Calcium Homeostasis, Transporters, and Blockers in Health and Diseases of the Cardiovascular System.

Calcium is a highly positively charged ionic species. It regulates all cell types' functions and is an important second messenger that controls and triggers several mechanisms, including membrane stabilization, permeability, contraction, secretion, mitosis, intercellular communications, and in the activation of kinases and gene expression. Therefore, controlling calcium transport and its intracellular homeostasis in physiology leads to the healthy functioning of the biological system. However, abnormal extracellular and intracellular calcium homeostasis leads to cardiovascular, skeletal, immune, secretory diseases, and cancer. Therefore, the pharmacological control of calcium influx directly via calcium channels and exchangers and its outflow via calcium pumps and uptake by the ER/SR are crucial in treating calcium transport remodeling in pathology. Here, we mainly focused on selective calcium transporters and blockers in the cardiovascular system.

Nanophotonics Enable Targeted Photothermal Silencing of Nociceptor Neurons.

The sensory nervous and immune systems work in concert to preserve homeostasis. While this endogenous interplay protects from danger, it may drive chronic pathologies. Currently, genetic engineering of neurons remains the primary approach to interfere selectively with this potentially deleterious interplay. However, such manipulations are not feasible in a clinical setting. Here, this work reports a nanotechnology-enabled concept to silence subsets of unmodified nociceptor neurons that exploits their ability to respond to heat via the transient receptor potential vanilloid type 1 (TRPV1) channel. This strategy uses laser stimulation of antibody-coated gold nanoparticles to heat-activate TRPV1, turning this channel into a cell-specific drug-entry port. This delivery method allows transport of a charged cationic derivative of an N-type calcium channel blocker (CNCB-2) into targeted sensory fibers. CNCB-2 delivery blocks neuronal calcium currents and neuropeptides release, resulting in targeted silencing of nociceptors. Finally, this work demonstrates the ability of the approach to probe neuro-immune crosstalk by targeting cytokine-responsive nociceptors and by successfully preventing nociceptor-induced CD8+ T-cells polarization. Overall, this work constitutes the first demonstration of targeted silencing of nociceptor neuron subsets without requiring genetic modification, establishing a strategy for interfering with deleterious neuro-immune interplays.

Loss of fragile X mental retardation protein precedes Lewy pathology in Parkinson's disease.

Parkinson's disease (PD) is the most common neurodegenerative movement disorder and is characterized by the progressive loss of dopaminergic (DA) neurons in the substantia nigra pars compacta (SNc) and the gradual appearance of α-synuclein (α-syn)-containing neuronal protein aggregates. Although the exact mechanism of α-syn-mediated cell death remains elusive, recent research suggests that α-syn-induced alterations in neuronal excitability contribute to cell death in PD. Because the fragile X mental retardation protein (FMRP) controls the expression and function of numerous neuronal genes related to neuronal excitability and synaptic function, we here investigated the role of FMRP in α-syn-associated pathological changes in cell culture and mouse models of PD as well as in post-mortem human brain tissue from PD patients. We found FMRP to be decreased in cultured DA neurons and in the mouse brain in response to α-syn overexpression. FMRP was, furthermore, lost in the SNc of PD patients and in patients with early stages of incidental Lewy body disease (iLBD). Unlike fragile X syndrome (FXS), FMR1 expression in response to α-syn was regulated by a mechanism involving Protein Kinase C (PKC) and cAMP response element-binding protein (CREB). Reminiscent of FXS neurons, α-syn-overexpressing cells exhibited an increase in membrane N-type calcium channels, increased phosphorylation of ERK1/2, eIF4E and S6, increased overall protein synthesis, and increased expression of Matrix Metalloproteinase 9 (MMP9). FMRP affected neuronal function in a PD animal model, because FMRP-KO mice were resistant to the effect of α-syn on striatal dopamine release. In summary, our results thus reveal a new role of FMRP in PD and support the examination of FMRP-regulated genes in PD disease progression.

The neuronal calcium ion channel activity of constrained analogues of MONIRO-1.

Structural modifications of the neuronal calcium channel blocker MONIRO-1, including constraining the phenoxyaniline portion of the molecule and replacing the guanidinium functionality with tertiary amines, led to compounds with significantly improved affinities for the endogenously expressed CaV2.2 channel in the SH-SY5Y neuroblastoma cell line. These analogues also showed promising activity towards the CaV3.2 channel, recombinantly expressed in HEK293T cells. Both of these ion channels have received attention as likely targets for the treatment of neuropathic pain. The dibenzoazepine and dihydrobenzodiazepine derivatives prepared in this study show an encouraging combination of neuronal calcium ion channel inhibitory potency, plasma stability and potential to cross the blood-brain-barrier.

The effect of an L/N-type calcium channel blocker on intradialytic blood pressure in intradialytic hypertensive patients.

BACKGROUND: Intradialytic hypertension (HTN), which is one of the poor prognostic markers in patients undergoing hemodialysis, may be associated with sympathetic overactivity. The L/N-type calcium channel blocker, cilnidipine, has been reported to suppress sympathetic nerves activity in vivo. Therefore, we hypothesized that cilnidipine could attenuate intradialytic systolic blood pressure (SBP) elevation. METHODS: Fifty-one patients on chronic hemodialysis who had intradialytic-HTN (SBP elevation ≥10 mmHg during hemodialysis) and no fluid overload were prospectively randomized into two groups: control and cilnidipine groups. Cilnidipine group patients took cilnidipine (10 mg/day) for 12 weeks. The primary endpoint was the change in the intradialytic SBP elevation before and after the 12-week intervention. RESULTS: Before the intervention, no differences were observed in age, sex or pre-dialytic SBP (148.5 ± 12.9 vs. 148.3 ± 19.3 mmHg) between the two groups. Intradialytic SBP elevation was unchanged in the control group. Cilnidipine significantly lowered the post-dialytic SBP with an attenuation of the intradialytic SBP elevation from 12.0 ± 15.4 mmHg to 4.8 ± 10.1 mmHg. However, the observed difference in the intradialytic SBP elevation by cilnidipine did not reach statistical significance (group×time interaction effect p = 0.25). Cathecolamine levels were unaffected by the intervention in both groups. CONCLUSION: Cilnidipine lowers both the pre- and post-dialytic SBP and might attenuate intradialytic SBP elevation. Therefore, cilnidipine may be effective in lowering SBP during HD in patients with intradialytic-HTN.

Discovery and optimization of a novel series of pyrazolyltetrahydropyran N-type calcium channel (Cav 2.2) blockers for the treatment of pain.

A novel series of pyrazolyltetrahydropyran N-type calcium channel blockers are described. Structural modifications of the series led to potent compounds in both a cell-based fluorescent calcium influx assay and a patch clamp electrophysiology assay. Representative compounds from the series were bioavailable and showed efficacy in the rat CFA and CCI models of inflammatory and neuropathic pain.

Synthesis and evaluation of aminobenzothiazoles as blockers of N- and T-type calcium channels.

Both N- and T-type calcium ion channels have been implicated in pain transmission and the N-type channel is a well-validated target for the treatment of neuropathic pain. An SAR investigation of a series of substituted aminobenzothiazoles identified a subset of five compounds with comparable activity to the positive control Z160 in a FLIPR-based intracellular calcium response assay measuring potency at both CaV2.2 and CaV3.2 channels. These compounds may form the basis for the development of drug leads and tool compounds for assessing in vivo effects of variable modulation of CaV2.2 and CaV3.2 channels.

Agmatine suppresses peripheral sympathetic tone by inhibiting N-type Ca(2+) channel activity via imidazoline I2 receptor activation.

Agmatine, a putative endogenous ligand of imidazoline receptors, suppresses cardiovascular function by inhibiting peripheral sympathetic tone. However, the molecular identity of imidazoline receptor subtypes and its cellular mechanism underlying the agmatine-induced sympathetic suppression remains unknown. Meanwhile, N-type Ca(2+) channels are important for the regulation of NA release in the peripheral sympathetic nervous system. Therefore, it is possible that agmatine suppresses NA release in peripheral sympathetic nerve terminals by inhibiting Ca(2+) influx through N-type Ca(2+) channels. We tested this hypothesis by investigating agmatine effect on electrical field stimulation (EFS)-evoked contraction and NA release in endothelium-denuded rat superior mesenteric arterial strips. We also investigated the effect of agmatine on the N-type Ca(2+) current in superior cervical ganglion (SCG) neurons in rats. Our study demonstrates that agmatine suppresses peripheral sympathetic outflow via the imidazoline I2 receptor in rat mesenteric arteries. In addition, the agmatine-induced suppression of peripheral vascular sympathetic tone is mediated by modulating voltage-dependent N-type Ca(2+) channels in sympathetic nerve terminals. These results suggest a potential cellular mechanism for the agmatine-induced suppression of peripheral sympathetic tone. Furthermore, they provide basic and theoretical information regarding the development of new agents to treat hypertension.

Long-term effects of L- and N-type calcium channel blocker on uric acid levels and left atrial volume in hypertensive patients.

Left ventricular (LV) diastolic dysfunction is associated with hypertension and hyperuricemia. However, it is not clear whether the L- and N-type calcium channel blocker will improve LV diastolic dysfunction through the reduction of uric acid. The aim of this study was to investigate the effects of anti-hypertensive therapy, the L- and N-type calcium channel blocker, cilnidipine or the L-type calcium channel blocker, amlodipine, on left atrial reverse remodeling and uric acid in hypertensive patients. We studied 62 patients with untreated hypertension, randomly assigned to cilnidipine or amlodipine for 48 weeks. LV diastolic function was assessed with the left atrial volume index (LAVI), mitral early diastolic wave (E), tissue Doppler early diastolic velocity (E') and the ratio (E/E'). Serum uric acid levels were measured before and after treatment. After treatment, systolic and diastolic blood pressures equally dropped in both groups. LAVI, E/E', heart rate and uric acid levels decreased at 48 weeks in the cilnidipine group but not in the amlodipine group. The % change from baseline to 48 weeks in LAVI, E wave, E/E' and uric acid levels were significantly lower in the cilnidipine group than in the amlodipine group. Larger %-drop in uric acid levels were associated with larger %-reduction of LAVI (p < 0.01). L- and N-type calcium channel blocker but not L-type calcium channel blocker may improve LV diastolic function in hypertensive patients, at least partially through the decrease in uric acid levels.

Analgesic effect of a broad-spectrum dihydropyridine inhibitor of voltage-gated calcium channels.

Voltage-activated calcium channels are important facilitators of nociceptive transmission in the primary afferent pathway. Consequently, molecules that block these channels are of potential use as pain therapeutics. Our group has recently reported on the identification of a novel class of dihydropyridines (DHPs) that included compounds with preferential selectivity for T-type over L-type channels. Among those compounds, M4 was found to be an equipotent inhibitor of both Cav1.2 L- and Cav3.2 T-type calcium channels. Here, we have further characterized the effects of this compound on other types of calcium channels and examined its analgesic effect when delivered either spinally (i.t.) or systemically (i.p.) to mice. Both delivery routes resulted in antinociception in a model of acute pain. Furthermore, M4 was able to reverse mechanical hyperalgesia produced by nerve injury when delivered intrathecally. M4 retained partial activity when delivered to Cav3.2 null mice, indicating that this compound acts on multiple targets. Additional whole-cell patch clamp experiments in transfected tsA-201 cells revealed that M4 also effectively blocks Cav3.3 (T-type) and Cav2.2 (N-type) currents. Altogether, our data indicate that broad-spectrum inhibition of multiple calcium channel subtypes can lead to potent analgesia in rodents.

Discovery of a 1-isopropyltetrahydroisoquinoline derivative as an orally active N-type calcium channel blocker for neuropathic pain.

N-type calcium channel blockade is a promising therapeutic approach for the treatment of neuropathic pain. Starting from lead compound (S)-1, we focused our optimization efforts on potency for N-type calcium channel inhibition and improvement of CYP inhibition profile. 2-{[(1-Hydroxycyclohexyl)methyl]amino}-(1R)-(1-isopropyl-6-methoxy-3,4-dihydroisoquinolin-2(1H)-yl)ethanone oxalate ((R)-5r) was identified as a novel orally active small-molecule N-type calcium channel inhibitor with reduced CYP inhibition liability. Oral administration of (R)-5r improved mechanical allodynia in a spinal nerve ligation model of neuropathic pain in rats with an ED50 value of 2.5 mg/kg.

Discovery of an 8-methoxytetrahydroisoquinoline derivative as an orally active N-type calcium channel blocker for neuropathic pain without CYP inhibition liability.

In lead optimization efforts starting from the tetrahydroisoquinoline (S)-1, we identified 2-{[(2R)-2-hydroxypropyl]amino}-1-[(1S)-8-methoxy-1-phenyl-3,4-dihydroisoquinolin-2(1H)-yl]ethanone ((1S)-8t) as a novel orally active small-molecule N-type calcium channel blocker without CYP inhibition liability. CYP3A4 inhibition profile was improved by reducing the lipophilicity of compound (S)-1. Moreover, introduction of a methoxy group to the C-8 position of tetrahydroisoquinoline led to identification of (1S)-8t, which eliminated CYP2D6 inhibition liability. Oral administration of (1S)-8t exerted efficacy in a rat spinal nerve ligation (SNL) model of neuropathic pain with an ED50 value of 2.8 mg/kg.

Discovery and SAR of novel tetrahydropyrrolo[3,4-c]pyrazoles as inhibitors of the N-type calcium channel.

A novel series of substituted tetrahydropyrrolo[3,4-c]pyrazoles were investigated as blockers of the N-type calcium channel (Cav2.2 channels), a chronic pain target.

Recent progress in the discovery and development of N-type calcium channel modulators for the treatment of pain.

The importance of voltage-gated calcium channels (VGCCs) in basic physiological processes such as cardiac and neurological function has generated intense interest in these proteins as targets of pharmacological intervention. N-type calcium channels are a subset of VGCCs distinguished by their physiology, pharmacology and significance to the pathology of chronic pain. Despite decades of investigation, only a single drug targeting N-type channel function has entered the marketplace. This review will summarize our current understanding of the biology, physiology and pharmacology of N-type calcium channels and the implication of these features for therapeutic intervention. From this basis of understanding, recent efforts to discover and develop peptide based and small molecule modulators of N-type calcium channel function will be discussed.

Discovery and SAR of a novel series of 2,4,5,6-tetrahydrocyclopenta[c]pyrazoles as N-type calcium channel inhibitors.

A novel series of substituted 2,4,5,6-tetrahydrocyclopenta[c]pyrazoles were investigated as N-type calcium channel blockers (Cav2.2 channels), a chronic pain target. One compound was active in vivo in the rat CFA pain model.

Discovery of novel tetrahydroisoquinoline derivatives as orally active N-type calcium channel blockers with high selectivity for hERG potassium channels.

N-type calcium channels represent a promising target for the treatment of neuropathic pain. The selective N-type calcium channel blocker ziconotide ameliorates severe chronic pain but has a narrow therapeutic window and requires intrathecal administration. We identified tetrahydroisoquinoline derivative 1a as a novel potent N-type calcium channel blocker. However, this compound also exhibited potent inhibitory activity against hERG channels. Structural optimizations led to identification of (1S)-(1-cyclohexyl-3,4-dihydroisoquinolin-2(1H)-yl)-2-{[(1-hydroxycyclohexyl)methyl]amino}ethanone ((S)-1h), which exhibited high selectivity for hERG channels while retaining potency for N-type calcium channel inhibition. (S)-1h went on to demonstrate in vivo efficacy as an orally available N-type calcium channel blocker in a rat spinal nerve ligation model of neuropathic pain.

An orally available Cav2.2 calcium channel inhibitor for the treatment of neuropathic pain.

BACKGROUND AND PURPOSE: Neuropathic pain affects up to 10% of the global population and is caused by an injury or a disease affecting the somatosensory, peripheral, or central nervous system. NP is characterized by chronic, severe and opioid-resistant properties. Therefore, its clinical management remains very challenging. The N-type voltage-gated calcium channel, Cav2.2, is a validated target for therapeutic intervention in chronic and neuropathic pain. The conotoxin ziconotide (Prialt®) is an FDA-approved drug that blocks Cav2.2 channel but needs to be administered intrathecally. Thus, although being principally efficient, the required application route is very much in disfavour. EXPERIMENTAL APPROACH AND KEY RESULTS: Here, we describe an orally available drug candidate, RD2, which competes with ziconotide binding to Cav2.2 at nanomolar concentrations and inhibits Cav2.2 almost completely reversible. Other voltage-gated calcium channel subtypes, like Cav1.2 and Cav3.2, were affected by RD2 only at concentrations higher than 10 µM. Data from sciatic inflammatory neuritis rat model demonstrated the in vivo proof of concept, as low-dose RD2 (5 mg·kg-1) administered orally alleviated neuropathic pain compared with vehicle controls. High-dose RD2 (50 mg·kg-1) was necessary to reduce pain sensation in acute thermal response assessed by the tail flick test. CONCLUSIONS AND IMPLICATIONS: Taken together, these results demonstrate that RD2 has antiallodynic properties. RD2 is orally available, which is the most convenient application form for patients and caregivers. The surprising and novel result from standard receptor screens opens the room for further optimization into new promising drug candidates, which address an unmet medical need.

A comprehensive review on ziconotide.

Managing severe chronic pain is a challenging task, given the limited effectiveness of available pharmacological and non-pharmacological treatments. This issue continues to be a significant public health concern, requiring a substantial therapeutic response. Ziconotide, a synthetic peptide initially isolated from Conus magus in 1982 and approved by the US Food and Drug Administration and the European Medicines Agency in 2004, is the first-line intrathecal method for individuals experiencing severe chronic pain refractory to other therapeutic measures. Ziconotide produces powerful analgesia by blocking N-type calcium channels in the spinal cord, which inhibits the release of pain-relevant neurotransmitters from the central terminals of primary afferent neurons. However, despite possessing many favorable qualities, including the absence of tolerance development, respiratory depression, and withdrawal symptoms (largely due to the absence of a G-protein mediation mechanism), ziconotide's application is limited due to factors such as intrathecal administration and a narrow therapeutic window resulting from significant dose-related undesired effects of the central nervous system. This review aims to provide a comprehensive and clinically relevant summary of the literatures concerning the pharmacokinetics and metabolism of intrathecal ziconotide. It will also describe strategies intended to enhance clinical efficacy while reducing the incidence of side effects. Additionally, the review will explore the current efforts to refine the structure of ziconotide for better clinical outcomes. Lastly, it will prospect potential developments in the new class of selective N-type voltage-sensitive calcium-channel blockers.

Calcium influx through N-type channels and activation of SK and TRP-like channels regulates tonic firing of neurons in rat paraventricular thalamus.

The thalamus is a major relay and integration station in the central nervous system. While there is a large body of information on the firing and network properties of neurons contained within sensory thalamic nuclei, less is known about the neurons located in midline thalamic nuclei, which are thought to modulate arousal and homeostasis. One midline nucleus that has been implicated in mediating stress responses is the paraventricular nucleus of the thalamus (PVT). Like other thalamic neurons, these neurons display two distinct firing modes, burst and tonic. In contrast to burst firing, little is known about the ionic mechanisms modulating tonic firing in these cells. Here we performed a series of whole cell recordings to characterize tonic firing in PVT neurons in acute rat brain slices. We found that PVT neurons are able to fire sustained, low-frequency, weakly accommodating trains of action potentials in response to a depolarizing stimulus. Unexpectedly, PVT neurons displayed a very high propensity to enter depolarization block, occurring at stimulus intensities that would elicit tonic firing in other thalamic neurons. The tonic firing behavior of these cells is modulated by a functional interplay between N-type Ca(2+) channels and downstream activation of small-conductance Ca(2+)-dependent K(+) (SK) channels and a transient receptor potential (TRP)-like conductance. Thus these ionic conductances endow PVT neurons with a narrow dynamic range, which may have fundamental implications for the integrative properties of this nucleus.

Comparison of the antialbuminuric effects of L-/N-type and L-type calcium channel blockers in hypertensive patients with diabetes and microalbuminuria: the study of assessment for kidney function by urinary microalbumin in randomized (SAKURA) trial.

OBJECTIVE: To clarify whether the L-/N-type calcium channel blocker (CCB) cilnidipine is more renoprotective than the L-type CCB amlodipine in patients with early-stage diabetic nephropathy. METHODS: In this prospective, multicenter, open-labeled, randomized trial, the antialbuminuric effects of cilnidipine and amlodipine were examined in renin-angiotensin system (RAS) inhibitor-treated patients with hypertension (blood pressure [BP]: 130-180/80-110 mmHg), type 2 diabetes, and microalbuminuria (urinary albumin to creatinine [Cr] ratio [UACR]: 30-300 mg/g). RESULTS: Patients received cilnidipine (n = 179, final dose: 10.27 ± 4.13 mg/day) or amlodipine (n = 186, 4.87 ± 2.08 mg/day) for 12 months. Cilnidipine and amlodipine equally decreased BP. The UACR values for the cilnidipine and amlodipine groups were 111.50 ± 138.97 and 88.29 ± 63.45 mg/g, respectively, before treatment and 107.93 ± 130.23 and 89.07 ± 97.55 mg/g, respectively, after treatment. The groups showed similar changes for the natural logarithm of the UACR, serum Cr, and estimated glomerular filtration rate. CONCLUSIONS: Cilnidipine did not offer greater renoprotection than amlodipine in RAS inhibitor-treated hypertensive patients with type 2 diabetes and microalbuminuria.