Novel TRPV1 channel blocker developed by high-throughput screening of marine natural products

Abstract

The invention relates to a novel TRPV1 channel blocker developed by high-throughput screening of marine natural products, and hexa-epoxyanthrone separated from marine bacteria. In particular, these chrexanthophycins are potent TRPV1 inhibitors, which are potent as analgesic agents. The present application also relates to a method of treating pain, comprising applying an analgesic comprising these chrexanthophycins to a pain site of a patient.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of U.S. Provisional Application Serial No. filed on April 27, 2023.

[0003] 63 / 498,526, the entire contents of which are incorporated herein by reference, including any tables, figures, or drawings. Background Art

[0004] Pain is a protective reflex of the body, but pain can seriously affect the quality of life of an individual. Statistics show that about 60% of elderly people over 65 years old, 70% of cancer patients and 95% of spinal cord injury patients suffer from chronic pain syndrome. Existing analgesics including opioids, nonsteroidal anti-inflammatory drugs (NSAIDs), and alcohol have significant side effects. For example, opioids can cause addiction, drug tolerance, respiratory depression and epilepsy, while NSAIDs cause gastrointestinal bleeding and increase the risk of cardiovascular events. Therefore, the development of safe and effective analgesics with minimal side effects is an urgent medical need. Summary of the invention

[0005] An embodiment of the present application relates to an analgesic comprising a hexacyclic xanthone in a carrier. The hexacyclic xanthone is selected from chrexanthomycin A (cA), chrexanthomycin B (cB), chrexanthomycin C (cC), chrexanthomycinF (cF) or any combination thereof. In one embodiment, the analgesic further comprises capsazepine. The therapeutic effect of the compound of the present application is comparable to that of capsazepine. In a preferred embodiment, the concentration of the hexacyclic xanthone is about 0.75 μM to 20 μM. In a preferred embodiment, the carrier can be a solution comprising a solvent, the solvent including but not limited to dimethyl sulfoxide (DMSO).

[0006] Embodiments of the present application relate to a method for treating pain, comprising applying an analgesic as described herein to a patient's painful area. In one embodiment, the analgesic comprises a hexacyclic xanthone in a carrier. The hexacyclic xanthone is selected from chrexanthomycin A (cA), chrexanthomycin B (cB), chrexanthomycin C (cC), chrexanthomycin F (cF) or any combination thereof. In a preferred embodiment, the carrier can be a solution comprising a solvent, the solvent including but not limited to DMSO. In a preferred embodiment, the applying comprises applying by injection, topical administration or any other means. BRIEF DESCRIPTION OF THE DRAWINGS

[0007] Figure 1 The chemical structures of chrexanthomycins according to embodiments are shown.

[0008] Figure 2A Schematic representation of FM4-64 dye uptake by TRPV1 analyzed using Zeiss Celldiscoverer 7 is shown.

[0009] Figure 2B The steps of capsaicin-induced TRPV1 activation analyzed by dye uptake assay are shown.

[0010] Figure 3A Shown is a bar graph comparing the inhibitory properties of chrexanthomycins of different structures on TRPV1 expressed by the n2 / n1 ratio.

[0011] Figure 3B Shown is a bar graph of the n2 / n1 ratio using a cell-based model, representing the inhibitory effects of cC and cF on TRPV1 relative to capsaicin.

[0012] Figure 4 Dose-response curves of % inhibition versus concentration are shown for cC (upper panel) and cF (lower panel).

[0013] Figure 5A Shown are representative current signal recordings of cC inhibiting TRPV1 channels, where 20 μM of drug application was performed after 10 nM capsaicin activation of the membrane patch, while the potential was maintained at +60 mV during capsaicin activation and during the application of drug cC to the membrane patch, and where intervals I, II and III represent 3 second intervals of baseline, capsaicin activation and drug inhibition periods.

[0014] Figure 5B Shown are representative current signal recordings of cF inhibiting TRPV1 channels, where 20 μM of drug application was performed after 10 nM capsaicin activation of the membrane patch, while the potential was maintained at +40 mV during capsaicin activation and during the application of drug cF to the membrane patch, and where intervals I, II and III represent 3 second intervals of baseline, capsaicin activation and drug inhibition periods.

[0015] Fig. 6A Shown are the changes in relative macroscopic current before and after the addition of cC (left) and cF (right).

[0016] Figure 6B Shown are bar graphs comparing the changes in relative macroscopic current between the capsaicin activated state (before) and after addition of cF or cC.

[0017] Figure 6C A bar graph showing the time that mice licked the painful sites induced by capsaicin- / TRPV1 treated with capsaicin, cC and cF. A bar graph showing the time that mice licked the painful sites induced by capsaicin through activation of TRPV1 channels treated with capsaicin, cC and cF. DETAILED DESCRIPTION

[0018] The embodiments of the present application relate to novel hexacyclic xanthones with strong pain suppressing effects, whose IC 50 The values ​​ranged from 0.75 μM to 2.1 μM and there was no significant cytotoxicity. In a mouse model, these xanthones showed efficacy similar to capsaicin in blocking capsaicin-induced pain perception. The unique pharmacophore of these new hexacyclic xanthones compared to known TRPV1 modulators suggests a new binding mode between the ligand and the ion channel. These new antagonists can be used as innovative analgesics.

[0019] In the screening of potential TRPV1 modulators, it was found that Figure 1 The hexacyclic xanthone chrexanthomycin shown, in particular chrexanthomycin A, chrexanthomycin B, chrexanthomycin C and chrexanthomycinF (cA, cB, cC, cF) showed an inhibitory effect on TRPV1. These compounds were identified during a screening of marine bacterial metabolites. In order to screen potential regulators of TRPV1, a high-throughput screening system based on FM4-64 dye uptake was established using HEK293 cells transfected with hTRPV1-pIRES-eGFP and cultured in 96-well plates. The cells were incubated with 5 μM FM4-64 dye and 100 μg / ml test compounds for 3 minutes, washed, imaged using Zeiss Cell discoverer 7, and analyzed using Image J ( Figure 2A ). Because of the plasmid structure of hTRPV1-pIRES-eGFP, TRPV1 and eGFP are expressed simultaneously in one cell, so the number of GFP-positive cells, which is an indicator of the number of TRPV1-expressing cells, is calculated as n1. The degree of dye uptake is directly related to the activity of the TRPV1 channel, so the number of cells that are both GFP-positive and dye-uptake-positive is calculated as n2 ( Figure 2B ). The ratio (n2 / n1) is a functional indicator of TRPV1 channel activity.

[0020] Depend on Figure 1As can be seen, cA, cB, and cF contain a 4',5'-anhydroglucuronic acid moiety and a hemiketal group at ring A of the hexacyclic xanthone core, while the six-membered ring system of cF is bent in a different orientation than cA and cB. All four members of the chrexanthomycins have an inhibitory effect on TRPV1, with a significant decrease in the relative n2 / n1 ratio compared to the vehicle control (DMSO). Among the four chrexanthomycins, cC with a glucuronic acid moiety exhibited the lowest relative n2 / n1 ratio compared to its dehydrated product cA or other chrexanthomycins with anhydroglucuronic acid moieties ( Figure 3A ), which suggests that the addition of a hydroxyl group could favor the inhibitory effect and make cC the most potent TRPV1 inhibitor.

[0021] The extent of the effects of cC and cF on TRPV1 channel function compared with capsaicin and capsaicin in HEK293 cells transfected with human TRPV1 and cultured in 96-well plates was compared by comparing the n2 / n1 ratios of the groups incubated with cC and cF with those incubated with capsaicin alone and with capsaicin. Figure 3B ). The average n2 / n1 ratio of the capsaicin group was 0.254, while the ratios of the cC, cF, and capsaicin groups were 0.0307, ​​0.068, and 0.0667, respectively. Post hoc analysis of variance (ANOVA) showed that the n2 / n1 ratios of the cC, cF, and capsaicin groups were significantly different from the n2 / n1 ratio of the capsaicin group. No significant differences were observed between the capsaicin group and the drug groups. In addition, the n2 / n1 ratio of the cC group was slightly lower than that of the capsaicin group, but this difference was not significant in the ANOVA test. However, the t-test showed that there was a significant difference between cC and capsaicin (p=0.00937). This suggests that cC is a TRPV1 inhibitor superior to capsaicin in a cell-based model.

[0022] The half-maximal inhibitory concentration (IC) of cC in cell-based models 50 )

[0023] The cell-based model enables in vitro quantitative examination of the effectiveness of cC in inhibiting TRPV1 channels. The test involves mixing 1 μM of capsaicin with various concentrations of cC and 5 μM of FM4-64, and adding the mixture to each well for incubation for 3 minutes. The results of different concentrations of cC standardized with a DMSO-only control (without capsaicin) were compared with a group without cC, which showed that the TRPV1 channel was activated in large quantities and was set to 100% activation. Dose-response curves drawn with eight different cC concentrations (including 0.2 μM, 0.5 μM, 0.75 μM, 1 μM, 2 μM, 5 μM, 10 μM and 20 μM) showed a flattening trend at high concentrations (10 μM or 20 μM) of cC, where it was tested once at high concentrations, and the inhibitory effect curves of cC at relatively low concentrations (less than or equal to 5 μM) were as shown in Figure 2. Figure 4 shown.

[0024] Figure 4 The IC of cC in a cell-based system is shown. 50 In the range of 0.75 μM to 1 μM, and the figure shows that cC performs best in the concentration range of 0.75 μM to 1 μM. However, at concentrations greater than 1 μM, the inhibitory effect of cC decreases slightly and then increases again with further increases in concentration. For the IC 50 , in the 2 μM group, 50% TRPV1 inhibition was not achieved, which is consistent with the IC 50 In a higher range than.

[0025] Effects of Chrexanthomycin C on TRPV1 channels in patch clamp assay

[0026] High-throughput drug screening assays based on cellular FM4-64 dye uptake can identify potential TRPV1 inhibitors, however this assay cannot conclusively distinguish the specific mechanism of blockade due to many factors, such as drug-induced membrane potential changes caused by other channels on the cell membrane, pinocytosis, and pore formation. Factors such as these can lead to changes in FM4-64 dye uptake. High-resolution patch clamp experiments using an inside-out ex vivo recording configuration can investigate the potential inhibitory effects of cC and cF on TRPV1, where changes in patch clamp macroscopic currents before and after drug application indicate drug effectiveness.

[0027] Most compounds that showed inhibitory effects in the high-throughput dye uptake screen also showed similar effects in patch clamp studies, indicating that cC and cF effectively inhibited TRPV1 channels in patch clamp macroscopic current recordings, with cC having a better inhibitory effect than cF ( Figure 5A and Figure 5B), which is consistent with the dye uptake assay. Capsaicin-activated TRPV1 expression membranes showed a decrease in macroscopic current after applying the candidate drug, indicating that the capsaicin-activated TRPV1 channel was inhibited by the candidate drug and its activity was reduced. In order to confirm that the inhibitory effect is specific to the TRPV1 channel, the channel current change curve (trace) recorded in the 3-second interval shows that this current record is a typical TRPV1 channel activation current curve. This shows that after adding capsaicin and drugs, the TRPV1 channel changes specifically.

[0028] Fig. 6A The combination of macroscopic currents of a single membrane patch is shown. Specifically, this shows that after the addition of cC and cF, the macroscopic current showed a downward trend, with the application of cC causing the current to drop more significantly. In the capsaicin-activated state of the TRPV1 channel, after the addition of cC, the average relative macroscopic current dropped to 0.285. After performing a t-test, we found a significant difference between capsaicin and cC in terms of the relative macroscopic current drop (p = 6.980*10 -9 )( Figure 6B ). In conclusion, the results indicate that cC exhibits a significant TRPV1 inhibitory effect during the capsaicin-activated state.

[0029] Blocking capsaicin-induced pain in a mouse model

[0030] The inhibitory effect of cC on TRPV1 was demonstrated in vitro using dye uptake assays and patch clamp electrophysiology. In vivo behavioral experiments injected the drug mixed with capsaicin into the right hind paws of 8 one-month-old wild-type female mice. The mixture was analyzed using high-performance liquid chromatography (HPLC), and it was confirmed that capsaicin did not react with cC. The total time spent by mice licking their paws (which is a typical nociceptive behavior) was recorded as an indicator of TRPV1 activation and compared with a positive control group injected with capsaicin alone. The efficacy of the injected drug was associated with a reduction in licking time. As another group of negative controls, mice were injected with a mixture of capsaicin and capsaicin to compare with the inhibitory effects of cC and cF.

[0031] The mice were observed for one month after the injection to check for any adverse side effects, and no obvious health problems were found, indicating that both drugs are safe. The average licking time of the capsaicin-only group was over 50 seconds, while the average licking times of the cC and capsaicin groups were 19 seconds and 18 seconds, respectively. Post hoc ANOVA showed that there was a significant difference in licking time between the capsaicin-only group and the group injected with cC and capsaicin. In contrast, no significant difference was found between cC and capsaicin ( Figure 6C ), indicating that cC is as effective as capsaicin in blocking capsaicin-induced pain perception.

[0032] The following are examples illustrating the process of implementing the present application. These examples should not be construed as limiting. Unless otherwise indicated, all percentages are by weight and all solvent mixture ratios are by volume.

[0033] Materials and methods

[0034] strain

[0035] The strains were purchased from Thailand Bioresource Research Center and China Marine Microbial Culture Collection Center, or isolated from coastal waters of the Red Sea.

[0036] Fermentation and extraction

[0037] The strains were cultured in 125 ml conical flasks containing 50 ml of different culture media at two different temperatures, room temperature and 30°C, with stirring at 180 rpm for 1 week, wherein the culture media were AM4 (soybean powder 20 g / L, bacterial peptone 2 g / L, glucose 20 g / L, soluble starch 5 g / L, yeast extract 2 g / L, NaCl 4 g / L, K2HPO4 0.5 g / L, MgSO4·7H2O 0.5 g / L, CaCO3 2 g / L, 3% sea salt, and the pH was set to 7.8), GYM (yeast extract 4 g / L, malt extract 10 g / L, glucose 4 g / L, 3% sea salt, and the pH was set to 7.2 to 7.4), and AM6 (soluble starch 20 g / L, glucose 10 g / L, bacterial peptone 5 g / L, yeast extract 5 g / L, CaCO3 5g / L, 3% sea salt, pH set to 7.2 to 7.5), and 10 to 15 glass beads (3 mm in diameter). The bacterial culture was extracted three times with equal volumes of ethyl acetate to obtain a crude extract. The crude extract of S. chrestomyceticus BCC 24770 cultured in AM5 medium at 30°C showed significant regulatory activity and was cultured on a large scale to accumulate sufficient amounts.

[0038] Isolation and purification

[0039] About 60 g of crude extract was obtained from 30 L of culture broth and separated by reverse phase C18 column chromatography and eluted with 20%, 40%, 60%, 80% and 100% acetonitrile / water to obtain different fractions. The medium polarity fraction was enriched in bioactive TRPV1 modulators. The 40% fraction was further purified by semi-preparative HPLC (Waters 2695 Separations Module; Milford, USA) and eluted with an isothermal mobile phase at 3 ml min -1Elution was performed at a flow rate of . (Solution A: acetonitrile; Solution B: Milli-Q water.) Rinse with acetonitrile at a concentration of 33% for 50 min. The eluent was monitored at a UV wavelength of 230 nm (Waters 2998 Photodiode Array Detector). The compounds were collected, dried with nitrogen, and dissolved in dimethyl sulfoxide (DMSO) for further biological evaluation. Semi-preparative HPLC was performed using a C18 column with a size of 250×10 mm and a particle size designed to be 5 μm.

[0040] Structure determination

[0041] The results were performed on 500 MHz and 800 MHz Varian NMR spectrometers. 1 H NMR was acquired on a 200 MHz Varian NMR spectrometer. 13 C NMR spectrum. Standard 2D NMR experimental spectra were collected at 25°C, including HSQC, HMBC and COSY. MS data were recorded on a Bruker ultrafleXtreme ultra-high resolution TOF LC-MS system. HRMS m / z = 621.1235 [M+Na] + (The calculated value is C 31 H 25 O 14 , 621.1239, δ=-0.64ppm).

[0042] Reagents and plasmids

[0043] Capsaicin and capsaicin were from Sigma-Aldrich. The expression vector hTRPV1-IRES-EGFP was generated by subcloning human TRPV1 cDNA into the selected competent cells; Top10 was used due to its high transformation efficiency. The mixture of Top10 cells and hTRPV1-IRES-EGFP was heat shocked, and the mixture was evenly spread on a kanamycin culture dish and incubated for 12 to 18 hours. After incubation, colonies were formed and expanded by transferring the selected colonies to LB medium. The mixture was cultured in a shaker for another 12 to 18 hours. After colony expansion, plasmids were extracted using the TIANprep MidiPlasmid Kit (TIANGEN).

[0044] Cell culture and transfection

[0045] HEK293T (RRID: CVCL_1926) cells were obtained from ATCC and were considered to be authenticated by ATCC, so no further authentication was performed. The cell line was routinely tested negative for mycoplasma contamination and maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum and 100 U / mL penicillin / streptomycin (Life Technologies) at 37°C, 95% air, 5% CO2. Cell transfection was performed using PEI (3 μL / μg plasmid).

[0046] High-throughput FM4-64 dye uptake assay and imaging

[0047] HEK293 cells transfected in 96-well plates were divided into 8 groups, each containing 12 wells. 10 to 11 wells of each group were set as experimental groups, in which cells were incubated with a compound mixture. 1 to 2 wells of each group were set as control groups, in which cells were incubated with control solutions A or B. The same group was operated simultaneously to reduce the difference between the wells. The cells in DMEM were washed once with CaCl2-HBSS buffer to remove residual culture medium. Subsequently, cells were incubated at room temperature (RT) for 2 min to 3 min with a compound mixture or control solution. After FM4-64 incubation, cells were washed three times with CaCl2-HBSS buffer to remove FM4-64 residues. Fluorescence imaging of a group of cells was performed on Zeiss Celldiscoverer 7.

[0048] To distinguish GFP and FM4-64 fluorescent dyes, we used a 470nm LED light source to excite the GFP channel and a 567nm light to excite the FM4-64 channel. A 5x objective (Plan Apo, dry objective, autocorrection, NA 0.35), an Orca camera (Orca Flash 4.0v3), a CO2 module S1 culture system, and Zenblue software were used for image recording. Each image included a GFP channel, an FM4-64 channel, and a bright field channel. And 4 non-overlapping images were taken for each well using the Tile function of Zenblue.

[0049] Image analysis for drug screening

[0050] ImageJ software is used to analyze all images. The same threshold is applied to all images in the same experimental group, to select GFP positive and dye uptake positive cells. The number of cells is calculated by the "Analyzeparticle" function in ImageJ. The "AND" function in the image calculator is used to select GFP positive and dye uptake positive cells. The number of GFP positive cells (cells expressed by TRPV1) is calculated as N1, and the number of cells as both GFP positive and dye uptake positive is calculated as N2. The ratio of N1 / N2 represents the percentage of cells expressed by TRPV1 that absorbs FM4-64 fluorescent dye, and is used to analyze the function of TRPV1.

[0051] Patch clamp electrophysiology studies

[0052] 3T3 cells were recorded using an in-vivo voltage clamp facing outward. A patch electrode with a resistance of 4MΩ±1.1MΩ was pulled on a glass pipette puller (DMZ Zeitz-Puller). First, 3T3 cells were transferred to a patch container to minimize the use of drugs and transfected with hTRPV1-IRES-EGFP. Before the assay, the cell culture medium and the pipette culture medium were immediately replaced with PBS. The culture dish was connected to the ground electrode via a salt bridge. A glass pipette filled with PBS was inserted into the patch electrode. A positive pressure of about 40mmHg was applied to the pipette and immersed in the cell culture medium. When the holding potential was 0mV, the offset was adjusted to close to 0pA. The pipette was moved into the cell, showing green fluorescence, and was almost not attached to the cell membrane. Then, the positive pressure in the pipette was removed, and the holding potential was changed to -80mV, forming a tight seal between the cell membrane and the pipette. The sealing resistance should be >=1GΩ. The sealed membrane was separated to form a membrane patch in an inside-out configuration. Recording was started at a positive holding potential.

[0053] The membrane patch was activated by 10 nM capsaicin to fully activate the TRPV1 channel. After full activation, 20 μM of drug (cC) was applied to test its ability to inhibit TRPV1. Drugs were applied in 100 μL PBS in the form of drug stock solutions with the required volume for use. Drugs were applied to reach a concentration of 20 μM, with DMSO solutions in the culture medium below 0.2% v / v.

[0054] The channel current change curves were collected using the pClamp10 software suite and the patch clamp macroscopic currents were recorded. The signals were amplified by Axon Axopatch 200B (Axon Instruments) and digitized by Axon Digidata 1550B (AxonInstruments). The channel current change curves were exported in .abf file format and analyzed using pyabf and python to filter out the channel current change curves and calculate the macroscopic currents before and after drug application. The macroscopic currents were summarized and the drug efficacy was elucidated by comparing the changes in macroscopic currents.

[0055] Mouse

[0056] The mice used were 4- to 6-week-old C57B / 6J mice provided by the APCF of the Hong Kong University of Science and Technology. All animal procedures were approved by the Research Practice Committee of the Hong Kong University of Science and Technology (Ethics Agreement No.: 2016074).

[0057] Capsaicin-induced pain perception assay

[0058] Mice were subjected to a capsaicin-induced pain perception assay, in which 10 μL of capsaicin (300 μM) or solvent control was injected into the top of the hind paw using a fine (30-G) needle; capsaicin was dissolved in 0.3% by volume ethanol and 3% by volume dimethyl sulfoxide (DMSO). The mice were gently wrapped in the hands of the researchers and kept in a supine position with the muzzle of the mice close to the little finger of the researchers; in this state, the mice remained calm during the injection process. The behavior of the mice was recorded using a digital camera (GoPro5).

[0059] The observation cage was made of a transparent large cage liner from APCF with bedding and food on the bottom. The mice were placed in the observation cage for 10 minutes at least 1 hour before the experiment to reduce the stress caused by the unfamiliar environment. During the experiment, the experimenter grabbed the mouse's neck with the index finger and thumb of his left hand to capture the mouse. The mouse was then gradually picked up and its tail was fixed with the little finger so that its right hind paw stepped on the ring finger or middle finger of the experimenter. Drugs were prepared in 1.5 mL test tubes. For the control group, a 1 mM DMSO solution stock solution of capsaicin was diluted to 100 μM using PBS. For the test group, in addition to capsaicin, the drugs originally dissolved in DMSO were diluted to the desired concentration.

[0060] The injection was performed by shaking and rotating the test tube containing the drug to obtain a uniform concentration of the drug. A BD ultra-fine 6mm needle was used to aspirate 10μL of the drug from the needle. After removing excess bubbles, 10μL of the drug was injected into the back of the right hind paw of the captured mouse. The needle was slowly withdrawn to prevent blood and drug from leaking from the wound. After the injection, the mouse was placed in an observation cage for 5 minutes to record pain behavior. The video was captured using a camera configured with HD 30fps. Environmental noise, light and temperature were constant throughout the recording. The behavior of the mice was identified by experienced people who consistently plotted pain behavior graphs in seconds over a 5-minute period.

[0061] After the experiment, the mice were transferred to their original cages and provided with warmth to help them recover. The mice were observed for another month.

[0062] statistics

[0063] All data are presented as mean ± SEM; n represents the number of independent biological replicates. Unless otherwise stated, Student's two-tailed t-test was used for statistical analysis, and P < 0.05 was considered statistically significant.

[0064] All patents, patent applications, provisional applications, and publications cited herein are hereby incorporated by reference in their entirety, including all figures and tables, to the extent not inconsistent with the explicit teachings of this specification.

[0065] It should be understood that the examples and embodiments described herein are for illustrative purposes only, and various modifications or changes based on the examples and embodiments described herein will be suggested to those skilled in the art, and will be included in the spirit and authority of the present application and the scope of the appended claims. In addition, any element or limitation of any invention or embodiment thereof disclosed herein may be combined with any and / or all other elements or limitations disclosed herein (alone or in any combination) or any other invention or embodiment thereof, and all such combinations are within the scope of the present application, but are not limited thereto.

[0066] References

[0067] Brandt, MR, Beyer, CE, & Stahl, SM (2012). TRPV1 antagonists and chronic pain: Brandt beyond thermal perception. Pharmaceuticals, 5(2), 114-132.

[0068] Turk,D.C.,&Melzack,R.(2011).The measurement of pain and theassessment of people experiencing pain.

[0069] Moran,M.M.,&Szallasi,A.(2018).Targeting nociceptive transientreceptor potential channels to treat chronic pain:current state of thefield.British journal of pharmacology,175(12),2185-2203.

[0070] Yam,M.F.,Loh,Y.C.,Tan,C.S.,Khadijah Adam,S.,Abdul Manan,N.,&Basir,R.(2018).General pathways of pain sensation and the major neurotransmittersinvolved in pain regulation.International journal of molecular sciences,19(8),2164.

[0071] Doly,S.,Fischer,J.,Salio,C.,&Conrath,M.(2004).The vanilloid receptor-1is expressed in rat spinal dorsal horn astrocytes.Neuroscience letters,357(2),123-126.

[0072] Cao,E.,Liao,M.,Cheng,Y.,&Julius,D.(2013).TRPV1structures in distinctconformations reveal activation mechanisms.Nature,504(7478),113-118.

[0073] Zhao,R.,&Tsang,S.Y.(2017).Versatile roles of intracellularlylocatedTRPV1 channel.Journal of Cellular Physiology,232(8),1957-1965.

[0074] Breese,N.M.,George,A.C.,Pauers,L.E.,&Stucky,C.L.(2005).Peripheralinflammation selectively increases TRPV1 function inIB4-positive sensoryneurons from adult mouse.Pain,115(1-2),37-49.

[0075] Ji,R.R.,Samad,T.A.,Jin,S.X.,Schmoll,R.,&Woolf,C.J.(2002).p38 MAPKactivation by NGF in primary sensory neurons afterinflammation increasesTRPV1 levels and maintains heathyperalgesia.Neuron,36(1),57-68.

[0076] Akbar,A.,Yiangou,Y.,Facer,P.,Brydon,W.G.,Walters,J.R.,Anand,P.,&Ghosh,S.(2010).Expression of the TRPV1 receptordiffers in quiescentinflammatory bowel disease with or withoutabdominal pain.Gut,59(6),767-774.

[0077] Moran,M.M.,&Szallasi,A.(2018).Targeting nociceptivetransient receptorpotential channels to treat chronic pain:current stateof the field.Britishjournal of pharmacology,175(12),2185-2203.

[0078] Brandt,M.R.,Beyer,C.E.,&Stahl,S.M.(2012).TRPV1antagonists and chronicpain:beyond thermalperception.Pharmaceuticals,5(2),114-132.

[0079] Nilius,B.,&Szallasi,A.(2014).Transient receptor potentialchannels asdrug targets:from the science of basic research to the artofmedicine.Pharmacological reviews,66(3),676-814.

[0080] Carroll,A.R.,Copp,B.R.,Davis,R.A.,Keyzers,R.A.,&Prinsep,M.R.(2019).Marine natural products.Natural productreports,36(1),122-173.

[0081] Haefner,B.(2003).Drugs from the deep:marine natural productsas drugcandidates.Drug discovery today,8(12),536-544.

[0082] Jiménez,C.(2018).Marine natural products in medicinalchemistry.ACSmedicinal chemistry letters,9(10),959-961.

[0083] Molinski,T.F.,Dalisay,D.S.,Lievens,S.L.,&Saludes,J.P.(2009).Drugdevelopment from marine natural products.Nature reviewsDrug discovery,8(1),69-85.

[0084] Montaser,R.,&Luesch,H.(2011).Marine natural products:a newwave ofdrugs.Future medicinal chemistry,3(12),1475-1489.

[0085] Mehbub,M.F.,Lei,J.,Franco,C.,&Zhang,W.(2014).Marinesponge derivednatural products between 2001 and 2010:trends andopportunities for discoveryof bioactives.Marine drugs,12(8),4539-4577.

[0086] Khalifa,S.A.,Elias,N.,Farag,M.A.,Chen,L.,Saeed,A.,Hegazy,M.E.F.,...&El-Seedi,H.R.(2019).Marine natural products:A sourceof novel anticancerdrugs.Marine drugs,17(9),491.

[0087] Lin,Z.,Reilly,C.A.,Antemano,R.,Hughen,R.W.,Marett,L.,Concepcion,G.P.,...&Schmidt,E.W.(2011).Nobilamides A–H,long-acting transient receptorpotential vanilloid-1(TRPV1)antagonistsfrom mollusk-associatedbacteria.Journal of medicinalchemistry,54(11),3746-3755.

[0088] Wu,Y.,Yeh,F.L.,Mao,F.,&Chapman,E.R.(2009).Biophysicalcharacterization of styryl dye-membraneinteractions.Biophysical journal,97(1),101-109.

[0089] Nishikawa,S.(2011).Fluorescent AM1-43 and FM1-43 probes fordentalsensory nerves and cells:Their labeling mechanisms andapplications.JapaneseDental Science Review,47(2),150-156.

[0090] Meyers,J.R.,MacDonald,R.B.,Duggan,A.,Lenzi,D.,Standaert,D.G.,Corwin,J.T.,&Corey,D.P.(2003).Lighting up the senses:FM1-43 loading of sensory cellsthrough nonselective ionchannels.Journal of Neuroscience,23(10),4054-4065.

[0091] Gaffield,M.A.,&Betz,W.J.(2006).Imaging synaptic vesicleexocytosis andendocytosis with FM dyes.Nature protocols,1(6),2916-2921.

[0092] Kornreich,B.G.(2007).The patch clamp technique:principlesandtechnical considerations.Journal of Veterinary Cardiology,9(1),25-37.

[0093] Chen,C.C.,Cang,C.,Fenske,S.,Butz,E.,Chao,Y.K.,Biel,M.,...&Grimm,C.(2017).Patch-clamp technique to characterize ionchannels in enlargedindividual endolysosomes.Nature Protocols,12(8),1639-1658.

[0094] Phillips,E.,Reeve,A.,Bevan,S.,&McIntyre,P.(2004).Identification ofspecies-specific determinants of the action of theantagonist capsazepine andthe agonist PPAHV on TRPV1.Journal ofBiological Chemistry,279(17),17165-17172.

[0095] Abbas,M.A.(2020).Modulation of TRPV1 channel function bynaturalproducts in the treatment of pain.Chemico-biologicalinteractions,330,109178.

[0096] Kym,P.R.,Kort,M.E.,&Hutchins,C.W.(2009).Analgesicpotential of TRPV1antagonists.Biochemical pharmacology,78(3),211-216.

[0097] Cheng,A.,Liu,C.,Ye,W.,Huang,D.,She,W.,Liu,X.,...&Qian,P.Y.(2022).Selective C9orf72 G-Quadruplex-Binding Small MoleculesAmelioratePathological Signatures of ALS / FTD Models.Journal ofMedicinal Chemistry,65(19),12825-12837.

[0098] Cao,X.,Zhou,Z.,Tian,Y.,Liu,Z.,Cheng,K.O.,Chen,X.,...&Huang,P.(2021).Opposing roles of E3 ligases TRIM23 and TRIM21 inregulation of ion channelANO1 protein levels.Journal of BiologicalChemistry,296.

Claims

1. An analgesic comprising a hexacyclic xanthone in a carrier.

2. The analgesic according to claim 1, wherein the hexacyclic xanthone is selected from chrexanthomycin A (cA), chrexanthomycin B (cB), chrexanthomycin C (cC), chrexanthomycin F (cF) or any combination thereof.

3. The analgesic according to claim 1, further comprising capsaicin. The analgesic according to claim 1 , wherein the concentration of the hexacyclic xanthone is 0.75 μM to 20 μM.

5. The analgesic according to claim 1, wherein the carrier is a solution comprising a solvent. The analgesic according to claim 5 , wherein the solvent comprises dimethyl sulfoxide (DMSO).

7. A method for treating pain, comprising applying the analgesic according to claim 1 to a painful site of a patient.

8. The method of claim 7, wherein the hexacyclic xanthone is selected from chrexanthomycin A (cA), chrexanthomycin B (cB), chrexanthomycin C (cC), chrexanthomycin F (cF), or any combination thereof.

9. The method of claim 7, wherein the carrier is a solution comprising a solvent.

10. The method of claim 9, wherein the solvent comprises DMSO.

11. The method of claim 7, wherein the applying comprises injecting.

12. The method of claim 7, wherein said applying comprises topical administration.

Images