Involvement of leukotriene B4 in murine dermatitis models.

Article abstract of DOI:10.1016/S0006-2952(97)00464-4

Leukotriene B4 (LTB4) is a product of the 5-lipoxygenase pathway of arachidonic acid (AA) metabolism. LTB4 is a potent chemotactic factor for neutrophils and has been postulated to play an important role in a variety of pathological conditions including rheumatoid arthritis, psoriasis, and inflammatory bowel disease. To investigate the role of LTB4 in dermatitis, we used S-(4-dimethylaminobenzyl)-N-[(2S)-3-mercapto-2-methylpropionyl]-L- cysteine (SA6541), a potent leukotriene A4 (LTA4) hydrolase inhibitor. SA6541 inhibited LTB4 production with an IC50 value of 270 nM in vitro. 5-Hydroperoxyeicosatetraenoic acid (5-HPETE) or AA injection induced LTB4 production and neutrophil influx in mouse ear. SA6541 inhibited 5-HPETE- and AA-induced LTB4 production and neutrophil influx in mouse ear when administered orally at a dose of 50 mg/kg. SA6541 also inhibited 5-HPETE-induced prostaglandin E2 (PGE2) production, probably by an indirect effect through the inhibition of LTB4 production. These results suggest that LTB4 may be important in the pathogenesis of dermatitides such as psoriasis.

Full text of DOI:10.1016/S0006-2952(97)00464-4

Biochemical Pharmacology, Vol. 55, pp. 297–304, 1998.
© 1998 Elsevier Science Inc. All rights reserved.
ISSN 0006-2952/98/$19.00 ϩ 0.00
PII S0006-2952(97)00464-4
Involvement of Leukotriene B4 in Murine
Dermatitis Models
Fumio Tsuji,* Yurika Miyake, Masato Horiuchi and Shiro Mita
DISCOVERY RESEARCH DIVISION, SANTEN PHARMACEUTICAL CO., LTD., OSAKA 533, JAPAN
ABSTRACT. Leukotriene B4 (LTB4) is a product of the 5-lipoxygenase pathway of arachidonic acid (AA)
metabolism. LTB4 is a potent chemotactic factor for neutrophils and has been postulated to play an important
role in a variety of pathological conditions including rheumatoid arthritis, psoriasis, and inflammatory bowel
disease. To investigate the role of LTB4 in dermatitis, we used S-(4-dimethylaminobenzyl)-N-[(2S)-3-mercapto-
2-methylpropionyl]-L-cysteine (SA6541), a potent leukotriene A4 (LTA4) hydrolase inhibitor. SA6541
inhibited LTB4 production with an IC value of 270 nM in vitro. 5-Hydroperoxyeicosatetraenoic acid
50
(5-HPETE) or AA injection induced LTB4 production and neutrophil influx in mouse ear. SA6541 inhibited
5-HPETE- and AA-induced LTB4 production and neutrophil influx in mouse ear when administered orally at
a dose of 50 mg/kg. SA6541 also inhibited 5-HPETE-induced prostaglandin E2 (PGE2) production, probably by
an indirect effect through the inhibition of LTB4 production. These results suggest that LTB4 may be important
in the pathogenesis of dermatitides such as psoriasis. BIOCHEM PHARMACOL 55;3:297–304, 1998. © 1998 Elsevier
Science Inc.
KEY WORDS. SA6541; leukotriene A4 hydrolase inhibitor; leukotriene B4; dermatitis; psoriasis; mice
LTB4,† a product of the action of 5-LO on AA, induces
chemokinetic, chemotactic, and aggregation responses in
polymorphonuclear leukocytes [1, 2], degranulation [3, 4],
and increased adhesion of these cells to endothelial cell
monolayers [5]. Although neutrophils were initially identi-
fied as a major source of LTB4, many different cell types
have subsequently been shown to produce this molecule
(e.g. monocytes/macrophages, keratinocytes, lymphocytes,
and mast cells) under conditions associated with patho-
physiological stimulation [6–10]. In addition to activating
neutrophils, LTB4 also has been shown to be an autacoid
capable of inducing the expression of various cytokines and
their receptors in monocytes and lymphocytes [11–15].
These in vivo results suggest a potentially broader role for
LTB4 in inflammatory processes, and implicate it as a
probable endogenous mediator of inflammatory processes.
Evidence supporting a role for LTB4 in in vivo inflammatory
processes has also come from preclinical animal models.
Exogenous LTB4 was shown to induce polymorphonuclear
leukocyte infiltration in animals [16–19]. LTB4 has been
found in a number of pathological fluids associated with the
development of inflammation [20, 21], and both blockers of
LTB4 synthesis (i.e. 5-LO inhibitors) and antagonists of
LTB4 receptors have been reported to inhibit the develop-
ment of inflammatory processes [22–24]. Furthermore, some
types of inflammatory responses were shown to be reduced
markedly in 5-LO knockout mice [25, 26]. In addition,
administration of exogenous LTB4 induces inflammatory
responses in normal volunteers [27–30], and biologically
relevant levels of LTB4 and its metabolites have been
reported in numerous human tissues and fluids in patholog-
ical states [31–36]. Thus, prevention of LTB4 synthesis via
inhibition of either 5-LO or LTA4 hydrolase, or, alterna-
tively, inhibiting its action by blocking LTB4 receptors
have been considered viable modes of action for novel
anti-inflammatory drugs. LTA4 hydrolase produces LTB4
from LTA4. To examine the role of LTB4 in dermatitis, we
investigated the effects of S-(4-dimethylaminobenzyl)-N-
[(2S)-3-mercapto-2-methylpropionyl]-L-cysteine (SA6541)
(see Table 1), a newly synthesized potent LTA4 hydrolase
inhibitor, in murine dermatitis models. In the present study,
we used two models of epidermal inflammation involving
LTB4 production in mice.
MATERIALS AND METHODS
Animals
Inbred male ICR mice, 6- to 7-weeks-old, were purchased
from Japan SLC Inc. They were housed under a 12-hr
light–dark cycle (lights on at 7:00 a.m.), with room
temperature maintained at 23 Ϯ 1°, and humidity at 55 Ϯ
10%. Food and water were available ad lib. All experiments
were conducted in accordance with the recommendations
of the Declaration of Helsinki.
* Corresponding author: Dr. Fumio Tsuji, Discovery Research Division,
Santen Pharmaceutical Co., Ltd., 3-9-19 Shimoshinjo, Higashiyodogawa-
ku, Osaka 533, Japan. Tel. 81-6-321-7034; FAX: 81-6-321-2955.
† Abbreviations: AA, arachidonic acid; 12(R)-HETE, 12(R)-hydroxyei-
cosatetraenoic acid; 5-HPETE, 5-hydroperoxyeicosatetraenoic acid; 5-LO,
5-lipoxygenase; LTA4, leukotriene A4; LTB4, leukotriene B4; MPO,
myeloperoxidase; PGB2, prostaglandin B2; and PGE2, prostaglandin E2
Received 31 March 1997; accepted 29 July 1997.

298
F. Tsuji et al.
FIG. 1. Synthesis of SA6541. (a) 47% HBr aq., 120°, 1 hr; (b) N-t-butoxycarbonylcysteine, N,N-diisopropylethylamine, CH2Cl2,
room temperature, 2.5 hr; (c) 4 N HCl/dioxane, anisole, room temperature, 1 hr; (d) dicyclohexylcarbodiimide, CH2Cl2, room
temperature, 4 hr; (e) CH2Cl2, N,N-dimethylformamide, triethylamine, room temperature, 2 days; and (f) 28% NH3 aq., room
temperature, 1 hr.
Reagents
(pH 7.8) and dialyzed against two changes of the same
buffer. LTA4 hydrolase activity was determined as follows.
The standard reaction mixture contained 60 mM HEPES
buffer, 3 mM dithiothreitol, pH 7.8, and enzyme in a total
volume of 150 ␮L with or without LTA4 hydrolase inhib-
itor. After preincubation at 26° for 1 min, LTA4 (in an
ethanol solution, containing less than 50 mM lithium
hydroxide) was added to a final concentration of 60 ␮M.
After a 1-min incubation at 26°, the reaction was termi-
nated by the addition of acidic acetonitrile:ethanol:acetic
acid (150:50:3, 100 ␮L). PGB2 was added as an internal
standard for HPLC analysis. The mixture was kept at Ϫ20°
for 30 min, followed by centrifugation at 10,000 ϫ g for 5
min at 4°. The supernatant (25 ␮L) was analyzed with a
TSK ODS-80TS column (4.6 ϫ 75 mm, Tosoh Co. Ltd.)
using acetonitrile:methanol:water:acetic acid (900:300:800:
1.8, pH 5.6, containing 0.05% EDTA) as the mobile phase
(flow rate 1.0 mL/min). The absorbance at 270 nm was
monitored, and the amount of LTB4 formed was calculated
from the peak area ratio LTB4/PGB2.
SA6541, an inhibitor of LTA4 hydrolase, was synthesized
by the Central Research Laboratories of the Santen Phar-
maceutical Co., Ltd. Figure 1 shows the route of SA6541
synthesis. 4-Dimethylaminobenzaldehyde (Nacalai Tesque),
N-t-butoxycarbonylcystine (Kokusan Chemical Works), thio-
benzoic acid (Sigma–Aldrich Japan), methylacrylic acid
(Sigma–Aldrich Japan), and p-nitrophenol (Nacalai
Tesque) were purchased from the sources shown for
SA6541 synthesis. Indomethacin, a cyclooxygenase inhib-
itor, was purchased from the Sigma Chemical Co. SA6541
and indomethacin were suspended in saline containing 1%
carboxymethylcellulose (vehicle) for oral administration.
5-HPETE (Funakoshi) and sodium AA (Sigma) were used
to induce dermatitis. 5-HPETE is an intermediary metabo-
lite of 5-LO from AA to LTA4. These agents were sus-
pended in phosphate-buffered saline. To determine the in
vitro IC values of LTA4 hydrolase, SA6541 and bestatin
50
(Sigma) were dissolved in saline. LTA4 was prepared as
described [37].
Enzyme Assay
5-HPETE- or AA-Induced Mouse Ear
Dermatitis Model
LTA4 hydrolase was purified from guinea pig lung and used
in the enzyme assay according to the method of Ohishi et al.
[37]. Briefly, guinea pig lung was homogenized in 3 vol. of
50 mM phosphate-buffered saline containing 1 mM EDTA
(pH 7.4). The homogenate was centrifuged at 100,000 ϫ g
for 60 min, and the resultant supernatant was subjected to
ammonium sulfate fractionation. The precipitates between
40 and 70% saturation were collected and dissolved in 20
mM Tris–acetate buffer containing 1 mM dithiothreitol
Mice were challenged intradermally with 25 ␮L of 5-
HPETE (5 ␮g) or AA (2 ␮g) in the external ear, using a
microsyringe with a 30-gauge needle. At various time
points after intradermal challenge, the animals were killed
by an overdose of anesthetic, and injected sites were
punched out with a 6-mm gasket punch and then placed in
tubes containing 0.75 mL of 0.5% hexadecyltrimethylam-
monium bromide/80 mM phosphate buffer (pH 5.4) for

LTB4 in Dermatitis
299
measurement of MPO activity or 1 mL of ethanol for
measurement of LTB4, PGE2, or leukotriene C4/D4/E4.
Administration of SA6541 or Indomethacin
in the Dermatitis Models
Animals in the control group were given orally the vehicle
only. Other groups were dosed orally with SA6541 or
indomethacin at 1 mL suspension/100 g body weight.
SA6541 was suspended in vehicle at a concentration of 1
mg/mL (when administered at a dose of 10 mg/kg) or 5
mg/mL (when administered at a dose of 50 mg/kg). Indo-
methacin was suspended in vehicle at a concentration of
0.5 mg/mL. One hour after oral dosing, mice were chal-
lenged intradermally with 25 ␮L of 5-HPETE (5 ␮g) or AA
(2 ␮g) using a microsyringe with a 30-gauge needle. For
measurement of MPO activity, the mice were dosed orally
with these drugs again 2 hr after intradermal challenge.
Animals were killed by an overdose of anesthetic, and
injected sites were punched out 4 or 8 hr after challenge.
For measurement of LTB4, PGE2, or leukotriene C4/D4/E4,
injected sites were punched out 30 min after challenge.
FIG. 2. Inhibition curves of LTA4 hydrolase by SA6541 and
bestatin. Each point indicates the mean of duplicate measure-
ments in a single experiment.
Measurement of LTB4, PGE2 or Leukotriene C4/D4/E4
Measurement of MPO Activity
The samples were homogenized and centrifuged at 12,000 ϫ g
for 15 min. The supernatants were evaporated to dryness
and dissolved in 3 mL of 0.1 M citrate buffer (pH 3.0).
Solutions were loaded onto C18 SepPak (Waters) columns,
which were then washed with 25% methanol in water
containing 1% acetic acid. LTB4, PGE2, or leukotriene
C4/D4/E4 was eluted from these columns by 80% methanol
in water containing 1% acetic acid, and subsequently
lyophilized to remove the organic solvent. The recoveries of
LTB4 and PGE2 in this extraction procedure were 89 and
96%, respectively. LTB4, PGE2, or leukotriene C4/D4/E4
was quantified using an appropriate enzyme immunoassay
kit (Amersham). The amounts of LTB4, PGE2, or leukotri-
ene C4/D4/E4 are expressed as units of pg/cm2 by conversion
using the area of the punched-out ear tissue (0.2826 cm2).
MPO activity was measured according to the method of
De Young et al. [38]. The samples were homogenized and
subjected to two freeze–thaw cycles, and then centrifuged at
12,000 ϫ g for 15 min. Aliquots of 30 ␮L of the superna-
tant were diluted with 100 ␮L of phosphate-buffered saline
and 75 ␮L of 0.22 M phosphate buffer in 96-well plates, and
then mixed with 35 ␮L of enzyme substrate containing
tetramethylbenzidine/hydrogen peroxide (Amersham) for 5
min at 37°. The reaction was terminated by adding 30 ␮L
of 1.0 M sulfuric acid, and the absorbance of each sample
was measured with a microtiter plate reader at 450 nm.
Activity is expressed as units of OD/min/cm2 by conversion
using the area of the punched-out ear tissue (0.2826 cm2).
TABLE 1. Inhibitory effects of SA6541 and bestatin on
LTA4 hydrolase
Statistical Analysis
Dunnett’s multiple comparison test (Statistical Library,
Yukms Corp.) was used for statistical analysis of the results.
RESULTS
Inhibition of LTA4 Hydrolase by SA6541
The chemical structures and IC values of SA6541 and
50
bestatin, a known inhibitor of LTA4 hydrolase [39, 40], are
shown in Table 1. Figure 2 shows the LTA4 hydrolase
inhibitory curves by SA6541 and bestatin. SA6541 inhib-
ited LTA4 hydrolase in a concentration-dependent manner
similar to bestatin, but the inhibition by SA6541 was more
potent than that by bestatin. The inhibitory potency of
SA6541 for LTA4 hydrolase was 200-fold stronger than
Compounds
SA6541
IC50*(M)
2.7 ϫ 10Ϫ7
5.5 ϫ 10Ϫ5
Bestatin
* The IC values were obtained from graphs of inhibition percentage versus the log
50
of concentration.
that of bestatin in terms of IC .
50

300
F. Tsuji et al.
FIG. 3. Effects of intradermally injected 5-HPETE on neutro-
phil accumulation [(a) dose–response and (b) time–course] and
(c) time–course of changes in LTB4 concentration in mouse ear.
Values are means ؎ SEM of 4 animals. (a) MPO activity was
measured 4 hr after 5-HPETE injection. (b) MPO activity was
measured at each time point after 5-HPETE (5 ␮g) injection.
(c) LTB4 concentration was measured at each time point after
5-HPETE (5 ␮g) injection.
FIG. 4. Effects of intradermally injected AA on neutrophil
accumulation [(a) dose–response and (b) time–course] and (c)
time–course of changes in PGE2 concentration in mouse ear.
Values are means ؎ SEM of 4 animals. (a) MPO activity was
measured 4 hr after AA injection. (b) MPO activity was
measured at each time point after AA (2 ␮g) injection. (c) PGE2
concentration was measured at each time point after AA (2 ␮g)
injection.
course of changes in LTB4 concentration in the mouse ear
is shown in Fig. 3c. There was an immediate sharp increase
of LTB4 concentration and then a return to the low level 2
hr after 5-HPETE injection.
5-HPETE-Induced Inflammation
The effects of intradermally injected 5-HPETE on neutro-
phil accumulation in the mouse ear are shown in Fig. 3a.
5-HPETE induced neutrophil accumulation at doses above
1 ␮g 4 hr after injection. The time–course of neutrophil
accumulation in the mouse ear following intradermal in-
jection of 5-HPETE (5 ␮g) is shown in Fig. 3b. Neutrophil
accumulation gradually increased until 24 hr. The time–
AA-Induced Inflammation
The effects of intradermally injected AA on neutrophil
accumulation in the mouse ear are shown in Fig. 4a. AA

LTB4 in Dermatitis
301
FIG. 5. Effects of SA6541 on 5-HPETE-induced neutrophil
accumulation. Values are means ؎ SEM of 6–16 animals. The
samples used for measurement of MPO activity were collected 8
hr after 5-HPETE (5 ␮g) injection. Drugs were administered
orally 1 hr before and 2 hr after 5-HPETE injection. Key:
(**)P < 0.01 vs the control group (by Dunnett’s multiple
comparison test).
induced neutrophil accumulation in a dose-dependent
manner 4 hr after injection. The time–course of neutrophil
accumulation in the mouse ear following intradermal in-
jection of AA (2 ␮g) is shown in Fig. 4b. The neutrophil
accumulation increased to a peak at about 8 hr after AA
injection and then decreased thereafter. The time–course
of changes in PGE2 concentration in the mouse ear is
shown in Fig. 4c. There was an immediate sharp increase of
PGE2 concentration and then a return to the low level 2 hr
after AA injection. The time–course of changes in LTB4
concentration in the mouse ear showed almost the same
pattern as 5-HPETE-induced inflammation (data not shown).
Effects of SA6541 on 5-HPETE-Induced Inflammation
The effects of SA6541 on 5-HPETE-induced neutrophil
accumulation and increases of LTB4, PGE2, or leukotriene
C4/D4/E4 concentration in the mouse ear are shown in Figs.
5 and 6, respectively. SA6541 (50 mg/kg ϫ 2) significantly
inhibited neutrophil accumulation 8 hr after 5-HPETE
injection and also inhibited the increases in LTB4 and
PGE2 concentration 30 min after 5-HPETE injection.
However, SA6541 had no effect on the increase in leuko-
triene C4/D4/E4 concentration. On the other hand, indo-
methacin had no effect on neutrophil accumulation or the
increases in LTB4 or leukotriene C4/D4/E4 concentrations,
but it inhibited the increase in PGE2 concentration.
FIG. 6. Effects of SA6541 on 5-HPETE-induced production of
(a) LTB4 (b) PGE2, or (c) leukotriene C4/D4/E4. Values are
means ؎ SEM of 4–6 animals. The samples were collected 30
min after 5-HPETE (5 ␮g) injection. Drugs were administered
orally 1 hr before 5-HPETE injection. Key: (**)P < 0.01 vs the
control group (by Dunnett’s multiple comparison test).
However, SA6541 had no effect on the increase in the level
of PGE2. On the other hand, indomethacin had no effect on
neutrophil accumulation or increase in LTB4 concentration,
but it inhibited the increase in PGE2 concentration.
Effects of SA6541 on AA-Induced Inflammation
The effects of SA6541 on AA-induced neutrophil accumu-
lation and increases in LTB4 and PGE2 concentrations in
the mouse ear are shown in Figs. 7 and 8, respectively.
SA6541 (50 mg/kg ϫ 2) significantly inhibited neutrophil
accumulation 8 hr after AA injection and also inhibited the
increase in LTB4 concentration 30 min after AA injection.
DISCUSSION
We showed that the newly synthesized agent SA6541 is a
potent LTA4 hydrolase inhibitor. Orally administered

302
F. Tsuji et al.
FIG. 7. Effects of SA6541 on AA-induced neutrophil accumu-
lation. Values are means ؎ SEM of 8–12 animals. The samples
used for the measurement of MPO activity were collected 8 hr
after AA (2 ␮g) injection. Drugs were administered 1 hr before
and 2 hr after AA injection. Key: (*)P < 0.05 vs the control
group (by Dunnett’s multiple comparison test).
SA6541 was efficacious in two models of epidermal inflam-
mation in mice. There have been few reports concerning
orally active LTA4 hydrolase inhibitors. As epidermal
inflammation models, we used 5-HPETE- or AA-induced
dermatitis. 5-HPETE is an intermediary metabolite of 5-LO
from AA to LTA4, and LTB4 production from 5-HPETE
requires 5-LO and LTA4 hydrolase [41]. LTB4 production
and neutrophil accumulation were evoked by 5-HPETE.
However, there was little production of PGE2 after intrad-
ermal injection of 5-HPETE, and, therefore, the 5-LO
pathway of AA metabolism was important in this model.
After oral administration, SA6541 blocked 5-HPETE-
induced production of LTB4 and neutrophil accumulation.
However, SA6541 had no effect on increases in leukotriene
C4/D4/E4 concentrations. On the other hand, indometha-
cin did not block 5-HPETE-induced production of LTB4 or
neutrophil accumulation. Therefore, LTB4 may be the
main chemotactic factor for neutrophil accumulation in
this model.
LTB4 and PGE2 production and neutrophil accumula-
tion were evoked by AA. After oral administration,
SA6541 blocked AA-induced production of LTB4 and
neutrophil accumulation but did not inhibit PGE2 produc-
tion. These results suggest that SA6541 has no inhibitory
effect on cyclooxygenase. Thus, SA6541 may be a specific
inhibitor of LTA4 hydrolase in the AA cascade. The
inhibitory effect of SA6541 on neutrophil accumulation
after AA injection was weaker than that after 5-HPETE
injection, while SA6541 almost completely blocked LTB4
production. These observations suggest that LTB4 is merely
one of the chemotactic factors for neutrophil accumulation
in AA-induced dermatitis. 12(R)-HETE is another candi-
date for the chemotactic factor in AA-induced dermatitis.
12(R)-HETE was found previously in human psoriatic skin,
and it has been suggested that 12(R)-HETE acting as the
LTB4 receptor agonist on neutrophils may be relevant to
recruitment of neutrophils in this disease [42, 43]. In guinea
FIG. 8. Effects of SA6541 on AA-induced production of (a)
LTB4 or (b) PGE2. Values are means ؎ SEM of 4 animals. The
samples were collected 30 min after AA (2 ␮g) injection. Drugs
were administered orally 1 hr before AA injection. Key:
(**)P < 0.01 vs the control group (by Dunnett’s multiple
comparison test).
pig skin, 12(R)-HETE injected intradermally causes influx
of neutrophils [44].
As shown in Figs. 5 and 6, a greater inhibitory effect of
SA6541 was seen on neutrophil infiltration than on LTB4
production. One possible explanation for these results is
that there may be a threshold concentration of LTB4 for
neutrophil infiltration. As shown in Fig. 3, 1 ␮g of
5-HPETE scarcely induced neutrophil infiltration, whereas
at 10 ␮g significant induction was seen. However, we
consider that 1 ␮g of 5-HPETE probably induces LTB4
production. Therefore, slight inhibition of LTB4 produc-
tion might cause marked inhibition of neutrophil infiltra-
tion.
While SA6541 had no inhibitory effect on cyclooxge-
nase, it inhibited PGE2 production after 5-HPETE injec-
tion. In our preliminary studies, LTB4 injected intrader-
mally induced PGE2 production in the mouse ear. For
example, LTB4 (500 pg/ear) injection induced PGE2 pro-
duction at about 60 pg/cm2 30 min after challenge. There-
fore, PGE2 production after 5-HPETE injection may be
mediated by LTB4 production from 5-HPETE. The inhibi-
tion of PGE2 production by SA6541 in the 5-HPETE-

LTB4 in Dermatitis
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induced dermatitis model may be achieved via inhibition of
LTB4 production.
For clinical application, orally active LTA4 hydrolase
inhibitors may be useful for the treatment of rheumatoid
arthritis, psoriasis, and inflammatory bowel disease. Ford-
Hutchinson [45] reported that there was no convincing
evidence for the presence of 5-LO in skin. However, we
consider that 5-LO may be present in skin on the grounds
that LTB4 production from 5-HPETE was observed in the
mouse ear. In addition, we have shown that LTB4 is
important for neutrophil accumulation in the mouse ear.
In conclusion, LTB4 may be important in the pathogen-
esis of dermatitides such as psoriasis.
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