WO2002015926A1 - c-mpl LIGAND-CONTAINING MEDICINAL COMPOSITIONS FOR INCREASING PLATELETS AND ERYTHROCYTES - Google Patents

c-mpl LIGAND-CONTAINING MEDICINAL COMPOSITIONS FOR INCREASING PLATELETS AND 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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French (fr)
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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Priority to AU2001280161A priority Critical patent/AU2001280161A1/en
Publication of WO2002015926A1 publication Critical patent/WO2002015926A1/en

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    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50Medicinal 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
    • A61K47/51Medicinal 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
    • A61K47/56Medicinal 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
    • A61K47/59Medicinal 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
    • A61K47/60Medicinal 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
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00Drugs for disorders of the blood or the extracellular fluid
    • A61P7/06Antianaemics
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00Medicinal 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

Abstract

Medicinal compositions containing a therapeutically effective amount of c-mpl ligand for increasing platelets and erythrocytes in patients with myelodysplastic syndrome or hypoplastic anemia.

Description

明 細 c -m p 1リガンドを含有する、 血小板及び赤血球を増加させるための医薬組成 物  A pharmaceutical composition for increasing platelets and erythrocytes, comprising the c-mp1 ligand
発明の分野 Field of the invention
本発明は、 c—m p 1リガンドを含む、 血小板及び赤血球を増加させるための 医薬組成物に関する。 さらに具体的には、 本発明は、 骨髄異形成症候群または再 生不良性貧血患者において c一 m p 1 リガンドを含む、 血小板及び赤血球を増加 させるための医薬組成物に関する。 発明の背景  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. Background of the Invention
骨髄異形成症候群及び再生不良性貧血は、 いずれも造血幹細胞の障害による骨 髄機能不全による血球減少状態を特徴とする疾患である。 骨髄異形成症候群は、 骨髄が一般に正ないし過形成を示すと共に血球の異形成像が認められ、 造血幹細 胞に質的異常があるために有効な血球産生が行われない病態(いわゆる無効造血) を総称する症候群で、 慢性かつ不可逆的に経過し、 急性非リンパ性白血病の発症 あるいは骨髄不全に伴う感染症及び出血による死亡等予後不良である (吉田弥太 郎ら、 骨髄異形成症候群. 新内科学大系、 中山書店 1992 : 18 : 384)。 治療法とし て確立されたものはなく、 移植可能な年齢の患者に対しては同種骨髄移植が施行 されたり、高リスク群である芽球増加を伴う RA(RAEB)及び移行期の RAEB (RAEB-t) 等では白血病に準じて化学療法が施行される。 その他の患者では血球減少に対し て、 造血刺激のために蛋白同化ホルモン及び男性ホルモン療法ならびにサイトカ イン療法、 または輸血が施行される。  Both 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. Academic system, Nakayama Shoten 1992: 18: 384). There is no established treatment, and allogeneic bone marrow transplantation is performed for patients of transplantable age, RA with high blasts (RAEB), which is a high-risk group, and RAEB in transition (RAEB In -t) etc., chemotherapy is administered according to leukemia. In other patients, anabolic and androgen therapy and cytokine therapy or blood transfusion are used to stimulate hematopoiesis for cytopenia.
一方再生不良性貧血は、 骨髄は低形成で異型細胞 (芽球) の増加を伴わないこ とから、 造血幹細胞の減少ならびに造血微小環境の異常に由来する疾患と考えら れている(浦部晶夫、再生不良性貧血、新内科学大系、 中山書店 1992 ; 18 : 365)。 近年は骨髄不全の原因として免疫的な関与が示唆されており、 治療法としては移 植可能な年齢の重症患者に対しては同種骨髄移植が選択され、 それ以外の重症患 者及び中等症患者に対しては免疫抑制療法としてステロイ パルス療法、 シクロ スポリン投与、 抗胸腺細胞免疫グロプリン及び抗リンパ球グロプリン投与が施行 される。 また、 骨髄異形成症候群と同様に血球減少に対して、 造血刺激のために 蛋白同化ホルモン及び男性ホルモン療法ならびにサイトカイン療法、 または輸血 が施行される。 On the other hand, 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). In recent years, immunological involvement has been suggested as a cause of bone marrow failure. 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. In addition, as in myelodysplastic syndrome, for cytopenia, anabolic hormone and androgen therapy and cytokine therapy or blood transfusion are administered to stimulate hematopoiesis.
骨髄異形成症候群または再生不良性貧血患者に対し貧血症状を改善するために、 これまで顆粒球コロニー刺激因子 (G- CSF)、 エリトロポェチン (EP0)、 顆粒球単 球コロニ一刺激因子(GM-CSF)、 インターロイキン類 (IL- 1、 IL- 3、 IL- 6等) など の造血因子による臨床効果が検討されてきた。 それにもかかわらず、 貧血症状の 改善に関しては、 赤芽球系特異的な造血因子である EP0であってもその有効率は 低く約 15%〜約 35 %に過ぎなかった (後記の比較例参照)。 もちろん EP0以外の 造血因子による改善効果は全く認められないか、 ほとんど認められない。  To improve anemia symptoms in patients with myelodysplastic syndrome or aplastic anemia, granulocyte colony stimulating factor (G-CSF), erythropoietin (EP0), granulocyte monocyte colony stimulating factor (GM-CSF) ), Interleukins (IL-1, IL-3, IL-6, etc.) have been studied for their clinical effects. Nevertheless, with regard to the improvement of anemia symptoms, the effective rate of EP0, which is an erythroid-specific hematopoietic factor, was as low as only about 15% to about 35% (see the comparative example below). ). Of course, no or no improvement effect by hematopoietic factors other than EP0 was observed.
また、 c- mpl リガンドとして本発明でも用いられているポリエチレングリコー ル修飾遺伝子組換えヒト巨核球増殖分化因子(PEG- rHuMGDF)または遺伝子組換え トロンボポェチン(rHuTPO)の臨床試験が実施されているが、 これまで赤血球系の 造血に対する影響はないと報告されている(Basser L et al. , "Enhancement of plate le t recovery af ter myelos聊 ress ive chemotherapy by recombinant human megakaryocyte growth and deve lopment factor in pat ients wi th advanced cancer, " J. Cl in. Oncol. 2000 ; 18 : 2852他)。  In addition, clinical trials of polyethylene glycol-modified recombinant megakaryocyte proliferation / differentiation factor (PEG-rHuMGDF) or recombinant thrombopoietin (rHuTPO), which are also used in the present invention as c-mpl ligands, have been conducted. So far it has been reported that there is no effect of the erythroid on hematopoiesis (Basser L et al., "Enhancement of plate le t recovery af ter myelos lia ress ive chemotherapy by recombinant human megakaryocyte growth and deve lopment factor in patents wi th advanced cancer, "J. Clin. Oncol. 2000; 18: 2852 et al.).
このような状況にあって、 本発明は、 骨髄異形成症候群または再生不良性貧血 患者において血小板および赤血球増加作用を示す医薬組成物を提供することを目 的とする。 発明の概要  In such a situation, 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. Summary of the Invention
本発明者らは、 上記の課題を解決するために鋭意検討を重ねた結果、 意外にも c -m p 1リガンドが血小板増加作用のみならず骨髄異形成症候群または再生不 良性貧血患者の貧血症状を顕著に改善しうることを見出した。  The present inventors have conducted intensive studies to solve the above-mentioned problems, and as a result, surprisingly, 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.
したがって本発明は以下のものを含む。 本発明は、 治療上有効量の c一 m p 1 リガンドを含む、 骨髄異形成症候群また は再生不良性貧血患者において血小板および赤血球を増加させるための医薬組成 物を提供する。 Therefore, 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.
具体的には、 c一 m p lリガンドは、 配列番号 1のアミノ酸配列のうち少なく とも 7位〜 1 5 1位のアミノ酸残基を含むアミノ酸配列を有するポリペプチド、 その変異体、 またはそれらの誘導体からなり、 かつ トロンポポェチン (thrombopoet in; T P O) 活性を有するものである。 ここで 「トロンポポェチン 活性」 とは、 哺乳動物 (好ましくはヒト) において巨核球前駆細胞の増殖および 分化を促進するか、 あるいは該動物生体内で特異的に血小板の産生を刺激または 増強する活性を指す。  Specifically, 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. As used herein, 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. .
本発明の実施態様において、 該 c一; m p 1 リガンドが、 配列番号 1のアミノ酸 配列 1〜3 3 2からなるポリペプチド、 その変異体またはそれらの誘導体であつ て、 かつトロンポポェチン活性を有するもの、 あるいは、 配列番号 1のアミノ酸 配列 1〜 1 6 3からなるポリペプチド、 その変異体またはそれらの誘導体であつ て、 かつ卜ロンポポェチン活性を有するものを例示することができる。  In an embodiment of the present invention, 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; Alternatively, 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.
本発明の実施態様において、 該変異体が、 配列番号 1のアミノ酸配列において 少なくとも 1つのアミノ酸の置換、 欠失、付加および/または揷入を含み、 かつ卜 ロンポポェチン活性を有するポリペプチドである。  In an embodiment of the present invention, 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.
本発明の実施態様において、 該誘導体が、 水溶性ポリマーで修飾されたポリべ プチドまたはその変異体である。 水溶性ポリマーは、 少なくとも該ポリペプチド またはその変異体の N末端に結合されうる。 該水溶性ポリマーの好ましい例はポ リエチレングリコールである。  In an embodiment of the invention, 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.
本発明の実施態様において、 該 C- mpl リガンドは、 配列番号 2のアミノ酸配列 からなる組換えポリぺプチドの N末端に 1分子のポリエチレングリコールが結合 された PEG- rHuMGDF (後述の実施例参照)である。 この PEG- rHuMGDFをタンパク重 量で約 2. 5 g (約 143pmol) /kg/日の用量で約 2週間にわたって骨髄異形成症候 群または再生不良性貧血患者に投与したときには、 投与開始後約 2週間までは網 赤血球数の継続的な増加傾向は認められず、 約 2〜約 5週間で継続的に増加して 最大値に達し、 その最大値は投与開始日の値の約 1. 5〜約 2. 5倍であり、 投与開 始後約 10〜約 12週間後にほぼ投与前の値に復する、 という治験結果が得られ、 またこの治験例において、 投与開始の約 2週間後にヘモグロビン濃度が増加しは じめ、投与開始の約 8〜約 12週後に最大値に達し、その最大値は投与開始日の値 に比べて血液 I dLあたり約 2〜約 3. 5gの増加を示す、 という結果が得られた(後 述の実施例参照)。 In an embodiment of the present invention, 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. When 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. Approximately 2.5 times About 10 to 12 weeks after the start of treatment, the results showed that the values almost returned to those before administration.In this study, the hemoglobin concentration increased about 2 weeks after the start of treatment, and The maximum value was reached after about 8 to about 12 weeks, and the maximum value showed an increase of about 2 to about 3.5 g per blood IdL compared to the value at the start of administration (see below). See Examples).
本明細書中で使用する 「c— m p 1 リガンド」 は、 C- mpl (TP0受容体; Vigon, I. e t al. , Proc. Nat l. Acad. Sc i. USA 89 : 5640-5644 (1992) )に対して結合し、 か つ作用する物質である。  As used herein, "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).
本明細書中で使用する 「治療上有効量」 とは、 骨髄異形成症候群および再生不 良性貧血をもつ患者に対して、 巨核球前駆細胞の増殖および分化を促進するかま たは生体内で特異的に血小板の産生を刺激もしくは増強することによって血小板 を増加させる(所謂トロンポポェチン活性)とともに、 有意の赤血球増加作用を提 供する用量を意味する。  As used herein, 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.
本明細書中で使用する 「変異体」 とは、 配列番号 1のアミノ酸配列において少 なくとも 1つのアミノ酸の置換、 欠失、付加および/または挿入を含み、 かつ、 卜 ロンポポェチン活性をもつものを意味する。 また 「誘導体」 とは、 水溶性ポリマ ― (例えばポリエチレングリコール) 付加、 グリコシル化、 リン酸化、 硫酸化な どの化学修飾を含むタンパク質、 ポリペプチドまたはペプチドを意味する。 図面の簡単な説明  As used herein, the term "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. means. The term “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. BRIEF DESCRIPTION OF THE FIGURES
図 1は、 骨髄異形成症候群患者 (実施例 1 ) における血小板数の推移を示すグ ラフである。患者に対しタンパク重量で 2. 5 S (約 143pmol) /kg/日の PEG- rHuMGDF を 1日目から 14日目まで 14日間投与し、投与開始後 28日目までは週 3回、その 後 42日目までは週 1回血小板数を測定した。なお図に示された期間中に血小板輸 血は施行されなかった。  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.
図 2は、 骨髄異形成症候群患者 (実施例 1 ) におけるヘモグロビン濃度の推移 を示すグラフである。 患者に対しタンパク重量で 2. 5 g (約 143pmol) /kg/日の 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を 1日目から 14日目まで 14日間投与し、投与開始後 28日目までは 週 3回、その後 42日目までは週 1回ヘモグロビン濃度を測定した。なお図に示さ れた期間中に赤血球輸血は施行されなかった。 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.
図 3は、 骨髄異形成症候群患者 (実施例 1 ) における網赤血球数の推移を示す グラフである。 患者に対しタンパク重量で 2. 5 g (約 143pmol) /kg/日の PEG-rHuMGDFを 1 日目から 14日目まで 14日間投与し、投与開始後 28日目までは 週 3回、その後 42日目までは週 1回網赤血球数を測定した。なお図に示された網 赤血球数は、 測定された赤血球 1000個あたりの網赤血球の比率 (VD()) を赤血球 数に乗じて算出された値である。 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 () ).
図 4は、 骨髄異形成症候群患者 (実施例 2 ) における血小板数の推移を示すグ ラフである。患者に対しタンパク重量で 2. 5 g (約 143pmol) /kg/日の PEG- rHuMGDF を 1 日目から 14日目まで 14日間投与し、投与開始後 28日目までは週 3回、その 後 42日目までは週 1回血小板数を測定した。なお図に示された期間中に血小板輸 血は施行されなかった。  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.
図 5は、 骨髄異形成症候群患者 (実施例 2 ) におけるヘモグロビン濃度の推移 を示すグラフである。 患者に対しタンパク重量で 2. 5 / g (約 143pmol) /kg/日の PEG-rHuMGDFを 1 日目から 14日目まで 14日間投与し、投与開始後 28日目までは 週 3回、その後 42日目までは週 1回ヘモグロビン濃度を測定した。なお図に示さ れた期間中に赤血球輸血は施行されなかった。  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.
図 6は、 骨髄異形成症候群患者 (実施例 2 ) における網赤血球数の推移を示す グラフである。 患者に対しタンパク重量で 2. 5 g (約 143pmol) /kg/日の PEG-rHuMGDFを 1 日目から 14日目まで 14日間投与し、投与開始後 28日目までは 週 3回、その後 42日目までは週 1回網赤血球数を測定した。なお図に示された網 赤血球数は、 測定された赤血球 1000個あたりの網赤血球の比率 ( Q) を赤血球 数に乗じて算出された値である。 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. Note that 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.
図 7は、 再生不良性貧血患者における血小板数の推移を示すグラフである。 患 者に対しタンパク重量で 2. 5 z g (約 143pmol) /kg/日の PEG - rHuMGDFを 1日目から 14日目まで 14日間投与し、 投与開始後 28日目までは週 3回、 その後 42日目ま では週 1回血小板数を測定した。 なお図に示された期間中に血小板輸血は施行さ れなかった。  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.
図 8は、 再生不良性貧血患者におけるヘモグロビン濃度の推移を示すグラフで ある。 患者に対しタンパク重量で . 5 /i g (約 143 11101) /1¾/日の?£6-]:11 60?を 1 日目から 14日目まで 14 日間投与し、 投与開始後 28 日目までは週 3回、 その後 4 日目までは週 1回ヘモグロビン濃度を測定した。なお図に示された期間中に赤 血球輸血は施行されなかった。 Figure 8 is a graph showing changes in hemoglobin concentration in aplastic anemia patients. is there. .5 / ig (about 143 11101) / 1¾ / 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.
図 9は、 再生不良性貧血患者における網赤血球数の推移を示すグラフである。 患者に対し 2. 5 x g (約 143pmo l) /kg/日の PEG-rHuMGDFを 1 日目から 14 日目まで 14日間投与し、 投与開始後 28日目までは週 3回、 その後 42日目までは週 1回網 赤血球数を測定した。 なお図に示された網赤血球数は、 測定された赤血球 1000 個あたりの網赤血球の比率 。) を赤血球数に乗じて算出された値である。 発明の詳細な説明  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. ) Is a value calculated by multiplying by the number of red blood cells. Detailed description of the invention
本発明の医薬組成物の有効成分である c一 m p 1 リガンドとして、 骨髄異形 成症候群または再生不良性貧血患者において血小板および赤血球増加作用を示す 任意のタンパクもしくはペプチド性物質または非タンパク性物質が使用できる。 タンパク性 c— m p 1 リガンドの例には、 (1) トロンポポェチン(TP0 または MGDF)、 その変異体およびその誘導体 (W095/18858または日本国特表平 9- 508262 号公報、 日本国特許第 2991640号公報、 日本国特許第 2991630号公報および日本 国特許第 2996729 号公報など)、 ( 2) 巨核球の刺激剤としてのァゴニスト抗体 (W099/03495, W099/10494など) 、 (3) 造血レセプ夕一ァゴニストなどの他の夕 ンパク質 (画/ 23888 または日本国特表平 11-500308 号公報、 W097/12978, W097/12985または日本国特表平 11- 510062号公報、 W098/17810, W096/34016また は日本国特表平 8- 532372号公報、 TO00/24770など) などが含まれる。  As the active ingredient of the pharmaceutical composition of the present invention, 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. it can. Examples of 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. 2996729), (2) agonist antibody as a megakaryocyte stimulant (W099 / 03495, W099 / 10494, etc.), (3) Hematopoietic receptor Yuichi Other proteins such as agonists (Paint / 23888 or Japanese Patent Publication No. 11-500308, W097 / 12978, W097 / 12985 or Japanese Patent Publication No. 11-510062, W098 / 17810, W096 / 34016 Or Japanese Patent Publication No. 8-532372, TO00 / 24770, etc.).
ペプチド性 c一 m p 1 リガンドの例には、 即 1 レセプターに結合可能なぺプチ ド性化合物が含まれる (W096/40189, W096/40750, W098/25965 , 日本国特開平 10- Π492号公報、 W099/42127, WO00/24770など)。  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.).
非タンパク性 c一 m p 1リガンドの例には、 (1) ベンゾジァゼピン誘導体 (日 本国特開平 11-1477号公報、 日本国特開平 11- 152276号公報など)、 (2)他の低分 子系 mp lレセプ夕一リガンド(W099/1 1262、W099/22733、W099/22734、W000/35446、 WO00/28987など) などが含まれる。 特に好ましい c一 mp 1リガンドは、 トロンボポェチン、 その変異体およびそ の誘導体である。本発明で使用しうるトロンポポェチンの変異体および誘導体は、 血小板産生を特異的に刺激又は増大させる生物活性を有することが必要である。 Examples of non-proteinaceous c-mp1 ligands 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.) and the like. 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.
トロンポポェチンとしてはたとえば配列番号 1のアミノ酸配列 1〜332 を有す るポリペプチドまたはその変異体を挙げることができる。 本発明では、 部位特異 的突然変異誘発法やポリメラーゼ連鎖反応(PCR) を含む公知の変異導入法(米国 特許第 4, 518, 584参照) を用いることによって、 トロンポポェチンの種々の変異 体を得ることができる。  Thrombopoetin includes, for example, a polypeptide having the amino acid sequence of 1 to 332 of SEQ ID NO: 1 or a mutant thereof. In the present invention, 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.
例えば、 そのような変異体の例は、 配列番号 1のアミノ酸配列において少なく とも 7位〜 1 5 1位のアミノ酸配列を含むトランケーシヨン型タンパク質、 例え ば 1〜244、 1〜2 3 1、 1〜2 1 1、 1〜1 9 1、 1〜1 7 7、 1〜1 74、 1〜1 7 1、 1〜; 1 70、 ;!〜 1 6 9、 1〜: L 63、 1〜; 1 5 8、 1〜: L 5 7、 1 〜1 56、 1〜1 5 5、 1〜1 54、 1〜1 53、 1〜1 5 1、 7〜 1 6 3などを 含む。 また本発明で使用可能な他の変異体の非限定例として、 次のものを挙げる ことができる。  For example, 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 h r 33, T h r 333, S e r 334, l i e 335, G 1 y 336, T y r 337, P r o 338, Ty r 339, A s p 340, V a 1341 , P r o 342, A s p 343, Ty r 344, A 1 a 345, G 1 y 346, V a 1347, H i s 348, H i s 349, H i s 35。, H i s 351, H i s 352, H i s 353] TPO (1-332)、 [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),
[A s n25, L y s m, T h r 333, S e r 334,· l i e 335, G 1 y 336 , T y r 337, P r o 338, Ty r 339, A s p 340, V a 1 m, P r o 342, A s p 343, Ty r 344 , A 1 a 345, G 1 y 346, V a 1 u H i s 348, H i s 349, H i s 35°, H i s 351, H i s 352, H i s 353] TPO (1-332), [A sn 25 , Lys m , T hr 333 , Ser 334 , lie 335 , G 1 y 336 , Tyr 337 , Pro 338 , Ty r 339 , Asp 340 , Va 1 m , P ro 342 , A sp 343, Ty r 344 , A 1 a 345, G 1 y 346, V a 1 u H is 348, H is 349, H is 35 °, H is 351, H is 352, H is 353] TPO ( 1-332),
[A s n25〕 TPO (1-332), [A sn 25 ] TPO (1-332),
[Th r33] TPO (1-332), [Th r 33 ] TPO (1-332),
[△H i s 33] TPO (1- 163)、 [△ H is 33 ] TPO (1-163),
[ΔΑ Γ g117] TP O (1-163), [ΔΑ Γ g 117 ] TP O (1-163),
[AG 1 y116] TPO (1-163), [AG 1 y 116 ] TPO (1-163),
[H i s33, Th r33', P r o34] TPO (1-163), [H i s 33 A 1 a33'; P r o34] TPO (1-163), [H is 33 , Thr 33 ', Pro 34 ] TPO (1-163), [H is 33 A 1 a 33 '; Pro 34 ] TPO (1-163),
[H i s 33 G 1 y33': P r o34, S e r 38] TPO (1-163), [H is 33 G 1 y 33 ' : Pro 34 , Ser 38 ] TPO (1-163),
[G 1 y 16, As n116', A r g'17] TPO (1—163) 、 [G 1 y 16 , As n 116 ′, Ar g ′ 17 ] TPO (1—163),
[G 1 y I6, A 1 a1I6>, A r gIIT] TPO (1-163)、 [G 1 y I6 , A 1 a 1I6> , A rg IIT ] TPO (1-163),
[G 1 y 16, G 1 y116', A r 117] TPO (1-163)、 [G 1 y 16 , G 1 y 116 ′, A r 117 ] TPO (1-163),
[A 1 a , V a 13, A r g129] TPO (1—163) 、 [A 1 a, V a 1 3, A rg 129] TPO (1-163),
[A 1 a V a 13, A r g133] TPO (1-163), [A 1 a V a 1 3 , A rg 133] TPO (1-163),
[A 1 a V a 13, A r 143] TPO (1-163)、 [A 1 a V a 1 3 , A r 143] TPO (1-163),
[A 1 a V a 13, L e u82] TPO (ト 163) 、 [A 1 a V a 1 3 , L eu 82] TPO ( DOO 163),
[A 1 a V a 13, L e u146] TPO (1-163), [A 1 a V a 1 3 , L eu 146] TPO (1-163),
[A 1 a V a 13, P r o148] TPO (1-163)、 [A 1 a V a 1 3 , P ro 148] TPO (1-163),
[A 1 a V a 13, A r g59] TPO (1-163)、 及び [A 1 a V a 1 3 , A rg 59] TPO (1-163), and
[A 1 a V a 13, A r 115] TPO (1-163) 0 [A 1 a V a 1 3 , A r 115] TPO (1-163) 0
上記の式において、 例えば [As n25] TPO (卜 332)は、 TP〇のアミノ酸配 列 1〜332 (酡列番号 1) の 25位のアミノ酸がァスパラギン (Asn) に置換さ れていることを示す。 また例えば [AH i s33] TPO (1- 163)は、 トランケ一 シヨン型アミノ酸配列 1〜 163 (配列番号 2) の 33位のヒスチジン (His) が 欠失されていることを示す。 さらに例えば [H i s 33, Th r33', P r o34] TP O (1-163)は、 トランケ一シヨン型アミノ酸配列 1〜163 (配列番号 2) の 33 位および 34位のアミノ酸がそれぞれヒスチジン(His)、 プロリン(Pro)に置換さ れ、 かつそれらのアミノ酸の間にトレォニン(Thr)が揷入(または付加) されてい ることを示す。 In the above formula, for example, [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〇 is replaced with asparagine (Asn) Is shown. For example, [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. Furthermore, for example [H is 33, Th r 33 ', P ro 34] 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.
TPOタンパク質またはその変異体にはさらに、 アミノ酸配列 [Me t"2-L y s— 、 [Me t"1] または [G 1 y"1] を含有させることができる。特に Met_Lys は、 目的タンパク質を細菌細胞内で発現させて精製した後、 ジぺプチダーゼ (例 えばカテブシン C) 等の酵素による処理によって除去することができる。 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 ]. In particular, 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).
あるいは、 TPOタンパク質のアミノ酸配列における特異的突然変異体は、 グ リコシル化の起こる部位 (セリン、 スレオニン、 またはァスパラギン等) の改変 も含み得る。 グリコシル化が起こらないかまたは部分的なグリコシル化しか起こ らないのは、 ァスパラギン残基を含むグリコシル化認識部位において、 あるいは o -結合炭水化物が付加することによって修飾された分子の部位において、ァミノ 酸の置換または欠失が起こった結果である。 ァスパラギン残基を含むグリコシル 化認識部位は、 適切な細胞性グリコシル化酵素によって特異的に認識されるトリ ペプチド配列から構成される。 このトリペプチド配列は、 Asn- Xaa- Thr またはAlternatively, 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であり、 ここで Xaaはプロリン以外のあらゆるアミノ酸である。 さ まざまなアミノ酸の置換または欠失が、 グリコシル化認識部位の 1 番目または 3 番目のアミノ酸位置のうちいずれか一方または両方において起こった場合 (およ び/または 番目の位置においてアミノ酸欠失がおきた場合)、 改変されたトリ ペプチド配列においてはグリコシル化が起こらない。 これに対して、 TPOタン パク質の立体構造 (またはコンホメーシヨン) を大きく変えない程度でかつ生物 学的活性を実質的に維持しうる範囲で、 TPOタンパク質のアミノ酸配列中に新 たにグリコシル化認識部位を導入することもできる。 そしてグリコシル化の結果 得られる TPOタンパク質は、 天然 (特にヒト) と同じ炭水化物鎖のみならず非 天然の炭水化物鎖を有しうる。 Asn-Xaa-Ser, where Xaa is any amino acid except proline. When 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. In contrast, 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. And 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.
本発明で使用しうる上記のような TP Oおよびその変異体からなる c一 mp C-mp comprising TPO and its variants as described above that can be used in the present invention.
1 リガンドは、 公知の方法 (日本国特許第 2991640号公報、 冊 95/26746など) ま たは慣用の遺伝子組換え技術(Sambrooket al., Molecular Cloning, A Laboratory1 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).
Manual, Cold Spring Harbor Laboratory Press, 1989、 およひ Ausubelet al. ,Manual, Cold Spring Harbor Laboratory Press, 1989, Osube Ausubelet al.,
Current Protocols in Molecular Biology (Current Protocols, U. S. A. , 1993) ) を用いて得ることができる。 トロンポポェチン活性を有するタンパク質をコード する D N Aは、本出願人によって独立行政法人産業技術総合研究所(日本国茨城県 つくば巿東 1丁目 1番地 1中央第 6)、 あるいは中華人民共和国 (CCTCC) ま たは中華民国 (F I RD 1)、 の寄託機関に寄託されている細胞株、例えば FERCurrent Protocols in Molecular Biology (Current Protocols, U.S.A., 1993)). 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
M BP— 4565、 CCTCC-M 95002, F I RD 1 940083 (ベ クタ一 PEF 18 S— A2 a)、 FERM BP— 4564、 CCTCC— M95M BP—4565, CCTCC-M 95002, F I RD 1 940083 (Vector PEF 18 S—A2a), FERM BP—4564, CCTCC—M95
001、 F I RD I 940082 (ベクター pHT l— 23 1)、 FERM BP 一 46 17、 CCTCC一 M95004、 F I RD 1 940085 (ベクター p001, FIRD I 940082 (vector pHT l-231), FERM BP-14617, CCTCC-M95004, FIRD 1940085 (vector p
HTF 1)、 FERM BP— 46 16、 CCTCC -M 95003, F I RD I 940084 (ベクター pHGT l)、 FERM BP— 4988、 CCTCC一 C 95004、 F I RD 1 960023 (CH028Z1/1Z3— C 6)、 また は FERB BP— 4989、 CCTCC_C 95005、 F I RD I 9600 24 (CH0163T— 63— 79— C I) から単離することができる。 このよ うな DNAを出発原料として適当な発現用ベクター (プラスミドなど) に組み込 んだ後、 このベクターで原核 (大腸菌など) または真核宿主細胞 (チャイニーズ ハムスター卵巣細胞など) を形質転換し、 得られた宿主の培養により目的の c一 mp 1 リガンドを製造することができる。 c一 mp 1 リガンドは、 一般的にタン パク質の精製に用いられる手段、 例えばイオン交換クロマトグラフィー、 レクチ ンァフィニティ一クロマトグラフィ一、卜リアジン色素吸着クロマトグラフィー、 疎水相互クロマトグラフィー、 ゲル濾過クロマトグラフィー、 逆相クロマトグラ フィ一、 へパリンァフィ二ティ一クロマトグラフィー、 硫酸化ゲルクロマトダラ フィ一、 ハイド口キシルアパタイトクロマトグラフィー、 抗体ァフィ二ティーク 口マトグラフィ一、 等電点クロマトグラフィー、 金属キレーティングクロマトグ ラフィー、 分取電気泳動法、 等電点電気泳動法などの手段の一つ以上を組み合わ せることによって、 精製可能である。 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), or FERB BP—4989, CCTCC_C 95005, FI RD I 9600 24 (CH0163T—63— 79—CI). After incorporating such DNA into a suitable expression vector (such as a plasmid) as a starting material, the vector is used to transform prokaryotic (such as E. coli) or eukaryotic host cells (such as Chinese hamster ovary cells) and obtained. By culturing the obtained host, the desired c-mp1 ligand can be produced. 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.
本発明で使用しうる TPOタンパク質の誘導体の例は、 少なくとも 1つの水溶 性ポリマー部分に結合した c—mp 1リガンドタンパク質である。 水溶性ポリマ 一は、例えばポリエチレングリコール、モノメトキシーポリエチレンダリコール、 デキストラン、 ポリ (N-ビニルピロリドン) ポリエチレングリコール、 プロピレ ングリコールホモポリマー、 ポリプロピレンォキシド /エチレンォキシドコポリ マー、 ポリオキシェチル化ポリオール (すなわちグリセロール) およびポリビニ ルアルコールからなる群から選択できる。 好ましくは、 少なくとも 1つのポリェ チレングリコール(PEG)分子に結合した PEG化 c—mp 1リガンド分子である。さ らに好ましくは、 1 つのポリエチレングリコール分子をタンパクの N末端に結合 したモノぺグ化 c _mp 1リガンド分子である。  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. Preferably, it is a 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.
c -mp 1リガンドの PEG化は、 当業界で周知となっている PEG化反応を利用 して達成され得る (たとえば、 Focus on Growth Factors 3 (2) :4- 10 (1992) 、 EP 0 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
154316、 EP 0401 384参照)。 好ましくは、 反応性ポリエチレングリコール分子 を用いるァシル化またはアルキル化反応を介して、 PEG化を行うのがよい。 PEG の好ましい平均分子量は約 5kDa〜約 50kDa であり、 約 12kDa 〜約 25kDa の間で あればなおよい。 水溶性ポリマ一対 c _ m p 1 リガンドタンパク質の反応モル比 は、 一般に 1 : 1から 100 : 1ならよく、 ポリ PEG化の場合 1 : 1〜20 : 1、 モノ PEG化 では 1 : 1 〜5 : 1 であればなおよい。 154316, EP 0401 384). Preferably, 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. In general, 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.
ァシル化による PEG化には、 一般に、 ポリエチレングリコール の活性エステ ル誘導体と、 c一 m p 1リガンドタンパク質との反応を含む。好適な活性化 PEGェ ステルは、 PEGを N -ヒドロキシスクシンイミドへエステル化したものである。 ァ シル化による PEG付加反応では、 一般に、 多数の PEGが付加した c _ m p 1 リガ ンド産物が生じ、 そこではァシル結合基を介してリジン ε—ァミノ基に PEG付加 が起こる。 結合はアミドであることが好ましい。 また、 生じる産物は、 実質上、 PEG1個の付加物、 PEG2個の付加物または PEG3個の付加物のみ (例えば >95%)で あることが好ましい。 必要に応じて、 透析、 塩析、 限外濾過法、 イオン交換クロ マトグラフィ一、 ゲル濾過クロマトグラフィーおよび電気泳動を含む標準的技術 その他の技術を適用することによって、 混合物または未反応種の中から、 より精 製された PEG 付加種を分離することができる。  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 ε-amino group via an acyl-bonding group. Preferably, the bond is an amide. It is also preferred that 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.
アルキル化による PEG付加には、還元アルキル化反応を利用することができる。 1個の PEGが付加した産物を得るための還元アルキル化を介した誘導体化では、 リジン残基における ε—ァミノ基と、タンパク質における Ν -末端残基の 一アミ ノ基との間における pKa 値の差を利用することができる。  For the addition of PEG by alkylation, a reductive alkylation reaction can be used. For derivatization via reductive alkylation to obtain a product with one PEG attached, the pKa value between the ε-amino group at the lysine residue and one amino group at the Ν-terminal residue in the protein Can be used.
本発明で使用しうる PEG化 c一 m p 1 リガンドの具体例は、 W 095/26746 の Specific examples of PEGylated c-mp1 ligands that can be used in the present invention are described in W 095/26746.
Tab l e 10に記載されるような mono-MePEG- MGDFである。 Mono-MePEG-MGDF as described in Table 10
本発明の医薬組成物は、 治療上有効量の c一 m p 1 リガンドに加えて、 賦形 剤、 担体または希釈剤を含むことができ、 投与方法に応じて種々の投薬剤形 (例 えば錠剤、 溶液剤、 懸濁剤、座剤、 点鼻薬など) としうる。 また徐放性を付与した 製剤とすることもできる。徐放性製剤は腸溶性物質および/またはユードラジッド 等の疎水性ポリマー物質を親水性物質と組合わせ、 それらに薬剤を単に分散させ て多粒状化するかまたは多層形態とし、 必要に応じて疎水性被覆剤でコーティン グすることによって得ることができる。また必要に応じて、防腐剤、界面活性剤、 可溶化剤、 酸化防止剤、 等張化剤などの添加剤を含有させてもよい。 また本発明 の医薬組成物は、 凍結乾燥または乾燥された剤形であってもよく、 使用時に医薬 上許容可能な希釈剤に溶解して対象患者に投与しうる。 凍結乾燥製剤には安定化 剤として糖類や界面活性剤を含有させることができる。 希釈剤の例は、 種々の pH およびイオン強度から成る緩衝剤 (例えばトリス-塩酸、 酢酸塩、 燐酸塩)、 生理 食塩水などを含む。 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. Also, if necessary, preservatives, surfactants, Additives such as a solubilizing agent, an antioxidant, and a tonicity agent may be contained. Further, 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. Examples of diluents include buffers of different pH and ionic strength (eg, Tris-HCl, acetate, phosphate), saline, and the like.
界面活性剤の例は Tween 20、 Tween 80、 Pluronic F68、 胆汁酸塩などである。 可溶化剤の例はグリセロール、 ポリエチレングリコールなどである。 酸化防止剤 の例はァスコルビン酸、 メタ重亜硫酸ナトリウムなどである。 防腐剤の例はチメ 口サール、 ベンジルアルコール、 パラベンなどである。 等張化剤の例はラクトー ス、 マンニトールなどである。 その他の添加剤として、 容器の表面に吸着しない ようにするためのアルブミン、 ゼラチン等のタンパク質、 プロテアーゼ阻害剤、 消化管吸収促進剤などを挙げることができる。  Examples of surfactants are Tween 20, Tween 80, Pluronic F68, bile salts and the like. Examples of solubilizers are glycerol, polyethylene glycol and the like. Examples of antioxidants are ascorbic acid, sodium metabisulfite and the like. Examples of preservatives are thimeru sal, benzyl alcohol, parabens and the like. Examples of 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.
本発明の医薬組成物は、 有効成分とポリエチレングリコールのような水溶性ポ リマ一との共有結合、 有効成分と金属イオンとの錯体化、 あるいはポリ乳酸、 ポ リグリコール酸、 ヒドロゲルなどのような重合化合物の粒状製剤中またはその表 面上への、 あるいはリボソーム、 ミクロエマルジヨン、 ミセル、 単層もしくは多 層小胞、 赤血球ゴースト、 またはスフエロプラスト中への有効成分の取り込み、 などの形態をとることができる。  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. 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.
本発明の医薬組成物は、 非経口 (静脈内、 動脈内、 直腸内、 皮下、皮内、 経肺、 経鼻など) および経口を含めた種々の投与経路で投与しうる。 投与量は、 有効成 分として通常約 0. 01 ^ g /kg体重〜約 1000 M g /kg体重、好ましくは 0. 05 g/kg 体重〜 300 ^ g/kg体重、さらに好ましくは 1 g/kg体重〜 100 ^ g/kg体重である。 投薬量は年齢、病状、体重、 性別、患者の食事状態及び投与経路等に応じて変化し 得るし、 また一日一回または数回に分けて約 1〜2 週間あるいはそれ以上に亘っ て投与することができる。 c—m p 1 リガンドは、上記の用量範囲に限定されず、 副作用を発現しない範囲でかつ目的の治療効果を達成しうる投薬量で組成物中に 含有しうる。 後述の実施例では N末端が単一のポリエチレングリコールで修飾さ れた配列番号 2のァミノ酸配列からなるポリべプチド PEG - rHuMGDFをタンパク重 量で約 2. 5 g (約 143pmol) /kg/日の用量で使用したときには、 他の付加的造血 因子を含有させなくとも、 骨髄異形成症候群または再生不良性貧血患者の臨床試 験において所望の血小板増加効果および赤血球増加効果(すなわち貧血改善効果) をともに達成することができた。 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. In the examples described below, 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).
本発明の組成物には、 c—m p 1 リガンドに加えてエリスロポエチン (EP0)、 顆粒球コロニー刺激因子 (G- CSF)、 顆粒球マクロファ一ジコロニー刺激因子 (GM-CSF) , マクロファージコロニー剌激因子 (M- CSF)、 コロニー刺激因子 (CSF) - 1、 インタ一ロイキン (IL) -1、 IL-2、 IL_3、 IL-4、 IL - 5、 IL - 6、 IL— 7、 IL - 8、 IL - 9、 IL- 10、 IL - 11、 IL - 12、 IL - 13、白血球遊走阻止因子(LIF)、幹細胞因子(SCF)、 インタ一フエロン α, β ' ァのような因子の 1つ以上を付加的造血因子として含有 せることが可能である。  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 α, β ' Can be included as an additional hematopoietic factor.
本発明により、 配列番号 1のアミノ酸配列のうち少なくとも 7位〜 1 5 1位の アミノ酸残基を含むアミノ酸配列を有するポリペプチド、 その変異体、 またはそ れらの誘導体からなりかつトロンポポェチン活性を有する治療上有効量の c一 m p 1リガンドを含む上記の医薬組成物を、 骨髄異形成症候群または再生不良性貧 血患者に投与することによって、 血小板及び赤血球を増加させるために該患者を 治療することができる。 たとえば、 該患者に対し c _m p 1 リガンド (後述実施 例の PEG- rHuMGDF) をタンパク重量で約 2. 5 g (約 143pmol) /kg/日の用量で約 2週間にわたって投与したとき、 投与開始後約 2週間までは網赤血球数の継続的 な増加傾向は認められないが約 2〜約 5週間で継続的に増加して最大値に達し、 その最大値は投与開始日の値の約 1. 5〜約 2. 5倍となる。 また、 投与開始の約 2 週間後にヘモグロビン濃度が増加しはじめ、投与開始の約 8〜約 12週後に最大値 に達し、その最大値は投与開始日の値に比べて血液 I dLあたり約 2〜約 3. 5gの増 加を示す。 本発明の治療法は、 本発明の医薬組成物を骨髄異形成症候群または再 生不良性貧血患者に投与する場合、 投与後ある一定のタイムラグ (約 1〜約 2週 間) の後に網赤血球数およびヘモグロビン濃度が増加し始め最大値に達するとい う特徴を有している。 使用する C -即 1リガンドの種類、 投与量によって網赤血球 数およびヘモグロビン濃度の時間あたりの増加量が変化するが、 治療効果の推移 は類似の傾向を示すと考えられる。 実施例 According to the present invention, 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. Can be. For example, when c_mp1 ligand (PEG-rHuMGDF in Examples described later) was administered to the patient at a dose of about 2.5 g (about 143 pmol) / kg / day of protein for about 2 weeks, 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. In addition, 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. When the pharmaceutical composition of the present invention is administered to a patient with myelodysplastic syndrome or aplastic anemia, 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. Example
本発明を以下の実施例、比較例および参考例によってさらに具体的に説明する。 本実施例で使用した薬剤、 投与方法および治験方法は以下のとおりである。 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.
1 . 薬剤及び投与方法 1. Drugs and administration method
ポリエチレンダリコール修飾遺伝子組換えヒト巨核球増殖発育因子 (以下、 Polyethylene Dalicol Modified Recombinant Human Megakaryocyte Growth Factor (hereinafter referred to as
PEG- rHuMGDFという) は、 国際公開 W095/26746に記載の方法を用いて、 配列番号 2のァミノ酸配列からなるポリべプチドを遺伝子組換え技術により大腸菌で生産 し、 その N末端にポリエチレングリコール (平均分子量 20kDa) を化学的に結合 させて得られたものである。 1バイアル (l. OmL) あたり PEG- rHuMGDFをタンパク 量として 250 ^ g含む注射剤を、 希釈用溶液(PEG- rHuMGDFを含まない溶媒) で 10 倍希釈後、 患者体重 1kgあたり 2. (タンパク量として) を 1 日 1回 14日間 静脈内投与した。 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.
2 . 治験方法  2. Investigation method
本試験の実施に先立って、 実施計画の内容に関して実施施設の治験審査委員会 で審議され承認された。 また、 本試験の実施にあたっては実施計画、 日本国薬事 法第 14条第 3項及び第 80条の 2に規定する基準、 ヘルシンキ宣言ならびに医薬 品の臨床試験の実施に関する基準 (GCP) 及びその省令を遵守した。  Prior to the implementation of this study, the contents of the implementation plan were discussed and approved by the clinical trial review board of the site. In conducting this study, the implementation plan, the standards stipulated in Article 14, Paragraph 3 and Article 80-2 of the Pharmaceutical Affairs Law of Japan, the Declaration of Helsinki and the standards (GCP) for conducting clinical trials of pharmaceuticals and their ministerial ordinances Was observed.
バイタルサイン、 自覚症状及び他覚所見は、 治験薬投与期間中は毎日、 投与終 了後経過観察第 1週及び第 2週は週 3回以上、 経過観察第 3週及び第 4週は週 1 回以上実施した。 また、 投与開始前 6週間及び治験薬投与期間 ·経過観察期間中 の輸血 (血小板及び赤血球) についても調査した。 血液学的検査は、 治験薬投与 期間ならびに経過観察第 1週及び第 2週は週 3回以上、 経過観察第 3週及び第 4 週は週 1回以上実施した。 血液生化学検査、 凝固 ·線溶系検査及び尿検査は、 投 与開始日及び投与 2日目以降の全期間で週 1回実施した。血中 PEG-rHuMGDF濃度 は、 投与開始日の投与開始前、 投与 15分後、 2時間後及び 8時間後、 投与 2日目 の投与開始前、 投与 4日目、 7日目、 10日目の投与開始前及び投与 15分後、 投与 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. In addition, blood transfusions (platelets and red blood cells) during the 6 weeks before the start of treatment and during the study drug administration period and follow-up period were also investigated. 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
14日目の投与開始前、 投与 15分後、 2時間後及び 8時間後、 投与終了翌日及び投 与終了 2週間後の血清を用い、 株式会社エスアールエル ·ティジンバイオにて測 定された。 また、 PEG_rHuMGDF及び PEG-rHuMGDFのタンパク部分に対する抗体 価 (MA法) ならびに TP0中和抗体 (生物活性中和試験) は、 投与開始前及び投 与終了 2週間後の血清を用い、 株式会社エスァ一ルエル ·ティジンバイオにて測 定された。 Before the start of administration on day 14, 15 minutes, 2 hours and 8 hours after administration, using serum from the day after administration and 2 weeks after administration, measured by SRL Tijin Bio Co., Ltd. Was decided. The antibody titer against the protein portion of PEG_rHuMGDF and PEG-rHuMGDF (MA method) and the TP0 neutralizing antibody (biological activity neutralization test) were determined using the serum before the start of administration and two weeks after the end of administration. Measured at Luel Tijin Bio.
ぐ実施例 1 > 骨髄異形成症候群患者における試験例 Example 1> Test cases in patients with myelodysplastic syndrome
1 . 対象患者  1. Target patients
本症例 (49歳、 男性) は平成 6年 11月に骨髄異形成症候群 (FAB分類で不応性 貧血 (RA) ) と診断され、 平成 6年 12月から平成 7年 1月までは活性型ビタミン D3 (アルファカルシドロール) 2 g/日及びレノグラスチム 100 i g/日を投与され た。 また、 平成 7年 1月から 2月にプリモポラン 10mg/日投与、 平成 7年 3月に インターロイキン (IL_) 11投与、 平成 7年 5月にステロイドパルス療法及びそ れに引き続き 10月まで維持量のステロイド投与を受けた。以上のいずれの治療に 対しても効果は認められなかった。 This case (49-year-old man) was diagnosed with myelodysplastic syndrome (FAB classification refractory anemia (RA)) in November 1994, and active vitamins from December 1994 to January 1995. D 3 (alpha Cal Sid roll) 2 g / day and lenograstim 100 ig / day was administered. Primopolane 10 mg / day was administered from January to February 1995, interleukin (IL_) 11 was administered in March 1995, steroid pulse therapy in May 1995, and the maintenance dose was continued until October. Received steroids. No effect was observed for any of the above treatments.
平成 11年 3月から 7月までの血小板数は平均で約 13, 000/mm3で血小板輸血は 施行されていなかった。選択基準(20歳以上 75歳以下、 P. S.が 0〜2)を満たし、 除外基準(重篤な合併症 ·感染症ならびに骨髄線維化を有する患者、 GOT及び GPT が施設正常値の 3倍以上、血清クレアチニンが 2. Omg/dL以上、血栓症の既往を有 し血栓塞栓症を起こす恐れのある患者、 生物学的製剤に過敏症の患者、 皮内反応 試験陽性及び妊娠 ·授乳中の婦人) に当てはまらなかったため、 本人より文書に よる同意を得て本試験に症例登録された。 The number of platelets from March 1999 to July did not platelet transfusion is in force in about 13, 000 / mm 3 on average. Exclusion criteria (Patients with severe complications, infection and bone marrow fibrosis, GOT and GPT more than 3 times the normal value of the facility, 2. Serum creatinine 2. Omg / dL or more, patients with a history of thrombosis who may cause thromboembolism, hypersensitivity to biological products, positive intradermal reaction test and pregnant / nursing women) The patient was enrolled in this study with written consent from the individual.
2 . 血小板数の変動 2. Fluctuations in platelet count
本症例においては、 血小板数は図 1の通り一過性の増加を示した。 2. 5 i g/kg/ 日の PEG- rHuMGDFを患者に投与した 14日間は血小板数の持続的な増加傾向は認 められなかったが、 投与開始後 16日目から 28日目の間に血小板数は 16, 000/mm3 から 21, 000/mm3まで増加、 その後も増加傾向は持続し投与開始後 42 日目には血 小板数 25, 000/mi3に達した。その後、投与開始後 57日目には血小板数 22, 000/mm3 と増加傾向が維持されたが、 投与開始後 71 日目には血小板数 12, 000/nmi3と投与 前と同様まで復した。 In this case, 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.
なお、 本症例では出血傾向は認められず、 投与開始前 6週間及び投与開始後 6 週間に血小板輸血は施行されなかった。 No bleeding tendency was observed in this case, and 6 weeks before and 6 No weekly platelet transfusions were performed.
3 . 赤血球系の変動  3. Fluctuations in the erythroid system
図 2にヘモグロビン濃度の推移を、 図 3に網赤血球数の推移を示した。 本症例 においては、 2. 5 i g/kg/日の PEG- rHuMGDF 投与開始日にはヘモグロビン濃度 10. Og/dL, 網赤血球数 59, 400/mm3であったが、 投与開始後 14 日目から網赤血球 数の持続的な増加傾向が認められ、 投与開始後 35 日目に網赤血球数 97, 900/mm3 に達した後、 投与開始後 Π 日目には網赤血球数 59, 700/mm3と投与前と同様まで 復した。 これに対し、ヘモグロビン濃度は投与開始後 28日目までは持続的な増加 傾向は認められなかったが、投与開始後 35日目から 57日目の間に 10. 8g/dLから 12. lg/dLまで増加し、 その後も維持された。 Figure 2 shows the change in hemoglobin concentration, and Figure 3 shows the change in reticulocyte count. In this case, 2. 5 ig / kg / hemoglobin concentration in PEG-rHuMGDF administration start day day 10. Og / dL, but were reticulocyte number 59, 400 / mm 3, 14 days after initiation of administration from sustained increase in reticulocyte count was observed after reaching the reticulocyte number 97, 900 / mm 3 in 35 days after the start of administration, reticulocyte number 59 in Π day after the start of administration, 700 / It was restored to mm 3 as before administration. On the other hand, 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.
他の赤血球系パラメ一夕に関してもヘモグロビン濃度と同様の推移を示した。 赤血球数は投与開始日に 270X 104/mm3であったが、投与開始後 57日目には 319 X lOVmm3まで増加した。 また、 へマトクリットも投与開始日に 30. 6%であったが、 投与開始後 57日目には 36. 2%まで増加した。 ただし、 赤血球恒数である平均赤血 球容積、 平均赤血球ヘモグロビン量及び平均赤血球ヘモグロビン濃度は PEG- rHu MGDF投与前 ·中 ·後を通じて一定の範囲内の変動に留まった。 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. However, 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.
4 . 白血球系の変動  4. Leukocyte fluctuation
白血球数は PEG- rHuMGDF投与前 ·中 ·後を通じて一定の範囲内の変動に留まつ た。 また、 白血球分画の変動も PEG_rHuMGDF投与前 ·中 ·後を通じて一定の傾向 は認められなかった。  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.
5 . 血中 PEG_rHuMGDF濃度  5. Blood PEG_rHuMGDF concentration
血中 PEG- rHuMGDF濃度は、 PEG- rHuMGDF及び内因性 TP0を検出する ELISA法 にて測定した。 投与開始前の内因性 TP0濃度は PEG- rHuMGDF換算で 1, 595pg/mL と健常人と比較し高値であった。投与 1日目の投与 15分後には 60, 730pg/niLまで 上昇したが投与 2 日目の投与前には 7, 838pg/mL まで低下した。 投与 15分後の The blood PEG-rHuMGDF concentration was measured by an ELISA method for detecting PEG-rHuMGDF and endogenous TP0. Before the start of administration, 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濃度は投与 4日目に 78, 980pg/mL、 投与 7日目に 75, 620pg/mL, 投 与 10日目に 81, 320pg/mL, 投与 14日目に 81, 960pg と、 14日間の投与中に定 常状態に達した。 また、 投与前 (投与 24時間後) の PEG- rHuMGDF濃度も、 投与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. In addition, the PEG-rHuMGDF concentration before administration (24 hours after administration)
4日目に 11, 100pg/mL, 投与 7日目に 15, 120pg/mL、 投与 10日目に 12, 250pg/mL, 投与 14日目に 13, 780pg/mL,投与終了翌日に 13, 940pg と、 14日間の投与中に 定常状態に達した。 投与終了 1週間後の内因性 TP0濃度は PEG-rHuMGDF換算で 1, 464pg/mLと投与前値にほぼ復していた。 11, 100 pg / mL on Day 4, 15, 120 pg / mL on Day 7, 12, 250 pg / mL, on Day 10 The dose reached 13,780 pg / mL on day 14 and 13,940 pg on the day after administration, reaching a steady state during the 14-day administration. One week after the end of the administration, the endogenous TP0 concentration was 1,464 pg / mL in terms of PEG-rHuMGDF, almost reverting to the value before administration.
<実施例 2 > 骨髄異形成症候群患者における試験例 <Example 2> Test example in myelodysplastic syndrome patients
1 . 対象患者  1. Target patients
本症例 (54歳、 女性) は昭和 59年 7月に骨髄異形成症候群 (FAB分類で RA) と診断され、 同年ェポェチンアルファ (rHuEPO) 20, 000単位/日を 4週間投与さ れた。 また、 平成 3年 8月から 10月まで再び rHuEPOを漸増法で 3, 000〜24, 000 単位/日投与されたが、 いずれも効果は認められなかった。 また、 平成 11年 1月 から酢酸メテノロンを投与されている。  This case (54-year-old woman) was diagnosed with myelodysplastic syndrome (RA according to FAB classification) in July 1984, and was administered epoetin alfa (rHuEPO) 20,000 units / day for 4 weeks in the same year . From August to October 1991, rHuEPO was again administered in an ascending manner from 3,000 to 24,000 units / day, but no effect was observed in any case. Also, metenolone acetate has been administered since January 1999.
平成 11年 6月から 10月までの血小板数は平均で約 21, 000/mm3で血小板輸血は 施行されていなかった。選択基準(20歳以上 75歳以下、 P. S.が 0〜2) を満たし、 除外基準(重篤な合併症 ·感染症ならびに骨髄線維化を有する患者、 GOT及び GPT が施設正常値の 3倍以上、血清クレアチニンが . Offlg/dL以上、血栓症の既往を有 し血栓塞栓症を起こす恐れのある患者、 生物学的製剤に過敏症の患者、 皮内反応 試験陽性及び妊娠 ·授乳中の婦人) に当てはまらなかったため、 本人より文書に よる同意を得て本試験に症例登録された。 Platelet count from June 1999 to October did not platelet transfusion is in force in about 21, 000 / mm 3 on average. Exclusion criteria (Patients 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.
2 . 血小板数の変動  2. Fluctuations in platelet count
本症例においては、 血小板数は図 4の通り一過性の増加を示した。 2. 5 ^ g/kg/ 日の PEG- rHuMGDFを患者に投与した 14日間は血小板数の持続的な増加傾向は認 められなかったが、 投与開始後 21 日目から 28日目の間に血小板数は 28, 000/mm3 から 39, OOO/mm3まで増加した後、 投与開始後 52 日目まで血小板数 38, 000/mm3と 増加傾向が維持されたが、 投与開始後 80 日目には血小板数 16, 000/mm3と投与前 と同様まで復した。 In this case, 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. after the number of platelets was increased from 28, 000 / mm 3 to 39, OOO / mm 3, platelet count 38 to 52 days after the start of administration, 000 / mm 3 and an increase tendency is maintained, after the initiation of administration 80 days The eyes returned to a platelet count of 16,000 / mm 3 , the same as before administration.
なお、 本症例では投与前から時折歯肉からの少量出血または下肢の紫斑が認め られたが、 特に投与開始前 6週間及び投与開始後 6週間に血小板輸血は施行され なかった。  In this case, small bleeding from the gingiva or purpura of the lower limb were occasionally observed before administration, but platelet transfusion was not performed particularly 6 weeks before administration and 6 weeks after administration.
3 . 赤血球系の変動 図 5にヘモグロビン濃度の推移を、 図 6に網赤血球数の推移を示した。 本症例 においては、 2. 5 g/kg/日の PEG- rHuMGDF 投与開始日にはヘモグロビン濃度 6. 8g/dL、 網赤血球数 44, 300/匪3であったが、 投与開始後 11 日目の網赤血球数 49, 600/mm3から持続的な増加傾向が認められ、 投与開始後 28 日目に網赤血球数 71, 400/min3に達した後、 投与開始後 80 日目には網赤血球数 37, 700/min3まで復し た。 これに対し、ヘモグロビン濃度は投与開始後 16日目までは持続的な増加傾向 は認められなかったが、 投与開始後 18 日目から 80 日目の間に 7. 4g/dL から 9. Og/dLまで増加し、 その後も維持された。 3. Fluctuations in the erythroid system Figure 5 shows changes in hemoglobin concentration, and Figure 6 shows changes in reticulocyte count. In this case, 2. 5 g / kg / hemoglobin concentration in PEG-rHuMGDF administration start day day 6. 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. On the other hand, 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.
他の赤血球系パラメータに関してもヘモグロビン濃度と同様の推移を示した。 赤血球数は投与開始日に 164Χ ΐ04/ηιπι3であったが、投与開始後 57日目には 222 Χ lOVmm3まで増加した。 また、 へマトクリットも投与開始日に 20. 4%であったが、 投与開始後 57日目には 26. 7%まで増加した。 ただし、 赤血球恒数である平均赤血 球容積、 平均赤血球ヘモグロビン量及び平均赤血球ヘモグロビン濃度は PEG- rHu MGDF投与前 ·中 ·後を通じて一定の範囲内の変動に留まった。 The other erythroid parameters showed the same transition as the hemoglobin concentration. Red blood cell count was 164Χ ΐ0 4 / ηιπι 3 start date administration, but 57 days after the start of administration was increased to 222 Χ lOVmm 3. Hematocrit was also 20.4% on the first day of treatment, but increased to 26.7% on the 57th day after treatment. However, 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.
なお、 自覚症状としては投与前からだるさ及び易疲労感がみとめられ、 P. S.は 1であったが、 投与終了後貧血の改善に伴って P. S.は 0に改善された。  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.
4 . 白血球系の変動  4. Leukocyte fluctuation
白血球数は PEG- rHuMGDF投与前 ·中 ·後を通じて一定の範囲内の変動に留まつ た。 また、 白血球分画の変動も PEG-rHuMGDF投与前 ·中 ·後を通じて一定の傾向 は認められなかった。  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.
5 . 血中 PEG- rHuMGDF濃度  5. PEG-rHuMGDF concentration in blood
投与開始前の内因性 TP0濃度は PEG- rHuMGDF換算で 1653pg/mLと健常人と比較 し高値であった。投与 15分後の PEG- rHuMGDF濃度は 14日間の投与中に定常状態 に達した。 また、 投与前 (投与 24時間後) の PEG- rHuMGDF濃度も 14日間の投与 中に定常状態に達した。 投与終了 2週間後の内因性 TP0濃度は投与前値にほぼ復 していた。  Before the start of administration, 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. Two weeks after the end of administration, the endogenous TP0 concentration almost returned to the value before administration.
<実施例 3 > 再生不良性貧血患者における試験例 <Example 3> Test example in patients with aplastic anemia
1 . 対象患者 本症例 (48歳、 男性) は平成 9年 2月に再生不良性貧血と診断され、 平成 9年 3月から平成 1 1年 7月までシクロスポリン 400〜300nig/日及び平成 11年 1月まで フィルグラスチム 150 z g/日を投与された。 平成 9年 9月時点でシクロスポリン 及びフィルグラスチムの効果は認められなかったため、平成 9年 1 1月にさらに抗 胸腺細胞免疫グロブリンを投与された。 その結果反応 (Good Response) が認めら れ、 赤血球及び血小板輸血が不要となった。 1. 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.
平成 1 1年 9月から 1 1月までの血小板数は平均で 19, 000/mm3で血小板輸血は施 行されていなかった。 選択基準 (16歳以上 65歳未満、 P. S.が 0〜2) を満たし、 除外基準(重篤な合併症 ·感染症ならびに骨髄線維化を有する患者、 GOT及び GPT が施設正常値の 3倍以上、血清クレアチニンが 2. Omg/dL以上、血栓症の既往を有 し血栓塞栓症を起こす恐れのある患者、 生物学的製剤に過敏症の患者、 皮内反応 試験陽性及び妊娠 ·授乳中の婦人) に当てはまらなかったため、 本人より文書に よる同意を得て本試験に症例登録された。 1989 platelet count from 1 September until 1 January is platelet transfusion on average at 19, 000 / mm 3 had not been enforced. Selection criteria (16 years old to less than 65 years old, PS 0-2), exclusion criteria (serious complications, patients with infection and bone marrow fibrosis, GOT and GPT more than 3 times 2. Serum creatinine 2. Omg / dL or more, patients with a history of thrombosis who may cause thromboembolism, hypersensitivity to biological products, positive intradermal reaction test and pregnant / nursing women) The patient was enrolled in this study with written consent from the individual.
2 . 血小板数の変動  2. Fluctuations in platelet count
本症例においては、 血小板数は図 7のような増加を示した。 2. 5 z g/kg/日の PEG-rHuMGDFを患者に投与した 14 日間は血小板数の持続的な増加傾向は認めら れなかったが、投与開始後 17日目から 29日目の間に血小板数は 22, 000/mm3から 36, 000/匪3まで増加した後、 投与開始後 68 日目まで血小板数 33,000/πυη3と増加 傾向が維持された。 In this case, 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.
なお、 本症例では外傷による左眼下の紫斑以外に出血傾向は認められず、 投与 開始前 6週間及び投与開始後 6週間に血小板輸血は施行されなかった。  In this case, no bleeding tendency was observed other than purpura below the left eye due to trauma, and platelet transfusion was not performed 6 weeks before and 6 weeks after the start of treatment.
3 . 赤血球系の変動  3. Fluctuations in the erythroid system
図 8にヘモグロビン濃度の推移を、 図 9に網赤血球数の推移を示した。 本症例 においては、 2. 5 ^ g/kg/日の PEG-rHuMGDF 投与開始日にはヘモグロビン濃度 Figure 8 shows changes in hemoglobin concentration, and Figure 9 shows changes in reticulocyte count. In this case, the hemoglobin concentration on the first day of PEG-rHuMGDF administration at 2.5 ^ g / kg / day
5. 6g/dL、 網赤血球数 30, 200/mm3であったが、 投与開始後 10 日目の網赤血球数5. 6 g / dL, was the reticulocyte number 30, 200 / mm 3, reticulocyte count of 10 days after initiation of administration
39, 800/mm3から持続的な増加傾向が認められ、 投与開始後 47 日目に網赤血球数39, 800 / mm 3 showed a continuous increasing trend, and the reticulocyte count on the 47th day after starting treatment
72, 500/mm3に達した後、 投与開始後 68 日目には網赤血球数 39, 500/mm3まで復し た。 これに対し、ヘモグロビン濃度は投与開始後 19日目までは持続的な増加傾向 は認められなかったが、 投与開始後 1 日目から 68 日目の間に 6. 2g/dL から 8. 9g/dLまで増加した。 After reaching 72,500 / mm 3 , the reticulocyte count returned to 39,500 / mm 3 68 days after the start of administration. On the other hand, 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.
他の赤血球系パラメ一夕に関してもヘモグロビン濃度と同様の推移を示した。 . 赤血球数は投与開始日に 145 X l04/mm3であったが、投与開始後 57日目には 225 X lOVmm3まで増加した。 また、 へマトクリットも投与開始日に 17. 1%であったが、 投与開始後 57日目には 27. 1%まで増加した。 ただし、 赤血球恒数である平均赤血 球容積、 平均赤血球ヘモグロビン量及び平均赤血球ヘモグロビン濃度は PEG-rHu MGDF投与前 · 中 ·後を通じて一定の範囲内の変動に留まった。 The changes in hemoglobin concentration were similar for other erythroid parasites. . Red blood cell count was 145 X l0 4 / mm 3 at the starting date of administration, but 57 days after the start of administration was increased to 225 X lOVmm 3. Hematocrit was 17.1% on the first day of administration, but increased to 27.1% on the 57th day after administration. However, the average erythrocyte volume, the average erythrocyte hemoglobin amount and the average erythrocyte hemoglobin concentration, which are the erythrocyte constants, remained within a certain range before, during, and after administration of PEG-rHu MGDF.
4 . 白血球系の変動  4. Leukocyte fluctuation
白血球数は PEG- rHuMGDF投与前 ·中 ·後を通じて一定の範囲内の変動に留まつ た。 また、 白血球分画の変動も PEG- rHuMGDF投与前 ·中 *後を通じて一定の傾向 は認、められなかった。  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.
5 . 血中 PEG- rHuMGDF濃度  5. PEG-rHuMGDF concentration in blood
投与開始前の内因性 TP0濃度は PEG- rHuMGDF換算で 1056pg/mLと健常人と比較 し高値であった。 本症例においても骨髄異形成症候群の症例と同様に、 投与直後 及び投与前(投与 24時間後)の PEG- rHuMGDF濃度は 14日間の投与中に定常状態 に達し、 投与終了後は投与前値に復した。 ぐ結果の考察 >  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. In this case, as in the case of myelodysplastic syndrome, the PEG-rHuMGDF concentration immediately after administration and before administration (24 hours after 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>
1 . 骨髄異形成症候群患者及び再生不良性貧血患者における血小板数増加作用 本報告の骨髄異形成症候群患者及び再生不良性貧血患者の血小板数は、 症例登 録前の平均で 10, 000〜20, 000/讓 3程度で安定しており、 PEG- rHuMGDF投与開始後1. Platelet count increase in myelodysplastic syndrome patients and aplastic anemia patients The platelet counts in myelodysplastic syndrome patients and aplastic anemia patients in this report averaged 10,000 to 20 000 before registration. 000 / Yuzuru has been stable at about 3, PEG- rHuMGDF administration after the start
2週間は継続的な増加傾向は認められなかったが、 その後 2週間で継続的な増加 が認められ最大値 (プラト一) に到達した。 血小板数の最大値はいずれの症例に おいても症例登録前の平均値の 1. 9倍であった。 この血小板数増加は一過性のも ので、 4週間程度維持された後 2〜4週後(PEG- rHuMGDF投与開始後 10〜12週後) にほぼ投与前に復した。 No continuous increase was observed for 2 weeks, but a continuous increase was observed in the following 2 weeks, reaching the maximum value (Plat-1). The maximum platelet count in all cases was 1.9 times the average before registration. Since the increase in platelet count was transient, it was maintained for about 4 weeks and returned almost 2 to 4 weeks later (10 to 12 weeks after the start of PEG-rHuMGDF administration) before administration.
これまで実施された単回静脈内投与による第 I相試験では、前述の通り投与 11 日後に最大値に到達し、癌患者を対象とした反復皮下投与による第 I相試験では、 In the phase I study with a single intravenous dose performed to date, the maximum value was reached 11 days after administration as described above, and in the phase I study with repeated subcutaneous administration in cancer patients,
10日間の反復皮下投与の 6日目で血小板数の増加が始まり、 12〜18日目に最大値 に到達した ( Basser RL e t al. , "Thrombopo ie t ic e f fec t s of pegyl at ed recombinant human megakaryocyt e growth and deve l opment f ac tor (PEG-rHuMGDF) in pat ient s wi th advanced cancer, " Lance t 1996 ; 348 : 1279)。 上記の骨髄異 形成症候群患者及び再生不良性貧血患者における血小板数の増加はこれらの場合 とは異なっており、 PEG- rHuMGDFが作用してから血小板数が増加するまでに健常 人あるいは正常な骨髄機能を有する癌患者と比べて時間がかかることが示唆され た。 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.
2. 骨髄異形成症候群患者及び再生不良性貧血患者における貧血改善作用  2. Anemia ameliorating effect in myelodysplastic syndrome patients and aplastic anemia patients
投与開始日のヘモグロビン濃度及び網赤血球数は、 骨髄異形成症候群患者の実 施例 1では 10. Og/dL及び 59, 400/腿3、 実施例 2では 6. 8g/dL及び 44, 300/薦 3、 再生不良性貧血患者の実施例 3では 5. 6g/dL及び 30, 200/mm3と、貧血の重症度と してはそれぞれ grade 1、 grade 3ならびに grade 4に分類され、 ヘモグロビン濃 度の低い症例では網赤血球数も低かった。 Hemoglobin concentration and reticulocyte count of administration initiation day, the actual Example 1 of myelodysplastic syndrome 10. Og / dL and 59, 400 / thigh 3, Example 2, 6. 8 g / dL and 44, 300 / Como 3, example in 3 5. 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.
網赤血球数は血小板数と同様に、 PEG- rHuMGDF投与開始後 2週間は継続的な増 加傾向は認められなかったが、その後 2〜5週間で継続的な増加が認められ最大値 に到達した。網赤血球数の最大値は投与開始日の 1. 6〜2. 4倍であった。 この網赤 血球数増加は一過性のもので、 PEG- rHuMGDF投与開始後 10〜12週後にほぼ投与 前に復した。 網赤血球数の増加開始時期は血小板数の増加開始時期と同様かやや 早く、 PEG- rHuMGDFの作用が巨核球系及び赤芽球系で似ていることが考えられた。 ヘモグロビン濃度は網赤血球数が増加を始めた 1〜3週間後に増加を始め、赤芽 球系造血が PEG- rHuMGDF投与により刺激されたことが示唆された。その後網赤血 球数が減少中あるいはほぼ投与前値に復した PEG- rHuMGDF投与開始後 8〜 12週後 に、 ヘモグロビン濃度は最大値 (プラトー) に到達した。 観察されたへモグロビ ン濃度の最大値は投与開始日に比べて 2. 1〜3. 4g/dL増加していた。また、最も重 症度の高かった再生不良性貧血の実施例において、 最大のヘモグロビン濃度の増 加が認められた。 本実施例に記載した観察期間内はヘモグロビン濃度の増加は維 持されており、 血小板に比べ赤血球の寿命がはるかに長いことが、 血小板数及び ヘモグロビン濃度の増加の維持期間の差に現れたものと考えられた。  The reticulocyte count, like the platelet 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.
PEG- rHuMGDFの赤血球系に与える影響に関して、 前臨床試験においては、 正常 なマウス (Ulich TR et al., "Systemic hematologic effects of PEG-rHuM P L ligand-induced megakaryocyte hyperplasia in mice, " Blood 1996; 87: 5006; Kabaya K et al. , "In vivo effects of pegylated recombinant human megakaryocyte growth and development factor on hematopoiesis in normal mice," Stem Cells 1996; 14:651) またはラット(Harada K et al. , "A mild transient decrease of peripheral red blood cell counts induced by a suprap armacological doses of pegylated human megakaryocyte growth and development factor in rats," J Pharin Pharmacol 1999; 51: 841)に PEG- rHu MGDF を投与した際に赤血球系パラメ一夕の減少が認められた。 また、 単回静脈 内投与による第 I相試験では赤血球系パラメータに関して顕著な変動は認められ なかった。 In preclinical studies, the effect of PEG-rHuMGDF on erythroid (Ulich TR et al., "Systemic hematologic effects of PEG-rHuM PL ligand-induced megakaryocyte hyperplasia in mice," Blood 1996; 87: 5006; Kabaya K et al., "In vivo effects of pegylated recombinant human megakaryocyte growth and development factor on hematopoiesis in normal mice, "Stem Cells 1996; 14: 651) or rat (Harada K et al.," A mild transient decrease of peripheral red blood cell counts induced by a suprap armacological doses of pegylated human megakaryocyte growth and When PEG-rHu MGDF was administered to the development factor in rats, "J Pharin Pharmacol 1999; 51: 841), a decrease in erythroid parameters was observed. No significant changes were observed in erythroid parameters in a single intravenous phase I study.
これらの知見に対し、 grade 1〜4の貧血を呈している骨髄異形成症候群患者及 び再生不良性貧血患者においては、 赤芽球系造血が刺激され、 網赤血球数の増加 及びヘモグロビン濃度等の赤血球系パラメ一夕の増加が認められた。 これまで骨 髄異形成症候群患者由来の骨髄細胞を in vitroで IL- 3、 IL-6、 エリス口ポェチ ン(EP0)、顆粒球単球コロニー刺激因子(GM- CSF)、幹細胞因子(SCF)及び PEG- rHu MGDF で培養することで、 巨核球コロニーのみならず赤芽球コロニー及び顆粒球 単球コロニーの増加が認められているが (Fontenay- RoupieMet al., "Analysis of megakaryocyte growth and development factor (thrombopoietin) effects on blast cell and megakaryocyte growth in myelodysplasia, " Leuk Res 1998; 22: 527)、本発明者らは骨髄異形成症候群患者に PEG-rHuMGDFを単独投与することで、 in vivo で血小板数のみならず赤血球系のパラメータも増加することを初めて見 出した。  In contrast to these findings, 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. To date, 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). And PEG-rHu MGDF, the increase of erythroid and granulocyte monocyte colonies as well as megakaryocyte colonies was observed (Fontenay- Roupie Metal et al., "Analysis of megakaryocyte growth and development factor (thrombopoietin) effects on blast cell and megakaryocyte growth in myelodysplasia, "Leuk Res 1998; 22: 527), by administering PEG-rHuMGDF alone to myelodysplastic syndrome patients, only the platelet count in vivo It was also found for the first time that the parameters of the erythroid system also increased.
骨髄異形成症候群患者及び再生不良性貧血患者における貧血改善効果に関して は、 後述の比較例に記載のように、 rHuEPOの第 II相及び第 III相試験の結果が 示されている。 ヘモグロビン濃度 10g/dL 未満の骨髄異形成症候群患者に対して rHuEPOを漸増法で計 8週間 3000〜24000単位/日投与した場合 (浦部晶夫ほか、 遺伝子組換えヒ卜エリ トロポェチンの骨髄異形成症候群に伴う貧血に対する第 Regarding the anemia ameliorating effect in myelodysplastic syndrome patients and aplastic anemia patients, the results of the phase II and III studies of rHuEPO are shown as described in Comparative Examples below. RHuEPO administered to myelodysplastic syndrome patients with hemoglobin concentration of less than 10 g / dL by escalating method for 3000 weeks to 24,000 units / day for a total of 8 weeks (Urabe Akio et al., Genetically modified human erythropoietin The second against anemia
II 相臨床試験、 臨床血液 1993; 34: 928)、 貧血改善効果は投与前非輸血例では 5/9例(55.6%)で、輸血例では 2/25例(8.0%)で認められ、全体の有効率は 20.6% であった。 また、 同様に rHuEPO 24000単位/日を 8週間投与した場合 (浦部晶夫 ほか、 骨髄異形成症候群に伴う貧血に対する KRN5702 (遺伝子組換えヒトエリス ロポェチン) の効果、 診断と治療 1993; 81: 2025)、 貧血改善効果は投与前非輸 血例では 4/10例 (40.0%) で、 輸血例では 9/32例 (28.1%) で認められ、 全体の 有効率は 31.0¾であった。いずれの試験でも低リスク群の RA及び環状鉄芽球増加 を伴う RA (RARS) では有効率が高く、 高リスク群の RAEB、 RAEB-t及び慢性骨髄 単球性白血病 (dMoL) では有効率は低くなる傾向が認められた。 Phase II clinical study, clinical blood 1993; 34: 928) In 5 of 9 cases (55.6%), in transfusion cases, it was observed in 2/25 cases (8.0%), and the overall effective rate was 20.6%. Similarly, when rHuEPO was administered at 24000 units / day for 8 weeks (Akira Urabe et al., Effect of KRN5702 (recombinant human erythropoietin) on anemia associated with myelodysplastic syndrome, diagnosis and treatment 1993; 81: 2025). The improvement was observed in 4/10 patients (40.0%) in non-transfusion patients before administration and in 9/32 patients (28.1%) in transfusion patients, with an overall efficacy rate of 31.0%. In all studies, the efficacy rate was high in RA in the low-risk group and RA with increased sideroblasts (RARS), and in the high-risk group in RAEB, RAEB-t, and chronic myelomonocytic leukemia (dMoL). A tendency to decrease was observed.
一方、 ヘモグロビン濃度 10g/dL未満の再生不良性貧血患者に対して rHuEPOを 漸増法で計 8週間 3000〜24000単位/日投与した場合(浦部晶夫ほか、遺伝子組換 ぇヒトエリスロポエチンの再生不良性貧血に対する効果一第 II相臨床試験一、臨 床血液 1993; 34: 1002)、貧血改善効果は投与前非輸血例では 5/9例(55.6%)で、 輸血例では 5/20例(25.0%)で認められ、 全体の有効率は 34.5%であった。 また、 同様に rHuEPO 24000単位/日を 8週間投与した場合 (浦部晶夫ほか、 再生不良性 貧血に対する KRN5702 (遺伝子組換えヒトエリスロポエチン)の第 ΠΙ相臨床試験、 診断と治療 1993; 81: 2397)、貧血改善効果は投与前非輸血例では 3/7例(42.9%) で、 輸血例では 3/24例 (12.5%) で認められ、 全体の有効率は 19.4%であった。 いずれの試験でも重症度が高くなると有効率は低くなる傾向が認められた。  On the other hand, 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%. Similarly, when rHuEPO was administered at 24000 units / day for 8 weeks (Akira Urabe et al., Phase II clinical trial of KRN5702 (recombinant human erythropoietin) for aplastic anemia, diagnosis and treatment 1993; 81: 2397) The effect of improving anemia was observed in 3/7 (42.9%) of non-transfusion patients before administration and in 3/24 (12.5%) of transfusion patients, and the overall efficacy rate was 19.4%. In all tests, the efficacy rate tended to decrease as the severity increased.
これら骨髄異形成症候群患者及び再生不良性貧血患者においては、 へモグロビ ン濃度と内因性 EP0濃度は負の相関関係を示し (浦部晶夫ほか、 遺伝子組換えヒ トエリスロポエチンの骨髄異形成症候群に伴う貧血に対する第 Π相臨床試験、臨 床血?仪 1993; 34: 928; Urabe A et al. , Serum erythropoietin titers in hematological m lignancies and related diseases, Int J Cell Cloning 1992; In these myelodysplastic syndrome patients and aplastic anemia patients, hemoglobin levels and endogenous EP0 levels show a negative correlation (Akio Urabe et al., Anemia Associated with Myelodysplastic Syndrome of Recombinant Human Erythropoietin). 1993; 34: 928; Urabe A et al., Serum erythropoietin titers in hematological mlignancies and related diseases, Int J Cell Cloning 1992;
10: 333)、 輸血例のような重症度の高い患者では内因性 EP0濃度がより高くなる 傾向にある。 また、 重症度の高い患者ほど EP0反応性を示す赤芽球系の造血前駆 細胞が少ないと考えられる (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は赤芽球系に分化した前駆細胞に 作用し、赤血球の分化'成熟を促進する(Krantz SB. Erythropoietin. Blood 1991; 77: 419)。 EPOあるいは EPO受容体遺伝子をノックアウトしたマウスは、 赤芽球 系前駆細胞は存在するものの赤芽球及び赤血球の産生は抑制され、 貧血により胎 内で死亡する (WuHet al. , "Generation of committed erythroid BFU-E and CFU-E progenitors does not re uire erythropoietin or the erythropoietin receptor., " Cell 1995; 83: 59)。 EPO受容体遺伝子をノックアウトしたマウス の胎児肝細胞を in vitroで TP0を含むサイトカインの組合せで培養した場合は赤 芽球系前駆細胞コロニーが形成されたが、 TP0 を含まないサイト力インの組合せ または TP0 単独で培養した場合は形成されなかった (Kieran 匿 et al. , Thrombopoietin rescues in vitro erythroid colony format ion from mouse embryos lacking the erythropoietin receptor" Proc Natl Acad Sci USA 1996; 93: 9126)。 10: 333), Endogenous EP0 levels tend to be higher in more severe patients such as transfusion cases. In addition, 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 uire erythropoietin or the erythropoietin receptor., "Cell 1995; 83:59). When fetal hepatocytes of a mouse knocked out of the EPO receptor gene were cultured in vitro with a combination of cytokines containing TP0, erythroid progenitor cell colonies were formed, but a combination of cytokinin without TP0 or It was not formed when cultured alone with TP0 (Kieran et al., Thrombopoietin rescues in vitro erythroid colony format ion from mouse embryos lacking the erythropoietin receptor "Proc Natl Acad Sci USA 1996; 93: 9126).
これらの結果を考え合わせると、 TP0 は骨髄異形成症候群患者及び再生不良性 貧血患者においては、 造血前駆細胞に作用して巨核球系前駆細胞と同様に赤芽球 系前駆細胞を増加させ、 その結果濃度が上昇している内因性 EP0と協調して赤血 球系の造血が亢進した可能性が考えられた。  Taken together, these results indicate that TP0 acts on hematopoietic progenitors in myelodysplastic syndrome patients and aplastic anemia patients to increase erythroid progenitors as well as megakaryocytic progenitors. The results suggest that erythroid hematopoiesis may have been enhanced in coordination with endogenous EP0 whose concentration is increasing.
3. 血中 TP0濃度 3. Blood TP0 concentration
血中 TP0 濃度は血小板数と負の相関関係にあることが既に知られている (Tahara T et al. , A sensitive sandwich EL ISA for measure ing thrombopoietin in human serum: serum thrombopoietin levels in healthy volunteers and in patients withhaemapoietic disorders, Br J Haematol 1996; 93: 783; Ichikawa N et al. , "Regulation of serum thrombopoietin levels by platelets and megakaryocytes in patients with aplastic anemia and idiopathic thrombocytopenic purpura, " Thromb Haemost 1996; 76: 156; Tamura H et al. , It is already known that blood TP0 concentration is negatively correlated with platelet count (Tahara T et al., A sensitive sandwich ELISA for measure ing thrombopoietin in human serum: serum thrombopoietin levels in healthy volunteers and in patients withhaemapoietic disorders, Br J Haematol 1996; 93: 783; Ichikawa N et al., "Regulation of serum thrombopoietin levels by platelets and megakaryocytes in patients with aplastic anemia and idiopathic thrombocytopenic purpura," Thromb Haemost 1996; 76: 156; Tamura H et al.,
Plasma thrombopoietin (TPO) levels and expression of TPO receptor on platelets in patients with myelodysplastic syndromes, Br J Haematol 1998;Plasma thrombopoietin (TPO) levels and expression of TPO receptor on platelets in patients with myelodysplastic syndromes, Br J Haematol 1998;
103: 778; Schrezenmeier H et al. , "Thrombopoietin serum levels in patients with aplastic anaemia: correlation with platelet count and persistent elevation in remission, " Br J Haematol 1998; 100: 571 ; Zwierzina H et al., "Endogenous serum thrombopoiet in concentrations in patients with myelodysplastic syndromes," Leukemia 1998; 12: 59)。 また、 健常人を対象と した第 I相試験では投与前 PEG- rHuMGDF濃度は 50pg/mL程度であった。本報告の 症例は血小板数が約 13, 000/賺 3、 21, 000/讓3及び 19, 000/腿3と健常人のほぼ 20 分の 1程度の極めて高度の血小板減少を呈しているのと同時に、 投与前 PEG- rHu MGDF濃度で表される内因性 TP0濃度は 1595pg/mL、 1653pg 及び 1056pg/mLと 健常人と比較して 20〜30倍の高値を示した。 14日間の PEG- rHuMGDF投与中に血 中 PEG- rHuMGDF濃度は定常状態に達し、 トラフレベルは約 10ng/mLであった。 in vitroで PEG- rHuMGDFを健常人骨髄細胞に添加して培養した際の巨核球コロニー 形成は lOng/mLでブラトーに達したが、 骨髄異形成症候群患者の骨髄細胞ではよ り高濃度の PEG- rHuMGDF で巨核球コロニー形成が刺激されたことから、 2.5 / g/kg/日の PEG- rHuMGDFの投与により観察された血中 PEG- rHuMGDF濃度は本症例 血小板産生を刺激する濃度に達していると考えられた。 103: 778; Schrezenmeier H et al., "Thrombopoietin serum levels in patients with aplastic anaemia: correlation with platelet count and persistent elevation in remission," Br J Haematol 1998; 100: 571; Zwierzina H et al., "Endogenous serum thrombopoiet in concentrations in patients with myelodysplastic syndromes," Leukemia 1998; 12:59). In a phase I study in healthy subjects, the PEG-rHuMGDF concentration before administration was about 50 pg / mL. 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 /賺3, 21, 000 / Yuzuru 3 and 19, 000 / thigh 3 and healthy persons At the same time, 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. During the 14-day PEG-rHuMGDF administration, the blood PEG-rHuMGDF concentration reached a steady state and the trough level was about 10 ng / mL. In vitro, 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.
4. 総括 4. Summary
骨髄異形成症候群患者及び再生不良性貧血患者に 2.5^g/kg/日の PEG - rHuM 2.5 ^ g / kg / day PEG-rHuM for patients with myelodysplastic syndrome and aplastic anemia
GDFを 14日間投与することで、 血小板数が一過性に増加するのと同時に、 網赤血 球数も一過性に増加し、 さらにその後ヘモグロビン濃度等の赤血球系パラメ一夕 の増加が認められた。 これまでの研究において、 c— mp 1リガンドタンパク質 は in vitroで EP0及びその他のサイトカインと相乗的に未分化の造血前駆細胞か らの赤芽球系造血を刺激すること(Kobayashi M et al. , "Recombinant human thrombopoiet in (m l ligand) enhances proliferation of erythroid progenitors, " Blood 1995; 86: 2494; Tanimukai S et al. , "Recombinant human c -即 1 ligand (thrombopoiet in) not only acts on megakaryocyte progenitors, but also on erythroid and multipotential progenitors in vitro, " Exp HematolWhen GDF was administered for 14 days, the platelet count transiently increased at the same time as the reticulocyte count transiently increased, followed by an increase in erythroid parameters such as hemoglobin concentration. Was done. In previous studies, c-mp1 ligand protein stimulated erythroid hematopoiesis from undifferentiated hematopoietic progenitors in vitro synergistically with EP0 and other cytokines (Kobayashi M et al., "Recombinant human thrombopoiet in (ml ligand) enhances proliferation of erythroid progenitors," Blood 1995; 86: 2494; Tanimukai S et al., "Recombinant human c-immediately 1 ligand (thrombopoiet in) not only acts on megakaryocyte progenitors, but also on erythroid and multipotential progenitors in vitro, "Exp Hematol
1997; 25: 1025; Yoshida M et al. , "Thrombopoiet in alone stimulates the early proliferation and survival of human erythroid, myeloid and multipotential progenitors in serum-free culture, " Br J Haematol 1997; 98: 254; B i rkiann1997; 25: 1025; Yoshida M et al., "Thrombopoiet in alone stimulates the early proliferation and survival of human erythroid, myeloid and multipotential progenitors in serum-free culture," Br J Haematol 1997; 98: 254; B i rkiann
J et al. , "Effects of recombinant human thrombopoiet in alone and in conbination with erythropoietin and early-acting cytokines on human mobilized purified CD34+ progenitor cells cultured in serum-depleted medium, " Stem Cells 1997; 15: 18) 、 X線等による骨髄抑制状態の動物におい て血小板数のみならず網赤血球数及び赤血球系パラメータの回復促進ならびに改 善をすること力示されて!^る(Kaushansky K et al. , "Thrombopoiet in expands erythroid progenitors, increases red cell production, and enhances erythroid recovery after myelos卿 ressive therapy, " J Clin Invest 1995; 96: 1683; Neelis KJ et al. , "Prevention of thrombocytopenia by thrombopoietin in myelos卿 ressed rhesus monkeys accompanied by prominent erythropoietic stimulation and iron depletion, " Blood 1997; 90: 58; Neelis KJ et al. ,J et al., "Effects of recombinant human thrombopoiet in alone and in conbination with erythropoietin and early-acting cytokines on human mobilized Purified CD34 + progenitor cells cultured in serum-depleted medium, "Stem Cells 1997; 15:18), promoted recovery of not only platelet counts but also reticulocyte counts and erythroid parameters in bone marrow-suppressed animals by X-rays, etc. (Kaushansky K 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.,
"Simultaneous administration of TPO and G-CSF after cytoreductive treatment of rhesus monkeys prevents thrombocytopenia, accelerates platelet and red cell reconstitution, alleviates neutropenia, and promotes the recovery of immature bone marrow cells, " Exp Hematol 1997; 25: 1084; Shibuya K et al. , lultilineage hematopoiet ic recovery by a single injection of pegylated recombinant human megakaryocyte growth and development factor in myelosuppressed mice, " Blood 1998; 91: 37)。 本発明者らは今回、 PEG-rHuM"Simultaneous administration of TPO and G-CSF after cytoreductive treatment of rhesus monkeys prevents thrombocytopenia, accelerates platelet and red cell reconstitution, alleviates neutropenia, and promotes the recovery of immature bone marrow cells," Exp Hematol 1997; 25: 1084; Shibuya K et al., lultilineage hematopoietic recovery by a single injection of pegylated recombinant human megakaryocyte growth and development factor in myelosuppressed mice, "Blood 1998; 91: 37).
GDFが骨髄異形成症候群患者及び再生不良性貧血患者において、 血小板数のみな らず網赤血球数及びヘモグロビン濃度等の赤血球系パラメータの改善に作用する ことを初めて見出した。 本症例では安全性に問題は認められず、 PEG- rHuMGDFの 投与は骨髄異形成症候群患者及び再生不良性貧血患者の血小板減少症及び貧血の 改善に有用であることが示された。 For the first time, 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. In this case, there were no safety issues, indicating that administration of PEG-rHuMGDF is useful for improving thrombocytopenia and anemia in patients with myelodysplastic syndrome and aplastic anemia.
c— mp 1リガンドに関して in vitroで EP0及びその他のサイトカインと相 乗的に未分化の造血前駆細胞からの赤芽球系造血を刺激することは示されていた が、 invitroでの骨髄細胞の反応性と臨床試験成績との間に乖離があるため、 c 一 mp 1リガンドが骨髄異形成症候群及び再生不良性貧血患者で貧血改善効果を もたらすことは予期されなかった。 また造血因子のうち、 骨髄異形成症候群及び 再生不良性貧血で実際に臨床適応となっているのは好中球減少に対する G- CSFの みであり、 貧血改善効果に対して臨床適応となっている造血因子はない。 骨髄異 形成症候群及び再生不良性貧血患者の貧血を改善できる造血因子が事実上ないこ とから、 c一 mp 1リガンドの貧血改善効果の意義は大きいと考えられる。 比較例 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. Of the hematopoietic factors, only G-CSF for neutropenia is the only clinical indication for myelodysplastic syndrome and aplastic anemia, and clinical indication for the amelioration of anemia. There are no hematopoietic factors. 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
1. 骨髄異形成症候群に対する造血因子の臨床効果(Geissler RG et al., 1. Clinical effect of hematopoietic factor on myelodysplastic syndrome (Geissler RG et al.,
"Clinical use of hematopoietic growth factors in patients with myelodysplastic syndromes, " Int J Hematol 1997; 65: 339) "Clinical use of hematopoietic growth factors in patients with myelodysplastic syndromes," Int J Hematol 1997; 65: 339)
これまでに顆粒球コロニー刺激因子 (G- CSF)、 顆粒球単球コロニー刺激因子 So far granulocyte colony stimulating factor (G-CSF), granulocyte monocyte colony stimulating factor
(GM- CSF)、 インターロイキン (IL_) 3、 IL- 6、 エリトロポェチン (EP0) の臨床 効果が検討されている。 貧血改善効果に関しては、 G- CSF に関しては殆ど認めら れず、 GM- CSFに関しても殆ど認められなかった。 IL-3に関しては 1つの試験を除 いて貧血改善効果は殆ど認められず、 全体の有効率は 12% (10/85例) であった。(GM-CSF), interleukin (IL_) 3, IL-6, and erythropoietin (EP0) have been studied for their clinical effects. The anemia ameliorating effect was hardly observed for G-CSF, and was hardly observed for GM-CSF. Except for one test, IL-3 showed almost no anemia ameliorating effect, and the overall efficacy rate was 12% (10/85 subjects).
IL-6 に関しては貧血改善効果は全く認められなかった(Gordon MS et al. , "A phase I trial of recombinant human interleukin-6 in patients with myelodysplastic syndromes and thrombocytopenia, " Blood 1995; 85: 3066)。 赤芽球系特異的な造血因子である EP0の貧血改善効果に関しては様々な用法 ·用 量で検討が加えられたが、 全体の有効率は 24.3% (125/515例) であった。 但し有 効性の判定基準は試験によって統一されていないため、これらのうち 17試験につ いて有効性の判定基準をヘモグロビン濃度 1.5g/dL以上の増加に統一して再判定 したところ、 有効率は 16% ( 33/205 例) であった(Hells tr m-Lindberg E,No anemia ameliorating effect was observed for IL-6 (Gordon MS et al., "A phase I trial of recombinant human interleukin-6 in patients with myelodysplastic syndromes and thrombocytopenia," Blood 1995; 85: 3066). The effects of EP0, an erythroid-specific hematopoietic factor, on improving anemia were examined in various dosage forms and dosages, but the overall efficacy rate was 24.3% (125/515 cases). However, since the efficacy criteria were not standardized by the tests, the efficacy criteria were re-evaluated in 17 of these studies with the hemoglobin concentration increased to 1.5 g / dL or more. Was 16% (33/205 cases) (Hells trm-Lindberg E,
"Efficacy of erythropoietin in the myelodysplastic syndromes: a meta-analysis of 205 patients from 17 studies, " Br J Haematol 1995; 89: 67)。 ェポェチンアルファ (rHuEPO)の第 Π相(浦部晶夫ほか、遺伝子組換えヒトエリス ロポェチンの骨髄異形成症候群に伴う貧血に対する第 Π 相臨床試験、 臨床血液"Efficacy of erythropoietin in the myelodysplastic syndromes: a meta-analysis of 205 patients from 17 studies," Br J Haematol 1995; 89: 67). Phase II study of epoetin alfa (rHuEPO) (Akio Urabe et al., Phase II clinical trial of recombinant human erythropoietin for anemia associated with myelodysplastic syndrome, clinical blood
1993; 34: 928)及び第 III相(浦部晶夫ほか、 骨髄異形成症候群に伴う貧血に対す る KRN5702 (遺伝子組換えヒトエリトロポェチン) の効果、 診断と治療 1993; 81:1993; 34: 928) and Phase III (Akio Urabe et al., Effect of KRN5702 (recombinant human erythropoietin) on anemia associated with myelodysplastic syndrome, diagnosis and treatment 1993; 81:
2025)試験の結果でも、 第 II相試験全体の有効率は 20.6% (7/34例)、 第 III相試 験全体の有効率は 31.0% (13/42例) であった。 但し rHuEPO投与前非輸血例でへ モグロビン濃度 2g/dL以上の増加が認められたか、 投与前輸血例で輸血を必要と しなくなつた症例 (著明改善) は、 第 II相試験では 4/34例 (11.8%)、 第 IE相試 験でも 6/42例 (14.3« であった。 表 1に rHuEPOの第 II相及び第 ΠΙ相試験の 結果をまとめた。 2025) From the results of the study, the efficacy rate for the entire Phase II study was 20.6% (7/34 subjects), and the efficacy rate for the entire Phase III study was 31.0% (13/42 subjects). However, if the hemoglobin concentration increased by 2 g / dL or more in non-transfused patients before rHuEPO administration, or if transfusion was not required in transfused patients before administration (marked improvement), 4 / 34 cases (11.8%), phase IE trial In the study, 6/42 patients (14.3%). Table 1 summarizes the results of the rHuEPO phase II and phase III studies.
表 1 table 1
骨髄異形成症候群における rHuEPOの貧血改善効果 (浦部ら、 上掲、 より引用)  Improvement of anemia by rHuEPO in myelodysplastic syndrome (Urabe et al., Cited above)
著明改善:非輸血例でヘモグロビン濃度 2g/dL以上の増加、 輸血例で輸血を必要としな くなっ广'  Significant improvement: hemoglobin concentration increased by 2 g / dL or more in non-transfusion cases, blood transfusion no longer required transfusion.
改善:非輸血例でヘモグロビン濃度 lg/dL以上 2g/dL未満の増加、輸血例で輸血量が 50% 以上減少  Improvement: 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
やや改善:非輸血例でへモグロピン濃度 0.5g/dL以上 lg/dL未満の増加、 輸血例で輸血 量の減少が 50%未満  Moderate improvement: Hemoglobin concentration increased from 0.5g / dL to less than lg / dL in non-transfusion cases, transfusion volume decreased less than 50% in transfusion cases
無効:非輸血例でへモグロビン濃度 0.5g/dL未満の増加または輸血施行、 輸血例で輸血 量の減少を認めない  Ineffective: No increase in hemoglobin concentration of less than 0.5 g / dL or blood transfusion in non-transfusion cases, no decrease in transfusion volume in transfusion cases
2. 再生不良性貧血に対する造血因子の臨床効果( Kojima S, "Use of hematopoietic growth factors for treatment of aplastic anemia, Bone Marrow Transplant 1996; 18, Suppl 3: S36; Marsc JCW, "Hematopoietic growth factors in the pathogenesis and for the treatment of aplastic anemia, " Semin Hematol 2000; 37: 81) 2. Clinical effects of hematopoietic factors on aplastic anemia (Kojima S, "Use of hematopoietic growth factors for treatment of aplastic anemia, Bone Marrow Transplant 1996; 18, Suppl 3: S36; Marsc JCW," Hematopoietic growth factors in the pathogenesis and for the treatment of aplastic anemia, "Semin Hematol 2000; 37: 81)
これまでに G- CSF、 GM-CSF, IL- 3、 IL-1、 IL- 6、 EP0 の臨床効果が検討されて いる。 貧血改善効果に関して G-CSFでは殆ど認められなかった。 GM- CSFに関して は 4.5% (3/66例) で貧血改善効果が報告されている。 IL- 3、 IL- 1及び IL- 6に関 しては貧血改善効果は全く認められなかった。 赤芽球系特異的な造血因子である EP0の貧血改善効果に関しては、 rHuEPOの第 II相(浦部晶夫ほか、 遺伝子組換え ヒ卜エリ トロポェチンの再生不良性貧血に対する効果一第 II相臨床試験一、臨床 血液 1993; 34: 1002)及び第 ΙΠ 相 (浦部晶夫ほか、 再生不良性貧血に対する KRN5702 (遺伝子組換えヒトエリスロポエチン) の第 III相臨床試験、 診断と治療 1993; 81: 2397) 試験の結果が示されている。 第 II相試験全体の有効率は 34.5%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. For GM-CSF, 4.5% (3/66 cases) reported an anemia ameliorating effect. As for IL-3, IL-1 and IL-6, no anemia improving effect was observed at all. Regarding the anemia ameliorating effect of EP0, an erythroid-specific hematopoietic factor, the effect of rHuEPO on phase II (Akio Urabe et al., Effect of recombinant human erythrotropin on aplastic anemia-Phase II clinical trials 1 , Clinical Blood 1993; 34: 1002) and Phase I (Akio Urabe et al., For aplastic anemia) The results of a phase III clinical trial of KRN5702 (recombinant human erythropoietin), diagnosis and treatment 1993; 81: 2397) are shown. Effectiveness rate of the entire phase II study is 34.5%
(10/29例)、第 III相試験全体の有効率は 19.4 (6/31例)であった。但し rHuEPO 投与前非輸血例でヘモグロビン濃度 2g/dL以上の増加が認められたか、 投与前輪 血例で輸血を必要としなくなつた症例 (著明改善) は、 第 II相試験では 4/29例(10/29 subjects), the overall efficacy rate of the phase III study was 19.4 (6/31 subjects). However, in non-transfusion patients before rHuEPO administration, 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
(13.8%)、 第 ΠΙ相試験でも 4/42例 (12.9%) であった。 (13.8%) and 4/42 (12.9%) in the phase III study.
表 2に rHuEPOの第 II相及び第 III相試験の結果をまとめた。 表 2  Table 2 summarizes the results of the Phase II and III studies of rHuEPO. Table 2
再生不良性貧血における rHuEPOの貧血改善効果 (浦部ら、 上掲、 より引用)  RHuEPO improves anemia in aplastic anemia (Urabe et al., Cited above)
著明改善:非輸血例でヘモグロビン濃度 2g/dL以上の増加、 輸血例で輸血を必要としな くなつ  Marked improvement: hemoglobin concentration increased by 2 g / dL or more in non-transfusion cases, blood transfusion cases no longer required blood transfusion
改善:非輸血例でへモグロピン濃度 lg/dL以上 2g/dL未満の増加、輸血例で輸血量が 50% 以上減少  Improvement: 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
やや改善:非輸血例でヘモグロビン濃度 0.5g/dL以上 lg/dL未満の増加、 輸血例で輸血 量の減少が 50 未満  Slight improvement: hemoglobin concentration increased from 0.5g / dL to less than lg / dL in non-transfusion cases, transfusion volume decreased less than 50 in transfusion cases
無効:非輸血例でへモグロビン濃度 0.5 /dL未満の増加または輸血施行、 輸血例で輸血 量の減少を認めない 上記造血因子のうち GM- CSF及び IL- 3 について in vitroでは、 単独あるいは EP0 を共存させることで、 健常人由来あるいは骨髄異形成症候群及び再生不良性 貧血患者由来の骨髄細胞の赤血球系造血が誘導あるいは増幅されることが報告さ れてレ る (Goodman JW et al. , ^lnterleukin 3 promotes erythroid burst formation in serum-free" cultures without detectable erythropoietin, " Proc Natl Acad Sci USA 1985; 82: 3291; Migliaccio A et al. , Bruno M, and Migliaccio G. Evidence for direct action of human biosynthetic (recombinant) GM-CSF on erythroid progenitors in serum-free culture. Blood 1987; 70: 1867; Migliaccio G et al. , "In vitro differentiation of human granulocyte/macrophage and erythroid progenitors: comparative analysis of t a influence of recombinant human erythropoietin, G - CSF, GM-CSF, and IL-3 in serum-sup lemented and serum-deprived cultures, " Blood 1988; 72: 248; Sonoda Y et al. , 'Erythroid burst-promoting activity of purified recombinant human GM-CSF and interleukin-3: studies with anti-GM-CSF and anti-IL-3 sera and studies in serum-free cultures, Blood 1988; 72: 1381 ; Ottmann OG et al. , "Stimulation of human hematopoietic progenitor cell proliferation and differentiation by recombinant human inierleukin 3. Comparison and interactions with recombinant human granulocyte-macrophage and granulocyte colony-stimulating factors, " Exp Hematol 1989; 17: 191; Aoki A and Shibata A. , "In vitro study of erythropoiesis in patients with aplastic anemia and myelodysplastic syndromes: a possible tool for prospective determination of the clinical effectiveness of growth factors, " Hematol Pathol 1992; 6: 143)。 これら in vitroでの骨髄細胞に対する作用から 予想さ、 臨床試験での GM- CSF及び IL- 3の貧血改善効果は上記の通り非常に限ら れた有れる結果に反して効率でしかなかった。 参考例 No effect: No increase in hemoglobin concentration of less than 0.5 / dL or blood transfusion was not performed in non-transfusion cases, and no decrease in blood transfusion volume was observed in transfusion cases. It has been reported that coexistence induces or amplifies erythroid hematopoiesis of bone marrow cells derived from healthy individuals or from patients with myelodysplastic syndrome and aplastic anemia (Goodman JW et al., ^ Lnterleukin 3 promotes erythroid burst formation in serum-free "cultures without detectable erythropoietin," Proc Natl Acad Sci USA 1985; 82: 3291; Migliaccio A et al., Bruno M, and Migliaccio G. Evidence for direct action of human biosynthetic (recombinant) GM-CSF on erythroid progenitors in serum-free culture.Blood 1987; 70: 1867; Migliaccio G et al., "In vitro differentiation of human granulocyte / macrophage and erythroid progenitors: comparative analysis of ta influence of recombinant human erythropoietin, G- CSF, GM-CSF, and IL-3 in serum-suplemented and serum-deprived cultures, "Blood 1988; 72: 248; Sonoda Y et al., 'Erythroid burst-promoting activity of purified recombinant human GM-CSF and interleukin -3: studies with anti-GM-CSF and anti-IL-3 sera and studies in serum-free cultures, Blood 1988; 72: 1381; Ottmann OG et al., "Stimulation of human hematopoietic progenitor cell proliferation and differentiation by recombinant human inierleukin 3.Comparison and interactions with recombinant human granulocyte-macrophage and granulocyte colony-stimulating factors, "Exp Hematol 1989; 17: 191; Aoki A and Shibata A.," In vitro study of erythropoiesis in patients with aplastic anemia and myelodysplastic syndromes : A possible tool for prospective determination of the clinical effectiveness of growth factors, "Hematol Pathol 1992; 6: 143). As expected from these in vitro effects on bone marrow cells, the anemia improving effect of GM-CSF and IL-3 in clinical trials was only efficient, contrary to the very limited results described above. Reference example
<参考例 1> モノ PEG化 c一 mp 1 リガンド(PEG- rHuMGDF)の作製  <Reference Example 1> Preparation of mono-PEGylated c-mp1 ligand (PEG-rHuMGDF)
下記のモノ PEG 化 c — mp 1 リガンド(PEG-rHuMGDF)の製法例は国際公開 画/ 26746に記載される方法に基いている。  The following method for producing mono-PEGylated c-mp1 ligand (PEG-rHuMGDF) is based on the method described in WO / 26746.
(1) 組換えヒト MGDF (r-HuMGDF) の大腸菌での発現  (1) Expression of recombinant human MGDF (r-HuMGDF) in E. coli
r-HuMGDFを E. coliで発現させるために、 成熟タンパク質の初めの 163 アミ ノ酸をコ一ドしている DNA配列を、 E. の最適コドンを使って、 化学的に合 成した。 さらに、 アミノ酸のメチォニンとリジンをコードしている DNA配列を、 遺伝子の 5'末端に付加した。したがって、この DNA配列がコードしている r - HuMGDF タンパク質は、 Met-Lysで始まる全長 165アミノ酸で構成されている(W095/26746 の図 25参照)。 To express r-HuMGDF in E. coli, the DNA sequence encoding the first 163 amino acids of the mature protein was chemically synthesized using the optimal codon of E. In addition, 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遺伝子の合成は、 数ステップで達成した。 最初に、 遺伝子の隣接フラ グメントに相当する相補的オリゴヌクレオチド (長さ 60-70bp)を、 E. co/ /の最 適コドンを使って、 化学的に合成した。 この合成に際して、 アミノ酸のメチォ二 ンとリジンのコドンを、 成熟遺伝子の 5'末端に置き、 ストップ, コドンを遺伝子 の 3'末端に置いた。 さらに、 制限酵素 Xbalと Hindl llの切断サイトを、 それぞ れ、 遺伝子の 5'と 3'末端に置き、 合成リボゾームの結合部位を、 初めのメチォ二 ンの上流の適した場所に置いた。 次に、 各遺伝子フラグメントの相補的なオリゴ ヌクレオチドをァニールした。さらに、これら個々の合成遺伝子フラグメントを、 ポリメラ一ゼ' チェイン' リアクション (PCR) を用いて増幅させた。 増幅させた フラグメントを適当なベクタ一にサブクロ一ニングし、配列を確認した。次に、完 全長の r- HuMGDF 遺伝子を再構築できるように、 個々のフラグメントを連結し、 適当なベクターにサブクローニングした。 最後に、 再構築した遺伝子の配列を確 認した。  Synthesis of the r_HuMGDF gene was achieved in a few steps. First, complementary oligonucleotides (60-70 bp in length) corresponding to flanking fragments of the gene were chemically synthesized using the optimal codon of E.co//. During this synthesis, 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. In addition, 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. Next, the complementary oligonucleotide of each gene fragment was annealed. In addition, 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.
5'と 3'末端において、それぞれ、 Xbalと Hindl l lの制限部位に隣接している、 合成 r-Hu C— M P Lリガンド遺伝子フラグメントは、リポソ一ム結合部位、 ATG開 始コドン、 成熟した Met- Lys r- HuMGDFタンパク質をコードしている配列、 そして ストップコドンを持っている。  At the 5 'and 3' ends, 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.
上記のフラグメントを、 ラクトース誘導性発現ベクターである pAMGl lの Xbal および Hindl l lの両部位にクローニングした。 pAMGl lベクタ一は、 pRIOO-由来 の複製起点を持つ、 低コピー数のプラスミドである。 発現プラスミド pAMGl lは、 PCR重複オリゴ' 変位誘発による、 一連の部位特異的な塩基変換を作ることによ つて、 プラスミド pCFM1656 (ATCC# 69576, 1994. 2. 24寄託) から、 誘導すること ができる。 プラスミ ド複製プロモーター PcopB の 5'に近接した Bgl l l 部位 (plasmid bp # 180) から始め、 プラスミド複製遺伝子に向かうことにより、 塩 基対の変換は次のようになる:  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:
PAMGl l bp # PCFM1656での bp pAMGl lで置換された bp PAMGl l bp # bp in PCFM1656 bp replaced with pAMGl l
# 204 T/A C/G  # 204 T / A C / G
# 428 A/T G/C # 509 G/C A/T # 428 A / TG / C # 509 G / CA / T
# 617 - - 2G/Cb を挿入  # 617--Insert 2G / Cb
# 679 G/C T/A  # 679 G / C T / A
# T/A C/G  # T / A C / G
# 994 G/C A/T  # 994 G / C A / T
#讓 A/T C/G  # Judge A / T C / G
#1007 C/G T/A  # 1007 C / G T / A
#1028 A/T T/A  # 1028 A / T T / A
#1047 C/G T/A  # 1047 C / G T / A
#1178 G/C T/A  # 1178 G / C T / A
#1466 G/C T/A  # 1466 G / C T / A
#2028 G/C b を欠失  # 2028 G / C b deleted
#2187 C/G T/A  # 2187 C / G T / A
#2480 A/T T/A  # 2480 A / T T / A
#2499-2502 AGTG GTCA # 2499-2502 AGTG GTCA
TCAC CAGT  TCAC CAGT
#2642 TCCGAGC bpを欠失 # 2642 TCCGAGC bp deleted
AGGCTCG  AGGCTCG
#3435 G/C A/T # 3435 G / C A / T
#3446 G/C A/T  # 3446 G / C A / T
#3643 A/T T/A  # 3643 A / T T / A
#4489-4512 以下の bpを揷入 # 4489-4512 Import bp below
GAGCTCACTAGTGTCGACCTGCAG (配列番号 3 ) GAGCTCACTAGTGTCGACCTGCAG (SEQ ID NO: 3)
CTCGAGTGATCACAGCTGGACGTC (配列番号 4 ) そして、 各単一の Aat l l と Cl alの切断部位間の DNA 配列を、 次のオリゴヌクレ ォチドで置換すると: CTCGAGTGATCACAGCTGGACGTC (SEQ ID NO: 4) and the DNA sequence between each single Aatll and Clal cleavage site is replaced by the following oligonucleotide:
Aatll (#4358) Aatll (# 4358)
5 ' CTCATAATTTTTAAAAAATTCATTTGACAAATGCTAAAATTCTT- 3 ' TGCAGAGTATTAAAAATTTTTTAAGTAAACTGTTTACGATTTTAAGAA-  5 'CTCATAATTTTTAAAAAATTCATTTGACAAATGCTAAAATTCTT- 3' TGCAGAGTATTAAAAATTTTTTAAGTAAACTGTTTACGATTTTAAGAA-
-GATTAATATTCTCAATTGTGAGCGCTCACAATTTAT 3' (配列番号 5 ) -GATTAATATTCTCAATTGTGAGCGCTCACAATTTAT 3 '(SEQ ID NO: 5)
-CTAATTATAAGAGTTAACACTCGCGAGTGTTAAATAGC 5 ' (配列番号 6 )  -CTAATTATAAGAGTTAACACTCGCGAGTGTTAAATAGC 5 '(SEQ ID NO: 6)
Clal (#4438) pAMGl lにクローニングされた r- HuMGDFの発現は、 以下の配列をもつ Ps4 のよ うな、 合成ラクトース誘導性プロモーターによって、 推進される: Clal (# 4438) The expression of r-HuMGDF cloned into pAMGll is driven by a synthetic lactose-inducible promoter, such as Ps4, which has the following sequence:
5 ' GACGTCTCATAATTTTTAAAAAATTCATTTGACAAATGCTAAA-5 'GACGTCTCATAATTTTTAAAAAATTCATTTGACAAATGCTAAA-
-ATTCTTGATTAATATTCTCAATTGTGAGCGCTCACAATTTATCGAT 3' (配列番号 7 ) この Ps4 プロモ一ターは、 E. coli l ac l 遺伝子の産物であるラクト一ス' リ プレツーサー(Lac l)によって、 抑制される。 -ATTCTTGATTAATATTCTCAATTGTGAGCGCTCACAATTTATCGAT 3 '(SEQ ID NO: 7) This Ps4 promoter is suppressed by a lactose' repressor (Lac1) which is a product of the E. coli lac1 gene.
pAMGl l-r-HuMGDF プラスミドは、 続いて l acl Qアレルを含んでいる R coli K - 12株に形質転換される。 l acl qアレルは、 Laclの発現を増大する l ac lプロモ —ター内の変異であり、 Ps4 プロモーターからタンパク質の発現のより厳密な制 御をもたらす。 したがって、 この株では、 ラクト一スがなければ、 r- HuMGDFの発 現は Laclによって抑制される。 ラク! スを加えると、 Ps4プロモーターのオペ レーターサイ卜に結合している Lac lタンパク質が減少し、 Ps4 から r- HuMGDFの 転写が始まる。 この参考例で使用された . co 宿主細胞は、 ATCC# 69717 とし て ATCC (米国) に寄託されている。  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.
£ coli宿主 ATCC# 69717 は、 pAMGl卜 r- HuMGDF プラスミドで形質転換し、 次 のような発酵手法にしたがって、生育させた。 Lur ia肉汁に接種された . co /株 は、 およそ 12時間、 30°Cでインキュベートされる。 菌株はその後、 無菌的にパッ チ- 培地 (20 g/Lイースト' エキス; 3. 4 g/L クェン酸; 15 g/L K2HP04; 15 mlThe 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
Dow P2000 ; 5 g/Lグルコース; 1 g/L MgS04 - 7H20 ; 5. 5 ml/L微量金属類; 5. 5 ml/L ビタミン類) の入った発酵槽に移される。パッチ · フェイズのプロセスは培養が、Dow P2000; 5 g / L glucose; 1 g / L MgS0 4 - 7H 2 0; 5. 5 ml / L trace metals; 5. proceeds to enter the fermenter with 5 ml / L vitamins). The patch phase process involves culturing,
600nmで 5. 0+/-1. 0の最適密度に達するまで続けられる。 流加培養期は、 第 1の 流動培地 (f eed med ium) (700 g/Lグルコース ; 6. 75 g/L MgS04 · 7H20)の供給 開始で、 始められる。 流量は確立されたスケジュールにあわせて 2時間毎に調節 する。 培養が 600nmで 20〜25 の最適密度に達すると、 第 2 の流動培地(129 g/L トリプチカーゼ' ペプトン; 258 g/L イースト' エキス) の供給開始が始まる。 初めの流動培地が調節され続けたのに対し、 第 2の流動培地は、 一定流量を維持 する。全発酵中を通じて、温度はおよそ 30°Cを維持する。培養は、必要に応じて、 酸や塩基を添加して pHを 7 に維持する。 望ましい溶存酸素レベルを、 発酵槽の 攪拌、 空気注入および酸素注入の各速度を調節して維持する。 培養の最適密度が 600nmで 57〜63 に達すると、 第 3 の流動培地の供給が始まる。 第 3 の流動培地 (300 g/L ラクトース) は一定流量で発酵槽に導入される; 第 1の流動培地の供 給は中止し、 第 2の流動培地の流量は新たな一定流量に変える。 発酵は第 3の流 動培地の供給が始まってから、 およそ 10時間で終わる。発酵の終わりに、 培養は 15+/- 5 °Cに冷却する。菌株は遠心分離によって収集される。得られたペーストは、 - 60°C以下でパックしたまま保存する。 Continue until an optimum density of 5.0 +/- 1.0 at 600 nm is reached. Fed-batch phase, 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. When the culture reaches an optimal density of 20-25 at 600 nm, 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. When the optimal density of the culture reaches 57-63 at 600 nm, the supply of the third flowing medium starts. The third fluid medium (300 g / L lactose) 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. At the end of the fermentation, the culture is cooled to 15 +/- 5 ° C. Strains are collected by centrifugation. The obtained paste is stored as packed below -60 ° C.
上記のように E co7/で産生した rHuMGDFの精製は次のように行った。  Purification of rHuMGDF produced by Eco7 / as described above was performed as follows.
l, 800gのセル · ペーストを約 18リットルの lOmM EDTAに懸濁させ、 15, OOOps i で高圧ホモジナイザーに通した。 破砕されたセル懸濁液を遠心分離し、 得られた ペレツト 10 リットルの 10mM EDTA に再懸濁させた。 懸濁液を遠心分離し、 得ら れた 200gのペレツ卜を 2リットルの lOmM Tr i s、8Mグァニジン塩酸塩、 10mMDTT、 5inM EDTA, pH8. 7 に溶解させた。 この溶液を、 200 リットルの 10mM CAPS 、 3M 尿素、 30% グリセロール、 3mMシス夕ミン、 ImMシスティン、 ρΗΙΟ. 5で穏やかに 希釈した。  l, 800 g of cell paste was suspended in about 18 liters of lOmM EDTA and passed through a high-pressure homogenizer at 15, OOOpsi. The crushed cell suspension was centrifuged and resuspended in 10 liters of the resulting pellet in 10 mM EDTA. The suspension was centrifuged, and the obtained 200 g of the pellet was dissolved in 2 liters of 10 mM Tris, 8 M guanidine hydrochloride, 10 mM DTT, 5 in M EDTA, pH 8.7. This solution was gently diluted with 200 liters of 10 mM CAPS, 3 M urea, 30% glycerol, 3 mM cis-min, ImM cysteine, ρΗΙΟ.5.
希釈した溶液を室温で 16時間、 穏やかに撹拌し、 pHを 6. 8に調節した。 pHを 調節した溶液を清浄化して、 lOmMリン酸ナトリウム、 1. 5M尿素、 15%グリセロー ル、 pH6. 8 で平衡化した リットルの CM Sepharose カラムに加えた。 添加後、 カラムは 10mMリン酸ナトリウム、 15%グリセロール、 pH7. 2で洗った。 MGDFは 0 から 0. 5Mの NaClの勾配、 10mMリン酸ナトリウム、 pH 7. 2 で溶出させた。  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.
CM溶出液は、 分子量 10, 000カットオフメンブレンを用いて、 濃縮し、 10mMリ ン酸ナトリウム、 pH6. 5にバッファ一交換した。 約 2nig/mlに濃縮した溶液をカテ プシン C (500 : 1のモル比) で室温、 90分間処理した。 この溶液を、 10mMリン酸 ナトリウム、 15% グリセロール、 pH7. 2 で平衡化した 1. 2 リットルの SP High 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
Performance Sepharoseカラムに添加した。添加後、 MGDF (1-163)は 0. 1から 0. 25M の NaC lの勾配、 lOmMリン酸ナトリウム、 pH7. 2で溶出させた。 SP High Performance カラムからの溶出液に、 0.讓になるように硫酸アンモニゥムを加えた。 この溶出 液を、 10mMリン酸ナトリウム、 0. 6M硫酸アンモニゥム、 pH7. 2で平衡化した 1. 6 リットルの Phenyl Toyopearl カラム(東ソ一製)に添加した。 MGDF (1-163)ピー クは 0. 6 から 0Mの硫酸アンモニゥムの勾配、 10mMリン酸ナトリウム、 pH7. 2 で 溶出させた。 Phenyl Toyopearl溶出液は、 分子量 10, 000カツトオフメンブレン を用いて、 濃縮し、 10mM Tris、 5Γノルビトール、 pH7. 5にバッファー交換した。 ( 2 ) N-末端 ーァミノ基に付着部位を有する、 モノメトキシ—ポリエチレング リコール MGDFコンジュゲート (PEG- rHuMGDF) の調製 Added to Performance Sepharose column. After addition, MGDF (1-163) was eluted with a gradient of 0.1 to 0.25 M NaCl, 10 mM sodium phosphate, pH 7.2. Ammonium sulfate was added to the eluate from the SP High Performance column so as to be 0.1%. This elution The solution was added to a 1.6-liter Phenyl Toyopearl column (manufactured by Tosoichi) equilibrated with 10 mM sodium phosphate, 0.6 M ammonium sulfate, pH 7.2. MGDF (1-163) peak was eluted with a gradient of 0.6 to 0 M ammonium sulfate, 10 mM sodium phosphate, pH 7.2. The Phenyl Toyopearl eluate was concentrated using a 10,000 cut-off membrane with a molecular weight of 10,000, and the buffer was exchanged with 10 mM Tris, 5Γ norbitol, pH 7.5. (2) Preparation of monomethoxy-polyethylene glycol MGDF conjugate (PEG-rHuMGDF) having an attachment site at the N-terminal amino group
20mM NaCNBH 3を含む lOOmM リン酸ナトリウム (pH5 ) 中に上記 rHuMGDF (2 ml, 2. 5 mg/ml)を溶解した冷却(4°C) 撹拌溶液に、 5倍モル過剰のメトキシポリエ チレングリコールアルデヒド(MePEG) (平均分子量 20kDa) を加えた。 反応混合物 の撹拌は、 同じ温度で続けた。 20 mM NaCNBH 3 above rHuMGDF (2 ml, 2. 5 mg / ml) in lOOmM sodium phosphate (pH 5) containing a cooling obtained by dissolving (4 ° C) to a stirred solution of 5-fold molar excess of Metokishiporie Chi glycol aldehyde (MePEG) (average molecular weight 20 kDa) was added. Stirring of the reaction mixture was continued at the same temperature.
反応中におけるタンパク質の修飾度合いは、 Superdex 200 HR 10/30 カラム (Pharmac ia Biotech) を用いた、 SEC HPLC によってモニタ一した。 溶離は 0. 1M リン酸ナトリウムバッファー、 pH6. 9 を用いて、 流速 0. 7ml/min で行った。  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.
16時間後、 SEC HPLC分析は、 タンパク質の初めの量の約 90% が修飾されてい ることを示した。 この時点で反応混合物のタンパク質濃度は、 滅菌水で希釈する ことによって、 lmg/mlに下げ、 0. 5M酢酸で反応混合物の pHを 4 に調整した。 mono-MePEG (20kDa) -rHuMGDF コンジユゲートは、 SP Sepharose HP (Pharmac ia Biotech) イオン交換樹脂を用いたイオン交換クロマトグラフィーによって、過剰 の MePEGと他の反応副生物から分離した。  After 16 hours, SEC HPLC analysis showed that about 90% of the original amount of protein was modified. At this point, the protein concentration of the reaction mixture was reduced to 1 mg / ml by diluting with sterile water, and the pH of the reaction mixture was adjusted to 4 with 0.5 M acetic acid. The mono-MePEG (20 kDa) -rHuMGDF conjugate was separated from excess MePEG and other reaction by-products by ion exchange chromatography using SP Sepharose HP (Pharmacia Biotech) ion exchange resin.
反応混合物をカラムに添加(2. 5 ing/nil (樹脂)') し、 未反応の MePEGを 3カラ ム容量の開始バッファー A (20mMリン酸ナトリウム、 pH7. 2、 15%グリセロール) で 溶出させた。 その後、 MePEG- MGDFコンジュゲートを、 20カラム容量で、 終バッフ ァー B (バッファー A中、 1M NaCl) の、 0%から 25% の直線勾配を用いて溶出し た。 溶出液は 280nmでモニターした。 poly- MePEG- rHuMGDFコンジュゲートを含ん でいるフラクションをプールし、 濃縮、 滅菌濾過した。 コンジュゲートの均一性 は、 4〜20% のプレキャスト · 勾配' ゲル (N0VEX) を用いた SDS-PAGE (ナトリウ ム · ドデシル'スルフアート ·ポリアクリルアミドゲル電気泳動) で判定し、 1本 のメジャーバンドとして検出した。 <参考例 2〉 全長ヒト TPOの発現 Add the reaction mixture to the column (2.5 ing / nil (resin) ') and elute unreacted MePEG with 3 column volumes of starting buffer A (20 mM sodium phosphate, pH 7.2, 15% glycerol). Was. The MePEG-MGDF conjugate was then eluted with 20 column volumes using a linear gradient of final buffer B (1M NaCl in buffer A) from 0% to 25%. The eluate was monitored at 280 nm. Fractions containing poly-MePEG-rHuMGDF conjugate were pooled, concentrated and sterile filtered. 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
下記の全長ヒト ΤΡΟの発現例は国際公開 W095/21919 に記載される方法に基い ている。 The following expression examples of full-length human are based on the method described in International Publication W095 / 21919.
(1) ヒト TPO完全長 c DNAプラスミド (pHTP l) の作製  (1) Preparation of human TPO full-length cDNA plasmid (pHTPl)
配列番号 6のように予想されたヒト TPO c DNAのアミノ酸コーディング領 域を全て持つ、 動物細胞発現べクタ一の構築を行なった。  An animal cell expression vector having all the amino acid coding regions of human TPO cDNA predicted as shown in SEQ ID NO: 6 was constructed.
P CRによりヒト TPO c DNAコ一ディング領域を全てカバーする D N A断 片を以下の様に作製した。  A DNA fragment covering the entire human TPO cDNA coding region by PCR was prepared as follows.
用いたプライマーのヌクレオチド配列は次の通りである。 The nucleotide sequences of the primers used are as follows.
h T P 0- I : 5'-TTGTGACCTCCGAGTCCTCAG- 3, (配列番 号 8);  hTP0-I: 5'-TTGTGACCTCCGAGTCCTCAG-3, (SEQ ID NO: 8);
S A: 5, - C AGGTAT C C GGGGATTTGGT C- 3, (配列番号 9 ) hTPO-P: 5,一 TGCGTTTCCTGATGCTTGTAG- 3' (配列番 号 10);  S A: 5, -C AGGTAT C C GGGGATTTGGT C-3, (SEQ ID NO: 9) hTPO-P: 5, TGCGTTTCCTGATGCTTGTAG-3 '(SEQ ID NO: 10);
h T P O-KO: 5, - GAGAGAGCGGCCGCTTACCCTTCCTG AGACAGATT-3' (配列番号 11)。  hTPO-KO: 5, -GAGAGAGCGGCCGCTTACCCTTCCTG AGACAGATT-3 '(SEQ ID NO: 11).
クロ一ン p E F 18 S- HL 34 (W095/21919の実施例 16) の 300ngを铸 型として 1回目の P CRを行なった。 プライマ一 hTPO- I並びに SA各 0.5μ Μを用い、 Vent RTM DNA polymerase (New England BioLabs社製) 1ュニットを 使用して反応 (96°C1分間、 62°C1分間、 72°C1分間という反応を 30サイクル行 なった後 72 7分間)を行なった。反応溶液の組成は以下の通り。最終濃度で 10mM KCK 10inM(NH4) 2S04、 20mM Tris-HCl (pH8.8), 2mM MgS04, 0.1% Triton X- 100、 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 μm of each primer, hTPO-I and 0.5 μl 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,
市販のヒト正常肝臓由来ポリ (A) + RNA (Clontecii社製) lpgを 70°Cで 10分間加熱後氷上で急冷し、 10mM DTT、 500 M dNTPmix、 25ng random primer Commercially available 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
(宝酒造社製)、 10ユニット RNase Inhibitor (ベーリンガーマンハイム社製)、(Takara Shuzo), 10 units RNase Inhibitor (Boehringer Mannheim),
200ュニット Superscript-1 M II RNaseH- (LIFE TECHNOLOGIES社製)を加え、 37°C で 1時間保温し c DNAを合成した。合成した c DN A反応液の 20分の 1量を錶 型として使用して 2回目の P CRを行なった。プライマー h TP O-P並びに hT PO- KO各 2.5 M、 2.5 ユニットの AmiDliTa(iT m DNA polymerase (宝酒造社 製) を用いて反応 (95°C1分間、 58°C 1分間、 72°C 1分間の反応を 30サイクル) を行なった。 200 units Superscript- 1 M II RNaseH- (manufactured by LIFE TECHNOLOGIES) was added, and the mixture was incubated at 37 ° C for 1 hour to synthesize cDNA. One-twentieth volume of the synthesized cDNA reaction solution A second PCR was performed using the mold. Primer h TP OP and hT PO- KO each 2.5 M, 2.5 units of AmiDliTa (i T m DNA polymerase (manufactured by Takara Shuzo) reaction (95 ° C1 minutes using, 58 ° C 1 min, of 72 ° C 1 minute The reaction was performed for 30 cycles).
第 1及び第 2回の P C R溶液を 1 %ァガロースゲル電気泳動にかけ、 それぞれ 予想された大きさの主要なバンドをプレップ- A-ジーン DNA精製キット (バイオ ラッド社製)を用いて精製した。それぞれの精製量のうち各 20分の 1量を铸型と して 3回目の P CRを実施した。 Vent R™ DNA polymerase (New England BioLabs 社製) 1ユニットを使用して反応 (96°C2分間加熱後、 96°C2分間、 72°C2分間、 という反応を 3サイクル行なった後 72°C7分間) を行なった。 この反応液にそれ ぞれ 1 Mとなるように hTPO- I並びに hTPO- KOを加えた後、 96°C2 分 間の加熱を行ない、 そののち 96°C1分間、 62°C1分間、 72°C 1分間の反応を 25サ ィクル行なったあと 72°Cでさらに 7分間反応させた。反応液を等量の水飽和フエ ノ一ル-クロロホルムで 1回抽出後、さらに等量のクロ口ホルムで 1回抽出したの ち、 エタノール沈殿(0.3M酢酸ナトリゥム、 0.5 zlベーリンガーマンハイム社製 グリコーゲン、 2.5 倍量エタノール存在下) を行なって DNAを回収した。 回収 した DN Aを制限酵素 BamHI並びに Not Iで消化後 1 %ァガロースゲル電気泳動に かけ、予想された大きさの主要なバンドをプレツプ- A-ジーン DNA精製キット(バ ィォラッド社製) を用いて精製したのち、 予め同様に制限酵素 BamHI並びに Notl で消化した pBluescriptll SK+ベクター (Stratagene社) に連結後、 コンビテン トハイ E. coliDH5 (東洋紡績社製) を形質転換した。 得られたコロニーより 4ク ローンを選びプラスミド DNAを調製した。 精製したプラスミド DNAについては Tad Dye DeoxyT M Terminater Cycle Seauening Kit (アプライドバイオシステ ムズ社製) を用い、 アプライドバイオシステムズ社製 373ADNAシークェンサ一に よりシークェンスし、 BamHIから Notl にかけての領域に塩基配列の置換がなく、 予想通りの TPOcDNA配列を持つことが確認できたクローン pBLTPを得た。 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 ™ 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. After adding hTPO-I and hTPO-KO to this reaction solution to 1 M, respectively, heat at 96 ° C for 2 minutes, then 96 ° C for 1 minute, 62 ° C for 1 minute, 72 ° C After performing the reaction for 1 minute for 25 cycles, the reaction was further carried out at 72 ° C for 7 minutes. The reaction solution was extracted once with an equal volume of water-saturated phenol-chloroform, extracted once more with an equal volume of chloroform, and then precipitated with ethanol (0.3 M sodium acetate, 0.5 zl Boehringer Mannheim Glycogen). , In the presence of 2.5 volumes of ethanol) to recover DNA. 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. Using purified for plasmid DNA Tad Dye Deoxy TM Terminater Cycle Seauening Kit ( manufactured by Applied Bio System James Corp.), and Sequence More Applied Biosystems 373ADNA Shikuensa one, replacement of the nucleotide sequence in the region of the over the Notl from BamHI However, a clone pBLTP which was confirmed to have the expected TPO cDNA sequence was obtained.
P BLTPを制限酵素 EcoRI並びに BamHIで消化後 1 %ァガ口一スゲル電気泳 動にかけ、 高分子量のバンドをプレップ- A-ジーン DNA精製キット (バイオラッ ド社製)を用いて精製した。同様に pEF 1 8 S-HL 34も制限酵素処理し 45 Ob のバンドを精製した。 それぞれの DNAを連結しコンビテントハイ E. coli DH5 (東洋紡績社製) を形質転換した。 得られたコロニーよりプラスミド DNAを 調製し、 ヒト TPO c DNAのインサートを含むクローン p B LTENを得た。 得られた p BL TENを制限酵素 EcoRI並びに Notlで消化後、 1 %ァガロースゲ ル電気泳動にかけ、約 1200bpのバンドをプレップ- A-ジ一ン爾 A精製キット (バ ィォラッド社製) を用いて精製した後、 同様に制限酵素処理した発現ベクター p EF 1 8 Sに連結しコンビテントハイ E. coliDH5 (東洋紡績社製) を形質転換し た。 得られたコロニーよりプラスミド DNAを調製し、 ヒト TPOc DNAのコ —ディング領域を全て含むクローン pHTP 1を得た。 このクローンのプラスミ ド DNAを大量に調製し以下の実験に使用した。 プラスミド DNAの調製は本質 的に Molecular Cloning (Sambrookら、 Cold Spring Harbor Laboratory Press、 1989) に記載されているようにして実施した。 After digesting PBLTP with restriction enzymes EcoRI and BamHI, it was subjected to 1% agarose gel electrophoresis, and a high molecular weight band was purified using Prep-A-Gene DNA purification kit (manufactured by BioRad). Similarly, pEF 18 S-HL 34 was treated with a restriction enzyme. The Ob band was purified. The respective DNAs were ligated and transformed into a competent high E. coli DH5 (manufactured by Toyobo Co., Ltd.). Plasmid DNA was prepared from the obtained colonies to obtain a clone pBLTEN containing an insert of human TPO cDNA. 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).
(2) CHO細胞内でのヒト TPO発現用の哺乳動物発現プラスミド、 pDEF2 0 2 -hTPO-P 1の構築  (2) Construction of mammalian expression plasmid, pDEF202-hTPO-P1, for human TPO expression in CHO cells
マウス DHFRミニ遺伝子を含むプラスミド pMGl、 1 pgを制限酵素 EcoRI と BamHIで処理した後、ァガロースゲル電気泳動しマウス DHFRミニ遺伝子を含む断 片 (約 .5kbp) を回収した。  After treating 1 pg of the plasmid pMGl containing the mouse DHFR minigene with the restriction enzymes EcoRI and BamHI, agarose gel electrophoresis was performed to recover a fragment (about 0.5 kbp) containing the mouse DHFR minigene.
回収した断片を 50mM Tris- HC1 (pH7.5), 7mM MgCl2、 lmM j8-メルカプトエタ ノール、 0.2inM dNTPからなる反応液 25 1 中に溶解し、 Klenowフラグメント 2 単位を加え、 室温で 30分間反応させ、 DNA の末端を平滑化した。 次いで、 フエ ノールノクロロホルム処理、 エタノール沈殿後、 10mMTri_HCl (pH8.0)、 lmM EDTA からなる TE溶液 10/ lに溶解した。 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.
次に、 得られたマウス DHFR ミニ遺伝子を含む断片と動物細胞用発現べクタ一 PEF 1 8Sを制限酵素 Smalで処理した後、アルカリフォスファターゼ(宝酒造製) で脱リン酸化してえられたベクター DNAを T4DNAリガーゼ(宝酒造製) で結合さ せ、 発現べクタ一 pDEF20 2を得た。  Next, 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. Was ligated with T4 DNA ligase (Takara Shuzo) to obtain an expression vector pDEF202.
次に、 このベクター pDEF20 2を制限酵素 EcoRIと Spelで処理し、 ァガロース ゲル電気泳動で大きい方のベクターフラグメントを回収したのち、 このフラグメ ントとヒト TPO cDNA(Plクローン)を含むプラスミド pHTP 1を制限酵素 EcoRI と Spelで処理して得られたヒト TPOcDNA(Plクローン)とを T4DNAリガーゼ(宝 酒造製) で結合させ、 発現べクタ一 PDEF202- hTPO- P1得た。 このプラスミドは SV40の複製開始領域、 ヒトェロンゲーションファクター 1一アルファプロモー ター、 SV40初期ポリアデニル部位、 マウス DHFRミニ遺伝子、 PUC1 8の複製開 始領域、 j3-ラクタマ一ゼ遺伝子(Ampつを含み、 ヒトェロンゲーシヨンファクタ 一 1一アルファプロモーター下流にヒト TPO cDNAが接続されている。 Next, 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.
(3) CHO細胞でのヒト TPOの発現  (3) Expression of human TPO in CHO cells
CHO細胞 (dhir-株、 Urlaubと Chasin; Proc. Natl. Acad. Sci.USA; 77巻 4 2 16頁、 1980) を 6 cm径のプレー卜 (Falcon社製) 中 10 %牛胎児血清を 含む α最小必須培地 (a-MEM (—)、 チミジン、 ヒポキサンチン添加) で培養増 殖させ、 これをリン酸カルシウム法 (CellPhect、 フアルマシア社製) によって形 質転換した。 すなわち、 上記 (1) で調製した PDEF202- MP0-P1プラスミド 1 0 iigにバッファ一 A: 1 20 lおよび H20: 1 2 0 u\ を加え混合したのち、 室温で 10分間放置した。 つぎに、 この溶液にバッファー B : 1 20 ^1を加え、 再度混合したのち、 室温で 30分間放置した。 この]) NA溶液をプレートに滴下し たのち、 C02インキュベータ一中で 6時間培養した。プレートから培地を除去し、 α-ΜΕΜ (一) にて 2回洗浄後、 10 %ジメチルスルフォォキシド含有 - MEM (-) を添加し、 室温で 2分間処理した。 次いで、 10 %透析牛胎児血清含有非選択培 地 (前出 - MEM (—)、 ヒポキサンチン、 チミジン添加) を添加して 2日間培養し たのち、 10%透析牛胎児血清含有選択培地 (α- MEM (—)、 ヒポキサンチン、 チ ミジン無添加) での選択をおこなった。 選択は細胞をトリプシン処理した後、 6 cm径プレート 1枚あたりを、 10 cm径プレート 5枚あるいは 24ゥエルプレート 20枚に分割したのち、 2日ごとに選択培地にて培地交換を行いながら培養を続 行する事により実施した。 細胞が増殖してきたプレートあるいはゥエルについて はその培養上清中のヒト TP0活性を M— 07 eアツセィ及び B a/F 3アツセィ (W095/21919) を用いて測定したところ、 いずれのアツセィ系においても活性が 認められた。 培養培地中にヒト TP0を分泌する細胞を、 25nMのメソトレキセ一 トを含む選択培地でさらに選択し、 高レベルのヒト TPOを産生する細胞クロー ンを単離した。 プラスミド PDEF 202— hTP〇— P lによって形質転換された C HO細 胞株(CHO- DUKXBll)は 1 995年 1月 3 1日付で独立行政法人産業技術総合研究 所 (日本国茨城県つくば市東 1丁目 1番地 1中央第 6) に受託番号 FERM B P-4988として寄託されている。 またこの細胞株は中華人民共和国及び中華 民国の寄託機関にそれぞれ受託番号 CCTCC- C95004、 FIRDI 960023として寄託され ている。 配列表フリーテキスト 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 α 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. Next, 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 α-ΜΕΜ (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. For selection, after trypsinizing the cells, divide one 6 cm diameter plate into 5 10 cm diameter plates or 20 24-well plates, and culture every 2 days while changing the medium with the selection medium This was implemented by continuing. The human TP0 activity in the culture supernatant of the plates or wells on which the cells had grown was measured using M-07 e Atssay and Ba / F3 Atssay (W095 / 21919). Activity was observed. Cells secreting human TP0 in the culture medium were further selected on a selective medium containing 25 nM methotrexate, and cell clones producing high levels of human TPO were isolated. 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
配列番号 2-人工配列の説明:配列番号 1のアミノ酸配列 1〜 163  SEQ ID NO: 2—Description of Artificial Sequence: Amino Acid Sequence of SEQ ID NO: 1 to 163
配列番号 3-人工配列の説明:ィンサ一卜 (センス鎖)  SEQ ID NO: 3—Description of Artificial Sequence: Insert (Sense Strand)
配列番号 4-人工配列の説明:インサート (アンチセンス鎖)  SEQ ID NO: 4-Description of artificial sequence: insert (antisense strand)
配列番号 5-人工配列の説明:ィンサート (センス鎖)  SEQ ID NO: 5-Description of artificial sequence: insert (sense strand)
配列番号 6-人工配列の説明:インサ一卜 (アンチセンス鎖)  SEQ ID NO: 6-Description of artificial sequence: insert (antisense strand)
配列番号 7-人工配列の説明:プロモーター  SEQ ID NO: 7-Description of artificial sequence: promoter
配列番号 8-人工配列の説明:プライマー  SEQ ID NO: 8-Description of artificial sequence: primer
配列番号 9-人工配列の説明: プライマー  SEQ ID NO: 9-Description of Artificial Sequence: Primer
配列番号 10-人工配列の説明:プライマー  SEQ ID NO: 10—Description of Artificial Sequence: Primer
配列番号 11-人工配列の説明:プライマー 本明細書で引用した全ての刊行物、特許および特許出願の全体を参考として本 明細書に取り入れるものとする。 また、 本明細書は、 本願の優先権の基礎である 日本国特許出願第 2000-254473号の明細書および図面に記載される内容を包含す るものとする。  SEQ ID NO: 11-Description of Artificial Sequence: Primers All publications, patents and patent applications cited herein are hereby incorporated by reference in their entirety. This description includes the contents as disclosed in the description and drawings of Japanese Patent Application No. 2000-254473, which is a priority document of the present application.

Claims

請求の範囲 The scope of the claims
1 . 配列番号 1のアミノ酸配列のうち少なくとも 7位〜 1 5 1位のアミノ酸残 基を含むアミノ酸配列を有するポリペプチド、 その変異体、 またはそれらの誘導 体からなり、 かつトロンポポェチン活性を有する治療上有効量の c一 m p 1 リガ ンドを含む、 骨髄異形成症候群または再生不良性貧血患者において血小板及び赤 血球を増加させるための医薬組成物。  1. Therapeutic comprising a polypeptide having an amino acid sequence containing at least the 7th to 15th amino acid residues in the amino acid sequence of SEQ ID NO: 1, a mutant thereof, or a derivative thereof, and having a thrombopoietin activity A pharmaceutical composition for increasing platelets and erythrocytes in a patient with myelodysplastic syndrome or aplastic anemia, comprising a c-mp1 ligand in an effective amount.
2 . 前記 c— m p 1 リガンドが、 配列番号 1のアミノ酸配列 1〜3 3 2からな るポリペプチド、 その変異体またはそれらの誘導体であって、 かつトロンポポェ チン活性を有するものである、 請求項 1に記載の医薬組成物。  2. The c-mp1 ligand is a polypeptide consisting of the amino acid sequence 1-332 of SEQ ID NO: 1, a mutant thereof or a derivative thereof, and having thrombopoietin activity. 2. The pharmaceutical composition according to 1.
3 . 前記 c一 m p 1リガンドが、 配列番号 1のアミノ酸配列 1〜1 6 3からな るポリペプチド、 その変異体またはそれらの誘導体であって、 かつトロンポポェ チン活性を有するものである、 請求項 1に記載の医薬組成物。  3. The c-mp1 ligand is a polypeptide consisting of the amino acid sequence 1-163 of SEQ ID NO: 1, a mutant thereof or a derivative thereof, and having thrombopoietin activity. 2. The pharmaceutical composition according to 1.
4 . 前記変異体が、 配列番号 1のアミノ酸配列において少なくとも 1つのアミ ノ酸の置換、 欠失、付加および/または挿入を含み、 かつトロンポポェチン活性を 有するポリペプチドである、 請求項 1〜3のいずれか一項に記載の医薬組成物。  4. The variant according to claims 1 to 3, wherein the mutant is a polypeptide comprising substitution, deletion, addition and / or insertion of at least one amino acid in the amino acid sequence of SEQ ID NO: 1 and having thrombopoietin activity. The pharmaceutical composition according to any one of the preceding claims.
5 . 前記誘導体が、 水溶性ポリマ一で修飾されたポリベプチドまたはその変異 体である、 請求項 1〜 3のいずれか一項に記載の医薬組成物。 5. The pharmaceutical composition according to any one of claims 1 to 3, wherein the derivative is a polypeptide modified with a water-soluble polymer or a mutant thereof.
6 . 前記水溶性ポリマーがポリエチレングリコ一ルである、 請求項 5に記載の 医薬組成物。  6. The pharmaceutical composition according to claim 5, wherein the water-soluble polymer is polyethylene glycol.
7 . 前記水溶性ポリマーが、 少なくとも前記ポリペプチドまたはその変異体の N末端に結合されている、請求項 5または 6に記載の医薬組成物。  7. The pharmaceutical composition according to claim 5, wherein the water-soluble polymer is bound to at least the N-terminus of the polypeptide or a variant thereof.
8 . 前記 c-mp 1 リガンドが、配列番号 2のァミノ酸配列からなる組換えポリぺプ チドの N末端に 1分子のポリエチレングリコールが結合された PEG_rHuMGDFであ る、 請求項 1に記載の医薬組成物。  8. The medicament according to claim 1, wherein the c-mp1 ligand is 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. Composition.
4 Four
PCT/JP2001/007283 2000-08-24 2001-08-24 c-mpl LIGAND-CONTAINING MEDICINAL COMPOSITIONS FOR INCREASING PLATELETS AND ERYTHROCYTES WO2002015926A1 (en)

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