Inhibition Of T-type Calcium Channels Research Articles

606 Small molecule blockade of T-type calcium channels by DX416 inhibits itch and reduces corresponding inflammation in acute and chronic itch mouse models

The processing of itch signals is regulated by many different ion channels. Among those are T-type calcium channels which are important regulators that contribute to the firing behavior of pruriceptors, or itch sensing neurons. The human genome encodes three different T-type channels: Cav3.1, Cav3.2, Cav3.3. It has been shown recently that pharmacological blockade of T-type calcium channels inhibits acute itch and that immune cells can express Cav 3 channels with roles in effector function. Thus, we hypothesized that local administration of the novel small molecule T-Type calcium channel inhibitor, DX416, would decrease cutaneous itch both through reducing pruriceptors signals triggering itch and decreasing local inflammatory immune responses potentiating itch.

T-type calcium channel inhibition restores sensitivity to MAPK inhibitors in de-differentiated and adaptive melanoma cells

BackgroundDrug resistance remains as one of the major challenges in melanoma therapy. It is well known that tumour cells undergo phenotypic switching during melanoma progression, increasing melanoma plasticity and resistance to mitogen-activated protein kinase inhibitors (MAPKi).MethodsWe investigated the melanoma phenotype switching using a partial reprogramming model to de-differentiate murine melanoma cells and target melanoma therapy adaptation against MAPKi.ResultsHere, we show that partially reprogrammed cells are a less proliferative and more de-differentiated cell population, expressing a gene signature for stemness and suppressing melanocyte-specific markers. To investigate adaptation to MAPKi, cells were exposed to B-Raf Proto-Oncogene (BRAF) and mitogen-activated protein kinase kinase (MEK) inhibitors. De-differentiated cells became less sensitive to MAPKi, showed increased cell viability and decreased apoptosis. Furthermore, T-type calcium channels expression increased in adaptive murine cells and in human adaptive melanoma cells. Treatment with the calcium channel blocker mibefradil induced cell death, differentiation and susceptibility to MAPKi in vitro and in vivo.ConclusionIn summary, we show that partial reprogramming of melanoma cells induces de-differentiation and adaptation to MAPKi. Moreover, we postulated a calcium channel blocker such as mibefradil, as a potential candidate to restore sensitivity to MAPKi in adaptive melanoma cells.

Ethosuximide improves chronic pain-induced anxiety- and depression-like behaviors

Chronic pain is a heavy burden disease. Current treatments are generally weakly effective or associated with adverse effects. New therapeutic approaches are therefore needed. Recent studies have suggested T-type calcium channels as an attractive target for the treatment of chronic pain. In this perspective, it was decided to perform a preclinical evaluation of the efficacy of ethosuximide, a T-type channel blocker used clinically as an antiepileptic, as a novel pharmacological treatment for chronic pain. Assessment of the effect of ethosuximide was thus made in both nociception and pain-related comorbidities as anxiety and depression are frequently encountered in chronic pain patients. Our results show that such symptoms occurred in three animal models of chronic pain designed to reflect traumatic neuropathic, chemotherapy-induced neuropathic and inflammatory pain conditions. Administration of ethosuximide reduced both chronic pain and comorbidities with a marked intensity ranging from partial reduction to a complete suppression of symptoms. These results make ethosuximide, and more broadly the inhibition of T-type calcium channels, a new strategy for the management of uncontrolled chronic pain, likely to improve not only pain but also the accompanying anxiety and depression.

Scholarinine A, a N3 type caged-monoterpene indole alkaloid as Cav3.1 T-type calcium channel inhibitor from Alstonia scholaris

Scholarinine A (1), a new N3-type caged monoterpene indole alkaloid was isolated from the branches and leaves of Alstonia scholaris. Structurally, 1 is featured by unique 6/5/6/5/5/6 carbocyclic skeleton bearing a novel imidazolidine ring E. The structure of 1 was determined by extensive spectroscopic analyses and X-ray crystallography. Biological studies exhibited that compound 1 is a potent Cav3.1 T-type calcium channel inhibitor with an IC50 value of 4.28 μM. In addition, 1 enhanced the neurite outgrowth of nerve growth factor (NGF) mediated PC12 cells at 10 μM.

Hyphenone A, the first 3,3-diisoprenylated bicyclic polyprenylated acylphloroglucinols as Cav3.1 T-type calcium channel inhibitor from Hypericum henryi

Hyphenone A (1), a new type of bicyclic polyprenylated acylphloroglucinols (BPAPs) featured with an unprecedented 3,3-diisoprenylated 1,3,5-trione core, were characterized from the roots of Hypericum henryi together with two new congeners (hyphenones B–C, 2–3) and five known biosynthetic related PPAPs. Biogenetically, 1 should be derived from a novel 3,3,5,5-tetraisoprenylated MPAPs precursor. Their structures were elucidated by comprehensive spectroscopic data and X-ray diffraction. Moreover, 1–3 were identified as the first PPAPs type Cav3.1 T-type calcium channel (TTCC) inhibitors, with IC50 values of 7.07, 6.19, and 5.47 μM, respectively.

A potential role for T-type calcium channels in homocysteinemia-induced peripheral neuropathy.

Homocysteinemia is a metabolic condition characterized by abnormally high level of homocysteine in the blood and is considered to be a risk factor for peripheral neuropathy. However, the cellular mechanisms underlying toxic effects of homocysteine on the processing of peripheral nociception have not yet been investigated comprehensively. Here, using a rodent model of experimental homocysteinemia, we report the causal association between homocysteine and the development of mechanical allodynia. Homocysteinemia-induced mechanical allodynia was reversed on pharmacological inhibition of T-type calcium channels. In addition, our in vitro studies indicate that homocysteine enhances recombinant T-type calcium currents by promoting the recycling of Cav3.2 channels back to the plasma membrane through a protein kinase C-dependent signaling pathway that requires the direct phosphorylation of Cav3.2 at specific loci. Altogether, these results reveal an unrecognized signaling pathway that modulates the expression of T-type calcium channels, and may potentially contribute to the development of peripheral neuropathy associated with homocysteinemia.

Rapid-acting antidepressant ketamine, its metabolites and other candidates: A historical overview and future perspective.

Major depressive disorder (MDD) is one of the most disabling psychiatric disorders. Approximately one‐third of the patients with MDD are treatment resistant to the current antidepressants. There is also a significant therapeutic time lag of weeks to months. Furthermore, depression in patients with bipolar disorder (BD) is typically poorly responsive to antidepressants. Therefore, there exists an unmet medical need for rapidly acting antidepressants with beneficial effects in treatment‐resistant patients with MDD or BD. Accumulating evidence suggests that the N‐methyl‐D‐aspartate receptor (NMDAR) antagonist ketamine produces rapid and sustained antidepressant effects in treatment‐resistant patients with MDD or BD. Ketamine is a racemic mixture comprising equal parts of (R)‐ketamine (or arketamine) and (S)‐ketamine (or esketamine). Because (S)‐ketamine has higher affinity for NMDAR than (R)‐ketamine, esketamine was developed as an antidepressant. On 5 March 2019, esketamine nasal spray was approved by the US Food and Drug Administration. However, preclinical data suggest that (R)‐ketamine exerts greater potency and longer‐lasting antidepressant effects than (S)‐ketamine in animal models of depression and that (R)‐ketamine has less detrimental side‐effects than (R,S)‐ketamine or (S)‐ketamine. In this article, the author reviews the historical overview of the antidepressant actions of enantiomers of ketamine and its major metabolites norketamine and hydroxynorketamine. Furthermore, the author discusses the other potential rapid‐acting antidepressant candidates (i.e., NMDAR antagonists and modulators, low‐voltage‐sensitive T‐type calcium channel inhibitor, potassium channel Kir4.1 inhibitor, negative modulators of γ‐aminobutyric acid, and type A [GABAA] receptors) to compare them with ketamine. Moreover, the molecular and cellular mechanisms of ketamine’s antidepressant effects are discussed.

SYNTHESIS OF 1,3,4-OXADIAZOLES AS SELECTIVE T-TYPE CALCIUM CHANNEL INHIBITORS.

Neuropathic pain, epilepsy, insomnia, and tremor disorder may arrive from an increase of intracellular Ca2+ concentration through a dysfunction of T-type Ca2+ channels. Thus, T-type calcium channels could be a target in drug discovery for the treatments of neuropathic pain and epilepsy. From rational drug design approach, a group of 2,5-disubstituted 1,3,4-oxadiazole molecules was synthesized and their selective T-type channel inhibitions were evaluated. The synthetic strategy consists of a short sequence of three reactions: (i) condensation of thiosemicarbazide with acid chlorides; (ii) ring closing by 1,3-dibromo-5,5- dimethylhydantoin; and (iii) coupling with various acid chlorides. 5-Chloro-N-(5- phenyl-1,3,4-oxadiazol-2-yl)thiophene-2-carboxamide (11) was found to selectively inhibit T-type Ca2+ channel over Na+ and K+ channels in mouse dorsal root ganglion neurons and/or human embryonic kidney (HEK)-293 cells and to suppress seizure-induced death in mouse model. Consequently, compound 11 is a useful probe for investigation of physiologic and pathophysiologic roles of the T-channel, and provides a basis to develop a novel therapeutic to treat chronic neuropathic and inflammatory pains.

Inhibition of T-Type calcium channels in mEC layer II stellate neurons reduces neuronal hyperexcitability associated with epilepsy

Temporal lobe epilepsy (TLE) is a form of adult epilepsy involving the entorhinal cortex (EC). Layer II neurons of the medial EC (mEC) are spared and become hyperexcitable in TLE. Studies have suggested a role for T-type calcium channels (T-type Ca2+ channels) in facilitating increases in neuronal activity associated with TLE within the hippocampus. We sought to determine if T-type Ca2+ channels play a role in facilitating neuronal hyperexcitability of layer II mEC stellate neurons in TLE.TLE was induced in rats by electrical stimulation of the hippocampus to induce status epilepticus (SE). Brain slices were prepared from rats exhibiting spontaneous seizures and compared with age-matched control rats. Action potentials (APs) were evoked either by current injection steps or via presynaptic stimulation of mEC deep layers. The selective T-type Ca2+ channel antagonist, TTA-P2 (1 μM), was applied to determine the role of T-type Ca2+ channels in maintaining neuronal excitability. Quantitative PCR techniques were used to assess T-type Ca2+ channel isoform mRNA levels within the mEC layer II.TLE mEC layer II stellate neurons were hyperexcitable compared to control neurons, evoking a higher frequency of APs and generating bursts of APs when synaptically stimulated. TTA-P2 (1 μM) reduced firing frequencies in TLE and control neurons and reduced AP burst firing in TLE stellate neurons. TTA-P2 had little effect on synaptically evoked AP’s in control neurons. TTA-P2 also inhibited rebound APs evoked in TLE neurons to a greater degree than in control neurons. TLE tissue had almost a 3-fold increase in Cav3.1 mRNA compared to controls. Cav3.2 or Cav3.3 levels were unchanged.These findings support a role for T-type Ca2+ channel in establishing neuronal hyperexcitability of mEC layer II stellate neurons in TLE. Increased expression of Cav3.1 may be important for establishing neuronal hyperexcitability of mEC layer II neurons in TLE.

Structural hybridization of pyrrolidine-based T-type calcium channel inhibitors and exploration of their analgesic effects in a neuropathic pain model

Highly effective and safe drugs for the treatment of neuropathic pain are urgently required and it was shown that blocking T-type calcium channels can be a promising strategy for drug development for neuropathic pain. We have developed pyrrolidine-based T-type calcium channel inhibitors by structural hybridization and subsequent assessment of in vitro activities against Cav3.1 and Cav3.2 channels. Profiling of in vitro ADME properties of compounds was also carried out. The representative compound 17h showed comparable in vivo efficacy to gabapentin in the SNL model, which indicates T-type calcium channel inhibitors can be developed as effective therapeutics for neuropathic pain.

Efficacy and safety of a T-type calcium channel blocker in patients with neuropathic pain: A proof-of-concept, randomized, double-blind and controlled trial.

T-type calcium channels have been shown to play an important role in the initiation and maintenance of neuropathic pain and represent a promising therapeutic target for new analgesic treatments. Ethosuximide (ETX), an anticonvulsant and a T-type channel blocker has shown analgesic effect in several chronic pain models but has not yet been evaluated in patients with neuropathic pain. This proof-of-concept, multicentre, double-blind, controlled and randomized trial compared the efficacy and safety of ETX (given as add-on therapy) to an inactive control (IC) in 114 patients with non-diabetic peripheral neuropathic pain. After a 7-day run-in period, eligible patients aged over 18years were randomly assigned (1:1) to ETX or IC for 6weeks. The primary outcome was the difference between groups in the pain intensity (% of change from the baseline to end of treatment) assessed in the intention-to-treat population. This study is registered with EudraCT (2013-004801-26) and ClinicalTrials.gov (NCT02100046). The study was stopped during the interim analysis due to the high number of adverse events in the active treatment group. ETX failed to reduce total pain and showed a poor tolerance in comparison to IC. In the per-protocol analysis, ETX significantly reduced pain intensity by 15.6% (95% CI -25.8; -5.4) from baseline compared to IC (-7.8%, 95% CI -14.3; -1.3; p=0.033), but this result must be interpreted with caution because of a small subgroup of patients. Ethosuximide did not reduce the severity of neuropathic pain and induces, at the doses used, many adverse events. This article shows that ETX is not effective to treat neuropathic pain. Nevertheless, per-protocol analysis suggests a possible analgesic effect of ETX. Thus, our work adds significant knowledge to preclinical and clinical data on the benefits of T-type calcium channel inhibition for the treatment of neuropathic pain.

Synthesis and biological evaluation of pyrrolidine-based T-type calcium channel inhibitors for the treatment of neuropathic pain

The treatment of neuropathic pain is one of the urgent unmet medical needs and T-type calcium channels are promising therapeutic targets for neuropathic pain. Several potent T-type channel inhibitors showed promising in vivo efficacy in neuropathic pain animal models and are being investigated in clinical trials. Herein we report development of novel pyrrolidine-based T-type calcium channel inhibitors by pharmacophore mapping and structural hybridisation followed by evaluation of their Cav3.1 and Cav3.2 channel inhibitory activities. Among potent inhibitors against both Cav3.1 and Cav3.2 channels, a promising compound 20n based on in vitro ADME properties displayed satisfactory plasma and brain exposure in rats according to in vivo pharmacokinetic studies. We further demonstrated that 20n effectively improved the symptoms of neuropathic pain in both SNL and STZ neuropathic pain animal models, suggesting modulation of T-type calcium channels can be a promising therapeutic strategy for the treatment of neuropathic pain.

Amyloid Beta Peptides Block New Synapse Assembly by Nogo Receptor-Mediated Inhibition of T-Type Calcium Channels

Compelling evidence links amyloid beta (Aβ) peptide accumulation in the brains of Alzheimer's disease (AD) patients with the emergence of learning and memory deficits, yet a clear understanding of the events that drive this synaptic pathology are lacking. We present evidence that neurons exposed to Aβ are unable to form new synapses, resulting in learning deficits invivo. We demonstrate the Nogo receptor family (NgR1-3) acts as Aβ receptors mediating an inhibition of synapse assembly, plasticity, and learning. Live imaging studies reveal Aβ activates NgRs on the dendritic shaft of neurons, triggering an inhibition of calcium signaling. We define T-type calcium channels as a target of Aβ-NgR signaling, mediating Aβ's inhibitory effects on calcium, synapse assembly, plasticity, and learning. These studies highlight deficits in new synapse assembly as a potential initiator of cognitive pathology in AD, and pinpoint calcium dysregulation mediated by NgRs and T-type channels as key components. VIDEO ABSTRACT.

Protective effect of quercetin on bupivacaine-induced neurotoxicity via T-type calcium channel inhibition

Purpose: To determine the effect of quercetin on bupivacaine-induced neurotoxicity and to investigate the mechanisms involved. Methods: Cultured SH-SY5Y cells were divided into five treatment groups: control group with no drug, bupivacaine treatment group, quercetin group, bupivacaine--quercetin combination treatment group, and bupivacaine-mibefradil combination treatment group. Cell morphology in each group was examined by microscopy while cell viability was assessed by 3-(4, 5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2- tetrazolium bromide (MTT) assay after 24 h incubation. Cytosolic calcium ion concentration was determined by flow cytometry while Cav3.1 protein expression was evaluated by western blot. Results: Quercetin (50 μM) significantly ( p < 0.05) protected SH-SYS5 cells from bupivacaine-induced cell apoptosis and also significantly reduced intracellular calcium ion concentration ( p < 0.01) by approximately 40 %. Cav3.1 protein expression was normalized following quercetin treatment. Conclusion: These results show that quercetin reduces the neurotoxicity induced by bupivacaine, possibly through inhibition of T-type calcium channel. This finding implies a novel mechanism for neuroprotective effect of quercetin, and its potential for treating toxicity arising from the use of local anesthetic agents. Keywords: Quercetin, Bupivacaine, Local anaesthetic, Neuroprotection, Neurotoxicity, T-type calcium channel

Lycoplanine A, a C16N Lycopodium Alkaloid with a 6/9/5 Tricyclic Skeleton from Lycopodium complanatum.

Lycoplanine A (1), a Lycopodium alkaloid with a 6/9/5 tricyclic ring skeleton fused with the γ-lactone ring and featuring an unusual 1-oxa-6-azaspiro[4.4]nonane moiety and an unprecedented 3-azabicyclo[6.3.1]dodecane unit, was isolated from the club moss Lycopodium complanatum. The structure and absolute configuration of 1 were identified by a combination of NMR spectroscopic analysis and single-crystal X-ray diffraction. Biological studies showed that 1 is a potent Cav3.1 T-type calcium channel (TTCC) inhibitor with an IC50 value of 6.06 μM.

Synthesis and diabetic neuropathic pain-alleviating effects of 2N-(pyrazol-3-yl)methylbenzo[d]isothiazole-1,1-dioxide derivatives

A novel series of fused-benzensulfonamide 2-N-(pyrazol-3-yl)methylbenzo[d]isothiazole-1,1-dioxide derivatives was designed and synthesized as metabolically stable T-type calcium channel inhibitors. Several compounds, 9, 10, and 17, displayed potent T-type channel inhibitory activity. Among them, compounds 10 and 17 showed good metabolic stability in human liver microsomes, and low hERG channel and CYP450 inhibition. Compound 10 exhibited diabetic neuropathic pain-alleviating effects in a streptozotocin-induced peripheral diabetic neuropathy (PDN) model. The maximum efficacy of compound 10, which was 3-fold more potent than gabapentin, was observed at 1h after administration, and co-administration of compound 10 with gabapentin showed a considerable synergic effect.

Neuritin Enhances Synaptic Transmission in Medial Prefrontal Cortex in Mice by Increasing CaV3.3 Surface Expression.

Neuritin is a neurotrophic factor involved in neural development and synaptic plasticity. However, its role in modulating synaptic transmission remains unclear. Here, we investigated the effects of neuritin on miniature excitatory postsynaptic currents (mEPSCs) and glutamate release in the medial prefrontal cortex (mPFC) in mice. Incubation of mPFC slices with neuritin for 45 min significantly increased mEPSC frequency and glutamate release as measured by high-performance liquid chromatography, which was mimicked by insulin and abrogated by an insulin receptor (IR) inhibitor. Neuritin-induced upregulation of synaptic transmission was correlated with activation of ERK, and inhibition of mitogen-activated protein kinases/extracellular signal-regulated kinases (MEK/ERK) activity attenuated the neuritin-induced increase in mEPSC frequency and glutamate release. T-type calcium channel inhibitors but not the L-type inhibitor abolished the inward calcium current and the effects of neuritin on mEPSC frequency and glutamate release. Western blotting of membrane proteins showed that neuritin promoted surface expression of CaV3.3 α-subunit, which was also eliminated by inhibition of IR or MEK/ERK activity. The effects of neuritin on mEPSC frequency, glutamate release, and CaV3.3 α-subunit expression were inhibited by an intracellular protein-transport inhibitor. These results confirm involvement of the IR and ERK signaling pathway, and provide novel insights into the mechanisms of neuritin function in synaptic transmission.

0129 LOCAL THALAMIC RETICULAR NUCLEUS INHIBITION OF T-TYPE CALCIUM CHANNELS REDUCES SLEEP SPINDLES IN MICE

Parvalbumin (PV)-containing GABAergic thalamic reticular neurons (TRN) express Cav3.3 T-type calcium channels that are thought to generate burst firing necessary for spindle generation. Cav3.3 is a risk gene for schizophrenia, where spindle abnormalities are present. The role of Cav3.3 in the control of sleep and spindles has been examined using global Cav3.3 knockout mice but not with acute, local pharmacological blockade. Therefore, here we examined the inhibitory effect of a selective T-type inhibitor, TTA-P2, in TRN on sleep and spindles. In vitro recordings were made from identified TRN PV neurons in slices from mice (13-22d) expressing a red fluorescent protein in PV neurons. In vivo, mice (N=8) were implanted with bilateral microdialysis cannulae targeting TRN (AP -0.7, ML ±1.3, DV -4.0) and electrodes (EEG/EMG) for sleep-wake recordings. A custom Matlab script detected individual spindles (10-15Hz) in the EEG, allowing analysis of drug effects on spindle density (spindles/min) during NREM sleep. Data was compared with the ACSF-day values and related to the histological location of the probe. TRN-PV neurons exhibited low-threshold spikes/inward currents after removal of hyperpolarizing currents/voltage steps respectively, which were blocked by TTA-P2 (3 µM, n=4), confirming the expression of low threshold T-type Ca channels in TRN PV neurons. In 6/8 mice with probe locations in TRN (uni- or bi-lateral), TTA-P2 infusion at ZT2-6 significantly decreased (-27.0 ± 11.6%, p=0.008) spindle density (ACSF: 4.25 ± 0.19; TTA-P2: 3.13 ± 0.37) without any significant effect on amplitude or duration. There was no effect on NREM sleep duration, delta (0.5-4Hz) or slow-wave activity (0.5–1.5Hz). Localized pharmacological inhibition of T-type calcium channels within TRN selectively decreased NREM spindle density without an effect on NREM sleep. These results contrast with those observed with a constitutive, global knockout of Cav3.3, where sleep fragmentation was observed in addition to reduced spindles (Astori et al., 2011). Veterans Affairs Medical Research Service Awards, 5I01BX001356, I01BX001404, 1IK2BX002130, R21-NS079866, RO1-MH039683, PO1-HL095491, R21 NS093000, R03 MH107650, JTM received partial salary compensation and funding from Merck MISP, but has no conflict of interest with this work.

Effects of T-type calcium channel inhibitors on monocrotaline-induced pulmonary arterial hypertension in rats

Objective To investigate the effects of T-type calcium channel inhibitors (ProTx-1, micromolar Ni2+ and Mibefradil) on Monocrotaline(MCT)-induced pulmonary arterial hypertension (PAH) in rats. Methods Forty male Sprague-Dawley rats were randomly divided into 5 groups: normal control group, MCT group, ProTx-1 group, micromolar Ni2+ group and Mibefradil group (8 cases in each group). The right ventricular systolic pressure (RVSP), the right ventricular hypertrophy index (RVHI), and the index of pulmonary vascular remodeling(MA%) were measured on day 28 after MCT-treatment.Western blot was used to detect the expression of proliferating cell nuclear antigen(PCNA) and Cleaved Caspase-3 in pulmonary artery. Results (1)RVSP and RVHI in MCT group were significantly higher than those in the other 4 groups (F=21.55, P 0.05). (2)MA% in normal control group[(23.43±1.95)%] was lower than that in MCT group[(80.42±4.30)%], ProTx-1 group[(60.35±3.83)%], micromolar Ni2+ group[(62.44±3.81)%] and Mibefradil group[(58.66±4.23)%](F=216.2, P 0.05), however, they were all signi-ficantly lower than that in MCT group.(3)The expression of PCNA in MCT group was higher than that in normal control group, meanwhile, 3 intervention groups were significantly lower than that in MCT group.The expression of Cleaved Caspase-3 in MCT group was higher than that in normal control group, nevertheless, 3 intervention groups showed no significant changes compared with MCT group, respectively. Conclusions T-type calcium channel inhibitors could ameliorate the progression of MCT-PAH in rats, mainly through suppressing the proliferation of pulmonary artery smooth muscle cells. Key words: Pulmonary arterial hypertension; Monocrotaline; Voltage dependent calcium channel; Proliferation; Apoptosis

Cav2.2 and Cav3.1 calcium channel inhibitors from Valeriana jatamansi Jones

In China, the roots and rhizomes of Valeriana jatamansi Jones are traditionally used to treat gastrointestinal and rheumatic pain. In the present study, a number of N- and T-type calcium channel inhibitors were initially isolated and identified.