JP5883901B2 - Pharmaceutical composition and polya extract for the treatment of diseases induced by immune disorders - Google Patents

Description

The present invention relates to a novel use of lanostane compounds in the treatment of diseases induced by immune disorders such as allergies.

Description of Background Art Immunoglobulin E (IgE) is a type of immunoglobulin (or “antibody”) molecule. IgE is present in human serum at lower concentrations than other immunoglobulins: IgG, IgM, IgA and IgD. IgE is thought to have a protective role against parasites, but has been clearly established as a necessary or even useful role, at least in developed countries where parasitic infection is not a significant problem Absent. IgE is known as a mediator of allergic rhinitis (“hay fever”), asthma, hives, and immediate hypersensitivity allergic reactions including food and drug allergies.

  In an IgE-mediated allergic reaction, IgE is secreted by B cells and then binds to the FcεRI receptor present on the surface of basophils, mast cells and Langerhans cells through the Fc portion. If IgE bound to the surface of these cells now contacts and binds to the allergen, this causes cross-linking of the bound IgE molecule and the lower receptor, pharmacological mediators such as histamine, serotonin, leukotrienes, and slow anaphylaxis Trigger the release of reactive substances. These mediators cause pathological manifestations of allergic reactions.

  Some patients with a history of some or all IgE-mediated allergic conditions also suffer from painful skin symptoms called atopic dermatitis.

  Taking asthma as an example of allergic diseases, the immune reaction time can be divided into an immediate asthmatic reaction and a delayed asthmatic reaction. An immediate asthmatic response is a reaction caused by inflammatory mediators released from mast cells that occur within 15-30 minutes after contact with an allergen. When the patient is again exposed to the same allergen, the allergen identifies and binds to IgE bound to mast cells, activates the mast cells, and the granules in the cells contain more histamine, leukotrienes, IL-2, Degranulated to release inflammatory substances including cytokines such as IL-4, IL-5 and GM-CSF, and chemoattractant factors, thereby increasing vascular permeability, Airway smooth muscle contracts. These inflammatory substances, cytokines and chemotactic factors affect not only immediate asthmatic responses but also late-onset responses.

  Reactions caused by eosinophils and neutrophils are associated with late-onset reactions. Four to six hours after the immediate response, cytokines and chemotactic factors released from mast cells will attract inflammatory cells such as eosinophils and neutrophils, resulting in invasion. In addition, cytokines such as eotaxin secreted from epithelial cells, endothelial cells and fibroblasts are a major part of chemotactic factors, which further bring eosinophils or Th2 immune cells into sites of inflammation in the lung bronchi Move. Many inflammatory proteins such as MBP (major basic protein), ECP (eosinophil cationic protein), eosinophil-derived neurotoxin, EPO (eosinophil peroxidase) and leukotrienes are preferred when activated. It will be secreted from the acid sphere. These inflammatory proteins stimulate airway smooth muscles to contract and increase vascular permeability, causing airway edema so that the epithelial tissue of the airway is directly damaged, and thus epithelial cells lose their integrity. The airway secretes excess mucus, which not only obstructs or narrows the airway, but also causes neutrophil infiltration and mast cell degranulation, which increases eosinophil infiltration. As a result, the degree of asthma becomes more serious.

  The applicant of the present application disclosed a pharmaceutical composition useful in enhancing the immunity of the human body in Taiwan Patent Application No. 92113393 (Taiwan Patent Publication No. 200425900, published on December 1, 2004). The composition comprises an active ingredient lanostane compound. A Poria extract for enhancing the immunity of the human body is also disclosed, the extract comprising 5-60% by weight of lanostane compound and not secolanostane. Extracts are prepared from metabolite, sclerotium, or fermentation product of pinehod (Poria cocos (Schw) Wolf).

  Substances or methods that can treat allergies are an important issue not only for researchers but also for the general public. So far, there has been no report that lanostane compounds are effective in treating allergies.

SUMMARY OF THE INVENTION The primary object of the present invention is to provide a novel use of lanostane compounds in the treatment of diseases induced by immune disorders such as allergies.

  Another object of the present invention is to provide a pharmaceutical composition for the treatment of diseases (eg allergies) induced by immune disorders comprising a lanostane compound as an active ingredient.

  Another object of the present invention is to provide a method for treating diseases (eg allergies) induced by immune disorders using lanostane compounds.

  A pharmaceutical composition capable of treating a disease (for example, allergy) induced by an immune disorder in a mammal (for example, a human), wherein lanostane represented by the following chemical formula (I):

Wherein R ₁ is H or CH ₃ ; R ₂ is OCOCH ₃ , ═O or OH; R ₃ is H or OH; R ₄ is —C (═CH ₂ ) —C (CH ₃ ) ₂ R _a (where R _a is H or OH), or —CH═C (CH ₃ ) —R _b (where R _b is CH ₃ or CH ₂ OH); R ₅ is A pharmaceutical composition comprising H or OH; and R ₆ is CH ₃ or CH ₂ OH; or a pharmaceutically acceptable salt thereof in an amount effective to treat said disease.

  The present invention also provides the use of lanostane of formula (I) above in the preparation of a medicament for the treatment of diseases induced by immune disorders in mammals.

  Preferably, the allergy is allergic rhinitis, allergic conjunctivitis, allergic asthma, atopic dermatitis, throat allergy, atopic eczema, or rheumatoid arthritis. More preferably, the allergy is allergic asthma.

  Preferably, lanostane having the chemical formula (I) is

Or

It is.

  Preferably, the pharmaceutical composition comprises lanostane (I) or a pharmaceutically acceptable salt thereof at 0.1 to 60% by weight of the composition.

  The pharmaceutical compositions for the treatment of allergies according to the invention can generally be administered to patients orally, for example in the form of tablets, pills, soft capsules, hard capsules, fine granules, powders or pellets. The pharmaceutical composition of the present invention may be in the form of a liquid that can be administered orally or parenterally as an aqueous solution. The pharmaceutical composition of the present invention can be dispersed in a miscible solvent such as ethanol, water and administered parenterally in the form of a spray, lotion, ointment, tincture, emulsion or ampoule.

  Preferably, the pharmaceutical composition is administered orally.

  A further object of the present invention is the use of polya extract as a raw material for said lanostane (I). The polya extract contains 1-60% by weight of lanostane (I) and is substantially free of cecolanostan.

Preferably, the polya extract is prepared by a method comprising the following steps:
a) extraction of metabolites, fermentation products or sclerotia particles of matsuhod with water, methanol, ethanol or mixed solvents thereof;
b) concentrating the liquid extract obtained in step a);
c) introducing the concentrate obtained in step b) into a silica gel column;
d) elute the silica gel column with a less polar eluent, collect the eluate obtained; and e) concentrate the eluate to produce a concentrated eluate.

  Preferably, the concentrated eluate obtained in step e) has a chromatographic value (Rf) developed by a mixed solvent of dichloromethane: methanol = 96: 4 and detected by an ultraviolet lamp and iodine vapor, by thin layer chromatography. ) Is 0.1 or more.

  Preferably, the extraction in step a) is performed using 95% ethanol.

  Preferably, the extraction in step a) is performed by extracting the metabolite, fermentation product or mycorrhizal particles of matsuhod with boiling water; adding a base to the resulting aqueous extraction solution until the pH value is 9 to 11; Collecting the aqueous solution; adding acid to the basic aqueous solution until a pH value of 4-6 is reached, forming a precipitate; collecting the precipitate; extracting the precipitate with ethanol; and collecting the liquid extract .

  Preferably, the concentrate obtained in step b) is further extracted with a two-phase solvent containing 95% v / v aqueous methanol solution and n-hexane in a volume ratio of 1: 1, and the methanol layer is extracted from the two-phase solvent extraction mixture. And the methanol layer is concentrated and fed to a silica gel column in step c) to form the concentrate used.

  Preferably, the less polar eluent in step d) is a mixed solvent comprising dichloromethane and methanol in a volume ratio of 96.5: 3.5.

  Preferably, the polya extract comprises 5 to 35% by weight of the lanostane (I).

  Preferably, the pharmaceutical composition of the present invention further comprises a diluent, excipient or carrier.

BEST MODE FOR CARRYING OUT THE INVENTION In the following experiments conducted here, ovalbumin (OVA) was used as an allergen to induce mice to become allergic, and the appearance of mouse OVE-specific IgE antibodies. It was confirmed that the mice suffered from asthma induced from. During the experimental period, different groups of mice were fed with polya extract or pure lanostane compound, and after one month of breeding, each mouse was tested for airway hypersensitivity, thereby determining the severity of asthma in the mice. An important index that reflects We have also observed different immune cells in the mouse bronchoalveolar lavage fluid (BALF), which are particularly affected by eosinophils and neutrophils. Furthermore, eotaxin, which is a chemotactic factor, was an important observation factor for observing whether the secretion of cytokines changed dramatically in the present invention. From the observation of the above three pharmacological or inflammatory substances, airway hypersensitivity, inflammatory cells and chemotactic factor (eotaxin), polya extract and lanostane compound are excellent drugs for the prevention and treatment of asthma I understand.

  The polya extract disclosed in the present invention that promotes nutrient uptake by mammals (eg, humans) can be prepared in a manner similar to that disclosed in Taiwan Patent Publication No. 200425900, which A crude extract is obtained by extraction by a conventional extraction method, and the crude extract is chromatographed by chromatography using a low polarity fraction of lanostane (using an eluent of dichloromethane: methanol = 96: 4) and secolanostane (secolanostane). ) In a high-polar fraction (using an eluent of dichloromethane: methanol 90:10 and 0: 100), when developed by thin layer chromatography with a mixed solvent of dichloromethane: methanol = 96: 4 The lanostane fraction has a chromatographic value (Rf) of 0.1 or more. Incidentally detected, also secolanostane fraction is detected at Rf is less than 0.1. Some lanostane is separated by eluting the lanostane fraction by silica gel column chromatography. The eluent used here is dichloromethane: methanol = 97: 3 to 95: 5.

  The following examples are presented to further illustrate the present invention and should not be used to limit the scope of the present invention.

  Percentages and other amounts described herein are by weight unless otherwise specified. The percentages are selected in an arbitrary range such that the total is 100%.

Example 1:
Polya powder was produced from 30 kg of Poria cocos (Schw) Wolf grown in China. The polya powder was extracted with 120 L of 95% ethanol over 24 hours. This mixture was filtered to obtain a filtrate. The residue was extracted three more times and filtered. The filtrates were combined and concentrated to obtain a dry extract in an amount of 265.2 g. The dry extract was partitioned and extracted using a two-phase extractant (n-hexane: 95% methanol = 1: 1), from which the methanol layer was removed and then concentrated to a dry solid in an amount of 246.9 g. Got. Separation of the dry solid was performed using a silica gel column packed with silica gel having a weight 10 to 40 times that of the dry solid. The silica gel having a diameter of 70 to 230 mesh is Silica Gel 60 manufactured by Merck. The column was eluted using the following eluents in order: dichloromethane: methanol = 96: 4 mixed solvent; dichloromethane: methanol = 90: 10 mixed solvent; and pure methanol. The eluate was tested by thin layer chromatography (TLC), where a UV lamp and iodine vapor were used for detection, and a mixed solvent of dichloromethane: methane = 96: 4 was used as a developing solution. Eluates with similar composition in TLC were mixed.

  From the elution performed using a mixed solvent of dichloromethane: methanol = 96: 4, an amount of 78 g of PCM was obtained. The PCM showed 6 spot marks in thin layer chromatography. The eluate obtained by elution with dichloromethane: methanol = 90: 10 and pure methanol eluent was mixed to obtain a PCW portion in an amount of 168 g.

  The PCM portion was further separated using an eluent of dichloromethane: methanol = 96.5: 3.5 and a similar silica gel column, and K1 (K1-1 and K1-2), K2 (K2-1 and K2 -2), purified lanostane components of K3, K4, K4a, K4b, K5, K6a and K6b were obtained. The details of the separation step and identification analysis data are described in the above-mentioned Taiwan Patent Publication No. 200425900.

  The K1-K6b compound has the following structure:

  The amounts of the lanostane compounds K1 to K6b separated from the PCM part are listed in the table below. The PCM portion contains about 15% by weight of lanostane compounds K1 to K6b.

Example 2:
100 kg of polya was boiled in 800 kg of water for 3 hours, then allowed to cool to 50 ° C., adjusted to a pH value of 11 using 5N NaOH solution, and then the solution was stirred for 3 hours . A centrifuge was used to separate the liquid from the solid, and an additional 800 kg of water was added to the separated solid. The above procedure was repeated, including adjusting the pH value to 11 with NaOH, stirring and removing the solids by centrifugation. The resulting two liquids were combined and concentrated in vacuo at 50 ° C. to a 100 kg solution, and then the pH value was adjusted to 6.5 with 3N HCl to produce a precipitate. The precipitate was separated from the solution, followed by washing with 40 L H ₂ O and centrifuging to recover the precipitate; the precipitate was spray dried with 8 L of water to give 380 g of powder. Thereafter, the powder was extracted three times with 4 L of ethanol, and the extracted solutions were combined and concentrated to obtain 238.9 g of an ethanol extract. 238.9 g of this ethanolic extract was found to be free of secolanostan compound from TLC analysis of Example 1 and was then separated by HPLC, and for each gram of extract, 185.93 mg K2, 20.34 mg K3, K4 Gave 15.82 mg and K1 4.52 mg. In other words, 1 gram of extract contains about 226.07 mg of lanostane compound.

Example 3:
Experiments in the treatment of asthmatic mice using polya extract or pure lanostane compounds In this example, mice are induced to become allergic by using ovalbumin (OVA) as an allergen, and the mouse's OVE-specificity From the appearance of specific IgE antibodies, it was confirmed that mice suffered from induced asthma. During the experimental period, different groups of mice were fed with polya extract or pure lanostane compound, and after one month of breeding, each mouse was tested for airway hypersensitivity, thereby determining the severity of asthma in the mice. An important index that reflects In this example, different immune cells in mouse bronchoalveolar lavage fluid (BALF) were also observed as to whether they were specifically affected by eosinophils and neutrophils. Furthermore, eotaxin, a chemotactic factor, was an important observation factor for observing whether the secretion of cytokines changed dramatically in this example.

experimental method:
(1) Experimental animals: Inbred BALB / c female mice were bred using standard research food as appropriate. The room temperature was maintained at 19-24 ° C. and the relative humidity was 50-70%. Animal experiments were conducted in accordance with the guidelines for the protection and use of laboratory animals at Kennin University, Taiwan. BALB / c mice were divided into 10 groups of 8 mice.

(2) Establishment of an experimental asthma animal model: The immunization protocol has been described in the past (Sy, LB, et al. Propolis extractives an immunoregulatory activity an immunity in inflammatory inflammatory : 1053-1060. (2006)) and modified. Briefly, BALB / c mice were 10 μg / ml and 30 μg / ml OVA (albumin, chicken eggs, A-5503, Sigma) supplemented with 2 mg of adjuvant aluminum hydroxide (Al (OH) ₃ , 77161, Pierce). Were immunized at 8 weeks of age and 10 weeks of age, respectively. Mice after receiving the second intraperitoneal injection were placed in the chamber and all mice were exposed to 8 ml of 2% OVA aerosol for 20 minutes using an ultrasonic nebulizer (DeVilbiss Pulmo-Aide, 5650D, USA). Inhaled.

  (3) Grouping of experimental animals: 10 groups of OVA-sensitized asthmatic mice were treated with the untreated asthma group (As); the polya extract prepared in Example 2 and the pure compounds K1, K2 and K3. Treatment groups (1PCE, 1K1, 1K2, 1K3, 2PCE, 2K1, 2K2, and 2K3); and a control drug group (Pred). Each mouse in the treatment group of the 1PCE group received 0.0372 mg of the PCE ethanol extract prepared in Example 2 daily (1PCE). 2PCE means a mouse that received twice the dose of PCE (0.0744 mg) as compared to the 1PCE group. Each mouse in the 1K1, 1K2, and 1K3 treatment groups received 0.0087 mg of K1, K2, and K3, respectively, daily. 2K1, 2K2 and 2K3 mean mice that received twice the dose (0.0174 mg) of K1, K2 and K3 compared to the 1K1, 1K2 and 1K3 groups. Each mouse in the Pred group received 0.1 mg prednisolone per day for 5 consecutive days prior to lethality. The PCE, K1, K2, K3 and prednisolone compounds were first dissolved in ethanol and finally dissolved in a phosphate buffer solution (PBS). Each mouse was forcibly administered with a total volume of 0.4 mL each time.

(4) Airway hypersensitivity test:
Airway hyperresponsiveness (AHR) values increase in proportion to the severity of the asthma, and thus a decrease in AHR values is an important indicator for determining whether asthma has alleviated. For each mouse in all groups, the AHR test was performed on the day following the 29th day of dosing in the treatment group, with the last exposure (inhalation). AHR was tested using the Buxco system (Biosystem XA; Buxco Electronics Inc. Sharon, CT, USA), where mice were placed in chambers with different concentrations (25 mg / ml and 50 mg / ml) of PBS or methacholine aerosol. It was introduced into the chamber using an ultrasonic nebulizer. Three minutes after introduction, the average value of AHR per minute was recorded. Airway contraction induced by methacholine was more pronounced when methacholine concentration was increased. Penh (pause of enhancement) values were calculated by collecting data derived from the system's transducer (differential pressure transducer, Buxco) and preamplifier (MAX II, Buxco). The relative increase in Penh was calculated as Penh% = Penh _methacholine / Penh _PBS ; where Penh _methacholine is the mean value of Penh after the mouse inhaled methacholine aerosol for 3 minutes and Penh _PBS is 3 It is the average value of Penh after inhaling for a minute.

(5) Bronchoalveolar lavage fluid (BALF) and lung tissue structure:
All groups of mice that completed the AHR analysis were sacrificed the next day. Immediately the lungs were passed through a tracheal cannula (18 GA, angiocath, BD) Hanks balanced salt solution containing 2% fetal bovine serum (FBS) and 2 mM 2Na-EDTA (HBSS, SH30016.01, Hyclone, USA). Washed 3 times with 1 ml. About 3 ml of BALF was collected. The BALF collected at the first time was centrifuged at 1500 rpm for 5 minutes at 4 ° C. The collected BALF supernatant was stored at -20 ° C to determine the cytokine concentration. The BALF collected in the second and third rounds were combined and centrifuged under the same conditions as the BALF collected in the first round. Cells obtained by decanting the supernatant were tapped to separate them, and cells obtained from the first centrifugation were added thereto. The total cell number was determined using 0.5 ml of CM-10, with the cell density adjusted to 3 × 10 ⁵ cells / ml. In subsequent experiments, a Cytospin centrifuge (Cytospin 4 Cytocentrifugation, Thermo Shandon, USA) was used for cell immobilization. 200 μl of the cell suspension per mouse was centrifuged at 500 rpm for 4 minutes, and a glass slide was prepared on which the cell suspension was dried in air. The cells were stained with Liu staining solution (Liu A and Liu B, Delta, 232, Japan). The cells were observed under a magnification of 1000 times using a microscope (Olympus, BX41TF, Japan) equipped with an oil immersion lens. 200 leukocytes were counted on each slide, including 4 different cells: eosinophils, neutrophils, lymphocytes and monocytes. Experimental results were expressed as a percentage of a specific subpopulation of lymphocytes relative to total BALF cells.

(6) Eotaxin concentration measurement:
The eotaxin concentration in the bronchoalveolar lavage fluid was measured by sandwich-ELISA using a kit marketed under the trade name R & D. Briefly, an ELISA plate was first coated with specific Abs and left overnight at 4 ° C. Prior to performing the experiments, the plates were treated with 1% PBS-BSA and washed. The supernatant was then added to the ELISA plate, which was maintained at room temperature for 2 hours, after which biotin-conjugated Abs was added. After further maintaining at room temperature for 2 hours, avidin-bound HRP was then added and left for a further 2 hours. The substrate tetramethylbenzidine (TMB) was then added for coloring and the absorption wavelength was measured at OD450 wavelength. The concentration was calculated by interpolation based on standard values.

(7) Biological statistical analysis:
All data were expressed as mean ± SD. Because of the statistically significant differences, all analyzes were performed using Student's t-test compared to the As group treatment. A p value <0.05 was considered significant.

result:
1. From Table 1, it can be seen that as the methacholine concentration increases, the value of AHR also increases in the group. The 2K2 group showed the lowest AHR value compared to the As group, and when subjected to stronger methacholine (50 mg / mL) stimulation, the 1K1, 2K2, and 1K3 groups also had significantly lower AHR values than the As group. Show. Higher AHR values indicate that mice suffer from more severe airway hyperresponsiveness. The obtained results showed that the matsuhod extract and components of the present invention have an effect of preventing / treating asthma. The Pred group showed lower AHR values, but not statistically significant relative to the As group. Therefore, the matsuhod extract and components of the present invention are more advantageous in the prevention / treatment effect of asthma from the viewpoint of airway contraction caused by asthma, compared to general medical steroids for the treatment of asthma.

  2. From the results shown in Table 2, it can be seen that the As group has the highest percentage of eosinophils in BALF of OVA-sensitized mice, and the Pred group has the lowest percentage but no statistical advantage. However, the percentage of lymphocytes in the Pred group is significantly higher than that in the As group. Nevertheless, mice administered 1PCE, 2PCE, 1K1 and 2K1 have a significantly lower percentage of neutrophils in BALF compared to the As group. This result indicates that the components of Matsuhod can weaken the infiltration of inflammatory cells in lung tissue.

  3. During the active phase of asthma, many types of inflammatory proteins are involved in local inflammation. In this example, eotaxin concentration in BALF was analyzed and the results are shown in Table 3. Many types of cells can secrete eotaxin, including epithelial cells on the airway bronchus, endothelial cells and fibroblasts. Secreted eotaxin can induce eosinophils or Th2 cells into the airway bronchus (Rakin et al., Eotaxin and eosinophil recruitment: implication for human disease. Mol Med Today 6: 20-27.). From the data in Table 3, it can be seen that mice administered with 1PCE, 1K1, 2K1, and 1K3 have significantly lower eotaxin concentrations in BALF than the As group. No significant difference in eotaxin concentration was observed in the Pred group mice compared to the As group. This result supports that the components of matsuhod disclosed in the present invention can prevent infiltration of airway inflammatory cells by inhibiting secretion of inflammatory protein eotaxin. Therefore, the component of Matsuhod of the present invention is less effective in asthma in that it weakens the infiltration of inflammatory cells (eosinophils or Th2 lymphocytes) received in the lung tissue as compared with a general medical steroid (Pred) for treating asthma. More advantageous in terms of preventive / therapeutic effects.

Claims (7)

A therapeutic agent for allergy in mammals comprising lanostane represented by the following chemical formula (I) or a pharmaceutically acceptable salt thereof as an active ingredient:
R ₁ is H or CH ₃ ; R ₂ is OCOCH ₃ , ═O or OH; R ₃ is H or OH; R ₄ is —C (═CH ₂ ) —C (CH ₃ ) be a ₂ R _a (where R _a is H or OH) or -CH = C (CH ₃₎ is -R _b (where R _b is CH ₃ or CH ₂ OH); R ₅ is H Or OH; and R ₆ is CH ₃ or CH ₂ OH. The allergy therapeutic agent according to claim 1, wherein the allergy is allergic rhinitis, allergic conjunctivitis, allergic asthma, atopic dermatitis, throat allergy, atopic eczema, or rheumatoid arthritis. The allergy therapeutic agent according to claim 2, wherein the allergy is allergic asthma. The lanostane (I) is
Or
The allergy therapeutic agent of any one of Claims 1-3 which is these. The allergy therapeutic agent of any one of Claims 1-4 which contains the said lanostane (I) or those pharmaceutically acceptable salt in 0.1 to 60 weight%. The allergy therapeutic agent of any one of Claims 1-5 administered orally. The allergy therapeutic agent according to any one of claims 1 to 6, wherein the mammal is a human.