Canadian Patents Database / Patent 2457540 Summary

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(12) Patent Application: (11) CA 2457540
(54) English Title: USE OF ANNELLATED PYRROLE COMPOUNDS IN THE TREATMENT OF ARTICULAR CARTILAGE OR SUBCHONDRAL BONE DEGENERATION
(54) French Title: UTILISATION DE COMPOSES DE PYRROLE ANNELES DANS LE TRAITEMENT DE LA DEGENERESCENCE D'UN CARTILAGE ARTICULAIRE OU D'UN OS SOUS-CHONDRAL
(51) International Patent Classification (IPC):
  • A61K 31/40 (2006.01)
(72) Inventors :
  • PELLETIER, JEAN-PIERRE (Canada)
  • MARTEL-PELLETIER, JOHANNE (Canada)
(73) Owners :
  • MERCKLE GMBH (Germany)
  • ASCENTIA PHARMA INC. (Canada)
(71) Applicants :
  • MERCKLE GMBH (Germany)
  • ASCENTIA PHARMA INC. (Canada)
(74) Agent: DEETH WILLIAMS WALL LLP
(74) Associate agent:
(45) Issued:
(86) PCT Filing Date: 2002-08-29
(87) Open to Public Inspection: 2003-03-13
Examination requested: 2007-08-09
(30) Availability of licence: N/A
(30) Language of filing: English

(30) Application Priority Data:
Application No. Country/Territory Date
2,356,099 Canada 2001-08-30
60/315,773 United States of America 2001-08-30

English Abstract




Treating or preventing degeneration or destruction of articular cartilage
and/or subchondral bone in the affected joint of a mammal is accomplished by
administering a compound of formula (I), wherein the variables have the
meanings given in the present description. A preferred compound of formula (I)
is formula (II). This treatment ameliorates, diminishes, actively treats,
reverses or prevents any injury, damage or loss of articular cartilage or
subchondral bone subsequent to sa id early stage of said degeneration.


French Abstract

On effectue le traitement ou la prévention de la dégénérescence ou de la destruction d'un cartilage articulaire et/ou d'un os sous-chondral d'une articulation atteinte chez un mammifère en administrant à celui-ci un composé représenté par la formule (I), dans laquelle les variables ont les significations données dans les pièces descriptives de la demande. Un composé préféré de formule (I) est représenté par la formule (.alpha.). Ce traitement atténue, réduit, traite activement, inverse ou prévient les lésions, dommages ou pertes d'un cartilage articulaire ou d'un os sous-chondral une fois passée la phase initiale de cette dégénérescence.


Note: Claims are shown in the official language in which they were submitted.




42
CLAIMS
1. Use of one or more than one compound of Formula (I):
Image
wherein
X represents
CR8R9, S, O, NR12 or C(O);
A represents
CR10R11 or a bond between X and the atom carrying radicals R6
and R7;
the first of radicals R1, R2, R3 represents
aryl, optionally substituted with one or more than one substituents
independently selected among the group consisting of halogen, alkyl,
halogenoalkyl, alkoxy, aryloxy, halogenoalkoxy, alkylthio, hydroxy,
nitro, alkylsulfinyl, alkylsulfonyl, sulfamoyl, N-alkylsulfamoyl, N,N-di-
alkylsulfamoyl, alkylsulfonamido and alkylsulfon-N-alkylamido; or
an aromatic or non-aromatic, mono- or bicyclic, optionally
benzoannellated, heterocyclic group having 1, 2 or 3 heteroatoms
independently selected from N, O and S and optionally being
substituted with one or more than one substituents independently
selected among the group consisting of halogen, alkyl,
halogenoalkyl, alkoxy, aryloxy, halogenoalkoxy, alkylthio, hydroxy,
nitro, alkylsulfinyl, alkylsulfonyl, sulfamoyl, N-alkylsulfamoyl, N,N-di-
alkylsulfamoyl, alkylsulfonamido and alkylsulfon-N-alkylamido;




43

the second of radicals R1, R2, R3 represents
alkyl, optionally substituted with one or more than one substituents
independently selected among the group consisting of halogen,
cycloalkyl, alkoxy, trifluormethoxy, hydroxy and trifluormethyl;
cycloalkyl, optionally substituted with one or more than one
substituents independently selected among the group consisting of
halogen, alkyl, halogenoalkyl, cycloalkyl, alkoxy, halogenalkoxy and
hydroxy;
aryl, optionally substituted with one or more than one substituents
independently selected among the group consisting of halogen, alkyl,
halogenoalkyl, alkoxy, aryloxy, halogenoalkoxy, alkylthio, hydroxy,
nitro, alkylsulfinyl, alkylsulfonyl, sulfamoyl, N-alkylsulfamoyl, N,N-di-
alkylsulfamoyl, alkylsulfonamido and alkylsulfon-N-alkylamido; or
an aromatic or non-aromatic, mono- or bicyclic, optionally
benzoannellated, heterocyclic group having 1, 2 or 3, heteroatoms
independently selected from N, O and S and optionally being
substituted with one or more than one substituents independently
selected among the group consisting of halogen, alkyl,
halogenoalkyl, alkoxy, aryloxy, halogenoalkoxy, alkylthio, hydroxy,
nitro, alkylsulfinyl, alkylsulfonyl, sulfamoyl, N-alkylsulfamoyl, N,N-di-
alkylsulfamoyl, alkylsulfonamido and alkylsulfon-N-alkylamido;
the third of radicals R1, R2, R3 represents
H, alkyl, halogenoalkyl, hydroxyalkyl, -CHO, -COOH, halogen, cyano,
alkylsulfonyl, sulfamoyl or B-Y,
wherein
B represents alkylene or alkenylene, optionally substituted with
hydroxy or alkoxy;
Y represents -COOH, SO3H, OPO(OH)2, OP(OH)2, -CHO or
tetrazolyl; or
the second and the third of radicals R1, R2, R3 represent,
together with the atom they are attached to, saturated or unsaturated
cycloalkyl;


44
R4-R11, which may be the same or different, represent
hydrogen, alkyl, hydroxyalkyl, alkoxyalkyl, hydroxy, COOH or
acyloxy, where vicinal radicals may also represent bonds or geminal
radicals, together with the C atom they are attached to, may also
represent carbonyl or cycloalkyl;
R12 represents
hydrogen, alkyl or phenyl,
and optical isomers, physiologically acceptable salts and derivatives
thereof,
for treating or preventing degeneration or destruction of articular
cartilage and/or subchondral bone.
2. The use according to Claim 1, wherein the first and the second of
radicals R1, R2, R3 independently represent an optionally substituted
aryl or aromatic heterocyclic residue.
3. The use according to Claim 1, wherein the third of radicals R1, R2, R3
represents COOH or B-Y, wherein Y is COOH and B represents
alkylene.
4. The use according to Claim 2, wherein the third of radicals R1, R2, R3
represents COOH or B-Y, wherein Y is COOH and B represents
alkylene.
5. The use according to Claim 1 wherein said compound of formula (I) is
[6-(4-chlorophenyl)-2,2-dimethyl-7-phenyl-2,3-dihydro-1H-pyrrolizine-5-
yl]-acetic acid of the formula (Ia)
Image
a physiologically acceptable salt or a physiologically hydrolysable ester
thereof.
6. The use according to any one of Claims 1 to 5, wherein the
degeneration or destruction of articular cartilage and/or subchondral



45
bone comprises injury, damage or loss of articular cartilage and/or
subchondral bone.
7. The use according to any of Claims 1 to 5, for the treatment of an early
stage of said degeneration or destruction.
8. The use according to any of Claims 1 to 5, for treating a mammal whose
status presently or prospectively is in a condition of said degeneration or
destruction.
9. The use according to Claim 8, wherein said status of said mammal as
presently or prospectively being in said conditon is determined by one or
more of the following:
(A) positive results from the clinical examination and evaluation of
the joints of said mammal, including measurement of hip
dysplasia progression;
(B) performance of any invasive surgical procedure on one or more
joints of said mammal;
(C) positive results from an examination of one or more joints of said
mammal using noninvasive procedures including radiographic
and magnetic resonance imaging (MRI); and
(D) positive results from any biochemical test performed on body
fluids or joint tissue of said mammal with respect to one or more
of the following substances:
(1) increased interleukin-1 beta (IL-1.beta.);
(2) increased tumor necrosis factor alpha (TNF.alpha.);
(3) increased ratio of IL-1.beta. to IL-1 receptor antagonist protein (IL-
1Ra);
(4) increased expression of p55 TNF receptors (p55 TNF-R);
(5) increased interleukin-6 (IL-6); increased leukemia inhibitory
factor (LIF);
(6) unchanged or decreased insulin-like growth factor-1 (IGF-1);
(7) decreased transforming growth factor beta (TGF.beta.); unchanged
or decreased platelet-derived growth factor (PDGF);
(8) unchanged or decreased basic fibroblast growth factor (b-FGF);
(9) increased keratan sulfate;
(10) increased matrix metalloproteases (MMPs) including stromelysin;


46
(11) increased ratio of matrix metalloproteases (MMPs) including
stromelysin, to tissue inhibitor of metalloproteases (TIMP);
(12) increased osteocalcin;
(13) increased alkaline phosphatase;
(14) increased cAMP responsive to hormone challenge;
(15) increased urokinase plasminogen activator (uPA);
(16) increased cartilage oligomeric matrix protein;
(17) presence of type-II specific collagen neoepitopes and
(18) increased collagenase.
10. The use according to any of Claims 1 to 5, wherein said treatment or
prevention comprises administering said compound in an amount of
about 1-10 mg/kg/day.
11. The use according to any of Claims 1 to 5, wherein said treatment or
prevention includes administering in addition to one or more than one
compound of Formula (I) one or more members selected from the group
consisting essentially of polysulfated glycosaminoglycan (PSGAG),
glucosamine, chondroitin sulfate (CS), hyaluronic acid (HA), pentosan
polysulfate (PPS), doxycycline, and minocycline.
12. A pharmaceutical composition, comprising:
(A) one or more than one compound of Formula (I) as defined in
claim 1; and
(B) one or more members selected from the group consisting
essentially of
(1) polysulfated glycosaminoglycan (PSGAG), glucosamine,
chondroitin sulfate (CS), hyaluronic acid (HA), pentosan
polysulfate (PPS), doxycycline, and minocycline.
13. A pharmaceutical composition according to Claim 12, wherein said
compound of formula (I) is [6-(4-chlorophenyl)-2,2-dimethyl-7-phenyl-
2,3-dihydro-1H-pyrrolizine-5-yl]-acetic acid of the formula (Ia)
Image


47
a physiologically acceptable salt or a physiologically hydrolysable ester
thereof.
14. A method of treating or preventing degeneration or destruction of
articular cartilage and/or subchondral bone in one or more joints of a
mammal in need of such treatment, comprising administering to said
mammal an amount therapeutically effective for treating or preventing
degeneration or destruction of articular cartilage and/or subchondral
bone,
of one or more than one compound of Formula (I) as defined in any one
of Claims 1 to 5.

Note: Descriptions are shown in the official language in which they were submitted.


CA 02457540 2004-02-10
USE OF ANNELLATED PYRROLE COMPOUNDS IN THE TREATMENT OF
ARTICULAR CARTILAGE OR SUBCHONDRAL
BONE DEGENERATION
The present invention relates to the use of annellated pyrrole compounds and
in particular of ML3000, salts or derivatives thereof, in mammals as a means
of
treating and preventing cartilage and/or subchondral bone injury and loss in
the
inflamed joints of such mammals. Such damage to the cartilage and/or
subchondral
bone is a natural sequelae of the process of osteoarthritis and its aftermath
when it
occurs in the mammal. The ability to achieve this unexpected effect is
referred to as
"chondroprotection".
BACKGROUND OF THE INVENTION
Nonsteroidal antiphlogistika (NSAIDs), such as acetylsalicylic acid (ASA),
diclofenac, indomethacin, ibuprofen and naproxen, are widely used in the
clinic. From
a pharmacological point of view they act as inhibitors of the cyclooxygenase
(COX).
Pyrrolizines which pharmacologically act similar, are known from numerous
publications. For instance, antiphlogistically active pyrrolizines are
described in Arch.
2o Pharm. 319, 65-69 (1986); 319, 231-234 (1986); 318, 661-663 (1985); 318,
663-664
(1985); 319, 500-505 (1986); 319, 749-755 (1986); 327, 509-514 (1994); 330,
307-312
(1997) as well as in J. Med. Chem. 1987, 30, 820-823 and 1994, 37, 1894-1897.
Further pyrrolizines can be taken from US 5,260,451 (corresponding to EP
0397175) as well as from WO 95/32970; WO 95/32971; and WO 95/32972. These
compounds are represented by the structural formula
Are Are
R4 ~ ~g ~X i
I ~ Ar2 /~- Ar2
R5 N~ R7 N
R3 R5 R6 R4 R3
and share an annellated diarylpyrrol moiety as well as a third acidic residue
R3. The
compounds are characterized by a high lipophilicity, good bioavailability and
half-lifes
in the medium range, s. Drugs of the Future, 1995, 20 (10):1007-1009.
Further pyrrolizines of similar constitution are described in DE 198 45 446.6
and WO 01/05792. Moreover, alkylsulfinylbenzoyl and alkylsulfonylbenzoyl
substituted
pyrrolizines, according to US 4,232,038, are said to have anti-inflammatory,
analgetic


CA 02457540 2004-02-10
2
and antipyretic properties. According to DE 196 24 290.8 and DE 196 24 289.4
certain
compounds of this type have a lipid-reducing action.
ML3000 ([2,2-dimethyl-6-(4-chlorophenyl)-7-phenyl-2,3-dihydro-1 H-
pyrrolizine-5 yl]-acetic acid) of the Formula (la)
CI
is a non-antioxidant balanced dual inhibitor of COX and 5-Lipoxygenases (5-
LO) (3). The drug is a nonselective inhibitor of COX, inhibiting both COX-1
and COX-
2. This drug has analgetic, antipyretic and anti-inflammatory activity, and
has been
demonstrated to have potent anti-inflammatory action in a number of animal
models
including carrageenan-induced paw edema in the rat, and rat adjuvant arthritis
(4).
Osteoarthritis (OA) is the most common of musculosceletal diseases. It mainly
affects the weight-bearing diarthrodial joints such as the hip and knee, but
also affects
other joints such as interphalangual joints and the spine. The structural
changes of this
disease include the progressive erosion of the articular cartilage, the
formation of
osteophytes and, at the clinical stage of the disease, a variable degree of
synovial
inflammation. Also associated with these changes is a significant remodeling
of the
subchondral bone, which, according to several studies, is believed to be
predominantly
an excessive bone resorption in the early stage of the disease, followed by
excessive
bone formation leading to bone sclerosis and an increased thickening of the
subchondral bone.
The mechanisms leading to the development and progression of
structural changes seen in osteoarthritis (OA) are multiple and complex, and
remain
largely unknown. They involve not only cartilage, where a number of
morphological
changes are observed, but also the synovial membrane which is the site of an
inflammatory reaction of variable degree and severity (1 ). There are a number
of
pathways believed to be responsible for the catabolism of cartilage matrix
including the
upregulation of soluble factors, e.g., interleukin-1 (IL-1), tumor necrosis
factor-a (TNF-
3o a), and prostaglandins, which can induce loss of articular cartilage.
Direct injury to
chondrocytes also stimulates matrix metalloprotease (MMP) activity, e.g.,


CA 02457540 2004-02-10
3
collagenases, stromelysins and gelatinases, and the production of various
inflammatory mediators (2).
It has to be considered that metabolic processes continuously occur in any
given joint that are necessary for its repair and normalization subsequent to
it being
subjected to an insult such as a traumatic injury.
Accordingly, in order for a compound to be an acceptable chondroprotective
agent it must first of all be capable of sustaining such chondrocyte metabolic
activity,
i.e., of not inhibiting or interfering with the cellular replication and
biosynthesis of matrix
components which are part of the healing process. In this regard, the skilled
artisan
1o will recognize that many NSAIDs display a marked inhibitory action on the
biosynthesis
of the principal components of the extracellular matrix.
At the same time an acceptable chondroprotective agent must be capable of
counteracting the degradative action of mediators such as various cytokines,
prostaglandins and proteinases on the cartilage. Accordingly, it has been
accepted in
the art that potential chondroprotective drugs should be evaluated both as to
their
positive effects on anabolic pathways as well as to their ability to inhibit
catabolic
processes. Catabolic events which have typically been monitored include, inter
alia,
the release and inhibition of matrix degrading enzymes, effects on
prostaglandin and
leukotriene biosynthesis, and the ability of the drug to inhibit IL-1 mediated
degradation of articular cartilage.
A number of drugs like NSAIDs, with activity directed at inhibiting COX
enzymes, have been in use for many years. Although they may effectively reduce
the
symptoms of the osteoarthritis such as pain, they have shown limited ability
in
reducing the in vivo progression of experimental OA (8,9). While treatment
with
Tenidap and Carprofen, two NSAIDs with both cyclooxygenase-1 (COX-1 ) and COX-
2
inhibitory activity, were shown to exhibit anti-OA effects (8,9) other NSAIDs,
such as
diclofenac or ASA, were ineffective (10) or even demonstrated to accelerate
cartilage
damage in the experimental dog model of OA (11 ). Similarly, in humans, a
recent
study in knee OA patients has demonstrated that, based on X-ray criteria,
treatment
with tiaprofenic acid, a further NSAID, over a 5-year period could not retard
the
progression of cartilage damage and that indomethacin even accelerated its
progression (15).
Surprisingly, it has been found that certain annellated pyrrole compounds,
such
as ML3000, significantly reduce the development of lesions in experimental dog
OA.
The protective effect of these compounds was particularly evident in the
reduction in
the development of cartilage lesions. This phenomenon was associated not only
with


CA 02457540 2004-02-10
4
a significant inhibition of both PGE2 and LTB4 production, but also with an in
situ
reduction in two major catabolic factors involved in cartilage degradation,
namely IL-1 f3
and collagenase-1.
SUMMARY OF THE INVENTION
Thus, the present invention relates to the use of annellated pyrrole compounds
represented by the general formula (I):
R1
,X
R2
R7
R6 ~R4 R3
R5
wherein
X represents
CR8R9, S, O, NR12 or C(O);
A represents
CR10R11 or a bond between X and the atom carrying radicals R6
and R7;
the first of radicals R1, R2, R3 represents
aryl, optionally substituted with one or more than one substituents
independently selected among the group consisting of halogen, alkyl,
halogenoalkyl, alkoxy, aryloxy, halogenoalkoxy, alkylthio, hydroxy,
nitro, alkylsulfinyl, alkylsulfonyl, sulfamoyl, N-alkylsulfamoyl, N,N-di-
alkylsulfamoyl, alkylsulfonamido and alkylsulfon-N-alkylamido; or
an aromatic or non-aromatic, mono- or bicyclic, optionally
benzoannellated, heterocyclic group having 1, 2 or 3 heteroatoms
independently selected from N, O and S and optionally being
substituted with one or more than one substituents independently
selected among the group consisting of halogen, alkyl,
halogenoalkyl, alkoxy, aryloxy, halogenoalkoxy, alkylthio, hydroxy,


CA 02457540 2004-02-10
vitro, alkylsulfinyl, alkylsulfonyl, sulfamoyl, N-alkylsulfamoyl, N,N-di-
alkylsulfamoyl, alkylsulfonamido and alkylsulfon-N-alkylamido;
the second of radicals R1, R2, R3 represents
5 alkyl, optionally substituted with one or more than one substituents
independently selected among the group consisting of halogen,
cycloalkyl, alkoxy, trifluormethoxy, hydroxy and trifluormethyl;
cycloalkyl, optionally substituted with one or more than one
substituents independently selected among the group consisting of
halogen, alkyl, halogenoalkyl, cycloalkyl, alkoxy, halogenalkoxy and
hyd roxy;
aryl, optionally substituted with one or more than one substituents
independently selected among the group consisting of halogen, alkyl,
halogenoalkyl, alkoxy, aryloxy, halogenoalkoxy, alkylthio, hydroxy,
vitro, alkylsulfinyl, alkylsulfonyl, sulfamoyl, N-alkylsulfamoyl, N,N-di-
alkylsulfamoyl, alkylsulfonamido and alkylsulfon-N-alkylamido; or
an . aromatic or non-aromatic, mono- or bicyclic, optionally
benzoannellated, heterocyclic group having 1, 2 or 3, heteroatoms
independently selected from N, O and S ~ and optionally being
substituted with one or more than one substituents independently
selected among the group consisting of halogen, alkyl,
halogenoalkyl, alkoxy, aryloxy, halogenoalkoxy, alkylthio, hydroxy,
vitro, alkylsulfinyl, alkylsulfonyl, sulfamoyl, N-alkylsulfamoyl, N,N-di-
alkylsulfamoyl, alkylsulfonamido and alkylsulfon-N-alkylamido;
the third of radicals R1, R2, R3 represents
H, alkyl, halogenoalkyl, hydroxyalkyl, -CHO, -COOH, halogen, cyano,
alkylsulfonyl, sulfamoyl or B-Y,
wherein
B represents alkylene or alkenylene, optionally substituted with
hydroxy or alkoxy;
Y represents -COOH, S03H, OPO(OH)Z, OP(OH)2, -CHO or
tetrazolyl; or
the second and the third of radicals R1, R2, R3 represent,


CA 02457540 2004-02-10
6
together with the atom they are attached to, saturated or unsaturated
cycloalkyl;
R4-R11, which may be the same or different, represent
hydrogen, alkyl, hydroxyalkyl, alkoxyalkyl, hydroxy, COOH or
acyloxy, where vicinal radicals may also represent bonds or geminal
radicals, together with the C atom they are attached to, may also
represent carbonyl or cycloalkyl;
R12 represents
hydrogen, alkyl or phenyl,
and optical isomers, physiologically acceptable salts and derivatives thereof,
for treating or preventing degeneration or destruction of articular cartilage
and/or subchondral bone.
The term "alkyl, alkoxy etc." includes linear or branched alkyl groups, such
as
CH3, CZHS, n-propyl, CH(CH3)2, n-butyl, CH(CH3)-CzHS, isobutyl, C(CH3)3; n-
pentyl or n-
hexyl, in particular GH3, CZHS or CH(CH3)2, preferably having - unless
otherwise stated
- 1 to 8, in particular 1 to 6 and more preferably 1 to 4 carbon atoms; as a
substituent
of a radical R1 to R12 "alkyl, alkoxy etc." preferably comprises 1 to 4 carbon
atoms.
Substituted "alkyl, alkoxy etc." includes in particular:
halogenoalkyl, i.e., alkyl, which is partially or completely substituted with
fluoro,
chloro, bromo and/or iodo, e.g. CHZF, CHF2, CF3, CH2GI, 2-fluoroethyl, 2-
chloroethyl or
2,2,2-trifluoroethyl; as a substituent of a radical R1 to R12 halogenoalkyl
preferably
means CHFZ and especially CF3;
halogenoalkoxy, i.e., alkoxy, which is partially or completely substituted
with
fluoro, chloro, bromo and/or iodo, e.g. halogenoalkoxy residues corresponding
to the
afore-mentioned halogenoalkyl residues; as a substituent of a radical R1 to
R12
halogenoalkoxy preferably means OCHF2 and especially OCF3;
alkoxyalkyl, i.e., alkyl substituted by alkoxy, e.g. -CH2-OCH3 or 2-
Methoxyethyl;
hydroxyalkyl, i.e., alkyl which is - preferably mono - substituted by hydroxy,
e.g., hydroxymethyl or 2-hydroxyethyl;


CA 02457540 2004-02-10
7
trifluoromethylalkyl, i.e. alkyl, which is - preferably mono - substituted by
trifluoromethyl, e.g., the residues as described in respect of hydroxyalkyl
which are
substituted with trifluormethyl instead of hydroxy;
trifluoromethoxyalkyl, i.e. alkyl, which is - preferably mono - substituted by
trifluoromethoxy, e.g., the residues as described in respect of hydroxyalkyl
which are
substituted with trifluormethxy instead of hydroxy;
cycloalkylalkyl, i.e., alkyl, which is - preferably mono - substituted by
cycloalkyl, e.g. the residues as described in respect of hydroxyalkyl which
are
substituted with cyclopropyl, cyclobutyl, cyclopentyl or cyclohexyl instead of
hydroxy.
The term "cycloalkyl" includes mono- or bicyclic alkyl groups, such as
cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, etc., preferably having -
unless
otherwise stated - 3 to 9, in particular 3 to 7 and more preferably 5 or 6
carbon atoms.
The term "alkylene" includes linear or branched alkylene groups, such as
methylene and ethylene, preferably having - unless otherwise stated - 1 to 8,
in
particular 1 to 6 and more preferably 1 to 4 carbon atoms. If alkylene is
substituted
with hydroxyl or alkoxy, monosubstitution is preferred.
The term "alkenylene" includes linear or branched, mono- or polyunsaturated
alkylene groups, such as ethenylene, preferably having - unless otherwise
stated - 2
to 8, in particular 2 to 6 and more preferably 2 to 4 carbon atoms. If
alkenylene is
substituted with hydroxyl or alkoxy, monosubstitution is preferred.
Acyloxy means -OCOR, wherein R represents alkyl or aryl. Preferred
examples are acetyloxy and benzoyloxy.
-COOAIkyI means alkoxycarbonyl, such as CO-OCH3, CO-OC2H5, CO-OCH2
CZH5, CO-OCH(CH3)2, n-butoxycarbonyl, CO-OCH(CH3)-C2H5, CO-OCH~-CH(CH3)2,
CO-OC(CH3)3, in particular CO-OCH3, CO-OC2H5, CO-OCH(CH3)Z or CO-OCH2
CH(CH3)Z.
-COOAIkPhenyl means an alkoxycarbonyl group which is substituted on the
alkyl moiety with phenyl, such as benzyloxycarbonyl.
Alkylthio means -S-Alkyl and is also referred to as alkylsulfanyl or
alkylmercapto, such as SCH3, SCZHS, SCH~-C2H5, SCH(CH3)2, n-butylthio, 1
methylpropylthio, 2-methylpropylthio, SC(CH3)3. Methylthio is preferred.
Alkylsulfinyl means -S(O)-Alkyl and is also referred to as alkylsulfoxo, such
as
SO-CH3, SO-C2H5, n-propylsulfinyl, 1-methylethylsulfinyl, n-butylsulfinyl, 1
methylpropylsulfinyl, 2-methylpropylsulfinyl, 1,1-dimethylethylsulfinyl.
Methylsulfinyl is
preferred.


CA 02457540 2004-02-10
8
Alkylsulfonyl means -S(O)z-Alkyl and is also referred to as alkylsulfone, such
as SOz-CH3, SOz-CZHS, n-propylsulfonyl, SOz-CH(CH3)z, n-butylsulfonyl, 1-
methylpropylsulfonyl, 2-methylpropylsulfonyl, SOz-C(CHa)3. Methylsulfonyl is
preferred.
Sulfamoyl means -S(O)zNHz and is also referred to as amidosulfonyl or sulfonic
acid amid.
N-Alkylsulfamoyl means mono-substituted sulfamoyl -S(O)zNH-Alkyl, e.g.
-S(O)zNH-CH3.
N,N-Dialkylsulfamoyl means di-substituted sulfamoyl -S(O)zN-(Alkyl)z, wherein
the N-bounded alkyl residues may be the same or different, e.g. -S(O)zN(CH3)z.
Alkylsulfonamido means -NHS(O)z-Alkyl, such as NHSOz-CH3, NHSOz-C2H5, n-
propylsulfonamido, NHSOz-CH(CH3)z, n-butylsulfonamido, 1-
methylpropylsulfonamido,
2-methylpropylsulfonamido, NHSOz-C(CH3)3. Methylsulfonamido is preferred.
Alkylsulfon-N-alkylamido means -N(Alkyl)S(O)z-Alkyl, wherein the N- and the
S-bounded alkyl residues may be the same or different, e.g N(CH3)SOz-CH3.
Carbonyl, CHO, -COOH, -S03H means >C=0, formyl, carboxy,
carboxycarbonyl and sulfo, respectively.
"Aryl" preferably means naphthyl and in particular phenyl.
The term "halogen" includes a fluoro, chloro, bromo or iodo atom. Usually
fluoro and chloro, and in some cases also bromo are preferred.
2o "Heterocyclic residues" include in particular 5- or 6-membered heterocyclic
residues which may be aromatic or non-aromatic, mono- or bicyclic, and/or
benzoannellated. Examples are nitrogen-containing heterocyclic residues, such
as
pyrrolyl, imidazolyl, pyrazolyl, pyridazinyl, pyrazinyl, indolyl, chinolinyl,
especially pyridyl,
pyrimidyl and isochinolinyl. The aromatic residues also include heterocyclic
residues
which contain an oxygen or a sulfur atom, such as thienyl, benzothienyl,
furanyl and
especially benzofuranyl. Also included are heterocyclic residues which contain
2 or more
than 2 different heteroatoms, such as thiazolyl, isothiazolyl, thiadiazolyl,
isoxazolyl and
oxazolyl. Thienyl, ~ pyridyl and thiazolyl are preferred aromatic
,heterocyclic residues.
Non-aromatic residues include nitrogen-containing heterocyclic residues, such
as
pyrrolidinyl, piperidinyl and piperazinyl. This also includes heterocyclic
residues which
contain 2 or more than 2 different heteroatoms, such as morpholinyl.
Substituted residues, in particular alkyl, cycloalkyl, aryl and heteroaryl,
are
preferably mono-, di- or tri-substituted.


CA 02457540 2004-02-10
9
The [a]-annelland may be 6- or especially 5-membered, heterocyclic or
especially alicyclic, if alicyclic, then unsaturated or especially saturated,
and/or
substituted or unsubstituted.
The [a]-annellated pyrrole compounds of Formula (I) include in particular
those
wherein X represents CR8R9 and A represents a bond between X and the atom
carrying radicals R6 and R7 (pyrrolizines); X represents CR8R9 and A
represents
CR10R11 (indolizines); X represents NR12 and A represents a bond between X and
the atom carrying radicals R6 and R7 (pyrrolo[1,2-a]imidazoles); X represents
S and A
represents a bond between X and the atom carrying radicals R6 and R7
(pyrrolo[2,1
b]thiazoles); X represents S and A represents CR10R11 (pyrrolo[2,1-b]1,3-
thiazines);
X represents O and A represents CR10R11 (pyrrolo[2,1-b]1,3-oxazines); X
represents
O and A represents a bond between X and the atom carrying radicals R6 and R7
(pyrrolo[2,1-b]oxazoles), residues not mentioned having the meanings given
above.
If the [a]-annelland is a 5-membered unsaturated residue, especially R4 and
R6 represent a bo'r~d, such as, e.g., in pyrrolizine, pyrrolo[2,1-b]imidazole
and
pyrrolo[2,1-b]thiazole. If the [a]-annelland is a 6-membered unsaturated
residue,
especially R4 and R6, such as, e.g., in pyrrolo[2,1-b]1,3-thiazine,
pyrrolo[2,1-b]1,3
oxazine or 5,6-dihydroindolizine, and optionally also R8 and R10, such as,
e.g., in
indolizine, represent a bond.
Without being bound to a specific [a]-annelland, according to a particular
embodiment of the invention, R4-R7 which may be the same or different
represent
hydrogen or alkyl. According to a further particular embodiment of the
invention, at
least one of radicals R4, R5, R6 and R7 represents hydroxyalkyl, in particular
hydroxymethyl, and the remaining radicals among R4, R5, R6 and R7
independently
represent H or alkyl. According to this embodiment it is preferred that R4 is
hydroxyalkyl, in particular hydroxymethyl, and R5 is H or alkyl, and R6, R7
independently are H or alkyl. According to a further particular embodiment of
the
invention, one of radicals R8 and R9 represents H, alkyl, hydroxyalkyl or
alkoxyalkyl
and the other represents hydroxyl, alkoxy, carboxyl or acyloxy, or R8 and R9
together
with the C atom they are attached to, represent a carbonyl group.
6,7-Dihydro-5H-pyrrolizines are especially useful, i.e. compunds of Formula
(I),
wherein X represents CR8R9, A represents a bond between X and the atom
carrying
radicals R6 and R7, and R4, R5, R6, R7, R8, R9 which may be the same or
different,
have the meanings as given above and preferably represent hydrogen or alkyl.
6,7-
Dihydro-5H-pyrrolizines wherein R4 to R9 are hydrogen or at least one or two
of


CA 02457540 2004-02-10
radicals R4 to R9, for instance R6 und/oder R7, represent alkyl, in particular
methyl,
are especially preferred.
According to an important aspect of the present invention, compounds of
Formula (I), wherein the first and the second of radicals R1, R2, R3,
preferably R1 and
5 R2, independently represent an II-electron-rich system selected from aryl
and
aromatic heterocyclic residues, in particular phenyl, optionally substituted
with one or
more than one substituents that in particular are independently selected among
the
group consisting of halogen, alkyl and halogenoalkyl, in particular CF3, R1
being
preferably unsubstituted phenyl and R2 being preferably 4-substituted phenyl,
are
10 especially useful.
According to a further important aspect of the invention, compounds of Formula
(I), wherein the third of radicals R1, R2, R3, preferably R3, represents an
acidic
residue such as COOH or B-Y, wherein Y is COOH and B preferably represents
alkylene, or represents a precursor of an acidic residue such as B-Y, wherein
Y is
tetrazolyl, are especially useful.
The use of [6-(4-chlorophenyl)-2,2-dimethyl-7-phenyl-2,3-dihydro-1 H-
pyrrolizine-5-yl]-acetic acid (ML3000) represented by Formula (la):
its physiologically acceptable salts and derivatives, e.g., physiologically
hydrolysable esters, is especially preferred.
Physiologically acceptable salts include acid or base addition salts.
Acid addition salts are, for instance, salts of compounds of Formula (I) with
inorganic acids, such as hydrochloric acid, sulfuric acid, nitric acid or
phosphoric acid,
or with organic acids, in particular carboxylic acids, e.g. acetic acid,
tartaric acid, lactic
acid, citric acid, malic acid, amygdalic acid, ascorbic acid, malefic acid,
fumaric acid,
gluconic acid or sulfonic acid, e. g. methanosulfonic acid, phenylsulfonic
acid and
toluenesulfonic acid, and the like.
Base addition salts are, for instance, salts of compounds of Formula (I) with
inorganic bases, such as sodium or potassium hydroxide or with organic bases,
such
as mono-, di- or triethanolamine, and the like.


CA 02457540 2004-02-10
11
Physiologically acceptable derivatives include in particular prodrugs of the
compounds of formula (I) which are reconverted in vivo to the compounds of
formula
(I) or an active form thereof (metabolite). Examples are hydrolysable esters
of the
compounds of formula (I) wherein the third of radicals R1, R2, R3 represents
an acidic
residue, e.g. alkyl (the third of radicals R1, R2, R3 comprising the
functionality
COOAIkyI), aralkyl (the third of radicals R1, R2, R3 comprising the
functionality
COOAlkaryl, e.g., COOAIkPhenyl), pivaloyloxymethyl, acetoxymethyl, phthalidyl,
indanyl and methoxymethyl esters thereof.
According to a particular aspect, the present invention relates to the use of
chondroprotective agents which are selected among the compounds of Formula
(I).
The term "chondroprotective agent" as used herein will be understood to refer
to those compounds whose chief site of action is the cartilage. It will also
be
appreciated that such chondroprotective agents may also possess anti-
inflammatory
action with regard to' the synovium, may positively impact the biosynthesis of
cells in
subchondral bone and other connective tissues such as synovial fibroblasts,
and may
mediate inflammatory cell migration so as to impede the inflammatory process.
The present invention provides methods of treatment, and pharmaceutical
compositions useful therein as well as suitable packaging therefor, which are
applicable to mammals which suffer from or in the future may suffer from
injury,
2o damage or loss of articular cartilage and/or subchondral bone in one or
more joints of
such a mammal.
Using the compounds of Formula (I) has particular advantages over other
NSAIDs, especially those more established in use, which may actually
exacerbate the
progress of osteoarthritis, especially when long-term application is
indicated. It is
surprising that the compounds of Formula (I) are useful in treating or
preventing such
articular cartilage damage while simultaneously having no adverse impact on
the
course of inflammation in the mammal joint involved.
The, ability of the compounds of formula (I) to'reverse the disease process
which ultimately leads to articular cartilage and/or subchondral bone
destruction and
loss has far-reaching implications for the safe and effective treatment of
mammals,
especially those which are in the early stages of articular cartilage and/or
subchondral
bone degeneration or destruction.
As used herein, the term "mammal(s)" denotes any mammal, preferably
humans, cat, dog or horse, of which there are a large number of different
breeds.
In accordance with the present invention, treating or preventing the
degeneration or destruction of articular cartilage and/or subchondral bone in
one or


CA 02457540 2004-02-10
12
more joints of a mammal in need of such treatment, comprises administering to
said
mammal an amount therapeutically effective for treating or preventing said
degeneration or destruction of articular_ cartilage and/or subchondral bone,
of one or
more than one compound of Formula (I).
Said treatment or prevention especially comprises ameliorating, diminishing,
actively treating, reversing or preventing any degeneration or destruction,
e.g. injury,
damage or loss, of articular cartilage and/or subchondral bone, especially
subsequent
to said early stages of said degeneration or destruction. The expression
"treating or
preventing" as used herein with reference to the administration of the
chondroprotective compounds of the present invention, is intended to refer to
both the
therapeutic objective of said administration as well as the therapeutic
results actually
achieved by said administration. As above-discussed, the extent of therapy
accomplished by administration of said compounds may range from an
amelioration to
a significant diminishing of the course of the disease, and beyond to active
treatment
of the disease, including a reversal of the disease process. The .higher
degrees of
therapeutic effectiveness result in the prevention of any injury, damage or
loss of
articular cartilage and/or subchondral bone subsequent to the early stages of
degeneration in said articular cartilage and/or subchondral bone.
The expression "the early stages of degeneration in articular cartilage and/or
subchondral bone" is intended to mean the very beginning of the initial
pathologic
changes in the articular cartilage and/or subchondral bone which define and
are the
result of a disease process.
Cartilage is a fibrous connective tissue existing in several forms, e.g.,
hyaline
cartilage, elastic cartilage, and fibrocartilage. It is a connective tissue
comprising
water, collagen and proteoglycans which together create a unique fiber-
reinforced
water gel which is stiff but resilient and has considerable shock-absorbing
capacity.
Articular cartilage is cartilage to be found in the joints of mammals. It
comprises living
cells (chondrocytes) which generate 'and are surrounded by the interstitial
materiel
generally referred to as the extracellular matrix. Chondrocytes producing the
extracellular matrix of the cartilage are highly active, and the integrity of
this matrix is
maintained by an equilibrium between the actions of the catabolic cytokines IL-
1a, (3
and TNFa and the anabolic cytokines IGF and TGF(3. IL-1a, (3 and TNFa act by
inducing the production of specific matrix degrading metalloproteases, while
IGF and
TGF~3 act as growth factors by inducing the production of the macromolecular
building
blocks of cartilage, collagen and the proteoglycans. Other cytokines and their


CA 02457540 2004-02-10
13
inhibitors, as well as tissue inhibitors of metalloprotease (TIMP), also
influence this
equilibrium, referred to as matrix homeostasis.
The term "metalloprotease" as used herein is intended to refer to the matrix
metalloproteases (MMPs), especially including those in this family of enzymes
which
usually exhibit elevated concentrations during articular cartilage
degeneration, i.e., the
stromelysins, the collagenases, and the gelatinases. Collagenase is generally
responsible for the degradation of native collagen; stromelysin is generally
responsible
for the degradation of the proteoglycans; and gelatinase is generally
responsible for
the degradation of denatured collagen. An enzyme with MMP properties,
aggrecanase, is also included within this term, since it is responsible for
the proteolysis
of cartilage proteoglycan aggregates which are present during the early stages
of
cartilage degeneration. The three collagenases present in articular cartilage
during
the early stages of degeneration are collagenase-1 (MMP-1 ), collagenase-2
(MMP-8),
and collagenase-3 (MMP-13). Of the three stromelysins, stromelysin-1 (MMP-3),
stromelysin-2 (MMP-10), and stromelysin-3 (MMP-11 ), only stromelysin-1
appears in
articular cartilage during the early stages of its degeneration.
Since osteoarthritis is defined as the failure of the diarthrodial (movable,
synovial-lined) joint, it follows that in such a joint there will always be
found at least two
movable bony surfaces that would meet but for the fact that they are
surrounded by
the synovial membrane, which secretes synovial fluid, a transparent alkaline
viscid
fluid which fills the joint cavity, and articular cartilage, which is
interposed between the
articulating bony surfaces, usually in place of the synovial membrane at that
point.
The earliest gross pathologic finding in osteoarthritis is softening of the
articular
cartilage in habitually loaded areas of the joint surface, which in the case
of the knee
joint of the mammal, especially in models of osteoarthritis involving
transection of the
cruciate ligament in the knee joint, consists of the femoral condyle and the
tibial
plateau. With progression of osteoarthritis the integrity of the cartilage
surface is lost
and the articular cartilage thins, with vertical clefts extending into the
depth of the
cartilage in a process called fibrillation. Joint motion may cause fibrillated
cartilage to
shed segments that expose the bone underneath (subchondral), which then
undergoes sclerosis. Subchondral cysts also develop which may be filled with
synovial
fluid. At the joint margins osteophytes (bone spurs) form.
Changes in the subchondral bone also play a role in the pathology of cartilage
degeneration and destruction. Studies of the joints of mammals, especially
dogs,
which have undergone anterior cruciate ligament transection reveals
subchondral
sclerosis and osteopenia, i.e., bone loss in the subchondral trabeculae.
Subsequent to


CA 02457540 2004-02-10
14
these changes, there is a thickening of the subchondral plate. The loss of
subchondral bone increases the mechanical strain on the overlying articular
cartilage,
leading to its degeneration. The subsequent thickening of the subchondral
plate
negatively affects intrinsic repair mechanisms and thereby contributes to the
progression of cartilage breakdown.
The breakdown of the extracellular matrix of the cartilage is accompanied by
mitotic division of the chondrocytes which then form in clusters. There is a
reduction in
the glycosaminoglycan components of the cartilage and patchy proteoglycan
depletion. In many areas fibrocartilage, characterized by an extracellular
matrix of
thick, compact parallel collagenous bundles, replaces hyaline cartilage.
However, it
should be noted that these and the above-described pathologic changes in the
articular cartilage are characteristic of later stages of osteoarthritis, and
that
hypertrophy, i.e., thickening of the articular cartilage occurs first, as
shown by the
cruciate-deficient mammal, especially dog knee joint model. Cartilage
thickening
results from increased water content, an increase in proteoglycan synthesis,
and an
increase in both the content and concentration of proteoglycans in the
articular
cartilage. This stage of hypertrophic repair of the articular cartilage may
persist for
some time, but the repair cartilage tissue which is formed lacks the
resiliency and
resistance to mechanical stress possessed by normal hyaline cartilage.
Eventually,
proteoglycan production subsides and the chondrocytes are no longer able to
maintain
their extracellular matrix. This end stage results in full-thickness loss of
articular
cartilage.
The early stages of the pathologic changes leading to cartilage injury and
loss
involve attempted repair through increased synthesis of matrix macromolecules.
The
makeup of the repair cartilage is deficient however, due to altered
composition and
distribution of the glycosaminoglycan component and a change in its capacity
to
aggregate with the hyaluronic acid component. Particles released during these
pathologic changes may also lead to inflammatory changes in the synovial
membrane.
However, despite this ongoing pathology, the initial stages of cartilage
injury and loss
may be asymptomatic with relatively little pain. Accordingly, an appropriate
objective is
to identify those extracellular matrix components and cytokines for which
measurable
changes may be identified which profile a mammal subject in the early stages
of
cartilage injury and loss before focal cartilage loss can be identified
radiographically.
Meeting this objective will permit diagnostic classification of mammals which
are
candidates for early pharmacological intervention before significant cartilage
degeneration occurs.


CA 02457540 2004-02-10
Said pathologic changes in the articular cartilage include changes in the
composition, form and density of the articular cartilage from that present
before the
onset of said disease process, which result in a degradation of the beneficial
properties of said articular cartilage including strength, resilience,
elasticity,
5 conformational integrity and stability, viability, and the ability to
successfully resist
various kinds of mechanical stress, especially the ability to absorb
mechanical shocks.
These pathologic changes in composition especially include changes in the type
and
amount of glycosaminoglycans and collagen fibers present in the articular
cartilage.
Pathologic changes in the subchondral bone include sclerosis thereof,
1o increasing density with decreasing resilience and elasticity thereof, and a
diminishing
ability to successfully resist various kinds of mechanical stress, especially
the ability to
absorb mechanical shocks. These pathologic changes especially include improper
repair of trabecular microfractures with trabecular thickening, and pathogenic
changes
in osteoblastic metabolite production and differentiated phenotype.
15 Synovitis, i.e., inflammation of the synovium, the synovial membrane, can
contribute to the pathology of cartilage injury and loss. Synovial
inflammation is
characterized by extensive infiltration of the synovial fluid by mono-nuclear
cells, by
synovial membrane cell hyperplasia, and by lymphoid aggregates. Synovitis
contributes significantly to cartilage injury in rheumatoid and other
inflammatory
arthropathies. The role of synovial inflammation in the early stages of OA are
less well
understood, however synovitis is present at the clinical stage of OA.
According to one aspect of the present invention, the use of compounds of
Formula (I) is directed to treating or preventing the degeneration or
destruction of
articular cartilage and/or subchondral bone, wherein said degeneration or
destruction
is associated with osteoarthritis. In particular, said use is directed to
treating or
preventing the pathologic changes invloved therewith. Thus, the present
invention also
relates to the treatment of osteoarthritis, wherein said treatment is
accompanied by a
therapeutically useful impact oE1 articular cartilage andlor subchondral bone.
Treating or preventing the degeneration or destruction of articular cartilage
3o and/or subchondral bone may also comprise administering in addition to one
or more
than one compound of Formula (I), one or more members selected from the group
consisting essentially of polysulfated glycosaminoglycan (PSGAG), glucosamine,
chondroitin sulfate (CS), hyaluronic acid (HA), pentosan polysulfate (PPS),
doxycycline, and minocycline.
Further, the compounds of Formula (I) of the present invention may also be
combined with other therapeutically active ingredients which would be readily
apparent


CA 02457540 2004-02-10
16
to the skilled artisan in this field, and which will usually be determined by
the
circumstances under which the therapeutic agent of the present invention is
administered. For instance, where a joint has become seriously infected at the
same
time by microorganisms, e.g., bacteria, fungi, protozoa, virus and the like,
the active
ingredient of the present invention will desirably be administered in
combination with
one or more antibiotic, antifungal, antiprotozoal, antiviral or similar
therapeutic agents.
Also, the active ingredient of the present invention may be administered in
combination
with NSAIDs as well with inhibitors of other mediators of inflammation.
Additional
classes of such inhibitors and examples thereof include, e.g., H~ -receptor
antagonists;
kinin-B~ - and BZ -receptor antagonists; prostaglandin inhibitors such as PGD-
, PGF-
PGIz-, and PGE-receptor antagonists; thromboxane AZ (TXA2)-inhibitors; PAF-
receptor antagonists; gold in the form of an aurothio group together with
various
hydrophilic groups; immunosuppressive agents, e.g., cyclosporine,
azathioprine, and
methotrexate; anti-inflammatory glucocorticoids, e.g., dexamethasone; broad-
~ spectrum antiparasitic antibiotics, e.g., the avermectins and the
milbemycins;
penicillamine; hydroxychloroquine; anti-gout agents, e.g., colchicine,
xanthine oxidase
inhibitors, e.g., allopurinol, and uricosuric agents, e.g., probenecid,
sulfinpyrazone, and
benzbromarone.
Because the , early stages of articular cartilage degeneration are prevalent
among geriatric mammals, it will be appreciated by those skilled in the art
that the
compounds of Formula (I) may also be administered in combination with
therapeutic
agents intended for the treatment of disease conditions, syndromes and
symptoms
which are also found in abundance in older mammals. Such therapeutic agents
and
the conditions which they are used to treat include, e.g., cognitive
therapeutics to
counteract memory loss and impairment; and antidyskinetic/antiparkinsonian
agents,
e.g., selegeline. Another large class of such therapeutic agents includes anti-

hypertensives and other cardiovascular drugs intended to offset hypertension,
myocardial ischemia including angina, congestive heart failure, and myocardial
infarction, e.g., diuretics, vasodilators such as hydralazine, (3-adrenergic
receptor
antagonists such as propranolol, angiotensin-II converting enzyme inhibitors
(ACE-
inhibitors) such as enalapril used to treat geriatric mammals with mitral
insufficiency,
and enalapril alone and in combination with neutral endopeptidase inhibitors,
angiotensin II receptor antagonists such as losartan, renin inhibitors,
calcium channel
blockers such as nifedipine, sympatholytic agents such as methyldopa, a2-
adrenergic
agonist such as clonidine, a-adrenergic receptor antagonists such as prazosin,
and


CA 02457540 2004-02-10
17
HMG-CoA-reductase inhibitors (anti-hypercholesterolemics) such as lovastatin
or
atorvastatin. Still other classes of such therapeutic agents include
antineoplastic
agents, especially antimitotic drugs including the vinca alkaloids such as
vinblastine
and vincristine, for treating various cancers; therapeutic agents for treating
renal
failure; anti-obesity drugs for treating excess weight problems in mammals;
anti-
parasitic drugs for treating both endo- and ecto-parasites which commonly
afflict
mammals; and anti-pruritic drugs for treating various types of pruritis in
mammals.
Other types of drugs which can be used in combination with the anti
inflammatory agents of the present invention include growth hormone
secretagogues;
strong analgesics; local and systemic anesthetics; and H2-receptor antagonists
and
other gastroprotective agents. It will be recognized by those of ordinary
skill in this art
that some of the above combinations of therapeutic agents will be used most
frequently to treat various acute conditions in mammals, e.g., bacterial
infections
occurring simultaneously with degenerative joint disease. However, there would
be an
equal if not greater interest on the part of such skilled persons in treating
chronic
conditions in mammals.
In accordance with a regimen which would be used for this purpose, it is
contemplated that the compounds of Formula (I) would be administered in
combination
with other medications used on a regularly scheduled basis for treating
chronic
conditions such as hyperlipidemia. It is also envisioned that administration
in
combinations could assume a number of different forms and still be within the
scope of
the present invention. For example, the compounds of Formula (I) might simply
be
formulated with one or more of the other therapeutic agents which are to form
the
intended combination, into a convenient dosage form, such as an oral tablet,
containing all of the drugs forming the combination. Varying half-lives for
the different
drugs could be accommodated by the person skilled in preparing formulations by
creating controlled-release forms of said drugs with different release times
so that
relatively uniform dosing was achieved. A medicated feed used as the dosage
form .
could also be prepared in accordance with well known principles in the art of
formulation, in which the drugs used in the combination were simply present
together
in admixture in the feed composition. The present invention also contemplates
co-
administration in which the combination of drugs is achieved by the
simultaneous
administration of the drugs to be given in combination. Such co-administration
could
even be by means of different dosage forms and routes of administration. The
present
invention further contemplates the use of such combinations in accordance with
different but regular and continuous dosing schedules whereby desired plasma
levels


CA 02457540 2004-02-10
18
of the drugs involved were maintained in the mammal being treated, even though
the
individual drugs making up the combination were not being administered to said
mammal simultaneously. All such combinations would be well within the skill of
the art
to devise and administer.
When the compounds of Formula (I) are to be used as active ingredients in the
methods and compositions of the present invention, they can be incorporated
into
standard pharmaceutical dosage forms. Thus, the present invention also relates
to
pharmaceutical compositions comprising a pharmaceutically acceptable carrier
and an
amount therapeutically effective for treating or preventing said degeneration
or
destruction of articular cartilage and/or subchondral bone, of a compound of
Formula
(I) as above-defined. For example, they are useful when administered in
systemic or
local, oral or parenteral applications and for this purpose are combined with
the usual
pharmaceutical excipients, diluents and adjuvants, e.g., organic and inorganic
inert
carrier materials such as water, gelatin, lactose, starch, magnesium stearate,
talc,
vegetable oils, gums, polyalkyleneglycols, etc. These pharmaceutical
preparations
can be employed in a solid form, e.g., as tablets, capsules, and especially in
combination with or for admixture with a palatable food item suitable for
mammals; or
they can be administered in liquid form, e.g., as solutions and elixirs.
Pharmaceutical
excipients and adjuvants which can be added include preservatives,
antioxidants,
antimicrobial agents and other stabilizers; wetting, emulsifying, and
suspending
agents, and anticaking compounds; fragrance and coloring additives;
compositions for
improving compressibility, or to create a delayed-, sustained-, or controlled-
release of
the active ingredient; and various salts to change the osmotic pressure of the
pharmaceutical preparation or to act as buffers. Particular dosage forms which
have
been used with success include a 5% mixed-micelle solution of ML3000 for
intravenous injection, a 3% palatable paste, and oral tablets.
The therapeutically effective amount of a compound of Formula (I) as defined
may be administered systemically to said mammal, wherein . said systemic
administration comprises: (1 ) injection or infusion into suitable body
tissues or cavities
of a pharmaceutical composition containing said compound in suitable liquid
form such
as aqueous solutions, emulsions or suspensions for intraarterial, intra- or
transdermal
(including subcutaneous), or intraspinal especially intrathecal and most
commonly
intramuscular or intravenous delivery thereof; or for serving as a depot for
delivery
thereof; (2) instillation into suitable body tissues or cavities of a
pharmaceutical
composition containing said compound in suitable solid form, e.g., comprising
a matrix
of bio-compatible and bio-erodible materials in which particles of a solid


CA 02457540 2004-02-10
19
chondroprotective compound of Formula (1) are dispersed, or in which,
possibly,
globules or isolated cells of a liquid chondroprotective compound of Formula
(1) are
entrapped, for serving as a solid implant composition for delayed-, sustained-
, and/or
controlled-release delivery thereof; or (3) ingestion or administration of a
pharmaceutical composition containing said compound in suitable solid or
liquid form
for transdermal delivery thereof, for instance a transdermal patch or a
subepidermal
(subcuticular) implant, for peroral delivery thereof.
A substantial number of the dosage forms described herein may be formulated
so as to provide controlled-, sustained-, and/or delayed release of the active
ingredient
from said dosage form.
A useful controlled release dosage form of ML3000 in accordance with the
present invention is one which maintains a ML3000 plasma level greater than
100
ng/mL for most of the day after a single oral dose at 5 mg/kg. Preferred oral
controlled
release dosage forms of ML3000 in accordance with the present invention are
ones
which maintain a plasma ML3000 concentration greater than 100 ng/mL for a
period of
time greater than that for which an immediate release dosage form of ML3000
maintains a comparable plasma level, when said immediate release dosage farm
and
controlled release dosage form are administered at the same dose.
Immediate release ML3000 dosage forms containing doses of 2.5 and 5 mg/kg
maintain a plasma ML3000 concentration above 100 and 200 ng/mL for 8 hours,
respectively.
Preferred peroral dosage forms for systemic administration are solids, e.g.,
palatable oral compositions such as fast dissolving palatable wafers, tablets,
capsules,
caplets, efc., and liquids, e.g., solutions, suspensions, emulsions, etc.
Pharmaceutical
compositions of special types suitable for oral administration to mammals may
be
used, and include, but are not limited to such items as an oral paste to be
delivered to
the back of the tongue of the mammal being treated, a granular form to be
delivered
through incorporation in the mammal's food, and a..chewable form wherein the
active
ingredient is consumed along with the palatable chew, or a chewable form which
may
deliver the active ingredient by leaching from the body of the chew which is
not
consumed, during mastication by the mammal being treated.
Said therapeutically effective amount of a compound of Formula (I) as defined
may also be administered locally to said mammal, wherein said local
administration
comprises: (1) injection or infusion into a local site of degeneration or
destruction of
articular cartilage and/or subchondral bone of a pharmaceutical composition
containing
said compound of formula (I) in suitable liquid form for delivery thereof,
including


CA 02457540 2004-02-10
components which provide delayed-release, controlled-release, and/or sustained-

release of said compound into said local site; or for serving as a depot for
delivery
thereof wherein said composition provides storage of said compound and
thereafter
delayed-, sustained-, and/or controlled-release thereof; or (2) instillation
of a
5 pharmaceutical composition containing said compound in suitable solid form
for
serving as a solid implant for delivery thereof, said composition optionally
providing
delayed-, sustained-, and/or controlled-release of said compound to said local
site.
Local administration is focused on suitable articular tissues into which the
chondroprotective compound of Formula (I) may be injected, infused, implanted,
10 deposited, inserted, or instilled. Such administration may include, but is
not limited to,
that which is intraarticular, intrachondrial, intracostal, intraligamentous,
intramedulary,
intramuscular, intraosteal, intrapelvic, intraspinal, intrasternal,
intrasynovial, intratarsal,
intrathecal, or intravenous.
Pharmaceutical compositions in liquid form containing the chondroprotective
15 compound of Formula (I) offer the advantage of permitting injections of the
liquid into
or in close proximity to the articular site. By injection of the compound of
Formula (I)
directly into the joint, it is possible to achieve a high concentration of
said compound in
a short period of time, thus not only substantially enhancing access of said
compound
to the joint tissues, and thus the therapeutic activity of the compound of
Formula (I),
20 but also at the same time minimizing the occurrence of untoward adverse
reactions
that might otherwise occur. The result is a high local concentration of the
compound
of Formula (I) with a correspondingly low systemic carryover concentration.
Injections may also be made of pharmaceutical compositions containing the
chondroprotective compound of Formula (I), where the pharmaceutical
composition is
in delayed-release, controlled-release, or sustained-release form. These
formulations
of recognized composition may be a solids, semi-solids, gels or other
liquid/solid
combinations in which an erodible matrix or series of coatings is used to
provide a
continuous release of the compound of Formula (I) at a predetermined rate or
at
variable rates if desired. The terms "extended-release" and "long-acting" as
well as
others are used to describe these formulations. All of these employ various
combinations of bioerodible polymers, e.g., various cellulosic polymers, and
natural
materials, e.g., corn ~ starch and magnesium stearate, to obtain slow and/or
uniform
dispensing of the compound of Formula (I) contained within the matrix. These
pharmaceutical compositions may be injected into the articular site if
suitably liquid or
suspendable, or may be delivered by other means if more solid in nature.


CA 02457540 2004-02-10
21
The therapeutically effective amount for treating or preventing articular
cartilage and/or subchondral bone degeneration or destruction, of the compound
of
Formula (I), is administered to a mammal being treated in an amount expressed
as
milligrams per kilogram of body weight of said mammal, per day: "mg/kg/day".
The
expression "per day" as used herein should not be interpreted as necessarily
requiring
that any particular dosage form be administered on a daily basis to the mammal
being
treated. The expression "per day" is merely an indication of the smallest
convenient
but arbitrary segment of time which is being used as part of the overall unit
for
measuring the dose of chondroprotective compound being administered. The dose,
i.e., the therapeutically effective amount of a compound of Formula (I) for
treating or
preventing articular cartilage and/or subchondral bone degeneration or
destruction will
usually range from about 0.1 mg/kg/day to about 20.0 mg/kg/day, preferably
from
about 0.1 mg/kg/day to about 12.0 mg/kg/day, more preferably from about 0.5
mg/kg/day to about 10.0 mg/kg/day, and most preferably from about 0.5
mglkg/day to
about 8.0 mg/kg/day. Typical dosage forms and amounts for ML3000 would include
oral administration of ML3000 at a dose rate of 2.5-5.0 mg/kg/day of body
weight.
It is necessary for the skilled artisan, not only to determine the preferred
route
of administration and the corresponding dosage form and amount, but said
artisan
must also determine the dosing regimen, i.e., the frequency of dosing. In
general
2o terms it is most likely that the choice will be between once-a-day (s.i.d.)
dosing and
twice-a-day (b.i.d.) dosing, and that the former wilt provide more rapid and
profound
therapy, while the latter will provide less profound but more sustained
therapy.
However, this generalization does not take into account such important
variables as
the specific type of articular cartilage or subchondral bone degeneration or
destruction
involved, the specific therapeutic agent involved and its pharmacokinetics,
and the
specific patient (mammal) involved. For an approved product in the
marketplace,
much of this information is already provided by the results of clinical
studies carried out
to obtain sucf~, approval. In other cases, such informati8n may be obtained in
a
straightforward manner in accordance with the teachings and guidelines
contained in
3o the instant specification taken in light of the knowledge and skill of the
artisan. The
results which are obtained can also be correlated with data from corresponding
evaluations of an approved product in the same assays.
It is also contemplated that in accordance with the present invention there
will
also be provided a package suitable for use in commerce for treating or
preventing the
degeneration or destruction of articular cartilage and/or subchondral bone in
one or
more joints of a mammal in need of such treatment, comprising a suitable outer


CA 02457540 2004-02-10
22
carton and an inner container removably housed therein; enclosed in said
container a
suitable dosage form of a compound of Formula (I) as described hereinabove;
and
associated with said carton or container printed instructional and
informational
material, which may be attached to said carton or to said container enclosed
in said
carton, or displayed as an integral part of said carton or container, said
instructional
and informational material stating in words which convey to a reader thereof
that said
active ingredient, when administered to a mammal in a condition of
degeneration or
destruction of articular cartilage and/or subchondral bone in one or more
joints thereof,
will ameliorate, diminish, actively treat, reverse or prevent any injury,
damage or loss
of articular cartilage or subchondral bone. In a preferred embodiment said
package
comprising carton and container as above-described will conform to all
regulatory
requirements relating to the sale and use of drugs for the treatment of
animals,
including especially said instructional and informational material.
It is also contemplated that in accordance with the present invention there
will
further be provided a package of the type described immediately above,
comprising a
suitable container as described; enclosed in said container an oral dosage
form of a
compound of Formula (I); and associated with said container printed
instructional and
informational material as above-described.
The method of the present invention can be further defined to comprise two
2o basic steps: (I) establishing the status of a candidate mammal as presently
or
prospectively being in a condition of degeneration or destruction of articular
cartilage
and/or subchondral bone in one or more joints of said mammal, thereby
confirming
that said mammal is in need of such treatment; and thereupon (II) treating or
preventing said condition by administering to said mammal an amount
therapeutically
effective for treating or preventing said degeneration or destruction of
articular
cartilage and/or subchondral bone, of a chondroprotective compound of Formula
(I).
The various aspects of Step (II) have already been discussed above in detail.
Accordingly, the aspects of Step (I) will now be discussed in detail.
As far as diagnosis is concerned, it is expedient to establish the status of a
mammal which is a candidate for treatment in accordance with the present
invention
as to whether or not the mammal is presently or prospectively in a condition
of
degeneration or destruction of articular cartilage and/or subchondral bone in
one or
more joints of said mammal. The expression "presently or prospectively" as
used
herein is intended to mean that in accordance with the below-discussed methods
of
making that determination, it is possible to identify a candidate mammal as
either
being presently in need of such treatment, or as very likely or expected to be
in need


CA 02457540 2004-02-10
23
of such treatment in the short term future. Prospective need of treatment may
be
established by those determinations of positive factors which from the
experience of
the artisan lead directly to the condition of articular cartilage and/or
subchondral bone
degeneration or destruction. For example, the artisan may establish from
clinical
examination of a mammal that it has incipient hip dysplasia, and may confirm
this
conclusion with radiographic evidence from which it may be determined in
accordance
with established methods of measurement that the mammal will develop hip
dysplasia
within the short term future.
The status of said mammal as presently or prospectively being in said
condition
of degeneration or destruction and especially in said early stages, and thus
in need of
such treatment, is in particular determined by:
(A) positive results from the clinical arthroscopic examination and evaluation
of
the joints of the candidate mammal. The diagnosis of incipient or realized hip
dysplasia has already been discussed. Other clinical symptomology and signs
would
include those gained from direct examination of the joints of the candidate
mammal;
(B) performance of any invasive surgical procedure on one or more,joints of
the
candidate mammal which would be under most circumstances be sufficient reason
by
itself to conclude that treatment was needed. This follows from the fact that
invasive
surgery on the joint of a mammal inevitably degrades the ability of that joint
to bear its
accustomed load as efficiently as before surgery.. The increased mechanical
stress on
the joint would, in the experience of the skilled artisan, lead directly to
the early stages
of articular cartilage and/or subchondral bone degeneration. Such surgery on
the joint
would also produce an effusion of blood and other fluids containing cytokines
and
other factors which are causative agents of inflammation, and would thereby
permit
their migration and absorption into the solid tissues of the joint, including
the cartilage
and/or subchondral bone. The artisan would appreciate that this would also
lead
directly to the early stages of articular cartilage and/or subchondral bone
degeneration;
(C) positive results from an examination of one or more joints of said mammal
using noninvasive procedures including radiographic and magnetic resonance
imaging
(MR/). The latter technique is better for evaluating soft tissues than is the
former. MR/
is a technique for multiplanar body imaging that shows increased soft tissue
contrast
resolution. Since MRI can visualize soft tissue changes, it is suitable for
imaging the
pathology of the early changes in articular cartilage and subchondral bone
degeneration;
(D) positive results from any biochemical test performed on body fluids or
joint
tissue of the candidate mammal with respect to one or more of the following


CA 02457540 2004-02-10
24
substances: increased interleukin-1 beta (IL-1 (3); increased tumor necrosis
factor
alpha (TNFa); increased ratio of IL-1 (3 to IL-1 receptor antagonist protein
(IL-1 Ra);
increased expression of p55 TNF receptors (p55 TNF-R); increased interleukin-6
(IL-
6); increased leukemia inhibitory factor (LIF); unchanged or decreased insulin-
like
growth factor-1 (IGF-1 ); decreased transforming growth factor beta (TGF(3);
unchanged or decreased platelet-derived growth factor (PDGF); unchanged or
decreased basic fibroblast growth factor (b-FGF); increased keratan sulfate;
increased
stromelysin; increased ratio of stromelysin to tissue inhibitor of
metaNoproteases
(TIMP); increased osteocalcin; increased alkaline phasphatase; increased CAMP
1o responsive to hormone challenge; increased urokinase plasminogen activator
(uPA);
increased cartilage oligomeric matrix protein; and increased collagenase.
IL-1, which occurs as IL-1 a and IL-1 (3, is a catabolic cytokine which
mediates
articular cartilage injury and loss in mammal joints. It acts by suppressing
the
synthesis of type 11 collagen found in articular cartilage while promoting the
synthesis
of type I collagen characteristic of fibroblasts; by inducing the production
of enzymes
involved in matrix degradation; and by suppressing the ability of chondrocytes
to
synthesize new proteoglycans. The number of IL-1 receptors on the surface of
chondrocytes in artieular cartilage in the early stages of degeneration which
must be
occupied in order to elicit catabolic enzyme production is only one-fourth as
great as
2o that required normally (1% vs. 4%). 1L-1 and its modulator IL-1Ra are
produced in an
autocrine and paracrine fashion by the same synovial macrophages, and IL-1 Ra
production may be increased in the presence of granulocyte macrophage colony-
stimulating factor (GM-CSF). However, there is a significant disparity between
1L-1
and IL-1 Ra potency, with approximately 130-fold more IL-1 Ra being required
to
abolish the effects of IL-1, as measured in chondrocytes and cartilage
explants. Any
imbalance between IL-1 and IL-1 Ra will further exacerbate the degeneration of
articular cartilage.
Consequently, it is also an appropriate objective to measure levels of IL-1
and
IL-1Ra and their ratios in mammals in the early stages of articular cartilage
degeneration, and the same values in mammals not so afflicted so that
measurable
changes may be identified which profile a mammal subject in the early stages
of
cartilage injury and loss before focal cartilage loss can be identified
radiographically.
These results provide diagnostic classification of mammals which are
candidates for
early pharmacological intervention before significant cartilage degeneration
occurs.
Furthermore, the proportion of IL-1a and IL-1(3-secreting macrophages
occurring in


CA 02457540 2004-02-10
the synovial fluid and synovial tissue of a joint in the early stages of
articular cartilage
degeneration can be detected and is significantly greater than the proportion
of similar
cells isolated from synovial fluid and synovial tissue from normal joints,
i.e., joints
which are not in the early stages of articular cartilage degeneration. Here
again, these
5 results provide diagnostic classification of mammals which are candidates
for early
pharmacological intervention before significant cartilage degeneration occurs.
Further still, changes in subchondral bone occur before gross alterations in
the
articular cartilage become apparent because cytokines responsible for
initiating and
maintaining the inflammatory process gain access to the lower layers of
cartilage
1o through microcracks across the calcified zone. The metabolism of the
chondrocytes
involved is adversely affected, and in addition the chondrocytes in the.middle
zone of
the articular cartilage produce many cytokines, including those responsible
for initiating
and maintaining the inflammatory process. These chondrocytes, acting in an
autocrine fashion, thus contribute to the destruction of their own
extracellular matrix.
15 The increased water content of the articular cartilage also facilitates
this process by
increasing diffusion of the inflammatory cytokines throughout the matrix. It
is,
consequently, an appropriate objective to measure levels of various
inflammatory
cytokines produced by chondrocytes, synovial cells, and/or subchondral
osteocytes in
mammals, especially canines during the process of articular cartilage
degeneration,
20 and the same values in mammals not so afflicted so that measurable changes
may be
identified which profile a mammal subject in the early stages of cartilage
injury and
loss before focal cartilage loss can be identified radiographically. These
results
provide diagnostic ,classification of mammals which are candidates for early
pharmacological intervention before significant cartilage degeneration occurs.
25 Tumor necrosis factor alpha (TNFa) has only one-tenth the potency of IL-1
with
regard to the degeneration of articular cartilage, but its concentration in
synovial fluid
significantly increases in the knee joints of mammals, especially with
sectioned
cruciate ligaments compared to the opposite, unoperated knee. There is also
enhanced expression of p55 TNF receptors (TNF-R) on chondrocytes isolated from
articular cartilage present in such knee joints. Accordingly, since TNFa plays
a role in
the pathologic changes which take place in the early stages of cartilage
injury and
loss, it is likewise an appropriate objective to measure levels of TNFa and
TNF-R in
the joints of mammals in the early stages of articular cartilage degeneration,
and the
same values in mammals not so afflicted so that measurable changes may be
identified which profile a mammal subject in the early stages of cartilage
injury and


CA 02457540 2004-02-10
26
loss before focal cartilage loss can be identified radiographically. These
results
provide diagnostic classification of mammals which are candidates for early
pharmacological intervention before significant cartilage degeneration occurs.
Interleukin-6 (IL-6) is a multifunctional cytokine, but plays an inflammatory
role
and is found in elevated levels in joints and synovial fluid from damaged as
compared
to control limbs. IL-6 is also responsible for enhanced expression of TNF-R on
chondrocytes and increased proteoglycan production by chondrocytes, as well as
induction of glycosaminoglycan release. Measurement of IL-6 levels in joints,
synovial
fluid and chondrocytes of mammal joints in the early stages of articular
cartilage injury
and loss, compared to control, can be used as a diagnostic tool for
identifying
mammals that are appropriate candidates for pharmacological treatment, before
any
focal cartilage loss is evident from radiographic examination.
Leukemia inhibitory factor (LIF) is produced by monocytes, granulocytes, T
cells, fibroblasts, and other cell types associated with inflammatory
conditions.
Synoviocytes and chondrocytes synthesize and secrete LIF in the presence of IL-
1 ~3
and TNFa. Thus, measurement of comparative increases in levels of LIF can be
used
diagnostically to select mammal candidates for pharmacologic treatment of the
early
stages of articular cartilage injury and loss.
The degeneration, injury and loss of articular cartilage in mammals is caused
by an imbalance between the cytokines that drive the above-described catabolic
processes and those cytokines which are responsible for maintaining the
synthetic and
proliferative responses of the chondrocytes in the cartilage. Insulin-like
growth factor
(IGF-1 ), transforming growth factor beta (TGF(3), platelet-derived growth
factor
(PDGF), and fibroblast growth factor, e.g., basic fibroblast growth factor
(bFGF), are
all mitogenic with respect to the chondrocytes and stimulate matrix synthesis
in
articular cartilage.
Insulin-like growth factor (IGF) exists as types I and II, and IGF-I is a
potent
mediator of cartilage synthesis. Furthermore, it reduces degradation and
promotes
synthesis of proteoglycans even in the presence of IL-1 ~i and TNFa. Serum
levels of
IGF-1 are maintained by high-affinity binding proteins (IGF-BPs) and IGF-1 is
important in both bone and cartilage turnover. Levels of IGF-1 compared to
control
permit diagnostic evaluation of mammal candidates for early pharmacologic
treatment
of articular cartilage degeneration.
Transforming growth factor (TGF~i) is produced by chondrocytes and is a
powerful mitogen for the turnover of both cartilage and bone. Further, it
stimulates the


CA 02457540 2004-02-10
27
synthesis of matrix and has anti-inflammatory activity. It also inhibits the
degradation
of the matrix by stimulating protease inhibitor production, and blocking
collagenase
and metalloprotease release. Further still, it promotes cartilage repair by
stimulating
production of collagen, fibronectin, inhibitors of plasminogen activators, and
tissue
inhibitors of metalloproteases (TIMP) by various cells in the mammal joint.
Synovial
fluid levels of TGF(3 are low in the joints of mammals in the early stages of
articular
cartilage injury and loss. Consequently, levels of TGF~i compared to control
permit
diagnostic evaluation of mammal candidates for early pharmacologic treatment
of
articular cartilage degeneration.
With the progressive degeneration, i.e., catabolism of the articular cartilage
in
the mammal joint, a number of metabolites are produced which are useful as
markers
of the cartilage degeneration, both as to its occurrence and as to its
advance. For
example, degradation of cartilage by IL-1a and IL-1(3 or TNFa releases
glycosaminoglycans (GAGS), which can be measured in the synovial fluid of a
mammal being tested. Furthermore, GAG levels change after treatment so that it
is
possible to monitor the course of pharmacologic intervention, using synovial
fluid GAG
levels as a marker of articular cartilage turnover.
Since the degradation of articular cartilage involves collagen as well as the
other cartilage components, several collagen products serve as markers of
cartilage
degradation in mammal, especially canine articular cartilage injury and loss.
Type-II
specific collagen breakdown products, e.g., 20-30 amino acid neoepitopes, can
be
identified in body fluids such as synovial fluid, plasma, serum or urine. The
presence
of neoepitopes in these body fluids may be used as indicators of OA onset and
progression.
Keratan sulfate is a particular GAG which has an epitope, 5D4, whose levels in
synovial fluid can be used as a marker of early articular cartilage injury and
loss.
Conversely, levels of chondroitin sulfate, another particular GAG, expressed
as a
number of epitopes, is associated with anabolic events in the articular
cartilage of
mammals in the early stages of cartilage injury and loss. Levels of these
epitopes in
synovial fluid, particularly 3B3, 7D4 and 846, can be determined by specific
monoclonal antibodies which recognize them. The 3B3 epitope is expressed on
chondroitin sulfate chains of cartilage during repair and the remodeling of
the
extracellular matrix, and consequently its levels in synovial fluid correlate
inversely with
those of the above-mentioned 5D4. The expression of 3B3 in newly synthesized
PGs
in the superficial and upper middle layer of the articular cartilage mean that
3B3 is


CA 02457540 2004-02-10
28
associated with early changes in the articular cartilage of mammals in the
early stages
of cartilage degeneration. Accordingly, the determination of 3B3 levels in the
synovial
fluid of test mammals and comparison of these levels with control values
permits the
creation of a diagnostic profile of a mammal that is an appropriate candidate
for early
pharmacologic treatment.
Further markers of cartilage anabolic activity are the propeptides of type II
procollagen (PIIP). Type II is the major collagen of articular cartilage and
it is
produced by the chondr0cytes as procollagen. During the process of collagen
fibril
formation, the noncollagenous aminopropeptide and carboxypropeptide are
cleaved
and released into body fluids, where they can be measured as reflection of
anabolic
activity in the articular cartilage. Levels of carboxy-PIIP will be raised and
its synovial
fluid levels correlate with radiographic evidence of changes in the cartilage.
Accordingly, measurement of carboxy-PIIP levels in synovial fluid and
comparison with
controls permits identification of mammal candidates for early pharmacologic
treatment.
An imbalance in the stromelysin/TIMP ratio in the articular cartilage and
joint
fluids of mammals in the early stages of articular cartilage degeneration is
also useful
in identifying such mammals. Altered joint loading following injury causes the
production of excess stromelysin, an enzyme produced by chondrocytes and
synoviocytes under the influence of IL-1. The concentrations of stromelysin
are also
higher in fibrillated cartilage than they are in cartilage more distal from
the lesion
involved. The increased levels of stromelysin may occur for only a fairly
short period
of time, but where the damage to the joint transcends the tidemark zone of the
articular cartilage, and reaches into the subchondral bone, there is a
substantial
likelihood of subsequent articular cartilage degeneration, usually preceded by
a
stiffening of the subchondral bone.
Further, in the cruciate-deficient mammal model used in detecting the early
stages of articular cartilage degeneration, there is an increased number of~
cells
involved in the synthesis of stromelysin, IL-1a, IL-1(3, and three oncogene
proteins, c-
MYC, c-FOS, and c-JUN. In the synovium these are found mainly in the
superficial
synovial lining cells, while in the cartilage the cells are the chondrocytes
on the
superficial and middle layers and the cells in the fibrillated areas of the
tibial plateau.
Further, stromelysin and IL-1 diffuse into the cartilage matrix of the tibial
plateau.
Stromelysin, which degrades components of connective tissue including
proteoglycans
and type IX collagen, is actively synthesized in the synovium of mammals in
the early


CA 02457540 2004-02-10
29
stages of articular cartilage degeneration, and is the primary proteolytic
enzyme
involved in the cartilage destruction. Increased levels of stromelysin mRNA
are
detectable in the synovia of such mammals, as are increased levels of
collagenase
mRNA. Increased levels of both isoforms of IL-1, but especially IL-1(i,
stimulate the
increased synthesis of stromelysin by enhancing synovial fibroblast induction
of
stromelysin and collagenase gene expression. At the same time, IL-1 does not
induce
mRNA of tissue inhibitor of metalloprotease (T1MP) and the levels of this
inhibitor
remain unchanged while the detectable levels of metalloproteases in the
synovium are
dramatically increased.
The metalloproteases are secreted by chondrocytes as proenzymes which
must be activated before degradation of extracellular matrix macromolecules
can take
place. Activation involves an enzymatic cascade in which serine proteases
including
the piasminogen activatorlplasmin system play a key role.
The integrity of the articular cartilage in a mammal joint depends upon the
adequacy of the support which it receives from the bony bed which it covers,
i.e., the
structural properties of the underlying subchondral bone. Alterations in this
bony bed
precede degradative changes in the articular cartilage. These alterations
include
increased stiffening of the subchondral bone, accompanied by loss of shock-
absorbing
capacity. These subchondral bone changes are caused by inappropriate repair of
trabecular microfractures which result, in turn, from excessive loading of the
joint.
Trabecular thickening of the subchondral bone is part of a bone alteration
leading to
increased bone mineral density and/or volume in affected joints, which in turn
is
caused by a bone cell defect in the osteoblasts, resulting in altered
phenotypic
characteristics in these osteoblast-like cells of the subchondral bone.
These alterations in subchondral bone density are not only evidence of an
imbalance in the bone remodeling process, but also are a key ingredient in
eventual
focal cartilage loss. Bone sclerosis is also due to dysregulation of this bone
remodeling process. Further, site-related differences in oste~blast metabolism
occur
which lead to the production of different cartilage-degrading molecules. These
3o changes in osteoblast metabolites in turn lead to corresponding changes in
chondrocyte metabolism, rendering them more susceptible to cytokine-induced
activity
of the types above-described. This osteoblastic anomaly and differentiated
phenotype
is characterized by divergent production levels of osteocalcin, alkaline
phosphatase,
CAMP responsive to hormone challenge, urokinase plasminogen activator (uPA),
and
insulin-like growth factor 1 (IGF-1 ).


CA 02457540 2004-02-10
Further evidence of subchondral bone activity involvement in eventual
articular
cartilage degeneration is joint space narrowing which may be measured by bone
scintigraphy. These changes in subchondral bone activity are accompanied by
corresponding changes in specific bone cell metabolites, e.g., osteocalcin.
5 Osteocalcin is a vitamin K-dependent, calcium binding bone protein which is
the most
abundant noncollagen protein in bone. Increased levels of osteocalcin are a
marker of
bone turnover in various disease states, including particularly the early
stages of
articular cartilage degeneration. Body fluid, especially synovial fluid levels
of
osteocalcin directly correlate to subchondral bone changes as measured by
10 scintigraphy.
In addition to markers of subchondral bone activity as indicators of the early
stages of articular cartilage degeneration in mammals, metabolites from
cartilage and
synovium activity are also useful as markers which indicate the early stages
of such
cartilage degeneration. For example, detection of increased serum levels of
cartilage
15 oligomeric matrix protein serves as a marker of cartilage turnover.
Similarly, detection
of high levels of hyaluronate in body fluids, especially serum serves as a
marker of
synovial inflammation. In both cases, the increased body fluid, especially
serum levels
of these metabolite markers indicate the early stages of articular cartilage
degeneration.
20 The expression "body fluid" as used herein is intended to include all of
those
accessible body fluids usable as clinical specimens which may contain a
compound
being tested for in sufficient concentration in said fluid to be within the
limits of
detection of the test device or assay being used. Body fluids will thus
include whole
blood, serum, plasma, urine, cerebrospinal fluid, synovial fluid, and
interstitial and
25 other extracellular fluids. Accordingly, the afore-described measurements
usually are
conducted in vitro on a specimen (sample) that has been obtained from the
candidate
mammal.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1: Table listing macroscopic grading of cartilage changes of
femoral condyles and tibial plateaus;
Figure 2: Table listing histological grading of synovial membrane;
Figure 3: Table listing cartilage cell score for collagenase-1 (MMP-1 ) and
synovial membrane cell scores for IL-1 f3 in osteoarthritic dogs;


CA 02457540 2004-02-10
31
Figure 4: Histologic grading of dog osteoarthritic cartilage from femoral
condyles and tibial plateaus;
Figure 5: Levels of PGE2 in the synovial fluids and LTB4 in cultured
synovial explants of osteoarthritic dogs.
Figure 6: Osteocalcin release by dog primary osteoblasts;
Figure 7: Alkaline phophatase activity in dog primary osteoblasts;
Figure 8: ' IGF-1 production by dog primary osteoblasts;
Figure 9: uPA activity by dog primary osteoblasts.
Figure 10: PGE2 production by dog primary osteoblasts;
DESCRIPTION OF PREFERRED EMBODIMENTS
In order to further demonstrate the methods and compositions of the present
invention, there are presented in the paragraphs which follow specific
descriptive
examples of typical procedures which may be employed in carrying out said
methods.
However, said examples are intended to be illustrative only and should not be
taken as
in any way a limitation of the present invention, for which purpose the
present claims
are appended hereto.
EXAMPLE 1
The therapeutic effectiveness of ML3000 on the development of lesions in the
experimental osteoarthritis (OA) dog model was studied. In particular, the
action of
ML3000 on the synthesis of collagenase-1 (MMP-1 ) in cartilage and interleukin-
113 (IL-
1 (3) in synovial membrane as well as on alkaline phophatase activity, IGF-1
production,
osteocalcin release, PEGZ and uPA activity in or by primary osteoblasts was
determined.
Experimental groups
Twenty-one adult crossbred dogs (2-3 years old), weighing 20-25 kg each,
were used in this study. Surgical sectioning of the anterior cruciate ligament
(ACL) of
the right knee through a stab wound was performed on all dogs as previously
described (5,6). Prior to surgery, the animals were anesthetized intravenously
with
pentobarbital sodium (25 mg/kg) and incubated. Following surgery, the dogs
were kept
at a housing farm where they were free to exercise in a large pen.


CA 02457540 2004-02-10
32
The dogs were randomly separated into 3 treatment groups. Group 1 (n = 7
dogs) was made up of dogs that received placebo (encapsulated methylcellulose)
treatment (OA dogs); Groups 2 and 3 (n = 7 dogs per group) received
encapsulated
ML3000 twice daily for 8 weeks at total doses of 2.5 and 5 mg/kg,
respectively,
beginning the day following surgery. The medication was administered 7
days/week
throughout the duration of the study. All dogs were killed 8 weeks after
surgery.
All dogs in each experimental group completed the study. No clinical
signs of drug toxicity, including those related to the gastrointestinal tract,
were noted in
the group of dogs treated with ML3000. The level of daily activity was similar
in all
dogs from the 3 experimental groups, and there was no change in the body
weight of
the dogs during the study period.
Statistical analysis
Values are expressed as mean ~ SEM. Statistical analysis was done
using the Mann-Whitney U test. P values less than 0.05 were considered
significant.
Macroscopic grading
Immediately after killing, the right knee of each dog was dissected,
placed on ice, and the synovial fluid aspirated. Each knee was examined by 2
independent, blinded observers (DVJ, JCF) for gross morphologic changes
including
the presence of osteophyte formation and cartilage lesions as previously
described
(5,6). The degree of osteophyte formation was graded by measuring the maximal
width (mm) of the spur of each femoral condyle. The cartilage changes on the
medial
and lateral femoral condyles and tibial plateaus were graded separately under
a
dissecting microscope (Stereozoom; Bausch & Lomb, Rochester, NY).
The area of the articular surface changes was measured and expressed
in mm2. The depth of erosion was graded on a scale of 0-4, as follows: 0 =
surface
appears normal, 1 = minimal fibrillation or a slight yellowish discoloration
of the
surface, 2 = erosion extended into superficial or middle layers, 3 = erosion
extended
into deep; layers, and'4 = erosion extended to the subcliondral bone.
Osteophytes. In placebo-treated OA dogs, osteophytes were present in 93%
of the condyles, and their widths measured 4.50 ~ 0.66 mm. In dogs treated
with 2.5
mg/kg/day and 5 mg/kg/day ML3000, osteophytes were present in 93% and 86%
respectively. The width measurements of osteophytes in the two treated groups
were
marginally smaller compared with the placebo-treated OA group (3.57 ~ 0.56 and
3.86
~ 0.66 respectively), and these differences did not reach statistical
significance.


CA 02457540 2004-02-10
33
Cartilage. In the placebo-treated OA dogs, cartilage lesions of moderately
severe grade and size were present on both condyles and plateaus with more
severe
lesions on the plateaus (Figure 1 ). There was a significant reduction in the
size of
lesions on the condyles and plateaus of both groups treated with ML3000. In
the 2.5
mglkglday group, the size of lesions on femoral condyles was lower by 39% (P <
0.05)
and by 45% (P < 0.01 ) on tibial plateaus. In the group that received 5
mg/kg/day, the
size of the lesions on femoral condyles was also reduced (61 %, P < 0.04),
while the
effect on tibial plateaus was about the same as those found in the 2.5
mg/kglday
group (54%, P < 0.01 ). Both concentrations of ML3000 significantly reduced
the grade
of lesions on tibial plateaus to about the same extent. Although the lesions
on femoral
condyles of treated dogs showed a tendency towards a reduction in their grade,
it did
not reach statistical significance.
Synovial membrane. Synovium from placebo-treated OA dogs was
hypertrophic, demonstrated a red and yellowish discoloration and contained a
large
number of blood vessels. In dogs treated with ML3000, the synovia was thinner,
contained fewer blood vessels, and the discoloration was less intense compared
with
placebo-treated OA dogs.
Histologic grading
Histologic evaluation was performed on sagittal sections of cartilage
2o from the lesioned areas of each femoral condyle and tibial plateau as
described (7,8).
Specimens were dissected, fixed in TissuFix #2 (Laboratoires Gilles Chaput,
Montreal,
Quebec, Canada), and embedded in paraffin for histologic evaluation. Serial
sections
(5 um) were stained with Safranin O. The severity of the OA lesions was graded
on
scale of 0-14, by 2 independent observers (DJ, JF), using the
histologic/histochemical
scale of Mankin et al (12). This scale evaluates the severity of OA lesions
based on
the loss of staining with Safranin O (scale 0-4), cellular changes (scale 0-
3), invasion
of the tidemark by blood vessels (scale 0-1 ), and structural changes (scale 0-
6, where
0 = normal cartilage structure and 6 --,..erosion of the cartilage down to the
subchoridral
bone). This scoring system was based on the most severe histologic changes
within
each cartilage section.
Representative specimens of synovial membrane from the gutters of
the medial and lateral knee compartments were also dissected from underlying
tissue.
The specimens were fixed in TissuFix #2, embedded in paraffin, sectioned (5
Nm), and
stained with hematoxylin and eosin. Two synovial membrane specimens from each
compartment were examined, serial selections were made throughout the
specimens,


CA 02457540 2004-02-10
34
and each one scored separately. The highest score from each specimen was
recorded. The average was calculated and considered as a unit for the whole
knee.
The severity of synovitis was graded on a scale of 0-10 (13) by 2 independent
observers (DVJ, JCF) adding the scores for 3 histologic criteria: synovial
lining cell
hyperplasia (scale 0-2), villous hyperplasia (scale 0-3), and degree of
cellular
infiltration by mononuclear and polymorphonuclear cells (scale 0-5).
Cartilage. Cartilage from placebo-treated OA dogs presented morphologic
changes including fibrillation and fissures, hypercellularity and cloning, and
a loss of
Safranin O staining. Histologic scores for the lesions on the plateaus were
slightly
more severe than for those on the condyles (Figure 4). In the ML3000-treated
dogs,
the lesions on the condyles were less severe compared with the placebo-treated
OA
dogs, and statistical significance was obtained in the group treated with the
highest
dosage of the drug (5 mg/kg/day). This reduction in the histologic score was
largely
due to a decrease in severity of structural changes and loss of Safranin O
staining.
On tibial plateaus, a statistically significant decrease in the severity of
lesions (P <
0.002 and P < 0.02, respectively) was obtained with both doses of ML3000 (2.5
and 5
mg/kg/day). This results from a decrease in the loss of Safranin O staining
and ,
severity of structural changes in addition to a reduction in cell cloning.
Synovial membrane. Synovia from placebo-treated OA dogs was thick, had
numerous villi, and showed synovial lining cell hyperplasia. In the ML3000-
treated
dogs, there was a significant reduction in villous hyperplasia in the 5
mg/kglday group.
PGE2 measurement in synovial fluid
Synovial fluid taken at the time of sacrifice was centrifuged (14000 g, 15
minutes, 4°C), and supernatants used for PGE2 determination. The level
of PGE2 was
determined using a specific enzyme immunoassay (EIA) (Cayman Chemical, Ann
Arbor, MI).
Synovial fluids from placebo-treated OA dogs contained a high level of
PGE2 (652.8 ~ 149.9 pg/ml). Treatment with ML3000 at 2:5 and 5 mg/kg/day
markedly decreased this level (Figure 5).
LTB4 production in synovial explant culture
Representative specimens of synovial membrane from all dogs were
aseptically dissected from underlying tissue. The specimens were rinsed
several
times in Dulbecco's Modified Eagle's Medium (DMEM; Gibco-BRL Life
Technologies,
Burlington, Ontario, Canada), and 150 mg of tissue incubated (duplicate) for
48 hours
at 37°C in a humidified atmosphere of 5% COa/95% air in DMEM medium in
the


CA 02457540 2004-02-10
presence of 1 Ng/ml lipopolysaccharide (LPS; Sigma-Aldrich Canada, Oakville,
Ontario, Canada). The LTB4 was extracted from cultured synovium explants as
follows: membranes were homogenized in EIA buffer (1 M phosphate, pH 7.4,
containing 1 % BSA, 4 M NaCI, 10 mM EDTA and 0.1 % sodium azide) and
centrifuged
5 (30000 g, 20 minutes, 4°C). Supernatants were collected and the
levels of LTB4
determined using EIA (Cayman Chemical).
Cultured synovium from OA dogs treated with 1 pg/ml LPS contained a
significant amount of LTB4 (2.48 ~ 0.69 pg/mg tissue). Both groups treated
with
ML3000 demonstrated a statistically significant reduction in LTB4 levels
(Figure 5).
10 Immunohistochemistry
Cartilage and synovial membrane specimens were processed for
immunohistochemical analysis as previously described (7,14). Briefly,
specimens
were fixed in 4% neutral buffered formalin for 24 hours, then embedded in
paraffin.
Sections (5 pm) 'of paraffin-embedded specimens were placed on Superfrost Plus
15 slides (Fisher Scientific, Nepean, Ontario, Canada), deparaffinized in
toluene,
dehydrated in a graded series of ethanol, and preincubated with chondroitinase
ABC
(0.25 units/ml) in phosphate buffered saline (PBS; Sigma-Aldrich Canada) for
60
minutes at 37°C. After this, the specimens were washed in PBS, then
again in 0.3%
hydrogen peroxide/PBS for 30 minutes. Slides were further incubated with a
blocking
2o solution (Dako Diagriostics, Mississauga, Ontario, Canada) and 5% skim milk
for 60
minutes, blotted and then overlaid with the primary monoclonal antibody
against
collagenase-1 (100 pg/ml, dilution 1:500, Oncogene Research Products,
Cambridge,
MA) (cartilage) or the primary antibody against human IL-1 f3 (1 Ng/ml,
dilution 1:50;
Biosource International, Camarillo, CA) (synovial membrane) for 18 hours at
room
25 temperature in a humidified chamber.
Each slide was washed 3 times in PBS (pH 7.4) and stained using the
avidin-biotin complex method (Vectastain ABC kit; DAKO Diagnostics Canada).
This
~.xnethod entails incubation in the presence of tie biotin-conjugated
secondary antibody
for 30 minutes at room temperature followed by the addition of the avidin-
biotin-
30 peroxidase complex for 30 minutes. All incubations were carried out in a
humidified
chamber and the colour developed with a 3,3'-diaminobenzidine (DAKO
Diagnostics
Canada) containing hydroxide peroxide. Slides were counterstained with neutral
red
(cartilage) or hematoxylin/eosin (synovium).
To determine the specificity of staining, 3 control procedures were
35 employed according to the same experimental protocol: 1 ) use of absorbed
immune


CA 02457540 2004-02-10
36
serum (1 hour, 37°C) with a 20-fold molar excess of recombinant
collagenase-1 or IL
1 f3; 2) omission of the primary antibody; and 3) substitution of the primary
antibody
with an autologous preimmune serum. The purified antigens used in our study
were
human recombinant collagenase-1 (Oncogene Research Products) or human
recombinant IL-1 (3 (Genzyme, Cambridge, MA, USA).
Several sections were made from each block of cartilage, and slides
from each specimen, were processed for immunohistochemical analysis. Each
section
was examined under a light microscope (Leitz Orthoplan; Wild Leitz, St.
Laurent,
Quebec, Canada) and photographed with Kodak Ektachrome 64 ASA film (Kodak,
Rochester, NY).
Cartilage (Figure 3). In placebo-treated dogs, a large number of chondrocytes
in the superficial layers of cartilage specimens stained positive for
collagenase-1 on
both the condyles and plateaus. In the ML3000-treated OA dogs, both condyles
and
plateaus presented a significant decrease in the chondrocyte cell score for
coilagenase-1 - the effect of which was slightly more pronounced in the group
treated
with the highest dosage of the drug.
Synovial membrane (Figure 3). The examination of the synovium samples
from placebo-treated OA dogs showed the presence of a large number of lining
cells
staining strongly positive for IL-1 f3. In dogs treated with ML3000, there was
a dose-
dependent and significant decrease in the number of cells showing positive
staining for
IL-1 f3.
Morphometric analysis
Cartilage. Quantification of the different antigens in cartilage was done
using a
published method (7,14). The presence of the antigen was estimated by
determining
the number of chondrocytes staining positive in the upper (superficial and
upper
intermediate layers) , zone of cartilage. In this zone, cartilage was divided
into 3
microscopic fields (X 40; Leitz Diaplan), and averaged. For each arthritic
specimen, it
was ensured prior to evaluation that an intact cartilage surface could be
detected and
used as a marker for validation of morphometric analysis. The total number of
chondrocytes and the number of chondrocytes staining positive for the specific
antigen
were determined. The final results were expressed as the percentage of
chondrocytes
staining positive for the antigen (cell score) with the maximum score being
100%.
Each slide was subjected to double blind evaluation resulting in a variation
of < 5%.
The data obtained from the medial and lateral condyles and tibial plateaus
were
considered as independent for the purpose of statistical analysis.


CA 02457540 2004-02-10
37
Synovial membrane. For synovial membrane analysis, a cell score of the
different specimens was determined for each section using our published method
(8).
Each specimen was divided into 5 microscopic fields (X 40) at the synovial
lining level.
The percentage of cells staining positive for the specific antigen was
evaluated in each
field as described above for cartilage. Cell count scores were given
separately for the
synovial lining cells and mononuclear cell infiltrate with the maximum for
each area
being 100%.
Osteoblast analysis
The proximal end of the tibia was removed as below-described, rinsed in a cold
physiological saline solution, and planed on ice prior to and throughout
dissection.
Medial tibial plateaus was extracted to prepare explants and primary bone cell
cultures; no marginal cortical bone tissue was included. The overlying
cartilage was
first removed from the tibial plateaus, and plug explants were dissected out
exclusively
from the midportion of the medial plateau. The trabecular bone tissue was then
dissected away from the subchondral bone plate. All manipulations were
performed
under a magnifying microscope to ensure complete removal of cartilage and
trabecular
bone.
The samples were used to prepare primary cell cultures as described in (16),
with minor modifications. Bone samples were cut into small pieces (2 mm2)
prior to
2o their sequential digestion in the presence of 1 mg/ml type I collagenase
(Sigma) in
Ham's F-12/Dulbecco's modified Eagle's medium (DMEM; Sigma) without serum, at
37°C for 20, 20, and 240 minutes. This treatment removed both adherent
and
remaining bone marrow cells from the cortical bone pieces.
After washing with the same medium, the digested bone pieces were cultured
in BGJ medium containing 20% fetal bovine serum (FBS; Wisent, St. Bruno,
Quebec,
Canada). This medium was replaced every 2 days until cells are observed in the
Petri
dishes, at which time the culture medium was replaced with fresh medium
containing
10% FBS.:~At confluence, cells were passaged once at a:.-ratio of 25,000
cells/cm2 and
are grown in 24-well plates (Falcon, Lincoln Park, NJ) for 5 days prior to
assay. Cells
obtained under these culture conditions showed an osteoblast-like cell
phenotype, as
noted in (16). Conditioning is performed for the last 2 days of culture, in
the presence
or absence of 50nM 1,25(OH)2D3 (1,25-dihydroxyvitamin D) for maximal
stimulation, in
Ham's F-12/DMEM , containing 2% charcoal-stripped FBS, which yields maximal
stimulation of alkaline phosphatase activity and osteocalcin secretion, as
noted (16).
The medium was collected at the end of the incubation and frozen at -
80°C prior to


CA 02457540 2004-02-10
38
assay. Cells were then washed twice with phosphate buffered saline (PBS), pH
7.4,
and solubilized in alkaline phosphatase buffer (100 mM glycine, 1 mM MgCl2, 1
mM
ZnCIZ, 1 % Triton X-100; pH 10.5) for 60 minutes with agitation at
4°C.
Osteocalcin release
Osteocalcin release was measured in conditioned Ham's F-12/DMEM (1:1 )
prepared for the last 2 days of culture of osteoblast-like cells as described
in (16),
containing 2% charcoal-treated FBS, and in the presence of 50 nM 1,25(OH)2D3
or
vehicle (0.1 % ethanol). Nascent osteocalcin was determined using a specific
enzyme
immunoassay (Biomedical Technologies, Stoughton, MA). The detection limit of
this
assay is 0.5 ng/ml, and 2% charcoal-treated FBS contains <0.1 ng/ml
osteocalcin.
Results are shown in Figure 6.
Alkaline phosphatase activity
Cellular alkaline phosphatase activity was determined, on cells used for
osteocalcin release, as the release of p-nitrophenol hydrolyzed from p-
nitrophenyl
phosphate (12.5 mM final concentration) at 37°C for 30 minutes after
solubilizing the
cells in alkaline phosphatase buffer as above-described. Alkaline phosphatase
was
determined immediately on aliquots. Protein determination was performed by the
bicinchoninic acid method described in Smith, P. K.; Krohn, R. L; Hermanson,
G. T.;
Mallia, A. K.; Gartner, F. H.; Provenzano, M. D.; et al.; "Measurement of
Protein Using
Bicinchoninic Acid", Anal Biochem, 150, 1985, 76-85. Results are shown in
Figure 7.
Evaluation of uPA and IGF-1 in primary osteoblasts
For evaluation of uPA and IGF-1, the conditioned media from confluent
osteoblast-like cells fed with Ham's F-12/DMEM, without PBS, but containing 1
insulin-transferrin-selenium mix (ITS, Sigma) for the last 2 days of culture.
First, uPA
levels were determined by specific enzyme-linked immunosorbent assay (ELISA;
American Diagnostics, Greenwich, CT). There was then used the procedure
described in Leprince, P.; Rogister, B.; Moonen, G. A.; "Colorimetric Assay
for the
Simultaneous Measurement of Plasminogen Activators and Plasminogen Ativat~ir
Inhibitors in Serum-Free Conditioned Media from Cultured Cells", Anal Biochem,
177,
1989, 341-346, to determine the activity of uPA via the hydrolysis of the
specific
substrate DL-Val-Leu-Arg-p-nitroanilide (Sigma), which releases p-nitroaniline
that can
be detected at 405 nm. PAI-1 levels are determined by ELISA, using materials
available from American Diagnostics (Greewich, CT). IGF-1 was determined using
a
high-sensitivity ELISA (Diagnostic Systems Laboratories, Webster, TX) that
does not
cross-react with insulin. Internal control studies are performed with the
media alone


CA 02457540 2004-02-10
39
containing 1 °lo ITS, and any values obtained should be below the limit
of detection.
For the conditioned medium of cell culture samples, 3 or 4 supernatants are
pooled,
lyophilized, and then reconstituted in PBS buffer, pH 7.4. Samples are then
treated
according to the method described in Mohan, S.; Bautista, C. M.; Herring, S.
J.;
Linkhart, T. A.; Baylink, D. J.; "Development of Valid Methods to Measure
Insulin-Like
Growth Factors-I and -II in Bone Cell-Conditioned Medium, Endocrinology, 126,
1990,
2534-42. Results are shown in Figure 8 and 9.
Evaluation of PGE2 in primary osteoblasts
PGE2 in primary osteoblasts was determined essentially in the same manner
as described above for synovial fluid. Results are shown in Figure 10.
From the above results it can be concluded that ML3000, a balanced dual
inhibitor of COX/5-LO, can significantly reduce the development of early
experimental
OA at the same time as inhibiting in vivo the production of PGEZ and LTB4. The
protective effect of the drug is believed largely related to the marked
inhibition of major
OA pathophysiological pathways - namely the excess synthesis of IL-1 f3 and
collagenase-1. Some of these effects appeared to be linked to the inhibition
of the
excess production of,LTB4.
REFERENCES
1. Pelletier JP, Martel-Pelletier J, Howell DS. Etiopathogenesis of
osteoarthritis.
In: Koopman WJ, editor. Arthritis & Allied Conditions. A Textbook of
Rheumatology. 14th ed. Baltimore: Lippincott Williams & Wilkins; 2000. p.
2195-245.
2. Martel-Pelletier J, Di Battista JA, Lajeunesse D. Biochemical factors in
joint
articular tissue degradation in osteoarthritis. In: Reginster JY, Pelletier
JP,
Martef-Peifetie~- J, Henrotin Y, editors. Osteoarthritis: Clinical and
experimental
aspects. Berlin: Springer-Verlag; 1999. p. 156-87.
3. Laufer S, Tries S, Augustin J, Dannhardt G. Pharmacological profile of a
new
pyrrolizine derivative inhibiting the enzymes cyclo-oxygenase and 5-
lipoxygenase. Arzneimittelforschung 1994;44:629-36.
4. Laufer S, Tries S, Augustin J, Elsasser R, Albrecht W, Guserle R, et al.
Acute
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7-


CA 02457540 2004-02-10
phenyl-2,3-dihydro-1 H-pyrrolizine-5-yl]-acetic acid. Arzneimittelforschung
1995;45:27-32.
5. Fernandes JC, Martel-Pelletier J, Otterness IG, Lopez-Anaya A, Mineau F,
Tardif G, et al. Effects of tenidap on canine experimental osteoarthritis: I.
5 Morphologic and metalloprotease analysis. Arthritis Rheum 1995;38:1290-303.
6. Pelletier JP, Mineau F, Raynauld JP, Woessner JF Jr, Gunja-Smith Z, Martel-
Pelletier J. Intraarticular injections with methylprednisolone acetate reduce
osteoarthritic lesions in parallel with chondrocyte stromelysin synthesis in
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Currie
MG, et al. Selective inhibition of inducible nitric oxide synthase in
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osteoarthritis is associated with reduction in tissue levels of catabolic
factors. J
Rheumatol 1999;26:2002-14.
8. Fernandes JC, Martel-Pelletier J, Jovanovic D, Tardif G, Di Battista JA,
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15 Coman V, et al. The effects of tenidap on canine experimental
osteoarthritis. II:
Study of the expression of collagenase-1 and interleukin-1 beta by in situ
hybridization. J Rheumatol 1998;25:951-8.
9. Pelletier JP, Lajeunesse D, Jovanovic DV, Lascau-Coman V, Jolicoeur FC,
Hilal G, et al. Carprofen simultaneously reduces progression of morphological
2o changes in cartilage and subchondral bone in experimental dog
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J Rheumatol 2000;27:2893-902.
10. Pelletier JP, Martel-Pelletier J. In vivo protective effects of
prophylactic
treatment with tiaprofenic acid or intraarticular corticosteroids on
osteoarthritic
lesions in the experimental dog model. J Rheumatol Suppl 1991;27:127-30.
25 11. Palmoski MJ; Brandt KD. In vivo effect of aspirin on canine
osteoarthritic
cartilage. Arthritis Rheum 1983;26:994-1001.
12. Mankin HJ, Dorfman H, Lippiello L, Zarins A. Biochemical and metabolic
abnormalities in articular cartilage from osteoarthritic human hips. II.
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13. Pelletier JP, Martel-Pelletier J, Ghandur-Mnaymneh L, Howell DS, Woessner
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14. Moldovan F, Pelletier JP, Hambor J, Cloutier JM, Martel-Pelletier J.
35 Collagenase-3 (matrix metalloprotease 13) is preferentially localized in
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CA 02457540 2004-02-10
41
deep layer of human arthritic cartilage in sifu: In vitro mimicking effect by
transforming growth factor beta. Arthritis Rheum 1997;40:1653-61.
15. Huskisson EC, Berry H, Gishen P, Jubb RW, Whitehead J. Effects of
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16. Lajeunesse, D.; Busque, L.; Menard, P.; Brunette, M. G.; Bonny, Y.;
"Demonstration of an Osteoblast Defect in Two Cases of Human Malignant
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Clin Invest, 98, 1996, 1835-1842.

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ASCENTIA PHARMA INC.
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PELLETIER, JEAN-PIERRE
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