CN1882355A - 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 erythropoietin 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

A long-acting erythropoietin that retains the tissue protective activity of endogenous erythropoietin

References to related applications

This application claims priority to US Provisional Patent Application No. 60/409,020, filed September 9, 2002, the contents of which are incorporated herein by reference.

field of invention

The present invention relates to a modified long-acting erythropoietin that better maintains tissue protection. In particular, the present invention relates to chemically modified long-acting erythropoietin, which not only prolongs its half-life in serum, but also maintains the tissue protective function of the natural protein in vivo. The present invention also relates to using the long-acting erythropoietin of the present invention to treat anemia and related diseases. Finally, the present invention relates to a detection method for analyzing whether erythropoietin has tissue protection ability.

Background of the invention

Naturally occurring or endogenous erythropoietin (EPO) is a glycoprotein hormone produced primarily by the kidney. Endogenous EPO contains 165 amino acids and has a molecular weight (in humans) of about 30,000 to about 34,000 Daltons. The sugar group in EPO, which consists of 3 N-linked and 1 O-linked oligosaccharide chains, accounts for approximately 40% of the molecular weight of the entire protein. N-linked oligosaccharide chains bind to the amino nitrogens of asparagine at positions 24, 38 and 83, while O-linked oligosaccharide chains bind to the oxygen atom of a serine residue at position 126. EPO may exist in 3 forms: alpha, beta and asialo form (Asialo). The α and β forms have the same potency, biological activity, and molecular weight, but differ slightly in the composition of carbohydrates, while the asialo form is the α or β form in which the terminal sialic acid in the sugar chain has been removed, the terminal Sialic acid shields sugar chains from being recognized by the liver.

Until recently, the primary function of endogenous EPO was to stimulate the proliferation and maturation of responsive bone marrow erythroid precursors, along with other growth factors, and to maintain an individual's hematocrit (percentage of whole blood containing red blood cells) . The process of red blood cell production, called erythropoiesis, is a precisely regulated physiological mechanism that optimizes the number of red blood cells to meet tissue oxygenation needs without impeding circulation. For example, EPO increases red blood cell production by stimulating the endocytosis of precursor cells into mature red blood cells in the bone marrow when oxygen delivery by red blood cells decreases, and the resulting red blood cells then enter the circulation. When the number of red blood cells in the circulatory system exceeds that required for normal tissue circulation, the EPO in the circulatory system decreases. Therefore, when the body is in a healthy state, the concentration of EPO in the blood plasma is very low, just enough to stimulate the replacement of red blood cells that are normally lost due to aging. The normal range for plasma EPO is from 0.01 to 0.03 units/ml.

Given that the majority of EPO in an individual is produced by the kidneys, loss of renal function, such as chronic renal failure (CRE), results in reduced amounts of EPO and often anemia. Also, anemia may be caused by other chronic diseases, such as cancer, or treatments associated with these diseases, such as chemotherapy. Thus, it has been demonstrated that administration of recombinant EPO (described in detail below), which has substantially the same biological activity as endogenous EPO, can effectively restore hematocrit levels to individuals with reduced red blood cells.

In addition to its role in maintaining hematocrit levels in chronic conditions, recombinant EPO has been used to increase red blood cell levels prior to elective or scheduled surgery, thereby reducing or eliminating the need for blood transfusions. For example, the administration of recombinant EPO can relieve patients' concerns about contracting viruses or pathogens through blood transfusion, or solve the problem of religious restrictions on blood transfusion.

Further, some recent evidence suggests that EPO, as a member of the cytokine superfamily, has other important therapeutic properties mediated through its interaction with the EPO receptor (EPO-R). For example, EPO and its receptors play an important role in attenuating tissue damage because the interaction of EPO and its receptors provides a compensatory response that alters the cellular microenvironment of hypoxia, as well as regulates programmed cellular processes resulting from metabolic stress. die. In fact, patients with chronic renal failure and/or cancer treated with EPO generally experience improved sensory recovery or intellectual acuity, an effect that has previously been attributed to increases in patients' hematocrit. More recently, however, these improvements have been attributed to the tissue protective and enhanced activity of EPO, as described in International Application No. WO/02053580 and US Patent Publication Nos. 2002/0086818 and 2003/0072737.

Recent studies have also shown that systemically administered EPO can cross the blood-brain barrier because the capillaries that form the blood-brain barrier also express EPO receptors. This therefore provides an anatomical basis for receptor-mediated endocytosis from the peripheral circulation to the brain.

Recombinant human EPO (epoetin α), which can be obtained commercially, such as PROCRIT(R) (from Ortho Biotech Inc., Raritan, NJ), and EPOGEN(R) (from Amgen, Inc., Thousand Oaks, CA), has been used in It is used to treat anemia caused by end-stage renal disease, to treat HIV-infected patients with AZT (azidovidine) therapy, and to counteract the effects of chemotherapy. Although there are many therapeutic effects of recombinant human EPO, the most important use of recombinant human EPO so far is to alleviate chronic anemia. In this regard, recombinant human EPO is typically administered at an initial dose of 50-150 units/kg three times per week intravenously or subcutaneously over approximately 6 to 8 weeks to restore the patient's guideline hematocrit range. After the patient achieves the desired hematocrit level, for example in an amount between about 30% and about 36%, said hematocrit level can be maintained by maintaining the dose of EPO without iron deficiency or complications, The dosage is a sufficient amount and administered at a frequency appropriate to maintain the normal hematocrit level achieved with the initial dose of EPO. Although dosage requirements may vary according to the needs of the individual patient, generally the maintenance dosage may be administered about three times per week (or less frequently if the dosage is larger).

The dose and frequency of administration of recombinant EPO will depend in part on the half-life of the molecule, which may be limited while the molecule is in the body. For example, intravenously administered EPOGEN ^(R) has been reported to be cleared at a rate consistent with first order kinetics with a circulating half-life in the range of about 4-13 hours in adult and pediatric patients with CRF. Therefore, the dosage and efficiency of administration are adjusted in order to achieve a therapeutic effect taking into account the relatively short half-life of recombinant EPO.

In addition, because recombinant EPO is injected intravenously or subcutaneously, it often requires a nurse or physician to administer recombinant EPO to the patient. This brings more inconvenience to the patient, which is another reason why the extended half-life of the molecule should be considered. Therefore, research to increase the half-life of recombinant EPO has received attention in the past decade, based on the premise that an extended half-life can reduce the dose required while still providing the same or improved therapeutic effect.

In fact, recent experiments on human EPO have shown that there is a direct relationship between the sugar content of sialic acid in EPO, its circulating half-life, and in vivo activity. As described in PCT Publication No. WO95/05465, sialic acid residues are added to the ends of sugar chains and prevent the recognition of galactose by the liver. Typically, each N-linked oligosaccharide chain contains up to 4 sialic acids and each O-linked oligosaccharide chain contains up to 2 sialic acids. Thus, an unmodified EPO polypeptide may contain a total of up to 14 sialic acids.

Over time, these sialic acid residues may break off from the protein, exposing galactose residues that can be recognized by the liver. Once the liver recognizes the galactose residue, the protein is filtered from the blood. Therefore, it is believed that gradually increasing the sialic acid contained in each EPO molecule can better avoid the exposure of galactose residues and correspondingly increase its biological activity (by testing the erythropoiesis-stimulating Ability to increase hematocrit in normal mice). Since unmodified EPO only contains 14 sialic acid sites, such a structure limits the extension of the half-life of EPO. This led to the hypothesis that engineered EPO analogs containing additional oligosaccharide chains might have enhanced biological activity. Additional oligosaccharide chains containing termini can be modified with sialic acid residues by providing additional sites for glycosylation. See PCT Publication Nos. WO91/05867, WO94/09257 and WO01/81405.

For example, a modified EPO analog may contain at least one additional N-linked sugar chain and/or at least one additional O-linked sugar chain. In particular WO 01/81405 discloses the addition of N-linked sugar chains at amino acids at positions 30, 51, 57, 69, 88, 89, 136 and/or 138 of the molecule. The modified EPO molecule can contain 1-4 additional glycosylation sites at any position, so that the EPO molecule can add 2-16 sialic acid residues. Additionally, attempts to extend the half-life of EPO have involved attaching polyethylene glycols (PEGs) to the amino acid backbone of the EPO protein. See, PCT Publications WO0032772, WO0102017, WO03029291; US Publication Nos. US2003077753, US20030120045, US200301666566; and US Patent Nos. 6,583,272 and 6,340,272. Fusion constructs linking other proteins to EPO have also been used to extend the half-life of EPO. See, US Publication nos. 20040009902, 20030124115, 20030113871, and US Patent No. 6,242,570.

Although these efforts to increase the serum half-life of EPO have proven successful and proved useful in maintaining hematocrit levels, no attention has been paid to the possible effects of these additional modifications on other functions of EPO.

Therefore, there is a need to provide modified EPO with extended serum half-life (long-acting) and maintaining the function of endogenous EPO. In particular, there is a need in the art for a long-acting EPO compound, which has erythrocyte-stimulating and tissue-protecting functions, and a pharmaceutical composition for treating anemia and/or related diseases. Additionally, there is a need for assays for determining whether a particular EPO is antagonistic to the tissue protective capacity of endogenous EPO.

Summary of the invention

The present invention relates to a method of modulating human hematocrit levels comprising the steps of providing a tissue protective erythropoietin product with a longer serum half-life than recombinant human erythropoietin (rhuEPO) and administering a therapeutic an effective amount of this erythropoietin product (hereinafter referred to as long-acting EPO). In one technical solution, the step of providing long-acting EPO further comprises the step of modifying recombinant EPO on at least one N-linked oligosaccharide chain or O-linked oligosaccharide chain using at least one chemical modification, the chemical modification described herein Modifications include oxidation, sulfation, phosphorylation, PEGylation, or combinations thereof.

Alternatively, the step of administering a therapeutically effective amount of long-acting EPO may comprise administering a molar amount of long-acting EPO lower than that of rhuEPO to achieve a comparable hematocrit.

In one embodiment, the serum half-life of the long-acting EPO is at least 20% longer than the serum half-life of rhuEPO. In another technical solution, the serum half-life of the long-acting EPO is at least 40% longer than that of rhuEPO.

The invention also relates to an artificial erythropoietin product (long-acting EPO) comprising at least one erythropoietin derivative in which at least one N-linked oligosaccharide chain or at least one O-linked oligosaccharide chain has At least one chemical modification The chemical modification is derived from oxidation, sulfuration, phosphorylation, PEGylation or a combination thereof, and the long-acting EPO described herein has a longer serum half-life than rhuEPO. The long-acting EPO preferably has a tissue protective function.

In one embodiment, the at least one chemical modification comprises oxidation of at least one N-linked oligosaccharide chain or at least one O-linked oligosaccharide chain to provide at least one additional acidic residue. For example, at least one chemical modification can include sulfation at at least one N-linked oligosaccharide chain or at least one O-linked oligosaccharide chain to provide long-acting EPO with increased negative charge. In another embodiment, the at least one chemical modification comprises phosphorylation of at least one N-linked oligosaccharide chain or at least one O-linked oligosaccharide chain to provide long-acting EPO with increased negative charge. on the other hand. The at least one chemical modification comprises the addition of polyethylene glycol chains to at least one N-linked oligosaccharide chain or at least one O-linked oligosaccharide chain.

The present invention also relates to a method for preparing long-acting EPO with prolonged serum half-life and tissue protection function, comprising the steps of: providing at least one erythropoietin or a derivative of erythropoietin; Endogenous or recombinant erythropoietin or erythropoietin derivatives undergo oxidation, sulfation, phosphorylation, PEGylation, or combinations thereof.

The step of modifying may also include substituting at least one vicinal hydroxyl group on at least one N-linked oligosaccharide chain or at least one O-linked oligosaccharide chain with at least one acid residue. In one embodiment, the step of substituting at least one vicinal hydroxyl group on at least one N-linked oligosaccharide chain or at least one O-linked oligosaccharide chain with at least one acid residue may further comprise replacing at least one acid residue with at least one The acid residues are substituted for at least a plurality of ortho hydroxyl groups on at least one N-linked oligosaccharide chain or at least one O-linked oligosaccharide chain.

In another technical solution, the step of modifying further includes the following steps: providing an organic solvent; dissolving erythropoietin or a derivative of erythropoietin in the solvent to form a solution; providing at least one condensing agent; providing at least one sulfate donor; and at least one condensing agent and at least one sulfate donor mixed into the above solution. In another technical scheme, the step of modifying further includes the steps of: providing an organic solvent; dissolving the erythropoietin or erythropoietin derivatives into a solution formed in all organic solvents; providing at least a condensing agent; providing phosphoric acid; and mixing at least one condensing agent and at least one phosphoric acid into said solution.

In another technical scheme, the step of modifying further includes the steps of: providing an organic solvent; dissolving erythropoietin or a derivative of erythropoietin in the organic solvent to form a first solution; providing at least one oxidizing agent; adding at least one oxidizing agent to the first solution to form a second solution; providing at least one polyethylene glycol chain; mixing the at least one polyethylene glycol chain into the second solution. The step of providing at least one polyethylene glycol chain may comprise providing at least one polyethylene glycol chain comprising at least one primary amino group at the end of the chain.

The present invention also relates to a method of treating anemia in a patient at risk of tissue damage comprising the step of providing at least one N-linked oligosaccharide chain or at least one O-linked oligosaccharide chain with at least one A chemically modified long-acting EPO, wherein the chemical modification includes oxidation, sulfation, phosphorylation, PEGylation, or a combination thereof; administering a therapeutically effective amount of long-acting EPO, the administration molar amount of the long-acting EPO described here Lower than rhuEPO, but comparable to target hematocrit, the long-acting EPO described here has tissue protective properties.

In this aspect of the invention, the long-acting EPO preferably has a longer serum half-life than rhuEPO. In one embodiment, its serum half-life is at least 20% greater than the serum half-life of rhuEPO. On the other hand, its serum half-life is at least 40% longer than that of rhuEPO.

The present invention also relates to pharmaceutical compositions comprising at least one therapeutically effective amount of long-acting EPO having at least one chemical modification on at least one N-linked oligosaccharide chain or at least one O-linked oligosaccharide chain , the chemical modification is derived from oxidation, sulfation, phosphorylation, PEGylation, or a combination thereof, at least one long-acting EPO described herein has a longer serum half-life than recombinant erythropoietin and has tissue protection function. In one embodiment, the pharmaceutical composition further comprises at least one pharmaceutically acceptable carrier. The at least one pharmaceutically acceptable carrier can comprise at least one diluent, adjuvant, excipient, carrier, or a combination thereof.

In another embodiment, the pharmaceutical composition further comprises at least one wetting agent, emulsifying agent, pH buffering agent, or a combination thereof. In another embodiment, the pharmaceutical composition further comprises at least one tissue protective factor.

Description of drawings

Further features and advantages of the present invention will become clearer after detailed description in conjunction with the following drawings, and the relevant drawings are described as follows:

Figure 1 shows data demonstrating that hyperglycosylated EPO analogs are able to cross the blood-brain barrier, as evidenced by cerebrospinal fluid sampling; and

Figure 2 is a comparison of the protective effectiveness of different forms of EPO against cell death induced by exposure to trimethyltin.

Detailed description of the invention

The present invention relates to the application of long-acting EPO, which is an EPO molecule with an extended serum half-life (long-acting), which is derived from chemical modification of the sugar chains connected to the amino acid backbone of EPO, thereby enabling Maintain the function of endogenous EPO. As described in the Background Art, efforts to extend the half-life of EPO have generally focused on adding substances to the amino acid backbone of EPO—addition of sugar chains, PEGs, proteins, etc. However, it is believed that these added substances affect the function of the EPO analog such that, for example, this function is compromised for a longer half-life. Although known EPO analogs with longer half-lives than recombinant EPO have erythropoietic activity, these analogs do not retain the other recently discovered therapeutic functions of EPO, eg, tissue protective activity.

For example, a 17 amino acid fragment corresponding to positions 30-47 of EPO, also known as the O'Brien peptide, has been shown to have tissue protective activity in vitro, but no erythropoietic activity in vitro. Campana, W.M., Misasi, R. & O'Brien, J.S., Int. J. Mol. Med., 1, 235-41 (1998). Thus, it is believed that modified EPO analogs having added substances within the O'Brien peptide sequence do not possess tissue protective activity in other in vitro assays. In addition, since the three-dimensional orientation of EPO plays an important role in its function, addition of substances to the molecule may affect the overall function of the resulting EPO analog.

Therefore, the present invention relates to a long-acting EPO having at least one of erythropoietic activity, tissue protective activity, transcytosis ability or a combination thereof. Preferably, the long-acting EPO of the present invention has at least one of tissue protection activity, endocytosis and translocation and erythropoietic activity.

In a specific aspect, the long-acting EPO of the invention has a serum half-life that is at least about 20% longer than recombinant EPO. In another embodiment, the long-acting EPO of the present invention has a serum half-life that is at least about 30% longer than recombinant EPO. In yet another embodiment, the long-acting EPO described herein has a serum half-life that is at least about 40% longer than recombinant EPO.

Briefly, long-acting EPOs according to the present invention include EPO analogs with sugar chains altered by at least one modification compared to natural (endogenous) EPO, preferably natural human EPO. In one embodiment, the long-acting EPOs of the present invention undergo various modifications to sugar chains.

In one embodiment, the long-acting EPO molecules of the present invention are prepared by oxidizing the vicinyl hydroxyl groups of natural EPO sugar chains to acidic residues. In another embodiment, the sialic acid residues on the EPO molecule are replaced with more stable acidic residues. In yet another embodiment, sulfuration and/or phosphorylation of the sugar chains of EPO results in the long-acting EPO of the present invention. In yet another embodiment, polyethylene glycol can be added to the sugar chain of EPO to obtain the long-acting EPO of the present invention. Any combination of the aforementioned modifications is also included in the present invention. Also, as mentioned above, the present invention also encompasses compositions, including pharmaceutical compositions, comprising one or more of the aforementioned long-acting EPO molecules.

The long-acting EPO molecules described in the present invention are expected to be used in pharmaceutical compositions, which can treat anemia and related diseases, especially diseases caused by including but not limited to: acute renal failure, sepsis, HIV, chemotherapy, etc. resulting complications.

The present invention also relates to a method for treating anemia and related diseases, as well as a kit for the treatment process. The term "treatment" as used herein refers to drug treatment and prophylactic or preventive measures, the purpose of which is to prevent or slow down (reduce) the targeted pathological condition or disorder. Those in need of treatment include those already with the disorder as well as those susceptible to the disorder, or those in which the disorder is to be prevented. The present invention includes that the long-acting EPO can be used for chronic administration, acute treatment, and/or intermittent administration. For the purposes herein, "chronic administration" refers to administration in a continuous mode relative to the acute mode of administration, in order to facilitate long-term maintenance of the initial therapeutic effect (activity), and "intermittent administration" is not continuous without interruption carried out, but cyclically.

The long-acting EPO of the present invention, and its use, can be applied to any mammal. As used herein, the term "mammal" refers to any animal classified within the class Mammalia, including humans, domestic and captive animals, and zoological, sporting, or pet animals such as dogs, cats, cows, horses, sheep, Pigs, goats, rabbits, etc. A preferred mammal is a human. Administration of the long-acting EPO of the present invention includes, but is not limited to, oral, intravenous, intranasal, local, intracavity, inhalation or parenteral administration, the latter including intravenous, arterial, subcutaneous, intramuscular, intraperitoneal , submucosa, intradermal, and combinations thereof.

The present invention also relates to the use of the long-acting EPO according to the present invention as a carrier for other molecules into the region of the body having EPO receptors. For example, because some molecules have low blood-brain barrier penetration ability, linking these molecules to the long-acting EPOs molecules of the present invention provides a safe and effective transport system for these molecules into the brain. And, as will be discussed in detail later, because other regions of the body express EPO receptors, such as the retina, heart, and lungs, the long-acting EPO of the present invention may be an ideal transport system for molecules with low penetration in these regions. Role.

Further, the present invention relates to assay methods for determining whether a specific EPO maintains the function of endogenous EPO. For example, assays described herein can determine whether modified EPO has tissue protective activity, ie, is an agonist of endogenous EPO. Here, the term "agonist" is used in its broadest sense to include those molecules that mimic the biological activity of native EPO. In like manner, the term "antagonist" includes in its broadest sense those molecules which partially or fully block, inhibit, or neutralize the activity of native EPO. In one embodiment, the presence of a particular EPO is detected in an in vitro assay, such as a P19 cell and/or mouse motor neuron assay. In another embodiment, the assays of the present invention include evaluating the activity of a particular EPO in vivo using various assays such as focal ischemia in rats, retinal ischemia in rats, spinal injury, and bicuculline seizure model.

Function

As described in the background section, various attempts to increase the half-life of EPO have been successful, as this resulted in EPO analogs with longer half-lives and retained erythropoietic activity. Also, as described, EPO analogs with longer half-lives mainly include additions to the amino acid sequence of EPO. The addition includes sugar chains, PEGs and protein sequences. However, it is foreseeable that these additions may affect other therapeutic activities of EPO, such as tissue protection function and molecular endocytosis and translocation. Without being bound by any particular theory, based on the importance of the O'Brien peptide sequence to the tissue protective function, the addition of sugar chains to the O'Brien peptide sequence may affect its function. In addition, added sugar chains, whether within or outside the O'Brien peptide sequence, are believed to affect the three-dimensional orientation of the molecule. For example, in the three-dimensional configuration of the molecule, an added sugar chain may block a region of the molecule that is essential for its function. Further, it is believed that added substances may also affect the function of glycoproteins. While those skilled in the art may be familiar with some methods of adding or attaching additional carbohydrates, polyethylene glycols, proteins, etc. to the amino acid backbone of EPO, added carbohydrates will represent other additions to the amino acid backbone of EPO.

organizational protection

In order to evaluate the possibility that increased sugar chains affect the function of glycoproteins, the present inventors studied various forms of EPO with 5 N-linked sugar chains (compared to 3-N-linked sugar chains of recombinant EPO) analog. Specifically, the present inventors used an EPO analog with an additional glycosylation site at amino acid position 32, which resulted in approximately 3-fold increased half-life compared to recombinant EPO (epoetin α).

Although this EPO analog was present in the cerebrospinal fluid after systemic injection (Figure 1), it was surprisingly found to have no tissue protective activity in subsequent in vitro P19 assays (Figure 2). This lack of tissue protective activity was unexpected. In addition, loss of tissue protective activity may cause complications in the treatment of anemic patients if these patients have other conditions requiring tissue protective capabilities. For example, if these non-tissue-protective EPO analogs compete with endogenous EPO with tissue-protective activity for those receptors that elicit tissue-protective responses, the degree of injury resulting from injury may actually be suppressed by the action of such EPO. aggravated. In fact, if a patient suffering a stroke is administered such an EPO analog, the infarct volume resulting from the stroke may actually be greater compared to an individual not treated with the EPO analog.

Without being bound by any particular theory, these findings reveal that at least one additional EPO receptor type is functionally present in neural tissue, that its signaling is distinct from that of red blood cell precursors, and that there is an EPO-like There is a danger that drugs may antagonize the ability of endogenous EPO to bind to this form of the receptor. The distinctly different biological activities of endogenous EPO and these EPO analogs reveal that the receptors signal through functionally differential EPO receptors corresponding to different domains of the EPO molecule. In fact, although the EPO receptor gene protein sequence has been reported to be identical to that expressed by erythroid precursors, the binding affinity of neural EPO receptors in vitro is much lower than that of proerythroid EPO receptors. See, e.g., Masuda, S., et al., J Biol Chem, 268, 11208-16 (1993). Possibly, these differences are caused by accessory proteins and may indicate that different signaling pathways are used than those activated during erythrocyte maturation. Interestingly, this difference in affinity is not modified by complete deglycosylation of EPO, which is not a surprise if neuroactive binding occurs in the AB loop domain of normally non-glycosylated EPO result. In addition, EPO produced by astrocytes (possibly the same product produced by other cells such as neurons) is also smaller than that produced by the kidney. Masuda, S., J. Bio Chem, 269, 19488-93 (1994). This difference appears to be caused by differences in glycosylation programs. Whether there is a difference in the affinity of asialo's natural ligands for these known receptor proteins has not been determined, but there is clearly a relationship.

In addition to the presence of EPO receptor-modified accessory proteins, the EPO receptor is a complex group that exists in many altered forms, including truncated soluble receptors. Yamaji, R., et al., Eur JBiochem, 239, 494-500; Yamaji, R., et al., Bichim Biophys Acta, 1403, 169-78 (1998); Barron, C., et al., Gene, 147, 263- 8 (1994); Chin, K., et al., Brain Res Mol Brain Res, 81, 29-42 (2000); Fujita, M., et al., Lukemia, 11 Suppl 3, 444-5 (1997); Westenfelder, C., Biddle, D.L. Baranowski, R&.L., Kid.Internat., 55, 808-820 (1999). Whether any such form promotes the neural activity of EPO has not yet been determined.

Further, as discussed in the Background Art, O'Brien peptides exhibit tissue protective activity in vitro, but no erythropoietic activity in vitro. In fact, analysis of EPO analogs containing added sugar chains within O'Brien peptides revealed that the EPO analogs lack tissue protective functions. This result suggested that certain modifications to the O'Brien peptide, such as adding sugar chains, affected the function of the protein. O'Brien peptide-modified EPO analogues may act as antagonists of endogenous EPO located in vivo because they partially or completely block the ability of endogenous EPO to bind to EPO receptors. Therefore, the use of such EPO analogues runs the risk of increasing the degree of injury resulting from the injury. Thus, in other in vitro assays, such as rat motor neuron assays, and in vivo assays such as rat focal ischemia assays, bicuculline seizure assays, rat retinal ischemia assays, and spinal cord injury assays, it is believed that EPO analogs modified on O'Brien peptides will lack tissue protection.

receptor-mediated endocytosis

Using the same EPO analog as above, that is, an EPO analog with 5 N-linked sugar chains (compared to the 3 N-linked sugar chains of recombinant EPO), the inventors studied the passage of this analogue through blood The capacity of the brain barrier. After systemic injection (Figures 1 and 2), EPO analogues appeared in the cerebrospinal fluid. Without being bound by any particular theory, it is believed that the EPO analogs can cross the intact blood-brain barrier, since the capillaries forming the blood-brain barrier also express EPO receptors and are receptor-mediated transport from the peripheral circulation to the brain. The endocytosis and translocation function of the animal provides an anatomical basis. Thus, other systemically administered EPO analogs are also believed to be able to cross the blood-brain barrier, as well as other barriers with capillaries expressing EPO receptors.

Overall, because prior art EPO analogs maintain erythropoietic activity at the expense of at least some of the functions of endogenous EPO, there is a need in the art for a long-acting EPO that maintains endogenous All known functions of sexual EPO. Advantageously, the long-acting EPO of the present invention not only increases the longer serum half-life compared with recombinant EPO, but also maintains all functions of endogenous EPO, namely tissue protection function and endocytosis transport ability. Various methods of modifying EPO to obtain such beneficial proteins are provided in the following sections.

Modification of natural EPO

The long-acting EPO of the present invention can be prepared by various methods. In general, long-acting EPO can be obtained by chemically modifying the sugar chains attached to EPO. Here, the term "sugar chain" refers to N-linked and O-linked oligosaccharide chains present on endogenous EPO, increased N-linked and O-linked oligosaccharide chains present on EPO analogs, And any other sugar chains attached to EPO, especially sugar chains (Sugar chains).

In one embodiment, endogenous or recombinant EPO is modified to prevent any interference with the tissue protective capacity of endogenous EPO. Additionally, according to the present invention, it is contemplated that the EPO analogues be modified to provide additional glycosylation sites that are not located near the O'Brien peptide, ie the amino acid sequence at positions 30-47. Here, the term "EPO analogue" refers to a modified EPO molecule having at least 1 added N-linked sugar chain and/or at least 1 added O-linked sugar chain. In one embodiment, the EPO analog used for modification does not contain any increased glycosylation sites within 5 amino acids of the O'Brien peptide. In another embodiment, the EPO analog does not contain any increased glycosylation sites within 3 amino acids of the O'Brien peptide. In yet another embodiment, the EPO analog does not contain any increased glycosylation sites within the O'Brien peptide,

EPO analogues can also be used for modification as described in the present invention, provided that the analogues are considered in three-dimensional space and it is confirmed that the added sugar chain does not hinder the O'Brien peptide or reduce the tissue protective ability. On the other hand, the use of EPO analogs is contemplated for the modification of the present invention, provided that the glycosylation method does not inhibit the tissue protective function of the peptide. In yet another aspect, the EPO analog modified according to the modification method provided by the present invention has one sugar chain (or less) in the O'Brien peptide. For example, endogenous EPO has a sugar chain at amino acid 38, and an additional sugar chain within the O'Brien peptide has been shown to inhibit the tissue protective function of this protein. Therefore, EPO analogs with one or no sugar chains on the O'Brien peptide can be used for modification. In one embodiment, the sugar chain incorporated at amino acid position 38 can be reattached to other positions in the peptide.

Non-limiting examples of modification methods according to the present invention include (1) providing additional acidic residues on the sugar chain through oxidation of adjacent hydroxyl groups; (2) replacing sialic acid residues with more stable residues; (3) increasing the negative charge of erythropoietin by sulfation and/or phosphorylation; and/or (4) terminating sugar chains with more complex molecules. Therefore, the modification of EPO sugar chains may include oxidation, sulfation, phosphorylation, and/or PEGylation and other steps, which will be described in detail later and further illustrated in Example 1.

Oxidation of sugar chains and substitution of sialic acid residues

Chemical modification of the long-acting EPO of the present invention may include oxidation on the sugar chains (sugars) of the EPO to provide additional acidic residues. In one aspect, sialic acid residues can be replaced with more stable acidic residues. This type of modification results in an increased half-life of the molecule relative to endogenous EPO, as the liver screens the galactose chains and removes the associated protein from the circulation, while the galactose chains are protected from Detected. More significant modifications on the sugar chains of EPO lead to more prolongation of the serum half-life of the long-acting EPOs of the present invention. For example, when more ortho hydroxyl groups were replaced by acids, this resulted in a greater increase in serum half-life.

While those of ordinary skill in the art know several suitable methods for altering the galactose unit of erythropoietin, one suitable method involves (1) oxidation of a sugar molecule with an ortho hydroxyl group with periodate to an aldehyde; and (2) oxidation of the aldehyde to an acid. Reagents suitable for the oxidation of sugar chains to aldehydes are well known to those of ordinary skill in the art, including, but not limited to, periodates, such as sodium periodate, and sugar oxidases, such as galactase. In addition, the skilled person knows suitable reagents to convert aldehydes, such as the Quantitative Benedict Solution (commercially available from Fisher). On the other hand, sugar molecules can be oxidized by sodium periodate and further treated with quantitative Benedict's reagent (Fisher) to convert the aldehyde to acid.

On the other hand, EPO isomer EPO, which has about 0-13 sialic acid residues, or an EPO analogue, in which at least one sugar chain lacks a sialic acid residue, is oxidized by galactitol. The asialo form of EPO can be used in this aspect of the invention, ie the alpha or beta form of EPO with all the individual acids removed from the ends of the sugar chains. It is preferred to use asialo erythropoietin. Once EPO is oxidized, other oxidizing agents such as quantitative Benedict's reagent are used to convert the aldehyde to acid.

In another aspect, ruthenium tetroxide can be used to generate acids on sugar chains. Since the system modifies the galactose chains, even if the resulting acid involved is removed from the EPO molecule, the molecule should still be able to evade hepatic clearance because the constituent galactose chains screened by the liver are no longer present.

increase negative charge

In another aspect of the present invention, the long-acting EPO of the present invention can be prepared by adding sulfate and/or phosphate to the EPO molecule, which will increase the negative charge of the molecule, thereby increasing the half-life of the molecule. That is, the negative charge of the EPO molecule can be increased by sulfation, which involves the transfer of sulfo groups from sulfate donors. And, it is also possible to increase the negative charge by introducing a phosphate group into the sugar.

A suitable method for sulphating insulin is described in S. Pongor et al., Preparation of High-Potency, Non-aggregating Insulins Using a Novel Sulfation Procedure, Diabetes, Vol.32, No.12, December 1983. For example, sulfation of insulin can be performed in an organic solvent, such as dimethylformamide (DMF), in the presence of a condensing agent, such as N,N'-dicyclohexyldiimine (DCC), and a sulfate supply. body. The degree of sulfation can be controlled within 8 times by changing the amount of condensing agent. Although the traditional preparation of sulfated insulin results in a loss of the main biological activity of insulin, the biological activity of sulfated insulin prepared using the Ponger procedure is between 78% and 87% of that of unmodified insulin.

Similar procedures are used for EPO, and one of ordinary skill in the art can control the amount of sulfation and thus the serum half-life of chemically modified EPO. For example, the negative charge of EPO can be increased by dissolving EPO or an EPO analogue in at least one water-soluble carbodiimide, preferably DCC, at a temperature of about 4°C, and adding sulfuric acid to the protein. DCC is the preferred sulfate donor and those skilled in the art will readily know of other suitable sulfate donors for use in the present invention.

A similar procedure can be used to control the phosphorylation of EPO, using phosphoric acid (H ₃ PO ₄ ) as the phosphate donor. Also, while phosphoric acid is preferred, the skilled artisan can readily select other phosphate donors for phosphorylation of EPO.

Sugar chain ends are terminated with PEGs

The sugar chain of EPO can also be modified by adding at least one polyethylene glycol (PEG), which has a long and safe clinical history. The molecular formula is as follows:

PEG can also be methoxylated PEG (mPEG), which has the following molecular formula:

In one embodiment, the PEG is an amino PEG, preferably with a primary amino group (mPEG- _NH2 ) at the terminus of the methylated PEG. PEG chains terminated with primary amino groups are very useful functionalized polymers. The amino-terminal groups of mPEG-NH ₂ are more prone to acylation reactions than the hydroxyl groups of general PEGs, and they are also prone to amino reduction reactions (reductive amination reactions). In another embodiment, the PEG is an electrophilic activated PEG, such as mPEG-propyl succinate (mPEG-SPA) or mPEG-butyl succinate (mPEG-SBA), both of which are available from Nektar Therapeutics of Birmingham, Alabama The company bought it. In yet another embodiment, the PEG is methoxylated PEG-hydrazide.

Alternatively, at least one PEG can be added by oxidation with periodate (discussed above), followed by cyanoborohydride and aminoPEG. For example, EPO in solution is first oxidized with a periodate, such as sodium periodate, at room temperature for a preset period of time to produce aldehydes on the sugar chains. A suitable periodate is sodium meta-periodate, commercially available from Sigma Corporation. Periodate is then removed by buffer exchange, while the oxidized sialic acid groups on the N-linked oligosaccharide chains of EPO can be contacted with at least one aminoPEG in the presence of cyanoborohydride. Suitable PEGs that may be used include, but are not limited to, methoxylated PEG-hydrazide, which is commercially available from Nektar Therapeutics of Birmingham, Alabama.

On the other hand, at least one PEG can be added by attaching a PEG group to a terminal galactose residue after oxidation with galactase. For example, the asialo form of EPO (exposing the terminal galactose residue) in buffer is first contacted with galactose oxidase (commercially available from Sigma) to generate aldehydes in the sugar chains. The buffer is then removed by buffer exchange while simultaneously contacting the oxidized galactose residue with at least one aminoPEG in the presence of cyanoborohydride.

The methods described above are not limiting, and other methods can also be used to prepare the compounds of the present invention. For example, the skilled artisan will recognize that these chemical modifications can be used to make long-acting forms of other EPO derivatives such as the tissue protective cytokines disclosed in International Publication No. WO/02053580 and U.S. Patent Publication Nos. 2002/0086816 and 2003/0072737 , which is hereby incorporated by reference in its entirety.

Preparation of EPO molecules

Various host expression vector systems can be used to produce EPO to produce long-acting EPO molecules according to the invention. Such host expression vectors represent a class of vehicles by which EPO of interest is produced and subsequently purified, and also represent a class of cells that, when transformed with the appropriate nucleotide coding sequence Or upon transfection, the modified erythropoietin gene product can be expressed in situ. These include, but are not limited to, bacteria, insects, plants, mammalian host systems, such as, but not limited to, insect cell systems infected with recombinant viral expression vectors (such as baculoviruses) containing sequences encoding long-acting EPO products; Plant cell systems transformed with recombinant plasmid expression vectors (e.g., Ti plasmid) for sequences of erythropoietin-related molecules or transfected with recombinant virus expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV); or lactation Animal cell systems, including human cell systems, such as HT1080, COS, CHO, BHK, 293, 3T3, which contain recombinant expression constructs containing promoters derived from mammalian cell genomes, such as metallothionein promoters Promoters, or promoters derived from mammalian viruses, such as the late promoter of adenovirus and the 7.5K promoter of vaccinia virus.

In addition, the selected host cell strain can regulate the expression of the inserted sequence, or modify and process the gene product in a desired characteristic manner. Such modification and processing of the protein product may be very important for the function of the protein. Those skilled in the art know that different host cells have specific mechanisms for post-translational processing and modification of proteins and gene products. Appropriate cell lines or host systems can be selected to ensure correct processing and modification of the expressed foreign protein. In this regard, eukaryotic host cells with the correct processing of primary transcripts, cellular machinery for glycosylation and phosphorylation of gene products can be used. Such mammalian host cells include human host cells including, but not limited to, HT1080, CHO, VERO, BHK, HeLa, COS, MDCK, 293, 3T3, and WI38.

For long-term, high-yield production of recombinant proteins, a stable expression system is preferred. For example, cell lines can be engineered that stably express the gene product of the recombinant EPO molecule. Instead of using expression vectors that contain viral origins of replication, host cells can use DNA controlled by appropriate expression regulatory elements, such as promoters, enhancers, sequences, transcription terminators, polyadenylation sites, etc., and selectivity Mark conversions. After the introduction of exogenous DNA, engineered cells can be grown in nutrient-rich media for 1 to 2 days, and then transferred to selective media. A selectable marker in the recombinant plasmid confers selection resistance and allows stable integration of the plasmid into the chromosome of the cell and growth into foci, which can then be cloned and amplified into cell lines. This approach can advantageously be used to engineer cell lines expressing the gene product of an EPO mutein-related molecule. Such engineered cell lines may be particularly useful in screening and evaluating compounds that affect the endogenous activity of EPO-related molecule gene products.

Alternatively, expression of the endogenous EPO mutein gene in a cell line or microorganism can be characterized by inserting a heterologous DNA regulatory element into the genome of a stable cell line or cloned microorganism such that the inserted regulatory element is operatively linked to endogenous erythropoiesis. Genetic modification of the mutein. For example, endogenous EPO mutein genes, which are usually "transcriptionally silent", that is, usually do not express the EPO gene, or are expressed at very low levels in cell lines, can be promoted by insertion into said cell lines or microorganisms. A regulatory sequence for the expression of a gene product. Alternatively, the transcriptionally silenced endogenous EPO gene can be activated by inserting hybrid regulatory elements capable of functioning in multiple cell types.

Heterologous regulatory units can be inserted into stable cell lines or cloned microorganisms to allow efficient linkage to the endogenous erythropoietin protein gene using techniques such as targeted homologous recombination that are within the reach of those skilled in the art are well known and are described in French Patent No. 2646438, US Patent Nos. 4,215,051 and 5,578,461, and International Publication Nos. WO93/09222 and WO91/06667, the disclosures of which are incorporated herein in their entirety.

pharmaceutical composition

The present invention also relates to pharmaceutical compositions comprising the long-acting EPO according to the present invention. Since the long-acting EPOs of the present invention advantageously possess erythropoietic activity as well as tissue protective and endocytic transport capabilities, they are expected to treat anemia and related diseases in individuals who are also at risk of various tissue damages such as stroke and myocardial infarction . In addition, it is expected that the long-acting EPOs of the present invention can be used in the treatment of anemia and related diseases in individuals who also experience mental system deterioration such as Alzheimer's disease, Parkinson's disease and the like. Further, the long-acting EPOs of the present invention can be used to treat anemia in individuals suffering from conditions resulting from the normal aging process (eg, balance problems leading to falls, easy bruising, etc.). Also, the present invention relates to the use of long-acting EPOs according to the present invention as carriers of other molecules, wherein said molecules are difficult to penetrate the barrier of capillaries with EPO receptors.

For example, any of the aforementioned long-acting EPOs may be included in the pharmaceutical compositions of the present invention. In addition, various forms of EPO analogs can be included in the pharmaceutical compositions of the present invention in admixture with at least one tissue protective cytokine, which will be discussed in detail below.

The pharmaceutical composition of the present invention contains a therapeutically effective amount of long-acting EPO, preferably in a purified form. The formulation should be compatible with the mode of administration. Prepared in a form for ease of administration. In other words, the pharmaceutical composition of the present invention contains the long-acting EPO of the present invention in such an amount that its targeted condition can be treated with the correct dosage and strategy. Also, as will be discussed in detail later, the drug combination should be administered in non-toxic doses.

The pharmaceutical compositions of the present invention may contain a therapeutically effective amount of a long-acting EPO compound and a suitable amount of a pharmaceutically acceptable carrier so as to provide a form for proper administration to a patient. In one embodiment, the term "pharmaceutically acceptable" means approved by a regulatory agency of the federal or state government or listed in the United States Pharmacopoeia or other recognized foreign pharmacopoeia for use in mammals, especially humans. The term "carrier" refers to a diluent, adjuvant, excipient or pharmaceutical carrier with which the therapeutic agent is administered. Such pharmaceutical carriers can be sterile liquids, such as aqueous and oily saline solutions, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Saline solution is a preferred carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dry powdered skim milk, glycerin , propylene glycol, water, ethanol, etc.

The pharmaceutical compositions of the present invention may also contain small amounts of lubricants or emulsifiers, or pH buffering agents. These compositions can be prepared in the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained-release preparations and the like.

The compositions of the present invention can be prepared in neutral or salt form. Pharmaceutically acceptable salts include those formed with free amino groups, such as those derived from hydrochloric acid, phosphoric acid, acetic acid, oxalic acid, tartaric acid, etc., and those formed with free carboxyl groups, such as those derived from sodium, potassium, ammonium, Salts of calcium, ferric hydroxide, isopropylamine, triethylamine, 2-ethylaminoethanol, histidine, procaine, etc.

Compositions containing long-acting EPO

As previously mentioned, any of the long-acting EPOs described herein may be used in pharmaceutical compositions. In one embodiment, long-acting EPO produced by oxidation of ortho hydroxyls is included in the pharmaceutical composition of the present invention. In another embodiment, the pharmaceutical composition according to the present invention comprises at least one long-acting EPO resulting from the substitution of sialic acid residues with more stable residues. In yet another embodiment, the long-acting EPO contained in the pharmaceutical composition is obtained by increasing the negative charge of EPO by sulfation and/or phosphorylation. In yet another embodiment, the long-acting EPO contained in the pharmaceutical composition of the present invention is produced by terminating the sugar chain with a more complex molecule, such as a PEG chain.

In addition, the present invention relates to the use of mixtures of long-acting EPOs produced by any of the methods described herein in pharmaceutical compositions described herein. For example, a pharmaceutical composition according to the invention may comprise at least one long-acting EPO formed by substituting sialic acid with a more stable residue, and at least one long-acting EPO formed by increasing the negative charge of EPO by sulfation and/or phosphorylation. long-acting EPO.

transport system

As previously mentioned, the long-acting EPOs of the present invention are advantageously capable of crossing the capillary barrier with EPO receptors. Accordingly, in another embodiment of the present invention is a delivery system that uses the long-acting EPOs of the present invention as carriers for molecules that have low barrier penetration into targeted regions of the body containing EPO receptors . Such a transport system advantageously provides a new and safe method of transport across intact barriers.

In one embodiment, a delivery system comprising the long-acting EPOs of the present invention and at least one molecule with a low brain-penetrating ability provides a novel and safe delivery method across the intact blood-brain barrier. That is, the long-acting EPOs of the present invention allow those molecules with low brain penetration ability to act as molecular "Trojan horses", thereby enhancing the ability of the brain to uptake small or large molecular diagnostic agents or therapeutic molecules.

In fact, an important problem in the treatment of human brain tumors stems from the need to deliver therapeutic agents to specific regions of the brain, distribute them within and target brain tumors. Molecules that may otherwise be useful in diagnosis and therapy either fail to cross the blood-brain barrier (BBB) in the brain immediately adjacent to the tumor, or the blood-tumor barrier (BTB) in sufficient amounts. Therefore, new delivery strategies that are specific to the brain and able to traverse the vasculature are needed. For example, antibody molecules used for diagnosis or therapy cannot cross BTB in sufficient quantities due to their size. Therefore, the long-acting EPOs of the present invention can be used as carriers to transport these molecules across the BBB or BTB. An example of a molecule that can be used with the long-acting EPOs of the invention is an antisense oligonucleotide, which is typically used to inhibit oncogene signaling or to image brain gene expression in vivo. In addition, the long-acting EPOs of the present invention can be included in various gene therapy agents (viral or non-viral formulations), which are generally too large to cross BTB without assistance.

Further, the long-acting EPOs of the present invention can be used as carrier-mediated transporters of various chemotherapeutic agents. Because drug-active efflux transporters expressed on the BBB and BTB actively return chemotherapeutic agents from the brain to the blood, distribution of these agents to the brain may be inhibited or prevented. Partly for these reasons, most of the chemotherapeutic molecules traditionally used to treat cancers located outside the central nervous system (CNS) are ineffective for the treatment of brain tumors. Therefore, the use of the long-acting EPO of the present invention as a carrier for these chemotherapeutic agents is useful not only in delivering the agent to the brain, but also in retaining the agent in the brain for treatment. In another embodiment, the long-acting EPO can be used with drugs that inhibit active efflux transporters to further ensure the uptake of these chemotherapeutic agents that normally reflux from the brain into the blood.

In addition, the invention also encompasses the use of modified EPO as a carrier for molecules that have low penetration in vivo to express other regions of the EPO receptor. Non-limiting examples of such cells include retinal, muscle, heart, lung, liver, kidney, small intestine, adrenal cortex, adrenal medulla, capillary endothelium, testis, ovary, pancreas, bone, skin, and endometrial cells. In particular, responding cells include, but are not limited to, neuronal cells; retinal cells: photoreceptor cells (rods and cones); nerve junctions, bipolar cells, horizontal cells, amacrine cells, and Müeller cells; muscles cells; heart cells: cardiomyocytes, pacemaker cells, sinus node cells, sinus node cells, and connective tissue (AV node and bundle of his) cells; lung cells; liver cells: hepatocytes, astrocytes, and Kupffer cells ; kidney cells: glomeruli, renal epithelial cells, and tubulointerstitial cells; small intestinal cells: goblet cells, intestinal glands (crypts), and enteroendocrine cells; adrenal cortex cells: glomerulus cells, fascicular cells, and reticular cells; adrenal medulla cells: chromaffin cells; capillary cells: podocytes; testicular cells: Leydig cells, Sertoli cells, and sperm cells and their precursors; ovarian cells: Graffian follicles and primordial follicle cells; pancreas Cells: Langerhans islet cells, α-cells, β-cells, γ-cells, and F-cells; bone cells: osteoprogenitors, osteoclasts, and osteoblasts; skin cells; endometrial cells: endometrial stroma and endometrial cells; and stem cells and endothelial cells present in the aforementioned organs.

Compositions mixing EPO analogues and tissue protective cytokines

As previously mentioned, the pharmaceutical composition of the present invention may comprise EPO analogues having at least one increased N-linked sugar chain and/or at least one O-linked sugar chain (exhibiting prolonged serum half-life but lacking tissue protective activity) and at least one tissue protective cytokine. For example, EPO analogs with at least 2 added N-linked sugar chains, one of which is located on the O'Brien peptide, together with tissue protective cytokines, can form compositions according to the invention. In another embodiment, the pharmaceutical composition of the present invention may comprise at least one tissue protective cytokine and at least one EPO containing an additional sugar chain, wherein by considering the analog in three-dimensional space, it is known that the Sugar chains block the O'Brien peptide sequence. In yet another embodiment, the pharmaceutical composition of the present invention comprises at least one tissue protective cytokine and at least one EPO analog which results in It has no tissue protection function.

EPO analogs with relocated glycosylation sites are contemplated for use in the pharmaceutical compositions of the present invention. Without being bound by any particular theory, it is believed that if the naturally occurring glycosylation site at amino acid 38 is relocated to a position other than the 30-47 amino acid segment of the EPO analog, then the EPO analog Tissue protection will be enhanced compared to EPO analogs with a glycosylation site at position 38. Therefore, the pharmaceutical composition of the present invention may contain EPO analogs that relocate the glycosylation site at amino acid 38 to other sites in the molecule. Relocated glycosylation sites may occur at positions 51, 57, 69, 88, 89, 136 or 138, as indicated in PCT Publication No. WO 01/81405. In one embodiment, the O'Brien peptide sequence contains 1 or fewer sugar chains.

Suitable tissue protective cytokines for use in this aspect of the invention are preferably those that lack action on the bone marrow but retain endogenous tissue protective effects, but any cytokine with tissue protective capabilities may also be used in the present invention. For example, suitable tissue protective cytokines include those derived from guanidinylation, amidinylation, carbamylation (carbamylation), picric acidification, acylation (acetylation or succinylation), nitration, or EPOs produced by chemically modifying their combinations. In addition, EPO molecules with at least one arginine, lysine, tyrosine, tryptophan, cysteine residue or hydroxyl group modified are also expected to be useful as tissue protective cytokines according to this aspect of the invention .

Also, additional tissue protective cytokines used in the present invention can be replaced by arginine, lysine, tyrosine, tryptophan by limited proteolysis, removal of amino groups and/or introduction of mutations by molecular biology techniques or cysteine residues as described in molecular biology techniques disclosed in Satake et al., 1990, Biochim. Biophs. Acta 1038:125-9, incorporated herein in its entirety. For example, suitable tissue protective cytokines include at least one or more mutated EPO at C7S, R10I, V11S, L12A, E13A, R14A, R14B, R14E, R14Q, Y15A, Y15F, Y15I, K20A, K20E, E21A, C29S, C29Y, C33S, C33Y, P42N, T44I, K45A, K45D, V46A, N47A, F48A, F48I, Y49A, Y49S, W51F, W51N, Q59N, E62T, L67S, L70A, D96R, S100R, S100E, S100A S100T，G101A，G101I，L102A，R103A，S104A，S104I，L105A，T106A，T106I，T107A，T107L，L108K，L108A，S126A，F142I，R143A，S146A，N147K，N147A，F148Y，L149A，R150A，G151A，K152A， L153A, L155A, C160S, I6A, C7A, B13A, N24K, A30N, H32T, N38K, N83K, P42A, D43A, K52A, K97A, K116A, T132A, I133A, T134A, K140A, P148A, R150B, W, G151A, K15 Point mutations at G158A, C161A, and/or R162A. Examples of the above-mentioned modifications are described in co-pending US Patent Publication Nos. 2003/0104998, 2002/00861816 and 2003/0072737, which are hereby incorporated by reference in their entirety. In the mutein nomenclature used herein, the amino acid that is altered is described by the one-letter code of the native amino acid, followed by its position in the EPO molecule, followed by the one-letter code of the substituted amino acid. For example, S100E refers to the replacement of serine at position 100 with glutamic acid in the human EPO molecule.

In another embodiment, the tissue protective cytokine may comprise one or more of the above point mutations, provided that the point mutations do not include I6A, C7A, K20A, P42A, D43A, K45D, K45A, F48A, Y49A, K52A, K49A , S100B, R103A, K116A, T132A, I133A, K140A, N147K, N147A, R150A, R150E, G151A, K152A, K154A, G158A, C161A, or R162A.

In yet another embodiment, the tissue protective cytokine may comprise a combination of point mutations, such as K45D/S100E, A30N/H32T, K45D/R150E, R103E/L108S, K140A/K52A, K140A/K52A/K45A, K97A/K152A, K97A /K152A/K45A, K97A/K152A/K45A/K52A, K97A/K152A/K45A/K52A/K140A, K97A/K152A/K45A/K52A/K140A/K154A, N24K/N38K/N83K, and N24K/Y15A. In yet another embodiment, the tissue protective cytokine does not comprise any of the above combinations. In another embodiment, the tissue protective cytokine may comprise any one of the above point mutations, provided that the point mutations do not comprise any of the following combinations of mutations: N24K/N38K/N83K and/or A30N/H32T.

Certain modifications or combinations of modifications may affect the flexibility of the mutein's ability to bind to its receptor, such as the EPO receptor or a second receptor. Examples of such modifications or combinations of modifications include, but are not limited to, K152W, R14A/Y15A, I6A, C7A, D43A, P42A, F48A, Y49A, T132A, I133A, T134A, N147A, P148A, R150A, G151A, G158A, C161A, and R162A. Associated mutations deleterious to human growth hormone are well known to those skilled in the art. Accordingly, in one embodiment, the tissue protective cytokine does not comprise one or more modifications or combinations of modifications that may affect the flexibility of the mutein's ability to bind to its receptor. Further discussion of such tissue protective cytokines is contained in co-pending U.S. Application No. 10/612,665, filed July 1, 2003, entitled "Recombinant Tissue Protective Cytokines and Encoding Nucleic Acid Thereof for Protection, Restoration , and Enhancement of Responsive Cells, Tissues, and Organs", the entire disclosure of which is incorporated herein by reference.

Finally, any cytokine superfamily with tissue protective capacity can be used as long as it does not affect the erythropoietic activity or serum half-life of long-acting EPO. Examples include, but are not limited to, interleukin-3 (IL-3), interleukin-5 (IL-5), granulocyte-macrophage colony-stimulating factor (GMCSF), pigment epithelium-derived factor (PEDF), vascular endothelial growth factor ( VEGF).

In another aspect of the present invention, the pharmaceutical composition of the present invention comprises an EPO analog with at least one increased N-linked sugar chain and/or at least one increased O-linked sugar chain (with extended serum half-life but lacks tissue protective activity) and at least one small molecule with tissue protective function. Suitable small molecules include, but are not limited to, steroids (such as laza bases and glucocorticoids), antioxidants (such as coenzyme Q ₁₀ , alpha lipoic acid, and NADH), anticatabolic enzymes (such as glutathione peptide peroxidase, superoxide dismutase, catalase, synthetic catalytic scavengers, and mimics), indole derivatives (such as indoleamines, carbazoles, and carbolines), nitric acid neutralizers, Adenosine/adenosine agonists, phytochemicals (flavans), herbal extracts (gingko and turmeric), vitamins (vitamins A, E, and C), oxidase electron acceptor inhibitors (such as xanthene oxidase electron acceptor inhibitors), minerals (such as copper, zinc, and magnesium), NSAIDS (such as acetylsalicylic acid, naproxen, and ibuprofen), and combinations thereof. In addition, the pharmaceutical composition of the present invention contains EPO analogs, tissue protective cytokines, and small molecules with tissue protective activity.

The amount of tissue protective cytokines and/or small molecules present in the pharmaceutical compositions of the present invention is preferably sufficient to maintain or exceed the activity produced by endogenous EPO in neural or other responsive cell systems. In one embodiment, the amount of the tissue protective cytokine and/or small molecule is sufficient to enhance the tissue protective capacity of the individual by protecting, maintaining, or enhancing the viability and function of erythropoietic responsive cells. For example, the pharmaceutical compositions described in this aspect of the invention preferably contain an effective, non-toxic amount of the tissue protective cytokine, eg, about 1 ng or more. In one embodiment, the amount of tissue protective cytokine present in the pharmaceutical composition of the present invention is about 5 mg or less. In another embodiment, the tissue protective cytokine is present in the pharmaceutical composition of the present invention in an amount of about 500 ng to 5 mg. In yet another embodiment, the pharmaceutical composition contains about 1 μg to 5 mg of the tissue protective cytokine, preferably about 500 μg to 5 mg. In another embodiment, the amount of tissue protective cytokine present in the pharmaceutical composition of the present invention is greater, about 1 mg to 5 mg. It is well known to those skilled in the art that the dosage of the pharmaceutical composition administered to a patient depends on factors such as, but not limited to, the patient's physical condition and the frequency of administration. The dosage of the medicine will be discussed in detail later.

Treatment and Administration

The aforementioned long-acting EPOs and pharmaceutical compositions containing the long-acting EPOs are expected to be useful in the therapeutic or preventive treatment of anemia, human diseases involving anemia or symptoms of anemia, or diseases or treatments that cause anemia. In general, the long-acting EPOs of the present invention allow for less frequent administration or use of lower doses of erythropoietin to treat the above-mentioned diseases without compromising the patient's ability to recover from other tissue injuries, especially is damage to other EPO responsive cells, tissues, or organs. Non-limiting examples of such EPO responsive cells include retinal, muscle, heart, lung, liver, kidney, small intestine, adrenal cortex, adrenal medulla, capillary endothelium, testis, ovary, pancreas, bone, skin and endometrial cells . In particular, responding cells include, but are not limited to, neuronal cells; retinal cells: photoreceptor cells (rods and cones); nerve junctions, bipolar cells, horizontal cells, amacrine cells, and Müeller cells; muscles cells; heart cells: cardiomyocytes, pacemaker cells, sinus node cells, sinus node cells, and connective tissue (AV node and bundle of his) cells; lung cells; liver cells: hepatocytes, astrocytes, and Kupffer cells ; renal cells: mesangial, renal epithelial, and tubulointerstitial cells; small intestinal cells: goblet cells, intestinal glands (crypts), and enteroendocrine cells; adrenal cortical cells: glomerular cells, fascicular cells, and reticular cells; adrenal medullary cells: chromaffin cells; capillary cells: podocytes; testicular cells: Leydig cells, Sertoli cells, and sperm cells and their precursors; ovarian cells: Graffian follicles and primordial follicle cells ; pancreatic cells: Langerhans islet cells, α-cells, β-cells, γ-cells, and F-cells; bone cells: osteoprogenitors, osteoclasts, and osteoblasts; skin cells; endometrial cells: inner membrane matrix and endometrial cells; and stem cells and endothelial cells present in the above-mentioned organs. Individuals predisposed to stroke, spinal cord injury, peripheral nerve injury, diabetic neuropathy, retinopathy, including but not limited to macular edema, diabetic nephropathy, etc. may also benefit from the use of long-acting EPO. Other Disease and Tissue Protective Effects of Long-Acting EPO Discussion Copending U.S. Patent Application No. 10/188,905, filed July 3, 2002 and Application Serial No. 10/612,665, filed July 1, 2003, It is hereby incorporated by reference in its entirety.

The present invention contemplates that the long-acting EPOs can be used for systemic or chronic administration, acute treatment and/or intermittent administration. In one embodiment, the pharmaceutical composition of the present invention can be administered chronically to protect or enhance target cells, tissues or organs. In another embodiment, the pharmaceutical composition of the present invention may be administered acutely, ie as monotherapy during the injury. In another embodiment, the pharmaceutical compositions described herein may be administered in a cyclical fashion.

Administration of the composition can be parenteral, e.g., by intravenous injection, intraperitoneal injection, intraarterial, intramuscular, intradermal, or subcutaneous administration; by inhalation; transmucosal, e.g., oral, nasal, rectal, intravaginal, lingual Submucosal, submucosa, and transdermal; or combinations thereof. Preferably, the administration of the pharmaceutical composition of the present invention is parenteral. The dose for such administration is about 0.01 pg to about 5 mg, preferably about 1 pg to about 5 mg. In another embodiment, the dose is from about 500 pg to about 5 mg. In yet another embodiment, the dose is from about 500 ng to about 5 mg. In yet another embodiment, the dose is from about 1 [mu]g to about 5 mg. For example, the dosage may be from about 500 μg to about 5 mg. In another embodiment, the dosage may be from about 1 mg to about 5 mg.

The pharmaceutical compositions of the present invention suitable for parenteral administration contain aqueous or nonaqueous sterile injectable solutions or suspensions, which may contain antioxidants, buffers, bacterial inhibitors and solutes that make the composition Essentially isotonic with the blood of the subject. In this aspect of the invention, the pharmaceutical composition may also contain water, ethanol, polyols, glycerin, vegetable oils, and mixtures thereof. Pharmaceutical compositions adapted for parenteral administration may be presented in unit-dose or multi-dose containers, for example, hermetically sealed ampoules and vials, and may be stored lyophilized (freeze-dried), in which case they need only be ordered immediately A sterile liquid carrier, such as sterile saline solution for injection, is added just before use. Extemporaneous injections and suspensions can be formulated as sterile powders, granules and tablets. In one embodiment, the autoinjector containing the long-acting EPOs injection of the present invention can be used for emergency use in ambulances, emergency rooms and battlefields.

In one embodiment, the composition of the present invention can be prepared into a pharmaceutical composition suitable for intravenous administration to humans according to conventional procedures. For example, the pharmaceutical compositions can be formulated in sterile isotonic aqueous buffer. If desired, the pharmaceutical composition may also contain a solubilizer and/or a local anesthetic such as lidocaine to relieve pain at the injection site. The ingredients may be presented alone or mixed together in unit dosage form, such as lyophilized powder or water-free concentrate in sealed containers, such as ampoules or sachets labeled with the amount of active ingredient. When the pharmaceutical compositions described herein are administered by infusion, an infusion bottle containing sterile pharmaceutical grade water or saline solution may be used to dispense the drug. Also, when the pharmaceutical composition of the present invention is administered in the form of injection, an ampoule containing a sterile saline solution may be used to mix the ingredients before administration.

Pharmaceutical compositions suitable for oral administration can be presented in the form of: capsules or tablets; powders or granules; solutions, syrups or suspensions (liquids with or without water); edible foams (foams or whips); emulsifiers ; or a combination thereof. Oral formulations may contain from about 10% to about 95% by weight active ingredient. In one embodiment, the weight percent of the active ingredient in the oral formulation is from about 20% to about 80%. In another embodiment, the weight percent of the active ingredient in the oral formulation is from about 25% to about 75%.

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

Additionally, active agents intended for oral administration can be coated or compounded with materials that delay disintegration and/or absorption of the active agent within the gastrointestinal tract. For example, glyceryl monostearate, glyceryl distearate, or a combination thereof may be mixed with or coated with the active agent. Thus, sustained release of the active agent can be maintained for many hours, preventing degradation of the active agent in the stomach if desired. Oral pharmaceutical compositions can be formulated according to specific pH or enzymatic conditions to facilitate the release of the active agent at a specific site in the gastrointestinal tract.

Pharmaceutical compositions suitable for transdermal administration may be provided in discrete patches that remain in intimate contact with the epidermis of a subject for an extended period of time. Additionally, pharmaceutical compositions adapted for topical administration may be presented in the form of ointments, creams, suspensions, lotions, powders, solutions, pastes, gels, sprays, aerosols, oils , eye drops, lozenges, pastilles, and mouthwashes, and combinations thereof. When administered topically to the skin, mouth, eyes, or other external tissues, topical ointments or creams are preferred. Also, when present in an ointment, the active ingredient, long-acting EPO, may be employed with a wax or a water-miscible ointment base. Alternatively, the active ingredient may be formulated in a cream with an oil-in-water or water-in-oil base. When topical administration is in the form of eye drops, the pharmaceutical compositions described herein preferably contain the active agent dissolved or suspended in a suitable carrier such as an aqueous solvent.

Pharmaceutical compositions suitable for nasal and pulmonary administration may contain solid carriers such as powders (preferably having a particle size in the range of about 20 to about 500 microns). The powder may be administered by nasal inhalation quickly from a container containing the powder near the nose. In another embodiment, the pharmaceutical composition for nasal administration of the present invention contains a liquid carrier, such as nasal spray or nasal drops. Preferably, the pharmaceutical composition of the present invention is administered directly in the nasal cavity.

Direct pulmonary inhalation can be accomplished by deep inhalation through an oral cannula inserted into the throat and other specially designed devices including, but not limited to, pressurized aerosols, nebulizers, or nebulizers, which can be controlled by specialized Designed to provide a predetermined amount of active ingredient. Preferably, the pharmaceutical composition is administered by deep inhalation directly into the throat.

Pharmaceutical compositions adapted for rectal administration may be presented as suppositories or enemas. In one embodiment, the suppositories of the present invention contain from about 0.5% to 10% by weight of the active ingredient. In another embodiment, the suppository contains from about 1% to about 8% by weight of the active ingredient. In another embodiment, the suppository contains from about 2% to about 6% by weight of the active ingredient. In this aspect of the invention, the pharmaceutical composition of the invention may contain usual binders and carriers, such as triglycerides.

Pharmaceutical compositions adapted for vaginal administration may be presented as pessaries, tampons, creams, gels, pastes, foams or sprays.

The pharmaceutical composition of the present invention can also be administered by infusion, organ injection or topical administration. In these embodiments, the pharmaceutical composition preferably contains about 0.01 pM to about 30 pM, preferably about 15 pM to about 30 nM of the long-acting EPO of the present invention. In one embodiment, the infusion solution is a University of Wisconsin (UW) solution (having a pH of about 7.4 to about 7.5 and an osmolarity of about 320 mOSm/l) containing about 1 U/ml to about 25 U/ml of EPO; 5% hydroxyethyl starch (preferably having a molecular weight of from about 200,000 to about 300,000 and being substantially free of ethylene glycol, 2-chloroethanol, sodium chloride, and acetone), 25 mM KH ₂ PO ₄ , 3 mM glutathione 5 mM adenosine; 10 mM glucose; 10 mM HEPES buffer; 5 mM magnesium gluconate; 1.5 mM _CaCl2 ; 105 mM sodium gluconate; 200,000 units of penicillin; 40 units of insulin; 16 mg of dexamethasone; UW solutions are described in detail in US Patent No. 4,798,824, which is hereby incorporated by reference in its entirety. In another embodiment, the UW solution may contain about 0.01 pg/ml to about 400 ng/ml, preferably 40 ng/ml to about 300 ng/ml of recombinant tissue protective cytokine.

It may be desirable to administer pharmaceutical compositions described herein topically to an area in need of treatment. Such administration may be accomplished by: local infusion during surgery; topical application, such as with a wound dressing after surgery; by injection; by catheter; by suppository; The entry means may be perforated, non-porous, or gelatinous materials, including membranes, such as silica gel membranes, or fibers.

In addition, the long-acting EPO of the present invention can be delivered by a controlled release system as mentioned above with regard to the transdermal administration. For example, the peptide is administered using intravenous infusion, implanted osmotic pumps, transdermal patches, liposomes, or other means. In one embodiment, a pump can be used, as described in Saudek et al., 1989, N. Engl. J. Med. 321:574. In another embodiment, the compound may be delivered using a vehicle, particularly a liposome, as described in International Publication No. WO 91/04014 and US Patent No. 4,704,355, which are hereby incorporated by reference in their entirety. In another embodiment, polymeric materials can be used to prepare controlled release systems, such as those described in Howard et al., 1989, J. Neurosurg. 71:105.

Such a controlled release system can be placed close to the therapeutic target, ie the target cell, tissue or organ, thus requiring only a fraction of the systemic dose. See, for example, Goodson, Medical Applications of Contolled Release, vol. 2, pp. 115-138, 1984. Other controlled release systems useful in the present invention are described in the review by Langer, Science 249:1527-1533,1990.

dose

Based on the general knowledge of those skilled in the art, those skilled in the art can select the preferred effective and non-toxic doses suitable for the above administration methods. Examples of these factors include the particular form of long-acting EPO; the pharmacokinetic parameters of the EPO, such as bioavailability, metabolism, half-life, etc. (provided to the skilled person); the condition or disease to be treated; patient's weight; method of administration; frequency of administration, ie, chronic, acute, intermittent administration; concomitant medications; and other factors known to affect the effectiveness of the administered agent. Thus, the precise dosage should be determined according to the judgment of the practitioner and the circumstances of the particular patient.

For example, the Physicians Desk Reference (PDR) indicates that, depending on the patient population treated with EPO, different hematocrit levels are considered to avoid toxicity. Physicians Desk Reference, ^54th Ed., 519-525 and 2125-2131 (2000). In fact, for patients with CRF, the PDR recommends administering EPO doses to achieve a non-toxic target hematocrit of 30% to 36%. In contrast, for cancer patients undergoing chemotherapy, the PDR teaches to adjust the dose to different hematocrit levels, ie if the hematocrit level exceeds 40%. The PDR suggests that professionals monitor a patient's hematocrit during treatment with EPO and, if the patient's hematocrit reaches or exceeds the upper end of a predetermined range, adjust the dose and/or discontinue treatment to avoid toxicity. Thus, a skilled practitioner, in light of the teachings of the present invention, should be able to administer EPO in doses sufficient to achieve therapeutic effect while avoiding any toxic complications.

In one embodiment, the long-acting EPO of the present invention is administered chronically or systemically at a dose of about 0.1 μg/kg body weight to about 100 μg/kg body weight each time. For example, in the treatment of cancer patients receiving chemotherapy, a dose of about 1 μg/kg body weight to about 5 μg/kg body weight is administered once weekly. In another embodiment, the dose of the long-acting EPO is about 5 μg/kg body weight to about 50 μg/kg body weight each time. In yet another embodiment, the dosage of the long-acting EPO is about 10 μg/kg body weight to about 30 μg/kg body weight. In yet another embodiment, the long-acting EPO is administered in an amount of about 1 μg/kg body weight or less. For example, about 0.45 [mu]g/kg body weight to about 0.75 [mu]g/kg body weight of long-acting EPO may be effective when administered once weekly to treat anemia in CRF patients.

An effective dose is preferably sufficient to achieve serum levels of long-acting EPO greater than about 10,000 mU/ml (80 ng/ml). In one embodiment, the effective dose is capable of achieving a serum level of long-acting EPO of about 15,000 mU/ml (120 ng/ml) or higher. In another embodiment, the effective dose is capable of achieving a serum level of long-acting EPO of about 20,000 mU/ml (160 ng/ml). Detection is preferably performed at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 hours or a combination thereof after administration. The administered dose may be repeated as deemed necessary by one of ordinary skill in the art. For example, as long as clinically necessary, the administration can be repeated every day, or after an appropriate interval, such as every 1 to 12 weeks, preferably every 1 to 3 weeks.

Since the long-acting EPOs of the present invention have an extended half-life, their activity in vivo will also be increased. For example, when a mammalian patient is undergoing systemic chemotherapy for cancer, including radiotherapy, administering the long-acting EPO pharmaceutical composition of the present invention during treatment can reduce symptoms associated with anemia, and the frequency and dose of its administration are less than Existing recombinant EPO compositions.

And, as mentioned above, when the pharmaceutical composition of the present invention contains the long-acting EPO or EPO analogs of the present invention in a mixed form with tissue protective cytokines, the composition can be used for the treatment of tissue Anemia and related disorders in patients at risk of injury. For example, patients suffering from anemia who are also at high risk of heart disease can be treated with the composition of the present invention instead of existing EPO analogues to avoid the increased risk of damage due to treatment.

treatment kit

The present invention also provides a pharmaceutical kit or kit comprising one or more containers filled with one or more components of the pharmaceutical composition of the present invention. In one embodiment, an effective amount of long-acting EPO and a pharmaceutically acceptable carrier may be packaged in a single dose bottle or other container.

When the pharmaceutical composition of the present invention is used for parenteral administration, for example, the composition can be preserved under freeze-dried conditions. Thus, a kit may include a lyophilized composition, a sterile liquid carrier, and a syringe for injection. In one embodiment, the kit includes ampoules containing sufficient lyophilized material for several treatments so that the administerer can weigh out the specified amount of material and add the specified amount of carrier for each treatment. In another embodiment, the kit includes a plurality of ampoules, each containing a specified amount of lyophilized material, and a plurality of containers, each containing a specified amount of carrier, so that the administerer need only dispense one The contents of the ampoule and a carrier container are mixed for each treatment without measuring or weighing. In another embodiment, the kit comprises an autoinjector containing the EPO solution of the present invention for injection. In another embodiment, the kit includes at least one ampoule containing a lyophilized composition, at least one container containing a carrier solution, at least one container containing a local anesthetic, and at least one syringe (or the like). Ampoules and containers are preferably hermetically sealed.

When administering the pharmaceutical composition of the present invention by infusion, the kit preferably includes at least one ampoule containing the pharmaceutical composition and at least one infusion bottle containing sterile pharmaceutical grade water or saline solution.

Kits according to the present invention may also include at least one oral cannula or special device suitable for direct pulmonary inhalation such as a pressurized aerosol, nebulizer, or nebuliser. In this aspect of the invention, the kit may comprise a device for direct pulmonary inhalation containing the pharmaceutical composition, or the device and at least one ampoule containing the aqueous or oily solution of the long-acting EPO of the present invention .

When the long-acting EPO pharmaceutical composition of the present invention is suitable for oral, transdermal, rectal, vaginal, or nasal administration, the kit preferably includes at least one ampoule containing the active ingredient and at least one administration aid. Examples of administration aids include, but are not limited to, weighing spoons (for oral administration), sterile cleansing pads (for transdermal administration), and nasal aspirators (for nasal administration). Such kits may contain a single dose of long-acting EPO (acute treatment) or multiple doses of long-acting EPO (chronic treatment).

Additionally, the kit may be provided with one or more types of solutions. For example, the long-acting EPO pharmaceutical composition of the present invention can be prepared in albumin solution and polysorbate solution. If the kit contains a polysorbate solution, the words "albumin-free" preferably appear on the label of the container and on the main panel of the kit.

Additionally, the kit includes a statement in the form prescribed by a governmental agency regulating the manufacture, use, or sale of drugs or biological products, which statement reflects approval by such agency for manufacture, use, or sale in humans.

EPO detection test

The present invention also relates to assays for determining the erythropoietic and tissue protective abilities of the long-acting EPOs described herein and the EPO analogs used in several pharmaceutical compositions described herein. For example, the erythropoietic activity of long-acting EPOs can be detected using the TF-1 assay, which is described in more detail in Example 2. The tissue protective activity of EPO compounds can be tested using in vitro and in vivo assays, which are discussed in more detail below. In addition, it is contemplated that the present invention also includes assays that can be used not only to determine whether a particular EPO compound has tissue protective activity, but also to determine whether the EPO compound acts as an antagonist of endogenous EPO.

The detection method described in the present invention is preferably designed to be completed in a short time using a minimum amount of EPO mixture. Also, the detection methods provided herein are non-limiting, as those of ordinary skill in the art will appreciate other methods for detecting the erythropoietic and tissue protective activities of EPO compounds.

Erythropoietic activity assay

The erythropoietic properties, ie, the ability to control hematocrit levels, of a particular EPO compound can be determined by a variety of assays. In one embodiment, the TF1 cell line can be used to determine whether an EPO compound has erythropoietic activity. The cells can be pelleted, washed, and resuspended at a concentration of 10 ⁵ cells in 1 ml of culture medium supplemented with a certain concentration of recombinant EPO and the EPO compound of interest. Individual cultures can be maintained for 24 hours while the number of cells is determined using formazan reagent (CellTiter; Promega, Madison, WI).

The activity of an EPO compound of interest can first be assessed in vivo by observing its effect on hemoglobin concentration using female BALA/c mice. Animals were administered 500 U/kg-bw of EPO, the EPO compound of interest, or an equal volume of vehicle subcutaneously three times per week for a total of three weeks (interval sufficient to observe an erythropoietic response). An EPO compound is determined to have erythropoietic activity if it increases serum hemoglobin concentrations in mice.

Further assessment of activity can be obtained in vitro using TF1 erythroleukemia cells. If the relative number of TF1 cells exceeds the control group, then the EPO compound of interest has erythropoietic activity. In addition, those of ordinary skill in the art know that other assays for detecting erythropoietic activity can also be used. For example, the European Pharmacopoeia describes at least two methods for testing the erythropoietic activity of EPO compounds, including the hypoxic mouse test and the reticulocyte test.

Detection of tissue protection ability based on EPO receptor

In one embodiment, the detection of tissue protective ability according to the present invention is based on the tissue protective receptor of EPO. Once the sequence of the tissue protective receptor has been isolated, various assays can be used to determine the tissue protective capacity of a particular EPO compound. As is known to those of ordinary skill in the art, the type of assay used depends primarily on the weight of the EPO compound.

For example, the detection method can be a competitive assay or a sandwich assay or a steric hindrance assay. Competitive assays rely on the competitive binding of tracer analogs to a limited number of binding sites on the binding ligand for the assay sample analyte. Here, the term "analyte" refers to the EPO compound of interest whose tissue protective activity is to be detected. The term "binding ligand" refers to any protein that binds an analyte, in particular the EPO receptor. "Tracer" refers to a labeled reagent, eg, labeled analyte analog, immobilized analyte analog, labeled binding ligand, immobilized binding ligand, and steric conjugate. The tracer herein can have any detectable function that does not affect the binding of the analyte to its binding partner. Non-limiting examples include moieties that are directly detectable, such as fluorescent dyes, chemiluminescent agents, and radioactive labels, and moieties that must be reacted or derivatized to be detected, such as enzymes. Suitable tracers may be radioactive isotopes P ³² , C ¹⁴ , I ¹²⁵ , H ³ , I ¹³¹ , and mixtures thereof; fluorophores such as rare earth chelates, fluorescein, fluorescein derivatives, rhodamine, if Damamine derivatives, dansyl chloride, umbelbelliferase (firefly luciferase and bacterial luciferase (US Patent No. 4,737,456)), luciferin, 2,3-dihydrophthalazinediones, horseradish peroxide enzyme (HRP), alkaline phosphatase, β-galactosidase, glucoamylase, lysozyme, sugar oxidase (glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase), and utilization Oxidation of dye precursors by hydrogen peroxide such as HRP, lactoperoxidase, microperoxidase and mixtures thereof conjugated heterocyclic oxyalcohols (uricase and xanthine oxidase); biotin/avidin; Spin tags; phage tags; stable free radicals; and combinations thereof. In one embodiment, the tracer is at least one of horseradish peroxidase or alkaline phosphatase.

Methods for coupling tracers to proteins or polypeptides are known to those skilled in the art. For example, coupling agents such as dialdehyde, carbodiimide, bismaleimide, diimidazole, diazotized p-diaminobenzidine, etc. can be used to combine with the above-mentioned fluorescent agent, chemiluminescent agent , and enzymatic labels, some of which are described in US Pat. Nos. 3,940,475 and 3,645,090, which are hereby incorporated by reference in their entirety.

In sandwich assays, immobilization of the reactants is required, that is, the separation of the bound ligand from any analyte free in solution, which can be accomplished by precipitating the bound ligand or analyte analog prior to the detection step, as in Adsorption to water-insoluble matrices or surfaces by covalent coupling, such as glutaraldehyde cross-linking (US Pat. No. 3,720,760), or immobilization of ligands or analogs after the detection step, such as by immunoprecipitation.

Thus, the binding ligand can be insolubilized before or after the competition reaction, and tracer or analyte bound to the binding ligand is separated from unbound tracer and analyte. Separation can use decantation (where the bound ligand is precipitated beforehand) or centrifugation (where the bound ligand is precipitated after a competitive reaction). The amount of sample analyte is inversely proportional to the amount of bound tracer as determined by the amount of labeled substance. A dose-response curve of a known amount of analyte can be drawn and compared with test results to quantitatively determine the amount of analyte in the sample. When an enzyme is used as a tracer, the detection method is usually referred to as an ELISA system.

In a sequential sandwich assay, for example, the immobilized binding ligand is used to adsorb the test sample analyte, the test sample is washed away, the bound analyte is used to adsorb the labeled binding ligand, and the residual tracer is then combined with the binding agent. material separation. The amount of bound tracer is proportional to the analyte in the test sample. In a "simultaneous" sandwich assay, there is no need to separate the test sample prior to the addition of the labeled binding ligand.

Competitive and sandwich assays use a phase separation step as an essential part of the assay, but sterically hindered assays are performed in a single reaction mixture. Another competitive assay method, called "homologous" assay, does not require phase separation. In such detection methods, a conjugate of the enzyme and the analyte is prepared and used such that when the anti-analyte is attached to the analyte, the presence of the anti-analyte affects the activity of the enzyme. Bifunctional organic bridges are used to couple tissue protective receptors to enzymes such as peroxidase. Conjugates used with EPO are selected such that binding of EPO inhibits or enhances the activity of the marker enzyme. This type of detection method is often referred to as EMIT.

Steric conjugates are used in sterically hindered methods of homology detection. These conjugates can be synthesized by covalently linking a small molecular weight hapten to the small analyte, such that the anti-hapten antibody is substantially incapable of binding to the conjugate simultaneously with the anti-analyte. Analyte present in the test sample will bind the anti-analyte, allowing the anti-hapten to bind to the conjugate, resulting in a change in the properties of the conjugated hapten, e.g. a change in fluorescence when the hapten is a fluorophore .

Further information regarding the detection of tissue protective capabilities is described in co-pending U.S. Patent Application No. 10/188,905, filed July 3, 2002 and Application Serial No. 60/456,891, filed 2002 The patent application dated April 25 is hereby incorporated by reference in its entirety.

function detection

In the absence of the tissue protective receptor sequence, the tissue protective function of EPO can be determined by functional assays in vivo and in vitro. Preferably, one of ordinary skill in the art can determine the tissue protective activity of an EPO compound using either in vitro or in vivo assays alone, but in some cases both will be required to ensure that the compound exhibits the same tissue protective activity both in vivo and in vitro. protective activity.

In practice, those of ordinary skill in the art can use the combination of the detection methods disclosed in the present invention to determine whether the EPO analog with at least one increased N-linked sugar chain and/or at least one increased O-linked sugar chain is Has tissue protective activity. First, in vitro assays such as P19 cell and rat motor neuron assays can be used to determine whether the EPO of interest has tissue protective activity. Then, in vivo assays such as the rat focal ischemia model, the bicuculline convulsion model, or the spinal cord injury model can be used to validate the results of the in vitro assays.

In vitro models of the present invention include, but are not limited to, those models used to determine the absence of the above-mentioned EPO analog tissue protective activity: P19 cell assay, rat motor neuron assay, and cDNA microarray, which will be described later and in Examples 2 for a more detailed discussion. These examples are non-limiting, as those skilled in the art are aware of other suitable in vitro assay models for determining the tissue protective activity of EPO compounds. In general, an EPO compound will be considered tissue protective if the EPO compound maintains or enhances cell viability compared to a control. The erythropoietin will be considered antagonistic if it severely affects the viability of the cells in the assay compared to the control.

A. In Vitro Assay Based on P19 Cell Line

In one embodiment, the assay for in vitro tissue protective activity is based on the P19 cell line. For example, P19 cells can be maintained in DMEM supplemented with 2 mM L-glutathione, 100 U/ml penicillin G, 100 μg/ml streptomycin sulfate (Gibco) and 10% fetal bovine serum (Hyclone Laboratories). Not differentiated, which contains 1.2g/lNaHCO ₃ 10mM hepes buffer. In addition to replacing fetal bovine serum with 5 μg/ml insulin, 100 μg/ml transferrin, 20 nM progesterone, 100 μM putrescine, and 30 nM Na ₂ SeO ₃ (Sigma), the serum-free medium can contain The same composition as the above-mentioned culture solution.

Cells reacted with 50% confluency were treated overnight with recombinant EPO and/or EPO compound of interest, dissociated with trypsin, rinsed in serum-free medium and placed in 25 ^cm tissue culture In the flask, reach a final density of 10 ⁴ cells/cm ² in serum-free medium (or with pretreatment supplements). Cell viability was detected by trypan blue exclusion.

As is well known to those skilled in the art, after deprivation of serum in undifferentiated neuron-like P19 cells, addition of recombinant EPO can prevent cell death. For example, recombinant EPO rescued up to 50% of neuron-like cells from death if used at a concentration of 0.1 U/ml to 100 U/ml. Therefore, in order to determine that the EPO compound of interest has tissue protective activity, it must rescue more P19 cells from death than the control group, preferably about 25% to about 50% of the cells must be rescued, most preferably About 40% to about 50% cells.

B. In Vitro Rat Motor Neuron Analysis

In another embodiment, the rat motor neuron assay is used to detect the tissue protective activity of EPO compounds of interest in vitro. For example, primary motor neurons can be obtained from the spinal cord of 15 day old Sprauge Dawley rat embryos and purified using immunopanning. Cells are preferably seeded at low density (20,000 cells/cm ² ) on coverslips in 24 mm well plates pre-treated with poly-DL-ornithine (orinthine) and N-trimethyllysine inner salt. Coated, and containing complete medium (Neurobasal, B27 (2%), 0.SmM L-glutamine, 2% horse serum, 25 μM 2-mercaptoethanol, 25 μM glutamic acid, 1% penicillin and streptavidin element, 1 ng/ml of BDNF). After a period of time, preferably about 6 days, EPO (10 U/ml) and the EPO compound of interest (10 U/ml) or excipients can be added to the culture medium (preferably in the detection of viable neuronal cell density about 5 days ago). Then the culture medium was removed, the cells were mixed with PBS containing 4% paraformaldehyde for 40 minutes, permeabilized with 0.2% Triton X-100, blocked with PBS containing 10% calf serum, and anti-phosphorylated neurofilament ( SMI-32; 1:9000) was incubated overnight and developed by the avidin-biotin method using diaminobenzidine. The viability of motor neurons can be assessed morphologically by counting SMI-32 positive cells.

Mixed primary cultures of motor neurons characteristically undergo apoptosis under maintenance culture conditions. It has been shown that the addition of recombinant EPO (10 U/ml) to the culture medium 5 days before the detection of cell number significantly increased the number of primary motor neurons on day 5. Thus, to be considered to have tissue protective activity, the EPO compound of interest preferably rescues at least the same number of motor neurons as compared to the control group. In one embodiment, an EPO compound of interest is considered to have tissue protective activity if it rescues more motor neurons than a control under maintenance culture conditions.

C. cDNA Microarray-Based In Vitro Assays

Another method for testing the tissue protective activity of EPO compounds of interest is cDNA microarray. This assay can be used to determine whether EPO and an EPO compound of interest affect gene expression in P19 cells differently. mRNA isolated from undifferentiated P19 cells can display different patterns of gene regulation, as determined by mouse 1200cDNA microarrays, depending on the degree of exposure to EPOs. For example, the expression of 1,200 genes in P19 cells can be detected by using a nylon membrane array from Clontech (Atlasmouse 1.2). Cells ( ¹⁰⁷ /sample) can be treated overnight with saline, recombinant EPO, EPO compound of interest (1 mU/ml), or a mixture thereof. Cells were then lysed for RNA extraction or serum was removed for 3 hours (always present with the same cytokines added during pretreatment). After standard total RNA extraction by column chromatography, polyA+RNA can be purified using on-column DNase-treated columns. Probes were subsequently constructed in the presence of [ ^P32 ]-ATP. The labeled probes, preferably having 20 million counts or more, can hybridize to cDNA nylon membranes at 68°C. The film is developed and exposed to x-ray film. The intensity of the radioactive signal can be detected with a Phosphor Imager and analyzed with the Atlas Image 2.0 computer program (Clontech).

In vivo assays of the present invention include, but are not limited to, assays for evaluating tissue protective activity of EPO compounds, such as site-directed ischemia models and intra-hippoeampal bicuculline models. Additionally, in vivo models for evaluating tissue protective activity include spinal cord injury assays. Further, various detection methods disclosed in International Publication No. WO/02053580 and US Patent Publication Nos. 2002/0086816 and 2003/0072737 can be used in the present invention.

D. In Vivo Analysis Based on Ischemia Model

In one embodiment, the assay for detecting the tissue protective ability of a particular EPO compound is based on an ischemia model. For example, male Sprague-Dawley rats (-250 g) can be used in a three vessel ischemia model. Briefly, rats were anesthetized with pentobarbital (60 mg/kg-bw) and a core temperature of 37°C was maintained using a water bath. The right carotid artery was ligated with two sutures and severed. The laryngeal orifice near the right orbit allows visualization of the mid-cerebrovascular vessels, which allow anesthesia of the nasal vessel ends. To create a penumbra around the fixed MCA wound, the carotid artery on the other side was pulled with forceps to occlude it for 1 hr. At the onset of reversible carotid occlusion, saline, recombinant EPO (5000 U/kg-bw) or the EPO compound of interest (5000 U/kg-bw) can be administered.

After 24 hours, the brains were removed and continuous 1-mm thick sections were excised from the entire brain using a brain surgery device (Harvard Apparatus). Each fraction was then incubated in 154 mM NaCl containing 2% triphenyltetrazolium chloride for 30 minutes at 37°C. Injury volume can be determined using a computerized image analysis system (MCID, Imaging Research, St. Catharines, Ontario, Canada). In such an assay, an EPO compound of interest is considered to have neuroprotective activity if it improves infarct volume due to MCA ischemia in rats to the same or greater extent than recombinant EPO.

E. In vivo detection of a bicuculline-based convulsion model in the hippocampus

In another embodiment, the tissue protective ability of EPO compounds can be tested in vivo by the bicuculline assay in the hippocampus. For example, male Sprangue-Dawley rats (250–280 g) are housed under conditions of constant temperature (23°C) and relative humidity (60%), given adequate food and water, and provided with a fixed 12-hour day/night cycle . Rats were surgically implanted with cannulas and electrodes under stereotaxic guidance as described in Vezzani, A., et al., J. Neurosci, 19, 5054-65 (1999). Briefly, rats were anesthetized with Equithesin (1% pentobarbital/4% chloral hydrate; 3 ml/kg i.p.). Two helical electrodes are placed on either side of the parietal cortex and a ground wire is placed under the sinuses. Bipolar Nicadmium wire insulated electrodes (60 μm) can then be implanted on both sides of the gyri of the dorsal hippocampus (diaphragmatic electrodes), and a cannula (22-gauge) placed unilaterally on top of the dura can be implanted for Drugs were infused intrahippocampus and intracerebroventricularly. The positional coordinates of the implanted electrode based on the breech should be: (mm) anterior-posterior -3.5; both sides 2.4 and 3 subdural, using a pressure gauge set at -2.5. Paxinos, G. & Watston, C., The Rat Brain in Stereotaxic Coordinates, Academic Press, New York (1986). Electrodes can be attached to multi-hole sockets (March Electronics, NY) and, attached to injection containers, the skull is protected with acrylic dental adhesive.

The experiment is preferably performed when the animal is fully recovered 3 to 7 days after surgery. Animals were then intraperitoneally administered recombinant EPO or the EPO compound of interest (both 5000 U/kg-bw) or vehicle, once 24 hours before induction of bicuculline onset, and again 30 minutes before induction. The procedure for recording EEG and intracerebral drug injection has been described in Vezzani, A., et al., J. Pharmacol Exp Ther, 239, 256-63 (1986). Briefly, animals were housed in Plexiglass cages (25 x 25 x 60) for a minimum of 10 minutes before starting EEG recordings (4-channel EEG polygraph, model BP8, Battaglia Rangoni, Bologna, Italy). About 15 to about 30 minutes later, EEG recordings were performed for a duration of 120 minutes following infusion with 0.8 nmol/0.5 μl of bicuculline methodide. All injections were administered to non-anesthetized rats with a needle (28-gauge) extending 3 mm below the cannula.

Convulsions can be detected by EEG analysis, which has previously been shown to provide a sensitive measure of the anticonvulsant activity of drugs. Vezzani, A., et al., J. Pharmacol Exp Ther, 239, 256-63 (1986). For the purposes of this test, a tic consists of at least two of the following changes occurring simultaneously in all four leads of recordings: high frequency and/or multispike complex and/or high voltage simultaneous peaks (highvoltage synchronized spike) or wave type movement. Synchronous spikes mixed with twitches can be observed. The parameters chosen for quantification of convulsions are preferably the latency to the first convulsion (convulsion onset), the total elapsed time of the epileptic response (determined by adding together the duration of the ictal events; duration of convulsions), and the duration of seizures recorded on the EEG. peak response during (twitch response).

A model of intrahippocampal bicuculline twitches using EEG responses as numbers has been shown to be a sensitive and specific predictor of the drug's anti-convulsant ability. Vezzani, A., et al., J. Pharmacol Exp Ther, 239, 256-63 (1986). Thus, to be considered tissue protective, the long-acting EPO of interest should reduce the frequency and severity of convulsions to an equal or greater extent than recombinant EPO.

F. In vivo detection based on an acute reversible glaucoma rat model

In another in vivo assay of the present invention, the rat model of acute reversible glaucoma can be used to test the tissue protective ability of a specific EPO compound of interest. For example, because retinal cells are very sensitive to ischemia, many of these cells die after 30 minutes of ischemic stress. Therefore, to test whether a peripherally administered EPO compound of interest exhibits tissue protective activity sufficient to protect cells sensitive to ischemia, an acute, reversible glaucoma rat model can be used, as described in Rosenbaum et al., Vis. Res. 37: 3443-51, 1997. Specifically, saline can be injected into the preocular cavity of adult male rats, and the pressure can exceed the systemic arterial blood pressure and be maintained for 60 minutes. Rats were then administered saline and 5000 U EPO/kg body weight intraperitoneally 24 hours before ischemia was induced and continued as daily doses for an additional 3 days.

Dark-adapted rats can be examined 1 week after treatment by electroretinography to determine whether the EPO compound of interest has tissue protective activity. If the EPO has tissue protective activity, there should be good retention of activity in electroretinograms compared to saline treated rats.

G. Myocardial infarction detection

Myocardial infarction assays can also be used in the present invention to detect whether EPO compounds have tissue protective activity in general or in the heart in particular. For example, 5000 U/kg body weight of EPO can be administered to adult male rats 24 hours before the rats are anesthetized and ready for coronary artery ligation. An additional dose of EPO may be administered at the beginning of the procedure while the left main coronary artery is ligated for 30 minutes and then released. After the treatment, the same dose of EPO was administered every day for 1 week, and the cardiac function of the rats was detected at the same time. Rats that received a blank injection (saline) showed a large increase in left terminal diastolic pressure, predicting an enlarged, stiff heart, second only to myocardial infarction, whereas animals administered long-acting EPO, If the long-acting EPO has tissue protective activity, then the animal should show unimpaired cardiac function compared to the blank control group (significant difference at P (0.01 level).

H. Spinal Cord Injury Detection

Spinal cord injury assays can also be used in the present invention to evaluate the tissue protective activity of specific EPO compounds of interest. In particular, rat spinal cord compresses can be used in the present invention. Wistar rats (female) weighing from about 180 g to about 300 g are preferably used. Before the operation, the animals are preferably fasted for 12 hours before the operation, imprisoned in a humane manner, and anesthetized by intraperitoneal injection of pentobarbital sodium (40 mg/kg-bw). After skin infiltration (0.25% bupivacaine), a total monolevel (T-3) laminectomy was performed through a 2 cm incision with the aid of a dissecting microscope. Traumatic spinal cord injury was induced by epidural temporary aneurysm clips by applying a closure force of 0.6 Newton (65 g) to the spinal cord for 1 min. After the clips were removed, the skin incision was sutured, and the rat was fully recovered from anesthesia and returned to its cage. Rats were monitored using bladder palpation at least twice daily until spontaneous emptying resumed.

Animals in the control group were administered usual saline (by intravenous injection) immediately after the incision was sutured. The remaining animals were administered 16 mg/kg-bw of the EPO compound of interest intravenously. The motor neurological function of the rats was then evaluated using a locomotor rating scale. On this scale, animals are scored on a scale from 0 (no hindlimb movement observed) to 21 (normal gait). Rats were assessed for loss of function at 1 hour, 12 hours, 12 hours, 48 hours, 72 hours, and 1 week after injury by the same examiner blinded to the treatment the animals were given. If long-acting EPO has tissue protective activity, rats administered this EPO should heal from injury faster and better than saline-injected rats.

I. Detection of Rabbit Spinal Cord Ischemia

In another embodiment, a rabbit spinal cord ischemia assay is used to measure tissue protective capacity. For example, New Zealand white rabbits (36, 8-12 months old, male) weighing 1.5kg-2.5kg can be used in this assay. Animals were fasted for 12 hours and managed humanely. Anesthesia was induced by 3% bromochlorothane dispersed in 100% oxygen and maintained at 0.5-1.5% bromochloroethane dispersed in 50% oxygen and 50% air in the mixed gas. Insert an IV catheter (22-gauge) into the left ear vein. Ringers lactate solution was infused at a rate of 4 ml/kg body weight (bw) per hour during the procedure. Before the operation, Cefazoline (10ml/kg-bw) was injected intravenously to prevent infection. The animal was placed in the right recumbent position, the skin was treated with povidone-iodine, infiltrated with bupivacaine (0.25%), and a lateral skin incision was made at the 12th segment parallel to the spine. After incision of the skin and subcutaneous thoracolumbar fascia, the longissimus psoas and lumbar iliocostalis muscles were removed. The abdominal artery was exposed through a left retroperitoneal approach, moving a little below the left renal artery. A PE-60 tube was wrapped around the artery from the distal left renal artery, and both ends were threaded through a larger rubber tube. Abdominal arteries were atraumatically ligated for 20 minutes by pulling on the PE tubing.

Heparin (400 IU) was administered as an intravenous bolus at the abdominal artery ligation. After 20 minutes of ligation, the rubber tube and catheter were removed, the incision was sutured and the animal was monitored until healed, while the neurological function of the animal was tested sequentially. Immediately after the abdominal artery ligation was released, control animals were given intravenous injection of usual saline. Immediately after reperfusion, another group of animals was injected intravenously with 6.5 μg/kg-bw of the EPO compound of interest (n=6 for each group).

Motor function was evaluated according to the criteria of Drummond and Moore at 1 hour, 24 hours, and 48 hours after reperfusion by an examiner blinded to the treatment of the animals. Each animal was scored from 0 to 4 according to the following scale: 0 = paralysis of lower limbs, no obvious hindlimb movement; 1 = low hindlimb movement, only weak movement against gravity; 2 = moderate hindlimb movement And has strong resistance to gravity but no ability to extend legs from under body; 3 = good athletic ability with ability to extend legs and jump from under body, but not normal; 4 = normal athletic ability. Twice daily manual bladder emptying was performed in paralyzed animals.

If long-acting EPO has tissue protective activity, animals administered this EPO should heal from injury faster and better than saline-injected animals.

J. Other possible assays for detecting tissue protective effects of EPO and its analogs

As noted previously, several tissues possess EPO receptors and, thus, may respond to the tissue protective capabilities of EPO. Thus, depending on the intended clinical use of the EPO compound of interest, the skilled person will recognize that similar in vitro assays involving these additional responder cells, or in vivo assays involving the corresponding organs, may also be performed. For example, in vitro assays based on serum depletion can be performed using cardiac, retinal, and Leydig cells in a manner similar to that described above for P19 assays.

In vivo assays can also be directed to specific organs. For example, to evaluate the effect of EPO on retinal cells, one of ordinary skill in the art can perform the above-mentioned retinal ischemia assay. In addition, one of ordinary skill in the art can readily modify the myocardial infarction assay described above in order to evaluate the effect of EPO analogs on cardiomyocytes. Those of ordinary skill in the art can choose an appropriate detection method or model for evaluating whether a specific EPO has tissue protective activity on erythropoietic responsive cells, tissues or organs.

Example

The embodiments described below are only used to explain the preferred embodiments of the present invention, and should not be construed as limiting the present invention, and the scope of the present invention is defined by the appended claims.

Example 1: Chemically Modified EPO

A. Oxidation of sugar chains

The sugar chain portion of EPO can be converted into an acid using the procedure described below. EPO and enough sodium periodate to provide the oxidation required (the greater the amount of sodium periodate, the higher the degree of oxidation) can be placed in 100 mM sodium acetate buffer. The solution was then ice-bathed for 20 minutes and dialyzed against distilled water. The product was then removed from the dialysis tube and collected into a clean tube (Product I).

Quantitative Benedict's solution (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) can be dissolved in distilled water to a final volume of 1 Litre. Then add a few drops of methylene blue to the Quantitative BenedictSolution.

Product I can then be added to this quantitative Benedict solution until the color of the solution becomes clear, indicating that the solution is fully oxidized. The solution can then be desalted and concentrated using an Ultrafree Centrifugal Filter Unit. The sample (product II) can then be further dialyzed against distilled water.

B. Oxidation of the asialo form of EPO by glucose oxidase

50 to 500 μg of asialo-form EPO, 10 μl of 1 U/μl glucose oxidase, and 100 μl of 10 mM sodium phosphate buffer can be mixed in a 15 ml conical centrifuge tube (total volume 110 μl). The mixture can then be incubated at 37°C for 2 hours while being extensively dialyzed against distilled water. The product can then be removed from the dialysis tube and collected in a clean tube (Product III).

Quantitative Benedict's solution (above) can be dissolved in distilled water to a final volume of 1 liter. A few drops of methylene blue can then be added to the quantitative Benedict's solution.

Product III can then be added to this quantitative Benedict solution until the color of the solution becomes clear, indicating complete oxidation of the solution. The solution was then desalted and concentrated using a centrifugal filter unit (Ultrafree Centrifugal Filter Unit). The sample (product IV) can then be further extensively dialyzed against distilled water.

C. Sulfation of EPO

EPO can be dissolved in N,N-dimethylformamide (DMF-SA) at 4°C. Then N,N'-dicyclohexylcarbodiimide (DCC) dissolved in DMF was added, and the solution was shaken at 4°C for 4 hours. Crushed ice can be added and the pH adjusted to 7.5 with 10N NaOH. The volume of the solution can be adjusted and centrifuged at 1000 xg for 15 minutes in a type HN-S2 centrifuge tube (DAMONIEC, Needham Hts., Massachusetts). The supernatant can then be extensively dialyzed. For more descriptions of sulfation see S. Pongor et al., Preparation of High-Potency, Non-aggregating Insulins Using a Novel Sulfation Procedure, Diabetes, Vol.

D. Connect PEG chain to EPO

EPO can be modified by attaching PEG chains to oxidized sugar chains, as obtained in A above (product I). The degree of modification can be controlled by adjusting the concentration of periodate in the oxidation.

When preparing PEG-EPO coupling products, EPO (2-4 mg/ml in 50 mM sodium acetate) can be oxidized first with 1 mM-10 mM sodium m-periodate (Sigma) at room temperature for 30 minutes. The phosphate buffer was then removed by buffer exchange in 100 mM sodium acetate, pH 5.4.

Methoxy-PEG-hydrazide (Nektar Therapeutics) of various molecular weights can then be added in 5- to 100-fold molar excess (polymer:protein). The intermediate hydrazide linkage can then be reduced by adding 15 mM sodium cyanoborohydride (Sigma) and reacting overnight at 4°C. The conjugate obtained can then be fractionated/purified using techniques well known in the art.

E. Linking PEG Chains to Asialo-EPO

The asialo form of EPO can be modified by attaching a PEG chain to a newly generated terminal galactose residue after oxidation with galactose oxidase, such as those obtained in B above (product III).

Recombinant human EPO (rhuEPO) can be desialylated with sialidase (Prozyme, Inc) according to the manufacturer's manual. Chemical modification is preferably determined by subjecting the reaction products to SDS polyacrylamide gel electrophoresis. The stained product bands should show that the modified EPO has an apparent molecular weight of about 31 kDa, while the unmodified EPO has a molecular weight of about 34 kDa. The sialic acid retained in EPO is preferably less than 0.1 mol/mol EPO.

After obtaining the asialo form of EPO, the galactose residues of newly exposed EPO (2–4 mg/ml in 10 mM sodium phosphate buffer) can be treated with 100 units of galactose oxidase (Sigma) per ml of EPO solution. (dissolved in PBS) for oxidation. The reaction mixture was then incubated at 37°C for 2 hours.

Phosphate buffer was then removed by buffer exchange in 100 mM sodium acetate, pH 5.4. Methylated-PEG-hydrazide (Nektar Therapeutics) of various molecular weights was then added in 5- to 100-fold molar excess (polymer:protein). The intermediate hydrazide linkage is then preferably further reduced by adding 15 mM sodium cyanoborohydride (Sigma) overnight at 4°C. The resulting conjugate can then be fractionated/purified using techniques well known in the art.

F. Linking PEG chains to asialo-EPO

The asialo form of EPO can be modified by attaching a PEG chain to a newly generated terminal galactose residue after oxidation with galactose oxidase, such as those obtained in B above (product III).

RhuEPO (1 mg) can be treated with sialidase (Seikagaku Corporation of Japan, 1 U of lyophilized powder dissolved in 100 μL of 75 mM _NaPO4 (pH 6.5)) at a ratio of 1 mgEPO to 0.05 units of sialidase (5 μL) Desialylation. Then 5 units (5 μL) of galactose oxidase (450 μL dissolved in 75 mM NaPO ₄ (pH 6.5) (Sigma)) were added to the mixture.

The phosphate buffer was then removed by buffer exchange in 100 mM sodium acetate pH 5.4. Then PEG-NH ₂ (750 molecular weight, Nektar Therapeutics) and 15 mM sodium cyanoborohydride (Sigma) were added to the mixture and reacted overnight at 4°C. Preferably, PEG-NH ₂ is added in a 250-fold molar excess (polymer:protein) (80 mg of PEG-NH ₂ ). The resulting conjugate is then fractionated/purified using techniques well known in the art.

Example 2. Function detection

A. Erythropoiesis Stimulation Assay

The erythropoiesis-stimulating ability, ie, the ability to regulate hematocrit levels, of a particular EPO compound is determined by the following assay.

TF1 is a human erythroid leukemia cell that is completely dependent on growth factors, including EPO. Kitamura, et al., Blood 73, 375-80. The TF1 cell line was obtained from ACTT and treated with 2 mM L-glutamine, 10 mM Hepes, 1 mM sodium pyruvate, 4.5 g/L glucose, 1.5 g/L sodium bicarbonate, 5 ng/ml GM-CSF, and 10% fetal calf Serum RPMI1640 has been preserved until the time of experiment. TF1 cells obtained from the active growth phase were pelleted, washed three times with culture medium alone, and resuspended at a concentration of ¹⁰⁵ cells in 1 ml of culture medium, with or without GM-CSF, and supplemented with specific concentrations of EPO or an EPO analogue having at least one added N-linked sugar chain and/or at least one added O-linked sugar chain. Each culture was maintained for 24 hours and the number of cells was determined using formazan reagent (CellTiter; Promega, WI) according to the manufacturer's manual.

Furthermore, the activity of a chemically modified EPO compound was first evaluated in vivo by observing its effect on hemoglobin concentration in female BALB/c mice. Animals were administered subcutaneously with 500 U/kg-bw of EPO, the EPO compound of interest, or an equal volume of vehicle 3 times a week for a total of 3 weeks (an interval sufficient to observe an erythropoietic response). An EPO compound is considered to have erythropoietic activity if it increases serum hemoglobin concentrations in mice. Further activity assays were obtained in vitro using TF1 erythroid leukemia cells. The study confirmed that EPO was considered to have erythropoietic activity if the increase in relative TF1 cell numbers exceeded that of the control group.

Those of ordinary skill in the art know that these and other assays, such as the hypoxic mouse assay and the reticulocyte assay (Ph.Eur.), are also suitable for use in the present invention to determine the erythropoiesis of long-acting tissue protective cytokines active.

B. Tissue Protective Activity Assay

The tissue protective function of the EPO analogs having at least one increased N-linked sugar chain and/or at least one added O-linked sugar chain was examined by the following method.

Neuronal cultures were established from the hippocampus of 18-day-old rat embryos. The brain was removed and dissected from the meninges, and the hippocampus was isolated. Cells were then dispersed in 2.5% trypsin solution, incubated at 37°C for 5 min, and then titrated. The cell suspension was diluted with serum-free neurobasal medium containing 1% B-27 supplement (Gibco, Rockville, MD, USA), and placed on a cover slip at a density of 80,000 cells per cover slip. Cover the coverslip with polyguanylic acid. Cells were then pretreated with EPO overnight and then exposed to 5 μM TMT for 24 hours with or without 1) EPO, 2) having at least one increased N-linked glycan and/or at least one increased O-linked glycan , or 3) the asialo form of EPO. Cultures of between 10 and 14 days were then used in vitro.

Cell viability can be determined by 3-(4,5-dimethyl-thiazol-2-yl)-2,5-biphenyltetrazolium bromide (MTT) assay. Denziot, E., and Lang, R. 1986. Rapid Colormetric Assay for Cell Growth and Survial. Modifications to thetetrazolium dye procedure giving improved reliability. J Immunol Methods 89:271-277. Briefly, MTT tetrazolium salt was dissolved in serum-free medium to a final concentration of 0.75 mg/ml and added to cells at the end of treatment for 3 hours at 37°C. The broth was then removed and the formazan was extracted with 1N HCl:isopropanol (1:24). Absorbance at 560 nm was read on a microplate reader.

As described in Figures 1 and 2, EPO analogs having at least one added N-linked sugar chain and/or at least one added O-linked sugar chain did not have a tissue protective function.

The scope of the invention described and claimed herein is not to be limited by the specific embodiments disclosed herein, since these specific embodiments are merely illustrative of several aspects of the invention. Any equivalent embodiments are also intended to be within the scope of this invention. Indeed, various modifications of the invention in addition to those described herein will become apparent to those of ordinary skill in the art from reading the foregoing description. Such modifications also fall within the scope of the appended claims. All documents cited herein are incorporated by reference in their entirety for all purposes.

Claims (27)

1. A method for regulating human hematocrit levels, comprising the steps of: providing an erythropoietin product that has a longer serum half-life than rhuEPO and that includes tissue protection; and A therapeutically effective amount of the erythropoietin product is administered. 2. The method according to claim 1, said step of providing erythropoietin products further comprising the steps of: At least one N-linked oligosaccharide chain or O-linked oligosaccharide chain of rhuEPO is modified with at least one chemical modification, wherein said chemical modification comprises oxidation, sulfation, phosphorylation, PEGylation, or combination. 3. The method of claim 1, wherein said step of administering a therapeutically effective amount of the erythropoietin product comprises administering the erythropoietin product in a lower molar amount than rhuEPO to achieve a comparable target hematocrit. 4. The method of claim 1, wherein the serum half-life is at least about 20% greater than the serum half-life of rhuEPO. 5. The method of claim 4, wherein the serum half-life is at least about 40% longer than the serum half-life of rhuEPO. 6. Artificial erythropoietin products, including: At least one erythropoietin derivative, wherein at least one N-linked oligosaccharide chain or at least one O-linked oligosaccharide chain has at least one chemical modification derived from oxidation, sulfation, phosphorylation , PEGylated, or a mixture thereof, and wherein the erythropoietin product has a longer serum half-life than rhnEPO. 7. The erythropoietin product according to claim 6, wherein said erythropoietin product has a tissue protective function. 8. The erythropoietin product of claim 6, said at least one chemical modification comprising oxidation of at least one N-linked oligosaccharide chain or at least one O-linked oligosaccharide chain to provide at least one increased acid residues. 9. The erythropoietin product of claim 6, said at least one chemical modification comprising sulfation of at least one N-linked oligosaccharide chain or at least one O-linked oligosaccharide chain to stimulate erythropoietin in the erythrocyte An increased negative charge is provided on the progenitin product. 10. The erythropoietin product of claim 6, said at least one chemical modification comprising phosphorylation of at least one N-linked oligosaccharide chain or at least one O-linked oligosaccharide chain to stimulate erythropoietin in the erythrocyte An increased negative charge is provided on the progenitin product. 11. The erythropoietin product of claim 6, said at least one chemical modification comprising adding at least one polyethylene glycol to at least one N-linked oligosaccharide chain or at least one O-linked oligosaccharide chain alcohol chain. 12. A method for preparing an erythropoietin product with extended serum half-life and tissue protective activity, comprising the steps of: providing at least one erythropoietin or erythropoietin derivative; and On at least one N-linked oligosaccharide chain or at least one O-linked oligosaccharide chain of at least one endogenous or recombinant erythropoietin by oxidation, sulfation, phosphorylation, PEGylation, or a combination thereof grooming. 13. The method of claim 12, said modifying step further comprising substituting at least one vicinal hydroxyl group on at least one N-linked oligosaccharide chain or at least one O-linked oligosaccharide chain with at least one acid residue A step of. 14. The method of claim 13, said step of substituting at least one vicinal hydroxyl group on at least one N-linked oligosaccharide chain or at least one O-linked oligosaccharide chain with at least one acid residue further Including: replacing multiple ortho hydroxyl groups on at least one N-linked oligosaccharide chain or at least one O-linked oligosaccharide chain with multiple acid residues. 15. The method of claim 12, said modifying step further comprising the steps of: providing an organic solvent; dissolving the erythropoietin or erythropoietin derivative in the organic solvent to form a solution; providing at least one condensing agent; providing at least one sulfate donor; and The at least one condensing agent and at least one sulfate donor are mixed into the solution. 16. The method of claim 12, wherein said modifying step further comprises the steps of: providing an organic solvent; dissolving the erythropoietin or erythropoietin derivative in the organic solvent to form a solution; providing at least one condensing agent; provide phosphoric acid; and The at least one condensing agent and at least one phosphoric acid are mixed into the above solution. 17. The method of claim 12, said modifying step further comprising the steps of: providing an organic solvent; dissolving erythropoietin or erythropoietin derivatives in the organic solvent to form a first solution; providing at least one oxidizing agent; adding the at least one oxidizing agent to the first solution to form a second solution; providing at least one polyethylene glycol chain; and mixing said at least one polyethylene glycol chain into a second solution. 18. The method of claim 17, said step of providing at least one polyethylene glycol chain comprising providing at least one polyethylene glycol chain having at least one primary amino group at the end of the chain. 19. A method of treating anemia in a patient at risk of tissue damage comprising the steps of: Providing erythropoietin products having at least one chemical modification on at least one N-linked oligosaccharide chain or O-linked oligosaccharide chain, wherein said chemical modification comprises oxidation, sulfation, phosphorylation, PEGylation , or a combination thereof; administering a therapeutically effective amount of said erythropoietin product, wherein said erythropoietin product is administered in a lower molar amount than rhuEPO to achieve a comparable target hematocrit, The erythropoietin product described therein has the function of protecting cells. 20. The method of claim 19, said erythropoietin product having a longer serum half-life than rhuEPO. 21. The method of claim 20, wherein the serum half-life is at least about 20% greater than the serum half-life of rhuEPO. 22. The method of claim 21, wherein the serum half-life is at least about 40% greater than the serum half-life of rhuEPO. 23. A pharmaceutical composition comprising: A therapeutically effective amount of at least one erythropoietin derivative having at least one chemical modification on at least one N-linked oligosaccharide chain or at least one O-linked oligosaccharide chain derived from oxidation , sulfation, phosphorylation, PEGylation, or a combination thereof, The at least one erythropoietin derivative has a longer serum half-life than recombinant erythropoietin and has tissue protection function. 24. The pharmaceutical composition of claim 23, further comprising at least one pharmaceutically acceptable carrier. 25. The pharmaceutical composition of claim 24, wherein said at least one pharmaceutically acceptable carrier comprises at least one diluent, adjuvant, excipient, carrier, or a mixture thereof. 26. The pharmaceutical composition of claim 23, further comprising at least one wetting agent, emulsifying agent, pH buffering agent, or a combination thereof. 27. The pharmaceutical composition of claim 23, further comprising at least one tissue protective cytokine.