Margaric acid reduces PIEZO2-mediated pain

Abstract

The present invention describes a method for treating pain by administering a therapeutically effective amount of a pharmaceutical composition containing margaric acid to a subject in need of pain treatment. Also described are pharmaceutical compositions, such as topical and transdermal compositions, containing margaric acid and a pharmaceutically acceptable excipient. Further described are compositions for treating pain, containing margaric acid, eicosapentaenoic acid, and a pharmaceutically acceptable excipient.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application No. 62 / 976,014, filed February 13, 2020, which is incorporated herein by reference in its entirety.

[0002] The present disclosure relates to methods of treating pain using dietary fatty acids. [Background technology]

[0003] Skin is innervated by sensory neurons that express mechanosensitive ion channels, allowing for the detection and discrimination of painful and pleasant touch. The PIEZO2 mechanosensitive ion channel is highly expressed in sensory neurons and Merkel cells that mediate mild touch (i.e., brushing) and vibration. Importantly, studies have also shown that PIEZO2 contributes to tactile allodynia (i.e., when innocuous sensations become painful under inflammatory conditions).

[0004] Mechanosensitive ion channels are known to be regulated by the mechanical properties of membrane proteins, intracellular proteins, and extracellular proteins, as well as cytoskeletal elements. Several lines of evidence suggest that PIEZO2 channels interact with cellular components to mediate their physiological roles. For example, association of PIEZO2 with stomatin-like protein 3 and cholesterol increases sensitivity to mechanical stimuli, sensitization by inflammatory substances such as bradykinin, and regulation by phosphoinositide lipids. Interestingly, PIEZO2 requires cytoskeletal elements such as actin and tubulin for normal function. Collectively, these data suggest a complex interplay that works cooperatively to regulate PIEZO2 function.

[0005] The recent finding that Piezo2-deficient humans and knockout mice fail to develop sensitization and pain responses to innocuous touch after skin inflammation suggests that targeting this receptor may be a viable strategy for treating tactile allodynia.

[0006] What is needed are chemical compounds that interact with Piezo receptors and their use to mediate the function of Piezo receptors. Summary of the Invention

[0007] In one aspect, a method for treating pain comprises administering to a subject in need of pain treatment a pharmaceutical composition comprising a therapeutically effective amount of margaric acid.

[0008] In another embodiment, the pharmaceutical composition comprises margaric acid and a pharmaceutically acceptable excipient.

[0009] In yet another aspect, a composition for treating pain comprises margaric acid, eicosapentaenoic acid, and a pharmaceutically acceptable excipient. [Brief explanation of the drawings]

[0010] [Figure 1A-F]Margaric acid (MA) inhibits heterologously expressed mouse PIEZO2 currents in N2APiezo1- / - cells. Figure 1a shows representative whole-cell patch clamp recordings (at -60 mV) evoked by mechanical stimulation of control and MA-treated N2APiezo1- / - cells transfected with Piezo2 variant 2 (V2). Figure 1b shows the normalized current density evoked by maximum displacement of MA-treated N2APiezo1- / - cells transfected with Piezo2 V2. A Boltzmann function (Equation (2)) was fitted to the data (IC50 = 190.6 ± 14.7 SEM). Circles indicate the mean ± SD. n is indicated above the x-axis of panel c. The box plot in Figure 1c shows the mean, median, and 75th-25th percentiles of the displacement threshold required to elicit PIEZO2 V2 currents in control and N2APiezo1- / - cells. n is indicated above the x-axis. One-way ANOVA and Bonferroni test. Figure 1d shows representative PIEZO2 currents (at -60 mV) in control and MA (50 μM per day, 4 days)-treated N2APiezo1- / - cells transfected with Piezo2 V2. Figure 1e shows the PIEZO2 V2 current density evoked by maximal displacement in control and MA (50 μM per day, 18 h, 4 days)-treated N2APiezo1- / - cells. n is indicated above the x-axis. One-way ANOVA and Bonferroni test. The box plot in Figure 1f shows the mean, median, and 75th-25th percentiles of the displacement threshold required to elicit PIEZO2 V2 currents in control and MA (50 µM per day, 4 days)-treated N2APiezo1- / - cells transfected with PIEZO2 V2. n is indicated above the x-axis. Unpaired t-test. Asterisks indicate values ​​significantly different from the control (***p<0.001). ns indicates values ​​not significantly different from the control. [Figure 2A-G]This supports the finding that MA inhibits heterologously expressed mouse PIEZO2 currents in N2APiezo1- / - cells. Figure 2a shows the current densities evoked by maximum displacement in control and MA (1, 25, 50, 100, 200, 300, 400, and 600 μM)-treated N2APiezo1- / - cells transfected with a Piezo2 variant (V2). Bars represent mean values ​​+ SD. n is indicated above the x-axis. Unpaired t-test, unpaired t-test with Welch's correction, and Mann-Whitney test. Figure 2b shows the time constants of PIEZO2 V2 inactivation evoked by maximum displacement in control and MA (1, 25, 50, 100, 200, 300, 400, and 600 μM)-treated N2APiezo1- / - cells. Bars represent mean values ​​± SD. n is indicated above the x-axis. One-way ANOVA and Bonferroni test. Figure 2c shows the time constant of PIEZO2 V2 inactivation induced by maximum displacement in control and MA (50 μM / day, 4 days)-treated N2APiezo1- / - cells. Bars represent mean ± SD. n is indicated above the x-axis. Unpaired t-test. Figure 2d shows representative currents (at -60 mV) from control and MA (25 μM / day, 8 days)-treated N2APiezo1- / - cells transfected with Piezo2 V2. Figure 2e shows PIEZO2 V2 current densities induced by maximum displacement in control and MA (25 μM / day, 8 days)-treated N2APiezo1- / - cells. n is indicated above the x-axis. Unpaired t-test. Figure 2f shows the mean, median, and 75th to 25th percentiles of the displacement threshold required to elicit PIEZO2 V2 currents in control and MA (25 μM per day for 8 days)-treated N2APiezo1- / - cells transfected with Piezo2 V2. n is indicated above the x-axis. Unpaired t-test. Figure 2g shows the time constant of PIEZO2 V2 inactivation induced by maximal displacement in control and MA (25 μM per day for 8 days)-treated N2APiezo1- / - cells. Bars represent mean values ​​+ SD. n is indicated above the x-axis. Unpaired t-test. Asterisks indicate values ​​significantly different from the control (***p<0.001 and **p<0.01). ns indicates no significant difference from the control. [Figure 3A-I]Supporting this finding, MA inhibits heterologously expressed mouse PIEZO2 currents in N2APiezo1- / - cells. Figure 3a is a schematic diagram of the absence (black) and / or presence (white) of the Piezo2 variant (V) exon. Figure 3b shows representative whole-cell patch clamp recordings from control and MA (300 μM; 18 h)-treated N2APiezo1- / - cells transfected with Piezo2 V14. Figure 3c shows the current density evoked by the maximum displacement in control and MA (300 μM; 18 h)-treated N2APiezo1- / - cells transfected with Piezo2 V14. Bars represent mean ± SD. n is indicated above the x-axis. Mann-Whitney test. The box plot in Figure 3d shows the mean, median, and 75th to 25th percentiles of the displacement threshold required to evoke currents in control and MA (300 µM; 18 h)-treated N2APiezo1- / - cells transfected with Piezo2 V14. n is indicated above the x-axis. Unpaired t-test. Figure 3e shows the time constant of inactivation evoked by maximal displacement in control and MA (300 µM; 18 h)-treated N2APiezo1- / - cells transfected with Piezo2 V14. Bars are mean ± SD. n is indicated in m. Figure 3f shows representative whole-cell patch clamp recordings in control and MA (300 µM; 18 h)-treated N2APiezo1- / - cells transfected with Piezo2 V16. Figure 3g shows the current density evoked by maximum displacement in control and MA (300 µM; 18 h)-treated N2APiezo1- / - cells transfected with Piezo2 V16. Bars represent mean ± SD. n is indicated above the x-axis. Unpaired t-test with Welch's correction. Box plots in Figure 3h show the mean, median, and 75th-25th percentiles of the displacement threshold required to evoke currents in control and MA (300 µM; 18 h)-treated N2APiezo1- / - cells transfected with Piezo2 V16. n is indicated above the x-axis. Mann-Whitney test.Figure 3i shows the time constant of inactivation induced by maximum displacement in control and MA (300 μM; 18 h)-treated N2APiezo1- / - cells transfected with Piezo2 V1. Bars represent mean ± SD. n is indicated above the x-axis. Unpaired t-test. Asterisks indicate values ​​significantly different from the control (**p<0.01 and *p<0.05). ns indicates no significant difference from the control. [Figure 4A-F]Latrunculin A enhances PIEZO2 inhibition by MA. Figure 4a shows the normalized current density induced by maximum displacement in MA (1, 10, 25, 50, 100, and 300 μM; 18 h)-supplemented N2A cells (expressing endogenous Piezo1) and N2APiezo1- / - cells transfected with Piezo2 (1, 25, 50, 100, 200, 300, 400, and 600 pM; 18 h) supplemented with MA. A Boltzmann function (Equation (2)) was fitted to the data (PIEZO1 IC50 = 28.3 ± 3.48 EM; PIEZO2 IC50 = 190.6 ± 14.7 SEM). Symbols represent mean ± SD. Figure 4b shows the normalized current density evoked by the maximum displacement of MA (1, 25, 50, 100, 200, 300, 400, and 600 μM; 18 h)-supplemented N2A Piezo1 cells transfected with Piezo2, with or without latrunculin A treatment. A Boltzmann function (Equation (2)) was fitted to the data (MA IC50 = 190.6 ± 14.7 SEM; MA + LatA IC50 = 75.4 ± 13.3 SEM). Circles represent mean ± SD. Figure 4c shows the normalized current density evoked by the maximum displacement of MA (1, 10, 25, 50, 100, and 300 μM; 18 h)-supplemented N2A cells (expressing endogenous Piezo1). A Boltzmann function (Equation (2)) was fitted to the data (MA IC50 = 28.3 ± 3.4 SEM; MA + LatA IC50 = 25.6 ± 8.4 SEM). Triangles represent mean ± SD. Figure 4d shows a ribbon representation of the PIEZO2 monomer (PDB ID: 6KG7; gray). Residues exchanged with those in PIEZO1 are highlighted. Figure 4e shows the inhibition of N2A and N2APiezo1- / - cells transfected with Piezo2 and Piezo2-Piezo1 beam chimeras by MA (100 μM) supplementation. n is indicated above the x-axis. Unpaired t-test and Mann-Whitney test. Figure 4f shows the normalized current density evoked by the maximum displacement of MA (100 μM; 18 h)-supplemented N2A cells (expressing endogenous Piezo1) and N2APiezo1− / − cells transfected with Piezo2 and Piezo2-Piezo1 beam chimeras, with and without latrunculin treatment.n is indicated above the x-axis. Unpaired t-test (for PIEZO1) and Mann-Whitney test (for PIEZO2 and PIEZO2-Piezo1 beam chimeras). Asterisks indicate values ​​significantly different from the control (**p<0.01 and ***p<0.001). ns indicates values ​​not significantly different from the control. [Figure 5A-D] We also show that latrunculin A potentiates PIEZO2 inhibition by MA. Figure 5a shows representative whole-cell patch-clamp recordings (at -60 mV) evoked by mechanical stimulation of control and MA (1, 25, 50, 100, 200, 300, 400, and 600 μM)-supplemented N2APiezo1- / - cells transfected with Piezo2 V2, with or without latrunculin A treatment. Figure 5b shows the current density evoked by maximum displacement of control and MA (1, 25, 50, 100, 200, 300, 400, and 600 μM)-supplemented N2APiezo1- / - cells transfected with Piezo2 V2, with or without latrunculin A treatment. n is indicated above the x-axis. Unpaired t-test, Mann-Whitney test, and unpaired t-test with Welch's correction. Figure 5c shows representative whole-cell patch-clamp recordings (at -60 mV) evoked by mechanical stimulation of control and MA (1, 10, 25, 50, 100, and 300 μM)-supplemented N2A cells (expressing endogenous Piezo1) with or without latrunculin A treatment. Figure 5d shows the current densities evoked by maximum displacement of control and MA (1, 10, 25, 50, 100, and 300 μM)-supplemented N2A cells (expressing endogenous Piezo1) with or without latrunculin A treatment. n is indicated above the x-axis. Unpaired t-test with Welch's correction, unpaired t-test, Mann-Whitney test. Asterisks indicate values ​​significantly different from the control (***p<0.001, **p<0.01, and *p<0.05). ns indicates no significant difference from the control. [Figure 6A-E]We also demonstrate that latrunculin A potentiates PIEZO2 inhibition by MA. Figure 6a shows representative whole-cell patch clamp recordings (at -60 mV) evoked by mechanical stimulation in control and MA (100 μM; 18 h)-treated N2APiezo1- / - cells transfected with a Piezo2-Piezo1 beam chimera, with or without latrunculin A (1 μM, 1 h). Figure 6b shows the current-voltage correlation of PIEZO2-PIEZO1 chimera mechano-dependent currents determined by whole-cell patch clamp experiments. Circles represent mean ± SD. n = 6. The box plot in Figure 6c shows the mean, median, and 75th-25th percentiles of the displacement threshold required to evoke currents in control and MA (100 μM; 18 h)-treated N2APiezo1- / - cells transfected with a Piezo2-Piezo1 beam chimera, with or without latrunculin A (1 μM, 1 h). n is indicated on the x-axis. Mann-Whitney test (control vs. MA and control vs. MA + LatA) and unpaired t-test (MA vs. MA + LatA). Figure 6d shows the time constants of inactivation induced by maximum displacement of transfected Piezo2 V2 and Piezo2-Piezo1 beam chimeras in N2APiezo1- / - cells. Bars represent mean ± SD. n is indicated above the x-axis. Mann-Whitney test. Figure 6e shows the current densities induced by maximum displacement of control, MA (100 μM, 18 h)-treated N2APiezo1- / - cells transfected with Piezo2-Piezo1 beam chimeras in the presence or absence of latrunculin A (1 μM, 1 h). Bars represent mean ± SD. n is indicated above the x-axis. Mann-Whitney test (control vs. MA + LatA and MA vs. MA + LatA) and unpaired t-test (control vs. MA). Asterisks indicate values ​​significantly different from the control (**p<0.01 and ***p<0.001). ns indicates not significantly different from the control. [Figure 7A-F]MA is shown to decrease mechanically activated currents in MCC13 and mouse DRG neurons. Figure 7a shows representative whole-cell patch-clamp recordings (-60 mV) induced by mechanical stimulation of fast (τ < 10 ms), intermediate (10 < t < 30 ms), and slow (t > 30 ms) inactivation currents in control (top) and MA (300 μM)-treated (bottom) MCC13. Figure 7b shows the current density induced by the maximum displacement in control and MA (300 μM)-treated MCC13 cells. Bars are mean ± SD. n is shown above the x-axis. Unpaired t-test. The box plot in Figure 7c shows the mean, median, and 75 - 25 percentiles of the displacement thresholds required to induce mechanical currents in control and MA (300 μM)-treated MCC13 cells. n is shown above the x-axis. Unpaired t-test with Welch's correction. Figure 7d shows representative whole-cell patch-clamp recordings (-60 mV) induced by mechanical stimulation of fast (τ < 10 ms), intermediate (10 < t < 30 ms), and slow (τ > 30 ms) inactivation currents in control (top) and MA (300 μM)-treated (bottom) DRG neurons. Figure 7e shows the current density induced by the maximum displacement in control and MA (300 μM)-treated DRG neurons. Bars are mean ± SD. n is shown above the x-axis. Unpaired t-test. Figure 7f shows the mean, median, and 75 - 25 percentiles of the displacement thresholds required to induce mechanical currents in control and MA (300 μM)-treated DRG neurons. n is shown above the x-axis. Unpaired t-test with Welch's correction. Asterisks indicate values significantly different from control (***p < 0.001, **p < 0.01, and *p < 0.05). n.s. indicates values not significantly different from control. [Figure 8A-D]We demonstrate that MA reduces action potentials evoked by mechanical stimulation in mouse DRG neurons. Figure 8a shows representative current-clamp recordings of membrane potential changes evoked by mechanical stimulation (up to 10 and 15 μm indentations, respectively) in control and MA (300 μM)-treated DRG neurons. Figure 8b shows the membrane potential peak versus mechanical indentation for independent control (n = 10) and MA-treated (n = 7) DRG neurons. The upper panel shows box plots depicting the mean, median, and 75th to 25th percentiles of the displacement threshold required to evoke action potentials in these neurons. Unpaired t-test. Figure 8c shows representative current-clamp recordings of membrane potential changes evoked by a 1 Hz mechanical stimulus train in control and MA-treated DRG neurons. The inset shows the gradual decrease in membrane potential with increasing pulse number. Figure 8d shows raster plots showing the number of evoked action potentials per sweep versus mechanical indentation (left) and action potentials evoked by a 12 μm indentation (right) in control (n = 6) and MA-treated (n = 6) DRG neurons. Bars indicate the stimulus that evoked the action potential. Columns and rows represent cells and sweeps, respectively. Asterisks indicate values ​​significantly different from the control (***p < 0.001). [Figure 9A-G]This shows that MA does not alter the electrical excitability of mouse DRG neurons. Figure 9a shows representative whole-cell patch-clamp recordings of control and MA (300 μM)-treated DRG neurons subjected to stepwise depolarization from a holding potential of -80 mV. Figure 9b shows the normalized inward current density evoked by stepwise depolarization from a holding potential of -80 mV in control (n = 7) and MA (n = 7; 300 μM)-treated DRG neurons. Circles represent mean ± SD. Figure 9c shows the normalized outward current density evoked by stepwise depolarization from a holding potential of -80 mV in control (n = 7) and MA (n = 7; 300 μM)-treated DRG neurons. Circles represent mean ± SD. Figure 9d shows membrane potential values ​​recorded immediately after achieving the whole-cell configuration from control and MA (300 μM)-treated DRG neurons. n is indicated above the x-axis. Mann-Whitney test. Figure 9e shows representative current-clamp recordings of membrane potential changes evoked by current injection in control and MA (300 μM)-treated DRG neurons. Figure 9f shows action potential amplitudes evoked by current injection from control and MA (300 μM)-treated DRG neurons. n is indicated above the x-axis. Unpaired t-test with Welch's correction. The box plot in Figure 9g shows the mean, median, and 75th-25th percentiles of the minimum injected currents that evoked action potentials from control and MA (300 μM)-treated DRG neurons. n is indicated above the x-axis. Mann-Whitney test. ns indicates values ​​not significantly different from the control. [Figure 10A-D]MA is shown to restore normal mechanical responses in sensitized mouse DRG neurons. Figure 10a shows representative whole-cell patch-clamp traces of mechanically activated currents after perfusion of control and MA (300 μM; 18 h)-treated DRG neurons with bath solution (60 s) and then bath solution containing bradykinin (BK; 1 μM). Figure 10b shows the current density induced by 10-μm displacement in control and MA (300 μM; 18 h)-treated DRG neurons perfused successively with bath solution (60 s) and then bath solution containing bradykinin (BK; 300 s, 1 μM). Bars are mean ± SD. Data samples are paired. n is shown above the x-axis. Paired t-test, unpaired t-test, Mann-Whitney test, and Wilcoxon signed-rank test. Figure 10c shows representative whole-cell patch-clamp recordings (at -60 mV) of mechanically evoked rapid (t < 10 ms), intermediate (10 < t < 30 ms), and slow (τ > 30 ms) inactivation currents in control, BK (1 μM, 18 h), and BK+MA (1 μM and 300 μM, 18 h each)-treated DRG neurons. Figure 10d shows the current density of mechanically evoked (at -60 mV) mechanically activated currents induced by the maximum displacement of rapid (τ < 10 ms), intermediate (10 < t < 30 ms), and slow (t > 30 ms) inactivation currents in control, BK (1 μM, 18 h), and BK+MA (1 μM and 300 μM, both 18 h)-treated DRG neurons. Bars are mean ± SD. Unpaired t-test. n is shown above the x-axis. Asterisks indicate values significantly different from control (***p < 0.001, **p < 0.01, and *p < 0.05). n.s. indicates no significant difference from control. [Figure 11] Also, MA is shown to restore normal mechanical responses in sensitized mouse DRG neurons. Figure 11a shows the current (fold change) in control and MA (300 μM; 18 h)-treated DRG neurons perfused successively with bath solution for 60 s and then bath solution containing bradykinin (BK; 1 μM) for 300 s. n is shown below the bars. Unpaired t-test. n.s. indicates no significant difference from control. [Figure 12A-D] We demonstrate that administration of MA prevents tactile allodynia in mice. Figure 12a is a schematic diagram illustrating the repetitive injury paradigm. Mice were tested for baseline withdrawal thresholds to punctate mechanical (von Frey) and radiant heat (Hargreaves) stimuli. Subsequently, one hind paw was injected daily for 1 week with a small amount of saline or saline containing MA (5 mM). Mechanical and thermal withdrawal thresholds were then measured again with or without acute inflammation induced by application of mustard oil (AITC). Figure 10b shows that repeated saline injections for 7 days induce an exaggerated response to mechanical stimuli. Under baseline conditions, when the force from the von Frey filament reached 0.2–0.4 g, mice began to withdraw their paw in the majority of trials (out of 10) (open circles). In contrast, after repetitive injury, mice withdrew their paw much more frequently, even to a very light filament (0.02 g) (filled circles). A significant hyperresponsiveness is observed for all filaments above 0.02 g until a maximal response is elicited (a 10 / 10 response for 0.6–1 g). These allodynia-like responses are not observed when mice are treated with MA (compare open circles with filled circles). **p>0.01; n = 6 mice per treatment group (n = 3 males and 3 females). Figure 12c shows that repeated saline injections decrease the latency to radiant heat focused on the injured and contralateral paws. The inclusion of MA in the injection solution has no effect on this hyperresponsiveness to heat (n = 6 mice per treatment group, n = 3 males and 3 females). Figure 12d shows that acute topical administration of mustard oil (AITC) does not increase the hyperresponsiveness to punctate stimuli one week after MA treatment (compare circles with squares). In contrast, saline-injected mice treated with AITC responded much more frequently to the low force filament ((squares) n = 6 mice per treatment group, n = 3 males and 3 females). [Figures 13A-H]We demonstrate that MA reduces mechanically activated currents in human iPSC-derived neurons. Figure 13a shows a photomicrograph of a human iPSC-derived neuron in the whole-cell patch clamp configuration prepared for mechanical stimulation. Figure 13b shows representative whole-cell patch clamp traces of mechanically activated currents in control and MA-treated iPSC-derived neurons (300 and 600 μM for 18 h and 50 μM daily for 5 days). Figure 13c shows the current density evoked by maximum displacement in control and MA-treated iPSC-derived neurons (300 and 600 μM for 18 h and 50 μM daily for 5 days). Bars represent mean ± SD. n is indicated above the x-axis. Unpaired t-test. The box plot in Figure 13d shows the mean, median, and 75th-25th percentiles of the displacement threshold required to evoke currents in control and MA-treated iPSC-derived neurons (300 and 600 μM for 18 h and 50 μM daily for 5 days). n is indicated above the x-axis. Figure 13e shows the time constant of PIEZO2 inactivation induced by maximum displacement in control and MA (300 and 600 μM for 18 hours, and 50 μM daily for 5 days)-treated iPSC-derived neurons. Bars represent mean ± SD. n is indicated above the x-axis. Unpaired t-test. Figure 13f shows representative whole-cell patch clamp recordings of control and MA (300 μM)-treated iPSC-derived neurons subjected to stepwise depolarization from a membrane potential of -80 mV. Figure 13g shows the normalized inward current density induced by stepwise depolarization from -80 mV in control (n = 8) and MA (n = 8; 300 μM)-treated DRG neurons. Circles represent mean ± SD. Figure 13h shows the normalized outward current density induced by stepwise depolarization from -80 mV in control (n = 8) and MA (n = 8; 300 μM)-treated DRG neurons. Circles represent mean values ​​± SD. Asterisks indicate values ​​significantly different from the control (***p<0.001 and **p<0.01). ns indicates values ​​not significantly different from the control. [Figure 14A-D]We also demonstrate that MA reduces mechanically activated currents in human iPSC-derived neurons. Figure 14a shows representative whole-cell patch clamp recordings (at -60 mV) evoked by mechanical stimulation in control and MA (300 μM)-supplemented N2APiezo1- / - cells transfected with human Piezo2. Figure 14b shows the current density evoked by maximum displacement in control and MA (300 μM; 18 h)-treated N2APiezo1- / - cells transfected with human Piezo2. Bars represent mean ± SD. n is indicated above the x-axis. Unpaired t-test. Figure 14c shows the mean, median, and 75th-25th percentiles of the displacement threshold required to evoke currents in MA (300 μM; 18 h)-treated N2APiezo1- / - cells transfected with human Piezo2. n is indicated above the x-axis. Unpaired t-test. Figure 14d shows the time constant of inactivation induced by maximum displacement in control and MA (300 μM; 18 h)-treated N2APiezo1- / - cells transfected with human Piezo2. Bars represent mean ± SD. n is indicated above the x-axis. Unpaired t-test. Asterisks indicate values ​​significantly different from the control (***p<0.001). ns indicates no significant difference from the control. [Figures 15A-G]We further demonstrate that MA reduces mechanically activated currents in rat DRG neurons. Figure 15a is a photomicrograph showing a rat DRG neuron in the whole-cell patch clamp configuration prepared for mechanical stimulation. Figure 15b shows representative whole-cell patch clamp traces of mechanically activated currents in control and MA (300 μM, 18 h)-treated rat DRG neurons. Figure 15c shows the current density evoked by maximum displacement in control and MA (300, 18 h)-treated rat DRG neurons. Bars represent mean ± SD. n is indicated above the x-axis. Unpaired t-test. The box plot in Figure 15d shows the mean, median, and 75th to 25th percentiles of the displacement threshold required to evoke currents in control and MA (300 μM, 18 h)-treated rat DRG neurons. n is indicated above the x-axis. Unpaired t-test. Figure 15e shows representative current-clamp recordings of membrane potential changes evoked by mechanical stimulation (maximum indentation of 9 and 13 μm, respectively) in control and MA (300 μM)-treated rat DRG neurons. Figure 15f shows the membrane potential peak versus mechanical indentation for independent control (n = 11) and MA-treated (n = 7) DRG neurons. The upper panel shows box plots indicating the mean, median, and 75th to 25th percentiles of the displacement threshold required to evoke action potentials in these neurons. Mann-Whitney test. Figure 15g shows resting membrane potential values ​​recorded immediately after achieving whole-cell current-clamp configuration from control and MA (300 μM)-treated rat DRG neurons. n is indicated above the x-axis. Mann-Whitney test. Asterisks indicate values ​​significantly different from the control (**p < 0.01). ns indicates no significant difference from the control. [Figures 16A-G]We further demonstrate that MA reduces mechanically activated currents in rat DRG neurons. Figure 16a shows the current-voltage relationship of mechanical currents in rat DRG neurons determined by whole-cell patch clamp experiments. Circles represent mean ± SD. n = 3. Figure 16b shows the time constants of inactivation of currents evoked by maximum displacement in control and MA (300 μM, 18 h)-treated rat DRG neurons. Bars represent mean ± SD. n is indicated above the x-axis. Unpaired t-test. Figure 16c shows representative current-clamp recordings of membrane potential changes evoked by a train of mechanical pulses in control and MA-treated rat DRG neurons. Figure 16d shows the number of evoked action potentials per sweep versus mechanical indentation in control (n = 6) and MA-treated (n = 6) rat DRG neurons. Figure 16e shows action potential amplitudes measured from the resting potential to the peak membrane potential in control and MA (300 μM at 18 h)-treated rat DRG neurons. Bars represent mean ± SD. n is indicated above the x-axis. Unpaired t-test. Figure 16f shows the normalized outward current density evoked by step depolarization from a holding potential of -80 mV in control (n = 8) and MA (n = 6; 300 μM)-treated rat DRG neurons. Circles represent mean ± SD. Figure 16g shows the normalized inward current density evoked by step depolarization from a holding potential of -80 mV in control (n = 8) and MA (n = 6; 300 μM)-treated DRG neurons. Circles represent mean ± SD. Asterisks indicate values ​​significantly different from the control (**p < 0.01). ns indicates not significantly different from the control. [Figures 17A-D]We demonstrate that an ω3-enriched diet decreases the time constant of PIEZO2 inactivation in mouse DRG neurons. Figure 17a shows representative PIEZO2 currents (at -60 mV) evoked by mechanical stimulation of DRG neurons isolated from WT mice fed a control or ω3-enriched diet. Figure 17b shows the PIEZO2 time constant of inactivation evoked by maximum displacement of DRG neurons isolated from WT mice fed a control or ω3-enriched diet. Bars represent mean ± SD. n is indicated above the x-axis. Unpaired t-test. Figure 17c shows the current density evoked by maximum displacement of DRG neurons isolated from control and ω3-enriched diets. Bars represent mean ± SD. n is indicated above the x-axis. Unpaired t-test. Figure 17d shows the box plots showing the mean, median, and 75th to 25th percentiles of the displacement threshold required to evoke mechanical currents in DRG neurons isolated from control and ω3-enriched diets. n is indicated above the x-axis. Unpaired t-test with Welch's correction. Asterisks indicate values ​​that are significantly different from the control (*p<0.05). ns indicates values ​​that are not significantly different from the control. [Figures 18A-D]We demonstrate that eicosapentaenoic acid (EPA) supplementation decreases the time constant of PIEZO2 inactivation in rat DRG neurons. Figure 18a shows representative whole-cell patch clamp traces of mechanically activated currents in control and EPA (200 μM, 18 h)-treated rat DRG neurons. Figure 18b shows the time constant of PIEZO2 inactivation evoked by maximal displacement in control and EPA (200 μM, 18 h)-treated rat DRG neurons. Bars represent mean ± SD. n is indicated above the x-axis. Unpaired t-test. Figure 18c shows the current density evoked by maximal displacement in control and EPA (200 μM, 18 h)-treated rat DRG neurons. Bars represent mean ± SD. n is indicated above the x-axis. Unpaired t-test. The box plot in Figure 18d shows the mean, median, and 75th to 25th percentiles of the displacement threshold required to elicit mechanical activation in control and EPA (200 μM, 18 h)-treated rat DRG neurons. n is indicated above the x-axis. Unpaired t-test with Welch's correction. Asterisks indicate values ​​significantly different from the control (**p<0.01). ns indicates values ​​not significantly different from the control. [Figure 19A-C] Figure 19a shows that EPA supplementation reverses the phenotype of PIEZO2 arthrogryposis mutations. Figure 19a shows a ribbon representation of mouse PIEZO2 monomers, highlighting the mutations that cause arthrogryposis in humans. Figure 19b shows representative normalized macroscopic currents (at -60 mV) evoked by maximal displacement in N2A cells transfected with PIEZO2 arthrogryposis mutants S2691R and E2727 deletion, with or without EPA supplementation (left and right, respectively). Figure 19c shows the time constants of PIEZO2 inactivation evoked by maximal displacement in arthrogryposis mutants S2691R and E2727 deletion, with or without EPA supplementation. Bars represent mean ± SD. Unpaired t-test with Welch's correction. Asterisks indicate values ​​significantly different from the control (**p<0.01 and ***p<0.001). ns indicates no significant difference from the control. [Figure 20A-I]EPA supplementation reduces the inactivation time constant of heterologously expressed mouse PIEZO2 currents in N2APiezo1- / - cells. Figure 20a shows representative whole-cell patch clamp recordings from control and EPA (200 μM; 18 h)-treated N2APiezo1- / - cells transfected with PIEZO V2. Figure 20b shows the time constant of inactivation induced by maximum displacement in control and EPA (200 μM; 18 h)-treated N2APiezo1- / - cells transfected with PIEZO V2. Bars represent mean ± SD. n is indicated above the x-axis. Unpaired t-test. Box plots in Figure 20c show the mean, median, and 75th-25th percentiles of the displacement threshold required to evoke currents in control and EPA (200 μM; 18 h)-treated N2APiezo1- / - cells transfected with PIEZO V2. n is indicated above the x-axis. Unpaired t-test. Figure 20d shows the current density evoked by maximum displacement in control and EPA (200 μM; 18 h)-treated N2APiezo1− / − cells transfected with PIEZO V2. Bars represent mean ± SD. n is indicated above the x-axis. Mann-Whitney test. Figure 20e shows representative whole-cell patch clamp recordings from control and EPA (200 μM; 18 h)-treated N2APiezo1− / − cells transfected with PIEZO V16. Figure 20f shows the time constant of inactivation evoked by maximum displacement in control and EPA (200 μM; 18 h)-treated N2APiezo1− / − cells transfected with PIEZO V16. Bars represent mean ± SD. n is indicated above the x-axis. Unpaired t-test. The box plot in Figure 20g shows the mean, median, and 75th-25th percentiles of the displacement threshold required to elicit currents in control and EPA (200 µM; 18 h)-treated N2APiezo1- / - cells transfected with PIEZO V16. n is indicated above the x-axis. Unpaired t-test. Figure 20h shows the current density elicited by maximal displacement in control and EPA (200 µM; 18 h)-treated N2APiezo1- / - cells transfected with PIEZO V16.Bars represent mean ± SD. n is indicated above the x-axis. Mann-Whitney test. Figure 20i shows representative whole-cell patch clamp recordings of control and EPA (200 μM; 18 h)-treated N2APiezo1- / - cells transfected with PIEZO V14. Figure 20j shows the time constant of maximum displacement-induced inactivation in control and EPA (200 μM; 18 h)-treated N2APiezo1- / - cells transfected with PIEZO V14. Bars represent mean ± SD. n is indicated above the x-axis. Unpaired t-test. Box plots in Figure 20k show the mean, median, and 75th-25th percentiles of the displacement threshold required to evoke currents in control and EPA (200 μM; 18 h)-treated N2APiezo1- / - cells transfected with PIEZO V14. n is indicated above the x-axis. Unpaired t-test. Figure 20l: Current density evoked by maximum displacement in control and EPA (200 μM; 18 h)-treated N2APiezo1- / - cells transfected with PIEZO V14. Bars represent mean ± SD. n is indicated above the x-axis. Mann-Whitney test. Asterisks indicate values ​​significantly different from the control (***p<0.001). ns indicates not significantly different from the control. [Figure 21A-D]We show that the combination of margaric acid and eicosapentaenoic acid (MA and EPA, respectively) reduces PIEZO2 currents and enhances inactivation in cultured mouse DRG neurons. Figure 21a shows representative whole-cell patch clamp recordings (at -60 mV) of PIEZO2 currents evoked by mechanical stimulation in control (left) and MA (300 μM) + EPA (200 μM)-treated (right) DRG neurons. Figure 21b shows the PIEZO2 current density evoked by maximum displacement in control and MA (300 μM) + EPA (200 μM)-treated DRG neurons. Bars represent mean ± SD. Figure 21c shows the displacement threshold required to evoke PIEZO2 currents in control and MA (300 μM) + EPA (200 μM)-treated DRG neurons. Box plots show the mean (square), median (bisect), box boundaries (75th-25th percentiles), outlier range (whiskers) by a factor of 1.5, and minimum and maximum data points. Figure 21d shows the time constants of PIEZO2 inactivation induced by maximal displacement in control and MA (300 µM) + EPA (200 µM)-treated DRG neurons. Bars represent mean ± SD. n is indicated above the v axis. p values ​​are indicated above the bars and boxes.

[0011] The above-discussed features and other features will be appreciated and understood by those skilled in the art from the following detailed description, drawings, and appended claims. DETAILED DESCRIPTION OF THE INVENTION

[0012] We previously explored how fatty acids affect mechanical signaling. Margaric acid (MA; heptadecanoic acid; C17:0), an esterified saturated fatty acid, inhibits PIEZO1 channels by increasing membrane structural order and stiffness when enriched in the plasma membrane, thereby increasing the mechanical threshold required to activate the channel. Given the overall similarity of PIEZO1 and PIEZO2, we reasoned that MA might reduce PIEZO2 function and thus have therapeutic potential. However, unlike PIEZO1, which can be activated solely by changes in membrane tension, PIEZO2 requires an intact cytoskeleton for normal function and can only be gated in cell-associated or whole-cell patch-camp configurations. Therefore, it remains to be determined whether MA can efficiently regulate and reduce PIEZO2 activity.

[0013] As shown herein, using electrophysiological and behavioral approaches, we identified that MA reduces PIEZO2 function under normal and inflammatory conditions. We found that MA potently reduces PIEZO2 currents in a wide range of cell types, from mice and rats to humans, by increasing the mechanical stimulus required to activate the channel. Notably, MA supplementation in combination with latrunculin A treatment (i.e., a toxin that disrupts actin polymerization) revealed that PIEZO2 mechanosensitivity depends on both plasma membrane and cytoskeletal elements. Results from PIEZO2-PIEZO1 chimeras indicate that PIEZO2 beams (large intracellular domains that run parallel to the membrane and are thought to be important for force sensing) attenuate the membrane's effect on Piezo2 gating. Importantly, we identified that MA efficiently reduces action potential firing evoked by mechanical stimulation, but not by current injection, in dorsal root ganglion (DRG) neurons, suggesting that MA may blunt touch responses in vivo. Furthermore, MA reduces PIEZO2 currents enhanced by the pain-inducing agent bradykinin, indicating that it may be particularly useful for reducing the enhanced touch response during inflammation. We found that MA selectively reduces the enhanced touch response seen during repetitive injury in a mouse model without affecting the heat response.

[0014] In one aspect, a method for treating pain comprises administering to a subject in need of pain treatment a pharmaceutical composition comprising a therapeutically effective amount of margaric acid.

[0015] Exemplary types of pain to be treated include inflammatory pain, pain due to nerve injury, neuropathic pain, chronic pain, intractable cancer pain, complex regional pain syndrome, surgical or postoperative pain, dental pain, pain due to skin injury, back pain, headache, migraine, allodynia, and hyperalgesia. In certain embodiments, the pain is chronic. In other embodiments, the pain is acute. The pain can be mild or severe.

[0016] Exemplary pain indications include the treatment or prevention of surgical or postoperative pain from various surgical procedures (including amputation and cardiac surgery), dental pain / tooth extraction, cancer pain, muscle pain, breast pain, pain from skin injury, lower back pain, headaches of various etiologies, including migraine, menstrual pain, tactile allodynia, and hyperalgesia.Pain can be somatic (either nociceptive or neuropathic), acute and / or chronic.Peripheral neuropathies that can be treated with margaric acid include mononeuropathy, multiple mononeuropathy, and polyneuropathy (including axonal neuropathy and demyelinating neuropathy).It encompasses both sensory and motor neuropathy. Neuropathy or neuropathic pain can be associated with several peripheral neuropathies of various etiologies, including trauma-induced neuropathies (those caused by physical injury (e.g., blunt trauma, abrasion, or burns) or medical conditions, physical injury to the brain, physical injury to the spinal cord, or stroke associated with brain injury); neurological disorders associated with neurodegeneration; and post-operative neuropathies and neuropathic pain (including, e.g., from shingles, diabetes, etc.), infectious and viral neuropathies (e.g., leprosy, Lyme disease, herpes viruses, etc.). (and more specifically, including that caused by varicella zoster virus (which can result in postherpetic neuralgia)), human immunodeficiency virus (HIV, which can result in HIV neuropathy), or papillomavirus, or any other pathogen-induced nerve damage); toxin-induced neuropathies (including, but not limited to, alcoholism, vitamin B6 poisoning, hexacarbon poisoning, amiodarone, chloramphenicol, disulfuram, isoniazid, gold, lithium, metronidazole, misonidazole, nitrofurantoin);Drug-induced neuropathies (therapeutic agent-induced neuropathies, especially a) chemotherapy-induced neuropathies (caused by anticancer drugs such as taxol, taxotere, cisplatin, nocodazole, vincristine, vindesine, and vinblastine), and b) antiviral neuropathies (caused by antivirals, e.g., ddI, DDC, d4T, foscarnet, dapsone, metronidazole, and isoniazid)); vitamin deficiency-induced neuropathies (including those caused by vitamin B12 deficiency, vitamin B6 deficiency, and vitamin E deficiency); hereditary neuropathies (including, but not limited to, Friedreich's ataxia, familial amyloidotic polyneuropathy, Tangier disease, and Fabry disease); diabetic neuropathy Neuropathies resulting from metabolic disorders such as thyroid and renal failure and hypothyroidism; neuropathies secondary to tumor invasion, autoimmune neuropathies (including those resulting from Guillain-Barré syndrome), chronic inflammatory demyelinating polyneuropathy, monoclonal gammopathy of undetermined significance and polyneuropathy, and multiple sclerosis; other neuropathies and neuropathic pain (e.g., inflammation-induced nerve injury, neurodegeneration, post-traumatic neuralgia, central neuropathic pain syndromes (e.g., phantom limb pain, pain, complex regional pain syndrome (including, but not limited to, reflex sympathetic dystrophy, causalgia)), neoplasm-associated pain, vasculitic / vasculopathic neuropathies; and sciatica, as well as idiopathic neuropathies.

[0017] Neuropathic pain may manifest as allodynia, hyperalgesic pain, or phantom pain. In another embodiment, the neuropathy may instead result in a loss of pain sensitivity.

[0018] There are three types of allodynia: Tactile allodynia (also called static allodynia) is pain caused by touch, such as when clothing touches the skin or when someone lightly touches the arm. Mechanical allodynia (also called dynamic allodynia) is pain caused by movement across the skin, such as when drying the skin with a towel or rubbing it with a bed sheet. Thermal allodynia is pain caused by heat or cold that does not damage tissue. People with allodynia experience pain in response to stimuli that are generally considered to be innocuous.

[0019] Fibromyalgia is a disease characterized by chronic, excruciating generalized pain as its core symptom, accompanied by various comorbid symptoms such as insomnia, general fatigue, and depression. Fibromyalgia is often accompanied by tactile allodynia. Fibromyalgia may also be accompanied by mechanical and thermal allodynia.

[0020] Additional medical conditions associated with allodynia include chronic inflammation, migraine, trigeminal neuralgia, postherpetic neuralgia, peripheral neuropathy, diabetic neuropathic pain, chronic fatigue syndrome, and complex regional pain syndrome.

[0021] Inflammatory pain includes inflammatory joint pain, inflammatory musculoskeletal pain, pain due to injury, arthritis pain, and complex regional pain syndrome.

[0022] The agent may be administered via routes commonly used to administer pharmaceuticals for treating pain, including, but not limited to, oral, parenteral (including subcutaneous, intradermal, intramuscular, intravenous, intraarticular, and intramedullary), transmucosal (including intranasal), transdermal, and topical (including transdermal, buccal, sublingual, and intraocular) routes. For example, intravenous delivery may be via bolus injection or infusion, which may be continuous for less than a minute to several hours. In certain embodiments, a course of treatment involves administration by a combination of routes.

[0023] Pharmaceutical compositions can contain various pharmaceutically acceptable additives, including, but not limited to, carriers, excipients, binders, stabilizers, antimicrobial agents, antioxidants, diluents, and / or supports.

[0024] As used herein, topical administration refers to non-systemic administration. This includes applying a compound disclosed herein externally to the epidermis or instilling such a compound into the ear, eye, or nose so that the compound does not significantly enter the bloodstream. In contrast, systemic administration refers to oral, intravenous, intraperitoneal, and intramuscular administration.

[0025] In one embodiment, the topical or transdermal dose of margaric acid provides 0.1-20 mg / kg of margaric acid.

[0026] Formulations for transdermal administration include liquid or semi-liquid preparations (e.g., liquids, gels, lotions, creams, ointments, or pastes) suitable for penetration through the skin to the site of pain. Formulations for transdermal administration may contain an excipient to solubilize margaric acid. Drops suitable for administration to the eyes, ears, or nose may also be used as topical formulations. Margaric acid for transdermal or topical administration may comprise, for example, 0.01% to 10% w / w (weight %), 0.2 to 10% w / w, or 0.5 to 25% w / w of the formulation.

[0027] A topical patch is a topical formulation that is configured to deliver an active agent topically or transdermally to a subject when applied topically to the skin surface of the subject. The formulation may include two or more layers, and the two or more layers may include at least an adhesive matrix and a backing material.

[0028] Formulations of the compounds disclosed herein suitable for oral administration may be presented as discrete units such as capsules, cachets or tablets each containing a predetermined amount of the active ingredient, as a powder or granules, as a solution or suspension in an aqueous liquid or non-aqueous liquid, or as an oil-in-water or water-in-oil liquid emulsion.

[0029] Pharmaceutical preparations that can be used orally include tablets and capsules (for example, capsules made of gelatin and soft capsules) known in the art.Tablets can be made by compression or molding, optionally with one or more accessory ingredients.Compressed tablets can be prepared by compressing the active ingredient in free form, such as powder or granules, in a suitable machine, optionally mixed with a binder, an inert diluent, or a lubricant, surfactant, or dispersant.Molded tablets can be made by molding a mixture of powdered compounds moistened with an inert liquid diluent in a suitable machine.

[0030] Formulations for injection may be presented in unit dosage form (e.g., ampoules or multi-dose containers) containing an added preservative. The compositions may take the form of a suspension, solution, or emulsion in an oily or aqueous vehicle and may contain excipients such as suspending, stabilizing, and / or dispersing agents. The formulations may be presented in unit-dose or multi-dose containers (e.g., sealed ampoules and vials) and may be stored in powder form or freeze-dried (lyophilized) condition requiring only the addition of a sterile liquid carrier (e.g., saline or sterile pyrogen-free water) immediately prior to use.

[0031] In addition to the formulations described previously, the compounds can also be formulated as a depot preparation. Such long acting formulations can be administered by implantation (for example subcutaneously or intramuscularly) or by intramuscular injection.

[0032] In one embodiment, the transdermal pharmaceutical composition comprises margaric acid and a pharmaceutically acceptable excipient.

[0033] In another embodiment, the composition for treating pain comprises margaric acid, eicosapentaenoic acid, and pharmaceutically acceptable excipients.Eicosapentaenoic acid (EPA; 20:5) is a dietary long-chain omega-3 fatty acid that is believed to be involved in reducing inflammation.When combined with MA, EPA is expected to further reduce tactile inflammatory pain and tactile pain.

[0034] In one embodiment, the topical formulation comprises 0.49-0.55% by weight margaric acid, 0.49-0.55% by weight eicosapentaenoic acid, 25.05-27.69% by weight primary solvent, 23.77-26.27% by weight co-solvent, and 45.19-49.95% by weight thickener, all weights based on the total weight of the topical formulation. In a further embodiment, the primary solvent is sesame oil, the co-solvent is paraffin oil, and the thickener is castor oil.

[0035] The present invention is further illustrated by the following non-limiting examples. [Example]

[0036] method Cell culture and electrophysiology: Piezo1 knockout mice N2A (N2A Piezo1- / - ) cells were a gift from Dr. Gary R. Lewin. Human Merkel cell carcinoma cell line (MCC13 cells; Cell Bank Australia reference number: CBA1338) was obtained from Sigma. DRG neurons were obtained from sacrificed mice. N2A Piezo1- / - Cells were cultured in Dulbecco's modified Eagle's medium (DMEM), 5% penicillin-streptomycin, and 10% fetal bovine serum (FBS). MCC13 cells were cultured in RPMI1640 (2 mM L-glutamine + 25 mM HEPES; Sigma), 5% penicillin-streptomycin, and 10% FBS. DRG neurons were cultured in DMEM, 1% penicillin-streptomycin, 1% MEM vitamin solution, 1% L-glutamine, and 10% horse serum. Prior to electrophysiological measurements, N2A Piezo1- / - , MCC13, and DRG neurons overnight (

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[0037] For whole-cell recordings, the bath solution contained 140 mM NaCl, 6 mM KCl, 2 mM CaCl, 1 mM MgCl, 10 mM glucose, and 10 mM HEPES (pH 7.4; 300 mOsm). For voltage-clamp recordings, the pipette solution contained 140 mM CsCl, 5 mM EGTA, 1 mM CaCl, 1 mM MgCl, and 10 mM HEPES (pH 7.2); and for current-clamp recordings, the solution contained 140 mM KCl, 6 mM NaCl, 2 mM CaCl, 1 mM MgCl, 10 mM glucose, and 10 mM HEPES (pH 7.4; 300 mOsm). MA and bradykinin acetate (Sigma) were dissolved in the bath solution for 2 min at a final concentration of 300 μM and 5 min at 1 μM, respectively, to be perfused during the experiment. For long-term exposure experiments, bradykinin was supplemented to the medium and added to the cells 18–24 h before recording. For cytoskeleton disruption experiments, N2A Piezo1- / - Recordings were made after incubation for 1 hour in medium supplemented with 1 μM latrunculin A (Cayman Chemicals). Pipettes were made from borosilicate glass (Sutter Instruments) and flame-polished before use to reach a resistance of 3–5 MΩ.

[0038] During mechanical stimulation, currents were recorded at a constant voltage (-60 mV, voltage clamp), and voltages were recorded without current injection (current clamp). Both variables were sampled at 100 kHz and low-pass filtered at 10 kHz using a MultiClamp™ 700B amplifier and Clampex (Molecular Devices, LLC). To measure voltage-dependent currents, a square pulse protocol consisting of 20 mV incremental steps starting from -80 mV for 40 ms at 500 ms intervals was used with P / 4 subtraction. To record action potentials evoked by current injection, 20 pA incremental steps for 40 ms at 500 ms intervals were injected. In both cases, variables were sampled at 20 kHz and low-pass filtered at 10 kHz. Leak currents before mechanical stimulation were subtracted offline from current traces, and data were digitally filtered at 2 kHz with ClampFit (Molecular Devices, LLC). Recordings in which the leak current exceeded 200 pA, the access resistance exceeded 10 MΩ, and the gigaseal did not withstand at least six consecutive steps of mechanical stimulation were excluded from analysis.

[0039] Mechanical stimulation: N2A for indentation assay Piezo1- / - MCC13 cells, DRG neurons, and human iPSC374-derived neurons were mechanically stimulated with a heat-polished, blunted glass pipette (3-4 µm) driven by a piezo servo controller (E625, Physik Instrumente). The blunted pipette was attached to a micromanipulator at an approximately 45° angle and positioned 3-4 µm above the cell without indentation. Displacement measurements were obtained using a square pulse protocol consisting of 1 µm incremental indentation steps, each lasting 200 ms with a 2 ms ramp at 10 s intervals. The mechanical activation current threshold for each experiment was defined as the indentation step eliciting the first current deflection from baseline. In current-clamp experiments, the mechanical threshold was defined as the indentation step eliciting the first action potential.

[0040] For pulse train assays, a 13 s sweep with a train rate of 1 Hz square pulses lasting 200 ms was used. Subsequent sweeps were in 1 μm increments. Only cells that did not detach throughout the entire stimulation protocol were included in the analysis. The piezo servo controller was automated using a MultiClamp™ 700B amplifier via Clampex (Molecular Devices, LLC).

[0041] N2A Piezo1- / - Transfection of cells: N2A Piezo1- / - Cells were diluted to 75–200 ng ml -1 mmPiezo2 variants (2, 14, and 16) or 1 ng·ml -1 Cells were co-transfected with a PIEZO2-PIEZO1 chimera cloned into pcDNA3.1 and GFP391pMO and recorded 48 hours later using Lipofectamine® 2000 (Thermo Fisher Scientific) according to the manufacturer's instructions. Fatty acids were replenished 18–24 hours before recording unless otherwise noted.

[0042] Primary culture of DRG neurons: Primary cultures of DRG neurons were obtained from 8-12 week-old male C57BL / 6 mice. Mice were anesthetized with isoflurane and then sacrificed by cervical dislocation. DRGs were dissected and kept on ice in Hank's balanced salt solution 1x (HBSS without CaCl2 and MgCl2). DRGs were then incubated with 1 mg / mL collagenase B (Sigma) in HBSS at 37°C and 5% CO2 for 1 hour and then dissociated in serum-free medium. The cell suspension was centrifuged at 800 rpm for 8 minutes. The resulting pellet was resuspended in DMEM complete medium containing 1% penicillin-streptomycin, 1% 402MEM vitamin solution, 1% L-glutamine, and 10% horse serum. Cells were cultured on coverslips pretreated with poly-L-lysine. All cultured neurons were used after 18-24 hours.

[0043] Mouse Behavior: MA Solution: MA was prepared in sterile Dulbecco's phosphate-buffered saline containing 70 g / L fatty acid-free bovine serum albumin (BSA; Sigma) to a final concentration of 5 mM MA. The solution was filtered to remove undissolved material.

[0044] Subjects: Male and female C57B16 / J mice aged 8-12 weeks (n=12; control n=6; male=3, female=3) and MA (n=6; male=3, female=3) were used per experiment. Mice were housed under a 12-hour light / dark cycle and had free access to food and water.

[0045] Treatment: Animals were divided into two groups (control and MA) and injected into the plantar left hind paw 7 days prior to behavioral assessment. Injections were performed as follows: Mice were restrained by the tail, foot, and shoulder muscles. Compounds were delivered to the plantar surface of each mouse's hind paw using a 3 / 10 cc insulin syringe. A beveled needle was inserted at a 35-40° angle, and 20-50 μl of compound was injected subcutaneously. The needle was held in place for a few seconds, then removed, and the animals were returned to their home cages. All female animals were handled and treated before males to reduce stress.

[0046] Before all behavioral assays, animals were allowed to habituate to the testing apparatus for approximately 30 min. To induce acute inflammation, AITC (1 mM) was applied to the paw using a small paint brush.

[0047] Mechanical threshold (Von Frey test): Prick mechanical allodynia was measured using Von Frey monofilaments (Stoelting, Inc.) at various force levels (0.008-1 g). Animals were placed in a small enclosure on a wire mesh, and the filament was punctured into the plantar hind paw, and the response was recorded. A withdrawal response was recorded when the animal lifted, shook, or licked its paw in response to the stimulus. No response was indicated if the animal did not move in response to the stimulus. Each filament was pressed against the plantar paw until the fiber bent slightly. The amount of force required to bend the fiber depended on the thickness of each monofilament. A response was indicated by 5 withdrawal responses to the filament. For each subject, testing was stopped when 10 withdrawals were recorded for any particular filament.

[0048] Mechanical pain (pinprick): Animals were placed in a small enclosure on a wire mesh surface and the plantar surface of a hind paw was punctured at a 45° angle with a needle (25G 5 / 8 inch). Responses were recorded, and 10 stimuli were presented per hind paw.

[0049] Thermal threshold (Hargreaves test): Animals were placed in a small enclosure on a glass plate heated to 32°C (IITC). A focused radiant heat source was applied to the plantar surface of the hind paw. The latency to withdraw each mouse's paw was recorded with three stimulus presentations administered per paw. Mice were restrained by the tail, foot, and shoulder muscles. Compounds were delivered to the plantar surface of each mouse's hind paw using a 3 / 10 cc insulin syringe. A beveled needle was inserted at a 35-40° angle, and 20-50 μl of compound was injected subcutaneously. The needle was held in place for a few seconds, then removed, and the animal was returned to its home cage. All female animals were handled and treated before males to reduce stress.

[0050] Human iPSC-derived neurons: For the generation of human peripheral sensory neuron cultures, a version of the healthy control WTC11 iPSC line was used. This line was previously engineered to carry a doxycycline-inducible NGN2-BRN3A construct, which allows for rapid and efficient differentiation of sensory neurons. Undifferentiated iPSCs were maintained in E8 flex medium (Invitrogen) on polystyrene plates coated with Matrigel® (Coming). Medium was changed every 1–3 days, and cells were passaged every 4–7 days with Accutase® (Invitrogen) and seeded overnight with 10 μM ROCK inhibitor Y-27632 (Tocris). For sensory neuron differentiation, iPSCs were plated at 20,000 cells (cm) in neural differentiation medium (NDM) on Matrigel®-coated plates. 2 ) -1 After 48 hours, the cells were then plated onto dishes coated with polyethyleneimine (Sigma-Aldrich) and laminin (Invitrogen) at 50,000 cells·cm 2 ) -1 NDM consisted of 1:1 DMEM / F12 and Neurobasal™ medium supplemented with N2, B27, and GlutaMAX™ at the manufacturer's recommended dilution. 2 μg ml -1 The medium contained 100 μM doxycycline (Clontech) and was supplemented with 10 μM Y-27632 for the first 48 hours. From day 8 onwards, the following neurotrophic factors (all from R&D Systems) were added at 10 ng / ml each: BDNF, GDNF, β-NGF, and NT-3. Full medium changes were performed every other day until day 8, followed by half-medium changes every other day for the remainder of the period. Prior to electrophysiological recordings, a subset of dishes was supplemented with MA at 300 or 600 μM for 18 hours or 50 μM for 5 days. All recordings were performed on neurons cultured for 14–16 days.

[0051] Data analysis: Results are expressed as mean + SD (unless otherwise noted). Box plots show the 25th-75th percentile range, mean, median, and outliers with a coefficient of 1.5. Data were plotted using OriginPro (from OriginLab). The time constant of inactivation, τ, was obtained by fitting a single exponential function (1) between the peak value of the current and the end of stimulation.

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[0052] Example 1: Margaric acid inhibits PIEZO2 currents in N2A cells First, we characterized PIEZO2 channels in transfected neuro-2a (N2A) cells using an electrically driven piezo glass probe. We previously identified that the plasma membrane of N2A cells is enriched in MA after overnight incubation, promoting high bending stiffness, as determined by mass spectrometry and atomic force microscopy. Importantly, we determined that PIEZO1 exhibits reduced activity in this membrane environment. To determine whether PIEZO2 can also be regulated by membrane mechanical properties, we transfected Piezo2 variant V2 and N2A cells. Piezo1- / - After supplementing the culture medium of cells (i.e., cells lacking the Piezo1 gene) with MA at concentrations ranging from 1 to 600 μM overnight, the mechanical currents were measured. 50We found that MA inhibited PIEZO2 currents in a concentration-dependent manner at a concentration of 190.6 ± 14.7 μM (mean ± SEM; Fig. 1b) (Fig. 1a, b, Fig. 2a). Furthermore, MA increased the displacement threshold required to elicit PIEZO2 currents by 3-fold (Fig. 2b) compared to control cells (Fig. 1c) without affecting the time constant of inactivation.

[0053] When overnight supplementation was used, MA concentrations higher than 100 μM were required to reduce PIEZO2 currents (Figure 1b). Fatty acids can accumulate when consumed in large amounts through the diet. Similarly, we previously demonstrated that MA can accumulate in the plasma membrane when supplemented at low concentrations in cell culture media for several days. Therefore, a daily supplementation protocol was implemented to inhibit PIEZO2 activity with lower doses of MA. Indeed, over a 4-day period, N2A Piezo1- / - Supplementing cells with only 50 μM MA reduced PIEZO2 current by 65% ​​(Figure 1d, e). Continuous supplementation with low doses of MA increased the displacement threshold without altering PIEZO2 inactivation, as seen at higher concentrations overnight (Figure 1f and Figure 2c). Supplementation with 25 μM per day for 8 days yielded similar results (Figure 2d-g). Prolonged culture times demonstrated that MA concentration could be further reduced. Notably, MA also inhibited the activity of two other Piezo2 variants (V14 and 16) that are particularly abundant in the trigeminal ganglion, indicating that MA likely affects the majority of alternatively spliced ​​isoforms of this channel (Figure 3a-i). Together, our results demonstrate that MA inhibits PIEZO2 current by increasing the mechanical threshold required for activation. Thus, like PIEZO1, PIEZO2 is less active in stiff membranes (≥78 pN).

[0054] Example 2: PIEZO2 function depends on the plasma membrane and cytoskeleton. When comparing the activities of PIEZO1 and PIEZO2 with increasing concentrations of MA, the IC 50 was 28.3 ± 3.4 μM, and the IC of PIEZO2 50The IC value was determined to be 190.6 ± 14.7 μM (mean 127 ± SEM; Figure 4a, Figure 5a-d). Several lines of evidence suggest that in inside-out patches, PIEZO2 requires the cytoskeleton for activation, whereas PIEZO1 can only be activated by membrane tension. These different characteristics may explain why approximately 7 times more MA is required to inhibit PIEZO2 channels than PIEZO1 channels. We previously demonstrated that disrupting actin filaments does not affect the bending stiffness of the plasma membrane in untreated or MA-enriched N2A cells. Therefore, to determine the contribution of the actin cytoskeleton in PIEZO2 gating, we treated MA-enriched cells with latrunculin A and compared their mechanically evoked responses to cells treated with MA alone. Latrunculin A treatment resulted in a significant leftward shift in the MA dose-response profile of PIEZO2 (IC). 50 = 75.4 ± 13.3 μM, mean ± SEM, Figure 4b), which is close to that of Piezo1 (Figure 4a). Meanwhile, the MA dose-response profile of PIEZO1 was similar in control and latrunculin-treated cells (IC 50 = 28.3 μM ± 3.4 (control cells, 25.6 μM ± 8.4 (latrunculin-treated cells, mean ± SEM; Figures 4c and 5c, d), indicating that the mechanism of MA-mediated inhibition of PIEZO1 currents is solely dependent on the plasma membrane. These results imply that the cytoskeleton is an important determinant of the differential responses of PIEZO2 and PIEZO1 to MA.

[0055] Unlike PIEZO1, PIEZO2 requires a rigid plasma membrane (i.e., MA 24The effect of MA-mediated enrichment is more pronounced when the cytoskeleton is pharmacologically disrupted (Figure 4b, c). Next, we wondered whether modifying the PIEZO2 intracellular region (which likely interacts with cytoskeletal elements) could enhance MA-mediated inhibition, similar to the effect observed with latrunculin A treatment. Both PIEZO1 and PIEZO2 contain a 90-Å-long intracellular helix called the beam (i.e., connecting the transmembrane blade and the central pore), and we reasoned that this could also tether the channel to the cytoskeleton. Notably, the sequence identity between the PIEZO1 and PIEZO2 beams is low (35%), which could therefore explain the different responses of these channels to MA inhibition. To test this hypothesis, we engineered a PIEZO2 chimera and replaced its beam with that of PIEZO1 (Figure 4d). The PIEZO2-PIEZO1 beam chimera exhibits similar functional properties to PIEZO2, including reversal potential (7.7 mV for PIEZO2 vs. 5.6 mV for the chimera) and displacement threshold (6.27 ± 1.35 µM for PIEZO2 vs. 6.9 ± 0.8 µM for the chimera, mean ± SD; Figures 6a-c). However, the inactivation time constant of the chimera is faster than that of PIEZO2 (7.59 ± 1.96 ms for PIEZO2 vs. 1.91 ± 0.46 ms for the chimera, mean ± SD; Figures 2c and 6d). Notably, transferring the PIEZO1 beam to PIEZO2 yielded channels that were much more sensitive to MA inhibition (100 µM, overnight), similar to that seen with PIEZO1 (Figure 4e). This chimera required higher mechanical stimuli to open when expressed in cells supplemented with MA (100 μM overnight; Fig. 6c), an effect that was not modulated by latrunculin A treatment (Fig. 4c, f). Together, our results emphasize that PIEZO2 mechanosensitivity depends on a synergistic interaction between plasma membrane mechanics and interactions with cytoskeletal elements.

[0056] Example 3: Margaric acid reduces mechanical currents in sensory cells Piezo2 is expressed in Merkel cells and their innervating afferents and has been shown to transduce skin indentations and whisker movements into electrical signals. Given the above results in heterologous expression systems, we asked whether MA could reduce PIEZO2 currents in cells mediating touch sensation. To this end, we measured the effect of MA on PIEZO2 activity in a human Merkel cell carcinoma cell line (MCC13) and acutely cultured mouse DRG neurons. Similar to dissociated Merkel cells, MCC13 cells exhibited mechanosensitive currents with variable inactivation kinetics (Figure 7a). These mechanical currents have been shown to be mediated by PIEZO2. Similar to experiments using transiently transfected N2A cells, MA supplementation in MCC13 cells reduced endogenous PIEZO2 currents (Figures 7a, b) by increasing the displacement threshold (Figure 7c). Similarly, cultured mouse DRG neurons also exhibit mechanical currents with variable inactivation kinetics. However, in this case, only rapidly adapting currents (t < 10 ms) were assigned to PIEZO2. Notably, MA supplementation reduced the magnitude of mechanically induced currents in all DRG neurons (Fig. 7f), including those known to be mediated by PIEZO2 (Fig. 7d, e), by increasing the displacement threshold. Together, these findings explain our heterologous expression results and demonstrate that MA reduces endogenous mechanical currents in diverse cell types known to be involved in mechanosensing. Example 4: Margaric acid reduces action potential firing evoked by mechanical stimulation

[0057] Touch detection relies on mechanosensitive ion channels expressed in sensory nerve terminals. These channels transduce mechanical stimuli into electrical signals, depolarizing neurons and subsequently generating action potentials that propagate toward the central nervous system. PIEZO2 mediates the majority of mechanically activated excitatory currents in mouse DRG neurons. Because MA reduces mechanical currents (including PIEZO2 currents), we sought to determine whether this saturated fatty acid also impairs the ability of DRG neurons to elicit mechanically activated action potentials. Indeed, we found that MA completely inhibited action potential generation in mouse DRG neurons when the indentation step was less than 12 μm (Figure 8a, left and center panels). Nevertheless, we were able to elicit action potentials in MA-treated neurons when using larger indentation steps [12 μm (red step) for MA vs. 7–12 μm for control, Figure 8a, right panel and Figure 8b]. Furthermore, by stimulating DRG neurons with a series of 1 Hz mechanical stimulation trains, we found that MA-enriched neurons elicited fewer action potentials than controls (<12 μm; Figure 8c), even with larger indentations (>12 μm; Figure 8d). Interestingly, MA-enriched neurons also showed a gradual decrease in membrane potential with increasing indentation pulse number (Figure 8c, inset). This suggests that it is more difficult to open mechanosensitive channels in MA-treated neurons after repeated stimulation. Taken together, MA supplementation increases the mechanical threshold required for DRG neurons to fire action potentials.

[0058] Mechanically driven depolarization in DRG neurons induces voltage-gated Na+ receptors, which are important for generating action potentials. + Channel and K + To determine whether MA impairs the function of ion channels downstream of mechanical activation, we recorded voltage-gated currents in the presence or absence of MA. We measured voltage-activated inward Na channels in control and MA-enriched DRG neurons. +Current and outward K + We found no significant differences in current magnitude (Fig. 9a-c). Furthermore, MA supplementation did not alter the membrane potential when measured in the whole-cell configuration (Fig. 9d). Notably, we found no differences in action potential firing evoked by current injection between control and MA-enriched neurons (Fig. 9e, f).

[0059] Overall, our results indicate that MA does not alter the electrical excitability of DRG neurons, but instead specifically reduces action potential firing evoked by mechanical stimulation.

[0060] Example 5: Margaric acid counteracts PIEZO2 bradykinin sensitization Tissue injury is often accompanied by the accumulation of algesic-inflammatory substances such as bradykinin, eicosanoids, and protons. These inflammatory molecules bind to or interact with various membrane proteins, activating intracellular signaling cascades and increasing sensitivity to sensory stimuli, leading to allodynia or hyperalgesia. It has been demonstrated that mechanically induced PIEZO2 currents are enhanced downstream of bradykinin β2 receptor activation in DRG neurons. Therefore, molecules that reduce PIEZO2 sensitization may be beneficial for treating mechanical allodynia. Given that MA significantly reduced PIEZO2 currents in DRG neurons (Figures 7d-f), we wondered whether MA supplementation would also reduce bradykinin-mediated PIEZO2 sensitization. Similar to previous reports, we found that acute bradykinin perfusion sensitized mechanically activated currents in DRG neurons (a 2.5-fold increase; Figures 10a-b and 10b). As expected, MA supplementation reduced mechanical currents, even after bradykinin sensitization (Fig. 10a, bottom). Notably, currents recorded in MA-supplemented neurons after bradykinin administration (Fig. 10a and Fig. 11) were similar to those in control DRG neurons (3.36 ± 1.68 pA / pF for control vs. 3.64 ± 1.96 pA / pF for bradykinin with MA, mean ± SD; Fig. 10b). Similar findings were observed with longer exposure to bradykinin. Overnight incubation with bradykinin enhanced the magnitude of all mechanically evoked currents in DRG neurons (Fig. 10c, middle panel and 10d). However, combining overnight incubation with bradykinin and MA restored current densities to those of control neurons (Fig. 10c, d). Taken together, these results support that enriching the plasma membrane with MA can counteract bradykinin-induced mechanical sensitization and reduce mechanical currents to non-inflammatory levels, making MA a promising molecule for reducing mechanical hypersensitivity.

[0061] Example 6: Margaric acid reduces PIEZO2-mediated mechanical allodynia Previous studies by our group and others have demonstrated that PIEZO2 is required for mechanical allodynia in both mice and humans. A key prediction from our data is that MA administration protects against this type of pain. Acute subcutaneous injection of MA did not affect baseline touch responses (Figure 12a, b). This likely reflects the time required for fatty acids to be incorporated into membranes and accumulate to regulate PIEZO2 function. Therefore, we designed an experiment to examine nociceptive responses after chronic exposure to MA (Figure 12a). Mice injected daily with small amounts of saline into their hind paws showed significant hypersensitivity to punctate touch within 7 days (repeated injury model; Figure 12b).

[0062] Surprisingly, mice failed to develop this type of enhanced withdrawal response when MA was included in the injections (Figure 12b). Importantly, this type of mild injury or daily exposure to MA had no effect on thermal withdrawal latency measured by the Hargreaves radiant heat test (Figure 12c). Allyl isothiocyanate (AITC), an irritating compound found in wasabi and mustard oil, induces pain by activating nociceptors. We induced enhanced neuroinflammatory pain by topically applying AITC to the left hind paw of mice. Normally, AITC induces immediate hind paw withdrawal from even the lightest von Frey filament (<0.16 g; Figure 12d). However, mice treated with MA for 7 days responded with sensitivity similar to their basal response from the previous week (Figure 12d). Collectively, these results provide proof of concept that reducing PIEZO2 function with MA is a feasible pharmacological approach to treat mechanical allodynia.

[0063] Example 7: Margaric acid reduces mechanical currents in human iPSC-derived neurons Our previous results demonstrated that enriching the plasma membrane with MA has an inhibitory effect on mouse PIEZO2 function in vitro, ex vivo, and in vivo. These results offer a clear potential explanation. Therefore, we explicitly tested the effects of MA on human sensory neurons. Recently, we developed a platform for robustly and reproducibly reprogramming human induced pluripotent stem cells (iPSCs) into well-characterized neurons with functional and transcriptional characteristics indicative of low-threshold mechanoreceptors (Figure 13a). Notably, all of these in vitro-derived tactile neurons possess mechanically evoked currents that are entirely dependent on PIEZO2 expression. Overnight incubation of human iPSCs with MA (300 and 600 μM) significantly reduced endogenous PIEZO2 currents (Figures 13b-c). Furthermore, we demonstrated that MA-supplemented N2A neurons significantly reduced the PIEZO2 currents. Piezo1- / - We confirmed these results by measuring the mechanical currents of human PIEZO2 transfected into cells (Figure 14a-b). Furthermore, supplementing human iPSC270-derived neurons with 50 μM MA daily for 5 days (a strategy similar to that used in mice) significantly reduced PIEZO2 currents (Figure 13b-c). As expected, MA increased the mechanical threshold required to activate the human channel without altering the inactivation time constant, mirroring the results obtained with the mouse orthologue (Figure 13d-e and Figure 14c-d). Similar to mouse cultured DRG neurons, MA significantly reduced voltage-activated inward Na currents when compared to control human iPSC-derived neurons. + Current and outward K + The α-amyloid fatty acid (MA) did not alter the mechanically activated currents (Figure 13f-h). These findings indicate that MA specifically affects mechanically activated currents while maintaining the electrical excitability of human sensory neurons. Because MA is commonly found in foods such as dairy fat, rye, and fish, we speculate that including this fatty acid as a dietary supplement or topical ointment may be a strategy to alleviate mechanical allodynia in humans.

[0064] Example 8: MA reduces mechanically activated currents in rat DRG neurons. Figures 15 and 16 further demonstrate that MA reduces mechanically activated currents in rat DRG neurons. PIEZO2 is an essential transduction channel for tactile discrimination, vibrating, and proprioception. Mice and humans lacking Piezo2 experience severe mechanical and proprioceptive deficits and fail to develop tactile allodynia. Bradykinin, a pain-inducing agent released during inflammation, enhances PIEZO2 activity. Molecules that reduce PIEZO2 function can ameliorate tactile allodynia. Here, we find that margaric acid (MA), a dietary fatty acid, reduces PIEZO2 function in a dose-dependent manner. Chimeric analysis demonstrates that the PIEZO2 channel is a critical region regulating MA-mediated channel inhibition. MA reduces neuronal action potential firing evoked by mechanical stimulation in mouse and rat neurons, bradykinin-induced PIEZO2 enhancement, and PIEZO2 currents in tactile neurons derived from human induced pluripotent stem cells. Finally, we demonstrate that MA alleviates post-injury hypersensitivity to touch in mice. Our findings report a natural product that inhibits PIEZO2 function, identify a critical region for channel inhibition, and provide evidence for a new avenue for treating tactile allodynia and other forms of pain.

[0065] Example 9: Combination of MA and EPA As shown in Figure 17, an ω3-enriched diet reduces the time constant of PIEZO2 inactivation in mouse DRG neurons. As shown in Figure 18, eicosapentaenoic acid (EPA) supplementation reduces the time constant of PIEZO2 inactivation in rat DRG neurons. As shown in Figure 19, EPA supplementation abolishes the phenotype of PIEZO2 arthrogryposis mutation. As shown in Figure 20, EPA supplementation abolishes the N2A Piezo1- / - Heterologous expression in mouse PIEZO2 cells decreases the time constant of inactivation of PIEZO2 currents.

[0066] Example 10: MA and EPA have synergistic effects on PIEZQ2 function in mouse DRG neurons.

[0067] It has previously been demonstrated that MA and EPA decrease PIEZO2 currents in mouse and rat dorsal root ganglion neurons, a human Merkel cell carcinoma cell line (MCC13), and human induced pluripotent stem cell (iPSC)-derived tactile receptor neurons. Importantly, several Piezo2-related human diseases result from increased PIEZO2 function due to decreased inactivation current. Figure 21 shows that combining MA and EPA impaired PIEZO2 function, affecting two of its unique characteristics: 1) decreasing current while increasing displacement threshold, and 2) enhancing its inactivation. As previously proposed, individual or combined fatty acids can be used to develop different ointment / lotion formulations to counteract mechanical allodynia. Example 11: Exemplary Topical Formulations

[0068] Exemplary MA formulations are provided in Tables 1 and 2. Table 3 provides the process. [Table 1] [Table 2] [Table 3]

[0069] Consideration Mechanosensory ion channels are essential for detecting innocuous, pleasant, alarming, or painful stimuli. PIEZO2, through its expression in specialized epithelial cells (Merkel cells) and peripheral sensory neurons, has emerged as a key molecular detector for certain aspects of mild touch (vibration perception and tactile discrimination). Importantly, PIEZO2 is also essential for experiencing touch-induced pain after injury or under chronic inflammation, a common condition known as tactile allodynia that remains difficult to treat. Specifically, pain-inducing agents (e.g., bradykinin) produced in response to tissue injury enhance PIEZO2 responses. In an ideal scenario, novel therapeutic approaches would be developed to specifically counteract this type of pain without impairing normal touch function. In this study, we demonstrated that application of margaric acid, a natural product found in dairy products and several food sources, such as mutton fat, rye, and fish, effectively reduced Piezo2 function.

[0070] Previously, we demonstrated that MA can be efficiently enriched in various cell types and consequently alter the activation profile of PIEZO1 channels by increasing the structural order and stiffness of the plasma membrane. Interestingly, previous studies have suggested that PIEZO1 and PIEZO2 have distinct gating mechanisms. While both are likely sensitive to membrane tension, only PIEZO2 function depends on an intact cytoskeleton. Whether PIEZO2 activation also depends on the mechanical properties of the plasma membrane is less understood. Our data indicate that PIEZO2 activation is impaired by membrane stiffness, and that the beam domain and cytoskeleton counteract the effects of membrane tension. This highlights the beam as a key region for coordinating MA-mediated channel inhibition. Placing our data in a larger context supports the idea that PIEZO2 functions as part of a force-bearing center. In this model, PIEZO2 function is tightly controlled by a platform comprising the plasma membrane (fatty acid tails and polar head groups), stomatin-like proteins, cytoskeletal elements (actin and microtubules), and extracellular tethers (e.g., adhesion foci).

[0071] How selective is MA for mechanosensitivity? Our data suggest that MA significantly alters membrane potential, current-evoked action potentials, and voltage-activated inward Na+ transport. + Current and outward K +We show that MA reduces the ability of DRG neurons to fire action potentials upon mechanical stimulation without affecting currents. Similarly, MA reduces the mechanical activation of PIEZO2 while leaving other ionic currents intact in iPSC-derived neurons. Furthermore, subcutaneous injection of MA did not affect the ability of mice to sense heat stimuli. These results strongly support the idea that this dietary fatty acid specifically reduces the mechanical excitability of neurons. Therefore, MA appears to have many preferable properties over other identified mechanoreceptor antagonists (e.g., the tarantula peptide GsMTx-4) that failed to inhibit mechanical currents from DRG neurons. In addition, the conopeptide analog NMB-1 has been shown to inhibit only moderate and mildly inactivating mechanosensitive currents, but not rapid ones. Importantly, unlike peptide toxins, MA can inhibit all mechanosensitive currents in mouse and rat DRG neurons, as well as human iPSC-derived neurons.

[0072] The recent discovery that PIEZO2 is required for tactile allodynia in mice and humans has made this channel a promising target for treating inflammatory conditions. Our data show that MA can counteract the bradykinin-induced enhancement of PIEZO2 by reducing mechanical currents to non-inflammatory levels. Thus, MA appears to offer a potential approach to reducing post-inflammatory mechanical hypersensitivity. Notably, under our behavioral conditions, MA selectively reduces tactile allodynia without significantly affecting baseline touch sensitivity. Current experimental and clinical studies support therapies that target peripheral sensory receptors while avoiding systemic delivery. Due to its high hydrophobicity, MA is a candidate for the development of topical lotion and / or cream treatments to attenuate inflammatory pain.

[0073] Use of the terms "a," "an," and "the" and similar referents (especially in the context of the claims that follow) should be construed to encompass both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The terms "first," "second," etc., as used herein, are not meant to denote any particular order but are merely a convenient way of indicating a plurality (e.g., multiple layers). The terms "comprising," "having," "including," and "containing" should be construed as open-ended terms (i.e., meaning "including, but not limited to"), unless otherwise indicated. The recitation of ranges of values ​​is intended merely to serve as a shorthand method of individually referring to each separate value falling within the range set forth herein, unless otherwise indicated, and each separate value is incorporated herein as if it were individually listed herein. All range endpoints are included within the range and are independently combinable. All methods described herein can be performed in any suitable order unless otherwise indicated herein or clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., "etc.") is intended merely to better illustrate the invention and does not limit the scope of the invention unless otherwise asserted. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the invention as used herein.

[0074] Although the present invention has been described with reference to exemplary embodiments, those skilled in the art will recognize that various changes can be made and equivalents can be substituted for elements thereof without departing from the scope of the invention. In addition, many modifications can be made to adapt a particular situation or material to the teachings of the invention without departing from essential scope thereof. Therefore, it is not intended that the invention be limited to the particular embodiment disclosed as the best mode contemplated for carrying out this invention, but the invention is intended to include all embodiments falling within the scope of the appended claims. The invention includes any combination of the above-described elements in all possible variations thereof unless otherwise indicated herein or clearly contradicted by context.

Claims

1. A pharmaceutical composition for use in the treatment of pain, comprising a therapeutically effective amount of margaric acid, said pharmaceutical composition being a topical composition, said topical composition being administered by topical administration.

2. The composition for use according to claim 1, wherein the topical dose of margaric acid is 0.1 to 20 mg / kg of margaric acid.

3. 2. The composition for use according to claim 1, wherein the pain is chronic or acute or mild or severe.

4. 2. The composition for use according to claim 1, wherein the pain is inflammatory pain, pain due to nerve injury, neuropathic pain, chronic pain, intractable cancer pain, complex regional pain syndrome, surgical or post-operative pain, dental pain, pain due to skin injury, lower back pain, headache, migraine, or hyperalgesia.

5. 5. The composition for use according to claim 4, wherein the pain is neuropathic pain.

6. The composition for use according to claim 5, wherein the neuropathic pain is allodynia.

7. 7. The composition for use according to claim 6, wherein the allodynia is tactile allodynia, mechanical allodynia, or thermal allodynia.

8. A composition for use as described in claim 7, wherein the allodynia is tactile allodynia or mechanical allodynia.

9. The subject is a patient suffering from fibromyalgia, chronic inflammation, migraine, trigeminal neuralgia, postherpetic neuralgia, peripheral neuropathy, or Neuropathy, diabetic neuropathic pain, chronic fatigue syndrome, or complex regional pain syndrome 9. The composition for use according to claim 7 or 8, which is affected by the group.

10. 5. The composition for use according to claim 4, wherein the pain is inflammatory pain.

11. The composition for use in treating inflammatory pain, inflammatory joint pain, inflammatory musculoskeletal pain, pain due to injury, arthritis pain, and complex regional pain syndrome, according to claim 10.

12. The composition for use according to claim 1 , wherein the pharmaceutical composition further comprises eicosapentaenoic acid.

13. The composition for use according to claim 1 , wherein the composition is a topical composition for instillation into the eyes.

14. 10. The composition for use according to claim 1, wherein the topical pharmaceutical composition comprises margaric acid at 0.01% to 10%, 0.2 to 10%, or 0.5 to 25% by weight.

15. 2. The method for use according to claim 1, wherein the composition is a topical composition in the form of drops. composition.

16. The composition for use according to claim 1 , wherein the composition is an intraocular composition.

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