Steroid blockers of t-type calcium channels for treatment of pain

Abstract

Among the various aspects of the present disclosure is the provision of neuroactive steroid compositions and methods of use thereof. Disclosed herein are neuroactive steroid compositions that inhibit a voltage-gated calcium ion channel for use in the treatment of pain. Methods to treat pain that includes administering a neuroactive steroid composition to a subject in need is also disclosed.

Description

[0001] STEROID BLOCKERS OF T-TYPE CALCIUM CHANNELS FOR TREATMENT OF PAIN

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims the benefit under 35 U.S.C. § 119(e) of U.S. Provisional Patent Application No. 63 / 695,628, filed September 17, 2024, the entire disclosure of which is incorporated herein by reference.

[0004] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0005] This invention was made with government support under MH122379 and GM 141802 awarded by the National Institutes of Health and under 5I01 BX004763- 04 awarded by the U.S. Department of Veterans Affairs. The government has certain rights in the invention.

[0006] FIELD OF THE INVENTION

[0007] The present disclosure generally relates to neuroactive steroid compounds, pharmaceutically acceptable salts of neuroactive steroid compounds, compositions comprising neuroactive steroid compounds, compositions comprising pharmaceutically acceptable salts of neuroactive steroid compounds, methods of preparing neuroactive steroid compounds, methods of preparing pharmaceutically acceptable salts of neuroactive steroid compounds, methods for preventing or treating pain by administering neuroactive steroid compounds, methods of preventing or treating pain by administering pharmaceutically acceptable salts of neuroactive steroid compounds, methods for preventing or treating pain by administering a composition comprising neuroactive steroid compounds, and methods of preventing or treating pain by administering a composition comprising pharmaceutically acceptable salts of neuroactive steroid compounds.

[0008] BACKGROUND OF THE INVENTION

[0009] The use of opioids in the operating rooms and clinics has been steadily increased over the last decades making them one of the most commonly prescribed classes of drugs in the USA. This is due to the fact that most commonly used injectable general anesthetics (GAs) do not provide a level of analgesia that is required for surgical procedures, necessitating the use of other agents such as opioid analgesics in the perioperative period. Although opioids are very effective in treating the acute pain associated with surgical procedures, they are only partially effective for more chronic painful disorders, and their use is associated with side effects including constipation, urinary retention, impaired cognitive function, respiratory depression, tolerance and addiction. It is estimated that more than 12 million people in United States abused opioids in 2010 resulting in more overdose deaths than heroin and cocaine combined (National Center for Health Statistics, 2012). Other currently available medications have either limited efficacy or serious side effects. For example, regional anesthesia and use of local anesthetics may not be suitable if early mobility after surgery is desirable. Furthermore, non-specific effects of local anesthetics such as numbness may prevent early neurological assessment after surgery. Thus, further research into new therapeutic modalities for treatment of pain in the perioperative period is warranted.

[0010] The important role of voltage-gated calcium channels (VGCCs) in pain processing has been recognized for a while since calcium (Ca2+) is the major trigger for the release of synaptic vesicles from neuronal presynaptic terminals in response to noxious stimulation. An increase of intracellular Ca2+in pain sensing neurons (nociceptors) can also influence the excitability of these cells. Previous studies have shown that the blockade of Cav3.2 isoform of T-type VGCCs in nociceptive dorsal root ganglion (DRG) neurons by 50-reduced neuroactive steroids (NASs) underlies their potent anti-nociceptive effects in animals with paw skin incision. Specifically, it was demonstrated that 30-OH [(30,50,170)-3- hydroxyandrostane-17-carbonitrile] in addition to hypnotic properties, also displays excellent analgesia in a clinically relevant rodent model of skin incision when administered intrathecally, systemically or peripherally. In addition, it has been shown that NASs that inhibit Cav3.2 channels are effective in alleviating mechanical hyperalgesia post-surgery when administered preemptively whereas morphine provides dose-dependent pain relief only when administered once the pain had developed. Thus, 30-OH and related NASs may represent a novel class of compounds having desirable and unique analgesic properties following systemic and intrathecal delivery, or the site of surgical tissue injury. However, limited aqueous solubility and potent hypnotic / sedative effects linked to their effect on GABAA receptors may hinder future development of NASs for novel pain therapies. For example, although 30-OH lacks any direct effect on synaptic and extra- synaptic GABAA receptors, it is demonstrated that the sex-specific hypnotic effect of 30-OH is largely mediated by its peripheral metabolism into an active metabolite that is a potent positive allosteric modulator (PAM) of neuronal GABAA receptors.

[0011] SUMMARY OF THE INVENTION

[0012] Among the various aspects of the present disclosure is the provision of neuroactive steroid compositions and methods of use thereof.

[0013] Briefly, therefore, the present disclosure is directed to neuroactive steroid compositions that inhibit a voltage-gated calcium ion channel for use in the treatment of pain.

[0014] The present disclosure includes methods to treat pain. In one aspect, the method comprises administering a neuroactive steroid composition to a subject in need. In one aspect, the method comprises administering a therapeutically effective amount of a compound selected from: or a pharmaceutically acceptable salt thereof. In another aspect, the method comprises administering a therapeutically effective amount of a compound selected from: or a pharmaceutically acceptable salt thereof, to inhibit a voltage-gated calcium ion channel.

[0015] Other objects and features will be in part apparent and in part pointed out hereinafter.

[0016] DESCRIPTION OF THE DRAWINGS

[0017] Those of skill in the art will understand that the drawings, described below, are for illustrative purposes only. The drawings are not intended to limit the scope of the present teachings in any way.

[0018] Figure 1 A shows the baseline assessment of the mechanical sensitivity using an electronic Von Frey apparatus.

[0019] Figure 1 B shows post-drug injection assessment of the mechanical sensitivity using an electronic Von Frey apparatus up to 60 minutes post-drug injection into the plantar service of the hind paw of intact adult female mice.

[0020] Figure 1 C shows the Open Field Test using ANY-maze video tracking software to determine the time spent in the outer and the inner zone. Total distance travelled and the velocity were also tracked by ANY-maze software. The observation lasted 15 minutes post-intraperitoneal drug injection in intact adult female mice.

[0021] Figure 2 shows the chemical structures of parent neuroactive steroids, 30- OH and ECN, and their newly synthesized analogs, B372 and YX23, respectively.

[0022] Figure 3A shows the families of original current traces obtained using the I- V protocols from the same HEK293 cell before and after application of 10 mM B372. Cells were maintained at a holding potential (Vh) of -90 mV and subjected to multiple depolarizing steps (Vt) ranging from -70 mV to +25 mV in 5 mV increments. The duration of the voltage step was 320 ms. Note typical criss-cross patterns caused by faster inactivation of currents at more depolarized test potentials. Insets represent calibration bars.

[0023] Figure 3B shows the average l-V graph from 21 cells using the same protocol depicted in FIG. 3A. Averaged data points represent mean+SEM in baseline pre-drug conditions and following application of 10 pM B372 in external solution. Note that B372 inhibited peak current amplitudes over the wide range of test potentials.

[0024] Figure 3C shows bar graphs comparing the percent of current inhibition by 10 pM B372 and 10 pM 3[3b-OH. Both drugs inhibit on average 48.2+5.5% and 48.2+4.2% of baseline peak currents (p>0.05, two-tailed t-test).

[0025] Figure 3D shows bar graphs comparing percent of current inhibition by 10 pM YX23 and 10 pM ECN. Both drugs inhibit on average 45.1 +5.2% and 47.4+5.2% of base-line peak currents (p>0.05, two-tailed t-test).

[0026] Figure 3E shows a concentration-response curve for the inhibition of Cav3.2 currents by escalating concentrations of B372. Each circle symbol is an average mean+SEM of multiple cells (n=5-26). The solid line is the best fit of Hill-Langumir function yielding an estimated ICso of 3.5 pM, slope steepness factor of 4.2 and maximal current inhibition of 48.6%.

[0027] Figure 3F shows a concentration-response curve for the inhibition of Cav3.2 currents by escalating concentrations of YX23. Each circle symbol is an average mean+SEM of multiple cells (n=5-8). The solid line is the best fit of Hill-Langumir function yielding an estimated ICso of 2.4 pM, slope steepness factor of 1 .9 and maximal current inhibition of 47.0%.

[0028] Figure 4A shows the average Cav3.2 current steady-state inactivation curves from multiple experiments. Top open circles represent the control conditions; bottom open circles represent the conditions after bath applications of 60 pM YX23 in the same cells (n=9). Top filled squares represent the control conditions; bottom filled squares represent the conditions after bath applications of 60 pM B372 in the same cells (n=6). Solid lines are fitted using Boltzmann equation, giving half-maximal availability (Vso), which occurred at -73.0 mV with a slope of 8.0 mV in control conditions. In contrast, Vso was -80.1 mV with a slope k of 10.1 mV in the conditions after YX23 was applied. In another set of experiments, it was found half-maximal availability (Vso), which occurred at -70.2 mV with a slope k of 6.5 mV in control conditions. In contrast, Vso was shifted to more negative potential of -90.0 mV with a slope k of 8.8 mV in the conditions after B372 was applied.

[0029] Figure 4B shows a scatter plot of Vso values in control conditions (filled circles) and after applications of YX23 (filled squares) obtained in individual cells by using steady-state inactivation curves as depicted in FIG. 4A. The average Vso in the control conditions occurred at -72.7+2.8 mV and in the presence of YX23 at - 82.9+4.1 mV (***, p=0.0009).

[0030] Figure 4C shows a scatter plot of Vso values in control conditions (filled circles) and after applications of B372 (filled squares) obtained in individual cells by using steady-state inactivation curves as depicted in FIG. 4A. The average Vso in the control conditions occurred at -68.8+4.2 mV and in the presence of B372 at - 87+4.8 mV (*, p=0.028).

[0031] Figure 5A shows a dose-response experiment with escalating doses of B372 (from 60 to 160 mg / kg given systemically; injection volume of 10 pl / g of body weight, i.p.) (right line) using loss of righting reflex (LORR) in the WT female adult mice showed about a 2-fold rightward shift with B372 (the EDso of 92 mg / kg, i.p.) compared to published EDso for 3[3-OH (48 mg / kg, i.p.) (n=9 adult female mice per group).

[0032] Figure 5B shows an Open Field Test using B372 at 60 mg / kg, i.p. (injection volume of 10 pl / g of body weight). The time spent in the Inner Zone and Outer Zone (Thigmotaxis) during a 15-min observation post-B372 injection were measured. There was no significant difference compared to vehicle-treated controls.

[0033] Figure 5C shows an Open Field Test using B372 at 60 mg / kg, i.p. (injection volume of 10 pl / g of body weight). The average velocity of female adult mice movements during a 15-min observation post-B372 injection was measured and no difference in the average velocity of their movements when compared to the vehicle controls was found.

[0034] Figure 5D shows an Open Field Test using B372 at 60 mg / kg, i.p. (injection volume of 10 pl / g of body weight). The total distance traveled of female adult mice during a 15-min observation post-B372 injection was measured and no difference in the total distance traveled when compared to the vehicle controls (n=9-10 mice per group) was found.

[0035] Figure 6A shows a small amount (20pl) of YX23 (at 10 pM) injected intraplantary (i.pl.) into the receptive field of sensory neurons of an adult WT mouse left paw and paw withdrawal response (PWRs) to punctate mechanical stimuli as a bending force (in gm) were recorded before injection (0 time point) and at different time points after the injection (10, 20, 40 and 60 min) with diminished PWRs as evidenced by significantly increased mechanical thresholds in injected paws at 20 and 40 minutes when compared to injections of a vehicle (**, p<0.01 ) (n=11 female mice per data point).

[0036] Figure 6B shows injections of either YX23 or vehicle did not change PWRs in contralateral non-injected paws indicating the lack of a systemic effect (n=11 female mice per data point).

[0037] Figure 6C shows a small amount (20pl) of YX23 (at 10 pM) injected intraplantary (i.pl.) into the receptive field of sensory neurons of an adult Cav3.2 KO mouse left paw and paw withdrawal response (PWRs) to punctate mechanical stimuli were recorded before injection (0 time point) and at different time points after the injection (10, 20, 40 and 60 min) with no significant difference in mechanical sensitivity at any time point after the XY23 injection when compared to vehicle controls (n= 8 female mice per data point).

[0038] Figure 6D shows injections of either YX23 or vehicle did not change PWRs in contralateral non-injected paws of adult Cav3.2 KO mouse indicating the lack of a systemic effect (n= 8 female mice per data point).

[0039] Figure 7A shows a small amount (20pl) of B372 (at 100 pM) injected intraplantary (i.pl.) into the receptive field of sensory neurons of an adult WT mouse left paw and paw withdrawal response (PWRs) to punctate mechanical stimuli as a bending force (in gm) were recorded before injection (0 time point) and at different time points after the injection (10, 20, 40 and 60 min) with diminished PWRs as evidenced by significantly increased mechanical thresholds in injected paws at 20 and 40 minutes when compared to injections of a vehicle (**, p<0.01 ) (n=10 female mice per data point).

[0040] Figure 7B shows injections of either B372 or vehicle did not change PWRs in contralateral non-injected paws indicating the lack of a systemic effect (n=10 female mice per data point).

[0041] Figure 7C shows a small amount (20pl) of B372 (at 100 pM) injected intraplantary (i.pl.) into the receptive field of sensory neurons of an adult Cav3.2 KO mouse left paw and paw withdrawal response (PWRs) to punctate mechanical stimuli were recorded before injection (0 time point) and at different time points after the injection (10, 20, 40 and 60 min) with no significant difference in mechanical sensitivity at any time point after the B372 injection when compared to vehicle controls (n= 6 female mice per data point).

[0042] Figure 7D shows injections of either B372 or vehicle did not change PWRs in contralateral non-injected paws of adult Cav3.2 KO mouse indicating the lack of a systemic effect (n= 6 female mice per data point).

[0043] DETAILED DESCRIPTION OF THE INVENTION

[0044] The present disclosure is based, at least in part, on the discovery that two newly synthesized neuroactive steroids analogs have diminished potential for metabolic conversion to positive allosteric modulators of GABAA receptors. Although we have shown that 3[3-OH neuroactive steroids can be an effective hypnotic (Fine-Raquet et al., Exp Biol Med, 2023, 248(7), 578-587), efficacious analgesic (Todorovic et al., Mol Pharmacol, 2004, 66(5), 1223-1235) and potent voltage-dependent Cav3.2 inhibitors (Todorovic et al., Channels, 2007, 1 (4), 238- 245), in vivo conversion to potent GABAA positive allosteric modulators, 3a-OH neuroactive steroids confound interpretation of how much of the analgesic effect is due to Cav3.2 inhibition and how much is due to GABAA modulation. To avoid the confounding effect of these dual actions and excessive sedation / hypnosis in vivo neuroactive steroid analog, B372, a 5a-steroid epimer of 3[3-OH, was synthesized.

[0045] The second neuroactive steroid analog of interest, YX23 is an analogue of (3[3,5a,17[3)-17-hydroxyestrane-3-carbonitrile (ECN), a neuroactive steroid previously shown to be a potent voltage-dependent CaV3.2 inhibitor (Todorovic et al., Channels, 2007, 1 (4), 238-245). ECN does not have a 3-OH group and, unlike 3[3-OH cannot be metabolized in vivo to a GABAA modulator. YX23 also does not have a 3[3-OH and by replacing the cyano group of ECN with a methoxy group in the 3(3 position of the steroid ring, YX23 was made a more soluble analogue compared to ECN. When the LogP (lipophilicity vs. hydrophilicity) of ECN vs. YX23 was calculated, it was found that LogP value for ECN is 4.04 vs LogP of 3.59 for YX23 suggesting higher hydrophilicity of YX23 and its higher solubility. In this study the hypnotic and analgesic properties of B372 and YX23 were examined. ELECTROPHYSIOLOGY STUDIES

[0046] To examine the hypothesis that B372 and YX23 are Cav3.2 inhibitors a patch-clamp study using stably transfected human embryonic kidney (HEK) 293 cells was performed. For screening purpose, these neuroactive steroids were dissolved in 100% DMSO as 10-20 mM stock solutions and for actual recordings they were freshly diluted in an external solution to final concentrations ranging from 1 -60 pM. Maximal concentration of DMSO in external solutions used for recordings was 0.3%, which has previously been demonstrated has minimal effect on Cav3.2 currents (Todorovic et al., Neuron, 2001 , 31 (1 ), 75-85). The current-voltage (l-V) protocol for recordings of recombinant Cav3.2 currents is depicted in FIG. 3A, which shows two families of inward currents generated from a holding potential (Vh) of -90 mV and a wide range of test potentials (Vt) from -80 mV to +10 mV in incremental steps of 5 mV (n=17 cells). Top traces in FIG. 3A show baseline predrug currents, and bottom traces show currents after application of 10 pM B372. The peak inward currents from this representative experiment were plotted on the l-V curve depicted in FIG. 3B. Note that B372 inhibited about 50% of peak current amplitudes over the range of potentials with maximal inhibition occurring between Vt -40 and Vt -20 mV. Importantly, the patch clamp experiments have established that B372 is an efficacious Cav3.2 inhibitor similar to 3[3-OH with both compounds blocking on average about 50% of peak current at 10 pM concentrations (FIG. 3C).

[0047] Similar patch-clamp studies with YX23 have shown that YX23, at 10 pM similarly inhibits baseline Cav3.2 currents by about 45% (p<0.001 by two-tailed Student t-test, n=6 cells) (FIG. 3D). Importantly, the patch-clamp experiments have established that YX23 is an efficacious Cav3.2 inhibitor like ECN with both compounds blocking on average about 45% of peak current at 10 pM concentrations (FIG. 3D). From the concentration-response experiments using l-V protocols and escalating concentrations of neuroactive steroids the maximal block of peak T-currents for each analogue, slope of the curve and an ICso for current inhibition were estimated. FIG. 3E depicts average data points from multiple experiments for B372 which inhibited about 48.6% of peak T-currents with an ICso of 3.5 pM and slope factor of 4.2. Similarly, FIG. 3F depicts average data points from multiple experiments for YX23 which inhibited about 47.0% of peak T- currents with an IC50 of 2.4 pM and slope factor of 1.9. Previous studies have demonstrated that both 3[3-OH and ECN inhibited T-currents in a voltagedependent manner (Todorovic et al., Mol Pharmacol, 2004, 66(5), 1223-1235). Hence, any state-dependent features of Cav3.2 current inhibition by YX23 and B372 was evaluated. Drug binding to inactivated states of ion channels is an important property since it allows for tissue selectivity based on differences in membrane potentials, where more depolarized membranes will have T-channels which cycle through the inactivated state more often vs. less excitable tissue. Transitions from closed to inactivated states can be measured using long prepulses at different potentials, producing what are commonly referred to as steadystate inactivation curves. The steady-state inactivation curves and resulting current availability in multiple cells using a standard double-pulse protocol with 3.6 s-long pre-pulses to variable voltages (from -110 to -45 mV) and Vt to -30 mV was assessed.

[0048] FIG. 4A shows that when compared to control predrug conditions both neuroactive steroids decreased T-current amplitudes over all tested preconditioning potentials. Furthermore, when compared to control predrug conditions, both YX23 and B372 had a great effect on the voltage-dependent kinetics of channel inactivation, as determined by a hyperpolarizing shift in steadystate inactivation curves of about 10-20 mV as shown on FIG. 4B and FIG. 4C, respectively. These data suggest that B372 and YX23 bind to and stabilize inactive states of the Cav3.2 T-channel and thus are more potent blockers at depolarized membrane potentials. For example, in FIG. 4A the data shows that 60 pM YX23 inhibits only about 30% of maximal T-current at -110 mV, while the same concentration inhibits about 90% T-current at preconditioning potentials of -65 mV. Based on these data, it can be concluded that structural modifications of 3[3-OH and ECN did not have an apparent adverse impact on their ability to inhibit Cav3.2 current.

[0049] SEDATIVE / HYPNOTIC ASSESSMENT

[0050] A recent study showed that a systemic injection of 3[3-OH produced potent hypnotic effect as measured with loss of righting reflex (LORR) in the range of doses from 20 to 120 mg / kg, i.p. (Manzella et al., Br J Amaesth, 2023, 130(2), 154- 164). Hence, in order to assess the hypnotic potential of its newly synthesized epimer, B372, a dose-response experiment with escalating doses of B372 (from 60 to 160 mg / kg given systemically) (i.p.) was performed. The LORR in the WT female adult mice (n=9 per group) was measured (FIG. 5A). For ease of comparison, a dotted line was included to show the dose response to 3[3-OH that was previously published (Manzella et al., Br J Amaesth, 2023, 130(2), 154-164). The resulting 2-fold rightward shift with B372 (the EDso of 92 mg / kg, i.p.) compared to published EDso for 3[3-OH (48 mg / kg, i.p.) suggests the structural modification of 3[3-OH greatly diminished sedative / hypnotic potency of B372.

[0051] To further assess systemic effects of B372, an Open Field Test was conducted using B372 at 60 mg / kg, i.p. (injection volume of 10 pl / g of body weight) (n=9-10 mice per group) (FIG. 5B). When the times spent in the Inner Zone and Outer Zone (Thigmotaxis) during a 15-min observation post-B372 injection were measured, it was concluded that there was no significant difference compared to vehicle-treated controls (n=9-10 mice per group). Similarly, no difference in the average velocity of their movements (FIG. 5C) or total distance traveled (FIG. 5D) when compared to the vehicle controls was found suggesting lack of significant sedative effects or changes in the overall motor abilities of the mice.

[0052] Similarly, the results indicated that YX23, at 100 mg / kg, i.p. had no appreciable effect on any of the observed open field behaviors when compared to vehicle-controls (FIG. 5B, FIG. 5C and FIG. 5D). In addition, unlike B372, no appreciable signs of sedation or lack of movement when compared to vehicle controls was observed. Hence, no LORR studies were performed.

[0053] ANALGESIC PROPERTIES ASSESSMENT

[0054] To assess the analgesic properties of chosen neuroactive steroids, studies of mechanical sensitivity using an electronic von Frey apparatus applied to the plantar surface of the hind paw in mice were conducted.

[0055] It was previously reported that local analgesic properties of other neuroactive steroids when a small amount (20pl) is injected intraplantary (i.pl.) into the receptive field of sensory neurons of an adult rodent paw correlates well with their Cav3.2 blocking potency (Pathirathna et al., Pain, 2005, 114(3), 429-443). Hence, herein YX23 was injected into the left paw of wild type (WT) mice and PWRs to punctate mechanical stimuli as a bending force (in gm) measured before injection (0 time point) and at different time points after the injection (10, 20, 40 and 60 min) in both injected (left) (FIG. 6A) and non-injected (right) (FIG. 6B) hind paws. For these i.pl. injections, the YX23 was dissolved in 15% 2-hydroxypropyl-[3- cyclodextrin solution in pH-balanced saline (pH = 7.4 to avoid tissue irritation) in 20 pl volume at 10 pM concentration. As shown in FIG. 6A it was found that the injection of the YX23, at 10 pM showed diminished PWRs as evidenced by significantly increased mechanical thresholds in injected paws at 20 and 40 minutes when compared to injections of a vehicle (**, p<0.01 ). Importantly, injections of either YX23 or vehicle did not change PWRs in contralateral noninjected paws (FIG. 6B) indicating the lack of a systemic effect (n=11 female mice per data point).

[0056] To further assess the role of Cav3.2 channels in peripheral nociception, the effect of YX23 in Cav3.2 KO mice was examined. As shown in FIG. 6C, no significant difference in mechanical sensitivity at any time point after the XY23 injection when compared to vehicle controls (n= 8 female mice per data point) was found. The finding was similar in the contralateral paw (FIG. 6D) suggesting that YX23’s antinociceptive effects are indeed largely mediated by the peripheral CaV3.2 channels.

[0057] To examine the antinociceptive properties of B372, B372 was injected into the left paw of wild type (WT) mice and PWRs to punctate mechanical stimuli as a bending force (in gm) were measured before injection (0 time point) and at different time points after the injection (10, 20, 40 and 60 min) in both injected (left) (FIG. 7A) and non-injected (right) (FIG. 7B) hind paws. For these i.pl. injections, the B372 (similarly to YX23) was dissolved in 15% 2-hydroxypropyl-[3-cyclodextrin solution in pH-balanced saline (pH = 7.4 to avoid tissue irritation) in 20 pl volume at 100 pM concentration. As shown in FIG. 7A the injection of the B372, at 100 pM showed diminished PWRs as evidenced by significantly increased mechanical thresholds in injected paws at 20 and 40 minutes when compared to injections of a vehicle (**, p<0.01 ). Importantly, injections of either B372 or vehicle did not change PWRs in contralateral non-injected paws (FIG. 7B) indicating the lack of a systemic effect (n=10 mice per data point).

[0058] To further assess the role of Cav3.2 channels in peripheral nociception the effect of B372 in Cav3.2 KO mice was also examined. As shown in FIG. 7C, no significant difference in mechanical sensitivity at any time point after the B372 injection when compared to vehicle controls (n= 6 mice per data point) resulted. The finding was similar in the contralateral paw (FIG. 7D) suggesting that YX23’s antinociceptive effects are indeed largely mediated by the peripheral Cav3.2 channels.

[0059] The present disclosure is directed to methods of preventing or treating pain comprising administering a therapeutically effective amount of a compound selected from: or a pharmaceutically acceptable salt thereof. In some embodiments, the pain is acute or chronic. In some embodiments, the pain is acute. In some embodiments, the pain is chronic.

[0060] The present disclosure is also directed to methods of preventing or treating pain comprising administering a therapeutically effective amount of a compound selected from: or a pharmaceutically acceptable salt thereof, which inhibits a voltage-gated calcium ion channel. In some embodiments, the pain is acute or chronic. In some embodiments, the pain is acute. In some embodiments, the pain is chronic. In some embodiments, the voltage-gated calcium ion channel is selected from the group consisting of Cav1 , Cav2.1 , Cav2.2, Cav2.3, Cav3.1 , Cav3.2, and Cav3.3. In some embodiments, the voltage-gated calcium ion channel is Cav1. In some embodiments, the voltage-gated calcium ion channel is Cav2.1. In some embodiments, the voltage-gated calcium ion channel is Cav2.2. In some embodiments, the voltage-gated calcium ion channel is Cav2.3. In some embodiments, the voltage-gated calcium ion channel is Cav3.1. In some embodiments, the voltage-gated calcium ion channel is Cav3.2. In some embodiments, the voltage-gated calcium ion channel is Cav3.3.

[0061] In some embodiments, the methods further comprise diminishing positive allosteric modulation of GABAa receptors.

[0062] In some embodiments, the compound is

[0063] In some embodiments, the compound is

[0064] The present disclosure is also directed to a compound selected from: treating pain in a mammal.

[0065] The present disclosure is also directed to a compound selected from: manufacture of a medicament for preventing or treating pain in a mammal.

[0066] The present disclosure is also directed to the use of a compound selected The present disclosure is also directed to the use of a compound selected from: the manufacture of a medicament for preventing or treating pain in a mammal.

[0067] The present disclosure is also directed to a compound of Formula I or a pharmaceutically acceptable salt thereof.

[0068] The present disclosure is also directed to pharmaceutical composition comprising the compound of Formula I, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

[0069] CHEMICAL AGENTS: The term “solvate” is intended to mean a solvate form of a specified compound that retains the effectiveness of such a compound. Examples of solvates include compounds of the invention in combination with, for example: water, isopropanol, ethanol, methanol, dimethylsulfoxide (DMSO), ethyl acetate, acetic acid, or ethanolamine. The term “mmol”, as used herein, is intended to mean millimole. The term

[0070] “equiv”, as used herein, is intended to mean equivalent. The term “mL”, as used herein, is intended to mean milliliter. The term “g”, as used herein, is intended to mean gram. The term “kg”, as used herein, is intended to mean kilogram. The term “pg”, as used herein, is intended to mean micrograms. The term “h”, as used herein, is intended to mean hour. The term “min”, as used herein, is intended to mean minute. The term “M”, as used herein, is intended to mean molar. The term "pL", as used herein, is intended to mean microliter. The term “pM”, as used herein, is intended to mean micromolar. The term “nM”, as used herein, is intended to mean nanomolar. The term “N”, as used herein, is intended to mean normal. The term “amu”, as used herein, is intended to mean atomic mass unit. The term “°C”, as used herein, is intended to mean degree Celsius. The term “wt / wt”, as used herein, is intended to mean weight / weight. The term “v / v”, as used herein, is intended to mean volume / volume. The term “MS”, as used herein, is intended to mean mass spectroscopy. The term “HPLC”, as used herein, is intended to mean high-performance liquid chromatography. The term “RT”, as used herein, is intended to mean room temperature. The term "e.g.", as used herein, is intended to mean example. The term “N / A”, as used herein, is intended to mean not tested.

[0071] As used herein, the expression “pharmaceutically acceptable salt” refers to pharmaceutically acceptable organic or inorganic salts of a compound of the invention. Preferred salts include, but are not limited, to sulfate, citrate, acetate, oxalate, chloride, bromide, iodide, nitrate, bisulfate, phosphate, acid phosphate, isonicotinate, lactate, salicylate, acid citrate, tartrate, oleate, tannate, pantothenate, bitartrate, ascorbate, succinate, maleate, gentisinate, fumarate, gluconate, glucaronate, saccharate, formate, benzoate, glutamate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate, or pamoate (i.e., 1,1'- methylene-bis-(2-hydroxy-3-naphthoate)) salts. A pharmaceutically acceptable salt may involve the inclusion of another molecule such as an acetate ion, a succinate ion, or other counterion. The counterion may be any organic or inorganic moiety that stabilizes the charge on the parent compound. Furthermore, a pharmaceutically acceptable salt may have more than one charged atom in its structure. Instances where multiple charged atoms are part of the pharmaceutically acceptable salt can have multiple counterions. Hence, a pharmaceutically acceptable salt can have one or more charged atoms and / or one or more counterion. As used herein, the expression “pharmaceutically acceptable solvate” refers to an association of one or more solvent molecules and a compound of the invention. Examples of solvents that form pharmaceutically acceptable solvates include, but are not limited to, water, isopropanol, ethanol, methanol, DMSO, ethyl acetate, acetic acid, and ethanolamine. As used herein, the expression “pharmaceutically acceptable hydrate” refers to a compound of the invention, or a salt thereof, that further can include a stoichiometric or non-stoichiometric amount of water bound by non-covalent intermolecular forces.

[0072] FORMULATION

[0073] The agents and compositions described herein can be formulated by any conventional manner using one or more pharmaceutically acceptable carriers or excipients as described in, for example, Remington’s Pharmaceutical Sciences (A.R. Gennaro, Ed.), 21st edition, ISBN: 0781746736 (2005), incorporated herein by reference in its entirety. Such formulations will contain a therapeutically effective amount of a biologically active agent described herein, which can be in purified form, together with a suitable amount of carrier so as to provide the form for proper administration to the subject.

[0074] The term "formulation" refers to preparing a drug in a form suitable for administration to a subject, such as a human. Thus, a "formulation" can include pharmaceutically acceptable excipients, including diluents or carriers.

[0075] The term "pharmaceutically acceptable" as used herein can describe substances or components that do not cause unacceptable losses of pharmacological activity or unacceptable adverse side effects. Examples of pharmaceutically acceptable ingredients can be those having monographs in United States Pharmacopeia (USP 29) and National Formulary (NF 24), United States Pharmacopeial Convention, Inc, Rockville, Maryland, 2005 ("USP / NF"), or a more recent edition, and the components listed in the continuously updated Inactive Ingredient Search online database of the FDA. Other useful components that are not described in the USP / NF, etc. may also be used.

[0076] The term “pharmaceutically acceptable excipient,” as used herein, can include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic, or absorption-delaying agents. The use of such media and agents for pharmaceutically active substances is well known in the art (see generally Remington’s Pharmaceutical Sciences (A.R. Gennaro, Ed.), 21st edition, ISBN: 0781746736 (2005)). Except insofar as any conventional media or agent is incompatible with an active ingredient, its use in the therapeutic compositions is contemplated. Supplementary active ingredients can also be incorporated into the compositions.

[0077] A "stable" formulation or composition can refer to a composition having sufficient stability to allow storage at a convenient temperature, such as between about 0 °C and about 60 °C, for a commercially reasonable period of time, such as at least about one day, at least about one week, at least about one month, at least about three months, at least about six months, at least about one year, or at least about two years.

[0078] The formulation should suit the mode of administration. The agents of use with the current disclosure can be formulated by known methods for administration to a subject using several routes which include, but are not limited to, parenteral, pulmonary, oral, topical, intradermal, intratumoral, intranasal, inhalation (e.g., in an aerosol), implanted, intramuscular, intraperitoneal, intravenous, intrathecal, intracranial, intracerebroventricular, subcutaneous, intranasal, epidural, intrathecal, ophthalmic, transdermal, buccal, and rectal. The individual agents may also be administered in combination with one or more additional agents or together with other biologically active or biologically inert agents. Such biologically active or inert agents may be in fluid or mechanical communication with the agent(s) or attached to the agent(s) by ionic, covalent, Van der Waals, hydrophobic, hydrophilic, or other physical forces.

[0079] Controlled-release (or sustained-release) preparations may be formulated to extend the activity of the agent(s) and reduce dosage frequency. Controlled- release preparations can also be used to effect the time of onset of action or other characteristics, such as blood levels of the agent, and consequently affect the occurrence of side effects. Controlled-release preparations may be designed to initially release an amount of an agent(s) that produces the desired therapeutic effect, and gradually and continually release other amounts of the agent to maintain the level of therapeutic effect over an extended period of time. In order to maintain a near-constant level of an agent in the body, the agent can be released from the dosage form at a rate that will replace the amount of the agent being metabolized or excreted from the body. The controlled-release of an agent may be stimulated by various inducers, e.g., change in pH, change in temperature, enzymes, water, or other physiological conditions or molecules.

[0080] Agents or compositions described herein can also be used in combination with other therapeutic modalities, as described further below. Thus, in addition to the therapies described herein, one may also provide to the subject other therapies known to be efficacious for the treatment of the disease, disorder, or condition.

[0081] THERAPEUTIC METHODS

[0082] Also provided is a process of treating, preventing, or reversing pain in a subject in need thereof comprising administering a therapeutically effective amount of a neuroactive steroids, so as to alleviate pain symptoms.

[0083] Methods described herein are generally performed on a subject in need thereof. A subject in need of the therapeutic methods described herein can be a subject having, diagnosed with, suspected of having, or at risk for developing a pain disorder. A determination of the need for treatment will typically be assessed by a history, physical exam, or diagnostic tests consistent with the disease or condition at issue. Diagnosis of the various conditions treatable by the methods described herein is within the skill of the art. The subject can be an animal subject, including a mammal, such as horses, cows, dogs, cats, sheep, pigs, mice, rats, monkeys, hamsters, guinea pigs, and humans or chickens. For example, the subject can be a human subject.

[0084] Generally, a safe and effective amount of a neuroactive steroid is, for example, an amount that would cause the desired therapeutic effect in a subject while minimizing undesired side effects. In various embodiments, an effective amount of a neuroactive steroid described herein can substantially inhibit pain, slow the progress of pain, or limit the development of pain

[0085] According to the methods described herein, administration can be parenteral, pulmonary, oral, topical, intradermal, intramuscular, intraperitoneal, intravenous, intratumoral, intrathecal, intracranial, intracerebroventricular, subcutaneous, intranasal, epidural, ophthalmic, buccal, or rectal administration.

[0086] When used in the treatments described herein, a therapeutically effective amount of a neuroactive steroid can be employed in pure form or, where such forms exist, in pharmaceutically acceptable salt form and with or without a pharmaceutically acceptable excipient. For example, the compounds of the present disclosure can be administered, at a reasonable benefit / risk ratio applicable to any medical treatment, in a sufficient amount to treat pain.

[0087] The amount of a composition described herein that can be combined with a pharmaceutically acceptable carrier to produce a single dosage form will vary depending upon the subject or host treated and the particular mode of administration. It will be appreciated by those skilled in the art that the unit content of agent contained in an individual dose of each dosage form need not in itself constitute a therapeutically effective amount, as the necessary therapeutically effective amount could be reached by administration of a number of individual doses.

[0088] Toxicity and therapeutic efficacy of compositions described herein can be determined by standard pharmaceutical procedures in cell cultures or experimental animals for determining the LDso (the dose lethal to 50% of the population) and the EDso, (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index that can be expressed as the ratio LD50 / ED50, where larger therapeutic indices are generally understood in the art to be optimal.

[0089] The specific therapeutically effective dose level for any particular subject will depend upon a variety of factors including the disorder being treated and the seventy of the disorder; activity of the specific compound employed; the specific composition employed; the age, body weight, general health, sex and diet of the subject; the time of administration; the route of administration; the rate of excretion of the composition employed; the duration of the treatment; drugs used in combination or coincidental with the specific compound employed; and like factors well known in the medical arts (see e.g., Koda-Kimble et al. (2004) Applied Therapeutics: The Clinical Use of Drugs, Lippincott Williams & Wilkins, ISBN 0781748453; Winter (2003) Basic Clinical Pharmacokinetics, 4thed., Lippincott Williams & Wilkins, ISBN 0781741475; Sharqel (2004) Applied Biopharmaceutics & Pharmacokinetics, McGraw-Hill / Appleton & Lange, ISBN 0071375503). For example, it is well within the skill of the art to start doses of the composition at levels lower than those required to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved. If desired, the effective daily dose may be divided into multiple doses for purposes of administration. Consequently, single-dose compositions may contain such amounts or submultiples thereof to make up the daily dose. It will be understood, however, that the total daily usage of the compounds and compositions of the present disclosure will be decided by an attending physician within the scope of sound medical judgment.

[0090] Again, each of the states, diseases, disorders, and conditions, described herein, as well as others, can benefit from compositions and methods described herein. Generally, treating a state, disease, disorder, or condition includes preventing, reversing, or delaying the appearance of clinical symptoms in a mammal that may be afflicted with or predisposed to the state, disease, disorder, or condition but does not yet experience or display clinical or subclinical symptoms thereof. Treating can also include inhibiting the state, disease, disorder, or condition, e.g., arresting or reducing the development of the disease or at least one clinical or subclinical symptom thereof. Furthermore, treating can include relieving the disease, e.g., causing regression of the state, disease, disorder, or condition or at least one of its clinical or subclinical symptoms. A benefit to a subject to be treated can be either statistically significant or at least perceptible to the subject or to a physician.

[0091] Administration of a neuroactive steroid can occur as a single event or over a time course of treatment. For example, a neuroactive steroid can be administered daily, weekly, bi-weekly, or monthly. For treatment of acute conditions, the time course of treatment will usually be at least several days. Certain conditions could extend treatment from several days to several weeks. For example, treatment could extend over one week, two weeks, or three weeks. For more chronic conditions, treatment could extend from several weeks to several months or even a year or more. Treatment in accord with the methods described herein can be performed prior to, concurrent with, or after conventional treatment modalities for pain.

[0092] A neuroactive steroid can be administered simultaneously or sequentially with another agent, such as an antibiotic, an anti-inflammatory, or another agent. For example, a neuroactive steroid can be administered simultaneously with another agent, such as an antibiotic or an anti-inflammatory. Simultaneous administration can occur through the administration of separate compositions, each containing one or more of a neuroactive steroid, an antibiotic, an antiinflammatory, or another agent. Simultaneous administration can occur through the administration of one composition containing two or more of a neuroactive steroid, an antibiotic, an anti-inflammatory, or another agent. A neuroactive steroid can be administered sequentially with an antibiotic, an anti-inflammatory, or another agent. For example, a neuroactive steroid can be administered before or after administration of an antibiotic, an anti-inflammatory, or another agent.

[0093] ADMINISTRATION

[0094] Agents and compositions described herein can be administered according to methods described herein in a variety of means known to the art. The agents and composition can be used therapeutically either as exogenous materials or as endogenous materials. Exogenous agents are those produced or manufactured outside of the body and administered to the body. Endogenous agents are those produced or manufactured inside the body by some type of device (biologic or other) for delivery within or to other organs in the body.

[0095] As discussed above, administration can be parenteral, pulmonary, oral, topical, intradermal, intratumoral, intranasal, inhalation (e.g., in an aerosol), implanted, intramuscular, intraperitoneal, intravenous, intrathecal, intracranial, intracerebroventricular, subcutaneous, intranasal, epidural, intrathecal, ophthalmic, transdermal, buccal, and rectal.

[0096] Agents and compositions described herein can be administered in a variety of methods well-known in the arts. Administration can include, for example, methods involving oral ingestion, direct injection (e.g., systemic or stereotactic), implantation of cells engineered to secrete the factor of interest, drug-releasing biomaterials, polymer matrices, gels, permeable membranes, osmotic systems, multilayer coatings, microparticles, implantable matrix devices, mini-osmotic pumps, implantable pumps, injectable gels and hydrogels, liposomes, micelles (e.g., up to 30 pm), nanospheres (e.g., less than 1 pm), microspheres (e.g., 1-100 pm), reservoir devices, a combination of any of the above, or other suitable delivery vehicles to provide the desired release profile in varying proportions. Other methods of controlled-release delivery of agents or compositions will be known to the skilled artisan and are within the scope of the present disclosure.

[0097] Delivery systems may include, for example, an infusion pump which may be used to administer the agent or composition in a manner similar to that used for delivering insulin or chemotherapy to specific organs or tumors. Typically, using such a system, an agent or composition can be administered in combination with a biodegradable, biocompatible polymeric implant that releases the agent over a controlled period of time at a selected site. Examples of polymeric materials include polyanhydrides, polyorthoesters, polyglycolic acid, polylactic acid, polyethylene vinyl acetate, and copolymers and combinations thereof. In addition, a controlled release system can be placed in proximity of a therapeutic target, thus requiring only a fraction of a systemic dosage.

[0098] Agents can be encapsulated and administered in a variety of carrier delivery systems. Examples of carrier delivery systems include microspheres, hydrogels, polymeric implants, smart polymeric carriers, and liposomes (see generally, Uchegbu and Schatzlein, eds. (2006) Polymers in Drug Delivery, CRC, ISBN-10: 0849325331 ). Carrier-based systems for molecular or biomolecular agent delivery can: provide for intracellular delivery; tailor biomolecule / agent release rates; increase the proportion of biomolecule that reaches its site of action; improve the transport of the drug to its site of action; allow colocalized deposition with other agents or excipients; improve the stability of the agent in vivo prolong the residence time of the agent at its site of action by reducing clearance; decrease the nonspecific delivery of the agent to nontarget tissues; decrease irritation caused by the agent; decrease toxicity due to high initial doses of the agent; alter the immunogenicity of the agent; decrease dosage frequency, improve taste of the product; or improve shelf life of the product. KITS

[0099] Also provided are kits. Such kits can include an agent or composition described herein and, in certain embodiments, instructions for administration. Such kits can facilitate the performance of the methods described herein. When supplied as a kit, the different components of the composition can be packaged in separate containers and admixed immediately before use. Components include, but are not limited to a neuroactive steroid, solubilizers, sterile packaging, and any combination thereof. Such packaging of the components separately can, if desired, be presented in a pack or dispenser device which may contain one or more unit dosage forms containing the composition. The pack may, for example, comprise metal or plastic foil such as a blister pack. Such packaging of the components separately can also, in certain instances, permit long-term storage without losing the activity of the components.

[0100] Kits may also include reagents in separate containers such as, for example, sterile water or saline to be added to a lyophilized active component packaged separately. For example, sealed glass ampules may contain a lyophilized component and in a separate ampule, sterile water, sterile saline each of which has been packaged under a neutral non-reacting gas, such as nitrogen. Ampules may consist of any suitable material, such as glass, organic polymers, such as polycarbonate, polystyrene, ceramic, metal or any other material typically employed to hold reagents. Other examples of suitable containers include bottles that may be fabricated from similar substances as ampules, and envelopes that may consist of foil-lined interiors, such as aluminum or an alloy. Other containers include test tubes, vials, flasks, bottles, syringes, and the like. Containers may have a sterile access port, such as a bottle having a stopper that can be pierced by a hypodermic injection needle. Other containers may have two compartments that are separated by a readily removable membrane that upon removal permits the components to mix. Removable membranes may be glass, plastic, rubber, and the like.

[0101] In certain embodiments, kits can be supplied with instructional materials. Instructions may be printed on paper or other substrate, and / or may be supplied as an electronic-readable medium or video. Detailed instructions may not be physically associated with the kit; instead, a user may be directed to an Internet web site specified by the manufacturer or distributor of the kit.

[0102] A control sample or a reference sample as described herein can be a sample from a healthy subject. A reference value can be used in place of a control or reference sample, which was previously obtained from a healthy subject or a group of healthy subjects. A control sample or a reference sample can also be a sample with a known amount of a detectable compound or a spiked sample.

[0103] The methods and algorithms of the invention may be enclosed in a controller or processor. Furthermore, methods and algorithms of the present invention, can be embodied as a computer-implemented method or methods for performing such computer-implemented method or methods, and can also be embodied in the form of a tangible or non-transitory computer-readable storage medium containing a computer program or other machine-readable instructions (herein “computer program”), wherein when the computer program is loaded into a computer or other processor (herein “computer”) and / or is executed by the computer, the computer becomes an apparatus for practicing the method or methods. Storage media for containing such computer programs include, for example, floppy disks and diskettes, compact disk (CD)-ROMs (whether or not writeable), DVD digital disks, RAM and ROM memories, computer hard drives and back-up drives, external hard drives, “thumb” drives, and any other storage medium readable by a computer. The method or methods can also be embodied in the form of a computer program, for example, whether stored in a storage medium or transmitted over a transmission medium such as electrical conductors, fiber optics or other light conductors, or by electromagnetic radiation, wherein when the computer program is loaded into a computer and / or is executed by the computer, the computer becomes an apparatus for practicing the method or methods. The method or methods may be implemented on a general-purpose microprocessor or on a digital processor specifically configured to practice the process or processes. When a general-purpose microprocessor is employed, the computer program code configures the circuitry of the microprocessor to create specific logic circuit arrangements. Storage medium readable by a computer includes medium being readable by a computer per se or by another machine that reads the computer instructions for providing those instructions to a computer for controlling its operation. Such machines may include, for example, machines for reading the storage media mentioned above.

[0104] Compositions and methods described herein utilizing molecular biology protocols can be according to a variety of standard techniques known to the art (see e.g., Sambrook and Russel (2006) Condensed Protocols from Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, ISBN-10: 0879697717; Ausubel et al. (2002) Short Protocols in Molecular Biology, 5th ed., Current Protocols, ISBN-10: 0471250929; Sambrook and Russel (2001 ) Molecular Cloning: A Laboratory Manual, 3d ed., Cold Spring Harbor Laboratory Press, ISBN-10: 0879695773; Elhai, J. and Wolk, C. P. 1988. Methods in Enzymology 167, 747-754; Studier (2005) Protein Expr Purif. 41 (1 ), 207-234; Gellissen, ed. (2005) Production of Recombinant Proteins: Novel Microbial and Eukaryotic Expression Systems, Wiley-VCH, ISBN-10: 3527310363; Baneyx (2004) Protein Expression Technologies, Taylor & Francis, ISBN-10: 0954523253).

[0105] Definitions and methods described herein are provided to better define the present disclosure and to guide those of ordinary skill in the art in the practice of the present disclosure. Unless otherwise noted, terms are to be understood according to conventional usage by those of ordinary skill in the relevant art.

[0106] In some embodiments, numbers expressing quantities of ingredients, properties such as molecular weight, reaction conditions, and so forth, used to describe and claim certain embodiments of the present disclosure are to be understood as being modified in some instances by the term “about.” In some embodiments, the term “about” is used to indicate that a value includes the standard deviation of the mean for the device or method being employed to determine the value. In some embodiments, the numerical parameters set forth in the written description and attached claims are approximations that can vary depending upon the desired properties sought to be obtained by a particular embodiment. In some embodiments, the numerical parameters should be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. Notwithstanding that the numerical ranges and parameters setting forth the broad scope of some embodiments of the present disclosure are approximations, the numerical values set forth in the specific examples are reported as precisely as practicable. The numerical values presented in some embodiments of the present disclosure may contain certain errors necessarily resulting from the standard deviation found in their respective testing measurements. The recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually recited herein. The recitation of discrete values is understood to include ranges between each value.

[0107] In some embodiments, the terms “a” and “an” and “the” and similar references used in the context of describing a particular embodiment (especially in the context of certain of the following claims) can be construed to cover both the singular and the plural, unless specifically noted otherwise. In some embodiments, the term “or” as used herein, including the claims, is used to mean “and / or” unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive.

[0108] The terms “comprise,” “have” and “include” are open-ended linking verbs. Any forms or tenses of one or more of these verbs, such as “comprises,” “comprising,” “has,” “having,” “includes” and “including,” are also open-ended. For example, any method that “comprises,” “has” or “includes” one or more steps is not limited to possessing only those one or more steps and can also cover other unlisted steps. Similarly, any composition or device that “comprises,” “has” or “includes” one or more features is not limited to possessing only those one or more features and can cover other unlisted features.

[0109] All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided with respect to certain embodiments herein is intended merely to better illuminate the present disclosure and does not pose a limitation on the scope of the present disclosure otherwise claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the present disclosure. Groupings of alternative elements or embodiments of the present disclosure disclosed herein are not to be construed as limitations. Each group member can be referred to and claimed individually or in any combination with other members of the group or other elements found herein. One or more members of a group can be included in, or deleted from, a group for reasons of convenience or patentability. When any such inclusion or deletion occurs, the specification is herein deemed to contain the group as modified thus fulfilling the written description of all Markush groups used in the appended claims.

[0110] All publications, patents, patent applications, and other references cited in this application are incorporated herein by reference in their entirety for all purposes to the same extent as if each individual publication, patent, patent application, or other reference was specifically and individually indicated to be incorporated by reference in its entirety for all purposes. Citation of a reference herein shall not be construed as an admission that such is prior art to the present disclosure.

[0111] Having described the present disclosure in detail, it will be apparent that modifications, variations, and equivalent embodiments are possible without departing from the scope of the present disclosure defined in the appended claims. Furthermore, it should be appreciated that all examples in the present disclosure are provided as non-limiting examples.

[0112] EXAMPLES

[0113] The following non-limiting examples are provided to further illustrate the present disclosure. It should be appreciated by those of skill in the art that the techniques disclosed in the examples that follow represent approaches the inventors have found function well in the practice of the present disclosure, and thus can be considered to constitute examples of modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments that are disclosed and still obtain a like or similar result without departing from the spirit and scope of the present disclosure. EXAMPLE 1 - NEUROACTIVE STEROID ANALOGS AND VOLTAGE-DEPENDENT BLOCKERS OF CAV3.2 CURRENTS, B372 AND YX23, ARE EFFECTIVE ANALGESICS WITH DIMINISHED SEDATIVE PROPERTIES IN INTACT FEMALE MICE

[0114] In this study two neuroactive steroids, B372 and YX23 were shown to be effective analgesics in intact animals when injected into the receptive field of sensory neurons of a hind paw at doses that do not cause systemic analgesic effects. Furthermore, by modifying the chemical structure of their parent neuroactive steroids, 3[3-OH and ECN, respectively, the analogs when injected systemically, do not cause hypnosis thus making them potentially more useful in the outpatient clinical setting where analgesia, without heavy sedation, is a desirable therapeutic goal.

[0115] Voltage Gated Calcium Channels (VGCCs) serve essential functions in action potential formation, cellular excitability regulation, and synaptic transmission control. These channels are classified based on their activation thresholds: high- voltage-activated (HVA) channels and low-voltage-activated (LVA) channels, also known as transient or T-type Ca2+channels (Catterall et al., Annu Rev Cell Dev Biol, 2000, 16, 521-555). Different genes encode the a1 subunits that constitute the channel pores. The Cav1 family produces L-type channels, while the Cav2 family includes three subtypes: Cav2.1 (P / Q-type), Cav2.2 (N-type), and Cav2.3 (R-type) channels. Research has demonstrated that N-type channel blockers significantly contribute to presynaptic inhibition within the spinal cord's dorsal horn. Furthermore, knockout studies using Cav2.1 and Cav2.3 deficient mice have revealed altered pain responses, indicating these channels' involvement in nociceptive processing (Saegusa et al., Proc Natl Acad Sci, 2000, 97(11 ), 6132- 6137). The cloning of T-channel pore-forming a1 subunits has identified three distinct subtypes: Cav3.1 (G-type), Cav3.2 (H-type), and Cav3.3 (l-type) (Perez- Reyes et al., Physiol Rev, 2003, 83(1 ), 117-161 ). Among these, Cav3.2 channels have been established as critical mediators of nociception (Todorovic et al., Neuron, 2001 , 31 (1 ), 75-85; Bourinet et al., EMBO J, 2005, 24(2), 315-324; Choi et al., Genes Brain Behav, 2007, 6(5), 425-431 ; and Jacus et al., J Neurosci, 2012, 32(27), 9374-9382), including findings from recent investigations involving plantar skin incision models (Tat et al., Cells, 2020, 9(12); Joksimovic et al., Sci Signal, 2018, 11 (545); and Joksimovic et al., Mol Neurobiol, 2020, 57(1 ), 208-216). The current research contributes significantly to the existing body of evidence supporting Cav3.2 channels as viable therapeutic targets. The study demonstrates that novel synthesized compounds (NASs) that selectively block Cav3.2 channels provide effective analgesia in WT animals while showing no effect in Cav3.2 KO mice. This validates the specificity of these inhibitors and reinforces the central role of Cav3.2 channels in pain transmission pathways.

[0116] Neuroactive steroids are potent modulators of neuronal activity by causing a variety of behavioral and neuroendocrine changes in humans and animals (e.g., general anesthesia, an-algesia, cognitive and mood disturbances) (Zorumski et al., IDrugs, 2000, 3(9), 1053-1063 and Zorumski et al., Neurobiol Stress, 2019, 11 , 100196). Interestingly, it is traditionally believed that these effects on neurosensory processing and neuronal excitability are primarily mediated by their potent modulation of GABAA receptors. For example, neuroactive steroid, alphaxalone ((3a, 5a)-3-hydroxypregnane-11 ,20-dione) which effectively potentiates GABAA- gated currents is also a good analgesic (Pathirathna et al., Pain, 2005, 114(3), 429-443) but importantly, a potent general anesthetic, a common property of positive allosteric GABAA modulators. However, as previously reported, alphaxalone is also a potent inhibitor of T-currents in DRG neurons suggesting that its analgesic effects could also be due to its blocking effect on T-channels (Pathirathna et al., Pain, 2005, 114(3), 429-443). Although hypnotic effects combined with potent analgesic action could be very beneficial in surgical setting where general anesthesia is a desired state, in the outpatient clinical setting the goal of pain alleviation without serious sedation is of paramount importance to assure good quality of life. Hence, there is a need for the development of effective analgesics that are not only non-addictive but also non-sedative / non-hypnotic.

[0117] Of importance for this study presented herein is the previous discovery that a neuroactive steroid with a 5a configuration at the steroid A,B ring fusion, [(+)- ECN] [(3[3,5a,17[3)-17-hydroxyestrane-3-carbonitrile] (see structure on FIG. 2), is a potent volt-age-dependent blocker of Cav3.2 T-channels in rat dorsal root ganglion (DRG) neurons (Todorovic et al., Mol Pharmacol, 1998, 54(5), 918-927). Importantly, ECN only weakly inhibits recombinant Cav2.3 currents (Nakashima et al., Neuropharmacology, 1999, 38(6), 843-855) and has very little effect on voltage gated Na+, K+, N- and L-type HVA Ca2+channels, glutamate, and GABA-gated channels (23Todorovic et al., Mol Pharmacol, 1998, 54(5), 918-927). Furthermore, it has been shown that the analgesic efficacy of alphaxalone, ECN and related Sa- reduced steroids are correlated with their ability to inhibit T-currents in DRG neurons (Pathirathna et al., Pain, 2005, 114(3), 429-443). Several synthetic 5|3- reduced steroid analogues have been identified that lack any direct effect on GABAA currents but potently and completely inhibit Cav3.2 currents in DRG cells and exhibit potent analgesic effects in vivo (Todorovic et al., Mol Pharmacol, 2004, 66(5), 1223-1235). One of the most potent and efficacious steroid analogues in this group, 3[3-OH ((3[3,5[3,17[3)-3-hydroxyandrostane-17-carbonitrile) (FIG. 2) is a voltage-dependent and selective blocker of T-currents in acutely dissociated DRG cells (Todorovic et al., Mol Pharmacol, 2004, 66(5), 1223-1235). However, limited aqueous solubility (ECN) and potent hypnotic / sedative effects (3[3-OH) linked to 3[3-OH effects on GABAA receptors may hinder future development of these neuroactive steroids for novel pain therapies. Specifically, although 3[3-OH lacks any direct effect on synaptic and extra-synaptic GABAA receptors, previously published reports show that a sex-specific hypnotic effect of 3[3-OH after intraperitoneal (i.p.) injections is largely mediated by its peripheral metabolism into an active metabolite, 3a-OH that is a potent positive allosteric modulator of neuronal GABAA receptors (Manzella et al., Br J Anaesth, 2023, 130(2), 154-164). Herein, using two novel neuroactive steroid analogs, B372 and YX23 with more favorable pharmacokinetic and pharmacodynamic properties, as well as better water solubility, their analgesic potential as neuronal Cav3.2 inhibitors in pain pathways is explored. It is believed that the introduction of novel neuroactive steroids analogs as presented in this study is an important step toward an intended therapeutic goal, i.e. these novel neuroactive steroids analogs may get us a step closer to promising therapeutic strategy that is non-habit forming and non-sedative.

[0118] This study focuses on female mice since sex differences in pain perception and response to pain are well documented, i.e. females are in general more sensitive to a variety of painful conditions thus more commonly requiring the use of an array of analgesics and psychological pain coping strategies (Smith et al., J clin Invest, 2025, 135(11 )). In addition, based on previous work, sex-specific hypnotic effect of some neuroactive steroids (i.e. 3[3-OH) depends on their peripheral metabolism that seems to be more pronounced in female sex (Manzella et al., Br J Anaesth, 2023, 130(2), 154-164) thus resulting in a higher propensity for sedative / hypnotic side effects that could have a negative impact on daily functioning and the quality of life.

[0119] Although not of interest for this study, it is noteworthy that previously examined neuroactive steroid analogs with T-channel blocking properties, 3[3-OH and ECN are also recognized as potentially useful drugs for the treatment of chronic pain (Joksimovic et al., Front Pharmacol, 2018, 9, 1127). When injected systemically or intrathecally they were reported to ameliorate the development of allodynia, both mechanical and thermal. Further studies with B372 and YX23 in the setting of chronic pain could be a promising next step especially since chronic use of opioids is controversial and often contraindicated in this patient population.

[0120] EXAMPLE 2 - MATERIALS AND METHODS

[0121] Animals

[0122] Adult female C57BL / 6J mice were purchased from Jackson Laboratories. Animals were housed on a 14:10 light-dark cycle and given access to food and water ad libitum. Cav3.2 knock-out (KO) mice were purchased from Jackson Laboratories and then bred in-house. All experiments were approved by the Institutional Animal Care and Use Committee (IACUC) at the University of Colorado Anschutz Medical Campus and adhered to the National Institutes of Health (NIH) Guide for the Care and Use of Laboratory Animals.

[0123] Drug preparation

[0124] Synthesis of YX23 (3B,5a,17B)-3-Methoxyestran-17-ol.

[0125] (5a, 17|3)-17-Hydroxyestran-3-one (2).

[0126] Small pieces of lithium (630 mg) were added to a three-neck round bottom flask. The flask was equipped with a jacketed condenser containing a dry ice / acetone coolant. Anhydrous NH3 was condensed into the flask to obtain liquid NH3 (~150 mL) at -78 °C. THF (50 mL) was added, followed by the 17(3- hydroxyestr-4-en-3one (1 , 5 g, 18.2 mmol) dissolved in THF and added in three portions (50 mL + 10 mL + 10 mL). After 1 h, solid NH4CI (10 g) was added, the reaction was allowed to warm to room temperature (ca. 23 °C) and the ammonia allowed to evaporate overnight. Water was added to the flask contents and the product was extracted into EtOAc (200 mL x 2). The combined extracts were dried over anhydrous Na2SO4 and filtered. The solvent was removed on a rotary evaporator under reduced pressure and the residue was purified by flash column chromatography (silica gel, eluted with 20% EtOAc in hexanes) to give product 2 (3.47 g, 69%):1H NMR (400 MHz, CDCI3) 5 3.65-3.61 (m, 1 H), 0.75 (s, 3H), 2.40- 0.67 (m, 24H);13C NMR (100 MHz, CDCI3) 5 211.9, 81.8, 49.9, 48.6, 47.7, 45.7, 43.6, 43.0, 41.2, 41.0, 36.5, 33.8, 30.5, 30.4, 30.1 , 25.7, 23.1 , 11.0.

[0127] (5a, 17|3)-17-((tert-Butyldimethylsilyl)oxy)-estran-3-one (3).

[0128] To a solution of steroid 2 (1 .57 g, 5.7 mmol) in DMF (20 mL) was added TBSCI (1 .29 g, 8.5 mmol) and imidazole (0.77 g, 11 .4 mmol) at room temperature (~23 °C). After 16 h, saturated aqueous NaHCOs was added and the product was extracted into EtOAc (100 mL). The EtOAc was washed with brine (100 mL x 3), dried over anhydrous Na2SO4 and filtered. The solvent was removed on a rotary evaporator under reduced pressure and the residue was purified by flash column chromatography (silica gel, eluted with 10% EtOAc in hexanes) to give product 3 (2.10 g, 94 %):1H NMR (400 MHz, CDCI3) 5 3.56-3.52 (m, 1 H), 2.36-0.65 (m, 23H), 0.86 (s, 9H), 0.72 (s, 3H), 0.00 (s, 3H), -0.01 (s, 3H);13C NMR (100 MHz, CDCI3) 5 211 .9, 81 .7, 49.5, 48.6, 47.9, 45.7, 43.7, 43.3, 41 .3, 41 .0, 36.9, 33.9, 30.8, 30.5, 30.2, 25.8 (3C), 25.8, 23.3, 18.0, 11.3, -4.5, -4.9. (3(3 , 5a, 17|3)-17-((tert-Butyldimethylsilyl)oxy)-estran-3-ol (4).

[0129] To a solution of steroid 3 (2.1 g, 5.4 mmol) in THF (50 mL) was added Lithium tri-te / Y-butoxyaluminum hydride (7 mL, 1.0 M in THF, 7 mmol) at -40 °C. After 1.5 h, water was added and the reaction was allowed to rise to room temperature (~23 °C). After 16 h, the product was extracted into dichloromethane (100 mL x 3). The combined extracts were washed with water (100 mL), dried over anhydrous Na2SO4 and filtered. The solvent was removed on a rotary evaporator under reduced pressure and the residue was purified by flash column chromatography (silica gel, eluted with 20% EtOAc in hexanes) to give product 4 (1.90 g, 90%):1H NMR (400 MHz, CDCh) 5 3.56-3.51 (m, 1 H), , 2.36-0.65 (m, 25H), 0.87 (s, 9H), 0.70 (s, 3H), 0.00 (s, 3H), -0.01 (s, 3H);13C NMR (100 MHz, CDCh) 5 81.8, 70.5, 49.8, 48.2, 46.2, 43.4, 43.3, 41.3, 41.2, 37.1 , 35.7, 33.5, 30.9, 30.6, 28.4, 25.8 (3C), 25.6, 23.3, 18.1 , 11.3, -4.5, -4.9.

[0130] (3(3 , 5a, 17[3)-3-Methoxy-17-((tert-butyldimethylsilyl)oxy)-estrane (5).

[0131] A suspension of sodium hydride (60% in mineral oil, 0.6 g, 15 mmol) and steroid 4 (1.90 g, 4.8 mmol) in THF (100 mL) was refluxed under N2 for 1 h. lodomethane (1.5 mL, 22.5 mmol) was added and the reaction was stirred for 2 h. After cooling to room temperature (~23 °C), water was slowly added and the product was extracted into EtOAc (100 mL x 2). The combined extracts were dried over anhydrous Na2SO4 and filtered. The solvent was removed on a rotary evaporator under reduced pressure and the residue was purified by flash column chromatography (silica gel, eluted with 10% EtOAc in hexanes) to give product 5 (1.82 g, 93%):1H NMR (400 MHz, CDCh) 5 3.56-3.52 (m, 1 H), 3.34 (s, 3H), 3.13- 3.08 (s, 1 H), 2.10-0.53 (m, 23H), 0.87 (s, 9H), 0.70 (s, 3H), 0.00 (s, 3H), 0.00 (s, 3H);13C NMR (100 MHz, CDCh) 5 81.9, 79.1 , 55.5, 49.8, 48.3, 46.6, 43.4, 41.3, 41.2, 39.6, 37.1 , 33.6, 32.1 , 30.9, 30.6, 28.4, 25.8 (3C), 25.6, 23.4, 18.1 , 11.3, - 4.5, -4.8. (3[3,5a,17[3)-3-Methoxyestran-17-ol (6, YX23).

[0132] To a solution of steroid 5 (1 .82 g, 4.47 mmol) in THF (30 mL) was added TBAF (6.7 mL, 1.0M in THF, 6.7 mmol) at room temperature. The reaction was refluxed for 2 h and the solvent was removed on a rotary evaporator under reduced pressure. The residue was purified by flash column chromatography (silica gel, eluted with 20% EtOAc in hexanes) to give product 6 (YX23) (1 .30 g, 100%):1H N MR (400 MHz, CDCh) 5 3.64-3.59 (m, 1 H), 3.33 (s, 3H), 3.13-3.08 (s, 1 H), 2.10-0.55 (m, 24H), 0.73 (s, 3H);13C NMR (100 MHz, CDCh) 5 81.9, 79.1 ,

[0133] 55.5, 50.1 , 48.1 , 46.5, 43.0, 41.2, 41.1 , 39.5, 36.7, 33.5, 32.1 , 30.5, 30.4, 28.4,

[0134] 25.5, 23.2, 11.0.

[0135] Synthesis of B372 (3B,5a,17B)-3-Hvdroxyandrostan-17-carbonitrile

[0136] (3[3,5a)-3-(Methoxymethoxy)-androstan-17-one (2).

[0137] To a solution of epiandrosterone (1 , 2 g, 6.9 mmol) in dichloromethane (40 mL) was added chloromethyl methyl ether (13.8 mmol) and ( / -Pr)2NEt (20.7 mmol) at 23 °C. After 16 h, the solvent was removed and the residue was purified by flash column chromatography (silica gel, eluted with 20% EtOAc in hexanes) to give steroid 2 (2.15g , 93%):1H NMR (400 MHz, CDCh) 5 4.68-4.66 (m, 2H), 3.51 -3.45 (m, 1 H), 3.35 (s, 3H), 2.46-2.39 (m, 1 H), 2.10-2.00 (m, 1 H), 1.95-0.90 (m, 20H), 0.84 (s, 3H), 0.82 (s, 3H);13C NMR (100 MHz, CDCh) 5 221.4, 94.5, 76.1 , 55.1 ,

[0138] 54.4, 51.3, 47.7, 44.8, 36.9, 35.8, 35.7, 35.1 , 35.0, 31.5, 30.8, 28.6, 28.4, 21.7,

[0139] 20.4, 13.7, 12.2. (3[3,5a)-3-(Methoxymethoxy)-androst-16-ene-17-ol, 17-(1 , 1 , 1 trifluoromethanesulfonate) (3).

[0140] To a solution of steroid 2 (2.15 g, 6.4 mmol) in THF (~ 80 mL) was added potassium hexamethyldisilazide (25 mL, 12.5 mmol) and A / -phenyl bis(trifluoromethanesulfonimide) (5 g, 14 mmol) at -78 °C. After 1 h, the reaction was slowly warmed to 23 °C and after stirring overnight, the reaction was quenched with water and brine. The product was extracted into EtOAc (150 mL x 3). The solvent was removed, and the residue was purified by flash column chromatography (silica gel, eluted with 10% EtOAc in hexanes) to give steroid 3 (2.57 g, 86%):1H NMR (400 MHz, CDCh) 5 5.53 (s, 1 H), 4.65 (s, 2H), 3.50-3.45 (m, 1 H), 3.34 (s, 3H), 2.20-2.16 (m, 1 H), 1.98-0.72 (m, 19H), 0.93 (s, 3H), 0.82 (s, 3H);13C NMR (100 MHz, CDCh) 5 159.3, 121.0 (q, J = 291.4 Hz), 114.4, 94.5, 76.0, 55.0, 54.7, 54.2, 45.0, 44.8, 36.7, 35.8, 35.1 , 33.4, 32.6, 30.8, 28.5, 28.5, 28.4, 20.4, 15.2, 12.1.

[0141] (3[3,5a)-3-(Methoxymethoxy)-androst-16-ene-17-carbonitrile (4).

[0142] To a solution of steroid 3 (2.57 g, 5.5 mmol) was added Cu (I) I (100 mg), NaCN (940 mg) and tetrakis(triphenylphosphine)palladium (260 mg) under a nitrogen atmosphere. The reaction was refluxed for 2 h and cooled to room temperature. Aqueous saturated NaHCCh (40 mL) and water (100 mL) were added and the product was extracted into EtOAc (100 ml x 3). The combined organic layers were washed with brine (100 mL), dried over anhydrous Na2SO4, filtered and the solvent was removed. The residue was purified by flash column chromatography (silica gel, eluted with 10% EtOAc in hexanes) to give steroid 4 (1 .65 g, 87%):1H NMR (400 MHz, CDCh) 5 6.6 (s, 1 H), 4.67 (s, 2H), 3.51 -3.46 (m, 1 H), 3.36 (s, 3H), 2.36-2.29 (m, 1 H), 2.11 -2.04 (m, 1 H), 1.91 -0.72 (m, 18H), 0.90 (s, 3H), 0.84 (s, 3H);13C NMR (100 MHz, CDCh) 5 147.4, 127.4, 115.9, 94.5, 76.0,

[0143] 55.8, 55.1 , 54.6, 48.2, 44.9, 36.7, 35.8, 35.1 , 34.0, 33.9, 32.8, 31.7, 28.5, 28.4,

[0144] 20.8, 16.3, 12.1.

[0145] (3(3 , 5a, 17[3)-3-(Methoxymethoxy)-androstan-17-carbonitrile (5).

[0146] To a solution of steroid 4 (1 .65 g, 4.8 mmol) in EtOAc (150mL) was added Pd / C (10%, 200 mg) in a Parr hydrogenation flask and hydrogenation was continued at 55 psi H2 overnight. The mixture was filtered through Celite and washed with EtOAc. The solvent was removed and the residue was purified by flash column chromatography (silica gel, eluted with 20-50% EtOAc in hexanes) to give steroid 5 (1.63 g, 98%):1H NMR (400 MHz, CDCh) 5 4.67 (s, 2H), 3.51 -3.46 (m, 1 H), 3.36 (s, 3H), 2.28-2.24 (m, 1 H), 2.11 -2.08 (m, 1 H), 1.96-0.65 (m, 21 H), 0.90 (s, 3H), 0.82 (s, 3H);13C NMR (100 MHz, CDCh) 5 121.4, 94.5, 76.1 , 55.1 , 54.3, 54.0, 44.7, 44.4, 40.2, 37.1 , 36.9, 35.8, 35.6, 35.1 , 31.9, 28.6, 28.5, 26.5, 24.5, 20.8, 14.3, 12.2.

[0147] (3[3,5a,17[3)-3-Hydroxyandrostan-17-carbonitrile (6, B372).

[0148] To a solution of the steroid 5 (1 .63 g, 4.7 mmol) in methanol (40 mL) was added acetyl chloride (2 ml) at room temperature. After 2 h, water (20 mL) was added and the product was extracted into dichloromethane (100 mL x 2). The combined organic layers were washed with brine (50 mL x 3), dried over anhydrous Na2SO4 and the solvent removed. The residue was purified by flash column chromatography (silica gel, eluted with 20% EtOAc in hexanes) to give B372 (1.31 g, 93%):1H NMR (400 MHz, CDCh) 5 3.56-3.54 (m, 1 H), 2.26-1.90 (m, 1 H), 1 .79-0.61 (m, 23H ), 0.88 (s, 3H), 0.79 (s, 3H);13C NMR (100 MHz, CDCh) 5 121.3, 70.9, 54.2, 53.9, 44.6, 44.3, 40.1 , 37.9, 37.0, 36.8, 35.7, 35.4, 31.8, 31.2, 28.3, 26.4, 24.4, 20.8, 14.2, 12.2.

[0149] All neuroactive steroids used in this study were dissolved in 15% (2- hydroxypropyl)-[3-cyclodextnn solution (Santa Cruz Biotechnology Inc., Dallas, TX, USA). Drugs were delivered either via intraperitoneal injection (i.p.) at a volume of 10 pl / g or intraplantar injection at a volume of 20pl.

[0150] Behavioral tests:

[0151] Electronic Von Frey

[0152] Mechanical sensitivity was assessed using an electronic Von Frey apparatus (Ugo Basile, Stoelting, USA). The withdrawal threshold was defined as the minimum force (in grams) required to elicit a clear withdrawal response. The testing took place over two consecutive days.

[0153] Day 1: Baseline Assessment

[0154] Mice were habituated to the testing room in their home cages for 30 minutes prior to testing. Animals were then placed individually in clear plexiglass chambers on an elevated mesh platform allowing access to the plantar surface of the hind paws. After a 30-m inute acclimation period to the testing apparatus, mechanical sensitivity was assessed by applying the electronic von Frey filament to the mid- plantar region of each hind paw. Measurements were taken at three time points (30, 35, and 40 minutes after placement in the apparatus), with one application per hind paw at each time point. The schematic diagram of the testing protocol is presented in FIG. 1A.

[0155] Day 2: Post-injection Assessment

[0156] The baseline assessment protocol from Day 1 was repeated, followed by a 2-hour rest period in their home cages. Von Frey testing begins with mice receiving a 20p I intraplantar hind paw injection of either neuroactive steroid or vehicle. Immediately after injection, mice were placed in the testing apparatus. Mechanical sensitivity was assessed in both the injected and contralateral hind paws at 10-, 20-, 40-, and 60-minutes post-injection, with three applications per hind paw at each time point. The schematic diagram of the testing protocol is presented in FIG. 1B

[0157] Open Field Test

[0158] Anxiolytic effects of the neuroactive steroids were assessed using the open field test. Mice behavior was tracked using ANY-maze video tracking software (Stoelting Co., Wood Dale, IL, USA). The testing apparatus consisted of a square container (40cm x 40cm). Using ANY-maze, the arena was divided into 2 zones. The outer 1 / 3 was classified as the outer zone and the inner 2 / 3 was classified as the inner zone. Total distance travelled and average velocity was also tracked by ANY-maze software. The schematic diagram of the testing protocol and apparatus is presented in FIG. 1C.

[0159] Mice were habituated to the testing room 30 minutes before the test starts. The animals then received either drug or vehicle via i.p. injection at a volume of 10 pl / g body weight. Thirty minutes post injection, mice were placed within the testing apparatus and allowed to roam freely for 15 minutes. The arena was thoroughly cleaned with 70% ethanol between subjects to eliminate olfactory cues.

[0160] Electrophysiology:

[0161] Human embryonic kidney (HEK-293) cells were stably transfected to express human Cav3.2 channels. Cells were grown in Dulbecco’s modified Eagle’s medium and Ham’s F-12 Nutrient Mixture supplemented with 10% fetal bovine serum (FBS), 100 U / ml penicillin, and 0.1 mg / ml streptomycin (0.1 mg / ml), and incubated at 37°C with 5% CO2. Media exchange was performed every 2 days, with regular splitting after every 4-7 days (at 85%-90% confluency). Prior to recording, cells were split and plated onto poly-D-lysine-coated glass coverslips and allowed to adhere for 2-24 h in an incubator.

[0162] For recordings in HEK-293 cells, electrodes were pulled from borosilicate microcapillary tubes to a final resistance of 3-5 MQ. To record T-currents, electrodes were filled with internal solution comprising 135 mM tetramethylammonium (TMA) hydroxide, 10 mM ethylene glycol tetraacetic acid (EGTA), 2 mM MgCL, and 40 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES), and titrated in a plastic dish to a pH of 7.15-7.25 using hydrofluoric acid. The HEK-293 culture medium was exchanged with external solution comprising 152 mM tetraethylammonium (TEA) chloride, 10 mM HEPES, and 2 mM CaCL, and titrated to a pH of 7.4 with TEA hydroxide. Gigaohm seals were reached prior to cell opening to obtain the whole-cell configuration. Membrane resistance, access resistance, and cell capacitance were monitored during the formation of the patch and the subsequent recordings. To obtain current-voltage (IV) curves, cells were maintained at a holding potential (Vh) of -90 mV and subjected to multiple depolarizing steps (Vt) ranging from -70 mV to +25 mV. The duration of the voltage step was 320 ms. To obtain steady-state inactivation curves, cells were held at Vh = -90 mV, hyperpolarized or depolarized (-110 mV to -40 mV) for a duration of 3.5 s, and then brought to a Vt = -30 mV.

[0163] Data and statistical analysis for patch-clamp experiments.

[0164] Statistical comparisons in the in vitro experiments were made using paired t-test. All data are expressed as mean ± standard error of the mean (SEM); p values are reported only when statistically significant (<0.05). The percent reductions in peak current at various concentrations of neuroactive steroids were used to generate concentration-response curves. Mean values were fit to the following Hill-Langmuir function:

[0165] P l([Drug])=P lmax / ( 1 +(ICso / [Drug]) / i) (1 ) where Plmax is the maximal percent inhibition of peak current by the drug, ICso is the concentration that produces 50% inhibition, and h is the apparent Hill- Langmuir coefficient for inhibition. The fitted values are reported with > 95% linear confidence limits.

[0166] The voltage-dependence of steady-state inactivation was fit to the Boltzmann equation: l(V) = lmax / (1 + exp[(V - Vso) / k]), where Imax is the maximal current amplitude, V50 is the half-maximal inactivation voltage, and k (units of millivolts) represents the slope factor.

[0167] Data and statistical analysis for in vivo experiments:

[0168] Two-tailed independent samples t-tests and one-way ANOVA followed by Tukey’s post hoc test were used to compare groups when equal variances were assumed, a was set at 0.05, thus p-values <0.05 were considered statistically significant. Data were graphed as mean ± SEM, and level of significance indicated by elbow connectors with asterisks. GraphPad Prism 9.3 (GraphPad Software Inc., San Diego, CA, USA) was used for all analyses.

Claims

CLAIMSWhat is claimed is:1 . A method of preventing or treating pain comprising administering aor a pharmaceutically acceptable salt thereof.

2. The method of claim 1 , wherein the pain is acute or chronic.

3. A method of preventing or treating pain comprising administering a therapeutically effective amount of a compound selected from:or a pharmaceutically acceptable salt thereof, to inhibit a voltage-gated calcium ion channel.

4. The method of claim 3, wherein the pain is acute or chronic.

5. The method of claim 3, wherein the voltage-gated calcium ion channel is selected from the group consisting of Cav1 , Cav2.1 , Cav2.2, Cav2.3, Cav3.1 , Cav3.2, and Cav3.3.

6. The method of claim 3, further comprising diminishing positive allosteric modulation of GABAa receptors.

7. The method of any one of claims 1 to 6, wherein the compound is od of any one of claims 1 to 6, wherein the compound is9. A compound selected from:manufacture of a medicament for preventing or treating pain in a mammal.11 . Use of a compound selected from:preventing or treatingpain in a mammal.

12. Use of a compound selected from:the manufacture of a medicament for preventing or treating pain in a mammal.

13. A compound of Formula Ior a pharmaceutically acceptable salt thereof.

14. A pharmaceutical composition comprising the compound of claim 13, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

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