Note: Descriptions are shown in the official language in which they were submitted.
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Use of CSAIDs in Rhinovirus infection
Field of Invention
The present invention relates to the use of a CSBP/p38 inhibitor in the
treatment of a
CSBP/p38 mediated disease.
Background of the Invention
Human rhinovirus (HRV), the most frequent cause of the common cold, is
increasingly associated with more serious sequelae including exacerbation's of
asthma, chronic bronchitis, COPD, otitis media, and sinusitis (Gern et al.,
Clin
Micro Reviews 12(1): 9-18 (1999); Pitkaranta, and Hoyden, Annals of Medicine
30
(6): 529-537 (1998); Seemungal et al, ATS abstract "Rhinoviruses are
associated
with exacerbation's of COPD" (1998)). Recent published studies in adults and
adolescents, using PCR to assist in viral detection, have shown that up to 50
to 80%
of asthma exacerbation's are associated with upper respiratory tract virus
infection,
and that rhinovirus is the most common virus (Atmar et al, Archives of
Internal
Medicine. 158 (22): 2453-9 (1998); .Iohnston, SL., British Medical Journal
310:
1225-9 (1995)). HRV infects nasal epithelial cells; recent evidence suggests
the
virus may also infect bronchial epithelium. Prodromal cold symptoms are
apparent
within 24 hours post-infection, peak on days 2 through 5, and resolve within
seven
to fourteen days; but can be more protracted in some individuals. Symptoms are
believed to arise more from the host's response to infection, than an acute
cytotoxic
effect, since only a small fraction of upper respiratory epithelial cells are
demonstrably infected, and there is minimal epithelial cell damage (Winther et
al,
JAMA 256: 1763-1767 (1986). Increased intranasal levels of kinins, IL-1, IL-8,
IL-
6, IL-1 l, and neutrophils are found in normal individuals infected with
rhinoviruses.
A correlation between IL,-8 concentration in nasal secretions with local
myeloperoxidase levels and with symptom severity has been demonstrated in
several
recent studies (Grieff, et al. Eur Respir J 13: 41-47 (1999); Teren, et al. Am
J Respir
Crit Care Med 155: 1362-1366 (199, Turner, et al. Clin Infect Dis 26: 840-846
(1998). Intranasal concentrations of IL-1 and IL-6 have been correlated with
symptom severity as well (Proud et al, J. Infect. Dis.169:1007-1013 (1994);
Zhu et
al, J. Clin. Invest. 97:421-430 (1990. Experimental rhinovirus infection also
results in enhanced immediate and late phase allergic reactions, and in
increased
infiltration of T lymphocytes and eosinophils into the lower airways. In
atopics and
CA 02385722 2002-03-15
WO 01/19322 PCT/US00/25386
asthmatics, these effects persist for up to 2 months post - infection (Gern
and Busse,
Am J Respir Crit Care Med, 152: 540-S45 (1995). Human bronchial epithelial
cell
lines have been shown to produce IL,-1, II,-6, IL-8, IL-11 and GM-CSF in
response
to rhinovirus infection (Subauste et al, J Clin Invest, 96: 549-557 (1995);
Gern et
al., supra, 1999). Early production of cytokines by rhinovirus - infected
epithelial
cells may therefore be responsible for triggering recruitment of neutrophils,
T cells
and activated eosinophils into the upper and lower airways.
In addition, IL-l, IL-6, and IL-8 are also produced in response to infection
with other respiratory viruses (influenza, respiratory syncytial virus) which
can
cause the common cold and associated sequelae.
By interfering with the biochemical processes of epithelial cells resulting
from virus infection there represents a viable new therapeutic target by an
inhibitor
of CSBP/p38. This invention is directed to the novel discovery of treatment of
this
therapeutic target.
Summary of the Invention
The present invention relates to the use of a CSBP/p38 kinase inhibitor for
the treatment, including prophylaxis, of the common cold, or respiratory viral
infection caused by human rhinovirus infection (HRV), other enteroviruses,
coronavirus, influenza virus, parainfluenza virus, respiratory syncytial
virus, or
adenovirus infection in a human in need thereof which method comprises
administering to said human an effective amount of a CBSP/p38 inhibitor.
Another aspect of the present invention is a method of treating, including
prophylaxis of influenza induced pneumonia in a human in need thereof which
method comprises administering to said human an effective amount of a CBSP/p38
inhibitor
The present invention also relates to the use of the CSBP/p38 kinase
inhibitor for the treatment, including prophylaxis, of inflammation associated
with a
viral infection of a human rhinovirus (HRV), other enteroviruses, coronavirus,
influenza virus, parainfluenza virus, respiratory syncytial virus, or
adenovirus.
Brief Description of the Drawings
Figure 1 demonstrates Cytokine Production by Rhinovirus infected BEAS-
2B cells. Culture supernatants were collected 72 hours post-infection of BEAS-
2B
cells with rhinovirus-39 (MOI 1). Uninfected cells served as controls. Protein
concentrations in supernatants were determined by ELISA (R&D Systems). Results
represent the mean concentration values obtained from 6 experiments.
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Figure 2 demonstrates Inhibition of cytokines by CSAll7s: BEAS-2B
cultures infected with rhinovirus-39 were cultured in the presence of various
concentrations of drug. Cytokine levels in supernatant were determined 72
hours
post-infection using commercially available ELISA kits. Results are expressed
as
inhibition from infected untreated cultures. Cytokine concentrations in
infected
control cultures were 4902 pg/ml IL-6, 4520 pg/ml IL-8, and 28 pg/ml GM-CSF.
Figure 3 demonstrates Tyrosine phosphorylation of p38 kinase by rhinovirus
infection. BEAS-2B cells were incubated with rhinovirus-39 for various times
as
indicated. Cell lysates were separated by 10% SDS-polyacrylamide gel,
transferred
to nitrocellulose membrane and probed with specific antibody to phosphorylated
p38
kinase (A) or total p38 kinase (B). Amounts of p38 kinase were quantitated by
image analyzer and are presented as volumes based on densitometer scans.
Figure 4 demonstrates Tyrosine phosphorylation of p38 kinase by rhinovirus
infection. BEAS-ZB cells were incubated with various doses (MOI) of rhinovirus-
39 for 30 minutes. Cell lysates were separated by 10% SDS-polyacrylamide gel,
transferred to nitrocellulose membrane and probed with specific antibody to
phosphorylated p38 kinase or total p38 kinase. Amounts of p38 kinase were
quantitated by image analyzer and are presented as relative amounts of total
or
phosphorylated p38 kinase compared to control cells incubated with media alone
(fold increase).
Figure 5 demonstrates the effect of Compound VI, 1-(1,3-Dihydroxyprop-2-
yl)-4-(4-fluorophenyl)-5-[2-phenoxypyrimidin-4-yl]imidazole on improvement of
pulmonary function with increasing doses. BALB/c mice were treated from days 3-
8
post-infection with a sub-lethal dose of Influenza A. Pulmonary resistance was
determined using whole body plethysmography.
Figure 6 demonstrates the effect of Compounds V, 1-(4-Piperidinyl)-4-(4-
fluorophenyl)-5-(2-methoxy-4-pyrimidinyl)imidazole and Compound VI, on
prevention of weight loss in animals in an in vivo influenza model.
Figure 7 demonstrates the efficacy of of Compounds V and VI at improving
arterial blood oxygen levels (%Sp02) upon treatment. Sp02 was determined using
daily pulse oximetry.
Detailed Description of the Invention
IL-l, TNF, and other cytokines affect a wide variety of cells and tissues and
these cytokines as well as other leukocyte derived cytokines are important and
critical inflammatory mediators of a wide variety of disease states and
conditions.
-3-
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The inhibition of these cytokines is of benefit in controlling, reducing and
alleviating
many of these disease states.
In particular, the present invention is directed to the treatment of a viral
infection in a human, which is caused by the human rhinovirus (HRV), other
enterovirus, coronavirus, influenza virus, parainfluenza virus, respiratory
syncytial
virus, or an adenovirus. In particular the invention is directed to
respiratory viral
infections that exacerbate asthma (induced by such infections), chronic
bronchitis,
chronic obstructive pulmonary disease, otitis media, and sinusitis. While
inhibiting
IL-8 or other cytokines may be beneficial in treating a rhinovirus may be
known,
the use of an inhibitor of the p38 kinase for treating HRV or other
respiratory viral
infections causing the common cold is believed novel.
It should be noted that the respiratory viral infection treated herein may
also be associated with a secondary bacterial infection, such as otitis media,
sinusitis, or pneumonia.
For use herein treatment may include prophylaxis for use in a treatment
group susceptible to such infections. It may also include reducing the
symptoms of,
ameliorating the symptoms of, reducing the severity of, reducing the incidence
of, or
any other change in the condition of the patient, which improves the
therapeutic
outcome.
The mechanism of action for inhibition of a cytokine by a cytokine
suppressive anti-inflammatory drug (CSAID) versus inhibition of virus -
induced
IL-8 production in airway epithelial cells is believed to be different. In the
rhinovirus system, IL-8 production, and CSAID inhibition of IL-8 synthesis is
independent of IL-1 and TNF production, whereas the published studies have
focused on IL-1 and TNF - induced IL-8 production.
It should be noted that the treatment herein is not directed to the
elimination or
treatment of the viral organism itself but is directed to treatment of the
respiratory viral
infection that exacerbates other diseases or symptoms of disease, such as
asthma (induced
by such infections), chronic bronchitis, chronic obstructive pulmonary
disease, otitis
media, and sinusitis.
The present invention will demonstrate that CSAID inhibitors are useful in
the treatment of symptoms associated with HRV, including exacerbations of
underlying conditions such as asthma, COPD, sinusitis and otitis media amongst
others.
A preferred virus for treatment herein is the human rhinovirus infection
(HRV) or respiratory syncytial virus (RSV).
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Another aspect of the present invention is a method of treating, including
prophylaxis of influenza induced pneumonia in a human in need thereof which
method comprises administering to said human an effective amount of a CBSP/p38
inhibitor. Therefore, for this usage, a preferred virus for treatment is the
influenza
virus.
Lastly, another aspect of the present invention relates to the use of a
CSBP/p38 kinase inhibitor for the treatment, including prophylaxis, of
inflammation
associated with a viral infection of a human rhinovirus (HRV), other
enteroviruses,
coronavirus, influenza virus, parainfluenza virus, respiratory syncytial
virus, or
adenovirus. Preferably the viral infection is HRV or RSV, or the influenza or
parainfluenza virus.
Suitable CSAll7 compounds are well known in the art, and an assay for
determining CBSP/p38 inhibition is also readily available using assays
disclosed
in the below noted patents or applications. For instance, see US Patents
5,716,972, US 5,686,455, US 5,656,644, US 5,593,992, US 5,593,991, US
5,663,334, US 5,670,527, US 5,559,137, 5,658,903, US 5,739,143, US 5,756,499,
and US 5,716,955; WIPO publications WO 98/25619, WO 97/25048, WO
99/01452, WO 97/25047, WO 99/01131, WO 99/01130, WO 97/33883, WO
97/35856, WO 97/35855, WO 98/06715, WO 98/07425, WO 98/28292, WO
98/56377 , WO 98/07966 , WO 99/01136 , WO 99/17776 , WO 99/01131 , WO
99/01130, WO 99/32121, WO 00/26209, WO 99/58502, WO 99/58523, WO
99/57101, WO 99/61426, WO 99/59960; WO 99/59959, WO 00/18738, WO
00/17175, WO 99/17204, WO 00/20402, WO 99/64400, WO 00/01688, WO
00/07980, WO 00/07991, WO 00/06563, WO 00/12074, WO 00/12497, WO
00/31072, WO 00/31063, WO 00/23072, WO 00/31065, WO 00/35911, WO
00/39116, WO 00/43384, WO 00/41698, WO 97/36587, WO 97/47618, WO
97/16442, WO 97/16441, WO 97/12876, WO 98/7966, WO 98/56377, WO
98/22109, WO 98/24782, WO 98/24780, WO 98/22457, WO 98/52558, WO
98/52941, WO 98/52937, WO 98/52940, WO 98/56788, WO 98/27098 , WO
99/00357, WO 98/47892, WO 98/47899, WO 99/03837, WO 99/01441, WO
99/01449, WO 99/03484, WO 95/09853, WO 99/15164, WO 98/50356; WO
95/09851, WO 95/09847, WO 95/09852, WO 92/12154, WO 94/19350, DE
19842833, JP 2000 86657 and De Laszlo et al., Bioorg. Med. Chem. Lett 8 (1998)
2689-2694 whose disclosures are all incorporated herein by reference in their
3 5 entirety.
Preferred compounds of this invention include those contained in WO
99/01131, and a representative genus is described below. Also preferred for
use
herein are the compounds disclosed in WO 99/61426 Scios, Inc.; and those
compounds disclosed in WO 98/27098 containing the compound known as VX-745;
(also known as 5-(2,6-Dichloro-phenyl)-2-(2,4-difluoro-phenylsulfanyl)-1,7,8a-
-5-
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WO 01/19322 PCT/US00/25386
triaza-naphthalen-6-one), the Johnson & Johnson compound RWJ-68354 disclosed
in
WO 98/47899, RPR compound RPR-200765A, the Zeneca compound ZM 336372
disclosed in WO 99/15164; the Sugen compound SU 4984 disclosed in WO
98/50356. A review of various inhibitors of p38 kinase is taught in Boehm et
al.,
Exp. Opin Ther. Patents 10(1):25-37 (2000).
Compounds of Formula (I) are represented by the formula:
R2
R
N
N
R4 (I)
wherein
R1 is 4-pyridyl, pyrimidinyl, 4-pyridazinyl, 1,2,4-triazin-5-yl, quinolyl,
isoquinolinyl, or quinazolin-4-yl ring, which ring is substituted with Y-Ra
and
optionally with an additional independent substituent selected from C 1 _4
alkyl,
halogen, hydroxyl, C 1 _4 alkoxy, C 1 _4 alkylthio, C 1 _4 alkylsulfinyl,
CH20R12,
amino, mono and di- C1_6 alkyl substituted amino, an N-heterocyclyl ring which
ring has from 5 to 7 members and optionally contains an additional heteroatom
selected from oxygen, sulfur or NR15, N(R10)C(O)Rb or NHRa;
Y is oxygen or sulfur;
R4 is phenyl, naphth-1-yl or naphth-2-yl, or a heteroaryl, which is optionally
substituted by one or two substituents, each of which is independently
selected,
and which, for a 4-phenyl, 4-naphth-1-yl, 5-naphth-2-yl or 6-naphth-2-yl
substituent, is halogen, cyano, nitro, C(Z)NR7R17, C(Z)OR16,
(CR1pR20)vCORl2, SRS, SORS, OR12, halo-substituted-C1-4 alkyl, C1-4
alkyl, ZC(Z)R12, NRlpC(Z)R16, or (CR1pR20)v~lOR20 and which, for other
positions of substitution, is halogen, cyano, C(Z)NR13R14, C(Z)OR3,
(CR1pR20)m"COR3, S(O)mR3, OR3, halo-substituted-C1-4 alkyl, C1_4 alkyl,
(CR10R20)m"NR10C(Z)R3~ NR10S(O)m'R8~ NR10S(O)m'~7R17~ ZC(Z)R3
or (CR1pR20)m"~13R14~
Z is oxygen or sulfur;
n is an integer having a value of 1 to 10;
m is 0, or the integer 1 or 2;
m' is an integer having a value of 1 or 2,
m" is 0, or an integer having a value of 1 to S;
-6-
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WO 01/19322 PCT/US00/25386
v is 0, or an integer having a value of 1 or 2;
R2 is -C(H) (A) (R22);
A is an optionally substituted aryl, heterocyclyl, or heteroaryl ring, or A is
a substituted
C 1-10 alkyl;
R22 is an optionally substituted C1_10 alkyl;
Ra is aryl, arylC 1 _6alkyl, heterocyclic, heterocyclylC 1 _6 alkyl,
heteroaryl,
heteroarylC 1 _6alkyl, wherein each of these moieties may be optionally
sub stituted;
Rb is hydrogen, C1_6 alkyl, C3_~ cycloalkyl, aryl, arylC1_4 alkyl, heteroaryl,
heteroarylC 1 _4alkyl, heterocyclyl, or heterocyclylC 1 _4 alkyl, wherein each
of
these moieties may be optionally substituted;
R3 is heterocyclyl, heterocyclylCl_10 alkyl or Rg;
RS is hydrogen, C 1 _4 alkyl, C2_4 alkenyl, C2_4 alkynyl or NR~R1 ~, excluding
the
moieties SRS being SNR~R1~ and SORS being SOH;
R6 is hydrogen, a pharmaceutically acceptable cation, C1_10 alkyl, C3_~
cycloalkyl,
aryl, arylC 1 _4 alkyl, heteroaryl, heteroarylC 1 _4alkyl, heterocyclyl,
aroyl, or
C1-10 alkanoyl;
R~ and R1~ is each independently selected from hydrogen or C1_4 alkyl or R~
and
R1~ together with the nitrogen to which they are attached form a heterocyclic
ring of 5 to 7 members which ring optionally contains an additional heteroatom
selected from oxygen, sulfur or NR15;
Rg is C 1 _ 10 alkyl, halo-sub stituted C 1 _ l p alkyl, C2_ 10 alkenyl, C2_
10 alkynyl, C3 _~
cycloalkyl, C5_~ cycloalkenyl, aryl, arylC1_10 alkyl, heteroaryl,
heteroarylCl_10
alkyl, (CR10R20)nORll~ (CR10R20)nS(O)mRl8~ (CR10R20)nNHS(O)2R18~
(CR10R20)nNR13R14~ wherein the aryl, arylalkyl, heteroaryl, heteroaryl alkyl
may be optionally substituted;
R9 is hydrogen, C(Z)R11 or optionally substituted C1-10 alkyl, S(O)2Rlg,
optionally substituted aryl or optionally substituted aryl-C 1 _4 alkyl;
R10 and R20 is each independently selected from hydrogen or C1_4 alkyl;
R11 is hydrogen, C1_10 alkyl, C3_~ cycloalkyl, heterocyclyl, heterocyclyl
C 1-1 Oalkyl, aryl, arylC 1 _ 10 alkyl, heteroaryl or heteroarylC 1 _ 10
alkyl, wherein
these moieties may be optionally substituted;
R12 is hydrogen or R16;
R13 and R14 is each independently selected from hydrogen or optionally
substituted
3 5 C 1 _q alkyl, optional 1y substituted aryl or optionally substituted aryl-
C 1 _4 alkyl,
or together with the nitrogen which they are attached form a heterocyclic ring
of
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WO 01/19322 PCT/US00/25386
to 7 members which ring optionally contains an additional heteroatom selected
from oxygen, sulfur or NR9 ;
R15 is Rlp or C(Z)-C1_4 alkyl;
R16 is C1_4 alkyl, halo-substituted-C1-4 alkyl, or C3_~ cycloalkyl;
Rlg is C1_l0 alkyl, C3_~ cycloalkyl, heterocyclyl, aryl, aryll-l0alkyl,
heterocyclyl,
heterocyclyl-C1-l0alkyl, heteroaryl or heteroaryll_l0alkyl;
or a pharmaceutically acceptable salt thereof.
R2 is a substituted alkyl derivative. It is recognized that the first
methylene
carbon in this chain is a tertiary carbon, and it will contain one hydrogen
moiety. This
methylene group will have has two additional substituents, an R22 moiety and
an A
moiety, -C(H)(A)(R22). Both A and R22 may not be unsubstituted C1_10 alkyl
moieties.
In a preferred embodiment, R2 is a -C(AA1)(A) moiety, wherein AA1 is the
R22 moiety, but is specifically the side chain residue (R) of an amino acid,
as is further
described herein.
Suitably, A is an optionally substituted C3_~cycloalkyl, aryl, heteroaryl, or
heterocyclic ring, or A is a substituted C1_10 alkyl moiety.
When A is an aryl, heteroaryl and heterocyclic ring, the ring may be
substituted
independently one or more times, preferably, 1 to 3 times by C 1 _ 10 alkyl;
halogen;
halo substituted C1-10 alkyl, such as CF3; (CR10R20)tORl l~ (CR10R20)t~13R14~
especially amino or mono- or di-C1_4 alkylamino; (CR10R20)tS(O)mRl8~ wherein m
is 0, 1 or 2; SH; NR10C(Z)R3 (such NHCO(C1-10 alkyl)); or NR10S(O)mRg (such as
NHS02(C 1 _ 10 alkyl)).
Suitably, t is 0, or an integer of 1 to 4.
When A is an optionally substituted cycloalkyl it is as defined below with the
R22 substitution.
When A is an optionally substituted heterocyclyl ring, the ring is preferably
a
morpholino, pyrrolidinyl, piperazinyl or a piperidinyl ring.
When A is an optionally substituted aryl moiety, it is preferably a phenyl
ring.
When A is an optionally substituted heteroaryl ring, it is as defined below in
the definition section.
When A is a substituted C1_10 alkyl moiety, the alkyl chain may be straight or
branched. The chain is substituted independently 1 or more times, preferably 1
to 3
times by halogen, such as fluorine, chlorine, bromine or iodine;
halosubstituted C1_10
alkyl, such as CF3; C3_~cycloalkyl, C1-10 alkoxy, such as methoxy or ethoxy;
hydroxy substituted C1-10 alkoxy; halosubstituted C1-10 alkoxy, such as
OCF2CF2H;
_g_
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WO 01/19322 PCT/US00/25386
OR11; S(O)mRlg (wherein m is 0, 1 or 2); NR13R14; C(Z)1VR13R14~
S(O)m'~13R14~ ~23C(Z)R11~ ~S(O)2R18~ C(Z)R11~ OC(Z)R11~ C(Z)OR11;
C(Z)~l lOR9~ N(OR6)C(Z)~13R14~ N(OR6)C(Z)Rl 1; C(-NOR6)Rl 1
NR23C(=NR19)NR13R14~ OC(Z)NR13R14~ NR23C(Z)~13R14~ or
NR23C(Z)OR10.
Preferably A is a C3_~ cycloalkyl, or a C1_6 alkyl, more preferably a C1-2
alkyl, i. e. a methylene or ethylene moiety, more preferably a methylene
moiety which
is substituted by one of the above noted groups.
Preferably, when A is a C1_10 alkyl, it is substituted by OR11 where R11 is
preferably hydrogen, aryl or arylalkyl; NR13R14; OC(Z)R11; or C(Z)ORl 1.
More preferably, A is substituted by OR11 where R11 is hydrogen.
Suitably, R22 is a C 1 _ 10 alkyl chain, which chain may be straight or
branched
and which may be optionally substituted independently, one or more times,
preferably
1 to 3 times, by halogen, such as fluorine, chlorine, bromine or iodine; halo
substituted
C 1 _ 10 alkyl; C 1 _ 10 alkoxy, such as methoxy or ethoxy; hydroxy
substituted C 1 _ 10
alkoxy; halosubstituted C1-10 alkoxy, such as OCF2CF2H; OR11; S(O)mRlg;
~13R14~ C(Z)NR13R14~ S(O)m'~13R14~ ~23C(Z)R11~ ~S(O)2R18~
C(Z)R11; OC(Z)Rl l; C(Z)OR11; C(Z)NR110R9; N(OR6)C(Z)NR13R14~
N(OR6)C(Z)R11; C(=NOR6)R11; NR23C(=NR19)~13R14~ OC(Z)NR13R14;
NR23C(Z)NR13R14; NR23C(Z)OR10; optionally substituted C3_~ cycloalkyl;
optionally substituted aryl, such as phenyl; optionally substituted
heteroaryl; or an
optionally substituted heterocyclic. The optional substituents on these
cycloalkyl, aryl,
heteroaryl, and heterocyclic moieties are as defined herein below.
It is noted that those R22 substituent groups which contain carbon as the
first
connecting group, i.e. C(Z)OR11; C(Z)NR110R9 C(Z)R11, C(Z)NR13R14, and
C(=NOR6)R11, may be the sole carbon in alkyl chain. Therefore, the R22 group
may,
for instance, be a carboxy, an aldehyde, or an amide, as well as being a
substituent off
a methylene unit, such as carbamoylmethyl, or acetamidomethyl.
Preferably R22 is a C 1 _6 unsubstituted or substituted alkyl group, such as a
C 1 _3
alkylene, such as methyl, ethyl or isopropyl, or a methylene or ethylene
moiety
substituted by one of the above noted moieties, or as noted above those
substituent
groups which contain a carbon may substitutent for the first methylene unit of
the alkyl
chain, such as carboxy, C(O)OR11, C(O)NR13R14, or R22 is an optionally
substituted
aryl group, such as a benzyl or phenethyl. In other words, R22 can be an
optionally
substituted alkyl group, or R22 can be C(Z)OR11, C(Z)NR11 OR9~ C(Z)Rl l,
C(Z)NR13R14, or C(=NOR6)R11.
-9-
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WO 01/19322 PCT/US00/25386
Preferably R22 is a C1_6 unsubstituted or substituted alkyl group, more
preferably a C1_2 alkylene chain, such as a methylene or ethylene moiety, more
preferably methylene.
Preferably the alkyl chain is substituted by OR11, where Rl 1 is preferably
hydrogen, aryl or arylalkyl; S(O)mRlg, where m is 0 and Rlg is a C1_6 alkyl;
or an
optionally substituted aryl, i.e. a benzyl or phenethyl moiety.
More preferably, R22 is phenyl, benzyl, CH20H, or CH2-O-aryl.
Preferably, one or both of A and R22 contain hydroxy moieties, such as in C 1
_6
alkyl OR11, wherein R11 is hydrogen, i.e.CH2CH20H.
Suitably, when AA1 is the (R) side chain residue of an amino acid, it is a C1-
6
alkyl group, which may be straight or branched. This means the R group off the
core
amino acid of the structure R-C(H)(COOH)(NH2). The R residue term is for
example,
CH3 for alanine, (CH3)2CH- for valine, (CH3)2CH-CH2-for leucine, phenyl-CH2-
for
phenylalanine, CH3-S-CH2-CH2- for methionine, etc. All generally recognized
primary amino acids are included in this groups, such as but not limited to,
alanine,
arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid,
glycine,
histidine, isoleucine, leucine, lysine, methionine, phenylalanine, serine,
threonine,
tryptophan, tyrosine, valine, hydroxylysine, methylhistidine, and other
naturally
occurring amino acids not found in proteins, such as (3-alanine, y-
aminobutyric acid,
homocysteine, homoserine, citrulline, ornithine, canavanine, djenkolic acid,
and [3-
cyanoalanine, or other naturally occurring non-mammalian amino acids.
Preferably AA1 is the residue of phenylalanine, or alanine.
Preferably, A is a hydroxy substituted C1_10 alkyl, and R22 is a C1-10 alkyl
or a
hydroxy substituted C 1 _ 10 alkyl.
For further definitions please refer to the descriptions in WO 99/0113 l, or
in
WO 99/01136, supra.
A preferred compound for use of 1-(1,3-Dihydroxyprop-2-yl)-4-(4-
fluorophenyl)-5-(2-phenoxypyrimidin-4-yl)imidazole, or a pharmaceutically
acceptable salt thereof.
Other suitable compounds for use herein include but are not limited to, trans-
1-(4-Hydroxycyclohexyl)-4-(4-fluorophenyl)-S-[(2-methoxy)pyrimidin-4-
yl]imidazole; 1-(4-Piperidinyl)-4-(4-fluorophenyl)-5-(2-methoxy-4-
pyrimidinyl)imidazole; or (4-Fluorophenyl)-2-(4-methylsulfmylphenyl)-5-(4-
pyridyl)-imidazole.
Methods of using and dosage amounts are the same as those disclosed in the
references cited above. See for instance, Adams et al., US patent 5,756,499,
issued
26 May 1998. In order to use a compound of formula (I) or a pharmaceutically
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acceptable salt thereof in therapy, it will normally be formulated into a
pharmaceutical composition in accordance with standard pharmaceutical
practice.
For all methods of use disclosed herein (or the compounds of Formula (I) and
other CSAID compounds), suitably, the daily oral dosage regimen will be from
about 0.1 to about 80 mg/kg of total body weight, preferably from about 0.2 to
30
mg/kg, more preferably from about 0.5 mg to l5mg. The daily parenteral dosage
regimen about 0.1 to about 80 mg/kg of total body weight, preferably from
about 0.2
to about 30 mg/kg, and more preferably from about 0.5 mg to lSmg/kg. The daily
topical dosage regimen will preferably be from 0.1 mg to 150 mg, administered
one
to four, preferably two or three times daily. The daily inhalation dosage
regimen
will preferably be from about 0.01 mg/kg to about 1 mg/kg per day.
The novel use of CSAID compounds herein may also be used in association
with the veterinary treatment of mammals, other than humans, in need of
inhibition
of CSBP/p38 or cytokine inhibition or production for treatment of influenza
pneumonia, and other sequelae associated with viral infection.
The CSBP/p38 inhibitor may also be administered with a second therapeutic
agent. The second therapeutic agent may be an antiviral agent such as
ribavirin,
amantidine, rimantidine, Pleconaril,AG 7088 or BTA-188; it may also be an
antiviral agent such as an influenza neuraminidase inhibitor, such as
zamanivar
(Relenza), oseltamivir (Tamiflu) or RWJ-270201; it may be an antihistamine,
such
as Benadryl~, chlorpheneramine and salts thereof, brompheneramine or salts
thereof, and the generally accepted non-sedating antihistamines, such as
loratadine
(Clarion~), descarboethoxyloratadine (DCL), fexofenadine (Allegra~), and
cetirizine hydrochloride (Zyrtec~) etc., a decongestant, such as
phenylpropanolamine and salts thereof, pseudoephedreine or salts thereof;
steroids,
such as dexamethasone, prednisone, or prenisolone, etc.; various antibiotics,
such as
the quinolones, cephalosporins, ~i-lactamase inhibitors, etc.; anti-
inflammatory
agents, such as an NSAID, a COX-1 or COX-2 inhibitor, ASA, or indomethacin,
etc. It is recognized that the above noted agents may be administerd as
immediate
release, or as extended release dosage forms, either together with a suitable
CSAID
compound, or seperately. The compositions may be administered sequentially, in
combination with, or contemporaneously with a CSAID agent. The administration
route of the second agent may also differ from that of the CSAID agent, and
hence
the dosing schedule may vary accordingly.
Cetirizine HCl manufacture and dosing is described in US Patent 4,525,358;
fexofenadine manufacture and dosing is described in US Patents 4,524,129; US
5,375,693; US 5,578,610; US 5,855,912; US 5,932,247; and US 6,037,353.
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Loratadine and DCL manufacture and dosing are described in US patent
4,282,233;
US 4,371,516; US 4,659,716; US 4,863,931; US 5,314,697; and US 5,595,997.
Zamanivar dosing is disclosed in US Patents 4,627,432; US 4,778,054; US
4,811,731; US 5,035,237; US 5,360,817; and US 5,648,379. Oseltamivir dosing is
disclosed in US Patents 'CTS 5,763,483; US 5,866,601; and US 5,952,375.
The CSPB/p38 inhibitor may be administered systemically or non-
systemically, such as orally, bucally, topically (intranasal) or via
inhalation
(aerosol), or both topically and via inhalation. As noted above, the second
therapeutic agent may be administered by any suitable means, including
parenteral,
suppository, etc. which means of administration is not necessarily by the same
route,
nor concurrent therewith.
As used herein "topically" shall include non-systemic administration. This
includes the application of a compound externally to the epidermis or the
buccal
cavity and/or the instillation of such a compound into the ear, eye and nose.
As used herein "systemic administration" refers to oral, intravenous,
intraperitoneal and intramuscular administration, subcutaneous intranasal,
intrarectal, or intravaginal.
It will be recognized by one of skill in the art that the optimal quantity and
spacing of individual dosages of a CSBP/p38 inhibitor will be determined by
the
nature and extent of the condition being treated, the form, route and site of
administration, and the particular patient being treated, and that such
optimums can
be determined by conventional techniques. It will also be appreciated by one
of skill
in the art that the optimal course of treatment, i.e., the number of doses of
a
CSBP/p38 inhibitor given per day for a defined number of days, can be
ascertained
by those skilled in the art using conventional course of treatment
determination tests.
Methods:
Cell lines, rhinovirus serotype 39, and influenza virus A/PR/8/34 were
purchased from American Type Culture Collection (ATCC). BEAS-2B cells were
cultured according to instructions provided by ATCC using BEGM (bronchial
epithelial growth media) purchased from Clonetics Corp. HELA cell cultures,
used
for detection and titration of virus, were maintained in Eagle's minimum
essential
media containing 10% fetal calf serum, 2mM 1-glutamine, and 10 mM HEPES
buffer (MEM).
A modification of the method reported by Subauste et al., Supra, for in vitro
infection of human bronchial epithelial cells with rhinovirus was used in
these
studies. BEAS-2B cells (2x105/well) were cultured in collagen-coated wells for
24
hours prior to infection with rhinovirus. Rhinovirus serotype 39 was added to
cell
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cultures for one hour incubation at 34°C after which inoculum was
replaced with
fresh media and cultures were incubated for an additional 72 hours at
34°C.
Supernatants collected at 72 hours post-infection were assayed for cytokine
protein
concentration by ELISA using commercially available kits (R&D Systems). Virus
yield was also determined from culture supernatants using a microtitration
assay in
HELA cell cultures (Subauste et al., supra 1995). In cultures treated with p3
8
kinase inhibitors, drug was added 30 minutes prior to infection. Stocks of
compounds were prepared in DMSO (10 mM drug) and stored at -20°C.
For detection of p38 kinase, cultures were incubated in basal media without
growth factors and additives to reduce endogenous levels of activated p38
kinase.
Cells were harvested at various timepoints after addition of rhinovirus.
Detection of
tyrosine phosphorylated p38 kinase by immunoblot was analyzed by a
commercially
available kit and was performed according to the manufacturer's instructions
(PhosphoPlus p38 MAPK Antibody Kit: New England BioLabs Inc.).
In some experiments, BEAS-2B cells were infected with influenza virus
(strain A/PR/8/34) in place of rhinovirus. Culture supernatant was harvested
48 and
72 hour post-infection and tested by ELISA for cytokine as described above.
Cells and Virus: Influenza A/PR/8/34 sub type H1N1 (VR-95 American Type
Culture Collection, Rockville, MD) was grown in the allantoic cavity of 10 day
old
chicken eggs. Following incubation at 37°C, and refrigeration for 2 1/2
hours at
4°C, allantoic fluid was harvested, pooled, and centrifuged (1,000 rcf;
15 min; 4°C)
to remove cells. Supernatent was aliquoted and stored at -70°C. The
titer of the
stock culture of virus was 1.0 x 101° Tissue Culture Infective Dose/ml
(TC117so)
Inoculation procedure: Four-six week old female Balb/cAnNcrlBr mice were
obtained from Charles River, Raleigh, NC. Animals were infected intranasally.
Mice were anesthetized by intraperitioneal injection of Ketamine (40mg/kg;
Fort
Dodge Labs, Fort Dodge, Ia) and Xylazine (5 mg/kg; Miles, Shawnee Mission, Ks)
and then inoculated with 100 TCID50 of PR8 diluted in PBS in 20 u1. Animals
were
observed daily for signs of infection. All animal studies were approved by
SmithKline Beecham Pharmaceuticals Institutional Animal Care and Use
Committee.
hirus titration: At various times post infection, animals were sacrificed and
lungs
were aseptically harvested. Tissues were homogenized, in vials containing 1
micron
glass beads (Biospec Products, Bartlesville, OK) and 1 ml. of Eagles minimal
essential medium. Cell debris was cleared by centrifugation at 1,000 rcf for
15
minutes at 4°C, and supernatants were serially diluted on Madin-Darby
canine
kidney (MDCK) cells. After 5 days of incubation at 37°C (5% C02), SO
p,1 of 0.5%
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chick red blood cells were added per well, and agglutination was read after 1
hour at
room temperature. The virus titer is expressed as 50% tissue culture infective
dose
(TCIDSO) calculated by logistic regression.
ELISA: Cytokine levels were measured by quantitative ELISA using commercially
available kits. Ear samples were homogenized using a tissue minser in PBS.
Cell
debris was cleared by centrifugation at 14,000 rpm for 5 minutes. The cytokine
concentrations and thresholds were determined as described by the
manufacturer;
IL-6, IFN-y, and KC (R&D Systems, Minneapolis, MN).
Myeloperoxidase Assay: Myeloperoxidase (MPO) activity was determined
kinetically
as described by Bradley et al. (1982). Briefly, rabbit cornea were homogenized
in
Hexadecyl Trimethyl-Ammonium Bromide (HTAB) (Sigma Chemical Co. St. Louis,
Mo) which was dissolved in 0.5 m Potassium phosphate buffer (J.T. Baker
Scientific,
Phillipsburg, NJ). Following homogenization, the samples were subjected to
freeze-
thaw-sonication (Cole-Parmer 8853, Cole-Parmer, Vernon Hills, Il) 3 times.
I S Suspensions were then cleared by centrifugation at 12,500 x g for 15
minutes at 4oC.
MPO enzymatic activity was determined by colormetric change in absorbance
during a
reaction of O-Dianisidine dihydrochloride (ODI) 0.175 mg/ml (Sigma Chemical
Co.
St. Louis, Mo) with .0002% Hydrogen peroxide (Sigma Chemical Co. St. Louis,
Mo).
Measurements were performed by using a Beckman Du 640 Spectrophotometer
(Fullerton, Ca.) fitted with a temperature control device. 50 u1 of material
to be
assayed was added to 950 u1 of ODI and change in absorbance was measured at a
wave length of 460 nm for 2 minutes at 25°C.
Whole Body Plethysomography: Influenza virus infected mice were placed into a
whole body plethysomograph box with an internal volume of approximately 350-
ml.
A bias airflow of one 1/min was applied to the box and flow changes were
measured
and recorded with a Buxco XA data acquisition and respiratory analysis system
(Buxco Electronics, Sharon, CT). Animals were allowed to acclimate to the
plethysmograph box for 2 min. before airflow data was recorded. Airway
measurements were calculated as Penh (enhanced pause). Penh has previously
been
shown as an index of airway obstruction and correlates with increased
intrapleural
pressure. The algorithm for Penh calculation is as follows: Penh =
[(expiratory time
/ relaxation time)-1] x (peak expiratory flow / peak inspiratory flow) where
relaxation time is the amount of time required for 70% of the tidal volume to
be
expired.
Determination of arterial oxygen saturation. A Nonin veterinary hand held
pulse
oximeter 8500V with lingual sensor (Nonin Medical, Inc., Plymouth MN) was used
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to determine daily arterial oxygen saturation %Sp02 as described (Sidwell et
al.
1992 Antimicrobial Agents and Chemotherapy 36:473-476).
Results:
Inhibition of cytokine production by specific inhibitors of p38 MAP kinase:
Consistent with published reports, IL-6, IL-8, and GM-CSF were detected 72
hours
post-infection of BEAS-2B cells with rhinovirus-39 (multiplicity of infection;
MOI
1.0) (figure 1). Production of IL-6, IL,-8, and GM-CSF was not mediated
through
IL-1 or TNF produced in response to rhinovirus infection since addition of
neutralizing antibodies to IL-1 and TNF to the infected cultures did not
reduce the
amount of IL-6, IL-8 or GM-CSF produced (not shown). Productive infection of
cells was confirmed by titering infectious supernatants from BEAS-2B cells on
HELA monolayers. There was low but consistent replication of virus during the
72
hour culture period resulting in 1.22 + 0.3 logl0 TCID50 increase over the
initial
input inoculum (n=6 experiments). To investigate the role of p38 kinase signal
transduction in rhinovirus-induced cytokine production by epithelial cells,
specific
p38 kinase inhibitors SB203580, Compound II, Compound III, and an inactive
analog, SKF106978, were tested for their ability to inhibit cytokine
production in
rhinovirus-infected BEAS-2B cell cultures. The compound (4-Fluorophenyl)-2-(4-
methylsulfinylphenyl)-5-(4-pyridyl)-imidazole is alternatively referred to as
SB
203580 and may be found in US patent 5,656,644. The compound traps-1-(4-
Hydroxycyclohexyl)-4-(4-fluorophenyl)-5-[(2-methoxy)pyrimidin-4-yl]imidazole
also known as Compound II may be found in WO 97/25048. The compound 4-(4-
Fluorophenyl-5-[(2-phenylamino)pyrimidin-4-yl]-1-(piperdin-4-yl)imidazole,
also
known as Compound III may be found in US Patent 5,658,903. The compound 2-
(4-Methylsulfinyl)-3-[4-(2-methylpyridyl]-6,7-dihydro[SH]pyrrolo[ 1,2-
a]imidazole,
is also known as SB 106978. Concentrations of IL-8, IL-6 and GM-CSF in culture
supernatants from infected cells treated with inhibitors were all lower than
those
from untreated infected cultures (figure 2). IL-6 was the most sensitive to
inhibition
with significant inhibition (40%) being observed with SB 203580 concentrations
as
low as 30 nM . GM-CSF was the least sensitive to inhibition by SB 203580, with
an
IC50 of approximately 4 uM.
Another inhibitor of p38 kinase, Compound II, was slightly more potent in
inhibiting GM-CSF with an ICSO of approximately 1 uM. Compound II was
comparable to SB 203580 in inhibiting IL-6 and IL,-8 production. As expected
based on the relative potency of these compounds in specific binding to p38
kinase
(data shown in legend box), cytokine inhibition was greatest with Compound
III,
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with an IC50 value < 10 nM for IL-6, while SKF106978 was inactive at all
concentrations tested. Maximum effect obtained by any of the p38 kinase
inhibitors
against any of the three cytokines was 50% -70% inhibition. The inhibition of
cytokine production by CSAIDs was not due to general cell cytotoxicity as
determined by standard XTT assays (CC50 > 40 uM for all compounds tested)
(Roehm et al., J. of Immunol. Methods 142:257-265 (1991).
These compounds also did not exhibit direct antiviral activity as assessed
using a standard HELA cell antiviral assay (MIC50 > 10 uM for all compounds
tested) (Andries et al., Journal of Virology 64(3):1117-1123 (1990) or by
measuring
virus yield in the RV-infected BEAS-ZB cultures directly (not shown).
Activation of p38 kinase by rhinovirus infection:
The presence of tyrosine phosphorylated p38 kinase was measured by immunoblot
at
various times after the addition of virus to BEAS-2B cultures. Rhinovirus
infection of
BEAS-2B cells resulted in an increase in phosphorylated p38 kinase that was
both
dose and time-dependent. Increases in phosphorylated p38 kinase were evident
by 15
minutes post exposure to rhinovirus-39 (MOI 10), appeared to peak by 30
minutes
after addition of virus and remained elevated 60 minutes post-infection
(figure 3). In
addition, rhinovirus-induced tyrosine phosphorylation of p38 kinase was dose-
dependent (figure 4). When cells were cultured in the absence of virus, there
was no
increase in the amount of tyrosine phosphorylation of p38 kinase at any of the
timepoints tested. Overall levels of p38 kinase protein were comparable
between all
the groups indicating that virus infection caused phosphorylation of p38
kinase
without de novo synthesis of protein (figures 3 and 4).
Effects on in vitro influenza virus infection:
Exposure of BEAS-2B cells with influenza virus (A/PR/8/34; MOI 1.0) also
resulted
in elaboration of 1L-8 and IL-6 as measured 48 - 72 hours post-infection,
although
the secreted protein levels were lower than that obtained with rhinovirus
infection.
Consistent with observations in rhinovirus-infected cells, treatment of
influenza
infected-BEAS-2B cells with p38 kinase inhibitors, Compound IV, 1-(4-
Piperidinyl)-4-(4-fluorophenyl)-5-[(2-methylphenyl)amino]pyrimidin-4-
yl]imidazole and Compound II, 1-traps-4-hydroxycyclohexyl)-4-(4-fluorophenyl)-
5-[(2-methoxy)pyrimidin-4-yl]imidazole, was also effective in inhibiting IL-6
and
3 5 IL-8 production.
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Effects on in vivo influenza virus infection:
Five (5) independent and reproducible studies demonstrated the efficacy of
therapeutic dosing with Compound V, 1-(4-Piperidinyl)-4-(4-fluorophenyl)-5-(2-
methoxy-4-pyrimidinyl)imidazole and Compound VI, 1-(1,3-Dihydroxyprop-2-yl)-
4-(4-fluorophenyl)-5-[2-phenoxypyrimidin-4-yl]imidazole at improving clinical
disease in the murine influenza pneumonia model. BALB/c mice were dosed orally
b.i.d. on days 3-8 post influenza AlPR8 and monitored daily for weight loss,
pulmonary functions and arterial blood oxygen levels %Sp02. The antiviral
Tamiflu
was used as control and demonstrated 47% improvement in pulmonary functions
(p<0.01 days 5-12), 64% improved %Sp02 (p<0.01 days 5-18), and prevention of
weight loss relative to placebo. The optimal dose of Compound VI was 10 mg/kg
leading to 39% improvement in pulmonary functions (p<0.01 days 5-12), 30%
improvement in %Sp02 (p<0.01 days 5-13, p<0.05 days 14-15), and a similar
effect
on weight loss as Tamiflu treatment. Efficacy was observed in doses as low as
1
mg/kg: 27% improvement in pulmonary functions (p<0.01 days 6-9), 11.6% (p<0.01
Days 7-13). At 0.1 mg/kg, we observed 19% improvement in pulmonary functions
(p<0.05 day 7,8) but no effect on %Sp02 or weight loss. At 10 mg/kg, Compound
VI was equally effective to Compound V at 30 mg/kg. Samples were collected for
evaluation of virus titers and cytokines in lung homogenates. A non-
significant
trend for inhibition of lung cytokines IL-6, KC, IFN-gamma, and RANTES was
observed. There was no negative effect on lung virus titers.
No negative effect on immunity to secondary influenza virus infection: In two
studies, mice treated with Compound VI or Compound V during acute PR/8 (H1N1)
influenza infection were protected from a lethal challenge with the same virus
as
demonstrated by 100% survival and normal pulmonary functions. All control
primary infection animals died by Day 7. Thus, the CSAIDS have no effect on
immunity to a homologous challenge.
All publications, including but not limited to patents and patent
applications,
cited in this specification are herein incorporated by reference as if each
individual
publication were specifically and individually indicated to be incorporated by
reference
herein as though fully set forth.
The above description fully discloses the invention including preferred
embodiments thereof. Modifications and improvements of the embodiments
specifically disclosed herein are within the scope of the following claims.
Without
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further elaboration, it is believed that one skilled in the art can, using the
preceding
description, utilize the present invention to its fullest extent. Therefore
the
Examples herein are to be construed as merely illustrative and not a
limitation of the
scope of the present invention in any way. The embodiments of the invention in
which an exclusive property or privilege is claimed are defined as follows.
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