WO2002015926A1 - Compositions medicinales contenant des ligands c-mpl, destinees a l'augmentation des plaquettes et des erythrocytes - Google Patents

Compositions medicinales contenant des ligands c-mpl, destinees a l'augmentation des plaquettes et des erythrocytes Download PDF

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WO2002015926A1
WO2002015926A1 PCT/JP2001/007283 JP0107283W WO0215926A1 WO 2002015926 A1 WO2002015926 A1 WO 2002015926A1 JP 0107283 W JP0107283 W JP 0107283W WO 0215926 A1 WO0215926 A1 WO 0215926A1
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administration
day
patients
peg
rhumgdf
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PCT/JP2001/007283
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Japanese (ja)
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Norio Komatsu
Akio Urabe
Masami Bessho
Hideaki Mizoguchi
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Kirin Beer Kabushiki Kaisha
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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
    • A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • A61K47/59—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
    • A61K47/60—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/06—Antianaemics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • a pharmaceutical composition for increasing platelets and erythrocytes comprising the c-mp1 ligand
  • the present invention relates to a pharmaceutical composition for increasing platelets and erythrocytes, comprising a c-mpl ligand. More specifically, the present invention relates to a pharmaceutical composition for increasing platelets and erythrocytes, comprising a c-mpl ligand in a patient with myelodysplastic syndrome or aplastic anemia.
  • myelodysplastic syndrome and aplastic anemia are diseases characterized by cytopenia due to bone marrow dysfunction due to hematopoietic stem cell damage.
  • Myelodysplastic syndrome is a condition in which the bone marrow generally shows normal or hyperplasia and a dysplastic image of blood cells is observed, and effective blood cell production is not performed due to qualitative abnormalities in hematopoietic stem cells (so-called ineffective hematopoiesis).
  • the disease is chronic and irreversible and has a poor prognosis such as acute nonlymphocytic leukemia or death due to infection and bleeding associated with bone marrow failure (Yataro Yoshida et al., Myelodysplastic Syndrome.
  • aplastic anemia is considered to be a disease caused by a decrease in hematopoietic stem cells and abnormal hematopoietic microenvironment because bone marrow is hypoplastic and does not increase the number of atypical cells (blasts) (Akio Urabe) , Aplastic anemia, Shinshin University of Science, Nakayama Shoten 1992; 18: 365).
  • Allogeneic bone marrow transplantation is selected for critically ill patients at the age of transplantation, and sterol pulse therapy, cyclosporine administration, anti-thymocyte immunoglobulin and immunosuppressive therapy for other severe and moderately ill patients.
  • Anti-lymphocyte globulin administration is performed.
  • myelodysplastic syndrome for cytopenia, anabolic hormone and androgen therapy and cytokine therapy or blood transfusion are administered to stimulate hematopoiesis.
  • G-CSF granulocyte colony stimulating factor
  • EP0 erythropoietin
  • GM-CSF granulocyte monocyte colony stimulating factor
  • Interleukins IL-1, IL-3, IL-6, etc.
  • an object of the present invention is to provide a pharmaceutical composition having a platelet and erythrocytosis effect in a patient with myelodysplastic syndrome or aplastic anemia.
  • c-mp 1 ligand not only has a thrombocytopenic effect but also anemia symptoms of myelodysplastic syndrome or regenerative poor anemia patients. It has been found that it can be significantly improved.
  • the present invention includes the following.
  • the present invention provides a pharmaceutical composition for increasing platelets and erythrocytes in a patient with myelodysplastic syndrome or aplastic anemia, comprising a therapeutically effective amount of a c-mp1 ligand.
  • the c-mpl ligand is obtained from a polypeptide having an amino acid sequence containing at least the amino acid residues at positions 7 to 151 of the amino acid sequence of SEQ ID NO: 1, a variant thereof, or a derivative thereof. And has a thrombopoetin (TPO) activity.
  • TPO thrombopoetin
  • the term “thrompopoietin activity” refers to an activity that promotes the growth and differentiation of megakaryocyte progenitor cells in a mammal (preferably a human), or specifically stimulates or enhances the production of platelets in the body of the animal. .
  • the c-type; mp1 ligand is a polypeptide consisting of the amino acid sequence 1-332 of SEQ ID NO: 1, a variant thereof or a derivative thereof, and having a thrombopoietin activity;
  • a polypeptide consisting of the amino acid sequence 1-163 of SEQ ID NO: 1, a variant thereof or a derivative thereof and having trombopoietin activity can be exemplified.
  • the variant is a polypeptide comprising at least one amino acid substitution, deletion, addition and / or insertion in the amino acid sequence of SEQ ID NO: 1 and having a trombopoietin activity.
  • the derivative is a polypeptide modified with a water-soluble polymer or a variant thereof.
  • a water-soluble polymer can be attached to at least the N-terminus of the polypeptide or a variant thereof.
  • a preferred example of the water-soluble polymer is polyethylene glycol.
  • the C-mpl ligand is a PEG-rHuMGDF in which one molecule of polyethylene glycol is bound to the N-terminus of a recombinant polypeptide consisting of the amino acid sequence of SEQ ID NO: 2 (see Examples described later). It is.
  • this PEG-rHuMGDF was administered to a myelodysplastic syndrome group or aplastic anemia patient at a protein weight of about 2.5 g (about 143 pmol) / kg / day for about 2 weeks, No continuous increase in reticulocyte count was observed up to the week, and it increased continuously in about 2 to about 5 weeks and reached a maximum value, which was about 1.5 to 1.5 times the value on the first day of administration.
  • c-mp1 ligand refers to C-mpl (TP0 receptor; Vigon, I. et al., Proc. Natl. Acad. Sci. USA 89: 5640-5644 (1992 ) A substance that binds to and acts on).
  • a "therapeutically effective amount” refers to the promotion or proliferation of megakaryocyte progenitor cells in patients with myelodysplastic syndrome and reproductive anemia. It means a dose that increases platelets by stimulating or enhancing the production of platelets (so-called thrombopoietin activity) and provides a significant erythrocytosis effect.
  • variant refers to a mutant having at least one amino acid substitution, deletion, addition and / or insertion in the amino acid sequence of SEQ ID NO: 1 and having trombopoietin activity.
  • derivative refers to a protein, polypeptide or peptide containing a chemical modification such as addition of a water-soluble polymer (eg, polyethylene glycol), glycosylation, phosphorylation, or sulfation.
  • FIG. 1 is a graph showing the change in platelet count in myelodysplastic syndrome patients (Example 1). Patients were administered 2.5 S (approximately 143 pmol) / kg / day of PEG-rHuMGDF by protein weight for 14 days from day 1 to day 14, and three times a week until day 28 after the start of administration, Platelet counts were measured weekly until day 42. Platelet transfusion was not performed during the period shown in the figure.
  • FIG. 2 is a graph showing changes in hemoglobin concentration in a patient with myelodysplastic syndrome (Example 1). 2.5 g (about 143 pmol) / kg / day of protein weight for patients
  • PEG-rHuMGDF was administered for 14 days from day 1 to day 14, and the hemoglobin concentration was measured three times a week until day 28 after administration and once a week until day 42. Shown in the figure Erythrocyte transfusions were not performed during this period.
  • FIG. 3 is a graph showing the change in reticulocyte count in a myelodysplastic syndrome patient (Example 1).
  • Patients were administered PEG-rHuMGDF at a protein weight of 2.5 g (approximately 143 pmol) / kg / day for 14 days from day 1 to day 14, and three times a week until day 28 after the start of treatment, and then 42 Until the day, reticulocyte count was measured once a week.
  • the reticulocyte count shown in the figure is a value calculated by multiplying the reticulocyte count by the measured ratio of reticulocytes per 1000 red blood cells (VD () ).
  • FIG. 4 is a graph showing the change in platelet count in myelodysplastic syndrome patients (Example 2). Patients were administered 2.5 g (approximately 143 pmol) / kg / day of PEG-rHuMGDF by protein weight for 14 days from day 1 to day 14, and three times a week until day 28 after the start of dosing. Platelet counts were measured weekly until day 42. Platelet transfusion was not performed during the period shown in the figure.
  • FIG. 5 is a graph showing changes in hemoglobin concentration in a myelodysplastic syndrome patient (Example 2).
  • Patients were administered PEG-rHuMGDF at a protein weight of 2.5 / g (approximately 143 pmol) / kg / day for 14 days from day 1 to day 14, three times a week until day 28 after the start of treatment, and Hemoglobin levels were measured weekly until day 42. Erythrocyte transfusion was not performed during the period shown in the figure.
  • FIG. 6 is a graph showing the change in reticulocyte count in a myelodysplastic syndrome patient (Example 2).
  • Patients were administered PEG-rHuMGDF at a protein weight of 2.5 g (approximately 143 pmol) / kg / day for 14 days from day 1 to day 14, and three times a week until day 28 after the start of treatment, and then 42 Until the day, reticulocyte count was measured once a week.
  • the reticulocyte count shown in the figure is a value calculated by multiplying the reticulocyte count per 1000 measured red blood cells ( Q ) by the red blood cell count.
  • FIG. 7 is a graph showing changes in platelet count in patients with aplastic anemia. Patients were administered 2.5 zg (approximately 143 pmol) / kg / day of PEG-rHuMGDF in terms of protein weight for 14 days from day 1 to day 14, and three times a week until day 28 after administration. Platelet counts were measured weekly until day 42. Platelet transfusion was not performed during the period shown in the figure.
  • Figure 8 is a graph showing changes in hemoglobin concentration in aplastic anemia patients. is there. .5 / ig (about 143 11101) / 13 ⁇ 4 / day in protein weight for the patient? £ 6-]: 11 60? Was administered for 14 days from the first day to the 14th day, and hemoglobin concentration was measured three times a week until the 28th day and once a week until the 4th day. Erythrocyte transfusion was not performed during the period shown in the figure.
  • FIG. 9 is a graph showing the change in reticulocyte count in aplastic anemia patients.
  • Patients received 2.5 xg (approximately 143 pmol) / kg / day of PEG-rHuMGDF for 14 days from day 1 to day 14, three times a week until day 28 after administration, and then day 42 Up to once a week, reticulocyte counts were measured.
  • the reticulocyte count shown in the figure is the ratio of reticulocytes per 1000 red blood cells measured.
  • any protein or peptide substance or a non-protein substance exhibiting a platelet and erythrocytosis effect in a patient with myelodysplastic syndrome or aplastic anemia is used as the c-mp1 ligand.
  • proteinaceous c-mp1 ligands include: (1) thrompopoetin (TP0 or MGDF), its mutants and derivatives (W095 / 18858 or Japanese Patent Publication No. 9-508262, Japanese Patent No. 2991640) Gazettes, Japanese Patent No. 2991630 and Japanese Patent No.
  • Examples of the peptidic c-mp1 ligand include peptide compounds capable of binding to the 1 receptor immediately (W096 / 40189, W096 / 40750, W098 / 25965, Japanese Patent Application Laid-Open No. 10-492, W099 / 42127, WO00 / 24770, etc.).
  • non-proteinaceous c-mp1 ligands examples include (1) benzodiazepine derivatives (JP-A-11-1477, JP-A-11-152276, etc.), and (2) other low molecular weight systems.
  • mpl receptor Yuichi ligand W099 / 1 1262, W099 / 22733, W099 / 22734, W000 / 35446, WO00 / 28987, etc.
  • Particularly preferred c-mp1 ligands are thrombopoietin, variants and derivatives thereof. Mutants and derivatives of thrombopoietin that can be used in the present invention need to have a biological activity that specifically stimulates or increases platelet production.
  • Thrombopoetin includes, for example, a polypeptide having the amino acid sequence of 1 to 332 of SEQ ID NO: 1 or a mutant thereof.
  • various mutants of thrompopoetin can be obtained by using known mutagenesis methods including site-directed mutagenesis and polymerase chain reaction (PCR) (see US Pat. No. 4,518,584). Can be.
  • an example of such a variant is a truncation-type protein comprising at least the amino acid sequence of positions 7 to 151 in the amino acid sequence of SEQ ID NO: 1, e.g., 1-244, 1-231, 1-211, 1-191, 1-177, 1-174, 1-171, 1-; 170,; ⁇ 169, 1 ⁇ : L63, 1 ⁇ ; 158, 1 ⁇ : L57, 1-156, 1-155, 1-154, 1-153, 1-155 1, 7 to 16 3 etc. are included.
  • Non-limiting examples of other mutants that can be used in the present invention include the following.
  • T hr 33 , T hr 333 , Ser 334 lie 335 , G y 336 , Tyr 337 , Pro 338 , Tyr 339 , Asp 340 , Va 1 341 , Pro 342 , Asp 343 , Ty r 344 , A 1a 345 , G 1 y 346 , Va 1 347 , H is 348 , H is 349 , H is 35 . , H is 351 , H is 352 , H is 353 ] TPO (1-332),
  • [As n 25 ] TPO (group 332) is that the amino acid at position 25 in amino acid sequence 1 to 332 (column number 1) of TP ⁇ is replaced with asparagine (Asn) Is shown.
  • [AH is 33 ] TPO (1-163) indicates that histidine (His) at position 33 of the truncation amino acid sequence 1 to 163 (SEQ ID NO: 2) has been deleted.
  • TP O (1-163) is 33-position and 34-position amino acid histidine respective Toranke one Chillon amino acid sequence 1-163 (SEQ ID NO: 2) (His) or proline (Pro), and threonine (Thr) is inserted (or added) between these amino acids.
  • the TPO protein or a variant thereof may further include the amino acid sequence [Me t " 2 -L ys-, [Me t" 1 ] or [G 1 y " 1 ].
  • Met_Lys contains the target protein. After expression in bacterial cells and purification, they can be removed by treatment with an enzyme such as a dipeptidase (eg, cathepsin C).
  • specific mutants in the amino acid sequence of the TPO protein may also include alterations in the site of glycosylation (such as serine, threonine, or asparagine). No or only partial glycosylation occurs Not as a result of amino acid substitution or deletion at the glycosylation recognition site containing the asparagine residue, or at the site of the molecule modified by the addition of an o-linked carbohydrate.
  • the glycosylation recognition site containing the asparagine residue is composed of a tripeptide sequence that is specifically recognized by the appropriate cellular glycosylation enzyme. This tripeptide sequence is Asn- Xaa- Thr or
  • Asn-Xaa-Ser where Xaa is any amino acid except proline.
  • Xaa is any amino acid except proline.
  • various amino acid substitutions or deletions occur at one or both of the first and third amino acid positions of the glycosylation recognition site (and / or the amino acid deletion at the first position). Glycosylation does not occur in the modified tripeptide sequence.
  • a new glycosylation recognition sequence in the amino acid sequence of the TPO protein has been added to the extent that the three-dimensional structure (or conformation) of the TPO protein is not significantly changed and the biological activity can be substantially maintained. Sites can also be introduced.
  • the TPO protein obtained as a result of glycosylation can have not only the same carbohydrate chains as natural (particularly humans) but also non-natural carbohydrate chains.
  • C-mp comprising TPO and its variants as described above that can be used in the present invention.
  • the ligand can be prepared by a known method (Japanese Patent No. 2991640, Vol. 95/26746, etc.) or a conventional gene recombination technique (Sambrook et al., Molecular Cloning, A Laboratory).
  • the DNA encoding the protein having a thrombopoietin activity can be obtained from the National Institute of Advanced Industrial Science and Technology (Tsukuba-Higashi 1-1, Ibaraki, Japan, Central No. 6) or the People's Republic of China (CCTCC) by the applicant. Is a cell line deposited with the depositary institution of the Republic of China (FI RD 1), such as FER
  • HTF 1 HTF 1
  • FERM BP—46 16 CCTCC-M 95003
  • FI RD I 940084 vector pHGT 1
  • FERM BP—4988, CCTCC-C 95004 FI RD 1 960023 (CH028Z1 / 1Z3—C 6)
  • FERB BP—4989, CCTCC_C 95005 FI RD I 9600 24 (CH0163T—63— 79—CI).
  • a suitable expression vector such as a plasmid
  • the vector is used to transform prokaryotic (such as E. coli) or eukaryotic host cells (such as Chinese hamster ovary cells) and obtained.
  • the c-mp1 ligand can be obtained by any of the means commonly used for protein purification, such as ion exchange chromatography, lectin affinity chromatography, triazine dye adsorption chromatography, hydrophobic mutual chromatography, gel filtration chromatography, reverse Phase chromatography, heparin affinity chromatography, sulfated gel chromatography, hide-mouth xylapatite chromatography, antibody affinity mouth chromatography, isoelectric focusing, metal chelating chromatography, min Purification can be achieved by combining one or more means such as preparative electrophoresis and isoelectric focusing.
  • An example of a derivative of a TPO protein that can be used in the present invention is a c-mp1 ligand protein attached to at least one water-soluble polymer moiety.
  • Water-soluble polymers include, for example, polyethylene glycol, monomethoxy-polyethylene dalicol, dextran, poly (N-vinylpyrrolidone) polyethylene glycol, propylene glycol homopolymer, polypropylene oxide / ethylene oxide copolymer, polyoxyethylated polyol (ie, Glycerol) and polyvinyl alcohol.
  • PEGylated c-mp1 ligand molecule attached to at least one polyethylene glycol (PEG) molecule. More preferred is a monogated c_mp1 ligand molecule in which one polyethylene glycol molecule is bound to the N-terminus of the protein.
  • PEGylation of the c-mp1 ligand can be accomplished using PEGylation reactions well known in the art (eg, Focus on Growth Factors 3 (2): 4-10 (1992), EP 0
  • a reactive polyethylene glycol molecule PEGylation may be performed via an acylation or alkylation reaction using
  • the preferred average molecular weight of PEG is from about 5 kDa to about 50 kDa, and more preferably between about 12 kDa to about 25 kDa.
  • the reaction molar ratio of the water-soluble polymer to the c_mp1 ligand protein may be 1: 1 to 100: 1, 1: 1 to 20: 1 for polyPEGylation, 1: 1 to 5: monoPEGylation. 1 is even better.
  • PEGylation by acylation generally involves the reaction of an active ester derivative of polyethylene glycol with a c-mpl ligand protein.
  • a preferred activated PEG ester is PEG esterified to N-hydroxysuccinimide.
  • the PEG addition reaction by acylation generally produces a c_mp1 ligand product to which a large number of PEGs have been added, where PEG addition occurs to the lysine ⁇ -amino group via an acyl-bonding group.
  • the bond is an amide.
  • the resulting product is substantially only one PEG adduct, two PEG adducts or three PEG adducts (eg> 95%).
  • Standard or other techniques including dialysis, salting-out, ultrafiltration, ion-exchange chromatography, gel filtration chromatography, and electrophoresis, as needed, can be used to determine the mixture or unreacted species. Thus, more refined PEG-added species can be separated.
  • a reductive alkylation reaction For the addition of PEG by alkylation, a reductive alkylation reaction can be used.
  • the pKa value between the ⁇ -amino group at the lysine residue and one amino group at the ⁇ -terminal residue in the protein Can be used.
  • PEGylated c-mp1 ligands that can be used in the present invention are described in W 095/26746.
  • the pharmaceutical composition of the present invention can contain an excipient, a carrier or a diluent in addition to a therapeutically effective amount of the c-mp1 ligand, and various dosage forms (for example, tablets) depending on the administration method. , Solutions, suspensions, suppositories, nasal drops, etc.). It can also be a preparation with sustained release. Sustained-release preparations combine a hydrophobic polymeric substance, such as an enteric substance and / or eudrazide, with a hydrophilic substance and then simply disperse the drug into them to form a multiparticulate or multi-layered form, and if necessary, a hydrophobic form. It can be obtained by coating with a hydrophilic coating agent.
  • the pharmaceutical composition of the present invention may be in a lyophilized or dried dosage form, and may be administered to a subject patient by dissolving in a pharmaceutically acceptable diluent at the time of use.
  • the freeze-dried preparation can contain a saccharide or a surfactant as a stabilizer.
  • diluents include buffers of different pH and ionic strength (eg, Tris-HCl, acetate, phosphate), saline, and the like.
  • surfactants are Tween 20, Tween 80, Pluronic F68, bile salts and the like.
  • solubilizers are glycerol, polyethylene glycol and the like.
  • antioxidants are ascorbic acid, sodium metabisulfite and the like.
  • preservatives are thimeru sal, benzyl alcohol, parabens and the like.
  • tonicity agents are lactose, mannitol and the like.
  • Other additives include proteins such as albumin and gelatin for preventing adsorption on the surface of the container, protease inhibitors, and gastrointestinal absorption promoters.
  • the pharmaceutical composition of the present invention comprises a covalent bond between the active ingredient and a water-soluble polymer such as polyethylene glycol, a complexation of the active ingredient with a metal ion, or a compound such as polylactic acid, polyglycolic acid, or hydrogel.
  • a water-soluble polymer such as polyethylene glycol
  • a complexation of the active ingredient with a metal ion or a compound such as polylactic acid, polyglycolic acid, or hydrogel.
  • Incorporation of the active ingredient into or on the surface of a granular formulation of the polymerized compound or into ribosomes, microemulsions, micelles, mono- or multilamellar vesicles, erythrocyte ghosts, or spheroplasts Can be taken.
  • the pharmaceutical composition of the present invention can be administered by various routes including parenteral (intravenous, intraarterial, rectal, subcutaneous, intradermal, pulmonary, nasal, etc.) and oral.
  • the dose is usually about 0.01 g / kg body weight to about 1000 mg / kg body weight, preferably 0.05 g / kg body weight to 300 g / kg body weight, more preferably 1 g / kg body weight as active ingredient.
  • kg body weight ⁇ 100 ⁇ g / kg body weight.
  • Dosage may vary according to age, medical condition, weight, sex, patient's diet and administration route, etc., and may be administered once or several times a day for about 1 to 2 weeks or more. can do.
  • the c-mp 1 ligand is not limited to the above dose range, and may be contained in the composition in a range that does not cause side effects and at a dosage that can achieve the intended therapeutic effect.
  • the polypeptide PEG-rHuMGDF consisting of the amino acid sequence of SEQ ID NO: 2 whose N-terminus is modified with a single polyethylene glycol is When used at a dose of about 2.5 g (about 143 pmol) / kg / day, it is desirable in clinical trials of patients with myelodysplastic syndrome or aplastic anemia without the inclusion of other additional hematopoietic factors. Were able to achieve both the effect of increasing platelets and the effect of increasing erythrocytes (ie, the effect of improving anemia).
  • the composition of the present invention includes c-mp 1 ligand, erythropoietin (EP0), granulocyte colony stimulating factor (G-CSF), granulocyte macrophage colony stimulating factor (GM-CSF), macrophage colony stimulating factor (M-CSF), colony stimulating factor (CSF) -1, interleukin (IL) -1, IL-2, IL_3, IL-4, IL-5, IL-6, IL-7, IL-8, One or more of the following factors: IL-9, IL-10, IL-11, IL-12, IL-13, leukocyte migration inhibitory factor (LIF), stem cell factor (SCF), interferon ⁇ , ⁇ ' Can be included as an additional hematopoietic factor.
  • G-CSF granulocyte colony stimulating factor
  • GM-CSF granulocyte macrophage colony stimulating factor
  • M-CSF macrophage colony stimulating factor
  • CSF colony stimulating factor
  • a polypeptide having an amino acid sequence containing at least the amino acid residues at positions 7 to 151 in the amino acid sequence of SEQ ID NO: 1, a variant thereof, or a derivative thereof, and having a thrombopoietin activity Treating a patient with myelodysplastic syndrome or aplastic anemia by administering the above-mentioned pharmaceutical composition comprising a therapeutically effective amount of the c-mp1 ligand to increase platelets and erythrocytes.
  • a pharmaceutical composition comprising a therapeutically effective amount of the c-mp1 ligand to increase platelets and erythrocytes.
  • c_mp1 ligand PEG-rHuMGDF in Examples described later
  • administration was started. There is no continuous increase in reticulocyte count until about 2 weeks thereafter, but it increases continuously and reaches a maximum in about 2 to about 5 weeks, and the maximum is about 1 of the value on the day of administration. 5 to about 2.5 times.
  • hemoglobin concentration began to increase about 2 weeks after the start of administration, and reached a maximum value about 8 to about 12 weeks after the start of administration, and the maximum value was about 2 to 2 per blood IdL compared to the value on the day of administration. It shows an increase of about 3.5g.
  • the therapeutic method of the present invention may provide a reticulocyte count after a certain time lag (about 1 to about 2 weeks) after the administration. And the characteristic that hemoglobin concentration starts to increase and reaches a maximum value.
  • the increase in reticulocyte count and hemoglobin concentration over time varies depending on the type and dosage of the C-immediate ligand used, but it is thought that the trend of the therapeutic effect shows a similar tendency.
  • the present invention will be more specifically described by the following examples, comparative examples, and reference examples.
  • the drugs, administration methods and clinical trial methods used in this example are as follows.
  • PEG-rHuMGDF is a method described in International Publication W095 / 26746, in which a polypeptide comprising the amino acid sequence of SEQ ID NO: 2 is produced in Escherichia coli by a genetic recombination technique, and a polyethylene glycol (N-terminal) is produced. It is obtained by chemically bonding an average molecular weight of 20 kDa). Injection containing 250 ⁇ g of PEG-rHuMGDF per vial (l. OmL) as a protein was diluted 10-fold with a diluting solution (solvent not containing PEG-rHuMGDF), and then diluted per 1 kg of patient weight 2. ) was administered intravenously once a day for 14 days.
  • Vital signs, subjective symptoms, and objective findings should be observed daily during the study drug administration period, follow-up after the end of treatment should be at least three times a week for the first and second weeks, and weekly for the third and fourth weeks of follow-up Performed more than once.
  • blood transfusions plates and red blood cells
  • Hematological examinations were performed at least three times a week during the study drug administration period and the first and second weeks of follow-up, and at least once a week during the third and fourth weeks of follow-up.
  • Blood chemistry tests, coagulation and fibrinolysis tests, and urinalysis were performed once a week on the first day of administration and on the whole period after the second day of administration.
  • Blood PEG-rHuMGDF concentrations were measured before administration on the first day of administration, 15 minutes, 2 hours and 8 hours after administration, before administration on day 2 of administration, and on days 4, 7, and 10 of administration. Before start of administration and 15 minutes after administration
  • Example 1 Test cases in patients with myelodysplastic syndrome
  • the platelet count showed a transient increase as shown in FIG. 2.
  • a continuous increase in platelet count was not observed for 14 days when patients received PEG-rHuMGDF at 5 ig / kg / day, but platelet counts did not increase between days 16 and 28 after the start of treatment.
  • the number increased from 16,000 / mm 3 to 21,000 / mm 3 and continued to increase thereafter, reaching platelet counts of 25,000 / mi 3 on the 42nd day after administration. Thereafter, although a tendency increased platelet count 22, 000 / mm 3 in 57 days after the start of administration was maintained condensate until as before administration to platelet number 12, 000 / nmi 3 to 71 days after initiation of administration did.
  • Figure 2 shows the change in hemoglobin concentration
  • Figure 3 shows the change in reticulocyte count.
  • the hemoglobin concentration did not show a continuous increasing tendency up to 28 days after the start of treatment, but from 10.8 g / dL to 12.lg / d between days 35 and 57 after the start of treatment. It increased to dL and was maintained thereafter.
  • the changes in hemoglobin concentration were similar for other erythroid parasites.
  • the red blood cell count was 270 ⁇ 10 4 / mm 3 on the first day of administration, but increased to 319 ⁇ 10 Vmm 3 on the 57th day after the start of administration. Hematocrit also increased from 30.6% on the first day of treatment to 36.2% on day 57 after treatment.
  • the mean erythrocyte volume, mean erythrocyte hemoglobin amount and mean erythrocyte hemoglobin concentration which are the erythrocyte constant, remained within a certain range before, during and after administration of PEG-rHu MGDF.
  • the leukocyte count remained within a certain range before, during, and after administration of PEG-rHuMGDF.
  • the leukocyte fraction did not show any change before, during or after PEG_rHuMGDF administration.
  • the blood PEG-rHuMGDF concentration was measured by an ELISA method for detecting PEG-rHuMGDF and endogenous TP0.
  • the endogenous TP0 concentration was 1,595 pg / mL in terms of PEG-rHuMGDF, which was higher than that in healthy subjects.
  • the dose increased to 60,730 pg / niL 15 minutes after administration on day 1 of administration, but decreased to 7,838 pg / mL before administration on day 2 of administration. 15 minutes after administration
  • PEG-rHuMGDF concentrations were 78,980 pg / mL on day 4 of administration, 75,620 pg / mL on day 7 of administration, 81,320 pg / mL on day 10 of administration, and 81,960 pg / mL on day 14 of administration. A steady state was reached during the course of the day.
  • the PEG-rHuMGDF concentration before administration 24 hours after administration
  • Exclusion criteria Principals with severe complications, infection and bone marrow fibrosis, GOT and GPT more than 3 times the institutional normal value, For patients with serum creatinine greater than or equal to .Offlg / dL, who have a history of thrombosis and who may cause thromboembolism, patients who are hypersensitive to biologics, women with positive intracutaneous reaction tests, and pregnant / nursing women) Since this was not the case, the patient was enrolled in this study with written informed consent.
  • the platelet count showed a transient increase as shown in FIG. 2.5
  • Patients receiving PEG-rHuMGDF at 5 ⁇ g / kg / day did not show a continuous increase in platelet count for 14 days, but between 21 and 28 days after the start of treatment.
  • the eyes returned to a platelet count of 16,000 / mm 3 , the same as before administration.
  • FIG. 5 shows changes in hemoglobin concentration
  • Figure 6 shows changes in reticulocyte count.
  • 8 g / dL was the reticulocyte number 44, 300 / negation 3, after the initiation of administration on day 11 observed sustained increase from reticulocyte number 49, 600 / mm 3 of, after reaching the reticulocyte number 71, 400 / min 3 to 28 days after the start of administration, the 80 days after the start of administration halftone and welfare to erythrocyte number 37, 700 / min 3.
  • the hemoglobin concentration did not show a continuous increasing tendency up to 16 days after the start of administration, but from 7.4 g / dL to 9.Og / It increased to dL and was maintained thereafter.
  • the other erythroid parameters showed the same transition as the hemoglobin concentration.
  • Red blood cell count was 164 ⁇ ⁇ 0 4 / ⁇ 3 start date administration, but 57 days after the start of administration was increased to 222 ⁇ lOVmm 3.
  • Hematocrit was also 20.4% on the first day of treatment, but increased to 26.7% on the 57th day after treatment.
  • the mean erythrocyte volume, mean erythrocyte hemoglobin amount and mean erythrocyte hemoglobin concentration which are the erythrocyte constant, remained within a certain range before, during and after administration of PEG-rHu MGDF.
  • P.S. As subjective symptoms, laxity and feeling of fatigue were observed before administration, and P.S. was 1. However, P.S. was improved to 0 with the improvement of anemia after administration.
  • the leukocyte count remained within a certain range before, during, and after administration of PEG-rHuMGDF. Also, there was no constant tendency in the change in leukocyte fraction before, during, and after administration of PEG-rHuMGDF.
  • the endogenous TP0 concentration was 1653 pg / mL in terms of PEG-rHuMGDF, which was higher than that in healthy subjects.
  • PEG-rHuMGDF concentration 15 minutes after administration reached a steady state during 14 days of administration.
  • the PEG-rHuMGDF concentration before administration 24 hours after administration also reached a steady state during the 14-day administration.
  • the endogenous TP0 concentration almost returned to the value before administration.
  • Target patients This case (48 years old, male) was diagnosed with aplastic anemia in February 1997, and from March 1997 to July 1999 cyclosporine 400-300 nig / day and from January 1999 Grastim was administered at 150 zg / day. No effect of cyclosporine or filgrastim was observed as of September 1997, and anti-thymocyte immunoglobulin was further administered in January 1997. As a result, a good response was observed, and transfusion of red blood cells and platelets was unnecessary.
  • the platelet count increased as shown in Figure 7. 2.A continuous increase in platelet counts was not observed for 14 days after administration of PEG-rHuMGDF to patients at 5 zg / kg / day, but platelet counts did not increase between days 17 and 29 after the start of treatment. number 22, 000 / mm 3 to 36, 000 was increased to / negation 3, increasing the platelet count 33,000 / Paiupushiron'ita 3 trend was maintained up to 68 days after the start of administration.
  • Figure 8 shows changes in hemoglobin concentration
  • Figure 9 shows changes in reticulocyte count.
  • the reticulocyte count returned to 39,500 / mm 3 68 days after the start of administration.
  • the hemoglobin concentration did not show a continuous increasing tendency until day 19 after the start of treatment, but it increased from 6.2 g / dL between days 1 and 68 after the start of treatment. 8. Increased to 9g / dL.
  • the leukocyte count remained within a certain range before, during, and after administration of PEG-rHuMGDF. In addition, no change was observed in the leukocyte fraction throughout the period before and during PEG-rHuMGDF administration.
  • the endogenous TP0 concentration before the start of administration was 1056 pg / mL in terms of PEG-rHuMGDF, which was higher than that in healthy subjects.
  • the PEG-rHuMGDF concentration immediately after administration and before administration reached a steady state during the 14-day administration, and reached the pre-administration value after the end of administration. Restored. Discussion of results>
  • Platelet counts begin to increase on day 6 of repeated 10-day subcutaneous administration and peak at days 12-18 (Basser RL et al., "Thrombopoie t ic ef fects of pegyl at ed recombinant human megakaryocyt e growth and deve l opment f ac tor (PEG-rHuMGDF) in patients wis advanced cancer," Lance t 1996; 348: 1279).
  • the increase in platelet counts in patients with myelodysplastic syndrome and aplastic anemia described above is different from these cases, and is normal or normal bone marrow function between PEG-rHuMGDF and platelet count increase. It is suggested that it takes more time than a cancer patient who has the disease.
  • Example 1 of myelodysplastic syndrome 10
  • 6 g / dL and 30, 200 / mm 3 of aplastic anemia patients are classified into each and with the severity of anemia grade 1, grade 3 and grade 4, hemoglobin conc Reticulocyte counts were lower in less severe cases.
  • the reticulocyte count did not show a continuous increase trend for 2 weeks after the start of PEG-rHuMGDF administration, but continued to increase for 2 to 5 weeks and reached the maximum value .
  • the maximum value of reticulocyte count was 1.6 to 2.4 times the day of administration. This increase in reticulocyte count was transient and almost returned before administration 10 to 12 weeks after the start of PEG-rHuMGDF administration.
  • the time at which the reticulocyte count began to increase was similar to or slightly earlier than the time at which the platelet count began to increase, suggesting that the action of PEG-rHuMGDF was similar in megakaryocyte and erythroid cells.
  • Hemoglobin concentration began to increase 1 to 3 weeks after the reticulocyte count began to increase, indicating that erythroid hematopoiesis was stimulated by PEG-rHuMGDF administration. Thereafter, the hemoglobin concentration reached the maximum value (plateau) 8 to 12 weeks after the start of PEG-rHuMGDF administration, in which the reticulocyte count was decreasing or almost returned to the value before administration. The maximum observed hemoglobin concentration increased by 2.1 to 3.4 g / dL from the day of administration. In addition, in the example of the most severe aplastic anemia, the largest increase in hemoglobin concentration was observed. The increase in hemoglobin concentration was maintained during the observation period described in this example, and the longer life span of erythrocytes compared to platelets was reflected in the difference between the platelet count and the maintenance period of the increase in hemoglobin concentration. It was considered.
  • erythroid hematopoiesis was stimulated in patients with myelodysplastic syndrome and aplastic anemia with grade 1 to 4 anemia, and increased reticulocyte count and hemoglobin concentration. An increase in erythroid parameters was observed.
  • bone marrow cells from patients with myelodysplastic syndrome have been treated in vitro with IL-3, IL-6, erythropoietin (EP0), granulocyte monocyte colony stimulating factor (GM-CSF), and stem cell factor (SCF).
  • rHuEPO administered to aplastic anemia patients with hemoglobin concentrations of less than 10 g / dL by a gradual increase method for 3000 weeks to 24000 units / day for a total of 8 weeks (Akio Urabe et al., Genetically Modified 1) Phase II clinical study 1, clinical blood 1993; 34: 1002), the anemia ameliorating effect was 5/9 (55.6%) in non-transfusion patients before administration and 5/20 (25.0%) in transfusion patients. ), And the overall effective rate was 34.5%.
  • Endogenous EP0 levels tend to be higher in more severe patients such as transfusion cases.
  • patients with higher severity are considered to have fewer erythroid hematopoietic progenitor cells showing EP0 reactivity (Aoki I et al., "Responsiveness of bone marrow erythropoietic stem cells (CFU-E and BFU-E) to recombinant human erythropoietin (rh-Ep) in vitro in aplastic anemia and myelodys lastic syndrome, "Al J Hematol 1990; 35: 6).
  • EP0 is a progenitor cell differentiated into erythroid lineage And promotes erythroid differentiation 'maturation (Krantz SB. Erythropoietin. Blood 1991; 77: 419). Mice knocked out of EPO or the EPO receptor gene have erythroid progenitor cells but suppress erythroid and erythrocyte production and die in utero due to anemia (WuHet al., "Generation of committed erythroid"). BFU-E and CFU-E progenitors does not re erythropoietin or the erythropoietin receptor., "Cell 1995; 83:59).
  • TP0 acts on hematopoietic progenitors in myelodysplastic syndrome patients and aplastic anemia patients to increase erythroid progenitors as well as megakaryocytic progenitors.
  • TPO Plasma thrombopoietin
  • the cases of this report and has a very high degree of thrombocytopenia approximately about one-twentieth of the number of platelets is about 13, 000 / ⁇ 3, 21, 000 / Yuzuru 3 and 19, 000 / thigh 3 and healthy persons
  • the endogenous TP0 concentration represented by the pre-administration PEG-rHu MGDF concentration was 1595 pg / mL, 1653 pg, and 1056 pg / mL, which were 20 to 30 times higher than those in healthy subjects.
  • the blood PEG-rHuMGDF concentration reached a steady state and the trough level was about 10 ng / mL.
  • PEG-rHuMGDF was added to healthy human bone marrow cells and cultured to form megakaryocyte colonies, which reached a plateau at lOng / mL, whereas bone marrow cells from patients with myelodysplastic syndrome had higher concentrations of PEG- Since the megakaryocyte colony formation was stimulated by rHuMGDF, the blood PEG-rHuMGDF concentration observed after administration of 2.5 / g / kg / day of PEG-rHuMGDF reached a level that stimulated platelet production in this case. it was thought.
  • Neelis KJ et al. "Thrombopoiet in expands erythroid progenitors, increases red cell production, and enhances erythroid recovery after myelos ressive therapy," J Clin Invest 1995; 96: 1683
  • Neelis KJ et al. "Prevention of thrombocytopenia by thrombopoietin in myelos Sir ressed rhesus monkeys accompanied by prominent erythropoietic stimulation and iron depletion," Blood 1997; 90: 58; Neelis KJ et al.,
  • GDF was found to be effective in improving erythroid parameters such as reticulocyte count and hemoglobin concentration, as well as platelet count, in patients with myelodysplastic syndrome and aplastic anemia.
  • erythroid parameters such as reticulocyte count and hemoglobin concentration, as well as platelet count
  • PEG-rHuMGDF is useful for improving thrombocytopenia and anemia in patients with myelodysplastic syndrome and aplastic anemia.
  • c-mp1 ligand has been shown to synergize in vitro with EP0 and other cytokines to stimulate erythroid hematopoiesis from undifferentiated hematopoietic progenitors, but bone marrow cell responses in vitro Due to the discrepancy between gender and clinical trial performance, it was not expected that c-mp1 ligand would have an ameliorating effect on anemia in patients with myelodysplastic syndrome and aplastic anemia.
  • G-CSF for neutropenia is the only clinical indication for myelodysplastic syndrome and aplastic anemia, and clinical indication for the amelioration of anemia.
  • Virtually no hematopoietic factors can improve anemia in patients with myelodysplastic syndrome and aplastic anemia. Therefore, it is considered that the significance of the anemia ameliorating effect of the c-mp1 ligand is great. Comparative example
  • G-CSF granulocyte colony stimulating factor
  • GM-CSF interleukin
  • IL_ interleukin
  • IL-6 interleukin-6
  • EP0 erythropoietin
  • Hemoglobin concentration increased from lg / dL to less than 2 g / dL in non-transfusion cases, transfusion volume decreased by 50% or more in transfusion cases
  • G-CSF The clinical effects of G-CSF, GM-CSF, IL-3, IL-1, IL-6, and EP0 have been studied.
  • G-CSF showed little effect on anemia improvement.
  • GM-CSF 4.5% (3/66 cases) reported an anemia ameliorating effect.
  • IL-3, IL-1 and IL-6 no anemia improving effect was observed at all.
  • phase II Akio Urabe et al., Effect of recombinant human erythrotropin on aplastic anemia-Phase II clinical trials 1 , Clinical Blood 1993; 34: 1002
  • Phase I Akio Urabe et al., For aplastic anemia
  • the overall efficacy rate of the phase III study was 19.4 (6/31 subjects).
  • hemoglobin concentration increased by 2 g / dL or more, or in patients who did not require blood transfusion in pre-administration ring blood patients (significant improvement), 4/29 patients in the phase II study
  • Table 2 summarizes the results of the Phase II and III studies of rHuEPO.
  • RHuEPO improves anemia in aplastic anemia (Urabe et al., Cited above)
  • Hemoglobin concentration increased from lg / dL to less than 2 g / dL in non-transfusion cases, transfusion volume decreased by more than 50% in transfusion cases
  • PEG-rHuMGDF mono-PEGylated c-mp1 ligand
  • the DNA sequence encoding the first 163 amino acids of the mature protein was chemically synthesized using the optimal codon of E.
  • a DNA sequence encoding the amino acids methionine and lysine was added to the 5 'end of the gene. Therefore, the r-HuMGDF protein encoded by this DNA sequence is composed of a total of 165 amino acids starting with Met-Lys (W095 / 26746). See Figure 25).
  • r_HuMGDF gene was achieved in a few steps.
  • complementary oligonucleotides 60-70 bp in length
  • flanking fragments of the gene were chemically synthesized using the optimal codon of E.co//.
  • the amino acid methionine and lysine codons were placed at the 5 'end of the mature gene, and the stop and codons were placed at the 3' end of the gene.
  • the cleavage sites for the restriction enzymes Xbal and Hindlll were placed at the 5 'and 3' ends of the gene, respectively, and the binding site for the synthetic ribosome was placed in a suitable location upstream of the first methionine.
  • each gene fragment was annealed.
  • these individual synthetic gene fragments were amplified using polymerase 'chain' reaction (PCR). The amplified fragment was subcloned into an appropriate vector and the sequence was confirmed. The individual fragments were then ligated and subcloned into a suitable vector so that the full-length r-HuMGDF gene could be reconstructed. Finally, the sequence of the reconstructed gene was confirmed.
  • the synthetic r-HuC-MPL ligand gene fragment flanking the Xbal and HindIII restriction sites, respectively, contains the liposomal binding site, the ATG initiation codon, and the mature Met- It has a sequence encoding the Lys r-HuMGDF protein and a stop codon.
  • the above fragment was cloned into both the Xbal and HindIII sites of the lactose-inducible expression vector pAMGII.
  • the pAMGll vector is a low copy number plasmid with a pRIOO-derived origin of replication.
  • the expression plasmid pAMGll can be derived from plasmid pCFM1656 (ATCC # 69576, deposited on Feb. 24, 1994) by making a series of site-specific base changes by inducing PCR overlapping oligos. . Starting at the Bglll site (plasmid bp # 180) close to the 5 'of the plasmid replication promoter PcopB and going to the plasmid replication gene, the conversion of the base pair is as follows:
  • This Ps4 promoter is suppressed by a lactose' repressor (Lac1) which is a product of the E. coli lac1 gene.
  • the pAMGl l-r-HuMGDF plasmid is subsequently transformed into the R coli K-12 strain containing the lacl Q allele.
  • the lacl q allele is a mutation in the lacl promoter that increases Lacl expression, resulting in tighter control of protein expression from the Ps4 promoter. Therefore, in this strain, in the absence of lactose, expression of r-HuMGDF is suppressed by Lacl. Easy! When added, the Lacl protein bound to the Ps4 promoter operator site decreases, and the transcription of r-HuMGDF from Ps4 starts.
  • the .co host cell used in this reference example has been deposited with the ATCC (USA) as ATCC # 69717.
  • the E. coli host ATCC # 69717 was transformed with the pAMGl r-HuMGDF plasmid and grown according to the following fermentation procedure.
  • the .co / strain inoculated in Luria broth is incubated for approximately 12 hours at 30 ° C.
  • Strain is then aseptically patch - medium (20 g / L yeast 'extract; 3. 4 g / L Kuen acid; 15 g / LK 2 HP0 4 ; 15 ml
  • the first fluid medium (f eed med ium); at the start of the supply (700 g / L glucose 6. 75 g / L MgS0 4 ⁇ 7H 2 0), initiated.
  • the flow rate is adjusted every 2 hours according to the established schedule.
  • the start of supply of the second flowing medium (129 g / L trypticase 'peptone; 258 g / L yeast' extract).
  • the second fluid medium maintains a constant flow rate while the first fluid medium continues to be adjusted I do. Throughout the entire fermentation, the temperature is maintained at approximately 30 ° C.
  • the culture is maintained at pH 7 by adding acids and bases as needed.
  • the desired dissolved oxygen level is maintained by adjusting the fermenter agitation, air injection and oxygen injection rates.
  • the third fluid medium 300 g / L lactose
  • the third fluid medium is introduced into the fermentor at a constant flow rate; the supply of the first fluid medium is stopped and the flow rate of the second fluid medium is changed to a new constant flow rate. Fermentation ends approximately 10 hours after the start of the supply of the third fluid medium.
  • the culture is cooled to 15 +/- 5 ° C. Strains are collected by centrifugation. The obtained paste is stored as packed below -60 ° C.
  • the diluted solution was gently stirred at room temperature for 16 hours and the pH was adjusted to 6.8.
  • the pH adjusted solution was clarified and applied to a liter CM Sepharose column equilibrated with 10 mM sodium phosphate, 1.5 M urea, 15% glycerol, pH 6.8. After loading, the column was washed with 10 mM sodium phosphate, 15% glycerol, pH 7.2.
  • MGDF was eluted with a gradient of 0 to 0.5 M NaCl, 10 mM sodium phosphate, pH 7.2.
  • the CM eluate was concentrated using a 10,000 molecular weight cut-off membrane, and buffer-exchanged to 10 mM sodium phosphate, pH 6.5.
  • the solution concentrated to about 2 nig / ml was treated with cathepsin C (molar ratio of 500: 1) at room temperature for 90 minutes. This solution was equilibrated with 10 mM sodium phosphate, 15% glycerol, pH 7.2, 1.2 liters of SP High
  • the degree of protein modification during the reaction was monitored by SEC HPLC using a Superdex 200 HR 10/30 column (Pharmacia Biotech). Elution was performed using 0.1 M sodium phosphate buffer, pH 6.9, at a flow rate of 0.7 ml / min.
  • the homogeneity of the conjugate was determined by SDS-PAGE (sodium / dodecyl / sulfate / polyacrylamide gel electrophoresis) using a 4-20% precast-gradient 'gel (N0VEX) and determined as one major band. Detected. ⁇ Reference Example 2> Expression of full-length human TPO
  • a DNA fragment covering the entire human TPO cDNA coding region by PCR was prepared as follows.
  • the nucleotide sequences of the primers used are as follows.
  • hTP0-I 5'-TTGTGACCTCCGAGTCCTCAG-3, (SEQ ID NO: 8);
  • hTPO-KO 5, -GAGAGAGCGGCCGCTTACCCTTCCTG AGACAGATT-3 '(SEQ ID NO: 11).
  • a first PCR was performed using 300 ng of clone p EF 18 S-HL34 (Example 16 of W095 / 21919) as a type III. Using 1 ⁇ m of each primer, hTPO-I and 0.5 ⁇ l of SA, 1 unit of Vent RTM DNA polymerase (manufactured by New England BioLabs) (reaction at 96 ° C for 1 minute, 62 ° C for 1 minute, 72 ° C for 1 minute) After 30 cycles, 72 minutes). The composition of the reaction solution is as follows. The final concentration of 10mM KCK 10inM (NH 4) 2 S0 4, 20mM Tris-HCl (pH8.8), 2mM MgS0 4, 0.1% Triton X- 100,
  • poly (A) + RNA manufactured by Clontecii
  • lpg derived from normal human liver is heated at 70 ° C for 10 minutes, quenched on ice, 10 mM DTT, 500 M dNTPmix, 25 ng random primer
  • the first and second PCR solutions were subjected to 1% agarose gel electrophoresis, and major bands having the expected sizes were purified using a Prep-A-Gene DNA purification kit (Bio-Rad).
  • a third PCR was performed with each 1/20 of each purified amount as type III. Reaction using one unit of Vent R TM DNA polymerase (New England BioLabs) (Heating at 96 ° C for 2 minutes, then performing 3 cycles of 96 ° C for 2 minutes, 72 ° C for 2 minutes, and then 72 ° C for 7 minutes) was performed.
  • the collected DNA is digested with the restriction enzymes BamHI and NotI and subjected to 1% agarose gel electrophoresis.
  • the major band of the expected size is purified using the Prep-A-Gene DNA Purification Kit (Viorad). After that, the resultant was ligated to pBluescriptll SK + vector (Stratagene) previously digested with restriction enzymes BamHI and Notl in the same manner, and then transformed into competent high E. coli DH5 (Toyobo Co., Ltd.). Four clones were selected from the obtained colonies, and plasmid DNA was prepared.
  • the obtained pBLTEN is digested with restriction enzymes EcoRI and Notl, and then subjected to 1% agarose gel electrophoresis, and a band of about 1200 bp is purified using Prep-A-Gene A purification kit (manufactured by Biorad). After that, it was ligated to an expression vector pEF18S similarly treated with a restriction enzyme, and transformed into a competent high E. coli DH5 (manufactured by Toyobo Co., Ltd.). Plasmid DNA was prepared from the obtained colonies, and a clone pHTP1 containing the entire coding region of human TPOc DNA was obtained. A large amount of plasmid DNA of this clone was prepared and used in the following experiments. Preparation of plasmid DNA was performed essentially as described in Molecular Cloning (Sambrook et al., Cold Spring Harbor Laboratory Press, 1989).
  • agarose gel electrophoresis was performed to recover a fragment (about 0.5 kbp) containing the mouse DHFR minigene.
  • the recovered fragment is dissolved in a reaction solution 25 1 consisting of 50 mM Tris-HC1 (pH 7.5), 7 mM MgCl 2 , lmM j8-mercaptoethanol, and 0.2 inM dNTP, 2 units of Klenow fragment is added, and the mixture is added at room temperature for 30 minutes. The reaction was performed to blunt the ends of the DNA. Next, the resultant was treated with phenol / chloroform and precipitated with ethanol, and then dissolved in a 10 / l TE solution containing 10 mM Tri_HCl (pH 8.0) and lmM EDTA.
  • the obtained fragment containing the mouse DHFR minigene and the expression vector PEF 18S for animal cells were treated with the restriction enzyme Smal, and then dephosphorylated with alkaline phosphatase (Takara Shuzo) to obtain the vector DNA.
  • dephosphorylated with alkaline phosphatase (Takara Shuzo) to obtain the vector DNA.
  • T4 DNA ligase (Takara Shuzo) to obtain an expression vector pDEF202.
  • this vector pDEF202 is treated with the restriction enzymes EcoRI and Spel, the larger vector fragment is recovered by agarose gel electrophoresis, and the plasmid pHTP1 containing this fragment and human TPO cDNA (Pl clone) is restricted.
  • Enzyme EcoRI And human TPO cDNA (Pl clone) obtained by treatment with Spel were ligated with T4 DNA ligase (Takara Shuzo) to obtain an expression vector PDEF202-hTPO-P1.
  • This plasmid contains the replication initiation region of SV40, human alpha-promoting factor 11 alpha promoter, the early polyadenyl site of SV40, the mouse DHFR minigene, the replication initiation region of PUC18, the j3-lactamase gene (including Amp, The human TPO cDNA is connected downstream of the long factor alpha promoter.
  • CHO cells (dhir-strain, Urlaub and Chasin; Proc. Natl. Acad. Sci. USA; 77, 4216, 1980) containing 10% fetal bovine serum in a 6 cm diameter plate (Falcon)
  • the culture was grown in ⁇ minimum essential medium (a-MEM (-), thymidine, and hypoxanthine added), and this was transformed by the calcium phosphate method (CellPhect, manufactured by Pharmacia). That is, the (1) buffer foremost prepared PDEF202- MP0-P1 plasmid 1 0 iig in A: 1 20 l and H 2 0: 1 2 0 After u ⁇ added mixture was allowed to stand at room temperature for 10 minutes.
  • Buffer B 120 ⁇ 1 was added to this solution, mixed again, and allowed to stand at room temperature for 30 minutes. The]) After dropping NA solution to the plate and cultured for 6 hours in a C0 2 incubator scratch. The medium was removed from the plate, washed twice with ⁇ - ⁇ (1), added with 10% dimethyl sulfoxide-containing MEM (-), and treated at room temperature for 2 minutes. Then, a non-selective medium containing 10% dialyzed fetal calf serum (see above-with MEM (-), hypoxanthine, and thymidine) was added and cultured for 2 days. -MEM (—), hypoxanthine, thymidine-free) were selected.
  • the CHO cell strain (CHO-DUKXBll) transformed with the plasmid PDEF 202—hTPII—Pl was established on January 31, 1995, by the National Institute of Advanced Industrial Science and Technology (Higashi, Tsukuba, Ibaraki, Japan). Deposit number FERM BP-4988 at 1-chome, 1-chome No. 6). This cell line has been deposited at the depository institutions of the People's Republic of China and the Republic of China under the accession numbers CCTCC-C95004 and FIRDI 960023, respectively. Sequence listing free text
  • SEQ ID NO: 2 Description of Artificial Sequence: Amino Acid Sequence of SEQ ID NO: 1 to 163

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Abstract

L'invention concerne des compositions médicinales renfermant une quantité efficace sur la plan thérapeutique du ligand c-mpl et destinées à accroître des plaquettes et des érythrocytes chez des patients souffrant du syndrome myélodysplasique ou d'anémie hypoplasique.
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WO2022114603A1 (fr) * 2020-11-24 2022-06-02 재단법인대구경북과학기술원 Nouvel anticorps anti-c-mpl et son utilisation

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