WO2005025606A1 - Erythropoietines a action prolongee pouvant maintenir une activite de protection tissulaire d'une erythropoietine endogene - Google Patents

Erythropoietines a action prolongee pouvant maintenir une activite de protection tissulaire d'une erythropoietine endogene Download PDF

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Publication number
WO2005025606A1
WO2005025606A1 PCT/US2004/007133 US2004007133W WO2005025606A1 WO 2005025606 A1 WO2005025606 A1 WO 2005025606A1 US 2004007133 W US2004007133 W US 2004007133W WO 2005025606 A1 WO2005025606 A1 WO 2005025606A1
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WIPO (PCT)
Prior art keywords
epo
erythropoietin
linked oligosaccharide
tissue protective
life
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PCT/US2004/007133
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English (en)
Inventor
Anthony Cerami
John Smart
Michael Brines
Carla Cerami
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Warren Pharmaceuticals, Inc.
The Kenneth S. Warren Institute, Inc
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Publication date
Priority claimed from PCT/US2003/028073 external-priority patent/WO2004022577A2/fr
Application filed by Warren Pharmaceuticals, Inc., The Kenneth S. Warren Institute, Inc filed Critical Warren Pharmaceuticals, Inc.
Priority to AU2004260543A priority Critical patent/AU2004260543A1/en
Priority to US11/075,957 priority patent/US20050176627A1/en
Publication of WO2005025606A1 publication Critical patent/WO2005025606A1/fr
Priority to IL174178A priority patent/IL174178A0/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/18—Growth factors; Growth regulators
    • A61K38/1816—Erythropoietin [EPO]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • A61K47/59—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
    • A61K47/60—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/475—Growth factors; Growth regulators
    • C07K14/505—Erythropoietin [EPO]

Definitions

  • the present invention relates to long acting erythropoietins that advantageously maintain tissue protective capabilities after modification.
  • the present invention relates to long acting erythropoietins that are chemically modified in a way that not only increases the serum half-life, but also maintains the tissue protective function of the native protein in vivo.
  • the present invention also relates to the treatment of anemia and anemia related diseases with the long acting erythropoietins of the present invention.
  • the present invention is directed to assays useful in the determination of whether an erythropoietin exhibits tissue protective capabilities.
  • Naturally occurring or endogenous erythropoietin is a glycoprotein hormone produced mainly in the kidney.
  • Endogeneous EPO includes 165 amino acids and has a molecular weight (in humans) of about 30,000 to about 34,000 daltons.
  • the glycosyl residues in EPO which consist of three N-linked and one O-linked oligosaccharide chains, are responsible for about 40 percent of the protein's total weight.
  • the N-linked oligosaccharide chains are bonded to amide nitrogens of asparagine at positions 24, 38 and 83, while the O-linked oligosaccharide chain is bonded to the oxygen at the serine residue located at position 126.
  • the EPO protein may occur in three forms: a, ⁇ , and asialo.
  • the and ⁇ forms have the same potency, biological activity, and molecular weight, but differ slightly in the carbohydrate components, while the asialo form is an a or ⁇ form with the terminal sialic acid, which shields the carbohydrate chain from being recognized by the liver, removed from the carbohydrate chains.
  • the principle function of endogeneous EPO was to act in concert with other growth factors to stimulate the proliferation and maturation of responsive bone marrow erythroid precursor cells and maintain an individual's hematocrit (percent of whole blood that contains red blood cells).
  • the process of producing the red blood cells is called erythropoiesis, which is a precisely controlled physiological mechanism that optimizes the number of red blood cells for proper tissue oxygenation without impeding circulation. For example, when oxygen transport by red blood cells is reduced, EPO will increase red blood cell production by stimulating the conversion of precursor cells in the bone marrow into mature red blood cells, which are then released into the circulation. When the number of red blood cells in circulation is over that needed for normal tissue circulation, EPO in circulation is decreased. Thus, when the body is in a healthy state, EPO is present in very low concentrations in plasma, which is sufficient to stimulate replacement of red blood cells lost normally though aging. Plasma EPO levels normally range from 0.01 Units/ml to 0.03 Units/ml.
  • recombinant EPO (discussed in more detail below), which has substantially the same biological effects as endogeneous EPO, has been proven useful in restoring hematocrit levels in individuals with decreased red blood cells.
  • recombinant EPO has been used to boost red blood cell levels prior to elective or scheduled surgeries, thereby reducing or eliminating the need to transfuse blood.
  • EPO EPO receptor
  • EPO-R EPO receptor
  • EPO and its receptor may play an important role in attenuating tissue injury because the interaction between EPO and the receptor provides compensatory responses that serve to improve hypoxic cellular microenvironment, as well as modulate programmed cell death caused by metabolic stress.
  • Recombinant human EPO (epoetin alfa), which has been commercially available under tradenames 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, AZT (zidovudine) therapy in HlV-infected patients, and chemotherapy. While the therapeutic effects of recombinant human EPO are numerous, to date the principal application of recombinant human EPO has been to address chronic anemia.
  • recombinant human EPO is typically administered in an initial dose of between 50-150 units/kg three times per week for about six to eight weeks either by an intravenous or subcutaneous injection in order to restore the suggested hematocrit range within the patient.
  • a desired hematocrit level such as an amount falling within from about 30 percent to about 36 percent, that level may be sustained by maintenance doses of EPO, an amount sufficient to and administered with a frequency suitable for maintaining the normal hematocrit levels achieved by the initial doses of EPO, in the absence of iron deficiency and concurrent illnesses.
  • dosage requirements may vary according to the patient's individual needs, typically maintenance dosages may be administered about three times a week (less if larger doses are provided).
  • the dosage amount and frequency of the administration of recombinant EPO is determined in part upon the half-life of the molecule, which may be limited when the molecule is in vivo.
  • intravenously administered EPOGEN® is reportedly eliminated at a rate consistent with first order kinetics with a circulating half-life ranging from approximately 4 to 13 hours in adult and pediatric patients with CRF.
  • the dosage amount and frequency of dosing must be tailored to account for the relatively short half- life of the recombinant EPO.
  • recombinant EPO is administered either by an intravenous or subcutaneous injection, a nurse or physician often is required to administer recombinant EPO to a patient.
  • the N-linked oligosaccharide chains typically have up to 4 sialic acids per chain, and the O-linked oligosaccharide chains have up to 2 sialic acids per chain.
  • an unmodifed EPO polypeptide may accommodate up to a total of 14 sialic acids.
  • these sialic acid residues may be cleaved from the protein, thereby exposing the galactose residues to detection by the liver. Once the liver detects the galactose residues, the protein is filtered from the blood.
  • a stepwise increase in sialic acid content per EPO molecule is believed to better shield the galactose residues to provide a corresponding stepwise increase in biological activity (measured by the ability of equimolar concentrations of isolated erythropoietin isoforms 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 led to the hypothesis that an EPO analog engineered to contain additional oligosaccharide chains would have enhanced biological activity. By providing these additional glycosylation sites, additional oligosaccharide chains having terminal ends may then be modified with sialic acid residues. See PCT Publication Nos.
  • a modified EPO analog may have at least one additional N-linked carbohydrate chain and/or at least one additional O-linked carbohydrate chain.
  • WO01/81405 discloses the addition of N-linked carbohydrate chains to the molecule at amino acids at 30, 51, 57, 69, 88, 89, 136 and/or 138.
  • the modified EPO molecules may have anywhere from 1 to 4 additional glycosylation sites, which permit the addition of 2 to 16 sialic acid residues to the molecule.
  • attempts to extend the half-life of EPO have involved the attachment of polyethylene glycols (PEGs) to the amino acid backbone of the EPO protein.
  • PEGs polyethylene glycols
  • the present invention is directed to a method for regulating the hematocrit level in humans including the steps for providing an erythropoietin product having a longer serum half-life than recombinant human erythropoietin (rhuEPO) and including tissue protective functionality and administering a therapeutically effective amount of the erythropoietin product, hereinafter referred to as long acting EPO.
  • rhuEPO recombinant human erythropoietin
  • the step of providing a long acting EPO further includes the step of modifying recombinant erythropoietin 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 includes oxidation, sulfation, phosphorylation, PEGylation, or a combination thereof.
  • the step of administering a therapeutically effective amount of the long acting EPO may include administering the long acting EPO at a lower molar amount than rhuEPO to obtain a comparable target hematocrit.
  • the serum half-life of the long acting EPO is at least about 20 percent longer than the serum half-life of rhuEPO. In another embodiment, the serum half-life of the long acting EPO is at least about 40 percent longer than the serum half-life of rhuEPO.
  • the present invention is also directed to a man-made erthyropoietin product (long acting EPO) including at least one erythropoietin derivative, wherein at least one N-linked oligosaccharide chain or at least one O-linked oligosaccharide chain has at least one chemical modification as a result of oxidation, sulfation, phosphorylation, PEGylation, or mixtures thereof, and wherein the long acting EPO has a longer serum half-life than rhuEPO.
  • the long acting EPO preferably has tissue protective functionality.
  • the at least one chemical modification includes 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.
  • the at least one chemical modification may include sulfation of at least one N-linked oligosaccharide chain or at least one O-linked oligosaccharide chain to provide an increased negative charge on the long acting EPO.
  • the at least one chemical modification includes phosphorylation of at least one N- linked oligosaccharide chain or at least one O-linked oligosaccharide chain to provide an increased negative charge on the long acting EPO.
  • the at least one chemical modification includes addition of at least one polyethylene glycol chain to at least one N-linked oligosaccharide chain or at least one O-linked oligosaccharide chain.
  • the present invention also relates to a method for preparing a long acting EPO having an extended serum half-life and tissue protective activity including the steps of: providing at least one erythropoietin or erythropoietin derivative; and modifying at least one N-linked oligosaccharide chain or at least one O-linked oligosaccharide chain on the at least one endogenous or recombinant erythropoietin or erythropoietin derivative by oxidation, sulfation, phosphorylation, PEGylation, or a combination thereof.
  • the step of modifying may further include the step of replacing at least one vicinal hydroxyl on at least one N-linked oligosaccharide chain or at least one O-linked oligosaccharide chain with at least one acid residue.
  • the step of replacing at least one vicinal hydroxyl on 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 vicinal hydroxyls on the least one N-linked oligosaccharide chain or at least one O-linked oligosaccharide chain with a plurality of acid residues.
  • the step of modifying further includes the steps of: providing an organic solvent; dissolving the erythropoietin or erythropoietin derivative in the organic solvent to form a solution; providing at least one condensing agent; providing at least one sulfate donor; and mixing the at least one condensing agent and the at least one sulfate donor into the solution.
  • the step of modifying further includes the steps of: providing an organic solvent; dissolving the erythropoietin or erythropoietin derivative in the organic solvent to form a solution; providing at least one condensing agent; providing phosphoric acid; and mixing the at least one condensing agent and the at least one phosphoric acid into the solution.
  • the step of modifying further includes the steps of: providing an organic solvent; dissolving the erythropoietin or erythropoietin derivative in the organic solvent to form a first solution; providing at least one oxidizing agent; adding the at least one oxidizing agent to the first solution to form a second solution; providing at least one polyethylene glycol chain; and mixing the at least one polyethylene glycol chain into the second solution.
  • the step of providing at least one polyethylene glycol chain may include providing at least one polyethylene glycol chain with at least one primary amino moiety at an end of the chain.
  • the present invention also relates to a method for treating anemia in patients at risk for tissue damage including the steps of: providing a long acting EPO 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 includes oxidation, sulfation, phosphorylation, PEGylation, or a combination thereof; administering a therapeutically effective amount of the long acting EPO, wherein the long acting EPO is administered at a lower molar amount than rhuEPO to obtain a comparable target hematocrit, wherein the long acting EPO has tissue protective functionality.
  • the long acting EPO preferably has a longer serum half-life than rhuEPO.
  • the serum half-life is at least about 20 percent longer than the serum half-life of rhuEPO. In another embodiment, the serum half-life is at least about 40 percent longer than the serum half-life of rhuEPO.
  • the present invention further relates to a pharmaceutical composition including: a therapeutically effective amount of at least one long acting EPO, 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 oxidation, sulfation, phosphorylation, PEGylation, or mixtures thereof, wherein the at least one long acting EPO has a longer serum half-life than recombinant erythropoietin and has tissue protective functionality.
  • the pharmaceutical composition further includes at least one pharmaceutically acceptable carrier.
  • the at least one pharmaceutically acceptable carrier may include at least one diluent, adjuvant, excipient, vehicle, or mixtures thereof.
  • the pharmaceutical composition further includes at least one wetting agent, emulsifying agent, pH buffering agent, or a combination thereof.
  • the pharmaceutical composition further includes at least one tissue protective cytokine.
  • FIG. 1 is an illustration of data demonstrating that an EPO analog having an additional carbohydrate chain within the O'Brien peptide sequence is able to cross the blood-brain barrier as shown by a cerebral spinal fluid sampling; and
  • FIG. 2 is a comparison of the effectiveness of EPO versus a long acting EPO analog in protecting against cell death triggered by exposure to trimethyl tin.
  • the present invention is directed to the use of long acting EPO, EPO molecules having an extended serum half-life (long acting) that result from chemically modifying the carbohydrate chains attached to the amino acid backbone of EPO so that the functionality of endogenous EPO is maintained.
  • long acting EPO EPO molecules having an extended serum half-life (long acting) that result from chemically modifying the carbohydrate chains attached to the amino acid backbone of EPO so that the functionality of endogenous EPO is maintained.
  • efforts to extend the half-life of EPO have generally been focused on additions to the amino acid backbone of EPO — the addition of carbohydrate chains, PEGs, proteins, etc.
  • these additions are believed to effect the functionality of the EPO analog such that, for example, the functionality is compromised to achieve the longer half-life.
  • EPO analogs with longer half-lives than recombinant EPO that have erythropoietic activity do not retain other recently discovered therapeutic benefits of EPO, e.g., tissue protective activity.
  • tissue protective activity e.g., a 17 amino acid fragment of EPO corresponding to amino acids 30-47, also referred to as the O'Brien peptide, has been shown to have tissue protective activity in vitro, but has no erythropoietic activity in vitro. Campana, W.M., Misasi, R. & O'Brien, J.S., Int. J. Mol. Med., 1, 235-41 (1998).
  • the present invention relates to a long acting EPO with at least one of erythropoietic activity, tissue protective activity, transcytosis capability, or a combination thereof.
  • the long acting EPO of the present invention has erythropoietic activity and at least one of tissue protective activity or transcytosis capability.
  • the long acting EPO of the present invention has a serum half-life that is at least about 20 percent longer than the serum half-life of recombinant EPO. In another embodiment, the serum half-life of the long acting EPO of the present invention is at least about 30 percent longer than the half-life of recombinant EPO. hi still another embodiment, the long acting EPO of the present invention has a serum half-life that is at least about 40 percent longer than the serum half-life of recombinant EPO.
  • the long acting EPOs of the present invention include EPO analogs with carbohydrate chains that are altered with at least one modification as compared to a native (endogenous) EPO, preferably as compared to native human EPO.
  • the long acting EPOs of the present invention undergo a plurality of modifications to the carbohydrate chains.
  • the vicinyl hydroxyls on the carbohydrate chain of a native EPO are oxidized into acid residues to produce the long acting EPOs of the present invention.
  • the sialic acid residues on the EPO are replaced with less labile acid residues.
  • sulfation and/or phosphorylation of the carbohydrate chain of an EPO results in a long acting EPO according to the present invention.
  • the long acting EPO of the present invention results from the addition of polyethylene glycol to the carbohydrate chain of EPO.
  • the present invention also embraces compositions, including pharmaceutical compositions, which include one or more of the aforementioned long acting EPOs.
  • the long acting EPOs of the present invention are contemplated for inclusion in pharmaceutical compositions for treating anemia and related diseases, especially those with complications resulting from illnesses 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 used for the treatment procedure.
  • treatment refers to both therapeutic treatment and prophylactic or preventative measures, wherein the object is to prevent or slow down (lessen) the targeted pathologic condition or disorder.
  • Those in need of treatment include those already with the disorder as well as those prone to have the disorder or those in whom the disorder is to be prevented.
  • the present invention contemplates the use of the long acting EPOs for chronic administration, acute treatment, and/or intermittent administration.
  • chronic administration refers to administration of the agent(s) in a continuous mode as opposed to an acute mode, so as to maintain the initial therapeutic effect (activity) for an extended period of time and "intermittent administration” is treatment that is not consecutively done without interruption, but rather is cyclic in nature.
  • the long acting EPOs of the present invention, and the uses thereof, are applicable for any mammal.
  • the term "mammal” refers to any animal classified as a mammal, including humans, domestic and farm animals, and zoo, sports, or pet animals, such as dogs, cats, cattle, horses, sheep, pigs, goats, rabbits, etc.
  • the mammal is human.
  • the administration of the long acting EPOs of the present invention include, but is not limited to, oral, intravenous, intranasal, topical, intraluminal, inhalation or parenteral administration, the latter including intravenous, intraarterial, subcutaneous, intramuscular, intraperitoneal, submucosal, intradermal, and combinations thereof.
  • the present invention further relates to the use of long acting EPOs of the present invention as a carrier for other molecules into areas of the body that have EPO receptors.
  • long acting EPOs of the present invention may act as a transport system for molecules having poor penetration through such areas.
  • the present invention is directed to assays for determining whether a particular EPO maintains the functionality of endogenous EPO.
  • an assay of the present invention may determine whether a modified EPO is tissue protective, i.e., an agonist with regard to endogenous EPO.
  • a modified EPO is tissue protective, i.e., an agonist with regard to endogenous EPO.
  • the term "agonist” is used in the broadest sense and includes molecules that mimic the biological activity of a native EPO.
  • the term “antagonist” is used in the broadest sense, and includes any molecule that partially or fully blocks, inhibits, or neutralizes the biological activity of native EPO.
  • testing of a particular EPO occurs in an in vitro assay, such as a PI 9 cell and/or rat motoneuron assay.
  • the assay of the present invention involves the evaluation of a particular EPO in vivo using various assays such as the rat focal ischemia, rat retinal ischemia, spinal chord trauma, and bicuculline seizure models.
  • EPO analogs have a longer half-life and maintain erythropoietic activity.
  • EPO analogs with longer half-lives have mainly included additions to the amino acid sequence of EPO.
  • Such additions include carbohydrate chains, PEGs, and protein sequences.
  • these additions may interfere with other therapeutic benefits of EPO, such as the tissue protective functionality and the transcytosis capability of the molecule.
  • the placement of the added carbohydrate chains in the O'Brien peptide sequence may affect functionality based on importance of this peptide with regard to tissue protective functionality.
  • the added carbohydrate chains are believed to have an affect on the three-dimensional orientation of the molecule.
  • the additional carbohydrate chains may block an area of the molecule that is essential to the functionality.
  • the method of addition may also have an affect on the functionality of the glycoprotein.
  • tissue protective activity may produce complications when used for treatment in anemia patients if those patients have other conditions requiring the tissue protective capability.
  • tissue protective EPO analog competes with tissue protective endogenous EPO for the receptor that triggers the tissue protective response
  • the extent of injury resulting from a trauma may actually be exacerbated due to the use of such EPO analogs.
  • the infarct volume resulting from the stroke may actually be greater than in an individual not treated with an EPO analog.
  • the EPO receptor is a complex gene for which a number of edited versions, including a truncated, soluble receptor, exist. Yamaji, R., et al., Eur JBiochem, 239, 494-500; Yamaji, R., et al., Bichim
  • the O'Brien peptide has been shown to have tissue protective activity in vitro, but no erythropoietic activity in vitro.
  • an assay performed on an EPO analog that contains an added carbohydrate chain within the O'Brien peptide sequence demonstrated that the EPO analog lacked tissue protective capabilities.
  • certain modifications to the O'Brien peptide sequence such as the addition of carbohydrate chains, interferes with the functionality of the protein.
  • An EPO analog with modifications to the O'Brien peptide sequence likely acts as an antagonist towards the endogenous EPO located within the body because it partially or fully blocks the endogenous EPO's ability to bind to the EPO receptor.
  • EPO analogs with modifications to the O'Brien peptide sequence would also lack tissue protective capabilities in other in vitro assays such as the rat motoneuron assay and in in vivo assays such as the rat focal ischemic, bicuculline seizure, rat retinal ischemia, and spinal cord trauma assays.
  • EPO analog Compared to the 3 N-linked carbohydrate chains of recombinant EPO, the inventors studied the analog's ability to traverse the blood brain barrier.
  • the EPO analog appeared within the cerebral spinal fluid after systemic injection ( Figure 1). Without being bound to any particular theory, it is believed that the EPO analog is able to cross the intact blood brain barrier because the capillaries forming the blood brain barrier also express the EPO receptor and provide an anatomical basis for receptor-mediated transcytosis from the peripheral circulation into the brain.
  • the present invention is directed to a long acting EPO of the present invention that not only increases the serum half-life as compared to recombinant EPO, but also maintains the functionality of endogenous EPO, i.e., the tissue protective functionality and/or the transcytosis capability.
  • a long acting EPO of the present invention that not only increases the serum half-life as compared to recombinant EPO, but also maintains the functionality of endogenous EPO, i.e., the tissue protective functionality and/or the transcytosis capability.
  • the long acting EPOs of the present invention may be formed in a variety of ways.
  • the long acting EPOs may be generated by chemically modifying the carbohydrate (sugar) chains attached to naturally occurring or recombinant human EPO.
  • carbohydrate chains refer to the N-linked and O-linked oligosaccharide chains found in endogenous EPO, the additional N-linked and O-linked oligosaccharide chains found in EPO analogs, and any other carbohydrate chains, specifically sugar chains, attached to EPO.
  • endogenous or recombinant EPO is used for modification so as to prevent any interference with the tissue protective capabilities of endogenous EPO.
  • EPO analogs are contemplated for modification according to the present invention providing the additional glycosylation sites are not located near the O'Brien peptide, i.e., the 30-47 amino acid sequence.
  • the term "EPO analogs" 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.
  • an EPO analog used for modification does not include any additional glycosylation sites within about 5 amino acids of the O'Brien peptide sequence.
  • the EPO analog does not include any additional glycosylation sites within about 3 amino acids of the O'Brien peptide sequence.
  • the EPO analog does not include any additional glycosylation sites within the O'Brien peptide sequence.
  • An EPO analog may also be used for modification according to the present invention provided that the analog is reviewed in three-dimensional space and it is confirmed that none of the additional carbohydrate chains block the O'Brien peptide or cause a loss of tissue protective functionality.
  • an EPO analog is contemplated for use in modification according to the present invention providing the method of glycosylation does not inhibit the tissue protective functionality of the peptide.
  • an EPO analog may be used for modification according to the present invention providing that there is one carbohydrate chain (or less) in the O'Brien peptide.
  • endogenous EPO contains a carbohydrate chain at the 38 amino acid and an additional carbohydrate chain within the O'Brien peptide sequence has been shown to inhibit the tissue protective activity of the protein (See Example 2 and Figure 2).
  • an EPO analog having one carbohydrate chain or no carbohydrate chains in the O'Brien peptide sequence is contemplated for use in modification.
  • the carbohydrate chain attached to the 38 amino acid may be relocated to somewhere else on the protein.
  • Nonlimiting examples of modifications according to the present invention include (1) providing additional acid residues on the carbohydrate chains through oxidation of vicinal hydroxyls; (2) replacing the sialic acid residues with less labile residues; (3) increasing the negative charge on erythropoietin by sulfation and/or phosphorylation; and/or (4) terminating the carbohydrate chains with more complex molecules.
  • the modifications to the carbohydrate chains of EPO may include oxidation, sulfation, phosphorylation, and/or PEGylation, among other procedures, which will be described in greater detail below and further illustrated in prophetic Example 1.
  • a chemically modified long acting EPO of the present invention may include an EPO in which the carbohydrates (sugars) are oxidized to provide additional acid residues. Jh another embodiment, the sialic acid residues are replaced with less labile acid residues. Modifications of this type result in an increased half-life of the molecule, as compared to endogenous EPO, because the galactose chains for which the liver screens for and removes the associated protein from circulation are protected from detection. A more substantial chemical modification to the carbohydrate chains on the EPO leads to a greater increase in the serum half-life of the long acting EPOs of the present invention.
  • one suitable method involves (1) modifying the sugar molecules with vicinal hydroxyls with periodate to form aldehydes; and (2) oxidizing the aldehydes to generate acids.
  • Reagents suitable for oxidizing the sugar chain to form aldehydes include, but are not limited to, periodates, such as sodium periodate, and sugar oxidases, such as galactose oxidase.
  • reagents for transforming the aldehydes such as Quantitative Benedict Solution (commercially available from Fisher).
  • the sugar molecules are oxidized with sodium periodate and further treated with Quantitative Benedict Solution (Fisher) to convert the aldehydes into acids.
  • Quantitative Benedict Solution Fisher
  • an EPO isomer, EPO having about 0-13 sialic acid residues, or an EPO analog, having at least one carbohydrate chain that lacks a sialic acid residue is subjected to oxidation using galactose oxidase.
  • EPO asialo form of EPO
  • an ⁇ or ⁇ form of EPO with all the single acids removed from the terminal ends of the carbohydrate chains.
  • this asialoerythropoietin is used.
  • the EPO is subjected to another oxidative agent, such as Quantitative Benedict Solution, to transform the aldehydes into acids.
  • a ruthenium tetroxide system may be used to generate the acids on the carbohydrate chains.
  • 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 half-life of the molecule.
  • the negative charge of the EPO molecule may be increased by sulfation, which involves the transfer of a sulfuryl group from a sulfate donor.
  • the negative charge may also be increased by introducing a phosphoric group into a carbohydrate.
  • One suitable method for sulfation of insulin is discussed in S.
  • the bioactivity of the sulfated insulin made with the Ponger process varied between 78 percent and 87 percent of unmodified insulin.
  • EPO electroactive polymer
  • one of ordinary skill in the art may control the amount of sulfation and therefore the serum half-life of the chemically modified EPO.
  • the negative charge of EPO may be increased by adding sulfates to the protein by dissolving EPO or an EPO analog in at least one water soluble carbodiimide, preferably DCC, at a temperature of about 4°C.
  • DCC is preferred as the sulfate donor
  • phosphoric acid H 3 PO 4
  • skilled artisans would be able to readily select other phosphate donors to effect phosphorylation of EPO.
  • the carbohydrate chains of EPO may 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:
  • PEG polyethylene glycol
  • the PEG may also be a methoxy PEG (mPEG) having the following general formula:
  • the PEG is an amino PEG, preferably a methoxy PEG with primary amino groups at the termini (mPEG— NH 2 ).
  • Polyethylene glycol chains with primary amino groups at the termini are very useful functionalized polymers.
  • the amino end groups on mPEG- NH 2 are more reactive toward acylating agents than the hydroxyl groups that are present on conventional PEGs and they also readily undergo reductive amination reactions.
  • the PEG is an electrophilically activated PEG, such as mPEG-succinimidyl propionate (mPEG-SPA) or mPEG-succinimidyl butanoate (mPEG-SBA), both of which are commercially available from Nektar Therapeutics of Birmingham, Alabama.
  • the PEG is a methoxy PEG-hydrazide. Jh one embodiment, the addition of the at least one PEG is achieved via oxidation with periodate (as disclosed above), followed by the use of cyanoborohydride and an amino PEG.
  • EPO in solution may be first oxidized with a periodate, e.g., sodium periodate, for a predetermined period of time at room temperature, which produces aldehydes in the carbohydrate chains.
  • a periodate e.g., sodium periodate
  • a suitable periodate is sodium meta-periodate, which is commercially available from Sigma.
  • the periodate may then be removed by buffer exchange, at which time the oxidized sialic acid groups on N-linked oligosaccharide groups of EPO may be subjected to at least one amino PEG in the presence of cyanoborohydride.
  • Suitable PEGs for use include, but are not limited to, methoxy-PEG-hydrazides, which are commercially available from Nektar Therapeutics.
  • the addition of the at least one PEG is performed by the attachment of PEG groups to terminal galactose residues after oxidation with galactose oxidase.
  • an asialo form of EPO (having exposed terminal galactose residues) in buffer is first subjected to galactose oxidase (commercially available from Sigma) to generate aldehydes in the carbohydrate chains.
  • the buffer may then be removed by buffer exchange, at which time the oxidized galactose residues may be subjected to at least one amino PEG in the presence of cyanoborohydride.
  • the methods provided above are not intended to be limiting as these or other methods may be used to prepare the compounds of the invention.
  • EPO Molecules A variety of host-expression vector systems may be utilized to produce EPO for the manufacture of long acting EPOs of the invention. Such host-expression systems represent vehicles by which the EPOs of interest maybe produced and subsequently purified, but also represent cells that may, when transformed or transfected with the appropriate nucleotide coding sequences, exhibit the modified erythropoietin gene product in situ.
  • bacteria include but are not limited to, bacteria, insect, plant, mammalian, including human host systems, such as, but not limited to, insect cell systems infected with recombinant virus expression vectors (e.g., baculovirus) containing the long acting EPO product coding sequences; plant cell systems infected with recombinant virus expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with recombinant plasmid expression vectors (e.g.
  • recombinant virus expression vectors e.g., baculovirus
  • plant cell systems infected with recombinant virus expression vectors e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV
  • recombinant plasmid expression vectors e.g.
  • Ti plasmid containing erythropoietin-related molecule coding sequences; or mammalian cell systems, including human cell systems, e.g., HT1080, COS, CHO, BHK, 293, 3T3, harboring recombinant expression constructs containing promoters derived from the genome of mammalian cells, e.g., metallothionein promoter, or from mammalian viruses, e.g., the adenovirus late promoter; the vaccinia virus 7.5K promoter.
  • a host cell strain may be chosen that modulates the expression of the inserted sequences, or modifies and processes the gene product in the specific fashion desired.
  • Such modifications and processing of protein products may be important for the function of the protein.
  • different host cells have specific mechanisms for the post-translational processing and modification of proteins and gene products. Appropriate cell lines or host systems can be chosen to ensure the correct modification and processing of the foreign protein expressed.
  • eukaryotic host cells that possess the cellular machinery for proper processing of the primary transcript, glycosylation, and phosphorylation of the gene product may be used.
  • mammalian host cells including human host cells, include but are not limited to HT1080, CHO, VERO, BHK, HeLa, COS, MDCK, 293, 3T3, and WI38.
  • cell lines that stably express the recombinant EPO molecule gene product may be engineered.
  • host cells can be transformed with DNA controlled by appropriate expression control elements, e.g., promoter, enhancer, sequences, transcription terminators, polyadenylation sites, and the like, and a selectable marker.
  • appropriate expression control elements e.g., promoter, enhancer, sequences, transcription terminators, polyadenylation sites, and the like, and a selectable marker.
  • engineered cells may be allowed to grow for 1-2 days in an enriched media, and then are switched to a selective media.
  • the selectable marker in the recombinant plasmid confers resistance to the selection and allows cells to stably integrate the plasmid into their chromosomes and grow to form foci that in turn can be cloned and expanded into cell lines.
  • This method may advantageously be used to engineer cell lines that express an EPO mutein-related molecule gene product.
  • Such engineered cell lines may be particularly useful in screening and evaluation of compounds that affect the endogenous activity of the EPO-related molecule gene product.
  • an endogenous EPO mutein gene within a cell line or microorganism may be modified by inserting a heterologous DNA regulatory element into the genome of a stable cell line or cloned microorganism such that the inserted regulatory element is operatively linked with the endogenous erythropoietin mutein gene.
  • an endogenous EPO mutein gene that is normally "transcriptionally silent", i.e., an EPO gene that is normally not expressed, or is expressed only at very low levels in a cell line may be activated by inserting a regulatory element that is capable of promoting the expression of an expressed gene product in that cell line or microorganism.
  • a transcriptionally silent, endogenous EPO gene may be activated by insertion of a promiscuous regulatory element that works across cell types.
  • a heterologous regulatory element may be inserted into a stable cell line or cloned microorganism, such that it is operatively linked with an endogenous erythropoietin gene, using techniques, such as targeted homologous recombination, which are well known to those of skill in the art, and also described French Patent No. 2646438, U.S. Patent Nos. 4,215,051 and 5,578,461, and International Publication Nos. WO93/09222 and WO91/06667, the entire disclosures of which are incorporated by reference herein.
  • compositions including the long acting EPOs of the present invention.
  • the long acting EPOs of the present invention advantageously have erythropoietic activity, as well as tissue protective capability and/or transcytosis capability, they are contemplated for treatment of anemia and related diseases in individuals also at risk for various tissue injuries, such as stroke and myocardial infarction.
  • the long acting EPOs of the present invention are contemplated for treatment of anemia and related diseases in individuals also experiencing deterioration of mental faculties, such as Alzheimer's, Parkinson's and the like.
  • the long acting EPOs of the present invention are contemplated for the treatment of anemia in individuals subject to conditions resulting from the normal aging process, e.g., balance problems leading to falling, easy bruising, and the like.
  • 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 across barriers with capillaries having EPO receptors.
  • any of the long acting EPOs discussed above may be included in pharmaceutical compositions of the invention.
  • various non-tissue protective EPO analogs may be included in pharmaceutical compositions of the invention in a blend with at least one tissue protective cytokine, a cytokine, preferably a chemically modified or mutated EPO, that exhibits tissue protective effects without erythropoietic effects, which will be discussed in greater detail below.
  • the pharmaceutical compositions of the invention contain a therapeutically effective amount of the long acting EPO, preferably in purified form.
  • the formulation should suit the mode of administration.
  • the pharmaceutical compositions of the invention include an amount of the long acting EPO of the invention such that the targeted condition is treatable provided the proper dose and strategy is employed.
  • the pharmaceutical composition should be delivered in a non-toxic dosage amount.
  • compositions of the invention may include a therapeutically effective amount of the long acting EPO compound and a suitable amount of a pharmaceutically acceptable carrier so as to provide the form for proper administration to the patient.
  • pharmaceutically acceptable means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized foreign pharmacopeia for use in animals, and more particularly in humans.
  • carrier refers to a diluent, adjuvant, excipient, or vehicle with which the therapeutic is administered.
  • Such pharmaceutical carriers can be sterile liquids, such as saline solutions in water and oils, including those of 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 dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions.
  • Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like.
  • the pharmaceutical compositions of the invention may also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. These compositions can 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 or salt forms.
  • Pharmaceutically acceptable salts include those formed with free amino groups such as those derived from hydrochloric, phosphoric, acetic, oxalic, tartaric acids, etc., and those formed with free carboxyl groups such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxides, isopropylamine, triethylamine, 2-ethylamino ethanol, histidine, procaine, etc.
  • any of the long acting EPOs of the present invention are contemplated for use in pharmaceutical compositions.
  • a long acting EPO produced from oxidation of vicinal hydroxyls is included in the pharmaceutical composition of the invention
  • the pharmaceutical composition of the invention includes at least one long acting EPO that is a result of replacing the sialic acid residues with less labile residues.
  • the long acting EPO included in the pharmaceutical composition is a result of increasing the negative charge on EPO by sulfation and/or phosphorylation.
  • 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.
  • the present invention contemplates the use of a mixture of long acting EPOs produced by any of the methods of the present invention in the pharmaceutical compositions of the invention.
  • the pharmaceutical composition of the invention may include at least one long acting EPO that is a result of replacing the sialic acid residues with less labile residues and at least one long acting EPO that is the result of increasing the negative charge on EPO by sulfation and/or phosphorylation.
  • the long acting EPOs of the present invention advantageously are able to traverse barriers with capillaries having EPO receptors.
  • another aspect of the present invention is a transport system using the long acting EPOs of the present invention as carriers for molecules with poor barrier penetration into a targeted area of the body having EPO receptors.
  • Such transport systems advantageously provide a novel and safe method of delivery across the intact barriers.
  • the transport system includes the long acting EPOs of the present invention and at least one molecule with poor brain penetration to provide a novel and safe method of delivery across the intact blood brain barrier.
  • the long acting EPOs of the present invention may act as molecular "trojan horses” for molecules with poor brain penetration so as to enhance brain uptake of either small or large molecule diagnostics or therapeutic molecules.
  • an important problem in the treatment of human brain tumors is posed by the need to deliver therapeutic agents to specific regions of the brain, distributing them within and targeting them to brain tumors.
  • the molecules that might otherwise be effective in diagnosis and therapy either do not cross the blood-brain barrier (BBB) in the brain adjacent to the tumor or do not cross the blood-tumor barrier (BTB) in adequate amounts.
  • BBB blood-brain barrier
  • BTB blood-tumor barrier
  • the long acting EPOs of the present invention may be used as a carrier for such molecules to allow traversal of the BBB or BTB.
  • a molecule that may be used with the long acting EPOs of the present invention is an anti-sense oligonucleotide, which is typically used either to inhibit oncogenic signals or to image gene expression of the brain in vivo.
  • the long acting EPOs of the present invention may be included in various gene therapies (viral or nonviral formulations), which are often too large to cross the BTB without aid.
  • the long acting EPOs of the present invention may be used as carrier- mediated transporter for various chemotherapeutic agents. Because drug-active efflux transporters, which are expressed at the BBB and the BTB, actively efflux chemotherapeutic agents from the brain back to the blood, the distribution of these agents in the brain may be inhibited or prevented. It is partly for these reasons that most of the classical 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 a long acting EPO of the present invention as a carrier for such chemotherapeutic agents may be useful not only in carrying the agents into the brain, but also keeping the agents within the brain for therapy.
  • the long acting EPO may be joined with a drug that inhibits the active efflux transporter to further ensure the uptake of chemotherapeutic agents that are normally effluxed from brain to blood.
  • the present invention also contemplates the use of long acting EPO as carriers for molecules with poor penetration in other areas of the body having EPO receptors.
  • Non- limiting examples of such cells include retinal, muscle, heart, lung, liver, kidney, small intestine, adrenal cortex, adrenal medulla, capillary endothelial, testes, ovary, pancreas, bone, skin, and endometrial cells.
  • responsive cells include, without limitation, neuronal cells; retinal cells: photoreceptor (rods and cones), ganglion, bipolar, horizontal, amacrine, and Mueller cells; muscle cells; heart cells: myocardium, pace maker, sinoatrial node, sinoatrial node, sinus node, and junction tissue cells (atrioventricular node and bundle of his); lung cells; liver cells: hepatocytes, stellate, and Kupffer cells; kidney cells: mesangial, renal epithelial, and tubular interstitial cells; small intestine cells: goblet, intestinal gland (crypts) and enteral endocrine cells; adrenal cortex cells: glomerulosa, fasciculate, and reticularis cells; adrenal medulla cells: chromaffin cells; capillary cells: pericyte cells; testes cells: Leydig, Sertoli, and sperm cells and their precursors; ovary cells: Graffian follicle and primordial follicle; muscle
  • a pharmaceutical composition according to the present invention may include a non-tissue protective EPO analog having at least one additional N-linked carbohydrate chain and/or at least one additional O-linked carbohydrate chain (that exhibits an extended serum half-life but lacks tissue protective activity) in a blend with at least one tissue protective cytokine.
  • a non-tissue protective EPO analog having at least two additional N-linked carbohydrate chains, wherein one of the additional carbohydrate chains is located in the O'Brien peptide sequence, in combination with a tissue protective cytokine may form a composition of the invention.
  • the pharmaceutical composition of the invention may include at least one tissue protective cytokine and at least one non-tissue protective EPO analog that contains additional carbohydrate chains that are known, from reviewing the analog in three-dimensional space, to block the O'Brien peptide sequence.
  • a pharmaceutical composition of the invention includes at least one tissue protective cytokine and at least one EPO analog having noor greatly reduced tissue protective functionality as a result of addition of carbohydrate chains, PEGs or proteins to the amino acid backbone of the EPO protein.
  • An EPO analog with a relocated glycosylation site is contemplated for use in the pharmaceutical compositions of the present invention.
  • the pharmaceutical composition of the invention may include an EPO analog with a relocated glycosylation site, from the 38 amino acid, to elsewhere on the molecule.
  • the relocated glycosylation site may occur at amino acids 51, 57, 69, 88, 89, 136 or 138, as suggested in PCT Publication No. WO 01/81405.
  • the O'Brien peptide sequence contains 1 or less carbohydrate chains.
  • Suitable tissue protective cytokines for use with this aspect of the present invention are preferably those cytokines that lack an effect on the bone marrow but maintain the tissue protective effect of endogenous EPO, however any cytokine that exhibits tissue protective capability is contemplated for use with the present invention.
  • suitable tissue protective cytokines include chemically modified EPOs generated by guanidination, amidination, carbamylation (carbamoylation), trinitrophenylation, acylation (acetylation or succinylation), nitration, or mixtures thereof.
  • EPO molecules with a modification of at least one arginine, lysine, tyrosine, tryptophan, or cysteine residue or carboxyl groups are also contemplated for use as tissue protective cytokines according to this aspect of the present invention.
  • additional tissue protective cytokines for use with the present invention may be obtained by limited proteolysis, removal of amino groups, and/or mutational substitution of arginine, lysine, tyrosine, tryptophan, or cysteine residues by molecular biological techniques as disclosed in Satake et al, 1990, Biochim. Biophys. Ada 1038:125-9, which is incorporated by reference herein in its entirety.
  • tissue protective cytokines include at least one or more mutated EPOs having a site mutation at C7S, R10L VI IS, L12A, El 3 A, R14A, R14B, R14E, R14Q, Y15A, Y15F, Y15I, K20A, K20E, E21A, C29S, C29Y, C33S, C33Y, P42N, T44I, K45A, K45D, V46A, N47A, F48A, F48I, Y49A, Y49S, W51F, W51N, Q59N, E62T, L67S, L70A, D96R, S100R, S100E, S100A, S100T, G101A, G101L L102A, R103A, S104A, SI 041, L105A, T106A, T106I, T107A, T107L, L108K, L108A, S126A, F142I, R143A, S146
  • the tissue protective cytokine may include one or more of the above site mutations, providing that the site mutations do not include I6A, C7A, K20A, P42A, D43A, K45D, K45A, F48A, Y49A, K52A, K49A, S100B, R103A, Kl 16A, T132A, II 33 A,
  • the 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, K97A/K152A/K45A/K52A, K97A/K152A/K45A/K52A/K140A, K97A/K152A/K45A/K52A/K140A, K97A/K152A/K45A/K52A/K140A, K97A/K152A/K45A/K52A/K140A, K97A/K152A/K45A/K52A/K140A/K154A,
  • tissue protective cytokines do not include any of the above combinations.
  • tissue protective cytokines may include any of the above-referenced site mutations providing that the site mutations do not include any of the following combinations of substitutions: N24K/N38K N83K and/or A30N/H32T. Certain modifications or combinations of modifications may affect the flexibility of the mutein' s ability to bind with its receptor, such as an EPO receptor or secondary receptor.
  • 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. Further discussion of such tissue protective cytokines is included in co-pending U.S. Patent Application No.
  • a pharmaceutical composition according to the present invention may include an EPO analog having at least one additional N-linked carbohydrate chain and/or at least one additional O-linked carbohydrate chain (that exhibits an extended serum half-life but lacks tissue protective activity) in a blend with at least one small molecule that exhibits tissue protective functionality.
  • Suitable small molecules include, but are not limited to, steroids (e.g., lazaroids and glucocorticoids), antioxidants (e.g., coenzyme Q 10 , alpha lipoic acid, and NADH), anticatabolic enzymes (e.g., glutathione peroxidase superoxide dimutase, catalase, synthetic catalytic scavengers, as well as mimetics), indole derivatives (e.g., indoleamines, carbazoles, and carbolines), nitric acid neutralizing agents, adenosine / adenosine agonists, phytochemicals (flavanoids), herbal extracts (ginko biloba and turmenic), vitamins (vitamins A, E, and C), oxidase electron acceptor inhibitors (e.g., xanthene oxidase electron inhibitors), minerals (e.g., copper, zinc, and magnesium), NSAIDS (e.g.,
  • a pharmaceutical composition of the invention may include an EPO analog, a tissue protective cytokine, and a small molecule with tissue protective activity.
  • the tissue protective cytokines and/or small molecules are preferably present in the pharmaceutical compositions of the invention in an amount sufficient to maintain or exceed the same activity in neural or other responsive cellular systems as elicited by endogenous EPO.
  • the tissue protective cytokine and/or small molecule is present in an amount sufficient to enhance the tissue protection of the individual by protecting, maintaining, or enhancing the viability and function of erythropoietic responsive cells within the individual.
  • the pharmaceutical composition of this aspect of the present invention preferably includes an effective, non-toxic amount of the tissue protective cytokine, e.g., about 1 ng or greater.
  • the tissue protective cytokine is present in the pharmaceutical composition in an amount of about 5 mg or less.
  • the tissue protective cytokine is present in the pharmaceutical composition in an amount of about 500 ng to 5 mg.
  • the pharmaceutical composition includes about 1 ⁇ g to 5 mg of the tissue protective cytokine, preferably about 500 ⁇ g to 5mg.
  • a larger amount of the tissue protective cytokine is present in the pharmaceutical composition of the invention, e.g., about 1 mg to 5 mg.
  • the amount of pharmaceutical composition administered to a patient depends on a number of factors including, but not limited to, the condition of the patient and the dosing frequency. This will be discussed in greater detail below with regard to dosing.
  • the aforementioned long acting EPOs and pharmaceutical compositions including the long acting EPOs are intended for the therapeutic or prophylactic treatment of anemia, human diseases that either involve anemia or anemic conditions, or diseases or methods of treatment that result in anemia.
  • the long acting EPOs of the present invention permit less frequent dosing or the use of smaller doses of erythropoietin to treat the above diseases without jeopardizing a patient's ability to recover from other tissue injuries, in particular injuries to other EPO responsive cells, tissues, or organs.
  • Non-limiting examples of such EPO responsive cells include retinal, muscle, heart, lung, liver, kidney, small intestine, adrenal cortex, adrenal medulla, capillary endothelial, testes, ovary, pancreas, bone, skin, and endometrial cells.
  • responsive cells include, without limitation, neuronal cells; retinal cells: photoreceptor (rods and cones), ganglion, bipolar, horizontal, amacrine, and Mueller cells; muscle cells; heart cells: myocardium, pace maker, sinoatrial node, sinoatrial node, sinus node, and junction tissue cells (atrioventricular node and bundle of his); lung cells; liver cells: hepatocytes, stellate, and Kupffer cells; kidney cells: mesangial, renal epithelial, and tubular interstitial cells; small intestine cells: goblet, intestinal gland (crypts) and enteral endocrine cells; adrenal cortex cells: glomerulosa, fasciculate, and reticularis cells; adrenal medulla cells: chromaffin cells; capillary cells: pericyte cells; testes cells: Leydig, Sertoli, and sperm cells and then precursors; ovary cells: Graffian follicle and primordial follicle and primordial
  • the pharmaceutical compositions of the invention are administered chronically to protect or enhance the target cells, tissue or organ, hi another embodiment, the pharmaceutical compositions of the invention may be administered acutely, i.e., for a single treatment during injury. In yet another embodiment, the pharmaceutical compositions of the invention are administered in a cyclic nature.
  • the administration of the composition may be parenteral, e.g., via intravenous injection, intraperitoneal injection, intra-arterial, intramuscular, intradermal, or subcutaneous administration; via inhalation; transmucosal, e.g., oral, nasal, rectal, intravaginal, sublingual, submucosal, and transdermal; or combinations thereof.
  • the administration of the pharmaceutical composition of the invention is parenteral.
  • Such adminstration may be performed in a dose amount of about 0.01 pg to about 5 mg, preferably about 1 pg to about 5 mg.
  • the dose amount is about 500 pg to about 5 mg.
  • the dose amount is about 1 ng to about 5 mg.
  • the dose amount is about 500 ng to about 5 mg.
  • the dose amount is about 1 ⁇ g to about 5 mg.
  • the dose amount may be about 500 ⁇ g to about 5 mg.
  • the dose amount maybe about 1 mg to about 5 mg.
  • compositions of the invention adapted for parenteral administration include aqueous and non-aqueous sterile injectable solutions or suspensions, which may contain antioxidants, buffers, bacteriostats and solutes that render the compositions substantially isotonic with the blood of an intended recipient.
  • the pharmaceutical compositions may also include water, alcohols, polyols, glycerine, vegetable oils, and mixtures thereof.
  • compositions adapted for parenteral administration may be presented in unit-dose or multi-dose containers, for example sealed ampules and vials, and may be stored in a lyophihzed (freeze-dried) condition requiring only the addition of a sterile liquid carrier, e.g., sterile saline solution for injections, immediately prior to use.
  • a sterile liquid carrier e.g., sterile saline solution for injections, immediately prior to use.
  • Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets.
  • an autoinjector comprising an injectable solution of a long acting EPO of the invention may be provided for emergency use by ambulances, emergency rooms, and battlefield situations.
  • the pharmaceutical composition of the invention is formulated in accordance with routine procedures as a pharmaceutical composition adapted for intravenous administration to human beings.
  • the pharmaceutical composition may be in the form of a solution in sterile isotonic aqueous buffer.
  • the pharmaceutical composition may also include a solubilizing agent and/or a local anesthetic such as lidocaine to ease pain at the site of the injection.
  • the ingredients may be supplied either separately or mixed together in unit dosage form, for example, as a dry lyophihzed powder or water-free concentrate in a hermetically-sealed container such as an ampule or sachette indicating the quantity of active agent.
  • an infusion bottle with sterile pharmaceutical grade water or saline may be used for dispensing the composition.
  • an ampule of sterile saline may be provided to mix the ingredients may be mixed prior to administration.
  • Pharmaceutical compositions adapted for oral administration may be provided as capsules or tablets; powders or granules; solutions, syrups or suspensions (in aqueous or non-aqueous liquids); edible foams or whips; emulsions; or combinations thereof.
  • the oral formulation may include about 10 percent to about 95 percent by weight active ingredient. In one embodiment, the active ingredient is included in the oral formulation in an amount of about 20 percent to about 80 percent by weight.
  • the oral formulation includes about 25 percent to about 75 percent by weight of the active ingredient.
  • Tablets or hard gelatin capsules may include lactose, starch or derivatives thereof, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, stearic acid or salts thereof.
  • Soft gelatin capsules may include vegetable oils, waxes, fats, semi-solid, liquid polyols, or mixtures thereof. Solutions and syrups may include water, polyols, sugars, or mixtures thereof.
  • an active agent intended for oral administration may be coated with or admixed with a material that delays disintegration and/or absorption of the active agent in the gastrointestinal tract.
  • the active agent may admixed or coated with glyceryl monostearate, glyceryl distearate, or a combination thereof.
  • the sustained release of an active agent may be achieved over many hours and, if necessary, the active agent can be protected from being degraded within the stomach.
  • Pharmaceutical compositions for oral admimstration may also be formulated to facilitate release of an active agent at a particular gastrointestinal location due to specific pH or enzymatic conditions.
  • Pharmaceutical compositions adapted for transdermal administration may be provided as discrete patches intended to remain in intimate contact with the epidermis of the recipient for a prolonged period of time.
  • compositions adapted for topical administration may be provided as ointments, creams, suspensions, lotions, powders, solutions, pastes, gels, sprays, aerosols, oils, eye drops, lozenges, pastilles, and mouthwashes and combinations thereof.
  • topical administration is intended for the skin, mouth, eye, or other external tissues.
  • a topical ointment or cream is preferably used.
  • the active ingredient i.e., the long acting EPO, may be employed with either a paraffinic or a water-miscible ointment base.
  • the active ingredient may be formulated in a cream with an oil-in-water base or a water-in-oil base.
  • the pharmaceutical compositions of the invention preferably include the active ingredient, which is dissolved or suspended in a suitable carrier, e.g., in an aqueous solvent.
  • suitable carrier e.g., in an aqueous solvent.
  • Pharmaceutical compositions adapted for nasal and pulmonary adminisfration may include solid carriers such as powders (preferably having a particle size of about 20 microns to about 500 microns). Powders may be administered by rapid inhalation through the nose from a container of powder held close to the nose.
  • pharmaceutical compositions intended for nasal administration according to the present invention may include liquid carriers, e.g., nasal sprays or nasal drops.
  • the pharmaceutical compositions of the invention are administered into the nasal cavity directly.
  • Direct lung inhalation may be accomplished 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 so as to provide predetermined dosages of the active ingredient.
  • Pharmaceutical compositions intended for lung inhalation may include aqueous or oil solutions of the active ingredient.
  • the pharmaceutical compositions of the invention are administered via deep inhalation directly into the oropharynx.
  • Pharmaceutical compositions adapted for rectal administration may be provided as suppositories or enemas.
  • a suppository of the invention includes about 0.5 percent to 10 percent by weight of active ingredient.
  • the suppository includes about 1 percent to about 8 percent by weight active ingredient.
  • the active ingredient is present in the suppository in an amount of about 2 percent to about 6 percent by weight,
  • the pharmaceutical compositions of the invention may include traditional binders and carrier, such as triglycerides.
  • Pharmaceutical compositions adapted for vaginal administration may be provided as pessaries, tampons, creams, gels, pastes, foams or spray formulations.
  • the pharmaceutical compositions of the invention may also be administered by use of a perfusate, injection into an organ, or locally administered.
  • the pharmaceutical composition preferably has about 0.01 pM to about 30 pM, preferably about 15 pM to about 30 nM, of the long acting EPO of the present invention.
  • the perfusion solution 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/1), which contains about 1 U/ml to about 25 U/ml EPO; 5 percent hydroxyethyl starch (preferably having a molecular weight from about 200,000 to about 300,000 and substantially free of ethylene glycol, ethylene chlorohydrin, sodium chloride, and acetone), 25 mM KH 2 PO , 3 mM glutathione; 5 mM adenosine; 10 mM glucose; 10 mM HEPES buffer; 5 mM magnesium gluconate; 1.5mM CaCl 2 ; 105 mM sodium gluconate; 200,000 units penicillin; 40 units insulin; 16
  • the UW solution is discussed in detail in U.S. Patent No. 4,798,824, which is incorporated in its entirety by reference herein.
  • 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 desirable to administer the pharmaceutical compositions of the invention locally to the area in need of treatment.
  • Such administration may be achieved by local infusion during surgery; topical application, e.g., in conjunction with a wound dressing after surgery; by injection; by means of a catheter; by means of a suppository; or by means of an implant, said implant being of a porous, non-porous, or gelatinous material, including membranes, such as silastic membranes, or fibers.
  • a long acting EPO of the present invention may be delivered in a controlled-release system.
  • the polypeptide maybe administered using intravenous infusion, an implantable osmotic pump, a transdermal patch, liposomes, or other modes of admimstration.
  • a pump may be used, such as discussed in Saudek et al, 1989, N. Engl. J. Med. 321 :574.
  • the compound can be delivered in a vesicle, in particular a liposome, such as described in International Publication No. WO 91/04014 and U.S. Patent No. 4,704,355, the entire disclosures of which are incorporated by reference herein.
  • polymeric materials may be used to produce a controlled-release system, such as those materials discussed in Howard et al , 1989, J. Neurosurg. 71 : 105.
  • Such controlled release systems may be placed in proximity of the therapeutic target, i.e., the target cells, tissue or organ, thus requiring only a fraction of the systemic dose. See, e.g., Goodson, Medical Applications of Controlled Release, vol. 2, pp. 115-138, 1984. Other controlled release systems contemplated for use with the present invention are discussed in the review by Langer, Science 249:1527-1533, 1990.
  • Dosing Selection of the preferred effective and non-toxic dose for the administration methods above will be determined by a skilled artisan based upon factors known to one of ordinary skill in the art. Examples of these factors include the particular form of long acting EPO; the pharmacokinetic parameters of the EPO, such as bioavailability, metabolism, half-life, etc. (provided to the skilled artisan); the condition or disease to be treated; the benefit to be achieved in a normal individual; the body mass of the patient; the method of administration; the frequency of administration, i.e., chronic, acute, intermittent; concomitant medications; and other factors well known to affect the efficacy of administered pharmaceutical agents. Thus the precise dosage should be decided according to the judgment of the practitioner and the circumstances of the particular patient.
  • the Physicians Desk Reference shows that, depending on the patient population being treated with EPO, different hematocrit levels are targeted to avoid toxicity.
  • Physicans Desk Reference 54 th Ed., 519-525 and 2125-2131 (2000). In fact, in patients with
  • the PDR recommends dosing EPO to achieve non-toxic target hematocrits ranging from 30 percent to 36 percent.
  • the PDR teaches to adjust the dosage at a different hematocrit level, i.e., if the hematocrit level exceeds 40 percent.
  • the PDR shows that practitioners monitor the patient's hematocrit during therapy with EPO and, to avoid toxicity, adjust the dose and/or withhold treatment if the patient's hematocrit approaches or exceeds the upper limits of a target range. Therefore, the skilled practitioner, armed with the teachings of the present invention, should be able to administer doses of EPO sufficient to achieve a therapeutic effect while avoiding any toxicity complications.
  • the long acting EPO of the present invention is administered chronically or systemically at a dosage of about 0.1 ⁇ g/ kg body weight to about 100 ⁇ g /kg body weight per administration.
  • a dosage of about 0.1 ⁇ g/ kg body weight to about 100 ⁇ g /kg body weight per administration is contemplated for once weekly dosing in the treatment of cancer patients receiving chemotherapy.
  • the dosage of the long acting EPO is about 5 ⁇ g /kg body weight to about 50 ⁇ g /kg-body weight per administration, i still another embodiment, the long acting EPO is administered in an amount of about 10 ⁇ g /kg body weight to about 30 ⁇ g /kg body weight per administration.
  • the long acting EPO is administered in an amount of about 1 ⁇ g/ kg body weight or less.
  • about 0.45 ⁇ g/ kg body weight to about 0.75 ⁇ g/ kg body weight of long acting EPO may be effective when administered once weekly for treatment of anemia in CRF patients.
  • the effective dose is preferably sufficient to achieve serum levels of the long acting EPO of greater than about 10,000 mU/ml (80 ng/ml).
  • the effective dose achieves a serum level of the long acting EPO of about 15,000 mU/ml (120 ng/ml) or greater.
  • the effective dose achieves a serum level of the long acting EPO of about 20,000 mU/ml (160 ng/ml).
  • the serum levels are preferably measured and achieved at about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 hours, or combinations thereof post-administration. Dosages maybe repeated as deemed necessary by one of ordinary skill in the art. For example, administration may be repeated daily, as long as clinically necessary, or after an appropriate interval, e.g., 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 is also increased.
  • the administration of the long acting EPO pharmaceutical compositions of the invention during therapy may decrease the anemic concerns with less frequent and smaller doses than compared to the frequency and amount of present recombinant EPO compositions.
  • the pharmaceutical compositions of the invention include a long acting EPO of the present invention or an EPO analog in a blend with a tissue protective cytokine
  • the compositions may be used to treat anemia and related diseases in patients that are also at risk for tissue injury.
  • a patient with anemia that is also a high risk for heart disease maybe treated with the pharmaceutical compositions of the invention instead of the EPO analogs currently available so as to prevent the risk of increased damage from treatment.
  • the invention also provides a pharmaceutical pack or kit that includes one or more containers filled with one or more of the ingredients of the pharmaceutical compositions of the invention, hi one embodiment, the effective amount of the long acting EPO and a pharmaceutically acceptable carrier may be packaged in a single dose vial or other container.
  • the pharmaceutical composition of the invention is adapted for parenteral administration, for example, the composition may be stored in a lyophihzed condition.
  • the kit may include the lyophihzed composition, a sterile liquid carrier, and a syringe for injections.
  • the kit includes an ampule containing enough lyophihzed material for several treatments such that the administrator would weigh out a specific amount of material and add a specific amount of carrier for each treatment session.
  • the kit may contain a plurality of ampules each containing specific amounts of the lyophihzed material and a plurality of containers each containing specific amounts of carrier, such that the administrator need only mix the contents of one ampule and one carrier container for each treatment session without measuring or weighing.
  • the kit contains an autoinjector including an injectable solution of a long acting EPO of the invention.
  • the kit contains at least one ampule with the lyophihzed 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 containers are preferably hermetically-sealed.
  • the kit preferably includes at least one ampule with the pharmaceutical composition and at least one infusion bottle with sterile pharmaceutical grade water or saline.
  • a kit according to the present invention may also include at least one mouthpiece or specially adapted devices for direct lung inhalation such as pressurized aerosols, nebulizers, or insufflators.
  • the kit may include the device for direct lung inhalation, which contains the pharmaceutical composition, or the device and at least one ampule of aqueous or oil solutions of the long acting EPO of the present invention.
  • the kit preferably includes at least one ampule containing the active ingredient and at least one administration aid.
  • administration aids include, but are not limited to, measuring spoons (for oral administration), sterile cleaning pads (for transdermal administration, and nasal aspirators (for nasal administration).
  • kits may include a single dose of the long acting EPO (acute treatment) or a plurality of doses (chronic treatment).
  • the kit may be outfitted with one or more types of solutions.
  • the long acting EPO pharmaceutical compositions of the invention may be made in an albumin solution and a polysorbate solution. If the kit includes the polysorbate solution, the words "Albumin free” preferably appear on the container labels as wells as the kit main panels.
  • the kit may also include a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use or sale for human administration.
  • the present invention also relates to assays to determine the erythropoietic and tissue protective capabilities of the long acting EPOs of the present invention, as well as the EPO analogs used in several of the pharmaceutical compositions of the present invention.
  • the erythropoietic affect of a long acting EPO may be verified through the use of a TF-1 assay, which is discussed below and in Example 2.
  • the tissue protective properties of EPO compounds may be examined using in vitro assays and in vivo assays, which are discussed in greater detail below.
  • the present invention also contemplates tests for determining not only whether a particular 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 completed within a short period of time using a minimal amount of the EPO compound.
  • the assays provided herein are intended to be non-limiting, as one of ordinary skill in the art would recognize other assays useful for determining the erythropoietic and tissue protective capabilities of EPO compounds.
  • the erythropoietic attributes, i.e., the ability to control hematocrit levels, of a particular EPO compound may be determined using various assays.
  • the potency of the EPO compound of interest may first be assessed in vivo by observing its effect on the hemoglobin concentration using female BALB/c mice. Animals are administered 500 U/kg-bw EPO, the EPO compound of interest, or an equal volume of vehicle subcutaneously three times a week for a total of three weeks (a time interval sufficient to observe an erythropoietic response). An EPO compound is determined to be erythropoietic if it raises the serum hemoglobin concentration of the mice.
  • TF1 erythroleukemia cells Further assessment of potency may be obtained in vitro using TF1 erythroleukemia cells.
  • a TF1 cell line may be used to determine whether a particular EPO compound has erythropoietic activity.
  • the cells may be pelleted, washed, and resuspended at a concentration of 10 5 cells in 1 ml of medium, with recombinant EPO and an EPO compound of interest added at specific concentrations.
  • the individual cultures may be maintained for 24 hours, at which time the cell number is determined using a formazan reaction product (CellTiter; Promega, Madison, WI).
  • CellTiter Promega, Madison, WI
  • European Pharmocopeia discusses at least two assays useful in determining the erythropoietic activity of an EPO compound, which include exhypoxic mouse assays and reticulocyte assays.
  • the tissue protective capability assays of the present invention are based on the tissue protective receptor for EPO. Once the sequence for the tissue protective receptor is isolated, a variety of assays may be used to determine a particular EPO compound's tissue protective capability. As known to those of ordinary skill in the art, the type of assay employed largely depends on the weight of the EPO compound. For example, the assays may be competitive assays or sandwich assays or steric inhibition assays. Competitive assays rely on the ability of a tracer analogue to compete with the test sample analyte for a limited number of binding sites on a common binding partner.
  • analyte refers to the EPO compound of interest to be tested for tissue protective activity.
  • binding partner refers to any protein that binds to the analyte (typically the EPO receptors).
  • tracer refers to labeled reagants, such as labeled analyte analogue, immobilized analyte analogue, labeled binding partner, immobilized binding partner and steric conjugates. The tracer used herein may be any detectable functionality that does not interfere with the binding of analyte and its binding partner.
  • Nonlimiting examples include moieties that may be detected directly, such as fluorochrome, chemiluminescent, and radioactive labels, as well as moieties that must be reacted or derivatized to be detected, such as enzymes.
  • Suitable tracers may be the radioisotopes P , C , I , H , I , and mixtures thereof; fluorophores, such as rare earth chelates, fluorescein, fluorescein derivatives, rhodamine, rhodamine derivatives, dansyl, umbelliferone luciferase (firefly luciferase and bacterial luciferase (U.S. Pat. No.
  • the tracer is at least one of horseradish peroxidase or alkaline phosphatase.
  • coupling agents such as dialdehydes, carbodiimides, dimaleimides, bis-imidates, bis-diazotized benzidine, and the like may be used to tag with the above-described fluorescent, chemiluminescent, and enzyme labels, some of which are described in U.S. Patent Nos. 3,940,475 and 3,645,090, the entire disclosures of which are incorporated by reference herein.
  • Immobilization of reagents i.e., separating the binding partner from any analyte that remains free in solution, is required for a sandwich assay, and may be accomplished by either insolubilizing the binding partner or analyte analogue before the assay procedure, as by adsorption to a water-insoluble matrix or surface (U.S. Patent No. 3,720,760), by covalent coupling, such as glutaraldehyde cross-linking, or by insolubilizing the partner or analogue afterward, e.g., by immunoprecipitation.
  • the binding partner may be insolubilized before or after the competition and the tracer and analyte bound to the binding partner are separated from the unbound tracer and analyte. This separation may be accomplished by decanting (where the binding partner was preinsolubilized) or by centrifuging (where the binding partner was precipitated after the competitive reaction).
  • the amount of test sample analyte is inversely proportional to the amount of bound tracer as measured by the amount of marker substance. Dose-response curves with known amounts of analyte may be prepared and compared with the test results to quantitatively determine the amount of analyte present in the test sample.
  • the assays When used with enzymes as tracers, the assays are typically referred to as ELISA systems. Jh sequential sandwich assays, for example, an immobilized binding partner is used to adsorb test sample analyte, the test sample is removed as by washing, the bound analyte is used to adsorb labeled binding partner, and bound material is then separated from residual tracer. The amount of bound tracer is directly proportional to test sample analyte. In "simultaneous" sandwich assays, the test sample is not separated before adding the labeled binding partner.
  • Competitive and sandwich methods employ a phase-separation step as an integral part of the method, whereas steric inhibition assays are conducted in a single reaction mixture.
  • a conjugate of an enzyme with the analyte is prepared and used such that when anti-analyte binds to the analyte the presence of the anti-analyte modifies the enzyme activity.
  • the tissue protective receptor is conjugated with a bifunctional organic bridge to an enzyme such as peroxidase. Conjugates are selected for use with the EPO so that binding of the EPO inhibits or potentiates the enzyme activity of the label.
  • This type of assay is typically referred to as EMIT. Steric conjugates are used in steric hindrance methods for homogeneous assay.
  • conjugates are synthesized by covalently linking a low-molecular-weight hapten to a small analyte such that antibody to hapten substantially is unable to bind the conjugate at the same time as anti-analyte.
  • the analyte present in the test sample will bind anti-analyte, thereby allowing anti-hapten to bind the conjugate, resulting in a change in the character of the conjugate hapten, e.g., a change in fluorescence when the hapten is a fluorophore.
  • tissue protective capability assays is discussed in co-pending U.S. Patent Application No. 10/188,905, filed July 3, 2002 and in Application Serial No.
  • the tissue protective capabilities of an EPO may be determined using functional assays, both in vivo and in vitro.
  • one of ordinary skill in the art would perform a single in vitro or in vivo assay to determine the tissue protective capabilities of an EPO compound, but in certain instances it may be necessary to perform both to assure that the compound exhibits the same tissue protective capabilities in vitro and in vivo.
  • Jh practice one of ordinary skill in the art would be able to determine whether an EPO analog having at least one additional N-linked carbohydrate chain and/or at least one additional O-linked carbohydrate chain, using a combination of assays disclosed by the present invention.
  • in vitro tests such as the PI 9 cell and rat motoneuron assays could be used to determine whether the EPO compound of interest exhibited tissue protective capabilities.
  • in vivo studies such as the rat focal ischemia, bicuculline seizure, or spinal cord trauma models could be used to verify the results of the in vitro testing.
  • in vitro models contemplated by the present invention include, but are not limited, to those used to determine the lack of tissue protective capabilities of the EPO analogs above: the P19 cell assay, rat motoneuronal cell assay, and the cDNA microarray, which are discussed in greater detail below and further illustrated in Example 2.
  • the examples are intended to be non-limiting as one of ordinary skill in the art would recognize that there are other suitable in vitro assays for determining the tissue protective capabilities of EPO compounds.
  • the EPO compound would be considered tissue protective if, in comparison to a control, it maintained or enhanced the viability of the cell.
  • the erythropoietin would be considered antagonistic if, in comparison to the control, it detrimentally affected the viability of the cells within the assay.
  • the in vitro tissue protective capability assay is based on a P 19 cell line.
  • P19 cells may be maintained undifferentiated in DMEM supplemented with 2 mM L-glutamine, 100 U/ml penicillin G, 100 ⁇ g/ml streptomycin sulfate (Gibco) and 10 percent fetal bovine serum (Hyclone Laboratories), which contains 1.2 g 1 NaHCO 10 mM hepes buffer.
  • Serum-free medium may contain the same components as above, with the exception of 5 ⁇ g/ml of insulin, 100 ⁇ g/ml of fransferrin, 20 nM progesterone, 100 ⁇ M pufrescine and 30 nM Na 2 SeO 3 (Sigma) in place of the fetal bovine serum.
  • Cells that react with 50 percent confluency 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 2 tissue culture flasks at a final density of 10 4 cells/cm 2 in serum-free medium alone, or with the pretreatment additions. Cell viability may be determined by trypan blue exclusion.
  • recombinant EPO can prevent cell loss after serum withdrawal in undifferentiated neuronal-like P19 cells.
  • recombinant EPO rescues up to 50 percent of the neuronal-like cells from death if used in a concentration of 0.1 U/ml to 100 U/ml.
  • the EPO compound of interest must rescue more PI 9 cells from death than the control, preferably it must rescue about 25 percent to about 50 percent of the cells, most preferably about 40 percent to about 50 percent of the cells.
  • a rat motoneuron assay is used to determine the tissue protective capability of an EPO compound of interest in vitro.
  • primary motoneurons may be obtained using spinal cords from 15-day Sprauge Dawley rat embryos and purified by immunopanning.
  • the cells are preferably seeded at low density (20000 cells/cm 2 ) onto glass coverslips in 24 mm well plates precoated with poly-DL-orinthine and laminin and containing complete culture medium (Neurobasal, B27 (2 percent), 0.5 mM L-glutamine, 2 percent horse serum, 25 ⁇ M 2-mercaptoethanol, 25 ⁇ M glutamate, 1 percent penicillin and streptomycin, lng/ml BDNF).
  • complete culture medium Neurospore, B27 (2 percent
  • EPO lOU/ l
  • EPO compound of interest 10 U/ml
  • vehicle may be added to the cultures (preferably about 5 days before determination of surviving neuronal density).
  • the medium may then be discarded and the cells may be fixed with 4 percent paraformaldehyde in PBS for 40 minutes, permeabilized with 0.2 percent Triton X-100, blocked with 10 percent fetal calf serum in PBS, incubated with antibodies against non-phosphorylated neurofilaments (SMI-32; 1 :9000) overnight, and visualized using the avidin-biotin method with diaminobenzidine.
  • the viability of motoneurons may be assessed morphologically by counting SMI-32 positive cells. Mixed primary cultures of motoneurons characteristically undergo apoptosis during maintenance culture conditions.
  • the EPO compound of interest preferably salvages at least the same number of motoneurons as the control.
  • the EPO compound of interest is considered tissue protective if a greater number of motoneurons are saved from death during maintenance culture conditions as compared to the confrol.
  • This assay may be used to determined if recombinant EPO and the EPO compound of interest modify gene expression differently in PI 9 cells.
  • mRNA isolated from undifferentiated PI 9 cells can show a different pattern of gene modulation estimated from a mouse 1200 cDNA microarray, depending upon the exposure to the EPOs.
  • the expression of 1,200 genes in PI 9 cells may be measured by the use of nylon membrane arrays from Clontech (Atlas mouse 1.2). Cells (10 7 /sample) may be treated overnight with saline, recombinant EPO, an EPO compound of interest (lmU/ml), or mixtures thereof.
  • the cells are then lysed for RNA extraction or subjected to serum deprivation for 3 hours (always in the presence of the same cytokine added during prefreatment).
  • polyA + RNA may be purified.
  • Probes may then be constructed in the presence of [P 32 ]-ATP.
  • the labeled probes having preferably 20 million counts or higher, may be hybridized to the cDNA nylon membranes at 68° C. The membranes are washed and exposed to x-ray film. The intensity of radioactive signals may be measured with a Phosphor J ager 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, the tissue protective assays used to evaluate EPO compounds such as the focal ischemia model and infra-hippocampal biculline model. Jh addition, an in vivo model for evaluating tissue protection includes spinal cord injury assays. Furthermore, the various assays disclosed within International Publication No. WO/02053580 and U.S. Patent Publication Nos. 2002/0086816 and 2003/0072737 are contemplated for use with the present invention. D. In Vivo Assay Based on Focal Ischemia Model In one embodiment, the in vivo assay used to determine tissue protective capabilities of a particular EPO compound is based on a focal ischemia model.
  • male Sprague- Dawley rats ( ⁇ 250 gm) may be used with a three vessel focal ischemi model.
  • the rats may be anesthetized with pentobarbital (60 mg/kg-bw) and maintained at a core temperature of 37° C using a water blanket.
  • the right carotid may be occluded by two sutures and transected.
  • a burr hole adjacent and rostral to the right orbit allows visualization of the middle cerebral artery, which may be cauterized distal to the rhinal artery.
  • the contra-lateral carotid artery may be occluded for 1 hour using traction provided by fine forceps.
  • recombinant EPO (5000U/kg-bw) or the EPO compound of interest (5000 U/kg-bw) may be administered at the onset of the reversible carotid occlusion.
  • the brains are removed and serial 1-mm thick sections are cut through the entire brain using a brain matrix device (Harvard Apparatus). Each section may then be subsequently incubated in a solution of 2 percent triphenyltetrazolium chloride (w/v) in 154 mM NaCl for 30 minutes at 37° C.
  • the volume of injury may be determined using a computerized image analysis system (MCID, Imaging Research, St. Catharines, Ontario, Canada).
  • the EPO compound of interest is considered neuroprotective if it ameliorates the infarct volume due to the rat MCA focal ischemia to the same or greater extent as the recombinant EPO.
  • the tissue protective capability of an EPO compound is determined in vivo with infra-hippocampal biculline experiments.
  • male Sprague- Dawley rats 250-280 g are housed at a constant temperature (23° C) and relative humidity (60 percent) with free access to food and water and a fixed 12 hour light/dark cycle.
  • the rats are surgically implanted with cannula and electrodes under stereotaxic guidance as described in Vezzani, A., et al., J. Neurosci, 19, 5054-65 (1999).
  • rats may be anesthetized using Equithesin (1 percent Phenobarbital / 4 percent chloral hydrate; 3ml/kg i.p.).
  • Two screw electrodes are placed bilaterally over the parietal cortex, along with a ground lead positioned over the nasal sinus.
  • Bipolar nichrome wire insulated electrodes 60 ⁇ m may then be implanted bilaterally into the denate gyrus of the dorsal hippocampus (septal pole) and a cannula (22-gauge) may be unilaterally positioned on top of the dura for the intrahippocampal or intracerebroventricular infusion of drugs.
  • the coordinates from bregma for implantation of the electrodes should be: (mm) antero-posterior-3.5; lateral 2.4 and 3 below dura with the nose bar set at -2.5. Paxinos, G. & Watson, C, The Rat Brain in Stereotaxic Coordinates, Academic Press, New York (1986).
  • the electrodes may be comiected to a multipin socket (March Electronics, NY) and, together with the injection cannula, secured to the skull by acrylic dental cement.
  • the experiments are preferably carried out three to seven days after surgery when the animals have fully recovered.
  • Animals are then administered recombinant EPO or the EPO compound of interest (both 5000 U/kg-bw) or vehicle intraperitoneally 24 hours and again at 30 minutes before the induction of bicuculline seizures.
  • the procedures for recording the EEG and intracerebral injection of drugs have been previously described Vezzani, A., et al, J. Pharmacol Exp Ther, 239, 256-63 (1986). Briefly, the animals are allowed to acclimatize in a Plexiglass cage (25x25x60 cm) for a minimum of 10 minutes before initiating the EEG recording (4-channel EEG polygraph, model BP8, Battaglia Rangoni, Bologna, Italy).
  • EEG recordings are made continuously for 120 minutes after 0.8 nmol/0.5 ⁇ l bicuculline methiodide infusion. All the injections were made to unanesthetised rats using a needle (28-gauge) protruding 3 mm below the cannula. Seizures may be measured by EEG analysis, which has previously been shown to provide a sensitive measure of the anticonvulsant activity of drugs. Vezzani, A., et al., J. Pharmacol Exp Ther, 239, 256-63 (1986).
  • seizures consist of the simultaneous occurrence of at least two of the following alterations in all four leads of recordings: high frequency and/or multispike complexes and/or high voltage synchronized spike or wave activity. Synchronous spiking may be observed intermixed with seizures.
  • the parameters chosen to quantify seizures are preferably the latency to the first seizure (seizure onset), the total time spent in epileptic activity (determined by adding together the duration of ictal episodes; seizure duration), and the spiking activity during the EEG recording period (seizure activity).
  • an acute reversible glaucoma rat model may be used to determine the tissue protective capability of particular EPO compounds of interest. 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 peripherally- administered EPO compounds of interest exhibit tissue protective activities sufficient to protect cells sensitive to ischemia, an acute, reversible glaucoma rat model may be used as described by Rosenbaum et al, Vis. Res. 37: 3443-51, 1997.
  • saline maybe injected into the anterior chamber of the eye of adult male rats to a pressure above systemic arterial pressure and maintained for 60 minutes. Animals are then administered saline or 5000 U EPO /kg body weight intraperitoneally 24 hours before the induction of ischemia, and continued as a daily dose for three additional days. Electroretinography may be performed on dark-adapted rats one week after treatment to determine whether the EPO compound of interest possesses tissue protective activity. If the long acting EPO is tissue protective, there should be good preservation of activity on the electroretinogram, in contrast to animals treated with saline alone.
  • Myocardial Infarction assays are also contemplated for use with the present invention to determine whether an EPO compound exhibits tissue protective activity in general or within the heart specifically.
  • EPO 5000 U/kg body weight
  • An additional dose of EPO may be given at the start 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 given daily for one week after treatment, at which time the animals are studied for cardiac function.
  • the animals are preferably fasted for 12 hours before surgery, and humanely restrained and anesthesized with an intraperitoneal injection of thiopental sodium (40 mg/kg-bw).
  • thiopental sodium 40 mg/kg-bw.
  • T-3 complete single level laminectomy is performed through a 2 cm incision with the aid of a dissecting microscope.
  • Traumatic spinal cord injury is induced by the exfradural application of a temporary aneurysm clip exerting a 0.6 newton (65 grams) closing force on the spinal cord for 1 minute. After removal of the clip, the skin incision is closed and the animals allowed to recover fully from anethesia and returned to their cages.
  • the rats are monitored continuously with bladder palpation at least twice daily until spontaneous voiding resumed.
  • Animals in a control group receive normal saline (via intravenous injection) immediately after the incision is closed.
  • the remaining animals receive the EPO compound of interest in an amount of 16 micro grams/kg-bw iv.
  • the motor neurological function of the rats is then evaluated using a locomotor rating scale. In this scale, animals are assigned a score ranging from 0 (no observable hindlimb movements) to 21 (normal gait).
  • the rats are tested for functional deficits at 1 hour, 12 hours, 24 hours, 48 hours, 72 hours, and 1 week after injury by the same examiner who is blind to the treatment each animal receives. If the long acting EPO of interest is tissue protective, the rats that are given the EPO should exhibit a quicker and beter overall recovery from the injury than the rats that are given the saline injection.
  • rabbit spinal cord ischemia testing allows testing for tissue protective capability.
  • 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 humanely restrained. Anesthesia induction is via 3 percent halothane in 100 percent oxygen and maintained with 0.5 percent to 1.5 percent halothane in a mixture of 50 percent oxygen and 50 percent air.
  • An intravenous catheter 22 gauge is placed in the left ear vein. Ringers lactate is infused at a rate of 4 ml/kg body weight (bw) per hour during the surgical procedure.
  • cefazoline 10 mg/kg-bw is administered intravenously for prophylaxis of infection.
  • the animals are placed in the right lateral decubitus position, the skin prepared with povidone iodine, infiltrated with bupivacaine (0.25 percent) and a flank skin incision is made parallel to the spine at the 12th costal level.
  • the longissimus lumborum and iliocostalis lumborum muscles are retracted.
  • the abdominal aorta is exposed via a left retroperitoneal approach and mobilized just inferior to the left renal artery.
  • a piece of PE-60 tubing is looped around the aorta immediately distal to the left renal artery and both ends passed through a larger rubber tube. By pulling on the PE tubing, the aorta is non-traumatically occluded for 20 minutes. Heparin (400 IU) is administrated 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 full recovery, at which time, they are serially assessed for neurological function. A control group of animals receives normal saline intravenously immediately after release of aortic occlusion.
  • Motor function is assessed according to the criteria of Drummond and Moore by an investigator blind to the treatment at 1 hour, 24 hours, and 48 hours after reperfusion.
  • the urinary bladder is evacuated manually in paraplegic animals twice a day.
  • the animals that are given the EPO should exhibit a quicker and better overall recovery from the injury than the animals receiving the saline injection.
  • J. Other Potential Assays for Testing the Tissue Protective Effects of EPO and its Analogs As briefly discussed earlier, several types of tissues possess EPO receptors and, therefore, may be responsive to the tissue protective affects of EPO. Thus, depending upon the proposed clinical application for the EPO compound of interest, a skilled artisan would recognize that similar in vitro assays involving these additional responsive cells may be performed, or in vivo assays involving the associated organs may also be performed.
  • in vitro assays based on serum deprivation can be performed using myocardial, retinal, and Leydig cells and a protocol similar to that outlined above for the PI 9 assay.
  • In vivo assays can be directed to individual organs as well.
  • to evaluate an EPO's affect upon retinal cells one of ordinary skill in the art may perform the retinal ischemia assay described above.
  • to evaluate an EPO analog's effect upon myocardial cells a skilled artisan could readily modify the myocardial infarction model discussed above.
  • Those of ordinary skill in the art will be sufficiently skilled to select the appropriate assay or model to evaluate whether a particular EPO possesses tissue protective activities with regard to an erythropoietic responsive cell, tissue or organ.
  • Example 1 Chemically Modified EPO
  • EPO Oxidation of Sugar Chains
  • the sugar units of EPO may be converted into acids by the following procedure.
  • EPO and an amount of sodium periodate sufficient to provide the amount of oxidation desired may be placed within a 100 mM sodium acetate buffer. This solution may then be incubated on ice for about 20 minutes and dialyzed thoroughly using distilled water. The product may then be removed from the dialysis tubing and collected into a fresh tube (Product I).
  • a Quantitative Benedict Solution (18 g copper sulfate, lOOg sodium carbonate
  • EPO EPO may be dissolved in N,N-dimethylformamide (DMF-SA) at 4° C.
  • N,N'-dicyclohexyl carbodiimide (DCC) dissolved in DMF may then be added and the solution shaken for 4 hours at 4° C.
  • Cracked ice may be added and the pH may be adjusted to 7.5 with IO NaOH.
  • the volume of the solution may be adjusted and the sample may be centrifuged for 1000 x g for 15 minutes in a type HN-S2 centrifuge (DAMONIEC, Needham Hts., Massachusetts). The supernatant may then be extensively dialyzed. More information regarding sulfation is discussed in S.
  • PEG-EPO conjugates may be prepared by first oxidizing EPO (2-4 mg/ml in 50 mM sodium acetate) for 30 minutes at room temperature with 1 mM to 100 mM sodium meta- periodate (Sigma).
  • the phosphate buffer may then be removed by buffer exchange in 100 mM sodium acetate, ph 5.4.
  • Methoxy-PEG-hydrazide of various molecular weights (Nektar Therapeutics) may then be added at a 5 fold to 100 fold molar excess (polymer: protein).
  • the intermediate hydrazine linkage may then be further reduced by the addition of 15 mM sodium cyanoborohydride (Sigma) and allowed to react overnight at 4°C.
  • the resultant conjugates may then be fractionated / purified by techniques known in the art.
  • An asialo form of EPO may be modified through the attachment of PEG chains to newly created terminal galactose residues after oxidiation with galactose oxidase, such as those obtained above in B (Product III).
  • Recombinant human EPO rhuEPO
  • Sialidase A Prozyme, Inc.
  • the chemical modification is preferably confirmed by running the reaction product on a SDS polyacrylamide gel. Staining the resultant bands should show that the modified EPO has an apparent molecular weight of about 31 kDa, while the unmodified EPO has a molecular weight of about 34 kDa.
  • the sialic acid residues remaining on the EPO are preferably less than 0.1 mole/mole of EPO.
  • the PEG-NH 2 is preferably added at a 250 fold molar excess (polymer: protein) (80 mg of PEG-NH 2 ).
  • the resultant conjugates may then be fractionated / purified by techniques known in the art.
  • the erythropoietic attributes, i.e., the ability to control hematocrit levels, of a long acting EPO compound can be determined using the following assay.
  • TF1 is a human erythroleukemic cell line with complete dependence on growth factors, including EPO. Kitamura, et al, Blood 73, 375-80.
  • TF1 cells were obtained from ATCC and maintained in RPMI 1640 with the following: 2mM L-glutamine, 10 mM Hepes, 1 mM sodium pyruvate, 4.5 g/L glucose, 1.5 g/L sodium bicarbonate, 5 ng/ml GM-CSF, and 10 percent fetal bovine serum until experimentation.
  • TF1 cells obtained during active growth were pelleted, washed three times with medium alone, and resuspended at a concentration of 10 5 cells in 1 ml of medium, with or without GM-CSF, with EPO or an EPO analog having at least one additional N- linked carbohydrate chain and/or at least one additional O-linked carbohydrate chain added at specific concentrations.
  • the potency of a chemically modified EPO compound can be assessed in vivo by observing its effect on the hemoglobin concentration using female BALB/c mice. Animals were administered 500 U/kg-bw EPO, the EPO compound of interest, or an equal volume of vehicle subcutaneously three times a week for a total of three weeks (a time interval sufficient to observe an erythropoietic response). These or an EPO compound are determined to be erythropoietic if they raise the serum hemoglobin concentration of mice.
  • tissue protective attributes of an EPO analog having at least one additional N-linked carbohydrate chain and/or at least one additional O-linked carbohydrate chain were determined using the following assay. Neuronal cultures were established from the hippocampus of 18-day rat fetuses. Brains were removed and freed from meninges and the hippocampus was isolated. Cells were then dispersed by incubation for 5 minutes at 37° C in a 2.5 percent trypsin solution followed by titration.
  • the cell suspension was diluted in serum-free Neurobasal media containing 1 percent B- 27 supplement (Gibco, Rockville, MD, USA) and plated onto polyornithine-coated coverslips at a density of 80,000 cells per coverslip.
  • Cells were then pre-treated with EPO overnight and then exposed with or without 1) EPO (100 ng/ml), or 2) an EPO analog having at least one additional N-linked carbohydrate chain and/or at least one additional O-linked carbohydrate chain within the O'Brien peptide sequence(100 ng/ml) to 5 ⁇ M trimethyl tin (TMT) for 24 hours. Cultures were used between 10 and 14 days in vitro.
  • MTT 3-(4,5-dimethyl-thiazol-2-yl)-2,5- diphenyltetrazolium bromide

Abstract

L'invention concerne des méthodes destinées à augmenter le taux d'hématocrites d'un individu tout en maintenant les activités de protection tissulaire d'une érythropoïétine endogène, par administration d'un composé pharmaceutique contenant une érythropoïétine modifiée chimiquement à action prolongée. L'invention concerne en outre de nouvelles érythropoïétines modifiées chimiquement à action prolongée, des méthodes de production des érythropoïétines de l'invention, et des compositions contenant ces érythropoïétines.
PCT/US2004/007133 2002-09-09 2004-03-08 Erythropoietines a action prolongee pouvant maintenir une activite de protection tissulaire d'une erythropoietine endogene WO2005025606A1 (fr)

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AU2004260543A AU2004260543A1 (en) 2002-09-09 2004-03-08 Long acting erythropoietins that maintain tissue protective activity of endogenous erythropoietin
US11/075,957 US20050176627A1 (en) 2002-09-09 2005-03-09 Long acting erythropoietins that maintain tissue protective activity of endogenous erythropoietin
IL174178A IL174178A0 (en) 2002-09-09 2006-03-08 Long acting erythropoietins that maintain tissue protective activity of endogenous erythropoietin

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USPCT/US03/28073 2003-09-09
PCT/US2003/028073 WO2004022577A2 (fr) 2002-09-09 2003-09-09 Erythropoietines a action longue qui maintiennent l'activite protectrice du tissu d'une erythropoietine endogene

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WO2020081479A1 (fr) 2018-10-15 2020-04-23 Amgen Inc. Dispositif d'administration de médicament comprenant un mécanisme d'amortissement
WO2020081480A1 (fr) 2018-10-15 2020-04-23 Amgen Inc. Procédé d'assemblage de plate-forme pour dispositif d'administration de médicament
WO2020091956A1 (fr) 2018-11-01 2020-05-07 Amgen Inc. Dispositifs d'administration de médicament avec rétraction partielle de l'organe d'administration de médicament
WO2020091981A1 (fr) 2018-11-01 2020-05-07 Amgen Inc. Dispositifs d'administration de médicament à rétraction partielle d'élément d'administration de médicament
WO2020092056A1 (fr) 2018-11-01 2020-05-07 Amgen Inc. Dispositifs d'administration de médicament à rétraction d'aiguille partielle
WO2020219482A1 (fr) 2019-04-24 2020-10-29 Amgen Inc. Ensembles et procédés de vérification de stérilisation de seringue
WO2021041067A2 (fr) 2019-08-23 2021-03-04 Amgen Inc. Dispositif d'administration de médicament doté de composants configurables de mise en prise de protection d'aiguille et méthodes associées
WO2022246055A1 (fr) 2021-05-21 2022-11-24 Amgen Inc. Procédé d'optimisation d'une recette de remplissage pour un récipient de médicament

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