T-type calcium channel （IC50 = 2.7 μM); (L-type calcium channel（IC50 = 18.6 μM)

Mibefradil has IC50 values of 2.7 and 18.6 μM, respectively, which indicate its reversible inhibition of T-type and L-type currents[2]. Mibefradil (20 µM) significantly depolarizes the membrane potential (from -83±1 mV to -71±5 mV), slows the repolarization rate (44±16%), and lowers the amplitude of the excitatory node potential (37±10%)[3].

After four weeks of therapy, the 24-26-week-old C57BL/6J mice had different hearing thresholds. When compared to the saline-treated group, the hearing threshold at 24 kHz was considerably lower in the groups treated with benidipine and mibefradil (P<0.05)[3]. Rats given either Ethosuximide or Mibefradil in the spinal cord and DRG exhibit noticeably reduced CaV3.2 expression in comparison to the saline-treated group[4].

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Seven days after SNL, CaV3.2 protein levels were upregulated in ipsi-lateral L5/6 spinal cord and dorsal root ganglia (DRG) in immunofluorescence and Western blotting studies. Compared with the saline-treated group, rats receiving mibefradil or ethosuximide showed significant lower CaV3.2 expression in the spinal cord and DRG. No obvious histopathologic change in hematoxylin-eosin and toluidine blue staining were observed in all tested groups. Conclusion: In this study, we demonstrate that SNL-induced CaV3.2 upregulation in the spinal cord and DRG was attenuated by intrathecal infusion of mibefradil or ethosuximide. No obvious neurotoxicity effects were observed in all the tested groups. Our data suggest that continuous intrathecal infusion of TCC blockers may be considered as a promising alternative for the treatment of nerve injury-induced pain.[4]

The effects of Mibefradil/Ro 40-5967, a nondihydropyridine Ca++ channel blocker, on low-voltage activated (T-type) and high-voltage activated (L-type) Ca++ channels were compared. L-type barium currents were measured in Chinese hamster ovary cells stably transfected with the alpha 1 subunit of the class Cb Ca++ channel. T-type barium currents were investigated in human medullary thyroid carcinoma cells. The Ba++ currents of human medullary thyroid carcinoma cells were transient, activated at a threshold potential of -50 mV with the maximum at -14 +/- 3.2 mV and blocked by micromolar Ni++. The T- and L-type current inactivated with time constants of 33.4 +/- 4.1 and 416 +/- 26 msec at maximum barium currents, respectively. Ro 40-5967 inhibited reversibly the T- and L-type currents with IC50 values of 2.7 and 18.6 microM, respectively. The inhibition of the L-type current was voltage-dependent, whereas that of the T-type current was not. Ro 40-5967 blocked T-type current already at a holding potential of -100 mV. The different types of block, i.e., voltage-dependent vs. tonic block, may contribute to the pharmacological profile of Ro 40-5967 in intact animals.[1]

Smooth muscle cells in whole mouse deferens were loaded with the Ca(2+) indicator Oregon Green 488 BAPTA-1 AM and viewed with a confocal microscope. Ryanodine (10 microM) decreased the amplitude of NCTs by 45 +/- 6 %. Cyclopiazonic acid slowed the recovery of NCTs (from a time course of 200 +/- 10 ms to 800 +/- 100 ms). Caffeine (3 mM) induced spontaneous focal smooth muscle Ca(2+) transients (sparks). Neither of the T-type Ca(2+) channel blockers NiCl2 (50 microM) or mibefradil dihydrochloride (10 microM) affected the amplitude of excitatory junction potentials (2 +/- 5 % and -3 +/- 10 %) or NCTs (-20 +/- 36 % and 3 +/- 13 %). In about 20 % of cells, NCTs were associated with a local, subcellular twitch that remained in the presence of the alpha1-adrenoceptor antagonist prazosin (100 nM), showing that NCTs can initiate local contractions. Slow (5.8 +/- 0.4 microm s(-1)), spontaneous smooth muscle Ca(2+) waves were occasionally observed. Thus, Ca(2+) stores initially amplify and then sequester the Ca(2+) that enters through P2X receptors and there is no amplification by local voltage-gated Ca(2+) channels.[2]

Male Sprague-Dawley rats (200-250 g) were used for right L5/6 SNL to induce neuropathic pain. Intrathecal infusion of saline or TCC blockers [mibefradil (0.7 μg/h) or ethosuximide (60 μg/h)] was started after surgery for 7 days. Fluorescent immunohistochemistry and Western blotting were used to determine the expression pattern and protein level of CaV3.2. Hematoxylin-eosin and toluidine blue staining were used to evaluate the neurotoxicity of tested agents.[4]

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The aim of the present study was to investigate the protective effect of T-type calcium channel blockers against presbycusis, using a C57BL/6J mice model. The expression of three T-type calcium channel receptor subunits in the cochlea of 6-8-week-old C57BL/6J mice was evaluated using reverse transcription-quantitative polymerase chain reaction. The results confirmed that the three subunits were expressed in the cochlea. In addition, the capacity of T-type calcium channel blockers to protect the cochlear hair cells of 24-26-week-old C57BL/6J mice was investigated in mice treated with mibefradil, benidipine or saline for 4 weeks. Differences in hearing threshold were detected using auditory brainstem recording (ABR), while differences in amplitudes were measured using a distortion product otoacoustic emission (DPOAE) test. The ABR test results showed that the hearing threshold significantly decreased at 24 kHz in the mibefradil-treated and benidipine-treated groups compared with the saline-treated group. The DPOAE amplitudes in the mibefradil-treated group were increased compared with those in the saline-treated group at the F2 frequencies of 11.3 and 13.4 kHz. Furthermore, the DPOAE amplitudes in the benidipine-treated group were increased compared with those in the saline-treated group at an F2 frequency of 13.4 kHz. The loss of outer hair cells (OHCs) was not evident in the mibefradil-treated group; however, the stereocilia of the inner hair cells (IHCs) were disorganised and sparse. In summary, these results indicate that the administration of a T-type calcium channel blocker for four consecutive weeks may improve the hearing at 24 kHz of 24-26-week-old C57BL/6J mice. The function and morphology of the OHCs of the C57BL/6J mice were significantly altered by the administration of a T-type calcium channel blocker; however, the IHCs were unaffected.[3]

Absorption
The bioavailability after a single dose is 70%. After multiple doses, the proportion of first-pass metabolism of mibepidil decreases, with a steady-state bioavailability of approximately 90%. Food does not affect the rate or extent of absorption of mibepidil.

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Metabolism/Metabolites
Mibepidil is metabolized via two pathways: esterase-catalyzed hydrolysis of the ester side chain (producing alcohol metabolites) and cytochrome P450 3A4-catalyzed oxidation (the importance of this pathway decreases during long-term administration). The pharmacological effects of the metabolites are approximately 10% of those of the parent drug mibepidil.

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Biological Half-Life
17 to 25 hours at steady state.

Protein binding rate: ≥ 99%, mainly binding to α1-acidic glycoprotein.

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Oral LD50 in rats >800 mg/kg, Cardiovascular Drug Review, 9(4), 1991
Intravenous LD50 in rats 23 mg/kg, Cardiovascular Drug Review, 9(4), 1991
Oral LD50 in mice >800 mg/kg, Cardiovascular Drug Review, 9(4), 1991
Intravenous LD50 in mice 35 mg/kg, Cardiovascular Drug Review, 9(4), 1991

[1]. Martin RL, Lee JH, Cribbs LL, Perez-Reyes E, Hanck DA. Mibefradil block of cloned T-type calcium channels. J Pharmacol Exp Ther. 2000 Oct;295(1):302-8.

[2]. The Ca(++)-channel blocker Ro 40-5967 blocks differently T-type and L-type Ca++ channels. J Pharmacol Exp Ther. 1994 Dec;271(3):1483-8.

[3]. The sources and sequestration of Ca(2+) contributing to neuroeffector Ca(2+) transients in the mouse vas deferens. J Physiol. 2003 Dec 1;553(Pt 2):627-35.

Mibepidil belongs to the tetrahydronaphthyl class of drugs and is a T-type calcium channel blocker. Mibepidil was withdrawn from the market in 1998 due to potential harmful interactions with other drugs. It is a benzimidazole-substituted tetrahydronaphthyl compound that selectively binds to and inhibits T-type calcium channels. Indications: Used to treat angina pectoris and hypertension. Pharmacodynamics: Mibepidil belongs to the calcium channel blocker class of drugs. Calcium channel blockers affect the movement of calcium ions into heart and vascular cells. Therefore, they can dilate blood vessels, increase blood and oxygen supply to the heart, and reduce the burden on the heart. Mibepidil is a benzimidazole-substituted tetrahydronaphthyl compound that selectively binds to and inhibits T-type calcium channels. Mechanism of Action: Mibepidil is a tetrahydronaphthol calcium channel blocker that inhibits the influx of calcium ions through T (low voltage) and L (high voltage) calcium channels in myocardial and vascular smooth muscle, with higher selectivity for T channels. Mibepidil causes vasodilation, reducing peripheral vascular resistance and thus lowering blood pressure. Long-term use of mibepidil can slightly increase cardiac output. Mibepidil slows conduction in the sinoatrial and atrioventricular nodes, resulting in a slight decrease in heart rate and a slight prolongation of the PR interval. Studies have also shown that mibepidil can slightly prolong the corrected sinoatrial node recovery time and AH interval, and increase the Wenckebach point. The mechanism by which mibepidil relieves angina is not fully understood, but it is generally believed that its mechanism of action is related to reducing heart rate, total peripheral resistance (afterload), and the heart rate-systolic blood pressure product at any exercise intensity. The result of these effects is a reduction in cardiac load and myocardial oxygen consumption.

585.25364

1049728-52-0

Mibefradil dihydrochloride;116666-63-8;Mibefradil;116644-53-2

16219666

Typically exists as solid at room temperature

IZSWBBGKLWFDOC-YKXHUFBBSA-N

InChI=1S/C29H38FN3O3.2ClH.H2O/c1-20(2)28-23-12-11-22(30)18-21(23)13-14-29(28,36-27(34)19-35-4)15-17-33(3)16-7-10-26-31-24-8-5-6-9-25(24)32-26;;;/h5-6,8-9,11-12,18,20,28H,7,10,13-17,19H2,1-4H3,(H,31,32);2*1H;1H2/t28-,29-;;;/m0.../s1

[(1S,2S)-2-[2-[3-(1H-benzimidazol-2-yl)propyl-methylamino]ethyl]-6-fluoro-1-propan-2-yl-3,4-dihydro-1H-naphthalen-2-yl] 2-methoxyacetate;hydrate;dihydrochloride

Mibefradil (dihydrochloride hydrate); Mibefradil dihydrochloride hydrate;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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 (Please use freshly prepared in vivo formulations for optimal results.)