Pharmacological Blockade of TRPM8 Ion Channels Alters Cold and Cold Pain Responses in Mice

TRPM8 (Transient Receptor Potential Melastatin-8) is a cold- and menthol-gated ion channel necessary for the detection of cold temperatures in the mammalian peripheral nervous system. Functioning TRPM8 channels are required for behavioral responses to innocuous cool, noxious cold, injury-evoked cold hypersensitivity, cooling-mediated analgesia, and thermoregulation. Because of these various roles, the ability to pharmacologically manipulate TRPM8 function to alter the excitability of cold-sensing neurons may have broad impact clinically. Here we examined a novel compound, PBMC (1-phenylethyl-4-(benzyloxy)-3-methoxybenzyl(2-aminoethyl)carbamate) which robustly and selectively inhibited TRPM8 channels in vitro with sub-nanomolar affinity, as determined by calcium microfluorimetry and electrophysiology. The actions of PBMC were selective for TRPM8, with no functional effects observed for the sensory ion channels TRPV1 and TRPA1. PBMC altered TRPM8 gating by shifting the voltage-dependence of menthol-evoked currents towards positive membrane potentials. When administered systemically to mice, PBMC treatment produced a dose-dependent hypothermia in wildtype animals while TRPM8-knockout mice remained unaffected. This hypothermic response was reduced at lower doses, whereas responses to evaporative cooling were still significantly attenuated. Lastly, systemic PBMC also diminished cold hypersensitivity in inflammatory and nerve-injury pain models, but was ineffective against oxaliplatin-induced neuropathic cold hypersensitivity, despite our findings that TRPM8 is required for the cold-related symptoms of this pathology. Thus PBMC is an attractive compound that serves as a template for the formulation of highly specific and potent TRPM8 antagonists that will have utility both in vitro and in vivo

PBMC inhibits cold-evoked TRPM8 responses.

<p><b>A</b>) Representative images of HEK293T cells expressing mTRPM8. Pseudocolored images of the 340/380 nm Fura-2 ratio (R_340/380) show low basal Ca²⁺ before cooling the bath solution, which evoked a robust increase in intracellular Ca²⁺. After treating the cells for five minutes with vehicle (top row) or 25 nM PBMC (bottom row), cells again displayed low basal Ca²⁺ levels. A second cooling of the bath resulted in a calcium increase in vehicle, but not PBMC-treated cells. <b>B</b>) Average changes in the R_340/380 of vehicle-washed cold-responding cells show that, under these conditions, the second cold pulse resulted in a robust influx of calcium into the intracellular space which was only slightly smaller than that seen for the first cold pulse. <b>C</b>) Average changes in the R_340/380 of cells washed with PBMC show that the drug abolished the second calcium influx. <b>D</b>) 25 nM PBMC significantly inhibited cold responses in HEK293T cells transfected with TRPM8 as compared with vehicle controls. Data are presented as the average value of the second response as a percentage of the first compared for vehicle- (black bars; 82.0±1.0) and PBMC- (grey bars; 0.4±0.4) treated cells (Student's t-test, ***p<0.001).</p

PBMC affects thermoregulation in a dose-dependent manner.

<p><b>A</b>) Injection of 10 mg/kg icilin resulted in an average increase in core body temperature of 1.6°C as measured by thermal telemetry. This hyperthermic response to icilin was not present in TRPM8^-/- animals, which only exhibited a mild (<0.5°C) and transient (<30 minutes) increase, similar to that observed with vehicle (<b>C</b>). <b>B</b>) Injection of 1 mg/kg capsaicin resulted in a robust hypothermic response (∼4°C drop) in wildtype and TRPM8^-/- animals. <b>C</b>) Injection of vehicle (20% DMSO/80% saline (DS)) resulted in no change in core body temperature in either genotype beyond a small spike in body temperature within 30 minutes of injection. <b>D</b>) Intraperitoneal injection of warmed 10% Solutol/20% PEG-200/saline (SPS) vehicle resulted in no changes in core body temperature besides the injection spike in either genotype. <b>E</b>) Injection of warmed 10 mg/kg PBMC resulted in a subtle hypothermic effect (<1°C drop) in wildtype mice which resolved within three hours of injection, whereas TRPM8^-/- mice remained unaffected. <b>F</b>) Injection of warmed 20 mg/kg PBMC resulted in a profound hypothermic response (>6°C drop within 45 minutes) in wildtype animals, which did not occur in TRPM8^-/- mice. Arrows indicate injection time. Some error bars were omitted for clarity and all data are from 4–8 animals. Bars denote data that was statistically different (* p<0.05) between wildtype and TRPM8^-/- mice.</p

PBMC inhibits menthol-evoked TRPM8 responses.

<p><b>A</b>) Representative images of HEK293T cells expressing mTRPM8. Pseudocolored images of the 340/380 nm (excitation) Fura-2 ratio (R_340/380) show low basal Ca²⁺ before application of 200 µM menthol, which evoked a robust increase in intracellular Ca²⁺. A second application of menthol resulted in a second increase in intracellular Ca²⁺ after a ten minute treatment with vehicle (top row) but not after treatment with 25 nM PBMC (bottom row). <b>B</b>) Average changes in the Fura-2 ratio of vehicle-washed menthol-responding cells show that the second menthol pulse resulted in a robust calcium influx, albeit to a smaller degree than that of the first pulse. <b>C</b>) Average changes in the Fura-2 ratio of cells perfused with PBMC show that the drug abolished the second calcium increase.</p

PBMC reduces acute cold-evoked behavioral responses.

<p><b>A</b>) In the acetone evaporative cooling assay, wildtype mice exhibited an average response score of 2.2±0.1, while treatment with 10 mg/kg PBMC reduced this score to 1.8±0.1 (Student's t-test, **p<0.01 vs. baseline) and treatment with 20 mg/kg PBMC reduced it further to 1.4±0.1 (Student's t-test, ***p<0.001 vs. baseline). <b>B</b>) Every mouse given 10 mg/kg PBMC showed a decrease in acetone response scores ranging from 0.1 (orange stars, violet hexagons) to 1.1 (black triangle).</p

PBMC inhibits TRPM8 currents.

<p><b>A</b>) Representative whole-cell voltage clamp recording from a mTRPM8-expressing HEK293T cell. Currents, at both positive and negative potentials were measured during a voltage ramp from −80 mV to +80 mV (1 V/s) and evoked with 500 µM menthol (in the absence of extracellular calcium, with 10 mM EGTA in the pipette), followed by the addition of 0.25 nM then 2.5 nM PBMC for five minutes per concentration. Menthol was present in the perfusate for the duration of the drug application (green bars) and a perfusion artifact was inserted to demarcate the solution change. <b>B</b>) Current-voltage relationships at the time points indicated in <b>A</b>. <b>C</b>) Normalized currents plotted against a range of PBMC concentrations. Reduction of TRPM8 currents by PBMC was dose-dependent at +80 mV (squares) and −80 mV (circles). The calculated IC₅₀ values were 0.6 nM and 0.4 nM at positive and negative voltages, respectively (n = 6–8 cells per data point).</p

PBMC reduces cold hypersensitivity in CFA and CCI but not oxaliplatin pain models.

<p><b>A</b>) In the CFA model of inflammatory pain, wildtype animals showed an increase in behavioral response scores which peaked at 3.5±0.3 at two days post-injury, whereas scores for TRPM8^-/- mice did not change significantly from the baseline (BL) of 1.9±0.4 (ANOVA, p>0.05). <b>B</b>) PBMC (10 mg/kg) administered to wildtype mice on day two post-CFA-injection resulted in a significant decrease in scores to 2.5±0.2 (Student's t-test, **p<0.01). The effect of the drug was gone within 24 hours, with PBMC-treated animals average score of 3.0±0.1, which was not significantly different from the 2.7±0.4 score of vehicle-treated animals (Student's t-test, p>0.05). <b>C)</b> In the CCI model of neuropathic pain, wildtype animals exhibited a response score of 4.1±0.1 by day six post-injury, which remained constant through day eight. TRPM8^-/- mice exhibited no significant increase in responses over baseline, with scores reaching 1.7±0.3 by day six post-injury (ANOVA, p>0.05). <b>D)</b> Treatment of CCI-wildtype animals with 10 mg/kg PBMC resulted in a decrease in score on day seven post-injury to 3.0±0.1, which was significantly lower than 4.0±0.2 in vehicle-treated animals (Student's t-test, **p<0.01). Scores of PBMC-injected animals increased to 4.1±0.2 by 24 hours post-treatment, which was the same score for vehicle-treated animals. <b>E)</b> In the oxaliplatin-induced model of neuropathic pain, wildtype animals showed a peak score of 3.3±0.1 by day three, which remained constant through day seven at 3.2±0.2. TRPM8^-/- animals showed no significant changes in score over the seven day period (ANOVA, p>0.05). <b>F)</b> Wildtype animals treated on day three post-injury with 10 mg/kg showed a slight reduction (3.0±0.1) in acetone score as compared to vehicle-treated animals (3.3±0.1) although this decrease was not statistically significant (Student's t-test, p = 0.08).</p

PBMC shifts the voltage dependence of TRPM8 channel gating.

<p><b>A</b>) Representative whole-cell TRPM8 current traces in response to the indicated voltage step protocol. Traces show activity before and after application of 1 mM menthol and after application of 0.5 nM PBMC while still in the presence of 1 mM menthol. <b>B</b>) Steady-state activation curves under basal, menthol, and menthol + PBMC conditions. The normalized conductance (G/G_max) was plotted against voltage and the addition of PBMC resulted in a reduction of the normalized conductance. Lines represent Boltzmann functions fitted to the data (n = 6–8 cells). <b>C</b>) Average voltages (mV) of half-maximal normalized conductance (V_1/2) obtained from the Boltzmann functions in B. The addition of PBMC with menthol shifted the V_1/2 from 79.1±21.1 mV to 171.2±16.0 mV (Student's t-test, **p<0.01), towards the baseline value of 218.2±18.2 mV.</p

Structure of PBMC.

<p>1-phenylethyl-4-(benzyloxy)-3-methoxybenzyl(2-aminoethyl)carbamate.</p