WO2013013613A1 - G-csf二聚体在制备治疗神经退行性疾病药物中的应用 - Google Patents

G-csf二聚体在制备治疗神经退行性疾病药物中的应用 Download PDF

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WO2013013613A1
WO2013013613A1 PCT/CN2012/079106 CN2012079106W WO2013013613A1 WO 2013013613 A1 WO2013013613 A1 WO 2013013613A1 CN 2012079106 W CN2012079106 W CN 2012079106W WO 2013013613 A1 WO2013013613 A1 WO 2013013613A1
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Prior art keywords
csf
dimer
monomer
disease
stimulating factor
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PCT/CN2012/079106
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English (en)
French (fr)
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武栋栋
黄智华
黄予良
严孝强
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健能隆医药技术（上海）有限公司
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Priority to CN201280036654.9A priority Critical patent/CN103732240B/zh
Priority to US14/233,739 priority patent/US9642917B2/en
Priority to CA2842969A priority patent/CA2842969C/en
Priority to AU2012289433A priority patent/AU2012289433B2/en
Priority to JP2014521926A priority patent/JP5947891B2/ja
Priority to EP12817542.9A priority patent/EP2737905B1/en
Publication of WO2013013613A1 publication Critical patent/WO2013013613A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A61K47/642—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent the peptide or protein in the drug conjugate being a cytokine, e.g. IL2, chemokine, growth factors or interferons being the inactive part of the conjugate
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P21/00—Drugs for disorders of the muscular or neuromuscular 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
    • A61P25/14—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
    • A61P25/28—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

Definitions

  • G-CSF dimer in preparing medicine for treating neurodegenerative diseases
  • the invention relates to the field of biological and medical technology. More specifically, the present invention relates to a novel G-CSF dimer and its use in the treatment of neurodegenerative diseases. Background technique
  • G-CSF Human granulocyte colony stimulating factor
  • monocytes fibroblasts and endothelial cells.
  • G-CSF plays three major biological functions in the body: 1. It acts on neutrophil precursor cells and bone marrow stem cells. Drives the differentiation, proliferation and maturation of neutrophils. 2. Activate mature neutrophils to participate in the immune response. 3. Synergize with other blood growth factors such as Stem Cel l Factor, Flt_3 ligand, and GM-CSF to exert hematopoietic function.
  • the receptor for G-CSF has been confirmed mainly in the bone marrow hematopoietic stem cell Sca + Lin-Th l lOT , the precursor cell CD34 + , and the differentiated neutrophil precursor cells (commi tted granulocyte precursor) Neutrophils.
  • the human neutrophil-stimulating growth factor receptor (G-CSF Receptor, G-CSFR) is a single-stranded, high-affinity specific receptor for G-CSF containing 812 amino acids.
  • G-CSF G-CSFR complex It exists in a 2:2 ratio, that is, a state in which two ligands and two receptors are combined.
  • Each G-CSF molecule binds to one receptor molecule, and when both receptors bind to the G-CSF ligand and interact close to each other to form a 2:2 dimer, the carboxyl terminal of the G-CSF receptor
  • JAK2 Janus tyros ine kinase
  • Neurodegenerative disease is a state of neuronal cell loss in the brain and spinal cord. It is a chronic, progressive neurological disease, including Alzheimer's disease, Alzheimer's disease, AD, and Parkinson's disease. (Parkinson's disease, PD), Huntington disease, amyotrophic lateral sclerosis, spinal muscular atro hy, spinal cerebellum Spinal cerebellar ataxias, etc., which are characterized by neurodegenerative diseases and apoptosis, leading to abnormal behavior and dysfunction of patients, causing premature death of neurological diseases. The pathogenesis of neurodegenerative diseases is still unclear, and there are still no effective methods and drugs to prevent and treat.
  • Existing treatment methods include treating PD by taking or intravenously supplementing human brain neuron deficiency, such as levodopa, but , levodopa does not effectively control the natural pathological process of PD, can not affect the rate of degeneration of dopaminergic neurons, and has adverse side effects, such as switching and dyskinesia, its therapeutic effect can only last for about two years, long-term Use can also damage neurons, plus neuronal apoptosis; for acetylcholine deficiency in AD brain, increase its concentration with cholinesterase inhibitors, this method also can not control the development of the disease.
  • GDNF neurotrophic factor
  • the object of the present invention is a more effective drug for treating neurodegenerative diseases, a preparation method thereof and use thereof.
  • a dimer of colony stimulating factor G-CSF for the preparation of a composition for the treatment or prevention of a neurodegenerative disease.
  • the neurodegenerative disease is selected from the group consisting of: Parkinson's disease, Alzheimer's disease, Huntington's disease, amyotrophic lateral sclerosis, spinal muscular atrophy, primary lateral sclerosis, spinocerebellar ataxia .
  • the dimer of the colony stimulating factor G-CSF is a human colony stimulating factor G-CSF dimer.
  • the structure of the colony stimulating factor G-CSF dimer is as shown in Formula I:
  • Ml is the first monomer of human colony stimulating factor G-CSF
  • M2 is the second monomer of human colony stimulating factor G-CSF
  • L is a joint member between the first monomer and the second monomer that connects the first monomer and the second monomer together, wherein, the G-CSF dimer retains the biological activity of G-CSF, and its serum half-life is more than twice the serum half-life of the first monomer or the second monomer.
  • joint element L is selected from the group consisting of:
  • Y is a carrier protein
  • Z is a short peptide of no, or 1-30 amino acids
  • "-" is a chemical bond or a covalent bond, and more preferably, "-" is a peptide bond.
  • amino acid sequence of the dimer of the human colony stimulating factor G-CSF is as shown in SEQ ID NO: 1.
  • first monomer and the second monomer are the same.
  • amino acid sequences of the first monomer and the second monomer are shown in SEQ ID NO: 8.
  • the amino acid of the dimer of the human colony stimulating factor G-CSF is composed of two G-CSF-Fc complexes.
  • the G-CSF-Fc complex is a G-CSF monomer having an Fc fragment).
  • the amino acid sequence of the G-CSF-Fc complex is as shown in SEQ ID NOS: 2 to 7.
  • the dimer of the colony stimulating factor G-CSF is prepared by the following steps:
  • the G-CSF dimer comprises two G-CSF-Fc complexes, and the amino acid sequence of the G-CSF-Fc complex is as shown in SEQ ID NOS: 2 to 7.
  • the composition is a pharmaceutical composition, a health care product composition or a food composition.
  • the pharmaceutical composition is a solid preparation or a liquid preparation.
  • the pharmaceutical composition contains: 0.01 to 99% by weight of a colony stimulating factor G-CSF dimer and a pharmaceutically acceptable excipient or carrier.
  • the excipient or carrier is selected from the group consisting of: cellulose and its derivatives, gelatin, talc, solid lubricants, calcium sulfate, vegetable oils, polyols, emulsifiers, wetting agents, colorants, flavoring agents, stabilizers, Antioxidant, preservative, pyrogen-free water.
  • the G-CSF dimer-containing drug is used to increase the concentration of dopamine in the striatum; to prevent loss of dopaminergic nerve fibers; and/or to prevent the loss of dopaminergic neurons.
  • a medicament for treating a neurodegenerative disease comprising a colony stimulating factor G-CSF dimer as an active ingredient.
  • the neurodegenerative disease is selected from the group consisting of: Parkinson's disease, Alzheimer's disease, Huntington's disease, amyotrophic lateral sclerosis, spinal muscular atrophy, primary lateral sclerosis, spinocerebellar ataxia .
  • the dimer of the colony stimulating factor G-CSF is a human colony stimulating factor G-CSF dimer.
  • the medicament comprises: 0.01 - 99% by weight of a human colony stimulating factor G-CSF dimer and the balance of a pharmaceutically acceptable excipient or carrier.
  • the structure of the colony stimulating factor G-CSF dimer is as shown in formula I:
  • Ml is the first monomer of human colony stimulating factor G-CSF
  • M2 is the second monomer of human colony stimulating factor G-CSF
  • L is a joint member between the first monomer and the second monomer that connects the first monomer and the second monomer together
  • the G-CSF dimer retains the biological activity of G-CSF and has a serum half-life of more than twice the serum half-life of the first monomer or the second monomer.
  • a method of treating a neurodegenerative disease comprising the step of: administering a dimer of colony stimulating factor G-CSF to a subject in need of treatment.
  • the neurodegenerative disease is selected from the group consisting of: Parkinson's disease, Alzheimer's disease, Huntington's disease, amyotrophic lateral sclerosis, spinal muscular atrophy, primary lateral sclerosis, spinocerebellar ataxia .
  • the subject comprises a mammal (e.g., a human).
  • G-CSF dimer molecules in the present invention can significantly increase the concentration of dopamine in the striatum of PD model animals, significantly inhibit the loss of dopaminergic nerve fibers, significantly increase the number of dopaminergic neurons in the PD model animals, and increase dopamine.
  • the function of neurons can significantly improve the learning and memory ability of AD model rats, protect neurons, reduce neuronal cell apoptosis in hippocampus, and alleviate the symptoms of dementia.
  • the dimer G-CSF of the present invention has a prolonged serum half-life and effectively prevents the loss of neurons, thereby enabling more effective treatment of neurodegenerative diseases.
  • Figure 1 shows a schematic representation of a G-CSF dimer of the invention. Where "-" indicates the linker peptide, G-CSF The ellipse represents the G-CSF monomer.
  • FIGS. 2a and 2b show schematic views of a G-CSF dimer of the invention.
  • "-" indicates an amino acid linker peptide
  • G-CSF oval indicates a G-CSF monomer
  • an oval labeled "C” in Fig. 2a indicates a carrier protein
  • G-CSF is located at the N-terminus of the carrier protein, It is shown in 2b that two Fc are paired by a disulfide bond.
  • FIGs 3a and 3b show schematic diagrams of a G-CSF dimer of the invention. Wherein, "-" indicates an amino acid linker peptide, and G-CSF oval indicates a G-CSF monomer.
  • the oval labeled "C” in Fig. 3a indicates a carrier protein, and G-CSF is located at the C-terminus of the carrier protein. It is shown in 3b that the two Fc are paired by a disulfide bond.
  • Figure 4 shows the change in mouse striatum dopamine concentration.
  • Figure 5A shows a picture of immunohistochemical staining of mouse striatum TH-positive nerve fibers.
  • Figure 5B shows immunohistochemical densitometry values of mouse striatum TH positive nerve fibers.
  • Figure 6A shows immunohistochemical staining of TH-positive neurons in the substantia nigra pars compacta of mice.
  • Figure 6B shows the results of TH positive cell count analysis in the substantia nigra parsing of mice.
  • the inventors have prepared a G-CSF dimer for the first time through extensive and intensive research, and unexpectedly found that: by the G-CSF dimer of the present invention, the half-life in vivo can be prolonged, the kinetics of the drug can be improved, and the injection can be reduced. Frequency; significantly enhances drug activity in the body, relieves symptoms of neurodegenerative diseases, and promotes recovery of neurodegenerative diseases. On the basis of this, the present invention has been completed. G-CSF dimer
  • the structure of the G-CSF dimer of the present invention is as shown in Formula I.
  • Ml is the first monomer of human colony stimulating factor G-CSF
  • M2 is the second monomer of human colony stimulating factor G-CSF
  • L is a joint member between the first monomer and the second monomer that connects the first monomer and the second monomer together
  • the G-CSF dimer retains the biological activity of G-CSF and has a serum half-life of more than twice the serum half-life of the first monomer or the second monomer.
  • the biological activity includes:
  • first monomer and the second monomer are the same, the first monomer and the second monomer
  • amino acid sequence is shown in SEQ ID NO: 8.
  • joint element L is selected from the group consisting of:
  • Y is a carrier protein
  • Z is a short peptide of no, or 1-30 amino acids
  • carrier proteins include (but are not limited to): human
  • Fc fragment of IgG(l, 2,3,4), or human albumin (albumin) Fc fragment of IgG(l, 2,3,4), or human albumin (albumin)
  • G-CSF is located at the C-terminus of the available carrier protein and may also be located at the N-terminus of the carrier protein.
  • linker refers to a short peptide that functions between a G-CSF monomer and a G-CSF monomer.
  • the length of the linker peptide is not particularly limited.
  • the linker peptide is typically 5-50 amino acids in length. In general, the linker peptide does not affect or significantly affect the formation of the correct folding and spatial conformation of the G-CSF monomer and the G-CSF monomer.
  • Some examples of linked peptides include (but are not limited to):
  • the linker peptide has an amino acid sequence selected from the group consisting of:
  • a hydrophobic amino acid Gly and Pro consisting of a 3 - 15 amino acid sequence, such as Gly-Pro-Gly-Pro-Gly-Pro; or a 3-20 amino acid sequence consisting of Gly and Ser, such as GSGG.
  • the amino acid sequence encoded by the multiple cloning site is usually 5-20, preferably 10-20 amino acids, examples of which include (but are not limited to): TGLQPTRGIDDITSPVD;
  • an amino acid sequence derived from a protein other than the G-CSF monomer such as an amino acid sequence derived from IgG or albumin;
  • a preferred linker peptide is GSGGGSGGGGSGGGGS (positions 175-190 in SEQ ID NO: 1).
  • a preferred linker peptide is ASTKGP (positions 175-180 in SEQ ID NO: 4).
  • amino acid sequences which do not affect the activity of the G-CSF monomer may be added to the N-terminus or C-terminus of the fusion protein.
  • these added amino acid sequences facilitate expression (eg, signal peptides) and facilitate purification (eg, 6 X His sequence, Saccharomyces cerevisiae alpha-factor signal peptide cleavage site (Glu-Lys-Arg), or may facilitate fusion Protein activity.
  • SEQ ID NO: 1 is a sequence of G-CSF dimer, the structure is shown in Figure 1, wherein the 1-174th position is a G-CSF monomer, and the 175th-190th position is a linker peptide, 191-364 The position is another G-CSF monomer.
  • SEQ ID NO: 2 is the sequence of the G-CSF monomer carrying the Fc fragment constituting the G-CSF dimer, wherein the 1-174 position is a G-CSF monomer, and the 175-190 position is a linker peptide,
  • the 191-418 position is an Fc fragment of human IgG2.
  • two G-CSF monomers bearing an Fc fragment form a dimer by disulfide pairing of Fc.
  • SEQ ID NO: 3 is a sequence of a G-CSF monomer having an Fc fragment constituting a G-CSF dimer, wherein positions 245-418 are G-CSF monomers, and positions 229-244 are linker peptides, The 1 -228 position is an Fc fragment of human IgG2. Two G-CSF monomers carrying an Fc fragment form a dimer by the disulfide pairing of Fc.
  • SEQ ID NO: 4 is the sequence of the G-CSF monomer carrying the Fc fragment constituting the G-CSF dimer, wherein the 1-174 position is a G-CSF monomer, and the 175-180 position is a linker peptide, 181-403 is an Fc fragment of human IgG2.
  • Two G-CSF monomers bearing an Fc fragment form a dimer by the disulfide pairing of Fc.
  • SEQ ID NO: 5 is a sequence of a G-CSF monomer having an Fc fragment constituting a G-CSF dimer, wherein
  • Positions 230-403 are G-CSF, positions 224-229 are linker peptides, and positions 1 -223 are Fc fragments of human IgG2.
  • Two G-CSF monomers bearing an Fc fragment form a dimer by the disulfide pairing of Fc.
  • SEQ ID NO: 6 is the sequence of the G-CSF monomer carrying the Fc fragment constituting the G-CSF dimer, wherein the 1-172 position is G-CSF, and the 175-190 position is a linker peptide, 191- Position 413 is an Fc fragment of human IgG2. Two G-CSF monomers bearing an Fc fragment form a dimer by the disulfide pairing of Fc.
  • SEQ ID NO: 7 is the sequence of the G-CSF monomer carrying the Fc fragment constituting the G-CSF dimer, wherein the positions 240-413 are G-CSF, and the positions 224-239 are the linker peptide, the first - The 223 position is an Fc fragment of human IgG2.
  • Two G-CSF monomers bearing an Fc fragment form a dimer by the disulfide pairing of Fc.
  • SEQ ID NO: 9 is the cDNA sequence of SEQ ID NO: 2.
  • SEQ ID NO: 10 is the cDNA sequence of SEQ ID NO: 6.
  • the G-CSF dimer provided by the present invention has the same molar G-CSF molecular concentration (G-CSF dimer concentration is 0.1-1000 ng/mL, preferably 1-1 ng/mL; G-CSF The monomer concentration is 0.04 ⁇ 400ng/ml, preferably 0.4 ⁇ 40 ng/mL.
  • the protective effect on MPP+ induced PC 12 cells is more obvious; the stronger activated dopaminergic neurons
  • the biological activity of STAT3 can significantly improve MPTP-induced animal behavioral abnormalities; can significantly increase MPTP-induced concentration of dopamine in animal striatum; can significantly inhibit MPTP-induced massive loss of dopaminergic neurons; with stronger activation
  • the DNA sequence encoding the G-CSF dimer or fusion protein of the present invention may be all artificially synthesized.
  • the coding DNA sequences of the G-CSF first monomer and/or the G-CSF second monomer can also be obtained by PCR amplification or synthesis, and then spliced together to form a DNA sequence encoding the fusion protein of the present invention.
  • the G-CSF dimer coding sequence can be engineered, for example, using host cell-preferred codons to eliminate sequences that are detrimental to gene transcription and translation.
  • a G-CSF dimer gene is detected using a mammalian cell-preferred codon, and a computer DNA software is used to exclude a gene that is not conducive to gene transcription and translation, including a sub-shearing site, a transcription termination sequence, and the like.
  • the DNA sequence encoding the novel fusion protein of the present invention After obtaining the DNA sequence encoding the novel fusion protein of the present invention, it is ligated into a suitable expression vector and transferred to a suitable host cell. Finally, the transformed host cells are cultured, and the novel fusion protein of the present invention is obtained by separation and purification.
  • vector as used herein includes plasmids, cosmids, expression vectors, cloning vectors, viral vectors and the like.
  • various carriers known in the art such as commercially available carriers can be used.
  • a commercially available vector is selected, and then a nucleotide sequence encoding a novel fusion protein of the present invention is operably linked to an expression control sequence to form a protein expression vector.
  • operably linked refers to a condition in which portions of a linear DNA sequence are capable of affecting the activity of other portions of the same linear DNA sequence. For example, if the signal peptide DNA is expressed as a precursor and is involved in the secretion of the polypeptide, then the signal peptide (secretion leader sequence) DNA is operably linked to the polypeptide DNA; if the promoter controls the transcription of the sequence, then it is operably linked to A coding sequence; if the ribosome binding site is placed at a position that enables translation, then it is operably linked to the coding sequence.
  • “operably linked to” means adjacent, and for secretory leader sequences means adjacent in the reading frame.
  • the term "host cell” includes prokaryotic cells and eukaryotic cells.
  • prokaryotic host cells include Escherichia coli, Bacillus subtilis and the like.
  • eukaryotic host cells include yeast cells, insect cells, and mammalian cells.
  • the host cell is a eukaryotic cell, more preferably a mammalian cell.
  • the cell After obtaining the transformed host cell, the cell can be cultured under conditions suitable for expression of the fusion protein of the present invention, thereby expressing the fusion protein.
  • the expressed fusion protein is then isolated.
  • the method for preparing a G-CSF dimer of the present invention comprises:
  • G-CSF dimer comprises two G-CSF-Fc complexes, and the amino acid sequence of the G-CSF-Fc complex is as shown in SEQ ID NOS: 2 to 7.
  • the G-CSF dimer of the present invention has an excellent serum half-life
  • the pharmaceutical composition of the present invention wherein the G-CSF dimer is the main active ingredient can be used for treating a nerve damage disease; and protecting neurons.
  • the neurological injury disease is selected from the group consisting of Parkinson's disease (PD), Alzheimer's disease (AD), Huntington's disease (HD), amyotrophic lateral sclerosis (ALS), and spinal cord muscle.
  • PD Parkinson's disease
  • AD Alzheimer's disease
  • HD Huntington's disease
  • ALS amyotrophic lateral sclerosis
  • spinal cord muscle spinal cord muscle.
  • the diseases mentioned in the present invention include those diseases with neurodegenerative diseases, and although the neurological diseases can be caused by various causes and can occur in various sites or nerves, the damaged nerves caused by the therapeutic drugs of the present invention are themselves Improvements in repair and function suggest that the therapeutic agents of the present invention are effective in treating and ameliorating different types of neurodegenerative diseases.
  • the neurological diseases referred to in the present invention include, but are not limited to, Parkinson's disease (PD), Alzheimer's disease (AD), Huntington's disease (HD), amyotrophic lateral sclerosis (ALS), and spinal cord muscle.
  • SMA Atrophic disease
  • PLS primary lateral sclerosis
  • SCA spinocerebellar ataxia
  • SMA spinal muscular atrophy
  • SMON Friedreich Disorders
  • amyloidosis amyloidosis
  • SMON subacute spinal optic neuropathy
  • the therapeutic agent of the present invention is medically administered to a suitable site of administration by a suitable method of administration, the choice of site of administration being dependent on the condition being treated and the condition being treated.
  • a suitable method of administration for those diseases mainly associated with degenerative brain, the therapeutic drug of the present invention is administered to the brain; for those diseases with focal striatum degeneration, the drug can be administered to the striatum, for those A disease with systemic neurodegenerative disease that can be administered systemically.
  • a preferred mode of administration is a suitable method such as injection.
  • the medicament of the present invention is administered to a site where neurodegeneration occurs, as well as arterial, intravenous or subcutaneous injection.
  • compositions of the present invention comprise a G-CSF dimer of the invention in a safe and effective amount and a pharmaceutically acceptable excipient or carrier.
  • safe, effective amount means: The amount of the compound is sufficient to significantly improve the condition, rather than causing serious side effects.
  • the safe and effective amount is determined according to the specific conditions such as the age, condition, and course of treatment of the subject.
  • the pharmaceutical composition contains 1 to 1000 mg of the G-CSF dimer/agent of the present invention, preferably 0.05 to 300 mg of the G-CSF dimer/agent of the present invention, and more preferably 0.3 to 200 mg of the present invention G- CSF dimer/agent.
  • “Pharmacologically acceptable excipient or carrier” means: one or more compatible solid or liquid fillers or gels which are suitable for human use and which must be of sufficient purity and of sufficiently low toxicity .
  • “compatibility” it is meant herein that the components of the composition are capable of intermixing with the compounds of the invention and with each other without significantly reducing the efficacy of the compound.
  • Examples of pharmaceutically acceptable excipients or carriers are cellulose and its derivatives (such as sodium carboxymethylcellulose, sodium ethylcellulose, cellulose acetate, etc.), gelatin, talc, solid lubricants (such as stearic acid, magnesium stearate), calcium sulfate, vegetable oils (such as soybean oil, sesame oil, peanut oil, olive oil, etc.), polyols (such as propylene glycol, glycerin, mannitol, sorbitol, etc.), emulsifiers (such as Tween) ®), wetting agents (such as sodium lauryl sulfate), colorants, flavoring agents, stabilizers, antioxidants, preservatives, pyrogen-free water, etc.
  • cellulose and its derivatives such as sodium carboxymethylcellulose, sodium ethylcellulose, cellulose acetate, etc.
  • gelatin such as sodium carboxymethylcellulose, sodium ethylcellulose, cellulose acetate, etc.
  • the G-CSF dimer of the present invention When administered, it can be administered orally, rectally, parenterally (intravenously, intramuscularly or subcutaneously), Partial administration.
  • Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules.
  • the active compound is mixed with at least one conventional inert excipient (or carrier), such as sodium citrate or dicalcium phosphate, or mixed with: (a) a filler or compatibilizer, for example, Starch, lactose, sucrose, glucose, mannitol and silicic acid; (b) binders, for example, hydroxymethylcellulose, alginate, gelatin, polyvinylpyrrolidone, sucrose and acacia; (c) humectants, For example, glycerin; (d) a disintegrant, for example, agar, calcium carbonate, potato starch or tapioca starch, alginic acid, certain complex silicates, and sodium carbonate; (e) a slow solvent such as paraffin; (f) Absorbing accelerators, for example, quaternary amine compounds; (g) wetting agents, such as cetyl alcohol and glyceryl monostearate; (h) adsorbents, for example, sodium citrate
  • the dosage form may also contain a buffer.
  • Solid dosage forms such as tablets, troches, capsules, pills, and granules can be prepared with coatings and shells such as enteric coatings and other materials known in the art. They may contain opacifying agents and the release of the active compound or compound in such compositions may be released in a portion of the digestive tract in a delayed manner. Examples of embedding components that can be employed are polymeric and waxy materials. If necessary, the active compound may also be in microencapsulated form with one or more of the above-mentioned excipients.
  • Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups or elixirs.
  • the liquid dosage form may contain inert diluents conventionally employed in the art, such as water or other solvents, solubilizers and emulsifiers, for example, ethanol, isopropanol, ethyl carbonate, ethyl acetate, propylene glycol, 1 , 3-butanediol, dimethylformamide and oils, especially cottonseed oil, peanut oil, corn germ oil, olive oil, castor oil and sesame oil or a mixture of these substances.
  • inert diluents conventionally employed in the art, such as water or other solvents, solubilizers and emulsifiers, for example, ethanol, isopropanol, ethyl carbonate, ethyl acetate, propylene glycol, 1 , 3-butanediol, dimethyl
  • compositions may contain adjuvants such as wetting agents, emulsifying and suspending agents, sweetening agents, flavoring agents, and flavoring agents.
  • adjuvants such as wetting agents, emulsifying and suspending agents, sweetening agents, flavoring agents, and flavoring agents.
  • the suspension may contain a suspending agent, for example, ethoxylated isostearyl alcohol, polyoxyethylene sorbitol and sorbitan ester, microcrystalline cellulose, aluminum methoxide and agar or a mixture of these and the like.
  • a suspending agent for example, ethoxylated isostearyl alcohol, polyoxyethylene sorbitol and sorbitan ester, microcrystalline cellulose, aluminum methoxide and agar or a mixture of these and the like.
  • compositions for parenteral injection may comprise a physiologically acceptable sterile aqueous or nonaqueous solution, dispersion, suspension or emulsion, and sterile powder for reconstitution into sterile injectable solutions or dispersions.
  • Suitable aqueous and nonaqueous vehicles, diluents, solvents or vehicles include water, ethanol, polyols and suitable mixtures thereof.
  • Dosage forms of the G-CSF dimer of the present invention for topical administration include ointments, powders, patches, propellants and inhalants.
  • the active ingredient is mixed under sterile conditions with a physiologically acceptable carrier and any preservatives, buffers, or, if necessary, propellants.
  • the G-CSF dimer of the present invention may be administered alone or in combination with other pharmaceutically acceptable compounds.
  • a pharmaceutical composition e.g., a human
  • the dose at the time of administration is a pharmaceutically effective effective dose for 60 kg of body weight. 5 ⁇ 100mg ⁇
  • the dosage is usually 0. 01 ⁇ 300mg, preferably 0. 5 ⁇ 100mg.
  • specific doses should also consider factors such as the route of administration, the health of the patient, etc., which are within the skill of the skilled physician.
  • the main advantages of the invention are:
  • the G-CSF dimer of the present invention consists of SEQ ID NO: 1, or a G-CSF-Fc complex selected from SEQ ID NOs: 2-7 forms a dimer as shown in Figures 1-3.
  • the preparation method is as follows:
  • the gene sequence of the human G-CSF monomer is ligated to the Fc gene fragment of IgG2 using the cDNA sequence of the whole gene synthesis G-CSF-Fc complex (as shown in SEQ ID NO: 10 or SEQ ID NO: 9), 5 '
  • the Hindlll site was introduced, and the elements required for mammalian cell expression, such as Kozak sequence and signal peptide sequence, were introduced into the EcoRI site at the 3' end, cloned into the pUC 19 plasmid, designated pG-CSF-Fc, and transformed into E. coli TG l .
  • the pUC 19 plasmid was digested with Hind III and EcoRI, and the G-CSF-IgG2 Fc fragment of about 1400 bp was recovered and ligated with the pcDNA3 (Invitrogen) expression plasmid digested with Hind III and EcoRI to construct the expression plasmid pEX-G-CSF- Fc.
  • the expression plasmid pEX-G-CSF-Fc was linearized, purified, electroporated and transformed into CHO cells, screened, and detected by ELISA. Cell lines with higher protein yield were selected, cryopreserved cells, and cell banks were prepared.
  • an expression plasmid containing the cDNA sequence encoding SEQ ID NOs: 2-7 can be constructed, and CHO cells can be transfected by linearization to express G-CSF dimer, and the expression amount can be detected by ELISA, and cells with higher protein yield can be selected. Strain, and prepare a cell bank.
  • One cell ( ⁇ lx 10 7 cells/ml) was taken from the cell bank, resuscitated in a 10 cm culture dish, cultured in 10 mL of basal medium, and cultured at 37 ° C, 5% C0 2 for 24 hr.
  • Seed amplification 10 mL culture was passaged into 30-40 mL medium, cultured to a cell concentration of 1.0-1.5 x 10 6 cells/mL and the survival rate was ⁇ 90%, and gradually expanded to 300-400 mL of medium. Place in a shake flask at 37 ° C, 5% C0 2 . Culture, speed 120 rpm.
  • Cell can production (30-100L): When the cell concentration in the 3L-10L cell tank reaches 1.0 - 3.0 X 10 6 cells/ml and the survival rate is ⁇ 90%, inoculate the culture in the 3-10 L cell tank to 30 -100L cell tank, the pH of the medium was controlled at 6.8, the dissolved oxygen was about 50%, the rotation speed was 65-100 rpm, and the culture was started for 12-48 hr. The glucose concentration in the control medium ( ⁇ lg/L) was started, and the culture was carried out by fed.
  • the cell supernatant is collected (containing G-CSF-Fc complex, G-CSF dimer,
  • G-CSF-Fc multimers and metabolites cell supernatants are collected and purified by filtration and multi-stage gel chromatography, for example, rProtein A Sepharose FF (GE Healthcare, cat# 17- 1279-04) 50 mM citric acid/sodium citrate, 0.2 M NaCl, pH 3.7-3.8 buffer eluted to give >90% purity of G-CSF dimer followed by Capto Adhere chromatography with 50 mM NaAc/HAC , 0.2 M NaCl, pH ⁇ 4.5-5.0 buffer elution, then SP Sepharose FF (GE Heathcare Cat #17-0729-04), 10 mM PB (pH 6.0 ⁇ 0.1) buffer equilibration, lOmM PB, 0.2M NaC pH 7.2 ⁇ 0.1) eluent, the eluate was virus removed by low pH, filtered, etc., and finally G-CSF dimer was obtained.
  • G-CSF dimer can activate STAT3 of M-NFS-60 cells in vitro and stimulate the proliferation of M-NFS-60 cells (ED50 0.1-10 ng/mL).
  • Example 2 In vivo half-life of G-CSF dimer
  • Rats were injected subcutaneously with G-CSF dimer (dimer consisting of two sequences of G-CSF-Fc complex as shown in SEQ ID NO: 3) 100 g/kg.
  • the half-life of the monomeric G-CSF in the rat is about 2 hours.
  • G-CSF dimers were administered (dimers consisting of two sequences such as G-CSF as shown in SEQ ID NO: 6).
  • the dose of the Fc complex is 30 60 120 240 ug/kg, single administration, 0.5 1 , 2, 4 8 16 24, 36, 48 72, 96 hours after the administration, 6 (120 hrs), 7 9 11 13, and 15 days, blood collection serum was stored frozen in a -70 ° C refrigerator. Blood concentration was measured by ELISA (ELISA, Quantikine human G-CSF ELISA kit, R&D System, Inc. Minneapolis, Min, Cat: PDCS50). The test results were analyzed using a non-compartmental model for pharmacokinetic parameters (software WinNonlin v 5.2 Pharsight Corporation, USA) and the results are shown in Table 2 below.
  • PC12 cells are a rat pheochromocytoma cell line that can synthesize, metabolize, and deliver dopamine in vitro and can be used as an in vitro model to screen for active compounds.
  • PC12 cells were seeded in a 96-well plate at 40,000 cells/well.
  • the medium composition DMEM, 10% horse serum.
  • Penicillin-Streptomycin MPP + (Sigma) was added to a final concentration of 30 3000 ⁇ , and G-CSF was added to 0.4 ng/mL 4 ng/mL 40 ng/mL G-CSF dimer to 1 ng/mL 10 ng/mL. After lOOng/mL o culture for 24 hr, Fluorimetric cell viability assay was used. The results showed that after MPP + treatment, the survival rate of PC12 cells decreased with the increase of MPP concentration. At the same MPP concentration, the protective effect of G-CSF dimer on PC12 cells under the same molar G-CSF concentration. Significantly better than G-CSF monomer.
  • PC12 cells were seeded in a 96-well plate at 40,000 cells/well.
  • Medium composition DMEM, 10% horse serum + 5% FCS
  • Penicillin-Streptomycin MPP+ Sigma was added to a final concentration of 1 100 ⁇ , and G-CSF monomer was added to 0.4 ng/mL 4 ng/mL 40 ng/mL G-CSF dimer to 1 ng/mL 10 ng/mL. 100 ng/mL. to cultivate
  • tyrosine hydroxylase (TH) was detected by immunohistochemistry, and TH cells were counted in the substantia nigra.
  • the results showed that in the G-CSF dimer treatment group, the number of TH cells in the substantia nigra was significantly higher than that in the G-CSF treatment group. Under the same molar G-CSF molecular concentration, the protective effect of G-CSF dimer on PC12 cells was significantly better than that of G-CSF monomer.
  • the molecular weight ratio of the G-CSF monomer to the G-CSF dimer (the G-CSF dimer selected from the G-CSF-Fc complex represented by the sequence of SEQ ID NOS: 2-7) is about 1 : 5, 1 mole of G-CSF dimer contains 2 moles of G-CSF monomer molecule, therefore, the mass ratio of G-CSF monomer to G-CSF dimer is about 1 in the same molar G-CSF molecule.
  • G-CSF monomer contains a molar concentration of G-CSF molecules equivalent to the molar concentration of G-CSF molecules contained in Ing / mL G-CSF dimer; 4 ng / mL of G-
  • the molar concentration of G-CSF molecules contained in the CSF monomer corresponds to the molar concentration of G-CSF molecules contained in the 10 ng/mL G-CSF dimer; the molarity of G-CSF molecules contained in the 40 ng/mL G-CSF monomer
  • the concentration corresponds to the molar concentration of the G-CSF molecule contained in the 100 ng/mL G-CSF dimer.
  • Example 5 Activation of STAT3 by dopaminergic neuron cells by G-CSF dimer
  • the whole brain of fetal rats of 14-day-old SD rats was placed in pre-cooled D-Hanks solution, substantia nigra under a dissecting microscope, cut into 1 mm 3 size, 10 mL 0.125% trypsin was added, and placed Digested into the incubator at 37 °C for 15 min, then the aspirate tissue was transferred to a centrifuge tube containing pre-cooled DMEM + 10% FBS to terminate digestion, pipetted several times with a pipette, and after standing, the supernatant was taken and sucked to another Inside a centrifuge tube. Repeat the above steps 2 to 3 times.
  • the serum-free neuronal basic medium neurobasal invitrogen, Cat. No.
  • Cell lysate (Cell Signaling Technology, Cat. No. 9803; main components: 20 mM Tris-HCl (H 7.5), 150 mM NaCl, 1 mM Na 2 EDTA, 1 mM EGTA, 1 % Triton, 2.5 mM sodium pyrophosphate , 1 mM beta-glycerophosphate, 1 mM Na 3 V0 4 , 1 ⁇ leupeptin, 1 mM PMSF) Cells were lysed on ice for 20 minutes and scraped with cell scrapers. The cell lysate was collected, centrifuged at 12,000 rpm for 10 minutes at 4 °C. The supernatant was aspirated and the protein concentration was determined. Another supernatant of ⁇ was used to detect changes in STAT phosphorylation levels using the STAT3 [pY705] ELISA kit (invitrogen, Cat. No. KH00481).
  • the G-CSF dimer (G-CSF-D) has a stronger activated dopaminergic neuron STAT3 than the G-CSF monomer. Biological activity.
  • Example 6 Therapeutic effect of G-CSF dimer on MPTP-induced animal PD model
  • MPTP I-methyl-4-phenyl-1,2,3,6-tetrahydropyridine
  • the experiment selected C57/BL6J mice, male, 20 ⁇ 22g, 12-14 weeks, and the animals were kept at room temperature 24 ⁇ 2.
  • the environment of C maintain a light-dark 12-hour cycle, give plenty of information, and drink freely.
  • mice were randomly divided into 5 groups, 10 in each group, which were solvent control group, MPTP model group, MPTP+G-CSF. 4 ( ⁇ g/kg group, MPTP+GCSF-D 4 ( ⁇ g/kg group, MPTP+GCSF-D l OO g/kg group, wherein GCSF-D is selected from sequences such as SEQ ID NO: 2-7 A G-CSF dimer composed of the G-CSF-Fc complex shown.
  • MPTP was intraperitoneally injected at a dose of 30 mg/kg for 5 consecutive days. After one day of recovery (from day 7), MPTP+G-CSF 4 ( ⁇ g/kg group was given by G-CSF 4 ( ⁇ g/kg subcutaneous injection). Drug, G-CSF was administered daily once a day for 5 days, ie from day 7 to day 1; MPTP+GCSF-D 4 ( ⁇ g/kg group was administered by 4 ( ⁇ g/kg subcutaneously) GCSF-D was administered on days 7 and 9 respectively; MPTP+GCSF-D lOO g/kg group was administered subcutaneously at 100 g/kg, and GCSF-D was administered on days 7 and 9 respectively; solvent The control group was given an equal volume of physiological saline.
  • the pole test is used to detect typical behavioral symptoms in PD - a slowing of movement (Matsuura et al, 1997; Araki et al, 2001; Kato et al, 2004)
  • T-tum time to turn
  • T-LA locomotion activity time
  • mice were sacrificed by decapitation.
  • the striatum was weighed out and placed in a 1.5 ml centrifuge tube and immediately placed in crushed ice.
  • the sample was added to a 300 ⁇ l ice water bath sample solution (0.2 ⁇ perchloric acid, 0.2 mM sodium metabisulfite, 0.01% EDTA-2Na, and 0.3 ⁇ DHBA as an internal standard) per 10 mg of sample.
  • the above mixture was ultrasonically pulverized using an ultrasonic system, and then centrifuged at 10,000 g for 20 min at 4 °C. The supernatant was taken and filtered through a 0.22 ⁇ aqueous phase filter to measure the concentration of striatal dopamine by high performance liquid chromatography.
  • the primary antibody was monoclonal mouse anti-THC1: 1000, CHEMICON), and was incubated with brain slices at the striatum and midbrain at 4 ° C overnight, washed with PBS three times, and then incubated with biotinylated secondary antibody for 1 h at room temperature.
  • SABC complex incubation at room temperature DAB color development, ethanol gradient dehydration, xylene transparent, neutral gum seal. Black matter TH positive cell count, striatum TH positive staining optical density scan.
  • the whole brain of the fetal rat of the SD rat at 17 days of pregnancy was placed in pre-cooled D-Hanks solution, and the hippocampus was taken under a dissecting microscope. Cut into 1 mm 3 size, add 10 mL of 0.125% trypsin, and digest it in a 37 °C incubator for 15 min, then aspirate the tissue and transfer to a centrifuge tube containing pre-cooled DMEM + 10% FBS to terminate digestion. Blow several times with a pipette. After standing, take the supernatant and suck into another centrifuge tube. Repeat the above steps 2 to 3 times.
  • the serum-free neuronal basic medium neurobasal invitrogen, Cat. No.
  • the cell lysate was collected and centrifuged at 12,000 rpm for 10 minutes at 4 ° C. The supernatant was aspirated and the protein concentration was determined. The supernatant of ⁇ was used for STAT3 [pY705] ELISA reagent.
  • the box (invitrogen, Cat. No. KH00481) detects changes in STAT phosphorylation levels.
  • the G-CSF dimer (G-CSF-D) has a stronger dimeric G-CSF that activates hippocampal neuron STAT3 compared to the G-CSF monomer.
  • Biological activity The G-CSF dimer on induced apoptosis of PC12 cells
  • PC 12 cells can grow with protuberances after induction by nerves (NGF), and have neuronal characteristics.
  • NNF nerves
  • ⁇ ⁇ amyloid
  • ⁇ induces apoptosis in PC 12 cells and can simulate AD model in vitro.
  • PC 12 cells were cultured in basal medium (DMEM, 10% FCS, 1% Penicillin-Streptomycin), trypsinized, resuspended in medium containing NGF 50 ng/mL, and the cell concentration was adjusted to 2 ⁇ 10 4 cells/well.
  • basal medium DMEM, 10% FCS, 1% Penicillin-Streptomycin
  • trypsinized resuspended in medium containing NGF 50 ng/mL
  • 96-well plates were incubated at 37 ° C in a 5% CO 2 incubator for 24 hr.
  • ⁇ was added to a final concentration of 1-100 ⁇ /L with different concentrations of G-CSF monomer and G-CSF-D (GCSF-D was selected from G-sequences selected from sequences such as SEQ ID NOs: 2-7).
  • CSF-Fc complex The composition of G-CSF dimer), the final concentration of G-CSF monomer was 0.4, 4, 40 ng / mL, and the final concentration of G-CSF-D was 1, 10, 100 ng / mL, respectively.
  • GCSF-D is composed of a G-CSF-Fc complex selected from the sequences as SEQ ID NOS: 2-7.
  • the drug was administered on the 3rd day after the animal model was established.
  • ⁇ +G-CSF 40 ⁇ ⁇ /13 ⁇ 4 group was administered G-CSF by G-CSF 40 ⁇ ⁇ /13 ⁇ 4 subcutaneous injection, administered daily for 5 days;
  • Ap+GCSF-D 4 ⁇ g/kg group was administered by subcutaneous injection of 40ug/kg, GCSF-D was administered on the 3rd and 5th day respectively;
  • ⁇ +GCSF-D 10 ⁇ g/kg group was injected subcutaneously at 100g/kg
  • GCSF-D was administered once on days 3 and 5; the solvent control group was given an equal volume of physiological saline.
  • Both the G-CSF treatment group and the G-CSF dimer treatment group improved the learning consolidation and reproducibility of model rats.
  • the expression of NeuN positive neurons in the hippocampus of the model group was decreased.
  • the number of NeuN positive neurons in the G-CSF treatment group and the G-CSF dimer treatment group increased, compared with the G-CSF monomer treatment group, the G-CSF dimer treatment group (10) ( ⁇ g/kg) The number of NeuN positive neurons was significantly increased.
  • PPTP I-methyl-4-phenyl-1,2,3,6-tetrahydropyridine
  • MPTP I-methyl-4-phenyl-1,2,3,6-tetrahydropyridine
  • PD Jinsen's disease
  • TH Tyrosine hydroxylase
  • MPTP+G-CSF-D 3 ( ⁇ g/kg group: intraperitoneal injection of MPTP at a dose of 30 mg/kg for 5 consecutive days, after one day of animal recovery (ie from day 7), subcutaneous injection at 30 g/kg G-CSF-D was administered once on days 7, 9, and 11 respectively;
  • MPTP+G-CSF-D lOO g/kg group MPTP was intraperitoneally injected at a dose of 30 mg/kg for 5 consecutive days. After one day of recovery (ie from day 7), G was given by subcutaneous injection at 100 g/kg. -CSF-D, administered once on days 7, 9, and 11, respectively;
  • MPTP model group MPTP was intraperitoneally injected at a dose of 30 mg/kg for 5 consecutive days. After one day of recovery, an equal volume of solvent (0.5% mouse serum/PBS) was given from day 7;
  • Normal control group An equal volume of physiological saline was administered for 5 days, and after the animal was recovered for one day, an equal volume of solvent (0.5% mouse serum/PBS) was administered from the 7th day.
  • the G-CSF dimer (G-CSF-D) consists of two sequences of G-CSF-Fc as shown in SEQ ID NO: 6. Animals were sacrificed on day 12, and the concentration of dopamine in the striatum was measured, and the dopaminergic nerve fibers in the striatum and the dopaminergic neurons in the substantia nigra were observed. a. Detecting the concentration of dopamine in the striatum
  • mice were sacrificed by decapitation.
  • the striatum was removed and weighed and placed in a 1.5 ml centrifuge tube and immediately placed in crushed ice.
  • the sample was added to a 300 ⁇ l ice water bath sample solution (0.2 ⁇ perchloric acid, 0.2 mM sodium metabisulfite, 0.01% EDTA-2Na, and 0.3 ⁇ DHBA as an internal standard) per 10 mg of sample.
  • the above mixture was ultrasonically pulverized using an ultrasonic system, and then centrifuged at 10,000 g for 20 min at 4 °C. The supernatant was taken and filtered through a 0.22 ⁇ aqueous phase filter to measure the concentration of striatal dopamine by high performance liquid chromatography.
  • Figure 5A shows a picture of immunohistochemical staining of mouse striatum TH-positive nerve fibers. After injection of MPTP for 5 consecutive days, the density of TH-positive nerve fibers in striatum was reduced. Treatment with G-CSF dimer could increase the number of TH-positive nerve fibers in a dose-dependent manner. It can be seen that G-CSF dimer is induced by MPTP. The loss of dopaminergic nerve fibers has a significant protective effect.
  • Figure 5B shows immunohistochemical densitometry values of mouse striatum TH positive nerve fibers.
  • the results indicate that MPTP can cause a significant decrease in the density of TH-positive nerve fibers in the striatum (### p ⁇ 0.001).
  • the G-CSF dimer significantly inhibited the MPTP-induced decrease in the density of striatal TH-positive nerve fibers.
  • the G-CSF dimer administration group showed a dose-dependent relationship, which was significantly different from the MPTP model group (* p ⁇ 0.05).
  • Figure 6A shows immunohistochemical staining of TH-positive neurons in the substantia nigra pars compacta of mice. After mice were injected with MPTP for 5 consecutive days, the TH-positive neurons in the substantia nigra pars compacta were largely lost. The treatment of G-CSF dimer could restore the number of TH-positive neurons. It can be seen that the G-CSF dimer acts on MPTP-induced dopaminergic neurons. A large number of meta-losses have significant protection.
  • Figure 6B shows the results of TH positive cell count analysis in the substantia nigra parsing of mice.
  • the number of TH-positive neurons in the substantia nigra pars compacta was significantly reduced after 5 days of MPTP injection (### p ⁇ 0.001), which was about 49% of the normal control group, indicating that MPTP caused intrahealoid A large number of dopaminergic neurons are lost.
  • the G-CSF dimer group significantly inhibited MPTP-induced loss of TH-positive neurons in the substantia nigra pars compacta, and dose-dependently increased the number of TH-positive cells in the substantia nigra.
  • the G-CSF dimer administration group showed a dose-dependent relationship, which was significantly different from the MPTP model group (*** p ⁇ 0.001), and the G-CSF dimer 30 g/kg dose group was in the substantia nigra.
  • TH-positive dopamine neurons were approximately 86% of the normal control group, and G-CSF dimer 100 ⁇ g/kg dose group had 99% of TH-positive dopamine neurons in the substantia nigra, which was equivalent to 99% of the normal control group. Level.
  • G-CSF dimer can obviously protect MPTP-induced loss of dopaminergic nerve fibers, and can also significantly protect MPTP-induced large loss of dopaminergic neurons. Comparative Example 1 Therapeutic effect of G-CSF monomer on MPTP-induced PD animal model
  • mice were injected intraperitoneally with MPTP at 30 mg/kg for 5 days to obtain a PD mouse model; 250 g/kg G-CSF (Neupogen, Amgen) was administered continuously after one day of recovery.
  • G-CSF Neurogen, Amgen
  • Model group Number of TH-positive dopaminergic neurons in the substantia nigra pars compacta before the first dose.
  • Normal group Mice not injected with MPTP.
  • TH-positive dopaminergic neurons in the substantia nigra pars compacta recovered to approximately 70%, 80%, and 77% of the normal group, respectively.
  • the G-CSF dimer of the present invention was administered intermittently three times at a dose of 30 g/kg or 100 g/kg each time, on the first day after the end of administration, Dopamine TH-positive neurons in the substantia nigra in the 30 g/kg group and 100 g/kg group recovered to 86% and 99% of the normal control group, respectively.
  • TH-positive dopaminergic neurons were substantially completely restored to the normal control group on the first day after the end of administration.
  • the total dose of G-CSF dimer 100 g/kg was 300 ⁇ ⁇
  • the total dose of G-CSF monomer in Comparative Example 1 was 1750 ⁇ ⁇ .
  • the molecular weight ratio of G-CSF monomer to G-CSF dimer is about

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Abstract

本发明公开了G-CSF二聚体在制备治疗神经退行性疾病药物中的应用。本发明使用G-CSF二聚体分子，能够显著增加PD模型动物黑质多巴胺能神经元的数量，提高多巴胺能神经元的功能。此外，G-CSF二聚体分子能够显著减少海马区神经元细胞的凋亡，改善AD模型大鼠学习记忆能力。本发明的二聚体G-CSF的血清半衰期延长，有效阻止神经元的丢失，从而能够更有效地治疗神经退行性疾病。

Description

G-CSF二聚体在制备治疗神经退行性疾病药物中的应用 技术领域
本发明涉及生物和医药技术领域。 更具体地， 本发明涉及一种新型的 G-CSF二聚体 及其在治疗神经退行性疾病中的用途。 背景技术
人粒细胞集落刺激因子 (G-CSF)是一个含有 204个氨基酸组成并含有 30个氨基酸信 号肽的糖蛋白。 成熟的 G-CSF蛋白被分泌到细胞外不含信号肽由 174个氨基酸构成，分子 量为 18 - 20 kDa。 人体主要由单核细胞， 成纤维细胞和内皮细胞分泌。
G-CSF在生物体内主要发挥三大生物学功能： 1. 作用于嗜中性粒前体细胞和骨髓干 细胞。 驱动嗜中性粒细胞的分化、 增殖和成熟。 2. 激活成熟的嗜中性细胞参与免疫反 应。 3. 协同其他血液生长因子， 如 Stem Cel l Factor , Flt_3配体， GM-CSF发挥造血 功能。
G-CSF的受体已证实主要存在于骨髓造血干细胞 Sca+Lin— Th llOT， 前体细胞 CD34+， 以 及定向分化的嗜中性粒前体细胞 (commi tted granulocyte precursor)禾口成熟的嗜中性 粒细胞。 人促嗜中性粒细胞刺激生长因子受体 （G-CSF Receptor, G-CSFR) 是一个单链 的， 对 G-CSF有高亲合力的特异性受体， 含有 812个氨基酸。
Tamada 等人获得 G_CSF： G-CSFR复合物的晶体结构， 并进行了 2. 8埃衍射分析 (PNAS , 2008 , Vol. 103 : 3135-3140)， 结果显示， G-CSF： G-CSFR复合物是以 2： 2 比 例存在， 即 2个配体和 2个受体结合的状态存在。 每一个 G-CSF分子结合一个受体分子， 当两个都结合了 G-CSF配体的受体， 相互靠近相互作用而形成 2 : 2 二聚体时， G-CSF受 体的羧基端才能激活下游的信号分子 JAK2 (Janus tyros ine kinase)。 JAK2再通过激活 STAT3启动转录基因， 而刺激细胞增殖。
2003年 Schabi tz W. R等报道（Stroke , 2003 , 34： 745-751)重组人 G-CSF对神经细 胞在缺血性动物模型中具有保护功能。 2006年， Shyu等报道 (CMAJ, 2006， 174 : 927-933) 在急性中风病人治疗中， 每天注射重组人 G-CSF连续 5天， 显示一定疗效。 皮下给药后， G-CSF在大鼠体内的半衰约 2hr， 经皮下注射人体时， 半衰期只有 3. 5hr， 因此， 病人需 每天注射药物， 影响病人的生活质量。
神经退行性疾病 (neurodegenerative disease)是大脑和脊髓的神经元细胞丧失的状态， 是一种慢性、进行性神经系统疾病， 主要包括阿尔茨海默氏病 CAlzhemier's disease, AD)、 巾白金森氏病（Parkinson's disease，PD)、 亨廷顿舞蹈病（Huntington disease) 肌萎縮侧索硬 化症 (amyotrophic lateral sclerosis), 脊骨遺肌萎縮病 (spinal muscular atro hy) 脊骨遺小脑共 济失调 (spinal cerebellar ataxias)等， 其共同特征是发生神经元的退行性病变和凋亡， 导 致病人行为异常和功能障碍， 引起过早死亡的神经性疾病。 神经退行性疾病的发病机制 尚不清楚， 至今还没有有效的方法和药物来防治， 现有治疗方法包括， 通过服用或静脉 注射补充人大脑神经元匮乏的物质治疗 PD， 如左旋多巴， 但是， 左旋多巴并不能有效的 控制 PD的自然病变过程， 不能影响多巴胺能神经元的变性速度， 还具有不良的副作用， 如开关现象和运动障碍， 其治疗作用也只能维持两年左右， 长期使用还可损害神经元， 加上神经元的凋亡； 针对 AD脑内乙酰胆碱不足， 用胆碱酯酶抑制剂提高其浓度， 该方 法同样不能控制疾病的发展。
目前， 治疗帕金森症疾病的药物多数用于减轻症状， 如使用多巴胺替代物（左旋多 巴或多巴胺激动剂） ， 其中， 左旋多巴 （L-DOPA ) 作为多巴胺合成前体补充脑内多巴 胺， 是目前最常用且最有效的 PD治疗药物， 但长期使用该药物易出现疗效降低和严重的 副作用， 甚至可能发生 "开 -关"现象。 另外， 阻止多巴胺能神经元细胞的丢失也是治疗 PD的主要策略之一， 目前研究最多的为神经营养因子 （neurotrophic factor, GDNF ) ， 但 在临床试验中 GDNF未显示出药效且具有一系列的副作用如恶心、 厌食、 体重下降等 ( Neurology, 2003, 69:69-73 ) 。 G-CSF用于治疗帕金森症疾病也有报道， 但不论在 PD动 物模型中还是临床试验中， G-CSF的给药剂量均很高， 疗效作用慢， 给药频率高， 给药 周期长， 易降低患者的依从性， 非常不利于病人使用。
然而， 本领域迫切需要开发更有效的治疗神经退行性疾病的药物。 发明内容
本发明的目的就是一种更有效的治疗神经退行性疾病的药物及其制法和用途。 在本发明的第一方面， 提供集落刺激因子 G-CSF的二聚体在制备治疗或预防神经退 行性疾病的组合物中的用途。
所述神经退行性疾病选自： 帕金森氏病、 阿尔茨海默氏病、 亨廷顿舞蹈病、 肌萎縮侧 索硬化病， 脊髓肌萎縮病、 原发性侧索硬化病， 脊髓小脑共济失调。
所述集落刺激因子 G-CSF的二聚体为人集落刺激因子 G-CSF二聚体。
根据本发明， 所述集落刺激因子 G-CSF二聚体的结构如式 I所示：
M1-L-M2 式 I
式中，
Ml是人集落刺激因子 G-CSF的第一单体；
M2是人集落刺激因子 G-CSF的第二单体；
L是位于所述的第一单体和第二单体之间的，将所述第一单体和第二单体连接在一起的 接头元件， 其中， 所述的 G-CSF二聚体保持了 G-CSF的生物活性， 并且其血清半衰期是所述第一单 体或第二单体的血清半衰期的 2倍以上。
进一步地， 所述的接头元件 L选自下组：
(i) 3-50个氨基酸的短肽；
(ii) 式 Π所示的多肽元件：
-Z-Y-Z- 式 II
式中，
Y为载体蛋白；
Z为无、 或 1-30个氨基酸的短肽；
"-"为化学键或共价键， 更佳地， "-"为肽键。
在另一优选例中， 所述人集落刺激因子 G-CSF的二聚体的氨基酸序列如 SEQ ID ΝΟ: 1 所示。
在另一优选例中， 所述的第一单体和第二单体是相同的。 所述的第一单体和第二单体 的氨基酸序列如 SEQ ID NO:8所示。
根据本发明， 所述人集落刺激因子 G-CSF的二聚体的氨基酸由两个 G-CSF-Fc复合物
(G-CSF-Fc复合物即为带 Fc片段的 G-CSF单体)构成。优选的，所述 G-CSF-Fc复合物的氨基酸 序列如 SEQ ID NO:2〜7所示。
在另一优选例中， 所述集落刺激因子 G-CSF的二聚体采用以下步骤制备：
(a)采用包含编码 G-CSF-Fc复合物的 DNA序列的表达载体转化哺乳动物细胞，其中， 所述 DNA序列如 SEQ ID NO: 10或 SEQ ID NO: 9所示；
(b)培养所述哺乳动物细胞， 使其表达 G-CSF-Fc复合物和 G-CSF二聚体； 和
(c)分离纯化所述 G-CSF二聚体；
其中，所述 G-CSF二聚体包含两个 G-CSF-Fc复合物，所述 G-CSF-Fc复合物的氨基酸 序列如 SEQ ID NO: 2〜7所示。
根据本发明， 所述组合物为药物组合物、 保健品组合物或食品组合物。
所述的药物组合物为固体制剂或液体制剂。
所述的药物组合物含有： 0.01-99wt%集落刺激因子 G-CSF的二聚体和药理上可以接受 的赋形剂或载体。
所述的赋形剂或载体选自： 纤维素及其衍生物、 明胶、 滑石、 固体润滑剂、 硫酸钙、 植物油、 多元醇、 乳化剂、 润湿剂、 着色剂、 调味剂、 稳定剂、 抗氧化剂、 防腐剂、 无热 原水。
在另一优选例中， 所述的含 G-CSF二聚体的药物用于提高纹状体内多巴胺的浓度； 防 止多巴胺能神经纤维的丢失； 和 /或防止多巴胺能神经元的丢失。 在本发明的第二个方面， 提供一种治疗神经退行性疾病的药物，含有作为活性成分的 集落刺激因子 G-CSF二聚体。
所述神经退行性疾病选自： 帕金森氏病、 阿尔茨海默氏病、 亨廷顿舞蹈病、 肌萎縮侧 索硬化病， 脊髓肌萎縮病、 原发性侧索硬化病， 脊髓小脑共济失调。
所述集落刺激因子 G-CSF的二聚体为人集落刺激因子 G-CSF二聚体。
所述的药物含有： 0.01 -99^%人集落刺激因子 G-CSF的二聚体和余量的药理上可以接 受的赋形剂或载体。
所述集落刺激因子 G-CSF二聚体的结构如式 I所示：
M1-L-M2 式 I
式中，
Ml是人集落刺激因子 G-CSF的第一单体；
M2是人集落刺激因子 G-CSF的第二单体；
L是位于所述的第一单体和第二单体之间的，将所述第一单体和第二单体连接在一起的 接头元件，
其中， 所述的 G-CSF二聚体保持了 G-CSF的生物活性， 并且其血清半衰期是所述第一单 体或第二单体的血清半衰期的 2倍以上。
在本发明的第三个方面， 提供一种治疗神经退行性疾病的方法， 包括步骤： 给需要治疗的对象施用集落刺激因子 G-CSF的二聚体。
所述神经退行性疾病选自： 帕金森氏病、 阿尔茨海默氏病、 亨廷顿舞蹈病、 肌萎縮侧 索硬化病， 脊髓肌萎縮病、 原发性侧索硬化病， 脊髓小脑共济失调。
在另一优选例中， 所述的对象包括哺乳动物 (如人)。
本发明使用 G-CSF二聚体分子， 能够显著增加 PD模型动物纹状体内多巴胺的浓度， 显 著抑制多巴胺能神经纤维的丢失， 能够显著增加 PD模型动物黑质多巴胺能神经元的数量， 提高多巴胺能神经元的功能。 此外， G-CSF二聚体分子能够显著改善 AD模型大鼠学习记忆 能力， 保护神经元， 减少海马区神经元细胞的凋亡， 减轻痴呆的症状。 本发明的二聚体 G-CSF的血清半衰期延长， 有效阻止神经元的丢失， 从而能够更有效地治疗神经退行性疾 病。
应理解， 在本发明范围内中， 本发明的上述各技术特征和在下文（如实施例）中具体 描述的各技术特征之间都可以互相组合， 从而构成新的或优选的技术方案。 限于篇幅， 在此不再一一累述。 附图说明
图 1显示了本发明一种 G-CSF 二聚体的示意图。 其中， " -" 表示连接肽， G-CSF 椭圆形表示 G-CSF单体。
图 2a和图 2b显示了本发明一种 G-CSF 二聚体的示意图。 其中， " -"表示氨基酸连 接肽， G-CSF椭圆形表示 G-CSF单体， 图 2a中标记为 " C " 的椭圆形表示载体蛋白， 并且 G-CSF 位于载体蛋白的 N-末端， 图 2b中显示两个 Fc通过二硫键配对。
图 3a和图 3b显示了本发明一种 G-CSF 二聚体的示意图。 其中， " -"表示氨基酸连 接肽， G-CSF椭圆形表示 G-CSF单体， 图 3a中标记为 " C " 的椭圆形表示载体蛋白， 并 且 G-CSF 位于载体蛋白的 C-末端， 图 3b中显示两个 Fc通过二硫键配对。
图 4显示了小鼠纹状体多巴胺浓度的变化。
图 5A显示了小鼠纹状体 TH阳性神经纤维免疫组化染色图片。
图 5B显示了小鼠纹状体 TH阳性神经纤维免疫组化光密度值。
图 6A显示了小鼠黑质致密部 TH阳性神经元免疫组化染色图片。
图 6B显示了小鼠黑质致密部 TH阳性细胞计数分析结果。 具体实施方式
本发明人经过广泛而深入的研究， 首次制备了一种 G-CSF二聚体， 并意外发现： 通 过本发明的 G-CSF二聚体， 能够延长体内半衰期， 改善药物的动力学， 减少注射频率； 显著增强体内药物活性， 减轻神经退行性疾病的症状， 促进神经退行性疾病的恢复。 在 此基础上， 完成了本发明。 G-CSF二聚体
本发明的 G-CSF二聚体的结构如式 I所示。
M1-L-M2 式 I
式中，
Ml是人集落刺激因子 G-CSF的第一单体；
M2是人集落刺激因子 G-CSF的第二单体；
L是位于所述的第一单体和第二单体之间的，将所述第一单体和第二单体连接在一起的 接头元件，
其中， 所述的 G-CSF二聚体保持了 G-CSF的生物活性， 并且其血清半衰期是所述第一单 体或第二单体的血清半衰期的 2倍以上。
所述的生物活性包括：
(a) . 作用于嗜中性粒前体细胞和骨髓干细胞， 驱动嗜中性粒细胞分化、 增殖和成熟；
(b) 激活成熟的嗜中性细胞参与免疫反应。
在另一实施方式中， 所述第一单体和第二单体是相同的， 所述第一单体和第二单体 的氨基酸序列如 SEQ ID NO: 8所示。
在另一实施方式中， 所述的接头元件 L选自下组：
(i) 3-50个氨基酸的短肽；
(ϋ) 式 Π所示的多肽元件：
-Z-Y-Z- 式 II
式中，
Y为载体蛋白；
Z为无、 或 1-30个氨基酸的短肽；
"-"为化学键或共价键， 更佳地 "-"为肽键。
G-CSF二聚体代表性的结构如图 1-3中所示。 其中， 载体蛋白包括 (但并不限于)： 人
IgG(l , 2,3,4) 的 Fc 片段、 或人白蛋白 (albumin)
G-CSF 位于可用载体蛋白的 C-末端， 也可用位于载体蛋白的 N-末端。
如本文所用， 术语 "连接肽" （linker)是指位于 G-CSF单体和 G-CSF单体之间的、 起 连接作用的短肽。 连接肽的长度没有特别限制。 连接肽的长度通常为 5-50个氨基酸。 通 常， 连接肽不影响或不显著影响 G-CSF单体和 G-CSF单体形成正确的折叠和空间构象。 一些连接肽的例子包括 (但并不限于)：
较佳地， 所述的连接肽具有选自下组的氨基酸序列：
(a) 疏水性氨基酸 Gly和 Pro构成的 3 - 15个氨基酸序列，例如 Gly-Pro-Gly-Pro-Gly-Pro； 或者由 Gly和 Ser构成的 3-20个氨基酸序列， 如 GSGG。
(b) 多克隆位点所编码的氨基酸序列。该序列通常为 5-20个，较佳地 10-20个氨基酸， 其例子包括 (但并不限于）： TGLQPTRGIDDITSPVD；
(c) 来自于 G-CSF单体之外的蛋白的氨基酸序列，例如来自于 IgG或白蛋白的氨基酸 序列；
(d)由 (a)、 （b)和 (c)组合形成的氨基酸序列。
一种优选的连接肽是 GSGGGSGGGGSGGGGS (SEQ ID NO: 1中第 175-190位）。
一种优选的连接肽是 ASTKGP(SEQ ID NO: 4中第 175-180位）。
此外， 在融合蛋白的 N端或 C末端还可添加其他不影响 G-CSF单体活性的氨基酸序 列。较佳地， 这些添加的氨基酸序列有利于表达 (如信号肽)， 有利于纯化 (如 6 X His序列、 酿酒酵母 α -因子信号肽切割位点 (Glu-Lys-Arg)， 或可促进融合蛋白的活性。 序列说明
SEQ ID NO: l为 G-CSF二聚体的一种序列， 结构如图 1所示，其中第 1-174位为 G-CSF 单体， 第 175-190位为连接肽， 第 191-364位为另一 G-CSF单体。 SEQ ID NO:2为构成 G-CSF二聚体的带有 Fc片段的 G-CSF单体的序列， 其中第 1- 174 位为 G-CSF单体， 第 175-190位为连接肽， 第 191 -418位为人 IgG2的 Fc片段。 如图 2所示， 二个带有 Fc片段的 G-CSF单体， 通过 Fc的二硫健配对作用构成二聚体。
SEQ ID NO:3为构成 G-CSF二聚体的带有 Fc片段的 G-CSF单体的序列， 其中第 245-418位为 G-CSF单体， 第 229-244位为连接肽， 第 1 -228位为人 IgG2的 Fc片段。 二个带 有 Fc片段的 G-CSF单体， 通过 Fc的二硫健配对作用构成二聚体。
SEQ ID NO:4为构成 G-CSF二聚体的带有 Fc片段的 G-CSF单体的序列， 其中第 1- 174 位为 G-CSF单体， 第 175- 180位为连接肽， 第 181 -403位为人 IgG2的 Fc片段。 二个带有 Fc 片段的 G-CSF单体， 通过 Fc的二硫健配对作用构成二聚体。
SEQ ID NO:5为构成 G-CSF二聚体的带有 Fc片段的 G-CSF单体的序列， 其中第
230-403位为 G-CSF , 第 224-229位为连接肽， 第 1 -223位为人 IgG2的 Fc片段。 二个带有 Fc 片段的 G-CSF单体， 通过 Fc的二硫健配对作用构成二聚体。
SEQ ID NO:6为构成 G-CSF二聚体的带有 Fc片段的 G-CSF单体的序列， 其中第 1- 174 位为 G-CSF , 第 175- 190位为连接肽， 第 191 -413位为人 IgG2的 Fc片段。 二个带有 Fc片段 的 G-CSF单体， 通过 Fc的二硫健配对作用构成二聚体。
SEQ ID NO:7为构成 G-CSF二聚体的带有 Fc片段的 G-CSF单体的序列， 其中第 240-413位为 G-CSF , 第 224-239位为连接肽， 第 1 -223位为人 IgG2的 Fc片段。 二个带有 Fc 片段的 G-CSF单体， 通过 Fc的二硫健配对作用构成二聚体。
SEQ ID NO :9为 SEQ ID NO:2的 cDNA序列。
SEQ ID NO: 10为 SEQ ID NO:6的 cDNA序列。 本发明提供的 G-CSF二聚体， 与具有相同摩尔 G-CSF分子浓度条件下 （G-CSF二聚 体浓度为 0.1〜1000ng/mL， 优选的为 1 - l OOng/mL ; G-CSF单体浓度为 0.04〜400ng/ml，优选 的为 0.4〜40 ng/mL ) G-CSF单体相比， 对 MPP+诱导的 PC 12细胞的保护作用更明显； 具有更 强的激活多巴胺能神经元 STAT3的生物学活性； 能显著改善 MPTP诱导的动物行为学异 常； 能显著提高 MPTP诱导的动物纹状体多巴胺的浓度； 可以明显抑制 MPTP诱导的多巴胺 能神经元的大量丢失； 具有更强的激活海马神经元 STAT3的生物学活性； 抑制 Α β诱导 所致的 PC 12细胞的凋亡； 改善 AD模型动物的学习记忆能力。 制备方法
编码本发明 G-CSF二聚体或融合蛋白的 DNA序列， 可以全部人工合成。 也可用 PCR 扩增或合成的方法获得 G-CSF第一单体和 /或 G-CSF第二单体的编码 DNA序列， 然后将其 拼接在一起， 形成编码本发明融合蛋白的 DNA序列。 为了提高宿主细胞的表达量， 可以对 G-CSF二聚体编码序列进行改造， 例如采用宿 主细胞偏好的密码子， 消除不利于基因转录及翻译的序列。 在本发明的一个实施例中， 就采用哺乳动物细胞偏好的密码子， 并采用计算机 DNA软件对 G-CSF二聚体基因进行检 测， 排除在基因中不利于基因转录及翻译的序列， 包括内含子剪切位点， 转录终止序列 等。
在获得了编码本发明新融合蛋白的 DNA序列之后，将其连入合适的表达载体，再转 入合适的宿主细胞。 最后， 培养转化后的宿主细胞， 通过分离纯化得到本发明的新的融 合蛋白。
如本文所用， 术语 "载体"包括质粒、 粘粒、 表达载体、 克隆载体、 病毒载体等。 在本发明中，可选用本领域已知的各种载体如市售的载体。比如，选用市售的载体， 然后将编码本发明新融合蛋白的核苷酸序列可操作地连于表达调控序列， 可以形成蛋白 表达载体。
如本文所用， "可操作地连于"指这样一种状况， 即线性 DNA序列的某些部分能够 影响同一线性 DNA序列其他部分的活性。例如， 如果信号肽 DNA作为前体表达并参与多 肽的分泌， 那么信号肽 (分泌前导序列) DNA就是可操作地连于多肽 DNA; 如果启动子控 制序列的转录， 那么它是可操作地连于编码序列； 如果核糖体结合位点被置于能使其翻 译的位置时， 那么它是可操作地连于编码序列。 一般， "可操作地连于" 意味着相邻 近， 而对于分泌前导序列则意味着在阅读框中相邻。
在本发明中， 术语 "宿主细胞"包括原核细胞和真核细胞。 常用的原核宿主细胞的 例子包括大肠杆菌、 枯草杆菌等。 常用的真核宿主细胞包括酵母细胞， 昆虫细胞、 和哺 乳动物细胞等。 较佳地， 该宿主细胞是真核细胞， 更佳地是哺乳动物细胞。
在获得转化的宿主细胞后， 可在适合表达本发明融合蛋白的条件下培养该细胞， 从 而表达出融合蛋白。 然后再分离出表达的融合蛋白。
在一实施例中， 本发明的 G-CSF二聚体的制备方法包括：
(a)采用包含编码 G-CSF-Fc复合物的 DNA序列的表达载体转化哺乳动物细胞，其中， 所述 DNA序列如 SEQ ID NO: 10或 SEQ ID NO: 9所示；
(b)培养所述哺乳动物细胞， 使其表达 G-CSF-Fc复合物和 G-CSF二聚体； 和
(c)分离纯化所述 G-CSF二聚体；
其中，所述 G-CSF二聚体包含两个 G-CSF-Fc复合物，所述 G-CSF-Fc复合物的氨基酸 序列如 SEQ ID NO: 2〜7所示。 药物组合物和施用方法
由于本发明 G-CSF二聚体具有优异的血清半衰期， 因此本发明 G-CSF二聚体以及含有 本发明 G-CSF二聚体为主要活性成分的药物组合物可用于治疗神经损伤疾病； 以及保护神 经元。 其中， 所述的神经损伤疾病选自下组： 帕金森氏病 (PD)、 阿尔茨海默氏病 (AD)、 亨廷顿舞蹈病 (HD)、 肌萎縮侧索硬化病 (ALS)， 脊髓肌萎縮病 (SMA)、 原发性侧索硬化 病 (PLS)， 脊髓小脑共济失调 (SCA)。
本发明提及的疾病包括那些伴有神经退行性病变的疾病， 尽管神经疾病可以使各种 原因引起的以及可以在各种部位或神经中出现， 但是本发明的治疗药物引起的受损神经 本身的修复和功能的改善提示本发明的治疗药物在治疗和改善不同类型的神经退行性 的疾病是有效的。 本发明所指的神经疾病包括， 但不仅限于， 帕金森氏病 (PD)、 阿尔茨 海默氏病 (AD)、 亨廷顿舞蹈病 (HD)、 肌萎縮侧索硬化病 (ALS)， 脊髓肌萎縮病 (SMA)、 原发性侧索硬化病 (PLS)， 脊髓小脑共济失调 (SCA)脊髓小脑变性、 脑硬化、 纹状体黑质 变性、 脊髓性肌萎縮 (SMA)、 Friedreich 共济失调， 淀粉样变、 亚急性脊髓视神经病 (SMON), 以及涉及一种治疗这些疾病的方法， 和在生产用于治疗这些疾病的治疗药物 中的用途。
通过适当的施用方法将本发明的治疗药物医用到合适的施用部位， 施用部位的选择 依赖于治疗的疾病和治疗的症状。 例如， 对于那些主要伴有大脑退行性的疾病， 将本发 明的治疗药物施用到大脑中； 对于那些具有局灶性的纹状体退行性的疾病， 可以将药物 施用到纹状体， 对于那些具有全身性神经退行性的疾病， 可以全身施用。 优选的施用方 式是诸如注射的合适方法。 优选的将本发明的药物施用到神经退行性发生的部位， 以及 动脉、 静脉或皮下注射。
本发明的药物组合物包含安全、有效量范围内的本发明 G-CSF二聚体及药理上可以接 受的赋形剂或载体。 其中 "安全、 有效量 "指的是： 化合物的量足以明显改善病情， 而不 至于产生严重的副作用。 安全、 有效量根据治疗对象的年龄、 病情、 疗程等具体情况来确 定。 通常， 药物组合物含有 l-1000mg本发明 G-CSF二聚体 /剂， 较佳地 0.05-300 mg本发明 G-CSF二聚体 /剂， 更佳地， 含有 0.3-200mg本发明 G-CSF二聚体 /剂。
"药理上可以接受的赋形剂或载体 "指的是： 一种或多种相容性固体或液体填料或凝 胶物质， 它们适合于人使用， 而且必须有足够的纯度和足够低的毒性。 "相容性"在此指 的是组合物中各组份能与本发明的化合物以及它们之间相互掺和，而不明显降低化合物的 药效。药理上可以接受的赋形剂或载体部分例子有纤维素及其衍生物 (如羧甲基纤维素钠、 乙基纤维素钠、 纤维素乙酸酯等)、 明胶、 滑石、 固体润滑剂 (如硬脂酸、 硬脂酸镁)、 硫 酸钙、 植物油 (如豆油、 芝麻油、 花生油、 橄榄油等)、 多元醇 (如丙二醇、 甘油、 甘露醇、 山梨醇等)、 乳化剂 (如吐温 ®)、 润湿剂 (如十二烷基硫酸钠)、 着色剂、 调味剂、 稳定剂、 抗氧化剂、 防腐剂、 无热原水等。
施用本发明 G-CSF二聚体时， 可以口服、 直肠、 肠胃外 (静脉内、 肌肉内或皮下)、 局 部给药。 用于口服给药的固体剂型包括胶囊剂、 片剂、 丸剂、 散剂和颗粒剂。 在这些固体 剂型中， 活性化合物与至少一种常规惰性赋形剂 (或载体)混合， 如柠檬酸钠或磷酸二钙， 或与下述成分混合： （a) 填料或增容剂， 例如， 淀粉、 乳糖、 蔗糖、 葡萄糖、 甘露醇和硅 酸； （b) 粘合剂， 例如， 羟甲基纤维素、 藻酸盐、 明胶、 聚乙烯基吡咯烷酮、 蔗糖和阿拉 伯胶； （c) 保湿剂， 例如， 甘油； （d) 崩解剂， 例如， 琼脂、 碳酸钙、 马铃薯淀粉或木薯 淀粉、 藻酸、 某些复合硅酸盐、 和碳酸钠； （e) 缓溶剂， 例如石蜡； （f) 吸收加速剂， 例 如， 季胺化合物； （g) 润湿剂， 例如鲸蜡醇和单硬脂酸甘油酯； （h) 吸附剂， 例如， 高岭 土； 和 (i) 润滑剂， 例如， 滑石、 硬脂酸钙、 硬脂酸镁、 固体聚乙二醇、 十二烷基硫酸钠， 或其混合物。 胶囊剂、 片剂和丸剂中， 剂型也可包含缓冲剂。 固体剂型如片剂、 糖丸、 胶 囊剂、 丸剂和颗粒剂可采用包衣和壳材制备， 如肠衣和其它本领域公知的材料。它们可包 含不透明剂， 并且，这种组合物中活性化合物或化合物的释放可以延迟的方式在消化道内 的某一部分中释放。可采用的包埋组分的实例是聚合物质和蜡类物质。必要时， 活性化合 物也可与上述赋形剂中的一种或多种形成微胶囊形式。
用于口服给药的液体剂型包括药学上可接受的乳液、 溶液、 悬浮液、 糖浆或酊剂。 除 了活性化合物外， 液体剂型可包含本领域中常规采用的惰性稀释剂， 如水或其它溶剂， 增 溶剂和乳化剂， 例知， 乙醇、 异丙醇、 碳酸乙酯、 乙酸乙酯、 丙二醇、 1，3-丁二醇、 二甲 基甲酰胺以及油， 特别是棉籽油、 花生油、 玉米胚油、 橄榄油、 蓖麻油和芝麻油或这些物 质的混合物等。
除了这些惰性稀释剂外，组合物也可包含助剂，如润湿剂、乳化剂和悬浮剂、甜味剂、 矫味剂和香料。
除了活性化合物外， 悬浮液可包含悬浮剂， 例如， 乙氧基化异十八烷醇、 聚氧乙烯山 梨醇和脱水山梨醇酯、 微晶纤维素、 甲醇铝和琼脂或这些物质的混合物等。
用于肠胃外注射的组合物可包含生理上可接受的无菌含水或无水溶液、分散液、悬浮 液或乳液，和用于重新溶解成无菌的可注射溶液或分散液的无菌粉末。适宜的含水和非水 载体、 稀释剂、 溶剂或赋形剂包括水、 乙醇、 多元醇及其适宜的混合物。
用于局部给药的本发明 G-CSF二聚体的剂型包括软膏剂、 散剂、 贴剂、 喷射剂和吸入 剂。 活性成分在无菌条件下与生理上可接受的载体及任何防腐剂、缓冲剂， 或必要时可能 需要的推进剂一起混合。
本发明 G-CSF二聚体可以单独给药， 或者与其他药学上可接受的化合物联合给药。 使用药物组合物时， 是将安全有效量的本发明 G-CSF二聚体适用于需要治疗的哺乳 动物（如人）， 其中施用时剂量为药学上认为的有效给药剂量， 对于 60kg体重的人而言， 每次给药剂量通常为 0. 01〜300mg， 优选 0. 5〜100mg。 当然， 具体剂量还应考虑给药途 径、 病人健康状况等因素， 这些都是熟练医师技能范围之内的。 本发明的主要优点在于：
1. 更强的体内生物半衰期。
2. 显著提高 PD模型动物纹状体中多巴胺的浓度， 显著抑制 PD模型动物纹状体多 巴胺能神经纤维的丢失和黑质多巴胺能神经元的丢失， 提高多巴胺能神经元的功能。
3. 显著抑制海马区神经元细胞的凋亡， 改善 AD模型动物的学习记忆能力。
4. 在神经退行性疾病中具有显著的神经保护作用， 有效治疗神经退行性疾病。 下面结合具体实施例， 进一步阐述本发明。 应理解， 这些实施例仅用于说明本发明 而不用于限制本发明的范围。 下列实施例中未注明具体条件的实验方法， 通常按照常规 条件如 Sambrook等人， 分子克隆： 实验室手册 (New York: Cold Spring Harbor Laboratory Press , 1989)中所述的条件， 或按照制造厂商所建议的条件。 实施例 1 G-CSF二聚体的制备
本发明的 G-CSF二聚体由 SEQ ID NO : l构成， 或由选自 SEQ ID NOs: 2-7的 G-CSF-Fc 复合物形成如附图 1-3的二聚体。 制备方法如下：
a . G-CSF 二聚体表达细胞株的构建
采用全基因合成 G-CSF-Fc复合物的 cDNA序列（如 SEQ ID NO: 10或 SEQ ID NO: 9所 示）， 将人 G-CSF单体的基因与 IgG2的 Fc基因片段连接， 5 '引入 Hindlll 位点， 以及哺乳 动物细胞表达所需要的元件如 Kozak序列和信号肽序列， 3 '端引入 EcoRI位点， 克隆到 pUC 19质粒， 命名为 pG-CSF-Fc， 转化 E. coli TG l。 用 Hind III和 EcoRI酶切 pUC 19质粒， 回收约 1400bp的 G-CSF-IgG2Fc片段， 与经 Hind III和 EcoRI酶切后的 pcDNA3 (Invitrogen) 表达质粒相连接， 构建表达质粒 pEX-G-CSF-Fc。 表达质粒 pEX-G-CSF-Fc经线性化， 纯 化， 电穿孔转化 CHO细胞， 筛选， ELISA 法检测表达量， 筛选出蛋白产量较高的细胞 株， 冻存细胞， 制备细胞库。
根据上述的方法可构建含有编码 SEQ ID NOs: 2-7cDNA序列的表达质粒， 通过线性 化转染 CHO细胞， 表达 G-CSF二聚体， ELISA 法检测表达量， 筛选出蛋白产量较高的细 胞株， 并制备细胞库。
b . 细胞规模培养
从细胞库取一支细胞 (；〜 lx 107 cells/ml)， 复苏于 10cm培养皿， 培养于 10mL基础培 养基， 37°C， 5% C02培养 24hr。
种子扩增： 将 10mL培养物传代至 30-40mL培养基中， 培养至细胞浓度达 1.0- 1.5 x 106 cells/mL且存活率≥90%时， 逐级扩大至 300-400mL培养基中， 置于摇瓶中 37°C， 5% C02. 培养， 转速 120rpm。
细胞罐中扩增 (3L-10L) ： 细胞浓度达 1.0 - 3.0 X 106 cells/mL且存活率≥ 90%时， 将 300-400mL培养物接种至 3-10 L细胞罐中培养， 培养基 pH控制在 6.8， 溶解氧约 50%， 转 速 65-100rpm。
细胞罐生产 (30-100L): 3L-10L细胞罐中的细胞浓度达 1.0 - 3.0 X 106 cells/ml且存活 率≥90%时， 将 3-10 L细胞罐中的培养物接种至 30-100L细胞罐， 培养基 pH控制在 6.8， 溶 解氧约 50%， 转速 65-100rpm， 培养 12-48hr开始控制培养基中的葡萄糖浓度 (<lg/L)， 采 用补料培养。
c G-CSF二聚体的分离纯化
细胞罐中培养结束后， 收集细胞上清（含有 G-CSF-Fc复合物、 G-CSF二聚体、
G-CSF-Fc多聚体及代谢物)， 细胞上清收集后经过滤、多级凝胶层析纯化， 例如， rProtein A Sepharose FF (GE Healthcare, cat# 17- 1279-04)捕获， 用 50 mM 柠檬酸 /柠檬酸钠， 0.2M NaCl,pH 3.7-3.8的缓冲液洗脱，得到纯度>90%的 G-CSF二聚体，接下来采用 Capto Adhere 层析， 用 50 mM NaAc/HAC ,0.2 M NaCl ,ρΗ 4.5-5.0的缓冲液洗脱， 再用 SP Sepharose FF (GE Heathcare Cat #17-0729-04)， 10mM PB (pH 6.0±0.1)缓冲液平衡， lOmM PB ，0.2M NaC pH 7.2±0.1)洗脱液， 洗脱液经低 pH除病毒， 过滤等， 最终获得 G-CSF二聚体。
分离纯化的 G-CSF二聚体的纯度 > 95% (采用反向 HPLC分析)， 分子量为 47±5 Kd (采 用还原型 SDS-PAGE分析)， 0糖基化占整个分子量的 2-10%， 等电点为 5.8-6.8， 紫外吸收 光谱为 280nm。 G-CSF二聚体在体外能激活 M-NFS-60细胞的 STAT3 , 刺激 M-NFS-60细胞 的增殖 (ED50为 0.1-10 ng/mL)。 实施例 2 G-CSF二聚体体内半衰期
大鼠皮下单次注射 G-CSF二聚体（二聚体由二个序列如 SEQ ID NO:3所示的 G-CSF-Fc复合物构成） 100 g/kg， 药代动力学参数如下表 1所示 （n=6)。 单体 G-CSF在大 鼠的半衰期约 2小时。
表 1 药代动力学参数
参数 单位 平均值 SD
AUC(O-t) ng/mL*h 4234.8 640.3
MRT(O-t) h 21.6 1.4
tl/2 h 7.7 1.2
CLz/F L/h/kg 0.024 0.003
Cmax ng/mL 162.2 30.2 实施例 3 G-CSF二聚体在人体内的药代动力学
24个健康受试者随机分成 4组， 分别为 30 60 120 240ug/kg剂量组， 给予 G-CSF 二聚体 (二聚体由二个序列如 SEQ ID NO:6所示的 G-CSF-Fc复合物构成)剂量分别为 30 60 120 240ug/kg,单次给药，于给药后 0.5 1 , 2, 4 8 16 24, 36, 48 72, 96 小时， 第 6(120 hrs), 7 9 11 13，和 15天， 采血分离血清冻存于 -70°C冰箱。 采用 ELISA法检测血药浓度 (ELISA, Quantikine human G-CSF ELISA kit, R&D System, Inc. Minneapolis, Min, Cat: PDCS50)。检测结果采用非房室模型分析药代动力学参数 (软件 WinNonlin v 5.2 Pharsight Corporation, USA), 结果如下表 2所示。
表 2 药代动力学参数
参数 （n=6) 3(Wkg 6(^g/kg 12(Wkg 240μ§/1¾
C (ng/mL) 21.3 (10.3) 44.6 (17.7) 219.9 (76.6) 759 (160)
T (h, median & range) 8 (8-16) 8 (8-16) 16 (16-36) 36 (36) tl/2 (h) 43.9 (4.3) 56.1 (23.3) 59.3 (23.5) 62.8 (10.8)
AUC(o-inf) (ng.h/mL) 778 (213) 1847 (686) 8349 (2769) 46664 (17258)
CL/F (mL/h/kg) 41.4 (12.8) 36.8 (14.6) 18.5 (7.7) 5.7 (2.0) 另外， 在该项临床研究中， G-CSF二聚体显示出了良好的安全性和耐受性。 实施例 4 G-CSF二聚体对 MPP+诱导的 PC12细胞神经毒性的保护作用
PC12细胞是一种大鼠嗜铬细胞瘤细胞系， 体外培养能合成、 代谢、 传递多巴胺， 可作为体外模型筛选具有活性的化合物。
实验将 PC12细胞按 40000个 /孔种于 96孔板， 培养基成分： DMEM , 10%马血清
+5%FCS 1% Penicillin-Streptomycin MPP+(Sigma)加入至 30 3000μηι终浓度， 分别加 入 G-CSF至 0.4ng/mL 4ng/mL 40ng/mL G-CSF二聚体至 lng/mL 10ng/mL lOOng/mL o 培养 24hr后， 采用荧光细胞存活分析 (Fluorimetric cell viability assay)。 结果表明， MPP+ 处理后， PC12细胞的存活率随着 MPP浓度的提高而降低， 在相同 MPP作用浓度时， 相同 摩尔 G-CSF分子浓度下， G-CSF二聚体对 PC12细胞的保护作用明显优于 G-CSF单体。
PC12细胞按 40000个 /孔种于 96孔板， 培养基成分： DMEM, 10%马血清 +5%FCS
1% Penicillin-Streptomycin MPP+(Sigma)加入至 1 100μηι终浓度， 分别加入 G-CSF单体 至 0.4 ng/mL 4 ng/mL 40ng/mL G-CSF二聚体至 1 ng/mL 10 ng/mL 100ng/mL。 培养
24hr后， 免疫组化检测酪氨酸羟化酶 (TH)， 黑质 TH阳性细胞计数。 结果表明， G-CSF二 聚体处理组， 黑质 TH阳性细胞数明显多于 G-CSF处理组。 在相同摩尔 G-CSF分子浓度条 件下， G-CSF二聚体对 PC12细胞的保护作用明显优于 G-CSF单体。 G-CSF单体与 G-CSF二聚体 (；选自序列如 SEQ ID NO:2-7所示的 G-CSF-Fc复合物构成 的 G-CSF二聚体）的分子量比值为约 1 :5, 1摩尔 G-CSF二聚体包含 2摩尔 G-CSF单体分子，因 此，在相同摩尔 G-CSF分子时， G-CSF单体与 G-CSF二聚体的质量比为约 1 :2.5，即 0.4ng/mL 的 G-CSF单体所含的 G-CSF分子摩尔浓度相当于 Ing/mL G-CSF二聚体所含的 G-CSF分子 摩尔浓度； 4ng/mL的 G-CSF单体所含的 G-CSF分子摩尔浓度相当于 10ng/mL G-CSF二聚 体所含的 G-CSF分子摩尔浓度； 40ng/mL的 G-CSF单体所含的 G-CSF分子摩尔浓度相当于 lOOng/mL G-CSF二聚体所含的 G-CSF分子摩尔浓度。 实施例 5 G-CSF二聚体对多巴胺能神经元细胞 STAT3的激活作用
取孕 14天 SD大鼠的胎鼠的全脑放入预冷的 D-Hanks液中， 在解剖显微镜下取黑质， 剪碎成 1 mm3大小， 加入 10 mL 0.125%胰酶， 并放入 37 °C孵箱内消化 15 min， 随后吸出 组织转移到装有预冷的 DMEM + 10%FBS的离心管内终止消化， 用移液枪吹打数次， 静 置后取上清并吸到另一支离心管内。 重复上述步骤 2〜3次。 用无血清神经元基础培养基 neurobasal(invitrogen,货号： 21 103049) + 无血清添加剂 B27(invitrogen，货号： 17504044) 培养 8天， 每 2天换液一次。 培养至第八天， 分别用不同浓度 G-CSF二聚体 (G-CSF-D)和 G-CSF单体 (G-CSF二聚体的终浓度： 1、 10、 lOO ng/mL; G-CSF的终浓度： 0.4、4、40 ng/mL) 处理神经元 15分钟 （Schneider A et al.. J Clin Invest 2005， 1 15(8):2083-2098)。 将培养基完 全吸出，细胞用 PBS洗两遍，按照细胞裂解液使用说明步骤裂解细胞。用细胞裂解液 (Cell Signaling Technology,货号： 9803； 主要成分： 20 mM Tris-HCl ( H 7.5)， 150 mM NaCl, 1 mM Na2EDTA， 1 mM EGTA, 1 % Triton, 2.5 mM sodium pyrophosphate , 1 mM beta-glycerophosphate, 1 mM Na3V04， 1 μ^τηΐ leupeptin, 1 mM PMSF)冰上裂解细胞 20 分钟， 用细胞刮子刮取细胞。 收集细胞裂解液， 12000rpm， 4°C离心 10分钟。 吸取上清， 测定蛋白浓度。 另取 ΙΟΟμΙ的上清用 STAT3 [pY705] ELISA 试剂盒 (invitrogen, 货号： KH00481)检测 STAT磷酸化水平变化。
在相同摩尔数 G-CSF分子浓度下， G-CSF二聚体 (G-CSF-D)与 G-CSF 单体相比， 二 聚体的 G-CSF 具有更强的激活多巴胺能神经元 STAT3的生物学活性。 实施例 6 G-CSF二聚体对 MPTP诱导的动物 PD模型的治疗作用
i -甲基—4-苯基 -1， 2， 3， 6-四氢毗啶 (MPTP), 可以特异性的损伤多巴胺能神经元， 使黑质多巴胺能神经元大量丢失， 出现类似帕金森氏病的症状。
实验选取 C57/BL6J 小鼠， 雄性， 20〜22g， 12-14周， 动物伺养于室温 24 ± 2。C 的 环境中， 维持光-暗 12小时循环交替， 给以充足的伺料， 自由饮水。
50只小鼠随机分成 5组，每组 10只，分别为溶剂对照组、 MPTP模型组、 MPTP+G-CSF 4(^g/kg组、 MPTP+GCSF-D 4(^g/kg组、 MPTP+GCSF-D l OO g/kg组， 其中 GCSF-D为由 选自序列如 SEQ ID NO :2-7所示的 G-CSF-Fc复合物构成的 G-CSF二聚体。
MPTP以 30mg/kg的剂量腹腔注射， 连续给予 5天， 恢复一天后 (从第 7天起) MPTP+G- CSF 4(^g/kg组按 G-CSF 4(^g/kg皮下注射给药， 每天给药 G-CSF, 每天一次， 连续 5天， 即从第 7〜1 1天； MPTP+GCSF-D 4(^g/kg组按 4(^g/kg皮下注射给药， 于第 7、 9天分别给 药 GCSF-D—次； MPTP+GCSF-D lOO g/kg组按 lOO g/kg皮下注射给药， 于第 7、 9天分别 给药 GCSF-D—次； 溶剂对照组给予等体积的生理盐水。
第 12天对动物进行以下评价。
a. PD小鼠的行为学评价
在本实验于 MPTP末次给药后第 10天进行行为学检测。 方法： 爬杆法 (pole test)用于 检测 PD中典型行为学症状一运动徐缓 (Matsuura etal, 1997; Araki et al, 2001 ; Kato et al, 2004)
将小鼠头向上轻柔的放在粗糙的杆顶 (直径 8 mm， 高 55cm)。 小鼠从头向上调整至头 完全向下的时间记录为 T-tum (time to turn)，小鼠从向下运动至四肢全部到达杆底的时间 记录为 T-LA (locomotion activity time), 超过 30 s 按照 30 s 记录。 每只小鼠重复检测 5次 取平均值。
结果表明， G-CSF二聚体能显著改善 MPTP诱导的小鼠行为学异常， 而且， 在同摩 尔 G-CSF分子的浓度下， G-CSF二聚体显示出比 G-CSF更好的效果。
b.检测紋状体内多巴胺的浓度
方法： 小鼠断头处死， 将纹状体取出称重后装在 1.5 ml的离心管中， 并立即置于碎 冰中。 样本每 10 mg 加入 300 μΐ 冰水浴中的样品处理液 (0.2 Μ 高氯酸、 0.2 mM焦亚硫 酸钠、 0.01% EDTA-2Na，同时含有 0.3 μΜ DHBA 作为内标）。 以上混合液使用超声仪超 声粉碎， 随后将其在 4°C 下 10,000 g 离心 20 min。 取其上清液并用 0.22μΜ 水相滤膜过 滤， 采用高效液相色谱法检测纹状体多巴胺的浓度。
结果表明， G-CSF二聚体能显著提高 MPTP诱导的小鼠纹状体多巴胺的浓度，而且， 在同摩尔 G-CSF分子的浓度下， G-CSF二聚体显示出比 G-CSF更好的效果， 具有显著性 差异。
c.黑质内多巴胺能神经元的情况观察
方法： 10%水合氯醛麻醉， 4%多聚甲醛灌流后取脑。 4%多聚甲醛后固定 24h， 再将 样本转入 10%， 20%, 30%的蔗糖溶液中梯度脱水至样本沉底， 在 -20°C 冰冻切片机中做 中脑与纹状体部位冠状切片， 小鼠脑切片厚度为 20μηι。 ΤΗ 为多巴胺能能神经元的特异 性标记。 一抗为单克隆小鼠抗 THC1 : 1000， CHEMICON), 与纹状体和中脑部位的脑片 4°C 共同孵育过夜， PBS 洗三遍后用生物素化二抗， 室温孵育 lh。 SABC 复合物室温孵育 DAB显色， 乙醇梯度脱水， 二甲苯透明， 中性树胶封片。 黑质 TH阳性细胞计数， 纹状体 TH 阳性染色光密度扫描。
结果表明， G-CSF二聚体可以明显保护 MPTP诱导的多巴胺能神经元的大量丢失， 而且， 在同摩尔 G-CSF分子的浓度下， G-CSF二聚体显示出比 G-CSF更好的效果， 具有 显著性差异。 实施例 7 G-CSF二聚体对海马神经元细胞 STAT3的激活作用
取孕 17天 SD大鼠的胎鼠的全脑放入预冷的 D-Hanks液中， 在解剖显微镜下取海马。 剪碎成 1 mm3大小， 加入 10 mL 0.125%胰酶， 并放入 37 °C孵箱内消化 15 min， 随后吸出 组织转移到装有预冷的 DMEM + 10%FBS的离心管内终止消化， 用移液枪吹打数次， 静 置后取上清并吸到另一支离心管内。 重复上述步骤 2〜3次。 用无血清神经元基础培养基 neurobasal(invitrogen, 货号： 21 103049)+ 无血清添加剂 B27(invitrogen， 货号： 17504044) 培养 8天， 每 2天换液一次。 培养至第八天， 分别用不同浓度 G-CSF二聚体（由两个选自序 列如 SEQ ID NO:2-7所示的 G-CSF-Fc复合物构成)和 G-CSF(G-CSF二聚体的终浓度： 1、 10、 100 ng/mL; G-CSF单体的终浓度： 0.4、 4、 40 ng/mL)处理神经元 15分钟 （Schneider A et al.J Clin Invest 2005, 1 15(8):2083-2098 将培养基完全吸出， 细胞用 PBS洗两遍， 按照细胞裂解液使用说明步骤裂解细胞。用细胞裂解液 (Cell Signaling Technology,货号： 9803； 主要成分： 20 mM Tris-HCl ( H 7.5)， 150 mM NaCl, 1 mM Na2EDTA， 1 mM EGTA, 1 % Triton, 2.5 mM sodium pyrophosphate, 1 mM beta-glycerophosphate, 1 mM Na3V04, 1 g/ml leupeptin, 1 mM PMSF)冰上裂解细胞 20分钟， 用细胞刮子刮取细胞。 收集细胞裂解液， 12000rpm， 4°C离心 10分钟。 吸取上清， 测定蛋白浓度。 另取 ΙΟΟμΙ的 上清用 STAT3 [pY705] ELISA 试剂盒 (invitrogen, 货号： KH00481)检测 STAT磷酸化水 平变化。
在相同摩尔数 G-CSF分子浓度下， G-CSF二聚体 (G-CSF-D)与 G-CSF 单体相比， 二 聚体的 G-CSF 具有更强的激活海马神经元 STAT3的生物学活性。 实施例 8 G-CSF二聚体对 诱导的 PC12细胞凋亡的保护作用
PC 12细胞用神经因此 (NGF)诱导后可长出突起， 具有神经细胞特性， Αβ(β淀粉样蛋 白)诱导 PC 12细胞凋亡可以体外模拟 AD模型。
将 PC 12细胞培养于基础培养基 (DMEM， 10%FCS , 1% Penicillin- Streptomycin) , 胰 酶消化， 重悬于含 NGF 50ng/mL的培养基中， 细胞浓度调整为 2X104cells/孔加入 96孔板， 于 37°C， 5%C02培养箱中培养 24hr。 加入 Αβ至终浓度 1-100 μηιοΙ/L, 分别用不同浓度的 G-CSF单体和 G-CSF-D(GCSF-D为由选自序列如 SEQ ID NO:2-7所示的 G-CSF-Fc复合物 构成的 G-CSF二聚体）处理， G-CSF单体的终浓度分别为 0.4、 4、 40ng/mL，G-CSF-D的终 浓度分别为 1、 10、 lOOng/mL, 模型孔加入等体积的 PBS， 阴性对照孔不加 Αβ， 继续培 养 24hr。 Hochest染色观察细胞形态学， 或 MTT法检测 PC12细胞的增殖。
与阴性对照孔相比， 模型孔的 PC12细胞细胞核荧光染色明显不均匀， 可见到细胞凋 亡引起的致密浓染的高荧光细胞核， G-CSF单体和 G-CSF二聚体处理的 PC12细胞细胞核 荧光染色均匀， 未见到明显的致密浓染的高荧光细胞核。 G-CSF二聚体能抑制 NGF分化 后 Αβ诱导所致的 PC12细胞的凋亡， 保护神经细胞。 实施例 9 G-CSF二聚体对 诱导的动物 AD模型的治疗作用
SD大鼠雄性， 180〜220g， 动物伺养于室温 24±2 °C 的环境中， 维持光-暗 12 小时 循环交替， 给以充足的伺料， 自由饮水。
50只大鼠随机分成 6组， 分别为溶剂对照组、 Αβ模型组、 Αβ+G-CSF 40μ§/1¾组、 Αβ+GCSF-D 4(^g/kg组、 Αβ+GCSF-D lOO g/kg组， 每组 10只。 其中， GCSF-D为由选自 序列如 SEQ ID NO:2-7所示的 G-CSF-Fc复合物构成。
实验大鼠用 1%戊巴比妥钠 (40mg/kg)麻醉后， 固定头部， 备皮消毒， 沿颅顶中线作
2cm切口， 分离骨膜暴露头骨， 于前囟向后 3.0mm， 中线左右各旁开 2.2mm， 用牙科钻打 开颅骨， 垂直进微量进样器针 2.8mm， 模型组和各药物处理组将 5μ1 Αβ1-40(β淀粉样蛋 白）(2μ§/μ1)溶液注入， 假手术组注射 5ul生理盐水。
动物造模后第 3天开始给药， Αβ+G-CSF 40μ§/1¾组按 G-CSF 40μ§/1¾皮下注射给药 G-CSF, 每天给药， 连续 5天； Ap+GCSF-D 4(^g/kg组按 40ug/kg皮下注射给药， 于第 3、 5天分别给药 GCSF-D—次； Αβ+GCSF-D 10(^g/kg组按 lOO g/kg皮下注射给药 GCSF-D, 于第 3、 5天分别给药一次； 溶剂对照组给予等体积的生理盐水。
第 10天进行 Morris 水迷宫行为测试行为学。
行为学测试完成后， 10%水合氯醛麻醉， 4%多聚甲醛灌流后取脑。 4%多聚甲醛后 固定 24h， 再将样本转入 10%， 20%, 30%的蔗糖溶液中梯度脱水至样本沉底，在 -20°C 冰 冻切片机中做海马部位冠状切片， 切片厚度为 20μηι。 NeuN (神经元核抗原)为神经元的特 异性标记物，一抗与海马部位的脑片 4°C 共同孵育过夜， PBS 洗三遍后用生物素化二抗， 室温孵育 lh。 SABC 复合物室温孵育 lh。 DAB显色， 乙醇梯度脱水， 二甲苯透明， 中 性树胶封片。 海马 NeuN 阳性细胞计数。
G-CSF处理组和 G-CSF二聚体处理组均能改善模型大鼠的学习巩固和再现能力。 与 假手术组比较， 模型组大鼠海马区 NeuN 阳性神经元细胞数表达降低。 与模型组比较， G-CSF处理组和 G-CSF二聚体处理组 NeuN 阳性神经元细胞数均有升高，与 G-CSF单体处 理组比较， G-CSF二聚体处理组（10(^g/kg)NeuN 阳性神经元细胞数明显升高。 实施例 10 G-CSF二聚体对 MPTP诱导的 PD动物模型的治疗作用
i -甲基—4-苯基 -1， 2， 3， 6-四氢毗啶（MPTP) ， 可以特异性的损伤多巴胺能神经元， 使黑质多巴胺能神经元大量丢失， 从而出现类似帕金森氏病 （PD ) 的症状。 酪氨酸羟化 酶（TH)是多巴胺能神经元的特异性标记， 可以用于定量检测黑质多巴胺能神经元的数 实验选取雄性 C57/BL6J 小鼠， 22〜30g， 12-14周， 随机分成 4组， 分别为：
MPTP+G-CSF-D 3(^g/kg组： 以 30 mg/kg的剂量腹腔注射 MPTP, 连续给予 5天， 动物 恢复一天后 （即从第 7天起） 按 30 g/kg皮下注射给予 G-CSF-D, 分别于第 7、 9、 11天分 别给药一次；
MPTP+G-CSF-D lOO g/kg组： 以 30 mg/kg的剂量腹腔注射 MPTP, 连续给予 5天， 动 物恢复一天后 （即从第 7天起） 按 100 g/kg皮下注射给予 G-CSF-D, 分别于第 7、 9、 11 天分别给药一次；
MPTP模型组： 以 30mg/kg的剂量腹腔注射 MPTP, 连续给予 5天， 动物恢复一天后， 从第 7天起给予等体积的溶剂 （0.5%鼠血清 /PBS ) ；
正常对照组： 给予等体积的生理盐水， 连续给予 5天， 动物恢复一天后， 从第 7天起 给予等体积的溶剂 （0.5%鼠血清 /PBS ) 。
所述 G-CSF二聚体 （G-CSF-D) 由二个序列如 SEQ ID NO:6所示的 G-CSF-Fc构成。 第 12天处死动物， 检测纹状体内多巴胺浓度、 观察纹状体内多巴胺能神经纤维与黑 质内多巴胺能神经元的情况。 a.检测紋状体内多巴胺的浓度
方法： 小鼠断头处死， 将纹状体取出称重后装在 1.5ml 的离心管中， 并立即置于碎 冰中。 样本每 10 mg 加入 300 μ1 冰水浴中的样品处理液 （0.2 Μ 高氯酸、 0.2 mM焦亚硫 酸钠、 0.01% EDTA-2Na， 同时含有 0.3 μΜ DHBA 作为内标） 。 以上混合液使用超声仪 超声粉碎， 随后将其在 4°C下 10,000 g 离心 20 min。 取其上清液并用 0.22μΜ 水相滤膜过 滤， 采用高效液相色谱法检测纹状体多巴胺的浓度。
结果如图 4所示，小鼠连续 5天注射 MPTP后，纹状体中多巴胺的浓度的大幅度下降， 使用 G-CSF二聚体治疗可以剂量依赖的提高纹状体多巴胺的浓度。
结果表明， 与正常对照组相比， MPTP可以引起纹状体中多巴胺的浓度的大幅度下 降 （### p < 0.001 ) 。 G-CSF二聚体能显著抑制 MPTP诱导的小鼠纹状体多巴胺的含量降 低， 提高纹状体中多巴胺的浓度。 而且， G-CSF二聚体给药组显示出剂量依赖关系， 与
MPTP模型组相比均具有显著性差异 （* p < 0.05 ) 。 b.紋状体内多巴胺能神经纤维与黑质内多巴胺能神经元的情况观察
方法： 10%水合氯醛麻醉， 4%多聚甲醛灌流后取脑。 4%多聚甲醛后固定 24h， 再将 样本转入 10%， 20%, 30%的蔗糖溶液中梯度脱水至样本沉底， 在一 20°C 冰冻切片机中 做纹状体与中脑部位冠状切片， 小鼠脑切片厚度为 20μηι， TH免疫组化分析。 一抗为单 克隆小鼠抗 TH抗体 （1 : 1000， Sigma) ， 与纹状体和中脑部位的脑片 4°C 共同孵育过夜， PBS 洗三遍后用生物素化二抗 (羊抗鼠）， 室温孵育 lh。 SABC 复合物室温孵育 lh。 DAB 显色， 乙醇梯度脱水， 二甲苯透明， 中性树胶封片。进行纹状体 TH阳性染色光密度扫描， 黑质致密部 TH阳性细胞计数等测试。
结果如图 5A、 图 5B、 图 6A和图 6B所示：
图 5A显示了小鼠纹状体 TH阳性神经纤维免疫组化染色图片。 小鼠连续 5天注射 MPTP后， 纹状体 TH阳性神经纤维的密度降低， 使用 G-CSF二聚体治疗可以剂量依赖的 增加 TH阳性神经纤维数量， 可见 G-CSF二聚体对 MPTP诱导的多巴胺能神经纤维的丢失 有显著保护作用。
图 5B显示了小鼠纹状体 TH阳性神经纤维免疫组化光密度值。 结果说明， MPTP可以 引起纹状体 TH阳性神经纤维的密度的大幅度下降 （### p < 0.001 ) 。 而 G-CSF二聚体可 以明显抑制 MPTP诱导的纹状体 TH阳性神经纤维的密度减少。 G-CSF二聚体给药组显示 出剂量依赖关系， 与 MPTP模型组相比均具有显著性差异 （* p < 0.05 ) 。
图 6A显示了小鼠黑质致密部 TH阳性神经元免疫组化染色图片。 小鼠连续 5天注射 MPTP后， 黑质致密部 TH阳性神经元大量丢失， 使用 G-CSF二聚体治疗可以恢复 TH阳性 神经元数量， 可见 G-CSF二聚体对 MPTP诱导的多巴胺能神经元的大量丢失有显著保护 作用。
图 6B显示了小鼠黑质致密部 TH阳性细胞计数分析结果。 与正常对照组相比， 连续 5 天注射 MPTP后， 黑质致密部 TH阳性神经元的数量显著减少 （### p < 0.001 ) ， 大约为 正常对照组的 49%， 说明 MPTP导致黑质内多巴胺能神经元的大量丢失。 而 G-CSF二聚体 组可以明显抑制 MPTP诱导的黑质致密部 TH阳性神经元数量的丢失， 可剂量依赖地增加 黑质 TH-阳性细胞数。 G-CSF二聚体给药组显示出剂量依赖关系， 与 MPTP模型组相比均 具有显著性差异 （*** p < 0.001 ) ， G-CSF二聚体 30 g/kg剂量组黑质内 TH阳性多巴胺神 经元大约为正常对照组的 86 %， G - C S F二聚体 100 μ g/k g剂量组黑质内 T H阳性多巴胺神经 元大约为正常对照组的 99%， 相当于正常对照组的水平。
可见， G-CSF二聚体可以明显保护 MPTP诱导的多巴胺能神经纤维的丢失， 还可以 明显保护 MPTP诱导的多巴胺能神经元的大量丢失。 对比例 1 G-CSF单体对 MPTP诱导的 PD动物模型的治疗作用
实验方法 (；参见 US7723302): 对小鼠以 30 mg/kg腹腔注射 MPTP, 连续给予 5天， 获 得 PD小鼠模型； 动物恢复一天后给予 250 g/kg G-CSF (Neupogen, Amgen) ， 连续给 药 7天， 最后一次给药后 （即给药结束后） 的不同时间观察黑质致密部 TH阳性多巴胺能 神经元的数量。
模型组： 第一次给药前的黑质致密部 TH阳性多巴胺能神经元的数量。
正常组： 未注射 MPTP的小鼠。
给药结束后第 1、 7、 14天黑质致密部 TH阳性多巴胺能神经元大约分别恢复至正常 组的 70%、 80%、 77%。 讨论：
如实施例 10所述， 本发明的 G-CSF二聚体， 每次以 30 g/kg或 100 g/kg的剂量， 间 断性地给药三次后， 在结束给药后的第 1天， 30 g/kg组、 100 g/kg组中黑质内多巴胺 TH 阳性神经元分别大约恢复至正常对照组的 86%、 99%。 尤其是， 100 g/kg组， 在给药结 束后的第 1天， 其 TH阳性多巴胺能神经元基本完全恢复至正常对照组水平。
而对比例 1中，即使采用 250 g/kg 剂量的 G-CSF, 连续给药 7天， 在结束给药后的第 1天， TH阳性多巴胺能神经元也仅仅只能恢复至正常组的 70 %。
从总给药剂量上看： G-CSF二聚体 100 g/kg组的总给药剂量为 300 μ§, 对比例 1中 G-CSF单体的总给药剂量为 1750 μ§。
从 G-CSF单体摩尔分子浓度来看： G-CSF单体与 G-CSF二聚体的分子量比值约为
1 :5， 因此， 本发明的 G-CSF二聚体 100 g/kg组中， G-CSF单体摩尔分子浓度仅为对比例 1的 1/15。
可见， 采用本发明所述的 G-CSF二聚体， 在治疗过程中， 给药剂量更低， 疗效更好、 更快， 且给药频率低， 非常有利于提高患者的依从性。 在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引 用作为参考那样。 此外应理解， 在阅读了本发明的上述讲授内容之后， 本领域技术人员 可以对本发明作各种改动或修改， 这些等价形式同样落于本申请所附权利要求书所限定 的范围。

Claims

权 利 要 求
1. 集落刺激因子 G-CSF的二聚体在制备治疗或预防神经退行性疾病的组合物中的用 途。
2. 如权利要求 1所述的用途， 其特征在于， 所述神经退行性疾病选自： 帕金森氏病、 阿 尔茨海默氏病、 亨廷顿舞蹈病、 肌萎縮侧索硬化病， 脊髓肌萎縮病、 原发性侧索硬化病， 脊 髓小脑共济失调。
3. 如权利要求 1或 2所述的用途， 其特征在于， 所述集落刺激因子 G-CSF的二聚体为人集 落刺激因子 G-CSF二聚体。
4. 如权利要求 1或 2所述的用途， 其特征在于， 所述集落刺激因子 G-CSF的二聚体的结 构如式 I所示：
M1-L-M2 式 I
式中，
Ml是人集落刺激因子 G-CSF的第一单体；
M2是人集落刺激因子 G-CSF的第二单体；
L是位于所述的第一单体和第二单体之间的， 将所述第一单体和第二单体连接在一起的 接头元件，
其中， 所述的 G-CSF二聚体保持了 G-CSF的生物活性， 并且其血清半衰期是所述第一单 体或第二单体的血清半衰期的 2倍以上。
5. 一种治疗神经退行性疾病的药物， 其特征在于， 含有作为活性成分的集落刺激因子
G-CSF的二聚体。
6. 如权利要求 5所述的药物， 其特征在于， 所述神经退行性疾病选自： 帕金森氏病、 阿 尔茨海默氏病、 亨廷顿舞蹈病、 肌萎縮侧索硬化病， 脊髓肌萎縮病、 原发性侧索硬化病， 脊 髓小脑共济失调。
7. 如权利要求 5或 6所述的药物， 其特征在于， 所述集落刺激因子 G-CSF的二聚体为人集 落刺激因子 G-CSF二聚体。
8. 如权利要求 5或 6所述的药物， 其特征在于， 所述集落刺激因子 G-CSF的二聚体的结 构如式 I所示：
M1-L-M2 式 I
式中，
Ml是人集落刺激因子 G-CSF的第一单体；
M2是人集落刺激因子 G-CSF的第二单体； L是位于所述的第一单体和第二单体之间的， 将所述第一单体和第二单体连接在一起的 接头元件，
其中， 所述的 G-CSF二聚体保持了 G-CSF的生物活性， 并且其血清半衰期是所述第一单 体或第二单体的血清半衰期的 2倍以上。
9. 一种治疗神经退行性疾病的方法， 其特征在于， 包括步骤：
给需要治疗的对象施用集落刺激因子 G-CSF的二聚体。
10. 如权利要求 9所述的方法， 其特征在于， 所述神经退行性疾病选自： 帕金森氏病、 阿 尔茨海默氏病、 亨廷顿舞蹈病、 肌萎縮侧索硬化病， 脊髓肌萎縮病、 原发性侧索硬化病， 脊 髓小脑共济失调。
PCT/CN2012/079106 2011-07-25 2012-07-24 G-csf二聚体在制备治疗神经退行性疾病药物中的应用 WO2013013613A1 (zh)

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