MXPA05002617A - Long acting erythropoietins that maintain tissue protective activity of endogenous erythropoietin. - Google Patents

Long acting erythropoietins that maintain tissue protective activity of endogenous erythropoietin.

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Publication number
MXPA05002617A
MXPA05002617A MXPA05002617A MXPA05002617A MXPA05002617A MX PA05002617 A MXPA05002617 A MX PA05002617A MX PA05002617 A MXPA05002617 A MX PA05002617A MX PA05002617 A MXPA05002617 A MX PA05002617A MX PA05002617 A MXPA05002617 A MX PA05002617A
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Mexico
Prior art keywords
epo
erythropoietin
linked
oligosaccharide chain
product
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MXPA05002617A
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Spanish (es)
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John Smart
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Warren Pharmaceuticals Inc
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Publication of MXPA05002617A publication Critical patent/MXPA05002617A/en

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Abstract

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

Description

PROLONGED ACTION ERYTHROPOYETINES THAT KEEP THE PROTECTIVE ACTIVITY OF ERYTHROPOYETINE TISSUES ENDOGENOUS REFERENCE TO RELATED APPLICATIONS This application claims the benefit of the provisional application ÜS No. 60 / 409,020, filed on September 9, 2002, which is incorporated in its entirety as a reference here.
FIELD OF THE INVENTION The present invention relates to long-acting erythropoietins which advantageously maintain the protective capabilities of the tissue after modification. In particular, the present invention relates to long-acting erythropoietins that are chemically modified in such a way that it increases the serum half-life, but also maintains the protective function of the natural protein tissue in vivo. The present invention also relates to the treatment of anemia, and diseases related to anemia, with the long-acting erythropoietins of the present invention. Finally, the present invention is directed to the assays useful for the determination of the protective capacities of the tea which has an erythropoietin.
BACKGROUND OF THE INVENTION Natural or endogenous erythropoietin (EPO) is a glycoprotein hormone produced mainly in the liver. In endogenous EPO it consists of 165 amino acids and has a molecular weight (in humans) of around 30,000 to about 34,000 daltons. The glycosyl residues in the EPO, which consist of three oligosaccha chains linked to N and one linked to 0, are responsible for approximately 40% of the total weight of the proteins. The N-linked oligosaccha chains bind to the asparagine amide nitrogens at positions 24, 38 and 83, while the oligosaccha chain linked to 0 binds to the oxygen in the salt residue located at position 126. The EPO protein can be found in three ways: a, ß and so on. The forms and ß have the same potency, biological activity and molecular weight, but differ slightly in the carbohydrate components, while the form thus is an α or β form in which the terminal sialic acid (carbohydrate) has been eliminated.
So far, the main function of endogenous EPO is to act in concert with other growth factors to stimulate the proliferation and maturation of erythroid precursor cells in the bone marrow, respondents, and maintain an individual's hematocrit (the percentage of blood). complete that contains red blood cells). The process of producing red blood cells is called erythropoiesis, which is a precisely regulated physiological mechanism that optimizes the number of red blood cells for proper oxygenation of tissues without impeding circulation, for example, when oxygen transport is reduced By red blood cells, EPO increases the production of red blood cells by stimulating the conversion of bone marrow precursor cells into mature red blood cells, which then are released into the circulation. When the number of red blood cells in the circulation exceeds those necessary for normal tissue circulation, EPO is reduced in the circulation, so when the body is in a healthy state, EPO is present in very low concentrations in the blood. plasma, which is enough to stimulate the replacement of red blood cells that are normally lost by aging. Plasma EPO levels normally range from 0.01 units / mL to 0.03 units / mL.
Since the kidney produces most of the EPO for an individual, the loss of renal function, such as chronic kidney failure (CRF), leads to impaired production of EPO and often leads to anemia. Similarly, anemia can result from other chronic conditions such as cancer or treatments associated with these diseases, such as chemotherapy. Thus, the administration of recombinant EPO (which is described in more detail below), which has considerably the same biological effects as endogenous EPO, has been shown to be useful in reestablishing hematocrit levels in individuals with red blood cells. diminished.In addition to the function of recombinant EPO to maintain hematocrit levels for chronic conditions, recombinant EPO has been used to boost red blood cell levels before elective or scheduled surgeries, thereby reducing or eliminating the need for transfusion blood For example, recombinant EPO can be administered to solve aspects about the patient receiving a virus or pathogen from the blood supply or to solve the religious limitations regarding blood transfusions.
Moreover, some evidence recently suggests that EPO, as a member of the cytokine superfamily, has other important therapeutic attributes, which are mediated by the interaction with the EPO receptor (EPO-). For example, EPO and its receptor can play an important role in attenuating tissue injury because the interaction between EPO and the receptor provides compensatory responses that serve to improve the hypoxic cellular microenvironment, as well as modulate programmed cell death caused by metabolic stress. . In fact, patients with chronic renal failure and / or cancer have usually experienced a feeling of improved well-being and improved mental acuity after treatment with EPO, an effect that was previously attributed to the patient's increased ectocritus. However, in recent times, these improvements have been attributed to the protective and tissue enhancing effects of EPO, as described in International Publication No. WO / 0253580 and US Patent Publications Nos. 2002/0086816 and 2003 / 0072737.
Recent studies have also suggested that EPO administered systemically can cross the intact blood-brain barrier because the capillaries that form the blood-brain barrier also express the receptor for EPO. As such, an anatomical basis for transitosis mediated by the receptor is effected from the peripheral circulation to the brain.
Recombinant EPO (epoetin alfa), which is currently commercially available under the trademarks PROCRIT® (from Ortho Biotech Inc., Raritan, NJ) and EPOGEN® (from Amgen, Inc., Thousand Oaks, CA), has been used to treat anemia resulting from end-stage renal disease, to treat patients infected with HIV when used together with AZT therapy (zidovudine), and to counteract the effects of chemotherapy. Although the therapeutic effects of recombinant EPO are numerous, to date the main application of recombinant EPO has been to solve chronic anemia. In this regard, recombinant EPO is usually administered at an initial dose of 50-150 units / kg three times per week for approximately 6 to 8 weeks by an intravenous or subcutaneous injection to restore the suggested range of the hematocrit in the patient . After the patient reaches a desired hematocrit level, such as an amount that falls within from about 30% to about 35%, this level can be maintained by maintenance EPO therapy in the absence of iron deficiency and concomitant diseases.
Although the dose requirement may vary according to the patient's individual needs, common maintenance doses may be administered approximately 3 times a week (less if larger doses are provided).
The amount of the dosage and the frequency of the administration of the recombinant EPO is determined in part by the half-life of the molecule, which can be limited when the molecule is in vivo. For example, EPOGEN® administered intravenously is reportedly eliminated at a rate consistent with first-order kinetics with a circulating half-life ranging from approximately 4 to 13 hours in adults and pediatric patients with CRF. Thus, for it to have a therapeutic effect, the amount of dose and the frequency of the dose should be designed to take into account the relatively short life span of recombinant EPO.
In addition, because recombinant EPO is administered by an intravenous or subcutaneous injection, a nurse or physician is often asked to administer recombinant EPO to a patient. This presents an additional inconvenience for a patient, and is still another reason because it may be desirable to prolong the half-life of the molecule. As such, efforts to increase the half-life of recombinant EPO have gained the attention of research in the past decade based on the assumption that a prolonged half-life would decrease dose requirements while providing the same or better benefits therapeutic In fact, recent experiments in human EPO showed that there is a direct relationship between the content of carbohydrate containing sialic acid from EPO, its circulating half-life and bioactivity in vivo. As described in PCT Publication No. WO 95/05465, residues of sialic acid crown the ends of the sugar chains and prevent the liver from detecting galactose. The N-linked oligosaccharide chains usually have up to 4 sialic acids per chain, and the O-linked oligosaccharide chains have up to 2 sialic acids per chain. Thus, an unmodified EPO polypeptide can accommodate up to a total of 14 sialic acids.
Over time, these sialic acid residues can dissociate from the protein, thus exposing the galactose chains for detection in the liver. Once the liver detects the galactose chains, the protein is filtered from the blood. As such, a progressive increase in the content of sialic acid per molecule of EPO is considered the best defense of galactose chains to provide a corresponding progressive increase in biological activity (measured by the ability of equimolar concentrations of erythropoietin isoforms) isolated to raise the hematocrit of normal mice). Since unmodified EPO contains only 14 sialic acid sites, this approach may have limited ability to extend the half-life of EPO. This leads to the hypothesis that an EPO analogue engineered to contain additional oligosaccharide chains would improve biological activity. By providing these additional glycosylation sites, additional oligosaccharide chains having terminal ends can then be modified with sialic acid residues. See PCT Publication No. WO 91/05867, WO 94/09257, and WO 01/81405.
For example, a modified EPO analog may have at least one additional N-linked carbohydrate chain and / or at least one additional 0-linked carbohydrate chain. Specifically, WO 01/81405 discloses the addition of N-linked carbohydrate chains to the molecule at amino acids 30, 51, 57, 69, 88, 89 135 and / or 138. Modified EPO molecules can have anywhere from 1 to 4 additional glycosylation sites, which allows the addition of 2 to 16 sialic acid residues to the molecule.
However, although efforts to increase the serum half-life of EPO have shown very good results and are useful for maintaining hematocrit levels, no attention has been paid to the effect that these additional glycosylation sites may have on other functions. of the EPO.
Thus, it would be beneficial to provide a modified EPO with a prolonged serum half-life (prolonged action) that maintains the functionality of the endogenous EPO. In particular, there is a need in the art for a long-acting EPO compound with erythropoietic functionality and tissue protective functionality for use in pharmaceutical compositions for treating individuals with anemia and / or related diseases. In addition, tests to determine if a specific EPO is antagonistic to the tissue protective capabilities of endogenous EPO would be desirable.
BRIEF COMPENDI OF THE INVENTION The present invention is directed to a method for regulating the level of hematocrit in humans, which includes the steps of providing an erythropoietin product with a longer half-life in serum than recombinant human erythropoietin (rhuEPO) and which includes the protective functionality of tissue and the administration of an effective therapeutic amount of the erythropoietin product. In one embodiment, the step of providing an erythropoietin product further includes the step of modifying the recombinant erythropoietin with at least one chemical modification to at least one of the oligosaccharide chains linked to N or the oligosaccharide chain linked to 0, wherein the chemical modification it consists of oxidation, sulphation phosphorylation, PEGylation or a combination of these.
In addition, the step of administering an effective therapeutic amount of the erythropoietin product may include administration of the erythropoietin product in a molar amount less than rhuEPO to obtain a comparable chosen hematocrit.
In one embodiment, the serum half-life is at least approximately 20% longer than the serum half-life of rhuEPO. In another embodiment, the serum half-life is at least about 40% longer than the serum half-life of rhuEPO.
The present invention is also directed to a synthetic erythropoietin product that includes 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 as a result of the oxidation, sulfation, phosphorylation PEGylation or mixtures thereof, and wherein the erythropoietin product has a longer serum half-life than rhuEPO. The erythropoietin product preferably has tissue protective functionality.
In one embodiment, the at least one chemical modification includes the oxidation of at least one N-linked oligosaccharide chain or at least one O-linked oligosaccharide chain, to provide at least one additional acid residue. For example, the at least one chemical modification may consist of sulfation of at least one N-linked oligosaccharide chain or at least one O-linked oligosaccharide chain to obtain an increased negative charge in the EPO product. In another embodiment, the at least one chemical modification consists of the phosphorylation of at least one oligosaccharide chain linked to N or at least one oligosaccharide chain linked to O to obtain an increased negative charge on the EPO product. In yet another embodiment, the at least one chemical modification consists in the addition of at least one polyethylene glycol chain to at least one N-linked oligosaccharide chain or at least one oligosaccharide chain linked to 0.
The present invention also relates to a method for preparing an erythropoietin product having a prolonged serum half-life and the protective activity of the tissue, the method includes the steps of: disposing of at least one erythropoietin or erythropoietin derivative; and modifying at least one oligosaccharide chain linked to N or at least one oligosaccharide chain linked to 0 in at least one endogenous or recombinant erythropoietin by oxidation, sulfation, phosphorylation, PEGylation, or a combination thereof.
The step of modifying further may consist in the step of substituting at least one nearby hydroxyl on at least one N-linked oligosaccharide chain or at least one oligosaccharide chain linked to 0 with at least one acid residue. In one embodiment, the step of substituting at least one near hydroxyl in at least one N-linked oligosaccharide chain or at least one O-linked oligosaccharide chain with at least one acid residue further includes replacing a plurality of hydroxyls nearby in at least one chain. oligosaccharide linked to N or at least one oligosaccharide chain linked to 0 with a plurality of acid residues.
In another embodiment, the step of modifying further, consists of the steps of: disposing of an organic solvent; dissolving the erythropoietin or erythropoietin derivative in the organic solvent to form a solution; having at least one condensing agent; provide at least one sulfate donor; and mixing the at least one condensing agent and the at least one sulfate donor in the solution. In still another embodiment, the step of modifying further includes the steps of: disposing of an organic solvent; dissolving the erythropoietin or erythropoietin derivative in the organic solvent to form a solution, having at least one condensing agent; provide phosphoric acid; and mixing the at least one condensing agent and the at least one phosphoric acid in the solution.
In still another embodiment, the step of modifying further includes the steps of: disposing of an organic solvent, dissolving the erythropoietin or the erythropoietin derivative in the organic solvent to form a first solution; provide at least one oxidizing agent; adding the at least one oxidizing agent to the first solution to form a second solution; having at least one polyethylene glycol chain; and mixing at least one polyethylene glycol chain in the second solution, the step of having at least one polyethylene glycol chain may include providing at least one polyethylene glycol chain with at least one primary amino moiety at one end of the chain.
The present invention also relates to a method for treating anemia in patients at risk of tissue damage, which includes the steps of: providing an erythropoietin product with at least one chemical modification to the N-linked oligosaccharide chains or the oligosaccharide chain linked to Or, wherein the chemical modification consists of sulfation oxidation, phosphorylation, PEGylation or a combination thereof, administering an effective therapeutic amount of the erythropoietin product, wherein the erythropoietin product is administered in a molar amount less than rhuEPO to obtain a chosen hematocrit comparable, where the erythropoietin product has tissue protective functionality.
In this aspect of the invention, the erythropoietin product preferably has a longer serum half-life than rhuEPO. In one embodiment, the serum half-life is at least about 20% longer than the serum half-life than rhuEPO. In another embodiment, the serum half-life is at least about 40% longer than the serum half-life of rhuEPO.
The present invention further relates to a pharmaceutical composition containing: an effective therapeutic amount of at least one erythropoietin derivative, wherein at least one oligosaccharide chain linked to N or at least one oligosaccharide chain linked to 0 has at least one chemical modification as a result of oxidation, sulfation, PEGylation phosphorylation, or mixtures thereof, wherein at least one erythropoietin derivative has a longer half-life in serum than recombinant erythropoietin, and has the protective functionality of the tissue. In one embodiment, the pharmaceutical composition further contains at least one carrier accepted for pharmaceutical use. The at least one carrier accepted for pharmaceutical use can consist of at least one diluent, adjuvant, excipient, vehicle or mixtures thereof.
In another embodiment, the pharmaceutical composition further contains at least one wetting agent, emulsifying agent, pH buffer, or a combination thereof. In still another embodiment, the pharmaceutical composition further contains at least one tissue protective cytosine.
BRIEF DESCRIPTION OF THE FIGURES Other features and advantages of the invention can be ascertained from the following detailed description that is provided in connection with the drawings described below: Figure la is a comparison of the efficacy of different forms of EPO in the protection against cell death activated by exposure to trimethyltin; Y Figure Ib is a comparison of the efficacy of different forms of EPO in the protection against cell death activated by exposure to trimethyltin.
DETAILED DESCRIPTION OF THE INVENTION The present invention is directed to the use of EPO molecules having a prolonged serum half-life (prolonged action) that is chemically modified with carbohydrate chains so as to maintain the functionality of the endogenous EPO. As described in the background, actions to prolong the half-life of EPO have usually focused on adding more carbohydrate chains to the EPO molecule to protect the galactose chains against exposure. However, it is considered that the added carbohydrate chains effect the functionality of the EPO analog such that, for example, the functionality is compromised to obtain the longer half-life. Although EPO analogs with longer half-lives than recombinant EPO having erythropoietic activity are known, these analogs do not retain other newly discovered therapeutic benefits of EPO, the protective activity of the tissue.
For example, it has been shown that a 17 amino acid fragment of EPO corresponding to amino acids 30-47, also known as the O'Brien peptide, has tissue protective activity in vitro, but has no erythropoietic activity in vitro. Bell, W. M. Misasi, R S O'Brien, J. S., Int. J. Mol. Med. 1, 235-41 (1998). Therefore, it is believed that a modified EPO molecule having additional glycosylation sites within the O'Brien peptide may not have tissue protective activity in other in vitro assays. In addition, because the three-dimensional orientation of the EPO molecule is important for functionality, the addition of glycosylation sites to the molecule may interfere with the overall functionality.
Thus, the present invention relates to a long-acting EPO with at least one erythropoietic activity, the protective activity of the tissue, transitosis capacity, or a combination of these. Preferably, the long-acting EPO of the present invention has erythropoietic activity and at least tissue protective activity or transitosis capacity.
In one embodiment, the long-acting EPO of the present invention has a serum half-life that is at least about 20% longer than the serum half-life of the recombinant EPO. In another embodiment, the serum half-life of the long-acting EPO of the present invention is at least about 30% longer than the half-life of the recombinant EPO. In yet another embodiment, the long-acting EPO of the present invention has a serum half-life that is at least about 40% longer than the serum half-life of the recombinant EPO.
In summary, the long-acting EPOs of the present invention consist of EPO molecules with carbohydrate chains that are altered with at least one modification as compared to natural (endogenous) EPO, preferably compared to natural human EPO. In one embodiment, the long-acting EPOs of the present invention undergo a plurality of modifications in the carbohydrate chains.
In another embodiment, the proximal hydroxyls in the carbohydrate chain of a natural EPO are oxidized into residues to produce the long-acting EPO molecules of the present invention. In another embodiment, the sialic acid residues in the EPO are replaced with less labile acid residues. In yet another embodiment, sulfation and / or phosphorylation of the carbohydrate chain in an EPO leads to a long-acting EPO according to the present invention. In yet another embodiment, the long-acting EPO of the present invention results from the addition of polyethylene glycol to the carbohydrate chain of EPO. Any combination of the aforementioned modifications is also contemplated in the present invention. As already mentioned, the present invention also encompasses compositions, including pharmaceutical compositions, which contain one or more of the above-mentioned long-acting EPO molecules.
The long-acting EPO molecules of the present invention are contemplated for inclusion in pharmaceutical compositions for treating anemia and related diseases, especially those with complications resulting from diseases such as, but not limited to, acute renal failure, sepsis, HIV , chemotherapy and the like.
The present invention is also directed to methods for treating anemia and related diseases, as well as kits that are used for the treatment procedure. When used herein, the term "treatment" refers to therapeutic treatment and prophylactic or preventive measures, wherein the goal is to avoid or slow down (reduce) the target pathological condition or disorder. People in need of treatment include those who already have the disorder as well as those who are likely to have the disorder or those in whom the disorder can be avoided. The present invention contemplates the use of long-acting EPO for chronic administration, acute treatment and / or intermittent administration. For the purpose of this description, "chronic administration" refers to the administration of the agent (s) in a continuous mode, as opposed to an acute mode, to maintain the initial therapeutic effect (activity) for a prolonged time and " intermittent administration "is the treatment that is not performed consecutively without interruption, but rather in a cyclical manner.
The long-acting EPOs of the present invention, and their uses, can be used in any mammal. When used herein, the term "mammal" refers to any animal classified as a mammal, including humans, domestic and farm animals, and zoo animals, for sports or pets, such as dogs, cats, cattle, horses, sheep, pigs, goats, rabbits, etc. Preferably, the mammal is human. The long-acting EPO administration of the present invention includes, but is not limited to, oral, intravenous, intranasal, topical, intraluminal administration by parenteral inhalation, the latter including intravenous, subcutaneous, intramuscular, intraperitoneal, submucosa , intradermal and combinations of these.
The present invention further relates to the use of the long-acting EPO molecules of the present invention as a carrier for other molecules in the areas of the body that have receptors for EPO. For example, because some molecules have poor penetration through the blood-brain barrier, the binding of these molecules to the long-acting EPOs of the present invention provides a safe and efficient transport system of these molecules to the brain. And, as described in more detail below, because other areas of the body express EPO receptors like the retina, the heart, the lungs, the long-acting EPO molecules of the present invention can act as a transport system for the molecules that have poor penetration through the areas.
In addition, the present invention is directed to assays to determine whether an EPO maintains the functionality of endogenous EPO. For example, an assay of the present invention can determine whether a modified EPO is tissue protective, i.e., an agonist with respect to endogenous EPO's. When used herein, the term "agonist" is used in the broadest sense to include molecules that mimic the biological activity of a natural EPO. Likewise, the term "antagonist" is used in the broadest sense and includes any molecule that partially or completely blocks, inhibits or neutralizes the biological activity of natural EPO. In one embodiment, tests for a specific EPO are carried out in an in vitro assay, such as a test of P19 cells and / or rat motoneurons. In another embodiment, the assay of the present invention consists in the evaluation of a specific EPO in vivo using various assays such as focal rat ischemia, rat retinal ischemia, spinal cord trauma and bicuculline epilepsy models.
Functionality As described in the background section, some attempts to increase the half-life of EPOs have had good results in that EPO analogues have a longer half-life and maintain erythropoietic activity. And, as described, EPO analogs with longer half-lives have mainly EPO molecules included with carbohydrate chains added to the amino acid sequence. However, it is anticipated that these additional carbohydrate chains may interfere with other therapeutic benefits of EPOs, such as the protective functionality of the tissue and the ability of transitosis of the molecule. Without adhering to any specific theory, the placement of the added carbohydrate chains in the O'Brien peptide may affect the functionality based on the importance of this peptide with respect to the protective functionality of the tissue. In addition, the added carbohydrate chains, whether they are within the O'Brien peptide or outside the O'Brien peptide, are considered to have an effect on the three-dimensional orientation of the molecule. For example, in the three-dimensional configuration of the molecule, additional carbohydrate chains can block an area of the molecule that is paramount to functionality. In addition, it is considered that the glycosylation method (or addition of the carbohydrate chains) may have an effect on the functionality of the glycoprotein.
Tissue Protective Capacity To assess the possibility that additional carbohydrate chains can affect the functionality of the glycoprotein, the present invention studied forms of EPO analogues having N-linked carbohydrate chains (as compared to carbohydrate chains linked to 3 N Recombinant EPO In particular, the inventors used an EPO analogue, wherein the analogue has additional glycosylation sites at the 32 amino acids, which gives rise to approximately a half-life three times greater than the recombinant EPO (epoetin alfa).
Although this EPO analogue appeared within the brain spinal fluid after systemic injection (Figures la) it was surprising to note that it did not protect the tissue when evaluated in a subsequent P19 assay in vitro (Figure Ib). This absence of tissue protective activity was unexpected. In addition, the absence of tissue protective activity can produce complications if it is used for treatment in patients with anemia if these patients have other conditions that require the protective capacity of the tissue. For example, if the non-protective EPO analogue competes with tissue-protective endogenous EPO by the receptor that activates the protective response of the tissue, the degree of injury resulting from a trauma can actually be exacerbated by the use of these analogues. of EPO. In fact, if a patient under treatment with an EPO analogue such as this suffers from a stroke, the volume of infarction resulting from the stroke may actually be larger than in an individual not treated with an EPO analogue.
While not wishing to adhere to any specific theory, this finding suggests that at least one additional version of an EPO receptor functionally exists in neural tissues for which signaling differs from that of erythrocyte precursors, and there is a risk that certain EPO analogs can antagonize the ability of endogenous EPO to bind to this version of the receptor. The very different biological activities between endogenous EPO and these EPO analogs suggests that receptor signaling is observed by functionally different EPO receptors that respond to different domains of the EPO molecule. In fact, while it has been documented that the gene protein sequence of the EPO receptor is identical to that expressed by the erythroid precursors, the binding affinity of the neuronal-type receptor for EPO in vitro is much lower than the pro-erythrocyte EPO receptor. . See, for example, Masuda, S., et al., J. Biol Chem. 268, 11208-16 (1993). It is presumed that these differences arise from the accessory proteins and may indicate that different days of signaling are used to those activated in the erythrocyte maturation program. It is important to note that this difference in affinity is not modified by complete deglycosylation of EPO, a result that is not unexpected if the naturally active binding is found in the normally non-glycosylated AB loop region of EPO. Id. In addition, the EPO produced by the astrosites (which is perhaps the same product produced by other cells such as neurons) is also a smaller version than that produced by the kidney. Masuda, S., J. Bio CHem, 269, 19488-93 (1994). It seems that the difference is the result of a different degree of glycosylation. Id. It is not yet determined if the natural ligand of sialiate possesses differences in affinity with these known receptor proteins, but it is of obvious importance.
In addition to the presence of accessory proteins that modify EPO receptors, the EPO receptor is a complex gene for which there are a number of edited versions, including a truncated one, of the soluble receptor. Yamaji, R., et al., Eur J. Biochem 239, 494-500; Yamaji R., et al., Bichim Biophys Acta, 1403, 169-78 (1998); Barron, C., et al., Gene, 147, 2S3-8 (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). Neither has it been determined if any of these versions favors the neuronal effects of EPO.
Furthermore, as described in the background, it has been shown that the O'Brien peptide has protective activity of the tissue in vitro, but not erythropoietic activity in v tro. In fact, an assay performed on an EPO analogue containing an added carbohydrate chain within the O'Brien peptide showed that the EPO analog lacked tissue protective capabilities this result that some modifications to the O'Brien peptide as the addition of the carbohydrate chains, interferes with the functionality of the protein. An EPO analogue with modifications to the O'Brien peptide likewise acts as an antagonist towards endogenous EPO located within the body because it partially or completely blocks the ability of endogenous EPO to bind to the EPO receptor. As such, the risk of increasing the degree of injury resulting from trauma is probably with the use of an EPO analog such as this. Thus, it is considered that EPO analogs with modifications to the O'Brien peptide would also lack tissue protective capacities in other in vitro assays, such as the rat motor neuron test and in vivo assays such as focal ischemia. in rat, bicuculline epilepsy, retinal ischemia in rat and spinal cord trauma trials.
Receiver-mediated Transitosis Using the same EPO analogue as above, ie, an EPO analogue with 5 N-linked carbohydrate chains (compared to the three N-linked carbohydrate chains of the recombinant EPO), the inventors studied the analogue ability to cross the blood-brain barrier. The EPO analogue appeared within the brain spinal fluid after systemic injection (Figures la and Ib). Without adhering to any specific theory, it is considered that the EPO analogue can cross the intact blood-brain barrier because the capillaries that form the blood-brain barrier also express the EPO receptor and provide an anatomical basis for receptor-mediated transitosis. from the peripheral circulation and to the brain. As such, other EPO analogues administered systemically are also considered to be able to cross the blood-brain barrier, as well as other barriers with capillaries that express the EPO receptor.
In summary, because EPO analogs of the prior art have been shown to maintain erythropoietic activity at the expense of at least some of the functionality of endogenous EPO, there is a need in the art for a long-acting EPO that maintains all known functionality of endogenous EPO. Advantageously, the present invention is directed to a long-acting EPO of the present invention that not only increases the serum half-life in comparison with the recombinant EPO, but also maintains the functionality of the endogenous EPO, that is, the protective functionality of the tissue and the capacity for transitosis. Some methods to modify EPO to offer a beneficial protein like this are provided in the next section.
Modification of natural EPO The long-acting EPO of the present invention can be prepared in different forms. In general, long-acting EPO can be generated by chemical modification of the carbohydrate (sugar) chains linked to EPO. When used herein, the term "carbohydrate chains" refers to the N-linked and O-linked oligosaccharide chains found in endogenous EPO, the N-linked and O-linked oligosaccharide chains found in the analogs of EPO and any other carbohydrate chain, specifically, sugar chains, linked to EPO.
In one embodiment, endogenous or recombinant EPO is used for modification to avoid interference with the tissue protective capabilities of endogenous EPO. In addition, EPO analogs are contemplated for modification in accordance with the present invention that provide additional glycosylation sites are not located near the O'Brien peptide, ie, the 30-47 amino acid sequence. When used herein, the term "EPO analogues" refers to modified EPO molecules that have at least one additional N-linked carbohydrate chain and / or at least one additional O-linked carbohydrate chain. In one embodiment, an EPO analog used for modification does not include any additional glycosylation site in the approximately 5 amino acid stretch of the O'Brien peptide. In another embodiment, the EPO analog has no additional glycosylation site in the approximately 3 amino acid stretch of the O'Brien peptide. In yet another embodiment, the EPO analog has no additional glycosylation site in the O'Brien peptide.
An EPO analog can also be used for modification according to the present invention provided that the analog is checked in the three-dimensional space and it is confirmed that none of the additional carbohydrate chains blocks the O'Brien peptide or causes a loss of functionality protective of the woven. In another aspect, it is contemplated that an EPO analogue may be used in the modification in accordance with the present invention so long as the glycosylation method does not inhibit the protective functionality of the peptide tissue. In yet another aspect, an EPO analogue can be used for modification according to the present invention provided that there is a carbohydrate chain (or less) in the O'Brien peptide. For example, an endogenous EPO contains a carbohydrate chain at all 38 amino acids, and it has been shown that an additional carbohydrate chain within the O'Brien peptide inhibits the protective activity of tissue of the protein. Thus, an EPO analog having a carbohydrate chain or no carbohydrate chain in the O'Brien peptide can be used in the modification. In one embodiment, the carbohydrate chain attached to the 38 amino acids can be relocated to some other part of the protein.
Examples of the modifications that do not limit and are in accordance with the present invention consist in: (1) providing additional acidic residues in the carbohydrate chains by oxidation of the nearby hydroxyls; (2) substitute sialic acid residues with skilled residues; (3) increase the negative charge in erythropoietin by sulfation and / or phosphorylation; and / or (4) terminate the carbohydrate chains with more complex molecules. Thus, modifications to the carbohydrate chains of EPO may include sulfation oxidation, phosphorylation and / or PEGylation, among other methods, which will be described in more detail below and further shown in Example 1.
Oxidation of sugar chains? substitution of sialic acid residues A chemically modified long-acting EPO of the present invention may consist of an EPO wherein the carbohydrates (sugars) are oxidized to obtain additional acidic residues. In another embodiment, the sialic acid residues are substituted with less labile acid residues, modifications of this type result in an increased half-life of the molecule, compared to an endogenous EPO, because the galactose chains for which the liver detects and removed the associated protein from the circulation are protected from detection. A more considerable chemical modification to the carbohydrate chains in EPO leads to a greater increase in the serum half-life of the long-acting EPOs of the present invention. For example, when the closest hydroxyls are replaced by acids, a larger increase in serum half-life results.
Although a person with ordinary skill in the art would be aware of the various convenient methods for converting the galactose and erythropoietin units, one convenient method is to: (1) modify the sugar molecules with nearby hydroxyls with periodate to form aldehydes; and (2) oxidizing the aldehydes to have acids. Suitable reagents for oxidizing the sugar chains to form aldehydes are those known to those skilled in the art and include, but are not limited to, periodates, such as sodium periodate, and sugar oxidases, such as galactose oxidase. In addition, those skilled in the art will become aware of the convenient reagents for transforming the aldehydes, such as the quantitative solution of Benedict (commercially available from Fisher). In one embodiment, the sugar molecules are oxidized with sodium periodate and are also treated with Benedict's Quantitative Solution (Fisher) to convert the aldehydes to acids.
In another embodiment, an EPO isomer, one having approximately 0-13 sialic acid residues, or an EPO analog, having at least one carbohydrate chain lacking a sialic acid residue, is subjected to oxidation using galactose oxidase. Such a form of EPO can be used in accordance with this aspect of the invention, ie, a α or β form of the EPO with the terminal carbohydrate (sialic acid) removed. Preferably, asialoerythropoietin is used. Once oxidized, the EPO is subjected to another oxidizing agent, such as the quantitative solution of Benedict converting the aldehydes into acids.
In yet another embodiment, a ruthenium tetroxide system can be used to obtain the acids in the sugar chain. Since those modifications include the galactose chain even if the acids involved in these transformations are removed from the molecule, the molecule must be able to evade deletion by the liver since a galactose chain, the component that the liver detects, will not be exposed.
Increasing Necrotizing Care In another aspect of the present invention, a long-acting EPO of the present invention is formed by adding sulfates and / or phosphates to the EPO molecule, which will increase the negative charge of the molecule and thereby increase the average life of the molecule. In other words, the negative charge of the EPO molecule can be increased by sulfation, which includes the transfer of a sulphuryl group from a sulfate donor, including the protein glycolipids, glycosaminoglycans and steroids. And, the negative charge can also be increased by introducing a phosphoric group into a carbohydrate.
A convenient method for insulin sulfation 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, an insulin sulfation was carried out in an organic solvent, such as dimethylformamide (DMF), in the presence of condensation agents, such as?,? '- dicyclohexylcarbodiimide ( DCC) and a sulfate donor. The degree of sulphation can be regulated in a range of 800% by modifying the amount of the condensing agent. Although the sulfated insulin prepared in normal form gave rise to a greater loss of the bioactivity of the insulin, the bioactivity the sulfated insulin prepared with the Pongor process was between 78% and 87% of the unmodified insulin.
By using a similar procedure for EPO, one skilled in the art can regulate the amount of sulphation and thus the serum half-life of the chemically modified EPO. For example, the negative charge of EPO can be increased by adding sulfates to the protein by dissolving the EPO or an EPO analogue in at least one water-soluble carbodiimide, preferably DCC, at a temperature of about 4 ° C. Although DCC is preferred as the sulfate donor, those skilled in the art will readily become aware of other suitable sulfate donors for use with the present invention.
Similar procedures can be used to regulate the phosphorylation of EPO using phosphoric acid (H3PO4) as the phosphate donor. Again, although phosphoric acid is preferred, experts will readily be able to choose other phosphate donors to carry out the phosphorylation of EPO.
Termination of carbohydrate chains with PEG The carbohydrate chains of EPO can also be modified by the addition of at least one polyethylene glycol (PEG), a compound with a long and safe clinical history, which has the following general formula: The PEG may also be a methoxy PEG (mPEG) having the following general formula: In one embodiment, the PEG is an amino PEG, preferably a PEG methoxy with primary amino groups in the terminal (mPEG-NH2). The polyethylene glycol chains with primary amino groups on the terminals are very useful functionalized polymers. The amino end groups in mPEG-NH2 are more reactive to the acylating agents than the hydroxyl groups that are present in the normal PEGs, and also easily undergo reductive amination reactions. In another embodiment, PEG is an electrophilically activated PEG, such as mPEG-succinimidyl propionate (mPEG-SPA) or mPEG-succinimidyl butanoate (mPEG-SBA), which are commercially available from Nectar Therapeutics of Birmingham, Alabama. In still another embodiment, the PEG is a methoxy PEG hydrazide.
In one embodiment, the addition of at least one PEG is achieved by oxidation with periodate (as already described), followed by the use of cyanoborohydride and an amino PEG. For example, the EPO in solution can first be oxidized with a periodate, for example sodium periodate, for a predetermined time at room temperature, which produces aldehydes in the carbohydrate chains. A convenient periodate is sodium metaperiodate, which is available commercially from Sigma. The periodate then the periodate can be removed by buffer exchange, at which time the oxidized sialic acid groups in the N-linked oligosaccharide groups of the EPO can be subjected to at least one amino PEG in the presence of cyanoborohydride. Convenient PEGs for use include, but are not limited to, methoxy-PEG-idrazides, which are commercially available from Nectar Therapeutics.
In another embodiment, the addition of at least one PEG is carried out by the binding of the PEG groups to the terminal galactose residues after oxidation with galactose oxidase. For example, such a form of EPO (having the terminal galactose residues exposed) in buffer is first subjected to galactose oxidase (available commercially from Sigma) to generate aldehydes in the carbohydrate chains. The buffer can then be removed by buffer exchange, at which time the oxidized galactose residues can be subjected to at least one amino PEG in the presence of cyanoborohydride.
The aforementioned methods are not intended to be limiting since it is possible to use these and other methods to prepare the compounds of the invention. For example, an expert will appreciate the applicability of these chemical modifications to create long-acting versions of other EPO derivatives, such as tissue protective cytokines described in International Publication No. WO / 02053580 and US Patent publications. Nos. 2002/0086816 and 2003/0072737, which are incorporated herein by reference in their entirety.
Production of EPO molecules To produce the long-acting EPO molecules and EPO-related molecules of the invention it is possible to use various host-expression vector systems. These host-expression systems represent vehicles by which it is possible to produce the long-acting EPOs of interest and subsequently can be purified, but also represent cells that can, when transformed or transfected with the appropriate nucleotide coding sequences, display the erythropoietin gene product. modified in situ. These include, but are not limited to, host systems of bacteria, insects, plants, mammals, including humans, such as, but not limited to, insect cell systems infected with recombinant virus expression vectors (e.g. baculovirus) containing the coding sequences of the long-acting EPO product; plant cell systems infected with recombinant virus expression vectors (eg cauliflower mosaic virus, CaMV, tobacco mosaic virus, TMV) or transformed with recombinant plasmid expression vectors (eg Ti plasmid) containing coding sequences of molecules related to erythropoietin; or mammalian cell systems, including human cell systems, for example HT1080, COS CHO BHK, 293, 3T3, recombinant host expression constructs containing promoters obtained from the genome of mammalian cells, for example the metallothionein promoter, or of mammalian virus, for example the adenovirus late promoter, the 7.5K promoter of the vaccinia virus.
In addition, it is possible to choose a strain of host cells that modulates the expression of the inserted sequences, or modify and process the gene product in the specific manner desired. Modifications and processing like these of protein products may be important for protein function. As those skilled in the art know, different host cells have specific mechanisms for post-translational processing and modification of proteins and gene products. Suitable cell lines and host systems can be chosen to ensure the correct modification and procedure of the foreign protein expressed. For this purpose, it is possible to use eukaryotic host cells that possess the cellular machinery for the proper processing of the primary transcript, the glycosylation and phosphorylation of the gene product. Mammalian host cells such as these, including human host cells, may be, but are not limited to, HT1080, CHO, VERO, BHK, HeLa, COS, MDCK, 293, 3T3, and W138.
Stable expression is preferred for high yield and long term production of recombinant proteins. For example, it is possible to manipulate cell lines that stably express the gene product molecules related to tissue-protective cytosine, recombinant. Instead of using expression vectors containing viral replication origins, the host cells can be transformed with DNA controlled by suitable expression control elements, for example, promoter, enhancer, sequences, transcription terminators, polyadenylation sites and the like, and a selectable marker. After the introduction of strange DNA, the manipulated cells can be allowed to grow for 1-2 days in an enriched medium, and then they are changed to a selective medium. The selectable marker in the recombinant plasmid confers resistance to selection and allows cells to stably integrate the plasmid into their chromosomes and grow to form foci that in turn can be cloned and extended into cell lines. This method can conveniently be used to manipulate cell lines that express the mutein-related molecule gene product of EPO. Cell lines manipulated like these can be particularly useful in the screening and evaluation of compounds that affect the endogenous activity of the EPO-related molecule gene product.
Otherwise, the characteristic expression of a mutein gene of endogenous EPO within a cell line or microorganism can be modified by inserting a heterologous DNA regulatory element into the genome of a stable cell line or cloned microorganism so that the regulatory element inserted is operably linked to the mutein gene of endogenous erythropoietin. For example, an endogenous EPO mutein gene that is normally "silent transcription", ie, an EPO gene that is not normally expressed, or is expressed only at very low levels in a cell line, can be activated by inserting a regulatory element that can favor the expression of a gene product expressed in this cell line or microorganism. Otherwise, an endogenous EPO gene, of silent transcription, can be activated by the insertion of a promiscuous regulatory element that functions through cell types.
It is possible to insert an ether regulatory element into a stable cell line or cloned microorganism, so that it is operably linked to an endogenous erythropoietin gene, using techniques, such as targeted homologous recombination, which is well known to experts in the art, and is also described in French Patent No. 2646438, US Patent Nos. 4,215,051 and 5,578,461, and International Publication No. 0 93/09222 and WO 91/06667, the full descriptions of which are incorporated as reference in the present.
Pharmaceutical Compositions The present invention also relates to pharmaceutical compositions containing long-acting EPO molecules of the present invention. Because the long-acting EPOs of the present invention advantageously have erythropoietic activity, as well as tissue protective capacity and transitosis capacity, they are considered for the treatment of anemia and related diseases in individuals also at risk of different tissue lesions, such as cerebrovascular accident and myocardial infarction. In addition, the long-acting EPOs of the present invention are considered for the treatment of anemia and related diseases in individuals who also experience impairment of mental faculties, such as Alzheimer's disease, Parkinson's and the like. Moreover, the long-acting EPOs of the present invention are considered for the treatment of anemia in individuals subject to states resulting from the normal aging process, for example balance problems leading to falls, contusions and the like. Moreover, the present invention relates to the use of the long-acting EPOs of the present invention as carriers for other molecules that have poor penetration through barriers with capillaries having EPO receptors.
For example, any of the long-acting EPO described above can be included in pharmaceutical compositions of the invention. In addition, some EPO analogs may be included in the pharmaceutical compositions of the invention in a combination with at least one tissue protective cytosine, which will be described in greater detail below.
The pharmaceutical compositions of the invention contain an effective therapeutic amount of the modified EPO, preferably in purified form. The formulation must be adapted to the mode of administration. In other words, the pharmaceutical compositions of the invention contain an amount of the modified EPO of the invention so that the chosen state can be treated provided that the appropriate dose and strategy is employed. And, as described in more detail below, the pharmaceutical composition must be delivered in a non-toxic dose amount.
The pharmaceutical compositions of the invention may contain an effective therapeutic amount of the long-acting EPO compound and a convenient amount of a carrier accepted for pharmaceutical use to provide the proper form of administration to the patient. In a specific modality, the term "accepted for pharmaceutical use" means approved by a regulatory institution of the federal or state government or listed in the United States Pharmacopoeia or other generally recognized foreign pharmacopoeia, for use in animals, and more specifically in humans. The term "carrier" refers to a diluent, adjuvant, excipient or vehicle with which the therapeutic is administered. Pharmaceutical carriers such as these may be sterile liquids, such as saline solutions in water and oils, including petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. A saline solution is a preferred carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous solutions of dextrose and glycerol can also be used as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients may be starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, calcium carbonate, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, anhydrous skim milk, glycerol, propylene. glycol, water, ethanol and the like.
The pharmaceutical compositions of the invention may also have minor amounts of wetting or emulsifying agents, or buffering agents. These compositions may take the form of solutions, suspensions, emulsion, tablets, pills, capsules, powders, sustained-release formulations, and the like.
The compounds of the invention can be formulated as neutral forms or salts. The salts accepted for pharmaceutical use can be those that are formed with free amino groups such as those obtained from hydrochloric, phosphoric, acetic, oxalic, tartaric acids, etc., and those formed with free carboxyl groups such as those obtained from sodium, potassium hydroxides. , ammonium, calcium, iron, isopropylamine, triethylamine, 2-ethylaminoethanol, histidine, procaine, and so on.
Composition that contains EPO of action Orolon.cja.da. As briefly mentioned in the foregoing, any of the long-acting EPOs of the present invention is considered for use in pharmaceutical compositions. In one embodiment, a long-acting EPO produced from the oxidation of the nearby hydroxyls is included in the pharmaceutical composition of the invention. In another embodiment, the pharmaceutical composition of the invention contains at least one long-acting EPO which is a result of replacing the sialic acid residues with less labile residues. In yet another embodiment, the long-acting EPO contained in the pharmaceutical composition is a result of increasing the negative charge on the EPO by sulfation and / or phosphorylation. In still another embodiment, a long-acting EPO produced by terminating the carbohydrate chains with more complex molecules, e.g. PEG chains, is included in the pharmaceutical compositions of the invention.
In addition, the present invention contemplates the use of a mixture of the long-acting EPOs produced by any of the methods of the present invention in the pharmaceutical compositions of the invention. For example, the pharmaceutical composition of the invention can obtain at least one long-acting EPO which is a result of replacing sialic acid residues with less labile residues and at least one long-acting EPO which is the result of increasing the negative charge in EPO by sulfation or phosphorylation, or both.
Transport system As described above, the long-acting EPOs of the present invention can advantageously cross the barriers with capillaries having receptors for, EPO. Thus, another aspect of the present invention is a transport system that uses the long-acting EPOs of the present invention as carriers for molecules with poor barrier penetration in a chosen area of the body having receptors for EPO. Transport systems such as these advantageously provide a novel and safe method of supply through intact barriers.
In one embodiment, the transport system includes the long-acting EPOs of the present invention and at least one molecule with poor penetration into the brain to provide a novel and safe delivery method through the intact blood-brain barrier. In other words, the long-acting EPOs of the present invention may allow poorly penetrating molecules in the brain to act as "Trojan horse" molecules to enhance brain uptake of small or large molecules of diagnostic therapeutic molecules.
In fact, a major problem in the treatment of human brain tumors is presented by the need to deliver therapeutic agents to specific areas of the brain, distribute them within and direct them to brain tumors. Molecules that might otherwise be effective in diagnosis and therapy do not cross the blood-brain barrier (BBB) in the brain next to the tumor or do not cross the blood-tumor barrier (BST) in adequate amounts. So that, there is a need for novel delivery strategies that are unique to the brain and that derive the vasculature. For example, antibodies that could be used as diagnostic or therapeutic molecules do not cross BST in sufficient quantities to be effective because of their size. As such, the long-acting EPOs of the present invention can be used as a carrier for these molecules to allow BHE or BST to pass through. An example of a molecule that can be used with the long-acting EPO of the present invention is an antisense oligonucleotide, which is usually used to inhibit oncogenic signals or to image brain gene expression in vivo. In addition, the long-acting EPO of the present invention can be included in different gene therapies (viral or non-viral formulations) which are often too large to cross the BST without help.
In addition, the long-acting EPOs of the present invention can be used as carrier-mediated carriers for different chemotherapeutic agents. Because the transporters perform the active effusion of the drug, which are expressed in the BHE and the BST, the chemotherapeutic agents that perform active brain effusion return to the blood, the distribution of these agents in the brain can be inhibited or prevented. It is partly for these reasons that most of the customary chemotherapeutic molecules that have been used to treat cancer outside the central nervous system (CNS) are ineffective in the treatment of brain tumors. Thus, the use of the long-acting EPO of the present invention as a carrier for these chemotherapeutic agents may be useful not only to carry the agents into the brain, but also to maintain the agents within the brain for therapy. In another embodiment, the long-acting EPO may be linked with a medicament that inhibits the transporter that performs the active effusion to further ensure the uptake of the chemotherapeutic agents that normally emanate from the brain to the blood.
In addition, the present invention also contemplates the use of modified EPO as carriers for molecules with poor penetration in other areas of the body that have receptors for EPO. Examples that do not limit these cells include retinal, muscle, cardiac, lung, liver, kidney, small intestine, adrenal cortex, adrenal medulla, capillary endothelium, testes, ovary, pancreas, bone, skin and endometrium cells. In particular, sensitive cells include, without limitation, neuronal cells; the cells of the retina: photoreceptor (rods and cones), ganglion, bipolar, horizontal, amadrina and Müeller cells; muscle cells; heart cells: myocardium, pacemaker, sinoatrial node, sinus node and joint tissue cells (atrioventricular node and atrioventricular trunk); lung cells; liver cells, hepatocytes, stellate cells and Kupffer cells; renal cells: mesangial cells, renal epithelial cells and tubular interstitial cells; cells of the small intestine: calciform cells, cells of the intestinal gland (crypts) and enteric endocrine cells; cells of the adrenal cortex: glomerular cells; Fasciculate and reticular: cells of the adrenal medulla: chromaffin cells; capillary cells: periterial cells; testicle cells: Leydig cells, Sertoli and sperm cells and their precursors; ovarian cells: Graffian follicle cells and primordial follicle cells; pancreatic cells: islets of Langerhans, cells a, b cells, cells? and F cells; bone cells: osteoprogenitors, osteoclasts and osteoblasts; skin cells; endometrial cells: endometrial stroma and endometrial cells; as well as the primordial and endothelial cells present in the aforementioned organs.
Mixed composition EPO Analogue and Tissue Protective Cytosine As mentioned in summary above, a pharmaceutical composition according to the present invention may contain an EPO analog having at least one additional N-linked carbohydrate chain and / or at least one an additional 0-linked carbohydrate chain (having a prolonged serum half-life but lacking tissue protective activity) in a combination with at least one tissue protective cytokine. For example, an EPO analog having at least two additional N-linked carbohydrate chains, wherein one of the additional carbohydrate chains is located on the O'Brien peptide, in combination with a tissue protective cytokine, can form a composition of the invention. In another embodiment, the pharmaceutical composition of the invention can have at least one tissue protective cytokine and at least one EPO analog containing additional carbohydrate chains that are known, from the review of the analog in three-dimensional space, to block O'Brien's peptide. In still another embodiment, a pharmaceutical composition of the invention contains at least one tissue protective cytokine and at least one EPO analog that does not have tissue protective functionality as a result of the method of adding the additional carbohydrate chains to the protein.
An EPO analogue with a relocated glycosylation site is contemplated for use in the pharmaceutical compositions of the present invention. Without adhering to any specific theory, it is considered that if the natural glycosylation site at amino acid 38 were relocated to any part in an EPO analog, outside of amino acid segment 30-47, the tissue protective capabilities of the EPO analogue would increase in comparison with an EPO analogue with a glycosylation site at amino acid 38. Thus, the pharmaceutical composition of the invention can include an EPO analogue with a relocated glycosylation site, from amino acid 38 to anywhere in the molecule. The relocated glycosylation site can be found at amino acids 51, 57, 69, 88, 89, 136 or 138, as suggested in PCT Publication WO 01/81405. In one embodiment, the O'Brien peptide contains one or fewer carbohydrate chains. In another embodiment, the O'Brien peptide has two or more carbohydrate chains.
The tissue protective cytokines, suitable for use with this aspect of the present invention, are preferably those cytokines that lack an effect on the bone marrow, but maintain the protective effect of the tissue of the endogenous [sic], notwithstanding any cytokine which shows tissue protective capacity is contemplated for use with the present invention. For example, convenient tissue protective cytokines consist of chemically modified EPOs, obtained by guanidination, amidination, carbamylation (carbamoylation), trinitrophenylation, acylation (acetylation or succinylation), nitration, or mixtures thereof. In addition, EPO molecules with a modification of at least one residue of arginine, licina, tyrosine, tryptophan or cysteine or carboxyl groups are also contemplated for use as tissue protective cytokines in accordance with this aspect of the present invention.
Moreover, other tissue protective cytokines for use with the present invention can be obtained by limited proteolysis, elimination of amino groups and / or mutational substitution of arginine, lysine, tyrosine, tryptophan or cysteine residues, by molecular biology techniques such as this one. described in Satake et al., 1990, Biochim, Biophys. Acta 1038: 125-9, which is incorporated as a reference in the present in its integrity. For example, convenient tissue protective cytokines include at least one or more mutated EPO having a site mutation in C7S, 10I, 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, SIOOR, S100E, S100A, S100T, G101A, G101I, G101I, L102A, R103A, S104A, S104I, L105A, T106A, T10SI, T107A, T107L, L108K, L108A; S126A, F142I, R143A, S146A, N147K, N147A, F148Y, L149A; R150A, G151A, K152A, L153A, L55A, C160S, I6A, C7A, B13A, W24K, A30N, H32T, N38K, N83K, P42A, D43A, K52A, K97A, K116A, T132A, I133A, T134A, 140A, P148A, R150B, G151A, 152W, K154A, G158A, C161A and / or R162A. Examples of the aforementioned modifications are described in copending US Patent Publications Nos. 2003/0104988, 2002/0086816 and 2003/0072737, which are incorporated by reference in their entireties. In the nomenclature of the mutein used herein, the changed amino acid is represented by the first one-letter code of the natural amino acid, followed by its position in the EPO molecule, followed by the one-letter code of the replacement amino acid. For example, S100E refers to a human EPO molecule in which, at amino acid 100, serine has been changed to a glutamic acid.
In another embodiment, the tissue protective cytokine may include one or more above site mutations, provided that the site mutations do not include 16A, C7A, K20A, P42A, D43A, K45D, K45A, F48A, Y49A,. K52A, K49A, S100B, R103A, K116A, T132A, I133A, K140A, N147K, N147A, R150A, R15E, G151A, K152A, K154A, G158A, C161A, or R162A.
In yet another embodiment, tissue protective cytokines may include combinations of site mutations, such as K45D / S100E, A30N / H32T, K45D / R150E, R103E / L108S, K140A / K52A, K140A / K52A / K45A, K97A / K152A , K97A / K152A / K45A, 97A / K152A / K45A / K52A, K97A / 152A / K45A / 152A / K140A, K97A / K152A / K45A / K52A / K140A / K154A, N24K / N38K / N38K and N24K / Y115A. In yet another embodiment, tissue protective cytokines do not include any of the above combinations. In another embodiment, tissue protective cytokines can include any of the aforementioned site mutations provided that the site mutations do not include any of the following substitution combinations: N24K / N38K / N83K and / or A30N / H32T.
Some modifications or combinations of modifications may affect the flexibility of the mutein's ability to bind with its receptor, such as a receptor for EPO or secondary receptor. Examples of these modifications or combinations of modifications include, but are not limited to, K152W, 14A / Y15A, 16A, C7A, D43A, P42A, F48A, Y49A, T132A, I133A, T134A, N147A, N147A, P148A, R150A, G151A, G158A, C161A and R162A. People with ordinary skill in the art know that the corresponding mutations are detrimental to human growth hormone. Thus, in one embodiment, the tissue protective cytokine does not include one or more of the modifications or combinations of modifications that may affect the flexibility of the mutein's ability to bind with its receptor. Another discussion of these tissue protective cytokines is included in co-pending US Patent Application, Proxy File No. 10165-022-999, filed July 1, 2003, entitled "Protective tissue cytokines, recombinants, and nucleic acids encoding these for protection, restoration and enhancement of sensitive cells, tissues or organs ", the complete description of which is incorporated herein by reference.
Finally, any of the cytokine superfamily that shows protective tissue capabilities can also be used as long as it does not interfere with the erythropoietic effects 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 (G CSF), the pigment-epithelium factor (PEDF) ) and vascular endothelial growth factor (VEGF).
In another aspect of the present invention, a pharmaceutical composition according to the present invention may contain an EPO analog having at least one additional 3ST-linked carbohydrate chain and / or at least one additional 0-linked carbohydrate chain (which exhibits a prolonged serum half-life but lacking tissue protective activity) in a combination with at least one small molecule that exhibits tissue protective functionality. Convenient small molecules include, but are not limited to, steroids (e.g. lazaroids and glucocorticoids), antioxidants (e.g. coenzyme Q10, alpha lipoic acid and NADH), anticatabolite enzymes (e.g. glutathione peroxidase superoxide dismutase, catalase, catalytic scavengers synthetics, as well as mimetics), indole derivatives (for example indole amines, carbazoles and carbolines), nitric acid neutralizing agents, adenosine / adenosine agonists, cytochemicals (flavonoids), herbal extracts (ginko biloba and turmenic), vitamins (vitamins A, E and C), inhibitors of electron oxidase receptors (e.g. santeno oxidase inhibitors), minerals (e.g. copper, zinc and magnesium), NSAIDS (e.g. aspirin, naproxen and ibuprofen), and combinations of these. In addition, a pharmaceutical composition of the invention may include an EPO analogue, a tissue protective cytosine and a small molecule in tissue protective activity.
Tissue and / or small molecule protective cytokines are preferably present in the pharmaceutical compositions of the invention in an amount sufficient to maintain or overcome the same activity in neuronal or other sensitive cellular systems as that shown by endogenous EPO. In one embodiment, the tissue protective cytokine and / or the small molecule is present in an amount sufficient to increase the weave protection of the individual by protecting, maintaining or increasing the viability and function of the responding erythropoietic cells within the individual. For example, the pharmaceutical composition of this aspect of the present invention preferably includes an effective, non-toxic, amount of tissue protective cytokine, for example, about 1 ng or greater. In one embodiment, the tissue protective cytokine will be present in the pharmaceutical composition in an amount of about 5 mg or less. In another embodiment, the tissue protective cytokine will be present in the pharmaceutical composition in an amount of about 500 ng to 5 mg. In still another embodiment, the pharmaceutical composition contains about 2 μg to 5 mg of the tissue protective cytokine, preferably about 500 μg to 5 mg. In another embodiment, a larger amount of the tissue protective cytokine is present in the pharmaceutical composition of the invention, for example, about 1 mg to 5 mg. As is known to those skilled in the art, the amount of the pharmaceutical composition administered to a patient depends on various factors such as, but not limited to, the condition of the patient and the frequency of the dose. This will be described in more detail below with respect to the dose.
Treatment and methods of administration The aforementioned long-acting EPO and the pharmaceutical compositions containing the long-acting EPO are intended for therapeutic or prophylactic treatment of anemia, diseases in humans that include anemia or anemia states, or diseases or treatment methods that result in anemia. In general, the long-acting EPOs of the present invention allow less frequent dosing or the use of smaller doses of erythropoietin to treat the above diseases without compromising the possibility of patients recovering from other tissue lesions.
The present invention contemplates the use of long-acting EPOs for systemic or chronic administration, acute treatment and / or intermittent administration. In one embodiment, the pharmaceutical compositions of the invention are administered chronically to protect or augment the target cells, tissue or organs. In another embodiment, the pharmaceutical compositions of the invention can be administered in acute form, i.e., for a single treatment during the injury. In still another embodiment, the pharmaceutical compositions of the invention are administered in a cyclic fashion.
Administration of the composition may be parenteral, for example by intravenous injection, intraperitoneal injection, intraarterial, intramuscular, intradermal or subcutaneous administration; by inhalation; through the mucosa, for example, oral, nasal, rectal, intravaginal, sublingual, submucosal and transdermal; or combinations of these. Preferably the administration of the pharmaceutical composition of the invention is parenteral. Administration like this can be carried out in a dose amount of about 0.01 pg to about 5 mg, preferably about 1 pg to about 5 mg. In one embodiment, the amount of the dose is about 500 pg to about 5 mg. In another embodiment, the amount of the dose is about 1 ng to about 5 mg. In yet another embodiment, the amount of the dose is about 500 ng to about 5 mg. In yet another embodiment, the amount of the dose is about 1 μg to about 5 mg. For example, the amount of the dose may be about 500 ^ ig to about 5 mg. In another embodiment, the amount of the dose may be about 1 mg to about 5 mg.
The pharmaceutical compositions of the invention adapted for parenteral administration include sterile, aqueous and non-aqueous injectable solutions or suspensions, which may contain antioxidants, buffering solutions, bacteriostats and solutes which convert the compositions considerably isotonic with the blood of the patient to whom they are intended. . In this aspect of the invention, the pharmaceutical compositions may also include water, alcohols, polyols, glycerin, vegetable oils, and mixtures thereof. Pharmaceutical compositions adapted for parenteral administration may be present in single-dose or multi-dose containers, for example in sealed vials and ampoules, and may be stored in a freeze-dried state (freeze drying) requiring only the addition of a sterile liquid carrier. , sterile saline for injections, immediately before use. Solutions and suspensions for extemporaneous injection can be prepared from sterile powders, granules and tablets. In one embodiment, an auto injector containing an injectable solution of a long-acting EPO of the invention can be provided for emergency use in ambulances, emergency rooms and battlefield situations.
In one embodiment, the pharmaceutical composition of the invention is formulated according to the usual procedures as a pharmaceutical composition adapted for intravenous administration to humans. For example, the pharmaceutical composition may be in the form of a solution in sterile isotonic aqueous buffer. When necessary, the pharmaceutical composition can also include a solubilizing agent and / or a local anesthetic such as lidocaine to relieve pain at the injection site. The ingredients may be supplied separately or mixed together in the unit dosage form, for example, as an anhydrous lyophilized powder or concentrate without water in a sealed container such as a vial or sachet indicating the amount of the active ingredient . When the pharmaceutical compositions of the invention are to be administered by infusion, a sterile, pharmaceutical grade water or saline infusion bottle can be used to dose the composition. And, when the pharmaceutical composition is to be administered by injection, a sterile saline ampule can be provided to mix the ingredients before administration.
Pharmaceutical compositions adapted for oral administration can be provided as capsules or tablets; powders, or granules; solutions, syrups or suspensions (in aqueous or non-aqueous liquids); edible foams; emulsions, or combinations of these. The oral formulation may contain about 10% to about 95% by weight of active ingredient. In one embodiment, the active ingredient is included in the oral formulation in an amount of from about 20% to about 80% by weight. In still another embodiment, the formulation contains about 25% to about 75% by weight of the active ingredient.
Hard gelatine tablets or capsules may contain lactose, starch or derivatives thereof, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, stearic acid or salts thereof. Soft gelatine capsules may contain vegetable oils, waxes, semi-solid fats, liquid polyols or mixtures thereof. The solutions and syrups may contain water, polyols, sugar or mixtures thereof.
In addition, an active agent intended for oral administration can be coated with or mixed with a material that retards the disintegration and / or absorption of the active agent in the gastrointestinal tract. For example, the active agent can be mixed or coated with glyceryl monostearate, glyceryl distearate, or a combination thereof. Thus, the sustained release of an active agent can be achieved for many hours, and if necessary, it is possible to protect the active agent from degradation within the stomach. Pharmaceutical compositions for oral administration can also be formulated to facilitate the release of an active agent at a specific gastrointestinal site by the specific pH or the enzymatic condition.
Pharmaceutical compositions adapted for transdermal administration can be arranged as small patches intended to remain in intimate contact with the epidermis of the recipient for a prolonged time. In addition, pharmaceutical compositions adapted for topical administration may be made available as ointments, creams, suspensions, lotions, powders, solutions, pastes, gels, sprays, aerosols, oils, eye drops, tablets, lozenges and mouth rinses and combinations thereof. . When topical administration is intended for the skin, mouth, or os or other external tissues, it is preferred to use an ointment or topical cream. And, when formulated in an ointment, the active ingredient, i.e., the long-acting EPO can be employed with a paraffinic or water miscible base. Otherwise, the active ingredient may be formulated in a cream with an oil-in-water base or a water-in-oil base. When topical administration is in the form of ophthalmic drops, the pharmaceutical compositions of the invention preferably contain the active ingredient, dissolved or suspended in a convenient carrier, for example, in an aqueous solvent.
The pharmaceutical compositions adapted for nasal and pulmonary administration may contain solid carriers as powders (preferably with a particle size of about 30 microns to about 500 microns). Powders can be administered by rapid inhalation through the nose from a powder container that is held close to the nose. In another embodiment, the pharmaceutical compositions proposed for nasal administration in accordance with the present invention may include liquid carriers, for example nasal sprays or nasal drops. Preferably, the pharmaceutical compositions of the invention are administered to the nasal cavity directly.
Direct inhalation to the lung can be carried out by deep inhalation through a mouthpiece into the oropharynx and other specially adapted devices including, but not limited to, pressurized aerosols, nebulizers or insufflators, which can be constructed to provide predetermined doses of the active ingredient. Pharmaceutical compositions intended for inhalation to the lung may include aqueous or oily solutions of the active ingredient. Preferably, the pharmaceutical compositions of the invention are administered by deep inhalation directly to the oropharynx.
Pharmaceutical compositions adapted for rectal administration can be provided as suppositories or enemas. In one embodiment, the suppositories of the invention contain about 0.5% and 10% by weight of the active ingredient. In another embodiment, suppository includes about 1% to about 8% by weight of the ingredient. In still another embodiment, the active ingredient is present in an amount of from about 2% to about 6% by weight. In this aspect of the invention, the pharmaceutical compositions of the invention may contain the usual binders and carriers, such as triglycerides.
Pharmaceutical compositions adapted for vaginal administration can be arranged as pessaries, tampons, creams, gels, pastes, foams or aerosol formulations.
The pharmaceutical compositions of the invention can also be administered by the use of a perfusate, injection into an organ, or administered locally. In embodiments such as these, the pharmaceutical composition preferably has about 0.01 pM to about 30 pM, preferably from about 15 pM to about 30 nM, of the long-acting EPO of the present invention. In one embodiment, the solution for perfusion is the University of Wisconsin (UW) solution (with a pH of about 7.4 to about 7.5 and an osmolality of about 320 mOSm / L), which contains about 1 ü / mL to about 25 U / mL of EPO; 5% hydroxyethyl starch (preferably with a molecular weight of from about 200,000 to about 300,000 and substantially free of ethylene glycol, ethylene chlorohydrin, sodium chloride and acetone), 25 mM K¾P04, 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; and 12 mg of phenol red. The UW solution is described in detail in US Patent No. 4,798,824, which is incorporated herein by reference herein. In another embodiment, the UW solution may contain about 0.01 pg / mL to about 400 ng / mL, preferably about 40 ng / mL to about 300 ng / mL of recombinant, tissue-protective cytokine.
It may be necessary to administer the pharmaceutical compositions of the invention locally in the area in need of treatment. Such administration can be obtained by local infusion during surgery; topical application, for example together with a wound dressing after surgery; by injection; by means of a catheter; by means of a suppository; or by means of an implant; the implant being a porous, non-porous or gelatinous material, including membranes, such as silastic membranes, or fibers.
In addition, as briefly described above with respect to transdermal administration, a long-acting EPO of the present invention can be delivered in a system with release control.
For example, the polypeptide can be administered using intravenous infusion, an implantable osmotic pump, a transdermal patch, liposomes or other modes of administration. In one embodiment, it is possible to use a pump, as described in Saudek et al., 1989, N. Engl. J. Med. 321: 574. In another embodiment, the compound can be administered in a vesicle, in particular a liposome as described in international publication WO 91/04014 and US Patent No. 4,704,355, the full descriptions of which are incorporated by reference herein. In another embodiment, it is possible to use polymeric materials to produce a controlled release system, such as those materials described in Howard et al., 1989, J. Weurosurg. 71: 105.
Controlled release systems such as these can be placed in proximity to the therapeutic target, ie, target cells, tissues or organs, thus requiring only a fraction of the systemic dose. See, for example, Goodson, Medical Applications of Controlled Relay, vol. 2, 115-138, 1984. Other controlled-release systems contemplated for use with the present invention are described in the review by Langer, Science 249: 1527-1533, 1990.
Dosage The selection of the effective and non-toxic dose, preferred for the above administration methods will be determined by one skilled in the art based on factors known to those skilled in the art. Examples of these factors include the specific form of long-acting EPO; the pharmacokinetic parameters of EPO, such as bioavailability, metabolism, half-life, etc. (which are provided to the expert individual), - the condition or disease to be treated; the benefit that can be achieved in a normal individual; the patient's body mass; the method of administration; the frequency of administration, that is, chronic, acute, intermittent, concomitant medications; and other well-known factors that can affect the effectiveness of the pharmaceutical agents that are administered. Thus, the precise dosage must be decided according to the practitioner's judgment and the specific patient's case.
For example, the Physicians Desk Reference (PDR) shows that, depending on the population of patients treated with EPO, different levels of hematocrit are chosen to avoid toxicity. Physicans Desk Reference, 54th Ed., 519-525 and 2125-2131 (2000). In fact, in patients with CRF, the PDR recommends dosing EPO to obtain non-toxic target hematocrit ranging from 30% to 36%. In contrast, for cancer patients on chemotherapy, the PDR teaches' to adjust the dose to a different hematocrit level, that is, if the hematocrit level is greater than 40%. The PDR shows that practitioners monitor the patient's hematocrit during EPO therapy and, to avoid toxicity, adjust the dose and / or maintain the treatment if the patient's hematocrit approaches or exceeds the upper limits of a target range. Therefore, the skilled worker, guided by the teachings of the present invention, should be able to administer sufficient doses of EPO to obtain a therapeutic effect while avoiding complications due to toxicity.
In one embodiment, the long-acting EPO of the present invention is administered chronically or systemically in a dose of about 0.1 g / kg of body weight to about 100 μg / kg of body weight per administration. For example, about 1 μg / kg of body weight to about 5 g / kg of body weight is contemplated for dosing once a week in the treatment of cancer patients receiving chemotherapy. In another embodiment, the dose of the long-acting EPO is about 5 g / kg of body weight to about 50 μg of body weight per administration. In still another embodiment, the long-acting EPO is administered in an amount of about 50 [mu] g / kg body weight to about 30 g / kg body weight per administration. In still 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 g / kg of body weight to about 0.75 μg / kg of body weight of the long-acting EPO may be effective if administered once a week for the treatment of anemia in patients with CRF.
The effective dose is preferably sufficient to improve serum levels of long-acting EPO greater than about 10,000 mU / mL (80 ng / mL). In one embodiment, the effective dose achieves a serum level of the long-acting EPO of about 15.00 mü / mL (120 ng / mL) or greater. In another embodiment, the effective dose achieves a serum level of the long-acting EPO of approximately 20,000 mü / mL (160 ng / mL). Serum levels or concentrations are preferably measured and achieved at about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 hours, or combinations of these after administration. The doses may be repeated as deemed necessary by the person skilled in the art. For example, the administration may be repeated daily, whenever clinically necessary, or after an appropriate interval, for example every 1 to 12 weeks, preferably every 1 to 3 weeks.
Because the long-acting EPOs of the present invention have an increased serum half-life, their effectiveness in the body also increases. For example, when a mammalian patient is subjected to systemic chemotherapy for cancer treatment, including radiation therapy, administration of the long-acting EPO pharmaceutical compositions of the invention during therapy can decrease anemic problems with less frequent doses and smaller compared to the frequency and quantity of the recombinant EPO compositions present.
And, as already mentioned, when the pharmaceutical compositions of the invention include a long-acting EPO of the present invention or an EPO analogue in combination with a tissue protective cytokine, the compositions can be used to treat anemia and related diseases. in patients who are also at risk of tissue injury. For example, a patient with anemia who is also at high risk of heart disease can be treated with the pharmaceutical compositions of the invention instead of the currently available EPO analogs and to prevent the risk of increased damage from the treatment.
Kits for treatment The invention also provides a pharmaceutical package or kit that includes one or more containers filled with one or more of the ingredients of the pharmaceutical compositions of the invention. In one embodiment, the effective amount of the long-acting EPO and the accepted carrier for pharmaceutical use can be packaged in a vial for a single dose or other container.
When the pharmaceutical composition of the invention is adapted for parenteral administration, for example, the composition can be stored in a lyophilized state. Thus, the kit can include the lyophilized composition, a sterile liquid carrier and syringe for injections. In a modality, the kit includes a vial containing enough freeze-dried material for several treatments so that the one administering weighs a sufficient amount of material and adds a sufficient amount of the carrier for each transfer of treatment. In another embodiment, the kit may contain a plurality of ampules each containing specific amounts of the lyophilized material and a plurality of packages each containing specific amounts of carrier, so that the administrator needs only to mix the contents of a vial and a container of carrier for each transfer of treatments without measuring or weighing. In . still another embodiment, the kit contains an autoinjector that includes an injectable solution of a long-acting EPO of the invention. In still another embodiment, the kit contains at least one vial with the lyophilized composition, at least one container of carrier solution, at least one container with a local anesthetic and at least one syringe (or the like). The ampules and packages are preferably sealed.
When the pharmaceutical compositions of the invention are to be administered by infusion, the kit preferably contains at least one ampoule with the pharmaceutical composition and at least one bottle of infusion with sterile, pharmaceutical grade water or saline.
A kit according to the present invention can also contain at least one buccal piece or devices specially adapted for direct inhalation to the lung, such as aerosols under pressure, nebulizers or insufflators. In this aspect of the invention, the kit can contain the device for direct inhalation to the lung, which contains the pharmaceutical composition, or the device and at least one ampoule of aqueous or oily solutions of the long-acting EPO of the present invention.
When the pharmaceutical composition of the long-acting EPO of the invention is adapted for oral, transdermal, rectal, vaginal or nasal administration, the kit preferably contains at least one ampoule containing the active ingredient and at least one auxiliary for administration. Examples of adjuvants for administration include, but are not limited to, measuring spoons (for oral administration), sterile cleaning pads (for transdermal administration), and nasal aspirators (for nasal administration). These kits may contain a single dose of the long-acting EPO (acute treatment) or a plurality of doses (chronic treatment).
In addition, the kit can be equipped with one or more types of solutions, for example, the long-acting EPO pharmaceutical compositions of the invention can be prepared in an albumin solution and a polysorbate solution. If the kit contains the polysorbate solution, the words "albumin-free" will preferably appear on the package labels as well as on the main faces of the kit.
In addition, the kit may also contain a notice in the form prescribed by a governmental institution that regulates the manufacture, use and sale of pharmaceutical or biological products, whose notice shows the approval of the institution for the manufacture, use or sale for administration to humans .
EPO Assays The present invention also relates to assays for determining the erythropoietic and protective tissue capacities of the long-acting EPOs of the present invention, as well as EPO analogs that are used in the various pharmaceutical compositions of the EPO. the present invention. For example, the erythropoietic effect of a long-acting EPO can be verified by the use of a TF-1 assay, which will be described in greater detail in example 2. The protective properties of the tissue of the EPO composition can be examined using assays in vitro and in vivo assays, which are described in more detail below. In addition, the present invention also contemplates tests to determine not only whether a specific EPO compound has tissue protective activity, but also whether the EPO compound acts as an antagonist with respect to endogenous EPO.
The assays of the invention are preferably designed to be carried out in a short time using a minimum amount of the EPO compound. Furthermore, the assays provided herein are suggested to be non-limiting, in view of one skilled in the art will be aware of other assays useful for determining the erythropoietic and protective tissue capacities of EPO compounds.
Erythropoetic activity assays The erythropoietic attributes, ie the ability to regulate hematocrit levels, of a specific EPO compound can be determined using some assays. In one embodiment, a TF-1 cell line can be used to determine whether a specific EPO compound has erythropoietic activity. It is possible to pack, wash and resuspend the cells at a concentration of 10 5 cells in 1 mL of medium, with the recombinant EPO and an EPO compound of interest added in specific concentrations. Individual cultures can be maintained for 24 hours, at which time the number of cells is determined using a formazan reaction product (CellTiter, Promega, Madison, I).
The potency of the EPO compound of interest is first tested in vivo by observing its effect on hemoglobin concentration using female BALB / c mice. The animal is administered 500 U / kg of body weight of the EPO, the EPO compound of interest, or an equal volume of vehicle subcutaneously, three times a week for a total of 3 weeks (sufficient time to observe a erythropoietic response). It is determined that an EPO or erythropoietic compound increases the hemoglobin concentration in the serum of the mice.
Another potency assay can be obtained using in vitro TFI erythroleukemia cells. The EPO compound of interest is erythropoietic if the relative number of TFI cells increases beyond the control. In addition, those skilled in the art will appreciate that other assays are available to determine erythropoietic activity. For example, the European Pharmacopoeia describes at least two assays useful for determining the erythropoietic activity of an EPO compound, which include tests in ex hypoxic mice and reticulocyte assays.
Tests of tissue protective capacity based on EPO receptors In one embodiment, the tissue protective capacity assays of the present invention rely on the tissue protective receptor for EPO. Once the sequence for the tissue protective receptor is isolated, it is possible to use a variety of assays to determine the tissue protective capacity of a specific EPO compound. As is known to those skilled in the art, the type of assay employed depends to a large extent on the weight of the EPO compound.
For example, the assays can be competitive assays or sandwich assays or steric inhibition assays. Competitive assays depend on the ability of a tracer analog to compete with the analyte in the test sample for a limited number of binding sites in a common binding counterpart. As used herein, the term "analyte" refers to the EPO compound of interest and that is to be tested for the protective activity of the tissue. The term "binding partner" refers to any protein that binds to the analyte (usually the receptors for EPO). When used herein, "tracer" refers to labeled reagents, such as the analogue of the labeled analyte, analog of the immobilized analyte, labeled tagged part, immobilized binding counterpart, and steric conjugates. The tracer that is used herein can be any functionality that can be detected and that does not interfere with the binding of the analyte and its binding counterpart. Non-limiting examples include portions that can be detected directly, such as labeled fluorochrome, chemiluminescent and radioactive, as well as portions that can react or be derived to be detected, such as enzymes. Suitable tracers may be radioisotopes P32, C14, I125, H3, I131, and mixtures thereof; fluorophores, such as rare earth chelates, fl orescein, fluorescein derivatives, rhodamine, rhodamine derivatives, dansyl, umbeliferota luciferase (firefly luciferase and bacterial luciferase (US Patent No. 4,737,456)), luciferin, 2,3-dihydroftalazindiones , horseradish peroxidases (HRP), alkaline phosphatase, β-galactosidase, glucoamylase, lysozyme, saccharide oxidases (glucose oxidase, galactose oxidase and glucose-6-phosphate dehydrogenase), heterocyclic oxidases (uricase and xanthine oxidase) coupled with an enzyme that uses hydrogen peroxide to oxidize a dye precursor such as HRP, lactoperoxidase, microperoxidase and mixtures thereof; biotin / avidin; centrifugation labels, bacteriophage labels, stable free radicals; and combinations of these. In one embodiment, the tracer is at least horseradish peroxidase or alkaline phosphatase.
One skilled in the art knows the methods for covalently joining the tracers to the proteins or polypeptides. For example, coupling agents such as dialdehydes, carbodiimides, dimaleimides, bisimidates, bis-diazotized benzidine and the like can be used for labeling with the fluorescent, chemiluminescent and enzyme labels mentioned above, some of which are described in US Patent Nos. 3,940,475, and 3,645,090, the full descriptions of which are incorporated by reference herein.
The immobilization of the reagents, that is, the separation of the binding part from any analyte that is free in solution, is necessary for a sandwich assay, and can be carried out by insolubilizing the counterpart of the binding or analog of the analyte before the test procedure, or by absorption to a water-insoluble matrix or surface (US Patent No. 3,720,760), by covalent coupling, such as crosslinking glutaraldehyde, or by insolubilizing the counterpart or analog thereof, for example by immunoprecipitation .
Thus, the binding counterpart can be insolubilized before or after the competition, and the tracer and the analyte bound to the binding part are separated from the unbound tracer and analyte. This separation can be carried out by decanting (when the counterpart was preinsolubilized) or by centrifugation (when the counterpart of the binding precipitated after the competitive reaction). The amount of the analyte in the test sample is inversely proportional to the amount of bound tracer, as measured by the amount of the marking substance. It is possible to prepare dose-response curves with the known amounts of the analyte and compare them with the results of the test to quantitatively determine the amount of analyte present in the test sample. When used with enzymes as tracers, assays are usually called ELISA systems.
A successive sandwich test, for example, a counterpart of the immobilized binding is used to absorb the analyte in the test sample, the test sample is washed out, the bound analyte is used to absorb the counterpart of the labeled linkage, and the bound material is then separated from the residual tracer. The amount of bound tracer is directly proportional to the analyte in the test sample. In the "simultaneous" sandwich tests, the test sample does not separate before adding the labeled binding counterpart.
The competitive and sandwich methods employ a step of phase separation, an integrated part of the method, while the steric inhibition assays are carried out in a single reaction mixture. Other competitive assay species, called a "homogeneous" assay, however, do not need phase separation. In an assay like this, a conjugate of an enzyme with the analyte is prepared and used so that when the antianalyte binds to the analyte, the presence of the antianalyte modifies the activity of the enzyme. The tissue protective receptor is conjugated with a bifunctional organic bridge to an enzyme such as peroxidase. The conjugates are selected for use with EPO so that the binding of EPO inhibits or potentiates the enzymatic activity of the tag. This type of assay is commonly known as EMIT.
For the homogeneous test, steric conjugates are used in steric hindrance methods. These conjugates are synthesized by covalently attaching a low molecular weight hapten to a small analyte so that the antibody to the hapten is substantially unable to bind the conjugate at the same time as the antianalyte. The analyte present in the test sample will bind the antianalyte, thereby allowing the antihaptenum to bind by conjugating, resulting in a change in the character of aptene, conjugated, for example, a change in fluorescence when the hapten is a fluorophore .
More information related to weaving protective capacity tests is described in copending US Patent Application No. 10 / 188,905, filed July 3, 2002 and in serial application No. 60 / 456,891, filed on October 25, 2002. April 2003, which are incorporated herein by reference in their entirety.
Function Assays In the absence of sequence identification for the tissue-protective receptor, the tissue protective capabilities of an EPO can be determined using functional, in vivo and in vitro assays. Preferably, a person with ordinary skill in the art would conduct a single in vitro or in vivo assay to determine the protective capabilities of an EPO compound tissue, but in some cases, it may be necessary to perform both to ensure that the compound present the same tissue protective capacities in vitro and in vivo.
During practice, a person with ordinary skill in the art will be able to determine whether an EPO analogue has at least one additional N-linked carbohydrate chain and / or at least one additional 0-linked carbohydrate chain, using a combination of assays described by the present invention. First, in vitro tests, such as assays on P19 cells and mouse motoneurons, could be used to determine if the EPO compound of interest presents the protective capabilities of tissue. Then, in in vivo studies, such as rat focal ischemic models, epilepsy with bicuculline, or spinal cord trauma, they could be used to verify the results of in vitro tests.
The in vitro models contemplated by the present invention include, but are not limited to, those which are used to determine the lack of the protective capacities of the tissue of the above hyperglycosylated erythropoietin: the P19 cell assay, the rat motor neuron cell assay and the cDNA microassay, which are described in more detail below and are further shown in Example 2. The examples are intended not to limit the invention, since one skilled in the art will realize that there are other in vitro assays suitable for determining the tissue protective capabilities of EPO compounds. In general, the EPO compound would be considered protective of the tissue if, in comparison with a control, it maintains or increases the viability of the cell. Erythropoietin will be considered an antagonist if, in comparison with a control, it affects in a detrimental way the viability of the cells within the assay.
A. In vitro assay based on the line, of P19 cells In one embodiment, the in vitro tissue protective capacity assay is based on a P19 cell line. For example, P19 cells can be maintained undifferentiated in DMEM supplemented with 2 mM L-glutamine, penicillin G 100 ü / mL, streptomycin sulfate 100 μg / mL (Gibco) and 10% fetal bovine serum (Hyclone Laboratories), which Contain 1.2 g / L of NaHCO3 buffer hepes 10 mM. The serum-free medium may contain some components as above, with the exception of 5 μg / mL of insulin, 100 μg / mL of transferrin, 20 nM progesterone, putrecin 100 μ? and 30 nM Na2Se03 (Sigma) instead of fetal bovine serum.
Cells that react with 50% confluence are treated overnight with recombinant EPO and / or an EPO compound of interest, dissociated with trypsin, washed in serum-free medium and plated in 25 cm tissue culture flasks at a final density of 104 cells / cm2 in serum-free medium only, or with additions prior to treatment. The viability of the cells can be determined by trypan blue exclusion.
As is known to the person skilled in the art, the addition of recombinant EPO can prevent cell loss after removing the serum in undifferentiated neuronal type P19 cells. For example, recombinant EPO rescues up to 50% of neuronal cells from death if used in a concentration of 0.1 ü / mL to 100 U / mL. Thus, to be considered protective of the tissue, the EPO compound of interest should rescue more P19 cells from death compared to the control, preferably it should rescue approximately 25% to approximately 50% of the cells, more preferably around 40% up to about 50% of the cells.
B. In vitro test in rat motoneurons. In another embodiment, a rat motor neuron test is used to determine the protective capacity of the tissue, in vitro, of an EPO compound of interest. For example, it is possible to obtain primary motoneuoronas using spinal cord from Spraugue Dawley 15-day rat embryos and purify them by immuno-parenchyma. The cells are preferably seeded at a low density (20,000 cells / cm 2) on glass objects covered in 24 mm well plates pre-coated with poly-DL-ornithine and laminin and with a complete culture medium content (neurobasal, P17 (2%), 0.5 mM L-glutamine, 2% horse serum, 25 μ 2-mercaptoethanol, 25 μ glutamate, 1% penicillin and streptomycin, 1 ng / mL BDNF). After a time, preferably around 6 days, the EPO (10 U / mL) and the EPO compound of interest (10 U / mL) or vehicle can be added to the cultures (preferably around 5 days before the determination of the neuronal density survivor). The medium is then discarded and the cells can be fixed with 4% formaldehyde in PBS for 40 minutes, permeabilized with 0.2% Triton X-100, blocked with 10% fetal bovine serum in PBS, incubated with antibodies against neurofilaments. non-phosphorylated SMI-32; 1: 9000) overnight, and are observed using the avidita-biotin method with diaminobenzidine. The viability of motoneurons can be assayed morphologically by counting SMI-32 positive cells.
The mixed primary cultures of motoneurons characteristically suffer apoptosis during maintenance culture conditions. The addition of recombinant EPO (10 U / mL) to the culture medium 5 days before the cell number assessment, as demonstrated, significantly increases the number of primary motor neurons observed at 5 days. Thus, to be considered protective of the tissue, the recombinant EPO compound of interest preferably rescues at least the same number of motor neurons compared to a control. In one embodiment, the EPO compound of interest is considered protective of the tissue if more motoneurons are saved from death during maintenance culture conditions compared to the control.
C. In vitro assay based on cDNA microarray. Another in vitro assay to determine the protective tissue capacity of an EPO compound of interest is a cDNA microarray. This arrangement can be used to determine whether the recombinant EPO and the EPO compound of interest modify gene expression differently in P19 cells. The mRNA isolated from undifferentiated P19 cells may show a different model of gene modulation estimated from a microarray of 1200 mouse cDNA, depending on the exposure of the EPO. For example, the expression of 1200 genes in pl9 cells can be measured by using Clontech nylon membrane tests (Atlas Mouse 1.2). Cells (10 / sample) can be treated overnight with saline, recombinant EPO, an EPO compound of interest (1 mü / mL), or a mixture of these. The cells are then lysed for A extraction or subjected to serum deprivation for 3 hours (always in the presence of the same cytokine added during the previous treatment). After extraction of the normal total RNA by column chromatography, with treatment of the DNase in the column, it is possible to purify the polyA + RNA. It is then possible to build probes in the presence of [p32] -ATP. Labeled probes, preferably with beads of 20 million or greater, can be hybridized to nylon cDNA membranes at 68 ° C. The membranes are washed and exposed to X-ray film. The intensity of the radioactive signals can be measured with a Phosphor Imager and analyzed with the Atlas Image 2.0 computer program (Clontech).
In vivo assays contemplated by the present invention include, but are not limited to, tissue protection assays that are used to evaluate EPO compounds such as the focal ischemia model and the intra hippocampal biculin model. In addition, an in vivo model to assess tissue protection includes spinal cord injury trials. In addition, the various assays described in International Publication No. WO / 02053580 and US Patent Publications Nos. 2002/0086816 and 2003/0072737 are contemplated for use with the present invention.
D. In vivo assays based on focal ischemia model In one embodiment, the in vivo assay that is used to determine the tissue protective capabilities of a specific EPO compound is based on a model of focal ischemia. For example, male Sprague-Dawley rats (~ 250 g) can be used with a 3-vessel focal ischemia model. In short, rats can be anesthetized with pentobarbital (60 mg / kg body weight) and kept at an internal temperature of 37 ° C using a layer of water. The right carotid can be included for two fractures and cut transversely. A drill hole together with a rostrate in the right orbit allows observation of the middle cerebral artery, which can be cauterized distal to the nasal artery. To produce a penumbra surrounding this fixed MCA lesion, the carotid versus lateral artery can be occluded for 1 hour using traction provided by fine forceps. At the beginning of the reversible occlusion of the carotid it is possible to administer saline, recombinant EPO (5000 U / kg of body weight) or the EPO compound of interest (5000 U / kg of body weight).
After 24 hours, the brains are separated and serial sections 1 mm thick are cut through the entire brain using a brain matrix device (HarvardApparatus). Each section can then be subsequently incubated in a solution of 2% triphenyltetrasodium chloride (w / v) in 154 mM NaCl for 30 minutes at 37 ° C. The volume of the lesion can be determined using a computerized system for image analysis (MCID, Imaging Research, St. Catharines, Ontario, Canada). In this assay, the EPO compound of interest is considered to be neuroprotective if it reduces the infarct volume due to the focal MCA ischemia in the rat to the same or greater degree than the recombinant EPO.
E .. In vivo assay based on the intra-hippocampal epithelial epithelium model In another embodiment, the protective tissue capacity of an EPO compound is determined in vivo with intra-hippocampal biculin experiments. For example, male Sprague-Dawley rats (250-280 g) are housed at a constant temperature (23 ° C) and relative humidity (60%) with free access to food and water and a fixed cycle of 12 light / dark hours. The rats are surgically implanted with cannula and electrodes under stereotaxic guidance as described in Besan A., et al., J. Neurosci, 19, 5054-65 (199). In summary, rats are anesthetized using Equithesin (1% pentobartital / 4% doral hydrate, 3 mL / kg ip). Two screw electrodes are placed bilaterally on the parietal cortex, together with a ground connection placed on the nasal sinus. Isolated microbipolar wire electrodes (60 μp?) Are then implanted bilaterally in the dentate gyrus of the dorsal hippocampus (ceptal pole) and a cannula (22 gauge) can be unilaterally placed in the upper part of the dura for infusion of drugs intrahipocampo intra-ventricular brain. The coordinates of the bregma for the implantation of the electrodes must be (mm) antero-posterior-3.5; lateral 2.4 and 3 below the dura with the nasal bar set to minus 2.5. Paxinos, G. & Watson, C., The at Brain in Stereotaxic Coordinates, Academic Press, New York (1986). The electrodes can be connected to a multi-contact plug (March Electronics, NY) and, together with the injection cannula, secured to the skull by acrylic dental cement.
The experiments are preferably carried out 3 to 7 days after surgery when the animals have fully recovered. The animals are then administered recombinant EPO or the EPO compound of interest (both at 5000 U / kg of body weight) or vehicle intraperitoneally 24 hours and again at 30 minutes before induction of seizures with bicuculline. Methods for recording the EEG and intracellular injection of drugs have been previously described by Venazzi A., et al., J. Pharmacol Exp. Ther 239, 256-63 (1986). In summary, the animals are allowed to acclimate in Plexiglas cages (25x25x60 cm) for a minimum of 10 minutes before starting the EEG recording (4-channel EEG polygraph, model DP8, Battaglia Rangoni, Bologna Italy). After approximately 15 minutes to approximately 30 minutes, the EEG is recorded continuously for 120 minutes after infusion of bicuculline methylodide 0.8 mmol / 0.5 μ? . all injections were made to non-anesthetized rats using a needle (caliber 28) protruding 3 mm below the cannula.
Seizures can be measured by EEG analysis, which has previously shown that it provides a sensitive measure of the anticonvulsant activity of drugs. Vezzani A., et al., J. Pharmacol. Exp Ther, 239, 256-63 (1986). For the purpose of this test, seizures consist of the simultaneous presence of at least two of the following alterations in all four record conductors: high frequency and / or multi-spike complexes and / or high-voltage synchronized spike and wave activity. The formation of the synchronous spike can be observed between mixed with seizures. The parameters chosen to quantify seizures are preferably the latency of the first seizures (onset of seizure), the total time elapsed in epileptic activity (determined by adding together the duration of the ictal episodes, duration of the seizure), and the spiking activity during the period of EEG registration (seizure activity).
The seizure model with bicuculline intrahipocampo using EEG activity as a reading has been shown to be a sensitive and specific predictor of the anticonvulsant potency of the drugs. Vezzani A., et al., J. Pharmacol. Exp. Ther 239, 256-63 (1986). Thus, to be considered protective of tissue, the EPO compound of interest should reduce the frequency and severity of seizures to the same degree or greater than recombinant EPO.
F. In Vivo Assay Based on the Rat Model with Reversible Acute Glaucoma In yet another in vivo assay according to the present invention, a rat model with reversible acute glaucoma can be used to determine the protective capacity of the tissue of the specific EPO compounds that they are interesting For example, because retinal cells are very sensitive to ischemia, many of these cells will die after 30 minutes of ischemic stress. As such, to test whether EPO compounds of interest, administered peripherally, show sufficient tissue protective activities to protect cells responsive to ischemia, a rat model with reversible, acute glaucoma, as described by Rosenbaum et al. ., Vis. Res. 37: 3443-51, 1997. In particular, saline can be injected into the anterior chamber of the eye of adult male rats at a pressure above the systemic arterial pressure and maintained for 60 minutes. The animals are then administered saline or 5000 ü EPO / kg body weight intraperitoneally 24 hours before induction of ischemia, and is continued as a daily dose for 3 more days.
It is possible to perform electroretinography in rats adapted to darkness one week after treatment to determine if the EPO compound of interest possesses tissue protective activity. If the EPO is protective of the tissue, there should be good conservation of the activity in the electroretinogram, contrary to the animals treated with saline only.
G. Myocardial Infarction Assays
[0104] Myocardial infarction assays are also contemplated for use with the present invention to determine whether an EPO compound exhibits the protective activity of the tissue in general or specifically in the heart. For example, adult male rats can receive EPO (5000 U / kg of body weight) 24 hours before being anesthetized and prepared for coronary artery occlusion. One more dose of EPO may be given at the beginning of the procedure, at which time the left main coronary artery is occluded for 30 minutes and then released. The same dose of EPO is provided daily for one week after treatment, at which time the animals are studied for cardiac function. Animals receiving a substitute injection (saline) will demonstrate a large increase in diastolic pressure at the left end, indicative of a rigid, dilated heart secondary to myocardial infarction, while animals receiving the EPO compound of interest should show no decrease in cardiac function, compared to the controls worked in a similar way (significant difference at the p <0.01 level) if the EPO is protective of the tissue.
H. Spinal cord injury assays It is also possible to use spinal cord injury assays with the present invention to evaluate the protective abilities of the specific EPO compounds of interest. In particular, the use of rat spinal cord compression is contemplated with the present invention. Wistar rats (female) weighing approximately 180 g to about 300 g are preferably used in this study. Animals are preferably fasted for 12 hours before surgery, subjected to human fastening and anesthetized with an intraperitoneal injection of sodium thiopental (40 mg / kg body weight). After skin infiltration (bupivacaine 0.25%), a single complete level (T-3) of laminectomy is performed through a 2 cm incision with the help of a dissecting microscope. Traumatic spinal cord injury is induced by the extradural application of a temporary aneurysm clip by exerting a closing force of 0.6 newton (65 grams) on the spinal cord for 1 minute. After removing the clip, the incision of the skin is closed and the animals are allowed to recover completely from the anesthesia and return to their cages. The rats are monitored continuously with vedic palpation at least twice a day until the spontaneous avoidance is restarted.
The animals in a control group receive normal saline (by intravenous injection) immediately after the incision is closed, the remaining animals receive the EPO compound of interest in an amount of 16 micrograms / kg of body weight iv. The neurological motor function of the rats is then evaluated using a locomotive classification scale. In this scale, the animals are assigned in a register that ranges from 0 (unobserved movements of the hind limbs) to 21 (normal gait). Rats are tested for functional deficiencies at the time, 12, 24 hours, 48 hours, 72 hours and one week after the injury by the same examiner who is blind to the treatment each animal receives. If the EPO compound of interest is tissue protector, the rats receiving EPO should show a better and better overall recovery of the lesion compared to rats that received saline injection.
J. Assessment of spinal cord ischloraemia in rabbits In another modality, ischemia tests in the rabbit spinal cord allow evaluation of the protective capacity of the tissue. For example, New Zealand white rabbits (36, 8-12 months old, male) weighing 1.5 kg to 2.5 kg are used in this study. The animals are fasted for 12 hours and held humanely. The induction of anesthesia is through 3% halothane in 100% oxygen and maintained with 0.5% to 1.5% halothane in a mixture of 50% oxygen and 50% air. An intravenous catheter (22 gauge) is placed in the vein of the left ear. Ringer's lactate is infused at a rate of 4 mL / kg of body weight per hour during the surgical procedure. Before the operation, cefazolin 10 mg / kg of body weight is administered intravenously for prophylaxis of infection. The animals are placed in the right lateral decubitus position, the skin is prepared with povidone iodide, it is infiltrated with bupivacaine (0.25%) and an incision is made of the skin on the flank parallel to the spine at the twelfth rib level. After the incision of the skin and the subcutaneous thoracolumbar fascia, the longissimus lumborum and iliocostalis lumborum muscles retreat. The abdominal aorta is exposed by a left retroperitoneal approach and is mobilized just inferior to the left renal artery. A piece of PE-60 tubing is wrapped around the aorta immediately distal to the left renal artery and both ends pass through a larger cork tube. When pulling the PE tubing, the aorta is occluded without trauma for 20 minutes.
Heparin (400 IU) is given as an intravenous bolus before aortic occlusion. After 20 minutes of occlusion, the tube and catheter are removed, the incision is closed and the animals are monitored until complete recovery, at which time they are evaluated in series for neurological function. A control group of animals receives normal saline intravenously immediately after releasing the aortic occlusion. Another group of animals receives 6.5 body weight of the EPO compound of interest intravenously, immediately after the impact (N-6 for each group).
Motor function is assessed according to the criteria of Drummond and Moore by a researcher blinded to treatment at the time, 24 hours, and 48 hours after reperfusion. Each animal is assigned a register from 0 to 4 as follows: 0 = paraplegic without evident lower limb motor function; 1 = poor limb motor function, only weak antigravity movement; 2 = moderate function of the lower extremities with good antigravity resistance but inability to bring the legs to the body; 3 = excellent motor function with the ability to pull the legs under the body and jump, but not normally; 4 = normal motor function. The urinary bladder is evacuated manually in the paraplegic animals twice a day.
If the EPO compound of interest protects the tissue, the animals that received the EPO should show a general recovery of the lesion faster and better than the animals that received the saline injection.
As described in summary in the foregoing, some tissue types possess receptors for EPO and, therefore, may be responsive to the tissue protective effects of EPO. Thus, depending on the clinical application proposed for the EPO compound of interest, one skilled in the art should recognize that it is possible to perform similar in vitro assays involving these additional responder cells, or in vivo assays involving associated organs can also be performed. . For example, "in vitro assays based on serum deprivation can be carried out using myocardial, retinal and Leydig cells and a protocol similar to that described above for the P19 assay.
In vivo assays can be targeted to individual organs as well. For example, to evaluate an effect of EPO on the cells of the retina, one skilled in the art can perform the retinal ischemia test described above. Furthermore, to evaluate an effect of an EPO analog on myocardial cells, an expert could easily modify the model of myocardial infarction described above. Those skilled in the art will have sufficient skills to choose the appropriate test or model to assess whether a specific EPO possesses tissue protective activities with respect to a cell, tissue or organ that elicits an erythropoietic response.
EXAMPLES The following examples are only illustrations of the preferred embodiments of the present invention, and should not be considered as limiting the invention, the scope of which is defined by the claims.
Example 1: Chemically modified EPO A oxidation of the sugar chains.
The EPO sugar units can be converted to acids by the following procedure. EPO and a quantity of sodium periodate sufficient to provide the desired amount of oxidation (the greater the amount of sodium periodate the greater the degree of oxidation) can be placed in a 100 mM sodium acetate buffer. This solution is then incubated on ice for approximately 20 minutes and dialyzed perfectly using distilled water. The product is then removed from the dialysis tubing and collected in a new tube (product I).
A quantitative Benedict 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 until a final volume of one liter. Then several drops of methylene blue are added to the Benedxct quantitative solution.
Then the product I can be added to the Benedict quantitative solution until the color of the solution becomes clear indicating that the solution is completely oxidized. Then the solution is desalinated and concentrated using an ultra free centrifugal filtration unit. The sample (product II) can then also be dialyzed perfectly using distilled water.
B. Oxidation of the asialo form of EPO with galactose oxidase 50 to 500 μg of the asialo form of EPO, 10 μL of galactose oxidase 1 U / uL, and 100 μL of 10 mM sodium phosphate buffer can be mixed in a 15 mL conical centrifuge tube (total volume 110 μ?). This mixture can then be incubated for 2 hours at 37 ° C, at which time the solution can be perfectly dialyzed using distilled water. The product can be removed from the dialysis tubing and collected in a new tube (product III).
A quantitative solution of Benedict (as already described) dissolves in distilled water to a final volume of one liter. Then several drops of methylene blue are added to the Benedict quantitative solution.
The product III is added to the Benedict quantitative solution until the color of the solution becomes clear indicating that the solution is completely oxidized. The solution is then desalted and concentrated using an ultra free centrifugal filtration unit. Then the sample (product IV) can be dialyzed perfectly using distilled water).
C. Sulfation of EPO EPO is dissolved in?,? - dimethylformamide (DMF-SA) at 4 ° C. Then add?,? -dicyclohexylcarbodiimide (DCC) dissolved in DMF, and the solution is stirred for 4 hours at 4 ° C. Crushed ice can be added and the pH adjusted to 7.5 with 10 N NaOH. The volume of the solution is adjusted and the sample is centrifuged 100 xg for 15 minutes in a H-type H2 centrifuge (DAMONIEC, Needham Hts. Massachusetts). Then the supernatant is dialysed extensively. More information regarding sulphation is described in S. Pongor et al. , Preparation of High-Potency, Non-aggregating Insulins üsing a Novel Sulfatation Procedure, Diabetes, Vol. 32, No. 12, December 1983, the integrity of which is incorporated as a reference here.
D. Union of PEG chains to EPO EPO can be modified by linking PEG chains to oxidized carbohydrates, such as those that were obtained earlier in step A (product I). The degree of modification can be regulated by varying the concentration of periodate during oxidation.
PEG-EPO conjugates can be prepared by first oxidizing the EPO (2-4 mg / mL in 50 mM sodium acetate) for 30 minutes at room temperature with 1 mM to 100 mM sodium metaperiodate (Sigma). Then the phosphate buffer is removed by buffer exchange in 100 mM sodium acetate, pH 5.4.
Then methoxy-PEG-hydrazide of different molecular weights (Nektar Therapeutics) is added in a molar excess of 5 to 100 times (polymer: protein). The intermediate linkage of hydrazine can then be further reduced by the addition of 15 mM sodium cyanoborohydride (Sigma) and the reaction overnight at 4 ° C. The resulting conjugates are then fractionated / purified by the known techniques.
E. Binding of PEG chains to asiatic EPO An asialo form of EPO can be modified by binding of the PEG chains to the newly created terminal galactose residues after oxidation with galactose oxidase, as was obtained earlier in B (product III ).
Recombinant human EPO (rhuEPO) can be desalted using Sialidase A (Prozyme, Inc.) according to the manufacturer's protocol. The chemical modification is preferably confirmed by running the reaction product on a SDS polyacrylamide gel. Staining of the resulting bands should show that the modified EPO has an apparent molecular weight of approximately 31 kDa. While the unmodified EPO has a molecular weight of approximately 34 kDa. The sialic acid residues remaining in the EPO are preferably less than 0.1 mol / mol EPO.
After the asialo form of EPO is obtained, the galactose residues freshly exposed in EPO (2-4 mg / mL in 10 mM sodium phosphate buffer) can be oxidized with 100 units of galactose oxidase in PBS (Sigma) by mL of EPO solution. The reaction mixture can then be incubated at 37 ° C for 2 hours.
The phosphate buffer is then removed by buffer exchange in 100 mM sodium acetate, pH 5.4. Then methoxy-PEG-hydrazide of different molecular weights (Nektar Therapeutics) is added in a molar excess of 5 times to 100 times (polymer: protein). The intermediate hydrazine linkage is then preferably further reduced by the addition of 15 mM sodium cyanoborohydride (Sigma) and allowed to react overnight at 4 ° C. the resulting conjugates can then be fractionated / purified by known techniques.
F. Binding of PEG chains to the EPO soya A similar form of EPO can be modified by the binding of the PEG chains to the newly created terminal galactose residues after oxidation with galactose oxidase, as previously obtained in B (product III ).
RhuEPO (1 mg) can be disalized using neuraminidase (Seikagaku Corporation of Japan, 1 μl of lyophilized powder are dissolved in 100 μM of 75 mM NaP04 (6.5)) in a ratio of 1 mg of EPO to 0.05 units of neuronaraidase (5 mg). μ $). Then 5 units (5 μ $) of galactose oxidases (450 μ ?. dissolved in 75 mM NaP04 (pH 6.5) (Sigma)) are added to the mixture.
The phosphate buffer is then removed by buffer exchange in 100 mM sodium acetate, pH 5.4. Then PEG-NH2 (molecular weight 750, Nektar Therapeutics) and 15 mM sodium cyanoborohydride (Sigma) are added to the mixture and left to react overnight at 4 ° C. PEG-NH2 is preferably added in a 250-fold molar excess (polymer: protein) (80 mg of PEG-NH2). The resulting conjugates can then be fractionated / purified by the known techniques.
Example 2: Functional assays A. Erythropoietic assay In the following test, the erythropoietic attributes, that is, the ability to control hematocrit levels, of a specific EPO compound were determined.
TF1 is a line of human erythroleukemic cells with complete dependence on growth factors, including EPO. Kitamura et al., Blood 73, 375-80. TF1 cells were obtained from the ATCC and maintained in RPMI 1640 with the following: 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 of GM-CSF and 10) of fetal bovine serum until the moment of the experiment. TF1 cells obtained during active growth were packed, washed three times with medium alone and resuspended at a concentration of 10 5 cells 1 ml of medium, with or without GM-CSF, with EPO or an EPO analog having at least one chain additional N-linked carbohydrate and / or at least one additional O-linked carbohydrate chain added at specific concentrations. The individual cultures were maintained for 24 hours and the number of cells was determined using a formazan reaction product (CellTaiter; Promega, WI). According to the manufacturer's protocol.
First, the potency of the EPO compound was evaluated in vivo by observing its effect on the concentration of hemoglobin using female BALB / c mice. The animals were administered 500 U / kg of body weight of the EPO, the EPO compound of interest or an equivalent volume of vehicle, subcutaneously, three times a week, for a total of 3 weeks (a sufficient time to observe a erythropoietic response). An EPO compound is determined to be erythropoietic if it raises the concentration of hemoglobin in the serum of the mouse. Another assessment of potency was obtained in vitro using erythroleukemic TF1 cells. Studies confirmed that an EPO is erythropoietic if the relative TFI cell number increases beyond the control.
Those skilled in the art will appreciate that other assays, such as the test in ex hypoxic mice and the meticulosite assay (European Pharmacopeia) can conveniently be used in the present invention to determine erythropoietic activity.
B. Tissue Protection Assay
[0111] The following test was used to determine the tissue protection attributes of an EPO analogue, at least one additional N-linked carbohydrate chain and / or at least one additional O-linked carbohydrate chain.
Neuronal cultures were established from the hippocampus of rat fetuses of 18 days. The brains were removed and released from the meninges and the hippocampus was isolated. The cells were then dispersed by incubation for 5 minutes at 37 ° C in a 2.5% trypsin solution followed by titration. The cell suspension was diluted in serum-free neurobasal medium containing 1% supplemental B-27 (Gibco, Rackville, MD USA) and plated onto polyornithine-coated objects at a density of 80,000 cells per object cover. The cells were then pretreated with EPO overnight and then exposed with or without: 1) EPO, 2) an EPO analogue having at least one additional N-linked carbohydrate chain and / or at least one carbohydrate chain linked to Or add, or 3) a form of EPO to TMT 5 μ for 24 hours. The cultures were used between days 10 and 14 in vi tro.
The viability of the cells was measured by the assay with 3- (4-bromide., 5-dimethylthiazol-2-yl) -2,5-diphenyltriazolium (MTT). Densito, F. , and Lang, R. 1986. Rapad Colormetric improved reability. J. Immunol Methods 89: 271-277. Briefly, the tetrazolium salt of MTT was dissolved in serum-free medium to a final concentration of 0.75 mg / mL and added to the cells at the end of the treatment for 3 hours at 37 ° C. The medium was then removed and the formazan was extracted with 1N HCl: isopropanol (1:24). The absorbance at 560 nm was read on a microplate reader.
As seen in Figure 1A, the EPO analog having at least one additional N-linked carbohydrate chain and / or at least one additional O-linked carbohydrate chain did not show a tissue protective function.
The invention described and claimed herein is not limited in scope by the specific embodiments described herein, since these embodiments are intended to exemplify the different aspects of the invention. Any equivalent mode is intended to be within the scope of this invention. In fact, various modifications of the invention in addition to those shown and described herein will be apparent to those skilled in the art from the aforementioned description. These modifications are also intended to fall within the scope of the appended claims. All references mentioned herein are incorporated by reference herein in their entirety for all purposes.

Claims (27)

  1. CLAIMS 1. A method for regulating the level of hematocrit in humans, comprising the steps of: providing an erythropoietin product having a serum media longer than rhuEPO and containing the protective functionality of the tissue; and administering an effective therapeutic amount of the erythropoietin product.
  2. 2. The method of claim 1, wherein the step of providing an erythropoietin product further comprises the step of: modifying the rhuEPO with at least one chemical modification to at least one of the N-linked oligosaccharide chains or the O-linked oligosaccharide chain, wherein the chemical modification consists of oxidation, sulfation, phosphorylation, PEGylation or a combination of these.
  3. 3. The method of claim 1, wherein the step of administering an effective therapeutic amount of the erythropoietin product consists in administering the erythropoietin product in a molar amount less than rhuEPO to obtain a comparable target hematocrit.
  4. 4. The method of claim 1, characterized in that the serum half-life is at least about 20% greater than the serum half-life of rhuEPO.
  5. 5. The method of claim 1, characterized in that the serum half-life is at least about 40% greater than the serum half-life of rhuEPO.
  6. 6. A synthetic erythropoietin product comprising: at least one erythropoietin derivative, wherein at least one oligosaccharide chain linked to N or at least one O-linked oligosaccharide chain has at least one chemical modification as a result of oxidation, sulfation, phosphorylation, PEGylation, or mixtures thereof, and wherein the erythropoietin product has a longer half-life in serum than rhuEPO.
  7. 7. The erythropoietin product of claim 6, characterized in that the erythropoietin product has tissue protective functionality.
  8. 8. The erythropoietin product of claim 6, characterized in that at least one chemical modification consists in the oxidation of at least one oligosaccharide chain linked to N or at least one oligosaccharide chain linked to 0, to obtain at least one additional acid residue.
  9. 9. The erythropoietin product of claim 6, characterized in that at least one chemical modification consists in sulfation of at least one oligosaccharide chain linked to N or at least one oligosaccharide chain linked to O to obtain an increased negative charge in the EPO product.
  10. 10. The erythropoietin product of claim 6, characterized in that at least one chemical modification consists of the phosphorylation of at least one oligosaccharide chain linked to N or at least one oligosaccharide chain linked to 0 to obtain an increased negative charge in the EPO product.
  11. 11. The erythropoietin product of claim 6, characterized in that at least one chemical modification comprises the addition of at least one polyethylene glycol chain or at least one oligosaccharide chain linked to N or at least one oligosaccharide chain linked to 0.
  12. 12. A method for preparing an erythropoietin product having a prolonged serum life and tissue protective activity comprises the steps of: providing at least one erythropoietin or erythropoietin derivative; and modifying at least one oligosaccharide chain linked to N or at least one oligosaccharide chain linked to 0 in at least one endogenous or recombinant erythropoietin by oxidation, sulfation, phosphorylation, PEGylation, or a combination thereof.
  13. 13. The method of claim 12, characterized in that the step of modifying further comprises the step of substituting at least one near hydroxyl in at least one N-linked oligosaccharide chain or at least one O-linked oligosaccharide chain with at least one acid residue .
  14. 14. The method of claim 13, characterized in that the step of substituting at least one nearby hydroxyl in at least one N-linked oligosaccharide chain or at least one O-linked oligosaccharide chain with at least one acid residue further comprises replacing a plurality of nearby hydroxyls on at least one N-linked oligosaccharide chain or at least one O-linked oligosaccharide chain with a plurality of acid residues.
  15. 15. The method of claim 12, characterized in that the modification step further comprises the steps of: disposing of an organic solvent; dissolving the erythropoietin or the erythropoietin derivative in the organic solvent to form a solution; having at least one condensing agent; have at least one sulphate donor; and mixing the at least one condensing agent and at least one sulfate donor in the solution.
  16. 16. The method of claim 12, characterized in that the modification step further comprises the steps of: disposing of an organic solvent; dissolving erythropoietin or the erythropoietin derivative in the organic solvent to form a solution; having at least one condensing agent; dispose of phosphoric acid; and mixing the at least one condensing agent and at least one phosphoric acid in the solution.
  17. 17. The method of claim 12, characterized in that the modification step further comprises the steps of: disposing an organic solvent, - dissolving the erythropoietin or the erythropoietin derivative in the organic solvent to form a first solution; have at least one oxidizing agent; adding at least one oxidizing agent to the first solution to form a second solution; have at least one sulphate donor; and mixing the at least one polyethylene glycol chain; and mixing at least one polyethylene glycol chain in the second solution.
  18. 18. The method of claim 17, characterized by the step of having at least one polyethylene glycol chain consists in providing at least one polyethylene glycol chain with at least one primary amino moiety at one end of the chain.
  19. 19. A method for treating anemia in patients at risk of tissue damage comprises the steps of: providing an erythropoietin product with at least one chemical modification to at least one of the oligosaccharide chains linked to N by the oligosaccharide chain linked to 0, wherein Chemical modification consists of oxidation, sulfation, phosphorylation, PEGylation, or a combination of these; administering an effective therapeutic amount of the erythropoietin product, wherein the erythropoietin product is administered in a molar amount less than rhuEPO to obtain a comparable target hematocrit; wherein the erythropoietin product has tissue protective functionality.
  20. 20. The method of claim 19, characterized in that the erythropoietin product has a longer serum half-life than rhuEPO.
  21. 21. The method of claim 20, characterized in that the serum half-life is at least about 20% longer than the serum half-life of rhuEPO.
  22. 22. The method of claim 21, characterized in that the serum half-life is at least about 40% longer than the serum half-life of the rhuEPO.
  23. 23. A pharmaceutical composition containing: an effective therapeutic amount of at least one erythropoietin derivative, wherein at least one oligosaccharide chain linked thereto or at least one O-linked oligosaccharide chain has at least one chemical modification as a result of oxidation, sulfation , phosphorylation, PEGylation or mixture of these; wherein at least one erythropoietin derivative has a longer half-life in serum than recombinant erythropoietin and has tissue protective functionality.
  24. 24. The pharmaceutical composition of claim 23, further comprises at least one carrier accepted for pharmaceutical use.
  25. 25. The pharmaceutical composition of claim 24, characterized in that at least one carrier accepted for pharmaceutical use consists of at least one diluent, adjuvant, excipient, vehicle or mixtures thereof.
  26. 26. The pharmaceutical composition of claim 23, further comprises at least one wetting agent, emulsifying agent, pH buffer, or a combination thereof.
  27. 27. The pharmaceutical composition of claim 23, further comprises at least one tissue protective cytokine.
MXPA05002617A 2002-09-09 2003-09-09 Long acting erythropoietins that maintain tissue protective activity of endogenous erythropoietin. MXPA05002617A (en)

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US7718363B2 (en) 2003-04-25 2010-05-18 The Kenneth S. Warren Institute, Inc. Tissue protective cytokine receptor complex and assays for identifying tissue protective compounds
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CA3079319A1 (en) 2005-08-05 2007-02-15 Araim Pharmaceuticals, Inc. Tissue protective peptides and uses thereof
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CN101671388B (en) * 2008-09-09 2013-01-02 曹国栋 Blood brain barrier penetrable erythropoietin (EPO) and application thereof
RU2475273C1 (en) * 2012-04-02 2013-02-20 Юлия Николаевна Козлова Method of obtaining polymeric cement of medical purpose
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