U.S. patent application number 14/699563 was filed with the patent office on 2015-09-17 for use of cyclodextrin for treatment and prevention of bronchial inflammatory diseases.
The applicant listed for this patent is UNIVERSITE DE LIEGE. Invention is credited to Didier Cataldo, Brigitte Evrard, Jean-Michel Foldart, Agnes Noel.
Application Number | 20150258133 14/699563 |
Document ID | / |
Family ID | 48870741 |
Filed Date | 2015-09-17 |
United States Patent
Application |
20150258133 |
Kind Code |
A1 |
Cataldo; Didier ; et
al. |
September 17, 2015 |
USE OF CYCLODEXTRIN FOR TREATMENT AND PREVENTION OF BRONCHIAL
INFLAMMATORY DISEASES
Abstract
The invention provides the use of a cyclodextrin compound for
the manufacturing of a medicament for the treatment or prevention
of bronchial inflammatory diseases, particularly for asthma and
chronic obstructive pulmonary disease (COPD).
Inventors: |
Cataldo; Didier; (Olne,
BE) ; Evrard; Brigitte; (Embourg, BE) ; Noel;
Agnes; (Durbuy, BE) ; Foldart; Jean-Michel;
(Trooz, BE) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
UNIVERSITE DE LIEGE |
ANGLEUR |
|
BE |
|
|
Family ID: |
48870741 |
Appl. No.: |
14/699563 |
Filed: |
April 29, 2015 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
13804626 |
Mar 14, 2013 |
9034846 |
|
|
14699563 |
|
|
|
|
13478743 |
May 23, 2012 |
|
|
|
13804626 |
|
|
|
|
12846241 |
Jul 29, 2010 |
|
|
|
13478743 |
|
|
|
|
11664999 |
Apr 10, 2007 |
7829550 |
|
|
PCT/EP2005/054966 |
Sep 30, 2005 |
|
|
|
12846241 |
|
|
|
|
Current U.S.
Class: |
514/58 |
Current CPC
Class: |
A61K 9/0078 20130101;
A61P 11/00 20180101; A61K 31/724 20130101; Y10S 514/826 20130101;
A61K 9/0073 20130101 |
International
Class: |
A61K 31/724 20060101
A61K031/724; A61K 9/00 20060101 A61K009/00 |
Foreign Application Data
Date |
Code |
Application Number |
Oct 10, 2004 |
EP |
04104957.8 |
Claims
1-3. (canceled)
4. A method for the treatment of COPD (chronic obstruction
pulmonary disease) in a host mammal in need of such treatment,
comprising the step of administering in an aerosol, a
pharmaceutical composition consisting essentially of hydroxypropyl
beta-cyclodextrin in a concentration of from 10-75 mM.
Description
FIELD OF THE INVENTION
[0001] The present invention relates to the use of cyclodextrin
compound for the treatment and prevention of bronchial inflammatory
diseases, including chronic obstructive pulmonary disease
(COPD).
BACKGROUND OF THE INVENTION
[0002] Compounds for the treatment and prevention of bronchial
inflammatory disesases including COPD are classified in the art as
bronchodilator also called reliever medications or
nonbronchodilators antinflammatory agents referred to as controller
agents, on the basis of their pharmacodynamic effects.
[0003] Short-acting bronchodilators such as inhaled beta agonist or
anticholinergics are considered reliever medications.
Corticosteroids, cromolyn sodium, nedocromil sodium,
sustained-release theophylline and long-acting beta agonist are
considered controller medications, since they are used to achieve
and maintain control of symptoms and are used daily on a long-term
basis.
[0004] Among reliever medication, inhaled .beta.2-adrenergic
agonists are drugs for relief of symptoms due to acute airway
obstruction. They have a rapid onset of action and a 3-6 h duration
of activity. Unfortunately they have side effects such as
tachycardia, palpitations and tremor that often disappear during
chronic administration.
[0005] Anticholinergic agents induce airway smooth muscle
relaxation. Their activity is not as effective as beta agonists in
asthma but is more prolonged (6 to 8 hours).
[0006] Among controller medications, glucocorticosteroids are
effective agents with anti-inflammatory effects. Unfortunately,
their side effects include adrenal suppression, osteoporosis,
growth suppression, weight gain, hypertension, diabetes, dermal
thinning, cataracts, myopathy and psychotic actions. These effects
are dose related and are usually seen with systemic administration.
Local side effects, including oral candidiasis and dysphonia may
occur at lower doses of inhaled glucocorticoids.
[0007] Cromolyn sodium and nedocromil sodium are also classified as
controller agents, because of their similar clinical profile. They
inhibit bronchoconstriction induced by neurally mediated
events.
[0008] Theophylline is generally considered as a bronchodilator but
has weak bronchodilator activity in therapeutic doses. It may also
have anti-inflammatory properties. The dose-related adverse effects
of theophylline are nausea, nervousness, anxiety and
tachycardia.
[0009] Lipoxygenase inhibitors and leukotriene receptor agonists
are also controller agents. They alter the pathological effects of
leukotrienes derived from the 5-lipoxygenation of arachidonic acid.
They can inhibit the bronchospastic effects of allergens, exercise,
cold dry air, and aspirin allergy. Both are efficacious in
alleviating symptoms and improving pulmonary function during 4-6
weeks of therapy in patients with moderate asthma.
[0010] There is therefore a need for improved compounds which can
be used for the treatment or prevention of bronchial inflammatory
diseases including COPD.
BRIEF SUMMARY OF THE INVENTION
[0011] It is now surprisingly found that cyclodextrin is useful as
active component for the treatment or prevention of bronchial
inflammatory diseases, including COPD.
[0012] The invention therefore provides the use of cyclodextrin
compound for the treatment or prevention of bronchial inflammatory
disease including COPD in a host mammal in need of such
treatment.
[0013] By cyclodextrin compound, one means cyclodextrin as well as
their pharmaceutically acceptables salts, enantiomeric forms,
diastereoisomers and racemates.
[0014] By cyclodextrin, one means cyclic oligosaccharides produced
by enzymatic degradation of starch such as described in
"Cyclodextrin Technology, J Szejtli, Kluwer Academic Publishers
1998, pp 1-78", and which are composed of a variable number of
glucopyrannose units (n), mostly 6, 7 or 8. These cyclodextrins are
respectively named .alpha., .beta. and .gamma. cyclodextrins
(.alpha. CD, .beta. CD, .gamma.CD).
##STR00001##
[0015] Cyclodextrin is also represented by CD hereafter.
[0016] Cyclodextrin compound according to the invention is
cyclodextrin per se, alkyl-cyclodextrin (R-CD) wherein R is methyl,
ethyl, propyl and butyl; carboxyalkyl-cyclodextrin(CR-CD),
etherified-cyclodextrin (RO-CD), sulfoalkyl-cyclodextrin (SR-CD),
hydroxyalkyl-cyclodextrin(HR-CD), glucosyl-cyclodextrin, di and
triglycerides-cyclodextrin or a combination thereof and their
pharmaceutically acceptable salts which are at least water soluble
in an amount of 0.5 gr/100 ml at 25.degree. C.
[0017] The water-soluble cyclodextrin compound preferably used in
the present invention refers to a cyclodextrin compound having
water solubility of at least that of .beta.-cyclodextrin (1.85
g/100 ml).
[0018] Examples of such water-soluble cyclodextrin compound are
sulfobutylcyclodextrin, hydroxypropylcyclodextrin,
maltosylcyclodextrin, and salts thereof. In particular,
sulfobutyl-.beta.-cyclodextrin, hydroxypropyl-.beta.-cyclodextrin,
maltosyl-.beta.-cyclodextrin, and salts thereof
[0019] Other preferred cyclodextrin compound according to the
invention are methylcyclodextrins (products of the cyclodextrins
methylation) such as 2-O-methyl.beta.-cyclodextrin;
dimethylcyclodextrin (DIMEB) (preferably substituted in 2 and in
6); trimethylcyclodextrin (preferably substituted in 2, 3 and
6);
"random methylated" cyclodextrins (RAMEB or RM) (preferably
substituted at random in 2, 3 and 6, but with a number of 1.7 to
1.9 methyl by unit glucopyrannose), hydroxypropylcyclodextrins
(HPCD), hydroxypropylated cyclodextrins preferably substituted
randomly mainly in position 2 and 3 (HP-.beta.CD, HP-.gamma. CD)),
sulfobutylethercyclodextrins (SBECD), hydroxyethyl-cyclodextrins,
carboxymethylethylcyclodextrins, ethylcyclodextrins, cyclodextrins
amphiphiles obtained by grafting hydrocarbonated chains in the
hydroxyl groups and being able to form nanoparticles, cholesterol
cyclodextrins and triglycerides-cyclodextrins obtained by grafting
cyclodextrins monoaminated (with a spacer arm) as described in
Critical Review in Therapeutic drug Carrier Systems, Stephen D.
Bruck Ed, Cyclodextrin-Enabling Excipient; their present and future
use in Pharmaceuticals, D. Thomson, Volume 14, Issue 1 p 1-114
(1997)
[0020] Most preferred cyclodextrins compounds are [0021]
.beta.-cyclodextrin with optionally a chemical function grafted on
the glucopyrannose units such as
hydroxypropyl-.beta.cyclodextrin(HP.beta.CD),
sulfonylbutylether-.beta.cyclodextrin(SBE.beta.CD), random
methylated-.beta.cyclodextrin(RM.beta.CD),
dimethyl-.beta.cyclodextrin(DIME.beta.CD),
trimethyl-.beta.cyclodextrin(TRIME.beta.CD), hydroxybutyl
.beta.cyclodextrin(HB.beta.CD), glucosyl .beta.cyclodextrin,
maltosyl .beta.cyclodextrin and 2-O-methyl
.beta.cyclodextrin(Crysmeb), or a combination thereof and their
pharmaceutically acceptable salts.
[0022] The cyclodextrin compounds according to the invention are
produced by the well-known enzymatic degradation of starch such as
the method described in "Cyclodextrin Technology, J Szejtli, Kluwer
Academic Publishers 1998, pp 1-78, followed by grafting of an
appropriate chemical group.
[0023] The invention further provides the use of such cyclodextrin
compound for the manufacturing of a medicament for the treatment or
prevention of bronchial inflammatory diseases to a patient in need
of such treatment.
[0024] According to the invention the cyclodextrin compound has to
be administered to the patient over several months or years
(especially in case of prevention). The cyclodextrin compound is
administered preferably as aerosol, with non-toxic doses ranging
between nanomolar and molar concentrations.
[0025] The present invention relates to a method used for treating
bronchial inflammatory diseases, preferably asthma and chronic
obstructive pulmonary disease (COPD) in a host mammal in need of
such treatment, e.g., a patient suffering from such a disease, by
the application of a cyclodextrin compound according to the
invention in a pharmaceutically effective amount. The present
invention provides cyclodextrins for controlling inflammation in
COPD and COPD-related diseases.
[0026] In COPD patients, there is a significant neutrophilic
inflammation in the bronchial walls leading to progressive
destruction of airways structures by the repeated productions of
proteases and oxidants (oxygen reactive species). To date, marketed
therapies are not able to adequately decrease or prevent this
neutrophilic inflammation in COPD patients. In particular, it is
well know that inhaled or oral steroids display no efficiency
against neutrophilic inflammation. For example, a study of S.
Culpitt et al (Am J Respir Crit Care Med (1999) 160: 1635-1639)
performed in COPD patients reports the lack of efficacy of high
doses inhaled steroids in COPD-related neutrophilic inflammation
and chemotactic agents for neutrophils (mainly IL-8 in humans).
[0027] It is well established that neutrophils are the major
inflammatory cells in COPD and that these cells are attracted in
the airways by potent chemotactic agents such as IL-8 in human (V.
Murugan et al, Exp Lung Res. 2009 August; 35(6):439-85). In
rodents, the cytokine system is similar but chemotactic agents
responsible for neutrophils chemotaxis are slightly different and
include CXCL-1 (also named KC) as a major chemoattractant playing a
similar role to human IL-8 especially documented in the context of
tobacco smoke exposure (V. Lagente Clin Exp Pharmacol Physiol. 2008
May; 35(5-6):601-5). Lipopolysaccharide (LPS), a component of
external membrane of gram-negative bacteria, is able to induce a
neutrophilic inflammation with characteristics similar to that
observed in COPD and is therefore considered as a suitable model of
COPD. Pathways activated by LPS that lead to neutrophilic
inflammation are similar to those observed in COPD and include
CXCL-1 production (A. Roos Biochem Biophys Res Commun. 2012 Jun.
22; 423(1):134-9).
[0028] Asthma is an inflammatory disease of the bronchial tree
related or not to an allergen exposure. This inflammation provokes
symptoms in patients by stimulating the bronchial smooth muscles to
contract, enhancing the mucus secretion, and inducing bronchial
morphological changes thought to be an aggravating factor regarding
the course of the disease. Airway hyperresponsiveness is a hallmark
of the disease and is responsible for most of symptoms. Bronchial
tree is a very complex tissue with many cell types (as for example
epithelial cells, smooth muscle cells, inflammatory cells, nerves,
mucus producing cells, fibroblasts) and the bronchial remodelling
events which comprise many aspects mainly consist in a deposition
of extracellular matrix components in the bronchial walls, a smooth
muscle hyperplasia and a hyperplasia of the mucus producing cells.
The use of cyclodextrin compounds according to the invention
inhibits the inflammatory cells influx in the compartments of
bronchoalveolar lavage and peribronchial tissue and inhibits the
hyperresponsiveness defined as an abnormal response to stimulating
agents such as methacholine. The disease and current treatments are
reviewed in, e.g., GINA Workshop Report, Global Strategy for Asthma
Management and Prevention (NIH Publication No. 02-3659) and Fabbri,
L. M., and Hurd, S. S., Eur. Respir. J. 22 (2003) 1-2.
[0029] The invention therefore further relates to a method for
treating bronchial inflammatory diseases in a patient suffering
from such a disease, using a cyclodextrin compound according to the
invention in a therapeutically effective amount.
[0030] The invention preferably further relates to a method for
treating emphysema in a patient suffering from such a disease,
using cyclodextrin compounds according to the invention. In such a
disease, the alveolar walls are destroyed by proteolytic processes
and this destruction impairs the transfer of oxygen to the blood.
In such a disease, physiological problems also occurs because of
the derived hyperinflation which causes abnormalities in the
ventilation by causing a dysfunction of respiratory muscles and
because of a hypertension in pulmonary arteries leading to cardiac
failure in advanced stages.
[0031] The invention preferably further relates to a method for
treating chronic obstructive pulmonary disease (COPD) in a patient
suffering from such a disease, using cyclodextrin compounds
according to the invention. In such a disease, the bronchial walls
of small airways are remodelled by proteolytic processes and this
remodelling and fibrosis induce an airway obstruction which can be
measured by spirometry. In such a disease, physiological problems
also occurs because of the derived hyperinflation which causes
abnormalities in the ventilation/perfusion ratio and causes
hypoventilation and eventually CO2 accumulation.
[0032] According to the invention the cyclodextrin compound has to
be administered over several months or years, to the patient in
need of such a therapy. The cyclodextrin compounds are administered
preferably by the aerosolization of a liquid or powder composition,
with non-toxic doses ranging between micro and molar concentrations
per kg and day.
[0033] A further preferred object of the invention is a
pharmaceutical composition of cyclodextrin compound according to
the invention for the treatment of bronchial inflammatory diseases,
and its use, containing a cyclodextrin or a salt thereof and
preferably a water-soluble cyclodextrin derivative (water soluble
being defined as a solubility of at least 0.5 g/100 ml water at
25.degree. C.).
[0034] The pharmaceutical compositions are aqueous compositions
having physiological compatibility. The compositions include, in
addition to cyclodextrin or a salt thereof, auxiliary substances,
buffers, preservatives, solvents and/or viscosity modulating
agents. Appropriate buffer systems are based on sodium phosphate,
sodium acetate or sodium borate. Preservatives are required to
prevent microbial contamination of the pharmaceutical composition
during use. Suitable preservatives are, for example, benzalkonium
chloride, chlorobutanol, methylparabene, propylparabene,
phenylethyl alcohol, sorbic acid. Such preservatives are used
typically in an amount of 0.01 to 1% weight/volume.
[0035] The cyclodextrin compound of the present invention exhibits
its effects through either oral administration, parenteral
administration or topical administration, and it is preferably
formed into a composition for parenteral administration,
particularly an injection composition or topical administration,
particularly an aerosol composition. Such aerosol composition is
for example a solution, a suspension, a micronised powder mixture
and the like. The composition is administered by using a nebulizer,
a metered dose inhaler or a dry powder inhaler or any device
designed for such an administration.
[0036] Examples of galenic compositions include tablets, capsules,
powders, granules and the like. These may be produced through well
known technique and with use of typical additives such as
excipients, lubricants, and binders.
[0037] Suitable auxiliary substances and pharmaceutical
compositions are described in Remington's Pharmaceutical Sciences,
16th ed., 1980, Mack Publishing Co., edited by Oslo et al.
Typically, an appropriate amount of a pharmaceutically-acceptable
salt is used in the composition to render the composition isotonic.
Examples of pharmaceutically acceptable substances include saline,
Ringer's solution and dextrose solution. pH of the solution is
preferably from about 5 to about 8, and more preferably from about
7 to about 7.5.
[0038] A preferred pharmaceutical composition for nebulization
comprises cyclodextrin (CD), NaCl and water. The solution is
prepared by dissolving CD in 100 ml of purified water, adding NaCl
by stirring so as to dissolve them and complete with water so as to
obtain 200 ml of solution. Preferably the solution is sterilized by
filtration through a 0.22 .mu.m polypropylene membrane or by a
steam sterilization process.
[0039] Especially preferred composition is a combination of (for
200 ml of solution): [0040] 10-50 g CD, preferably 20 gCD,
preferably HP.beta.CD; sodium chloride 1.2-1.5 g, preferably 1.42 g
(isotonicity) and water, preferably pyrogen-free, sterile, purified
water ad 200 ml.
[0041] Such a composition is useful for the treatment of bronchial
inflammatory diseases.
[0042] Most preferred composition is a combination of
2-O-methyl.beta.CD with sodium chloride 1.2-1.5 g, preferably 1.42
g (isotonicity) and water, preferably pyrogen-free, sterile,
purified water ad 200 ml.
[0043] In summary, the present invention provides a method for the
treatment of COPD (Chronic obstruction pulmonary disease) in a host
mammal in need of such treatment, comprising the step of
administering an effective amount of cyclodextrin compound to the
mammal, wherein the cyclodextrin compound is selected from the
group consisting of b-cyclodextrin, hydroxypropyl-bcyclodextrin,
sulfolbutylether-bcyclodextrin, random methylated-bcyclodextrin,
dimethyl-bcyclodextrin, trimethyl-bcyclodextrin, hydroxypropyl
b-cyclodextrin, hydroxybutyl bcyclodextrin, glucosyl-bcyclodextrin,
maltosyl-bcyclodextrin, 2-O-methyl-bcyclodextrin or a combination
thereof and their pharmaceutically acceptable salts. Preferably,
the cyclodextrin compound is hydroxypropyle-beta-cyclodextrine and
its pharmaceutically acceptable salts. Preferably, the mode of
administration is inhalation.
[0044] The following examples, references, and figures are provided
to aid the understanding of the present invention. It is understood
that modifications can be made in the procedures set forth without
departing from the invention.
BRIEF DESCRIPTION OF THE DRAWINGS
[0045] FIGS. 1-2 Effects of inhalation of cyclodextrin compound on
BAL eosinophil percentage (FIG. 1) and peribronchial inflammation
score (FIG. 2). Controls are mice exposed to ova by inhalation and
placebo by inhalation (OVA)
[0046] FIG. 3 Effects of inhalation of cyclodextrin compound on
peribronchial eosinophils reported here as a number/mm of
epithelial basement membrane.
[0047] FIG. 4 Airway responsiveness measurements: Enhanced Pause
(Penh) was measured in OVA exposed mice during 5 minutes after a 2
minutes inhalation of cyclodextrin or Placebo (OVA) and increasing
doses of methacholine (Mch).
[0048] FIG. 5 Measurement of cytokines by Elisa. Eotaxin was
measured by incubation in a wheel coated with a primary antibody
specifically dedicated to the recognition of the protein and after
rinsing, a second antibody against eotaxin, coupled with horse
radish peroxydase was used to quantify the amounts of eotaxin in
the solution.
[0049] FIG. 6 Measurement of allergen specific IgE levels in
serum.
[0050] FIGS. 7A-B Measurement of eotaxin and IL-13 in Bal and lung
protein extracts. IL-13 was measured by incubation in a wheel
coated with a primary antibody specifically dedicated to the
recognition of the protein and after rinsing, a second antibody
against IL-13, coupled with horse radish peroxydase was used to
quantify the amounts of eotaxin in the solution.
[0051] FIG. 8 Airway responsiveness measurements: Enhanced Ponse
(Penh) was measured in mouses after receiving crysmeb or placebo
treatment FIGS. 9A-B Peribronchial inflammation score measured in
histology when treated with various Crysmeb concentration compared
to placebo (FIG. 9A) and to other cyclodextrin and fluticasone
(FIG. 9B)
[0052] FIG. 10 Airway responsiveness measurements: comparison of
cyclodextrin compounds with placebo and Fluticasone. Measurements
of methacholine-induced airway response in mice exposed 7 days to
allergens and receiving an inhaled therapy 30 min before the
allergen exposition.
[0053] FIG. 11 Levels of IL-13 measured by Elisa in lung protein
extracts.
[0054] FIG. 12 Measurement of CXCL-1 by ELISA in whole lung tissue
extracts (crushed in liquid nitrogen) ***p<0.01 (n=8/group).
[0055] FIG. 13 Measurements of CXCL-1 levels in lung protein
extracts of mice exposed to aerosolized placebo (PBS) or
hydroxypropyl-.beta.-cyclodextrin (HPBeta) at 15 mM
(n=6/group).
[0056] FIG. 14 Measurements of peribronchial inflammation by
histology (see <<experimental protocol>> section for
description of the scoring system) (n=6/group).
DETAILED DESCRIPTIONS OF THE INVENTION
Example 1
Use of Compositions Containing HP-.beta.-Cyclodextrin for Therapy
of Allergen-Induced Airway Inflammation and Bronchial
Hyperresponsiveness in a Mouse Model of Asthma
Materials
[0057] HP-.beta.-CD (degree of substitution=0.64) was obtained from
Roquette (France). .alpha.- and HP-.gamma.-CD were obtained from
Wacker Chemie Gmbh (Germany). Apyrogenic phosphate buffered saline
(PBS) was purchased from Bio-Wittaker (Verviers, Belgium).
Methacholine was from Sigma-Aldrich (Germany).
[0058] All other materials were of analytical grade. Sterile water
for injection was used throughout this study. Sterile, apyrogenic
and isotonic CD solutions were prepared at 1, 7.5 and 50 mM for
HP-.beta.-CD and .alpha.-CD and at 50 mM for HP-.gamma.-CD.
Cyclodextrins were tested following the Bacterial Endotoxin Test
described in USP XXVI using Limulus Amebocyte Lysate (LAL).
Osmolalities of all the solutions were measured by a Knauer
Automatic semi-micro Osmometer and adjusted to the value of 300
mOsm/kg by the addition of an adequate amount of NaCl. A terminal
sterilization of the solutions was performed by steam sterilization
process.
Methods
[0059] Aerosol was produced by using an ultrasonic nebuliser
SYSTAM, the vibration frequency of which is 2.4 MHz with variable
vibration intensity and ventilation levels. Vibration intensity was
fixed in position 6 and the ventilation level was 25 (t1/2)
Umin.
[0060] Characterization of Nebulized Aerosol
[0061] Aerosol size distribution emitted from CDs solutions was
determined with a laser size analyzer Mastersizer (Malvern, Orsay,
France). Ten milliliters of each solution were directly nebulized
in the laser beam. The mouth piece was held at 1 cm from the center
of the laser beam. The resulting aerosol was aspirated on the
opposite side of the beam. Environmental temperature and relative
humidity were maintained constant, that is to say at 20.degree. C.
and 40-45%. Triplicates of each measurement were performed and
compared to controls of PBS. The results are expressed as the
percentage of droplets comprised in the range 0.5 to 5.79 .mu.m and
the median diameter. The concentration of droplets in the air
evaluated by the obscuration percentage of the laser beam was in
the same range for each experiment (15-25%). A comparison of the
MMAD, the GSD and the percentage of droplets comprised in the range
of 0.5 to 5.79 .mu.M of all the CDs solutions with the
corresponding values for PBS demonstrated that the presence of CDs
in the solution did not influence the droplet size distribution in
the aerosols. A fraction of droplets comprised in the range of 0.5
to 5.79 .mu.m close to 65% was obtained in each experiment.
[0062] Sensitisation, Allergen Exposure and Therapeutic
Protocols.
[0063] In order to study modulation of airway inflammation, males
BALB/c mice of 6 to 8 weeks old were sensitized by intraperitoneal
injection of 10 .mu.g ovalbumin (OVA) (Sigma Aldrich, Schnelldorf,
Germany) emulsified in aluminum hydroxyde (AlumInject; Perbio,
Erembodegem, Belgium) on days 1 and 8. Mice were subsequently
exposed to allergens by daily inhalation of an aerosol of OVA 1%,
for 30 minutes, generated by ultrasonic nebulizer (Devilbiss 2000),
from day 21 to 27. Mice were subjected to inhalation of .alpha.-CD,
HP-.beta.-CD 1, 7.5, 50 mM and HP-.gamma.-CD 50 mM 30 minutes
before OVA inhalation. Mice were sacrificed performed on day 28 as
previously reported by Cataldo and al in Am. J. Pathol 2002;
161(2):491-498.
[0064] Bronchoalveolar Lavage Fluid (BAL)
[0065] Immediately after assessment of airway responsiveness, and
24 hours after the last allergen exposure.
[0066] Mices were sacrificed and a bronchoalveolar lavage was
performed using 4.times.1 ml PBS-EDTA 0.05 mM (Calbiochem,
Darmstadt, Germany) as previously described by Cataldo D D, Tournoy
K G, Vermaelen K et al. in Am J Pathol 2002; 161(2):491-498. Cells
were recovered by gentle manual aspiration. After centrifugation of
bronchoalveolar fluid (BALF) (1200 rpm for 10 minutes, at 4.degree.
C.), the supernatant was frozen at -80.degree. C. for protein
assessment and the cell pellet was resuspended in 1 ml PBS-EDTA
0.05 mM. The differential cell counts were performed on
cytocentrifuged preparations (Cytospin) after staining with
Diff-Quick (Dade, Belgium).
[0067] Pulmonary histology and tissue processing After BAL, the
thorax was opened and the left main bronchus was clamped. The left
lung was excised and frozen immediately at -80.degree. C. for
protein and mRNA extraction. The right lung was infused with 4 ml
paraformaldehyde 4%, embedded in paraffin and used for histology.
Sections of 5 .mu.m thickness were cut off from paraffin and were
stained with haematoxylin-eosin. The extent of peribronchial
inflammation was estimated by a score calculated by quantification
of peribronchial inflammatory cells, as previously described by
Cataldo D D, Tournoy K G, Vermaelen K et al. in Am J Pathol 2002;
161(2):491-498. A value of 0 was adjudged when no inflammation was
detectable, a value of 1 when there was occasionally inflammatory
cells, a value of 2 when most bronchi were surrounded by a thin
layer (1 to 5 cells) of inflammatory cells and a value of 3 when
most bronchi were surrounded by a thick layer (>5 cells) of
inflammatory cells. Since 5-7 randomly selected tissue sections per
mouse were scored, inflammation scores are expressed as a mean
value and can be compared between groups. After Congo Red staining,
the eosinophilic infiltration in the airway walls was quantified by
manual count and reported to the perimeter of epithelial basement
membrane defining an eosinophilic inflammatory score.
[0068] The left lung was crushed using a Mikro-Dismembrator (Braun
Biotech International, Gmbh Melsungen, Germany). For proteins
extraction, the crushed lung tissue was incubated overnight at
4.degree. C. in a solution containing 2M urea, 1M NaCl and 50 mM
Tris (pH 7.5) and subsequently centrifuged 15 minutes at
16.000.times.g. The supernatant was stored at -80.degree. C.
[0069] Bronchial responsiveness measurement Twenty-four hours after
the last allergen exposure, the bronchial hyper responsiveness was
determined by measuring the Penh (Enhanced Pause) using a
barometric plethysmograph (Emka technologies, Paris) as proposed by
Hamelmann, E., et al., Am. J Respir. Crit. Care Med. 156 (1997)
766-775). The Penh was measured at baseline and 5 min after the
inhalation of increasing doses (25, 50, 75 and 100 mM) of
methacholine (Mch).
Measurements of Cytokines by ELISA
[0070] Eotaxin and IL-13 levels were assessed using commercial
ELISAs (R&D systems, Abingdon, UK). Eotaxin was measured by
incubation in a wheel coated with a primary antibody specifically
dedicated to the recognition of the protein and after rinsing, a
second antibody against eotaxin, coupled with horse radish
peroxydase was used to quantify the amounts of eotaxin in the
solution
Measurement of Allergen Specific Serum IgE
[0071] At the end of the experiment, blood was drawn from the heart
for measurement of OVA specific serum IgE. Microtiter plates were
coated with OVA. Serum was added followed by a biotinylated
polyclonal rabbit anti-mouse IgE (S. Florquin, ULB, Brussels,
Belgium). A serum pool from OVA-sensitized animals was used as
internal laboratory standard; 1 unit was arbitrarily defined as
1/100 dilution of this pool.
Measurement of Eotaxin and IL-13 in Bal and Lung Protein
Extracts.
[0072] IL-13 was measured by incubation in a wheel coated with a
primary antibody specifically dedicated to the recognition of the
protein and after rinsing, a second antibody against IL-13, coupled
with horse radish peroxydase was used to quantify the amounts of
eotaxin in the solution.
Statistical Analysis
[0073] Results of BAL cell count, pulmonary histology, cytokines
and mRNA levels were expressed as mean+/-SEM and the comparison
between the groups was performed using Mann-Whitney test.
Mann-Whitney test was performed using GRAPHPAD INSTAT version 3.00
for WINDOWS 95 (GRAPHPAD SOFTWARE, San Diego, Calif., USA,
WWW.GRAPHPAD.Com.). P values <0.05 were considered as
significant.
Pharmacological Results:
[0074] Inflammatory cells in the BAL.
[0075] After allergen exposure, eosinophil counts were
significantly decreased after the inhalation of HP-.beta.-CD and
HP-.gamma.-CD at the dose of 50 mM. There was a dose dependent
decrease in BAL eosinophils with the HP-.beta.-CD 1, 7.5 and 50 mM
inhalation. Other inflammatory cells were not present in different
amounts in the BAL after HP-.beta.-CD inhalation when compared to
placebo. On the contrary, .alpha.-CD inhalation led to a tendency
to increase the number of eosinophils in BAL after allergen
exposure (FIG. 1).
Peribronchial Inflammation
[0076] After allergen exposure, mice treated with placebo displayed
a significant increase in peribronchial inflammation as quantified
by the peribronchial inflammation score. Mice treated with
HP-.beta.-CD 1, 7.5, and 50 mM and HP-.gamma.-CD 50 mM were shown
to have decreased inflammation score when compared to placebo
treated mice. .alpha.-CD inhalation did not reduce the
peribronchial inflammation score (FIG. 2).
Peribronchial Eosinophil Infiltration
[0077] As demonstrated previously, the allergen exposure did induce
a significant increase in the number of eosinophils detectable in
the peribronchial area. All CD tested induced a decrease of this
infiltration and this decrease reached statistical significance for
.alpha.-CD, HP-.beta.-CD 1, 7.5, and HP-.gamma.-CD 50 mM (FIG.
3).
Bronchial Responsiveness
[0078] The inhalation of HP-.beta.-CD 50 mM reduced the
methacholine-induced Penh increase (FIG. 4). When measuring the
area under the methacholine dose-response curve (A.U.C) for
different CDs, HP-.beta.-CD 50 mM was the only to show a
significant decrease (FIG. 5).
Cytokine Measurements in BAL and Lung Protein Extracts
[0079] When compared to placebo exposed mice, all doses of
HP-.beta.-CD tested induced a decrease in levels of eotaxin
measured by ELISA in lung protein extracts (FIG. 7a). IL-13 levels
were decreased in BAL after HP-.beta.-CD exposure and, on the
contrary, were increased after .alpha.-CD exposure (FIG. 7b).
Measurements of Allergen-Specific IgE in Serum
[0080] There were no significant differences between the groups for
the allergen sensitization as assessed by the similar levels of OVA
specific IgE measured by ELISA in serum (FIG. 6).
Example 2
Use of Compositions Comprising 2-O-Methyl-Cyclodextrin for Therapy
of Allergen-Induced Airway Inflammation and Bronchial
Hyperresponsiveness in a Mouse Model of Asthma
Materials
[0081] Materials are identical to example 1 with the exception of
the cyclodextrin compound which is here 2-O-methyl-cyclodextrin,
KLEPTOSE CRYSMEB.RTM., a product commercialised by Roquette. It
has, on average, 4 methyl groups per native cyclodextrin molecule
and is characterized by an average molecular weight of 1135 and a
average molar degree of substitution of 0.57.
[0082] Sterile, apyrogenic and isotonic CD solutions were prepared
with 10, 20, 50 and 75 mM for 2-O-methyl-cyclodextrin.
Cyclodextrins were tested following the Bacterial Endotoxin Test
described in USP XXVI using Limulus Amebocyte Lysate (LAL).
Osmolalities of all the solutions were measured by a Knauer
Automatic semi-micro Osmometer and adjusted to the value of 280-300
mOsm/kg by the addition of an adequate amount of NaCl. A terminal
sterilization of the solutions was performed by steam sterilization
process.
Methods
[0083] Same methods are used as in example 1 but in the present
example we did expose mice to aerosolized CRYSMEB (10, 20, 50, 100
or 200 mM) in a standard exposure box (20.times.30.times.15 cm) for
30 min/day during 7 days.
Pharmacological Results:
Inflammatory Cells in the BAL.
[0084] The cellular composition of the bronchoalveolar lavage was
not significantly altered by the exposure to CRYSMEB. In
particular, there were no differences regarding eosinophil and
neutrophil counts (see table 1).
[0085] Bronchoalveolar lavage eosinophilia was significantly
decreased in the groups treated by CRYSMEB. The decrease in lavage
eosinophilia was comparable with that obtained with different
concentrations of HP-beta-cyclodextrins or fluticasone, a commonly
used inhalation steroid used as a reference therapy (table 2)
Peribronchial Inflammation
[0086] After allergen exposure, mice treated with placebo displayed
a significant increase in peribronchial inflammation as quantified
by the peribronchial inflammation score. Mice treated with CRYSMEB
20 mM were shown to have decreased inflammation score when compared
to placebo treated mice (FIG. 9A). Peribronchial inflammation score
was measured and was significantly decreased in every treatment
group as compared to placebo (FIG. 9B)
Bronchial Responsiveness
[0087] The inhalation of CRYSMEB 10 mM reduced the
methacholine-induced Penh increase (FIG. 10).
[0088] The responsiveness to methacholine was increased after
allergen exposure and placebo and was significantly reduced by the
treatment with CRYSMEB in an extent comparable to that obtained
with fluticasone therapy (FIG. 10)
Cytokine Measurements in BAL and Lung Protein Extracts
[0089] In order to unveil mechanisms implicated in the
pharmacological effect of CRYSMEB, we measured IL-13, a major Th2
cytokine implicated in the airway hyperresponsiveness and
inflammation. We found that levels of IL-13 measured by ELISA in
whole lung protein extracts were significantly decreased by the
exposure to CRYSMEB as well as fluticasone and HP-beta-CD 50 mM.
(see FIG. 11)
Example 3
Pharmaceutical Composition to be Administered in an Aerosol to a
Patient in Need of Treatment for Bronchial Inflammatory Disease
[0090] HP betaCD 75 mM Solution osmolality is 308 mOs/kg. pH is
7.2.
[0091] The concentration of CD compound can be modified in function
of the requirements. It is preferred to adjust the tonicity by
addition of NaCl.
[0092] A preferred composition for nebulization is:
[0093] For 200 ml of solution: [0094] HP.beta.CD exempt from
pyrogenic 20.15 g (75 mM) [0095] Sodium chloride 1.42 g
(isotonicity) [0096] Pyrogen-free, sterile, purified water, q.s. ad
200 ml [0097] a) Weigh 20.15 g of HP.beta.CD exempt from pyrogenic
(3.2% H.sub.2O, from ROQUETTE) and dissolve them in 100 ml of
purified water. [0098] b) Weigh 1.42 g of sodium chloride and add
them to solution (a) by energetically stirring so as to dissolve
them. [0099] c) Complete with water so as to obtain 200 ml of
solution. [0100] Sterilize by filtration through a 0.22 .mu.m
polypropylene membrane.
TABLE-US-00001 [0100] TABLE 1 differential cell counts in the
bronchoalveolar lavage measured after the exposure to different
concentrations of inhaled CRYSMEB. PLACEBO Crysmeb 20 mM Crysmeb 50
mM Crysmeb 75 mM Epithelial cells (%) 15.9714 .+-. 5.154 29.9 .+-.
5.909 36.1375 .+-. 4.52 30.8875 .+-. 1.349 Eosinophils (%) 0.0428
.+-. 0.0428 0.0375 .+-. 0.0375 0.1125 .+-. 0.0789 0.0375 .+-.
0.0375 Neutrophils (%) 0.1285 .+-. 0.236 0.0375 .+-. 0.1061 0.2
.+-. 0.3505 0.0375 .+-. 0.1061 Lymphocytes (%) 0.1857 .+-. 0.1421
0.425 .+-. 0.1485 0.275 .+-. 0.1161 0.075 .+-. 0.0491 Macrophages
(%) 83.5857 .+-. 1.179.degree. 69.5 .+-. 5.956 63.1625 .+-. 4.695
68.9 .+-. 1.334
TABLE-US-00002 TABLE 2 differential cell counts in the
bronchoalveolar lavage measured after the exposure to different
concentrations of inhaled CRYSMEB. HPBeta-CD HPBeta-CD HPBeta-CD
CRYSMEB PLACEBO Fluticasone 1 mM 10 mM 50 mM 10 mM Epithelial 3.86
.+-. 2.551 22.85 .+-. 5.343 25.08 .+-. 3.413 34.44 .+-. 3.723 45.04
.+-. 5.534 36.83 .+-. 5.644 cells (%) Eosinophils (%) 53.08 .+-.
4.683 32.92 .+-. 7.306* 34.2 .+-. 7.705* 27.24 .+-. 4.98* 12.18
.+-. 4.366* 8.84 .+-. 2.946* Neutrophils (%) 3.03 .+-. 1.333 1.85
.+-. 1.093 0.36 .+-. 0.1563 0.83 .+-. 0.5838 0.72 .+-. 0.3992 1.26
.+-. 0.4587 Lymphocytes (%) 3.62 .+-. 1.576 1.68 .+-. 0.7115 0.48
.+-. 0.1869 0.21 .+-. 0.08571 0.12 .+-. 0.12 0.214 .+-. 0.1079
Macrophages (%) 36.25 .+-. 5.016 40.52 .+-. 3.122 39.75 .+-. 5.427
37.114 .+-. 3.878 41.86 .+-. 9.043 52.714 .+-. 6.49 Total cells
220.42 .+-. 81.709 75.92 .+-. 11.922 74.92 .+-. 14.396 114.43 .+-.
33.245 37.33 .+-. 10.683 131.93 .+-. 33.637 (10.sup.4/ml)
Example 4
Used Inhaled Hydroxypropyl-.beta.-Cyclodextrin to Address Allergen-
or LPS-Induced Inflammation in Rodents
Experimental Protocol:
[0101] In ovalbumin (OVA)-induced inflammation model, mice were
immunized by intraperitoneal injection of OVA (10 .mu.g) (Sigma
Aldrich, Schnelldorf, Germany) and aluminium hydroxyde on days 0
and 7. From days 21 to 25, mice were exposed to inhalation of 1%
OVA or PBS (phosphate buffer saline) for 30 minutes. Airway
hyperresponsiveness was measured on day 26 before sacrifice. Mice
were either exposed to aerosolized hydroxypropyl-beta-cyclodextrin
(15 mM) or PBS for 30 min 6 hours before allergen exposure.
[0102] After sacrifice, thorax was opened and the right lungs were
excised and snap frozen in liquid nitrogen for protein extraction.
The left lung was insufflated at constant pressure with 4%
paraformaldehyde and embedded in paraffin for further histological
analysis. A peribronchial inflammation score was applied on each
hematoxylin-eosin stained slide as previously reported (Cataldo et
al Am J Pathol 2002). A value from 0 to 2 was adjudged to each
bronchus. Score of 0 corresponded to bronchi without inflammation;
score 1 to occasional mononuclear cells observed around bronchi,
and score 2 to 1 to 5 layer(s) of inflammatory cells around
bronchi. Six bronchi per mice were counted and statistical analysis
was performed by using GraphPad Program. Lung tissues were crushed
and total protein extracts were prepared by incubating crushed lung
tissues in a 2M urea solution. Tissue lysates were centrifuged for
15 min at 16100.times.g. ELISA for CXCL-1 on lung protein extracts
were assessed using antibodies from R&D Systems and the R&D
Duoset.RTM. Elisa Development kit (R&D Systems, Minneapolis,
Minn., USA).
Results:
[0103] We already reported that overall inflammation was lower as
well as bronchial responsiveness to methacholine. As allergens are
able to generate neutrophilic inflammation by activation of
chemotactic factors, we addressed the potential efficacy of these
compounds to decrease levels of neutrophil chemoattractants in
mouse lungs after allergen exposure. Levels of CXCL-1, a major
neutrophil chemoattractant were significantly decreased in lung
protein extracts of cyclodextrins-treated mice (HPb in FIG. 12) as
compared to placebo-exposed mice (OVA in FIG. 12). In summary,
there is a decreased production of chemotactic agents for
neutrophils after exposure to cyclodextrins in an allergen-induced
inflammation model in mice.
Example 5
Used Inhaled Hydroxypropyl-.beta.-Cyclodextrin to Address Allergen-
or LPS-Induced Inflammation in Rodents
[0104] Experimental Protocol:
[0105] In lipopolysaccharide (LPS)-induced inflammation model, mice
were exposed to 3 .mu.g LPS diluted in 100 .mu.l of PBS or PBS
(phosphate buffer saline) only administered by direct tracheal
instillations at day 1 and 5 of the protocol. Mice were daily
exposed either to aerosolized hydroxypropyl-beta-cyclodextrin (15
mM) or PBS for 30 min from day 0 to 5 (6 hours after LPS or sham
exposure at days 1 and 5). Mice were sacrificed at day 6.
[0106] After sacrifice, thorax was opened and the right lungs were
excised and snap frozen in liquid nitrogen for protein extraction.
The left lung was insufflated at constant pressure with 4%
paraformaldehyde and embedded in paraffin for further histological
analysis. A peribronchial inflammation score was applied on each
hematoxylin-eosin stained slide as previously reported (Cataldo et
al Am J Pathol 2002). A value from 0 to 2 was adjudged to each
bronchus. Score of 0 corresponded to bronchi without inflammation;
score 1 to occasional mononuclear cells observed around bronchi,
and score 2 to 1 to 5 layer(s) of inflammatory cells around
bronchi. Six bronchi per mice were counted and statistical analysis
was performed by using GraphPad Program.
[0107] Lung tissues were crushed and total protein extracts were
prepared by incubating crushed lung tissues in a 2M urea solution.
Tissue lysates were centrifuged for 15 min at 16100.times.g. ELISA
for CXCL-1 on lung protein extracts were assessed using antibodies
from R&D Systems and the R&D Duoset.RTM. Elisa Development
kit (R&D Systems, Minneapolis, Minn., USA).
Results:
[0108] Levels of CXCL-1 (a chemotactic agent for neutrophils) were
significantly decreased after treatment of animals inhaled with
hydroxypropyl-.beta.-cyclodextrin as compared to placebo-treated
mice (FIG. 13). Peribronchial inflammation was significantly lower
in the hydroxypropyl-.beta.-cyclodextrin-treated group as compared
to placebo-exposed animals (FIG. 14).
CONCLUSION
[0109] Overall, these data demonstrate that
hydroxypropyl-.beta.-cyclodextrin is able to decrease the
production of CXCL-1, a potent chemotactic agent for neutrophils in
various inflammatory conditions and LPS-induced peribronchial
inflammation suggesting a usefulness of this compound for treatment
of COPD.
* * * * *