JPH0749375B2 - Myelodysplastic syndrome therapeutic agent - Google Patents

Description

TECHNICAL FIELD The present invention relates to a therapeutic agent for myelodysplastic syndrome containing human monocyte-macrophage colony stimulating factor (hereinafter referred to as human M-CSF) as an active ingredient.

[Technical background and conventional technology]

Myelodysplastic syndrome is a disease in which qualitative and quantitative abnormalities are observed in one or more of three hematopoietic systems of erythroid system, granulocyte and monocyte system, and platelet system. Qualitative and quantitative abnormalities are widely observed in bone marrow and peripheral blood such as macrocytosis, ring-shaped iron blasts, megaloblastic erythropoietic hematopoiesis, neutrophil hematopoiesis and megakaryocyte disorders, and chromosomal abnormalities. The clinical course is characterized by anemia, infection due to abnormal production of phagocytes and dysfunction, and bleeding due to thrombocytopenia and impaired platelet function. Myelodysplastic syndrome has 1) unresponsive anemia (RA), 2) unresponsive anemia and ringed sideroblast increase (3) unresponsive anemia and blast increase (RAEB), 4) chronic myelomonocytic monocytes depending on the condition. It can be divided into 5 types of inflammatory leukemia (CMML) and 5) blast crisis RAEB. Patients of all types become myelogenous leukemia, which is a fatal disease after several months and years of follow-up. Convert. Myelodysplastic syndromes larger differences by disease type, Ara-C minor therapy, although VD ₃ therapy is being performed, the outcome is not always good, it has been desired to establish a useful therapy . Among colony stimulating factors, which are one of hematopoietic factors, there is a factor (M / CSF) that acts on monocyte-macrophage stem cells, and its protein and gene structure have been clarified (Japanese Patent Laid-Open No. 64-22899). . This human M-CSF also acts on mature human monocyte-macrophage to promote its functional activation and production of various cytokines (Motoyoshi K et al. 17: 68-71 (19).
89)), and clinically granulocytopenia (Motoyoshi K et al., Ex.
perimental Hematology Volume 14, 1069-1075, 1986) and bone marrow transplant (Masaoka T et al Bone Marrow Transplantati
on.3: 121-127 (1988), the utility is clarified, and the expectation as a medicine is great. The safety of this human M-CSF has already been confirmed in clinical trials, and it has been revealed that there are almost no side effects (Motoyoshi
K et al lmmunobiolgy 172, 205-212, 1986). But,
The availability of human M-CSF as a therapeutic agent for myelodysplastic syndrome has been left unstudied.

[Objective and Summary of the Invention] Myelodysplastic syndrome is a malignant and serious disease that transforms into a fatal myelogenous leukemia within a few months and a few years as described above. Currently, clinically useful therapeutic methods and drugs are Absent. The present invention uses human M-CSF for myelodysplastic syndrome,
As a result of investigation as a therapeutic agent thereof, it was found that administration of human M-CSF causes reduction and disappearance of blast cells and restoration of normal leukocyte and erythrocyte count, which are the most problematic in myelodysplastic syndrome. Was completed.

The present invention is a therapeutic agent for myelodysplastic syndrome containing human M-CSF as an active ingredient. Human M-CSF includes human urine and human M
-It is possible to use human M-CSF prepared from the culture medium of CSF-producing cells or the culture medium of human M-CSF gene recombinant cells.

[Technical Structure of the Invention] Human M-CSF according to the present invention can be prepared by a known method (JP-A-64).
-22899 gazette), and was freeze-dried and prepared. That is, an anti-human M-CSF antibody obtained by immunizing a rabbit with purified human M-CSF was added with 0.1 M phosphate buffer (p
It was dialyzed in H7.0) and adjusted to a concentration of 20 mg / ml. 200 ml of the antibody solution was added to 100 g of formyl-cellulofine previously washed with distilled water and 0.1 M phosphate buffer, and the mixture was stirred at room temperature for 2 hours and then sodium cyanoborohydride.
700 mg was added and the mixture was further stirred for 16 hours, and formyl-cellulofine and anti-human M-CSF antibody were bound to prepare an antibody-bound support. After binding, the mixture was washed with 0.2 M Tris-hydrochloric acid buffer solution, 200 ml of Tris buffer solution containing 500 mg of sodium cyanoborohydride was further added, and the mixture was stirred at room temperature for 4 hours to inactivate unreacted groups. Next, the antibody-bound support was added with 0.5 M NaCl.
It was thoroughly washed with 0.02 M phosphate buffer containing The antibody-bound support bound 29.2 mg of anti-CSF antibody per gram of support. Next, 1000 L of human urine was concentrated with an ultra-concentrator and desalted, then adsorbed on DEAE-cellulose to remove non-adsorbed contaminants, and eluted with 0.3 M NaCl solution to give 0.5 M in the eluate.
Human M-CSF by adding sodium chloride to the concentration
A solution containing was prepared. The specific activity of this human M-CSF was 2 × 10 ⁵ units / mg. This solution containing human M-CSF (total amount 500 ml) was added to 100 g of the above antibody-bound support, and the mixture was stirred overnight at 10 ° C. or lower and subjected to batch chromatography. After stirring, pass through a glass filter to collect the antibody-bound support, 0.02M containing 0.5M NaCl.
The antibody-bound support was thoroughly washed with phosphate buffer. After washing, add 500 ml of 0.2M acetate buffer (pH 2.5), 10 ℃, 1
After stirring for time, human M-CSF was eluted. The pH of the eluate
After adjusting to 7.0, it is concentrated and desalted with an ultrapermeable membrane, and human MC
The SF fraction was obtained. This fraction was subjected to high-performance liquid chromatography using a linear concentration gradient of acetonitrile 0-100 (pH 2.0) containing 0.1% trifluoroacetic acid on a reverse phase column of Hi-Pour 214TP (Vydac, diameter 2.2 x 25 cm) to obtain human M-CS.
F was collected and freeze-dried to obtain 3.2 mg of human M-CSF. The specific activity of purified human M-CSF was 1.4 × 10 ⁸ units / mg, and the purity by SDS-PAGE was 96% or more. The physicochemical properties of the obtained human M-CSF are as follows.

a) Molecular weight It is a homodimer composed of the same subunits, and the molecular weight measured by sodium dodecyl sulfate polyacrylamide gel electrophoresis is 70,000 to 90,000 daltons. The molecular weight of the unit measured by sodium dodecyl sulfate polyacrylamide gel electrophoresis is 35,000 to 45,000 daltons.

b) Amino acid sequence of subunits The subunit proteins constituting the homodimer are shown below.
It has an amino acid sequence of 214 to 238, and the 122nd and 140th asparagine are asparagine (Asn) -x.
-Has a typical N-glycoside binding site represented by threonine (Thr) / serine (Ser), where x represents any amino acid.

Glu-Glu-Val-Ser-Glu-Tyr-Cys-Ser-His-Met-
lle-Gly-Ser-Gly-His-Leu-Gln-Ser-Leu-Gln-
Arg-Leu-lle-Asp-Ser-Gln-Met-Glu-Thr-Ser-
Cys-Gln-lle-Thr-Phe-Glu-Phe-Val-Asp-Gln-
Glu-Gln-Leu-Lys-Asp-Pro-Val-Cys-Tyr-Leu-
Lys-Lys-Ala-Phe-Leu-Leu-Val-Gln-Asp-lle-
Met-Glu-Asp-Thr-Met-Arg-Phe-Arg-Asp-Asn-
Thr-Pro-Asn-Ala-lle-Ala-lle-Val-Gln-Leu-
Gln-Glu-Leu-Ser-leu-Arg-Leu-Lys-Ser-Cys-
Phe-Thr-Lys-Asp-Tyr-Glu-Glu-His-Asp-Lys-
Ala-Cys-Val-Arg-Thr-Phe-Tyr-Glu-Thr-Pro-
Leu-Gln-Leu-Leu-Glu-Lys-Val-Lys-Asn-Val-
Phe-Asn-Glu-Thr-Lys-Asn-leu-Leu-Asp-Lys-
Asp-Trp-Asn-lle-Phe-Ser-Lys-Asn-Cys-Asn-
Asn-Ser-Phe-Ala-Glu-Cys-Ser-Ser-Gln-Asp-
Val-Val-Thr-Lys-Pro-Asp-Cys-Asn-Cys-Leu-
Tyr-Pro-Lys-Ala-lle-Pro-Ser-Ser-Asp-Pro-
Ala-Ser-Val-Ser-Pro-His-Gln-Pro-Leu-Ala-
Pro-Ser-Met-Ala-Pro-Val-Ala-Gly-Leu-Thr-
Trp-Glu-Asp-Ser-Glu-Gly-Thr-Glu-Gly-Ser-
Ser-Leu-Leu-Pro-Gly-Glu-Gln-Pro-Leu-His-
Thr-Val-Asp-Pro-Gly-Ser-Ala-Lys-Gln-Arg-
Pro-Pro-Arg-Ser-Thr-Cys-Gln-Ser-Phe-Glu-
Pro-Pro-Glu-Thr-Pro-Val-Van-Lys-c) Isoelectric point The isoelectric point (pI) measured by polyacrylamide gel isoelectric focusing method and sucrose density gradient isoelectric focusing method is 3.
1 to 3.7.

d) Circular dichroism spectrum The far-UV external CD spectrum measured by a circular dichroism disperser has a wavelength of 208n.
It has a minimum peak at m and 222 nm, respectively, and contains an α-helix structure.

e) Thermal stability No biological activity is lost by heating at 60 ± 0.5 ° C for 60 minutes.

f) Infrared absorption spectrum wavenumber 1680 cm ^-1, the intensity absorbed in 1200 cm ^-1 and 1130 cm ^-1, wave number
It has an infrared absorption spectrum showing moderate absorption at 1540 cm ^-1 1430 cm ^-1 and 1070 cm ^-1 .

Human M-CSF exhibiting such physicochemical properties can usually be administered by parenteral administration such as intravenous, intraarterial, intramuscular, subcutaneous and intraperitoneal administration. Formulations for administration include
Examples include injections and infusions, and these preparations can be prepared by a method known per se. For example, human M-CSF can be prepared by adding human M-CSF to an appropriate buffer, aseptically filling, aseptically filling in a glass vial, sealing, and freeze-drying if necessary.

Human M-CSF has the property of being adsorbed on glass, plastic, sterile membranes and the like. This adsorption can be prevented by a surfactant, human serum albumin or gelatin, and the stability thereof can be remarkably improved by formulating with these. The concentration of the surfactant when formulated is 0.2 μg / m
l or more, human serum albumin, gelatin concentration is 1mg / m
l or more is desirable.

The dose of human M-CSF for myelodysplastic syndrome may vary depending on the age and symptoms of the patient, but is usually 0.4 μg to 16 μg.
μg / kg body weight / day, usually 1.6 μg / 8 μg / kg body weight / day.

An example of the present invention using human M-CSF obtained by the above method will be described below.

Example-1, human M-CS for patients with myelodysplastic syndrome
Therapeutic effect of F (1) Method for preparing a therapeutic agent for myelodysplastic syndrome of the present invention (hereinafter referred to as "the present agent") Human M-CSF and a stabilizer shown in Table 1 were added to a 20 mM phosphate buffer solution having a pH of 7.2. Then, human M-CSF was prepared at a concentration of 100 μg / ml. Aseptic with a nitrocellulose-based aseptic membrane,
Aseptically fill 1 ml into glass vials. This product was prepared by freeze-drying and sealing.

(2) Stability of this drug The stability of this drug was determined by measuring the M-CSF activity by a soft agar method using mouse bone marrow cells. The results are shown in Table 1. The surfactant Tween 80 was prepared at a concentration of 10 µg / ml or more, and human serum albumin or gelatin was prepared at a concentration of 1 mg / ml or more. At 70% or more at the start of the test (immediately after production), it was stable.

(3) Therapeutic effect of this drug on patients with myelodysplastic syndrome (1) The therapeutic effect of this drug on patients with myelodysplastic syndrome is examined by measuring changes in the number of myeloblasts, normal white blood cells, and red blood cells in peripheral blood did. A 40-year-old patient with myelodysplastic syndrome was prepared with a buffer containing human serum albumin 5 mg / ml. As an active ingredient, human M-CSF, 1.6 μg / kg, body weight / day, continuously 14
Intravenous drip was administered every day. After administration, the number of blast cells and leukocytes in peripheral blood were measured over time, and the therapeutic effect of this drug on myelodysplastic syndrome patients was examined.

As shown in Figure 1, the number of blast cells in peripheral blood was
100 / mm ³ in which it was but decreased after administration of this drug, from about 40 / mm ³ next 10 days after the administration, became 10 / mm ³ in 30 days after administration. The number of myeloblasts was 0 / mm ³ even after 100 days.
Met. The number of normal leukocytes and neutrophils in peripheral blood after administration of this drug was 700 / mm ³ and 500 / mm ³ before administration, indicating leukopenia, but it was 750 / mm ^{3 on the} 10th day after administration. And 500 / m
m ³ was 900 / mm ³ , and 600 / mm ³ on the 30th day, and 1800 / mm ³ and 900 / mm ³ on the 90th day, which were almost restored to normal values.

In FIG. 1, the horizontal axis represents the period expressed in days, and the vertical axis represents the number of blast cells (●-●), the number of white blood cells (○-○), and the number of neutrophils (△-△) in the peripheral blood. . From these results, it became clear that this drug is useful as a therapeutic agent for myelodysplastic syndrome.

Example-2 Therapeutic effect of human M-CSF on pediatric myelodysplastic syndrome patients The therapeutic effect of this agent on pediatric myelodysplastic syndrome patients was investigated using the same agent obtained in the same manner as in Example-1. 2.4 μg / k of this drug as an active ingredient in a 3-year-old myelodysplastic syndrome patient
Intravenous drip infusion was performed for 9 consecutive days at g / body weight / day. After treatment, peripheral leukocyte count, neutrophil count, erythrocyte count, and blast cell ratio in bone marrow cells before and after administration of this drug were measured to examine the therapeutic effect of this drug.

As shown in Fig. 2, the number of red blood cells before administration of this drug was 184 × 10 ⁴ / mm ³ , the number of white blood cells was 2000 / mm ^3, and the number of neutrophils was 180 / mm ³ , but the number of red blood cells was 208 × 10 5 days after the start of administration. Increased to ⁴ / mm ³ , white blood cell count 2600 / mm ³ and granulocyte count 290 / mm ^3, and 10 days after the start of administration, red blood cell count 308 × 10 ⁴ / mm ³ , white blood cell count 3100 / mm ³ and granulocyte Number 530
/ mm ³ increased. In addition, the proportion of blast cells in the bone marrow cells before the start of administration was 23%, but it was 7% after the administration, showing a marked decrease in the proportion of blast cells.

In FIG. 2, the horizontal axis represents the period expressed in days, and the vertical axis represents the white blood cell count (◯ − ◯), neutrophil count (Δ−Δ), and red blood series (□ − □) in the peripheral blood. From these results, it became clear that this drug is useful as a therapeutic agent for myelodysplastic syndrome.

〔The invention's effect〕

(1) It is possible to provide a drug having an effective therapeutic effect on the disease by reducing or eliminating the number of blast cells of myelodysplastic syndrome which is a refractory disease and increasing normal cells in the peripheral blood.

[Brief description of drawings]

Fig. 1 is a graph showing changes in peripheral blood leukocyte count, neutrophil count, and myeloblast count in patients with myelodysplastic syndrome treated with this drug, and Fig. 2 in children with myelodysplastic syndrome treated with this drug. It is a graph which shows a peripheral blood red blood cell count, a white blood cell count, a neutrophil count, and a ratio of a bone marrow blast cell.

Claims (1)

[Claims] 1. A therapeutic agent for myelodysplastic syndrome comprising human monocyte-macrophage colony stimulating factor as an active ingredient.