U.S. patent application number 09/833274 was filed with the patent office on 2001-12-27 for therapeutic agent for dermatitis.
Invention is credited to Fujita, Akihiro, Yoshii, Haruo.
Application Number | 20010056097 09/833274 |
Document ID | / |
Family ID | 18624080 |
Filed Date | 2001-12-27 |
United States Patent
Application |
20010056097 |
Kind Code |
A1 |
Yoshii, Haruo ; et
al. |
December 27, 2001 |
Therapeutic agent for dermatitis
Abstract
The present invention provides a therapeutic agent for
dermatitis, particularly a therapeutic agent for atopic dermatitis,
which is very safe and which shows few adverse side-effects in
comparison to, for example, steroidal agents. The present invention
relates to a therapeutic agent containing a compound represented by
the following formula (I) or a pharmaceutically acceptable salt or
hydrate thereof as an effective ingredient: 1 wherein R is hydrogen
or a halogen. The therapeutic agent for dermatitis according to the
present invention effectively and in a dose-dependent manner
suppresses antigen-induced swelling in a mouse ear, a recognized
animal model for atopic dermatitis, and suppresses the
antigen-induced flare-up reaction in mice which occurred with the
swelling reaction. In addition, no adverse reaction in the skin are
observed.
Inventors: |
Yoshii, Haruo; (Katoh-gun,
JP) ; Fujita, Akihiro; (Katoh-gun, JP) |
Correspondence
Address: |
HOLLANDER LAW FIRM, P.L.C.
SUITE 305
10300 EATON PLACE
FAIRFAX
VA
22030
|
Family ID: |
18624080 |
Appl. No.: |
09/833274 |
Filed: |
April 12, 2001 |
Current U.S.
Class: |
514/259.4 |
Current CPC
Class: |
Y10S 514/862 20130101;
A61P 17/00 20180101; Y10S 514/86 20130101; Y10S 514/863 20130101;
A61K 31/519 20130101; Y10S 514/858 20130101; Y10S 514/864 20130101;
Y10S 514/861 20130101; Y10S 514/859 20130101 |
Class at
Publication: |
514/258 |
International
Class: |
A61K 031/519 |
Foreign Application Data
Date |
Code |
Application Number |
Apr 13, 2000 |
JP |
111,809/2000 |
Claims
What is claimed is:
1. A therapeutic agent for dermatitis comprising a compound
represented by the following formula (I) or a pharmaceutically
acceptable salt or hydrate thereof as an effective ingredient:
4wherein R is hydrogen or a halogen.
2. The therapeutic agent for dermatitis according to claim 1,
wherein the agent is a therapeutic agent for atopic dermatitis.
3. The therapeutic agent for dermatitis according to claim 1,
wherein the agent is intended for external use.
4. The therapeutic agent for dermatitis according to claim 1,
wherein R is hydrogen.
5. A therapeutic agent for dermatitis which comprises
7-amino-3-(2-chlorobenzyl)-1,2,3,4-tetrahydro-1-phenylpyrido(2,3-d)pyrimi-
din-2,4-dione.
6. The therapeutic agent for dermatitis according to claim 1 which
is a formulation of a liquid, a suspension or emulsion, a plaster,
an ointment, a cataplasm, a liniment or a lotion.
7. The therapeutic agent for dermatitis according to claim 1 which
is an ointment having from about 0.1% by weight to about 15% by
weight based on the total weight of the ointment, of said compound
represented by formula (I).
8. The therapeutic agent for dermatitis according to claim 7 which
is an ointment having from about 0.3% by weight to about 10% by
weight based on the total weight of the ointment, of said compound
represented by formula (I).
9. A method for the treatment of dermatitis comprising
administering to a patient in need of such treatment a
pharmaceutically effective amount of at least one compound
represented by the following formula (I) or a pharmaceutically
acceptable salt or hydrate thereof: 5wherein R is hydrogen or
halogen.
10. The method according to claim 9, wherein the dermatitis is
atopic dermatitis.
11. The method according to claim 9, wherein R is hydrogen.
12. The method according to claim 9, wherein R is chloride and is
substituted at the opposition.
13. The method according to claim 9 comprising applying said
compound represented by formula (I) externally to an affected area
of the skin.
14. The method according to claim 13 comprising applying said
compound represented by formula (I) externally to an affected area
of the skin from one to six times a day until the affected area
heals.
15. The method according to claim 9, wherein said compound
represented by formula (I) is formulated into a liquid, a
suspension or emulsion, a plaster, an ointment, a cataplasm, a
liniment or a lotion.
16. The method according to claim 15, wherein the formulation is an
ointment.
17. The method according to claim 16, wherein the ointment contains
from about 0.1% by weight to about 15% by weight based on the total
weight of the ointment, of said compound represented by formula
(I).
18. The method according to claim 17, wherein the ointment contains
from about 0.3% by weight to about 10% by weight based on the total
weight of the ointment, of said compound represented by formula
(I).
19. The method according to claim 9, wherein the patient is
suffering from dermatitis selected from the group consisting of
contact dermatitis, atopic dermatitis, seborrheic dermatitis,
nummular eczema, Vidal's lichen, stasis dermatitis, dyshidrotic
eczema, asteatosis eczema dermatitis, and autosensitization eczema.
Description
FIELD OF THE INVENTION
[0001] The present invention relates to medical uses of
7-amino-3-benzyl-1-phenylpyrido[2,3-d]pyrimidin-2,4-dione
derivatives as a therapeutic agent for dermatitis, particularly as
a therapeutic agent for atopic dermatitis.
BACKGROUND OF THE INVENTION
[0002] Dermatitis, which usually has the same meaning as eczema, is
an inflammation reaction of the skin to various external and
internal causes and is the most common disease among the skin
diseases. Typical clinical features in acute stage dermatitis
include swelling erythema, followed by the formation of papules and
serous papules on the erythema. After the formation of vesicles in
the skin, pustules form, followed by the erosion, crusting and
desquamation of the skin. Only then does the skin begin to heal.
When dermatitis turns chronic, thickening, lichenification and
pigmentation of the skin all result and, in most cases, accompanied
by itching. Histologically, dermatitis is characterized in swelling
among epidermal cells (in a spongy state) during the acute stage.
Contact dermatitis, atopic dermatitis, seborrheic dermatitis,
nummular eczema, Vidal's lichen, stasis dermatitis, dyshidrotic
eczema, asteatosis eczema dermatitis, autosensitization eczema,
etc. are included among recognized categories of dermatitis.
[0003] Although atopic dermatitis is thought to occur by atopy,
i.e. by an allergic reaction in which immunoglobulin E (IgE)
participates, a definite cause of its onset is still unknown.
Atopic dermatitis is often accompanied by a high level of IgE in
the blood and by eosinophilia. Atopy itself belongs to a type I
allergic response but, since atopic dermatitis is a reaction
similar to eczema and contact dermatitis from a pathological
viewpoint, the participation of a type IV allergic response
(delayed type) has been suggested. It has also been suggested that
a delayed type reaction accompanied by infiltration of eosinophils
and lymphocytes plays an important role in the onset of atopic
dermatitis and its change to chronic dermatitis. See Iwamoto, et
al.: J. Leukoc. Biol., 52, pages 572-578 (1992); Frigas, et al.: J.
Allergy Clin. Immunol., 77, pages 527-537 (1986), etc.
[0004] Symptoms of atopic dermatitis vary with age of the subject.
At from about the second to the sixth month from birth, weeping
eczema of the face appears first and then it gradually expands to
the legs and arms and to the trunk. A strong itch is also
characteristic. The symptoms are classified into a baby period
(younger than 2 years old), an infant period (2-12 years old) and
an adult period. During the baby period, the onset is mostly
limited to the face but, gradually the skin of trunk becomes dry
resulting in follicular papules (atopic skin). In infants and small
children, the lesions become dry and thick areas form in the pits
of the elbow and knee (lichenification). There are many cases where
atopic dermatitis heals by about 12 years of age but when it
carries over into an adult period, it becomes more severe. Even if
the atopic dermatitis once attenuates in severity, recurrence and
the worsening of symptoms are often seen and there are even some
cases which require a long period to complete healing or else a
complete healing is not achieved.
[0005] External application of a steroidal agent (ointment or the
like) is the most effective therapy to date and no therapeutic
method to replace it has yet been established. However, steroid
preparations which are frequently used to treat dermatitis,
including atopic dermatitis, are the pharmaceuticals which have a
very active clinical effect and which also cause a great variety of
adverse reactions. There have been various reports on the side
effects of steroid preparations and, in the case of agents for
external use such as ointments, direct harmful effects such as the
thinning, shrinking and flushing of the skin have become a
widespread social problem. The severe adverse reactions caused by
steroid preparations used as remedies for dermatitis and atopic
dermatitis have led to an eager demand in patients and in the
medical field for safer pharmaceuticals which have fewer side
effects.
[0006] The compounds which are used as a therapeutic agent for
dermatitis according to the present invention are disclosed in
Japanese Patent Laid-Open Publication 2000-119272 and in
corresponding U.S. patent application Ser. No. 09/418982 to Ogino
et al. Compounds having a pyrido[2,3-a]pyrimidine structure, have
been reported as having an anti-allergic action. See Japanese
Patent Laid-Open Publication Sho-63/45279 and U.S. Pat. No.
4,808,587 to Go et al. Further, compounds having a
7-aminopyrido[2,3-d]pyrimidine structure have a bronchodilating
action. See Japanese Patent Publications 06-159322, 06-159323, and
06-159324 (Hei-8/3046, Hei-8/3164 and Hei-8/3165) and corresponding
U.S. Pat. No.5,776,942 to Furukawa et al.
[0007] None of the references discussed above disclose a medical
use or a method for treating dermatitis or atopic dermatitis using
a compound made according to the present invention. The present
invention solves the above-mentioned problems by providing a
therapeutic agent for dermatitis, particularly a therapeutic agent
for atopic dermatitis, which is safer and which exhibits fewer
adverse side-effects than existing therapeutic agents for
dermatitis or atopic dermatitis.
SUMMARY OF THE INVENTION
[0008] The present inventors have carried out an intensive
investigation of 7-aminopyrido[2,3-d]pyrimidine derivatives and
have found that
7-amino-3-benzyl-1-phenylpyrido[2,3-d]pyrimidin-2,4-dione
derivatives are highly effective as therapeutic agents for
dermatitis, particularly for atopic dermatitis, and that these
therapeutic agents for dermatitis cause few adverse reactions in
the skin.
[0009] The present invention provides a therapeutic agent for
dermatitis which contains a compound represented by the following
formula (I), or a pharmaceutically acceptable salt or hydrate
thereof, as an effective ingredient: 2
[0010] In the formula, R is hydrogen or a halogen.
[0011] Compounds represented by the above formula (I) include the
pharmaceutically acceptable salts thereof such as acid addition
salts, salts with alkali metals, salts with alkaline-earth metals,
salts with other metals or salts with bases.
[0012] The present invention includes any and all steric isomers
such as cis-trans isomers, optical isomers, conformational isomers
and hydrates of the compounds of formula (I).
[0013] A pharmaceutical composition which is used as a therapeutic
agent for dermatitis according to the present invention can be
prepared by combining a compound represented by the above formula
(I) with a pharmaceutically acceptable carrier or diluent.
DETAILED DESCRIPTION OF THE INVENTION
[0014] The present invention provides a therapeutic agent and a
method of using a therapeutic agent which comprises
7-amino-3-benzyl-1-phenylpyrido- [2,3-d]pyrimidin-2,4-dione
derivatives for treating dermatitis, and particularly for atopic
dermatitis. Specifically, the present invention provides a
therapeutic agent for dermatitis which contains a compound
represented by the following formula (I), or a pharmaceutically
acceptable salt or hydrate thereof, as an effective ingredient:
3
[0015] In the formula, R is hydrogen or a halogen. A "halogen"
includes, but is not limited to, fluorine, chlorine, bromine or
iodine.
[0016] Preferred embodiments of the present invention are:
[0017] (1) A therapeutic agent for dermatitis containing a compound
represented by the above formula (I) or a pharmaceutically
acceptable salt or hydrate thereof as an effective ingredient.
[0018] (2) The therapeutic agent for dermatitis according to
paragraph (1), wherein the agent is a therapeutic agent for atopic
dermatitis.
[0019] (3) The therapeutic agent for dermatitis according to
paragraphs (1) or (2), wherein the agent is for external use.
[0020] (4) The therapeutic agent for dermatitis according to any of
paragraphs (1) to (3), wherein R is hydrogen in formula (I).
[0021] (5) The therapeutic agent for dermatitis according to any of
paragraphs (1) to (3), wherein R is substituted at the o-position
in formula (I).
[0022] (6) The therapeutic agent for dermatitis according to
paragraph (5), wherein R is chloride.
[0023] Especially preferred compounds for use in treating
dermatitis are:
[0024]
7-amino-3-benzyl-1,2,3,4-tetrahydro-1-phenylpyrido[2,3-d]pyrimidin--
2,4-dione (compound 1)
[0025]
7-amino-3-(2-chlorobenzyl)-1,2,3,4-tetrahydro-1-phenylpyrido[2,3-d]-
pyrimidin-2,4-dione (compound 2)
[0026] The most preferred compound is the compound 1.
[0027] Compounds represented by the above formula (I) include the
pharmaceutically acceptable salts thereof such as acid addition
salts with hydrochloric acid, sulfuric acid, nitric acid,
hydrobromic acid, phosphoric acid, perchloric acid, thiocyanic
acid, boric acid, formic acid, acetic acid, haloacetic acid,
propionic acid, glycolic acid, citric acid, tartaric acid, succinic
acid, gluconic acid, lactic acid, malonic acid, fumaric acid,
anthranilic acid, benzoic acid, cinnamic acid, p-toluenesulfonic
acid, naphthalenesulfonic acid or sulfanilic acid; salts with
alkali metal such as sodium or potassium, salts with an
alkaline-earth metal such as calcium or magnesium, or salts with
other metals such as aluminum; or salts with bases such as ammonia
or organic amines. Those salts may be manufactured by known methods
from the compounds of the present invention in a free state or may
be mutually converted among the salts. The present invention
includes any and all steric isomers such as cis-trans isomers,
optical isomers, conformational isomers and hydrates of the
compounds of formula (I).
[0028] A pharmaceutical composition which is used as a therapeutic
agent for dermatitis according to the present invention can be
prepared by combining a pharmaceutically effective amount of a
compound represented by the above formula (I) with an appropriate
pharmaceutically acceptable carrier or diluent. In manufacturing
the pharmaceutical composition, any of various conventional methods
may be used. Optimally, a therapeutic agent for dermatitis
comprises a preparation for external use such as a liquid,
suspension/emulsion, plaster, ointment, cataplasm, liniment or
lotion. For prescription, the compound of the above formula (I) may
be used as a pharmaceutically acceptable salt or hydrate, or it may
be combined with other pharmaceutically active ingredient(s).
Methods for the manufacture of preparations for external use are
mentioned in detail, for example, in General Rule for
Pharmaceutical Preparations, Commentary to the 13th Revision of the
Japanese Pharmacopoeia (published by Hirokawa Shoten, 1996), the
disclosure of which is herein incorporated by reference in its
entirety.
[0029] Ointment preparations may be roughly classified into fat/oil
type ointments, emulsified ointments, water-soluble ointments and
suspended ointments according to the type of the base (vehicle)
used therefor. An ointment may comprise, for example, fats, fatty
oils, lanolin, vaseline, paraffins, waxes, resins, plastics,
glycols, higher alcohols, glycerol, water, emulsifiers, suspending
agents or other appropriate additives as a diluent, carrier or as a
vehicle. Manufacture of an ointment comprises, for example, adding
the compound of the present invention to the appropriate additives,
diluents, carriers or vehicles followed by mixing to make the
mixture homogeneous.
[0030] In manufacturing a cataplasm, a powder of the compound of
the above formula (I) may be admixed with an essential oil
component to give a muddy product. Depending upon the type and
state of the disease to be treated, the ordinary skilled artisan
can manufacture other types of preparations which are optimum for
the desired therapy.
[0031] A therapeutic agent according to the present invention, or a
pharmaceutical composition which is used as a therapeutic agent for
dermatitis according to the present invention may be used to treat
animal or human subjects who have been diagnosed with or who are
known to be in need of treatment for dermatitis or eczema, such as
one or more conditions selected from the group consisting of
contact dermatitis, atopic dermatitis, seborrheic dermatitis,
nummular eczema, Vidal's lichen, stasis dermatitis, dyshidrotic
eczema, asteatosis eczema dermatitis, and autosensitization
eczema.
[0032] The preferred dosage of the compound of the present
invention varies depending upon the subject to be administered
(age, body weight, symptoms, etc. of the patient), form of the
preparation, method for the administration, term for the
administration, etc. For example, to achieve the desired effect,
the compound of the present invention may be administered as an
ointment containing, by weight based on the total weight of the
ointment, from about 0.1% to about 15% of the compound of the
present invention. The ointment may be applied on the affected area
once to several times per day, for example, 5 or 6 times daily
until the affected area heals.
[0033] Compounds represented by the above formula (I) can be
manufactured by methods disclosed in: a) Japanese Patent Laid-Open
Sho-63/45279, corresponding U.S. Pat. No. 4808587 to Go et al. and
corresponding publication EP 0243311 B; b) Japanese Patent
publications 06-159322, 06-159323 and 06-159324 (Hei-8/3049,
Hei-8/3164, Hei-8/3165), corresponding U.S. Pat. No. 5,776,942 to
Furukawa et al., and corresponding European patent publication EP
0696590 A; and c) Japanese Patent Laid-Open Publication
2000-119272, corresponding U.S. patent application Ser. No.
09/418982 to Ogino et al., and corresponding European patent
publication EP 0994113 A or by a similar method. The disclosure of
each of said Japanese and European patent publications, U.S. Pat.
Nos. 4,808,587 and 5,776,942, and U.S. application Ser. No.
09/418,982 to Ogino et al are each herein incorporated by reference
in their entireties.
[0034] Methods for the production of the compounds of the present
invention are further illustrated in detail by way of the following
non-limiting examples. The starting materials may be purchased from
Aldrich Chemical Co., Inc.; Furuka Chemical Inc.; Lancaster
Synthesis Inc.; Maybridge Chemical Co., Ltd.; or Tokyo Kasel K.K.
or may be synthesized by known methods mentioned in the literature
such as J. Org. Chem., 16, 1879 (1951); J. Am. Chem. Soc., 75, 114
(1953); etc. In the following examples all parts, percentages and
ratios are by weight, all temperatures are in .degree. C., and all
reactions are conducted at about atmospheric pressure and at about
room temperature unless indicated to the contrary:
EXAMPLE 1
Preparation of Ointment
[0035] (1) Manufacture of 6-Amino-1-phenyluracil
[0036] Phenylurea (68.1 g, 0.5 mol) and ethyl cyanoacetate (53 mL,
0.5 mol) were added to a solution prepared by mixing methanol (400
mL) with potassium tert-butoxide (67.3 g, 0.6 mol). The mixture was
heated under reflux for 5.5 hours and the solvent was evaporated in
vacuo until a residue was formed. The residue was then dissolved in
hot water (2 L). Glacial acetic acid was added to the solution
until the solution became acidic and a uracil derivative was
obtained as a bulky and yellowish precipitate. The precipitate was
collected by filtration, washed several times with water and dried
at 50.degree. C. to give 6-amino-1-phenyluracil (67.4 g) in a 66%
yield.
[0037] Mp:>280.degree. C.
[0038] .sup.1H-NMR (DMSO-d.sub.6) .delta.: 4.67(s,1H), 6.08(s,2H),
7.31(d,2H,J=7 Hz), 7.47-7.54(m,3H), 10.43(s,1H)
[0039] IR (KBr): 3478, 3334, 2981, 1712, 1634, 1476, 1387, 1299,
704 cm.sup.-1
[0040] MS (EI) m/z: 203[M.sup.+], 160, 132, 77
[0041] (2) Manufacture of
7-Amino-1,2,3,4-tetrahydro-1-phenylpyrido[2,3-d]-
pyrimidin-2,4-dione
[0042] 6-Amino-1-phenyluracil (30.5 g, 150 mmol) and propene
3-methoxycyanide (25 mL, 225 mmol) were added to a solution
prepared by mixing tert-butanol (300 mL) with potassium
tert-butoxide (18.7 g, 225 mmol). The mixture was heated at
110.degree. C. for 12 hours with stirring until the butanol was
evaporated. Water (200 mL) was then added to the evaporation
residue and the resulting mixture was acidified with acetic acid.
The precipitate was collected by filtration and washed with hot
ethanol to give
7-amino-1,2,3,4-tetrahydro-1-phenylpyrido[2,3-d]pyrim- idin
-2,4-dione (20 g) in a 53% yield.
[0043] Mp:>300.degree. C.
[0044] .sup.1H-NMR (DMSO-d.sub.6) .delta.: 6.27(d,1H,J=9 Hz),
6.89(s,2H), 7.20-7.87(m,5H), 7.88(d,1H,J=9 Hz), 11.26(s,1H)
[0045] IR (KBr): 3038, 1709, 1605, 1417, 789 cm.sup.-1
[0046] MS (EI) m/z: 254[M.sup.+], 211, 77
[0047] (3) Manufacture of
7-amino-1,2,3,4-tetrahydro-1-phenylpyrido[2,3-d]-
pyrimidin-2,4-dione (Compound 1)
[0048] A suspension prepared by adding
7-amino-3-benzyl-1,2,3,4-tetrahydro
-1-phenylpyrido[2,3-d]pyrimidin-2,4-dione (3 g, 12 mmol) and
ammonium sulfate (500 mg) to hexamethyldisilazane (100 mL) was
refluxed at room temperature and atmospheric pressure for 24 hours.
The solvent was evaporated and the residue was dissolved in 100 mL
of L(-)-5,6,7,8-tetrahydrofolic acid (THF). Into this solution was
dropped a solution prepared by adding benzyl bromide (2 mL, 18
mmol) and 1M Bu4NF to THF (14 mL, 14 mmol) under reflux. The mixed
solution was stirred for 2 hours and concentrated in vacuo. The
residue was purified by silica gel column chromatography
(benzene:acetone=5:1) and recrystallized from methanol to give
7-amino-3-benzyl-1,2,3,4-tetrahydro-1-phenylpyrido[2,3-d-
]pyrimidin-2,4-dione [compound 1] (1 g) in a 27% yield.
[0049] Mp:261-262.degree. C.
[0050] .sup.1H-NMR (DMSO-d.sub.6) .delta.: 5.08(s,2H),
6.32(d,1H,J=8 Hz), 6.98(s,2H), 7.22-7.49(m,10H), 7.94(d,1H,J=8
Hz)
[0051] IR (KBr): 3498, 3392, 1695, 1655, 1618, 787, 700
cm.sup.-1
[0052] Anal. Calcd for C.sub.20H.sub.16N.sub.4O.sub.2: C, 69.76; H,
4.68; N, 1 6.27
[0053] Found: C, 69.81; H, 4.77; N, 16.13
[0054] MS (EI) m/z: 344[M.sup.+], 211, 91
[0055]
7-amino-3-(2-chlorobenzyl)-1,2,3,4-tetrahydro-1-phenylpyrido[2,3-d]-
pyrimidin-2,4-dione (compound 2) may also be prepared by utilizing
the above methods of subparagraphs (1) to (3), for example by
employing 2-chlorobenzylbromide as a reactant instead of
benzylbromide.
[0056] (4) Preparation of an Ointment
[0057] The compound 1 or 2, prepared as above, was thoroughly
suspended in white vaseline in a mortar to prepare an ointment
having a final concentration, by weight, of about 0.3%, about 1%,
about 3% or about 10% of compound 1 or 2. As a control, an ointment
consisting only of a base was prepared. As a positive control, an
ointment containing 0.12% of betamethasone valerate (a steroid
preparation) was prepared.
EXAMPLE 2
Preparation of Animal Model for Atopic Dermatitis
[0058] Seven week old BALB/c female (SPF) mice of were selected as
subjects. According to a method of Tanaka, et al. (Allergy, 46,
pages 42-48(1997)), egg white albumin (final concentration: 2
.mu.g/mL) and aluminum hydroxide gel (final concentration: 10
mg/mL) were suspended in physiological saline and 0.5 mL of the
suspension was administered to effect intraperitoneal sensitization
on the first day of the test. On the 14th day of the test, an
additional sensitization was carried out under the same conditions.
Further, on the 28th day of the test, 25 .mu.L of egg albumin (a 20
.mu.g/mL solution in physiological saline) was intradermally
administered to the right ear of a mouse to induce a swelling
reaction.
[0059] This swelling reaction reached its peak at 4 hours after the
challenge by the antigen. Even after 24 to 48 hours, a significant
swelling reaction was noted as compared with the control in which
antigen was merely challenged (not sensitized). In this model, the
reaction at 1-4 hour(s) after the challenge is thought to be a
swelling reaction of an immediate type caused by various mediators
from mast cells and it seems that inflammation cells, such as
eosinophils and lymphocytes, do not participate in the reaction. On
the contrary, the swelling from 24 to 48 hours after the challenge
is thought to be the so-called delayed-type swelling reaction
accompanied by infiltration of eosinophils, neutrophils and
lymphocytes. In addition, a flare-up reaction having a peak at 24
hours after the challenge appeared on the ear of the
antigen-induced site. Like swelling, flare-up is an important
symptom of inflammation. Although the mechanism of the flare-up
reaction is ambiguous, it may be a flare-up reaction of a delayed
type in view of the time course of its expression. As such, this
model animal in which the inflammation of both immediate and
delayed types are induced is appropriate as a model animal for
atopic dermatitis. In the following experiment, the effectiveness
of the therapeutic agent for dermatitis according to the present
invention was tested using the said model animal.
[0060] The following Examples 3 to 6 show the results of an
investigation of the effectiveness and safety of the therapeutic
agent for dermatitis according to the present invention. In the
following Examples, the result is given by a mean value .+-.
standard deviation and a statistical library (Yukms) was used to
evaluate the statistical accuracy of the differences observed
between the subject groups. A Student's two-sided unpaired t-test
was used to analyze the differences between the vehicle-only
control group and the challenge-only group or the untreated group,
while Dunnett's two-sided multiple comparison test or the
Mann-Whitney test was used to analyze the differences between the
control group and the test drug group.
[0061] In Examples 3 to 6 below, the letter "n" refers to the
number of mice or other animals tested in each subject group. A
"challenge" is defined as an exposure of a subject to the antigen
which causes dermatitis. Such a challenge usually does not result
in a swelling reaction or dermatitis unless the challenge follows
an initial sensitization, usually an intradermal exposure, of a
subject to the antigen which causes dermatitis.
EXAMPLE 3
Suppressive Action Against an Antigen-Induced Swelling Reaction in
a Mouse Ear
[0062] Each of a vehicle (control) and a test agent (containing the
compound of the present invention or a positive control) was
applied to the antigen-induced site (right auricle) of a mouse. A
swelling reaction was induced in accordance with Example 2 at 2
hours before and 4 hours after the challenge. After the challenge,
the thickness of the swelling induced ear was measured by a Dial
Thickness Gauge (manufactured by Ozaki Seisakusho). The intensity
of the reaction was expressed in terms of an increase in the
auricle thickness by deducting the thickness (early value) measured
before the challenge from the thickness of the swelling induced
ear. The suppression rate of the test drug for ear swelling was
calculated by the following formula:
Suppression Rate (%)=100.times.[(Ear Thickness Increase of
Vehicle-Only-Control)-(Ear Thickness Increase of the Test
Group)]/[(Ear Thickness Increase of the Vehicle-Only-Control)-(Ear
Thickness Increase of the Challenge-Only-Group)]
[0063] Test results are shown in Tables 1 and 2. The therapeutic
agent for dermatitis according to the present invention
significantly and dose-dependently suppressed the antigen-induced
swelling reaction in a mouse ear.
1 TABLE 1 Ear Thickness Ear Thickness Ear Thickness Ear Thickness
Ear Thickness Increase at 1 Increase at 4 Increase at 8 Increase at
24 Increase at 48 hr after hr after hr after hr after hr after
Challenge .times. Challenge .times. Challenge .times. Challenge
.times. Challenge .times. 0.01 mm 0.01 mm 0.01 mm 0.01 mm 0.01 mm
(Suppression (Suppression (Suppression (Suppression (Suppression n
Rate) Rate) Rate) Rate) Rate) Challenge 2 6.5 .+-. 0.5# 4.0 .+-.
1.0### 3.5 .+-. 1.5### 2.0 .+-. 2.0### 2.0 .+-. 2.0### Only No- 5
9.0 .+-. 1.1 25.2 .+-. 0.9 19.2 .+-. 0.6 14.0 .+-. 1.5 9.2 .+-. 1.6
Treatment- Control Vehicle- 5 9.8 .+-. 0.6 25.2 .+-. 0.6 20.4 .+-.
1.1 17.0 .+-. 1.0 11.2 .+-. 0.9 Only- Control Positive 3 8.3 .+-.
0.7 16.3 .+-. 2.0** 12.0 .+-. 1.2** 5.0 .+-. 1.0** 2.0 .+-. 1.0**
Control (45.5%) (42.0%) (49.7%) (80.0%) (100.0%) (Steroid
Preparation, 0.12% Ointment) Compound 4 6.3 .+-. 1.4* 18.0 .+-.
0.9** 16.0 .+-. 0.4* 9.3 .+-. 1.1** 6.8 .+-. 1.0 1 (106.1%) (34.0%)
(26.0%) (51.3%) (47.8%) (1% Ointment) Compound 4 5.3 .+-. 0.5**
21.3 .+-. 0.9* 15.8 .+-. 1.4* 7.8 .+-. 0.9** 5.3 .+-. 0.9* 1
(136.4%) (18.4%) (27.2%) (61.3%) (64.1%) (3% Ointment) Average .+-.
Standard Error Significant Difference from Base-Only-Control: *P
< 0.05, **P < 0.01 (Dunnett Multiple Comparison) #P <
0.05, ###P < 0.001 (Student's t-Test)
[0064]
2 TABLE 2 Ear Thickness Ear Thickness Increase at 4 hr Increase at
24 hr after Challenge .times. after Challenge .times. 0.01 mm 0.01
mm n (Suppression Rate) (Suppression Rate) Challenge Only 1 2.0 1.0
Vehicle-Only-Control 4 24.5 .+-. 1.7 16.5 .+-. 1.6 Positive Control
(Steroid 4 11.0 .+-. 0.6** 6.3 .+-. 2.4* Preparation, 0.12% (60.0%)
(65.8%) Ointment) Compound 1 4 13.3 .+-. 0.3** 9.3 .+-. 1.7 (3%
Ointment) (49.8%) (46.5%) Compound 1 4 20.3 .+-. 2.4 13.5 .+-. 1.3
(0.3% Ointment) (18.7%) (19.4%) Compound 2 4 19.5 .+-. 3.2 11.8
.+-. 2.6 (1% Ointment) (22.2%) (30.3%) Compound 2 4 19.3 .+-. 2.4
8.0 .+-. 1.4* (3% Ointment) (23.1%) (54.8%) Average .+-. Standard
Error Significant Difference from Base-Only-Control: *P < 0.05,
**P < 0.01 (Dunnett Multiple Comparison)
EXAMPLE 4
Suppressive Action Against an Antigen-Induced Flare-Up Reaction in
a Mouse
[0065] Each of a vehicle (control) and an agent for the test (the
therapeutic agent for dermatitis according to the present invention
or a positive control) was applied to the antigen-induced site
(right auricle) of a mouse to induce a swelling reaction in
accordance with Example 2 at 2 hours before and 4 hours after the
challenge. The right ear was observed by naked eye and the degree
of flare-up was scored according to the following standard:
[0066] 0: no flare-up
[0067] 1: in such a degree that a flare-up was confirmed, the color
is clear red or pale red and the size was small
[0068] 2: between score 3 and score 1
[0069] 3: color of the flare-up was clearly red and the size of the
flare-up was big
[0070] An example of the result is shown in Table 3. The
therapeutic agent for dermatitis according to the present invention
significantly suppressed the antigen-induced flare-up reaction in
mice.
3 TABLE 3 Flare-up Score at Flare-up Score at 24 4 hr after
Challenge hr after Challenge n (Suppression Rate) (Suppression
Rate) Vehicle-Only-Control 23 0.8 .+-. 0 2.5 .+-. 0.2 Positive
Control (Steroid 20 0.6 .+-. 0 1.5 .+-. 0.2*** Preparation, 0.12%
(25.0%) (40.0%) Ointment) Compound 1 22 0.8 .+-. 0 1.7 .+-. 0.2*
(3% Ointment) (0.0%) (32.0%) Average .+-. Standard Error
Significant Difference from Base-Only-Control: *P < 0.05, ***P
< 0.001 (Mann-Whitney test)
EXAMPLE 5
Suppressive Action Against Eosinophil Infiltration Into the
Antigen-Induced Ear of a Mouse
[0071] The degree of easinophil infiltration into the ear of a
mouse was determined by measuring eosinophil peroxidase activity in
a skin tissue homogenate and using the measurement as an index.
Each of the vehicle (control) and the test drug (the therapeutic
agent for dermatitis according to the present invention or a
positive control) was applied to the antigen-induced site (right
auricle) where a swelling reaction was induced according to Example
2 at 2 hours before and 4 hours after the challenge. 24 hours after
the challenge, the right ear was cut, ear tissues were pooled in
one group and a homogenate of the ear tissues was prepared
according to the method of Holliday, et al. (Toxicology, 106, pages
237-242 (1996)). This homogenate was centrifuged at 20,000 rpm for
20 minutes (using KR-20000T, Kubota) and the peroxidase activity in
the supernatant fluid was measured using o-phenylenediamine
dihydrochloride (Sigma) as a substrate. By measuring the absorbance
of radiation having a wavelength of 492 nm, the resulting reaction
curve was substantially linear relative to the dosage of the test
drug and the degree of eosinophil infiltration was compared by
defining the eosinophil infiltration of the control as 100%.
[0072] The therapeutic agent for dermatitis according to the
present invention suppressed eosinophil infiltration into an
antigen-induced mouse ear in a dose-dependent manner. The
eosinophil infiltration suppressing action of a 3% ointment of the
compound 1 occurred in the same degree as that of the steroid
preparation of the positive control.
EXAMPLE 6
Influence of Repeated Application on the Ear Thickness of a Normal
Mouse
[0073] The therapeutic agent for dermatitis according to the
present invention or the positive control (steroid preparation) was
applied twice daily to the right ear of a normal mouse for a total
of nine times and, 2 hours after the final application, the
thickness of the ear was measured.
[0074] Test results are shown in Table 2. As shown in the above
test result, the therapeutic agent for dermatitis according to the
present invention showed excellent ear swelling suppressive action,
flare-up suppressive action and eosinophil infiltration suppressive
action in a 3% ointment. And, even with the repeated application of
a 10% ointment which was considerably in a higher concentration
than the above, no influence on the skin was noted at all. However,
the repeated application of a positive control (steroid
preparation) significantly reduced the auricle thickness.
4 TABLE 4 Ear Thickness .times. 0.01 mm n (Suppression Rate) No
Treatment 6 22.2 .+-. 0.4 Vehicle-Only-Control 6 22.5 .+-. 0.2
Positive Control (Steroid 6 18.3 .+-. 0.5** Preparation, 0.12%
Ointment) Compound 1 6 22.3 .+-. 0.2 (3% Ointment) Compound 1 6
22.3 .+-. 0.5 (10% Ointment) Average .+-. Standard Error
Significant Difference from Base-Only-Control: **P < 0.01
(Dunnett Multiple Comparison)
EXAMPLE 7
Suppressive Action Against Chemotaxis in Guinea Pig Eosinophils in
the Abdominal Cavity
[0075] The suppressive effect of the compounds of the present
invention on the leukotriene B.sub.4 or LTB.sub.4 induced
chemotaxis of guinea pig eosinophils in the abdominal cavity were
investigated using a modified Boyden chamber method. The guinea pig
eosinophils in the abdominal cavity were prepared using Hartley
male guinea pigs according to the conventional method. The
chemotaxis of the eosinophils was induced by 10.sup.-/ M of
leukotriene B.sub.4 or LTB.sub.4 in 37.degree. C. for 2 hours in
the presence or absence of the test drug (the therapeutic agent for
dermatitis of the present invention). After the chemotaxis cells
were fixed and stained using the Giemsa stain, the number of
chemotaxis cells was counted in 10 visual fields of a microscope
(.times.400). The experiments were repeated several times and the
suppression rate of the test drug was calculated by the following
formula:
Suppression Rate (%)=100.times.[(the number of chemotaxis
eosinophils in control group)-(the number of chemotaxis eosinophils
in the test drug group)]/[(the number of chemotaxis eosinophils in
control group)-(the number of spontaneous chemotaxis
eosinophils)]
[0076] The 50% inhibitory concentration (IC.sub.50) was calculated
from the regression line obtained by the least square method based
on the calculated suppression rates.
[0077] As a result of 5 trials, Compound 1 showed a remarkable
suppressive action against eosinophil chemotaxis (IC.sub.50 of
Compound 1: 1.9.+-.0.4 .mu.mol/L).
EXAMPLE 8
Inhibitory Action Against Phosphodiesterase
[0078] The inhibitory action of the compounds of the present
invention against activities of various phosphodiesterase (PDE)
isozymes were investigated using the conventional method. Each of
the inhibitory action of the compounds of the present invention on
the activities of phosphodiesterase type II (PDE II) partially
purified from human platelets, phosphodiesterase type III (PDE II)
partially purified from human platelets, phosphodiesterase type IV
(PDE IV) partially purified from human U937 promonocytic leukemia
cells and phosphodiesterase type V (PDE V) partially purified from
human platelets were measured.
[0079] A 100 .mu.M concentration of the present compound 1
exhibited the following suppression activities against PDE II, III,
IV and V: 6%, 22%, 82% and 15%, respectively. As shown above, the
results clearly indicate a PDE IV selective inhibitory effect.
Furthermore, this PDE IV inhibitory effect was dose dependent.
[0080] As shown in the above animal experiment,
7-amino-3-benzyl-1-phenylp- yrido[2,3-d]pyrimidin-2,4-dione
derivatives according to the present invention significantly and
dose-dependently suppressed the antigen-induced auricle swelling
reaction in mice, which was used as an atopic dermatitis model, and
also significantly suppressed the antigen-induced flare-up reaction
in mice accompanied therewith. The compounds also had a suppressive
action against eosinophil infiltration into the antigen-induced
auricle of a mouse. Accordingly, the
7-amino-3-benzyl-1-phenylpyrido[2,3-d]pyrimidin-2,4-dione
derivatives of the present invention are useful as a therapeutic
agent for dermatitis such as atopic dermatitis, eczema and contact
dermatitis.
[0081] Further, a direct adverse reaction in the skin, such as
thinning of the skin noted in the case of steroid preparations, was
not observed at all in the therapeutic agent for dermatitis
according to the present invention. Said therapeutic agent was very
safe.
[0082] As mentioned hereinabove, the therapeutic agent for
dermatitis according to the present invention is useful as a
therapeutic agent for atopic dermatitis and dermatitis, and is a
very safe pharmaceutical agent causing little adverse reaction in
subjects treated with said agent. Accordingly, the therapeutic
agent for dermatitis according to the present invention
unexpectedly solves the technical problems in the art and is highly
useful as an agent for which there has been a brisk demand from
patients and the medical field.
* * * * *