U.S. patent application number 09/779401 was filed with the patent office on 2001-11-15 for use of pde-4-specific inhibitors to reduce the severity of a bacterial infection after a respiratory viral infection.
Invention is credited to DeMarsh, Peter L., Dillon, Susan B., Woodnutt, Gary.
Application Number | 20010041739 09/779401 |
Document ID | / |
Family ID | 26877137 |
Filed Date | 2001-11-15 |
United States Patent
Application |
20010041739 |
Kind Code |
A1 |
DeMarsh, Peter L. ; et
al. |
November 15, 2001 |
Use of PDE-4-specific inhibitors to reduce the severity of a
bacterial infection after a respiratory viral infection
Abstract
This invention relates to a method for the prophylaxis of or
reducing the severity of post-viral bacterial infection by
administering a PDE 4-specific inhibitor prior to or during the
course of a viral infection or thereafter during the course of the
bacterial infection.
Inventors: |
DeMarsh, Peter L.; (West
Chester, PA) ; Dillon, Susan B.; (Alamo, CA) ;
Woodnutt, Gary; (Chester Springs, PA) |
Correspondence
Address: |
GLAXOSMITHKLINE
Corporate Intellectual Property - UW2220
P.O. Box 1539
King of Prussia
PA
19406-0939
US
|
Family ID: |
26877137 |
Appl. No.: |
09/779401 |
Filed: |
February 8, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60181385 |
Feb 9, 2000 |
|
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|
Current U.S.
Class: |
514/519 ;
514/210.04 |
Current CPC
Class: |
A61K 31/00 20130101;
A61K 31/277 20130101 |
Class at
Publication: |
514/519 ;
514/210.04 |
International
Class: |
A61K 031/277; A61K
031/407 |
Claims
What is claimed is:
1. A method for reducing the severity of or preventing a bacterial
infection of the respiratiory tract following a respiratory viral
tract infection by administering an effective amount of a PDE
4-specific inhibitor alone or in combination with a
pharmaceutically acceptable excipient prior to or during the course
of the viral infection, during the course of both the viral
infection and the bacterial infection following thereafter, or
during the course of the bacterial infection alone.
2. The method of claim 1 wherein the PDE 4 inhibitor is
administered during the course of the viral infection.
3. The method of claim 1 wherein the PDE 4 inhibitor is
administered during the course of the viral infection and during
the course of the bacterial infection.
4. The method of claim 1 wherein the PDE 4 inhibitor is
administered during the course of the bacterial infection.
5. The method of claim 3 wherein an antibiotic is administered
during the course of the bacterial infection.
6. The method of claim 4 wherein an antibiotic is administered
during the course of the bacterial infection.
7. The method of claim 3 wherein an antiviral is administered
during the course of the viral infection.
8. The method of claim 4 wherein an antiviral is administered
during the course of the viral infection.
9. The method of claim 1 wherein the inhibitor is administered
orally as a controlled-release tablet.
10. The method of claim 1 wherein the inhibitor is administered
intranasally or by aerosol to the respiratory tract.
Description
AREA OF THE INVENTION
[0001] This invention relates to a method for the prophylaxis of or
reducing the severity of post respiratory viral infection in the
respiratory tract by bacteria by administering a PDE 4-specific
inhibitor prior to or during the course of a viral infection or
thereafter during the course of the bacterial infection.
BACKGROUND
[0002] Primary respiratory tract viral infections have been shown
to lead to increased susceptibility to secondary bacterial
infections [Loosli C. G. 1973, Med. 52, 369-384; Stuart-Harris C.
H., Laird J., Tyrell D. A., Kelsall M. H., Franks Z. C., Pownall M.
1949, J. Hyg. 47; 434; and Stuart-Harris C. H. 1966, Brit. Med. J.
149; 217]. The mechanisms of this susceptibility are poorly
understood. And there is no prophylactics or treatments available
for reducing or moderating this susceptibility. Herein there is
provided a model for studying this phenomenon and a demonstrated
method for reducing this susceptibility by administering a PDE 4
specific inhibitor.
[0003] Streptococcus pneumoniae is regarded as one of the most
common causes of community-acquired pneumonia and is an important
cause of illness and death in industrialized countries. S.
pneumoniae colonizes the human nasophararynx and in some
circumstances the organisms spread to cause upper or lower
respiratory tract infection. The mechanisms underlying the
transition from a benign colonization to disease are not well
understood. A number of mechanisms have been proposed but the
nature of this transition remains to be discovered.
[0004] Epidemiological studies have demonstrated that viral
infections pre-dispose the host lung to bacterial pneumonia and
secondary bacterial pneumonia is the most frequent complication of
influenza infection in man [Loosli C. G. 1973, Med. 52, 369-384;
Stuart-Harris C. H., Laird J., Tyrell D. A., Kelsall M. H., Franks
Z. C., Pownall M. 1949, J. Hyg. 47; 434; and Stuart-Harris C. H.
1966, Brit. Med. J. 149; 217]. Investigators have demonstrated in
vitro that cells stimulated with either cytokines or virus show
enhanced bacterial adherence and that bacterial adherence to host
tissues is an essential step in bacterial colonization and
infections [Jones W. T., Menna J. H. 1982, Infect. Immun. 38;
791-794; Jiang Z., Nagata N., Molina E., Bakaletz L. O., Hawkins
H., Patel J. A. 1999, Infect. Immun. 67; 187-192; Sanford B. A.,
Shelokov A., Ramsay M. A. 1978, J. Infect. Dis. 137; 176-181;
Geelen S., Bhattacharyya C., Tuomanen E. 1993, Infect. Immun. 61;
1538-1543; and Hakansson A., Kidd A., Wadell G., Sabharwal H.,
Svanborg C. 1994, Infect. Immun. 62, 2707-2714.]
[0005] Phase variation of pneumococci has been demonstrated to be
one of the virulence mechanisms of respiratory tract infections.
For S. pneumoniae the transparent phenotype has been shown to
adhere to lung epithelial cells and to be more virulent in an
infant rat colonization model than the opaque phenotype [Kim J. O.,
Weiser J. N. 1998, J. Infect. Dis. 177:368-377; and Weiser J. N.,
Austrian R., Sreenivasan P. K., Masure H. R. 1994, Infect. Immun.
62; 2582-2589]. The mechanism of the altered virulence of S.
pneumoniae phenotypes has been shown to be linked to the cell wall
and the production of the capsular glycoprotein CbpA [Berube L. R.,
et al, 1999, Micro. Path. 26; 65-75 and Geelen S. et al, 1993,
Infect. Immun., 61; 1538-1543].
[0006] Here-in there is described a murine model of sub-lethal
influenza infection followed by Streptococcus pneumoniae
co-infection. Also disclosed is the finding that administrating a
PDE 4-specific inhibitor reduces the susceptibility of a mammal to
post-viral bacterial infection.
SUMMARY OF THE INVENTION
[0007] This invention relates to a means for reducing the severity
of or preventing a bacterial infection of the respiratiory tract
following a respiratory viral tract infection by administering an
effective amount of a PDE 4-specific inhibitor alone or in
combination with a pharmaceutically acceptable excipient prior to
or during the course of the viral infection, during the course of
both the viral infection and the bacterial infection following
thereafter, or during the course of the bacterial infection
alone.
DESCRIPTION OF THE FIGURES
[0008] FIG. 1 is a graph of the S. pneumoniae LD.sub.50
determination in non-infected vs. sub-lethally influenza infected
mice.
[0009] FIG. 2 is a chart of the survival following S. pneumoniae
challenge following treatment with a PDE 4 inhibitor.
[0010] FIG. 3 is a chart of the colony-forming-units/mouse
following a S. pneumoniae challenge following treatment with a PDE
4 inhibitor.
SPECIFIC EMBODIMENTS
[0011] Respiratory viral infections and course of treatment
[0012] This invention can be used in those situations where a
respiratory viral infection is or will most likely occur. It can be
used regardless of the type of viral material causing the
respiratory infection and regardless of the locus of the infection,
be it in the upper respiratory tract or the lower tract or both.
Numerous viruses are known to be involved in respiratory viral
infections, including but not limited to Rhinovirus, Influenza,
Respiratory Syncytial Virus, Parainfluenza, Adenovirus, Enterovirus
and Coronavirus.
[0013] The PDE4 inhibitor is administered in several ways: i)
during the course of the respiratory viral infection, starting at
any time and running for less that the full length of the viral
infection; ii) during the whole of the course of the respiratory
viral infection or some part thereof, and during the whole of the
course of the bacterial infection, or some part thereof; or iii) at
the start of or during the course of the bacterial infection or
some part thereof. In addition, the inhibitor can be administered
prior to the onset of a respiratory viral infection, and preferably
continued for some period during the course of the infection.
Furthermore, an inhibitor can be administered intermitently during
the respiratory viral infection phase, or over the course of both
the viral infection and the subsequent bacterial infection.
[0014] PDE 4 Inhibitors
[0015] The inhibitors of interest in this invention are PDE 4
inhibitors. Of particular interest are those which are specific for
PDE 4. A preferred group of inhibitors are those that have an
IC.sub.50 ratio (high/low binding) of about 0.1 or greater as
further described in U.S. Pat. No. 5,998,428; this patent is
incorporated herein in full by reference as if fully set forth
herein. A preferred standard for PDE 4-specific inhibitors which
can be used in this invention is one where the compound has an
IC.sub.50 ratio of about 0.1 or greater; said ratio being the ratio
of the IC.sub.50 value for competing with the binding of 1 nM of
[.sup.3H]R-rolipram to a form of PDE 4 which binds rolipram with a
high affinity over the IC.sub.50 value for inhibiting the PDE 4
catalytic activity of a form which binds rolipram with a low
affinity using 1 .mu.M[.sup.3H]-cAMP as the substrate.
[0016] Other PDE 4 inhibitors that may be included in these
formulations include those set out in U.S. Pat. No. 5,552,438
issued Sep. 3, 1996. This patent and the compounds it discloses are
incorporated herein in full by reference. The compound of
particular interest, which is disclosed in U.S. Pat. No. 5,552,438,
is cis-4-cyano-4-[3-(cyclopentyloxy-
)-4-methoxyphenyl]cyclohexane-1-carboxylic acid (Ariflo.RTM.) and
its salts, esters, pro-drugs or physical forms. Other PDE 4
inhibitors which may be of interest include: 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 PDE 4 inhibitor identified as CI-1018 (PD-168787;
Parke-Davis/Warner-Lambert); a benzodioxole derivative Kyowa Hakko
disclosed in WO 9916766; V-1 1294A 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; and a
compound identified as T-440 (Tanabe Seiyaku; Fujii, K. et al. J
Pharmacol Exp Ther, 1998, 284(1): 162). Preferred compounds of this
invention are those which have an IC.sub.50 ratio of greater than
0.5, and particularly those compounds having a ratio of greater
than 1.0. The most preferred compounds are roflumilast and
cis-4-cyano-4-[3-(cyclop-
entyloxy)-4-methoxyphenyl]cyclohexane-1-carboxylic acid.
[0017] In addition, one or more of these PDE 4 inhibitors can be
combined with other methods for treating influenza and/or bacterial
infections. For example, a PDE 4 inhibitor could be co-administered
with a treatment for influenza during the course of the viral
infection. Examples of such drugs are alpha and beta interferon,
Inosine pranobex, moroxydine hydrochloride, zanamivir, rimantadine,
rimantadine hydrochloride, oseltamivir phosphate and the like.
[0018] If the PDE 4 inhibitor is administered during the course of
the bacterial infection, an anti-infective agent may be
co-administered at the same time or in conjunction with the PDE 4
inhibitor.
[0019] As regards antibiotics, they are a very diverse class of
compounds which often classified and discussed in groups. Herein
those of greatest interest will be the drugs and preparations which
treat infections in the lung. S. pneumoniae is often found in
patients with pneumonia. It is sensitive to the penicillins,
benzylpenicillin, amoxycillin, or ampicillin; to cephalosporins; to
erythromycin; or to co-trimoxazole. Mycoplasma pneumoniae is also
an important cause of community-acquired pneumonia. Erythromycin or
tetracycline are the antibacterials of choice against M.
pneumoniae; erythromycin is usually recommended when mycoplasmal
pneumonia is suspected. Bacteria that may be responsible for
community-acquired pneumonia include Haemophilus influenzae and,
more recently, Moraxella (Branhamella) catarrhalis especially in
patients with chronic lung disease; Legionella pneumophila (see
Legionnaires' Disease); Chlamydia psittaci (see Psittacosis);
Chlamydia pneumoniae (formerly known as the TWAR strain of
Chlamydia psittaci); and Coxiella burnetii (see Q fever). A fuller
discussion of pulmonary infections and antibiotics for their
treatment can be found in the likes of Tomes CPS.TM. System from
Micromedex. This publication is available on the Web courtesy of
Micromedex, Inc., The section relating to antibiotics relevant to
this invention has the URL
[0020] http://www.tomescps.com/DKS/DATAJMT/MTM1-z.htm#l
-a5-328-k.
[0021] For the purposes of this invention, it is preferred to
administer the PDE 4 inhibitorin an amount between about 1 mg to
200 mg, more preferably 5 to 100 mg, and most preferably between 5,
or 10 to 60 mg of the active ingredient. Additional preferred
dosage amounts are about within these ranges are 10, 15, 20, 30,
40, 50, 60, 70, 80 or 90 mg per preparation. The inhibitor can be
administered by any route. A perferred formulation will be an oral
immediate release tablet or a oral controlled release tablet. The
inhibitor may be administered up to 6 times per day, though a twice
or once a day dosing regime is preferred.
[0022] Assay
[0023] The assay used here-in is a murine model where the subject
is administered a sub-lethal influenza infection followed by
Streptococcus pneumoniae co-infection and then evaluated for
survival. In this model, mice are infected with a dose of influenza
virus that leads to a non-lethal self-limiting infection. Mice that
had recently cleared an influenza infection required 4 loglo fewer
S. pneumoniae to cause mortality than normal non-influenza
challenged animals. While mice dosed with saline required 10.sup.4
cfu of S. pneumoniae to show significant mortality, influenza
challenged animals showed significant mortality with as few as
280-cfu pneumococcl.
[0024] Mice: Male CBA/J mice obtained from Jackson Laboratories
(Bar Harbor, Me.) 3 to 4 weeks of age were utilized. Mice were
housed 10 per cage in standard laboratory plastic caging and fed
lab chow and water ab libitum. All animal procedures were performed
in accordance with the highest standards for the humane handling,
care and treatment of research animals.
[0025] Preparation 1
[0026] Bacteria: Two separate S. pneumoniae clinical isolates
(strains #1629 and #0100993) were individually animal-passaged in
mice and subsequently recovered and plated onto sheep red blood
cell trypticase soy agar (TSA) then stored frozen at -70.degree. C.
The re-isolated organisms were grown overnight on chocolate TSA
(Becton Dickinson, Md.) and harvested in saline. The concentration
of the stock was determined by absorbance (560 nm) using a
spectrophotometer (Beckman DU 640B, Calif.). Inoculum doses were
based on viable counts determined by scoring colony-forming units
on sheep reb blood TSA.
[0027] Preparation 2
[0028] Virus: Influenza type A virus strain A/PR/8/34 sub type H1N1
(American Type Culture Collection, Rockville, Md.) was propagated
in the allantoic cavity of 10 day old fertilized chicken eggs. The
eggs were incubated for 48 hrs at 37.degree. C., refrigerated for 2
1/2 hours at 4.degree. C. then the allantonic fluid was harvested.
Pooled allantoic fluid was centrifuged (1,000 rcf; 15 min;
4.degree. C.) to remove cells, and then divided into aliquots for
storage at -70.degree. C. The titer of the stock culture of virus
was 1.0.times.10.sup.9 TCID.sub.50/ml.
[0029] Preparation 3
[0030] Mice were anesthetized with ketamine/xylazine (40 mg/kg;10
mg/kg) and inoculated intranasally (IN) with 50 .mu.l of influenza
virus or vehicle control. Mice that were co-infected with bacteria
were anesthetized as above then infected IN with 25 .mu.l of
bacterial suspension.
[0031] Preparation 4
[0032] Virus Titrations: Lungs were removed aseptically and placed
in vials containing 1 micron glass beads (Biospec Products,
Bartlesville, Okla.) and 1 ml. of Eagles minimal essential medium
with penicillin and streptomycin. The lungs were homogenized for 45
seconds using a mini-bead beater (Biospec Products, Bartlesville,
Okla.); the vials were then centrifuged at 1,000 rcf for 15 minutes
at 4.degree. C., and the lung supernatants were frozen at
-20.degree. C. Virus was quantitated in an in vitro microassay
using Madin-Darby canine kidney (MDCK) cells (r). Serial dilutions
of lung homogenates (in medium plus 2.5 ug/ml trypsin) were added
to round-bottom microtiter wells containing adherent MDCK cells.
After 5 days of incubation at 37.degree. C. (5% CO.sub.2), 50 .mu.l
of 0.5% 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
(TCID.sub.50) calculated by logistic regression (s).
[0033] Preparation 5
[0034] Bacterial Titrations: Lungs were harvested as above and
homogenized (Stomacher 80 Seward Medical, London, England) in 1.0
ml of saline. Serial dilutions were made in saline and inoculated
onto sheep red blood cell TSA for enumeration.
[0035] Preparation 6
[0036] Histochemistry: Whole lungs were inflated-fixed with 10%
formalin in phosphate-buffered saline and immersed in fixative for
24 hrs at 4.degree. C. Tissues were processed for paraffin
sectioning using an automated tissue processor. Paraffin-embedded
lungs were serially sectioned at 6 .mu.m and prepared for
hemotoxylin and eosin (H&E) staining.
[0037] Preparation 7
[0038] Cytokine analysis: Bronchiolar alveolar lavage fluid (BALF)
cytokine levels were measured by quantitative ELISA using
commercially available kits. Mice were sacrificed and the trachea
exposed, a cannula was inserted and ligated. By syringe each lung
was flushed three times with 1 ml of normal saline. The BALF was
centrifuged at 2000 rcf for 1 min. and the supernatant was stored
at -70.degree. C. The cytokine concentrations and thresholds were
determined as described by the manufacturer; TNF-.alpha., IL-6,
IFN-.gamma., and KC (R&D Systems, Minneapolis, Minn.) IL-10,
IL-1.beta. (Biosource International, Camarillo, Calif.).
[0039] Preparation 8
[0040] Determination of pulmonary functions: Barometric
plethysmography was conducted on unrestrained mice using whole body
plethysmography (Buxco, Troy, N.Y.), as described (Hamelmann et al,
1997, Noninvasive measurement of airway responsiveness in allergic
mice using barometric plethysmography, Amer. J. of Resp. Crit. Care
Med. 156, 766-775.). Briefly, individual chambers were calibrated
prior to each reading, mice were placed in the main chamber, and
pressure differences between the main chamber and a reference
chamber were recorded for 2 minutes. Using the Biosystem Pulmonary
Analyzer software (Buxco) the resultant box pressure was used to
calculate the phases of the respiratory cycle, tidal volumes, and
enhanced pause (Penh). Penh value is a function of the proportion
of maximal expiratory to maximal inspiratory box pressure signals
and the timing of expiration. Uninfected or Influenza A/PR/8/34
infected mice dosed with Ariflo.RTM. at 10 or 30 mg/Kg/day (days
0-8) were evaluated daily for 16 days.
EXAMPLE 1
[0041] Sub-lethal Influenza Infection Model.
[0042] Initial 21-day survival studies determined that mice
challenged intranasally (IN) with as few as 350 TCID.sub.50 of
influenza strain A/PR/8134 resulted in 80% to 90% of the mice
succumbing to the infection. Lower doses of virus resulted in over
95% of the animals surviving. From these studies a dose range of 10
to 50 TCID.sub.50 was selected as a sub-lethal dose for use in
co-infection models. To confirm that mice challenged with this low
inocula have a self-limiting infection, mice were challenged IN
with 10 to 50 TCID.sub.50 and sacrificed 2 hrs after infection, on
days 1 to 11 and day 14. The lungs were harvested and the viral
titers determined. Two hours after infection there was no virus
detectable from the lungs of the mice. However, by day 1 after
infection there was detectable virus in the lung homogenates. The
virus titers peaked on day 5 with 4.8.times.10.sup.5 TCID.sub.50 in
the lungs. The virus titers decreased after day 5 with all the
animals being clear of virus by day 9 after infection.
[0043] Histopathology from these animals show a progressive lower
respiratory tract infection with heavy thickening of the alveoli by
day 6 after infection. There is interstitial pneumonitis with
capillary thrombosis and necrosis of the of the alveolar walls.
Both alveolar type I and II cells lining the alveolar spaces
slough. While the virus was reduced to undetectable levels by day 9
after infection, considerable consolidation of the lungs remains.
By day 15 after infection, although some consolidated areas remain,
there are increased areas of improved lung.
[0044] Acute Influenza A infection results in a reduction in
pulmonary functions. Whole body plethysmography is currently
accepted as a measurement for pulmonary resistance in allergic or
infected mice (Haczku et al. 1997; Hamelmann et al. 1997a;
Hamelmann et al. 1997b; Schwarze et al. 1998; van Schaik et al.
1998). Penh was used in this study as a parameter representing
pulmonary functions. Uninfected or Influenza A/PR/8/34 infected
mice were evaluated daily for 16 days. Penh values were elevated
above baseline (0.4-0.5) in all animals between days 5-16 post
infection.
[0045] The wet and dry lung weights from sub-lethal influenza
challenged mice were evaluated as follows: The wet and dry lung wts
were evaluated from mice sub-lethally challenged with influenza
virus; n=4 per group. The wet lungs weights, indicated an
increasing pneumonitis with significant increases in wet weight as
early as day 2 after infection followed by a rapid increase over
days 5 and 6. The dry lung weights did not reach statistically
significant increases in weight until day 6 after infection.
Otherwise the dry lung wts reflected the wet lung weights. The wet
lung weights showed small but statistically significant increases
as early as day 2 with larger increases on days 5 to 6. The dry
lung weights did not show significant increases until day 6 after
infection. Both the wet and dry weights remained elevated after the
virus cleared, day 9.
[0046] The cytokine response to influenza challenge was evaluated
in bronchiolar lavage fluid (BALF), increases were seen in IL-6,
IFN.gamma., and the chemokine KC (Table 1). All showed peak levels
on day 8 after challenge with levels decreasing to pre-infection
levels by day 10. TNF.alpha., IL-10, IL-1.beta., and IL-2 were also
evaluated, however no significant elevation in levels ware found
for these cytokines at any time point.
1TABLE 1 Table I. Cytokine Concentration (pg/ml) in the BALF from
Sub-lethally Infected Mice Day 1 Day 3 Day 6 Day 8 Day 10 Day 13
Day 15 IL-6 120 .+-. 15 118 .+-. 12 448 .+-. 221 575 .+-. 170* 279
.+-. 194 116 .+-. 6 128 .+-. 10 IFN-.gamma. 34 .+-. 10 33 .+-. 16
692 .+-. 328* 2392 .+-. 783* 87 .+-. 49 30 .+-. 5 30 .+-. 2 KC 16
.+-. 2 24 .+-. 7 112 .+-. 38* 206 .+-. 52* 73 .+-. 42 42 .+-. 11 23
.+-. 13 TNF-.alpha. 40 .+-. 21 16 .+-. 10 19 .+-. 2 33 .+-. 15 16
.+-. 5 13 .+-. 1 22 .+-. 13 IL-10 272 .+-. 59 177 .+-. 96 255 .+-.
86 242 .+-. 117 148 .+-. 35 107 .+-. 49 208 .+-. 112 IL-1.beta. 84
.+-. 29 203 .+-. 90 130 .+-. 19 128 .+-. 89 99 .+-. 79 61 .+-. 58
105 .+-. 41 IL-2 2 .+-. 1 2 .+-. 1 2 .+-. 1 2 .+-. 1 2 .+-. 1 2
.+-. 1 1 .+-. 1 * P < 0.05
EXAMPLE 2
[0047] Establishment of Bacterial Co-Infection Model
[0048] In initial studies mice were infected IN with a sub-lethal
dose of influenza virus or non-virally infected vehicle controls.
On day 9 after infection the mice were given a second challenge IN
with S. pneumoniae (strain #0100993). The non-virally infected mice
were resistant to infection requiring 2.8.times.10.sup.7 cfu/mouse
S. pneumoniae to lead to no animals surviving. The calculated
LD.sub.50 for non-virally infected mice challenged with S.
pneumoniae was 4.7.times.10.sup.5 cfu/mouse. In contrast, the mice
challenged with a sub-lethal dose of influenza were sensitive to a
second challenge with S. pneumoniae with all the mice co-infected
with 2.8.times.10.sup.2 cfu/mouse of S. pneumoniae dying. The
LD.sub.50 for the virus infected mice was only 66 cfu/mouse. These
results replicated with a second strain of S. pneumoniae (strain #
1629), the non-virus infected animals showed a S. pneumoniae
LD.sub.50 of 2.4.times.10.sup.5 cfu/mouse while the virus
challenged animals showed an LD.sub.50 of only 47 cfu/mouse.
[0049] Initial studies in the bacterial co-infection model were
performed on day 9 after virus challenge because that was the first
day the animals were influenza virus culture negative. A study was
done to determine the length of time that the increased sensitivity
to infection with S. pneumoniae was sustained. In this study mice
were challenged with influenza as above and then separate groups
were challenged with 375 cfu of S. pneumoniae on day 7 through 15
(FIG. 1). Mice were challenged with a sub-lethal dose of influenza
virus; n=10 per group. On days 7 to 15 after infection individual
groups of mice were challenged with 375 cfu/mouse of S. pneumoniae.
The mice challenged with pneumococci just before of just after the
virus is cleared, days 7 to 9, showed high susceptibility to the
secondary infection. The mice challenged with pneumococci at latter
time points, day 10 to 15, showed increasing resistance to
infection. The animals did not return to normal levels of
pneumococcal resistance at any time point tested up to 15 days. The
mice co-infected with bacteria on days 7 to 9 all had a low
survival rate. Mice co-infected on later days showed improved
survival, although this improvement did not make the virus
challenged animals as resistant to infection as normal saline
challenged animals.
[0050] To determine if the decrease in survival demonstrated in
co-infected mice was due to bacterial pneumonia, mice were infected
with virus as above and challenged on day 9 with S. pneumoniae
(strain # 0100993) 492 cfu/mouse. Mice were sacrificed at 2, 24 and
48 hrs post bacterial challenge and whole lungs were homogenized
and the number of S. pneumoniae determined for each animal. Two hrs
after infection there were few recoverable organisms from the lungs
of the mice. However, by 24 hrs post infection the number of
recovered S. pneumoniae were 8.9.times.10.sup.5 cfu/mouse, at 48
hrs the numbers had climbed to 2.1.times.10.sup.6 cfu/mouse. Later
time points could not be taken since the animals start to succumb
to the infection on day 3 after infection with all the animals
dying by day 5. These results replicated with a second strain of S.
pneumoniae (strain # 1629), with 3.8.times.10.sup.5 cfu/mouse being
recovered from the lungs at 24 hrs after infection climbing to
1.6.times.10.sup.8 cfu/mouse at 48 hrs post bacterial
challenge.
EXAMPLE 3
[0051] Pretreatment With a PDE 4 Inhibitor
[0052] In this study the mice were treated orally with 10 or 30
mg/kg of
cis-4-cyano-4-[3-(cyclopentyloxy)-4-methoxyphenyl]cyclohexane-1-carboxyli-
c acid once daily on days 0 to 9. The vehicle was 2%
methylcellulose. These mice were infected with a sub-lethal dose of
influenza virus IN as described above. On day 9 after influenza
infection the mice were challenged IN with S. pneumoniae (strain
#0100993) prepared as described above. The mice were then followed
for survival. The mice treated with control diluent had an
LD.sub.50 of 72 and Ariflo (10, 30 mg/kg) treated mice had a
LD.sub.50 of 252 and 221 cfu/mouse respectively of S. pneumoniae.
See FIG. 3 for survival rates.
* * * * *
References