U.S. patent application number 10/067662 was filed with the patent office on 2002-11-07 for carbon monoxide dependent guanylyl cyclase modifiers and methods of use.
Invention is credited to Glasky, Alvin J., Rathbone, Michel P..
Application Number | 20020165242 10/067662 |
Document ID | / |
Family ID | 27403169 |
Filed Date | 2002-11-07 |
United States Patent
Application |
20020165242 |
Kind Code |
A1 |
Glasky, Alvin J. ; et
al. |
November 7, 2002 |
Carbon monoxide dependent guanylyl cyclase modifiers and methods of
use
Abstract
Disclosed herein are methods and associated compositions and
medicaments directed generally to the control of cellular and
neural activity and for selectively and controllably inducing the
in vivo genetic expression of one or more naturally occurring
genetically encoded molecules in mammals. More particularly, the
present invention selectively activates or derepresses genes
encoding for specific naturally occurring molecules such as
proteins or neurotrophic factors and induces the endogenous
production of such naturally occurring compounds through the
administration of carbon monoxide dependent guanylyl cyclase
modulating purine derivatives. The methods of the present invention
may be used to affect a variety of cellular and neurological
functions and activities and to therapeutically or prophylactically
treat a wide variety of neurodegenerative, neurological, cellular,
and physiological disorders.
Inventors: |
Glasky, Alvin J.; (Tustin,
CA) ; Rathbone, Michel P.; (Hamilton, CA) |
Correspondence
Address: |
ATTN: Louis C. Cullman
OPPENHEIMER WOLFF & DONNELLY LLP
Suite 700
840 Newport Center Drive
Newport Beach
CA
92660
US
|
Family ID: |
27403169 |
Appl. No.: |
10/067662 |
Filed: |
February 4, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10067662 |
Feb 4, 2002 |
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08878656 |
Jun 19, 1997 |
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6350752 |
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08878656 |
Jun 19, 1997 |
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08492929 |
Jul 20, 1995 |
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08492929 |
Jul 20, 1995 |
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08488976 |
Jun 8, 1995 |
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5801184 |
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08488976 |
Jun 8, 1995 |
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08280719 |
Jul 25, 1994 |
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5447939 |
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Current U.S.
Class: |
514/263.35 |
Current CPC
Class: |
A61K 31/522 20130101;
A61P 37/00 20180101; A61K 31/52 20130101; A61K 31/708 20130101;
A61P 25/28 20180101; A61K 31/7076 20130101; A61P 25/00
20180101 |
Class at
Publication: |
514/263.35 |
International
Class: |
A61K 031/522 |
Claims
What is claimed is:
1. A method for the treatment of mammalian disease conditions
associated with cellular damage due to oxidative stress, said
method comprising the step of inducing the in vivo production of at
least one naturally occurring endogenous antioxidant by treating a
mammalian subject with an effective amount of at least one carbon
monoxide dependent guanylyl cyclase modulating purine
derivative.
2. The method of claim 1 wherein said carbon monoxide dependent
guanylyl cyclase modulating purine derivative is selected from the
group consisting of guanosine,
4-[[3-(1,6-dihydro-6-oxo-9H-purin-9-yl)-1-oxo-pr-
opyl]amino]benzoic acid, and inosine pranobex.
3. The method of claim 1 wherein said effective amount of said at
least one carbon monoxide dependent guanylyl cyclase modulating
purine derivative produces a treating concentration of at least 1
.mu.M.
4. The method of claim 2 wherein said mammalian subject is treated
by orally administering said at least one carbon monoxide dependent
guanylyl cyclase modulating purine derivative.
5. The method of claim 2 wherein said mammalian subject is treated
by injecting said at least one carbon monoxide dependent guanylyl
cyclase modulating purine derivative.
6. The method of claim 1 wherein said mammalian disease condition
is Alzheimer's disease and related degenerative disorders.
7. The method of claim 1 wherein said mammalian disease condition
is old age benign forgetfulness and related disorders.
8. The method of claim 1 wherein said mammalian disease condition
is aging related loss of neurons or neuronal connectivity and
related deterioration of sensory, motor, reflex, or cognitive
abilities.
9. The method of claim 1 wherein said mammalian disease condition
is Parkinson's disease and related disorders.
10. The method of claim 1 wherein said mammalian disease condition
is spino-cerebellar atrophy.
11. The method of claim 1 wherein said mammalian disease condition
is motor neuronopathy or Amyotrophic Lateral Sclerosis.
12. The method of claim 1 wherein said mammalian disease condition
is damage to neurons or their processes by physical agents.
13. The method of claim 1 wherein said mammalian disease condition
is damage to neurons by ischemia, anoxia, hypoxia, or
hypoglycemia.
14. The method of claim 1 wherein said mammalian disease condition
is damage to neurons by chemical agents.
15. The method of claim 1 wherein said mammalian disease condition
is trauma to the heart, brain or spinal cord.
16. The method of claim 1 wherein said mammalian disease condition
is epilepsy or seizures.
17. The method of claim 1 wherein said mammalian disease condition
is peripheral neuropathy.
18. The method of claim 1 wherein said mammalian disease condition
is learning disability.
19. The method of claim 1 wherein said mammalian disease condition
is cerebral palsy.
20. The method of claim 1 wherein said mammalian disease condition
is psychiatric disorder.
21. The method of claim 1 wherein said mammalian disease condition
is memory disorder.
22. The method of claim 1 wherein said mammalian disease condition
is Huntington's disease.
23. The method of claim 1 wherein said endogenous antioxidant is a
bile pigment.
24. The method of claim 23 wherein said bile pigment is selected
from the group consisting of biliverdin or bilirubin.
25. The method of claim 23 wherein said bile pigment is produced
through the additional step of degrading heme with heme
oxygenase.
26. A method for inducing the in vivo production of heme oxygenase
in a mammal, said method comprising the step of treating a mammal
with an effective amount of at least one carbon monoxide dependent
guanylyl cyclase modulating purine derivative.
27. The method of claim 26 wherein said carbon monoxide dependent
guanylyl cyclase modulating purine derivative is selected from the
group consisting of guanosine,
4-[[3-(1,6-dihydro-6-oxo-9H-purin-9-yl)-1-oxo-pr-
opyl]amino]benzoic acid, and inosine pranobex.
28. The method of claim 26 wherein said effective amount of said at
least one carbon monoxide dependent guanylyl cyclase modulating
purine derivative produces a treating concentration of at least 1
.mu.M.
29. The method of claim 27 wherein said mammal is treated by orally
administering said at least one carbon monoxide dependent guanylyl
cyclase modulating purine derivative.
30. The method of claim 27 wherein said mammal is treated by
injecting said at least one carbon monoxide dependent guanylyl
cyclase modulating purine derivative.
31. The method of claim 26 further comprising the additional step
of inducing the degrading of heme in said mammal with said heme
oxygenase to endogenously produce bile pigment and carbon
monoxide.
32. The method of claim 31 further comprising the additional step
of modulating guanylyl cyclase in said mammal with said
endogenously produced carbon monoxide.
33. The method of claim 31 further comprising the additional step
of neutralizing or sequestering free radicals in said mammal with
said endogenously produced bile pigment.
34. The method of claim 31 further comprising the additional step
of inducing a reduction in the blood pressure of said mammal with
said endogenously produced carbon monoxide.
Description
RELATED APPLICATIONS
[0001] The present invention is a continuation-in-part of
co-pending application Ser. No. 08/488,976, filed Jun. 8, 1995,
which is a continuation-in-part of co-pending application Ser. No.
08/280,719, filed Jul. 25, 1994.
FIELD OF THE INVENTION
[0002] The present invention relates in general to the control of
cellular and neural activity and to the treatment of cellular and
neural disorders. More particularly, the present invention is
directed to methods and associated compositions and medicaments for
the modification of mammalian cellular and neural activity through
the administration of carbon monoxide dependent guanylyl cyclase
modulating purine derivatives which selectively and controllably
induce the in vivo genetic expression of naturally occurring
genetically encoded molecules including neurotrophic factors. The
methods, compositions, and medicaments of the present invention may
be used to affect a variety of cellular and neurological activities
and to therapeutically or prophylactically treat a wide variety of
physiological, neurodegenerative, and neurological disorders.
BACKGROUND OF THE INVENTION
[0003] The evolution of the central nervous system in mammals was a
natural response to an increasingly complex environment requiring
solutions to difficult problems. The resulting structure is an
intricate biochemical matrix that is precisely controlled and
attenuated through an elaborate system of chemically modulated
regulatory pathways. Through an elaborate series of highly specific
chemical reactions, these pathways oversee and direct every
structural and operational aspect of the central nervous system
and, through it, the organism itself. Normally the complex
interplay of the various control systems cooperates to produce a
highly efficient, versatile central nervous system managed by the
brain. Unfortunately, when the biochemical matrix of the central
nervous system is damaged, either through age, disease or other
reasons, the normal regulatory pathways may be incapable of
effectively compensating for the loss. In such cases it would be
highly desirable to modify or supplement the neural mechanisms to
prevent or compensate for such disorders. That is the focus of the
present invention.
[0004] More specifically, the mammalian brain is composed of
approximately ten billion nerve cells or "neurons" surrounded by a
even greater number of support cells known as neuroglia or
astrocyte cells. Neurons, like other cells of the body, are
composed of a nucleus, a cytoplasm and a surrounding cell membrane.
However, unlike other cells, neurons also possess unique, fiberlike
extensions allowing each individual nerve cell to be networked with
literally thousands of other nerve cells to establish a neural
infrastructure or network. Communication within this intricate
network provides the basis for all mental processes undertaken by
an organism.
[0005] In each nerve cell, incoming signals are received by neural
extensions known as "dendrites" which may number several thousand
per nerve cell. Similarly, neural information is projected along
nerve cell "axons" which may branch into as many as 10,000
different nerve endings. Together, these nerve cell axons and
dendrites are generally termed "neurites" through which each
individual neuron can form a multitude of connections with other
neurons. As a result, the number of possible neural connections in
a healthy brain is in the trillions, giving rise to tremendous
mental capacity. Conversely, when the connections within the neural
network break down as nerve cells die or degenerate due to age,
disease, oxidative stress, or direct physical insult, the mental
capacity of the organism can be severely compromised.
[0006] The connection of the individual axons with the dendrites or
cell bodies of other neurons takes place at junctions or sites
known as "synapses." It is at the synapse that the individual
neurons communicate with each other through the flow of chemical
messengers across the synaptic junction. The majority of these
chemical messengers, or "neurotransmitters," are small peptides,
catecholamines or amino acids. When the appropriate stimulus is
received by a neural axon connection, the neurotransmitters diffuse
across the synapse to the adjacent neuron, thereby conveying the
stimulus to the next neuron along the neural network. Based upon
the complexity of the information transferred between the nerve
cells, it is currently believed that between 50 and 100 distinct
neurotransmitters are used to transmit signals in the mammalian
brain.
[0007] Quite recently, it was discovered that nitric oxide (NO) and
carbon monoxide (CO) may function as neurotransmitters. These
gaseous molecules appear to participate in a number of neuronal
regulatory pathways affecting cell growth and interactions. In the
brain, as well as in other parts of the body, CO is produced by the
enzyme "heme oxygenase II" (HO). Whether produced from the HO
enzyme or from other sources, it is believed that when CO diffuses
into a neuron it induces a rise in a secondary transmitter molecule
known as "cyclic guanosine monophosphate" (cGMP), by modulating an
enzyme known as "guanylate cyclase" or "guanylyl" cyclase. Thus, CO
acts as a signaling molecule in the guanylyl cyclase regulatory
pathway. The resultant increase in cGMP levels appears to modify
several neurotropic factors as well as other neuronal factors which
may induce, promote or modify a variety of cellular functions
including cell growth, protection, and intercellular
communication.
[0008] Neurotrophic factors are molecules that exert a variety of
actions stimulating both the development and differentiation of
neurons and the maintenance of cellular integrity and are required
for the survival and development of neurons throughout the
organism's life cycle. Generally, neurotrophic factors may be
divided into two broad classes: neurotrophins and pleiotrophins.
Pleiotrophins differ from the neurotrophins in that they lack a
molecular signal sequence characteristic of molecules that are
secreted from cells and they also affect many types of cells
including neurons. Two effects of neurotrophic factors are
particularly important: (i) the prevention of neuronal death and
(ii) the stimulation of the outgrowth of neurites (either nascent
axons or dendrites). In addition, it appears that CO-induced
neurotrophic factors may reduce the membrane potential of nerve
cells making it easier for the neurons to receive and transmit
signals.
[0009] Many of today's researchers believe that memory is
associated with the modification of synaptic activity, wherein the
synaptic connections between particular groups of brain neurons
become strengthened or facilitated after repeated activation. As a
result, these modified connections activate much easier. This type
of facilitation is believed to occur throughout the brain but may
be particularly prominent in the hippocampus, a brain region which
is crucial for memory. The stimulation of neuronal pathways within
the hippocampus can produce enhanced synaptic transmission through
these pathways for many days following the original stimulation.
This process is known as long term potentiation (LTP).
[0010] More particularly, long term potentiation is a form of
activity-dependent synaptic electrical activity that is exhibited
by many neuronal pathways. In this state, generally accepted as a
type of cellular memory, nerve cells are more responsive to
stimulation. Accordingly, it is widely believed that LTP provides
an excellent model for understanding the cellular and molecular
basis of synaptic plasticity of the type that underlies learning
and memory in vertebrates, including man.
[0011] NO and CO are currently the leading candidates for messenger
substances that facilitate LTP because inhibitors of these
compounds retard the induction of potentiation. The ability to
modify neural activity and to increase the ease of LTP using these
or other signal transducers could potentially increase learning
rates and cognitive powers, possibly compensating for decreased
mental acuity. Prior to the present invention, there were no known
methods or agents which could operate on the cellular level in vivo
to reliably modify cellular and neural regulatory pathways so as to
facilitate the LTP of neurons.
[0012] In contrast to the enhanced mental capacity provided by long
term potentiation, mental functions may be impeded to varying
degrees when the neuronal network is disrupted through the death or
dysfunction of constituent nerve cells. While the decline in mental
abilities is directly related to the disruption of the neural
network, it is important to remember that the disruption is
occurring on an individual cellular level. At this level the
deleterious effects associated with neuronal disruption may be
brought about by any one of a number of factors including
neurodegenerative diseases and disorders, heart attack, stroke,
aging, trauma, and exposure to harmful chemical or environmental
agents.
[0013] Among the known neurological diseases which adversely impact
neuronal function are Alzheimer's disease and related disorders,
Parkinson's disease, motor neuropathic diseases such as Amyotrophic
Lateral Sclerosis, cerebral palsy, multiple sclerosis, and
Huntington's disease. Similar problems may be brought about by loss
of neuronal connectivity due to normal aging or through damage to
neurons from stroke, heart attack, or other circulatory
complications. Direct physical trauma or environmental factors
including chemical agents, heavy metals and the like may also
provoke neuronal or cellular distress, dysfunction, or death.
[0014] Accumulated cellular damage due to oxidative free radicals
is believed to be one of the critical factors in a variety of
cellular and neurodegenerative diseases including Amyotrophic
Lateral Sclerosis, Parkinson's disease, Alzheimer's disease,
cancer, and aging. Most cells possess a variety of protective
mechanisms that guard against cytotoxic free radicals. For example,
high levels of glutathione may protect against free radical
oxidation. Neurons are deficient in this antioxidant source.
[0015] Whatever the cause of the neural disorder or dysfunction,
the general inability of damaged nerve cells to undergo substantial
regrowth or regeneration under natural conditions has led to the
proposal that neurotrophic factors be administered to nerve cells
in order to help restore neuronal function by stimulating nerve
growth and function. Similarly, stimulating neuritogenesis, or the
growth of neurites, by administering neurotrophic factors may
contribute to the ability of surviving neurons to form collateral
connections and thereby restore neural function.
[0016] At present, prior art techniques and compounds have not been
effective or practical to directly administer neurotrophic factors
to a patient suffering from a neural disorder. In part, this is due
to the complex molecular interaction of the neurotrophic factors
themselves and to the synergistic regulation of neural cell growth
and neuritogenesis. Neurotrophic factors are the result of a long
chemical cascade which is exquisitely regulated on the molecular
level by an intricate series of transmitters and receptors.
Accordingly, neuronal cells are influenced by a concert of
different neurotrophic factors, each contributing to different
aspects of neuronal development at different times. Neurotrophic
factors are, effectively, the tail end of this cascade and thus are
one of the most complex components of the regulatory pathway. As
such, it was naive for prior art practitioners to assume that the
unattenuated administration of single neurotrophic factors at
random times (from the cells viewpoint) could substantially improve
cell activity or regeneration. In contrast, modification of the
regulatory pathway earlier in the cascade could allow the proper
growth factors to be produced in the correct relative amounts and
introduced into the complex cellular environment at the appropriate
time.
[0017] Other practical considerations also preclude the prior art
use of neurotrophic factors to stimulate the regeneration of the
neuronal network. Neurotrophic factors (including neurotrophins and
pleiotrophins) are large proteins and, as such, are not amenable to
normal routes of medical administration. For example, these
proteins cannot be delivered to a patient or subject orally as the
patient's digestive system would digest them before they reached
the target neural site. Moreover, due to their relatively large
size, the proteins cannot cross the blood brain barrier and access
the most important neurological site in the body. Alternatively,
the direct injection of neurotrophic factors into the brain or
cerebrospinal fluid crudely overcomes this difficulty but is
fraught with technical problems of its own which have thus far
proven intractable. For example, direct infusion of known
neurotrophins into the brain has proven impractical as it requires
administration over a period of years to provide therapeutic
concentrations. Further, direct injection into the brain has been
associated with dangerous swelling and inflammation of the nerve
tissue after a very short period of time. Thus, as theoretically
desirable as the direct administration of neurotrophic factors to a
patient may be, at the present time, it is unfeasible.
[0018] Accordingly, it is a general object of the present invention
to provide methods and associated compositions and medicaments for
effectively modifying mammalian cells, neurons, cellular activity,
or neural activity to achieve a variety of beneficial results.
These results include protection against oxidative stress and
damage by free radicals and more generalized physiological
responses such as reductions in mammalian blood pressure.
[0019] Thus, it is another object of the present invention to
provide methods and associated compositions and medicaments for
treating mammalian neurological diseases and cellular
disorders.
[0020] It is yet another object of the present invention to provide
methods and associated compositions and medicaments for inducing
long term changes in the membrane potential of a mammalian
neuron.
[0021] It is still another object of the present invention to
provide methods and associated compositions and medicaments for
inducing the in vivo physiological production and administration of
genetically encoded molecules and neurotrophic factors within
cells.
[0022] It is a further object of the present invention to provide
methods and associated compositions and medicaments for enhancing
the neurotogenic effects of neurotrophic factors in a physiological
environment.
SUMMARY OF THE INVENTION
[0023] These and other objects are accomplished by the methods,
compositions, and medicaments of the present invention which, in a
broad aspect, provide for the selective inducement of the in vivo
genetic expression and resultant production of naturally occurring
genetically encoded molecules including neurotrophic factors, and
for the modification of cellular and neural activity through the
treatment of mammalian cells and neurons with at least one carbon
monoxide dependent, guanylyl cyclase modulating, purine
derivative.
[0024] As will be appreciated by those skilled in the art, the in
vivo activation or derepression of genetic expression and the
exemplary modifications of cellular and neural activity brought
about by the methods, compositions, and medicaments of the present
invention may be expressed in a variety of forms or combinations
thereof. For example, the treatment of a mammalian cell or neuron
through the teachings of the present invention may result in the
cell's direct self-administration of the in vivo expressed
molecule(s) through the enhanced cellular production of various
naturally occurring genetically encoded compounds, such as proteins
and neurotrophic factors, or in the stimulation of the activity of
those compounds and their subsequent effect on naturally occurring
cellular or neuronal metabolism, function, development, and
survival. These subsequent effects can include protection from free
radical oxidation and cellular destruction, stabilization of cell
receptors against other factors, the endogenous production of
carbon monoxide and antioxidant compounds, and even reductions in
blood pressure via carbon monoxide activated cellular mechanisms.
The methods and medicaments of the present invention may also
stimulate the growth, development and survival of the cell or
neuron directly without the deleterious effects of prior art factor
methodology. Further, the present invention may be used to lower or
change the membrane potential of the cell, increasing its
plasticity and inducing long term potentiation.
[0025] Exemplary carbon monoxide dependent guanylyl cyclase
modulating purine derivatives useful for practicing the present
invention include guanosine, inosine pranobex and
4-[3-(1,6-dihydro-6-oxo-9-purin-9-yl)-1-o- xopropyl]amino]benzoic
acid (AIT-082). Unlike prior art compounds, these compounds may be
administered directly to a patient either orally or through
injection or other conventional routes. These exemplary compounds
are nontoxic and will cross the blood-brain barrier as well.
[0026] In a further, more specific aspect, the methods and
compositions of the present invention may be used for the treatment
or prophylactic prevention of neurological diseases and other
cellular disorders, including those brought about by disease,
oxidative stress, age, trauma or exposure to harmful chemical
agents. By promoting the survival, growth and development of
individual neurons and cells, the present invention facilitates the
regeneration and development of the neural network and alleviates
the manifestations of cellular and neural dysfunction.
[0027] Of course, those skilled in the art will appreciate that
pharmaceutical compositions and medicaments may be formulated
incorporating effective concentrations of the carbon monoxide
dependent guanylyl cyclase modifying purine derivatives of the
present invention along with pharmaceutically acceptable excipients
and carriers. These pharmaceutical compositions may be administered
orally, transdermally, topically or by injection. Moreover, as the
active agents used in the methods of the present invention can
cross the blood-brain barrier, they do not have to be injected or
infused directly into the brain or central nervous system.
[0028] In yet another aspect, the methods and compositions of the
present invention may be used to induce long term changes in the
membrane potential of a mammalian neuron. These long term
potentiation changes may lead to increased membrane plasticity with
a corresponding enhancement of cellular memory. In turn, this
enhanced cellular memory may elevate the mental capacity of the
subject leading to faster learning and increased retention of
material.
[0029] Other objects, features and advantages of the present
invention will be apparent to those skilled in the art from a
consideration of the following detailed description of preferred
exemplary embodiments thereof taken in conjunction with the data
expressed in the associated figures which will first be described
briefly.
BRIEF DESCRIPTION OF THE DRAWINGS
[0030] FIG. 1 is a graphical representation of murine plasma
concentration following administration of the purine derivative
AIT-082 in accordance with the present invention;
[0031] FIG. 2 is a graphical representation of the effect of
atropine, a cholinergic antagonist, on memory enhancement in mice
by the purine derivative AIT-082;
[0032] FIG. 3 is a graphical representation of nerve growth factor
mediated neurotogenic response in neuronal cells grown in vitro
with various concentrations of the purine derivative AIT-082;
[0033] FIGS. 4A, 4B and 4C are graphical comparisons of the effects
of selective inhibitors and the purine derivative AIT-082 on nerve
growth factor mediated neurotogenic response; FIG. 4A shows the
neurotogenic response of cells grown in the presence of
methemoglobin, a carbon monoxide scavenger; FIG. 4B shows the same
response of cells grown in the presence of methylene blue, a
guanylyl cyclase inhibitor FIG. 4C shows the response of cells
grown in the presence of zinc protoporphyrin IX, a carbon monoxide
scavenger;
[0034] FIGS. 5A and 5B are graphical comparisons of nerve growth
factor mediated neurotogenic response for cells grown in the
presence of the purine derivative AIT-082 and various
concentrations of nitric oxide inhibitors;
[0035] FIG. 6 is a graphical comparison of cyclic GMP production in
neuronal cells grown in culture with the purine derivative AIT-082
and without AIT-082;
[0036] FIG. 7 is a graphical representation of the effects of
different doses of the purine derivative AIT-082 on learning as
measured in Swiss Webster mice using a winshift memory test;
[0037] FIG. 8 is a graphical comparison of the duration of action
of the purine derivative AIT-082 measured over time for single
doses of 60 mg/kg and 30 mg/kg;
[0038] FIG. 9 is a graphical comparison of learning abilities of
age-induced memory deficit Swiss Webster mice treated with the
purine derivative AIT-082 and the drug physostigmine;
[0039] FIG. 10 is a graphical comparison of learning abilities of
age-induced memory deficit C57BL/6 mice treated with the purine
derivative AIT-082 and the drug physostigmine;
[0040] FIG. 11 is a graphical comparison of age-induced memory
deficit prophylaxis in mice treated with the purine derivative
AIT-082 and untreated mice;
[0041] FIGS. 12A and 12B are graphical comparisons of the
production of nerve growth factor by murine cortical astrocytes in
response to the addition of purine derivatives as measured using an
ELISA assay; FIG. 12A illustrates measured nerve growth factor
concentrations for neurons grown in the presence of different
concentrations of guanosine triphosphate and FIG. 12B illustrates
nerve growth factor concentrations for cells grown in the presence
of various concentrations of guanosine;
[0042] FIGS. 13A and 13B are graphical comparisons of the
production of various neurotrophic factor mRNA by murine cortical
astrocyte cells grown in the presence and absence of guanosine at
different times; FIG. 13A illustrates mRNA levels of nerve growth
factor (NGF) and FIG. 13B illustrates mRNA levels of fibroblast
growth factor (FGF);
[0043] FIGS. 14A, 14B and 14C are graphical comparisons of
neurotogenic responses to different concentrations of purine
derivative in the presence and absence of nerve growth factor; FIG.
14A illustrates neurotogenic response to various purine derivatives
at different concentrations in the presence of nerve growth factor,
FIG. 14B illustrates neurotogenic response in the absence of nerve
growth factor and FIG. 14C illustrates neurotogenic response to
individual purine derivatives and combinations of purine
derivatives in the presence and absence of nerve growth factor;
[0044] FIGS. 15A, 15B and 15C are graphical comparisons of nerve
growth factor mediated neurotogenic responses in neurons grown in
the presence of various concentrations of different purine
derivatives; FIG. 15A illustrates neurotogenic response to various
concentrations of inosine; FIG. 15B illustrates the same
neurotogenic response to various concentrations of hypoxanthine and
FIG. 15C illustrates the neurotogenic response of neuronal cells
exposed to different concentrations of xanthine;
[0045] FIG. 16 is a graphical representation of nerve growth factor
mediated neuritogenesis measured for neuronal cells grown at
various concentrations of the purine derivative AIT-034;
[0046] FIG. 17 is a graphical comparison of neurotogenic response
of neuronal cells grown at various concentrations of guanosine
triphosphate and adenosine triphosphate with and without nerve
growth factor;
[0047] FIG. 18 is a graphical comparison of nerve growth factor
mediated neurotogenic response to monophosphate, diphosphate, and
triphosphate purine derivatives of guanosine and adenosine;
[0048] FIG. 19 is a graphical comparison of cyclic GMP produced in
neuronal cells grown in the presence of different concentrations of
the purine derivative guanosine;
[0049] FIGS. 20A, 20B and 20C are graphical comparisons of nerve
growth factor mediated neurotogenic responses of cells grown with
and without the purine derivative guanosine in the presence of
various concentrations of three different inhibitors; FIG. 20A
illustrates the neurotogenic response of cells grown in the
presence of methylene blue, a guanylyl cyclase inhibitor, FIG. 20B
illustrates the neurotogenic response of cells grown in the
presence of various concentrations of LY83583, also an inhibitor of
guanylyl cyclase, FIG. 20C illustrates the neurotogenic response of
cells grown in the presence of various concentrations of atrial
natriuretic factor, a hormone which interacts with guanylyl
cyclase;
[0050] FIG. 21 is a graphical representation of nerve growth
factor-mediated neurotogenic responses for neurons grown in the
presence of sodium nitrate, an inorganic nitric oxide donor;
[0051] FIGS. 22A and 22B are graphical comparisons of nerve growth
factor mediated neurotogenic response of neurons grown in the
presence of nitric oxide donors and scavengers of nitric oxide and
carbon monoxide; FIG. 22A shows the neurotogenic response of cells
grown in the presence of various combinations of nitric oxide
donors and hemoglobin and FIG. 22B shows the neurotogenic response
of cells grown in the presence of various combinations of nitric
oxide donors and methemoglobin;
[0052] FIG. 23 is a graphical comparison showing the nerve growth
factor mediated neurotogenic response of cells grown in various
concentrations of hemoglobin with or without the purine derivative
guanosine;
[0053] FIG. 24 is a graphical comparison showing the nerve growth
factor mediated neurotogenic response of cells grown in various
concentrations of L-nitro arginine methylester (L-NAME) with and
without the purine derivative guanosine;
[0054] FIG. 25 is a graphical comparison of the nerve growth factor
mediated neurotogenic response for cells grown in the presence of
various concentrations of zinc protoporphyrin IX (ZnPP), an
inhibitor of CO synthesis, with and without guanosine;
[0055] FIG. 26 is a negative control for the graphical comparison
shown in FIG. 25 and is a graphical comparison of nerve growth
factor mediated neurotogenic response for cells grown in various
concentrations of copper protoporphyrin IX (CUPP), with and without
the purine derivative guanosine; and
[0056] FIG. 27 is a graphical representation of the nerve growth
factor mediated neurotogenic response for neuron cells grown in the
presence of various concentrations of the purine derivative inosine
pranobex.
DETAILED DESCRIPTION
[0057] In a broad aspect, the present invention is directed to
methods and associated compositions and medicaments for use in
uniquely treating mammalian cells and neurons to modify cellular or
neural activity. More specifically, the present invention is
directed to the use of effective purine derivatives to modulate the
carbon dioxide dependent guanylyl cyclase regulatory system within
cells or neurons to produce a variety of beneficial results,
including the inducement of in vivo genetic expression of naturally
occurring neurotrophic factors and the resultant direct
administration of such naturally occurring genetically encoded
molecules to a mammal, the endogenous production of antioxidant
compounds and carbon monoxide, and the resultant ability to reduce
mammalian blood pressure. Preventing degenerative cellular
destruction and treating disease conditions associated with
cellular damage due to oxidative stress by free radicals also can
be achieved with the methods and compositions of the present
invention.
[0058] In exemplary embodiments illustrative of the teachings of
the present invention, particular purine derivatives were used to
induce genetic expression of encoded molecules, to stimulate
neuritogenesis, to enhance neuronal growth and to modify the
membrane potential of neurons to produce increased learning
capabilities in mammals. Exemplary studies and treatments were
performed as discussed below using various dosages and routes of
administration of selected exemplary purine derivatives
representative of compositions that are effective with the methods
of the present invention. Of course, those skilled in the art will
recognize that the present invention is not specifically limited to
the particular compositions, dosages or routes of administration
detailed below.
[0059] Depending upon the particular needs of the individual
subject involved, the compositions used may be administered as
medicaments in various doses and regimens to provide effective
treatment concentrations based upon the teachings of the present
invention. What constitutes an effective amount of the selected
composition will vary based upon such factors including the
activity of the selected purine derivative, the physiological
characteristics of the subject, the extent and nature of the
subject's neurodegradation or disorder and the method of
administration. Exemplary treatment concentrations which have
proven effective in modifying neural activity range from less than
1 .mu.M to concentrations of 500 mM or more. Generally, initial
doses will be modified to determine the optimum dosage for
treatment of the particular mammalian subject. The compositions may
be administered using a number of different routes including
orally, topically, transdermally, intraperitoneal injection or
intravenous injection directly into the blood stream. Of course,
effective amounts of the purine derivatives may also be
administered through injection into the cerebrospinal fluid or
infusion directly into the brain, if desired.
[0060] The methods of the present invention may be effected using
purine derivatives administered as medicaments to a mammalian
subject either alone or in combination as a pharmaceutical
formulation. Further, the purine derivatives may be combined with
pharmaceutically acceptable excipients and carrier materials such
as inert solid diluents, aqueous solutions or non-toxic organic
solvents. If desired, these pharmaceutical formulations may also
contain preservatives and stabilizing agents and the like.
[0061] The methods and medicaments of the present invention provide
for the controlled long term modification of various types of
cellular or neural activities including the in vivo production of
naturally occurring genetically encoded molecules such as heme
oxygenase and neurotrophic growth factors (including neurotrophins,
pleiotrophins and cytokines), the direct administration of such in
vivo produced molecules, enhancing the effects of these molecules
and neurotrophic factors, and the stimulation of cell growth,
function, protection, and development. Further, the present
invention may be used to promote neuritogenesis, to form collateral
nerve circuits, to enhance the production of cyclic purine
nucleotides, to enhance synapse formation and to alter the membrane
potential of the neuron. These effects may be extremely beneficial
in treating neurodegeneration and in increasing learning capacity.
similarly, inducing the in vivo production of naturally occurring
endogenous antioxidant compounds may be extremely useful in
treating and preventing disease conditions associated with
oxidative damage including cancer, aging, and a variety of
neurological disorders.
[0062] For obvious practical and moral reasons, initial work in
humans to determine the efficacy of experimental compositions and
methods with regard to such afflictions is unfeasible. Accordingly,
in the early development of any drug or therapy it is standard
procedure to employ appropriate animal models for reasons of safety
and expense. The success of implementing laboratory animal models
is predicated on the understanding that the cellular or
neurophysiology of mammals is similar. Thus, a cellular or
neurotropic response in a member of one species, for example, a
rodent, frequently corresponds to the same reaction in a member of
a different species, such as a human. Only after the appropriate
animal models are sufficiently developed will clinical trials in
humans be carried out to further demonstrate the safety and
efficacy of a therapeutic agent in man.
[0063] With regard to neurodegenerative diseases and disorders and
to their clinical effects, the mouse model closely resembles the
human pathology of these conditions in many respects. Accordingly,
it is well understood by those skilled in the art that it is
appropriate to extrapolate the mouse or "murine" model to humans
and to other mammals. As with humans, mice are susceptible to
learning disorders resulting from neuronal degradation, whether due
to traumatic injury, oxidative damage, age, disease or harmful
chemical agents. Just as significantly, neurotropic factors appear
to act in substantially the same manner in a murine model as they
do in humans with remarkably similar neuronal reactions.
Accordingly, for purposes of explanation only and not for purposes
of limitation, the present invention will be primarily demonstrated
in the exemplary context of mice as the mammalian subject. Those
skilled in the art will appreciate that the present invention may
be practiced with other mammalian subjects, including humans, as
well.
[0064] As will be shown by the data herein, several purine
derivatives have been found to work effectively in accordance with
the teachings of the present invention. In particular, the data
shows that guanosine appears to work well in stimulating the
production of neurotrophic factors and enhancing neuritogenesis.
Similarly another exemplary purine derivative,
4-[3-(1,6-dihydro-6-oxo-9-purin-9-yl)-1-oxopropyl]amino]benzo- ic
acid (AIT-082) has been shown to stimulate the in vivo activation
or derepression of naturally occurring genes and the resultant
production of naturally occurring genetically encoded molecules
such as neurotrophic factors. It also induces the endogenous
production of heme oxygenase which in turn induces the endogenous
production of carbon monoxide and bile pigment antioxidant
compounds. AIT-082 also increases neuritogenesis, enhances the
effects of neurotrophic factors and alters the membrane potential
of neurons thereby facilitating long term potentiation of the
cells.
[0065] AIT-082 is disclosed in U.S. Pat. No. 5,091,432 issued Feb.
25, 1992 to a co-inventor of the present application and
incorporated herein by reference. Yet another exemplary composition
which has been shown to be suitable for use in the present
invention is inosine pranobex or isoprinosine. Inosine pranobex, a
mixture of inosine and DIP-PacBa at a 1:3 molar ratio was found to
enhance neuritogenesis and the effects of neurotrophic factors in
vitro. The different embodiments of the present invention presented
above demonstrate the applicability of using various purine
derivatives to modify cellular and neural activity through
modulating the carbon monoxide dependent guanylyl cyclase
system.
[0066] Exemplary preferred embodiments of the present invention
involve the treatment of cells or neurons with AIT-082 or
4-[3-(1,6-dihydro-6-oxo- -9-purin-9-yl)-1-oxopropyl]amino]benzoic
acid. As discussed previously, AIT-082 is a unique derivative of
the purine hypoxanthine containing a para-aminobenzoic acid moiety.
It is rapidly absorbed after oral administration and, after
crossing the blood brain barrier, enters the brain unchanged. It
may be detected at levels as high as 3.3 ng/mg brain tissue 30
minutes after oral administration. AIT-082 induces the in vivo
genetic expression of naturally occurring genetically encoded
molecules including heme oxygenase and neurotrophic factors. As a
result, the present invention is able to induce the direct
self-administration of these compounds to the treated cells and to
stimulate the associated metabolic pathways and resultant
physiological effects. It also stimulates neurite outgrowth from
neuronal cells when added alone to the cultures as well as
enhancing the neurotogenic effects of neurotrophic factors such as
nerve growth factor (NGF). More importantly, AIT-082 enhances
working memory in old, memory deficient mice after either
intraperitoneal and oral administration.
[0067] The neurotogenic activity of AIT-082 is inhibited by
hemoglobin, by Methylene Blue, and by ZnPP, all scavengers of CO,
but not by CuPP or by other inhibitors of nitric oxide synthase.
Screening tests for in vitro activity at known neurotransmitter and
neuromodulator receptors were negative. ZnPP is also known as an
inhibitor of heme oxygenase I which identifies the site of action
of this exemplary guanylyl cyclase modulating purine derivative.
Heme oxygenase is a known heat shock (stress) protein. Heat shock
proteins have been demonstrated to regulate the conformation,
intracellular transport, and degradation of intracellular proteins.
They are also involved in maintaining cellular viability during
stress. Some in the art believe that decreased levels or
functioning of heat shock (stress) proteins may be one of the
factors leading to increased deposition of abnormally folded
proteins and cell death in the brains of Alzheimer's patients.
Thus, the ability of AIT-082 to induce the in vivo production of
heme oxygenase evidences the impact of the present invention on a
variety of protective cellular pathways, indicating the therapeutic
applicability of the present invention to the treatment of heart
attack and stroke in addition to treating other disease
conditions.
[0068] A further understanding of the present invention will be
provided to those skilled in the art from the following
non-limiting examples which illustrate exemplary protocols for the
identification, characterization and use of purine derivatives in
accordance with the teachings of the present invention.
EXAMPLE 1
Plasma Levels of AIT-082 in Mice
[0069] Adult C57BL/6 mice were administered 30 mg/kg of AIT-082 in
saline i.p. The animals were sacrificed by decapitation at 30, 45,
60 and 90 minutes after administration of AIT-082. Blood was
collected in heparinized tubes, mixed and centrifuged at 2000 rpm
for 15 minutes. The plasma supernatant was removed and stored at
-70.degree. C. until analysis. A high pressure liquid
chromatography system was developed for the analytical measurement
of AIT-082 in plasma and brain tissue. The assay developed was
selective for AIT-082 in the presence of a number of closely
related purine molecules. The sensitivity of the method was 0.1
microgram of AIT-082 per ml of plasma and 0.1 microgram of AIT-082
per milligram of brain tissue (wet weight).
[0070] The results of these determinations are shown in Table A and
graphically represented in FIG. 1 where plasma levels of AIT-082
are provided at 30, 45 and 60 minutes after administration of 30
mg/kg i.p. to C57BL/6 mice. From the data, it was estimated that
the blood level of AIT-082 reached its peak at approximately 45
minutes and a plasma elimination half-time of approximately 12
minutes with the k.sub.e1=3.45 hr.sup.-1.
1TABLE A Plasma Levels of AIT-082 Time Level (min) (.mu.g/ml .+-.
S.E. 15 42 .+-. 6 30 108 .+-. 13 45 437 .+-. 131 60 86 .+-. 24 90
20 .+-. 12
EXAMPLE 2
AIT-082 Crosses the Blood Brain Barrier
[0071] Brain tissue was analyzed from two animals receiving 30
mg/kg i.p. of AIT-082 and sacrificed 30 minutes after drug
administration. The brains were rapidly removed and chilled on ice.
Brain tissue was dissected into cortex and remainder of the brain.
Brain tissue (approx. 250-300 mg wet weight) was homogenized with
5.0 ml of saline using a Brinkman Polytron tissue grinder and
stored at -70.degree. C. until analysis. Brain homogenates were
deproteinized by ultrafiltration through Gelman Acrodisc filters;
first through a 1.2 micron filter and then through a 0.2 micron
filter. A 30 .mu.l sample was injected into the HPLC for analysis
as above. A standard curve was prepared by the addition of known
quantities of AIT-082 to brain homogenates from untreated animals.
Analysis of the brain tissue indicated that AIT-082 was detected in
both the cortex sample and the remaining brain samples from both
animals. The results are shown directly below in Table B.
2TABLE B Brain Tissue Levels of AIT-082 Brain wt Level of AIT-082
Sample # Brain Region (mg) (ng/mg brain tissue) S3 Cortex 181 2.8
S3 Remainder 153 3.3 S4 Cortex 146 3.4 S4 Remainder 217 2.3
[0072] This demonstration of the presence of AIT-082 in the brain
tissue after 30 minutes is critical in that it indicates that
AIT-082 crosses the blood-brain barrier without degradation.
EXAMPLE 3
AIT-082 Interacts with the Cholinergis System
[0073] Because of the finding that there is a severe loss of
cholinergic neurons in the hippocampus in Alzheimer's disease
patients, there has been considerable interest in the effect on
memory of compounds which alter the activity of this system.
Support for the cholinergic hypothesis of memory comes from studies
using lesions or a stroke model. Lesions of the CA1 region of the
hippocampus appear to specifically disrupt working memory. In the
stroke model, occlusion of the vertebral and carotid arteries (30
minutes) produces specific cell loss in the CA1 region of the
hippocampus and a loss of working memory. In these models in aged
rats, physostigmine, a cholinesterase inhibitor, has been shown to
improve memory. THA, another drug which increases cholinergic
function, was shown to improve memory in aged monkeys. The
observation that AIT-082 improves memory in the same general manner
as physostigmine and THA raises the question of whether AIT-082
might have some effect on the cholinergic system.
[0074] To elucidate the mechanisms by which AIT-082 improves
memory, attempts were made to block its actions by
co-administration of the short-acting cholinergic antagonist
atropine to mice and subjecting them to simple learning tests.
Atropine reportedly has the ability to block the effects of
physostigmine and THA. Mice were injected with AIT-082 (30 mg/kg) 2
hr prior to testing on days 1 through 4. Atropine (0.5 mg/kg) (28),
was injected 1/2 hour prior to testing or 1.5 hours after AIT-082
on day 3 only. All injections were i.p. After a reference run to
determine where the reward was placed in a T-maze, the mice were
retested to determine if they could remember the location of the
reward. The percentage of correct responses is graphically
represented in FIG. 2.
[0075] FIG. 2 demonstrates that atropine blocked the memory
enhancing activity of AIT-082 on day 3 and that the effect was
transient since the enhancing effects of AIT-082 reappeared on day
4 when no atropine was administered. This observation suggests that
a cholinergic mechanism may be involved in the action of
AIT-082.
EXAMPLE 4
Effect of AIT-082 onN Acetylcholine Receptors
[0076] The interaction of AIT-082 with acetylcholine receptors was
determined by interference with the binding of QNB (3-quinuclidinyl
benzilate) in mouse tissue using the method of Fields [J.Biol.
Chem. 253(9): 3251-3258, 1978]. There was no effect of AIT-082 in
this assay.
[0077] In the study, mice were treated with AIT-082 at 30 mg/kg 2
hours prior to sacrifice, decapitated and the tissue processed to
obtain membranes containing the acetylcholine receptors. When these
tissues were assayed in vitro, there was no effect of AIT-082 on
affinity (Kd) for QNB when AIT-082 was administered under the same
conditions as utilized in testing for effects on memory. There was
a change in the number of receptors (B max) in cortex and striatum,
with the cortex showing a decrease and the striatum an increase in
acetylcholine binding sites. These data are consistent with the
hypothesis that there is an increase input to the cortex as a
result of AIT-082 being administered to the animals. Typically, an
increased input will result in down regulation of the
receptors.
EXAMPLE 5
Effect of AIT-082 on Receptor Ligand Binding in Vitro
[0078] AIT-082 was evaluated for its ability to inhibit ligand
binding to 38 isolated receptors. The receptors screened and their
ligands were:
[0079] Adenosine
[0080] Amino Acids:
[0081] Excitatory Amino Acids (glycine, kainate, MK-801, NMDA, PCP,
quisqualate and sigma);
[0082] Inhibitory Amino Acids (benzodiazepine, GABA-A, GABA-B, and
glycine)
[0083] Biogenic Amines (dopamine-1, dopamine-2, serotonin-1,
serotonin-2)
[0084] Calcium Channel Proteins (nifedipine, omegaconotoxin,
chloride, potassium)
[0085] Peptide Factors (ANF, EGF, NGF)
[0086] Peptides: (angiotensin, arg-vasopressin-V1 and V2, bombesin,
CCK central and peripheral, neurotensin, NPY, somatostatin,
substance K, substance P, VIP)
[0087] Second Messenger Systems:
[0088] Adenyl Cyclase
[0089] Protein Kinase (phorbol ester and inositol triphosphate)
[0090] The testing was conducted under contract at Nova Labs
(Baltimore, Md.). AIT-082 had no activity in any of the in vitro
assays conducted.
[0091] Accordingly, while AIT-082 acts through the cholinergic
nervous system (atropine blocks its activity), AIT-082 appears to
act through a mechanism that does not involve direct interaction
with acetylcholine receptors. It is of importance to note that in
vitro, AIT-082 does not bind to the adenosine receptor.
[0092] AIT-082 was evaluated in a series of psychopharmacological
tests that were established in order to more fully evaluate the
scope of its central nervous system activity. Among the tests
utilized were:
[0093] (a) motor coordination, by the accelerating Rota-Rod
treadmill,
[0094] (b) exploratory and home cage locomotor activity, by the
Stoelting activity monitor,
[0095] (c) anxiolytic activity, by the elevated Plus maze, and
[0096] (d) nocioception.
[0097] AIT-082 was compared with standard reference drugs.
EXAMPLE 6
AIT-082 Increases Motor Coordination in Mice
[0098] Motor coordination was measured using an accelerating
Rota-Rod treadmill for mice (Ugo Basile Co.). At various times
after treatment with saline or drug, mice were placed on the
Rota-Rod, which accelerates to maximum speed over a 5 minute
period. The time in seconds at which the subject falls off was
recorded in Table C directly below. Each animal was tested 3 times
and the mean time was recorded.
3TABLE C Effect of AIT-082 on Roto-rod performance AIT dose Time
(mg/kg) (sec) Control 123 .+-. 64 0.005 162 .+-. 93 0.05 207* .+-.
73 0.5 184* .+-. 76 30.0 187* .+-. 68 60.0 229* .+-. 80 *p <
0.05, t-test vs controls
[0099] Subjects receiving AIT-082 showed improved motor
coordination by remaining on the roto-rod for longer periods of
time when compared to control (saline) or low doses (0.005
mg/kg).
EXAMPLE 7
AIT-082 Does not Inhibit Exploratory Activity
[0100] To measure exploratory behavior, subjects received saline or
AIT-082 administration, were placed in a novel large cage
(25.times.48.times.16 cm, W.times.L.times.H), and movement was
measured at one-minute intervals for 30 minutes. The large cage
(San Diego Instruments, San Diego, Calif.) was equipped with
vertical detectors and rearing movements were also recorded. No
effects were noted with respect to exploratory activity indicating
that the subjects were not incapacitated.
EXAMPLE 8
AIT-082 Does not Inhibit Locomotor Activity
[0101] To measure home cage locomotor activity, the home cage was
placed on a platform of an activity monitor (Stoelting
Instruments). Home cage locomotor activity movements were recorded
at one minute intervals for 15 minutes. Subjects received saline or
AIT-082 and were returned to their home cages. Ten minutes after
injection, the home cage was replaced on the platform of the
activity monitor. Home cage locomotor activity movements were
recorded at one minute intervals for 30 minutes. During the first
five minutes, grooming activity was also monitored and recorded.
The results are shown in Table D directly below.
4TABLE D Effect of AIT-082 on locomotor activity Movements AIT dose
(mean .+-. S.D.) (mg/kg) Pre-drug Post-drug Difference Control 1633
.+-. 434 1385 .+-. 492 248 .+-. 492 0.005 1884 .+-. 230 1375 .+-.
563 509 .+-. 429 0.05 1718 .+-. 606 1508 .+-. 456 209 .+-. 340 0.5
1610 .+-. 349 1320 .+-. 689 290 .+-. 435 30.0 1440 .+-. 264 1098
.+-. 189 342 .+-. 267 60.0 1690 .+-. 223 634* .+-. 223 1056* .+-.
154 *p < 0.05, t-test vs controls
[0102] As shown by the data in Table D, at the high dose (60
mg/kg), subjects may have become more habituated to their
environment and exhibited less movement after treatment with
AIT-082. Otherwise, no effects were noted.
EXAMPLE 9
AIT-082 Does not Substantially Increase Anxiety
[0103] A Plus maze was constructed of black plexiglass consisting
of two opposite-facing open arms (30.times.5 cm, L.times.W) and two
opposite facing closed arms (30.times.5.times.15 cm,
L.times.W.times.H). The walls of the closed arms were clear
plexiglass and the four arms were connected by a central area
5.times.5 cm. The entire Plus maze was mounted on a base 38 cm
above the floor. Testing consisted of placing the subject at one
end of one of the open arms. The time the subject took to leave the
start position (the first 10 cm of the open arm) was recorded. The
time it took for the subject to enter halfway into one of the
closed arms was also recorded. When the subject arrived at the
half-way point in the closed arm, the three-minute test session
began. During the three-minute test session, the number of times
the subject entered the open arms was recorded. An entry was
defined as placing at least two paws onto the platform of the open
arm. There was a slight anxiogenic effect of AIT-082 at 30 mg/kg,
but this was not observed at a higher dose (60 mg/kg) or at the
lower doses (0.005 to 0.5 mg/kg).
EXAMPLE 10
AIT-082 Does not Efffect Nocioception
[0104] Mice were placed on an electric hot plate (Omnitech) at
55.degree. C. and the latency time until the subject licked his
hind paw was measured. If there was no response by 45 seconds, the
trial was terminated. By this test there was no effect of AIT-082
on nocioception.
EXAMPLE 11
AIT-082 Is not Toxic
[0105] Preliminary acute toxicity tests in rats and mice of AIT-082
have demonstrated that the LD.sub.50 is in excess of 3000 mg/kg
when administered by the oral or intraperitoneal route. AIT-082 has
been evaluated under Panlabs's General Pharmacology Screening
Program (Panlabs, 11804 North Creek Parkway South, Bothwell, Wash.
98011) and the results indicated an absence of any toxicity when
measured in their standard profile of 79 different test
systems.
[0106] By the nature of the chemical structure of AIT-082, it is
not anticipated that the compound will be metabolized into any
toxic metabolites.
[0107] In conclusion, there were few deleterious effects of AIT-082
on a variety of psychopharmacological tests except for a slight
anxiogenic effect at one dose. There was an increase in motor
coordination (roto-rod test) over a range of doses (0.05 to 60
mg/kg) and possibly a learning or habituation effect at one dosage
(60 mg/kg) in the locomotor test.
[0108] Following psychopharmacological characterization of this
exemplary compound, further studies were conducted to demonstrate
the neurogenic effects of the present invention.
EXAMPLE 12
AIT-082 Promotes Neuritogenesis in PC12 Cells
[0109] Much of the work performed in the characterization of the
compounds of the present invention involved the use of PC12 cells.
These cells are derived from a rat pheochromocytoma and when grown
in the presence of NGF, extend neurites, cease cell division and
assume many characteristics of sympathetic neurons. When cultured
in the absence of nerve growth factor (NGF), few PC12 cells have
neurites greater than one cell diameter. Addition of saturating
concentrations of NGF for 48 hours stimulates neurite outgrowth in
about 20-35% of the cells. Because they constitute a homogeneous
population of neuronal-like cells, without contaminating astroglia
type cells, it is possible to study the direct effects of the
purine based compounds on neurite outgrowth in these cells.
[0110] To demonstrate neuronal modification by the exemplary
compounds of the present invention, a dose response curve of
AIT-082 was generated measuring the stimulation of neuritogenesis
in PC12 cells. Cells cultured in RPMI 1640 with 1.5% horse serum
and 1.5% fetal bovine serum were replated onto poly-ornithine
coated 24-well culture plates (2.5.times.10.sup.4 cells per well).
AIT-082 and NGF were added to the various cultures immediately upon
plating. After 48 hours, medium was removed and the cells
immediately fixed in 10% formalin and PBS for 10 minutes. Cells and
neurites were counted within 2 days of fixation.
[0111] A neurite was defined as a process extending from the cell
at least 1 cell body diameter in length and displaying a growth
cone at its tip. For each treatment, 2 representative microscope
fields were counted from each of 6 sister cultures receiving
identical treatments. The total number of cells counted per well
(approximately 100 cells) and the total number of cells containing
neurites in each well were used to determine fraction of
neurite-bearing cells. The mean values (.+-.SEM) were then
determined for each of the treatments. To facilitate comparison
neurite outgrowth was expressed relative to the proportion of cells
bearing neurites in the presence of NGF alone (NGF=100%). The
effects of compounds with and without NGF were compared by analysis
of variance (ANOVA) followed by Tukey's test for significance.
[0112] The results are shown in FIG. 3 where the curve represents
different levels of AIT-082 plus saturating concentrations (40
ng/ml) 2.5 S NGF. The center horizontal line represents control
values for cells cultured in the presence of 40 ng/ml NGF alone.
Upper and lower horizontal lines are indicative of confidence
limits of NGF alone as determined using standard statistical
methods.
[0113] As shown in FIG. 3, AIT-082 stimulates neuritogenesis and
enhances NGF-stimulated neuritogenesis in PC12 cells at low
concentrations (1 .mu.M). Analysis of the data shows that AIT-082
was as effective as NGF in promoting neuritogenesis in PC12 cells
and enhanced the optimal effects of NGF by 30%. For the purposes of
comparison, and as will be discussed in more detail below, inosine
and hypoxanthine are weakly effective in stimulating neuritogenesis
and in enhancing NGF-stimulated neuritogenesis in PC12 cells but
are effective at lower concentrations of 30-300 nM. Guanosine
produces a significant effect similar to AIT-082 but at a higher
concentration of 30-300 .mu.M.
EXAMPLE 13
Effect of Inhibitors on AIT-082 Neuritogenesis
[0114] Age-related memory loss has been associated with loss of
NGF-dependent basal forebrain neurons. It can be ameliorated by
i.c.v. infusion of NGF. The effect of AIT-082 on neuritogenesis
alone and with NGF were studied using the protocol of Example 12.
In order to study the mechanism by which AIT-082 exerts its
effects, a series of experiments were conducted in which inhibitors
were utilized to block or modify specific biochemical processes.
All of the cultures contained NGF at optimal dose (40 ng/ml) so the
series without AIT-082 added represented the effect of the
inhibitors on NGF activity. Where indicated, AIT-082 was added at
10 .mu.M, its apparent, presently understood, optimal dose. Three
selective inhibitors were used.
[0115] The results of these studies are shown below in Table E
below, and FIGS. 4A, 4B, and 4C graphically present the proportion
of cells bearing neurites after 48 hours culture under the
conditions indicated. The base line value was cells grown without
NGF or AIT-082.
5TABLE E Effect of AIT-082 and selective inhibitors on
neuritogenesis alone and with NGF Con- AIT-082 AIT-082 + Inhibitor
centration along.sup.1 NGF alone NGF None 0.2 .+-. 0.02 0.2 .+-.
0.02 0.26 .+-. 0.01 Methemoglobin 0 0.2 .+-. 0.02 0.26 .+-. 0.01 1
.mu.M 0.2 .+-. 0.02 0.17 .+-. 0.02 Methylene Blue 0 0.2 .+-. 0.02
0.26 .+-. 0.01 5 .mu.M 0.24 .+-. 0.03 0.10 .+-. 0.01 Zn 0 0.20 .+-.
0.02 0.26 .+-. 0.01 Protoporphyrin IX 1 .mu.M 0.22 .+-. 0.03 0.13
.+-. 0.01 .sup.1Proportion of cells bearing neurites
[0116] Methemoglobin (MHb) captures and removes nitric oxide (NO)
and carbon monoxide (CO) from the culture media. MHb had no effect
on NGF activity but inhibited the action of AIT-082, implying that
either NO or CO is involved in the action of AIT-82.
[0117] Methylene blue (MB) inhibits soluble guanylyl cyclase, the
enzyme which produces cyclic GMP (cGMP) a intracellular substance
which, as previously discussed, is involved in the second messenger
system of nerve impulse transmission. MB had no effect on NGF
activity but inhibited the action of AIT-082, implying that
guanylyl cyclase is involved in the mechanism of action of
AIT-082.
[0118] Zinc protoporphyrin IX (ZnPP) is an inhibitor of heme
oxygenase II (HO) which in turn produces CO. ZnPP had no effect on
NGF activity but inhibited the action of AIT-082. This identifies a
potential site of action of AIT-082 as involving the production of
HO. It also identifies the resultant production of CO as part of
the mechanism of action of AIT-082. These results are indicative of
a number of important aspects and features of the present
invention.
[0119] As previously discussed, Ho is a heat shock (stress)
protein. These proteins are believed to be part of protective
mechanisms necessary to maintain cellular viability during stress
(see, e.g. Georgopoulos, C. and W. J. Welch, Role of the major heat
shock proteins as molecular chaperones. Annu. Rev. Cell Biol.,
1993. 9: p. 601-634). In addition to regulating the confirmation,
intracellular transport, and degradation of intracellular proteins,
Ho is also strongly implicated in the production of potent
antioxidant compounds produced by the degradation of heme through
the enzymatic activity of HO. In mammalian tissue the sole source
of the protective antioxidant bile pigments biliverdin and
bilirubin is heme degraded by HO. There is substantial evidence
that these bile pigments play an important physiological role in
cellular antioxidant defense mechanisms (Stocker, P., et al.,
Bilirubin is an antioxidant of possible physiological importance.
Science, 1987. 235: p. 1043-1047). Moreover, HO is found in the
brain, and its level is known to greatly increase after heat shock
(Ewing, J. F., S. N. Haber, and M. D. Maines, Normal and
heat-induced patterns of expression of heme oxygenase-1 (HSP32) in
rat brain: hyperthermia causes rapid induction of mRNA and protein.
J. Neurochem., 1992. 58: p. 1140-1149).
[0120] Consistent with this understanding, a number of
neurotrophins exert their neuro-protective effects by stimulating
endogenous defenses against oxidative stress and damage by free
radicals (Williams, L. R., Oxidative stress, age-related
neurodegeneration, and the potential for neurotrophic therapy, in
Cerebrovasc. Brain Metab. Rev. 1995, Raven Press, Ltd.: New York,
N.Y. p. 55-73. Mattson, M. P., B. Cheng, and V. L. Smith-Swintosky,
Mechanisms of neurotrophic factor protection against calcium and
free radical-mediated excitotoxic injury: implications for treating
neurodegenerative disorders. Exp. Neurol., 1993. 124: p. 89-95).
Because cytotoxic free radicals are suspected in the etiology of a
variety of neurodegenerative diseases, it is reasonable to conclude
that the present invention is able to stimulate the production or
activity of HO to produce protective antioxidant compounds which
function to prevent degenerative cell destruction by oxidative free
radicals through the neutralization or sequestration of these toxic
oxidative compounds.
[0121] Currently, it is believed by many skilled in the art that
ALS, Parkinson's disease, and Alzheimer's disease may result from
an inability to protect against accumulated cellular damage be free
radicals. Some practitioners skilled in the art have experimented
with the treatment of ALS through the administration of
neurotrophins (DiStefano, P. S., Neurotrophic factors in the
treatment of motor neuron disease and trauma. Exp. Neurol., 1993.
124: p. 56-59. Thoenen, H., R. A. Hughes, and M. Sendtner, Trophic
support of motorneurons: Physiological, pathophysiological, and
therapeutic implications. Exp. Neurol., 1993. 124: p. 47-55).
Because the present invention is able to endogenously produce these
protective compounds it provides an effective treatment for these
and other degenerative cellular conditions. This protective ability
is particularly important for the treatment of neurons because,
unlike most cells which possess a variety of protective mechanisms,
such as high levels of glutathione, neurons are deficient in this
antioxidant source.
[0122] The ability of the present invention to stimulate the
activity or production of HO has at least one additional direct
physiological benefit in mammals. When heme is degraded by HO into
the bile pigments bilirubin and biliverdin, Co is also produced.
Thus, the ability of the present invention to stimulate HO
production and activity also provides the present invention with
the ability to stimulate the in vivo cellular production of CO. CO
is known as an activator of soluble guanylate cyclase and relaxes
vascular smooth muscle via a cGMP-dependent mechanism (Graser, T.,
Y. P. Verdernikov, and D. S. Li, Study of the mechanism of carbon
monoxide induced endothelium-independent relaxation in porcine
coronary artery and vein. Biomed. Biochim. Acta, 1990. 49: p.
293-296. Morita, T., et al., Smooth muscle cell-derived carbon
monoxide is a regulator of vascular cGMP. Proc. Natl. Acad. Sci.
USA, 1995. 92: p. 1475-1479). Recently, it was demonstrated by
others in the art that withdrawal of the blood pressure lowering
effects of CO through inhibition of HO resulted in an increase in
mammalian blood pressure (Johnson, R. A., et al., A heme oxygenase
product, presumably carbon monoxide, mediates a vasodepressor
function in rats. Hypertension, 1995. 25: p. 166-169). Accordingly,
it is reasonable to conclude that the ability of the present
invention to stimulate the production of CO in vivo will reduce
mammalian blood pressure in addition to providing increased
antioxidant protection. The ability of the present invention to
directly induce the in vivo production of naturally occurring
cellular compounds producing these dramatic physiological effects
is unprecedented in the art.
EXAMPLE 14
Effect of Nitric Oxide Inhibitors on AIT-082
[0123] Nitric oxide is produced by the action of the enzyme nitric
oxide synthetase (NOS). Two chemicals that have been shown to
selectively inhibit NOS are N-nitro-L-arginine methyl ester
(L-NAME) and N-nitro-L-arginine (NOLA). Different levels of these
chemicals were administered simultaneously with AIT-082 and
neuritogenesis in PC12 was measured using the protocol of Example
12. The results for L-NAME are presented in Table F while the
results for NOLA are presented in Table G. Both tables are shown
directly below with graphical representations of the data presented
in FIGS. 5A and 5B.
6TABLE F The effect of L-NAME on neuritogenesis AIT- Concentration
of L-NAME (.mu.M) 082 None 0.1 1.0 10.0 0 0.246 .+-. 0.017 0.259
.+-. 0.027 0.257 .+-. 0.013 0.251 .+-. 0.013 10 0.254 .+-. 0.008
0.220 .+-. 0.010 0.302 .+-. 0.027 0.254 .+-. 0.018 .mu.M 100 0.309
.+-. 0.027 0.257 .+-. 0.016 0.232 .+-. 0.019 0.289 .+-. 0.006
.mu.M
[0124]
7TABLE G The effect of NOLA on neuritogenesis AIT- Concentration of
NOLA (.mu.M) 082 None 0.1 1.0 10.0 0 0.246 .+-. 0.017 0.259 .+-.
0.009 0.311 .+-. 0.016 0.305 .+-. 0.017 10 0.254 .+-. 0.008 0.277
.+-. 0.016 0.312 .+-. 0.029 0.298 .+-. 0.019 .mu.M 100 0.309 .+-.
0.027 0.279 .+-. 0.027 0.295 .+-. 0.028 0.310 .+-. 0.023 .mu.M
[0125] As shown by the data in Tables F and G, neither of these
inhibitors of NOS were active in blocking the effect of AIT-082 on
neuritogenesis. These results indicate that NO was not involved in
the mechanism of action of AIT-082.
EXAMPLE 15
EFFECT OF AIT-082 ON cGMP LEVELS IN PC-12 CELLS
[0126] To demonstrate CO-dependent guanylyl cyclase modification,
cyclic guanosine monophosphate (cGMP) levels in PC12 cells were
measured following addition of AIT-82. Initially, PC-12 cells were
primed with 40 ng/ml NGF for 3 days in low serum medium (1.5% horse
serum+5% fetal calf serum). Cells were seeded onto assay plates in
low serum medium containing 40 ng/ml NGF and incubated for 1 hour.
The medium was changed to low arginine medium (80 .mu.M) with no
serum and NGF and papaverine (100 .mu.M) where indicated. Test
compounds were added for the indicated time and the reaction was
stopped by adding 5% TCA containing 10,000 dpm of .sup.3H-cGMP.
cGMP was assayed by the radioimmunoassay method of Maurice [Mol.
Pharmacol. 37: 671-681, 1990]. TCA was purified by adding powdered
charcoal (5 g) and filtering the mixture through Whatman #1 paper.
This removed contaminants in the TCA that otherwise interfere with
the radioimmunoassay (RIA) of CGMP.
[0127] It was necessary to purify the cGMP from cAMP and other
contaminants before radioimmunoassay since these other materials
can interfere with the assay. Briefly, the TCA solution was applied
to Dowex columns (50W-8X, 200-400 mesh) and eluted. A neutral
alumina column was then placed under each Dowex column. The cGMP
was eluted from the Dowex columns into neutral alumina columns by
adding 4 mL of 0.05M HCl to each Dowex column. The neutral alumina
columns were then sequentially rinsed with 2 ml of HCl, 4 mL water
and finally with 0.2M sodium acetate (pH 6.2). The cGMP collected
for the RIA, eluted in 1 mL of sodium acetate with a recovery
between 50-65%. The cGMP was assayed using a Dupont RIA kit. The
results are graphically presented in FIG. 6.
[0128] As shown in FIG. 6, the addition of AIT-082 increased the
production of cGMP in PC12 cells indicating that AIT-082 acts by
modifying the activity of the carbon monoxide-dependent enzyme
guanylyl cyclase.
EXAMPLE 16
Effect of AIT-082 on Genetic Expression of Neurothrophin mRNA
[0129] To demonstrate that AIT-082 induced the in vivo genetic
expression and resultant cellular production of neurotrophins,
naturally occurring, genetically encoded molecules, as well as
enhancing their activity, the following experiment was performed.
Induction of neurotrophin mRNA was determined by northern blot
analysis of astrocytes cultured with AIT-082, NGF, or both. The
cells were harvested and RNA extracted at 24 hours after
treatment.
[0130] More particularly, astrocytes from the cerebral cortex of
NIH Swiss mice (Harlan) were isolated. Briefly, newborn pups (0-24
hours) were decapitated. Their brains were removed under aseptic
conditions and were placed in modified Dulbecco's medium (DMEM)
containing 20% heat-inactivated horse serum (Hyclone)--("complete
medium"). The neopallium was then dissected from each cerebral
hemisphere and minced into 1 mm cubes.
[0131] The astrocytes were then isolated by mechanical
dissociation. The cubes were vortexed at maximum speed for one
minute. The cell suspension was then passed first through 75 mm
Nitex then through 10 mm Nitex. The resulting cell suspension was
diluted in complete medium to a final concentration of one brain
per 10 ml of complete medium. Ten milliliters of the diluted cell
suspension, was added to each 100 mm Falcon tissue culture plate
(Fisher). After 3 days the medium was replaced with 10 ml fresh
complete medium and subsequently was replaced twice weekly with
DMEM containing 10% heat inactivated horse serum--("growth
medium"). After two weeks in culture the astrocytes formed a
confluent monolayer.
[0132] For RNA extraction, astrocytes were trypsinized. The
astrocytes were then replated onto 100 mm PORN coated plates at a
cell density of 10.sup.6 cells per plate (10 ml growth medium).
After 2 hrs PBS, Guo, or GTP at 10 mM were added to the appropriate
plates. Total RNA was harvested from 1.5.times.10.sup.7 cells for
each treatment, 4 and 24 hrs after treatment using TRIzol reagent
and supplier protocol (GIBCO BRL/Life Technologies, Inc.). For slot
blots, total RNA was bound to Hybond-N filters (Amersham/United
States Biochemicals) as described in Transfer and Immobilization of
Nucleic Acids and Proteins to S & S Solid Supports (S and S
protocols: Schleicher & Schuell, New Hampshire, USA). Northern
blots were also performed using 10-20 mg total RNA from each
sample. These were electrophoresed in 1% agarose gels containing
formaldehyde and blotted onto Hybond-N filters according to S and S
protocols.
[0133] The blots were probed with P.sup.32-labelled cDNA (NGF, NT-3
and BDNF probes) or oligonucleotide probe (FGF-2) by hybridization
in Piperazine-N,N'-bis-[2-ethanesulfonic acid] (PIPES) buffer (50
mM PIPES, pH 6.8; 50 mM NaH.sub.2PO.sub.4; 0.1 M NaCl; 5% SDS and 1
mM EDTA) overnight at 50.degree. C. The blots were then washed
twice with (2.times.SSC, 0.1% SDS) wash buffer at room temperature
for 20 minutes each, and then with (0.1.times.SSC, 0.1% SDS) wash
buffer twice at 52.degree. C. for 20 minutes each. 1.times.SSC is
0.15M NaCl and 15 mM sodium citrate, pH 7.0. Damp membranes were
wrapped in Saran wrap and autoradiography was performed using
Hyperfilm-MP (Amersham/USB) and a cassette with intensifying
screens. Various concentrations (0.25 to 4 mg of total RNA), as
determined by spectrophotometry, of each sample were blotted and
probed so that quantification could be done after insuring a linear
film response. Quantification was performed using MCID Image
Analysis (St. Catherine's, Ontario, Canada).
[0134] To provide probes, a cDNA clone of the mouse NGF gene in the
plasmid pGEM.NGF(+), and cDNA clones of human NT-3 and BDNF in
Bluescript were isolated. After isolation, the cDNA probes were
labeled with .sup.32P-dCTP (ICN Biomedicals Canada, Ltd.) using a
Random Primed DNA Labeling Kit (Boehringer Mannheim Biochemica) as
described in the kit.
[0135] A 40-mer antisense oligonucleotide was synthesized (MOBIX,
McMaster University) as a probe for FGF-2. This was complementary
to the 5' end of mouse FGF-2 coding sequence on the mRNA. The oligo
was 5' end-labeled using polynucleotide kinase, One-Phor-All
buffer, and the protocol supplied by Pharmacia Biotech Inc., and
ATPgP.sup.32 (ICN Biomedicals Canada, Ltd.).
[0136] The results of the study for the production of four
different neurotrophic factors are shown below in Table H.
8TABLE H Northern Blot analysis of neurotrophin mRNAs from
Astrocytes Neurotrophin NGF AIT-082 AIT-082 (100 mM) + mRNA Control
40 ng/ml 100 mM NGF (40 ng/ml) NGF - - ++ + FGF-2 + - ++ + BDNF + +
+ + NT-3 - - ++ + The conditions which produced a detectable amount
of each of the neurotrophin mRNAs are indicated by a "+", with a
"++" indicating that at least twice the detectable amount was
present. Those blots which were negative are indicated by a
"-".
[0137] The results indicate that AIT-082 induced the expression of
mRNAs for several neurotrophic factors, including NGF. More
importantly, these data clearly establish that AIT-082 selectively
and controllably induced the in vivo genetic expression of at least
one naturally occurring genetically encoded molecule in a mammal
treated in accordance with the teachings of the present invention.
Administering this exemplary purine derivative selectively induced
the expression of mRNA encoding three of the four identified
neurotrophic factors, NGF, FGF-2, and NT-3, but did not induce
activation or derepression of the gene encoding for BDNF mRNA. This
selective control coupled with the ease of administration provided
by the compounds and methods o the present invention effectively
overcomes the limitations of the prior art. Rather than
administering these molecular compounds directly to cells through
complex and potentially dangerous techniques, the present invention
is able to treat a mammalian patient utilizing traditional,
noninvasive drug delivery routes that induce the treated cells to
express the genetic material encoding the desired compounds
resulting in their direct in vivo delivery and administration.
Though potentially useful in conjunction with modified genes or
other molecular biology techniques, with the present invention,
genetic modification is unnecessary.
[0138] It has been shown previously that, within the hippocampus
from Alzheimer's patients, there is an altered program of gene
expression leading to aberrant levels of mRNA for neurotrophic
factors. A number of animal and clinical studies have demonstrated
that administration of single neurotrophins are inadequate to treat
neurodegenerative disease. Accordingly, the ability of the
compounds of the present invention to stimulate the production of
multiple neurotrophin mRNAs within cells substantially increases
their potential as treatments for a variety of neurodegenerative
diseases by providing a method for the effective direct
administration of these naturally occurring genetically encoded
molecules to a patient through the induction of their in vivo
genetic expression.
[0139] The preceding examples show that AIT-082 stimulates
neuritogenesis in vitro in PC12 cells alone and enhances the effect
of nerve growth factor (NGF). Further, the neurotogenic effect of
AIT-082 was reduced by methemoglobin (which captures and removes
nitric oxide and carbon monoxide), methylene blue (which inhibits
guanylyl cyclase), and by zinc protoporphyrin IX (an inhibitor of
heme oxygenase, which produces carbon monoxide). The neurotogenic
effect of AIT-082 was unaffected by L-NAME or NOLA, inhibitors of
NO production. In addition, AIT-082 stimulated the production of a
number of different neurotrophic factors as evidenced by increased
mRNA levels of these factors in astrocytes after AIT-082
administration in vitro. Moreover, since AIT-082 is orally active
and rapidly passes the blood-brain barrier as shown in Example 2,
it has significant therapeutic potential as an NGF-mimetic agent in
Alzheimer's disease and in other neurodegenerative and cellular
diseases.
[0140] In view of the previous results, studies were performed to
demonstrate the effectiveness of using AIT-082 to treat exemplary
neurodegenerative diseases. Loss of memory represents the core
symptom of Alzheimer's disease as it does in a number of other
neural afflictions. Specifically working (or episodic) memory is
impaired in Alzheimer's disease, amnesia, aging and after
hippocampal lesions in monkeys. The effects of AIT-082 in
ameliorating this memory loss was used to demonstrate the efficacy
of the compounds of the present invention with respect to the
treatment of neurodegenerative diseases.
EXAMPLE 17
Comparison of Memory Trace in Different Mice Strains
[0141] The win-shift T-maze paradigm has been shown to specifically
model working memory in rodents and is a widely accepted method.
The rodent's natural behavior is to forage for food when hungry and
therefore it will not return to the same location after it has
consumed any food that was present. This model was not designed to
account for all of the vast data on memory. Data from hypoxia and
ischemia studies, procedures which selectively damage CA1
hippocampal cells, produce deficits in working memory while other
types of memory are not affected. This strongly suggest that there
are several types of memories which have different anatomical sites
and most likely different neurochemical inputs. Accordingly, while
the win-shift model may not account for all neurochemical inputs
involved in working memory, the model does provide a useful art
accepted tool in designing pharmacological experiments to provide
information on the mechanism by which memory can be modified.
[0142] Male Swiss Webster mice six months (young adult) and eleven
months (old) of age, obtained from the National Institute on Aging,
were maintained in individual cages, on a 22 hour light/dark cycle
with continuous access to water. Food was limited so that the mice
stabilized at 80% of free feeding weight. Mice were weighed and
handled daily for one week. The win-shift model was run as
described in the literature and consists of a T-maze in which the
correct response alternates after each correct trial. The interval
between trials is varied and allows for the determination of the
longest period between trials that a subject can remember the
correct response on the previous trial. This allows the measure of
the duration of the memory trace. A score of 5 (5 correct responses
per 10 trials, 50% correct) is considered chance; that is, the
animal does not remember which box it selected for positive reward
on the previous trial. The reward goal box is alternated after each
correct trial. Ten trials per mouse are run each day. If the animal
establishes a spatial learning set (right side only), they would
return to the same goal each trial and have a correct response rate
of significantly less than 50% correct. The latency time to leave
the start box is recorded as a measure of motivation, the running
time (the time from leaving the start box to reaching the goal box)
is recorded as a measure of performance, and the number of correct
responses as a measure of memory.
[0143] The data in Table I illustrate the effect of increasing the
inter-trial interval in young adult mice without any drug
treatment.
9TABLE I Effect of inter-trial interval in win-shift
paradigm.sup.(1) Inter-trial Interval (seconds) 30 60 90 120 150
Swiss Webster mice 7.5* 7.5* 5.0 C57BL/6 mice 7.0* 7.4* 7.0* 7.8
5.6 .sup.(1)Score is the mean number of correct responses per 10
trials. Saline was administered 1 hour prior to testing. *p <
0.05. Data analysis following significant ANOVA, a Dunnett test was
run comparing drug tested groups with controls. An Arc Sign
transformation was performed on percentage data.
[0144] From the data in Table I, it can be seen that Swiss Webster
mice are capable of remembering the win-shift strategy when the
inter-trial delay interval is 30 or 60 seconds. Few mice with
saline treatment scored above chance (50%) with the 90-second
inter-trial delay interval. These data indicate that the "memory
trace" in these animals disappears between 60 and 90 seconds All
drug evaluation tests in normal adult Swiss Webster mice were
conducted with the 90-second inter-trial interval except where
indicated otherwise. In C57BL/6 mice, the duration of the memory
trace was 120 seconds.
EXAMPLE 18
Effect of AIT-082 on Memory Trace Duration
[0145] The activity of AIT-082 was compared with tacrine (THA) and
physostigmine (PHY), experimental anticholinesterase agents which
enhance memory in animals. The drugs were also evaluated for their
effects on locomotor activity. In the win-shift memory paradigm,
AIT-082 was evaluated for its ability to induce tolerance after 18
days of drug administration. In addition AIT-082 was tested for its
activity to modify learning in a modified T-maze discrimination
task.
[0146] The drugs used in this example are
4-[[3-(1,6-dihydro-6-oxo-9-purin- -9-yl)-1-oxopropyl]amino] benzoic
acid (AIT-082), as an exemplary potassium salt, tacrine
hydro-chloride (tetrahydroaminoacridine, THA, Sigma Chemical Co.,
St. Louis, Mo.), and physostigmine, hemisulfate salt (PHY, Sigma
Chemical Co., St. Louis, Mo.). The drugs were dissolved in saline
and prepared fresh daily. All injections were made at a volume of
0.1 ml/10 grams body weight. When testing drug effects,
intraperitoneal (i.p.) injections of AIT-082 or THA were made one
hour prior to the start of testing. Due to its shorter duration of
action, PHY was injected 30 minutes prior to testing. Control
subjects receive a similar injection of saline (vehicle).
[0147] To determine the duration of the memory trace, subjects were
administered drug or saline and 30 minutes (PHY) or 1 hour (AIT-082
or THA) later they were given a single reference run with the milk
reward in both goal boxes. After the indicated inter-trial delay,
subjects were returned to the start box and given the first test
trial with the milk reward only in the goal box opposite to the one
entered on the previous correct trial. The subjects were given 10
trials with the reward alternating only after correct
responses.
[0148] To determine if tolerance to the biological effects of
AIT-082 developed, drug or saline was administered daily for 18
days prior to the testing in the standard win-shift paradigm.
[0149] Subjects were also trained in the same T-maze used for the
win-shift model discussed above. As in the win-shift method,
subjects were shaped and then given a single reference run in which
reward was available in both goal boxes. The subject was only
allowed to consume the milk reward in the goal box selected. On the
next run, the reward and thus the correct response was in the same
goal box selected for the reference run and was not alternated. The
subject was required to learn that there was no shift in the goal
box for the correct response. The subjects were given 10 trials per
day and continued until the subject had at least 8 out of 10
correct responses on two consecutive days. The number of days to
reach this criteria of performance was recorded. After the subject
reached criteria, the goal box for the correct response was
reversed. The number of days taken to reach criteria on reversal
was recorded.
[0150] The results of the T-maze learning task and win-shift memory
test are presented in Table J directly below.
10TABLE J Effect of AIT-082, THA and PHY at 90-Second Inter-trial
Interval in Swiss Webster Mice Type of Test.sup.(1) Control THA
AIT-082 PHY Dosage (mg/kg) Saline 1.25 0.5 30.0 0.125 Win-shift
Memory Test Correct responses 4.6 7.1* 6.5* 8.2* 6.5 (Correct
responses/10 trials) Latency time (seconds) 2.68 8.22* 1.95 2.03
Running time (seconds) 1.95 3.65* 2.20 1.95 2.65 Locomotor
Activity.sup.(2) 343 671* 323 378 N/T T-maze Learning(days to reach
criteria) Learning 3.6 N/T.sup.(3) 3.0 3.3 N/T Reversal 4.2
N/T.sup. 3.78 3.5 N/T Tolerance 4.9 N/T.sup. N/T 7.6* N/T (Correct
responses/10 trials) .sup.(1)at least 8 animals were run per group.
.sup.(2)Spontaneous movements per hour. .sup.(3)Not tested.
*Indicates p < 0.05. Data analysis following significant ANOVA,
a Dunnett test was run comparing drug tested groups with controls.
An Arc Sign transformation was performed on percentage data and
latencies were transformed to reciprocal time scores or speed
scores.
[0151] As shown by the data in Table J, AIT-082 treatment resulted
in an increased number of correct responses (memory) compared to
saline control. While the effect was in the same range as with THA
and PHY, both THA and PHY also increased latency time (prolonged
the time to leave the start box, evidencing decreased motivation)
and THA increased spontaneous locomotor activity. AIT-082 had no
effect on learning or reversal and no tolerance developed to the
memory enhancing effect of AIT-082 after 18 days of pre-treatment.
Only AIT-082 enhanced memory function without affecting learning,
motivation, performance and locomotor activity. Similar data have
been observed with oral administration of AIT-082.
EXAMPLE 19
Effect of AIT-082 Dosage on Memory Trace Duration
[0152] The dose response and duration of action of AIT-082 was
studied in young adult Swiss Webster mice. The results are
presented as the percent correct response over chance; chance being
50% correct. As shown in FIG. 7, AIT-082 is active in improving
memory in normal adult Swiss Webster mice over a dose range from
0.5 to 60 mg/kg, with the optimal effect at 20 to 30 mg/kg.
Further, as shown in FIG. 8, the onset of action is rapid (1 hour,
data not shown) and lasts for more than seven days after a single
dose of 60 mg/kg. Those skilled in the art will appreciate that the
extended duration of the drug's effects will substantially lower
the frequency of administration providing benefits in terms of
patient compliance and cost.
EXAMPLE 20
Effect of AIT-082 on Memory Trace Duration in C57BL/6 Mice
[0153] Previous work has established that normal adult Swiss
Webster mice have a memory trace duration of 60 seconds in the
win-shift paradigm which may be increased by the administration of
AIT-082. In order to further demonstrate the applicability and
operability of the methods and compositions of the present
invention, an alternative strain of mice having a different
duration of memory trace was administered AIT-082, using the
preceding protocol. The results are shown in Table K directly
below.
11TABLE K Duration of Memory Trace in C57BL/6 Mice Treatment Groups
Control AIT-082 Physostigmine Inter- (Saline) (30 mg/kg) (0.125
mg/kg) trial No. above No. above No. above interval chance/ chance/
chance/ (sec) Total.sup.# Correct.diamond. Total.sup.#
Correct.diamond. Total.sup.# Correct.diamond. 30 3/5 70 .+-. 11**
60 3/5 70 .+-. 16** 90 4/5 70 .+-. 6** 120 4/5 78 .+-. 16** 150 1/5
56 .+-. 10 180 2/7 58 .+-. 12 4/6 70 .+-. 15** 3/6 65 .+-. 16* 210
4/6 78 .+-. 15** 1/6 53 .+-. 9 240 0/6 50 .+-. 6 270 0/6 50 .+-. 6
.sup.#= No. subjects above chance (60% correct)/Total No. subjects
tested .diamond.= Mean score .+-. S.E. **= p < 0.01 (t-test
against chance) *= p < 0.05 (t-test against chance)
[0154] Typically, in the win-shift foraging paradigm, C57BL/6 mice
have a duration of memory trace of 120 seconds. As shown in Table
K, at 30 mg/kg i.p., AIT-082 prolonged the duration of the memory
trace to over 210 seconds. While physostigmine also prolonged the
duration of the memory trace from 120 to 180 seconds in this model,
it was not as active as AIT-082.
EXAMPLE 21
Treatment of Age Induce Memory Disorders Using AIT-082
[0155] In light of the preceding results, studies were performed to
demonstrate that AIT-082 improves memory in mammals with neuronal
disorders as well as in healthy subjects. Twelve-month old male
Swiss Webster mice were screened for performance in the win-shift
foraging test. Subjects were tested at various time delays,
beginning at 10 seconds and increasing the inter-trial time
interval to 30, 60, 90 and 120 seconds. The results for untreated
mice are shown in Table L directly below.
12TABLE L Age-induced Working Memory Deficits in Swiss Webster Mice
Duration of No. of % Degree of Memory Memory Trace Subjects of
Subjects Impairment less than 10 seconds 6 25% Severe 10 seconds 8
33 Moderate 30 seconds 10 42 Mild Total 24 100
[0156] The results in Table L demonstrate that individual subjects
can be classified by the degree of working memory impairment.
Subjects with severe impairment could not remember the correct
response at 10 seconds while subjects with mild deficit could
remember the correct response with a 30 second inter-trial interval
but not at 60 seconds. Subjects with a moderate deficit could
remember the correct response with a 10 second inter-trial interval
but not at 30 seconds. Thus, the win-shift model can detect
age-induced impairments in working memory. As will be appreciated
by those skilled in the art, this observation is important since it
provides the ability to use age-matched subjects with varying
degrees of impairment for evaluation of potential therapeutic
agents.
[0157] Following the establishment of a baseline, six subjects in
each of the three groups were treated with AIT-082 (30 mg/kg, one
hour before testing) or physostigmine (0.125 mg/kg, 30 minutes
before testing) using the win-shift foraging test. The results are
presented in Table M directly below and graphically represented in
FIG. 9.
13TABLE M Effect of AIT-082 and PHY on the duration of memory trace
in Swiss Webster mice with age-induced deficits Inter-trial Degree
of Interval Control AIT-082 PHY Deficit (sec) (Saline) 30 mg/kg
0.125 mg/kg Mild 60 0/6 6/6* 5/6* 90 4/6 3/6 120 2/6 2/6 150 1/6
2/6 180 1/6 1/6 210 0/6 0/6 Moderate 30 0/6 4/6* 1/6 60 2/6 0/6 90
0/6 Severe <10 0/6 0/6 0/6 Data is presented as the number of
subjects performing significantly above chance/total number of
subjects; *Indicates p < 0.05 (t-test)
[0158] Six subjects had a severe deficit with no memory trace, they
could not remember the task at 10 seconds. None of these subjects
showed memory restoration with either AIT-082 or PHY treatment. In
the six subjects with a moderate memory deficit who had a duration
of memory trace of 10 seconds, AIT-082 increased the duration of
the memory trace to greater than 30 seconds in 4 subjects (67% of
the subjects) and increased the memory trace to greater than 60
seconds in two subjects (50%). In the six subjects with a mild
memory deficit who had a duration of memory trace of 30 seconds,
AIT-082 increased the duration of the memory trace in 2 subjects to
60 seconds, in 2 subjects to 90 seconds and in one subject each to
120 and 180 seconds. PHY increased the duration of the memory trace
from 10 seconds to 30 seconds in only one animal in the moderate
deficit group. In the mild deficit group, PHY increased the
duration of the memory trace in 2 subjects to 60 seconds, in one
subject to 90 seconds and in two subjects to at least 180 seconds.
Thus, AIT-082 is more active than physostigmine in the moderate
deficit group and at least as active in the mild deficit group.
EXAMPLE 22
Treatment of Age Deficit Memory Disorders Using AIT-082
[0159] Twelve-month old male C57BL/6 mice were screened for
performance in the win-shift foraging test. Subjects were tested at
various inter-trial time intervals. Subjects who could not perform
to criteria (>60% correct) at 10 seconds delay were classified
as having a severe deficit. Subjects who performed to criteria at
10 seconds but not at 30 seconds were classified as having a
moderate degree of deficit and subjects who performed to criteria
at 30 seconds but not at 60 seconds were classified as having mild
deficit. As in Example 21, subjects in each group were treated with
either AIT-082 or PHY to determine the extent to which the working
memory trace was prolonged. The results are presented in Table N
directly below and graphically represented in FIG. 10.
14TABLE N Effect of AIT-082 and PHY on the duration of memory trace
in C57BL/6 mice with age-induced deficits Inter-trial Degree of
Interval Control AIT-082 PHY Deficit (sec) (Saline) 30 mg/kg 0.125
mg/kg Mild 60 0/6 4/4* 7/8* 90 2/4* 3/8 120 2/8 150 2/8 180 2/8 210
0/8 Moderate 10 6/6 6/6* 6/6 30 0/6 4/6 1/6 60 1/6 0/6 90 0/6
Severe <10 0/6 0/6 0/6 Data is presented as the number of
subjects performing significantly above chance/total number of
subjects; *Indicates p < 0.05 (t-test)
[0160] In the mild deficit group, AIT-082 prolonged the duration of
the memory trace from 30 to 90 seconds, and from 10 to 30 seconds
in the moderate deficit group. While PHY prolonged memory in the
mild group, it was ineffective in the moderate group. Therefore
AIT-082 restored working memory deficits in both normal mice and
mice with age induced neuronal disorder for both Swiss Webster and
C57BL/6 strains. Specifically, the results show that AIT-082
restores working memory in mice with mild and moderate memory
deficits. Based on the other Examples previously provided it is
reasonable to conclude that it accomplishes this restoration by
modifying the carbon monoxide dependent guanylyl cyclase
system.
EXAMPLE 23
Prophylaxis of Age Deficit Memory Disorders Using AIT-082
[0161] It has been observed that age-induced memory deficits
typically begin to manifest themselves in mice between 14 and 16
months of age. Therefore, we began treating mice at 14 months of
age with AIT-082 (30 mg/kg/day) in their drinking water. The
animals were measured monthly for their memory using the win-shift
foraging tests previously described. The results are shown in FIG.
11 and show that the administration of AIT-082 delayed the onset
and severity of memory deficits
EXAMPLE 24
Prophylaxis of Alchool-Induced Deficit Memory Disorders Using
AIT-82
[0162] In order to demonstrate the broad applicability of the
present invention with respect to different types of
neurodegenerative disorders, AIT-082 was used to retard the
production of alcohol induced memory deficit. Six month old male
C57BL/6 mice were evaluated in the win-shift model in combination
with treatment with ethanol, a non-specific memory suppressant, and
AIT-082. Subjects were treated with saline (control) or AIT-082 (30
mg/kg. i.p.) 1 hour prior to testing. Ethanol was administered at a
dose of 0.5 gm/kg i.p. ten minutes prior to testing. The results of
a pilot study are presented in Table O directly below.
15TABLE O Working memory deficit produced by ethanol and its
reversal by AIT-082 Treatment Ethanol + Control Ethanol AIT-082
Correct trials.sup.1,2 8.08 .+-. 0.29 6.5 .+-. 26* 7.89 .+-. 0.54
.dagger. Latency time(sec).sup.2 1.24 .+-. 0.17 1.18 .+-. 0.10 1.77
.+-. 0.27 Running time(sec).sup.2 1.44 .+-. 0.35 1.17 .+-. 0.08
3.22 .+-. 0.61*.dagger. Number of subjects 13 13 9 .sup.1Indicates
mean number of correct responses per 10 trials; .sup.2Indicates
mean values .+-.S.E.; *Indicates p < 0.05 (t-test) compared to
control; .dagger.Indicates p < 0.05 (t-test) compared to
ethanol.
[0163] The results in Table O demonstrate that it is possible to
identify a dose of a blocking agent that can produce a memory
deficit as measured in the win-shift model. Ethanol was selected as
a non-specific blocking agent and its effects were reversed by
administration of AIT-082 prior to the treatment with ethanol.
Therefore it would appear feasible to evaluate other more specific
blocking agents which have activity at specific receptor sites.
[0164] In addition to AIT-082 other purine derivatives are believed
to play a role in neuronal survival, synaptogenesis and recovery of
function following injury or cell death in the central nervous
system. For example, similarities between guanosine and AIT-082
indicate that AIT-082 and guanosine act through comparable
mechanisms. That is, both appear to act as carbon monoxide
dependent guanylyl cyclase modulators. Further, it is known that
after cells are damaged, they leak massive amounts of both purine
nucleosides and nucleotides to the extracellular space. The
extracellular concentration of guanosine in the region of a focal
brain injury may reach 50 mM and is elevated up to five fold for at
least seven days. Therefore, following injury, astrocytes or glia
and neurons are exposed to high extracellular concentrations of
guanosine.
[0165] Accordingly, the following studies were undertaken in order
to demonstrate the effectiveness of using other exemplary purine
derivatives such as guanosine to modulate the carbon monoxide
dependent guanylyl cyclase system.
EXAMPLE 25
Astrocytes Produce Tropic Factors Upon Exposure to Guanosine and
GTP
[0166] Astrocytes appear to proliferate in response to
extracellular guanosine or guanosine triphosphate (GTP). GTP or
guanosine may also stimulate the release of trophic factors from
cultures of neocortical astrocytes from neonatal mouse brains.
Astrocytes were incubated with different concentrations of
guanosine of GTP respectively. Neurotrophin immunoreactivity in the
culture medium from treated cells was then measured by ELISA.
[0167] Briefly, 96 well Falcon plates (Fisher) were coated with 1
mg/ml of sheep mono-specific anti-NGF IgG (affinity column
purified) contained in 0.1M sodium carbonate buffer pH 9.6. After
an overnight incubation at 4.degree. C. blocking solution (PBS with
10% goat serum) was added to remove excess antibody. After a four
hour incubation at room temperature the plates were washed 3 with
PBS containing 0.05% Tween 20. The conditioned media and standard
2.5S HPLC purified NGF were added and incubated overnight. The next
day plates were washed 3 times with PBS-0.05% Tween 20. The
secondary antibody, rabbit mono-specific anti-NGF IgG conjugated
with b-galactosidase (Pierce-SPDP method) (1:500 dilution) was
added. The plates were incubated overnight at 4.degree. C. The next
day the plates were washed 3 times with PBS-0.05% Tween 20. To each
well substrate, 0.2 mM 4-methylumbelliferyl-b-galactoside (MUG) in
0.1M phosphate buffer (1 mM MgCl.sub.2 pH 7.2) was added. After a 4
hour incubation at room temperature the reaction was stopped by the
addition of 0.1M glycine, pH 10.3. Samples were then read using
Microfluor ELISA reader (excitation 360 nm; emission 450 nm). The
sensitivity of this assay was 10 pg/well NGF.
[0168] The ELISA assay detected neurotrophins NGF and NT-3 with
almost equal affinity and BDNF with 100 times less affinity. As
shown in FIGS. 12A and 12B, both guanosine and GTP increased the
amount of NGF-like immunoreactivity in the culture medium. The
astrocytes exposed to the various levels of guanosine produced a
much stronger response than those exposed to equivalent
concentrations of GTP.
EXAMPLE 26
Astrocytes Produce Neurotrophic Factors Upon Exposure to
Guanosine
[0169] In order to confirm the results of the previous assay, mRNA
levels of the tropic factors FGF-2 and NGF were measured in
astrocytes which had been exposed to guanosine. The mRNA levels
were measured using the same protocol used previously in Example
16. As shown in FIGS. 13A and 13B, the addition of guanosine
increased NGF and FGF-2 mRNA at 4 hours and at 24 hours,
respectively, after it was added to astrocytes. The observed
increase in neurotrophin mRNA is important following brain injury
or recovery from brain injury when the extracellular concentration
of guanosine is considerably high. As cells are exposed to a high
concentration of guanosine for several days following brain injury,
this data indicates that guanosine may be responsible for some of
the recovery of function.
[0170] As previously discussed, an agent that can penetrate the
blood brain barrier and increase concentrations of neurotrophic
factors as measured here by mRNA levels should have a substantial
positive effect on neuronal survival and on the formation of
collateral nerve circuits. In turn, this should enhance functional
recovery in many different neurological diseases or after damage to
the nervous system.
EXAMPLE 27
Neurons Undergo Neuritogenesis Upon Exposure to GUANOSINE
[0171] In addition to changes in glia or astrocytes, important
neuronal changes also take place following focal brain injury.
Neuritic processes of surviving neurons may undergo neuritogenesis.
Accordingly, based on previous results using AIT-082, studies were
performed to demonstrate that guanosine may also modify carbon
monoxide guanylyl cyclase to stimulate neuritogenesis. As
previously discussed, because PC12 cells constitute a homogeneous
population of neuronal-like cells, without contaminating
astroglia-type cells, the direct effects of the exemplary purine
derivatives of the present invention on neurite outgrowth in these
cells can be observed easily. Accordingly, PC12 cells were exposed
to guanosine and adenosine with and without NGF and monitored as in
Example 12. The effects of exposure to purine derivatives with NGF
are shown in FIG. 14A while exposure without NGF is shown in FIG.
14B. A direct comparison of the effects of these purine derivatives
with and without the presence of NGF is shown for each compound in
FIG. 14C.
[0172] As shown in FIG. 14A, guanosine, but not adenosine, enhanced
the neurite outgrowth induced by NGF in PC12 cells after 48 hours.
The enhancement was significant over that of NGF alone at guanosine
concentrations of 30 and 300 mM. Adenosine did not enhance NGF
induced neurite outgrowth at any concentration. This indicates that
neurite outgrowth induced by purines is not just a generalized
phenomenon. 5'-N-ethylcarboxamidoadenosine (NECA), an adenosine
A.sub.1 and A.sub.2receptor agonist, also enhanced neuritogenesis,
but not to the same extent as guanosine.
[0173] On their own, in the absence of NGF, both adenosine and
guanosine slightly increased the proportion of cells with neurites
as shown in FIG. 14B. The effects of guanosine at both 30 and 300
mM was greater than adenosine at the same concentrations. In the
presence of (NECA), there was little stimulation of neurite
outgrowth. Because the effects of the compounds in the presence of
NGF were much more readily scored and less variable from experiment
to experiment than with the compounds alone, most of the data for
enhancement of neurite outgrowth was determined in the presence of
NGF.
[0174] The comparative data shown in FIGS. 14A and 14B and
emphasized in FIG. 14C show that guanosine causes some neurite
extension, but can also react synergistically to enhance the
trophic effects of NGF. Adenosine, although slightly enhancing
neurite outgrowth on its own does not enhance the effects of NGF.
Interestingly, NECA but not adenosine could synergistically enhance
the actions of guanosine, both in the presence and absence of NGF
as shown in FIG. 14C. The fact that adenosine did not increase
NGF-dependent neurite outgrowth in PC12 cells but that guanosine
did, suggests that they interact differently with PC12 cells.
Adenosine would interact with adenosine receptors, such as the
A.sub.2 purinoceptor. This would activate adenylate cyclase which
increases intracellular cAMP. NECA apparently acts in this manner.
But the effects of NECA were synergistic with those of guanosine.
This indicates that guanosine and NECA use different signalling
pathways to enhance neurite outgrowth.
EXAMPLE 28
Various Purine Derivatives Provide Different Rates of
Neuritogenesis
[0175] In view of the previous results, other exemplary purine
derivatives were examined to demonstrate the specificity of those
compounds which modulate carbon monoxide dependent guanylyl cyclase
to modify neural activity. Specifically, different concentrations
of the purine derivatives inosine, hypoxanthine and xanthine were
tested in the presence of NGF using the protocol of Example 12 to
demonstrate their ability to modify neural activity.
[0176] As shown in FIG. 15A, inosine only slightly enhanced neurite
outgrowth over that produced in cells treated with NGF alone. This
was true for concentrations of inosine ranging from 0.3 to 300 mM.
FIG. 15A also shows that the action of inosine on the enhancement
of neurite outgrowth was much less effective than that of
guanosine.
[0177] FIGS. 15B and 15C also show that hypoxanthine and xanthine
each produced results similar to that of inosine on NGF-induced
neuritogenesis. In FIG. 15C xanthine, in concentrations from 0.3 to
30 mM (300 mM was toxic to the cells), only slightly enhanced
NGF-induced neurite outgrowth. FIG. 15B shows that hypoxanthine
showed the greatest, although still modest, enhancement at
concentrations of 0.3 and 300 mM, although other concentrations had
no significant enhancement. Even though some enhancement of neurite
outgrowth was observed with hypoxanthine, the relative amount of
enhancement was not nearly as great as was the effect of guanosine.
These results indicate that inosine, xanthine and hypoxanthine do
not modulate the carbon monoxide-dependent guanylyl cyclase system
to modify neural activity but rather influence other signaling
mechanisms.
EXAMPLE 29
Effects of AIT-34 on Neuritogenesis
[0178] To demonstrate the effects of compounds similar to AIT-082
on neuritogenesis, PC12 cells were exposed to AIT-34, otherwise
known as 3(1,6dihydro-6-oxo-9h
purin-9-yl)-N-[3-(2-oxopyrrolidin-1-yl) propyl]propanamide, during
growth and monitored according to Example 12. As shown in FIG. 16,
different concentrations of AIT-034 did not enhance NGF-induced
neuritogenesis as is observed with AIT-082.
EXAMPLE 30
Effects of ATP and GTP on Neuritogenesis
[0179] To further demonstrate that purine derivatives having
different functional groups may be used in accordance with the
teachings of the present invention, PC12 cells were exposed to
adenosine triphosphate (ATP) and guanosine triphosphate (GTP) and
monitored for neuritogenesis using the protocol of Example 12.
[0180] In a manner very similar to the actions of adenosine and
guanosine on neurite outgrowth in PC12 cells, their corresponding
nucleotides ATP and GTP had parallel effects on neurite outgrowth.
As shown in FIG. 17, ATP did not enhance neuritogenesis in either
NGF treated cells or on its own. In sharp contrast, GTP at 30 and
300 mM, did enhance neuritogenesis in the presence of NGF and
further elicited neurite outgrowth on its own.
[0181] However, as shown in FIG. 18, GTP did not appear to be
acting as a source from which guanosine was released in a
controlled manner. If GTP was hydrolyzed to guanosine diphosphate
(GDP), guanosine monophosphate (GMP) and finally to guanosine by
ectoenzymes, one would predict that GDP and GMP would also enhance
neurite outgrowth from PC12 cells. Yet, neither GDP nor GMP were
effective alone or with NGF in eliciting neurite outgrowth. By way
of comparison, the adenine-based compounds all had an inhibitory
effect.
EXAMPLE 31
Guanosine But not GTP Increases cGMP in PC12 Cells
[0182] Based on the previous examples, a study was conducted to
demonstrate the neurotogenic mechanisms of GTP and guanosine
respectively. Guanosine and GTP have been shown to increase
intracellular cyclic 3',5'-guanosine monophosphate (cGMP) in
arterial smooth muscle. Since cGMP analogues have been reported to
stimulate neurite outgrowth from neuroblastoma cells it was
possible that both guanosine and GTP might exert their effects
through increasing intracellular cGMP. As shown in FIG. 19,
guanosine increased intracellular cGMP in PC12 cells as determined
by radioimmunoassay using the protocol detailed in Example 15. Such
an increase would be expected of a carbon monoxide dependent
guanylyl cyclase modulator. In contrast, it was found that GTP did
not increase levels of cGMP, indicating that any GTP-stimulated
neuritogenesis occurs by another mechanism.
EXAMPLE 32
Use of Non-Selective Inhibitors of Guanylyl Cyclase Reduces
Guanosine Neuritogenesis
[0183] To demonstrate that guanosine modifies the carbon
monoxide-dependent guanylyl cyclase system, studies were conducted
to show that increased levels of intracellular cGMP were necessary
for guanosine to enhance NGF's neurotogenic effects on PC12 cells.
In particular, different concentrations of three inhibitors of
guanylyl cyclase were added to PC12 cells with guanosine.
Neuritogenesis was then determined using the protocol of Example
12.
[0184] Methylene Blue (MB) inhibits soluble guanylyl cyclase (sGC),
the enzyme that synthesizes cGMP. As shown in FIG. 20A the addition
of MB (0.1-5 mM) to cultures of PC12 cells abolished the
synergistic effects of guanosine with NGF. Conversely, MB had no
effect on NGF-stimulated neurite outgrowth.
[0185] LY83583 inhibits both particulate and sGC. FIG. 20B shows
that the neurite outgrowth response elicited by guanosine was
inhibited by LY83583, but the response elicited by NGF was
unaffected. The mechanism by which LY83583 inhibits guanylyl
cyclase is unresolved, but is likely indirect, involving
glutathione metabolism. Therefore, two non-selective inhibitors of
guanylyl cyclase, each with a different mechanism of action,
attenuated the neurotogenic action of guanosine.
[0186] These data indicate that guanosine and NGF act through
different mechanisms. They also indicate that increases in
intracellular cGMP were necessary, although possibly not
sufficient, for guanosine to exert its neurotogenic effects.
[0187] To test whether increases in cGMP were sufficient to cause
neurite outgrowth, atrial natriuretic factor (ANF) was added to
cell cultures in a manner similar to that used for guanosine. ANF
is a hormone whose only known signal transduction pathway is
through activation of particulate guanylyl cyclase. As shown in
FIG. 20C, ANF, like guanosine, enhanced NGF-stimulated neurite
outgrowth from PC12 cells indicating that increased intracellular
cGMP production, induced by carbon monoxide dependent guanylyl
cyclase or other mechanisms assisted in stimulating neurite
outgrowth.
EXAMPLE 33
Nitric Oxide or Carbon Monoxide Promotes Guanosine
Neuritogenesis
[0188] Because guanosine increased intracellular cGMP as shown in
Example 31, studies were performed to demonstrate whether its
signal could be transduced through production of NO or CO. If NO
was involved, then addition of nitric oxide donors that liberate NO
should mimic the effects of guanosine.
[0189] PC12 cells were grown for 48 hours in the presence of sodium
nitroprusside (SNP) or sodium nitrite (SN), both of which liberate
NO. Alone, neither SNP nor SN elicited neurite outgrowth from PC12
cells. However, like guanosine, both SNP and SN enhanced
NGF-mediated neurite outgrowth in a synergistic manner as shown for
the addition of SN in FIG. 21. Further confirming the effect, FIGS.
22A and 22B show that the neurotogenic properties of the NO donors
were inhibited by both hemoglobin (Hb) and methemoglobin (MB). Both
are substances which scavenge NO and CO with high affinity and
preclude these agents from being used as signal transmitters.
[0190] Accordingly, if NO or CO mediates the neurotogenic effects
of guanosine, then these effects should be reduced by addition of
hemoglobin to the cultures. The expected effect is clearly shown in
FIG. 23 where Hb (0.1-1 mM) inhibited the neurotogenic effects of
guanosine but not those of NGF. This indicates that the
neurotogenic action of guanosine, but not that of NGF, requires
synthesis of NO or CO.
[0191] Several facts indicate that it is CO rather than NO which
interacts with guanosine to modify neural activity. For example, if
the effects of guanosine were mediated through NO, then addition of
guanosine to the PC12 cells should stimulate cNOS in PC12 cells to
produce NO. However, cNOS had not been reported in PC12 cells and
untreated (guanosine and NGF naive) PC12 cells did not stain for
diaphorase, an enzyme that co-localizes with NOS. Since cNOS is
calcium/calmodulin-sensitive, its activity should increase after
adding a calcium ionophore, thus leading to increased cGMP levels.
Addition of the ionophore A23187 to cultures of PC12 cells failed
to elicit an increase in cGMP.
EXAMPLE 34
Carbon Monoxide, Not Nitric Oxide, Mediades the Effects of
Guanosine on Neuritogenesis
[0192] Based on the results of the previous examples, studies were
performed to demonstrate that the purine derivatives of the present
invention, including guanosine, modulate the carbon
monoxide-dependent guanylyl cyclase system to modify neural
activities.
[0193] As in Example 6 where it was shown that carbon monoxide
mediates the effects of AIT-082 through the use of inhibitors, the
same techniques demonstrate that guanosine also interacts with the
carbon monoxide dependent system. Specifically, as shown in FIG.
24, the cNOS inhibitor L-nitro arginine methyl ester (L-NAME) did
not affect the ability of guanosine to enhance NGF-mediated neurite
outgrowth. These data confirm that cNOS was not involved in the
signal transduction pathway that mediated the neurotogenic effects
of guanosine on PC12 cells.
[0194] To further demonstrate that CO, rather than NO, mediated the
neurotogenic effects of guanosine, zinc protoporphyrin IX (ZnPP),
which inhibits heme oxygenase and hence inhibits CO synthesis, was
added to the cells during growth. As shown in FIG. 25, ZnPP
abolished the neurotogenic effects of guanosine, but did not affect
those of NGF. In contrast, a related protoporphyrin derivative,
copper protoporphyrin IX (CuPP), does not inhibit heme oxygenase.
Accordingly, FIG. 26 shows that copper protoporphyrin IX did not
reduce the ability of guanosine to enhance NGF-dependent neurite
outgrowth from PC12 cells. As with AIT-082, these data indicate
that guanosine increased CO synthesis. In turn, CO activated sGC
and increased intracellular GMP, thereby promoting
neuritogenesis.
EXAMPLE 35
Inosine Pranobex Enhances Neuritogenesis
[0195] To provide further evidence of the scope and operability of
the present invention, neurotogenic studies were performed using
inosine pranobex. Specifically, inosine pranobex is a mixture of
inosine and DIP-PacBa at a 1:3 molar ratio. Various concentrations
of this compound were added to PC12 cells with NGF which were then
monitored according to the protocol of Example 12.
[0196] As shown in FIG. 27, inosine pranobex substantially enhanced
the amount of neurite outgrowth of the treated cells. The curve
shown in FIG. 27 represents the different levels of inosine
pranobex plus saturating concentrations of NGF while the horizontal
lines represent the NGF control with attendant confidence levels.
Here the treated cells are above the control baseline at most of
the selected concentrations.
[0197] The modification of cellular and, more specifically, neural
activity in accordance with the teachings of the present invention
may be used to treat a wide variety of cellular and
neurodegenerative diseases in order to provide recovery of cellular
or neural function. Thus, the present invention may be used to
treat cellular and neurodegeneration from any cause including
oxidative stress, disease, trauma, age, and exposure to harmful
physical or chemical agents. Similarly, the methods and medicaments
disclosed herein may be used to treat neurological diseases
including, but not limited to, Alzheimer's Disease and related
degenerative disorders, Parkinson's disease and related disorders
such as striatonigral degeneration, spino-cerebellar atrophies,
motor neuronopathies or "motor system diseases" including
Amyotrophic Lateral Sclerosis, Werdnig-Hoffmann disease,
Wohlfart-Kugelberg-Welander syndrome and hereditary spastic
diplegia, damage to neurons by ischemia (as in strokes), anoxia, or
hypoglycemia (as, for example, after prolonged circulatory arrest),
Huntington's disease, cerebral palsy, multiple sclerosis,
psychiatric disorders including affective disorders, schizophrenia,
epilepsy and seizures, peripheral neuropathies from any cause,
learning disabilities and disorders of memory. Also, damage to
neurons or their processes by physical agents such as radiation or
electrical currents or by chemical agents including alcohol,
aluminum, heavy metals, industrial toxins, natural toxins and legal
or illegal drugs may be treated. The methods may further be used to
treat victims of trauma to the brain or spinal cord resulting in
neuronal damage or age related conditions such as benign
forgetfulness and deterioration of sensory, motor, reflex or
cognitive abilities due to loss of neurons or neuronal
connectivity. Simply administering an effective dosage of at least
one of the carbon monoxide dependent guanylyl cyclase modulating
purine derivatives of the present invention to a subject suffering
from any of the foregoing cellular or neural disorders will induce
intracellular changes producing restoration of function.
[0198] Specifically, modification of the carbon monoxide dependent
guanylyl cyclase system in accordance with the teachings of the
present invention produces changes in neural activity in neurons
and glia cells including astrocytes. For example, using the present
invention the neural activity of astrocytes may be modified to
synthesize various neurotrophic factors and cytokines including
fibroblast growth factor (FGF), nerve growth factor (NGF), brain
derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3). These
factors can influence the sprouting of neuritic processes from
surviving neurons as well as promote the development of new cells.
New synapses may then form and provide some recovery of function.
These neurotrophic factors also play a neuroprotective role. Thus,
inducing their production can ameliorate further neural damage.
[0199] Numerous purine derivatives may be used in accordance with
the teachings of the present invention. However, the ability to
modify neural activity by modulating the carbon monoxide dependent
guanylyl cyclase system is not a general property of all purines or
purine derivatives. For example, as shown in the data below,
inosine, adenosine, hypoxanthine and xanthine were all relatively
ineffective at modifying neural activity. Other purine derivatives
which failed to modify neural activity include
3-(6-amino-9H-purin-9-yl)propionic acid, ethyl ester (AIT-0026),
3-(1,6-dihydro-6-oxo-9H-purin-9-yl)-N-{3-(2-oxopyrolidin-1-yl)propyl]prop-
anamide (AIT-0034) and propentofylline. Moreover, while other
purines and purine derivatives such as
5'-N-ethylcarboxamidoadenosine (NECA) were shown to stimulate
neurite outgrowth, they did not do so by modulation of the carbon
monoxide dependent guanylyl cyclase mechanism. Accordingly, the
scope of the invention is defined by the functional reactivity of
purine derivatives which modify cellular or neural activity as
described herein and as shown by the data presented. Of course,
those skilled in the art will appreciate that functionally
equivalent isomers, analogs and homologs of the compounds of the
present invention may be substituted.
[0200] Those skilled in the art will further appreciate that the
present invention may be embodied in other specific forms without
departing from the spirit or central attributes thereof. In that
the foregoing description of the present invention discloses only
exemplary embodiments thereof, it is to be understood that other
variations are contemplated as being within the scope of the
present invention. Accordingly, the present invention is not
limited to the particular embodiments which have been described in
detail herein. Rather, reference should be made to the appended
claims as indicative of the scope and content of the present
invention.
* * * * *