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Title:
NOVEL USE OF ANGELAN FROM ANGELICA GIGAS FOR TREATING DIABETES MELLITUS
Document Type and Number:
WIPO Patent Application WO/2001/060386
Kind Code:
A1
Abstract:
The present invention relates to a novel use of angelan from Angelica gigas for treating diabetes mellitus, more particularly, a composition for treatment and prevention of diabetes mellitus comprising angelan isolated from Angelica gigas as an active element. Angelan isolated from Angelica gigas can prevent destruction of beta cells and also treat or prevent diabetes mellitus.

Inventors:
KIM HWANMOOK (KR)
HAN SANGBAE (KR)
LEE CHANGWOO (KR)
LEE KIHOON (KR)
JEON YOUNGJIN (KR)
AHN KYUNGSEOP (KR)
Application Number:
PCT/KR2000/000880
Publication Date:
August 23, 2001
Filing Date:
August 10, 2000
Export Citation:
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Assignee:
KOREA RES INST OF BIOSCIENCE (KR)
KIM HWANMOOK (KR)
HAN SANGBAE (KR)
LEE CHANGWOO (KR)
LEE KIHOON (KR)
JEON YOUNGJIN (KR)
AHN KYUNGSEOP (KR)
International Classes:
A61K31/715; (IPC1-7): A61K31/715; A61K31/726
Foreign References:
KR990031277A
Other References:
Y.J. JEON ET AL.: "Activation of NF-kappa B/Rel in angelan-stimulated macrophages", IMMUNOPHARMACOLOGY, vol. 43, no. 1, 1999, pages 1 - 9
S.B. HAN ET AL.: "Characteristic immunostimulation by angelan isolated from angelica gigas nakai", IMMUNOPHARMACOLOGY, vol. 40, no. 1, 1998, pages 39 - 48
Attorney, Agent or Firm:
Lee, Duckrog (Yeil Bldg. 700-19, Yorksam-dong Kangnam-ku Seoul 135-080, KR)
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Description:
NOVEL USE OF ANGELAN FROM ANGELICA GIGAS FOR TREATING DIABETES MELLITUS TECHNICAL FIELD The present invention relates to a novel angelan from Angelica gigas for treating diabetes mellitus, more particularly, a composition for treatment and prevention of diabetes mellitus comprising angelan as an active component.

BACKGROUND ART Diabetes mellitus and its complications are diseases with a high fatality rate in the world, together with cancer and cardiovascular diseases. According to a report issued by the National Commission on Diabetes, the fatality rate of diabetes is still continuously increasing. Diabetic patients, compared to normal persons, are highly susceptible to suffer from such complications as blindness, kidney disease and heart disease. Now, by the insulin therapy the acute or fatal symptoms of diabetes can be controlled, but the long-term complications reduce life expectancy.

Diabetes mellitus is classified into insulin-dependent diabetes mellitus (Type 1) and non-insulin-dependent diabetes mellitus (Type 2). Insulin-dependent diabetes mellitus is caused by damage of insulin-producing pancreatic beta cells, which leads to decrease of the amount of insulin and finally results in hyperglycemia.

Most insulin-dependent diabetes mellitus is the consequence of progressive beta-cell destruction during an asymptomatic period, often extending over many years.

In the prediabetic period, circulating islet-cell autoantibodies and insulin autoantibodies may be detected. Insulin-dependent diabetes mellitus has been regarded as an autoimmune disease, and this hypothesis has been strengthened by the studies on the nonobese diabetic (NOD) mice and the BioBreeding (BB) rats. Both of these animals develop insulin-dependent diabetes mellitus spontaneously and their diabetic syndromes share many pathological features with that of humans with insulin-dependent diabetes mellitus. NOD mice, which can naturally fall into insulin-dependent diabetes mellitus, may usually start to expose the symptoms of diabetes in 12-14 weeks, and 80% of all mice have the symptoms. Lymphocytes may be activated by unknown factors, and permeate into pancreatic ducts. In the islet, the lymphocytes destroy beta cells and then bring about diabetes. Insulin acts on muscle, liver or fat cells to promote glucose metabolism and to lessen the level of glucose in blood. The diminution of insulin in blood by destruction of beta cells leads to deactivation of glucose metabolism in muscle or liver cell, resulting in diabetes mellitus. It has been reported in many studies that beta cell antigen, macrophages, helper T cells, cytotoxic T cells (CTLs), and the like are concerned with the outbreak of diabetes mellitus, and that oxygen free radicals or cytokines from lymphocytes are responsible for destruction of beta cells.

Isolation and characterization of angelan has already been reported (Immunopharmacology 40,39-48,1998). In the component analysis of the immunostimulating fraction of hydrothermal extract from Angelica gigas, polysaccharide content occupies most of the fraction components, or 80-90% (w/w) ; proteins are 7-8% ; and uronic acids are 15.5-68%. Calcium and magnesium ion is contained relatively in a large quantity, and Fe, Al, Mn, Zn, K, Na, P, and S is also included. The polysaccharide composition of angelan was examined. Each purified

sample was hydrolyzed with 2 M trifluoroacetic acid, and then analyzed using TLC and ion-exchange HPLC. From the analysis, the polysaccharide was composed of galacturonic acid, galactose, and arabinose in a large quantity, and contained mannose, rhamnose, and xylose in a small quantity. It was proved that angelan contains galacturonic acid, galactose, and arabinose as major components, and that the immunostimulating activity of angelan originates from these pectic polysaccharides and inorganic compounds.

Angelan activated macrophages and natural killer cells to augment the expression of IL-6 and IFN-gamma, and Th cells were secondarily affected by angelan (Immunopharmacology 40,39-48,1998). Particularly, angelan enhanced the immune function of macrophages strongly. When macrophages was treated with angelan, the expression of NO, iNOS, IL-1, IL-6 was greatly increased, which was proved to result from activation of NF-kB transcription factor (Immunopharmacology 43,1-9,1999).

Also, angelan selectively increased the proliferation of B cells, but had no influence on the proliferation of T cells. Angelan potentiated the in vivo T-dependent antibody response and showed a strong anti-cancer effect. Consequently, we can see that angelan is an immunopotentiating agent to increase immune functions and can be used as an anti-cancer agent.

Diabetes research has been directed toward prevention and cure of insulin- dependent diabetes mellitus. Studies on prevention of insulitis and treatment of diabetes has mainly utilized the experimental models of diseases in laboratory animals such as NOD mice, and most therapeutic strategies for treatment of diabetes mellitus are directed to suppression and regulation of autoimmune response in order to prevent beta- cell destruction. Autoimmune disease, insulin-dependent diabetes mellitus results from

increasing abnormalities of cellular immunity.

Various immunotherapies for preventing destruction of pancreatic beta-cells have been attempted. Neonatal thymectomy is the method of suppressing the outbreak of diabetes mellitus in NOD mice by killing T lymphocytes. Also, it has been known that depletion of macrophages or T cells using antibodies to T cell-dependent antigens represses diabetes. Some reports discloses that diabetes mellitus can be prevented by controlling production and reaction of free radical, for example, NO released from lymphocytes by antioxidants such as nicotinamide, vitamin E, probucol, MDL29311, and U78518F, as has been reported.

To date, researches on immunosuppressive therapy are continued. However, treatment of diabetes mellitus utilizing glucocorticoids and cyclophosphamide has proved to be largely unsuccessful. Although studies on the use of cyclosporin A, rapamycin, and FK506 in diabetes appear to be encouraging, generalized immunosuppression involves potential complications including infections and drug- induced kidney and liver damage. Furthermore, curing with long-term administration can occasionally induce cancer. Studies on immunosuppressive therapy for diabetes mellitus is progressing, and the suppression of diabetes of NOD mice using cytokines such as IL-4 and IL-10 has been reported. Also, such immuno-regulatory agents as OK- 432, LZ-8, BCG, and CFA have been reported to control diabetes mellitus of NOD mice, but their mechanism has yet to be established. Although diabetes mellitus can be treated with immunosuppressive agents, the side effects such as the excessive immunosuppression and toxicity to liver or kidney can be occurred.

The inventors achieved this invention, confirming that angelan as a polysaccharide from Angelica gigas has an excellent effect on prevention and treatment

of diabetes mellitus without any side effect.

DISCLOSURE OF THE INVENTION The purpose of the present invention is to provide a composition comprising angelan from Angelica gigas as an active component for prevention and treatment of diabetes mellitus.

After the hydrothermal extraction from Angelica gigas, the ethanol precipitation was applied to the filtrate to collect the polymer fraction, and then two kinds of polysaccharides, or acidic and neutral polysaccharides were obtained with an anion-exchange resin, DEAE-cellulose adsorption method, wherein acidic one of the two was named as angelan. The purpose of the invention was achieved by examining the effect of angelan for prevention and treatment of diabetes mellitus, in administrating angelan to NOD mice.

The present invention consists of following steps: the step of comparing the urine glucose level of angelan-injected NOD mice with that of control group, not receiving angelan, in order to verify the diabetes-suppressive effect of angelan from Angelica gigas ; the step of observing the transition of pancreatitis to insulitis in angelan-injected NOD mice, comparing to the case of control group, to verify the insulitis-suppressive effect of angelan; the step of measuring the expression level of cytokine mRNA in the separated spleen of NOD mice after a two-day intraperitoneal injection of the inventive angelan, to confirm the diabetes-suppressive activity of angelan based on its regulation of the cytokine production; the step of transplanting splenocytes of non-angelan-injected mice to NOD. Scid mice and measuring the

occurring level of diabetes, to confirm that the inventive angelan regulates immune responses and suppresses the outbreak of diabetes; the step of checking it by analyzing difference between the blood glucose level of ICR mice with and without intraperitoneal injection of streptozotocin that angelan cannot inhibit the beta cell destruction by streptozotocin, to confirm that the inventive angelan has selective regulation of immune responses resulting in suppression of diabetes mellitus; and the step of examining their weight and mortality to investigate the side effects of angelan on NOD mice.

The inventors purified angelan from Angelica gigas using previously disclosed method. The minced root was heated in boiling water for 1 hr and was filtered using the fourfold of gauzes and filtering papers (No. 4), and then the supernatant was precipitated two times with 3 volumes of ethanol at 4°C for 3 hr, followed by centrifugal separation, resulting in collection of the brown polymer fraction. This polymer fraction was easily obtained using the ethanol precipitation method, and after boiling the re- dissolved fraction in water for 20 mins, the precipitation of denatured proteins was not observed. Considering these results, the polymer fraction was presumed to comprise plenty of non-proteinic polymers. Also, most of the colored matter of the fraction was adsorbed by DEAE-cellulose resin. Two kinds of polysaccharides, or acidic and neutral polysaccharides were prepared with the DEAE-cellulose adsorption method. Both the two contained a little protein, and the acidic one was named as angelan, of which molecular weight is above 1 Mdalton.

The inventors examined therapeutic effects of angelan from Angelica gigas on diabetes mellitus. Doses of the inventive angelan were 30 mg/kg/day every other day with intraperitoneal injection. The dose level, route, and schedule of administration, however may vary depending on the condition of the subject.

Angelan suppressed completely the crisis of diabetes mellitus of NOD mice.

Prevention and cure of diabetes mellitus by angelan was examined by a test, in which the urine or blood glucose level had been compared to that of control and significant drop was observed. Angelan inhibited lymphocyte infiltration into pancreatic ducts, to suppress insulitis. Activation of T lymphocytes and macrophages plays an important role in autoimmune disease system. In activation of T cells, IL-2 cytokine has a significant role. Thus it has been reported that the inhibition of IL-2 production leads to the suppression of activating T cells, consequently resulting in the treatment of the autoimmune diseases. Immunosuppressive agents such as cyclosporin A inhibit the expression of IL-2 and suppress the activation of T cells. IL-12, the cytokine which macrophages produce, stimulates Thl cells to increase the production of IL-2. Angelan suppressed the expression of IL-2 and IL-12 in NOD mice. The decrease of IL-2 and IL- 12 production played an important role in treating diabetes mellitus.

To date, researches on immunosuppressive therapy are continued. However, treatment of diabetes mellitus utilizing glucocorticoids and cyclophosphamide has proved to be largely unsuccessful. Although studies on the use of cyclosporin A in diabetes appear to be encouraging, the generalized immunosuppression involves potential complications including infections and drug-induced kidney and liver damage.

Furthermore, curing with long-term administration can occasionally induce cancer.

The inventive angelan was proved to treat diabetes mellitus without any side effect. Because of diabetes, NOD mice have their weight decrease and come to die in 2 weeks after symptoms of diabetes. NOD mice, receiving angelan, have their weight not decrease and any mice were not found dead. This result addresses that the angelan removed diabetes and prevented the death of the mice, as well as has no side effect in

animal treatment. Angelan has no toxicity to kidney, liver and lung.

Insulin-dependent diabetes mellitus can be induced by streptozotocin.

Streptozotocin destroys selectively pancreatic beta cells, and diabetes mellitus induced by streptozotocin is similar with diabetes naturally occurring in NOD mice. NOD mice mediate the autoimmune responses to break the beta cells, whereas streptozotocin does not do that. Angelan could not suppress the destruction of beta cells occurring by streptozotocin. This result shows that angelan is a substance which regulates selectively the immune responses to suppress the outbreak of diabetes mellitus.

NOD mice are the best disease animal models in studies on insulin-dependent diabetes. NOD mice can naturally fall into insulin-dependent diabetes mellitus and the symptoms are very similar with those of human. The effects of angelan on preventing and treating of diabetes were examined using the NOD mouse model.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 shows prevention and cure of diabetes mellitus by administrating angelan. Diabetic mice were selected according to the urine glucose level, and the percentage is based on the total mice (10 heads). Open circles (o) are of control NOD mice and closed circles (o) are of NOD mice receiving angelan.

FIG. 2 illustrates decrease of blood glucose by administration of angelan. After we isolated the plasma from NOD mouse, the blood glucose was measured with the blood biochemical analyzer (Cibacorning, USA).

FIG. 3 illustrates the decline of insulitis by administration of angelan.

FIG. 4 illustrates the decline of insulitis by administration of angelan. Control

NOD mice (A) suffered from severe insulitis, but NOD mice receiving angelan (B, C, D) had their insulitis decreased.

FIG. 5 shows the change of expression pattern of lymphocyte's cytokines by administration of angelan. A is of 6-week-old NOD mouse without diabetes, B is of 11- week-old NOD mouse with diabetes, and C is of 11-week-old NOD mouse receiving angelan.

FIG. 6 illustrates the occurring level of diabetes mellitus in transplanting spleen cells from NOD mice to NOD. Scid mice (Open circles (o) are of the group transplanted with splenocytes of NOD. Scid mice with diabetes crisis, and closed circles (-) are of the group transplanted with splenocytes of NOD mice receiving angelan).

FIG. 7 illustrates the effect of angelan on the experimental group with streptozotocin-induced diabetes. Blood glucose values were measured with Accutrend sensor system (Boehringer Mannheim, Germany).

FIG. 8 shows that angelan has no side effect. NOD mice receiving angelan showed no drop of their weight and were never led to death.

EXAMPLES The following examples are offered to illustrate this invention and are not meant to be construed in any way as limiting the scope of this invention.

Example 1 Prevention and cure of clinical diabetes by administration of angelan Angelan was intraperitoneally injected in 30 mg/kg from 8 weeks to 24 weeks

of age, and the urine glucose was measured with uropaper (Eiken Chemical Co. Ltd., Japan) every week. In control group of NOD mice, not receiving angelan, the urine glucose was first detected from 15 weeks of age, and at 24 weeks, 80% of the mice had the urine glucose detected (FIG. 1). Experimental group of NOD mice with administration of angelan didn't have the urine glucose detected, and this result shows complete suppression of clinical diabetes by administration of angelan. Average blood glucose of non-angelan-injected NOD mice was 500 mg/dl, which means diabetes mellitus crisis, and that of angelan-injected NOD mice was 100 mg/dl, which means the normal condition (FIG. 2). Consequently, it was proved that angelan could prevent clinical diabetes in NOD mice completely.

Example 2 Suppression of insulitis by administration of angelan In insulin-dependent diabetes mellitus, insulitis may be induced by lymphocyte infiltration into pancreatic ducts and destruction of beta cells thereby. Macrophages and T cells are the major infiltrating lymphocytes, and these immune cells destroy beta cells.

The pancreas from both the angelan-injected and the non-injected NOD mice were preserved in formalin and to preparation. After staining with hematoxylin/eosin, the lymphocyte infiltration level was measured. The value of infiltration, or 1,2, and 3 points were respectively given to 25,50, and 75% of insulitis progression, and the average value of insulitis progression was calculated (FIG. 3). The NOD mice, not receiving angelan had more than 2 points, which means over 50% of infiltration, while the infiltration level of the angelan-injected mice was less than 0.5 point, which means below 10% of infiltration. This result showed that angelan could suppress the

lymphocyte infiltration into pancreatic ducts to debilitate insulitis. FIG. 4 shows the result of pathological tests: A indicates the condition of insulitis of non-angelan-injected mice, showing that most of pancreatic ducts were infiltrated by lymphocytes; B, C, and D show that lymphocyte infiltration was reduced by administration of angelan. This experiment's results demonstrated that angelan suppressed insulitis, resulting in prevention of occurrence of diabetes mellitus.

Example 3 Inhibition of cytokine production by angelan Th cells and macrophages have been known to play an important role in the pathogenesis of diabetes mellitus. According to unknown mechanism, macrophages are activated, and the activated cells produce IL-12. Then IL-12 activates Thl cells to promote production of IL-2 and IFN-gamma, and these cytokines activate cytotoxic T cells (CTLs) and macrophages. The activated CTLs and macrophages destroy beta cells and reduce production of insulin, resulting in occurrence of diabetes mellitus. In this manner, beta cells are disrupted through multi-steps of various mechanisms. Cytokines have a significant part in these immune responses, and control of cytokine production is expected to be the prevention and cure of diabetes mellitus. FIG. 5 illustrates the change of cytokine expression of lymphocytes by angelan. Angelan was intraperitoneally administered to NOD mice from 8 weeks age every other day. In splenocytes from 11- week-old mice, the cytokine mRNA expression level was measured using RT-PCR (the reverse transcription polymerase chain reaction). A is of 6-week-old mouse, B is of 11- week-old one, and C denotes the cytokine expression of 11-week-old mouse receiving angelan. 6-week-old one is of normal condition without the symptoms of diabetes or

insulitis, and 11-week-old one is at the starting point of insulitis. In other words, the 11- week-old mouse is in the situation that the immunologic functions are abnormally activated and lymphocyte infiltration is started. At the age of 11 weeks when the functions are brisk, the immunologic functions of mouse's lien cells and their regulation by angelan were measured. In 11-week-old mouse, the mRNA expression of IL-2 increased intensively, and by administration of angelan, the expression of IL-2 was strongly suppressed. The intense increase of IL-2 expression in spleen cells from 11- week-old NOD mouse means that IL-2 plays an important role in occurrence of diabetes, and the suppression by angelan shows that the diabetes-suppressing effect of angelan mainly originates from angelan's suppression of IL-2 expression. Angelan suppressed the immune response of Thl cells by inhibiting the mRNA expression of IL-12. From above results, it becomes clear that angelan regulates the cytokine production of lymphocytes, resulting in suppressing the occurrence of diabetes mellitus.

Example 4 Transplantation test to NOD. Scid mouse To examine that prevention and cure of insulin-dependent diabetes by angelan is the result of its regulation of immunologic reactions, the test of transplantation to NOD. Scid mouse was performed. NOD. Scid mouse is a mouse without T lymphocytes, so never suffer from diabetes. The splenocytes from NOD mice used in Example 1 were transplanted to NOD. Scid mice, and symptoms of diabetes mellitus were observed. All of the NOD. Scid mice, which the splenocytes of the 25-week-old NOD mice not receiving angelan of the mice in Example 1 had been transplanted to, fell into some symptoms of diabetes mellitus after 6 weeks (FIG. 6). This result shows that T cells in

the transplanted splenocytes destroyed beta cells of NOD. Scid mice to cause diabetes mellitus, and consequently proves that diabetes mellitus in NOD mouse is induced by the immune responses. NOD. Scid mice, to which the splenocytes of NOD mice receiving angelan had been transplanted, didn't show the symptoms of diabetes. This result confirmed that angelan regulates the immune responses to suppress disruption of beta cells by T cells.

Example 5 Effects of angelan on the streptozotocin-induced diabetes Streptozotocin, which can induce Insulin-dependent diabetes mellitus, destroys selectively pancreatic beta cells, and diabetes mellitus induced by streptozotocin is similar with diabetes naturally occurring in NOD mice. NOD mice mediate the autoimmune responses to break the beta cells, whereas streptozotocin does not do that.

ICR mouse received 60 mg/kg of streptozotocin three times with intraperitoneal injection. Streptozotocin was used in solution of citrate buffer (pH 4.2,4°C). The blood glucose level of ICR mouse treated with streptozotocin was 450 mg/dl, whereas that of normal ICR mouse without streptozotocin was 200 mg/dl (FIG. 7). Angelan couldn't inhibit the destruction of beta cells by streptozotocin. This result proves that angelan cannot suppress the destruction of beta cells by chemical compounds, but can suppress diabetes mellitus of NOD mouse by selectively regulating immune responses.

Example 6 Side effects of angelan In NOD mice receiving angelan from 8 to 24 weeks age, their weight and

mortality were checked in order to examine if angelan has some negative functions (FIG.

8). Since NOD mice receiving angelan showed no drop of their weight and were never led to death, angelan was proved to have no side effect.

INDUSTRIAL APPLICABILITY Through the aforementioned examples, it has been addressed that angelan is a therapeutic agent for prevention and cure of diabetes mellitus by suppressing destruction of beta cells. Therefore, angelan in the present invention is greatly useful for the medical and pharmaceutical industries relating to prevention and cure of diabetes mellitus.