THE USE OF HUMAN BCDF FOR PREPARING A COMPOSITION FOR TREATING THROMBOCYTOPENIA

Abstract: Human B cell differentiation factor or its biological equivalent is useful for preparing a composition for treating thrombocytopenia.

Description: The present invention relates to the use of human B cell differentiation factor (shortly "h BCDF") for preparing compositions for treating thrombocytopenia (platelet deficiency). More particularly, the present invention relates to the preparation of compositions for treating thrombocytopenia comprising human BCDF as the effective ingredient. Factors for differentiating mature B cells into antibody-producing cells in humans and mice are collectively called B cell differentiation factor (BCDF). Human BCDF possesses activity which is important in the human body. Extensive research work in the recent past has led to the determination of the DNA sequence coding for BCDF and of the amino acid sequence of BCDF (Japanese Patent Application OPI Nos. 42688/88 and 56291/88). Further, human BCDF has been successfully produced using E. coli (Japanese Patent Application OPI No. 157996/88). It has been found that human BCDF can be used as an immunotherapeutic agent which is effective in the treatment of infectious diseases and cancers (Japanese Patent Application No. 289007/87, OPI 63527/89). The present inventors also found that human BCDF can be an effective supporting agent for bone marrow transplantation therapy (Japanese Patent Application No. 289007/87 and European patent application no. 89110835.9 (=EP-A-350641). However, no report has hitherto been found on pharmaceutical effects of human BCDF on thrombocytopoiesis in order to cure platelet deficiency. It was also proposed to designate human BCDF as BSF-2 or interleukin 6 (IL-6) (Nature, 324 , 73 (1986), EMBO. J., 6, 1219 (1987)). In the present specification, however, the conventional term "BCDF" is used. Furthermore, human BCDF as used according to the present invention has no interferon activity and is thus different from IFN-β₂ having interferon activity (Published European patent application no. 0220574). Firstly, a short outline of thrombocytopoiesis and thrombocytopenia will be given and furthermore, the therapy currently applied for thrombocytopenia will be described briefly. Platelets are one kind of blood cells which play an important role for the hematostatic mechanism in living bodies. Platelets adhere and coagulate on damaged tissue and at the same time, release intracellular components which induce a series of coagulation reactions. As different forms of thrombocytopenia which causes reduction in the count of platelet, there are known hereditary thrombocytopenia, idiopathic thrombocytopenic purpura, aplastic anemia, etc. A clinically more important form of thrombocytopenia is secondary thrombocytopenia induced by general irradiation with X rays, or administration of drugs having an action of preventing hematopoiesis, or by similar treatments. In many cases, secondary thrombocytopenia is caused by chemotherapy, radiotherapy, bone marrow transplantation, etc. applied to a cancer patient which results in poor formation of bone marrow megakaryocytes. Secondary thrombocytopenia is a dangerous disease which impedes the recovery of the patient and sometimes causes death by bleeding. A therapy for thrombocytopenia which is currently most frequently applied involves platelet transfusion in order to keep the platelet count at a value of more than 20,000/µl. However, in the case of bone marrow transplantation, for example, several months are required to increase the platelet count to the normal level so that the transfusion of platelets inconsistent with HLA (human leukocyte antigen) is repeatedly required. As the result, anti-platelet antibody is formed and even after transfusion of platelets, the platelet count does not increase in many cases. In addition, there is also a danger of causing infection with cytomegalovirus, etc. Taking into account the fact that interstitial pneumonia is one of the three most frequent causes of postoperative death, it is preferred to keep the number of platelet transfusions as small as possible. Thrombocytopoiesis is the process in which megakaryocytic progenitor cells derived from multipotential stem cells are transformed into megakaryocytes via megakaryoblasts and platelets are then produced from megakaryocytes. It is considered that this physiological thrombocytopoiesis would be controlled by humoral factors. Therefore, if thrombocytopoiesis of the patient himself can be promoted by supplementing such humoral factors from an external source, bleeding would be prevented without transfusion of platelets. It can thus be expected that such administration of appropriate humoral factors would be effective for a radical treatment of thrombocytopenia. As the humoral factors that participate in thrombocytopoiesis, it is said that factors such as thrombopoietin (TPO), megakaryocyte stimulating factor (MSF), thrombocytopoiesis stimulating factor (TSF), etc. would accentuate maturation and differentiation of megakaryocyte in vivo but the substances themselves are not yet elucidated ("IGAKU-NO-AYUMI" Progress in Medicine), 143 , 528 (1987). On the other hand, it has been reported that megakaryocyte colony stimulating factor (Meg-CSF) and megakaryocyte colony potentiator (Meg-POT) are required for the formation of megakaryocyte colonies in vitro (Medical Immunology, 14 , 463 (1987)). In recent years, it has been revealed as to Meg-CSF that IL-3, GM-CSF, EPO, etc. have Meg-CSF activity ("JIKKEN IGAKU" Experimental Medicine, 5 , 822 (1987)). However, with respect to the other factors, their presence is merely suggested by various physiological activity assay systems. Sparrow et al, Leukemia Research, Vol. 11, No. 1, pp. 31-36 (1987) discloses that Interleukin-3 stimulates the in vitro proliferation and differentiation of bone marrow precursor cells. This article mentions that a second factor which can be found in mouse lung conditioned medium or serum-free P388D1 cell conditioned medium, called megakaryocyte-potentiator, per se does not stimulate colony formation, but acts in synergy with interleukin-3 to increase the cell size. The article does not, however, give any information about the nature or about the process of producing and isolating this factor. The amino acid sequences, DNA sequences and other parameters of these factors are, of course, also unclear. Therefore, it has not yet been possible to industrially produce pure products appropriate to be used as drugs. More important, no report is found to the fact that the administration of these factors, in vivo would increase the count of platelets in peripheral blood. There has not been found any factor having an activity of stimulating thrombocytopoiesis and promoting the platelet production of the patient himself when administered to the patient. Accordingly, an object of the present invention is to provide a composition for promoting thrombocytopoiesis of a patient suffering from thrombocytopenia and thus providing an agent for treating thrombocytopenia. As a result of extensive investigations to solve the foregoing problem, the present inventors have found that (1) human BCDF can augment megakaryocytic colony formation in vitro synergistically with IL-3, etc. and can induce maturation and differentiation of megakaryocytes by itself, and (2) in vivo administration of human BCDF can increase the platelet count in peripheral blood. Thus the present invention has been accomplished. The present invention is directed to the use of human B cell differentiation factor or its biological equivalent for preparing a composition for treating thrombocytopenia. The human BCDF in accordance with the present invention has the following amino acid sequence (I) or (II) shown, for example, in Japanese Patent Application OPI Nos. 42688/88 and 56291/88 and Japanese Patent Application No. 289007/87. Amino acid sequence (I): or, Amino acid sequence (II): Amino acid sequence (I) is that of human BCDF and amino acid sequence (II) is a polypeptide having a structure obtained by adding one Ala to human BCDF at the N-end thereof (hereafter referred to as human Ala-BCDF). However, human BCDF used in the present invention has not necessarily the structure shown by the amino acid sequence (I) or (II) described above. In the present invention there may be used biological equivalents of h BCDF which are derivatives of amino acid sequence (I) or amino acid sequence (II), obtainable by amino acid substitution, amino acid deletion, amino acid insertion or amino acid addition, which derivatives have human BCDF activity. That is, those having a structure formed by adding one or a plurality of amino acids to human BCDF at the N-end and/or C-end thereof and those having a structure in which one or a plurality of amino acids in the human BCDF structure are replaced by other amino acids can also be used as the human BCDF of the present invention, as long as they have human BCDF activity. Preferably, human BCDF or human Ala-BCDF is used. The content of the human BCDF or its biological equivalent in accordance with the present invention is 0.0001 to 100 wt%, preferably 0.1 to 1.0 wt%, in the composition for treating thrombocytopenia. Furthermore, the composition for treating thrombocytopenia of the present invention comprising as the effective ingredient the human BCDF may also contain a usual stabilizer such as serum albumin and an excipient such as mannitol. Furthermore, the composition for treating thrombocytopenia of the present invention may also contain, as auxiliary agent(s) one or more cytokines other than human BCDF, for example, IL-3, IL-1, IL-4, IL-5, G-CSF, GM-CSF, M-CSF, EPO and Meg-CSF, in addition to the human BCDF. When such auxiliary agent(s) is/are incorporated, the effects of the composition for treating thrombocytopenia can be increased synergistically. This is because IL-1, IL-3, IL-4, IL-5, G-CSF, GM-CSF, M-CSF, EPO and Meg-CSF can potentiate the promotion of hematopoietic functions. The amount of these auxiliary agents to be added is not particularly limited but may be in a range of 0.0001 to 200000 wt%, respectively, based on the human BCDF as 100 %. As already stated, the addition amount of these auxiliary agents is not necessarily limited to the range described above but may be appropriately determined depending upon conditions and age of the patient. The auxiliary agents such as IL-3 need not always be administered as a drug together with human BCDF at the same time. That is, these auxiliary agents may be administered at an appropriate time prior to or after administration of the composition for treating thrombocytopenia containing human BCDF as the effective ingredient. Of course, the composition for treating thrombocytopenia produced by the present invention may be used in combination with platelet transfusion therapy. The composition for treating thrombocytopenia may be administered as an intravenous injection or as an intramuscular or subcutaneous injection. That is, the composition may be administered in any mode. For preparing medical compositions of human BCDF, the methods described in Japanese Patent Application Nos. 289007/87 and 147594/88 can be applied. Human BCDF used in the present invention may be any BCDF produced by human T cells, B cells or fibroblasts by known methods (Proc. Natl. Acad. Sci., USA, 82, 5490 (1985) or by culturing transformants obtained by transforming an appropriate host such as E. coli, yeast, monkey cells (COS cells), hamster cells or bacteria by a recombinant DNA comprising a gene coding for human BCDF and a suitable vector. Furthermore, human BCDF purified from blood or urine samples can also be used. With respect to details of these production methods, reference is made to Japanese Patent Application OPI Nos. 115024/86, 42688/88, 56291/88 and 157966/88. The composition for treating thrombocytopenia produced in the present invention containing human BCDF as the effective ingredient accelerates maturation and differentiation of megakaryocytes and increases the platelet count in peripheral blood. By these activities, the composition is effective for treatment of a patient suffering from thrombocytopenia, especially a cancer patient after chemotherapy, radiotherapy and bone marrow transplantation therapy and a patient with weak resistance to viral infection upon platelets transfusion. Example 1 Acceleration of megakaryocyte colony formation by human BCDF Bone marrow cells were prepared from the femurs of C57BL/6 mouse in a conventional manner. Among the bone marrow cells, 7.5 x 10⁴ of non-adherent cells were cultured in 1 ml of Iskove's modification of Dulbecco's medium containing 1 % bovine serum albumin, 360 µg/ml of iron-saturated human transferrin, 0.98 µg/ml of cholesterol, 100 U/ml of penicillin-streptomycin and 0.3 % agar. To the medium were supplemented human BCDF having various concentrations as shown in Table 1 and 2.5 % pokeweed mitogen stimulated murine spleen cells conditioned medium (simply referred to as PWM-SCM; containing Meg-CSF activity). After 7 days in culture at 37 °C, the agar discs were transferred to glass slides and fixed with 2 % (v/v) glutaraldehyde. Megakaryocyte colonies were counted after histochemical staining for acetylcholinesterase (AchE). The results are shown in Table 1. Furthermore, 20 U/ml of mouse Il-3 (manufactured by Genzyme Co., Ltd.) was supplemented in place of PWM-SCM.The results are shown in Table 2. In the case of human BCDF alone, the ability of forming megakaryocyte colonies was poor but human BCDF concentration-dependently induced the formation of megakaryocyte colonies in the copresence of PWM-SCM or mouse IL-3 having Meg-CSF activity. A similar effect was noted also with human bone marrow cells. That is, compared to the addition of human IL-3 (100 U/ml) alone, the count of megakaryocyte colonies increased by 2.5 times by allowing human BCDF (100 ng/ml) to coexist with human IL-3 (100 U/ml). Example 2 Promotion of maturation/differentiation of megakaryocytes by human BCDF Mouse bone marrow cells prepared by the method of Example 1 were inactivated by intrinsic cholinesterase and 1 x 10⁵ of cells were cultured in 0.2 ml of serum-free liquid medium containing various concentrations of human BCDF. After incubation at 37 °C for 4 days, the count of megakaryocytes in the bone marrow cells was determined by the count of cells positive to AchE which is an enzyme specific to mouse megakaryocyte. Furthermore, the degree of maturation/-differentiation was examined using as indices the diameter of megakaryocyte and the AchE activity. As shown in Tables 3 and 4, human BCDF concentration-dependently increases the diameter of megakaryocyte and the AchE activity by itself. Further in the single cell culture system where isolated single megakaryocytes were set up in 1 µl of liquid medium for one day, the diameter of megakaryocytes was significantly increased by the addition of 200 ng/ml of human BCDF, as shown in Table 5. The foregoing results reveal that human BCDF directly acts on megakaryocytes to induce maturation/differentiation of megakaryocytes. Liquid culture, determination of AchE activity and single cell culture were carried out in accordance with the methods described in BLOOD, 67 , 1512 (1986) and J. Clin. Invest., 79 , 286 (1987). Example 3 Maturation of mouse bone marrow megakaryocytes by administration of human BCDF in vivo and increase of platelet count in peripheral blood. Human BCDF, 5 to 10 µg, was dissolved in 100 µl of 1 % mouse serum containing PBS. The solution was intraperitoneally administered to 4 to 7 C57BL/6 mice (male, 6 to 7 weeks age) twice a day in 12 hour intervals for 5 days. As the control group, human BCDF inactivated by heat treatment at 100 °C for 40 minutes was similarly administered. Four hours after the final administration, the femur was removed from the mice. The femur was fixed with 10 % formaline, dehydrated, embedded in plastic and longitudinally cut in sections and stained with hematoxylin-eosin to examine the effect of human BCDF on bone marrow megakaryocytes. As the result, as shown in Table 6, the number of megakaryocytes did not increase significantly as compared to the control group but the diameter of megakaryocytes significantly increased in the group administered with human BCDF. Thus this fact showed BCDF-induced stimulation of megakaryocyte maturation of megakaryocyte. In a similar manner, 0.5 to 10 µg of human BCDF was intraperitoneally administered to 4 to 8 mice for 5 days. Four hours after the final administration, blood was collected from the heart by a syringe treated with EDTA and the platelet count was measured with a blood autoanalyzer (manufactured by Coaltar Co., Ltd.). As shown in Table 7, the count of platelet increased dependently on dose of human BCDF. Furthermore, as the experimental result of collecting blood with passage of time, the effect of human BCDF became remarkable on day 4 after the administration, as shown in Table 8. Example 4 Recovery of platelet count in peripheral blood of mice with thrombocytopenia by administration of human BCDF in vivo . Human BCDF, 70 µg (10 µg/mouse/day x 7 days) was continuously administered to five (5) DBA/2 mice (female, 8 week age) by an osmosis pump manufactured by Alza Co., Ltd. and implanted subcutaneously. Peripheral blood was collected from the heart of mice with passage of time and the platelet count in blood was measured with an automatic hematological analyzer (Toa Medical Electronic Co., Ltd.). As compared to the control group administered with human albumin (80 ± 6 x 10⁴/µl), the platelet count was 97 ± 14 x 10⁴/µl in the group administered with human BCDF for 7 days, showing a statistically significant increase. Furthermore, when human BCDF inactivated by heat treatment at 100 °C for 40 minutes was administered, no significant increase of the platelet count was noted. The effect of continuous perfusion with human BCDF was also noted in mice with thrombocytopenia induced by administration of a chemotherapeutic agent. That is, in mice in to which 150 mg/kg of 5-FU was intravenously injected, the platelet count increased in the group subcutaneously administered with human BCDF by an osmosis pump from 5 hours after administration of 5-FU, compared to the control group. This result was shown in Table 9. Further in mice irradiated with 4.0 Gy X ray, a significant acceleration of recovery of the platelet count was also noted in the group subcutaneously administered with human BCDF by an osmosis pump after irradiation with X ray, compared to the control group. This result was shown in Table 10. Example 5 Increase of platelet count in peripheral blood in monkey by administration of human BCDF in vivo Human BCDF was dissolved in 1 % heat-inactivated autologous serum containing PBS. The solution was subcutaneously administered twice daily to cynomolgus monkeys (Macaca fascicularis, female, weighing 2.8 to 3.8 kg) in a dose of 2 ml each/time in 12 hours intervals every day for 14 consecutive days. Blood was collected from the vein and the blood cell count was measured with an automatic hematological analyzer (Toa Medical Electronic Co., Ltd.). As shown in Table 11, the platelet count increased with passage of time by administration of human BCDF in doses of 5 µg/kg/day and 10 µg/kg/day (administered by dividing into two portions a day) and increased by 2 to 2.3 times at maximum of the level prior to the administration. The increase was reverted to the original level prior to the administration in about 2 weeks after the administration. Also by administration in a dose more than 20 to 80 µg/kg/day, the platelet count similarly increased by 2.2 to 2.5 times. The platelet derived from BCDF-administered monkey showed normal coagulation ability. Furthermore, by morphological examination of bone marrow, stimulation of bone marrow megakaryocytopoiesis was noted by administration of human BCDF. On the other hand, no increase of the leukocyte count was noted in a dose of 5 to 40 µg/kg/day, as shown in Table 11 . For increasing the number of leukocytes such as granulocytes administration in a dose greater than 80 µg/kg/day was required. Furthermore, any increase in the erythrocyte count was not found in a dose of 5 to 500 µg/kg/day. Such a specific platelet-increasing activity of human BCDF was also recognized in the case of intravenous administration. As stated above, it has been revealed that human BCDF exhibits the activity of increasing the platelet count also in monkey by administration of human BCDF in vivo . Human BDCF was subcutaneously administered every day from Day 0 to Day 13 (dividing into two portions a day).

Claims: The use of human B cell differentiation factor having the following amino acid sequence (I) or (II), or of a biological equivalent thereof, for preparing a composition for the treatment of thrombocytopenia, wherein said biological equivalent thereof is a derivative of said acid amino sequence (I) or (II), obtainable by amino acid substitution, amino acid deletion, amino acid insertion or amino acid addition, which derivative has human BCDF activity, Amino acid sequence (I) : Amino acid sequence (II) : The use according to claim 1,wherein the composition further comprises in addition to human BCDF one or more auxiliary component(s) selected from at least one of interleukin 3, interleukin 1, interleukin 4, interleukin 5, granulocyte colony stimulating factor, granulocyte-macrophage colony stimulating factor, macrophage colony stimulating factor, erythropoetin and megakaryocyte colony stimulating factor. The use according to any of the claims 1 or 2, wherein said human B cell differentiation factor is produced in a prokaryote.

Inventors: AKIYAMA, YUKIO, HIRANO, TOSHIO, DR., ISHIBASHI, TOSHIYUKI, KIMURA, HIDEO, KISHIMOTO, TADAMITSU, OKANO, AKIRA, AKIYAMA, YUKIO, C/O CENTRAL RESEARCH LABORATORIES, OKANO, AKIRA, C/O CENTRAL RESEARCH LABORATORIES
Applicants: AJINOMOTO CO., INC., KISHIMOTO, TADAMITSU
Application year: 1990
Main classification: A61K37
IPC codes: C07K14, A61K38, A61P7