U.S. patent application number 11/418806 was filed with the patent office on 2006-11-16 for method for improving respiratory function and inhibiting muscular degeneration.
Invention is credited to Yang D. Teng.
Application Number | 20060258667 11/418806 |
Document ID | / |
Family ID | 22696535 |
Filed Date | 2006-11-16 |
United States Patent
Application |
20060258667 |
Kind Code |
A1 |
Teng; Yang D. |
November 16, 2006 |
Method for improving respiratory function and inhibiting muscular
degeneration
Abstract
The present invention provides a method for improving
respiratory function and inhibiting muscular degeneration (e.g.,
dystrophy and atrophy). Alternative embodiments of the invention
provide a method of inhibiting motor neuron apoptosis and the
subsequent muscular degeneration associated with the denervation of
muscular tissue resulting from neuron death.
Inventors: |
Teng; Yang D.; (Wellesley,
MA) |
Correspondence
Address: |
HAMILTON, BROOK, SMITH & REYNOLDS, P.C.
530 VIRGINIA ROAD
P.O. BOX 9133
CONCORD
MA
01742-9133
US
|
Family ID: |
22696535 |
Appl. No.: |
11/418806 |
Filed: |
May 5, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10244087 |
Sep 13, 2002 |
7071194 |
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11418806 |
May 5, 2006 |
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PCT/US01/40291 |
Mar 14, 2001 |
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10244087 |
Sep 13, 2002 |
|
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60189241 |
Mar 14, 2000 |
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Current U.S.
Class: |
514/252.15 ;
514/649 |
Current CPC
Class: |
A61K 31/506 20130101;
A61K 2300/00 20130101; A61K 2300/00 20130101; A61K 45/06 20130101;
A61K 31/137 20130101; A61K 31/137 20130101; A61K 31/506
20130101 |
Class at
Publication: |
514/252.15 ;
514/649 |
International
Class: |
A61K 31/506 20060101
A61K031/506; A61K 31/137 20060101 A61K031/137 |
Goverment Interests
GOVERNMENT SUPPORT
[0002] The invention was supported, in whole or in part, by grants
PO1-NS-28130 and RO1-NS-35647 from the National Institute for
Health. The Government has certain rights in the invention.
Claims
1. A method of improving respiratory function in an individual with
abnormal respiratory function comprising administering an agent
selected from the group consisting of: a serotonin (5HT) receptor
type 1A agonist and a .beta.2-adrenergic agonist.
2. The method of claim 1 wherein the individual with abnormal
respiratroy function is afflicted with ALS.
3. The method of claim 1 wherein the individual with abnormal
respiratory function suffers from a spinal cord injury.
4. The method of claim 1 wherein the serotonin (5HT) receptor type
1A agonist is selected from the group consisting of 8-OH-DPAT and
buspirone.
5. The method of claim 1 wherein the .beta.2-adrenergic agonist is
selected from the group consisting of clenbuterol and
salbutanol.
6. A method of inhibiting motor neuron apoptosis in an individual
comprising administering a .beta.2-adrenergic agonist selected from
the group consisting of clenbuterol and salbutanol.
7. The method of claim 6 wherein the individual in whom motor
neuron apoptosis is inhibited has a disorder selected from the
group consisting of: ALS and spinal cord injury.
8. A method of inhibiting muscular degeneration in an individual
comprising administering an agent selected from the group
consisting of: a serotonin (5HT) receptor type 1A agonist and a
.beta.2-adrenergic agonist.
9. The method of claim 8 wherein the individual is afflicted with
ALS.
10. The method of claim 8 wherein the individual suffers from a
spinal cord injury.
11. The method of claim 8 wherein the serotonin (5HT) receptor type
1A agonist is selected from the group consisting of 8-OH-DPAT and
buspirone.
12. The method of claim 8 wherein the .beta.2-adrenergic agonist is
selectd from the group consisting of clenbuterol and
salbutanol.
13. A method of inhibiting denervation of muscle in an individual
afflicted with ALS comprising administering a .beta.2-adrenergic
agonist selected from the group consisting of clenbuterol and
salbutanol.
14. A method of inhibiting denervation of muscle in an individuals
suffering from a spinal cord injury comprising administering a
.beta.2-adrenergic agonist selected from the group consisting of
clenbuterol and salbutanol.
15. A method of preventing respiratory abnormalities in an
individual afflicted with ALS comprising administering a serotonin
(5HT) receptor type 1A agonist in combination with a
.beta.2-adrenergic agonist.
16. A method of preventing muscular degeneration in an individual
afflicted with ALS comprising administering a serotonin (5HT)
receptor type 1A agonist in combination with a .beta.2-adrenergic
agonist.
Description
RELATED APPLICATIONS
[0001] This application is a continuation of U.S. application Ser.
No. 10/244,087, filed Sep. 13, 2002, which is a continuation of PCT
Application Serial No. PCT/US01/40291, filed Mar. 14, 2001, which
claims the benefit of U.S. Provisional Application Ser. No.
60/189,241, filed Mar. 14, 2000, the entire teachings of which are
incorporated herein by reference.
BACKGROUND OF THE INVENTION
[0003] It is well known that pulmonary pathophysiology is one of
the most significant factors associated with the morbidity and
mortality of individuals afflicted with either acute or chronic
spinal cord injuries (SCI). Furthermore, abnormalities of
respiratory function and failure of the respiratory system are
leading causes of mortality in the late stages of amylotrophic
lateral sclerosis (ALS).
[0004] ALS (also known as Lou Gehrig's disease) is a progressive
disease of the nervous system. ALS specifically and progressively
damages motor neurons, and the resulting denervation of muscular
tissue in turn mediates muscular degeneration (e.g., dystrophy and
atrophy). More specifically, muscular degeneration results from
neuronal death, which occurs primarily by apoptosis, and the
resulting denervation of muscles that normally receive axons from
the affected motor neurons. The progressive muscular degeneration
results in deficits in somatomotor function and speech and
eventually is manifest as respiratory failure. Hence, it is crucial
to identify effective therapies to prevent motor death and muscular
degeneration. To date, SCI- and ALS-induced respiratory
abnormalities have been neither successfully managed or treated due
to a lack of effective therapeutic agents.
SUMMARY OF THE INVENTION
[0005] The present invention provides a method for improving
respiratory function and inhibiting muscular degeneration (e.g.,
dystrophy and atrophy). Alternative embodiments of the invention
provide a method of inhibiting motor neuron apoptosis and the
subsequent muscular degeneration associated with the denervation of
muscular tissue resulting from neuron death. The methods provided
herein can be used to improve the respiratory function of
individuals who have abnormal respiratory function either as a
consequence of a spinal cord injury or amyotrophic lateral
sclerosis.
[0006] One embodiment of the invention provides a method of
improving respiratory function in an individual with abnormal
respiratory function comprising administering either a serotonin
(5HT) receptor type 1A agonist or a Beta2-adrenergic agonist. In
particular embodiments the serotonin (5HT) receptor type 1A agonist
is 8-OH-DPAT. In an alternative embodiment the serotonin (5HT)
receptor type 1A agonist is buspirone. In a second alternative
embodiment the invention provides a method of improving respiratory
function by administering a Beta2-adrenergic agonist selected from
the group consisting of clenbuterol and salbutamol. An alternative
embodiment of the instant invention provides a method of preventing
respiratory abnormalities in an individual afflicted with ALS
comprising administering a serotonin (5HT) receptor type 1A agonist
in combination with a .beta.-2 adrenergic agonist.
[0007] The invention also provides a method of inhibiting motor
neuron apoptosis in an individual comprising administering a
Beta2-adrenergic agonist selected from the group consisting of
clenbuterol and salbutamol.
[0008] The invention further provides a method of inhibiting
muscular degeneration in an individual comprising administering an
agent selected from the group consisting of a serotonin (5HT)
receptor type 1A agonist (e.g., 8-OH-DPAT or buspirone) or a
Beta2-adrenergic agonist (e.g., clenbuterol and salbutamol). An
alternative embodiment of this aspect of the invention provides a
method of preventing muscular degeneration in an individual
afflicted with ALS comprising administering a serotonin (5HT)
receptor type 1A agonist in combination with a .beta.-2 adrenergic
agonist.
[0009] The invention also further provides a method of inhibiting
denervation of muscles in an individual who is either afflicted
with ALS or suffering from a spinal cord injury comprising
administering either a serotonin (5HT) receptor type 1A agonist or
a Beta2-adrenergic agonist.
BRIEF DESCRIPTION OF THE DRAWINGS
[0010] FIG. 1. Non-invasive measurements of respiratory function in
conscious rats a. Schematic presentation of the restrained
head-out-plethysmograph system for rodents. b. The animals breathe
from a funnel fixed in the front wall of a box made of an opaque
material. The box surrounds the front-two thirds of the cylinder of
the plethysmograph, and the rear outlet of the box is covered with
a piece of bath towel (illustrated by a dash line). The animals are
exposed to the room air for baseline recordings then to an air
mixture containing 7% CO.sub.2 (mixed with 60% O.sub.2 and 33%
N.sub.2) for 5 minutes and recording of respiratory activity is
continued for another 2 minutes (a total recording duration of 7
minutes). After a new baseline is obtained by allowing the animals
to breathe room air for 20 minutes, the rats are allowed for other
procedures and recordings determined by each experiment
specifically (see Method for details).
[0011] FIG. 2. Effects of incomplete contusive SCI at T8 on
respiratory function at 24 hours post injury. a. Plethysomograph
tracings of respiratory flow rate (unit: ml/sec) obtained from
conscious rats breathing room air 24 hours prior to spinal cord
injury and 24 hours after injury; b. Plethysmograph tracings of
respiratory flow rate (unit: ml/sec) obtained from conscious rates
breathing air containing 7% CO.sub.2 24 hours piro to spinal cord
injury and 24 hours after injury.
[0012] FIG. 3. Time-dependent effect of 8-OH-DPAT in minute
ventilation in T8 rats at 24 hours post injury. Curves represent
the average minute ventilation (Ve) for rats before SCI and after
SCI (10 g.times.2.5 cm weight drop), and then prior to S--OH-DPAT
administration and after the drug injection (250 .mu.g/kg in 0.5
ml/rat) at 24 hours post injury (p.i.; n=3). SCI resulted in a
significant drop in baseline Ve (i.e. breathing room air), and the
injury also significantly diminished Ve response to 7% CO2
challenge. 8-OH-DPAT treatment significantly improved baseline Ve
at 4 minutes post drug injection (*P<0.05 compared to pre-injury
baseline VE; #P<0.05 compared to pre-injury Ve under 7% CO.sub.2
challenge; repeated measures ANOVA followed by Tukey's procedure).
SCI rats (n=3) showed a time-dependent decline of their minute
ventilation (Ve) in response to breathing 7% CO.sub.2
(.tangle-solidup.), or under room air breathing (.diamond.)
subsequent to a single dose of 8-OH-DPAT ( P<0.05 compared to Ve
under 7% CO.sub.2 challenge at 20 minutes after the administration
of 8-OH-DPAT; one way ANOVA followed by Tukey's procedure). Note:
the decline speed is much greater in the baseline conditions (i.e.
breathing room air) than that under CO.sub.2 challenge.
[0013] FIG. 4. Effect of buspirone treatment on respiratory
function under baseline conditions or challenged by air mixtures of
7% CO.sub.2 at 24 hours after SCI. Plethysmograph tracings of
respiratory flow rate (unit: ml/sec) obtained from conscious rats
breathing room air or an air mixture containing 7% CO.sub.2 24
hours prior to spinal cord injury and 24 hours p.i. Also given is
respiratory flow rate tracing recorded before buspirone injection
and after its administration when the animal was breathing room air
or challenged by an air mixture containing 7% CO.sub.2.
[0014] FIG. 5. Antagonistic effect of p-MPPI on 8-OH-DPAT-induced
respiratory improvement unconscious rats at 24 hours after SCI.
Plethysmograph tracings of respiratory flow rate (unit: ml/sec)
obtained from conscious rats breathing room air or an air mixture
containing 7% CO.sub.2 24 hours prior to spinal cord injury and 24
hours p.i. Also given is respiratory flow rate tracing recorded
before buspirone injection and after drug administration.
[0015] FIG. 6. A schematic representation of the effects of the
intraperitoneal administration of buspirone at a dose of 3.0 mg/kg
on the respiratory function (e.g., tidal volume and respiratory
rate) of SOD1 mice.
[0016] FIG. 7. A schematic representation of the effects of the
subcutaneous administration of buspirone at a dose of 3.0 mg/kg on
the respiratory function (e.g., tidal volume and respiratory rate)
of SOD1 mice.
DETAILED DESCRIPTION OF THE INVENTION
[0017] Because of damages to the long axons of bulbospinal premotor
neurons, high level injuries between lower brainstem and C4 result
in pentaplegia that required immediate respirator support (Prakash,
1989). In contrast, patients with SCI between T1 and S1 have been
observed for losing control of intercostal and abdominal muscles,
leading to a diminished ability to generate inspiratory and
expiratory movements. These patients could experience an alarming
sense of difficulty breathing (dyspnea) (Prakash, 1989). Unlike
high level injuries, SCI between T1 and S1 may spare some of the
axonal connection between bulbospinal premotor neurons to phrenic
nucleus (i.e. somatomotor neurons at C3 to C5, Feldman and
McCrimmon, 1999). Hence, we believe that breathing dysfunction of
such patients would be better managed with drugs that stimulate
respiration. This rationale is based on that drug treatment can be
easily executed in a timing manner, and it can prevent
complications that frequently occur in the process of ventilator
support (Mansel and Norman, 1990). However, historically
respiratory disorders caused by lower thoracic SCI were much less
studied in experimental models, and therefore such treatments are
still not available. Recently, using a clinically relevant animal
model of SCI, we reported that incomplete contusion of SCI at T8
produced significant respiratory abnormalities (Teng et al., 1998a
and and 1999). The deficits consist of an abnormally lower tidal
volume (Vt) and higher respiratory rate (f) in conscious rats at 24
hours and 7 days post injury (p.i.) relative to values observed
prior to SCI. Moreover, T8 SCI diminished ventilatory response to
the respiratory stimulating effect of 7% CO.sub.2 (Teng et al.,
1999). The abnormal repriatroy pattern in SCI rats is conforming to
that found in patients with lower thoracic SCI (Prakash, 1989). We
consequently decided to seek drug therapies for respiratory
malfunctions in conscious SCI rats. We hypothesized that
8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT) and buspirone,
agonists of 5HT.sub.1A receptors may improve respiratory function
after contusion SCI in conscious rats, because our previous
discovery showed these drugs counteracted respiratory dysfunction
induced by morphine overdose (Ferreira et al., 1998).
[0018] To test this hypothesis, we used a head-out plethysmograph
system (Dorato et al., 1983; Teng et al., 1999) to examine the
effects of SCI at T8 on respiration in conscious rats, and the
effect of treatment with 8-OH-DPAT or buspirone on respiratory
deficits. For examining the specificity of the beneficiary effect
of 8-OH-DPAT on respiratory function after SCI, we also conducted a
time-course study for 8-OH-DPAT. In addition, the specific
antagonism of 8-OH-DPAT effects was evaluated by pre-treating SCI
rats with
4-(2'-methoxyphenyl)-1-[2'-[N-(2''-pyridinyl)-p-iodobenzamido]ethyl]piper-
azine (p-MPPI), an in vivo competive antagonist of 5HT.sub.1A
receptors, to determine if the effects of 8-OH-DPAT could be
prevented.
[0019] In this study, we have fulfilled our task to examine whether
5HT.sub.1A receptor agonistic drugs, 8-OH-DPAT and buspirone
(Middlemiss and Fozard, 1983; Hamon et al., 1986; Hoyer and
Schoeffter, 1991) could reverse respiratory malfunctions resulting
from traumatic SCI. We first demonstrate that incomplete contusion
injury at T8 causes significant abnormalities of respiration, which
is consistent with previous findings from others and ourselves
(Baker et al., 1979; Teng et al., 1998a and 1999). Respiratory
disorders that are discerned in conscious rats at 24 hours and 1
week after SCI include rapid and shallow pattern of repiration
under baseline conditions (i.e. room air ventilation), and a
dramatically diminished ventilatory response to breathing CO.sub.2
that is abnormally concentrated (i.e. 7% vs the physiological
0.4%). A single treatment of 8-OH-DPAT administered i.p. at 24
hours after injury counteracts SCI-induced respiratory
abnormalities in a time-related manner. Identical results are also
achieved by 8-OH-DPAT treatment at 7 days p.i. At 24 hours p.i.,
pre-injury levels of respiratory parameters are also reinstated by
treatment of buspirone, a partial 5HT.sub.1A receptor agonist
(Hoyer and Schoeffter, 1991). In addition, pre-treatment of the SCI
rats wit p-MPPI, a specific antagonist of 5HT.sub.1A receptors
(Thielen et al., 1990) prevents 8-OH-DPAT from counteracting
SCI-induced respiratory abnormalities. The results suggest that the
beneficial effect of 8-OH-DPAT and buspirone in treating
SCI-triggered respiratory deficits is mediated through 5HT.sub.1A
receptors.
[0020] This is the first report that systematic administration of
5HT.sub.1A agonistic drugs promptly and efficiently restores
respiratory abnormalities resulting from contusion SCI to normal.
Nevertheless, previous reports from other groups show that
8-OH-DPAT and buspirone are capable to reverse respiratory
depression such as an apneustic respiration (an abnormal breathing
pattern characterized by an over-elongated inspiration phase). The
apneusis was induced by antagonist (i.e. MK-801 and Ketamine) of
the NMDA-subtype receptors of the excitatory amino acids (Lalley et
al., 1994; Wilken et al., 1997), pentobarbital (Lalley et al.,
1994), or hypoxia (Lalley et al., 1994). Our group has also shown
that 8-OH-DPAT and buspirone administered i.v. counteract
respiratory depression (mostly, apnea) triggered by morphine
overdose (Ferreira et al., 1998). With detailed data generated
exclusively from anesthetized animals, those studies have
illuminated the possibility to use 5HT.sub.1A agonistic drugs to
treat respiratory depression in conscious animals.
[0021] SCI in general severely damages respiratory function
(Prakash, 1989). Moreover, most of the morbidity and mortality at
both acute and chronic stages after human SCI is due to respiratory
dysfunction (Slaok and Shuoart, 1994; Frankel et al., 1998). Thus,
therapeutic strategies are urgently needed to improve respiration
after SCI. However, historically there has been very limited
experimental information on SCI-resulted respiratory malfunction,
still less investigations for potential drug treatments to
reinstate proper respiration. A main reason for this reality is
that little has been done to examine effects of SCI on respiration
in clinically relevant animal models. Recently, by using a
plethysmograph, wer were able to evaluate respiration in conscious
rats before and repeatedly after a standardized SCI (Teng et al.,
1998a and 1999). Furthermore, in the current study, we successfully
demonstrate that both 8-OH-DPAT and buspirone can restore
SCI-induced respiratory abnormalities to normal. Our finding is
consistent with an earlier report: buspirone taken orally (8
mg/day) reverses apneusis in a child after a surgery to remove a
brainstem tumor (Wilken et al., 1997).
[0022] The rapid and shallow respiratory pattern that was found in
rats after SCI was associated with the loss of ventral motor
neurons at and near the T8 injury site (Teng et al., 1998 b and
1999). Ventral motoneurons at thoracic levels innervate both the
intercostal (motneurons at T.sub.1-T.sub.13) and abdominal muscles
(motoneurons at T.sub.5-L.sub.3; Holstege, 1991). The intercostal
muscles have an important respiratory function and their paralysis
causes significant alteration in the elastic properties of the
lungs and reduces the outward elastic recoil of the rib cage
(Gibson et al., 1977; Troyer and Heilporn, 1980). Therefore,
patients with quadriplegia caused by SCI below C5 with detectable
intercostal electromyographic activity had much better respiratory
function than those who lost it (Troyer and Heilporn, 1980).
Consequently, respiratory impairments were expected for T8 SCI due
to loss of thoracic motoneurons, as well as the loss of white
matter containing supraspinal control pathways to respiratory
motoneurons below the injury site. Indeed, we have demonstrated
that basic fibroblast growht factor (FGF2), a neurotrophic factor
administered into the injury epicenter at 5 minutes p.i. prevents
respiratory abnormalities from happening through reducing neuronal
losses (Ten et al., 1999).
[0023] The mechanism(s) and site(s) whereby 5HT.sub.1A agonistic
drugs act to improve respiration following SCI remain to be further
investigated. However, the beneficial effects of 8-OH-DPAT and
buspirone on p.i. respiration that we observed can not be owned to
possible neuronal protection for two reasons. First, by 24 hours
and 7 days p.i., major neuronal loss at or near injury epicenter
already completed in the spinal cord (Noble and Wrathall, 1985 and
1989; Crowe et al., 1997; Teng et al., 1998b). Correspondingly,
effects of 8-OH-DPAT in the current study display as a
time-dependent phenomenon (FIG. 3), which is contrasted with that
of FGF2 (Teng et al., 1999). Secondly, there are no evidences that
5HT.sub.1A agonistic drugs spare neurons or neural tissues after
traumatic injury. Hence, another possibility for these drugs to
work is to act on surviving spinal motoneurons. Yet published data
concerning whether 5HT.sub.1A agonists postsynaptically stimulate
spinal somatomotor neurons do not support this notion. Jackson and
White (1990) reported that iontophoretically delivered 8-OH-DPAT
into the ventral horn inhibited the glutamate-evoked firing of
motoneurons while similarly applied agonists for 5HT.sub.1B,
5HT.sub.1C and 5HT.sub.2 augmented it. In a SCI-related study,
increased serotonergic inervation of phrenic motoneurons (located
at C3 to C5 of the cervical spinal cord) is identified to be
accountable for long-term facilitation of respiratory motor output
triggered by episodic hypoxia at 28 days after cervical dorsal
rhizotomy (Kinkead et al., 1998). Again, the effect is mediated by
5HT.sub.2 receptors in this case since it is blocked by
pretreatment of ketanserin, a 5HT.sub.2 antagonist (Bach and
Mitchell, 1996; Kinkead et al., 1998). Thus, it is conceivable that
in our study 8-OH-DPAT and buspirone are not acting on spinal
motoneurons. In fact, sites of action for the respiratory effect of
5HT.sub.1A agonists were confined within the brainstem to certain
degree in literatures describing their cardiovascular effects and
their counteraction mechanisms on apneusis. Work by Fozard et al.,
(1987) show that in conscious, spontaneous hypertension rats,
8-OH-DPAT causes dose-related falls in blood pressure and heart
rate. The same effects are not observed in pithed rats. Moreover,
the response to 8-OH-DPAT is blocked by intracisternal injection of
8-MeO-CIEPAT, an irreversible 5HT.sub.1A receptor antagonist
(Fozard et al., 1987). Another report reveals that application of
5-methoxy-N,N-dimethyltrptamine, a 5HT.sub.1A agonist to the dorsal
surface of the medulla oblongata reverses apneusis produced by
pentobarbital (Lalley, 1994). Under this circumstance, the most
feasible target for the drug is neurons in the dorsal respiratory
group of the medulla. In our earlier study of 5HT.sub.1A agonist
reversal of morphine-induced apnea (Ferreira et al., 1998), we
believe that 5HT.sub.1A agonist drugs may act directly on brainstem
respiratory rhythm generating centers: the Pre-Botzinger Complex to
re-start respiration which was suppressed by morphine (Smith et
al., 1991). Indeed, it has been reported that direct application of
5HT to the medulla area embodying Pre-Botzinger Complex increments
the frequency of burst discharge of those neurons (Al-Zubaidy et
al., 1996; Onimaru et al., 1998). Further, our present data
indicate that 5HT.sub.1A agonists improve respiration by directly
stimulating respiratory neurons and not by other mechanisms such as
augmenting sensitivity to CO.sub.2. Table 2 exhibits that 8-OH-DPAT
treatment does not affect ventilatory response to 7% CO.sub.2 in
normal rats. Thus, despite we did not directly investigate the
mechanism(s) and location(s) for 5HT.sub.1A agonists to improve
SCI-triggered respiratory dysfunction, our data from 8-OH-DPAT
time-course and p-MPPI antagonism experiments clearly suggest that
the effect is mediated through 5HT.sub.1A receptors. Furthermore,
existing evidences indicate that the most likely site(s) for
8-OH-DPAT buspirone to restore p.i. respiratory function to normal
is respiratory neurons in the brainstem. Thereafter, we postulate
that by stimulating the brainstem respiratory premotoneurons both
pre- and post-synoptically, 5HT.sub.1A agnistic drugs can increase
the activity of phrenic motoneurons which are essentially undamaged
by T8 injury. Subsequently, the enchanced diaphragm contraction can
compensate activity loss of intercostal muscles resulting from
neuronal death in the thoracic spinal cord. We're currently testing
this hypothesis by comparing pre-8-OH-DPAT phrenic nerve outflow to
that recorded after drug administration in anesthetized rats at
different time points p.i.
[0024] Our data have provided the first description of the reversal
of SCI-induced respiratory abnormalities in conscious rats by
systemic administration of 5HT.sub.1A receptor agonistic drugs. The
study also indicates that the respiratory improving effect is
generated through specific interaction between these drugs and
5HT.sub.1A receptors. Morever, we have shown that buspirone, a
human clinical drug is highly effective for restoring respiratory
function to normal after contusion SCI. In conclusion, our results
demonstrate that 5HT.sub.1A receptor agonistic drugs can be used to
improve respiratory function in conscious rats subsequent to
SCI.
[0025] The data presented herein demonstrate that specific agonists
of serotonin (5HT) receptor type 1A such as 8-OH-DPAT and buspirone
counteract (e.g., ameliorate) abnormalities mediated by motoneuron
loss in spinal cord injury (Teng et al., 1999; 2000). It is well
known that the same category drugs can ameliorate respiratory
suppression that is caused by morphine overdose, glutamate
antagonists, and sleep disorders (Ferreira et al., 1998; Rappaport
et al., 1988). Considering the fact that ALS-related respiratory
abnormalities are also triggered by muscle dysfunction due to
denervation, we determined whether buspirone could mitigate
respiratory deficits in SOD1 mice (a mutant mouse-model for
familial ALS, an inherited form of ALS), (Example 6). The data
summarized in FIGS. 6 and 7 demonstrates that buspirone treatment
significantly increases tidal volume and simultaneously reduces
respiratory frequency. These results clearly demonstrate that 5HT
1A agonists can be used to improve respiratory abnormalities that
are resulted from ALS pathophysiology.
[0026] While not wishing to be bound by theory, it is believed that
5HT 1A agonists improve respiration through their proper
stimulation of respiratory premotoneurons (i.e. neurons in brain
stem). Since these drugs are currently used clinically for
anxiolytic purpose we think that they can be applied to ameliorate
late stage ALS patients' respiratory stress (e.g. ALS patients at
late stage often complain for lack of air) as well as their emotion
imbalance. Further, we hypothesize that proper stimulation of
motoneurons (i.e. neurons in brain stem and spinal cord) may delay
their degeneration caused by ALS pathology and pathophysiology.
This opportunity will be further enhanced by co-application of
.beta.2-adrenergic agonists because the later can preserve muscle
function and increase expression of neural trophic factors. Hence,
it is very likely that synergistic or additive effects may be
observed between 5HT 1A agonists and .beta.2-adrenergic agonists in
mitigating ALS symptoms and retarding development of ALS pathology.
Together, they can form a new strategy to treat ALS, improve life
quality, and elongate life span for ALS patients.
[0027] .beta.2-Adrenergic agonists are highly potential
counteracting drugs to for the muscle fiber degeneration, since
these compounds have been demonstrated to increase mussel strength
by either induce muscle hypertrophy (Bardsley et al., 1992; Emery
et al., 1984; Rothwell and Stock, 1985) or retarding denervation
atrophy (Maltin et al., 1987; Zeman et al., 1987). In terms of
general pharmacology, clenbuterol is the most widely studied agent
among the .beta.2-adrenergic agonists. Clenbuterol is an extemely
potent and selective .beta.2-adrenergic agonist with a long
duration of action and has been shown to increase muscle mass in
innervated (Agbenyega et al., 1990; Emery et al., 1984; maltin et
al., 1987), denervated (Maltin et al., 1986, Zeman et al., 1988),
and dystrophic muscles (Rothwell and Stock, 1985; Zeman et al.,
1994). Long-term studies show that clenbuterol given orally 1.0-1.5
mg/kg body weight/day significantly increase soleus (SOL) weight
and SOL muscle weight to body weight ratio in both normal control
mice and muscular dystrophic (i.e. mdx mutant mice) mice during a
one year treatment period. In addition, there is a 22% increase in
myosin concentration of mdx diaphram (DIA), correlating well with
enhanced normalized active tension in mdx DIA. Another long-term
study reveals that clenbuterol increases absolute and relative
muscle masses in mdx mice. The larger SOL muscle also produces
larger absolute forces. Twitch contraction time is significantly
faster following clenbuterol administration, supported by
fiber-type transitions toward fast-twitch fibers. On the other
hand, in chronically spinal cord injured rats, enervation-caused
muscle atrophy is also reserved by oral administration of
clenbuterol (Khan et al., 1999; Zeman et al., 1999). Furthermore,
clenbuterol is demonstrated to be neuroprotective (i.e. reducing
the cortical infarct volume in Long-Evans rats as measured 7 days
after permanent occlusion of the middle cerebral artery) by
increasing mRNA expression for neural trophic factors such as nerve
growth factor (NGF), basic fibroblast growth factor (bFGF), and
transforming growth factor-beta 1 (TGH-beta1) in cortical and
hippocampal tissue (i.e. neurons and glial cells; Culmsee et al.,
199a and b). This kind of neural growth factor up-regulation is
thought as the mechanism for the inhibition effects of clenbuterol
on neuronal apoptsis.
[0028] Although what causes neuronal death in ALS is still not
completely understood, we do know that most neurons die from
apoptosis in ALS. We also know that neural growth factors such as
NGF and bFGF can inhibit neuronal apoptosis both in vitro and in
vivo. Thus, it is reasonable to hypothesize that .beta.2-adrenergic
agonists such as clenbuterol can be used to prevent or reduce
neuronal loss of ALS. In addition, the anti-muscle degeneration
effects of clenbuterol, especially its robust effect on
enervation-related muscular dystrophy may significantly minimize
the impacts fo the direct killer of ALS: loss of muscular function.
As clenbuterol is related to a number of compounds that are
currently used to treat asthmatics, its lont-term use may not be
associated with a long list of side effects. Indeed both
clenbuterol (Maltin et al., 1993) and another .beta.2-adrenergic
agonist, salbutamol (Martineau et al., 1992), have recently been
used in investigations on human subjects for their effects on
muscles. Therefore, clenbuterol and other .beta.2-adrenergic
agonists may become successful therapeutic agents for ALS patients
who currently do not have any effective treatments.
[0029] The following Examples are offered for the purpose of
illustrating the present invention and are not to be construed to
limit the scope of the invention.
Methods and Materials Used in Examples
[0030] Spinal cord injury. Female Sprague-Dawley rats (250-280 g
and 360-390 g; Taconic, Germantown, N.Y.) were anesthetized with 4%
chloral hydrate (360 mg/kg, i.p.). An incomplete spinal courd
contusion injury was produced at T8 with a weight drop device (10
g.times.2.5 cm) as previously described (Wrathall et al., 1985).
After SCI, manual expression of bladders was performed twice daily
until a reflex bladder was established. Animal care also included
housing the rats in pairs to reduce isolation-induced stress,
maintaining ambient temperature at 22-25.degree. C., and using
highly absorbent bedding. No prophylactic antibiotics were
given.
[0031] Monitoring of respiratory parameters by plethysmograph.
Experiments were conducted in unanesthetized, awake, spontaneously
breathing rats at 24 hours prior to SCI, at 24 hours p.i. and
weekly afterwards at 1, 2, 3, and 4 weeks p.i. (1) Acclimatization
of the Animals. We found that correct plethysmograph-recordign of
respiratory parameters of conscious rats required animal training
for acclimatization. Animals were placed in the body cylinder of
the plethysmograph (FIG. 1a) for 60 minutes per day for at least 5
days. This procedure led them to become used to the environment.
Upon acclimatization, rats remained quietly in the cylinder
allowing for the acquisition of data without physical signs of
stress (i.e. defecation, urination, and bloody secretions in the
eyes and nose) and motion artifacts.
[0032] (2) Non-Invasive Measurements of Respiratory Rate, Tidal
Volume and Minute Ventilation. Non-invasive measurements of
respiratory function in conscious rats were performed with a
restrained head-out plethysmograph specially designed for rodents
(BUXCO Electronics, Inc., Sharori, Conn.) (FIG. 1a). The
plethymograph apparatus has a neck seal that prevents leakage of
air from between the animal's neck and the plethymograph opening.
Displacement of the thoracic wall produced by the animal's
respiratory movements causes changes in the cylinder pressure,
which results in air flowing across a pneumotachograph located on
the wall of the cylinder. The pressure drop across the
pneumotachograph is measured with a pressure transducer and is
proportional to the flow. This signal is amplified and integrated
into volume. From measurements of volume and flow, a computer and
appropriate software provides respiratory parameters, such as
respiratory rate (f), tidal volume (Vt), minute ventilation (Ve),
peak inspiratory flow, peak expiratory flow, inspiratory time (Ti),
expiratory time (Te), and accumulated volume. An additional opening
on the wall of the box allows volume calibration by injecting and
removing air from the box with a calibrated syringe.
[0033] The noise level in the laboratory was kept to a minimum in
order not to startle the animals. Further, the animals were
visually isolated from the investigators by means of a chamber made
of an opaque material that surrounded and covered the front-end of
the body plethysmograph (FIG. 1b). Baseline recordings lasted for 4
minutes.
[0034] (3) Measurement of Ventilatory Response to Carbon Dioxide.
For measurement of the ventilatory response to CO.sub.2, animals
were exposed to air containing 7% CO.sub.2. The animals breathed
from a funnel fixed in the front wall of a chamber made of an
opaque material (FIG. 1b). The animals were exposed to the gas
mixture containing 7% CO.sub.2 (mixed with 60% O.sub.2 and 33%
N.sub.2) for 5 minutes with recording of respiratory activity for
another 2 minutes (a total recording duration of 7 minutes).
[0035] Drug Administration. The 5HT.sub.1A receptor agonists,
8-OH-DPAT and buspirone (both purchased from RBI, Natick, Mass.)
were dissolved in 0.9% saline (pH adjusted to 7.4). Both agonists
were administered intraperitoneally in 0.5 ml final injecting
volume per rat (i.p.) and in doses of 250 .mu.g/kg for 8-OH-DPAT
and 1.5 mg/kg for buspirone respectively. The 5HT.sub.1A receptor
antagonist, p-MPPI (RBI, Natick, Mass.) was also dissolved in 0.9%
saline and given i.p. in a dose of 3 mg/kg (pH 7.4 and final volume
of 0.5 ml). The doses of the above drugs were decided based on our
earlier report that demonstrated that 5HT.sub.1A agonists could
reverse morphine-induced respiratory depression (Ferreira et al.,
1998). Vehicle solution (VEH) was 0.9% saline and also injected at
i.p. (pH 7.4; volume: 0.5 ml/rat).
[0036] Experimental protocol. SCI surgical procedures were
performed only after animals finished at least 5 days
plethysmograph acclimatization (see above) and at 24 hours after
plethysmograph data acquisition for pre-injury respiratory
parameters. Test of functional deficits were performed at 24 hours
prior to SCI, and at 24 hours and weekly afterwards for 4 weeks
p.i. to determine a proper degree of SCI was achieved (Gale et al.,
1985; Basso et al., 1995).
[0037] Baseline respiratory function was measured under room air
ventilation and after the animal was stabilized inside a boy
cylinder (FIGS. 1a and b) for 30 minutes at each time point prior
to SCI and after injury. Immediately following the evaluation of
baseline respiration, the animals were let to breathe air
containing 7% CO.sub.2 for 7 minutes to monitor their ventilatory
response to CO.sub.2 stimulus (Teng et al., 1998a and 1999). For
VEH and 8-OH-DPAT studies, at 24 hours p.i., respiratory function
of a SCI rat was first evaluated by plethysmograph for baseline
performance as well as respiratory response to 7% CO.sub.2
challenge. Twenty-four minutes post the end of CO.sub.2 breathing,
the rat was removed from the body cylinder (FIG. 1a) after a new
baseline was recorded for 4 minutes. The animal was then injected
with saline VEH (0.5 ml, i.p.) and immediately put back into the
cylinder in a smooth manner for continuing respiratory monitoring
(the procedure took about 1.2 minutes average). Baseline
respiration (i.e. under room air ventilation) was examined for
another 10 minutes, and at the end, ventilatory response was
evaluated when the animal was challenged by 7% CO.sub.2 for 7
minutes. Twenty-four minutes post the end of CO.sub.2 stimulus
(including a recording of a new baseline for 4 minutes), the rat
was again taken out from the body cylinder subsequent to a new
baseline recording for 4 minutes. Therewith the animal was injected
with 8-OH-DPAT (250 .mu.g/kg in 0.5 ml, i.p.), and immediately sent
back into the cylinder for continuing respiratory monitoring (the
procedure took about 1.2 minutes in average). Following the drug
administration, baseline respiration (i.e. under room air
ventilation) was examined constantly for another 23 minutes. At the
end of the 23.sup.rd minute, ventilatory response was evaluated
once more when the rat was challenged with 7% CO.sub.2 for 7
minutes. Similar procedures for the 8-OH-DPAT study were repeated
at 7 days p.i. except that no saline VEH treatment was given. In
the time-course study for the respiratory effect of 8-OH-DPAT,
recordings of baseline respiratory function (for 4 minutes) and
ventilatory response for 7% CO.sub.2 (for 7 minutes) were repeated
hourly for up to 5 hours after the administration of 8-OH-DPAT. In
experiments of p-MPPI antagonism of 8-OH-DPAT effects, p-MPPI (3
mg/kg in 0.5 ml/rat, i.p.) was given at 20 minutes before the
administration of 8-OH-DPAT. Baseline respiratory function was
examined beginning at 4 and 18 minutes after p-MPPI injection (each
lasted for 2 minutes). Baseline recording was performed again at 4
and 8 minutes following i.p. 8-OH-DPAT (each lasted for 2 minutes),
and at the end of the 10.sup.th minute after 8-OH-DPAT, ventilatory
response to breathing 7% CO.sub.2 was measured. For the study of
the buspirone effects, similar sequential procedures as those in
the 8-OH-DPAT experiment were followed. However, the 7% CO.sub.2
challenge was given at 10 minutes after i.p. injection of buspirone
(1.5 mg/kg in 0.5 ml), and neither time-course nor antagonism study
was performed for buspirone.
[0038] All animals survived the study. Experimental data are
expressed as mean.+-.SEM. Statistical significance was defined at
the p<0.05 level. The statistical tests used are described below
and also specified in the figure legends. All experimental
procedures were carried out in strict accordance with the
Laboratory Animal Welfare Act, Guide for the Care and Use of
Laboratory Animals (NIH, DHEW Publication No. 78-23, Revised 1978)
after review and approval by the Animal Care and Use Committee of
Georgetown University.
[0039] Statistical analyses. Respiratory data were analyzed
statistically using repeated measures ANOVA, followed by Tukey's or
Dunn's test for multiple comparisons between groups used in
previous studies (e.g., Wrathall et al., 1994; Teng and Wrathall,
1997; Teng et al., 1999). The same statistical tests were used for
analyzing respiratory data from drug treatment studies.
[0040] Contusion spinal cord injury (SCI) at T8 produces
respiratory abnormalities in conscious rats breathing room air
challenged with CO.sub.2. In seeking ways to improve respiration in
SCI animals, we tested drugs that stimulate serotonin 1A
(5HT.sub.1A) receptors, based on our findings that those agents can
counteract respiratory depression produced by morphine overdose.
Respiratory function was measured with a head-out plethysmograph
system in conscious rats.
EXAMPLE 1
Treatment with the Serotonin 1A Receptor (5HT.sub.1A) Agonist
8-OH-DPAT Improves Respiratory Function in Spinal Cord Injured
Rats
[0041] Respiratory function was evaluated when rats were breathing
room air for the data baseline respiration. In addition, rats were
challenged with air mixtures containing 7% CO.sub.2, as described
in Methods. This was done to determine the effect of SCI on the
central chemoreceptor-mediated respiratory responses to high
concentration of CO.sub.2.
[0042] Twenty-four hours after we examined respiratory function in
rats prior to injury to establish normal parameters, rats were
subjected to SCI. At 24 hours p.i. and 7 days p.i., all SCI rats
were tested behaviorally for their hindlimb reflexes and
coordinated use of hindlimbs, including a detailed examination of
open field locomotion (Gale et al., 1985; Basso et al., 1995;
Wrathall et al., 1994; Teng and Wrathall, 1997). We found that
behavioral deficits proper to this degree of SCI as well as
post-injury time points (i.e. at 24 hours or 7 days p.i.; Wrathall
et al., 1994; Teng and Wrathall, 1997) existed in all SCI rats
(data not shown). Further, no significant differences in behavioral
deficits were found among the SCI rats (repeated measures ANOVA,
P>0.05). Thereupon SCI rats were randomized to receive either
8-OH-DPAT (250 .mu.g/kg in 0.5 ml/rat, i.p.), a 5HT.sub.1A
agonistic drug (Middlemiss and Fozard, 1983; Harmon et al., 1986)
or VEH solution (0.5 ml/rat, i.p.). The administration was started
at 24 minutes after the end of the first CO.sub.2 challenge:
following a recording of a new baseline for 4 minutes (see Methods
for details).
[0043] Contusion SCI at thoracic 8 vertebral level caused a
significant decrease in Vt along with a significant increase in f
at 24 hours p.i. (Table 1; FIG. 2) and 7 days p.i. (Table 1)
compared to pre-injury respiratory parameters. Rats at 24 hours and
7 days after SCI demonstrated a pattern of breathing which was more
shallow and rapid than prior to injury (FIG. 2). Our data
demonstrated again that SCI at T8 produced significant impairments
on respiration as evaluated in conscious rats (Teng et al., 1999).
At 24 hours after SCI, while VEH treatment did not alter the
abnormal respiratory pattern resulting from SCI (Table 1),
8-OH-DPAT administration promptly and successfully reversed the
injury-triggered respiratory abnormalities. For example at 22
minutes after i.p. injection of 8-OH-DPAT, Vt was changed
significantly from post-SCI level of 0.66.+-.0.03 to 0.80.+-.0.06
(unit: ml; P<0.05, repeated measures ANOVA with Tukey's
procedure; Table 1), a value that was statistically
indistinguishable compared to Vt prior to SCI (0.90.+-.0.02, unit:
ml; P>0.05, repeated measures ANOVA with Tukey's procedure;
Table 1). At the same time, treatment of 8-OH-DPAT also brought f
that was significantly increased by SCI (131.6.+-.5.7 vs
90.8.+-.3.7, unit: breaths/min: P<0.05, repeated measures ANOVA
with Tukey's procedure; Table 1) back to normal (98.5.+-.3.7,
units: breaths/min; P>0.05, repeated measures ANOVA with Tukey's
procedure; Table 1). Nonetheless, treatment with 8-OH-DPAT did
initially drive f even higher than the original p.i. f levels,
which lasted for about 20 minutes (Table 1; some data not shown).
The decrease in Vt and increase in f presented till 7 days p.i.
(Table 1). Once again, 8-OH-DPAT treatment restored this abnormal
pattern of breathing to normal at 7 days p.i. (Table 1). Vt and f
were recovered to normal starting at 14 days after injury (n=2,
data not shown), consistent with what we reported previously for
the chronic recovery of respiratory function occurring in this
model of SCI (Teng et al., 1999).
[0044] The SCI rats showed a dramatic decrease in the ventilatory
response to CO.sub.2. The Ve when breathing air containing 7%
CO.sub.2 was significantly decreased at 24 hours p.i. as compared
to that observed prior to the injury (161.7.+-.14.9 vs
250.4.+-.17.0, unit: ml/min; P<0.05, repeated measures ANOVA
with Tukey's procedure; Table 1). The abnormalities of ventilatory
response to 7% CO.sub.2 were still significant at 7 days p.i.
(Table 1). Beginning at 14 days p.i., the response to 7% CO.sub.2
recovered to pre-injury levels (data not shown). The severely
impaired ventilatory response to 7% CO.sub.2 in the SCI animals at
24 hours and 7 days after SCI was normalized by the treatment of
8-OH-DPAT in the same rats that did not show any significant Ve
improvement following VEH administration (Table 1). TABLE-US-00001
TABLE 1 Respiratory Parameters of Conscious Rats That Received
Systemic Saline and 8OH-DPAT at 24 Hours after SCI 24 h p.i.: Prior
to SCI 24 h after 24 h p.i.: Post 7% CO2 post Experimental and
Treatments T8 SCI Saline (i.p.) Saline (i.p.) Group Baseline 7% CO2
Baseline 7% CO2 Baseline Baseline T8 SCI Rats That Ti 0.20 .+-.
0.01 0.19 .+-. 0.01 Ti 0.20 .+-. 0.01 0.17 .+-. 0.01 Ti 0.19 .+-.
0.01 Ti 0.16 .+-. 0.01 Received Te 0.50 .+-. 0.03 0.22 .+-. 0.01 Te
0.28 .+-. 0.01.dwnarw. 0.25 .+-. 0.03 Te 0.34 .+-. 0.03.dwnarw. Te
0.26 .+-. 0.03 8OHDPAT Tv 0.90 .+-. 0.02 1.64 .+-. 0.09 Tv 0.66
.+-. 0.03.dwnarw. 1.09 .+-. 0.03.dwnarw. Tv 0.68 .+-. 0.03.dwnarw.
Tv 1.04 .+-. 0.07.dwnarw. (250 .mu.g/kg, i.p.) f 90.8 .+-. 3.70
153.5 .+-. 7.59 f 131.6 .+-. 5.7.uparw. 148.0 .+-. 10.9 f 125.4
.+-. 5.6.uparw. f 150.0 .+-. 9.8 (n = 5) Ve 81.6 .+-. 3.94 250.4
.+-. 17.0 Ve 86.5 .+-. 6.42 161.7 .+-. 14.9.dwnarw. Ve 85.9 .+-.
6.60 Ve 157.6 .+-. 17.5.dwnarw. 24 h p.i.: 7% CO2 24 h p.i.: 3
& 4 min post 24 h p.i.: 21 & at 24 h p.i.: 8-OH-DPAT 22 min
post 24 min after Experimental Pre-8-OH-DPAT (250 .mu.g/kg, i.p.)
8-OH-DPAT 8-OH-DPAT Group Baseline Baseline Baseline 7% CO2 T8 SCI
Rats That Ti 0.18 .+-. 0.01 Ti 0.18 .+-. 0.01 Ti 0.18 .+-. 0.01 Ti
0.17 .+-. 0.01 Received Te 0.31 .+-. 0.02.dwnarw. Te 0.25 .+-.
0.02.dwnarw. Te 0.47 .+-. 0.02 Te 0.18 .+-. 0.01 8OHDPAT Tv 0.57
.+-. 0.02.dwnarw. Tv 0.96 .+-. 0.08 Tv 0.80 .+-. 0.06 Tv 1.52 .+-.
0.08 (250 .mu.g/kg, i.p.) f 125.6 .+-. 3.6.uparw. f 155.5 .+-.
6.7.uparw. f 98.5 .+-. 5.18 f 175.9 .+-. 9.7 (n = 5) Ve 83.8 .+-.
3.8 Ve 149.9 .+-. 15.6.uparw. Ve 81.3 .+-. 10.9 Ve 267.5 .+-. 21.8
7 days after 7 d p.i.: 21 & 7 d p.i.: 7% CO2 T8 SCI 7 d p.i.: 3
& 4 min post 22 min post at 31' & 32' post Experimental (n
= 3) 8-OH-DPAT 8-OH-DPAT 8-OH-DPAT Group Baseline 7% CO2 Baseline
Baseline 7% CO2 T8 SCI Rats That Ti 0.18 .+-. 0.01 0.18 .+-. 0.00
Ti 0.16 .+-. 0.01 Ti 0.15 .+-. 0.01 Ti 0.16 .+-. 0.01 Received Te
0.28 .+-. 0.03.dwnarw. 0.18 .+-. 0.02 Te 0.26 .+-. 0.03.dwnarw. Te
0.32 .+-. 0.02.dwnarw. Te 0.17 .+-. 0.01 8OHDPAT Tv 0.72 .+-.
0.03.dwnarw. 1.39 .+-. 0.08.dwnarw. Tv 1.11 .+-. 0.05 Tv 0.79 .+-.
0.04 Tv 1.79 .+-. 0.35 (250 .mu.g/kg, i.p.) f 134.4 .+-.
10.2.uparw. 167.3 .+-. 11.7 f 155.2 .+-. 8.9.uparw. f 131.7 .+-.
7.7.uparw. f 184.6 .+-. 6.6.uparw. (n = 5) Ve 96.3 .+-. 5.1 253.6
.+-. 41.3 Ve 172.1 .+-. 17.1.uparw. Ve 103.0 .+-. 2.1.uparw. Ve
327.2 .+-. 53.7.uparw. .uparw. or .dwnarw.: Significantly higher or
lower compared to pre-SCI values; P < 0.05, one way ANOVA
followed by Tukey's or Dunn's test for multiple comparisons.
EXAMPLE 2
Time-Course Study of the Effects of 8-OH-DPAT on minute Ventilation
(V.sub.e)
[0045] Considering the relative short systematic half life of
8-OH-DPAT in rats (T1/2: .about.50 minutes; Kleven and Koek, 1998),
we decided to conduct a time-course study to determine if the
respiratory effects of 8-OH-DPAT was time-related and thus, a
dose-dependent event. At 24 hours p.i., the enhancing effect of
8-OH-DPAT on ventilatory response to 7% CO.sub.2 challenge
decreased in a time-dependent manner, with the values recorded at 5
hours after the drug injection being slightly and not significantly
higher than those collected at 24 hours p.i. and before 8-OH-DPAT
treatment (FIG. 3). Therefore, a single dose treatment of 8-OH-DPAT
normalized ventilatory response to breathing 7% CO.sub.2 for more
than 2 hours after the drug administration (FIG. 3). Interestingly,
although 8-OH-DPAT-treatment also showed a time-dependent
improvement of baseline Ve (i.e. under room air breathing),
however, the effect was significant for only 1 hour (FIG. 3).
EXAMPLE 3
Effects of 8-OH-DPAT on the Respiratory Function of Normal Rats
[0046] Normal rats without SCI demonstrated highly consistent
respiratory parameters under baseline conditions and 7% CO.sub.2
challenge relative to data collected at 24 hours pre-injury in the
above SCI studies (compare Table 2 to Table 1) as well as to those
reported earlier by our group (Teng et al., 1998a and 1999). Saline
VEH injection (0.5 ml/per rat, i.p.) did not change respiratory
parameters either in baseline conditions or under 7% CO.sub.2
breathing compared to those obtained before VEH treatment (Table
2). In contrast, treatment of 8-OH-DPAT quickly and significantly
enhanced respiratory function (Table 2). However, 8-OH-DPAT
treatment only increased baseline Vt for 4 minutes in normal rats
before it dropped back to previous levels (Table 2). On the other
hand, the stimulating effect of 8-OH-DPAT on f lasted till the last
minute of the observation (i.e. 23 minutes post drug
administration) and with a strong potency (Table 2). This
phenomenon of a stronger potency of 8-OH-DPAT on f than Vt obtained
in normal animals was also noticed in SCI rats at 14, 21 and 28
days p.i. when the drug was given to chronic SCI rats with an
already recovered respiratory function (data not shown).
Strikingly, 8-OH-DPAT treatment in normal rats did not
significantly alter ventilatory response to 7% CO.sub.2 challenge
starting at 24 minutes after the administration of 8-OH-DPAT (Table
2). This result brings up a sharp contrast between the effect of
8-OH-DPAT on CO.sub.2-triggered ventilatory response in normal rats
and animals with acute SCI (i.e. at 24 hours and 7 days p.i.; Table
1 and 2). TABLE-US-00002 TABLE 2 Respiratory Parameters of Normal
Conscious Rats That Received Systemic 8OH-DPAT Pre- 3 & 4 min
8-OH-DPAT post Experimental Prior to Treatments Post Saline (i.p.)
(250 .mu.g/kg, i.p.) 8-OH-DPAT Group Baseline 7% CO2 Baseline 7%
CO2 Baseline Baseline Normal Ti 0.23 .+-. 0.01 0.18 .+-. 0.02 Ti
0.24 .+-. 0.03 0.17 .+-. 0.01 Ti 0.24 .+-. 0.01 Ti 0.19 .+-. 0.04
Conscious Rats Te 0.54 .+-. 0.04 0.22 .+-. 0.04 Te 0.51 .+-. 0.04
0.21 .+-. 0.02 Te 0.69 .+-. 0.06 Te 0.25 .+-. 0.09.dwnarw. (n = 3)
Tv 0.89 .+-. 0.05 1.43 .+-. 0.04 Tv 0.90 .+-. 0.09 1.37 .+-. 0.21
Tv 1.04 .+-. 0.07 Tv 1.14 .+-. 0.08.uparw. f 84.9 .+-. 7.58 169.9
.+-. 6.57 f 87.2 .+-. 4.57 161.1 .+-. 10.2 f 71.6 .+-. 6.02 f 177.5
.+-. 36.2.uparw. Ve 75.1 .+-. 4.75 242.5 .+-. 44.5 Ve 78.6 .+-.
6.97 224.5 .+-. 42.5 Ve 74.0 .+-. 4.65 Ve 198.6 .+-. 32.9.uparw. 7%
CO2 at 11 & 12 min 21 & 22 min 31' & 32' after
Experimental post 8-OH-DPAT post 8-OH-DPAT 8-OH-DPAT Group Baseline
Baseline 7% CO2 Normal Ti 0.16 .+-. 0.02 Ti 0.18 .+-. 0.02 Ti 0.19
.+-. 0.01 Conscious Rats Te 0.30 .+-. 0.07.dwnarw. Te 0.41 .+-.
0.08.dwnarw. Te 0.23 .+-. 0.03 (n = 3) Tv 0.92 .+-. 0.05 Tv 0..84
.+-. 0.02 Tv 1.50 .+-. 0.14 f 149.8 .+-. 26.5.uparw. f 134.4 .+-.
21.2.uparw. f 155.6 .+-. 18.5 Ve 138.6 .+-. 27.6.uparw. Ve 112.9
.+-. 15.2.uparw. Ve 239.3 .+-. 48.9 .uparw. or .dwnarw.:
Significantly higher or lower compared to pre-SCI values; P <
0.05, one way ANOVA followed by Tukey's or Dunn's test for multiple
comparisons.
EXAMPLE 4
Buspirone Treatment Improves Respiratory Function in Spinal-Cord
Injured Rats
[0047] Treatment with buspirone, a partial agonist of 5HT.sub.1A
receptors (Hoyer and Schoeffter, 1991) also reversed the abnormal
respiratory function resulting form T8 SCI at 24 hours p.i.: SCI
reduced Vt values (0.74.+-.0.02 vs 1.09.+-.0.04, unit: ml;
P<0.05, repeated measures ANOVA with Tukey's procedure) were
incremented rapidly by buspirone treatment (1.5 mg/kg in 0.5 ml/per
rat, i.p.) to levels indiscernible from pre-injury readings (FIG. 4
and Table 3). The effect of buspirone on Bt sustained up to 9
minutes after the drug administration. Unlike 8-OH-DPAT, the
initial stimulating effect of buspirone on f was rather milder and
more transient relative to that of 8-OH-DPAT (Table 1 and Table 3).
Buspirone treatment restored f to normal range beginning at 6
minutes after the dosing (Table 3; some data not shown). In
addition, treatment of buspirone normalized ventilatory, response
to 7% CO.sub.2 challenge that was started at 9 minutes after
buspirone administration (1.01.+-.0.11 vs 1.09.+-.0.04, unit:
ml/min; P>0.05, repeated measures ANOVA, with Tukey's
procedure). TABLE-US-00003 TABLE 3 Respiratory Parameters of
Conscious SCI Rats That Received Buspirone at 24 Hours after SCI
Pre-buspirone Experimental Prior to T8 SCI 24 hours Post SCI (1.5
mg/kg, i.p.) Group Baseline 7% CO2 Baseline 7% CO2 Baseline T8 SCI
Rats That Ti 0.18 .+-. 0.02 0.17 .+-. 0.01 Ti 0.22 .+-. 0.01 0.17
.+-. 0.02 Ti 0.23 .+-. 0.04 Received Te 0.40 .+-. 0.01 0.20 .+-.
0.01 Te 0.21 .+-. 0.02.dwnarw. 0.20 .+-. 0.02 Te 0.23 .+-.
0.01.dwnarw. buspirone Tv 1.09 .+-. 0.04 1.62 .+-. 0.08 Tv 0.74
.+-. 0.02.dwnarw. 1.12 .+-. 0.11.dwnarw. Tv 0.74 .+-. 0.04.dwnarw.
(1.5 mg/kg, i.p.) f 104.4 .+-. 3.2 169.1 .+-. 7.7 f 142.2 .+-.
9.6.uparw. 168.5 .+-. 14.2 f 134.7 .+-. 13.2.uparw. (n = 3) Ve
114.8 .+-. 5.7 274.0 .+-. 23.8 Ve 105.8 .+-. 8.7 190.2 .+-.
29.8.dwnarw. Ve 100.5 .+-. 14.7 7% CO2 at 3 & 4 min 8 & 9
min 15 & 16 min after Experimental post buspirone post
buspirone buspirone Group Baseline Baseline 7% CO2 T8 SCI Rats That
Ti 0.19 .+-. 0.01 Ti 0.20 .+-. 0.03 Ti 0.17 .+-. 0.01 Received Te
0.27 .+-. 0.03.uparw. Te 0.40 .+-. 0.05 Te 0.21 .+-. 0.03 buspirone
Tv 1.17 .+-. 0.13 Tv 1.01 .+-. 0.11 Tv 1.46 .+-. 0.14 (1.5 mg/kg,
i.p.) f 143.5 .+-. 11.9.uparw. f 101.2 .+-. 5.5 f 162.6 .+-. 17.4
(n = 3) Ve 171.3 .+-. 30.1 Ve 101.8 .+-. 11.4 Ve 240.9 .+-. 44.8
.uparw. or .dwnarw.: Significantly higher or lower compared to
pre-SCI values; P < 0.05, one way ANOVA followed by Tukey's or
Dunn's test for multiple comparisons.
EXAMPLE 5
The 5HT.sub.IA-Receptor Antagonist p-MPPI Specifically Reverses the
8-OH-DPAT-Mediated Improvement of Respiratory Function in Spinal
Cord Injured Rats
[0048] Through testing whether a specific 5HT.sub.1A-receptor
antagonist, p-MPPI (Theilen et al., 1990) could efficiently block
the stimulus effect of 8-OH-DPAT on respiratory function, we
further studied the specificity of 8-OH-DPAT effects. The p-MPPI
antagonism of 8-OH-DPAT was first studied by a series of dose
titration experiments (data not shown). We found that a dose of 2
mg/kg (in 0.5 ml/per rat, i.p.) could substantially block the
stimulating effects of 8-OH-DPAT on respiratory function. We also
tested the effect of p-MPPI (3 mg/kg in 0.5 ml/rat, i.p.) per se on
respiratory function in normal rats (n=3). No significant impacts
of p-MPPI treatment were found on respiratory function of the
normal rats, except that administration of p-MPPI resulted in a
small but not significant increase in Vt, f and Ve (data not
shown). Therefore, in the definitive study, this dose of p-MPPI (3
mg/kg in 0.5 ml/rat, i.p.) was given at 20 minutes before the
administration of 8-OH-DPAT. Pre-treatment of p-MPPI significantly
suppressed the effects of 8-OH-DPAT on respiration at 24 hours p.i.
(FIG. 5). Pre-treatment of p-MPPI stabilized post-SCI baseline Vt,
f and Ve at routine p.i. levels regardless of the later injection
of 8-OH-DPAT (Table 4). In addition, the stimulating effects of
8-OH-DPAT on ventilatory response to 7% CO.sub.2 at 24 hours p.i.
were significantly blocked by treatment of p-MPPI (FIG. 5; Table
4). TABLE-US-00004 TABLE 4 Respiratory Parameters of Conscious SCI
Rats That Pre-treated with p-MPPI before Administration of 8OH-DPAT
3 & 4 min post 19 & 20 min Experimental Prior to SCI 24
hours Post SCI p-MPPI post p-MPPI Group Baseline 7% CO2 Baseline 7%
CO2 Baseline Baseline Normal Rats with Ti 0.27 .+-. 0.02 0.22 .+-.
0.01 Ti 0.26 .+-. 0.05 0.22 .+-. 0.02 Ti 0.33 .+-. 0.01 Ti 0.28
.+-. 0.04 Saline i.p. Te 0.52 .+-. 0.00 0.32 .+-. 0.06 Te 0.35 .+-.
0.01.dwnarw. 0.30 .+-. 0.01 Te 0.28 .+-. 0.02.dwnarw. Te 0.54 .+-.
0.10 (n = 3) Tv 1.48 .+-. 0.02 2.37 .+-. 0.12 Tv 0.66 .+-.
0.04.dwnarw. 0.84 .+-. 0.03.dwnarw. Tv 0.75 .+-. 0.08.dwnarw. Tv
0.77 .+-. 0.08.dwnarw. f 78.5 .+-. 1.64 114.9 .+-. 13.9 f 101.8
.+-. 7.2 118.8 .+-. 3.8 f 103.2 .+-. 5.4.uparw. f 76.2 .+-. 7.7 Ve
116.9 .+-. 2.4 269.4 .+-. 21.9 Ve 67.4 .+-. 8.7.dwnarw. 99.9 .+-.
6.1.dwnarw. Ve 76.9 .+-. 4.6.dwnarw. Ve 57.8 .+-. 4.1.dwnarw. 7%
CO2 at 3 & 4 min 9 & 10 min 16' & 17' after
Experimental post 8-OH-DPAT post 8-OH-DPAT 8-OH-DPAT Group Baseline
Baseline 7% CO2 Normal Rats with Ti 0.32 .+-. 0.02 Ti 0.31 .+-.
0.02 Ti 0.19 .+-. 0.02 Saline i.p. Te 0.32 .+-. 0.08.dwnarw. Te
0.28 .+-. 0.02.dwnarw. Te 0.32 .+-. 0.05 (n = 3) Tv 0.85 .+-.
0.14.dwnarw. Tv 0.82 .+-. 0.11.dwnarw. Tv 1.15 .+-. 0.21.dwnarw. f
99.4 .+-. 9.1.uparw. f 107.5 .+-. 1.9.uparw. f 126.8 .+-. 14.4 Ve
82.5 .+-. 10.9.dwnarw. Ve 88.3 .+-. 13.3.dwnarw. Ve 143.1 .+-.
28.1.dwnarw. .dwnarw. or .uparw.: Significantly higher or lower
compared to pre-SCI values; P < 0.05, one way ANOVA followed by
Tukey's or Dunn's test for multiple comparisons. Note: This is the
only group of rats with body weight that ranged between 360 and 390
grams. Thus, the higher pre-SCI Vt values and lower f were due to
body size as previously described (Teng et al., 1999).
Summary
[0049] T8 SCI rats (n=5) showed decreased tidal volume (Vt:
0.9.+-.0.02 to 0.66.+-.0.03 ml: P<0.05) and increased
respiratory rate (f; 90.8.+-.3.7 to 131.6.+-.5.7; P<0.05) under
room air ventilation at 24 hours post injury (p.i.). Moreover,
these animals exhibited a diminished response to the respiratory
stimulating effect of 7% CO.sub.2: minute ventilation (Ve) changed
from 250.4.+-.17.0 ml/min prior to SCI to 161.7.+-.14.9 ml/min at
24 hours p.i. (P<0.05). Similar respiratory deficits were also
observed in the SCI rats at 7 days p.i. (n=3). Treatment with the
5HT.sub.1A receptor agonist, 8-hydroxy-2-(di-n-propylamino)tetralin
(8-OH-DPAT, 250 .mu.g/kg; i.p.) at 24 hours or 7 days p.i.
normalized Vt, f and the respiratory response to 7% CO.sub.2.
Results identical to those of 8-OH-DPAT were obtained with another
5HT.sub.1A receptor agonist, buspirone (1.5 mg/kg, i.p.; n=3). In
contrast, saline vehicle administration (i.p.; n=5) showed no
beneficial effects on SCI-impaired respiration. Finally,
pretreatment with a specific antagonist of 5HT.sub.1A receptors,
4-(2'-methoxyphenyl)-1-[2'-[N-(2''-pyridinyl)-p-iodobenzainido]ethyl]pipe-
razine (p-MPPI, 3 mg/kg, i.p.; n=3) given 20 min before 8-OH-DPAT
administration, prevented 8-OH-DPAT from restoring respiration to
normal. Our results demonstrate that drugs which stimulate
5HT.sub.1A receptors improve respiratory function in conscious rats
after SCI.
EXAMPLE 6
Buspirone Treatment Improves Respiratory Function in a Mutant Mouse
Model of Familial ALS
[0050] Based on the data presented above in Example 4 and the
observation that both SCI-related and ALS-related respiratory
abnormalities are triggered by muscle dysfunction (e.g.,
degeneration) which is mediated by denernvation, we determined
whether buspirone treatment could ameliorate respiratory
abnormalities (e.g., deficits in tidal volume and respiratory
rates) in SOD1 mice. SOD1 mice are mutant mice which provide an
animal model for an inherited form of ALS which is more commonly
referred to as familial ALS. Respiratory function was determined
prior to (e.g., pre) and after (e.g., post) buspirone
administration. Baseline tidal volumes and respiratory rates were
determined prior to the intraperitoneal or subcutaneous
administration of buspirone at a dose of 3.0 mg/kg. These
parameters of respiratory function were subsequently reevaluated 2
minutes after drug treatment. The data presented in FIGS. 6 and 7
demonstrate that the subcutaneous administration of buspirone
significantly improved the respiratory function (e.g., increased
tidal volume and decreased respiratory rate) of the treated
mice.
[0051] These results are consistent with the theory that 5HT 1A
agonists can be used to counteract (e.g., ameliorate) the
respiratory abnormalities mediated by ALS pathophysiology. While
not wishing to be bound by theory, it is believed that 5HT 1A
agonists improve respiratory function by stimulating respiratory
premotoneurons in the brain stem and spinal cord. Consistent with
this theory, it is further hypothesized that the proper stimulation
of motoneurons, for example by the administration of of a 5HT 1A
agonist such as buspirone, could delay the muscular degeneration
(e.g., dystrophy and atrophy) associated with the
pathophysiological consequences of ALS. It si also hypothesized
that the coadministration of a .beta.2-adrenergic agonist in
combination with a 5HT 1A agonist could mediate a synergistic
effect which will further inhibit ALS-related muscular
degeneration. The predicted synergism is predicated on the ability
of .beta.2-adrenergic agonists to promote muscle hypertrophy and to
increase the expression of neural growth (e.g. NGF and bFGF) and
trophic factors. Thus, a treatment strategy comprising the
coadministration of a 5HT 1A agonist (e.g., 8-OH-DPAT or buspirone)
in combination with a .beta.2-adrenergic agonist (e.g., clenbuterol
or salbutanol) provides a novel therapeutic strategy for the
treatment of ALS.
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[0097] While this invention has been particularly shown and
described with references to preferred embodiments thereof, it will
be understood by those skilled in the art that various changes in
form and details may be made therein without departing from the
scope of the invention encompassed by the appended claims.
* * * * *