Application of atalantia buxifolia alkali in preparation of medicine for inhibiting itching perception of human and / or animals

Abstract

The invention provides application of Atalafoline in preparation of a medicine for inhibiting itching perception of people and / or animals, and belongs to the technical field of biomedicine. The atalantia buxifolia alkali achieves the purpose of inhibiting pruritus induction by inhibiting activation of a human or animal TRPV3 ligand gated ion channel. The atalantia buxifolia alkali can enhance the resistance of human beings or animals to itching, and can be used as an active component to be developed into a medicine or a skin care product with an itching relieving effect so as to relieve the itching of the human beings or the animals. Meanwhile, the atalantia buxifolia alkali acts on a receptor TRPV3 or a functional acquired mutant thereof through targeting, and can be applied to research of related ion channel diseases and preparation of related therapeutic drugs.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the application of fentanyl in the preparation of drugs that inhibit the perception of itch in humans and / or animals. Background Technology

[0002] Pruritus is an extremely uncomfortable skin sensation that urgently requires scratching for relief. As a common dermatological condition, pruritus is the most frequent clinical manifestation of skin diseases such as atopic dermatitis (AD), urticaria, contact dermatitis, nodular prurigo, fungal infections, and psoriasis. It is also a common symptom of many systemic diseases, such as kidney disease, biliary tract disease, thyroid disease, malignant tumors, and AIDS. Currently, the neural pathways and molecular mechanisms related to pruritus are gradually being discovered, including histamine-dependent and histamine-independent pruritus pathways, and the receptors, neurotransmitters, and nerves involved in these pathways. Transforming receptor potential cation channel subfamily V member 3 protein (TRPV3) is a non-selective cation channel protein most abundantly expressed in keratinocytes of the skin. It participates in physiological processes such as keratinocyte proliferation, skin barrier formation, sensory transmission, and hair growth, playing a crucial role in maintaining skin homeostasis. TRPV3 is associated with the occurrence and development of pruritus. Recent studies have shown that functionally enhancing TRPV3 point mutations induce severe scratching behavior in mice, rats, and humans. Knockout TRPV3 mice show a significant reduction in scratching response. TRPV3 plays an important role in the occurrence and development of skin inflammation and pruritus, including histamine-dependent and histamine-independent pruritus pathways, and is closely related to inflammatory skin diseases such as atopic dermatitis, Olmsted syndrome, and psoriasis.

[0003] Atalafoline, molecular formula C 17 H 17 NO6, molecular weight 331.32. Atalantia buxifolia (Poir. Oliv.), a plant in the Rutaceae family, is an alkaloid derived from this plant. Experimental studies have shown that Atalantia buxifolia has good expectorant, antitussive, and antiasthmatic effects. However, there are currently no reports on Atalantia buxifolia or its active ingredient atalantia buxifolia participating in itch perception. Summary of the Invention

[0004] In view of this, the purpose of this invention is to provide a new use for euphorbia alkaloids, specifically the application of euphorbia alkaloids in the preparation of drugs that inhibit the perception of itch in humans and / or animals. Euphorbia alkaloids inhibit the 2-APB activation of TRPV3 ligand-gated ion channels in humans and / or animals, thereby inhibiting the perception of itch in humans and / or animals and improving the resistance of humans and / or animals to itch.

[0005] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0006] This invention provides the application of fenestrated alkaloids in the preparation of drugs that inhibit the perception of itch in humans and / or animals.

[0007] Preferably, the itch includes histamine-dependent itch and / or histamine-independent itch.

[0008] Preferably, the indications for inhibiting the perception of itch in humans and / or animals include at least one of the following: skin inflammation, acute pruritus, chronic pruritus, and neuropruritus.

[0009] This invention provides an antipruritic drug with fenestrated alkaloids as the active ingredient, and also includes pharmaceutically acceptable excipients.

[0010] Preferably, the concentration of the eurygnetine is 0.25 μM or higher.

[0011] This invention provides an antipruritic skin care product, which uses citrus alkaloids as the active ingredient and also includes skin care excipients.

[0012] Preferably, the concentration of the eurygnetine is 0.25 μM or higher.

[0013] This invention provides the application of fenestrated alkaloids in the preparation of reagents for in vitro inhibition of TRPV3 ion channels in cells.

[0014] This invention provides an inhibitor of the TRPV3 ion channel, the inhibitor comprising fenestrated alkaloids and cell buffer.

[0015] Preferably, the concentration of the inhibitor, fentanyl erythroside, is 0.25 μM or higher.

[0016] This invention provides the application of fentanyl in the preparation of drugs that inhibit itch perception in humans and animals. Fentanyl can significantly inhibit the activation of TRPV3 (Transient Receptor Potential Vanilloid 3) ion channels and their gain-of-function mutants, specifically 2-aminoethoxydiphenylborate (2-APB), in different species. Electrophysiological experiments show that fentanyl can act on TRPV3 receptors in other species, including humans. Inhibition of TRPV3, a receptor closely related to chronic pruritus on the animal skin surface, desensitizes itch perception for a certain period. Experiments in mouse models demonstrate that both injection and application of fentanyl can enhance mice's tolerance to histamine-induced acute pruritus (VEH), significantly reducing the number of scratching behaviors. Simultaneously, injection and application of fentanyl can dose-dependently inhibit spontaneous scratching behavior in a mouse pruritus model (AEW), significantly enhancing the resistance of mice to chronic pruritus. In summary, the aforementioned fenestrated alkaloid enhances the resistance to pruritus in species such as mice, and can be developed as a potential antipruritic agent or skin care product for use in humans and other animals to relieve pruritus. Secondly, fenestrated alkaloid targets the receptor TRPV3 and its gain-of-function mutants, and can be applied to the research of related ion channelopathies and as a potential therapeutic drug. Attached Figure Description

[0017] Figure 1 The results of the effect of eurygia on TRPV3 are shown in Figure A, where A represents the current result of mTRPV3 being inhibited by 50 μM eurygia; B represents the dose-response curve of eurygia on mTRPV3 fitted by the Hill equation (n=6).

[0018] Figure 2 The diagrams show the 2-APB activation currents of citrus alkaloids inhibiting TRPV3 in different species, where A is the activation current of citrus alkaloids inhibiting hTRPV3 and B is the activation current of citrus alkaloids inhibiting ratTRPV3.

[0019] Figure 3 The graph shows the results of electrophysiological experiments to detect the activity of euphorbia benzoin against multiple TRPV3 gain-of-function mutants. A, B, and C are representative current graphs of 1 μM euphorbia benzoin acting on the TRPV3_M672I, TRPV3_W692C, and TRPV3_A628T channels, respectively. D is the inhibition rate of 1 μM euphorbia benzoin against TRPV3 gain-of-function mutants.

[0020] Figure 4The figures show the results of dongfengjie alkaloids in relieving pruritus in mice. Figures A and B show the statistical results of the number of scratches and the total movement distance of mice in chronic and acute pruritus models after administration, respectively. **p<0.01, ****p<0.0001; Figures C and D show the results of dongfengjie alkaloids in toxicity detection of HaCat cells and HEK293T cells, respectively. Detailed Implementation

[0021] This invention provides the application of euphorbia alkaloids in the preparation of drugs that inhibit the perception of itch in humans and / or animals, wherein the structural formula of euphorbia alkaloids is shown in Formula I:

[0022]

[0023] In this invention, the alkaloid is preferably an alkaloid derived from Atalantia buxifolia (Poir. Oliva), a plant belonging to the Rutaceae family. This invention does not impose any particular limitation on the extraction method of the alkaloid; any extraction method well-known in the art can be used. In one embodiment of the present invention, the extraction is preferably carried out according to the method of Qin DK: the roots of Dongfeng orange are identified and collected from Hainan Province, my country. The Dongfeng orange roots are air-dried and pulverized into powder, and then extracted with ethanol to obtain an ethanol extract. The ethanol extract is dissolved in acid water and then extracted with chloroform to collect a chloroform extract. After being dissolved in benzene, it is subjected to acidic alumina column chromatography, and eluted with benzene and ethyl acetate (volume ratio of 9:1) to obtain brown columnar crystals. After repeated recrystallization with benzene and dilute ethanol, light yellow needle crystals are obtained to obtain Dongfeng orange alkaloid (Qin DK (1986). Yaoxuexue bao=Actapharmaceutica Sinica,21(9),683-685.). In the present invention, the purity of the Dongfeng orange alkaloid is preferably ≥95%. The molecular formula of the Dongfeng orange alkaloid is C 17 H 17 NO6 has a molecular weight of 331.32.

[0024] In this invention, the itch includes histamine-dependent itch and / or histamine-independent itch.

[0025] In this invention, the indications for inhibiting itch perception in humans and / or animals preferably include at least one of the following: skin inflammation, acute pruritus, chronic pruritus, and neurogenic pruritus. The acute pruritus is preferably histamine-induced acute pruritus, and the chronic pruritus is preferably 2,4-dinitrofluorobenzene (DNFB)-induced atopic dermatitis-related chronic pruritus. The indications for inhibiting itch perception in humans and / or animals also preferably include at least one of the following: atopic dermatitis, Olmsted syndrome, psoriasis, eczema, and chronic urticaria, wherein Olmsted syndrome preferably includes the TRPV3_A628T mutant Olmsted syndrome.

[0026] In this invention, the fenestrated alkaloid preferably significantly inhibits the activation of TRPV3 ligand-gated ion channels of different species by specifically targeting the receptor TRPV3. TRPV3, as a receptor closely related to chronic pruritus on the skin surface of humans or animals, is inhibited, which will suppress the sensation of pruritus for a certain period of time, thereby reducing the perception of pruritus in humans and animals and improving the resistance of humans or animals to pruritus.

[0027] In one embodiment of the present invention, patch-clamp technique was used to demonstrate that eurypticine inhibits the activation of 2-aminoethoxydiphenylborate (2-APB) in the mTRPV3 channel, and the degree of inhibition is concentration-dependent. The dose-dependent inhibition data of eurypticine on the mTRPV3 channel were fitted using the Hill function equation to obtain the IC50 of eurypticine inhibiting mTRPV3. 50 The concentration is 0.25 μM. Furthermore, in another embodiment of the present invention, the toxicity of euphorbia pekinensis to HaCat cells expressing endogenous TRPV3 channels and HEK293T cells without TRPV3 channel expression was detected by CCK8 assay. The CCK8 results showed that as the concentration of euphorbia pekinensis increased to 200 μM, it had no significant effect on the death or number of HaCat and HEK293T cells. Therefore, the effective concentration of euphorbia pekinensis for inhibiting itch perception in humans and animals is 0.25 μM or higher, more preferably 0.3–200 μM, further preferably 0.4–50 μM, even more preferably 0.45–20 μM, and most preferably 0.5 μM.

[0028] In one embodiment of the present invention, a mouse model experiment was conducted. The results showed that, regardless of whether it was acute or chronic pruritus, the resistance to pruritus could be improved after injection or application of the aforementioned euphorbia cinnamic acid. Furthermore, the present invention constructed recombinant TRPV3 plasmids for rats, mice, and humans, and used patch-clamp technology to verify that euphorbia cinnamic acid inhibits 2-APB activation of the TRPV3 channel in mammals, demonstrating that euphorbia cinnamic acid has good universality in different species.

[0029] Based on the fact that euphorbia alkaloid has the effect of inhibiting the perception of itch in humans and / or animals, the present invention provides an antipruritic drug, which uses euphorbia alkaloid as the active ingredient and also includes pharmaceutically acceptable excipients.

[0030] In this invention, the concentration of the eurygnetine is 0.25 μM or higher.

[0031] In this invention, the source and preparation method of the fenestrated alkaloid are preferably the same as described above, and will not be repeated here. The concentration of the fenestrated alkaloid in the antipruritic drug is preferably the same as described above, and the antipruritic drug preferably also includes excipients. The excipients preferably include buffers and / or chelating agents; more preferably, they include at least one of the following: sodium chloride, hydroxyethylpiperazine ethanesulfonic acid, and ethylenediaminetetraacetic acid.

[0032] Based on the fact that the aforementioned euphorbia alkaloid has the effect of inhibiting the perception of itch in humans and / or animals, the present invention provides an antipruritic skin care product, which uses euphorbia alkaloid as the active ingredient and also includes skin care excipients.

[0033] In this invention, the concentration of the eurygnetine is 0.25 μM or higher.

[0034] In this invention, the active ingredient of the skincare product includes the aforementioned antipruritic drug. This invention does not impose any particular limitation on the dosage form of the antipruritic skincare product; conventional dosage forms in the art can be used. The antipruritic skincare product of this invention is preferably applied to humans and animals via topical application or injection to relieve itching and to overcome acute and chronic itching in a short period of time.

[0035] Based on the fact that euphorbia benzoyl inhibits the activation of mTRPV3 channels, this invention provides an application of euphorbia benzoyl in the preparation of reagents for in vitro inhibition of intracellular TRPV3 ion channels.

[0036] In this invention, the fentanyl alkaloid specifically targets the receptor TRPV3 and its gain-of-function mutant (TRPV3_A628T), thereby inhibiting intracellular TRPV3 ion channels. Olmsted Syndrome (OS), a relatively rare human genetic disorder recently discovered clinically, is also closely related to gain-of-function mutations in TRPV3 channels. OS patients exhibit severe keratosis of the hands, feet, and perioral area, accompanied by baldness and severe itching. Multiple TRPV3 mutants exist in this patient population; several mutation sites have been identified, including G573A, G573S, G573C, Q580P, W692C, W692G, and L673F. The mutated TRPV3 channels remain open, leading to intracellular Ca2+ ion channel depletion. 2+Increased concentrations induce apoptosis, thereby promoting disease development. This invention overexpressed mouse TRPV3 receptor (mTRPV3) in the HEK293T cell line and, using patch-clamp technique, discovered that the target of the fenestrated alkaloid is the TRPV3 receptor, inhibiting its 2-APB activation. Furthermore, by constructing 15 TRPV3 gain-of-function mutants associated with OS, including W521S, G568C, and G573S, this invention found that fenestrated alkaloid had no activity against the TRPV3_G573S, TRPV3_M672I, and TRPV3_W692C mutants, but exhibited inhibitory activity only against the TRPV3_A628T mutant. Therefore, the fenestrated alkaloid inhibits TRPV3 channel activation by acting on the TRPV3 receptor, and specifically inhibits the activity of the TRPV3_A628T mutant.

[0037] In this invention, the source and preparation method of the fenestrated alkaloid are preferably the same as described above, and will not be repeated here. The concentration of the fenestrated alkaloid in the inhibitor is preferably the same as described above, and the inhibitor preferably further includes a buffer and / or a chelating agent; more preferably, it includes at least one of the following: sodium chloride, hydroxyethylpiperazine ethanesulfonic acid, and ethylenediaminetetraacetic acid. This invention does not specifically limit the dosage form of the inhibitor.

[0038] This invention provides an inhibitor of the TRPV3 ion channel, the inhibitor comprising fenestrated alkaloids and cell buffer.

[0039] In this invention, the concentration of the inhibitor, fentanyl erythrone, is above 0.25 μM.

[0040] In this invention, the source and preparation method of the fenestrated alkaloid are preferably the same as described above, and will not be repeated here. The concentration of the fenestrated alkaloid in the inhibitor is preferably the same as described above, and the inhibitor preferably further includes a buffer and / or a chelating agent; more preferably, it includes at least one of the following: sodium chloride, hydroxyethylpiperazine ethanesulfonic acid, and ethylenediaminetetraacetic acid. This invention does not specifically limit the dosage form of the inhibitor.

[0041] The following examples illustrate the application of the euphorbia alkaloid provided by the present invention in the preparation of drugs that inhibit the perception of itch in humans and / or animals, but these examples should not be construed as limiting the scope of protection of the present invention.

[0042] 1. Sources of recombinant plasmids for human, rat, and mouse TRPV3 receptors:

[0043] Based on existing reference genomes on NCBI, TRPV3 receptor sequences from humans, rats, and mice were synthesized (see Table 1). The synthesized cDNAs were then cloned into the BamHI / EcoRI multiple cloning site of the pEGFPN1 plasmid vector to obtain recombinant plasmids containing the human hTRPV3 receptor gene, the mouse mTRPV3 receptor gene, and the rat rat TRPV3 receptor gene, respectively. All recombinant plasmids were synthesized by Shanghai Sangon Biotech Co., Ltd.

[0044] Table 1. TRPV3 receptor sequence information in humans, rats, and mice.

[0045] Species GeneID Gene number Human hTRPV3 receptor 79054 NP_076985.4 mouse mTRPV3 receptor 171382 NP_599013.1 rat rat TRPV3 receptor 171384 XP_017451750.1

[0046] Example 1

[0047] Electrophysiological experiments on the targeting effects of fentanyl and pruritus-related receptors such as mTRPV3.

[0048] Cell culture of HEK293T cells without TRPV3 channel expression: Observe cell morphology under an inverted microscope. When the cell density reaches 80%, passage can be performed. Aspirate the culture medium, add 1 mL of trypsin, digest for about 20 seconds, and then aspirate the trypsin. When the cells adhering to the bottom of the flask gradually detach, add cell culture medium (DMEM medium supplemented with 10% fetal bovine serum + 0.1% penicillin-streptomycin) to stop the digestion. Pipette the cells to completely detach them and suspend them evenly, taking care to avoid generating air bubbles and contamination. Aliquot the cell suspension into new T25 culture flasks according to experimental requirements, add 6 mL of culture medium, shake to mix, and then return to the incubator for further culture.

[0049] Recombinant plasmid transfection: HEK293T cells were transfected using the Lipofectae 2000 Transfection Kit (Invitrogen, USA) to transfect the hTRPV3 receptor recombinant plasmid, mTRPV3 receptor recombinant plasmid, and ratTRPV3 receptor recombinant plasmid, respectively. Specifically, HEK293T cells in logarithmic growth phase were selected and transferred to small dishes for culture one day in advance. After adhesion, transfection could be performed. Before transfection, the DMEM medium (Dulbecco's modified Eagle's medium) was replaced with serum-reduced medium (Opti-MEM medium). 6 μL of Lipo2000, 4 μg of recombinant plasmids carrying the target genes (mTRPV3, hTRPV3, ratTRPV3), and 1 μg of EGFP (pcDNA3.1 vector) plasmid were added to 200 μL of OptiMEM medium, mixed well, and incubated for 20 min. The mixture was then added to the small dishes containing the cells to be transfected. After 4 h, the Opti-MEM medium was replaced with DMEM medium. The DMEM medium was supplemented with 10% fetal bovine serum and 1% penicillin / streptomycin. After transfection, HEK293T cells expressing hTRPV3, mTRPV3, and ratTRPV3 receptors were obtained.

[0050] HEK293T cells expressing the mTRPV3 receptor were subjected to patch-clamp experiments at room temperature (20–25 °C).

[0051] WPI 0.86mm thin-walled borosilicate glass capillaries were selected as the glass electrode material. The glass electrodes were fabricated in five steps using a stretching apparatus (P-97, Shutter). After thermal polishing, the electrode tip diameter was 1.5–3.0 μm. Electrode fluid was then injected into the glass electrode after fabrication. The initial resistance of the glass electrode was 1.5–2.5 MΩ. After a high-impedance king ohm (GΩ) seal was formed between the electrode and the cell membrane, the fast capacitance of the electrode was compensated. Then, a short but strong negative voltage was applied to rapidly break the cell membrane clamped in the electrode, followed by compensation of the slow capacitance of the cell. After establishing whole-cell recording mode, the cells were clamped at 0 mV. After stabilizing the cells for 10 min, current recording was started using an appropriate pulse voltage (80 / -80 mV). Drug perfusion was performed using a Biolab RS200, with a drug switching rate of 50 ms. The series resistance (Rs) remained constant within the range of 5 to 8 MΩ throughout the experiment, and the system series resistance compensation was generally between 30% and 60%.

[0052] Electrophysiological recording: The software was set to Whole-cell mode to compensate for cell fast capacitance, series resistance, and leakage current. Cells were slowly lifted to the lower edge of the glass tube of the rapid drug delivery switching system (RSC-200) and rinsed with isotonic electrode fluid. During the experiment, different channels were opened to align the corresponding glass tubes with the cells, allowing for control of different drug delivery conditions (1 μM, 10 μM, 25 μM, 50 μM, 100 μM). The RSC-200's channel switching signal was connected to the amplifier's A / D channel, and all data were recorded through the amplifier in PatchMaster software. Throughout the cell clamping, drug delivery, and data recording process, the seal between the cells and the glass electrode needed to be kept stable.

[0053] Record the electrode solutions for the TRPV3 channel: 130 mM NaCl, 3 mM HEPES, 0.2 mM EDTA, adjusted to pH 7.2 with NaOH. Drugs used in the experiment must be prepared using the above-mentioned electrode solutions.

[0054] The results are as follows Figure 1 As shown, citronella tinctoria inhibits 2-APB activation of the mTRPV3 channel in a concentration-dependent manner. The dose-dependent inhibition data of citronella tinctoria on the mTRPV3 channel were obtained by fitting the Hill function equation, yielding the IC50 of citronella tinctoria for inhibiting mTRPV3. 50 The concentration was 0.25 μM. These patch-clamp electrophysiological results indicate that fenestrated alkaloids are an effective inhibitor of the TRPV3 channel.

[0055] Example 2

[0056] Universality of fentanyl in mammals

[0057] Electrophysiological experiments were performed on HEK293T cells expressing the hTRPV3 receptor and HEK293T cells expressing the ratTRPV3 receptor, respectively, using the same methods as in Example 1.

[0058] Whole-cell patch-clamp recordings showed that fenestrated alkaloids inhibited 2-APB activation of TRPV3 receptors in different species and exhibited inhibitory activity against TRPV3 channels in both humans and rats (see [link to study]). Figure 2 ). As an inhibitor of different species, fenestrated alkaloids can be used as a drug and antipruritic for potential TRPV3 channel diseases.

[0059] Example 3

[0060] Experiment on inhibition of mTRPV3 gain-of-function mutant by fenestrated pine

[0061] 1. Preparation of mTRPV3 gain-of-function mutants

[0062] The point mutation experiment was used to investigate whether fenestrated alkaloids inhibit mTRPV3 channel gain-of-function mutants.

[0063] All mutated mTRPV3 channels were constructed using homologous recombination and obtained using the Fasta rapid site-directed mutagenesis kit from Cyprom. Constructing a single-amino acid mutation requires introducing the mutant base into the homologous complementary region of the primer during DNA amplification.

[0064] The primer design principle is as follows: the 5' complementary region of the forward and reverse amplification primers contains a 21bp reverse complementary region, the non-complementary region of the primers is 18bp in length, and the required point mutation site is located in the homologous complementary region.

[0065] A single-base site-directed mutation was introduced into the mTRPV3 receptor recombinant plasmid. Primers were designed, and the mTRPV3 receptor recombinant plasmid was amplified by reverse PCR. The reaction was carried out using a centrifuge tube containing 1 μl template DNA sequence, 8.5 μl deionized water, 2 μl 2×Max buffer, 0.5 μl ldNTP mixture, 1 μl 5' PCR primer, 1 μl 3' PCR primer, and 0.5 μl DNA polymerase. Primers for the mTRPV3 gain-of-function mutant are shown in Table 1, where F represents the upstream primer and R represents the downstream primer.

[0066] Table 1 Primers for mTRPV3 gain-of-function mutants

[0067]

[0068]

[0069] Amplify in a PCR instrument according to the following program: 95℃ pre-denaturation for 30s; 95℃ denaturation for 15s, 60℃ annealing for 15s, 72℃ extension for 9min, 30 cycles; 72℃ extension for 5min.

[0070] Recombinant reaction: Take 2 μl of Dpnl digestion product, 2 μl of CEⅡ buffer, 1 μl of Exnase Ⅱ and 5 μl of deionized water, react at 37℃ for 30 min, and then cool in an ice water bath for 5 min. The ligation product was transformed into 100 μL LDH5α competent cells, plated and cultured overnight, then single colonies were picked, amplified by shaking culture, and sequenced for verification, resulting in 15 mTRPV3 gain-of-function mutants: TRPV3_W521S, TRPV3_G568C, TRPV3_G568V, TRPV3_G573S, TRPV3_G573V, TRPV3_G573A, TRPV3_G573C, TRPV3_Q580P, TRPV3_A628T, TRPV3_M672I, TRPV3_L673F, TRPV3_A675T, TRPV3_W692C, and TRPV3_W692F.

[0071] The inhibitory activity of fentanyl against 2-APB activation of 15 mTRPV3 gain-of-function mutant channels was detected using the method described in Example 1. The results showed that fentanyl had no activity against 14 mutants, including TRPV3_G573S, TRPV3_M672I, and TRPV3_W692C, and only exhibited inhibitory activity against the TRPV3_A628T mutant. Figure 3 This suggests that eurypticin may become a lead compound targeting specific types of OS diseases.

[0072] Example 4

[0073] Evaluation of the antipruritic activity and safety of fentanyl

[0074] I. The effect of euphorbialine on relieving chronic pruritus in mice

[0075] 2,4-Dinitrofluorobenzene (DNFB)-induced model of atopic dermatitis and chronic pruritus: Three days before the experiment, the hair on the right posterior neck of C57BL / 6 mice was removed with paraffin. The mice were placed in transparent observation cages and randomly divided into 4 groups. The model was established using 2,4-dinitrofluorobenzene reagent (DNFB). On day 4, 100 μl of 0.5% DNFB (dissolved in acetone / olive oil = 4:1, v / v) was applied topically to the hair-removed area on the right posterior neck of the mice to sensitize them. From day 8 onwards, the mice received topical application of 50 μl of 0.2% DNFB once daily for 5 consecutive days, resulting in the model group (DNFB). 100 μl of acetone / olive oil mixture served as the blank control group (VEH).

[0076] To explore the antipruritic activity of fenestrated baicalein, 50 μL LDMSO (0.05%), 50 μL fenestrated baicalein (0.2 mg / kg), and 50 μL fenestrated baicalein (0.5 mg / kg) were subcutaneously injected into the shaved area 0.5 h before DNFB sensitization, using physiological saline as the solvent. On day 12, the number of times mice in different treatment groups scratched within 1 hour was observed and counted. A scratching behavior was defined as a mouse lifting its forepaw or hind paw off the ground to scratch the treated area on the right side of its neck until the paw was placed back on the ground.

[0077] In the neck and back induced model, mice in the DNFB group exhibited intense scratching behavior, while the scratching behavior in the acetone / olive oil control group was not obvious. The number of itching occurrences within 1 hour in the DNFB model group (75.2, n=5) was significantly higher than that in the blank control group (VEH6, n=5). Treatment with 0.2 mg / kg and 0.5 mg / kg of fenestrated dermatitis significantly reduced the number of scratching behaviors in the DNFB model group to 49 (n=5) and 20.6 (n=5) times, respectively, indicating that fenestrated dermatitis can dose-dependently reduce spontaneous scratching behavior in mice. These animal experimental results indicate that fenestrated dermatitis alleviates chronic itching in mice with DNFB-induced atopic dermatitis by directly inhibiting the TRPV3 channel. Figure 4 (A)

[0078] II. Dongfeng Citrusine as an adjunct to relieve acute pruritus in mice

[0079] Histamine-induced acute pruritus model: Three days before the experiment, the hair on the right posterior neck of C57BL / 6 mice was removed with paraffin. One hour before the experiment, the mice were placed in transparent observation cages and randomly divided into four groups: two control groups and two experimental groups. The two control groups received a subcutaneous injection of 50 μL DMSO (0.05%) at the shaved area, while the two experimental groups received subcutaneous injections of 50 μL fenestrated alkaloid (0.2 mg / kg) and 50 μL fenestrated alkaloid (0.5 mg / kg), respectively, at the shaved area. Then, the two control groups received injections of saline and 50 μL histamine (100 μM), respectively, at the same location, while the two experimental groups received injections of 50 μL histamine (100 μM). The number of times the mice scratched within 30 minutes was observed and counted. A scratching behavior was defined as the mouse lifting its forepaw or hind paw off the ground to scratch the treated area on the right posterior neck until the paw was placed back on the ground.

[0080] Within 30 minutes after intradermal injection of histamine, the number of spontaneous scratching events increased to 68.2 (n=6), compared to 13.6 (n=6) in the control group. Conversely, different concentrations of fentanyl could dose-dependently inhibit histamine-induced pruritus. The scratching events in mice treated with 0.2 mg / kg and 0.5 mg / kg fentanyl decreased to 30.5 (n=6) and 11.2 (n=6), respectively. These results further confirm that fentanyl alleviates histamine-induced acute pruritus in mice by inhibiting the TRPV3 channel. Figure 4 (B)

[0081] III. Dongfeng guanine has no cytotoxicity.

[0082] The cytotoxicity of fentanyl to HaCat cells with endogenous TRPV3 channel expression and HEK293T cells without TRPV3 channel expression was detected using the CCK8 assay: HaCat cells and HEK293T cells were digested with trypsin, centrifuged, and resuspended in serum-containing medium. Cells were then counted using a hemocytometer to obtain a count of 5 × 10⁶ cells. 3 ~5×10 4 Single-cell suspensions of 100 μl / ml were seeded into 96-well plates, and the plates were pre-cultured in an incubator for approximately 24 hours (37°C, 5% CO2). Different concentrations of citric acid (1 μM, 10 μM, 50 μM, 100 μM, and 200 μM) were added to each well, with three replicates for each concentration group, and the plates were incubated for 24 hours. 10 μl of CCK-8 solution was added to each well. After adding the reagents, the plates were gently shaken to aid mixing, and the plates were incubated for 1 hour. The absorbance (OD value) at 450 nm was measured using a microplate reader.

[0083] The CCK8 experiment results showed that as the concentration of fentanyl increased, even reaching a maximum of 200 μM (200 times the effective concentration detected by electrophysiological assay), fentanyl did not cause death or a decrease in cell number in HaCat and HEK293T cells. Figure 4 (C and D) It can be seen that euphorbia alkaloids have no cytotoxicity.

[0084] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. Other embodiments can be obtained based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. Use of psilocybin in the preparation of a medicament for inhibiting the perception of itch in a human and / or an animal.

2. Use according to claim 1, characterized in that, The itch includes histamine-dependent itch and / or histamine-independent itch.

3. Use according to claim 1, characterized in that, The indication of inhibiting the perception of itch in a human and / or an animal includes at least one of the following: skin inflammation, acute itch, chronic itch, and neuropathic itch.

4. An antipruritic medicament, characterized in that, is a preparation for external use, which comprises psilocybin as an active ingredient, and a pharmaceutically acceptable adjuvant.

5. The antipruritic medicament of claim 4, wherein, The concentration of the psilocybin is 0.25 μM or more.

6. An anti-itch skin care product, characterized by, is a preparation for external use, which comprises psilocybin as an active ingredient, and a skin care adjuvant.

7. The antipruritic skin care product according to claim 6, characterized in that, The concentration of the psilocybin is 0.25 μM or more.

8. Use of psilocybin in the preparation of an agent for inhibiting the TRPV3 ion channel of a cell in vitro.

9. An inhibitor of a TRPV3 ion channel, characterized in that, The agent includes psilocybin and a cell buffer.

10. The inhibitor of claim 9, wherein The concentration of the psilocybin in the agent is 0.25 μM or more.

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