HK1077005A - Modulation of excitable tissue function by peripherally administered erythropoietin - Google Patents

Description

Modulation of stressed tissue function by peripherally administered erythropoietin

no marking

1. Field of the invention

The present invention relates to the use of peripherally administered erythropoietin and other erythropoietin receptor activity modulators or EPO-activated receptor modulators to positively affect the function of stressed tissues. This includes protecting stressed tissues such as neurons and cardiac tissue from neurotoxins, oxygen deficiency and other adverse stimuli, and enhancing the function of stressed tissues, such as for aiding learning and memory. The invention also relates to methods of transporting substances across endothelial cell barriers by association with erythropoietin molecules, erythropoietin receptor activity modulators or other EPO-activated receptor modulators.

2. Background of the invention

A wide variety of acute and chronic conditions and diseases result from stress tissue damage and dysfunction caused by external and internal stimuli. Such stimuli include lack of adequate oxygenation or glucose, neurotoxins, consequences of aging, infectious agents, and trauma. For example, the damaged stressed tissue may be the result of seizures and chronic seizures, 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, among others.

It is known that: a reduction in the energy supply available to the brain, such as glucose or oxygen, can lead to deep impairment of brain function, including cognitive impairment. Many (but not all) neurons in the central nervous system are highly vulnerable to metabolic limitations such as oxygen deficiency, hypoglycemia, stress, and/or prolonged intense excitation. In these cases, the electrochemical gradient of these cells often collapses, leading to irreversible neuronal damage and cell death. The current view is that this general mechanism is the common ultimate pathway for a number of common and debilitating neurological degenerative diseases including stroke, epilepsy and Alzheimer's disease.

Although the consequences of the effects of limited energy substrates on brain function are well known, little research has been conducted on the effects of enhancing energy release in otherwise normal brain. The current data strongly suggest: enhanced release of both glucose and oxygen significantly improves complex cognitive function in both animal models and normal human subjects (Kopf et al, 1994, behavioural and neurobiology 62: 237-. Furthermore, it has been demonstrated that more and more neuropeptides produced in the brain directly provide improvement in normal brain cognitive function. The physiological basis for these enhancements ultimately depends on the remodeling of neuronal interconnections by synaptic changes.

The cellular structure of brain tissue shows great plasticity and undergoes continuous remodeling. These processes, mediated by many nutritional molecules, not only occur after damage, but also play a prominent role in learning, memory and cognitive function. Although the prototypical neurotrophin is Nerve Growth Factor (NGF), increasing amounts of cytokines have been recognized to exert trophic functions in the brain (Hefti et al, 1997, annual assessment of pharmacology and toxicology 37: 239-67).

Recently, many independent researchers have recognized that neural tissue expresses high concentrations of EPO and its receptor (EPO-R; Digicaliobiogllu et al, 1998, Proc. Natl. Acad. Sci. USA 92: 3717-20; Juul et al, pediatric Res. 43: 40-9; Marti et al, 1997, International Kidney science 51: 416-8; Morishita et al, 1997, neuroscience 76: 105-16). Although it appears that EPO and its receptor protein are each products of a single gene, CNS variants are significantly smaller. The physiological implications of this observation are not clear, but the quality differences do appear to alter biological activity. For example, in studies on patients, researchers have concluded that EPO is not thought to be transported from the periphery to the brain (Marti et al, 1997 supra). However, to date, this possibility of EPO has not been evaluated using any direct study. Although brain EPO is approximately 15% less than kidney EPO (due to differences in sialylation), brain EPO is more active in erythrocyte colony stimulation at low ligand concentrations (Masuda et al, 1994, J. Biochem. 269: 19488-93). On the other hand, CNS receptors show a much lower affinity for deglycosylated EPO than peripheral receptors, which is 30% higher (Konishi et al, 1993, encyclopedia 609: 29-35; Masuda et al, 1993, J. Biochem.268: 11208-16).

In the brain, expression of EPO has been found in astrocytes, and increased expression and release of EPO can be induced by oxygen deficiency and other metabolic stressors (Marti et al, 1996, J. European neuroscience 8: 666-76; Masuda et al, 1993, J. biochem 268: 11208-16; Masuda et al, 1994, J. biochem. 269: 19488-93), or even by occupancy of other receptors such as the insulin-like growth factor family (Masuda et al, 1997, brain research 746: 63-70). Neurons are a target of this EPO secretion because 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 appears to be unmodulated during metabolic stress (Digicaligioglu et al, 1995, Proc. Natl. Acad. Sci. USA 92: 3717-20).

Recent studies have shown that EPO can be impressively protected against hypoxic neuronal damage in vitro as well as in vivo when injected directly into the ventricles of the brain (Morishita et al, 1997, neuroscience 76: 105-16; Sadamoto et al, 1998, Commission on biochemistry and biophysics 253: 26-32; Sakanaka et al, 1998, Proc. Natl. Acad. Sci. USA 95: 4635-40). Konishi et al (1993, brain Studies 609: 29-35) have demonstrated that EPO promotes the survival of cholinergic neurons in vivo when injected directly into the ventricles of adult rats. Centrally administered EPO in the ventricles also successfully prevented spatial learning disorders associated with ischemic injury in rats (Sadamoto et al, 1998, Comm. Biochem. BioPhysics 253: 26-32). One recent publication suggests: only the 17-amino acid portion of EPO is required for these neurotrophic effects in cultured neural cells (Campana et al, 1998, J. International molecular medicine 1: 235-41).

For many years, the only clear physiological role of Erythropoietin (EPO) has been its control of the production of red blood cells. Recently, there has been some evidence that EPO exerts other important physiological functions as a member of the cytokine superfamily, which are mediated through interaction with the erythropoietin receptor (EPO-R). These effects include mitogenesis, modulation of calcium influx into smooth muscle and nerve cells, and effects on intermediary metabolism. EPO is believed to provide a compensatory response that may improve the microenvironment of hypoxic cells. Although studies have established that intracranial injection of EPO protects neurons against hypoxic neuronal damage, intracranial administration is an impractical and unacceptable route of administration for medical applications, particularly for normal individuals. In addition, previous studies of anemic patients administered EPO have concluded that: peripherally administered EPO is not transported into the brain (Marti et al, 1997 supra).

References cited or discussed herein are not to be understood as prior art to the present invention.

3. Brief summary of the invention

The present invention relates to compositions and methods for modulating the function of stressed tissues in mammals, and methods and compositions for delivering drugs to stressed tissues. The present invention is based, in part, on applicants' discovery that: erythropoietin (EPO), administered systemically and at high doses, is specifically absorbed by the brain. In particular, the applicant has found that: EPO, delivered at high doses, can cross the blood brain barrier where it enhances cognitive function and protects nervous tissues from stressful states such as oxygen deficiency.

Erythropoietin and EPO (used interchangeably herein), modulators of EPO receptor activity, and EPO-activated receptor modulators refer to compounds that, when administered systemically (outside the blood-brain barrier), activate the EPO-activated receptor of electrically stressed tissues to enhance and/or protect against injury and death. Thus, EPO can refer to any form of erythropoietin that modulates stressed tissue, as well as EPO analogs, fragments, and mimetics thereof. In preferred embodiments, erythropoietin exhibits increased specificity for the brain EPO receptor for use in the methods of the invention. In another embodiment, the erythropoietin is non-erythropoietic. In yet another embodiment, erythropoietin is administered at a dose greater than that necessary to maximally stimulate erythropoiesis.

The present invention provides pharmaceutical compositions in dosage unit form suitable for modulating excitable tissue, enhancing cognitive function, or delivering compounds across endothelial tight junctions, each dosage unit containing an effective, non-toxic amount of EPO, an EPO receptor activity modulator, an EPO-activated receptor modulator, or a combination thereof, in the range of about 50,000 to 500,000 units, and a pharmaceutically acceptable carrier. In one embodiment, the effective non-toxic amount of EPO in the pharmaceutical composition comprises 50,000 to 500,000 units of EPO. In another embodiment, the effective non-toxic amount of EPO of the pharmaceutical formulation is a dose effective to achieve a circulating concentration of EPO of greater than 10,000mU/ml serum. In another embodiment, the circulating concentration of EPO is achieved at about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 hours after EPO administration. In another embodiment, the invention provides a pharmaceutical kit comprising an effective amount of EPO in one or more containers for modulating excitable tissue, enhancing cognitive function, or transporting a compound across an endothelial tight junction.

The present invention provides a method of modulating the function of stressed tissue in a mammal comprising peripherally administering to said mammal an effective amount of erythropoietin. The stressed tissue may be a normal tissue or an abnormal diseased tissue. In one embodiment, the stressed tissue is neuronal tissue of the central nervous system. In other embodiments, the stressed tissue is selected from neuronal tissue of the peripheral nervous system and cardiac tissue.

In one embodiment, a method is provided for enhancing the function of a mammalian excitable tissue, particularly normal and abnormal excitable tissues, by peripheral administration of an effective amount of EPO or an EPO receptor activity modulator. Enhancement of the function of stressed tissue leads to, for example, enhanced learning, associative learning, or memory. Non-limiting examples of conditions or diseases that may be treated with this aspect of the invention include mood disorders, anxiety disorders, depression, autism, attention deficit hyperactivity disorder, alzheimer's disease, aging and cognitive dysfunction.

In another embodiment, modulation of excitable tissue provides protection from lesions resulting from damage to excitable tissue, e.g., neurons on central nerve cells, peripheral nerve cells, or cardiac tissue. Injuries that may produce such lesions include, but are not limited to, hypoxia, seizures, neurodegenerative diseases, neurotoxin intoxication, multiple sclerosis, hypotension, cardiac arrest, radiation or hypoglycemia. In one embodiment, the disorder is the result of hypoxia, and may be prenatal or postpartum hypoxia, asphyxia, congestion, near drowning, post-operative 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 anesthesia. In the case where the pathology is a seizure, non-limiting examples thereof may be epilepsy, convulsions or chronic seizures. In the case where the disorder 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, alcoholism, retinal ischemia, aging, glaucoma, or neuronal loss. In another embodiment, administration of EPO can be used to prevent injury or tissue damage during a surgical procedure such as, for example, tumor resection or aneurysm repair.

In yet another embodiment, a method of promoting transcytosis of a molecule across a mammalian endothelial cell barrier by administering a composition of molecules that bind erythropoietin is provided. The binding between the molecule to be transported and the EPO can be, for example, a labile covalent bond, a stable covalent bond or a non-covalent binding to the binding site of the molecule. In one embodiment, the endothelial cell barrier may be a blood brain barrier, a blood eye barrier, a blood testis barrier, a blood ovary barrier, or a blood placenta barrier.

The invention also provides compositions for transporting molecules across endothelial cell barriers via transcytosis comprising said molecules 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 the composition is a hormone, neurotrophic factor, antimicrobial agent, radiopharmaceutical, antisense compound, antibody, immunosuppressive agent, toxin, or anticancer agent. Suitable molecules for delivery using the methods of the invention include, but are not limited to, hormones, such as growth hormone, antibiotics, anti-cancer agents, and toxins.

These and other aspects of the invention will be better appreciated with reference to the following drawings and detailed description.

4. Brief description of the drawings

Morris water maze test. A. Results of Morris aqueous maze test performed with mice receiving peripheral EPO or saline (beam) daily. B. Subjects receiving EPO performed significantly better than subjects treated with SHAM. Regression line (R)²0.88) showed a slope (0.68) significantly different from slope 1, clearly favoring the EPO group.

FIGS. 2A-C. conditional taste diversion assay. A. The effect of peripheral sham and EPO treatment on water consumption in mice undergoing the conditioned taste shift test was compared. Water consumption is expressed as the percentage of volume consumed by control mice that were not rendered diseased with lithium chloride. B and C demonstrate that EPO-enhanced learning is healthy because EPO subjects can tolerate much greater cravings than controls in avoiding water containing disease-associated cues to continue to spend more time searching for water.

Figures 3A-B.A, experimental results demonstrating that peripherally administered EPO pretreatment reduced the severity of seizures and protected mice from kainic acid neurotoxin induced convulsions and death. The numbers in parentheses below each bar indicate the number of animals receiving each kainate dose. B shows that the protective effect of peripherally administered EPO increases with daily administration of EPO. C indicates that the onset of EPO action is delayed, characterized by induction of the gene expression program.

Fig. 4A-B depict the protective effect of rhEPO against ischemic brain injury (focal stroke). A. Systemic administration of EPO at various times after induction of cerebral ischemia reduced the size of the infarct. B. The role of two forms of EPO in protecting the brain from injury was compared in this model: recombinant human EPO (rhepo) and 17 amino acid EPO derivatives (17-mer) demonstrated that some EPO analogs are not effective for neuroprotection.

Fig. 5 depicts the protective effect of rhEPO against blunt trauma delivered into the cerebral cortex.

FIGS. 6A-B depict the protective effect of EPO on ischemic heart injury. A. Creatine Kinase (CK) activity, an indicator of myocardial cell damage. B. Myeloperoxidase (MPO) activity, a measure of inflammation.

Figure 7 shows that treatment of mice with EPO delayed and reduced neurological symptoms resulting from experimental allergic encephalitis, a model of multiple sclerosis.

FIGS. 8A-B.A, the minimal effective dose of EPO that provides neuroprotection in a rat focal stroke model. B. EPO serum concentrations at various time points following intraperitoneal administration of 5000U rhEPO to female Balb/c mice.

FIGS. 9A-C.A, EPO-R immunolocalization on and around capillaries. B. Biotinylated EPO administered IP to mice was found in peripheral capillaries in close proximity to the brain at 5 hours. C. After 17 hours, biotin labeling could be found in specific neurons.

5. Detailed description of the invention

The present invention provides compositions and methods for modulating stressed tissue function, such as, for example, enhancing cognitive function and protecting stressed cells from toxic stimuli, using Erythropoietin (EPO). In particular, the invention provides compositions comprising EPO and methods of using them for prophylactic and therapeutic treatments, including drug release. Stressed tissues as used herein include, but are not limited to, neuronal tissue of the central and peripheral nervous systems and cardiac tissue.

The invention described herein provides methods for modulating the function of stressed tissues by peripheral administration of EPO, or EPO receptor activating molecules or molecules exhibiting EPO-activated receptor activity, as well as using any molecule that mimics EPO activity by acting through an otherwise non-canonical EPO receptor. Without being bound by any particular mechanism of action, such molecules can signal via the EPO receptor, for example, triggering a signal transduction cascade that ultimately activates a gene expression program leading to protection or enhancement of the function of the stimulated tissue. Molecules that interact with the EPO receptor and modulate the activity of the receptor, referred to herein as EPO or EPO receptor activity modulators, may be used within the scope of the invention to protect or enhance stressed tissue function. These molecules may be, for example, naturally occurring, synthetic, or recombinant forms of EPO molecules, as described above, or other molecules that are not necessarily similar in any way to EPO except for modulation of EPO receptor activity, as described herein. These molecules can be used in combination for various purposes as described herein.

The compositions and methods described herein may be used to treat and/or protect normal or abnormal tissue, such as neurons of the central nervous system, neurons of the peripheral nervous system, or cardiac tissue. In particular, in section 5.1 below, EPO compositions useful in the practice of the present invention are described. In section 5.2.1, methods of using such EPO compositions to enhance functions of stressed tissues such as learning, memory, and other cognitive functions are described, while in section 5.2.2, methods of protecting stressed tissues from damage and injury are described. The surprising ability of EPO to pass tight junctions of capillary endothelial cells is also described in section 5.2.3 below, and this finding provided a means of transporting compounds across such barriers. Finally, in section 5.3, diseases that can be targeted using the methods of the invention are described, while in section 5.4, methods of administration and effective dosages of such EPO compositions are described.

5.1 compositions comprising erythropoietin

EPO compositions suitable for use in the present invention include any erythropoietin compound that, when administered peripherally, activates the EPO-activated receptor to modulate, i.e., enhance the function of, protect against damage or injury to, or deliver the compound to, stressed tissues. Erythropoietin is a glycoprotein hormone whose molecular weight in humans is 34 to 38 kD. The mature protein contains 166 amino acids, with the glycosyl residues making up about 40% of the weight of the molecule. EPO forms useful in the practice of the invention include the following naturally occurring, synthetic and recombinant forms of the molecule: erythropoietin, erythropoietin analogs, erythropoietin mimetics, erythropoietin fragments, hybrid erythropoietin molecules, erythropoietin receptor binding molecules, erythropoietin agonists, renal erythropoietin, brain erythropoietin, oligomers and polymers thereof, muteins thereof, and the like. The terms "erythropoietin" and "EPO" may be used interchangeably or in combination.

Provided herein are synthetic and recombinant molecules, such as brain EPO and kidney EPO, recombinant mammalian forms of EPO, as well as naturally occurring, tumor-derived, and recombinant isoforms thereof, such as recombinantly expressed molecules and those produced by homologous recombination. In addition, the invention includes molecules comprising peptides that bind to the EPO receptor, as well as recombinant constructs or other molecules having some or all of the structural and/or biological properties of EPO, including fragments and multimers of EPO or fragments thereof. EPO herein comprises molecules with altered EPO receptor binding activity, preferably with increased receptor affinity, particularly when used to enhance transport across endothelial cell barriers. Also included herein are muteins comprising molecules having an additional or reduced number of glycosylation sites. As noted above, the terms "erythropoietin," "EPO," and "mimetic" and other terms are used interchangeably herein to refer to the stressed tissue protective and enhancing molecules associated with EPO and molecules that are capable of crossing the endothelial tight junctions and thus serving as the release means for other molecules. In addition, molecules produced using transgenic animals are also included herein. It should be noted that: EPO molecules included herein do not necessarily resemble EPO structurally or in any other way, as described above, except for the ability to interact with the EPO receptor or modulate EPO receptor activity or activate the EPO-activated signal cascade, etc.

By way of non-limiting example, forms of EPO useful in the practice of the present invention include EPO muteins, such as those described in U.S. patent 5,457,089 and U.S. patent 4,835,260, with altered amino acids at the carboxy terminus; EPO isoforms with different numbers of sialic acid residues per molecule, such as described in us patent 5,856,292; polypeptides described in U.S. Pat. No.4,703,008; agonists described in us patent 5,767,078; peptides that bind to the EPO receptor as described in U.S. Pat. nos. 5,773,569 and 5,830,851; small molecule mimetics that activate the EPO receptor as described in U.S. patent 5,835,382; and EPO analogs as described in WO 9505465, WO 9718318 and WO 9818926. All of the above references are incorporated herein as if such publications were referring to various alternative forms or methods for preparing such forms of erythropoietin of the present invention.

EPO is commercially available (commercially available under the trade mark proclt from Ortho Biotech, and EPOGEN from Amgen, inc., Thousand Oaks, CA).

In another embodiment of the invention, the EPO molecules contained herein include hybrid EPO molecules that can be prepared to contain EPO receptor modulating activity as well as another activity, such as the activity of growth hormone. Such hybrid molecules with multiple domains thus have the ability to interact with the EPO receptor-also with the activity of another molecule such as a hormone. Methods for preparing such molecules having two domains are known to those skilled in the art. One property of such molecules is the transport across the endothelial cell barrier provided by the EPO receptor activity modulating domain, and the activity of another molecule at the target site, as will be described in more detail in section 5.2.3 below.

Any of the above compounds can be tested using the assays described herein to identify EPO compounds that are capable of modulating, i.e., enhancing the function of, protecting against damage or injury to, or delivering the compound to, a stressed tissue. For example, the ability of an EPO compound to enhance functions of stressed tissues, such as learning, memory, and other cognitive functions, can be tested 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 conditional taste diversion test, an example of which is described in detail in section 7. In addition, the above-described EPO compounds can be tested using the assays described in section 5.2.2 to identify EPO compounds that can protect stressed tissues from damage and injury. The examples described in sections 8, 9, 10, 11 and 12 provide specific examples of these assays. Assays such as those described in sections 5.2.3 and 9 below can also be used to identify the ability of an EPO compound to transport compounds across endothelial tight junctions, such as the blood-brain barrier. Thus, EPO compositions suitable for use in the present invention include any and all compounds capable of modulating, i.e., enhancing the function of, protecting against damage or injury to, or delivering the compound to, stressed tissue via EPO-activated receptor signaling when administered peripherally.

5.2 methods of prophylactic and therapeutic use of the invention

In various embodiments of the invention, the EPO compositions may be used to protect stressed tissues from injury or hypoxic stress, to enhance the function of stressed tissues, or to transport compounds across endothelial tight junctions of stressed tissues. As noted above, the present invention is based, in part, on the discovery that: EPO molecules can be transported from the luminal surface to the surface of the basement membrane of endothelial cells of capillaries, including, for example, the brain, retina, and testis, where endothelial cells are tightly associated. Without wishing to be bound by any particular theory, after transcytosis of EPO, EPO can interact with EPO receptors on stressed tissues, such as neurons of the central nervous system, the peripheral nervous system, or cardiac tissues, and receptor binding can trigger a signaling cascade that leads to activation of the extent of gene expression in stressed tissues, resulting in protection of cells from damage such as neurotoxins, hypoxia, and the like. Thus, methods of protecting stressed tissue from damage or hypoxic stress, enhancing the function of stressed tissue, and transporting compounds across tight junctions of stressed tissue are described in detail below.

5.2.1 methods of enhancing stress tissue function

In one aspect, the invention relates to methods of enhancing stressed tissue function by administering an EPO molecule that activates a gene expression program that enhances stressed tissue function. The enhancement of the function of the stress organization improves the learning, associative learning and memory ability. Many diseases and conditions can be treated using this method, and the method can be used to enhance cognitive function in the absence of any condition or disease. These applications of the present invention are described in more detail below, including enhancing learning and training in human and non-human mammals.

Conditions and diseases that may be treated by the methods of this aspect of the invention include any condition or disease that would benefit from an increase in neuronal function. Examples of such disorders include central nervous system disorders 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 that may be enhanced by the methods of the present invention are set forth in section 5.3.

In one embodiment, for example, an EPO molecule may be administered to a subject or patient suffering from a disorder that results in a loss of cognitive function, such as Alzheimer's disease, for example.

The ability of EPO to enhance cognitive function can be tested in experimental animals using any of the methods described herein or any other art-recognized model of learning or cognitive function. As described in the examples described in sections 6 and 7, peripheral administration of erythropoietin has been demonstrated to enhance learning and cognitive function in normal experimental animals in several well-defined learning models. Examples of such learning models are the Morris water maze test, which is given as an example in section 6, and the conditional taste diversion (CTA) test, which is given as an example in section 7. In one embodiment, for example, the animal is tested for cognitive function following EPO administration using a very sensitive, well known standard assay, the conditional taste transfer (CTA) assay. CTA was used to test the ability of animals to learn to associate disease with a novel stimulus, such as taste, so that the animal will avoid the novel taste when later receiving the novel stimulus again. CTA affects the brain at various cortical and sub-cortical levels. The association of incremental and decremental messages that produce aversive behavior can be attenuated or enhanced by effecting changes to any of the interconnected elements. In the form of associative learning, the strength of CTA is determined by a variety of variables, including novelty of oral stimuli (e.g., non-novel stimuli cannot be aversively adjusted), degree of "disease" produced (toxicity), number of repetitions (training), opposing motivation (such as craving), etc., to name a few. Although there are many chemical and physical agents that can produce CTA in a dose-dependent manner, lithium chloride can reliably produce discomfort and anorexia. Like a naturally occurring disease, lithium produces CTA by stimulating the above pathways, including cytokine release.

Enhancement of stress cell functions, such as cognitive functions, provides many benefits to individuals in educational and work environments and can improve the ability to train and educate non-human mammals.

5.2.2 methods for protecting stressed tissues from injury

In another embodiment, the invention relates to a method of protecting a mammal from a lesion resulting from damage to stressed tissue. The protective effect is achieved by administering to the mammal an amount of erythropoietin effective to protect stressed tissue from damage by a peripheral route of administration. As shown in detail in the examples of section 8 below, EPO administered prior to the kainic acid toxin is a significant neuroprotective agent to mice, increasing seizure threshold and preventing death. EPO has a strong and durable neuroprotective effect. It is worth noting that: the positive effect seen in this context occurs in a time which is too short for an increase in the hematocrit to be achieved by the administration of EPO, which is a result of the erythropoietic activity of EPO. In addition, as has been seen above, embodiments of the present invention include EPO that lacks the ability to increase hematocrit.

In one embodiment, the present invention may be advantageously used for acute and chronic prevention and treatment of neurological disorders as described above, and for enhancing cognitive function of normal or diseased brain. As has been seen above, neuronal damage and death in the central nervous system is severe and often leads to death, which is high in both 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 acute dosing regimens for treating acute events.

In one embodiment, for example, the methods of the invention can be used to protect a mammal from damage caused by radiation damage to the brain.

In another embodiment, serious conditions that may be treated or prevented according to the present invention are the prevention and treatment of the uterus in prenatal hypoxic conditions, treatment after birth to protect the brain from hypoxic damage that persists during production, and the prevention and treatment of asphyxiation, drowning and other conditions in which the central nervous system is at risk of neurotoxic damage due to hypoxia or other neurotoxic stimuli. It is well known that individuals suffering from oxygen deficiency during childbirth, or as a result of a non-fatal hypoxic event or event, may develop a lifelong neurological deficit. Hypoxia and/or interruption of cerebral blood flow that may occur after trauma or during surgical procedures also carries the risk of causing lifelong neurological deficits.

Post-operative cognitive dysfunction, including shortages following use of a cardio-pulmonary machine, may also be treated with the methods provided herein. In addition, the method of the present invention may be used to treat hypoxia caused by carbon monoxide poisoning or inhalation of smoke.

In another embodiment, EPO is used to protect cardiac tissue from continued damage during ischemia, infarction, inflammation, or trauma.

These are non-limiting examples of stressed tissue damage that may be treated according to the present invention. Timely and early treatment of these patients can be performed by mobile medical emergency healthcare professionals to begin treatment as soon as possible once the likelihood of neurological damage is determined. The risk of neurologic damage induced by labor can be reduced by prophylactic treatment of the fetus prior to or during labor. These and other utilities and conditions will be recognized by the skilled artisan.

5.2.3 methods of delivery of Compounds

The invention also relates to methods of facilitating transport of molecules across endothelial cell barriers in mammals by administering compositions comprising specific molecules in combination with erythropoietin. It has been seen above that the inventors herein have discovered that heretofore unexpected and surprising activity of peripherally administered EPO on neuronal tissue in excitable tissues, such as the central nervous system, the peripheral nervous system, or cardiac tissue, identifies EPO as a molecule capable of crossing the tight junctions of such excitable tissues, such as the blood-brain barrier. Thus, EPO can be used as a carrier for transporting other molecules across the blood-brain barrier and other similar barriers.

In one embodiment, EPO receptor binding molecules comprising molecules conjugated to EPO molecules can be used to transport those molecules across the blood-brain barrier. Such molecules may thus be carried on EPO for release across the BBB. In another embodiment, the antibody or other binding partner of the molecule may be associated with EPO, or with a modulator of EPO receptor activity, whereby the molecule to be transported is bound to the binding partner by non-covalent binding, which in turn is further bound to the transportable EPO molecule. In another embodiment, EPO receptor binding molecules comprising antibodies to EPO receptors are useful in the methods described herein. Such antibodies provide a transport carrier that can carry other molecules, most in the same way: antibodies to transferrin receptor have been used to obtain the ability to cross the blood-brain barrier (Pardridge et al, 1991, "Selective transport of anti-transferrin receptor antibodies across the blood-brain barrier in vivo" [ J. Pharmacology & Experimental therapeutics 27: 66).

The skilled artisan will recognize various ways to associate the molecule with EPO and the other agents described above, covalently, non-covalently, and otherwise; in addition, the assessment of the efficacy of the composition can be readily determined using experimental systems. Binding of the molecules to EPO and analogs can be achieved in a number of ways, including labile covalent binding, cross-linking, and the like. In one embodiment, for example, the association between the molecule to be transported across the barrier and erythropoietin may be a labile covalent bond, in which case the molecule is released from the conjugate with EPO after crossing the barrier. In one embodiment, a biotin/avidin interaction may be used. In another embodiment, as mentioned above, the hybrid molecule may be prepared by recombinant or synthetic means, e.g., it comprises a domain of the molecule having the desired pharmacological activity and a domain capable of causing modulation of EPO receptor activity.

The molecule may be conjugated to the EPO or EPO receptor activity modulator via a multifunctional molecule, i.e. a multifunctional cross-linker. The term "multifunctional molecule" as used herein includes molecules having one functional group capable of more than one successive reaction, such as formaldehyde, as well as molecules having more than one reactive group. The term "active group" as used herein refers to a functional group on a cross-linking agent that reacts with a functional group on a molecule (e.g., a peptide, protein, carbohydrate, nucleic acid, specific hormone, antibiotic, or anti-cancer agent to be transported across the endothelial cell barrier) so as to form a covalent bond between the cross-linking agent and the molecule. The term "functional group" retains its standard meaning in organic chemistry. The multifunctional molecules that can be used are preferably biocompatible linkers, i.e., they are non-carcinogenic, non-toxic, and substantially non-immunogenic in vivo. Multifunctional crosslinkers such as those known in the art and those described herein can be readily tested in animal models to determine their biocompatibility. The multifunctional molecule is preferably a bifunctional molecule. The term "bifunctional molecule" as used herein refers to a molecule having two active groups. The bifunctional molecule may be a heterobifunctional molecule or a homobifunctional molecule. Heterobifunctional crosslinkers allow vector conjugation. It is particularly preferred for the multifunctional molecule to be sufficiently water soluble to allow the crosslinking reaction to occur in aqueous solutions, such as buffered aqueous solutions at pH6-8, and to maintain the resulting conjugate water soluble for more efficient biodistribution. In general, the multifunctional molecule is covalently bonded to an amino or thiol functional group. However, reactive multifunctional molecules with other functional groups, such as carboxylic acid or hydroxyl groups, are also included in the present invention.

The homobifunctional molecule has at least two reactive functional groups, which are identical. Reactive functional groups on homobifunctional molecules include, for example, aldehyde groups and reactive ester groups. Homobifunctional molecules having aldehyde groups include, for example, glutaraldehyde and octanediol. The use of glutaraldehyde as a crosslinking agent is disclosed by Poznansky et al, science 223, 1304-charge 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 disulfonic and disulfonic ester salts, such as their sodium and potassium salts. These homobifunctional reagents are commercially available from Pierce, Rockford, Illinois.

Heterobifunctional molecules have at least two different reactive groups. The active groups react with different functional groups present, for example, on EPO and on the molecule. The two different functional groups that react with the reactive groups on the heterobifunctional crosslinkers are typically amino groups, such as the epsilon amino groups of lysine; sulfhydryl groups, such as cysteine; carboxylic acid esters on carboxylic acids, such as aspartic acid; or a hydroxyl group, such as the hydroxyl group on serine.

When the active group of the heterobifunctional molecule forms a covalent bond with an amino group, the covalent bond will typically be an amido or imino bond. The reactive group forming a covalent bond with an amino group may be, for example, an activated carboxylate group, a halocarbonyl group, or an ester group. The preferred halocarbonyl group is chlorocarbonyl. The ester group is preferably an active ester group, such as, for example, an N-hydroxy-succinimide ester group.

The other functional group is typically a thiol group, a group capable of being converted to a thiol group, or a group that forms a covalent bond with a thiol group. The covalent bond will typically be a thioether bond or a disulfide bond. The reactive group forming a covalent bond with a thiol group may be, for example, a double bond that reacts with a thiol group or an activated disulfide bond. The reactive group containing a double bond capable of reacting with a thiol group is a maleimide group, although others are possible, such as acrylonitrile. The reactive disulfide group may be, for example, a 2-pyridyldisulfide group or a 5, 5' -disulfide-bis- (2-nitrobenzoic acid) group. Some examples of heterobifunctional reagents containing an active disulfide bond include N-succinimidyl 3- (2-pyridyl-dithio) propionate (Carlsson et al, 1978, J. Biochem. 173: 723-737), sodium 5-4-succinimidyloxycarbonyl-. alpha. -methylbenzyl thiosulfate, and 4-succinimidyloxycarbonyl-. alpha. -methyl (2-pyridyl-dithio) toluene. N-succinimidyl 3- (2-pyridyldithio) propionate is preferred. Some examples of heterobifunctional reagents containing a reactive group with a double bond reactive with a thiol group include succinimidyl 4- (N-maleimidomethyl) cyclohexane-1-carboxylate and inter-succinimidyl maleimidobenzoate.

Other heterobifunctional molecules include succinimidyl 3- (maleimido) propionate, sulfosuccinimidyl 4- (p-maleimido-phenyl) butyrate, sulfosuccinimidyl 4- (N-maleimidomethyl-cyclohexane) -1-carboxylate, maleimidobenzoyl-N-hydroxy-succinimidyl ester. The sulfonic acid sodium salt of succinimidyl-m-maleimidobenzoate is preferred. Many of the heterobifunctional reagents described above and their sulfonates are commercially available from Pierce.

The skilled person can readily determine the need for a conjugation that will be reversible or labile as described above. The conjugates can be tested in vitro for EPO receptor activity modulating activity, as well as for the desired pharmacological activity. If the conjugate retains both properties, it can then be tested for its suitability in vivo. If the conjugate molecule requires activity separate from EPO, it will preferably be unstably bound or reversibly bound to EPO. The instability profile can also be tested using standard in vitro procedures prior to performing in vivo tests.

Additional information on how to make and use these and other multifunctional reagents can be obtained from the following list of publications or other approaches available in the art: carlsson et al, 1978, J. Biochem 173: 723-737; cumber et al, 1985, methodology in enzymology 112: 207-224; jue et al, 1978, biochemistry 17: 5399-5405; sun et al, 1974, biochemistry 13: 2334-; blattler et al, 1985, biochemistry 24: 1517-; liu et al, 1979, "biochemistry" 18: 690-697; youle and Neville, 1980, proceedings of the national academy of sciences of the united states 77: 5483-5486; lenner et al, 1981, proceedings of the national academy of sciences USA 78: 3403-; jung and Moroi, 1983, journal of biochemistry and biophysics 761: 162; caulfield et al, 1984, biochemistry 81: 7772-7776; staros, j.v., 1982, biochemistry 21: 3950-; yoshitake et al, 1979, Eur. J. Biochem.101: 395-; yoshitake et al, 1982, journal of biochemistry 92: 1413-; pilch and Czech, 1979, J. Biochem.254: 3375-3381; novick et al, 1987, journal of biochemistry 262: 8483-; lomant and Fairbanks, 1976, journal of molecular biology 104: 243- > 261; hamada and Tsuruo, 1987, analytical biochemistry 160: 483-488; and Hashida, 1984, journal of applied biochemistry 6: 56-63. In addition, Means and Feeney, 1990, bioconjugate chemistry 1: the method of crosslinking is reviewed in 2-12. Barriers that are traversed using the above-described methods and compositions of the invention include, but are not limited to, the blood-brain barrier, the blood-eye barrier, the blood-testis barrier, the blood-ovary barrier, and the blood-placenta barrier.

Candidate molecules for transport across endothelial cell barriers include, for example: hormones such as growth hormone, neurotrophic factors, antibiotics or antifungal agents such as those commonly rejected by the brain and other barrier organs, peptide radiopharmaceuticals, antisense drugs, antibodies to biologically active agents, drugs, and anticancer 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 β 1), transforming growth factor beta 2(TGF β 2), transforming growth factor beta 3(TGF β 3), interleukin 1, interleukin 2, interleukin 3 and interleukin 6, AZT, antibodies to tumor necrosis factor, and immunosuppressive agents such as cyclosporine.

In another embodiment, recombinant chimeric toxin molecules comprising EPO can be used for therapeutic release of toxins to treat viral or proliferative diseases, such as cancer. Compounds that may be fused to EPO to construct a chimeric toxin suitable for this embodiment include, but are not limited to, toxic substances such as Pseudomonas exotoxin, diphtheria toxin, and ricin, among others.

5.3 target conditions

As described above, the EPO compositions provided herein and methods of their use can be used to treat and prevent diseases caused by hypoxic conditions that adversely affect stressed tissues, such as in central nervous system tissues, peripheral nervous system tissues, or cardiac tissues, such as, for example, the brain, heart, or retina. Thus, the present invention may be used to treat or prevent damage to stressed tissues caused by hypoxic conditions in a wide variety of conditions and events. Non-limiting examples of such conditions and events are provided below.

In examples of protection of neuronal tissue pathologies that may be treated in accordance with the present invention, such pathologies include those resulting from a reduction in oxygenation of neuronal tissue. Any condition that reduces the availability of oxygen to neuronal tissue, leading to stress, damage and ultimately neuronal cell death can be treated with the methods of the invention. These conditions, collectively referred to as hypoxia and/or ischemia, result from or include, but are not limited to, stroke, vascular occlusion, prenatal or postpartum hypoxia, asphyxia, congestion, near drowning, carbon monoxide poisoning, inhalation of smoke, trauma, including surgery and radiation therapy, pulseless, 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 anesthesia.

In one embodiment, for example, EPO can be administered to prevent damage or tissue damage resulting from the risk of damage or tissue damage during a surgical procedure such as, for example, tumor resection or aneurysm repair.

Other pathologies caused or resulting from hypoglycemia that can be treated by the methods described herein include insulin overdose, also known as iatrogenic hyperinsulinemia, insulinomas, growth hormone deficiency, adrenocortical insufficiency, drug overdose, and certain tumors.

Other pathologies resulting from stress neuronal tissue damage include seizures, such as epilepsy, convulsions, or chronic seizures. Other conditions and diseases that may be treated include such diseases: such as stroke, multiple sclerosis, hypotension, cardiac arrest, alzheimer's disease, parkinson's disease, cerebral palsy, brain or spinal trauma, AIDS dementia, age-related loss of cognitive function, memory loss, amyotrophic lateral sclerosis, seizures, alcoholism, retinal ischemia, glaucomatous optic nerve damage, and neuronal loss.

The methods of the invention may be used to treat diseases of and damage to retinal tissue. Such conditions include, but are not limited to, macular degeneration, retinal detachment, retinitis pigmentosa, arteriosclerotic retinopathy, hypertensive retinopathy, retinal artery occlusion, retinal vein occlusion, hypotension, and diabetic retinopathy.

In another embodiment, the methods of the invention can be used to protect or treat damage resulting from radiation damage to stressed tissue.

Other utility of the methods of the invention is in the treatment of neurotoxin intoxication such as domoic acid oyster intoxication, neurolathyrism and guam disease, amyotrophic lateral sclerosis, and parkinson's disease.

As mentioned hereinabove, the present invention also relates to a method of enhancing the function of stressed tissues in a mammal by peripheral administration of an erythropoietin. Many diseases and conditions can be treated using this method, and the method can be used to enhance cognitive function in the absence of any condition or disease. These applications of the present invention are described in more detail below, including enhancing learning and training in human and non-human mammals.

Conditions and diseases involving the central nervous system that may be treated by the methods of this aspect of the invention include, but are not limited to, mood disorders, anxiety disorders, depression, autism, attention deficit hyperactivity disorder, and cognitive dysfunction. These conditions may benefit from an increase in neuronal function.

Other patients that may be treated in accordance with the teachings of the present invention include sleep disruptions, such as sleep apnea and performance-related patients; subarachnoid and aneurysm bleeding, hypotensive shock, concussive injury, septic shock, anaphylactic shock, and various sequelae of encephalitis and meningitis, for example, encephalitis associated with connective tissue diseases such as lupus. Other uses include preventing or protecting against neurotoxin intoxication such as domoic acid oyster intoxication, neurolathyrism and guam disease, amyotrophic lateral sclerosis, parkinson's disease; post-operative treatment of emboli or ischemic injury; irradiating the whole brain; sickle cell crisis; and convulsions.

Another group of conditions that can be treated by the methods of the present invention include inherited or acquired mitochondrial dysfunction, which is the cause of a variety of neurological diseases characterized by neuronal damage and death. For example, Leishmaniasis (subacute necrotic encephalomyelitis) is characterized by progressive visual loss and encephalomyelitis, resulting from neuronal loss and myopathy. In these cases, defective mitochondrial metabolism does not provide enough high energy substrates to supply fuels for the metabolism of stressed cells. Modulators of EPO receptor activity optimize the function of failure in various mitochondrial diseases.

As mentioned above, hypoxic conditions can adversely affect stressed tissues. Stressed tissues include, but are not limited to, central nervous system tissues, peripheral nervous system tissues, and cardiac tissues. In addition to the conditions described above, the methods of the present invention may also be used to treat inhalation toxicities such as inhalation of carbon monoxide and smoke, severe asthma, adult respiratory distress syndrome, and congestion and near drowning. Other conditions that may cause hypoxic conditions or otherwise induce stress tissue damage include hypoglycemia that may occur with improper administration of insulin, or hypoglycemia with insulin-producing neoplasms (insulinomas).

It is believed that various neuropsychological conditions resulting from damage to stressed tissue can be treated with the present method. Chronic conditions in which neuronal damage may be involved and which can be treated by the present invention include conditions involving the central and/or peripheral nervous system including age-related cognitive function loss and senile dementia, chronic seizures, alzheimer's disease, parkinson's disease, dementia, memory loss, amyotrophic lateral sclerosis, multiple sclerosis, tuberous sclerosis, hepatolenticular degeneration, cerebral and progressive supranuclear palsy, guam's disease, lewy body dementia, prion diseases such as spongiform encephalomyelopathy, e.g. creutzfeldt-jakob disease, huntington's chorea, myotonic dystrophy, Freidrich ataxia and other ataxia, and tourette's syndrome, seizure disorders such as epilepsy and chronic seizures, stroke, brain or spinal trauma, AIDS dementia, alcoholism, autism, local defects in the retina, glaucoma, autonomic function disorders such as hypertension and sleep disorders, and neuropsychiatric disorders including, but not limited to, schizophrenia, schizoaffective disorder, attention deficit disorder, dysthymia, major depression, mania, obsessive-compulsive disorder, substance-induced psychotic disorders, anxiety, panic disorder, and unipolar and bipolar disorders. Other neuropsychiatric and neurodegenerative diseases include, for example, those listed in the american psychiatric society's Diagnostic and Statistical Manual (DSM), the most prevalent versions of which are incorporated herein by reference in their entirety.

In another embodiment, recombinant chimeric toxin molecules comprising EPO can be used for therapeutic release of toxins to treat proliferative disorders, such as cancer, or viral disorders, such as subacute sclerosing panencephalitis.

5.4 pharmaceutical formulations and administration

In accordance with the present invention, EPO, analogs, mimetics, erythropoietin fragments, hybrid erythropoietin molecules, erythropoietin receptor binding molecules, erythropoietin agonists, renal erythropoietin, brain erythropoietin, muteins thereof, and analogs thereof, can be administered parenterally, transmucosally, e.g., orally, nasally, rectally, intravaginally, sublingually, submucosally, or transdermally. Parenteral administration, such as by intravenous or intraperitoneal injection, is preferred, and also includes, but is not limited to, intraarterial, intramuscular, intradermal, and subcutaneous administration. The preferred route of administration of the small molecule mimetic of EPO is by the oral route.

Subjects for whom peripheral administration of EPO is an effective treatment regimen are preferably humans, but can be any animal, preferably mammals. Thus, as one of ordinary skill in the art can readily appreciate, the methods and pharmaceutical compositions of the present invention are particularly suited for administration to any animal, particularly a mammal, including, but in no way 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 field or in zoos), research animals, such as mice, rats, rabbits, goats, sheep, pigs, dogs, cats, and the like. As has been seen above, domesticated animals, including pets and work animals, are candidates for both the neuroprotective benefits of the present invention and for the enhancement of cognitive function. Neurological damage caused by hypoxia, as well as acute and chronic disorders including epilepsy, is common in such animals and is therefore a candidate for treatment. In the foregoing, it is also seen that cognitive enhancement in non-human animals is a benefit of the present invention, and thus the maintenance of learning, training and learning behaviors can be enhanced, supplemented and maintained using the teachings of the present invention. Thus, the pet owner's expense and psychological stress is reduced. For example, the time required to train dogs and other domestic animals is reduced. In addition, wild animals, which are generally difficult to train, can also be better trained using the method of the invention.

5.4.1 formulations and effective dosages

The invention also provides a pharmaceutical composition. Pharmaceutical compositions comprising EPO and a modulator of EPO receptor activity can be administered to a patient in therapeutically effective doses to protect stressed tissue from damage, to enhance the function of stressed tissue, or to deliver a compound to stressed tissue. Applicants have found that elevated doses of EPO are preferred for modulating and protecting stressed tissues from damage.

The selection of a preferred effective dose will be determined by the skilled artisan upon consideration of several factors, which will be known to those of ordinary skill in the art. These factors include the particular form of erythropoietin and its pharmacokinetic parameters such as bioavailability, metabolism, half-life, etc., which will be determined during the usual development procedures normally used to obtain regulatory approval for a pharmaceutical compound. Other factors to be considered in terms of dosage include the condition or disease to be treated or the benefit to be achieved in normal individuals, the physical condition of the patient, the route of administration, whether the administration is acute or chronic, concurrent medication, and other factors that are well known to affect the efficacy of the administered agent. The precise dosage should therefore be determined at the discretion of the physician and in accordance with the specifics of each patient, e.g., the condition and immune status of the individual patient as determined by standard clinical techniques.

In one embodiment, the invention provides a pharmaceutical composition in dosage unit form suitable for modulating excitable tissue, enhancing cognitive function or transporting a compound across an endothelial tight junction, each dosage unit comprising an effective non-toxic amount of 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 in the range of 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, an effective non-toxic amount of EPO is in the range of about 50,000 to 500,000 units.

In one embodiment, such a pharmaceutical composition of EPO can be administered systemically to protect stressed tissue from damage, to enhance the function of stressed tissue, or to deliver a compound to stressed tissue. Such administration may be parenteral, transmucosal, e.g., oral, nasal, rectal, intravaginal, sublingual, submucosal, or transdermal, preferably parenteral, e.g., by intravenous or intraperitoneal injection, and also includes, but is not limited to, intraarterial, intramuscular, intradermal, and subcutaneous administration.

In a preferred embodiment, EPO can be administered systemically at a dose of 2000-10000 units/kg body weight, preferably about 2000-5000 units/kg body weight, most preferably 5000 units/kg body weight per administration. Such an effective dose should be sufficient to provide a serum concentration of EPO of greater than about 10,000, 15,000, or 20,000mU/ml serum after EPO administration. Such serum concentrations may be achieved at about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 hours after administration. These doses may be administered repeatedly as needed. For example, the administration may be repeated daily, or after appropriate intervals, for example once every 1 to 12 weeks, preferably once every 3 to 8 weeks, as long as clinically necessary. In one embodiment, an effective amount of EPO and a pharmaceutically acceptable carrier can be packaged in a single dose vial or other container. In one embodiment, EPO is non-erythropoietic, i.e., it produces the activity described herein but does not cause an increase in hemoglobin concentration or hematocrit. In another embodiment, EPO is administered at a dose greater than that required to maximally stimulate erythropoiesis.

The pharmaceutical compositions of the present invention may comprise a therapeutically effective amount of the compound and a pharmaceutically acceptable carrier. In a particular 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 carrier with which a therapeutic agent is administered. Such pharmaceutical carriers can be sterile liquids, water and oil solutions, such as saline, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. When the pharmaceutical composition is administered intravenously, saline solution is the preferred carrier. 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 compositions may also contain minor amounts of wetting or emulsifying agents, or pH buffering agents, if desired. These compositions may take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained release formulations and the like. The compositions may be formulated as suppositories with conventional binders and carriers such as triglycerides. The compounds of the present invention may be formulated in neutral or salt form. Pharmaceutically acceptable salts include those formed with free amino groups such as those derived from hydrochloric, phosphoric, acetic, oxalic, tartaric acids, and the like, and those formed with free carboxyl groups such as those derived from sodium hydroxide, potassium hydroxide, ammonium hydroxide, calcium hydroxide, ferric hydroxide, isopropylamine, triethylamine, 2-ethylamino ethanol, histidine, procaine, and the like. Examples of suitable pharmaceutical carriers are described in e.w. martin, "Remington pharmaceutical science". Such compositions will contain a therapeutically effective amount of the compound, preferably in purified form, in combination with a suitable amount of carrier to provide the appropriate form of administration to the patient. The formulation should be compatible with the mode of administration.

Pharmaceutical compositions suitable for oral administration may be formulated as capsules or tablets; making into powder or granule; making into solution, syrup or suspension (in aqueous or non-aqueous liquid); forming edible foams or whipping agents (whiss); or made into emulsion. Tablets or hard gelatine capsules may contain lactose, starch or derivatives thereof, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, stearic acid or salts thereof. Soft gelatin capsules may contain vegetable oils, waxes, fats, semi-solid or liquid polyols, and the like. Solutions and syrups may contain water, polyols and sugars.

The active agent intended for oral administration may be coated with or mixed with a substance that delays disintegration and/or absorption of the active agent in the gastrointestinal tract (e.g., glyceryl monostearate or glyceryl distearate may be employed). Thus, a sustained release of the active agent can be achieved over several hours and, if desired, the active agent can be protected from degradation in the stomach. Pharmaceutical compositions for oral administration may be formulated to facilitate release of the active agent at a particular gastrointestinal site according to particular pH or enzymatic conditions.

Pharmaceutical compositions suitable for transdermal administration may be formulated as discrete patches intended to remain in intimate contact with the epidermis of the recipient for an extended period of time. Pharmaceutical compositions suitable for topical administration may be formulated as ointments, creams, suspensions, lotions, powders, solutions, pastes, gels, sprays, aerosols or oils. For topical administration to the skin, mouth, eye or other external tissue, a topical ointment or cream is preferably used. When formulated as an ointment, the active ingredient may be used 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 suitable for topical administration to the eye include eye drops. In these compositions, the active ingredient may be dissolved or suspended in a suitable carrier, such as an aqueous solvent. Pharmaceutical compositions suitable for topical administration in the mouth include lozenges, pastilles and mouthwashes.

Pharmaceutical compositions suitable for nasal administration may comprise a solid carrier such as a powder (preferably having a particle size in the range of 20 to 500 microns). The powder may be administered by nasal inhalation, i.e. by rapid inhalation through the nose from a powder container placed in the vicinity of the nose. In another aspect, compositions suitable for nasal administration may comprise a liquid carrier, such as a nasal spray or nasal drops. These compositions may comprise aqueous or oily solutions of the active ingredient. Compositions for administration by inhalation may be presented in a specially adapted apparatus, including but not limited to a pressurized aerosol, nebulizer or insufflator, which may be adapted to provide a predetermined dose of the active ingredient. In a preferred embodiment, the pharmaceutical composition of the invention is administered to the lungs via nasal administration.

Pharmaceutical compositions suitable for rectal administration may be formulated as suppositories or enemas. Pharmaceutical compositions suitable for vaginal administration may be formulated as pessaries, tampons, creams, gels, pastes, foams or spray formulations.

Pharmaceutical compositions suitable for parenteral administration include aqueous and non-aqueous sterile injectable solutions or suspensions, which may contain antioxidants, buffers, bacteriostats and solutes which render the composition substantially isotonic with the blood of the intended recipient. Other ingredients that may be present in such compositions include, for example, water, alcohols, polyols, glycerin, and vegetable oils. Compositions suitable for parenteral administration may be presented in unit-dose or multi-dose containers, for example sealed ampoules and vials, and may be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example sterile saline solution for injections, immediately prior to use. Injectable solutions and suspensions for the preparation at the time may be prepared using sterile powders, granules and tablets.

In a preferred embodiment, the composition is formulated in accordance with routine procedures as a pharmaceutical composition suitable for intravenous administration to a human. Generally, compositions for intravenous administration are solutions in sterile isotonic aqueous buffers. If desired, the composition may also include solubilizing agents and local anesthetics such as lidocaine to reduce pain at the injection site. Typically, the components are separated or mixed together and made into unit dosage forms, e.g., as a dry lyophilized powder or as a water-free concentrate, in a sealed container such as an ampoule or sachet indicating the amount of active agent. When the composition is intended for administration by infusion, it may be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline. When the composition is administered by injection, an ampoule of sterile water for injection or saline may be provided so that the ingredients may be mixed prior to administration.

Suppositories usually contain the active ingredient in the range of 0.5% to 10% by weight; oral formulations typically contain 10% to 95% of the active ingredient.

The invention also provides a pharmaceutical pack or kit comprising one or more containers filled with one or more of the ingredients of the pharmaceutical composition of the invention. These containers are optionally accompanied by instructions in a form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which instructions reflect approval by a regulatory agency of manufacture, use or sale for the purpose of administering drugs to humans.

5.4.2 methods of administration

The present invention provides compositions and methods for peripheral administration of EPO to enhance function or protect stressed tissues and to deliver compounds to such tissues. As seen above, the present invention is based in part on the discovery that: peripherally administered EPO has direct neuroprotective or neuroenhancing properties in stressed tissues, such as central nervous system tissue, peripheral nervous system tissue or cardiac tissue. "stressed 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 methods for their administration.

The present invention provides methods for the administration of EPO and modulators of EPO receptor activity using routes of administration other than directly into the central nervous system, these different routes being collectively referred to by the terms "peripheral" and "systemic" routes. Peripheral administration includes oral or parenteral administration, such as intravenous, intraarterial, subcutaneous, intramuscular, intraperitoneal, rectal, submucosal, or intradermal administration. Other routes of administration of the agents described herein may also be used. Acute and chronic administration is provided herein.

In one embodiment, for example, EPO can be released with a controlled release system. For example, the polypeptide may be administered using intravenous infusion, implantable osmotic pumps, transdermal patches, 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, New England journal of medicine 321: 574). In another embodiment, the compounds may be delivered using carriers, particularly liposomes (see Langer, science 249: 1527. 1533 (1990); Treat et al, liposomes in the treatment of infectious diseases and cancer, Lopez-Berestein and Fidler (ed.), Liss, New York, 353. 365 (1989); WO 91/04014; U.S. Pat. No.4,704,355; Lopez-Berestein, supra, 317. 327. incorporated generally by reference above). In another embodiment, polymeric materials may be used [ see "medical applications for controlled release", Langer and Wise (ed.), CRC Press: boca Raton, Florida, 1974; controllable bioavailability of drugs: design and presentation of drugs, Smolen and Ball (ed.), Wiley: NewYork (1984); ranger and Peppas, J. Macro science review and Macro chemistry 23: 61, 1953; see also Levy et al, 1985, science 228: 190; during et al, 1989, neurologic discipline 25: 351, a step of; howard et al, 1989, J.Neuro-surgery 71: 105).

In yet another embodiment, the controlled release system may be placed near the target of treatment, i.e., the brain, so that only a small fraction of the systemic dose is required (see, e.g., Goodson, 115 and 138, in controlled release medicine, Vol.2, supra, 1984). Other controlled release systems are discussed in the review of Langer (1990, science 249: 1527-.

In another embodiment, the EPO, when appropriately formulated, may be administered by nasal, buccal, rectal, vaginal or sublingual route.

In a particular embodiment, topical administration of the EPO compositions of the present invention to an area in need of treatment is contemplated; this can be achieved in some way: such as, but not limited to, local infusion during surgery, local application, e.g., in conjunction with a post-operative wound dressing, by injection, via a catheter, via a suppository, or via an implant that is a porous, non-porous, or gelatinous material, including membranes, such as silicone rubber membranes, or fibers.

The invention may be better understood by reference to the following non-limiting examples, which are intended as illustrations of the invention. The following examples are presented in order to more fully illustrate the preferred embodiments of the invention. They should in no way be construed, however, as limiting the broad scope of the invention.

As described hereinafter, the studies conducted by the inventors herein are standard, worldwide accepted tests conducted in animal models that are predictive of prophylactic and therapeutic benefits.

6. Example 1: peripherally administered EPO enhanced cognitive function

In this example, a spatial localization experiment called the Morris water maze test showed EPO-induced enhancement of mouse cognitive function. In this test, a small transparent platform was placed in one quarter of a swimming pool with opaque water. Mice placed in the pool must swim until they reach a sub-surface resting platform that is not visible to swimming mice. The test involves measuring the time it takes for the animals to reach the platform (i.e. the length of time they spend swimming). In successive trials, the time taken for each mouse to reach the platform will decrease as a function of their location learned. This type of learning experiment involves the hippocampus, as hippocampal lesions impede learning in this experiment.

The experiment was carried out in a round black pond with a diameter of 150 cm. Four points are arbitrarily assigned north, south, east and west. Each quarter portion gives some visual cues to distinguish: e.g., flashing lights, bright bands arranged in a square, etc., to determine the direction of the mouse in the pool. The platforms are arbitrarily placed in a quarter circle. The test involved placing the animal's head forward in a quarter circle of the pool and releasing it. The test length was 90 seconds in total. If the animal fails to reach the platform, it is placed on the platform for an additional 15 seconds. The subject was allowed to rest for 1 hour and then placed in another quadrant for testing. All 4 quadrants were used in a 1 day trial and animals were tested for 12 consecutive days (i.e., 48 trials total).

The experiment itself consisted of intraperitoneal injection of 5000U/kg of recombinant human EPO (sold under the trade name PROCRIT by Ortho-Biotech) per mouse 4 hours before the start of the day of the experiment, on each of the 12 day trials. Control animals were sham injected with saline.

Learning was assessed by measuring the length of time each mouse was on the platform. As shown in fig. 1A, the results were plotted according to the time on the platform for the EPO-treated and sham groups. The results show that both groups of animals spend more time on the platform on each successive test day, i.e. they learn to reach the platform faster, but EPO-treated animals do so much faster than the sham group. Thus, EPO-treated animals had a faster "learning curve" than the sham group. Regression line (R) when results are expressed as the difference between EPO-treated and sham-treated groups and the results of EPO-and sham-treated groups are compared²0.88) showed a slope (0.68) significantly different from slope 1, clearly favoring the EPO group (fig. 1B).

7. Example 2: peripheral administration of EPO enhances conditioned taste diversion by the academic society

The conditioned taste transfer (CTA) assay performed in this example demonstrates that: EPO significantly affected the ability of the mice to remember and learn to avoid the unpleasant taste sensation, which in the case of this experiment is the disease-causing substance. In this example, lithium chloride was used to produce CTA, since lithium chloride reliably produces discomfort and anorexia in a dose-dependent manner. Like a naturally occurring disease, lithium produces CTA by stimulating pathways described above, including cytokine release.

Female Balb/c mice were trained to limit their total daily water intake to 5 minutes once a day and learned to keep balance by drinking sufficient water during this period. Animals were divided into groups and given sham control (saline) or EPO (5000U/kg), injected Intraperitoneally (IP), 4 hours prior to the administration of the novel saccharin-vanilla solution. After drinking the sweet liquid, the animals received saline or a pathogenic dose of lithium (20mg/kg, 0.15M LiCl, IP injection) immediately. Thereafter, animals were treated in three groups. The first group (control) did not inject lithium after drinking water. The second group was injected with lithium and EPO. The third group (sham) was injected with saline (without EPO) and lithium.

Conditioned taste transfer was assessed by measuring the reduction in drinking water followed by exposure to a pathogenic solution-neochic saccharin-vanilla liquid. After 5 days of recovery from lithium or sham treatment, the same novel saccharin-vanilla solution was again administered to the animals that were water deficient. A plot of the group 2 and group 3 results compared to group 1 (control) is shown in fig. 2A. Day 2 represents the baseline of water consumption after habituation test cage. On day 3, animals were injected intraperitoneally with saline or EPO (5000U/kg), 4 hours later given a novel saccharin-vanilla solution, followed by treatment with lithium or sham saline (arrows). At day 3, this treatment resulted in a small reduction in fluid consumption in all groups, a side effect that had previously been demonstrated for injections and novel fluids. For the control group, the first test to determine CTA after recovery showed no reduction in water consumption. However, animals receiving lithium showed virtually complete aversion to the liquid despite its lack of water (day 4). Continued water deficit eventually abolished CTA (days 5-9), but animals characterized by EPO-received recovery significantly later, as shown by the filled circles in fig. 2A.

The robustness of the CTA established herein can be better appreciated by considering the degree of water deficit on each experimental day, since EPO-treated animals tolerating water deficit are approximately twice as many as sham-injected subjects (fig. 2B). Although the EPO group showed significantly stronger CTA, the animals in this group approached the drinking tube more rapidly than the sham group, as shown in fig. 2C. The strength of CTA was shown by repeated injections of lithium alone (without EPO), which resulted in attenuated CTA, which was more attenuated in the EPO group (fig. 2A, day 10). These data show that EPO pretreatment is associated with a significant boost of CTA produced with lithium.

8. Example 3: peripherally administered EPO protects the brain from damage by excitotoxins

This example demonstrates that EPO crosses the blood brain barrier and has neuroprotective effects on mice treated with kainic acid neurotoxin. There are many compounds in nature that exhibit specific toxicity to neurons. These molecules typically interact with the endogenous receptors for the amino acid transmitter glutamate, subsequently causing over-stimulation and neuronal damage. One of these: the kainate, a substance widely used in the study of neuronal damage due to excitotoxicity, is a glutamate analog. Kainate is a potent neurotoxin that specifically destroys neurons, particularly those located in areas with high density of kainate receptors such as the hippocampus, which induces seizures, brain damage and death.

The following neurotoxicity studies were performed with mice using kainate. This model is used to assess the protective benefit of treatment on diseases such as psychomotor epilepsy. Experimental animals such as rats and mice are injected parenterally to cause partial (limbic) seizures in a dose-dependent manner, which may then spread and cause death. The experiments described in this section were performed to test whether peripherally administered EPO crossed the blood brain barrier and, if so, whether EPO had an effect on neuronal energy balance, in particular whether it had a neuroprotective effect against kainate.

For this purpose, female Balb/c mice (average weight 15-20gm) were predicted to be injected intraperitoneally with 5000U/kg of recombinant human erythropoietin (rhEPO; sold under the trademark PROCRIT by the company Ortho-Biotech) or with saline (sham) and also with IP (substance/kg body weight) at a specific concentration at a specific time point before, during or after the receipt of kainate (Sigma Chemical). The subjects were then monitored and scored for the development of seizure activity at 20 minutes after receiving kainate. Each test was terminated 60 minutes after kainate administration. As shown in figure 3A, EPO pretreatment significantly reduced the severity of seizures and delayed the onset of seizure status in mice treated with kainate. Comparison between EPO-and sham-treated animals showed a significant reduction in mortality in animals receiving kainate at doses ranging from 20-30mg/kg, indicating neuroprotection resulting from pretreatment with EPO. The numbers in parentheses below each bar indicate the number of animals receiving each kainate dose.

The dose dependence of EPO in providing neuroprotection against kainate is shown in fig. 3B. Mice were administered EPO (5000U/kg; daily IP injection for up to 5 days). The neuroprotective effect of each dose of EPO was assessed by determining the survival after administration of kainate (20mg/kg), which resulted in a mortality rate of approximately 50% for control animals (no EPO administration; see FIG. 3A). Bars indicate an increase in survival of EPO-treated animals compared to sham-injected animals. As shown in FIG. 3B, neuroprotection was increased with another dose of 5000U/kg EPO.

The neuroprotection provided by EPO is characterized by a delay in onset, characterized by activation of a gene expression program. Figure 3C shows that a single dose of EPO administered at kainate administration (20mg/kg) did not provide any immediate protection against EPO-associated delay in death (in minutes) caused by the seizure, while EPO administered 24 hours prior to kainate improved the latency and severity of the seizure and the time to death. This effect lasts for a maximum of 7 days.

9. Example 4: peripherally administered EPO protects the brain from damage due to ischemia

Previous in vivo studies using the gerbil sphere reperfusion model have shown that blocking blood flow to the brain results in cell death in the brain, and EPO injected directly into the cerebral cortex protects the brain from such cell death (Sakanaka et al, 1998, proceedings of the american national academy of sciences 95: 4635). The experiments described in this example show for the first time that peripherally released EPO defends against neuronal cell death in vivo in animal models of ischemia.

The following experiments were performed using a middle cerebral artery occlusion model, which is an art-recognized model of ischemic focal stroke. In the experimental protocol, male rats (body weight 250gm) were anesthetized with phenobarbital and maintained at 37 ℃. The carotid artery was visually inspected and the ipsilateral carotid artery was permanently closed. The ipsilateral Middle Cerebral Artery (MCA) was visually examined and cauterized at its origin. The contralateral artery was closed with forceps for 1 hour. Animals were sacrificed 24 hours later, and brains were removed and cut into 1mm series of sections. Viable tissue was visually examined by visualization of viable tissue from necrotic areas by in situ triphenyltetrazolium reduction. Cell death occurred both in the central and peripheral edge portions of ischemia.

Using this MCA model, EPO was administered by peripheral injection at various times prior to and immediately following injury, and the volume of injury was quantified using computer-assisted image analysis. The results of this analysis, shown in figure 4A, demonstrate the effect of treatment with EPO at the following times after stroke: 24 hours before stroke, at the time of stroke, and at hours 3, 6, and 9 after stroke. As shown in fig. 4A, EPO protects tissues from necrotic damage when administered up to 6 hours after stroke.

Interestingly and in contrast, 17-mers derived from EPO that have previously been reported to have neuro-activity, promote axonal growth in vitro and myelination of nerve cells ex vivo (Campana et al, 1998, J. International molecular medicine 1: 235-41; U.S. Pat. No. 5,700,909 published 1997 on 12/23) did not protect against injury in this system (FIG. 4B, "17-mers"). Thus, this model, as well as other methods provided by the present invention for determining the effect of EPO on stress tissue function, can be used to identify modulators of EPO and EPO receptor activity that can be used to modulate stress tissue function, such as to protect it from damage or enhance learning and cognition.

10. Example 5: peripherally administered EPO protects brain from blunt trauma

In one model of mechanical trauma, the cortical impact model, the mouse brain was protected from blunt trauma by pretreatment with systemic EPO administration. To create trauma, an air-filled driven piston (Clippard Valves) with a diameter of 3mm that accurately releases the blow to the skull was used. Each mouse was anesthetized and safely placed in a stereotactic apparatus to prevent head movement. The scalp is incised to determine the position of bregma, which is the reference point for the initial strike position with the piston. The position of the piston was then adjusted by moving the piston 2mm from the bregma tail and 2mm from the bregma front and the impact was performed by using a precise nitrogen pulse. This device allows to precisely select the piston velocity (4m/s) and the impact displacement (2 mm).

Mice were treated with EPO (5000U/kg) 24 hours before, at and 3, 6 or 9 hours after the injury and continued daily dosing. Mice were sacrificed 10 days later, and then the brains were examined and the volume of brain necrosis was determined. In sham-treated mice, large areas of necrosis were observed (fig. 5) with extensive mononuclear cell infiltration. In contrast, when animals were pretreated with EPO or administered EPO no more than 3 hours after injury, the animals were protected from such injury and only few monocytes were detected in the area of injury.

11. Example 6: peripherally administered EPO protects myocardium from ischemic injury

This example demonstrates the role of EPO in protecting cardiac tissue against hypoxic injury. For this purpose, rats were predicted with EPO (5000U/kg) 24 hours before the operation according to Latini et al (1999, J. cardiovascular Pharmacology 31: 601-8). Subsequently, the subject was anesthetized, placed on assisted ventilation and a thoracotomy performed. The heart and its internal circulation are identified, and a removable suture is placed around the nearest portion of the left anterior descending coronary artery, which is then ligated. Another dose of EPO (5000U/kg) was then administered and the occlusion was maintained for 30 minutes. At this point, the ligature was released and the animals were maintained under deep anaesthesia for a further 6 hours and then sacrificed. Immediately after death, the heart was removed and the affected area fraction (AAR) as well as the unaffected area fraction (septa) were removed and prepared for biochemical analysis. Two parameters were evaluated: creatine Kinase (CK) (lower CK, less tissue viability) and myeloperoxidase, which are measures of myocardial survival, are products of monocyte infiltration. The results are shown in fig. 6A and 6B. As shown in these figures, treatment with EPO produced sustained CK activity, consistent with increased tissue viability, and decreased MPO activity relative to controls, whether in infarct size (AAR) or in the perfused Left Ventricular (LV) free inner wall, indicating significantly reduced infiltration by inflammatory cells.

12. Example 7: peripherally administered EPO reduces experimental allergic encephalitis

Experimental allergic (or autoimmune) encephalomyelitis (EAE) in rats is an art-recognized animal model of Multiple Sclerosis (MS). Various models of EAE have been developed for immunological, virological, toxicity and trauma parameters in order to understand the characteristics of MS.

To test whether EPO protects against symptoms of EAE, the following experiment was performed. 6-8 week old female Lewis rats (Charles River, Calco, Italy) were immunized under ether shallow anesthesia by injecting 50. mu.g of an aqueous solution of guinea pig myelin basic protein (MBP; Sigma, St. Louis, MO) emulsified with an equal volume of complete Freund's adjuvant (CFA, Sigma) with 7mg/ml heat killed Mycobacterium tuberculosis added to H37Ra (Difco, Detroit, MI) to a final volume of 100. mu.l into two hindfoot pads.

After treatment, rats were assessed daily for signs of Experimental Autoimmune Encephalomyelitis (EAE) and scored as follows: 0, no disease; 1, tail weakness; 2, ataxia; 3, hind limbs were completely paralyzed and urinary incontinence. Body weight was also monitored. Rats were initially administered EPO (5000U/kg, IP, once daily) on day 3 post-immunization for a period of 18 days. Control rats received only vehicle alone. As shown in fig. 7, the EPO-treated rats showed an improvement in score (i.e., lower score) and an improvement in disease duration. In addition, a significant delay in the onset of symptoms was noted in the EPO-treated rats.

13. Example 8: minimal effective dose and pharmacokinetics of EPO required to protect stressed tissues

The optimal and effective dose of EPO was assessed using the above described focal ischemic stroke animal model. As shown in fig. 8A, EPO doses less than 450 units/kg body weight are not reliable in their effectiveness in protecting stressed tissues from necrotic injury. As shown in figure 8B, in an animal study, IP injection of four female subject mice at a dose of approximately 5000 units/kg body weight produced circulating concentrations of EPO in the serum of greater than 20,000mU/ml within 5 hours after administration, greater than 10,000mU at 10 hours after administration, but less than 5 units/ml at 24 hours after administration.

14. Example 9: erythropoietin-mediated CNS release

The experiments described below show that EPO conjugated molecules are successfully transported across the blood brain barrier and their localization within the basement membrane. As shown in FIG. 9A, brain sections were stained with an antibody to EPO receptor (EPO-R), which showed that brain capillaries expressed high levels of EPO-R. To investigate whether EPO can be transported across the blood brain barrier, EPO was labeled with biotin as described below. The substance containing rhEPO was concentrated using a Centricon-10 filter (Millipore) and the recovery was measured by reading the absorbance at a wavelength of 280 nm. Next, 0.2mg of long-armed biotin (Vector Labs) was dissolved in 100. mu.l of DMSO, added to the concentrated rhEPO solution and immediately vortexed. The mixture was then incubated at room temperature for 4 hours while stirring gently and protected from light. Unbound biotin was removed from the solution by using a Centricon-10 chromatography column. Animals were then IP dosed with biotinylated EPO and sacrificed 5 hours later. Brain sections were labeled with avidin coupled to peroxidase and diaminobenzidine was added until sufficient reaction product was generated for visualization with light microscopy. EPO coexisted in the same capillaries positively stained for EPO-R (fig. 9B). At later time points, the biotin label appears to be localized in a particular neuron (e.g., at 17 hours, fig. 9C). In contrast, if cold EPO was added in 100-fold excess to the labeled EPO, all specific staining was eliminated. The results demonstrate that systemically administered conjugated EPO compounds are successfully transported across the blood brain barrier.

The successful transport of systemically administered EPO-biotin conjugates across the blood-brain barrier into the brain demonstrates that other therapeutic compounds can be transported across the blood-brain barrier in a similar manner by complexing EPO with the desired compound. As an example, brain derived neurotrophic factor (BNF) can be covalently coupled to EPO via carbodiimide coupling using standard procedures. After purification, the conjugate can be administered to an animal by intraperitoneal injection. The positive effect of BNF on the central nervous system can be measured relative to control animals to assess successful transport of the molecule in association with EPO, whereas non-conjugated BNF has no central nervous system activity.

The present invention is not to be limited in scope by the specific embodiments described which are intended as 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 also intended to fall within the scope of the appended claims.

All references mentioned herein are incorporated herein by reference in their entirety.

Claims (27)

1. A pharmaceutical composition in dosage unit form suitable for modulating excitable tissue, enhancing cognitive function or transporting a compound across endothelial tight junctions, comprising per dosage unit an effective, non-toxic amount of EPO in the range of about 50,000 to 500,000 units, an EPO receptor activity modulator, an EPO-activated receptor modulator, or a combination thereof, and a pharmaceutically acceptable carrier.

2. The pharmaceutical composition of claim 1 wherein the effective non-toxic amount of EPO comprises 50,000 to 500,000 units of EPO.

3. The pharmaceutical composition of claim 1 wherein the effective non-toxic amount of EPO is a dose effective to achieve circulating concentrations of EPO greater than 10,000mU/ml serum.

4. The pharmaceutical composition of claim 3, wherein the circulating concentration of EPO is measured about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 hours after EPO administration.

5. A pharmaceutical kit with one or more containers containing the pharmaceutical composition of claim 2.

6. A method of protecting a mammal from a condition resulting from damage to stressed tissue comprising peripherally administering to said mammal an effective amount of EPO, an EPO receptor activity modulator, or an EPO-activated receptor modulator to protect stressed tissue.

7. The method of claim 6, wherein the injury is the result of: seizure, multiple sclerosis, stroke, hypotension, cardiac arrest, ischemia, myocardial infarction, inflammation, age-related loss of cognitive function, radiation damage, cerebral palsy, neurodegenerative disorders, alzheimer's disease, parkinson's disease, lygod's disease, AIDS dementia, memory loss, amyotrophic lateral sclerosis, alcoholism, mood disorders, anxiety disorders, attention deficit disorder, autism, creutzfeldt-jakob disease, brain or spinal cord trauma, heart-lung bypass, glaucoma, retinal ischemia, or retinal trauma.

8. The method of claim 6, wherein the injury is the result of hypoxia.

9. The method of claim 8, wherein the hypoxia is prenatal or postpartum hypoxia, asphyxia, congestion, near drowning, post-operative cognitive dysfunction, carbon monoxide poisoning, smoke inhalation, chronic obstructive pulmonary disease, emphysema, adult respiratory distress syndrome, hypotensive shock, septic shock, anaphylactic shock, insulin shock, sickle cell crisis, cardiac arrest, dysrhythmia, nitrogen anesthesia, or local tissue hypoxia.

10. A method of enhancing normal or abnormal excitable tissue function in a mammal comprising peripherally administering to said mammal a peripherally effective excitable tissue enhancing amount of EPO, an EPO receptor activity modulator, an EPO-activated receptor modulator, or a combination thereof.

11. The method of claim 10, wherein said enhancing the function of stressed tissue enhances associative learning or memory.

12. The method of claim 10 wherein said enhancing the function of stressed tissue is useful in the treatment of mood disorders, anxiety disorders, depression, autism, attention deficit hyperactivity disorder, alzheimer's disease, aging or cognitive dysfunction.

13. The method of claim 6 or 10, wherein the excitable tissue is central nervous system tissue, peripheral nervous system tissue, or cardiac tissue.

14. The method of claim 6 or 10, wherein said administering comprises oral, topical, intraluminal or by inhalation or parenteral administration.

15. The method of claim 14, wherein the parenteral administration is intravenous, intraarterial, subcutaneous, intramuscular, intraperitoneal, submucosal, or intradermal.

16. The method of claim 6 or 10, wherein said administration is acute or chronic.

17. The method of claim 6 or 10 wherein the EPO is non-erythropoietic.

18. The method of claim 6 or 10 wherein the EPO is administered at a dose greater than that required to maximally stimulate erythropoiesis.

19. A method of promoting transcytosis of a molecule across an endothelial cell barrier in a mammal comprising administering to said mammal a composition comprising said molecule in association with EPO, an EPO receptor activity modulator, an EPO-activated receptor modulator, or a combination thereof.

20. The method of claim 19, wherein said association is a labile covalent bond, a stable covalent bond, or a non-covalent association with a binding site of said molecule.

21. The method of claim 19, wherein said endothelial cell barrier is a blood brain barrier, a blood eye barrier, a blood testis barrier, a blood ovary barrier, or a blood placenta barrier.

22. The method of claim 19, wherein the molecule is a receptor agonist or antagonist hormone, a neurotrophic factor, an antimicrobial agent, a radiopharmaceutical, an antisense compound, an antibody, an immunosuppressive agent, a toxin, or an anticancer agent.

23. The method of claim 6, 10 or 19, wherein 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, renal erythropoietin, 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.

24. The method of claim 23, wherein said EPO receptor binding molecule is an antibody to the erythropoietin receptor.

25. A composition for transporting a molecule across an endothelial cell barrier via transcytosis, comprising said molecule in association with an EPO, an EPO receptor activity modulator, or an EPO-activated receptor modulator.

26. The composition of claim 25, wherein 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.

27. The composition of claim 25, wherein the molecule is a receptor agonist or antagonist hormone, a neurotrophic factor, an antimicrobial agent, a radiopharmaceutical, an antisense compound, an antibody, an immunosuppressive agent, a toxin, or an anticancer agent.