U.S. patent application number 10/478432 was filed with the patent office on 2004-09-16 for therapeutic composition for repairing chondropathy.
Invention is credited to Horikiri, Yuji, Sakurai, Naoki, Takagi, Toshiki, Takigawa, Masaharu, Tamura, Takashi, Yanaka, Noriyuki.
Application Number | 20040180900 10/478432 |
Document ID | / |
Family ID | 18998464 |
Filed Date | 2004-09-16 |
United States Patent
Application |
20040180900 |
Kind Code |
A1 |
Takigawa, Masaharu ; et
al. |
September 16, 2004 |
Therapeutic composition for repairing chondropathy
Abstract
A composition for regenerative treatment of cartilage disease,
which comprises a PDE4 inhibitor as an active ingredient,
specifically a composition comprising a PDE4 inhibitor and a
biocompatible and biodegradable polymer is provided, which
composition, when formulated into a form suited to administer
locally to affected cartilage region, such as microsphere
preparation, can provide a pharmaceutical composition showing an
excellent effect in regenerative treatment of cartilage.
Inventors: |
Takigawa, Masaharu;
(Okayama-shi, JP) ; Sakurai, Naoki; (Solona Beach,
CA) ; Takagi, Toshiki; (Itami-shi, JP) ;
Yanaka, Noriyuki; (Higashihiroshima-shi, JP) ;
Horikiri, Yuji; (Kawanishi-shi, JP) ; Tamura,
Takashi; (Amagasaki-shi, JP) |
Correspondence
Address: |
FINNEGAN, HENDERSON, FARABOW, GARRETT & DUNNER
LLP
1300 I STREET, NW
WASHINGTON
DC
20005
US
|
Family ID: |
18998464 |
Appl. No.: |
10/478432 |
Filed: |
November 21, 2003 |
PCT Filed: |
May 22, 2002 |
PCT NO: |
PCT/JP02/04930 |
Current U.S.
Class: |
514/252.16 ;
424/489; 514/262.1 |
Current CPC
Class: |
A61K 31/381 20130101;
A61K 31/4709 20130101; A61K 31/40 20130101; A61K 31/4425 20130101;
A61K 31/501 20130101; A61K 31/5025 20130101; A61P 43/00 20180101;
A61K 31/4453 20130101; A61K 31/502 20130101; A61K 31/4409 20130101;
A61P 19/10 20180101; A61K 31/5377 20130101; A61K 9/1647 20130101;
A61K 31/50 20130101; A61P 19/02 20180101; A61K 31/44 20130101; A61K
31/423 20130101; A61K 31/551 20130101; A61K 31/277 20130101; A61K
31/27 20130101; A61K 31/42 20130101; A61P 19/00 20180101; A61K
31/4985 20130101; A61P 19/08 20180101; A61K 31/4418 20130101; A61K
31/522 20130101; A61K 31/343 20130101; A61K 31/519 20130101; A61K
31/4166 20130101 |
Class at
Publication: |
514/252.16 ;
514/262.1; 424/489 |
International
Class: |
A61K 031/519; A61K
009/14 |
Foreign Application Data
Date |
Code |
Application Number |
May 23, 2001 |
JP |
2001-154048 |
Claims
1. A composition for regenerative treatment of cartilage disease,
which comprises a PDE4 inhibitor as an active ingredient.
2. The composition according to claim 1, which is for local
administration and is prepared so as to release the PDE4 inhibitor
gradually at the affected region.
3. The composition according to claim 2, which comprises a
biocompatible and biodegradable polymer.
4. The composition of any one of claims 1-3, which is for
regenerative treatment of osteoarthrosis.
5. The composition according to claim 3, wherein the biocompatible
and biodegradable polymer is water-insoluble.
6. The composition according to claim 5, which is in the form of
microsphere preparation.
7. The composition according to claim 6, wherein the particle size
of a microsphere is 0.1-150 .mu.m.
8. The composition according to claim 6 or 7, wherein the PDE4
inhibitor content is 0.0001-80% by weight.
9. The composition according to any one of claims 5 to 8, wherein
the water-insoluble biocompatible and biodegradable polymer is a
hydroxy fatty acid polyester.
10. The composition according to claim 9, wherein the
water-insoluble biocompatible and biodegradable polymer is one or
more polymers selected from the group consisting of poly lactic
acid, lactic acid-glycolic acid copolymer and 2-hydroxybutyric
acid-glycolic acid copolymer.
11. The composition according to claim 9 or 10, wherein the
water-insoluble biocompatible and biodegradable polymer has an
average molecular weight of 2000-800000.
12. The composition according to any one of claims 6 to 11, which
is an injectable microsphere preparation prepared by dispersing at
a concentration of 0.0001-1000 mg/ml microspheres in an aqueous
solution containing a dispersant.
13. The composition according to claim 12, which comprises a
dispersant at a concentration of 0.01-2% by weight.
14. The composition according to claim 12 or 13, wherein the
dispersant is one or more selected from the group consisting of
polyoxyethylene sorbitan fatty acid ester, polyethylene castor oil,
carboxymethyl cellulose sodium, sodium alginate, dextran and sodium
hyaluronate.
15. The composition according to any one of claims 1 to 14, wherein
the PDE4 inhibitor is a selective PDE4 inhibitor.
16. The composition according to any one of claims 1 to 15, wherein
IC.sub.50 of the PDE4 inhibitor is less than 100 nM.
17. The composition according to any one of claims 1 to 16, wherein
the PDE4 inhibitor is a compound having a partial structure having
PDE4 inhibitory activity as follows: (A) naphthalene or an
analogous chemical structure thereof; or (B)
3-cyclopentyloxy-4-methoxyphenyl or an analogous chemical structure
thereof.
18. The composition according to any one of claims 1 to 17, wherein
the PDE4 inhibitor is a compound having a partial structure of
naphthalene or isoquinoline skeleton having PDE4 inhibitory
activity or a pharmaceutically acceptable salt thereof.
19. The composition according to claims 18, wherein the PDE4
inhibitor is
2,3-bis(hydroxymethyl)-6,7-diethoxy-1-[1-(2-metoxyethyl)-2-oxo-4-pyridyl]-
naphthalene or
2,3-bis(hydroxymethyl)-6,7-diethoxy-1-[2-(4-(3-pyridyl)-1(2-
H)-phthaladinon-2-yl)-4-pyridyl]naphthalene or a pharmaceutically
acceptable salt thereof.
20. A composition for preparing an injectable preparation, which is
obtainable by suspending microspheres set forth in any one of
claims 6-11 into an aqueous solution containing an aggregation
inhibitor, and lyophilizing the resultant suspension.
Description
TECHNICAL FIELD
[0001] The present invention relates to a composition for
regenerative treatment of cartilage disease, specifically, to a
pharmaceutical composition for regenerative treatment of cartilage
disease such as osteoarthrosis (degenerative joint disease),
chondrodystrophy, degenerative discopathy or meniscus injury.
BACKGROUND ART
[0002] Cartilage is considerably elastic that plays a role in the
construction of skeleton together with bone and the protection of
internal organs. Cartilage tissue consists of chondrocytes and
cartilage matrix surrounding the same.
[0003] Cartilage is formed by mesenchyme-originated chondroblasts
which cells produce matrix in circumference in the process of cell
division and growth. The cartilage matrix consists of amorphous
matrix and fibrous components, and is classified into the following
groups according to the ratio of components: (1) hyaline cartilage
(articular cartilage, costicartilage, thyroid cartilage etc.); (2)
fibrocartilage (discus intervertebrali, pubic symphysis etc.); and
(3) elastic cartilage (pharynx lid cartilage, cartilage of acoustic
meatus, auricular cartilage, etc.) IGAKU-DAIJITEN, 18th edition,
published by Nanzando, pp. 1542.
[0004] The main components of cartilage matrix are proteoglycan and
collagen (Type II, Type IX and the like) It is known that
proteoglycan participates in the imbibition (swelling) nature
peculiar to cartilage tissue, and collagen in the rigidity of
cartilage against the tension and shearing force.
[0005] In proteoglycan of cartilage matrix, it is considered that
glucosaminoglycans such as chondroitin sulfate, keratan sulfate are
connected with a core protein of about 220,000 molecular weight to
form macromolecules, wherein glucosaminoglycans hydrates many water
molecules, which contributes to the imbibition nature of cartilage.
The Bone, Vol. 4, pp. 8 (1994).
[0006] Articular cartilage has a calcification layer at the
transmigration region with bone tissue, and, after the completion
of growth, nutrients are supplied to chondrocytes from synovial
fluid and are hardly supplied directly from blood. In addition,
articular cartilage is formed from hyaline cartilage of high cell
differentiation degree, and hence is a sensitive organ with
extremely low regenerative ability.
[0007] The surface of articular cartilage is covered by highly
viscous synovial fluid, and by virtue of lubrication mechanism of
lubricant comprising as a principal component hyaluronic
acid-protein complex, the smooth joint kinematics is maintained.
However, it is considered that there is so-called durability in
articular cartilage, and alteration of joints with aging is
unavoidable physiological phenomenon.
[0008] Examples of known diseases caused by cartilage disorder
include osteoarthrosis, chondrodystrophy, degenerative discopathy
or meniscus injury.
[0009] Among them, osteoarthrosis is a disease wherein a
proliferative change of bone and articular cartilage occurs on the
basis of a regressive change of tissue constituting a joint,
mainly, articular cartilage, finally leading to a remarkable
morphological change of the joint, which disease has markedly
increased with the aging of population. In particular, the knee
joint anthropathy can prevent patients from maintaining the
standing position or walking normally as the pathology progresses,
and lead to the significant decrease of their ADL (Ability of Daily
Life) which possibly results in a bedridden condition.
[0010] Treatment of osteoarthrosis can be classified mainly into
conservative treatment and surgical therapy. Conservative treatment
is carried out by the following methods, for example, (1)
administration of non-steroidal antiinflammatory analgesic; (2)
thermotherapy; (3) control of weight; (4) therapy with braces; (5)
intra-articular infusion of steroidal antiinflammatory analgesic;
(6) intra-articular infusion of hyaluronate formulation. In cases
wherein conservative treatment is ineffective, or the disease is in
progressed or terminal stage, surgical therapy is conducted by (a)
arthroscopic irrigation surgery; (b) high tibial osteotomy or (c)
artificial joint replacement, and the like. Senility and Disorder,
Vol. 10, 2nd. issue, pp. 61-69, (1997) & 6th issue, pp. 66-77
(1997).
[0011] There are various compounds having PDE4 inhibitory activity,
which can suppress the release of inflammatory mediator by
inhibiting the PDE4 activity. J. Mol. Cell. Cardiol., 12
(Suppl.II), S61 (1989).
[0012] It is described that a compound having PDE4 inhibitory
activity suppresses the production of TNF-.alpha. which is a
cytokine released from mononuclear phagocytes in response to
immunostimulants, and is useful in treatment of various
inflammatory diseases caused by TNF-.alpha.. JP 2000-503678A, JP
2000-502724A, JP 2000-510105A, JP 2000-514804A, 2000-502350A, JP
2000-501741A, and the like.
[0013] However, it has not been known that PDE4 inhibitor is
effective for reparative treatment of cartilage diseases at
all.
DISCLOSURE OF INVENTION
[0014] As stated above, cartilage is known to have extremely low
regenerative ability, and it was considered that, once damaged, the
regeneration thereof is almost impossible. The conventional
pharmacotherapy was only conservative treatment which restrains the
progressing of disorder. Accordingly, it has long been demanded the
development of pharmacotherapy and/or pharmaceutical agent that
enables to conduct regenerative treatment of cartilage
diseases.
[0015] The present inventors have first found that PDE4 is produced
by chondrocytes and then compounds having PDE4 inhibitory activity
show activity on cartilage diseases. The inventors have intensively
studied and found that the said PDE4 inhibitors are useful in
regenerative treatment of cartilage diseases, and established the
present invention.
[0016] The present invention provides a composition for
regenerative treatment of cartilage disease, which comprises a PDE4
inhibitor as an active ingredient. In particular, the present
invention provides a pharmaceutical preparation suited to
administer locally to the site of cartilage disease, specifically,
a composition for regenerative treatment of cartilage disease in
the form of microsphere preparation.
BRIEF DESCRIPTION OF DRAWINGS
[0017] FIG. 1 is a graph showing the cAMP hydrolyzing activity in
each fraction obtained by fractionating rabbit articular
chondrocyte extract by Mono Q Sepharose column chromatography, in
the presence of Compound (44) .largecircle.) and absence of
Compound (44) (.circle-solid.).
[0018] FIG. 2 is a copy of microphotograph showing the results of
observation under microscope of regeneration of old rabbit
articular cartilage in the presence of microsphere containing
Compound (1) or free of Compound (1).
[0019] FIG. 3 is a graph showing the cAMP or cGMP hydrolyzing
activity in each fraction obtained by fractionating human articular
chondrocyte extract by Mono Q Sepharose column chromatography.
[0020] FIG. 4 is a graph showing the inhibitory activity
(IC.sub.50) of PDE4 inhibitor toward fractions 28-30 that showed
potent cAMP hydrolyzing activity as demonstrated in FIG. 3.
[0021] FIG. 5 is a graph showing the in vitro drug elution
characteristics of microspheres obtained in Examples 1, 2 and
3.
[0022] FIG. 6 is a graph showing the time-course of plasma
concentration of Compound (1) administered to a rat intravenously.
Data are shown by mean .+-. standard deviation (n=3).
[0023] FIG. 7 is a graph showing the time-course of plasma
concentration of an active ingredient following the subcutaneous
injection of microsphere dispersion obtained in Example 1-(5),
2-(2) or 3-(2) into a rat. Data are shown by mean .+-. standard
deviation (n=5).
[0024] FIG. 8 is a graph showing the time-course of Compound (1)
remaining in the preparation following the subcutaneous injection
of microsphere dispersion obtained in Example 2-(2) into a rat.
Data are shown by mean.+-.standard deviation (n=5).
[0025] FIG. 9 is a graph showing the time-course of Compound (2)
remaining in the preparation following the subcutaneous injection
of microsphere dispersion obtained in Example 6-(5) or 7-(2) into a
rat. Data are shown by mean.+-.standard deviation (n=4).
BEST MODE FOR CARRYING OUT THE INVENTION
[0026] The composition of the present invention for regenerative
treatment of cartilage disease can enhance the expression of
cartilage matrix protein encoding gene and thereby showing superior
matrix production promoting effect on cartilage especially on
articular cartilage that has extremely low regenerative activity,
and cure cartilage diseases through the regeneration of
cartilage.
[0027] As herein used, the term "regenerative treatment of
cartilage disease" refers to treatment not only for arresting the
progress of cartilage disease but also for restoring a cartilage
undergone deformation and/or detrition due to illness, lesion, or
the like to the original state.
[0028] The pharmaceutical composition of the present invention can
be prepared by combining a PDE4 inhibitor as an active ingredient
and a conventional pharmaceutically acceptable excipient or a
diluting agent therefor. Preferred pharmaceutical composition is a
sustained release composition for local administration, which
contains a PDE4 inhibitor(s) and a biocompatible and biodegradable
polymer(s). The composition for local administration is preferably
in the form of depot formulation, and more preferably in the form
of microsphere, which microsphere can be formulated as an
injectable preparation.
[0029] Examples of PDE4 inhibitor usable as an active ingredient of
pharmaceutical compositions of the present invention include all
the compounds having PDE4 inhibitory activity, for example, those
described in JP 05-229987A (1993), JP 09-59255A (1997), JP
10-226685A (1998), EP 158380, WO/94/25437, U.S. Pat. No. 5,223,504,
WO/95/4045, EP 497564, EP 569414, EP 623607, EP 163965, U.S. Pat.
No. 5,605,914, WO/95/35282, WO/96/215, U.S. Pat. No. 5,804,588,
U.S. Pat. No. 5,552,438, WO/93/9118, WO/96/31485, EP 459505,
WO/97/22585, EP 738715, WO/91/16314, WO/96/218, WO/97/18208, EP
158380, WO/99/50270, EP 260817, WO/98/11113, WO/94/22852, EP
432856, U.S. Pat. No. 4193926, WO/98/13348, WO/96/6843, JP
2000-503678A (WO/98/14432), JP 2000-502724A (WO/98/9961), JP
2000-510105A (WO/97/40032), JP 2000-514804A (WO/98/2440), JP
2000-502350A (WO/97/23457), JP 2000-501741A (WO/97/2585), and the
like.
[0030] PDE can be classified into PDE1-5 according to the teaching
of "Trends in Pharmacological Sciences, vol. 11, pp. 150-155", and
PDE4 inhibitors suitable for the present composition for
regenerative treatment of cartilage disease are preferably
selective to PDE4 with higher inhibitory activity against PDE4
compared to others (PDE1-3, 5), more preferably have 10 times or
more inhibitory activity on PDE4 than on the other PDEs. The
inhibitory activity of such PDE4 inhibitor on PDE4 is particularly
preferably 50 times or more, and yet more preferably 100 times or
more of that on the other PDEs.
[0031] Preferable PDE4 inhibitors are compounds of which IC.sub.50
of PDE4 inhibitory activity is 0.1-1000 nM, preferably 0.1-100 nM,
more preferably less than 100 nM, when determined by a method
described in "Advances in Cyclic Nucleotide Research", vol. 10, pp.
69-92, 1979, Raven Press.
[0032] Specific examples of selective PDE4 inhibitors include
Compounds (1) to (57) represented by the following formulas or
pharmaceutically acceptable salts thereof. 123456789101112
[0033] The compounds having PDE4 inhibitory activity can be
classified into (A) to (D) below according to the chemical
structure, and a PDE4 inhibitor for the present invention can be
selected from these compounds appropriately; however, preferred
compounds belong to (A) and (B), in particular, (A).
[0034] (A) Compounds having naphthalene skeleton or a partial
structure analogous thereto [e.g., Compounds (1), (2), (38), (47),
and (52) to (57)];
[0035] (B) Compounds having 3-cyclopentyloxy-4-methoxyphenyl
structure or a partial structure analogous thereto [e.g., Compounds
(6), (9), (11), (12), (14), (17), (19), (20), (21), (24), (25),
(26), (27), (33), (34), (35), (39), (40), (44), (49), (50) and
(51)];
[0036] (C) Compounds having a xanthine skeleton or a partial
structure analogous thereto [e.g., Compounds (5), (7), (28), (29),
(30), (31), (32), (36), (37), (41), (43) and (46)]; and
[0037] (D) Compounds having a different structure from those
described in (A) to (C) above [e.g., Compounds (3), (4), (8), (10),
(13), (15), (16), (18), (22), (23), (42), (45) and (48)].
[0038] Examples of compounds of group (A) include those shown by
the following formulas (I) to (III) and pharmacologically
acceptable salts thereof. 13
[0039] Wherein R.sup.1 and R.sup.2 are the same or different and
each a hydrogen atom, a hydroxyl group, a cyclo-lower alkyloxy
group, or an optionally substituted lower alkoxy group, or bind
together at the ends to form a lower alkylenedioxy group;
[0040] R.sup.3 is an optionally substituted 6-membered
nitrogen-containing heterocyclic group; and
[0041] --OR.sup.4 and --OR.sup.5 are the same or different and each
an optionally protected hydroxyl group. JP 05-229987A, (1993).
14
[0042] Wherein R.sup.1' and R.sup.2' are the same or different and
each a hydrogen atom or an optionally protected hydroxyl group;
[0043] either of R.sup.3' and R.sup.4' is an optionally protected
hydroxy-substituted methyl group and the other is a hydrogen atom,
a lower alkyl group or an optionally protected hydroxy-substituted
methyl group; and
[0044] R.sup.5' and R.sup.6' are the same or different and each a
hydrogen atom, an optionally substituted lower alkyl group, an
optionally substituted phenyl group or an optionally protected
amino group, or bind together at the ends and form in association
with the adjacent nitrogen atom an optionally substituted
heterocyclic group. JP-09-59255A, (1993). 15
[0045] Wherein A is a group selected from those shown by the
formulas: 16
[0046] wherein R.sup.1" and R.sup.2" are the same or different and
each a hydrogen atom or an optionally protected hydroxyl group;
[0047] R.sup.31 is an optionally protected hydroxymethyl group;
R.sup.32 is a hydrogen atom, a lower alkyl group or an optionally
protected hydroxymethyl group; R.sup.33 is an optionally
substituted lower alkyl group; R.sup.41 is an optionally protected
hydroxymethyl group; R.sup.42 is an optionally protected
hydroxymethyl group; the dotted line represents the presence or
absence of a double bond; and
[0048] R.sup.5" and R.sup.6" are the same or different and each a
hydrogen atom or an optionally protected amino group, or bind
together at the ends and form in association with the adjacent
nitrogen atom an optionally substituted heterocyclic group.
JP-10-226685A (1998).
[0049] As a PDE4 inhibitor which is an active ingredient of the
present composition for regenerative treatment of cartilage
disease, among group (A), compounds having naphthalene or
isoquinoline skeleton and pharmaceutically acceptable salts thereof
are more preferred, and Compounds (1) and (2) and their
pharmaceutically acceptable salts are still more preferred.
[0050] Since PDE4 inhibitors may cause vomiting or gastric acid
secretion depending on dosage when acted systemically (Cellular
Signaling, 9 (3-4), pp. 227-236 (1997)), the present composition
for regenerative treatment of cartilage disease is preferably
applied locally to a vicinity of affected region (especially,
vicinity of articular cartilage), so that the drug concentration in
the systemic blood does not increase but the one at the affected
cartilage region is maintained. To establish this purpose, it is
preferred to formulate the composition into a sustained release
form which can advantageously reduce the frequency of
administration and also decrease the burden of patients.
[0051] Examples of preferred embodiments of the present composition
include depot preparations which gradually release a drug when
administered locally (e.g., pellet preparation, gel preparation,
matrix preparation, microsphere preparation, a sustained release
preparation obtained by adding a drug into an aqueous solution of a
biocompatible and biodegradable polymer, a preparation which is
designed to be a liquid at the time of administration and to form a
gel in a living body after administration, a preparation embedded
in various bases which are reported to be generally used in the
field of orthopedics, and the like.)
[0052] Examples of pellet preparations include a long-term
sustained release preparation obtainable by compressing a drug and
fine particles of lactic acid-glycolic acid copolymer of which
terminal carboxyl group is esterified by an alcohol, and the like.
(JP2001-187749A) Examples of gel preparations include those
obtained by dissolving into a phosphate buffer a drug and
hyaluronic acid which is chemically bound to polyethylene glycol
(Journal of Controlled Release, 59 (1999) pp. 77-86), and the
like.
[0053] Examples of matrix preparations comprising a drug include
those obtained by impregnating a drug into granular material of
collagen or fibrous membrane preparation, or by adding a drug to a
granular material of collagen or a reaction mixture for preparing a
fibrous membrane preparation, and the like (JP10-182499A (1998),
JP06-305983 (1994)).
[0054] Examples of a sustained release preparation obtained by
adding a drug into an aqueous solution of a biocompatible and
biodegradable polymer include those obtained by adding a drug into
an aqueous sodium hyaluronate solution, and the like.
[0055] Examples of a preparation designed to be a liquid at the
time of administration and to form a gel in a living body after
administration include those wherein a drug and a lactic
acid-glycolic acid copolymer are dissolved in
N-methyl-2-pyrrolidone (Journal of Controlled Release, 33 (1995)
pp. 237-243), or a preparation comprising a drug and a polymer that
exists as an solution at low temperature but forms a gel at body
temperature, such as a block co-polymer of lactic acid-glycolic
acid copolymer and polyethylene glycol and the like (ibid.,
27(1993), 139-147).
[0056] Examples of a preparation embedded in various bases which
are reported to be generally used in the field of orthopaedics
include those prepared by mixing a drug and a base (e.g.,
water-insoluble biocompatible and biodegradable polymer, polymethyl
methacrylate, hydroxyapatite, tricalcium phosphate or the like).
Biomaterials, vol. 21, pp. 2405-2412 (2000); and International
Journal of Pharmaceutics, vol. 206, pp. 1-12 (2000).
[0057] Preparations for local administration that release an
effective amount of PDE4 inhibitor gradually to a vicinity of
cartilage region with a lesion(s) (especially, vicinity of
articular cartilage) are preferred in the respect that the
administration frequency during the term required for regenerative
treatment of cartilage disease can be reduced.
[0058] Among depot preparations, in the case of microspheres
feasible for local administration by injection, the particle size
of such microspheres is preferably in the range suitable for
passing a needle, more preferably 0.01-150 .mu.m, particularly
preferably 0.1-100 .mu.m in the respect that the irritation at the
affection site can be reduced.
[0059] Since the present composition for regenerative treatment of
cartilage disease, which comprises a PDE4 inhibitor as an active
ingredient, is administered locally to a vicinity of cartilage
region with a lesion(s) (especially, vicinity of articular
cartilage), it would be preferable to make the dosage small.
Accordingly, the PDE4 inhibitor content in the composition such as
microsphere preparation can be preferably 0.0001-80% by weight,
more preferably 0.001-50% by weight, and further more preferably
0.01-50% by weight. The dose of a PDE4 inhibitor as an active
ingredient may vary depending on the kind of PDE4 inhibitor to be
used, the weight, age, conditions of the subject or a site to be
applied and is generally determined by a physician; however, for
local administration, the dose can usually be in the range of from
1 ng to 1 g per affected region.
[0060] The composition for regenerative treatment of cartilage
disease of the present invention can be prepared in a conventional
manner using a PDE4 inhibitor and a pharmaceutically acceptable
excipient or a carrier therefor. Preferred composition can be
prepared by combining a PDE4 inhibitor and a biocompatible and
biodegradable polymer.
[0061] Among them, the water-insoluble biocompatible and
biodegradable polymer is a water-insoluble biocompatible and
biodegradable polymer that requires at least 1000 ml of water to
dissolve 1 g of the polymer at 25.degree. C., and specific example
include hydroxy fatty acid polyesters and derivatives thereof (for
example, poly lactic acid, poly glycolic acid, poly citric acid,
poly malic acid, poly-.beta.-hydroxybutyric acid, ring-opening
polymerized .epsilon.-caprolactones, lactic acid-glycolic acid
copolymer, 2-hydroxybutyric acid-glycolic acid copolymer, block
copolymer of poly lactic acid and polyethylene glycol, block
copolymer of poly glycolic acid and polyethylene glycol, and block
copolymer of lactic acid-glycolic acid copolymer and polyethylene
glycol, etc.), polymers of alkyl .alpha.-cyanoacrylates (e.g.,
polybutyl-2-cyanoacrylate, etc.), polyalkylene oxalate (e.g.,
polytrimethylene oxalate, polytetramethylene oxalate, etc.),
polyortho-esters, polycarbonates (e.g., polyethylene carbonate,
polyethylenepropylene carbonate, etc.), polyortho-carbonates,
polyamino acids (e.g., poly-.gamma.-L-alanine,
poly-.gamma.-benzyl-L-glut- amic acid,
poly-.gamma.-methyl-L-glutamic acid, etc.), hyaluronic acid esters.
One or more of these polymers can be used. Other biocompatible and
biodegradable polymers include sodium hyaluronate, chondroitin
sulfate, collagen, gelatin, fibrin, and the like.
[0062] Among the water insoluble biocompatible and biodegradable
polymers above, hydroxy fatty acid polyesters are particularly
preferred. Above all, those of which average molecular weight
ranging in between 2000 and about 800000 are more preferred, those
ranging in between 2000 and about 200000 are especially preferred
and those ranging in between 5000 and 50000 are most preferred.
[0063] In addition, among the hydroxy fatty acid polyesters above,
poly lactic acid, lactic acid-glycolic acid copolymer and
2-hydroxybutyric acid-glycolic acid copolymer are more preferred.
The molar ratio of lactic acid and glycolic acid in a lactic
acid-glycolic acid copolymer is preferably 90:10 to 30:70, more
preferably 80:20 to 40:60, and the molar ratio of 2-hydroxybutyric
acid and glycolic acid in a .sup.2-hydroxybutyric acid-glycolic
acid copolymer is preferably 90:10 to 30:70, more preferably 80:20
to 40:60.
[0064] When formulating a PDE4 inhibitor above into a depot
preparation, it can be carried out appropriately depending on the
intended embodiment, optionally after pulverizing a PDE4 inhibitor
if necessary.
[0065] Pulverization of PDE4 inhibitor can be carried out using any
one of conventional methods for producing fine particles including
mechanical pulverization methods such as jet mill, hammer mill,
convolution ball mill, jar ball mill, beads mill, shaker mill, rod
mill and tube mill pulverizations, or so-called crystallization
method wherein a drug is first dissolved in a solvent and then
recrystallized by adjusting pH, changing temperature, or altering
the constitution of solvent, and recovering the particles by
centrifugation, filtration, or the like.
[0066] When preparing the above-mentioned various types of
formulations of the present pharmaceutical composition, any
appropriate process can be used depending on the selected PDE4
inhibitor.
[0067] For example, microsphere preparation can be prepared by the
following methods. In case that a salt of a PDE4 inhibitor shows
low incorporation rate into a microsphere, it may be converted into
corresponding free form using an acid or a base prior to the
preparation of microspheres.
[0068] (1) In-Water Drying Method
[0069] In this method, a drug is added to a solution of
water-insoluble biocompatible and biodegradable polymer in a
water-immiscible organic solvent of which boiling point is lower
than water (water-insoluble polymer solution), and the resultant
organic phase is dispersed into an aqueous phase to give an O/W
emulsion, which is followed by removal of the organic solvent. This
method can be conducted in a manner similar to those described in,
for example, JP 56-19324B (1981), JP 63-91325A (1988), JP
08-151321A (1996), Kajeev Jain et al., "Controlled Drug Delivery by
Biodegradable Poly (Ester) Devices: Different Preparative
Approaches", Drug Development and Industrial Pharmacy, vol. 24(8),
pp. 703-727, 1998, JP 60-100516A (1985), JP 62-201816A (1987), JP
09-221417A (1997) and JP 06-211648A (1994).
[0070] (2) Phase Separation Method
[0071] In this method, into a solution of water-insoluble
biocompatible and biodegradable polymer in an organic solvent is
dissolved or dispersed a drug, or is dispersed an aqueous solution
of the drug. A hardening agent is then added gradually with
stirring to obtain solid precipitations. This method can be
conducted in a manner similar to those described in, for example,
JP 60-67417A (1985), U.S. Pat. No. 5503851, U.S. Pat. No. 5000886,
Eur. J. Pharm. Biopharm. vol. 42 (1), pp.16-24 (1996) and the
forecited Jain et al. (ibid.)
[0072] (3) Spray Drying Method
[0073] In this method, to a solution of water insoluble
biocompatible and biodegradable polymer in an organic solvent is
dissolved or dispersed a drug, or is dispersed an aqueous solution
of the drug. The resultant solution or dispersion is then sprayed
via a nozzle into a drying chamber of a spray drier to volatilize
the organic solvent in the fine droplets in a very short time. This
method can be conducted in a manner similar to those described in,
for example, JP 01-155942A (1989), JP 05-194200A (1993), JP
05-70363A (1993), JP 08-151321A (1996), JP 09-221417A (1997), U.S.
Pat. No. 5,922,253, "Spray Drying Handbook" (John Wiley & Sons,
New York 1984), Partick B. Deasy, "Microcapsulation and Related
Drug Processes" (Marcel Dekker, Inc., New York 1984) and the
forecited Jain et al. (ibid), and the like.
[0074] (4) Solvent Diffusion Method
[0075] In this method, a solution of a drug and a water insoluble
biocompatible and biodegradable polymer in a water miscible organic
solvent is added to an aqueous solution of protective colloid,
followed by emulsification with stirring to yield fine particles.
This method can be conducted in a manner similar to those described
in, for example, JP 05-58882A (1993), JP 09-110678A (1997) and
International Journal of Pharmaceutics, vol. 187, pp. 143-152
(1999).
[0076] In the aforementioned "In-Water Drying Method", different
preparation processes may be employed depending on the type of
organic phase though they all can be conducted in a conventional
manner. Examples of organic phase include the followings.
[0077] (a) An organic phase wherein a drug is directly dissolved or
dispersed in a solution of a water-insoluble, biocompatible and
biodegradable polymer. This, when dispersed in an aqueous phase,
gives O/W emulsion (JP 56-19324B (1981), JP 63-91325A (1988), JP
06-32732A (1994), JP 08-151321A (1996), JP 06-32732A (1994), and
the forecited Jain, etc.)
[0078] (b) An organic phase which is W/O emulsion wherein an
aqueous solution of a drug is dispersed in a solution of a
water-insoluble, biocompatible and biodegradable polymer. The W/O
emulsion, when dispersed in an aqueous phase, gives (W/O)/W
emulsion (JP 60-100516A (1985), JP 62-201816A (1987), JP 09-221417A
(1997), and the forecited Jain, etc.)
[0079] (c) An organic phase which is O/O emulsion, which uses two
or more water-insoluble, biocompatible and biodegradable polymers,
wherein a drug is dissolved or dispersed in a polymer solution that
is dispersed in the other(s). The O/O emulsion, when dispersed in
an aqueous phase, gives (O/O)/W emulsion (JP 06-211648A
(1994)).
[0080] By using any of the organic phases above, the emulsification
can be achieved by a conventional method, for example, the
intermittent shaking method, the method using a mixer such as a
propeller shaker or a turbine shaker, the colloidal mill method,
the homogenizer method and the ultrasonication method.
[0081] Examples of organic solvent usable in these methods include
halogenated hydrocarbons (methylene chloride, chloroform, carbon
tetrachloride, chloroethane, dichloroethane, trichloroethane,
etc.), aliphatic esters (ethyl acetate, butyl acetate, etc.),
aromatic hydrocarbons (benzene, etc.), aliphatic hydrocarbons
(n-hexane, n-pentane, cyclohexane, etc.), ketones (methylethyl
ketone, etc.), ethers (diethyl ether, diisopropyl ether, methyl
isobutyl ether, etc.)
[0082] In preparation of emulsion above, an emulsifier may be added
to an aqueous phase to stabilize emulsion, which emulsifier
includes, for example, anionic surfactants (sodium oleate, sodium
stearate, sodium lauryl sulfate, etc.), nonionic surfactants
{polyoxyethylene sorbitan fatty acid ester [Tween80, Tween 60
(Nikko Chemicals, Co., Ltd.)], polyethylene castor oil derivatives
[HCO-60, HCO-50 (Nikko Chemicals, Co., Ltd.)],
polyvinylpyrrolidone, polyvinyl alcohol, carboxymethyl cellulose,
methyl cellulose, lecithin, gelatin, etc.
[0083] Further, when one or more other ingredients are incorporated
in addition to PDE4 inhibitor, the former can be preferably added
to the organic phase at the time of preparation of O/W emulsion. To
obtain a microsphere preparation with an elevated concentration of
medicinal ingredient, it is necessary to prepare an organic phase
containing an active ingredient at high concentration. For this
purpose, an osmoregulatory agent may be included in an aqueous
phase to prevent the outflow of an active ingredient into an
aqueous phase (JP 2608245).
[0084] The O/W emulsion obtained in the above-mentioned manner is
then subjected to in-water-drying to remove organic solvent present
in emulsion to give microspheres.
[0085] Organic solvent can be removed from emulsion in a
conventional manner such as heating, placing under reduced
pressure, blowing air, or the like, and for example, a method where
a solvent is distilled off in an open system (JP 56-19324B (1981),
JP 63-91325A (1988), JP 08-151321A (1996), JP. 06-211648A (1994))
or in a closed system (JP 09-221418A (1997)) can be employed. In
addition, a method where a solvent is extracted and removed by
means of a large quantity of outside water phase (JP-2582186) can
also be used.
[0086] Further, the following methods can be appropriately used
depending on the PDE4 inhibitor.
[0087] A method wherein a solution containing a drug, a
biodegradable polymer and a water-miscible good solvent (Solvent A:
acetone, tetrahydrofuran, etc.) for the said polymer is first added
to a homogeneous mixed solution comprising a poor solvent (Solvent
B: water, ethanol, etc.) for the said polymer, which is miscible
with solvent A, and a poor solvent (Solvent C: glycerin, etc.) for
the said polymer, which is immiscible with solvent A. The mixture,
upon emulsification, gives emulsion wherein the polymer solution
constitutes the dispersed-phase and the homogeneous mixed solution
constitutes the continuous-phase. The solvent A is then removed
from the dispersed phase (WO/01/80835).
[0088] A method for preparing microspheres from emulsion by
in-water-drying method, in which emulsion an organic phase
containing an organic solvent with a boiling point lower than water
(methylene chloride, ethyl acetate, etc.) and a water insoluble
polymer is emulsified in an aqueous phase, comprising (1) employing
a device equipped with a gas separation membrane (permeable
evaporation membrane, porous membrane, etc.), (2) providing
emulsion to be subjected to the in-water-drying to one side of the
gas separating membrane, and (3) distilling off the organic solvent
in emulsion to the other side of the gas separating membrane
(WO/01/83594).
[0089] Furthermore, the organic solvent remaining in microspheres
can be removed by heating microspheres in an aqueous phase at
temperature higher than the boiling point of the organic solvent
(JP 2000-239152A) or heating the microspheres to dry after coating
with an additive of high melting point (JP 09-221417A (1997)).
[0090] The resultant microspheres are recovered by centrifugation,
filtration or sieving, washed to remove substances attached on the
surface such as additives in the water-phase, and subjected to
lyophilization optionally after combining with an aggregation
inhibitor to prevent the agglomeration of microspheres, for
example, sugar, sugar alcohol or inorganic salt, preferably
lactose, mannitol or sorbitol. It is preferred to use a sieve to
obtain microspheres of an intended particle size, and it is more
preferred to use a sieve allowing particles of, for example, 150
.mu.m or below to pass so as to improve the syringeability when the
microsphere preparation is used as injectable solution.
[0091] For preparing microspheres by "Phase Separation Method",
amphiphilic solvents such as acetone, acetonitrile, tetrahydrofuran
and dioxane in addition to the organic solvents used in the
"In-water Drying Method" above can be used. A PDE4 inhibitor and
optionally one or more additional ingredients, or a solution
thereof, are dissolved or dispersed in an organic solution of water
insoluble polymer in any one of these organic solvents to form an
organic phase. The organic phase is added gradually to a solvent
(disperse medium) immiscible with the organic solvent above, for
example, silicon oil, liquid paraffin, sesame oil, soybean oil,
corn oil, cotton seed oil, coconuts oil, linseed oil, with stirring
to form O/O emulsion. If desired, a surfactant may be added to the
disperse medium. The water insoluble polymer can be solidified by
cooling the emulsion or evaporating the solvent in the organic
phase by heating. Alternatively, a hardening agent such as hexane,
cyclohexane, methyl ethyl ketone, octamethyl-cyclotetrasiloxane or
the like can be added gently to emulsion with stirring, or vice
versa, to separate out the water insoluble polymer from emulsion
thereby forming microspheres.
[0092] The resultant microspheres are recovered by centrifugation,
filtration or sieving, washed with hexane or purified water to
remove solvents, additives, etc. attached on its surface, and
optionally subjected to air-drying, vacuum-drying, or
lyophilization. Alternatively, it can be lyophilized after adding
an aggregation inhibitor in a manner similar to that used in the
above-mentioned in-water-drying method.
[0093] Examples of internal organic phase in the phase separation
method include the following embodiments.
[0094] (a) An organic phase wherein a drug is directly dissolved or
dispersed in a solution of a water-insoluble, biocompatible and
biodegradable polymer.
[0095] (b) An organic phase which is W/O emulsion wherein an
aqueous solution of a drug is dispersed in a solution of a
water-insoluble, biocompatible and biodegradable polymer.
[0096] (c) An organic phase which is O/O emulsion, which uses two
or more water-insoluble, biocompatible and biodegradable polymers,
wherein a drug or a solution thereof is dissolved or dispersed in a
polymer solution that is dispersed in the other(s).
[0097] Further, the preparation of microspheres by "Spray Drying
Method" is conducted using the same organic solvent as the
above-mentioned phase separation method. To an organic solvent is
dissolved a water insoluble biocompatible and biodegradable
polymer, and a PDE4 inhibitor and optionally one or more additional
ingredients, or a solution thereof, are dissolved or dispersed in
the solution, and sprayed via a nozzle into a drying chamber of a
spray drier to volatilize the organic solvent to form
microspheres.
[0098] For the present invention, any commercially available spray
dryers, for example, such as Pulvis Mini Spray GS31 (YAMATO
Scientific Co., Ltd.), Mini Spray Dryer (Shibata Scientific
Technology Ltd.), can be used.
[0099] The resultant microspheres are then worked-up in a manner
similar to that used in the in-water drying method to yield the
desired microsphere preparation.
[0100] Examples of water-miscible organic solvents usable in the
"Solvent Diffusion Method", include acetone, methanol, ethanol or a
mixture thereof, which may further contain a volatile solvent
(methylene chloride, chloroform) in which a drug can dissolve, if
necessary. Examples of colloid protective agent include polyvinyl
alcohol.
[0101] When the microsphere preparation of the present composition
for regenerative treatment of cartilage disease, which comprises a
PDE4 inhibitor as an active ingredient, is administered to a
vicinity of affected region (especially, in the articular
cartilage), it can be preferably applied locally, more preferably,
into articular cartilage as injection or implant.
[0102] An injectable preparation of microspheres can be prepared by
dispersing/suspending microspheres obtained by the present
invention at a concentration of 0.0001-1000 mg/ml, preferably
0.0005-800 mg/ml, more preferably 0.001-500 mg/ml into an aqueous
solution containing a dispersant.
[0103] Examples of dispersant include nonionic surfactants such as
polyoxyethylene sorbitan fatty acid ester (Tween80, Tween60, Nikko
Chemicals Co., Ltd.), polyethylene castor oil (HCO-60, HCO-50,
Nikko Chemicals Co., Ltd.), cellulose-derived dispersants such as
carboxymethyl cellulose sodium, sodium alginate, dextran, sodium
hyaluronate, and the like. These dispersants can serve to improve
the dispersibility of microspheres and stabilize the elution of an
active ingredient. A dispersant can generally be added to a
composition at a concentration of 0.01-2 % by weight preferably
0.05-1 % by weight.
[0104] The injectable preparation above may optionally contain a
preservative (methylparaben, propylparaben, benzyl alcohol,
chlorobutanol, sorbic acid, boric acid, amino acid, polyethylene
glycol, etc.), an isotonizing agent (sodium chloride, glycerin,
sorbitol, glucose, mannitol, etc.), a pH modifier (sodium
hydroxide, potassium hydroxide, hydrochloric acid, phosphoric acid,
citric acid, oxalic acid, carbonic acid, acetic acid, arginine,
lysine, etc.), a buffer (sodium hydrogen phosphate, potassium
hydrogen phosphate, etc.) or the like.
[0105] If necessary, a steroid antiinflammatory analgesic or
non-steroidal antiinflammatory analgesic may be dissolved or
dispersed in the injectable preparation. Examples of steroidal
antiinflammatory analgesic include dexamethasone, triamcinolone,
triamcinolone acetonide, halopredone, paramethasone,
hydrocortisone, prednisolone, methylprednisolone, betamethasone,
and the like. Examples of non-steroidal antiinflammatory analgesic
include ibuprofen, ketoprofen, indomethacin, naproxen, piroxicam,
and the like.
[0106] In addition to the above-mentioned suspension, the
microsphere injection containing PDE4 inhibitor can be in the form
of a kit for preparing an injectable preparation at the time of
use, which kit comprises a solid preparation of an aggregation
inhibitor and microspheres, a dispersant and injectable distilled
water.
[0107] The solid preparation used in a kit can be prepared by
suspending microspheres in an aqueous solution containing an
aggregation inhibitor, and subjecting the suspension to
lyophilization, vacuum drying, spray drying, and/or the like. The
lyophilization is especially preferred.
[0108] When preparing a solid preparation, a dispersant can be
added to an aqueous solution containing aggregation inhibitor
(mannitol, sorbitol, lactose, glucose, xylitol, maltose, galactose,
sucrose, etc.) in order to improve the re-dispersibility into
injectable distilled water, thereby yielding a solid preparation of
good dispersibility. If necessary, it can be formulated into a kit
for preparing an injectable preparation, in which a steroidal
antiinflammatory analgesic and/or a non-steroidal antiinflammatory
analgesic as well as a dispersant are combined.
[0109] The present composition for regenerative treatment of
cartilage disease, which comprises a PDE4 inhibitor as an active
ingredient, can be used in treatment of various warm blood mammals
such as human, a domestic animal (a horse, a bull, a sheep, a pig),
a pet (a dog, a cat), and the like. The composition for
regenerative treatment of cartilage disease can be used in
regenerative treatment of various cartilage diseases such as
osteoarthrosis, chondrodystrophy, degenerative discopathy, meniscus
injury or the like, and be preferably used in regenerative
treatment of osteoarthrosis.
EXAMPLES
[0110] The following Experimental Examples, Examples and Test
Examples are provided to further illustrate the present invention.
Throughout the following examples, a compound with a given number
is the same compound indicated by the same number in the list above
which shows specific examples of preferred compounds with chemical
structure.
Experimental Example 1
Increase of Matrix Production of Articular Chondrocytes
[0111]
1 Test Compounds Compound(1) (10.sup.-5 M or 10.sup.-4 M);
Compound(2) (10.sup.-6 M or 10.sup.-5 M); Compound(9) (10.sup.-6 M
or 10.sup.-5 M); Compound(11) (10.sup.-6 M); Compound(21)
(10.sup.-6 M or 10.sup.-5 M); Compound(27) (10.sup.-6 M or
10.sup.-5 M); Compound(44) (10.sup.-5 M or 10.sup.-4 M);
[0112] (Isolation and Maintenance of Articular Chondrocytes)
[0113] Four NZW line rabbits (Kitayama Labes., Co Ltd.; male;
4-week-old) were sacrificed with bleeding under ether anesthesia
and femur knee joints were collected aseptically. The collected
knee joints were stored in phosphate buffer (pH 7.2) containing
0.2% glucose and only the cortical layer of knee joint was scrapped
with a surgical knife into a 50 ml tube containing phosphate buffer
(pH 7.2) containing 0.2% glucose. The collected knee joint cortical
layer was cut into as small sections as possible on a dish with a
razor and shaken at 37.degree. C. for 15 minutes in phosphate
buffer containing 0.2% glucose, supplied with
10.times.trypsin-ethylenediamine tetraacetic acid (EDTA).multidot.4
Na salt (GIBCO; Cat. No. 15400-054) (50 ml, with 100 mg of trypsin
and 40 mg of EDTA.multidot.4Na; pH 7.2). After shaking, the sample
was centrifuged (1,400 rpm) to collect precipitates, and the
precipitates were washed twice with 40 ml of phosphate buffer
containing 0.2% glucose. The washed precipitates were combined with
40 ml of serum-free .alpha.-minimum essential medium (MEM: GIBCO;
Cat. No. 12571-063) containing 60 mg of collagenase for cell
diffusion (Wako Pure Chemical Industries, Ltd., 034-10533) and
transferred to a 100 ml beaker containing a sterilized stirrer bar.
Under stirring with the stirrer bar, the collagenase digestion was
carried on for about 1 hour in a CO.sub.2 incubator at 37.degree.
C. Cartilage fragments were removed from the collagenase-treated
cells using a 40 .mu.M Cell Strainer ([FALCON; Cat. No.2340]). To
the residual treated cells was added 10 ml of .alpha.-MEM medium
containing 10% fetal calf serum (FCS), and centrifuged (1,400 rpm).
The precipitates were washed with 10% FCS-.alpha.MEM medium twice,
suspended in appropriate volume of the same medium and the
resultant suspension was seeded into collagen type II (Wako Pure
Chemical Industries, Ltd., 033-13901)-coated plates (48 well) at
20,000 cells/well. On the next day, the medium was replaced with
10% FCS-.alpha.MEM medium.
[0114] (Increase of Matrix Production)
[0115] When cells reached to confluent after the medium exchange
procedure above, the medium for test group was replaced with test
compound-containing medium (including 0.1% dimethylsulfoxide as a
vehicle). As a medium to which a test compound is added, 10%
FCS-.alpha.MEM medium containing 0.2 mM ascorbic acid was used. The
day on which test compound-containing medium was added for the
first time was defined as "day 1". The medium exchange with the
same medium was again conducted at day 3 and the cultivation
continued until day 5. As to the control group, the medium was
exchanged at the same time using the same medium as the test group
except that it is free of test compound (containing vehicle only),
and the cultivation was carried out in the same manner. After
completion of cultivation, the supernatant was removed from the
culture medium. Cells were fixed by addition of 0.25 ml of neutral
buffer containing 4% paraformaldehyde and incubation for 2 hours.
Cells were washed three times with 1 ml of phosphate buffer (pH
7.2) and then stained for 4 hours with 0.1% Alcian blue 8GX (Sigma;
A3157) dissolved in 0.1 M hydrochloric acid, which Alcian blue
selectively stains cartilage matrix proteoglycan. After staining,
the cells were washed 3 times with 1 ml of phosphate buffer (pH
7.2). Alcian blue which had stained cartilage matrix was dissolved
with 0.25 ml of aqueous 6 M guanidine hydrochloride solution and a
portion of the solution was used to determine the absorbance at 620
nm. The amounts of Alcian blue used for staining was calculated
from the absorbance, which in turn was used for the estimation of
the amount of matrix (proteoglycan). The results are shown in Table
1.
2 TABLE 1 Proteoglycan Test Compound Concentration (M) Production
(%) Vehicle -- 100 Compound(1) 1 .times. 10.sup.-5 133 1 .times.
10.sup.-4 144 Compound(2) 1 .times. 10.sup.-6 125 1 .times.
10.sup.-5 128 Compound(9) 1 .times. 10.sup.-6 128 1 .times.
10.sup.-5 138 Compound(11) 1 .times. 10.sup.-6 112 Compound(21) 1
.times. 10.sup.-6 115 1 .times. 10.sup.-5 120 Compound(27) 1
.times. 10.sup.-6 129 1 .times. 10.sup.-5 131 Compound(44) 1
.times. 10.sup.-5 125 1 .times. 10.sup.-4 116
[0116] As shown in Table 1 above, it was demonstrated that all the
test compounds (Compounds (1), (2), (9), (11), (21), (27) and (44))
having PDE4 inhibitory activity exert matrix production promoting
activity.
Experimental Example 2
Fractionation of cAMP-Hydrolyzing PDE Expressed in Articular
Cartilage
[0117] Four NZW line rabbits (Kitayama Labes., Co Ltd.; male;
4-week-old) were sacrificed with bleeding under ether anesthesia
and femur knee joints were collected aseptically. 15 The collected
knee joints were stored in phosphate buffer (pH 7.2) containing
0.2% glucose and only the cortical layer of knee joint portion was
scrapped with a knife into a 50 ml tube containing phosphate buffer
(pH 7.2) containing 0.2% glucose. The collected knee joint cortical
layer was cut into as small sections as possible on a dish with a
razor, washed with ice-cold phosphate buffer and homogenized with a
homogenizer (Polytron: Kinematica A.G.) in homogenization buffer
(20 mM Tris-HCl, pH 7.4, 2 mM magnesium acetate, 0.3 mM calcium
chloride, 1 mM dithiothreitol, 40 .mu.M leupeptin, 1.3 mM
benzamidine, 0.2 mM phenylmethylsulfonyl fluoride and 1 mM sodium
azide). The resultant homogenate was centrifuged (100,000.times.g,
60 minutes) to separate supernatant.
[0118] The supernatant was subjected to Mono Q Sepharose High
Performance column (Amersham Pharmacia Biotech) previously
equilibrated with an elution buffer (20 mM Tris-HCl, pH 7.4, 1 mM
calcium chloride, 1 mM dithiothreitol, 2 .mu.M leupeptin, 5 mM
benzamidine). After washing the column with 20 ml of elution
buffer, proteins were eluted into 1 ml fractions by sodium chloride
gradient under ice-cooling. Each fraction was subjected to the
determination of cAMP hydrolyzing activity (PDE activity) on as a
substrate.
[0119] The determination of PDE activity was performed by a
radio-labeled nucleic acid assay. That is, the reaction was
initiated by adding from 10 to 30 .mu.l of elution fraction to 500
.mu.l of assay buffer [50 mM Tris-HCl, pH 8.0, 5 mM magnesium
chloride, 4 mM 2-mercaptoethanol, 0.33 mg/ml fetal bovine albumin
(fatty acid-free, Sigma), 1 mM ethyleneglycol
bis(.beta.-aminoethylether)-N,N,N',N'-tetra-acetic acid] containing
1 .mu.M of unlabeled cAMP and 22 .mu.M of [.sup.3H]-cAMP (Amersham
Pharmacia Biotech). For the control group, no drug was added while
for the test group, Compound(44) was added at a final concentration
of 1.times.10.sup.-5 M. After incubation at 37.degree. C. for 30
minutes, the reaction was quenched by boiling for 1.5 minutes.
Then, 100 .mu.l of 1 mg/ml Crtalus atrox snake venom was added and
incubated at 37.degree. C. for 30 minutes. After addition of 500
.mu.l of methanol, the reaction mixture was subjected to Dowex
column (1.times.8-400). To each eluate was added a liquid
scintillation cocktail and the radio activity was measured. The
results are shown in FIG. 1.
[0120] FIG. 1 shows that there are four peaks showing strong cAMP
hydrolyzing activity. These four peaks fulfill the features of: (1)
having hydrolytic activity selective to cAMP; (2) said cAMP
hydrolyzing activity being free from the influence of cGMP; and (3)
said activity being strongly inhibited by Compound(44) that is a
selective PDE4 inhibitor, and hence were considered to be
PDE4-related peaks. It was reported that PDE4 includes four
subtypes, that is, PDE4A, PDE4B, PDE4C and PDE4D (Saldou et. al.,
Cellular Signaling, Vol.10, 427-440, 1998), and these four peaks
were assumed to be such subtypes or splicing variants originated
therefrom.
Experimental Example 3
Expression of A Gene Encoding Cartilage Matrix Protein
[0121] Total RNA was extracted with ISOGEN (Nippon Gene Co., Ltd.)
from rabbit knee articular chondrocytes cultured for 4 days
according to the same manner as Experimental Example 1 in the
presence of Compound (1). at a final concentration of
1.times.10.sup.-4 M or 1.times.10.sup.-5 M, and 15 .mu.g of the
total RNA was dissolved in 4.5 .mu.l of a sterilized water. This
solution was combined with 2 .mu.l of 5.times.MOPS buffer, 3.5
.mu.l of formaldehyde and 10 .mu.l of formamide, and denatured at
90.degree. C. for 15 minutes. The mixture was then electrophoresed
on 1% agarose gel in the presence of formaldehyde. After completion
of electrophoresis, RNA was transferred to a nylon membrane
(Amersham Pharmacia Biotech) overnight by capillary method. The RNA
was fixed to the nylon membrane by UV crosslinking and subjected to
prehybridization at 60.degree. C. for 2 hours in 50 ml of
hybridization solution (6.times.SSC, 5.times.Denhart's solution,
0.5% SDS, 100 pg/ml heat-denatured salmon sperm DNA, free of 50%
formamide).
[0122] Then, DNA probes of mouse type II collagen gene and human
aggrecan (typical protein consisting proteoglycan) gene were
respectively radio-labeled with .alpha.[.sup.32P]dCTP using
Random-Prime Labeling Kit Ver.2 (Amersham Pharmacia Biotech). Each
probe (1.times.10.sup.8 dpm) and 5 ml of hybridization solution
were added to a prehybridized nylon membrane and sealed, and
allowed to hybridize at 60.degree. C. overnight. The nylon membrane
was washed with a solution containing 0.2.times.SSC and 0.2% sodium
dodecyl sulfate at 60.degree. C. for 40 minutes three times. The
nylon membrane was subjected to an autoradiography and exposed to
X-ray film using LAS-1000 (Fuji Photo Film Co., Ltd.) A relative
amount of each RNA was measured using Image Gause(Fuji Photo Film
Co., Ltd.) and corrected with 28S RNA (internal RNA: internal
control). The gene expression rate in test group was calculated by
assuming that in control group as 10.0%. The results are shown in
Table 2.
3 TABLE 2 Expression of Type II Expression of collagen gene (%)
aggrecan gene (%) Control 100 100 Compound(1) 278 2693 10.sup.-5 M
Compound(1) 406 4262 10.sup.-4 M
[0123] As shown in Table 2 above, in the test group wherein a PDE4
inhibitor (Compound (1)) was added, the expression of type II
collagen and aggrecan genes increased in a dose-dependent manner,
which indicated that a PDE4 inhibitor affects articular
chondrocytes so as to increase gene expression of type II collagen
and aggrecan, which are major components of articular cartilage
matrix, and thereby promoting the cartilage matrix production.
Experimental Example 4
Increase of Knee Articular Cartilage Matrix in Old Rabbit
[0124] (Acclimation)
[0125] Old JW line rabbits (Kitayama Labes., Co Ltd.; male;
37-week-old) were housed at room temperature (23.+-.2.degree. C.)
and is 50.+-.20% humidity. During the housing period, the rabbits
were free to access commercially available food (Oriental Bio;
CE-2)
[0126] (Increase of Articular Cartilage Matrix Production)
[0127] The rabbits were anesthetized by an intravenous injection of
Nembutal (Dainabot Co., Ltd.; 50 mg/kg/ml) into an ear vein. The
left knee articular cartilage portion was shaved and sterilized
with 70% aqueous ethanol. For the test groups, 250 .mu.l of the
test compound-containing microsphere dispersion (drug content: 2.5
mg) prepared in Example 2-(3) was injected intra-articularly with a
18 gage needle (Terumo Corporation). For the control group, 250
.mu.l of test compound-free microsphere dispersion prepared in
Control Example 1-(2) was injected intra-articularly. At 14 days
after administration, rabbits were sacrificed with bleeding under
Nembutal anesthesia. The knee joints were isolated, fixed in
neutral buffer containing 10% formaldehyde and decalcified with
aqueous 0.5 M EDTA-4Na solution to obtain sections. The sections
were stained with 0.1 M hydrochloric acid containing 0.10% Alcian
blue 8GX (Sigma; A3157) which selectively stains cartilage matrix
(proteoglycan) and the stainability between the test and control
groups was compared microscopically. As a result, the thickness of
the matrix layer (proteoglycan), which was stained with Alcian
blue, was more than three times in test groups compared with the
control groups.
Experimental Example 5
Regeneration of Knee Articular Cartilage Matrix in Old Rabbit
[0128] (Acclimation)
[0129] Old JW line rabbits (Kitayama Labes., Co Ltd.; male;
37-week-old) were housed at room temperature (23.+-.2.degree. C.)
and 50.+-.20% humidity in a rabbit cage (C type:
W370.times.D520.times.H330). During the housing period, the rabbits
were free to access commercially available food (Oriental Bio;
CE-2).
[0130] (Regeneration of Articular Cartilage)
[0131] The rabbits were anesthetized by an intravenous injection of
Nembutal (Abbott Laboratories; 50 mg/kg/ml) into an ear vein. The
left knee articular cartilage portion was shaved and sterilized
with 70% aqueous ethanol. The median ligament of left knee was
incised to expose the femur head and meniscus, and the bleeding
from neighbor tissue was stopped with sterilized cotton. A hole (2
mm diameter and 3 mm depth) was bored in the hollow at the middle
of femoral head (un-loaded portion) with a drill (TOYO Associates
LTD.: Mr. Meister). The hole was washed with sterilized saline to
remove bone scraps etc. generated during boring. The articular
capsule and median ligament were sutured with silk thread and
hemostasis and disinfection were conducted with sterilized cotton.
Nine days later, for the test groups, 250 .mu.l of the test
compound-containing microsphere dispersion (drug content: 2.5 mg)
prepared in Example 2-(3) was injected intra-articularly with a 18
gage needle (Terumo Corporation). For the control group, 250 ul of
test compound-free microsphere dispersion prepared in Control
Example 1-(2) was injected intra-articularly. Rabbits were
sacrificed 14 days after administration with bleeding under
Nembutal anesthesia. The knee joints were isolated, fixed in
neutral buffer containing 10% formaldehyde and decalcified with
aqueous 0.5 M EDTA-4Na solution to obtain sections. The degree of
regeneration at the hole was observed microscopically. The results
are shown in FIG. 2. As shown in FIG. 2, an advanced regeneration
of hole was confirmed clearly in the test groups compared with the
control group.
Experimental Example 6
Regenerative Healing Effects on Papain-Induced Gonarthrosis
[0132] (Acclimation)
[0133] Japanese White rabbits (Kitayama Labes., Co Ltd.; male;
13-week-old) were housed for 8 days at room temperature
(23.+-.2.degree. C.) and 50.+-.20% humidity. During the housing
period, the rabbits were fed with commercially available food (RC4,
Oriental Yeast, Co., Ltd.) at the rate of about 140 g/day.
[0134] (Regenerative Healing Effect)
[0135] The rabbits were anesthetized by an intravenous injection of
Nembutal (Abbott Laboratories; Lot. 791102) into an ear vein. The
both knee portions were shaved and sterilized with 70% aqueous
ethanol. The rabbits received injections of 0.5 ml of aqueous
saline solution containing 0.8% papain (Merck EC 3.4.22.2 lot
587644 019) twice into both knee joints at an interval of five
days. One week after the second injection, for the test group, the
microsphere dispersion prepared in Example 2-(3) (containing 0.2 or
2 mg of Compound(1)) was injected intra-articularly (left knee; 4-6
rabbits/group). For the control group, compound-free microsphere
dispersion prepared in Control Example 1-(2) was injected
intra-articularly (right knee; 4-6 rabbits/group) in the same
amount as the dispersion used in test group. Furthermore, for the
Artz-treated group, 0.3 ml of 1% aqueous hyaluronic acid sodium
salt solution (Artz, Kaken Pharmaceutical Co., Ltd.) was injected
intra-articularly (left knee; 2 rabbits per group). For the
non-Artz treated group, 0.3 ml of saline was injected
intra-articularly (right knee; 2 rabbits per group). To both of the
Artz-treated and non-Artz-treated group, the same intra-articular
injection as the above was conducted weekly four times in total.
Four weeks after the last injection, rabbits were sacrificed with
bleeding under ether anesthesia, knee joints were isolated and
fixed in neutral buffer containing 10% formaldehyde. As a result,
it was observed that the papain treatment caused an articular
cartilage degeneration characterized by the irregularity of
articular cartilage surface, decreased hematoxylin and eosin
stainability, cartilage matrix fibrosis and disappearance of
articular chondrocytes. Although a slight inhibition in the
decrease of hematoxylin and eosin stainability was observed in the
Artz-treated group, remarkable regenerating effects could not be
confirmed. On the other hand, in the test group, wherein
Compound(1)-containing microsphere was administered, showed great
improvement of the above-mentioned pathological symptoms. In the
control group or non-Artz treated group, recovering effects were
not observed at all.
Experimental Example 7
Fractionation of cAMP Hydrolytic PDE Expressed in Human Articular
Cartilage
[0136] From the human knee articular cartilage was isolated on
surgery of osteoarthrosis patients. Cartilage portion was scrapped
with a surgery knife, washed with ice-cold phosphate buffer and
stored at -80.degree. C. The cartilage tissues were milled at
-80.degree. C. and then, scattered into pieces with homogenizer
(Kinematica A.G., Polytron) in ice-cold homogenization buffer (20
mM Tris-HCl, pH 8.0, 1 mM ethylene glycol
bis(.beta.-aminoethylether)-N,N,N',N'-tetraacetic acid, 1 mM
dithiothreitol, 10 .mu.g/ml leupeptin, 5 mM benzamidine, 0.2 mM
phenylmethylsulfonyl fluoride, 1 mM sodium azide and 5 mM
mercaptoethanol). The resultant homogenate was centrifuged
(100,000.times.g; 30 minutes) to separate supernatant.
[0137] The above supernatant was loaded on a Mono Q Sepharose High
Performance column (Amersham Pharmacia Biotech) equilibrated with
elution buffer (20 mM Tris-HCl, pH 8.0, 1 mM ethylene glycol
bis(.beta.-aminoethylether)-N,N,N',N'-tetraacetic acid, 1 mM
dithiothreitol, 2 .mu.g/ml leupeptin, 5 mM benzamidine). After
washing the column with 20 ml of elution buffer, proteins were
eluted into 1 ml fractions by aqueous sodium chloride solution
(concentration gradient: 0 to 1000 mM, 70 ml) under ice-cooling.
Each fraction was subjected to the determination of hydrolytic
activity (PDE activity) toward cAMP and cGMP as a substrate.
[0138] The determination of PDE activity was performed by a
radio-labeled nucleic acid assay. That is, the reaction was
initiated by adding from 10 to 50 .mu.l of elution fraction to 500
.mu.l of assay buffer (50 mM Tris-HCl, pH 8.0, 5 mM magnesium
chloride, 4 mM 2-mercaptoethanol) containing 1 .mu.M of unlabeled
cAMP and 22 nM of [.sup.3H]-cAMP (Amersham Pharmacia Biotech).
[0139] After incubation at 37.degree. C. for 30 minutes, the
reaction was quenched by boiling for 1.5 minutes. Then, 100 .mu.l
of 1 mg/ml Crtalus atrox snake venom was added and incubated at
37.degree. C. for 30 minutes. After addition of 500 .mu.l of
methanol, the reaction solution was subjected to Dowex column
(1.times.8-400). To each eluate was added a liquid scintillation
cocktail and the radio activity was measured. The results are shown
in FIG. 3.
[0140] As shown in FIG. 3, it was revealed that, in a sample
solution prepared by treating human cartilage derived from
osteoarthrosis patient, no fractions having cGMP hydrolyzing
activity exist, while fractions with potent cAMP hydrolyzing
activity do.
[0141] The inhibitory activity of PDE4 inhibitor on fraction Nos.
28-30 containing potent cAMP hydrolyzing activity was measured
according to the above-mentioned radio-labeled nucleic acid assay.
The test compounds are Compound(1), Compound(2), Compound(11),
Compound(44) and Compound (27).
[0142] As a result of experiments, it was confirmed that
Compound(1) and Compound(2), in particular Compound(2) inhibits the
hydrolytic activity of the fraction strongly. Further, the
IC.sub.50 of Compound(1) and Compound (2) for the fraction(s),
which was measured according to a method described in Journal of
Medicinal Chemistry, vol.42, 1088-1099 (1999), was consistent with
the IC.sub.50 of PDE4 inhibition activity described in the same
literature. The results are shown in FIG. 4.
Experimental Example 8
Increase of Intracellular cAMP in Human Articular Chondrocytes
[0143] (Isolation of Articular Chondrocytes)
[0144] Human articular cartilage (articular cartilage of
degenerative malum coxae patient) was soaked in phosphate buffer
(pH 7.2) and only the cortical layer of the joint portion was
scrapped with a knife into a 50 ml tube containing same buffer. The
collected knee joint cortical layer was cut into as small sections
as possible on a dish with a razor and transferred to a centrifuge
tube.
[0145] To the centrifuge tube, phosphate buffer (pH 7.2) containing
1 mg/ml of hyaluronidase (SIGMA: Cat. No. H-3506) was added and
shaken at 37.degree. C. for 15 minutes. The precipitates were
separated by centrifugation (2,000 rpm, 5 minutes) and added to
Hank's balanced salt solution (GIBCO; Cat. No. 15050-065)
containing 0.25% trypsin and shaken at 37.degree. C. for 30
minutes.
[0146] After separation of precipitates by centrifugation (2,000
rpm, 5 minutes), a-minimum essential medium (GIBCO; Cat. No.
12571-063) containing 0.25% collagenase for cell diffusion (Wako
Pure Chemical Industries, Ltd., 034-10533) and 10% fetal calf serum
(GIBCO; Cat. No. 10099-141) were added to the precipitates and
shaken at 37.degree. C. overnight.
[0147] Cartilage fragments were removed using a 40 .mu.m Cell
Strainer (FALCON; Cat. No.2340), and .alpha.-minimum essential
medium containing 10% fetal calf serum was added to the
collagenase-treated cells and centrifuged (1,400 rpm, 10
minutes).
[0148] The precipitates were washed three times with
.alpha.-minimum essential medium containing 10% fetal calf serum
and suspended in the same medium to an appropriate volume and
seeded into 48-well plates (50,000 cells/well). On the next day,
the medium was replaced with .alpha.-minimum essential medium
containing 10% fetal calf serum. The .alpha.-minimum essential
medium used contained antibiotics (100 U/ml penicillin G and 100
.mu.g/ml streptomycin sulfate) and an antifungal (0.25 .mu.g/ml
amphotericin B) (GIBCO; Cat. No. 15240-062).
[0149] (Increase of Intracellular cAMP Concentration)
[0150] When cells reached to confluent after the medium exchange
procedure above, the medium for test group was replaced with
.alpha.-minimum essential medium containing 10% fetal calf serum
(including 0.1% dimethylsulfoxide) supplied with 1 .mu.M PGE.sub.2
(SIGMA, Cat. No. P-0409) and 10.sup.-6 M or 10.sup.-5 M test
compound. As to the control group, the medium was replaced with
.alpha.-minimum essential medium (including 0.1% dimethylsulfoxide)
containing 10% fetal calf serum supplied with 1 .mu.M PGE.sub.2
(SIGMA, Cat. No. P-0409).
[0151] After 30 minutes cultivation, medium was discarded. The
resulting culture was washed with phosphate buffer (pH 7.2) and
treated with 50% ethanol for 30 minutes. Ethanol was collected and
the ethanol extract was evaporated to dryness. The residue was
dissolved in an assay buffer attached with cAMP EIA system
(Amersham Pharmacia Biotech; Cat. No. RPN225) and cAMP
concentration was measured with said system. The results are shown
in Table 3.
4TABLE 3 Test cAMP Production Compound Concentration PGE.sub.2 (1
.mu.M) (picomol/well) Vehicle 0 + 0.4 Compound(2) 1 .times.
10.sup.-6 + 13.8 1 .times. 10.sup.-5 + 23.2 Compound(11) 1 .times.
10.sup.-5 + 10.6
Experimental Example 9
Matrix Production of Rabbit Articular Chondrocytes in the Presence
of IL-1
[0152] Four NZW line rabbits (Kitayama Labes., Co Ltd.; male;
4-week-old) were sacrificed with bleeding under ether anesthesia
and knee joints of femur side were collected aseptically. Only the
cortical layer was scrapped with a surgical knife in phosphate
buffer (pH 7.2) containing 0.2% glucose and then placed into a 50
ml tube containing phosphate buffered saline containing 0.2%
glucose. The collected knee joint cortical layer was cut into as
small sections as possible on a dish with a razor, combined with 50
ml of phosphate buffer (100 mg trypsin, 40 mg EDTA.multidot.4 Na;
pH 7.2) containing 0.2% glucose, supplied with
10.times.trypsin-ethylenediamine tetraacetic acid tetra sodium salt
(EDTA.multidot.4Na: GIBCO; Cat. No.15400-054) and shaken at
37.degree. C. for 15 minutes.
[0153] After shaking, the precipitates were collected by
centrifugation (1,400 rpm) and washed twice with 40 ml of phosphate
buffer (pH 7.2) with 0.2% glucose.
[0154] To the washed precipitates was added 40 ml of
.alpha.-minimum essential medium (MEM: GIBCO; Cat. No. 12571-063)
containing 60 mg of collagenase for cell diffusion (Wako Pure
Chemical Industries, Ltd., 034-10533) and antibiotics (200 U/ml
penicillin G and 200 ug/ml streptomycin sulfate) (GIBCO; Cat. No.
15140-122), and the mixture was transferred to a 100 ml beaker
containing a sterilized stirrer bar.
[0155] The incubation was conducted in a CO.sub.2 incubator at
37.degree. C. for 30 minutes under stirring with a stirrer bar.
Deoxyribonuclease I (Takara Shuzo Co., LTD.; Cat. No. 2210A) was
then added at a concentration of 70 U/ml. The cultivation was
conducted under the same condition for another 30 minutes. The
supernatant of the culture was collected in another vessel and the
remaining cartilage slips were then cultured again for about 30
minutes in freshly prepared a-minimum essential medium containing
60 mg of collagenase and 70 U/ml deoxyribonuclease I.
[0156] To the previously collected culture supernatant and the last
culture, from which cartilage slips had been removed using a 40
.mu.m Cell Strainer (FALCON; Cat. No.2340), 10 ml of
.alpha.-minimum essential medium (MEM: GIBCO; Cat. No. 12571-063)
containing 10% fetal calf serum (GIBCO Cat. No. 10099-141) and
antibiotics (200 U/ml penicillin G and 200 pg/ml streptomycin
sulfate) (GIBCO; Cat. No. 15140-122) was added and centrifuged
(1,400 rpm, 10 minutes).
[0157] The precipitates were washed twice with .alpha.-minimum
essential medium containing 10% fetal calf serum and antibiotics,
suspended with an appropriate volume of the same medium, and seeded
into 48-well plates (20,000 cells/well). On the next day, the
medium was replaced with the same medium.
[0158] (Increase of Matrix Production)
[0159] Following the above medium exchange procedures, when cells
reached to confluent, the medium of test group was replaced with a
medium (including 0.1% dimethylsulfoxide) supplied with 1 ng/ml
recombinant human IL-1.beta. (PEPRO TECH; Cat. No. 200-01B) and a
test compound. As the medium, .alpha.-minimum essential medium
containing fetal bovine serum and antibiotics (supra), and also 0.2
mM ascorbic acid was used. On the other hand, in the control group,
the medium was replaced with the same medium as the test group
except that a test compound was not added.
[0160] The day when IL-1.beta. containing medium was added was
defined as "day 1" and the cultivation was continued until day
3.
[0161] After the cultivation, the supernatant was removed from the
culture and cells were fixed by addition of 0.25 ml of 10% neutral
buffered formalin solution (Wako Pure Chemical Industries, Ltd.,
Cat. No. 060-01667).
[0162] The fixed cells were washed three times with 1 ml of
phosphate buffer (pH 7.2) and stained for 4 hours with 0.1% Alcian
blue 8GX (Sigma; A3157) ) dissolved in 0.1 M hydrochloric acid,
which Alcian blue selectively stains matrix (proteoglycan).
[0163] After staining, the cells were washed 3 times with 1 ml of.
phosphate buffer (pH 7.2). Alcian blue which had stained cartilage
matrix was dissolved with 0.25 ml of 6 M aqueous guanidine
hydrochloride solution and a portion of which was used to determine
the absorbance at 620 nm. The amount of Alcian blue used for
staining was calculated from the absorbance, which in turn was used
for the estimation of the amount of matrix (proteoglycan). The
relative percentage was calculated by assuming the proteoglycan
production in control group, wherein a test compound was not added,
to be 100%. The results are shown in Table 4.
5TABLE 4 Concentratio IL-1.beta. Proteoglycan Test Compound n (M)
(1 ng/ml) Production (%) Vehicle 0 + 100 Compound(2) 1 .times.
10.sup.-6 + 143 Compound No. 53 1 .times. 10.sup.-6 + 211 Compound
No. 56 1 .times. 10.sup.-6 + 168 Compound No. 52 1 .times.
10.sup.-6 + 209 Compound No. 57 1 .times. 10.sup.-6 + 169
Compound(11) 1 .times. 10.sup.-6 + 121
[0164] As shown in the table 4 above, in the presence of IL-1, the
test compound having PDE4 inhibitory activity increased the matrix
production. It is thought that IL-1 plays a important role in
cartilage matrix degradation, because IL-1 is expressed in synovial
fluid and cartilage cells of osteoarthrosis patients, and induces
the production and synthesis of matrix metalloproteinase (MMP),
which is matrix (such as cartilage matrix, proteoglycan) catabolic
enzyme (The Journal of Pharmacology and Experimental Therapeutics,
vol 277, pp. 1672-1675, 1966; Journal of Biochemistry, vol 123, pp.
431-439, 1998; Arthritis & Rheumatism, vol 44, pp. 585-594,
2001). Therefore, the results described above suggested that a PDE4
inhibitor, which is an active ingredient of the present invention,
has inhibitory activity against IL-1-related cartilage matrix
degradation.
Experimental Example 10
Regenerative Healing Effects on Gonarthrosis Induced by Partial
Excision of Medial Meniscus/Abscission of Bilateral Collateral
Ligaments
[0165] (Acclimation)
[0166] Japanese White rabbits (male; 10-week-old; 7 rabbits/group)
were housed for 16 days at room temperature (23.+-.2.degree. C.)
and 55.+-.15% humidity. During the housing period, the rabbits were
free to access commercially available food (Oriental Bio Service;
LRC4).
[0167] (Healing of Gonarthrosis)
[0168] Under ether anesthesia, the right knee joint of each rabbit
was excised and 1/2 portion of medial meniscus was isolated with
double-edged small straight scissors. The bilateral collateral
ligaments were also cut. After the operation, muscle and epidermal
tissues were sutured and sterilized. Two weeks from the operation,
under ether anesthesia, each rabbit of the test group (7
rabbits/group) was administered intra-articularly the
drug-containing microsphere prepared in Example 7-(1), which
contains 1 .mu.g of Compound (2). The rabbit of the control group
(7 rabbits/group) received the same amount of drug-free microsphere
prepared in Control Example 2. Six weeks after the operation, the
above-mentioned drug-containing or -free microsphere was again
administered. Ten weeks after the operation, under ether
anesthesia, rabbits were sacrificed by laparotomy with bleeding and
the tibial proximal end was isolated. The isolated tibial proximal
end was treated with India ink and then soaked into 10% neutral
buffered formalin solution for fixation.
[0169] (Experimental Results)
[0170] After wiping off the excess India Ink, the overhead surface
image of the formalin-fixed tibial proximal end was imported into
an analyzer, Image Analyzer (IMAGING RESEARCH, MICD imaging
analyzer), with a stereomicroscope (OLYMPUS OPTICAL Co.,Ltd. model
SZX12-2111). This analyzer was used to measure the area of medial
portion where India ink remains (the degenerated area). The gross
medial area was also measured and the percentage (%) (the
degenerated area rate) of the degenerated medial area in the gross
medial area was calculated. The results are shown in Table 5.
6 TABLE 5 Degenerated Area Drug Drug amount Rate (%) Control 0
.mu.g 18.86 .+-. 2.03 Compound(2) 1 .mu.g 10.16 .+-. 1.35
Example 1
[0171] (1) To 0.1 g of Compound(1) and 1.9 g of lactic
acid-glycolic acid copolymer (lactic acid: glycolic acid=50:50;
average molecular weight 20,000; PLGA5020: Wako Pure Chemical
Industries, Ltd.) was added 4.0 g of methylene chloride, and the
mixture was shaken for 30 minutes thoroughly to form an oil phase
(O).
[0172] (2) The oil phase was added to 8 ml of 0.5% aqueous solution
of polyvinyl alcohol (POVAL PVA-220C: Kuraray Co., Ltd.) and
emulsified at 25.degree. C. for 5 minutes with homogenizer
(Polytron, Kinematica A.G.) to form (O/W) emulsion, wherein the oil
phase is dispersed in the water phase.
[0173] (3) The emulsion was added to 1000 ml of distilled water,
stirred at 400 rpm with Three-one motor (Shinto Scientific Co.,
Ltd.) and subjected to in-water drying method at 25.degree. C. for
3 hours to remove methylene chloride.
[0174] (4) The resultant microsphere suspension was filtered
through 150 .mu.m filter to remove aggregates and filtered under
reduced pressure through 20 .mu.m filter to remove the water phase.
The resultant microsphere was combined with a little amount of
distilled water and lyophilized to give 1.6 g of microsphere.
[0175] Ten mg of the resultant microsphere was dissolved in 3 ml of
acetonitrile. The solution was combined with 7 ml of 0.5 M aqueous
sodium chloride solution, stirred with a mixer (Touch mixer MT-51:
YAMATO Scientific Co., Ltd.) and then centrifuged at 2000 rpm for 5
minutes to separate supernatant. A portion of supernatant was
loaded on FL-HPLC (column; Hypersil 5-ODS, diameter: 4 mm, length:
300 mm, GL Sciences, Inc., excitation wavelength: 315 nm,
fluorescence wavelength: 465 nm) and the drug concentration in the
supernatant was determined by comparing with a standard curve
prepared separately with a drug solution. On the basis of the
resultant concentration and the volume of supernatant, the drug
content in microsphere was estimated as 4.21%.
[0176] An adequate amount of resulting microsphere was dispersed in
a dilute solution of polyoxyethylene sorbitan fatty-acid ester
(Tween 80: Nikko Chemicals Co., Ltd.) The particle distribution was
measured with a particle size analyzer SALD-1100 (Shimadzu
Corporation), and the average particle size was calculated. The
average particle size was 57 .mu.m.
[0177] (5) The microsphere obtained in (4) above was added to
physiological saline (dispersion medium) containing 0.5%
carboxymethyl cellulose sodium (Nichirin Chemical Industries) and
0.1% polyoxyethylene sorbitan fatty acid ester (Tween 80: Nikko
Chemicals Co., Ltd.) at final drug concentration of 2.5 mg/ml, and
the mixture was stirred with a mixer (Touch mixer MT-51: YAMATO
Scientific Co., Ltd.) thoroughly to yield microsphere
dispersion.
Example 2
[0178] (1) Microsphere (1.6 g) was prepared in a manner similar to
that described in Example 1-(1) to (4) except that a mixture of
0.57 g of lactic acid-glycolic acid copolymer (lactic acid:glycolic
acid=50:50; average molecular weight 20,000; PLGA5020: Wako Pure
Chemical Industries, Ltd.) and 1.33 g of lactic acid polymer
(average molecular weight 20,000; PLA0020: Wako Pure Chemical
Industries, Ltd.) was used.
[0179] The drug content and the average particle size of
microsphere were measured in a manner similar to that described in
Example 1-(4) and proved to be 3.70% and 47.7 .mu.m,
respectively.
[0180] (2) The microsphere obtained in (1) above was treated in a
manner similar to that described in Example 1-(5) to give
microsphere dispersion (drug rate: 2.5 mg/ml).
[0181] (3) The microsphere obtained in (1) above was treated in a
manner similar to that described in Example 1-(5) to give
microsphere dispersion (drug rate: 10.0 mg/ml).
Example 3
[0182] (1) Microsphere (1.5 g) was prepared in a manner similar to
that described in Example 1-(1) to (4) except that lactic acid
polymer (average molecular weight 20,000; PLA0020: Wako Pure
Chemical Industries, Ltd.) was used.
[0183] The drug content and the average particle size of
microsphere were measured in a manner similar to that described in
Example 1-(4) and proved to be 3.73% and 52.2 .mu.m,
respectively.
[0184] (2) The microsphere obtained in (1) above was treated in a
manner similar to that described in Example 1-(5) to give
microsphere dispersion (drug rate: 2.5 mg/ml).
Example 4
[0185] (1) To 0.2 g of Compound(1) and 0.3 g of lactic acid polymer
(average molecular weight 20,000; PLA0020: Wako Pure Chemical
Industries, Ltd.) was added 1.0 g of methylene chloride, and the
mixture was shaken with a mixer (Touch mixer MT-51: YAMATO
Scientific Co., Ltd.) thoroughly to form an oil phase (O).
[0186] (2) The oil phase was added to 4 ml of 0.25% aqueous
solution of methyl cellulose (METOLOSE: Shin-Etsu Chemical Co.,
Ltd.) and emulsified at 25.degree. C. for 5 minutes with
homogenizer (Polytron, Kinematica A.G.) to form (O/W) emulsion,
wherein the oil phase is dispersed in the water phase.
[0187] (3) The emulsion was added to 400 ml of distilled water,
stirred at 400 rpm with Three-one motor (Shinto Scientific Co.,
Ltd.) and subjected to in-water drying method at 25.degree. C. for
3 hours to remove methylene chloride.
[0188] (4) The resultant microsphere suspension was filtered
through 150 .mu.m filter to remove aggregates and filtered under
reduced pressure through 20 .mu.m filter to remove water phase. The
resultant microsphere was combined with a little amount of
distilled water and lyophilized to give microsphere. The drug
content and the average particle size of microsphere were measured
in a manner similar to that described in Example 1-(4) and proved
to be 39.6% and 33.4 .mu.m, respectively.
Example 5
[0189] (1) To 0.05 g of Compound(1) and 0.45 g of lactic
acid-glycolic acid copolymer (lactic acid:glycolic acid=50:50;
average molecular weight 20,000; R202H: Boehringer Ingelheim Co.,
Ltd.) was added 1.0 g of methylene chloride, and the mixture was
shaken with a mixer (Touch mixer MT-51: YAMATO Scientific Co.,
Ltd.) thoroughly to form an oil phase (O).
[0190] (2) The oil phase was added to 40 ml of 0.5% aqueous
solution of polyvinyl alcohol (GOHSENOL EG-40: The Nippon Synthetic
Chemical Industry Co., Ltd.) and emulsified at 25.degree. C. for 4
minutes with homogenizer (Polytron, Kinematica A.G.) to form (O/W)
emulsion, wherein the oil phase is dispersed in the water
phase.
[0191] (3) Emulsion was poured into a cylindrical airtight
container (inside diameter: 110 mm; volume 1,000 ml) containing 400
ml of purified water, and methylene chloride was removed from the
container by stirring at 25.degree. C. and 400 rpm using 4-bladed
propeller (diameter: 50 mm, propeller R type: HEIDON) equipped with
Three-one motor (BL-600; HEIDON) while supplying nitrogen gas into
hollow fibers of cylinder-type hollow fiber membrane module made of
silicone rubber (NAGAYANAGI Co., Ltd.) inserted in the container
(gas flow rate is 2 L/minute). This procedure was conducted for 1
hour.
[0192] The cylindrical hollow fiber membrane module made of
silicone rubber used in this procedure is cylinder type NAGASEP
M60-1800 of the following specification.
7 Cylinder diameter 100 mm Cylinder length 120 mm .times. 120 mm
Membrane thickness of hollow fiber 60 .mu.m membrane Inside
diameter of hollow fiber 200 .mu.m membrane Outside diameter of
hollow fiber 320 .mu.m membrane Number of hollow fiber 1800
Effective membrane area of hollow 0.15 m.sup.2 fiber membrane
[0193] (4) The resulting microsphere suspension was filtered
through 150 .mu.m filter to remove aggregates and filtered under
reduced pressure through 20 .mu.m filter to remove water phase. The
resultant microsphere was combined with a little amount of
distilled water and lyophilized to give 0.26 g of microsphere. The
drug content and the average particle size of microsphere were
measured in a manner similar to that described in Example 1-(4) and
proved to be 3.07% and 71.7 .mu.m, respectively.
Example 6
[0194] (1) To 0.05 g of Compound(2) and 0.45 g of lactic
acid-glycolic acid copolymer (lactic acid:glycolic acid=50:50;
average molecular weight 20,000; RG502H: Boehringer Ingelheim Co.,
Ltd.), 2.5 g of methylene chloride was added and shaken with a
mixer (Touch mixer MT-51: YAMATO Scientific Co., Ltd.) thoroughly
to form an oil phase (0).
[0195] (2) The oil phase was added to 3 ml of 0.5% aqueous solution
of polyvinyl alcohol (POVAL PVA-220C: Kuraray Co., Ltd.) and
emulsified at 22.degree. C. for 5 minutes with homogenizer
(Polytron: Kinematica A.G.) to form (O/W) emulsion, wherein the oil
phase was dispersed in the water phase.
[0196] (3) The above procedures (1) and (2) were repeated five
times. The resultant emulsions (from 5 trials) were combined, added
to 1000 ml of distilled water, and stirred at 400 rpm with
Three-one motor (Shinto Scientific Co., Ltd.) to remove methylene
chloride by conducting in-water-drying at 25.degree. C. for 1.5
hours, at 40.degree. C. for 1 hour and at 25.degree. C. for 0.5
hours.
[0197] (4) The resultant microsphere suspension was filtered
through 150 .mu.m filter to remove aggregates and filtered under
reduced pressure through 20 .mu.m filter to remove water phase. The
resultant microsphere was combined with a little amount of
distilled water and lyophilized to give 2.3 g of microsphere.
[0198] Ten mg of the resultant microsphere was dissolved in 3 ml of
acetonitrile. The solution was combined with 6 ml of aqueous 0.5 M
sodium chloride solution, stirred with a mixer (Touch mixer MT-51:
YAMATO Scientific Co., Ltd.) and then centrifuged at 2000 rpm for 5
minutes to separate supernatant. A portion of supernatant was
loaded on UV-HPLC (column; Hypersil 5-ODS, diameter: 4 mm, length:
300 mm, GL Sciences, Inc., detection wavelength: 240 nm) and the
drug concentration in the supernatant was determined by comparing
with a standard curve prepared separately with a drug solution. On
the basis of the resultant concentration and the volume of
supernatant, the drug content in microsphere was estimated.
Further, the average particle size was measured in a manner similar
to that described in Example 1-(4). As a result, the drug content
was 9.9% and the average particle size was 26.4 .mu.m.
[0199] (5) The microsphere obtained in (4) above was treated in a
manner similar to that described in Example 1-(5) to give
microsphere dispersion (drug rate: 0.1 mg/ml).
Example 7
[0200] (1) Microsphere (2.2 g) was prepared in a manner similar to
that described in Example 6-(1) to (4) except that lactic
acid-glycolic acid copolymer (lactic acid:glycolic acid=75:25;
average molecular weight 20,000; PLGA7520: Wako Pure Chemical
Industries, Ltd.) was used and that 2.0 g of methylene chloride was
added.
[0201] The drug content and the average particle size of
microsphere were measured in a manner similar to that described in
Example 6-(4) and proved to be 10.1% and 27.0 .mu.m,
respectively.
[0202] (2) The microsphere obtained in (1) above was treated in a
manner similar to that described in Example 6-(5) to give
microsphere dispersion (drug rate: 0.1 mg/ml).
Control Example 1
Control of Example 2
[0203] (1) To 0.6 g of lactic acid-glycolic acid copolymer (lactic
acid:glycolic acid=50:50; average molecular weight 20,000;
PLGA5020: Wako Pure Chemical Industries, Ltd.) and 1.4 g of lactic
acid polymer (average molecular weight 20,000) was added 4.0 g of
methylene chloride, and the mixture was shaken for 30 minutes
thoroughly to form an oil phase (O). In accordance with the
procedures described in Example 1-(1) to (4), 1.7 g of microsphere
free of drug was obtained.
[0204] (2) The microsphere obtained in (1) above was treated in a
same procedures described in Example 1-(5) to prepare microsphere
dispersion, wherein the dispersed microsphere concentration in the
dispersion is the same as that of Example 2-(3).
Control Example 2
(Control of Example 7)
[0205] To 0.45 g of lactic acid-glycolic acid copolymer (lactic
acid:glycolic acid=75:25; average molecular weight 20,000;
PLGA7520: Wako Pure Chemical Industries, Ltd.) was added 2.0 g of
methylene chloride, and the mixture was shaken with a mixer (Touch
mixer MT-51: YAMATO Scientific Co., Ltd.) thoroughly to form an oil
phase. In accordance with the procedures described in Example 6-(2)
to (4), 2.2 g of microsphere free of drug was obtained.
Test Example 1
[0206] To 10 mg of microsphere in a test tube was added 10 ml of
phosphate buffer (pH 7.4) containing 0.05% Tween 80, and stirred
with a rotating cultivator at 25 rpm in an
air-temperature-controlled cabinet at 37.degree. C. When a defined
period of time passed from the initiation of stirring, test tube
was centrifuged (2000 rpm, 5 min) and of stirring, test tube was
centrifuged (2000 rpm, 5 min) and 9 ml of supernatant was sampled
and loaded on FL-HPLC (column; Hypersil 5-ODS, diameter: 4 mm,
length: 300 mm, GL Sciences, Inc., excitation wavelength: 315 nm,
fluorescence wavelength: 465 nm) and the drug content was
determined by comparing with a standard curve prepared separately
with a drug solution. On the basis of the result and the sampling
volume, the elution amount of drug was estimated.
[0207] Further, the estimation of elution amount of drug was
repeated regularly by adding 9 ml of phosphate buffer (pH 7.4) to
the test tube after sampling, and conducting the same procedures
under the same conditions, which comprises stirring, sampling, and
estimating.
[0208] After the final sampling, the remaining eluate was removed
from the test tube and the drug content in the residual microsphere
was determined according to the method described in Example
1-(4).
[0209] The above procedures were carried out on the microspheres
obtained in Examples 1 to 3. The results are shown in FIG. 5.
[0210] The elution rate was calculated based on the assumption that
the sum of drug eluted from and remained in the microsphere being
100%.
Test Example 2
[0211] Male SD rats (7-weeks-old, 3 rats/group, Japan SLC) were
conditioned for a week by housing at room temperature
(23.+-.2.degree. C.) under 12 hours light-dark cycle while feeding
with food and water ad libitum. Each rat then received
rapid-injection of Compound(1) (1 mg/ml) dissolved in physiological
saline containing 10% polyethylene glycol 400 ((Wako Pure Chemical
Industries, Ltd.) from femoral vein at 0.5 ml/animal (total drug
dosage: 0.5 mg/rat).
[0212] After drug administration, under ether anesthesia blood
samples were collected at regular time intervals from jugular vein
with a syringe containing heparin and centrifuged to obtain plasma
samples. To 0.1 ml of plasma were added 0.2 ml of internal standard
solution and 1 M dibasic potassium phosphate and then 7.0 ml of
chloroform. The mixture was shaken for 10 minutes and centrifuged
for 5 minutes to separate 5 ml of organic phase. The resultant
organic phase was evaporated to dryness at 40.degree. C. under
nitrogen atmosphere, re-dissolved in 0.5 ml of mobile phase and
then loaded on FL-HPLC (column; Hypersil 5-ODS, diameter: 4 mm,
length: 300 mm, GL Sciences, Inc., excitation wavelength: 315 nm,
fluorescence wavelength: 465 nm) to determine the plasma
concentration. The results are shown in FIG. 6.
Test Example 3
[0213] Male SD rats (7-weeks-old, 5 rats/group, Japan SLC) were
conditioned for a week by housing at room temperature
(23.+-.2.degree. C.) under 12 hours light-dark cycle while feeding
with food and water ad libitum. Each rat then received
subcutaneously microsphere dispersion obtained in Examples 1-(5),
2-(2) or 3-(2) from back at 2 ml per rat (total drug dosage: 5
mg/rat). After drug administration, under ether anesthesia, blood
samples were collected at regular time intervals from jugular vein
with a syringe containing heparin and centrifuged to obtain plasma
samples. The concentration of the compound in plasma was determined
in a manner similar to that described in Test Example 2. As a
result of formulating PDE4 inhibitor into microsphere, the maximum
plasma concentration of PDE4 inhibitor could be reduced to 1/25 to
1/100, even when compared with that achieved by intravenous
injection of saline containing only a tenth amount of PDE4
inhibitor (Test Example 2). The results are shown in FIG. 7.
Test Example 4
[0214] Male SD rats (7-weeks-old, 5 rat per group, Japan SLC) were
conditioned for a week by housing at room temperature
(23.+-.2.degree. C.) under 12 hours light-dark cycle while feeding
with food and water ad libitum. Each rat then received
subcutaneously microsphere dispersion obtained in Example 2-(2)
from back at 2 ml per rat (total drug dosage: 5 mg/rat).
[0215] At days 3, 7, 10, 14, 21 and 35 after drug administration,
microspheres were collected from the sites of administration. To
the collected microspheres, 5 ml of acetonitrile containing
internal control substance was added and dissolved with homogenizer
(Polytron: Kinematica A.G.) After centrifugation at 3,000 rpm, 5
minutes, 3 ml of supernatant was collected, combined with 7 ml of
0.5 M aqueous sodium chloride solution, stirred with a mixer (Touch
mixer MT-51: YAMATO Scientific Co., Ltd.) and then centrifuged at
2,000 rpm for 5 minutes to separate supernatant. A portion of
supernatant was filtrated through KC prep-omni 13 (Katayama
Chemistry Inc.) and loaded on FL-HPLC (column; Hypersil 5-ODS,
diameter: 4 mm, length: 300 mm, GL Sciences, Inc., excitation
wavelength: 315 nm, fluorescence wavelength: 465 nm). The drug
concentration was determined by comparing with a standard curve
prepared separately with a drug solution. On the basis of the
resultant concentration and the volume of supernatant, the residual
rate of a drug remaining in microsphere was calculated. The results
are shown in FIG. 8.
Test Example 5
[0216] Male SD rats (7-weeks-old, Japan SLC) were conditioned for a
week by housing at room temperature (23.+-.2.degree. C.) under 12
hours light-dark cycle while feeding with food and water ad
libitum. Each rat then received subcutaneously
Compound(2)-containing microsphere dispersions obtained in Examples
6-(5) and 7-(2) at 1 ml per rat (total drug dosage: 0.1 mg/rat)
from back.
[0217] Microspheres were collected at regular time intervals from
the administration site. To the collected microspheres, 10 ml of
acetonitrile was added and dissolved with homogenizer (Polytron:
Kinematica A.G.) After centrifugation at 3,000 rpm for 5 minutes, 3
ml of supernatant was collected, combined with 6 ml of 0.5 M
aqueous sodium chloride, stirred with a mixer (Touch mixer MT-51:
YAMATO Scientific Co., Ltd.) and then centrifuged at 2000 rpm for 5
minutes to separate supernatant. A portion of supernatant was
filtrated through KC prep-omni 13 (Katayama Chemistry Inc.) and
loaded on UV-HPLC (column; Hypersil 5-ODS, diameter: 4 mm, length:
300 mm, GL Sciences, Inc., detection wavelength: 240 nm). The drug
concentration was determined by comparing with a standard curve
prepared separately with a drug solution. On the basis of the
resultant concentration and the volume of supernatant, the residual
rate of a drug remaining in microsphere was calculated. The results
are shown in FIG. 9.
[0218] Industrial Applicability
[0219] Although only conservative treatment has been considered to
be available as drug treatment of cartilage disease, the present
composition for regenerative treatment of cartilage disease, which
comprises a PDE4 inhibitor as an active ingredient, especially when
administered locally to the affected cartilage region, makes it
possible to regenerate the cartilage without producing side effects
due to systemic action of PDE4 inhibitor, whereby exerts
regenerative therapeutic effects on cartilage diseases especially
osteoarthrosis. Still higher effect can be achieved by formulating
a composition containing a PDE4 inhibitor and a biocompatible and
biodegradable polymer into a depot preparation, especially into an
injectable microsphere preparation, administering the same locally
to an affected cartilage region thereby allowing efficacy to
last.
* * * * *