U.S. patent application number 11/893294 was filed with the patent office on 2008-01-17 for modulation of excitable tissue function by peripherally administered erythropoietin.
Invention is credited to Michael Brines, Anthony Cerami, Carla Cerami.
Application Number | 20080014193 11/893294 |
Document ID | / |
Family ID | 39182207 |
Filed Date | 2008-01-17 |
United States Patent
Application |
20080014193 |
Kind Code |
A1 |
Brines; Michael ; et
al. |
January 17, 2008 |
Modulation of excitable tissue function by peripherally
administered erythropoietin
Abstract
Methods and compositions are provided for protecting or
enhancing excitable tissue function in mammals by systemic
administration of an erythropoietin receptor activity modulator,
such as erythropoietin, which signals via an EPO-activated receptor
to modulate the function of excitable tissue. Excitable tissues
include central neuronal tissues, such as the brain, peripheral
neuronal tissues, retina, and heart tissue. Protection of excitable
tissues provides treatment of hypoxia, seizure disorders,
neurodegenerative diseases, hypoglycemia, and neurotoxin poisoning.
Enhancement of function is useful in learning and memory. The
invention is also directed to compositions and methods for
facilitating the transport of molecules across endothelial cell
tight junction barriers, such as the blood-brain barrier, by
association of molecules with an erythropoietin receptor activity
modulator, such as an erythropoietin.
Inventors: |
Brines; Michael;
(Woodbridge, CT) ; Cerami; Anthony; (New York
City, NY) ; Cerami; Carla; (Sleepy Hollow,
NY) |
Correspondence
Address: |
Jones Day
222 East 41st Street
New York City
NY
10017-6702
US
|
Family ID: |
39182207 |
Appl. No.: |
11/893294 |
Filed: |
August 14, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09717057 |
Nov 21, 2000 |
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11893294 |
Aug 14, 2007 |
|
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09547220 |
Apr 11, 2000 |
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09717057 |
Nov 21, 2000 |
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60129131 |
Apr 13, 1999 |
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Current U.S.
Class: |
424/130.1 ;
514/7.7 |
Current CPC
Class: |
A61K 38/1816 20130101;
A61P 25/00 20180101 |
Class at
Publication: |
424/130.1 ;
514/008 |
International
Class: |
A61K 38/22 20060101
A61K038/22; A61P 25/00 20060101 A61P025/00 |
Claims
1. A method for enhancing the function of normal or abnormal
excitable tissue in a mammal comprising administering peripherally
to said mammal a peripherally effective excitable tissue enhancing
amount of an EPO, an EPO receptor activity modulator, an
EPO-activated receptor modulator, or combination thereof.
2. The method of claim 1 wherein said enhancing the function of
excitable tissue results in the enhancement of associative learning
or memory.
3. The method of claim 1 wherein said enhancing the function of
excitable tissue is used in the treatment of mood disorders,
anxiety disorders, depression, autism, attention deficit
hyperactivity disorder, Alzheimer's disease, aging or cognitive
dysfunction.
4. The method of claim 1 wherein said excitable tissue is central
nervous system tissue or peripheral nervous system tissue.
5. The method of claim 1 wherein said administration comprises
oral, topical, intraluminal or by inhalation or parenteral
administration.
6. The method of claim 5 wherein said parenteral administration is
intravenous, intraarterial, subcutaneous, intramuscular,
intraperitoneal, submucosal or intradermal.
7. The method of claim 1 wherein said administration is acute or
chronic.
8. The method of claim 1 wherein said EPO is nonerythropoietic.
9. The method of claim 1 wherein said EPO is administered at a dose
greater than the dose necessary to maximally stimulate
erythropoiesis.
10. The method of claim 1, wherein said 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.
11. The method of claim 10 wherein said EPO receptor-binding
molecule is an antibody to the erythropoietin receptor.
Description
[0001] This application is a continuation application under 35
U.S.C. .sctn. 120 of U.S. application Ser. No. 09/717,057, filed
Nov. 21, 2000, which is a divisional of U.S. application Ser. No.
09/547,220, filed Apr. 11, 2000, now abandoned, which claims
benefit under 35 U.S.C. .sctn. 119(e) of U.S. provisional patent
application No. 60/129,131, filed Apr. 13, 1999, the entire
contents of each of which is incorporated herein by reference in
its entirety.
1. FIELD OF THE INVENTION
[0002] 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
[0003] 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.
[0004] 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 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.
[0005] 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.
[0006] 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).
[0007] 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 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).
[0008] 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).
[0009] 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 cholinerpic 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).
[0010] 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).
[0011] Citation or discussion of a reference herein shall not be
construed as an admission that such is prior art to the present
invention.
3. BRIEF SUMMARY OF THE INVENTION
[0012] 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.
[0013] 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 carrier. 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
1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 hours after the administration of
EPO. In another embodiment, the invention provides a pharmaceutical
kit comprising an effective 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.
[0014] 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.
[0015] 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.
[0016] 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 prevent injury or tissue
damage during surgical procedures, such as, for example, tumor
resection or aneurysm repair.
[0017] 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.
[0018] 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.
[0019] 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.
[0020] 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
[0021] FIG. 1A-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 (R.sup.2=0.88) shows a slope (0.68) significantly
different from a slope of 1, markedly in favor of the EPO
group.
[0022] 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.
[0023] 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.
[0024] 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.
[0025] FIG. 5 depicts the protective effect of rhEPO against blunt
trauma delivered to the cerebral cortex.
[0026] 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.
[0027] 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.
[0028] 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 5000 U of rhEPO was
administered intraperitoneally to female Balb/c mice.
[0029] FIG. 9A-C A. Immunolocalization of EPO-R on and around
capillaries. B. Biotinylated EPO administered IP to mice is found
at 5 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
[0030] 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.
[0031] 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 described
herein. These molecules may be used in combination for the various
purposes herein described.
[0032] 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
[0033] 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-occurring, 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.
[0034] 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 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.
[0035] 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. Pat. No. 5,457,089 and in U.S. Pat. No. 4,835,260; EPO
isoforms with various numbers of sialic acid residues per molecule,
such as described in U.S. Pat. No. 5,856,292; polypeptides
described in U.S. Pat. No. 4,703,008; agonists described in U.S.
Pat. No. 5,767,078; peptides which bind to the EPO receptor as
described in U.S. Pat. Nos. 5,773,569 and 5,830,851; small-molecule
mimetics which activate the EPO receptor, as described in U.S. Pat.
No. 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, Calif.).
[0036] 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 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.
[0037] 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
[0038] 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 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.
[0039] 5.2.1 Methods for Enhancing Excitable Tissue Function
[0040] 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.
[0041] 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.
[0042] 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.
[0043] 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 Morris 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
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.
[0044] 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.
[0045] 5.2.2 Methods for Protecting Excitable Tissue from
Injury
[0046] 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.
[0047] 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, hypotension and brain or spinal cord
trauma. The present invention provides for acute administration for
the treatment of acute events.
[0048] 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.
[0049] 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.
[0050] 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.
[0051] In another embodiment, EPO is used to protect cardiac tissue
from injury sustained during ischemia, infarction, inflammation, or
trauma.
[0052] 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.
[0053] 5.2.3 Methods for Delivery of Compounds
[0054] 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 molecule capable of crossing tight junctions of such
excitable tissue, such as the blood-brain barrier. As such, EPO is
useful as a carrier for delivering other molecules across the
blood-brain and other similar barriers.
[0055] In one embodiment, EPO receptor binding molecules comprising
molecules conjugated to an EPO molecule, may be used to transport
those molecules across the blood brain barrier. Such molecules can
thereby piggyback on EPO for delivery across the BBB.
[0056] 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 barrier
in vivo. J. Pharmacol. Exp. Therap. 27: 66).
[0057] 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,
biotin/avidin 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.
[0058] 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-cancer agent to
be delivered across an endothelial cell barrier) 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 viva. 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, polyfunctional molecules reactive with other
functional groups, such as carboxylic acids or hydroxyl groups, are
contemplated in the present invention.
[0059] 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, Ill.
[0060] 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,
e.g., 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.
[0061] 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 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.
[0062] 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-methyl(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.
[0063] 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.
[0064] 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 liability
characteristics may also be tested using standard in vitro
procedures before in vivo testing.
[0065] 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
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, 1983, Biochem. Biophys. Acta 761:162; Caulfield et
al., 1984, Biochem. 81:7772-7776; Staros, 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 barrier.
[0066] 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 .beta.1
(TGF.beta.1), transforming growth factor .beta.2 (TGF.beta.2),
transforming growth factor .beta.3 (TGF.beta.3), interleukin 1,
interleukin 2, interleukin 3, and interleukin 6, AZT, antibodies
against tumor necrosis factor, and immunosuppressive agents such as
cyclosporin.
[0067] 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
[0068] 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 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.
[0069] 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.
[0070] 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.
[0071] 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.
[0072] 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.
[0073] 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.
[0074] In another embodiment, the methods of the invention may be
used to protect or treat injury resulting from radiation damage to
excitable tissue.
[0075] 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.
[0076] 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.
[0077] 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.
[0078] A further group of conditions treatable by the methods of
the present invention include mitochondrial 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
mitochondrial 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 mitochondrial diseases.
[0079] 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 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).
[0080] 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 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, dysthyrnic 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.
[0081] 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
[0082] 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, 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.
[0083] 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.
[0084] 5.4.1 Formulation and Effective Dose
[0085] 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.
[0086] 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 parameters such as bioavailability, metabolism,
half-life, etc., which will have been 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.
[0087] 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.
[0088] 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.
[0089] 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, 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.
[0090] 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, ferric 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 carrier so as to provide the form for proper
administration to the patient. The formulation should suit the mode
of administration.
[0091] 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.
[0092] 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. Pharmaceutical 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
paraffinic 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.
[0093] Pharmaceutical compositions adapted for nasal administration
may comprise solid carriers such as powders (preferably having a
particle size in the range of 20 to 500 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 carriers, 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
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.
[0094] 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.
[0095] 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 multi-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.
[0096] 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.
[0097] Suppositories generally contain active ingredient in the
range of 0.5% to 10% by weight; oral formulations preferably
contain 10% to 95% active ingredient.
[0098] 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 container(s) can be a notice in the
form 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.
[0099] 5.4.2 Methods of Administration
[0100] 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.
[0101] 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.
[0102] 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 Langer,
supra; Sefton, 1987, CRC Crit. Ref. Biomed. Eng. 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 Langer, 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. Pat. 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, Langer and Wise (eds.),
CRC Press: Boca Raton, Fla., 1974; Controlled Drug Bioavailability,
Drug Product Design and Performance, Smolen and Ball (eds.), Wiley:
New 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).
[0103] 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).
[0104] In another embodiment, EPO, as properly formulated, can be
administered by nasal, oral, rectal, vaginal, or sublingual
administration.
[0105] 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.
[0106] 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.
[0107] 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
[0108] In this Example, a spatial navigation experiment, known as
the Morris Water Maze test, demonstrates EPO-induced enhancement of
cognitive function 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 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.
[0109] 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).
[0110] 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.
[0111] Learning was measured by measuring the length of time each
mouse spent on the platform. Shown in FIG. 1A, 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
(R.sup.2=0.88) shows a slope (0.68) significantly different from a
slope of 1, markedly in favor of the EPO group (FIG. 1B).
7. EXAMPLE 2
Peripherally Administered EPO Strengthens a Learned Conditioned
Taste Aversion
[0112] 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 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.
[0113] 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 LiCl,
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.
[0114] 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 FIG. 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 (5000 U/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 5 to 9), but was characterized by a markedly delayed recovery
by the animals which had received EPO, as shown by the filled
circles in FIG. 2A.
[0115] 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 (FIG. 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 FIG. 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 (FIG. 2A Day 10). These data show that
EPO pre-treatment is associated with a markedly potentiated CTA
produced by lithium.
8. EXAMPLE 3
Peripherally Adminstered EPO Protects Brain from an Excitotoxin
[0116] 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.
[0117] 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.
[0118] 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 FIG. 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.
[0119] The dose-dependency of EPO in providing neuroprotection from
kainate is shown in FIG. 3B. Mice were administered EPO (5000 U/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 FIG. 3A). Columns
indicate improvement in survival of EPO-treated subjects, compared
to sham-injected animals. As shown in FIG. 3B, neuroprotection
increases with additional dosages of 5000 U/kg of EPO.
[0120] The neuroprotection provided by EPO is characterized by a
delayed onset, characteristic of the activation of a gene
expression program. FIG. 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
[0121] 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.
[0122] 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.degree.
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 hours 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.
[0123] 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 quantified by
computer-assisted image analysis. The results of this analysis,
shown in FIG. 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 FIG. 4A, EPO protects tissue from necrotic injury when
administered up to 6 hours post stroke.
[0124] 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. Pat.
No. 5,700,909 issued Dec. 23, 1997), had no effect in protection
against injury in this system (FIG. 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
[0125] 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), 3 mm 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 2 mm caudal and 2 mm 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).
[0126] Mice were treated with EPO (5000 U/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 (FIG. 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.
11. EXAMPLE 6
Peripherally Administered EPO Protects Myocardium from Ischemic
Injury
[0127] This Example demonstrates the effect of EPO in protection of
heart tissue against hypoxic injury. To accomplish this, rats were
pretested with EPO (5000 U/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 (5000
U/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 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 FIG. 6A and
FIG. 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
[0128] 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.
[0129] 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 .mu.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, Mich.) in a final volume of 100
.mu.l.
[0130] 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 (5000 U/kg, IP, once
daily) starting on day 3 post-immunization and continued until day
18. Control rats received vehicles alone. As shown in FIG. 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
[0131] Optimum and effective dosages of EPO was assessed using the
animal model of focal ischemia stroke described above. As shown in
FIG. 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 FIG. 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
[0132] The experiments presented hereinbelow indicate the
successful transport of a molecule conjugated to EPO across the
blood-brain barrier and its localization inside basement membrane.
As shown in FIG. 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 was 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 280 nm. Next, 0.2
mg of long arm biotin (Vector Labs) was dissolved in 100 .mu.l 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 and 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 5 hours 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 (FIG. 9B). At later
time points, the biotin label appeared localized within specific
neurons (e.g., 17 hours, FIG. 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 barrier.
[0133] 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.
[0134] 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.
[0135] All references cited herein are incorporated by reference
herein in their entireties for all purposes.
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