Use of fluropropione for the preparation of a medicament for the treatment of skin inflammation and for the inhibition of the TRPV3 channel

Abstract

The application belongs to the technical field of medicine application, and relates to application of fluparoxane in preparation of medicines for treating skin inflammation and inhibiting TRPV3 channels. On one hand, a new use of fluparoxane medicines is developed, and fluparoxane can be used for treating skin inflammation. Fluparoxane also has good TRPV3 inhibiting activity, and can be used for preparing medicines for treating diseases related to enhanced TRPV3 ion channel activity. On the other hand, a new way for treating skin inflammation is provided. Therefore, the new use of fluparoxane provided by the application has great value for scientific research and clinical research.

Description

Technical fields:

[0001] This invention belongs to the field of pharmaceutical application technology, and relates to the application of fulvoacetone in the preparation of drugs for treating skin inflammation and in inhibiting TRPV3 channels. Background technology:

[0002] Flopropione (chemical name: trihydroxyphenylacetone) is an antispasmodic agent. Modern pharmacology shows that it mainly works by inhibiting catechol-O-methyltransferase and antagonizing serotonin receptors. Clinically, it is used to relieve spasmodic symptoms caused by pancreatic and biliary disorders.

[0003] TRPV3 is a temperature-sensitive (33–39°C) calcium-permeable, non-selective cation channel highly expressed in skin keratinocytes, tongue, oral cavity, nasal mucosa, and intestinal mucosa. Besides being activated by thermostimulation, TRPV3 is also a ligand-gated channel, activating with several chemical agonists, including natural monoterpenoid stimulants such as carvacrol and synthetic small molecule 2-APB. Additionally, it is involved in the activation of intracellular protons (H... + It can also activate TRPV3. When the TRPV3 channel is opened, extracellular divalent calcium ions enter the cell, increasing intracellular calcium levels. 2+ A significant increase in TRPV3 concentration allows it to participate in numerous signal transduction processes and regulate cellular function. Studies have shown that TRPV3 plays a crucial role in physiological and pathological processes such as skin sensation, skin barrier formation, skin inflammation, wound healing, hair growth, intestinal inflammation, myocardial apoptosis, and inflammatory responses. Abnormal channel function may lead to certain diseases. Current technology indicates that TRPV3 functional enhancement mutations are associated with symptoms such as itching and skin inflammation in DS-Nh mice (Gly573Ser) and WBN / Kob-Ht rats (Gly573Cys). The pathogenesis of human inherited keratotic diseases Olmsted Syndrome (OS) and focal palmoplantar keratosis (FPPK) is also closely related to acquired mutations in TRPV3. Persistent channel opening induces increased keratinocyte apoptosis, leading to the occurrence of OS and FPPK. These findings suggest that TRPV3 is a potential drug target for treating inflammatory, itchy, and keratotic skin diseases. Currently, no research reports have been found on the treatment of skin inflammation with fulvaline, nor have any reports been found on the relationship between fulvaline and TRPV3 channels. Summary of the Invention:

[0004] The purpose of this invention is to overcome the shortcomings of the existing technology and provide an application of fulvoacetone in the preparation of drugs for treating skin inflammatory diseases and in inhibiting TRPV3 channels, thus developing a new use for fulvoacetone.

[0005] To achieve the above objectives, the present invention provides the use of florfenicol in the preparation of a drug for treating skin inflammation, wherein florfenicol can improve the symptoms of DNFB-induced atopic dermatitis.

[0006] The chemical structural formula of the fulroacetone is:

[0007]

[0008] The present invention also provides the use of fulvin as a TRPV3 channel inhibitor to inhibit TRPV3 ion channel activity, and can be used to prepare drugs for treating diseases related to enhanced TRPV3 ion channel activity.

[0009] Compared with existing technologies, this invention, on the one hand, develops a new use for floxacin, enabling its use in treating skin inflammation. Floxacin also exhibits good TRPV3 inhibitory activity, allowing for the preparation of drugs to treat diseases related to enhanced TRPV3 ion channel activity. On the other hand, it also provides a new approach to treating skin inflammation. Therefore, the new use of floxacin provided by this invention has significant value for both scientific research and clinical studies. Attached image description:

[0010] Figure 1 This diagram illustrates the inhibitory effects of florfenicol on transient receptor potential vanillic acid-3 (TRPV3) calcium fluorescence and current, as per the present invention. A represents the chemical structure of florfenicol; B shows the inhibitory effect of florfenicol on the TRPV3 channel expressed in HEK293T cells during calcium fluorescence analysis (n=3, 3 parallel experiments); C shows the TRPV3 current recorded by whole-cell patch-clamp with 2-APB alone (50 μM) or in combination with 100 μM florfenicol; D shows the current-voltage curves of TRPV3 voltage increasing from -100 mV to +100 mV under different conditions, with 0 representing the baseline, 1 representing the use of 50 μM 2-APB alone, and 2 representing the use of 100 μM florfenicol simultaneously. Flopropione and 50 μM 2-APB, 3 is the eluent; E represents the inhibition of TRPV3 whole-cell currents by increasing flopropione concentration from 0.1 to 300 μmol / L; F is the Hill equation fitting for the dose-dependent inhibition of 2-APB-mediated TRPV3 currents by flopropione, with a half-inhibition concentration of 16.18 ± 5.67 μM (n = 5) and a Hill coefficient of 1.092; data are expressed as mean ± SEM.

[0011] Figure 2This is a schematic diagram of the experimental results of selective inhibition of TRPV3 by furoacetone according to the present invention. A shows the TRPV1 current (left) when activated by AITC and co-administered with furoacetone, and completely inhibited by barium chloride, as well as the current-voltage curve (right) showing the TRPV1 voltage rising from -100 to +100 mV under different conditions. 0 represents TRPV1 before administration, 1 represents AITC alone, 2 represents AITC and furoacetone used together, and 3 represents after complete inhibition by barium chloride. B shows the TRPV1 current (left) induced by capsaicin and co-administered with and eluted with furoacetone, as well as the current-voltage curve (right) showing the TRPV1 voltage rising from -100 to +100 mV under different conditions. 0 represents TRPV1 before capsaicin administration, 1 represents capsaicin alone, 2 represents capsaicin and furoacetone used together, and 3 represents after elution. C shows the TRPV1 current (left) induced by GSK101 and co-administered with furoacetone, and completely inhibited by barium chloride. The TRPV4 current under complete barium chloride inhibition, and the current-voltage curves (right) showing the TRPV4 voltage rising from -100 to +100 mV under different conditions, where 0 represents TRPV4 before GSK101 administration, 1 represents GSK101 alone, 2 represents GSK101 used in combination with 100 μM floxacin, and 3 represents TRPV4 after complete inhibition by barium chloride; D represents the TRPM8 current induced by menthol, as well as when used in combination with 100 μM floxacin and eluted, and the current-voltage curves (right) showing the TRPM8 voltage rising from -100 to +100 mV under different conditions, where 0 represents TRPM8 before menthol administration, 1 represents menthol alone, 2 represents menthol used in combination with 100 μM floxacin, and 3 represents TRPM8 after elution; E represents the average current inhibition analysis of floxacin on hTRPV3, hTRPA1, hTRPV1, hTRPV4, and hTRPM8 channels.

[0012] Figure 3 This is a schematic diagram of the experimental results used in this invention to verify whether floridone directly targets a single TRPV3 channel. A shows the single-channel current plot and fitted Gaussian histogram when 30 μM / L 2-APB is applied; B shows the single-channel current plot and fitted Gaussian histogram when 30 μM / L 2-APB and 50 μM / L floridone are applied simultaneously; C shows the single-channel current plot and fitted Gaussian histogram when the solvent is applied; the dashed lines represent the open channel state (O) and the closed channel state (C), respectively; D is a summary of the hTRPV3 single-channel conductivity after applying different modifiers (n = 4, ****P < 0.0001, compared with the solvent); E shows the average P of a single hTRPV3 channel in the presence of different TRPV3 modifiers. OPEN Summary of values ​​(n=5, **** P < 0.0001; data are expressed as mean ± SD; ****P < 0.0001, confirmed by one-way ANOVA.

[0013] Figure 4 This is a schematic diagram illustrating the experimental results of fuloacetone improving DNFB-induced AD symptoms according to the present invention. A is a flowchart of the experimental process for generating a mouse dorsal skin AD model through local application of DNFB and treatment with fuloacetone; B shows the dorsal skin phenotypic characteristics of mice in the blank control group, DNFB group, 0.1 mM fuloacetone group, 1 mM fuloacetone group, and 10 mM fuloacetone group for 9 consecutive days; C shows representative histological images (n = 3-4) of paraffin-embedded sections (6 μm) of dorsal skin from different groups of mice, with a bar of 500 μm; D shows the statistical analysis of the thickness of dorsal skin tissue sections from different groups of mice (n = 3, ...). **** P<0.0001 (one-way ANOVA), data are expressed as mean±SD.

[0014] Figure 5 This is a schematic diagram illustrating the experimental results of the topical application of fulvenom to inhibit DNFB-induced ear swelling, as per the present invention. A is a flowchart of fulvenom treatment for DNFB-induced ear swelling in mice; B shows the ear swelling observed in mice on day 9 of the experiment in the blank control group, DNFB group, 0.1 mM fulvenom group, 1 mM fulvenom group, and 10 mM fulvenom group; C shows the statistical changes in ear thickness in mice with DNFB-induced ear swelling treated with fulvenom (n = 6-10). * p<0.05, *** p<0.001, **** p<0.0001 (two-way ANOVA); data are expressed as mean ± SD. Detailed implementation method:

[0015] The present invention will now be described in further detail through specific embodiments and in conjunction with the accompanying drawings.

[0016] Example 1:

[0017] This embodiment involves identifying florfenicone as an inhibitor of TRPV3 using calcium fluorescence analysis and patch-clamp technique. The specific steps are as follows:

[0018] In this embodiment, HBSS cell balanced salt buffer was used as the drug preparation solvent to prepare three groups of compounds: positive control group (200 μM 2-APB group): HBSS solution containing 1.2 mM 2-APB (2-aminoethoxydiphenylborate), with a final drug concentration of 200 μM at actual action; blank control group (HBSS group): HBSS solution containing 0.2% (v / v) DMSO; fulvoacetone group (200 μM fulvoacetone): HBSS solution containing 1.2 mM fulvoacetone, with a final drug concentration of 200 μM at actual action. Human embryonic kidney cells HEK293T were used as the overexpression tool cells. hTRPV3 was overexpressed on the cells using transient transfection technology. Intracellular calcium ion flow was detected using Cal-520 fluorescent dye. The FlexStation 3 multi-mode microplate reader was used with a rapid kinetic fluorescence analysis module. The excitation wavelength was 485nm and the emission wavelength was 525nm. Readings were taken every 1.6s. Three groups of compounds were automatically added at 17s. The positive control drug (2-APB (200uM)) was automatically added at 100s. TRPV3 was activated using 2-APB. Data was collected up to 180s.

[0019] For agonists, the characteristic curve shows a significant increase in relative fluorescence intensity upon compound addition at 17s; for inhibitors, the characteristic curve shows no significant increase in fluorescence value upon compound addition at 17s, while the relative fluorescence intensity after addition of the positive control (2-APB) at 100s is significantly lower than that of the control group. Data processing results are as follows: Figure 1 As shown in B. From Figure 1 As shown in Figure B, the relative fluorescence intensity of the florofacetone group was significantly lower than that of the agonist 2-APB group after the addition of 2-APB (200 μM) at 100 s, indicating that florofacetone may have an inhibitory effect on the opening of TRPV3 channels excited by 2-APB.

[0020] To confirm the inhibitory effect of fulvoacetone on TRPV3 channels, whole-cell patch-clamp recordings of hTRPV3 currents expressed in HEK293T cells were performed using an AXON MultiClamp 700B amplifier and Clampex 10.6 software. Electrodes were pulled using a DMZ electrode puller. Cell perfusion fluid was administered via gravity perfusion. First, a channel agonist (extracellular solution containing 2-APB (50 μM) with NaCl 130 mM, EDTA 0.2 mM, HEPES 10 mM, pH 7.2-7.4) was added, followed by extracellular solution containing fulvoacetone (100 μmol / L) and 2-APB (50 μM). Finally, the cells were eluted with the extracellular solution. The membrane potential was maintained at 0 mV, and the ramp voltage was increased from -100 to +100 mV over 500 ms. The average of -80 mV and +80 mV was taken to represent the time-effect plot of the compound on the ion channel. The current was sampled at 20 kHz and filtered at 2.0 kHz. The results are as follows. Figure 1 As shown in C. From Figure 1 As can be seen from C, administration of 100 μmol / L floridone significantly inhibited the current of 2-APB-activated TRPV3, with an inhibition rate of 84.04 ± 2.37% (n = 6).

[0021] During the whole-cell patch-clamp recordings described above, voltage ramps were recorded in four groups: one group served as baseline; one group used 50 μM 2-APB alone (group 1); one group used both 100 μM florfenicol and 50 μM 2-APB simultaneously (group 2); and one group was eluted with extracellular fluid (group 3). Current-voltage curves showing the voltage rise from -100 mV to +100 mV were recorded for each of the four treatment groups. The results are as follows: Figure 1 As shown in D. From Figure 1 As can be seen from D, florfenicol inhibited the voltage at which 2-APB activated TRPV3.

[0022] To determine the inhibitory efficacy of floridone on TRPV3, whole-cell recordings of TRPV3 currents were performed using different concentrations of floridone (0.1-300 μM). The results are as follows: Figure 1 As shown in E. From Figure 1 E shows that the TRPV3 current is dose-dependently inhibited by florfenicol, and the Hill equation fitting for the dose-dependent inhibition of the 2-APB-mediated activated TRPV3 current by florfenicol is as follows: Figure 1 As shown in Figure F, the half-maximal inhibitory concentration (IC50) was 16.18 ± 5.67 μM (n = 5), and the Hill coefficient was 1.092. Data are expressed as mean ± SEM.

[0023] Example 2:

[0024] This embodiment involves an experiment to verify whether fulvin selectively inhibits the TRPV3 channel. The specific steps are as follows:

[0025] After confirming the inhibitory effect of florfenicol on TRPV3 channels, the study verified whether florfenicol selectively inhibited TRPV3 channels by investigating whole-cell currents in HEK 293T cells, which express other thermosensitive TRP channels, including hTRPV1, hTRPV4, hTRPM8, and hTRPA1 channels. In this embodiment, extracellular fluid (NaCl (130 mM), EDTA (0.2 mM), and HEPES (10 mM), pH 7.2-7.4) was used as the solvent to dissolve the drug. The specific experimental steps are as follows:

[0026] (1) TRPA1 channels were activated with AITC (300 μM), followed by the addition of floridone (100 μM) and AITC (300 μM), and then barium chloride (130 mM). The whole-cell current of TRPA1 was recorded when it was activated by 300 μM AITC and when it was completely inhibited by the combined application of AITC and floridone and then by barium chloride. The current-voltage curves of TRPA1 before (0) and after (1) administration of 300 μM AITC, and after the combined application of AITC and floridone (2) and complete inhibition by barium chloride (3) were also recorded as the voltage increased from -100 mV to +100 mV. The results are as follows: Figure 2 As shown in Figure A.

[0027] (2) TRPV1 channel activation was induced by capsaicin (1 μM), followed by the addition of florfenicol (100 μM) and capsaicin (1 μM), and then eluted with extracellular fluid. The TRPV1 current induced by 1 μM capsaicin, as well as the current during the combined application and elution of capsaicin and florfenicol (100 μM), was recorded. The current-voltage curves of TRPV1 before (0) and after (1) administration of 1 μM capsaicin, and after the combined application of capsaicin (1 μM) and florfenicol (100 μM) (2) and elution (3) were also recorded as the voltage increased from -100 mV to +100 mV. The results are as follows: Figure 2 As shown in B.

[0028] (3) The TRPV4 channel was activated using GSK101 (0.1 μM), followed by the addition of 100 μM floridone and GSK101 (0.1 μM), and then barium chloride (130 mM). The TRPV4 current was recorded when activated by 0.1 μM GSK101 and when 100 μM floridone and GSK101 were used together and then completely suppressed by 130 mM barium chloride. The current-voltage curves of TRPV4 before (0) and after (1) the application of 0.1 μM GSK101, and after the application of GSK101 with 100 μM floridone (2) and after complete suppression by barium chloride (3) were also recorded, showing the voltage rise from -100 mV to +100 mV. The results are as follows: Figure 2 As shown in C.

[0029] (4) The TRPM8 channel was induced by menthol (500 μM), followed by the addition of florfenicol (100 μM) and menthol (500 μM), and then eluted with extracellular fluid. The TRPM8 current induced by 500 μM menthol, as well as the current of menthol and florfenicol used together and eluted, were recorded. The current-voltage curves of TRPM8 before (0) and after (1) administration of 500 μM menthol, and after (2) administration of menthol and florfenicol used together and after (3) elution were also recorded as the voltage increased from -100 mV to +100 mV. The results are as follows: Figure 2 As shown in D.

[0030] The effect of 100 μM fulvin on the average current suppression of hTRPV3, hTRPA1, hTRPV1, hTRPV4, and hTRPM8 channels was analyzed by one-way ANOVA. The results are as follows: Figure 2 E is shown. The data is presented as mean ± SD, n = 3; **** p<0.0001.

[0031] from Figure 2 As shown in Figure A, simultaneous perfusion with 100 μM floridone and AITC solution reduced the TRPA1 current induced by 300 μM allyl isothiocyanate (AITC) by approximately 0.56907 ± 5.25485%. Figure 2 B shows that simultaneous perfusion of 100 μM floridone and capsaicin reduced the hTRPV1 current induced by 1 μM capsaicin by approximately 9.3246 ± 13.6502%. Similarly, the TRPV3 current inhibited by floridone (…) Figure 1 C) Compared to 100 μmol florfenicol, 0.1 μM GSK101 (GSK101, Figure 2 TRPV4 current induced by C and E or TRPM8 current induced by 500 μM menthol Figure 2 D and E) showed no significant inhibitory effect.

[0032] The above results demonstrate that florofacetone did not exhibit inhibitory effects on other thermosensitive TRP ion channels, indicating that it is a selective inhibitor of TRPV3. These results suggest that florofacetone is a highly selective antagonist of the TRPV3 channel.

[0033] Example 3:

[0034] This embodiment involves an experiment to verify whether floxacin directly targets a single TRPV3 channel. To further confirm whether floxacin can directly target a single TRPV3 channel, this embodiment performed single-channel recording and data fitting from the inside out on HEK293T cells overexpressing hTRPV3 under different regulatory conditions in the extracellular fluid of HEK293T cells. Three different regulatory agents were used: one was 2-APB (30 uM / L), one was simultaneous administration of 2-APB (30 uM / L) and floxacin (50 uM / L), and one was extracellular fluid administration. The results are as follows: Figure 3 As shown in AC, the single-channel conductivity of hTRPV3 after the administration of different modifiers and the average P of a single hTRPV3 channel in the presence of different TRPV3 modifiers are compared. OPEN The values ​​were analyzed, and the results are as follows: Figure 3 As shown in DE.

[0035] from Figure 3 As shown in A, D, and E, 2-APB, as a control, activated the channel at a clamping potential of -60mV, increasing the opening probability of a single TRPV3 channel with a single-channel conductance of 133.1±11.5pS.

[0036] from Figure 3 Figures B and E show that the opening probability of a single channel is significantly reduced by florfenicol, decreasing from 0.51±0.09 to 0.13±0.10 at -60 mV. Furthermore, through... Figure 3 As shown in Figure D, the channel conductance before and after florfenicol perfusion reveals that it did not affect the conductance of a single hTRPV3 channel. Similar results were obtained when performing single-channel recording from the inside out in a calcium-containing recording solution; that is, florfenicol reduced the opening probability of a single channel without altering its individual hTRPV3 channel conductance. These results indicate that florfenicol suppresses TRPV3 current by directly acting on individual channels and reducing their opening frequency.

[0037] Example 4:

[0038] This embodiment relates to an experiment involving the topical application of fulvetone to improve symptoms of 2,4-dinitrofluorobenzene (DNFB)-induced atopic dermatitis (AD). The specific steps are as follows:

[0039] (1) Construction of the AD model: The experimental mice were 6-week-old C57BL / 6N strain mice purchased from Beijing Vital River Laboratory Technology Co., Ltd. The backs of the mice were shaved one day in advance, covering an area of ​​approximately 1... * 1cm 2 After one day of adaptation, the mice were randomly divided into two groups: 5 mice served as the control group and received no further treatment, and 20 mice were used to model AD. 100 μL of 0.5% DNFB was applied topically, and no treatment was given for three days. On the fifth and sixth days, 50 μL of 0.2% DNFB was applied topically to induce atopic dermatitis. The expression level of TRPV3 was upregulated in the skin cells isolated from the lesions.

[0040] (2) Experimental Procedure: AD model mice were randomly divided into 4 groups of 5 mice each: a model group (DNFB, no treatment) and 3 treatment groups. The treatment groups were 0.1 mM fulvoacetone, 1 mM fulvoacetone, and 10 mM fulvoacetone. On the sixth day, the 3 treatment groups were treated with 100 μL of fulvoacetone HBSS solution of different concentrations (0.1 mM, 1 mM, and 10 mM) after a half-hour interval following the application of DNFB topically. This treatment was repeated for four consecutive days. The condition of the back skin of the mice was photographed and recorded daily, and H&E staining histological examination was performed on the dorsal skin tissue sections. The changes in the control group, model group, and treatment groups were summarized. The results are as follows: Figure 4 As shown.

[0041] from Figure 4 As can be seen from B, compared with the model group mice, the skin thickness and scab formation were alleviated to varying degrees in the three treatment groups with topical application of fulvaline.

[0042] from Figure 4 As shown in C and 4D, histological examination of H&E-stained dorsal skin tissue sections revealed that, compared with the DNFB group mice (n=3), topical application of fulvaline resulted in a dose-dependent reduction in epidermal thickness and a decrease in inflammatory cell infiltration in the dermis, consistent with the results of phenotypic observation.

[0043] Example 5:

[0044] This embodiment involves an experiment on the inhibitory effect of topical application of floxacin on ear swelling induced by DNFB. To confirm the effect of floxacin on reducing DNFB-induced skin inflammation, this embodiment utilizes a mouse model of DNFB-induced ear swelling to further verify the effect of floxacin on ear inflammation. The specific steps are as follows:

[0045] (1) Establishment of an ear swelling model: The experimental mice were 6-week-old C57BL / 6N strain mice purchased from Beijing Vital River Laboratory Technology Co., Ltd. They were randomly divided into two groups: 5 mice served as the control group with no further treatment, and 20 mice were used for ear swelling modeling. 100 μL of 0.5% DNFB was applied topically to the right ear of each mouse, with no treatment for three days. On the fifth and sixth days, 50 μL of 0.2% DNFB was applied topically. The right ear of the modeling mice showed significant swelling and epidermal inflammation.

[0046] (2) Experimental Procedure: Mice with ear swelling were randomly divided into four groups of five mice each: a model group (DNFB, no treatment) and three treatment groups. The treatment groups received 0.1 mM floxacin, 1 mM floxacin, and 10 mM floxacin, respectively. Half an hour after the last DNFB application, the three treatment groups received topical application of floxacin at different concentrations (0.1 mM, 1 mM, and 10 mM), respectively, for four consecutive days. Before the last floxacin treatment, the ear thickness of all mice was measured for the first time after the last DNFB application. Ear thickness was measured again at 24, 48, 72, and 96 hours after the first measurement, and the results were statistically analyzed. The results are shown below. Figure 5 As shown.

[0047] from Figure 5 As shown in B and 5C, measurements of ear appearance and thickness revealed that, compared to the DNFB group, mice treated with floridone topically for 4 days exhibited significantly reduced epidermal inflammation and ear swelling in the right ear. These results indicate that floridone can alleviate DNFB-induced ear inflammation.

Claims

1. The application of fulvoacetone in the preparation of drugs for treating atopic dermatitis.

2. The use of fulvetone according to claim 1 in the preparation of a drug for treating atopic dermatitis, characterized in that, Furoacetone, as a TRPV3 channel inhibitor, is used to selectively inhibit TRPV3 ion channel activity.

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