TW201929863A - 2[beta],3[alpha],5[alpha]-TRIHYDROXYL ANDROSTANE-6-KETONE FOR TREATMENT OF INFLAMMATORY REACTIONS - Google Patents

Abstract

Disclosed is an application of 2[beta],3[alpha],5[alpha]-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 2[beta],3[alpha],5[alpha]-trihydroxyl androstane-6-ketone inhibits the activation of microglial cells and macrophages by down-regulating the expression of inflammatory pathway key molecules NF-[kappa]B, and thus can used for treating inflammations.

Description

2β, 3α, 5α-trihydroxyandrost-6-one for the treatment of inflammatory reactions

The invention relates to a new medical application of 2β, 3α, 5α-trihydroxyandrost-6-one, and specifically relates to the application of 2β, 3α, 5α-trihydroxyandrost-6-one in the treatment of inflammatory reactions.

Macrophages are ubiquitous in blood, lymph, and tissues. They are one of innate immune cells. They are important cells involved in the natural immune response. They have many functions such as killing bacteria, phagocytosing pathogenic bacteria, presenting antigens, and secreting cytokines. It plays an important role in the removal of pathogenic microorganisms and senescent cells, the promotion and suppression of inflammatory responses, the induction of adaptive immune responses, and the repair and reconstruction of damaged tissues. It can maintain the homeostasis and also affect the formation and treatment of diseases. makes an important impact.

Different PAMP (pathogen-associated molecular patterns) and DAMP (damage-associated molecular patterns) can stimulate macrophages to polarize in different directions, forming M1 and M2 macrophages. For example, pro-inflammatory factors IFN-γ, TNF, and LPS stimulate M1 macrophages by IFN-γR, TLR and other receptors and their downstream signaling NF-κB. A large number of pro-inflammatory factors such as TNF-α, IL-1, NO, and reactive oxygen intermediates have strong antigen presenting ability. At the same time, M1 macrophages promote Th1 immune response, promote inflammatory response in the early stage of inflammation, and kill cells. Pathogens of infection. Polarization is induced by IL-4, IL-13, immune complexes, IL-10, glucocorticoids, etc. The activated M2 macrophages show a large number of anti-inflammatory factors such as IL-10 and TGF-β. Amino acid enzyme 1 ((Arg1), CD206, DC-SIGN, mannitol receptor, scavenger receptor CD163, CCR2, CXCR1 and other marker molecules show that M2 macrophages mainly activate Th2 type immune responses after polarization. It is mainly involved in pathological processes such as anti-inflammatory response, promoting tissue remodeling, fibrosis, and tumor development. M1 and M2 macrophages can switch to each other under specific microenvironments.

Macrophages polarized by M1 macrophages are thought to be involved in various disease processes such as inflammatory diseases, parasitic infections, asthma, cardiovascular diseases, tumors, etc., all of which play an important role.

Microglia is a type of glial cells in the central nervous system, which accounts for about 5-10% of the total glial cells. Microglia are widely considered to be macrophages in the brain and spinal cord, which are mononuclear phagocytes. Family, the main immune effector in the central nervous system.

As the immune effector cells resident in the central nervous system, microglial cells and their mediated neuroinflammation play a very important role in the process of central nervous system injury and disease outcome. Under normal circumstances, microglial cell bodies are small, with slender, highly branched processes with many spinous processes. Under physiological conditions, microglial cells are in a resting state and play an immune surveillance role. When the central nervous system is stimulated by factors such as inflammation, infection, and trauma, microglial cells can be rapidly activated and then mediate multiple immune responses. The activated microglial cells become larger, the cell body becomes round, the protrusions on the cell surface disappear, and the state becomes amoeba-like macrophages, which can quickly transfer and phagocytose apoptotic neurons, synapses, and cell debris. Maintain the homeostasis of the central nervous system and delay the development of neurodegenerative diseases.

Clinical and neuropathological studies have shown that activated microglial cells play an important role in neurodegenerative diseases such as Parkinson's disease, HIV encephalopathy, multiple sclerosis, and Alzheimer's disease. At the same time, excessive activation or uncontrollable microglial cells can cause neurotoxicity, and are important sources of pro-inflammatory factors and oxidative stress, such as nitric oxide (NO), oxygen free radicals, proteolytic enzymes, and 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 secreted explosively. In the later stage of inflammation caused by injury, neurotrophic factors such as BDNF are mainly secreted, which is beneficial to the nutrition and repair of neurons.

The inventors of the present invention have unexpectedly discovered that 2β, 3α, 5α-trihydroxyandrost-6-one inhibits the activation of LPS-induced microglial cells and macrophages by down-regulating the expression of the key molecule of the inflammatory pathway NF-kB, It can be used to treat inflammation.

According to one aspect of the present invention, the use of 2β, 3α, 5α-trihydroxyandrost-6-one, a deuterate thereof, or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for treating an inflammatory reaction in a patient is provided. In some implementations, the inflammatory response is an inflammatory response mediated by NF-κB signaling. In some implementations, the inflammatory response is a peripheral inflammatory response or a central nervous system inflammatory response. In some implementations, the peripheral inflammatory response manifests as macrophage polarization. In some implementations, the central nervous system inflammatory response manifests as activation of microglial cells. In some implementations, the medicament also includes another therapeutic agent. In some implementations, the patient is human.

Another aspect of the present invention provides a method for 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 deuterate thereof, or a pharmacology thereof. A pharmaceutically acceptable salt, or a pharmaceutical composition comprising 2β, 3α, 5α-trihydroxyandrost-6-one, a deuterate thereof, or a pharmaceutically acceptable salt thereof. In some implementations, the inflammatory response is an inflammatory response mediated by NF-κB signaling. In some implementations, the inflammatory response is a peripheral inflammatory response or a central nervous system inflammatory response. In some implementations, the peripheral inflammatory response manifests as macrophage polarization. In some implementations, the central nervous system inflammatory response manifests as activation of microglial cells. In some implementations, the patient is human.

Another aspect of the present invention provides 2β, 3α, 5α-trihydroxyandrost-6-one, a deuterate thereof, or a pharmaceutically acceptable salt thereof for use in the treatment of a patient's inflammatory response. In some implementations, the inflammatory response is an inflammatory response mediated by NF-κB signaling. In some implementations, the inflammatory response is a peripheral inflammatory response or a central nervous system inflammatory response. In some implementations, the peripheral inflammatory response manifests as macrophage polarization. In some implementations, the central nervous system inflammatory response manifests as activation of microglial cells. In some implementations, the patient is human.

Yet another aspect of the present invention provides a method for reducing or eliminating a patient's inflammatory response, the method comprising administering to a patient an effective amount of 2β, 3α, 5α-trihydroxyandrost-6-one, a deuterated compound thereof, or a medicament A scientifically acceptable salt, or a pharmaceutical composition comprising 2β, 3α, 5α-trihydroxyandrost-6-one, a deuterate thereof, or a pharmaceutically acceptable salt thereof. Another aspect of the present invention provides a method for reducing or eliminating a patient's NF-κB signaling-mediated inflammatory response, the method comprising administering to the patient an effective amount of 2β, 3α, 5α-trihydroxyandrost-6- A ketone, a deuterate thereof, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition including 2β, 3α, 5α-trihydroxyandrost-6-one, a deuterate thereof, or a pharmaceutically acceptable salt thereof. Another aspect of the present invention provides a method for 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 , A deuterate thereof or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising 2β, 3α, 5α-trihydroxyandrost-6-one, a deuterate thereof or a pharmaceutically acceptable salt thereof. Another aspect of the present invention provides a method for 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 deuterate thereof or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition including 2β, 3α, 5α-trihydroxyandrost-6-one, a deuterate thereof or a pharmaceutically acceptable salt thereof. Another aspect of the present invention provides a method for reducing or eliminating the activation of microglial cells in the inflammatory response of the central nervous system, which method comprises administering to a patient an effective amount of 2β, 3α, 5α-trihydroxyandrost-6 -A ketone, a deuterate thereof or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising 2β, 3α, 5α-trihydroxyandrost-6-one, a deuterate thereof or a pharmaceutically acceptable salt thereof.

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

The term "pharmaceutically acceptable" means that the substance does not have such properties, i.e., taking into account the disease or condition to be treated and the respective route of administration, these properties will enable rational and prudent medical practitioners to avoid giving to patients Take the 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 substance and the amount of the substance are effective in preventing, reducing or ameliorating one or more symptoms of a disease or disorder, and / or prolonging the survival of a subject being treated. .

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

2β, 3α, 5α-trihydroxyandrost-6-one and its pharmaceutically acceptable salts
2β, 3α, 5α-trihydroxyandrost-6-one is also referred to herein as "YC-10" or "the compound of the present invention", and its structural formula is shown by formula (I). YC-10 has been proven to have antitumor and neuroprotective effects.
(Formula I)

The compounds of the present invention may be formulated in the form of a pharmaceutically acceptable salt or in the form of a pharmaceutically acceptable salt. Expected pharmaceutically acceptable salt forms include, but are not limited to, mono, double, tertiary, and quaternary salts. Pharmaceutically acceptable salts are non-toxic in the amounts and concentrations at which they are administered. Without preventing it from exerting physiological effects, by changing the physical properties of the compounds, the preparation of such salts can facilitate pharmacological applications. Useful changes in physical properties 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 their sulfate, chloride, hydrochloride, fumarate, maleate, phosphate, aminosulfonate, acetate , Citrate, lactate, tartrate, mesylate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate, cyclohexylaminosulfonate and quinate salts. Pharmaceutically acceptable salts can be obtained from acids such as hydrochloric acid, maleic acid, sulfuric acid, phosphoric acid, aminosulfonic acid, acetic acid, citric acid, lactic acid, tartaric acid, malonic acid, methanesulfonic acid, ethanesulfonic acid Acids, benzenesulfonic acid, p-toluenesulfonic acid, cyclohexylaminosulfonic acid, fumaric acid and quinic acid.

When acidic functional groups such as carboxylic acids or phenols are present, pharmaceutically acceptable salts also include base addition salts, such as those containing benzathine penicillin, chloroprocaine, choline, diethanolamine, ethanolamine, tert-butyl Salts of amines, ethylenediamine, meglumine, procaine, aluminum, calcium, lithium, magnesium, potassium, sodium, ammonium, alkylamines and zinc. Such salts can be prepared using the appropriate corresponding base.

With standard techniques, pharmaceutically acceptable salts can be prepared. For example, the compound in the form of a free base is dissolved in a suitable solvent, such as an aqueous or water-alcoholic solution containing a suitable acid, 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 a particular compound is a base, the required pharmaceutically acceptable salt can be prepared by any suitable method available in the art, such as treating the free base with an inorganic or organic acid, such as Inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid and similar acids, such organic acids as acetic acid, maleic acid, succinic acid, mandelic acid, fumaric acid, malonic acid, pyruvate, oxalic acid, glycolic acid , Salicylic acid, pyranosidyl acid (such as glucuronic acid or galacturonic acid), alpha-hydroxy acids (such as citric or tartaric acid), amino acids (such as aspartic acid or glutamine) Acid), aromatic acid (such as benzoic acid or cinnamic acid), sulfonic acid (such as p-toluenesulfonic acid or ethanesulfonic acid), or the like.

Similarly, 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, secondary amine or tertiary amine), alkali metal hydroxide or 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-spermine), ammonia, primary amines, secondary amines, and tertiary amines And cyclic amines (such as hydroxyethylpyrrolidine, piperidine, morpholine, and piperazine), and inorganic salts, which are derived from sodium, calcium, potassium, magnesium, manganese, iron, copper, zinc, aluminum, and lithium.

A pharmaceutically acceptable salt of a compound may exist as a complex. Examples of complexes include 8-chlorotheophylline complexes (similar to, for example, theophylline: diphenhydramine 8-chlorotheophylline (1: 1) complexes; halohaining) and various including cyclic Dextrin complex.

The present invention is also intended to include a pharmaceutically acceptable deuterated compound or other non-radioactive substituted compound using the compound. Deuteration is the replacement of one or more or all of the hydrogen in the active molecular group with the isotope deuterium. Because it is non-toxic and non-radioactive, it is about 6-9 times more stable than the carbon-hydrogen bond. It 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 an excellent modification method.

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

Suitable dosage forms depend, in part, on the use or route of administration, such as oral, transdermal, transmucosal, 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 the delay in which the compound or composition exerts its effect.

Carriers or excipients can be used to produce the composition. Such carriers or excipients may be selected to facilitate administration of the compound. Examples of carriers include calcium carbonate, calcium phosphate, various sugars (such as lactose, glucose, or sucrose), or starch types, cellulose derivatives, gelatin, vegetable oils, polyethylene glycols, and physiologically compatible solvents. Examples of physiologically compatible solvents include sterile water for injection (WFI) solutions, saline solutions, 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 inhaled. In some implementations, injections or lyophilized powder injections are preferred. For oral administration, for example, the compounds can be formulated in conventional oral dosage forms such as capsules, tablets, and liquid preparations such as syrups, elixirs, and concentrated drops.

Pharmaceutical preparations for oral use are available, such as by combining the composition or its components with solid excipients, optionally grinding the resulting mixture, and processing the mixture of granules after adding suitable adjuvants (if necessary), Thus, tablets or dragees are obtained. Suitable excipients are in particular fillers, for example sugars, containing lactose, sucrose, mannitol or sorbitol; cellulose preparations, for example corn starch, wheat starch, rice starch, potato starch, gelatin, tragacanth, methyl fiber Cellulose, hydroxypropyl methyl cellulose, sodium carboxymethyl cellulose (CMC), and / or polyvinylpyrrolidone (PVP: povidone). If desired, disintegrating agents may be added, such as the cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate.

Alternatively, injections (parenteral) can be used, such as intramuscular, intravenous, intraperitoneal and / or subcutaneous. For injection, the composition of the invention or its components is formulated as a sterile liquid solution, preferably in a physiologically compatible buffer or solution, such as a saline solution, a Hank solution or a Ringer solution. In addition, the composition or its components may be formulated in a solid form and redissolved or suspended immediately before use. Also available in lyophilized powder form.

Administration can also be by transmucosal, topical or transdermal means. For transmucosal, topical or transdermal administration, a penetrant suitable for the barrier to be penetrated is 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. In addition, detergents can be used to promote penetration. Transmucosal administration can, for example, be by nasal spray or suppository (rectally or vaginally).

May be determined by standard procedures to be administered with an effective amount of the various components, consideration factors such as the compound IC _50, the biological half life of the compound, the age, the weight and size of the object, and related disorders. The importance of these and other factors is well known to those skilled in the art. Generally, the dosage will be between about 0.01 mg / kg and 50 mg / kg of the subject being treated, preferably between 0.1 mg / kg and 20 mg / kg. Multiple doses can be used.

The composition of the invention or its components can also be used in combination with other therapeutic agents for the same disease. Such combined use includes 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 implementations, the dosage of one or more compounds of the invention or other therapeutic agents used in combination can be modified, for example, by reducing the dosage of a compound or therapeutic agent relative to the compound or therapeutic agent used alone by methods known to those skilled in the art.

It is understood that the combination or combination includes use with other therapies, drugs, medical procedures, etc., where such other therapies or procedures may be at a different time than the composition of the invention or its components (e.g., in the short term Within (such as a few hours, such as 1, 2, 3, 4 to 24 hours) or over a longer period (such as 1 to 2 days, 2 to 4 days, 4 to 7 days, 1 to 4 weeks) or between The composition or component thereof of the present invention is administered at the same time. The combined use also includes use with a therapy or medical procedure (such as surgery) which is administered once or infrequently, and accompanied by the composition or component of the present invention Administration within a short or longer period of time before or after such other therapies or procedures. In some implementations, the invention is used to deliver the composition of the invention or its components and one or more other pharmaceutical therapeutic agents, It is delivered by the same or different routes of administration.

The combined administration of any route of administration comprises delivering the composition of the invention or its components and one or more other pharmaceutical therapeutic agents together in any formulation by the same route of administration, comprising the two compounds being chemically linked and Preparations that maintain their respective therapeutic activity when administered. In one aspect, these other drug therapies can be co-administered with a composition of the invention or a component thereof. By co-administration in combination with a formulation containing co-formulation or chemically linked compounds, or in a short period of time (e.g., within one hour, within two hours, within three hours, up to 24 hours ) Compounds are administered in the form of two or more separate preparations, which are administered by the same or different routes.

Co-administration of separate preparations includes co-administration of delivery via one device, such as the same inhalation device, the same syringe, etc., or from different devices in the short term relative to each other. Co-formulations of a compound of the invention and one or more additional drug therapies delivered by the same route of administration include preparing materials together so that they can be administered by a single device, containing different compounds in a formulation, or compounds Modified so that they are chemically linked together but still maintain their respective biological activities. Such chemically linked compounds may include linkers that separate two active ingredients, which linkers are substantially maintained in the body or may degrade in the body.

Examples
Example 1.
2β, 3α, 5α -trihydroxyandrost- 6- one inhibits inflammatory response of peripheral macrophages induced by LPS
1. Cells: Mouse macrophage cell line RAW 264.7, purchased from ATCC; primary cultured peritoneal macrophages.
2. The main reagents:
2β, 3α, 5α-trihydroxyandrost-6-one was synthesized by Guangzhou Sepute Pharmaceutical Technology Co., Ltd .; HP-β-CD was purchased from Xi'an Deli Biotechnology Co., Ltd .; lipopolysaccharide LPS (E. coli ( Escherichiacoli) 0111: B4, Sigma, catalog number L2630); antibodies: anti-p65 (Santa Cruz. Sc-8008); phosphorylated NF-κB p65 (Ser536) (93H1) rabbit mAb (CST, catalog number 3033); p38 antibody (CST, USA, Cat. No. 9212) (1: 1000); p-p38 antibody (Thr 180 / Tyr 182) (CST, USA, Cat. 9216) (1: 1000); Durbock's Modified Eagle's Medium (Dulbacco's Modified Eagle Medium) (Gibco, Cat. No. 10013608R); fetal bovine serum (FBS) (Gibco, Cat. No. 10091148); TRIzol® reagent (Life, 15596-018).
3. Main equipment: cell ultraclean bench (Thermo, MSC-ADVANTAGE); CO ₂ cell incubator (Thermo 3111, USA); inverted phase contrast / fluorescence microscope (Olympus IX71, Japan); desktop low-temperature 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 method
Western blot method was used to detect 2β, 3α, 5α -trihydroxyandrost- 6- one inhibiting LPS- induced phosphorylation of NF-κB .
Macrophage cell line RAW264.7 was administered with different concentrations (0.1mM, 0.5mM, 2.5mM, 10mM) of 2β, 3α, 5α-trihydroxyandrost-6-one or the positive drug dexamethasone (DXMS) 1 mM After 30 min of treatment, 100 ng / ml LPS was added for 30 min to stimulate the protein, and the proteins were collected for Western blotting detection. 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 BCA method. Take 20μg of protein sample, add 5 × SDS protein loading buffer, and boil for 5 minutes, then separate by 10% SDS-polyacrylamide gel electrophoresis; the separated protein is transferred to PVDF membrane by wet transfer method, and then 5% The skimmed milk powder was blocked at room temperature for 1 hour; the diluted primary antibody was added and incubated overnight at 4 ° C; TBST was washed 3 times for 5 min each time, and the corresponding secondary antibody was added; incubated at room temperature with shaking for 1 h, and TBST was washed 3 times for 5 min each. Chemiluminescence photochromography.

IF ( immunofluorescence ) detects that 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 mM, 0.5 mM, 2.5 mM) for 30 min, and then 100 ng / ml LPS was added. After stimulation for 30 min, immunofluorescence test was performed with 4% paraformaldehyde-fixed cells to detect the subcellular localization of p65. HP-β-CD is a 2β, 3α, 5α-trihydroxyandrost-6-one solvent. (DAPI staining shows nuclei in blue, p65 protein in green).

Reverse transcription PCR amplification (RT-PCR) :
Cell treatment: RAW264.7 was pretreated with different concentrations (0.1mM, 0.5mM, 2.5mM, 10mM) of 2β, 3α, 5α-trihydroxyandrost-6-one or positive drug dexamethasone (DXMS) for 1 uM for 30min Then, 100 ng / ml LPS was added, and after 6 hours of stimulation, 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) Total RNA extraction: follow Trizol extraction reagent instructions. After the cells were treated to the specified time point, the medium was aspirated and washed with PBS twice to wash the medium. Add 1 ml of triol to fully lyse the cells (the following reagents are calculated as 1 ml of Trizol). Add 200ul of chloroform, shake vigorously and mix well, and then stand at room temperature for 3min. Centrifuge at 12000g at 4 ° C for 15min, 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. Centrifuge at 12000g at 4 ° C for 10min and discard the supernatant. Add 500ul of pre-chilled 75% ethanol and centrifuge at 7500g at 4 ℃ for 10min. Discard the supernatant carefully. After air drying, an appropriate amount of DEPC water was added to dissolve the RNA precipitate. 2) RNA quantification: Use Nanodrop 2000 nucleic acid quantifier to quantify RNA and determine the OD ratio at 260 / 280nm wavelength. The ratio is considered to be of good quality within the range of 1.8-2.0. 3) Reverse transcription reaction: The total amount of RNA in each reaction system is 2ug, oligo dT 1ul, and the reaction system is adjusted to 13ul using DEPC water. After centrifugation, place at 65 ° C for 5 min. Immediately after pre-denaturation, place on ice, add 4ul of RT reaction buffer, 2ul of dNTP, and 1ul of reverse transcriptase. After centrifugation, perform reverse transcription reaction. The reverse transcription reaction conditions are: 42 ° C 60min-70 ° C 10min-4 ° C. 4) PCR amplification reaction parameters: The PCR amplification reaction system is: Taul enzyme premix 5ul, cDNA 1ul, primer 2ul, ddH2O 2ul. The cycle parameters are: holding phase: 95 ° C for 15min; cycle phase (40 cycles): 95 ° C for 10 seconds-56 ° C for 20 seconds-72 ° C for 30 seconds; melting curve phase: 95 ° C for 15 seconds-60 ° C for 60 seconds-95 ° C 15 seconds-60 ° C for 60 seconds.

IF detected 2β, 3α, 5α -trihydroxyandrost- 6- one inhibited nuclear translocation of NF-κB p65 subunit in primary peritoneal macrophages induced by LPS .
Primary peritoneal macrophages were given different concentrations (0.1 mM, 0.5 mM, 2.5 mM) of 2β, 3α, 5α-trihydroxyandrost-6-one or DXMS (dexamethasone) 1 uM for 30 min before adding 100 ng / ml LPS. After stimulation for 30 min, immunofluorescence test was performed with 4% paraformaldehyde-fixed cells to detect the subcellular localization of p65. HP-β-CD is 2β, 3α, 5α-trihydroxyandrost-6-one solvent. (DAPI in blue and p65 protein in green).

3 Experimental results:
2β, 3α, 5α- Trihydroxyandrogens -6- Peripheral macrophage cell line RAW264.7 in LPS Key molecules in induced inflammation signaling NF-kB Its activation has a negative regulatory effect and has an inhibitory effect on the performance of its downstream pro-inflammatory factors.

As shown in Figure 1, NF-κB was phosphorylated and activated by LPS-stimulated RAW264.7 cells, while 2β, 3α, 5α-trihydroxyandrost-6-one inhibited such up-regulation changes in a dose-dependent manner.

As shown in Figure 2, immunofluorescence showed that 30 minutes after LPS stimulation, 2β, 3α, 5α-trihydroxyandrost-6-one could block the NF-κB p65 subunit caused by LPS stimulation in RAW264.7 cells Nuclear shift.

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

2β, 3α, 5α- Trihydroxyandrogens -6- Ketones inhibit primary peritoneal macrophages LPS Key molecules in induced inflammation signaling NF-kB Into the nuclear.

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

Example 2.
2β, 3α, 5α -trihydroxyandrost- 6- one inhibits LPS- induced inflammatory response in central microglial cells
1) Cell: Mouse microglial cell line BV2, purchased from the Cell Resource Center of Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences; primary cultured microglial cells.
2) Main reagents:
HP-β-CD, purchased from Xi'an Deli Biotechnology Co., Ltd .; 2β, 3α, 5α-trihydroxyandrost-6-one was synthesized by Guangzhou Septor Pharmaceutical Technology Co., Ltd .; lipopolysaccharide LPS (E. coli 0111 : B4, Sigma, catalog number L2630); antibodies: anti-p65 (Santa Cruz. Sc-8008); phosphorylated NF-κB p65 (Ser536) (93H1) rabbit mAb (cell signaling technology, catalog number 3033); p38 antibody (CST, USA, Cat. No. 9212) (1: 1000); p-p38 antibody (Thr 180 / Tyr 182) (CST, USA, Cat. 9216) (1: 1000); Durbeck Modified Eagle's medium (Gibco, Cat. No. 10013608R); fetal bovine serum (FBS) (Gibco, Cat. No. 10091148); TRIzol® reagent (Life, 15596-018).
3) Main equipment:
Cell Purification Station (Thermo, MSC-ADVANTAGE); CO2 Cell Incubator (Thermo 3111, USA); Inverted Phase Contrast / Fluorescence Microscopy (Olympus IX71, Japan); Desktop Low Temperature High-Speed Centrifuge (Eppendrof, German); Chemiluminescence Imaging Instrument (Bio-Rad, USA); vertical plate electrophoresis instrument (Bio-Rad Mini-Protein II cells, USA); transfer cell (Bio-Rad Mini Tran-Biot Transfer Cell, USA)

2. Experimental method
2β, 3α, 5α -trihydroxyandrost- 6- one inhibited LPS- induced microglial BV2 activation. ( Figure 5)
Take BV2 cells in logarithmic growth phase, digest cells with 0.25% trypsin, adjust cell concentration, and inoculate on 6-well plates. After 24 hours, pretreat with 2β, 3α, 5α-trihydroxyandrost-6-one for 1 hour, and then add LPS (final concentration 100ng / ml); only add LPS treatment as a positive control; no treatment group is negative Contrast. After 24 hours of treatment, the amoeba-like cells were observed and counted under a phase-contrast microscope, and the data were analyzed statistically.

Western blotting method detects 2β, 3α, 5α -trihydroxyandrost- 6- one blocks LPS- induced phosphorylation activation of NF-κB , a key molecule in inflammation-related signaling in BV2 cells <br/> BV2 cells were adjusted to cell density and seeded in 6-well plates, and randomly divided into blank control group (without drug), LPS group, and different concentrations (0.1 mM, 0.5 mM, 2.5 mM, 10 mM) of 2β, 3α, 5α-trihydroxy The androster-6-one group and the DXMS (dexamethasone) positive control group. After 24 hours of cell seeding, pre-treat with 2β, 3α, 5α-trihydroxyandrost-6-one or DXMS for 30min, and then add LPS at a final concentration of 100ng / ml for 30min. Total cell protein was extracted with M-PER reagent, BCA Method to determine protein concentration. Take 20μg of protein sample, add 5 × SDS protein loading buffer, boil for 5 minutes, and then separate by 10% SDS-polyacrylamide gel electrophoresis; then transfer to PVDF membrane by wet transfer method, and use 5% skim milk powder chamber. Block at room temperature for 1 h; add diluted primary antibody and incubate at 4 ° C overnight; wash TBST 3 times for 5 min each time, add the corresponding secondary antibody, incubate at room temperature for 1 h, and wash 3 times for 5 min each. Chemiluminescence photochromography.

IF detected 2β, 3α, 5α -trihydroxyandrost- 6- one inhibited nuclear translocation of NF-κB p65 subunit in primary microglial cells induced by LPS .
Primary microglial cells were administered with different concentrations of 2β, 3α, 5α-trihydroxyandrost-6-one or DXMS (dexamethasone) 0.5 uM for 30 min, and then 100 ng / ml LPS was added. After stimulation for 30 min, immunofluorescence test was performed with 4% paraformaldehyde-fixed cells to detect the subcellular localization of p65. HP-β-CD is 2β, 3α, 5α-trihydroxyandrost-6-one solvent. (Blue is DAPI, green is p65 protein, and red is microglial marker Iba-1 protein).

3. Experimental results:
2β, 3α, 5α- Trihydroxyandrogens -6- Ketones down-regulate key molecules for inflammatory signaling NF-κB While inhibiting LPS Induced glial cell line BV2 Of activation.

As shown in FIG. 5, after 24 hours of LPS stimulation in BV2 cells, the cells showed obvious activated morphology, and 2β, 3α, 5α-trihydroxyandrost-6-one reduced the number of activated morphological cells in a dose-dependent manner.

As shown in Figure 6, NF-κB was phosphorylated and activated by BV2 cells after LPS stimulation, and 2β, 3α, 5α-trihydroxyandrost-6-one inhibited such up-regulation changes in a dose-dependent manner; compared with single addition of LPS Compared with 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 p65 total protein was also significantly down-regulated.

2β, 3α, 5α -trihydroxyandrost- 6- one inhibits the nuclear entry of NF-kB , a key molecule of LPS- induced inflammatory signaling in primary microglial cells .
As shown in Figure 7, NF-κB was phosphorylated and activated into the nucleus after stimulation with LPS in microglial cells isolated from primary culture. 2β, 3α, 5α-trihydroxyandrost-6-one significantly inhibited LPS stimulation. Nuclear shift of the NF-κB p65 subunit.

Our experimental results show that 2β, 3α, 5α-trihydroxyandrost-6-one inhibits the activation of LPS-induced microglial cells and macrophages by down-regulating the expression of the key molecule of the inflammatory pathway NF-κB, which strongly suggests that 2β, 3α, 5α-trihydroxyandrost-6-one has the effect of antagonizing the inflammatory response.

Figure 1. 2β, 3α, 5α-trihydroxyandrost-6-one blocks LPS-induced phosphorylation activation of NF-κB, a key molecule for inflammation-related signaling in RAW264.7 cells. Western blot was used to detect 2β, 3α, 5α-trihydroxyandrost-6-one inhibiting LPS-induced NF-κB phosphorylation.

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

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

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

Figure 5. 2β, 3α, 5α-trihydroxyandrost-6-one inhibits LPS-induced microglial BV2 activation. (A) Observe the morphology of BV2 cells by phase contrast microscopy; (B) ##: P <0.01 compared with the normal control group; **: P <0.01 compared with the LPS treated group.

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

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

Claims (7)

A use of 2β, 3α, 5α-trihydroxyandrost-6-one, its deuterate or a pharmaceutically acceptable salt thereof in the preparation of a medicament for treating an inflammatory response in a patient. The use according to claim 1, wherein the inflammatory response is an inflammatory response mediated by NF-κB signaling. The use according to claim 1, wherein the inflammatory response is a peripheral inflammatory response or a central nervous system inflammatory response. The use according to claim 3, wherein the peripheral inflammatory response is manifested as macrophage polarization. As claimed in claim 3, wherein the central nervous system inflammatory response is manifested by activation of microglial cells. As claimed in claim 1, wherein the medicament also includes another therapeutic agent. As used in claim 1, wherein the patient is a human.