KR20010074413A - Th2 adjuvant including Ovalbumin adsorbed in aluminium hydroxide for treating Behcet's disease - Google Patents

Abstract

PURPOSE: Th2 adjuvant including ovalbumin absorbed in aluminium hydroxide adjuvant is provided, which can be used for treating Behcet's disease as an inflammatory disease generated in the Asian countries and the Mediterranean Sea coastal countries. CONSTITUTION: The titled Th2 adjuvant contains ovalbumin absorbed in aluminium hydroxide adjuvant. The amount of aluminium hydroxide is 0.2 to 10mg per 60g weight and ovalbumin is 0.2 to 50 microgram per 60g weight. Cutaneous gangrene and inflammatory diseases show improved effect by injecting ovalbumin-aluminium.

Description

TF2 adjuvant including Ovalbumin adsorbed in aluminum hydroxide for treating Behcet's disease

The present invention relates to a Th2 adjuvant used in the treatment of Behcet's disease, and more particularly, to a Th2 adjuvant including ovalbumin adsorbed on aluminum hydroxide adjuvant used in the treatment of Behcet's disease.

In general, Behcet's disease is one of the most threatening diseases in countries around Asia and the Mediterranean, including Japan, Korea, and China, and is an inflammatory disease that affects many organs, including the eye. Treatment of patients with Behcet's disease progressed in several ways. These treatments include local, systemic or medical treatments, but they have not achieved satisfactory results. Several experiments have shown that immunological changes play an important role in the pathogenesis of Behcet's disease. Immune changes seen in Behcet's disease include T cell activity and abnormal function of white blood cells. Immunologically, mucosal lesions first penetrate CD4, CD8 cells, macrophages and dendritic cells, and then neutrophils. Because immunological processes are involved in a series of developments of Behcet's disease, several drugs that modulate the immune response have been used to treat Behcet's disease. These agents include corticosteroids, colchicine, cytotoxic agents and immunophilin ligands such as cyclosporin and the like. However, these drugs have a problem that can cause severe side effects when used for a long time.

The present invention has been made to solve the above problems, it is an object of the present invention to provide a new therapeutic material used to treat Behcet's disease.

In order to achieve the above object, the present invention provides a Th2 adjuvant comprising ovarianbumin adsorbed on aluminum hydroxide adjuvant used in the treatment of Behcet's disease.

In the field of immunology, "adjuvant" generally acts to unspecifically increase the immune response to a specific antigen, thereby reducing the frequency of injections as well as the amount of antigen required for a given vaccine, or by slowing the release of antigen to produce a vaccine. As used herein, the term "Th2 adjuvant" used in the present invention activates T helper type 2 (Th2) cells to increase the secretion of Th2 cytokines IL-4 and IL-10, resulting in excessive An adjuvant that functions to inactivate activated T helper type 1 cells.

Since the amount of aluminum hydroxide used in the present invention may be toxic at 10 mg or more per 60 grams of body weight and less effective at 0.2 mg or less, 0.2 to 10 mg per 60 grams of body weight is preferable, and more preferably 2 mg per 60 grams of weight is suitable.

Ovalbumin used in the present invention was used chick ovalbumin, the amount can be used 0.2 to 50 ㎍ per 60 grams of weight, but 2 ㎍ is appropriate. The amount of ovalbumin is determined by the amount of aluminum hydroxide, which helps the aluminum hydroxide to work efficiently, and the amount that shows the best effect in the smallest amount is 2 µg.

The administration method and duration of the present invention is administered by intraperitoneal injection by mixing 2 μg of Ovalbumin adsorbed on 2 mg of aluminum hydroxide adjuvant in 1 ml of physiological saline per 60 grams of body weight. Dosing is intraperitoneally 2-3 times at intervals of 1-3 weeks.

In another aspect, the present invention provides a method for treating Behcet's disease in mice using ovabubumin adsorbed on the aluminum hydroxide adjuvant.

Recently, there has been an interest in Th1 and Th2 in patient cytokines. To understand whether macrophages play a role in HSV-induced Behcet's disease mouse model, the inventors of the present invention found that ICR with crodronate (Cl ₂ MDP, lip-clodronate) encapsulated with liposomes to knock out macrophages. Mice was injected intraperitoneally. Behcet's disease occurred in 7 of the 25 HSV treated mice, whereas 20 mice treated with HSV bound with crodronate encapsulated with liposomes showed no Behcet's disease. To determine the possible role of cytokines affected by macrophage knockout in the development of Behcet's disease, mRNA expression in the spleen was examined by RT-PCR. The Th2 cytokine, IL-4, was expressed 21 days after the first crospolate encapsulated with liposomes and mice treated with HSV with crodronate encapsulated with liposomes and HSV. , But not in mice treated with HSV alone. The Th1 cytokines IL-2 and IFN- [gamma] were expressed in crodronated mice encapsulated with all experimental soldier liposomes, mice treated with HSV and mice treated with HSV and crodronate encapsulated with liposomes. Ovalbumin-aluminium was injected into Behcet's disease mice to see if Th2 cytokine relieves Behcet's disease. After Behcet's disease symptoms, 2 ug of Ovalbumin adsorbed on 2 mg of Al (OH) ₃ was injected intraperitoneally. Ovalbumin-aluminum immunity improved the symptoms of Behcet's disease in six of nine. For splenocyte reaction, splenocytes were separated 7 days after the first Ovalbumin-aluminum injection and splenocytes isolated from spleen tissue were cultured for 2 days with or without adding 0.17 ug / ml of Ovalbumin. By RT-PCR, IL-10 was detected in splenocytes of ovalbumin-aluminum immunized mice. IL-10 was also detected in the spleens of mice with improved symptoms of Behcet's disease by direct injection of 2 ug of Ovalbumin adsorbed on 2 mg of Al (OH) ₃ into the abdominal cavity.

In conclusion, macrophages knockout reduces the incidence of Behcet's disease induced by HSV through production of the Th2 cytokine, IL-4. Another cytokine induced by Ovalbumin-aluminum injection, IL-10, ameliorated the symptoms of Behcet's disease. Thus, Th2 cytokines play an important role in the improvement of HSV induced Behcet's disease symptoms in mouse models.

Chronic, recurrent inflammatory symptoms, a typical symptom of Behcet's disease, are associated with macrophage and cytokines secreted from the macrophages in the inflammatory process of Behcet's disease. Therefore, the inventors of the present invention thought that macrophage knockout had an effect on the incidence of Behcet's disease in a mouse model, and in order to knock out macrophages, the inventors of the present invention opted for crodronate intraperitoneal injection. Crodronate was used by Boehringer Mannheim, Germany.

Hereinafter, the present invention will be described in detail through non-limiting examples and experimental examples.

EXAMPLE

animal

Four to five week old male ICR mice were used in the present invention. Using the method of Hirata et al. ( Acta Otolaryngol Suppl 1993.503 : 79-81), the earlobe of the mouse was wounded using a needle and then herpes simplex at 1.0 x 10 ⁶ plaque forming unit / ml. Inoculated with virus type 1 (F strain). Viral inoculation was performed twice at 10 day intervals and 16 weeks of observation. As a control, culture medium was inoculated on the same site of the mouse. Then, for T cell and macrophage inactivation, crodronate, anti-mac1 and -mac2 antibodies and cyclosporin A encapsulated in liposomes were injected into the abdominal cavity. This knockout method followed the method of Encyclopaedia Yun ( Diabetes 1991. 40: 1568-1597). Mice were raised in a room with a light cycle of 12 hours starting at 8 am, 20-22 ° C. Mice had free access to food and water, and animals were photographed during the experiment.

Preparation of crodronate encapsulated with liposomes

Crodronate encapsulated with liposomes was prepared according to the method of Ruijin N ( J. Immunol Meth 1989. 124: 1-6). Briefly, 75 mg of Sigma's phosphatidyl choline and 11 mg of Sigma's cholesterol are dissolved in chloroform in a round bottom flask. After low vacuum rotary evaporation at 37 ° C., the lipid was dispersed by gentle rotation in 10 ml of PBS dissolved with 2.5 g of dichloromethylene diphosphonate (Cl ₂ MDP). The resulting liposomes were washed twice at 100,000 × g to remove free, unencapsulated crodronate, and then the liposomes were redispersed in 4 ml PBS.

Inactivate Macrophage

Mice were intraperitoneally treated with 2 mg / day of anti-mac 1 and -mac2 monoclonal antibodies two days before, one day before, and one and two days after infection. Another group of mice received crodronate encapsulated with 200 ul of liposomes two days before virus inoculation.

RNA extraction and RT-PCR

Total RNA was obtained by guanidium thiocyanate-phenol-chloroform extraction (Anal Biochem 1987. 162: 156-159). The spleen tissue was made up of 1 ml of 4M guanidine solution (Aldrich), 25 mM sodium citrate at pH 7.0, 0.5% sodium N-lauroyl sarcosinate (Fisher), and 0.1 M 2-mercaptoethanol (Sigma). After homogenization with extraction buffer, guanidium thiocyanate is involved in RNA extraction. To the sample was added 1/10 volume of chloroform: isoamyl alcohol (49: 1) and allowed to stand on ice for 5 minutes and then centrifuged at 10,000 xg for 15 minutes at 4 ° C. The RNA in the upper water layer was collected, precipitated with the same amount of isopropanol, and washed twice with 70% ethanol. RNA pellets were dissolved in distilled water, quantified by OD 260/280 crystals and identified under ethidium bromide stained agar gel. 2 ug of total RNA was reverse transcribed using cDNA kit (Gibco BRL), oligo dT primers and AMV reverse transcriptase to make cDNA for use as template in PCR amplification. 2 ul reverse transcriptase reaction was carried out in a reaction mixture consisting of 50 mM KCl (pH 8.4), 20 mM Tris-HCl, 2.5 mM MgCl, 200 uM dNTP, 2.5 U Taq polymerase (Gibco BRL) and 1.2 uM primer. Added. Specific primers for cytokines and beta-actin are as follows.

β-actin primer (Murray LJ, et al. Eur J Immunol 1990, 20: 163-170)

Sense: 5'-TGGAATCCTGTGGCATCCATGAAAC-3 '

Antisense: 5'-TAAAACGCAGCTCAGTAACAGTCCG-3 ',

IL-2 primer (Yokota T., et al. PNAS USA 1985. 82: 68-72)

Sense: 5'-TGATGGACCTACAGGAGCTCCTGAG-3 '

Antisense: 5'-GAGTCAAATCCAGAACATGCCGCAG-3 ',

IL-4 primers (Chan SY, et al., Transplantation 1995. 59: 1155-1161)

Sense: 5'-ACGCCATGCACGGAGATGGAT-3 '

Antisense: 5'-CAAGCATGGAGTTTTCC-3 ',

IL-10 primer (Moore KW, et al, Science 1990, 248: 1230-1233)

Sense: 5'-AGACTTTCTTTCAAACAAAGGACCAGCTGGA-3 '

Antisense: 5'-CCTGGAGTCCAGCAGACTCAATACACACTGC-3 ',

IFN-γ primers (Murray LJ, et al. Eur J Immunol 1990, 20: 163-170)

Sense: 5'-AGCGGCTGACTGAACTCAGATTGTAG-3 '

Antisense: 5'-GTCACAGTTTTCAGCTGTATAGGG-3 '.

Amplification was denatured at 94 ° C. for 5 minutes with Perkin Elmer Thermo Cycler 900, 30 seconds at 94 ° C .; 30 seconds at 56 ° C .; 35 cycles were performed according to the 1 minute profile at 72 ° C. The product was electrophoresed on 1.8% agarose gel and checked under UV.

Flow Cytometric Analysis of Intracellular Cytokines

Splenocytes were freshly obtained before intracellular cytokine staining. To allow cytokines to accumulate in the Golgi apparatus, 5 ug of brefeldin A (Sigma) was added during the last 4 hours of incubation. Cells were collected, washed with culture medium containing brefeldin A, fixed with 4% formaldehyde and washed with PBS containing 1% fetal bovine serum, 0.1% sodium azide and 0.1% saponin. Anti-IL-4, IL-10, IFN- ?? with avidin-Fluorochrome Stain with antibody (Caltag). Samples were collected at least 20,000 cells and analyzed on a flow cytometer (FACS Vantage, Becton Dickinson).

Adjuvant

a. Th2 adjuvant: Intraperitoneally mix Ovalbumin-Aluminum (2 mg Al (OH) ₃ and 2 ug Ovalbumin per 1 ml PBS) based on 60 grams of body weight.

b. Th1 adjuvant: 100 ul Freund's complete adjuvant (FCA) and 2 ug of ovalbumin (Immunology, 1998, 93:41) repeatedly until stable emulsion was formed through a double herb emulsifying needle based on 60 gram body weight. -48, Brewer JM) intraperitoneally mixed with 1ml PBS.

Experimental Example 1: Splenocyte Reaction

Mouse groups were injected once intraperitoneally with 2 ug of Ovalbumin emulsified in PBS, aluminum or FCA. After one week, the spleens were removed, and the splenocytes were separated and cultured. The spleens were inoculated with 0.17 ug / ml of Ovalbumin and propagated for 2 days, and cytokine production was examined by RT-PCR.

Mice with Behcet's disease symptoms induced by HSV inoculation were injected intraperitoneally with 2 ug of Ovalbumin emulsified in PBS, aluminum or FCA. When symptoms improved after Ovalbumin treatment, cytokine production in spleen tissues was investigated by RT-PCR.

For Th2 adjuvant, 2 ug of Ovalbumin adsorbed on 2 mg of Al (OH) ₃ adjuvant was injected intraperitoneally, one week after the first Ovalbumin-aluminum injection, the spleen was excised, and then the splenocyte suspension was 0.17. It consisted of two days of incubation with or without ug / ml of Ovalbumin.

For Th1 adjuvant, intraperitoneal injection of Ovalbumin-Front Complete Adjuvant (FCA), resected the spleen one week after the first Ovalbumin-Fread Complete Adjuvant injection, and then 0.17 ug / ml It consisted of incubating for 2 days with or without treatment of Ovalbumin.

By RT-PCR, IL-10 was detected in splenocytes media and Ovalbumin treated media of mice immunized with Ovalbumin-Aluminum.

Ovalbumin-Freundant adjuvant injected mice expressed only Th1 cytokine, IL-2 and IFN-γ. To see if Th2 cytokines relieve Behcet's disease symptoms, Ovalbumin-Aluminum was injected into Behcet's disease mice to induce Th2 cytokine production. The injection method consists of intraperitoneal injection of 2 ug of ovalbumin adsorbed on 2 mg of Al (OH) ₃ adjuvant at three week intervals after Behcet's disease symptoms occur. Ovalbumin-aluminum immunization improved Behcet's disease symptoms in 6 of 9 mice. All six improved had skin ulcers and inflammation on the face, nape, back and abdomen. After the Ovalbumin-Aluminum injection, all skin problems were resolved, and three unimproved had eye and nerve involvement. As a control, mice injected with Ovalbumin-Freundant Adjuvant did not improve or worsen dermatitis, eye and nerve invasion symptoms, and some died.

Number of ICR mice according to symptoms after HSV challenge and / or immune cell inactivation process Behcet's disease symptoms (% of mice) Culture medium 0/32 (0) HSV 14/50 (28.0) Liposome-Croconate + HSV 0/20 (0)

Results of Th1 / Th2 cytokine mRNA expression in the group treated with liposomes encapsulated with crodronate, HSV-encapsulated crodronate, or HSV only IL-2 IFN-γ IL-4 IL-10 β-actin a.Crodronate encapsulated in liposomes + + + - + b.Clipronate + HSV with liposome encapsulation + + + - + c.HSV + + - - +

The inoculation sequence in Table 2 is as follows. a is crodronate encapsulated with liposomes (day 0) → crodronate encapsulated with liposomes (10 days) → killed animals (21 days), b is crodronate encapsulated with liposomes (day 0) → HSV ( 2 days) → crodronate encapsulated with liposomes (10 days) → HSV (12 days) → experimental animal kills (21 days), c is HSV (0 days) → HSV (10 days) → killed experimental animals (21 days) ).

Splenocyte Response Results in In Vitro Cultures IL-4 IL-10 Improved number / total mouse count aluminum - - 0/3 Ovalbumin-Aluminum - + 6/9 Ovalbumin-FCA - - 0/3

[Experimental Example 2: Adaptive Transfer]

Adoptive transfer was performed to determine whether the symptoms of Behcet's disease induced by HSV influx the splenic cells of mice immunized with Th1 or Th2 adjuvant to improve the symptoms of Behcet's disease. Ovalbumin were injected into the vein of the aluminum processing splenocytes, ovalbumin -FCA treated spleen cells, separated by separation of normal mouse spleen cells, each 1 x 10 ⁷ of spleen cells to mouse BD. Behcet's disease mice injected with Ovalbumin-aluminum-treated splenocytes improved skin ulcer symptoms, while Behcet's disease mice injected with Ovalbumin-FCA-treated splenocytes or splenocytes of normal mice showed no deterioration or changes in skin ulcer symptoms. Did.

The results of this experiment are shown in Table 4.

Adaptive Transfer Results Ovalbumin-Aluminum Treated Splenocytes Treated with Behcet's Disease Mice Improvement Ovalbumin-FCA Treated Splenocytes in Behcet's Disease Mice worse

Ovalbumin-aluminum immunization improved Behcet's disease symptoms in six of the nine mice used in the experiment. All six dogs had skin ulcers and inflammation on the face, nape, back, and abdomen, but after the injection of Ovalbumin-Aluminum, all skin problems were resolved, and the results of the adaptive transfer were improved. .

Claims (3)

Th 2 adjuvant comprising ovalbumin adsorbed on aluminum hydroxide adjuvant used to treat Behcet's disease. The Th2 adjuvant of claim 1, wherein the amount of aluminum hydroxide is 0.2 to 10 mg per 60 grams of weight, adsorbed on aluminum hydroxide adjuvant. The Th2 adjuvant of claim 1, wherein the amount of the ovalbumin is 0.2 to 50 µg per 60 grams of body weight.