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* Research Business Area Dermatology, Research Laboratories, Schering AG, Berlin, Germany;
Center for Experimental Therapeutics and Reperfusion Injury, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA 02115; and Departments of
Medicinal Chemistry and
Immunology, Berlex Biosciences, Richmond, CA 94804
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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10-fold less potent than the LTB4R-Ant in blocking
responses to LTB4. A broad panel of cutaneous inflammation
models that display pathological aspects of psoriasis, atopic
dermatitis, and allergic contact dermatitis was used to directly
compare the topical efficacy of ATLa with that of LTB4R-Ant
and methylprednisolone aceponate. ATLa was efficacious in all models
tested: LTB4/Iloprost-, calcium ionophore-, croton oil-,
and mezerein-induced inflammation and trimellitic anhydride-induced
allergic delayed-type hypersensitivity. ATLa was efficacious in mouse
and guinea pig skin inflammation models, exhibiting dose-dependent
effects on edema, neutrophil or eosinophil infiltration, and epidermal
hyperproliferation. We conclude that the LXA4 and
aspirin-triggered LXA4 pathways play key
anti-inflammatory roles in vivo. Moreover, these results suggest
that ATLa and related LXA4 analogs may have broad
therapeutic potential in inflammatory disorders and could provide an
alternative to corticosteroids in certain clinical
settings. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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LXA4 and ATLs undergo rapid metabolic
inactivation by PG dehydrogenase-mediated oxidation and reduction
(8, 9). These metabolites have reduced affinity for ALX-R
and decreased anti-inflammatory potency. Chemical modifications to
the C15-C20 region of LXA4 and ATLs prevent
metabolic inactivation, thus providing stable analogs with superior
pharmaceutical characteristics (10). The stable lipoxin
analogs inhibit neutrophil transcellular migration, pathogen- and
TNF-
-induced epithelial cell IL-8 release, and vascular permeability
of mouse ear skin exposed to inflammatory stimuli (Ref. 1
and references therein). In TNF--induced dorsal air pouch
inflammation, stable LXA4 analogs have potent
local and systemic anti-inflammatory efficacy via down-regulation
of proinflammatory cytokine and chemokine networks (11, 12). More specifically, lipoxins inhibit cytokine-stimulated
IL-1
, macrophage inflammatory protein-2, and superoxide production
while stimulating the anti-inflammatory cytokine IL-4 in
neutrophils (11). Eosinophil-driven allergic reactions are
also inhibited by stable LXA4 analogs (13, 14). Lipoxins inhibit eosinophil chemotaxis, and IL-5 and
eotaxin secretion (13). Moreover, lipoxins inhibit
mesangial cell proliferation (15). Finally, lipoxins have
been shown to inhibit the transcription factor NF-
B, which is a
central regulator of inflammatory molecules and also is pivotal
for proliferation and anti-apoptosis (16). Thus,
an anti-proliferative effect can add to the anti-inflammatory
mechanisms of lipoxins that interfere with the activation and migration
of inflammatory cells.
Results to date suggest that the LXA4/ALX-R
pathway promotes counter-regulatory signals to diverse proinflammatory
mediators and that stable LXA4 analogs may have
therapeutic potential in inflammatory and autoimmune disease. Because
of the limited amounts of synthetic LXA4 analogs
available, a role for LXA4/ALX-R in regulating
cutaneous inflammation has not been studied systematically, and there
is limited information on the efficacy profile of
LXA4 analogs in industry standard animal models
that represent distinct mechanisms of clinically relevant cutaneous
inflammation. Quantitative results comparing the potency and efficacy
of a stable LXA4 analog to other
anti-inflammatory agents, including clinically relevant standards,
such as glucocorticoids, is also lacking. To address these points, the
topical efficacy of a stable ATL analog synthesized in bulk, namely
15-epi-16-p-fluorophenoxy-lipoxin
A4 methyl ester (ATLa; Fig. 1
), was tested in a variety of skin
inflammation models. The models were chosen to evaluate potential
utility in distinct dermatoses, since they exhibit pathological
features found in irritant contact dermatitis, psoriasis, allergic
contact dermatitis, urticaria, and atopic dermatitis. Anti-inflammatory
potency and efficacy were compared with those of a leukotriene
B4 (LTB4) receptor
antagonist (LTB4-R-Ant) and methylprednisolone
aceponate (MPA), a mid-potent glucocorticoid that is marketed in Europe
and frequently used for topical treatment of atopic dermatitis in
children. The present results indicate that ATLa displays broad topical
efficacy in all models of skin inflammation examined and proved to be
dose-dependent for inhibiting edema, leukocyte infiltration, and
epidermal hyperproliferation. ATLa gives a unique anti-inflammatory
profile, suggesting potential use in topical treatment of
dermatoses.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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) was synthesized using previously
described methods (17). Material of >95% purity was
qualified against a synthetic ATLa standard (provided by Dr. N. Petasis
(University of Southern California, Los Angeles, CA) and Dr. C. N.
Serhan (Brigham and Woman’s Hospital, Harvard University, Boston, MA))
using 1H nuclear magnetic resonance, HPLC
coinjection with photodiode array UV-visible detection (two methods),
and liquid chromatography-mass spectrometry. The
LTB4 receptor antagonist ZK-158252 (18, 19) (LTB4R-Ant), Iloprost, and
LTB4 for in vivo studies were synthesized at
Schering AG (Berlin, Germany). LTB4 for in vitro
studies was purchased from Cayman (Ann Arbor, MI).
Hexadecyltrimethylammonium bromide (HTAB), fMLP, calcium ionophore
A-23187, croton oil, mezerein, and trimellitic anhydride were obtained
from Sigma (St. Louis, MO). Human neutrophil chemotaxis and transcellular migration assays
Neutrophils from healthy volunteers were obtained as previously described (10) and suspended in RPMI 1640 (BioWhittaker, Walkersville, MD) at 1 x 106 cells/ml. FMLP (10 nM), 10 nM LTB4, or vehicle was added to the lower wells of a 48-well, 5-µm pore size chemotaxis chamber (NeuroProbe, Cabin John, MD). Neutrophils (50 µl) were placed in the upper wells, and the chamber was incubated (37°C, 5% CO2) for 1 h. Following incubation, filters were removed, and cells were scraped from the upper surface. The filters were fixed and stained with Diff-Quik (Dade Behring, Newark, DE). For each incubation, performed in triplicate, cells that migrated across the filter toward the lower surface were enumerated by light microscopy. To assess inhibition of migration, neutrophils were suspended in RPMI medium with vehicle or increasing concentrations of the compounds (ATLa or LTB4R-Ant) and incubated for 30 min at 37°C before placement in the chamber. Human neutrophil transmigration across confluent monolayers of human T84 epithelial cells was performed as previoulsy described (10).
Animal models
All animal studies were approved by the competent authority for labor protection, occupational health, and technical safety for the state and city of Berlin, Germany, and were performed in accordance with the ethical guidelines of Schering. Female NMRI mice (26–28 g) or pirbright white guinea pigs (Charles River, Wilmington, MA; 200–250 g) were housed according to institutional guidelines of the Schering animal facility. NMRI mice are outbred Swiss mice from Lynch to Poiley (National Institutes of Health, Bethesda, MD; albino, Aa, BB, cc, DD, histocompatibility H-2q). Eight to 11 animals were randomly allocated to the different treatment groups.
Skin inflammation models
Because of the acute character of the models, ATLa, LTB4R-Ant, or MPA was applied topically at the same time as the elicitation of the inflammatory reaction. Tissue weight (ears or dorsal skin punch biopsies) served as a criterion for edema formation. Peroxidase activity in skin homogenates served as a measure of total granulocyte (neutrophil and eosinophil) infiltration, and elastase activity served as a specific measure of neutrophil infiltration.
LTB4/Iloprost-induced inflammation
The stable PGI2 analog Iloprost enhances
LTB4-induced ear inflammation, leading to edema
and a neutrophilic infiltrate with a maximum reaction 24 h after
elicitation (20). Ten microliters of 0.003% (w/v)
LTB4 and 0.003% (w/v) Iloprost in
ethanol/isopropylmyristate (95/5), with or without the respective
anti-inflammatory agent, were applied dorsally to mouse ears.
Animals were euthanized with CO2 24 h after
application. Ears were cut off, weighed as an indicator for edema
formation, and snap-frozen. Ears (area, 1 cm2)
were homogenized in 2 ml of buffer containing 0.5% HTAB and 10 mM MOPS
(pH 7.0) in a Polytron(R) PT 3000 homogenizer (KINEMATICA, Lucerne,
Switzerland) set at maximum speed (30,000 rpm). Samples were
centrifuged, and 750 µl of supernatants were transferred to
96-deep-well plates (Beckman, Palo Alto, CA). The supernatants were
then used to determine peroxidase and elastase activity as a measure
for infiltrating granulocytes (peroxidase) and neutrophils (elastase),
as described below. Anti-inflammatory effects of a given compound were
defined as the percent inhibition of edema formation and peroxidase and
elastase activities.
Calcium ionophore-induced inflammation
The calcium ionophore A-23187, applied topically, induces acute
inflammation with edema and granulocyte infiltration that peaks at
24 h (21). Ten microliters of a 0.1% (w/v) solution of
A-23187 in ethanol/isopropylmyristate (95/5), with or without the
respective anti-inflammatory agent, were applied dorsally to mouse
ears. Animals were euthanized 24 h later, and the ears were
processed as described for the LTB4/Iloprost
inflammation model.
Croton oil-induced inflammation
The nonspecific contact irritant croton oil leads to acute inflammation and is characterized by edema formation and a mainly granulocytic cell infiltration into the skin (22). Ten microliters of 1% (v/v) croton oil in ethanol/isopropylmyristate (95/5), with or without the respective anti-inflammatory agent, were applied dorsally to mouse ears. Animals were euthanized at 24 h, and ears were processed as described for the LTB4/Iloprost inflammation model.
Mezerein-induced inflammation
Mezerein causes acute inflammation, with edema formation and
granulocyte infiltration within 24 h and epidermal
hyperproliferation within 72 h (23). Ten microliters of
0.03% (w/v) mezerein in ethanol/isopropylmyristate (95/5) with or
without the respective anti-inflammatory agent were applied
dorsally to mouse ears. For inflammation end points, animals were
euthanized at 24 h, and ears were processed as described for the
LTB4/Iloprost inflammation model. As an
indication for hyperproliferation, epidermal thickness was determined
morphometrically in Formalin-fixed, plastic-embedded, sectioned, and
toluidine blue-stained specimens obtained from the ears of mice
euthanized at 72 h.
Trimellitic anhydride-induced delayed-type hypersensitivity (DTH)
Sensitization with the occupational contact allergen trimellitic anhydride (TMA) induces a DTH reaction, with prominent eosinophil infiltration 24 h after challenge (24), which, in contrast to other types of acute contact dermatitis, is characterized by a mixed Th1/Th2 reaction. Mice were sensitized on days 0 and 1 by a single application of 50 µl of 3% (w/v) TMA in acetone/isopropylmyristate (80/20) onto a shaven area of 2 x 2 cm on the right flank. The DTH reaction was induced on day 5 by challenging the animals with a single application of 10 µl of 3% (w/v) TMA in acetone/isopropylmyristate (80/20) with or without the respective anti-inflammatory substance onto the dorsal sides of both ears. Animals were euthanized 24 h after challenge, and ears were processed as described for the LTB4/Iloprost inflammation model.
Peroxidase activity assay
Peroxidase activity as a measure of total granulocyte infiltration was measured as previously described (25). Briefly, tetramethylbenzidine (TMB) dihydrochloride was used as a sensitive chromogen substrate for peroxidase. To convert TMB into TMB dihydrochloride, 34 µl of 3.7% hydrochloric acid (equimolar) was added to 5 mg of TMB. Then 1 ml of DMSO was added. This stock solution was slowly added to sodium acetate-citric acid buffer (0.1 mol/L, pH 6.0) in a ratio of 1:100. Two hundred microliters of this TMB solution, 40 µl of the homogenized sample, and 25 µl of 1 mM H2O2 were added to a microtiter plate to start the reaction. The reaction was stopped after 30 min with 45 µl of 1 N H2SO4. Changes in OD were monitored at 450 nm at 25°C against the mixture of all solutions without the added sample homogenate. Absolute extinction numbers were used to express peroxidase activity.
Elastase activity assay
Elastase activity was measured by fluorescence of 7-amino-4-methyl-coumarin (AMC) that is released from the substrate MeO-Succ-Ala-Ala-Pro-Val-AMC (Bachem, Torrance, CA). Homogenized samples in HTAB were diluted 1/10 in cetrimide buffer (0.3% cetrimide, 0.1 M Tris, and 1 M NaCl, pH 8.5). The substrate MeO-Succ-Ala-Ala-Pro-Val-AMC (300 mM in DMSO) was diluted 1/100 in cetrimide buffer to a working concentration of 3 mM. In cetrimide buffer, diluted samples were pipetted in multiwell plates, and the reaction was started by addition of the AMC substrate at 37°C. The reaction was stopped after 1 h with ice-cold 100 mM Na2CO3, and samples were measured in a Spectra Max Gemine (Molecular Devices, Menlo Park, CA) at 380 nm and compared against a standard curve with the AMC standard 7-amino-4-methylcoumarin (5 mM in ethanol).
Statistical analysis
For all animal models statistical analysis was performed with
the so-called modified Hemm (inhibition) test, which was developed by
Schering’s Department of Biometrics based on the program SAS System
for Windows 6.12 (SAS Institute, Cary, NC). To determine the inhibitory
effect of anti-inflammatory compounds, the difference between the
respective mean value of the positive controls and the mean value of
the vehicle controls was set at 100%, and the percentile change by the
test substance was estimated: % change = [(mean
valuetreated group - mean
valuepositive group)/(mean
valuepositive group - mean
valuecontrol group)] x 100. To test whether the
change caused by the treatment is different from zero, a 95%
confidence interval was calculated under consideration of the variance
of observations within the entire experiment. If the interval did not
include zero, the hypothesis that there is no change was rejected at
the level of = 0.05. For each experiment
IC50 values were determined graphically.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A hallmark of inflammation is the movement of circulating
leukocytes into tissues via chemotaxis and transcellular migration
across endothelial and epithelial cell barriers (26).
Classical chemotactic mediators include the bacterially derived
N-formylated peptide, fMLP, and the proinflammatory
eicosanoid, LTB4. Human neutrophil chemotaxis to
fMLP (10 nM) was inhibited in a dose-dependent manner by ATLa with an
IC50 of 0.1 nM and a maximal effect at 10
nM (data not shown). ATLa was 100-fold more potent than the
specific LTB4 receptor antagonist
(LTB4R-Ant; IC50 = 10
nM). In contrast, LTB4R-Ant inhibited neutrophil
chemotaxis induced by LTB4 (10 nM) more potently
(IC50 = 1 nM) than ATLa
(IC50 = 10 nM). In addition to chemotaxis,
ATLa potently inhibited neutrophil transepithelial migration to 10 nM
fMLP or 10 nM LTB4 (Fig. 2). Inhibition was dose dependent, with
an IC50 of 1 nM against fMLP and 10 nM
against LTB4. As expected,
LTB4R-Ant inhibited
LTB4-induced transepithelial migration to
LTB4 (IC50 = 1 nM), but
was virtually ineffective on transepithelial migration to fMLP (<20%
inhibition at 1 µM). Thus, in addition to potently inhibiting human
neutrophil chemotaxis, ATLa potently inhibits neutrophil transmigration
through epithelial cell monolayers, a finding consistent with previous
in vitro studies (10). The present results correlate with
the finding that the receptor ALX-R is expressed on both human
neutrophils and human epithelial cells and may mediate
LXA4-dependent effects in both cell types
(1). Our results also suggest that ATLa might display
broader anti-inflammatory potential than
LTB4R-Ant, since neutrophil responses to more
than one mediator are potently attenuated.
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To characterize its anti-inflammatory potential for human skin
diseases, ATLa was examined in diverse models of skin inflammation. The
models are based on cutaneous reactions to exogenous stimuli that
provoke a variety of symptoms, including edema, neutrophil or
eosinophil infiltration, and epidermal hyperproliferation. The models
encompass features of irritant dermatitis, allergic contact dermatitis,
and some aspects of psoriasis and atopic dermatitis. A limitation of
the models is their acute character, which does not allow for the
assessment of therapeutic administration. Therefore, compounds were
tested in a preventive manner by coapplication with the proinflammatory
reagents. ATLa was compared with two reference compounds:
LTB4R-Ant and the topically active
glucocorticoid, MPA. Comparative data for ATLa and
LTB4R-Ant in all models are summarized in Table I. Data for MPA are provided in the text
and figures.
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As a direct correlate to antagonism of LTB4
responses in vitro (see above), ATLa was characterized in an
LTB4-dependent model. Topical ATLa
dose-dependently inhibited edema as well as neutrophil infiltration
(Fig. 3). IC50
values were 40 µg/cm2, and complete
inhibition was achieved at 300 µg/cm2 for both
efficacy end points (mean of three independent experiments). The
anti-inflammatory effects of ATLa in this model were comparable to
the selective LTB4R-Ant, which exhibited
IC50 in the range of 20–30
µg/cm2 for all parameters tested. The results
in this model are consistent with the potent in vitro functional
antagonism of LTB4-stimulated neutrophil
responses by both ATLa and LTB4R-Ant. Given their
known anti-inflammatory mechanism of action (27),
glucocorticoids such as MPA are not active in this direct model of
LTB4-induced inflammation and were not
tested.
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To examine the influence of ATLa on inflammation mediated by
endogenous LTB4 production, efficacy was tested
in calcium ionophore-induced inflammation in mice (Table I). ATLa
exerted dose-dependent efficacy on all end points in this model
(IC50 = <100–200
µg/cm2), with complete inhibition of cell
infiltration and 70% inhibition of edema at 1000
µg/cm2. ATLa was equipotent to
LTB4R-Ant in this model, but was 100- to
1000-fold less potent than MPA (IC50 = 0.1–10
µg/cm2). To demonstrate efficacy across
species, ATLa effects were also tested in calcium ionophore-induced
inflammation in the ears of guinea pigs (Table I). Inhibitory effects
were similar in extent to LTB4R-Ant. The data
confirm that ATLa has anti-inflammatory efficacy in two
species.
Croton oil- and mezerein-induced inflammation
Croton oil and mezerein are phorbol ester compounds that elicit an
inflammatory reaction triggered by protein kinase C activation. This
leads to the release of various proinflammatory mediators, which induce
edema, cellular infiltration, and epidermal hyperproliferation. In the
model of croton oil-induced inflammation, ATLa and
LTB4R-Ant inhibited edema formation and cell
infiltration dose-dependently, with IC50 values
in the range of 200–600 µg/cm2 (Table I). MPA
inhibited these parameters with higher potency than both ATLa and
LTB4R-Ant (IC50 = <0.2
µg/cm2). In the mezerein-induced inflammation
model, IC50 values for edema and cell
infiltration measured at 24 h were 100–150
µg/cm2 for ATLa (Table I and Fig. 4). LTB4R-Ant
showed similar potency to ATLa, with IC50 values
from 100–260 µg/cm2 for these end points. MPA
was significantly more potent for all inflammatory parameters
(IC50 = <0.1–0.75
µg/cm2). Edema formation was inhibited by
70–80%, and cellular infiltration was completely inhibited at a dose
of 1000 µg/cm2 for the eicosanoids and at 10
µg/cm2 for the glucocorticoid.
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5-fold (see Fig. 5A, i, 11 µm;
ii, 52 µm; and iv, 10 µm). ATLa caused
a potent, dose-dependent inhibition of epidermal hyperproliferation
(IC50 = 120 µg/cm2).
Complete inhibition of hyperproliferation was achieved at the highest
dose (1000 µg/cm2; Fig. 5A,
iii, 10 µm; and v; and Fig. 5B), with near-complete inhibition observed at 300
µg/cm2. ATLa was 3-fold more potent than
LTB4R-Ant (IC50 = 400
µg/cm2; Fig. 5B) on this end point.
The potency of MPA on epidermal hyperproliferation was not directly
compared with ATLa in these experiments, but is in the same same range
as its anti-inflammatory effects (data not shown).
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TMA stimulates a T cell-mediated inflammatory reaction with
a characteristic of DTH. Application of TMA to sensitized animals
evokes a mixed cellular infiltrate composed primarily of eosinophils,
but with some neutrophils. In this model MPA exerts very potent effects
on edema formation and neutrophil infiltration
(IC50 = <0.2 µg/cm2),
but weaker effects on eosinophil infiltration, as
measured with the peroxidase assay for total
granulocytes (IC50 = >10
µg/cm2). ATLa inhibited TMA-induced edema
formation and cell infiltration, with IC50 values
of 320–520 µg/cm2 (Fig. 6). Complete or almost complete
inhibition for all parameters was achieved with 1000
µg/cm2. In contrast to the potent and
dose-dependent effects of ATLa on eosinophil infiltration (parameter:
granulocyte infiltration), the LTB4R-Ant was only
able to produce 40% inhibition at the lowest dose tested and was
completely ineffective at other dosages. The
LTB4R-Ant was able to potently inhibit neutrophil
infiltration, as measured by the elastase assay. Together these results
suggest a differential and superior effectiveness of ATLa compared with
LTB4R-Ant with respect to eosinophil infiltration
in this allergic DTH reaction.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). Our present results establish the
following. 1) ATLa potently inhibited neutrophil chemotaxis and
transcellular migration, confirming earlier reports (10),
and was a broader inhibitor of neutrophil responses than our own potent
LTB4 receptor antagonist. 2) Topically applied
ATLa dose-dependently inhibited cutaneous inflammation evoked by
diverse inflammatory mediators, with complete inhibition of several
efficacy end points. 3) ATLa was efficacious in at least two species:
mouse and guinea pig. 4) ATLa potency in vivo was at least equivalent
to a potent LTB4 receptor antagonist, but
considerably less potent than MPA, a clinically used topical
glucocorticoid standard. 5) ATLa was shown for the first time to block
inflammation-dependent epidermal hyperproliferation and to inhibit a T
cell-dependent allergic cutaneous reaction. These findings confirm the
actions of ATLa and firmly establish that lipoxin analogs, such as
ATLa, are a novel therapeutic class that may be considered for topical
treatment of human dermatoses of diverse etiology. ATLa inhibition of neutrophil chemotaxis and transcellular migration to both LTB4 and fMLP in vitro demonstrates a broader influence for ATLa than LTB4R-Ant. In addition to potent LTB4 antagonism in vitro, ATLa exhibited dose-dependent topical efficacy in exogenous LTB4/Iloprost-induced inflammation, with potency equivalent to LTB4-R-Ant. Earlier studies with lipoxin analogs in this model did not establish dose-dependence or potency relative to a direct LTB4 antagonist (6). ATLa potency in this model was quite remarkable given that ATLa is not a direct LTB4 receptor antagonist (IC50 = >10 µM) (28).
To explore the broader effects of ATLa, efficacy was investigated in cutaneous reactions triggered by stimuli that induce endogenous release of various inflammatory mediators, including LTB4. Calcium ionophore-induced inflammation is more prominent compared with the LTB4/Iloprost model and is inhibited by LTB4 receptor antagonists (29). As expected, ATLa exhibited dose-dependent topical efficacy with similar potency to LTB4R-Ant, but was less potent than MPA. Importantly, ATLa was shown to inhibit ionophore-induced inflammation in two species: mouse and guinea pig.
Croton oil is an irritant that stimulates keratinocytes in vitro and in
vivo to release the inflammatory mediators IL-1, TNF-, IL-8, and
GM-CSF via protein kinase C stimulation (30, 31). As
expected, MPA showed anti-inflammatory effects at low doses,
whereas ATLa, although as efficacious as MPA, was 1000-fold less
potent. ATLa efficacy in this model is consistent with lipoxins
inhibiting TNF--induced epithelial cell IL-8 release
(32), and TNF--induced neutrophil infiltration and
cytokine/chemokine networks in the mouse air pouch (11).
Extensive ATLa inhibition of both inflammatory responses (24 h) and
epidermal hyperproliferation (72 h) in the mezerein-induced model is
particularly noteworthy. This is the first demonstration of an effect
of lipoxins on epidermal hyperproliferation. Epidermal
hyperproliferation is a frequent finding in some cutaneous disorders
and is prominent in psoriasis. The enhanced epidermal growth not only
contributes significantly to patient discomfort, but also complicates
disease treatment (33, 34). The inhibitory effect on
epidermal hyperproliferation might be a direct anti-proliferative
effect on keratinocytes, since lipoxins have been shown to antagonize
mitogenic effects and to be anti-proliferative (15).
Moreover, keratinocyte hyperproliferation appears to be induced by IL-6
and IL-8, both of which are inhibited by lipoxins (1, 32, 33, 35, 36). The strong inhibitory effect of ATLa on epidermal
hyperproliferation in combination with its ability to locally inhibit
chemokine networks suggest that ATLa could be an effective topical
treatment for psoriasis.
The occupational allergen TMA induces cutaneous and respiratory allergic reactions in man (37). TMA sensitizes and elicits a DTH reaction in animals, with a mixed Th1 and Th2 character (38). The pronounced efficacy of ATLa on edema formation as well as on neutrophil and eosinophil cell infiltration in this model is the first demonstration that lipoxin analogs can modulate a cutaneous, T cell-dependent allergic response. These results are consistent with earlier studies showing that lipoxin analogs inhibit eosinophil chemotaxis in vitro (39) and eosinophil-driven inflammation in vivo (13, 14). Taken together these findings suggest that ATLa could be explored for topical treatment of dermatoses with a prominent eosinophil component, such as allergic contact dermatitis or atopic dermatitis.
Lipoxins are produced in human asthmatics and potently attenuate human monocyte inflammatory responses (40). Moreover, ATLa potently attenuates airway hyper-reactivity and eosinophilia in murine allergic airway inflammation via multipronged suppression of inflammatory mediators (41). Such findings highlight the anti-inflammatory profile of lipoxins as being distinct from LTB4 receptor antagonists. The latter have shown poor efficacy in murine allergic airway inflammation (42) and in human asthma clinical trials (43).
In summary, the in vitro and in vivo characterization of ATLa shows a unique anti-inflammatory profile and suggests its utility for the topical treatment of inflammatory reactions in skin. While less potent than the clinically used topical glucocorticoid MPA, topical ATLa showed equivalent efficacy on most end points measured. To date there is no evidence to suggest that lipoxins would be expected to cause skin atrophy or systemic endocrinological side effects, which limit the long term use of several topical steroids. ATLa and related lipoxins analogs might thus offer an alternative approach to chronic treatment of dermatoses or treatment of skin reactions that are steroid resistant. Based on the similarity of cutaneous inflammatory reactions and their underlying mechanisms to inflammation in other organs, the impressive therapeutic effects of topically applied ATLa suggest potential utility in other inflammatory and autoimmune diseases. These include rheumatoid arthritis, multiple sclerosis, inflammatory bowel disease, acute respiratory distress syndrome, and ischemia/reperfusion injury. For these indications, further understanding of the systemic pharmacology, efficacy, and safety profile of ATLa and related lipoxin analogs is required.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. John F. Parkinson, Department of Immunology, Berlex Biosciences, Richmond, CA 94804. E-mail address: john_parkinson{at}berlex.com
3 Abbreviations used in this paper: LXA4, lipoxin A4; ALX-R, LXA4 receptor; AMC, 7-amino-4-methyl-coumarin; ATLa, 15-epi-16-p-fluorophenoxy-lipoxin A4 methyl ester; DTH, delayed-type hypersensitivity; HTAB, hexadecyltrimethylammonium bromide; LTB4, leukotriene B4; LTB4R-Ant, leukotriene B4 receptor antagonist; MPA, methylprednisolone aceponate; TMA, trimellitic anhydride; TMB, tetramethylbenzidine.
Received for publication July 23, 2002. Accepted for publication October 15, 2002.
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 T. Ohira, G. Bannenberg, M. Arita, M. Takahashi, Q. Ge, T. E. Van Dyke, G. L. Stahl, C. N. Serhan, and J. A. Badwey A Stable Aspirin-Triggered Lipoxin A4 Analog Blocks Phosphorylation of Leukocyte-Specific Protein 1 in Human Neutrophils J. Immunol., August 1, 2004; 173(3): 2091 - 2098. [Abstract] [Full Text] [PDF]
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 S. Fiorucci, E. Distrutti, A. Mencarelli, G. Rizzo, A. R. D. Lorenzo, M. Baldoni, P. del Soldato, A. Morelli, and J. L. Wallace Cooperation between Aspirin-Triggered Lipoxin and Nitric Oxide (NO) Mediates Antiadhesive Properties of 2-(Acetyloxy)benzoic Acid 3-(Nitrooxymethyl)phenyl Ester (NCX-4016) (NO-Aspirin) on Neutrophil-Endothelial Cell Adherence J. Pharmacol. Exp. Ther., June 1, 2004; 309(3): 1174 - 1182. [Abstract] [Full Text] [PDF]
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H. Schacke, A. Schottelius, W.-D. Docke, P. Strehlke, S. Jaroch, N. Schmees, H. Rehwinkel, H. Hennekes, and K. Asadullah Dissociation of transactivation from transrepression by a selective glucocorticoid receptor agonist leads to separation of therapeutic effects from side effects PNAS, January 6, 2004; 101(1): 227 - 232. [Abstract] [Full Text] [PDF]
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) was determined with
increasing compound concentration. Data are expressed as the mean
± SEM from three experiments, performed in triplicate.
) were compared
with those of LTB4-R-Ant (
), both of which were
coapplied with LTB4/Iloprost. Results are expressed as the
mean ± SEM of eight animals per group. *,
p < 0.05 vs vehicle control.
) and
to the effects of MPA (
