JP2928608B2 - Insulin-dependent diabetes mellitus preventive or therapeutic agent - Google Patents

Description

Description: TECHNICAL FIELD The present invention relates to a prophylactic or therapeutic agent for insulin-dependent diabetes mellitus containing lymphotoxin as an active ingredient.

[Prior Art] Diabetes is classified into insulin-dependent, non-insulin-dependent, and other types. Insulin-dependent diabetes often occurs at a young age and is also called juvenile diabetes or type I diabetes. Insulin-dependent diabetes mellitus can be considered as a phenomenon in which immunological unresponsiveness to a self antigen is disrupted by a factor that induces reactivity in the autologous pancreatic islet cells. The factors currently considered here include encounters between isolated autoantigens and the immune system, emergence of forbidden clones, T cell bypass by modified autoantigens or cross-reactive antigens, activation of non-specific lymphocytes, Malfunctions of suppressor T cells, and abnormalities of the anti-idiotype network.

Findings suggesting the involvement of the autoimmune mechanism in insulin-dependent diabetes are that there are many complications of autoimmune diseases such as Addison's disease and pernicious anemia, and that antibodies to pancreatic islets of Langerhans are found. Autoantibodies against pancreatic Langerhans islet cells are antibodies against the cytoplasm (Bottazo, GF et al .: Lancet)
2 , 1979 (1987)) and antibodies against cell membranes (Lernmark, A. et al.): New England Journal of Medicine (N. Engl. J. Med.) 29
9 , 375 (1978)). Antibodies against the cytoplasm show complement fixation, and not only β cells (insulin synthesis and secretion) but also α cells (glucagon synthesis and secretion) and δ cells (somatostatin synthesis and secretion) in the pancreatic islets of Langerhans
Also reacts with. On the other hand, an antibody against the cell membrane recognizes a membrane protein antigen on the β cell membrane and reacts mainly with β cells. Antibodies against cell membranes show diversity, inhibiting insulin secretion and impairing beta cells through the involvement of complement (Charles, MA et al .: Journal of Immunology (J. Immunol.) 130 , 1187 (198
3)).

However, animal models have reported that diabetes was caused by lymphocyte transfer (Wicke et al.
r, LS, Miller, BJ, Mullen, Y):. die Bee Tees (Diabetes) 35, 855 (1986 )), also anti-Thy1 · 2 (Harada et al. (Harada, M., Makino, S ):. Ekusuperi (. Exp.Anim) mental Animal 35, 501 (1986)), L3T4 antibody (sizzle, et al. (Shizuru, JA, Taylor-Edwards , C., et al..):
Sanence 240 , 659 (1988)), anti-class I
I, Ia antigen antibody (Boitard, C., Benedlac,
A., et al.): Proceedings of National.
Academy of Science of America (Pro
c. Natl. Acad. Sci. USA.) 85 , 9719 (1988)), the importance of not only the involvement of antibodies but also the involvement of T lymphocytes has been emphasized in the pathogenesis mechanism. It is also known that insulin-dependent diabetes is under some form of genetic control, but the details have not been analyzed yet.

[Problems to be Solved by the Invention] As described above, the onset mechanism of insulin-dependent diabetes mellitus is complicated, and at present the established knowledge has not yet been obtained, and an effective prevention or treatment method has been discovered. Absent. On the other hand, substances derived from T cells or B cells represented by lymphotoxin are known as cytotoxic cytokines that are considered to be effective in treating cancer. This cytotoxic cytokine initially acts on the immune system to modify its response, a so-called biological response
It had no activity as a modifier (BRM) and was thought to act directly on cancer cells to selectively destroy cancer cells (Japan Cancer Society, 43rd Annual Meeting, p. 273, abstract No. 965) , 1984).

On the other hand, it has been reported that Tumor necrosis factor (TNF), which is also a cytotoxic cytokine and is effective in treating cancer, is effective in preventing NOD mice in an animal model of insulin-dependent diabetes mellitus (Sato et al. Satoh, J. et al.): Journal of Clinical Investigation (J. Clin. Invest.) 84 , 1345
(1989)). However, the dose is large, and there are also concerns about side effects and the like.

[Means for Solving the Problems] The present inventors have conducted intensive studies to solve the above problems, and as a result, it has been found that lymphotoxin acts as an immunomodulator to prevent or treat insulin-dependent diabetes. It led to the present invention.

That is, the present invention relates to a prophylactic or therapeutic agent for insulin-dependent diabetes mellitus containing lymphotoxin as an active ingredient, and the effect of such lymphotoxin exceeded the effect of TNF.

[Examples] The lymphotoxin used in the present invention is initially released from lymphocytes stimulated with mitogens and antigens, and has broad cytotoxic activity against tumor cells, while inhibiting lymphocytes from normal cells and cultured cells. Was found as a biological factor showing little the same activity (Ruddle, NH, Waksman, BH): Scienc
e) 157, 1060 (1967) ). The lymphotoxin of the present invention can be used in vitro on LM cells of cultured cell mice.
It is a protein having a direct cytotoxic effect in o) and / or an antitumor effect in vivo (in vivo) on female A sarcoma-bearing mice, and has a sugar chain in its natural form. The lymphotoxin used in the present invention was prepared by introducing human lymphotoxin chromosomal DNA into Chinese hamster ovary cells and expressing it (Japanese Patent Application Laid-open No. Sho 62-62).
No. 11095). Aggarwal et al. Also published a journal of biological chemistry (J. Bi) from the culture supernatant of human B cells transformed with EB virus.
ol. Chem.) 259 , 686 (1984)). Recombinant lymphotoxin can also be prepared by a method using Escherichia coli or the like as a host cell. If present, it can be used with or without sugar chains.

This for the nature of the protein lymphotoxin has been reported by Agaruvaru (Aggarwal) et (Journal of Biological Chemistry (J.Biol.Chem.) 260, 2345 ( 1985) and 259, 686 (198
Four)).

As a method of administering lymphotoxin as an agent for preventing or treating insulin-dependent diabetes, any of the known administration methods may be used, but intravenous administration or intraperitoneal administration is particularly preferred.

The present invention will be described more specifically with reference to the following examples, but the present invention is not limited to the following examples as long as the gist is not exceeded.

(Test method) Lymphotoxin was prepared by the method disclosed in JP-A-62-11095. This is SDS electrophoresis and reverse phase HPLC
(High performance liquid chromatography) showed unity, and there was no contamination with other cytokines.

The efficacy of lymphotoxin for insulin-dependent diabetes mellitus was determined using insulin-dependent diabetes model mouse (NO
(D mouse) was judged based on whether or not improvement was observed in one or more of the various disease states already reported. Specifically, in vivo judgment was made on the presence or absence of excretion of sugar in urine and disappearance of pancreatitis.

Example 1 [Preventive Effect of Insulin-Dependent Diabetes Mellitus by Lymphotoxin] A four-week-old female NOD mouse (at the start of administration, body weight of 16 to 18 g) was treated with 500 LM of the lymphotoxin obtained by the above method in physiological saline. 0.1ml dissolved in unit / ml (50LM unit,
It was intraperitoneally administered three times a week at a rate of 0.25 μg / animal) / time, which was used as a preventive treatment group. For LM units, see Yamazaki et al. (Yama
zaki, S., Onishi, E. et al.), Japanese Journal of Medical Science of Biology ((Japan J. Med. Sci. Boil.) 39 , 105 (1986)) According to the method described in (1). A control group to which physiological saline containing no lymphotoxin was similarly administered was used. The administration period was 23 weeks and the judgment was made on the 27th week.
A case where sugar at 0 mg / dl or more was detected in urine was defined as positive.
The results are shown in Table 1.

[Effects of the Invention] Proceedings of Jacob, CO et al.
Of National Academy of Science of. America ((Proc.Natl.Acad.Sic.USA.) 87, 986 (1
According to 990)), when 3 μg / animal was administered to the tumor necrosis factor (TNF), the incidence of disease was 50%.
% In the present invention.
Disease administration was completely suppressed by administration of 0.25 μg / animal.

As described above, the preventive or therapeutic agent containing lymphotoxin as an active ingredient obtained according to the present invention is effective for insulin-dependent diabetes mellitus at a dose at which no toxic effect is observed.

Claims (1)

(57) [Claims] [1] A preventive or therapeutic agent for insulin-dependent diabetes mellitus comprising lymphotoxin as an active ingredient.