EA007812B1 - Long acting erythropoietins that maintain tissue protective activity of endogenous erythropoietin - Google Patents

Abstract

Methods for increasing the hematocrit of an individual while maintaining the tissue protective activities of endogenous through the administration of a pharmaceutical compound containing chemically modified long acting erythropoietin. Also disclosed are the new chemically modified long acting erythropoietins, methods of producing the chemically modified long acting erythropoietins, and compositions comprising the chemically modified long acting erythropoietins

Description

Cross reference related to related applications

This application claims priority by provisional application US No. 60/409020, registered September 9, 2002, which is incorporated into this description in full by reference.

Field of Invention

The present invention relates to long-acting erythropoietins that advantageously retain tissue protective properties after modification. In particular, the present invention relates to long-acting erythropoietins that are chemically modified in such a way that not only their serum half-life is extended, but also the tissue protective function of the native protein ίη νίνο is preserved. The present invention also relates to the treatment of anemia and related diseases with long-acting erythropoietins according to the present invention. In addition, the present invention relates to assays that are used to determine whether erythropoietin exhibits tissue protective properties.

BACKGROUND OF THE INVENTION

Natural or endogenous erythropoietin (EPO) is a glycoprotein-derived hormone produced primarily in the kidneys. Endogenous EPO consists of 165 amino acids and has a molecular weight (in humans) of approximately 30,000 to 34,000 Da. Glycosyl residues in the EPO molecule, which consist of three Ν-linked oligosaccharide chains and one O-linked oligosaccharide chain, account for approximately 40% of the total protein weight. Ν-linked oligosaccharide chains are attached to the amide nitrogen of the asparagine located at positions 24, 38 and 83, while the O-linked oligosaccharide chain is attached to oxygen at the serine residue located at position 126. The EPO protein can exist in three forms: , β and asialo. Forms α and β have the same efficiency, biological activity and molecular weight, but differ slightly in carbohydrate components, while the asialo form is an α or β form with terminal sialic acid removed from the carbohydrate chains, which protects the carbohydrate chain from recognition the liver.

Until recently, it was believed that the main function of endogenous EPO is to, together with other growth factors, stimulate the proliferation and maturation of erythroid bone marrow progenitor cells and maintain an individual's hematocrit (percentage of whole blood containing red blood cells). The process of erythrocyte formation is called erythropoiesis, which is strictly controlled by a physiological mechanism that determines the optimization of the number of red blood cells for characteristic tissue oxygenation without delaying blood circulation. For example, when oxygen transport by red blood cells decreases, EPO helps increase red blood cell production by stimulating the conversion of progenitor cells in the bone marrow to mature red blood cells, which are then released into the bloodstream. When the number of red blood cells in the bloodstream exceeds the amount necessary for normal circulation in the tissue, EPO in the bloodstream decreases. Thus, when the body is in a healthy state, EPO is present in plasma in very low concentrations, which are sufficient to stimulate the recovery of red blood cells lost during normal aging. Plasma EPO levels normally range from 0.01 to 0.03 units / ml.

It has been established that since the kidneys produce most of the EPO in an individual, impaired renal function, such as chronic renal failure (CHE), complicates the formation of EPO and often leads to anemia. Similarly, anemia can occur as a result of other chronic conditions, such as cancer, or treatments related to diseases such as chemotherapy. Thus, the introduction of recombinant EPO (discussed in more detail below), which has essentially the same biological effects as endogenous EPO, has been found to be beneficial in restoring the hematocrit in an individual with a reduced number of red blood cells.

In addition to the role of recombinant EPO in maintaining the hematocrit in chronic diseases, recombinant EPO is used to maintain red blood cell levels before an optional or planned surgery, thereby reducing or eliminating the need for blood transfusion. For example, recombinant EPO can be administered to a patient when a virus or pathogen enters the body during blood supply, or when there are problems with religious restrictions regarding blood transfusion.

In addition, some recent evidence suggests that EPO as a member of the superfamily of cytokines has other important therapeutic properties that are mediated by interaction with the EPO receptor (EPO-H). For example, EPO and its receptor can play an important role in reducing tissue damage, since the interaction between EPO and the receptor leads to compensatory reactions that contribute to the improvement of the hypoxic microenvironment of the cell, as well as the regulation of programmed cell death caused by metabolic stress. In fact, patients with chronic renal failure and / or cancer patients usually experience a sensation of improved health and increased mental acuity after treatment with erythropoietin - an effect that was previously attributed to an increase in the hematocrit in the patient. However, according to

- 1 007812 to the latest data, these phenomena are attributed to the protective and enhancing effects of EPO in the tissue, discussed in international publication No. ^ 0/02053580 and US patent publications No. 2002/0086816 and 2003/0072737.

Recent studies have shown that systemically administered EPO can cross the intact blood-brain barrier since capillaries forming the blood-brain barrier also express an EPO receptor. As such, the anatomical base for receptor-mediated transcytosis is created from peripheral circulation to the brain.

The recombinant human EPO (epoetin alpha), which was commercially available under the trade names PK0SK1T® (from 0g11yu Vyu1es11 1ps., Katyap, N1) and ΕΡ00ΕΝ® (from Atdep, 1ps., TNoicapb 0ak§, CA), was used to treat anemia, arising at the last stage of renal disease, for the treatment of HIV-infected patients when used together with therapy through ΑΖΤ (zidovudine), and to neutralize the effects of chemotherapy. Although recombinant human EPO has many therapeutic effects, the main use of recombinant human EPO has been directed to chronic anemia. In this regard, recombinant human EPO is usually administered at an initial dose of between 50-150 units / kg 3 times a week for 6 to 8 weeks, either by intravenous or subcutaneous injection, to restore the hematocrit in the patient. After the patient reaches the desired level of hematocrit, which is approximately 30 to 36%, this level can be maintained with maintenance doses of EPO, an amount sufficient to maintain and administered at a frequency sufficient to maintain normal hematocrit levels achieved with the initial doses of EPO , in the absence of iron deficiency and associated diseases. Although dosages may vary depending on the individual needs of the patient, usually maintenance dosages can be administered approximately 3 times per week (less if larger dosages are given).

The dosage amount and frequency of administration of the recombinant EPO is determined in part by the half-life of the molecule, which may be limited when the molecule is located νίνο. For example, the ΕΡ00ΕΝ® intravenously administered drug is reported to be eliminated at a rate consistent with first order reaction kinetics, with a half-life in the bloodstream ranging from approximately 4 to 13 hours in adult patients and children and adolescents with SLE. Thus, in order for the agent to be therapeutically effective, its dosage amount and dosage frequency must be compared with the relatively short half-life of the recombinant EPO.

In addition, since recombinant EPO is administered by intravenous or subcutaneous injection, a nurse or doctor is often required to introduce recombinant EPO to a patient. This circumstance causes inconvenience to the patient, and there is also another reason why it is desirable to extend the half-life of the molecule. Attempts to increase the half-life of recombinant EPO have aroused the interest of researchers in the last 10 years on the assumption that prolonging the half-life will make it possible to reduce the dosage, while providing the same or improved therapeutic effect.

In fact, recent experiments on human EPO have shown that there is a relationship between the carbohydrate composition of EPO containing sialic acid, its half-life in the bloodstream and the bioactivity of νίνο. As shown in PCT publication No. U095 / 05465. sialic acid residues close the ends of the sugar chains and prevent the liver from detecting galactose. Typically, Ν-linked oligosaccharide chains have up to 4 sialic acid residues per chain and O-linked oligosaccharide chains have up to 2 sialic acid residues per chain. Thus, an unmodified EPO polypeptide can contain a total of up to 14 sialic acid residues.

Over time, these sialic acid residues can be cleaved from the protein, and thus, open galactose residues are recognized by the liver. As the liver recognizes galactose residues, the protein is filtered from the blood. In fact, a gradual increase in sialic acid content per EPO molecule is believed to contribute to better screening of galactose residues to achieve a corresponding gradual increase in biological activity (measured by the ability of equimolar concentrations of the isolated erythropoietin isoforms to increase the hematocrit of a normal mouse). Since unmodified EPO contains only 14 sites of sialic acid, this approach has limited ability to extend the half-life of EPO. Such considerations lead to the assumption that the engineering EPO analog containing additional oligosaccharide chains obtained by engineering should have increased biological activity. By obtaining additional glycosylation sites, additional oligosaccharide chains having terminal ends can then be modified with sialic acid residues. See PCT Publication Nos. 91/05867, \\ '0 94/09257 and \\' 01/81405.

For example, a modified EPO analogue may have at least one additional O-linked carbohydrate chain and / or at least one additional O-linked carbohydrate chain. In particular, publication \ Y0 01/81405 describes the addition of Ν-linked carbohydrate chains to a molecule at amino acid positions 30, 51, 57, 69, 88, 89, 136 and / or 138. Modified molecules

- 2 007812

EPO can have from 1 to 4 additional glycosylation sites, which allow you to add from 2 to 16 sialic acid residues per molecule. In addition, attempts to increase the half-life of EPO include the addition of polyethylene glycols (PEG) to the amino acid backbone of the EPO protein. See PCT Publications XVO 00/32772, XVO 01/02017 and XVO 03/029291; U.S. Patent Publications No. I8 2003/077753, I8 2003/0120045, 2003/01666566; and U.S. Patent Nos. 6,583,272 and 6,340,272. Fusion constructs that attach other proteins to EPO have also been used to increase the half-life of EPO. See, for example, US Patent Publication No. I8 2004/000009902, 2003/0124115, 2003/0113871 and US Patent No. 6,242,570.

However, although attempts to extend the half-life of EPO in serum have been successful and useful in maintaining hematocrit levels, the researchers did not pay attention to the effects that these additional modifications could have on other EPO functions.

Thus, obtaining a modified EPO with an extended serum half-life (long-acting), which retains the functionality of endogenous EPO, is of great importance. In particular, in this area there is a need for combining long-acting EPO with erythropoietin functionality and tissue protective function for use in pharmaceutical compositions for the treatment of individuals suffering from anemia and / or related diseases. In addition, there is a need for analyzes that can be used to establish whether a single EPO is antagonistic to the tissue protective properties of endogenous EPO.

SUMMARY OF THE INVENTION

The present invention relates to a method for regulating the hematocrit level in humans, comprising the steps of producing an erythropoietin product having a longer half-life in serum than recombinant human erythropoietin (ThyEPO), and having a tissue protective function, and comprising administering a therapeutically effective amount of an erythropoietin product called here further EPO long-acting. In one aspect, the step of producing a long acting EPO further includes a step of modifying recombinant erythropoietin with at least one chemical modification of at least one of the Ν-linked oligosaccharide chains or O-linked oligosaccharide chain, where the chemical modifications include oxidation, sulfation phosphorylation, attachment of PEG chains or combinations thereof.

In addition, the step of administering a therapeutically effective amount of long-acting EPO may include administering a long-acting EPO at a lower molar amount than tycEPO to achieve a comparable hematocrit.

In one aspect, the half-life of a long-acting EPO in serum is at least about 20% longer than the half-life of serum EPO. In another aspect, the half-life of EPO long-acting in serum is at least about 40% longer than the half-life of serum EPO.

The present invention also relates to an artificially prepared erythropoietin product (long acting EPO) comprising at least one erythropoietin derivative, wherein at least one Ν-linked oligosaccharide chain or at least one O-linked oligosaccharide chain has at least one chemical modification as a result of oxidation, sulfation, phosphorylation, addition of PEG chains or combinations thereof, and where the specified long-acting EPO has a longer half-life in seconds serum than thyero. The resulting long-acting modification of EPO preferably exhibits tissue protective function.

In one aspect, the at least one chemical modification comprises oxidizing at least one Ν-linked oligosaccharide chain or at least one O-linked oligosaccharide chain to form at least one additional acid residue. For example, at least one chemical modification may include sulfation of at least one Ν-linked oligosaccharide chain or at least one O-linked oligosaccharide chain with an increase in negative charge on the EPO product. In another aspect, the at least one chemical modification comprises the phosphorylation of at least one Ν-linked oligosaccharide chain or at least one O-linked oligosaccharide chain with an increase in negative charge on a long-acting EPO product. In another aspect, the at least one chemical modification comprises the addition of at least one polyethylene glycol chain to at least one Ν-linked oligosaccharide chain or at least one Linked oligosaccharide chain.

The present invention also relates to a method for producing a long acting EPO having a longer serum half-life and exhibiting tissue protective activity, comprising the steps of producing at least one erythropoietin or an erythropoietin derivative; and modifying at least one Ν-linked oligosaccharide chain or at least one O-linked oligosaccharide chain in at least one endogenous molecule

- 3,007812 or a recombinant erythropoietin or erythropoietin derivative by oxidation, sulfation, phosphorylation, addition of PEG chains, or combinations thereof.

The modification step may also include the step of replacing at least one adjacent hydroxyl on at least one Ν-linked oligosaccharide chain or at least one O-linked oligosaccharide chain with at least one acid residue. In one aspect, the step of substituting at least one adjacent hydroxyl for at least one Ν-linked oligosaccharide chain or at least one O-linked oligosaccharide chain with at least one acid residue also includes replacing the plurality of adjacent hydroxyls in at least one Ν-linked oligosaccharide chain or at least one Linked oligosaccharide chain with a plurality of acid residues.

In another aspect, the modification step also includes the steps of preparing an organic solvent; dissolving erythropoietin or an erythropoietin derivative in an organic solvent to form a solution; preparing at least one condensing agent; preparing at least one sulfate donor and mixing at least one condensing agent and at least one sulfate donor in solution. In another aspect, the modification step also includes the steps of preparing an organic solvent; dissolving erythropoietin or an erythropoietin derivative in an organic solvent to form a solution; preparing at least one condensing agent; preparing phosphoric acid and mixing at least one condensing agent and at least one phosphoric acid in solution.

In another aspect, the modification step also includes the steps of preparing an organic solvent; dissolving erythropoietin or an erythropoietin derivative in an organic solvent to form a first solution; preparing at least one oxidizing agent; adding at least one oxidizing agent to the first solution to form a second solution; preparing at least one polyethylene glycol chain and mixing at least one polyethylene glycol chain in a second solution. The step of preparing at least one polyethylene glycol chain may include preparing at least one polyethylene glycol chain with at least one primary amine moiety at the end of the chain.

The present invention also relates to a method for treating anemia in patients at risk of tissue damage, comprising the steps of producing a long acting EPO with at least one chemical modification in at least one of the Ν-linked oligosaccharide chains or the O-linked oligosaccharide chain, where the modification includes oxidation, sulfation, phosphorylation, addition of PEG chains (PEGylation), or combinations thereof; the introduction of a therapeutically effective amount of EPO long-acting, where EPO long-acting is administered at a lower molar amount than g1ShEPO. to achieve a comparable hematocrit level, where long-acting EPO has a tissue protective function.

In this aspect of the invention, the long acting EPO preferably has a longer serum half-life than gypo EPO. In one aspect of the invention, the serum half-life is at least 20% longer than the serum half-life of huyEPO. In another aspect, the serum half-life is at least 40% longer than the serum half-life of huyEPO.

The present invention further relates to a pharmaceutical composition comprising a therapeutically effective amount of at least one long acting EPO, wherein at least one Ν-linked oligosaccharide chain or at least one O-linked oligosaccharide chain has at least one chemical modification as the result of oxidation, sulfation, phosphorylation, addition of PEG chains (PEGylation), or combinations thereof, where at least one long acting EPO has a longer duration flax serum half-life than recombinant erythropoietin and has tissue protective function. In one embodiment, the pharmaceutical composition further includes at least one pharmaceutically acceptable carrier. At least one pharmaceutically acceptable carrier may include at least one diluent, adjuvant, excipient, solvent, or mixtures thereof.

In another embodiment, the pharmaceutical composition further includes at least one moisturizing agent, emulsifying agent, pH buffering agent, or a combination thereof. In another embodiment, the pharmaceutical composition further includes at least one tissue protective cytokine.

Brief Description of the Figures

Further characteristics and advantages of the invention can be identified on the basis of the following detailed description, which is given with the graphic material presented below:

FIG. 1 is an illustration of evidence that a hyperglycosylated EPO analog is able to cross the blood-brain barrier, as shown by taking cerebrospinal fluid; and in FIG. 2 presents data comparing the efficiencies of various forms of EPO, manifested in

- 4 007812 protection against cell death initiated by exposure to trimethyltin.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to the use of long-acting EPO, EPO molecules with a longer serum half-life (long-acting), which are products of chemical modification related to carbohydrate chains attached to the amino acid backbone of EPO, so that the functionality of endogenous EPO is maintained. As described in the background to the invention, researchers' attempts to extend the half-life of EPO have focused on adding carbohydrate chains, PEG, proteins, etc. to the amino acid backbone of EPO. However, it is believed that these additives affect the functionality of the EPO analog in such a way that functionality is impaired and it becomes difficult to achieve a longer half-life. Although EPO analogues with longer half-lives than recombinant EPO exhibiting erythropoietin activity are known, these analogs do not retain other recently discovered therapeutic effects of EPO, for example, tissue protective activity.

For example, it was shown that the EPO fragment consisting of 17 amino acids corresponding to the amino acids at positions 30-47, also called the O'Vpei peptide, exhibits tissue protective activity of ίη νίίτο, but does not have the activity of erythropoietin ίη νίίτο. Satrap, ν.Μ., M18a81, K. & O'Vpep, 18., Ιηί. 1. Mo1. Meb., 1, 235-41 (1998). Therefore, it is believed that a modified EPO analog having additives within the sequence of the O'Bpep peptide may not exhibit tissue protective activity in other studies of ίη νίίτο. In addition, since the three-dimensional orientation of EPO is important for the manifestation of its functionality, additives in the molecule can disrupt the overall functionality of the resulting analogue of EPO.

Thus, the present invention relates to long-acting EPO molecules exhibiting at least one of the activities: erythropoietic activity, tissue protective activity, transcytosis ability, or a combination of activities. Preferably, the long acting EPO of the invention exhibits erythropoietin activity and at least one tissue protective activity or transcytosis ability.

In one aspect, the long acting EPO of the present invention has a serum half-life that is at least about 20% longer than the serum half-life of the recombinant EPO. In another aspect, the half-life in serum of EPO long-acting according to the present invention is at least about 30% longer than the half-life of recombinant EPO. In another aspect, the long-acting EPO of the present invention has a serum half-life that is at least about 40% longer than the serum half-life of the recombinant EPO.

Briefly, long-acting EPOs of the present invention include EPO analogues with carbohydrate chains that are altered by at least one modification compared to native (endogenous) EPO, preferably compared to native human EPO. In one aspect, the long acting EPO of the present invention undergoes multiple modifications of carbohydrate chains.

In one aspect, the adjacent hydroxyls of the carbohydrate chain of the native EPO are converted by oxidation to acid residues, with the formation of long acting EPO molecules according to the present invention. In another aspect, the sialic acid residues of the EPO molecule are replaced by less labile acid residues. In another aspect, sulfation and / or phosphorylation of the EPO carbohydrate chain results in the formation of a long acting EPO according to the present invention. In another aspect, the long acting EPO of the present invention is formed by the addition of polyethylene glycol to the EPO carbohydrate chain. Any combination of the above modifications is also contemplated by the present invention. And, as mentioned above, the present invention also encompasses compositions comprising pharmaceutical compositions that contain one or more of the aforementioned long acting EPO molecules.

The long acting EPO molecules of the present invention are contemplated for inclusion in pharmaceutical compositions for treating anemia and related diseases, especially those that have complications resulting from disorders such as, but not limited to, acute renal failure, sepsis, HIV, disorders due to chemotherapy, etc.

The present invention also relates to methods for treating anemia and related diseases, as well as to kits used for treatment. As used herein, the term “treatment” refers to both therapeutic treatment and prophylactic or preventive measures, where the goal is to prevent or ameliorate (slow down) an established pathological condition or disorder. Individuals in need of treatment include individuals who already have the disorder, as well as individuals who are predisposed to the disease or in whom the disease needs to be prevented. The present invention contemplates the use of long acting EPO for chronic administration, acute treatment and / or alternating administration. In the description, chronic administration refers to the administration of the agent (s) continuously, as opposed to the acute type, so that

- 5 007812 would preserve the initial therapeutic effect (activity) for an extended period of time, and alternating administration is a treatment that is not administered continuously without interruption, but rather is cyclical in nature.

Long-acting EPO according to the present invention is applied to any mammals. As used in the text, the term mammal refers to any animal classified as a mammal, including humans, domestic and farm animals, and zoological animals, sporting or domesticated animals such as dogs, cats, cattle, horses, sheep, pigs, goats, rabbits etc. Preferably, the mammal is a human. The administration of long-acting EPO according to the present invention includes, but is not limited to, oral, intravenous, intranasal, local, intraluminal administration, inhalation, or parenteral administration, the latter including intravenous, intra-arterial, subcutaneous, intramuscular, intraperitoneal administration , intradermal administration and combinations thereof.

The present invention also relates to the use of long acting EPO molecules according to the present invention as a carrier for other molecules in areas of the body that have EPO receptors. For example, since some molecules do not penetrate well through the blood-brain barrier, the binding of such molecules to long-acting EPO molecules according to the present invention leads to a reliable and efficient system for transporting these molecules to the brain. And, as will be discussed in more detail later, since other areas of the body, such as the retina, heart and lungs, express EPO receptors, the long-acting EPO molecules of the present invention can act as a system for transporting molecules having low penetration through such regions.

In addition, the present invention relates to studies to determine whether a single EPO preparation retains the functionality of endogenous EPO. For example, a study according to the present invention allows one to establish whether a modified EPO exhibits tissue protective function, i.e. is he an agonist of endogenous EPO. As used in the text, the term agonist is used in the broadest sense of the word, and it includes molecules that mimic the biological activity of native EPO. Similarly, the term antagonist is used in the broadest sense of the word, and it includes any molecule that partially or completely blocks, inhibits or neutralizes the biological activity of native EPO. In one aspect, a single EPO assay is performed using an анализаη νίίτο assay, such as an assay that includes P19 cells and / or rat motor neurons. In another aspect, the study of the present invention includes evaluating a single EPO ίη νίνο using various assays on models such as focal rat ischemia, rat retinal ischemia, spinal cord injury and an epileptic seizure caused by bicucullin.

Functionality.

As discussed in the Background to the invention, various attempts to extend the half-life of EPO have been successful in that the EPO analogues have a longer half-life and retain erythropoietin activity. And, as discussed, EPO analogues with longer half-lives mainly included additives to the amino acid sequence of EPO. Such additives include carbohydrate chains, PEG and protein sequences. However, it has been suggested that these additional carbohydrate chains may interfere with other therapeutic effects of EPO, such as tissue protective function and the ability of the molecule to transcytose. Without binding themselves to any particular theory, the inventors suggested that the inclusion of additional carbohydrate chains in the sequence of the 0'Bep peptide may affect functionality on the basis that the peptide is important for the manifestation of tissue protective function. In addition, it is believed that the added carbohydrate chains, whether they are within the sequence of the peptide 0'Vpep or outside the sequence of the peptide 0'Vpep. affect the three-dimensional orientation of the molecule. For example, in a three-dimensional configuration of a molecule, additional carbohydrate chains can block a portion of the molecule that is essential for the manifestation of the function. In addition, it is believed that the method of introducing additives may also affect the functionality of the glycoprotein. Although specialists in this field may be familiar with a number of ways to add or attach additional carbohydrates, polyethylene glycols, proteins, etc. to the amino acid skeleton of EPO, mention of additional carbohydrates will also apply to other additives to the amino acid skeleton of EPO.

Fabric resistant property.

In order to assess the possibility of the effect of additional carbohydrate chains on the functionality of the glycoprotein, the authors of the present invention studied the forms of EPO analogues that had 5 Ν’s linked carbohydrate chains (compared to 3 Ν-linked carbohydrate chains of recombinant EPO). In particular, the inventors used an EPO analogue, where the analogue had an additional glycosylation site at the amino acid at position 32, which led to an approximately three-fold increase in half-life compared to recombinant EPO (erythropoietin alpha).

- 6 007812

Although this EPO analog appeared in the cerebrospinal fluid after systemic injection (Fig. 1), it did not exhibit tissue protective function when evaluated by subsequent analysis on P19 ίη νίίτο cells (Fig. 2). This lack of tissue protective activity was unforeseen. In addition, the lack of tissue protective activity can cause complications in the treatment of patients with anemia, if patients have other diseases requiring tissue protection. For example, if an EPO analog that does not exhibit tissue protective activity competes with an endogenous EPO that has tissue protective activity for a receptor that initiates a tissue protective response, the degree of damage resulting from trauma can actually increase with the use of such EPO analogues. In fact, if the patient experienced a stroke when using such an analogue of EPO, then the volume of a heart attack as a result of the stroke may indeed be greater than that of an individual who was not treated with an analogue of EPO.

Although the authors did not want to bind themselves to any particular theory, the data obtained allowed them to assume that at least one additional variant of the EPO receptor functionally exists in nerve tissues, where signal transmission is different from signal transmission of red blood cell precursors, and that there is a risk that some EPO analogs can counteract the ability of endogenous EPO to bind to this receptor variant. The difference in biological activities between endogenous EPO and these EPO analogues indicates that receptor signaling is carried out through functionally different EPO receptors that respond to different domains of the EPO molecule. In fact, it has been shown that although the protein sequence encoded by the EPO receptor gene is identical to the sequence expressed by erythroid precursors, the neuronal-type receptor binding affinity for EPO ίη νίίτο is much lower than the EPO proerythrocyte receptor. See, for example, Makib, 8., e1 a1., Ί. ΒίοΙ. Sies., 268, 11208-16 (1993). Apparently, these differences arise due to additional proteins, and the results obtained may indicate that, rather, different signal transmission paths are realized than they are activated during the maturation of red blood cells. It is interesting that this difference in affinity does not change with complete deglycosylation of EPO, and this result is not unexpected, since active binding in the nervous tissue occurs in the normal non-glycosylated region of the loop of the AB EPO molecule. In the same place. In addition, EPO produced by astrocytes (which predominantly represents the same product produced by other cells, such as neurons), is a smaller variant than that produced by the kidneys. Makiba, 8., e1 a1., 1. Βίο1. Sies., 269, 19488-93 (1994). This difference is apparently the result of varying degrees of glycosylation. In the same place. Whether a desialized natural ligand exhibits differences in affinity for said known receptor proteins has not yet been established, but this is an important problem.

In addition to the existence of EPO - receptor modifying additional proteins, the EPO receptor is the product of a complex gene that encodes a number of known receptor variants, including a shortened, soluble receptor. Ouashar. B., e1 a1., Eig. ί. Vusyet., 239, 494-500; Ouatar. V., e1 a1., Vusyt. Vyryuk. Ls1a, 1403, 169-78 (1998); Waggop, S., e1 a1., Oepe, 147, 263-8 (1994); Syp, K., e1 a1., Vgash Vek. Μο1. Vgash Vek., 81, 29-42 (2000); Eschya, M., e1 a1., Leiketaa, 11 8irr1. 3, 444-5 (1997); \\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\ 's C. & Vaga ^ ukkb V., K1b. 1p! Egpa1., 55, 808-820 (1999). Whether any of these receptor variants contribute to the manifestation of the effects of EPO in the nervous tissue also needs to be clarified.

In addition, as discussed in the Background section of the invention, it was shown that the O'Vpep peptide exhibits tissue protective activity w νίίτο, but does not show the activity of erythropoietin 1n νίίτο. In fact, a study performed with an EPO analog that contains an additional carbohydrate chain in the O'Bpep peptide showed that the EPO analog lacks tissue protective properties. Based on the results, researchers suggest that some modifications of the O'Vpep peptide, such as the addition of carbohydrate chains, affect protein functionality. The EPO analog with modifications of the O'Bpep peptide probably acts as an antagonist with respect to the endogenous EPO of the body, since it partially or completely blocks the ability of endogenous EPO to bind to the EPO receptor. Essentially, the risk of increased injury due to injury is likely to be associated with the use of such an EPO analogue. Thus, it is believed that EPO analogues with modifications in the O'Vpep peptide should also lack tissue protective properties when tested using other assays, such as analyzes on rat motor neurons, and assays, such as assays using models of focal rat ischemia, an epileptic seizure caused by bicucullin, rat retinal ischemia, and spinal cord injury.

Receptor-mediated transcytosis.

The inventors studied the ability of an EPO analog to overcome the blood-brain barrier using the same EPO analog as mentioned above, i.e. EPO analog with 5 Ν-linked carbohydrate chains (as opposed to 3 3-linked carbohydrate chains of recombinant EPO). An EPO analog appeared in the cerebrospinal fluid after systemic injection (Figs. 1 and 2). Without binding themselves to any particular theory, the inventors believe that the EPO analog is able to overcome the intact blood-brain barrier, since capillaries also form the blood-brain barrier

- 7 007812 express the EPO receptor and create an anatomical basis for receptor-mediated transcytosis from peripheral circulation to the brain. In essence, it is believed that other systemically administered EPO analogues are also able to cross the blood-brain barrier, as well as other barriers with a network of capillaries expressing the EPO receptor.

As a result, it was shown that since the analogs of EPO of the prior art retain the activity of erythropoietin when at least one of the functions of endogenous EPO is lost, there is a need to create a long-acting EPO preparation in this area that retains all of the known functions of endogenous EPO. Advantageously, the present invention is directed to the production of a long-acting EPO according to the present invention, which not only prolongs the serum half-life compared to recombinant EPO, but also preserves the functionality of endogenous EPO, i.e., tissue protective function and transcytosis ability. Various methods for modifying EPO to produce such a protein with a beneficial effect are presented in the next section.

Modification of native EPO.

Long-acting EPO preparations according to the present invention can be obtained in various ways. In general, long acting EPOs can be prepared by chemical modification related to carbohydrate (sugar) chains attached to EPO. As used in the text, the term carbohydrate chains refers to Ν-linked and O-linked oligosaccharide chains found in endogenous EPO, additional дополнительн-linked and O-linked oligosaccharide chains found in EPO analogues, and to any other carbohydrate chains, especially sugar chains, attached to EPO.

In one aspect, endogenous or recombinant EPO molecules are used for modification to prevent any effect on the tissue protective properties of endogenous EPO. In addition, EPO analogues are proposed for modification according to the present invention, provided that additional glycosylation sites are not located near the O'Bpep peptide, i.e. near amino acid sequence 30-47. As used herein, the term EPO analogues refers to modified EPO molecules that have at least one additional Ν-linked carbohydrate chain and / or at least one additional O-linked carbohydrate chain. In another aspect, the EPO analog used for modification does not contain any additional glycosylation sites within approximately 5 amino acids of the O'Bpep peptide. In another aspect, the EPO analog does not contain any additional glycosylation sites within approximately 3 amino acids from the O'Bpep peptide. And in another aspect, the EPO analog does not contain any additional glycosylation sites in the O'Vpep peptide.

The EPO analogue can also be used for modification according to the present invention, provided that the analogue has an established three-dimensional configuration, and it is confirmed that none of the additional carbohydrate chains blocks the O'Vpep peptide and does not cause loss of tissue protective function. In another aspect, an EPO analog is contemplated for use in a modification of the present invention, provided that the glycosylation method does not inhibit the tissue protective function of the peptide. And in another aspect, an EPO analogue can be used for modification according to the present invention, provided that the O'Bpep peptide has one (or less) carbohydrate chain. For example, endogenous EPO contains a 38 amino acid carbohydrate chain, and an additional carbohydrate chain in the O'Bpep peptide has been shown to inhibit tissue protective activity of the protein. Thus, an EPO analog having one carbohydrate chain or no carbohydrate chains in the O'Bpep peptide is intended to be used for modification. In one aspect, a carbohydrate chain linked to a 38 amino acid can be moved to some other portion of the protein.

Non-limiting examples of modifications of the present invention include (1) the formation of additional acid residues on carbohydrate chains by oxidation of adjacent hydroxyls; (2) substitution of sialic acid residues with less labile residues; (3) an increase in the negative charge on the erythropoietin molecule by sulfation and / or phosphorylation; and / or (4) attaching more complex molecules to the ends of the carbohydrate chains. Thus, modifications to the EPO carbohydrate chains may include oxidation, sulfation, phosphorylation and / or PEGylation, along with other procedures that will be discussed in more detail below and further illustrated in Example 1.

Oxidation of sugar chains and substitution of sialic acid residues.

The chemically modified long acting EPO of the present invention may include EPO molecules in which carbohydrates (sugars) are oxidized to form additional acid residues. In another aspect, sialic acid residues are substituted with less labile acid residues. Modifications of this type lead to an increase in the half-life of the molecule compared to endogenous EPO, since the galactose chains, through which the liver recognizes, passes and removes the bound protein from the blood circulation, are protected from detection. A more substantial chemical modification of the carbohydrate chains of the EPO molecule leads to a more significant increase in the half-life of the long-acting EPO serum of the present invention. For example,

- 8 007812 the more adjacent hydroxyls are substituted by acids, the greater the increase in serum half-life.

Although an ordinary person skilled in the art knows the existence of several suitable methods for converting galactose units of erythropoietin, it should be noted that a suitable method includes (1) modifying sugar molecules with adjacent periodate hydroxides to form aldehydes; and (2) the oxidation of aldehydes to produce acids. Reagents suitable for the oxidation of the sugar chain to form aldehydes are known to those skilled in the art and include, but are not limited to, periodates such as sodium periodate and sugar oxidases such as galactose oxidase. In addition, those skilled in the art should be aware of suitable reagents for aldehyde conversion, such as the Benedict reagent (commercially available from BY1cg). In one aspect, the sugar molecules are oxidized with sodium periodate and then treated with Benedict's reagent (Ivieg) to convert the aldehydes to acids.

In another aspect, an EPO isomer having about 0-13 sialic acid residues, or an EPO analog having at least one carbohydrate chain that does not have a sialic acid residue, is oxidized using galactose oxidase. The asialo form of EPO can be used according to this aspect of the invention, i.e. The α- or β-form of EPO with all single sialic acid residues removed from the terminal sections of the carbohydrate chains. Preferably, such asialoerythropoietin is used. After oxidation, EPO is exposed to another oxidizing agent, such as Benedict's reagent, to convert aldehydes to acids.

In another aspect, a ruthenium tetroxide system can be used to form acids on carbohydrate chains. Provided that such a system modifies the galactose chain, even if the resulting acids are removed from the EPO molecule, the molecule should still continue to avoid the possibility of being removed by the liver, since the galactose chain, which is a component that the liver recognizes, will no longer exist.

Increase negative charge.

In another aspect of the present invention, a long-acting EPO molecule according to the present invention is formed by the addition of sulfates and / or phosphates to the EPO molecule, which leads to an increase in the negative charge of the molecule and thereby to an increase in the half-life of the molecule. In other words, the negative charge of the EPO molecule can be increased by sulfation, which involves the transfer of a sulfuryl group from a sulfate donor. And also the negative charge can be increased by introducing a phosphorus group into the carbohydrate.

A suitable method for insulin sulfation is discussed in the publication by Z. Ropdog e1 a1., Rgeragayop οί Nf11-Ro1epsu. ПηκυΙίηκ апηκυΙίηκ апηκυΙίηκ Iapd a Youe1 Ziyayop Rgosebige, 01аее1е5. Yo1. 32, Νο. 12, Bespieg 1983. For example, insulin sulfation was carried out in an organic solvent such as dimethylformamide (ΌΜΕ), in the presence of condensing agents such as Ν, Ν'-dicyclohexylcarbodiimide (BSS), and a sulfate donor. The degree of sulfation can be adjusted to a level of more than eight times by changing the amount of condensing agent. Although sulfated insulin obtained in the usual way loses the main bioactivity of insulin, the bioactivity of insulin sulfated by the Ropdeg method is between 78 and 87% of the activity of unmodified insulin.

Using the same procedure for EPO, an ordinary person skilled in the art will be able to control the degree of sulfation and, therefore, the half-life in serum of chemically modified EPO. For example, the negative charge of EPO can be increased by adding sulfates to the protein by dissolving EPO or an EPO analog in at least one water-soluble carbodiimide, preferably BSS, at a temperature of about 4 ° C. Although BSS is preferred as a condensing agent, those skilled in the art will readily identify other suitable condensing agents for use in the present invention.

Similar procedures can be used to regulate the degree of phosphorylation of EPO with phosphoric acid (H ₃ PO ₄ ) as a phosphate donor. Again, although phosphoric acid is preferred, those skilled in the art can easily select other phosphate donors for phosphorylation of EPO.

Attachment of PEG chains to the ends of carbohydrate chains.

EPO carbohydrate chains can be modified by the addition of at least one polyethylene glycol (PEG), a compound with a long and reliable clinical history, which has the following general formula:

PEG may be methoxyPEG (mPEG) having the following general formula:

- 9 007812

In one aspect, the PEG is an aminoPEG, preferably methoxyPEG with primary amino groups at the ends (mPEG-NOH ₂ ). Polyethylene glycol chains with primary amino groups at the ends are very suitable polymers exhibiting functional activity. The terminal amino groups of mPEG-ΝΗ are more active with respect to acylating agents than the hydroxyl groups that are present in ordinary PEG molecules, and they are also easily subjected to reductive amination reactions. In another aspect, PEG is an electrophilically activated PEG, such as mPEG-succinimidyl propionate (mPEG-BRL) or mPEG-succinimidylbutanoate (mPEG-BVL), both of which are commercially available from the company Tetareis alumina. In another aspect, PEG is a methoxyPEG hydrazide.

In one aspect, the addition of at least one type of PEG is achieved through periodate oxidation (as described above) followed by the use of cyanoborohydride and aminoPEG. For example, an EPO molecule in a solution may first be oxidized by a periodate, such as sodium periodate, for a predetermined period of time at room temperature, which leads to the formation of aldehydes in carbohydrate chains. A suitable periodate is sodium meta-periodate, which is commercially available from Bsh. Then the periodate can be removed by buffer exchange, then the oxidized sialic acid groups on the связанных-linked oligosaccharide groups of the EPO molecule can be exposed to at least one type of PEG - aminoPEG, in the presence of cyanoborohydride. Suitable PEG types for use include, but are not limited to, methoxy-PEG hydrazides, which are commercially available from Tkaretareys.

In another aspect, the addition of at least one type of PEG was performed by attaching groups of the PEG molecule to terminal galactose residues after oxidation with galactose oxidase. For example, the asialo-form of EPO (having unprotected terminal galactose residues) in the buffer is first exposed to galactose oxidase (commercially available from Bschsha) with the formation of aldehydes in carbohydrate chains. The buffer can then be removed by buffer exchange, after which the oxidized galactose residues can be exposed to at least one type of PEG aminoPEG in the presence of cyanoborohydride.

The methods described above are not considered to be limiting, since these or other methods can be used to prepare the compounds of the invention. For example, a person skilled in the art should recognize that these chemical modifications can be used to create variants of other derivatives of EPO long-acting, such as cytokines with tissue protective function described in international publication No. ^ 0/02053580 and US patent publications No. 2002/0086816 and 2003/0072737, which are fully incorporated into this description by reference.

Obtaining EPO molecules.

A number of expression vector host systems can be used to produce EPO for the production of long-acting EPO and EPO molecules according to the invention. Such host expression systems are carriers by which long acting EPO molecules can be produced and subsequently purified, but also are cells that, when transformed or transfected with appropriate nucleotide coding sequences, can expose the modified erythropoietin gene ίη З11и. These systems include, but are not limited to, cells of bacteria, insects, plants, mammals, including human host systems, such as, but not limited to, insect cell systems infected with expression vectors of a recombinant virus (e.g., baculovirus) containing sequences, long-acting EPO coding; plant cell systems infected with expression vectors of a recombinant virus (e.g., cauliflower mosaic virus, CaMU; tobacco mosaic virus, TMU) or transformed with expression vectors of a recombinant plasmid (e.g., T1 plasmid) containing sequences encoding erythropoietin-related molecules; or mammalian cell systems containing human cell systems, such as HT1080, C08, CHO, BHK, 293, 3T3, recombinant expression constructs containing promoters derived from the mammalian cell genome, such as the metallothionein promoter, or from mammalian viruses, such as the late adenovirus promoter; 7.5K cowpox virus promoter.

In addition, a host cell strain can be selected that regulates the expression of the introduced sequences or modifies and processes the gene product in a specific desired manner. Such modifications and processing of protein products may be important for protein function. As is known to those skilled in the art, various host cells have specific mechanisms for post-translational processing and modification of proteins and genes.

- 10 007812

Appropriate cell lines or host systems can be selected to ensure the correct modification and processing of the expressed foreign protein. For this purpose, eukaryotic host cells can be used that have a cellular apparatus for the characteristic processing of the primary transcript, glycosylation and phosphorylation of the gene product. Such mammalian host cells, including human host cells, include, but are not limited to, HT1080, CHO, UEKO, BHK, HeLa, CO8, MOSCOW, 293, 3T3 and No. 138.

For the production of recombinant proteins for a long time and in high yield, stable expression is preferred. For example, cell lines that stably express a recombinant gene product encoding an EPO molecule can be obtained by genetic engineering. Instead of using expression vectors, which include viral sources of replication, host cells can be transformed with DNA, the transformation being regulated by appropriate elements that control expression, for example, a promoter, enhancer, sequences, transcription terminators, polyadenylation sites, etc., and selective marker. After the introduction of foreign DNA, the cells obtained by genetic engineering grow for 1-2 days in an enriched medium, and then they are transferred to a selective medium. A selectable marker in a recombinant plasmid confers selection resistance and allows cells to stably integrate the plasmid into their chromosomes and grow with the formation of centers, which, in turn, can clone and increase their mass in cell lines. This method can be successfully used to obtain genetically engineered cell lines that express a gene product related to the mutein of the EPO molecule. Such genetically engineered cell lines may be particularly useful in screening and evaluating compounds that affect the endogenous activity of the gene product of a related EPO molecule.

Alternatively, expression characteristic of the endogenous mutein EPO gene in a cell line or microorganism can be modified by inserting a heterologous DNA regulatory element into the stable cell line genome or a cloned microorganism so that the introduced regulatory element efficiently binds to the endogenous erythropoietin mutein gene. For example, the endogenous mutein EPO gene is usually transcriptionally silent, i.e. the EPO gene, which is usually not expressed or is expressed only at a very low level in the cell line, can be activated by introducing a regulatory element that is capable of stimulating the expression of the expressed gene product in such a cell line or microorganism. Alternatively, the transcriptionally silent, endogenous EPO gene can be activated by introducing a heterogeneous regulatory element that works in different types of cells.

A heterologous regulatory element can be integrated into a stable cell line or a cloned microorganism so that it is operatively linked to the endogenous erythropoietin gene in a manner such as predetermined homologous recombination, which is well known to those skilled in the art and is also described in French Patent No. 2646438, US patent No. 4215051 and 5578461 and international publications No. 093/09222 and No. 091/06667, a full description of which is incorporated into this description by reference.

Pharmaceutical Compositions

The present invention also relates to pharmaceutical compositions containing long acting EPO molecules according to the present invention. Since the long acting EPO molecules of the present invention predominantly exhibit erythropoietic activity, as well as tissue protective properties and transcytosis, they are proposed for use in the treatment of anemia and related diseases in individuals at risk of various tissue damage, such as stroke and myocardial infarction. In addition, long-acting EPO molecules according to the present invention are proposed for use in the treatment of anemia and related diseases in individuals also experiencing mental impairment, such as Alzheimer's disease, Parkinson's disease, etc. In addition, long-acting EPO molecules according to the present invention are proposed for use in the treatment of anemia in individuals experiencing conditions resulting from a normal aging process, for example, problems of equal equilibrium, leading to a drop in, minor injuries, etc. In addition, the present invention relates to the use of long acting EPO molecules as carriers for other molecules that have low passability through barriers with capillaries having EPO receptors.

For example, any of the long-acting EPO molecules described above can be incorporated into pharmaceutical compositions of the invention. In addition, various EPO analogues can be incorporated into pharmaceutical compositions of the invention in admixture with at least one tissue protective cytokine, which will be discussed in more detail below.

The pharmaceutical compositions of the invention comprise a therapeutically effective amount of long acting EPO, preferably in purified form. The composition should be appropriate for the type of administration. In other words, the pharmaceutical composition according to the invention includes such an amount of long acting EPO according to the invention that the condition being treated is treatable provided that the appropriate dose and stratum are used

- 11 007812 gia. And, as will be discussed in more detail below, the pharmaceutical compositions must be delivered in a non-toxic dosage amount.

The pharmaceutical compositions of the invention may include a therapeutically effective amount of a long acting EPO and a suitable amount of a pharmaceutically acceptable carrier to provide an appropriate dosage form for administration to a patient. In a separate aspect, the term “pharmaceutically acceptable” means a compound approved by a federal regulatory authority, or located in the state government, or listed in the United States Pharmacopeia or other generally recognized foreign pharmacopeia for use in animals, and especially humans. The term “carrier” refers to a diluent, adjuvant, excipient or binder with which the therapeutic preparation is administered. Such pharmaceutical carriers may be sterile liquids, such as physiological saline solutions in water and oils, including oils from petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, and the like. Saline is the preferred carrier when the pharmaceutical composition is administered intravenously.

Saline solutions and aqueous solutions of dextrose and glycerol can also 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, skim milk powder, glycerin, propylene glycol, water, ethanol, etc. P.

The pharmaceutical compositions of the invention may also contain small amounts of a wetting agent, or emulsifier, or pH adjusting buffers. These compositions can be prepared in the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained release compositions, and the like.

Compounds according to the invention can be obtained in the form of neutral or salt forms. Pharmaceutically acceptable salts include salts in the formation of which free amino groups are involved, such as salts formed with hydrochloric, phosphoric, acetic, oxalic, tartaric acid, etc., and salts in which free carboxyl groups are involved, such as salts formed with hydroxides of sodium, potassium, ammonium, calcium, iron, as well as salts with isopropylamine, triethylamine, 2-ethylaminoethanol, histidine, procaine, etc.

Composition containing EPO long-acting.

As summarized above, any of the long acting EPO molecules of the present invention is contemplated for use in pharmaceutical compositions. In one aspect, a long-acting EPO molecule resulting from the oxidation of adjacent hydroxyls is included in a pharmaceutical composition according to the invention. In another aspect, the pharmaceutical composition of the invention includes at least one long acting EPO that results from the replacement of sialic acid residues with less labile residues. In another aspect, a long-acting EPO included in a pharmaceutical composition is formed by increasing the negative charge on the EPO molecule by sulfation and / or phosphorylation. In yet another aspect, a long-acting EPO variant formed by attaching more complex molecules, such as PEG chains, to the ends of carbohydrate chains is included in the pharmaceutical compositions of the invention.

In addition, the present invention contemplates the use of a mixture of long acting EPO molecules obtained by any of the methods of the present invention in pharmaceutical compositions of the invention. For example, a pharmaceutical composition according to the invention may include at least one long acting EPO that results from replacing sialic acid residues with less labile residues, and at least one long acting EPO that results from an increase in negative charge on the EPO molecule by sulfation and / or phosphorylation.

Transport system.

As previously discussed, long-acting EPO molecules of the present invention are capable of overcoming barriers with a network of capillaries having EPO receptors. Thus, another aspect of the present invention is a transport system using the long acting EPO of the present invention as carriers for molecules having low permeability with respect to a target region of the body having EPO receptors. Such transport systems advantageously represent a new and reliable method of delivery across intact barriers.

In one aspect, the transport system includes a long-acting EPO according to the present invention and at least one low brain permeability molecule in order to provide a new and reliable method of delivery across the intact blood-brain barrier. In other words, the long-acting EPO of the present invention helps to ensure that molecules with low brain permeability act as a molecular Trojan horse in order to increase the incorporation of small or large molecules into the brain for diagnostic or therapeutic purposes.

- 12 007812 molecules.

In fact, an important problem in the treatment of human brain tumors is dictated by the need to deliver therapeutic agents to certain areas of the brain, distribute them in brain tumors and target them to brain tumors. Molecules that may in other cases be effective in diagnosis and therapy, either cannot overcome the blood-brain barrier (BBB) in the areas of the brain adjacent to the tumor, or cannot cross the barrier between the circulatory system and the tumor (TBB) in appropriate amounts. Thus, there is still a need for new delivery strategies that will be unique to the brain and which will bypass the vasculature. For example, antibodies that could be used either as diagnostic or therapeutic molecules cannot overcome HTB in quantities sufficient for their effective action due to their size. Essentially, the long acting EPO of the present invention can be used as a carrier for such molecules to help overcome BBB or TBB. One example of a molecule that can be used with long-acting EPO according to the present invention is an antisense oligonucleotide, which is usually used either to inhibit oncogenic signals or to display gene expression in the brain ίη νΐνο. In addition, the long-acting EPO of the present invention can be incorporated into various gene therapy compositions (viral or non-viral compositions), which are often too large to overcome VTB without adjuvants.

In addition, the long-acting EPO of the present invention can be used as a carrier-mediated carrier for various chemotherapeutic agents. Since active drug carriers that are expressed in BBB and TBB effectively transfer chemotherapeutic agents from the brain back to the blood, the distribution of these agents in the brain can be inhibited or prevented. Partly for this reason, most of the classical chemotherapeutic molecules used to treat tumors outside the central nervous system (ΟΝ8) are not effective in treating brain tumors. Thus, the use of long-acting EPO according to the present invention as a carrier for such chemotherapeutic agents may prove to be useful not only in transferring agents to the brain, but also for preserving agents in the brain for therapeutic effect. In another aspect, a long-acting EPO may be associated with a drug that inhibits the active carrier to further ensure the utilization of chemotherapeutic agents that usually enter the brain from the blood.

In addition, the present invention also contemplates the use of modified EPO as carriers for molecules with low permeability to other regions of the body having EPO receptors. Non-limiting examples of such cells include cells of the retina, muscle, heart, lung, liver, kidney, small intestine, adrenal cortex, adrenal medulla, capillary endothelium, testis, ovary, pancreas, bone, skin, and endometrium. In particular, sensitive cells include, without limitation, neural cells; retinal cells: photoreceptor (rods and cones), ganglion, bipolar, horizontal cells, retinal cells lacking an axon, and Mueller cells; muscle cells; heart cells: myocardium, pacemaker, sinus-atrial node, sinus node and connective tissue cells (atrioventricular node and its ligament); lung cells; liver cells: hepatocytes, stellate cells and Kupffer cells; kidney cells: mesangium, renal epithelial cells and tubular interstitiocyte; small intestine cells: goblet cells, intestinal crypt cells (crypts) and small intestinal endocrine cells; cells of the adrenal cortex: cells of the glomerular zone of the adrenal cortex, bundle cells and reticular cells; adrenal medulla cells: chromaffin cells; capillary cells: pericytes; testicular cells: Leydig cells, Sertoli and sperm cells and their predecessors; ovarian cells: graph vial follicular cells and primary ovarian follicle cells; pancreatic cells: islets of Langerhans, α-cells, β-cells, γ-cells and P-cells; bone cells: preosteoblasts, osteoclasts and osteoblasts; skin cells; endometrial cells: endometrial stroma and endometrial cells; as well as stem and endothelial cells present in the above organs.

Composite mixture of EPO analog and tissue protective cytokine.

As briefly mentioned above, the pharmaceutical composition according to the present invention may include an EPO analog having at least one additional Ν-linked carbohydrate chain and / or at least O-linked carbohydrate chain (which has an extended serum half-life, but which lacks tissue protective activity), in a mixture with at least one tissue protective cytokine. For example, an EPO analog having at least two additional Ν-linked carbohydrate chains, where one of the additional carbohydrate chains is located in the 0'Bep peptide, in combination with a tissue protective cytokine can form a composition according to the invention. In another aspect, the pharmaceutical composition according to the invention may include at least one tissue protective cytokine and at least one EPO analogue containing additional carbohydrate chains, which, as is known from the established three-dimensional conformation of the analogue, block the sequence of the 0'Bep peptide. In another aspect, pharmaceutical

The 13007812 composition according to the invention includes at least one tissue protective cytokine and at least one EPO analog that does not exhibit tissue protective function as a result of the addition of additional carbohydrate chains to the protein.

An EPO analog with a displaced glycosylation site is contemplated for use in the pharmaceutical compositions of the present invention. Without binding themselves to any particular theory, the inventors believe that if the natural glycosylation site of amino acid 38 is moved to another location of the EPO analog molecule, outside the 30-47 amino acid segment, the tissue protective properties of the EPO analog should increase compared to the EPO analog, whose glycosylation site is located at amino acid 38. Thus, the pharmaceutical composition according to the invention may include an EPO analog with a displaced glycosylation site, from amino acid 38 to another site in the molecule. The displaced glycosylation site may be located at amino acids 51, 57, 69, 88, 89, 136 or 138, as proposed in PCT publication No. \ ¥ O 01/81405. In one aspect, the O'Vpep peptide sequence contains 1 or less carbohydrate chains.

Suitable tissue protective cytokines used according to this aspect of the present invention are preferably those cytokines that do not affect the bone marrow, but they retain the tissue protective effect of endogenous EPO, however, any cytokine that exhibits tissue protective properties is contemplated for use in accordance with the present invention. For example, suitable tissue protective cytokines include chemically modified EPO obtained by guanidation, amidation, carbamylation (carbamoylation), trinitrophenylation, acylation (acetylation or succinylation), nitration, or combinations thereof. In addition, EPO molecules with the modification of at least one residue of arginine, lysine, tyrosine, tryptophan or cysteine or carboxyl groups are also proposed to be used as tissue protective cytokines according to this aspect of the present invention.

In addition, additional tissue protective cytokines for use in accordance with the present invention can be obtained by limited proteolysis, removal of amino groups and / or mutational substitution of residues of arginine, lysine, tyrosine, tryptophan or cysteine using molecular biological methods, as described in publication 8a1ake e1 a1. , 1990, VyusYt. VyurRuk. Ac1a 1038: 125-9, which is incorporated herein by reference in its entirety. For example, suitable tissue protective cytokines include at least one or more mutated EPOs having a mutation site at C78, Β10Ι, U118, L12L, E13A, B14A, B14B, B14E, B140, Υ15Α, Υ15Ρ, Υ15Ι, K20A, K20E, E21A , C298, C29 + C338, C33 + Ρ42Ν, Τ44Ι, K45A, Κ45Ό, U46A, Ν47Α, Ρ48Α, Ρ48Ι, Υ49Α, Υ498, No. 51Ρ, No. 51Ν, Ο59Ν, E62T, 67678, 7070A, Ό96Β, 8100, 8100, 8100, 8100, 8100, 8100, 8100, 8100, 8100, 8100, 8100 , 6101A, 6101Ι, L102A, B103A, 8104Α, 8104Ι, Α105Α, T106A, Τ106Ι, T107A, T107B, b108K, Ε108Α, 8126Α, Ρ142Ι, B143A, 8146Α, Ν147Α, Ρ147Α, Ρ147Α, Ρ147Υ, Ρ147Α, Ρ147Α, , L155A, C1608, Ι6Α, C7A, B13A, Ν24Κ, Α30Ν, H32T, Ν38Κ, Ν83Κ, P42A, Ό43Α, K52A, K97A, K116A, T132A, Ι133Α, T134A, K140A, P148A, 154, 154A, B14A, 152A, B14+ K154A, C158A, C161A and / and and B162A. Examples of the above modifications are described in US patent publications pending simultaneously, No. 2003/0104988, 2002/0086816 and 2003/0072737, which are incorporated herein by reference in their entirety. In the description, the replaced amino acid is first denoted by a single-letter code denoting a native amino acid, then its position in the EPO molecule is indicated, and then the amino acid to be replaced is indicated by a single-letter code. For example, 8100E refers to a human EPO molecule in which, at position 100, serine is replaced by glutamic acid.

In another aspect, the tissue protective cytokine may include one or more of the above mutation sites, provided that the mutation sites do not contain Ι6Α, C7A, K20A, P42A, Ό43Α, Κ45Ό, K45A, Ρ48Α, Υ49Α, K52A, K49A, 8100B , B103A, K116A, T132A, Ι133Α, K140A, Ν147Κ, Ν147Α, B150A, B150E, 6151A, K152A, K154A, 6158A, C161A or B162A.

In another aspect, tissue protective cytokines may include combinations of mutation sites, such as ^ 5 ^ 8100¾ Α30Ν / Η32Τ, Κ45 ^ / Κ150Е, B103E / S088, K140A / K52A, K140A / K52A / K45A, K97A / K152A, K97A / K152 / K45A, K97A / K152A / K45A / K52A, K97A / K152A / K45A / K52A / K140A, K97A / K152A / K45A / K52A / K140A / K154A, Ν24Κ / Ν38Κ / Ν83Κ and Ν24Κ / Υ15Α. In yet another aspect, tissue protective cytokines do not contain any of the above combinations. In another aspect, tissue protective cytokines may contain any of the above mutation sites, provided that the mutation sites do not contain any of the following combinations of substitutions: Ν24Κ / Ν38Κ / Ν83Κ and / or Α30Ν / Η32Τ.

Some modifications or combinations of modifications may affect the ability of a mutein to bind to its receptor, such as an EPO receptor or a secondary receptor. Examples of such modifications or combinations of modifications include, but are not limited to, 1 (152+, Β14Α / Υ15Α, Ι6Α, C7A, Ό43Α, P42A, Ρ48Α, Υ49Α, T132A, Ι133Α, T134A, Ν147Α, P148A, B150, , C161A and B162A. Corresponding mutations in human growth hormone are known to those skilled in the art as harmful. Thus, in one aspect, the tissue protective cytokine does not include one or more modifications or combinations of modifications that may affect the ability

- 14 007812 mutein to bind to its receptor. A discussion of such tissue protective cytokines is included in the parallel patent application US No. 10 / 612,665, filed July 1, 2003, entitled Recombinant tissue protective cytokines and nucleic acids encoding them to protect, restore and increase their sensitive cells, tissues and organs, the universal disclosure of which incorporated herein by reference.

In conclusion, any of the superfamilies of cytokines that exhibit tissue protective properties can also be used because they do not affect the effects of erythropoietin or the half-life of long-acting EPO serum. Examples include, but are not limited to, interleukin-3 (1L-3), interleukin-5 (1L-5), colony stimulating granulocyte and macrophage factor (CMC8E), pigment epithelium factor (PEEE), and vascular endothelial growth factor (UECE) )

In another aspect of the present invention, the pharmaceutical composition of the present invention may include an EPO analog having at least one additional Ν-linked carbohydrate chain and / or at least one additional O-linked carbohydrate chain (which has an extended serum half-life, but which has no tissue protective activity), in a mixture with at least one small molecule that exhibits tissue protective property. Suitable small molecules include, but are not limited to, steroids (e.g. lazaroids and glucocorticoids), antioxidants (e.g. coenzyme p ₁₀ , alpha lipoic acid and и), anti-catabolic enzymes (e.g. glutathione peroxidase, superoxide dismutase, catalase, synthetic catalytic cleaners as well as mimetics), indole derivatives (e.g. indolamines, carbazoles and carbolines), nitric acid, neutralizing agents, adenosine / adenosine agonists, substances of plant origin (flavonoids), herbal extracts (gink long biloba and turmeric), vitamins (vitamins A, E, and C), electron-withdrawing oxidase inhibitors (e.g., electronic xanthine oxidase inhibitors), minerals (e.g., copper, zinc, and magnesium), Ν8 (8 (e.g., aspirin, naproxen, and ibuprofen) and their combinations. In addition, the pharmaceutical composition according to the invention may include an EPO analog, a tissue protective cytokine, and a small molecule exhibiting tissue protective function.

Tissue-protective cytokines and / or small molecules are preferably present in the pharmaceutical compositions of the invention in an amount sufficient to maintain or increase the same activity in neural or other sensitive cellular systems as manifested by endogenous EPO. In one aspect, the tissue protective cytokine and / or small molecule is present in an amount sufficient to enhance the protection of an individual's tissue by protecting, maintaining, or enhancing the viability and function of erythropoietin-sensitive individual cells. For example, a pharmaceutical composition according to this aspect of the present invention preferably includes an effective, non-toxic amount of tissue protective cytokine, for example about 1 ng or more. In one aspect, a tissue protective cytokine is present in the pharmaceutical composition in an amount of about 5 mg or less. In another aspect, a tissue protective cytokine is present in the pharmaceutical composition in an amount of from about 500 ng to 5 mg. In another aspect, the pharmaceutical composition comprises from about 1 μg to 5 mg of tissue protective cytokine, preferably from about 500 μg to 5 mg. In an alternative aspect, a greater amount of tissue protective cytokine is contained in the pharmaceutical composition of the invention, for example, from about 1 to 5 mg. As is known to specialists in this field, the amount of pharmaceutical composition administered to a patient depends on a number of factors, including, but not limited to, the patient's condition and frequency of dosing. Regarding dosing, a discussion will be described in more detail below.

Methods of treatment and administration.

The aforementioned EPO long-acting and pharmaceutical compositions containing EPO long-acting, are intended for therapeutic or prophylactic treatment of anemia, human diseases, including either anemia or anemic conditions, or diseases or methods of treatment that lead to anemia. In general, the long-acting EPO of the present invention facilitates the less frequent administration or use of smaller doses of erythropoietin for the treatment of the above diseases, enabling patients to recover from other tissue injuries, in particular injuries of other EPO-dependent cells, tissues or organs. Non-limiting examples of such EPO-dependent cells include retinal, muscle, heart, lung, liver, kidney, small intestine, adrenal cortex, adrenal medulla, capillary endothelium, testes, ovaries, pancreas, bone, skin and endometrial cells. In particular, EPO-dependent cells include, without limitation, neuronal cells; retinal cells; photoreceptors (rods and cones), ganglia, bipolar, horizontal, amacrine cells and Mueller cells; muscle cells; heart cells: myocardial cells, pacemaker cells; cells of the sinus atrium; sinus node and connective tissue cells (atrioventricular node and His bundle); lung cells; liver cells: hepatocytes, stellate cells and Kupffer cells; renal cells: mesangial, renal epithelial cells and interstitial cells of the renal tubules; small intestine cells: goblet cells, cells of the intestinal lymph nodes (crypt)

- 15 007812 and enteric endocrine cells; adrenal cortex cells: glomerular cells, bundle cells and reticular cells; adrenal medulla cells: chromaffin cells; kapmllar cells: pericytes; testis cells: Leydig, Sertoli cells and their predecessors; testicular cells: cells of the graaf vesicle and cells of the primary ovarian follicle; pancreatic cells: Langerhans islet cells, α cells, β cells, γ cells and P cells; bone cells; bone cell precursors, osteoclasts and osteoblasts; skin cells; endometrial cells: endometrial stromal cells and endometrial cells; as well as stem and endothelial cells present in the above organs. Individuals susceptible to stroke, spinal cord injury, peripheral nerve damage, diabetic nephropathy, retinal diseases, including, without limitation, macular edema, diabetic retinopathy, etc., may also be shown to use long acting EPO. In addition, the use of long-acting EPO in the treatment of diseases and for obtaining a tissue protective effect is also discussed in the jointly filed July 3, 2002, US patent application No. 10 / 188,905, as well as in application No. 10 / 512,665, filed July 1, 2003, both of which all their fullness are incorporated into this description by reference.

The present invention contemplates the use of long acting EPO for systematic or chronic administration, acute administration and / or alternating administration. In one aspect, the pharmaceutical compositions of the invention are administered chronically to protect or heal target cells, tissue, or organ. In another aspect, the pharmaceutical compositions of the invention can be administered acutely, i.e. for single treatment during damage. In another aspect, the pharmaceutical compositions of the invention are administered in a cyclic manner.

Administration of the composition may be parenteral, for example, by intravenous injection, intraperitoneal injection, intraarterial, intramuscular, intradermal or subcutaneous administration; through inhalation; through the mucous membrane, for example, oral, nasal, rectal, intravaginal, sublingual, submucosal and transdermal; or combined. Preferably, the administration of the pharmaceutical composition of the invention is parenteral. Such administration can be carried out in a dose of from about 0.01 pg to 5 mg, preferably from 1 pg to 5 mg. In one aspect, the dose is from about 500 pg to 5 mg. In another aspect, the dose is from about 1 ng to 5 mg. In another aspect, the dose is from about 500 ng to 5 mg. In yet another aspect, the dose is from about 1 μg to 5 mg. For example, the dose may be from about 500 μg to 5 mg. In another aspect, the dose may be from about 1 to 5 mg.

Pharmaceutical compositions of the invention adapted for parenteral administration include aqueous and non-aqueous sterilized injectable solutions or suspensions, which may contain antioxidants, buffers, bacteriostatic agents and dissolved substances, which render the compositions substantially isotonic with respect to the blood of a given recipient. In this aspect of the invention, pharmaceutical compositions may also include water, alcohols, polyols, glycerin, vegetable oils, and mixtures thereof. Pharmaceutical compositions adapted for parenteral administration can be presented in single dose containers or in multiple doses, for example, hermetically sealed ampoules and vials, and can be stored in a lyophilized (dried at a temperature below 0 ° C) condition, for which injection only addition is required sterilized liquid carrier, such as sterilized saline, immediately before use. Injectable solutions and suspensions prepared for immediate administration can be prepared from sterilized powders, granules and tablets. In one aspect, an automatic injection device containing an injectable long-acting EPO solution according to the invention is adapted for emergency cases in ambulances, emergency rooms and in combat situations.

In one aspect, a pharmaceutical composition according to the invention is prepared according to conventional methods as a pharmaceutical composition adapted for intravenous administration to humans. For example, the pharmaceutical composition may be in the form of a solution in a sterilized isotonic aqueous buffer. When necessary, the pharmaceutical composition may also include a solubilizing agent and / or a local anesthetic, such as lidocaine, to relieve pain at the injection site. The ingredients can be added either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or anhydrous concentrate in a hermetically sealed container, such as an ampoule or small bag, which indicates the amount of active ingredient. When the pharmaceutical compositions of the invention are to be administered by infusion, an infusion bottle with sterilized water intended for pharmaceutical purposes or with physiological saline can be adapted to dispense the composition. And, when the pharmaceutical composition is to be administered by injection, the ampoule with sterilized saline can be used for mixing before administration.

Pharmaceutical compositions adapted for oral administration may be prepared in the form of capsules or tablets; powders or granules; solutions; syrups or suspensions (in aqueous

- 16 007812 or non-aqueous liquids); edible foams or whipped cream; emulsions, or combinations thereof. The oral composition may include from about 10 to 95 wt.% The active ingredient. In one aspect, the active ingredient is included in the oral composition in an amount of from about 20 to 80 wt.%. In another aspect, the oral composition comprises from about 25 to 75 wt.% The active ingredient.

Tablets or hard gelatine capsules may include lactose, starch or derivatives thereof, magnesium stearate, sodium sugar, cellulose, magnesium carbonate, stearic acid and its salts. Soft gelatine capsules may include vegetable oils, waxes, fats, semi-solid, liquid polyols, or mixtures thereof. Solutions and syrups may include water, polyols, sugars, or mixtures of these ingredients.

In addition, the active ingredient intended for oral administration may be coated with a material or mixed with a material that slows down the disintegration and / or absorption of the active ingredient in the gastrointestinal tract. For example, the active ingredient may be mixed or coated with glyceryl monostearate, glyceryl distearate, or combinations thereof. Thus, sustained release of the active ingredient can be achieved over many hours and, if necessary, the active ingredient can be protected from degradation in the stomach. Pharmaceutical compositions for oral administration can also be prepared to facilitate the release of the active ingredient in a separate section of the gastrointestinal tract under conditions of a specific pH and enzymatic reaction.

Pharmaceutical compositions adapted for transdermal administration may be in the form of separate patches intended for prolonged contact with the epidermis of the recipient. In addition, pharmaceutical compositions adapted for topical administration may be formulated as ointments, creams, suspensions, lotions, powders, solutions, pastes, gels, sprays, aerosols, oils, eye drops, lozenges, lozenges and mouthwashes and their combinations. When the topical administration is for the skin, mouth, eyes or other external tissues, it is preferable to apply topically an ointment or cream. And when the pharmaceutical composition is prepared as an ointment, then the active ingredient, i.e. EPO long-acting, can be used with paraffin or water-miscible ointment base. Alternatively, the active ingredient may be contained in a cream with an oil-in-water or water-in-oil base. When topical administration is by eye drops, the pharmaceutical compositions of the invention preferably include an active ingredient that is dissolved or suspended in a suitable carrier, for example, in an aqueous solvent.

Pharmaceutical compositions adapted for administration to the nose and lungs may include solid carriers, such as powders (preferably having a particle size of from about 20 to about 500 microns). Powders can be administered by rapid inhalation through the nose from a powder container that is kept close to the nose. As an alternative aspect, pharmaceutical compositions intended for administration to the nose according to the present invention may include liquid carriers, for example, nasal sprays or nasal drops. Preferably, the pharmaceutical compositions of the invention are administered directly into the nasal cavity.

Direct inhalation into the lungs may be accompanied by deep inhalation through the tip into the mouth of the pharynx and be carried out using other specially adapted devices, including but not limited to pressurized aerosols, nebulizers or insufflators that can be designed to deliver predetermined dosages of the active ingredient . Pharmaceutical compositions intended for inhalation into the lungs may include aqueous or oily solutions of the active ingredient. Preferably, the pharmaceutical compositions of the invention are administered by deep inhalation directly into the mouth of the pharynx.

Pharmaceutical compositions adapted for rectal administration may be formulated as suppositories or enemas. In one aspect, the suppositories according to the invention include from about 0.5 to 10 wt.% The active ingredient. In another aspect, the suppository includes from about 1 to 8 wt.% The active ingredient. In another aspect, the active ingredient is contained in a suppository in an amount of from about 2 to 6 wt.%. In this aspect of the invention, the pharmaceutical compositions of the invention may include binders and carriers, such as triglycerides.

The pharmaceutical compositions of the invention adapted for vaginal administration may be prepared as pessaries, tampons, creams, gels, pastes, foams or spray formulations.

The pharmaceutical compositions of the invention can also be administered by perfusion, injection into an organ, or topical administration. In such aspects, the pharmaceutical composition preferably contains from about 0.01 to 30 pM, preferably from about 15 pM to 30 nM, of a long acting EPO according to the present invention. In one aspect, the perfusion solution is a solution (with a pH of about 7.4 to about 7.5 and an osmotic pressure of about 320 mOOSm / L) Ishueguayu οί HUBeopmp (U). which contains from about 1 U / ml to 25 U / ml EPO; 5% hydroxyethyl starch (preferably having a molecular weight of from about

- 17 007812

200,000 to 300,000 and mostly free of ethylene glycol, ethylene chloride, sodium chloride and acetone), 25 mM KH ₂ PO ₄ , 3 mM glutathione; 5 mM adenosine; 10 mM glucose; 10 mM ΗΕΡΕδ buffer; 5 mM magnesium gluconate; 1.5 mM CaCl ₂ ; 105 mM sodium gluconate; 200,000 units of penicillin; 40 units of insulin; 16 mg of dexamethasone and 12 mg of phenol red. The solution of IA is described in detail in US patent No. 47 98824, which is incorporated into this text in its entirety by reference. In another aspect, the solution i \\ 'may contain from about 0.01 pg / ml to 400 ng / ml, preferably from about 40 to 300 ng / ml of a recombinant tissue protective cytokine.

In some situations, it is desirable to administer the pharmaceutical compositions of the invention locally to the area to be treated. Such administration can be achieved by local infusion during surgery; topical application, for example, together with a wound dressing after surgery; by injection; through a catheter; by a suppository or by an implant, which may be a porous, non-porous or gelatinous material including membranes, such as silastic membranes, or fibers.

Furthermore, as briefly described above with respect to transdermal administration, the long acting EPO of the present invention can be delivered in a controlled release system. For example, a polypeptide may be administered by intravenous infusion, an implantable osmotic pump, transdermal patch, liposomes, or other modes of administration. In one aspect, a pump may be used, such as described in 8ib e1 a1., 1989, публикации. ip1. I. Furniture 321: 574. In another aspect, the compound can be delivered in a vial, in particular in a liposome, such as described in international publication No. XV 0 91/04014 and US patent No. 4704355, the universal disclosure of which is incorporated herein by reference. In another aspect, polymeric materials can be used to create a controlled release system, such as the materials described in the publication No ^ atb e1 a1., 1989, I. No. igokigd. 71: 105.

Such controlled release systems can be placed close to a therapeutic target, i.e. target cells, tissue or organ, which requires only part of the systemic dose. See, for example, the publication Soobkop, Meb1ca1 ArrNsabopk oG Sop1to11eb Ke1eake, νο1. 2, pp. 115-138, 1984. Other controlled-release systems that are contemplated to be used according to the present invention are described in review, 8c1epse 249: 1527-1533, 1990.

Dosage

The selection of a preferably effective and non-toxic dose for the administration methods described above will be determined by a qualified experimenter in the art based on factors known to those of ordinary skill in the art. Examples of such factors include a separate form of long acting EPO; pharmacokinetic parameters of EPO, such as bioavailability, metabolism, half-life, etc. (as determined by a qualified experimenter); the condition or disease being treated; beneficial effects that should be achieved in a normal individual; body weight of the patient; route of administration; frequency of administration, i.e. chronic, acute, alternating administration; concomitant therapeutic treatment and other factors that are well known to affect the effectiveness of the administered pharmaceuticals. Thus, the exact dosage should be established according to the decision of the practitioner and the conditions of the individual patient.

For example, the doctor’s desk reference (RNuyapk Zekk KeGegeps (ΡΌΚ)) indicates that depending on the patient population being treated with EPO, different hematocrit levels are achieved to avoid toxicity. RNu81s1apk Eekk KeGegepse, 54 ¹¹ 'Ε6., 519-525 apb 21252131 (2000). In fact, in patients with SSC, ΡΌΚ recommends dosing of EPO to achieve non-toxic target hematocrit numbers ranging from 30 to 36%. In contrast, for cancer patients taking chemotherapy, in в it is recommended to adjust the dosage at different hematocrit levels, i.e. if the hematocrit level exceeds 40%. In ΡΌΚ it is indicated that practitioners monitor the hematocrit in patients during EPO therapy and, in order to avoid toxicity, adjust the dose and / or stop treatment if the patient's hematocrit reaches or exceeds the upper limits of a given area. Therefore, a qualified practitioner who has gained knowledge and experience on the basis of the present invention will be able to administer doses of EPO sufficient to achieve a therapeutic effect, and at the same time avoid any complications associated with toxicity.

In one aspect, the long acting EPO of the present invention is administered chronically or systemically at a dosage of from about 0.1 to 100 μg / kg body weight per administration. For example, approximately 1 to 5 μg / kg of body weight is proposed for dosing once a week in the treatment of cancer patients receiving chemotherapy. In another aspect, the dosage of long acting EPO is from about 5 to about 50 μg / kg body weight per administration. In another aspect, a long acting EPO is administered in an amount of about 10 to 30 μg / kg body weight per administration. In yet another aspect, long acting EPO is administered in an amount of about 1 μg / kg body weight or less. For example, dosages of approximately 0.45 to 0.75 μg / kg of body weight of long acting EPO may be effective once a week for the treatment of anemia in patients with SLE.

- 18 007812

Preferably, an effective dose is sufficient to achieve serum levels of long-acting EPO that are higher than about 10,000 mU / ml (80 ng / ml). In one aspect, the effective dose in the serum reaches a long-acting EPO level of approximately 15,000 mU / ml (120 ng / ml) or higher. In another aspect, the effective dose reaches a serum level of long-acting EPO of approximately 20,000 mU / ml (160 ng / ml). Serum EPO levels are preferably measured at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 hours, or combinations thereof after administration. Dosages may be repeated if an ordinary person skilled in the art deems it necessary. For example, administration may be repeated daily, as long as it is clinically necessary, or after an appropriate interval, for example, every 1-12 weeks, preferably every 1-3 weeks.

Since long-acting EPO according to the invention have an extended serum half-life, their effectiveness in the body also increases. For example, when a sick mammal undergoes systemic chemotherapy to treat cancer, including radiation therapy, the administration of pharmaceutical compositions containing long-acting EPO in the treatment process of the present invention can reduce anemic conditions by decreasing the frequency of administration of doses and the number of doses compared to the frequency and the number of currently used compositions containing recombinant EPO.

And, as described above, when the pharmaceutical compositions of the invention include a long acting EPO of the present invention or an EPO analog mixed with a tissue protective cytokine, the compositions can be used to treat anemia and related diseases in patients at risk of tissue damage. For example, a patient with anemia who has a high risk of heart disease can be treated with the pharmaceutical compositions of the invention instead of the EPO analogues currently available to prevent the damage that the treatment can cause.

Kits for treatment.

The invention also relates to a pharmaceutical bag or kit that includes one or more containers filled with one or more ingredients of the pharmaceutical compositions of the invention. In one aspect, an effective amount of a long acting EPO and a pharmaceutically acceptable carrier may be packaged in one dose vial or in another container.

When the pharmaceutical composition according to the invention is adapted for parenteral administration, the composition can be stored, for example, in a lyophilized state. Thus, the kit may include a lyophilized composition, a sterile liquid carrier, and an injection syringe. In one aspect, the kit includes an ampoule containing a sufficient amount of lyophilized material for several treatments, the healthcare provider administering the drug must weigh a certain amount of material and add a certain amount of carrier for each treatment session. In another aspect, the kit may contain many ampoules, each of which contains a certain amount of lyophilized material, and many containers, each of which contains a certain amount of carrier, so that the healthcare provider administering the drug only needs to mix the contents of one ampoule and one container for each treatment session without measurement and weighing. In another aspect, the kit comprises an automatic administration device comprising an injectable long acting EPO solution according to the invention. In another aspect, the kit contains at least one ampoule with a lyophilized composition, at least one container with a solution as a carrier, at least one container with a local anesthetic, and at least one syringe (etc.). Preferably, the ampoules and containers are sealed.

In cases where the pharmaceutical compositions of the invention are administered by infusion, the kit preferably includes one ampoule with a pharmaceutical composition and at least one infusion bottle with sterilized water or sterilized saline for pharmaceutical purposes.

The kit according to the invention may also include at least one tip or specially adapted devices for direct inhalation into the lungs, such as pressurized aerosols, nebulizers or insufflators. In one aspect of the invention, the kit may include a device for direct inhalation into the lungs that contains a pharmaceutical composition, or a device and at least one ampoule of a long-acting EPO aqueous or oily solution according to the present invention.

In the case where the pharmaceutical composition according to the invention, containing long-acting EPO, is adapted for oral, transdermal, rectal, vaginal or nasal administration, the kit preferably includes one ampoule containing the active ingredient and at least one auxiliary agent for administration. Examples of administration aids include, but are not limited to, scoops (for oral administration), sterile cleansing soft pads (for transdermal administration), and nasal aspirators (for nasal administration). Such kits may include a single dose of long acting EPO (acute

- 19 007812 administration) or multiple doses (chronic administration).

In addition, the kit may be provided with one or more types of solutions. For example, pharmaceutical compositions of the invention containing long-acting EPO can be prepared in an albumin solution and a polysorbate solution. If the kit includes a polysorbate solution, it is preferable that the words free from albumin be written on the labels of the containers, as well as on the main panels of the kits.

In addition, the kit may also include a message in the form prescribed by the state authority regulating the production, use or sale of pharmaceuticals or biological products, which message notifies the body of the manufacture, use or sale of the drug for administration to a person.

The study of EPO.

The present invention also relates to studies to determine the erythropoietic and tissue protective properties of long-acting EPO according to the present invention, as well as EPO analogs used in some pharmaceutical compositions of the present invention. For example, the long-acting erythropoietic effect of EPO can be determined using the TP-1 assay, which will be discussed in more detail in Example 2. The tissue protective properties of EPO compounds can be tested using ίη νίίτο and ίη νίνο analyzes, which are discussed in more detail below. In addition, the present invention also provides tests to determine not only whether a single EPO compound exhibits tissue protective activity, but also whether the EPO compound acts as an antagonist against endogenous EPO.

The assays of the invention are preferably designed to be performed in a short period of time using a minimum amount of EPO compound. In addition, the developed assays are not intended to limit the invention, since other assays used to determine the erythropoietic and tissue protective properties of EPO compounds are known to those skilled in the art.

Erythropoietic activity assays.

Properties of erythropoietin, i.e. the ability to regulate hematocrit levels in a single EPO compound can be determined using various assays. In one aspect, TP1 cell line can be used to determine if a particular EPO compound has erythropoietic activity. Cells can be precipitated, washed, and resuspended at a concentration of 10 ⁵ cells in 1 ml of medium with recombinant EPO and an EPO compound of interest at certain concentrations. Individual cultures can be maintained for 24 hours, while the number of cells is determined using the reaction product of formazan (Ce11T11eg: Rgoteda, ΜαόΝοη, VI).

The ability of the EPO compound of interest can be evaluated first by ίη νίνο by determining its effect on hemoglobin concentration using female VLV / s mice. The animals are administered 500 U / kg of EPO body weight of interest to the EPO compound or an equal volume of vehicle subcutaneously 3 times a week for a total of 3 weeks (time interval sufficient to observe an erythropoietic reaction). It was found that the EPO compound exhibits erythropoietic activity if it increases the concentration of hemoglobin in mouse serum.

Further assessment of the ability can be obtained ίη νίίτο using TP1 erythroleukemia cells. The EPO compound of interest is erythropoietic if the relative number of TP1 cells becomes higher than the control. In addition, those skilled in the art will recognize that other assays used to determine erythropoietic activity are available. For example, the European Pharmacopoeia describes at least two assays used to determine the erythropoietic activity of EPO compounds, which include mouse studies after hypoxia and reticulocyte assays.

Studies of tissue protective properties based on the EPO receptor.

In one aspect, studies of tissue protective properties according to the present invention are based on the tissue protective EPO receptor. Since the sequence for the tissue protective receptor is isolated, a number of assays can be used to determine the tissue protective properties of an individual EPO compound. As is known to those skilled in the art, the type of assay used is highly dependent on the weight of the EPO compound.

For example, studies may include a competitive assay, or a sandwich assay, or a steric inhibition assay. Competitive analysis is based on the ability of an analog containing a labeled atom to compete with the analyte in the test sample for a limited number of binding sites on a common binding partner. As used herein, the term “analyte” refers to an EPO compound of interest that is tested for tissue protective activity. The term binding partner refers to any protein that binds to an analyte (usually EPO receptors). As used herein, the term labeled substance refers to labeled reagents, such as labeled assay analog, immobilized assay analog, labeled binding partner, immobilized binding partner, and steric conjugates. The labeled substance used in the invention may have any detectable function that does not affect the binding of the analyte and its binding

- 20 007812 partner. Non-limiting examples include fragments that can be directly determined, such as fluorochrome, fluorescent and radioactive labels, as well as fragments that must be reacted or produced to be detected, such as enzymes. Suitable labeled substances may be the radioactive isotopes P ³² , C ¹⁴ , I ¹²⁵ , Η ³ , I ¹³¹ and mixtures thereof; fluorophores, such as rare earth chelates, fluorescein, fluorescein derivatives, rhodamine, rhodamine derivatives, dansil, umbelliferonluciferase (firefly luciferase and bacterial luciferase (US Patent No. 4,737,456)), luciferin, 2,3-dihydrofthalazidazinazone, , β-galactosidase, glucoamylase, lysozyme, saccharidoxidase (glucose oxidase, galactose oxidase and glucose-6-phosphate dehydrogenase), heterocyclic oxidases (uratoxidase and xanthine oxidase) associated with an enzyme that uses hydrogen peroxide yes oxidation dyestuff precursor such as ΗΚΡ, lactoperoxidase, microperoxidase, and mixtures thereof; biotin / avidin; spin tags labeled with bacteriophages; stable free radicals; and their combinations. In one aspect, the labeled substance is at least one of horseradish peroxidase or alkaline phosphatase enzymes.

A skilled experimenter is aware of covalent binding methods for labeled substances with proteins or polypeptides. For example, binding agents, such as dialdehydes, carbodiimides, dimaleimides, bis-imidates, bis-diazotized benzidine, and the like, can be used to bind to the fluorescent, chemiluminescent, and enzymatic labels described above, some of which are described in US Pat. 3940475 and 3645090, the universal disclosure of which is incorporated herein by reference.

Reagent immobilization, i.e. separation of the binding partner from any analyte that remains free in the solution is required for sandwich analysis and may be accompanied by the conversion to insoluble form of either the binding partner or the analyzed analogue prior to the analysis procedure by adsorption on a water-insoluble matrix or surface (US patent No. 3720760), covalent binding, such as cross-linking with glutaraldehyde, or later converting into an insoluble form of a partner or analogue, for example, by immunoprecipitation itation.

Thus, the binding partner can be converted to an insoluble form before or after a competitive reaction, and the labeled substance and analyte associated with the binding partner are separated from the unbound labeled substance and analyte. This separation may be accompanied by decantation (where the binding partner was previously converted to insoluble form) or centrifugation (where the binding partner was precipitated after a competitive reaction). The amount of analyte in the test sample is inversely proportional to the amount of bound labeled substance, as measured by the amount of marker compound. Dose response curves plotted with known amounts of analyte were compared with the results of the study to quantify the analyte present in the test sample. When enzymes are used as labeled substances, the assays are commonly referred to as E18A.

In a subsequent sandwich analysis, for example, an immobilized binding partner is used to adsorb the analyte present in the test sample, the test sample is removed by washing, the bound analyte is used to adsorb the labeled binding partner, and the bound material is then separated from the residual labeled substance. The amount of bound labeled substance is directly proportional to the amount of analyte in the test sample. In a simultaneous sandwich assay, the test sample is not separated until a labeled binding partner is added.

In the competitive method and sandwich analysis, the phase separation stage is used as an integral part of the methods, while the study of steric inhibition is carried out in an undivided reaction mixture. Another type of competitive analysis, called homogeneous analysis, however, does not require phase separation. In such an assay, an enzyme conjugate with an analyte is prepared and used in such a way that when the antianalyzed substance is bound to the analyte, the presence of the antianalyzed substance changes the enzymatic activity. The tissue protective receptor is conjugated via a bifunctional organic bridge to an enzyme such as peroxidase. Conjugates are selected for use with EPO in such a way that EPO binding leads to inhibition or potentiation of the enzymatic activity of the label. This type of analysis is commonly called EM1T.

Steric conjugates are used in steric hindrance research methods used for homogeneous analysis. These conjugates are synthesized by covalently linking a low molecular weight hapten to a small molecule of the analyte in such a way that the anti-hapten antibody is basically unable to bind the conjugate for the same time as the anti-analyte. The analyte present in the test sample will bind to the anti-analyte, thereby facilitating the binding of the antigapten to the conjugate, resulting in a change in the properties of the conjugated hapten, for example, a change in fluorescence when the hapten is a fluorophore.

- 21 007812

More detailed information regarding studies of tissue protective properties is set forth in parallel patent application US No. 10/188905, registered July 3, 2002, and in application No. 60/456891, registered April 25, 2003, both of which are included in this text in their entirety.

Function research.

Since there is no information on the identification of the sequence of the tissue protective receptor, the tissue protective properties of EPO can be determined by examining the function of both ίη νονο and ίη νίίτο. Preferably, one skilled in the art should perform one ίη νίίτο or ίη νίνο analysis to determine the tissue protective properties of the EPO compound, but in some cases both analyzes may be required to make sure that the compound exhibits the same tissue protective properties of ίη νίίτο and ίη νίνο.

In practice, one skilled in the art should be able to determine whether an EPO analog having at least one additional Ν-linked carbohydrate chain and / or at least one additional O-linked carbohydrate chain exhibits tissue protective properties using a combination of assays disclosed by the present invention . First, ίη νίίτο tests, such as P19 cell model analysis and rat motor neuron analysis, can be performed to determine if the EPO compound of interest exhibits tissue protective properties. Ίη νίνο studies, such as studies using rat focal ischemia models, bicucullin-induced epileptic seizures, or spinal cord injury, can be used to confirm the results of the ίη νίίτο test.

The ίη νίίτο models contemplated by the present invention include, but are not limited to, models used to determine the lack of tissue protective properties of hyperglycosylated erythropoietin: P19 cell analysis, analysis of rat motor neurons and cDNA microarrays, which are described in more detail below and are also illustrated in Example 2. The examples are not intended to limit the invention, as one skilled in the art should recognize that there are other suitable analyzes ίη νίίτο for determining tissue protection total properties of EPO compounds. In general, an EPO compound should be considered as having tissue-protective properties if it maintains or enhances cell viability compared to control. Erythropoietin should be considered as an antagonist if it has a detrimental effect on cell viability in the analysis used in comparison with the control.

A. ίη νίίτο Analysis Based on the P19 Cell Line

In one aspect, the analysis of the тканη νίίτο tissue protective properties is based on the P19 cell line. For example, P19 cells can remain undifferentiated in a medium enriched with 2 mM L-glutamine, 100 U / ml penicillin C, 100 μg / ml streptomycin sulfate (Όίόοο) and 10% fetal bovine serum (Ηνοίοπο ΕηόοπιΙοπα). which contains 1.2 g / l No. 1 HCO ₃ . 10 mM HEPE8 buffer. The serum-free medium may contain the same components as above, with the exception of 5 μg / ml insulin, 100 μg / ml transferrin, 20 nM progesterone, 100 μM putrescine and 30 nM No. 8cO ₃ (81g) instead of fetal bovine serum.

Cells with 50% confluency are treated overnight with recombinant EPO and / or the EPO compound of interest, treated with trypsin for dissociation, washed in serum-free medium and placed in 25 cm ² tissue culture vials at a final density of 10 ⁴ cells / cm ² in serum-free medium - cells per se or pre-treated with additives. Cell viability can be determined by the exclusion of trypan blue.

As is known to qualified experimenters, the addition of recombinant EPO can prevent cell loss after taking serum and placing it in undifferentiated neuron-like P19 cells. For example, recombinant EPO helps to preserve up to 50% of neuron-like cells from death if it is used in a concentration of from 0.1 to 100 U / ml. Thus, in order to claim that EPO has a tissue-protective property, the EPO compound of interest should prevent the death of a large number of P19 cells and enclose in comparison with the control, preferably it should enclose approximately 25 to 50% of the cells, most preferably approximately 40 to 50% of the cells.

B. Analysis of ίη νίίτο rat motor neurons.

In another aspect, an analysis of rat motor neurons is used to determine the tissue protective properties of the EPO ίη νίίτο compound of interest. For example, primary motor neurons can be isolated from the spinal cord of 15-day-old rat embryos 8aida Ea \ u1su and purified by immunopanning. Cells are preferably seeded at low density (20,000 cells / cm ² ) in 24 mm well plates precoated with poly-E-antinthine and laminin and containing complete culture medium (No. IgO8a1, B27 (2%), 0.5 mM L-glutamine , 2% horse serum, 25 μM 2-mercaptoethanol, 25 μM glutamate, 1% penicillin and streptomycin, 1 ng / ml ΒΌΝΕ). After a period of time, preferably after about 6 days, EPO (10 U / ml) and the EPO compound of interest (10 U / ml) or vehicle can be added to the cultures (preferably about 5 days before determining the density of surviving neuronal cells). Then

- 22 007812 medium is removed and the cells are fixed with 4% paraformaldehyde in PB8 for 40 min, treated with 0.2% Triton X-100 to violate the permeability of the cell membrane, blocked with 10% fetal calf serum in PB8, incubated with antibodies against non-phosphorylated neurofilaments (8ΜΙ-32; 1: 9000) during the night and examined visually using the method based on the use of avidin / biotin with diaminobenzidine. The viability of motor neurons can be evaluated morphologically by counting 8ΜΙ-32 positive cells.

Mixed primary cultures of motor neurons undergo apoptosis while maintaining culture conditions. The addition of recombinant EPO (10 U / ml) to the culture medium 5 days before estimating the number of cells was shown to significantly increase the number of primary motor neurons observed on day 5. Thus, in order to claim that the EPO compound has a tissue protective property, the EPO compound of interest should preferably remain viable in at least the same number of motor neurons as in the control. In one aspect, an EPO compound of interest is considered to have tissue protective properties if a greater number of motor neurons are protected from death while maintaining culture conditions compared to the control.

C. Analysis of ίη νίίτο based on a cDNA microchip.

Another analysis of ίη νίίτο for determining the tissue protective properties of the EPO compound of interest is a cDNA microchip. Such an analysis can be used if the recombinant EPO and the EPO compound of interest change the expression of the gene in P19 cells in different ways. mRNA isolated from undifferentiated P19 cells can have a different character of gene modulation, established using a 1200 mouse cDNA microchip, depending on the effect of EPO. For example, the expression of 1200 genes in P19 cells can be determined using chip nylon membranes from C1op1cc11 (mouse atlas 1.2). Cells (10 ⁷ / sample) can be treated overnight with physiological saline, recombinant EPO, the EPO compound of interest (1 mU / ml) or mixtures thereof. The cells are then lysed for RNA extraction or serum removed for 3 hours (always in the presence of the same cytokine added during pretreatment). After standard extraction of total RNA using column chromatography, with DNA-ase treatment on the column, polyA + RNA was purified. Then the probes were obtained in the presence of [P ³² ] ATP. Labeled probes having preferably 20 million impulses or higher can be hybridized with cDNA present on nylon membranes at 68 ° C. The membranes are washed and held on an x-ray film. The intensity of the radioactive signals can be measured using a phosphorus indicator and analyzed using the Abak 1tade 2.0 computer program (S1op1ss11).

The ίη νίνο assays reviewed in the present invention include, but are not limited to, tissue protective function tests performed to evaluate EPO compounds using models such as focal ischemia model and epileptic seizure model caused by the introduction of bicucullin into the hippocampus. In addition, the ίη νίνο model, used to evaluate tissue protective function, includes a spinal cord injury model. In addition, various assays disclosed in international publication No. ^ 0/02053580 and US patent publications No. 2002/0086816 and 2003/0072737 are contemplated for use in the present invention.

I. Tests ίη νίνο based on the model of focal ischemia.

In one aspect, the ίη νίνο analysis used to determine the tissue protective properties of an individual EPO compound is based on a focal ischemia model. For example, male 8gadie-OaMeu rats (~ 250 g) can be used to obtain a model of three-vascular focal ischemia. Briefly, rats are anesthetized with pentobarbital (60 mg / kg body weight) and internal body temperature is maintained at 37 ° C. using water heating. The right carotid artery can be pinched with two sutures and dissected. A trepanation hole adjacent to the right orbit and directed at it, helps to visualize the middle cerebral artery, which can be cauterized distally relative to the nasal artery. In order to get partial shade surrounding this fixed MCA lesion, the contralateral carotid artery can be pinched for 1 h by extension using pointed tweezers. Saline, recombinant EPO (5000 U / kg body weight) or the EPO compound of interest (5000 U / kg body weight) can be administered at the beginning of a reversible occlusion of the carotid artery.

After 24 hours, the brain is removed and 1 mm thick sections are made throughout the brain using a matrix device for the brain (Natuatb Lrrata Shk). Each section is subsequently incubated in a 2% solution of triphenyltetrazolium chloride (w / v) in 154 mm No. 101 for 30 minutes at 37 ° C. The extent of damage can be determined using a computerized image analysis system (ISS, 1taddsd Vekeatsb, 8ί. SaFatshek, Οηΐηπο. Samba). In this analysis, an EPO compound of interest is considered to have neuroprotective properties if it contributes to a reduction in infarct volume due to MCA of focal rat ischemia to the same or greater extent than recombinant EPO.

E. Analysis of ίη νίνο based on a model of an epileptic seizure caused by the introduction of bicullin into the hippocampus.

In another aspect, the tissue protective property of EPO compounds is determined by experiments.

- 23 007812 ίη νίνο in a model of an epileptic seizure caused by the introduction of bicullin into the hippocampus. For example, male rats of the 8gadiye-Oayeteu line (250-280 g) are kept at a constant temperature (23 ° C) and relative humidity (60%), with free access to food and water, and fixed in a stationary state with a 12-hour light / darkness. Rats are surgically implanted with a cannula and electrodes under stereotactic control, as described in the publication Uehhapk A., e1 a1., Ί. No. ICGO5CE, 19, 5054-65 (1999). Briefly, rats are anesthetized with equitezin (1% phenobarbital / 4% chloral hydrate; 3 ml / kg ip). Two screw electrodes are placed bilaterally above the cerebral cortex in the crown of the head, along with a grounded wire located above the nasal cavity. Bipolar isolated electrodes with nichrome wire (60 μm) are then implanted bilaterally into the dentate gyrus of the dorsal hippocampus (septal pole) and the cannula (22 gauge) is placed on one side on the upper part of the dura mater for drug infusion into the hippocampus or the ventricle of the brain. The coordinates from Bregma for implantation of the electrodes should be as follows (mm): front-back - 3.5; lateral - 2.4 and 3; below the dura mater with a nasal plate - 2.5. Rah1pok, 6. & EDa1kop, S., THe Ea1 Vgat ίη 81geo1ah1s Soogbta1ek, Asabeshu Rgekk, Уονν Wogk (1986). The electrodes are connected to a multi-channel outlet (Magsy E1ec1goshs5, ΝΥ) and, together with an injection cannula, are attached to the skull with acrylic dental cement.

The experiments are preferably carried out from 3 to 7 days after surgery, when the animals are fully restored. Then, recombinant EPO or an EPO compound of interest (both in an amount of 5000 U / kg body weight) or vehicle is administered intraperitoneally, 24 hours, and again 30 minutes before the induction of seizures caused by bicucullin. The procedures for recording EEC and intracerebral injection of drugs were previously described in the publication Uehhapk A., e1 a1., 1 P11agshaso1. Exp Tieg., 239, 256-63 (1986). Briefly, animals are allowed to acclimatize in Plexiglass cages (25x25x60 cm) for a minimum of 10 minutes prior to the start of the EEC recording (4-channel EEC detector, model BP8, VyadNa Kapdosh, Vo1odpa, Ia1u). After approximately 15-30 minutes, EEC recordings are made continuously for 120 minutes after infusion with 0.8 nmol / 0.5 μl of bicucullinmethiodide. All injections are given to non-anesthetized rats with a needle (28 gauge) 3 mm below the cannula.

Seizures can be measured by an EEC analysis, with which, as shown earlier, a sensitive measurement of the anticonvulsant activity of drugs can be made. Uechhapk A., e1 a1., 1 Ryagtaso1. Exp Tieg., 239, 256-63 (1986). In this analysis, seizures consist of the simultaneous manifestation of at least two of the following changes in all four branches for registration: high frequency, and / or complexes with many peaks, and / or synchronized peak activity, or high voltage waves. Synchronous peak mode alternates with seizures. The parameters selected for quantifying seizures are preferably latent with respect to the first seizure (onset of seizure), total time spent in a state of epileptic activity (determined by adding together the duration of the ictal episodes; duration of the seizure), and peak activity during the EEC recording period (seizure intensity).

A model of an epileptic seizure caused by the introduction of bicullin into the hippocampus, using EEC activity as an indication of data, has been shown to be a sensitive and specific model predicting the ability of drugs intended for seizures. Uechhapk A., e1 a1., 1 Ryagtaso1. Exp Tieg., 239, 256-63 (1986). Thus, in order to consider the tested long-acting EPO as having a tissue-protective property, it must reduce the frequency and severity of seizures to the same degree or higher than the recombinant EPO.

E. Analysis of ίη νί \ Ό based on a model of acute reversible rat glaucoma.

In another analysis of ίη νί \ Ό according to the present invention, the rat acute reversible glaucoma model is used to determine the tissue protective properties of individual EPO compounds of interest. For example, since retinal cells are very sensitive to ischemia, many of these cells will die after 30 minutes of ischemic stress. Essentially, in order to test whether the peripherally administered test EPO compounds exhibit tissue protective activities sufficient to protect ischemic sensitive cells, the rat acute reversible glaucoma model is used for this purpose, as described in Cockpit E1 A1, Y15. Kek., 37: 3443-51, 1997. In particular, saline can be injected into the anterior chamber of the eye of adult male rats in order to increase systemic blood pressure and maintain for 60 minutes. Then the animals are injected with saline or 5000 U EPO / kg of body weight intraperitoneally 24 hours before the induction of ischemia and continue as a daily dose for 3 additional days.

Electroretinography is performed on dark-adapted rats 1 week after treatment to determine if the EPO compound of interest has tissue protective activity. If EPO has a tissue protective property, signs of good protective activity should appear on the electroretinogram, as opposed to animals treated only with physiological saline.

C. Analysis for myocardial infarction.

Tests based on myocardial infarction studies are also being considered for use on

- 24 007812 to the present invention, to establish whether the EPO compound exhibits tissue protective activity in general or, in particular, in the heart. For example, adult male rats are given EPO (5000 U / kg body weight) 24 hours before the rats are anesthetized and prepared for occlusion of the coronary artery. An additional dose of EPO is given at the beginning of the procedure, at which time the left main coronary artery is squeezed for 30 minutes and then released. The same dose of EPO is given daily for 1 week after treatment, at which time cardiac function is studied in animals. Animals that receive a fictitious injection (saline) will experience a significant increase in left end diastolic pressure, indicating an enlarged, hard heart, in addition to myocardial infarction, while animals receiving a long-acting EPO should not experience a decrease cardiac function compared with the control receiving a fictitious injection (significant difference at p <0.01), if long-acting EPO has tissue protective properties.

H. Studies of spinal cord injuries.

Spinal injury studies can also be used in the present invention to evaluate the tissue protective functions of the test single EPO compound. In particular, a rat spinal cord compression model is contemplated for use in the present invention. Preferably, rat No. 181ag (females) rats weighing about 180 to 300 g are used in this study. The animals are preferably kept on a starvation diet for 12 hours before surgery and are kept in a humane state and anesthetized by intraperitoneal injection of sodium thiopental (40 mg / kg body weight). After skin infiltration (bipivacaine 0.25%), a complete one-stage (T-3) laminectomy is performed by means of a 2 cm incision using a microscope used in such cases. Traumatic damage to the spinal cord is achieved by extradural application of a temporary clamp for aneurysm, exerting a pressure of 0.6 Newton (65 g) on the spinal cord for 1 min. After removing the clamp, the incision in the skin is closed, and the animals are left to fully recover from anesthesia, and they are returned to the cells. Rats are constantly checked by palpation of the bladder at least 2 times a day until spontaneous emptying of the bladder resumes.

Animals in the control group receive normal saline (via intravenous injection) immediately after the skin incision is closed. The remaining animals receive the test EPO compound in an amount of 16 μg / kg body weight (intravenously). Then assess the motor neurological function of rats using the scale of standards of motor ability. On this scale, animals are rated from 0 (no hind limb movements) to 21 (normal gait). Functional abnormalities in rats are examined at the times of 1 h, 12 h, 24 h, 48 h, 72 h and 1 week after injury by the same expert who has no idea about the treatment that the animal receives. If long-acting EPO exhibits tissue protective properties, rats that are given EPO should faster and better achieve full recovery from damage than rats that receive saline injection.

I. Research of rabbit spinal cord ischemia.

In another aspect, a study on a model of rabbit spinal cord ischemia reveals a tissue protective property. For example, New Zealand white rabbits (36, 8-12 months old, males) weighing 1.5 to 2.5 kg are used in this study. Animals are kept on a hungry diet for 12 hours and, without causing damage, are kept stationary. Anesthesia is carried out using 3% halotane in 100% oxygen and is supported by halotane from 0.5 to 1.5% in a mixture of 50% oxygen and 50% air. An intravenous catheter (22 gauge) is placed in the vein of the left ear. Ringer's lactate is administered by infusion at a rate of 4 ml / kg body weight per hour during surgery. Before surgery, kefazolin in an amount of 10 mg / kg of body weight is administered intravenously to prevent infection. The animals are placed lying on their right side, the skin treated with povidonyodin is impregnated with bipivacaine (0.25%) and the skin incision on the side is made parallel to the incision on the spine at the level of 12 ribs. After dissection of the skin and subcutaneous pectoral fascia, the long lumbar muscle and the ileo-rib muscle are removed. The abdominal aorta is exposed through access in the retroperitoneal region on the left side and mobilized just below the left renal artery. A piece of PE-60 tube is twisted in a loop around the aorta directly distally to the left renal artery and both ends are passed through a large rubber tube. Due to stretching on the PE tube, the aorta is clogged without tavmatic damage for 20 minutes.

Heparin (400 IU) is administered as an intravenous bolus to block the aorta. After a 20-minute blockage, the tube and catheter are removed, the incision is closed, and the animals are checked until complete recovery is achieved, after which the state of the neurological function is assessed in animals. The control group of animals receives normal saline intravenously immediately after the release of the aorta from clogging. Another group of animals receives 6.5 μg / kg body weight of the EPO compound of interest intravenously immediately after reperfusion (n = 6 for each group).

According to the Igittopb and Mooge criterion, the motor function is evaluated by a researcher who has no idea about the treatment performed, at times 1, 24 hours and 48 hours after reperfusion. A score of 0 to 4 is assigned to each animal as follows: 0 = paraplegia with implicit

- 25 007812 motor function of the lower extremities; 1 = low motor function of the lower extremities, only weak antigravity movement; 2 = moderate lower limb function with good anti-gravity stability but inability to pull the legs under the body; 3 = excellent motor function with the ability to pull the legs under the body and jump, but abnormally; 4 = normal motor function. The bladder is empty in paraplegic animals manually 2 times a day.

If the long-acting EPO of interest has a tissue-protective property, animals that are given EPO should faster and better achieve full recovery from the damage than animals receiving saline injections.

1. Other potential studies to test the tissue protective effects of EPO and its analogues.

As briefly described earlier, some types of tissues have EPO receptors and therefore may be sensitive to tissue protective effects due to the action of EPO. Thus, depending on the intended clinical use of the EPO compound of interest, a person skilled in the art will recognize that similar ίη νίίτο assays including these additional EPO sensitive cells can be performed or ίη νΐνο assays involving related organs can also be performed. For example, ίη νίΐΓο analyzes based on serum depletion can be performed using myocardial cells, retinal cells and Leydig cells, and a procedure similar to that described above for analysis can be performed on P19 cells.

Ίη νΐνο analyzes can also be directed to individual organs. For example, to assess the effects of EPO on retinal cells, one of ordinary skill in the art can perform the retinal ischemia study described above. In addition, in order to evaluate the effect of EPO on myocardial cells, a qualified experimenter in this field can easily modify the model of myocardial infarction described above. Those skilled in the art will be qualified to select the appropriate assay or model to assess whether a single EPO has tissue protective activity against cells, tissue, or organs that are sensitive to erythropoietin.

Examples

The following examples only illustrate preferred aspects of the present invention and are not intended to limit the invention, the scope of which is defined by the attached claims.

Example 1. Chemically modified EPO.

A. Oxidation of sugar chains.

Sugar units of EPO can be converted into acids according to the following procedure. EPO and an amount of sodium periodate sufficient to achieve the desired oxidation state (the higher the amount of sodium periodate, the higher the oxidation state) was placed in 100 mM sodium acetate buffer. This solution was then kept on ice for approximately 20 minutes and carefully dialyzed using distilled water. Then the product was removed from the dialysis tube and collected in a fresh tube (product I).

Benedict's reagent (18 g of copper sulfate, 100 g of sodium carbonate (anhydrous), 200 g of potassium citrate, 125 g of potassium thiocyanate, 25 g of potassium ferrocyanide) was dissolved in distilled water to a final volume of 1 liter. Then, a few drops of methylene blue were added to the Benedict reagent solution.

Then, product I was added to the Benedict reagent solution until the solution became clarified, which indicates that the solution was completely oxidized. Then, the solution was desalted and concentrated using ITAHGEN SEChpGuide1 and IHe. Then the sample (product II) was carefully dialyzed using distilled water.

B. Oxidation of the asial form of EPO by galactose oxidase.

The asialo-form of EPO in an amount of 50 to 500 μg, 10 μl of 1 U / μl of galactose oxidase and 100 μl of 10 mM sodium phosphate buffer was mixed in a 15 ml conical centrifuge tube (110 μl total volume). Then this mixture was incubated for 2 hours at 37 ° C, after which time the solution was carefully dialyzed using distilled water. The product was removed from the dialysis tube and collected in a fresh tube (product III).

Benedict's reagent (as described above) was dissolved in distilled water to a final volume of 1 liter. A few drops of methylene blue were then added to the solution.

Product III was added to the Benedict reagent solution until the solution was clarified, indicating that the solution was completely oxidized. Then, the solution was desalted and concentrated using ITAHFGHGHGHGHZDA! E111eg IP. Then the sample (product IV) was carefully dialyzed using distilled water.

C. Sulfation of EPO.

EPO was dissolved in Ν, Ν-dimethylformamide (ΌΜΡ-8Α) at 4 ° C. Then, Ν, Ν'-dicyclohexylcarbodiimide (ESS) dissolved in добав was added to the solution, and the solution was shaken for 4 h at 4 ° С. Crushed ice was added and the pH was adjusted to pH 7.5 with 10 n Ν αΟΗ. The volume of the solution was adjusted and the sample was centrifuged at 1000 xd for 15 min in a ΗΝ-82 type centrifuge (IAMOMES, No. 6 6 Ηΐ.., Makkasikeyek). Then the supernatant was carefully dialyzed. More detailed information

- 26 007812 regarding sulphation is described in publication 8. Ropdog c1 a1., Rrégayop o £ Sdy-Po1epsu, ηοη-αββΓΟβαΙίηβ 1p8i11P8 Iapd a Nous1 8i1 £ aiop Prgosebye, P1ae1e8, Uo1. 32, no. 12, Esset 1983, the full scope of which is incorporated herein by reference.

Ό. Connection of PEG chains to EPO.

The EPO molecule can be modified by attaching PEG chains to oxidized carbohydrates, such as those obtained in section A above (product Ι). The degree of modification can be controlled by changing the concentration of the periodate during oxidation.

PEG-EPO conjugates were first prepared by oxidation of EPO (2-4 mg / ml in 50 mM sodium acetate) using sodium meta-periodate (81 dt) in an amount of 1 to 100 mM for 30 minutes at room temperature. The phosphate buffer was then removed by buffer exchange in 100 mM sodium acetate, pH 5.4.

Methoxy-PEG-hydrazine of various molecular weights (No. Klag Tyegareiysk) was then added at 5100-fold molar excess (polymer: protein). The intermediate hydrazine bond was then restored by adding 15 mM sodium cyanoborohydride (81 dtA) and allowed to react overnight at 4 ° C. The resulting conjugates were then fractionated / purified by methods known in the art.

E. Attachment of PEG chains to the asialo form of EPO.

The asialo form of EPO was modified by attaching PEG chains to the newly created terminal galactose residues after oxidation with galactose oxidase, the preparation of the asialo form oxidation product (product ΙΙΙ) is described in section B. above.

Recombinant human EPO (gyyEPO) was treated with sialidase A (Rgohute, 1 ps.) To remove sialic acid in accordance with the technical protocol. To confirm the implementation of chemical modification, the reaction product was subjected to EF in a polyacrylamide gel with SDS. Staining of the bands on the electrophoregram showed that the modified EPO has a molecular weight of approximately 31 kDa, while the unmodified EPO has a molecular weight of approximately 34 kDa. The sialic acid residues remaining in the EPO molecule are approximately less than 0.1 mol / mol EPO.

After obtaining the asialo form of EPO, the newly discovered access galactose residues on the EPO molecule (2-4 mg / ml in 10 mM sodium phosphate buffer) were oxidized by 100 units of galactose oxidase in PB8 (81 dtA) in ml of EPO solution. Then the reaction mixture was incubated at 37 ° C for 2 hours

Then the phosphate buffer was removed by buffer exchange in 100 mm sodium acetate, pH 5.4. Then, methoxy-PEG hydrazide of various molecular weights (No. Klag Tyegareiysk) was added with a molar excess of 5 to 100 times (polymer: protein). Then the intermediate hydrazine bond is preferably reduced by adding 15 mM sodium cyanoborohydride (81 dt) and allowed to react overnight at 4 ° C. The resulting conjugates were then fractionated / purified by methods known in the art.

P. Attachment of PEG chains to the asialo-form of EPO.

The asialo form of EPO was modified by attaching PEG chains to the newly created terminal galactose residues after oxidation with galactose oxidase, the preparation of the asialo form oxidation product (product ΙΙΙ) is described in section B. above.

Ya111.1 EPO (1 mg) was treated to remove sialic acid with a neuraminidase (8e1kadaki Sogrogaiop o £ 1arap, 1 unit of lyophilized powder was dissolved in 100 μl of 75 mM NaPO ₄ (pH 6.5)) at a ratio of 1 mg EPO at 0.05 units of neuraminidase (5 μl). 5 units (5 μl) of galactose oxidase (450 μl was dissolved in 75 mM NaPO ₄ (pH 6.5) (81 dtA)) then added to the mixture.

Then the phosphate buffer was removed by buffer exchange in 100 mm sodium acetate, pH 5.4. Then, PEG-ΝΗ (750 molecular weight, No. 1 <Tyagareibsk) and 15 mM sodium cyanoborohydride (81 dt) were added to the mixture and allowed to react overnight at 4 ° C. PEG-ΝΗ _{2 was} added preferably at a 250-fold molar excess (polymer: protein) (80 mg PEG-ΝΗ :). The resulting conjugates were then fractionated / purified by methods known in the art.

Example 2. Functional research.

A. Analysis of erythropoietic activity.

Properties of erythropoietin, i.e. the ability of a single EPO compound to regulate hematocrit levels was determined using the following assay.

TP1 is an erythroleukemia cell line that is completely dependent on growth factors, including EPO. Kyatiga, e1 a1., B1oob 73, 375-80. TP1 cells were obtained from ATCC and maintained in KPM1 1640 medium with the following components: 2 mM L-glutamine, 10 mM Nerek, 1 mM sodium pyruvate, 4.5 g / L glucose, 1.5 g / L sodium bicarbonate, 5 ng / ml CM-C8P and 10% fetal bovine serum before the experiment. TP1 cells obtained in the active growth phase were besieged, washed 3 times only with medium and resuspended to a concentration of 10 ⁵ cells in 1 ml of medium, in the presence or absence of CM-C8P, in the presence of EPO or an EPO analogue with at least one additional A Ν-linked carbohydrate chain and / or at least one additional O-linked carbohydrate chain added at certain concentrations. Individual cultures were maintained for 24 hours and the number

- 27 007812 cells were determined using the reaction product formazan (Ce11TIeg; Roscheda, Mashzop, XVI) in accordance with the manufacturer's protocol.

In addition, the ability of a chemically modified EPO compound was initially assessed ίη νίνο by determining its effect on hemoglobin concentration using female mouse line VLV / s. The animals were injected with 500 U / kg of EPO body weight of interest of the EPO compound or an equal volume of the carrier subcutaneously 3 times a week for 3 weeks (this interval is sufficient to observe an erythropoietic response). Such compounds or an EPO compound are considered as an erythropoietin compound if they increase the concentration of hemoglobin in the serum of mice. Further assessment of the ability of the EPO compound was performed by ίη νίίτο using TE1 erythroleukemia cells. Studies have confirmed that EPO is an erythropoietin product if the relative number of TE1 cells is increased compared to the control.

Those of skill in the art will recognize that these and other assays, such as those performed on a mouse model after hypoxia, and reticulocyte analysis (Eigoreap Ratschasore1a), are also suitable for use in the present invention to determine the erythropoietic activity of long-acting tissue protective cytokines.

B. Study of tissue protective function.

The tissue protective properties of an EPO analog having at least one additional O-linked carbohydrate chain and / or at least one additional O-linked carbohydrate chain were determined using the following analysis.

Used cultures of neurons derived from the hippocampus of 18-day-old rat fetuses. The brain was removed and freed from the meningeal membranes of the brain and the hippocampus was isolated. Then the cells were dispersed by incubation for 5 min at 37 ° C in a 2.5% trypsin solution, followed by titration. The cell suspension was diluted in a basic serum-free medium of IgObase1 containing 1% B-27 of an additional component (C1bco, Eoskusche, MO, I8A), and placed on polyornithine-coated coverslips at a density of 80,000 cells on a coverslip. Then the cells were pretreated with EPO overnight and then kept in the presence or absence of 1) EPO, 2) an EPO analog having at least one additional Ν-linked carbohydrate chain and / or at least one additional 0-linked carbohydrate chain, or 3) asialo-forms of EPO, in 5 μM TMT for 24 hours. Cultures were used between 10 and 14 days ίη νίΙΐΌ.

Cell viability was determined by analysis using 3- (4,5-dimethylthiazol-2-yl) 2,5-diphenyltetrazolium bromide (MTT). Oyem / YuR E., Amb Bamd, K. 1986. Kar1b Co1o1pe1ps Azzau Gog Ce11 Sgo \\ 111 amb 8ιΐΓνίνη1. MobShsaBosh 1о 1Ье ίеί ^ аζο1 ^ иш бе ргосебиге §ίνίη§ ипротеб геЫШУ. ί. Ittiηο1. Meshobz 89: 271-277. Briefly, the MTT tetrazolium salt was dissolved in serum-free medium to a final concentration of 0.75 mg / ml and added to the cells at the end of the treatment for 3 hours at 37 ° C. Then the medium was removed and formazan was extracted with a mixture of 1 n HCl: isopropanol (1:24). Absorption at 560 nm was measured using a microplate reader.

As shown in FIG. 1 and 2, an EPO analog having at least one additional Ν-linked carbohydrate chain and / or at least one additional O-linked carbohydrate chain did not exhibit tissue protective function.

The claimed invention described herein should not be limited to certain aspects, since these aspects are considered as an illustration of some embodiments of the invention. Any equivalent aspects are intended to be included within the scope of this invention. In fact, various changes to the invention, introduced in addition to those that have been demonstrated and described in the text, will be apparent to those skilled in the art from the above description. Such modifications should also be included in the scope of the attached claims. All references cited in the text in their entirety are incorporated into this description by reference.

Claims (27)

CLAIM 1. The method of regulating the hematocrit number in humans, which includes the stage of obtaining an erythropoietin product, which has a longer half-life in serum than fatty, and exhibits tissue protective function; and administering a therapeutically effective amount of said erythropoietin product. 2. The method of claim 1, wherein the step of producing an erythropoietin product further comprises the step of modifying gbIERO by means of at least one chemical modification of at least one of the Ν-linked oligosaccharide chains or the O-linked oligosaccharide chain, where the chemical modification includes oxidation, sulfation, phosphorylation, pegylation, or combinations thereof. 3. The method according to claim 1, wherein the step of administering a therapeutically effective amount of an erythropoietin product comprises administering an erythropoietin product in a lower molar amount than ishro to achieve a comparable specified hematocrit number. 4. The method according to claim 1, where the half-life of erythropoietin in serum is at least about - 28 007812 is 20% longer than the half-life of serum HYiERO. 5. The method according to claim 4, where the half-life of erythropoietin in serum is at least about 40% longer than the half-life of hyERO in serum. 6. Produced erythropoietin product comprising at least one erythropoietin derivative, where at least one Ν-linked oligosaccharide chain or at least one O-linked oligosaccharide chain has at least one chemical modification as a result of oxidation, sulfation, phosphorylation , PEGylation or combinations thereof, and where the erythropoietin product has a longer half-life than serum gyyOrO. 7. Erythropoietin product according to claim 6, where the erythropoietin product exhibits tissue protective function. 8. The erythropoietin product of claim 6, wherein at least one chemical modification involves oxidizing at least one Ν-linked oligosaccharide chain or at least one O-linked oligosaccharide chain to form at least one additional acid residue. 9. The erythropoietin product of claim 6, wherein at least one chemical modification includes sulphating at least one Ν-linked oligosaccharide chain or at least one O-linked oligosaccharide chain to produce an increased negative charge on the EPO product. 10. The erythropoietin product of claim 6, wherein at least one chemical modification includes phosphorylating at least one Ν-linked oligosaccharide chain or at least one O-linked oligosaccharide chain to produce an increased negative charge on the EPO product. 11. The erythropoietin product of claim 6, wherein the at least one chemical modification comprises adding at least one polyethylene glycol chain to at least one linked oligosaccharide chain or at least one O-linked oligosaccharide chain. 12. A method of producing an erythropoietin product having an extended half-life in serum and tissue protective activity, comprising the steps of producing at least one erythropoietin or an erythropoietin derivative and modifying at least one связанной-linked oligosaccharide chain or at least one O-linked oligosaccharide chains in a molecule of at least one endogenous or recombinant erythropoietin by oxidation, sulfation, phosphorylation, PEGylation, or combinations thereof. 13. The method according to claim 12, wherein the modification step further comprises the step of replacing at least one adjacent hydroxyl on at least one Ν-linked oligosaccharide chain or on at least one O-linked oligosaccharide chain with at least one acid residue . 14. The method according to claim 13, wherein the step of replacing at least one contiguous hydroxyl on at least one-linked oligosaccharide chain or on at least one O-linked oligosaccharide chain by at least one acid residue further includes replacing a plurality of adjacent hydroxyls on at least one Ν-linked oligosaccharide chain or on at least one O-linked oligosaccharide chain with many acid residues. 15. The method of claim 12, wherein the modification step further comprises the steps of preparing an organic solvent; dissolving erythropoietin or erythropoietin derivative in an organic solvent to form a solution; preparing at least one condensing agent; preparing at least one sulfate donor; and mixing at least one condensing agent and at least one sulfate donor in solution. 16. The method of claim 12, wherein the modification step further comprises the steps of preparing an organic solvent; dissolving erythropoietin or erythropoietin derivative in an organic solvent to form a solution; preparing at least one condensing agent; preparing phosphoric acid and mixing at least one condensing agent and at least one phosphoric acid in solution. 17. The method of claim 12, wherein the modification step further comprises the steps of preparing an organic solvent; dissolving erythropoietin or an erythropoietin derivative in an organic solvent to form a first solution; preparing at least one oxidizing agent; - 29 007812 add at least one oxidizing agent to the first solution with the formation of the second solution; preparing at least one polyethylene glycol chain; and mixing at least one polyethylene glycol chain with a second solution. 18. The method according to claim 17, wherein the step of preparing at least one polyethylene glycol chain comprises preparing at least one polyethylene glycol chain with at least one primary amine fragment at the end of the chain. 19. A method of treating anemia in patients at risk of tissue damage, comprising the steps of preparing an erythropoietin product with at least one chemical modification on at least one of the Ν-linked oligosaccharide chains or at least an O-linked oligosaccharide chain, where the chemical modification includes oxidation, sulfation, phosphorylation, pegylation, or combinations thereof; administering a therapeutically effective amount of an erythropoietin product, wherein the erythropoietin product is administered at a lower molar amount than gayerO, to achieve a comparable specified hematocrit number; where the erythropoietin product exhibits tissue protective function. 20. The method according to claim 19, where the erythropoietin product has a longer half-life in serum than gyiERO. 21. The method according to claim 20, where the half-life of erythropoietin in serum is at least about 20% longer than the half-life of hyyero in serum. 22. The method according to p. 21, where the half-life is at least about 40% longer than the half-life of hyperOI in the serum. 23. Pharmaceutical composition comprising a therapeutically effective amount of at least one erythropoietin derivative, where at least one-linked oligosaccharide chain or at least one O-linked oligosaccharide chain has at least one chemical modification as a result of oxidation, sulfation, phosphorylation , PEGylation or combinations thereof, where at least one erythropoietin derivative has a longer serum half-life than recombinant erythropoietin, and It is a fabric protective function. 24. The pharmaceutical composition according to item 23, further comprising at least one pharmaceutically acceptable carrier. 25. The pharmaceutical composition according to claim 24, wherein the at least one pharmaceutically acceptable carrier comprises at least one diluent, adjuvant, excipient, binder, or mixtures thereof. 26. The pharmaceutical composition of claim 23, further comprising at least one wetting agent, an emulsifying agent, a pH buffering agent, or combinations thereof. 27. The pharmaceutical composition according to item 23, further comprising at least one tissue protective cytokine.