U.S. patent application number 09/919195 was filed with the patent office on 2001-12-20 for methods and compositions for the treatment and prevention of lung disease.
Invention is credited to Chandraratna, Roshantha A., Massaro, Donald, Massaro, Gloria DeCarlo.
Application Number | 20010053758 09/919195 |
Document ID | / |
Family ID | 22438919 |
Filed Date | 2001-12-20 |
United States Patent
Application |
20010053758 |
Kind Code |
A1 |
Massaro, Gloria DeCarlo ; et
al. |
December 20, 2001 |
Methods and compositions for the treatment and prevention of lung
disease
Abstract
Methods and compositions for the treatment of lung disease, such
as emphysema and/or bronchopulmonary dysplasia, in a mammal. Also
disclosed are methods promoting the formation of alveolar septa and
increasing the gas-exchange surface area of a mammalian lung, and
for the prevention and/or treatment of alveolar destruction.
Inventors: |
Massaro, Gloria DeCarlo;
(Washington, DC) ; Massaro, Donald; (Washington,
DC) ; Chandraratna, Roshantha A.; (Laguna Hills,
CA) |
Correspondence
Address: |
Carlos A. Fisher
ALLERGAN, INC.
2525 Dupont Drive
Irvine
CA
92623
US
|
Family ID: |
22438919 |
Appl. No.: |
09/919195 |
Filed: |
July 31, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09919195 |
Jul 31, 2001 |
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09548897 |
Apr 13, 2000 |
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60129213 |
Apr 14, 1999 |
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Current U.S.
Class: |
514/1 ;
514/312 |
Current CPC
Class: |
A61K 31/381 20130101;
A61K 31/00 20130101; A61P 11/00 20180101; C07D 333/24 20130101 |
Class at
Publication: |
514/1 ;
514/312 |
International
Class: |
A61K 031/00; A61K
031/47 |
Claims
What is claimed is:
1. A composition for the treatment or prevention of alveolar
destruction in a mammal comprising a pharmaceutically effective
amount of an RAR .beta. antagonist having RAR specific modulating
activity.
2. The composition of claim 1 wherein said RAR.beta. antagonist is
not specific to RAR.alpha..
3. The composition of claim 1 wherein said RAR.beta. antagonist is
not specific to RAR.gamma..
4. The composition of claim 1 wherein said RAR.beta. antagonist is
not specific to RAR.alpha. or RAR.gamma..
5. The composition of claim 1 wherein said composition further
comprises said RAR.beta. antagonist in dissolved form.
6. The composition of claim 5 wherein said RAR.beta. antagonist is
not specific to RAR.alpha..
7. The composition of claim 5 wherein said RAR.beta. antagonist is
not specific to RAR.gamma..
8. The composition of claim 5 wherein said RAR.beta. antagonist is
not specific to RAR.alpha. or RAR.gamma..
9. An aerosol for pulmonary delivery of a pharmaceutical
composition, said pharmaceutical composition comprising an RAR
.beta. antagonist having specific RAR modulating activity.
10. The aerosol of claim 9 wherein said RAR.beta. antagonist is not
specific to RAR.alpha..
11. The aerosol of claim 9 wherein said RAR.beta. antagonist is not
specific to RAR.gamma..
12. The aerosol of claim 9 wherein said RAR.beta. antagonist is not
specific to RAR.alpha. or RAR.gamma..
13. A method for the treatment or prevention of alveolar
destruction in a mammal comprising the step of administering a
therapeutically effective amount of an RAR .beta. antagonist
specific RAR modulating activity to said mammal.
14. The method of claim 13, wherein said RAR.beta. antagonist is
not specific to RAR.alpha..
15. The method of claim 13 wherein said RAR.beta. antagonist is not
specific to RAR.gamma..
16. The method of claim 13 wherein said RAR.beta. antagonist is not
specific to RAR.alpha. or RAR.gamma..
17. The method of claim 13, wherein said composition is
administered in the form of an inhalant.
18. The method of claim 17 wherein said RAR.beta. antagonist is not
specific to RAR.alpha..
19. The method of claim 17 wherein said RAR.beta. antagonist is not
specific to RAR.gamma..
20. The method of claim 17 wherein said RAR.beta. antagonist is not
specific to RAR.alpha. or RAR.gamma..
21. A method to increase the gas-exchange surface area of a
mammalian lung in a mammal in need thereof comprising the step of
administering a therapeutically effective amount of an RAR .beta.
antagonist having specific RAR modulating activity to said
mammal.
22. The method of claim 21, wherein said RAR.beta. antagonist is
not specific to RAR.alpha..
23. The method of claim 21 wherein said RAR.beta. antagonist is not
specific to RAR.gamma..
24. The method of claim 21 wherein said RAR.beta. antagonist is not
specific to RAR.alpha. or RAR.gamma..
25. The method of claim 21, wherein said composition is
administered in the form of an inhalant.
26. The method of claim 25 wherein said RAR.beta. antagonist is not
specific to RAR.alpha..
27. The method of claim 25 wherein said RAR.beta. antagonist is not
specific to RAR.gamma..
28. The method of claim 25 wherein said RAR.beta. antagonist is not
specific to RAR.alpha. or RAR.gamma..
29. The RAR.beta. antagonist of any of the foregoing claims,
comprising the structural formula: 2wherein a) X is selected from
the group consisting of CR.sub.2, O, S, and NR; b) R' and R" are
each independently selected from the group consisting of H and
lower alkyl; c) Ar and Ar' are each independently a single ring
aryl moiety; and d) B is selected from the group consisting of
--CR'CH--, --CHCR'--, --COO--, --OOC--; --COHN--; --NHOC--;
--CSHN--; and --NHSC--.
30. The RAR.beta. antagonist of claim 29 wherein Ar and Ar' are
each independently selected from the group consisting of
substituted or unsubstituted phenyl, furyl, thienyl and pyridyl
groups.
Description
FIELD OF THE INVENTION
[0001] This invention concerns the use of retinoic acid receptor
(RAR) antagonists for the inhibition of alveolar destruction and/or
to promote the formation of alveoli in mammalian lung tissue
deficient in adequate numbers of functional alveoli.
BACKGROUND OF THE INVENTION
[0002] Among aerobic animals, the lung functions to provide an
interface for the exchange of gases between blood and the
atmosphere. The agents of this exchange are numerous small sacs
termed alveoli (in adult humans about 300,000,000 per lung) that
provide a gas permeable-liquid impermeable barrier between the gas
and liquid phases. Between the alveoli are numerous capillaries
carrying deoxygenated blood to the lung from the tissues and
oxygenated blood from the alveoli to the tissues. The partial
pressure of oxygen in the lungs is approximately 100 mm Hg at sea
level; at this pressure the binding of oxygen by hemoglobin in the
erythrocytes is favored. The alveoli thus provide a means for
presenting the oxygen to hemoglobin to permit the conversion of
deoxyhemoglobin to hemoglobin. Because the exchange occurs at the
surface of the gas/blood barrier, alveoli have evolved as a means
for providing extremely high surface area in a compact overall
area, thus maximizing possible gas exchange. Lack of adequate gas
exchange would lead to disability which could progress to
death.
[0003] Diseases that result in fewer alveoli therefore are quite
serious, and are common causes of inadequate oxygenation and
resultant disability and death. Among such diseases are
brochopulmonary dysplasia (BPD) and emphysema. BPD is a disease of
prematurely born infants, and is characterized mainly by a failure
of the infant to form a sufficient number of appropriately-sized
alveoli. Emphysema, a disease of middle and advanced age, appears
to be due to progressive proteinase-induced alveolar
destruction.
[0004] The process of alveoli formation is reasonably well
understood from a gross developmental standpoint, and seems to be
similar in rat, mouse, and human, the major species studied. The
process includes the subdivision (septation) of the saccules that
constitute the gas-exchange region of the immature lung. Septation
results in the formation of smaller, more numerous gas-exchange
structures (alveoli). The timing of the onset and cessation of
septation vary among species, but both onset and cessation are
critical to the formation of alveoli of the size and number needed
for adequate oxygenation.
[0005] The molecular basis of the initiation and cessation of
alveoli formation are not as well understood as the structural
events and timing accompanying alveoli development. Knowledge of
the molecular signals that initiate and end septation, and that
govern the spacing of septa relative to the O.sub.2-demand, are
virtually unknown; however, several lines of evidence suggest that
certain retinoids (retinoic acid and its derivatives) may play a
key signaling role. In Massaro et al., Nature Medicine 3:675
(1997), hereby incorporated by reference herein, rats were treated
with elastase, causing destruction of alveolar walls in a manner
similar to that seen in pulmonary emphysema. Treatment of the rats
with all-trans-retinoic acid (ATRA), an agonist of all RAR
isotypes, appeared to reverse this destruction. Similarly,
treatment of newborn rats (which are born with immature lungs
lacking an adult complement of alveoli) with ATRA induced the
formation of an increased number of alveoli in rats without
enlarging the lung. See Massaro et al., Am. J. Physiol. 270: L305
(1996) incorporated by reference herein.
[0006] ATRA can have a multiplicity of physiological effects. The
retinoid receptors, when bound by an appropriate ligand, are
mediators of various life processes, including reproduction,
metabolism, differentiation, hematopoiesis, and embryogenesis.
[0007] There is therefore a need for methods and compositions that
provide a practicable means for inhibiting alveolar destruction
and/or promoting the formation of alveoli in a postnatal aerobic
animal, particularly a mammal such as a human. Additionally, there
is a need for therapeutic methods that are able to more
specifically treat such a condition without a high likelihood of
serious side effects.
BRIEF DESCRIPTION OF THE DRAWINGS
[0008] FIG. 1A is a photomicrograph of a histological section of
the lung of a 14-day old rat injected intraperitoneally daily from
age 3 to 13 days with cottonseed oil (control).
[0009] FIG. 1B is a photomicrograph of a histological section of
the lung of a 14-day old rat injected intraperitoneally daily from
age 3 to 13 days with an RAR.beta. agonist dissolved in cottonseed
oil.
[0010] FIG. 2A is a photomicrograph of a histological section of
the lung of a 4-day wild-type mouse.
[0011] FIG. 2B is a photomicrograph of a histological section of
the lung of a 4-day mouse genetically lacking RAR.beta..
[0012] FIG. 2C is a photomicrograph of a histological section of
the lung of a 21-day wild-type mouse.
[0013] FIG. 2D is a photomicrograph of a histological section of
the lung of a 21-day mouse genetically lacking RAR.beta..
SUMMARY OF THE INVENTION
[0014] The present invention is directed to methods and
compositions for promoting the formation of alveoli in mammalian
lung tissue. In one embodiment the invention comprises a
therapeutic method for inducing the formation of alveoli in
mammalian lung tissue by administration of a composition comprising
a therapeutically effective amount of an ligand that is an
RAR.beta. antagonist or inverse agonist. In a preferred aspect of
this embodiment, the RAR.beta. receptor antagonist or inverse
agonist has specific RAR modulating activity at the RAR.beta.
receptor, and is not specific to the RAR.alpha. receptor. In
another preferred aspect, the RAR.beta. receptor antagonist or
inverse agonist has specific RAR modulating activity at the
RAR.beta. receptor and is not specific to the RAR.gamma. receptor.
In a particularly preferred aspect of this embodiment, the
RAR.beta. receptor antagonist or inverse agonist has specific RAR
modulating activity at the RAR.beta. receptor and is not specific
to either the RAR.alpha. or the RAR.gamma. receptor. Even more
preferably, the ligand is specific to RAR.beta. isotypes.
Preferably such a ligand is a retinoid.
[0015] By "specific RAR modulating activity" is meant that such a
compound has a disassociation constant (K.sub.D) (the ligand
concentration at which 50% of the RAR receptors are complexed with
the ligand) at an RAR receptor at least 10 times, preferably at
least 25 times, even more preferably at least 50 times, and most
preferably at least 100 times greater than the K.sub.D for the
binding of the same ligand to an RXR receptor. Determination of the
K.sub.D at an RAR or RXR receptor for a given ligand is a routine
matter. Membrane preparations of host cells expressing a cloned RAR
or RXR receptor, and the amino acid and nucleotide sequences of
such receptors, has been described in various publications
available, and within the knowledge of, the person or ordinary
skill in the art. For example, U.S. Pat. No. 5,776,699, to Klein et
al., describes assays employing RAR and RXR, and PCT Publication
No. W093/11755 discloses ligand binding assays. These references
are now incorporated by reference herein in their entirety.
[0016] By "agonist" is meant a retinoid receptor ligand that will
cause the activation of transcription at a gene having an
appropriate retinoid receptor response element.
[0017] By "antagonist" is meant a retinoid receptor ligand that
will inhibit the activation of transcription by the retinoid
receptor at a gene having an appropriate retinoid receptor response
element in the presence of an agonist of the retinoid receptor.
[0018] By "inverse agonist" is meant a retinoid receptor ligand
that will inhibit the expression of transcription at a gene having
an appropriate retinoid receptor response element beyond a basal
expression level existing in the absence of an agonist of the
retinoid receptor.
[0019] In another embodiment, the invention is directed to
therapeutic compositions for the treatment of an emphysemic mammal,
or of a mammal suffering from bronchopulmonary dysplasia,
comprising a therapeutically effective amount of an ligand that is
an RAR.beta. antagonist or inverse agonist. In a preferred aspect
of this embodiment, the RAR.beta. receptor antagonist or inverse
agonist has specific RAR modulating activity at the RAR.beta.
receptor, and is not specific to the RAR.alpha. receptor. In
another preferred aspect, the RAR.beta. receptor antagonist or
inverse agonist has specific RAR modulating activity at the
RAR.beta. receptor and is not specific to the RAR.gamma. receptor.
In a particularly preferred aspect of this embodiment, the
RAR.beta. receptor antagonist or inverse agonist has specific RAR
modulating activity at the RAR.beta. receptor and is not specific
to either the RAR.alpha. or the RAR.gamma. receptor. Even more
preferably, the ligand is specific to RAR.beta. isotypes.
Preferably such a ligand is a retinoid. It is contemplated that the
RAR.beta. antagonist or inverse agonist may be used either as the
only active ingredient in the composition, or in combination with
one or more additional therapeutically active ingredient. In one
aspect, the additional therapeutically active ingredient is a
retinoid; in a preferred aspect, an additional therapeutically
active ingredient is another RAR-active ligand, for example,
all-trans-retinoic acid.
[0020] By "RAR-active retinoid" is meant that the retinoid has
agonist, inhibitory, or inverse agonist (negative hormone) activity
at an RAR receptor.
[0021] By "therapeutically effective amount" is meant that the
amount of the RAR-specific therapeutic agent is sufficient, either
as the result of a single dose, or as the result of multiple doses
over the term of therapy, to decrease the rate of alveolar
destruction in an emphysemic mammal, or to promote the growth of
alveolar septa in said mammal.
DETAILED DESCRIPTION OF THE INVENTION
[0022] The present invention is directed to compositions and
methods for the treatment or prevention of alveolar destruction
and/or to promote the formation of alveoli is mammalian lungs
deficient in adequate numbers of functional alveoli. Such methods
and compositions involve RAR-active retinoids, therapeutic
compositions containing such agents, and methods for their use.
[0023] The retinoid receptors are part of the
steriod/thyroid/vitamin D superfamily of nuclear receptors. The
retinoid receptors include the retinoic acid receptors (RAR) and
the retinoid X receptor (RXR). The RAR and RXR receptors are single
chain polypeptides containing a number of structural domains in
common: a ligand binding domain, a sequence-specific DNA binding
domain, and a leucine zipper motif. In the presence of ligand, the
single RAR or RXR chains can, by virtue of the leucine zipper, form
dimers. The RAR chain is believed to exist in vivo exclusively as
an RAR/RXR heterodimer. RXR may form heterodimers with RAR or other
members of the superfamily, such as the vitamin D receptor and the
thyroid receptor.
[0024] Retinoid receptor dimers are effective transcription factors
regulating gene transcription by binding to retinoic acid response
elements (RAREs) or retinoid X response elements (RXREs) present in
(or near) the promoters of retinoid responsive genes, or by
negatively regulating the enhancer functions of other transcription
factors.
[0025] Described RAR isotypes include RAR.beta., RAR.alpha., and
RAR.gamma., and described RXR isotypes include RAR.beta.,
RAR.alpha., and RAR.gamma.. Within each receptor class, these
isotypes have sequence homology, but are encoded by different
genes. Within each isotype several isoforms have been described;
these isoforms differ in their N terminals and are generated by
alternative splicing and/or differential usage of more than one
promoter. See e.g., Nagpal & Chandraratna, Current Pharm.
Design 2:295-316 (1996) and Mangelsdorf et al., The Retinoid
Receptors in The Retinoids: Biology, Chemistry and Medicine Ch.
8(2d ed. Sporn et al. eds. 1994), both of which are hereby
incorporated by reference herein.
[0026] Depending both upon the ligand and the nature (e.g., RAR or
RXR; isotype; isoform) of the monomer chains contained in the
dimers, an enormous variety of biological responses regulated by
the retinoid receptors are possible.
[0027] Ligands specific to RAR (e.g., ATRA) or RXR (e.g., TTNB),
and to specific RAR isotypes have been described. See e.g., Nagpal
& Chandraratna, Current Pharm. Design 2:295-316 (1996).
Therefore, the design and/or selection of RAR-specific ligands, and
of RAR isotype-specific ligands is well within the ability of the
person of ordinary skill in the art.
[0028] By "specific" to a given retinoid receptor is meant that the
disassociation constant (K.sub.D) for the binding of the ligand to
a given target receptor or receptor isotype or isoform is at least
10 times lower than the K.sub.D value for the ligand and a
non-target receptor or receptor isotype or isoform. K.sub.D is
defined as the concentration of ligand at which 50% of the
receptors are ligand bound. Even more preferably, K.sub.D is at
least 25 times lower for the target receptor than for untargeted
receptors. Most preferably, K.sub.D is at least 50, or at least
100, times lower for the target receptor than for untargeted
receptors.
[0029] An aspect of the present invention comprises compositions
for the treatment or prevention of alveolar destruction and/or the
promotion of alveolar formation in a mammal. Such compositions
comprise a ligand that is an RAR.beta. antagonist or inverse
agonist. In a preferred aspect of this embodiment, the RAR.beta.
receptor antagonist or inverse agonist has specific RAR modulating
activity at the RAR.beta. receptor, and is not specific to the
RAR.alpha. receptor. In another preferred aspect, the RAR.beta.
receptor antagonist or inverse agonist has specific RAR modulating
activity at the RAR.beta. receptor and is not specific to the
RAR.gamma. receptor. In a particularly preferred aspect of this
embodiment, the RAR.beta. receptor antagonist or inverse agonist
has specific RAR modulating activity at the RAR.beta. receptor and
is not specific to either the RAR.alpha. or the RAR.gamma.
receptor. Even more preferably, the ligand is specific to RAR.beta.
isotypes. Preferably such a ligand is a retinoid. Alveolar
destruction may be the result of a pathological condition such as
emphysema. Treatment to promote alveolar formation may be in
response to a condition such as bronchopulmonary dysplasia
(BPD).
[0030] Another aspect of the invention is methods for the treatment
of treatment or prevention of alveolar destruction and/or the
promotion of alveolar formation in a mammal, comprising
administering a therapeutic amount of a composition comprising a
retinoid receptor ligand that is an RAR.beta. antagonist or inverse
agonist. In a preferred aspect of this embodiment, the RAR.beta.
receptor antagonist or inverse agonist has specific RAR modulating
activity at the RAR.beta. receptor, and is not specific to the
RAR.alpha. receptor. In another preferred aspect, the RAR.beta.
receptor antagonist or inverse agonist has specific RAR modulating
activity at the RAR.beta. receptor and is not specific to the
RAR.gamma. receptor. In a particularly preferred aspect of this
embodiment, the RAR.beta. receptor antagonist or inverse agonist
has specific RAR modulating activity at the RAR.beta. receptor and
is not specific to either the RAR.alpha. or the RAR.gamma.
receptor. Even more preferably, the ligand is specific to RAR.beta.
isotypes. Preferably such a ligand is a retinoid.
[0031] The above-mentioned embodiments and aspects are clearly
useful for the treatment of medical conditions, such as emphysema
and BPD, which have been heretofore difficult or impossible to
treat without a lung transplant. As indicated above, use of the
RAR.beta. antagonist or inverse agonist in combination with another
agent with activity in treatment of alveolar deficiencies, such as
all-trans-retinoic acid, is useful as well. Such methods and
compositions may provide a synergistic therapeutic effect and/or
permit the use of lower effective doses of ATRA or another
therapeutic agent (and the RAR.beta. antagonist or reverse
agonist), thus reducing the prospect of undesired side effects
resulting from the use higher concentrations of either agent when
used alone.
[0032] The compositions of the present invention can be
administered in any therapeutically effective manner or form, and
in conjunction with any pharmacologically effective vehicle. For
example, in a particularly preferred aspect, the compositions of
the invention may be administered in the form of an inhalant as a
powdered or liquid aerosol. Such a formulation may comprise the
active agent solubilized in a micronized hydrophobic/hydrophilic
emulsion. Such compositions are well known to those of skill in the
art.
[0033] Alternatively, the compositions may be administered
systemically, such as intravenously by infusion, or by
intraperitoneal injection. For intravenous administration, the
necessary composition of pharmacologically effective infusion
solutions, such as the proper electrolyte balance and tonicity are
well known to those of skill in the art, and therefore formulation
of the compositions of the present invention with such solutions
would be well within the ability of such a person with the
disclosure of this application. Similarly, administration of drugs
by intraperitoneal injection is well known, and pharmacological
vehicles are well known.
[0034] Synthesis of candidate compounds having Specific RAR
modulating activity is well-known in the art. For example, among
other references, the synthesis of RAR ligands having antagonist
and/or inverse agonist activity is disclosed in commonly owned U.S.
Pat. Nos. 5,739,338; 5,728,846; 5,760,276; 5,877,207; the
disclosures of all of which is incorporated by reference herein.
Additionally, the construction of combinatorial libraries of
compounds suitable for screening as RAR-selective ligands is now
commonplace and well known to those of skill in the art.
[0035] Likewise, methods of screening candidate compounds for
Specific RAR modulating activity is routine and well-known in the
art. For example, U.S. Pat. No. 5,455,265, incorporated by
reference herein, describes a chimeric receptor transactivation
assay which tests for RAR-agonist activity in the RAR-.alpha.,
RAR-.beta., RAR-.gamma., RXR-.alpha. receptor subtypes. Briefly,
this assay employs chimeric proteins containing an RAR
ligand-binding domain and a heterologous polypeptide segment having
the ability to bind to a response element (RE), in turn
facilitating transcription of a specific, measurable target gene,
such as chloramphenicol acetyltransferase (CAT). Only an agonist of
the given RAR isotype whose ligand binding domain comprises part of
the chimeric protein will permit the activation of CAT
transcription and expression. These results can be compared with
those obtained using chimeric proteins having non-target ligand
binding moieties. For example, those ligands able to stimulate CAT
synthesis when used in combination with chimeric proteins having
RAR binding domains, but not those having RXR ligand binding
domains, will be RAR specific agonists.
[0036] Variation of this transactivation assay permits testing
ligands as RAR antagonists, or antagonists of a given target
isotype. A competitive assay involving the use of a stimulatory
concentration of a known agonist of a given receptor (for example,
ATRA is known to be an agonist of all RAR isotypes), and measuring
the reduction in a reporter gene expression (e.g., CAT expression)
as a function of the added candidate compound.
[0037] Additionally, straightforward receptor binding studies can
be performed as competition, rather than transactivation assays.
For example, and without limitation, membrane preparations
containing cloned retinoid receptors can be used, and the receptors
loaded with a known radiolabeled ligand. The release of
radioactivity from these preparations as a function of test
compound concentration can be determined. The lower the Kd for a
given ligand, the more likely the ligand will be effective as a
modulator of receptor activity. Further disclosure is available in,
e.g., U.S. Pat. No. 5,776,699, previously incorporated by reference
herein.
EXAMPLE 1
[0038] Involvement of RAR Receptors in Alveolus Formation
[0039] In most mammals new alveoli are formed postnatally; thus the
lung of many newborn mammals is immature, and not merely a smaller
version of the adult lung. For example, in humans alveolus
formation can continue up to the age of 20.
[0040] The study of alveolus development in newborn mammals, in
this case newborn rats, therefore provides an opportunity to study
the effect of various agents on alveolus formation. The rate of
increase in specific lung volume (expressed as cm.sup.3/100 g body
weight) in rats is greatest within the first 10 days following
birth, after which it increases at a less steep rate.
[0041] However, the lung's efficiency is determined not merely by
the volume of air that can be contained in the lung, but by the
alveolar surface area, which is a function of tissue growth within
the lung. Burri et al., Anat. Rec. 178:711-730 (1973) and Burri,
Anat. Rec. 180: 77-98 (1974) performed extensive studies of the
postnatal rat lung and demonstrated that three developmental phases
can be shown. In the first stage (day 1-4) there is a massive
expansion of the lung's volume. In the second stage, there is an
increase in lung tissue proliferation due to septation, including
alveolar and capillary surface areas. In the final stage (day 21 to
adult) there is a period of concomitant tissue redistribution,
lengthening of the septa and alveolar growth.
[0042] In this example, healthy 3-day old Sprague-Dawley rat pups
were injected intraperitonally with either cottonseed oil (carrier)
or with cottonseed oil containing 1 .mu.l/gram of a retinoid ligand
specific to RAR receptors and not to RXR receptors at a dosage of
500 micrograms per kg. This retinoid, termed AGN 193174, has
RAR.beta. agonist activity and the following structure: 1
[0043] The expression of the RAR.beta. receptor has been associated
in certain cases with diminished cell replication. Following the
initial injection, the rat pups were subjected to identical daily
injections until day 13. A set of rat pups were sacrificed at day 4
and at day 21 by anesthesia with phenobarbitol sodium and scission
of the abdominal aorta. Following sacrifice, rat lungs were fixed
and histological sections prepared essentially as described in
Massaro et al., Am. J. Physiol. 270: L305 (1996), incorporated by
reference herein. The histological sections were viewed and
photographs taken under light microscopy.
[0044] The histological sections, replicated in FIGS. 1A and 1B,
show that at age 14 days alveoli were larger, and the degree of
sepatation less, in rats that had been treated with a RAR.beta.
agonist daily from age 3 through 13 days (the sepatation stage of
lung development), than in rats treated with cottonseed oil
alone.
[0045] The differences in alveolar dimensions between treated and
untreated rat pups were so pronounced, they were quantitated by
measuring the distance between alveolar walls (Lm) and the
surface-to-volume ratio (S/V) of the gas-exchange structures
(septa). The Lm in cottonseed oil-treated rats was 80 .mu.m.+-.2.8
.mu.m (N=4) compared to 110 .mu.m.+-.7.3 .mu.m (N=3) in RAR.beta.
agonist-treated rats (N=3; P=0.03 by Mann Whitney nonparametric
analysis). The S/V was 504 .mu.m.sup.-1.+-.18 .mu.m.sup.-1 (N=4) in
cottonseed oil-treated rats compared to 365 .mu.m.sup.-1.+-.23
.mu.m.sup.-1 (N=3) in RAR.beta. agonist-treated rats (P=0.03). Lung
volume was the same in both rat groups but surface area was larger
(634 cm.sup.2 .+-.9 cm.sup.2) in cottonseed oil-treated rats than
in RAR.beta. agonist-treated rats (492 cm.sup.2 .+-.28cm.sup.2,
P=0.03).
[0046] These data demonstrate that treatment of immature rats with
a RAR.beta. agonist inhibits septation of the saccules of the
gas-exchange region, resulting in larger but fewer alveoli and a
lower surface to volume ratio, and clearly suggest that agonist
(positive) stimulation of the RAR.beta. receptor results in the
down-regulation of naturally occurring, spontaneous alveolus
formation. A clear implication of this finding is the inverse is
also true: that antagonist (neutralizing) or inverse agonist
(negative) activity at the RAR.beta. receptor is required for
adequate sepation to occur. Thus, therapeutic treatment of mammals
with an RAR antagonist or inverse agonist will promote the growth
of septa.
[0047] It is quite surprising, in light of the previous finding
that ATRA (an RAR-specific agonist of RAR.beta., RAR.alpha., and
RAR.gamma.) stimulates septation, that a RAR.beta. agonist is able
to actually repress septation, and that therefore repression of
signal transduction at RAR.beta. through the action of an RAR.beta.
antagonist will promote septation.
EXAMPLE 2
[0048] Studies Mice lacking RAR.beta. Receptors
[0049] This example employed a strain of mice that were uniformly
lacking a functional gene for the RAR.beta. receptor (RAR.beta.
knock-out or k.o. mice). As controls a founder strain of mice,
identical to the RAR.beta. k.o. mice but for the genetic lesion
causing the lack of RAR.beta., were also examined. Animals from
each group were sacrificed at 4 days and at 21 days, and
histological sections of the lungs of animals from each group
prepared and inspected as in Example 1. Photomicrographs are shown
in FIG. 2A-2D and additional results are shown below in Table
1.
1TABLE 1 n Sa Age va .times. 10.sup.-4 Mice cm.sup.2 Days
.mu.m.sup.3 Na .times. 10.sup.-6 Na/kg .times. 10.sup.-6 Wild-type
3 4 17.6 .+-. 0.9 0.50 .+-. 0.03 193 .+-. 4.06 62 .+-. 2.7
RAR.beta. k.o. 3 4 8.9 .+-. 0.6 0.95 .+-. 0.09 362 .+-. 16.0 65
.+-. 2.9 P <0.05 <0.05 <0.05 NS Wild-type 4 21 3.82 .+-.
0.15 5.35 .+-. 0.49 586 .+-. 38.3 215 .+-. 11.4 RAR.beta. k.o. 4 21
2.38 .+-. 0.10 9.74 .+-. 0.05 918 .+-. 53.0 242 .+-. 9.6 P 0.02
0.02 0.02 NS
[0050] In Table 1, n is the number of animals sampled for each
measurement, va is the mean volume of an individual alveolus, Na is
the number of alveoli, and Sa is the alveolar surface area. Lung
volume was measured by water displacement, va was measured by the
point- sample intercepts method, and Sa was measured by point and
intersection counting. Na was calculated.
[0051] The data show that lung volume did not differ between
RAR.beta. k.o. and wild-type mice at either age, but at both ages
alveoli were smaller and more numerous in RAR.beta. k.o. mice than
in wild-type mice. These intergroup differences in alveolar
dimensions were easily apparent in histological sections of lungs
of 4-day old mice (FIG. 2A and 2B) but less apparent in sections of
lungs of 21-day old mice (FIG. 2C and 2D). Alveolar surface area
was not different between groups at 4 days, and slightly greater in
the RAR.beta. k.o. mice at day 14.
[0052] These data therefore provide strong evidence that alveolus
formation is promoted in vivo by inhibition of the RAR.beta.
receptor. Negative regulation, as opposed to mere inhibition, of
the same receptor may also be involved in alveolus formation.
[0053] The data also indicate that the increase in lung air volume
seen in the first few days is an event independent from the
presence or absence of RAR receptors or the presence or absence of
an RAR modulating ligand, as shown in FIGS. 2A-2D. The septation
stage of lung development can therefore be uncoupled from the
increase in lung volume seen immediately post birth. Thus,
modulating of RAR receptors can be used to specifically affect
septation without modulating other events in alveolar
formation.
[0054] Thus, signal transduction through RAR receptors appears to
be an endogenous modulator of alveolus formation. The generation of
RAR.beta. antagonists will permit the pharmacological blockade of
RAR.beta. signaling, with resulting will induce alveolus formation.
In addition, treatment with a RAR.beta. antagonist or inverse
agonist plus all-trans retinoic acid may well augment the effect of
either agent alone, allowing the use of lower doses of these
drugs.
[0055] It will be recognized that the foregoing examples and
preferred embodiments are exemplary only, and that the invention is
defined soely by the claims that conclude this specification.
* * * * *