Note: Descriptions are shown in the official language in which they were submitted.
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MODULATION OF EXCITABLE TISSUE FUNCTION
BY PERIPHERALLY ADMINISTERED ERYTHROPOIETIN
1. FIELD OF THE INVENTION
The present invention is directed to the use of peripherally administered
erythropoietin and other erythropoietin receptor activity modulators or EPO-
activated
receptor modulators to positively affect excitable tissue function. This
includes the
protection of excitable tissue, such as neuronal and cardiac tissue, from
neurotoxins,
hypoxia, and other adverse stimuli, and the enhancement of excitable tissue
function, such
as for facilitating learning and memory. The present invention is further
drawn to methods
for transport of substances across endothelial cell barriers by association
with an
erythropoietin molecule, erythropoietin receptor activity modulator or other
EPO-activated
receptor modulators.
2. BACKGROUND OF THE INVENTION
Various acute and chronic conditions and diseases originate from excitable
tissue
damage and dysfunction brought about by external and internal stimuli. Such
stimuli
include lack of adequate oxygenation or glucose, neurotoxins, consequences of
aging,
infectious agents, and trauma. For example, excitable tissue may be subjected
to damage as
a consequence of seizures and chronic seizure disorders, convulsions,
epilepsy, stroke,
Alzheimer's disease, Parkinson's disease, central nervous system injury,
hypoxia, cerebral
palsy, brain or spinal cord trauma, AIDS dementia and other forms of dementia,
age-related
loss of cognitive function, memory loss, amyotrophic lateral sclerosis,
multiple sclerosis,
hypotension, cardiac arrest, neuronal loss, smoke inhalation and carbon
monoxide
poisoning.
It is widely understood that decreases in energy supply available to the
brain,
such as glucose or oxygen, results in a profound impairment of brain function,
including
cognition. Many (but not all) neurons in the central nervous system are easily
damaged
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while working under metabolically-limited conditions, e.g., hypoxia,
hypoglycemia, stress,
and/or prolonged, strong excitation. Under these circumstances, the
electrochemical
gradients of these cells often collapse, resulting in irreversible neuronal
injury and cell
death. Current opinion favors this general mechanism as a common final pathway
for a
wide range of common and debilitating degenerative neurological diseases
including stroke,
epilepsy, and Alzheimer's disease.
Although the consequences of limited energy substrate on brain function are
well
known, the effects of improving energy delivery in an otherwise normal brain
has been less
extensively explored. Current data suggest strongly that improved delivery of
either
glucose or oxygen markedly improves complex cognitive function in both animal
models
and in normal human subjects (Kopf et al., 1994, Behavioral and Neural Biology
62:237-
243; Li et al., 1998, Neuroscience 85:785-794; Moss et al., 1996,
Psychopharmacology
124:255-260). Further, a growing list of neuropeptides produced within the
brain have
been demonstrated to directly provide an improvement in cognitive function in
normal
brain. The physiological basis of these enhancements ultimately depends upon
remodeling
of neuronal interconnections through synaptic changes.
Brain tissue cytoarchitecture exhibits extreme plasticity and undergoes
continuous remodeling. These processes, mediated by many trophic molecules,
occur not
only following injury, but also play a prominent role in learning, memory, and
cognitive
function. Although the prototype neurotrophin is nerve growth factor (NGF), an
increasing
number of cytokines have been recognized to perform trophic functions in the
brain (Hefti
et al. 1997, Annu. Rev. Pharmacol. Toxicol. 37:239-67).
Recently, a number of independent investigators have recognized that nervous
tissue expresses high levels of both EPO and its receptor (EPO-R;
Digicaylioglu et al.,
1998, Proc. Natl. Acad. Sci. USA 92:3717-20; Juul et al., Pediatr. Res. 43:40-
9; Marti et al.,
1997, Kidney Int. 51:416-8; Morishita et al., 1997, Neuroscience 76:105-16).
Although it
appears that EPO and its receptor proteins are each the products of single
genes, the CNS
versions are significantly smaller. The physiological meaning of this
observation has not
been clarified, but the mass differences do appear to modify biological
activity. For
example, in studies of human patients, investigators have concluded that EPO
is not
transported into the brain from the periphery (Marti et al., 1997, supra). To
date, however,
this possibility has not been evaluated for EPO by any direct study. Although
brain EPO is
about 15% smaller than renal EPO (due to differences in sialylation), brain
EPO is more
active in erythroid colony stimulation at low ligand concentrations (Masuda et
al., 1994, J.
Biol. Chem. 269:19488-93). On the other hand, the CNS receptor exhibits a much
lower
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affinity for deglycosylated EPO than the 30% larger peripheral receptor
(Konishi et al.,
1993, Brain Res. 609:29-35; (Masuda et al., 1993, J. Biol. Chem. 268:11208-
16).
In the brain, EPO expression has been found in astrocytes, and increased EPO
expression and release can be induced by hypoxia and other metabolic stressors
(Marti et
al., 1996, Eur. J. Neurosci. 8:666-76; Masuda et al., 1993, J. Biol. Chem.
268:11208-16;
Masuda et al., 1994, J. Biol. Chem. 269:19488-93) or even by occupancy of
other receptors
such as insulin-like growth factor family (Masuda et al., 1997, Brain Res.
746:63-70).
Neurons are one target for this secreted EPO as they express EPO-R in a highly
cell type-
specific manner (Morishita et al., 1997, Neuroscience 76:105-16). In contrast
to EPO
itself, EPO-R density does not appear to be modulated during metabolic stress
(Digicaylioglu et al., 1995, Proc. Natl. Acad. Sci. USA 92:3717-20).
Recent study has demonstrated that EPO impressively protects against hypoxic
neuronal injury in vitro, as well as in vivo when injected directly into the
cerebral ventricles
(Morishita et al., 1997, Neuroscience 76:105-16; Sadamoto et al., 1998,
Biochem. Biophys.
Res. Commun. 253:26-32; Sakanaka et al., 1998, Proc. Natl. Acad. Sci. USA
95:4635-40).
Konishi et al. (1993, Brain Res. 609:29-35) have demonstrated that EPO
promotes the in
vivo survival of cholinergic neurons in adult rats when injected directly into
the cerebral
ventricles. EPO administered centrally into the cerebral ventricles also
successfully
prevents ischemic injury-related deficits in spatial learning in rats
(Sadamoto et al., 1998,
Biochem. Biophys. Res. Commun. 253:26-32). A recent publication suggests that
only a
17-amino acid portion of EPO is needed for these neurotrophic effects in
cultured neural
cells (Campana et al., 1998, Int. J. Mol. Med. 1:235-41).
For many years, the only clear physiological role of erythropoietin (EPO) had
been its control of the production of red blood cells. Recently, several lines
of evidence
suggest that EPO, as a member of the cytokine superfamily, performs other
important
physiologic functions which are mediated through interaction with the
erythropoietin
receptor (EPO-R). These actions include mitogenesis, modulation of calcium
influx into
smooth muscle and neural cells, and effects on intermediary metabolism. It is
believed that
EPO provides compensatory responses that serve to improve hypoxic cellular
microenvironments. Although studies have established that EPO injected
intracranially
protects neurons against hypoxic neuronal injury, intracranial administration
is an
impractical and unacceptable route of administration for therapeutic use,
particularly for
normal individuals. Furthermore, previous studies of anemic patients given EPO
have
concluded that peripherally-administered EPO is not transported into the brain
(Marti et al.,
1997, supra).
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Citation or discussion of a reference herein shall not be construed as an
admission that such is prior art to the present invention.
S 3. BRIEF SUMMARY OF THE INVENTION
The present invention is directed to compositions and methods for modulating
excitable tissue function in mammals, as well as methods and compositions for
drug
delivery to excitable tissues. The invention is based, in part, on the
Applicants' discovery
that erythropoietin (EPO), administered systemically and at a high dosage, is
specifically
taken up by the brain. In particular, the Applicants have found that EPO,
delivered in high
doses, can cross the blood-brain barrier, where it can enhance cognitive
function, and
protect neural tissue from damage resulting from stressful conditions, such as
hypoxia.
Erythropoietin and EPO, used interchangeably herein, and EPO receptor activity
modulators, and EPO-activated receptor modulators refer to compounds, which,
when
administered systemically (outside the blood-brain barrier), are capable of
activating EPO-
activated receptors of electrically excitable tissues to enhance and/or
protect from injury and
death. Thus, EPO can refer to any form of erythropoietin that can modulate
excitable tissue,
as well as EPO analogs, fragments and mimetics thereof. In a preferred
embodiment, for
use in the methods of the present invention, the erythropoietin displays
increased specificity
for the brain EPO receptor. In another embodiment, the erythropoietin is
nonerythropoietic.
In yet another embodiment, the erythropoietin is administered at a dose
greater than the dose
necessary to maximally stimulate erythropoiesis.
The present invention provides a pharmaceutical composition in dosage unit
form
adapted for modulation of excitable tissue, enhancement of cognitive function
or delivery of
compounds across endothelial tight junctions which comprises, per dosage unit,
an effective
non-toxic amount within the range from about 50,000 to 500,000 Units of EPO,
an EPO
receptor activity modulator, an EPO-activated receptor modulator, or a
combination thereof,
and a pharmaceutically acceptable Garner. In one embodiment, the effective non-
toxic
amount of EPO in said pharmaceutical composition comprises 50,000 to 500,000
Units of
EPO. In another embodiment, the effective non-toxic amount of EPO of said
pharmaceutical preparation is a dose effective to achieve a circulating level
of EPO of
greater than 10,000 mU/ml of serum. In another embodiment, the circulating
level of EPO
is achieved about l, 2, 3, 4, S, 6, 7, 8, 9, or 10 hours after the
administration of EPO. In
another embodiment, the invention provides a pharmaceutical kit comprising an
effective
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amount of EPO for modulation of excitable tissue, enhancement of cognitive
function or
delivery of compounds across endothelial tight junctions packaged in one or
more
containers.
The present invention provides a method for modulating the function of
excitable
tissue in a mammal, comprising administering peripherally to said mammal an
effective
amount of an erythropoietin. The excitable tissue may be normal tissue or
abnormal,
diseased tissue. In one embodiment, the excitable tissue is neuronal tissue of
the central
nervous system. In other embodiments, the excitable tissue is selected from
the group
consisting of neuronal tissue of the peripheral nervous system and heart
tissue.
In one embodiment, a method is provided for the enhancement of excitable
tissue
function in a mammal, in particular, both normal and abnormal, excitable
tissue, by
administering peripherally an effective amount of EPO or an EPO receptor
activity
modulator. Enhancement of excitable tissue function provides enhancement of,
for
example, learning, associative learning, or memory. Non-limiting examples of
conditions
or diseases treatable by this aspect of the present invention include mood
disorders, anxiety
disorders, depression, autism, attention deficit hyperactivity disorder,
Alzheimer's disease,
aging and cognitive dysfunction.
In another embodiment, the modulation of excitable tissue provides protection
from pathology resulting from injury to excitable tissue, for example, to
neurons of the
central nervous system, peripheral nervous system, or heart tissue. Such
pathology may
result from injuries including, but not limited to hypoxia, seizure disorders,
neurodegenerative diseases, neurotoxin poisoning, multiple sclerosis,
hypotension, cardiac
arrest, radiation, or hypoglycemia. In one embodiment, the pathology is a
result of hypoxia,
and may be prenatal or postnatal oxygen deprivation, suffocation, choking,
near drowning,
post-surgical cognitive dysfunction, carbon monoxide poisoning, smoke
inhalation, chronic
obstructive pulmonary disease, emphysema, adult respiratory distress syndrome,
hypotensive shock, septic shock, insulin shock, anaphylactic shock, sickle
cell crisis, cardiac
arrest, dysrhythmia or nitrogen narcosis. In the instance wherein the
pathology is a seizure
disorder, it may be, by way of non-limiting example, epilepsy, convulsions or
chronic
seizure disorder. In the instance wherein the pathology is a neurodegenerative
disease, it
may be, for example, stroke, Alzheimer's disease, Parkinson's disease,
cerebral palsy, brain
or spinal cord trauma, AIDS dementia, age-related loss of cognitive function,
memory loss,
amyotrophic lateral sclerosis, seizure disorders, alcoholism, retinal
ischemia, aging,
glaucoma or neuronal loss. In another embodiment, administration of EPO may be
used to
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prevent injury or tissue damage during surgical procedures, such as. for
example, tumor
resection or aneurysm repair.
In yet another embodiment, methods are provided for facilitating the
transcytosis
of a molecule across an endothelial cell barrier in a mammal by administration
of a
composition of a molecule in association with erythropoietin. The association
between the
molecule to be transported and EPO may be, for example, a labile covalent
bond, a stable
covalent bond, or a noncovalent association with a binding site for the
molecule. In one
embodiment, the endothelial cell barriers may be the blood-brain barrier, the
blood-eye
barrier, the blood-testes barrier, the blood-ovary barrier or the blood-
placenta barrier.
The invention further provides a composition for transporting a molecule via
transcytosis across an endothelial cell barrier comprising said molecule in
association with
an EPO, an EPO receptor activity modulator, or an EPO-activated receptor
modulator. In
one embodiment, the EPO is erythropoietin, an erythropoietin analog, an
erythropoietin
mimetic, an erythropoietin fragment, a hybrid erythropoietin molecule, an
erythropoietin
receptor-binding molecule, an erythropoietin agonist, a renal erythropoietin,
a brain
erythropoietin, an oligomer thereof, a multimer thereof, a mutein thereof, a
congener
thereof, a naturally-occurring form thereof, a synthetic form thereof, a
recombinant form
thereof, or a combination thereof. In another embodiment, the molecule of said
composition
is a hormone, a neurotrophic factor, an antimicrobial agent, a
radiopharmaceutical, an
antisense compound, an antibody, an immunosuppressant, a toxin, or an anti-
cancer agent.
Suitable molecules for transport by the method of the present invention
include, but are not
limited to hormones, such as growth hormone, antibiotics, anti-cancer agents,
and toxins.
These and other aspects of the present invention will be better appreciated by
reference to the following Figures and Detailed Description.
4. BRIEF DESCRIPTION OF THE FIGURES
FIG. IA-B. Morris Water Maze test A. The results of a Morris Water Maze test
performed in mice receiving either EPO or saline (SHAM) administered
peripherally each
day. B. Subjects receiving EPO performed significantly better than SHAM
treated subjects.
The regression line (R2=0.88) shows a slope (0.68) significantly different
from a slope of 1,
markedly in favor of the EPO group.
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FIGS. 2A-C. Conditioned Taste Aversion test A. Comparison of peripheral
sham and EPO treatment on water consumption in mice undergoing Conditioned
Taste
Aversion testing. Water consumption is expressed as a percentage of the volume
consumed
by control mice, which were not made ill with lithium chloride. B and C
illustrate that the
EPO-enhanced learning is robust, as EPO subjects tolerated much greater thirst
than
controls in avoidance of water containing the illness-associated cue yet spent
more time
seeking water.
FIG. 3A-B A. The results of an experiment which demonstrates that
peripherally-administered EPO pretreatment reduces seizure severity and
protects mice from
convulsions and death by the neurotoxin kainate. The numbers in parentheses
under each
column indicate the number of animals receiving each kainate dose. B shows
that the
protective effects of peripherally-administered EPO increase with daily
administration of
EPO. C illustrates that the onset of action of EPO is delayed, characteristic
of the induction
of a gene expression program.
FIG. 4A-B depicts the protective effect of rhEPO against ischemic brain injury
(focal stroke). A. Systemic administration of EPO given at various times after
the
induction of brain ischemia reduces infarct size. B. Comparison of two forms
of EPO in
protecting brain from injury in this model: recombinant human (rhEPO) and 17
amino acid
EPO derivative (17-mer) illustrates that some EPO analogs are ineffective for
neuroprotection.
FIG. 5 depicts the protective effect of rhEPO against blunt trauma delivered
to
~e cerebral cortex.
FIG. 6A-B depicts the protective effect of EPO from ischemic heart injury. A.
Creatine kinase (CK) activity, an indicator of damage to the myocardial cells.
B.
Myeloperoxidase (MPO) activity, a measure of inflammation.
FIG. 7 shows that treatment of mice with EPO delays and reduces the
neurological symptoms produced by an experimental allergic encephalitis, a
model of
multiple sclerosis.
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FIG. 8A-B A. The minimum effective dose of EPO to provide
neuroprotection in a focal stroke model performed in rats. B. Serum levels of
EPO at
various time points after SOOOU of rhEPO was administered intraperitoneally to
female
Balb/c mice.
FIG. 9A-C A. Immunolocalization of EPO-R on and around capillaries. B.
Biotinylated EPO administered IP to mice is found at S hours within the brain
immediately
surrounding capillaries. C. After 17 hours, the biotin label can be found to
be within
specific neurons.
5. DETAILED DESCRIPTION OF THE INVENTION
The present invention provides compositions and methods for the use of
erythropoietin (EPO) for modulating excitable tissue function, such as, for
example,
enhancing cognitive function and protecting excitable cells from toxic
stimuli. In particular,
the invention provides compositions comprising EPO, as well as methods for
their use in
prophylactic and therapeutic treatments, including drug delivery. As used
herein, excitable
tissue, includes, but is not limited to, neuronal tissue of the central and
peripheral nervous
systems, and cardiac tissue.
The invention described herein provides methods for modulating excitable
tissue
function by peripheral administration of EPO, or an EPO receptor activating
molecule or a
molecule exhibiting EPO-activated receptor activity, as well as any molecule
that mimics
the activity of EPO by acting through other, non-classical EPO receptors.
Without being
bound by any particular mechanism of action, such a molecule may signal via
the EPO
receptor, for example, initiates a signal transduction cascade ultimately
activating a gene
expression program resulting in the protection or enhancement of excitable
tissue function.
Molecules capable of interacting with the EPO receptor and modulating the
activity of the
receptor, herein referred to as EPO or EPO receptor activity modulators, are
useful in the
context of the present invention for the protection or enhancement of
excitable tissue
function. These molecules may be, for example, naturally-occurring, synthetic,
or
recombinant forms of EPO molecules, describe above, or other molecules which
may not
necessarily resemble EPO in any manner, except to modulate EPO receptor
activity, as
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described herein. These molecules may be used in combination for the various
purposes
herein described.
The compositions and methods described herein can be used to treat and/or
protect both normal tissue or abnormal tissue, for example, neurons of the
central nervous
system, neurons of the peripheral nervous system, or heart tissue. In
particular, in Section
5.1, below, EPO compositions useful for practice with invention are described.
In Section
5.2.1, methods are described for the use of such EPO compositions for
enhancing the
function of excitable tissue, such as learning, memory, and other aspects of
cognitive
function, and, in Section 5.2.2, methods for protecting excitable tissue from
damage and
injury are described. Also described in Section 5.2.3 below, the discovery of
the unexpected
ability of EPO to cross capillary endothelial cell tight junctions provides
methods for
delivery of compounds across such barriers. Finally, described in Section 5.3
are conditions
that can be targeted using the methods of the invention, and in Section 5.4,
methods of
administration and effective dosages of such EPO compositions are described.
5.1 COMPOSITIONS COMPRISING ERYTHROPOIETIN
EPO compositions suitable for use with the invention include any
erythropoietin
compound that, when administered peripherally, is capable of activating EPO-
activated
receptors to modulate, i.e. enhance the function of, protect from damage or
injury, or deliver
compounds to, excitable tissue. Erythropoietin is a glycoprotein hormone which
in humans
has a molecular weight of 34 to 38 kD. The mature protein comprises 166 amino
acids, and
the glycosyl residues comprise about 40% of the weight of the molecule. The
forms of EPO
useful in the practice of the present invention encompass naturally-occurnng,
synthetic and
recombinant forms of the following molecules: erythropoietin, erythropoietin
analogs,
erythropoietin mimetics, erythropoietin fragments, hybrid erythropoietin
molecules,
erythropoietin receptor-binding molecules, erythropoietin agonists, renal
erythropoietin,
brain erythropoietin, oligomers and multimers thereof, muteins thereof, and
congeners
thereof. The term "erythropoietin" and "EPO" may be used interchangeably or
conjunctively.
Synthetic and recombinant molecules, such as brain EPO and renal EPO,
recombinant mammalian forms of EPO, as well as its naturally-occurring, tumor-
derived,
and recombinant isoforms, such as recombinantly-expressed molecules and those
prepared
by homologous recombination are provided herein. Furthermore, the present
invention
includes molecules including peptides which bind the EPO receptor, as well as
recombinant
constructs or other molecules which possess part or all of the structural
and/or biological
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properties of EPO, including fragments and multimers of EPO or its fragments.
EPO herein
embraces molecules with altered EPO receptor binding activities, preferably
with increased
receptor affinity, in particular as pertains to enhancing transport across
endothelial cell
barriers. Muteins comprising molecules which have additional or reduced
numbers of
glycosylation sites are included herein. As noted above, the terms
"erythropoietin," 'EPO,"
and "mimetics" as well as the other terms are used interchangeably herein to
refer to the
excitable tissue protective and enhancing molecules related to EPO as well as
the molecules
which are capable of crossing endothelial tight junctions and as such are
useful as a delivery
means for other molecules. Furthermore, molecules produced by transgenic
animals are
also encompassed here. It should be noted that EPO molecules as embraced
herein do not
necessarily resemble EPO structurally or in any other manner, except for
ability to interact
with the EPO receptor or modulate EPO receptor activity or activate EPO-
activated
signaling cascades, as described herein.
By way of non-limiting example, forms of EPO useful for the practice of the
present invention include EPO muteins, such as those with altered amino acids
at the
carboxy terminus described in U.S. Patent 5,457,089 and in U.S. Patent
4,835,260; EPO
isoforms with various numbers of sialic acid residues per molecule, such as
described in
U.S. Patent 5,856,292; polypeptides described in U.S. Patent 4,703,008;
agonists described
in U.S. Patent 5,767,078; peptides which bind to the EPO receptor as described
in U.S.
Patents 5,773,569 and 5,830,851; small-molecule mimetics which activate the
EPO
receptor, as described in U.S. Patent 5,835,382; and EPO analogs described in
WO
9505465, WO 9718318, and WO 9818926. All of the aforementioned citations are
incorporated herein to the extent that such disclosures refer to the various
alternate forms or
processes for preparing such forms of the erythropoietins of the present
invention.
EPO can be obtained commercially (under the trademarks of PROCRIT,
available from Ortho Biotech, and EPOGEN, available from Amgen, Inc., Thousand
Oaks,
CA).
In a further embodiment of the present invention, the EPO molecules embraced
herein include hybrid EPO molecules that may be prepared which comprise the
EPO
receptor modulating activity as well as another activity, for example, that of
growth
hormone. Such hybrid molecules with multiple domains thus possess the ability
to interact
with the EPO receptor- as well as having the activity of another molecule such
as a
hormone. Methods of preparation of such molecules with two domains are known
to one
skilled in the art. As will be described in more detail in Section 5.2.3
below, one feature of
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such molecules is transport across endothelial cell barriers provided by the
EPO receptor
activity modulating domain, and activity of the other molecule at the target
site.
Any of the compounds described above may be tested to identify EPO
compounds capable of modulating excitable tissue, i. e. enhance the function
of, protect from
damage or injury, or deliver compounds thereto, using the assays described
herein. For
example, EPO compounds may be tested for their ability to enhance the function
of
excitable tissue, such as learning, memory, and other aspects of cognitive
function using the
methods described in Section 5.2.1. Examples of in vivo assays for cognitive
function
include the Morris Water Maze test, an example of which is described in
Section 6, and the
Conditioned Taste Aversion test, an example of which is described in detail in
Section 7. In
addition, the EPO compounds described above may be tested using assays
described in
Section 5.2.2, to identify EPO compounds capable of protecting excitable
tissue from
damage and injury. The Examples described in Sections 8, 9, 10, 11, and 12
provide
specific examples of such assays. EPO compounds may also be assayed for their
capacity to
delivery of compounds across epithelial tight junctions, such as the blood-
brain barrier,
using assays such as those described in Section 5.2.3 and Section 9, below.
Thus, EPO
compositions suitable for use with the invention include any and all compounds
that, when
administered peripherally, are capable of signaling through EPO-activated
receptors to
modulate excitable tissue, i.e. enhance the function of, protect from damage
or injury, or
deliver compounds thereto.
5.2 METHODS FOR PROPHYLACTIC AND THERAPEUTIC USE OF THE
INVENTION
In various embodiments of the invention, EPO compositions can be used for
protecting excitable tissue from injury or hypoxic stress, enhancing the
function of excitable
tissue, or for delivery of compounds across endothelial tight junctions of
excitable tissue.
As described above, the invention is based, in part, on the discovery that EPO
molecules can
be transported from the luminal surface to the basement membrane surface of
endothelial
cells of the capillaries of organs with endothelial cell tight junctions,
including, for example,
the brain, retina, and testis. While not wishing to be bound by any particular
theory, after
transcytosis of EPO, EPO can interact with an EPO receptor on excitable
tissue, such as, for
example, neurons of the central nervous system, the peripheral nervous system,
or heart
tissue, and receptor binding can initiate a signal transduction cascade
resulting in the
activation of a gene expression program within the excitable tissue, resulting
in the
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protection of the cell from damage, such as by neurotoxins, hypoxia, etc.
Thus, methods
for protecting excitable tissue from injury or hypoxic stress, enhancing the
function of
excitable tissue, and delivering compounds across tight junctions of excitable
tissue are
described in detail hereinbelow.
5.2.1 METHODS FOR ENHANCING EXCITABLE TISSUE FUNCTION
In one aspect, the present invention is directed to a method for enhancing the
function of excitable tissue by administration of an EPO molecule capable of
activating a
gene expression program that enhances excitable tissue function. Enhancement
of excitable
tissue function provides enhancement of learning, associative learning, and
memory.
Various diseases and conditions are amenable to treatment using this method,
and further,
this method is useful for enhancing cognitive function in the absence of any
condition or
disease. These uses of the present invention are described in further detail
below, and
include enhancement of learning and training in both human and non-human
mammals.
Conditions and diseases treatable by the methods of this aspect of the present
invention include any condition or disease that can benefit from enhancement
of neuronal
function. Examples of such disorders include disorders of the central nervous
system
including, but not limited, to mood disorders, anxiety disorders, depression,
autism,
attention deficit hyperactivity disorder, and cognitive dysfunction. Other non-
limiting
examples of cognitive functions which can be enhanced using the methods of the
invention
are described in Section 5.3.
In one embodiment, for example, an EPO molecule may be administered to a
subject or patient who is suffering from a disorder resulting in loss of
cognitive functions,
such, for example, as Alzheimer's Disease.
The ability of EPO to enhance cognitive functions can be tested in
experimental
animals using any of the methods described herein, or any other art-accepted
learning or
cognitive function model. As described in the Examples presented in Sections 6
and 7,
peripherally-administered erythropoietin was discovered to enhance learning
and cognitive
function as demonstrated by several well established learning models in normal
experimental animals. Examples of such learning models are the Moms water maze
test,
an example of which is given in Section 6 and the conditioned taste aversion
(CTA) test, an
example of which is given in Section 7. In one embodiment, for example, the
conditioned
taste aversion (CTA), a very sensitive, well known, standard test is used to
test an animal's
cognitive function after administration of EPO. CTA is used to test an
animal's ability for
learning to associate illness with a novel stimuli, such as taste, such that
the animals avoid
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the novel taste upon subsequent re-exposure to the novel stimuli. CTA involves
the brain at
a variety of cortical and subcortical levels. The association which links
ascending and
descending information together producing aversive behavior can be either
attenuated or
strengthened by changes affecting any of the interconnecting units. As a form
of associative
learning, the strength of CTA is determined by a large number of variables
including
novelty of the oral stimulus (e.g., non-novel stimuli cannot be aversively
conditioned),
degree of "illness" produced (toxicity), number of repetitions (training),
countering drives
(such as thirst) to name a few. Although a wide variety of chemical and
physical agents can
produce CTA in a dose-dependent manner, lithium chloride reliably produces
malaise and
anorexia. Like a naturally occurring illness, lithium produces a CTA by
stimulating the
pathways described above, including cytokine release.
Enhancement of excitable cell function, for example, cognitive function,
offers
numerous benefits to individuals in the educational and work environment, and
to enhance
the ability to train and educate non-human mammals.
5.2.2 METHODS FOR PROTECTING EXCITABLE TISSUE FROM
INJURY
In another embodiment, the present invention is directed toward a method for
protecting a mammal from pathology resulting from injury to excitable tissue.
Protection
is provided by administering to a mammal by a peripheral route of
administration an amount
of erythropoietin effective to protect the excitable tissue from injury. As is
shown in detail
in the example in Section 8, below, EPO administered in advance of the toxin
kainate is
markedly neuroprotective in mice, raising seizure threshold and preventing
death. The
neuroprotective effect EPO is large and is sustained. It is notable that the
positive effects
seen herein occur within too short of a time relative to the administration of
an EPO to be a
result of an increase in hematocrit as a consequence of the erythropoietic
activity of EPO.
Furthermore, as noted above, an embodiment of the present invention comprises
an EPO
which lacks the ability to increase hematocrit.
In one embodiment, the present invention may be used advantageously both in
the acute and chronic prophylaxis and treatment of neurological disorders, as
described
herein, and in enhanced cognitive function of the normal or the diseased
brain. As noted
above, damage and death of neurons in the central nervous system is a serious
and often
lethal occurrence responsible for a high degree of morbidity and mortality in
the population.
Acute neurological damage may occur during or as a result of seizures,
convulsions,
epilepsy, stroke, hemorrhage, central nervous system injury, hypoxia,
hypoglycemia,
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hypotension and brain or spinal cord trauma. The present invention provides
for acute
administration for the treatment of acute events.
In one embodiment, for example, the methods of the invention may be used to
protect a mammal from injury resulting from radiation damage to the brain.
In another embodiment, a serious condition treatable or preventable in
accordance with the present invention is prophylaxis and treatment in utero of
prenatal
hypoxic conditions, post-birth treatment to protect the brain from hypoxic
injury sustained
during birth, as well as in suffocation, drowning, and other conditions
wherein the central
nervous system is at risk for neurotoxic damage as a result of oxygen
deprivation or
exposure to other neurotoxic stimuli. As is well known, individuals who suffer
from
hypoxia during labor, or as a consequence of non-fatal hypoxic accidents or
incidents may
suffer a lifelong neurologic deficit. Hypoxia and/or cessation of cerebral
blood flow, which
may occur post-trauma or during surgical procedures, also carries a risk of
causing lifelong
neurologic deficit.
1 S Postoperative cognitive dysfunction, including deficits following the use
of a
heart-lung machine, are also treatable by the methods provided herein.
Furthermore, the
present methods may be applied to the treatment of hypoxia resulting from
carbon
monoxide poisoning or smoke inhalation.
In another embodiment, EPO is used to protect cardiac tissue from injury
sustained during ischemia, infarction, inflammation, or trauma.
These are non-limiting examples of damage to excitable tissue treatable in
accordance with the present invention. Acute and early treatment of these
disorders may be
carried out by mobile medical emergency health care professionals such that
treatment may
be started as soon as suspicion of potential for neurologic damage is
ascertained. Risk of
neurologic damage induced by labor may be reduced by prophylactically
treatment of the
fetus before or during labor. These and other utilities and situations will be
recognized by
the skilled artisan.
5.2.3 METHODS FOR DELIVERY OF COMPOUNDS
The present invention is further directed to a method for facilitating the
transport
of a molecule across an endothelial cell barrier in a mammal by administering
a composition
which comprises the particular molecule in association with erythropoietin. As
noted
above, the inventors herein discovered the heretofore unexpected and
surprising activity of
peripherally-administered EPO on excitable tissue, such as nervous tissue in
the central
nervous system, the peripheral nervous system, or heart tissue, identifying
EPO as a
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molecule capable of crossing tight junctions of such excitable tissue, such as
the blood-brain
barrier. As such, EPO is useful as a carnet for delivering other molecules
across the blood-
brain and other similar barriers.
In one embodiment, EPO receptor binding molecules comprising molecules
S conjugated to an EPO molecule, may be used to transport those molecules
across the blood
brain barner. Such molecules can thereby piggyback on EPO for delivery across
the BBB.
In another embodiment, an antibody or other binding partner to the molecule
may be
associated with EPO, or with an EPO receptor activity modulator, thus
associating the
molecule to be transported by noncovalent binding to the binding partner,
which is further
associated with the transportable EPO molecule. In another embodiment, EPO
receptor-
binding molecules comprising antibodies to the EPO receptor are useful for the
method
described here. Such antibodies provide a transport carrier on which other
molecules may
hitchhike, much in the same fashion that antibodies to the transferrin
receptor have been
used to gain access across the blood-brain (Pardridge et al., 1991, Selective
transport of an
antitransferrin receptor antibody through the blood-brain barner in vivo. J.
Pharmacol. Exp.
Therap. 27: 66).
The skilled artisan will be aware of various means for associating molecules
with
EPO and the other agents described above, by covalent, non-covalent, and other
means;
furthermore, evaluation of the efficacy of the composition may be readily
determined in an
experimental system. Association of molecules with EPO and analogs may be
achieved by
any number of means, including labile, covalent binding, cross-linking, etc.
In one
embodiment, for example, the association between the molecule to be
transported across the
barrier and the erythropoietin may be a labile covalent bond, in which case
the molecule is
released from association with the EPO after crossing the barrier. In one
embodiment,
biotinlavidin interactions may be employed. In another embodiment, as
mentioned above,
a hybrid molecule may be prepared by recombinant or synthetic means, for
example, which
includes both the domain of the molecule with desired pharmacological activity
and the
domain responsible for EPO receptor activity modulation.
A molecule may be conjugated to an EPO or EPO receptor activity modulator
through a polyfunctional molecule, i.e., a polyfunctional crosslinker. As used
herein, the
term "polyfunctional molecule" encompasses molecules having one functional
group that
can react more than one time in succession, such as formaldehyde, as well as
molecules with
more than one reactive group. As used herein, the term "reactive group" refers
to a
functional group on the crosslinker that reacts with a functional group on a
molecule (e.g.,
peptide, protein, carbohydrate, nucleic acid, particularly a hormone,
antibiotic, or anti-
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cancer agent to be delivered across an endothelial cell barner) so as to form
a covalent bond
between the cross-linker and that molecule. The term "functional group"
retains its standard
meaning in organic chemistry. The polyfunctional molecules which can be used
are
preferably biocompatible linkers, i.e., they are noncarcinogenic, nontoxic,
and substantially
non-immunogenic in vivo. Polyfunctional cross-linkers such as those known in
the art and
described herein can be readily tested in animal models to determine their
biocompatibility.
The polyfunctional molecule is preferably bifunctional. As used herein, the
term
"bifunctional molecule" refers to a molecule with two reactive groups. The
bifunctional
molecule may be heterobifunctional or homobifunctional. A heterobifunctional
cross-linker
allows for vectorial conjugation. It is particularly preferred for the
polyfunctional molecule
to be sufficiently soluble in water for the cross-linking reactions to occur
in aqueous
solutions such as in aqueous solutions buffered at pH 6 to 8, and for the
resulting conjugate
to remain water soluble for more effective bio-distribution. Typically, the
polyfunctional
molecule covalently bonds with an amino or a sulfhydryl functional group.
However,
1 S polyfunctional molecules reactive with other functional groups, such as
carboxylic acids or
hydroxyl groups, are contemplated in the present invention.
The homobifunctional molecules have at least two reactive functional groups,
which are the same. The reactive functional groups on a homobifunctional
molecule
include, for example, aldehyde groups and active ester groups.
Homobifunctional
molecules having aldehyde groups include, for example, glutaraldehyde and
subaraldehyde.
The use of glutaraldehyde as a cross-linking agent was disclosed by Poznansky
et al.,
Science 223, 1304-1306 (1984). Homobifunctional molecules having at least two
active
ester units include esters of dicarboxylic acids and N-hydroxysuccinimide.
Some examples
of such N-succinimidyl esters include disuccinimidyl suberate and dithio-bis-
(succinimidyl
propionate), and their soluble bis-sulfonic acid and bis-sulfonate salts such
as their sodium
and potassium salts. These homobifunctional reagents are available from
Pierce, Rockford,
Illinois.
The heterobifunctional molecules have at least two different reactive groups.
The reactive groups react with different functional groups, e.g., present on
the EPO and the
molecule. These two different functional groups that react with the reactive
group on the
heterobifunctional cross-linker are usually an amino group, eg., the epsilon
amino group of
lysine; a sulfhydryl group, e.g., the thiol group of cysteine; a carboxylic
acid, e.g., the
carboxylate on aspartic acid; or a hydroxyl group, e.g., the hydroxyl group on
serine.
When a reactive group of a heterobifunctional molecule forms a covalent bond
with an amino group, the covalent bond will usually be an amido or imido bond.
The
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reactive group that forms a covalent bond with an amino group may, for
example, be an
activated carboxylate group, a halocarbonyl group, or an ester group. The
preferred
halocarbonyl group is a chlorocarbonyl group. The ester groups are preferably
reactive ester
groups such as, for example, an N-hydroxy-succinimide ester group.
The other functional group typically is either a thiol group, a group capable
of
being converted into a thiol group, or a group that forms a covalent bond with
a thiol group.
The covalent bond will usually be a thioether bond or a disulfide. The
reactive group that
forms a covalent bond with a thiol group may, for example, be a double bond
that reacts
with thiol groups or an activated disulfide. A reactive group containing a
double bond
capable of reacting with a thiol group is the maleimido group, although
others, such as
acrylonitrile, are also possible. A reactive disulfide group may, for example,
be a 2-
pyridyldithio group or a 5,5'-dithio-bis-(2-nitrobenzoic acid) group. Some
examples of
heterobifunctional reagents containing reactive disulfide bonds include N-
succinimidyl 3-
(2-pyridyl-dithio)propionate (Carlsson et al., 1978, Biochem J., 173:723-737),
sodium 5-4-
succinimidyloxycarbonyl-alpha-methylbenzylthiosulfate, and 4-
succinimidyloxycarbonyl-
alpha-methyb(2-pyridyldithio)toluene. N-succinimidyl 3-(2-
pyridyldithio)propionate is
preferred. Some examples of heterobifunctional reagents comprising reactive
groups having
a double bond that reacts with a thiol group include succinimidyl 4-(N-
maleimidomethyl)cyclohexane-1-carboxylate and succinimidyl m-
maleimidobenzoate.
Other heterobifunctional molecules include succinimidyl 3-
(maleimido)propionate, sulfosuccinimidyl 4-(p-maleimido-phenyl)butyrate,
sulfosuccinimidyl 4-(N-maleimidomethyl- cyclohexane)- 1 -carboxylate,
maleimidobenzoyl-N-hydroxy-succinimide ester. The sodium sulfonate salt of
succinimidyl
m-maleimidobenzoate is preferred. Many of the above-mentioned
heterobifunctional
reagents and their sulfonate salts are available from Pierce.
The need for the above-described conjugated to be reversible or labile may be
readily determined by the skilled artisan. A conjugate may be tested in vitro
for both the
EPO receptor activity modulation activity, and for the desirable
pharmacological activity. If
the conjugate retains both properties, its suitability may then be tested in
vivo. If the
conjugated molecule requires separation from the EPO for activity, a labile
bond or
reversible association with EPO will be preferable. The lability
characteristics may also be
tested using standard in vitro procedures before in vivo testing.
Additional information regarding how to make and use these as well as other
polyfunctional reagents may be obtained from the following publications or
others available
in the art: Carlsson et al., 1978, Biochem. J. 173:723-737; Cumber et al.,
1985, Methods in
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Enzymology 112:207-224; Jue et al., 1978, Biochem. 17:5399-5405; Sun et al.,
1974,
Biochem. 13:2334-2340; Blattler et al., 1985, Biochem. 24:1517-152; Liu et
al., 1979,
Biochem. 18:690-697; Youle and Neville, 1980, Proc. Natl. Acad. Sci. U.S.A.
77:5483-
5486; Lerner et al., 1981, Proc. Natl. Acad. Sci. U.S.A. 78:3403-3407; Jung
and Moroi,
S 1983, Biochem. Biophys. Acta 761:162; Caulfield et al., 1984, Biochem.
81:7772-7776;
Status, J.V., 1982, Biochem. 21:3950-3955; Yoshitake et al., 1979, Eur. J.
Biochem.
101:395-399; Yoshitake et al., 1982, J. Biochem. 92:1413-1424; Pilch and
Czech, 1979, J.
Biol. Chem. 254:3375-3381; Novick et al., 1987, J. Biol. Chem. 262:8483-8487;
Lomant
and Fairbanks, 1976, J. Mol. Biol. 104:243-261; Hamada and Tsuruo, 1987, Anal.
Biochem. 160:483-488; and Hashida, 1984, J. Applied Biochem. 6:56-63.
Additionally,
methods of cross-linking are reviewed by Means and Feeney, 1990, Bioconjugate
Chem.
1:2-12. Barriers which are crossed by the above-described methods and
compositions of the
present invention include but are not limited to the blood-brain barrier, the
blood-eye
barrier, the blood-testes barrier, the blood-ovary barrier, and the blood-
placenta barner.
Candidate molecules for transport across an endothelial cell barrier include,
for
example, hormones such as growth hormone, neurotrophic factors, antibiotics or
antifungals
such as those normally excluded from the brain and other barriered organs,
peptide
radiopharmaceuticals, antisense drugs, antibodies against biologically-active
agents,
pharmaceuticals, and anti-cancer agents. Non-limiting examples of such
molecules include
growth hormone, nerve growth factor (NGF), brain-derived neurotrophic factor
(BNF),
ciliary neurotrophic factor (CTF.), basic fibroblast growth factor (bFGF),
transforming
growth factor X31 (TGF~31), transforming growth factor (32 (TGF~32),
transforming growth
factor ~i3 (TGF~33), interleukin 1, interleukin 2, interleukin 3, and
interleukin 6, AZT,
antibodies against tumor necrosis factor, and immunosuppressive agents such as
cyclosporin.
In another embodiment, recombinant chimeric toxin molecules comprising EPO
can be used for therapeutic delivery of toxins to treat a viral disorder or
proliferative
disorder, such as cancer. Compounds that could be fused to EPO to construct a
chimeric
toxin suitable for this embodiment include, but are not limited to, toxic
substances, such as
pseudomonas exotoxin, diphtheria toxin, and ricin, among others.
5.3 TARGET CONDITIONS
As described above, the EPO compositions and methods for their use provided
herein can be used to treat and prevent conditions arising from hypoxic
conditions, which
adversely affect excitable tissues, such as excitable tissues in the central
nervous system
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tissue, peripheral nervous system tissue, or cardiac tissue such as, for
example, brain, heart,
or retina. Therefore, the invention can be used to treat or prevent damage to
excitable tissue
resulting from hypoxic conditions in a variety of conditions and
circumstances. Non-
limiting examples of such conditions and circumstances are provided
hereinbelow.
In the example of the protection of neuronal tissue pathologies treatable in
accordance with the present invention, such pathologies include those which
result from
reduced oxygenation of neuronal tissues. Any condition which reduces the
availability of
oxygen to neuronal tissue, resulting in stress, damage, and finally, neuronal
cell death, can
be treated by the methods of the present invention. Generally referred to as
hypoxia and/or
ischemia, these conditions arise from or include, but are not limited to
stroke, vascular
occlusion, prenatal or postnatal oxygen deprivation, suffocation, choking,
near drowning,
carbon monoxide poisoning, smoke inhalation, trauma, including surgery and
radiotherapy,
asphyxia, epilepsy, hypoglycemia, chronic obstructive pulmonary disease,
emphysema,
adult respiratory distress syndrome, hypotensive shock, septic shock,
anaphylactic shock,
insulin shock, sickle cell crisis, cardiac arrest, dysrhythmia, and nitrogen
narcosis.
In one embodiment, for example, EPO may be administered to prevent injury or
tissue damage resulting from risk of injury or tissue damage during surgical
procedures,
such as, for example, tumor resection or aneurysm repair.
Other pathologies caused by or resulting from hypoglycemia which are treatable
by the methods described herein include insulin overdose, also referred to as
iatrogenic
hyperinsulinemia, insulinoma, growth hormone deficiency, hypocortisolism, drug
overdose,
and certain tumors.
Other pathologies resulting from excitable neuronal tissue damage include
seizure disorders, such as epilepsy, convulsions, or chronic seizure
disorders. Other
treatable conditions and diseases include diseases such as stroke, multiple
sclerosis,
hypotension, cardiac arrest, Alzheimer's disease, Parkinson's disease,
cerebral palsy, brain
or spinal cord trauma, AIDS dementia, age-related loss of cognitive function,
memory loss,
amyotrophic lateral sclerosis, seizure disorders, alcoholism, retinal
ischemia, optic nerve
damage resulting from glaucoma, and neuronal loss.
The methods of the invention may be used to treat conditions of, and damage
to,
retinal tissue. Such disorders include, but are not limited to macular
degeneration, retinal
detachment, retinitis pigmentosa, arteriosclerotic retinopathy, hypertensive
retinopathy,
retinal artery blockage, retinal vein blockage, hypotension, and diabetic
retinopathy.
In another embodiment, the methods of the invention may be used to protect or
treat injury resulting from radiation damage to excitable tissue.
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A further utility of the methods of the present invention is in the treatment
of
neurotoxin poisoning, such as domoic acid shellfish poisoning, neurolathyrism,
and Guam
disease, amyotrophic lateral sclerosis, and Parkinson's disease.
As mentioned above, the present invention is also directed to a method for
enhancing excitable tissue function in a mammal by peripheral administration
of
erythropoietin. Various diseases and conditions are amenable to treatment
using this
method, and further, this method is useful for enhancing cognitive function in
the absence of
any condition or disease. These uses of the present invention are described in
further detail
below, and include enhancement of learning and training in both human and non-
human
mammals.
Conditions and diseases treatable by the methods of this aspect of the present
invention directed to the central nervous system include but are not limited
to mood
disorders, anxiety disorders, depression, autism, attention deficit
hyperactivity disorder, and
cognitive dysfunction. These conditions benefit from enhancement of neuronal
function.
Other disorders treatable in accordance with the teachings of the present
invention include sleep disruption, for example, sleep apnea and travel-
related disorders;
subarachnoid and aneurysmal bleeds, hypotensive shock, concussive injury,
septic shock,
anaphylactic shock, and sequelae of various encephalitides and meningitides,
for example,
connective tissue disease-related cerebritides such as lupus. Other uses
include prevention
of or protection from poisoning by neurotoxins, such as domoic acid shellfish
poisoning,
neurolathyrism, and Guam disease, amyotrophic lateral sclerosis, Parkinson's
disease;
postoperative treatment for embolic or ischemic injury; whole brain
irradiation; sickle cell
crisis; and eclampsia.
A further group of conditions treatable by the methods of the present
invention
include mitochondria) dysfunction, of either an hereditary or acquired nature,
which are the
cause of a variety of neurological diseases typified by neuronal injury and
death. For
example, Leigh disease (subacute necrotizing encephalopathy) is characterized
by
progressive visual loss and encephalopathy, due to neuronal drop out, and
myopathy. In
these cases, defective mitochondria) metabolism fails to supply enough high
energy
substrates to fuel the metabolism of excitable cells. An EPO receptor activity
modulator
optimizes failing function in a variety of mitochondria) diseases.
As mentioned above, hypoxic conditions adversely affect excitable tissues. The
excitable tissues include, but are not limited to, central nervous system
tissue, peripheral
nervous system tissue, and heart tissue. In addition to the conditions
described above, the
methods of the present invention are useful in the treatment of inhalation
poisoning such as
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carbon monoxide and smoke inhalation, severe asthma, adult respiratory
distress syndrome,
and choking and near drowning. Further conditions which create hypoxic
conditions or by
other means induce excitable tissue damage include hypoglycemia that may occur
in
inappropriate dosing of insulin, or with insulin-producing neoplasms
(insulinoma).
Various neuropsychologic disorders which-are believed to originate from
excitable tissue damage are treatable by the instant methods. Chronic
disorders in which
neuronal damage may be involved and for which treatment by the present
invention is
provided include disorders relating to the central nervous system and/or
peripheral nervous
system including age-related loss of cognitive function and senile dementia,
chronic seizure
disorders, Alzheimer's disease, Parkinson's disease, dementia, memory loss,
amyotrophic
lateral sclerosis, multiple sclerosis, tuberous sclerosis, Wilson's Disease,
cerebral and
progressive supranuclear palsy, Guam disease, Lewy body dementia, prion
diseases, such as
spongiform encephalopathies, e.g., Creutzfeldt-Jakob disease, Huntington's
disease,
myotonic dystrophy, Freidrich's ataxia and other ataxias, as well as Gilles de
la Tourette's
1 S syndrome, seizure disorders such as epilepsy and chronic seizure disorder,
stroke, brain or
spinal cord trauma, AIDS dementia, alcoholism, autism, retinal ischemia,
glaucoma,
autonomic function disorders such as hypertension and sleep disorders, and
neuropsychiatric
disorders that include, but are not limited to schizophrenia, schizoaffective
disorder,
attention deficit disorder, dysthymic disorder, major depressive disorder,
mania,
obsessive-compulsive disorder, psychoactive substance use disorders, anxiety,
panic
disorder, as well as unipolar and bipolar affective disorders. Additional
neuropsychiatric
and neurodegenerative disorders include, for example, those listed in the
American
Psychiatric Association's Diagnostic and Statistical manual of Mental
Disorders (DSM), the
most current version of which is incorporated herein by reference in its
entirety.
In another embodiment, recombinant chimeric toxin molecules comprising EPO
can be used for therapeutic delivery of toxins to treat a proliferative
disorder, such as cancer,
or viral disorder, such as subacute sclerosing panencephalitis.
5.4 PHARMACEUTICAL PREPARATIONS AND ADMINISTRATION
According to the invention, EPO, its analogues, mimetics, erythropoietin
fragments, hybrid erythropoietin molecules, erythropoietin receptor-binding
molecules,
erythropoietin agonists, renal erythropoietin, brain erythropoietin, muteins
thereof, and
congeners thereof, may be introduced parenterally, transmucosally, e.g.,
orally, nasally,
rectally, intravaginally, sublingually, submucosally or transdermally.
Preferably,
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administration is parenteral, e.g., via intravenous or intraperitoneal
injection, and also
including, but is not limited to, intra-arterial, intramuscular, intradermal
and subcutaneous
administration. The preferred route of administration of small molecule EPO
mimetics is by
the oral route.
A subject in whom peripheral administration of EPO is an effective therapeutic
regiment is preferably a human, but can be any animal, preferably a mammal.
Thus, as can
be readily appreciated by one of ordinary skill in the art, the methods and
pharmaceutical
compositions of the present invention are particularly suited to
administration to any animal,
particularly a mammal, and including, but by no means limited to, domestic
animals, such
as feline or canine subjects, farm animals, such as but not limited to bovine,
equine, caprine,
ovine, and porcine subjects, wild animals (whether in the wild or in a
zoological garden),
research animals, such as mice, rats, rabbits, goats, sheep, pigs, dogs, cats,
etc. As noted
above, domesticated animals, including pets and work animals, are candidates
for both the
neuroprotective benefits of the present invention, as well as the enhancement
of cognitive
function. Neurological damage arising from hypoxia, and well as acute and
chronic
disorders including epilepsy, are common among such animals, and thus are
candidates for
treatment. Also as noted above, cognitive enhancement in non-human animals is
a benefit
of the present invention, in that learning, training, and retention of learned
behavior may be
enhanced, reinforced, and maintained using the teachings of the present
invention. As such,
the expense and psychological strain to the pet owner is reduced. For example,
the time
required for training dogs and other domestic animals is reduced. Furthermore,
wild
animals typically difficult to train may be better candidates for training by
the methods of
the present invention.
5.4.1 FORMULATION AND EFFECTIVE DOSE
The present invention also provides pharmaceutical compositions
Pharmaceutical compositions comprising EPO and EPO receptor activity
modulators can be
administered to a patient at therapeutically effective doses to protect
excitable tissue from
damage, enhance the function of excitable tissue, or to deliver a compound to
excitable
tissue. The Applicants have discovered that an elevated dose of EPO is
preferred to
modulate excitable tissue, and to protect against injury thereto.
Selection of the preferred effective dose will be determined by a skilled
artisan
based upon considering several factors which will be known to one of ordinary
skill in the
art. Such factors include the particular form of erythropoietin, and its
pharmacokinetic
p~,~eters such as bioavailability, metabolism, half life, etc., which will
have been
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established during the usual development procedures typically employed in
obtaining
regulatory approval for a pharmaceutical compound. Further factors in
considering the dose
include the condition or disease to be treated or the benefit to be achieved
in a normal
individual, the body mass of the patient, the route of administration, whether
administration
is acute or chronic, concomitant medications, and other factors well known to
affect the
efficacy of administered pharmaceutical agents. Thus the precise dosage should
be decided
according to the judgment of the practitioner and each patient's
circumstances, e.g.,
depending upon the condition and the immune status of the individual patient,
according to
standard clinical techniques.
In one embodiment, the invention provides a pharmaceutical composition in
dosage unit form adapted for modulation of excitable tissue, enhancement of
cognitive
function or delivery of compounds across endothelial tight junctions which
comprises, per
dosage unit, an effective non-toxic amount within the range from about 50,000
to 500,000
Units, 60,000 to 500,000 Units, 70,000 to 500,000 Units, 80,000 to 500,000
Units, 90,000
to 500,000 Units, 100,000 to 500,000 Units, 150,000 to 500,000 Units, 200,000
to 500,000
Units, 250,000 to 500,000 Units, 300,000 to 500,000 Units, 350,000 to 500,000
Units,
400,000 to 500,000 Units, or 450,000 to 500,000 Units of EPO, an EPO receptor
activity
modulator, or an EPO-activated receptor modulator and a pharmaceutically
acceptable
carrier. In a preferred embodiment, the effective non-toxic amount of EPO is
within the
range from about 50,000 to 500,000 Units.
In one embodiment, such a pharmaceutical composition of EPO may be
administered systemically to protect excitable tissue from damage, enhance the
function of
excitable tissue, or to deliver a compound to excitable tissue. Such
administration may be
parenterally, transmucosally, e.g., orally, nasally, rectally, intravaginally,
sublingually,
submucosally or transdermally. Preferably, administration is parenteral, e.g.,
via
intravenous or intraperitoneal injection, and also including, but is not
limited to, intra-
arterial, intramuscular, intradermal and subcutaneous administration.
In a preferred embodiment, EPO may be administered systemically at a dosage
between 2000-10000 Units /kg body weight, preferably about 2000-5000 Units/kg-
body
weight, most preferably 5000 Units/kg-body weight, per administration. This
effective
dose should be sufficient to achieve serum levels of EPO greater than about
10,000, 15,000,
or 20,000 mU/ml of serum after EPO administration. Such serum levels may be
achieved
at about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 hours post-administration. Such
dosages may be
repeated as necessary. For example, administration may be repeated daily, as
long as
clinically necessary, or after an appropriate interval, e.g., every 1 to 12
weeks, preferably,
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every 3 to 8 weeks. In one embodiment, the effective amount of EPO and a
pharmaceutically acceptable carrier may be packaged in a single dose vial or
other
container. In one embodiment, an EPO is nonerythropoietic, i.e., it is capable
of exerting
the activities described herein but not causing an increase in hemoglobin
concentration or
hematocrit. In another embodiment, an EPO is given at a dose greater than that
necessary to
maximally stimulate erythropoiesis.
The pharmaceutical compositions of the invention may comprise a
therapeutically effective amount of a compound, and a pharmaceutically
acceptable carrier.
In a specific embodiment, the term "pharmaceutically acceptable" means
approved by a
regulatory agency of the Federal or a state government or listed in the U.S.
Pharmacopeia or
other generally recognized pharmacopeia for use in animals, and more
particularly in
humans. The term "carrier" refers to a diluent, adjuvant, excipient, or
vehicle with which
the therapeutic is administered. Such pharmaceutical carriers can be sterile
liquids, such as
saline solutions in water and oils, including those of petroleum, animal,
vegetable or
synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and
the like. A
saline solution is a preferred carrier when the pharmaceutical composition is
administered
intravenously. Saline solutions and aqueous dextrose and glycerol solutions
can also be
employed as liquid carriers, particularly for injectable solutions. Suitable
pharmaceutical
excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice,
flour, chalk, silica
gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim
milk, glycerol,
propylene, glycol, water, ethanol and the like. The composition, if desired,
can also contain
minor amounts of wetting or emulsifying agents, or pH buffering agents. These
compositions can take the form of solutions, suspensions, emulsion, tablets,
pills, capsules,
powders, sustained-release formulations and the like. The composition can be
formulated as
a suppository, with traditional binders and carriers such as triglycerides.
The compounds of
the invention can be formulated as neutral or salt forms. Pharmaceutically
acceptable salts
include those formed with free amino groups such as those derived from
hydrochloric,
phosphoric, acetic, oxalic, tartaric acids, etc., and those formed with free
carboxyl groups
such as those derived from sodium, potassium, ammonium, calcium, fernc
hydroxides,
isopropylamine, triethylamine, 2-ethylamino ethanol, histidine, procaine, etc.
Examples of
suitable pharmaceutical carriers are described in "Remington's Pharmaceutical
Sciences" by
E. W. Martin. Such compositions will contain a therapeutically effective
amount of the
compound, preferably in purified form, together with a suitable amount of
Garner so as to
provide the form for proper administration to the patient. The formulation
should suit the
mode of administration.
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Pharmaceutical compositions adapted for oral administration may be provided as
capsules or tablets; as powders or granules; as solutions, syrups or
suspensions (in aqueous
or non-aqueous liquids); as edible foams or whips; or as emulsions. Tablets or
hard gelatine
capsules may comprise lactose, starch or derivatives thereof, magnesium
stearate, sodium
saccharine, cellulose, magnesium carbonate, stearic acid or salts thereof.
Soft gelatine
capsules may comprise vegetable oils, waxes, fats, semi-solid, or liquid
polyols etc.
Solutions and syrups may comprise water, polyols and sugars.
An active agent intended for oral administration may be coated with or admixed
with a material that delays disintegration and/or absorption of the active
agent in the
gastrointestinal tract (e.g., glyceryl monostearate or glyceryl distearate may
be used). Thus,
the sustained release of an active agent may be achieved over many hours and,
if necessary,
the active agent can be protected from being degraded within the stomach.
Pharmaceutical
compositions for oral administration may be formulated to facilitate release
of an active
agent at a particular gastrointestinal location due to specific pH or
enzymatic conditions.
ph~aceutical compositions adapted for transdermal administration may be
provided as discrete patches intended to remain in intimate contact with the
epidermis of the
recipient for a prolonged period of time. Pharmaceutical compositions adapted
for topical
administration may be provided as ointments, creams, suspensions, lotions,
powders,
solutions, pastes, gels, sprays, aerosols or oils. For topical administration
to the skin,
mouth, eye or other external tissues a topical ointment or cream is preferably
used. When
formulated in an ointment, the active ingredient may be employed with either a
para~nic or
a water-miscible ointment base. Alternatively, the active ingredient may be
formulated in a
cream with an oil-in-water base or a water-in-oil base. Pharmaceutical
compositions
adapted for topical administration to the eye include eye drops. In these
compositions, the
active ingredient can be dissolved or suspended in a suitable carrier, e.g.,
in an aqueous
solvent. Pharmaceutical compositions adapted for topical administration in the
mouth
include lozenges, pastilles and mouthwashes.
Pharmaceutical compositions adapted for nasal administration may comprise
solid carriers such as powders (preferably having a particle size in the range
of 20 to S00
microns). Powders can be administered in the manner in which snuff is taken,
i.e., by rapid
inhalation through the nose from a container of powder held close to the nose.
Alternatively, compositions adopted for nasal administration may comprise
liquid Garners,
e.g., nasal sprays or nasal drops. These compositions may comprise aqueous or
oil solutions
of the active ingredient. Compositions for administration by inhalation may be
supplied in
specially adapted devices including, but not limited to, pressurized aerosols,
nebulizers or
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insufflators, which can be constructed so as to provide predetermined dosages
of the active
ingredient. In a preferred embodiment, pharmaceutical compositions of the
invention are
administered via the nasal cavity to the lungs.
Pharmaceutical compositions adapted for rectal administration may be provided
as suppositories or enemas. Pharmaceutical compositions adapted for vaginal
administration may be provided as pessaries, tampons, creams, gels, pastes,
foams or spray
formulations.
Pharmaceutical compositions adapted for parenteral administration include
aqueous and non-aqueous sterile injectable solutions or suspensions, which may
contain
antioxidants, buffers, bacteriostats and solutes that render the compositions
substantially
isotonic with the blood of an intended recipient. Other components that may be
present in
such compositions include water, alcohols, polyols, glycerine and vegetable
oils, for
example. Compositions adapted for parenteral administration may be presented
in unit-dose
or mufti-dose containers, for example sealed ampules and vials, and may be
stored in a
freeze-dried (lyophilised) condition requiring only the addition of a sterile
liquid carrier,
e.g., sterile saline solution for injections, immediately prior to use.
Extemporaneous
injection solutions and suspensions may be prepared from sterile powders,
granules and
tablets.
In a preferred embodiment, the composition is formulated in accordance with
routine procedures as a pharmaceutical composition adapted for intravenous
administration
to human beings. Typically, compositions for intravenous administration are
solutions in
sterile isotonic aqueous buffer. Where necessary, the composition may also
include a
solubilizing agent and a local anesthetic such as lidocaine to ease pain at
the site of the
injection. Generally, the ingredients are supplied either separately or mixed
together in unit
dosage form, for example, as a dry lyophilized powder or water-free
concentrate in a
hermetically sealed container such as an ampule or sachette indicating the
quantity of active
agent. Where the composition is to be administered by infusion, it can be
dispensed with an
infusion bottle containing sterile pharmaceutical grade water or saline. Where
the
composition is administered by injection, an ampule of sterile water for
injection or saline
can be provided so that the ingredients may be mixed prior to administration.
Suppositories generally contain active ingredient in the range of 0.5% to 10%
by
weight; oral formulations preferably contain 10% to 95% active ingredient.
The invention also provides a pharmaceutical pack or kit comprising one or
more
containers filled with one or more of the ingredients of the pharmaceutical
compositions of
the invention. Optionally associated with such containers) can be a notice in
the form
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prescribed by a governmental agency regulating the manufacture, use or sale of
pharmaceuticals or biological products, which notice reflects approval by the
agency of
manufacture, use or sale for human administration.
5.4.2 METHODS OF ADMINISTRATION
The present invention provides compositions and methods for peripheral
administration of EPO to enhance function or protect excitable tissues, and to
deliver
compounds to such tissues. As noted above, the present invention is based, in
part, on the
discovery that peripherally administered EPO has direct neuroprotective or
neuroenhancement properties in excitable tissue, such as tissue of the central
nervous
system, peripheral nervous system, or heart tissue. Excitable tissue, as used
herein,
includes, but is not limited to, neuronal tissue of the central and peripheral
nervous systems,
and cardiac tissue. This section describes such compounds, and their methods
for their of
administration.
The present invention provides for administration of EPO and EPO receptor
activity modulators by routes of administration other than directly into the
central nervous
system, and the terms "peripheral" and "systemic" subsumes these various
routes.
Peripheral administration includes oral or parenteral administration, such as
intravenous,
intraarterial, subcutaneous, intramuscular, intraperitoneal, rectal,
submucosal or intradermal
administration. Other routes are useful for the administration of the agents
described herein.
Both acute and chronic administration are provided herein.
In one embodiment, for example, EPO can be delivered in a controlled-release
system. For example, the polypeptide may be administered using intravenous
infusion, an
implantable osmotic pump, a transdermal patch, liposomes, or other modes of
administration. In one embodiment, a pump may be used (see Larger, supra;
Sefton, 1987,
CRC Crit. Ref. Biomed. Erg. 14:201; Buchwald et al., 1980, Surgery 88:507;
Saudek et al.,
1989, N. Engl. J. Med. 321:574). In another embodiment, the compound can be
delivered
in a vesicle, in particular a liposome (see Larger, Science 249:1527-1533
(1990); Treat et
al., in Liposomes in the Therapy of Infectious Disease and Cancer, Lopez-
Berestein and
Fidler (eds.), Liss, New York, pp. 353-365 (1989); WO 91/04014; U.S. Patent
No.
4,704,355; Lopez-Berestein, ibid., pp. 317-327; see generally ibid.). In
another
embodiment, polymeric materials can be used [see Medical Applications of
Controlled
Release, Larger and Wise (eds.), CRC Press: Boca Raton, Florida, 1974;
Controlled Drug
Bioavailability, Drug Product Design and Performance, Smolen and Ball (eds.),
Wiley: New
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York (1984); Ranger and Peppas, J. Macromol. Sci. Rev. Macromol. Chem. 23:61,
1953;
see also Levy et al., 1985, Science 228:190; During et al., 1989, Ann. Neurol.
25:351;
Howard et al., 1989, J. Neurosurg. 71:105).
In yet another embodiment, a controlled release system can be placed in
proximity of the therapeutic target, i. e. , the brain, thus requiring only a
fraction of the
systemic dose (see, e.g., Goodson, pp. 115-138 in Medical Applications of
Controlled
Release, vol. 2, supra, 1984). Other controlled release systems are discussed
in the review
by Langer (1990, Science 249:1527-1533).
In another embodiment, EPO, as properly formulated, can be administered by
nasal, oral, rectal, vaginal, or sublingual administration.
In a specific embodiment, it may be desirable to administer the EPO
compositions of the invention locally to the area in need of treatment; this
may be achieved
by, for example, and not by way of limitation, local infusion during surgery,
topical
application, e.g., in conjunction with a wound dressing after surgery, by
injection, by means
of a catheter, by means of a suppository, or by means of an implant, said
implant being of a
porous, non-porous, or gelatinous material, including membranes, such as
sialastic
membranes, or fibers.
The present invention may be better understood by reference to the following
non-limiting Examples, which are provided as exemplary of the invention. The
following
examples are presented in order to more fully illustrate the preferred
embodiments of the
invention. They should in no way be construed, however, as limiting the broad
scope of the
invention.
As is described hereinbelow, the studies that were performed by the inventors
herein are standard, universally-accepted tests in animal models predictive of
prophylactic
and therapeutic benefit.
6. EXAMPLE 1: PERIPHERALLY ADMINISTERED EPO ENHANCES
COGNITIVE FUNCTION
In this Example, a spatial navigation experiment, known as the Morris Water
Maze test, demonstrates EPO-induced enhancement of cognitive ftmction in mice.
In this
test, a small transparent platform is placed in one quadrant of a swimming
pool with opaque
water. Mice placed into this swimming pool must swim until they reach the
resting
platform below the surface, which is invisible to the swimming mice. The test
consists of
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measuring the time the animals take to get to the platform (i.e., the length
of time they spend
swimming). On successive trials, the time each mouse takes to reach the
platform will
decrease as a function of them learning its location. This type of learning
experiment
involves the hippocampus, as hippocampal lesions prevent learning in this
test.
Experiments were carried out in a circular black pool, 150 cm in diameter.
Four
points were arbitrarily assigned: north, south, east and west. Distinctive
visual cues were
applied to each of these four quadrants: e.g., flashing lights, bright tape
applied in squares
etc., to orient the mice in the pool. A platform was arbitrarily placed in one
quadrant. A
trial consisted of placing the animal head-first in one quadrant of the pool
and releasing it.
The trial length was 90 seconds total. If the animal did not make it to the
platform, she was
placed on it for an additional 15 seconds. The subjects were rested for an
hour, then placed
in another quadrant for testing. All 4 quadrants were used over the course of
a day's trials,
and the animals were tested on 12 successive days (i.e., a total of 48
trials).
The experiment itself consisted of injecting each mice with 5000 U/kg
recombinant human EPO (sold under the tradename of PROCRIT, Ortho-Biotech,
Inc.) by
intraperitoneal injection, 4 hours before the beginning of the day's testing,
each day for the
12 trial days. Control animals were sham-injected with saline.
Learning was measured by measuring the length of time each mouse spent on the
platform. Shown in Figure 1 A, the results are plotted as the time spent on
the platform by
the EPO-treated group and the sham group. As the results indicate, both groups
of animals
spent more time on the platform, i. e. they learned to reach the platform
faster, on each
successive trial day, but the EPO-treated animals did so faster than the sham
group. Thus
EPO-treated animals have a much faster "learning curve" than the sham group.
When
results were expressed as the difference between the EPO-treated and the sham-
treated
groups, and the results of the EPO and the sham-treated group were compared,
the
regression line (R2=0.88) shows a slope (0.68) significantly different from a
slope of 1,
markedly in favor of the EPO group (Figure 1 B).
7. EXAMPLE 2: PERIPHERALLY ADMINISTERED EPO STRENGTHENS A
LEARNED CONDITIONED TASTE AVERSION
The Conditioned Taste Aversion (CTA) test performed in this Example
demonstrates that EPO dramatically affects the ability of mice to remember,
and learn to
avoid, an unpleasant taste sensation, in this case an illness-provoking
substance. In this
example, lithium chloride is used to produce CTA, because lithium chloride
reliably
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produces malaise and anorexia in a dose-dependent manner. Like a naturally
occurring
illness, lithium produces a CTA by stimulating the pathways described above,
including
cytokine release.
Female Balb/c mice were trained to limit their total daily water intake to a
single
minute drinking period per day, and learned to drink enough water during this
period to
remain at equilibrium. Animals were divided into groups and administered
either a sham
control (saline) or EPO (5000 U/kg), injected intraperitoneally (IP), 4 hours
before
presentation of a novel saccharin-vanilla liquid. Immediately after finishing
drinking the
sweet liquid, animals received either saline or an illness-producing dose of
lithium (20
mg/kg of a 0.15 M LiCI, delivered IP). Thereafter, three groups of animals
were followed.
The first group (control) did not receive lithium after drinking. The second
group received
both lithium and EPO. The third group (sham) received saline (without EPO) and
lithium.
Conditioned Taste Aversion was measured by measuring the reduction in
drinking upon subsequent exposure to the illness-producing solution, novel
saccharin-
vanilla liquid. After a 5-day recovery from the lithium or sham treatment,
water-deprived
animals were presented again with the same novel saccharin-vanilla liquid. The
results
plotted for groups 2 and 3, compared to 1 (control) are shown in Figure 2A.
Day 2
represents the baseline consumption of water after habituation to the test
cage. On Day 3,
animals received an intraperitoneal injection of either saline or EPO
(SOOOU/kg) 4 hrs
before presentation of the novel saccharin-vanilla fluid, followed by
treatment with lithium
or a sham saline (arrow). This treatment resulted in a small decrease in fluid
consumption
in all groups on Day 3, a previously documented adverse effect of the
injection and novelty
of the fluid. After recovery, the first test for the establishment of a CTA
showed no
decrease in consumption for controls. However, animals having received lithium
demonstrated a virtually complete aversion to the fluid, in spite of being
water deprived
(Day 4). Continued deprivation of water eventually produced an extinction of
the CTA
(Days S to 9), but was characterized by a markedly delayed recovery by the
animals which
had received EPO, as shown by the filled circles in Figure 2A.
The robustness of the CTA established herein is better appreciated by
considering
the degree of water deficit present on each test day, as the EPO-treated
animals tolerated a
water deficit approximately twice that of sham-injected subjects (Figure 2B).
In spite of the
markedly accentuated CTA demonstrated by the EPO group, the animals in this
group more
readily approached the drinking tube compared to the sham group, as shown in
Figure 2C.
The strength of the CTA was demonstrated by a repeat injection of lithium
alone (without
EPO) which produced an attenuated CTA which was greater in the EPO group
(Figure 2A
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Day 10). These data show that EPO pre-treatment is associated with a markedly
potentiated
CTA produced by lithium.
8. EXAMPLE 3: PERIPHERALLY ADMINISTERED EPO PROTECTS BRAIN
FROM AN EXCITOTOXIN
This Example demonstrates that EPO crosses the blood brain barrier and has a
neuroprotective effect in mice treated with the neurotoxin kainate. Many
compounds exist
in nature which exhibit toxicity specifically for neurons. These molecules
typically interact
with endogenous receptors for the amino acid transmitter glutamate,
subsequently causing
excessive stimulation and neuronal injury. One of these, kainate, a substance
widely used to
study neuronal injury due to excitotoxicity, is an analogue of glutamate.
Kainate is a potent
neurotoxin which specifically destroys neurons, particularly those located in
regions with a
high density of kainate receptors, such as the hippocampus, and induces
seizures, brain
injury, and death.
The following neurotoxicity studies were performed with mice using kainate.
This model is used to assess the protective benefit of treatments for
conditions such as
temporal lobe epilepsy. Parenteral injections in experimental animals such as
rats and mice
elicit partial (limbic) seizures in a dose-dependent manner, which then may
generalize and
cause death. The experiments presented in this section were performed to test
whether
peripherally-administered EPO crosses the blood brain barrier, and if so,
whether EPO has
an effect on neuronal energy balance, and specifically, it has neuroprotective
effects against
kainate.
To this end, female Balb/c mice (weighing on average 15-20 gm) were pretested
with 5000 U/kg of recombinant human erythropoietin (rhEPO; sold under the mark
PROCRIT, Ortho-Biotech, Inc.) or saline (sham) given intraperitoneally at
specific time
points before, at or after receiving kainate (Sigma Chemical), also IP, at
specific
concentrations (mass/kg-body weight). Subjects were then monitored and graded
for the
development of seizure activity at 20 minutes after receiving kainate. Each
trial was
terminated 60 minutes after the kainate dose. As shown in Figure 3A, EPO
pretreatment
dramatically reduces seizure severity and delays the onset of status
epilepticus in mice
treated with kainate. The comparison between EPO- and sham-treated animals
demonstrates a significantly lower death rate in animals receiving kainate
dosages in the 20-
30 mg/kg range, indicating neuroprotection afforded by pretreatment with EPO.
The
numbers in parentheses under each column indicate the number of animals
exposed to each
kainate dose.
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The dose-dependency of EPO in providing neuroprotection from kainate is
shown in Figure 3B. Mice were administered EPO (SOOOU/kg; IP daily for up to
five days).
The neuroprotective effect of each dose of EPO was assessed by determining
survival after
administration of kainate (20 mg/kg), which produces an approximate 50 %
mortality for
control animals (no EPO; see Figure 3A). Columns indicate improvement in
survival of
EPO-treated subjects, compared to sham-injected animals. As shown in Figure
3B,
neuroprotection increases with additional dosages of SOOOU/kg of EPO.
The neuroprotection provided by EPO is characterized by a delayed onset,
characteristic of the activation of a gene expression program. Figure 3C shows
the EPO-
related delay (in minutes) in death from seizures of a single dose of EPO
given at the time
of kainate administration (20 mg/kg) does not provide any immediate
protection, whereas
EPO given 24 hours before kainate improves the latency and severity of
seizures and time to
death. This effect lasts for up to 7 days.
9. EXAMPLE 4: PERIPHERALLY ADMINISTERED EPO PROTECTS BRAIN
FROM DAMAGE DUE TO ISCHEMIA
Previous in vivo studies using a global reperfusion model in the gerbil, have
indicated that stopping blood flow to the brain results in cell death in the
brain, and that
EPO injected directly into the cerebral ventricles protects the brain from
such cell death
(Sakanaka et al., 1998, Proc. Natl. Acad. Sci. U.S.A. 95:4635). The
experiments presented
in this Example, for the first time, show that EPO delivered peripherally
protects the neural
cell death in vivo in an animal model of ischemia.
The following experiment was performed using the middle cerebral artery
occlusion model, an art-accepted model of ischemic focal stroke. In the
protocol, male rats
(250 gm of body weight) were anesthetized with phenobarbital, and maintained
at 37°C.
The carotid arteries were visualized, and the ipsalateral carotid artery
permanently occluded.
The ipsalateral middle cerebral artery (MCA) was visualized and cauterized at
its origin.
The contralateral artery was occluded by clamping for 1 hour. Animals were
sacrificed 24
ho~.s later, and the brain removed and sectioned into 1 mm serial sections.
Viable tissue
was visualized by in situ triphenyltetrazolium reduction to visualize live
tissue from necrotic
regions. The ischemic core, and the surrounding penumbra, undergoes cell
death.
Using this MCA model, EPO was administered by parenteral injections at
various times before and immediately after the injury, and the volume of the
injury was
qu~tified by computer-assisted image analysis. The results of this analysis,
shown in
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Figure 4A, indicated the effect of treatment with EPO at the following times
after the stroke:
24 hours before the stroke, at the time of the stroke, and 3, 6, and 9 hours
after the stroke.
As shown in Figure 4A, EPO protects tissue from necrotic injury when
administered up to
6 hours post stroke.
Interestingly and in contrast, a 17-mer derived from EPO, which had been
previously reported to have neurotropic activity, promoting neurite growth in
vitro and
nerve cell myelination ex vivo (Campana et al., 1998, Int. J. Mol. Med. 1:235-
41; U.S.
Patent 5,700,909 issued Dec. 23, 1997), had no effect in protection against
injury in this
system (Figure 4B, "17-mer"). Thus, this model, as well as the other methods
for assaying
the effect of EPO on excitable tissue function provided by the present
invention, can be used
to identify EPO and EPO receptor activity modulators which can be used to
modulate
excitable tissue function, such as protection from injury, or enhancement of
learning and
cognition.
10. EXAMPLE 5: PERIPHERALLY ADMINISTERED EPO PROTECTS BRAIN
FROM BLUNT TRAUMA
In a model of mechanical trauma, the cortical impact model, pretreatment with
systemically-administered EPO protects mouse brain from blunt trauma. To
induce trauma a
pneumatically-driven piston (Clippard Valves), 3mm in diameter which can
precisely
deliver a blow to the skull was employed. Each mouse was anesthetized and
placed
securely in a sterotaxic device, to prevent the head from moving. A scalp
incision was
made in order to determine the location of the bregma, which is the reference
point with
which the piston was initially positioned. The piston was then adjusted by
moving it 2mm
caudal and 2mm ventral to bregma and the impact made by use of a precise pulse
of
nitrogen. This device allows for a precise selection of piston velocity (4
m/s) and impact
displacement (2 mm).
Mice were treated with EPO (SOOOU/kg) 24 hours before, at the time of injury,
3,
6, or 9 hours later and continued as daily dosages. Mice were sacrificed 10
days after the
procedure, and the brains subsequently examined and the volume of brain
necrosis
determined. In sham-treated mice, a large area of necrosis was observed
(Figure 5), and
with abundant infiltration of monocytes. In contrast, animals are protected
from such
damage, and few mononuclear cells were detected in the area of injury, when
animals are
pre-treated with EPO or given EPO up to 3 hours after injury.
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11. EXAMPLE 6: PERIPHERALLY ADMINISTERED EPO PROTECTS
MYOCARDIUM FROM ISCHEMIC INJURY
This Example demonstrates the effect of EPO in protection of heart tissue
against
hypoxic injury. To accomplish this, rats were pretested with EPO (SOOOU/kg) 24
hours
before the procedure performed as per Latini et al., (1999, J. Cardiovasc.
Pharmacol.
31:601-8). Subsequently, subjects were anesthetized, placed on assisted
ventilation and a
thoracotomy performed. The heart and its intrinsic circulation is identified
and a removable
suture placed around the most proximal portion of the left anterior descending
coronary
artery and then ligated. An additional dose of EPO (SOOOU/kg) was then given
and the
occlusion maintained for 30 minutes. At this time, the ligature was loosened
and the animal
is maintained under deep anesthesia for an additional 6 hours and subsequently
sacrificed.
Immediately after death, the heart was removed and a portion of the affected
region (AAR)
as well as unaffected region (septum) was removed and prepared for biochemical
analyses.
Two parameters were assessed, creatine kinase (CK) as a measure of the
survival of
1 S myocardium (the lower the CK, the less viable the tissue) and
myeloperoxidase, which is a
product of mononuclear cell infiltrate. The results are shown in Figure 6A and
Figure 6B.
As indicated in these figures, treatment with EPO results in maintained CK
activity,
consistent with an increase in tissue viability, and decreased MPO activity,
relative to the
control, in both the infarct area (AAR) and the perfused left ventricle (LV)
free wall,
indicating that there is significantly less infiltration by inflammatory
cells.
12. EXAMPLE 7: PERIPHERALLY ADMINISTERED EPO ATTENUATES
EXPERIMENTAL ALLERGIC ENCEPHALITIS
Experimental allergic (or autoimmune) encephalomyelitis (EAE) in rats, is an
art
accepted animal model for multiple sclerosis (MS). Various animal models with
EAE have
been developed applying immunologic, virologic, toxic and traumatic parameters
in order to
understand features of MS.
To test whether EPO protects against symptoms of EAE, the following
experiment was performed. Female Lewis rats, 6-8 weeks of age (Charles River,
Calco,
Italy) were immunized under light ether anesthesia by injecting into both hind
footpads 50
~.g of guinea pig myelin basic protein (MBP; Sigma, St. Louis, MO) in water,
emulsified in
equal volumes of complete Freund's adjuvant (CFA, Sigma) with 7 mg/ml of heat-
killed
Mycobacterium tuberculosis added to H37Ra (Difco, Detroit, MI) in a final
volume of 100
p1.
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After treatments, rats were assessed daily for signs of experimental
autoimmune
encephalomyelitis (EAE) and scored as follows: 0, no disease; 1, flaccid tail;
2, ataxia; 3,
complete hind limb paralysis with urinary incontinence. Body weights were also
monitored.
Rats were administered EPO (SOOOU/kg, IP, once daily) starting on day 3 post-
s immunization and continued until day 18. Control rats received vehicles
alone. As shown
in Figure 7, rats treated with EPO demonstrated an improvement in score (i.e.,
a lower
number) and in the duration of the disease. In addition, a marked delay in the
onset of
symptoms was noticed in rats treated with EPO.
13. EXAMPLE 8: MINIMUM EFFECTIVE DOSE AND PHARMACOKINETICS OF
EPO REQUIRED FOR PROTECTION OF EXCITABLE TISSUE
Optimum and effective dosages of EPO was assessed using the animal model of
focal ischemia stroke described above. As shown in Figure 8A, an EPO dosage of
less than
450 Units/kg body weight was not reliably effective in protecting excitable
tissue from
necrotic injury. As shown in Figure 8B, in animal studies, a dose of
approximately 5000
Units/kg-body weight delivered IP to four female mouse subjects resulted in a
circulating
level of EPO greater than 20,000 mUnits/ml of serum within 5 hours after its
administration, greater than 10,000 mUnits after 10 hours post administration,
but less than
5 Units/ml 24 hours after administration.
14. EXAMPLE 9: CNS DELIVERY MEDIATED BY ERYTHROPOIETIN
~e experiments presented hereinbelow indicate the successful transport of a
molecule conjugated to EPO across the blood-brain barner and its localization
inside
basement membrane. As shown in Figure 9A, brain sections were stained with
antibodies
for EPO receptor (EPO-R), which shows that brain capillaries express high
levels of EPO-
R. In order to study whether EPO is can be transported across the blood-brain
barrier, EPO
~'~'~ labeled with biotin as follows. The volume containing rhEPO was
concentrated using a
Centricon-10 filter (Millipore), and recovery measured by reading the
absorbance reading at
a wavelength of 280nm. Next, 0.2mg of long arm biotin (Vector Labs) was
dissolved in
100 ~.1 of DMSO, added to the concentrated rhEPO solution and vortexed
immediately.
This mixture was then incubated at room temperature for four hours, while
gently stirring
~d Protected from light. Unbound biotin was removed from the solution by using
a
Centricon-10 column. Biotinylated EPO was then administered to animals IP, and
S hours
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WO 00/61164 PCT/US00/10019
later the animals were sacrificed. Brain sections were labeled with avidin
coupled to
peroxidase, and diaminobenzidine added until sufficient reaction product
developed for
observation by-light microscopy. EPO was found along the same capillaries that
stained
positive for EPO-R (Figure 9B). At later time points, the biotin label
appeared localized
S within specific neurons (e.g., 17 hours, Figure 9C). In contrast, if cold
EPO was added in
1000 time excess to labeled EPO, all specific staining was eliminated. The
results
demonstrate the successful delivery of a systemically administered conjugated
EPO
compound across the blood brain barner.
Successful delivery of a systemically administered EPO-biotin conjugate
across the blood brain barrier into the brain demonstrates that other
therapeutic compounds
can be delivered across the blood-brain barrier in similar fashion, by
complexing EPO to the
compound of interest. As one example, brain-derived neurotrophic factor (BNF)
can be
covalently coupled to EPO by carbodiimide coupling using standard procedures.
After
purification, the conjugate can administered to animals via intraperitoneal
injection.
Positive effects of BNF on the central nervous system can be measured relative
to control
animals, to measure the successful transport of this molecule in association
with EPO, in
contrast to the lack of a central nervous system activity by unconjugated BNF.
The invention is not to be limited in scope by the specific embodiments
described which are intended as single illustrations of individual aspects of
the invention,
and functionally equivalent methods and components are within the scope of the
invention.
Indeed various modifications of the invention, in addition to those shown and
described
herein will become apparent to those skilled in the art from the foregoing
description and
accompanying drawings. Such modifications are intended to fall within the
scope of the
appended claims.
All references cited herein are incorporated by reference herein in their
entireties for all purposes.
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