WO2003059249A2

WO2003059249A2 - Nivalenol as specific inhibitor to transcriptional factor c-maf and pharmaceutical compositions comprising the same

Info

Publication number: WO2003059249A2
Application number: PCT/KR2003/000099
Authority: WO
Inventors: Chung-Yeon Choi; Yoon-Seok Chang; Yoon-Keun Kim; Kyeong-Eob Min; Sang-Hyeon Jo; In-Gyu Kim; Ju-Hong Jeon; Sang-Chul Park; Heun-Soo Kang
Original assignee: Metabolic Engineering Laboratories Co., Ltd.
Priority date: 2002-01-16
Filing date: 2003-01-16
Publication date: 2003-07-24
Also published as: WO2003059249A3; KR100485271B1; KR20030062137A; AU2003206148A8; AU2003206148A1

Abstract

The present invention relates to a pharmaceutical composition for treatment or prophylaxis of Th2 cytokine-related diseases, which comprises (a) a pharmaceutically effective dose of nivalenol as an inhibitor for the activity of IL-4, especially via c-Maf transcription factor inhibition, and (b) a pharmaceutically acceptable carrier.

Description

NIVALENOL AS SPECIFIC INHIBITOR TO TRANSCRIPTIONAL FACTOR C-MAF AND PHARMACEUTICAL COMPOSITIONS COMPRISING THE SAME

BACKGROUND OF THE INVENTION FIELD OF THE INVENTION

The present invention relates to a novel use of nivalenol and particularly, to its transcriptional inhibitor to c-Maf, a well-known transcriptional factor for

Th2 immune response, and pharmaceutical compositions comprising it.

DESCRIPTION OF THE RELATED ART

Asthma is an allergic disorder that mainly involves bronchus and its clinical manifestations are dyspnea, cough and wheezing. Despite of currently available medication, the morbidity and mortality of asthma is increasing worldwide, which demands the development of new therapeutic modality. Asthma is characterized by the reversible obstruction, airway hypersensitivity, and chronic eosinophilic inflammation of airway. T cell plays a central role during the course of allergic inflammation while eosinophil acts as an effector cell .

Nivalenol is low molecular trichothecene mycotoxin endogenously expressed by Fusarium spp . and represented by the following formula I .

Nivalenol, a fungal toxin contaminating crop grains and inhibiting protein expression, has been found to inhibit function of bone marrow and non-specifically increase IgA expression (Hinoshita, F. et al . , Nephron, 75:469- 478 (1997) ) . Recently, it was reported that nivalenol decreased antigen-specific IgE production in ovalbumin specific TCR transgenic mice (Choi, JY. et al, Toxicology and Applied Pharmacology 2000) . c-Maf, a well-known transcription factor having leucine zipper, was found to play tissue-specific role on the expression of interleukin-4 (IL-4) by enhancing IL-4 expression rather than to function in the initial signaling pathway (I-Cheng Ho et al . Cell , 85:973-983(1996)). Some attempts have been made to treat Th2 -related diseases by modifying the Th2 immune response. For instance, U.S. Pat. No. 6,086,898 discloses the conversion of Th2 type immunity into Thl type using Listeria adjuvant, whereas U.S. Pat. No. 5,958,671 discloses the method to isolate regulatory molecules of T cell subsets using Maf family proteins . Throughout this application, various patents and publications are referenced and citations are provided in parentheses. The disclosures of these patents and publications in their entitles are hereby incorporated by references into this application in order to more fully describe this invention and the state of the art to which this invention pertains.

SUMMARY OF THE INVENTION

The present invention describes that nivalenol effectively inhibits the activity of c-Maf transcription factor, thereby lowering the Th2 immunity.

Another object of this invention is to illustrate pharmaceutical compositions comprising nivalenol, as the active component, and appropriate carriers as supplementary components .

Other objects and advantages of the present invention^' will become apparent from the detailed description to follow taken in conjugation with the appended claims and drawings .

BRIEF DESCRIPTION OF THE DRAWINGS Fig.l represents alleviating efficacy of composition according to the present invention on bronchial hyperresponsiveness .

Fig. 2 represents the proportion of eosinophils in BAL (bronchoalveolar lavage) fluid depending on composition of the present invention.

Fig. 3a represents decrease of serum antigen-specific IgE depending on composition of the present invention.

Fig. 3b represents decrease of serum antigen-specific IgGl depending on composition of the present invention.

Fig. 3c represents change of serum antigen-specific IgG2a amount depending on composition of the present invention.

Fig. 4a represents decrease of IL-4 expression in splenocytes depending on composition of the present invention.

Fig. 4b represents decrease of IL-5 expression in splenocytes depending on composition of the present invention. Figs. 5a-5b represent Th2 cell-specific function of composition of the present invention (but do not function in undifferentiated T cell) .

Fig. 6a represents inhibition of IL-5 expression in D10G4.1 Th2 cell line depending on composition of the present invention. Fig. 6b represents change of IL-6 amount in D10G4.1 Th2 cell line depending on composition of the present invention,

Fig. 7 represents inhibition of the transcriptional activity of IL-4 promoter in D10G4.1 Th2 cell line depending on composition of the present invention.

Fig. 8 represents in vi tro inhibition of c-Maf transcription factor activity in NIH3T3 cell line depending on composition of the present invention.

Fig. 9 represents no cellular toxicity of composition of the present invention at shown concentration by MTT assay.

DETAILED DESCRIPTION OF THIS INVENTION

In one aspect of this invention, there is a nivalenol as a novel inhibitor against the transcriptional activity of IL-4, specifically an activity of c-Maf transcription factor.

Paracelsus, the father of pharmacology, stated that

"All substances are poisons; there is none which is not a poison. The right dose differentiates a poison and a remedy." In other words, all medicines are good remedy under appropriate treatment but toxin under abuse. It is common in pharmaceutical history to find the novel use of previous toxin as a good medicine. On the basis of the above pharmaceutical idiosyncrasy, the present invention describes the novel pharmacological function of nivalenol, a fungal toxin, as an inhibitor of the transcriptional activity of IL-4, specifically c-Maf transcription factor. As a major target of the nivalenol of the present invention, c-Maf, a leucine zipper transcription factor, plays its pivotal role on IL-4 expression in a tissue- specific manner by amplifying the signal for IL-4 expression. Various immune-related diseases such as allergy and asthma are caused by imbalance of Th2 cytokines . We have screened a number of inhibitors acting on the upstream signaling pathway of Th2 cytokine expression, and discovered that a well-known fungal toxin, nivalenol, effectively inhibits c-Maf transcription factor, thereby impeding Th2 cytokine expression.

Another aspect of this invention provides therapeutic or preventive compositions for Th2 cytokine-related diseases, comprising (a) a pharmaceutically effective dosage of nivalenol as inhibitor of c-Maf transcription factor, and (b) a pharmaceutically acceptable carrier.

Nivalenol, an active ingredient in the present composition, inhibits the activity of c-Maf resulting in inhibition of IL-4 expression in mature Th2 lymphocytes. Consequently, this invention provides evidence to show that nivalenol is a powerful candidate to treat Th2-related diseases .

Hereafter, the term "Th2 cytokines" refers to specific cytokines expressed in T helper 2 lymphocytes, such as IL-4, IL-5, IL-10 and IL-13, preferably IL-4 and IL-5 and preferably IL-4.

The Examples presented below illustrate that nivalenol inhibits IL-4 expression in mature Th2 lymphocytes. In a preferred embodiment of the present invention, nivalenol functions as a specific inhibitor to IL-4 expression in mature Th2 lymphocytes .

Th2-related diseases include allergic diseases, cancer as well as infectious diseases. Among them, allergic diseases have been ascribed to the nature of IgE expression. The present invention showed that nivalenol inhibits the binding of c-Maf transcription factor to IL-4 promoter and, therefore, the IL-4 production is inhibited. As a result, the expression of IgE is impeded. Thus, nivalenol has a potency for treatment or prophylaxis of allergic diseases. In the most preferred embodiment, the pharmaceutical composition of nivalenol in the present invention is effective particularly in the treatment of asthma.

Asthma is a chronic inflammatory disorder of the airways in which many cells and cellular elements play a role, in particular, mast cells, eosinophils, T lymphocytes, macrophages, neutrophils and epithelial cells (www.nhlbi.nih.gov/nhlbi/lung/asthma/prof/ashgdln.pdf) . In the past, asthmatic incidences increased at the rate of 5.6% in 1981, 10.0% in 1990 and 15% in 1997, respectively, among Korean children. Hereafter, "asthma" and "bronchial asthma" are used as equivalent terms. In bronchial asthma, a representative of chronic and allergic inflammatory diseases, bronchial inflammation is mainly under the control of CD4⁺ T lymphocytes and a decline of Th2 immune response owing to the action of allergen causes bronchial inflammation.

Current therapies for asthma largely configure the protection from contacting allergens, desensitization and drug treatment of steroid-based medicines. Anti- inflammatory drugs, developed recently, have failed to offer any significant advantage in both mortality and motility. Furthermore, there has not been any significant reduction in the incidence of severe asthmatics, which require steroid treatment . In fact, many asthmatic patients are still suffering from adverse side effects arising from chronic treatment with steroid drugs. A new therapeutic strategy is urgently in demand, which would effectively treat the asthmatic diseases without the afore-mentioned adverse side effects. Recent inventions, U.S. Pat. No. 5,871,734 and U.S. Pat. No. 5,767,065 disclose an asthmatic treatment employing antibodies to VLA-4 (Very Late Antigen-4 targeting IL-4 receptor) . Another asthmatic treatment employing Listeria adjuvant is described in U.S. Pat. No. 6,086,868. In many cancer patients, an increase in Th2 cytokines, resulting from the conversion of Thl immunity into Th2 immunity (Yamamura, M. et al . , J^". Clin . Invest . , 91:1005- 1010(1993); Pisa, P. et al . , P. N.A . S . USA., 89:7708- 7712(1992)), have been observed. Hence, the composition of the present invention described herein may be also effective in treating certain types of cancer. Additionally, various infectious diseases including AID/HIV infection, tuberculosis, Leishmaniasis, schistosomiasis and nematode infection also manifest an increase of Th2 cytokines and conversion of Thl immunity to Th2. (Shearer, G. M. et al . , Prog. Chem. Immunol . 54:21-43(1992); Clerici, M. et al . , Immunology Today 14:107-111(1993); Fauci, A.S. Science, 239:617-623(1988); Locksley, R. M. et al . , Immunoparasi tology Today 1 :A58-A61 (1992) ; Pearce, E. J. , et al., J^". Exp. Med. 173:159-166(1991); Grzych, J-M., et al . J^". Immunol . 141:1322-1327(1991); Kullberg, M. C, et al . , J^". Immunol . 148:3264-3270(1992); Bancroft, A. J. , et al . , J^". Immunol . 150:1395-1402(1993); Pearlman, E., et al . , Infect . Immun . 61:1105-1112(1993); and Else, K. J. , et al . , J. Exp . Med. 179:347-351(1994)). Hereafter, the term "prevention" or "prophylaxis" is meant to (a) prevention of disease from occurring in an animal, preferably mammal, more preferably, human which may be predisposed to the diseases but has not yet been diagnosed as having it; (b) an inhibition of the disease, i.e., arresting its development; and (c) an alleviation or relief of its symptom.

The pharmaceutically acceptable carrier contained in the present composition, may be varied. Examples of the carrier comprise conventional ones such as lactose, dextrose, sucrose, sorbitol, mannitol, starch, gum acacia, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxy benzoate, propylhydroxy benzoate, talc, stearic acid, magnesium and mineral oil without restriction thereto. The pharmaceutical compositions of this invention, may additionally contain wetting agent, sweetening agent, emulsifying agent, suspending agent, preservatives, flavors, perfumes, lubricating agent, or mixtures of these substances.

The pharmaceutical compositions of this invention may be administered orally or parenterally, and the parenteral administration comprises intravenous injection, subcutaneous injection and intramuscular injection. The correct dosage of the pharmaceutical formulation varies depending upon the mode of application, age, body- weight, sex of the patient, diet, time of administration, condition of the patient, drug combinations, reaction sensitivity and severity of the disease. It is understood that the ordinary, skilled physician will readily be able to determine and prescribe a correct dosage of this pharmaceutical composition. In an embodiment of the present invention, a preferred dosage of the present composition is the administration of once-daily with 1 mg nivalenol/kg body weight of an adult.

According to the conventional techniques known to those skilled in the art, the pharmaceutical compositions of this invention can be formulated with pharmaceutical acceptable carrier as described above and several forms e.g. a unit dosage form. Non-limiting examples of the formulations include a solution, a suspension or an emulsion, an extract, an elixir, a powder, a granule, a tablet or a capsule, and additional dispersing agent or stabilizer.

The following specific examples are intended to be illustrative of the invention and should not be construed as limiting the scope of the invention as defined by appended claims.

Example I: Therapeutic efficacy of nivalenol on bronchial asthma in asthmatic mouse model

1-1 : Induction of bronchial asthma in mice

Female BALB/c mice with age of 6-7 wks were purchased from Daehan biolink, Inc. (Korea) and were maintained under the specific pathogen free (SPF) condition. Mice were immunized by two intraperitoneal injections of 200 βi PBS solution containing 20 βg ovalbumin and 2 mg alum for 2 wks. Subsequently, on days 21, 22 and 23 the mice were inhaled with 1% ovalbumin by nebulization for 30 min

1-2: Treatment with nivalenol

Nivalenol (Sigma, USA) was administered orally, and the asthmatic symptoms including bronchial hyperresponsiveness, eosinophilic inflammation and increase of serum-specific antibody were evaluated after day 1 through 20, both in the control and nivalenol treated group, respectively.

1-3 : Methacholine bronchial provocation test (MBPT)

Bronchial responsiveness to methacholine was analyzed by measuring Penh (enhanced pause) in a Korean type Total Body Plethysmography (Allmedicus, Inc. Korea).

In the course of measuring bronchial responsiveness, fresh air was supplied through a bias-flow in an effort to keep mice from hypoxia, and main chamber of pneumotachograph was connected with outside to compensate change of surroundings caused by dust, noise, temperature, etc. Although airway contraction causes increase both of peak expiratory pressure (PEP) and peak inspiratory pressure (PIP) , the increase of PEP is superior and the expiratory time-relation time (Te-RT) is elongated comparing to relaxation time (RT) leading to the elongation of pause. In some experiments, Penh (enhanced pause) is the preferred index in measuring airway contraction. At 24 hrs after the last insufflation of ovalbumin, Penh was measured for 3 min employing the animal Total Body Plethysmography. Methacholine (2.5, 6.25, 12.5, 25 or 50 mg/dl; Sigma) was then insufflated for 3 min, and Penh was measured for 3 min. The results represent the average percentage increase of Penh by methacholine against average basal Penh (Fig. 1) . In Fig. 1, "AW" refers asthmatic mice treated with water, "AN" indicates asthmatic mice treated with nivalenol and "water" means normal mice treated with water.

In Fig. 1, PC200 is methacholine concentration (fflg/dL) required to achieve 200% increase of Penh and the lower PC200 indicates higher bronchial hyperresponsiveness . As shown in Fig. 1, nivalenol treatment significantly alleviates bronchial hyperresponsiveness (p<0.05).

1-4: Bronchoaveolar lavage (BAL) Following a methacholine bronchial provocation the mice were rested for 24 hrs (48 hrs after last ovalbumin insufflation) , and then mice were anesthetized with 50 mg/ mi ketamine (Yuhan corp., Korea) . After sampling 500 βi of blood from inferior vena cava the mice were then operated for ventrotomy. Catheter was inserted into airway using 24 gauge catheter. After ligation, bronchial aveoli were repeatedly rinsed with 0.5 mi sterile saline.

The bronchoaveolar lavage was then centrifuged for 5 min at 1000 rpm at 4^°C. The cells were resuspended in 2 mi of 10% FBS-IMDM, and counted on a hemacytometer . The resuspended cells were attached onto slide glass by mean of cytospin and stained with Diff Quick dye. A minimum of 300 inflammatory cells were classified into eosinophil, macrophage and lymphocyte, respectively, under microscope at magnification of 1000 x (Fig. 2) .

As shown in Fig. 2, average eosinophil number was slightly decreased without a significant statistical difference when compared with that of the negative control (p<0.05) .

1-5 : Effect of nivalenol on anti-ovalbumin antibody expression

The serum concentration of IgE antibody specific to ovalbumin was assessed by ELISA as follows. Ovalbumin was dissolved in bicarbonate buffer (Sigma. C-3041) to make 20 mg/mi , and coated 100 βi/vιe.11 onto a 96-well plate (Nunc . 442404) over night at 4^°C. The plate was rinsed 3-5 times with PBS containing 0.05% Tween-20 (Showa Chemicals Inc.), and incubated with 200 £/well of 3% BSA (Sigma. A-2153) in 0.05% Tween-20-PBS for 1-2 hrs at 37^°C.

Sample of 50-100 βi was diluted with 0.1% BSA in 0.05% Tween-20-PBS, added 100 βi per well and reacted with ovalbumin for 2 hrs at 37°C. After rinsing 3-5 times with 0.05% Tween-20-PBS, 100 βi of 2 mg/mi biotin-conjugated anti-mouse IgE antibody (Pharmingen, 02122D) was added for 1 hr at 37^°C. Finally, 100 β/well of SaV-HRP (Pharmingen. 13047E) diluted to 1:1000 in dilution buffer was reacted for 30 min at 37^°C . 100 βi/wel± of OPD substrate solution (Sigma, P-6912) containing 14.7 βi of phosphate-citrate buffer (Sigma, P-

4809) and 7.35 βi of H₂0₂ (Sigma, H-1009; light-protected) was added and reacted for 30 min. 50 /z£/well of 12.5% H₂S0₄

(Matsumoen Chemicals, Ltd.) was added to stop the reaction and the absorbance was measured at 490 nm using Microplate reader (Molecular Devices, THERMO max) . In Fig. 3a-3c, "AW" refers to the asthmatic mice treated with water, "AN" asthmatic mice treated with nivalenol and "water" normal mice treated with water, respectively. As shown in Fig. 3a-3c, the levels of serum antigen- specific IgE and IgGl were significantly decreased because of a treatment with nivalenol (p<0.05), but no significant change was observed in IgG2a in both groups (p<0.05) .

1-7: Effect of nivalenol on cytokine expression in splenocytes

Mice were treated with either ovalbumin alone or ovalbumin with nivalenol (0 , 30 , 60 , 120 or 250 ng/m) for 2 days and their splenocytes were removed and isolated. The expressions of interferon-γ (IFN-γ ), interleukin-4 (IL-4) and interleukin-5 (IL-5) were respectively measured as shown in Fig. 4a, 4b. In Fig. 4, "AW" denotes splenocytes isolated from asthmatic mice treated with water, and "AN" splenocytes isolated from asthmatic mice treated with nivalenol, respectively.

As shown in Fig. 4a and 4b, IL-4 expression was completely suppressed by nivalenol, while IL-5 expression decreased in a dose-dependent manner. undifferentited T(EL4) and differentiated T 2(D10G4.1) cell Example II: Specific effect of nivalenol on Th2 lymphocytes

The specific effects of nivalenol on Th2 lymphocytes were studied by cell cultures in which undifferentiated

T(EL4) cell line and differentiated Th2(D10G4.1) cell line were employed. After mixing each cell line with an appropriate amount of anti-Q CD3 monoclonal antibody (Pharmingen), a various amount of nivalenol (0 , 30 , 60 , 120 or 250 ng/mi) was added. The culture media were harvested after 24 hrs. And IL-4 level in the medium was assessed by a solid-phase sandwich ELISA as follows. Monoclonal antibody (Pharmingen) to IL-4 was coated on plate, the plate was blocked with 3% bovine serum albumin (BSA) , and prediluted 10 βi of culture media (100 /t /well) was reacted for 1 hr at RT . After rinsing the plate, polyclonal antibody against IL-4 conjugated with alkaline phosphatase was used as secondary antibody (Pierce) . The intensity of color was analyzed on a Microplate reader (Molecular Devices) .

As shown in Fig. 5a and 5b, IL-4 expression was increased with EL4 cells in the presence of anti-α CD3 monoclonal antibody without nivalenol treatment. By contrast, nivalenol treatment seems to have eliminated the effect. IL-4 expression by anti-α CD3 monoclonal antibody was suppressed to non-detectable level by nivalenol treatment . As shown in Fig. 6a and 6b, the increased IL-5 expression in D10G4.1 cell line by anti-α CD3 monoclonal antibody was also suppressed in a dose-dependent manner with respect to nivalenol concentration. Although IL-6 expression was increased by anti-α CD3 monoclonal antibody in D10G4.1 cells, nivalenol did not appear to affect the IL-6 expression.

Example III: Effect of nivalenol on IL-4 transcription

The effect of nivalenol on IL-4 transcription was evaluated by transforming the reporter vector into D10G4.1 cell line by electroporation (Neumann, E. et al . , EMBO J. , 1:841(1982)) . The cells were lysed and the supernatant was analyzed for luciferase activity using Luciferase activity analysis kit (Promega) . The luciferase activity after nivalenol treatment was also measured.

As shown in Fig. 7, luciferase activity of the transformed D10G4.1 cells was increased 30-fold in the presence of anti-α CD3 monoclonal antibody. However, addition of nivalenol dramatically suppressed the luciferase activity. The results indicate that the specific effect of nivalenol on IL-4 ascribes to its inhibitory effect on IL-4 promoter.

Example IV: Effect of nivalenol on c-maf transcription factor

The reporter vector employed in Example III was transfected into NIH3T3 cell line (KCLB, Korea) using

Lipo ectamine (Invitrogen) . In addition, c-maf was cotransfected into the transformed NIH3T3 cells following the manufecturer' s instructions. The NIH3T3 cells that had been cotransfected with reporter vector and c-maf gene were treated with nivalenol, and their luciferase activities were measured.

As shown in Fig. 8, overexpression of c-Maf transcription factor has increased the luciferase activity approximately 3 -fold. Nivalenol treatment appeared to have neutralized the increased luciferase activity to the basal level .

The results show that nivalenol suppressed the enhanced activity of IL-4, which was brought about by the c-Maf overexpression.

Example V: Toxicity of nivalenol

NIH3T3 cells were cultured, supplemented with 10% FBS- DMEM (Invitrogen) , in 24-well plate, in the presence of a various amount of nivalenol for 48 hrs. (Fig. 9) .

Subsequently, MTT (3- [4 , 5-dimethylthiazol-2-yl] -2 , 5- diphenyl) was added for 3 hrs at 37^°C, and the color development was evaluated using Microplate reader (Molecular Devices) . As shown in Fig. 9, MTT activities were similar in the samples with or without nivalenol even in the presence of a dosage that can inhibit c-Maf activity.

Thus, nivalenol would not induce any cellular toxicity at the working dosage level . Having described a preferred embodiment of the present invention, it is to be understood that variants and modifications thereof falling within the spirit of the invention may become apparent to those skilled in this art, and the scope of this invention is to be determined by appended claims and their equivalents.

Claims

What is claimed is :

1. A nivalenol compound as an inhibitor to the activity of c-Maf transcription factor, whereby IL-4 transcription is inhibited.

2. A pharmaceutical composition for treatment or prevention of Th2 cytokine-related diseases, which comprises (a) a pharmaceutically effective dose of nivalenol as an inhibitor of c-Maf transcription factor; and (b) a pharmaceutically acceptable carrier.

3. The pharmaceutical composition according to claim 2, wherein the nivalenol inhibits IL-4 expression of Th2 lymphocytes by inhibiting c-Maf transcription factor.

4. The pharmaceutical composition according to claim 2, wherein the Th2 cytokine-related diseases are allergic diseases, cancers or infectious diseases.

5. The pharmaceutical composition according to claim 4, wherein the allergic disease is bronchial asthma.

6. The pharmaceutical composition according to claim 2, wherein the infectious disease is HIV infection, tuberculosis, Leishmaniasis, schistosomiasis or nematode infection.