Protection, restoration, and enhancement of erythropoietin- responsive cells, tissues and organs

Priority of the provisional application No. 60/259,245 filed on December 29. 2000, and which is incorporated herein by reference in its entirety, is sought under 35 U.S.C. § 119 (e) (i).

BACKGROUND OF THE INVENTION

For a long time, the only understandable physiological role of erythropoietin was to control red blood cell production. More recently, more evidence suggests that erythropoietin, as a member of the cytokine superfamily, performs other important physiological functions through indirect interaction with the erythropoietin receptor (erythropoietin-R). These effects include mitogenesis, modulation of calcium uptake into smooth muscle and nerve cells, and effects on metabolic intermediates. Erythropoietin is believed to provide subsequent responses that serve to improve the hypoxic cell microenvironment as well as to modulate programmed cell death caused by metabolic stress. Although studies have found that erythropoietin injected intracranially protects neurons from hypoxic nerve injury, intracranial administration is an impractical and unacceptable method of administration for therapeutic purposes, especially in normal individuals. In addition, previous studies in anemic patients receiving erythropoietin have shown that peripherally administered erythropoietin is not transported in

52276 Β brain (Marti et al., 1997, Kidney Int. 51: 416-8; Juui et al., 1999, Pediatr. Res. 46: 543-547; Buemi et al., 2000, Nephrol. Dial. Transplant. 15 : 422-433.).

Various modified forms of erythropoietin are described whose activities are directed to enhance the erythropoietin activity of molecules, such as the amino acids altered at the carboxy position described in U.S. Patent 5,457,089 and U.S. Patent 4,835,260; erythropoietin isoforms with different number of sialic acid residues per molecule, as described in U.S. Patent 5,856,292; the polypeptides described in U.S. Patent 4,703,008; agonists described in U.S. Pat Patent 5,767,078; erythropoietin receptor binding peptides as described in U.S. Patent 5,773,569 and U.S. Patent 5,830,851; and small molecule imitations as described in U.S. Patent 5,835,382.

The present invention relates to the use of erythropoietin for the protection, maintenance, augmentation, or regeneration of cells and associated cells, tissues and organs responsive to erythropoietin in situ as well as ex vivo, and to the transport of erythropoietin across the endothelial cell barrier to protect and augment cells and associated cells , tissues and organs responsive to erythropoietin, and which are distal to the vasculature system, or retain associated molecules.

SUMMARY OF THE INVENTION

In one aspect the present invention relates to the use of erythropoietin for the preparation of pharmaceutical compositions for protecting, maintaining, enhancing or restoring the function or viability of mammalian cells and their associated erythropoietin-responsive cells, tissues and organs. In one particular aspect, mammalian cells and their associated cells, tissue or organ are distal to the vasculature system due to a solid endothelial cell banner. In another particular aspect,

52276 Β Cells, tissues, organs or other parts of the body are isolated from the mammalian body, such as those destined for transplantation. In non-limiting examples, erythropoietin-responsive cells or tissues may be from nerves, retina, muscle, heart, lung, liver, kidney, small intestine, adrenal cortex, adrenal medulla, capillary endothelium, testis, ovary, pancreas or endometrial cells or tissue. These applications of erythropoietin-responsive cells are illustrative only. In one aspect, the erythropoietin-responsive cells or their associated cells, tissues, or organs are not excitatory cells, tissues, or organs, or do not contain predominantly excited cells or tissues. In a particular embodiment, the mammalian cell, tissue or organ to which the aforementioned erythropoietin derivative has been administered are those that have or will spend a period of time in at least one condition that is detrimental to the viability of the cell, tissue or organ. Such conditions include traumatic in-situ hypoxia or metabolic dysfunction, surgically induced in-situ hypoxia or metabolic dysfunction, or in-situ exposure to toxins, the latter of which may be associated with chemotherapy or radiation therapy. In a certain form of performance, unfavorable conditions are the result of cardio-pulmonary bypass (heart-lung apparatus), which is used for certain surgical procedures.

Erythropoietins are useful for the therapeutic or prophylactic treatment of diseases of the central nervous system, CNS or peripheral nervous system in humans, which have primary neurological or psychiatric symptoms, as well as for eye diseases, cardiovascular diseases, cardiopulmonary diseases. respiratory diseases, renal, urinary and reproductive diseases, gastrointestinal diseases and endocrine and metabolic abnormalities.

The present invention also relates to pharmaceutical compositions comprising certain erythropoietin derivatives for administration to mammals, in particular humans. Such pharmaceutical compositions may be

52276 Β formulated for oral, intranasal, or parenteral administration, or may be in the form of a perfusion solution to maintain the viability of cells, tissues or organs ex vivo.

Erythropoietin derivatives used for the above purposes may be any native erythropoietin, or erythropoietin analog, imitation of erythropoietin, and a fragment of erythropoietin, a hybrid molecule of erythropoietin, a molecule that binds erythropoietin and receptor, erythropoietin agonist, renal erythropoietin, erythropoietin erythropoietin its multimer, its mutein, its related compound, its naturally occurring form, its synthetic form, its recombinant form, a glycosylated variant thereof, a deglycosylated variant thereof, or a combination thereof. Any form of erythropoietin that is effective for erythropoietin-responsive cells is encompassed in this aspect of the invention.

Other erythropoietin derivatives used for the aforementioned purposes and pharmaceutical compositions include both native erythropoietins and erythropoietins that have been modified by at least one modification compared to native erythropoietin, and preferably compared to native human erythropoietin. The at least one modification may be a modification of at least one amino acid from erythropoietin molecules, or a modification of at least one carbohydrate from erythropoietin molecules. Of course, erythropoietin molecules useful for these purposes may have a large number of modifications compared to the native molecule, such as multiple modifications of a portion of molecules with amino acids, multiple modifications of a portion of molecules with carbohydrates, or at least one modification of a portion of molecules with amino acids and at least one modification of part of the molecule with carbohydrates. The modified erythropoietin molecule retains its ability to protect, maintain, increase or restore the function or viability of mammalian cells

52276 Β responsive to erythropoietin, but other features of erythropoietin molecules that are not related to the above, such as the desired characteristic, may be absent compared to the native molecule. In a preferred embodiment, the erythropoietin derivative is not erythropoietin.

In one embodiment, the erythropoietin of the present invention comprises portions of sialic acid. In a preferred embodiment, the modified erythropoietin is asialoerythropoietin, and preferably human asialoerythropoietin. In another embodiment, the modified erythropoietin has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 parts of sialic acid.

In another embodiment, the modified erythropoietin does not contain N- or O-linked carbohydrates.

In a third embodiment, the modified erythropoietin has a reduced carbohydrate content due to the reaction of erythropoietin and its native carbohydrates with at least one glycosidazoin.

In a fourth embodiment, a portion of the modified carbohydrate erythropoietin molecule has at least one glycosylation pattern in animals other than mammals based on the expression of recombinant erythropoietin in animal cells other than mammals. In a preferred embodiment, the modified erythropoietins are expressed in insect or plant cells.

In a fifth embodiment, the modified erythropoietin contains one or more oxidized carbohydrates which may also be chemically reduced. Priority form code

52276 Β derivatives, the modified erythropoietin is oxidized by the periodate; in another preferred embodiment, the periodate-oxidized erythropoietin is chemically reduced by borohydride salts such as sodium borohydride or sodium cyanoborohydride.

In a sixth embodiment, the modified erythropoietin for the aforementioned applications comprises at least one or more arginine residues. In one embodiment, the modified entropoetin comprises a portion of glyoxal on one or more arginine residues, such as an arylglyoxal or alkylglyoxal portion. In another embodiment, the at least one arginine residue is modified by reaction with a vicinal diketone such as, but not limited to, 2,3butanedione or cyclohexanedione.

In a seventh embodiment, the modified erythropoietin comprises at least one or more modified lysine residues or a modified N-terminal amino group from erythropoietin molecules, such modifications as those obtained by reacting the lysine residue or Nterminal amino group with an amino group modifying agent. The modified lysine residue can be further chemically reduced. In a preferred embodiment, the erythropoietin is subjected to biotinylation or carbamylation via one or more lysine groups. In another preferred embodiment, the lysine is reacted with an aldehyde or reducing sugar to form an imine, which can be stabilized by reduction with sodium cyanoborohydride to give an N-alkylated lysine such as glucitolyl lysine, or which in the case of reducing sugars may be stabilized by Amadori or Heyns rearrangement to give alpha-deoxy-alpha-amino sugar such as alpha-deoxy-alpha-fructosilysin. In another preferred embodiment, the lysine group is carbamylated, based on reaction with a cyanate ion, alkyl-carbamylated, aryl-carbamylated, or aryl-thiocarbamylated saalkyl

52276 Β isocyanate, aryl-isocyanate, or aryl-isothiocyanate, or the lysine group can be acylated with a reactive alkylcarboxylic or arylcarboxylic acid derivative, as in reaction with acetic anhydride, amberhydride anhydride and fital acid anhydride. The at least one lysine group may also be modified with trinitrophenyl by reaction with nitrobenzenesulfonic acid, or preferably a dream salt. In another embodiment, the lysine residues may be modified by reaction with a glyoxal derivative, such as a reaction with glyoxal; methylglyoxal or 3-deoxyglucozone to give the corresponding alpha-carboxylated derivatives.

In an eighth embodiment, the at least one residue of the erythropoietin brosine can be modified at the aromatic ring position by an electrophilic agent, such as by nitration or iodination.

In a ninth embodiment, at least one aspartic acid or glutamic acid residue from erythropoietin may be modified by a reaction, such as a reaction with a carbodiimide, followed by a reaction with an amine, such as, but not limited to, glycinamide.

In a tenth embodiment, at least the tritophane residue from the erythropoietin is modified by a reaction, such as a reaction with n-bromosuccinimide or n-chlorosuccinimide.

In an eleventh embodiment, a modified erythropoietin molecule is obtained from which at least one erythropoietin amino group is removed by a reaction, such as a reaction with ninhydrinorrt followed by reduction of the resulting carbonyl group by reaction with

52276 Β borohydride.

In a twelfth embodiment, a modified erythropoietin having at least one free site on at least one of the cystine bonds in the erythropoietin molecule is obtained by reaction with a reducing agent such as dithiothreitol, followed by reaction of the following sulfhydryls with sayodoacetamide, iodoacetic acid or other prevented the re-establishment of disulfide bonds.

In a thirteenth embodiment, a modified erythropoietin is obtained in which at least one of a number of amino acids, such as leucine, is substituted with at least one lysine, arginine, tryptophan, tyrosine, or cysteine ​​residue from erythropoietin, using methods from the molecular biology.

In a fourteenth embodiment, the modified erythropoietin is subjected to limited chemical proteolysis that affects specific residues, for example, to separate tritophane residues. The erythropoietin fragments thus obtained are included herein.

As noted above, the erythropoietin used for the purposes set forth herein may have at least one of the aforementioned modifications, but may also have more than one of said modifications. An example of a modified erythropoietin with one modification on the part of the carbohydrate molecule and with one modification on the part of the molecule with amino acids is asialoerythropoietin whose lysine residues are biotinylated or carbamylated. The present invention also encompasses compositions, including pharmaceutical compositions, that contain one or more of the aforementioned erythropoietins.

52276 Β

In another aspect of the present invention, there is provided a method of protecting, maintaining, enhancing or restoring the function or viability of erythropoietin-responsive mammalian cells and their associated cells, tissues and organs, by administering an effective amount of any one or more of the aforementioned erythropoietins. In one particular aspect of this method, mammalian cells responsive to erythropoietin and their associated cells, tissue or organ are distal to the vasculature system due to a solid endothelial cell barrier. In another particular aspect, the cells, tissues, organs, or other parts of the body are isolated from the mammalian body, as if intended for transplantation. In non-limiting applications, erythropoietin-responsive cells or tissues may be from the nervous system, retina, muscle, heart, lungs, liver, kidney, small intestine, adrenal cortex, adrenal medulla, capillary endothelium, testis, ovary, pancreas or endometrial cells or tissue. These examples of erythropoietin-responsive cells are illustrative only. In a particular embodiment, the erythropoietin-responsive cells or their associated cells, tissues or organs are not excited cells, tissues or organs, or do not contain predominantly excited cells or tissues. In another particular embodiment, the mammalian cell, tissue or organ in which the aforementioned erythropoietin derivative may be administered are those that have or will spend a period of time in at least one condition that is detrimental to the viability of the cell, tissue or organ. Such conditions may include traumatic in-situ hypoxia or metabolic dysfunction, surgically induced in-situ hypoxia or metabolic dysfunction, or in-situ exposure to toxins, the latter of which may be associated with chemotherapy or radiation therapy. In one embodiment, the present invention provides protection against adverse conditions resulting from cardiopulmonary bypass.

In another aspect of the present invention, each of the foregoing erythropoietins as well as any other erythropoietin molecules, including native human erythropoietin, can be used to prepare

52276 Β pharmaceutical compositions for ex-vivo treatment of cells, tissues and organs to protect, maintain, enhance or restore the function or viability of cells responsive to erythropoietin and their associated cells, tissues and organs in mammals. Such ex-vivo processing is useful, for example, for storing cells, tissues or organs for transplantation, whether for autotransplantation or for xenotransplantation. Cells, tissue or organ may be bathed in a solution containing erythropoietin, or the perfusion solution may be infused into the organ via blood vessels or otherwise, to maintain cellular function during a period in which the cells, tissue or organ are not connected to the donor's blood vessels. or recipient. The administration of the perfusion solution can be performed on the donor before the organ is removed, as well as on the removed organ and the recipient. Moreover, the above-mentioned administration of any erythropoietin is useful whenever a cell, tissue or organ is isolated from an individual's blood vessels. thus essentially existing ex vivo over a period of time, the term isolated referring to the restriction or blockage of blood vessels from or towards a cell, tissue, organ or part of the body, such as is performed during surgery, including, in particular, cardiopulmonary bypass surgery; connecting blood vessels, cells, tissues, organs or parts of the body; removing a cell, tissue, organ or part of the body from the body of a mammal, such as before performing a xenotransplant or before and during autotransplantation; or traumatic amputation of a cell, tissue, organ, or body part. Thus, this aspect of the invention relates to the use of erythropoietin perfusion in situ and gh in vivo. Ex vivo, erythropoietin may be present in a solution to store cells, tissues or organs. In any case, continuous perfusion, pulsed perfusion, infusion, bath, injection, or catheterization may be used in this mode.

In another aspect, the invention relates to a method of protecting, maintaining, enhancing, or restoring the viability of a mammalian cell, tissue, organ, or body part comprising a cell or

52276 Β erythropoietin-responsive tissue in which a cell, tissue, organ or body part is isolated from a mammalian body This procedure involves exposing an isolated mammalian cell, organ tissue or body part to a certain amount of erythropoietin over a period of time effective to protect, maintain, increase , or renew the aforementioned ability to live. In non-limiting examples, the term isolated refers to the restriction or blockage of blood vessels from or to the forearm, tissue, organ or part of the body, such as is performed during surgery, including, in particular, cardiopulmonary bypass surgery; connecting blood vessels, cells, tissues, organs or parts of the body; removing a cell, tissue, organ or part of the body from the body of a mammal, such as before performing a xenotransplant or rge and during autotransplantation; or traumatic amputation of a cell, tissue, organ, or body part. Thus, this aspect of the invention relates to the use of erythropoietin perfusion in situ and ex vivo. Eh vivo, erythropoietin may be present in a solution for storing cells, tissues or organs. In any case, continuous perfusion, pulsed perfusion, infusion, bath, injection, or catheterization may be used in this mode of operation.

In the aforementioned isolation or ex-vivo embodiment, a useful entropoetin may be any of the above-mentioned erythropoietins, including any native erythropoietin, or erythropoietin analog, an imitation of erythropoietin, and an erythropoietin fragment, erythropoietin hybrid molecule, erythropoietin binding molecule, and erythropoietin binding molecule. agonist, renal erythropoietin, cerebral erythropoietin, its oligomer, its multimer, its mutein, a related compound, its naturally occurring form, its synthetic form, its recombinant form, its glycosylated variant, its deglycosylated variant, or a combination thereof. Any form of erythropoietin that may be effective for erythropoietin-responsive cells is encompassed in this aspect of the invention. Other erythropoietins include, but are not limited to, asialoerythropoietin, N-deglycosylated erythropoietin, O-deglycosylated

52276 Β erythropoietin, erythropoietin with reduced carbohydrate content, erythropoietin with altered glycosylation patterns, erythropoietin with oxidized carbohydrates and then reduced, erythropoietin modified with arylglyoxal, erythropoietin modified with cycloalkylaminoxyal, erythropoietin modified with 2,3- alkylovan-lysyl-erythropoietin, glucitolyl lysine erythropoietin, alpha-deoxy-alpha-fructosyl Iisine-erythropoietin, carbamylated erythropoietin, acetylated erythropoietin, succinylated erythropoietin, alpha-carboxyalkyl erythropoietin, nitrated erythropoietin, iodinated erythropoietin, all of which are not limited to Human erythropoietin is preferred; most preferred is native human erythropoietin. In another embodiment, human asialoerythropoietin is preferred. In another embodiment, human phenylglyoxal erythropoietin is preferred.

In non-limiting examples, the aforementioned ex-vivo erythropoietin-responsive cells or tissue may be or consist of nerves, retina, muscle, heart, lung, liver, kidney, small intestine, adrenal cortex, adrenal medulla, capillary endothelium, testis, ovary , pancreatic or endometrial cells or tissues These examples of erythropoietin-responsive cells are illustrative only.

All of the above procedures and applications are preferably applied to human beings, but may be easily used for any mammal, such as, but not limited to, pets, domesticated animals, domestic animals, and zoo animals. Methods of administration of the aforementioned pharmaceutical compositions include oral, intravenous, intranasal, topical, intraluminal, inhalation, or parenteral administration, wherein this

52276 Β The latter includes intravenous, intraarterial, subcutaneous, intramuscular, intraperitoneal, submucosal or intradermal administration. For ex-vivo administration, it is preferable to use a perfusion solution or a bath solution. This involves isolating an isolated part of the vascular system in situ.

In another aspect of the present invention, each of the aforementioned erythropoietins is useful for preparing a pharmaceutical composition for repairing a dysfunctional cell, tissue or organ, when administered upon the onset of a disease or condition responsible for the dysfunction. In a non-limiting example, administration of a pharmaceutical composition comprising erythropoietin restores cognitive function in animals that have previously experienced brain trauma, even when administered long after (e.g., \ n days, five days, a week, a month or longer) as the trauma subsided. Erythropoietins useful for such administration include any of the above mentioned erythropoietins or any native erythropoietin, or erythropoietin analog, imitation of entropoetin, and a fragment of erythropoietin, a hybrid molecule of erythropoietin, a molecule that binds erythropoietin and a receptor, erythropoietin, erythropoietin erythropoietin, its oligomer, its multimer, its mutein, its related compound, its naturally occurring form, a synthetic form thereof, a recombinant form thereof, a glycosylated variant thereof, a deglycosylated variant thereof, or a combination thereof Any form of erythropoietin that is effective for erythropoietin-responsive cells is encompassed by this aspect of the invention. Other erythropoietin derivatives suitable for the above purposes and pharmaceutical compositions include both native erythropoietins as well as erythropoietins that have been modified by at least one modification compared to native erythropoietin, and preferably compared to human erythropoietin. At least one modification may be a modification of at least one amino acid from erythropoietin molecules, or modify at least one carbon

52276 Β Hydrates from erythropoietin molecules. Of course, erythropoietin molecules suitable for these purposes may have a large number of modifications compared to the native molecule, such as multiple modifications of a portion of molecules with amino acids, multiple modifications of a portion of molecules with carbohydrates, or at least one modification of a portion of molecules with amino acids and at least one modification of part of the molecule with carbohydrates. The modified erythropoietin molecule retains its ability to protect, maintain, enhance, or restore the function or viability of erythropoietin-responsive mammalian cells, but other features of erythropoietin molecules unrelated to the aforementioned, such as a desired characteristic, may be absent compared to sanitary molecule. Preferably human erythropoietin, most preferably native human erythropoietin. In another embodiment, human asialoerythropoietin is preferred.

In another embodiment, the present invention provides methods of administering the aforementioned erythropoietin to repair a dysfunctional cell, tissue or organ, when administered after the onset of a disease or condition responsible for the dysfunction. In a non-limiting example, methods of administering a pharmaceutical composition comprising erythropoietin restore cognitive function in animals that have previously experienced brain trauma, even when administered long after (e.g., tn days, five days, a week, a month, or longer) after the trauma subsided. Erythropoietins useful for such methods include any of the above-mentioned erythropoietins or any native erythropoietin, or erythropoietin analog, imitation of erythropoietin, and a fragment of erythropoietin, a hybrid molecule of erythropoietin, a molecule that binds erythropoietin and a receptor, erythropoietin, erythropoietin entropoetin, its oligomer, its multimer, its mutein, its related compound, its naturally occurring form, its synthetic form, its recombinant form, its glycosylated variant,

52276 A deglycosylated variant thereof, or a combination thereof, Any form of erythropoietin that is effective for erythropoietin-responsive cells in this aspect of the invention. Other erythropoietin derivatives suitable for the aforementioned purposes and pharmaceutical compositions include both native erythropoietins as well as erythropoietins that have been modified by at least one modification compared to native erythropoietin, and preferably compared to human erythropoietin. The at least one modification may be a modification of at least one amino acid from erythropoietin molecules, or a modification of at least one carbohydrate from erythropoietin molecules. Of course, erythropoietin molecules suitable for these purposes may have a large number of modifications compared to the native molecule, such as multiple modifications of part of the molecules with amino acids, multiple modifications of part of molecules with carbohydrates, or at least one modification of part of molecules with amino acids and at least at least one modification of a portion of the carbohydrate molecule. The modified erythropoietin molecule retains its ability to protect, maintain, enhance or restore the function or viability of mammalian cells responsive to erythropoietin, but other features of erythropoietin molecules that are not related to the aforementioned, such as a desired feature, may be absent compared to native molecule. Human erythropoietin is preferred, and native human erythropoietin is most preferred. In another embodiment, human asialoerythropoietin is preferred.

In another aspect of the present invention, methods are provided for accelerating molecule transcytosis through an endothelial cell barrier in a mammal by administering a mixture of molecules with erythropoietin such as: erythropoietin not containing sialic acid moieties; non-carbohydrate erythropoietin with N-bond or O-bond; erythropoietin having a reduced carbohydrate content due to the reaction of native erythropoietin with at least one glycosidase;

52276 Β erythropoietin with a portion of a carbohydrate-containing erythropoietin molecule having at least one glycosylation pattern in animals other than mammals based on the expression of recombinant erythropoietin in animal cells other than mammals; erythropoietin having at least one or more oxidized carbohydrates which may also be chemically reduced;

erythropoietin having at least one or more modified arginine residues; erythropoietin having at least one or more modified lysine residues or a modification of the N-terminal amino group of the erythropoietin molecule; entropoetin having at least one modified tyrosine residue; erythropoietin having at least one modified aspartic acid or glutamic acid residue; erythropoietin having at least one modified tryptophan residue; erythropoietin whose at least one amino group has been removed; erythropoietin having at least one free site on at least one of the cystine bonds in the erythropoietin molecule;

erythropoietin having at least one amino acid substitution; or incomplete erythropoietin.

The association between the transported molecule and the erythropoietin may be, for example, a labial covalent bond, a stable covalent bond or a non-covalent association with the molecule binding position. The endothelial cell barrier can be a blood-brain barrier, a blood-blood barrier, a blood-testis barrier, a blood-ovarian barrier, and a blood-placenta barrier. A molecule suitable for transport by the method of the present invention includes hormones, such as growth hormone, antibiotics and anticancer agents.

Another aspect of the present invention provides a composition for accelerating transcytosis of a mammalian endothelial cell barrier molecule in a mammal, said composition comprising said molecule in association with an erythropoietin such as erythropoietin which does not contain sialic acid moieties;

52276 Β N-linked or O-linked carbohydrate-free erythropoietin; erythropoietin having a reduced carbohydrate content due to the reaction of native erythropoietin with at least one glycosidase; erythropoietin with a portion of a carbohydrate-containing erythropoietin molecule having at least one glycosylation pattern in animals other than mammals based on the expression of recombinant erythropoietin in animal cells other than mammals; erythropoietin having at least one or more oxidized carbohydrates which may also be chemically reduced;

erythropoietin having at least one or more modified arginine residues; erythropoietin having at least one or more modified lysine residues or a modification of the N-terminal amino group of erythropoietin molecules; erythropoietin having at least one modified tyrosine residue; erythropoietin having at least one modified aspartic acid or glutamic acid residue; erythropoietin having at least one modified tryptophan residue; erythropoietin whose at least one amino group has been removed; erythropoietin having at least one free site on at least one of the cystine bonds in the erythropoietin molecule;

erythropoietin having at least one amino acid substitution; or incomplete erythropoietin.

The association may be, for example, an stable covalent bond, a stable covalent bond or a non-covalent association with a molecule binding position. The endothelial cell barrier may be a blood-brain barrier, a blood-eye barrier, a blood-testicular barrier, a blood-ovarian barrier, and a blood-placental barrier. A molecule suitable for transport by the method of the present invention includes hormones, such as growth hormone , antibiotics and anticancer agents.

(In another aspect of the present invention, any of the aforementioned erythropoietins is useful for preparing a pharmaceutical composition for accelerating transcytosis of molecules through endothelial cell

52276 Β a barrier in mammals by administering a mixture of molecules with erythropoietin such as: erythropoietin that does not contain parts of sialic acid; non-carbohydrate erythropoietin with N-bond or O-bond carbohydrates; erythropoietin having a reduced carbohydrate content based on the reaction of native erythropoietin with at least one glycosidase; erythropoietin with a portion of a carbohydrate-containing erythropoietin molecule having at least one glycosylation pattern in animals other than mammals based on the expression of recombinant erythropoietin in animal cells other than mammals; entropoetin having at least one or more oxidized carbohydrates which may also be chemically reduced; . erythropoietin having at least one or more modified arginine residues; erythropoietin having at least one or more modified lysine residues or modification of the N-terminal amino group of erythropoietin molecules; erythropoietin having at least one modified tyrosine residue; an entropy that has at least one modified aspartic acid or glutamic acid residue; erythropoietin having at least one modified tryptophan residue; erythropoietin whose at least one amino group has been removed; erythropoietin having at least one free site at at least one of the cystine bonds in the erythropoietin molecule; erythropoietin having at least one amino acid substitution; or incomplete erythropoietin.

The association may be, for example, a labile covalent bond, a stable covalent bond or a non-covalent association with a molecule binding position. The endothelial cell barrier can be a blood-brain barrier, a blood-eye barrier, a blood-testis barrier, a blood-ovarian barrier, and a blood-placenta barrier. A molecule suitable for transport by the method of the present invention includes hormones, such as growth hormone, antibiotics and anticancer agents

52276 Β

These and other aspects of the present invention will be better appreciated based on the reference to the following figures and detailed description

52276 Β

BRIEF DESCRIPTION OF PICTURES

Figure 1 represents the translocation of parenterally administered erythropoietin into cerebrospinal fluid.

Figure 2 shows the protection of the myocardium from ischemic damage by erythropoietin after temporary vascular occlusion.

Figure 3 shows the maintenance of cardiac function prepared for transplantation using erythropoietin.

Figure 4 compares the efficacy of in-vitro erythropoietin and asialoerythropoietin on the viability of serum-deprived R1 9 cells

Figure 5 is another experiment comparing the efficacy of in-vitro erythropoietin and asialoerythropoietin on the viability of serum-deprived R1 9 cells.

Figure 6 compares the efficacy of in-vitro erythropoietin and erythropoietin modified phenylglyoxal capacity for serum Ρ19 viable cells.

Figure 7 shows the protection of erythropoietin and asialoentropoietin in a model of focal cerebral ischemia in rats.

Figure 8 shows the dose response comparing the efficacy of human erythropoietin and

52276 Β Human asialoerythropoietin in middle cerebral artery occlusion in a model of ischemic stroke.

Figure 9 shows the effect of biotinylated erythropoietin and asialoerythropoietin in the R19 analysis.

Figure 10 shows the activity of iodinated erythropoietin in the Ρ19 analysis.

Figure 11 presents the effects of erythropoietin treatment on a rat glaucoma model.

Figure 12 shows the extent of erythropoietin retention of retinal function in a rat glaucoma model.

Figure 13 represents the restoration of cognitive function after brain trauma through erythropoietin administration that began five days after trauma.

Figure 14 represents the restoration of cognitive function after brain trauma through erythropoietin administration that began thirty days after trauma.

Figure 15 presents the efficacy of human asiaioerythropoietin in cerebral toxicity in a kainate model.

52276 Β

DETAILED DESCRIPTION OF THE INVENTION

The term erythropoietin-responsive cell refers to a mammalian cell whose function or viability may be maintained, induced, increased, regenerated, or otherwise benefit from erythropoietin exposure. Non-limiting examples of such cells include nerve, retina, muscle, heart, lung, liver, kidney, small intestine, adrenal cortex, adrenal medulla, capillary endothelium, testis, ovary, pancreas, or endometrial cells. Moreover, such erythropoietin-responsive cells and the consequent action of erythropoietin may be expanded to indirectly provide protection or augmentation to other cells not directly responsive to erythropoietin, or to tissues and organs containing such cells that are not erythropoietin-responsive. These other cells, tissues, or organs that indirectly benefit from an increase in erythropoietin-responsive cells are present as part of cells, tissues, or organs, as associated cells, tissues, or organs. Thus, the beneficial effects of erythropoietin as described herein may be obtained by the presence of a small number or a proportion of erythropoietin-responsive cells in a tissue or organ, for example, excited or nervous tissue present in such tissue, or Levdig cells from the testis, which make up testosterone. In one aspect, the erythropoietin-responsive cells or their associated cells, cells, or organs are not excited cells, tissues, or organs, or do not contain predominantly excited cells or tissues.

The methods of carrying out the present invention provide local or systematic protection or augmentation of cells, tissues and organs in the mammalian body, in a wide range of normal and adverse conditions, or protection of those intended for transfer to another mammalian body. In addition, recovery or regeneration of dysfunction is provided . As mentioned above, the ability of erythropoietin to cross a solid endothelial cell banner and exert positive effects on erythropoietin-responsive cells (as well as other cell types) distal to the vasculature system provides

52276 Β the ability to prevent and also treat very different conditions and diseases that otherwise cause cell and tissue damage in animals, including humans, and what is more, the success of those surgical procedures that have not been tried before and in which the risk has traditionally outweighed the benefits. The duration and extent of important adverse conditions caused by the end use, such as high-dose chemotherapy, radiation therapy, prolonged ex-νίνο transplant survival, and prolonged periods of surgically induced ischemia, can be performed using the advantages of the present invention. However, the invention is not so limited, but includes as one aspect, methods and compositions in which erythropoietin-responsive target cells are distal to the vasculature system due to a cellular endothelial barrier or endothelial solid bonds. In general, the present invention relates to any cells responsive to erythropoietin and associated cells, tissues and organs to which erythropoietin exposure may benefit. In addition, cell, tissue or organ dysfunction can be restored or regenerated after an acute adverse event (such as trauma) through exposure to erythropoietin.

Thus, the present invention relates generally to the use of erythropoietin for the preparation of pharmaceutical compositions for the aforementioned purposes in which cell function is maintained, initiated, increased, regenerated, or the action of the compositions is otherwise effective. The invention also relates to methods of maintaining, increasing, initiating or regenerating cellular function by administering to a mammal an effective amount of erythropoietin, as described herein. The invention further relates to methods for maintaining, initiating, enhancing, or regenerating cellular function ex vivo by exposing cells, tissues, or organs to erythropoietin. The invention also relates to erythropoietin-containing perfusion compositions used to preserve organs or tissues.

The various methods of work of the present invention use a pharmaceutical composition that includes at least

52276 Β erythropoietin in an amount effective for a particular mode and duration of exposure to exert positive effects or effects on a cell responsive to erythropoietin present in, or removed from, the mammalian body. When it is necessary for erythropoietin to cross the endothelial cell barrier for the target cell, tissues or organs for which therapy is prescribed, the pharmaceutical composition contains erythropoietin in a concentration that is able to exert the desired effects on erythropoietin-responsive cells after crossing the cell endothelial banner. Molecules capable of interacting with the erythropoietin receptor and modulating receptor activity are useful in the context of the present invention. These molecules may be, for example, natural, synthetic, or recombinant forms of erythropoietin molecules, as described above, or other molecules that may not necessary to be similar to erythropoietin in any way except to modulate the activity of erythropoietin-responsive cells, as described herein.

Erythropoietin is a glycoprotein hormone that in humans has a molecular weight of about kDa. The fully formed protein contains 165 amino acids, and glycosyl residues make up about 40% of the weight of molecules. The forms of erythropoietin useful for carrying out the present invention include natural, synthetic and recombinant forms of the following molecules. and other mammals that are responsive to erythropoietin: erythropoietin, asialoerythropoietin, deglycosylated erythropoietin, erythropoietin analogues, erythropoietin imitations, erythropoietin hybrid molecules, erythropoietin and receptor binding molecules, erythropoietin agonists, renal erythropoietin, their erythropoietin forms of erythropoietin used to practice the present invention include proteins that are functionally equivalent gene products. Such an equivalent erythropoietin gene product includes mutant erythropoietins, which may contain deletions, including inteme deletions, additions, including additions by which

52276 Β they receive fused proteins, or conservative substitutions of amino acid residues in and / or near the amino acid sequence, but the consequence is a silent change, which means that the change produces functionally equivalent erythropoietin. Such amino acid substitutions can be performed based on the similarity of polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphipathic (containing both hydrophilic and hydrophobic groups) nature of the residue in question. For example, non-polar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, and methionine; half-neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine; positively charged (basic) amino acids include arginine, lysine, and histidine; and negatively charged (acidic) amino acids include aspartic and glutamic acid. Alternatively, non-conservative amino acid changes, as well as larger insertions and deletions can be applied to create functionally altered erythropoietin mutants. Such mutants can be used to alter erythropoietin properties in a desired direction. For example, in one embodiment, the erythropoietin useful in carrying out the present invention may be an erythropoietin mutant altered in one or more amino acids into four functional erythropoietin domains that act on receptor binding: VLQRY and / or TK.VNFYAW and / or SGLRSLTTL and / or SNFLR . In another embodiment, erythropoietins may be used that contain mutations in the vicinity of the molecules that act on the kinetics or receptor binding properties of the molecules.

The term erythropoietin as well as single erythropoietin may be used alternately or together, and various analogs, moieties, hybrid molecules, agonists, muteins, and other forms as described above include variants in the extent and position of glycosylation of erythropoietin, including native, deglycosylated, asylated, and other partially glycosylated forms of erythropoietin. Non-limiting examples of such variants have been described by Tsuda et al.,

52276 Β

1990, Eur. J. Biochem. 188: 405-411, and incorporated herein by reference. In addition, various host systems can be used to express and produce recombinant erythropoietin, including, but not limited to, the cellular systems of bacteria, yeast, insects, plants, mammals, including humans. For example, recombinant erythropoietin produced in bacteria that does not produce glycosylated or sialylated product, can be used to obtain non-glycosylated erythropoietin forms. Alternatively, recombinant erythropoietin can be produced in other glycosylate-producing systems, e.g., plants, including human cells.

As noted above, the present invention encompasses any and all erythropoietin receptor modulator molecules capable of exhibiting positive activity against erythropoietin-responsive cells, regardless of any structural affinity of that molecule with erythropoietin.

In addition, erythropoietin itself can be modified to adapt its activities to a specific tissue or tissues. There are several non-limiting strategies that can be performed to achieve the desired tissue specificity and these include, modifications that shorten the periodic half-life and thus reducing the time erythropoietin may react with erythroid precursors, or modification of the primary structure of erythropoietin molecules. One approach to reducing the periodic half-life is to remove or modify the glycosylation moieties, of which entropoetin has three saN-bonds and one with an 0-bond. Such variants of glycosylated erythropoietin can be obtained in a number of ways. For example, sialic acids at the end of sugar chains can be removed by specific sialidases, depending on the chemical bond by which sialic acid is attached to the sugar chain.

52276 Β The glycosylated structure can be removed in different ways using other enzymes that break down specific bonds. Methods for modifying the primary structure are numerous and include substitution of specific amino acids, chemical modification of amino acids, or addition of other structures that interfere with the interaction of erythropoietin with any of its receptors. The use of such forms of erythropoietin is fully encompassed herein. In a preferred embodiment, the non-erythropoietin half-life of the present invention is shortened by about 90% compared to that of native erythropoietin.

Some of these molecules will mimic the effects of erythropoietin itself in other tissues or organs. For example, 17-merco contains the amino acid sequence 31-47 of native erythropoietin is inactive for erythropoiesis but is fully active for nerve cells in vitro (Campana & O ' Brien, 1998: Int. J. Mol. Med. 1: 235-41).

In addition, erythropoietin derivative molecules preferred for the uses described herein can be obtained by guanidination, amidination, carbamylation (carbamoylation), trinitrophenylation, acetylation, succinylation, nitration, or modification of residues and 1 and carboxino groups of arginine, lirosine, or cysteine, among other methods, as a limited proteolyzer by removal of amino groups, and / or mutational substitution of arginine, lysine, tyrosine, tryptophan residues, or cysteine ​​by molecular biology methods, to obtain erythropoietins that maintain an appropriate level of activity for specific organs and tissues but not for others, such as erythrocytes (e.g., Satake et al .; 199.0, Biochim. Biophvs. Acta 1038: 125-9; incorporated herein by reference in its entirety). One non-limiting example. as described below, is a modification of erythropoietin arginine residues by reaction with a glyoxal such as phenylglyoxal (according to

52276 Ta Takahashi Protocol, 1977, J. Biochem. 81: 395-402). As will be seen later, such a molecule of erythropoietin fully retains its neurotrophic effect. Such erythropoietin molecules have been fully adopted for the various applications and mixtures described herein.

Synthetic and recombinant molecules, such as brain erythropoietin and renal erythropoietin, recombinant forms of erythropoietin in mammals, as well as natural, tumor-derived, and recombinant isoforms, such as recombinant-expressed homologous molecules, are provided herein. In addition, the present invention encompasses molecules including erythropoietin receptor binding peptides, as well as recombinant compounds or other molecules possessing part or all of the structures and / or biological properties of erythropoietin, including erythropoietin fragments and multimers or fragments thereof. Erythropoietin herein includes molecules with altered erythropoietin receptor binding activities, preferably with increased receptor affinity, particularly with respect to increased transport across endothelial cell barriers. Included herein are muteins that contain molecules that have an increased or decreased number of glycosylation sites. As mentioned above, the terms erythropoietin and imitations, as well as other terms herein, are used interchangeably to indicate erythropoietin-responsive cell molecules with erythropoietin-related protective and enhancing properties, as well as molecules capable of breaking the endothelial cell barrier. In addition, molecules produced by transgenic animals are included herein. It should be noted that erythropoietin molecules as included herein are not necessarily similar in structure to erythropoietin in any other way than in their ability to react with the erythropoietin receptor or to modulate erythropoietin receptor activity or to activate erythropoietin-activating signaling cascades, such as described here.

52276 Β

In non-limiting examples, forms of erythropoietin useful for carrying out the present invention include erythropoietin muteins, such as those with altered amino acids at the carboxy position described in U.S. Patent 5,457,089 and U.S. Patent 4,835,260;

asialoerythropoietin and erythropoietin isoforms with different numbers of sialic acid residues in the molecule, as described in U.S. Patent 5,856,298; the polypeptides described in U.S. Patent 4,703,008; the agonists described in U.S. Patent 5,767,078; erythropoietin receptor binding peptides as described in U.S. Patent 5,773,569 and U.S. Patent 5,830,851; imitations that contain a small number of molecules and that activate the erythropoietin receptor, as described in U.S. Pat Patent 5,835,382; and the erythropoietin analogs described in WO 9505465, WO 9718318, and WO 9818926. All of the foregoing references are incorporated herein by the extent that such disclosures relate to various alternative forms or methods for preparing such forms of erythropoietin of the present invention.

Erythropoietin can be purchased, for example, under the trademark PROCRIT, from Ortho Biotech Inc., Raritan, NJ, and EPOGEN, from Amgen, Inc., Thousand Oaks, CA.

The activity (in units) of erythropoietin (erythropoietin) and entropoetin-like molecules is traditionally defined on the basis of its ability to stimulate red cell production in rodent models (and as obtained by international erythropoietin standards). One unit (U) of regular erythropoietin (MW of 3434,000) is 88 ng of protein (1 mg of protein is approximately 125,000 U). However, since the effect on erythropoiesis is secondary to the activities desired here and need not necessarily be a feature of the particular erythropoietins of the present invention, the definition of activity based on entropoietic activity is inappropriate. So, like here

52276 Β used, the unit of activity of erythropoietin or erythropoietin-like molecules is defined as the amount of protein required to elicit the same activity in erythropoietin-responsive nervous or other cellular systems as erythropoietin induced by the WHO international standard in the same system. One skilled in the art will readily determine units of erythropoietin that are not erythropoietin or similar molecules, following the instructions provided herein.

In connection with the aforementioned erythropoietin modifications used herein, the following discussion will be extended to the various erythropoietins of the present invention.

An erythropoietin of the present invention need not contain portions of sialic acid, as far as asialoerythropoietin is concerned. Preferably, the erythropoietin of the present invention is human asialoerythropoietin. In an alternative embodiment, the erythropoietin of the present invention may have at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 sialic acid residues. Asyaloerythropoietin can be obtained by desialylating erythropoietin using sialidase, as described in the manufacturer's package for a product called Sialvdase A from ProZyme Inc., San Leandro, Califomia. Typically, PR0ZYME® GLYCOPRO® sequence quality SIALYDASE A N (N-acetylneuraminate glycohydrolase, EC 3.2.1.18) is used to separate all terminal sialic acid residues that are not reduced from complex carbohydrates and glucoproteins, such as erythropoietin. It also separates sialic acids with a branched string (bound together by a common residue). Sialidase A was isolated from the Arthrohacter ureafaciens clone.

One erythropoietin may have at least a reduced number of N-linked carbohydrates. In order

52276 Β removal of carbohydrates with N-bond, erythropoietin can be treated with hydrazine, according to, for example, the methods described by Hermentin et al., 1996, Glycobiology 6 (2): 217-30. As stated above, erythropoietin has three N-linked hydrocarbon moieties, the present invention encompassing those erythropoietins having two, one or no N-linked carbohydrates.

An erythropoietin of the present invention may have at least a reduced carbohydrate content due to treatment of native erythropoietin with at least one glucosidase. For example, the procedure of Chen and Evangelista, 1998, Electrophoresis 19 (15): 2639-44 can be followed. In addition, the elimination of O-linked carbohydrates can be achieved using the procedures described by Hokke et al., 1995, Eur. J. Biochem.228 (3): 981-1008.

A portion of carbohydrate erythropoietin molecules may have at least one glycosylation pattern in mammalian animals based on the expression of recombinant erythropoietin in animal cells other than mammals. Preferably, the erythropoietins are expressed in insect or plant cells. In a non-limiting example, the expression of erythropoietin in insect cells by the baculovirus expression system can be performed in accordance with the work of Quelle et al., 1989, Blood 74 (2): 652-657. Another method is described in U.S. Patent 5,637,477. Expression in the plant system can be performed according to the mode of operation of the author Matsumoto et al., 1993, Biosci. Biotech. Biochem. 57 (8): 1249-1252. Alternatively, non-glycosylated forms of erythropoietin will be obtained by expression in the bacterium. These are only examples of methods used to prepare the erythropoietin of the present invention and do not constitute any limitation.

One entropoetin of the present invention may have at least one or more oxidized carbohydrates that may also be chemically reduced. For example, erythropoietin may be

52276 Β erythropoietin oxidized by periodate; Periodate-oxidized erythropoietin can also be chemically reduced by a borohydride salt such as sodium borohydride or sodium cyanoborohydride. Oxidation by the peiodate of borohydides can be performed, for example, by the methods described by the authors Linsley et al., 1994, Anal. Biochem.219 (2): 207-17. Chemical reduction following periodate oxidation can be performed according to the methods of TopeŠ and Meints, 1978, J. Supramol. Struct. 8 (1): 67-78.

One erythropoietin for the aforementioned applications may have at least one or more modified arginine residues. For example, the modified erythropoietin may contain an R-glyoxal moiety on one or more arginine residues, wherein R may be an anl, heteroaryl, lower alkyl, lower alkoxy, or cycloalkyl group, or an alpha-deoxyglycitolyl group. As used herein, the term lower alkyl means a straight or branched saturated aliphatic hydrocarbon group preferably containing 1-6 carbon atoms. Examples of these groups are methyl, ethyl, isopropyl, isobutyl, butyl, pentyl, hexyl groups and the like. The term alkoxy means a lower alkyl group as defined above, which is attached to the rest of the molecule via oxygen. Examples of alkoxy groups include methoxy, ethoxy, propoxy, isopropoxy and the like. The term cycloalkyl refers to cyclic alkyl groups having three to about 8 carbon atoms, which include, for example, cyclopropyl, cyldobutyl, cyclohexyl groups and the like. The term "aryl" refers to phenyl and naphthyl groups. The term "heteroaryl" refers to heterocyclic groups containing 4-10 ring members and 1-3 heteroatoms selected from the group consisting of oxygen, nitrogen and sulfur. Examples include, but are not limited to, isoxazolyl, phenylisoxazolyl, furyl, pyrimidinyl, quinolyl, tetrahydroquinolyl, pyridyl, imidazolyl, pyrrolidinyl, 1,2,4-triazoyl, thiazolyl, thierul group, and the like. The R group may be substituted, such as a 4-trihydroxybutyl group from 3-deoxyglucozone. Typical examples of R-glyoxal compounds are glyoxal, methylglyoxal, 3-deoxyglucozone and phenylglyoxal. Preferred R

52276 Β Glyoxal compounds are methylglyoxal or phenylglyoxal. Rtpeg procedure ίά such modification can be found in the authors Werber et al., 1975, Isr. J. Med. Sci. 11 (11): 1169-70, which uses phenylglyoxal.

In the following example, the arginine residue can be modified by reaction with a vicinal diketone such as 2,3-butanedione or cyclohexanedione, preferably in a buffer that forms a borate solution (50 mmol concentration) at pH 8-9. The method for the second modification with 2,3-butanedione can be performed by Riordan, 1973, Biochemistry 12 (20): 39153923; and the procedure with cyclohexanone rgeta to the work of Patthy et al., 1975, J. Biol. Chem 250 (2): 565-9.

An erythropoietin of the present invention may contain at least one or more modified lysine residues or a modification of the N-terminal amino group of erythropoietin molecules, such modifications as those obtained by reacting the lysine residue with an amino group modifying agent. In another embodiment, the lysine residues can be modified by reaction with glyoxal derivatives, such as a reaction with glyoxal, methylglyoxal and 3-deoxyglucozone to give alpha-carboxyalkyl derivatives. Examples are the reaction with glyoxal to give carboxymethyl lysine as in Glomb and Monnier, 1995, J. Biol. Chem. 270 (17): 10017-26, or with methylglyoxal to give (1-carboxyethyl) lysine as in Degenhardt et al., 1998, Cell. Mol. Biol. (Noisy-le-grand) 44 (7): 1139-45. The modified lysine residue can be further chemically reduced. On the other hand, erythropoietin can be biotinylated via 1 isine groups, according to the procedure described in Example 5, in which the d-biotinoyl-e-aminocapric acid -N-hydroxysuccinimide ester was reacted with sentropoetin and then the unreacted biotin removed by gel filtration on Centricon 10 column, as described by the author

52276 Β

Wojchowski and Caslake, 1989, Blood 74 (3): 952-8. In this paper, the authors use three different ways to biotinylate erythropoietin, each of which can be used for the applications listed herein. Biotin can be added to: (1) portions of sialic acid, (2) carboxylated groups, or (3) amino groups.

In another preferred embodiment, the lysine can be reacted with an aldehyde or reducing sugar to form an imine, which can be stabilized by reduction with sodium cyanoborohydride to give an N-alkylated lysine such as glucitolyl lysine, or which in the case of reducing sugars can be stabilized by Amadori or Heyns rearrangement to give alpha-deoxy-alpha-amino sugar which is alpha-deoxy-alpha-fructosyl lysine. An example of the preparation of a fructosyl lysine-modified protein by incubation with 0.5 M glucose in sodium phosphatan buffer solution pH 7.4 for 60 days is described by Makita et al., 1992, J. Biol. Chem. 267: 5133-5138. In another example, the lysine group may be carbamylated, for example, by reaction with a cyanate ion, or may be alkyl- or arylcarbamylated or -thiocarbamylated with alkyl- or aryl-isocyanate or -isothiocyanate, or may be acetylated by a reactive alkyl- or arylcarboxylic acids, such as by reaction with anhydride of acetic, amber or phthalic acid. Modifications of lysine groups with 4-sulfophenylisothiocyanate or acetic anhydride are suitable, both described by Gao et al., 1994, Proc Natl Acad Sci USA 91 (25): 12027-30. Lysine groups can also be modified by trinitrophenyl reaction with trinitrobenzenesulfonic acid or preferably with its salts. Such procedures are described below in Example 5.

At least one tyrosine residue from erythropoietin can be modified in the aromatic ring position by means of an electrophilic reagent, for example, by nitration or iodination. U

52276 In a non-limiting example, erythropoietin may be reacted with tetranitromethane (Nestler et al., 1985, J. Biol. Chem. 260 (12): 7316-21; or may be iodinated as described in Example 5.

At least one aspartic or glutamic acid residue from erythropoietin can be modified, for example, by reaction with a carbodiimide followed by reaction with an amine, such as, but not limited to, glycinamide. Examples of such modifications can be found in Example 5.

In another example, the tryptophan residue from erythropoietin can be modified, for example, by reaction with n-bromosuccinimide or n-chlorosuccinimide, according to the methods described by Josse et al., Chem Biol Interact 1999 Mau 14; 119-120.

In another example, the erythropoietin molecule can be obtained by removing at least one amino group, which can be achieved by reaction with ninhydrin followed by reduction of the next carbonyl group by reaction with borohydride.

In another example, erythropoietin having at least one free site in at least one cystine bond in the erythropoietin molecule is obtained by reaction with a reducing agent such as dithiothreitol, followed by reacting the obtained sulfhydryls with iodoacetamide, iodosycetic acid or another electrophilic reagent. re-formation of disulfide bonds.

Erythropoietin having at least one of a number of amino acids substituted is obtained,

52276 Β such as leucine, with at least one residue of lysine, arginine, tryptophan, tyrosine or cysteine ​​from erythropoietin, using molecular biology methods.

Modified erythropoietin can be obtained by subjecting erythropoietin to limited chemical proteolysis that affects specific residues, for example, to separate tryptophan residues. The erythropoietin fragments thus obtained are encompassed by the present invention.

As stated above, the erythropoietin used for the purposes of the present invention may have at least one of the aforementioned modifications, but may also have more than one of the aforementioned modifications. An example of a modified erythropoietin with one modification on the part of the carbohydrate molecules and one modification on the part of the molecules with amino acids, can be asialoerythropoietin whose lysine residues are biotinylated or carbamylated.

Various erythropoietin molecules and pharmaceutical compositions containing them for the uses described herein are encompassed by the present invention. Such erythropoietin molecules include, but are not limited to, asialoerythropoietin, N-deglycosylated erythropoietin, O-deglycosylated erythropoietin, erythropoietin with reduced carbohydrate content, erythropoietin with altered glycosylation patterns, erythropoietin with oxidized erythropoietin alkylglyoxal, 2,3-butanedione-modified erythropoietin, cyclohexanedione-modified erythropoietin. biotinylova erythropoietin, N-alkylovan-lysyl-erythropoietin, glucitolyl lysine erythropoietin, alpha-deoxy-alpha-fructosyl lysine-erythropoietin, carbamylated erythropoietin, acetylated erythropoietin, succinylated erythropoietin, succinylated erythropoietin, still non-limiting

52276 Β examples based on the doctrine presented here. The above-mentioned modified forms based on human erythropoietin are preferred.

Moreover, certain of the above erythropoietins are novel and the invention relates to such compounds as well as to pharmaceutical compositions containing them. In a non-limiting example, such novel erythropoietins include peiodaine-oxidized erythropoietin, glucitolyl lysine erythropoietin, fructosyl lysine erythropoietin, 3-deoxyglucozone erythropoietin, and carbamylated asialoerythropoietin.

The various vector systems present in the host can be used to prepare erythropoietin and erythropoietin-like molecules of the present invention. Such systems present in the host are carriers by which the erythropoietins of the present invention can be produced and then purified, but also cells that can, when transformed or transfected with the appropriate nucleotide coding sequences, represent a modified erythropoietin gene product in situ. These include, but are not limited to, the following host systems: bacteria, insects, plants, mammals, including the human host system, such as, but not limited to, insect cell systems infected with recombinant expressed virus vectors (e.g., baculovirus) that contain coding sequences of a modified erythropoietin product; plant cell systems infected with recombinant expressed virus vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with recombinant vectors present in the plasma (e.g., Ti plasmid) containing coding sequences of erythropoietin-like molecules;

or mammalian cell systems, including human cell systems. (e.g. HTI080, COS, CHO, BHK, 293, ZTZ) protecting recombinant expressed compounds containing promoters derived from a mammalian cell genome e.g., a metallothionein promoter) or from a mammalian virus (e.g., a promoter

52276 Β adenovirus; 7.5K virus vaccine promoter).

In addition, the host cell type may be selected to modulate the expression of the inserted sequences, or to modify the gene product in a desired specific manner. Such modifications (e.g., glycosylation) and processing (l /? L, separation) of protein products may be important for protein function. Different host cells have specific and characteristic mechanisms for post-translation and modification of proteins and gene products. Appropriate cell lines or systems hosts can be selected to ensure proper modification and processing of the expressed foreign proteins. Finally, eukanotic host cells having a cellular mechanism for proper processing of the primate transcript, glycosylation, and phosphorylation of the gene product may be used. Such mammalian host cells, including human host cells, include, but are not limited to, ΗΤ1080, CHO, VERO, BHK. , COS, MDCK, 293, ZTZ, and WI38.

For the production of recombinant proteins, long-term and with high yields, stable expression is desirable. For example, cell lines that stably express erythropoietin-like gene product molecules can be designed. Preferably, instead of using expressed vectors containing viral replication sources, host cells are transformed with DNA controlled by appropriate expression control elements (e.g., promoter, amplifier, sequences, transcription terminators, polyadenylation sites, etc.), and selective marker After insertion of foreign DNA, the projected cells can be left to grow for 1-2 days in an enriched medium, and then transferred to a selective environment. A selective marker in a recombinant plasmid provides resistance to preselection and allows cells to stably integrate the plasmid into their

52276 Β chromosomes and to grow to form foci that can be cloned and expanded into cell lines. This method can be usefully used to design cell lines that express erythropoietin-like gene product molecules. Thus designed cell lines may be particularly useful for the protection and evaluation of compounds that affect the endogenous activity of erythropoietin-like gene product molecules.

Alternatively, the expressed characteristic of the endogenous erythropoietin gene within a cell line or microorganism can be modified by inserting a heterologous DNA regulatory element into the genome of a stable cell line or cloned microorganism so that the inserted regulatory element is operably linked to the endogenous erythropoietin gene. For example, an endogenous erythropoietin gene that is normally transcriptionally silent, e.g., an erythropoietin gene that is not normally expressed, or is only slightly expressed in a cell line, can be activated by inserting a regulatory element capable of initiating the expression of a normally expressed gene product in that cell line or microorganism.

Alternatively, a transcriptionally silent, endogenous erythropoietin gene can be activated by inserting a common regulatory element that acts on cell types

The heterologous regulatory element can be inserted into a stable cell line or cloned microorganism so as to be operably linked to the endogenous entropoetin gene, using a method such as targeted homologous recombination, which is well known to those skilled in the art and described e.g. French patent No. Patent No. 2646438 (Pasteur Institute), U.S. Pat. 4,215,051 (Chappel); U.S. Pat. 5,578,461 (Sherwin et al.); International application no. PCT / US92 / 09627 (W093 / 09222) (Selden et al.) And International Application no. PCT / US90 / 06436 (WO91 / 06667) (Skoultchi et al.), Of which

52276 Β each attached here by reference.

In one embodiment of the present invention, an erythropoietin-related molecule that has an insufficient number of sialin residues, or no sialin residues at all, can be produced in a mammalian cell, including a human cell. Such cells can be designed to have insufficient, or deficient, enzymes that add sialic acid, prg .. β -galactoside ά 2,3 sialyltransferase (ά 2,3 sialyltransferase) and β-galactoside ά 2,6 sialyltransferase (ά 2 , 6 sialyltransferase) activity. In one embodiment, a mammalian cell is used in which one or both genes are released, the ά 2,3 sialyltransferase gene and / or the ά 2,6 sialyltransferase gene. Such deletions can be made using the so-called. genetic knock-out methods, which are well known in the art. In another embodiment, Chinese Hamster Ovary (CHO) cells deficient in dihydrofolate reductase (DHFR) have been used as host cells to produce recombinant erythropoietin-like molecules. CHO cells do not express the enzyme ά 2.6 sialyltransferase and therefore do not add sialic acid to the 2.6 bond of N-linked oligosaccharides from the glucoproteins produced in these cells. As a result, recombinant proteins produced in CHO cells lack sialic acid in the 2.6 bond from galactose (Sasaki et al. (1987; Takeuchi et al.; Mutsaers et al., Eur. J. Biochem. 156, 651 (1986)). Takeuchi et al., J. Chromotgr 400, 207 (1987) In one embodiment, in order to produce a host cell for the production of asialoerythropoietin, a gene with the code ά 2,3 sialyltransferase in CHO cells was deleted. Such CHO cells knocked out of 2,3 sialyltransferase have no sialyltransferase activity at all, and as a result they are used for recombinant expression and production of asialoerythropoietin.

In another embodiment, asia or glycoproteins can be produced by disrupting transport

52276 Β sialic acids in the Golgi apparatus, as reported by the author (Eckhardt et al., 1998, J. Biol. Chem. 273: 20189-95). By applying methods well known to those skilled in the art (prg, Oelmann et al., 2001, J. Biol. Chem. 276: 26291-300), mutagenesis of the CMP-sialic acid sugar nucleotide transporter can be achieved to produce cell mutants. ovaries of the Chinese hamster. These cells are unable to add sialic acid residues to glycoproteins such as erythropoietin and produce only asialoerythropoietin. Transfected mammalian cells that produce erythropoietin also produce cytosolic sialidase, which, if released into the culture medium, very efficiently degrades asialoerythropoietin (eg, Gramer et al., 1995 Biotechnology 13: 692-698). Using methods well known to those skilled in the art (e.g., from information provided by Ferrari et al., 1994, Glycobiology 4: 367-373), cell lines can be transfected, mutated, or otherwise induced to produce sialidases. In this way, asialoerythropoietin can be produced during the preparation of asialoerythropoietin.

In one aspect of the present invention, a pharmaceutical composition, as described above, comprising erythropoietin may be administered to a mammal in any manner that provides a sufficient level of erythropoietin in the vascular system that allows translocation across the endothelial cell barrier and effective effects in erythropoietin-responsive cells. . When used for tissue or organ perfusion, similar results are desirable. In the case where erythropoietin is used for ex-vivo perfusion, erythropoietin may take any form of erythropoietin, such as the aforementioned erythropoietins, but is not limited to and may contain native erythropoietins, including human erythropoietin. In case the cells or tissue are not related to the vascular system and / or the application is done by bathing the cells or tissues with the compositions of the present invention, the pharmaceutical composition provides an effective amount of erythropoietin that is effective for the cells.

52276 Β responsive to entropoetin. Endothelial cell barriers through which erythropoietin can be translocated include solid bonds, perforated bonds, hollow bonds, and all other types of endothelial barriers present in mammals. A solid bond endothelial cell barrier is preferred, but the invention is not so limiting.

The aforementioned erythropoietins are generally useful for the therapeutic or prophylactic treatment of diseases of the central nervous system or peripheral nervous system in humans who have receiving neurological or psychiatric symptoms, as well as for eye diseases, cardiovascular diseases, cardiopulmonary diseases, respiratory diseases, kidney, unname and reproductive diseases. , gastrointestinal diseases and endocrine and metabolic abnormalities.

In particular, such conditions and diseases include conditions of hypoxia, which adversely affect excited tissues, such as excited tissues in central nervous system tissue, peripheral nervous system or in cardiac tissue or in retinal tissue such as, for example, brain, heart or retina / eye. Therefore, the invention can be used to treat or prevent excited tissue injury that occurs as a result of a state of hypoxia under various conditions and circumstances. Non-limiting examples of such conditions and circumstances are shown herein in the table below.

In an example of protection against nerve tissue pathologies that can be treated in accordance with the present invention, such pathologies include those arising from reduced oxidation of nerve tissues. Any condition that reduces the availability of oxygen in the nervous tissue, resulting in stress, injury and ultimately nerve cell death, can be treated using the methods of the present invention. Commonly known as hypoxia and / or ischemia, these conditions result from or include, but are not limited to, stroke, vascular occlusion, prenatal or postnatal oxygen loss, suffocation, suffocation,

52276 Β strangulation, carbon monoxide poisoning, smoke inhalation, injuries, including surgery and radiation therapy, asphyxia, epilepsy, hypoglycaemia, chronic obstructive pulmonary disease, emphysema, adult respiratory distress syndrome, hypotensive shock, septic shock, anaphylactic shock, anaphylactic shock diseased cells, cardiac arrest, dysrhythmia, nitrogen anesthesia, and neurological deficits caused by bypass procedures for the heart and lungs.

In one embodiment, for example, a specific EPO composition may be administered to prevent injury or tissue injury due to the risk of injury or tissue injury during surgical procedures, such as, for example, tumor resection or aneurysm recovery. Other pathologies that are caused or caused by hypoglycemia and can be treated with the procedures described herein include insulin overdose, also known as iatrogenic hyperinsulinemia, insulin, growth hormone deficiency, cortisone depletion, drug overdose, and certain tumors.

Other pathologies that originate from excited nerve tissue include seizures, such as epilepsy, convulsions, or chronic seizures. Other treatable conditions and diseases include diseases such as stroke, multiple sclerosis, hypotension, cardiac arrest, Alzheimer's disease, Parldson's disease, cerebral palsy, brain or spinal cord injuries, AIDS dementia, loss of cognitive function age, memory loss, amyotrophic lateral sclerosis, seizures, alcoholism, retinal ischemia optic nerve injury caused by glaucoma and nerve cell loss

Specific compositions and methods of the present invention may be used to treat conditions and injuries of retinal tissue. Such disorders include, but are not limited to, retinal ischemia,

52276 Β macular degeneration, retinal detachment, retinitis pigmentosis, arteriosclerotic retinopathy, hypertensive retinopathy, retinal artery occlusion, retinal vein occlusion, hypotension and diabetic retinopathy.

In another embodiment, the principles of the methods of the present invention can be applied to protect or treat injuries resulting from injuries to excited tissue during radiation. Another benefit of the methods of the present invention is in the treatment of neurotoxin poisoning, such as shellfish poisoning, neurolatirism and Guam disease, amyotrophic Iateral sclerosis and Parkinson's disease. '

As noted above, the present invention also relates to a method of enhancing excited tissue function in a mammal by peripherally administering erythropoietin as described above. Various diseases and conditions are available for treatment using this procedure, moreover, this procedure is useful for increasing the function of cognition in the absence of any condition or disease. These applications of the present invention are described in more detail below and include increasing learning and exercise opportunities in both humans and other mammals.

Conditions and diseases that are treated by methods based on this aspect of the present invention, which relate to, but are not limited to, mood disorders, mood disorders, anxiety disorders, depression, autism, attention deficit hyperactivity disorder, and cognitive dysfunction. These conditions are effectively affected by increasing the function of nerve cells. Other disorders that can be treated in accordance with the doctrine of the present invention include disturbed sleep, for example, sleep apnea and travel disorders; subarachnoid and aneurysmal bleeding, hypotensive shock, stroke injury, septic shock, anaphylactic shock, and the consequences of various encephalitis and meningitis, for example, connective tissue diseases such as lupus.

52276 Β

Other uses include precaution or protection against neurotoxin poisoning, such as shellfish poisoning, neurolatirism, and Guam's disease, amyotrophic lateral sclerosis, Parkinson's disease; postoperative treatment of injuries caused by embolism or ischemia; irradiating the whole brain of a diseased cell crisis; and eclampsia

Another group of conditions that can be treated by the methods of the present invention include mitochondrial dysfunction, whether inherited or acquired, which causes various neurological diseases characterized by neuronal injury and death. For example, Leigh's disease (subacute necrotic encephalopathy) is characterized by progressive vision loss and encephalopathy, due to a decrease in neurin content and myopathy. In these cases, defective mitochondrial metabolism fails to supply enough high-energy substrates to stimulate the metabolism of excited cells. A modulator of erythropoietin receptor activity optimizes this deficient function in various mitochondrial diseases. As mentioned above, hypoxia conditions adversely affect excited tissues. Excited tissues include, but are not limited to, central nervous system tissue, peripheral nervous system tissue, and cardiac tissue. In addition to the conditions described above, the methods of the present invention are useful for treating inhalation poisoning such as inhalation of carbon monoxide and smoke, severe asthma, adult respiratory distress syndrome, suffocation and strangulation. Other conditions that affect hypoxia or otherwise cause injury excited tissue include hypoglycemia that may occur with inadequate insulin dosing, or with insulin-producing neoplasms (insulin).

Various neuropsychological disorders believed to originate from excited tissue injury are treated by emergency procedures Chronic disorders involving nerve cell injury and for

52276 Β which treatments provided by the present invention include disorders related to the central nervous system and / or peripheral nervous system including age-related loss of cognitive function, chronic seizures, Alzheimer's disease, Parkinson's disease, dementia, memory loss, amyotrophic lateral sclerosis, multiple sclerosis, tuberous sclerosis, Wilson's disease, cerebral and supranuclear palsy, Guam's disease, body dementia according to Lewy, prion disease, such as spongiform encephalopathies, e.g., Creutzfeldt-Jakob disease, Huntington's disease, myotonic dystrophy, Freidrich's ataxia and other ataxias, as well as Gilles de Ia Tourette's syndrome, seizures such as epilepsy and chronic seizures, cap , brain or spinal cord injury, AIDS dementia, alcoholism, autism, retinal ischemia, glaucoma, autonomic dysfunction disorders such as hypertension and sleep disorders, and neuropsychiatric disorders including, but not limited to, schizophrenia, schizoaffective disorders, attention deficit disorder, dystrophic disorder, major depressive disorder, mania, obsessive-compulsive disorder, psychoactive substance use disorders, anxiety, panic disorder, as well as unipolami and bipola affective disorders. Additional neuropsychiatric and neurodegenerative disorders include, for example, those listed in the book American Psychiatric Association's Diagnostic and Statistical Manual of Mental Disorders (DSM), the latest version of which is incorporated herein in its entirety.

52276 Β

In another embodiment, the recombinant erythropoietin-containing chimeric toxin molecules can be used for the therapeutic administration of a toxin for the treatment of a reproductive disorder, such as cancer, or a viral disorder, such as subacute sclerotic panencephalitis.

The following table lists the exempla, non-limiting indications for various conditions and diseases that can be treated with the aforementioned erythropoietins.

<td>cell, tissue, or organ</td><td>dysfunction. or pathology</td><td>condition Hi disease</td><td>type</td>

<td>heart</td><td>ischemia</td><td>coronary artery disease</td><td>acute, chronic, stable,</td>

<td></td><td></td><td>myocardial infarction</td><td>Dresler's syndrome</td>

<td></td><td></td><td>angina</td><td></td>

<td></td><td></td><td>congenital heart disease</td><td>valvular cardiomyopathy</td>

<td></td><td></td><td>Prinzmetal's angina</td><td></td>

<td></td><td></td><td>heart rupture</td><td>septal perforation of the aneurysm</td>

<td></td><td></td><td>angeiitis</td><td></td>

<td></td><td>arrhythmia</td><td>tachy-, bradyarrhythmia supraventricular, ventricular conduction abnormalities</td><td>stable, unstable hypersensitive carotid sinus node</td>

<td></td><td>congestive heart failure</td><td>left, right, biventriculama</td><td>cardiomyopathies, such as idiopathic familial, infectious, metabolic, accumulation disease, defects, connective tissue disorders, infiltration and granuloma neurovascular</td>

<td></td><td></td><td>myocarditis</td><td>autoimmune, infectious, idiopathic</td>

<td></td><td></td><td>so pulmonary</td><td></td>

<td></td><td>blunt and penetrating trauma</td><td></td><td></td>

<td></td><td>toxins</td><td>cocaine</td><td></td>

52276 Β

<td>“'Τ ^1,1 ......¹ ' Cell, tissue or organ</td><td>dysfunction or pathology</td><td>condition Ui disease</td><td>Type</td>

<td></td><td></td><td></td><td></td>

<td>vasculature</td><td>hypertension</td><td>primama, seconds</td><td></td>

<td></td><td>decompression sickness</td><td></td><td></td>

<td></td><td>fibromuscular hyperplasia</td><td></td><td></td>

<td></td><td>aneurysm</td><td>dissecting, perforated, enlarged</td><td></td>

<td></td><td></td><td></td><td></td>

<td>lungs</td><td>obstructive</td><td>asthma, chronic bronchitis, emphysema, and airflow obstruction</td><td></td>

<td></td><td>ischemic lung disease</td><td>pulmonary embolism, pulmonary thrombosis, fatty embolism</td><td></td>

<td></td><td>pulmonary diseases of the environment</td><td></td><td></td>

<td></td><td>ischemic lung disease</td><td>pulmonary embolism pulmonary thrombosis</td><td></td>

<td></td><td>interstitial lung disease</td><td>idiopathic pulmonary fibrosis</td><td></td>

<td></td><td>congenital</td><td>cystic fibrosis</td><td></td>

<td></td><td>pulmonary cor</td><td></td><td></td>

<td></td><td>trauma</td><td></td><td></td>

<td></td><td>pneumonia and pneumonitis</td><td>infectious, parasitic, toxic, traumatic, burns. aspiration</td><td></td>

<td></td><td>Sarcoidosis</td><td></td><td></td>

<td></td><td></td><td></td><td></td>

<td>pancreas</td><td>endocrine</td><td>diabetes, type I and II</td><td>beta cell dysfunction, diabetic dysfunction</td>

<td></td><td></td><td>other pancreatic endocrine cell dysfunctions</td><td></td>

<td></td><td>exocrine</td><td>exocrine pancreatic dysfunction</td><td>pancreatitis</td>

<td></td><td></td><td></td><td></td>

<td>bone</td><td>osteopenia</td><td>primama seconds</td><td>hypogonadism immobilization</td>

52276 Β

<td>cell, tissue go organ</td><td>dysfunction or pathology</td><td>condition or disease</td><td>dp</td>

<td></td><td></td><td></td><td>postmenopausal age-related hyperparathyroidism hyperryreoidism calcium, magnesium deficiency. phosphorus and / or vitamin D</td>

<td></td><td>osteomyelitis</td><td></td><td></td>

<td></td><td>avascular necrosis</td><td></td><td></td>

<td></td><td>trauma</td><td></td><td></td>

<td></td><td>Paget's disease</td><td></td><td></td>

<td></td><td></td><td></td><td></td>

<td>leather</td><td>alopecia</td><td>areata totalis</td><td>prima, seconds, pattern of male pattern baldness</td>

<td></td><td>vitiligo</td><td>localized generalized</td><td>in seconds</td>

<td></td><td>diabetic ulceration</td><td></td><td></td>

<td></td><td>peripheral vascular disease</td><td></td><td></td>

<td></td><td>Burns</td><td></td><td></td>

<td></td><td></td><td></td><td></td>

<td>autoimmune disorders</td><td>lupus erythematosus, Sjögren's, rheumatoid arthritis, glomerulonephritis, angiitis</td><td></td><td></td>

<td></td><td>Langerhans' histiocytosis</td><td></td><td></td>

<td></td><td></td><td></td><td></td>

<td>eye</td><td>optic neuritis</td><td></td><td></td>

<td></td><td>blunt penetrating injuries, infections, Sarcoid, Sickle C disease, retinal ablation, temporal arteritis</td><td></td><td></td>

<td></td><td></td><td></td><td></td>

<td>fetal and embryonic disorders</td><td>asphyxia</td><td></td><td></td>

<td></td><td>ischemia</td><td></td><td></td>

52276 Β

<td>cell, tissue, or organ</td><td>dysfunction or paiology</td><td>condition or disease</td><td>ίψ</td>

<td></td><td></td><td></td><td></td>

<td>CNS</td><td>chronic fatigue syndrome, acute and chronic hypoosmolar hyperosmolar syndromes, AIDS dementia, electric shock</td><td></td><td></td>

<td></td><td>encephalitis</td><td>rabies, herpes</td><td></td>

<td></td><td>meningitis</td><td></td><td></td>

<td></td><td>subdural hematoma</td><td></td><td></td>

<td></td><td>nicotine addiction</td><td></td><td></td>

<td></td><td>drug use and discontinuation of drug use</td><td>cocaine, heroin, kgek, manhuana, LSD, PCP, multiple drug use, ecstasy, opioids, sedates, amphetamines, caffeine</td><td></td>

<td></td><td>obsessive-compulsive disorder</td><td></td><td></td>

<td></td><td>spinal stenosis, transverse myelitis, Gmllian Barre, trauma, nerve root compression, tumor compression, heat stroke</td><td></td><td></td>

<td>ENT</td><td>buzzing Meniere's syndrome hearing loss</td><td></td><td></td>

<td></td><td>traumatic injury, barotrauma</td><td></td><td></td>

<td>kidney</td><td>renal failure</td><td>acute, chronic</td><td>vascular / ischemic, intestinal disease, diabetic kidney disease, nephrolic syndromes, infections</td>

<td></td><td>Henoch S. Purpura</td><td></td><td></td>

<td></td><td></td><td></td><td></td>

<td>transverse striated muscles</td><td>autoimmune disorders</td><td>miasterua gravis dermatomyositis polymyositis</td><td></td>

52276 Β

<td>cell, tissue, or organ</td><td>dysfunction or pathology</td><td>condition or disease</td><td></td>

<td></td><td></td><td></td><td></td>

<td></td><td>myopathies</td><td>hereditary-metabolic, endocrine and toxic</td><td></td>

<td></td><td>heat stroke</td><td></td><td></td>

<td></td><td>crash syndrome</td><td></td><td></td>

<td></td><td>rhabdomyositis</td><td></td><td></td>

<td></td><td>mitochondrial disease</td><td></td><td></td>

<td></td><td>infection</td><td>necrotizing fasciitis</td><td></td>

<td></td><td></td><td></td><td></td>

<td>sexual dysfunction</td><td>central and peripheral</td><td>seconds of impotence due to drug use</td><td></td>

<td></td><td></td><td></td><td></td>

<td>liver</td><td>hepatitis</td><td>viral, bacterial, parasitic</td><td></td>

<td></td><td>ischemic disease</td><td></td><td></td>

<td></td><td>cirrhosis, fatty liver</td><td></td><td></td>

<td></td><td>inflammatory / metabolic diseases</td><td></td><td></td>

<td></td><td></td><td></td><td></td>

<td>gastromtestmalm tract</td><td>ischemic disease</td><td></td><td></td>

<td></td><td>inflammatory disease</td><td></td><td></td>

<td></td><td>necrotizing enterocolitis</td><td></td><td></td>

<td></td><td></td><td></td><td></td>

<td>organ transplantation</td><td>treatment of donor and recipient</td><td></td><td></td>

<td></td><td></td><td></td><td></td>

<td>reproductive tract</td><td>infertility</td><td>vascular autoimmune uterine abnormalities, implantation disorders</td><td></td>

<td></td><td></td><td></td><td></td>

<td>endocrine system</td><td>hyper- and hypofunction of the gland</td><td></td><td></td>

As mentioned above, these diseases, disorders or conditions merely illustrate the numerous benefits provided by the erythropoietins of the present invention. Accordingly, the present invention in general

52276 Β provides therapeutic or prophylactic treatment for the consequences of mechanical trauma or human disease. Therapeutic or prophylactic treatment for diseases, disorders or conditions in the central nervous system of the CNS and / or peripheral nervous system is preferred. Therapeutic or rgophylactic treatment is provided for diseases, disorders or conditions that have a psychiatric component. Therapeutic or prophylactic treatment of diseases, disorders or conditions, including, but not limited to, those containing ophthalmic, cardiovascular, cardiopulmonary, respiratory, renal, urinary, reproductive, gastrointestinal, endocrine, or metabolic components is provided.

In one embodiment, such a pharmaceutical composition with erythropoietin may be administered systemically to protect or enhance target cells, tissues or organs. Such administration may be parenteral, by inhalation, or transmucosal, e.g., oral, nasal, rectal, intravaginal, sublingual, submucosal, or transdermal. Preferably, administration is parenteral, e.g., by intravenous or intraperitoneal injection, and also includes, but is not limited to, intra-arterial, intramuscular, intradermal, and subcutaneous administration.

For other routes of administration, such as administration of a perfusion solution, injection into an organ, or other topical administration, a pharmaceutical composition with similar erythropoietin levels as described herein is provided. A level of about 15pM-30 nM is preferred.

The pharmaceutical compositions of the present invention may consist of a therapeutically effective amount of a compound and a pharmaceutically acceptable carrier. In a specific embodiment, the term pharmaceutically acceptable means that it is approved by the state administration regulatory agency

52276 Β or that it is listed in the US Pharmacopeia or in another generally recognized foreign pharmacopoeia for use in animals and especially in humans. The term carrier refers to a diluent, adjuvant, excipient, or vehicle when the therapy is administered. Such pharmaceutical carriers may be sterile liquids, such as saline solutions in water and oils, including oils derived from petroleum, animals, plants or synthetic by the way, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. The saline solution is the preferred carrier when the pharmaceutical composition is administered intravenously. Saline and aqueous solutions of dextrose and glycerin can be used as liquid carriers, especially for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerin, propylene, glycol and water, . If desired, the mixture may contain small amounts of wetting or emulsifying agent, or pH buffers. These mixtures may be in the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained release formulations and the like. The mixture may be formulated as a suppository, usually diarrhea preventants and carriers such as triglycerides. The compounds of this invention may be formulated as neutral or in salt form. Pharmaceutically acceptable salts include those formed with free amino groups, such as those derived from hydrochloric, phosphomic, acetic, oxalic, tartaric acid, etc., and those formed with free carboxyl groups, such as those derived from sodium, potassium , ammonium, calcium, iron hydroxide, isopropylamine, triethylamine, 2-ethylamino ethanol, histidine, procaine, etc. Examples of suitable pharmaceutical carriers are described in the book Remington Pharmaceutical Sciences, by EW Martin. Such compositions contain therapeutically effective amounts of the compound, preferably in purified form, together with a suitable amount of carrier to provide a form suitable for administration.

52276 Β to the patient. The formulation should correspond to the method of application.

Pharmaceutical compositions adapted for oral administration may be in the form of capsules or tablets; powders or granules; solutions, syrups or suspensions (in water or liquids other than water); edible foams or creams; or emulsion. Hard gelatin tablets or capsules may contain lactose, starch or derivatives thereof, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, stearic acid or its salts. Soft gelatin capsules may contain vegetable oils, waxes, fats, semi-solid or liquid polyols, etc. Solutions or syrups may contain water, polyols and sugars.

The active agent intended for oral administration may be coated or mixed with a substance that slows down the disintegration and / or absorption of the active agent in the gastrointestinal tract (e.g., glycenyl monostearate or glycenyl distearate may be administered). In this way, a sustained release of the active agent over many hours can be achieved, and if necessary, the active agent can be protected from degradation in the stomach. Pharmaceutical compositions for oral administration may be formulated so as to accelerate the release of the active agent at a particular site in the gastrointestinal tract depending on the specific pH or state of the enzyme.

Pharmaceutical compositions adapted for transdermal application may be in the form of separate patches intended to remain in intimate contact with the recipient's epidermis for an extended period of time. Pharmaceutical compositions adapted for topical administration may be in the form of ointments, creams, suspensions, lotions, powders, solutions, pastes, gels, sprays, aerosols or oils. For topical application to the skin, mouth, eye or other external tissues, it is desirable to use a local ointment or cream. When formulated in an ointment, the active ingredient may be employed with

52276 Β ointment base, which is paraffinic or miscible with water. Alternatively, the active ingredient may be formulated in a cream with a base containing oil in water or water in oil. Pharmaceutical compositions adapted for topical application to the eye include eye drops. In these mixtures the active ingredient may be dissolved or suspended in a suitable carrier, for example, in an aqueous solvent. Pharmaceutical compositions for topical application to the mouth include diamond-shaped tablets, lozenges and mouthwashes.

Pharmaceutical compositions adapted for nasal and pulmonary administration may consist of solid carriers such as powders (preferably the particle size is in the range of 20 to 500 microns). The powders can be applied in the manner in which snuff is sniffed, i.e., by rapid inhalation through the nose from a powder container held to the nose. Alternatively, compositions adapted for nasal application may consist of liquid carriers, i.e., nasal sprays or drips. Alternatively, inhalation directly into the lungs can be performed by deep inhalation or insertion of a tube into the oropharynx. These mixtures may be aqueous or oily solutions of the active ingredient. Mixtures for administration by inhalation may be provided with specially adapted devices including, but not limited to, pressurized aerosols, nebulizers or blowing devices, which may be designed to deliver predetermined doses of the active ingredient. In a preferred embodiment, the pharmaceutical compositions of the present invention are administered to the nasal cavity directly or to the lungs via the nasal cavity or oropharynx.

Pharmaceutical compositions adapted for rectal administration may be in the form of suppositories or enemas. The pharmaceutical compositions intended for vaginal administration may be in the form of pessaries, tampons, creams, gels, pastes, foams or spray formulations.

52276 Β

Pharmaceutical compositions adapted for patenteral administration include aqueous and non-aqueous sterile injectable solutions or suspensions, which may contain antioxidants, buffers, bacteriostats and solutes, which make the compositions substantially isotonic with the intended recipient's blood. Other components may be present in such mixtures including water, alcohols, polyols, glycerin and vegetable oils, for example. Mixtures adapted for parenteral administration may be in single-dose or multi-dose containers, for example, in sealed ampoules or vials, and may be stored in a refrigerated dried (lyophilized) state requiring only the addition of a sterile liquid carrier, e.g., sterile saline solution for injection, immediately before use. Improvised injection solutions and suspensions can be made from sterile powders, granules and tablets. In one embodiment, an auto-injector containing an erythropoietin solution for injection may be used for emergency care in ambulances, emergency rooms and in war situations, and even for self-administration at home, especially where there is a possibility of traumatic amputation, such as is the reckless use of a mower. The likelihood that cells and tissues in a severed foot or toe will survive reconnection can be increased by administering erythropoietin to multiple sites in the torn area as soon as possible, even when medical personnel arrive at the scene or an injured person with a severed toe under the auspices of the ambulance.

In a preferred embodiment, the compositions are formulated according to routine procedures as a pharmaceutical composition adapted for intravenous administration to humans. Typically, the compositions for intravenous administration are solutions in sterile isotonic aqueous buffer. When necessary, the composition may also contain a solubilizing agent and a local anesthetic such as lidocaine to relieve pain at the injection site. (In general, the ingredients are delivered separately

52276 Β or mixed together in the form of a single dose, for example, as a dry lyophilized powder or anhydrous concentrate in a hermetically sealed container such as an ampoule or sachet, on which the amount of active ingredient is indicated. When the mixture is administered by infusion, an infusion bottle containing sterile water or saline of pharmaceutical quality may be added. When the mixture is administered by injection, an ampoule of sterile saline may be added to it so that the ingredients can be mixed before administration.

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

The perfusion mixture can be used for use in baths where organs are kept for transplantation, for in situ perfusion, or for application to the vasculature system of the donor organ, other than the organ is separated from the body. Such pharmaceutical compositions may contain erythropoietin levels or a form of erythropoietin that is not suitable for acute or chronic, local or systemic administration to an individual, but will serve for intended functions in a corpse, organ bath, organ perfusate or in situ perfusate prior to removal or reduction of levels. erythropoietin contained in them, before the treated organ or tissue is exposed or returned to normal circulation. The erythropoietin for this aspect of the invention may be any erythropoietin, such as naturally occurring forms such as human erythropoietin, or any of the erythropoietins described hereinbefore, such as asialoerythropoietin and phenylglyoxal-erythropoietins, in the sense of non-limiting examples.

The present invention also provides a pharmaceutical package or carton comprising one or more containers filled with one or more ingredients of the pharmaceutical compositions of the present invention. Optional, with

52276 Β such a court or courts may contain an advertisement in the form prescribed by the state administration agency which regulates the preparation, use or sale of pharmaceutical or biological products, and which advertisement by the agency gives approval for the preparation, application or excessive application.

In another embodiment, for example, erythropoietin may be delivered in a controlled release system. For example, the polypeptide may be administered by intravenous infusion, osmotic implantation pump, transdermal patch, liposome, or other routes of administration. In one embodiment, a pump can be used (see Langer, supra: Sefton, 1987, CRC Crit. Ref. Biomed. Eng. 14.201; Buchvvald et al. 1980, Surgery 88: 507; Saudek et al., 1989, N. Engl J. Med. 321: 574). In another embodiment, the compound can be delivered in a carrier, particularly in a liposome (see Langer, Science 249: 1527-1533 (1990); Treai et al., Liposomes in the Tsegar of Infectious Disease and Cancer, Lopez Berestein and Fidler). ), Liss, New York, pp. 353-365 (1989); WO 91/04014; U.S. Patent No. 4,704,355; Lopez-Berestein, ibid., Pp. 317-327; see general ibid.). K. from another embodiment, polymeric materials can be used (see 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; see also Levv et al., 1985, Science 228: 190; et al., 1989, Ann. Neurol. 25: 351; Howard et al., 1989, J. Neurosurg. 71: 105).

In another embodiment, the controlled release system may be located close to the therapeutic target, i.e., the target cell, tissue, or organ, thus requiring only a portion.

52276 Β systemic doses (see, e.g., Goodson, pp. 115-138 in Medical Applications of Controlled Release, vol. 2, supra, 1984). Other controlled release systems have been discussed in a review written by Langer (1990, Science 249: 1527-1533).

In another embodiment, erythropoietin, when properly formulated, may be administered nasally, orally, rectally, vaginally or sublingually.

In a specific embodiment, it may be desirable to administer the erythropoietin mixtures of the present invention topically to the area to be treated; this can be achieved by, for example, but not limited to, local infusion during surgery, topical application, e.g., when dressing a wound after surgery, injection, catheter, suppository or implant, said implant may be made of a porous, non-porous, or gelatinous material, including membranes, such as silicone membranes or fibers.

The choice of the preferred effective dose will be determined by one skilled in the art based on consideration of a number of factors known to one of ordinary skill in the art. Such factors include a particular form of erythropoietin, and its pharmacokinetic parameters such as bioavailability, metabolism, half-life, etc., which will be determined during the usual process for obtaining legal approval for a pharmaceutical compound. Other factors to consider when determining the dose include the condition or disease being treated or the condition achieved in the normal individual, the patient's body weight, the route of administration, whether administration is acute or chronic, concomitant medications, and other factors. which are well known to affect the efficacy of the administered pharmaceutical agent. Thus precise dosing is a must

52276 Β determined in accordance with the assessment of the physician and the condition of each patient, eg, depending on the condition and immune system of the individual patient, according to standard clinical procedures.

In another aspect of the present invention, there is provided a perfusion solution for perfusion and preservation of organ transplants, wherein the perfusion solution comprises an amount of erythropoietin capable of protecting cells and associated cells, tissues and organs responsive to erythropyetin. Transplantation includes, but is not limited to, xenotransplantation, in which an organ (including cells, tissue, or other part of the body) is separated from the donor and transplanted into a different recipient; and autotransplantation, in which an organ is removed from one part of the body and replaced with another, including surgical procedures on the table, in which one organ can be removed and ex vivo cut, repaired, or otherwise manipulated, such as removal tumor, and restore the original site. In one embodiment, the perfusion solution is known as a (UW) solution made by the University of Vinsonins and protected (U.S. Patent No. 4,798,824), containing from about 1 to about 25 U / ml erythropoietin, 5% starch hydroxyethium (molecular weight) from about 200,000 to about 300,000 and which does not contain ethylene glycol, ethylene chlorohydrin, sodium chloride and acetone at all); 25 mM KH₂BY₄; ZtM glutathione; 5mM adenosine; 10 mM glucose; 10 mM HEPES buffer; 5mM magnesium gluconate; 1.5mM CaCl₂, 105mM sodium gluconate; 200,000 units of penicillin; 40 units of insulin; 16mg dexamethasone; I2mg phenol red and has a pH of 7.4-7.5 and an osmolality of about 320 mOSm / l. The solution is used to save the kidneys and pancreas from corpses before transplantation. Applying the solution may extend storage above the 30-hour limit, which is recommended for storing kidneys from corpses. This particular perfusion solution is merely an illustration of a number of such solutions that can be adapted for this application by adding an effective amount of erythropoietin. In another embodiment, the perfusion solution contains from about 5

52276 Β up to about 35 U / ml erythropoietin, or from about 10 to about 30 U / ml erythropoietin. As mentioned above, any form of erythropoietin can be used in this aspect of the invention

While the preferred recipient of erythropoietin for the purposes set forth herein is man, the methods of the present invention apply equally to other mammals, particularly domestic animals, domesticated animals, pets, and zoo animals. However, the invention is not so limiting and may be useful. on each mammal.

In further aspects of the ex-vivo invention, any erythropoietin such as, but not limited to, the erythropoietins described above, as well as native erythropoietins or an analog thereof, an imitation of erythropoietin, and a fragment of erythropoietin, a hybrid molecule of erythropoietin, an erythropoietin binding molecule, and erythropoietin agonist, renal erythropoietin, cerebral erythropoietin, its oligomer, its multimer, its mutein, a related compound, its naturally occurring form, its synthetic form, its recombinant form, its glycosylated variant, its deglycosylated variant, or a combination thereof.

In another aspect of the present invention, methods and compositions for enhancing the viability of cells, tissues or organs not isolated from the vasculature system by an endothelial cell barrier are shown by exposing cells, tissues or organs directly to an erythropoietin-containing pharmaceutical composition, either by application or in contact of a pharmaceutical composition containing entropoetin with the vascular system of tissues or organs. Increased activity of erythropoietin-responsive cells in the treated tissue or organ is responsible for the positive effects.

As described above, the present invention is based in part on the discovery that molecules

52276 Β Erythropoietin can be transported from the luminal surface to the surface of the basement membrane of endothelial cells from the capillaries of organs with strong connections of endothelial cells, including, for example, the brain, retina and testes. Thus, erythropoietin-responsive cells across the barrier represent acceptable targets for the effective effects of erythropoietin, just as other cell, tissue or organ types that contain or depend wholly or partially on erythropoietin-responsive cells represent the targets of the methods of the present invention. Without wishing to be bound by a particular theory, after erythropoietin transcytosis, erythropoietin may react with the erythropoietin receptor to an erythropoietin-responsive cell, which may be, for example, from nerves, retina, muscle, heart, lung, liver, kidney, small intestine, adrenal cortex, adrenal medulla, capillary endothelium, testis, ovary, pancreas or endometrial cells or tissues, and receptor binding can initiate a signaling transduction cascade that results in activation of erythropoietin-responsive gene or tissue expression programs within the cell, resulting in protection of the cell, tissue, or organ from damage that may be caused by toxins, chemotherapeutic agents, radiation therapy, hypoxia, etc. Thus, methods for protecting tissue containing ertropoietin-responsive cells from injury or stress due to hypoxia, and for enhancing the function of such tissue, are described in detail below.

In one embodiment of the present invention, a mammalian patient undergoes systemic chemotherapy for the treatment of cancer, including radiation therapy, which most commonly has adverse effects, such as damage to nerves, lungs, heart, ovaries or testicles. The administration of a pharmaceutical composition comprising erythropoietin as described above is performed before and during chemotherapy and / or radiation therapy, in order to protect various tissues and organs from damage caused by chemotherapeutic agents, such as testicular protection. Treatment may be continued until periodic chemotherapy levels fall below

52276 Β potential danger to the mammalian body.

In another embodiment, it has been planned to remove various organs from the body of a car accident victim for transplantation to a large number of recipients, some of whom have had to be transported remotely and over an extended period of time. Prior to organ harvesting, the victim was infused with a pharmaceutical composition containing erythropoietin, as described herein. Organs taken for transport were placed in an erythropoietin-containing perfusion solution as described herein, and stored in an erythropoietin-containing bath. Certain organs were continuously infused using a pulsating perfusion device, using an erythropoietin-containing perfusion solution according to the present invention. During transport, there was a minimal decline in organ function and after transplantation and re-perfusion of organs in situ.

In another embodiment, surgery on the heart valve required temporal cardioplegia and arterial occlusion. Before the operation, the patient was given an infusion of 500 U of erythropoietin per kg of body weight. Such treatment prevented cell damage due to hypoxia and ischemia, especially after repeated perfusion.

In another embodiment, in any surgical procedure, such as cardiopulmonary bypass, erythropoietin present in nature or any erythropoietin of the present invention may be used. In one embodiment, the administration of the erythropoietin-containing pharmaceutical composition as described above is performed before, during, and / or after bypass surgery, to protect the function of the brain, heart, and other organs.

52276 Β

In the preceding examples in which the erythropoietin of the present invention, including naturally occurring entropoetin, is used for ex-vivo administration, or for the treatment of erythropoietin-responsive cells such as cells from nerves, retina, muscle, heart, lung, liver, the kidneys, small intestine, adrenal cortex, adrenal medulla, capillary endothelium of the testis, ovaries, pancreas or endometrial cells or tissues, in the present invention there is provided a single dose pharmaceutical composition adapted to protect or augment erythropoietin-responsive cells, tissues or organs distal to the vasculature system containing, in a single dose, an effective non-toxic amount in the range of about 50,000 to 500,000 U, 60,000 to 500,000 U , 70,000 to 500,000 U, 80,000 to 500,000 U, 90,000 to 500,000 U, 100,000 to 500,000 U, 150,000 to 500,000 U, 200,000 to 500,000 U, 250,000 to 500,000 U, 300,000 to 500,000 U, 350,000 to 500,000 U, 400,000 to 500,000 U, or 450,000 to 500,000 U of erythropoietin, an erythropoietin receptor modulator, or an erythropoietin-activated receptor modulator and a pharmaceutically acceptable carrier In a preferred embodiment, an effective, non-toxic amount of erythropoietin is in the range of about 50,000 to 500,000 U. In a preferred embodiment, the erythropoietin of the aforementioned mixture is not erythropoietin.

In another aspect of the present invention, it has been found that erythropoietin administration restores cognitive function in animals that have suffered brain trauma. After a delay of 5 or 30 days with erythropoietin administration, it was still possible to restore function compared to seemingly treated animals, indicating the ability of erythropoietin to regenerate. or restore brain activity In this way, the invention relates to the use of erythropoietin in the preparation of a pharmaceutical composition for the treatment of brain trauma and other cognitive dysfunctions, including treatment long after injury (e.g., three days, five days a week a month or longer). The invention also relates to a method of treating a cognitive dysfunction that accompanies an injury by administering an effective amount

52276 Erythropoietin Any of the erythropoietins described herein can be used in this aspect of the invention.

In addition, this recovery aspect of the invention relates to the use of any of the erythropoietins of the present invention to prepare a pharmaceutical composition for repairing tissue or organ cell dysfunction, when treatment is initiated after and long after the original injury has occurred. for dysfunction. Moreover, treatment with the erythropoietin of the present invention may comprise the course of a disease or condition during the acute phase as well as the chronic phase.

In the case where the erythropoietin of the present invention has erythropoietin activity, in a preferred embodiment, the erythropoietin may be administered systemically in doses between about 300 and about 10,000 U / kg body weight, preferably about 500 to 5,000 U / kg body weight, and most preferably about 1,000 U / kg body weight per administration This effective dose should be sufficient to achieve serum erythropoietin levels greater than about 10,000,15,000, or 20,000 mU / ml serum after erythropoietin administration. Such serum levels can be achieved after about 1,2,3,4,5,6,7,8,9, or 10 hours after administration. Such doses may be repeated as needed. For example, administration may be repeated daily, for as long as clinically necessary, or after a suitable time interval, e.g., from 1 to 12 weeks, preferably from up to 3 weeks. In one embodiment, an effective amount of an erythropoietin and a pharmaceutically acceptable carrier may be packaged as a single dose in a vial or other container. In another embodiment, the erythropoietin useful for the purposes set forth herein is not erythropoietin, i.e., it is capable of exhibiting the activities described herein but is unable to cause an increase in hemoglobin or hematocrit. Such a non-erythropoietin form of entropoetin is desirable in cases where it is intended that the methods of the present invention be applied chronically. In another embodiment, the erythropoietin is administered in a dose greater than necessary to

52276 Et maximally stimulated erythropoiesis. As stated above, the erythropoietin of the present invention does not necessarily have erythropoietin activity and therefore said dose expressed in hematopoietic units is merely an example of erythropoietins that are erythropoietin; in the text above, molar equivalents are given for the doses applied to each erythropoietin.

The present invention further relates to a method of accelerating the transport of molecules across an endothelial cell barrier in a mammal by administering a composition comprising a particular molecule in association with erythropoietin as described hereinabove. As described above, strong connections between endothelial cells in certain organs in the body create a barrier to the entry of certain molecules. For the treatment of various barrier organ conditions, agents for accelerating the passage of pharmaceutical agents are preferred. The erythropoietin of the present invention is useful as a carrier for transporting other molecules across the blood-brain membrane and other similar membranes. A mixture containing a molecule to cross the barrier with erythropoietin was prepared and transcytosis of the mixture across the barrier was achieved by peripheral application of the mixture. The association between the molecule to be transported across the barrier and the erythropoietin may be a labile covalent bond, in which case the molecule is released from the association with the erythropoietin after crossing the barrier. If the desired pharmacological activity of the molecule is maintained or remains unchanged in association with erythropoietin, such a complex can be administered.

Various methods for associating the molecules with the erythropoietin of the present invention and other agents described above, by covalent, non-covalent, and other means, will be known to one skilled in the art; moreover, the evaluation of the efficiency of the mixture can be quickly determined in the experimental system. The association of molecules with erythropoietin can be achieved at

52276 Β various ways, including labile, covalent bonding, cross-linking, etc. Biotin / avidin interactions may be used. As mentioned above, a hybrid molecule can be obtained by recombinant or synthetic methods, for example, one that contains both a domain of molecules with the desired pharmacological activity and a domain responsible for modulating erythropoietin receptor activity.

The molecule can be conjugated to erythropoietin via a polyfunctional molecule, i.e., a polyfunctional cross-linking reagent. As used herein, the term polyfunctional molecule includes molecules with one functional group that can react sequentially multiple times, such as formaldehyde, as well as molecules having more than one reactive group. As used herein, the term reactive group refers to a functional group in a cross-linking reagent that reacts with a functional group on a molecule (e.g., a peptide, protein, carbohydrate, nucleic acid, especially a hormone, antibiotic, or anticancer agent that should be transferred across the endothelial cell barrier) so as to form a covalent bond between the crosslinking reagent and that molecule. The term functional group retains its standard meaning in organic chemistry. The polyfunctional molecules that can be used are preferably biocompatible binding reagents, i.e., those that do not cause cancer, are non-toxic and are truly non-immunogenic in vivo. Polyfunctional crosslinking reagents, known in the art and described herein, can be readily tested on animal models to determine their biocompatibility. A polyfunctional molecule is predominantly bifunctional. As used herein, the term bifunctional molecule refers to a molecule with two reactive groups. A bifunctional molecule can be heterobifunctional or homobifunctional. The hetrobifunctional crosslinking reagent allows vector conjugation. It is particularly desirable for a polyfunctional molecule to be sufficiently soluble in

52276 Β water so that the cross-linking reaction can take place in aqueous solutions, such as aqueous solutions with buffer adjusted to pH 6 to 8, and for the resulting conjugate compound it is desirable to remain soluble in water for more efficient bio-distribution. Typically, a polyfunctional molecule is linked by covalent bonds to an amino or sulfhydryl functional group. However, polyfunctional molecules that react with other functional groups, such as acid carboxylic groups or hydroxyl groups, have been tested in the present invention.

Homobifunctional molecules have at least two reactive functional groups, which are the same. Reactive functional groups on the homobifunctional molecule include, for example, aldehyde groups and active ester groups. Homobifunctional molecules with aldehyde groups include, for example, glutaraldehyde and subaraldehyde. The use of glutaraldehyde as a crosslinking reagent has been discovered by Poznansk et al., Science 223, 1304-1306 (1984). Homobifunctional molecules having at least two active ester units include esters of dicarboxylic acids and N-hydroxysuccinimide. Certain examples of such N-succinimidyl esters include disuccinimidyl suberate and dithiobis- (succinimidyl propionate), and their soluble bis-sulfonic acid and soluble bis-sulfonate salts, such as their sodium and potassium salts. . These homobifunctional reagents are available from Pierce, Rockford, Illinois.

Heterobifunctional molecules have at least two different reactive groups. The reactive groups react with different functional groups, e.g., which are present on the erythropoietin and the molecule. These two different functional groups that react with the reactive group on the heterobifunctional crosslinking reagent are usually amino groups, e.g., a fifth amino group from lysine; a sulfhydryl group, e.g., a thiol group from

52276 Iste cysteine; carboxylic acid, e.g., carboxylate on aspartic acid; or a hydroxyl group, e.g., a hydroxyl group on serine.

Of course, the various erythropoietin molecules of the present invention do not always have the appropriate reactive groups available for use with a particular cross-linking reagent; however, a person skilled in the art is well aware of the choice of these reagents based on the available crosslinking groups for erythropoietins of the present invention.

When a reactive group from a heterobifunctional molecule forms a covalent bond with an amino group, that covalent bond is usually an amido or imido bond. The reactive group that forms a covalent bond with the amino group may be, for example, an activated carboxyl group, a halocarbonyl group or an ester group. Preferred halocarbonyl groups are chlorocarbonyl groups Preferred ester groups are reactive ester groups such as, for example, the N-hydroxy-succinimide ester group

Another typical functional group is either a thiol group, a group that can be converted to a thiol group, or a group that forms a covalent bond with a thiol group. A covalent bond is usually a thioether or disulfide bond. The reactive group that forms a covalent bond with the thiol group may be, for example, a double bond reacting with thiol groups or activated disulfide. Such a reactive group containing a double bond capable of reacting with a thiol group is a maleimido group, although others are possible, such as an aclonitrile group. The reactive disulfide group may be, for example, a 2-pyridyldithio group or a 5,5'-dithio-bis- (2-nitrobenzoic acid) group. Certain examples of heterobifunctional reagents containing reactive disulfide bonds include N-succinimidyl 3- (2-pyridyldithio) propionate (Carlsson, et al., 1978, Biochem J.,

52276 Β

173: 723-737), sodium S-4-succinimidyloxycarbonyl-alpha-methylbenzylthiosulfate, and 4-succinimidyloxycarbonyl-alpha-methyl- (2-pyridyldithio) toluene. N-succinimidyl 3- (2-pyridyldithio) propionate preferred Some examples of heterobifunctional reagents containing reactive groups with a double bond reacting with a thiol group include succinimidyl 4- (N-maleimidomethyl) cyclohexane-1-carboxylate and succinimidyl m-succinimidyl m. Other heterobifunctional molecules include succinimidyl 3- (maleimido) propionate, sulfosuccinimidyl 4- (p-maleimido-phenyl) butyrate, sulfosuccinimidyl 4- (N-rleleimidomethylcyclohexane) -1-carboxylate, maleimidobenzoyl-N-hydroxy-. It is preferred to use succinimidyl m-maleimidobenzoate sodium sulfonate. Many of the aforementioned heterobifunctional reagents and their sulfonate salts are available from Pierce Chemical Co., Rockford, Illinois USA.

Those skilled in the art can readily determine whether conjugated compounds need to be reversible or labile. The conjugated compounds can be tested in vitro for erythropoietin, and for the desired pharmacological activity. If the conjugated compound retains both properties, its suitability can then be tested in vivo. If the conjugated compound requires separation from erythropoietin due to activity, then a labile bond or a reversible bond with erythropoietin is preferred. Labile characteristics can also be tested using standard in vitro procedures before performing an in vivo test.

Additional information on how these and other polyfunctional reagents are made or used can be obtained from the following publications or other available papers in this field:

Carlsson, J. et al. 1978, Biochem. J. 173: 723-737.

52276 Β

Cumber, JA et al., 1985, Methods in Enzymology 112: 207-224.

Jue, R. et all., 1978, Biochem 17: 5399-5405.

Sun, TT et al., 1974, Biochem. 13: 2334-2340.

Blattler, WA et al., 1985, Biochem. 24: 1517-152.

Liu, FT et al., 1979, Biochem. 18: 690-697.

Youle, RJ. and Neville, DM Jr., 1980, Proc. Natl. Acad. Sci. USA 77: 5483-5486.

Lemer, RA et al., 1981, Proc. Natl. Acad. Sci. USA 78: 3403-3407.

Jung, SM and Moroi, M., 1983, Biochem. Biophys. Acta761: 162.

Caulfield, MP et al., 1984, Biochem. 81: 7772-7776.

Staros, JV, 1982, Biochem. 21: 3950-3955.

Yoshitake, Si dr., 1979, Eur. J. Biochem. 101: 395-399.

Yoshitake, S. et al., 1982, J. Biochem. 92: 1413-1424.

Pilch, PF and Czech, MP, 1979, J. Biol. Chem. 254: 3375-3381.

Novick, D. et all., 1987, J. Biol. Chem. 262: 8483-8487.

Lomant, AJ. and Fairbanks, G., 1976, J. Mol. Biol. 104: 243-261.

Hamada, H. and Tsumo, T., 1987, Anal. Biochem. 160: 483-488.

Hashida, S. et al., 1984, J. Applied Biochem. 6: 56-63.

In addition, cross-linking procedures are given in a review by Means and Feeney, 1990, Bioconjugate Chem. 1: 2-12.

Barriers to be overcome by the methods described herein and the compositions of the present invention include, but are not limited to, the blood-brain barrier, the blood-eye barrier, the blood-testis barrier, the blood-ovarian barrier, and the blood-uterine barrier.

52276 Β

Molecules that are candidates for transport across the endothelial cell barrier include, for example, hormones such as growth hormone, neurotrophic factors, antibiotics or antifungals such as those normally excluded from the brain and other barrier organs, peptide pharmaceuticals for radiation, antisense drugs , antibodies against biologically active agents, pharmaceutical preparations and anticancer agents. Non-limiting examples of such molecules include growth hormone, nerve growth factor (NGF), brain neutrophil factor (BDNF), cilia neurotrophic factor (CNTF), fibroblastic growth factor (bFGF), transforming growth factor βΐ (TGFpi), transforming growth factor β2 (TGFP). ), transforming growth factor β3 (TGFP3), interleukin 1, interleukin 2, interleukin 3, and interleukin 6, AZT, antibodies against tumor necrosis factor and immunosuppressive agents such as cyclosporine.

The present invention also relates to a composition comprising a molecule to be transported by transcytosis via a solid bond endothelial cell barrier and erythropoietin, as described above. The invention further relates to the use of compounds conjugated between molecules and erythropoietin as described above, for the preparation of a pharmaceutical composition for transporting molecules across a barrier, as described above.

The present invention may be better understood with reference to the following non-limiting examples, which are provided by way of example. The following examples are provided in order to more fully illustrate preferred embodiments of the present invention. However, they should in no way be construed as limiting the broad scope of the present invention.

52276 Β

Example 1

ERYTHROPOETIN CROSSES A SOLID BLOOD BARRIER-CEREBROSPINAL LIQUID

Adult male Sprague-Dawley rats were given anesthesia and recombinant human erythropoietin was administered intraperitoneally. Cerebrospinal fluid (CSF) samples were taken from the magnet system at 30-minute intervals for 4 hours and the erythropoietin concentration was determined using a sensitive and specific enzyme-linked immunosorbent assay. As illustrated in Figure 1, the initial concentration of erythropoietin in CSF is 8 mU / ml. After a delay of several hours, erythropoietin levels measured in CSF began to rise for 2.5 hours and later differed significantly from the initial concentration at p &lt;0.01 levels. The highest level of about 100 mU / ml is in the range known to be exhibits protective effects in vitro (0.1 to 100 mU / ml). The time to reach the highest level is about 3.5 hours, which is a significant delay compared to the highest serum levels (less than 1 hour). The results of this experiment illustrate that significant levels of erythropoietin can be transmitted via a solid cell bond through parenteral administration of erythropoietin in the form of a large pill in appropriate concentrations.

Example 2

MAINTAINING THE FUNCTION OF THE HEART PREPARED FOR TRANSPLANTATION

Male Wistar rats weighing 300 to 330 g were given erythropoietin (5000 U / kg body weight) or a 24-hour heart rate transporter for ex vivo tests, performed according to the protocol of Delcayre et al., 1992, Amer. J. Physiol. 263: H1537-45. They are animals

52276 Β sacrificed with pentobarbital (0.3mL), and heparin (0.2mL) was administered intravenously. The hearts were initially allowed to equilibrate for 15 min. The balloon of the left ventricle was then inflated to a volume that gives a final diastolic pressure of 8 mm Hg. The pressure curve of the volume in the left ventricle was constructed by increasing the volume of the balloon in aliquots of 0.02 ml. The zero volume is defined as a point whose final diastolic pressure in the left ventricle was equal to zero. Upon completion of the volume-pressure curve, the left ventricular balloon was inflated to return the final diastolic pressure to 8 mmHg and the control period was extended by 15 min, after checking the coronary fluid. The heart was then stopped with 50 mL of Celsior + molecules to rest at 4 ° C under a pressure of 60cm H₂A. The heart was then removed and stored for 5 hours at 4 ° C in a plastic container filled with the same solution and surrounded by beaten ice.

Upon completion of storage, the heart was transferred to a Langendorff apparatus. The balloon catheter was placed again in the left ventricle and inflated again to the same volume as in the period before ischemia. The heart was resuspended for at least 2 hours at 37 ° C. The pressure is set to 50cm H₂O for 15min re-flow, then again at 100cm H₂Oh for the next 2 hours. Artificial heart stimulation (320 beats per minute) was re-established. Isovolumetric measurements of contraction indices and diastolic pressure were taken in triplicate at 25,45,60,120 minutes from the start of re-perfusion. At that time, the curves of the highest values ​​for pressure and volume were made and the fluid was collected by the corona during 45 min of re-perfusion, in order to determine the creatinine kinase leakage. The two treatment groups were compared using a non-binding t-test, and a regression line using end-diastolic pressure data was used to determine the agreement curves. As shown in Figure 2, there was a significant improvement in left ventricular pressure after treatment with erythropoietin, as

52276 Β and improvement in the volume-pressure curve and reduced creatine kinase excretion.

Example 3

ERYTHROPOETIN PROTECTS THE MYOCARDIUM FROM ISCHEMIC INJURY.

Adult male rats were given recombinant human erythropoietin (5000 U / kg body weight) for 24 hours before being anesthetized and prepared for coronary artery occlusion. At the beginning of the procedure, an additional dose of erythropoietin was given and the left main coronary artery was occluded for 30 minutes and then released. The same dose of erythropoietin was given daily for one week after occlusion. Cardiac function was then examined in the animals. As Figure 3 shows, animals that received a false injection (saline) showed a large increase in left end-diastolic pressure, which is indicative of an enlarged, stiff heart that is second in myocardial infarction. In contrast, animals receiving erythropoietin did not experience a decrease in cardiac function compared to sham control (the difference was significant at p &lt;0.01 levels).

Example 4

ERYTHROPOETIN MOLECULES

Native erythropoietin can be modified to adapt its activities to a specific tissue or tissues. Several non-limiting strategies that can be implemented to achieve the desired tissue specificity include modifications that remove or modify

52276 Β glycosylated moieties, of which erythropoietin has three with N-bond and one with O-bond. Such variants of glycosylated erythropoietin can be produced in several ways. For example, sialic acids at the ends of sugar chains can be removed by specific sialidases depending on the chemical bond by which the sialic acid is attached to the sugar chain. Alternatively, glycosylated structures can be separated in various ways using other enzymes that cleave specific bonds. To confirm these principles, recombinant human erythropoietin was desialized using Sialidase A (Prozyme Inc.) according to the manufacturer's protocol. Successful chemical modification was confirmed by passing the reaction product through SDS polyacrylamide gel and staining the obtained bands, which showed that chemically modified erythropoietin had an apparent molecular weight of -31 kD as expected, compared to unmodified erythropoietin, which had a ratio of ~ 34 kD sialic acid residues which have been chemically determined to be &lt;0.1 mol / mol erythropoietin

In the second modification, in which amino acid residues from erythropoietin were modified, arginine residues were modified using phenylglyoxal according to the protocol of the author Takahashi (1977, J. Biochem. 81: 395-402), and the modification was performed at different time periods lasting 0.5 up to 3 hours at room temperature. The reaction was completed by dialysis of the reaction mixture in water. The use of such modified forms of erythropoietin is fully encompassed by the present invention.

Asyaloerythropoietin and phenylglyoxalerythropoietin were just as effective as native entropoietin for nerve cells in vitro as shown in Figures 4-6. An in vitro study was performed using nerve cell-like embryonic cancer cells (P19),

52276 Β enduring aptosis after serum isolation. Twenty-four hours before serum removal, 1-1000 ng / ml erythropoietin or modified erythropoietin was added to the cultures. The next day, the medium was removed, the cells were washed with fresh serum-free medium, and test medium (serum-free) was added. is back in culture for the next 48 hours. Tetrazolium reduction analysis was performed to determine the number of viable cells (CellTiter 96; Promega, Inc.). As Figures 4-5 show, asialoerythropoietin exhibits the same power as erythropoietin itself to prevent cell death. Phenylglyoxal-modified erythropoietin was examined by analysis of Ρ19 nerve cell-like cells, as described above. As shown in Figure 6, this chemically modified erythropoietin has fully preserved its effect on the protection of nerve cells.

Retention of nerve cell protection activity in vivo was confirmed using a focal ischemia model in rats in which a reversible lesion in the middle cerebral artery is performed as previously described (Brines et al., 2000, Proc. Nat. Acad. Sd. USA 97 : 10526-31). Adult male Sprague-Dawley rats were administered asialoerythropoietin or erythropoietin (5000 U / kg body weight intraperitoneally) or vehicle at the onset of arterial occlusion. The animals were sacrificed twenty-four hours later and their brains were separated for examination. Serial sections were cut and stained with tetrazolium salts to identify living parts of the brain. As shown in Figure 7, asialoerythropoietin was as successful as native erythropoietin in protecting nerve cells from one-hour ischemia. Figure 8 shows the results of another model of focal ischemia in which a comparative dose response with erythropoietin and asialoerythropoietin was performed. With a minimum dose of 250 U / kg, asialoerythropoietin provided protection, while unmodified erythropoietin did not provide protection.

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Prirner 5

MODIFICATION OF THE PRIMARY STRUCTURE OF ERYTHROPOETIN AND THE ABILITY TO PROTECT NERVE CELLS

A number of mutant erythropoietin molecules that do not bind to the erythrocyte erythropoietin receptor and thus do not support erythropoiesis in vivo or in vitro have been described. Some of these molecules will, in addition, mimic the effects of erythropoietin itself in other tissues or organs. For example, 17-mer containing amino acid sequences of 31-47 native erythropoietin is inactive for erythropoiesis but is fully active for nerve cells in vitro (Campana &amp; O'Brien, 1998: Int. J. Mol. Med. 1: 235-41).

Entropoietin derivatives which are preferred for the uses described herein can be produced by quanidination, amidination, trinitrophenylation, acetylation, succinylation, nitration, or by modification of arginine or carboxyl group residues, as mentioned above, among other processes, to retain erythropoietin. for specific organs and tissues, but not for others, such as erythrocytes. When erythropoietin was subjected to these reactions, it was found that the resulting molecule lacked both in vivo and in vitro erythropoietin activity (e.g., Satake et al .; 1990, Biochim. Biophys. Acta 1038: 125-9). The following are some examples for obtaining modified erythropoietins

Biotinylation of free amino groups from erythropoietin. 0.2 mg of D-biotinoyl-e-aminocapric acid -N-hydroxysuccinimide ester (Boehringer Mannheim # 1418165) was dissolved in 100 ml of DMSO. This solution was mixed with 400 ml of PBS containing approximately 0.2 mg

52276 Β erythropoietin in a tube covered with foil. After incubation for 4 hours at room temperature, the unreacted biotin was separated by gel filtration on a Centricon 10 column. As shown in Figure 10, this biotinylated erythropoietin protects P 19 cells from serum secretion.

In a scientific paper entitled Biotinylated recombinant human erythropoietins: bioactivity and benefit as a ligand receptor by Wojchowski et al., Blood, 1989, 74 (3): 952-8, the authors apply three different biotinylation procedures for erythropoietin. Biotin was added to (1) the sialic acid moieties (2) of the carboxyl group (3) and the amino group (3). The authors use an analysis of spleen cell proliferation in mice to show that (1) the addition of biotin to sialic acid moieties does not inhibit the biological activity of erythropoietin (2) the addition of biotin to carboxyl groups leads to significant biological inactivity of erythropoietin (3) the addition of biotin to amino groups has result in complete biological inactivity of erythropoietin. These procedures and modifications are fully incorporated herein. Figure 9 shows the activity of biotinylated erythropoietin and asialoerythropoietin in an analysis with seramically starved Ρ19 cells.

Entropoietin iodination. Method 1 - Zmcajoda .. One zmcejoda (Pierce, Rockford, II) was incubated in 100 μl of PBS (20 mM sodium phosphate, 0.15Μ NaCl, pH7.5) with a content of 1 mCi of free Na¹²⁵J during the 5th minute. Then, 100 [mu] g of entropoietin in 100 [mu] l of PBS was added to the mixture. After an incubation period of ten minutes at room temperature, the reaction was stopped by removing 200 ul of solution from the reaction vessel (leaving iodine beads inside). Excess iodine was removed by gel filtration on a Centricon 10 column. As shown in Figure 11, the iodo-erythropoietin produced in this way successfully protected Ρ19 cells from serum secretion.

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Method 2 - Chloramine T. 100 μg erythropoietin in 100 μl PBS was added in 500 μl Na^l25J and mixed together in an ependorf tube. Then 25 [mu] l of chloramine T (2 mg / ml) was added and the mixture was incubated for 1 minute at room temperature. Then 50 μl of buffer solution (2.4 mg / ml sodium metabisulfite, 10 mg / ml tyrosine, 10% glycerin, 0.1% xylene in PBS) was added to stop chloramine T. Iodothyrosine iodinated erythropoietin was then separated by gel filtration on a Centricon 10 column.

Lysine modifications: carbamylation: erythropoietin (100ug) was modified with potassium cyanate as described by Plapp et al., (Deoxyribonuclease A activity from bovine pancreatic with modified amino groups 1971, J. Biol. Chem. 246, 939845).

Trinitrophenylation: erythropoietin (100 [mu] g) modification with 2,4,6-trinitrobenzenesulphonate as described by Plapp et al., (Deoxyribonuclease A activity from bovine pancreatic self-modified amino groups 1971, J. Biol. Chem. 246, 939-845).

Acetylation: Erythropoietin (100 [mu] g) was incubated in 0.3M phosphate buffered saline (pH7.2) containing the same amount of acetic anhydride at 0 ° C for 1 hour. The reaction was stopped by dialysis in distilled water.

Succinylation: erythropoietin (100 [mu] g) in 0.5 M NaHCO3 (pH 8.0) was incubated with 15 moles of excess amber anhydride at 15 ° C for 1 hour. The reaction was stopped by dialysis in distilled water.

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Arginine modifications: erythropoietin was modified with 2,3 butanedione as described in Riordan (Functional arginyl residues in carboxypeptidase A. Modifications with butanedione Riordan JF, Biochemistry 1973, 12 (20): 3915-3923).

Erythropoietin was modified with cyclohexanone as in the work of Patthy et al., (Identification of functional arginine residues in ribonuclease A and lysozyme Patthy, L, Smith EL, J. Biol. Chem 1975 250 (2): 565-9).

Erythropoietin modified with phenylglyoxal as described in the work of the author Werber et al., (Reports: Carboxypeptidase B: modification of functional arginyl residues Werber. MM, Sokolovsky M IsrJMedSci 1975 11 (11): 1169-70).

Tyrosine modifications: erythropoietin (100 [mu] g) was incubated with tetranitromethane as previously described by Nestler et al., In Stimulation of steroidogenesis from rat ova using high density lipoproteins modified with Nestler's 3rd tetranitromethane, Chacko GCK. J Biol Chem 1985 Jun 25; 260 (12): 7316-21).

Modifications of glutamic acid (and aspartic acid): In order to modify carboxyl groups, erythropoietin (100 ug) was incubated with 0.02 M EDC in 1M glycinamide at pH 4.5 at room temperature for 60 minutes as described by Carrawav et al. , in Modification of the carboxyl group in chymotrypsin and chymotrypsinogen Carraway KL, Spoerl P, Koshland DE Jr. J Mol Biol 1969 Mau 28; 42 (1): 133-7.

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Modifications of tryptophan residues: Erythropoietin (100 [mu] g) was incubated with 20 [mu] M n-bromosuccinimide in 20 mM potassium phosphate buffer solution (pH 6.5) at room temperature as described by Ali et al., J Biol Chem. 1995 Mar3; 270 (9): 4570-4. The number of oxidized tryptophan residues was determined using the method described in the work of the author Korotchkina (Korotchkina, LG et al., Protein Ehrg Purif. 1995 Feb; 6 (l): 79-90).

Removal of amino groups: In order to remove amino groups, erythropoietin (100 [mu] g) was incubated in a solution of PBS (pH 7.4) containing 20 mM ninhydrin (Pierce Chemical, Rockford, II) at 37 ° C for two hours as described in the work of the author Kokkini et al., (Kokkini, G., et al., Modification of hemoglobin by ninhydrin Blood, Vol. 556, No. 4 1980: 701-705). The reduction of the obtained aldehyde was performed by reacting the product with sodium borohydride or lithium aluminum hydride. Specifically, erythropoietin (100 [mu] g) was incubated with 0.1M sodium borohydride in PBS solution for 30 minutes at room temperature. The reduction was completed by cooling the samples with ice for 10 minutes and dialysis in PBS solution, three times, overnight. (Kokkini, G., Blood, Vol. 556, No. 4 1980: 701-705). Reduction with lithium aluminum hydride was performed by incubating erythropoietin (100 ug) with 0.1Μ lithium aluminum hydride in PBS solution for 30 minutes at room temperature. The reduction was completed by cooling the samples with ice for 10 minutes and dialysis in PBS solution, three times, overnight.

Disulfide reduction and stabilization: erythropoietin (100 μg) was incubated with 500 mM DTT for 15 minutes at 60 ° C. Then, 20 mM iodoacetamide in water was added to the mixture, and incubation was performed for 25 minutes, at room temperature in the dark.

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Limited proteolysis: Erythropoietin may undergo limited chemical proteolysis that affects specific residues. Erythropoietin was reacted with 2- (2-nitrophenylsulfenyl) -3-methyl-3'-bromoindolenine, which is a specific reagent for separating tryptophan residues, in excess of 50 times in 50% acetic acid for 48 hours in the dark at room temperature in pipes under nitrogen pressure. .. The reaction was completed by the addition of tryptophan and desalting.

Example 6

PROTECTION AGAINST RETINAL ISCHEMIA WITH PERIPHERALLY APPLIED ERYTHROPOETIN.

Retinal cells are very sensitive to ischemia, so many of them die after 30 minutes from ischemic stress. Then, subacute or chronic ischemia expresses a worsening of vision that accompanies a number of common human diseases, such as diabetes mellitus, glaucoma and macular degeneration. Nowadays, there is no effective therapy that can protect cells from ischemia. There is a solid endothelial barrier between the blood and the retina, which excludes the largest number of large molecules. To examine whether peripherally administered erythropoietin would protect ischemic susceptible cells, a model of reversible glaucoma in rats was used as described by Rosenbaum et al., (1997; Vis. Res. 37: 3443-51). Specifically, saline was injected into the anterior chamber of the eye in adult male rats to a pressure above systemic arterial pressure and such pressure was maintained for 60 minutes. Animals were administered saline or 5000 U erythropoietin / kg body weight intraperitoneally 24 hours before induction of ischemia, and this was extended as a daily dose for the next 3 days. Electroretinography was performed on rats adapted to darkness one week after treatment. Figures 11 and 12 show that erythropoietin administration affected the well

52276 Β The electroretinogram (ERG) (Panel D) is preserved, in contrast to animals that received only saline (Panel C), in which the function was preserved to a very small extent. Figure 11 compares the electroretinogram of a- and b-wave amplitudes for groups of animals that received erythropoietin and groups that received saline, and shows the significant protection provided by erythropoietin.

Example 7

RESTORATING EFFECT OF ERYTHROPOETIN ON REDUCED KNOWLEDGE FUNCTION CAUSED BY BRAIN INJURY

When examining the ability of erythropoietin to restore cognitive function in mice after brain injury, female Balb / c mice were exposed to blunt object brain injury as described by Brines et al. PNAS 2000, 97, 10295-10672, and after five days intraperitoneal daily administration of 5000 U / kg body weight of erythropoietin was started. Twelve days after injury, the cognitive function in the Morris maze with water was examined in animals, four times a day. Although both grape animals, treated and those not treated in the test, performed poorly (with a swimming time of about 80 seconds out of a possible 90 seconds), Figure 13 shows that the animals receiving erythropoietin behaved better (negative in this experiment). the value is better). Even if the start of erythropoietin treatment is delayed until 30 days after trauma (Figure 14), recovery of cognitive function is also seen.

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Example 8

ΚΑΓΝΑΤΝΙ MODEL

In the kainate neurotoxic model, in which asialoerythropoietin was administered according to the protocol of Brines et al.,. Proc. Nat. Acad. Sci. USA 2000.97; 10295-10672 at a dose of 5000U / kg body weight given intraperitoneally 24 hours before administration of 25 mg / kg kainate, it was shown to be as effective as erythropoietin, as seen from the time to death values ​​(Figure 15). .

The present invention is not limited to the scope of the described specific embodiments which are intended only to illustrate certain aspects of the present invention, and the functionally equivalent methods and components are within the scope of the present invention. Various modifications of the present invention, in addition to those shown and described herein, will be apparent to those skilled in the art from the foregoing description and the accompanying drawings. Such modifications are within the scope of the additional claims.

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