Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
CA 02398184 2002-08-07
WO 01/58441 PCT/USO1/03972
Method and Compositions for Treating an Inflammatory Disease
Area of the Invention
This invention relates compositions and methods for preventing or treating an
inflammatory diseases by administering a phosphodiesterase 4 inhibitor in
combination with
an inhibitor of prostaglandin synthesis, NSAIDs being exemplary.
Background of the Invention
Identification of novel therapeutic agents for treating pulmonary diseases is
made
difficult by the fact that multiple mediators are responsible for the
development of the
disease. Thus, it seems unlikely that eliminating the effects of a single
mediator could have
a substantial effect on all three components of chronic asthma. An alternative
to the
"mediator approach" is to regulate the activity of the cells responsible for
the
pathophysiology of the disease.
One such way is by elevating levels of cAMP (adenosine cyclic 3',5'-
monophosphate). Cyclic AMP has been shown to be a second messenger mediating
the
biologic responses to a wide range of hormones, neurotransmitters and drugs;
[Krebs
Endocrinology Proceedings of the 4th International Congress Excerpta Medica,
17-29,
1973]. When the appropriate agonist binds to specific cell surface receptors,
adenylate
cyclase is activated, which converts Mg+2-ATP to cAMP at an accelerated rate.
Cyclic AMP modulates the activity of most, if not all, of the cells that
contribute to the
pathophysiology of extrinsic (allergic) asthma. As such, an elevation of cAMP
would
produce beneficial effects including: 1) airway smooth muscle relaxation, 2)
inhibition of
mast cell mediator release, 3) suppression of neutrophil degranulation, 4)
inhibition of
basophil degranulation, and 5) inhibition of monocyte and macrophage
activation. Hence,
compounds that activate adenylate cyclase or inhibit phosphodiesterase should
be effective
in suppressing the inappropriate activation of airway smooth muscle and a wide
variety of
inflammatory cells. The principal cellular mechanism for the inactivation of
cAMP is
hydrolysis of the 3'-phosphodiester bond by one or more of a family of
isozymes referred to
as cyclic nucleotide phosphodiesterases (PDEs).
It has been shown that a distinct cyclic nucleotide phosphodiesterase (PDE)
isozyme, PDE IV, is responsible for cAMP breakdown in airway smooth muscle and
inflammatory cells. [Torphy, "Phosphodiesterase Isozymes: Potential Targets
for Novel
Anti-asthmatic Agents" in New Drugs for Asthma, Barnes, ed. IBC Technical
Services Ltd.,
1989]. Research indicates that inhibition of this enzyme not only produces
airway smooth
muscle relaxation, but also suppresses degranulation of mast cells, basophils
and neutrophils
along with inhibiting the activation of monocytes and neutrophils. Moreover,
the beneficial
effects of PDE IV inhibitors are markedly potentiated when adenylate cyclase
activity of
target cells is elevated by appropriate hormones or autocoids, as would be the
case in vivo.
-1-
CA 02398184 2002-08-07
WO 01/58441 PCT/USO1/03972
Thus PDE IV inhibitors would be effective in the lung, where levels of
prostaglandin E2 and
prostacyclin (activators of adenylate cyclase) are elevated. Such compounds
would offer a
unique approach toward the pharmacotherapy of bronchial asthma and possess
significant
therapeutic advantages over agents currently on the market.
In addition, it could be useful to combine therapies in light of the fact that
the
etiology of many pulmonary diseases involves multiple mediators. In this
invention there is
presented the combination of a PDE 4 inhibitor and a non-steroidal anti-
inflammatory for
treating an inflammatory disease or a condition treatable by a PDE4-selective
inhibitor
which has associated with it an inflammatory component related to the
synthesis of
prostaglandins.
Summary of the Invention
In a first aspect this invention relates to a method for treating an
inflammatory
disease in a mammal by administering to a patient in need thereof an effective
amount of a
PDE 4-specific inhibitor and an effective amount of a non-steriodal anti-
inflammatory agent
wherein the drugs are administered concomitantly, or separately and
sequentially where the
sequential administration is close in time or remote in time.
Detailed Description of the Invention
The combination therapy contemplated by this invention comprises administering
a
PDE4 inhibitor with a non-steroidal anti-inflammatory agent to treat an
inflammatory
disease. The compounds may be administered together in a single dosage form.
Or they
may be administered as two different formulations. To illustrate, both drugs
may be
provided separately as oral formulations, or one may be an oral preparation or
as a
suppository or by injection or as an intravenous drip. They may be
administered at the same
time. Or they may be administered close in time or remotely, such as where one
drug is
administered in the morning and the second drug is administered in the
evening.
The PDE4-specific inhibitor useful in this invention may be any compound that
is
known to inhibit the PDE4 enzyme or which is discovered to act in as PDE4
inhibitor, and
which are only PDE4 inhibitors, not compounds which inhibit other members of
the PDE
family as well as PDE4. Generally it is preferred to use a PDE4 antagonists
which has an
IC50 ratio of about 0.1 or greater as regards the IC50 for the PDE IV
catalytic form which
binds rolipram with a high affinity divided by the IC50 for the form which
binds rolipram
with a low affinity.
PDE inhibitors like theophylline and pentoxyfyllin inhibit all or most all PDE
isozymes indiscriminately in all tissues. These compounds exhibit side
effects, apparently
because they non-selectively inhibit all PDE isozyme classes in all tissues.
The target
disease may be effectively treated by such compounds, but unwanted secondary
effects may
be exhibited which, if they could be avoided or minimized, would increase the
overall
-2-
CA 02398184 2002-08-07
WO 01/58441 PCT/USO1/03972
therapeutic effect of this approach to treating certain diseases. For example,
clinical studies
with the selective PDE 4 inhibitor rolipram, which was being developed as an
antidepressant, indicate it has psychotropic activity and produces
gastrointestinal effects,
e.g., pyrosis, nausea and emesis.
For purposes of this disclosure, the cAMP catalytic site which binds R and S
rolipram with a low affinity is denominated the "low affinity" binding site
(LPDE 4) and the
other form of this catalytic site which binds rolipram with a high affinity is
denominated the
"high affinity" binding site (HPDE 4). This term "HPDE4" should not be
confused with the
term "hPDE4" which is used to denote human PDE4.
Initial experiments were conducted to establish and validate a [3H]R-rolipram
binding assay. Details of this work are given in Example 1 below.
To determine whether both the high affinity binding activity and the low
affinity
binding activity resided in the same gene product, yeast were transformed by
known
methods and the expression of recombinant PDE 4 was followed over a 6 hour
fermentation
period. Western blot analysis using an antibody directed against PDE 4
indicated that the
amount of PDE 4 expressed increased with time, reaching a maximum after 3 hour
of
growth. In addition, greater than 90% of the immunoreactive product was in the
high speed
(100,000 x g) supernatant of yeast lysates. [3H]R-Rolipram binding and PDE
activity were
monitored along with protein expression. PDE 4 activity was co-expressed with
rolipram-
binding activity, indicating that both functions exist on the same gene
product. Similar to
results with the Western plot analysis, greater than 85% of the rolipram-
inhibitable PDE
activity and [3H]-rolipram binding activity was found to be present in the
yeast supernatant
fraction.
Overall, most of the recombinant PDE 4 expressed in this system exists as LPDE
4
and only a small fraction as HPDE 4. Consequently, inhibition of recombinant
PDE 4
catalytic activity primarily reflects the actions of compounds at LPDE 4.
Inhibition of PDE
4 catalytic activity can thus be used as an index of the potency of compounds
at LPDE 4.
The potency of compounds at HPDE 4 can be assessed by examining their ability
to
compete for [3H]R-rolipram. To develop SARs for both the low affinity and high
affinity
rolipram binding sites, the potencies of selected compounds were determined in
two assay
systems. Results from experiments using standard compounds were tabulated. As
expected, certain compounds were clearly more potent in competing with [3H]R-
rolipram at
the site for which rolipram demonstrated high affinity binding as compared
with the other
site, the one at which rolipram is a low affinity binder. SAR correlation
between high
affinity binding and low affinity binding was poor and it was concluded that
the SAR for
inhibition of high affinity [3H]R-rolipram binding was distinct from the SAR
for binding to
the low affinity rolipram binding site.
-3-
CA 02398184 2002-08-07
WO 01/58441 PCT/USO1/03972
It is now known that there are at least two binding forms on human monocyte
recombinant PDE 4 (hPDE 4) with which inhibitors interact. One explanation for
these
observations is that hPDE 4 exists in two distinct forms. One binds the likes
of rolipram and
denbufylline with a high affinity while the other binds these compounds with a
low affinity.
The preferred PDE4 inhibitors of use in this invention will be those compounds
which have
a salutary therapeutic ratio, i.e., compounds which preferentially inhibit
cAMP catalytic
activity where the enzyme is in the form that binds rolipram with a low
affinity, thereby
reducing the side effects which apparently are linked to inhibiting the form
which binds
rolipram with a high affinity. Another way to state this is that the preferred
compounds will
have an IC50 ratio of about 0.1 or greater as regards the IC50 for the PDE 4
catalytic form
which binds rolipram with a high affinity divided by the IC50 for the form
which binds
rolipram with a low affinity.
A further refinement of this standard is that of one wherein the PDE4
inhibitor has
an IC50 ratio of about 0.1 or greater; said ratio is the ratio of the IC50
value for competing
with the binding of 1nM of [3H]R-rolipram to a form of PDE 4 which binds
rolipram with a
high affinity over the IC50 value for inhibiting the PDE IV catalytic activity
of a form
which binds rolipram with a low affinity using 1 microM[3H]-cAMP as the
substrate. A
further review explanation with of this test can be found in co-pending U.S.
patent
5,998,428 the text of which is incorporated herein by reference to the extent
that text is
necessary to the practice of this invention.
Most preferred are those PDE4 inhibitors which have an IC50 ratio of greater
than
0.5, and particularly those compounds having a ratio of greater than 1Ø A
preferred
compound is cis 4-cyano-4-(3-cyclopentyloxy-4-methoxyphenyl)cyclohexan-I-
carboxylic
acid (Ariflo~). In addition, the following PDE4 inhibitors may be useful in
the practice of
this invention: AWD-12-281 from Astra (Hofgen, N. et al. 15th EFMC Int Symp
Med
Chem (Sept 6-10, Edinburgh) 1998, Abst P.98); a 9-benzyladenine derivative
nominated
NCS-613 (INSERM); D-4418 from Chiroscience and Schering-Plough; a
benzodiazepine
PDE4 inhibitor identified as CI-1018 (PD-168787; Parke-Davis/Warner-Lambent);
a
benzodioxole derivative Kyowa Hakko disclosed in WO 9916766; V-11294A from
Napp
(Landells, L.J. et al. Eur Resp J [Annu Cong Eur Resp Soc (Sept 19-23, Geneva)
1998]
1998, 12(Suppl. 28): Abst P2393); roflumilast (CAS reference No 162401-32-3)
and a
pthalazinone (WO 9947505) from Byk-Gulden; or a compound identified as T-440
(Tanabe
Seiyaku; Fuji, K. et al. J Pharmacol Exp Ther,1998, 284(1): 162).
The non-steroidal anti-inflammatory drugs (NSAIDs) which may be useful in
this invention are those which inhibit prostaglanding synthesis. It is
believed that
NSAIDs act through inhibition of cyclo-oxygenase-1 (COX-1) and cyclo-
oxygenase-2 (COX-2). Numerous drugs fall into this category. By way of
example,
-4-
CA 02398184 2002-08-07
WO 01/58441 PCT/USO1/03972
one or more of the following NSAIDs can be use herein: aspirin, carprofen,
choline
salicylate, ketoprofen, Mg salicylate, salicylaminde, salsalate, , sodium
salicylate,
sodium thiosalicylate, meclofenamate sodium, oxyphenbutazone, phenylbutazone,
indomethacin, piroxicam, sulindac, tolmetin and tolmetin sodium, mefenamic
acid,
zomerpirac, ibuprofen, fenoprofen, naproxen and naproxen sodium, diclofenac,
flurbiprofen, ketoprofen, ketorolac, trometamol, celecoxib, diflunisal, and
nabumatone. All are available from commercial sources or are well described in
the
medical and other scientific literature.
The combined analgesic and anti-inflammatory effects of NSAIDs and
PDE4-specific inhibitors make this combination particularly useful for the
symptomatic relief of painful and/or inflammatory conditions including
rheumatic
disorders such as rheumatoid arthritis, osteoarthritis, and the
spondyloarthropathies,
and also in peri-articular disorders, and soft-tissue rheumatism. The
combination
may also be useful in treating pulmonary diseases involving an inflammatory
condition.
It is contemplated that both active agents would be administered at the same
time,
or very close in time. Alternatively, one drug could be taken in the morning
and one later in
the day. Or in another scenario, one drug could be taken twice daily and the
other once
daily, either at the same time as one of the twice-a-day dosing occurred, or
separately:
Preferably both drugs would be taken together at the same time.
The present compounds and pharmaceutically acceptable salts which are
active when given orally can be formulated as syrups, tablets, capsules,
controlled-
release preparation or lozenges. A syrup formulation will generally consist of
a
suspension or solution of the compound or salt in a liquid carrier for
example,
ethanol, peanut oil. olive oil, glycerin or water with a flavoring or coloring
agent.
Where the composition is in the form of a tablet, any pharmaceutical carrier
routinely used for preparing solid formulations may be used. Examples of such
carriers include magnesium stearate, terra alba, talc, gelatin, acacia,
stearic acid,
starch, lactose and sucrose. Where the composition is in the form of a
capsule, any
routine encapsulation is suitable, for example using the aforementioned
carriers in a
hard gelatin capsule shell. Where the composition is in the form of a soft
gelatin
shell capsule any pharmaceutical carrier routinely used for preparing
dispersions or
suspensions may be considered, for example aqueous gums, celluloses, silicates
or
oils, and are incorporated in a soft gelatin capsule shell.
Typical parenteral compositions consist of a solution or suspension of a
compound or salt in a sterile aqueous or non-aqueous carrier optionally
containing a
-5-
CA 02398184 2002-08-07
WO 01/58441 PCT/USO1/03972
parenterally acceptable oil, for example polyethylene glycol,
polyvinylpyrrolidone,
lecithin, arachis oil or sesame oil.
Typical compositions for inhalation are in the form of a solution, suspension
or emulsion that may be administered as a dry powder or in the form of an
aerosol
using a conventional propellant such as fluroinated hydrocarbons such as
trichlorofluoromethane.
Preferably the composition for the PDE4 inhibitors is a unit dosage form
such as a tablet or capsule, or a controlled release preparation. While NSAIDs
are
normally taken by mouth, some of them such as diclofenac, ketoprofen,
ketorolac,
piroxicam, and tenoxicam can be given by intramuscular injection. Ketorolac
and
tenoxicam can also be given by intravenous injection.
The active ingredient may be administered from 1 to 6 times a day, sufficient
to exhibit the desired activity. Preferably, the active ingredient is
administered
about once or twice a day, more preferably twice a day.
As for the amount of drug administered, it is believed that for the PDE4
inhibitors
will be administered in an amount of between 1 and 200 micrograms per day per
adult
human. NSIADs be administered in conformity with approved labeling.
Example 1 -- Phosphodiesterase and Rolipram Bindin_~~
Example 1A
Isolated human monocyte PDE 4 and hrPDE (human recombinant PDE4) was
determined to exist primarily in the low affinity form. Hence, the activity of
test
compounds against the low affinity form of PDE 4 can be assessed using
standard assays for
PDE 4 catalytic activity employing 1 microM [3H]CAMP as a substrate (Torphy et
al., J. of
Biol. Chem., Vol. 267, No. 3 pp1798-1804, 1992).
Rat brain high speed supernatants were used as a source of protein and both
enantionmers of [3H]-rolipram were prepared to a specific activity of 25.6
Ci/mmol.
Standard assay conditions were modified from the published procedure to be
identical to the
PDE assay conditions, except for the last of the cAMP: SOmM Tris HCl (pH 7.5),
5 mM
MgCl2, and 1 nM of [3H]-rolipram (Torphy et al., J. of Biol. Chem., Vol. 267,
No. 3
pp1798-1804, 1992). The assay was run for 1 hour at 30° C. The reaction
was terminated
and bound ligand was separated from free ligand using a Brandel cell
harvester.
Competition for the high affinity binding site was assessed under conditions
that were
identical to those used for measuring low affinity PDE activity, expect that
[3H]-cAMP and
5' AMP were not present.
-6-
CA 02398184 2002-08-07
WO 01/58441 PCT/USO1/03972
Example 1 B
Measurement of Phosuhodiesterase Activity
PDE activity was assayed using a [3H]CAMP SPA or [3H]cGMP scintillation
proximity analysis (SPA) enzyme assay as described by the supplier (Amersham
Life
Sciences). The reactions were conducted in 96-well plates at room temperature,
in 0.1 ml of
reaction buffer containing (final concentrations): 50 mM Tris-HCI, pH 7.5, 8.3
mM MgCl2,
1.7 mM EGTA, [3H]CAMP or [3H] cGMP (approximately 2000 dpm/pmol), enzyme and
various concentrations of the inhibitors. The assay was allowed to proceed for
1 hr and was
terminated by adding 50 ~1 of SPA yttrium silicate beads in the presence of
zinc sulfate.
The plates were shaken and allowed to stand at room temperature for 20 min.
Radiolabeled
product formation was assessed by scintillation spectrometry. Activities of
PDE3 and PDE7
were assessed using 0.05 ~tM [3H]CAMP, whereas PDE4 was assessed using 1 uM
[3H]CAMP as a substrate. Activity of PDE1B, PDE1C, PDE2 and PDES activities
were
assessed using 1pM [3H]cGMP as a substrate.
f3HlR-rolipram binding assay
The [3H]R-rolipram binding assay was perFormed by modification of the method
of
Schneider and co-workers, see Nicholson, et al., Trends Pharmacol. Sci., Vol.
12, pp.l9-27
(1991) and McHale et al., Mol. Pharmacol., Vol. 39, 109-113 (1991). R-rolipram
binds to
the catalytic site of PDE4 see Torphy et al., Mol. Pharmacol., Vol. 39, pp.
376-384 (1991).
Consequently, competition for [3H]R-rolipram binding provides an independent
confirmation of the PDE4 inhibitor potencies of unlabeled competitors. The
assay was
performed at 30°C for 1 hr in 0.5 p1 buffer containing (final
concentrations): 50 mM,Tris-
HCI, pH 7.5, 5 mM MgCl2, 0.05% bovine serum albumin, 2 nM [3H]R-rolipram (5.7
x 104
dpm/pmol) and various concentrations of non-radiolabeled inhibitors. The
reaction was
stopped by the addition of 2.5 ml of ice-cold reaction buffer (without [3H]-R-
rolipram) and
rapid vacuum filtration (Brandel Cell Harvester) through Whatman GF/B filters
that had
been soaked in 0.3% polyethylenimine. The filters were washed with an
additional 7.5-ml
of cold buffer, dried, and counted via liquid scintillation spectrometry.
Example 2 - Preparation of a Controlled Release Tablet
A controlled-release formulation was prepared using the ingredients set out in
Table
1.
Table 1
Table Ingredients
Ingredient % w/w
Ariflo~ 3.3
Dibasic Calcium Phosphate88.5
(anh drous)
CA 02398184 2002-08-07
WO 01/58441 PCT/USO1/03972
Carbomer 934P 3.3
Carbomer 941 P 1.6
Ma nesium Stearate 1.0
O adr White OY-S-9603 2.4
Purified water ~s.
Blending and compression techniques:
Blendinc .
Excipients and drug were placed in a blender and mixed. The magnesium stearate
was then added and mixed for an additional 3 minutes. During the blending
process,
excipients and drug were mixed, passed through a screen and then mixed again.
Com rep sslon
Approximately 350 mg of each mix was compressed into tablets. A target tablet
strength of 10 kp was used.
Opadry White was suspended in the purified water and that suspension was used
to
coat the tablets; water was removed during the coating process an ddid not
form part of the
final product.
Example 3 - Preparation of an Immediate Release Tablet
Immediate release tablets were prepared by standard means and contained the
ingredients set out in Table 2.
I S Table 2
Immediate Release Tablets
Ingredients Quantity Quantity Quantity
5.0 10.0 ( 15.0
Lactose Monohydrate 113.0 108 103
Microcrystalline Cellulose70.0 70.0 70.0
Sodium Starch Glycolate10.0 10.0 10.0
Magnesium Stearate 2.0 2.0 2.0
Opadry White OY-S-96035.0 5.0 5.0
Total Tablet Weight 205.0 205 205
(mg)
Example 4 - Treatment of Arthritis
A patient diagnosed with arthritis and experiencing pain due to an
inflammation of a
joint is given a controlled-release tablet containing 30mg of Ariflo~ prepared
as per
Example 2 and a 500mg tablet of Relafen (nabumetone) twice daily. Treatment is
continued
until such time as the inflammation goes into remission. ,
_g-
