NNC 55-0396

IC50: 6.8 μM for Cav3.1 T-type channel

NNC 55-0396 is a T-type calcium channel antagonist.

T-type calcium channel activity is reported to be a critical component of the spontaneous voltage oscillation of inferior olive neurons occurring at a frequency similar to that of essential tremor.

In vitro: NNC 55-0396 showed no effect against high voltage calcium channels at 100 μM, but inhibited T-type channels in HEK293 cells with a potency comparable to that of mibefradil (IC50 6.8 versus 10.1 μM) [1].

In vivo: NNC 55-0396 was better tolerated than mibefradil in the horizontalwire test. To assess for a potential interaction with harmaline in the inverted wire grid test, mice was given NNC 55-0396, followed by harmaline or vehicle. NNC 55-0396 ameliorated harmaline-induced test deficits. In the GABAA α1-null model, NNC 55-0396 at 12.5 mg/kg suppressed harmaline-induced tremor by half by 20–100 min, whereas mibefradil at the same dose did not significantly affect tremor. In contrast to mibefradil, NNC 55-0396 was well tolerated and suppresses tremor, and exerts less cytochrome P450 inhibition [1].

Clinical trial: N/A

Reference:
[1] Quesada A,Bui PH,Homanics GE,Hankinson O,Handforth A. Comparison of mibefradil and derivative NNC 55-0396 effects on behavior, cytochrome P450 activity, and tremor in mouse models of essential tremor. Eur J Pharmacol.2011 May 20;659(1):30-6.

Ca2+ channel inhibitor NNC 55-0396 inhibits voltage-dependent K+ channels in rabbit coronary arterial smooth muscle cells.[Pubmed:24989838]

J Pharmacol Sci. 2014;125(3):312-9. Epub 2014 Jul 1.

We demonstrated the inhibitory effect of NNC 55-0396, a T-type Ca(2+) channel inhibitor, on voltage-dependent K(+) (K(V)) channels in freshly isolated rabbit coronary arterial smooth muscle cells. NNC 55-0396 decreased the amplitude of K(V) currents in a concentration-dependent manner, with an IC(50) of 0.080 muM and a Hill coefficient of 0.76.NNC 55-0396 did not affect steady-state activation and inactivation curves, indicating that the compound does not affect the voltage sensitivity of K(V) channel gating. Both the K(V) currents and the inhibitory effect of NNC 55-0396 on K(V) channels were not altered by depletion of extracellular Ca(2+) or intracellular ATP, suggesting that the inhibitory effect of NNC 55-0396 is independent of Ca(2+)-channel activity and phosphorylation-dependent signaling cascades. From these results, we concluded that NNC 55-0396 dosedependently inhibits K(V) currents, independently of Ca(2+)-channel activity and intracellular signaling cascades.

Blockade by NNC 55-0396, mibefradil, and nickel of calcium and exocytotic signals in chromaffin cells: implications for the regulation of hypoxia-induced secretion at early life.[Pubmed:25622555]

Eur J Pharmacol. 2015 Mar 15;751:1-12.

Adrenal chromaffin cells (CCs) express high-voltage activated calcium channels (high-VACCs) of the L, N and PQ subtypes; in addition, T-type low-VACCs are also expressed during embryo and neonatal life. Effects of the more frequently used T channel blockers NNC 55-0396 (NNC), mibefradil, and Ni2+ on the whole-cell Ba2+ current (IBa), the K+-elicited [Ca2+]c transients and catecholamine secretion have been studied in adult bovine CCs (BCCs) and rat embryo CCs (RECCs). NNC, mibefradil, and Ni2+ blocked BCC IBa with IC50 of 1.8, 4.9 and 70 muM, while IC50 to block IBa in RECCs were 2.1, 4.4 and 41 muM. Pronounced blockade of K+-elicited [Ca2+]c transients and secretion was also elicited by the three agents. However, the hypoxia-induced secretion (HIS) of catecholamine in RECCs was blocked substantially (75%) with thresholds concentrations of NCC (IC20 to block IBa); this was not the case for mibefradil and Ni2+ that required higher concentrations to block the HIS response. Thus, out of the three compounds, NNC seemed to be an adequate pharmacological tool to discern the contribution of T channels to the HIS response, without a contamination with high-VACC blockade.

T-type calcium channel antagonists, mibefradil and NNC-55-0396 inhibit cell proliferation and induce cell apoptosis in leukemia cell lines.[Pubmed:25989794]

J Exp Clin Cancer Res. 2015 May 21;34:54.

BACKGROUND: T-type Ca(2+) channels are often aberrantly expressed in different human cancers and participate in the regulation of cell cycle progression, proliferation and death. METHODS: RT-PCR, Q-PCR, western blotting and whole-cell patch-clamp recording were employed to assess the expression of T-type Ca(2+) channels in leukemia cell lines. The function of T-type Ca(2+) channels in leukemia cell growth and the possible mechanism of the effect of T-type Ca(2+) channel antagonists on cell proliferation and apoptosis were examined in T-lymphoma cell lines. RESULTS: We show that leukemia cell lines exhibited reduced cell growth when treated with T-type Ca(2+) channel inhibitors, mibefradil and NNC-55-0396 in a concentration-dependent manner. Mechanistically, these inhibitors played a dual role on cell viability: (i) blunting proliferation, through a halt in the progression to the G1-S phase; and (ii) promoting cell apoptosis, partially dependent on the endoplasmic reticulum Ca(2+) release. In addition, we observed a reduced phosphorylation of ERK1/2 in MOLT-4 cells in response to mibefradil and NNC-55-0396 treatment. CONCLUSIONS: These results indicate that mibefradil and NNC-55-0396 regulate proliferation and apoptosis in T-type Ca(2+) channel expressing leukemia cell lines and suggest a potential therapeutic target for leukemia.

NNC 55-0396, a T-type Ca2+ channel inhibitor, inhibits angiogenesis via suppression of hypoxia-inducible factor-1alpha signal transduction.[Pubmed:25471482]

J Mol Med (Berl). 2015 May;93(5):499-509.

UNLABELLED: Mitochondrial respiration is required for hypoxia-inducible factor (HIF)-1alpha stabilization, which is important for tumor cell survival, proliferation, and angiogenesis. Herein, small molecules that inhibit HIF-1alpha protein stability by targeting mitochondrial energy production were screened using the Library of Pharmacologically Active Compounds and cell growth assay in galactose or glucose medium. NNC 55-0396, a T-type Ca(2+) channel inhibitor, was selected as a hit from among 1,280 small molecules. NNC 55-0396 suppressed mitochondrial reactive oxygen species-mediated HIF-1alpha expression as well as stabilization by inhibiting protein synthesis in a dose-dependent manner. NNC 55-0396 inhibited tumor-induced angiogenesis in vitro and in vivo by suppressing HIF-1alpha stability. Moreover, NNC 55-0396 significantly suppressed glioblastoma tumor growth in a xenograft model. Thus, NNC 55-0396, a small molecule targeting T-type Ca(2+) channel, was identified by the systemic cell-based assay and was shown to have antiangiogenic activity via the suppression of HIF-1alpha signal transduction. These results provide new insights into the biological network between ion channel and HIF-1alpha signal transduction. KEY MESSAGE: HIF-1alpha overexpression has been demonstrated in hypoxic cancer cells. NNC 55-0396, a T-type Ca(2+) channel inhibitor, inhibited HIF-1alpha expression via both proteasomal degradation and protein synthesis pathways. T-type Ca(2+) channel inhibitors block angiogenesis by suppressing HIF-1alpha stability and synthesis. NNC 55-0396 could be a potential therapeutic drug candidate for cancer treatment.

Ca(v)3.2 T-type Ca2+ channel-dependent activation of ERK in paraventricular thalamus modulates acid-induced chronic muscle pain.[Pubmed:20685979]

J Neurosci. 2010 Aug 4;30(31):10360-8.

Treatments for chronic musculoskeletal pain, such as lower back pain, fibromyalgia, and myofascial pain syndrome, remain inadequate because of our poor understanding of the mechanisms that underlie these conditions. Although T-type Ca2+ channels (T-channels) have been implicated in peripheral and central pain sensory pathways, their role in chronic musculoskeletal pain is still unclear. Here, we show that acid-induced chronic mechanical hyperalgesia develops in Ca(v)3.1-deficient and wild-type but not in Ca(v)3.2-deficient male and female mice. We also show that T-channels are required for the initiation, but not maintenance, of acid-induced chronic muscle pain. Blocking T-channels using ethosuximide prevented chronic mechanical hyperalgesia in wild-type mice when administered intraperitoneally or intracerebroventricularly, but not intramuscularly or intrathecally. Furthermore, we found an acid-induced, Ca(v)3.2 T-channel-dependent activation of ERK (extracellular signal-regulated kinase) in the anterior nucleus of paraventricular thalamus (PVA), and prevention of the ERK activation abolished the chronic mechanical hyperalgesia. Our findings suggest that Ca(v)3.2 T-channel-dependent activation of ERK in PVA is required for the development of acid-induced chronic mechanical hyperalgesia.

NNC 55-0396 [(1S,2S)-2-(2-(N-[(3-benzimidazol-2-yl)propyl]-N-methylamino)ethyl)-6-fluoro-1,2, 3,4-tetrahydro-1-isopropyl-2-naphtyl cyclopropanecarboxylate dihydrochloride]: a new selective inhibitor of T-type calcium channels.[Pubmed:14718587]

J Pharmacol Exp Ther. 2004 Apr;309(1):193-9.

Mibefradil is a Ca2+ channel antagonist that inhibits both T-type and high-voltage-activated Ca2+ channels. We previously showed that block of high-voltage-activated channels by mibefradil occurs through the production of an active metabolite by intracellular hydrolysis. In the present study, we modified the structure of mibefradil to develop a nonhydrolyzable analog, (1S, 2S)-2-(2-(N-[(3-benzimidazol-2-yl)propyl]-N-methylamino)ethyl)-6-fluoro-1,2,3,4-t etrahydro-1-isopropyl-2-naphtyl cyclopropanecarboxylate dihydrochloride (NNC 55-0396), that exerts a selective inhibitory effect on T-type channels. The acute IC(50) of NNC 55-0396 to block recombinant alpha(1)G T-type channels in human embryonic kidney 293 cells was approximately 7 microM, whereas 100 microM NNC 55-0396 had no detectable effect on high-voltage-activated channels in INS-1 cells. NNC 55-0396 did not affect the voltage-dependent activation of T-type Ca2+ currents but changed the slope of the steady-state inactivation curve. Block of T-type Ca2+ current was partially relieved by membrane hyperpolarization and enhanced at a high-stimulus frequency. Washing NNC 55-0396 out of the recording chamber did not reverse the T-type Ca2+ current activity, suggesting that the compound dissolves in or passes through the plasma membrane to exert its effect; however, intracellular perfusion of the compound did not block T-type Ca2+ currents, arguing against a cytoplasmic route of action. After incubating cells from an insulin-secreting cell line (INS-1) with NNC 55-0396 for 20 min, mass spectrometry did not detect the mibefradil metabolite that causes L-type Ca2+ channel inhibition. We conclude that NNC 55-0396, by virtue of its modified structure, does not produce the metabolite that causes inhibition of L-type Ca2+ channels, thus rendering it more selective to T-type Ca2+ channels.

NNC 55-0396, Mibefradil derivative, is a highly selective T-type calcium channel blocker; displays IC50 values of 6.8 and > 100 μM for inhibition of Cav3.1 T-type channels and HVA currents respectively in INS-1 cells.

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