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. 2011 Nov;80(5):900-10.
doi: 10.1124/mol.111.073205. Epub 2011 Aug 5.

TTA-P2 is a potent and selective blocker of T-type calcium channels in rat sensory neurons and a novel antinociceptive agent

Wonjoo Choe  1 , Richard B Messinger, Emily Leach, Veit-Simon Eckle, Aleksandar Obradovic, Reza Salajegheh, Vesna Jevtovic-Todorovic, Slobodan M Todorovic
Affiliations

TTA-P2 is a potent and selective blocker of T-type calcium channels in rat sensory neurons and a novel antinociceptive agent

Wonjoo Choe et al. Mol Pharmacol. 2011 Nov.

Abstract

Several agents that are preferential T-type calcium (T-channel) blockers have shown promise as being effective in alleviating acute and chronic pain, suggesting an urgent need to identify even more selective and potent T-channel antagonists. We used small, acutely dissociated dorsal root ganglion (DRG) cells of adult rats to study the in vitro effects of 3,5-dichloro-N-[1-(2,2-dimethyl-tetrahydro-pyran-4-ylmethyl)-4-fluoro-piperidin-4-ylmethyl]-benzamide (TTA-P2), a derivative of 4-aminomethyl-4-fluoropiperdine, on T currents, as well as other currents known to modulate pain transmission. We found that TTA-P2 potently and reversibly blocked DRG T currents with an IC(50) of 100 nM and stabilized channel in the inactive state, whereas high-voltage-activated calcium and sodium currents were 100- to 1000-fold less sensitive to channel blocking effects. In in vivo studies, we found that intraperitoneal injections of 5 or 7.5 mg/kg TTA-P2 reduced pain responses in mice in phases 1 and 2 of the formalin test. Furthermore, TTA-P2, at 10 mg/kg i.p., selectively and completely reversed thermal hyperalgesia in diabetic rats treated with streptozocin but had no effect on the nociceptive response of healthy animals. The antihyperalgesic effects of TTA-P2 in diabetic rats were completely abolished by administration of oligonucleotide antisense for Ca(V)3.2 isoform of T channels. Thus, TTA-P2 is not only the most potent and selective blocker of T channels in sensory neurons yet described, but it also demonstrates the potential for the pharmacological effectiveness of this approach in addressing altered nociceptive responses in animal models of both inflammatory and neuropathic pain.

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Figures

Fig. 1.
Fig. 1.
Structure of TTA-P2 compound evaluated in this study.
Fig. 2.
Fig. 2.
TTA-P2 selectively inhibits T currents in acutely dissociated adult rat sensory neurons. A, traces of T current in a representative DRG cell before and after (black traces) and during (gray trace) bath application of 1 μM TTA-P2, which reversibly inhibited most of the peak inward current. Currents were evoked from a holding potential (Vh) of − 90 mV and stepping to test potential (Vt) of − 30 mV. Bars indicate calibration. B, temporal record from the same cell presented in A. The gray bar indicates duration of TTA-P2 application. C, traces of HVA current from another DRG cell before (black trace) and during (gray trace) the bath application of 10 μM TTA-P2. TTA-P2 inhibited less than 10% of peak current. Currents were evoked from Vh of − 50 mV and stepping to Vt of − 10 mV. Bars indicate calibration. D, traces of recombinant CaV2.3 current from a representative HEK 293 cell before (black trace) and during (gray trace) the bath application of 10 μM TTA-P2, which inhibited approximately 20% of the peak current. Currents were evoked from Vh of − 80 mV and stepping to Vt of − 20 mV. Bars indicate calibration. E, traces of total sodium current (INa+) from a representative DRG cell are shown on the left. Traces of TTX-resistant sodium current (INa+ TTXR) from another DRG cell are presented on the right. Note that there is little difference between baseline current (black traces) and during the bath application (gray traces) of 1 μM TTA-P2. Currents are evoked from Vh of − 90 mV and stepping to Vt of − 20 mV. Bars indicate calibration.
Fig. 3.
Fig. 3.
TTA-P2 inhibits T currents in DRG cells more potently than it does HVA currents in DRG cells and recombinant CaV2.3 currents in HEK 293 cells. The graphs illustrate concentration-response relationships for TTA-P2 inhibition of T currents in rat DRG cells (filled circles), HVA currents in DRG cells (filled squares), and recombinant CaV2.3 currents in HEK 293 cells (open triangles) (n = 4–15 cells per data point). The solid line is the best fit (eq. 1; see Materials and Methods) for T-current inhibition (IC50 = 0.11 ± 0.01 μM; slope coefficient, 0.8 ± 0.1; maximal inhibition, 90 ± 3% of the peak current), HVA current inhibition (IC50 = 165 ± 35 μM; slope coefficient, 1.3 ± 0.5; maximal inhibition constrained to 100%), and CaV2.3 current inhibition (IC50 = 35 ± 9 μM; slope coefficient, 0.9 ± 0.3; maximal inhibition constrained to 100%).
Fig. 4.
Fig. 4.
The effects of TTA-P2 on kinetic properties of T currents in rat DRG cells. A, traces represent families of averaged T currents evoked in the same DRG cells (n = 5) in control conditions (top) and during application of 100 nM TTA-P2 (bottom) by voltage steps from − 90 mV (Vh) to Vt from − 60 to − 20 mV in 10-mV increments. Bars indicate calibration. Inset, voltage-clamp protocol used to elicit currents. B, average I-V curves are shown from the same DRG cells shown in A before (open symbols) and during (filled symbols) bath application of 100 nM TTA-P2 using voltage steps in 5-mV increments. Note that TTA-P2 depressed the amplitude of T current similarly at most test potentials. C and D, we measured time-dependent activation (10–90% rise time; D) and inactivation τ (single-exponential fit of decaying portion of the current waveforms using eq. 4; C) from I-V curves in DRG cells shown in B over the range of test potentials from − 45 to − 20 mV. There are few differences between the control (open symbols) and TTA-P2 (filled symbols) groups with the exception of the 10–90% rise time at − 40 mV (D). *, p < 0.05. E, data points for channel conductance (G) are calculated from the graph presented in B by dividing current amplitudes with the driving force for ion permeation. Estimated reversal potential was taken to be 60 mV. Solid lines are fitted using eq. 2 (see Materials and Methods), giving half-maximal conductance (V50), which occurred at − 43.2 ± 0.6 mV with a slope k of 4.5 ± 0.6 mV in control conditions (open symbols). Likewise, V50 was − 43.6 ± 0.3 mV with a k of 3.5 ± 1.0 mV during TTA-P2 application (filled symbols). F, deactivating tail currents in controls (open symbols) and during application (filled symbols) of 100 nM TTA-P2 were fit with a single-exponential function. The resulting τ values are plotted (n = 5 cells). Points that are statistically significant are marked with an asterisk (p < 0.05). Inset, voltage-clamp protocol. Vertical lines in B–D and F represent S.E.M. of multiple determinations.
Fig. 5.
Fig. 5.
Effects of TTA-P2 on steady-state inactivation and recovery from inactivation of T currents in rat DRG cells. A, current availability curves at different conditioning potentials before and during application of 100 nM TTA-P2 to the same cells. B, the same data are normalized for clarity of presentation in the lower panel of this figure (n = 4 cells). Open symbols represent the control conditions; filled symbols represent the conditions during bath application of TTA-P2. Solid black lines are fitted using eq. 3 (see Materials and Methods), giving half-maximal availability (V50), which occurred at − 67.5 ± 0.3 mV with a slope k of 6.7 ± 0.3 mV in control conditions. In contrast, fitted V50 was −90 ± 1 mV with a k of 8.7 ± 1.0 mV in the conditions when TTA-P2 was applied. Vertical lines represent S.E.M. of multiple determinations. Inset, voltage-clamp protocol. C, TTA-P2 impairs recovery from inactivation in DRG cells. Symbols on the graph indicate averaged data from multiple cells (n = 5), and solid lines were fitted with a double-exponential equation (eq. 5; see Materials and Methods); yielding in predrug control: τ1, 2388 ± 300 ms; τ2, 155 ± 15 ms; TTA-P2: τ1, 1045 ± 181 ms; τ2, 43 ± 15 ms. Note that in the presence of TTA-P2, currents recovered (P2/P1) only to approximately 60% of control current (*, p < 0.05). If the fitting curve in the presence of TTA-P2 was constrained to 100% recovery, we obtained the following recovery τ values: τ1, 16,130 ± 4316 ms; τ2, 165 ± 50 ms (data not shown). Inset, voltage-clamp protocol for our double-pulse protocol.
Fig. 6.
Fig. 6.
TTA-P2 has antinocicpetive properties in the formalin pain model and has no effect on sensorimotor tests in mice. A, Adult mice experience significant analgesia after intraperitoneal injections of TTA-P2 at 5 mg/kg (open bars; n = 8) or 7.5 mg/kg (gray bars; n = 14) compared with experiments with vehicle (control, black bars; n = 15). Vertical bars indicate S.E.M.; the asterisks indicate a significance of p < 0.01 by Student's t test. B, histograms of average time in seconds in different sensorimotor tests using an inclined plane (left), platform (middle), and ledge (right) for mice given injections of 5 mg/kg TTA-P2 (white bars) or 7.5 mg/kg TTA-P2 (gray bars). Baseline measurements were taken 2 days before (controls, black bars). Note that black bars indicate controls with two different groups of animals receiving injections subsequently of either 5 or 7.5 mg/kg TTA-P2. Vertical bars indicate S.E.M. of multiple determinations. At either dose, TTA-P2 had no significant effect on either test, because p > 0.05 in comparisons of time points between controls and after injections of TTA-P2 (n = 6 mice in each group).
Fig. 7.
Fig. 7.
TTA-P2 effectively reverses hyperalgesia in diabetic STZ-treated rats but does not change baseline nociceptive thresholds in healthy rats. Top, baseline (B) PWLs in right paws (open bars) and left paws (filled bars) remain stable 1 h after intraperitoneal injections of TTA-P2 at 5, 7.5, or 10 mg/kg. Middle, dose-dependent antihyperalgesic effect of TTA-P2, which, at 10 mg/kg i.p., completely reversed thermal hyperalgesia in STZ-treated diabetic rats. Bottom, note that AS treatment also completely reversed diabetic hyperalgesia, as indicated by the apparent normalization of PWLs, and that subsequent applications of TTA-P2 at different doses did not change PWLs. Vertical bars indicate S.E.M. of multiple experiments (n = 4–8 per group). *, p < 0.05 baseline (B) versus treatment at different doses of TTA-P2; +, p < 0.05 for treatment with TTA-P2 versus new baseline in diabetic rats (STZ).
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