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5-Reduced Neuroactive Steroids Are Novel Voltage-Dependent Blockers of T-Type Ca²⁺ Channels in Rat Sensory Neurons in Vitro and Potent Peripheral Analgesics in Vivo

Department of Anesthesiology, University of Virginia School of Medicine, Charlottesville, Virginia (S.M.T., S.P., B.C.B., M.M.J., S.-H. K., V. J.-T.); and Departments of Molecular Biology and Pharmacology (X.J., K.R.N., D.F.C.) and Psychiatry (C.F.Z.), Washington University School of Medicine, St. Louis, Missouri

Received May 5, 2004; accepted July 26, 2004

	Abstract

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T-type Ca²⁺ channels are believed to play an important role in pain perception, and anesthetic steroids such as alphaxalone and allopregnanolone, which have a 5-configuration at the steroid A, B ring fusion, are known to inhibit T-type Ca²⁺ channels and cause analgesia in a thermal nociceptive model ( Soc Neurosci Abstr 29:657.9, 2003). To define further the structure-activity relationships for steroid analgesia, we synthesized and examined a series of 5-reduced steroids for their ability to induce thermal antinociception in rats when injected locally into the peripheral receptive fields of the nociceptors and studied their effects on T-type Ca²⁺ channel function in vitro. We found that most of the steroids completely blocked T-type Ca²⁺ currents in vitro with IC₅₀ values at a holding potential of -90 mV ranging from 2.8 to 40 µM. T current blockade exhibited mild voltage-dependence, suggesting that 5-reduced neuroactive steroids stabilize inactive states of the channel. For the most potent steroids, we found that other voltage-gated currents were not significantly affected at concentrations that produce nearly maximal blockade of T currents. All tested compounds induced dose-dependent analgesia in thermal nociceptive testing; the most potent effect (ED₅₀, 30 ng/100 µl) obtained with a compound [(3,5,17)-3-hydroxyandrostane-17-carbonitrile] that was also the most effective blocker of T currents. Compared with previously studied 5-reduced steroids, these 5-reduced steroids are more efficacious blockers of neuronal T-type Ca²⁺ channels and are potentially useful as new experimental reagents for understanding the role of neuronal T-type Ca²⁺ channels in peripheral pain pathways.

It is also becoming evident that certain neuroactive steroids can modulate voltage-gated ion channels (ffrench-Mullen et al., 1994; Nakashima et al., 1998). In particular, we previously reported that 5-reduced neuroactive steroids [e.g., alphaxalone and (3,5,17)-17-hydroxyestrane-3-carbonitrile, (+)-ECN] are potent blockers of voltage-gated Ca²⁺ channels in vitro (Todorovic et al., 1998). Voltage-gated Ca²⁺ channels are classified as low-voltage-activated (LVA or T-type) and high-voltage-activated (HVA) based on the membrane potentials at which the channels open (Bean, 1989; Hess, 1990; De Waard et al., 1996). T-type channels, which are of particular interest for this study, were first described in sensory neurons of the dorsal root ganglion (DRG) (Carbone and Lux, 1984) and activate with small membrane depolarizations, raising the possibility that they play crucial roles in the control of sensory neuron excitability (White et al., 1989). Although their unique biophysical properties (e.g., low activation threshold, slow deactivation, complete inactivation) make in vitro studies of T-currents relatively easy, the paucity of selective pharmacological tools for modulating their function impedes study of their function in vivo. Hence, despite their presence in peripheral sensory neurons (Schroeder et al., 1990; Scroggs and Fox, 1992; Cardens et al., 1995), the function of T-type Ca²⁺ channels in sensory processing in general, and in nociceptive processing in particular, remains poorly understood.

Recent evidence suggests that modulation of peripheral T channels influences somatic (e.g., thermal and mechanical) and visceral nociceptive inputs and that inhibition of T currents results in significant antinociception in a variety of animal pain models (Todorovic et al., 2001, 2002, 2003a, 2004; Kim et al., 2003). Therefore, T channels in peripheral nociceptors may be important, although previously unrecognized, targets for antinociceptive therapeutic agents.

The development of novel neuroactive steroids that are selective and potent blockers of neuronal T-type Ca²⁺ channels may greatly aid in revealing roles for these channels in sensory pathways (nociception in particular) and in the development of novel analgesics that might be safer and more effective for pain therapy. In the present study, we extend our earlier structure-activity studies of 5-reduced steroids to 5-reduced steroids (Fig. 1). We examined the in vitro effects of the compounds on T-type Ca²⁺ channels in freshly dissociated rat primary sensory neurons. In addition, we evaluated antinociceptive potential in vivo by measuring thermal nociception after injection of the steroids into peripheral receptive fields of sensory neurons. We found that 5-reduced steroids had activity in both experimental paradigms and that the potency of 5-reduced steroids to induce antinociception in vivo correlates well with their potency to block T currents in vitro.

View larger version (28K): [in this window] [in a new window]   Fig. 1. The structures of 5-reduced steroids evaluated in this study. Compounds with the 3-cyano group and 17-hydroxyl groups (3CN, 19-Nor3CN, 3CN, 19-Nor3CN) are shown on the left. Compounds with the 3-hydroxyl and 17-cyano groups (3OH, 19-Nor3OH, 3OH, 19-Nor3OH) are shown on the right.

	Materials and Methods

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Electrophysiological Methods, Solutions, and Current Isolation Procedures. Freshly dissociated DRG neurons from adult Sprague-Dawley rats of either sex (100–300 g) were obtained using enzymatic treatment and standard whole-cell, patch-clamp techniques as described elsewhere (Todorovic and Lingle, 1998; Todorovic et al., 1998). Glass coverslips with adherent DRG cells were transferred to a 35-mm culture dish with a total volume <1 ml. The solution application system consisted of multiple independently controlled glass capillary tubes; solution was removed from the opposite end of the chamber by constant suction. Manually controlled valves accomplished switching between solutions. Test solutions were maintained in all-glass syringes and allowed to flow by gravity. Use of glass syringes and capillary tubes minimized loss of lipophilic steroid compounds during perfusion. Changes in Ca²⁺ current amplitude in response to rapidly acting drugs or ionic changes were typically complete in 10 to 20 s. Switching between separate perfusion syringes, each containing control saline, resulted in no changes in Ca²⁺ current.

Most data from DRG cells were obtained from smaller diameter neurons (< 30 µm) with no visible processes. Voltage commands and digitization of membrane currents were done with Clampex 8.2 of the pClamp software package (Axon Instruments, Foster City, CA) running on an IBM-compatible computer. Membrane currents were recorded with an Axopatch 200B patch-clamp amplifier (Axon Instruments). Reported series resistance and capacitance values were taken from the amplifier settings. The average uncompensated series resistance was 7.6 ± 2.5 (mean ± S.D.) M, and the average membrane capacitance was 15 ± 4 pF in the 171 small DRG neurons studied. These were typically compensated 40 to 80% without oscillation in current traces. In 5 medium-sized DRG neurons (cell diameter 31–35 µm), the average membrane capacitance was 36 ± 2 pF, and series resistance was 1.4 ± 0.2 M. Although some medium-sized DRG neurons express "gigantic" T currents (e.g., Fig. 6A in Scroggs and Fox, 1992), we did not observe differences in the response to 5-reduced neuroactive steroids based on differences in the size of baseline T currents. To record T-currents, the membrane potential was held at -90 mV and stepped to -30 mV to evoke inward currents that inactivate almost completely during a 200-ms test pulse. The intracellular saline for recording T-currents consisted of 135 to 140 mM tetramethyl ammonium hydroxide, 10 mM EGTA, 40 mM HEPES, and 2 mM MgCl₂, titrated to pH 7.15 to 7.20 with hydrofluoric acid. In the presence of intracellular F^-, L-type HVA currents were blocked (Todorovic and Lingle, 1998), whereas N-type HVA currents were blocked by preincubation in 1 µM -conotoxin-GVIA. Because of the possibility of some residual HVA current contamination, all measurements of T-current amplitude in DRG cells were made from the peak of the inward current to the current remaining at the end of a 200-ms test step. For recording HVA Ca²⁺ currents, we used an intracellular solution containing 110 mM cesium methane sulfonate, 14 mM phosphocreatine, 10 mM HEPES, 9 mM EGTA, 5 mM Mg-ATP, and 0.3 mM Tris-GTP, pH adjusted to 7.15 to 7.20 with CsOH (standard osmolarity, 300 mOsM). When this internal saline was used for recording T-currents, most of the HVA current in these cells was blocked by preincubating cells with 1 µM -conotoxin-GVIA, 2 µM -conotoxin-MVIIC and including 5 µM nifedipine in the external solution to block N-, P/Q-, and L-type HVA, respectively. The amount of block of T current by steroids was not affected by using either internal saline (n = 2–4 cells for each steroid; data not shown). The standard extracellular saline for recording T- and HVA Ca²⁺ currents contained 152 mM tetraethylammonium chloride, 10 mM HEPES, and 10 mM BaCl₂, adjusted to pH 7.4 with tetraethylammonium hydroxide; osmolarity, 316 mOsM.

Voltage-gated Na⁺ currents were recorded using electrodes that were pulled from borosilicate glass and had resistances of 0.6 to 1.0 M when filled with pipette solution containing 140 mM CsF, 2 mM MgCl₂, 1 mM EGTA, 10 mM Na-HEPES (pH adjusted to 7.3 mM with CsOH, osmolarity adjusted to 310 mM mOsM using sucrose). Cells were superfused with solution containing 30 mM NaCl, 110 mM CoCl₂, 3 mM KCl, 1 mM CaCl₂, 0.1 mM CdCl₂, 2 mM MgCl₂, and 10 mM HEPES (pH adjusted to 7.4 with NaOH). On establishing whole-cell configuration, a series resistance compensation of 75% was applied, and cells were held at -90 mV. Cells that showed evidence of poor voltage control, as reflected by the shape of the current-voltage curve, were excluded from the study.

The standard external solution for recording voltage-gated K⁺ currents contained 140 mM NaCl, 5 mM KCl, 2 mM MgCl₂, 2.0 mM CaCl₂, 10 mM glucose, and 10 mM HEPES, pH 7.4, with NaOH. The standard pipette solution used to record voltage-gated K⁺ currents contained 110 mM KCl, 14 mM phosphocreatine, 10 mM HEPES, 9 mM EGTA, 5 mM Mg-ATP, and 0.3 mM Tris-GTP, 2 mM QX 314, pH adjusted to 7.2 with CsOH. To record voltage-gated K⁺ currents cells were held at -60 mV and depolarized to +60 mV by a 150-ms depolarizing step.

Analysis of Current Blockade. The percentage reduction in peak Ca²⁺ inward current carried by Ba²⁺ ions at a given steroid concentration was used to generate concentration-response curves. For each of these curves, all points are averages of multiple determinations obtained from at least five different cells. On all plots, vertical bars indicate standard errors. Mean values on all concentration-response curves were fit to the following function: PB ([Neurosteroid]) = PB_max /(1 + (IC₅₀/[Neurosteroid]) ^nH), where PB_max is the maximal percentage block of peak T current, IC₅₀ is the concentration that produces 50% maximal inhibition, and n_H is the apparent Hill coefficient for blockade. Fitted values are typically reported with 95% linear confidence limits.

The voltage-dependence of steady-state activation and inactivation was described by the Boltzmann distribution, I(V) = I_max/(1 + exp[-(V- V₅₀)/k]), where I_max represents maximal activatable current, V₅₀ represents the voltage where half of the current is activated or inactivated, and k (units of millivolts) represents the voltage dependence of distribution.

Steroids and Reagents. The synthesis, spectroscopic and physical properties of 3OH, 19-Nor3OH, 3OH, and 19-Nor3OH have been reported previously (Han et al., 1996). The starting material for the preparation of 3CN and 3CN was (5,17)-17-hydroxyandrostan-3-one. The 17-hydroxy group of this starting material was esterified using pyridine/acetic anhydride, and the 3-cyano group was introduced using a method described previously (Han et al., 1996). The resultant epimeric 3-cyanosteroids were separated by column chromatography on silica gel, and the 17-acetate group was hydrolyzed to regenerate the 17-hydroxy group of the final products, 3CN and 3CN. Likewise, 19-Nor3CN and 19-Nor3CN were prepared from (5,17)-17-hydroxyestran-3-one. The spectroscopic and physical properties of the previously unknown 3-cyanosteroids are given below.

3CN: colorless crystals, m.p., 176 to 178°C; IR, 3485, 2945, 2931, 2868, 2248, 1459, 1450, 1071, 1055 cm^-1; ¹H NMR (300 MHz, CDCl₃) 3.64 (1 H, t, J = 8.7 Hz, CHOH), 2.42 (1 H, tt, J = 12.3 Hz, 3.9 Hz, CHCN), 0.94 (3H, s), 0.71 (3 H, s); ¹³C NMR (75 MHz, CDCl₃) 122.92, 81.77, 50.90, 42.98, 42.52, 40.58, 36.69, 35.95, 35.71, 34.60, 30.43, 30.14, 28.76, 26.53, 25.71, 24.67, 23.51, 23.23, 20.26, 10.94. Anal. calculated for C₂₀H₃₁NO: C, 79.68; H, 10.36; N, 4.64. Found: C, 79.75; H, 10.51; N, 4.74.

3CN: m.p., 150 to 151°C; IR, 3496, 2931, 2867, 2238, 1451, 1377, 1330, 1249, 1055, 955 cm^-1; ¹H NMR (300 MHz, CDCl₃) 3.62 (1 H, t, J = 8.4 Hz, CHOH), 3.00 (1 H, s, w_1/2 = 11.5 Hz, CHCN), 1.01 (3H, s), 0.72 (3H, s); ¹³C NMR (75 MHz, CDCl₃) 122.54, 81.82, 51.02, 42.96, 40.50, 39.24, 36.77, 35.66, 35.11, 32.63, 30.44, 28.57, 27.82, 26.31, 25.81, 23.57, 23.22, 23.05, 20.33, 10.95. Anal. calculated for C₂₀H₃₁NO: C, 79.68; H, 10.36; N, 4.64. Found: C, 79.80; H, 10.51; N, 4.31.

19-Nor3CN: m.p., 150 to 151.5°C; IR, 3427, 2917, 2868, 2238, 1452, 1377, 1263, 1073, 1053 cm^-1; ¹H NMR (300 MHz, CDCl₃) 3.67 (1 H, t, J = 8.5 Hz, CHOH), 2.47 (1 H, tt, J = 12.3 Hz, 3.9 Hz, CHCN), 0.74 (3 H, s); ¹³C NMR (75 MHz, CDCl₃) 122.90, 81.92, 49.98, 43.15, 41.77, 39.96, 38.48, 36.66, 36.11, 31.35, 30.53, 30.09, 28.78, 27.07, 25.61, 25.23, 23.92, 23.21, 11.04. Anal. calculated for C₁₉H₂₉NO: C, 79.39; H, 10.17; N, 4.87. Found: C, 79.60; H, 9.97; N, 4.94.

19-Nor3CN: m.p., 148–149°C; IR, 3469, 2915, 2870, 2236, 1453, 1380, 1264, 1073, 1055 cm^-1; ¹H NMR (300 MHz, CDCl₃) 3.64 (1 H, t, J = 8.5 Hz, CHOH), 3.02 (1 H, s, w_1/2 = 10 Hz), 0.75 (3 H, s); ¹³C NMR (75 MHz) 122.55, 81.99, 50.11, 43.15, 41.73, 40.60, 38.40, 36.73, 32.83, 31.19, 30.56, 28.54, 27.87, 25.72, 25.35, 24.05, 23.20, 22.42, 11.02. Anal. calculated for C₁₉H₂₉NO: C, 79.39; H, 10.17; N, 4.87. Found: C, 79.50; H, 10.05; N, 4.95.

All steroids were dissolved in DMSO to make 10 to 30 mM stock solutions. Aliquots of the stock solutions were added to the standard external solution to achieve the final concentrations stated in the text. The final concentration of DMSO was less than 0.6% in these experiments; this concentration of DMSO did not affect I_Ba (data not shown, n = 5).

Behavioral Experiments. All experimental protocols were approved by the University of Virginia Animal Care and Use Committee, Charlottesville, VA, and in accordance with the Guide for the Care and Use of Laboratory Animals as adopted and promulgated by the US National Institutes of Health. Every effort was made to minimize animal suffering and the number of animals used.

Chemicals and Animals. Adult female Sprague-Dawley rats (weight, 300–330 g) were used for all in vivo experiments because female rats are less aggressive and easier to handle during pain testing. For behavioral experiments, most steroids were dissolved as a stock solution (in 100% DMSO) and diluted in saline so that the highest final DMSO concentration was 3%. Injections of 3% DMSO in saline did not affect latency for paw withdrawal in rats (n = 6 rats, data not shown). For experiments with 3OH and 3OH, 20% cyclodextrin in saline was used to dissolve steroids because higher doses of these steroids (e.g., 3 µg/100 µl) crystallized in DMSO and saline after several minutes. Intraplantar injections of 20% cyclodextrin and saline in control experiments did not change thermal paw withdrawal latencies (PWLs) in six animals (data not shown). Likewise, in three cells, 20% cyclodextrin did not affect T currents or the effects of 3 µM 3OH on T currents (data not shown).

Assessment of Thermal Nociception. The nociceptive response to thermal stimulation was measured using a paw thermal stimulation system consisting of a clear plastic chamber (10 x 20 x 24 cm) sitting on a clear, elevated glass floor and temperature regulated at 30°C (Hargreaves et al., 1988; Jevtovic-Todorovic et al., 1998, 2001; Jevtovic-Todorovic et al., 2003). Each animal was placed in the plastic chamber to accommodate for 15 min. A radiant heat source mounted on a movable holder beneath the glass floor was positioned to deliver a thermal stimulus to the plantar side of the hind paw. When the animal withdraws the paw, a photocell detects interruption of a light beam reflection and the automatic timer shuts off. This method has a precision of ± 0.05 s for the measurement of PWL. To prevent thermal injury, the light beam is automatically discontinued at 20 s if the rat fails to withdraw its paw.

To test the effects of neurosteroids in peripheral receptive fields, we injected 100 µl of test compounds intradermally in the ventral side of the right hind paw of animals. The noninjected side (left hind paw) was used as a control in each animal. All solutions were pH-balanced to 7.4 to avoid skin irritation. No signs of skin inflammation, discoloration, or irritation were noted at the sites of injection with test compounds. All doses are expressed in micrograms per 100 µl. At 10, 20, and 60 min after drug administration, the thermal stimulus was applied and PWLs were measured. The investigator assessing behavior measures was kept unaware of pharmacological interventions.

Statistical Analysis. Baseline values (B) were compared with thermal PWLs of noninjected and injected paws at various times during the testing as indicated in the figures (post-treatment values). In the data displayed, every point is an average of at least nine animals and values represent mean ± S.E.M. Statistical analysis was performed using an analysis of variance comparing withinsubject variables: paw condition (injected versus noninjected) and test session (before drug administration or 10, 20, and 60 min after treatment). Pair-wise comparisons were also conducted, and levels were adjusted using the Bonferroni procedure when appropriate.

Dose-response data were fit to the function: PI([Neurosteroid]) = PI_max/(1+(ED₅₀/[Neurosteroid])^nH), where PI_max is the maximal increase in PWLs caused by a drug in the injected versus noninjected paw 10 min after injection; the ED₅₀ is the dose that produces half-maximal increase in PWLs indicating an analgesic effect; and n_H is the apparent Hill coefficient indicating the slope of the curve. Fitted values are reported with 95% linear confidence limits. Fitting was done with Origin 7.0 software (OriginLab Corp., Northampton, MA).

	Results

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In Vitro Effects of 5-Reduced Neuroactive Steroids on T-Currents in Acutely Dissociated Rat DRG Neurons
Structure-Activity Studies. The DRG contains cell bodies of primary afferent (sensory) fibers with long processes that originate as sensory endings in the periphery and terminate in the dorsal horn of the spinal cord. Whole-cell recordings from dissociated DRG neurons of adult rats are used to study peripheral nociceptive mechanisms in vitro, because the small size of the peripheral nerve endings precludes direct measurement of the currents from the sensory endings. We limited our experiments to small-diameter (20–30 µm) and smaller medium-diameter (31–35 µm) acutely dissociated neurons because the majority of these cells are involved in peripheral nociceptive processing (Coderre et al., 1993; Levine et al., 1993; Snider and McMahon, 1998).

T-type Ca²⁺ currents were isolated as described under Materials and Methods and typically monitored with voltage steps to -30 mV from a holding potential of -90 mV. In our previous study with 5-reduced steroids, we found that neuroactive steroids with a cyano group at position 3 of the steroid were most potent in blocking isolated T currents [e.g., (+)-ECN] (Todorovic et al., 1998). Therefore, in our studies of 5-reduced neuroactive steroids, we initially focused on 5-reduced analogs with a cyano group at position 3 of the steroid (i.e., 3CN and 3CN).

3CN is an effective and concentration-dependent blocker of T currents in sensory neurons. Figure 2A shows representative tracings of the inhibitory effects of two 3CN concentrations—3 and 30 µM—on T-currents in DRG cells. The time course of the blocking effects of 3CN on T-currents demonstrates the stability of responses during the recordings and the fast kinetics (e.g., fast onset and offset) of T current blockade by 3CN (Fig. 2B). Similar fast kinetics were observed with other 5-reduced neuroactive steroids. The calculated IC₅₀ for T-current blockade is 11 ± 2 µM; the maximal blocking effect (81 ± 5%) is achieved with 60 µM, and the fitted concentration-response curve approaches 100% blockade (Fig. 2D).

View larger version (25K): [in this window] [in a new window]   Fig. 2. Inhibitory effects of 3CN and 3CN on T current in small-size DRG cells. A, traces of T-currents before (control), during, and after (wash) the application of two different concentrations of 3CN (3 and 30 µM). The application of 3CN depressed the current amplitude in a dose-dependent fashion but did not have an effect on the apparent kinetics of the current. B, the time course of T-current blockade in the same DRG cell induced by escalating concentrations of 3CN (from 1 to 60 µM), demonstrates a rapid and dose-dependent current blockade that was almost complete at 60 µM. The return to baseline was fast after each washout. The horizontal bars indicate the time of drug application. C, traces of T-current blockade in a DRG cell induced by two concentrations (3 and 60 µM) of 3CN demonstrates reversible and dose-dependent current blockade. D, concentration-response curves for T current blockade by 3CN () or 3CN (). The symbols indicate the average of multiple determinations ± S.E.M., with the solid line representing the best fit of the Hill equation. The fitted values for the curves are: 3CN, IC50 = 11 ± 2 µM, n = 1.0 ± 0.1, and max block = 94 ± 7% (n = 10 cells); 3CN, IC50 = 20 ± 10 µM, n = 1.0 ± 0.1, and max block = 84 ± 17% (n = 7 cells).

3CN, the 3-epimer of 3CN, was an effective although less potent (2-fold) blocker of T currents (Fig. 2C). The calculated IC₅₀ for 3CN was 20 ± 10 µM, with similar maximal blockade as estimated with 3CN (Fig. 2D). This result indicates that the stereochemistry of the cyano group at position 3 affects the potency of T current blockade.

Because the presence or absence of a methyl group (numbered carbon 19 according to the rules of steroid nomenclature) at steroid position 10 has been shown to affect the potency of steroid modulation of 5-steroids at GABA_A receptors (Han et al., 1996), we investigated the effect of this structural modification on the potency of T current blockade by 5-steroids. We found that 19-Nor3CN and 19-Nor3CN are potent blockers of T currents and, as indicated in Fig. 3, are more potent blockers of T currents than their structural analogs containing the methyl group at position 10, 3CN and 3CN, respectively. For example, 19-Nor3CN at 3 and 60 µM induced 24 and 80% blockade of T current (right tracing), respectively, whereas its analog, 3CN, induced less blockade at the same concentrations (10 and 65%, respectively) (Fig. 2C). Indeed, both 19-Nor3CN and 19-Nor3CN produced more potent blockade of T-currents than their structural analogs, as demonstrated by 3- and 2-fold leftward shifts from the concentration-response curves for 3CN and 3CN, respectively (the calculated IC₅₀ values for 19-Nor3CN and 19-Nor3CN was 6.3 ± 0.3 and 6.0 ± 1.0 µM, respectively) (Fig. 3B). These results indicate that a methyl group at position 10 has a negative effect on potency and that the stereochemistry of the cyano group at position 3 has no significant effect on the potency of these 19-norsteroids for block of T-channel current.

View larger version (26K): [in this window] [in a new window]   Fig. 3. The replacement of a methyl group with a hydrogen atom at position 10 of the 3-cyano,17-hydroxy steroid nucleus increases the potency of T-current blockade. A, raw traces on the left depict the effects of 19-Nor3CN at two concentrations, 3 and 60 µM, on T currents in a rat DRG neuron. Raw traces on the right depict the effects of 19-Nor3CN (an analog of 3CN with a hydrogen atom instead of a methyl group at position 10) on T currents at the same concentrations, 3 and 60 µM. Note that 19-Nor3CN and 19-Nor3CN induced T-current blockade of similar magnitude but 19-Nor3CN caused more profound blockade of T currents than 3CN (Fig. 2C) at each concentration (at 3 µM, 25 and 10% blockade, respectively, and at 60 µM, 80% and 65% blockade, respectively). B, left, concentration-response curve for 19-Nor3CN (fitted solid line was derived from the Hill equation). The calculated IC50 was 6.3 ± 0.7 µM, and the Hill coefficient was n = 1.0 ± 0.1, with a maximal block of T-current of 88 ± 3% (n = 7 cells). The dotted line, which represents the concentration-response curve for 3CN (the actual data points are presented in Fig. 2 D), demonstrates the lower potency of 3CN (3-fold rightward shift). Right, concentration response curve for 19-Nor3CN (an analog of 3CN with a hydrogen atom instead of a methyl group at position 10). Again, the solid line was derived from the Hill equation, and the calculated IC50 = 6.0 ± 0.9 µM with n = 1.0 ± 0.1 and the maximal block of T current = 89.9 ± 9.0% (n = 8 cells). The dotted line, which represents the concentration-response curve for 3CN (the actual data points are presented in Fig. 2D), indicates the lower potency of 3CN (about 2-fold rightward shift).

5-Reduced steroids that have a cyano group in the -configuration at position 17 and a hydroxyl group at position 3 are known modulators of GABA-A receptors. The steroid 3OH is a potent enhancer and the steroid 3OH is a potent activation-dependent blocker of GABA-mediated currents (Han et al., 1996; Wang et al., 2002). To determine whether steroids with this functional group substitution pattern were also modulators of T-channels, we evaluated 3OH, 3OH, and the corresponding 19-norsteroids (19-Nor3OH, 19-Nor3OH).

We found that the two structural analogs 3OH and 19-Nor3OH, which differ only by the presence or absence of a methyl group in position 10 (Fig. 1), have substantially different T-current blocking potential. 3OH was more potent in blocking T-currents than 19-Nor3OH (Fig. 4). For example, 3OH, at 60 µM, blocked 80% of the baseline T current (Fig. 4B), whereas 19-Nor3OH blocked only about 58% of the T current at the same concentration (Fig. 4A). The blockade of T current was concentration-dependent (Fig. 4C), and the calculated IC₅₀ was 8.2 ± 0.9 and 40 ± 10 µM for 3OH and 19-Nor3OH, respectively, with the fitted value for the maximal block of 98 ± 3% for both analogs (Fig. 4D).

View larger version (28K): [in this window] [in a new window]   Fig. 4. The replacement of a hydrogen atom with a methyl group at the 10 position of 3-hydroxy, 17-cyano steroid nucleus increases the potency of T-current blockade. Raw traces of T currents before (control), during, and after the application (wash) of 60 µM 19-Nor3OH (A) or 60 µM 3OH (B) (an analog of 19-Nor3OH with a methyl group instead of a hydrogen atom in position 10), demonstrate about 60 and 85% blockade of the peak current, respectively. C, the time course of T current blockade induced by two concentrations of 3OH, 1 and 60 µM, demonstrates a rapid and dose-dependent current blockade that was almost complete at 60 µM. The return to baseline was fast after drug washout. D, concentration-response curves for T current blockade by 3OH () or 19-Nor3OH (). The symbols indicate the average of multiple determinations ± S.E.M., with the solid line representing the best fit of the Hill equation. The fitted values for the curves are: 3OH, IC50 = 8.2 ± 0.9 µM, n = 1.0 ± 0.1, and max block = 100 ± 4% (n = 8 cells); 19-Nor3OH, IC50 = 40 ± 10 µM, n = 1.8 ± 0.3, and max block = 98 ± 11% (n = 8 cells).

Finally, we examined 19-Nor3OH and 3OH. 19-Nor3OH blocks about 25% of T currents at a concentration of 3 µM (Fig. 5A, left tracing), whereas 3OH blocks about 55% of T current in sensory neurons at the same concentration (Fig. 5A, right tracing). Figure 5B depicts the time course of T-current blockade caused by the three escalating concentrations of 3OH—1, 3, and 30 µM. At 30 µM, 3OH completely blocks T currents. At higher concentrations, the effects of 3OH were only partially reversible (Fig. 5B), presumably caused by slow washout of this compound. The blockade of T current was concentration-dependent, and the calculated IC₅₀ was 2.8 ± 0.6 and 6.0 ± 0.6 µM for 3OH and 19-Nor3OH, respectively (Fig. 5C). Overall, 3OH is the most potent blocker of T current in rat sensory neurons (i.e., 3OH is approximately 2- to 13-fold more potent than the other 5-reduced neuroactive steroids examined in this study).

View larger version (25K): [in this window] [in a new window]   Fig. 5. The replacement of a hydrogen atom with a methyl group at position 10 of the 3-hydroxy,17-cyano steroid nucleus increases the potency of T-current blockade. A, raw traces of T currents before (control), during, and after the application (wash) of 3 µM 19-Nor3OH (left) or 3 µM 3OH (right) (an analog of 19-Nor3OH with a methyl group instead of a hydrogen atom in position 10), demonstrate approximately 25% and more than 50% blockade of the peak current, respectively. B, the time course of T-current blockade in the same DRG cell induced by the three concentrations (1, 3, and 30 µM) of 3OH demonstrates potent and dose-dependent current blockade (about 55% current blockade with 3 µM, and a complete block with 30 µM). A full recovery of the current is observed only after 3 µM 3OH. Higher concentrations (e.g., 30 µM) are only partially reversible, presumably because of slow washout of this compound. C, concentration-response curves for T current blockade by 19-Nor3OH () or 3OH (). The symbols indicate the average of multiple determinations ± S.E.M., with the solid line representing the best fit of the Hill equation. The fitted values for the curves are: 19-Nor3OH, IC50 = 6.0 ± 0.6 µM, n = 1.0 ± 0.1, and max block = 99 ± 11% (n = 9 cells); 3OH, IC50 = 2.8 ± 0.6 µM, n = 1.0 ± 0.2, and max block = 100 ± 4% (n = 8 cells).

Mechanisms of Blockade of T-type Ca²⁺ Currents in Rat Sensory Neurons. We next determined whether 5-reduced steroids affect kinetic properties of T currents. Figure 6A depicts a family of inward currents evoked from a holding potential of -90 mV in the absence (left) and presence (right) of 3 µM 3OH. 3OH did not significantly alter the time course of T current activation, measured as 10-to-90% rise time (Fig. 6B, n = 5 cells), or inactivation (Fig. 6C; n = 5 cells per data point), measured by the fit of a single exponential function to the decaying phase of the current at potentials from -50 to -10 mV.

View larger version (28K): [in this window] [in a new window]   Fig. 6. The most potent 5-reduced neuroactive steroid, 3OH, causes voltage-dependent block of T currents. A, a family of T currents was evoked by voltage steps from a holding potential of -90 to -60 through -20 mV in a smaller medium-sized DRG cell before (left) and during (right) the application of 3OH. B, activation times, measured as the 10-to-90% rise time of peak currents at potentials from -50 to -10 mV in experiments similar to those presented in A (n = 5 cells), were not significantly different before (filled symbols) and during the application of 3 µM 3OH (. Vertical lines indicate SE. C, inactivation time constants () were determined from a single exponential fit of the decaying portions of T currents in the same cells as in B of this figure and plotted against test potential for control () and 3OH (). 3OH had very little effect on the time course of the inactivation. D, the steady state inactivation curves obtained in the absence () and presence of 3 µM 3OH () show a shift of 11 mV in the hyperpolarizing direction by 3OH (n = 5 cells). The solid lines are the best fit of the Boltzmann distribution with a calculated V50 of -69.0 ± 1.0 mV and a slope factor of 8.4 ± 0.6 mV under control conditions. The V50 in the presence of 3 µM 3OH was -80.0 ± 1.0 mV, with a slope factor of 10.0 ± 1.0 mV. E, 3OH has very little effect on the voltage-dependence of T current activation in DRG cells. The best fit of the Boltzmann equation before (solid line) and during (dotted line) the application of 3 µM3OH (n = 4 cells per data point) was obtained by measuring tail currents at the end of a 20-ms depolarizing pulse to activate T channels at different potentials as indicated on the x-axis (from Vh of -90 mV). Under these conditions, the tail current amplitude is directly proportional to the number of open channels at the end of the activating pulse. In the absence of 3OH (), half-maximal activation was -33.3 ± 1.2 mV with a slope factor of 9.2 ± 0.9 mV. In the presence of 3OH (, half-maximal activation was -34.9 ± 1.0 mV, and slope factor was 9.4 ± 0.8 mV.

Because many blockers of ion channels exhibit voltage-dependent features, we examined whether 3OH alters voltage-dependent inactivation of T channels at different potentials. For these studies, we selected 3OH, 3OH, and 3CN. T currents were evoked by a voltage step to -30 mV after a 5-s conditioning step at potentials from -110 to -50 mV in the presence and absence of a neuroactive steroid. This protocol defines the voltage-dependence of T current fractional availability in rat sensory neurons (Todorovic and Lingle, 1998). The normalized maximal current elicited from each conditioning potential is plotted as a function of the conditioning potential (Fig. 6D) (n = 6 cells). Based on the best fits using the Boltzmann equation, we found that, under control conditions, half availability (V₅₀) occurred at -69 mV with a slope factor of 8 mV. However, in the presence of 3 µM3OH, V₅₀ occurred at -80 mV with a slope factor of 10 mV, thus shifting the steady state inactivation to more negative potentials. Likewise, 3OH (n = 5 cells) and 3CN (n = 4 cells) also exhibited mild voltage-dependent block, shifting the steady-state inactivation to more negative potentials by about 10 mV (data not shown). These experiments indicate that 5-reduced neuroactive steroids exert more prominent blocking effects on T currents at more positive conditioning potentials. This may be functionally important because it suggests that these agents could be more effective under in vivo conditions when neuronal membrane potentials are in a more depolarized state (e.g., tissue injury, where the nociceptive fibers are more excitable and actively firing). On the other hand, there was very little effect of 3OH on the voltage dependence of activation (n = 4 cells, Fig. 6E).

Selectivity of Blockade of T-Type Ca²⁺ Currents in Rat Sensory Neurons. Various voltage-gated ion channels (e.g., HVA Ca²⁺ channels and voltage-gated Na⁺ and K⁺ channels) have been shown to play roles in nociceptive processing (Kirchhoff et al., 1992; Caterina and Julius, 1999; Silbert et al., 2003; Bell et al., 2004). In addition, some neuroactive steroids (e.g., 5-reduced steroids) are known to modulate HVA Ca²⁺ currents (ffrench-Mullen et al., 1994; Nakashima et al., 1998). To investigate whether 5-reduced steroids also block other voltage-gated ion channels, we examined the effects of 3OH, one of the most potent T-type Ca²⁺ currents blockers (Table 1), against HVA Ca²⁺ currents and voltage-gated Na⁺ and K⁺ channels. We studied the effects of this steroid on inward currents evoked by voltage steps from -70 mV to a test potential of -10 mV in cells that did not express T-type Ca²⁺ currents. Traces from a typical experiment with HVA currents are shown in Fig. 7A, and the time course of effect is depicted in Fig. 7B. At the lowest concentration (3 µM), 3OH did not significantly alter HVA currents (8 ± 4% block, p > 0.05, n = 10 cells); at the highest concentration (30 µM), 3OH blocked about 72 ± 8% of HVA currents (n = 4 cells). Note that the blocking effects of 3OH were substantially more pronounced upon T currents in DRG cells recorded under similar conditions. When the membrane potential was held at -70 mV (near the resting membrane potential) followed by membrane depolarization to -30 mV (test potential), most of the evoked T current was blocked by 3 µM 3OH (73 ± 6%, n = 7 cells) (Fig. 7C). The inhibitory effect of 3OH was concentration-dependent, as shown in Fig. 5D, and the calculated IC₅₀ for T-current blockade under these voltage conditions was 0.76 ± 0.16 µM, with maximal block approaching 100%. The calculated IC₅₀ for HVA current blockade was 10 ± 1 µM, with a fitted maximal block of 79 ± 1%. We also examined the effects of 10 µM 19-Nor3CN on HVA currents in 3 DRG cells and found no significant effect (3 ± 2% block, p > 0.05, data not shown). Thus, our data indicate that 5-reduced steroids are more than 10-fold less effective in blocking DRG HVA than T-type Ca²⁺ currents.

View this table: [in this window] [in a new window]   TABLE 1 Structure-activity relationships for the blockade of T-currents in rat DRG cells and peripheral antinociception by 5 -reduced neuroactive steroids A summary of effects of steroids used in this study on isolated T currents and peripheral thermal nociception.

View larger version (23K): [in this window] [in a new window]   Fig. 7. Effects of 3OH on LVA and HVA Ca2+ and voltage-dependent Na+ and K+ currents in rat DRG neurons. A, traces of HVA currents in a small DRG cell evoked from a holding potential of -70 to a test potential of 0 mV before and during the application of 3, 10, and 30 µM3OH. Note that 3OH, at 3 µM, had very little effect on peak HVA currents. At this concentration, 3OH almost completely blocked T currents evoked from the same holding potential (C). B, time course of the effects of escalating concentrations of 3OH upon HVA currents from the same cell depicted in A. Horizontal bars indicate times of drug application. Note the partial recovery after the application of 30 µM 3OH, which blocked about 80% of HVA current. C, at 3 µM, 3OH almost completely blocked T currents evoked by the membrane depolarization from -70 to -30 mV. D, the average concentration-response curves for the effects of 3OH on HVA (open symbols) and LVA currents (closed symbols) in experiments where holding potential was -70 mV (see traces in A and C). Symbols indicate the average of multiple determinations, vertical lines are S.E., and solid line is best fit of the Hill equation. The fitted values for the curves shown are: LVA (T) current: IC50 = 0.76 ± 0.20 µM, n = 1.0 ± 0.2, and max block = 100 ± 4% (n = 9 cells); HVA current: IC50 = 10 ± 1 µM, n = 1.6 ± 0.5, and max block = 79 ± 1% (n = 12 cells); E, traces from an experiment where voltage-gated Na+ currents were recorded in a DRG cell from a holding potential of -90 mV to a test potential of -10 mV. The left trace depicts the control, and the right trace depicts a very small effect of 3 µM 3OH upon peak of the inward current (about 4% block). F, traces of outward voltage-gated K+ currents in a different DRG cell evoked from Vh of -60 mV to Vt of +60 mV. At 3 µM, 3OH had no effect on the current.

At 3 µM, a concentration that blocks most of the T current, 3OH did not significantly affect either voltage-gated Na⁺ currents (4 ± 2% block, p > 0.05, n = 7 cells; Fig. 7E) or voltage-gated K⁺ currents (2 ± 1% increase, p > 0.05, n = 5 cells; Fig. 7F).

To examine the selectivity of another representative 5-reduced steroid that was also a very potent T-current blocker (Table 1), we studied 19-Nor3CN and found that this steroid, when applied at a concentration that caused near maximal blockade of T current (10 µM) (Fig. 3B, right), had no effect upon either HVA currents (3 ± 2% block, p > 0.05, n = 3 cells; data not shown), Na⁺ currents (3 ± 3% increase, p > 0.05, n = 7 cells; data not shown), or K⁺ currents (no change, n = 6 cells, data not shown).

In Vivo Effects of 5-Reduced Neuroactive Steroids on Peripheral Thermal Nociception in Adult Rats
Structure-Activity Studies. To determine whether 5-reduced neuroactive steroids modify nociceptive responses in vivo, we designed a series of in vivo studies whereby 100 µl of test compounds were injected directly into the peripheral receptive fields of skin nociceptors located in the hind paw of adult rats, and the latency to paw withdrawal in the presence of a radiant heat stimulus was measured (Hargreaves et al., 1988; Jevtovic-Todorovic et al., 1998, 2003; Todorovic et al., 2001, 2002, 2004).

Intraplantar injections of 5-reduced steroids into the right paw produced potent and dose-dependent antinociceptive responses in vivo at doses ranging from 0.003 to 3 µg/100 µl (Fig. 8). The maximum increase in PWLs (up to 60% from the pretreatment) was achieved 10 min after injection. This local analgesic effect lasted up to 60 min. Thermal PWLs on the noninjected (left) paw () remained unchanged during 60-min testing in all experiments, indicating a lack of systemic antinociceptive effect. The injection of the same volume of vehicle (3% DMSO dissolved in saline) had no effect on PWLs (n = 9 animals, data not shown).

View larger version (40K): [in this window] [in a new window]   Fig. 8. 5-reduced steroids induce time- and dose-dependent peripheral antinociception in thermal PWL testing. The plantar side of the right paw was injected with 100 µl of a test solution containing a given 5-reduced steroid at doses ranging from 0.003 to 3 µg, as indicated on the graphs. The peak antinociceptive effect was recorded at 10 min with a complete return to baseline by 60 min. The left (noninjected) paw showed stable withdrawal latencies throughout the testing (denoted control in the graphs). Baseline testing (B) was performed 2 days before actual drug testing and compared with 0 time PWLs to confirm the stability of PWLs recordings. Significant differences in PWLs between injected (right) and noninjected (left) paws are indicated by asterisks (*) with p < 0.05. The significant overall analysis of variance values and p values from subsequent pair-wise comparisons are listed in Table 2. The arrows indicate the time of injection (n = 9–12 animals per group).

Based on our in vivo study, the antinociceptive potency of the 3-cyano steroids is little influenced by the stereochemistry of the cyano group but is increased by the absence of the methyl group at position 10. For example, the maximal increase in PWLs at 10 min induced with 3 µg/100 µl 19-Nor3CN and 19-Nor3CN is about 6 s, whereas the maximal increase in PWLs induced with the same doses of 3CN and 3CN is about 3.5 to 4.0 s. On the other hand, although the antinociceptive potency of the 3-hydroxy steroids is once again little affected by the stereochemistry of the hydroxyl group, the presence of the methyl group at position 10 increases potency. For example, the maximal increase in PWLs at 10 min induced with 3 µg/100 µl of 3OH and 3OH is about 6 s, whereas the maximal increase in PWLs induced with 19-Nor3OH and 19-Nor3OH is about 4.5 s.

To further analyze the functional consequences of the presence or absence of the methyl group at position 10, we constructed dose-response curves (solid lines) that were plotted against the corresponding increases in PWLs (as measured by the right-left paw difference in PWLs at 10 min) (Fig. 7). As shown in Fig. 9, the antinociceptive dose-response curves of 19-Nor3CN and 19-Nor3CN were shifted approximately 2- to 2.5-fold to the left compared with 3CN and 3CN (Fig. 9, A and B). Even more impressively, the antinociceptive dose response curves of 3OH and 3OH were shifted approximately 4- to 10-fold to the left compared with 19-Nor3OH and 19-Nor3OH (Fig. 9, C and D). Note that the most potent blocker of T currents in rat sensory neurons in vitro, 3OH (IC₅₀ of 2.8 µM) (Fig. 5C) is also the most potent antinociceptive steroid in vivo (ED₅₀ of 0.03 µg/100 µl) (Fig. 9D); conversely, the least potent blocker of T currents in vitro, 19-Nor3OH (IC₅₀ of 40 µM) (Fig. 4D), is also the least potent antinociceptive steroid in vivo (ED₅₀ of 1.05 µg/100 µl; Fig. 9C).

View larger version (24K): [in this window] [in a new window]   Fig. 9. Dose-response curves for thermal antinociception after local intraplantar injections of 5-reduced neuroactive steroids. The average difference in PWLs (seconds) between injected and noninjected paws at 10 min (the maximal effect) was plotted against the corresponding doses of a given 5-reduced neuroactive steroid (in micrograms per 100 µl) and the best fits were obtained using the Hill equation (solid lines). A, 19-Nor3CN is a more potent antinociceptive agent than its analog, 3CN, as demonstrated by the leftward shift in the dose-response curve and a 2.5-fold decrease in the ED50 (0.3 ± 0.05 µg and 0.78 ± 0.15 µg). B, 19-Nor3CN is a slightly more potent antinociceptive agent than its analog, 3CN, as demonstrated by the small leftward shift in the dose-response curve and a 1.5-fold decrease in the ED50 (0.15 ± 0.04 and 0.25 ± 0.08 µg). C, 3OH is more potent than its analog, 19-Nor3OH, as demonstrated by the leftward shift in the dose-response curve and an over 4-fold decrease in the ED50 (0.24 ± 0.04 and 1.05 ± 0.26 µg). D, 3OH is substantially more potent than its analog, 19-Nor3OH, as demonstrated by a 12-fold decrease in the ED50 (0.03 ± 0.005 and 0.36 ± 0.08 µg). All values for ED50, Hill coefficient and the maximal increase in thermal PWLs by the steroid analogs are in Table 1.

Our structure-activity studies in vitro and in vivo suggest a strong correlation between the potency of 5-reduced neuroactive steroids for blocking T currents in vitro and their potency for antinociception in vivo. To further examine this correlation, we conducted linear regression analyses by plotting the ED₅₀ values for analgesia versus IC₅₀ values for T current blockade for each 5-reduced neuroactive steroid (Fig. 10A). We found that there is an excellent correlation (r = 0.78) (solid line on the graph), demonstrating a statistically significant relationship between antinociceptive effects in vivo and T current blockade in vitro (p = 0.02). Behavior pain testing is often evaluated using an analgesia index that allows comparison of multiple parameters in determining the potency of tested agents (e.g., Buerkle and Yaksh, 1996). When the analgesic effect of 5-reduced neuroactive steroids was expressed using the analgesia index (calculated as the ED₅₀/maximal increase in PWLs x 100), a measurement that includes both the potency and magnitude of the maximal antinociceptive response, we found that there is an even better correlation (r = 0.88) (solid line on the graph) between antinociceptive effects in vivo and T current blockade in vitro (p = 0.004) (Fig. 10B). Assuming that the nerve terminals and soma of sensory neurons express a similar repertoire of ion channels, our data would suggest that the ability of 5-reduced neuroactive steroids to block T currents in vitro may underlie, at least in part, their analgesic effects in vivo when they are injected into peripheral receptive fields of these neurons.

View larger version (25K): [in this window] [in a new window]   Fig. 10. Correlation between the antinociceptive effects of 5-reduced neuroactive steroids and T-current blockade. A, there is a statistically significant correlation between the antinociceptive potency of the steroids in vivo (expressed as ED50 values calculated at the peak effect -10 min) and their ability to block T currents in small sensory neurons in vitro (expressed as IC50 values). The solid line represents the best fit of the linear regression plot (r = 0.78 ± 0.30 and p = 0.02). B, there is a statistically significant correlation between the antinociceptive potency of the steroids in vivo, expressed as the analgesia index [ED50/maximal increase in PWLs (seconds) x 100] and their ability to block T currents in sensory neurons (expressed as the IC50 values). The solid line represents the best fit of the linear regression plot (r = 0.88 ± 0.31 and p = 0.004).

	Discussion

Top Abstract Materials and Methods Results Discussion References

 
We demonstrate that the newly synthesized 5-reduced neuroactive steroids examined in this study are potent blockers of the T-type Ca²⁺ channels in rat peripheral sensory neurons in vitro and very potent antinociceptive agents in vivo. Furthermore, we found an excellent correlation between the potency of T-current blockade in vitro and antinociceptive potency in vivo, strongly suggesting that T-type Ca²⁺ channels play an important role in peripheral somatic nociception. We also note that GABA_A receptor potentiation does not correlate with the antinociceptive activity of these 5-reduced neuroactive steroids because 3OH, a compound known to be a potent activation-dependent blocker of GABA_A receptors (Wang et al., 2002), was the most potent antinociceptive steroid identified.

Even though the existence of T channels was initially described in sensory neurons (Carbone and Lux, 1984) and subsequently confirmed in many in vitro studies using small size sensory neurons, most of which are nociceptors (Schroeder et al., 1990; Scroggs and Fox, 1992; Cardens et al., 1995; Todorovic et al., 2001), their role in nociception was not previously recognized. It has been shown that T-type Ca²⁺ channels in peripheral nociceptors can enhance both polymodal somatic nociceptive signals (Todorovic et al., 2001, 2002) and peripheral visceral nociceptive signals (Kim et al., 2003). Here, we not only show that the blockade of T currents caused by 5-reduced neuroactive steroids in vitro correlates very well with their in vivo antinociceptive potency, we also provide the first structure-activity data for the steroid effects. Compounds having either the 3-cyano and 17-hydroxyl groups (3CN, 3CN, 19-Nor3CN, and 19-Nor3CN) or the 3-hydroxyl and 17-cyano group (3OH, 3CN, 19-Nor3OH, and 19-Nor3CN) are effective T-channel blockers and antinociceptive agents. In addition, the stereochemistry of the group at position 3 on the steroid A-ring does not seem to be of major importance. Finally, the effect of the steroid 19-methyl group at position 10 of the steroid depends on the properties of the functional group at position 3. For example, the 19-norsteroids with the 3-cyano, 17-hydroxy groups are more potent than those that have the 19-methyl group, whereas the 19-norsteroids with the 3-hydroxy, 17-cyano groups are less potent than those having the 19-methyl groups.

As in our previous reports with 5-reduced steroids (Todorovic et al., 1998), we find that 5-reduced neuroactive steroids exhibit mild voltage-dependent blockade of T type Ca²⁺ channels. This voltage dependence may indicate their preference for inactive states of the channel, resulting in a higher fractional block of the channel at depolarized membrane potentials. For example, we found that the IC₅₀ for 3OH, the most potent T-current blocker, was decreased by about 4-fold at a holding potential of -70 mV compared with a holding potential of -90 mV (0.76 versus 3 µM, respectively). Although direct measurement of resting membrane potentials in skin nociceptive endings in vivo is not technically possible, measurement of the resting membrane potentials from the intact nociceptors in the nerve-DRG preparation in vitro indicates that their resting membrane potential is approximately at -74 mV (Todorovic and Anderson, 1992; Scroggs et al., 1994). Therefore, it is reasonable to propose that T channels in peripheral nociceptors are operational and highly susceptible to blockade by neuroactive steroids. It is noteworthy that most 5-reduced steroids examined in this study cause almost complete block of neuronal DRG T currents, whereas 5-reduced neuroactive steroids block T currents only partially (up to 40%; Todorovic et al., 1998).

There are reports in the literature that the modulation of HVA Ca²⁺ currents by some 5-reduced neuroactive steroids is G-protein dependent (ffrench-Mullen et al., 1994). However, it is unlikely that the modulation of T currents observed in our experiments is through G-protein-mediated pathways because the exclusion of ATP and GTP (two constituents necessary for maintenance of G-protein signaling pathways) in the intracellular solution did not influence the magnitude of T current blockade by 5-reduced neuroactive steroids. This observation suggests that related but structurally different steroids exhibit distinct mechanisms of blocking neuronal voltage-gated Ca²⁺ currents.

Previous studies suggest that other families of voltagegated ion channels (e.g., HVA Ca²⁺, K⁺, and Na⁺ channels) play important roles in nociceptor excitability. Our findings indicate that 3OH and 19-Nor3CN, the most potent representative T-current blockers in vitro, as well as the most potent representative peripheral analgesics in vivo, when applied at concentrations that effectively suppress T currents (e.g., 3 and 10 µM, respectively), do not significantly affect either HVA Ca²⁺-or voltage-gated Na⁺ and K⁺ currents in rat sensory neurons. However, further structure-activity studies will be necessary to establish the relative importance of T channels and other voltage- and ion-gated channels in the peripheral antinociceptive effects of 5-reduced neuroactive steroids.

Although the role of GABA_A receptors in mediating central analgesic effects of the neuroactive steroids was recently suggested (Nadeson and Goodchild, 2000), the role of GABA_A receptors in peripheral nociception remains poorly understood. It has been reported that local intraplantar injection of the GABAergic agent, muscimol, failed to increase analgesic threshold in vivo (Carlton et al., 1999). Based on these findings it seems that, despite the presence of GABA_A receptors on peripheral nociceptors (Carlton et al., 1999), GABA_A-mediated effects per se play a less significant role in peripheral nociception under physiological conditions. However, it seems that GABA_A-mediated responses play a more prominent role in the nociception caused by the peripheral tissue inflammation (caused by local intraplantar injections of formalin) (Carlton et al., 1999).

Local injections of 5-reduced neuroactive steroids cause prolongation of thermal PWLs in the injected paws but not in the contralateral (control) paws of rats, indicating that the observed antinociceptive effect results from a direct action on nociceptive nerve endings, rather than a systemic effect. In considering these novel 5-reduced steroids as local analgesics, it is important to note that T channels are preferentially located on the smaller size sensory neurons that play an important role in nociceptive transmission but not in other modalities of sensory transmission (e.g., touch, vibration) or motor transmission, making selective and potent blockade of T currents a desirable therapeutic objective. By blocking pain sensation preferentially, without causing undesirable motor weakness, locally injected 5-reduced neuroactive steroids may offer a breakthrough in safe and effective pain therapy. It is noteworthy that the potency of the 5-reduced steroids that were examined in vivo ranges from 0.03 (for 3OH) to 1 µg/100 µl (e.g., 19-Nor3OH). To our knowledge, this is one of the most potent groups of local analgesics yet described. For instance, using the same in vivo model of peripheral thermal nociception, we found that the clinically used voltage-gated Na⁺ channel blockers, phenytoin and carbamazepine, were 10-fold less potent than 3OH in inducing antinociception (Todorovic et al., 2003b). Furthermore, lidocaine, another local anesthetic that is also a Na⁺ channel blocker, is used clinically at concentrations 1000-fold higher. Thus, 5-reduced neuroactive steroids may be an important addition to the class of local analgesics commonly used for regional anesthesia. The steroids could also be amenable to delivery by direct applications in the form of skin patches or local infiltration (at the site of an acute tissue injury, e.g., thermal coagulation, sunburns) because these agents are highly lipid soluble and should be able to easily access peripheral nerve endings. In addition to the potential usefulness of T channel blockers for the treatment of short-term pain, recent studies suggest that local administration of T channels blockers can be beneficial in the treatment of chronic pain (Dogrul et al., 2003; Todorovic et al., 2004).

In summary, we report that the 5-reduced neuroactive steroids examined in this study are potent blockers of T-type Ca²⁺ channels in rat sensory neurons in vitro and potent peripheral antinociceptive agents in vivo. The correlation between T-channel block and antinociceptive activity strongly suggests that blockade of T-type Ca²⁺ currents in sensory neurons may underlie, at least in part, the potent analgesic properties of 5-reduced steroids. Thus these steroids are promising new tools for studying the role of T-type Ca^2+</