TW201440761A - Use of disodium norcantharidate for modulating the development of dendritic cells - Google Patents

Abstract

Novel Use of a small molecule, particularly, disodium norcantharidate, is disclosed herein. The disodium norcantharidate is useful as a lead compound for manufacturing a medicine or a pharmaceutical composition for ameliorating transplantation rejection.

Description

Use of disodium methyl succinic acid to regulate human dendritic cells

The present disclosure relates to a isolated from Wan phthalocyanine Coleoptera - Large cantharidin (Mylabris Phalerata) cantharidin (cantharidin, CA) derivative - demethylation cantharidic acid disodium (disodium norcantharidate, DNCTD, called "herein The novel use of the O6 compound"), which modulates the development and maturation of dendritic cells, is therefore a small molecule compound of a pharmaceutical or pharmaceutical composition potentially useful for the manufacture of anti-rejection drugs.

Mylabris refers to the dry worms from China's Mylabris Phalerata or Mylabris Cichorii , which have been used as Chinese herbal medicines for more than two thousand years. It is one of the medicinal herbs of many folk treatments.

It is known that cantharidin has anti-tumor activity, which can increase the number of white blood cells, but it also stimulates the urinary system to cause discomfort. The active ingredient in cantharidin is cantharidin (CA), but cantharidin is not easy to synthesize and has high toxicity. Therefore, people in this field have long hoped to find a derivative of cantharidin, which is still useful. Biologically active, but not as strong as CA. Accordingly, two derivatives of cantharidin, norcantharidin (NCTD) and disodium methylphylloate, were developed.

In the previous study, the inventors found that NCTD can develop dendritic cells in vitro, but its activity is poor, and it also promotes apoptosis. In vivo, NCTD can accumulate antigen-presenting cells and regulatory T cells. The donor donates the spleen and does not cause a strong immune response (see Hsieh et al., Transplantation 2011: 92(8), 848-857) and is therefore suitable for use as an immunosuppressive agent. Although previous studies have determined that NCTD is less cytotoxic than CA and is more suitable as an immunosuppressive agent than CA, NCTD still has a certain degree of cytotoxicity, which may cause apoptosis. Therefore, it is still necessary to develop a toxicity in this field. Lower cantharidin derivatives, used as immunosuppressive agents to make anti-rejection drugs.

The present disclosure is based, at least in part, on the discovery of cantharidin (CA) derivatives, which are cytotoxic and have modulating dendritic cell differentiation in humans. Mature characteristics come. The above findings suggest that disodium desmethyl decanoate in the present disclosure can be used as a lead compound for a therapeutic agent that reduces graft rejection.

Accordingly, a first object of the present disclosure is directed to the novel use of disodium desmethyl cinnamate to produce a pharmaceutical or pharmaceutical composition that inhibits differentiation or maturation of a dendritic cell. In the present disclosure, the inhibition refers to reducing the amount of expression of CD1a, CD40 or CD83 molecules on the surface of the dendritic cells.

Accordingly, an embodiment of the object of the present invention is to provide a pharmaceutical or pharmaceutical composition which inhibits differentiation or maturation of a dendritic cell. The pharmaceutical or pharmaceutical composition comprises a therapeutically effective amount of disodium succinate or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable Adjuvant.

The compound of the present invention, disodium desmethyl decanoate, is present in an amount of from about 0.1% to about 99% by weight based on the total weight of the pharmaceutical composition, based on the total weight of the pharmaceutical composition. In certain embodiments, the amount of the compound of the invention is at least about 1% by weight based on the total weight of the pharmaceutical composition. In a particular embodiment, the amount of the compound of the invention is at least about 5% by weight based on the total weight of the pharmaceutical composition. In other embodiments, the amount of the compound of the invention is at least about 10% by weight based on the total weight of the pharmaceutical composition. In further embodiments, the amount of the compound of the invention is at least about 25% by weight of the total weight of the pharmaceutical composition.

A second object of the present disclosure is directed to a method of inhibiting differentiation or maturation of a dendritic cell with disodium methyl succinate in vitro. The method comprises contacting the dendritic cells with a sufficient amount of sodium desmophyllin sodium or a pharmaceutically acceptable salt thereof in vitro. In one example, the sufficient amount of sodium norcantharidin is between about 1 μM and about 50 μM; preferably between about 5 μM and about 45 μM; more preferably between about 10 μM and about 40 μM; optimal is about 20 μM.

A third object of the present disclosure is to provide a method of reducing transplant rejection. The method comprises administering to a subject an effective amount of disodium desmethyl decanoate or a pharmaceutically acceptable salt thereof to inhibit differentiation or maturation of a human dendritic cell into T-lymphocytes in the subject. The individual can be a mammal, preferably a human.

These and other features of the present disclosure will be more apparent from the following detailed description and appended claims.

The above summary and the following detailed description are intended to be illustrative only, and are not intended to limit the scope of the disclosure Domain.

The above and other objects, features, advantages and embodiments of the present invention will become more <RTIgt; <RTIgt; </ RTI> <RTIgt; </ RTI> <RTIgt; </ RTI> <RTIgt; </ RTI> <RTIgt; Effect of human dendritic cell survival rate; Figure 2 depicts the effect of O6 on cell morphology of human dendritic cells, in which (A) is the control group and (B) is 20 μM, in accordance with an embodiment of the present invention. The O6-treated DC cells; FIG. 3 illustrates the effect of O6 on the expression of human dendritic cell surface antigen molecules according to an embodiment of the present invention; and FIG. 4 illustrates the O6-transformed body according to an embodiment of the present invention. Incubating the effects of DC cells and T cells, wherein (A) represents the effect on the proliferating T cells, (B) is the effect of the interferon-γ produced; and FIG. 5 illustrates an embodiment according to the present invention, The effect of O6 on the amount of cytokines secreted by DC cells, wherein (A) represents IL-10, (B) represents IL-12 and (C) represents the secretion amount of IL-23; and FIG. 6 shows an embodiment according to the present invention. Mode, with or without NCTD (0, 10 or 20 μM), CA (0, 1.25 or 2.5 μM) or O6 (0, 20 or 40 μM) versus PP2A in DC cells Effect of unit protein expression; Figure 7 is an embodiment of the invention with or without NCTD (0, 10 or 20 μM), CA (0, 1.25 or 2.5 μM) or O6 (0, 20 or 40 μM) Effect on the expression of p70 S6 kinase protein in DC cells; Figure 8 is an embodiment of the present invention, with or without Effects of NCTD (0, 10 or 20 μM), CA (0, 1.25 or 2.5 μM) or O6 (0, 20 or 40 μM) DC cells on the performance of PP2B A-unit protein; and Figure 9 is based on this An embodiment of the invention, the effect of p-STAT3 protein expression in DC cells treated with or without NCTD (0, 10 or 20 μM), CA (0, 1.25 or 2.5 μM) or O6 (0, 20 or 40 μM) .

The various features and elements in the figures are not drawn to scale, and are in the In addition, similar elements/components are referred to by the same or similar element symbols throughout the different drawings.

The description of the embodiments of the present invention is intended to be illustrative and not restrictive. The features of various specific embodiments, as well as the method steps and sequences thereof, are constructed and manipulated in the embodiments. However, other specific embodiments may be utilized to achieve the same or equivalent function and sequence of steps.

Although numerical ranges and parameters are used to define a broad range of values for the present invention, the relevant values in the specific embodiments have been presented as precisely as possible. However, any numerical value inherently inevitably contains standard deviations due to individual test methods. As used herein, "about" generally means that the actual value is within plus or minus 10%, 5%, 1%, or 0.5% of a particular value or range. Or, the word "about" means that the actual value falls within the acceptable standard error of the average value, depending on the technical field to which the present invention pertains. It depends on the consideration of the usual knowledge. Except for the experimental examples, or unless otherwise explicitly stated, all ranges, quantities, values, and percentages used herein are understood (eg, to describe the amount of material used, the length of time, the temperature, the operating conditions, the quantity ratio, and the like. Are all modified by "about". Therefore, unless otherwise indicated to the contrary, the numerical parameters disclosed in the specification and the appended claims are intended to be At a minimum, these numerical parameters should be understood as the number of significant digits indicated and the values obtained by applying the general carry method.

The scientific and technical terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the invention pertains, unless otherwise defined herein. In addition, the singular noun used in the specification encompasses the plural of the noun, and the plural noun of the noun is also included in the plural noun.

In the following, the term "inhibits or inhibiting" means applying the compound to dendritic cells or a body (ie, disodium desmethyl decanoate), To alter at least 45%, 50%, 55%, 60%, or 65% of dendritic cell morphology, including a decrease in the number of dendrites on each dendritic cell in the culture, dendritic cell surface antigens including CD1a, CD40 or The amount of CD83 is inhibited, or the ability to stimulate T-cell proliferation is reduced. Therefore, the term "inhibition" is also used herein to inhibit dendritic cell differentiation or maturation.

The term "an effective amount" means that the amount of dendrites on each dendritic cell is reduced and the dendritic cell surface antigen (including CD1a, CD40 or CD83) performance, or decreased dendritic cells stimulate T-cell proliferation The ability to produce, in other words, the effective amount can achieve the desired effect of inhibiting dendritic cell differentiation or maturation.

The words "compound", "composition", "agent" or "medicine or medicament" are used interchangeably herein and refer to when applied to a body (human Or an animal, a compound or composition capable of inducing a desired pharmaceutical and/or physiological response through local and/or systemic action.

The term "administered, administered" or "administration" as used herein refers to the direct administration of the compound or composition, or the administration of a prodrug, derivative, or analog of the active compound. Alternatively, a substantial amount of the active compound can be formed in the subject to be administered.

The words "subject" or "patient" are used interchangeably herein to mean an animal (including a human) that is treatable by the compounds and/or methods. "Individual" or "patient" is used herein to encompass both male and female genders unless otherwise specified. Thus "individual" or "patient" encompasses any mammal, preferably a human, which may benefit from treatment with the compound.

At least a part of the disclosure is a derivative of cantharidin (CA), which is a cytotoxic and has a modulating human dendrites, which is a derivative of cantharidin (CA), which is isolated from the insects of the genus Wanjing. The characteristics of cell differentiation or maturation. The above findings suggest that disodium desmethyl decanoate in the present disclosure can be used as a lead compound for a therapeutic agent that reduces graft rejection.

In the present disclosure, the cantharidin (CA) derivative, which is isolated from the insects of the genus Penicillium, is reduced, and the disodium methyl cinnamate (DNCTD, referred to as the O6 compound in the examples herein) has the following chemical structure:

Accordingly, a first object of the present disclosure is directed to the novel use of disodium desmethyl cinnamate to produce a pharmaceutical or pharmaceutical composition that inhibits differentiation or maturation of a dendritic cell. In the present disclosure, inhibition refers to reducing the amount of expression of CD1a, CD40 or CD83 molecules on the surface of dendritic cells.

One embodiment provided in accordance with the purpose of the present invention relates to a pharmaceutical or pharmaceutical composition which inhibits the differentiation or maturation of a dendritic cell. The pharmaceutical or pharmaceutical composition comprises a therapeutically effective amount of disodium succinic acid or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable adjuvant.

The compound of the present invention, disodium desmethyl decanoate, is present in an amount of from about 0.1% to about 99% by weight based on the total weight of the pharmaceutical composition, based on the total weight of the pharmaceutical composition. In certain embodiments, the amount of the compound of the invention is at least about 1% by weight based on the total weight of the pharmaceutical composition. In a particular embodiment, the amount of the compound of the invention is at least about 5% by weight based on the total weight of the pharmaceutical composition. In other embodiments, the amount of the compound of the invention is at least It is about 10% of the total weight of the pharmaceutical composition. In further embodiments, the amount of the compound of the invention is at least about 25% by weight of the total weight of the pharmaceutical composition.

A second object of the present disclosure is directed to a method of inhibiting differentiation or maturation of a dendritic cell with disodium decanoate in vitro. The method comprises contacting the dendritic cells with a sufficient amount of sodium desmophyllin sodium or a pharmaceutically acceptable salt thereof in vitro. In one example, the sufficient amount of sodium desmethylcantharidin is between about 1 μM and about 50 μM; preferably between about 5 μM and about 45 μM; more preferably between about 10 μM and about 40 μM; optimal is about 20 μM.

A third object of the present disclosure is to provide a method of reducing transplant rejection. The method comprises administering to a subject an effective amount of disodium desmethyl decanoate or a pharmaceutically acceptable salt thereof to inhibit differentiation or maturation of a human dendritic cell into T-lymphocytes in the subject. The individual can be a mammal, preferably a human.

In certain embodiments, disodium desmethyl succinate can be administered to a subject via oral, intravenous or intramuscular injection at an effective level of between about 1 and 100 mg/kg of body weight. An effective amount of disodium methylphylloate applied to the individual per day is about 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100 mg/kg body weight; preferably about 30- 70 mg/kg body weight, for example about 30, 40, 50, 60, 70 mg/kg body weight; more preferably about 50 mg/kg body weight. These doses can be administered in a single administration or divided into multiple administrations within one day.

In certain embodiments, the method further comprises administering Simultaneously, before or after demethylmethyl succinate is administered to the patient another drug known to inhibit dendritic cell differentiation or maturation, such as dexamethasone (DEX), cyclophosphamide, CTX), an immunosuppressive agent such as Methylprednisolone (MP).

Accordingly, the present disclosure provides a pharmaceutical or pharmaceutical composition that can be used to inhibit dendritic cell differentiation or maturation. The pharmaceutical or pharmaceutical composition comprises an effective amount of disodium desmethyl decanoate; and a pharmaceutically acceptable adjuvant thereof. Generally, the amount of disodium methylphyllinate is from about 0.1% to about 99% by weight based on the total weight of the pharmaceutical composition. In certain embodiments, the amount of disodium methotrexate is at least 1% by weight of the total pharmaceutical composition. In a particular embodiment, the amount of disodium methotrexate is at least 5% by weight of the total pharmaceutical composition. In other embodiments, the amount of disodium methotrexate is at least 10% by weight based on the weight of the overall pharmaceutical composition. In still other embodiments, the amount of disodium methylbehenate is at least 25% by weight of the total pharmaceutical composition.

In general, the compounds of the invention (i.e., disodium canregestinate) can be administered by oral (e.g., orally, in capsules, sachets, suspensions or elixirs), or parenterally, and the like. . Parenterally administered means include systemic administration such as intramuscular injection, intravenous injection, subcutaneous injection or intraperitoneal injection. Alternatively, it may be applied by means of a film, such as topical skin application or inhalation (such as intrabronchial, intranasal, intraoral or nasal drops); or intrarectal administration. May be administered alone or in combination with conventional drugs The adjuvant can be administered together. In a preferred embodiment, the compounds of the invention can be administered to an individual via oral means (e.g., through food).

If administered orally, the compound of the invention (ie, disodium desmethyl decanoate) can be formulated to contain various adjuvants (eg, microcrystalline cellulose, calcium carbonate, dicalcium phosphate, and glycine); Disintegrants (such as starch, alginic acid, and specific citrates); and granule binders (eg, polyvinylpyrrolidone, sucrose, gelatin, and acacia). In addition, lubricants such as magnesium stearate, sodium lauryl sulfate, and talc may also be included. Preferred materials associated therewith include sugars in lactose or milk as well as high molecular weight polyethylene glycols. The above solid dosage forms may also optionally include a coating or shell, such as a casing coating, and a coating to improve the rate of release of any of the pharmaceutically active ingredients. Examples of such coatings are well known to those skilled in the art. In one example, the pharmaceutical composition is formulated as a tablet. In another example, the pharmaceutical composition is a granule formulated to be filled in a soft or hard gelatin capsule or encapsulated in a biodegradable kit. When used in the form of oral suspensions and / or special effects (elixirs), the active ingredients may be combined with various sweeteners or flavors, colorants or dyes, and emulsifiers and/or suspensions may be added if desired. And diluents such as water, alcohol, propylene glycol, glycerin.

In certain embodiments, the pharmaceutical compositions of the invention are formulated as liquid dosage forms suitable for oral administration. Such liquid formulations may further include a buffer to maintain the pH. This liquid formulation can also be filled in a soft capsule. The liquid dosage form can be a solution, suspension, emulsion, microemulsion, precipitate or can carry a compound of the invention, a pharmaceutically acceptable derivative, isomer, metabolite, salt or solvate thereof Ren What is the liquid medium. The liquid can be designed to improve the solubility of the pharmaceutically acceptable salts of the compounds of the invention to form a drug-containing emulsion or dispersion. When such a dosage form is employed, it is formed by mixing the active compound with at least one pharmaceutically acceptable adjuvant, including, but not limited to, the above-described adjuvants.

If administered parenterally, the compound of the invention may be formulated as a liquid pharmaceutical composition which may be sterile solution or suspension which can be administered by intravenous, intramuscular, subcutaneous or intraperitoneal injection. liquid. Diluents which can be used in the manufacture of such sterile injectable solutions or suspensions include, but are not limited to, 1,3-butanediol, mannitol, water, Ringer's solution, isotonic sodium chloride solution. Fatty acids (e.g., oleic acid) and their glyceride derivatives, or natural pharmaceutically acceptable oils (e.g., olive oil or rapeseed oil) can also be used to make solutions or suspensions for injectable use. Alcohols or carboxymethylcellulose or similar dispersing agents for dilution may also be included in such oily solutions or suspensions. Other commonly used surfactants (e.g., Tweens or Spans series) or emulsifiers, or agents commonly used in the pharmaceutical arts to enhance bioavailability can be used.

The above pharmaceutical or pharmaceutical composition of the present invention may also be formulated into a variety of dosage forms suitable for mucosal application, such as buccal and/or sublingual pharmaceutical dosage units for delivery of a drug. Through the oral mucosa. A wide variety of biodegradable and pharmaceutically acceptable polymeric adjuvants can be used which provide the pharmaceutical compositions with acceptable adsorption and desired drug release patterns, as well as buccal and/or sublingual drugs. The active ingredient to be applied in the dosage unit Or other ingredients are compatible. In general, the above polymeric adjuvants comprise a hydrophilic polymer that adheres to the wetted surface of the oral mucosa. Examples of polymeric adjuvants include, but are not limited to, acrylic acid polymers and copolymers; hydrolyzed polyvinyl alcohol; polyethylene oxides; polyacrylates; Vinyl polymers and copolymers; polyvinylpyrrolidine; dextran; guar gum; pectins; starch; and cellulosic polymers.

It will be understood that the dosage of the compound of the invention will vary from individual to individual, not only because of the particular compound or composition employed, the route of administration, the compound (either alone or in combination with one or more drugs) Other factors may also be affected by other factors, such as the state or severity of the disease to be treated; the age, sex or weight of the patient, the health of the patient; the severity of the pathological condition to be treated, and the patient at the same time Other medical or special dietary content to be performed; and other factors conceivable by those of ordinary skill in the art; and the medical personnel responsible for care may ultimately determine the appropriate dosage based on these factors. The dosage and form of administration can be adjusted to provide a preferred therapeutic response. A therapeutically effective amount also refers to a toxic or detrimental effect of a compound or composition that is less than the therapeutic benefit it brings. In preferred instances, the compounds or compositions of the invention, when administered, should be administered in an appropriate dosage for a period of time to reduce the number and/or severity of symptoms.

The following examples are presented to illustrate certain aspects of the present disclosure and to assist those skilled in the art to understand and practice the present disclosure. However, the scope of the disclosure is not limited to these embodiments.

Example Example 1 Production of Disodium Dodecanoic Acid (DNCTD)

Disodium decanoic acid disodium (abbreviated as O6 compound in the following examples) was produced in the manner described in Scheme I below.

Wherein (i), (ii) or (iii) are treatments or conditions, respectively, wherein (i) is stirred in toluene at 80 ° C for 12 hours; (ii) is at 10 atmospheres of hydrogen at 10% Pd /C was used as a catalyst and reacted in tetrahydrofuran (THF) for 8 hours; (iii) was reacted with 0.1 N NaHCO ₃ in tetrahydrofuran (THF) at room temperature. Each of the processing conditions of (i), (ii) and (iii) above is described in detail as follows:

(i) Production of demethylnorsaldin (DHNCTD)

In a maleic ahnydric (11 g, 112.2 mmol), 300 ml of a solution of furan (30 g, 441.1 mmol) dissolved in toluene was added and stirred at 80 ° C. The solution was mixed for about 12 hours. After that, the reaction mixture was cooled to room temperature, and the product was collected by filtration, and then the collected product was washed with diethyl ether, and dried in vacuo to give demethyl-s-hydrosporin (DHNCTD) as a colorless solid (14.7 g, 88.5 m. , yield: 78.9%), its melting point is about 121-122 ° C, and its structure is identified by ¹ H-NMR and ¹³ C-NMR. The spectral data are as follows: ¹ H NMR (CDCl ₃ ) δ 6.58 (s, 2H, H-5,6), 5.47 (s, 2H, H-1, 4), 3.19 (s, 2H, H-2, 3); ¹³ C NMR (CDCl ₃ ) δ 170.1 (C=O), 137.2 ( C-5, C-6), 82.4 (C-1, C-4), 48.9 (C-2, C-3).

(ii) Production of demethylcantharidin (NCTD)

Add 10% Pd/C (470 mg) to a solution of the above-mentioned demethylhydrosporin (4.7 g, 28.3 mmol) in THF (200 ml), stir at 3 atmospheres of hydrogen, and at room temperature. The mixture was about 8 hours. The reaction mixture was filtered Celite 545 ^®, available vacuum dried to methyl cantharidin (norcantharidin, NCTD) of a colorless solid (4.3 g, yield: 91.4%) having a melting point of about 115-116 deg.] C, and by ¹ H The structure was identified by NMR and ¹³ C-NMR. The spectral data were as follows: ¹ H NMR (CDCl ₃ ) δ 5.05 (dd, J = 2.3, 2.2 Hz, 2H, H-1, 4), 3.18 (s, 2H, H) -2,3), 1.91 (m, 2H), 1.64 (m, 2H); ¹³ CNMR (CDCl ₃ ) δ 171.1 (C=O), 80.2 (C-1, C-4), 50.6 (C-2) , C-3), 28.1 (C-5, C-6).

(iii) manufacture of DNCTD (or O6 compound)

To a solution of the above-mentioned demethylcantharidin (NCTD) (480 mg, 2.9 mmol) in THF (50 ml) was added 0.1N aqueous sodium hydrogen carbonate (5.7 mL, 5.8 mmol). The reaction mixture was stirred at room temperature for about 1 hour, and dried in vacuo to give a white solid. The isolated compound was subjected to structural analysis and identification by ¹ H-NMR and ¹³ C-NMR. The spectral data are as follows: ¹ H NMR (D ₂ O) δ 4.73 (dd, J = 2.6, 2.5 Hz, 2H, H-1, 4), 2.93 (s, 2H, H-2, 3), 1.63 (m, 2H), 1.51 (m, 2H); ¹³ C NMR (D ₂ O) δ 178.1 (C=O), 79.1 (C-1, C-4), 54.0 (C-2, C-3), 28.3 ( C-5, C-6).

Example 2 Manufacture and cultivation of human dendritic cells (DCs) and their functional analysis

It is known that the cultivation of human monocytes or mouse bone marrow cells under the granulocyte-macrophage colony-stimulating factor (GM-CSF) induces the production of granulocytes, macrophages and derived from Dendritic cells of monocytes (see Liu and Nussenzweig, Immuno Rev., 2010 234:45-54). Accordingly, in the present experiment, monocytes derived from the blood surrounding the healthy person were collected first, and then CD14+ cells were isolated and purified using a commercially available miniMACS system. The isolated CD14+ cells were cultured in RPMI 1640 medium supplemented with 10% fetal calf serum (FCS), 100 ng/ml GM-CSF, and 50 ng/ml IL-4. The cells were maintained in a humidified environment at 37 ° C (the gas composition was 5% CO ₂ and 95% air).

In order to induce DCs to develop on the differentiation or maturation pathway, immature DCs were grown in a medium containing a combination of cytokines using a combination of 5 ng/ml TNF-α, 5 ng/ml. IL-1, 15 ng/ml of IL-6, and 1 μg/ml of prostaglandin E2. In some experiments, different concentrations of test compound (ie, 0, 1.25, 2.5, 5, 10, or 20 μM of O6) were added to the medium; The cultured cells are collected for a specific period of time for cell survival, cell type, cell surface antigen molecule expression, cellular immune function analysis, and further confirmation of the biological activity pathway of O6.

2.1 Cell survival analysis

In this example, the incubated DCs were collected on day 8 and the cells were stained with trypan blue dye and the number of cells was counted, with the dead cells appearing blue. The results are shown in Figure 1. It can be observed from the figure that even if the concentration of O6 added in the medium is as high as 20 μM, the cell viability is as high as 80%, indicating that O6 itself is not cytotoxic, which is similar to its analog, ie, known to be cytotoxic. CA or NCTD, which is not the same, also highlights the advantages of O6 as a therapeutic agent for dendritic cell development or anti-rejection compared to CA or NCTD.

2.2 Cell type analysis

In this example, the cell type analysis was performed by staining the DCs by Liu's staining method, and then observing the cell type by a microscope.

Liu staining The cells to be stained were transferred to a glass slide and covered with solution A (0.5 g of methylene blue and 1.7 g of Eosin yellow, both dissolved in 1,000 ml of methanol). After 45 seconds, solution B (1.3 g of azure, 1.4 g of methyl blue, 23.38 g of disodium hydrogen phosphate (Na ₂ HPO ₄ ) and 6.5 g of potassium dihydrogen phosphate (KH ₂ PO _{4 ) were added.} ), all dissolved in 1,000 ml of distilled water), wherein the addition amount of solution B and solution A is 2:1. The surface of the solution was lightly blown to make the two solutions homogeneously mixed. The slide was allowed to stand for about 90 seconds and then the dye was quickly washed away with running water. The microscope was then used to observe the appearance of the stained cells.

Results The cell type observed after the Liu staining method is shown in Fig. 2. It can be found that DC cells treated with 20 μM of O6 relative to the control group have less number of cells to be attached, and the number of the number of cells is also reduced or shortened, indicating that the DC cells have developed on the mature pathway.

2.3 Cell surface antigen molecular expression

The cell surface antigen molecule expression pattern is performed by flow cytometry analysis.

Cell flow analysis was performed on cell surface antigen molecules (including CD14, CD1a, CD83, respectively) by fluorescein isothiocyanate (FITC) and phycoerythrin (PE)-conjugated monoclonal antibodies. Two-color immunolabeling was performed on CD40, CD80, CD86, and the like. The antibodies used were as follows: anti-CD14 (IgG-FITC), anti-CD1a-PE, anti-CD80-PE, anti-CD83-PE, anti-CD40-PE, anti-CD86-PE, anti-human leukocyte antigen ( Human leukocyte antigen, HLA)-DR-PE, and anti-DC-SIGN-PE. During the operation, 1 x 10 ⁶ cells were injected into the FACS caliber flow cytometer for analysis and the results were analyzed using the CellQuest software equipped with the instrument.

The results showed that O6 did not significantly inhibit the expression of CD14, DC-SIGN, CD80, CD86 or anti-human leukocyte antigen (HLA)-DR relative to the control group; conversely, with the increase of O6 concentration, O6 treatment Significantly reduced changes in the expression pattern of DC cell surface antigen molecules CD1a, CD40, or CD83 (Fig. 3).

2.4 Analysis of cellular immune function

In this experiment, DC cells and T cells were cultured through allogeneic cells. To stimulate T cell proliferation, and measure the amount of secretions (eg, the amount of cytokine or interferon-γ) in the cells cultured in the cell culture supernatant to confirm cell function. The relevant operation methods and results are as follows.

Allogeneic Stimulation of T Cell Proliferation First, unattached cells in monocyte cultures were harvested as described in the Examples, and T cells were purified therefrom, and then purified by amplification using a commercially available miniMACS system kit. T cells. Thereafter, DC cells derived from monocytes and treated with the test compound O6 (or DDNCT) were collected and irradiated with 6 MeV X-rays (about 30 Gy) (dose of 4.0 Gy per minute), and then The irradiated DC cells were incubated with the above expanded T cells for about 5 days at a ratio of 1:10 or 1:30. Next, 5 μM carboxy fluorescein succinimidyl ester was added to the cell culture and left in the cell culture for about 18 hours. Next, the cultured cells were collected and analyzed by flow cytometry, and T cells were labeled with anti-CD4-PE and anti-CD25-FITC monoclonal antibodies.

Cytokine content determination DC cells and the above-mentioned T cells proliferated by allogeneic stimulation will secrete cytokines (such as IL-10, IL-12) and interferon-γ, respectively, during the incubation process. Therefore, they can be immunized by enzymes. Analysis was performed to measure the content of cytokines and interferon-γ in the supernatant of the cell culture.

Results As described above, DC cells treated with different concentrations of O6 (0, 1.25, 2.5, 5, 10 or 20 μM) and T cells (naïve T cells) were combined at a ratio of 1:10 or 1:30, respectively. The cultivation was carried out for about 5 days, and the results are shown in Fig. 4. It can be found that DC cells and T cells can be stimulated to stimulate T cell proliferation; after O6 treatment, their ability to stimulate T cell proliferation will be regulated (Fig. 4A); interferon-γ secretion mode is also the same ( 4B picture).

As for the amount of cytokines secreted by DC, the amount of IL-10 secreted will be inhibited as the concentration of O6 increases; the amount of IL-23 secreted will increase as the concentration of O6 increases; as for the secretion of IL-12 There is no significant change in the amount (Figure 5).

2.5 O6 biological activity pathway analysis

To confirm the biotransformation of O6 in cells, immunoblotting analysis was used to determine the expression of related proteins on different signaling pathways, including ubiquitous cells, in the presence or absence of O6 (0, 20 or 40 μM). a unit protein of phosphonate/threonine phosphatase or calmodulinin pathway IIA or IIB phosphatase (phosphotase 2A or 2B, PP2A or PP2B) and p70S6K protein on the mammalian rapamycin pathway.

Immunoblot analysis The incubated cells were lysed using a known lysis buffer and the total amount of protein was measured using the Bradford method. The extracted crude white (about 50-100 μg) was added to 0.15% of sodium dodecyl sulfate polyacrylamide gel, and the protein was separated by electrophoresis. Thereafter, the separated protein was transferred to a nitrocellulose membrane, and TBST buffer (0.8% sodium chloride, 0.02% potassium chloride, 25 mM Tris-HCl/pH 8.0, and 0.1%) containing 4% skim milk was used. Tween-20) to remove non-specific combinations. Various antibodies (including p-stat3, stat3, β-actin, PP2B A unit, PP2A C unit, p-p70 S6K, p70 S6K (purchased from Transduction Laboratories, Lexington, KY, USA)) were separately dissolved in 2% degreased The milk was in TRST buffer and incubated with the separated protein band at 4 ° C for at least one night. After washing, the cells were incubated with horseradish conjugated secondary antibodies for about 30 minutes at room temperature, and then the protein bands were detected using ECL chemical luminescence reagent (Amersham Pharmacia Biotech, US). The film was exposed by X-ray to facilitate visual observation of the expressed protein band.

The results are shown in Figures 6-9; it can be seen that the amount of C2 protein of PP2A and p70S6K was not inhibited by cells treated with 20 or 40 μM of O6 (Figures 6 and 7). Conversely, A of PP2B The amount of expression of the unit protein was inhibited (Fig. 8), indicating that the biological activity of O6 was achieved by regulating the C-unit protein of PP2A on the calmodulin pathway and the A unit of the PP2B protein.

In addition, the inventors further confirmed the effect of O6 by measuring the amount of expression of regulatory proteins such as stat3 and phosphorylated stat3. The experiment found that the phosphorylated stat3 was significantly decreased in DC cells treated with 20 or 40 μM of O6 compared to the control group (see Figure 9). This observation also confirmed that stat3 protein is involved in O6 promoting human DC cells. The process of maturity and differentiation.

It will be understood that the above-described embodiments and examples are merely illustrative, and that those skilled in the art can align various modifications. The description, examples, and materials set forth above are intended to be illustrative of the present invention and are illustrative of the invention. Although the disclosure has been disclosed above in the embodiments, it is not intended to limit the disclosure, any familiarity The scope of the present disclosure is defined by the scope of the appended claims, and the scope of the disclosure is subject to the scope of the appended claims.

Claims (8)

A use of disodium methylphyltreate which is useful for the manufacture of a medicament for inhibiting the differentiation or maturation of a dendritic cell. The use according to claim 1, wherein the inhibition is to reduce the amount of expression of the CD1a, CD40 or CD83 molecule on the surface of the dendritic cell. The use of claim 2, wherein the drug is a drug that slows transplant rejection. A method for inhibiting differentiation or maturation of a dendritic cell in vitro comprising contacting the dendritic cell with a sufficient amount of disodium methylbesylate or a pharmaceutically acceptable salt thereof. The method of claim 4, wherein the sufficient amount of disodium desmethyl decanoate is between about 1 μM and about 50 μM. The method of claim 5, wherein the amount of the disodium methylbehenate is 5 μM. The method of claim 5, wherein the amount of disodium methylbesylate is 20 μM. The method of claim 5, wherein the amount of disodium methylbesylate is 40 μM.