Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/475—Growth factors; Growth regulators
  • C07K14/505—Erythropoietin [EPO]
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P11/00—Drugs for disorders of the respiratory system
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P25/00—Drugs for disorders of the nervous system
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P25/00—Drugs for disorders of the nervous system
  • A61P25/04—Centrally acting analgesics, e.g. opioids
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P25/00—Drugs for disorders of the nervous system
  • A61P25/08—Antiepileptics; Anticonvulsants
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P25/00—Drugs for disorders of the nervous system
  • A61P25/14—Drugs for disorders of the nervous system for treating abnormal movements, e.g. chorea, dyskinesia
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P25/00—Drugs for disorders of the nervous system
  • A61P25/14—Drugs for disorders of the nervous system for treating abnormal movements, e.g. chorea, dyskinesia
  • A61P25/16—Anti-Parkinson drugs
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P25/00—Drugs for disorders of the nervous system
  • A61P25/18—Antipsychotics, i.e. neuroleptics; Drugs for mania or schizophrenia
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P25/00—Drugs for disorders of the nervous system
  • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P9/00—Drugs for disorders of the cardiovascular system
  • A61P9/04—Inotropic agents, i.e. stimulants of cardiac contraction; Drugs for heart failure
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P9/00—Drugs for disorders of the cardiovascular system
  • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis

Definitions

• polypeptide comprising a fusion of an amino acid sequence selected from the group of amino acid sequences as shown in SEQ ID NO 24, 26, 28, and
• polynucleotides which are at least 50% identical to a polynucleotide as defined in any one of (a) to (g) and which code for a polypeptide having cell protective and in particular neuroprotective activity but essentially no hematopoietic activity; and (i) polynucleotides the complementary strand of which hybridizes under stringent conditions to a polynucleotide as defined in any one of (a) to (h) and which code for a polypeptide having cell protective and in particular neuroprotective activity but essentially no hematopoietic activity; or the complementary strand of such a polynucleotide.
• a further aspect of the present invention is a homolog of an erythropoietin (EPO) variant encoding polynucleotide from another higher eukaryotic species.
• EPO erythropoietin
• polynucleotides encoding an EPO variant polypeptide, which comprises the N-terminal part of full length EPO including helix A and which lacks at least one of the following: (i) a fragment of at least 10 amino acids, preferably 1 1, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acids between helix A and helix B;
• the process of the present invention further comprises the step of modifying said EPO variant, wherein the modification is selected from the group consisting of oxidation, sulfation, phosphorylation, addition of oligosaccharides or combinations thereof.
• polypeptide comprising a fusion of an amino acid sequence selected from the group of amino acid sequences as shown in SEQ ID NO 24, 26, 28, and
• polynucleotides the complementary strand of which hybridizes under stringent conditions to a polynucleotide as defined in any one of (a) to (h) and which code for a polypeptide having cell protective and in particular neuroprotective activity but essentially no hematopoietic activity; or the complementary strand of such a polynucleotide.
• a deletion is still considered to be essentially the same if it involves 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 55, 56, 57, 58 or 61 more or less nucleotides as the respective deletion in SEQ ID NO 1, 3, 5, 7, 9, 1 1, 13, 15, 17, 19, or 21, which are also depicted in Fig. 1 and 2.
• helix D 20, 21, or 22 of helix D; wherein said variant has cell protective and in particular neuroprotective activity but essentially no hematopoietic activity.
• Am EPO variant polypeptide that exhibits essentially no hematopoietic activity is a polypeptide, which elicits in art known colony formation assays, an example of which is described below, at the same molar concentration as the rhEPO and wt mEPO, respectively, less than 10% of the CFU-E (Colony forming unit-erythroblast), preferably less than 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1 %.
• the respective CFU-E numbers are calculated for a given rhEPO, wt mEPO or EPO variant by subtracting from each value the number of CFU-E observed in a control reaction (without wt or EPO variant).
• polynucleotide molecules of the invention can be synthesized in vitro (for example, by phosphoramidite-based synthesis) or can be obtained from a cell, such as the cell of a mam- mal.
• Hybridization can also be used as a measure of homology between two nucleic acid sequences.
• a nucleic acid sequence encoding any of the EPO variants disclosed herein, or a derivative or fragment thereof, can be used as a hybridization probe according to standard hybridization techniques.
• the hybridization of an EPO variant probe to DNA or RNA from a test source is an indication of the presence of the relevant EPO DNA or RNA in the test source.
• Hybridization conditions are known to those skilled in the art and can be found in Current Protocols in Molecular Biology, John Wiley & Sons, N.Y., 6.3.1- 6.3.6, 1991.
• apoptosis is characterized by chromatin fragmentation, extravasation of cellular contents and eventually death of the cell. It has been recognized to play a role in many acute or chronic pathologic processes.
• Chronic neurodegenerative disorders that can be treated with the EPO variants of the present invention include, but are not limited to, dementias (e.g. Alzheimer's disease, vascular dementias), Pick's disease, diffuse Lewy body disease, progressive supranuclear palsy (Steel- Richardson syndrome), multiple sclerosis, multiple system atrophy (including Shy-Drager syndrome), chronic epileptic conditions associated with neurodegeneration, motor neuron diseases including amyotrophic lateral sclerosis, degenerative ataxias, cortical basal degeneration, ALS-Parkinson's-Dementia complex of Guam, subacute sclerosing panencephalitis, Huntington's disease, Parkinson's disease, synucleinopathies (including multiple system atrophy), primary progressive aphasia, striatonigral degeneration, Machado-Joseph dis- ease/spinocerebellar ataxia type 3 and olivopontocerebellar degenerations, Gilles De La Tourette's
• the intervals necessary will depend in part on the serum level of the EPO variant necessary to treat or ameliorate the respective disease and on the pharmacokinetic of the respective EPO variant, which will in part depend on modifications of EPO by, for example, PEG. It will be in the discretion of the practitioner to determine the exact duration, dose and type of EPO variant taking into consideration, e.g. the condition of the patient to be treated, the severity of the dondition etc.
• Suitable pharmaceutical excipi- ents include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like.
• the composition if desired, can also contain mi- nor amounts of wetting or emulsifying agents, or pH buffering agents. These compositions can take the form of solutions, suspensions, emulsion, tablets, pills, capsules, powders, sustained-release formulations and the like.
• the composition can be formulated as a suppository, with traditional binders and carriers such as triglycerides.
• compositions adapted for oral administration may be provided as capsules or tablets; as powders or granules; as solutions, syrups or suspensions (in aqueous or nonaqueous liquids); as edible foams or whips; or as emulsions.
• Tablets or hard gelatine capsules may comprise lactose, starch or derivatives thereof, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, stearic acid or salts thereof.
• Soft gelatine capsules may comprise vegetable oils, waxes, fats, semi-solid, or liquid polyols etc. Solutions and syrups may comprise water, polyols and sugars.
• compositions adapted for parenteral administration may be presented in unit-dose or multi-dose containers, for example sealed ampules and vials, and may be stored in a freeze-dried (lyophilized) condition requiring only the addition of a sterile liquid carrier, e.g., sterile saline solution for injections, immediately prior to use.
• a sterile liquid carrier e.g., sterile saline solution for injections, immediately prior to use.
• Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets.
• an autoinjector comprising an injectable solution of an EPO variant may be provided for emergency use by ambulances, emergency rooms, and battlefield situations, and even for self-administration in a domestic setting, particularly where the possibility of traumatic amputation may occur, such as by imprudent use of a lawn mower.
• the likelihood that cells and tissues in a severed foot or toe will survive after reattachment may be increased by administering an EPO variant to multiple sites in the severed part as soon as practicable, even before the arrival of medical personnel on site, or arrival of the afflicted individual with severed toe in tow at the emergency room.
• the composition is formulated in accordance with routine proce- dures as a pharmaceutical composition adapted for intravenous administration to human beings.
• compositions for intravenous administration are solutions in sterile isotonic aqueous buffer.
• the composition may also include a solubilizing agent and a local anesthetic such as lidocaine to ease pain at the site of the injection.
• the ingredients are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water-free concentrate in a hermetically-sealed container such as an ampule or sachette indicating the quantity of active agent.
• Such pharmaceutical compositions may comprise levels of an EPO variant or a form of an EPO variant not suitable for acute or chronic, local or system administration to an individual, but will serve the functions intended herein in a cadaver, organ bath, organ perfusate, or in situ perfusate prior to removing or reducing the levels of the EPO variant contained therein before exposing or returning the treated organ or tissue to regular circulation.
• polymeric materials can be used (see Medical Applications of Controlled Release (1974) Langer and Wise (eds.), CRC Press: Boca Raton, FIa.; Controlled Drug Bioavailability, Drug Product Design and Performance, (1984) Smolen and Ball (eds.), Wiley: N.Y.; Ranger and Peppas (1953) J. Macromol. Sci. Rev. Mac- romol. Chem.
• a controlled release system can be placed in proximity of the therapeutic target, i.e., the target cells, tissue or organ, thus requiring only a fraction of the systemic dose (see, e.g., Goodson (1984) 115-138 in Medical Applications of Controlled Re- lease, vol. 2).
• Other controlled release systems are discussed in the review by Langer (1990, Science 249: 1527-1533).
• EPO variant as properly formulated, can be administered by nasal, oral, rectal, vaginal, or sublingual administration.
• a perfusate or perfusion solution for perfusion and storage of organs for transplant, the perfusion solution including an amount of an phar- maceutic compositions effective to protect EPO variant-responsive cells and associated cells, tissues or organs.
• Transplant includes but is not limited to xenotransplantation, where a organ (including cells, tissue or other bodily part) is harvested from one donor and transplanted into a different re- cipient; and autotransplant, where the organ is taken from one part of a body and replaced at another, including bench surgical procedures, in which an organ may be removed, and while ex vivo, resected, repaired, or otherwise manipulated, such as for tumor removal, and then returned to the original location.
• the perfusion solution is the University of Wisconsin (UW) solution (U.S.
• 4,798,824 which contains from about 1 to about 25 U/ml erythropoietin, 5% hydroxyethyl starch (having a molecular weight of from about 200,000 to about 300,000 and substantially free of ethylene glycol, ethylene chlorohydrin, sodium chloride and acetone); 25 mM 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 .
• the pharmaceutical composition of the present invention is preferably administered in above outlined preferred and particular preferred doses on a daily basis.
• the pharmaceutical compositions in particular EPO variant polypeptide is preferably administered in above outlined preferred and particular preferred doses on a daily basis.
• the pharmaceutical composition will be administered once a stroke has been diagnosed.
• a first dose of the pharmaceutical composition is administered for the first time within 24 hours after the first symptoms of a stroke are evident, preferably within 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 hour or less.
• the administration is then continued for prefera- bly at least 7, more preferably at least 14 and more preferably for at least 21 days.
• the doses are administered preferably once a day and preferably in above indicated doses.
• Fig. 1 Comparison of EPO PCR products: Panel A depicts the DNA products of various PCR reactions performed with either pure plasmid comprising the different murine EPO variants or cDNA from mouse brain of kidney, which are separated on a 1.2% agarose gel. From the left to the right the lanes comprise: 1 kb molecular weight marker, the product of pure mK3, pure mG3, pure mG5, pure m301, pure mS, pure mWT, brain cDNA, kidney cDNA. Panel B depicts the DNA product of a PCR performed with cDNA from human brain. From the left to the right the lanes comprise 1 kb molecular weight standard and the PCR product of human brain cDNA.
• Fig. 2 Alignment of nucleotide sequences of the EPO variants identified in murine brain cDNA and "wild type" murine EPO, i.e. the sequence of the previously described EPO.
• Fig. 3 Alignment of nucleotide sequences of the EPO variants identified in human brain cDNA and "wild type" human EPO.
• Fig. 4 Alignment of the amino acid sequences of the EPO variants identified in mouse and human with the respective "wild type" EPO.
• Fig. 5 Hematopoietic activity of murine and human EPO and the EPO variants of the present invention.
• Panel A depicts the results of a colony forming assay for murine EPO and EPO variants
• Panel B depicts the of a colony forming assay for human EPO and EPO variants.
• Fig. 6 Experimental setup for neuroprotection assays with rhEPO and EPO-isomers.
• Fig 8 Panel A shows an experiment with 2 h 00 min, 2 h 15 min and 2 h 20 min OGD length with a protein concentration equalling 100 U/l hEPO. At all three time-points a protection rate of 40-50% for the human, but no protection with mEPO and rhEPO. Panel B shows an experiment with two different time-points (OGD length between two experiments varies according to density of neurons). At 2 h 45 min only weak protection is achieved with wtEPO (20-30%) compared to neuroguardians (60-70%). Full protection capacity of EPO is only observed at higher damage levels (3 h 15 min).
• Panel B shows a Western Blot of His-Tag-purified mouse wild type EPO (mEPO), human hS3 and hS4 EPO variants.
• mEPO was quantified with the EPO-mouse-ELISA. 130 pg mEPO were loaded onto the gel.
• primary antibody rabbit anti-rhEPO-Antik ⁇ rper; Santa-Cruz.
• Fig. 10 Alignment of the amino acid sequences of the EPO variants created recombinantly (alpha-helix mutants) and identified in vivo.
• SEQ ID NO 50 is the human alpha helix wild type sequence
• SEQ ID NO 51 is hAmA (point mutation Alanin);
• SEQ ID NO 52 is hAmE (point mutation glutamic acid);
• SEQ ID NO 53 is hA-10 (deletion mutant) and
• SEQ ID NO 54 is hA-20 (deletion mutant).
• Fig. 11 hEPO and hS3 mediated cytoprotection in a model of ischemia consisting of serum deprivation and hypoxia in H9c2 cardiac myoblasts.
• H9c2 cells were incubated in serum-deprived DMEM-medium either in normoxic or hypoxic conditions for 24h. Apoptosis was assessed 24h later by LDH assay. Data were normalized by setting the delta LDH release of untreated cells in normoxic and hypoxic conditions to 100%.
• A Column diagram representing the average values of normalized LDH release.
• hA 100/Ul Neuroprotection mediated by Erythropoietin Alpha-helix
• a nested PCR was performed in a Hybaid PCR machine in two steps, first PCR (3 min at 95 0 C; 35 cycles: 30 sec at 65°C, 1 min at 72°C,30 sec at 95°C; 10 min at 72 0 C; 4 0 C hold) and second PCR (3 min at 95°C; 5 cycles: 30 sec at 67°C, 1 min at 72°C, 30 sec at 95°C; 15 cycles: 30sec at 70 0 C, 1 min at 72°C, 30 sec at 95°C; 10 min at 72°C; 4°C).
• Pfu Turbo Hotstart DNA Polymerase (Stratagene) was used according to the manufacturer's protocol.
• the PCR product of the first step was diluted 1 :50 for the second PCR.
• a second cDNA synthesis protocol was performed using the Access RT-PCR System (Invitrogen) with the following parameters: 48°C 5 min; 94°C 2 min; 40 cycles: 94°C 30 sec, 65°C 1 min, 7O 0 C 2 min; 70°C 7 min; 4 0 C.
• the second PCR was performed as described above.
• the amplified full-length EPO cDNA and the EPO isomers were separated on a 1.2% TAE- agarose gel. A picture of the various PCR products is shown in Fig. Ia. The fragments were than purified using the Wizard SV-GeI Cleanup System (Promega) or the Gel Extraction Kit (Qiagen, Hilden, Germany). As Pfu Polymerase generates blunt end products, the cDNA was subcloned in the pCR-Blunt II-TOPO Vector using chemically competent ToplO One Shot Cells from (both Invitrogen).
• the nucleotide and peptide sequence of the EPO variant m301 corresponds to SEQ ID NO 19 and SEQ ID NO 20, respectively.
• the nucleotide and peptide sequence of the EPO variant mK3 corresponds to SEQ ID NO 21 and SEQ ID NO 22, respectively.
• murine EPO variants In comparison to murine EPO and rhEPO the murine EPO variants (mS and mG3-variant) lacked haematopoietic potential.
• Rat primary neuronal cultures were obtained from El 6 to early El 9 embryos of Wistar rats (Bundesinstitut fur Surgicallichen Medeau, Berlin, Germany). Cre-expressing mouse neurons were obtained from El 6 embryos of heterozygous transgenic mice expressing Cre-recombinase under the control of the tubulin ⁇ -1 promoter (provided by Dr. U. Schweitzer; Experimental Endocrinology, Charite). Murine and rat cultures were prepared according to a modified protocol from Brewer (1995) J Neurosci Res. 42: 674-83. Cerebral cortex was isolated after removal of meninges and rinsed twice in PBS (Biochrom, Berlin, Germany).
• N-Med modified Eagle's medium from Gibco with 10% fetal calf serum, 100 U penicillin plus streptomycin from Biochrom, 2 mM L- glutamine, 100 IE insulin/1, 10 mM HEPES and 44 mM glucose
• 24-well plates and 6-well plates were pretreated by over-night incubation at 4°C with poly-L- lysin from Biochrom (2.5 ⁇ g/ml in PBS). Rinsing of the wells with PBS was followed by Ih incubation at 37°C with coating medium (modified Eagle's medium with 5% FCS Gold from PAA, 1% Pen/Strep, 1OmM HEPES and 0,03 w/v collagen G from Biochrom), then the wells were carefully rinced twice with PBS. Volume and type of plating medium was chosen depending on experimental procedure.
• the neuroprotection induced by the murine EPO variants is more robust than that induced by EPO (rhEPO as well as wild type mouse EPO). For example, neuroprotection mediated by EPO can only be observed in a clearly defined window of OGD length (corresponding to a clearly defined damage level). At low concentration the neuroprotection by hS3 and hS4 was equal or better than the neuroprotection of wt hEPO. Overall, neuroprotection induced by the variants is stronger than that induced by rhEPO. In addition, variants have an higher neuroprotective potential than both wild type forms mEPO and hEPO, which were produced by the same procedure as the EPO variants. H9c2 - model of ischemia
• the rat BDIX heart myoblast cell line (obtained from European Collection of Cell Cultures) was cultured in DMEM (Biochrom) containing 4.5g/l glucose supplemented with 2mM L- glutamine, 10% inactivated fetal calf serum and 1 % penicillin-streptavidin. Subconfluent cultures (70%) were subcultured 1 :4. Cells were plated in 400 ⁇ l medium containing 12OpM hEPO or hS3 respectively in a density of 15,000 cells per well in 24- well plates and cultured for 48 hours.
• the cytoprotective potential of the EPO variants was shown exemplarily for purified hEPO and hS3 in a model of ischemia consisting of serum deprivation and hypoxia in H9c2 cardiac myoblasts (Figurel). LDH release was assessed as a marker of apoptotic cell death. We found significant cytoprotective capacities for both variants (approximately 20% and 25% for hEPO and hS3).

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