CN114028384B - Application of liquorice isoflavan derivative in preparation of drug for preventing, relieving or/and treating pruritus - Google Patents

Abstract

The invention discloses the application of a licorice isoflavane derivative in the preparation of drugs or health care products for preventing, alleviating or/and treating pruritus. Tests show that the licorice isoflavane derivative of the present invention is an antagonistic molecule of TRPV3. TRPV3 is the target to treat pruritus, and has obvious effects on prevention, relief, and treatment of pruritus, especially the treatment effect on non-histamine-dependent acute pruritus and chronic pruritus; Well, it has good medicinal prospects, and the licorice isoflavane derivatives of the present invention are a new class of TRPV3 antagonistic molecules, which can be used as lead compounds for further research and development of drugs for treating TRPV3-related diseases. The invention also provides a new drug source for preventing, alleviating and treating skin pruritus.

Description

Licorice isoflavane derivatives in the preparation of drugs for preventing, alleviating or/and treating pruritus Applications

technical field

The present invention relates to the field of medicine, and relates to a medicine for preventing, alleviating and/or treating pruritus, in particular to a natural product licorice isoflavane derivative component in the traditional Chinese medicine licorice used in medicines for preventing, alleviating and/or treating pruritus Applications.

Background technique

Glycyrrhizae isoflavanes are a class of natural products derived from traditional Chinese medicine licorice. Previous studies have revealed that licorice has a wide range of pharmacological effects. Specifically, licorice isoflavanes have been found to have antibacterial, anticancer, and antibacterial effects. Oxidation, anti-inflammatory, etc. On the basis of existing studies, it is of great research and application value to further explore the new functions of these compounds and the molecular mechanisms behind them.

Transient receptor potential ion channels (TRPs) are a class of non-selective cation channels that are widely expressed in many tissues and organs and participate in the regulation of various cellular functions. According to the homology of amino acid sequence, TRP channels are divided into seven subfamilies: TRPC (canonical), TRPV (vanilloid), TRPM (melastatin), TRPA (ankyrin), TRPN (NompC), TRPP (polycystin) and TRPML (mucolipin) ). By mediating the transmembrane flow of extracellular and extracellular cations, TRP channels play a key role in the absorption of calcium ions in cells, the guidance of neuronal growth cones, the growth and differentiation of keratinocytes, and the transmission of sensory signals. More importantly, a series of human diseases, including polycystic kidney disease, skeletal malformation, Olmsted syndrome, etc., have been proved to be caused by abnormal function of TRP channels.

For the TRPV subfamily, since the first member TRPV1 was successfully cloned in 1997, other members of the subfamily have been discovered and cloned successively. From the perspective of molecular sequence and structure, the sequences of the six members of the TRPV subfamily are highly homologous, and all have molecular structures similar to those of classical voltage-dependent potassium channels, that is, tetramers assembled from four subunits , in which each subunit contains 6 transmembrane regions, and the N-terminus containing the ankyrin repeat structure and the C-terminus containing the "TRP domain" located on the inner side of the cell. There is a ring structure between the 5th and 6th transmembrane region, which constructs the channel for ions to pass through, and is considered to be related to the ion selectivity of the channel. As non-selective cation channels, members of the TRPV subfamily all have higher selective permeability to divalent cations, among which Ca ²⁺ ions are the most important. Because TRPV channels are regulated by temperature, pH value and a series of endogenous and exogenous ligand molecules, they are widely involved in the transmission of sensory information in the body and have many important physiological functions such as regulating intracellular Ca ²⁺ balance.

The TRPV3 channel was first cloned in 2002, and it was found that warm temperature (31-39°C) can activate it. Subsequent studies have found more compounds that can activate TRPV3, including 2-APB and natural molecules such as camphor, menthol, and carvacrol. TRPV3 channels are widely expressed in various tissues and organs, among which various epithelial tissues are the most important, mainly including the epidermis of skin, oral cavity and gastrointestinal tract. Previous studies on mice have found that, on the one hand, TRPV3 knockout mice can suffer from temperature sensory impairment, abnormal curly hair, and damaged skin barrier; In both dermatitis and xeroderma models of chronic pruritus, the scratching response of mice was significantly reduced. On the other hand, transgenic mice expressing a TRPV3 enhanced-function mutant (p.G573S) developed severe hair loss and skin erythema, edema, erosion, desquamation, etc., accompanied by obvious spontaneous itching behavior. More importantly, our previous research found that the function-enhancing mutation of human TRPV3 can lead to a rare genetic skin disease - Olmsted syndrome. Hair loss and pain itching etc. These studies have shown that TRPV3 plays an important role in physiological processes such as skin barrier, hair development, temperature perception, inflammatory response, and pain and itching sensation conduction; and as an important target of skin drugs, especially its ability to treat pruritus potential, TRPV3 has increasingly become one of the most concerned TRP channels. However, due to the lack of effective research tools, including specific agonists and antagonists, in order to solve more important problems about TRPV3, finding and developing more drug molecules that regulate TRPV3 has become the focus of everyone's common attention.

For the functional study of ion channels, to evaluate the effect of a certain compound on it, traditionally mainly relies on the detection of electrophysiological properties. The single-cell patch-clamp recording technique has become the most widely used electrophysiological detection technique because of its direct and sensitive advantages. Specifically, the ion channel expressed on the cell membrane is captured by the high-impedance seal formed between the glass microelectrode and the cell membrane surface. The loop circuit composed of the amplifier, electrode and bath liquid is connected. When different stimuli are applied, the opening and closing status of the ion channel is reflected according to the current change in the circuit recorded under a certain artificially clamped cell membrane potential.

Itching, a common unpleasant sensation that induces a scratching response, is similar to pain but differs in that pain often prompts the person to avoid touching the affected area as much as possible. The generation process of itch sensation is also similar to that of pain sensation, relying on the detection of various itch-causing molecules by primary afferent neurons at the nerve endings of peripheral organs such as skin, external pruritogens, or secreted by skin cells, immune cells, etc. The endogenous itch-causing molecules in the body first activate the primary sensory neurons to generate nerve impulses, and then these nerve signals are transmitted to the dorsal horn of the spinal cord, and then relayed by the internal neurons of the spinal cord to the sensory center of the brain to generate itch sensation. It is generally believed that different pruritogens activate different groups of sensory neurons, and according to the response to histamine, the most common pruritogen, the pruritus responses induced by different pruritogens can be roughly divided into histamine-dependent and pruritus-dependent pruritus. Two types of non-histamine-dependent. At the same time, according to the duration of pruritus, it can be divided into two categories: acute pruritus and chronic pruritus. Compared with the warning significance of acute pruritus, persistent and debilitating chronic pruritus may accompany certain skin and systemic symptoms. It can occur when there are no other obvious diseases in the body, and it can become a purely annoying disease with no obvious physiological significance.

As a common clinical symptom in dermatology, pruritus is accompanied by a variety of diseases, such as atopic dermatitis, eczema, psoriasis, etc. At present, the neural and molecular mechanisms of histamine-dependent and non-histamine-dependent pruritus It has been gradually revealed, and accordingly many drugs for the treatment of skin itching have been developed, including antihistamine drugs mainly for histamine-dependent itching and refreshing drugs that can temporarily relieve itching, etc. These drugs may have a limited range of action, Or there are relatively large side effects, and there has been a lack of antipruritic drugs for non-histamine-dependent acute and chronic pruritus in clinical practice. Come new hope.

Contents of the invention

The object of the present invention is to provide a kind of licorice isoflavane for the deficiency of the existing medicine for treating itching, especially for the antipruritic medicine of non-histamine-dependent acute and chronic itching and its application. The application of derivatives in the preparation of medicines for treating pruritus. The licorice isoflavane derivatives of the present invention are TRPV3 antagonistic molecules, TRPV3 is used as a target to treat pruritus, and the therapeutic effect on non-histamine-dependent acute pruritus and chronic pruritus significantly.

To achieve the object of the present invention, one aspect of the present invention provides the use of a licorice isoflavane derivative in the preparation of a drug for preventing, alleviating or/and treating pruritus.

Wherein, the licorice isoflavane derivative is one or more of licorice isoflavane A, glycyrrhizidine or derivatives with 3-phenylchromane as the core, or its stereoisomers and their derivatives in The pharmaceutically acceptable salts and/or solvates are preferably Glycyrrhizin isoflavane A and Glycyrrhizidine.

Wherein, the structures of Glycyrrhizae isoflavane A and Glycyrrhizidine are as follows:

Using electrophysiological patch-clamp recording technology, natural product compounds derived from traditional Chinese medicine were tested in the HEK293T cell line overexpressing TRPV3 channels, and the ability of Glycyrrhizin isoflavan A and Glycyrrhizidine to antagonize TRPV3 was obtained in a dose-dependent manner.

Wherein, the medicine is composed of licorice isoflavane derivatives and a pharmaceutically acceptable carrier.

In particular, the purity of the licorice isoflavane derivatives is ≥ 1%, preferably more than 95% or 95-100% pure substance.

In particular, pharmaceutically acceptable carriers are generally accepted by health care professionals for this purpose and as inactive ingredients of the medicament. A compilation of pharmaceutically acceptable carriers can be found in "Handbook of Pharmaceutical Excipients, 2nd Edition, edited by A. Wade and P. J. Weller; published by the American Pharmaceutical Association, Washington and The Pharmaceutical Press, London, 1994) Find it in the reference book.

Wherein, the pruritus is histamine-dependent or/and non-histamine-dependent pruritus, preferably non-histamine-dependent pruritus.

In particular, said pruritus is acute pruritus or/and chronic pruritus.

Wherein, the medicine exists in the form of tablets, capsules, pills, powders, granules, syrups, emulsions, injections, sprays, gels, creams, cataplasms, and rubber plasters.

In particular, the medicament is in the form of tablets, capsules, pills, powders, granules, syrups.

Another aspect of the present invention provides a medicament for preventing, relieving and/or treating pruritus containing a licorice isoflavane derivative.

Wherein, the licorice isoflavane derivative is one or more of licorice isoflavane A, glycyrrhizidine or derivatives with 3-phenylchromane as the core, or its stereoisomers and their derivatives in The pharmaceutically acceptable salts and/or solvates are preferably Glycyrrhizin isoflavane A and Glycyrrhizidine.

In particular, the purity of the licorice isoflavane derivative is ≥ 1%, preferably more than 95% or 95-100% pure substance.

In particular, the ratio of the weight of the licorice isoflavan derivative to the total weight of the drug is (0.01-10):100, preferably (0.1-1):100.

The medicine can be made into various dosage forms by methods known in the art, such as tablet, capsule, pill, powder, granule, syrup, solution, injection, spray, aerosol, gel type, cream Potions, tinctures, cataplasms, rubber plasters or plasters, etc.

The present invention also provides a method for preventing, alleviating or/and treating pruritus, comprising administering a therapeutically effective dose of licorice isoflavane derivatives (licorice isoflavane A, glycyrrhizidine) to pruritus, and its therapeutically effective dose is 0.3-100 mg/kg.d, preferably 1-20 mg/kg.d, more preferably 10 mg/kg.d.

Unless otherwise stated, the term "therapeutically effective amount" used in the present invention is the amount of the drug that needs to produce an effective effect; "therapeutically effective amount" can be adjusted and changed, and is finally determined by medical personnel, and the factors considered include giving The nature of the drug route and preparation, the recipient's weight, age and other general conditions, as well as the nature and severity of the disease to be treated.

Compared with prior art, the present invention has following obvious advantages:

1. The present invention excavates new medicinal value to the known compound licorice isoflavane derivatives (licorice isoflavane A, licorice cidine), which is used to prevent, alleviate and treat pruritus, and can be prepared as a preventive , conditioning and/or treating pruritus medicines, thus opening up a new field for the application of Chinese herbal medicine licorice and natural product compounds in licorice.

2. The licorice isoflavane derivatives (licorice isoflavane A, licoricecidin) of the present invention have clear pharmacological effects, are effective in preventing, alleviating and treating pruritus, have quick effects, less toxic and side effects, and good safety. Has a good medicinal prospect.

3. The product of the present invention has abundant sources of raw materials, low price, safe clinical use, simple preparation process, can be made into various dosage forms, and has a small dosage and is easy to use, so it is easy to popularize.

4. The licorice isoflavane derivatives (licorice isoflavane A, licoricecidin) of the present invention are a new class of TRPV3 antagonistic molecules, which can be used as lead compounds for further research and development of drugs for treating TRPV3-related diseases.

The present invention adopts the non-histamine-dependent acute pruritus mouse model induced by polypeptide SLIGRL, and tests the ability of licorice isoflavane A and licoricedin to inhibit acute pruritus by counting the number of scratches within 30 minutes as the behavioral detection index. All of them can inhibit the acute pruritus induced by SLIGRL in a dose-dependent manner.

Atopic dermatitis chronic itching mouse model induced by calcipotriol was used, and the number of scratches per hour on different model building days was counted as an inspection index to test the inhibitory effect of licorice isoflavane A and licoricedin on chronic itching The results showed that all of them could inhibit chronic itching in atopic dermatitis model mice to some extent.

The present invention obtains a new class of antagonistic molecules based on the screening of the target molecule TRPV3, which exhibits the activity of inhibiting non-histamine-dependent acute pruritus and chronic pruritus, and can be used as a lead compound for further development of pruritus therapeutic drugs. research and development.

Description of drawings

Figure 1 is a diagram of the antagonism effect of different concentrations of Glycyrrhizae isoflavan A on the TRPV3 current induced by 2-APB, where A is a representative curve of the change of TRPV3 current recorded in the whole cell mode, including the stimulation of 100 μM 2-APB alone , and the changes when multiple concentrations (30, 100 and 300 μM) of Glycyrrhizae isoflavane A are superimposed simultaneously; B is the dose-effect relationship diagram of Glycyrrhizae isoflavane A antagonizing TRPV3 channel, which is calculated by S-type dose-response curve fitting It can be concluded that the IC ₅₀ value of Glycyrrhizae isoflavan A is 20.87 μM (LogIC ₅₀ =1.320±0.128, n=4-5), and the data corresponding to each concentration are expressed as the mean value ± SEM;

Figure 2 is a diagram of the antagonism effect of different concentrations of Glycyrrhizidine on the TRPV3 current induced by 2-APB, wherein A is a representative curve of recording the change of TRPV3 current in the whole cell mode, including when 100 μM 2-APB is stimulated alone, and Simultaneously superimpose the changes of Glycyrrhizidine at various concentrations (10, 30 and 100 μM); B is the dose-effect relationship diagram of Glycyrrhizidine antagonizing TRPV3 channel, calculated by S-type dose-response curve fitting, Glycyrrhizin isoflavane The IC ₅₀ value of A is 15.24 μM (LogIC ₅₀ =1.183±0.120, n=5-8), and the data corresponding to each concentration are expressed as the mean value ± SEM;

Figure 3 is a representative curve of TRPV3 current changes recorded in the membrane inside-out mode, including the current changes when 100 μM 2-APB is stimulated alone, and when different concentrations of Glycyrrhizin A and Glycyrrhizin are simultaneously superimposed In the figure, A is Glycyrrhizin isoflavane A; B is Glycyrrhizidine; finally 10 μM ruthenium red can completely block the TRPV3 current.

Figure 4 is a graph showing the effects of Glycyrrhizae isoflavane A and Glycyrrhizidine on TRPV1 and TRPA1 channels respectively; where: A and B are representative curves of TRPV1 current changes recorded in whole-cell mode, including administration of TRPV1 agonist alone Capsaicin, and the current changes when 100 μM Glycyrrhizin A (A) or Glycyrrhizidine (B) were superimposed at the same time, the final non-selective TRP channel antagonist ruthenium red can completely block the current; C, D is a representative curve of TRPA1 current changes recorded in the whole-cell mode, including the changes when the TRPA1 agonist AITC was administered alone, and when 100 μM Glycyrrhizin A (C) or Glycyrrhizidine (D) was superimposed at the same time In this case, the final non-selective TRP channel antagonist ruthenium red can completely block the current; E, F: Comparison of the blocking ability of Glycyrrhizin A (E) and Glycyrrhizidine (F) on TRPV3, TRPV1 and TRPA1 . ***P<0.001 by unpaired Student's t test. The above data of each group are expressed as mean ± SEM, n = 4-8.

Figure 5 shows the inhibitory effect of licorice isoflavan A on acute pruritus induced by SLIGRL; A-C: the experimental groups with 30, 100 and 300 μM licorice isoflavan A respectively and the corresponding control group mice without drug treatment The line graph of the number of scratches counted in a period of 5 minutes, using repeated measures analysis of variance, P>0.05 (A-B), **P<0.01 (C); D is the experiment of 30, 100 and 300 μM licorice isoflavane A The histogram of the total number of scratches within 30 min of mice in the corresponding control group. Using paired Student's t test, *P<0.05, **P<0.01, N.S.: P>0.05, the data of each group above are expressed as mean ± SEM, n=8;

Figure 6 is the inhibitory effect of Glycyrrhizidine on acute pruritus induced by SLIGRL; A-C: 30, 100 and 300 μM Glycyrrhizidine in the experimental group and the corresponding control group are line graphs of the number of scratches counted in a period of 5 minutes, Repeated measures analysis of variance, P>0.05 (A-B), *P<0.05 (C); D: The total number of scratching times within 30 minutes between the experimental group treated with 30, 100 and 300 μM Glycyrrhizidine and the corresponding control group histogram. Using paired Student's t test, **P<0.01, N.S.: P>0.05, the data of each group above are expressed as mean ± SEM, n=8;

Figure 7 is the inhibition percentage of different concentrations of licorice isoflavan A and glycyrrhizidine on the acute itching induced by SLIGRL, and the control chart of the percentage of acute itching of SLIGRL in TRPV3 KO mice relative to the wild type; 100 and 300μM licorice isoflavan and licorice cidine effect of the experimental group and the corresponding control group mice within 30min of the ratio of the total number of scratches, and the ratio of TRPV3 KO mice and wild mice within 30min of the total number of scratches, The above data of each group are expressed as mean ± SEM, n = 5-8.

Figure 8 is a schematic diagram of the operation flow for inducing AD model mice with calcipotriol and treating them with TRPV3 antagonists. The dotted arrows above the time line indicate the days when calcipotriol was used to model both ears of mice, and the black arrows below The number of days when 1‰ of licorice isoflavane A or licoricedine was applied to both ears of the mice was marked, and on the 0th, 4th, and 7th days, the video was recorded before modeling and administration;

Figure 9 is the chronic itching response of calcipotriol-induced AD model mice at different test time points; wherein:

A, C, E: The broken lines of counting the number of scratching times of the mice in the experimental group and the control model group on the 0th, 4th, and 7th day, respectively, with 1‰ licorice isoflavan A and licoricedin Figure; ANOVA with repeated measures, *P<0.05;

B, D, F: the histograms of the total number of scratches within 60 minutes of the mice in the experimental group superimposed with 1‰ licorice isoflavane A and licoricedin on the 0th, 4th, and 7th day and the model group as the control; Unpaired Student's t-test compared to control group, *P<0.05, N.S.: P>0.05. The above data of each group are expressed as mean ± SEM, n = 7-8;

Figure 10 is a histogram of the average thickness of the mouse ears of the experimental group superimposed with 1‰ Glycyrrhizin A and Glycyrrhizidine on the 0th, 4th, and 7th days and the model group without drug treatment; using unpaired Student's t test Compared with the control group, **P<0.01, ***P<0.001. The above data of each group are expressed as mean ± SEM, n = 7-8;

Figure 11 shows the observation results of HEK293T cells after different treatments under a fluorescence microscope; each row corresponds to the conditions marked on the left, under white light (bright field), green fluorescence (GFP) and red fluorescence (PI staining) channels Images taken from a typical field of view. After transient transfection of the TRPV3 G573S mutant plasmid, HEK293T cells showed GFP green fluorescence and a strong red fluorescence signal (PI staining), and the number of cells with normal morphology decreased; The fluorescent signal is stronger, but the red fluorescent signal becomes very weak; while adding different concentrations (10 and 20 μM) of Glycyrrhizin A and Glycyrrhizidine, the red fluorescent signal is weaker, and the scale bar is 100 μm;

Figure 12 shows HEK293T cells transiently transfected with TRPV3 G573S mutation channel under the same conditions, and tested the addition of different concentrations (10 and 20 μM) of Glycyrrhizin A, Glycyrrhizidine and Ruthen Red, and without adding any antagonists. The chemiluminescence intensity obtained from the cell viability; compared with the control group without any antagonist by unpaired Student's t test, **P<0.01, ***P<0.001. The above data of each group are expressed as mean ± SEM, n = 5.

Detailed ways

The present invention will be further described below in conjunction with specific embodiments, and the advantages and characteristics of the present invention will become clearer along with the description. However, these embodiments are only exemplary and do not constitute any limitation to the scope of the present invention. Those skilled in the art should understand that the details and forms of the technical solutions of the present invention can be modified or replaced without departing from the spirit and scope of the present invention, but these modifications and replacements all fall within the protection scope of the present invention.

The beneficial effects of the medicament of the present invention will be further described below through test examples, and these test examples include pharmacodynamic tests of the medicament of the present invention.

The licorice isoflavane A of the present invention is a white powder produced by Yunnan Xili Company, and its purity is 97% after being measured by the area normalization method of two detectors of high performance liquid chromatography, an ultraviolet detector and an evaporative light scattering detector. Batch number: BBP03331;

Glycyrrhiza Xidine: white powder, produced by Yunnan Xili Company, determined by the area normalization method of two detectors of high performance liquid chromatography, ultraviolet detector and evaporative light scattering detector, and its purity is 98%. Batch number: BBP03353.

The preparation method of the licorice isoflavane molecules described in the present invention is not particularly limited, and those skilled in the art can prepare the licorice isoflavane molecules described in the present invention according to the common knowledge in the field, or obtain this product from the market. Invention of the described licorice isoflavane molecules (licorice isoflavane A and licorice cidine).

Test Example 1 Effect of Licorice Isoflavanes on TRPV3

The effect of compounds on overexpressed human TRPV3 was evaluated by cell patch clamp method.

The HEK293T cell line was purchased from the ATCC Biological Resource Center in the United States, inoculated in complete DMEM medium after recovery, and cultured in a 37°C constant temperature incubator with saturated humidity and 5% CO ₂ .

Before transfection, seed HEK293T cells into 6-well plates, and when the cell density reaches 50-70%, use Lipofectamine 3000 transfection reagent to transfect the eukaryotic expression plasmid pCMV6-AC-GFP-TRPV3 containing human TRPV3 cDNA (U.S. Origene Company, product number RG211184).

The electrophysiological detection method adopts the whole cell or membrane inside-out (inside-out) recording mode, uses the HEKAEPC10 amplifier and PatchMaster recording software, and uses the P-97 microelectrode pulling instrument to pull an electrode with an impedance of 3-5MΩ. Currents were recorded with square wave voltages of ±80 mV (cell clamping potential 0 mV).

Take HEK293T cells transfected with TRPV3 plasmid for 16-24 hours, put them in a specially prepared bath solution (130mM NaCl, 3mM HEPES, 0.3mM EGTA, pH=7.4), the selection state is good, the surface is clean and has strong green fluorescence Signal cells, and then slowly move the electrode added with the inner solution (its components are the same as the bath solution) to slightly touch the surface of the cell membrane, and apply a slight negative pressure until the membrane is ruptured after forming a high-resistance seal, forming a whole-cell recording mode. Or lift the electrode up directly after forming a high-resistance seal, so that the membrane at the tip of the electrode is torn off, forming a membrane with the inside facing outside.

1) In the whole-cell recording mode, use the RSC-200 rapid exchange drug delivery system for gravity perfusion, first administer 100 μM 2-APB to activate the TRPV3 channel, and then superimpose different concentrations (300 μM, 100 μM, 30 μM) of licorice isoflavane A; or With different concentrations (100 μM, 30 μM, 10 μM) of Glycyrrhizidine, the amplitude of the current can be observed to decrease significantly. Among them, with the increase of the superimposed concentration of Glycyrrhizin isoflavane A, the magnitude of the current decrease continues to expand until it almost completely disappears (as shown in Figure 1A); the test results for Glycyrrhizidine are also similar, that is, in the whole cell mode , superposition of Glycyrrhizidine can reduce the current induced by 2-APB, and the magnitude of the current reduction is dose-dependent (as shown in Figure 2A).

By measuring the corresponding antagonistic abilities of Glycyrrhizae isoflavan A and Glycyrrhizidine on TRPV3 current at multiple concentrations, and using the S-type dose-effect curve to fit, it can be obtained that the IC ₅₀ value of Glycyrrhizae Isoflavan A is 20.87 μM (as shown in Figure 1B), the IC ₅₀ value of Glycyrrhizidine was 15.24 μM (as shown in Figure 2B).

2) In the membrane-inside-out recording mode, that is, only the current of the channel on a single membrane sheet is recorded, it is also observed that Glycyrrhizin isoflavane A and Glycyrrhizidine can dose-dependently reduce the current induced by 2-APB (such as Figure 3), indicating that their ability to inhibit 2-APB current does not depend on the assistance of intracellular molecules, but should act directly on the TRPV3 channel itself.

Test Example 2 Effect of Licorice Isoflavanes on TRPV1 or TRPA1

The effect of compounds on overexpressed human TRPV1 and TRPA1 was evaluated by cell patch clamp method.

The HEK293T cell line was purchased from the ATCC Biological Resource Center in the United States, inoculated in complete DMEM medium after recovery, and cultured in a 37°C constant temperature incubator with saturated humidity and 5% CO ₂ .

Before transfection, seed HEK293T cells into 6-well plates, and when the cell density reaches 50-70%, use Lipofectamine 3000 transfection reagent to transfect the eukaryotic expression plasmid pCMV6-AC-GFP-TRPV1 containing human TRPV1 cDNA (U.S. Origene Company, Cat. No. RG217653) or transfect the eukaryotic expression plasmid pCMV6-AC-GFP-TRPA1 containing human TRPA1 cDNA (U.S. Origene Company, Cat. No. RG219290).

The electrophysiological detection method adopts the whole cell or membrane inside-out recording mode, uses the HEKAEPC10 amplifier and PatchMaster recording software, uses the P-97 microelectrode puller to pull an electrode with an impedance of 3-5MΩ, and uses ±80mV square wave Voltage recording current (cell clamping potential 0 mV).

Take HEK293T cells transfected with TRPV1 or TRPA1 plasmid for 16-24 hours, put them in a specially prepared bath solution (130mMNaCl, 3mM HEPES, 0.3mM EGTA, pH=7.4), the selection state is good, the surface is clean and there is a strong green color Cells with fluorescent signals, then slowly move the electrode added with the inner solution (its components are the same as the bath solution) to slightly touch the surface of the cell membrane, and apply a slight negative pressure until a high-resistance seal is formed and the membrane is ruptured to form a whole-cell recording mode .

Using electrophysiological detection methods, by recording the effect of 100 μM licorice isoflavan A on the current induced by TRPV1 agonist capsaicin or TRPA1 agonist AITC in HEK293T cells overexpressing TRPV1 or TRPA1 in vitro, the results showed that licorice isoflavan A has TRPV1 had a weak blocking effect (blocking rate 31.9% ± 6.6%, Figure 4A), while almost no antagonism was observed for TRPA1 (3.3% ± 15.5%, Figure 4C), which were significantly lower than its antagonistic The effect of TRPV3 (88.0%±7.5%, FIG. 4E ), these results suggest that Glycyrrhizin isoflavan A has better action specificity on TRPV3 channel. The effect of 100 μM Glycyrrhizidine on TRPV1 and TRPA1 currents was also detected by using a similar electrophysiological test method, and the blocking degree of Glycyrrhizidine to TRPV1 and TRPA1 (TRPV1: 52.2% ± 10.1%, Fig. 4B; TRPA1: 8.4% ± 10.8%, Fig. 4D) is also significantly lower than its antagonism to TRPV3 (93.3% ± 5.8%, Fig. 4F), indicating that Glycyrrhizidine also has a certain specificity of action on TRPV3 channels.

For animal models of pruritus, the standard acute pruritus model uses intradermal injection of pruritus on the back of the mouse to produce pruritus, and the degree of pruritus is characterized by counting the number of scratches within a certain period of time after injection. Raising the hind foot to scratch the injection site until the hind foot landed again or putting it into the mouth for licking was counted as a scratching behavior.

Common pruritic agents include histamine, chloroquine, serotonin, compound 48/80, and peptide SLIGRL. As a newly published study shows, for the acute itching induced by intradermal injection of the PAR2 activator SLIGRL, the scratching response of mice knocked out of TRPV3 is significantly reduced. Therefore, the SLIGRL acute itch model was selected as a paradigm to evaluate the effect of TRPV3 antagonist molecules on suppressing acute itch under physiological conditions.

Test Example 3 Effect of Glycyrrhizae Isoflavan A on Acute Itching Model in Mice

24 of the wild-type C57BL/6J mice purchased at the age of 8-10 weeks (body weight 20-25g, half male and half female) (Speyford (Beijing) Experimental Animal Technology Co., Ltd.) were randomly divided into 3 groups, namely Divided into licorice isoflavan A high-dose group (300 μM), medium-dose group (100 μM), and low-dose group (30 μM), with 8 mice in each group, and the hair on the neck and back of the mice was removed at least 2-3 days in advance;

On the day of the formal experiment, put the mice into observation boxes (15cm×10cm×10cm) for about 5 minutes of adaptive activities; then pre-inject 50 μl of licorice with different concentrations (30 μM, 100 μM, 300 μM) into the skin behind the right ear. After 30 minutes, continue to inject 50 μl of normal saline containing 50 μg SLIGRL and superimposed corresponding concentration of licorice isoflavane A (that is, the physiological saline contains both SLIGRL and licorice isoflavane A, and licorice The concentration of isoflavane A corresponds to the concentration of the pre-injection respectively, that is, SLIGRL+licorice isoflavane A) to induce acute itching; immediately put the injected mice into the observation box, keep quiet conditions, and record with the camera for 30 minutes And after reviewing the video, the scratching behavior was counted in a period of 5 minutes.

The next day, pre-inject 50 μl of normal saline containing 0.1% DMSO (solvent) into the skin behind the left ear as a control group (no administration), and continue to inject 50 μl of saline containing 50 μg SLIGRL (also containing 0.1% DMSO) at the same position 30 minutes later. Physiological saline to induce acute itching; immediately put the injected mice into the observation box, keep a quiet condition, record with a camera for 30 minutes and review the video afterwards, and count the scratching behavior in a period of 5 minutes.

The results showed that under the superimposed use of 30, 100 or 300 μM licorice isoflavan A, the total number of scratches within 30 minutes after the injection of SLIGRL in mice was compared with that of the control group without administration (that is, only SLIGRL was injected on the left ear side, That is, the SLIGRL+DMSO group) decreased to varying degrees (Fig. 5D).

However, the difference between the 30 μM experimental group and the control group did not reach a significant level (P=0.1047), and as the effect concentration increased to 100 and 300 μM, the level of reduction in the total scratching times of mice in the experimental group within 30 minutes became more and more significant. This suggested that the ability of licorice isoflavan A to inhibit acute pruritus induced by SLIGRL was enhanced with the increase of concentration. It is worth mentioning that the broken line chart of counting the number of scratches in different periods (as shown in Figure 5A-C): Different concentrations of Glycyrrhizae isoflavane A made the number of scratches of the mice in the experimental group decay faster over time, while when When the concentration of superimposed licorice isoflavan A is high enough (300 μM), the number of scratches of mice in the experimental group is much lower than that of the control group without administration from the beginning, and the P value calculated by repeated measurement analysis of variance is also It is less than 0.01, which shows from another aspect that licorice isoflavan A does have an inhibitory effect on acute pruritus of SLIGRL.

The effect of test example 4 Glycyrrhizidine on mouse acute itching model

24 of the wild-type C57BL/6J mice purchased at the age of 8-10 weeks (body weight 20-25g, half male and half female) (Speyford (Beijing) Experimental Animal Technology Co., Ltd.) were randomly divided into 3 groups, namely Divided into Glycyrrhizidine high-dose group (300 μM), medium-dose group (100 μM), and low-dose group (30 μM), with 8 mice in each group, and the hair on the back of the neck of the mice was removed at least 2-3 days in advance;

In addition to pre-injecting 50 μl of physiological saline containing different concentrations (30 μM, 100 μM, 300 μM) of glycyrrhizidine intradermally behind the right ear on the day of the formal experiment, 30 minutes later continued to inject 50 μl of saline containing 50 μg SLIGRL and superimposed the corresponding concentration of glycyrrhizidine at the same position. Normal saline (i.e. normal saline includes both SLIGRL and Glycyrrhizidine, wherein the concentration of Glycyrrhizidine corresponds to the concentration of the pre-injection respectively, i.e. SLIGRL+Glycyrrhizidine) to induce acute pruritus, the rest are the same as those in the test Example 3 is the same.

As a result, it was found that for the experimental group mice superimposed with 30, 100 or 300 μM Glycyrrhizidine, the total number of scratches within 30 min was also observed compared with the control group without administration (that is, only SLIGRL was injected on the left ear side, that is, SLIGRL+DMSO group) decreased in different degrees (Fig. 6D). Among them, the difference between the 30 μM experimental group and the control group did not reach a significant level (P=0.0523), but when the concentration increased to 100 and 300 μM, the total number of scratches in the experimental group within 30 minutes was significantly lower than that of the control group. This means that Glycyrrhizidine has the same effect as Glycyrrhizin isoflavane A, and can also effectively inhibit the acute itching response induced by SLIGRL. The number of scratches counted by time intervals also obtained similar results to that of Glycyrrhiza isoflavan A (as shown in Figure 6A-C): the number of scratches of mice in each experimental group tended to decay faster with time, and when superimposed licorice After the concentration of cidine increased to 300μM, the difference between the two groups reached a significant level (P<0.05). The above results all prove that Glycyrrhizidine can also effectively inhibit acute pruritus induced by SLIGRL.

By calculating the ratio of the total number of scratches of mice in different experimental groups and their corresponding control mice within 30 minutes after injection of SLIGRL, it can be observed that with the increase of the concentration of licorice isoflavan A and glycyrrhizin The inhibitory effects were all stronger and stronger, showing a similar dose-dependence (Figure 7).

Considering that it has been reported that the acute pruritus induced by SLIGRL in TRPV3 KO mice (TRPV3 knockout mice) was significantly lower than that of the wild type, as a control on the other hand, it was also tried in TRPV3 KO mice (purchased from Jackson Laboratory ( Jackson Lab), the United States) injected the same dose of SLIGRL to induce acute itching, and as a result, the reduction in the scratching response of wild-type mice was equivalent to the reduction in high concentrations of licorice isoflavane A and licoricedin. Therefore: Glycyrrhizin isoflavan A and Glycyrrhizidine have completely antagonized TRPV3 at high concentrations, thereby achieving almost the same effect as directly knocking out TRPV3 in inhibiting SLIGRL acute pruritus.

Chronic pruritus is an important complication of many skin and systemic diseases, and there is a lack of clinically available treatment methods. Even scratching tends to aggravate skin damage and inflammation, leading to more itching, more scratching, and more scratching. The vicious cycle of itching seriously affects the patient's normal life and disease recovery process. Common skin diseases accompanied by chronic pruritus include: chronic eczema, atopic dermatitis (AD), psoriasis, xeroderma, and senile pruritus. Previous studies have shown that TRPV3 function-enhancing mutations can cause dermatitis symptoms in mice that are very similar to AD; and the latest published research proves that TRPV3 regulates the secretion of inflammatory factors such as TSLP in keratinocytes by interacting with PAR2 receptor protein , and then participate in the mediation of calcipotriol-induced symptoms such as chronic pruritus in AD model mice. Therefore, the calcipotriol-induced atopic dermatitis model was chosen as a paradigm for evaluating the ability of TRPV3 antagonist molecules to suppress chronic itch under physiological conditions.

Experimental Example 5 Effect of Licorice Isoflavan Compounds on Mouse Chronic Itching Model

22 wild-type C57BL/6J mice purchased at the age of 8-10 weeks (body weight 20-25g, half male and half female) (Speyford (Beijing) Experimental Animal Technology Co., Ltd.) were randomly divided into 3 groups, namely Divided into licorice isoflavan A group, licorice cidine group and model group, in which licorice isoflavan A group, model control group each had 7 rats, and licorice cidine group had 8 rats;

Dissolve calcipotriol in absolute ethanol, use 100 μM as the working concentration, use a 20 μl pipette gun to draw the calcipotriol solution and drop it on the auricles on both sides of the mouse, 20 μl for each ear, once a day, continuously induce 7 Day for atopic dermatitis (AD) modeling.

In order to test the effect of the compound on inhibiting chronic pruritus by antagonizing TRPV3, while using calcipotriol to induce the AD model, it was superimposed (with vaseline as a base) with a mass fraction of 1‰ of licorice isoflavan A or licoricedin plaster for treatment (i.e. Glycyrrhiza Isoflavan A group, Glycyrrhiza Citidin group), and the specific operation schedule of modeling and drug administration is shown in Figure 8: starting 5 days before the official modeling, the Glycyrrhiza Isoflavan A group, Glycyrrhiza Citidin group In the given group, 1‰ of licorice isoflavane or licoricedin plaster was applied to the auricles of both ears of the mice (about 15mg per ear) once a day; in the model group, 1‰ of DMSO plaster was applied to both ears of the mice every day Auricle (about 15mg per ear) once; during AD modeling, the licorice isoflavane A group and the licoricedine group continued to apply the medicine half an hour after the administration of calcipotriol every day, and the model group continued to apply the calcipotriol every day. Half an hour after administration, Vaseline matrix was applied until the end of modeling 7 days; on the 0th, 4th, and 7th days, video recordings were performed and the auricle thickness of both ears was tested.

During video recording, mice were placed in observation boxes (15cm×10cm×10cm), and after 5 minutes of adaptive activities, they were recorded with a camera for 60 minutes, and the surrounding environment was kept quiet. Afterwards, they reviewed the video and scratched each time period of 5 minutes. Counting of scratching behaviors.

Results Before formal AD modeling with calcipotriol (Day 0), the total number of scratches within 60 minutes of the experimental group (Glycyrrhizae isoflavan A group, Glycyrrhizidine group) and the model group were both very low, and there was no Significant difference (Fig. 9A-B); 4 days after modeling, the mice showed obvious symptoms of chronic itching. half of the model group, and the differences were significant (Figure 9C-D); and after 7 days of modeling, the symptoms of chronic itching in the mice were already very serious, and the total number of scratches in each experimental group within 60 minutes was still low at this time Compared with the model group, only the experimental group treated with 1‰ Glycyrrhizidine had a significant reduction (Fig. 9E-F). In addition, with the emergence of AD modeling process, in addition to chronic itching, there is also the continuous aggravation of mouse ear inflammation, which is reflected in the continuous increase of auricle thickness. As shown in Figure 10, on the 4th and 7th days, 1‰ Compared with the model group, the average ear thickness of mice in the licorice isoflavan A and licorice cidine experimental groups decreased significantly, suggesting that licorice isoflavan A and licorice cidine also have certain anti-inflammatory effects. It is worth noting that the average ear thickness of mice in the licorice isoflavan A experimental group has a tendency to decrease over time compared with the model group, suggesting that its anti-inflammatory effect may be limited and cannot always ensure good results , in comparison, the anti-inflammatory effect of licorice cidine is stronger.

Test Example 6 Effects of Glycyrrhizae Isoflavanes on TRPV3 Functional Enhancement Mutants

The licorice isoflavane compound of the present invention not only has an effect on the TRPV3 wild type, but also has a blocking effect on the TRPV3 function enhancing mutant, thereby expanding its range of action.

1. Qualitative test

It has been reported in previous studies that enhanced-function mutants of TRPV3 such as G573S can induce cell death. To this end, use the Novizym Mut Express II Fast Mutagenesis Kit to design the required site-directed mutagenesis primers according to the primer design principles as follows:

Upstream sequence: 5'-gtccatgagcatgtacagcgtcatgatccagaagg-3'

Downstream sequence: 5'-ctgtacatgctcatggactggaaaccccgcgta-3'

Point mutations were performed on the plasmid pCMV6-AC-GFP-TRPV3 according to the recommended operation steps of the point mutation kit to obtain a plasmid with a G573S mutation.

Use Thermo Fisher Lipofectamine 3000 Transfection Kit, and perform transfection according to the operation method recommended by the kit. The TRPV3 G573S mutation channel plasmid with GFP tag was transiently transfected in HEK293T cell line. During the transfection process, when the transfection solution was added, the antagonists ruthenium red (20 μM) and licorice isoflavan A (10 μM or 20 μM), Glycyrrhizidine (10 μM or 20 μM); after 12 hours of transfection, the cell culture medium was discarded, washed once with PBS, and then stained with Hoechst33342/PI cell apoptosis staining kit, after staining, it could be observed under the fluorescence microscope Many cells stained positive for PI ( FIG. 11 ) indicated that a large number of cells died.

It can be seen from Figure 11 that after the antagonist ruthenium red (20 μM) was added while transfecting the G573S mutant plasmid, the number of cells positive for PI staining became very small (as shown in Figure 11), indicating that the overexpressed TRPV3 G573S mutation Once the channel is blocked by the antagonist, it is difficult to induce cell death;

PI staining was performed on HEK293T cells overexpressing the G573S mutation channel that were added with 10 or 20 μM Glycyrrhizin A and Glycyrrhizidine at the time of transfection, and only a few cells with red fluorescence could be observed under a fluorescent microscope signal (as shown in Figure 11), indicating that the overexpressed TRPV3 G573S mutation channel is blocked by Glycyrrhizae isoflavane A and Glycyrrhizidine.

2. Quantitative test

In order to quantitatively test the effects of Glycyrrhizae isoflavane A and Glycyrrhizidine on the TRPV3 G573S mutant channel, the TRPV3 G573S mutant channel plasmid with GFP tag was transiently transfected in the HEK293T cell line, and the antagonist ruthenium was added during the transfection process. Red (10 μM or 20 μM), licorice isoflavane A (10 μM or 20 μM), glycyrrhizidine (10 μM or 20 μM), and then the cell suspension mixed with the above compounds and transfection solution was seeded into a 96-well plate for culture. After culturing for at least 12 hours, use the CellTiter-Glo Luminescent Cell Viability Assay kit to test the cell viability by detecting ATP with luciferase.

When testing, add 100 μl of the mixed CellTiter-Glo test solution to each well, and mix on a shaker for 2 minutes to lyse the cells; continue to stand at room temperature for at least 10 minutes to ensure that the reaction is complete; then use a multifunctional microplate reader to read in chemiluminescence mode .

It was found that, similar to the effect of ruthenium red, adding 10 or 20 μM of these two molecules while transfecting the G573S mutant plasmid could significantly increase the chemiluminescent intensity indicative of cell viability (as shown in Figure 12). Together with the above qualitative and quantitative detection results, it is shown that Glycyrrhizae isoflavane A and Glycyrrhizidine also have a strong blocking effect on TRPV3 function-enhancing mutants.

Embodiment 1 Licorice isoflavane A ointment

First melt 240g of white petrolatum, 160g of stearyl alcohol, 40g of glyceryl monostearate, and heat and melt in a water bath at 80°C to form an oil phase;

20g of sodium lauryl sulfate, 140g of glycerin, 4g of 4% ethylparaben, heated at 80°C and dissolved in 500ml of distilled water to prepare the water phase;

Slowly add the water phase to the oil phase, stir while adding, and condense into a milky base, add 100g of licorice isoflavane A, fill, sterilize, and repackage to make 1000 ointments, each containing licorice isoflavan Alkane 100 mg.

The amount of the added licorice isoflavane can be 1-200g, and each prepared ointment contains 1-200 mg of licorice isoflavane.

Glycyrrhizidine can also be used to replace licorice isoflavane to make licorice cidine ointment.

Embodiment 2 Licorice isoflavane A ointment

First white petrolatum 300g (usually 10-500g), paraffin 500g (usually 50-1000g), liquid paraffin 2000g (usually 500-8000g), glyceryl monostearate 1000g (usually 100-2000g), Span 80 50g (usually 5-100g), heat and melt in a water bath at 80°C to make an oil phase;

50g (usually 5-100g) of emulsifier OP, 10g (usually 1-20g) of 4% ethylparaben, heated and dissolved at 80°C in 1000ml (usually 100-2000ml) of distilled water to make an aqueous phase;

Slowly add the oil phase to the water phase, stir while adding, condense into a milky base, add 100g of licorice isoflavane A, fill, sterilize, package, and make 1000 ointments, each containing the compound licorice isoflavane Alkane 1-200mg.

The amount of the added licorice isoflavane can be 1-200g, and each prepared ointment contains 1-200 mg of licorice isoflavane.

Glycyrrhizidine can also be used to replace licorice isoflavane to make licorice cidine ointment.

Embodiment 3 Licorice isoflavane A ointment (preparation of oily matrix ointment)

Add 100g of licorice isoflavane A to 3000g (usually 50-5000g) of liquid paraffin and stir into a paste, add 5000g of Vaseline (usually 100-8000g), and grind until uniform. Fill and sterilize, and make 1000 ointments, each containing 100 mg of the compound licorice isoflavane A.

The amount of the added licorice isoflavane can be 1-200g, and each prepared ointment contains 1-200 mg of licorice isoflavane.

Glycyrrhizidine can also be used to replace licorice isoflavane to make licorice cidine ointment.

Embodiment 4 Licorice isoflavan A ointment (preparation of aqueous base ointment)

Add 5000ml of ethanol (usually 100-8000ml) to CMC-Na 200g (usually 30-300g), grind to make it wet, then add 5000ml of glycerin (usually 500-8000ml), continue to grind until uniform (no lumps), continue to add Add 200ml (usually 50-300ml) of sodium benzoate aqueous solution (containing 20g (usually 5-30g) sodium benzoate) while grinding, mixing, and swelling to obtain a water-soluble matrix; add licorice isoflavane A 100g, mix well, pour Pack and sterilize. Make 1000 ointments, each containing 100 mg of compound licorice isoflavane A.

The amount of the added licorice isoflavane can be 1-200g, and each prepared ointment contains 1-200 mg of licorice isoflavane.

Glycyrrhizidine can also be used to replace licorice isoflavane to make licorice cidine ointment.

The above-mentioned embodiments of the present invention are merely exemplary, and do not constitute any limitation to the scope of the present invention. Those skilled in the art should understand that the details and forms of the technical solutions of the present invention can be modified or replaced without departing from the spirit and scope of the present invention, but these modifications and replacements all fall within the protection scope of the present invention.

Claims (4)

1. The application of the liquorice isoflavone derivative as the only active effective component in preparing the medicine for preventing, relieving or/and treating the acute pruritus or the chronic pruritus which are not dependent on histamine is provided, wherein the liquorice isoflavone derivative is liquorice iso Huang Wanjia and liquorice cetirizine.

2. The use according to claim 1, wherein the medicament consists of a licorice isoflavan derivative and a pharmaceutically acceptable carrier.

3. The use according to claim 1 or 2, wherein the medicament is in the form of a tablet, capsule, pill, powder, granule, syrup, emulsion, injection, spray, gel, cream, cataplasma, or rubber patch.

4. The use according to claim 1 or 2, wherein the purity of the licorice isoflavan derivative is not less than 1%.

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