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Characterization of Novel Cannabinoid Based T-Type Calcium Channel Blockers with Analgesic Effects

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† Department of Physiology & Pharmacology, Hotchkiss Brain Institute, Cumming School of Medicine, University of Calgary, Calgary, AB T2N 1N4, Canada
‡ Core Laboratory for Neuromolecular Production, The University of Montana, Missoula, Montana 59812, United States
*G.W.Z., for biology: Mailing address: Department of Physiology & Pharmacology, Cumming School of Medicine, University of Calgary, 3330 Hospital Drive NW, Calgary, Canada T2N 4N1. Telephone: 403-220-8687. E-mail: [email protected]
*P.D., for chemistry: Telephone: 406-243-4362. Fax: 406-243-5228. E-mail: [email protected]
Cite this: ACS Chem. Neurosci. 2015, 6, 2, 277–287
Publication Date (Web):October 14, 2014
https://doi.org/10.1021/cn500206a

Copyright © 2014 American Chemical Society. This publication is licensed under these Terms of Use.

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Abstract

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Low-voltage-activated (T-type) calcium channels are important regulators of the transmission of nociceptive information in the primary afferent pathway and finding ligands that modulate these channels is a key focus of the drug discovery field. Recently, we characterized a set of novel compounds with mixed cannabinoid receptor/T-type channel blocking activity and examined their analgesic effects in animal models of pain. Here, we have built on these previous findings and synthesized a new series of small organic compounds. We then screened them using whole-cell voltage clamp techniques to identify the most potent T-type calcium channel inhibitors. The two most potent blockers (compounds 9 and 10) were then characterized using radioligand binding assays to determine their affinity for CB1 and CB2 receptors. The structure–activity relationship and optimization studies have led to the discovery of a new T-type calcium channel blocker, compound 9. Compound 9 was efficacious in mediating analgesia in mouse models of acute inflammatory pain and in reducing tactile allodynia in the partial nerve ligation model. This compound was shown to be ineffective in Cav3.2 T-type calcium channel null mice at therapeutically relevant concentrations, and it caused no significant motor deficits in open field tests. Taken together, our data reveal a novel class of compounds whose physiological and therapeutic actions are mediated through block of Cav3.2 calcium channels.

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T-type calcium channels are known for regulating neuronal and cardiac pacemaker activity. (1-4) They open in response to small membrane depolarizations that in turn trigger the initiation of action potentials. (1, 5, 6) Disruption of this sensitive signaling mechanism often leads to hyperexcitability disorders such as arrhythmia, epilepsy, and pain. (3, 7-21) The mammalian genome expresses three different types of T-type calcium channels, Cav3.1, Cav3.2, and Cav3.3, with specific cellular functions. (22) The Cav3.2 T-type channel isoform is particularly interesting due to its role in afferent pain signaling. Indeed, up-regulation of the Cav3.2 T-type channel isoform in primary afferent fibers has been linked to chronic pain disorders, whereas ablation of these channels mediates analgesia. (13, 14, 17) The development of selective T-type channel antagonists has not been a trivial undertaking, with only a few such small organic molecules having recently been identified. (15, 23-33) Even more challenging is the development of Cav3 isoform selective blockers due to the large degree of sequence similarity among these three Cav3 channel family members.
Many of the known organic molecules that have been shown to modulate T-type calcium channels have structures similar to endogenous anandamide-related molecules named lipoamino acids. (16, 23, 34) Lipoamino acids are known to interact with T-type calcium channels and several are also closely related to endocannabinoids. (16, 25, 34, 35) Therefore, it is not surprising that many of these T-type blockers also interact with cannabinoid (CB) receptors. (16, 25, 34, 35) We have previously shown that this mixed T-type/cannabinoid block has beneficial effects in inducing analgesia in animal models of inflammatory pain. (16, 34) However, interactions with CB receptors, particularly CB1 receptors, can have side effects that may affect mood and memory, in addition to their known psychoactive effects. (36, 37) Synthetic T-type calcium channel antagonists TTAP-1 based on substituted piperidines have been previously disclosed (Scheme 1).

Scheme 1

Scheme 1. Piperidine Containing T-type Ca2+ Inhibitors TTA-P1, Dual T-type Channel Blocker/Cannabinoid Agonist NMP7, and Chemical Optimization Plan 1 To Decrease Cannabinoid Receptor Affinities
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In previous studies, we synthesized and characterized a series of novel cannabinoid ligands with the primary structure bearing a carbazole scaffold such as compound NMP7. (16, 34) These compounds produced mixed cannabinoid receptor/T-type channel blockers that were found to be efficacious in animal models of inflammatory and neuropathic pain. Interestingly, from structure–activity relationships (SARs), we determined that tertiary amines were important for Cav3.2 block (16, 23, 34) and that the length of the linker attaching the tertiary amine to the carbazole scaffold affected binding to CB1 and CB2 receptors. (38) Some of these compounds appeared to preferentially and potently inhibit T-type channels in vitro. The goal of this study was to optimize Cav3.2 calcium channel selectivity of our series of carbazole derivatives.
Here we used the aforementioned compounds as a starting point for the development of a new series of compounds 1 (Figure 1) based on a carbazole scaffold with an added heterocyclic bearing a tertiary amine. We modified the chain length attached to the nitrogen of the carbazole, the length of the linker between the amide bond and the heterocycle ring and introduction of a lipophilic moiety attached to the heterocycle and characterized this set of compounds in vitro for their ability to blocking transiently expressed human Cav3.2 (hCav3.2) calcium channels and tested their affinities for cannabinoid receptors. The most potent and selective compound (9) was then tested in mouse models of inflammatory and neuropathic pain, revealing potent analgesia by virtue of its Cav3.2 channel blocking ability.

Figure 1

Figure 1. Percentage of whole cell current inhibition of human Cav3.2 (T-type) in response to 10 μM application of the compound series (n = 6 per compound). Note the potent and preferential block of Cav3.2 channels by compounds 9 and 10. Error bars reflect standard errors. For Cav3.2 channels, the holding and test potentials were respectively −110 and −20 mV.

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Chemistry

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The synthesis of the carbazoles derivatives is outlined in Scheme 2. Amidation under standard peptide coupling conditions (38) of N-alkylated carbazole-3-carboxylic derivatives 2 with Boc-protected amines afforded the desired amide derivatives 3 and 6. Deprotection of the Boc protecting group in the presence of TFA in dichloromethane followed by alkylation of the resultant compounds 4 and 8 with N-tert-butyl-2-chloroacetamide provided the corresponding desired compounds 9, 10, 13, 16, and 19 (Tables 1 and 2). Compound 20 was prepared by reductive amination of compound 4 using 3,3-dimethylbutyraldehyde (Table 2).

Scheme 2

Scheme 2. a

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Scheme aReagents and conditions: (a) corresponding amine, EDAC, HOBt, DMAP, DIPEA, DCM, 0°C to rt; (b) TFA, CH2Cl2; (c) N-tert-butyl-2-chloroacetamide, K2CO3, KI, n-butanol, reflux 3 h.

Table 1. Radioligand Competitive Binding Assays (mean ± SEM) for Carbazole-Based Analogues 7–10: Systematic Variation in the Linker Lengtha
Table a

Values are means of three experiments run in triplicate with standard deviation; n.b. no binding.

Table 2. Analogues of Compound 9: Systematic Variation in N-Alkyl Chain Length and in the Region Occupied by the Heterocycle

Results

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In Vitro Characterization of the Compound Series

The entire first set of 10 compounds was screened using whole-cell voltage clamp techniques for their ability to mediate tonic block of transiently expressed hCav3.2 channels (Figure 1 and Table 3). Next, we used radioligand binding assays to assess the affinities of these compounds for both CB1 and CB2 receptors (Table 1).
In the course of our initial exploratory work on the structure–activity relationship for this novel series of T-type channel blockers, we decided to determine the optimal linker length attached to the carbazole’s carbonyle (Table 1). We observed various degrees of inhibition of these channels, with compounds 9 and 10 being the most potent blockers of expressed hCav3.2. These two compounds mediated near complete inhibition at our standard test concentration of 10 μM (Figure 1). Interestingly, both of these compounds are very similar in structure, with both having a cyclic tertiary amine attached to the carbazole scaffold (Table 1). Previous work has indicated that this modification helps confer T-type channel blocking activity onto various organic molecules, (16, 28, 29, 34) in agreement with our data presented here. As shown in Table 1, compound 10 showed high affinity binding to CB1 receptors (15 nM), whereas its affinity for the CB2 receptor was approximately 100-fold lower (2 μM). Compound 9, however, did not bind to the two receptors with an affinity less than 5 μM for CB1 and CB2. The only difference between compounds 9 and 10 is the elongated chain attached to the nitrogen of the carbazole in 9 (Table 1). This type of modification has been shown to alter cannabinoid receptor binding, (16, 34, 38) but it has not been demonstrated whether this modification affects the interactions of these compounds with Cav3.2. Among the first series of compounds 5–10, replacement of a piperazine moiety by a methylpiperidine moiety appeared to be the most optimal for decreasing cannabinoid receptor affinity without impacting Cav3.2 calcium channel block. This striking difference in affinity for cannabinoid receptors between compounds bearing a piperazine moiety compared to a methylpiperidine moiety underscores the importance of chain length when developing compounds that preferentially target Cav3.2 calcium channels over cannabinoid receptors. Next, we determined the optimal chain length attached to the carbazole’s endocyclic nitrogen (Table 1 and Table 2, compounds 9, 13, and 16). Among the linear N-1 alkyl chains, a pentyl chain seemed to be the most optimal for occupying its interaction site Cav3.2 channels, because systematically decreasing the length from n-pentyl in 9 negatively impacted the respective Cav3.2 blocking activities. Replacement of the piperidine ring by a pyrolidine moiety (16) had a slight negative effect on Cav3.2 block, probably due to the lack of optimal ligand–receptor van der Waals contacts. Replacing the amide chain on the piperidine ring by an alkyl chain (34) dramatically decreased the Cav3.2 block.
As is clearly seen in the traces in Figure 2A, the slight structural modification in 9 compared to 10 does indeed impact hCav3.2 channel inhibition. The affinity of 9 versus 10 increased more than 2-fold with the IC50 of 9 and 10 being 1.48 and 3.68 μM, respectively (Figure 2B and Table 3). In addition, compound 10 shifted the half activation potential of hCav3.2 by −12 mV (Figure 2C and Table 3). There was no significant effect on half-inactivation potential (P = 0.143) (Figure 2D and Table 3). We then tested the Cav3 channel subtype selectivity of compound 9 using a single concentration of 3 μM. This concentration blocked hCav3.2 by 69.3 ± 4% (n = 8), which was significantly (P < 0.05) greater than that of either hCav3.1 (44.5 ± 7%; n = 5) or hCav3.3 (42.5 ± 5%; n = 5). Compound 9 was thus chosen for further testing in animal models of pain.

Figure 2

Figure 2. (A) Representative traces of hCav 3.2 before and after application of 3 μM compounds 10 and 9. (B) Dose–response relations for compound 9 and 10 block of hCav3.2 channels. The IC50 from the fit with the Hill equation was 1.48 and 3.68 μM, respectively (n = 6). (C) Effect of 3 μM compounds 9 and 10 on the steady state inactivation curve for Cav3.2 channels. (D) Effect of 3 μM compounds 9 and 10 on the current voltage relation for Cav3.2 channels. Note: Data in panels (B) and (C) were fitted with the Boltzmann equation, and data were obtained from 6 paired experiments.

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Table 3. Summary of Biophysical Parameters of hCav3.2 Calcium Channel in the Absence and the Presence of Compounds 10 and 9a
 V0.5act (mV) 3 μMVh (mV) 3 μMIC50 tonic (μM)
Wt hCav3.2–30.0–53.1 ± 1.67 
compound 9–29.7–58.2 ± 1.431.48 ± 0.2
compound 10–42.0*–58.1 ± 1.313.68 ± 0.5
a

Note that 3 μM of compound 10 produces a significant 12 mV negative shift in the half activation potential of hCav3.2 and although there is a trend for both compounds shift the half inactivation potential of the channel, it did not reach statistical significance.

Effects of Compound 9 in Vivo on Acute Pain

Given the Cav3.2 channel blocking property of compound 9, we hypothesized that this compound may affect pain transmission in animal models. Compound 9 was delivered by either intrathecal (i.t.) or intraperitoneal (i.p.) routes, and its effects on both the acute nociceptive and the slower inflammatory pain phases of the formalin test were evaluated. One-way ANOVA revealed that i.t. treatment of mice with compound 9 (1–10 μg/i.t., 20 min before) significantly decreased pain response time in both first (Figure 3A) and second (Figure 3B) phases (61 ± 8% and 76 ± 10% inhibition, respectively). I.p. treatment of mice with compound 9 (10–100 mg/kg, i.p., 30 min prior) also resulted in significantly (one-way ANOVA) reduced pain response time in both the first (Figure 3C) and second (Figure 3D) phases (47 ± 2% and 66 ± 48% inhibition, respectively). Importantly, systemic (via i.p.) treatment with compound 9 (30 mg/kg, i.p.) did not affect locomotor activity of mice assessed via an open-field test (Figure 4A), suggesting that the reduced response times observed in the previous formalin tests were not due to altered motor behavior. In order to investigate if the effects observed for compound 9 were specifically mediated via T-type channels, we performed a formalin test in CaV3.2 null mice that were treated either with vehicle or with compound 9 (10 μg/i.t.). The CaV3.2 null mice exhibited a lower mean response time when compared to wild-type mice, which is in agreement with previous data. (14, 16) As indicated in Figure 4B and C, they appear to be completely insensitive to i.t. treatment with compound 9 (10 μg i.t.) as revealed by two-way ANOVA, indicating that compound 9 mediates its analgesic effects specifically via Cav3.2 channels.

Figure 3

Figure 3. (A, B) Effect of increasing doses of intrathecal compound 9 on the first and second phases of formalin-induced pain. (C, D) Effect of increasing doses of intraperitoneal compound 9 (on the first and second phases of formalin-induced pain. Each bar represents the mean ± SEM (n = 6–8), and is representative of 2 independent experiments. Asterisks denote the significance relative to the control group (***P < 0.001, one-way ANOVA followed by Dunnett’s test).

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Figure 4

Figure 4. (A) Effect of 30 mg/kg intraperitoneal compound 9 on locomotor activity of wild type mice in the open field test. (B, C) Comparison of effect of 10 μg/i.t. intrathecal compound 9 on the first and second phases of formalin-induced pain in wild type and Cav3.2 knockout mice, respectively. Each bar represents the mean ± SEM (n = 6–7) and is representative of 2 independent experiments. Asterisks denote the significance relative to the control group ***P < 0.001 when comparing treatment; and #P < 0.05, for comparison between genotypes (two-way ANOVA followed by Tukey’s test). Note that control mice were of the same genetic background as the Cav3.2 null mice.

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Effect of Compound 9 on Chronic Neuropathic Pain

To verify whether compound 9 modulates pain transmission under neuropathic conditions, we analyzed mechanical withdrawal thresholds of mice with a partial sciatic nerve injury (PNI) and treated with compound 9 (30 mg/kg, i.p.) 14 days after nerve injury. As shown in Figure 5, sciatic nerve injury triggers mechanical hyperalgesia as confirmed by significant decrease of mechanical withdrawal thresholds when compared to baselines levels (Pre-PNI, P < 0.001). Two-way ANOVA revealed that systemic (i.p.) treatment of mice with compound 9 (30 mg/kg, i.p.) significantly attenuated the mechanical hyperalgesia induced by sciatic nerve injury when compared with the PNI + Control group for longer than 3 h after treatment. These data indicate that compound 9 treatment modulates pain transmission and mediates analgesia in this animal model of chronic neuropathic pain.

Figure 5

Figure 5. Blind analyses of the time course of treatment of neuropathic mice with vehicle or compound 9. Each circle represents the mean ± SEM (n = 6), and is representative of 2 independent experiments. (*P < 0.05, ***P < 0.001, two-way ANOVA followed by Tukey’s test). The dashed line and number symbols indicate the range of data points where injured animals significantly differed from the sham treated group (P < 0.001).

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Discussion

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T-type calcium channels are important contributors to a range of physiological functions (2-5, 32, 39-41) and it is well established that the Cav3.2 channel isoform plays important roles in the afferent pain pathway. (12, 14, 15, 19, 42-44) Finding specific and selective blockers of these channels has proven difficult as many of the well-known T-type channel blockers such as mibefradil or ethosuximide block other channels, which can then result in side effects. (12, 31, 45) In this study, we developed novel compounds with structures similar to some of the endogenous ligands that are known to interact with members of the T-type channel family (16, 23, 25, 34, 35) and then modified them to reduce their affinity for cannabinoid receptors while attempting to increase affinity for Cav3.2 channels.
We had previously synthesized a series of compounds based on endogenous cannabinoid ligands that targeted both CB receptors and T-type calcium channels. (16, 34) Using data obtained from these experiments, we designed additional compounds with an extra substituted tertiary amine attached to the carbazole scaffold. We then extended the chain length attached to the carbazole to one of the compounds (compound 9) to improve its selectivity for Cav3.2 channels over CB receptors. (16, 34) These data show that the length of the linker between the carbazole scaffold and the heterocyclic moiety is a key drug structural determinant that can be exploited to produce better and more selective Cav3.2 channel inhibitors. This compound blocked Cav3.2 channels with approximately 2-fold higher affinity than Cav3.1 and Cav3.3 channels. At this point we do not know if compound 9 affects other molecular targets such as high voltage activated channels or sodium channels. Nonetheless, the observation that their analgesic actions were abolished upon removal of Cav3.2 channels indicates that the primary biological target for compound 9 in the context of pain signaling is Cav3.2. The potent effects of compound 9 on pain response in injured wild type animals fits with the notion that Cav3.2 channels play an important role in the afferent pain pathway (17, 18, 20, 21, 42, 44, 46, 47) and also with a number of previous studies showing that Cav3.2 channel blockers are efficacious in various pain models. (12, 15, 20, 29) Calculated physiochemical properties such as topological polar surface area (tPSA), lipophilicity (clogP), and number of hydrogen bond donor and acceptor atoms are useful indicator of druglike properties. Poor oral absorption is expected for compounds with a TPSA more than 140 Å2, with a log D > 5, more than 5 hydrogen bond donors, and more than 10 hydrogen bond acceptors. Compound 9 has a TPSA value of 66.37 Å2, a molecular weight of 490.68 g/mol, a clogP value of 4.59, and less than five hydrogen bond acceptors or donors, suggesting a reasonable probability of good oral absorption and intestinal permeability.
Compound 9 blocked Cav3.2 channels in a concentration range that was similar to that previously reported for compounds such as NMP7 (34) and NMP181. (16) These two molecules, although structurally related to compound 9 did not discriminate among the three Cav3 family members, and mediated leftward shifts in the midpoint of the steady state inactivation curve. In contrast, compound 9 did not significantly affect voltage-dependent inactivation properties of Cav3.2 channels, suggesting that this compound does not interact strongly with inactivated channels. At this point, it is not clear which structural differences among these compounds are responsible for these differential effects on channel gating. While other types of T-type calcium channel blockers such as TTA-2 (48) or Z123212 (28) that are efficacious in various pain models are also known to promote voltage-dependent inactivation of Cav3.2 channels, our data showing efficacy of compound 9 in neuropathic and inflammatory pain indicate that such state-dependent inhibition is not a prerequisite for antihyperalgesic effects in vivo.
Altogether our data suggest that using a carbazole scaffold is an effective strategy for developing potent Cav3.2 calcium channel blockers for therapeutic intervention into inflammatory and neuropathic pain hypersensitivity. In addition, T-type channels are also associated with many other disorders, including epilepsy and cardiac hypertrophy; (2-4, 7-11, 18, 32, 49) therefore, the novel pharmacophores that we have identified here may prove useful toward treatment of these disorders.

Methods

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In Vitro Receptor Radioligand CB1 and CB2 Binding Studies

CB1 and CB2 radioligand binding data were obtained using National Institute of Mental Health (NIMH) Psychoactive Drug Screening Program (PDSP) resources as described earlier. (34, 50-52)

cDNA Constructs

Human Cav3.2 cDNA construct was kindly provided by Dr. Terrance Snutch (University of British Columbia, Vancouver, Canada).

tsA-201 Cell Culture and Transfection

Human embryonic kidney tsA-201 cells were cultured and transfected using the calcium phosphate method as described previously. (53) Transfected cells were then incubated 48 h at 37 °C and 5% CO2 and then resuspended with 0.25% (w/v) trypsin-EDTA (Invitrogen) and plated on glass coverslips a minimum of 3 to 4 h before patching.

Electrophysiology

Whole-cell voltage-clamp recordings from tsA-201 cells were performed at room temperature 2–3 days after transfection. External recording solution contained (in mM): 114 CsCl, 20 BaCl2, 1 MgCl2, 10 HEPES, 10 glucose, adjusted to pH 7.4 with CsOH. Internal patch pipet solution contained the following (in mM): 126.5 CsMeSO4, 2 MgCl2, 11 EGTA, 10 HEPES adjusted to pH 7.3 with CsOH. Internal solution was supplemented with 0.6 mM GTP and 2 mM ATP and mixed thoroughly just prior to use. Liquid junction potentials for the recording solutions were left uncorrected.
Tested compounds were prepared daily from DMSO stocks diluted in external solution. Using a custom built gravity driven microperfusion system, (53) diluted compounds were then applied rapidly and locally to the cells. Control vehicle experiments were performed to ensure that 0.1% DMSO had no effect on current amplitudes or on the half a ctivation and half-inactivation potentials (data not shown). Currents were measured using the whole-cell patch clamp technique and an Axopatch 200B amplifier in combination with Clampex 9.2 software (Molecular Devices, Sunnyvale, CA). After establishing whole cell configuration, cellular capacitance was minimized using the amplifier’s built-in analog compensation. Series resistance was compensated by at least 85% in all experiments. All data were digitized at 10 kHz with a Digidata 1320 interface (Molecular Devices) and filtered at 1 kHz (8-pole Bessel filter). Raw and online leak-subtracted data were both collected simultaneously. In current–voltage relation studies, the membrane potential was held at −110 mV and cells were depolarized from −80 to 20 mV in 10 mV increments. For steady-state inactivation studies, a 3.6 s conditioning prepulse of various magnitude (initial holding at −110 mV) was followed by a depolarizing pulse to −20 mV. Individual sweeps were separated by 12 s to permit recovery from inactivation between conditioning pulses. The duration of the test pulse was typically 200 ms and the current amplitude obtained from each test pulse was normalized to that observed at the holding potential of −110 mV.

Animals

During experiments, all efforts were made to minimize animal suffering according to the policies and recommendations of the International Association for the Study of Pain and all protocols used were approved by the Institutional Animal Care and Use Committee. For all experiments, either adult male C57BL/6J (wild-type) or CACNA1H knockout (Cav3.2 null) mice (20–25g) purchased from Jackson Laboratories were used. There were a maximum of five mice per cage (30 × 20 × 15 cm2), and access to food and water was unlimited. Temperature was kept at 23 ± 1 °C on a 12 h light/dark cycle (lights on at 7:00 a.m.). Intraperitoneal (i.p.) injections of drugs were a constant volume of 10 mL/kg body weight. Intrathecal (i.t.) injections used volumes of 10 μL and were performed using the method described previously (54) and carried out routinely in our laboratory. (16, 55) All drugs were dissolved in 1% or less DMSO, whereas control animals received PBS + 1% DMSO. For each test, a different group of mice were used and only one experiment per mouse was performed. In experiments examining the action of compound 9 on PNI-induced neuropathy, the observer was blind to the conditions tested.

Formalin Test

Before experiments were performed, mice were left to acclimatize for at least 60 min. Animals were then injected intraplantarily (i.pl.) in the ventral surface of the right hindpaw with 20 μL of a formalin solution (1.25%) made up in PBS. Following i.pl. injections of formalin, individual animals were placed immediately into observation chambers and monitored from 0 to 5 min (acute nociceptive phase) and 15–30 min (inflammatory phase). The time spent licking or biting the injected paw was then considered as a nociceptive response and recorded with a chronometer. Compound 9 was delivered by i.t. (20 min prior) or by i.p. (30 min prior) and its effect on both the nociceptive and inflammatory phases of the formalin test was evaluated.

Open-Field Test

Animals received compound 9 via i.p. (30 mg/kg) route 30 min before testing, and the ambulatory behavior of the treated animals was assessed in an open-field test as described previously. (56) Briefly, the apparatus consisted of a wooden box measuring 40 × 60 × 50 cm3 with a glass front wall. The floor was divided into 12 equal squares and the entire apparatus was placed in a sound free room. Animals were placed in the rear left square and left to explore freely and the number of squares crossed with all paws (crossing) in a 6 min time frame was counted. After each individual mouse session, the apparatus was then cleaned and dried with a 10% alcohol solution.

Partial Sciatic Nerve Injury (PNI)-Induced Neuropathic Pain

Before surgery, mice were anaesthetized with isoflurane (5% induction, 2.5% maintenance). Partial ligation of the sciatic nerve was performed by tying the distal 1/3 to 1/2 of the dorsal portion as previously described. (57) In sham-operated mice, the sciatic nerve was exposed without ligation and all wounds were closed and treated with iodine solution. After 14 days post surgery, mice received either compound 9 (30 mg/kg, i.p.) treatment or vehicle, while sham-operated animals received only vehicle (10 mL/kg, i.p.). Mechanical hyperalgesia was then evaluated in a time-dependent manner.

Evaluation of Mechanical Hyperalgesia

Mechanical hyperalgesia responses were recorded immediately before the surgeries (baselines), 14 days after the surgeries (0), and at various time points (0.5, 1, 2, 3 h) after treatment. Measurements were made using a Dynamic Plantar Aesthesiometer (DPA, Ugo Basile, Varese, Italy). Briefly, individual animals were placed in small enclosed testing arenas (20 cm × 18.5 cm × 13 cm, length × width × height) on top of a wire mesh floor and allowed to acclimate for a period of at least 90 min. The DPA device was then positioned so that the filament was directly under the plantar surface of the ipsilateral hind paw of the animal and tested three times per session.

Data Analysis and Statistics

Data were analyzed using Clampfit 9.2 (Molecular Devices). Origin 7.5 software (Northampton, MA) was used in the preparation of all figures and curve fittings. Current–voltage relationships were fitted with the modified Boltzmann equation: I = [Gmax(Vm – Erev)]/[1 + exp((V0.5act – Vm)/ka)], where Vm is the test potential, V0.5act is the half activation potential, Erev is the reversal potential, Gmax is the maximum slope conductance, and ka reflects the slope of the activation curve. Steady-state inactivation curves were fitted using the Boltzmann equation: I =1/(1 + exp((Vm – Vh)/k)), where Vh is the half-inactivation potential and k is the slope factor. Dose–response curves were fitted with the equation y = A2 + (A1 – A2)/(1 + ([C]/IC50)n), where A1 is initial current amplitude and A2 is the current amplitude at saturating drug concentrations, [C] is the drug concentration, and n is the Hill coefficient. Statistical significance was determined by paired or unpaired Student’s t tests and one-way or repeated measures ANOVA, followed by Dunnett’s test or Tukey’s multiple comparison tests. Significant values were set as indicated in the text and figure legends. All data are given as means ± standard errors.

Chemistry

All moisture sensitive reactions were performed in an inert, dry atmosphere of argon in flame-dried glassware. Air sensitive liquids were transferred via syringe or cannula through rubber septa. Reagent grade solvents were used for extraction and flash chromatography. THF was distilled from Na/benzophenone under argon; dichloromethane (CH2Cl2) and chloroform (CHCl3) were distilled from CaH2 under argon. All other reagents and solvents which were purchased from commercial sources were used directly without further purification. The progress of reactions was checked by analytical thin-layer chromatography (Sorbent Technologies, Silica G TLC plates w/UV 254). The plates were visualized first with UV illumination followed by charring with ninhydrin (0.3% ninhydrin (w/v), 97:3 EtOH-AcOH). Flash column chromatography was performed using prepacked Biotage SNAP cartridges on a Biotage Isolera One instrument. The solvent compositions reported for all chromatographic separations are on a volume/volume (v/v) basis. 1HNMR spectra were recorded at 400 or 500 MHz and are reported in parts per million (ppm) on the δ scale relative to tetramethylsilane as an internal standard. 13CNMR spectra were recorded at 100 or 125 MHz and are reported in parts per million (ppm) on the δ scale relative to CDCl3 (δ 77.00). Melting points were determined on a Stuart melting point apparatus from Bibby Scientific Limited and are uncorrected. High Resolution mass spectrometry (HRMS) was performed on a Waters/Micromass LCT-TOF instrument. All compounds were more than 95% pure.

N-((1-(2-(tert-Butylamino)-2-oxoethyl)piperidin-4-yl)methyl)-9-pentyl-9H-carbazole-3-carboxamide (9)

Under nitrogen atmosphere, a mixture of 9-pentyl-N-(piperidin-4-ylmethyl)-9H-carbazole-3-carboxamide 7 (16) (389 mg, 1.03 mmol), N-tert-butyl-2-chloroacetamide (185 mg, 1.24 mmol), potassium carbonate (427 mg, 3.09 mmol), and potassium iodide (160 mg, 0.96 mmol) in n-butanol (5 mL) was subjected to microwave irradiation at 110 °C for 3 h. The mixture was allowed to cool to room temperature, diluted with DCM (50 mL), and filtered. The organic solvents were evaporated in vacuo. The residue was purified on a Biotage KP-NH cartridge (amino-modified silica gel) using cyclohexane/EtOAc in different proportions to afford the title compound as a light yellow glass. Yield: 476 mg (94%). 1H NMR (400 MHz, CDCl3) δ 8.49 (d, J = 1.4 Hz, 1H), 8.01 (d, J = 7.9 Hz, 1H), 7.84 (dd, J = 8.6, 1.4 Hz, 1H), 7.41 (ddd, J = 8.3, 7.1, 1.1 Hz, 1H), 7.33 (d, J = 8.3 Hz, 1H), 7.29 (d, J = 8.6 Hz, 1H), 7.16 (ddd, J = 7.9, 7.1, 1.1 Hz, 1H), 7.00 (br s, 1H), 6.58 (t, J = 5.8 Hz, 1H), 4.19 (t, J = 7.2 Hz, 2H), 3.34 (t, J = 6.5 Hz, 2H), 2.81–2.67 (m, 4H), 2.00 (td, J = 11.7, 2.5 Hz, 2H), 1.83–1.74 (m, 2H), 1.71 (br d, J = 13.9 Hz, 2H), 1.64–1.48 (m, 1H), 1.31–1.22 (m, 15H), 0.78 (t, J = 6.9 Hz, 3H). 13C and DEPT NMR (101 MHz, CDCl3) δ 169.84 (C═O), 168.52 (C═O), 142.33 (C), 141.15 (C), 126.44 (CH), 125.28 (C), 124.81 (CH), 122.96 (C), 122.73 (C), 120.68 (CH), 119.85 (CH), 119.68 (CH), 109.23 (CH), 108.50 (CH), 62.73 (CH2), 53.89 (CH2), 50.51 (C), 45.57(CH2), 43.37 (CH2), 36.23 (CH), 30.61 (CH2), 29.48 (CH2), 28.93 (CH3), 28.76 (CH2), 22.57 (CH2), 14.08 (CH3). ESI: m/z 491.2 (M + H)+. HRMS calcd for C30H43N4O2 (M + H)+ 491.3386, found 491.3433.

N-tert-Butyl-2-[4-(9-pentyl-9H-carbazole-3-carbonyl)piperazin-1-yl]acetamide (10)

Under argon atmosphere, to a solution of tert-butyl 4-(9-pentyl-9H-carbazole-3-carbonyl)piperazine-1-carboxylate 8 (16) (360 mg, 1.03 mmol) and MeCN (10 mL) was added K2CO3 (427 mg, 3.09 mmol), KI (160 mg, 0.96 mmol), and N-tert-butyl-2-chloroacetamide (185 mg, 1.24 mmol). The reaction mixture was stirred at reflux for 3 h. The reaction mixture was cooled, diluted with DCM (30 mL), and filtered. The organic solvents were evaporated in vacuo. The organic layer was washed three times with brine (50 mL). The organic layer was then separated, dried over magnesium sulfate, filtered, and concentrated in vacuo. The crude product was purified by flash chromatography eluting with heptane/EtOAc (0–100%) to yield the title compound (437 mg, 92%) as yellow foam. 1H NMR (400 MHz, CDCl3) δ 8.19 (d, J = 1.2 Hz, 1 H), 8.09 (d, J = 7.6 Hz, 1 H), 7.38–7.55 (m, 4 H), 7.23–7.28 (m, 1 H), 6.96 (s, 1 H), 4.30 (t, J = 7.2 Hz, 2 H), 3.74 (br. s., 4 H), 2.96 (s, 2 H), 2.58 (br. s., 4 H), 1.86 (br. s., 2 H), 1.24–1.39 (m, 13 H), 0.81–1.01 (m, 3 H). 13C NMR (101 MHz, CDCl3) δ ppm 171.69 (C═O), 168.71 (C═O), 141.20 (C), 140.94 (C), 126.30 (CH), 125.60 (C), 125.19 (CH), 122.61 (C), 122.50 (C), 120.54 (CH), 120.14 (CH), 119.43 (CH), 109.07 (CH), 108.51 (CH), 77.44 (CH2), 77.33 (CH2), 77.13 (CH2), 76.81 (CH2), 62.27 (CH2), 53.48 (CH2), 50.64 (C), 43.24 (CH2), 29.38 (CH2), 28.83 (CH2), 28.66 (CH3), 22.48 (CH2), 13.98 (CH3). m/z 463.2 (M + H)+. HRMS calcd for C30H43N4O2 (M + H)+ 463.3073, found 463.2819.

tert-Butyl-4 ((9-Propyl-9H-carbazole-3-carboxamido)methyl)piperidine-1-carboxylate (11)

9-Propyl-9H-carbazole-3-carboxylic acid (16) (100 mg, 0.40 mmol), HOBt (62.32 mg, 0.48 mmol), DIPEA (0.13 mL, 0.79 mmol), DMAP (58.12 mg, 0.40 mmol), and EDAC (91.22 mg, 0.40 mmol) were added upon stirring to DCM (9 mL) under nitrogen. The obtained solution was cooled down on an ice–water bath, tert-butyl 4-(aminomethyl) piperidine-1-carboxylate (119.24 mg, 0.40 mmol) was added in one portion, and the resulting reaction mixture was then allowed to warm to room temperature and stirred for 16 h. The solvent was removed in vacuo, and the obtained residue was extracted with EtOAc (50 mL). The organic layer was washed three times with brine (25 mL). The organic layer was then separated, dried over magnesium sulfate, filtered, and concentrated in vacuo. The crude product was purified by flash chromatography eluting with heptane/EtOAc (0–100%) to produce the title compound 11 (158.3 mg, 89%) as yellow oil. 1H NMR (400 MHz, CDCl3) δ 8.57 (d, J = 1.4 Hz, 1H), 8.06 (d, J = 8.0 Hz, 1H), 7.89 (dd, J = 8.7, 1.4 Hz, 1H), 7.44 (ddd, J = 8.3, 7.1, 1.1 Hz, 1H), 7.31 (d, J = 8.3 Hz, 1H), 7.27 (d, J = 8.7 Hz, 1H), 7.19 (ddd, J = 8.0, 7.1, 1.1 Hz, 1H), 6.55 (t, J = 5.8 Hz, 1H), 4.26 (t, J = 7.2 Hz, 2H), 3.38 (t, J = 6.2 Hz, 2H), 3.10 (d, J = 12.1 Hz, 2H), 2.61 (td, J = 12.2, 2.5 Hz, 2H), 1.99 (s, 1H), 1.97–1.80 (m, 2H), 1.80–1.70 (m, 3H), 1.45 (s, 9H), 1.22 (qd, J = 12.8, 3.7 Hz, 2H), 0.97 (t, J = 7.4 Hz, 3H). 13C (101 MHz, CDCl3) δ 168.53, 154.77, 142.30, 141.16, 126.42, 125.42, 124.77, 122.97, 122.74, 120.74, 119.83, 119.68, 109.22, 108.53, 79.22, 45.22, 45.39, 36.91, 31.38, 28.41, 22.58, 12.10.

9-Propyl-N-(piperidin-4-ylmethyl)-9H-carbazole-3-carboxamide (12)

Trifluoroacetic acid (5 mL) was added to a solution of 11 (150 mg, 0.33 mmol) in CH2Cl2 (5 mL), and the reaction mixture was stirred at room temperature for 4 h. The mixture was evaporated, and the residue was basified with 2 N NaOH. The solution was extracted with CH2Cl2 three times. The extracts were dried with Na2SO4 and evaporated. The crude product was purified by flash chromatography eluting with heptane/EtOAc (0–100%) to produce the title compound 12 (95.5 mg, 83%) as dark yellow oil. 1H NMR (400 MHz, CDCl3) δ 8.56 (d, J = 1.5 Hz, 1H), 8.05 (d, J = 8.0 Hz, 1H), 7.90 (dd, J = 8.7, 1.5 Hz, 1H), 7.5 (ddd, J = 8.3, 7.2, 1.2 Hz, 1H), 7.31 (d, J = 8.3 Hz, 1H), 7.28 (d, J = 8.7 Hz, 1H), 7.17 (ddd, J = 8.0, 7.2, 1.2 Hz, 1H), 6.58 (t, J = 5.8 Hz, 1H), 4.25 (t, J = 7.1 Hz, 2H), 3.39 (t, J = 6.2 Hz, 2H), 3.09 (d, J = 12.1 Hz, 2H), 2.60 (td, J = 12.2, 2.5 Hz, 2H), 1.99 (s, 1H), 1.98–1.80 (m, 2H), 1.80–1.71 (m, 3H), 1.23 (qd, J = 12.8, 3.7 Hz, 2H), 0.96 (t, J = 7.5 Hz, 3H). 13C (101 MHz, CDCl3) δ 168.51, 142.32, 141.15, 126.40, 125.41, 124.79, 122.99, 122.73, 120.73, 119.82, 119.67, 109.21, 108.50, 46.47, 46.20, 45.37, 36.91, 31.37, 22.59, 12.10.

N-((1-(2-(tert-Butylamino)-2-oxoethyl)piperidin-4-yl)methyl)-9-propyl-9H-carbazole-3-carboxamide (13)

Under argon atmosphere, to a solution of 12 (90 mg, 0.26 mmol) and MeCN (2.5 mL) was added K2CO3 (107.8 mg, 0.78 mmol), KI (43.2 mg, 0.26 mmol), and N-tert-butyl-2-chloroacetamide (47.9 mg, 0.32 mmol). The title compound was prepared according to the analogous procedure described above for compound 9 to produce the title compound 13 (121.3 mg, 82%) as dark yellow oil. 1H NMR (400 MHz, CDCl3) δ 8.56 (d, J = 1.5 Hz, 1H), 8.05 (d, J = 8.0 Hz, 1H), 7.91 (dd, J = 8.7, 1.5 Hz, 1H), 7.44 (ddd, J = 8.2, 7.1, 1.1 Hz, 1H), 7.31 (d, J = 8.2 Hz, 1H), 7.28 (d, J = 8.7 Hz, 1H), 7.17 (ddd, J = 8.0, 7.1, 1.1 Hz, 1H), 6.98 (br s, 1H), 6.58 (t, J = 5.8 Hz, 1H), 4.26 (t, J = 7.1 Hz, 2H), 3.33 (t, J = 6.3 Hz, 2H), 2.82–2.67 (m, 4H), 2.03 (td, J = 11.8, 2.7 Hz, 2H), 1.86–1.76 (m, 2H), 1.7 (d, J = 13.7 Hz, 2H), 1.64–1.48 (m, 1H), 1.34 (s, 9H), 0.94 (t, J = 7.0 Hz, 3H). 13C and DEPT NMR (101 MHz, CDCl3) δ 169.84 (C═O), 168.5 (C═O), 142.34 (C), 141.17 (C), 126.41 (CH), 126.0 (C), 124.79 (CH), 122.94 (C), 122.71 (C), 120.68 (CH), 119.84 (CH), 119.69 (CH), 109.23 (CH), 108.51 (CH), 62.74 (CH2), 53.9 (CH2), 50.52 (C), 45.58 (CH2), 45.35 (CH2), 36.22 (CH), 30.61 (CH2), 28.90 (CH3), 22.76 (CH2), 12.18 (CH3). ESI: m/z 463.3 (M + H)+. HRMS calcd for C28H40N4O2 (M + H)+ 463.3073, found 463.3089.

tert-Butyl 4-((9-Butyl-9H-carbazole-3-carboxamido)methyl)piperidine-1-carboxylate (14)

Using 9-butyl-9H-carbazole-3-carboxylic acid (16) (100 mg, 0.37 mmol), HOBt (60.54 mg, 0.45 mmol), DIPEA (0.13 mL, 0.75 mmol), DMAP (54.73 mg, 0.45 mmol), tert-butyl 4-(aminomethyl) piperidine-1-carboxylate (112.22 mg, 0.45 mmol), and EDAC (85.85 mg, 0.45 mmol), the title compound was prepared according to the analogous procedure described above for compound 11, to produce the title compound 14 (147.3 mg, 85%) as a yellow oil. 1H NMR (400 MHz, CDCl3) δ 8.53 (d, J = 1.5 Hz, 1H), 8.04 (d, J = 7.9 Hz, 1H), 7.82 (dd, J = 8.6, 1.5 Hz, 1H), 7.43 (ddd, J = 8.3, 7.1, 1.1 Hz, 1H), 7.31 (d, J = 8.3 Hz, 1H), 7.27 (d, J = 8.6 Hz, 1H), 7.16 (ddd, J = 7.9, 7.1, 1.1 Hz, 1H), 6.57 (t, J = 5.8 Hz, 1H), 4.24 (t, J = 7.2 Hz, 2H), 3.37 (t, J = 6.2 Hz, 2H), 3.07 (d, J = 12.1 Hz, 2H), 2.61 (td, J = 12.2, 2.5 Hz, 2H), 2.00 (s, 1H), 1.98–1.80 (m, 2H), 1.80–1.71 (m, 3H), 1.45 (s, 9H), 1.43–1.32(m, 2H), 1.23 (qd, J = 12.8, 3.7 Hz, 2H), 0.90 (t, J = 7.4 Hz, 3H). 13C (101 MHz, CDCl3) δ 168.50, 154.75, 142.33, 141.16, 126.45, 125.43, 124.80, 122.97, 122.72, 120.73, 119.81, 119.68, 109.21, 108.50, 79.26, 45.47, 43.04, 36.90, 31.36, 28.41, 20.78, 14.13.

9-Butyl-N-(piperidin-4-ylmethyl)-9H-carbazole-3-carboxamide (15)

Trifluoroacetic acid (5 mL) was added to a solution of 14 (140 mg, 0.30 mmol) in CH2Cl2 (5 mL), and the title compound was prepared according to the analogous procedure described above for compound 12 to produce the title compound 15 (95.7 mg, 88%) as dark yellow oil. 1H NMR (400 MHz, CDCl3) δ 8.53 (d, J = 1.5 Hz, 1H), 8.02 (d, J = 7.9 Hz, 1H), 7.84 (dd, J = 8.7, 1.5 Hz, 1H), 7.41 (ddd, J = 8.3, 7.2, 1.1 Hz, 1H), 7.30 (d, J = 8.3 Hz, 1H), 7.27 (d, J = 8.7 Hz, 1H), 7.17 (ddd, J = 7.9, 7.2, 1.1 Hz, 1H), 6.58 (t, J = 5.8 Hz, 1H), 4.25 (t, J = 7.2 Hz, 2H), 3.39 (t, J = 6.2 Hz, 2H), 3.09 (d, J = 12.2 Hz, 2H), 2.60 (td, J = 12.2, 2.5 Hz, 2H), 1.99 (s, 1H), 1.98–1.80 (m, 2H), 1.80–1.71 (m, 3H), 1.43–1.32(m, 2H), 1.23 (qd, J = 12.8, 3.7 Hz, 2H), 0.90 (t, J = 7.4 Hz, 3H). 13C (101 MHz, CDCl3) δ 168.51, 142.32, 141.15, 126.45, 125.41, 124.79, 122.99, 122.73, 120.73, 119.82, 119.67, 109.21, 108.50, 46.47, 46.20, 43.03, 36.91, 31.37, 20.79, 14.12.

N-((1-(2-(tert-Butylamino)-2-oxoethyl)piperidin-4-yl)methyl)-9-butyl-9H-carbazole-3-carboxamide (16)

Under argon atmosphere, to a solution of 15 (90 mg, 0.25 mmol) and MeCN (2.5 mL) was added K2CO3 (103.7 mg, 0.75 mmol), KI (41.5 mg, 0.25 mmol), and N-tert-butyl-2-chloroacetamide (44.9 mg, 0.30 mmol). The title compound was prepared according to the analogous procedure described above for compound 9 to produce the title compound 16 (99.9 mg, 84%) as dark yellow oil. 1H NMR (400 MHz, CDCl3) δ 8.50 (d, J = 1.5 Hz, 1H), 8.01 (d, J = 7.9 Hz, 1H), 7.8 (dd, J = 8.6, 1.5 Hz, 1H), 7.4 (ddd, J = 8.3, 7.1, 1.1 Hz, 1H), 7.30 (d, J = 8.3 Hz, 1H), 7.27 (d, J = 8.6 Hz, 1H), 7.16 (ddd, J = 7.9, 7.1, 1.1 Hz, 1H), 7.01 (br s, 1H), 6.57 (t, J = 5.8 Hz, 1H), 4.24 (t, J = 7.2 Hz, 2H), 3.35 (t, J = 6.5 Hz, 2H), 2.83–2.68 (m, 4H), 2.00 (td, J = 11.7, 2.5 Hz, 2H), 1.82–1.72 (m, 2H), 1.73 (d, J = 13.8 Hz, 2H), 1.63–1.49 (m, 1H), 1.45–1.34 (m, 2H), 1.33 (s, 9H), 0.90 (t, J = 6.8 Hz, 3H). 13C and DEPT NMR (101 MHz, CDCl3) δ 169.83 (C═O), 168.57 (C═O), 142.34 (C), 141.14 (C), 126.45 (CH), 125.26 (C), 124.82 (CH), 122.94 (C), 122.73 (C), 120.68 (CH), 119.85 (CH), 119.68 (CH), 109.21 (CH), 108.50 (CH), 62.71 (CH2), 53.90 (CH2), 50.50 (C), 45.59(CH2), 43.00 (CH2), 36.26 (CH), 30.66 (CH2), 29.43 (CH2), 28.92 (CH3), 20.74 (CH2), 14.10 (CH3). ESI: m/z 477.2 (M + H)+. HRMS calcd for C29H41N4O2 (M + H)+ 477.3229, found 477.3217.

tert-Butyl 4-((9-Pentyl-9H-carbazole-3-carboxamido)methyl)pyrrolidine-1-carboxylate (17)

Using 9-pentyl-9H-carbazole-3-carboxylic acid (100 mg, 0.36 mmol), (16) HOBt (57.81 mg, 0.43 mmol), DIPEA (0.12 mL, 0.71 mmol), DMAP (52.29 mg, 0.43 mmol), (R)-3-(aminomethyl)-1-Boc-pyrrolidine (85.66 mg, 0.43 mmol), and EDAC (82.02 mg, 0.43 mmol), the title compound was prepared according to the analogous procedure described above for compound 11 using the amide coupling protocol, to produce the title compound 17 (141.8 mg, 86%) as a yellow oil. 1H NMR (400 MHz, CDCl3) δ 8.58 (d, J = 1.6 Hz, 1H), 8.02 (d, J = 8.0 Hz, 1H), 7.83 (dd, J = 8.6, 1.6 Hz, 1H), 7.42 (ddd, J = 8.3, 7.1, 1.1 Hz, 1H), 7.31 (d, J = 8.3 Hz, 1H), 7.28 (d, J = 8.6 Hz, 1H), 7.17 (ddd, J = 8.0, 7.1, 1.1 Hz, 1H), 6.57 (t, J = 5.8 Hz, 1H), 4.16 (t, J = 7.2 Hz, 2H), 3.22–3.12 (m, 2H), 2.34–2.21 (m, 2H), 2.12–1.98 (m, 1H), 1.81–1.67 (m, 3H), 1.45 (s, 9H), 1.38–1.20 (m, 4H), 0.80 (t, J = 6.9 Hz, 3H). 13C (101 MHz, CDCl3) δ 168.56, 154.75, 142.32, 141.13, 126.47, 125.25, 124.82, 122.92, 122.72, 120.69, 119.85, 119.68, 109.22, 108.50, 79.27, 54.28, 45.50, 39.78, 38.75, 36.26, 30.68, 29.45, 28.42, 20.75, 14.10.

9-Pentyl-N-(pyrrolidin-4-ylmethyl)-9H-carbazole-3-carboxamide (18)

Trifluoroacetic acid (5 mL) was added to a solution of compound 17 (135 mg, 0.29 mmol) in CH2Cl2 (5 mL), the title compound was prepared according to the analogous procedure described above for compound 12 to produce the title compound 18 (90.7 mg, 86%) as yellow oil. 1H NMR (400 MHz, CDCl3) δ 8.57 (d, J = 1.5 Hz, 1H), 8.03 (d, J = 7.9 Hz, 1H), 7.84 (dd, J = 8.6, 1.5 Hz, 1H), 7.41 (ddd, J = 8.3, 7.1, 1.1 Hz, 1H), 7.32 (d, J = 8.3 Hz, 1H), 7.27 (d, J = 8.6 Hz, 1H), 7.17 (ddd, J = 7.9, 7.1, 1.1 Hz, 1H), 6.57 (t, J = 5.84 Hz, 1H), 4.16 (t, J = 7.2 Hz, 2H), 3.22–3.13 (m, 2H), 2.34–2.22 (m, 2H), 2.12–1.99 (m, 1H), 1.82–1.68 (m, 3H), 1.38–1.20 (m, 4H), 0.82 (t, J = 6.9 Hz, 3H). 13C (101 MHz, CDCl3) δ 168.55, 142.33, 141.14, 126.46, 125.24, 124.82, 122.93, 122.72, 120.69, 119.85, 119.68, 109.22, 108.50, 54.27, 45.50, 39.79, 38.76, 36.26, 30.68, 29.44, 20.76, 14.11.

N-((1-(2-(tert-Butylamino)-2-oxoethyl)pyrrolidin-4-yl)methyl)-9-pentyl-9H-carbazole-3-carboxamide (19)

Under argon atmosphere, to a solution of compound 18 (85 mg, 0.23 mmol) and MeCN (2.5 mL) was added K2CO3 (95.4 mg, 0.69 mmol), KI (38.1 mg, 0.69 mmol), and N-tert-butyl-2-chloroacetamide (41.9 mg, 0.28 mmol). The title compound was prepared according to the analogous procedure described above for compound 10 to produce the title compound 19 (96.2 mg, 88%) as dark yellow oil. 1H NMR (400 MHz, CDCl3) δ 8.58 (d, J = 1.5 Hz, 1H), 8.02 (d, J = 7.9 Hz, 1H), 7.83 (dd, J = 8.6, 1.5 Hz, 1H), 7.41 (ddd, J = 8.3, 7.1, 1.1 Hz, 1H), 7.31 (d, J = 8.3 Hz, 1H), 7.27 (d, J = 8.6 Hz, 1H), 7.16 (ddd, J = 7.9, 7.1, 1.1 Hz, 1H), 7.01 (br s, 1H), 6.56 (t, J = 5.8 Hz, 1H), 4.16 (t, J = 7.2 Hz, 2H), 3.22–3.11 (m, 4H), 2.34–2.22 (m, 2H), 2.11–1.99 (m, 1H), 1.81–1.67 (m, 3H), 1.39–1.22 (m, 16H), 0.83 (t, J = 6.9 Hz, 3H). 13C and DEPT NMR (101 MHz, CDCl3) δ 169.83 (C═O), 168.57 (C═O), 142.34 (C), 141.14 (C), 126.45 (CH), 125.26 (C), 124.82 (CH), 122.94 (C), 122.73 (C), 120.68 (CH), 119.85 (CH), 119.68 (CH), 109.21 (CH), 108.50 (CH), 63.41 (CH2), 60.41 (CH2), 55.56 (CH2), 50.89 (C), 47.36(CH2), 44.07 (CH2), 36.67 (CH), 30.46 (CH2), 29.53 (CH2), 28.88 (CH3), 20.58 (CH2), 14.14 (CH3). ESI: m/z 477.5 (M + H)+. HRMS calcd for C29H41N4O2 (M + H)+ 477.3229, found 477.3251.

N-{[1-(3,3-Dimethylbutyl)piperidin-4-yl]methyl}-9-pentyl-9H-carbazole-3-carboxamide (20)

9-Pentyl-9H-carbazole-3-carboxylic acid (16) (100 mg, 0.27 mmol) was combined with cyanoborohydride (83.3 mg, 1.35 mmol/g) and 3,3-dimethylbutyraldehyde (0.06 mL, 0.31 mmol) and then suspended in anhydrous CH2Cl2(2 mL). The mixture was stirred at room temperature overnight. The reaction mixture was diluted with DCM (30 mL), and the organic layer was washed three times with brine (50 mL). The organic layer was then separated, dried over magnesium sulfate, filtered, and concentrated in vacuo. The crude product was purified by flash chromatography eluting with heptane/EtOAc (0–100%) to yield the title compound (88.4 mg, 71%). 1H NMR (400 MHz, CDCl3) δ 8.51 (d, J = 1.4 Hz, 1H), 8.04 (d, J = 7.8 Hz, 1H), 7.86 (dd, J = 8.6, 1.4 Hz, 1H), 7.42 (ddd, J = 8.2, 7.1, 1.1 Hz, 1H), 7.35 (d, J = 8.2 Hz, 1H), 7.29 (d, J = 8.6 Hz, 1H), 7.15 (ddd, J = 7.8, 7.1, 1.1 Hz, 1H), 7.01 (br s, 1H), 6.59 (t, J = 5.8 Hz, 1H), 4.20 (t, J = 7.2 Hz, 2H), 3.36 (t, J = 6.5 Hz, 2H), 2.69–2.54 (m, 4H), 2.01 (td, J = 11.7, 2.5 Hz, 2H), 1.83–1.74 (m, 4H), 1.64–1.48 (m, 1H), 1.33–1.23 (m, 8H), 0.91 (s, 9H), 0.77 (t, J = 6.9 Hz, 3H). 13C and DEPT NMR (101 MHz, CDCl3) δ 168.76 (C═O), 142.47 (C), 141.23 (C), 126.67 (CH), 125.48 (C), 124.18 (CH), 122.76 (C), 122.53 (C), 120.68 (CH), 119.85 (CH), 119.68 (CH), 109.23 (CH), 108.50 (CH), 53.89 (CH2), 52.31 (CH2), 45.67(CH2), 43.42 (CH2), 40.22 (CH2), 36.21 (CH), 30.54 (CH2), 30.12 (C), 29.42 (CH2), 29.34 (CH3), 28.71 (CH2), 22.56 (CH2), 14.03 (CH3). ESI: m/z 462.3 (M + H)+. HRMS calcd for C30H43N3O (M + H)+ 462.3484, found 462.3452.

Physicochemical Properties

Molecular properties of compound 9 were calculated using Instant JChem 6.3.3, 2014, ChemAxon, Budapest, Hungary.

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Author Information

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  • Corresponding Authors
    • Philippe Diaz - Core Laboratory for Neuromolecular Production, The University of Montana, Missoula, Montana 59812, United States;  Email: [email protected]
    • Gerald W. Zamponi - Department of Physiology & Pharmacology, Hotchkiss Brain Institute, Cumming School of Medicine, University of Calgary, Calgary, AB T2N 1N4, Canada;  Email: [email protected]
  • Authors
    • Chris Bladen - Department of Physiology & Pharmacology, Hotchkiss Brain Institute, Cumming School of Medicine, University of Calgary, Calgary, AB T2N 1N4, Canada
    • Steven W. McDaniel - Core Laboratory for Neuromolecular Production, The University of Montana, Missoula, Montana 59812, United States
    • Vinicius M. Gadotti - Department of Physiology & Pharmacology, Hotchkiss Brain Institute, Cumming School of Medicine, University of Calgary, Calgary, AB T2N 1N4, Canada
    • Ravil R. Petrov - Core Laboratory for Neuromolecular Production, The University of Montana, Missoula, Montana 59812, United States
    • N. Daniel Berger - Department of Physiology & Pharmacology, Hotchkiss Brain Institute, Cumming School of Medicine, University of Calgary, Calgary, AB T2N 1N4, Canada
  • Funding

    This work was supported by operating grants to G.W.Z. from the Canadian Institutes of Health Research and to P.D., S.W.M., and R.R.P. from the National Institutes of Health (NIH), Grant P30-NS055022. G.W.Z. is a Canada Research Chair. C.B. holds a T. Chen Fong studentship and an Alberta Innovates – Health Solutions (AI-HS) studentship award. V.M.G. held a MITACS Elevate fellowship. N.D.B. holds an AI-HS summer studentship award.

  • Notes
    The authors declare no competing financial interest.

Acknowledgment

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Radioligand binding assays were performed by the National Institute of Mental Health’s Psychoactive Drug Screening Program Contract # HHSN-271-2008-00025-C (NIMH/PDSP). NIMH/PDSP is directed by Bryan L. Roth M.D., Ph.D. at the University of North Carolina at Chapel Hill and Project Officer Jamie Driscol at NIMH, Bethesda MD. Marvin was used for drawing, displaying, and characterizing chemical structures, substructures, and reactions included in the Supporting Information, Marvin 5.11.5, 2013, ChemAxon (http://www.chemaxon.com).

References

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This article references 57 other publications.

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    A review. Two different Ca2+ channels exist in cardiomyocytes. Whereas the L-type Ca2+ channel is ubiquitous and the main source of Ca2+ for excitation-contraction coupling and pacemaker activity, the functional role of the T-type Ca2+ channel is diverse and depends on mammalian species, heart region, age, and various cardiac diseases. Two isoforms of T-type Ca2+ channel proteins in the heart, CaV3.1 and CaV3.2, are functionally expressed in embryonic hearts, but markedly diminish during development. In the adult heart, the T-type Ca2+ channel is almost undetectable in ventricular myocytes and is most prevalent in the conduction system, playing a functional role in facilitating pacemaker depolarization of the sinoatrial node. Interestingly, the T-type Ca2+ channel is re-expressed in atrial and ventricular myocytes under various pathol. conditions such as hypertrophy and heart failure, and contributes to abnormal elec. activity and excitation-contraction coupling, but the T-type channel provides a smaller contribution to the trigger for Ca2+ release than does the L-type Ca2+ channel. Instead, the T-type Ca2+ channel has been shown to play a crucial role in the process of pathol. cardiac hypertrophy. Increased Ca2+ influx via CaV3.2, the T-type Ca2+ channel, induces calcineurin/NFAT (nuclear factor of activated T-cell) hypertrophic signaling. Furthermore, new evidence has been accumulating on the regulatory mechanism of T-type Ca2+ channel expression, including the neuron restrictive silencer element-neuron restrictive silencer factor (NRSE-NRSF) system, MAP kinases, and cardiac homeobox transcription factor Csx/Nkx2.5. Here, the authors summarize present knowledge regarding cardiac T-type Ca2+ channels, and discuss their pathophysiol. significance in the heart.
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    Journal of Physiology (Oxford, United Kingdom) (2012), 590 (1), 109-118CODEN: JPHYA7; ISSN:0022-3751. (Wiley-Blackwell)
    Spontaneously active neurons typically fire either in a regular pattern or in bursts. While much is known about the subcellular location and biophys. properties of conductances that underlie regular spontaneous activity, less is known about those that underlie bursts. Here, we show that T-type Ca2+ channels localized to the site of action potential initiation in the axon initial segment play a pivotal role in spontaneous burst generation. In auditory brainstem interneurons, axon initial segment Ca2+ influx is selectively downregulated by dopaminergic signalling. This regulation has marked effects on spontaneous activity, converting the predominant mode of spontaneous activity from bursts to regular spiking. Thus, the axon initial segment is a key site, and dopamine a key regulator, of spontaneous bursting activity.
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    Jagodic, M. M., Pathirathna, S., Nelson, M. T., Mancuso, S., Joksovic, P. M., Rosenberg, E. R., Bayliss, D. A., Jevtovic-Todorovic, V., and Todorovic, S. M. (2007) Cell-specific alterations of T-type calcium current in painful diabetic neuropathy enhance excitability of sensory neurons J. Neurosci. 27, 3305– 3316
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    Cell-specific alterations of T-type calcium current in painful diabetic neuropathy enhance excitability of sensory neurons
    Jagodic, Miljen M.; Pathirathna, Sriyani; Nelson, Michael T.; Mancuso, Stefani; Joksovic, Pavle M.; Rosenberg, Ethan R.; Bayliss, Douglas A.; Jevtovic-Todorovic, Vesna; Todorovic, Slobodan M.
    Journal of Neuroscience (2007), 27 (12), 3305-3316CODEN: JNRSDS; ISSN:0270-6474. (Society for Neuroscience)
    Recent data indicate that T-type Ca2+ channels are amplifiers of peripheral pain signals, but their involvement in disorders of sensory neurons such as those assocd. with diabetes is poorly understood. To address this issue, we used a combination of behavioral, immunohistol., mol., and electrophysiol. studies in rats with streptozotocin (N-[methylnitrosocarbamoyl]-D-glucosamine)-induced early diabetic neuropathy. We found that, in parallel with the development of diabetes-induced pain, T-type c.d. increased by twofold in medium-size cells from L4-L5 dorsal root ganglia (DRG) with a depolarizing shift in steady-state inactivation. This not only correlated closely with more prominent afterdepolarizing potentials (ADPs) but also increased cellular excitability manifested as a lower threshold for burst firing in diabetic than in control cells. T-type currents and ADPs were potently inhibited by nickel and enhanced by L-cysteine, suggesting that the CaV3.2 T-type channel isoform was upregulated. Both control and diabetic DRG cells with ADPs stained pos. for isolectin B4, but only diabetic cells responded robustly to capsaicin, suggesting enhanced nociceptive function. Because increased excitability of sensory neurons may result in such pathol. perceptions of pain as hyperalgesia and allodynia, upregulation of T-type Ca2+ currents and enhanced Ca2+ entry into these cells could contribute to the development of symptoms in diabetic neuropathy.
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    Heron, S. E., Khosravani, H., Varela, D., Bladen, C., Williams, T. C., Newman, M. R., Scheffer, I. E., Berkovic, S. F., Mulley, J. C., and Zamponi, G. W. (2007) Extended spectrum of idiopathic generalized epilepsies associated with CACNA1H functional variants Ann. Neurol. 62, 560– 568
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    Extended spectrum of idiopathic generalized epilepsies associated with CACNA1H functional variants
    Heron, Sarah E.; Khosravani, Houman; Varela, Diego; Bladen, Chris; Williams, Tristiana C.; Newman, Michelle R.; Scheffer, Ingrid E.; Berkovic, Samuel F.; Mulley, John C.; Zamponi, Gerald W.
    Annals of Neurology (2007), 62 (6), 560-568CODEN: ANNED3; ISSN:0364-5134. (Wiley-Liss, Inc.)
    The relationship between genetic variation in the T-type calcium channel gene CACNA1H and childhood absence epilepsy is well established. The purpose of this study was to investigate the range of epilepsy syndromes for which CACNA1H variants may contribute to the genetic susceptibility architecture and det. the electrophysiol. effects of these variants in relation to proposed mechanisms underlying seizures. Exons 3 to 35 of CACNA1H were screened for variants in 240 epilepsy patients (167 unrelated) and 95 control subjects by single-stranded conformation anal. followed by direct sequencing. Cascade testing of families was done by sequencing or single-stranded conformation anal. Selected variants were introduced into the CACNA1H protein by site-directed mutagenesis. Constructs were transiently transfected into human embryo kidney cells, and electrophysiol. data were acquired. More than 100 variants were detected, including 19 novel variants leading to amino acid changes in subjects with phenotypes including childhood absence, juvenile absence, juvenile myoclonic and myoclonic astatic epilepsies, as well as febrile seizures and temporal lobe epilepsy. Electrophysiol. anal. of 11 variants showed that 9 altered channel properties, generally in ways that would be predicted to increase calcium current. Interpretation: Variants in CACNA1H that alter channel properties are present in patients with various generalized epilepsy syndromes. We propose that these variants contribute to an individual's susceptibility to epilepsy but are not sufficient to cause epilepsy on their own. The genetic architecture is dominated by rare functional variants; therefore, CACNA1H would not be easily identified as a susceptibility gene by a genome-wide case-control study seeking a statistical assocn.
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    Khosravani, H. (2003) Gating Effects of Mutations in the Cav3.2 T-type Calcium Channel Associated with Childhood Absence Epilepsy J. Biol. Chem. 279, 9681– 9684
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    Khosravani, H., Bladen, C., Parker, D. B., Snutch, T. P., McRory, J. E., and Zamponi, G. W. (2005) Effects of Cav3.2 channel mutations linked to idiopathic generalized epilepsy Ann. Neurol. 57, 745– 749
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    Effects of Cav3.2 channel mutations linked to idiopathic generalized epilepsy
    Khosravani, Houman; Bladen, Christopher; Parker, David B.; Snutch, Terrance P.; McRory, John E.; Zamponi, Gerald W.
    Annals of Neurology (2005), 57 (5), 745-749CODEN: ANNED3; ISSN:0364-5134. (Wiley-Liss, Inc.)
    Heron and colleagues (Ann Neurol 2004;55:595-596) identified three missense mutations in the Cav3.2 T-type calcium channel gene (CACNA1H) in patients with idiopathic generalized epilepsy. None of the variants were assocd. with a specific epilepsy phenotype and were not found in patients with juvenile absence epilepsy or childhood absence epilepsy. Here, we introduced and functionally characterized these three mutations using transiently expressed human Cav3.2 channels. Two of the mutations exhibited functional changes that are consistent with increased channel function. Taken together, these findings along with previous reports, strongly implicate CACNA1H as a susceptibility gene in complex idiopathic generalized epilepsy.
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    Powell, K. L., Cain, S. M., Ng, C., Sirdesai, S., David, L. S., Kyi, M., Garcia, E., Tyson, J. R., Reid, C. A., Bahlo, M., Foote, S. J., Snutch, T. P., and O’Brien, T. J. (2009) A Cav3.2 T-type calcium channel point mutation has splice-variant-specific effects on function and segregates with seizure expression in a polygenic rat model of absence epilepsy J. Neurosci. 29, 371– 380
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    Journal of Neuroscience (2009), 29 (2), 371-380CODEN: JNRSDS; ISSN:0270-6474. (Society for Neuroscience)
    Low-voltage-activated, or T-type, calcium (Ca2+) channels are believed to play an essential role in the generation of absence seizures in the idiopathic generalized epilepsies (IGEs). We describe a homozygous, missense, single nucleotide (G to C) mutation in the Cav3.2 T-type Ca2+ channel gene (Cacna1h) in the genetic absence epilepsy rats from Strasbourg (GAERS) model of IGE. The GAERS Cav3.2 mutation (gcm) produces an arginine to proline (R1584P) substitution in exon 24 of Cacna1h, encoding a portion of the III-IV linker region in Cav3.2.gcm segregates codominantly with the no. of seizures and time in seizure activity in progeny of an F1 intercross. We have further identified two major thalamic Cacna1h splice variants, either with or without exon 25.gcm introduced into the splice variants acts "epistatically," requiring the presence of exon 25 to produce significantly faster recovery from channel inactivation and greater charge transference during high-frequency bursts. This gain-of-function mutation, the first reported in the GAERS polygenic animal model, has a novel mechanism of action, being dependent on exonic splicing for its functional consequences to be expressed.
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    Zamponi, G. W., Lory, P., and Perez-Reyes, E. (2010) Role of voltage-gated calcium channels in epilepsy Pflügers Arch. 460, 395– 403
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    Role of voltage-gated calcium channels in epilepsy
    Zamponi Gerald W; Lory Philippe; Perez-Reyes Edward
    Pflugers Archiv : European journal of physiology (2010), 460 (2), 395-403 ISSN:.
    It is well established that idiopathic generalized epilepsies (IGEs) show a polygenic origin and may arise from dysfunction of various types of voltage- and ligand-gated ion channels. There is an increasing body of literature implicating both high- and low-voltage-activated (HVA and LVA) calcium channels and their ancillary subunits in IGEs. Cav2.1 (P/Q-type) calcium channels control synaptic transmission at presynaptic nerve terminals, and mutations in the gene encoding the Cav2.1 alpha1 subunit (CACNA1A) have been linked to absence seizures in both humans and rodents. Similarly, mutations and loss of function mutations in ancillary HVA calcium channel subunits known to co-assemble with Cav2.1 result in IGE phenotypes in mice. It is important to note that in all these mouse models with mutations in HVA subunits, there is a compensatory increase in thalamic LVA currents which likely leads to the seizure phenotype. In fact, gain-of-function mutations have been identified in Cav3.2 (an LVA or T-type calcium channel encoded by the CACNA1H gene) in patients with congenital forms of IGEs, consistent with increased excitability of neurons as a result of enhanced T-type channel function. In this paper, we provide a broad overview of the roles of voltage-gated calcium channels, their mutations, and how they might contribute to the river that terminates in epilepsy.
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  12. 12
    Barton, M. E., Eberle, E. L., and Shannon, H. E. (2005) The antihyperalgesic effects of the T-type calcium channel blockers ethosuximide, trimethadione, and mibefradil Eur. J. Pharmacol. 521, 79– 85
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    The antihyperalgesic effects of the T-type calcium channel blockers ethosuximide, trimethadione, and mibefradil
    Barton, Matthew E.; Eberle, Elizabeth L.; Shannon, Harlan E.
    European Journal of Pharmacology (2005), 521 (1-3), 79-85CODEN: EJPHAZ; ISSN:0014-2999. (Elsevier B.V.)
    The purpose of the present study was to explore the analgesic effects of the low voltage-activated T-type Ca2+ channel blockers ethosuximide, trimethadione, and mibefradil in persistent and acute nociceptive tests. The anticonvulsant effects of the compds. were also detd. in the i.v. pentylenetetrazol seizure model. Following i.p. administration, ethosuximide and trimethadione dose-dependently reversed capsaicin-induced mech. hyperalgesia. Similarly, the highest dose of mibefradil tested (30 μg, intracisternal) reversed capsaicin-induced mech. hyperalgesia. Ethosuximide and mibefradil produced statistically significant analgesic effects in both early and late phase formalin-induced behaviors and trimethadione reduced late phase behaviors. Addnl., ethosuximide and trimethadione produced antinociceptive effects in the rat-tail flick reflex test. In contrast, following intracisternal administration, mibefradil had no effect in the tail flick reflex test. In addn., the anticonvulsants ethosuximide and trimethadione increased the doses of pentylenetetrazol required to produce both first twitch and clonic seizures. In contrast however, mibefradil had no anticonvulsant effect. The present results demonstrate that the clin. used anticonvulsants ethosuximide and trimethadione provide analgesic effects at doses, which are anticonvulsant. Furthermore, the data further supports the idea that T-type Ca2+ channels may be important targets for treating persistent pain syndromes.
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    Bourinet, E., Alloui, A., Monteil, A., Barrere, C., Couette, B., Poirot, O., Pages, A., McRory, J., Snutch, T. P., Eschalier, A., and Nargeot, J. (2005) Silencing of the Cav3.2 T-type calcium channel gene in sensory neurons demonstrates its major role in nociception EMBO J. 24, 315– 324
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    Choi, S., Na, H. S., Kim, J., Lee, J., Lee, S., Kim, D., Park, J., Chen, C. C., Campbell, K. P., and Shin, H. S. (2007) Attenuated pain responses in mice lacking Ca(V)3.2 T-type channels Genes, Brain Behav. 6, 425– 431
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    Attenuated pain responses in mice lacking CaV3.2 T-type channels
    Choi, S.; Na, H. S.; Kim, J.; Lee, J.; Lee, S.; Kim, D.; Park, J.; Chen, C.-C.; Campbell, K. P.; Shin, H.-S.
    Genes, Brain and Behavior (2007), 6 (5), 425-431CODEN: GBBEAO; ISSN:1601-1848. (Blackwell Publishing Ltd.)
    Although T-type Ca2+ channels are implicated in nociception, the function of specific subtypes has not been well defined. Here, we compared pain susceptibility in mice lacking CaV3.2 subtype of T-type Ca2+ channels (CaV3.2-/-) with wild-type littermates in various behavioral models of pain to explore the roles of CaV3.2 in the processing of noxious stimuli in vivo. In acute mech., thermal and chem. pain tests, CaV3.2-/- mice showed decreased pain responses compared to wild-type mice. CaV3.2-/- mice also displayed attenuated pain responses to tonic noxious stimuli such as i.p. injections of irritant agents and intradermal injections of formalin. In spinal nerve ligation-induced neuropathic pain, however, behavioral responses of CaV3.2-/- mice were not different from those of wild-type mice. The present study reveals that the CaV3.2 subtype of T-type Ca2+ channels are important in the peripheral processing of noxious signals, regardless of modality, duration or affected tissue type.
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    Dogrul, A., Gardell, L. R., Ossipov, M. H., Tulunay, F. C., Lai, J., and Porreca, F. (2003) Reversal of experimental neuropathic pain by T-type calcium channel blockers Pain 105, 159– 168
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    Gadotti, V. M., You, H., Petrov, R. R., Berger, N. D., Diaz, P., and Zamponi, G. W. (2013) Analgesic effect of a mixed T-type channel inhibitor/CB2 receptor agonist Mol. Pain 9, 32
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    Analgesic effect of a mixed T-type channel inhibitor/CB2 receptor agonist
    Gadotti, Vinicius M.; You, Haitao; Petrov, Ravil R.; Berger, N. Daniel; Diaz, Philippe; Zamponi, Gerald W.
    Molecular Pain (2013), 9 (), 32CODEN: MPOAC5; ISSN:1744-8069. (BioMed Central Ltd.)
    Background: Cannabinoid receptors and T-type calcium channels are potential targets for treating pain. Here we report on the design, synthesis and analgesic properties of a new mixed cannabinoid/T-type channel ligand, NMP-181. Results: NMP-181 action on CB1 and CB2 receptors was characterized in radioligand binding and in vitro GTPγ[35S] functional assays, and block of transiently expressed human CaV3.2 T-type channels by NMP-181 was analyzed by patch clamp. The analgesic effects and in vivo mechanism of action of NMP-181 delivered spinally or systemically were analyzed in formalin and CFA mouse models of pain. NMP-181 inhibited peak CaV3.2 currents with IC50 values in the low micromolar range and acted as a CB2 agonist. Inactivated state dependence further augmented the inhibitory action of NMP-181. NMP-181 produced a dose-dependent antinociceptive effect when administered either spinally or systemically in both phases of the formalin test. Both i.t. and i.p. treatment of mice with NMP-181 reversed the mech. hyperalgesia induced by CFA injection. NMP-181 showed no antinocieptive effect in CaV3.2 null mice. The antinociceptive effect of intrathecally delivered NMP-181 in the formalin test was reversed by i.t. treatment of mice with AM-630 (CB2 antagonist). In contrast, the NMP-181-induced antinociception was not affected by treatment of mice with AM-281 (CB1 antagonist). Conclusions: Our work shows that both T-type channels as well as CB2 receptors play a role in the antinociceptive action of NMP-181, and also provides a novel avenue for suppressing chronic pain through novel mixed T-type/cannabinoid receptor ligands.
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    Jagodic, M. M., Pathirathna, S., Joksovic, P. M., Lee, W., Nelson, M. T., Naik, A. K., Su, P., Jevtovic-Todorovic, V., and Todorovic, S. M. (2008) Upregulation of the T-type calcium current in small rat sensory neurons after chronic constrictive injury of the sciatic nerve J. Neurophysiol. 99, 3151– 3156
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    Upregulation of the T-type calcium current in small rat sensory neurons after chronic constrictive injury of the sciatic nerve
    Jagodic, Miljen M.; Pathirathna, Sriyani; Joksovic, Pavle M.; Lee, WooYong; Nelson, Michael T.; Naik, Ajit K.; Su, Peihan; Jevtovic-Todorovic, Vesna; Todorovic, Slobodan M.
    Journal of Neurophysiology (2008), 99 (6), 3151-3156CODEN: JONEA4; ISSN:0022-3077. (American Physiological Society)
    Recent data indicate that peripheral T-type Ca2+ channels are instrumental in supporting acute pain transmission. However, the function of these channels in chronic pain processing is less clear. To address this issue, we studied the expression of T-type Ca2+ currents in small nociceptive dorsal root ganglion (DRG) cells from L4-5 spinal ganglia of adult rats with neuropathic pain due to chronic constrictive injury (CCI) of the sciatic nerve. In control rats, whole cell recordings revealed that T-type currents, measured in 10 mM Ba2+ as a charge carrier, were present in moderate d. (20 ± 2 pA/pF). In rats with CCI, T-type c.d. (30 ± 3 pA/pF) was significantly increased, but voltage- and time-dependent activation and inactivation kinetics were not significantly different from those in controls. CCI-induced neuropathy did not significantly change the pharmacol. sensitivity of T-type current in these cells to nickel. Collectively, our results indicate that CCI-induced neuropathy significantly increases T-type current expression in small DRG neurons. Our findings that T-type currents are upregulated in a CCI model of peripheral neuropathy and earlier pharmacol. and mol. studies suggest that T-type channels may be potentially useful therapeutic targets for the treatment of neuropathic pain assocd. with partial mech. injury to the sciatic nerve.
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    Obradovic, A., Hwang, S. M., Scarpa, J., Hong, S. J., Todorovic, S. M., and Jevtovic-Todorovic, V. (2014) CaV3.2 T-Type Calcium Channels in Peripheral Sensory Neurons Are Important for Mibefradil-Induced Reversal of Hyperalgesia and Allodynia in Rats with Painful Diabetic Neuropathy PloS One 9, e91467
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    Snutch, T. P. and David, L. S. (2006) T-type calcium channels: an emerging therapeutic target for the treatment of pain Drug Dev. Res. 67, 404– 415
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    Todorovic, S. and Jevtovic-Todorovic, V. (2014) Targeting of CaV3.2 T-type calcium channels in peripheral sensory neurons for the treatment of painful diabetic neuropathy Pflügers Arch. 466, 701– 706
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    Targeting of CaV3.2 T-type calcium channels in peripheral sensory neurons for the treatment of painful diabetic neuropathy
    Todorovic Slobodan M; Jevtovic-Todorovic Vesna
    Pflugers Archiv : European journal of physiology (2014), 466 (4), 701-6 ISSN:.
    Pain-sensing sensory neurons (nociceptors) of the dorsal root ganglion (DRG) can become sensitized (hyperexcitable) in response to pathological conditions such as diabetes, which in turn may lead to the development of painful peripheral diabetic neuropathy (PDN). Because of insufficient knowledge about the mechanisms for this hypersensitization, current treatment for painful PDN has been limited to somewhat nonspecific systemic drugs having significant side effects or potential for abuse. Recent studies have established that the CaV3.2 isoform of T-channels makes a previously unrecognized contribution to sensitization of pain responses by enhancing excitability of nociceptors in animal models of type 1 and type 2 PDN. Furthermore, it has been reported that the glycosylation inhibitor neuraminidase can inhibit the native and recombinant CaV3.2 T-currents in vitro and completely reverse mechanical and thermal hyperalgesia in diabetic animals with PDN in vivo. Understanding details of posttranslational regulation of nociceptive channel activity via glycosylation may facilitate development of novel therapies for treatment of painful PDN. Pharmacological targeting the specific pathogenic mechanism rather than the channel per se may cause fewer side effects and reduce the potential for drug abuse in patients with diabetes.
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  21. 21
    Zamponi, G. W., Lewis, R. J., Todorovic, S. M., Arneric, S. P., and Snutch, T. P. (2009) Role of voltage-gated calcium channels in ascending pain pathways Brain Res. Rev. 60, 84– 89
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    21
    Role of voltage-gated calcium channels in ascending pain pathways
    Zamponi, Gerald W.; Lewis, Richard J.; Todorovic, Slobodan M.; Arneric, Stephen P.; Snutch, Terrance P.
    Brain Research Reviews (2009), 60 (1), 84-89CODEN: BRERD2; ISSN:0165-0173. (Elsevier B.V.)
    A review. Voltage gated calcium channels (VGCCs) are well established mediators of pain signals in primary afferent neurons. N-type calcium channels are localized to synaptic nerve terminals in laminae 1 and 2 of the dorsal horn where their opening results in the release of neurotransmitters such as glutamate and substance P. The contribution of N-type channels to the processing of pain signals is regulated by alternate splicing of the N-type channel gene, with unique N-type channel splice variants being expressed in small nociceptive neurons. In contrast, T-type VGCCs of the Cav3.2 subtype are likely localized to nerve endings where they regulate cellular excitability. Consequently, inhibition of N-type and Cav3.2 T-type VGCCs has the propensity to mediate analgesia. T-type channel activity is regulated by redox modulation, and can be inhibited by a novel class of small org. blockers. N-type VGCC activity can be potently inhibited by highly selective peptide toxins that are delivered intrathecally, and the search for small org. blockers with clin. efficacy is ongoing. Here, we provide a brief overview of recent advances in this area, as presented at the Spring Pain Research conference (Grand Cayman, 2008).
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    Perez-Reyes, E. (2003) Molecular physiology of low-voltage-activated t-type calcium channels Physiol. Rev. 83, 117– 161
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    22
    Molecular physiology of low-voltage-activated T-type calcium channels
    Perez-Reyes, Edward
    Physiological Reviews (2003), 83 (1), 117-161CODEN: PHREA7; ISSN:0031-9333. (American Physiological Society)
    A review. T-type Ca2+ channels were originally called low-voltage-activated (LVA) channels because they can be activated by small depolarizations of the plasma membrane. In many neurons Ca2+ influx through LVA channels triggers low-threshold spikes, which in turn triggers a burst of action potentials mediated by Na+ channels. Burst firing is thought to play an important role in the synchronized activity of the thalamus obsd. in absence epilepsy, but may also underlie a wider range of thalamocortical dysrhythmias. In addn. to a pacemaker role, Ca2+ entry via T-type channels can directly regulate intracellular Ca2+ concns., which is an important second messenger for a variety of cellular processes. Mol. cloning revealed the existence of three T-type channel genes. The deduced amino acid sequence shows a similar four-repeat structure to that found in high-voltage-activated (HVA) Ca2+ channels, and Na+ channels, indicating that they are evolutionarily related. Hence, the α1-subunits of T-type channels are now designated Cav3. Although mRNAs for all three Cav3 subtypes are expressed in brain, they vary in terms of their peripheral expression, with Cav3.2 showing the widest expression. The electrophysiol. activities of recombinant Cav3 channels are very similar to native T-type currents and can be differentiated from HVA channels by their activation at lower voltages, faster inactivation, slower deactivation, and smaller conductance of Ba2+. The Cav3 subtypes can be differentiated by their kinetics and sensitivity to block by Ni2+. The goal of this review is to provide a comprehensive description of T-type currents, their distribution, regulation, pharmacol., and cloning.
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  23. 23
    Barbara, G., Alloui, A., Nargeot, J., Lory, P., Eschalier, A., Bourinet, E., and Chemin, J. (2009) T-type calcium channel inhibition underlies the analgesic effects of the endogenous lipoamino acids J. Neurosci. 29, 13106– 13114
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    23
    T-type calcium channel inhibition underlies the analgesic effects of the endogenous lipoamino acids
    Barbara, Guillaume; Alloui, Abdelkrim; Nargeot, Joel; Lory, Philippe; Eschalier, Alain; Bourinet, Emmanuel; Chemin, Jean
    Journal of Neuroscience (2009), 29 (42), 13106-13114CODEN: JNRSDS; ISSN:0270-6474. (Society for Neuroscience)
    Lipoamino acids are anandamide-related endogenous mols. that induce analgesia via unresolved mechanisms. Here, we provide evidence that the T-type/Cav3 calcium channels are important pharmacol. targets underlying their physiol. effects. Various lipoamino acids, including N-arachidonoyl glycine (NAGly), reversibly inhibited Cav3.1, Cav3.2, and Cav3.3 currents, with potent effects on Cav3.2 [EC50 ∼200 nM for N-arachidonoyl 3-OH-γ-aminobutyric acid (NAGABA-OH)]. This inhibition involved a large shift in the Cav3.2 steady-state inactivation and persisted during fatty acid amide hydrolase (FAAH) inhibition as well as in cell-free outside-out patch. In contrast, lipoamino acids had weak effects on high-voltage-activated (HVA) Cav1.2 and Cav2.2 calcium currents, on Nav1.7 and Nav1.8 sodium currents, and on anandamide-sensitive TRPV1 and TASK1 currents. Accordingly, lipoamino acids strongly inhibited native Cav3.2 currents in sensory neurons with small effects on sodium and HVA calcium currents. In addn., we demonstrate here that lipoamino acids NAGly and NAGABA-OH produced a strong thermal analgesia and that these effects (but not those of morphine) were abolished in Cav3.2 knock-out mice. Collectively, our data revealed lipoamino acids as a family of endogenous T-type channel inhibitors, suggesting that these ligands can modulate multiple cell functions via this newly evidenced regulation.
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  24. 24
    Bladen, C., Gunduz, M. G., Simsek, R., Safak, C., and Zamponi, G. W. (2013) Synthesis and Evaluation of 1,4-Dihydropyridine Derivatives with Calcium Channel Blocking Activity Pflügers Arch. 466, 1355– 1363
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    Chemin, J., Monteil, A., Perez-Reyes, E., Nargeot, J., and Lory, P. (2001) Direct inhibition of T-type calcium channels by the endogenous cannabinoid anandamide EMBO J. 20, 7033– 7040
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    Choe, Y. J., Seo, H. N., Jung, S. Y., Rhim, H., Kim, J., Choo, D. J., and Lee, J. Y. (2008) Synthesis and SAR study of T-type calcium channel blockers. Part II Arch. Pharm. 341, 661– 664
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    26
    Synthesis and SAR study of T-type calcium channel blockers. Part II
    Choe, Yun Jeong; Seo, Han Na; Jung, Soo Yeon; Rhim, Hyewhon; Kim, Jungahn; Choo, Dong Joon; Lee, Jae Yeol
    Archiv der Pharmazie (Weinheim, Germany) (2008), 341 (10), 661-664CODEN: ARPMAS; ISSN:0365-6233. (Wiley-VCH Verlag GmbH & Co. KGaA)
    3,4-Dihydroquinazoline derivs. have been known to be novel and potent T-type calcium channel blockers. From a systematic variation of 3,4-dihydroquinazoline deriv. I [(KYS05043), R1 = 4-PhC6H4NH, R2 = (CH2)5NH2, R3 = OMe], plausible SAR results were established. It was revealed that a 5-(dimethylamino)pentylamino group at R1, a biphenyl group at R2, and a benzyl amido group at R3 in the 3,4-dihydroquinazoline backbone are closely related with the channel selectivity (T/N-type) as well as the potency based on the discovery of I [(KYS05090), R1 = NMe(CH2)5NMe2, R2 = 4-PhC6H4, R3 = NHBn].
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  27. 27
    Furukawa, T., Miura, R., Honda, M., Kamiya, N., Mori, Y., Takeshita, S., Isshiki, T., and Nukada, T. (2004) Identification of R(−)-isomer of efonidipine as a selective blocker of T-type Ca2+ channels Br. J. Pharmacol. 143, 1050– 1057
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    27
    Identification of R(-)-isomer of efonidipine as a selective blocker of T-type Ca2+ channels
    Furukawa, Taiji; Miura, Reiko; Honda, Mitsuyoshi; Kamiya, Natsuko; Mori, Yasuo; Takeshita, Satoshi; Isshiki, Takaaki; Nukada, Toshihide
    British Journal of Pharmacology (2004), 143 (8), 1050-1057CODEN: BJPCBM; ISSN:0007-1188. (Nature Publishing Group)
    1 Efonidipine, a deriv. of dihydropyridine Ca2+ antagonist, is known to block both L- and T-type Ca2+ channels. It remains to be clarified, however, whether efonidipine affects other voltage-dependent Ca2+ channel subtypes such as N-, P/Q- and R-types, and whether the optical isomers of efonidipine have different selectivities in blocking these Ca2+ channels, including L- and T-types. 2 To address these issues, the effects of efonidipine and its R(-)- and S(+)-isomers on these Ca2+ channel subtypes were examd. electrophysiol. in the expression systems using Xenopus oocytes and baby hamster kidney cells (BHK tk-ts13). 3 Efonidipine, a mixt. of R(-)- and S(+)-isomers, exerted blocking actions on L- and T-types, but no effects on N-, P/Q- and R-type Ca2+ channels. 4 The selective blocking actions on L- and T-type channels were reproduced by the S(+)-efonidipine isomer. 5 By contrast, the R(-)-efonidipine isomer preferentially blocked T-type channels. 6 The blocking actions of efonidipine and its enantiomers were dependent on holding potentials. 7 These findings indicate that the R(-)-isomer of efonidipine is a specific blocker of the T-type Ca2+ channel.
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  28. 28
    Hildebrand, M. E., Smith, P. L., Bladen, C., Eduljee, C., Xie, J. Y., Chen, L., Fee-Maki, M., Doering, C. J., Mezeyova, J., Zhu, Y., Belardetti, F., Pajouhesh, H., Parker, D., Arneric, S. P., Parmar, M., Porreca, F., Tringham, E., Zamponi, G. W., and Snutch, T. P. (2011) A novel slow-inactivation-specific ion channel modulator attenuates neuropathic pain Pain 152, 833– 843
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    Jo, M. N., Seo, H. J., Kim, Y., Seo, S. H., Rhim, H., Cho, Y. S., Cha, J. H., Koh, H. Y., Choo, H., and Pae, A. N. (2007) Novel T-type calcium channel blockers: dioxoquinazoline carboxamide derivatives Bioorg. Med. Chem. 15, 365– 373
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    29
    Novel T-type calcium channel blockers: Dioxoquinazoline carboxamide derivatives
    Jo, Mi Na; Seo, Hee Jeong; Kim, Yoonji; Seo, Seon Hee; Rhim, Hyewhon; Cho, Yong Seo; Cha, Joo Hwan; Koh, Hun Yeong; Choo, Hyunah; Pae, Ae Nim
    Bioorganic & Medicinal Chemistry (2007), 15 (1), 365-373CODEN: BMECEP; ISSN:0968-0896. (Elsevier Ltd.)
    T-type calcium channel is one of therapeutic targets for the treatment of cardiovascular diseases and neuropathic pains. Since the withdrawal of mibefradil, a T-type calcium channel blocker, there have been a lot of efforts to develop T-type calcium channel blockers. A small mol. library of dioxoquinazoline carboxamide derivs. contg. 155 compds. was designed, synthesized, and biol. evaluated for T-type calcium channel blocking activity. Among those compds. synthesized, I shows the most potent T-type calcium current blocking activity with an IC50 value of 1.52 μM, which is comparable to that of mibefradil.
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  30. 30
    Kumar, P. P., Stotz, S. C., Paramashivappa, R., Beedle, A. M., Zamponi, G. W., and Rao, A. S. (2002) Synthesis and evaluation of a new class of nifedipine analogs with T-type calcium channel blocking activity Mol. Pharmacol. 61, 649– 658
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    Perez-Reyes, E., Van Deusen, A. L., and Vitko, I. (2009) Molecular pharmacology of human Cav3.2 T-type Ca2+ channels: block by antihypertensives, antiarrhythmics, and their analogs J. Pharmacol. Exp. Ther. 328, 621– 627
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    31
    Molecular pharmacology of human Cav3.2 T-type Ca2+ channels: block by antihypertensives, antiarrhythmics, and their analogs
    Perez-Reyes, Edward; Van Deusen, Amy L.; Vitko, Iuliia
    Journal of Pharmacology and Experimental Therapeutics (2009), 328 (2), 621-627CODEN: JPETAB; ISSN:0022-3565. (American Society for Pharmacology and Experimental Therapeutics)
    Antihypertensive drugs of the "calcium channel blocker" or "calcium antagonist" class have been used to establish the physiol. role of L-type Ca2+ channels in vascular smooth muscle. In contrast, there has been limited progress on the pharmacol. T-type Ca2+ channels. T-type channels play a role in cardiac pacemaking, aldosterone secretion, and renal hemodynamics, leading to the hypothesis that mixed T- and L-type blockers may have therapeutic advantages over selective L-type blockers. The goal of this study was to identify compds. that block the Cav3.2 T-type channel with high affinity, focusing on two classes of compds.: phenylalkylamines (e.g., mibefradil) and dihydropyridines (e.g., efonidipine). Compds. were tested using a validated Ca2+ influx assay into a cell line expressing recombinant Cav3.2 channels. This study identified four clin. approved antihypertensive drugs (efonidipine, felodipine, isradipine, and nitrendipine) as potent T-channel blockers (IC50 < 3 μM). In contrast, other widely prescribed dihydropyridines, such as amlodipine and nifedipine, were 10-fold less potent, making them a more appropriate choice in research studies on the role of L-type currents. In summary, the present results support the notion that many available antihypertensive drugs block a substantial fraction of T-current at therapeutically relevant concns., contributing to their mechanism of action.
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  32. 32
    Yamamoto, E., Kataoka, K., Dong, Y. F., Nakamura, T., Fukuda, M., Nako, H., Ogawa, H., and Kim-Mitsuyama, S. (2010) Benidipine, a dihydropyridine L-type/T-type calcium channel blocker, affords additive benefits for prevention of cardiorenal injury in hypertensive rats J. Hypertens. 28, 1321– 1329
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    32
    Benidipine, a dihydropyridine L-type/T-type calcium channel blocker, affords additive benefits for prevention of cardiorenal injury in hypertensive rats
    Yamamoto, Eiichiro; Kataoka, Keiichiro; Dong, Yi-Fei; Nakamura, Taishi; Fukuda, Masaya; Nako, Hisato; Ogawa, Hisao; Kim-Mitsuyama, Shokei
    Journal of Hypertension (2010), 28 (6), 1321-1329CODEN: JOHYD3; ISSN:0263-6352. (Lippincott Williams & Wilkins)
    Benidipine is a dihydropyridine calcium channel blocker inhibiting not only L-type but also T-type calcium channels. To elucidate potential additive benefit of benidipine for prevention of cardiorenal injury, we compared the cardiac and renal protective effects of equihypotensive doses of benidipine and cilnidipine in stroke-prone spontaneously hypertensive rats (SHRSP). SHRSP were divided into five groups, and were given vehicle, benidipine at 1 or 3 mg/kg per day, or cilnidipine at 1 or 3 mg/kg per day for 7 wk, and the protective effects against cardiorenal injury were compared among each group. Benidipine and cilnidipine at the same doses exerted comparable hypotensive effects on SHRSP throughout the treatment. Despite equihypotensive effects between both drugs, benidipine prevented cardiac hypertrophy, fibrosis, and inflammation to a greater extent than cilnidipine. Moreover, benidipine prevented glomerulosclerosis, tubulointerstitial injury, and renal inflammation more than cilnidipine. To elucidate the underlying mechanism of more beneficial effects of benidipine than cilnidipine, we compared the effects of these drugs on cardiac and renal oxidative stress, and aldosterone in SHRSP. Benidipine reduced both cardiac and renal NADPH oxidase activities in SHRSP more than cilnidipine, being assocd. with more attenuation of cardiac and renal superoxide by benidipine. Furthermore, serum aldosterone was significantly reduced by benidipine but not by cilnidipine. Benidipine exerted more protective effects against cardiorenal injury of hypertensive rats than cilnidipine, through more attenuation of oxidative stress than cilnidipine, and the redn. of aldosterone. Benidipine, via blockade of T-type calcium channels, seems to elicit additive benefits for prevention of hypertensive cardiorenal injury.
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    You, H., Altier, C., and Zamponi, G. W. (2010) CCR2 receptor ligands inhibit Cav3.2 T-type calcium channels Mol. Pharmacol. 77, 211– 217
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    You, H., Gadotti, V. M., Petrov, R. R., Zamponi, G. W., and Diaz, P. (2011) Functional characterization and analgesic effects of mixed cannabinoid receptor/T-type channel ligands Mol. Pain 7, 89
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    34
    Functional characterization and analgesic effects of mixed cannabinoid receptor/T-type channel ligands
    You, Haitao; Gadotti, Vinicius M.; Petrov, Ravil R.; Zamponi, Gerald W.; Diaz, Philippe
    Molecular Pain (2011), 7 (), 89CODEN: MPOAC5; ISSN:1744-8069. (BioMed Central Ltd.)
    Background: Both T-type calcium channels and cannabinoid receptors modulate signalling in the primary afferent pain pathway. Here, we investigate the analgesics activities of a series of novel cannabinoid receptor ligands with T-type calcium channel blocking activity. Results: Novel compds. were characterized in radioligand binding assays and in vitro functional assays at human and rat CB1 and CB2 receptors. The inhibitory effects of these compds. on transient expressed human T-type calcium channels were examd. in tsA-201 cells using std. whole-cell voltage clamp techniques and their analgesic effects in response to various administration routes (intrathecally, intraplantarly, i.p.) assessed in the formalin model. A series of compds. were synthesized and evaluated for channel and receptor activity. Compd. NMP-7 acted as non-selective CB1/CB2 agonist while NMP4 was found to be a CB1 partial agonist and CB2 inverse agonist. Furthermore, NMP-144 behaved as a selective CB2 inverse agonist. All of these three compds. completely inhibited peak Cav3.2 currents with IC50 values in the low micromolar range. All compds. mediated analgesic effects in the formalin model, but depending on the route of administration, could differentially affect phase 1 and phase 2 of the formalin response. Conclusions: Our results reveal that a set of novel cannabinioid receptor ligands potently inhibit T-type calcium channels and show analgesic effects in vivo. Our findings suggest possible novel means of mediating pain relief through mixed T-type/cannabinoid receptor ligands.
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    Chemin, J., Nargeot, J., and Lory, P. (2007) Chemical determinants involved in anandamide-induced inhibition of T-type calcium channels J. Biol. Chem. 282, 2314– 2323
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    Moreira, F. A., Grieb, M., and Lutz, B. (2009) Central side-effects of therapies based on CB1 cannabinoid receptor agonists and antagonists: focus on anxiety and depression Best. Pract. Res., Clin. Endocrinol. Metab. 23, 133– 144
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    Central side-effects of therapies based on CB1 cannabinoid receptor agonists and antagonists: focus on anxiety and depression
    Moreira, Fabricio A.; Grieb, Maximilian; Lutz, Beat
    Best Practice & Research, Clinical Endocrinology & Metabolism (2009), 23 (1), 133-144CODEN: BPRCE9 ISSN:. (Elsevier Ltd.)
    A review. Both agonists (e.g. Δ9-tetrahydrocannabinol, nabilone) and antagonists (e.g. rimonabant, taranabant) of the cannabinoid type-1 (CB1) receptor have been explored as therapeutic agents in diverse fields of medicine such as pain management and obesity with assocd. metabolic dysregulation, resp. CB1 receptors are widely distributed in the central nervous system and are involved in the modulation of emotion, stress and habituation responses, behaviors that are thought to be dysregulated in human psychiatric disorders. Accordingly, CB1 receptor activation may, in some cases, ppt. episodes of psychosis and panic, while its inhibition may lead to behaviors reminiscent of depression and anxiety-related disorders. The present review discusses these side-effects, which have to be taken into account in the therapeutic exploitation of the endocannabinoid system.
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    Witkin, J. M., Tzavara, E. T., and Nomikos, G. G. (2005) A role for cannabinoid CB1 receptors in mood and anxiety disorders Behav. Pharmacol. 16, 315– 331
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    Petrov, R. R., Knight, L., Chen, S. R., Wager-Miller, J., McDaniel, S. W., Diaz, F., Barth, F., Pan, H. L., Mackie, K., Cavasotto, C. N., and Diaz, P. (2013) Mastering tricyclic ring systems for desirable functional cannabinoid activity Eur. J. Med. Chem. 69, 881– 907
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    Chen, X. L., Bayliss, D. A., Fern, R. J., and Barrett, P. Q. (1999) A role for T-type Ca2+ channels in the synergistic control of aldosterone production by ANG II and K+ Am. J. Physiol. 276, F674– 683
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    Marger, F., Gelot, A., Alloui, A., Matricon, J., Ferrer, J. F., Barrere, C., Pizzoccaro, A., Muller, E., Nargeot, J., Snutch, T. P., Eschalier, A., Bourinet, E., and Ardid, D. (2011) T-type calcium channels contribute to colonic hypersensitivity in a rat model of irritable bowel syndrome Proc. Natl. Acad. Sci. U. S. A. 108, 11268– 11273
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    Moriguchi, S., Shioda, N., Yamamoto, Y., Tagashira, H., and Fukunaga, K. (2012) The T-type voltage-gated calcium channel as a molecular target of the novel cognitive enhancer ST101: Enhancement of long-term potentiation and CaMKII autophosphorylation in rat cortical slices J. Neurochem. 121, 44– 53
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    41
    The T-type voltage-gated calcium channel as a molecular target of the novel cognitive enhancer ST101: enhancement of long-term potentiation and CaMKII autophosphorylation in rat cortical slices
    Moriguchi, Shigeki; Shioda, Norifumi; Yamamoto, Yui; Tagashira, Hideaki; Fukunaga, Kohji
    Journal of Neurochemistry (2012), 121 (1 & 2), 44-53CODEN: JONRA9; ISSN:0022-3042. (Wiley-Blackwell)
    In this study, we report that spiro[imidazo[1,2-a]pyridine-3,2-indan]-2(3H)-one (ST101; previously coded as ZSET1446) targets T-type voltage-gated calcium channels in mediating improved cognition in the CNS. We prepd. rat somatosensory cortical and hippocampal slices, treated them with 0.01 to 100 nM ST101, and performed immunoblotting and electrophysiol. analyses using various voltage-gated calcium channel (VGCC) inhibitors. Treatment of rat cortical slices with a range of ST101 concns. significantly increased calcium/calmodulin-dependent protein kinase II (CaMKII) autophosphorylation following a bell-shaped dose-response curve, with 0.1 nM ST101 representing the maximally effective concn. protein kinase Cα autophosphorylation was also significantly increased by 0.1 nM ST101 treatment. ST101 treatment had a moderate effect on CaMKII autophosphorylation but no effect on hippocampal protein kinase Cα autophosphorylation in slice prepns. Consistent with increased cortical CaMKII autophosphorylation, AMPA-type glutamate receptor subunit 1 (Ser-831) phosphorylation as a CaMKII post-synaptic substrate was significantly increased by treatment with 0.1-1 nM ST101, whereas phosphorylation of the pre-synaptic substrate synapsin I (Ser-603) remained unchanged. Notably, enhanced CaMKII autophosphorylation seen following 0.1 nM ST101 treatment was significantly inhibited by pre-treatment with 1 μM mibefradil, a T-type VGCC inhibitor, but not with N-type (ω-conotoxin), P/Q-type (ω-agatoxin) or L-type (nifedipine) VGCC inhibitors. Similarly, 0.1 nM ST101 significantly potentiated long-term potentiation in cortical but not hippocampal slices. Enhanced long-term potentiation in cortical slices was totally inhibited by 1 μM mibefadil treatment. Finally, whole-cell patch-clamp anal. of Neuro2A cells over-expressing recombinant human CaV3.1 (α1G) T-channels and treated with 0.1 nM ST101 showed significant increases in T-type VGCC currents. These results indicate that T-type VGCCs are direct mol. targets for the novel cognitive enhancer ST101, a potential Alzheimer disease therapeutic.
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    Jevtovic-Todorovic, V. and Todorovic, S. M. (2006) The role of peripheral T-type calcium channels in pain transmission Cell Calcium 40, 197– 203
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    A review. Voltage-gated calcium channels are the primary mediators of depolarization-induced calcium entry into neurons. There is great diversity of calcium channel subtypes due to multiple genes that encode calcium channel α1 subunits, coassembly with a variety of ancillary calcium channel subunits, and alternative splicing. This allows these channels to fulfill highly specialized roles in specific neuronal subtypes and at particular subcellular loci. While calcium channels are of crit. importance to brain function, their inappropriate expression or dysfunction gives rise to a variety of neurol. disorders, including, pain, epilepsy, migraine, and ataxia. This Review discusses salient aspects of voltage-gated calcium channel function, physiol., and pathophysiol.
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    Journal of Biological Chemistry (2003), 278 (22), 20171-20178CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)
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    Gadotti, Vinicius M.; Zamponi, Gerald W.
    Molecular Pain (2011), 7 (), 59CODEN: MPOAC5; ISSN:1744-8069. (BioMed Central Ltd.)
    Cellular prion protein (PrPC) inhibits N-Methyl--Aspartate (NMDA) receptors. Since NMDA receptors play an important role in the transmission of pain signals in the dorsal horn of spinal cord, we thus wanted to det. if PrPC null mice show a reduced threshold for various pain behaviors. We compared nociceptive thresholds between wild type and PrPC null mice in models of inflammatory and neuropathic pain, in the presence and the absence of a NMDA receptor antagonist. 2-3 Mo old male PrPC null mice exhibited an MK-801 sensitive decrease in the paw withdrawal threshold in response both mech. and thermal stimuli. PrPC null mice also exhibited significantly longer licking/biting time during both the first and second phases of formalin-induced inflammation of the paw, which was again prevented by treatment of the mice with MK-801, and responded more strongly to glutamate injection into the paw. Compared to wild type animals, PrPC null mice also exhibited a significantly greater nociceptive response (licking/biting) after intrathecal injection of NMDA. Sciatic nerve ligation resulted in MK-801 sensitive neuropathic pain in wild-type mice, but did not further augment the basal increase in pain behavior obsd. in the null mice, suggesting that mice lacking PrPC may already be in a state of tonic central sensitization. Altogether, our data indicate that PrPC exerts a crit. role in modulating nociceptive transmission at the spinal cord level, and fit with the concept of NMDA receptor hyperfunction in the absence of PrPC.
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    Partial sciatic nerve injury in the mouse as a model of neuropathic pain: behavioral and neuroanatomical correlates
    Malmberg A B; Basbaum A I
    Pain (1998), 76 (1-2), 215-22 ISSN:0304-3959.
    The generation of knock-out and transgenic mice offers a promising approach to the identification of novel biochemical factors that contribute to persistent pain conditions. To take advantage of these mice, however, it is important to demonstrate that the traditional models of persistent pain, which were largely developed for studies in the rat, can be used in the mouse. Here, we combined behavioral and anatomical methods to characterize the pathophysiology of a partial nerve injury-evoked pain condition in the 'normal' mouse. In male C57BL6 mice we tied a tight ligature around 1/3 to 1/2 of the diameter of the sciatic nerve and evaluated the time-course and magnitude of the ensuing mechanical and thermal allodynia. We also used immunocytochemistry to analyze nerve injury-induced changes in substance P (SP) and NK-1 (SP) receptor expression in the spinal cord. As in the rat, partial nerve injury markedly decreased paw withdrawal thresholds to both mechanical and thermal stimuli on the injured side. We detected threshold changes one day after the injury. The thermal allodynia resolved by 49 days, but the mechanical allodynia persisted for the duration of the study (70 days). We found no changes contralateral to the nerve injury. Sympatholytic treatment with guanethidine significantly reduced both the thermal and mechanical allodynia. We observed a reduction of SP immunoreactivity in the superficial dorsal horn on the injured side at 7 and 14, but not at 3 or 70 days after the nerve injury, and we observed an increase of NK-1 receptor expression at 3, 7, 14 and 42, but not at 70 days after the injury. We conclude that partial injury to the sciatic nerve produces a comparable allodynia and neurochemical plasticity in the rat and mouse. These results establish a valuable model for future studies of the biochemical basis of neuropathic pain in mice with specific gene modifications.
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  23. Jeffrey R McArthur, Leonid Motin, Ellen C Gleeson, Sandro Spiller, Richard J Lewis, Peter J Duggan, Kellie L Tuck, David J Adams. Inhibition of human N‐ and T‐type calcium channels by an ortho ‐phenoxyanilide derivative, MONIRO‐1. British Journal of Pharmacology 2018, 175 (12) , 2284-2295. https://doi.org/10.1111/bph.13910
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  27. Jin Han Kim, Hui Rak Jeong, Da Woon Jung, Hong Bin Yoon, Sun Young Kim, Hyoung Ja Kim, Kyung-Tae Lee, Vinicius M. Gadotti, Junting Huang, Fang-Xiong Zhang, Gerald W. Zamponi, Jae Yeol Lee. Synthesis and biological evaluation of fluoro-substituted 3,4-dihydroquinazoline derivatives for cytotoxic and analgesic effects. Bioorganic & Medicinal Chemistry 2017, 25 (17) , 4656-4664. https://doi.org/10.1016/j.bmc.2017.07.010
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  • Abstract

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    Scheme 1

    Scheme 1. Piperidine Containing T-type Ca2+ Inhibitors TTA-P1, Dual T-type Channel Blocker/Cannabinoid Agonist NMP7, and Chemical Optimization Plan 1 To Decrease Cannabinoid Receptor Affinities
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    Figure 1

    Figure 1. Percentage of whole cell current inhibition of human Cav3.2 (T-type) in response to 10 μM application of the compound series (n = 6 per compound). Note the potent and preferential block of Cav3.2 channels by compounds 9 and 10. Error bars reflect standard errors. For Cav3.2 channels, the holding and test potentials were respectively −110 and −20 mV.

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    Scheme 2

    Scheme 2. a

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    Scheme aReagents and conditions: (a) corresponding amine, EDAC, HOBt, DMAP, DIPEA, DCM, 0°C to rt; (b) TFA, CH2Cl2; (c) N-tert-butyl-2-chloroacetamide, K2CO3, KI, n-butanol, reflux 3 h.

    Figure 2

    Figure 2. (A) Representative traces of hCav 3.2 before and after application of 3 μM compounds 10 and 9. (B) Dose–response relations for compound 9 and 10 block of hCav3.2 channels. The IC50 from the fit with the Hill equation was 1.48 and 3.68 μM, respectively (n = 6). (C) Effect of 3 μM compounds 9 and 10 on the steady state inactivation curve for Cav3.2 channels. (D) Effect of 3 μM compounds 9 and 10 on the current voltage relation for Cav3.2 channels. Note: Data in panels (B) and (C) were fitted with the Boltzmann equation, and data were obtained from 6 paired experiments.

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    Figure 3

    Figure 3. (A, B) Effect of increasing doses of intrathecal compound 9 on the first and second phases of formalin-induced pain. (C, D) Effect of increasing doses of intraperitoneal compound 9 (on the first and second phases of formalin-induced pain. Each bar represents the mean ± SEM (n = 6–8), and is representative of 2 independent experiments. Asterisks denote the significance relative to the control group (***P < 0.001, one-way ANOVA followed by Dunnett’s test).

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    Figure 4

    Figure 4. (A) Effect of 30 mg/kg intraperitoneal compound 9 on locomotor activity of wild type mice in the open field test. (B, C) Comparison of effect of 10 μg/i.t. intrathecal compound 9 on the first and second phases of formalin-induced pain in wild type and Cav3.2 knockout mice, respectively. Each bar represents the mean ± SEM (n = 6–7) and is representative of 2 independent experiments. Asterisks denote the significance relative to the control group ***P < 0.001 when comparing treatment; and #P < 0.05, for comparison between genotypes (two-way ANOVA followed by Tukey’s test). Note that control mice were of the same genetic background as the Cav3.2 null mice.

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    Figure 5

    Figure 5. Blind analyses of the time course of treatment of neuropathic mice with vehicle or compound 9. Each circle represents the mean ± SEM (n = 6), and is representative of 2 independent experiments. (*P < 0.05, ***P < 0.001, two-way ANOVA followed by Tukey’s test). The dashed line and number symbols indicate the range of data points where injured animals significantly differed from the sham treated group (P < 0.001).

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  • References

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    This article references 57 other publications.

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      A review. Two different Ca2+ channels exist in cardiomyocytes. Whereas the L-type Ca2+ channel is ubiquitous and the main source of Ca2+ for excitation-contraction coupling and pacemaker activity, the functional role of the T-type Ca2+ channel is diverse and depends on mammalian species, heart region, age, and various cardiac diseases. Two isoforms of T-type Ca2+ channel proteins in the heart, CaV3.1 and CaV3.2, are functionally expressed in embryonic hearts, but markedly diminish during development. In the adult heart, the T-type Ca2+ channel is almost undetectable in ventricular myocytes and is most prevalent in the conduction system, playing a functional role in facilitating pacemaker depolarization of the sinoatrial node. Interestingly, the T-type Ca2+ channel is re-expressed in atrial and ventricular myocytes under various pathol. conditions such as hypertrophy and heart failure, and contributes to abnormal elec. activity and excitation-contraction coupling, but the T-type channel provides a smaller contribution to the trigger for Ca2+ release than does the L-type Ca2+ channel. Instead, the T-type Ca2+ channel has been shown to play a crucial role in the process of pathol. cardiac hypertrophy. Increased Ca2+ influx via CaV3.2, the T-type Ca2+ channel, induces calcineurin/NFAT (nuclear factor of activated T-cell) hypertrophic signaling. Furthermore, new evidence has been accumulating on the regulatory mechanism of T-type Ca2+ channel expression, including the neuron restrictive silencer element-neuron restrictive silencer factor (NRSE-NRSF) system, MAP kinases, and cardiac homeobox transcription factor Csx/Nkx2.5. Here, the authors summarize present knowledge regarding cardiac T-type Ca2+ channels, and discuss their pathophysiol. significance in the heart.
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      Spontaneously active neurons typically fire either in a regular pattern or in bursts. While much is known about the subcellular location and biophys. properties of conductances that underlie regular spontaneous activity, less is known about those that underlie bursts. Here, we show that T-type Ca2+ channels localized to the site of action potential initiation in the axon initial segment play a pivotal role in spontaneous burst generation. In auditory brainstem interneurons, axon initial segment Ca2+ influx is selectively downregulated by dopaminergic signalling. This regulation has marked effects on spontaneous activity, converting the predominant mode of spontaneous activity from bursts to regular spiking. Thus, the axon initial segment is a key site, and dopamine a key regulator, of spontaneous bursting activity.
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      Journal of Neuroscience (2007), 27 (12), 3305-3316CODEN: JNRSDS; ISSN:0270-6474. (Society for Neuroscience)
      Recent data indicate that T-type Ca2+ channels are amplifiers of peripheral pain signals, but their involvement in disorders of sensory neurons such as those assocd. with diabetes is poorly understood. To address this issue, we used a combination of behavioral, immunohistol., mol., and electrophysiol. studies in rats with streptozotocin (N-[methylnitrosocarbamoyl]-D-glucosamine)-induced early diabetic neuropathy. We found that, in parallel with the development of diabetes-induced pain, T-type c.d. increased by twofold in medium-size cells from L4-L5 dorsal root ganglia (DRG) with a depolarizing shift in steady-state inactivation. This not only correlated closely with more prominent afterdepolarizing potentials (ADPs) but also increased cellular excitability manifested as a lower threshold for burst firing in diabetic than in control cells. T-type currents and ADPs were potently inhibited by nickel and enhanced by L-cysteine, suggesting that the CaV3.2 T-type channel isoform was upregulated. Both control and diabetic DRG cells with ADPs stained pos. for isolectin B4, but only diabetic cells responded robustly to capsaicin, suggesting enhanced nociceptive function. Because increased excitability of sensory neurons may result in such pathol. perceptions of pain as hyperalgesia and allodynia, upregulation of T-type Ca2+ currents and enhanced Ca2+ entry into these cells could contribute to the development of symptoms in diabetic neuropathy.
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      Heron, S. E., Khosravani, H., Varela, D., Bladen, C., Williams, T. C., Newman, M. R., Scheffer, I. E., Berkovic, S. F., Mulley, J. C., and Zamponi, G. W. (2007) Extended spectrum of idiopathic generalized epilepsies associated with CACNA1H functional variants Ann. Neurol. 62, 560– 568
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      Extended spectrum of idiopathic generalized epilepsies associated with CACNA1H functional variants
      Heron, Sarah E.; Khosravani, Houman; Varela, Diego; Bladen, Chris; Williams, Tristiana C.; Newman, Michelle R.; Scheffer, Ingrid E.; Berkovic, Samuel F.; Mulley, John C.; Zamponi, Gerald W.
      Annals of Neurology (2007), 62 (6), 560-568CODEN: ANNED3; ISSN:0364-5134. (Wiley-Liss, Inc.)
      The relationship between genetic variation in the T-type calcium channel gene CACNA1H and childhood absence epilepsy is well established. The purpose of this study was to investigate the range of epilepsy syndromes for which CACNA1H variants may contribute to the genetic susceptibility architecture and det. the electrophysiol. effects of these variants in relation to proposed mechanisms underlying seizures. Exons 3 to 35 of CACNA1H were screened for variants in 240 epilepsy patients (167 unrelated) and 95 control subjects by single-stranded conformation anal. followed by direct sequencing. Cascade testing of families was done by sequencing or single-stranded conformation anal. Selected variants were introduced into the CACNA1H protein by site-directed mutagenesis. Constructs were transiently transfected into human embryo kidney cells, and electrophysiol. data were acquired. More than 100 variants were detected, including 19 novel variants leading to amino acid changes in subjects with phenotypes including childhood absence, juvenile absence, juvenile myoclonic and myoclonic astatic epilepsies, as well as febrile seizures and temporal lobe epilepsy. Electrophysiol. anal. of 11 variants showed that 9 altered channel properties, generally in ways that would be predicted to increase calcium current. Interpretation: Variants in CACNA1H that alter channel properties are present in patients with various generalized epilepsy syndromes. We propose that these variants contribute to an individual's susceptibility to epilepsy but are not sufficient to cause epilepsy on their own. The genetic architecture is dominated by rare functional variants; therefore, CACNA1H would not be easily identified as a susceptibility gene by a genome-wide case-control study seeking a statistical assocn.
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      Khosravani, Houman; Bladen, Christopher; Parker, David B.; Snutch, Terrance P.; McRory, John E.; Zamponi, Gerald W.
      Annals of Neurology (2005), 57 (5), 745-749CODEN: ANNED3; ISSN:0364-5134. (Wiley-Liss, Inc.)
      Heron and colleagues (Ann Neurol 2004;55:595-596) identified three missense mutations in the Cav3.2 T-type calcium channel gene (CACNA1H) in patients with idiopathic generalized epilepsy. None of the variants were assocd. with a specific epilepsy phenotype and were not found in patients with juvenile absence epilepsy or childhood absence epilepsy. Here, we introduced and functionally characterized these three mutations using transiently expressed human Cav3.2 channels. Two of the mutations exhibited functional changes that are consistent with increased channel function. Taken together, these findings along with previous reports, strongly implicate CACNA1H as a susceptibility gene in complex idiopathic generalized epilepsy.
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      Low-voltage-activated, or T-type, calcium (Ca2+) channels are believed to play an essential role in the generation of absence seizures in the idiopathic generalized epilepsies (IGEs). We describe a homozygous, missense, single nucleotide (G to C) mutation in the Cav3.2 T-type Ca2+ channel gene (Cacna1h) in the genetic absence epilepsy rats from Strasbourg (GAERS) model of IGE. The GAERS Cav3.2 mutation (gcm) produces an arginine to proline (R1584P) substitution in exon 24 of Cacna1h, encoding a portion of the III-IV linker region in Cav3.2.gcm segregates codominantly with the no. of seizures and time in seizure activity in progeny of an F1 intercross. We have further identified two major thalamic Cacna1h splice variants, either with or without exon 25.gcm introduced into the splice variants acts "epistatically," requiring the presence of exon 25 to produce significantly faster recovery from channel inactivation and greater charge transference during high-frequency bursts. This gain-of-function mutation, the first reported in the GAERS polygenic animal model, has a novel mechanism of action, being dependent on exonic splicing for its functional consequences to be expressed.
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      Role of voltage-gated calcium channels in epilepsy
      Zamponi Gerald W; Lory Philippe; Perez-Reyes Edward
      Pflugers Archiv : European journal of physiology (2010), 460 (2), 395-403 ISSN:.
      It is well established that idiopathic generalized epilepsies (IGEs) show a polygenic origin and may arise from dysfunction of various types of voltage- and ligand-gated ion channels. There is an increasing body of literature implicating both high- and low-voltage-activated (HVA and LVA) calcium channels and their ancillary subunits in IGEs. Cav2.1 (P/Q-type) calcium channels control synaptic transmission at presynaptic nerve terminals, and mutations in the gene encoding the Cav2.1 alpha1 subunit (CACNA1A) have been linked to absence seizures in both humans and rodents. Similarly, mutations and loss of function mutations in ancillary HVA calcium channel subunits known to co-assemble with Cav2.1 result in IGE phenotypes in mice. It is important to note that in all these mouse models with mutations in HVA subunits, there is a compensatory increase in thalamic LVA currents which likely leads to the seizure phenotype. In fact, gain-of-function mutations have been identified in Cav3.2 (an LVA or T-type calcium channel encoded by the CACNA1H gene) in patients with congenital forms of IGEs, consistent with increased excitability of neurons as a result of enhanced T-type channel function. In this paper, we provide a broad overview of the roles of voltage-gated calcium channels, their mutations, and how they might contribute to the river that terminates in epilepsy.
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      The antihyperalgesic effects of the T-type calcium channel blockers ethosuximide, trimethadione, and mibefradil
      Barton, Matthew E.; Eberle, Elizabeth L.; Shannon, Harlan E.
      European Journal of Pharmacology (2005), 521 (1-3), 79-85CODEN: EJPHAZ; ISSN:0014-2999. (Elsevier B.V.)
      The purpose of the present study was to explore the analgesic effects of the low voltage-activated T-type Ca2+ channel blockers ethosuximide, trimethadione, and mibefradil in persistent and acute nociceptive tests. The anticonvulsant effects of the compds. were also detd. in the i.v. pentylenetetrazol seizure model. Following i.p. administration, ethosuximide and trimethadione dose-dependently reversed capsaicin-induced mech. hyperalgesia. Similarly, the highest dose of mibefradil tested (30 μg, intracisternal) reversed capsaicin-induced mech. hyperalgesia. Ethosuximide and mibefradil produced statistically significant analgesic effects in both early and late phase formalin-induced behaviors and trimethadione reduced late phase behaviors. Addnl., ethosuximide and trimethadione produced antinociceptive effects in the rat-tail flick reflex test. In contrast, following intracisternal administration, mibefradil had no effect in the tail flick reflex test. In addn., the anticonvulsants ethosuximide and trimethadione increased the doses of pentylenetetrazol required to produce both first twitch and clonic seizures. In contrast however, mibefradil had no anticonvulsant effect. The present results demonstrate that the clin. used anticonvulsants ethosuximide and trimethadione provide analgesic effects at doses, which are anticonvulsant. Furthermore, the data further supports the idea that T-type Ca2+ channels may be important targets for treating persistent pain syndromes.
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      Attenuated pain responses in mice lacking CaV3.2 T-type channels
      Choi, S.; Na, H. S.; Kim, J.; Lee, J.; Lee, S.; Kim, D.; Park, J.; Chen, C.-C.; Campbell, K. P.; Shin, H.-S.
      Genes, Brain and Behavior (2007), 6 (5), 425-431CODEN: GBBEAO; ISSN:1601-1848. (Blackwell Publishing Ltd.)
      Although T-type Ca2+ channels are implicated in nociception, the function of specific subtypes has not been well defined. Here, we compared pain susceptibility in mice lacking CaV3.2 subtype of T-type Ca2+ channels (CaV3.2-/-) with wild-type littermates in various behavioral models of pain to explore the roles of CaV3.2 in the processing of noxious stimuli in vivo. In acute mech., thermal and chem. pain tests, CaV3.2-/- mice showed decreased pain responses compared to wild-type mice. CaV3.2-/- mice also displayed attenuated pain responses to tonic noxious stimuli such as i.p. injections of irritant agents and intradermal injections of formalin. In spinal nerve ligation-induced neuropathic pain, however, behavioral responses of CaV3.2-/- mice were not different from those of wild-type mice. The present study reveals that the CaV3.2 subtype of T-type Ca2+ channels are important in the peripheral processing of noxious signals, regardless of modality, duration or affected tissue type.
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      Gadotti, V. M., You, H., Petrov, R. R., Berger, N. D., Diaz, P., and Zamponi, G. W. (2013) Analgesic effect of a mixed T-type channel inhibitor/CB2 receptor agonist Mol. Pain 9, 32
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      Analgesic effect of a mixed T-type channel inhibitor/CB2 receptor agonist
      Gadotti, Vinicius M.; You, Haitao; Petrov, Ravil R.; Berger, N. Daniel; Diaz, Philippe; Zamponi, Gerald W.
      Molecular Pain (2013), 9 (), 32CODEN: MPOAC5; ISSN:1744-8069. (BioMed Central Ltd.)
      Background: Cannabinoid receptors and T-type calcium channels are potential targets for treating pain. Here we report on the design, synthesis and analgesic properties of a new mixed cannabinoid/T-type channel ligand, NMP-181. Results: NMP-181 action on CB1 and CB2 receptors was characterized in radioligand binding and in vitro GTPγ[35S] functional assays, and block of transiently expressed human CaV3.2 T-type channels by NMP-181 was analyzed by patch clamp. The analgesic effects and in vivo mechanism of action of NMP-181 delivered spinally or systemically were analyzed in formalin and CFA mouse models of pain. NMP-181 inhibited peak CaV3.2 currents with IC50 values in the low micromolar range and acted as a CB2 agonist. Inactivated state dependence further augmented the inhibitory action of NMP-181. NMP-181 produced a dose-dependent antinociceptive effect when administered either spinally or systemically in both phases of the formalin test. Both i.t. and i.p. treatment of mice with NMP-181 reversed the mech. hyperalgesia induced by CFA injection. NMP-181 showed no antinocieptive effect in CaV3.2 null mice. The antinociceptive effect of intrathecally delivered NMP-181 in the formalin test was reversed by i.t. treatment of mice with AM-630 (CB2 antagonist). In contrast, the NMP-181-induced antinociception was not affected by treatment of mice with AM-281 (CB1 antagonist). Conclusions: Our work shows that both T-type channels as well as CB2 receptors play a role in the antinociceptive action of NMP-181, and also provides a novel avenue for suppressing chronic pain through novel mixed T-type/cannabinoid receptor ligands.
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    17. 17
      Jagodic, M. M., Pathirathna, S., Joksovic, P. M., Lee, W., Nelson, M. T., Naik, A. K., Su, P., Jevtovic-Todorovic, V., and Todorovic, S. M. (2008) Upregulation of the T-type calcium current in small rat sensory neurons after chronic constrictive injury of the sciatic nerve J. Neurophysiol. 99, 3151– 3156
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      Upregulation of the T-type calcium current in small rat sensory neurons after chronic constrictive injury of the sciatic nerve
      Jagodic, Miljen M.; Pathirathna, Sriyani; Joksovic, Pavle M.; Lee, WooYong; Nelson, Michael T.; Naik, Ajit K.; Su, Peihan; Jevtovic-Todorovic, Vesna; Todorovic, Slobodan M.
      Journal of Neurophysiology (2008), 99 (6), 3151-3156CODEN: JONEA4; ISSN:0022-3077. (American Physiological Society)
      Recent data indicate that peripheral T-type Ca2+ channels are instrumental in supporting acute pain transmission. However, the function of these channels in chronic pain processing is less clear. To address this issue, we studied the expression of T-type Ca2+ currents in small nociceptive dorsal root ganglion (DRG) cells from L4-5 spinal ganglia of adult rats with neuropathic pain due to chronic constrictive injury (CCI) of the sciatic nerve. In control rats, whole cell recordings revealed that T-type currents, measured in 10 mM Ba2+ as a charge carrier, were present in moderate d. (20 ± 2 pA/pF). In rats with CCI, T-type c.d. (30 ± 3 pA/pF) was significantly increased, but voltage- and time-dependent activation and inactivation kinetics were not significantly different from those in controls. CCI-induced neuropathy did not significantly change the pharmacol. sensitivity of T-type current in these cells to nickel. Collectively, our results indicate that CCI-induced neuropathy significantly increases T-type current expression in small DRG neurons. Our findings that T-type currents are upregulated in a CCI model of peripheral neuropathy and earlier pharmacol. and mol. studies suggest that T-type channels may be potentially useful therapeutic targets for the treatment of neuropathic pain assocd. with partial mech. injury to the sciatic nerve.
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    18. 18
      Obradovic, A., Hwang, S. M., Scarpa, J., Hong, S. J., Todorovic, S. M., and Jevtovic-Todorovic, V. (2014) CaV3.2 T-Type Calcium Channels in Peripheral Sensory Neurons Are Important for Mibefradil-Induced Reversal of Hyperalgesia and Allodynia in Rats with Painful Diabetic Neuropathy PloS One 9, e91467
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      There is no corresponding record for this reference.
    19. 19
      Snutch, T. P. and David, L. S. (2006) T-type calcium channels: an emerging therapeutic target for the treatment of pain Drug Dev. Res. 67, 404– 415
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      There is no corresponding record for this reference.
    20. 20
      Todorovic, S. and Jevtovic-Todorovic, V. (2014) Targeting of CaV3.2 T-type calcium channels in peripheral sensory neurons for the treatment of painful diabetic neuropathy Pflügers Arch. 466, 701– 706
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      Targeting of CaV3.2 T-type calcium channels in peripheral sensory neurons for the treatment of painful diabetic neuropathy
      Todorovic Slobodan M; Jevtovic-Todorovic Vesna
      Pflugers Archiv : European journal of physiology (2014), 466 (4), 701-6 ISSN:.
      Pain-sensing sensory neurons (nociceptors) of the dorsal root ganglion (DRG) can become sensitized (hyperexcitable) in response to pathological conditions such as diabetes, which in turn may lead to the development of painful peripheral diabetic neuropathy (PDN). Because of insufficient knowledge about the mechanisms for this hypersensitization, current treatment for painful PDN has been limited to somewhat nonspecific systemic drugs having significant side effects or potential for abuse. Recent studies have established that the CaV3.2 isoform of T-channels makes a previously unrecognized contribution to sensitization of pain responses by enhancing excitability of nociceptors in animal models of type 1 and type 2 PDN. Furthermore, it has been reported that the glycosylation inhibitor neuraminidase can inhibit the native and recombinant CaV3.2 T-currents in vitro and completely reverse mechanical and thermal hyperalgesia in diabetic animals with PDN in vivo. Understanding details of posttranslational regulation of nociceptive channel activity via glycosylation may facilitate development of novel therapies for treatment of painful PDN. Pharmacological targeting the specific pathogenic mechanism rather than the channel per se may cause fewer side effects and reduce the potential for drug abuse in patients with diabetes.
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    21. 21
      Zamponi, G. W., Lewis, R. J., Todorovic, S. M., Arneric, S. P., and Snutch, T. P. (2009) Role of voltage-gated calcium channels in ascending pain pathways Brain Res. Rev. 60, 84– 89
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      21
      Role of voltage-gated calcium channels in ascending pain pathways
      Zamponi, Gerald W.; Lewis, Richard J.; Todorovic, Slobodan M.; Arneric, Stephen P.; Snutch, Terrance P.
      Brain Research Reviews (2009), 60 (1), 84-89CODEN: BRERD2; ISSN:0165-0173. (Elsevier B.V.)
      A review. Voltage gated calcium channels (VGCCs) are well established mediators of pain signals in primary afferent neurons. N-type calcium channels are localized to synaptic nerve terminals in laminae 1 and 2 of the dorsal horn where their opening results in the release of neurotransmitters such as glutamate and substance P. The contribution of N-type channels to the processing of pain signals is regulated by alternate splicing of the N-type channel gene, with unique N-type channel splice variants being expressed in small nociceptive neurons. In contrast, T-type VGCCs of the Cav3.2 subtype are likely localized to nerve endings where they regulate cellular excitability. Consequently, inhibition of N-type and Cav3.2 T-type VGCCs has the propensity to mediate analgesia. T-type channel activity is regulated by redox modulation, and can be inhibited by a novel class of small org. blockers. N-type VGCC activity can be potently inhibited by highly selective peptide toxins that are delivered intrathecally, and the search for small org. blockers with clin. efficacy is ongoing. Here, we provide a brief overview of recent advances in this area, as presented at the Spring Pain Research conference (Grand Cayman, 2008).
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    22. 22
      Perez-Reyes, E. (2003) Molecular physiology of low-voltage-activated t-type calcium channels Physiol. Rev. 83, 117– 161
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      22
      Molecular physiology of low-voltage-activated T-type calcium channels
      Perez-Reyes, Edward
      Physiological Reviews (2003), 83 (1), 117-161CODEN: PHREA7; ISSN:0031-9333. (American Physiological Society)
      A review. T-type Ca2+ channels were originally called low-voltage-activated (LVA) channels because they can be activated by small depolarizations of the plasma membrane. In many neurons Ca2+ influx through LVA channels triggers low-threshold spikes, which in turn triggers a burst of action potentials mediated by Na+ channels. Burst firing is thought to play an important role in the synchronized activity of the thalamus obsd. in absence epilepsy, but may also underlie a wider range of thalamocortical dysrhythmias. In addn. to a pacemaker role, Ca2+ entry via T-type channels can directly regulate intracellular Ca2+ concns., which is an important second messenger for a variety of cellular processes. Mol. cloning revealed the existence of three T-type channel genes. The deduced amino acid sequence shows a similar four-repeat structure to that found in high-voltage-activated (HVA) Ca2+ channels, and Na+ channels, indicating that they are evolutionarily related. Hence, the α1-subunits of T-type channels are now designated Cav3. Although mRNAs for all three Cav3 subtypes are expressed in brain, they vary in terms of their peripheral expression, with Cav3.2 showing the widest expression. The electrophysiol. activities of recombinant Cav3 channels are very similar to native T-type currents and can be differentiated from HVA channels by their activation at lower voltages, faster inactivation, slower deactivation, and smaller conductance of Ba2+. The Cav3 subtypes can be differentiated by their kinetics and sensitivity to block by Ni2+. The goal of this review is to provide a comprehensive description of T-type currents, their distribution, regulation, pharmacol., and cloning.
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    23. 23
      Barbara, G., Alloui, A., Nargeot, J., Lory, P., Eschalier, A., Bourinet, E., and Chemin, J. (2009) T-type calcium channel inhibition underlies the analgesic effects of the endogenous lipoamino acids J. Neurosci. 29, 13106– 13114
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      23
      T-type calcium channel inhibition underlies the analgesic effects of the endogenous lipoamino acids
      Barbara, Guillaume; Alloui, Abdelkrim; Nargeot, Joel; Lory, Philippe; Eschalier, Alain; Bourinet, Emmanuel; Chemin, Jean
      Journal of Neuroscience (2009), 29 (42), 13106-13114CODEN: JNRSDS; ISSN:0270-6474. (Society for Neuroscience)
      Lipoamino acids are anandamide-related endogenous mols. that induce analgesia via unresolved mechanisms. Here, we provide evidence that the T-type/Cav3 calcium channels are important pharmacol. targets underlying their physiol. effects. Various lipoamino acids, including N-arachidonoyl glycine (NAGly), reversibly inhibited Cav3.1, Cav3.2, and Cav3.3 currents, with potent effects on Cav3.2 [EC50 ∼200 nM for N-arachidonoyl 3-OH-γ-aminobutyric acid (NAGABA-OH)]. This inhibition involved a large shift in the Cav3.2 steady-state inactivation and persisted during fatty acid amide hydrolase (FAAH) inhibition as well as in cell-free outside-out patch. In contrast, lipoamino acids had weak effects on high-voltage-activated (HVA) Cav1.2 and Cav2.2 calcium currents, on Nav1.7 and Nav1.8 sodium currents, and on anandamide-sensitive TRPV1 and TASK1 currents. Accordingly, lipoamino acids strongly inhibited native Cav3.2 currents in sensory neurons with small effects on sodium and HVA calcium currents. In addn., we demonstrate here that lipoamino acids NAGly and NAGABA-OH produced a strong thermal analgesia and that these effects (but not those of morphine) were abolished in Cav3.2 knock-out mice. Collectively, our data revealed lipoamino acids as a family of endogenous T-type channel inhibitors, suggesting that these ligands can modulate multiple cell functions via this newly evidenced regulation.
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    24. 24
      Bladen, C., Gunduz, M. G., Simsek, R., Safak, C., and Zamponi, G. W. (2013) Synthesis and Evaluation of 1,4-Dihydropyridine Derivatives with Calcium Channel Blocking Activity Pflügers Arch. 466, 1355– 1363
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    25. 25
      Chemin, J., Monteil, A., Perez-Reyes, E., Nargeot, J., and Lory, P. (2001) Direct inhibition of T-type calcium channels by the endogenous cannabinoid anandamide EMBO J. 20, 7033– 7040
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      There is no corresponding record for this reference.
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      Choe, Y. J., Seo, H. N., Jung, S. Y., Rhim, H., Kim, J., Choo, D. J., and Lee, J. Y. (2008) Synthesis and SAR study of T-type calcium channel blockers. Part II Arch. Pharm. 341, 661– 664
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      26
      Synthesis and SAR study of T-type calcium channel blockers. Part II
      Choe, Yun Jeong; Seo, Han Na; Jung, Soo Yeon; Rhim, Hyewhon; Kim, Jungahn; Choo, Dong Joon; Lee, Jae Yeol
      Archiv der Pharmazie (Weinheim, Germany) (2008), 341 (10), 661-664CODEN: ARPMAS; ISSN:0365-6233. (Wiley-VCH Verlag GmbH & Co. KGaA)
      3,4-Dihydroquinazoline derivs. have been known to be novel and potent T-type calcium channel blockers. From a systematic variation of 3,4-dihydroquinazoline deriv. I [(KYS05043), R1 = 4-PhC6H4NH, R2 = (CH2)5NH2, R3 = OMe], plausible SAR results were established. It was revealed that a 5-(dimethylamino)pentylamino group at R1, a biphenyl group at R2, and a benzyl amido group at R3 in the 3,4-dihydroquinazoline backbone are closely related with the channel selectivity (T/N-type) as well as the potency based on the discovery of I [(KYS05090), R1 = NMe(CH2)5NMe2, R2 = 4-PhC6H4, R3 = NHBn].
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    27. 27
      Furukawa, T., Miura, R., Honda, M., Kamiya, N., Mori, Y., Takeshita, S., Isshiki, T., and Nukada, T. (2004) Identification of R(−)-isomer of efonidipine as a selective blocker of T-type Ca2+ channels Br. J. Pharmacol. 143, 1050– 1057
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      27
      Identification of R(-)-isomer of efonidipine as a selective blocker of T-type Ca2+ channels
      Furukawa, Taiji; Miura, Reiko; Honda, Mitsuyoshi; Kamiya, Natsuko; Mori, Yasuo; Takeshita, Satoshi; Isshiki, Takaaki; Nukada, Toshihide
      British Journal of Pharmacology (2004), 143 (8), 1050-1057CODEN: BJPCBM; ISSN:0007-1188. (Nature Publishing Group)
      1 Efonidipine, a deriv. of dihydropyridine Ca2+ antagonist, is known to block both L- and T-type Ca2+ channels. It remains to be clarified, however, whether efonidipine affects other voltage-dependent Ca2+ channel subtypes such as N-, P/Q- and R-types, and whether the optical isomers of efonidipine have different selectivities in blocking these Ca2+ channels, including L- and T-types. 2 To address these issues, the effects of efonidipine and its R(-)- and S(+)-isomers on these Ca2+ channel subtypes were examd. electrophysiol. in the expression systems using Xenopus oocytes and baby hamster kidney cells (BHK tk-ts13). 3 Efonidipine, a mixt. of R(-)- and S(+)-isomers, exerted blocking actions on L- and T-types, but no effects on N-, P/Q- and R-type Ca2+ channels. 4 The selective blocking actions on L- and T-type channels were reproduced by the S(+)-efonidipine isomer. 5 By contrast, the R(-)-efonidipine isomer preferentially blocked T-type channels. 6 The blocking actions of efonidipine and its enantiomers were dependent on holding potentials. 7 These findings indicate that the R(-)-isomer of efonidipine is a specific blocker of the T-type Ca2+ channel.
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    28. 28
      Hildebrand, M. E., Smith, P. L., Bladen, C., Eduljee, C., Xie, J. Y., Chen, L., Fee-Maki, M., Doering, C. J., Mezeyova, J., Zhu, Y., Belardetti, F., Pajouhesh, H., Parker, D., Arneric, S. P., Parmar, M., Porreca, F., Tringham, E., Zamponi, G. W., and Snutch, T. P. (2011) A novel slow-inactivation-specific ion channel modulator attenuates neuropathic pain Pain 152, 833– 843
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      There is no corresponding record for this reference.
    29. 29
      Jo, M. N., Seo, H. J., Kim, Y., Seo, S. H., Rhim, H., Cho, Y. S., Cha, J. H., Koh, H. Y., Choo, H., and Pae, A. N. (2007) Novel T-type calcium channel blockers: dioxoquinazoline carboxamide derivatives Bioorg. Med. Chem. 15, 365– 373
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      29
      Novel T-type calcium channel blockers: Dioxoquinazoline carboxamide derivatives
      Jo, Mi Na; Seo, Hee Jeong; Kim, Yoonji; Seo, Seon Hee; Rhim, Hyewhon; Cho, Yong Seo; Cha, Joo Hwan; Koh, Hun Yeong; Choo, Hyunah; Pae, Ae Nim
      Bioorganic & Medicinal Chemistry (2007), 15 (1), 365-373CODEN: BMECEP; ISSN:0968-0896. (Elsevier Ltd.)
      T-type calcium channel is one of therapeutic targets for the treatment of cardiovascular diseases and neuropathic pains. Since the withdrawal of mibefradil, a T-type calcium channel blocker, there have been a lot of efforts to develop T-type calcium channel blockers. A small mol. library of dioxoquinazoline carboxamide derivs. contg. 155 compds. was designed, synthesized, and biol. evaluated for T-type calcium channel blocking activity. Among those compds. synthesized, I shows the most potent T-type calcium current blocking activity with an IC50 value of 1.52 μM, which is comparable to that of mibefradil.
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    30. 30
      Kumar, P. P., Stotz, S. C., Paramashivappa, R., Beedle, A. M., Zamponi, G. W., and Rao, A. S. (2002) Synthesis and evaluation of a new class of nifedipine analogs with T-type calcium channel blocking activity Mol. Pharmacol. 61, 649– 658
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      There is no corresponding record for this reference.
    31. 31
      Perez-Reyes, E., Van Deusen, A. L., and Vitko, I. (2009) Molecular pharmacology of human Cav3.2 T-type Ca2+ channels: block by antihypertensives, antiarrhythmics, and their analogs J. Pharmacol. Exp. Ther. 328, 621– 627
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      Molecular pharmacology of human Cav3.2 T-type Ca2+ channels: block by antihypertensives, antiarrhythmics, and their analogs
      Perez-Reyes, Edward; Van Deusen, Amy L.; Vitko, Iuliia
      Journal of Pharmacology and Experimental Therapeutics (2009), 328 (2), 621-627CODEN: JPETAB; ISSN:0022-3565. (American Society for Pharmacology and Experimental Therapeutics)
      Antihypertensive drugs of the "calcium channel blocker" or "calcium antagonist" class have been used to establish the physiol. role of L-type Ca2+ channels in vascular smooth muscle. In contrast, there has been limited progress on the pharmacol. T-type Ca2+ channels. T-type channels play a role in cardiac pacemaking, aldosterone secretion, and renal hemodynamics, leading to the hypothesis that mixed T- and L-type blockers may have therapeutic advantages over selective L-type blockers. The goal of this study was to identify compds. that block the Cav3.2 T-type channel with high affinity, focusing on two classes of compds.: phenylalkylamines (e.g., mibefradil) and dihydropyridines (e.g., efonidipine). Compds. were tested using a validated Ca2+ influx assay into a cell line expressing recombinant Cav3.2 channels. This study identified four clin. approved antihypertensive drugs (efonidipine, felodipine, isradipine, and nitrendipine) as potent T-channel blockers (IC50 < 3 μM). In contrast, other widely prescribed dihydropyridines, such as amlodipine and nifedipine, were 10-fold less potent, making them a more appropriate choice in research studies on the role of L-type currents. In summary, the present results support the notion that many available antihypertensive drugs block a substantial fraction of T-current at therapeutically relevant concns., contributing to their mechanism of action.
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    32. 32
      Yamamoto, E., Kataoka, K., Dong, Y. F., Nakamura, T., Fukuda, M., Nako, H., Ogawa, H., and Kim-Mitsuyama, S. (2010) Benidipine, a dihydropyridine L-type/T-type calcium channel blocker, affords additive benefits for prevention of cardiorenal injury in hypertensive rats J. Hypertens. 28, 1321– 1329
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      Benidipine, a dihydropyridine L-type/T-type calcium channel blocker, affords additive benefits for prevention of cardiorenal injury in hypertensive rats
      Yamamoto, Eiichiro; Kataoka, Keiichiro; Dong, Yi-Fei; Nakamura, Taishi; Fukuda, Masaya; Nako, Hisato; Ogawa, Hisao; Kim-Mitsuyama, Shokei
      Journal of Hypertension (2010), 28 (6), 1321-1329CODEN: JOHYD3; ISSN:0263-6352. (Lippincott Williams & Wilkins)
      Benidipine is a dihydropyridine calcium channel blocker inhibiting not only L-type but also T-type calcium channels. To elucidate potential additive benefit of benidipine for prevention of cardiorenal injury, we compared the cardiac and renal protective effects of equihypotensive doses of benidipine and cilnidipine in stroke-prone spontaneously hypertensive rats (SHRSP). SHRSP were divided into five groups, and were given vehicle, benidipine at 1 or 3 mg/kg per day, or cilnidipine at 1 or 3 mg/kg per day for 7 wk, and the protective effects against cardiorenal injury were compared among each group. Benidipine and cilnidipine at the same doses exerted comparable hypotensive effects on SHRSP throughout the treatment. Despite equihypotensive effects between both drugs, benidipine prevented cardiac hypertrophy, fibrosis, and inflammation to a greater extent than cilnidipine. Moreover, benidipine prevented glomerulosclerosis, tubulointerstitial injury, and renal inflammation more than cilnidipine. To elucidate the underlying mechanism of more beneficial effects of benidipine than cilnidipine, we compared the effects of these drugs on cardiac and renal oxidative stress, and aldosterone in SHRSP. Benidipine reduced both cardiac and renal NADPH oxidase activities in SHRSP more than cilnidipine, being assocd. with more attenuation of cardiac and renal superoxide by benidipine. Furthermore, serum aldosterone was significantly reduced by benidipine but not by cilnidipine. Benidipine exerted more protective effects against cardiorenal injury of hypertensive rats than cilnidipine, through more attenuation of oxidative stress than cilnidipine, and the redn. of aldosterone. Benidipine, via blockade of T-type calcium channels, seems to elicit additive benefits for prevention of hypertensive cardiorenal injury.
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    33. 33
      You, H., Altier, C., and Zamponi, G. W. (2010) CCR2 receptor ligands inhibit Cav3.2 T-type calcium channels Mol. Pharmacol. 77, 211– 217
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      There is no corresponding record for this reference.
    34. 34
      You, H., Gadotti, V. M., Petrov, R. R., Zamponi, G. W., and Diaz, P. (2011) Functional characterization and analgesic effects of mixed cannabinoid receptor/T-type channel ligands Mol. Pain 7, 89
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      Functional characterization and analgesic effects of mixed cannabinoid receptor/T-type channel ligands
      You, Haitao; Gadotti, Vinicius M.; Petrov, Ravil R.; Zamponi, Gerald W.; Diaz, Philippe
      Molecular Pain (2011), 7 (), 89CODEN: MPOAC5; ISSN:1744-8069. (BioMed Central Ltd.)
      Background: Both T-type calcium channels and cannabinoid receptors modulate signalling in the primary afferent pain pathway. Here, we investigate the analgesics activities of a series of novel cannabinoid receptor ligands with T-type calcium channel blocking activity. Results: Novel compds. were characterized in radioligand binding assays and in vitro functional assays at human and rat CB1 and CB2 receptors. The inhibitory effects of these compds. on transient expressed human T-type calcium channels were examd. in tsA-201 cells using std. whole-cell voltage clamp techniques and their analgesic effects in response to various administration routes (intrathecally, intraplantarly, i.p.) assessed in the formalin model. A series of compds. were synthesized and evaluated for channel and receptor activity. Compd. NMP-7 acted as non-selective CB1/CB2 agonist while NMP4 was found to be a CB1 partial agonist and CB2 inverse agonist. Furthermore, NMP-144 behaved as a selective CB2 inverse agonist. All of these three compds. completely inhibited peak Cav3.2 currents with IC50 values in the low micromolar range. All compds. mediated analgesic effects in the formalin model, but depending on the route of administration, could differentially affect phase 1 and phase 2 of the formalin response. Conclusions: Our results reveal that a set of novel cannabinioid receptor ligands potently inhibit T-type calcium channels and show analgesic effects in vivo. Our findings suggest possible novel means of mediating pain relief through mixed T-type/cannabinoid receptor ligands.
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    35. 35
      Chemin, J., Nargeot, J., and Lory, P. (2007) Chemical determinants involved in anandamide-induced inhibition of T-type calcium channels J. Biol. Chem. 282, 2314– 2323
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      There is no corresponding record for this reference.
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      Moreira, F. A., Grieb, M., and Lutz, B. (2009) Central side-effects of therapies based on CB1 cannabinoid receptor agonists and antagonists: focus on anxiety and depression Best. Pract. Res., Clin. Endocrinol. Metab. 23, 133– 144
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      Central side-effects of therapies based on CB1 cannabinoid receptor agonists and antagonists: focus on anxiety and depression
      Moreira, Fabricio A.; Grieb, Maximilian; Lutz, Beat
      Best Practice & Research, Clinical Endocrinology & Metabolism (2009), 23 (1), 133-144CODEN: BPRCE9 ISSN:. (Elsevier Ltd.)
      A review. Both agonists (e.g. Δ9-tetrahydrocannabinol, nabilone) and antagonists (e.g. rimonabant, taranabant) of the cannabinoid type-1 (CB1) receptor have been explored as therapeutic agents in diverse fields of medicine such as pain management and obesity with assocd. metabolic dysregulation, resp. CB1 receptors are widely distributed in the central nervous system and are involved in the modulation of emotion, stress and habituation responses, behaviors that are thought to be dysregulated in human psychiatric disorders. Accordingly, CB1 receptor activation may, in some cases, ppt. episodes of psychosis and panic, while its inhibition may lead to behaviors reminiscent of depression and anxiety-related disorders. The present review discusses these side-effects, which have to be taken into account in the therapeutic exploitation of the endocannabinoid system.
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    37. 37
      Witkin, J. M., Tzavara, E. T., and Nomikos, G. G. (2005) A role for cannabinoid CB1 receptors in mood and anxiety disorders Behav. Pharmacol. 16, 315– 331
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      Petrov, R. R., Knight, L., Chen, S. R., Wager-Miller, J., McDaniel, S. W., Diaz, F., Barth, F., Pan, H. L., Mackie, K., Cavasotto, C. N., and Diaz, P. (2013) Mastering tricyclic ring systems for desirable functional cannabinoid activity Eur. J. Med. Chem. 69, 881– 907
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    39. 39
      Chen, X. L., Bayliss, D. A., Fern, R. J., and Barrett, P. Q. (1999) A role for T-type Ca2+ channels in the synergistic control of aldosterone production by ANG II and K+ Am. J. Physiol. 276, F674– 683
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    40. 40
      Marger, F., Gelot, A., Alloui, A., Matricon, J., Ferrer, J. F., Barrere, C., Pizzoccaro, A., Muller, E., Nargeot, J., Snutch, T. P., Eschalier, A., Bourinet, E., and Ardid, D. (2011) T-type calcium channels contribute to colonic hypersensitivity in a rat model of irritable bowel syndrome Proc. Natl. Acad. Sci. U. S. A. 108, 11268– 11273
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    41. 41
      Moriguchi, S., Shioda, N., Yamamoto, Y., Tagashira, H., and Fukunaga, K. (2012) The T-type voltage-gated calcium channel as a molecular target of the novel cognitive enhancer ST101: Enhancement of long-term potentiation and CaMKII autophosphorylation in rat cortical slices J. Neurochem. 121, 44– 53
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      41
      The T-type voltage-gated calcium channel as a molecular target of the novel cognitive enhancer ST101: enhancement of long-term potentiation and CaMKII autophosphorylation in rat cortical slices
      Moriguchi, Shigeki; Shioda, Norifumi; Yamamoto, Yui; Tagashira, Hideaki; Fukunaga, Kohji
      Journal of Neurochemistry (2012), 121 (1 & 2), 44-53CODEN: JONRA9; ISSN:0022-3042. (Wiley-Blackwell)
      In this study, we report that spiro[imidazo[1,2-a]pyridine-3,2-indan]-2(3H)-one (ST101; previously coded as ZSET1446) targets T-type voltage-gated calcium channels in mediating improved cognition in the CNS. We prepd. rat somatosensory cortical and hippocampal slices, treated them with 0.01 to 100 nM ST101, and performed immunoblotting and electrophysiol. analyses using various voltage-gated calcium channel (VGCC) inhibitors. Treatment of rat cortical slices with a range of ST101 concns. significantly increased calcium/calmodulin-dependent protein kinase II (CaMKII) autophosphorylation following a bell-shaped dose-response curve, with 0.1 nM ST101 representing the maximally effective concn. protein kinase Cα autophosphorylation was also significantly increased by 0.1 nM ST101 treatment. ST101 treatment had a moderate effect on CaMKII autophosphorylation but no effect on hippocampal protein kinase Cα autophosphorylation in slice prepns. Consistent with increased cortical CaMKII autophosphorylation, AMPA-type glutamate receptor subunit 1 (Ser-831) phosphorylation as a CaMKII post-synaptic substrate was significantly increased by treatment with 0.1-1 nM ST101, whereas phosphorylation of the pre-synaptic substrate synapsin I (Ser-603) remained unchanged. Notably, enhanced CaMKII autophosphorylation seen following 0.1 nM ST101 treatment was significantly inhibited by pre-treatment with 1 μM mibefradil, a T-type VGCC inhibitor, but not with N-type (ω-conotoxin), P/Q-type (ω-agatoxin) or L-type (nifedipine) VGCC inhibitors. Similarly, 0.1 nM ST101 significantly potentiated long-term potentiation in cortical but not hippocampal slices. Enhanced long-term potentiation in cortical slices was totally inhibited by 1 μM mibefadil treatment. Finally, whole-cell patch-clamp anal. of Neuro2A cells over-expressing recombinant human CaV3.1 (α1G) T-channels and treated with 0.1 nM ST101 showed significant increases in T-type VGCC currents. These results indicate that T-type VGCCs are direct mol. targets for the novel cognitive enhancer ST101, a potential Alzheimer disease therapeutic.
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    42. 42
      Jevtovic-Todorovic, V. and Todorovic, S. M. (2006) The role of peripheral T-type calcium channels in pain transmission Cell Calcium 40, 197– 203
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      Orestes, P., Osuru, H. P., McIntire, W. E., Jacus, M. O., Salajegheh, R., Jagodic, M. M., Choe, W., Lee, J., Lee, S. S., Rose, K. E., Poiro, N., Digruccio, M. R., Krishnan, K., Covey, D. F., Lee, J. H., Barrett, P. Q., Jevtovic-Todorovic, V., and Todorovic, S. M. (2013) Reversal of neuropathic pain in diabetes by targeting glycosylation of Ca(V)3.2 T-type calcium channels Diabetes 62, 3828– 3838
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      Todorovic, S. M. and Jevtovic-Todorovic, V. (2011) T-type voltage-gated calcium channels as targets for the development of novel pain therapies Br. J. Pharmacol. 163, 484– 495
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      44
      T-type voltage-gated calcium channels as targets for the development of novel pain therapies
      Todorovic, Slobodan M.; Jevtovic-Todorovic, Vesna
      British Journal of Pharmacology (2011), 163 (3), 484-495CODEN: BJPCBM; ISSN:1476-5381. (Wiley-Blackwell)
      A review. It is well recognized that voltage-gated calcium (Ca2+) channels modulate the function of peripheral and central pain pathways by influencing fast synaptic transmission and neuronal excitability. In the past, attention focused on the modulation of different subtypes of high-voltage-activated-type Ca2+ channels; more recently, the function of low-voltage-activated or transient (T)-type Ca2+ channels (T-channels) in nociception has been well documented. Currently, available pain therapies remain insufficient for certain forms of pain assocd. with chronic disorders (e.g. neuropathic pain) and often have serious side effects. Hence, the identification of selective and potent inhibitors and modulators of neuronal T-channels may help greatly in the development of safer, more effective pain therapies. Here, the authors summarize the available information implicating peripheral and central T-channels in nociception. The authors also discuss possible future developments aimed at selective modulation of function of these channels, which are highly expressed in nociceptors.
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    45. 45
      Mullins, M. E., Horowitz, B. Z., Linden, D. H., Smith, G. W., Norton, R. L., and Stump, J. (1998) Life-threatening interaction of mibefradil and beta-blockers with dihydropyridine calcium channel blockers JAMA, J. Am. Med. Assoc. 280, 157– 158
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      Bourinet, E., Altier, C., Hildebrand, M. E., Trang, T., Salter, M. W., and Zamponi, G. W. (2014) Calcium-permeable ion channels in pain signaling Physiol. Rev. 94, 81– 140
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      Nelson, M. T., Joksovic, P. M., Perez-Reyes, E., and Todorovic, S. M. (2005) The endogenous redox agent L-cysteine induces T-type Ca2+ channel-dependent sensitization of a novel subpopulation of rat peripheral nociceptors J. Neurosci. 25, 8766– 8775
      Google Scholar
      47
      The endogenous redox agent L-cysteine induces T-type Ca2+ channel-dependent sensitization of a novel subpopulation of rat peripheral nociceptors
      Nelson, Michael T.; Joksovic, Pavle M.; Perez-Reyes, Edward; Todorovic, Slobodan M.
      Journal of Neuroscience (2005), 25 (38), 8766-8775CODEN: JNRSDS; ISSN:0270-6474. (Society for Neuroscience)
      Recent studies have demonstrated a previously unrecognized contribution of T-type Ca2+ channels in peripheral sensory neurons to pain sensation (nociception). However, the cellular mechanisms underlying the functions of these channels in nociception are not known. Here, in both acutely dissocd. and intact rat dorsal root ganglion neurons, we characterize a novel subpopulation of capsaicin- and isolectin B4-pos. nociceptors that also expresses a high d. of T-type Ca2+ currents. Using these "T-rich" cells as a model, we demonstrate that the endogenous reducing agent L-cysteine lowers the threshold for nociceptor excitability and induces burst firing by increasing the amplitude of T-type currents and shifting the gating parameters of T-type channels. These findings, which provide the first direct evidence of T-type Ca2+ channel involvement in the control of nociceptor excitability, suggest that endogenous T-type channel agonists may sensitize a unique subpopulation of peripheral nociceptors, consequently influencing pain processing under normal or pathol. conditions.
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    48. 48
      Francois, A., Kerckhove, N., Meleine, M., Alloui, A., Barrere, C., Gelot, A., Uebele, V. N., Renger, J. J., Eschalier, A., Ardid, D., and Bourinet, E. (2013) State-dependent properties of a new T-type calcium channel blocker enhance Ca(V)3.2 selectivity and support analgesic effects Pain 154, 283– 293
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      Simms, B. A. and Zamponi, G. W. (2014) Neuronal Voltage-Gated Calcium Channels: Structure, Function, and Dysfunction Neuron 82, 24– 45
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      49
      Neuronal Voltage-Gated Calcium Channels: Structure, Function, and Dysfunction
      Simms, Brett A.; Zamponi, Gerald W.
      Neuron (2014), 82 (1), 24-45CODEN: NERNET; ISSN:0896-6273. (Cell Press)
      A review. Voltage-gated calcium channels are the primary mediators of depolarization-induced calcium entry into neurons. There is great diversity of calcium channel subtypes due to multiple genes that encode calcium channel α1 subunits, coassembly with a variety of ancillary calcium channel subunits, and alternative splicing. This allows these channels to fulfill highly specialized roles in specific neuronal subtypes and at particular subcellular loci. While calcium channels are of crit. importance to brain function, their inappropriate expression or dysfunction gives rise to a variety of neurol. disorders, including, pain, epilepsy, migraine, and ataxia. This Review discusses salient aspects of voltage-gated calcium channel function, physiol., and pathophysiol.
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    50. 50
      Armbruster, B. N. and Roth, B. L. (2005) Mining the receptorome J. Biol. Chem. 280, 5129– 5132
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      Jensen, N. H. and Roth, B. L. (2008) Massively parallel screening of the receptorome Comb. Chem. High Throughput Screening 11, 420– 426
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      51
      Massively parallel screening of the receptorome
      Jensen, Niels H.; Roth, Bryan L.
      Combinatorial Chemistry & High Throughput Screening (2008), 11 (6), 420-426CODEN: CCHSFU; ISSN:1386-2073. (Bentham Science Publishers Ltd.)
      A review. The National Institute of Mental Health (NIMH) Psychoactive Drug Screening Program (PDSP) is a resource that provides free screening of novel compds. to academic investigators. This program differs from other public-sector screening programs in that compds. are screened against a large panel of transmembrane receptors, channels, and transporters, a selection that currently includes a large portion of the whole neuro-receptorome. This review discusses the research areas that can profit from this resource, exemplified by recent findings. The 1st area is the identification of side effects of medications. Examples include the identification of the histamine H1 receptor as being responsible for wt. gain under antipsychotic treatment and the assocn. of 5-HT2B receptor agonism with cardiac valvulopathy, which led to the removal of several medications. A 2nd area is the identification of mechanisms of actions of medications and natural products. Examples are the finding that the kappa opioid receptor is the pharmacol. target of the potent hallucinogen salvinorin A, that ephedrine and related compds. are not acting through direct sympathomimetic action, the identification of a strong dopaminergic action of WAY-100635, a compd. that had been used as a selective 5-HT1A antagonist, and the discovery that the metabolite desmethylclozapine activates M1 muscarinic receptors, an activity that might contribute to the clin. efficacy of the antipsychotic drug clozapine. A 3rd, relatively new area is the identification of inert compds. as agonists for engineered designer receptors that no longer respond to their natural ligand (DREADDs) but exhibit unchanged signaling properties.
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    52. 52
      Strachan, R. T., Ferrara, G., and Roth, B. L. (2006) Screening the receptorome: an efficient approach for drug discovery and target validation Drug Discovery Today 11, 708– 716
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      Feng, Z. P., Doering, C. J., Winkfein, R. J., Beedle, A. M., Spafford, J. D., and Zamponi, G. W. (2003) Determinants of inhibition of transiently expressed voltage-gated calcium channels by omega-conotoxins GVIA and MVIIA J. Biol. Chem. 278, 20171– 20178
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      53
      Determinants of Inhibition of Transiently Expressed Voltage-gated Calcium Channels by ω-Conotoxins GVIA and MVIIA
      Feng, Zhong-Ping; Doering, Clinton J.; Winkfein, Robert J.; Beedle, Aaron M.; Spafford, J. David; Zamponi, Gerald W.
      Journal of Biological Chemistry (2003), 278 (22), 20171-20178CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)
      The Conus magus peptide toxin ω-conotoxin MVIIA is considered an irreversible, specific blocker of N-type calcium channels, and is now in clin. trials as an intrathecal analgesic. Here, we have examd. the action of MVIIA on mutant and wild type calcium channels transiently expressed in tsA-201 cells. Although we have shown previously that mutations in a putative external EF-hand motif in the domain IIIS5-H5 region alters block by both ω-conotoxin GVIA and MVIIA (Feng, Z. P., et al., 2001), the introduction of five point mutations known to affect GVIA blocking (and located downstream of the EF-hand) affected MVIIA block to a smaller degree compared with GVIA. These data suggest that despite some overlap, MVIIA and GVIA block does not share identical channel structural determinants. At higher concns. (∼3 μM), MVIIA reversibly blocked L-, P/Q-, and R-type, but not T-type channels, indicating that the overall architecture of the MVIIA site is conserved in all types of high voltage-activated calcium channels. A kinetic anal. of the MVIIA effects on the N-type channel showed that MVIIA blocked resting, open, and inactivated channels. Although the development of MVIIA block did not appear to be voltage-, nor frequency-dependent, the degree of recovery from block strongly depended on the potential applied during washout. Interestingly, the degree of washout was highly variable and appeared to weakly depend on the holding potential applied during toxin application. We propose a model in which N-type calcium channels can form both reversible and irreversible complexes with MVIIA.
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    54. 54
      Hylden, J. L. and Wilcox, G. L. (1980) Intrathecal morphine in mice: A new technique Eur. J. Pharmacol. 67, 313– 316
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      54
      Intrathecal morphine in mice: a new technique
      Hylden, Janice L. K.; Wilcox, George L.
      European Journal of Pharmacology (1980), 67 (2-3), 313-16CODEN: EJPHAZ; ISSN:0014-2999.
      A simple, rapid technique for intrathecal injection by lumbar puncture in unanesthetized mice is described. Intrathecal 3H-labeled morphine sulfate (I sulfate) [64-31-3] was not found in significant quantities in either the midbrain or forebrain. Submicrogram quantities of morphine sulfate induced Straub tail response and tail-flick analgesia. These effects were dose related and antagonized by s.c. naloxone.
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    55. 55
      Gadotti, V. M. and Zamponi, G. W. (2011) Cellular prion protein protects from inflammatory and neuropathic pain Mol. Pain 7, 59
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      55
      Cellular prion protein protects from inflammatory and neuropathic pain
      Gadotti, Vinicius M.; Zamponi, Gerald W.
      Molecular Pain (2011), 7 (), 59CODEN: MPOAC5; ISSN:1744-8069. (BioMed Central Ltd.)
      Cellular prion protein (PrPC) inhibits N-Methyl--Aspartate (NMDA) receptors. Since NMDA receptors play an important role in the transmission of pain signals in the dorsal horn of spinal cord, we thus wanted to det. if PrPC null mice show a reduced threshold for various pain behaviors. We compared nociceptive thresholds between wild type and PrPC null mice in models of inflammatory and neuropathic pain, in the presence and the absence of a NMDA receptor antagonist. 2-3 Mo old male PrPC null mice exhibited an MK-801 sensitive decrease in the paw withdrawal threshold in response both mech. and thermal stimuli. PrPC null mice also exhibited significantly longer licking/biting time during both the first and second phases of formalin-induced inflammation of the paw, which was again prevented by treatment of the mice with MK-801, and responded more strongly to glutamate injection into the paw. Compared to wild type animals, PrPC null mice also exhibited a significantly greater nociceptive response (licking/biting) after intrathecal injection of NMDA. Sciatic nerve ligation resulted in MK-801 sensitive neuropathic pain in wild-type mice, but did not further augment the basal increase in pain behavior obsd. in the null mice, suggesting that mice lacking PrPC may already be in a state of tonic central sensitization. Altogether, our data indicate that PrPC exerts a crit. role in modulating nociceptive transmission at the spinal cord level, and fit with the concept of NMDA receptor hyperfunction in the absence of PrPC.
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    56. 56
      Kaster, M. P., Gadotti, V. M., Calixto, J. B., Santos, A. R., and Rodrigues, A. L. (2012) Depressive-like behavior induced by tumor necrosis factor-alpha in mice Neuropharmacology 62, 419– 426
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      Malmberg, A. B. and Basbaum, A. I. (1998) Partial sciatic nerve injury in the mouse as a model of neuropathic pain: Behavioral and neuroanatomical correlates Pain 76, 215– 222
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      57
      Partial sciatic nerve injury in the mouse as a model of neuropathic pain: behavioral and neuroanatomical correlates
      Malmberg A B; Basbaum A I
      Pain (1998), 76 (1-2), 215-22 ISSN:0304-3959.
      The generation of knock-out and transgenic mice offers a promising approach to the identification of novel biochemical factors that contribute to persistent pain conditions. To take advantage of these mice, however, it is important to demonstrate that the traditional models of persistent pain, which were largely developed for studies in the rat, can be used in the mouse. Here, we combined behavioral and anatomical methods to characterize the pathophysiology of a partial nerve injury-evoked pain condition in the 'normal' mouse. In male C57BL6 mice we tied a tight ligature around 1/3 to 1/2 of the diameter of the sciatic nerve and evaluated the time-course and magnitude of the ensuing mechanical and thermal allodynia. We also used immunocytochemistry to analyze nerve injury-induced changes in substance P (SP) and NK-1 (SP) receptor expression in the spinal cord. As in the rat, partial nerve injury markedly decreased paw withdrawal thresholds to both mechanical and thermal stimuli on the injured side. We detected threshold changes one day after the injury. The thermal allodynia resolved by 49 days, but the mechanical allodynia persisted for the duration of the study (70 days). We found no changes contralateral to the nerve injury. Sympatholytic treatment with guanethidine significantly reduced both the thermal and mechanical allodynia. We observed a reduction of SP immunoreactivity in the superficial dorsal horn on the injured side at 7 and 14, but not at 3 or 70 days after the nerve injury, and we observed an increase of NK-1 receptor expression at 3, 7, 14 and 42, but not at 70 days after the injury. We conclude that partial injury to the sciatic nerve produces a comparable allodynia and neurochemical plasticity in the rat and mouse. These results establish a valuable model for future studies of the biochemical basis of neuropathic pain in mice with specific gene modifications.
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