cayman Chemical Co.).
Isolated guinea pig trachea studies.
Tracheal spiral strips
were prepared and contractions were performed as described by Malo
et al. (1994). For most agonist-induced contractions, each
tissue was pretreated with 100 µM ABT-761 for 15 min before the
addition of a single concentration of LTD4, acetylcholine
(10 µM), histamine (10 µM), PGD2 (1 µM) or U-44069 (0.1 µM).
Determination of drug plasma concentrations.
Animals were
dosed and plasma concentrations of ABT-761 were determined as described
previously for A-79175 (Bell et al., 1995), with UV
detection at 275 nm.
Rat peritoneal anaphylaxis model.
Inhibition of leukotriene
formation in the rat was performed as previously described (Young
et al., 1991). Briefly, rats were passively sensitized to
bovine serum albumin, and 3 hr later they were challenged in the
peritoneal cavity with antigen. The peritoneal cavity was lavaged 15 min later, and the fluids were analyzed for leukotriene content by EIA.
Rat pleural inflammation model.
Pleural inflammation was
induced in male rats according to the method of Rao et al.
(1993). Animals were dosed with experimental compounds in 0.2% HPMC 1 hr before the intrapleural injection of the calcium ionophore A23187.
The rats were lightly anesthetized with Penthrane and injected
intrapleurally with 0.5 ml of 2% ethanol in injectable saline (Abbott
Laboratories) containing 20 µg of A23187. Thirty minutes later, the
animals were killed, and the pleural cavities were lavaged with
ice-cold saline. The lavage fluid was then added to ice-cold methanol
(final methanol concentration, 30%) to lyse cells and precipitate
protein. Eicosanoids were determined by EIA as described above.
AA-induced bronchospasm in the anesthetized guinea pig. Adult male albino Hartley-strain guinea pigs were anesthetized with an intraperitoneal injection of pentobarbital (20-24 mg/kg) and urethane (1.0 g/kg). After the induction of anesthesia, the trachea was surgically exposed and intubated with an endotracheal tube. All animals were permitted to spontaneously breathe room air.
Air-flow rate (ml/sec), tidal volume (ml/breath) and transpulmonary pressure (cm H2O) were recorded simultaneously according to previously published methods (Malo et al., 1994), and
Cdyn, a measurement of the status of peripheral airways,
was automatically calculated by an on-line computer. The jugular vein
was cannulated for the delivery of drugs and solutions to the animal.
The animal was kept warm during the experiment by being placed on a
heated table; the core temperature was monitored rectally.
Animals were orally dosed before anesthesia at the times noted. The
animals were pretreated with intravenous meclofenamic acid (10 mg/kg)
and propranolol (1 mg/kg) at 10 min before AA challenge. After the
pretreatments, the animals were challenged with intravenous arachidonic
acid (10 mg/kg). Measurements for compliance and conductance
calculations were made every 20 sec for a period of 15 min after AA
challenge.
Antigen-induced bronchospasm in the anesthetized guinea pig.
Bronchoconstriction in sensitized guinea pigs was induced and measured
as described in detail by Malo et al. (1994). For studies in
which the compound was delivered after antigen challenge, a slightly
different protocol was used. Immediately after ovalbumin (10 mg/ml, 30 sec) aerosol administration, the decrease in Cdyn was
observed. When a 20%, 40% or 60% decrease in Cdyn was
attained, an intravenous injection of ABT-761 or albuterol was given in 1 min. Measurements for compliance and conductance calculations were
made every minute for a period of 40 min after intravenous administration of ABT-761. Control responses were determined after the
same injection with only PEG-400.
Sephadex-induced eosinophilia in Brown Norway rats.
Eosinophilia was induced in Brown Norway rats by the injection of
Sephadex (Walsh, 1993; Walsh et al., 1994). Brown Norway rats weighing 130 to 150 g were orally dosed with ABT-761 in 0.2% HPMC (Abbott Laboratories). The animals were then injected in the
central tail vein with 1 ml of 0.5 mg/ml Sephadex G-200. Control animals received equal volumes of saline. For the next 3 days, the rats
were dosed either once or twice a day with either drug or HPMC. On day
3, rats were anesthetized by a nonlethal intraperitoneal injection of
25% urethane (~1 ml, 0.006 × body weight). Tracheal intubation
was performed, and the airways were lavaged with 2 × 5 ml of
phosphate-buffered saline without Ca++ containing 10 U/ml
of heparin. The fluid was recovered manually through gentle aspiration.
The fluid recovered from each lavage was pooled, and the volume was
measured. Total cell counts were performed using a Coulter counter
(model ZB1). A 1-ml aliquot of bronchoalveolar lavage fluid was added
to 2 ml of ice-cold methanol to precipitate the protein. This mixture
of methanol and bronchoalveolar lavage fluid was allowed to stand
overnight at 
20°C. Precipitated material was removed by
centrifugation, and levels of eicosanoids were determined as above. The
remaining bronchoalveolar lavage fluid was centrifuged (1500 × g); the cellular pellets were washed twice in a calcium-free
phosphate-buffered saline, resuspended in 5 ml of phosphate-buffered
saline and then centrifuged and stained with Wright-Giemsa for
differential counts.
Statistical methods. Percentage inhibition was computed by comparing individual values in treatment groups with the mean value of the control group. Statistical significance was determined using one-way analysis of variance and Duncan's multiple-range test. Linear regression was used to estimate IC50 and ED50 values.
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Results |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Characterization of ABT-761 as a selective 5-lipoxygenase inhibitor
in vitro.
ABT-761 (fig. 1) was tested
in a number of biochemical assays to validate its use in the pulmonary
models. Studies using cell lysates from RBL cells, a rich source of
5-lipoxygenase activity, indicated that the compound is a direct
inhibitor of 5-lipoxygenase. In three studies, ABT-761 inhibited the
formation of 5-HETE with an average IC50 value of 23 nM in
incubations using 6 µM AA as substrate (table 1). This
potency was greater than that observed for the standard inhibitors
zileuton (Carter et al., 1991) and ZD-2138 (McMillan
et al., 1992). The inhibitory potency of ABT-761 varied as a
function of substrate concentration, yielding IC50 values
of 23 to 151 nM for substrate concentrations of 6 to 65 µM,
respectively. These data suggest that ABT-761 is a direct competitive
inhibitor of 5-lipoxygenase.
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) but 2-fold less potent than ZD-2138. In other experiments, the
inhibitory activity of ABT-761 was found to be readily reversible by
simple centrifugation of the cells and resuspension of them in fresh
buffer (data not shown). These observations indicate that ABT-761 is a
potent, reversible inhibitor of 5-lipoxygenase product formation in
these cells.
The selectivity of the 5-lipoxygenase inhibition by ABT-761 compared
with inhibition of other eicosanoid pathways was examined in human
whole blood (Bell et al., 1995). As shown in table 1, ABT-761 weakly inhibited TXB2 and 12-HETE formation in
human whole blood challenged with the calcium ionophore A23187. The
IC50 values were 50 µM vs. TXB2
and 11 µM vs. 12-HETE. These values were ~300- and
~75-fold higher than the IC50 value for 5-lipoxygenase
inhibition, as indicated by inhibition of LTB4 in the same
donors. 15-HETE was also inhibited, but the inhibition was only 50% at
the highest concentration tested (100 µM). Taken together, these
results support the conclusion that ABT-761 is a selective inhibitor of
5-lipoxygenase.
Inhibition of ionophore-induced leukotriene formation from
cynomolgus monkey lung fragments.
Calcium ionophore A23187 (20 µM) induced the formation of significant amounts of LTB4
(45 ± 4.4 ng/g of tissue) from lung fragments taken from
cynomolgus monkeys. ABT-761 was found to inhibit the formation of
LTB4 in two experiments, with IC50 values of 9 and 95 nM. In the same two preparations, another 5-lipoxygenase inhibitor, A-79175, (Bell et al., 1995) gave
IC50 values of 28 and 280 nM, respectively.
Guinea pig trachea studies.
The effect of ABT-761 on the
contraction of guinea pig trachea induced by several agonists was
assessed to examine the specificity of the compound. The compound alone
had no effect on basal tone of the tracheal tissue. ABT-761 had no
effect on contraction of the guinea pig trachea induced by
acetylcholine, histamine, PGD2 or U-44069, a thromboxane
mimetic. The submaximal contractions induced by either 10 µM
acetylcholine or histamine, 1 µM PGD2 or 0.1 µM U-44069
in the absence or presence of 100 µM ABT-761 were similar. ABT-761
(100 µM) also had no effect on LTD4-induced contractions.
These results are consistent with binding data from studies in guinea
pig lung membranes in which concentrations of 
100 µM ABT-761 failed
to antagonize LTD4 binding (Nova Screen, data not shown).
Duration of action of ABT-761 in mice, rats and guinea pigs.
Our previous work with A-79175, zileuton and other
N-hydroxyurea-containing inhibitors (Bell et al.,
1995; Carter et al., 1991; Rubin et al., 1989)
indicated that glucuronidation of the hydroxyl group should be the
major route of metabolism. ABT-761 was glucuronidated in liver
microsomes from cynomolgus monkey, albeit very slowly. This slow rate
of metabolism in vitro translated to a long duration
in vivo. ABT-761 had an estimated oral half-life of 16 hours
in monkeys and 15 hours in humans (Brooks et al., 1995).
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Leukotriene inhibition studies in rodents.
An anaphylactic
reaction in the rat peritoneal cavity produces large amounts of
LTE4 and lesser amounts of LTB4 and
TXB2 (Young et al., 1991). ABT-761 was found to
be a potent inhibitor of leukotriene biosynthesis but not of
TXB2 in this model. When the compound was orally dosed as a
3-hr pretreatment, the compound gave dose-related inhibition of
LTE4 formation. An ED50 value of 1.4 mg/kg was
calculated from these data for LTE4 inhibition. In the same
experiment, the ED50 value for the inhibition of
LTB4 biosynthesis was 0.6 mg/kg (fig. 2).
There was no significant difference in the inhibition seen for the two
classes of leukotrienes. The potency of ABT-761 was ~3-fold greater
than that previously obtained for zileuton (Carter et al.,
1991). It was also more potent than ZD-2138 or Bay X 1005. In contrast
to the leukotriene inhibition seen with the compound, no inhibition of
TXB2 formation was seen at the highest dose tested (10 mg/kg).
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). ABT-761 administered orally inhibited the formation of LTE4 in this model in a dose-related fashion, yielding an
ED50 value of 3 mg/kg. Thus, ABT-761 was found to inhibit
leukotriene formation in both pleural and peritoneal cavities. Plasma
level determinations of ABT-761 from blood collected at the termination of these studies were consistent with the pharmacokinetic studies (table 2) in that doses of 1 to 10 mg/kg p.o. resulted in plasma levels
of 1 to 14 µM. In general, plasma levels needed to be >4 µM to
achieve >50% inhibition of leukotrienes in either cavity. These data
taken together with the biochemical and ex vivo results indicate that ABT-761 is a selective 5-lipoxygenase inhibitor with good
oral potency.
Bronchoconstriction models in guinea pigs.
Although there are
no animal models of human asthma that completely mimic this disease, a
number of models are available that appear to have similarities to the
bronchoconstrictive component of the disease (Malo et al.,
1994; Wegner et al., 1993). ABT-761 was used as an oral
agent in two of these, both in guinea pigs: AA-induced bronchospasm and
antigen-induced bronchospasm in actively sensitized animals.
). The initial phase of the
bronchoconstriction from 0 to 5 min appears to not be dependent on
leukotriene formation; selective inhibitors such as ABT-761 (fig.
3) and A-79175 do not block it. In
addition, fatty acids such as oleic acid that are not 5-lipoxygenase
substrates also induce the first portion of the bronchoconstrictive
response when injected intravenously but return to base line quickly.
In contrast, alternative substrates such as eicosapentaenoic acid gave
the same response as AA. In addition, bronchoconstriction induced by
intravenous injection of eicosapentaenoic acid was inhibited by the
selective 5-lipoxygenase inhibitor zileuton.
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). The pulmonary response in this model appears to be similar to
the bronchospasm observed after antigen challenge of allergic asthmatics in the clinical laboratory or during an acute asthmatic attack. The bronchoconstriction response in the guinea pig, however, is
more extensive than that normally experienced by the chronic asthmatic
because antigen challenge causes a 65% to 75% change in lung function
measured as Cdyn. When ABT-761 was administered orally 5 hr
before antigen challenge, the bronchospasm was inhibited. As shown in
figure 4, ABT-761 at 3 mg/kg gave nearly complete inhibition of the bronchospasm in animals pretreated with mepyramine and meclofenamic acid. At a higher dose of 10 mg/kg, a maximum inhibition of ~85% was observed, whereas a dose of 1 mg/kg gave ~50% inhibition (data not shown). This activity is superior to that
seen with zileuton previously (Malo et al., 1994) and, as shown in figure 4, is superior to the recently described
cysLT1 antagonist MK-476 (Jones et al., 1995). A
time course of predosing was also performed, and the compound gave
significant inhibition through 8 hr at a dose of 5 mg/kg (data not
shown). As in the rat leukotriene inhibition studies, ABT-761 was
linearly absorbed as determined by measuring plasma levels of compound
at the termination of the bronchospasm studies. Plasma levels of
ABT-761 were 4.5 to 5.5 µM at the 3 mg/kg dose and 12 to 15 µM at
the 10 mg/kg dose.
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Eosinophilic pulmonary inflammation in the rat.
Another
important aspect of asthma is a chronic inflammation of the lung
characterized by a dominant eosinophilic component. As with
bronchoconstriction, a number of animal models exist that have
characteristics similar to the human disease. The intravenous administration of Sephadex G-200 particles induces a lung eosinophilia in Brown Norway rats that is characterized by increases in the bronchoalveolar lavage fluid of cysteinyl leukotrienes on days 0 to 3 and in eosinophils on days 1 to 3 (Walsh et al., 1994; Namovic et al., 1996). At day 3, ~30% of the cells
lavaged from the lungs of rats receiving Sephadex were eosinophils
compared with 0.15% in the saline-treated controls. Monocytes are not
increased in this model, but neutrophils (day 1) and lymphocytes (day
3) are modestly increased (Namovic et al., 1996). When
ABT-761 was administered twice daily for 3 days, a dose-dependent
reduction in both the number of eosinophils found in the lavage fluid
and the levels of cysteinyl leukotrienes in the lavage fluid was
observed (fig. 6, a and b). In two other experiments, a
similar activity of ABT-761 was observed with significant inhibition of
cell influx and reduction of leukotrienes occurring at doses of 1 and 3 mg/kg (data not shown). In two of these experiments, no effect on PMNL number or lymphocyte number was observed at day 3. However, modest effects were seen on both cell types in a third experiment. As expected
from the previous rat studies, plasma levels of ABT-761 were maintained
throughout the 4-day study. Plasma levels of ABT-761 on day 4 before
dosing (3 mg/kg) were 5 µM and peaked at ~12 µM at 2 to 3 hours
after dosing.
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Discussion |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Human asthma is a complex disease with multiple initiating events
and multifactorial causes and symptomatic sequelae. Current research aimed at understanding the underlying causes of the disease has focused on bronchoconstriction, pulmonary inflammation and airway
hyperreactivity (Fischer et al., 1995; Israel, 1994; Kay, 1991) as important attributes of the disease. The recent availability of specific leukotriene modulators and success of some of those agents
in the treatment of chronic asthma (Israel et al., 1993) have clearly shown that leukotrienes are important factors in the
disease. However, the precise role of these potent mediators is not yet
completely defined, and new, more potent, longer-lasting agents may be
required to explore fully the role of leukotrienes in the disease.
ABT-761, as described in this study, appears to be such an agent.
ABT-761 was found to be a direct reversible inhibitor of 5-lipoxygenase
in broken cell preparations and against cellular leukotriene biosynthesis. The inhibition of 5-HETE formation in RBL lysates was
dependent on the concentration of substrate (AA) consistent with the
compound being a competitive inhibitor of 5-lipoxygenase. The compound
was a much weaker inhibitor of other eicosanoid-metabolizing enzymes,
such as cyclooxygenase and 12- and 15-lipoxygenase in calcium ionophore
A23187-challenged human whole blood. These data indicate that the
compound is similar in profile to zileuton (table 1) but ~4- to
5-fold more potent. The selectivity of ABT-761 was also somewhat
greater because it inhibited whole blood LTB4 formation at
a 5-fold lower concentration and TXB2 formation at approximately the same concentration. Comparisons of the molecule with
ZD-2138 (McMillan et al., 1992) and Bay X 1005 (Hatzelman et al., 1994) were also interesting (table 1). As expected
for a FLAP inhibitor (Hatzelman et al., 1994), Bay X 1005 did not inhibit the 5-lipoxygenase directly in RBL lysates but was
effective, albeit 3-fold less potent than ABT-761, against neutrophil
LTB4 biosynthesis. ZD-2138 also had a inhibitory profile
that was different than that of the two direct enzyme inhibitors,
zileuton and ABT-761; it was much less potent in the lysate assay than
in neutrophils or whole blood.
The whole blood eicosanoid data for ABT-761 data indicate a ~300-fold selectivity for the 5-lipoxygenase vs. thromboxane formation. Thus, when dosed in vivo, ABT-761 would be expected to completely block the formation of LTB4 and the cysteinyl leukotrienes LTC4, LTD4 and LTE4. It would not be expected to inhibit the formation of other eicosanoids. This inhibition should then be readily reversible when plasma concentrations of ABT-761 decrease over time.
ABT-761 was also found to be an effective specific inhibitor of leukotriene formation in lung tissue from monkeys. In contrast, it did not have nonspecific effects on lung tissue, as measured in the guinea pig. The compound lacked general bronchodilator activity, as tested in guinea pig tracheal preparations. It had no effect on basal tone of the tissue and failed to block contractions elicited by histamine, acetylcholine, the thromboxane agonist LTD4 or PGD2. These studies support the use of ABT-761 to probe the role of leukotrienes in vivo in pulmonary tissue.
ABT-761 was designed to be resistant to glucuronidation (Bell et
al., 1995; Brooks et al., 1995). This characteristic
translated to excellent bioavailability and long plasma half-lives in
several species. In addition, the compound was an orally bioavailable inhibitor in rodents, as demonstrated by potent inhibition of leukotriene production in the rat. Oral doses of 5 mg/kg inhibited leukotriene formation in the rat peritoneal cavity for several hours.
Cysteinyl leukotrienes and LTB4 were inhibited in parallel, consistent with the site of inhibition of ABT-761 being at
5-lipoxygenase. ABT-761 was more potent in the anaphylaxis assay than
the other inhibitors discussed above. It was 3-fold more potent than
zileuton and Bay X 1005 (table 3). Interestingly,
ZD-2138, which was the most potent inhibitor against in
vitro whole blood LTB4 formation, was inactive in this
model.
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ABT-761 was examined in three protocols of bronchoconstriction in the
guinea pig of increasing complexity. In an effort to simplify the
bronchospastic response to a single-mediator system, intravenous AA was
administered as a challenge. This model was somewhat successful in that
the bronchospasm observed was not histamine dependent as has been
observed for a significant part of the response to antigen (Malo
et al., 1994). The specific inhibitor ABT-761 was effective
in inhibiting most (~75%) of the bronchospasm observed.
Interestingly, FLAP antagonists were not effective in this model (Malo
et al., 1993), but the cysLT1 antagonist
ZD-198615 was effective (Malo et al., 1993). Moreover, an
initial bronchospastic response was observed in the animals at early
times (1-3 min) after challenge that was not leukotriene dependent on
the basis of several criteria described in Results.
ABT-761 was also examined in an antigen-challenge model using actively
sensitized animals. As described by us (Malo et al., 1994)
and others (Kallos and Kallos, 1984; Piper, 1977), in the presence of
an antihistamine and a cyclooxygenase inhibitor, the bronchoconstrictive response to aerosolized antigen in guinea pigs is
blocked by leukotriene modulators, including 5-lipoxygenase inhibitors,
cysLT1 receptor antagonists and FLAP antagonists (Malo et al., 1993) if administered before antigen challenge.
ABT-761 was very effective in blocking antigen-induced bronchospasm
giving nearly complete inhibition at oral doses of 3-10 mg/kg. Thus
the molecule was more potent than zileuton (Malo et al.,
1994), Bay X 1005 (Nagai et al., 1996) and the
cysLT1 receptor antagonist MK-476 recently described by
Jones et al. (1995) (fig. 5). Oral dosing of ABT-761 at 2 to
8 hr before antigen challenge resulted in a marked diminution of the
response. Inhibition was seen at plasma levels similar (4-15 µM) to
those required to inhibit leukotriene formation in the rat; this is
consistent with the proposal that the inhibition of bronchospasm was
derived from inhibition of leukotriene formation. In addition, these
observations confirm the leukotriene dependence of this guinea pig
model using the highly selective inhibitor ABT-761 and the selective
cysLT1 receptor antagonist MK-476.
Many asthmatics have a decreased overall lung function compared with
normal individuals, which can be partially reversed by the use of
beta agonists. In most patients, the disease is chronic. Drugs used in asthma treatment thus must be able to alleviate ongoing
bronchospasm as well as prevent pulmonary responses to a new insult.
Recent clinical data with zileuton indicate that pulmonary function in
asthmatics is improved acutely after a single dose of the compound
(Israel, 1994). Because the compound does not cause bronchodilatation
in normal subjects, the hypothesis has been proposed that ongoing
leukotriene formation is responsible for a portion of the bronchospasm
in these asthmatics and that inhibition of leukotriene formation would
improve pulmonary function. In response to these concepts, we tested
ABT-761 and albuterol for the effectiveness of these compounds against
an ongoing bronchospasm in the antigen-challenged guinea pig model.
These studies were done by inducing a compliance reduction of 40%
followed by an intravenous bolus of either albuterol or ABT-761.
Albuterol was able to significantly reverse the bronchoconstriction
when administered at 40% compliance; however, the effect of the
compound was substantially diminished when compliance had reached a
60% decrease before therapy. These data were not surprising given the
known bronchodilatory mode of action of albuterol; however, the data
did show that the bronchoconstrictive response to antigen is reversible
in this model. More unexpected was the response to intravenous ABT-761 after bronchospasm had been initiated. The compound was able to reverse
50% of the bronchospasm in response to antigen. Whether this is
because leukotrienes are responsible for 50% of the bronchospasm with
the other half being initiated by another mediator or because insufficient ABT-761 was delivered could not be determined because the
dose that was administered was the highest that was soluble in the
intravenous vehicle. The data obtained for ABT-761 in the guinea pig,
however, confirm the hypothesis that the bronchoconstrictive response
to antigen requires the continual formation of leukotrienes. Thus,
although the pulmonary response is more severe, the guinea pig model
appears to mimic the human asthmatic response (Israel, 1994) to the
extent that it also appears to require continual leukotriene formation
for continued bronchoconstriction to occur.
Pulmonary inflammation is another important characteristic of human
asthma (Kay, 1991). In contrast to other diseases, such as rheumatoid
arthritis, the inflammation in asthma is predominantly eosinophilic
(Martin et al., 1996). Several animal models of pulmonary inflammation have been reported. Recently, we modified one of these and
found it useful for the characterization of compounds (Namovic et
al., 1996). We have shown that the intravenous injection of
Sephadex particles in Brown Norway rats results in a specific eosinophilic inflammation. The eosinophil influx is temporally associated with the appearance of cysteinyl leukotrienes in
bronchoalveolar lavage fluid. ABT-761 administered orally twice daily
for the 3-day course of the response gave potent and parallel
inhibition of both the eosinophil influx as measured by bronchoalveolar
lavage fluid cells as well as by bronchoalveolar lavage fluid
leukotriene levels. Given the specificity of action of ABT-761 shown in
this report, we propose that the effect on eosinophil influx observed in this model has a leukotriene component. At this point it is unclear
whether the attraction of eosinophils to the lungs of treated rats is
dependent predominantly on LTB4, cysteinyl leukotrienes or
both. Both classes of leukotrienes have been shown to be chemotactic for human eosinophils, although data on rat eosinophils are
unavailable. We are inclined to believe that LTB4 is the
major eicosanoid chemoattractant because MK-476 and ZD-204219, two
selective cysLT1 receptor antagonists, are not as effective
in this model as leukotriene inhibitors (Namovic et al.,
1996). Whether these observations translate to other models of
pulmonary eosinophilia or to asthma itself remains to be explored.
In conclusion, ABT-761 appears to have the specificity, duration of action and tissue penetration required for effective attenuation of both the bronchoconstrictive and inflammation components of pulmonary disease. Given the early clinical success of leukotriene modulation with agents such as zileuton and zafirlucast, the more potent, longer-lasting 5-lipoxygenase inhibitor ABT-761 should prove to be a useful pharmacological and clinical agent.
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Acknowledgments |
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We gratefully acknowledge excellent technical support from Marian Namovic, George Grayson, Jimmie Moore, Pramila Bhatia, Robin Walsh, Denise Wilcox, Sandra Majest, Carole Goodfellow and Ellen Otis.
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Footnotes |
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Accepted for publication November 22, 1996.
Received for publication July 15, 1996.
1 P. Malo and R. Bell, unpublished observations.
Send reprint requests to: Dr. Randy L. Bell, Abbott Laboratories, Department 47K, Building AP9, 100 Abbott Park Road, Abbott Park, IL 60064.
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Abbreviations |
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LTB4, leukotriene B4; LTD4, leukotriene D4; 5-HETE, 5-hydroxyeicosatetraenoic acid; 12-HETE, 12-hydroxyeicosatetraenoic acid; 5-HPETE, 5-hydroperoxyeicosatetraenoic acid; RBL-1, rat basophilic leukemia cells; DMSO, dimethylsulfoxide; PBS, phosphate buffered saline; EIA, enzyme immunoassay; HPMC, hydroxypropyl methyl cellulose; AA, arachidonic acid; TXB2, thromboxane B2; Cdyn, dynamic compliance; PMNL, polymorphonuclear leukocytes; FLAP, 5-lipoxygenase activating protein.
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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