WO2019129182A1 - 2β,3α,5α-TRIHYDROXYL ANDROSTANE-6-KETONE FOR TREATMENT OF INFLAMMATORY REACTIONS - Google Patents

Abstract

Disclosed is an application of 2β,3α,5α-trihydroxyl androstane-6-ketone and a deuterated compound thereof or pharmaceutically acceptable salts thereof in the preparation of drugs for treating inflammatory reactions of patients. The present invention proves that the 2β,3α,5α-trihydroxyl androstane-6-ketone inhibits the activation of microglial cells and macrophages by down-regulating the expression of inflammatory pathway key molecules NF-κB, and thus can used for treating inflammations.

Description

2β,3α,5α-trihydroxyandrost-6-one for the treatment of inflammatory response Technical field

The invention relates to a new medical use of 2β, 3α, 5α-trihydroxyandrost-6-one, in particular to the application of 2β, 3α, 5α-trihydroxyandrost-6-one in the treatment of an inflammatory reaction.

Background technique

Macrophages are ubiquitous in blood, lymph and tissues. They are a kind of innate immune cells. They are important cells involved in the natural immune response. They have the functions of killing bacteria, phagocytizing pathogens, antigen presentation and secreting cytokines. The function plays an important role in the process of clearing, promoting and inhibiting inflammatory reaction of pathogenic microorganisms and senescent cells, inducing adaptive immune response and repairing and reconstructing damaged tissues, which can maintain the balance of the body and the formation of diseases. It plays an important role in the treatment process.

Different PAMP (pathogen-associated molecular patterns) and DAMP (injury-associated molecular patterns) can stimulate macrophages to polarize in different directions to form M1 and M2 macrophages. For example, pro-inflammatory factors IFN-γ, TNF and LPS produce M1 macrophage polarization through stimulation of IFN-γR, TLR and other receptors and their downstream signaling pathway NF-κB, respectively, and macrophages are expressed after polarization. Proinflammatory cytokines such as TNF-α, IL-1, NO, and reactive oxygen species have strong antigen-presenting ability, while M1-type macrophages promote Th1-type immune response, promoting inflammatory response in the early stage of inflammation, killing intracellular cells. Infected pathogen. It is induced by IL-4, IL-13, immune complex, IL-10, glucocorticoid, etc., and activated M2 macrophages express a large number of anti-inflammatory factors such as IL-10 and TGF-β. Lysin 1 ((Arg1), CD206, DC-SIGN, mannitol receptor, scavenger receptor CD163, CCR2, CXCR1 and other marker molecules are expressed, and M2 type macrophages mainly activate Th2 type immune response after polarization. It is mainly involved in anti-inflammatory response, promoting tissue remodeling, fibrosis, and tumor development. M1 and M2 macrophages can be converted into each other under specific microenvironments.

M1 macrophage-polarized macrophages are thought to be involved in various diseases such as inflammatory diseases, parasitic infections, asthma, cardiovascular diseases, and tumors, and all play important roles.

Microglia is a glial cell of the central nervous system, which accounts for 5-10% of the whole glial cells. Microglia are widely regarded as macrophages in the brain and spinal cord. The nuclear phagocytic cell family is the main immune effector in the central nervous system.

As immune effector cells resident in the central nervous system, microglia and their mediated neuroinflammation play a very important role in the damage of the central nervous system and the process of disease progression. Under normal conditions, the microglia have small cell bodies with elongated, highly branched branches and many spinous processes on the branches. Under physiological conditions, microglia are at rest and play an immune surveillance role. When the central nervous system is stimulated by factors such as inflammation, infection, and trauma, microglia can be rapidly activated to mediate multiple immune responses. The activated microglia becomes larger in volume, the cell body becomes rounded, the cell surface protrusion disappears, and it changes to the amoeba-like macrophage state, which can rapidly metastasize and phagocytose to clear apoptotic neurons, synapses and cell debris, etc. The homeostasis of the central nervous system delays the development of neurodegenerative diseases.

Clinical and neuropathological studies suggest that activated microglia play an important role in neurodegenerative diseases such as Parkinson's disease, HIV encephalopathy, multiple sclerosis, and Alzheimer's disease. At the same time, excessively activated or uncontrolled microglia can cause neurotoxicity and are important sources of pro-inflammatory factors and oxidative stress, such as nitric oxide (NO), oxygen free radicals, proteolytic enzymes, inflammatory factors such as interleukin-1 ( IL-1), tumor necrosis factor alpha (TNF-α) and gamma interferon (INF-γ), etc., cause brain tissue damage. At the same time, a large number of cytokines and cytotoxic substances are secretly secreted. In the late stage of inflammation caused by injury, neurotrophic factors such as BDNF are secreted, which is beneficial to the nutrition and repair of neurons.

Summary of the invention

The inventors of the present invention have surprisingly discovered that 2β,3α,5α-trihydroxyandrost-6-one inhibits LPS-induced activation of microglia and macrophages by down-regulating the expression of NF-κB, a key molecule of the inflammatory pathway, thereby Can be used to treat inflammation.

One aspect of the present invention provides the use of 2β, 3α, 5α-trihydroxyandrost-6-one, a progeny thereof or a pharmaceutically acceptable salt thereof for the preparation of a medicament for treating an inflammatory response in a patient. In some embodiments, the inflammatory response is an inflammatory response mediated by the NF-κB signaling pathway. In some embodiments, the inflammatory response is a peripheral inflammatory response or a central nervous system inflammatory response. In some embodiments, the peripheral inflammatory response is manifested by macrophage polarization. In some embodiments, the central nervous system inflammatory response is manifested by activation of microglia. In some embodiments, the medicament further comprises another therapeutic agent. In some embodiments, the patient is a human.

Another aspect of the invention provides a method of treating an inflammatory response in a patient, the method comprising administering to the patient an effective amount of 2β, 3α, 5α-trihydroxyandrost-6-one, a progeny thereof, or a pharmaceutically acceptable A salt acceptable, or a pharmaceutical composition comprising 2β, 3α, 5α-trihydroxyandrost-6-one, a progeny thereof, or a pharmaceutically acceptable salt thereof. In some embodiments, the inflammatory response is an inflammatory response mediated by the NF-κB signaling pathway. In some embodiments, the inflammatory response is a peripheral inflammatory response or a central nervous system inflammatory response. In some embodiments, the peripheral inflammatory response is manifested by macrophage polarization. In some embodiments, the central nervous system inflammatory response is manifested by activation of microglia. In some embodiments, the patient is a human.

A further aspect of the invention provides 2β, 3α, 5α-trihydroxyandrost-6-one, a progeny thereof or a pharmaceutically acceptable salt thereof for use in the treatment of an inflammatory response in a patient. In some embodiments, the inflammatory response is an inflammatory response mediated by the NF-κB signaling pathway. In some embodiments, the inflammatory response is a peripheral inflammatory response or a central nervous system inflammatory response. In some embodiments, the peripheral inflammatory response is manifested by macrophage polarization. In some embodiments, the central nervous system inflammatory response is manifested by activation of microglia. In some embodiments, the patient is a human.

A further aspect of the invention provides a method of reducing or eliminating an inflammatory response in a patient, the method comprising administering to the patient an effective amount of 2β, 3α, 5α-trihydroxyandrost-6-one, a progeny thereof or a pharmaceutical thereof An acceptable salt, or a pharmaceutical composition comprising 2β, 3α, 5α-trihydroxyandrost-6-one, a progeny thereof, or a pharmaceutically acceptable salt thereof. A further aspect of the invention provides a method of reducing or eliminating an inflammatory response mediated by a NF-κB signaling pathway in a patient, the method comprising administering to the patient an effective amount of 2β, 3α, 5α-trihydroxyandrosine-6- A ketone, a progeny thereof or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising 2β, 3α, 5α-trihydroxyandrost-6-one, a progeny thereof or a pharmaceutically acceptable salt thereof. A further aspect of the invention provides a method of reducing or eliminating a peripheral inflammatory response or a central nervous system inflammatory response in a patient, the method comprising administering to the patient an effective amount of 2β, 3α, 5α-trihydroxyandrost-6-one And a progeny thereof or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising 2β, 3α, 5α-trihydroxyandrost-6-one, a progeny thereof or a pharmaceutically acceptable salt thereof. A further aspect of the invention provides a method of reducing or eliminating macrophage polarization in a peripheral inflammatory response, the method comprising administering to a patient an effective amount of 2β, 3α, 5α-trihydroxyandrost-6-one, A progeny thereof or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising 2β, 3α, 5α-trihydroxyandrost-6-one, a progeny thereof or a pharmaceutically acceptable salt thereof. A further aspect of the invention provides a method of alleviating or eliminating activation of microglia in a central nervous system inflammatory response, the method comprising administering to a patient an effective amount of 2β, 3α, 5α-trihydroxyandrosine-6- A ketone, a progeny thereof or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising 2β, 3α, 5α-trihydroxyandrost-6-one, a progeny thereof or a pharmaceutically acceptable salt thereof.

DRAWINGS

Figure 1.2 β, 3α, 5α-trihydroxyandrost-6-one blocks the phosphorylation of NF-κB, a key molecule involved in LPS-induced inflammatory signaling pathways in RAW264.7 cells. Western blot analysis of 2β,3α,5α-trihydroxyandrost-6-one inhibited LPS-induced phosphorylation of NF-κB.

Figure 2. Immunofluorescence (IF) detection of 2β, 3α, 5α-trihydroxyandrost-6-one inhibits nuclear translocation of NF-κB p65 subunit in macrophages after LPS stimulation.

Figure 3. qRT-PCR detection of 2β,3α,5α-trihydroxyandrost-6-one inhibits multiple inflammatory factors in RAW264.7 cells after LPS stimulation (A: IL-6, B:iNOS, C:MCP-1 ) Expression of mRNA levels.

Figure 4. IF detection of 2β,3α,5α-trihydroxyandrost-6-one inhibits nuclear translocation of NF-κB p65 subunit in macrophages after LPS stimulation.

Figure 5.2 β, 3α, 5α-trihydroxyandrost-6-one inhibits LPS-induced activation of microglial BV2. (A) Phase contrast microscopy was used to observe the morphology of BV2 cells; (B)##: P<0.01 compared with normal control group; **: P<0.01 compared with LPS treated group.

Figure 6. Western blot analysis of 2β,3α,5α-trihydroxyandrost-6-one blocks LPS-induced phosphorylation of NF-κB, a key molecule of BV2 cell inflammation-related signaling pathway.

Figure 7. IF detection of 2β,3α,5α-trihydroxyandrost-6-one inhibits the entry of NF-kB, a key molecule of the LPS-induced inflammatory signaling pathway in primary microglia.

Detailed ways

As used herein, the term "composition" refers to a formulation suitable for administration to a prospective animal subject for therapeutic purposes, comprising at least one pharmaceutically active component, such as a compound. Optionally, the composition further comprises at least one pharmaceutically acceptable carrier or excipient.

The term "pharmaceutically acceptable" means that the substance does not have such characteristics, that is, taking into account the disease or condition to be treated and the respective route of administration, this property will prevent a rational and cautious medical practitioner from taking it to the patient. substance. For example, for injectables, such materials are generally required to be substantially sterile.

As used herein, the terms "therapeutically effective amount" and "effective amount" mean that the amount of the substance and substance is one to prevent, alleviate or ameliorate one or more symptoms of the disease or condition, and/or prolong the survival of the subject being treated. It's effective.

As used herein, "treatment" includes the administration of a compound of the present application, or a pharmaceutically acceptable salt thereof, to alleviate the symptoms or complications of a disease or condition, or to eliminate a disease or condition. The term "alleviation" as used herein is used to describe a process in which the signs or symptoms of a condition are reduced in severity. Symptoms can be alleviated without elimination. In one embodiment, administration of a pharmaceutical composition of the present application results in the elimination of signs or symptoms.

2β, 3α, 5α-trihydroxyandrost-6-one and pharmaceutically acceptable salts thereof

2?,3?,5?-trihydroxyandrost-6-one is also referred to herein as "YC-10" or "compound of the present invention" and has the structural formula shown in formula (I). YC-10 has been shown to have anti-tumor and neuroprotective effects.

The compounds of the invention may be formulated in the form of a pharmaceutically acceptable salt or in the form of a pharmaceutically acceptable salt. Desirable pharmaceutically acceptable salt forms include, but are not limited to, mono-, di-, tri-, tetra-, etc. salts. The pharmaceutically acceptable salts are non-toxic in the amounts and concentrations to which they are administered. The preparation of such salts can be facilitated by pharmacological applications by altering the physical properties of the compounds without preventing them from exerting physiological effects. Physically useful changes include lowering the melting point for transmucosal administration, and increasing solubility for administration of higher concentrations of the drug.

Pharmaceutically acceptable salts include acid addition salts such as those containing sulfates, chlorides, hydrochlorides, fumarates, maleates, phosphates, sulfamate, acetates, citrates, milks Salts of acid salts, tartrates, methanesulfonates, ethanesulfonates, besylate, p-toluenesulfonate, cyclamate and quinic acid salts. Pharmaceutically acceptable salts can be obtained from acids such as hydrochloric acid, maleic acid, sulfuric acid, phosphoric acid, sulfamic acid, acetic acid, citric acid, lactic acid, tartaric acid, malonic acid, methanesulfonic acid, ethanesulfonic acid, Benzenesulfonic acid, p-toluenesulfonic acid, cyclohexylsulfamic acid, fumaric acid and quinic acid.

When an acidic functional group such as a carboxylic acid or a phenol is present, the pharmaceutically acceptable salt also includes a base addition salt such as those containing benzathine, chloroprocaine, choline, diethanolamine, ethanolamine, tert-butylamine, ethylenediamine. , meglumine, procaine, aluminum, calcium, lithium, magnesium, potassium, sodium, ammonium, alkylamine and zinc salts. Such salts can be prepared using the appropriate corresponding base.

Pharmaceutically acceptable salts can be prepared by standard techniques. For example, the compound in its free base form is dissolved in a suitable solvent, such as an aqueous solution containing a suitable acid or a water-alcohol solution, and the solution is then evaporated for separation. In another example, a salt is prepared by reacting a free base and an acid in an organic solvent.

Thus, for example, if the particular compound is a base, the desired pharmaceutically acceptable salt can be prepared by any suitable method available in the art, for example, by treating the free base with a mineral or organic acid, such as hydrochloric acid. , hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid and similar acids, such as acetic acid, maleic acid, succinic acid, mandelic acid, fumaric acid, malonic acid, pyruvic acid, oxalic acid, glycolic acid, salicylic acid , pyranosidyl acid such as glucuronic acid or galacturonic acid, α-hydroxy acid such as citric acid or tartaric acid, amino acids such as aspartic acid or glutamic acid, aromatic acids such as benzoic acid or cinnamic acid, Sulfonic acid such as p-toluenesulfonic acid or ethanesulfonic acid or the like.

Likewise, if the particular compound is an acid, the desired pharmaceutically acceptable salt can be prepared by any suitable method, for example, by treating the free acid with an inorganic or organic base such as an amine (primary amine, A secondary or tertiary amine), an alkali metal hydroxide or an alkaline earth metal hydroxide or the like. Illustrative examples of suitable salts include organic salts derived from amino acids (such as L-glycine, L-lysine, and L-arginine), ammonia, primary, secondary, and tertiary amines, as well as cyclic amines (eg, Hydroxyethyl pyrrolidine, piperidine, morpholine and piperazine), as well as inorganic salts derived from sodium, calcium, potassium, magnesium, manganese, iron, copper, zinc, aluminum and lithium.

A pharmaceutically acceptable salt of the compound can exist as a complex. Examples of the complex include an 8-chlorotheophylline complex (similar to, for example, diphenhydramine: diphenhydramine 8-chlorotheophylline (1:1) complex; halo Haining) and various inclusions A cyclodextrin complex.

The invention also contemplates the use of pharmaceutically acceptable deuterated compounds or other non-radioactive substituted compounds using the compounds. Deuteration is the replacement of one or more or all of the hydrogen in the active molecular group of the drug into an isotope, because it is non-toxic and non-radioactive, and is about 6 to 9 times more stable than the carbon-hydrogen bond, which can block the metabolic site and prolong the drug. The half-life, which reduces the therapeutic dose without affecting the pharmacological activity of the drug, is considered to be an excellent modification method.

Pharmaceutical composition

In the present invention, "pharmaceutical composition" means a composition comprising YC-10 and a pharmaceutically acceptable carrier, wherein the compound and the pharmaceutically acceptable carrier are present in the composition in a mixed form. The composition will generally be used in the treatment of human subjects. However, they can also be used to treat similar or identical conditions in other animal subjects. As used herein, the terms "subject", "animal object" and like terms mean human and non-human vertebrate, such as mammals, such as non-human primates, competitive animals, and commercial animals, such as horses, cows, pigs, sheep, Rodents, and pets (such as dogs and cats).

Suitable dosage forms will depend, in part, on the route of administration or administration, for example, orally, transdermally, transmucosally, by inhalation or by injection (parenteral). Such dosage forms should enable the compound to reach the target cells. Other factors are well known in the art and include considerations such as toxicity and delay in the dosage form in which the compound or composition exerts its effects.

A carrier or excipient can be used to produce the composition. The carrier or excipient can be selected to facilitate administration of the compound. Examples of the carrier include calcium carbonate, calcium phosphate, various sugars (e.g., lactose, glucose or sucrose), or a starch type, a cellulose derivative, gelatin, vegetable oil, polyethylene glycol, and a physiologically compatible solvent. Examples of physiologically compatible solvents include sterile water for injection (WFI), saline solution, and glucose.

The composition or components of the composition can be administered by different routes, including intravenous, intraperitoneal, subcutaneous, intramuscular, oral, transmucosal, rectal, transdermal or inhalation. In some embodiments, injections or lyophilized powder injections are preferred. For oral administration, for example, the compounds can be formulated into conventional oral dosage forms such as capsules, tablets, and liquid preparations such as syrups, elixirs, and concentrated drops.

Pharmaceutical preparations for oral use can be obtained, for example by combining the composition or a component thereof with a solid excipient, optionally grinding the resulting mixture, and, if desired, processing a mixture of particles, if desired, thereby Get tablets or dragees. Suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol or sorbitol; cellulose preparations such as corn starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl fiber , hydroxypropyl methylcellulose, sodium carboxymethylcellulose (CMC) and/or polyvinylpyrrolidone (PVP: povidone). If necessary, a disintegrating agent such as crosslinked polyvinylpyrrolidone, agar or alginic acid or a salt thereof such as sodium alginate may be added.

Alternatively, injection (parenteral administration) may be used, such as intramuscular, intravenous, intraperitoneal, and/or subcutaneous. For injection, the compositions of the invention or components thereof are formulated as sterile liquid solutions, preferably in physiologically compatible buffers or solutions, such as saline solution, Hank's solution or Ringer's solution. Additionally, the compositions or components thereof can be formulated in solid form and reconstituted or suspended just prior to use. It is also possible to produce lyophilized powder forms.

Administration can also be by transmucosal, topical or transdermal means. For transmucosal, topical or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art and include, for example, for transmucosal administration, bile salts and fusidic acid derivatives. Additionally, detergents can be used to promote penetration. Transmucosal administration can, for example, be by nasal spray or suppository (transrectal or vaginal).

Effective amounts of the various components can be determined by standard procedures to be administered, for example, the considerations compound IC _50, the biological half life of the compound, the age, size and weight of the object-related disorders. The importance of these and other factors is well known to those of ordinary skill in the art. In general, the dosage will be between about 0.01 mg/kg to 50 mg/kg, preferably between 0.1 mg/kg and 20 mg/kg, of the subject being treated. Multiple doses can be used.

The compositions of the invention or components thereof may also be used in combination with other therapeutic agents that treat the same disease. Such combined use involves the administration of these compounds and one or more other therapeutic agents at different times, or the simultaneous use of such compounds and one or more other therapeutic agents. In some embodiments, the dosage of one or more compounds of the invention or other therapeutic agents used in combination may be modified, for example, by methods known to those skilled in the art to reduce the relative to the compound or therapeutic agent used alone. dose.

It is to be understood that the use or combination includes use with other therapies, drugs, medical procedures, and the like, wherein the other therapies or procedures may be at a different time than the compositions of the present invention or components thereof (eg, in the short term) (such as a few hours, such as 1, 2, 3, 4-24 hours) or for a longer period of time (such as 1-2 days, 2-4 days, 4-7 days, 1-4 weeks) or in this The composition of the invention or a component thereof is administered at the same time. The combined use also includes use with a therapy or medical procedure (such as surgery) that is administered once or infrequently, with the composition of the invention or a component thereof in the other Administration in a short or longer period of time before or after the therapy or procedure. In some embodiments, the invention is used to deliver a composition of the invention or a component thereof and one or more other pharmaceutical therapeutics, which pass Delivery by the same or different routes of administration.

Combination administration of any route of administration includes delivery of a composition of the invention or a component thereof and one or more other pharmaceutical therapeutics together in any formulation by the same route of administration, including chemically linking the two compounds and Formulations that maintain their respective therapeutic activity upon administration. In one aspect, the other drug therapy can be co-administered with a composition of the invention or a component thereof. A combination comprising co-administered co-formulation or a chemically linked compound, or a short-term (eg, within one hour, within 2 hours, within 3 hours, up to 24 hours) Compounds in the form of one or more separate formulations, which are administered by the same or different routes.

Co-administration of separate formulations includes co-administration of delivery via one device, such as the same inhalation device, the same syringe, etc., or administered by different devices in a short period of time relative to each other. A co-formulation of a compound of the invention and one or more additional pharmaceutical therapies delivered by the same route of administration comprises preparing the materials together such that they can be administered by a device, including combinations of different compounds in one formulation, or compounds They are modified such that they are chemically linked together but still retain their respective biological activities. Such chemically linked compounds can include a linker that separates the two active ingredients, which are substantially maintained in vivo or which may degrade in vivo.

Example

Example 1.

2β,3α,5α-trihydroxyandrost-6-one inhibits LPS-induced inflammatory response in peripheral macrophages

1. Cells: Mouse macrophage cell line RAW 264.7, purchased from ATCC; primary cultured peritoneal macrophages.

2. Main reagents:

2β,3α,5α-trihydroxyandrost-6-one was synthesized by Guangzhou Saipute Pharmaceutical Technology Co., Ltd.; HP-β-CD, purchased from Xi'an Deli Biotechnology Co., Ltd.; lipopolysaccharide LPS (Escherichiacoli 0111: B4, Sigma, Cat. L2630); antibody: anti-p65 (Santa Cruz. sc-8008); Phospho-NF-κB p65 (Ser536) (93H1) Rabbit mAb (CST, Cat. 3033); p38 antibody (CST, USA, Cat. 9212,) (1:1000); p-p38 antibody (Thr 180/Tyr 182) (CST, USA, Cat. 9216,) (1:1000); Dulbacco's Modified Eagle Medium (Gibco, Cat. 10013608R) ); fetal bovine serum (FBS) (Gibco, Cat. 10091148);

Reagent (Life, 15596-018).

3. Main equipment: Cell clean bench (Thermo, MSC-ADVANTAGE); CO ₂ cell incubator (Thermo 3111, USA); inverted phase contrast/fluorescence microscope (Olympus IX71, Japan); benchtop cryogenic high-speed centrifuge (Eppendrof, German) ); Chemiluminescence Imager (Bio-Rad, USA); Vertical Plate Electrophoresis (Bio-Rad Mini-Protein II cells, USA); Transfer Cell (Bio-Rad Mini Tran-Biot Transfer Cell, USA)

2. Experimental methods

Western blot analysis of 2β,3α,5α-trihydroxyandrost-6-one inhibited LPS-induced phosphorylation of NF-κB.

The macrophage cell line RAW264.7 was administered at different concentrations (0.1 μM, 0.5 μM, 2.5 μM, 10 μM) of 2β, 3α, 5α-trihydroxyandrost-6-one or the positive drug dexamethasone (DXMS) 1 μM pretreatment. After 30 min, after 30 min stimulation with 100 ng/ml LPS, the proteins were collected for Western blot analysis. Among them, hydroxypropyl-β-cyclodextrin (HP-β-CD) is a 2β, 3α, 5α-trihydroxyandrost-6-one solvent. Specifically, the total protein of the cells was extracted by M-PER reagent, and the protein concentration was determined by the BCA method. Take 20 μg of protein sample, add 5×SDS protein loading buffer, boil for 5 min, and separate by 10% SDS-polyacrylamide gel electrophoresis; the separated protein is transferred to PVDF membrane by wet transfer method, then 5% skim milk powder is used. Blocked at room temperature for 1 h; add the diluted primary antibody, incubate at 4 °C overnight; TBST wash 3 times, each 5 min, add the corresponding secondary antibody, incubate for 1 h at room temperature, wash 3 times with TBST for 5 min each time. Chemiluminescence method for color photographing.

IF (immunofluorescence) detection of 2β,3α,5α-trihydroxyandrost-6-one inhibits nuclear translocation of NF-κB p65 subunit in LPS-induced macrophages.

The macrophage cell line RAW264.7 was pretreated with 2β, 3α, 5α-trihydroxyandrost-6-one at different concentrations (0.1 μM, 0.5 μM, 2.5 μM) for 30 min, and then stimulated with 100 ng/ml LPS. After 30 min, 4% paraformaldehyde fixed cells were subjected to immunofluorescence assay to detect the subcellular localization of p65. HP-β-CD is a 2β, 3α, 5α-trihydroxyandrost-6-one solvent. (Blue for DAPI staining shows nuclei, green is p65 protein).

Reverse transcription PCR amplification (RT-PCR):

Cell treatment: RAW264.7 was treated with different concentrations (0.1μM, 0.5μM, 2.5μM, 10μM) of 2β, 3α, 5α-trihydroxyandrost-6-one or positive drug dexamethasone (DXMS) 1uM for 30min. After 6 hours of stimulation with 100 ng/ml LPS, total RNA was extracted for detection of inflammatory factors. HP-β-CD is a 2β, 3α, 5α-trihydroxyandrost-6-one solvent.

RNA extraction and amplification: 1) Extraction of total RNA: according to the instructions of the Trizol extraction reagent. After the cells were treated to the designated time point, the medium was aspirated, and the medium was washed twice with PBS. The lysed cells were thoroughly pipetted by the addition of 1 ml of Triol (the following reagents were calculated as 1 ml of Trizol). Add 200 ul of chloroform, mix vigorously by hand, and let stand for 3 min at room temperature. Centrifuge at 12000g for 15min at 4°C, take 400ul of the upper aqueous phase into a new tube, add 400ul of isopropanol, gently mix by hand, and let stand at room temperature for 20min. After centrifugation at 12000 g for 10 min at 4 ° C, the supernatant was discarded. Add 500 ul of pre-cooled 75% ethanol, centrifuge at 7500 g for 10 min at 4 ° C, and carefully discard the supernatant. After air drying, add appropriate amount of DEPC water to dissolve the RNA precipitate. 2) RNA quantification: RNA was quantified using a Nanodrop 2000 nucleic acid quantitation instrument, and the OD ratio at a wavelength of 260/280 nm was measured, and the ratio was considered to be good in the range of 1.8-2.0. 3) Reverse transcription reaction: The total amount of RNA in each reaction system was 2 ug, oligo dT 1 ul, and the reaction system was adjusted to 13 ul using DEPC water. After centrifugation and mixing, it was pre-denatured at 65 ° C for 5 min. Immediately after pre-denaturation, place on ice and add RT Reaction Buffer 4ul, dNTP 2ul, Reverse Transcriptase 1ul. The reverse transcription reaction was carried out after mixing by centrifugation. The reverse transcription reaction conditions were: 42 ° C 60 min - 70 ° C 10 min - 4 ° C. 4) PCR amplification reaction parameters: PCR amplification reaction system is: Taq enzyme premix 5 ul, cDNA 1 ul, primer 2 ul, ddH 2 O 2 ul. The cycle parameters were: Holding stage: 95 ° C 15 min; Cycling stage (40 cycles): 95 ° C 10 s - 56 ° C 20 s - 72 ° C 30 s; Melt Curve stage: 95 ° C 15 s - 60 ° C 60 s - 95 ° C 15 s - 60 ° C 60 s.

IF detection of 2β,3α,5α-trihydroxyandrost-6-one inhibited nuclear translocation of NF-κB p65 subunit in LPS-induced primary peritoneal macrophages.

Primary peritoneal macrophages were pretreated with different concentrations (0.1μM, 0.5μM, 2.5μM) 2β, 3α, 5α-trihydroxyandrost-6-one or DXMS (dexamethasone) 1uM for 30min, then added 100ng/ml After 30 min of LPS stimulation, the cells were fixed with 4% paraformaldehyde for immunofluorescence assay to detect the subcellular localization of p65. Among them, HP-β-CD is a 2β, 3α, 5α-trihydroxyandrost-6-one solvent. (Blue is DAPI and green is p65 protein).

3 experimental results:

2β,3α,5α-trihydroxyandrost-6-one has a negative regulatory effect on the activation of NF-kB, a key molecule of LPS-induced inflammatory signaling pathway in peripheral macrophage cell line RAW264.7, and its downstream pro-inflammatory factor The expression has an inhibitory effect.

As shown in Figure 1, NF-κB was activated by phosphorylation of RAW264.7 cells after LPS stimulation, while 2β, 3α, 5α-trihydroxyandrost-6-one dose-dependently inhibited this up-regulation.

As shown in Figure 2, immunofluorescence showed that 2β, 3α, 5α-trihydroxyandrost-6-one blocked the nucleus of NF-κB p65 subunit induced by LPS stimulation in RAW264.7 cells after 30 minutes of LPS stimulation. Shift.

Meanwhile, qRT-PCR results showed (Fig. 3) that 2β, 3α, 5α-trihydroxyandrost-6-one significantly inhibited the up-regulation of IL-6, iNOS, and MCP-1 mRNA expression levels induced by LPS.

2β,3α,5α-trihydroxyandrost-6-one inhibits the entry of NF-kB, a key molecule of the LPS-induced inflammatory signaling pathway in primary peritoneal macrophages.

As shown in Figure 4, NF-κB was phosphorylated and activated into the nucleus after LPS stimulation in primary isolated cultured peritoneal macrophages, while 2β, 3α, 5α-trihydroxyandrost-6-one significantly inhibited LPS stimulation. Nuclear arrest of the NF-κB p65 subunit.

Example 2.

2β,3α,5α-trihydroxyandrost-6-one inhibits LPS-induced inflammatory response in central microglia

1) Cells: Mouse microglia cell line BV2, purchased from the Cell Resource Center of Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences; primary cultured microglia.

2) Main reagents:

HP-β-CD, purchased from Xi'an Deli Biotechnology Co., Ltd.; 2β, 3α, 5α-trihydroxyandrost-6-one synthesized by Guangzhou Saite Pharmaceutical Technology Co., Ltd.; lipopolysaccharide LPS (Escherichiacoli0111: B4 , Sigma, Cat. L2630); Antibody: anti-p65 (Santa Cruz. sc-8008); Phospho-NF-κB p65 (Ser536) (93H1) Rabbit mAb (cell signaling technology, Cat. 3033); p38 antibody (CST , USA, Cat. 9212,) (1:1000); p-p38 antibody (Thr 180/Tyr 182) (CST, USA, Cat. 9216,) (1:1000); Dulbacco's Modified Eagle Medium (Gibco, Cat. 10013608R); fetal bovine serum (FBS) (Gibco, Cat. 10091148);

Reagent (Life, 15596-018).

3) Main equipment:

Cell clean bench (Thermo, MSC-ADVANTAGE); CO2 cell incubator (Thermo 3111, USA); inverted phase contrast/fluorescence microscope (Olympus IX71, Japan); benchtop cryostat (Eppendrof, German); chemiluminescence imager (Bio-Rad Mini-Protein II cells, USA); Bio-Rad Mini Tran-Biot Transfer Cell (USA)

2. Experimental methods

2β, 3α, 5α-trihydroxyandrost-6-one inhibits LPS-induced activation of microglial BV2. (Figure 5)

BV2 cells in the logarithmic growth phase were taken, cells were digested with 0.25% trypsin, and the cell concentration was adjusted and seeded on a 6-well plate. After 24 h, pretreatment with 2β, 3α, 5α-trihydroxyandrost-6-one for 1 hour, then adding LPS (final concentration of 100 ng/ml); only LPS treatment was used as a positive control; no treatment group was negative. Control. After treatment for 24 hours, the amoebic-like cells were observed under a phase contrast microscope and counted, and the data were statistically analyzed.

Western blot analysis of 2β,3α,5α-trihydroxyandrost-6-one blocked the phosphorylation of NF-κB, a key molecule involved in LPS-induced BV2 cell inflammation-related signaling pathway

The adjusted cell density of BV2 cells in the logarithmic growth phase was inoculated into 6-well plates and randomly divided into blank control group (no drug added), LPS group, 2β at different concentrations (0.1 μM, 0.5 μM, 2.5 μM, 10 μM). 3α, 5α-trihydroxyandrost-6-one group and DXMS (dexamethasone) positive control group. After inoculation for 24 hours, the cells were pretreated with 2β, 3α, 5α-trihydroxyandrost-6-one or DXMS for 30 min, then stimulated with a final concentration of 100 ng/ml LPS for 30 min. M-PER reagent was used to extract total cellular protein, determined by BCA method. Protein concentration. Take 20 μg of protein sample, add 5×SDS protein loading buffer, boil for 5 min, and then separate it by 10% SDS-polyacrylamide gel electrophoresis; then transfer to PVDF membrane by wet transfer, and block with 5% skim milk powder for 1 h at room temperature. Add the diluted primary antibody, incubate at 4 °C overnight; TBST wash 3 times, each 5 min, add the corresponding secondary antibody, incubate for 1 h at room temperature, wash 3 times with TBST for 5 min each time. Chemiluminescence method for color photographing.

IF detection of 2β,3α,5α-trihydroxyandrost-6-one inhibited nuclear translocation of NF-κB p65 subunit in LPS-induced primary microglia.

Primary microglia were treated with different concentrations of 2β, 3α, 5α-trihydroxyandrost-6-one or DXMS (dexamethasone) 0.5uM for 30 min, then stimulated with 100 ng/ml LPS. After 30 min, 4% paraformaldehyde fixed cells were subjected to immunofluorescence assay to detect the subcellular localization of p65. Among them, HP-β-CD is a 2β, 3α, 5α-trihydroxyandrost-6-one solvent. (Blue is DAPI, green is p65 protein, and red is microglia marker Iba-1 protein).

3. Experimental results:

2β,3α,5α-trihydroxyandrost-6-one inhibits LPS-induced activation of the microglial cell line BV2 by down-regulating NF-κB, a key molecule of the inflammatory signaling pathway.

As shown in Figure 5, after 24 hours of stimulation with LPS by BV2 cells, the cells showed a distinct activation pattern, while 2β, 3α, 5α-trihydroxyandrost-6-one dose-dependently reduced the number of cells in the activated form.

As shown in Figure 6, NF-κB was activated by phosphorylation of BV2 cells after LPS stimulation, while 2β, 3α, 5α-trihydroxyandrost-6-one dose-dependently inhibited this up-regulation; In the stimulation group, 0.5 μM 2β, 3α, 5α-trihydroxyandrost-6-one had significantly inhibited the phosphorylation of NF-kB p65 subunit Ser536 after LPS stimulation, and the p65 total protein was also significantly down-regulated.

2β,3α,5α-trihydroxyandrost-6-one inhibits the entry of NF-kB, a key molecule of LPS-induced inflammatory signaling pathway in primary microglia.

As shown in Figure 7, NF-κB was phosphorylated and activated into the nucleus after LPS stimulation in primary isolated cultured microglia, while 2β, 3α, 5α-trihydroxyandrost-6-one significantly inhibited LPS stimulation. Nuclear arrest of the NF-κB p65 subunit.

Our results show that 2β,3α,5α-trihydroxyandrost-6-one inhibits LPS-induced activation of microglia and macrophages by down-regulating the expression of NF-κB, a key molecule of the inflammatory pathway, strongly suggesting 2β 3α,5α-trihydroxyandrost-6-one has an antagonistic effect on inflammation.

Claims (7)

1. Use of 2β, 3α, 5α-trihydroxyandrost-6-one, its progeny or a pharmaceutically acceptable salt thereof for the preparation of a medicament for treating an inflammatory response in a patient.
2. The use according to claim 1, wherein the inflammatory response is an inflammatory response mediated by the NF-κB signaling pathway.
3. The use according to claim 1, wherein the inflammatory response is a peripheral inflammatory response or a central nervous system inflammatory response.
4. The use according to claim 3, wherein said peripheral inflammatory response is manifested by macrophage polarization.
5. The use according to claim 3, wherein the central nervous system inflammatory response is manifested by activation of microglia.
6. The use according to claim 1 wherein the medicament further comprises another therapeutic agent.
7. The use of claim 1 wherein the patient is a human.

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