Note: Descriptions are shown in the official language in which they were submitted.
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TITLE OF THE INVENTION
Nicotinic Receptor Agonists for the Treatment of Inflammatory Diseases
FIELD OF THE INVENTION
The present invention relates to the treatment of inflammatory diseases,
including a variety of pulmonary diseases, through the use or
administration of nicotinic receptor agonists.
BACKGROUND OF THE INVENTION
Although we breathe more than one cubic meter of air every hour, our lung
defense mechanisms usually deal with the large quantities of particles,
antigens, infectious agents and toxic gases and fumes that are present in
inhaled air. The interaction of these particles with the immune system and
other lung defense mechanisms results in the generation of a controlled
inflammatory response which is usually protective and beneficial. In
general, this process regulates itself in order to preserve the integrity of
the airway and alveolar epithelial surfaces where gas exchange occurs. In
some cases, however, the inflammatory response cannot be regulated
and the potential for tissue injury is increased. Depending on the type of
environmental exposure, genetic predisposition, and a variety of ill-defined
factors, abnormally large numbers of inflammatory cells can be recruited
at different sites of the respiratory system, resulting in illness or disease.
The inflammatory response to inhaled or intrinsic stimuli is characterized
by a non-specific increase in the vascular permeability, the release of
inflammatory and chemotactic mediators including histamine, eicosanoids,
prostaglandins, cytokines and chemokines. These mediators modulate the
expression and engagement of leukocyte-endothelium cell adhesion
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molecules allowing the recruitment of inflammatory cells present in blood.
A more specific inflammatory reaction involves the recognition and the
mounting of an exacerbated, specific immune response to inhaled
antigens. This reaction is involved in the development of asthma,
Hypersensitivity pneumonitis (HP) and possibly sarcoidosis. Dysregulation
in the repair mechanisms following lung injury may contribute to fibrosis
and loss of function in asthma, pulmonary fibrosis, chronic obstructive
pulmonary disease (COPD), and chronic HP.
It was previously reported that the incidence of HP is much lower among
current smokers than in non-smokers (1-4). Sarcoidosis is also less
frequent in smokers than in non smokers (5, 6). The mechanisms
underlying the beneficial effects of cigarette smoking on the development
of HP and other inflammatory diseases are still unknown but may be
linked to the immunomodulatory effect of nicotine. There are clinical
observations of asthma de novo or exacerbation after smoking cessation.
Proof of this is difficult to obtain and any protective effects of nicotine in
the prevention or treatment of asthma are likely overwhelmed by the
negative effects of tobacco smoke with its thousands of constituents.
The protective effect of smoking has also been reported in other diseases,
the most studied being ulcerative colitis, an inflammatory intestinal disease
(7, 8). Nicotine has been successfully used in the treatment of this disease
(9, 10). Other studies have looked at the possible therapeutic value of
nicotine in the treatment of Alzheimer's disease and Parkinson's disease
(11, 12).
Nicotinic receptors are pentamers made up of five polypeptide subunits
which act as ligand-gated ions channels. When the ligand binds to the
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receptor, a conformational change in the polypeptide occurs, opening a
central channel that allows sodium ion to move from the extracellular fluid
into the cytoplasm. Four types of subunits have been identified: a, R, y
and 5. The receptor can consist of any combination of these four types of
subunits (13). Recent work has shown that alveolar macrophages (AM)
can express the a-7 subunit (14), while bronchial epithelial cells express
the a-3, a-5 and a-7 subunits (15), and lymphocytes the a-2, a-5, a-7, (3-2
and R-4 subunits (14). Fibroblasts (16) and airway smooth muscles cells
(17) also express these receptors. Therefore, resident pulmonary cells
(AM, dendritic cells, epithelial cells, fibroblasts, etc.) and those recruited
in inflammatory diseases (lymphocytes, polymorphonuclear cells) express
nicotinic receptors.
Nicotinic receptor activation in lymphocytes affects the intracellular
signalization, leading to incomplete activation of the cell. In fact, nicotine
treatment upregulates protein kinase activity, which in turn upregulates
phospholipase A2 (PLA2) activity. PLA2 is responsible for cleaving
phosphoinositol-2-phosphate (PIP2) into inositol-3-phosphate (IP3) and
diacylglycerol (DAG) (13, 19). The continuous presence of 1P3 in the cell
would appear to result in the desensitization of calcium stores, leading to
their depletion (19). This observation could explain the fact that nicotine-
treated lymphocytes do not release enough calcium into the cytoplasm to
activate transcription factors such as NFk-B (20).
Nicotine, the major pharmacological component of cigarette smoke, is one
of the best known nicotinic receptor agonists (21). This natural substance
has well defined anti-inflammatory and immunosuppressive properties
(22), and may have anti-fibrotic properties (23). Exposure of animals to
smoke from cigarettes with high levels of nicotine is more
immunosuppressive than that from low-nicotine cigarettes (24). Moreover,
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treatment of rats with nicotine inhibits the specific antibody response to
antigens and induces T cell anergy (25). Although they are increased in
number, AM from smokers show a decreased ability to secrete
inflammatory cytokines in response to endotoxins ((20, 25, 26)) and
nicotine seems to be the responsible component of this inhibition (26)..
One study also showed that peripheral blood lymphocytes from smokers
express higher levels of FAS ligand (FASL) and that nicotine increases
FASL expression on lymphocytes from non-smokers, indicating that
nicotine may affect cell apoptosis (27). Nicotine was also shown to have
an inhibitory effect on the proliferation and extracellular matrix production
of human gingival fibroblasts in vitro (23). Of interest, nicotine treatment
seems to up-regulate the expression of nicotinic receptors (28).
Nicotinic agonists may down-regulate T cell activation, indeed, nicotine
has been shown to affect T cell expression of the co-stimulatory molecules
CD28 and CTLA4 (29).
The B7/CD28/CTLA4 co-stimulatory pathway plays a key regulatory role
in T-cell activation and homeostasis (30, 31). Two signaling pathways are
involved. A positive signal involves the engagement of B7 (CD80 ICD86)
molecules with T cell CD28 receptors which results in the potentiation of
T cell responses (proliferation, activation, cytokine expression, and
survival) (32). A negative signal involves B7 interactions with CTLA4 on
activated T cells, leading to a downmodulation of T cell responses (33,
34). The balance between CD28 and CTLA4 derived signals may alter the
outcome of T-cell activation.
In HP, it was previously reported that an upregulation of B7 molecule
expression on AM in patients with active HP (35) and in murine HP (36).
It was also shown that a blockade of the B7-CD28 co-stimulatory pathway
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in mice inhibited lung inflammation (36). These results also demonstrated
that the expression of B7 molecules on AM is lower in smokers than in
non-smokers and that an in vitro influenza virus infection is able to
upregulate B7 expression 'in normal human AM but not in AM from
smokers; whether this is due to nicotine or other substances present in
cigarette smoke is unknown (35). An up-regulation of the B7 molecules
has also been reported in asthma (37, 38) and sarcoidosis (39).
Epibatidine is the most potent nicotinic agonist known so far (40). It has
anti-inflammatory and analgesic properties. In fact, its analgesic potential
is two hundred times that of morphine (40). This molecule is also known
to inhibit lymphocyte proliferation in vitro (41). The binding of epibatidine
to the receptor is non-specific (42). Unfortunately, epibatidine has major
toxic side effects mostly on the cardiovascular and the central nervous
systems making it inappropriate for use as an anti-inflammatory drug to
treat pulmonary diseases (40).
Dimethylphenylpiperazinium (DMPP) is a synthetic nicotinic agonist that
is non-specific (13). Its potencjr for the receptor is about the same as
nicotine, depending on the kind of cells implicated in the stimulation (43).
Its advantage over nicotine and other nicotinic agonists is that its chemical
configuration prevents it from crossing the blood-brain barrier, thus
causing no addiction or other central nervous effects (13). The anti-
inflammatory properties of DMPP are not well described. However, it has
been shown that a chronic in vivo treatment could decrease the number
of white blood cells, decrease the cytokine production by splenocytes and
decrease the-activity of natural killer cells (44). The effect of DMPP on'
airway smooth muscle cells has also been tested. DMPP has an initial
short contractive effect which is followed by a relaxing effect when the
cells are in contact with the agonist for a longer period of time (45). This
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bronchodilatory effect would not in itself make DMPP a potentially useful
treatment of asthma, since more potent bronchodilators are currently
available on the market (B2 agonists). However, the properties of this
nicotinic receptor agonist are important since this drug could be safely
administered to asthmatics and COPD patients for its anti-inflammatories
properties. Moreover, there is no evidence that DMPP has any toxic effect
on major organs such as the heart, the brain, the liver or the lungs.
Despite advances in the treatment of inflammatory illnesses, including
pulmonary inflammatory diseases, treatment using available drugs or
agents frequently results in undesirable side effects. For example, the
inflammation of COPD is apparently resistant to corticosteroids, and
consequently the need for the development of new anti-inflammatory
drugs to treat this condition has been recognized (46).
Similarly, while corticosteroids and other immunosuppressive medications
have been routinely employed to treat pulmonary fibrosis, they have
demonstrated only marginal efficacy (47).
There is thus a need for new and reliable methods of treating inflammatory
diseases, including pulmonary inflammatory diseases, in a manner that
alleviates their symptoms without causing side effects.
SUMMARY OF THE INVENTION
In accordance with the present invention, there is provided a novel method
for treating inflammatory diseases. Specifically, a novel method is
described for treating pulmonary inflammatory diseases through the use
or administration of nicotinic receptor agonists.
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The idea of using nicotine or other nicotinic receptor agonists to treat
inflammatory pulmonary disease is novel. Despite the impressive anti-
inflammatory and immunosuppressive properties of nicotine and other
nicotinic receptor agonists, their usefulness in the treatment of allergic and
other inflammatory lung diseases has not previously been disclosed.
Nicotine itself is a safe substance that does not seem to have any long
term side effects (48,49). Smoke-related diseases of the lungs, heart and
arteries are not caused by nicotine but by the thousands of other
chemicals present in the inhaled smoke. The main problem is that nicotine
crosses the blood-brain barrier, inducing addiction. These are major
reasons for the lack of prior interest in nicotinic agonists in the treatment
of lung diseases. The harmful effects of cigarette smoking are obvious.
Although nicotine is not responsible for the toxic effects of cigarette
smoking (49), the association remains.
The present invention thus proposes the use nicotinic receptor agonists,
such as DMPP, to treat inflammatory lung diseases such as asthma,
COPD, interstitial pulmonary fibrosis (IPF), sarcoidosis, HP, and
bronchiolitis obliterans with organizing pneumonitis (BOOP). The drug
could be administered orally, or preferably by targeted delivery directly to
the lung by aerosolisation with different and preferred vehicules thus
minimizing any systemic effects.
The anti-inflammatory and immunosuppressive properties, as well as
minimal side effects, of nicotinic receptor agonists make these drugs
ideally suited for medical use in the treatment of a large variety of lung
diseases that are characterized by bronchial or interstitial inflammation.
These diseases include diseases such as asthma, COPD, IPF,
sarcoidosis, HP and BOOP.
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Other objects, advantages and features of the present invention will become
more
apparent upon reading the following non-restrictive description of preferred
embodiments
thereof, given by way of example only with reference to the accompanying
drawings.
In one aspect, the invention relates to the use of a nicotinic receptors
agonist selected
from nicotine, dimethylphenyl-piperazinium and epibatidine or their
derivatives thereof in
the manufacture of a medicament for the alleviation or prevention of a
pulmonary
inflammatory disease selected from the group consisting of asthma,
interstitial pulmonary
fibrosis (IPF), sarcoidosis, hypersensitivity pneumonitis (HP), chronic HP and
bronchiolitis obliterans with organizing pneumonitis (BOOP) in an animal.
In a further aspect, the invention relates to the use of a nicotinic receptors
agonist selected
from nicotine, dimethylphenyl-piperazinium and epibatidine or their
derivatives thereof
for alleviating or preventing a pulmonary inflammatory disease selected from
the group
consisting of asthma, interstitial pulmonary fibrosis (IPF), sarcoidosis,
hypersensitivity
pneumonitis (HP), chronic HP and bronchiolitis obliterans with organizing
pneumonitis
(BOOP)
In still a further aspect, the invention relates to the use of a
pharmaceutical composition
for alleviating or preventing a pulmonary inflammatory disease selected from
the group
consisting of asthma, interstitial pulmonary fibrosis (IPF), sarcoidosis,
hypersensitivity
pneumonitis (HP), chronic HP and bronchiolitis obliterans with organizing
pneumonitis
(BOOP), said composition comprising a nicotinic receptors agonist selected
from
nicotine, dimethylphenyl-piperazinium and epibatidine or their derivatives
thereof and a
pharmaceutically or physiologically acceptable carrier.
BRIEF DESCRIPTION OF FIGURES
Figure 1: Total and differential cell counts in BAL cells. There was a marked
inhibition
of total cell counts in nicotine treated mice due mainly to a decrease in the
lymphocyte
population.
Figure 2: IFN--y mRNA expression in isolated lung mononuclear cells. A
significant
inhibition of IFN-,ymRNA was observed.
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Figure 3: TNF-a mRNA expression was induced by a 24 h LPS stimulation. Results
are
expressed as a % of expression, 100% being attributed to the LPS alone group.
The
intensity of the band was obtained by dividing the intensity of the TNF-a band
by that of
0-actin. Treatment of stimulated cells with different doses (40 to 160 M for
nicotine and
DMPP) induced a drop of TNF-a mRNA expression. The greatest effect was
obtained
with the 40 M concentration of nicotine (a 98% reduction of expression),
while all doses
of DMPP caused a 60 to 50% reduction of expression.
Figure 4: TNF-a mRNA expression was induced by a 24 h SR stimulation. Results
are
expressed as described in fig. 5. Treatment of stimulated cells with different
doses (80
and 160 M for nicotine and 40 to 160 M for DMPP) induced a down-regulation
of
TNF-a mRNA expression. Only the 160 M dose of nicotine had an effect on mRNA
expression while the 40 and 80 M doses of DMPP induced up to 60% of reduction
of
TNF-a
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mRNA expression.
Figure 5: IL-10 mRNA expression was induced by a 24 h LPS stimulation.
Results are expressed as described in fig. 5. Treatment of stimulated cells
with different doses (40 to 160 pM for both nicotine and DMPP) induced
a down-regulation of IL-10 mRNA expression. The largest drop of
expression (a 87% reduction) occurred with 40 pM nicotine. DMPP
induced a 55 to 40% reduction of expression for all three doses.
Figure 6: IL-10 mRNA expression was induced by a 24 h SR stimulation.
Treatment of stimulated cells with different doses (80 and 160 pM for
nicotine and 40 to 80 pM for DMPP) induced a down-regulation of IL-10
mRNA expression. The greatest drop in mRNA expression with the
nicotine treatment occurred at 160 pM (60% drop of expression), and at
.15 80 pM (90 % drop of expression) with the DMPP treatment.
Figure 7: IFN-y mRNA expression was induced in RAW 264.7 cells by a
24 h LPS stimulation. Results are expressed as described in fig. 5.
Treatment of stimulated cells with different doses of DMPP induced a
reduction in IFN-y mRNA expression. The largest drop of expression (a
80% reduction) occurred with 40 pM DMPP.
Figure 8: a) The expression of CD 80 was induced with either LPS (38%)
or SR antigen (35%). Nicotine treatment (40iaM for 48h) reduced the
expression to 20% in LPS stimulated cells and 26% in SR stimulated cells.
b) The expression of CD 80 was induced with either LPS (38%) or SR
antigen (35%). DMPP treatment (40taM for 48h) reduced the expression
to 17% in LPS stimulated cells and 20% in SR stimulated cells.
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Figure 9: IFN-y mRNA expression in T lymphocytes isolated from BAL
performed on HP patients. DMPP treatment reduced expression of IFN-y
in these cells.
5- Figure 10: CD 86 expression in total cells from a BAL that was performed
on a normal patient. Cells that were treated with DMPP express 50% less
CD86 than non-treated cells.
Figure 11: BAL cells from DMPP, nicotine and epibatidine treated mice.
Treatment with nicotine and epibatidine had a significant inhibitory effect
on SR-induced inflammation after 24 hours.
Figure 12: A significant inhibitory effect of DMPP on lung inflammation
was found when we increased the number of animals.
Figure 13: TNF levels in BAL fluid from DMP-treated mice. DMPP
decreased significantly BALF TNF levels.
Figure 14: Effect of intra-peritoneal treatment with increasing doses of
DMPP on total cell accumulation in BAL of asthmatic mice. The number
of cells was highly elevated in OVA challenged and non-treated mice. The
DMPP treatment significantly reduced cell counts at the 0.5 and 2.0 mg/kg
doses.
Figure 15: Differential counts for the dose response. The OVA challenged
mice (OVA OVA) had more eoosinophils and lymphocytes in their BAL
compared to the control group (sal sal). The DMPP treatment significantly
reduced the presence of both osinophils and lymphocytes in BAL in all
groups (n = 8; p < 0.05).
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Figure 16: Second dose response for the DMPP IP treatment effect on
total cell accumulation in BAL of asthmatic mice. The number of cells was
highy elevated in OVA challenged and non-treated mice. The DMPP
treatment significantly reduced total cells at the 0.1 and 0.5 mg/kg doses.
Figure 17: Differential counts from the second dose response. The DMPP
treatment significantly reduced eosinophil and lymphocyte counts in the
0.1 and 0.5 mg/kg doses, 0.5 mg/kg being the most effective dose for the
anti-inflammatory effect of DMPP.
Figure 18: BAL IL-5 levels from control, asthmatic and treated mice. The
OVA challenges increased IL-5 levels in BAL, while the DMPP treatment
had a significant inhibitory effect on IL-5 levels in the 0.5 mg/kg treated-
group of mice.
Figure 19: Lung resistance after metacholine challenges from normal,
asthmatic and asthmatic treated with 0.5 mg/kg intranasal DMPP. DMPP
seems to reduce the % of augmentation of lung resistance compared to
asthmatic mice.
Figure 20: The provocative challenge dose of 200% lung resistance
augmentation (PC 200) was calculated. DMPP significantly reduced the
PC200 in treated-mice compared to asthmatic mice (p = 0.04 ; n = 6).
Figure 21: IL-4 mRNA expression was induced by a 24 h LPS stimulation.
Results are expressed as described in fig. 5. Cells were treated with
different doses (40 to 160 pM for both nicotine and DMPP). The nicotine
treatment induced a drop in the IL-4 mRNA expression (up to a 90%
reduction of expression in the 40pM group). DMPP treatment completely
blocked IL-4 mRNA expression in the LPS stimulated cells, at all doses.
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Figure 22: Effect of DMPP on blood eosinophil transmigration. DMPP
induces a dose-related inhibition of eosinophil transmigration across an
artificial basement membrane.
Figure 23: Effect of mecamylamine, a nicotinic antagonist, on the
inhibitory effect of DMPP on blood eosinophil transmigration.
Mecamylamine reverses the effect of DMPP, suggesting that nicotinic
receptor activation is necessary for the DMPP inhibitory effect.
Figure 24: Effect of other nicotinic agonists on transmigration of blood
eosinophils. Nicotine, epibatidine and cytosine all reduce blood eosinophil
transmigration.
Figure 25: Effect of DMPP on collagen IA mRNA expression by normal
human lung fibroblasts. DMPP inhibits collagen 1A mRNA expression in
a dose dependant manner.
Figure 26: Effect of nicotine on collagen 1A mRNA expression by human
lung fibroblasts. Nicotine inhibits collagen 1A mRNA expression at I and
10 pM while the higher doses have no inhibitory effect.
Figure 27: Effect of epibatidine, another nicotinic agonist, on collagen 1A
mRNA expression by human lung fibroblasts. Epibatidine also has an
inhibitory effect on collagen 1A mRNA expression.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
The presence of nicotinic receptors on inflammatory and pulmonary cells
has been described previously. However, the novelty of the present
invention resides in the observation that nicotinic receptor agonists appear
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to be useful in the treatment of inflammatory lung diseases, and in the
related discovery of the anti-inflammatory and immunosuppressive
properties of nicotinic agonists specifically directed against mechanisms
involved in the pathogenesis of such inflammatory pulmonary diseases as
asthma, HP, sarcoidosis, BOOP, IPF, and COPD. An example of this is
the effect of cigarette smoke on the expression of the B7 co-stimulatory
molecules.
Two animal models were used to study the effects of nicotinic antagonists
in inflammatory pulmonary diseases: an HP model and an asthma model.
With both of these models, the effects of nicotinic receptor agoriists (both
selective and non-selective) were studied on lung physiology, and
inflammation. In vitro studies were performed using isolated inflammatory
cells.from the animal studies or from patients as well as commercially
available cell lines in an attempt to understand the mechanisms by which
nicotinic agonists down-regulate inflammation.
Initially, experiments were conducted with non-specific agonists, i.e
agonists that bind to all nicotinic receptor subunits (nicotine,
dimethylphenylpiperazinium (DMPP) and epibatidine) (13, 42). A(i4
subunit specific agonist, cytisine (42), was also tested to see whether a
specific stimulation could also have anti-inflammatory effects.
For the purposes of the present application, the term "animal" is meant to
signify human beings, primates, domestic animals (such as horses, cows,
pigs, goats, sheep, cats, dogs, guinea pigs, mice, etc.) and other
mammals. Generally, this term is used to indicate living creatures having
highly developed vascular systems.
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For the purposes of the present invention, agonists or agents are molecules or
compounds
that bind to and modulate the function of the nicotinic receptor. Preferred
agents are
receptor-specific and do not cross the blood-brain barrier, such as DMPP.
Useful agents
may be found within numerous chemical classes, though typically they are
organic
compounds and preferably, small organic compounds. Small organic compounds
have a
molecular weight of more than 150 yet less than about 4, 500, preferably less
than about
1500, more preferably, less than about 500. Exemplary classes include
peptides,
saccharides, steroids, heterocyclics, polycyclics, substituted aromatic
compounds, and the
like.
Selected agents may be modified to enhance efficacy, stability, pharmaceutical
compatibility, and the like. Structural identification of an agent may be used
to identify,
generate, or screen additional agents. For example, where peptide agents are
identified,
they may be modified in a variety of ways as described above, e.g. to enhance
their
proteolytic stability. Other methods of stabilization may include
encapsulation, for
example, in liposomes, etc. The subject binding agents are prepared in any
convenient
way known to those skilled in the art.
For therapeutic uses, agents affecting nicotinic receptor function may be
administered by
any convenient means. Small organics are preferably administered orally; other
compositions and agents are preferably administered parenterally, conveniently
in a
pharmaceutically or physiologically acceptable carrier, e.g., phosphate
buffered saline, or
the like. Typically, the compositions are added to a retained physiological
fluid such as
blood or synovial fluid.
As examples, many such therapeutics are amenable to intravenous direct
injection or
infusion, parenteral, topical, inhaled/oral, sublingual, intraperitaneal,
intratracheal/nasal
administration e.g. through aerosol,
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intraocularly, or within/on implants (such as coliagen, osmotic pumps,
grafts comprising appropriately transformed cells, etc. with therapeutic
peptides. Generally, the amount administered will be empirically
determined, typically in the range of about 10 to 1000 pg/kg of the
recipient. For peptide agents, the concentration will generally be in the
range of about 50 to 500 pg/mI in the dose administered. Other additives
may be included, such as stabilizers, bactericides, etc. These additives will
be present in conventional amounts.
Nicotinic agonists would not replace all drugs that are currently used to
treat inflammatory lung diseases and the airflow obstruction that is often
associated with these diseases. Bronchodilators remain useful for the
immediate release of bronchospasms. However, bronchodilators have no
effect on the underlying cause or inflammation.
Corticosteroids are potent anti-inflammatory drugs. Their systemic use
causes major side effects that preclude their long-term uses whenever
possible. Inhaled poorly absorbed steroids are useful to treat airway
inflammation. At low doses these drugs have little or no side effects.
However, higher doses increase the risks for oral candidasis, vocal cords
paralysis, cataracts and osteoporosis. Inhaled steroids have no effects on
lung interstitium and have no anti-fibrotic properties (57).
More recent drugs, such as anti-leukotrienes, are useful in some
asthmatics (58) but have no effects in COPD and other lung diseases.
These drugs have anti-inflammatory properties limited to the components
of inflammation caused by leukotrienes (59). The treatment of interstitial
lung disease such as IPF, Sarcoidosis, HP, and BOOP basically rests on
the use of systemic corticosteroids. This treatment is effective in
controlling some of the inflammation but unfortunately induces serious
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side effects and does not reverse underlying fibrotic changes.
Immunosupressive agents such as cyclophosphamide and azathioprine
are sometimes tried in severe IPF but their therapeutic values are
unproven and at most, very limited (60). In essence, lung fibrosis is usually
progressive and untreatable, with most IPF patients dying of this condition
(61).
Nicotinic agonists may be useful as a steroid sparing or replacing drug. By
targeting their delivery to the lung phagocytes, these drugs could be
helpful in controlling both airway and interstitial inflammation. One major
advantage of nicotinic agonists over corticosteroids, besides having fewer
side effects, is the fact that these agonists have a direct effect on
fibroblasts and could therefore prevent or reverse fibrosis in the airways
and in the lungs, something corticosteroids cannot do. Interstitial fibrosis
is the hallmark if IPF, a major sequel of HP and sarcoidosis, and airway
efibrosis is a prevailing finding in chronic asthma (57).
Other substances are actively being studies as potential new treatments
for inflammatory lung diseases. Many cytokines are specifically targeted
(e.g. IL-5, IL-13, IL-16...) (62). It is believed that because of the
complexity of pathways involved in inflammation, any one specific cytokine
or other inflammatory mediator is unlikely to have a significant impact on
the treatment of these lung diseases. Nicotinic receptor agonists, not
unlike corticosteroids, have the advantage of targeting a broad spectrum
of the inflammatory response. Therein lies their potential in the treatment
of inflammatory lung diseases.
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EXAMPLES
1- Hypersensitivity-like inflammation
Effect of nicotinic agonists on long term-induced hypersensitivity
pneumonitis (HP) in mice.
Example 1: In vivo HP studies
The hypothesis is that the stimulation of nicotinic receptors with nicotine
down-regulates the immune response to HP antigens via inflammatory
cytokine suppression and inhibition of specific antigen-mediated cellular
activation.
This model was selected because, as mentioned previously, the incidence
of HP is lower in smokers than in non-smokers (50), and because this
model is well described. HP was induced by the administration of
Saccharopolyspora rectivirgula (SR) antigen, the causative agent of
farmer's lung (51), a form of HP. Mice were simultaneously treated with
intra-peritoneal (IP) nicotine, with doses ranging from 0.5 to 2.0 mg/kg,
twice a day. Nicotine administration significantly reduced the number of
total cells found in the bronchoalveolar lavage (BAL) of these mice. The
population that was the most affected by nicotine treatment were
lymphocytes (Fig. 1). Pulmonary macrophages and lymphocytes were
isolated, and stimulated with anti-CD3 + recombinant IL-2. The production
of IFN-y mRNA by these cells, a cytokine known to be_ involved in the
development of HP and other pulmonary inflammatory diseases (52), was
measured. Cells from nicotine treated animals showed significantly lower
expression of IFN-y mRNA than cells from non-treated animals (Fig. 2).
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Example 2: In vitro studies showing the effect of nicotinic agonists
on cytokine expression
To further clarify the mechanisms involved in suppressive effect of nicotine
in the in vivo model, an alveolar macrophage cell line was used.
The effect of nicotine or DMPP treatment on AMJ2-C11 cells was tested
on TNF-a, IL-10 mRNA expression by RT-PCR. These cytokines are
involved in the development of pulmonary inflammatory diseases such as
HP, asthma and sarcoidosis (52-55). Nicotine and DMPP treatments
showed a great decrease in TNF mRNA expression (up to a 98%
reduction of expression in LPS stimulated and treated with 40iaM nicotine),
but not in a dose-dependant manner (Fig. 3). Similar results were
observed with SR-stimulated cells (Fig. 4). This non-dose dependant
response can be explained by nicotinic receptor desensitization due to a
large quantity of agonist in the medium. IL-10 mRNA expression was also
impaired by nicotine and DMPP treatment. The best. down-regulation
occurred at a dosage of 40iaM nicotine (LPS stimulated; 88 % reduction
of mRNA expression; Fig. 5) and at a dosage of 80 pM DMPP (SR
stimulated; 87% mRNA expression reduction; Fig. 6). Once again, the
effect was not dose-dependant.
Another macrophage cell line (RAW 264.7, ATCC) was used to test the
effect of DMPP on IFN-y expression by RT-PCR, because AMJ2-C11 cells
did not appear to express IFN-y mRNA (data not shown). Cells were
stimulated with 50pg/ml of SR antigen and incubated with DMPP at doses
ranging from 40 to 160 pM. DMPP treatment reduced the expression of
1NF-y in these cells by up to 75% with the 40pM dose (Fig. 7). Once more,
the effect did not seem to be dose-dependant.
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Example 3: In vitro effects of nicotinic agonists on co-stimulatory
molecule expression
The effects of nicotine and DMPP on B7 (CD80) molecule expression
were tested in vitro. AMJ2-C11 cells (mouse alveolar macrophages, from
the ATCC) were incubated with 40 pM nicotine or DMPP and stimulated
with LPS (0.1 pg / ml) or SR antigen (50 pg / ml) for 48 hours. The
percentage of expression of CD80 in treated cells was about one half of
the expression found in LPS and SR stimulated non-treated cells (Figs. 8
(a) and (b)).
Example 4: Studies on human BAL cells (AM and lymphocytes)
Since one goal was to treat patients with DMPP or similar drugs, the effect
of this drug was verified on lymphocytes from patients with HP. BAL were
performed on patients with HP. Lymphocytes were isolated from the other
BAL cells, stimulated with PHA and incubated with DMPP. The dose-
response of DMPP were tested on cytokine mRNA production (by RT-
PCR) for IFN-y (Fig. 9).
A broncho-alveolar lavage was performed on a normal patient, and
alveolar macrophages were isolated. SR-stimulated and nicotine or DMPP
treated cells showed once again about half of the expression of CD86
than non-treated cells (Fig 10).
Example 5: Investigation of the effect of other nicotinic agonists on
the short term SR-induced acute inflammation
The intranasal instillation of Saccharopolyspora rectivirgula (SR) antigens,
the causative agent for farmer's lung, to mice, induces a prominent
inflammatory response in the lung. Neutrophils are the first inflammatory
cells recruited at the site of inflammation. Treatment of mice with DMPP
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(0.5mg/kg), nicotine (0.5mg/kg) and epibatidine (2 g/kg) had a marked
inhibitory effect on SR-induced inflammation (Fig. 11). Nicotinic agonists
were administered intra-nasally in 50 I volume every 6h and mice were
sacrificed 24 hr after SR instillation.
A significant inhibitory effect was observed with nicotine and epibatidine
but not with DMPP. However, after increasing the number of mice treated
or not treated with DMPP to 15, we did observe a significant inhibition
compared to the non-treated group (Fig. 12).
Levels of TNF (a pro-inflammatory cytokine) are lower in the broncho-
alveolar lavage of DMPP-treated mice (Fig. 13) indicating that the down-
regulation of inflammation may result from lower TNF concentrations.
It - Asthma-like inflammation
Example 6: In vivo asthma model
Similar experiments were performed in ovalbumine-sensitized mice. DMPP
allegedly decreases both the inflammatory response and the hyper-
responsiveness to inhaled allergens and methacholine.
Groups of Balb/c mice were sensitized by intra-peritoneal injection of 20 pg
OVA protein (chicken egg albumin; Sigma-Aldrich) emulsified in 2 mg aluminum
hydroxide in PBS. After 4 weeks, challenge doses of 1.5%/50p1 OVA were
administered intranasally. The challenge was performed daily for 3 consecutive
days and then the mice assessed for allergic inflammation of the lungs 24 h
after the last aerosol exposure. Groups of mice were treated with various
concentrations of DMPP during the challenge period. Broncho-alveolar lavage
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(BAL) was performed and the fluid centrifuged at 400 g to separate cells from
liquid (Figs. 14, 15, 16 and 17).
The supernatants were used to determine lung IL-5 levels. The total number of
BAL cells and differential cell counts were evaluated (Fig. 18).
The experiment was repeated with the optimal dose of DMPP to assess the
airway responsiveness.
Measurement of AHR
Airway hyper-reactivity (AHR) in response to metacholine was measured in
anesthetized, tracheotomized, ventilated mice using a computer-controlled
ventilator (FlexiVENT).
Increasing doses of metacholine ((0 mg/kg-32.5 mg/kg) were administered
through the jugular vein (Figs. 19, 20).
Example 7: Effect of agonist treatment on mRNA expression of IL-4
The effect of agonist treatment on mRNA expression of IL-4, a cytokine
that is well known to be involved in the development of asthma, was also
tested (53). Nicotine decreased IL-4 mRNA expression by up to 92 % with
40pM (Fig. 9) DMPP completely blocked IL-4 mRNA expression (Fig. 21).
As demonstrated previously, there was no IL-4 mRNA expression when
cells were stimulated with SR antigen (data not shown).
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Example 8: Action of various agonists on eosinophil transmigration
To further investigate the effect of nicotinic agonists on the down-
regulation of inflammation in asthma, we tested the action of various
agonists on eosinophil transmigration.
Infiltration of eosinophils and other inflammatory cells into lung tissues is
an important feature of asthma and the cause of airway inflammation and
hyper-responsiveness. The passage of inflammatory cells from the
circulation to the lung involves migration through the vascular
endothelium, the basement membrane, and extra-cellular matrix
components. Inflammatory cells cross the basement membrane by
producing proteinases. ln these preliminary in vitro experiments, we
investigated the effects of various nicotinic agonists on the migration of
purified blood eosinophils through an artificial basement membrane
(Matrigel coated chemotaxis chamber). DMPP induces a dose-related
inhibition of eosinophils transmigration (Fig. 22), while this effect is
reversed by the antagonist mecamylamine (MEC) (Fig. 23). This inhibitory
effect is further confirmed with other nicotinic agonists incuding nicotine,
epibatidine and cytosine (Fig. 24). Results are expressed as a percentage
of inhibition (agonists-treated cells) compared to the control condition
without the agonists.
These results suggest that nicotinic agonists down-regulate the synthesis
or activation of proteinases that degrade basement membrane
components, thus inhibiting the migration of eosinophils into lung mucosa.
Example 9: Effect of nicotinic agonists on coliagen production
Asthma is characterized by airway structural changes, including sub-
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epithelial collagen deposition, that may be a cause for the chronicity of the
disease. An imbalance between coliagen synthesis and its degradation by
fibroblasts may be involved in this process (56). In preliminary
experiments, we investigated the effects of nicotinic agonists on collagen
Al synthesis produced by primary normal fibroblasts. Collagen Al gene
expression was evaluated by RT-PCR.
The results are expressed percentage gene expression in agonists treated
cells compared to non-treated cells.
DMPP inhibits collagen Al gene expression in a dose-dependant manner
(Fig. 25). Nicotine has a slight inhibitory effect at 1 and 10 M, whereas
higher concentrations had no effects (Fig. 26), probably due to a
desensitization of the receptors. Lower doses may be necessary to
achieve an inhibition and will be tested. The inhibitory effect is also
observed with epibatidine (Fig. 27).
Although the present invention has been described hereinabove by way
of preferred embodiments thereof, it can be modified, without departing
from the spirit and nature of the subject invention as defined in the
appended claims.
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