JPH07103041B2 - Malignant tumor treatment adjuvant - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial field of use] The present invention relates to a therapeutic adjuvant for suppressing / reducing side effects after administration of a platinum complex antineoplastic agent.

[Background of Technology and Prior Art] Treatment of malignant tumors includes surgery, chemotherapy and radiation therapy. In recent years, the therapeutic effect has been improved due to the development of drugs used for chemotherapy and advances in surgical techniques and the like. However, there are few anti-malignant tumor agents that show remarkable therapeutic effects and few side effects, and the dose is limited, so that it is not possible to completely eliminate the malignant tumor. There is also a frequent relapse. Among the side effects, there are some which show remarkable toxicity to hematopoietic organs and various organs and cause organ damage or serious infectious diseases.

Platinum complex anti-neoplastic agents have excellent anti-tumor activity, (B.
Rosenberg et al., Nature, 205, 698 (1965)), which has the highest antitumor activity, cisdiaminedichloroplatium (cisplatin, CDDP) is a urinary tract malignant agent that has been difficult to treat with conventional chemotherapy. It has been reported to exert extremely high therapeutic effect on tumors, bladder tumors and gynecologic tumors (Merrin. CE Cancer Treat. Rep., 63, 1579.
(1979)). However, it is extremely toxic to the kidney and hematopoietic organs, and causes serious side effects such as renal failure, thrombocytopenia, leukopenia, and anemia, which is a major clinical problem.

On the other hand, a colony stimulating factor is a factor that stimulates the differentiation and proliferation of mammalian hematopoietic stem cells, and has acted on monocyte-macrophage colony stimulating factor (M-CSF) and granulocyte-monocyte stem cells to date. There are four known types of factors (GM-CSF), factors that act on granulocyte stem cells (G-CSF), and factors that act on pluripotent stem cells (Multi-CSF). Human macrophages colony-stimulating factor has been purified, and its protein and gene structure has been clarified (JP-A-64).
-22899), and its utility against granulocytopenia has been clarified, and it is highly expected as a medicine (Moto
yoshi Ket al, Experimental Hematology, vol 14, 1069
-1075 (1986)), clinical studies have already been conducted and its safety has been confirmed and it has been clarified that there are few side effects (Motoyoshi K et al, Immuno-biology, vol 172, 205-
212 (1986)).

However, human monocytes against the above-mentioned side effects of anti-neoplastic agents
The availability of macrophages colony-stimulating factor remains unexplored.

[Object and Summary of the Invention] As a result of research on a suppressor / reducer of side effects of CDDP, which has the highest antineoplastic activity among platinum complex antineoplastic agents, human monocyte-macrophage colony-stimulating factor was found to be a platinum complex antineoplastic agent. The present invention has been completed by finding that the renal damage and hematopoietic disorder caused by the agent are extremely reduced and that the mortality rate in the acute toxicity test of the platinum complex antineoplastic agent is reduced.

The present invention uses human monocyte-macrophage colony stimulating factor as an active ingredient. It is a therapeutic aid for the treatment of malignant tumors by administering platinum complex anti-malignant tumor agents. Further, the present invention is a side effect inhibitor of a platinum complex anti-malignant tumor agent containing human monocyte-macrophage colony stimulating factor as an active ingredient. Furthermore, the present invention is an inhibitor of renal disorders and / or hematopoietic disorders caused by administration of platinum complex anti-malignant tumor containing human monocyte-macrophage colony stimulating factor as an active ingredient.

[Technical Structure of the Invention] The human monocyte-macrophage colony stimulating factor (hereinafter referred to as human M-CSF) used in the present invention is a known method (JP-A-63-198700, JP-A-63-250400). Bulletin,
It was prepared by freeze-drying what was purified according to JP-A-64-22899. For example, human M-CSF derived from human urine was prepared by the method described in JP-A-63-250400 as follows. That is, an anti-human M-CSF antibody obtained by immunizing a rabbit with purified human M-CSF was dialyzed in a 0.1 M phosphate buffer (pH 7.0) to prepare a 20 mg / ml concentration. 200 ml of the antibody solution
Was washed with distilled water and 0.1 M phosphate buffer in advance.
After adding to 100 g of formyl-cellulophane and stirring at room temperature for 2 hours, adding 700 mg of sodium cyanoborohydride and further stirring for 16 hours, formyl-cellulophane and anti-human M-CSF antibody were bound to form an antibody-bound support. Prepared. After binding, the mixture was washed with 0.2 M Tris-hydrochloric acid buffer, 200 ml of Tris buffer containing 500 mg of sodium cyanoborohydride was added, and the mixture was stirred at room temperature for 4 hours to inactivate unreacted groups. The antibody-bound support was then washed extensively with 0.02M phosphate buffer containing 0.5M NaCl. The antibody-bound support bound 29.5 mg of anti-CSF antibody per gram of support. Next, 1,000 L of healthy human urine was concentrated by an ultrafiltration concentrator, desalted, and then adsorbed on DEAE-cellulose to remove non-adsorbed contaminants, and eluted with a 0.3 M NaCl solution, to the eluate. Human M-CSF was eluted by adding sodium chloride to a concentration of 0.5M, and sodium chloride was added to the eluate to a concentration of 0.5M to prepare a solution containing human M-CSF. The specific activity of this human M-CSF was 2 × 10 ⁵ units / mg. This solution containing human M-CSF (500 ml in total) was added to 100 g of the antibody-supported support, and the mixture was stirred overnight at 10 ° C. or lower and subjected to batch chromatography. After stirring, filter with a glass filter to collect the antibody-bound support, and add 0.5 MNa
The antibody-bound support was washed extensively with 0.02 M phosphate buffer containing Cl. After washing, 500 ml of 0.2M vinegar buffer (pH 2.5)
Was added and the mixture was stirred at 10 ° C. for 1 hour to elute human M-CSF. After adjusting the pH of the eluate to 7.0, the eluate was concentrated and desalted with an ultrafiltration membrane to obtain about 10 mg of purified human M-CSF. Purified human M-CSF
Has a specific activity of 5.2 × 10 ⁷ units / mg, and its purity by SDS-PAGE is
It was over 90%. The physicochemical properties of the obtained human M-CSF are as follows.

a) Molecular weight A homodimer consisting of the same subunit, which has a molecular weight of 70,000 to 90,000 daltons as measured by sodium dodecyl sulfate polyacrylamide gel electrophoresis and is dissociated with a reducing agent to eliminate the biological activity. The molecular weight measured by sodium dodecyl sulfate polyacrylamide gel electrophoresis is 35,000-45,000 daltons.

b) Amino acid sequence of subunits The subunit proteins that compose homodimers 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). Here, x represents an arbitrary 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-Grn-
Gru-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-Val-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 dichroic 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 .

As described above, human M-CSF derived from human urine can be expressed in advance without waiting for the manifestation of various disorders due to the administration of the platinum complex antineoplastic agent such as renal disorder, hematopoietic disorder, and toxicity. The drug can be administered immediately after administration of the antineoplastic agent or in combination with it in order to prevent it. In addition to human M-CSF derived from human urine, human M-CSF purified from the culture medium of various human cells may be used.

Human M-CSF is usually intravenous, intraarterial, intramuscular, subcutaneous,
It can be administered by parenteral administration such as intraperitoneal administration. Formulations for administration include injections and infusions,
These preparations can be prepared by a method known per se. For example, human M-CSF can be added to an appropriate buffer solution, subjected to aseptic filtration, aseptically filled into a glass vial, sealed, and optionally lyophilized to prepare a preparation.

The stability of human M-CSF is remarkably improved especially when it is formulated with human serum albumin and / or gelatin. The amount of human serum albumin or gelatin used is preferably 100 times or more the weight of human M-CSF.

Cisplatin (CDDP) is a typical example of the platinum complex anti-neoplastic agent targeted by the present invention. Besides this, carboplatin (CBDA), 254-
The therapeutic adjuvant of the present invention can also be applied to platinum complex anti-malignant tumor agents such as S and spiroplatin (DACCP). The dose of the platinum complex anti-neoplastic agent may vary depending on the patient's age and symptoms, but is usually 15 to 35 mg / mm ² (body surface area). The dose of human M-CSF for this may vary depending on the age and symptoms of the patient, but is usually 4 × 10 ^{4 to} 160.
× 10 ⁴ units / Kg body weight / day, preferably 16 × 10 ⁴ to 80 × 10 ⁴ units / Kg body weight / day.

[Examples] Examples of the present invention using human M-CSF are shown below.

Example 1 Effect of human M-CSF to reduce acute toxicity of platinum complex antineoplastic agent (1) Preparation of therapeutic adjuvant of the present invention (hereinafter referred to as "the present agent") In a 20 mM phosphate buffer solution of pH 7.2, Human M-CSF derived from human urine
And each protein shown in Table 1 was added, and the human M-CSF concentration was 10 μg / m 2.
This preparation was prepared by aseptically filtering it with a nitrocellulose-based sterile filtration membrane, and aseptically filling and sealing 1 ml in a glass vial.

(2) Stability of this drug The stability of this drug was measured by measuring the M-CSF activity using a soft agar method using mouse bone marrow cells. The results show that, as shown in Table 1, the biological activity of this drug containing human serum albumin or gelatin at 1 mg / ml or more is maintained at 70% or more at the start of the test (immediately after the production), and is considered stable.

(3) Acute toxicity-reducing effect of this drug CDDP1 platinum complex antineoplastic agent was added to 10 C ₃ H / HeN mice per group.
9 mg / Kg body weight (LD ₅₀ equivalent) was intraperitoneally administered. From the next day, the dose of human serum albumin added at 5.0 mg / ml was 100 × 10 ⁴ units / Kg ・ body weight, 500 × 10 ⁴ units / Kg ・ body weight, 1000 × 10 ⁴ units / Kg ・ body weight 5 After continuous intravenous administration for 7 days, the mortality of the mice was measured 7 days later.

As a result, as shown in Table 2, administration of this drug markedly reduced the mortality rate, and markedly reduced the toxicity of this product by the platinum complex anti-neoplastic agent. This decrease in mortality was dependent on the increased dose of this drug.

From this example, it is known that the present agent is effective as a therapeutic aid for reducing the toxicity of the agent when used in combination with administration of a platinum complex antineoplastic agent.

Example 2 Effect of the present agent on hematopoietic disorders and renal disorders caused by administration of platinum complex anti-malignant tumor agent Among the present agents obtained in the same manner as in Example 1, 5 mg of gelatin was added to perform the experiment.

The action of this drug on hematopoietic disorders and renal disorders caused by platinum complex anti-neoplastic agents was examined by the following method. The platinum complex antineoplastic agent cisplatin was intraperitoneally administered to C ₃ H / HeN consisting of 5 animals at a dose of 4 mg / Kg · body weight / day for 2 consecutive days. Cisplatin was administered this drug 320 × 10 ⁴ Units / kg · body weight gelatin 5mg added from the next day administered for 5 days in a continuous ip once daily. 1 day, 3 days, 5 days, 7 days after cisplatin administration
On the 9th, 9th, 11th and 14th days, the effects on the hematopoietic disorders and renal disorders were examined by measuring the white blood cell count, platelet count, serum BUN (urea nitrogen) amount and creatinine amount in the peripheral blood. The results are shown in FIGS. 1, 2 and 3.

In FIG. 1, the horizontal axis represents the number of days after CDDP administration, and the vertical axis represents the white blood cell count (× 10 ² / mm ³ ), Is the drug administration group, Indicates a control group.

In FIG. 2, the horizontal axis represents the number of days after administration of CDDP, and the vertical axis represents the platelet count (× 10 ⁴ / mm ³ ), Is the drug administration group, Indicates a control group.

In both figures, * indicates that there is a significant difference at a risk rate of 5%.

Peripheral blood leukocyte counts and platelet counts in mice treated with this drug were suppressed and their recovery was promoted compared with the non-administered group, and the effect of the platinum complex antineoplastic drug of this drug on hematopoietic disorders was observed. It was In addition, the increase in serum BUN and creatine levels was milder than that in the non-administered group, and it was clarified that this drug has an effect on renal damage.

[Effects of the Invention] (1) The present invention provides a therapeutic adjunct that reduces the side effects of platinum complex anti-malignant tumor agents and enables the effective use of the anti-malignant tumor effects.

(2) The present invention provides a therapeutic adjuvant that suppresses renal disorders and hematopoietic disorders caused by the administration of platinum complex anti-malignant tumor agents and enables effective use of the anti-malignant tumor effects.

(3) The present agent can prevent the side effect of the tumor agent by administering the agent simultaneously with or immediately after the administration of the platinum complex anti-neoplastic agent.

[Brief description of drawings]

FIG. 1 is a graph showing the leukocyte recovery-promoting action of CDDP-administered mice, and FIG. 2 is a graph showing the platelet recovery-promoting action of CDDP-administered mice.

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Hajime Yokota 2-5-1-406 Shirokane, Minato-ku, Tokyo (72) Inventor ▲ Yoshi ▼ Katsuo Taba 1-37-15 Minamigai, Higashiyamato-shi, Tokyo (56) ) Reference JP-A-1-207244 (JP, A) International Publication 87/06954 (WO, A)

Claims (4)

[Claims] 1. An inhibitor of renal damage caused by administration of a platinum complex anti-malignant tumor agent, which comprises a human monocyte-macrophage colony stimulating factor as an active ingredient. 2. An acute toxicity reducing agent resulting from administration of a platinum complex anti-malignant tumor agent, which comprises a human monocyte-macrophage colony stimulating factor as an active ingredient. 3. The renal disorder inhibitor according to claim 1 or 2, wherein the human monocyte-macrophage colony stimulating factor is a human urine-derived human monocyte-macrophage colony stimulating factor having the following physicochemical properties. Or acute toxicity reducing agent: a) Molecular weight A homodimer composed of the same subunit, the molecular weight measured by sodium dodecyl sulfate polyacrylamide gel electrophoresis is 70,000 to 90,000 daltons, and the organism is dissociated with a reducing agent. The molecular weight of the subunit whose activity has been lost was measured by sodium dodecyl sulfate polyacrylamide gel electrophoresis to be 35,000 to 45,000 daltons; b) Amino acid sequence of the subunit. The subunit proteins constituting the homodimer are shown below.
It has a 214 amino acid sequence, and the 122nd and 140th asparagine each have an N-glycoside binding site represented by asparagine (Asn) -x-threonine (Thr) / serine (Ser), and x is arbitrary. The amino acid of 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-Grn-
Gru-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 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 dichroic spectrum The far-UV external CD spectrum measured by a circular dichroism disperser has a wavelength of 208n.
It has minimum peaks at m and 222 nm, respectively, and contains α-helix structure; e) Thermal stability No biological activity is lost by heating at 60 ± 0.5 ℃ for 60 minutes; f) Infrared absorption spectrum Wavenumber 1680 cm ^-1 strength absorption wave number 1200 cm ^-1 and 1130 cm ^-1
It has an infrared absorption spectrum showing moderate absorption at 1540 cm ^-1 , 1430 cm ^-1 and 1070 cm ^-1 . 4. The renal disorder inhibitor according to claim 1, wherein the human monocyte-macrophage colony stimulating factor is administered at a dose of 16 × 10 ⁴ to 80 × 10 ⁴ units / kg body weight / day. Or an acute toxicity reducer.