KR20020000874A - Modulation of excitable tissue function by peripherally administered erythropoietin - Google Patents

Abstract

The present invention protects the function of excitatory tissue in mammals by systemically administering an erythropoietin receptor activity modulator, such as erythropoietin, which sends signals that modulate the function of excitatory tissue through an erythropoietin (EPO) -activated receptor. A method and composition for improving. Excited tissue includes central nervous tissue such as the brain, peripheral nervous tissue, retina and heart tissue. By protecting excitatory tissue, hypoxia, stroke seizure disorders, neurodegenerative diseases, hypoglycemia and neurotoxin poisoning can be treated. Improving the function of excited tissues is useful for improving learning and memory. The invention also relates to compositions and methods for facilitating the transport of molecules through endothelial cell occlusive membrane barriers such as the blood-brain barrier by binding any molecule with an erythropoietin receptor activity modulator such as erythropoietin.

Description

MODULATION OF EXCITABLE TISSUE FUNCTION BY PERIPHERALLY ADMINISTERED ERYTHROPOIETIN}

Various acute and chronic symptoms and diseases occur as the excitatory tissue is damaged and malfunctions by external and internal stimuli. Such stimuli include lack of adequate oxygen or glucose, neurotoxins, consequences of aging, infectious agents and trauma. For example, excitatory tissue can cause stroke or chronic stroke disorders, convulsions, epilepsy, seizures, Alzheimer's disease, Parkinson's disease, central nervous system damage, hypoxia, cerebral palsy, brain or spinal cord injury. , AIDS dementia and other types of dementia, loss of cognition, memory loss, amyotrophic lateral sclerosis, multiple sclerosis, hypotension, cardiac arrest, nerve cell damage, smoke inhalation and carbon monoxide poisoning Can be.

It is well known that as the supply of available energy sources (eg, glucose or oxygen) in the brain decreases, brain functions such as cognitive function are severely impaired. Numerous neurons (but not all neurons) of the central nervous system are easily damaged during exposure to metabolic constraints such as hypoxia, hypoglycemia, stress and / or persistent strong excitability. Under these circumstances, the electrochemical gradients of these neurons often collapse, causing irreversible damage and cell death. The current trend is to view this general mechanism as a common final pathway in which a variety of common and debilitating neurodegenerative diseases, including seizures, epilepsy and Alzheimer's disease, are caused.

The results of supplying limited energy source materials to brain function are well known, but the effects of improving the delivery of energy sources in the normal brain have not been extensively studied. Currently known data suggest that by improving glucose or oxygen transfer capacity, complex cognitive function can be greatly improved in both animal models and normal human subjects (Kopf et al., Behavioral and Neural Biology , 62). : 237-243, 1994 and Li et al., Neuroscience , 85 : 785-794, 1998 and Moss et al., Psychopharmacology , 124 : 255-260, 1996). In addition, it has been demonstrated that cognitive function in normal brain is directly improved as neuropeptides produced in the brain increase. The physiological basis of this cognitive enhancement ultimately depends on the remodeling of neuronal interconnections through synaptic changes.

Cell building of brain tissues is extremely flexible and undergoes continuous remodeling. These processes, mediated by numerous nutrient molecules, appear not only after injury but also play an important role in learning, memory and cognitive functions. Although circular neurotrophin is a nerve growth factor (NGF), it is known that many cytokines perform nutritional functions in the brain (Hefti et al., Annu. Rev. Pharmacol. Toxicol ., 37 : 239-267, 1997).

Recently, it has been recognized from many independent researchers that neural tissues express not only EPO but also its receptor (EPO-R) (Digicaylioglu et al ., Proc. Natl. Acad. Sci. USA , 92 : 3717-3720, 1998, Juul et al . , Pediatr. Res. , 43 : 40-49, Marti et al. , Kidney Int. , 51 : 416-418, 1997. And Morishita et al., Neuroscience , 76 : 105-116, 1997). Although EPO and its receptor proteins each appear to be the product of a single gene, EPO and its receptors produced in the central nervous system (CNS) are significantly smaller in size. The physiological meaning of these observations is not clear, but the mass differences appear to alter biological activity. For example, in a study of human patients, the researchers concluded that EPO is not transported from the periphery to the brain (see Martini et al., 1997, supra). However, to date no direct experimentation has examined this possibility for EPO. The brain's EPO is about 15% smaller than the kidney's EPO (due to differences in sialylation), but the brain's EPO is more active at stimulating red blood cell colonies even at low ligand concentrations (Masuda J. Biol. Chem. , 269 : 19488-19493, 1994). On the other hand, CNS receptors show lower activity against deglycosylated EPO than 30% larger peripheral receptors (Konishi et al., Brain Res ., 609 : 29-35, 1993) and Masda et al. See J. Biol. Chem. , 268 : 11208-11216, 1993).

In the brain, EPO has been shown to be expressed in astrocytes, and the expression and release of EPO is increased by hypoxia and other metabolic stress factors (Marty et al . , Eur. J. Neurosci. , 8 : 666-676, 1996, Masda et al ., J. Biol. Chem ., 268 : 11208-11216, 1993 and Masda et al ., J. Biol. Chem ., 269 : 19488-19493, 1994), or even May be increased by the presence of other receptors, such as the insulin-like growth factor family (see Masda et al . , Brain Res. , 746 : 63-70, 1997). Since neurons express EPO-R in a highly cell type-specific manner, it is one goal of this secreted EPO (see Morishita et al., Neuroscience , 76 : 105-116, 1997). Unlike EPO itself, the density of EPO-R does not seem to be regulated during metabolic stress (see Digicaioglu et al ., Proc. Natl. Acad. Sci. USA , 92 : 3717-3720, 1995). .

Recent studies have demonstrated that EPO injected directly into the ventricles provides excellent protection of neurons from hypoxic neuronal damage both in vitro and in vivo (Morishita et al., Neuroscience , 76 : 105-116, 1997). ], Sadamoto et al., Biochem. Biophys. Res. Commun. , 253 : 26-32, 1998, and Sakanaka et al. , Proc. Natl. Acad. Sci. USA, 95 : 4635-4640, 1998). Cornish et al ., Brain Res. , 609 : 29-35, 1993] demonstrated that EPO promotes in vivo survival of cholinergic neurons in mature rats when injected directly into the ventricles. EPO administered centrally to the ventricles also successfully prevents the lack of spatial learning ability due to ischemic injury in rats (see Sadamoto et al., Biochem. Biophys. Res. Commun ., 253 : 26-32, 1998). . Recent publications suggest that only 17 amino acids of EPO are required to exhibit this neurotrophic effect in cultured neurons (Campana et al., Int. J. Mol. Med. ., 1: 235-241, see 1998).

For many years, the only evident physiological role of erythropoietin (EPO) has been the function of controlling the production of red blood cells. Recently, a series of evidence suggests that EPO, as a member of the cytokine family, performs other important physiological functions mediated through interactions with erythropoietin receptors (EPO-R). These actions include mitogenesis, regulation of calcium influx into smooth muscle and neurons, and the effects on metabolism. EPO is believed to provide a targeted response that acts to improve the hypoxic intracellular microenvironment. Although numerous studies have established that intracranially injected EPO protects neurons from hypoxic neuronal damage, intracranial administration is an impractical and unacceptable route for therapeutic use, particularly to normal individuals. . In addition, previous studies in anemic patients administered EPO have concluded that peripherally administered EPO is not transported to the brain (see Marty et al., 1997, supra).

References cited or discussed herein will not be accepted as prior art in the present invention.

Summary of the Invention

The present invention relates to compositions and methods for modulating the function of excitatory tissue in mammals, as well as methods and compositions for delivering drugs to excitatory tissue. The present invention is based in part on the findings of Applicants that high doses of systemically administered erythropoietin (EPO) are specifically absorbed by the brain. In particular, Applicants have found that high-dose delivered EPO can cross the blood-brain barrier, thereby improving cognitive ability and protecting nerve tissue from damage caused by stress symptoms such as hypoxia. It was.

As used herein interchangeably, erythropoietin and EPO, and activity regulators of the EPO receptor, and EPO-activated receptor modulators, are systemically administered (administered outside the blood-brain barrier), electrical excitatory tissue It refers to compounds that can activate EPO-activated receptors to enhance these tissues and / or protect them from damage and cell death. That is, EPO may refer to any form of erythropoietin capable of modulating excitatory tissue as well as EPO analogs, fragments and mimics thereof. In a preferred embodiment for use in the methods of the invention, the erythropoietin of the invention exhibits increased specificity for the EPO receptor in the brain. In another embodiment, the erythropoietin is non-erythropoietic. In another embodiment, erythropoietin is administered at a higher dosage than is needed to maximize stimulation of erythropoiesis.

The present invention comprises a nontoxic effective amount of EPO, EPO receptor activity modulator, EPO-activated receptor modulator or mixtures thereof, in the range of about 50,000 to 500,000 units per dosage unit, and a pharmaceutically acceptable carrier. Pharmaceutical compositions in dosage unit form suitable for regulating excitatory tissue, enhancing cognitive function, or delivering a compound through an endothelial obstruction. In one embodiment, the nontoxic effective amount of EPO in the pharmaceutical composition comprises 50,000 to 500,000 units of EPO. In another embodiment, the non-toxic effective amount of EPO in the pharmaceutical composition is a dose effective to achieve a circulating amount of EPO greater than 10,000 milliunits in 1 ml serum. In another embodiment, such circulating amounts of EPO are achieved about 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 hours after administration of EPO. In another aspect, the present invention provides a pharmaceutical kit packaged in one or more containers of an amount of EPO effective to modulate excitatory tissue, enhance cognitive function, or deliver a compound through an endothelial closure membrane.

The present invention provides a method of modulating the function of excitatory tissue in a mammal, including peripherally administering an effective amount of erythropoietin to the mammal. Excited tissue may be normal tissue or abnormally diseased tissue. In one embodiment, the excitatory tissue is neuronal tissue of the central nervous system. In another embodiment, the excitatory tissue is selected from the group consisting of neuronal tissue and cardiac tissue of the peripheral nervous system.

In one embodiment, a method of enhancing the function of excitatory tissue in excitatory tissues of mammals, particularly normal and abnormal excitatory tissues, is provided by peripherally administering an effective amount of an EPO or EPO receptor activity modulator. When the function of the excitatory tissue is improved, for example, learning ability, associative learning ability or memory ability is improved. Non-limiting examples of symptoms or diseases that can be treated by 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, regulation of excitatory tissue provides a protective effect from lesions caused by damage to excitatory tissue, such as nerve cells of the central nervous system, nerve cells of the peripheral nervous system or heart tissue. Such lesions may result from injuries including but not limited to hypoxia, stroke seizure disorders, neurodegenerative diseases, neurotoxin poisoning, multiple sclerosis, hypotension, cardiac arrest, radiation or hypoglycemia. In one embodiment, the lesion results from hypoxia, prenatal or postpartum oxygen deficiency, asphyxia, airway obstruction, myopia, postoperative cognitive dysfunction, carbon monoxide poisoning, smoke inhalation, chronic obstructive pulmonary disease, emphysema, adult respiratory distress syndrome , Hypotension shock, septic shock, insulin shock, anaphylactic shock, sickle cell development, cardiac arrest, arrhythmia or nitrogen anesthesia. If the lesion is a stroke seizure disorder, non-limiting, for example, the lesion may be a convulsion or chronic stroke seizure disorder. If the lesion is a neurodegenerative disease, for example, the lesion may have seizures, Alzheimer's disease, Parkinson's disease, cerebral palsy, brain or spinal cord trauma, AIDS dementia, cognitive loss due to aging, amnesia, muscular dystrophy, stroke Seizure disorders, alcoholism, retinal ischemia, aging, glaucoma or neuronal damage. In another embodiment, administration of EPO can prevent tissue injury or damage during the surgical procedure, such as during tumor resection or aneurysm repair surgery.

In another embodiment, provided is a method of promoting transcytosis through the endothelial cell barrier of a molecule in a mammal by administering a composition of any molecule in combination with erythropoietin. The bond between the molecule to be transported and the EPO can be, for example, an unstable covalent bond, a stable covalent bond or a noncovalent association with the binding site of the molecule. In one aspect, the endothelial cell barrier can be a blood-brain barrier, a blood-eye barrier, a blood-testis barrier, a blood-ovary barrier or a blood-placental barrier.

The present invention also provides a composition for transporting said molecule by transcytosis through the endothelial cell barrier, comprising a molecule associated with an EPO, an EPO receptor activity modulator or an EPO-activated receptor modulator. In one embodiment, the EPO comprises erythropoietin, erythropoietin analogue, erythropoietin mimic, erythropoietin fragment, hybrid erythropoietin molecule, erythropoietin receptor-binding molecule, erythropoietin agonist, renal erythropoietin, Brain erythropoietin, oligomers thereof, multimers thereof, muteins thereof, their equivalents, natural forms thereof, synthetic forms thereof, recombinant forms thereof, or mixtures thereof . In another embodiment, the molecule of the composition is a hormone, neurotrophic factor, antibacterial agent, radiopharmaceutical, antisense compound, antibody, immunosuppressant, toxin or anticancer agent. Molecules suitable for transport by the methods of the present invention include, but are not limited to, hormones (eg, growth hormone), antibiotics, anticancer agents, and toxins.

These and other aspects of the invention will be better understood from the following figures and description.

The present invention relates to the use of peripherally administered erythropoietin (EPO) and other erythropoietin receptor activity modulators or EPO-activated receptor modulators to have a desirable effect on the function of excitatory tissue. The present invention includes protecting excitatory tissues, such as neuronal and heart tissues, from neurotoxins, hypoxia and other adverse stimuli, and enhancing the function of excitable tissues, such as learning and memory. The invention also relates to a method of transporting a substance through an endothelial cell barrier by binding to an erythropoietin molecule, an erythropoietin receptor activity modulator or other EPO-activated receptor modulator.

1A and 1B show Morris Water Maze test, FIG. 1A shows Morris Water Maze performed in mice receiving peripherally administered EPO or saline (Sham) every day. The results of the test are shown, and FIG. 1B shows that the subjects receiving EPO give significantly better results than the siamese subjects. A significant deviation (0.68) from 1 from the regression curve (R ² = 0.88) was found, indicating that the EPO group was highly advantageous.

Figures 2a to 2c shows a conditioned taste aversion test, Figure 2a compares the peripherally treated siamese group and the peripherally treated EPO group for water intake in mice subjected to the conditional taste avoidance test. Water intake is expressed as a percentage of volume ingested by control mice that have never felt pain by lithium chloride. 2B and 2C show that EPO subjects are more resistant to thirst by avoiding water containing pain-related signals than controls, and as EPO-enhanced learning ability is robust as water is searched for longer.

FIG. 3A shows experimental results demonstrating that peripherally administered EPO pretreatment reduces the severity of stroke seizure and protects the mouse from convulsions and death by neurotoxin kainate. The number in parentheses below each column refers to the number of animals that received each kainate dose. Figure 3b shows that the protective effect of peripherally administered EPO is increased by EPO administered daily. 3C shows that the onset of action of EPO is delayed depending on the induction characteristics of the gene expression program.

4A and 4B show the protective effect of recombinant human EPO (rhEPO) against ischemic brain injury (local seizures). 4A shows that infarct size can be reduced by systemic administration of EPO at various times after induction of cerebral ischemia. Figure 4b compares two forms of EPO, recombinant human EPO (rhEPO) and 17 amino acid EPO derivatives (17-mers) in protecting the brain from injury in this model, with some EPO analogs effective in neuroprotection. Not.

5 shows the protective effect of rhEPO against blunt trauma delivered to the cerebral cortex.

6A and 6B show the protective effect of EPO from ischemic heart injury, FIG. 6A shows creatine kinase (CK) activity indicating damage to cardiomyocytes, and FIG. 6B shows myeloperoxidase (MPO) indicating inflammation Activity.

FIG. 7 depicts delaying and alleviating neurological syndromes produced by experimental allergic encephalitis, a model of multiple sclerosis by treating mice with EPO.

8A shows the minimum effective amount of EPO to provide a neuroprotective effect in a local seizure model performed in rats. 8B depicts serum concentrations of EPO at various time points after intraperitoneal administration of 5,000 U of rhEPO to female Balb / c mice.

9A depicts immunolocalization of EPO-R on capillaries and around capillaries. FIG. 9B shows that biotinylated EPO administered intraperitoneally in mice is identified in the surrounding capillaries directly surrounding the brain after 5 hours. 9C shows that after 17 hours, biotin labels can be identified in certain neurons.

The present invention provides compositions and methods for the use of erythropoietin (EPO) to modulate excitatory tissue functions such as, for example, enhancing cognitive function and protecting excitatory cells from toxic stimuli. In particular, the present invention provides compositions comprising EPO as well as methods of their use in prophylactic and therapeutic regimens comprising drug delivery. As used herein, excitatory tissue includes, but is not limited to, neurocytic tissue of the central and peripheral nervous system, and heart tissue.

The invention described herein provides excitatory tissue by peripherally administering EPO, or EPO receptor active molecules or molecules exhibiting EPO-activated receptor activity, as well as any molecule that mimics EPO activity by acting through other atypical EPO receptors. It provides a way to adjust the function. While not wishing to be bound by specific mechanisms of action, these molecules capable of delivering signals through the EPO receptor may, for example, signal transduction cascades that ultimately activate gene expression programs that result in protecting or enhancing excitatory tissue function. Start the cascade. Molecules capable of interacting with EPO receptors and modulating the activity of receptors, referred to herein as EPO or EPO receptor activity modulators, are useful in the context of the present invention for protecting or enhancing excitatory tissue function. These molecules may be, for example, naturally occurring, synthetic or recombinant forms of EPO molecules as described above, or any other except for modulating EPO receptor activity as described herein. It may be another molecule that does not necessarily mimic EPO in a way. These molecules can be used in combination for the various purposes described herein.

The compositions and methods described herein can be used to treat and / or protect or protect both normal or abnormal tissues such as, for example, neurons of the central nervous system, neurons of the peripheral nervous system, or heart tissue. In particular, paragraph 5.1 below describes EPO compositions useful in the practice of the present invention. Paragraph 5.2.1 describes the use of the EPO composition to enhance excitatory tissue functions such as learning, memory, and other aspects of cognitive function, and paragraph 5.2.2 describes how to protect excitable tissue from damage or damage. This is described. The discovery of EPO's unexpected ability for capillary endothelial cell fusion membranes in paragraph 5.2.3 provides a method of delivering compounds across the barrier membrane. Finally, paragraph 5.3 describes the symptoms that can be targeted using the method of the invention, and paragraph 5.4 describes the effective dosage and method of administration of the EPO composition.

5.1 Compositions Comprising Erythropoietin

EPO compositions suitable for use in the present invention include EPO-activation to modulate excitatory tissue (i.e. enhance the function of the excitable tissue, protect it from damage or injury, or deliver the compound to the excitable tissue) when administered peripherally. Any erythropoietin compound capable of activating the receptor is included. Erythropoietin is a glycoprotein hormone having a molecular weight of 34 to 38 kD in humans. The mature protein contains 166 amino acids and the sugar residues make up about 40% of the molecular weight. EPO forms useful in the practice of the present invention include the following molecules that are naturally produced, synthetic or recombinant: erythropoietin, erythropoietin analogues, erythropoietin analogues, erythropoietin fragments, hybrid erythroses Poietin molecules, erythropoietin receptor-binding molecules, erythropoietin agonists, renal erythropoietin, brain erythropoietin, oligomers and multimers thereof, muteins thereof, and the like thereof. The terms "erythropoietin" and "EPO" may be used alternately or continuously.

Synthesized recombinant molecules, such as brain EPO and kidney EPO, recombinant mammalian forms of EPO, as well as naturally occurring and tumor-inducing recombinant isomers, such as recombinantly expressed molecules and homologous recombination. Provided herein are molecules produced by. Moreover, the present invention encompasses recombinant constructs or other molecules comprising some or all of the structural and / or biological specifics of EPO, including fragments and multimers or fragments thereof, as well as peptides that bind to the EPO receptor. EPO herein includes molecules that have modified EPO receptor binding activity, particularly suitable for enhancing delivery ability to endothelial cell barrier membranes, preferably comprising increased receptor affinity. Included herein are muteins comprising molecules with additional or reduced numbers of glycosylation sites. As noted above, the terms "erythropoietin", "EPO" and "analog" as well as other terms are used interchangeably herein to permeate endothelial fusion membranes as well as excitatory tissue protective and enhancing molecules associated with EPO. As such, and refers to molecules that are useful as a means of delivery to other molecules. Moreover, this includes molecules produced by genetically modified animals. EPO molecules as included herein are structurally or with EPO except for the ability to interact with the EPO receptor or to modulate EPO receptor activity or to activate an EPO-activated signal transduction cascade as described herein. It should be appreciated that they do not necessarily have to be identical in any other way.

By way of non-limiting example, forms of EPO useful in the practice of the present invention include, for example, EPO muteins, such as compounds having modified amino acids at the carboxy terminus described in US Pat. Nos. 5,457,089 and 4,835,260; EPO isomers having varying numbers of sialic acid residues per molecule as described in US Pat. No. 5,856,292; Polypeptides described in US Pat. No. 4,703,008; Agents described in US Pat. No. 5,767,078; Peptides bound to the EPO receptor as described in US Pat. Nos. 5,773,569 and 5,830,851; Small molecule analogs that activate the EPO receptor as described in US Pat. No. 5,835,382; And the EPO analogs described in WO 9505465, WO 9718318, and WO 9818926. All of the above cited references are incorporated herein to the extent that this description refers to various alternating forms or methods for preparing the erythropoietin of the present invention in this form.

EPO is commercially available [PROCRIT® and Amgen, Inc., available from Ortho Biotech, Thousand Oaks, CA, USA. (EPOGEN; registered trademark)].

In another embodiment of the invention, EPO molecules included herein include hybrid EPO molecules that can be prepared to include not only EPO receptor modulating activity, but also other activity, such as growth hormone activity. Said hybrid molecule with multiple domains not only includes the ability to interact with the EPO receptor in this manner, but may also have the activity of another molecule, such as a hormone. Methods of making such molecules having two domains are known to those skilled in the art. As described in more detail in section 5.2.3 below, one property of the molecule is its ability to deliver to endothelial cell barrier membranes provided by the EPO receptor activity regulatory domain, and the activity of other molecules at the target site.

Any of the compounds described above may contain EPO compounds capable of modulating excitatory tissue (ie, enhancing the function of the excitable tissue, protecting it from damage or injury, or delivering the compound to the excitable tissue) using the assays described herein. Can be tested to confirm. For example, EPO compounds can be tested using the methods described in paragraph 5.2.1 to test excitation tissue functions such as memory, and other aspects of cognitive function. Examples of in vivo assays for cognitive function include the example of the Morris Water Maze test described in paragraph 6 and the conditional taste avoidance test described in detail in paragraph 7. In addition, the EPO compounds described above can be tested using the assays described in paragraph 5.2.2 to identify EPO compounds that can protect excitable tissues from damage or damage. The examples described in paragraphs 8, 9, 10, 11 and 12 provide specific examples of such assays. In addition, EPO compounds can be assayed for their ability to deliver compounds to endothelial fusion membranes, such as cerebrovascular membranes, using assays as described in paragraphs 5.2.3 and 9 below. In this way, EPO compositions suitable for use in the present invention, when administered peripherally, are signaled through EPO-activated receptors to modulate excitatory tissue (i.e. enhance function of excitable tissue and protect it from damage or damage or Or any compound capable of delivering the compound to excitatory tissue).

5.2 Methods of Prevention and Therapeutic Use of the Invention

In various embodiments of the present invention, the EPO composition can be used to protect excitatory tissue from damage or hypoxic stress, to enhance the function of excitable tissue, or to deliver compounds to the endothelial fusion membrane of excitable tissue. As described above, the present invention allows in part the EPO molecules to be delivered from the luminal surface of the endothelial cells of the capillaries of organs with endothelial cell fusion membranes including, for example, the brain, retina, and testes to the basal membrane surface. Is based on the discovery that Without wishing to be bound by any particular theory, after transcytosis of EPO, EPO can interact with EPO receptors on excitatory tissues, such as, for example, the central nervous system, peripheral nervous system, or neurons of heart tissue, and receptor binding is signaled. Transduction cascades can be initiated to activate gene expression programs in excitatory tissue and to protect cells from damage such as neurotoxins, hypoxia and the like. In this manner, methods of protecting excitatory tissue from damage or hypoxic stress, enhancing excitatory function and delivering the compound to the fused membrane of excitable tissue are described in more detail below.

5.2.1 How to improve excitatory tissue function

In one aspect, the invention directs a method of enhancing excitatory tissue function by administering an EPO molecule capable of activating a gene expression program that enhances excitatory tissue function. Improvements in excitatory tissue function provide for improvement in learning, associative learning, and memory. The above methods can be used to treat a variety of diseases and symptoms, which are also useful for improving cognitive function even in the absence of any symptoms or diseases. These uses of the present invention are described in more detail below and include the improvement of learning and practice in both humans and non-human mammals.

Symptoms and diseases treatable by the method of this aspect of the invention are neurons

Any symptom or disease that may be beneficial by improving sexual function. Examples of such diseases include, but are not limited to, disorders of the central nervous system including mood disorders, anxiety disorders, depression, autism, peripheral deficit hyperactivity disorders, and cognitive dysfunctions. Another non-limiting example of cognitive function that can be improved using the method of the invention is described in paragraph 5.3.

In one embodiment, for example, the EPO molecule can be administered to a subject or patient suffering from a disease exhibiting a loss of cognitive function, such as Alzheimer's disease.

The ability of EPO to enhance cognitive function can be tested in experimental animals using any of the methods described herein, or learning or cognitive function models permitted in other fields. As described in the examples presented in paragraphs 6 and 7, peripherally administered erythropoietin was found to enhance learning and cognitive function as described by several well-established learning models in normal experimental animals. . Examples of such learning models include the Morris Water Maze test given in paragraph 6, and the Conditional Taste Avoidance (CTA) test given in paragraph 7. In one embodiment, for example, the Conditional Taste Avoidance (CTA) test, a very sensitive and well-known standard test, is used to test the cognitive function of animals after EPO administration. CTA is used to test an animal's ability to learn in association with a disease with a new stimulus such as taste, which, when subsequently reexposed to the new stimulus, avoids the new taste. CTA includes the brain at various cortical and subcortical levels. Associations linking rising and falling information indicative of an aspect to avoid pain may be weakened or enhanced by changes affecting interconnected units. In the form of associative learning, CTA intensity can be determined by oral stimuli (eg, new stimuli cannot be a condition to be avoided), the degree of "disease" produced (toxicity), number of repetitions (practice), opposite desires where only a few are known (eg , Thirst). Although a wide variety of chemical and physical agents may exhibit CTA in a dose dependent manner, lithium chloride exhibits discomfort and loss of appetite. Like naturally occurring diseases, lithium produces CTA by stimulating the pathways described above, including cytokine release.

Enhancement of excitatory cell function, such as cognitive function, provides the individual with a variety of benefits in an educational and working environment and improves the ability to practice and educate non-human mammals.

5.2.2 How to protect excitable tissue from damage

In another aspect, the present invention relates to a method of protecting a mammal from a pathological phenomenon indicating damage to excitatory tissue. Protection is provided by administering to the mammal by the peripheral route an amount of erythropoietin that is effective to protect excitatory tissue from damage. As set out in detail in the Examples in paragraph 8 below, EPO administered prior to the cinate toxin is markedly neuroprotective in mice, causing onset and preventing death. The neuroprotective effect of EPO is large and persistent. The positive effects shown herein occur over a very short time compared to administration of EPO, indicating the course of increasing erythrocyte volume fractions as a result of the erythropoietin activity of EPO. Moreover, as noted above, embodiments of the present invention include EPO lacking the ability to increase erythrocyte volume fraction.

In one aspect, the invention can be advantageously used to acute and chronic prevention and treatment of neurological diseases and to improve the cognitive function of normal or dead brains as described herein. As noted above, damage and necrosis of neurons in the central nervous system often results in severe and fatal incidences with high levels of morbidity and mortality in the population. Acute neurological damage may occur during or as a result of development, convulsions, epilepsy, seizures, bleeding, central nervous system damage, hypoxia, hypoglycemia, hypotension and brain or spinal trauma. The present invention provides acute administration for the treatment of acute cases.

In one aspect, for example, the present invention can be used to protect a mammal from damage resulting from irradiation damage to the brain.

In another embodiment, the serious symptoms treatable or protective according to the present invention include prenatal hypoxic symptoms in the uterus, postpartum treatment to protect the brain from hypoxia damage that persists in childbirth, as well as choking, confusion, and the central nervous system. Prevention and treatment of other symptoms at risk of neurotoxin damage as a result of exposure to oxygen deficiency or other neurotoxin stimulation. As is known, individuals suffering from hypoxia, or nonfatal hypoxia events or accidents during delivery, can suffer from neurological deficits over their lifetime. In addition, hypoxia and / or disruption of cerebrovascular flow, which may occur after trauma or during surgical operations, are accompanied by the risk of developing neurological deficits over life.

Postoperative cognitive dysfunction, including defects that appear after using cardiopulmonary system, is also treatable by the methods provided herein. Moreover, the present invention can be applied to the treatment of hypoxia which may be manifested by carbon monoxide poisoning or smoke inhalation.

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

These are examples of damage to excitable tissues treatable in accordance with the present invention. Rapid early treatment of these diseases can be performed by mobile emergency medical health professionals to begin treatment as soon as the potential for neurological damage is suspected. The risk of neurological damage induced by delivery can be reduced by prophylactic treatment of the fetus before or during delivery. These and other facilities and environments will be appreciated by those skilled in the art.

5.2.3 Methods of Delivery of Compounds

The present invention further relates to a method for facilitating delivery of molecules across barriers to endothelial cells in stray animals by administering a composition comprising a particular molecule that binds erythropoietin. As mentioned above, the inventors of the present invention have discovered the unexpected and surprising activity of EPO administered peripherally to excitable tissues such as the nervous or cardiac tissues in the central nervous system, peripheral nervous system, and excitatory tissues such as the blood-brain barrier. EPO was identified as a molecule capable of crossing the closed membrane of. As such, EPO is useful as a carrier for delivering other molecules across the blood-brain barrier and other similar barriers.

In one embodiment, EPO receptor binding molecules, including molecules bound to EPO molecules, can be used to deliver these molecules across the blood-brain barrier. Such molecules may be transported piggyback on EPO for delivery across BBB. In another embodiment, the antibody or other binding partner to the molecule can be bound to an EPO or an EPO receptor activity modulator, thereby binding to a molecule delivered by non-covalent association with the binding partner, and further binding to a deliverable EPO molecule. do. In other embodiments, EPO receptor binding molecules comprising antibodies against the EPO receptor are useful for use in the methods described above. Such antibodies may be used in a manner similar to the approach used for the transferrin receptor to cross the blood brain (Pardridge et al., 1991, Selective transport of an antitransferrin receptor antibody through the blood-brain barrier in vivo. J. Pharmacol. Exp. Therap. 27: 66), to provide delivery carriers that other molecules can piggyback.

Those skilled in the art will know a variety of ways to bind EPO and other agents and molecules described above by covalent, non-covalent and other methods. In addition, evaluation of the efficiency of the composition can be easily determined experimentally. Bonding of molecules with EPO and analogs can be accomplished by a number of methods including labile covalent bonds, crosslinks and the like. In one embodiment, for example, the bond between the molecule delivered through the barrier and erythropoietin may be an unstable covalent bond, in which case the molecule is liberated from the bond with EPO after crossing the barrier. In one embodiment, biotin / avidin interaction can be used. In other embodiments, as described above, the hybrid molecule can be prepared by a combination or synthetic method comprising, for example, both a region of a molecule having a desired pharmacological activity and a region capable of modulating EPO receptor activity.

Molecules can be bound to EPO or EPO receptor activity modulators through multifunctional molecules, ie multifunctional crosslinkers. The term "multifunctional molecule" as used herein includes molecules having one or more reactors as well as one functional group capable of reacting one or more times in succession, such as formaldehyde. As used herein, the term "reactor" refers to a cross-linking agent and a molecule between the molecules in response to a functional group on a molecule (e.g., peptides, proteins, carbohydrates, nucleic acids, in particular hormones, antibiotics or anticancer agents delivered across endothelial cell barriers). Refers to a functional group on a crosslinking agent that forms a covalent bond. The term "functional group" refers to the conventional meaning used in organic chemistry. Multifunctional molecules that can be used are preferably biocompatible crosslinkers, which are non-carcinogenic, nontoxic and substantially non-immunogenic crosslinkers in vivo. Multifunctional crosslinking agents, such as crosslinking agents known in the art and described herein, can be readily tested in animal models to determine their biocompatibility. Multifunctional molecules are preferably bifunctional. As used herein, the term “bifunctional molecule” refers to a molecule having two reactors. Bifunctional molecules may be heterodifunctional or homobifunctional. Heterobifunctional crosslinkers allow for vector bonding. In order for the crosslinker reaction to take place in an aqueous solution, such as an aqueous solution buffered at pH 6-8, the multifunctional molecules are sufficiently soluble in water in order to remain soluble in water for more effective bio-distribution. Particularly preferred. Typically, multifunctional molecules are covalently bonded to amino or sulfhydryl functional groups. However, multifunctional molecules that react with other functional groups, such as carboxylic acid or hydroxyl groups, are contemplated in the present invention.

Homofunctional molecules have the same two or more functional groups. Reactive functional groups on the homobifunctional molecule include, for example, an aldehyde group and an active ester group. Homofunctional molecules having an aldehyde group include, for example, glutaaldehyde and suvaraldehyde. The use of glutaaldehyde as a crosslinking agent is disclosed in Poznansky et al., Science 223, 1304-1306 (1984). Homofunctional molecules having two or more active ester units include esters of dicarboxylic acids and N-hydroxysuccinimides. Some examples of such N-succinimidyl esters are dissuccinimidyl suverate and dithio-bis- (succinimidyl propionate), and their soluble bis-sulfonic acid and bis-sulfonate salts, for example Sodium salts and potassium salts. Such homobiogenic agents are commercially available from Pierce, Rockford, Ill.

Heterodifunctional molecules have two or more reactors. The reactor reacts with different functional groups present in, for example, EPO and molecules. The two different functional groups which react with the reactor on the heterodifunctional crosslinker are typically amino groups, for example epsilon amino groups of lysine; Sulfhydryl groups such as thiol groups of cysteine; Carboxylates on carboxylic acids such as aspartic acid; Or a hydroxyl group, for example a hydroxyl group on serine.

If the reactive group of the heterodifunctional molecule forms a covalent bond with an amino group, the covalent bond will generally be an amido or imido bond. Reactive groups that form covalent bonds with amino groups can be, for example, activated carboxylate groups, halocarbonyl groups or ester groups. Preferred halocarbonyl groups are chlorocarbonyl groups. The ester group is preferably a reactive ester group, for example an N-hydroxy-succinimide ester group.

Other functional groups are typically thiol groups, groups that can be converted to thiol groups, or groups that form covalent bonds with thiol groups. The covalent bond will generally be a thioether bond or disulfide. Reactive groups that form covalent bonds with thiol groups can be, for example, double bonds that react with thiol groups or activated disulfides. Reactive groups containing double bonds capable of reacting with thiol groups are maleimido groups, but other reactive groups such as acrylonitrile are also possible. The reactive disulfide group can be, for example, a 2-pyridyldithio group or a 5,5'-dithio-bis- (2-nitrobenzoic acid) group. Some examples of heterodifunctional reagents containing reactive disulfide bonds include N-succinimidyl 3- (2-pyridyldithio) propionate (Carlsson et al ., 1978, Biochem J., 173: 723- 737]), sodium 5-4-succinimidyloxycarbonyl-α-methylbenzylthiosulfate and 4-succinimidyloxycarbonyl-α-methyl (2-pyridyldithio) toluene. Preference is given to N-succinimidyl 3- (2-pyridyldithio) propionate. Some examples of heterodifunctional reagents comprising reactive groups with double bonds reacting with thiol groups include succinimidyl 4- (N-maleimidomethyl) cyclohexane-1-carboxylate and succinimidyl m-maleimidobenzoate. Can be mentioned.

Other heterodifunctional molecules include succinimidyl 3- (maleimido) propionate, sulfosuccinimidyl 4- (p-maleimido-phenyl) butyrate, sulfosuccinimidyl 4- (N-maleimidomethyl-cyclo Hexane) -1-carboxylate and maleimidobenzoyl-N-hydroxy-succinimide ester are mentioned. The sodium sulfonate salt of succinimidyl m-maleimidobenzoate is preferred. Many of the aforementioned heterodifunctional reagents and sulfonate salts thereof are commercially available from Pierce.

Whether the conjugate needs to be reversible or unstable can be readily determined by the skilled person. Conjugates can be tested in vitro for both EPO uptake activity modulating activity and preferred pharmacological activity. If the conjugate possesses both properties, its suitability can then be tested in vivo. If conjugated molecules need to be separated from EPO for activity, unstable binding or reversible association with EPO is preferred. Instability properties may also be tested using standard in vitro methods prior to in vivo testing.

Additional information regarding the preparation and use of these and other multifunctional reagents can be obtained from the following publications, or other publications accessible in the art: Carlsson et al ., 1978, Biochem. J. 173: 723-737; Cumber et al ., 1985, Methods in Enzymology 112: 207-224; Jue et al ., 1978, Biochem. 17: 5399-5405; Sun et al ., 1974, Biochem. 13: 2334-2340; Blattler et al ., 1985, Biochem. 24: 1517-152; Liu et al ., 1979, Biochem. 18: 690-697; Youle and Neville, 1980, Proc, Natl. Acad. Sci. USA 77: 5483-5486; Lerner et al ., 1981, Proc. Natl. Acad. Sci. USA 78: 3403-3407; Jung and Moroi, 1983, Biochem. Biophys. Acta 761-162; Caulfield et al ., 1984, Biochem. 81: 7772-7776; Staros, JV, 1982, Biochem. 21: 3950-3955; Yoshitake et al ., 1979, Eur. J. Biochem. 101: 395-399; Yoshitake et al ., 1982, J. Biochem. 92: 1413-1424; Pilch and Czech, 1979, J. Biol. Chem. 254: 3375-3381; Novick et al., 1987, J. Biol. Chem. 262: 8483-8487; Lomant and Fairbanks, 1976, J. Mol. Biol. 104: 243-261; Hamada and Tsuruo, 1987, Anal. Biochem. 160: 483-488; And Hashida, 1984, J. Applied Biochem. 6: 56-63. Crosslinking methods are also described in Means and Feeney, 1990, Bioconjugate Chem. 1: 2-12. Barriers crossed by the methods and compositions of the present invention include, but are not limited to, blood-brain barrier, blood-ocular barrier, blood-testis barrier, blood-ovary barrier and blood-placental barrier.

Candidate molecules for delivery to the endothelial cell barrier include, for example, antibiotics or antifungal agents, peptide radiation drugs, antisense drugs, which are commonly excluded from hormones such as growth hormone, neurotropic factors, the brain and other barrier organs. , Antibodies against biologically active agents, drugs, and anticancer agents. Restrictive examples of such molecules include growth hormone, nerve growth factor (NGF), brain-derived neurotropic factor (BNF), ciliary neurotropic factor (CTF), primary fibroblast growth factor (bFGF), and conversion growth factor β1 (TGFβ1). , Converting growth factor β2 (TGFβ2), converting growth factor β3 (TGFβ3), interleukin 1, interleukin 2, interleukin 3 and interleukin 6, AZT, antibodies to tumor necrosis factor, and immunosuppressive agents (eg cyclosporin).

In another embodiment, recombinant chimeric toxin molecules comprising EPO can be used for therapeutic delivery of toxins to treat viral diseases or proliferative diseases such as cancer. Compounds that can be fused to EPO to constitute a suitable chimeric toxin for this embodiment include, but are not limited to, toxins such as Pseudomonas exotoxin, diphtheria toxin and lysine in particular.

5.3 Target Disease

As noted above, the EPO compositions and methods of use provided herein provide for hypoxic diseases that adversely affect excitatory tissues such as, for example, central nervous system tissues such as brain, heart and retina, peripheral nervous system tissues, or excitable tissues in cardiac tissues. It can be used to treat and prevent the resulting disease. Thus, the present invention can be used to treat or prevent excitatory tissue damage caused by hypoxia disease in a variety of diseases and situations. Non-limiting examples of such diseases and situations are provided below.

In the examples of protecting neurological lesions treatable in accordance with the present invention, such lesions include those due to reduced oxygenation of nerve tissue. Any disease that reduces the availability of oxygen to nerve tissue resulting in stress, injury and finally neuronal death can be treated by the methods of the present invention. These diseases, commonly referred to as hypoxia and / or ischemia, include stroke, vascular occlusion, prenatal or postpartum oxygen deficiency, choking, airway obstruction, near drowning, carbon monoxide poisoning, smoke inhalation, trauma (surgical and radiotherapy) , Housekeeping, epilepsy, hypoglycemia, chronic obstructive pulmonary disease, emphysema, adult respiratory distress syndrome, hypotension shock, septic shock, anaphylactic shock, insulin shock, sickle cell development, cardiac arrest, arrhythmia, and nitrogen anesthesia Or including, but not limited to.

In one embodiment, EPO can be administered to prevent injury or tissue damage due to injury or tissue damage risk during surgical procedures such as, for example, tumor resection or aneurysm repair.

Other lesions caused or caused by hypoglycemia that can be treated by the methods described herein include insulin overdose (also called pseudohuman hyperinsulinemia), insulinoma, growth hormone deficiency, cortisol hypoplasia, drug overdose. Dosing and specific tumors.

Other lesions due to damage to excitable nerve tissue include paroxysmal disorders such as epilepsy, convulsions or chronic paroxysmal disorders. Other treatable diseases and disorders include stroke, multiple sclerosis, hypotension, cardiac arrest, Alzheimer's disease, Parkinson's disease, cerebral palsy, brain or spinal cord trauma, AIDS dementia, age-related cognitive loss, memory loss, amyotrophic lateral sclerosis, Paroxysmal disorders, alcoholism, retinal ischemia, optic nerve damage due to glaucoma, and nerve loss.

The method of the invention can be used to treat diseases and damage of retinal tissue. Such diseases include, but are not limited to macular degeneration, retinal detachment, retinal pigmentation, atherosclerotic retinopathy, hypertensive retinopathy, retinal artery blockage, retinal vein blockage, hypotension, and diabetic retinopathy.

In another aspect, the methods of the present invention may be used to protect or treat injuries due to radiation damage to excitatory tissue.

Further usefulness of the methods of the present invention lies in the treatment of neurotoxin poisoning, for example Domosan shellfish poisoning, neurorolathyrism and Guam disease, amyotrophic lateral sclerosis, and Parkinson's disease.

As mentioned above, the present invention also relates to a method of enhancing excitatory tissue function in a mammal by peripheral administration of erythropoietin. Various diseases and disorders can be easily treated using this method, and this method is also useful for improving cognitive function without any disease or condition. This use of the invention is described in more detail below and includes the improvement of learning and training functions in both human and non-human mammals.

Diseases and disorders treatable by the methods of this aspect of the invention with respect to the central nervous system include, but are not limited to, mood disorders, anxiety disorders, depression, autism, attention deficit hyperactivity disorder, and cognitive dysfunction. These diseases are treated with an improvement in nerve function.

Other disorders treatable in accordance with the teachings of the present invention include sleep disorders, such as sleep apnea and travel related disorders, subarachnoid and aneurysm bleeding, hypotension shock, agitation, septic shock, aniphylactic shock, and various Encephalitis and meningitis, such as sequelae of connective tissue disease-associated encephalitis (eg lupus). Other uses include neurotoxin poisoning, such as Domosan shellfish poisoning, neuralitis and Guam disease, amyotrophic lateral sclerosis, and Parkinson's disease; Postoperative treatment of embolic or ischemic injury; Whole brain investigation; Sickle cell development; And prevention of or protection from eclampsia.

Additional groups of diseases treatable by the methods of the present invention include hereditary or acquired mitochondrial dysfunction caused by various neurological diseases represented by nerve damage and death. For example, Leigh disease (subacute necrotic encephalopathy) is characterized by progressive blindness and encephalopathy, and myopathy due to neuronal dropout. In these cases, defective mitochondrial metabolism does not provide enough high energy substrate to accelerate the metabolism of excitable cells. EPO receptor activity modulators optimize dysfunction in various mitochondrial diseases.

As mentioned above, hypoxic diseases adversely affect excitatory tissue. Excited tissues include, but are not limited to, central nervous system tissue, peripheral nervous system tissue, and heart tissue. In addition to the diseases described above, the methods of the present invention are useful for the treatment of inhalation poisoning such as carbon monoxide and smoke inhalation, severe asthma, adult respiratory distress syndrome, airway obstruction and myopathic death. Additional diseases that cause hypoxic disease or cause excitatory tissue damage by other means include hypoglycemia, or insulin-producing neoplasm (insulin species), which may occur upon improper administration of insulin.

Various neuropsychological disorders that are believed to result from excitatory tissue damage are treatable by the methods of the present invention. Chronic disorders that may be accompanied by nerve damage and to which treatment according to the invention may be applied include disorders relating to the central and / or peripheral nervous system, such as age-related cognitive loss and senile dementia, chronic paroxysmal disorders, Alzheimer's disease, Parkinson's disease, dementia, memory loss, amyotrophic lateral sclerosis, multiple sclerosis, nodular sclerosis, Wilson's disease, cerebral palsy and progressive nuclear palsy, Guam's disease, Lewy body dementia, prion disease, such as spongy encephalopathy (e.g. Creutzfeldt-Jakob disease Seizure disorders such as Gilles de la Tourette syndrome, epilepsy as well as Huntington's disease, myotonic dystrophy, Friedreich's ataxia and other ataxia, epileptic disorders, stroke, brain or spinal cord trauma, AIDS Sexual dementia, alcoholism, autism, retinal ischemia, glaucoma, autonomic dysfunction (e.g. hypertension and sleep disorders), and neuropsychiatric disorders (e.g. Schizophrenia, schizophrenia affect disorder, attention deficit disorder, mood disorder, major depressive disorder, mania, obsessive compulsive disorder, psychoactive substance use disorder, anxiety, panic disorder, and unipolar and bipolar affect disorder ) Is included. Additional neuropsychiatric and neurodegenerative disorders include, for example, those listed in the Diagnostic and Statistical Manual of Mental Disorders (DSM) of the American Psychiatric Association. The most recent version of this is incorporated herein by reference in its entirety.

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

5.4 Pharmaceutical Formulations and Administration

According to the present invention, EPO, analogs thereof, analogs, erythropoietin segments, hybrid erythropoietin molecules, erythropoietin receptor binding molecules, erythropoietin agonists, kidney erythropoietin, brain erythropoietin and muteins thereof, And their equivalents may be injected parenterally, transmucosally, for example orally, nasal, rectal, intravaginal, sublingual, submucosal or transdermal. Preferably administration is via parenteral, eg intravenous or intraperitoneal injection, and also includes, but is not limited to, intraarterial, intramuscular, intradermal and subcutaneous administration.

Subjects whose peripheral administration of EPO is an effective therapeutic regime are preferably humans, but may be any animal, preferably a mammal. Thus, as will be readily apparent to one of ordinary skill in the art, the methods and pharmaceutical compositions of the present invention are suitable for administration to any animal, particularly a mammal, such animals include livestock (e.g. cats and canines). ), Farm animals (e.g., but not limited to cattle, horses, goats, sheep and pigs), wild animals (whether in the wild or in the zoo), research animals (e.g. mice, mice, Rabbits, goats, sheep, pigs, dogs, cats, and the like). As mentioned above, domesticated animals, including pets and working animals, are candidates for both neuroprotective benefits and cognitive enhancement of the present invention. Acute and chronic disorders, including epilepsy as well as neurological damage due to hypoxia, are common in these animals and are therefore the subject of treatment. In addition, as described above, improving cognitive function in non-human animals is an advantage of the present invention in that retention of learning, training and learning behavior can be enhanced, augmented and maintained using the teachings of the present invention. This reduces the spending and psychological burden of the pet owner. For example, the time required to train dogs and other livestock is reduced. In addition, wild animals, which are typically difficult to train, may also be good candidates for training by the methods of the present invention.

5.4.1 Formulations and Effective Dosages

The invention also relates to pharmaceutical compositions. Pharmaceutical compositions comprising EPO and EPO receptor activity modulators may be administered to a patient in therapeutically effective dosages to protect excitatory tissue from damage, enhance the function of excitable tissue, or deliver the compound to excitatory tissue. Applicants have found that high doses of EPO are desirable for controlling excitatory tissue and protecting excitatory tissue damage.

The choice of the desired effect dosage will be determined by the skilled person by considering a number of factors known to those skilled in the art. These factors include certain forms of erythropoietin and their pharmacokinetic parameters such as bioavailability, metabolism, half-life, etc., which are common development processes typically used to obtain legal approval for pharmaceutical compounds. Is established. In addition, factors in consideration of dosage include the disease or condition to be treated or the benefits that can be achieved for a normal person, the body weight of the patient, the route of administration, whether the administration is to be carried out for a short time or for a long time, the agents used and the administration And other factors known to affect the effectiveness of a given drug. Therefore, the precise dosage should be determined according to standard clinical techniques, depending on the judgment of the medical staff and the situation of each patient, for example the condition and immune status of the individual patient.

In one embodiment, the present invention is modified to be suitable for the regulation of sensitive tissues, enhancement of cognitive function, or migration through endothelial obstructive membrane, wherein about 50,000 to 500,000 units, 60,000 to 500,000 units, 70,000 to 500,000 units, 80,000 to 80,000 per unit dose Nontoxic effects of 500,000 units, 90,000 to 500,000 units, 100,000 to 500,000 units, 150,000 to 500,000 units, 200,000 to 500,000 units, 250,000 to 500,000 units, 300,000 to 500,000 units, 350,000 to 500,000 units, 400,000 to 500,000 units, or 450,000 to 500,000 units A pharmaceutical composition is provided in dosage unit form comprising an amount of an EPO, an EPO receptor activity modulator or an EPO-activated receptor modulator, and a pharmaceutically acceptable carrier thereof.

In one embodiment, the pharmaceutical composition of such EPO can be administered systemically to prevent damage to sensitive tissues, to improve the function of sensitive tissues, or to transfer compounds to sensitive tissues. Such administration may be parenterally, mucosa, for example oral, nasal, rectal, vaginal, sublingual, submucosal or endothelial. Preferred administration is parenteral administration, for example intravenous injection or intraperitoneal injection, including but not limited to intraarterial, intramuscular, endothelial and subcutaneous administration.

In a preferred embodiment, the EPO is administered systemically at a dosage of 2000 to 10000 units / weight (kg), preferably about 2000 to 5000 units / weight (kg), most preferably 5000 units / weight (kg) per dose. It may also be administered. This effect dose should be sufficient to achieve excess serum levels above the serum amount of about 10,000, 15,000, or 20,000 milliunits / ml following administration of EPO. Such serum levels may be achieved about 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 hours after administration. Such administration may be repeated as necessary. For example, administration may be repeated daily if clinically necessary, or after a suitable time interval, for example every 1-12 weeks, preferably every 3-8 weeks. In one embodiment, the effective amount of EPO and their pharmaceutically acceptable carriers may be packaged in a single dose vial or other container. In one embodiment, EPO may not promote red blood cell production. That is, it may exert the aforementioned activity but may not cause an increase in the concentration of hemoglobin or hematocrit. In another embodiment, the EPO is provided at a higher dosage than is needed to maximally stimulate erythrocyte production.

The pharmaceutical composition of the invention may comprise a therapeutically effective amount of a compound and a pharmaceutically acceptable carrier thereof. In specific embodiments, the term "pharmaceutically acceptable" has been approved by a federal legal representative or has been described in the US Pharmacopoeia or other generally approved pharmacopoeia for use in animals and more specifically in humans. Means. The term "carrier" means a diluent, yazuvant, excipient, or vehicle with which the therapeutic drug is administered. Such pharmaceutical carriers may be sterile liquids such as saline solutions in oils and water, including petroleum based, animal based, vegetable or synthetic oils such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Saline solutions are preferred carriers when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions may specifically be used as liquid carriers in 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 scheme milk, glycerol, propylene, Glycol, water, ethanol and the like. If desired, the composition may contain small amounts of wetting or emulsifying agents or pH buffers. Such compositions may be in the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained release preparations and the like. The composition may be formulated as a suppository 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 salts formed with free amino groups such as those derived from hydrochloric acid, phosphoric acid, acetic acid, oxalic acid, tartaric acid, and the like, and sodium, potassium, ammonium, calcium, ferrous hydroxide, isopropylamine, triethylamine And salts formed with free carboxyl groups such as those derived from 2-ethylamino ethanol, histidine, procaine and the like. Examples of suitable pharmaceutical carriers are described in E. W. Martin, "Remington's Pharmaceutical Science". Such compositions will contain a therapeutically effective amount of the compound, preferably in tablet form, with an appropriate amount of carrier so as to provide the form for proper administration to the patient. The formulation should be suitable for the mode of administration.

Pharmaceutical compositions adapted for oral administration include capsules or tablets; Powder or granules; Solutions, syrups or suspensions (aqueous or non-aqueous solutions); Edible foams or whips; Or as an emulsion. Tablets or hard gelatin capsules may include lactose, starch or derivatives thereof, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, stearic acid or salts thereof. Soft gelatin capsules may include vegetable oils, waxes, fats, semisolid or liquid polyols, and the like. Solutions and syrups may include water, polyols and sugars.

Active agents intended for oral administration may be coated or mixed with materials that retard the disintegration and / or absorption of the active agent in the gastrointestinal tract (for example, glyceryl monostearate or glyceryl distearate may be used). . Thus, sustained release of the active agent may be maintained for several hours or, if necessary, may be avoided from degrading the active agent in the stomach. Pharmaceutical compositions for oral administration may be formulated to facilitate release of the active agent at specific locations in the gastrointestinal tract due to certain pH or enzymatic conditions.

Pharmaceutical compositions adapted for transdermal administration may be provided as individual patches that allow for intimate contact with the epidermis of the recipient for extended periods of time. Pharmaceutical compositions adapted for topical administration may be provided as ointments, creams, suspensions, lotions, powders, solutions, pastes, gels, sprays, aerosols or oils. For topical administration to the skin, mouth, eyes or other external tissues, topical ointments or creams are preferably used. When formulated in an ointment, the active ingredient may be formulated with a paraffin or water-miscible ointment base. Optionally, the active ingredient may be formulated as a cream with an oil-in-water based or water-in-oil based. Pharmaceutical compositions adapted for topical administration to the eye include eye drops. In such compositions the active ingredient may be dissolved or suspended in a suitable carrier such as a solvent. Pharmaceutical compositions adapted for topical administration in the oral cavity include lozenges, pastilles and gargles.

Pharmaceutical compositions adapted for nasal administration may comprise a solid carrier (preferably between 20 and 500 μm in diameter) such as powder. The powder may be administered in a nasal manner, i.e., by rapid inhalation through the nose from a powder container lying close to the nose. Optionally, the composition adapted for nasal administration may comprise a liquid carrier such as a nasal spray or nasal drops. Such compositions may comprise aqueous or oil solutions of the active ingredient. Compositions for administration by inhalation may also be supplied to specifically described devices, including but not limited to pressurized aerosols, nebulizers or blowers, which may be designed to provide a predetermined dosage of the active ingredient. In a preferred embodiment, the pharmaceutical composition of the present invention is administered to the lungs through the nasal cavity.

Pharmaceutical compositions adapted for rectal administration may be provided as suppositories or enemas. Pharmaceutical compositions for administration via the vagina may also be provided as a suppository, tampon, cream, gel, paste, foam or spray formulation.

Pharmaceutical compositions adapted for parenteral administration include aqueous and non-aqueous sterile injectable solutions, which may contain antioxidants, buffers, disinfectants and solutes that substantially keep the composition isotonic with the intended recipient's blood, or Suspensions. Other components that may be present in such compositions are, for example, water, alcohols, polyols, glycerin and vegetable oils. Compositions modified for parenteral administration may be present in single or multiple dose containers such as, for example, sealed ampoules and vials, and may only be added with a sterile liquid carrier (eg, sterilized for injection immediately before use). Only the addition of saline solution) may be stored in the required lyophilized state. Pregnancy injection solutions and suspensions may be prepared from sterile powders, granules, and tablets.

In a preferred embodiment, the composition can be formulated according to conventional methods as pharmaceutical compositions adapted for intravenous administration in humans. Typically, compositions for intravenous administration are solutions in sterile isotonic aqueous buffer. If desired, the composition may also include solubilizers and local anesthetics such as lidocaine to relieve pain at the injection site. Generally, the components are supplied in unit dosage form, for example as a dehydrated concentrate in a hermetically sealed container such as a dried lyophilized powder or an ampoule or a chasette representing a quantity of active agent. When the composition is administered by injection, it may be formulated into an injectable bottle containing sterile pharmaceutical water or saline. When the composition is administered by injection, ampoules of sterile water for injection or saline may be provided such that the ingredients are mixed before administration.

Suppositories generally contain 0.5% to 10% by weight of active ingredient, and oral preparations preferably contain 10 to 95% by weight of active ingredient.

The invention also provides a pharmaceutical pack or kit comprising one or more containers filled with one or more ingredients of the pharmaceutical compositions of the invention. Notice may be associated with such containers, optionally reflecting the approval of the manufacture, use, or sale of a human administered article in a form prescribed by a government agency involved in the manufacture, use, or sale of a pharmaceutical or biological product. .

5.4.2 Administration Method

The present invention provides compositions and methods for peripheral administration of EPO to improve or protect the action of sensitive tissues and to transfer the compounds to such tissues. As noted above, the present invention is based, in part, on the discovery that peripherally administered EPO exhibits direct neuroprotective or neuroenhancing properties in sensitive tissues such as central nervous system, peripheral nervous system or heart tissue. As used herein, susceptible tissues include, but are not limited to, nervous tissues and heart breakfast of the central and peripheral nervous systems. This section will describe these compounds and methods for their administration.

The present invention administers EPO and EPO receptor activity modulators by routes of administration other than direct administration to the central nervous system, and the terms "peripheral" and "systemic" encompass these various routes. Peripheral administration includes oral or parenteral administration, for example intravenous, intraarterial, subcutaneous, intramuscular, intraperitoneal, rectal, submucosal or transdermal administration. Other routes are also useful for the administration of the agents described herein. Short and long term administration is provided herein.

In one embodiment, for example, the EPO can be delivered to a controlled release system. For example, the polypeptide can be administered using intravenous runners, implantable osmotic pumps, transdermal patches, liposomes or other modes of administration. In other embodiments, pumps may be used (Langer, supra; Sefton, 1987, CRC Crit. Ref. Boimed. End. 14: 201; Buchwald et al., 1980, Surgery 88: 507; Saudek et al., 1989, N. Engl. J. Med. 321: 574). In other embodiments, the compound may be transferred to vesicles, specifically liposomes (Langer, Science 249: 1527-1533 (1990); Treat et al., In Liposomes in the Therapy of Infectious Disease and Cancer, Lopez-Berestein) and Fidler (eds.), Liss, New York, pp. 353-365 (1989); WO 91/04014; US Pat. No. 4,704,355; Lopez-Berestein, ibid., pp. 317-327; see generally ibid.] Reference). In other embodiments, polymeric materials can be used (Medical Applications of Controlled Release, Langer and Wise (eds), CRC Press: Boca Raton, Florida, 1974; Controlled Drug Bioavailability, Drug Product Design and Performance, Smolen and Ball ( eds.), Wiley: New York (1984); Ranger and Peppas, J. Macromol. Sci. Rev. Macromol. Chem. 23:61, 1953; Levy et al., 1985, Science 228: 190; During et al. , 1989, Ann. Neurol. 25: 351; Howard et al., 1989, J. Neurosurg. 71: 105).

In other embodiments, the controlled release system may require only a portion of the systemic dose located in the vicinity of the therapeutic target, ie in the brain (Goodson, pp. 115-138 in Medical Applications of Controlled Release, vol. 2, supra, 1984). Other controlled release systems are discussed in the review of Langer (see 1990, Science 249: 1527-1533).

In other embodiments, when properly formulated, EPO may be administered nasal, oral, rectal, vaginal or sublingual.

In specific embodiments, it may be desirable to administer the EPO composition of the invention locally to the area in need of treatment, which is not limited thereto, by injection, by means of a catheter, by means of suppositories, or by implantation. Post-operative topical injection, topical, with post-operative wound dressing by means of water (where the implant may be a porous, nonporous or gelatinous material and may include a membrane or fiber such as a sialic membrane) It may also be achieved by administration.

The invention may be better understood by reference to the following non-limiting examples, which are presented by way of example. The following examples are presented to more fully illustrate preferred embodiments of the present invention. However, the following examples should not be construed as limiting the broad scope of the invention.

As described below, the studies conducted by the inventors are universally acceptable standard tests for this model on which the prophylactic and therapeutic benefits are based.

6. Example 1: Peripherally administered EPO enhances cognitive function

In this example, a spatial navigation experiment known as the Morris Water Maze test is performed to demonstrate that the cognitive function of the mouse is enhanced by EPO. In this test, a small transparent platform is placed in a quarter section of a pool with opaque water. The mice placed in the pool must swim until they reach a resting platform beneath the surface invisible to the swimming mouse itself. This test consists of measuring the time it takes for the animal to reach the platform (ie, the time it takes for the animal to swim). In successive attempts, the time it takes for each mouse to reach the platform will be reduced according to the animal's ability to learn the platform's location. This type of learning experiment includes the hippocampus, since the learning in the test is inhibited if the hippocampus is damaged.

Experiments were performed in a circular black pool 150 cm in diameter. Four directions, namely, east, west, north and north, were arbitrarily designated. Distinct visual cues, such as flashlights, square-applied bright tapes, etc., were applied to each of the four zones to direct the mice in a direction in the pool. The platform was optionally positioned in quarter zone. This test consists of locating and releasing the animal in the quarter section of the cage to start the head. The test time was 90 seconds in total. If the animal did not reach the platform, it was placed on the platform for an additional 15 seconds. The animals were allowed to rest for 1 hour and then placed in another quarter zone for testing. All four quarter sections were used over the course of the 1 day test and the animals were tested for 12 consecutive days (ie a total of 48 trials).

The experiment was carried out with 5000U of recombinant human EPO (available under the trade name PROCRIT from Ortho-Biotech, Inc.) to each mouse 4 hours before the start of the day of the test, each day for a 12-day test period. / Kg intraperitoneal injection. Control animals were most injected with saline.

Learning was measured by measuring the time each mouse spent on the platform. As shown in FIG. 1A, the experimental results are plotted as time spent on the platform by the EPO treated and most treated groups. As the results indicate, both groups of animals spent more and more time on the platform. That is, both animal groups learned how to reach the platform more quickly in daily successive tests, but EPO treated animals learned more quickly than the most treated groups. Thus, EPO treated animals have a “learning curve” much faster than the most treated group. The results are expressed as the difference between the EPO treated group and the most processed group, and when comparing the results of the EPO treated group and the most processed group, the regression line (R ² = 0.88) has a slope that is significantly different from slope 1 (0.68). Which is very advantageous for EPO treated groups (FIG. 1B).

Example 2 Peripherally Administered EPO Enhances Conditional Taste Avoidance Behavior Learning

The conditional taste avoidance (CTA) test performed in this example demonstrates that EPO acts rapidly on the memory of mice, affecting learning to avoid unpleasant taste (in this case, the disease causing substance). do. In this example, lithium chloride is used to generate CTA because it surely causes discomfort and food decay depending on the dose. Like naturally occurring diseases, lithium generates CTA by stimulating the aforementioned pathways, including cytokine release.

Female Balb / c mice were trained to limit daily total water intake to 5 minutes of drinking once per day, and trained to maintain equilibrium by drinking sufficient water for the 5 minutes. The animals were divided into groups, administered virtual control (saline) or EPO (5000 U / kg) injected intraperitoneally (IP) and then given 4 hours saccharin-vanilla liquid. Immediately after completion of the drinking of the sweet liquid, the animal was administered saline or lithium (0.15 M LiCl 20 mg / kg, injected with IP) to cause disease. Thus, the three groups of animals are as follows. The first group (control) did not receive lithium after drinking. The second group received both lithium and EPO. The third group (virtual treatment) received saline (no EPO) and lithium.

Conditional taste avoidance measures were performed by measuring the reduction in drinking when continued exposure to a novel saccharin-vanilla liquid, a disease-causing solution. After 5 days recovery from lithium or hypothetical treatments, the new saccharin-vanilla liquid was again given to animals that did not receive water. The result of comparing group 2 and 3 and group 1 (control) is shown in FIG. 2A. On the second day, baseline consumption of water after habituation into the test cage is shown. On the third day, the animals were given new saccharin-vanilla liquid 4 hours after intraperitoneal injection of saline or EPO (5000 U / kg), followed by lithium or saline treatment (arrow). As a result of the treatment, on day 3, the fluid consumption in all groups was somewhat reduced (this is the aforementioned opposite effect on the injection and new of the fluid). After recovery, the first test to achieve CTA did not show this decrease in the control group. However, animals administered lithium showed a visually complete evasion behavior with respect to fluid despite the absence of water (day 4). As a result of the continued ingestion of water, CTA was extinguished (day 5 to day 9), but as indicated by the black circle in FIG. 2A, the recovery of the animals administered with EPO was markedly slow.

Since the resistance to water shortage in EPO treated animals is approximately twice that of virtually injected animals (FIG. 2B), the strength of CTA achieved herein is better assessed by considering the degree of water shortage that appears every test day. Despite the significantly enhanced CTA demonstrated by the EPO treated group, animals in the EPO treated group had more access to drinking than the virtual group, as shown in FIG. 2C. The strength of CTA was demonstrated by repeated injections of lithium only (no EPO), which resulted in more attenuated CTA in the EPO treated group (FIG. 2A, Day 10). These data show that EPO pretreatment is associated with significantly enhanced CTA generated by lithium.

8. Example 3: Peripherally administered EPO protects the brain from excitatory toxins

In this example, we want to demonstrate that EPO has a neuroprotective action in mice that cross the blood-brain barrier and are treated with the neurotoxin kainate. Many compounds are virtually toxic and present, particularly to neurons. Such molecules typically interact with endogenous receptors for the amino acid transporter glutamate and then cause excessive stimulation and neuronal damage. One of these, catenate, is an analog of glutamate as a substance widely used to study neuronal damage due to excitatory toxicity. Kinates are potent neurotoxins that destroy neurons, particularly neurons located in regions with high density kinate receptors, such as the hippocampus, causing seizures, brain damage and death.

The following neurotoxicity studies were performed with mice using kinates. The model is used to evaluate the protective benefits of treating conditions such as temporal lobe epilepsy. Parenteral injection into laboratory animals such as rats and mice can cause partial (limbic) seizures depending on the dose, leading to death. The experiments presented in this paragraph were performed as a test to see if peripherally administered EPO crossed the blood-brain barrier, and if so, EPO affects the energy balance of neurons and in particular neuroprotective action against kinates.

Eventually, 5000 U / kg of gene at a specific time before, immediately after or after administration of a specific concentration of canate (sigma chemical) intraperitoneally to female Balb / c mice (weighted on average 15-20 g). Recombinant human erythropoietin (rhEPO (commercially available under the trade name PROCRIT from Ortho-biotech, Inc.) or sham was preliminarily tested by intraperitoneal injection. The mice were then monitored and graded 20 minutes after mice were administered to observe the phenomenon of seizure activity. Each test was terminated 60 minutes after catenate administration. As shown in FIG. 3A, in mice treated with canate, pretreatment with EPO dramatically reduced seizure severity and delayed the onset of status epilepticus. Comparison between EPO-treated and saline-treated animals demonstrates a very low mortality rate when treated with EPO in animals receiving a canate dose of 20 mg / kg to 30 mg / kg, Pretreatment indicates that neuroprotection is possible. The values in parentheses in each column represent the number of animals exposed to each canate dose.

Dose-dependence of EPO in providing neuroprotection from canate is shown in FIG. 3B. Mice were given EPO (5000 U / kg; intraperitoneally up to 5 days). The neuroprotective effect on each EPO dose was assessed by measuring survival after dose of canate (20 mg / kg) showing a mortality of about 50% for control animals (no epo; shown in FIG. 3A). . The column shows an increase in survival of EPO-treated animals compared to saline-treated animals. As shown in FIG. 3B, neuroprotection is increased by additional administration of 5000 mg / kg EPO.

Neuroprotection provided by EPO is characterized by a delay in the invention and is characterized by the activation of gene expression programs. 3C shows that EPO-related death delays due to a single dose of EPO given immediately after catenate administration (20 mg / kg) did not provide any immediate protection, but EPO was administered 24 hours prior to catenate administration. Increases the time to incubation and severity of death and death. The effect lasts up to 7 days.

9. Example 4: Peripherally Administered EPO Prevents Brain Injury from Ischemia

Conventional in vivo studies, using a global reperfusion model, stop brain flow to the brain, resulting in brain cell death, and directly injecting EPO into the ventricle to stop the brain cell death. It was shown that it can be prevented (Sakanaka et al., 1998, Proc. Natl. Acad. Sci. U.S.A. 95: 4635. The tests shown in the examples of the present invention preferentially show that peripherally delivered EPO prevents neuronal cell death in vivo in animal models with ischemia.

The following tests were performed using a midbrain occlusion model, a seizure model of ischemic lesions accepted in the art. In the protocol, male rats (250 g weight) were anesthetized with phenobarbital and maintained at 37 ° C. Carotid arteries are seen and the ipsilateral carotid artery is permanently occluded. Ipsilateral midbrain artery (MAC) was visualized and destroyed at origin. The contralateral artery was occluded by clamping for 1 hour. Animals were sacrificed 24 hours later to remove brains and cut into 1 mm continuous sections. Viable tissues can be visualized using in situ triphenyltetrasolium reduction to view live tissues from necrotic tissues. The central and peripheral borders of ischemia undergo cell death.

Using the MCA model above, EPO was administered via parenteral injection at various times before injury and immediately after injury, and the injury volume was measured by computer-assisted image analysis. The results of this analysis are shown in FIG. 4B and show the effect of EPO treatment at the times described below before and after the seizure: 24 hours before the seizure, immediately after the seizure and after 3, 6 and 9 hours after the seizure. As shown in FIG. 4A, necrosis injury of tissues is prevented when EPO is administered within 6 hours after seizure.

Beneficially and conversely, 17-mer derived from EPO conventionally promotes the growth of neuritis and ex vivo myelination of neurons (Campana et al., 1998, Int) J. Mol. Med. 1: 235-41; US Patent No. 5,700,909, issued December 23, 1997, has been reported to have neurotropic activity, and the system (FIG. 4B, “17-mer”). There is no effect on the protection from injury, and therefore other models evaluating the effect of EPO on the action of the excitatory tissue provided by the present invention, as well as the above models, may be performed by excitatory tissue action, such as injuries. It can be used to identify EPO and EPO receptor activity modulators that can be used to modulate protection or improvement of learning and cognition.

10. Example 5: Peripherally administered EPO prevents blunt trauma of the brain

In models of mechanical trauma, cortical shock models, pretreatment with systemic-administered EPO prevents blunt trauma of the mouse brain. To induce damage, air-guided pistons were used, 3 mm in diameter and able to deliver air accurately to the skull. Each mouse was paralyzed and safely placed on a sterotaxic device to prevent head movement. Hair cutting was performed to measure the position of the bregma in which the piston was initially placed. The piston was then moved 2 mm towards the tail and 2 mm towards the abdomen to adjust for bregma and make an impact by using an accurate pulse of nitrogen. Such a device enables accurate selection of the piston speed (4 m / s) and impact movement (2 mm).

Mice were treated with EPO (5000 U / kg) 24 hours before, shortly after injury, 3, 6 or 9 hours after injury and continued to be administered daily. Mice were sacrificed 10 days after this procedure, then the brain was examined and the necrosis volume of the brain was measured. In saline-treated mice, necrosis of many sites was observed (FIG. 5) and was accompanied by infiltration of large amounts of monocytes. In contrast, when animals were pretreated with EPO or given EPO within 3 hours after injury, the animals were protected from the injury and little monocytes were detected at the site of injury.

Example 6 Peripherally Administered EPO Prevents Ischemia Damage of Myocardium

This example demonstrates the efficacy of EPO in preventing hypoxic damage of heart tissue. To accomplish this, mice were pre-tested with EPO (5000 U / kg) 24 hours before the procedure carried out according to Latini et al., (1999, J. Cardiovasc. Pharmacol. 31: 601-8). The subject was then anesthetized, placed in auxiliary ventilation and thoracotomy performed. The heart and its internal circulation were checked and the removable sutures were placed around the proximal junction of the left anterior descending coronary artery and ligated. Thereafter, EPO was further administered (5000 units / kg) and the blockage was maintained for 30 minutes. After 30 minutes, sutures were loosened and the animals were held for an additional 6 hours with deep anesthesia and sacrificed. Immediately after death, the heart was removed and a biochemical analysis was prepared by removing the affected local (AAR) as well as the unaffected local (diaphragm). Two parameters were evaluated: creatine kinase (CK), a measure of myocardial viability (lower CK results in lower tissue survival), and myeloperoxidase, the product of mononuclear cell infiltrates. The results are shown in FIGS. 6A and 6B. As shown in these figures, treatment with EPO maintains CK activity in both infarct area (AAR) and unsprayed left ventricular (LV) wall, thus resulting in higher tissue survival and lower MPO activity compared to the control. It indicates that the infiltration by inflammatory cells is considerably less.

12. Example 7: Peripherally Administered EP Attenuates Toxin of Experimental Allergic Encephalitis

Experimental allergic (or autoimmune) encephalomyelitis (EAE) in rats is an animal model of multiple sclerosis (MS) recognized in the art. Several animal models with EAE have been developed that apply immune, viral, toxin and traumatic parameters to understand the characteristics of MS.

To test whether EPO prevents EAE symptoms, the following experiment was performed. Heat-sterilized Mycobacterium tuberculosis (7 mg / ml) with an equal volume of Freund's complete adjuvant (CFA, Sigma) and added to H37Ra (Difco, Detroit, MI) to a final volume of 100 μl Female Lewis rats (Charles River, Calco, 6-8 weeks old) were injected with 50 μg of guinea pig myelin basic protein (MBP; Sigma, St.Louis, MO) in water into the hind paws under light ether anesthesia. , Italy).

After treatment, the signs of experimental autoimmune encephalomyelitis (EAE) in rats were assessed daily and scored as follows: 0, no disease; 1, soft tail; 2, ataxia; 3, complete hind limb paralysis with urinary incontinence. The body weight was also observed and recorded. Mice began to receive EPO (5000 U / kg, IP, once daily) three days after immunization and continued until day 18. Control rats received only excipients. As shown in FIG. 7, mice treated with EPO demonstrated an improved score (ie, fewer numbers) and improved resistance to disease. It was also found that initiation of symptoms was significantly delayed in mice treated with EPO.

13. Example 8: Minimum Effective Dose and Pharmacokinetics of EPO Required for Protection of Excited Tissue

The animal model of the focal ischemic attack was used to assess the optimal effective dose of EPO. As shown in FIG. 8A, an EPO dose of 450 units / kg body weight was certainly not effective in preventing necrotic damage of excitable tissues. As shown in FIG. 8B, in animal studies, IP was delivered to four female mouse subjects at a dose of about 5000 units / kg-body weight, resulting in a blood content of EPO of 20,000 serum within 5 hours after administration. It was greater than milliunits / ml and after 10 hours after administration it was greater than 10,000 milliunits / ml but after 24 hours of administration it became less than 5 units / ml.

14. Example 9: CNS Delivery Mediated by Erythropoietin

The experiments presented hereafter demonstrate successful delivery of the molecules bound to EPO to the blood-brain barrier and localization in the basement membrane. As shown in FIG. 9A, brain sections were stained with antibodies to the EPO receptor (EPO-R) showing that brain capillaries exhibit high levels of EPO-R. To study whether EPO can be moved across the blood-brain barrier, EPO was labeled with biotin as follows. The dose containing rhEPO was concentrated using a Centricon-10 filter (Millipore) and the recovery was measured by reading the absorbance scale at a wavelength of 280 nm. Next, 0.2 mg of long arm biotin (Vector Labs) was dissolved in 100 μl of DMSO, added to concentrated rhEPO solution and immediately vortexed. This mixture was then stirred slowly and incubated at room temperature for 4 hours while blocking light. Unbound biotin was removed from the solution using a Centricon-10 column. Biotinylated EPO was then administered to animal IP and animals were sacrificed after 5 hours. Brain sections were labeled with avidin bound to peroxidase and diaminobenzidine was added until sufficient reaction product was generated for observation under an optical microscope. EPO was found along the same capillaries stained with EPO-R positive (FIG. 9B). At later time points, the biotin content appeared to be localized within specific neurons (eg, FIG. 9C, 17 hours). In contrast, when cold EPO was added in excess of 1000 times the labeled EPO, all specific staining was removed. The results demonstrate that systemically administered binding EPO compounds are successfully delivered across the blood-brain barrier.

Successful delivery of systemically administered EPO-biotin conjugates to the brain across the blood-brain barrier indicates that other therapeutic compounds can also be delivered across the blood-brain barrier in a similar manner by complexing EPO to the desired compound. Demonstrate. As an example, brain-induced neuronal factor (BNF) can be covalently bound to EPO by carbodiimides using standard procedures. After purification, the binding can be administered to the animal via intraperitoneal injection. The positive effect of BNF on the central nervous system can be measured in relation to control animals by measuring the successful migration of this molecule associated with EPO, in contrast to the lack of central nervous system activity by unbound BNF.

The present invention is not to be limited in scope by the specific embodiments described which are intended as one description of the individual embodiments of the invention, and functionally equivalent methods and compositions are within the scope of the invention. Indeed, various modifications of the present invention will become apparent from the foregoing description and the accompanying drawings, as well as those shown and described herein. Such modifications are intended to fall within the scope of the appended claims.

All references cited herein are hereby incorporated by reference in their entirety for all purposes.

Claims (27)

Comprising about 50,000 to 500,000 units of EPO, an EPO receptor activity modulator, an erythropoietin (EPO) -activated receptor modulator or combinations thereof, and a pharmaceutically acceptable carrier, which are effective nontoxic ranges per dosage unit. A pharmaceutical composition in the form of a dosage unit suitable for the regulation of excitatory tissue, enhancement of cognitive function or delivery of a compound to the endothelial obstruction. The method of claim 1, A pharmaceutical composition wherein the effective nontoxic range of EPO comprises 50,000 to 500,000 units of EPO. The method of claim 1, A pharmaceutical composition wherein the effective nontoxic range of EPO is a dose effective to achieve circulating levels of EPO above 10,000 milliunits / ml (serum). The method of claim 3, wherein A pharmaceutical composition wherein circulating levels of EPO are measured at about 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours or 10 hours after EPO administration. A pharmaceutical kit with one or more containers comprising the pharmaceutical composition according to claim 2. A method of preventing mammalian lesions resulting from damage to excitatory tissue, comprising peripherally administering to the mammal an effective amount of EPO, an EPO receptor activity modulator, or an EPO-activated receptor modulator to protect the excitable tissue. . The method of claim 6, Injury may cause stroke seizure disorders, multiple sclerosis, stroke, hypotension, cardiac arrest, ischemia, myocardial infarction, inflammation, cognitive loss due to aging, radiation damage, cerebral palsy, neurodegenerative diseases, Alzheimer's disease, Parkinson's disease (Parkinson's disease), Leigh disease, AIDS dementia, amnesia, amyotrophic lateral sclerosis, alcoholism, mood disorders, anxiety disorders, attention deficit disorders, autism, Coroidfeldt-Jakob disease Creutzfeld-Jakob disease), brain or spinal cord injury, cardiopulmonary circuit, glaucoma, retinal ischemia or retinal damage. The method of claim 6, The damage is the result of hypoxia. The method of claim 8, Hypoxia may cause prenatal oxygen deficiency, asphyxiation, airway obstruction, near drowning, cognitive dysfunction after surgery, carbon monoxide poisoning, smoke inhalation, chronic obstructive pulmonary disease, emphysema, adult respiratory distress syndrome, hypotension shock, septic shock , Hypersensitivity shock, insulin shock, sickle cell development, cardiac arrest, arrhythmia, nitrogen anesthesia or local tissue hypoxia. Functioning the mammalian normal or abnormal excitatory tissue, including peripherally administering to the mammal an amount of EPO, an EPO receptor modulator, an EPO-activated receptor modulator, or a combination thereof that enhances peripheral effective excitatory tissue. How to improve. The method of claim 10, Enhancing the function of excitatory tissues enhances associative learning or memory. The method of claim 10, Enhancing the function of excitatory tissue is used to treat mood disorders, anxiety disorders, depression, autism, attention deficit hyperactivity disorder, Alzheimer's disease, aging or cognitive dysfunction. The method of claim 6 or 10, The excitatory tissue is central nervous system tissue, peripheral nervous system tissue or heart tissue. The method of claim 6 or 10, Wherein the administration comprises oral administration, topical administration, intraluminal administration, inhalation or parenteral administration. The method of claim 14, Parenteral administration includes intravenous, intraarterial, subcutaneous, intramuscular, intraperitoneal, submucosal or intradermal administration. The method of claim 6 or 10, The administration is acute or chronic. The method of claim 6 or 10, EPO is non-erythropoietic. A method in which EPO is administered at a dose above the dose necessary to maximize stimulation of red blood cell formation. Transcytosis of the molecule against the endothelial cell barrier, comprising administering to a mammal a composition comprising a molecule associated with an EPO, an EPO receptor activity modulator, an EPO-activated receptor modulator, or a combination thereof. how to promote transcytosis). The method of claim 19, The association is an unstable covalent bond, a stable covalent bond, or a non-covalent association with a binding site of a molecule. The method of claim 19, Endothelial cell barrier is a blood-brain barrier, a blood-eye barrier, a blood-testis barrier, a blood-ovary barrier or a blood-placental barrier. The method of claim 19, The molecule is a receptor agonist or antagonist hormone, neurotrophic factor, antimicrobial agent, radiation drug, antisense compound, antibody, immunosuppressant, toxin or anticancer agent. The method according to any one of claims 6, 10 and 19, EPO is an erythropoietin, erythropoietin analogue, erythropoietin-like agonist, erythropoietin segment, hybrid erythropoietin molecule, erythropoietin receptor binding molecule, erythropoietin agonist, kidney erythropoietin, brain erythropoietin, A method thereof which is an oligomer thereof, a polymer thereof, a mutein thereof, a homolog thereof, a natural acid form thereof, a synthetic form thereof, a recombinant form thereof, or a combination thereof. The method of claim 23, The EPO receptor binding molecule is an antibody against erythropoietin receptor. A composition for transporting molecules via transcytosis across an endothelial cell barrier, comprising a molecule associated with an EPO, an EPO receptor activity modulator or an EPO-activated receptor modulator. The method of claim 25, EPO is an erythropoietin, erythropoietin analogue, erythropoietin-like agonist, erythropoietin segment, hybrid erythropoietin molecule, erythropoietin receptor binding molecule, erythropoietin agonist, kidney erythropoietin, brain erythropoietin, A composition which is an oligomer thereof, a polymer thereof, a mutein thereof, an analogue thereof, a natural acid form thereof, a synthetic form thereof, a recombinant form thereof, or a combination thereof. The method of claim 25, A composition wherein the molecule is a receptor agonist or antagonist hormone, neurotrophic factor, antimicrobial agent, radiation drug, antisense compound, antibody, immunosuppressant, toxin or anticancer agent.