JPH02264729A - Adjuvant for malignant tumor therapy - Google Patents

Abstract

PURPOSE:To provide a malignant tumor therapy adjuvant inhibiting side effects caused by the administration of a platinic complete malyignant tumor remedy and capable of effectively utilizing the malignant tumor-resistant activity of the remedy by using a human monocyte-macrophage colony-stimulating factor (human M-CSF) as an active ingredient. CONSTITUTION:A therapeutic adjuvant inhibiting remal disoders and hematopoietic organ disoders caused by the administration of a platimic complex malignant tumor remedy (e.g. cisplatin or carbolplatin) contains a human urine- originated human M-CSF or the human M-CSF purified from the culture solution of various human cells as an active ingredient. The administration amount of the human M-CSF is 4X10<4>-100X10<4> units/kg body weight/day, preferably 16X10<4>-80X10<4> units/kg body weight/day based on the administration amount of the malignant tumor remedy in a dose of 15-35mg/mm<2> (body surface area). The preparation of the adjuvant together with human serum albumin and/or gelatin remarkably improves the stability of the human M-CSF.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Field of Application] The present invention relates to a therapeutic adjuvant that suppresses and alleviates side effects after administration of a platinum complex antineoplastic agent.

[Technical Background and Prior Art] Treatments for malignant tumors include surgical therapy, chemotherapy, and radiation therapy. In recent years, advances in the number of drugs used in chemotherapy and surgical techniques have improved the therapeutic effects thereof. However, among the anti-malignant tumor drugs, there are very few that show remarkable therapeutic effects and have few side effects. In some cases, the disease has not yet reached its peak, and reoccurrence is often observed.In addition, some side effects exhibit significant toxicity to the hematopoietic system and various organs, causing organ damage or serious infections.

Platinum complex antineoplastic agents have excellent antitumor activity, (
B. Rosenberg et al., 5ature, 2
05.698 (1965)), the highest anti-l1
Cisdiamine dichlorobratium (with ifi tumor activity)
Cisplatin, CDDP) is used to treat malignant tumors of the urinary tract and bladder 1 [IEl! It has been reported that it has an extremely high therapeutic effect on cancers and gynecological tumors (Herrin, C.E., Ca.
ncerTreat, Red,, 63.1579
(1979)).

However, it is extremely toxic to the kidneys and hematopoietic organs, causing serious side effects such as renal failure, thrombocytopenia, leukopenia, and anemia, and has become a major clinical problem.

On the other hand, the colony stimulating factor
It is a factor that stimulates the differentiation and proliferation of monocyte-macrophage colonies.
Factor acting on granulocyte-monocytic stem cells (GM-C8F)
, a factor that acts on granulocytic stem cells (G-C8F), a factor that acts on pluripotent stem cells (Hulti-CSF)
Four types are known.

Human macrophage colony-stimulating factor has been purified, and its protein and genetic structure have been clarified (
JP-A No. 64-22899), and its usefulness for treating granulocytopenia has been revealed, and there are great expectations as a medicine (Hotoyoshi et al., Exp.
er+1ienta+ t+e+eato+ogy,
vol 14.1069-1075 (1986))
, clinical trials have already been conducted and its safety has been confirmed and it has been revealed that there are almost no side effects (o
otoyosh+ et al, l1iuno-
biolooy, vol 172.205-212
(1986)).

However, the possibility of utilizing human monocyte-macrophage colony-stimulating factor for the above-mentioned 81 effects of anti-malignant 1ull agents remained unexamined.

[Objective and Summary of the Invention 1] As a result of research into agents for suppressing and mitigating the side effects of CDDP, which has the highest anti-malignant tumor activity among platinum complex antineoplastic agents, human monocyte-macrophage colony stimulating factor was found to be a platinum complex antineoplastic agent. The present invention was completed based on the discovery that the kidney damage and hematopoietic organ damage caused by platinum complex antineoplastic agents can be significantly alleviated, and the mortality rate in acute toxicity tests of platinum complex antineoplastic agents can be reduced.

The present invention is a therapeutic adjuvant for the treatment of malignant tumors by administering a platinum complex antineoplastic agent, which contains human monocyte-macrophage colony stimulating factor as an active ingredient.

Further, the present invention is an agent for suppressing side effects of a platinum complex antineoplastic agent containing human monocyte-macrophage colony stimulating factor as an active ingredient. Furthermore, the present invention is an agent for suppressing kidney damage and/or hematopoietic organ damage caused by administration of a platinum complex anti-malignant tumor containing human monocyte-macrophage colony stimulating factor as an active ingredient.

[Technical configuration of the invention] The human monocyte-macrophage colony stimulating factor (hereinafter referred to as human M-C8F) used in the present invention can be obtained by a known method (JP-A-63-198700, JP-A-63-25).
0400, JP-A No. 64-22899) was prepared by freeze-drying. For example, human M-C8F derived from human urine was prepared as follows by the method described in JP-A-63-250400. That is, anti-human M-C8F was obtained by immunizing rabbits with purified human M-C8F.
The C8F antibody was dialyzed in 0.1 M phosphate buffer (DH7,0) and prepared at 20 q/l at 1111 degrees. 200 II of the antibody solution was added to 100 g of phosphate buffer, which had been previously washed with distilled water and 0.1 M phosphate buffer. 7, added to olumilucerulofine and stirred at room temperature for 2 hours, then added 1 g of sodium cyanoborohydride 700I, and stirred for a further 16 hours to bind olumilucerulofine and anti-human M-C8F antibody, resulting in antibody binding support. A body was prepared. After binding, 0.
Tris washed with 2M Tris-HCl buffer and further containing 500q of sodium cyanoborohydride! lIi solution 20
0- was added and stirred at room temperature for 4 hours to inactivate unreacted groups.

The antibody-bound support was then thoroughly washed with 0.02M phosphate buffer containing 0.5M NaCl.

The antibody-bound support has 29.51 Rg of anti-C per gram of support.
It was bound to 8F antibody. Next, 1゜00OL of healthy human urine
After concentrating with an ultrafiltration concentrator and desalting, it is adsorbed onto DEAE-cellulose to remove non-adsorbed contaminants, resulting in 0.
Elute with 3M NaCl solution, add sodium chloride to the eluate to a concentration of 0.5M, and add human M-C8F.
was eluted, and sodium chloride was added to the eluate to a concentration of 0.5M to prepare a solution containing human M-C8F. The specific activity of this human M・-〇SF is 2×105 units/
It was η. This solution containing human M-C8F (total amount: 500 g) was added to 100 g of the above antibody-bound support, and the mixture was stirred overnight at 10° C. or lower to perform batch chromatography. After stirring, filter through a glass filter.
The antibody-bound support was collected and thoroughly washed with 0.02M phosphate buffer containing 0.5M NaCl. After washing, add 0.2M acetate buffer (pf12.5
) 500ae was added and stirred at 10°C for 1 hour to elute human M-C8F. After adjusting the pH of the eluate to 7.0, it was concentrated and desalted using an ultrafiltration membrane to obtain purified human M, -C8F.
About 10q was obtained. The specific activity of purified human M-C8F is 5.2
X10' units/q, purity by 5DS-PAGE method is 9
It was 0% or more. The physicochemical properties of the obtained human M-C8F are as follows.

a) A homodimer consisting of subunits with the same molecular weight, which has a molecular weight of 70,000 to 90,000 daltons as measured by sodium dodecyl sulfate polyacrylamide gel electrophoresis, and whose biological activity can be determined by dissociating with a reducing agent. The molecular weight of the deleted subunit measured by dodecyl Wiim sodium polyacrylamide gel electrophoresis was 35°00.
0 to 45,000 Daltons.

b) Amino acid sequence of subunits The subunit proteins constituting the 21 homozygotes have the following 214 to 238 amino acid sequences, with asparagine at positions 122 and 140 being asparagine (Asn)-x-threonine ( Thr)/Serine (3e
r) has a typical N-Gly'cyto binding site, denoted 't'.

Here, X represents any amino acid.

Giu-Gtu-Val-8er-Glu-Dingyr-C
ys-8er-H1s-F4et-11e-Gly-3
er-Gly-old 5-1Cu-Gin-3er-1au
-Gin-8ra-Laυ-l I e-Asp-3e
r-G l n-Hat-G Iu-Thr-3er-
Cys-G l n-1l e-Thr-Phe-G
1u-Phe-Val-^5p-Grn-Gru-G
ln-Leu-Lys-Asp-Pro-Vat-Cy
s-Tyr-Leu-Lys-Lys-^1a-Phe
-Leu-Leu-Vat-Gin-Asp-11e-
Het-Glu-Asp-Thr-Net-＾rg-p
he-Ara-Δ5p-Asn-Thr-Pro-^S
＾Ia-11e-Ala-11e-Va l-G I
n-Lau-G In-G lu-Leu-8er-1
eu-Arg-Leu-Lys-3er-Cys-Ph
e-Tbr-Lys-85p-Tyr-Glu-Glu
-His-^Sp-tys-AI a-Cys-Va
l -Arg-Thr-Phe-Tyr-G 1u-T
h r-P r.

-Leu-G l n-Leu-Leu-G lu -
Lys-Val-Lys-Asn-Val-P
he-^5n-Glu-Thr-'Lys-^sn-t
au-teu-asp-tys-Asp-rrp-As
n-11e-Phe-3er-Lys-Asn-Cys
-Asn-^s n -S e r -P h e-A
1a-G 1u-Cys-3er-8er-G In
-Asp-Va 1-Va l -Thr-Lys-P
TO-Asp-CVS-ASn-Cys-Lau-T
yr-P rO-L￥ S-A I a -11e-P
ro-3er-8er-Asp-Pro ro-A I a
-8er-Va I -3er-Pro ro-II i
5-Gln-Pro-Leu-Ala-Pro-8e
r-Hat-Ala-Pro-Vat-^1a-G l
y-Leu-Thr-Trp-G! u-Asp-
8e r-G I u-G I y-Tir-G I
u-Gly-3er-8er-Leu-Leu-Pro
-Gly-Giu-Gln-Pro-Leu-Former S-Dingh "-Val-ASD-Pro-GIV-8 (31"-
Aia-IJS-Gln-8rg-Pro-Pro-^
rg-3er-Dinghr-Cys-Gln-3er-Ph
e-Glu-Pr.

-Pro-G l 1l-Thr-Pro-Va l
-Val -LyS-C) Isoweighted polyacrylamide gel @weighted electrophoresis and sucrose! 5 Isoelectric point measured by frugal gradient isoelectric focusing method (1) I
) is 3.1 to 3.7.

d) Circular dichroism spectrum The far ultraviolet CD spectrum measured by a circular dichroism dispersion meter has a wavelength of 2.
There are minimum peaks at 08 nl and 2220-, respectively, which contain an α-helical structure.

e) Thermal stability No loss of biological activity when heated at 60±0.5°C for 60 minutes.

t) Infrared I @ collection spectrum wavenumber 1680e* 1200α-1 and 1130c
Intensity absorption at 11, wave number 4540a-' 1430
It has an infrared absorption spectrum showing moderate absorption at 1 and 107107 O'.

As described above, human M-C3F derived from human urine does not exhibit 01 effects such as various disorders caused by administration of platinum complex antineoplastic agents, such as kidney disorders, hematopoietic disorders, and toxicity. In order to suppress the expression, the anti-malignant tumor agent can be administered immediately after administration or in combination. In addition to human M-C8F derived from human urine, human M-C8F purified from the culture fluid of each human epithelium may be used.

Human M-C8F can usually be administered non-gently, such as intravenously, intraarterially, intramuscularly, subcutaneously, or intraperitoneally. Preparations for administration include injections, infusions, and the like, and these preparations can be prepared by methods known per se. For example, human M-C8F is added to an appropriate buffer solution, filtered aseptically, and aseptically filled into a glass vial. The preparation can be prepared by sealing and, if necessary, lyophilizing.

In particular, the stability of human M-C8F is significantly improved by formulating it with human serum albumin and/or gelatin. The amount of human serum albumin and gelatin used is
It is desirable that the weight is 100 times or more that of human M-C8F.

The platinum complex anti-malignant mts agents targeted by the present invention include:
Cisplatin (CDDP) is a typical example, but in addition to this, carpoplatin (CBDA),
254-8. The therapeutic adjuvant of the present invention can also be applied to platinum complex antineoplastic agents such as sviroplatin (DACCP). The dosage of platinum complex anti-malignant 811 agent may vary depending on the patient's age and symptoms, but it is usually 15 to 3
5q/JIII3 (body surface area). The dose of human M-C8F may vary depending on the age and symptoms of the patient, but is usually 4X10 units to 160X10'/
Kg Body weight per day Preferably 16X10'~80X104
Unit/Kg body weight/H.

[Example] Examples of the present invention using human M-C8F are shown below.

Example 1 Effect of human M-C8F on reducing gold 1 (11) Preparation of the therapeutic adjuvant of the present invention (hereinafter referred to as F1 solution) A 20-H phosphoric acid!I solution of pH 7.2 was added with human urine-derived Human M-C8F and each protein in Table 1 were added, and human M-C8F was added.
A preparation solution with a C8FI degree of 10 μg/d was prepared, aseptically filtered with a nitrocellulose-based sterile filtration membrane, and filled and sealed aseptically in a glass vial for 11 days to prepare the main solution.

(Stability of the 21 main rules) The stability of the main rules was determined by measuring M-C8F activity using the soft agar method using mouse bone marrow cells.
The biological activity of the main ingredients added above is maintained at 70% or more of that at the start of the test (immediately after manufacture) and is considered stable.

(3) Acute toxicity reduction effect according to the main rules Platinum complex antineoplastic agent CDDP 191115F/Kg・body weight (L
D so equivalent amount) was administered intraperitoneally. From the next day, 1115.0J of human serum albumin was added! 10 basic rules of y/d
0 x 10' units/Ky, weight, 500 x 10' units/K
The mice were administered intravenously continuously for 5 days at a dose of 1000 x 10' units/Kg body weight, and the mortality rate of the mice was measured after 7 days.

Conclusion As shown in Table 2, the mortality rate was significantly reduced by administering the solution, and the toxicity caused by the platinum complex antineoplastic agent according to the main rules was significantly reduced. This decrease in mortality was dependent on the increase in the main dose of M.

Table 1 Table 2 From this example, it is known that the main rule is effective as a therapeutic adjuvant that reduces the toxicity of a platinum complex anti-malignant am drug when used in combination with its administration.

ΔWai! Among the main rules obtained in the same manner as in Example 1, gelatin 519
It was carried out using the additive.

The effect of the main rule on hematopoietic organ damage and kidney damage caused by platinum complex antineoplastic agents was investigated using the following method. - Platinum complex anti-malignant Il1wA in C31→/HeN consisting of group of 5 animals
The drug cisplatin was administered intrathecally for 2 consecutive days at a dose of 4 days/7 days of body weight. From the day after cisplatin administration, 32 oxio'units/body weight of gelatin was intraperitoneally administered once a day for 5 consecutive days with the addition of 5sy gelatin. Peripheral blood white blood cell count, platelet count, and serum BUN (
The amount of urea (nitrogen) and creatinine were measured, and the effects on hematopoietic organ damage and kidney damage were investigated. The results are shown in Figure 1.
It is shown in FIG. 2 and Table 3.

In Figure 1, the horizontal axis represents the number of days after CDDP administration, the vertical axis represents the white blood cell count (X102/513), (U) represents the regular administration group, and (---Ichigoichi) represents the control group. show.

In Figure 2, the horizontal axis shows the number of days after CDDP injection, the vertical axis shows the platelet count (XIO'/3), (W) is the main administration group, and (-Tenichiban=) is the control. Indicates a group.

In both figures, the cross mark indicates a significant difference at a risk rate of 5%.

It was observed that the decline in peripheral blood white blood cell counts and platelet counts in mice administered with Honsho was suppressed and recovery was accelerated compared to the non-administered group, demonstrating the effect of Honsho's platinum complex antineoplastic agent on hematopoietic organ disorders. Furthermore, the increases in serum 8UNffi and creatine levels were milder than in the non-administered group, making it clear that the basic rule is also effective against kidney disorders.

Table 3

[Brief explanation of drawings]

FIG. 1 is a graph showing the effect of promoting white blood cell recovery in mice administered with CDDP, and FIG. 2 is a graph showing the effect of promoting platelet recovery in mice administered with CDDP. [Effects of the Invention] (1) The present invention provides a therapeutic adjuvant that reduces the side effects of a platinum complex anti-malignant Ha agent and makes it possible to effectively utilize its anti-malignant tumor effect. (ri) The present invention suppresses kidney damage and hematopoietic organ damage caused by the administration of platinum complex anti-malignant lls drugs, and the anti-malignant tS
We provide therapeutic adjuvants that enable effective utilization of the effects. (3) The main rule is that by administering the platinum complex anti-malignant SRS drug at the same time or directly after its administration, the side effects of the tumor drug can be prevented in advance.

Claims (1)

[Claims] A therapeutic adjuvant for the treatment of malignant tumors by administering a platinum complex antineoplastic agent, which contains human monocyte-macrophage colony stimulating factor as an active ingredient.