JP2709941B2 - Hematopoietic disease treatment adjuvant - Google Patents

Description

Description: TECHNICAL FIELD The present invention relates to an auxiliary agent for treating hematopoietic diseases, and more particularly to an agent for suppressing rejection during bone marrow transplantation.

(Conventional technology and its problems) In the treatment of various blood diseases such as leukemia, aplastic anemia, and immunodeficiency, bone marrow transplantation has been one of the coping methods. However, if the bone marrow donor and recipient do not have a common histocompatibility antigen (HLA), life-threatening immune reactions such as graft-host reaction (GVHD) and graft rejection can occur. Attempts have been made to avoid these undesirable reactions. GVHD is T from donor bone marrow
Can be prevented by removing the cells (Waldman H, Slavin
S, Pretice HG et al.) On the other hand, patients with transplanted T-cell depleted bone marrow are more likely to have graft rejection, especially for recipients with HL
This is remarkable when A does not match. Recipient graft rejection due to these genetic factors is one of the major obstacles to applying BMT to treat advanced aplastic anemia.

When certain mice are transplanted with a lethal dose of radiation, they reject parental, allogeneic H-2, or rat bone marrow cells ("hybrid", "allogeneic", "heterologous", respectively). Numerous studies have been conducted to elucidate the mechanisms of these three types of resistance. As a result, a rejection reaction that acts destructively on host blood cells and a host-graft reaction that is immunogenically specific have been described. Turned out to be very similar. BMT
Have been classified by Trentin et al. Into three classes: hybrid (hybrid), homologous (allo) and heterologous (xeno) resistant. There is a great difference between the strains of mice in their ability to reject exogenous bone marrow transplants. C57BL mice were H-2 ^b-type and a "resistant" to the bone marrow of DBA / 2 mice (H-2 ^b-type) and rats. C3H mice (H-2 ^k
Type) is "non-resistant" to B57BL mouse and rat bone marrow cells.

Considering the cells involved in the genetic resistance to bone marrow transplantation (BMT) in mice exposed to lethal dose, inhibitors of macrophage function, such as silica and carrageenan, show genetic resistance to bone marrow transplantation Therefore, it is considered that macrophages are involved.

In parenteral nutrition, an intravenous fat emulsion method is used clinically. It is known that administration of a fat emulsion accumulates fat in macrophages, which is thought to impair the function of macrophages and reticuloendothelial system. The present inventors have administered fat emulsion to mice to which a lethal dose has been administered, and have attempted to eliminate resistance to blood cells.

That is, an object of the present invention is to provide a therapeutic adjuvant that effectively suppresses rejection during bone marrow transplantation for treating hematopoietic diseases.

(Means for Solving the Problems) The present invention is an agent for suppressing rejection at the time of bone marrow transplantation comprising a fat emulsion, whereby the above object can be achieved.

As the oil component of the fat emulsion used in the present invention, for example, vegetable oils such as soybean oil, cottonseed oil, sesame oil, safflower oil, corn oil, peanut oil and olive oil are used, and preferably soybean oil is used. The soybean oil is preferably refined soybean oil, especially high-purity refined soybean oil containing at least 99.9% of triglycerides, diglycerides and / or monoglycerides. Such a high-purity refined soybean oil can be obtained by converting the refined soybean oil to a steam distillation method [HJLipe. J. Am. Oil Chemis
t. Soc., 27, 422-423 (1950)].

The oil component is usually emulsified using an emulsifier. Non-ionic surfactants, phospholipids, lecithin, hydrogenated lecithin and the like are used as emulsifiers. This does not limit its origin such as vegetable oil such as soybean oil, egg yolk, and animal oil.
Nonionic surfactants have a molecular weight of 2,000 to 20,000
For example, polyoxyethylene, polyoxypropylene copolymer, polyoxyethylene alkyl ether, polyoxyethylene alkyl allyl ether, etc. are used. The emulsifier may be used alone, or may be used as a mixture. Also,
The emulsion used in the present invention may further contain a known carbon number of 6 to
If desired, a small amount of 22 fatty acids, salts thereof (sodium salt, potassium salt, etc.), polyhydric alcohols and the like may be added.

The fat emulsion according to the present invention is generally an oil-in-water fat emulsion, and its particle size is preferably 1 μm or less.

Such a fat emulsion is produced by a known method or a method analogous thereto. That is, for example, the oil component 5 to 50
% (W / V), preferably 8 to 30% (W / V), an emulsifier having a weight ratio to the oil component of 1 to 50 (preferably 5 to 30),
Appropriate amount of water (however, oil component / water (weight ratio) is 0.05 to 0.43
And preferably 8 to 30% (W / V)].

The emulsification is specifically performed, for example, as follows.
That is, first of all, the required amount of oil component, emulsifier, and, if necessary, a co-stabilizer are mixed, and this is heated and dissolved at 30 to 80 ° C.
The mixture is homogenized with a homomixer, a pressurized injection homogenizer, an ultrasonic homogenizer, or the like, and then a required amount of water is added thereto, and the homogenizer is homogenized again. Thus, a very fine stable emulsion having an average particle size of 1.0 μ or less is produced (J. Am. Oil. Chem. Soc., 32.365-370).
(1950)].

(Experimental Examples) Hereinafter, the present invention will be described in detail with reference to experimental examples.

Materials and Methods Animals: 8-12 week old mice C57BL / 10SnSlc (B10: H- 2 b),
C57BL / 6SnSlc (B6: H- 2 b), DBA / 2CrSlc (DBA / 2: H-
^{2 d), C3H / HeSlc (} C3H: H-2 k) swim of 8-week-old rat F3
44 (all females) were purchased from Shizuoka Experimental Animal Farm (Hamamatsu City, Shizuoka Prefecture). Put laboratory animals in a laminar flow rack (Isolak: manufactured by Sanki Kagaku Kogyo Co., Ltd., Tokyo).
A mixture obtained by adding a crystalline penicillin GK salt (Meiji Seika Co., Ltd., Tokyo) at 200,000 U / L and a streptomycin sulfate (200 mg / L Meiji Seika Co., Ltd., Tokyo) was added.

Fat emulsion: One of the fat emulsions used clinically, Intralipos (Intralipos: manufactured by Midori Cross Co., Ltd.) 20%
Was used. Intralipos 20% is a nutritional supplement with a complex composition, including soybean oil (20%), yolk phospholipids (1.2%
%) And glycerin (2.5%) and is supplied as a suspension of fat particles. Intralipos 20% 2ml /
Mice / day were administered continuously for 1 to 3 days, and an experiment of genetic resistance to BMT was performed 2 to 4 hours after the last administration. The NK (natural killer) activity test was performed 24 hours after the last administration.

Bone marrow transplantation: McCulloch and Til for BMT methods and evaluation of transplant results
Performed according to the method of l. Recipient mice were irradiated with 8-9 gray gamma rays from a Co ⁶⁰ source immediately prior to transplantation. Bone marrow was collected from the femur of a normal mouse or the femur and tibia of a rat. Syngeneic, allogeneic and xenogeneic (rat) bone marrow cells, 5 × 10 ⁴ , 2-3 × 10 ⁵ , 4 ×
10 ⁶ were injected from the tail vein, and 8 days later, macroscopic colonies in the spleen of the recipient mouse were counted, and the implantation rate of bone marrow transplantation was calculated.

Natural killer cell activity: A chromium (Cr) release test was performed by the method of Kiessling et al. (Eur J Immunol 7: 655 (1971).
Lysis of YAC-1 lymphoma cells was measured by working cells of the spleen according to 7)). The test was performed by changing the original method to a 96-well microtiter dish. YAC-1 cells were statically cultured in RPM1-1640 medium supplemented with 10% fetal bovine serum and antibiotics (100 IU / ml penicillin, 50 μg / ml streptomycin). 1 × 10 ⁷ target cells (YAC-1 cells) were added to 200 μCi ⁵¹ C
Labeling was performed by culturing at 37 ° C. for 30 minutes in 0.5 ml of a medium containing r (sodium salt). Cells were washed three times with medium. The effector cells were isolated from the spleen of B10 mice by intravenous intralipos injection once and three times without treatment. Add 200 μl of each working cell suspension to the wells of the microtiter dish, and add to each well.
⁵¹ Cr-labeled target cells (10 ⁴⁾ were added. Each measurement was performed by taking three points. Place each dish in the presence of 5% carbon dioxide.
The mixture was incubated at 37 ° C. for 4 hours, and then centrifuged at 150 × g for 10 minutes.
Take 100 μl of the supernatant from each well, and place it on the gamma counter.
Measured for minutes. The control value and the maximum value of ⁵¹ Cr release were measured using cells that had been incubated without working cells and that had been incubated with distilled water, respectively. The results were expressed in% of dissolution as in the following equation.

Statistical processing: Student's t-test was followed.

Experimental results: Continuous intravenous injection of intralipos 20% into mice at 2 ml / mouse / day for 3 days was not lethal and had no significant effect on spleen weight of mice.

Effect of Intralipos 20% on BMT (Intralipos 20% with 57BL / 10 mice as a receptor
Table 1 shows the effects of the administration of.

When intraliposome 20% was intravenously injected into recipient mice for three consecutive days, syngeneic donor cells had no effect on implantation and growth in recipient spleens. Normal mice (C57BL / 10, H- ^2b ) irradiated with lethal dose were homozygous mouse DBA-2 (H-
Bone marrow cells of 2 ^d) or F344 rats (heterologous) rapidly rejected. Administration of Intralipos 20% to recipient mouse C57BL significantly reduced resistance to allogeneic and xenogeneic bone marrow cells. Rather than a single dose of Intralipos,
Three consecutive days of administration were more effective in suppressing allogeneic resistance in B10 mice. On the other hand, irradiated ^{C3H / He (H-2 k} )
The mice did not completely reject allogeneic C57BL / 6 (H- ^2b ) or heterologous bone marrow cells. However, intralipos administration also suppressed this moderate resistance of C3H mice to allogeneic bone marrow transplantation (Table 2).

Effect of Intralipos 20% on NK Cells Next, the cytolytic activity of NK cells, which are the main action cells in genetic resistance to BMT, was examined. Intralipos was administered intravenously, as can be seen in the results shown in Table 3.
In B10 mice, NK activity was significantly reduced. It was more effective to reduce NK activity for three consecutive days than for a single dose of Intralipos.

From the above experimental results, it was found that administration of Intralipos 20% suppressed allogeneic and xenogeneic resistance to bone marrow cell transplantation. It should be noted that there was no significant effect on the point that the transfected cells were implanted in the spleen and subsequently proliferated in mice that received 20% intralipos and received syngeneic bone marrow transplantation. This result indicates that injection of 20% intralipos into the recipient only affects homologous and heterologous resistance.

The mechanism of genetic resistance to BMT has not yet been fully elucidated. It has been reported that this genetic resistance to BMT involves antibody-dependent cell-mediated cytotoxicity (ADCC) and T cells. The fact that macrophage-toxic substances, such as silica and carrageenan, reduce spontaneous NK cell activity suggests that macrophages are required to maintain NK activity levels in normal young mice. There are also reports that NK cells play an important role in the body in genetic resistance to exogenous bone marrow transplantation. According to a report by Kiessling et al., Administration of silica or carrageenan to mice reduced cytotoxicity of NK cells by 39.9% and 56.3%, respectively. Macrophage-toxic silica and carrageenan also suppress genetic resistance to BMT. Therefore, silica and carrageenan are considered to reduce BMT resistance by acting on macrophages. This is because these substances accumulate in macrophages and impair the macrophage functions necessary for maintaining NK cell activity in the body. When intralipos 20% was intravenously injected for 3 consecutive days, the cytotoxicity of mouse spleen cells was reduced by 50-60% (Table 3). Intralipos 20 in reducing the cytotoxicity of NK cells
% Is considered to be similar to silica and carrageenan.

The intravenous injection of a fat emulsion used in the present invention is clinically used, and it is known that fat is taken up and accumulated in macrophages to inhibit the function of macrophages (Strunk RC et al., Am Leukocyte Biol 36). : 123; McCull
och EA et al. J Cell Comp Pyhsiol 61: 301). Mice injected with Intralipos had significantly reduced NK-cell activity of spleen cells. One of the ideas explaining this result is that the macrophage function was impaired and the NK-cell activity was reduced in the mice administered with Intralipos. However, significant amounts of intralipos are required to suppress macrophage function and NK-cell activity.

Reducing patient resistance to allogeneic bone marrow transplantation in humans is desirable, but presently difficult to achieve. The current drug administration and radioactive irradiation are dangerous and have reached a level where non-specific toxicity cannot be further increased. If genetic resistance to BMT could be controlled by more specific and less toxic methods, BMT would be more widely used. However, silica particles and carrageenan cannot be used in humans. Intravenous administration of fat emulsions, especially
It is a new measure as a pre-treatment for BMT (especially bone marrow transplantation without HLA).

Claims (1)

(57) [Claims] 1. An agent for suppressing rejection at the time of bone marrow transplantation comprising a fat emulsion.