KR20050054119A - Immunosuppressant comprising xanthorrhizol - Google Patents

Abstract

The present invention relates to a novel immunosuppressive agent comprising Xanthorrhizol isolated from the root extract of Curcuma xanthorrhiza (local name: Temu lawak), an Indonesian medicinal plant, and particularly, organ transplantation. The present invention relates to an immunosuppressive agent effective in controlling rejection reactions occurring in humans. The present inventors first confirmed that naxazole inhibits mouse mixed lymphocyte response, and then using T-cells transformed with recombinant gene fragments designed to know the expression level of interleukin-2 gene, naxazole is interleukin-2 It was found that the gene efficiently inhibits the expression of the gene, and in a mouse transplantation experiment, it was confirmed that the nasal sol significantly increased the survival rate of the heart-receptive mouse by effectively maintaining the activity of the transplanted heart.

Description

Immunosuppressants containing naxazoles {IMMUNOSUPPRESSANT COMPRISING XANTHORRHIZOL}

The present invention relates to novel uses of xanthorrhizol or derivatives thereof as immunosuppressive agents. The compound or the pharmaceutical composition containing the same as an active ingredient can be used for the treatment, diagnosis or prevention of rejection, autoimmune disease, inflammatory disease, etc., associated with organ or tissue transplantation.

Naxolizol is a substance isolated from Curcuma xanthorrhiza (local name: Temu lawak), which is widely used as a medicinal plant in Indonesia, and this plant is a gastrointestinal disease, liver dysfunction, constipation, bloody stool, diarrhea in Indonesian folk remedies. It has been used as a treatment for childhood fever, hemorrhoids and rashes. In recent years, research on this plant has been reported to be effective in anti-cancer, anti-inflammatory, hepatocyte protection, etc., but its use as an immunosuppressive agent is not known at all before the filing of the present invention.

Organ transplants are commonly performed in end-stage patients with renal failure, heart failure, liver failure and pulmonary insufficiency, especially in the kidney, heart, lung, liver, bone marrow, pancreas (islet cells), cornea, small intestine and skin allograft. In addition to being performed, xenograft using pig heart valves is also being performed. However, externally transplanted organs may cause various immune responses in transplanted patients, leading to graft rejection and graft-versus-host disease (GVHD).

Immune responses are mainly caused by T cells recognizing allogeneic antigens, and the main targets of immune responses that occur in transplant rejection reactions are different types of Major Histocompatibility Complex (MHC) I and MHC II antigens. When an acute transplant rejection occurs, antigen-bearing cells, such as dendrite cells or monocytes, from organ recipients migrate to the surrounding lymph nodes at the site of transplantation and are externalized by the recipient's CD4 + TH cells. It is recognized as a substance, and as a result, it stimulates the proliferation of TH cells of the recipient. When TH cells proliferate, cell populations, such as cytotoxic CD8 + T cells and CD4 + T cells, are generated and migrate and infiltrate the transplant site to mediate the transplant rejection reaction [Noelle, R.J. et al., FASEB 5 (13): 2770, 1991].

While acute graft rejection is a T cell-dependent response, the subsequent mechanism of graft destruction is an extensive series of processes, in which cytokine is secreted and interactions between CD4 + T cells and CD8 + cytotoxic T cells. Various combinations of lymphocytes, including antibody-forming B cells and other proinflammatory leukocytes, participate in the anti-allograft response. More specifically, transplanted cells bearing antigen are directly destroyed by cytotoxic CD8 + T cells, and interleukin-2 (IL-2; hereinafter interleukin) produced by activated CD4 + T cells is referred to as 'IL'. Abbreviated) activates CD8 + T cells and B cells. CD4 + T cells also produce cytokines that participate in graft destruction, such as interferon gamma (hereinafter abbreviated as 'IFN-γ') or interleukin-4 (IL-4). IFN-γ induces the expression of MHC I and MHC II molecules in transplant tissues, making them more easily attacked, increasing macrophage activity and affecting cells involved in the inflammatory response. In addition, they cause delayed-type hypersensitivity (abbreviated as DTH) and inflammation, causing nonspecific damage to the graft. It is known that this reaction is the main cause of the initial acute rejection that occurs within a few weeks after transplantation, the destruction of the graft tissue rapidly occurs within a few days if left untreated.

Therefore, the necessity of immunosuppressive agents to prevent such fatal transplant rejection symptoms has emerged strongly. The development of an effective immunosuppressive agent has enabled breakthroughs not only in organ transplantation but also in the treatment of autoimmune diseases, and contributed to the study of the mechanism of immune response in transplanted organs.

Immunosuppressants currently used to modulate allograft rejection include corticosteroids and cyclophosphos such as cyclosporine A (CsA), azathioprine, prednisone or methylprednison. Cyclophosphamide, and the like. Cyclosporin A is one of the most powerful and commonly used immunosuppressive agents that has revolutionized the field of organ transplant surgery, including FK506, rapamycin, mycophenolic acid, and 15-deoxyspergualin (15). Other immunosuppressants, such as deoxyspergualin, mizoribine, misoprostol, antibodies to OKT3 and IL-2 receptors, have also been used for the treatment or prevention of organ transplant rejection [Briggs, Immunology letters Jul. 29 (1-2): 89-94, 1991; FASEB 3: 3411, 1989]. As a result, these novel immunosuppressants have made a significant contribution to the survival of patients. However, since these inhibitors exhibit inhibitory functions in tissues other than immune tissues, there is a problem in that side effects such as toxicity to the kidney or liver are high.

Since the immune response that occurs in the rejection of transplantation depends mainly on T cells, drugs that inhibit the function of T cells show higher efficacy. For example, cyclosporin A and FK506 inhibit the transcription of cytokine genes to inhibit T cell activation, the mechanism of action of which is summarized as follows.

Cyclosporin A and FK506 bind to immunophillins such as Cyclophilins and FK506 binding proteins (FKBPs) to form drug-immunophilin complexes. This complex binds to and inhibits the enzyme activity of calcineurin, a calcium and calmodulin dependent serine / threonine phosphatase. When such an enzyme is inactivated, the transcription factor NF-AT in the cytoplasm cannot be translocated into the nucleus, and cytokine genes such as interleukin-2 are not expressed. Cyclosporin A, however, has the problem of showing toxicity and side effects even when used in an effective amount. FK506, on the other hand, is 10 to 100 times more potent than cyclosporin A in inhibiting in vitro interleukin-2 transcription and in vivo transplant rejection, but also exhibits side effects such as neurotoxicity and nephrotoxicity.

Another potent immunosuppressive agent, rapamycin, inhibits interleukin-2 dependent lymphocyte proliferation. The intracellular target mTOR of the rapamycin-FKBP complex has phosphoinositized kinase activity and the cell cycle proceeds as a result of mTOR signaling in response to cytokines.

In this way, all of the immunosuppressive agents affect the function of T cells. Cyclosporin A and FK506 interfere with the activation of T cells, while rapamycin inhibits the proliferation of T cells. However, targets of these inhibitors are also expressed in tissues other than immune tissues. Therefore, a substance that selectively inhibits a target molecule specifically expressed in immune cells, particularly T cells, will be an ideal and more efficient immunosuppressive agent, and its development is urgently required.

Accordingly, the present inventors pay attention to the fact that the extract of Curcuma xanthorrhiza inhibits the early rejection reaction and the expression of interleukin-2 more effectively than the conventional immunosuppressive agent, and confirms that the active ingredient is nagging sol. Was completed.

It is an object of the present invention to provide a novel use of naxazole as an immunosuppressant isolated from extracts of Curcuma xanthorrhiza.

In order to achieve the above object, the present inventors separated the nagging sol from Curcuma xanthorrhiza, an Indonesian medicinal plant. Separation, extraction, and purification of naxazole sol was in accordance with the method of the Republic of Korea Patent (Publication No. 2002-0074937).

First, the present inventors confirmed that naxazole inhibits mouse mixed lymphocyte reaction (MLR), using recombinant T cells designed to know the expression level of interleukin-2 gene, and using transformed T cells. It was confirmed that naxazole effectively suppressed the expression of the interleukin-2 gene. In addition, in the heart transplant experiments in mice, it was confirmed that nagging sol effectively maintains the activity of the transplanted heart, thereby significantly increasing the survival of heart-receptive mice.

Naxazoles are involved in the inhibition of gene expression of T cell proliferation factor, interleukin-2, inhibition of cell surface expression of CD69, inhibition of cell surface expression of interleukin-2 receptor, and signaling through T cell receptor (TcR). It showed activity such as selective inhibition of tyrosine phosphorylation to signal mediators, and suggests that this activity is an immunosuppressive effect induced by a mechanism different from cyclosporin A, which is widely known as an immunosuppressive agent.

Hereinafter, the present invention will be described in more detail with reference to Examples.

Example

Example 1 Determination of Naxazole Sol on T Cell Proliferation

Naxorisol was isolated and purified according to the Republic of Korea Patent Publication No. 2002-0074937 was used in the experiments described in this example and the following examples. T cells isolated from spleen cells of normal mice were suspended at 6 × 10 ⁶ / ml in RPMI 1640 medium (hereinafter referred to as “complete medium”) to which 10% fetal calf serum was added. 50 μl was aliquoted into a 96 well round bottemed plate. In the negative control group, 150 μl of each complete medium was added to the plate, and the experimental control group contained OKT3 and anti-clonal antibody OKT3, which specifically binds to CD3 epsilon chain and CD28 of T cell receptor. 50 μl of the solution in which 10 ug of each CD28 monoclonal antibody was dissolved and 100 μl of complete medium were added together. The experimental group was serially diluted (10, 10 ⁰ , 10 ^-1 , 10 ^-2 , 10 ^-3 , 10 ^-4 μg / ml) to the nasal sol in a complete medium, and this solution was added to each well. 50 μl and 100 μl of complete medium were added together to make 200 μl of the total amount per well. All plates were incubated for 3-4 days in 37 ° C., 5%, CO ₂ thermohygrostat (hereinafter referred to as “culture vessel”), and 10 μl (0.1 Ci) of 3H-TdR (Tritiated thymidine, 20.0 Ci / mmol). ) Was added to each well and incubated for 6 hours. After incubation, the cells of each well are adsorbed onto glass filter fibers with a cellharvester and dried, and then the amount of ³ H-TdR contained in the cells with a scintillation beta counter. The proliferation inhibitory response of T cells was examined by measuring. As shown in Figure 2a, nagging sol (●) showed a better immunosuppressive effect than CsA at low concentrations of less than 10 ^-1 ㎍ / ㎖ compared to CsA (○), the negative control standard (□) is T It did not inhibit the proliferation of the cells.

On the other hand, when T cell proliferation was induced using another T cell proliferation inducer Concanavalin A, the proliferation inhibitory ability of naxazole was confirmed. For the experimental control group, the experiment was performed in the same manner as in the above experiment except that 50 µl of a solution in which 40 µl / ml of concanavaline was dissolved instead of OKT3 and anti-CD28 monoclonal antibody. As shown in Figure 2b nagging sol (●) showed an immunosuppressive effect in the lower concentration range (10-2 ㎍ / ㎖) compared to CsA (○), negative control standard (□) is the proliferation of T cells Did not inhibit.

Example 2 Measurement of Inhibitory Activity of Naxazoles on Interleukin-2 Gene Expression

Human T cancer cells (Jurkat transfectant, Jurkat TAg) transformed with a recombinant gene fragment designed to indirectly determine the expression level of the interleukin-2 gene by combining a gene fragment that binds a transcriptional activator (NF-AT) and an alkaline phosphatase gene. ) Was suspended in RPMI 1640 medium containing 10% fetal bovine serum, 2 mM L-glutamine and 100 units penicillin, then laid down to 1x10 ⁵ / well, 4 M Ionomycin, 64 nM paramethoxyamphetamine ) And naxazole were treated at concentrations of 10 ⁻¹ , 10 ⁻² , 10 ⁻³ , 10 ⁻⁴ μg / ml and incubated for 24 hours. Next, the cells were precipitated by centrifugation, the culture solution was removed, and the precipitated cells were washed twice with a phosphate buffered saline solution, and each well was alkaline phosphatase test medium (1M diethanolamine, 0.5 mM MgCl ₂ , 150 μl of 10 mM homoarginine, 0.032 g 4-Nitrophenyl phosphate disodium salt hexahydrate) was added and reacted at 37 ° C. for 2 hours. Next, the degree of inhibition of expression of the interleukin-2 gene of naxazole was evaluated by measuring the absorbance at 410 nm using an ELISA reader. As shown in Figure 3 nagging sol (●) showed the same degree of immunosuppressive effect compared to CsA (○), negative control standard (□) did not inhibit the expression of the Il-2 gene in T cells. It was.

Example 3 Determination of Apoptosis Induction Capacity of Naxazole

The degree of apoptosis induction in human T cancer cells (Jurkat) by naxazole was determined. Cultured human T cancer cells 1x10 ⁶ cells / ml were washed 2 to 3 times with physiological saline and treated with naxazole at concentrations of 10, 1, 10 ^-1 , 10 ^-2 , 10 ^-3 , 10 ^-4 , 0 Thereafter, apoptosis induction of cells was confirmed on an agarose gel using a DNA fragmentation assay. As shown in FIG. 4, naxazoles effectively induced apoptosis from a concentration of 1 mg / ml.

Example 4 Combination Therapeutic Effects with Other Immunosuppressants

Cyclosporine A (CysA) and FK506 (FK), which are well known as immunosuppressive agents, and Totomycetin (IS-1), which have been shown to have immunosuppressive activity (PNAS August 6, 2002, vol. 99, no. 16, 10617-10622 ) And immunosuppressive ability in combination with naxazole sol (IS-2), respectively. The efficacy of the combination of naxazole / cyclosporin A (IS-2 / CysA), naxazole / FK506 (IS-2 / FK) and naxazole / totomycetin (IS-2 / IS-1) combinations was observed with cyclosporine A, FK506 and toto Compared with the case of using mycetin alone. When used in combination with naxazole as shown in Figure 5, it showed a much higher immunosuppressive effect compared to using CsA, FK506 or totomycetin alone.

Example 5 Inhibition of Tissue Rejection

In order to investigate the inhibitory effect of the nasal sol on the rejection induced by organ transplantation, the following cardiac transplantation experiments were conducted in mice: Anesthetized 8-10 week old Lewis mice The abdomen and the rib cage were incised, perfused 50 units (2 ml) of heparin through the lower eutrophic vein (IVC). Infusion was perfused to the myocardium via coronary artery. Observing the color of the heart, when the color of the heart turned white, the aorta, pulmonary artery, upper process vein and bilateral lungs were cut in turn, the heart was extracted, and stored in organ preservation solution. The anesthesia was then anesthetized, the abdominal cavity was dissected to expose the abdominal aorta and the inferior vena cava, and then the Satinsky clamp was used to block the inferior vena cava and the aorta together. Under 10-0 polypropylene sutures, the aorta of the heart of the donor mouse and the abdominal aorta of the recipient mouse, respectively, stored in the long-term preservation solution by continuous sutures, were single-sided sutures. After reperfusion, ventricular fibrillation was followed to observe the regular contraction of the atria and ventricles, and the abdominal wall was sutured with sutures. From the time of implantation in the electric heart-receiving mouse, the heart beat was palpated through the abdominal wall while injecting nasal sol injection preparation daily. At this time, when the heartbeat was not palpated at least two times, it was determined that the transplantation heart was rejected, and this period was compared with the case where cyclosporin A was administered.

As a result of measuring the activity of the transplanted heart of the recipient mouse, it was observed that naxazole increases the survival of the heart of the recipient mouse at a concentration similar to cyclosporin A, thereby significantly increasing survival compared to the control group. Therefore, it was confirmed that nagging sol significantly suppressed the rejection reaction caused by organ transplantation.

Table 1 shows the effects of naxazole and cyclosporin A on the cardiac transplant animal model described above.

Table 1

drug Concentration (mg / kg) Injection method Survival days Number of rats Cremophor sole 55 I.P.I.P. 910 43 CsA / Cremophor 5 I.P. > 100 2 Nagging Sol / PBS 5 I.P. 10 3 Nagging Sol / PBS 5 I.P. > 160 12 Nagging Sol / ME 3 I.V > 160 8

Example 6 Effect of Naxazole in Rheumatoid Arthritis

To induce arthritis in rats with Type II collagen (Type II collagen-induced rat arthritis test), Wistar rats (females, 150-200 g) were dissolved in 0.1 M acetic acid solution at 2 mg / ml. 0.5 ml of a solution emulsified overnight with bovine type II collagen and the same amount of incomplete Freund's adjuvant was injected into the right hind paw and tail. After 7 days rat tails were boosted again by injecting 0.2 ml of the same solution.

As described above, after 17 days of inducing arthritis with type II collagen, arthritis-induced rats were divided into four groups. In group 1, the progression of arthritis was examined without drug administration. Group 2 was administered steroid drugs, nasal sol in group 3, and methotrexate in group 4 at 1 mg / kg of rat body weight daily. . Steroid-based drugs are widely used in the treatment of inflammation, methotrexate is a drug that acts directly on immune cells and is most commonly used for rheumatoid arthritis, but it is known as a highly toxic drug. 6 is a result of verifying the drug efficacy by analyzing the amount of cytokine IL-4 involved in rheumatoid arthritis in the blood after 4 weeks of administration to naxazole, steroid-based drug, methotrexate. Naxazole has almost the same efficacy as methotrexate, and it is thought that the therapeutic effect of rheumatoid arthritis is superior to steroidal drugs.

As described above, the novel immunosuppressive agent comprising the naxazole sol of the present invention as an active ingredient inhibits mouse mixed lymphocyte response, effectively inhibits the expression of the interleukin-2 gene, and in particular, controls the rejection reaction occurring during organ transplantation. Effective in

1 shows the chemical formula of naxazole.

Figure 2a is a result of analysis on the proliferation inhibitory ability of naxazole sol when OKT3 and anti-CD28 monoclonal antibody induced the proliferation of Jurkat T cancer cells.

Figure 2b is an analysis of the proliferation inhibitory ability of nagging sol in the case of inducing proliferation of Jurkat T cancer cells using concanavalin A.

3 shows the results of measuring the inhibitory activity of naxazoles against interleukin-2 gene expression using transformed T cancer cells.

4 is a flow cytometry result of measuring the apoptosis inducing ability of naxazole sol using Annexin V and FITC fluorescence staining method.

5 is a graph showing the effect of combination treatment when naxazole is used in combination with cyclosporin A or FK506.

Figure 6 is a result confirming the inhibitory effect of tissue rejection reaction of naxazole sol by comparing the survival time of organ transplant mice.

Claims (5)

Pharmaceutical composition for the treatment or prevention of immune diseases containing xanthorizol or derivatives thereof as an active ingredient. 2. The pharmaceutical composition according to claim 1, wherein the immune disease is an autoimmune disease, a transplant rejection of an organ or tissue, a chronic transplant rejection, or a graft-versus-host disease. The pharmaceutical composition according to claim 2, wherein the autoimmune disease comprises lupus, rheumatoid arthritis, diabetes, myasthenia gravis, and multiple sclerosis. 4. A pharmaceutical composition according to any one of claims 1 to 3, wherein naxazole inhibits the synthesis or secretion of interleukin-1 or interleukin-4, or both. 4. A pharmaceutical composition according to any one of claims 1 to 3, which is used in combination with one or more other immunosuppressive agents comprising cyclosporin A or derivatives thereof and FK506 or derivatives thereof.