WO2008048646A1 - Utilisation de la protéine c humaine présentant un état de glycosylation et une teneur en acide sialique modifiés en tant que médicament - Google Patents

Utilisation de la protéine c humaine présentant un état de glycosylation et une teneur en acide sialique modifiés en tant que médicament Download PDF

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WO2008048646A1
WO2008048646A1 PCT/US2007/022180 US2007022180W WO2008048646A1 WO 2008048646 A1 WO2008048646 A1 WO 2008048646A1 US 2007022180 W US2007022180 W US 2007022180W WO 2008048646 A1 WO2008048646 A1 WO 2008048646A1
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protein
functional variant
human protein
human
apc
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PCT/US2007/022180
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Colleen Mullen
Nienwen Chow
Berislav V. Zlokovic
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Socratech L.L.C.
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    • 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
    • C07K14/745—Blood coagulation or fibrinolysis factors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • This invention relates to the use of an activated protein C (APC) with novel glycosylation, a prodrug thereof (e.g., zymogen forms), and/or a variant thereof as a neuroprotective or cytoprotective agent, an inhibitor of apoptosis or cell death, and/or a cell survival factor, especially for cells or tissues of the nervous system which are stressed or injured.
  • APC activated protein C
  • activated protein C is a serine protease which deactivates Factors V a and Vlll a .
  • Human protein C is primarily made in the liver as a single polypeptide of 461 amino acids.
  • This precursor is then post-translationally modified by (i) cleavage of a 42 amino acid signal sequence, (ii) proteolytic removal from the one-chain zymogen of the lysine residue at position 155 and the arginine residue at position 156 to produce the two-chain form (i.e., a light chain of 155 amino acid residues attached by disulfide linkage to a serine protease-containing heavy chain of 262 amino acid residues), (iii) carboxylation of the glutamic acid residues clustered in the first 42 amino acids of the light chain resulting in nine gamma- carboxyglutamic acid (GIa) residues, and (iv) glycosylation at four sites (one in the light chain and three in the heavy chain).
  • GIa gamma- carboxyglutamic acid
  • the heavy chain contains the serine protease triad of Asp257, His211 and Ser360. Similar to most other zymogens of extracellular proteases and the coagulation factors, protein C has a core structure of the chymotrypsin family, having insertions and an N-terminus extension that enable regulation of the zymogen and the enzyme. Of interest are two domains with amino acid sequences similar to epidermal growth factor (EGF). At least a portion of the nucleotide and amino acid sequences for protein C from human, monkey, mouse, rat, hamster, rabbit, dog, cat, goat, pig, horse, and cow are known, as well as mutations and polymorphisms of human protein C (see GenBank accession P04070). Other variants of human protein C are known which affect different biological activities.
  • EGF epidermal growth factor
  • Taylor & Esmon show that APC inhibits inflammatory stimuli disrupting cell permeability and normal coagulation processes such as sepsis.
  • Griffin et al. U.S. Patent 5,084,274
  • Grinnell et al. U.S. Patent 6,037,322
  • Griffin & Zlokovic U.S. Patent 7,074,402 and WO 2004/056309 disclose the use of APC as a neuroprotective agent.
  • Grinnell U.S. Patent 5,550,0366 teaches expression of human protein C using a papovavirus enhancer such as those from SV40 and BK.
  • Our invention does not require a papovavirus enhancer, a human host cell (e.g., adenovirus 5-transformed human embryonic kidney cell line 293), or expression of an immediate early protein of a large DNA virus (e.g., adenovirus, herpes simplex virus, pseudorabies virus) in the host cell.
  • Recombinant human protein C expressed in 293 host cells had higher anticoagulant activity because of an altered glycosylation pattern as compared to plasma-derived human protein C.
  • This APC is manufactured and marketed by EIi Lilly as XIGRIS® drotrecogin alfa. But the recombinant human protein C, prodrug thereof, and variant thereof of the present invention have a different glycosylation pattern from the prior art, and such pattern preferably does not increase APCs anticoagulant activity.
  • activated protein C APC
  • novel glycosylation, prodrug thereof, and/or variant thereof A need for pharmaceutical compositions that can act as neuroprotectants or cytoprotectants is addressed thereby. Further objectives and advantages of the invention are described below.
  • An object of the invention is production of human protein C with novel patterns of glycosylation.
  • This also provides a prodrug (i.e., zymogen human protein C) which is cleaved to a human activated protein C (APC); both have a glycosylation pattern different from that observed in natural (e.g., plasma) or prior art recombinant human protein C (e.g., human embryonic kidney cell lines).
  • variants e.g., derived from other species, mutants of human protein C, truncations of human protein C having at least 100 residues, at least 200 residues, at least 300 residues, or at least 400 residues contiguous in the wild- type amino acid sequence of human protein C
  • variants e.g., derived from other species, mutants of human protein C, truncations of human protein C having at least 100 residues, at least 200 residues, at least 300 residues, or at least 400 residues contiguous in the wild- type amino acid sequence of human protein C
  • glycosylation increase the ratio of neuroprotective activity to anticoagulant activity as compared to human protein C derived from human plasma and/or recombinant human embryonic kidney cell lines.
  • sialic acid content may distinguish human APC of this invention over native human APC and prior art recombinant APC.
  • Another object is use of one or more of the aforementioned carboxylated and glycosylated protein(s) to at least improve neuroprotection, cytoprotection, inhibition of apoptosis or cell death, and/or promotion of cell survival in aging, sepsis, mental retardation, and neurodegenerative diseases like Alzheimer's disease, amyotrophic lateral sclerosis, Down's syndrome, epilepsy, Hunting- ton's disease, ischemia, Parkinson's disease, spinal injury or trauma, stroke, etc.
  • the aforementioned carboxylated and glycosylated protein(s) to at least improve neuroprotection, cytoprotection, inhibition of apoptosis or cell death, and/or promotion of cell survival in aging, sepsis, mental retardation, and neurodegenerative diseases like Alzheimer's disease, amyotrophic lateral sclerosis, Down's syndrome, epilepsy, Hunting- ton's disease, ischemia, Parkinson's disease, spinal injury or trauma, stroke
  • An effective amount of APC, at least one prodrug (e.g., protein C and variants thereof), or at least one variant thereof (e.g., APC protease domain mutants with reduced anticoagulant activity) may be used to provide at least neuroprotection, to inhibit apoptosis or cell death, and/or to promote cell survival in stressed or injured brain cells and, more particularly, in stressed or injured brain endothelium and neurons.
  • At least one of the proteins may be used to prepare a medicament for neuroprotection, cytoprotection, inhibition of apoptosis or cell death, promotion of cell survival, vascular protection, or combinations thereof, or treatment of a neurodegenerative disease.
  • the invention provides a treatment for therapy or prophylaxis of a neurodegenerative disease, and the products used therein.
  • Pharmaceu- tical compositions may be manufactured and assessed in accordance therewith. Further aspects of the invention will be apparent to persons skilled in the art from the following detailed description and claims, and generalizations thereto.
  • the present invention is useful for treating many neurodegenerative diseases involving apoptosis and/or cell death in the central nervous system. Apoptosis and/or cell death are reduced as a result of practicing the invention. Injury due to ischemia or hypoxia may be prevented or at least mitigated. Similarly, injury from ultraviolet (UV) or gamma irradiation (i.e., physical insults of the environment), chemical contaminants and pollutants, or cutting the spinal cord or mechanically compressing it may be prevented or at least mitigated. In particular, neurotoxicity due to A ⁇ i -42 oligomers or overstimulation of N-methyl- D-aspartate (NMDA) receptors are useful model for neuronal cell injury and death that mimics the effects of neurodegenerative disease.
  • NMDA N-methyl- D-aspartate
  • Animal models with mutations in A ⁇ or SOD1 may be used to assess the effects of APC on behavior assays and pathology. Cytoprotection may be determined at the level of different cell types, organs or tissues, or whole organisms.
  • the present invention provides methods for protecting neuronal cells from cell death in a subject having or at risk of neurodegenerative disease.
  • the method includes administering an effective amount of activated protein C, a prodrug (e.g., zymogen), or a variant thereof to the subject, thereby providing neuroprotection to the subject.
  • the effective amount may be a low dose of APC or a variant thereof which is directly neuroprotective but with at least reduced anticoagulant activity as compared to prior art treatments.
  • Variants of APC with reduced anticoagulant activity have been described (Gale et al., J. Biol. Chem. 277:28836-28840, 2002).
  • diseases include, but are not limited to, aging, Alzheimer's disease, Down's syndrome, Hunting- ton's disease, epilepsy, ischemia, amyotrophic lateral sclerosis, mental retardation, Parkinson's disease, and stroke.
  • Treating neurodegenerative disease is clinically measurable by neurological or psychiatric tests; similarly, therapeutic effects on coagulation, fibrinolysis, thrombosis, and inflammation is clinically measurable.
  • Multiple sclerosis (MS) as well as other neuropathologies may also be treated; at least demyelination, impaired nerve conduction, or paralysis may be reduced thereby.
  • Neurological damage may be at least reduced or limited, and symptoms ameliorated thereby.
  • neurodegenerative diseases neuronal cells degenerate to bring about deterioration of cognitive function.
  • diseases and neurological deficiencies may bring about such degeneration including Alzheimer's disease, amyotrophic lateral sclerosis, Down's syndrome, Huntington's disease, Parkin- son's disease, hypoxia or ischemia caused by stroke, cell death caused by epilepsy, mental retardation and the like, as well as neurodegenerative changes resulting from aging.
  • neurodegenerative disease is used to denote conditions which result from loss of neurons, neuronal cell injury or loss, and/or injury of other types of brain cells such as oligodendrocytes, brain endothelial cells, other vascular cells, and/or other cell types in the nervous system which may bring about deterioration of a motor or sensory function of the nervous system, cognitive function, higher integrative intellectual functions, memory, vision, hearing etc.
  • Such degeneration of neural cells may be caused by Alzheimer's disease, amyotrophic lateral sclerosis, Huntington's disease, by pathological conditions caused by temporary lack of blood or oxygen supply to the brain, e.g., brought about by stroke; by epileptic seizures; due to chronic conditions such as amyotrophic lateral sclerosis, mental retardation and the like; as well as due to normal degeneration due to aging.
  • diseases such as Alzheimer's disease and stroke have both neurodegenerative and vascular components, with or without an inflammatory response, and thus can be treated by the methods of the invention.
  • the cell may be derived from brain vessels (e.g., an endothelial or smooth muscle cell) of a subject, especially from the endothelium of a brain vessel.
  • brain vessels e.g., an endothelial or smooth muscle cell
  • it may be a neuron, an astrocyte, a microglial cell, an oligodendrocyte, or a pericyte; a precursor or a progenitor cell thereof; or other types of differentiated cell from the subject's central or peripheral nervous system.
  • neuron includes hundreds of different types of neurons, each with distinct properties. Each type of neuron produces and responds to different combinations of neurotransmitters and neurotrophic factors. Neurons are thought not to divide in the adult brain, nor do they generally survive long in vitro.
  • the method of the invention provides for the protection from death or senescence of neurons from virtually any region of the brain and spinal cord.
  • Neurons include those in embryonic, fetal or adult neural tissue, including tissue from the hippocampus, cerebellum, spinal cord, cortex (e.g., motor or somatosensory cortex), striatum, basal forebrain (e.g., cholinergic neurons), ventral mesencephalon (e.g., cells of the substantia nigra), and the locus ceruleus (e.g., neuroadrenaline cells of the central nervous system).
  • cortex e.g., motor or somatosensory cortex
  • striatum e.g., basal forebrain (e.g., cholinergic neurons)
  • ventral mesencephalon e.g., cells of the substantia nigra
  • locus ceruleus e.g., neuroadrenaline cells of the central nervous system.
  • the present invention may be used to treat myocardial infarction, other heart diseases and their clinical symptoms, endothelial injury, adult respiratory distress syndrome (ARDS), and failure of the liver, kidney, or central nervous system (CNS).
  • ARDS adult respiratory distress syndrome
  • CNS central nervous system
  • diseases which benefit from the methodologies of the present invention such as for example, bronchitis, cardiac arrhythmias, cardiomyopathy, congestive heart failure, coronary arterial occlusion, diabetic neuropathy, graft or transplant rejection, and myocarditis.
  • Life-threatening local and remote tissue damage occurs during surgery, trauma, and stroke when major vascular beds are deprived for a time of oxygenation (ischemia) then restored with normal circulation (reperfusion). Cell death and tissue damage can lead to organ failure or decreased organ function.
  • Protein C refers to native genes and proteins belonging to this family as well as variants thereof (e.g., mutations and polymorphisms found in nature or artificially designed).
  • the chemical structure of the genes, and proteins may be a polymer of natural or non-natural nucleotides connected by natural or non- natural covalent linkages (i.e., polynucleotide) or a polymer of natural or non- natural amino acids connected by natural or non-natural covalent linkages (i.e., polypeptide). See Tables 1-4 of WIPO Standard ST.25 (1998) for a nonlimiting list of natural and non-natural nucleotides and amino acids.
  • Protein C genes and proteins may be recognized as belonging to this family by comparison to the human homolog PROC, use of nucleic acid binding (e.g., stringent hybridization under conditions of 400 mM NaCI, 40 mM PIPES pH 6.4, 1 mM EDTA, at 5O 0 C or 7O 0 C for an oligonucleotide; 500 mM NaHPO 4 pH 7.2, 7% SDS, 1 % BSA, 1 mM EDTA, at 45°C or 65°C for a polynucleotide of 50 bases or longer; and appropriate washing) or protein binding (e.g., specific immunoassay under stringent binding conditions of 50 mM Ths-HCI pH 7.4, 500 mM NaCI 1 0.05% TWEEN 20 surfactant, 1 % BSA, at room temperature and appropriate washing); or computer algorithms (Doolittle, Of URFS and ORFS, 1986; Gribskov & Devereux, Sequence Analysis Primer, 1991 ; and references
  • a “mutation” refers to one or more changes in the sequence of polynucleotides and polypeptides as compared to native protein C, and has at least one function that is more active or less active, an existing function that is changed or absent, a novel function that is not naturally present, or combinations thereof.
  • a “polymorphism” also refers to a difference in its sequence as compared to native protein C, but the changes do not necessarily have functional consequences. Mutations and polymorphisms can be made by genetic engineering or chemical synthesis, but the latter is preferred for non- natural nucleotides, amino acids, or linkages. The fusion of domains linked in their reading frames is another way of generating diversity in sequence or mixing-and-matching functional domains.
  • homologous protein C and protein S work best together and this indicates that their sequences may have coevolved to optimize interactions ( between the enzyme and its cofactor.
  • Exon shuffling or gene shuffling techniques may be used to select desirable phenotypes in a chosen background (e.g., separable domains with different biological activities, hybrid human/mouse sequences which locate the species determinants).
  • Percentage identity between a pair of sequences may be calculated by the algorithm implemented in the BESTFIT computer program (Smith & Waterman, J. MoI. Biol. 147:195-197, 1981 ; Pearson, Genomics 1 1 :635-650, 1991 ).
  • Another algorithm that calculates sequence divergence has been adapted for rapid database searching and implemented in the BLAST computer program (Altschul et al., Nucl. Acids Res. 25:3389-3402, 1997).
  • the protein C polynucleotide or polypeptide may be only about 60% identical at the amino acid level, 70% or more identical, 80% or more identical, 90% or more identical, 95% or more identical, 97% or more identical, or greater than 99% identical.
  • amino acid substitutions may also be considered when making comparisons because the chemical similarity of these pairs of amino acid residues are expected to result in functional equivalency in many cases.
  • Amino acid substitutions that are expected to conserve the biological function of the polypeptide would conserve chemical attributes of the substituted amino acid residues such as hydrophobicity, hydrophilicity, side-chain charge, or size.
  • the protein C polypeptide may be only about 80% or more similar, 90% or more similar, 95% or more similar, 97% or more similar, 99% or more similar, or about 100% similar.
  • the codons used may also be adapted for translation in a heterologous host by adopting the codon preferences of the host. This would accommodate the translational machinery of the heterologous host without a substantial change in chemical structure of the polypeptide.
  • Protein C and variants thereof may be used to determine structure-function relationships (e.g., alanine scanning, conservative or noncon- servative amino acid substitution, in vitro evolution).
  • structure-function relationships e.g., alanine scanning, conservative or noncon- servative amino acid substitution, in vitro evolution.
  • protein C folding and processing, secretion, receptor binding, signaling through EPCR, PAR-1 , and/or PAR-3, inhibition of p53 signaling, any of the other biological activities described herein, or combinations thereof may be related to changes in the amino acid sequence. See Wells ⁇ Bio/Technology 13:647-651 , 1995) and U.S. Patent 5,534,617.
  • Directed evolution by directed or random mutagenesis or gene shuffling using protein C may be used to acquire new and improved functions in accordance with selection criteria.
  • Mutant and polymorphic variant polypeptides are encoded by suitable mutant and polymorphic variant polynucleotides.
  • Structure-activity relationships of protein C may be studied (i.e., SAR studies) using variant polypeptides produced with an expression construct transfected in a host cell with or without expressing endogenous protein C.
  • mutations in discrete domains of protein C may be associated with decreasing or even increasing activity in the protein's function.
  • Gale et al. J. Biol. Chem. 277:28836-28840, 2002 have demonstrated that mutations in the surface loops of APC affect its anticoagualant activity. It was shown that APC mutants KKK191/193AAA (loop 37), RR229/230AA (calcium loop), RR306/312AA (autolysis loop), and RKRR306/314AAAA (autolysis loop) have reduced anticoagulant activity as compared to native APC. These APC mutants retain the anti-apoptotic activity of APC.
  • Zymogen protein C the precursor of activated protein C, is readily converted to activated protein C within the body by proteases. Protein C may be considered a prodrug form of activated protein C. Thus, the use of activated protein C is expressly intended to include protein C and variants thereof. Treatments with protein C would require appropriately larger doses known to those of skill in the art.
  • Recombinant forms of protein C can be produced with a selected chemical structure (e.g., native, mutant, or polymorphic).
  • a gene encoding human protein C is described in U.S. Patent 4,775,624 and can be used to produce recombinant human protein C as described in U.S. Patent 4,981 ,952.
  • Human protein C can be recombinantly produced in tissue culture and activated as described in U.S. Patent 6,037,322.
  • Natural human protein C can be purified from plasma, activated, and assayed as described in U.S. Patent 5,084,274. The nucleotide and amino acid sequence disclosed in these patents may be used as a reference for protein C.
  • human protein C or its mutants may be expressed from cognate nucleotide sequences. They may be transcribed using eukaryotic promoter (e.g., SV40 early or human cytomegalovirus immediate early promoter), enhancer (e.g., SV40 or human cytomegalovirus immediate early enhancer), and termination signal (e.g., SV40 late or bovine growth hormone polyadenylation site).
  • eukaryotic promoter e.g., SV40 early or human cytomegalovirus immediate early promoter
  • enhancer e.g., SV40 or human cytomegalovirus immediate early enhancer
  • termination signal e.g., SV40 late or bovine growth hormone polyadenylation site
  • a spliceable intron may also be included.
  • An expression cassette of the preceding paragraph may be transferred in a vector.
  • An integration vector also includes one or more signal(s) for site- specific integration or homologous recombination into the genome of a host cell.
  • An episomal vector also includes an origin of replication for autonomous replication; the episomal vector optionally may be amplified in a suitable host cell (e.g., nucleotide sequence encoding dihydrofolate reductase that is ampli- fiable by methotrexate treatment).
  • the vector may include another expression cassette for a selectable marker (e.g., resistance to gentamicin).
  • the vector may include a prokaryotic origin of replication for autonomous replication (e.g., f1 or permissive CoIEI ) and a prokaryotic selectable marker (e.g., resistance to ampicillin).
  • a prokaryotic origin of replication for autonomous replication e.g., f1 or permissive CoIEI
  • a prokaryotic selectable marker e.g., resistance to ampicillin
  • CHO cells Chinese hamster ovary (CHO) cells are known in the art. For example, they may be obtained from a publicly accessible cell depository (e.g., ATCC CCL-61 TM is the proline-requiring clone designated CHO-K1 ). Host cells may be grown in serum-free or defined medium; host cells may be adapted for suspension culture in a cell reactor (e.g., gas-permeable bag, microcarriers, roller or spinner bottle).
  • a cell reactor e.g., gas-permeable bag, microcarriers, roller or spinner bottle.
  • DHFR dihydrofolate reductase
  • ATCC CRL-9096TM is designated CHO/dhfr- and DXB11 is another well known clone
  • medium e.g., Iscove's modified Dulbecco's medium with 4 mM L-glutamine adjusted to contain 1.5 mg/mL sodium bicarbonate and supplemented by 0.1 mM hypoxanthine, 0.016 mM thymidine, 0.002 mM methotrexate, and 10% fetal bovine serum
  • standard culture conditions e.g., 95% air and 5% CO 2 atmosphere at 37 0 C.
  • the host cell may be infected (e.g., by a virus) or transfected (e.g., by electroporation or with cationic lipids) using an expression vector, and retention of an episomal vector may be achieved by positive selection (e.g., gentamicin).
  • Antibody specific for the recombinant protein may be used to select expressing host cells by panning on culture dishes, adhering to magnetic beads, or sorting by fluorescence.
  • hypoxanthine-thymidine containing (HT) medium may be used to select host cells containing a vector coexpressing recombinant protein and a dihydrofolate reductase (DHFR) gene, which is amplifiable by increasing levels of methotrexate.
  • DHFR dihydrofolate reductase
  • APC, prodrugs, or variants may be isolated from the cell (e.g., intracellular protein) or culture medium (i.e., secreted protein).
  • Recombinant protein may be purified by ion exchange, size exclusion, hydrophobicity interaction, and affinity chromatography. Precipitation and dialysis may be used to change the other chemicals in the composition.
  • Dosages, dosing protocols, and protein C variants that reduce bleeding in a subject as compared to activated protein C which is endogenous to subject are preferred.
  • the cytoprotective activity of protein C may thereby be maintained or increased while decreasing undesirable side effects of the adminis- tration of activated protein C (e.g., bleeding in the brain and other organs).
  • Activated protein C, a prodrug (e.g., zymogen), or a variant thereof may be used to formulate pharmaceutical compositions with one or more of the utilities disclosed herein. They may be administered in vitro to cells in culture, in vivo to cells in the body, or ex vivo to cells outside of a subject which may then be returned to the body of the same subject or another.
  • the cells may be removed from, transplanted into, or be present in the subject (e.g., genetic modification of endothelial cells in vitro and then returning those cells to brain endothelium).
  • the cell may be from the endothelium (e.g., an endothelial or smooth muscle cell), especially from the endothelium of a brain vessel. It may also be a neuron; a glial cell; a precursor, progenitor, or stem cell thereof; or another differentiated cell from the central or peripheral nervous system.
  • compositions which further comprise a pharmaceutically acceptable carrier and compositions which further comprise components useful for delivering the composition to a subject's brain are known in the art. Addition of such carriers and other components to the composition of the invention is well within the level of skill in this art. For example, a permeable material may release its contents to the local area or a tube may direct the contents of a reservoir to a distant location of the brain.
  • a pharmaceutical composition may be administered as a formulation which is adapted for direct application to the central nervous system, or suitable for passage through the gut or blood circulation. Alternatively, pharmaceutical compositions may be added to the culture medium. In addition to active compound, such compositions may contain pharmaceutically-acceptable carriers and other ingredients known to facilitate administration and/or enhance uptake.
  • a unit dose of the composition is an amount of APC or APC mutants which provides neuroprotection, cytoprotection, inhibits apoptosis or cell death, and/or promotes cell survival but does not provide a clinically significant anticoagulant, profibhnolytic, or antithrombotic effect, a therapeutic level of such activity, or has at least reduced activity in comparison to previously described doses of activated protein C. Measurement of such values are within the skill in the art: clinical laboratories routinely determine these values with standard assays and hematologists classify them as normal or abnormal depending on the situation.
  • compositions may be administered by any known route.
  • the composition may be administered by a mucosal, pulmonary, topical, or other localized or systemic route (e.g., enteral and parenteral).
  • a mucosal, pulmonary, topical, or other localized or systemic route e.g., enteral and parenteral
  • achieving an effective amount of activated protein C, prodrug, or functional variant in the central nervous system may be desired. This may involve a depot injection into or surgical implant within the brain.
  • "Parenteral" includes subcutaneous, intradermal, intramuscular, intravenous, intraarterial, intrathecal, and other injection or infusion techniques, without limitation.
  • Suitable choices in amounts and timing of doses, formulation, and routes of administration can be made with the goals of achieving a favorable response in the subject (i.e., efficacy), and avoiding undue toxicity or other harm thereto (i.e., safety).
  • "effective” refers to such choices that involve routine manipulation of conditions to achieve a desired effect (e.g., inhibition of apoptosis or cell death, promotion of cell survival, neuroprotection, cytoprotection, or combinations thereof).
  • “effective amount” refers to the total amount of activated protein C, prodrug (e.g., zymogen protein C) or functional variant that achieves the desired effect.
  • An "equivalent amount" of prodrug or functional variant with reduced anticoagulant activity can be determined by achieving the same or similar desired neuroprotective effect as the reference amount of activated protein C, but with reduced risk for bleeding due to reduced anticoa- gulant activity.
  • a bolus of the formulation administered only once to a subject is a convenient dosing schedule although achieving an effective concentration of activated protein C in the brain may require more frequent administration.
  • Treatment may involve a continuous infusion (e.g., for 3 hr after stroke) or a slow infusion (e.g., for 24 hr to 72 hr when given within 6 hr of stroke). Alternatively, it may be administered every other day, once or several times a week, or once or several times a month.
  • Dosages of active ingredients in a pharmaceutical composition can also be varied so as to achieve a transient or sustained concentration of the compound or derivative thereof in a subject and to result in the desired treatment outcome. But it is also within the skill of the art to start doses at levels lower than required to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved.
  • the amount of compound administered is dependent upon factors such as, for example, bioactivity and bioavailability of the compound (e.g., half-life in the body, stability, and metabolism); chemical properties of the compound (e.g., molecular weight, hydrophobicity, and solubility); route and scheduling of administration; and the like. It will also be understood that the specific dose level to be achieved for any particular subject may depend on a variety of factors, including age, health, medical history, weight, combination with one or more other drugs, and severity of disease.
  • a low dose may be used to prevent apoptosis or cell death and/or to promote cell survival.
  • APCs inhibition of thrombosis, clotting, and/or inflammation which were obtained at higher doses.
  • APCs anti-inflammatory effects are possibly mediated by down regulation of the NF- ⁇ B pathway.
  • homologous systems e.g., human native or recombinant APC administered to patients
  • a single bolus of APC may be sufficient to be directly neuroprotective without having a significant antithrombotic effect in brain circulation.
  • An illustrative amount may be calculated for a 70 kg adult human, and this may be sufficient to treat humans of between 50 kg and 90 kg.
  • the effective or equivalent amount may be packaged in a "unit dose" with written instructions for achieving one or more desired effects and/or avoiding one or more undesired effects.
  • the aforementioned formulations, routes of administration, and dosing schedules are merely illustrative of the techniques which may be used.
  • treatment refers to, inter alia, reducing or alleviating one or more symptoms of neurodegenerative disease.
  • standard therapy such as stroke treatment with a tissue plasminogen activator may be compared with and without activated protein C, a drug, or a variant thereof.
  • improvement in a symptom, its worsening, regression, or progression may be determined by objective or subjective measures.
  • the subject in need of treatment may be at risk for or already affected by neurodegenerative disease; treatment may be initiated before and/or after diagnosis.
  • an indication that treatment is effective may be improved neurological outcome, motor or sensory functions, cognitive functions, psychomotor functions, motor neurologic functions, higher integrative intellectual functions, memory, vision, hearing, etc.; reduced brain damage and injury as evidenced by noninvasive image analysis (e.g., MRI or brain perfusion imaging); or combinations thereof.
  • This effect may be confirmed by neuropathological analysis of brain tissue or diagnostic testing of blood or other biopsy tissue.
  • reduction in a neurodegenerative process by stabilizing brain endothelial cell functions and preventing their death will lead to improvements in the cerebral blood flow (CBF) and normalization of CBF regulatory functions.
  • CBF cerebral blood flow
  • apoptosis or a marker thereof e.g., DNA content and fragmentation
  • increased cell survival decreased cell death, or combinations thereof
  • an increase or decrease may be determined by comparison to treatment with or without activated protein C, a prodrug, or a variant thereof, or to the expected effects of untreated disease.
  • Treatment may also involve other existing modes of treatment and agents (e.g., protein S).
  • agents e.g., protein S.
  • combination treatment may be practiced (e.g., APC and tPA administered concurrently or sequentially).
  • Activated protein C may reduce organ damage in animal (i.e., in vivo) models and cell damage in tissue culture (i.e., in vitro) models of Alzheimer's disease, amyotrophic lateral sclerosis, ischemic injury, sepsis, spinal cord injury, and stroke. It may also reduce mortality in subjects afflicted by disease or progression in those at risk thereof. Neuroprotection can act directly on perturbed neurons to prevent cell injury and apoptosis. Activated protein C may interfere with N-methyl-D-aspartate (NMDA) apoptosis in cultured cortical neurons. Direct intracerebral infusions may significantly reduce NMDA excito- toxic brain lesions.
  • NMDA N-methyl-D-aspartate
  • BEC Human microvascular brain endothelial cells
  • Primary BEC are isolated from rapid (less than 3 hr) autopsies from neurologically normal young individuals after trauma. BEC are characterized and cultured. After FACS sorting using DiI-Ac-LDL, cells are greater than 98% positive for the endothelial markers von Willebrand factor and CD105, and negative for GFAP (astrocytes), CD11b (macrophages/microglia) and ⁇ -actin (smooth muscle cells). Early passage (P3-P5) cells are used for all studies.
  • Hypoxia model Hypoxia/aglycemia is used as an in vitro model of ischemic injury. Briefly, 0.7 x 10 6 BEC are seeded on 100 mm plate in RPM11640 medium supplemented with 20% fetal bovine serum, endothelial cell growth supply (30 ⁇ g/mL, Sigma), and heparin (5 U/mL, Sigma). Twenty-four hours later, the cells are washed twice with PBS and then transferred to serum-free Dulbecco's Modified Eagle Media (DMEM) medium without glucose and exposed to severe hypoxia (less than 2% oxygen) using an anaerobic chamber (Forma Scientific) equipped with a humidified, temperature-controlled incubator.
  • DMEM Dulbecco's Modified Eagle Media
  • the entire system is purged with 95% N 2 /5% CO 2 atmosphere.
  • the oxygen levels in the incubator are monitored by O 2 Fyrite (Forma Scientific).
  • Control BEC are maintained in DMEM supplemented with 20% oxygen and 5 mM glucose. In most studies, experiments are performed at 8 hr of culture when the hypoxic injury is already maximal.
  • LDH lactate dehydrogenase
  • TUNEL staining are performed to determine the apoptosis on acetone fixed cells.
  • Hoechst staining cells are stained for 5 min with 1 ⁇ g/mL of the fluorescent DNA-binding dye Hoechst 33,342 (Sigma). Images are obtained using an Olympus AX70 microscope.
  • In vivo animal model of stroke A modified intravascular MCAO technique is used to induce stroke.
  • Murine recombinant APC or vehicle are administered 10 min after the MCAO via the femoral vein.
  • CBF is monitored by laser Doppler flowmetry (Transonic Systems). The procedure is considered successful if a greater than or equal to 80% drop in CBF is observed immediately after placement of the suture.
  • Arterial blood gasses are measured before and during MCAO. Neurological studies are performed at 24 hr and animals are sacrificed at that time for neuropathological analysis. All animals survive at least 24 hr.
  • Neurological examinations are scored as follows: no neurological deficit (O), failure to extend left forepaw fully (1 ), turning to left (2), circling to left (3), unable to walk spontaneously (4), and stroke-related death (5).
  • Unfixed 1-mm coronal brain slices are incubated in 2% triphenyltetrazolium chloride in phosphate buffer (pH 7.4) and serial coronal sections are displayed on a digitizing video screen (Jandel Scientific). Brain infarct and edema volume are calculated with Swanson correction.
  • Endothelial dysfunction is critical during ischemic injury including ischemic brain damage.
  • APC exerts anti-apoptotic effects during ischemic brain damage.
  • BEC hypoxic brain endothelial cell
  • APC exerts a cytoprotective effect and prevents hypoxic injury.
  • Normoxic cells are rarely TUNEL-positive, while hypoxic BEC are mostly TUNEL-positive and also exhibited chromatin condensation and nuclear shrinkage.
  • the number of TUNEL-positive cells and cells with apoptotic nuclear changes are significantly reduced.
  • the volume of brain injury, change in the cerebral blood flow (CBF), and the deposition of cerebrovascular fibrin and neutrophils are determined after the establishment of MCAO.
  • APC activity can be investigated in models of neuronal injury.
  • APC is directly neuronal protective, its effects on N-methyl-D-aspartate (NMDA)-induced apoptosis in neuronal cultures and on NMDA-induced excito- toxic brain lesions in vivo produced by stereotactic NMDA microinjections into caudate nucleus are studied.
  • NMDA N-methyl-D-aspartate
  • Overstimulation of NMDA receptors is implicated in neurodegeneration in stroke and traumatic brain injury and is associated with neurodegenerative diseases including Alzheimer's disease and Huntington's disease.
  • Neuronal culture Primary neuronal cultures are established by dissecting cerebral cortex from fetal C57BL/6J mice at 16 days of gestation, treating tissue with trypsin for 10 min at 37 0 C, and dissociation cells by trituration. Dissociated cell suspensions are plated at 5 x 10 5 cells per well on 12-well tissue culture plates or at 4 x 10 6 cells per dish on 60 mm tissue culture dishes coated with poly-D-lysine, in serum-free Neurobasal medium plus B27 supplement (Gibco BRL). The medium suppresses glial growth to less than 2% of the total cell population. The absence of astrocytes is confirmed by the lack of glial fibrillary acidic protein staining. Cultures are maintained in a humidified 5% CO 2 incubator at 37 0 C for seven days before treatment. Medium is replaced every three days.
  • NMDA-induced apoptosis in neuronal culture For induction of neuronal apop- tosis, cultures are exposed for 10 min to 300 ⁇ M NMDA/5 ⁇ M glycine in Mg 2+ - free Earle's balanced salt solution (EBSS). Control cultures are exposed to
  • Detection of apoptosis and labeling for in situ DNA fragmentation Apoptotic cells are visualized by in situ terminal deoxynucleotidyl transferase-mediated digoxigenin-dUTP nick-end labeling (TUNEL) assay according to the manufacturer's instructions (Intergen Company). Cells are counterstained with the DNA- binding fluorescent dye, Hoechst 33342 (Molecular Probes) at 1 mg/mL for 10 min at room temperature to reveal nuclear morphology. The number of apop- totic cells is expressed as the percentage of TUNEL-positive cells of the total number of nuclei determined by Hoechst staining.
  • the cells are counted in 10 to 20 random fields (30 x magnification) by two independent observers blinded to experimental conditions. The number of cells under basal conditions (vehicle only) is subtracted from the number of apoptotic cells in control and experimental groups.
  • mice C57BL/6J mice, 23-25 g, male are anesthetized with i.p. ketamine and xylazine. Animals receive microinfusions into the right striatum of either vehicle, NMDA, NMDA + recombinant mouse
  • mice are sacrificed under deep anesthesia for analysis of excitotoxic lesions. Mice are transcardially perfused with PBS followed by 4% paraformaldehyde in 0.1 M of PBS, pH 7.4. The brains are removed and coronal sections at a 30 ⁇ m thickness are prepared using a vibratome. Every fifth section 1 mm anterior and posterior to the site of injection is stained with cresyl violet. The lesion area is identified by the loss of staining as reported. The lesion areas are determined by an image analyzer (Image-
  • APC has an anti-apoptotic effect on NMDA-perturbed cortical neurons.
  • APC IgG abrogated APCs neuroprotective activity.
  • Administration of APC to animals with N DMA-induced brain injury significantly reduces the lesion volume; the effect of APC was dose-dependent similar to the in vitro results.
  • APC mutants which lack wild-type levels of anticoagulant activity may retain normal neuroprotective activity.
  • Human APC protease domain mutants with low anticoagulant activity are assayed for their anti-apoptotic activity using NMDA-perturbed mouse neurons in vitro and in vivo as well as hypoxic human brain endothelial cells in vitro.
  • Two human APC mutants with alanine substitutions in either loop 37 (KKK191-193AAA, "3K3A-APC”) or the Ca ++ -binding loop (RR229/230AA, "229/30-APC”) have neuroprotective activity like native human APC ("rwt-APC"). None of these mutations affected APC amidolytic activity.
  • APC mutants with reduced anticoagulant activity retain wild-type levels of in vitro and in vivo neuroprotective activity, which acts directly on brain cells.
  • Such APC mutants are termed "functional mutants" because they are selectively deficient in APCs anticoagulant activity and therefore may have less risk of bleeding.
  • APC may have direct neuronal protective properties that do not depend on its systemic actions, and APC may prevent neuronal apoptosis by directly acting on perturbed neurons. Thus, APC may limit neuronal damage by preventing NMDA-induced neuronal apoptosis in neurodegenerative disorders associ- ated with over stimulation of NMDA receptors.
  • a transgenic mouse with one or more of the Swedish-type (K670N/M671 L), Dutch-type (E693Q), Iowa-type (D694N), and London-type (V717F) mutations in the APP gene is prepared by injection of pronuclei or transfection of embryonic stem cells.
  • the effect of treatment with APC is assessed by behavioral tests (e.g., hyperactivity, passive avoidance memory, water maze spatial memory acquisition and retention) and development of cerebral amyloid angiopathy, congophilic plaques, neuroritic and synaptic degeneration, and amyloid deposits.
  • a transgenic mouse with a H46R, L84V, and G93A mutations in the Cu/Zn superoxide dismutase (SOD1 ) gene is generated by pronuclear injection or embryonic stem cells transfection.
  • the effect of treatment with APC is assessed by motor activity (e.g., rotor-rod test), mortality, and degeneration of motor neurons.
  • DNA encoding native or mutant 3K3A human protein C was incorporated into the pcDNA3.1 expression vector. After amplifying the inserts and cloning them separately into the same cassette, their nucleotide sequences were verified to be correct.
  • the promoter and enhancer used to transcribe the protein are from the human cytomegalovirus immediate early region; a bovine growth hormone polyadenylation site is used for termination and mRNA processing.
  • Both native and mutant 3K3A human protein C use the native signal peptide of 42 amino acids.
  • CHO-K1 cells were transfected with the expression vector in plasmid form using liposome-mediated uptake and screened by fluorescence activated cell sorting (FACS as described by Brezinsky et al., J. Immunol. Meth. 277:141-155, 2003) to select clones producing high levels of properly processed protein.
  • CHO-K1 cells stably transfected with the expression vector were specifically bound with an anti-PC antibody (C3 for human light chain of protein C under nonreducing conditions as described by Heeb et al., Thromb. Res. 52:33-43, 1988), and then with a fluorescently-labeled secondary antibody to identify the clones expressing human protein C. Secreted human protein C on the surface of transfected cells were detected by such procedures.
  • Protein C undergoes extensive post-translational modification during its biosynthesis as a glycoprotein.
  • protein C is proteolytically processed to remove the 42 amino acid signal sequence, glycosylated at four N-linked glycosylation sites, wholly or partially ⁇ -carboxlyated at nine glutamic acid residues, excision of a furin consensus sequence (LK) to generate biologically functional protein as a disulfide bonded two-chain heterodimer, and beta hydroxylated at Asp 71.
  • LK furin consensus sequence
  • the ratio of the heterodimeric (HD) form to the single chain form of 3K3A was increased from 10%:90% to 40%:60%.
  • a chemically defined, serum free medium e.g., D CHOTM and CD Opti CHOTM
  • D CHOTM and CD Opti CHOTM from Invitrogen/Gibco as well as their chemically defined supplement (AGTTM feed) for the media
  • AGTTM feed chemically defined supplement
  • Growth in CD Opti CHOTM medium with free calcium not only increased overall expression (optimal cell density achieved with excess free calcium and AGTTM feed), but also resulted in a favorable ratio of heterodimer (HD) to single chain (SC) forms in the expression of 3K3A PC.
  • the ratio was estimated to be 85% HD to 15% SC, about that found in circulating plasma (i.e., native) PC.
  • Carbohydrate and sialic acid analysis were performed to compare post- translational modifications of XIGRIS APC recombinantly produced in a human embryonic kidney cell line and 3K3A APC recombinantly produced in a Chinese hamster ovary cell line. Differences between the recombinant human APC were found for both oligosaccharide profiles and sialic acid content.
  • N-linked carbohydrates from each glycoprotein sample were cleaved with N-Glycanase at a ratio of 1 :100 enzyme to substrate using about 100 ⁇ g each of XIGRIS APC and 3K3A APC. After their release by such enzymatic cleavage, glycans were extracted using cold ethanol and brought to dryness by centrifugal concentration. The recovered oligosaccharides were labeled with 2- aminobenzamide in the presence of sodium cyanoborohydride under acidic conditions. Subsequent to such derivatization, excess dye and other unused reagents were removed with a Glycoclean® S sample filtration cartridge.
  • Sialic acid from each glycoprotein sample was hydrolyzed with 0.5 M sodium bisulfite at 80 0 C. After its release by such hydrolysis, sialic acid was labeled with orthophenylenediamine at 80 0 C. Chromatographic peaks corresponding to sialic acid (glycolyl and acetyl) were integrated, and results were compared to a standard curve with linear range from 1.20 to 28.7 nmoles of sialic acid based on peak retention times. The ratio of sialic acid to protein (moles of sialic acid/moles of protein) was 8.1 for XIGRIS APC and 8.8 for 3K3A APC.
  • ELISA enzyme linked immunosorbent assay
  • Amidolytic Assay Amidolytic activity of 3K3A is determined by the hydrolysis rate of APC acting on the chromogenic substrate S-2366. An aliquot of APC was added to the chromogenic substrate S-2366 in TBS (pH 7.4). Hydrolysis of the substrate is monitored by the change in absorbance at 405 nm over 10 min. A standard curve of initial rate versus APC concentration was constructed, and the amidolytic activity of sample was derived in relation to the standard curve.
  • APTT Activated Partial Thromboplastin Time
  • 3K3A has lower antico- agulating activity compared to native PC. This assay was used to measure the anticoagulant activity of 3K3A.
  • PC deficient plasma was incubated with kaolin/ cephalin at 37°C, followed by the addition of APC. After incubation for 3 min, clotting was initiated by adding CaCI 2 and recorded using an START4 coagulo- meter.
  • BEC Human brain endothelial cells
  • Plasma derived human APC i.e., native
  • 3K3A APC were added into the culture medium well before oxygen/ glucose deprivation (OGD) treatment.
  • LDH release was measured with a kit using the manufacturer's instructions (Sigma, TOX-7).
  • ANOVA analysis of variance
  • Tukey post hoc test was used to compare the treatment effects of ordinal data between groups.
  • the neuroprotective potential of recombinant human 3K3A APC was evaluated using a middle cerebral artery occlusion (MCAO) induced transient ischemic stroke model in mouse. All procedures were conducted accordance to the National Institutes of Health's guidelines and approved by the Animal Care
  • mice Male C57BL/6 mice weighing 27-29 gm (Jackson Laboratory) were housed in a room at a temperature of 22 0 C with 12 hr dark and light cycles. Standard rat chow pellets and water were allowed ad libitum. Transient ischemia was induced according to the MCAO method. Mice were anesthetized with 100 mg/kg ketamine and 10 mg/kg xylazine. A midline incision was made in the ventral neck to expose the right common carotid artery. The external carotid artery was ligatured and dissected.
  • a 6-0 proline monofilament was advanced up to 9 mm from the lumen of external carotid artery into the internal carotid artery to block the right middle cerebral artery (MCA) for a period of 1 hr.
  • MCA right middle cerebral artery
  • the filament was pulled out completely.
  • the mice were killed and their brains were isolated to estimate infarct volume and brain swelling.
  • Coronal brain sections (1 mm thick) were cut and stained with 2% triphenyl tetrazolium chloride (TTC) solution in PBS for 5 min at 37°C. After fixing in 4% buffered formaldehyde overnight, sections were photographed using a digital camera (Sony DSC-S70). The area of infarction was measured in coronal brain sections by using Image pro plus image analysis software. Infarct areas of all sections were cumulated to calculate the total infarct area, and then multiplied by thickness of brain sections to calculate the volume of infarction. The volumes of both hemispheres were calculated separately, and brain swelling was calculated by subtracting the contralateral volume from the ipsilateral volume.
  • TTC triphenyl tetrazolium chloride
  • volume correction (infarct volume x contralateral volume) / ipsilateral volume.
  • Injury volume was calculated by adding the swelling volume to the volume of infarction. Unpaired student's t-test was used to compare two groups. A difference across the groups was considered statistically significant if p ⁇ 0.05.
  • mice were randomly divided into two groups. Vehicle (normal saline) or 3K3A APC (2 mg/kg, i.v.) treatment was done 5 min prior to MCAO. They were administered intravenously via the left femoral vein as a bolus injection. CHO produced human 3K3A APC produced a statistically significant decrease in infarction, swelling, injury, and neurological deficit score as compared to vehicle treated group when administered to treat a model of ischemic stroke. Recombi- nant human 3K3A APC is neuroprotective when administered to mice with MCAO induced transient ischemic stroke.
  • Vehicle normal saline
  • 3K3A APC 2 mg/kg, i.v.
  • EXAMPLE 9 EAhy926 endothelial cells were grown to confluency on a gelatin coated glass coverslip, and then incubated with 25 nM XIGRIS APC or CHO produced 3K3A APC (lot C). Cells were pretreated with APC for 2 hr prior to apoptosis induction by addition of 2 ⁇ M staurosporine for 4 hr as described by Mosnier et al. (Blood 104:1744-1740, 2004) with modifications. Cells were counted in 10 random fields at 2Ox magnification. The number of apoptotic (i.e., caspase-3 positive) cells was expressed as a percentage of the total number of nuclei. Our results indicate that 3K3A APC produced by CHO cells exhibits > 90% protection in this model. The protection is somewhat better than XIGRIS APC when used at the same concentration.
  • N2a Mouse neuroblastoma cells (N2a; ATTC CCL131 ) stably transfected with human SOD protein (G37R and G85R mutants) were obtained from Dr. Cleveland at UC San Diego. N2a cells were maintained in DMEM (high glucose with L-glutamine, Invitrogen 11995-065) supplemented with 100 U/ml penicillin and streptomycin, 10% fetal bovine serum, and 400 ⁇ g/ml geneticin G418. Differentiation was induced by replacing the media with low serum media (0.25% FBS) and adding 5 mM N 6 , 2'-O-dibutyryladenosine 3',5'-cyclic monophosphate sodium (db-cAMP) for three days.
  • DMEM high glucose with L-glutamine, Invitrogen 11995-065
  • fetal bovine serum 10% fetal bovine serum
  • db-cAMP 2'-O-dibutyryladenosine 3',5
  • Oxidative stress was induced by adding 100 ⁇ M xanthine (X) and 10 mU/ml xanthine oxidase (XO) for 4 hr. Meanwhile, 3K3A-APC was added. Cell viability was determined using a highly water-soluble tetrazolium salt 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5- (2,4-disulfophenyl)-2H-terazolium, monosodium salt (Donjido Molecular Technologies, WST-8) and the survival rate was represented as the viability percentage of non-treated cells.

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Abstract

La présente invention concerne une protéine C (par exemple, l'APC humaine) activée, un promédicament (par exemple, du zymogène) et/ou un variant qui présentent un état de glycosylation et/ou une teneur en acide sialique modifiés en raison du procédé de production. Ces substances peuvent être utilisées en tant qu'agents neuroprotecteurs ou cytoprotecteurs, ou pour le traitement d'une maladie neurodégénérative.
PCT/US2007/022180 2006-10-18 2007-10-18 Utilisation de la protéine c humaine présentant un état de glycosylation et une teneur en acide sialique modifiés en tant que médicament WO2008048646A1 (fr)

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WO2023119230A1 (fr) 2021-12-22 2023-06-29 L'oreal Compositions de modulation de la voie de coagulation et de la voie de nicotinamide-adénine dinucléotide et leurs procédés d'utilisation

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US5047335A (en) * 1988-12-21 1991-09-10 The Regents Of The University Of Calif. Process for controlling intracellular glycosylation of proteins
US5460953A (en) * 1990-02-23 1995-10-24 Eli Lilly And Company Vectors and compounds for expression of glycosylation mutants of human protein C
US5550036A (en) * 1986-04-09 1996-08-27 Eli Lilly And Company Method for co-amplification of human protein C genes in human cells
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US5047335A (en) * 1988-12-21 1991-09-10 The Regents Of The University Of Calif. Process for controlling intracellular glycosylation of proteins
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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2023119230A1 (fr) 2021-12-22 2023-06-29 L'oreal Compositions de modulation de la voie de coagulation et de la voie de nicotinamide-adénine dinucléotide et leurs procédés d'utilisation

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