KR20070032000A - Novel carbamylated epo and method for its production - Google Patents

Abstract

The present invention discloses a process for the preparation of novel carbamylated erythropoietin, compositions comprising the novel carbamylated erythropoietin, pharmaceutical compositions comprising the same and uses thereof.

New Carbamylated Erythropoietin

Description

New carbamylated EPO and its manufacturing method {NOVEL CARBAMYLATED EPO AND METHOD FOR ITS PRODUCTION}

The present invention relates not only to novel compounds, but also to methods of preparing such compounds. The novel compound carbamylated erythropoietin (CEPO) is characterized by carbamylation on all or most of the primary amines and N-terminal amino acids of the lysine of the molecule. The level of carbamylation of primary amines is low. Moreover, the novel compounds are free of aggregated proteins and polymers and are suitable for use in pharmaceutical compositions for the treatment of diseases in the central or peripheral nervous system and other tissues expressing major EPO receptors, for example. One other surprising advantage of the present preparation method is the fact that the present method provides a product containing little aggregated proteins and polymers than the product achieved from other known carbamylation methods described for erythropoietin.

Of carbamylated EPO Impairment of biological hematopoietic action is described by Satake, R. et al. (1990) Biochimica et Biophysica Acta; 1038: 125-129 and Mun, KC. and Golper, TA (2000) Blood Purif .; 18: 13-17. 2003 US patent application 20030072737 by Brines et al. Showed that loss of hematopoietic activity did not interfere with the tissue protective properties of EPO.

Carbamylation of proteins is well known as a side effect of using urea in the purification of proteins and as a result of high urea serum concentrations. This is caused by spontaneous decomposition of urea to cyanate. Cyanate is responsible for carbamylation of the primary amine of the protein, so that the N-terminus and lysine of the protein tend to be carbamylated (FIG. 1). In addition, other potential amino acid residues that are susceptible to carbamylation are arginine, cysteine, tyrosine, aspartic acid, glutamic acid and histidine, but the reaction is pH dependent and is not easily performed with N-terminal and lysine residues.

Studies to determine whether carbamylation of proteins can enhance or impair the biological action of proteins are described in Hoerkkoe, S. et al. (1992) Kidney International .; 41: 1175-1181, Plapp, B.V. Et al. (1971) Jour. Biol. Chem .; 246 (4): 939-945, Satake, R. et al. (1990) Biochimica et Biophysica Acta; 1038: 125-129 and Mun, K-C. and Golper, T. A. (2000) Blood Purif .; 18: 13-17]. They studied the biological effects of carbamylation of proteins by using KCNO as the cyanate source. They all observed a decrease or change in biological action as a result of increased carbamylation. The assessment of the degree of carbamylation was based on two analytical methods:

1. Determination of the reduction in free amino groups using trinitrobenzenesulfonic acid (TNBS) assay and

2. Amino acid analysis to determine lysine converted to homocitrulline residues.

Hoerkkoe S. et al. (1992) carbamylated low-concentration lipoprotein for up to 6 hours at 37 ° C. with 2 M KCNO, but did not yield fully carbamylated protein as measured by TNBS assay.

Plapp, BV et al. (1971) studied the effect of time and obtained almost completely carbamylated bovine pancreatic deoxyribonuclease A after treatment for 24 hours with 1 M KCNO at 37 ° C.

Mun, K-C. And Golper, T.A. (2000) studied the effect of time using a reaction time of up to 6 hours using 2 M KCNO. They also conducted all reactions of 6 hours at 37 ° C. and studied the effect of increasing KCNO concentrations. Mun, K-C. And Golper, T.A. (2000) could not prove the exact degree of carbamylation from the experimental design (see lines 33-35 on page 16).

Applicants have now found that the carbamylation of EPO in the present invention provides polymers and aggregates and makes them unsuitable as biopharmaceuticals. Applicants have also found that the information of the polymers and aggregates depends on the process conditions for carbamylation. Therefore, there was a need for the development of a method with optimal parameters regarding pH, time, cyanate concentration, temperature, protein concentration and most importantly the degree of protein polymerization. The present invention includes an optimal method of carbamylation that provides a product with a low degree of polymerization and aggregation, and furthermore, surprisingly, Applicants have found that the N-terminal and all lysine residues (all lysine residues occur in specific pH-ranges). It was found that the target fully carbamylated EPO was obtained. Subsequent steps of the process of the present invention were made to remove the formed aggregates and polymers. The resulting carbamylated pure EPO is a novel compound and is claimed as in this application together with a pharmaceutical composition comprising the compound.

It has already been illustrated that the degree of carbamylation depends on cyanate concentration and time. However, no method is described for obtaining a measurable carbamylation method for the preparation of a biopharmaceutical.

The presence of aggregates by suboptimal production is associated with the induction of antibodies. Thus, the presence of aggregates results in biopharmaceutical preparations that are not suitable for use in humans.

The method of carbamylation and purification described herein results in a protein characterized as fully carbamylated with the lowest formation of polymers or aggregates as possible, with minimal loss of final product. Therefore, it is a stage to grow economically.

Further processing of carbamylated proteins allows the product to be useful as a biopharmaceutical with only minimal risk of developing an immunological response to the protein due to aggregates and polymers.

Analytical methods for the evaluation of complete carbamylation include, in addition to amino acid analysis; Characterization of the decomposed and degraded products by TNBS and finally MALDI-TOF for free primary amino groups.

Fully carbamylated on the free amino groups at the N-terminus and lysine of the molecule, further aggregated or polymerized to a content of greater than 2.5%, with minimal over- or under-carbamylation The novel compounds of the invention, which are erythropoietins containing erythropoietins, can be used in the preparation of pharmaceutical compositions for the treatment of diseases that are responsive to the neuroprotective effects of native erythropoietin.

Summary of the Invention

The present invention relates to a scaled protein carbamylation process for the preparation of a biopharmaceutical. Moreover, the present invention relates to the products of the above methods, and to pharmaceutical compositions comprising the compounds, and to the use of such compositions.

The method of carbamylation and purification described herein results in a protein characterized as fully carbamylated with the lowest possible formation of polymers or aggregates and minimal loss of final product.

The carbamylation process has been optimized to provide carbamylated proteins with the lowest amount of polymers and aggregates, making the process economically viable. The final product further contains a limited amount of isoforms of over- and / or sub-carbamylated erythropoietin (less than or more than 9 carbamylated per molecule). Sub-carbamylated EPO contains less than 9 carbamyl residues, ie all eight lysines and N-terminus are not carbamylated. Sub-carbamylated EPO may have as little as 5 carbamyl residues and also does not have a standard red blood cell production action, making it suitable for use in the present invention. Over-carbamylated EPO will have more than 9 carbamyl residues and will have carbamylation at eight lysine residues and amino acids other than the N-terminus. CEPO may have as many as 15 carbamyl residues and also have the desired effect, i.e., may not have a standard red blood cell production. At least about 90% and almost 95% of the CEPO isomers are carbamylated at only eight lysine residues and at the N-terminus.

Further processing of carbamylated proteins removes up to 3% or 2.5% of aggregated and polymerized products, thereby providing a biopharmaceutical with only minimal risk of developing an immunological response to proteins due to aggregates and polymers As a useful product.

Analytical methods for the evaluation of carbamylation include amino acid analysis as well; Characterization of the degraded product and the product by TNBS and MALDI-TOF and LC-MS / MS for free primary amino groups.

Detailed description of the invention

Manufacturing method

Six steps constitute a method of carbamylation of proteins:

1. Concentration by Ultrafiltration.

2. Modification by carbamylation.

3. Desalting by gel filtration.

4. Purification by anion-exchange.

5. Ultrafiltration  And In diafiltration  By thickening and Buffer  exchange.

6. 0.22 μm filtration.

The starting material of the present carbamylation method is advantageously purified human EPO, but can be in the form of any EPO of animal or human type, and in non-limiting examples, asialo-EPO, mutation of human EPO, ie in amino acid sequence Molecules, EPO fragments, EPO peptides, other proteins, or multiple proteins introduced with a change in are synthetic, recombinant human EPO, such as mixtures of proteins, or biologically or chemically modified human EPO, if desired.

The first step of the method involves adjusting protein concentration by ultrafiltration, where the protein concentration is adjusted to maintain a low process volume. Protein concentrations of 0.05-10 mg / ml or 0.05-8 mg / ml are preferred embodiments. A more preferred embodiment is 0.05-7 mg / ml, most preferred 2-5 mg / ml. As the concentration is increased, aggregates are formed to increase. Ultrafiltration is performed using BioMax (Millipore) with a MWCO of 5 kDa. Other filters may be used. In addition, the solubility of the protein can be adjusted by adding stabilizers.

After the concentration step is complete, the protein solution is mixed with K-borate tetra hydrate, K-cyanate, pH 7-11 or pH 7-10. In a preferred embodiment, the pH is 8-10, most preferably 9.0. 10 minutes-30 days or 30 minutes-30 days or 1 hour-30 days or 1 hour-20 days or 1 hour-10 days or 1 hour-5 days or 1 hour-2 days or 1 hour-26 days or 18- For 26 days or preferably 22 hours-26 hours, most preferably 24 hours, the temperature is in the range of 0 ° C to 60 ° C or 0 ° C to 50 ° C or 0 ° C to 40 ° C or 0 ° C to 37 ° C, Preferred embodiments have a temperature interval of 30 ° C-34 ° C, preferably 32 ° C. However, if other process variables, namely temperature, cyanate concentration and protein concentration change, the preferred interval may change.

If the temperature is below the limit, the carbamylation will be slow and insufficient and the yield will be low. If the temperature limit is exceeded, the yield will be low due to the increased aggregation. Another important variable is time, because if time is reduced or if time is increased the formation of aggregates will be observed, resulting in low yields and thus incomplete carbamylation.

Thus, there is a process with coherent parameters, i.e. if the temperature is lowered, the reduced carbamylation reaction can be supplemented by increasing the cyanate concentration and / or reaction time. In addition, if the reaction time is reduced, the reduced carbamylation reaction can be supplemented by increasing the temperature and / or cyanate concentration. Finally, in processes with reduced cyanate concentrations, the reduced carbamylation reaction can be supplemented by increasing the reaction time and / or temperature.

Thus, in conclusion, a significant change in one of the important variables (time, temperature, cyanate concentration and protein concentration) is one or more other important variables in order to obtain fully carbamylated molecules with low formation of aggregates and polymers. It will include a change in.

The concentration of borate buffer may be 0.05-2 M, but in preferred embodiments 0.1-1, since cyanate inherently hydrolyzes and polymerizes under the acquisition of protons, and a lack of buffer capacity results in a shift in pH of the solution. M and most preferably 0.5 M.

In addition, the cyanate concentration is preferably in the range of 0.05-10 M or 0.05-8 M or 0.05-6 M or 0.05-4 M or 0.05-2 M, preferred embodiments are 0.05-1 M, most preferably 0.5 M.

Concentration of 0.5 M borate buffer is necessary to control the pH shift caused by proton acquisition of 0.5 M cyanate concentration in use. Methods using other salts of cyanates and borate can be used. In addition, reaction buffers other than borate may be used, such as carbonate buffer or phosphate buffer.

Desalting of the reaction mixture of protein and cyanate is carried out using chromatography gel filtration. A G-25 Fine (Amersham Biosciences) mattress is used. The holding time before the sample is applied to the column is controlled and should not exceed 2 hours, as this will lead to further carbamylation and polymer formation. Desalting and buffer exchange of proteins can be performed using dialysis, diafiltration or chromatography gel filtration. Other gel filtration mattresses can be used, for example, as crosslinked polysaccharides or crosslinked mixed polysaccharides, polyacrylamides, mattresses of polystyrene or mattresses of natural ceramics. Furthermore, the column height can vary at this stage.

The carbamylation step is carried out to obtain a product of less than 40% aggregates and less than 30% or less than 25% or less than 20% or less than 15% or less than 12.5% or less than 10% or less than 8% or less than 7% polymer. Can be adjusted.

Removal of aggregates and polymers is carried out by a purification step using anion exchange. It is observed that the carbamylated EPO can be separated from the residuals and aggregates / polymers of the starting material. Progression buffer A is: 0.3% Tris (25 mM), 0.3% (50 mM) NaCl, pH 8.5 ± 0.2, and elution buffer B is: 0.3% Tris (25 mM), 5.8% (1 M) NaCl, pH 8.5 ± 0.2. The gradient is carried out at 0-30% over 20 column volumes to obtain the desired separation. The purification step can result in products with less than 3% aggregates and less than 2.5% or less than 2% or less than 1.5% or less than 1% or less than about 0.5% polymer.

Elution, pooling and pooling of carbamylated EPO peaks affects the heterogeneity of the eluted protein, ie the distribution of isomers. That is, the amount of over- and sub-carbamylated CEPO will vary depending on the harvesting and pooling process. Narrow pooling will lower the content of over- and / or sub-carbamylated erythropoietin. Increasing the length of the gradient will allow selection of more defined products by excluding some species.

A composition of carbamylated EPO with less than about 40% by weight of the over- and sub-carbamylated isomers as measured by an ESI-mass spectrometer is one embodiment of the present invention. A more preferred embodiment is CEPO with less than about 35% by weight of over- and sub-carbamylated isomers as measured by an ESI-mass spectrometer. Even more preferred embodiments are CEPO with less than about 30% by weight of over- and sub-carbamylated isomers as measured by ESI-mass spectrometry. Even more preferred embodiments are CEPO with less than about 25% by weight of over- and sub-carbamylated isomers as measured by an ESI-mass spectrometer. Even more preferred embodiments are CEPO with less than about 20% by weight of over- and sub-carbamylated isomers, as measured by an ESI-mass spectrometer. Even more preferred embodiments are CEPO with less than about 15% by weight of over- and sub-carbamylated isomers, as measured by an ESI-mass spectrometer. Even more preferred embodiments are CEPO with less than about 10% by weight of over- and sub-carbamylated isomers, as measured by an ESI-mass spectrometer. Even more preferred embodiments are CEPO with less than about 5% by weight of over- and sub-carbamylated isomers as measured by an ESI-mass spectrometer. Even more preferred embodiments are CEPO with less than about 2% by weight of over- and sub-carbamylated isomers as measured by an ESI-mass spectrometer. Most preferred embodiments are CEPO with less than about 1% by weight of over- and sub-carbamylated isomers, as measured by an ESI-mass spectrometer.

In addition to affecting the total content of isomers, the pooling and pooling of carbamylated EPO peaks affects the distribution of over-carbamylated CEPO. It is preferred that the amount of over-carbamylated CEPO isomers is less than about 35% by weight as measured by an ESI-mass spectrometer. It is even more preferred that the amount of over-carbamylated CEPO isomers is less than about 30% by weight, as measured by ESI-mass spectrometry, and that the amount of over-carbamylated CEPO isomers is less than about 25% by weight. Even more preferred, less than about 20% by weight, even more preferably less than about 15% by weight. Most preferably the amount of over-carbamylated EPO should be about 10% by weight or less, about 5% by weight or less or about 1% by weight or less of the total CEPO.

Different run and elution buffers can be used as other anion exchange mattresses, and filled filters can be used. The mattress in a non-limiting embodiment is a mattress of crosslinked polysaccharide or crosslinked mixed polysaccharide, polyacrylamide, polystyrene or natural ceramic.

In addition to cation exchange, hydrophobic interaction chromatography, reverse phase chromatography, affinity chromatography and size exclusion chromatography can be used for purification.

In the next step for the adjustment of concentration and buffer, a diafiltration / superfiltration tangential flow filtration unit is used. Carbamylated EPO was adjusted to concentration> 0.5 mg / ml and the buffer was changed to 20 mM citrate, 100 mM NaCl buffer. Concentration and buffer changes are performed using BioMax (Millipore) with a MWCO of 5 kDa. Other filters may be used.

Finally, the purified biopharmaceutical drug component is 0.22 μm filtered using Millipak (Millipore) to reduce pathogens. Any 0.22 μm filter can be used.

Using this method, fully carbamylated EPO is obtained with less than 3% or preferably less than 2.5% aggregates as measured by SEC-HPLC. Complete carbamylation of eight lysine residues is demonstrated using amino acid analysis to determine converted lysine to homocyclin. Moreover, carbamylation was then performed using a TNBS assay for the determination of primary amines, thus showing complete carbamylation of lysine and the N-terminus.

In addition, complete characterization with MALDI-TOF determined the change in intrinsic mass of both PNGase treated proteins and proteins with N-glycans. In addition, MALDI-TOF peptide mass fingerprint analysis / LC-MS / MS analysis showed that all eight lysines and N-terminus were carbamylated. The very carbamylated amino acid was detected, and no modification of the glycan was detected. Moreover, over- and sub-carbamylated forms of EPO are obtained in the final product in reduced content. The product is new and claimed.

One embodiment of the invention is a composition obtained after the carbamylation step, but before anion exchange purification, wherein the aggregates and polymers are less than about 40 weight percent, or less than about 30 weight percent, or less than about 25 weight percent, or about 20 weight Carbamylated EPO that is less than%, or less than about 15 weight percent, or less than about 12.5 weight percent, or less than about 10 weight percent, or less than about 8 weight percent, or less than about 7 weight percent, and a substantial amount of cyanate do.

One further embodiment of the invention provides that the aggregates and polymers are less than about 3 weight percent, or less than about 2.5 weight percent, or less than about 2 weight percent, or less than about 1.5 weight percent, or less than about 1 weight percent or about 0.5 weight. A composition obtained after anion exchange purification, comprising less than% carbamylated EPO. In addition, the composition may comprise less than about 40 weight percent, or more preferably less than about 35 weight percent, or less than about 30 weight percent, or less than about 25 weight percent, or less than about 20 weight percent of total carbamylated EPO. , Or less than about 15 wt%, or less than about 10 wt%, or less than about 7.5 wt%, or less than about 5 wt%, or less than about 2 wt%, and most preferably less than about 1 wt% Or isomers consisting of sub-carbamylated EPO. In addition, the amount of over-carbamylated EPO in the composition is less than about 35 weight percent, or more preferably less than about 30 weight percent, or less than about 25 weight percent, or about 20 weight percent of total carbamylated EPO. Or less than about 15 weight percent, or less than about 10 weight percent, or less than about 7.5 weight percent, or less than about 5 weight percent, or less than about 2 weight percent, and most preferably less than about 1 weight percent. .

Pharmaceutical Compositions of the Invention

One aspect of the invention is the use of a compound of the invention for the preparation of a pharmaceutical composition for use in humans or mammals for the treatment of the conditions described below.

One embodiment of the invention provides less than about 3 weight percent, or more preferably less than about 2.5 weight percent, or less than about 2 weight percent, or less than about 1.5 weight percent, or less than about 1 weight percent of aggregates and polymers, and Most preferably a pharmaceutical composition comprising a therapeutically effective amount of carbamylated EPO of less than about 0.5% by weight, wherein the composition further comprises a total carbamylated amount of over- or sub-carbamylated EPO. Less than about 40 weight percent, more preferably less than about 35 weight percent, or less than about 30 weight percent, or less than about 25 weight percent, or less than about 20 weight percent, or less than about 15 weight percent, or about 10 weight percent of the EPO Isomers consisting of less than, or less than about 5 weight percent, or less than about 3 weight percent, or less than about 2 weight percent and most preferably less than about 1 weight percent. In addition, the amount of over-carbamylated EPO in the composition is less than about 35 weight percent, or more preferably less than about 30 weight percent, or less than about 25 weight percent, or about 20 weight percent of total carbamylated EPO. Less than about 15 weight percent, or less than about 10 weight percent, or less than about 5 weight percent, or less than about 3 weight percent, or less than about 2 weight percent and most preferably less than about 1 weight percent.

In the practice of one aspect of the present invention, a pharmaceutical composition as described above containing a compound of the present invention is applied to the vasculature to allow beneficial effects on translocation and reactive cells across the endothelial cell wall. It may be administrable to a mammal by any route that provides a sufficient concentration of a compound of the invention. When used to perfuse a tissue or organ, similar results are intended. When a cell or tissue is non-angiogenic and / or administered by washing the cell or tissue with a composition of the invention, the pharmaceutical composition provides the compound of the invention in an effective reactive cell-beneficial amount. Endothelial cell walls that can be transposed across the compounds of the present invention include tight junctions, perforated junctions, fenestrated junctions, and any other type of endothelial cell wall present in mammals. do. Preferred walls are endothelial cell tight junctions, but the invention is not so limited.

The above-mentioned compounds of the present invention are generally used in the central nervous system of humans with primary neurological or psychiatric syndromes, eye diseases, cardiovascular diseases, cardiopulmonary diseases, respiratory diseases, kidneys, urinary diseases and obstetric diseases, digestive diseases and endocrine abnormalities and metabolic abnormalities. Or for the therapeutic or prophylactic treatment of peripheral nervous system diseases. In particular, the symptoms and diseases include hypoxia symptoms that adversely affect excitatory tissues such as, for example, central nervous system tissues such as brain, heart, or retina / eye, peripheral nervous system tissues, or excitatory tissues of cardiac or retinal tissues. do. Thus, the compounds of the present invention can be used to treat or prevent damage to excitatory tissue due to hypoxia symptoms in a variety of conditions and environments. Non-limiting examples of such conditions and environments are provided in the table below.

In the example of the protection of a neural tissue condition treatable in accordance with the present invention, the condition includes those due to reduced oxidation of neural tissue. Any condition that reduces the availability of oxygen to neural tissue, resulting in stress, damage, and finally neuronal cell death, can be treated by the methods of the present invention. These symptoms, commonly referred to as hypoxia and / or ischemia, include trauma, fainting, epilepsy, including seizures, vascular obstruction, prenatal or postnatal oxygen deficiency, choking, choking, drowning accidents, carbon monoxide poisoning, smoke inhalation, surgery and radiotherapy Nerve caused by hypoglycemia, chronic obstructive pulmonary disease, emphysema, adult respiratory distress syndrome, hypotension shock, septic shock, anaflash shock, insulin shock, sickle cell development, cardiac arrest, arrhythmia, nitrogen poisoning, and cardiopulmonary bypass surgery It results from or includes, but is not limited to, a deficiency.

In one embodiment, for example, certain pharmaceutical compositions comprising a composition of the present invention are administered to prevent injury or tissue damage resulting from the risk of injury or tissue damage during surgical operations such as tumor resection or aortic aneurysm surgery. Can be. Other conditions caused or caused by hypoglycemia treatable by the methods described herein include overdoses of insulin, also called clinic hyperinsulinemia, insulin species, growth hormone deficiency, cortisol deficiency, drug overdosing, and certain tumors do.

Other conditions caused by excitatory nerve tissue damage include seizure diseases such as epilepsy, convulsions, or chronic paroxysmal diseases. Other treatable symptoms and diseases include seizures (ischemic seizures, subarachnoid hemorrhages, intracranial hemorrhages), multiple sclerosis, hypotension, cardiac arrest, Alzheimer's disease, Parkinson's disease, cerebral palsy, cerebral spinal cord trauma, AIDS dementia, age-related cognitive ability Diseases include, but are not limited to, age-related loss of cognitive function, memory loss, amyotrophic lateral sclerosis, paroxysmal disease, alcoholism, retinal ischemia, optic nerve damage due to glaucoma, and nerve loss.

Certain compositions and methods of the present invention can be used to treat disease symptoms such as physically or chemically induced inflammation or inflammation due to various traumas. The trauma includes vasculitis, chronic bronchitis, pancreatitis, osteomyelitis, rheumatoid arthritis, glomerulonephritis, optic neuritis, temporal arthritis, encephalitis, meningitis, transverse myelitis, dermatitis, polymyositis, necrotizing fasciitis, hepatitis, and necrotizing colitis Could be included.

It has been demonstrated that activated astrocytes can play a cytotoxic role on nerves by producing neurotoxins. Nitric oxide, reactive oxygen species, and cytokines are released from glial cells that respond to cerebral ischemia (Becker, KJ 2001. Targeting the central nervous system inflammatory response in ischemic Stroke.Curr Opinion Neurol 14: 349-353 and Mattson, MP, Culmsee, C., and Yu, ZF 2000. Apoptotic and Antiapoptotic mechanisms in Stroke.Cell Tissue Res 301: 173-187. The study further described that, in the neurodegenerative model, the activity of glial cells and the subsequent production of inflammatory cytokines depend on primary nerve damage (Viviani, B., Corsini, E., Galli, CL, Padovani, A). ., Ciusani, E., and Marinovich, M. 2000. Dying neural cells activate glia through the release of a protease product.Glia 32: 84-90 and Rabuffetti, M., Scioratti, C., Tarozzo, G., Clementi , E., Manfredi, AA, and Beltramo, M. 2000. Inhibition of caspase-1-like activity by Ac-Tyr-Val-Ala-Asp-chloromethyl ketone includes long lasting neuroprotection in cerebral ischemia through apoptosis reduction and decrease of proinflammatory cytokines. J Neurosci 20: 4398-4404). Inflammatory and glial cell activity is common in different types of neurodegenerative disorders, including cerebral ischemia, cerebral and experimental allergic encephalomyelitis, and disorders in which erythropoietin exerts a neuroprotective effect. Inhibition of cytokine production by erythropoietin could mediate at least some of its protective effects. However, unlike “standard” anti-inflammatory cytokines such as IL-10 and IL-13 that directly inhibit tumor necrosis factor production, erythropoietin appears to be active only in the presence of neuronal death.

While not wishing to be bound by any particular theory, it appears that the anti-inflammatory action can be hypothetically explained by a number of non-limiting theories. First, because erythropoietin prevents apoptosis, the inflammatory action induced by apoptosis will be prevented. In addition, erythropoietin can prevent the release of molecular signals from dying neurons that can act directly on glial cells to stimulate glial cells or reduce their response to the product. Another possibility is that erythropoietin targets a closer member of the inflammatory cascade (eg, caspase 1, free radicals or nitrogen intermediates) leading to both apoptosis and inflammation.

Moreover, erythropoietin typically appears to provide anti-inflammatory protection without the rebound effect associated with other anti-inflammatory compounds such as dexamethasone. Once again, it appears to be due to the effect of erythropoietin on multipurpose neurotoxins such as nitric oxide (NO), hoping not to be bound by any particular theory. Although activated astrocytes and microglia produce NO in the amount of neurotoxins that respond to various traumas, NO serves many purposes in the body, including coordinating essential physiological actions. Thus, although anti-inflammatory can be used to relieve inflammation by inhibiting NO or other neurotoxins, if anti-inflammatory has too long a half-life, anti-inflammatory can also be said to restore damage due to trauma that causes inflammation. May interfere with the chemical's role. It is assumed that the compounds of the present invention can alleviate inflammation while interfering with the recovery of neurotoxins such as NO.

Certain compositions and methods of the invention can be used to treat retinal tissue symptoms and damage to retinal tissue. Such disorders include, but are not limited to, retinal ischemia, macular degeneration, retinal detachment, retinitis pigmentosa, atherosclerotic retinopathy, hypertensive retinopathy, retinal artery blockage, retinal vein blockage, hypotension, and diabetic retinopathy Do not.

In another embodiment, the methods and principles of the present invention can be used to protect or treat damage due to radiation damage or chemotherapy induced damage to excitatory tissue. In a non-limiting example, it can be used to protect against damage caused by compounds such as taxanes, cisplatin and other chemotherapeutic agents that have the potential to induce peripheral neuropathy. Further usefulness of the methods of the present invention lies in the treatment of domoic acid clam poisoning, neurorolathyrism, and neurotoxin poisoning such as Guam disease, amyotrophic lateral sclerosis, and Parkinson's disease.

As mentioned above, the present invention also relates to a method of enhancing excitatory tissue function in a mammal by peripheral administration of a compound of the present invention as described above. Various diseases and symptoms can be treated using this method, and further, the method is useful for augmenting cognitive function without any symptoms and diseases. The use of the present invention is described in more detail below and includes the augmentation of learning and training in both human and non-human mammals.

Symptoms and diseases treatable by the method of this aspect of the present invention with respect to the central nervous system include, but are not limited to, mood disorders, anger disorders, depression, autism, attention deficit disorders, and cognitive impairment. The symptoms benefit from the expansion of nerve function. Other disorders treatable in accordance with the teachings of the present invention include, for example, sleep disturbances, sleep apnea, and travel-related disorders; Subarachnoid and aneurysm hemorrhage, hypotension shock, shaking, septic shock, anafloxi shock, and sequelae of various encephalitis and meningitis, such as cartilage tissue disease-related cerebrates such as lupus. Other uses include poisoning by neurotoxins, such as domoic clam poisoning, neurolattilism, and Guam disease, amyotrophic lateral sclerosis, Parkinson's disease; Postoperative treatment for embolic or ischemic injury; Whole brain irradiation; Sickle cell development; And prevention or protection from tracking.

A further group of symptoms treatable by the methods of the present invention include congenital or acquired mitochondrial dysfunction, which is the cause of various neurological diseases that are typified by nerve damage and death. For example, Leigh disease (subacute necrotic encephalopathy) is characterized by radical vision loss due to nerve drop out and encephalopathy and myopathy. In this case, defective mitochondrial metabolism fails to provide an energy substrate high enough to fuel metabolism of excitable cells. Erythropoietin receptor action modulators optimize the failure of action in various mitochondrial diseases. As mentioned above, hypoxia symptoms adversely affect excitatory tissue. Excitatory tissues include, but are not limited to, central nervous system tissue, peripheral nervous system tissue, and cardiac tissue. In addition to the symptoms described above, the methods of the present invention are useful for the treatment of inhalation poisoning such as carbon monoxide and smoke inhalation, severe asthma, adult respiratory distress syndrome, choking, and drowning accidents. In addition, symptoms that cause hypoxic symptoms or induce excitatory tissue damage by other methods include improper administration of insulin, or hypoglycemia that can occur in insulin-producing neoplasia (insulinoma).

Various neuropsychiatric disorders believed to occur due to excitatory tissue damage are treatable by the methods of the invention. Chronic disorders involving nerve damage and treatment provided by the present invention include age-related loss of cognitive function and senile dementia, chronic paroxysmal disease, Alzheimer's disease, Parkinson's disease, dementia, memory loss, amyotrophic lateral sclerosis, multiple sclerosis , Nodular sclerosis, Wilson's cerebral and eukaryotic nuclear palsy, Guam disease, Lewy dementia, prion disease, spongy encephalopathy, eg Creutzfeldt-Jakob disease, Huntington's disease, muscular dystrophy, Charcot-Marie-Tooth disease, Freidrich's ataxia And other ataxia, as well as seizure diseases such as Gilles de la Tourette's syndrome, epilepsy and chronic seizure disorders, seizures, cerebral spinal cord trauma, AIDS dementia, alcoholism, autism, autonomic nervous system such as retinal ischemia, glaucoma, hypertension and sleep disorders Dysfunction, and schizophrenia, schizoaffective disorder, attention deficit disorder hyperactivity, mood modulation disorder, major depression, manic, obsessive compulsive disorder, New active substance use disorders, as well as anger, panic disorder, including a single polarity, and bipolar disorder jeongdongseong including but not limited to neuropsychiatric disorders includes disorders related to the central nervous system and / or peripheral nervous system.

Additional neuropsychiatric and neurodegenerative disorders include, for example, those mentioned in the American Psychiatric Association's Diagnostic and Statistical Manual of Mental Disorders (DSM), the most current version, IV, which is hereby incorporated by reference in its entirety. do.

In another embodiment, recombinant chimeric toxin molecules comprising a compound of the present invention can be used for therapeutic delivery of toxins to treat proliferative disorders such as cancer, or viral disorders such as subacute sclerotic proencephalitis.

Table 1 lists additional exemplary, non-limiting guidelines regarding various symptoms and diseases that may be treated by the aforementioned compounds of the present invention.

As mentioned above, the disease, disorder or condition is merely illustrative of the range of benefits provided by the compounds of the present invention. Accordingly, the present invention generally provides a therapeutic or prophylactic treatment of the consequences of physical trauma in human disease. Therapeutic or prophylactic treatment of diseases, disorders or symptoms of the CNS and / or peripheral nervous system is preferred. Therapeutic or prophylactic treatment of a disease, disorder or condition is provided having a psychotic therapeutic component. Provided is a therapeutic or prophylactic treatment of a disease, disorder or condition including, but not limited to, ophthalmic, cardiovascular, cardiopulmonary, respiratory, kidney, urinary, obstetric, digestive, endocrine, or metabolic components do.

In one embodiment, the pharmaceutical composition comprising a compound of the present invention may be administered systemically to protect or enhance target cells, tissues, or organs. The administration can be administered parenterally, or by inhalation, for example, orally, nasal, rectally, intravaginally, sublingually, intramucosally, or intradermally. Preferably, the administration is parenteral, including but not limited to, for example, intravenous or intraperitoneal injection, and also intraarterial, intramuscular, intradermal, and subcutaneous administration.

For other routes of administration such as the use of perfusion, infusion into the trachea, or other topical administration, the pharmaceutical compositions will be provided to result in similar concentrations of the compounds of the invention as described above. A concentration of about 0.01 pM to 30 nM is preferred.

The pharmaceutical composition of the present invention may comprise a therapeutically effective amount of a compound, and a pharmaceutically acceptable carrier. In certain embodiments, the term “pharmaceutically acceptable” is approved by a federal or state regulatory authority or is in U.S. By encapsulation or other generally accepted foreign animal use, and more particularly in human use. The term "carrier" refers to a diluent, adjuvant, excipient, or vehicle with which the therapeutic agent is administered. The pharmaceutical carrier may be a sterile liquid such as saline in oil, including water and petroleum, animal oil, vegetable oil, or oils of synthetic origin, such as peanut oil, soy milk, mineral oil, sesame oil and the like. Saline is a preferred carrier when the pharmaceutical composition is administered intravenously. Saline and aqueous dextrose and glycerol solutions can also be used, in particular, as liquid carriers for infusion solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dry scheme milk, glycerol, propylene, glycol, Water, ethanol and the like. If desired, the composition may also contain small amounts of wetting or emulsifying agents, or pH buffers. The composition may take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained release preparations and the like. The compositions can be formulated as suppositories with traditional binders and carriers such as triglycerides. The compounds of the present invention may be formulated in neutral or salt form. Pharmaceutically acceptable salts include those formed with free amino groups such as those derived from hydrochloric acid, phosphoric acid, acetic acid, oxalic acid, tartaric acid and the like, and sodium, potassium, ammonium, calcium, ferric hydroxide, isopropylamine, triethyl And those formed with free carboxyl groups such as those derived from amines, 2-ethylamino ethanol, histidine, procaine and the like. Examples of suitable pharmaceutical carriers include those described in "Remington's Pharmaceutical Sciences" by E.W. Martin]. The composition will contain a therapeutically effective amount of the compound, preferably in purified form, together with a suitable amount of carrier to provide a form suitable for administration to a patient. The formulation will have to be adapted in the form of administration.

Pharmaceutical compositions adapted for oral administration include capsules or tablets; As a powder or granules; As a solution, syrup or suspension (in aqueous or non-aqueous liquid); As an edible form or whip; Or as an emulsion. Tablets or hard gelatin capsules may include lactose, starch or derivatives thereof, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, stearic acid or salts thereof. Soft gelatin capsules may include vegetable oils, waxes, fats, semi-solids, liquid polyols, and the like. Solutions and syrups may include water, polyols and sugars.

Active agents designed for oral administration are coated with or mixed with a substance that delays the degradation and / or absorption of the active agent in the gastrointestinal tract (eg, glyceryl monostearate or glyceryl distearate may be used). Can be. Thus, sustained release actives can be achieved over many hours and, if necessary, the actives can be protected from degradation in the stomach. Pharmaceutical compositions for oral administration may be formulated to facilitate release of the active agent at specific gastrointestinal tract locations due to certain pH or enzymatic symptoms.

 Pharmaceutical compositions adapted for transdermal administration may be presented as individual patches designed to be in direct contact with the recipient's envelope for an extended period of time. Pharmaceutical compositions adapted for topical administration may be provided as ointments, creams, lotions, powders, solutions, pastes, gels, sprays, aerosols or oils. For topical administration to the skin, mouth, eyes or other external tissues, topical ointments or creams are preferably used. When formulated in an ointment, the active ingredient can be used with paraffinic or water-miscible ointment bases. Alternatively, the active ingredient may be formulated as a cream with an oil-in-water base or an oil-in-water base. Pharmaceutical compositions adapted for topical administration to the eye include eye drops. In such compositions, the active ingredient may be dissolved or suspended in a suitable carrier such as an aqueous solvent. Pharmaceutical compositions adapted for topical administration to the mouth include long papers, pastilles, and mouthwashes.

Pharmaceutical compositions adapted for intranasal and pulmonary administration include solid carriers such as powders (preferably powders having a particle size in the range of 20 to 500 microns). The powder may be administered by way of rapid inhalation through the nose from the way the snuff is taken, ie from a container of powder located close to the nose. Alternatively, a composition adapted for intranasal administration may comprise a liquid carrier such as an intranasal spray or nasal mucus. Alternatively, direct inhalation into the lung can be achieved by installation in the central pharynx through deep inhalation or mouth-to-mouth sections. The composition may comprise an aqueous or oily solution of the active ingredient. Compositions administered by inhalation may be provided in specially adapted devices, including but not limited to pressurized aerosols, inhalers or inhalers, which may be constructed to provide a predetermined dosage of the active ingredient. In a preferred embodiment, the pharmaceutical compositions of the present invention are administered to the lungs either directly into the nasal cavity or through the nasal or central pharynx.

Pharmaceutical compositions adapted for rectal administration may be presented as suppositories or enemas. Pharmaceutical compositions adapted for vaginal administration may be presented as pessaries, tampons, creams, gels, pastes, foams or spray formulations.

Pharmaceutical compositions adapted for parenteral administration include aqueous and non-aqueous sterilization which may contain antioxidants, buffers, bacteriostatic agents, and solutes that substantially make the composition isotonic in the blood of a desired recipient. Injections or suspensions. Other components that may be present in the composition include, for example, water, alcohols, polyols, glycerin and vegetable oils. Compositions adapted for parenteral administration may be present in single- or multi-dose containers, such as, for example, sealed ampoules and vials, and require only the addition of a sterile liquid carrier such as sterile injectable saline immediately before use. Can be stored under freeze-dried (freeze-dried) conditions. Temporary infusion solutions and suspensions can be prepared from sterile powders, granules, and tablets. In one embodiment, an autoinjector comprising an injectable solution of a compound of the invention may be provided for urgent use by ambulances, emergency rooms, and war situations, and at home, in particular by indiscriminate use of lawn mowers. It can even be provided for self-administration in family life where the possibility of amputation of the same trauma may occur. The likelihood that cells and tissues of a severe foot or toe survive after recombination is as soon as practicable, even before the medical staff has reached the position or before an individual suffering from a severe foot has reached the emergency room. Can be increased by administering a compound of the invention to several of the more serious areas.

In a preferred embodiment, the composition is formulated according to routine procedures as a pharmaceutical composition adapted for intravenous administration to humans. Typically, the composition for intravenous administration is sterile isotonic aqueous buffer. If desired, the composition may also include a local anesthetic such as lidocaine and a dissolving agent to relieve pain at the infusion site. In general, the components are provided separately or together in a single dosage form in a sealed-packed container as a dry lyophilized powder or water-free concentrate, such as, for example, an ampoule or a stool indicating the amount of the active agent. . When the composition is administered by infusion, it may be formulated into an infusion bottle containing sterile pharmaceutical grade water or saline. When the composition is administered by injection, ampoules of sterile saline may be provided such that the active ingredient can be mixed prior to administration.

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

Perfusion compositions may be provided for use in an implanted organ bath, for in-situ perfusion, or for administration to an organ donor's vasculature prior to organ harvest. The pharmaceutical composition may comprise a compound of the invention at a concentration not suitable for acute or chronic, topical or systemic administration to an individual, but prior to exposing or returning the treated organ or tissue to the general circulation. It will provide the intended action herein in the body, organ bath, organ perfusion, or in situ perfusate before removing or reducing the concentration of a compound of the present invention contained therein.

The invention also provides a pharmaceutical pack or kit comprising one or more containers filled with one or more components of the pharmaceutical compositions of the invention. Optionally associated with the container (s) may be in the form prescribed by a government agency regulating the manufacture, use, or sale of a pharmaceutical or biological product, reflecting approval by the agency of manufacture, use, or sale for human administration. It can be attention.

In another embodiment, for example, the compounds of the present invention can be delivered in a sustained release system. For example, the polypeptide can be administered using intravenous infusion, implantable osmotic pumps, transdermal patches, liposomes, or other dosage forms. In one embodiment, pumps may be used (Langer, supra ; Sefton, 1987, CRC Crit. Ref. Biomed. Eng. 14: 201; Buchwald et. al ., 1980, Surgery 88: 507; Saudek et al ., 1989, N. Engl. J. Med. 321: 574). In another embodiment, the compound may be delivered in a vehicle, in particular liposomes (Langer, Science 249: 1527-1533 (1990); Treat et al ., in Liposomes in the Therapy of Infectious Diseases and Cancer, Lopez-Berestein and Fidler (eds.), Liss, New York, pp. 353-365 (1989); WO 91/04014; US Patent No. 4,704,355; Lopez-Berestein, ibid ., Pp. 317-327; see generally ibid . Reference). In another embodiment, polymeric materials can be used (Medical Applications of Controlled Release, Langer and Wise (eds.), CRC Press: Boca Raton, Florida, 1974; Controlled Drug Bioavailability, Drug Product Design and Performance, Smolen and Ball (eds.), Wiley: New York (1984); Ranger and Peppas, J. Macromol. Sci. Rev. Macromol. Chem. 23:61, 1953; see also Levy et. al ., 1985, Science 228: 190; During et al ., 1989, Ann. Neurol. 25: 351; Howard et al ., 1989, J. Neurosurg. 71: 105).

In yet another embodiment, the sustained release system can be located in close proximity to a therapeutic target, i.e., a target cell, tissue or organ, and therefore only requires a portion of the systemic dosage ( eg, Goodson, pp. 115-). 138 in Medical Applications of Controlled Release, vol. 2, supra , 1984). Other sustained-release systems are discussed in a review by Langer (1990, Science 249: 1527-1533).

In another embodiment, the compounds of the present invention may be suitably formulated and administered by intranasal, oral, rectal, vaginal, or sublingual administration.

In certain embodiments, it may be desirable to administer the compositions of the invention locally to the area in need of treatment; This can be done, for example, without limitation, by topical infusion during surgery, for example by topical application, with a catheter, suppository, or implant with the dressing of the wound site after surgery, wherein the implant is It is made of a porous, non-porous, or gelatinous material, including a membrane, such as a plastic membrane, or a fiber.

Selection of the preferred effective dosage will be determined by the skilled artisan based on consideration of various factors that will be known to those skilled in the art. Such elements include particular forms of the compounds of the present invention and their pharmacokinetic variables such as bioavailability, metabolism, half-life, etc., which may have been established during conventional development processes typically used to obtain regulatory approval of pharmaceutical compounds. will be. Additional factors to consider dosage include the benefits achieved in the symptom or disease or normal person being treated, the body mass of the patient, the route of administration, the concurrent medication, and the efficacy of the pharmaceutical agent administered, whether the administration is acute or chronic. Includes other factors that are well known to affect Therefore, the exact dosage will have to be determined according to the judgment of the attending physician and the condition of each patient, e.

In another aspect of the invention, a perfusate or perfusate is provided for perfusion and storage of an organ for transplantation, the perfusate being a substantial amount of a compound of the invention effective to protect reactive cells and related cells, tissues, or organs It includes. Transplantation is a heterogeneous organ transplantation that collects organs (including cells, tissues, or other body parts) from a donor and transplants them to another recipient; And allogeneic organ transplantation (tracheal can be removed, while cutting, repairing or otherwise proliferating, such as tumor removal in vitro, taking the organ in one part of the body and returning it to another part, including a bench surgical procedure) And then return to its original position). In one embodiment, the perfusate is erythropoietin about 1 to about 25 U / ml, 5% hydroxyethyl starch (molecular weight is about 200,000 to about 300,000, substantially free of ethylene glycol, ethylene chlorohydrin, sodium chloride and acetone) ; 25mM KH ₂ PO ₄ ; 3 mM glutathione; 5 mM adenosine; 10 mM glucose; 10 mM HEPES buffer; 5 mM magnesium gluconate; 1.5 mM CaCl ₂ ; 105 mM sodium gluconate; 200,000 units penicillin; 40 unit insulin; 16 mg dexamethasone; Contains 12 mg phenol red; University of Wisconsin (UW) solution (US Patent No. 4,798,824) having a pH of 7.4-7.5 and an osmoticity of about 320 mOSm / l. The solution is used to maintain the kidneys and pancreas of the body prior to transplantation. The solution can be used to extend preservation beyond the 30-hour limit recommended during renal preservation of the body. This particular perfusion is merely illustrative of a number of such solutions that may be suitable for this use, including effective amounts of the compounds of the present invention. In further embodiments, the perfusion solution may contain about 0.01 pg / ml to about 400 ng / ml of the compound of the present invention or about 40 to about 300 ng / ml of the compound of the present invention.

While the preferred recipients of the compounds of the present invention for purposes throughout this application are humans, the methods herein apply equally to other mammals, particularly livestock, livestock, pets and zoo animals. However, the present invention is not intended to be so limited, and the advantage may be applied to any animal.

Of the compounds of the present invention Therapeutic  And Preventive  Usage

As noted in Example 1 below, the presence of the erythropoietin receptor in the capillary endothelial cells of the human brain indicates that the target of the compound of the present invention is present in the human brain, and animal studies of the compound of the present invention directly Indicates that it can be transferred to the treatment or prevention of humans.

In another aspect of the invention, methods and compositions for enhancing the survival of cells, tissues, or organs that are not isolated from the vasculature by the endothelial cell wall directly comprise the compounds of the invention. By exposure to a pharmaceutical composition or by administering or contacting a pharmaceutical composition containing a compound of the invention to the vasculature of a tissue or organ. The enhanced action of reactive cells in treated tissues or organs contributes to the positive effects exerted.

As described above, the present invention is based on the discovery that some erythropoietin molecules can be transported from the lumen surface of the endothelial cells of capillaries of organs with endothelial cell tight junctions, including the brain, retina and testes, to the basal membrane surface. do. Thus, reactive cells across the wall are likely to be targets for the beneficial effects of the compounds of the invention, where other cell types or tissues or organs containing and relying in whole or in part on the reactive cells are targets of the methods of the invention. . Without wishing to be bound by any particular theory, after transcytosis of the compounds of the invention, the compounds of the invention may be found in the nerves, retina, muscle, heart, lung, liver, kidney, small intestine, adrenal cortex, adrenal medulla, capillary endothelium. May interfere with erythropoietin receptors on reactive cells such as cells, testes, ovaries, pancreas, bone, skin, or intrauterine cells, and receptor binding results in activation of gene expression programs within the reactive cells or tissues, and Or a signal transduction cascade that results in protecting the organ from damage such as toxins, chemotherapeutic agents, radiation therapy, hypoxia, and the like. Thus, methods of protecting reactive cell-containing tissues from injury or hypoxic stress and enhancing their function are described in detail herein below. As noted above, the methods of the present invention are equally applicable to humans as well as other animals.

In practicing one embodiment of the present invention, a mammalian patient receives systemic chemotherapy for cancer treatment, including radiation therapy, which has in common side effects such as nerve, lung, heart, ovary or prostate damage. Administration of a pharmaceutical composition comprising a compound of the present invention as described above is performed before or during chemotherapy and / or radiation therapy to protect various tissues and organs from damage by the chemotherapeutic agent, such as to protect the testicles. do. Treatment may continue until the circulating level of the chemotherapeutic agent drops below a level that poses a potential risk to the mammal's body.

In the practice of another embodiment of the present invention, various agencies have been planned to be collected from victims of motor vehicle accidents and implanted into numerous recipients, some of which required transportation over extended distances and periods. Prior to organ harvesting, the victim was infused with a pharmaceutical composition comprising a compound of the present invention as described herein. The embarked harvested organ was perfused with a perfusion containing the compound of the present invention as described herein and stored in a bath containing the compound of the present invention. Certain organs were continuously perfused with a pulsatile perfusion device, using a perfusion containing the compound of the present invention according to the present invention. Minimal degradation of organ function occurred during transport and upon transplantation and reperfusion of organs in situ.

In another embodiment of the present invention, surgical procedures for repairing the heart valves required temporary cardiac arrest and arterial occlusion. Prior to surgery, patients were injected with 4 μg of compounds of the invention per Kg body weight. This treatment particularly prevented hypoxic ischemic cell damage after reperfusion.

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

Use of the compounds of the invention for in vitro application or reactivity such as neural tissue, retinal tissue, heart, lung, liver, kidney, small intestine, adrenal cortex, adrenal medulla, capillary endothelial cells, testes, ovaries or intrauterine cells When used to treat cells or tissues, the present invention provides about 0.01 pg to 5 mg, 1 pg to 5 mg, 500 pg to 5 mg, 1 ng to 5 mg, 500 ng to per compound of the present invention per dosage unit. Reactive cells apart from the vasculature, including effective non-toxic amounts within the range of 5 mg, 1 μg to 5 mg, 500 μg to 5 mg, or 1 mg to 5 mg and comprising a pharmaceutically acceptable carrier Pharmaceutical compositions are provided in dosage unit forms adapted for the protection or enhancement of tissues or organs. In a preferred embodiment, the amount of the compound of the present invention is in the range of about 1 ng to 5 mg.

In a further aspect of the invention, EPO administration has been found to restore cognitive function in animals suffering from brain trauma. The compounds of the present invention will be expected to have the same cytoprotective effect as EPO. After a delay of 5 or 30 days, EPO could still restore function compared to sham-treated animals, indicating the ability of EPO to regenerate or restore brain function. Accordingly, the present invention also provides for the use of pharmaceutical compositions for the treatment of brain trauma and other cognitive impairments, including well treated after injury (eg, 3 days, 5 days, 1 week, 1 month or longer). It relates to the use of the compounds of the invention for the preparation. The invention also relates to a method of treating cognitive impairment after injury by administration of an effective amount of a compound of the invention. Any compound of the invention as described herein can be used in this aspect of the invention.

Moreover, this recovery aspect of the present invention relates to the use of any of the compounds of the present invention for the preparation of a pharmaceutical composition for repair of a cell, tissue or organ abnormality, wherein the treatment is initiated after the first injury causing the abnormality. . Moreover, treatment with the compounds of the present invention can determine the course of a disease or condition during the acute phase as well as during the chronic phase.

If the compound has an erythropoietic action, the compound may contain about 1 μg to about 100 μg / kg body weight, preferably about 5 μg to about 50 μg / kg body weight, and most preferably about 10 μg per dose. Systemically at a dosage of from about 30 μg / kg body weight. The effective dosage will have to be sufficient to achieve a serum concentration of the compound greater than about 10,000, 15,000, or 20,000 mU / ml (serum) after administration of the compound. The serum concentration may be achieved about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 hours after administration. The dosage can be repeated as necessary. For example, administration may be repeated for as long as clinically necessary, daily, or after appropriate intervals, for example every 1 to 12 weeks, but preferably every 1 to 3 weeks. Effective amounts of compounds and pharmaceutically acceptable carriers may be contained in single dose vials or other containers. However, the compounds of the present invention can impart activity described herein without causing erythropoiesis, ie without causing an increase in hemoglobin concentration or hematocrit. Such non-erythropoietic compounds are particularly preferred when the methods of the invention are to be provided chronically. In another embodiment, the compounds of the present invention are provided at a higher dosage than that of the corresponding dose (W / W) of natural erythropoietin required to maximize stimulation of erythropoiesis. As noted above, the compounds of the present invention have no erythropoietic action, and thus the dosages expressed in units are merely illustrative of the corresponding amounts of natural erythropoietin; The molar equivalents to the dosages herein are provided as applicable to any compound of the invention.

The invention may be better understood with reference to the following non-limiting examples, which serve as examples of the invention. The following examples are presented to more fully illustrate preferred embodiments of the invention. However, they should not in any way limit the broad scope of the invention.

Example  One

Carbamylated  Preparation of Erythropoietin

The starting material of the process of this example was purified recombinant human EPO.

First, protein concentration was adjusted by ultrafiltration to maintain a low process volume. Protein concentration was adjusted to 3 mg / ml. Ultrafiltration was performed using BioMax (Millipore) with a MWCO of 5 kDa.

After completion of the concentration step, the EPO solution was mixed with 0.5 M K-borate tetra hydrate 0.5 M K-cyanate and the solution was incubated at 32 ° C. for 24 hours.

Desalting of the reaction mixture of EPO and cyanate was carried out by gel filtration. Proteins were desalted with 25 mM Tris, 50 mM NaCl pH 8.5 buffer. G-25 Fine (Amersham Biosciences) resin was used.

A sample loading of approximately 20% column volume was used, using a flow rate of 90 cm / h on an approximately 15 cm high column.

Desalted carbamylated EPO was harvested for further processing.

At this time, the polymer / aggregate content was 7.3%.

The next step was the removal of aggregates and polymers carried out by the purification step using anion exchange. SOURCE 30Q (Amersham Biosciences) resin was used for purification. Carbamylated EPO was applied to the column at approximately 4.5 mg / ml. Progression buffer A was: 25 mM Tris, 50 mM NaCl pH 8.5, and elution buffer B was: 25 mM Tris, 1 M NaCl pH 8.5. The gradient was performed at 0-30% over 20 column volumes where the main peak of carbamylated EPO was collected and pooled.

The pool from the purification step was adjusted to concentration> 0.5 mg / ml and the buffer was changed to 20 mM citrate, 100 mM NaCl buffer using diafiltration / superfiltration tangential flow filtration units. Concentration and buffer changes were performed on 0.1m ² BioMax (Millipore) with a MWCO of 5 kDa.

Finally, the purified biopharmaceutical drug component was 0.22 μm filtered using a Millipak filter (Millipore) to reduce pathogens.

The process resulted in carbamylated EPO with properties that make it useful as a biopharmaceutical;

Polymer / aggregate content was 0.5% as determined by SEC-HPLC.

Carbamylated lysine was 100% as determined by amino acid analysis.

Concentration was above 0.5 mg / ml.

Characterization using MALDI-TOF was performed for determination of changes in intrinsic mass for both PNGase treated proteins and proteins with N-glycans. In addition, MALDI-TOF peptide mass fingerprint analysis / LC-MS / MS analysis was performed as follows:

1. CEPO and EPO were purified on POROS R1 column (POROS R1 reverse phase column material, PerSeptive Biosystems (1-1259-06)). The column material was stored in 50% HiPerSolv for HPLC VWR 152525R before use. The R1 column was equilibrated and washed in 5% formic acid (33015, Riedl de Haeon). Samples were eluted from the column using Agilent MALDI HCCA Quality Matrix Solution (G2037A). Intrinsic mass was determined by analysis on a Bruker® Reflex IV MALDI-TOF instrument.

2. 0.3 pmol CEPO and / or EPO were treated overnight with 1 unit of PNGase F and PNGase F / O-glycosidase. Total mass was determined using MALDI-TOF.

3. CEPO and EPO (1.5 pmol) were reduced in a solution with 50ul 10mM DTT, 50mM NH ₄ CO ₃ and then alkylated with an alkylated solution in 50ul 55mM iodoacetamide, 50mM NH ₄ C0 ₃ . Samples were purified on POROS R1 column prior to trypsin digestion. Some of the digested samples were purified on POROS R2 columns before MALDI-TOF analysis (POROS 50 R2 PerSeptive Biosystems (1-1159-05)). The R2 column was equilibrated and washed in 0.1% trifluoroacetic acid (99 +% spectral grade, Aldrich 302031-100 ml). Samples were eluted from the column with Agilent MALDI HCCA Quality Matrix Solution (G2037A). A pool of peptides obtained by trypsin digestion was treated with PNGase F, purified via POROS R2 column, and characterized by MALDI-TOF.

4. CEPO and EPO were reduced in solution with DTT and alkylated with iodoacetamide. Samples were purified on POROS R1 columns prior to Glu-C digestion. Some of the digested samples were purified on POROS R2 columns before MALDI-TOF analysis (POROS 50 R2 PerSeptive Biosystems (1-1159-05)). A pool of peptides obtained by Glu-C digestion was treated with PNGase F and purified via POROS R2 column.

5. To identify the possibility of having partially carbamylated CEPO, native EPO and CEPO were digested with Lys-C. Samples were analyzed using MALDI-TOF.

The conclusion was that eight lysines and the N-terminus were carbamylated. No other carbamylated amino acids were detected and no modification of the glycans was detected.

Reference :

MS  Common protein identification by:

Mann M, Hojrup P, Roepstorff P. ( 1993 ) Use of mass spectrometric molecular weight information to identify proteins in sequence databases , Biol Mass Spectrom 22 , 338-345

Yates, JR, Speicher S, Griffin PR, Hunkapiller T. ( 1993 ) peptide mass maps: a highly informative approach to proteins identification , Anal Bioche m 214 , 397-408

Intrinsic Mass:

Laugesen S, Roepstorff P. ( 2003 ) Combination of two matrices results in improved performance of MALDI MS for peptide mass mapping and protein analys is. J Am Soc Mass Spectrom. 14 (9), 992-1002.

decomposition/ Graffiti  :

Larsen MR, Hojrup P, Roepstorff P. ( 2005 ) Characterization of gel -separated glycoproteins using two - step proteolytic digestion combined with sequential microcolumns and mass spectrometry . Mol Cell Proteomics. 4 (2), 107-19.

To determine the extent and homogeneity of carbamylation of EPO, the specificity of carbamylation and the identity of carbamylation sites, and the presence of potential nonspecific modifications due to side reactions of the carbamylation and purification processes. The study was conducted. Samples were analyzed by peptide mapping using endoprotease LysC and trypsin for digestion and LC / MS analysis for total mass spectrometry and peptide evaluation of deglycosylated protein samples.

Example  2

Total mass spectrometry

The analysis was performed on three carbamylated EPO samples using the method of Example 1. All three samples were purified after carbamylation reaction using anion exchange, as described in Example 1. One of the CEPO samples (designated CEPO-CMC) was taken from a 1 gram preparation scale (concentration: 0.82 mg / ml; buffer: 20 mM Na-citrate, 0.3 mM citric acid, 0.1 M NaCl, pH 6.9-7.3) Prepared by CMC Biotech. The remaining two samples designated CEPO-1 and CEPO-2 were prepared from a 70 mg laboratory preparation scale (concentration: 1.1 mg / ml; buffer: 25 mM Tris, 0.2.M NaCl, pH 8.3-8.7). The CEPO samples were either unmodified or starting EPO (concentration: 0.82 mg / ml; buffer: 2 mM Na-citrate, 0.3 mM citric acid, 0.1 NaCl, pH6.9-7.3) and mock CEPO (K-sia EPO was performed with a carbamylation process without the addition of Nate) (concentration: 0.38 mg / ml; buffer: 20 mM Na-citrate, 0.3 mM citric acid, 0.1 M NaCl, pH 6.9-7.3).

ESI Mass spectrometer

Prior to total mass spectrometry, the samples were enzymatically deglycosylated. Each sample of the N- glycosidase F at 0.5 mg / ml protein concentration (Prozyme from Glyko), urea A. Pacifico Enschede The recombinant New lamina from (A. ureafaciens) claim (neuraminidase) and O- glycosidase Incubate overnight at 50 μg. Completion of the deglycosylation reaction was checked by SDS-PAGE by loading 3 μg of each sample into 12% Tris-glycine gel. The material remaining from each sample was used for mass spectrometry.

The deglycosylated sample was brought to a concentration of 4-5 M guanidinium hydrochloride by addition of an appropriate amount of guanidinium hydrochloride stock solution, followed by desalting with a buffer containing 2% formic acid and 40% acetonitrile. . Guanidinium hydrochloride was added to ensure high recovery of deglycosylated EPO and CEPO for mass spectral analysis. Mass measurements were performed using a Waters ESI-Q-Tof- or Waters ESI-LCT-mass spectrometer provided with an ESI-nanospray source of ionization. Evaluation of the data was performed by automated deconvolation and manual evaluation of certain peaks of interest using Mass Lynx 4.0 software. Evaluation of the relative proportions of different carbamylated CEPO species was performed by calculating the relative ratios based on the signal strengths recorded in the m / z spectrum.

Deglycosylation of the sample resulted in an N-linked sugar that was completely removed. However, the release of O-linked sugars was incomplete, especially for CEPO samples.

As expected, due to carbamylation, CEPO samples showed different mass spectra compared to EPO and Mok-CEPO samples. As shown in Table 2, the deconvolution of the spectra resulted in masses for the main peaks as theoretically expected for the various samples. In all CEPO samples, only highly carbamylated CEPO molecules were found, the main isomer being a totally carbamylated isomer with eight lysines and N-terminus, i. The CEPO sample showed isomerism containing additional isomers corresponding to 8, 10 and 11 carmarville residues bound to CEPO. A species containing eight carbamyl residues will be designated as sub-carbamylated and one or more carbamyl residues are lost. Species with 10 and 11 carbamyl residues will be designated as over-carbamylated and the extra carbamyl residues will bind non-specifically to amino acids other than lysine. There were some small signals in the spectra of all the samples, but none were considered non-specifically modified species. Some of the small signals were found in both EPO and CEPO samples and were considered contaminants already present in the starting EPO.

Table 3 shows the relative proportions of the various isomers. Sub-carbamylated CEPO ranges from about 1.5 to 5.5% of total CEPO depending on the sample, and over-carbamylated CEPO ranges from about 11 to 22% depending on the sample. CEPO-1 and CEPO-2 have similar distributions of isomers, while CEPO-CMC has few sub-carbamylated species compared to the other two samples. Different production scales result in products with different distributions, but because Table 3 shows the laboratory production scale, the production can be repeated in a similar process. As discussed previously, at any given production scale, the distribution can be adjusted by adjusting the pooling from the anion exchange column.

The numbers in Table 3 represent the minimum proportions of sub- and over-carbamylated CEPO. The reason for this is that the degree of carbamylation (8- to 11-fold) cannot specify the exact degree of sub, fully and over-carbamylated CEPO. For example, a CEPO molecule containing eight carbamyl residues, as determined by mass spectrometry, may have only seven carbamyl groups specifically bound to lysine, and the remaining carbamyl residues are non-another amino acid. -Will bind specifically. This situation would only be considered sub-carbamylated, even if there were non-specifically bound carbamyl groups. In contrast, a CEPO molecule containing ten carbamyl residues can specifically bind to only eight residues, and two bind non-specifically. In this situation, the CEPO isomers are only considered to be over-carbamylated, even though not all lysines are carbamylated.

Example  3

LysC  Peptide Mapping

Peptide map analysis of EPO and CEPO samples was performed using endoprotease LysC and trypsin for fragmentation. All peptide map analyzes were performed with deglycosylated peptides. Enzymatic glycosylation of the peptide was performed concurrently with the degradation of the endoprotease.

EPO and CEPO samples (about 150 μg each) were denatured and reduced by incubating with guanidinium hydrochloride and DTT. Free sulfidyl groups were alkylated with iodoacetic acid. The alkylated sample was desalted and the buffer was exchanged into the appropriate buffer using a single use gel filtration column.

Endoproteases, N-glycosidase and neuraminidase were added simultaneously to the alkylated EPO and CEPO samples. Samples were incubated overnight at 37 ° C. After incubation, about 5 μg of each digest was used for RP-HPLC / MS analysis using Phenomenix's Jupiter, C18 RP-column, coupled to ESI-LCT from Water. The UV signal and total ion number (TIC) at 220 nM in the mass spectrometer were recorded. TIC was evaluated for identification and quantification of the obtained peptides.

Since LysC will not be able to cleave carbamylated lysine, it is expected that if all lysines are carbamylated, no fragments will form with LysC to form, indicating the specificity of carbamylation. In the case of sub-carbamylation, certain segments of CEPO will have to form. Table 4 lists the sections theoretically formed from LysC digestion for EPO, fully and over-carbamylated CEPO and sub-carbamylated CEPO.

As expected, the LysC peptide patterns obtained for the EPO and CEPO samples digested with LysC were completely different. Degradation of the starting EPO and mok-CEPO with LysC resulted in the peptide pattern as expected. In both samples, all peptides K1 to K9 could be identified. Peptides K5 and K1 were cleaved in a partially nonspecific manner in both EPO and Mok-CEPO. No significant additional peak could be identified and no peaks were lost in the LysC map of Mok-CEPO compared to the starting EPO. From the data, it can be concluded that no significant non-specific covalent modification of the EPO protein occurred during the carbamylation and purification process.

The peptide map of the CEPO sample had a different peptide pattern from the starting EPO. There was one single main peak and several small peaks. As shown in Table 5, the mass obtained from the peaks correlated well with the expected mass for the sections from the LysC cleavage of the sub-carbamylated CEPO and for the uncut CEPO. Main peak (A) contained native, deglycosylated, fully carbamylated CEPO (9 × carbamyl) and over-carbamylated CEPO (10 × carbamyl) (Table 5). Four small peaks (B-E) contained sub-carbamylated CEPO (8x carbamyl), with different peaks containing sub-carbamylated CEPO not carbamylated in certain lysines. Peaks B and C contained sub-carbamylated CEPO specifically free of carbamylation at Lys45, while Peaks D and E contained sub-carbamylated CEPO specifically free of carbamylation at Lys97. (Table 5).

There was some difference in the relative proportions of the peaks for the different CEPO samples. CEPO samples 1 and 2 had more dominant peaks D and E than CEPO-CMC, indicating that they contained more sub-carbamylated CEPO species.

Technical limitations made it difficult to quantify the small peaks associated with the main peak. However, using peak regions from UV-detection as a crude guide for the relative proportions of the various species, sub-carbamylated CEPO can have less than 10% CEPO isomers in the sample, and CEPO-1 and CEPO- It can be estimated that 2 has about twice the amount of sub-carbamylated isomers than CEPO-CMC. Using the same procedure used for total mass spectrometry, the quantification of the over-carbamylated species located at peak A was about 21% for CEPO-CMC and 12% for CEPO-1 and CEPO-2.

In the end, the data from the LysC peptide mapping is in good agreement with the total mass spectrometry described in Example 2. CEPO-CMC contained little sub-carbamylated CEPO isomers, while CEPO-1 and CEPO-2 contained more over-carbamylated CEPO isomers. This peptide mapping also indicated that lysine 45 and lysine 97 can indicate the location of sub-carbamylation.

Example  4

Trypsin peptide Mapping

Decomposition of the sample using trypsin is described in Example 3.

Digestion of EPO and Mok CEPO with trypsin showed the peptide pattern as expected. In both samples, with the exception of some small di- and tri-peptides (such as T21), most of the peptides (T1 to T21) could be identified as expected for trypsin digestion. See Table 6. As in the case of LysC digestion, there were no significant additional peaks and no peaks lost from Mok-CEPO compared to the starting EPO. From the data, it was concluded that there was no non-specific covalent modification of the EPO protein during the carbamylation and purification process.

Peptide patterns of CEPO samples differed from unmodified EPO. Trypsin is usually cleaved at lysine and arginine, so for fully carbamylated EPO, only fragmentation will be expected to occur by cleavage of arginine. Thus, after the peptide pattern was obtained with trypsin, specific carbamylated sites and nonspecific carbamylated peptides could be identified. Peptides expected from trypsin digestion of CEPO molecules that are expected to cleave only in arginine (R) are shown in Table 6.

All peptides expected from trypsin cleavage were identified as major peaks in all CEPO samples except for the C-terminal peptide R13. The peptide was also not found in starting EPO or Mok-CEPO. Peaks of R1 to R12 due to specific cleavage of only arginine, which is described for a wide range of peptides, were detected in CEPO samples. Moreover, all lysine-containing peptides were detected almost exclusively in fully carbamylated form. The data indicate a high degree of specific carbamylation at the lysine and N-terminus.

In addition to the main peptide, six small peptides were also detected in all CEPO samples. Three of the small peptides were identified by mass spectrometry as a result of trypsin cleavage of the over-carbamylated CEPO and the remaining three were generated from the sub-carbamylated CEPO.

Table 7 lists all peptides identified in the trypsin map of the three CEPO samples analyzed. Highly abundant peptides formed from fully and specifically carbamylated EPO, R1 to R12, are in general font, and correctly carbamylated EPO is shown in bold form. Peptides formed mostly by cleavage of sub-carbamylated CEPO are shown in italics, and peptides formed by cleavage of over-carbamylated CEPO are underlined.

With respect to Table 7, the small peaks T9 and T10 containing peptides R6_a1 and R6_b1, respectively, are mostly derived from cleavage of sub-carbamylated CEPO molecules that are not carbamylated in Lys97. If the Lys45 amino acid is not carbamylated, peptide R4_b1 + 1xcarb is formed. Peptide R6_a1 (peak T9) is more abundant in CEPO-1 and CEPO-2 samples than CEPO-CMC, indicating that the former can have twice the amount of sub-carbamylated CEPO. Peptides R6_b1 and R4_b1 + 1xcarb were present in equal amounts in all CEPO samples.

It was difficult due to technical limitations to calculate the relative content of sub-carbamylated CEPO from the data. However, everything was observed together, presuming that the sub-carbamylated species was about 10% as deduced from total mass spectrometry and LysC peptide mapping.

Three different small peptides co-eluted with other peptides were hampered by a mass containing one extra carbamyl residue, ie, over-carbamylated CEPO. Peptides R10_2xcarb and R7_1xcarb were detected in trace amounts, where R12_3xcarb was found to have significant signal intensity. CEPO-CMC had 2-fold higher content of over-carbamylated CEPO species as CEPO-1 and CEPO-2. This is consistent with the results from total mass spectrometry. In addition, it can be concluded from the data that amino acid sequences 151-62 of EPO are non-specific carbamylation sites.

In addition, for the same reasons as in the quantification of sub-carbamylation, it was difficult to quantify over-carbamylated species. However, assuming that the ionization potency of peptides differing only in the degree of carbamylation was similar, the CEPO was calculated by the relative ionic count of R12-derivatives, wherein about 3-7% of the over-carbamylated species. This is lower than the amount of over-carbamylated isomers calculated by total mass spectrometry.

In general, the following can be concluded from the total mass spectrometry and peptide mapping data of EPO and CEPO.

From total mass spectrometry, CEPO samples appeared to have a higher degree of carbamylation. About 95-98% of all molecules are fully carbamylated and contain at least 9 carbamyl residues (see Table 3). LysC and trypsin mapping confirm a high degree of carbamylation at certain locations and almost 95% of the CEPO molecules are fully carbamylated at 8 lysines and N-terminus.

The data also showed isomers of the four CEPOs found. Species with 8, 9, 10 and 11 carbamyl residues were detected in the analyzed CEPO samples, with 9 carbamyl isomers predominant. The minor portion of the CEPO molecule containing eight carbamyl residues instead of nine was considered sub-carbamylated. For CEPO-1 and CEPO-2, the isomer was about 5% of the total and for CEPO-CMC, the isomer consisted of about 1.5% of the total CEPO molecules.

More significant proportions of CEPO molecules were over-carbamylated, ie they contained 10 or 11 carbamyl residues. Over-carbamylated CEPO ranges from about 11% for CEPO-1 and CEPO-2 to about 22% for CEPO-CMC.

Data from peptide mapping showed a high degree of specificity of carbamylation at the eight lysines and N-terminus. Moreover, peptide mapping generally confirmed the results of total mass spectrometry. At least about 90-95% of the CEPO molecules appeared to be specifically modified by carbamyl residues at both the lysine and N-terminus. However, the data also showed sub- and over-carbamylated CEPO species. Because of some technical limitations, it was difficult to determine the exact proportion of the sub-carbamylated species, but is estimated to be within the range of about 10% or less, consistent with the number found in the total mass spectrometry. Moreover, two distinct positions on EPO, Lys45 and Lys97 were identified as sites where the deficiency of carbamylation is most likely to occur.

In both settings of peptide mapping, over-carbamylated species were also found. In addition, due to technical limitations, it was difficult to quantify the exact amount of the over-carbamylated species. From the data obtained, it can be estimated that little was detected in the over-carbamylated species peptide mapping. Only 1/3 to 1/4 of the over-carbamylated species were identified by peptide mapping compared to total mass spectrometry. A valid explanation for this discrepancy is that not all over-carbamylated peptides have been identified in all or exact amounts. In the LysC map, significant amounts of uncleaved CEPO species containing 10 carbamyl residues were detected, and in trypsin mapping, three peptides were detected containing one extra carbamyl residue. Two of these fragments were detected only in trace amounts. The third peptide close to the C-terminus, the CEPO peptide amino acids 152-162, seems to account for over-carbamylation 1/3, and may be the site for non-specific carbamylation in EPO.

Significant amounts of other non-specific (no carbamyl related) modifications were not detected in the CEPO samples or the mock CEPO samples by any analysis performed.

1 shows the reaction of cyanates with the N-terminal and lysine residues of the protein.

Claims (126)

Measured by an ESI-mass spectrometer, comprising contacting a substantial amount of erythropoietin with a significant amount of cyanate for a temperature, pH, and period of time sufficient for the amine groups on the lysine and N-terminal amino acids of erythropoietin to be at least about 90% carbamylated. A method for preparing carbamylated erythropoietin protein having less than about 40% aggregated protein and less than about 40% by weight over- and sub-carbamylated protein as such. The method of claim 1, wherein the carbamylated erythropoietin protein is human erythropoietin. The method of claim 1, wherein the carbamylated erythropoietin protein has less than about 30% of the aggregated protein. The method of claim 1, wherein the carbamylated erythropoietin protein has less than about 20% of the aggregated protein. The method of claim 1 wherein the carbamylated erythropoietin protein has less than about 10% aggregated protein. The method of claim 1, wherein the carbamylated erythropoietin protein has less than about 30% by weight of the over- and sub-carbamylated protein. The method of claim 1, wherein the carbamylated erythropoietin protein has less than about 20% by weight of the over- and sub-carbamylated protein. The method of claim 1, wherein the carbamylated erythropoietin protein has less than about 10 weight percent of the over- and sub-carbamylated protein. The method of claim 1, wherein the carbamylated erythropoietin protein has less than about 30% by weight of the over-carbamylated protein. The method of claim 1, wherein the carbamylated erythropoietin protein has less than about 20% by weight of the over-carbamylated protein. The method of claim 1, wherein the carbamylated erythropoietin protein has less than about 10 weight percent of the over-carbamylated protein. The method of claim 1 wherein the concentration of erythropoietin protein in contact with cyanate is from about 0.05 mg / ml to about 10 mg / ml. The method of claim 1 wherein the concentration of erythropoietin protein in contact with cyanate is from about 2 mg / ml to about 5 mg / ml. The method of claim 1 wherein the concentration of cyanate is from about 0.05 M to about 10 M. The method of claim 1 wherein the concentration of cyanate is from about 0.05 M to about 2 M. The method of claim 1 wherein the temperature is in the range of about 0 ° C. to about 60 ° C. 6. The method of claim 1 wherein the temperature is in the range of about 30 ° C. to about 34 ° C. 7. The method of claim 1 wherein the pH is from about 7 to about 11. The method of claim 1 wherein the pH is from about 8 to about 10. The method of claim 1 wherein the time period is from about 10 minutes to about 30 days. The method of claim 1 wherein the time period is from about 1 hour to about 5 days. The method of claim 1, wherein the erythropoietin protein is contacted with cyanate in the presence of a buffer. The method of claim 22, wherein the buffer is borate. 23. The method of claim 22 wherein the concentration of the buffer is about 0.05 M to about 2 M. The method of claim 22 wherein the concentration of the buffer is from about 0.1 M to about 1 M. 23. The method of claim 22, wherein the concentration of the buffer is about 0.5 M. The method of claim 1 wherein the concentration of the erythropoietin protein in contact with the cyanate is from about 0.05 mg / ml to about 10 mg / ml, the concentration of cyanate is from about 0.05 M to about 10 M, and the temperature is from about 0 ° C. to about And a pH in the range of 60 ° C., about 7 to about 11, and a duration of about 10 minutes to 30 days. The method of claim 1 wherein the concentration of erythropoietin protein in contact with the cyanate is from about 2 mg / ml to about 5 mg / ml, the concentration of cyanate is from about 0.05 M to about 2 M, and the temperature is from about 30 ° C. to about And a pH in the range of 34 ° C., about 8 to about 10, and a duration of about 1 hour to 5 days. The method of claim 1 wherein the concentration of erythropoietin protein in contact with cyanate is about 3 mg / ml, the concentration of cyanate is about 0.5 M, the temperature is about 32 ° C., the pH is about 9.0, and the duration is about 24 How it is time. As measured by an ESI-mass spectrometer, comprising purifying carbamylated erythropoietin using anion exchange, cation exchange, hydrophobic interaction chromatography, reverse phase chromatography, affinity chromatography or size exclusion chromatography. A method of making carbamylated erythropoietin protein having less than about 3% aggregated protein and less than about 40% by weight over- and sub-carbamylated protein. 31. The method of claim 30, wherein the carbamylated erythropoietin protein is human erythropoietin. The method of claim 30, wherein at least about 90% of the amine residues on the lysine and N-terminal amino acids of erythropoietin are carbamylated. 31. The method of claim 30, wherein the carbamylated erythropoietin protein has less than about 2.5% protein aggregated. 31. The method of claim 30, wherein the carbamylated erythropoietin protein has less than about 0.5% aggregated protein. 31. The method of claim 30, wherein the carbamylated erythropoietin protein has less than about 30% by weight of the over- and sub-carbamylated proteins. 31. The method of claim 30, wherein the carbamylated erythropoietin protein has less than about 20 weight percent of the over- and sub-carbamylated protein. 31. The method of claim 30, wherein the carbamylated erythropoietin protein has less than about 10 weight percent of the over- and sub-carbamylated proteins. 31. The method of claim 30, wherein the carbamylated erythropoietin protein has less than about 30% by weight of the over-carbamylated protein. 31. The method of claim 30, wherein the carbamylated erythropoietin protein has less than about 20% by weight of the over-carbamylated protein. 31. The method of claim 30, wherein the carbamylated erythropoietin protein has less than about 10 weight percent of the over-carbamylated protein. A method of making a carbamylated erythropoietin protein having less than about 3% aggregated protein and less than about 40% by weight over- and sub-carbamylated protein as measured by an ESI mass spectrometer, comprising: (a) contacting a substantial amount of erythropoietin protein with a significant amount of cyanate for a temperature, pH, and period of time sufficient to carbamylize at least about 90% of the amine residues on the lysine and N-terminal amino acids of erythropoietin; And (b) Purifying the carbamylated erythropoietin protein using anion exchange, cation exchange, hydrophobic interaction chromatography, reverse phase chromatography, affinity chromatography or size exclusion chromatography. 42. The method of claim 41, wherein the carbamylated erythropoietin protein is human erythropoietin. 42. The method of claim 41, wherein the carbamylated erythropoietin protein has less than about 2.5% protein aggregated. 42. The method of claim 41, wherein the carbamylated erythropoietin protein has less than about 0.5% of aggregated protein. 42. The method of claim 41, wherein the carbamylated erythropoietin protein has less than about 30% by weight of the over- and sub-carbamylated proteins. 42. The method of claim 41, wherein the carbamylated erythropoietin protein has less than about 20% by weight of the over- and sub-carbamylated proteins. 42. The method of claim 41, wherein the carbamylated erythropoietin protein has less than about 10 weight percent of the over- and sub-carbamylated proteins. 42. The method of claim 41, wherein the carbamylated erythropoietin protein has less than about 30% by weight of the over-carbamylated protein. 42. The method of claim 41, wherein the carbamylated erythropoietin protein has less than about 20 weight percent of the over-carbamylated protein. 42. The method of claim 41, wherein the carbamylated erythropoietin protein has less than about 10 weight percent of the over-carbamylated protein. 42. The method of claim 41 wherein the concentration of erythropoietin protein in contact with cyanate is from about 0.05 mg / ml to about 10 mg / ml. 42. The method of claim 41 wherein the concentration of erythropoietin protein in contact with cyanate is from about 2 mg / ml to about 5 mg / ml. 42. The method of claim 41, wherein the concentration of erythropoietin protein in contact with cyanate is about 3 mg / ml. 42. The method of claim 41 wherein the concentration of cyanate is from about 0.05 M to about 10 M. 42. The method of claim 41 wherein the concentration of cyanate is from about 0.05 M to about 2 M. 42. The method of claim 41 wherein the concentration of cyanate is about 0.5 M. 42. The method of claim 41, wherein the temperature is in the range of about 0 ° C to about 60 ° C. 42. The method of claim 41 wherein the temperature is in the range of about 30 ° C to about 34 ° C. 42. The method of claim 41 wherein the temperature is about 32 ° C. 42. The method of claim 41, wherein the pH is about 7 to about 11. 42. The method of claim 41, wherein the pH is about 8 to about 10. 42. The method of claim 41, wherein the pH is about 9. The method of claim 41, wherein the period of time is from about 10 minutes to about 30 days. 42. The method of claim 41, wherein the time period is from about 1 hour to about 5 days. 42. The method of claim 41, wherein the duration is about 24 hours. 42. The method of claim 41, wherein the erythropoietin protein is contacted with cyanate in the presence of a buffer. 67. The method of claim 66, wherein the buffer is borate. 67. The method of claim 66, wherein the concentration of the buffer is about 0.05 M to about 2 M. 67. The method of claim 66, wherein the concentration of the buffer is about 0.1 M to about 1 M. 67. The method of claim 66, wherein the concentration of the buffer is about 0.5 M. The method of claim 41, wherein the concentration of erythropoietin protein in contact with the cyanate is from about 0.05 mg / ml to about 10 mg / ml, the concentration of cyanate is from about 0.05 M to about 10 M, and the temperature is from about 0 ° C. to about And a pH in the range of 60 ° C., about 7 to about 11, and a duration of about 10 minutes to 30 days. 42. The method of claim 41 wherein the concentration of erythropoietin protein in contact with the cyanate is from about 2 mg / ml to about 5 mg / ml, the concentration of cyanate is from about 0.05 M to about 2 M, and the temperature is from about 30 ° C to about And a pH in the range of 34 ° C., about 8 to about 10, and a duration of about 1 hour to 5 days. The method of claim 34, wherein the concentration of erythropoietin protein in contact with the cyanate is about 3 mg / ml, the concentration of cyanate is about 0.5 M, the temperature is about 32 ° C., the pH is about 9.0, and the duration is about 24 How it is time. Carbamylated at least about 90% of primary amines of lysine and amino terminal amino acids as measured by ESI-mass spectrometry, less than about 3% aggregated protein and about 40% by weight over- and sub-carbamylated protein Carbamylated erythropoietin protein having less than. 75. The carbamylated erythropoietin protein of claim 74, wherein the erythropoietin protein is human erythropoietin. 75. The carbamylated erythropoietin protein of claim 74, having less than about 2.5% aggregated protein. 75. The carbamylated erythropoietin protein of claim 74, having less than about 0.5% aggregated protein. 75. The carbamylated erythropoietin protein of claim 74, wherein the amount of aggregated protein is measured by SEC-HPLC. 75. The carbamylated erythropoietin protein of claim 74, having less than about 30% by weight of the over- and sub-carbamylated protein. 75. The carbamylated erythropoietin protein of claim 74, having less than about 20% by weight of the over- and sub-carbamylated protein. 75. The carbamylated erythropoietin protein of claim 74, having less than about 10% by weight of the over- and sub-carbamylated protein. 75. The carbamylated erythropoietin protein of claim 74, having less than about 30% by weight of the over-carbamylated protein. 75. The carbamylated erythropoietin protein of claim 74, having less than about 20% by weight of the over-carbamylated protein. 75. The carbamylated erythropoietin protein of claim 74, having less than about 10% by weight of the over-carbamylated protein. A compound comprising carbamylated erythropoietin protein having at least about 90% carbamylation of primary amines of lysine and amino terminal amino acids, less than about 40% aggregated protein, and a significant amount of cyanate. 86. The compound of claim 85, wherein the carbamylated erythropoietin protein is human erythropoietin. 86. The compound of claim 85, wherein the carbamylated erythropoietin protein has less than about 30% of the aggregated protein. 86. The compound of claim 85, wherein the carbamylated erythropoietin protein has less than about 20% of the aggregated protein. 86. The compound of claim 85, wherein the carbamylated erythropoietin protein has less than about 15% protein aggregated. 86. The compound of claim 85, wherein the carbamylated erythropoietin protein has less than about 10% of the aggregated protein. 86. The compound of claim 85, wherein the carbamylated erythropoietin protein has less than about 7% aggregated protein. 86. The compound of claim 85, wherein the amount of aggregated protein is measured by SEC-HPLC. At least about 90% carbamylation, less than about 3% aggregated protein, and over- and downstream of primary amines of lysine and amino terminal amino acids, as measured by an ESI mass spectrometer, the product of the method comprising the following steps: Carbamylated erythropoietin protein with less than about 40% by weight carbamylated protein: (a) contacting a substantial amount of erythropoietin protein with a significant amount of cyanate for a temperature, pH, and period of time sufficient to carbamylate at least about 90% of the amine residues on the lysine and N-terminal amino acids of the erythropoietin protein; And (b) Purifying the carbamylated erythropoietin protein using anion exchange, cation exchange, hydrophobic interaction chromatography, reverse phase chromatography, affinity chromatography or size exclusion chromatography. 94. The carbamylated erythropoietin protein of claim 93, wherein the erythropoietin protein is human erythropoietin. 94. The carbamylated erythropoietin protein of claim 93, having less than about 2.5% aggregated protein. 94. The carbamylated erythropoietin protein of claim 93, having less than about 0.5% aggregated protein. 94. The carbamylated erythropoietin protein of claim 93, wherein the amount of aggregated protein is measured by SEC-HPLC. 95. The carbamylated erythropoietin protein of claim 93, wherein the carbamylated erythropoietin protein has less than about 30% by weight of the over- and sub-carbamylated protein. 94. The carbamylated erythropoietin protein of claim 93, wherein the carbamylated erythropoietin protein has less than about 20% by weight of the over- and sub-carbamylated protein. 94. The carbamylated erythropoietin protein of claim 93, wherein the carbamylated erythropoietin protein has less than about 10% by weight of the over- and sub-carbamylated protein. 94. The carbamylated erythropoietin protein of claim 93, having less than about 20% by weight of the over-carbamylated protein. 94. The carbamylated erythropoietin protein of claim 93, having less than about 10% by weight of the over-carbamylated protein. 94. The carbamylated erythropoietin protein of claim 93, having less than about 5% by weight of the over-carbamylated protein. 94. The method of claim 93, wherein the method comprises a concentration of erythropoietin protein in contact with cyanate of about 3 mg / ml, a concentration of cyanate of about 0.5 M, a temperature of about 32 ° C., a pH of about 9.0, and a duration of time. Carbamylated erythropoietin protein comprising about 24 hours. A therapeutically effective amount of carbamylation having at least about 90% carbamylation of primary amines of lysine and amino terminal amino acids, less than about 3% aggregated protein and less than about 40% by weight of over- and sub-carbamylated protein. Pharmaceutical composition comprising an erythropoietin protein, and a pharmaceutically acceptable carrier. 107. The pharmaceutical composition of claim 105, wherein the carbamylated erythropoietin protein is human erythropoietin. 107. The pharmaceutical composition of claim 105, wherein the carbamylated erythropoietin protein has less than about 2.5% protein aggregated. 107. The pharmaceutical composition of claim 105, wherein the carbamylated erythropoietin protein has less than about 0.5% of aggregated protein. 107. The pharmaceutical composition of claim 105, wherein the carbamylated erythropoietin protein has less than about 30% by weight of the over- and sub-carbamylated protein. 107. The pharmaceutical composition of claim 105, wherein the carbamylated erythropoietin protein has less than about 20% by weight of the over- and sub-carbamylated protein. 107. The pharmaceutical composition of claim 105, wherein the carbamylated erythropoietin protein has less than about 10 weight percent of the over- and sub-carbamylated protein. 107. The pharmaceutical composition of claim 105, wherein the carbamylated erythropoietin protein has less than about 30% by weight of the over-carbamylated protein. 107. The pharmaceutical composition of claim 105, wherein the carbamylated erythropoietin protein has less than about 20% by weight of the over-carbamylated protein. 107. The pharmaceutical composition of claim 105, wherein the carbamylated erythropoietin protein has less than about 10% by weight of the over-carbamylated protein. 107. The pharmaceutical composition of claim 105, wherein the carbamylated erythropoietin protein has less than about 5% by weight of the over-carbamylated protein. 107. The pharmaceutical composition of claim 105, wherein the carrier is a diluent, adjuvant, or excipient. 108. A method of treating a chronic condition or disease comprising administering a pharmaceutical composition according to claim 105. 107. A method of treating subacute symptoms or disease comprising administering a pharmaceutical composition according to claim 105. 107. A method of treating acute symptoms or disease comprising administering a pharmaceutical composition according to claim 105. 105. A method of treating a central or peripheral nervous system disease, comprising administering a pharmaceutical composition according to claim 105. 123. The method of claim 120, wherein the disease is seizure, ischemic symptoms, spinal cord injury, traumatic brain injury, multiple sclerosis, amyotrophic lateral sclerosis, Alzheimer's disease, schizophrenia, or chemotherapy-induced neuropathy. Use of a compound according to claim 74 for the preparation of a pharmaceutical composition comprising said compound for the treatment of chronic conditions or diseases. Use of a compound according to claim 74 for the preparation of a pharmaceutical composition comprising said compound for the treatment of subchronic symptoms or diseases. Use of a compound according to claim 74 for the preparation of a pharmaceutical composition comprising said compound for the treatment of acute symptoms or disease. Use of a compound according to claim 74 for the preparation of a pharmaceutical composition comprising said compound for the treatment of diseases of the central or peripheral nervous system. 126. The use of claim 125, wherein the disease is seizure, ischemic symptoms, spinal cord injury, traumatic brain injury, multiple sclerosis, amyotrophic lateral sclerosis, Alzheimer's disease, schizophrenia, or chemotherapy-induced neuropathy.

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