U.S. patent application number 10/413953 was filed with the patent office on 2003-09-25 for compositions and methods for treating cystic fibrosis.
This patent application is currently assigned to Boehringer Ingelheim Pharma KG. Invention is credited to Anderskewitz, Ralf, Birke, Franz, Jennewein, Hans Michael, Jung, Birgit, Meade, Christopher J. Montague.
Application Number | 20030181518 10/413953 |
Document ID | / |
Family ID | 26245566 |
Filed Date | 2003-09-25 |
United States Patent
Application |
20030181518 |
Kind Code |
A1 |
Anderskewitz, Ralf ; et
al. |
September 25, 2003 |
Compositions and methods for treating cystic fibrosis
Abstract
A method of treating or preventing cystic fibrosis in a patient
in need of such treatment, the method comprising administering to
the patient a therapeutically effective amount of an LTB.sub.4
antagonist of formula (I) 1 or a tautomer thereof.
Inventors: |
Anderskewitz, Ralf;
(Laupheim, DE) ; Meade, Christopher J. Montague;
(Bingen, DE) ; Birke, Franz; (Ingelheim, DE)
; Jennewein, Hans Michael; (Wiesbaden, DE) ; Jung,
Birgit; (Schwabenheim, DE) |
Correspondence
Address: |
BOEHRINGER INGELHEIM CORPORATION
900 RIDGEBURY ROAD
P. O. BOX 368
RIDGEFIELD
CT
06877
US
|
Assignee: |
Boehringer Ingelheim Pharma
KG
Ingelheim
DE
|
Family ID: |
26245566 |
Appl. No.: |
10/413953 |
Filed: |
April 15, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10413953 |
Apr 15, 2003 |
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10050409 |
Jan 16, 2002 |
|
|
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6576669 |
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60266833 |
Feb 6, 2001 |
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Current U.S.
Class: |
514/486 ;
514/637 |
Current CPC
Class: |
Y02A 50/30 20180101;
A61P 11/00 20180101; A61K 31/27 20130101; A61P 43/00 20180101; A61K
45/06 20130101; A61P 1/18 20180101; A61K 31/155 20130101; A61P
11/12 20180101; Y02A 50/475 20180101; Y10S 514/851 20130101; A61K
31/155 20130101; A61K 2300/00 20130101; A61K 31/27 20130101; A61K
2300/00 20130101 |
Class at
Publication: |
514/486 ;
514/637 |
International
Class: |
A61K 031/325; A61K
031/155 |
Foreign Application Data
Date |
Code |
Application Number |
Jan 16, 2001 |
GB |
0101128 |
Claims
We claim:
1. A method of treating or preventing cystic fibrosis in a patient
in need of such treatment, the method comprising administering to
the patient a therapeutically effective amount of an LTB.sub.4
antagonist of formula (I) 3or a tautomer thereof.
2. The method according claim 1, wherein the therapeutically
effective amount of an LTB.sub.4 antagonist of formula (I) is
between 5 mg to 200 mg.
3. The method according to claim 1, wherein an additional active
ingredient selected from the group consisting of antibiotics,
LTA.sub.4 hydrolase inhibitors, 5-lipoxygenase inhibitors, and
agents that enhance mucus clearance, are administered
simultaneously or sequentially with the LTB.sub.4 antagonist of
formula (I).
4. The method according to claim 2, wherein an additional active
ingredient selected from the group consisting of antibiotics,
LTA.sub.4 hydrolase inhibitors, 5-lipoxygenase inhibitors, and
agents that enhance mucus clearance, are administered
simultaneously or sequentially with the LTB.sub.4 antagonist of
formula (I).
5. The method according to claim 1, wherein an additional active
ingredient selected from the group consisting of aminoglycoside
antibiotics, antibacterial peptides derived from or related to the
structure of defensins, and agents which inhibit the production or
action of neutrophil elastase, are administered simultaneously or
sequentially with the LTB.sub.4 antagonist of formula (I).
6. The method according to claim 2, wherein an additional active
ingredient selected from the group consisting of aminoglycoside
antibiotics, antibacterial peptides derived from or related to the
structure of defensins, and agents which inhibit the production or
action of neutrophil elastase, are administered simultaneously or
sequentially with the LTB.sub.4 antagonist of formula (I).
7. The method according to claim 1, wherein an additional active
ingredient selected from the group consisting of atreleuton,
zileuton, FK-706, CE 1037, EPI-HNE-4, alpha 1-antitrypsin,
ambroxol, gentamycin, amikacin, kanamycin, streptomycin, neomycin,
netimicin, colistin, iseganan, and tobramycin are administered
simultaneously or sequentially with the LTB.sub.4 antagonist of
formula (I).
8. The method according to claim 2, wherein an additional active
ingredient selected from the group consisting of atreleuton,
zileuton, FK-706, CE 1037, EPI-HNE-4, alpha 1-antitrypsin,
ambroxol, gentamycin, amikacin, kanamycin, streptomycin, neomycin,
netimicin, colistin, iseganan, and tobramycin are administered
simultaneously or sequentially with the LTB.sub.4 antagonist of
formula (I).
9. The method according to claim 1, wherein the LTB.sub.4
antagonist of formula (I) is administered orally.
10. The method according to claim 2, wherein the LTB.sub.4
antagonist of formula (I) is administered orally.
11. The method according to claim 3, wherein the LTB.sub.4
antagonist of formula (I) and the additional active ingredient are
administered orally.
12. The method according to claim 4, wherein the LTB.sub.4
antagonist of formula (I) and the additional active ingredient are
administered orally.
13. The method according to claim 5, wherein the LTB.sub.4
antagonist of formula (I) and the additional active ingredient are
administered orally.
14. The method according to claim 6, wherein the LTB.sub.4
antagonist of formula (I) and the additional active ingredient are
administered orally.
15. The method according to claim 7, wherein the LTB.sub.4
antagonist of formula (I) and the additional active ingredient are
administered orally.
16. The method according to claim 8, wherein the LTB.sub.4
antagonist of formula (I) and the additional active ingredient are
administered orally.
17. A medicament comprising: (A) an LTB.sub.4 antagonist of formula
(I) 4or a tautomer thereof; (B) one or more additional active
ingredients selected from the group consisting of antibiotics,
LTA.sub.4 hydrolase inhibitors, 5-lipoxygenase inhibitors, and
agents that enhance mucus clearance; and (C) optionally a
pharmaceutically acceptable carrier.
18. A medicament according to claim 17, wherein the weight ratio of
(A) to (B) is from 100:1 to 1:200.
19. A pharmaceutical kit comprising at least two separate unit
dosage forms (A) and (B): (A) an LTB.sub.4 antagonist of formula
(I) 5or a tautomer thereof, and optionally a pharmaceutically
acceptable carrier; and (B) one or more additional active
ingredients selected from the group consisting of antibiotics,
LTA.sub.4 hydrolase inhibitors, 5-lipoxygenase inhibitors, and
agents that enhance mucus clearance, and optionally a
pharmaceutically acceptable carrier.
20. A pharmaceutical kit comprising at least two separate
containers (A) and (B), (A) and (B) comprising: (A) an LTB.sub.4
antagonist of formula (I) 6or a tautomer thereof, and optionally a
pharmaceutically acceptable carrier; and (B) one or more additional
active ingredients selected from the group consisting of
antibiotics, LTA.sub.4 hydrolase inhibitors, 5-lipoxygenase
inhibitors, and agents that enhance mucus clearance, and optionally
a pharmaceutically acceptable carrier.
Description
RELATED APPLICATIONS
[0001] This application is a continuation of U.S. Ser. No.
10/050,409, filed Jan. 16, 2002. Benefit under 35 U.S.C. .sctn.
119(e) of prior provisional application Serial No. 60/266,833,
filed Feb. 6, 2001, is hereby claimed.
FIELD OF THE INVENTION
[0002] The invention relates to the use of a LTB.sub.4 antagonist
or a pharmaceutically acceptable salt thereof for the preparation
of a medicament for the treatment and/or prevention of diseases
caused by increased expression of mucin genes in the bronchial or
gastrointestinal epithelium.
BACKGROUND OF THE INVENTION
[0003] Cystic fibrosis (CF) is an inherited disease primarily due
to a defect in the cystic fibrosis trans-membrane regulating
protein (CFTR). This results in abnormal chloride transfer across
epithelial membranes. Symptoms appear in a number of organ systems,
but for most patients the most important pathological changes
associated with the CFTR defect are those in the lung. Patients
with cystic fibrosis produce excessive quantities of viscous mucus
which readily becomes infected. Recurring infections are associated
with worsening of the condition of the patient and an increased
high risk of death. Exactly how the CFTR defect causes increased
mucus production is not known. One hypothesis is that the CFTR
mutation causes changes in the tracheal epithelium which engender
chronic bacterial infection, particularly with Pseudomonas
aeruginosa. These bacteria stimulate expression of mucin genes,
such as MUC-2 and MUC-5. Overproduction of mucus, combined with
mucus dehydration related to the underlying chloride channel
defect, lead to formation of mucus plugs and eventually to lung
failure (J. D. Li, A. F. Dohrman, M. Gallup et al., Transcriptional
Activation of Mucin by Pseudomonas aeruginosa Lipopolysaccharide in
the Pathogenesis of Cystic Fibrosis Lung Disease, Proc. Natl. Acad.
Sci. U.S.A., 1997, 94:967-972; A. Dohrman, S. Miyata, M. Gallup et
al., Mucin Gene (MUC 2 and MUC 5AC) Upregulation by Gram-positive
and Gram-Negative Bacteria, Biochim. Biophys. Acta, 1998,
1406:251-259).
[0004] Cystic fibrosis is associated with markedly elevated levels
of leukotriene B.sub.4 in the epithelial lining fluid of the lung
(M. W. Konstan, R. W. Walenga, K. A. Hilliard, J. B. Hilliard,
Leukotriene B4 Markedly Elevated in the Epithelial Lining Fluid of
Patients with Cystic Fibrosis, Am. Rev. Respir. Dis., 1993,
148:896-901), and this elevation is also detectable in the sputum
(J. T. Zakrzewski, N. C. Barnes, P. J. Piper, J. F. Costello,
Detection of Sputum Eicosanoids in Cystic Fibrosis and in Normal
Saliva by Bioassay and Radioimmunoassay, Br. J. Clin. Pharmacol.,
1987, 23:19-27). The source of this LTB.sub.4 is unclear since both
inflammatory cells such as neutrophils which are much more abundant
in the CF lung as well as epithelial cells have the necessary
enzyme machinery to synthesize this leukotriene.
[0005] Similarly, the exact mechanism by which Pseudomonas
aeruginosa stimulates the increased expression of mucin genes in
the bronchial epithelium is also unclear. Certainly, both
clinically and experimentally, infection with Pseudomonas
aeruginosa is associated with neutrophil infiltration into the
lung, and products of activated neutrophils such as elastase,
TGFbeta and TNFalpha are known to be able to increase the amount of
mucin specific RNA in the bronchial epithelium either by
stimulating the transcription of the genes or by impeding RNA
degradation (K. Takeyama, C. Agusti, I. Ueki, J. Lausier, L. O.
Cardell, J. A. Nadel, Neutrophil-Dependent Goblet Cell
Degranulation: Role of Membrane-Bound Elastase and Adhesion
Molecules, Am. J. Physiol. 1998, 275:L294-L302; K. Takeyama, K.
Dabbagh, H. M. Lee et al., Epidermal Growth Factor System Regulates
Mucin Production in Airways, Proc. Natl. Acad. Sci. U.S.A., 1999,
96:3081-3086). However, there are also direct effects of
Pseudomonas aeruginosa on pulmonary epithelial cells, via bacterial
lipopolysaccharides (J. D. Li, A. F. Dohrman, M. Gallup et al.,
1997; loc. cit.), via bacterial proteases (J. D. Klinger, B.
Tandler, C. M. Liedtke, T. F. Boat, Proteinases of Pseudomonas
aeruginosa Evoke Mucin Release by Tracheal Epithelium, J. Clin.
Invest., 1984, 74:1669-1678), or via bacterial adhesins (J. K.
Ichikawa, A. Norris, M. G. Bangera et al., Interaction of
Pseudomonas aeruginosa with Epithelial Cells: Identification of
Differentially Regulated Genes by Expression Microarray Analysis of
Human cDNAs, Proc. Natl. Acad. Sci. U.S.A., 2000; 97:9659-9664).
The role of LTB.sub.4 in both the neutrophil-mediated and the
direct effects of Pseudomonas aeruginosa on the pulmonary
epithelium is unknown.
SUMMARY OF THE INVENTION
[0006] The present invention relates to the use of an LTB.sub.4
antagonist of formula (I), 2
[0007] a tautomer thereof or a pharmaceutically acceptable salt
thereof for the preparation of a medicament for the treatment
and/or prevention of diseases caused by increased expression of
mucin genes in the bronchial or gastrointestinal epithelium, in
particular diseases caused by Pseudomonas aeruginosa such as cystic
fibrosis.
[0008] Another aspect of the invention is a method for the
treatment of patients suffering from diseases caused by increased
expression of mucin genes in the bronchial or gastrointestinal
epithelium which method comprises administering to the patient in
need thereof an effective amount of a compound of formula (I), a
tautomer thereof or a pharmaceutically acceptable salt thereof.
[0009] The invention further provides a medicament containing,
separately or together,
[0010] (A) a compound of formula (I), a tautomer thereof or a
pharmaceutically acceptable salt thereof;
[0011] (B) one or more additional active ingredients selected from
the group consisting of antibiotics, LTA4 hydrolase inhibitors,
5-lipoxygenase inhibitors and agents that enhance mucus clearance;
and
[0012] (C) optionally a pharmaceutically acceptable carrier,
[0013] for simultaneous, sequential or separate administration in
the treatment of diseases caused by increased expression of mucin
genes in the bronchial or gastrointestinal epithelium.
[0014] Furthermore, the invention relates to a pharmaceutical kit
comprising at least two separate unit dosage forms (A) and (B):
[0015] (A) one of which comprises a composition containing a
compound of formula (I), a tautomer thereof or a pharmaceutically
acceptable salt thereof and optionally a pharmaceutically
acceptable carrier;
[0016] (B) one of which comprises a composition containing one or
more additional active ingredients selected from the group
consisting of antibiotics, LTA.sub.4 hydrolase inhibitors,
5-lipoxygenase inhibitors and agents that enhance mucus clearance
and optionally a pharmaceutically acceptable carrier.
DETAILED DESCRIPTION OF THE INVENTION
[0017] The pharmaceutically acceptable salts of the compound of
formula (I) include, for example salts of inorganic acids such as
hydrochloric, hydrobromic, sulfuric and phosphoric acids, and
organic acids such as fumaric, maleic, acetic, lactic, citric,
tartaric, ascorbic, succinic, glutaric, gluconic, tricarballylic,
oleic, benzoic, p-methoxybenzoic, salicylic, o- or
p-hydroxybenzoic, p-chlorobenzoic, methanesulfonic,
p-toluenesulfonic, and 3-hydroxy-2-naphthalene carboxylic acids.
Most preferred is the compound of formula (I) as such, i.e., in
form of the free base.
[0018] In a preferred embodiment of the present invention the
compound of formula I is used for the preparation of a medicament
for the prevention of goblet cell hyperplasia induced by products
of pathogen bacteria, in particular products derived from
Pseudomonas aeruginosa.
[0019] In addition, to being useful to inhibit Pseudomonas
aeruginosa-induced mucus production in cystic fibrosis patients,
the compound of formula (I) will also be useful, either alone or in
combination with other therapeutic agents such as antibiotics, for
the treatment of mucus hypersecretion associated with Pseudomonas
aeruginosa infection in patients without a defect in the CFTR
gene.
[0020] The compound of formula (I) will further be useful for the
treatment of mucoid enterocolitis, such as associated with
infection with Pseudomonas aeruginosa, as well as mucoid
enterocolitis associated with other pathogens such as Shigella.
Particularly useful will be the application of the compound of
formula (I) in combination with antibacterial therapy.
[0021] The compound of formula (I) can be used in the therapy of
cystic fibrosis either alone or in combination with other therapies
(B). It has now surprisingly been found that a significant
unexpected therapeutic benefit, particularly a synergistic
therapeutic benefit, in the treatment of diseases caused by
increased expression of mucin genes in the bronchial or
gastrointestinal epithelium can be achieved by combination therapy
using the compound of formula (I) and an active ingredient (B).
[0022] For instance, it is possible using this combination therapy
to reduce the dosages required for a given therapeutic effect
considerably compared with those required using treatment with (B)
alone, thereby minimizing possibly undesirable side effects.
[0023] Furthermore, this combination therapy exhibits both a fast
onset of action and a long duration of action, so that patients
feel a rapid improvement in their condition and a reduced need for
short-acting rescue medicaments.
[0024] Particularly useful may be combination of the compound of
formula (I) with antibiotic or antibacterial therapies (B1) for the
bacterial airway superinfection. These therapies will include, but
not be confined to those antibiotics listed with appropriate
dosages in Table 1 of the published consensus statement of G.
Doring et al., Antibiotic Therapy Against Pseudomonas aeruginosa in
Cystic Fibrosis: A European Consensus, Eur. Respir. J., 2000
16:749-767.
[0025] Also suitable for combination with the compound of formula
(I) are the antibiotic azithromycin and the antibiotic
duramycin.
[0026] The compound of formula (I) can also be combined with
antibacterial peptides derived from or related to the structure of
defensins. Since secreted mucus presents a barrier which can
prevent inhaled antibiotics or antibacterial peptides reaching
target bacteria in the airways at sufficient concentration for
effective antibacterial action, the prevention of mucin production
by the compound of formula (I) is particularly useful as a means of
enhancing the effectiveness of defensins, or antibiotics such as
colistin, iseganan, or tobramycin, which are preferably
administered by the inhaled route.
[0027] In view of the ability of the compound of formula (I) to
block Pseudomonas aeruginosa-induced mucus production, a further
particularly useful combination will be that of the compound of
formula (I) with agents that enhance mucus clearance (B2), such as
ambroxol. A detailed list of drugs which enhance mucus clearance is
given by E. Houtmeyers et al., Effects of Drugs on Mucus Clearance,
Eur. Respir. J., 1999, 14:452-467.
[0028] Another particularly useful combination is of the compound
of formula (I) with drugs that reduce mucus production (B3) by
other mechanisms than LTB.sub.4 inhibition. Such drugs include but
are not confined to drugs which inhibit the production or action of
neutrophil elastase such as FK-706, CE 1037, EPI-HNE-4, and alpha
1-antitrypsin.
[0029] Drugs which reduce the amount of LTB.sub.4 produced will
reduce the amount of LTB.sub.4 antagonist required to produce a
therapeutic effect. Such drugs include but are not confined to
LTA.sub.4 hydrolase inhibitors such as those described in U.S. Pat.
No. 5,723,492 and 5-lipoxygenase inhibitors such as atreleuton and
zileuton. The combination of the compound of formula (I) and a
compound from U.S. Pat. No. 5,723,492, or the compound of formula
(I) and a 5-lipoxygenase inhibitor are particularly useful for the
treatment of cystic fibrosis.
[0030] The weight ratio of the compound of formula (I) or salt
thereof (A) to (B) may be, in general, from 100:1 to 1:200, for
example from 75:1 to 1: 190, from 75:1 to 1:150, from 60:1 to
1:120, from 50:1 to 1:100, from 50:1 to 1:50, from 30:1 to 1:40,
form 20:1 to 1:20, from 10:1 to 1:15, from 8:1 to 1:10, from 4:1 to
1:10, or from 1:1 to 1:5. The two drugs (A) and (B) may be
administered separately in the same ratio.
[0031] The compound of formula (I) may be administered orally,
transdermally, by inhalation or parenterally. The compound of
formula (I) occurs as active ingredients in conventional
preparations, for example in compositions which consist essentially
of an inert pharmaceutical carrier and an effective dose of the
active substance, such as for example tablets, coated tablets,
capsules, powders, solutions, suspensions, emulsions, syrups,
suppositories, transdermal systems, etc. An effective dose of the
compounds according to the invention is between 0.01 and 100,
preferably between 0.1 and 50, most preferably between 5-30 mg/dose
for oral administration, and between 0.001 and 50, preferably
between 0.1 and 10 mg/dose for intravenous or intramuscular
administration. For inhalation, according to the invention,
solutions containing 0.01 to 1.0, preferably 0.1 to 0.5% active
substance are suitable. For administration by inhalation the use of
powders is preferred. It is also possible to use the compounds
according to the invention as a solution for infusion, preferably
in a physiological saline or nutrient saline solution.
[0032] The administration of the compound of formula (I) once or
twice a day for at least five days is preferred.
[0033] The compounds of formula (I) may be used on their own or in
conjunction with other active substances according to the
invention, optionally also in conjunction with other
pharmacologically active substances. Suitable preparations include
for example tablets, capsules, suppositories, solutions, elixirs,
emulsions or dispersible powders.
[0034] Suitable tablets may be obtained, for example, by mixing the
active substance(s) with known excipients, for example inert
diluents such as calcium carbonate, calcium phosphate or lactose,
disintegrants such as corn starch or alginic acid, binders such as
starch or gelatin, wetting agents, lubricants such as magnesium
stearate or talc and/or agents for delaying release, such as
carboxymethyl cellulose, cellulose acetate phthalate,
polyvinylpyrrolidone or polyvinyl acetate. The tablets may also
comprise several layers.
[0035] Coated tablets may be prepared accordingly by coating cores
produced analogously to the tablets with substances normally used
for tablet coatings, for example collidone or shellac, gum arabic,
talc, titanium dioxide or sugar. To achieve delayed release or
prevent incompatibilities the core may also consist of a number of
layers. Similarly the tablet coating may consist of a number or
layers to achieve delayed release, possibly using the excipients
mentioned above for the tablets.
[0036] Syrups or elixirs containing the active substances or
combinations thereof according to the invention may additionally
contain a sweetener such as saccharine, cyclamate, glycerol or
sugar and a flavor enhancer, e.g., a flavoring such as vanillin or
orange extract. They may also contain suspension adjuvants or
thickeners such as sodium carboxymethyl cellulose, wetting agents
such as, for example, condensation products of fatty alcohols with
ethylene oxide, or preservatives such as p-hydroxybenzoates.
[0037] Solutions for injection are prepared in the usual way, e.g.,
with the addition of preservatives such as p-hydroxybenzoates, or
stabilizers such as alkali metal salts of ethylenediamine
tetraacetic acid, and transferred into injection vials or
ampoules.
[0038] Capsules containing one or more active substances or
combinations of active substances may for example be prepared by
mixing the active substances with inert carriers such as lactose or
sorbitol and packing them into gelatin capsules.
[0039] Suitable suppositories may be made for example by mixing
with carriers provided for this purpose, such as neutral fats or
polyethyleneglycol or the derivatives thereof.
[0040] A therapeutically effective daily dose is between 0.1 mg and
800 mg, preferably 10 mg to 500 mg, in particular 100 mg to 300 mg
per adult.
[0041] It has now surprisingly been found that the potent, long
acting LTB.sub.4 antagonist of formula (I), is able to influence
the Pseudomonas aeruginosa-induced transcription of a mucin
gene.
[0042] The particular mucin gene here measured is muc-5AC RNA. This
is the main mucin induced in rat epithelium by inflammatory
stimuli. In man, studies with bronchial explants as well as
isolated bronchial epithelial cells have shown that the
transcription of this gene's human analogue, MUC-5AC, is
upregulated by Pseudomonas aeruginosa (A. Dohrman, S. Miyata, M.
Gallup et al., 1998, loc. cit.).
[0043] The LTB.sub.4 antagonist of formula (I) has also
surprisingly been found to be able to inhibit the increase in
number of goblet cells in the tracheal epithelium following
exposure to Pseudomonas aeruginosa toxin. Goblet cells are an
important source of mucin. Goblet cells hyperplasia is a feature of
cystic fibrosis (C. W. Bedrossian, S. D. Greenberg, D. B. Singer,
J. J. Hansen, H. S. Rosenberg, The Lung in Cystic Fibrosis: A
Quantitative Study Including Prevalence of Pathologic Findings
Among Different Age Groups, Hum. Pathol., 1976, 7:195-204) and can
also be induced experimentally in monkeys by exposure to
Pseudomonas aeruginosa (A. T. Cheung, R. B. Moss, G. Kurland, A. B.
Leong, W. J. Novick Jr., Chronic Pseudomonas aeruginosa
Endobronchitis in Rhesus Monkeys: II. A Histopathologic Analysis,
J. Med. Primatol., 1993, 22:257-262).
[0044] Biochemical Study of Effects of LTB.sub.4 Antagonist (1) on
Mucin RNA Production
[0045] Two different rat strains, BDE and F344/NHsd, were tested
because previous studies had shown strain-related differences in
reactivity to lipopolysaccharides.
[0046] Materials and Methods
1 Acronyms and Abbreviations CF cystic fibrosis FAM
6-carboxyfluorescein LPS lipopolysaccharide LTB.sub.4 leukotriene
B.sub.4, (5S,12R)-dihydroxy-6,14-cis-8,10-trans- eicosatetraenoic
acid PCR polymerase chain reaction TAMRA
6-carboxytetramethylrhodami- ne
[0047] Animals Used: Adult male rats, strain BDE/Han, average
weight about 330 g (range 340 g to 450 g), and adult male F344/NHsd
rats, average weight about 250 g (range 240 g to 260 g).
[0048] Animal Maintenance: The rats were housed in air-conditioned
rooms, at 21.degree. C.-25.degree. C., relative humidity between
50% and 65%, day-night cycle 12 hours. They were fed before the
start of the experiment and received tap water ad lib. 18 hours
prior to drug administration, food was withdrawn, but drinking
water remained available.
[0049] The compound of formula (I), namely carbamic acid,
[[4-[[3-[[4-[1-(4-hydroxyphenyl)-1-methylethyl]phenoxy]methyl]phenyl]meth-
oxy]phenyl]iminomethyl]ethyl ester, was synthesized as described in
the International Patent Application WO 96/02497 and jet-milled.
For administration to animals, the compound of formula (I) was
first dissolved in LABRASOL.RTM. and a 7% emulsion was then
prepared of this LABRASOL.RTM. solution in distilled water
(homogenized). LABRASOL.RTM. is composed of a defined mixture of
mono- di- and tri- glycerides and mono- and di-fatty acid esters of
polyethylene glycol. Source: Gattefosse, 69804 Saint-Priest,
France.
[0050] Lipopolysaccharide from Pseudomonas aeruginosa serotype 10,
purified by gel filtration, lot 50K4151. Purchased from Sigma,
catalogue number L-8643.
[0051] Anesthetic: Isoflurane (FORENE.RTM., Abbott).
[0052] Study Design
[0053] Exposure to Pseudomonas LPS: On the first day of the study
the rats were primed by injection of 10 .mu.g/kg/mL Pseudomonas
aeruginosa lipopolysaccharide i.p. or saline vehicle. Food was then
withdrawn. The following day the animals received the compound of
formula (I) in 7% LABRASOL.RTM. or 7% LABRASOL.RTM. vehicle alone,
and food was returned to the cages. 6 hours later the rats were
exposed for 30 minutes in groups of six to an aerosol of
Pseudomonas aeruginosa lipopolysaccharide. The aerosol was
generated from a 100 .mu.g/mL solution of P. aeruginosa LPS in
isotonic saline using a DeVilbiss type 646 jet nebulizer driven by
compressed air at 1.4 bar. The air was supplemented with 5% carbon
dioxide to prevent breath-holding behavior by the rats. 20 hours
after exposure the rats were killed by an overdose of FORENE.RTM..
A section of the trachea about 1 cm from the first bifurcation was
removed, shock-frozen in liquid nitrogen and stored at -80.degree.
C.
[0054] Extraction and Quantitation of RNA: Total RNA was isolated
with the RNeasy system (Qiagen, Germany). The kit method was
slightly modified by inclusion of an extra digestion step with
RNAase-free DNAase (Qiagen, Catalogue No. 79254, incubation 30
minutes at room temperature). Quality of the extracted RNA was
checked by agarose gel electrophoresis. Quantitation of muc-5AC RNA
was performed by the TAQMAN.RTM. real time PCR system using an ABI
Prism 7700 Detector (Perkin Elmer Corporation, California USA).
Probes were designed on the basis of gene bank sequence GI
"2315984". The forward probe was 5'-TGG GAA CCA TCA TCT ACA ACC
A-3', the reverse probe 5'-TCC TGA CTA ACC CCT TTG ACC A-3' and the
fluorogenic probe 5'-CCT TGA CGG CCA CTG TTA CTA TGC GAT GT-3',
labeled with the fluor FAM at the 5'-end and the quencher TAMRA at
the 3'-end. Ribosomal RNA was used as the housekeeping gene to
which all muc-5AC RNA measurements were compared (Taqman ribosomal
RNA control reagents (VIC.TM. Probe), Applied Biosystems). The
master mix was the standard TAQMAN.RTM. EZ RT-PCR core mix sold by
Applied Biosystems (Catalogue No. N808-0236). For muc-5AC assay it
was supplemented with 2 mM manganese and the forward and reverse
primers were both used at concentration 300 nM. The temperature
sequence (auto ramp) was as follows: 50.degree. C., 10 minutes
(reverse transcription); 60.degree. C., 30 minutes (DNA
polymerase); 95.degree. C., 5 minutes (separation of double strand
into two single strands); and 40 cycles of 20 seconds at 94.degree.
C. and 1 minute at 59.degree. C. (polymerase chain reaction). For
the ribosomal RNA assay the annealing temperature was 60.degree. C.
instead of 59.degree. C. On each plate, each sample was measured in
duplicate, first with the ribosomal RNA then the muc-5AC RNA. For
each sample and pair of plates a ratio of muc-5AC RNA to ribosomal
RNA was calculated. The plates were then repeated for all samples,
and the mean muc-5AC RNA to ribosomal RNA calculated from the
measurements on the two sets of plates. For standardization
purposes, one sample (from an RNA pool derived from rat tracheas
exposed to E. coli O55.B5 lipopolysaccharide) was measured on both
of the duplicate plates.
[0055] Statistical Analysis
[0056] For each rat strain the positive control were compared with
the negative control and the group treated with the compound of
formula (I) by one sided Wilcoxon tests. Because of the multiple
test situation the p values were adjusted according to
Bonferroni-Holm for each experiment to control the level of
significance (.alpha.=0.05) (9). Furthermore the two rat strains
were compared for each group with a two sided Wilcoxon test
(.alpha.=0.05). The statistical analysis was carried out with the
program SAS (SAS Institute Inc., Cary, N.C.), version 6.12.
[0057] Results
[0058] Exposure of rats of either the BDE strain or F344 strain to
Pseudomonas aeruginosa aerosol caused a marked increase in the
tracheal epithelial expression (relative to a 18S ribosomal RNA
housekeeping gene) of the muc-5AC gene (p=0.004). In the BDE rats
the increase in muc-5AC expression after Pseudomonas exposure was
more than 27 fold, in the F344 rats (largely because of higher
control values for muc-5AC expression in the untreated animals)
more than 10 fold. Prior treatment with the LTB.sub.4 antagonist of
formula (I) at 3 mg/kg p.o. reduced this increase to less than half
the value with only vehicle pre-treatment. This effect of the
compound of formula (I) was statistically significant whether the
BDE strain was studied (when p=0.003) or the F344 strain was
studied (p=0.010).
[0059] Histological Study of Effects of LTB.sub.4 Antagonist (I) on
Mucin-Containing Goblet Cells
[0060] This study was only carried out in BDE strain rats, these
having given good results in the previous biochemical study.
[0061] Materials and Methods
[0062] Animals Used: Adult male rats, strain BDE/Han, with
approximately the same average weight as in the biochemical study
(described above).
[0063] Animal Maintenance: The rats were housed as in the previous
study. As the duration of histological study was considerably
greater than that of the biochemical study, the rats were not
fasted.
[0064] The compound of formula (I) was synthesized and formulated
for administration to the rats as in the previous study.
[0065] Lipopolysaccharide from Pseudomonas aeruginosa serotype 10,
as in the previous study.
[0066] Anesthetic: Isoflurane (FORENE.RTM., Abbott).
[0067] Study Design
[0068] Exposure to Pseudomonas LPS and Section Preparation: The
technique for priming the rats and aerosol exposure to Pseudomonas
aeruginosa lipopolysaccharide was as described earlier. However, in
the histological study the rats were killed 96 hours after exposure
to the Pseudomonas aeruginosa lipopolysaccharide aerosol. The
longer period between exposing the mice to Pseudomonas aerosol and
killing them was necessary because histological changes occur more
slowly than changes in mRNA expression. Animals were given the
compound (I) in 7% LABRASOL.RTM. per os 5 hours before and 21, 45,
69, and 93 hours after the Pseudomonas aeruginosa aerosol. At
sacrifice, 3 hours after the last treatment with compound (I), the
complete lung was removed, fixed in 7% buffered formalin and
embedded in paraffin. The left main stem bronchus was used for
immunohistochemical staining. Lung sections were cut to include the
full length of the main intrapulmonary airway and stained
sequentially with hematoxylin and eosin or with Alcian blue
(AB)-periodic acid-Schiff (PAS) to evaluate the total epithelial
area and the area stained for intracellular mucous glycoconjugates,
respectively. Goblet cell production was determined by the volume
density of AB-PAS-stained mucous glycoconjugates on the epithelial
mucosal surface using an image analysis system (Soft Imaging
System, Munster, Germany). The number of AB-PAS staining goblet
cells and the total epithelial area were measured over a length of
2 mm of the basal lamina.
[0069] Results
[0070] Exposure of rats to Pseudomonas aeruginosa
lipopolysaccharide caused a marked increase in the number of goblet
cells per square millimeter epithelium. Treatment with 3 mg/kg p.o.
compound (I) reduced the effect of Pseudomonas aerosol treatment
(see table below). The mean number of goblets cells per square
millimeter was 507 in the negative control, 1669 in the Pseudomonas
aeruginosa treated positive control, and 408 in the group treated
with both Pseudomonas aeruginosa aerosol and compound (I) 3 mg/kg
per os.
2 Number of Goblet Cells/Square Treatment Millimeter Epithelium 137
66 1882 Negative Control 1183 (saline aerosol) 246 320 70 150 1928
1605 1641 Positive Control 713 (P. aeruginosa aerosol) 2553 1375
2301 1238 906 215 P. aeruginosa aerosol 519 plus 3 mg/kg p.o. 175
compound (I) administered 306 5 hours prior to the aerosol 530 and
daily thereafter 249 362
* * * * *