JPH06298666A - Agent for protecting marrow suppression - Google Patents

Abstract

PURPOSE:To obtain a treating agent effective for protecting the disorder of hematopoietic cell against the marrow suppression caused by chemotherapy or radiotherapy and preventing the decrease in leukocytes and platelets by using an interferon as an active component. CONSTITUTION:The agent contains an interferon as a main component. The interferon is properly compounded with pharmaceutical additives such as human serum albumin, gelatin, mannitol, sorbitol, lactose, trehalose and surfactant as other components and prepared in the form of a pharmaceutical preparation. The daily dose of the agent is 10-100,000,000 units per 1kg body-weight. The interferon to be used in the agent is obtained by purifying an interferon produced from an interferon-producing cell or by a gene recombination technique.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a therapeutic agent for efficiently protecting bone marrow cells from chemotherapy or radiation therapy.

[0002]

BACKGROUND OF THE INVENTION Myelosuppression is manifested as thrombocytopenia, leukopenia and anemia, and has become a therapeutic problem for cancer and leukemia with chemotherapy or radiation therapy for cancer and leukemia. Currently, for these myelosuppression, as a symptomatic treatment, red blood cell transfusion,
Only platelet transfusions have been applied. Currently, CSF (colony stimulating factor) is used for such leukopenia.
Although it is being applied as a drug for increasing white blood cells that have been reduced, there is no known drug that effectively protects myelosuppression.

Initial experiments have reported that certain bioactive substances of biological origin protect myelosuppression. IL-1 (interleukin-1) and MIP
-1α is an example (G, Damia, Cancer Res. 4082).
-4089, 52, 1992, B, Lord, Blood, 2605-2609, 79, 199
2). However, IL-1 has a strong inflammatory effect,
Side effects such as inflammatory and shock including headache and fever are concerned (Saito et al., Surgical treatment, 65,156-164,199
1). In addition, regarding MIP-1α, there are no reports other than initial experiments.

Interferon is a protein produced and secreted by animal cells upon viral infection.
Three types, β and γ, are known, and there are several subtypes in the α form. By interfering with the surface receptors of cells and changing the state of the cells, interferon blocks the growth of viruses and functions as an ecological defense mechanism in the early stages of virus infection. In addition, interferon has cell growth suppression and immune effector actions, and is also involved in tumor cell growth suppression (RMFriedman, interferon,
Tokyo Kagaku Dojin, 72-87, 1983).

On the other hand, the following effects of interferon on hematopoiesis are known. When interferon is administered 2-3 times a week or daily for the treatment of renal cancer, leukopenia of 3000 / ul or less is seen in 25% and thrombocytopenia of 100,000 or less is seen in about 10% of cancer patients (Noguchi, Medical History). , 164,389-392,1993). Recombinant interferon in mice for 1-2 weeks 1 million-10 million units / kg
The hematocrit was 46% to 38%.
%, Suggesting inhibition or prolongation of blood cell maturation (D. Orlic, Ann. NY Acad. Sci., 554,36-48,198
9). Thus, interferon has only been reported to act suppressively on hematopoiesis, and there has been no case where it was used as a blood cell protective agent in myelosuppression in chemical agents and radiotherapy.

[0006]

The present invention relates to chemotherapy,
It is an object of the present invention to provide a therapeutic drug that effectively protects hematopoietic cell damage against myelosuppression caused by radiotherapy and protects / prevents leukopenia and thrombocytopenia.

[0007]

[Means for Solving the Problems] The present inventors arrived at the present invention by making extensive studies in order to achieve the above object. That is, the present invention is a myelosuppressive protective agent containing interferon as an active ingredient. The present inventors prevent interferon administration during chemotherapy or radiation therapy to prevent myelosuppression caused by chemotherapy or radiation therapy, resulting in protection of reduction of blood cells such as myeloid cells, platelets and leukocytes. The present invention has been completed by discovering that it is prevented, that is, interferon, which originally acts to suppress myelocytomas, alleviates the degree of myelosuppression caused by chemotherapy or radiation therapy.

The examples of the present invention showed that administration of mouse interferon to mice and rats can effectively protect mice from myelosuppression caused by chemotherapy or radiotherapy. Since it is generally known that human interferon does not act on mice, the mouse and rat systems administered with mouse interferon are used in the examples, but this result can naturally be deduced to humans. Therefore, the drug effect observed in the mouse or rat system can also be expected in humans.

The interferons of the present invention are α, β,
interferon α, which includes γ and is obtained by the gene recombination method established as a general method at present.
Includes β and γ. There are three types of interferons, and α, β, and γ are known. Although the β type was used in the examples, the object of the present invention can be achieved by using any of these three types. The interferons of the invention are also not limited to human interferons, but also include the use of animal interferons for similar therapeutic uses in animals.

As means for producing interferon, there are means for purifying from cultured cells that originally produce interferon, and means for producing / purifying using interfering gene technology. For example, interferon used for the purpose of the present invention can also be introduced into animal cells such as hamster cells, mouse cells, monkey cells, insect cells, or yeast by linking and introducing the interferon gene downstream of a promoter or the like that functions in the host. Can be prepared.

Recombinant interferon can be produced by a known method. As an example, an example in which Escherichia coli is used as a host has been shown as an example, but other than this, it can be produced by using a widely known gene manipulation method. For example, prokaryotic cells such as Bacillus subtilis, yeast,
By introducing the interferon gene in the form of DNA or virus into animal cells such as hamster cells, mouse cells, monkey cells, human cells, insect cells, and insect bodies by linking the interferon gene downstream of a promoter that functions in the host Can also be prepared.

[0012] As an example, cell-derived interferon is secreted into the medium by treating cultured fibroblasts with an appropriate interferon-inducing agent such as poly I: C which is a synthetic nucleic acid. The fibroblast-derived interferon β can be purified by using affinity chromatography using a blue carrier. Interferon α can be obtained by culturing blood cells of white blood cell fraction or cell lines derived from blood cells and then treating with an inducer.

The myelosuppressive agent of the present invention contains interferon produced by the above-mentioned method as a main component.
As the other components, general pharmaceutical additives are selected. Of course, the object of the present invention can be achieved without additives. Additives are generally added primarily for stabilization.
Such a pharmaceutical additive is selected from proteins and / or saccharides described in the Japanese Pharmacopoeia that can be used as a pharmaceutical additive. Particularly preferably, it is selected from human serum albumin (HSA), gelatin, mannitol, sorbitol, lactose, trehalose, a surfactant and the like as appropriate or in combination, but it is not limited to these.

The present invention also includes simultaneous or sequential co-administration of interferon with other drugs, biological drugs, synthetic drugs and the like. Other drugs include
IL-1, IL-2, IL-3, IL-6, IL-7,
Interleukins such as IL-11, cytokines such as CSF (colony stimulating factor), EPO (erythropoietin), thrombopoietin, MSF (megakaryocytic stimulating factor), or known complementing or assisting the object of the present invention Selected from among the pharmaceutical products of.

In order to specifically achieve the myelosuppressive protective effect which is the object of the present invention, the composition containing interferon as the main component thus obtained is administered to a living body. The administration method is not particularly limited, but an appropriate one is selected from general injections such as intravenous injection, subcutaneous injection, intramuscular injection, and intravenous drip infusion. In some cases, transmucosal administration methods such as oral, nasal, pulmonary, and enteral administrations are also suitably performed.

The effective dose is 1K body weight per day
It is selected in the range of 10 to 100 million units per gram. It is preferably selected in the range of 1000 to 1 million units per 1 kg of body weight.

The above-mentioned dose varies depending on the symptoms and is not limited to these values. As a feature of the present invention, the dose is usually suitably selected to be a dose lower than the dose showing an increase in platelets in vivo, but is not limited to this.

The number of administrations is usually once or twice a day,
Alternatively, it is selected once every 2 to 3 days, but is not limited thereto. The timing at which the myelosuppressive action is observed varies slightly depending on the administration method, and the protocol for interferon administration is appropriately selected depending on the type and strength of chemotherapy or radiation therapy.

The subject to be administered is a patient whose body undergoes myelosuppression, that is, a treatment method or drug administration accompanied by a decrease in the number of platelets or a decrease in the number of white blood cells. As an administration time, an example in which interferon is administered before such treatment or drug administration is shown in the examples, but it is not particularly limited, and simultaneous or subsequent administration is also included in the present invention. The administration schedule of the present invention is appropriately selected depending on the nature of the therapeutic method such as a chemotherapeutic agent and the administration schedule.

[0020]

EXAMPLES The present invention will now be described in more detail and more specifically by way of examples, which, of course, are not intended to limit the present invention. Since it is generally known that human interferon does not act on mice, the examples show the results on mice and rats administered with mouse interferon. Because interferon is species-specific, mice need to be tested with mouse interferon. However,
The drug effect observed in mice and rats can naturally be expected in humans.

Example 1 Preparation of mouse interferon: The interferon of the present invention was prepared by the method of Tanaka et al. (Tanaka et a
l, J. Interferon Research, 6, 429-435). That is, an ATG encoding methionine was attached to the tip (5 ′ end) of the portion encoding the mature protein of mouse interferon β cDNA, and an expression plasmid for Escherichia coli ligated downstream of the tryptophan promoter of Escherichia coli was prepared. This plasmid was introduced (transformed) into Escherichia coli HB101 strain, and after culturing, the mouse interferon of interest was accumulated in E. coli cells by inducing with indole acrylic acid.

Purification of the mouse interferon β thus obtained was carried out according to a report by the present applicants (JP-A-61-224996, JP-A-61-249397). That is, crude mouse interferon extracted from Escherichia coli was purified by a combination of silica gel chromatography, blue dye chromatography, copper chelate chromatography and carboxymethyl cellulose chromatography.

Example 2 Protective effect in mitomycin-induced myelosuppression model: The interferon obtained in Example 1 was dissolved in physiological saline containing 0.1% homologous mouse serum, and once a day, 100,000 units per animal were prepared. C57BL / 6 mice (male, 6 weeks old) were subcutaneously administered for 5 days. Then, in order to induce myelosuppression, mitomycin C (Kyowa Hakko) was intraperitoneally administered once at a dose of 2 mg / kg. A physiological saline containing 0.1% allogeneic mouse serum was administered to the control group. For measurement, under ether anesthesia, 150 ul of whole blood was collected from the inferior vein, and 15% EDTA.
5 ul of 2K was added, and the number of white blood cells and the number of platelets were measured by an automatic blood cell counter Sysmex (Toa Medical Electronics).

The results are shown in Tables 1 and 2. As is clear from the table, in the group using interferon, the decrease in white blood cell count and platelet count could be suppressed after 5 days from the start of administration.

[0025]

[Table 1]

[Table 2]

Example 3 Protective effect in radiation-induced myelosuppression model: The interferon obtained in Example 1 was dissolved in physiological saline containing 0.1% homologous mouse serum, and 100,000 units per animal were administered once a day. C57
BL / 6 mice (male, 6 weeks old) were subcutaneously administered for 5 days.
Then, 9 Gy of radiation was applied once to induce myelosuppression. The control group was administered with physiological saline containing 0.1% autologous serum. The measurement was performed under ether anesthesia, and whole blood was taken from the inferior vertebral vein 15
Blood was collected from 0ul and 15% EDTA ・ 2K was added as an anticoagulant.
5ul was added and the number of white blood cells and the number of platelets were measured by an automatic blood cell counter Sysmex (Toa Medical Electronics). The results are shown in Tables 3 and 4. As is clear from the table, in the group using interferon, the decrease in white blood cell count and platelet count could be suppressed after 5 days from the start of administration.

[0027]

[Table 3]

[Table 4]

Example 4 Protective effect in nimustine-induced myelosuppression model: Example 1
The interferon obtained in 1. was dissolved in 0.1% allogeneic rat serum-containing physiological saline, and SD rats (male, 7 weeks old) were subcutaneously treated with Alza pump (Alza) at a dose of 1 million units / day for 7 days. It was used for continuous administration. Next, in order to induce myelosuppression, nimustine (Sankyo) was added on the 5th day of the experiment.
It was administered once at a dose of 25 mg / kg intravenously. In the control group
A physiological saline containing 0.1% allogeneic rat serum was administered. For measurement, under ether anesthesia, 250 ul of whole blood was collected from the inferior vein and 10 ul of 15% EDTA ・ 2K was added as an anticoagulant.
The white blood cell count and platelet count were measured with an automatic blood cell counter Sysmex (Toa Medical Electronics).

The results are shown in Tables 5 and 6. As is clear from the table, in the group using interferon, the decrease in white blood cell count and platelet count could be suppressed after 5 days from the start of administration.

[0030]

[Table 5]

[Table 6]

[0031]

INDUSTRIAL APPLICABILITY The present invention protects myelosuppression by chemotherapy and radiotherapy and is useful as a protective agent or therapeutic agent for leukopenia and thrombocytopenia.

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Wataru Takada 1111 Tehiro, Kamakura-shi, Kanagawa Toray Co., Ltd. Basic Research Laboratory (72) Inventor Kyoko Kasama 1111 Tehiro, Kamakura-shi, Kanagawa Toray Research Institute, Toray Co., Ltd.

Claims (1)

[Claims] 1. A myelosuppressive protective agent containing interferon as an active ingredient.