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WO1993016106A1 - Nouvel amplificateur de megakaryocites et production - Google Patents

Nouvel amplificateur de megakaryocites et production Download PDF

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Publication number
WO1993016106A1
WO1993016106A1 PCT/JP1993/000155 JP9300155W WO9316106A1 WO 1993016106 A1 WO1993016106 A1 WO 1993016106A1 JP 9300155 W JP9300155 W JP 9300155W WO 9316106 A1 WO9316106 A1 WO 9316106A1
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megakaryocyte
activity
amplification factor
cells
factor protein
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PCT/JP1993/000155
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English (en)
Japanese (ja)
Inventor
Shuhei Kondo
Hiroki Shigematsu
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Asahi Kasei Kogyo Kabushiki Kaisha
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Publication of WO1993016106A1 publication Critical patent/WO1993016106A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/475—Growth factors; Growth regulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention relates to a novel megakaryocyte mitogen and a method for producing the same. More specifically, the present invention relates to a novel megakaryocyte amplification factor protein having an activity of promoting proliferation of megakaryocytes, which are platelet precursor cells, and having an action of promoting platelet production, and its production by cell culture. About the method. The present invention also relates to a pharmaceutical composition containing the novel protein as a megakaryocyte amplification factor useful for prevention and treatment of diseases such as thrombocytopenia.
  • TPO Thrombopoetin
  • TPO which has the activity to promote megakaryocyte maturation, acts in the late stage.
  • Meg-CSF acts, progenitor cells repeat cell division, and megakaryocyte components increase.
  • TPO acts, and each megakaryocyte progenitor performs endamisis.
  • chromosome multiple is increased (up to 32 N)
  • the cytoplasm matures and increases, and platelet production begins.
  • TPO is also sometimes referred to as megakaryocyte potentiator (Meg-P0T).
  • the activity of Meg-CSF can be estimated by measuring the activity of megakaryocyte colony formation in soft agar culture of human or mouse bone marrow cells in vitro.
  • the activity of Meg-CSF is measured in urine of patients with aplastic anemia and patients with idiopathic thrombocytopenic purpura, in blood of patients with myelomegaly aplastic thrombocytopenia, and in kidney bean lectin-stimulated cells.
  • G It is found in leukocyte culture supernatant, mouse leukemia cell line WEHI-3 culture supernatant, etc.
  • interleukin 3 (interleukin is abbreviated as IL) acts non-specifically on many strains including megakaryocytes. It is becoming clear that it is ti-CSF. In addition, Meg-CSF in WEH 3 culture supernatant was completely consistent with IL-3, indicating that Meg-CSF activity in Many are attributed to IL-3. However, Meg-CSF, which has been shown to specifically act on the platelet system, is not yet known.
  • the activity of TPO can be estimated by measuring the strong effect of the colony forming activity of Meg-CSF and the effect of promoting the maturation of or megakaryocytes. Attempts have been made to prepare some factors having TP ⁇ -like activity. It is prepared from the culture supernatant of a human fetal kidney cell line and has the effect of promoting protein synthesis in megakaryocytic cells having a molecular weight of 15,000 on SDS-PAGE and an isoelectric point of 5.1. A Megakaryocyte Stimulatory Factor (MSF) and a method for its production have been reported (see US Pat. No. 4,894,440).
  • MSF Megakaryocyte Stimulatory Factor
  • IL-6 is also involved in the hematopoietic system and shows Meg-! ⁇ OT activity and megakaryocyte maturation promoting activity in vitro (Ishibashi, T. eta 1. rproc. Natl. Acad. Sc i. USAJ 86, 5953 (1989)), in
  • V i V 0 exhibits a platelet production promoting effect (Asano, S. eta 1. “Blo.d” 75, 1602 (1990)). Furthermore, it has been reported that IL-7, IL-11 and the like also have megakaryocyte amplification activity. However, the megakaryocyte-amplifying activity of these factors is weak, and it is unclear whether or not these are constitutive (healthy) hematopoietic factors inherent to living organisms.
  • the present inventors In order to find a novel megakaryocyte amplifying factor having the action of specifically and strongly promoting megakaryocyte amplification and the action of promoting platelet production in the above-mentioned technical background, the present inventors As a result of surprising studies, surprisingly, a completely new megakaryocyte amplification factor was found in the culture supernatant of human normal diploid cells, and by adding an appropriate production promoter to the culture medium, It has been found that a large amount of the factor is produced. Further, the factor was isolated and purified from the collected culture supernatant, and its properties were clarified, and its usefulness as a drug was demonstrated. The megakaryocyte amplification factor can also be expressed by applying genetic engineering technology. The present invention has been completed based on these findings.
  • one object of the present invention is to provide a substantially pure novel megakaryocyte amplification factor having a potent action.
  • Another object of the present invention is to cultivate animal cells in a medium, produce megakaryocyte amplifying factor in the culture solution, collect a culture supernatant from the culture solution, and use a megakaryocyte from the collected culture supernatant.
  • An object of the present invention is to provide a method for producing a megakaryocyte amplification factor, which comprises purifying a sphere amplification factor.
  • Still another object of the present invention is to increase the amount of the megakaryocyte amplification factor produced by adding the megakaryocyte amplification factor production promoter to the medium in the cell culture and culturing the cell.
  • a method for producing a megakaryocyte amplification factor is to provide a pharmaceutical composition containing a therapeutically effective amount of a megakaryocyte amplifying factor as an active ingredient, and a therapeutic method using the same.
  • a megakaryocyte amplification factor having an activity of activating megakaryocyte amplification and having an activity of increasing platelets in peripheral blood. More specifically, the present invention provides a substantially pure megakaryocyte amplification factor protein having an activity of activating megakaryocyte amplification and having the following properties.
  • megakaryocyte amplification factor proteins of the present invention include the following two substantially pure megakaryocyte amplification factor proteins that have the activity of activating megakaryocyte expansion and have the following properties: Can be mentioned.
  • animal cells are cultured in a medium, megakaryocyte amplification factor is produced in the culture medium, a culture supernatant is recovered from the culture medium, and the culture supernatant is recovered from the recovered culture supernatant.
  • a method for producing a megakaryocyte amplification factor comprising separating and purifying a megakaryocyte expansion factor.
  • the animal cell used in the method of the present invention has an activity of activating megakaryocyte amplification and increases platelets in peripheral blood.
  • Various cells having the ability to produce megakaryocyte expansion factor having the activity to be added can be used.
  • Normal diploid cells can be used advantageously, for example, cells from human kidney, intestine, lung, heart, ureter, skin, foreskin, tongue, thyroid, placenta, uterus, preferably human fetal kidney Cells from lung, foreskin, and even more preferably cells from human fetal lung can be used.
  • the megakaryocyte amplification factor can be separated and purified from these tissue extracts, but preferably, these cells are cultured in a suitable growth medium, and Then, the megakaryocyte amplification factor is produced, the culture supernatant is recovered from the culture solution, and it can be separated and purified from the recovered tissue culture solution. It is desirable that these cells be propagated by a culture method used for normal cell culture, for example, the method described in “Tissue culture” (Junnosuke Nakai et al., Asakura Shoten, edited by Showa 51) and used in the present invention.
  • the cells can produce megakaryocyte spreading factor by culturing them in a medium solution containing carbons, nitrogen source and, if necessary, inorganic salts and / or other additives.
  • the megakaryocyte produced in a culture solution is obtained by adding a megakaryocyte amplification factor production promoter, preferably an animal meat enzyme-decomposing peptone, and culturing the cells.
  • the amount of the sphere width factor can be greatly increased.
  • the concentration of animal meat enzyme-degrading peptone can be (! -4 W / V%, preferably 0.1-2 w / v%, based on the culture medium. , Which is commonly used for bacterial culture media Therefore, it is usually called proteose peptone, proteosebuton, or meat peptone.
  • the method for preparing this animal meat enzyme-degrading peptide is well-known, for example, according to the method described in "Bacterial Culture Studies Lecture Series 2" (Sakazaki Toshiichi, Naya Shoten, 1967). That is, as animal meat, meat or offal such as cows, pigs, nits, sheep, and whales are used, and beef is most commonly used. As enzymes for the degradation, trypsin, papine, pepsin, and no. There is creatine. These animal meats are crushed, mixed with water, and adjusted to a pH suitable for enzymatic degradation with sodium carbonate, concentrated hydrochloric acid and the like.
  • the enzyme is added to this and the enzyme is digested at 20-40 ° C for 1-20 days, usually at 37 ° C for 2-3 days. After digestion, heat to 100 ° C or higher to inactivate degrading enzymes and heat coagulate undigested proteins, remove them by filtration, and then concentrate, dry, and finely powder. . Concentration and drying methods include boiling into powder and concentrating at a low temperature using a vacuum drying device, followed by fine powdering. Commercial products include Proteose Peptone No. 1 (Pr. Te.se Pep t. No. 1), Proteose Peptone No. 2 and Proteose Peptone manufactured by Difco Inc. of the United States. No.
  • a megakaryocyte amplification factor by a cell culture method.
  • a suitable cell density favored properly in a density of 1 0 5 C el I s / ml, with 0. 1 ⁇ l Om g / ml of cell culture bead carrier Implantation, serum-containing 15-45 ° C, preferably 25-40.
  • serum-free conditions should be used.
  • Production culture is performed at a concentration of 3 to 4%, preferably 0.1 to 2 o / o.
  • the cells are allowed to grow sufficiently and are transferred to a production culture, preferably in a confluent state.
  • the number of culture days for production is usually 1 to 60 days, but can be more than 60 days.
  • the production rate of megakaryocytes is gradually slowed in the latter half of production, so the most efficient days are selected for industrial production. It is produced in solution from cells, and the amount of production is measured by the megakaryocyte amplification activity measurement method described in Reference Examples (a) and (b).
  • the megakaryocyte amplification factor of the present invention is obtained by expressing the megakaryocyte amplification factor in a suitable host cell by using a commonly used genetic technique, and collecting and purifying the megakaryocyte amplification factor. Can also be obtained.
  • RNA is extracted from cells from the kidney, lung, foreskin, and more preferably from cells from fetal human lung, and Liboli A + RNA is further purified.
  • Base click coater for appropriate expression like properly is to prepare c DNA library over using vectors and Po Li A ⁇ RNA and linker one for eukaryotic expression, suitable using this line bra rie A host cell, for example, Escherichia coli is transformed, and lipase DNA is prepared from the culture solution.
  • the plasmid is used to transfect a suitable host cell, preferably a cell derived from an animal, or more preferably, a monkey-derived c0s cell to obtain a megakaryocyte amplification factor gene.
  • a suitable host cell preferably a cell derived from an animal, or more preferably, a monkey-derived c0s cell to obtain a megakaryocyte amplification factor gene.
  • the megakaryocyte-amplifying factor can be produced by expressing, collecting and purifying it.
  • RNA RNA obtained by Pharmacia, Sweden, No. 27-4955-01
  • pcDL-SR ⁇ 296 may be used.
  • the resulting solution containing the cDNA library is divided into an appropriate number of pools, preferably 10 to 200, more preferably 50 to 100, and each of them is divided into E. coli MC1061 (ATCC533338). ). Incubate the transformed E. coli in the presence of ampicillin overnight. After collecting and lysing the cells, prepare plasmid DNA using Qiagen-tip-100 (manufactured by Qiagen, USA) according to the attached manual.
  • the obtained recombinant DNA is, for example, according to the Norredextran method (CURRENT PROTOCOLS IN MOLECULAR BIO LOGY 9.2.1.-9,2.6), suitable host cells, preferably monkey kidney cells, C0S1 cells (ATCC, CRL165) After that, the gene is expressed almost in the same manner as in the method described in WO 88/05053, Example 2. That is, under appropriate culture conditions, for example, a D-MEM medium containing 10% fetal bovine serum (manufactured by Flora Laboratory, USA). (: After culturing under conditions of 5% CO 2 for 40 hours, change to serum-free D-MEM medium, and collect the culture medium three times every two days.
  • the expression cells incorporating the megakaryocyte amplification factor gene can be screened using the activity of the megakaryocyte amplification factor as an index to clone the megakaryocyte amplification factor gene.
  • the megakaryocyte amplifying factor activity is measured by a method such as acetylcholinesterase activity measurement in liquid culture, and the pool containing the gene of this substance is used as an index. Can be narrowed down. Further, for the positive DNA, the E. coli is transformed again, and the obtained colonies (about 2000) are cultured as a group of about 10 cells, and the DNA is prepared in the same manner as above. In addition, introduction and expression into COS 1 cells and measurement of acetylcholinesterase activity can be performed to narrow down a secondary cDNA library.
  • Escherichia coli having a cDNA plasmid expressing the megakaryocyte amplifying factor activity is isolated. (Hayash i da, K. eta 1 "Hema topoiec Factor" 1, No. 2, 102-108 (1990)).
  • megakaryocyte amplification factor gene for example, Escherichia coli, yeast, monkey kidney cells (COS cells), Chinese hamster ovary cells (CH ⁇ cells), mouse Transfect host cells such as C127 cells, human fetal kidney cell line, silkworm cell SF9, etc., and express the megakaryocyte amplifying factor more efficiently, collect and purify it.
  • megakaryocyte amplification factor can be produced.
  • the culture supernatant is collected when the production of megakaryocyte expansion factor by the production cells reaches a desired production amount or days.
  • the method for separating and purifying the megakaryocyte amplification factor include methods usually used in protein chemistry, for example, adsorption using a carrier, salting out, electrophoresis, ion exchange, gel filtration, and the like.
  • a variety of chromatographic methods, etc. that apply ligand affinity can be used alone or in combination.
  • a chromatographic method preferably, a CM cell using Sepharose to which a carboxymethyl group is bonded is used.
  • the megakaryocyte amplification factor of the present invention is obtained from the fraction having the activity of the megakaryocyte amplification factor obtained during the purification using, for example, isoelectric focusing electrophoresis or ion exchange chromatography.
  • the fraction that is considered to have an electric point can be isolated by separating and further purifying it.
  • the novel megakaryocyte amplification factor thus obtained has an activity of activating megakaryocyte amplification and an activity of increasing platelets in peripheral blood.
  • the megakaryocyte amplifying factor is used as a reagent for studying the differentiation, proliferation and maturation of megakaryocytes from bone marrow stem cells or bone marrow megakaryocyte progenitor cells, or as a megakaryocyte amplifying factor alone or therapeutically.
  • An effective amount of the megakaryocyte amplification factor is added to at least one selected from pharmaceutically acceptable carriers, diluents and excipients to form a suitable dosage form, and the resulting drug is Can also be used.
  • the carrier, diluent and excipient those usually used in this field can be used.
  • the megakaryocyte amplifying factor IL-11, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, At least one factor selected from IL-11, GM-CSF, G-CSF, M-CSF, SCF, IFNs, L-IF, TNF and EPO, and a pharmaceutically acceptable carrier; At least one selected from diluents and excipients can be added to make a suitable dosage form and used as a pharmaceutical.
  • the megakaryocyte amplification factor of the present invention is useful for the treatment of certain thrombocytopenia, for example, thrombocytopenia after administration of anticancer drugs, thrombocytopenia after radiation therapy, thrombocytopenia due to megakaryocyte amplification factor deficiency, and aplastic anemia. It can be used to treat and prevent or prevent thrombocytopenia, thrombocytopenia after bone marrow transplantation, and thrombocytopenia in autoimmune diseases. It can also be used to treat leukemia. Further, it can be used as an alternative or adjuvant to platelet transfusion, or for growth culture of i.nvitro for bone marrow cells for transfusion.
  • the megakaryocyte amplification factor of the present invention can be used as an injection.
  • sucrose, glycerin, methylcellulose, a thickener such as carboxymethylcellulose, a pH adjuster for various inorganic salts, and the like can be added as additives.
  • the dosage of the megakaryocyte amplification factor of the present invention per adult dose varies depending on the age, sex, weight, symptoms, etc., but is generally about 0.1 ⁇ g to 100 mg per day. It can be administered once or several times as needed.
  • the megakaryocyte amplification factor activity of the novel protein obtained by the present invention was measured by the following two methods (a) and (b).
  • IMDM solution (Iscoes modei cation of Da 11 becco's med ium) used in the following method for preparing a bone marrow cell suspension was powdered IMDM (for 1 liter) (manufactured by Gibco, USA) 3 ⁇ 024 g of sodium bicarbonate, ⁇ -Menolecaptoethanol, 3.04 microliters, adjust ⁇ ⁇ to 7.1, then adjust to 1 liter, and further increase to 50 IU / It was prepared by adding Millirit Norrenicillin and 50 ⁇ g Z Milliliter Norest Streptomycin (both manufactured by Floraborate Lease Co., USA).
  • the femurs of 6- to 9-week-old C57BLZZ6 mice male were harvested, the upper part was cut, and 10 milliliters of 10 milliliters of IMD M solution was added. Using a plastic syringe (22 G needle), the bone marrow was pushed out vigorously into the 100 mm plastic dish from the knee joint side.
  • the bone marrow cell suspension obtained by suspending and dispersing the mice in a 2.5-milliliter IMDM solution per mouse was further subjected to the following colony assay.
  • the cell concentration was measured with a hemocytometer by staining with Trypan Blue (produced by Flora Bola Treasure Co., USA).
  • the acetyl cholesterol esterase staining solution used in the next experiment was 1.73 mM acetyl thiocholin iodide, 0.5 mM potassium ferricyanide, 5 mM sodium citrate, and 3 mM copper sulfate (all in Japan). (Manufactured by Wako Pure Chemical Industries, Ltd.), and dissolved in 400 milliliters of a 75 mM phosphate buffer of pH 6.0.
  • COS 1 cells As a culture supernatant of COS 1 cells containing IL-3, COS 1 cells (ATCCCRK165) were prepared using a plasmid in which mouse IL-3 cDNA was linked to the SV-40 promoter. With the method described in WO 88/05053, Example 2. The culture supernatant containing IL-3 expressed in the same manner was used.
  • the agar disk was transferred onto a slide glass, dried, and then fixed with 2% daltaraldehyde. After fixation, the cells were washed with phosphate buffered saline (PBS), and megakaryocytes were stained specifically with acetylcholinesterase staining solution. The number of colonies was counted as one colony consisting of 6 or more positive cells using an Olympus AHB microscope in three countries.
  • PBS phosphate buffered saline
  • the mouse bone marrow cell suspension prepared in the same manner as in the measurement in (a) above was added to diisopropylfluorophosphate (DFP) (Sigma, USA) to a final concentration of 0.4 mM.
  • DFP diisopropylfluorophosphate
  • the cell number was counted by a hemocytometer as described above.
  • CM Sepharose manufactured by Vanoremasia, Sweden
  • Adsorbed on a column (9 cm in diameter x 23.5 cm in height), 13.5 liters and 0.4 ml of the same equilibration buffer
  • 2OmM acetate buffer pH 4.0 containing M salt
  • 2M at pH 4.0 containing 0.75M salt 1OmM lysine hydrochloride
  • the adsorbed protein was eluted with 12 liters of mM acetate buffer.
  • approximately 5.5 liters of eluate was obtained as crude purified solution I containing megakaryocyte amplification factor activity.
  • Proteopeptide components contained in the culture supernatant in large amounts decreased to i% or less.
  • tissue plasminogen activator tissue plasminogen activator
  • E-1 about 200 milliliters of the same buffer containing 0.25 M salt (E-2), and the same buffer containing 0.5 M salt (E-3) To elute each. The flow rate was 200 milliliters / hour. Megakaryocyte amplification factor activity was observed in E-1, E-2, E-3 and all fractions, but megakaryocyte amplification factor with acidic isoelectric point was mainly in E-3 fraction. Recovered. That is, about 50 milliliters of the E-3 fraction was recovered as the roughly purified liquid IV at an activity recovery rate of about 10%.
  • Figure 1-(b) shows an example of the results of Q Sepharose column chromatography.
  • the crude solution IV 48 milliliters was concentrated to 3 milliliters using the above ultrafiltration hollow fiber, and this was previously concentrated to 50 mM sodium phosphate with a pH of 7.3.
  • Gel filtration was performed on a column of Sephacryl S—200 (manufactured by Pharmacia, Sweden) which had been sufficiently equilibrated with a buffer solution (diameter: 1.6; height: 900; 111).
  • the megakaryocyte amplification activity was recovered as a fraction having a peak at an eluted molecular weight of about 23 kd, i.e., a crudely purified solution V of about 25 milliliters at an activity recovery rate of 30 to 40%.
  • a crudely purified solution V of about 25 milliliters at an activity recovery rate of 30 to 40%.
  • Figure 1 shows an example of the results of Sephacryl S-200 column chromatography. Equivalent volume of purified water was added to crude solution V25 milliliter. Then, concentrated and desalted, and added with ampholine (3.5-10) (Pharmacia, Sweden) to a final concentration of 2%, followed by rotophoresis (Biorad, U.S.A.). Was carried out at a constant voltage of 12 bits for 5 hours.
  • An example of the results of isoelectric focusing is shown in Figure 1-(d). Megakaryocyte spreading activity had peaks at PH 3.5 and 4.9. ⁇ H 3. Active fractions near 5 and 4.9 were collected, respectively, to give purified samples 1 and 2 of megakaryocyte amplification factor. The total protein of purified sample 1 and purified sample 2 was 0.9 mg and 0.9 mg, respectively.
  • Table 2 shows the degree of purification in each purification step.
  • Example 2 Purification obtained in Example 2 using a Sephacryl S—200 HR (manufactured by Pharmacia, Sweden) column (diameter 1.6 cm ⁇ height 90 cm) previously equilibrated with PBS.
  • Sample 1 was developed with PBS (flow rate: 20 milliliter time), fractionated by 2 milliliters, and the megakaryocyte amplification factor activity of each fraction was measured.
  • the molecular weight of the substance was determined by comparing the elution positions of the fraction having megakaryocyte amplification factor activity and the low molecular weight marker protein kit for gel filtration (Pharmacia, Sweden). This substance was eluted with a peak near the molecular weight of 23,000.
  • the purified sample 1 obtained in Example 2 was applied to an isoelectric focusing column (1
  • Glycerol density gradient isoelectric focusing was performed using a 10-milliliter bottle (manufactured by Sho Kato, Japan) with a constant power of 3 bits for 40 hours.
  • As an amphoteric carrier 1% ampholine (Pharmacia, Sweden) was used. This substance had an isoelectric point in the pH range of 2.5 to 4.5.
  • the megakaryocyte amplification factor activity of this substance was examined by the soft agar culture method. Table 3 shows the results.
  • the Meg-POT activity of IL-16 (Genzam) was only 2.5 times higher than that of IL-13 alone, even at a high concentration of 200 ng / milliliter.
  • the purified preparation of this substance showed 14 times the activity of IL-13 alone.
  • the purified sample of this substance showed a greater number of cells per colony that strongly showed acetylcholinesterase activity.
  • the substance alone did not show megakaryocyte amplification factor activity.
  • the megakaryocyte amplifying factor activity of this substance was evaluated by a method of measuring acetylcholinesterase activity in liquid culture.
  • Figure 2 shows the results. In this method, the megakaryocyte amplification factor of this substance Child activity was demonstrated.
  • the purified sample 1 obtained in Example 2 was intraperitoneally administered to mice (C57BL male, 7 weeks old, 5 mice per group) for 5 consecutive days, blood was collected 3 hours after the final administration, and the platelet count and red blood cells were collected. The number was measured. As shown in Table 4, it was found that this substance significantly increased the platelet count at a risk factor (P) of 1% or less and exhibited a thrombopoietin effect. At this time, the number of red blood cells did not increase.
  • BSA serum serum albumin
  • Example 2 Using a Sephaacryl S-200 HR (manufactured by Pharmacia, Sweden) column (diameter 1.6 cm x height 90 cm) previously equilibrated with PBS, the purified sample 2 obtained in Example 2 was used.
  • the molecular weight of the substance was measured by comparing the elution positions of the fraction having megakaryocyte amplification factor activity and the low molecular weight marker protein kit for gel filtration (Pharmacia, Sweden). This substance was eluted with a peak near the molecular weight of 23,000.
  • Purified sample 2 was obtained using isoelectric focusing column (110 milliliters) (manufactured by Shoichi Kato Shoten, Japan) at 3 Watts of constant power. Glycerol density gradient isoelectric focusing was performed for 40 hours. As an amphoteric carrier, 1% ampholine (Pharmacia, Sweden) was used. This substance had an isoelectric point in the pH range of 3.9 to 5.9.
  • the megakaryocyte amplification activity of this substance was examined by the soft agar culture method. Table 3 shows the results. Meg—of IL-6 (manufactured by Genzym) POT activity was only 2.5 times that of IL-13 alone, even at the high concentration of 200 ng / milliliter. In contrast, the purified preparation of this substance showed 6 times the activity of IL-13 alone. Compared with the case of IL-3 alone, the purified sample of this substance showed a greater number of cells showing strongly acetylacetylcholinesterase activity per colony. This substance alone did not show megakaryocyte amplification factor activity.
  • the purified sample 2 obtained in Example 2 was intraperitoneally administered to mice (C57BL male, 7 weeks old, 5 animals per group) for 5 consecutive days, blood was collected 3 hours after the final administration, and the platelet count and The red blood cell count was measured. As shown in Table 4, it was found that this substance significantly increased the platelet count at a risk factor (P) of 1% or less and exhibited a thrombopoietin effect. At this time, the number of red blood cells did not increase. In Table 4, for Group 2, 2 ⁇ g per dose of this purified substance was added to 150 ⁇ g
  • Group B was administered as a control dissolved in PBS containing 1 ml / ml serum serum albumin (BSA).
  • Group 3 received BSA alone as a control.
  • the formulation examples of the pharmaceutical composition containing the megakaryocyte amplification factor of the present invention as an active ingredient and the method of preparing the pharmaceutical composition are shown, but the present invention is not limited to these formulation examples.
  • Purified megakaryocyte amplification factor of the present invention 1 mg Purified gelatin 20 mg Mannitol 100 mg sodium chloride 7.8 mg sodium phosphate 155.4 mg The above components are distilled for injection. Dissolve in 2 ml water and sterile vial Put in one, three and five. Primary drying at C for 0.75 Torr for 35 hours, followed by secondary drying at 3.0 ° C and a vacuum of 0.03 T0 rr for 5 hours. Manufactured. The obtained composition is dissolved in physiological saline or 500 ml of injection of glucose immediately before administration and used for intravenous drip infusion.
  • Purified megakaryocyte amplifying factor of the present invention 1 ⁇ g arubin 5 mg mannitol 25 mg sodium chloride 1.95 mg sodium phosphate 3.85 mg A vial for injection was produced in substantially the same manner as in Formulation Example 1.
  • FIGS. 11 (a) to 1-(d) are diagrams showing chromatograms and results of electrophoresis in each purification step of Example 2.
  • Fig. 11 (a) is the first-stage purified CM Sepharose chromatographic graph
  • Fig. 11 (b) is the fourth-stage Q Sepharose chromatographic graph
  • Fig. 11 (c) shows the results of the gel filtration chromatography at the fifth stage
  • Fig. 11 (d) shows the results of the isoelectric focusing at the sixth stage.
  • FIG. 2 shows that the megakaryocyte amplifying factors (purified samples 1 and 2) and the IL-6 megakaryocyte amplifying activity according to the present invention were compared with the acetylethylcholinesterase activity (AchEactivi) by liquid culture. ty) The results of evaluation by the measurement method are shown.
  • the megakaryocyte amplification factor protein of the present invention has an activity of promoting megakaryocyte amplification and increasing platelets in peripheral blood, and its activity is stronger than that of known factors having similar activities. It is. Therefore, the megakaryocyte amplification factor protein of the present invention can be effectively used alone or in the form of a pharmaceutical composition containing it as an active ingredient for the prevention and treatment of thrombocytopenia and the like. .

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Abstract

Une nouvelle protéine d'amplification de mégakaryocites, qui est pratiquement pure, est dotée d'un poids moléculaire de 23 000 ± 8 000, mesuré par filtration sur gel, et d'un point isoélectrique de 5,9 au moins, mesuré par électrophorèse isoélectrique. Sur le plan immunologique, elle se différencie de l'érythropoiétine, des interleukines 1α, 1β, 6, 7, et 11 de l'homme, ne présente aucune activité de stimulation des colonies de mégakaryocites mais permet d'activer l'amplification de ces mégakaryocites. On peut produire cette protéine en utilisant des techniques de culture de cellules et elle favorise l'amplifaction des mégakaryocites et accroît la teneur en plaquettes du sang périphérique, ce qui est efficace pour prévenir et traiter la thrombocytopénie et les affections semblables.
PCT/JP1993/000155 1992-02-07 1993-02-05 Nouvel amplificateur de megakaryocites et production WO1993016106A1 (fr)

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1994010312A1 (fr) * 1992-10-23 1994-05-11 Chugai Seiyaku Kabushiki Kaisha Codage de genes pour potentiateur de megacaryocyte
US5766581A (en) * 1994-03-31 1998-06-16 Amgen Inc. Method for treating mammals with monopegylated proteins that stimulates megakaryocyte growth and differentiation
US5795569A (en) * 1994-03-31 1998-08-18 Amgen Inc. Mono-pegylated proteins that stimulate megakaryocyte growth and differentiation

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
TW221061B (fr) * 1991-12-31 1994-02-11 Minnesota Mining & Mfg

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH04295500A (ja) * 1991-03-26 1992-10-20 Asahi Chem Ind Co Ltd 新規な巨核球増幅因子とその製法

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH04295500A (ja) * 1991-03-26 1992-10-20 Asahi Chem Ind Co Ltd 新規な巨核球増幅因子とその製法

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
BRITISH JOURNAL OF HAEMATOLOGY, Vol. 75, No. 3, (1990), N. BANU et al., "Tissue Sources of Murine Megakaryocyte Potentiator: Biochemical and Immunological Studies", p. 313-318. *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1994010312A1 (fr) * 1992-10-23 1994-05-11 Chugai Seiyaku Kabushiki Kaisha Codage de genes pour potentiateur de megacaryocyte
US5766581A (en) * 1994-03-31 1998-06-16 Amgen Inc. Method for treating mammals with monopegylated proteins that stimulates megakaryocyte growth and differentiation
US5795569A (en) * 1994-03-31 1998-08-18 Amgen Inc. Mono-pegylated proteins that stimulate megakaryocyte growth and differentiation

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AU3463493A (en) 1993-09-03

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