JPS623814B2 - - Google Patents

Description

[Detailed description of the invention]

This invention is directed to preparations containing human ceruloplasmin as a main component and used for the treatment of various malignant tumors, such as alkylating agents, antimetabolites, antibiotics,
It is a therapeutic agent that aims to prevent and treat side effects, mainly hematopoietic disorders, that are frequently observed when administered to the human body. Therefore, the dose of the antitumor agent was limited due to the occurrence of side effects, but with the application of this drug, it has become possible to increase the dose of the antitumor agent. Ceruloplasmin is a plasma protein that is synthesized in the liver as apoceruloplasmin, combines with copper absorbed by microsomes to become ceruloplasmin, and is released into the blood. The present invention discovers a new pharmaceutical use for ceruloplasmin, a copper-containing protein found in human plasma. The physiological role of ceruloplasmin is to transport copper, maintain copper balance in tissues, and oxidize Fe to Fe by its ferrooxidase activity.As a result, iron components are incorporated into apotransferrin, and transferrin Although its important role in iron metabolism has been demonstrated, the inventors further demonstrated that this substance, when administered prior to clinical use of various antineoplastic agents, typically The occurrence of side effects of the drug has been significantly suppressed, and thus, up until then, low-dose therapy had been unavoidable due to the side effects, but with the application of this drug, it has become possible to increase the dose, and it has also become effective as an anti-tumor drug. The present invention was completed based on the findings that the present invention is useful for the treatment of side effects mainly caused by hematopoietic dysfunction. This substance is obtained by subjecting Cohn-1 paste, a human serum fraction, to ethanol fractionation again, and then highly purifying it using ion exchange chromatography. [For example, Vox Sang, Vol.7.394~
405 (1962) and Vol.8.P.641-659 (1963)].
When formulating this product, ultraviolet irradiation treatment or
It is desirable to perform heat treatment at 60°C for 10 hours. After further sterilization, it is freeze-dried. The molecular weight of ceruloplasmin is 134,000 to 151,000, and each molecule contains 6 to 8 copper atoms. The electrophoretic mobility is equal to the position of α ₂ -globulin. The effects, effective dose, toxicity, etc. of human ceruloplasmin are shown below. (1) Preventive and therapeutic effects of ceruloplasmin on side effects of antineoplastic agents 1/10 dose and 1/30 of the LD ₅₀ value of antineoplastic agents
The amount was intravenously administered once to normal healthy mice (C3H/He), and the animals were observed for life or death for 30 days after administration. The mice in the control group were given physiological saline, and the mice in the experimental group were preliminarily given ceruloplasmin one week in advance, and those in the experimental group were given ceruloplasmin immediately after administration of the antitumor drug. A group was established for which administration was to begin. These drugs were administered intramuscularly on consecutive days. The effectiveness of ceruloplasmin was judged based on the average survival days. The number of animals in each experimental group was 10. The results are summarized in Table 1. In the group administered with ceruloplasmin, a significant prolongation of survival time was observed in all cases of cyclophosphamide, 6-mercaptopurine, and mitomycin, which were used as typical antitumor agents. We also investigated the effect of continuous administration of ceruloplasmin on the effects of these antitumor agents on experimentally transplanted tumors, but in all cases, we found almost no difference in their antitumor effects.
It was confirmed that ceruloplasmin did not reduce its antitumor effect. The above results demonstrate that ceruloplasmin can significantly reduce the side effects of antitumor agents without reducing their pharmacological effects. Additionally, based on these results, conventionally, the dose of anti-tumor drugs had to be reduced or discontinued due to their side effects, but with the combination of ceruloplasmin, the administration of anti-tumor drugs can be continued or It was shown that it is possible to increase the amount of

[Table] (2) Dosage, method of administration, and timing of administration When using this drug as a medicine, ceruloplasmin is isolated from human plasma and has no antigenicity to humans. This drug is used as an injection, and the dosage for humans is 5 mg to 150 mg. It is desirable to administer the drug every day or at appropriate intervals. This agent is easily water-soluble and can be used as an isotonic aqueous solution by adding sodium chloride, etc. The timing of administration is before, simultaneously with, and at the same time as various antitumor drugs.
However, it is more preferable to administer the drug 2 or 3 days before the administration. Furthermore, it is preferable to start administration as early as possible before the side effects of the antitumor agent appear. (3) Acute toxicity test Acute toxicity test of human ceruloplasmin
This study was conducted using rats and mice. The animals were 20±1 g d/d mice of both sexes (5
For each administration group, 10 male and female Wistar rats (5 weeks old) weighing 90±5 g were used per dose. The administration methods were four routes: intravenous injection, intraperitoneal injection, subcutaneous injection (dispensing equal amounts to 2 to 4 sites), and oral administration. Observations were made for toxicity symptoms and mortality up to 72 hours after drug administration, and the LD ₅₀ value (according to the Litchfield-Wilcoxon method) was calculated from the 72-hour mortality rate. Table 2 shows the _LD50 values in mice and rats.
Shown below.

【table】

[Table] (4) Stability test The stability of ceruloplasmin was measured using the oxidase activity of ceruloplasmin as an indicator when it was made into a freeze-dried product and when it was made into an isotonic aqueous solution, and the results are shown in Table 3. and 4.

【table】

[Table] When freeze-dried products are stored at -10°C, there is almost no decrease in titer even after storage for 15 months. 2～10
Approximately 70% of the titer is maintained even when stored at ℃ or for 15 months. It is also extremely stable even when stored in liquid form, and the titer decreases by only 30% after 30 days of storage at room temperature. Next, a production example of this invention substance will be shown. Production Example 1 Suspend 1 kg of -1 fraction paste obtained from human plasma by Cohn's alcohol fractionation method in the minimum volume of distilled water, and add 20% ethanol to the precipitated fraction.
% concentration to obtain the supernatant. Furthermore, ethanol was added to a concentration of 40% to obtain a precipitate fraction. Make an aqueous solution of this precipitated fraction and DEAE−
A ceruloplasmin fraction was obtained by adsorption and elution using Sephadex. For equilibration of DEAE-Sephadex and samples, 0.05M acetate buffer PH6.8 was used.
After adsorbing ceruloplasmin, wash with the same buffer containing 0.1M sodium chloride.
Ceruloplasmin was eluted with the same buffer containing 0.3M sodium chloride. With this production method, ceruloplasmin is 3.2~
5.0g was obtained. Formulation example 1 After irradiating the ceruloplasmin physiological saline solution with ultraviolet rays to inactivate any hepatitis viruses that may be present, the ceruloplasmin concentration was reduced to 7.5mg/
ml with physiological saline. After the liquid was sterilized, it was dispensed into 2 ml portions per vial and freeze-dried. This procedure produces a preparation containing 15 mg of ceruloplasmin and 18 mg of sodium chloride as an isotonic agent per vial. Dissolve in 2 ml of distilled water for injection before use.

Claims (1)

[Claims] 1. An agent for preventing and treating the side effects of an anti-malignant tumor drug whose main ingredient is human ceruloplasmin.