JPH05503302A - Megakaryocyte maturation factor - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed description of the invention] Megakaryocyte maturation factor The present invention relates to methods and pharmaceutical compositions for producing platelets. The present invention is more particularly Qualitatively, treatment of platelet disorders using factors that increase I of platelets in the circulating blood. Regarding.

The present invention also relates to pharmaceutical compositions containing factors that promote platelet production.

Light Transmission 2JIJL Multipotent hematopoietic stem cells give rise to various types of terminally differentiated blood cells. blood is , consisting of red blood cells, white blood cells, and platelets. Platelets are megakaryotic cells found mainly in the bone marrow. Derived from the fragmentation of large cells called spheres. Platelets play a major role in blood clotting and wound healing. Important role and role 6 Megakaryocytes undergo various stages of differentiation to produce mature platelets.

First, multipotent f# cells become involved in the formation of megakaryocytes, and cells and nuclei Through proliferation, a megakaryocyte precursor cell pool is formed. These progenitor cells are endogenous endoduplication diameter, highly lobulated ( aultilobula-ted) @ immature megakaryocytes characterized by micronuclei, i.e. 3 megakaryoblasts are formed. During the process of generation of mature megakaryocytes from megakaryoblasts, platelet characteristics Cytoplasmic granules containing numerous proteins are formed. Mature megakaryocytes are professionally plated. cytoplasmic extensions called proplatelee ic extension), which fragments and produces mature platelets. Ru.

Purification factors include those that stimulate the formation of mature megakaryocytes from megakaryocyte precursor cells in vitro. There are some that promote the differentiation of megakaryocytes by being stimulated. This type of factor is Granulocyte/macrophage colony stimulating factor (GM-C5F), granulocyte colony Stimulating factor (G-C5F) - erythropoietin (EPO), interleukin-3 (IL-3), interleukin-6 (IL, -6) and megakaryocyte colony Ma factor (Meg-CSF) (flofl+aan et al., Blood Ce 1ls 13.75-86 (1987); Mazur et al., Exp, Hem atol, 1i, 1128-1133 (1987); McNiece et al., Ex p, Hematol, 16.807-810 (1987); Lu et al., Br 1tJ, HenaLol, 70.149-156 (1988); l5hi bashi et al., Proc.

Natl, 8 cad, sci, U, s, ^, 86.5953-5957 (19 89)). U.S. Patent No. 4,894,440 describes megakaryocyte stimulating factor (MSF). A factor called . Purified MSF induces platelet proteoglycans in megakaryocytes. Stimulates the formation of platelet and platelet-specific proteins, such as platelet factor It is involved in the maturation of the cytoplasm of megakaryocytes, as evidenced by its ability to do so. purification It has been reported that IL-6 increases the amount of platelets in vivo (15h ibashi et al., Blood 74, 1241 (1989); 'Hill et al. J, Cl1n.

Invest, 85.1242-1247<1990)).

Thrombopoietic stimulation activity is associated with thrombocytopenia. was found in the plasma, serum and urine of human liver and kidney (HEK) MJ cells, as well as in the culture medium of human hepatokidney (HEK) MJ cells. ing. This activity is associated with thromboboetin or thrombopoiesis-stimulating factor (TSF), It is believed that this is due to factors that are considered to be important elements that support the maturation of megakaryocytes. (McDonald Ann, N, Y, ^cad, sci.

Quadruple, 1-24 (1937)), TSF derived from HE K cells was purified ( McDonald et al., J. 1. ab, Cl1n, Med, 106. l6Z-17 4 (1985)), the corresponding active substance derived from the plasma of thrombocytopenic ninjas is still unknown. Unpurified, the role of TSF in megakaryocyte formation remains to be established However, purified TSF alone does not stimulate megakaryocyte formation from progenitor cells. Itame (Lu et al., Br1tj, Ilematol, 70, 149-156 (1 988)), it is speculated that it plays a role in the later megakaryocyte molecular stage.

Suppression and reversal of megakaryocyte differentiation and maturation have also been observed.

Platelet factor IV (PF-IV) and transforming growth factor (TGF)-8 are giant Inhibits the formation of nuclear bulb precursor cells (Ish 1bash et al., Blood 69 , 1737-1741 (1987); (:ewirtz et al., J, Cl1n.

Invest, 83.1477-1486 (1989); Ran et al., Blo od 75, 1234-1239 (1990)'). Acts on microtubule formation Various compounds inhibit the formation of proplate red (Leven et al., Blo od 69, 1046-1052 (1987)), and serum-derived serine Thrombin, a sterase, induces contraction of proplate red extensions (ret-r Reverse megakaryocyte maturation by stimulating the action (Radley et al., Throibosis and) Iaemotosis 58,732- 736 (1987)).

Guinea pig megakaryocytes can differentiate in vitro under appropriate culture conditions and become blood cells. Forms long cytoplasmic extensions that are precursors of plates (Leven et al., supra; Handagama et al., JJet, Res.

48.1142-1146), these extensions called Proplate Red Furthermore, it has been observed that guinea pig platelets differentiate into small-sized anucleated cells. Ru. The formation of proplate red is an important step in the development of megakaryocytes into platelets. It's an elephant. Factors that affect this process play important roles in platelet production.

As mentioned above, it stimulates various stages of megakaryocyte differentiation and maturation and increases megakaryocyte number and A number of factors have been identified that promote this increase in magnitude. However, mature megakaryocytes The purified factor 3 that stimulates the process of further differentiation into proplate red-bearing cells is 1 Identification and identification of factors that stimulate the formation of proprelation trends, which have not been previously reported. Separation may be useful in the treatment of excessive bleeding due to platelet disorders.

The cytoplasm of mature megakaryocytes and platelets contains proteoglycans and platelet-specific proteins. Contains granules consisting of. Proteoglycans are covalently attached to the protein core It is a highly acidic polymer having at least one glycosaminoglycan. . Some proteoglycans are used to monitor the uronic acid content of glycosaminoglycans. Chondroitic acid purified from human platelets and bound to a protein core by It contained four sulfates z (01<ayana et al., BiochemJ, 233 ．． 73-81 (1986)), purified human platelet proteoglycan protein The core was sequenced (Perin et al., Biochem, J, 255.1007 -1013 (1988), Alliel et al., FEBS Letters 23 6, 123-126 (1988)).

This night's bacterium has a length of 131 amino acids and has eight Ser-gly configurations. It contained a region of 18 amino acids with repeat sequences. Iterative 5er gly array is already in the protein core region of another proteoglycan! ! Glico It was predicted that this is the binding site for saminoglycyl force. Human platelet 10 thioglycan =67 and 69 serine residues are thought to be modified with chondroitin silver sulfate. (Alliel et al., supra). Biology of human platelet proteoglycans Activity was not measured during or after purification.

PCT Publication No. W○9010060B by 5tevens et al. Three protein cores of secreted granule proteoglycans derived from the hematological cell line HL-60. The genomic and cDNA sequences for It has also been reported by ht et al. (Nuc, Δcids Res, 17.7 523 (1989)), and based on these DNA sequences, a composition of 131 amino acids was found. Molecular weight 17.60 containing a mature polypeptide and a signal peptide with 27 amino acids A protein π of 0 was assumed. This mature human secretory granule proteoglycan is It has the same amino acid sequence as that reported for platelet proteoglycans. 6 Biological studies of human secretory granule proteoglycans Activity is not described in the above-mentioned article by Stevens et al.

One of the objects of the present invention is to provide a hyperthermic therapy consisting of administering factors that promote the production of platelets. Another object of the present invention is to provide a method for treating excessive bleeding by increasing the amount of Proplate Red. It consists in the purification of factors thereby stimulating the production of platelets. Yet another object of the invention relates to the manufacture of pharmaceutical compositions containing factors that promote platelet production.

Light Encounter L Han Iichi The present invention provides a method for increasing the number of platelets, and a method for maturing megakaryocytes and promoting platelet development. The present invention relates to a method for treating platelet disorders using factors involved in platelet formation. Megakaryocytes are Mature and form proplate red, this proplate trend fragments and blood Release platelets. Changes in platelet left directly affect the amount of platelets produced. Affect.

The factor of the present invention that stimulates the formation of proplatelets from megakaryocytes stimulates megakaryocyte maturation. It is called a factor (M　M　F　). These factors increase platelet production and reduce excessive bleeding. Useful for treatment.

MkIF used for the treatment of platelet disorders is based on the amino acid sequence of natural MMF. A prokaryotic or can be a eukaryotic expression product and have a limited stability, solubility and circulation half-life. ing half-life), such as polyethylene glycol. They can be concretely modified with water-soluble polymers such as

Megakaryocyte maturation factor used alone or in combination with Ike's therapy to increase platelet volume It is possible. Another factor useful for use in combination with MMF is stem cell factor (SCF). ), GM-C3F, G-CSF, IL-3, IL-6, M e g -C3F , MSF and EPO.

The present invention provides a method for purifying factors that affect megakaryocyte maturation. MMF-containing substances One method for purifying megakaryocyte maturation factor from Contains one or more steps.

The present invention also provides methods for assaying megakaryocyte maturation factors.

This method involves incubating MMF (unpurified or purified) with megakaryocytes. and monitoring the formation of proplatelets from megakaryocytes. child In this assay, MMF stimulates the production of proplate red.

The present invention also relates to pharmaceutically acceptable compositions of purified and isolated megakaryocyte maturation factors. Ru. The present invention further provides SCF, GM-CSF, G-C3F, IL-3, IL-6, A macrokaryotic composition also comprising a pharmaceutically acceptable composition of Meg-CSFJiSF and EPO. Pharmaceutically acceptable compositions of sphere maturation factors are also provided.

Pa's, °t・ Figure 1A shows guinea pig megakaryocytes before the formation of a probe left. Shown in a stained state.

Figure 1B shows guinea pig megakaryocytes after formation of proplatelets. Shown with emsa staining.

Figure 2 shows the in vitro assay using human serum. This shows suppression of formation.

Figure 3 shows that proplate red formation is suppressed by trophic acid but not by triplicate. chymotrypsin, chymotrypsin, or trompositin. There is.

Figure 4 shows in vitro treatment with prothrombin and thrombin.

Figure 3 shows suppression of proplatelet formation in the assay.

Figure 5 shows in vitro results induced by prothrombin and thrombin. shows regression of proplate red formation.

Figures 6A and 6B show that inactivation of thrombin is a proplatelet. Effect on inhibition of formation and shrinkage of proplate pellets.

Figure 7 shows the D of human serum and pro-I thrombin that inhibits prototole formation. EAE chromatography is shown.

Figure 8 shows human serum that suppresses Propre-1 to redd formation and Pro-1 to thrombin. 5uperose-6 chromatography Figure '9 shows megakaryocytes, proplate red and the conversion of prothrombin to thrombin by platelets.

Figures 10A and 10B [J is guanidinium chloride lysate of human platelets and C Proplatelet formation in in vitro assay with HAPS lysate Shows irritation.

Figure 11 shows DEAE chroma of human platelets lysed in the presence of guanidinium chloride. showing the topography.

Figure 12 shows the concentration of MMF-III derived from a guanidinium chloride lysate of human platelets. MonoQ chromatography is shown.

Figure 13 shows MMF-I [derived from guanidinium chloride lysate of human platelets]. One minute of rose-6 chromatography is shown.

Figure 14 is derived from a guanidinium chloride lysate of human platelets. l1-chome 11F -m C4 reverse phase 5 pLca is shown.

Figure 15 shows DEAE chromatography of human platelets lysed in the presence of CHAPS buffer. It shows the graphics.

Figure 16 shows MMF-1 derived from human platelet CHAPS lysate. Shows Q chromatography.

Figure 17 shows the guanidinium chloride solution and CHAPS solution obtained by 5DS-PAGE. Analysis of lysate-derived MMF-1 [[ is shown.

Figures 18A and 18B show thrombin present at increasing concentrations, respectively. Proplay by MMF'-'''1 in in vitro assays Figure 2 shows stimulation of trett formation and inhibition of proplatelet contraction.

Figure 19 shows M　M　　　　’-”’ and MM　F for proplate red formation. A comparison of the activities of ``-''' is shown.

Figure 20 shows the activity of MMF 151 after removing chondroitin sulfate. Ru.

Figure 21 shows the blood of mice administered MMF'-'' at 4 μg/day or 20 μg/day. It shows the amount of platelets.

Figure 22 shows mice administered human recombinant IL-6 at 2 μg/day or 10 μg/day. It shows the amount of platelets in the body.

23!21 administers 20μg/day MMF 131, but 2ttgZ day I Administer L-6, or 20ttg/day 1'ii MF'-"' and 2μ Platelet levels are shown in mice treated with 3 combinations of IL-6 g/day.

Light 1! ”■1r Tomoichi Sara The present invention stimulates the maturation of megakaryocytes and the formation of proplate red, thereby The factor of the present invention related to megakaryocyte maturation factor (MMF>H) that increases the amount of circulating platelets is , even in the presence of inhibitory factors, megakaryocyte proplate red in vitro. It has the property of promoting the production of. As described in Example 2, one of said inhibitory factors is present in human serum.

MMF can be obtained from various sources such as human serum, urine, megakaryocytes and platelets. . The presence of MMF activity in megakaryocytes and platelets is illustrated in Examples 7 and 8. . However, all physiological substances that promote proplate red formation in vitro are M The term ``rMMF-containing substance that can be used as a source of MF'' refers to such physiological substances. means.

Factors that stimulate proplate red formation have never been disclosed. meganucleus Hematopoietic factors that promote the formation of spheres, such as G-C3F, GM-C3F, IL-3 and When IL-6 was tested by in vitro assay, the presence of serum inhibitory substances was detected. below did not stimulate the formation of proplate red. Therefore, the activity of these factors The activity is different from the activity of the agents of the invention.

The present invention also provides methods for assaying megakaryocyte maturation factors. This method is described in Example 1. As if it were not yet [! Alternatively, purified MMF can be incubated with megakaryocytes to remove consists of monitoring the formation of proplatelets. This method is a professional Certain proplate reds are preferably carried out in the presence of a totlet formation inhibitor. Formation inhibitors are present in human serum (see Example 2).

The present invention also relates to factors that suppress megakaryocyte maturation and progretlet formation. In this specification, these factors are referred to as megakaryocyte maturation inhibitors. Suppression of megakaryocyte maturation The substance inhibits the natural maturation of megakaryocytes to proplatelets and o has the property of stimulating contraction of proplate red extensions. Human serum is megakaryotic Suppressing bulb maturation (Example 2).

The anti-11 active substance present in human serum is an enzymatically inactive precursor of thrombin. (Example 5) Prothrombin prepared at 6μ is co-purified with prothrombin. Both thrombin and thrombin suppress the formation of propretends, but Thrombin exhibits suppressive activity at lower concentrations than prothrombin, and thrombin is found in human blood. Prothrombin in human serum is a megakaryocyte maturation inhibitor present in serum. It suppresses megakaryocytes after it is converted into nimbins. Is the isolated megakaryocyte prothrombin? (Example 6), except for thrombin and prothrombin. is a factor that suppresses proplatelet formation and increases the yield of proplatelet red. Factors that induce contraction are easily detected in in vitro assays.

The present invention also provides a method for purifying MMFcr). This method lyses human platelets and Human platelet lysate was subjected to two ion exchange chromatography steps (e.g. DE Throughout this operation, consisting of applying AE and MonoQ), Protein production from megakaryocytes in the presence of inhibitors of either serum or thrombin produced by M. The presence of MMF is detected by 1 in vitro maturation into platelets. platelet dissolves in the presence of guanidinium chloride or CHAPSMII solution (Example 8) However, other methods suitable for platelet lysis may be used, as described in Example 9. , platelet lysates obtained by either method were subjected to DEAE chromatography. Three distinct peaks of progradation/total formation activity are observed when Section 11 and Figure 15), these three peaks represent the salt concentration required for elution from the column. MMF-I, MMF-1 and MMF-I [[]. In Figure 15, MMF-1 containing fractions were assayed for proplate red formation. The physiological activities of MMF-1, MMF-n and MMF-m have not been investigated. This will be described in Example 12.

MMF-III is further purified by MonoQ chromatography (section 12 and Figure 16).

Two different biologically active forms of M, MP-I[I, have been purified from human platelets. Ru. As described in Example 10, guar chloride was added to inactivate platelet protease. When platelets are lysed in the presence of Nidinium, amino terminals starting with Y-P-T-Q MMF-I made by I having the end sequence is obtained. Platelets were collected using CHAPS [liquid present]. When dissolved in F-I[[ is obtained. Amino acid of MMF-■ derived from guanidinium chloride solution The sequence of 16 amino acids coming from the end of the protein is the amino acid sequence of purified human platelet proteoglycans. It is the same as the sequence of 16 amino acids coming from the min terminus (Hachiliel et al., supra). Reference HPerin et al., supra). Amino acid 1 of human platelet proteoglycan Thirty-one full-length sequences (AIliel et al., supra) were prepared using CHAPS lysis. The nine amino acids that match the first nine amino acids of MMF-Iff derived from It contains only internal sequences 58-67. Derived from guanidinium chloride solution MMF-In is hereafter referred to as MMF'-''. It is the same as Rikan.

MMF derived from CHAPS lysate contains the carboxylic acid of the full-length protein. A human platelet proteoglycan with a missing tip, corresponding to the terminal part. Yes, and hereinafter referred to as MMF58-1=1. Purified MMF'-'3' contains thrombin. Stimulating proplate red formation in vitro when present, thoron inhibit bottle-induced contraction of proplate red (Fig. 18A and Figure 18B).

Factors that have part or all of the amino acid sequence of human platelet proteoglycans are macrokaryotic. Having the ability to promote the maturation and formation of proplate red is due to this was not revealed until then. Human platelet proteoglycan is the first prisoner of platelets may act as a carrier to transport the vascular injury site to the site of vascular injury or as a supplementary subcomponent C1q (Okayama et al., supra). Perin et al., supra). However, human platelet proteoglycans The inviLro or invivo biological activity of the drug has not been clarified. Therapeutic benefits resulting from the administration of human platelet proteoglycans have also been described. Not yet.

The present invention has a part or all of the amino acid sequence of MMF1''31, and a mature giant It is also a megakaryocyte maturation factor that promotes the formation of proplate red from karyocytes. related. The factors described herein include bioactive peptide fragments and natural MMF The pro-H bioactive peptide contains the '-13' amino acid variation. 11 to form a protein core fragment with the ability to stimulate tread formation. Proteolytic hydrolysis of JMp1-131 was performed in situ by cellular proteases. 5 itu> or by the production of full-length purified MMF'-"'. For example, when compared based on the amount of uronic acid formed by rotease treatment, Made in MMF “-”’ is equivalent to full length MMF’-”’ +7) 7D7 rail It has tortogenic activity (Example 11).

In a preferred embodiment, MMF is a prokaryotic or eukaryotic expression product of foreign DNA. Ru. That is, MMF is preferably recombinant MMF, recombinant mouse MMF, and human MMF. This will be explained in Example 13 as MF l -1: l +. The foreign DNA to be inserted is Obtained by genomic or cDNA cloning or gene synthesis.

Expression of MMF can be carried out in prokaryotic host cells (A fungi) or eukaryotic host cells (yeast, nitrogen, insects, or occurs in mammalian cells).

The invention also relates to MMF analogs. These analogs are 1, M By manipulating the DNA sequence encoding the protein core of MF+-131, this core Generating nucleotide deletions, additions or substitutions within the code sequence, thereby creating an amino acid obtained by changing the amino acid sequence. These analogs can be prepared by means known to those skilled in the art. Manufactured in sequence.

Purified MMF having a different carbohydrate structure than natural MMF is also within the scope of the present invention. Ru. In order to obtain megakaryocyte maturation activity, glycosaminoglycans are added on MMF'-"'. It is important that side chains are present. This is purified M　M　F+　-121 Inactivated by treatment with chondroitinase ABC to remove bound carbohydrate chains (sugar H) It is clear from this (Example 11).

When carbohydrate structures change, they are called isoforms with different total charges. MMF molecules can be generated.

The MMF isoforms are separated from each other and determined by prior art isoelectric points; pneumophoresis or chromatography. - Purification by methods such as casing.

The present invention provides increased solubility, stability and/or circulating half-life compared to unmodified MMF. Chemically modified forms of MMF are also provided. Water-soluble polymer to MMF The covalent bond is between polyethylene glycol or polyethylene glycol and polypropylene. It may be a copolymer with pyrene glycol.

The polymer may be unsubstituted or substituted at one end with an alkyl group. These repairs Decorations and related modifications are described in U.S. Pat. No. 4,179,337 and are incorporated herein by reference. The specification incorporates this prior patent by reference.

The invention also relates to antibodies that specifically bind purified MMF. These antibodies are Antibodies directed against several antigenic determinants (polyclonal) or against a single determinant (monoclonal) and prepared by procedures known to those skilled in the art. These polyk Local and monoclonal antibodies can be purified, glycosylated or deglycosylated. Binds to MMF'-"' and MMFF1a-131. Antibodies against MMF , affinity chromatography to selectively remove MMF from culture media, serum or urine. Can be used in graphics. In addition, it specifically binds to MMF and can be used in vitro. Antibodies that inhibit proplatelet formation may stabilize or reduce the amount of circulating platelets. Used to treat conditions that result from excessive platelet production by reducing obtain.

The present invention describes the use of MMF alone or in combination with other therapies to treat platelet disorders. The methods and compositions of the present invention also provide methods for detecting subnormal levels of blood in circulating blood. Thrombocytopenia, a condition characterized by increased plate mass and the most common cause of abnormal bleeding It is useful in the treatment of. Thrombocytopenia is caused by (1) insufficient platelet production, (2) platelet destruction. (3) abnormal platelet distribution in the body. It's triggered. Specific disorders associated with thrombocytopenia are shown in Table 1 (Wint Robe et al., Clnical [Iematology-8th edition, pp.

1090-1127 (1981) @ Teru).

Table 1 Platelet disorders ■、Lack of platelet production A, many ＼( chemical and physical substances (ionizing radioactivity, antitumor drugs), aplastic anemia, congenital megakaryosis Globular hypoplasia, medullary wax process, and certain viral infections.

B,・self・ha Disorders caused by vitamin B12 or folic acid deficiency.

C1 mechanism Deficiency of thrombopoiesis, cyclic thrombocytopenia.

D, L]1 Many hereditary forms.

■ Promotion of platelet destruction A. In the process Idiopathic thrombocytopenic purpura, drug-induced antibodies, various hemolytic anemias, feto-maternal incompatibility sex, after blood transfusion.

B. In the process Kasabach-Merritt syndrome, thrombotic thrombocytopenic purpura, angry staining (viruses, bacteria, protozoa), massive blood transfusions.

■ Abnormal platelet distribution A, ff visceral disorder B. Hypothermic anesthesia The present invention is directed to the treatment of cancers caused by insufficient platelet production and, in some cases, increased platelet destruction. It is advantageously applied to thrombocytopenia, which can occur. Although the amount of mature megakaryocytes is normal When platelet abundance is low, platelet production is stimulated by the formation of proplatelets. MMF is used alone to increase volume.

If the lack of platelets is due to a lack of megakaryocytes, the amount of megakaryocytes and platelets To increase both, MMF is combined with one or more other hematopoietic factors, such as stem cells. Factors (SCF>, G-C3F, GM-CSF, IL-3, IL-6, Meg-C Use in combination with SF, MSF and EPO.

Platelet production deficiency is due to many processes. The most common are stem cells or giant Processes that reduce the karyocyte compartment, such as by myelosuppressive drugs or irradiation. bone marrow damage, aplastic anemia, congenital megakaryocyte aplasia, or myelodysplasia syndrome. . A purified factor called stem cell factor (SCF) is used in early hematopoietic cells, including megakaryocyte progenitor cells. Has the ability to stimulate the formation of progenitor cells. SCF is U.S. Patent Application No. 573,61 No. 6, which is incorporated herein by reference. stem cell deficiency Patients with thrombocytopenia due to thrombocytopenia receive a pharmaceutically effective amount of SCF I. It is treated by administering it in combination with MMF. Due to a deficiency in megakaryocyte mass Thrombocytopenia can be treated with therapeutically effective IG-C3F, GM-C3F, IL-3, IL -6, Meg-C3F, MSF or EPO in combination with a therapeutically effective amount of MMF Medication is achieved by administering the drug.

Platelet production deficiency occurs when the amount of mature megakaryocytes is normal, for example in megaloblastic hematopoiesis. or by inefficient thrombopoiesis, in which there are too many platelets but insufficient production of platelets. can occur.

In this case, therapeutically effective MMF I alone may be sufficient to increase platelet volume. can. Furthermore, abnormalities related to thrombopoiesis control, such as cyclic thrombocytopenia, Treat with a therapeutically effective amount of MMF.

Increased platelet destruction is due to platelet consumption (t) exceeding platelet production by megakaryocyte maturation. cause thrombocytopenia caused by via autoimmune response such as idiopathic thrombocytopenic purpura (ITP), which is characterized by increased platelet destruction. The disorder may be indicative of a decreased rate of platelet production. In such cases, therapeutically effective Treat ITP with MMF of I.

The present invention provides a therapeutically effective amount of MMF in a suitable diluent, adjuvant, solubilizer, It also relates to pharmaceutical compositions comprising together with preservatives and/or carriers. therapeutically effective amount MMF is an amount of MMF sufficient to increase the amount of circulating platelets in an animal. means. A therapeutically effective amount of MMF in a pharmaceutical composition depends on the half-life of the MMF formulation; As determined by one skilled in the art, taking into account variable factors such as the method of administration and the clinical condition to be treated. It can be done.

MMF pharmaceutical compositions can be prepared using a variety of MMF-compatible pH ranges and ionic strengths. dilution of buffers (e.g. Tris-HCl, acetate, phosphate), solubilization agents (e.g. Tween, Polysorbate), preservatives (e.g. Thime rosol, benzyl alcohol) as well as carriers (e.g. human serum alp Compositions containing MMF, including Min-3, can be treated in any manner appropriate to the condition to be treated, e.g. For example, it may be administered by continuous infusion, modified release formulation, or injection.

Preferred methods of administration will be apparent to those skilled in the art.

The present invention combines MMF with one or more other hematopoietic factors, such as SCF, G-C8F, G-C8F, Including M-CSF, IL-3, IL-6, Meg-C5F, MSF and EPO It also relates to compositions.

Megakaryocyte maturation inhibitors are used to stabilize or increase platelet mass . Excessive platelet concentrations can result in excessive blood clotting. This is caused by late night venous thrombosis and This is a condition observed in thrombosis associated with post-operative recovery. The maturation inhibitor alone or in combination with other therapeutic measures such as anticoagulants. Anticoagulant therapy currently Heparin and aspirin have been used.

In order to explain the present invention 3 in more detail, non-limiting examples are given below.

Example 1 Professional plate red In vitro assay for the formation of platelet precursors from megakaryocytes and Based on the observations of l'Jadley et al. and Leven et al. Guinea pig megakaryocytes were purified from bone marrow as described by N et al., supra. Approximately 5,00 0 megakaryocytes (measured with a hemocytometer) were mixed with 50 μM 2-mercaptoethanol and 10 0 μg/ml of mature inactivated bovine serum albumin (Sigma) was added. In 100 μl of 15coves medium (Gibco), a 96-well flat-bottom microplate was prepared. Placed in the wells of a black titer plate (Falcon>. 7% CO293 After incubation for 18 hours at 7°C, cells were treated with 10mM EDTA, 0.37% Fix the proplate levels in each well in formaldehyde and bright-field microscopy. The number of cells that had formed cells was determined. This data is based on pro plays per well. It is expressed as the number of trettes formed (PPF/well). Under these conditions, no other substances have been added to the medium. Guinea pig megakaryocytes produce cytoplasmic extensions (proplatelets) even if they do not Ru. Photomicrographs of megakaryocyte formation before and after incubation are shown in Figures 1A and 1A. It is shown in Figure 1B.

Example 2 Pro plate red deficiency in ゛ Prepared as described in Example 1 and incubated for 18 hours at 37°C in culture medium. When increasing the amount of human serum (Gibco) added to guinea pig megakaryocytes, in Addition of human serum suppressed proplate red formation in vitro (Figure 2) When I exceeded 0.03%, proplate red formation was completely suppressed.

Inhibition of proplatelet formation by human serum in this in vitro assay As shown in Table 2, the inhibition was only temporary when incubated in 0.1% human serum. Bated megakaryocytes did not form proplate red at 37°C for 18 hours. However, when incubated for 42 hours at 37°C, proplate red is formed.

Table 2 Proplatelet formation in human blood - Suppression 1! IIJL p PF /well Specific gravity 1 Specification i] Cultured in human serum 0189 Cultured in medium 371 322 Example 3 Prothrombin Pro platelets in thrombin! ” Old [ Serum-derived serine protease thrombin and 1nvitro propre The inhibition of treft formation was examined.

High-purity thrombin (Sigma) can be used as a proplatelet at concentrations below 10 ppH. showed an inhibitory effect on the formation of pores, and complete inhibition was observed at a concentration of 25 pM (Fig. 3). The inhibitory effect of thrombin is specific; the same concentration of serine brotease Rissin (derived from human urine from Calbioehem) or chymotrypsis (derived from human urine from Calbioche) was not observed.

Nanomolar levels of triazine and chymotrypsin are lethal to megakaryocytes. However, even if a similar amount of thrombin inhibits megakaryocyte differentiation, it does not affect their viability. Action: Thrombin-like serine protein derived from L mastication inhibiting peat 25 venom The tease thrombocytocin (Kirby et al., Biochemistry 18, 3564-3570 (1979), S (obtained from igma) was tested for inhibition of proplatelet formation. suppression No inhibition was observed and cell viability was maintained up to 2.8 nM.

Literature (Lottenberg et al., Methods Enzymol, 80.34 1-361 (1981)), the chromogenic substance S5-2238 (S i+ia) or Ch　o　m　oz　y　me　-P　c　a　(Bo Thrombin activity was determined by colorimetric assay using was detected. If thrombin is present in the complex mixture, specific anti-thrombin The specificity of the reaction was confirmed by the addition of the binding reagent hirudin. This assay The use of thrombin in many human serum suppressed proplate red formation. were detected, but the amounts were not sufficient to cause the degree of suppression observed. was too small (see Figure 2); in contrast, before thrombin treatment The precursor prothrombin is reported to exist in human serum at 1-2 μM) (Nano et al., Methods Enzymol, 80.286-303 ( 1981)).

Purified prothrombin was proplated in vitro when added to 2-5 nM. To inhibit tread formation (Fig. 4), complete inhibition was observed at 5 nM''C.

As with human serum, proplate levels with thrombin or prothrombin The suppression of the current is temporary.

3 in the presence of 0.35% human serum, 25 pM thrombin or 5 nM glothrombin No formation of proplate red was observed after 18 hours of incubation at 7°C. However, the suppressive effect had disappeared by the time 42:00 hours passed (Table 3).

Table 3 Proplate Red' for thrombin and prothrombin Furrow 1 wildebeest PPF/well Add 11 Kay 11 Human serum 3252 Thrombin 0227 Prothrombin 0163 None 229 312 In addition to inhibiting the formation of proplate red, thrombin and prothrombin also Dedifferentiation of platelets was induced. Example of approximately 5000 guinea pig megakaryocytes Incubate as in step 1 to form Proplate Red, and add thrombin or Proplate Red. Rothrombin was added to 66 pM or 5.7 nM, respectively, and the culture was returned to 37°C. did. The number of residual Proplate Red was counted at the time points shown in Figure 5.

Example 4 Thrombin was added to Proplate Red with approximately 40mM Tris (pH 8.0). ), 100mM NaCl, 2mM CaCI2 and 150μg/ml U Purified thrombin (12.5 μg/I) in serum albumin and a final concentration of 4.2 mM diisopropyl fluorophosphate (DFP, obtained from 5iBa) at room temperature. The mixture was reacted for 2 hours to inactivate the serine esterase activity. incubation After that, the thrombin reacted with DFP was added to 40mM Tris (pH 8,0) before use. ), was extensively dialyzed against 100 mM NaCl and 2 mM CaC12.

Inactivation of serine esterase activity is due to thrombin reacting with DFP being chromo-m This was confirmed by the fact that ozyme-Pca could no longer be used as a substrate.

Ability to inhibit proplate red formation between DFP-reacted thrombin and untreated thrombin force (Fig. 6A) and the ability to induce contraction of Proplay 1-Let extensions (Fig. 6B). Figure) was compared. Inactivated thrombin inhibits proplate red formation It shows 2% of the activity of thrombin, and in the induction of proplatelet contraction, thrombin showed 1.5% of the activity.

In addition to chemical inactivation of thrombin, neutralization of thrombin or prothrombin Add a substance to the proplatelet assay that interferes with the conversion of from to thrombin , the inhibition of proplate red formation by thrombin is avoided.

As shown in Table 4, 2.5mM EDTA, 0.04 units/ml heparin, 0 ．． Hirudin at C4 units/m+ or antithrombin■ at 0.10 units/m1 Add invitr.

Proplate red is formed.

Table 4 Many professional platelets in 0 bins Plate Lヱ Old 11- Human serum None 2 Human serum 2.5mM EDT A 1062 None None 230 Human serum None 2 Human serum 0.04 unit/end 1 Heparin 214 human serum 0.04 unit/ml Hirujin 1434 None None 255 Human serum None 24 Human serum 0.10 unit/ml antithrombin ■278 Example 5 The inhibitory active substance present in human serum of human I prothrombin is D EAE-Sepharose chromatography (Figure 7 and upperose 6) It was purified by chromatography (Figure 8). Snake venom 10 thrombinase ( Owen et al., Thrombosis Res, 3.705-714 (1973) (obtained from Sigma) by converting prothrombin to thrombin. Prothrombin I was measured and A. I checked.

Dialyze 20 ml of human serum against 40 mM Tris-MCI (pH 8,0). , a DEAE-cephalometer with a bed volume of 300 ml equilibrated with the same buffer. 2 ml/min to a space column (5 cm x 15 cm). Data bound to column Proteins were eluted with a linear NaCI gradient from O to IM in the same M buffer. Figure 7 The peak of Proplate Red inhibitory activity is shown in the prothrombin assay. It matched the peak obtained in . Pool the fractions corresponding to this peak and Concentrate, 20mM Tris-HCl, O,IM NaCl, 0.01% Polyester 5uperose-6 gel equilibrated in tyrene glycol 600 (pH 7.0) It was added to the filtration column at 0.75 ml/min. As shown in Figure 8, professional play The tread suppressing activity is due to the fact that the protein has a slightly larger molecular weight than bovine serum albumin. Elutes as a single broad peak that matches the peak obtained in the thrombin assay It was.

Example 6 to prothrombin) prothrombin to thrombin is converted to thrombin by an enzyme. It is physiologically inactive until it is converted to ion. Megakaryocytes convert prothrombin into thrombi Figure 9 shows the ability to convert into

Megakaryocytes were prepared in the same manner as in Example 1. Megakaryocytes were incubated as in Example 1. Proplatelet-forming megakaryocytes (PPF-Megs) were prepared. centrifugal Platelets were isolated from guinea pig bone marrow by fractionation. These platelets are 500xg After centrifugation, let the supernatant remain in the supernatant for 10 minutes and pelletize at 1.500Xg. . Specified number of guinea pig platelets, megakaryocytes (megs) or proplate red formation Prothrombin was added to giant FE spheres (PPP-Megs) at a final concentration of 143 μg/m. 1 and the culture was incubated for 1 hour at 37°C. Collect the culture supernatant, Thrombin was quantified using the chromophore S-2238 as described in Example 3.

Under these conditions, triggering occurs only when both cells and prothrombin are present. Produced by Longbin. Megakaryocytes and proplate red-forming megakaryocytes are prothrombotic. The conversion of thrombin to thrombin showed a similar effect, but this conversion required hundreds of megakaryocytes. I needed twice as many platelets.

Example 7 in with 0.035% human serum, 25 pM thrombin or 5 nM prothrombin. , 4°C or 37°C according to the conditions described for the in vitro assay. Incubated for 2 hours. Approximately 5,000 megakaryocytes were added and formed after 18 hours. We investigated the number of propleft lefts. Formation of proplate red is observed There wasn't. However, human serum, thrombin or prothrombin must first be Incubate with the karyocytes for 42 hours at 37°C, then add 5 μl of the reaction mixture to a fresh When transferred to fresh megakaryocyte cultures, extensive proplate red formation occurred after 18 hours. (Table 5).

Human serum, thrombin and The inhibitory activity of prothrombin was neutralized.

Table 5 The f-problem is...poor. Omihiro 11hiro i41 Human serum 0 62 327 Thrombin OO218 None 279 298 343 Megakaryocytes were incubated for 42 hours at 37°C in the same medium as in Example 1 without the presence of inhibitors. Cubated. Collect this conditioned medium and add it to CenLr1con-10 membrane. The concentrated medium was concentrated to 1/6 by centrifugation through a run filter, and 50 At 37°C with the same amount of new megakaryocytes and human serum or thrombin. Incubated for 18 hours. If megakaryocyte conditioned medium is used, Tread formation was observed, but inhibition occurred in the presence of non-conditioned medium (Table 6 ).

Table 6 ・T. Ra. No professional platelets on the wire. Not used. 205 Human serum not used 15 Human serum used 258 Thrombin not used 18 Use of thrombin 223 In these experiments, megakaryocytes were stimulated with protein levels by human serum or purified thrombin. It produces and secretes soluble factors that neutralize or functionally abolish the inhibition of rod formation. It has been proven that. These factors are called megakaryocyte maturation factors (M　M　F　　) .

Example 8 A human platelet lysate was prepared by stimulating a large number of proplatelets with human fl. By subjecting it to an in vitro proplate red formation assay, The presence of megakaryocyte maturation factor in platelets was investigated (Figure 10).

Human platelets derived from 1 donor were added to approximately 200 ml of platelet-rich plasma (PLA). Lelet rich plasa+a = PRP, purchased from HemaCare hand) containing approximately 3 to 4 1011 platelets in hand) It was obtained in the form of a plateletpheresis pack. small blood The plates were used within 24 hours after being removed from the container. This blood bag (blood b) ag) at a final concentration of 2 units/ml and incubated at 37°C. Incubated for 20 minutes. Transfer PRP to 50ml polypropylene tube Contaminant blood cells were removed by centrifugation at 120×g for 8 minutes at room temperature. Polymerize the supernatant. Transfer to a carbonate tube and centrifuge at 1,500Xg for 20 minutes to remove platelets. / It turned into a computer.

To perform platelet lysis in CHAPS, the resulting pellet was incubated at room temperature for 20 min. ．． Washed 3 times by centrifuging at 500 g, and diluted with 280 ml of Tyrode. Buffer J (137111M NaCI, 2.7mM KCI, 12mM Na HCO,,0,4s+HNaH2PO,,1s+Hgc+,,2mM CaCl 0.4% human serum albumin in 2.5.5J dextrose, pH 7.35 Wash 1+140ml of Tyrode with 2 units/m+ of avirase Wash 2 with 2 units/ml of avirase added to the buffer; 140 ml of Tiro Platelets were resuspended in Wash 3 consisting of DeM buffer and then subjected to a final centrifugation. was treated with 5 mM 3-(3-cholamidopropyl)-dimethyl on ice for 1 h. -ammonio-1-10 pansulfonate (CHAP S, Calbioc The platelets were lysed at 1.6×101° platelets/ml in 1.6×101° platelets/ml (obtained from Hera). lysate Centrifuge at 2,200Xg and dilute this external solution with 40mM Tris (pH 8). Platelet lysate was dialyzed with 150.00 exchanges (each exchange amount was 41). Clarification was achieved by centrifugation at 0×g for 60 minutes.

To perform platelet lysis in guanidinium chloride, the pellet was placed in 280 ml Tyrode's buffer with 22mM trisodium citrate and 0.4% human serum Wash with rubumin and 2#4/ml avirase 1 < pH 6,5) ; Add 2 units of 22 mM trisodium enoate to 140 ml of Tyrode's buffer. Wash 2 (pH 6,5) with 140 ml of avirase Wash 3 (pH 6) containing 22 mM trisodium citrate in Erode buffer , 5) and then subjected to a final centrifugation.

Platelet pellets were prepared in 50mM sodium acetate, 10mM EDTA, 1mM fluoride. Phenylmethylsulfonyl and 6M glycol chloride in 10mM 6-aminohexanoic acid. Add 8 x 10 g platelets/m+ to anidinium (pH 6.0) and stir gently. The mixture was solubilized at 4°C for 3 hours. This solution was mixed with 40mM Tris (pH 8), 1mM Dialyzed against PMSF while exchanging the external solution 4 times at 41/time, and Clarification was achieved by centrifugation at 0xg for 60 minutes.

Example 9 The trajectory of human beings Human platelet-derived MMF was purified using the following procedure. The presence of MMF during purification indicates the maturation Detected by in vitro proplate red formation in the presence of inhibitors .

A. Purification of MMF from guanidinium chloride-extracted platelets Guanidinium chloride of human platelets Example 8 Similarly prepared, this lysate was equilibrated with 40mM Tris-MCI (pH 8,0). 220 ml (2,6 cm x 40 cm) DEAE-Seph at a flow rate of 1 ml/min. Added to a wallose column. The column was washed with Tris M buffer and The total gradient volume was 800 m1, which was developed as a straight line NaCl gradient from 0 to IM. ), as shown in Figure 11, column fractions with proplate red forming activity tion assay, three independent peaks eluting at different NaCl concentrations, MMF-1, MMF-II and MMF-1[[ were observed.

Pool the fractions corresponding to MMF-I [[] and add 5mM sodium citrate. , 0.01% PEG600 (pH 5.0). Dialyzed pool (0,012A2.

/ml), 5mM sodium citrate, 0.01% PEG600 (Mono-Q FPLC column (0,5X5cm ) was added. The flow rate was adjusted to 0.5 ml/min. After washing with the same buffer, remove the The ram was developed with a straight Nacl gradient from O to IM, with a total gradient volume of 60 ml. ) and then washed with IM NaCl. As shown in Figure 12, professional play A broad peak corresponding to trett formation coincides with the absorption peak at 280 nm. child The fractions corresponding to the peaks were collected and the resulting pool (0,072A xs o/ml (18ml) was analyzed for purity, molecular weight and amino acid sequence.

B. Analysis of MMF purified from guanidinium chloride lysate Centricon- 200 μl of 2 ml Mono Q pool by ultrafiltration using IJ7 filtration device Concentrated to 40mM) Lis-HCl, 0.1mM NaC+, 2mM CaC 1z (pH 8°0) at a flow rate of 0.5 ml/min. ram (1 cm x 30 cm). As shown in Figure 13, the professional plate level At 230 nm, an activity peak corresponding to the cut-forming activity appears in the void volume. The peak and shoulder measured by the absorbance of . This is the MMF-m preparation Although the magnitude of MMF-1[I is heterogeneous, the activity levels of various forms of MMF-1 Add 2 ml of Mono-Q Boule to 1 ml of 0.1% trifluoro vinegar! *　 (TFA) and ultrafiltered using a Centrieon-10 filtration device. It was concentrated to 200 μm by filtration. The concentrated sample was diluted to 0.75% in 0.1% TFA. 4C-reverse phase high pressure liquid chromatography column (0,46X25cm Vydac C4 column 2147P54), and this column was A gradient of acetonitrile concentration in FA was developed.

As shown in Figure 14, the broad peak of 70 plate red formation activity was at 214 nm. This coincided with the absorbance peak at . This also represents a different form of active MMF-1 [[ It is something.

C. Purification of MMF from CHAPS lysates of human platelets as described in Example 8. CHAPS [extracted with buffer solution and normalized with 40mM Tris-HCl (pH 8,0)] As described for equilibrated platelet lysate and guanidinium chloride-extracted platelets. by DEAE-Sepharose and Mono-Q column chromatography. Purified. As shown in Figure 15, two distinct proplatelet-forming active pins MMF-I [and MMF-III are DEAE-Sepharose chromatograph Obtained by The third activity peak MMF-r is the flow-through (flo w-throBh) fraction but not detected in this preparation. It was. Activity peak near fraction 38 (MMF derived from guanidinium lysate) -I[[corresponding to] were pooled and applied to a Mono-Q column. Shown in Figure 16. As shown, the proplate red-forming activity has a broad peak in fractions 28 to 36. was eluted.

D, Analysis of purified MMF-1 [guanidinium chloride lysate by 5DS-PAGE] and an aliquot of the Mono-Q pool derived from the CHAPS lysates at 40mM of Tris-HCl (pH 8.0) over 24 hours at room temperature. was treated with chondroitinase ABC. Dry the sample at 5 peed-vae, Analyzed on a 12.5% SDS-polyacrylamide gel along with untreated samples (first Figure 7). Samples not treated with chondroitinase ABC were not detected in the gel. won. It was purified from a CHAPS lysate or a guanidinium chloride lysate. MMF-II is loaded with a considerable number of covalently bound carbohydrates that impede entry into the gel. This suggests that there was a Samples treated with chondroitinase were It migrated on 5DS-PAGE in the form of a band. This even after thorough digestion suggesting that not all carbohydrates can be removed from the protein core It is something.

Example 10 Of human blood! M derived from the decomposition product of guanidinium and CHAPS Guanidinium chloride lysate of human platelets and CHA MMF-I purified from PS lysate was purified using Biosystem. Using MS's model 470^ and model 473^ sequencers, the company's high-pressure liquid In parallel with online PTII analysis using a body chromatography system , subjected to N-terminal sequencing. Sequencing is done by comparing cycle-by-cycle chromatograms. So I went.

The following sequences were determined: MMF-1 [[: Y-P-T-Q-R-A-R -Y-Q-W-V-R-X-N-P-DCHAPS lysate-derived MMF-1 [[ :R-1-F-P-L-S-E-D-Y N-terminal amino acid determined for MMF-I from guanidinium chloride lysate The amino acid sequence is based on the N-terminal sequence of human platelet proteoglycans (AIliel et al., supra). (Perio et al., cited above). human platelet proteog MMF-I derived from guanidinium chloride lysate, which is the same as lycan −”’.

The N-terminal sequence of MMF-I from CHAPS lysate is a human platelet proteoglycan. The sequence of amino acids 58 to 66 of It was the same as the previous reference). MMF-I derived from CHAPS lysate is 74 amino acids at the carboxy terminus of the plate proteoglycan (i.e. MMF’-”’) The fragment is the same as that of MMp5g-1ff+.

Example 11 MMF'-'kol　MM)l''58-1 go I's pammf 1ko1 and MMF5m -1ff+ from the literature (Bitter and Muir, ^nal, Biochem , 4, 330-334 (1962)). I said yes. Amino acid sequence data for each form and amino acid yield obtained during sequencing The protein concentration was determined by the extinction coefficient calculated based on

MMF5g-131 contains approximately 140 μg of uronic acid per 1 μg of protein. M　M　　　p　　　-1:l　+ It contained 0 not g of uronic acid.

In its ability to inhibit thrombin-induced inhibition of propreto-nod formation MMF'-'' was then assayed (Figure 18A), and the purified thrombin was assayed for l5c. Serially diluted in oves medium or MMF=”’ preparation and added to culture wells. , and incubated for 3 hours at 37°C. MMF+-”’ is 0.1Bg/m I protein and 10 μg/ml of uronic acid, and thrombin was present at 0.35 It was present at ~1100p. Approximately 5000 megakaryocytes per well plus 18 After hours, the number of proplate reds in each well was counted.

Proplate red contraction induced by thrombin.

The purified tomato was assayed for its ability to inhibit MMF'-''t (Fig. Thrombin and MMF'-"' preparations were dispensed into culture wells as described above and incubated at 4°C. Incubated for 18 hours. The contents of the well contain Proplatelets. The number of remaining Proplate Red was counted after 10 minutes.

The inviLro platelet formation a sort described in Example 1 4. :M　M　　　　’-”’ and M　M　F5　m-131 with equivalent uronic acid Proplate red formation was examined (Figure 19).

In this assay, M　M　　　　　　　I　　　-121　and M　M　F5g-lj　1 It showed equivalent activity (per 1 Bg of uronic acid).

Bound carbohydrate (chondroitin sulfate) plays a role in the biological activity of MMF-I I looked into the role. M　M　　　　　”-’co1 derived from DEAE chromatography, 0.1 unit/ml chondroitinase A B C (Boehringer M 40mM Tris, 40mM # with or without annheim) The cells were incubated at 37° C. for 18 hours in sodium hydroxide (pH 8,0). processed Transfer M　M　F　-”’ to l5coves medium and perform proplatelet assay. It was added up to 50% by volume.

As is clear from the results shown in Figure 20, treatment with chondotroitinase ABC MMpS=-131 shows no detectable proplatelet-forming activity.

Example 12 Table 7 of DEAE ROMAT-F-T-K shows that human platelets were used in Example 8. Similarly, it was dissolved in CHAPSM solution and analyzed by DEAE chromatography in the same manner as in Example 9. The biological activities of MMF-1, MMF-ff and MMF-■ obtained by applying Showing a comparison.

Table 7 Characteristics of MMF pool derived from human platelet lysate uni plate Molecular weight, high 50,000 high 2, 50,000 Stability Sensitive to heat and pH Stable thrombin-induced roblet red shape Does it inhibit growth? From the prothrombin that was tested Thrombin-induced blockade Amide of thrombin Example 13 Ronin of Mass Hi MMF'-13' Unless otherwise specified, Maniat is et al.'s recombinant DNA Ni (Mole-cular 7.Col Spri ng Flabor Laboratory, pp, 212-246 (198 2)) was used.

A, Mouse MMF cDNA amplification and cloning cesium trifluoroacetate Bulleting protocol (Oka-yaa+a et al.-Meth, Enzyg+, 15 4.3-28 (1987)), the mouse cell line MC/9.5, namely MC/ 9 subclones (^TCCNo.

RNA was purified from CRL 8306). Mouse obesity (mast) cell secretory granules Published cDNA sequences of proteoglycans (Avraham et al., Proc. , Natl, ^cad, sei, UsA 86, 3763-3767 (198 9)), set the oligonucleotide primers M1 to M4 described below, and Synthesized using a pplied Biosystems DN^ synthesizer.

First strand cDNA synthesis was performed using MC/9.5 RNA as a template and antisense (a ntisense) Primer 5'-CTG^^TACATTGTTCCAC I started with ＾Ding CG-3° (Ml). The sequence of this primer is cDNA of mouse mast cell secreted granule proteoglycan at the 3' end of the quality coding region It is complementary to a portion of the A sequence. cDNA combination or manufacturer (Be thesda Re5erach Labo-ratories, (:aiat M-MLV reverse transcriptase enzyme This was done using raw materials.

The first Hc DN A from about 60 ng of RNA was added to the 5′ end of the coding region. An oligonucleotide that is a sense value (sense 5 trand) primer located at Reotide Brimer 5”-CT^^TCCAGACGCTGAGTGC^-3 ° Polymerase opening reaction (PCR) amplification using (Ml) (Saiki et al., 5c 239, 487-491 (1988). Ko The products of the PCB amplification were combined with nested primers and primers. 5’-GTGAGTCGACAGAGACCGTCACATTCA-3’ (M4) was used for further amplification. Primer M3 encodes mouse MMF-■ It contains the sequence 5'-CCACC-3' immediately before the sequence. An array like this It has already been shown that translation efficiency is high (Kozak, Nuc, ^Cid Res, 15.8125-8148 (1987)).

The products of PCR amplification using primers M3 and M4 were converted to B'amHI and 5al. I and vector pCD derivative pD S Ra 2 (Okaya ma et al.-Mo1. Ce11. Bio1.3, 280-298 (1983)) The ligation reaction was carried out to obtain a recombinant plasmid pDSRα2 (mu　M　MF　).

The DNA sequence of the mouse MMF insert was determined using the dideoxy method (Sanger et al., Proc. Nat　　　,^cad,sci,,USA.

1934-1938<1977). This mouse MMF array is what has been reported for mouse obese vesicle secreted granule groteoglycans (A vraham et al., supra).

B. Amplification and cloning of human l-1'' cDNA using human leukemia cells as described above. RNA was purified from the cell line (HEL, TCC No., TIB 810). Human portion Oligonucleotides were derived from the sequence of secreted granule proteoglycans (5tevens et al., supra). Creotide primers H1 to H4 were set.

1st armor c DNA combination or HEL RNA as a template and H) MMF anti sense primer From 5'-TGCTAACTAATTGCCTGGTGτ-3' (Hl) went.

PCR amplification was performed using primer H1 and 5'-GAGAGCTAGACTAAGTTGGTCA-3' (H2) It was carried out.

nestet and 5’-GCCGTAGTCGACAACC’! 'GGGAAAACCTCT The PCR product was further amplified using T-3' (H4).

The product of PCR amplification using primers H3 and H4 was purified by Hindl as described above. A recombinant plasmid was obtained by digesting with ll and 5alI and ligating to pDSRα2. pDsRα2<huMMF'-"') was obtained. Due to irreversible denaturation of supercoiled DNA Human MMF DNA was then isolated by the dideoxy method (Sanger et al., supra). The sequence was determined. The sequence of human MMF reports on human secretory granule proteoglycans. (Stevens et al., supra).

C. Expression of mouse and human MMF-I For this purpose, plasmid pDsRα2 (huMMF-1[[) or pDsRα2( MU　M　M　F　) ion) into CoS cells (Potter et al., P. roc, NaLl, ^cad, sci, UsA 81, 7161-7165 (1 984)) or Chinese hamster eggs by calcium phosphate co-precipitation. transfected into nest (CH○) cells (Higler et al., Ce11). 11, 223-232 (1977)),) Transfected CO8 cells was added to Dulbecco's modified essential medium (DMEM) supplemented with 1% fetal calf serum (FCS). ) for 2-5 days at 37°C. The conditioned medium was collected and prepared as in Example 1. In vitro proplate red formation was assayed as described.

Transfected CHOII cells were grown at low density (~105 cells/100 m DME supplemented with non-essential amino acids and 10% dialyzed FCS The cells were grown in M for 10-14 days at 37°C. Take a colony or Cells were treated with trypsin and transferred to fresh medium for an additional 10-14 days.

Collect the conditioned medium and in vitro proplate red form as in Example 1. was assayed for formation of trans. The infected CHO cell culture was grown in the presence of methotrexate, MMF expression was amplified.

Example 14 C of MMF'-+3' Designed to investigate the effect of administration of MMF'-1ff' on circulating platelet volume. The experiment was carried out in 6- to 8-week-old female Bal b/c mice (Charles R iver). All animals in one experiment were age-matched and littermates.

MMF'-'' was purified from human platelets in the same manner as in Example 1.Human recombinant IL- 6 was purified from CHO cell conditioned medium. 150mM NaCl-0.1% bovine 200 μl of MMF'-"' or IL-6 in serum albumin (BSA) at 8 o'clock Mice were injected subcutaneously twice a day at regular intervals for a total of 10 times. since the last injection After 3 hours, a small incision was made in the lateral caudal vein using a calibrated microtube. A 20 μm blood sample was taken from each animal. These samples were placed in a Sysmex machine. Electro-nic counter F-800 (To^Medical Electro-nic diluted directly into the diluent required for analysis in sCo, ). The obtained data are St. 5eheffe F test using atview512+ software program It was analyzed by Data with significance above 95% are marked with an asterisk.

MMF'-''' increases platelet volume when administered at 4 μg/day or 20 μg/day. (Fig. 21), when MM F+-1ff+ was administered at 20 μg/day, the A statistically significant 21% platelet increase was observed. IL-6 also 2μg 7 days or 10 days When administered at µg/day, it increased the amount of platelets, and at 10 µg/day, there was a statistically significant 34 % increase was observed (Figure 22).

20 μg/day of MMFl-Iff+ff combined with 2 μg of IL-6 administration on 7 days. When this test was performed, the number of platelets increased by 40%. This increase is M　M　p　+　-131 20 μg/day of IL-6 or 2 μg/day of IL-6 alone. This is statistically significant compared to the increase in weight. Under the conditions of this experiment, MMF'- Even if the amount of IL-6 used was further increased, the number of platelets increased significantly. That never happened. Other hematological parameters, such as white blood cell and red blood cell counts and Matocrit values were not affected by MMF'-'' or IL-6 treatment.

Although the preferred embodiment B1a of the present invention has been described above, there are various other variations as well. All of these variations, examples of which may occur to those skilled in the art, are included in the claims. ” shall be included within the scope of the present invention.

PP formation/well Suppression of PP formation % Number of PP formed/well PP formation/well PP formation/well PP formation/well Inhibition unit/ml (0-○) Inhibition unit/ml (o o) Prothrombin (ug/ml; ◇-◇) Generated thrombin (uq/ml) PP formation/unil PP formation/well PP formation/well (0-0) PP formation/well (o-o) A230(-> PP formation/well (0-0) 1. engineering, wear b (o 0) A280(-> PP formation/we, (o-o> PP formation/tsu=ru (0-0) FIG 21 FIG 22 FIo, 23 summary The present invention relates to a method of treating platelet disorders with factors that increase the amount of circulating platelets. do. The present invention provides methods for obtaining these factors, as well as pharmaceutical compositions containing platelet-producing factors. It also relates to products.

international search report 1--1-m-m, FCI'Z [1! R1107357Serial No, PCτ/LIS91/97:167^rt Unit 1814 The claxsg present a piurs work of mu tually fXcluffiLveindependent 1nvent 4ons as ioL ows: Note th@t the ollov ing are 1nd dependent and distinct abuse czespert4nent to the 1nvention of Gr oup X where ml ig the eratspecies a nd will be 5earched with eimiswg i4 and 1i-14in theevent thst no other f ees-repaid, Note that a 5earch of anyadditional 5 pieces within Group X r@quires payment of addittana■ al a eompogition of MMF and SCF (cl@ im Z or 6) 1bl a eompogition of N8F a nd G-C5F lc-2 am 2 or 6 n le) a co+spo *1tion of? 1MF and GW-C5F (cimiws+2 or 611dl a co+++po*1tion of MIIP an d XL-31c↓aims 2 or 617el a co+spogit 4on of MMF snd IL-6tclai+ms 2 or &+1 fl　a　composition　of　MMF　@nd　leg-C5F　 iela11m@2 or 617g1 a cow+pomition o f MMF and MSF (claim 2 or 61Bhl a co +spogltlon of MMF and EPOiclaim 2 o r　617jl　MMF　derived　fra＋＊　urine　Te1a 1 * 121; kl MMF derived fro+m megakmry oeytes + or plat book 1ets (claim 12J

Claims (1)

[Claims] 1. mammalian blood cell therapy comprising administering a therapeutically effective amount of megakaryocyte maturation factor. How to increase the number of boards. 2. A therapeutically effective amount of SCF, G-CSF, GM-CSF, IL-3, IL-6 2. The method of claim 1, also comprising administering , Meg-CSF, MSF or EPO. 3. Megakaryocyte maturation factor has part or all of the amino acid sequence of MMFI-13. 3. The method according to claim 1 or 2. 4. Claim 1 or 2, wherein the megakaryocyte maturation factor is a prokaryotic or eukaryotic expression product of a foreign DNA sequence. is the method described in 2. 5. a therapeutically effective amount of purified isolated megakaryocyte maturation factor and one or more pharmaceutically acceptable combinations containing the above adjuvants, diluents, solubilizers, preservatives or carriers; A product. 6. A therapeutically effective amount of SCF, G-CSF, GM-CSF-IL-3, IL-6 , Meg-CSF, MSF or EPO. 7. The megakaryocyte maturation factor has part or all of the amino acid sequence of MMF1-131. The composition according to claim 5 or 6. 8. Claim 5 or 5, wherein the megakaryocyte maturation factor is a prokaryotic or eukaryotic expression product of a foreign DNA sequence. is the composition described in 6. 9. Claim 5 or 6, wherein the megakaryocyte maturation factor is covalently bonded to the water-soluble polymer. Composition of. 10. The polymer is polyethylene glycol or polyethylene glycol and polyethylene glycol. selected from copolymers with propylene glycol; according to claim 9, wherein is unsubstituted or one end is substituted with an alkyl reed. Composition. 11. Incubating MMF with megakaryocytes in a proplatelet formation assay megakaryocyte maturation factor, which consists of monitoring megakaryocyte responses to MMF. Assay method. 12. Claim 1 wherein the MMF is derived from human serum, urine, megakaryocytes or platelets. The method described in 1. 13. One step of subjecting the MMF-containing substance to ion exchange chromatography A method for purifying megakaryocyte maturation factor from an MMF-containing substance, including the above. 14. 14. The method according to claim 13, wherein the MMF-containing substance is human platelets. 15. Mammalian blood due to inefficient thrombopoiesis or abnormalities in thrombopoiesis control A method for treating thrombocytopenia, the method comprising administering a therapeutically effective amount of megakaryocyte maturation factor. A method consisting of. 16. If the thrombocytopenia is due to megaloblastic hematopoiesis or cyclic thrombocytopenia. 16. The method according to claim 15. 17. A method for treating thrombocytopenia in a mammal due to increased platelet destruction, the method comprising: , administering a therapeutically effective amount of megakaryocyte maturation factor. 18. Claim 1: The thrombocytopenia is caused by idiopathic thrombocytopenia purpura. The method described in 7. 19. Thrombocytopenia in mammals due to a decrease in the cell or megakaryocyte compartment A therapeutic method for treating small children, which comprises a therapeutically effective amount of megakaryocyte maturation factor and a therapeutically effective amount of megakaryocyte maturation factor. SCF, G-CSF, GM-CSF, IL-3, IL-6, Meg-CSF, M A method comprising administering SF or EPO. 20. Claim 19, wherein the thrombocytopenia is caused by myelosuppressive drugs or irradiation. Method described. 21. 20. The thrombocytopenia according to claim 19, wherein the thrombocytopenia is caused by aplastic anemia. Method. 22. Claim 19, wherein the thrombocytopenia is caused by congenital megakaryocyte aplasia. Method described. 23. Claim 19, wherein the hypoplasia is caused by myelodysplastic syndrome. the method of.