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CELLULAR AND MOLECULAR

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

Department of Pharmacology, Tulane University Health Sciences Center, New Orleans, Louisiana (L.H., B.M.K., J.T.T., M.L.); Department of Medicinal Chemistry, Novo Nordisk A/S, Måløv, Denmark (T.M.T., J.B.H.); Department of Anesthesiology and Critical Care Medicine, Johns Hopkins University, Baltimore, Maryland (H.Z.); Department of Pharmacology, Medical College of Virginia, Richmond, Virginia (M.Z.); and Department of Neurology and Neurosurgery, Montreal Neurological Institute, McGill University, Montreal, Quebec, Canada (D.S.R.).

Received October 2, 2003; accepted December 5, 2003.

	Abstract

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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-tetrahydro-1-isopropyl-2-naphtyl cyclopropanecarboxylate dihydrochloride (NNC 55-0396), that exerts a selective inhibitory effect on T-type channels. The acute IC₅₀ of NNC 55-0396 to block recombinant ₁G T-type channels in human embryonic kidney 293 cells was 7 µM, whereas 100 µM 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.

Mibefradil, a tetralol derivative chemically distinct from other Ca²⁺ channel antagonists, has been reported to block T-type Ca²⁺ channel currents in many tissues, including heart (Madle et al., 2001), brain (McDonough and Bean, 1998), and vascular smooth muscle (Bian and Hermsmeyer, 1993; Mishra and Hermsmeyer, 1994; Schmitt et al., 1995). Mibefradil was also reported to block HVA Ca²⁺ channels, including ₁A, ₁B, ₁C, and ₁E (Bezprozvanny and Tsien, 1995; Jiménez et al., 2000). We previously demonstrated that mibefradil potently blocked HVA Ca²⁺ channels in a rat insulin-secreting cell line (INS-1 cells) via a mechanism involving intracellular hydrolysis of mibefradil to produce an active metabolite (Wu et al., 2000).

The cardiovascular effects of mibefradil are interesting; for instance, it decreases heart rate without a negative inotropic effect (Osterrieder and Holck, 1989; Cremers et al., 1997), and its action is not associated with the reflex activation of neurohormonal and sympathetic systems (Ernst and Kelly, 1998). These properties differ from other clinically important Ca²⁺ antagonists such as nifedipine, diltiazem, and verapamil, which in the heart selectively inhibit L-type (₁C) Ca²⁺ channels. However, it is unclear whether the unique effects of mibefradil are caused by the blockade of T-type Ca²⁺ channels since mibefradil also blocks the L-type Ca²⁺ currents in the cardiomyocytes (Leuranguer et al., 2001). Therefore, identifying a more selective T-type Ca²⁺ channel antagonist will be useful for the study of cardiac voltagegated calcium channels and may promote the development of a new class of therapeutically beneficial compounds.

In a previous study, we demonstrated that hydrolysis of the ester side chain of mibefradil resulted in a compound (des-methoxyacetyl mibefradil or Ro 40-5966) that exhibited an L-type Ca²⁺ channel-specific inhibitory effect (Wu et al., 2000). We thus proposed that modifications in this ester side chain, which decreased hydrolysis, might result in compounds with a lower potency in blocking L-type Ca²⁺ channels and selective action on T-type channels. To test this hypothesis, we examined the effects of several novel mibefradil derivates on T-type and HVA Ca²⁺ currents in whole-cell voltage-clamp recordings. We used HEK 293 cells stably transfected with the ₁G subtype of T-type Ca²⁺ channel (HEK/₁G) and INS-1 cells to test the effects of our new compounds on T-type and HVA Ca²⁺ channels, respectively. INS-1 cells express a variety of HVA Ca²⁺ currents, including P/Q-, N-, L-, and R-types (Horvath et al., 1998), with ₁D contributing most of the currents (Liu et al., 2002; Huang et al., 2004).

	Materials and Methods

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Cell Culture. INS-1 cells, an insulin-secreting cell line derived from rat pancreatic -cells (Asfari et al., 1992) were cultured in RPMI 1640 medium containing 10% fetal bovine serum, 100 U/ml penicillin, 100 µg/ml streptomycin, and 50 µM mercaptoethanol in an atmosphere of 5% CO₂ in air at 37°C for 2 to 5 days before recording.

Creation of HEK 293 Cell Lines Stably Expressing Recombinant T-Type Ca²⁺ Channels. An ₁G cDNA originally cloned from rat pancreatic -cells (Zhuang et al., 2000) in vector pcDNA3.1 hygro(-) (Invitrogen, Carlsbad, CA) was transfected into HEK cells using the FuGENE kit (Roche Diagnostics, Indianapolis, IN). Cell lines stably expressing Ca_v3.1 were obtained after transfection using standard cell cloning techniques (Freshney, 1983). HEK 293 cells stably transfected with ₁G cDNA (HEK 293/₁G) were incubated in Dulbecco's modified Eagle's medium, supplemented with 10% fetal bovine serum, 100 U/ml penicillin, 100 µg/ml streptomycin, and 0.5 mg/ml hygromycin-B in an atmosphere of 5% CO₂ in air at 37°C for 2 to 5 days before recording.

Electrophysiological Recording. Whole-cell recordings were carried out by the standard "giga-seal" patch-clamp technique. The whole-cell recording pipettes were made of hemocapillaries (Warner Instrument, Hamden, CT), pulled by a two-stage puller (PC-10; Narishige, Greenvale, NY), and heat-polished with a microforge (MF-200; World Precision Instruments, Sarasota, FL) before use. Pipette resistance was in the range of 2 to 5 M in our internal solution. The recordings were performed at room temperature (22–25°C). Electrical currents were recorded using an EPC-9 patch-clamp amplifier (HEKA, Lambrecht/Pfalz, Germany) and filtered at 2.9 kHz. Data were acquired with PULSE/PULSEFIT software (HEKA). Voltage-dependent currents were corrected for linear leak and residual capacitance by using an on-line P/n subtraction paradigm. In the whole-cell configuration, T-type Ca²⁺ currents were recorded at -20 or -10 mV when the holding potential was -70 mV. The HVA Ca²⁺ current was measured at 10 mV, with a holding potential of -40 mV.

Solutions. The extracellular solution used in the whole-cell Ca²⁺ current recording contained 10 mM CaCl₂, 110 mM tetraethylammonium-Cl, 10 mM CsCl, 10 mM HEPES, 40 mM sucrose, 0.5 mM 3,4-diaminopyridine, pH 7.3. The intracellular solution contained 130 mM N-methyl-D-glucamine, 20 mM EGTA (free acid), 5 mM 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetate, 10 mM HEPES, 6 mM MgCl₂, and 4 mM Ca(OH)₂, with pH adjusted to 7.4 with methanesulfonate. Mg-ATP (2 mM) was included in the pipette solution to minimize rundown of L-type Ca²⁺ currents. For perforated-patch experiments, 200 µg/ml nystatin was used. The pipette was filled with nystatin-containing intracellular solution, and gentle suction was used to achieve gigaohm resistance. The access resistance gradually decreased within 5 min after the gigaohm-seal formation, and then currents were recorded after stabilization. The extracellular solution contained 26 mM sucrose, 130 mM tetraethylammonium-Cl, 10 mM HEPES, 5 mM KCl, 2 mM CaCl₂, 2 mM MgCl₂, pH 7.3. The pipette solution contained 65 mM CsOH, 65 mM Cs-methanesulfonate, 20 mM sucrose, 10 mM HEPES, 10 mM MgCl₂, and 1 mM Ca(OH)₂, with pH 7.4.

Mass Spectrometric Analysis. Mass spectrometric analysis was performed on a PerSeptive Voyager-DE MALDI-TOF instrument (Applied Biosystems, Foster City, CA). Cultured INS-1 cells were treated with 20 µM NNC 55-0396 for various lengths of time under each experimental condition. After incubation, cells were washed with PBS three times, scraped into Eppendorf tubes with 1 ml of PBS, and centrifuged at 1000g for 5 min. The cell pellets were collected and redissolved by sonication (10 s) in 0.5 ml of PBS for mass spectrometric analysis. The sample was taken up in -cyano-4-hydroxcinnamic acid (Aldrich, Milwaukee, MI), which was used as the matrix. Ten microliters of each sample were mixed with 70 µl of the matrix. One microliter of the mixture was spotted on a plate for analysis on the MALDI-TOF instrument. Several positive ion spectra were recorded in the mass range m/z 820 to 99 at a mass resolution of 1000 and a scan speed of 2 s/decade. For NNC 55-0396, m/z 492 was the dominant ion (M + H). For calibration, a standard solution of 50 µM NNC 55-0396 was subjected to mass spectrometric analysis. The relative amount of NNC 55-0396 was determined by calibrating the intensities of NNC 55-0396 with the intensity of standard solution.

Statistics and Curve Fitting. Nonlinear regression analysis was used to fit concentration-response data to a sigmoid relationship, Y = 100/(1 + 10^((log IC₅₀ - X) slope), where slope is the Hill slope parameter, IC₅₀ is the concentration producing 50% blockage, and X is the drug concentration. Voltage-dependent activation and steady-state inactivation curves were generated by normalizing the currents with the maximal amplitude in each cell and fitting the data with the Boltzmann equation, 1/(1 + exp((V - V_1/2)/k), where k represents the slope and V_1/2 represents the voltage corresponding to half-activation of the channels. Student's t test was used to compare V_1/2 and k values determined from fits of the data with this equation. The data fitting and statistical analysis were performed with Prism version 4 (GraphPad Software, San Diego, CA). In the figures where the data are presented as symbols and error bars, the values are mean ± S.E.M.

General Chemical Procedure. Reagents, starting materials, and solvents were purchased from common commercial suppliers and were used as received. All dry solvents were dried over molecular sieves (0.3 or 0.4 nm). Evaporation was carried out on a rotary evaporator at bath temperatures <40°C and under appropriate vacuum. Flash chromatography was carried out on a Biotage FLASH 40 (Biotage AB, Uppsala, Sweden) using Biotage FLASH columns (KP-SIL 60 Å particle size, 32–63 µM). Melting points were determined with a Büchi B545 apparatus (Büchi, Flawil, Switzerland) and are uncorrected. Proton NMR spectra were recorded at ambient temperature using a Bruker AVANCE DPX 200 (Bruker, Newark, DE) (200 MHz) and DPX 300 (300 MHz), with tetramethylsilane as an internal standard for proton spectra. Chemical shifts are given in ppm (), and splitting patterns are designated as follows: s, singlet; d, doublet; dd, double doublet; t, triplet; dt, double triplet; q, quartet; quint, quintet; m, multiplet; and br, broad. The 70-electron-volt EI solid mass spectra were recorded on a Finnigan MAT TSQ 70 mass spectrometer (Thermo Finnigan, San Jose, CA). Reactions were followed by thin layer chromatography performed on silica gel 60 F254 (Merck, Darmstadt, Germany) or ALUGRAM SIL G/UV₂₅₄ (Macherey-Nagel, Düren, Germany) thin layer chromatography aluminum sheets.

Synthesis of NNC 55-0396. Methoxyacetic acid (2(S)-[2-[N-[3-(2-benzimidazolyl)propyl]-N-methylamino]ethyl]-6-fluoro-1(S)-isopropyl-1,2,3,4-tetrahydro-2-naphthyl ester dihydrochloride) (mibefradil, 0.570 g) in ethanol (96%, 5 ml) and aqueous sodium hydroxide (1 N, 5 ml) was refluxed for 2 h. The cold reaction mixture was concentrated in vacuo. The residue was partitioned between water and dichloromethane. The aqueous layer was extracted with dichloromethane. The combined organic layers were dried with sodium sulfate and concentrated to give 2-(2-[[3-(1-benzimidazol-2-yl)-propyl]methylamino]ethyl)-6-fluoro-1-isopropyl-1,2,3,4-tetrahydro-2-naphthalinol as a clear syrup (100%, 0.43 g). ¹H NMR (CDCl₃): 7.57 (br, 2H); 7.23 (m, 2H); 6.97 (m, 1H); 6.58 (m, 2H); 3.07–2.83 (m, 3H); 2.75 (m, 1H); 2.6 (m, 4H); 2.5–2.2 (s + m, 3H + 3H); 2.06 (quint, 2H); 1.81 (br dd, 1H); 1.50 (m, 2H); 1.20 (d, 3H); 0.53 ppm (d, 3H).

2-(2-{[3-(1-Benzimidazol-2-yl)-propyl]methylamino}ethyl)-6-fluoro-1-isopropyl-1,2,3,4-tetrahydro-2-naphthalinol (0.110 g) was dissolved in 1 ml of dichloromethane. Diisopropylethylamine (0.045 ml) and 0.071 ml of cyclopropanecarbonyl chloride was added. After stirring for 19 h, the reaction mixture aqueous saturated sodium hydrogencarbonate was added. The aqueous layer was extracted twice with dichloromethane. The combined organic layers were dried with sodium sulfate and concentrated in vacuo. The residue was purified by flash chromatography using dichloromethane/methanol 6:1 as an eluent to give the free base as a syrup. This product was dissolved in ethanol, and aqueous hydrochloride (1 N, 0.38 ml) was added. After stirring for 30 min, the mixture was concentrated. The residue was crystallized from ethyl acetate to give the title compound as a white powder (34%, 50 mg). Mp: 134 to 141°C. EI SP/MS: 491 (M+). ¹H NMR (DMSO— d6): 7.77 (m, 2H); 7.52 (m, 2H); 7.07 (m, 1H); 6.96 (broad d, 2H); 3.3 (m, 2H); 3.2 (m, 4H); 3.0 (m, 2H); 2.9 (m, 1H); 2.67 (s, 3H); 2.45 (m, 1H); 2.38 (m, 2H); 2.15–1.45 (m, 4H); 1.57 (m, 1H); 1.04 (d, 3H); 0.90 (m, 4H); 0.48 ppm (d, 3H).

Synthesis of (1S,2S)-2-(2-(N-[(3-Benzimidazol-2-yl)propyl]-N-methylamino)ethyl)-6-fluoro-1,2,3,4-tetrahydro-1-isopro-pyl-2-naphtyl valeroate dihydrochloride (NNC 55-0395). NNC 55-0395 was prepared by an analogous procedure: Mp 118 to 121°C. EI SP/MS: 507 (M+).¹H NMR (DMSO-d₆): 7.75 (m, 2H); 7.49 (m, 2H); 7.08 (m, 1H); 6.96 (br d, 2H); 3.15 (m, 4H); 2.95 (m, 3H); 2.7 (s, 3H); 2.48 (dt, 2H); 2.23 (m, 2H); 2.0 (m, 4H); 1.50 (p, 2H); 1.35 (quint, 2H); 1.02 (d, 3H); 0.90 (t, 3H); 0.35 ppm (d, 3H).

Synthesis of (1S,2S)-2-(2-(N-[(3-Benzimidazol-2-yl)propyl]-N-methylamino)ethyl)-6-fluoro-1,2,3,4-tetrahydro-1-isopro-pyl-2-naphtyl isobutyrate dihydrochloride (NNC 55-0397). NNC 55-0397 was prepared by an analogous procedure: Mp 114 to 117°C. EI SP/MS: 493 (M+). ¹H NMR (DMSO-d₆): 7.77 (m, 2H); 7.52 (m, 2H); 7.07 (m, 1H); 6.96 (br d, 2H); 3.43 (m, 1H); 3.3-3.05 (m, 5H); 2.97 (m, 2H); 2.88 (m, 1H); 2.70 (s, 3H); 2.61 (m, 1H); 2.45 (m, 1H); 2.3 (m, 2H);2.15–1.85 (m, 4H); 1.15 (d, 6H); 1.00 (d, 3H); 0.45 ppm (d, 3H).

	Results

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Synthesis of NNC 55-0396 and Other Mibefradil Analogs. We synthesized three analogs of mibefradil by replacing the ester chain (methoxyacetyl) group NNC 55-0395, NNC 55-0396, and NNC 55-0397 (Fig. 1). All compounds were synthesized from mibefradil in two steps. Alkaline hydrolysis of mibefradil and subsequent treatment with valeroyl chloride, cyclopropanecarbonyl chloride, and isobutyryl chloride gave the desired compounds NNC 55-0395, NNC 55-0396, and NNC 55-0397 upon workup, respectively.

View larger version (19K): [in this window] [in a new window]   Fig. 1. Synthesis of NNC 55-0396 and other analogs from mibefradil and chemical structures of NNC 55-0395, NNC 55-0396, and NNC 55-0397. The side chain of mibefradil inside the dashed box is replaced by the new structures indicated by arrows.

Pharmacological-Screening Effect of NNC 55-0395, NNC 55-0396, and NNC 55-0397 on T-Type and HVA Ca²⁺ Currents. We used perforated whole-cell patch-clamp to examine the effects of NNC 55-0395, NNC 55-0396, and NNC 55-0397 on HVA Ca²⁺ current in INS-1 cells. In these experiments, the resting membrane potential was held at -40 mV to eliminate T-type Ca²⁺ current. The inhibitory potency of the mibefradil analogs on HVA Ca²⁺ current was examined at dosages of 0.1, 1, 10, and 100 µM approximately 10 min after the drug perfusion. The results showed that both NNC 55-0395 and NNC 55-0397 had an inhibitory effect on HVA Ca²⁺ currents at 100 µM (Fig. 2, A and C), whereas no inhibition on the HVA Ca²⁺ current was detected with NNC 55-0396 at the same concentration (Fig. 2B).

View larger version (20K): [in this window] [in a new window]   Fig. 2. Pharmacological-screening effect of NNC 55-0395, NNC 55-0396, and NNC 55-0397 on HVA and T-type Ca2+ currents. A, B, and C, dose-dependent effects of the three compounds on HVA currents recorded with perforated whole-cell patch-clamp in INS-1 cells with a holding potential of -40 mV and a test pulse at 10 mV for 200 ms for every 30 to 40 s. The relative current values were obtained by normalizing the current amplitudes with respect to those recorded under the drug-free condition. D, E, and F, dose-dependent effects of the three compounds on T-type Ca2+ currents recorded with whole-cell patch-clamp in HEK 293/1G cells with a holding potential of -70 mV and a testing potential of -20 mV for 200 ms (n = 3–5 for each set of data).

Next, we determined the effects of NNC 55-0395, NNC 55-0396, and NNC 55-0397 on T-type Ca²⁺ current. These experiments were conducted by measuring the effects of the compounds on whole-cell current in HEK 293/₁G cells. The dose-dependent inhibition of compounds NNC 55-0395, NNC 55-0396, and NNC 55-0397 on T-type Ca²⁺ current was examined at dosages ranging from 1 to 100 µM. NNC 55-0396 and NNC 55-0397 blocked more than 50% of the T-type Ca²⁺ current at 8 µM (Fig. 2, E and F), whereas NNC 55-0395 inhibited less than 50% of the T-type Ca²⁺ current at 64 µM (Fig. 2D).

Both NNC 55-0395 and NNC 55-0397 blocked HVA Ca²⁺ channel currents in our screening experiments and were thus eliminated from further characterization. Compound NNC 55-0396, which blocked T-type Ca²⁺ current but not HVA Ca²⁺ currents, was selected for subsequent analysis.

Characterization of the Inhibitory Effects of NNC 55-0396 on T-Type Ca²⁺ Current. To further characterize NNC 55-0396, we used whole-cell patch-clamp and a bath perfusion system to examine its dose-dependent effects on T-type and HVA Ca²⁺ currents. At 8 µM, NNC 55-0396 reduced more than 50% of the peak of T-type Ca²⁺ current compared with the control in HEK 293/₁G cells (Fig. 3A); however, NNC 55-0396 at 100 µM did not block the HVA Ca²⁺ current in INS-1 cells (Fig. 3B), whereas this HVA Ca²⁺ current could be inhibited by 10 µM nifedipine, a selective L-type Ca²⁺ channel blocker (Fig. 3C). Pooled data of the effects of NNC 55-0396 on T-type and HVA Ca²⁺ currents are shown in Fig. 3D. After bathing HEK 293/₁G cells with NNC 55-0396 at 8 µM or bathing INS-1 cells with NNC 55-0396 at 100 µM for over 8 min, T-type Ca²⁺ currents were inhibited by 60%, whereas no decrease in the HVA Ca²⁺ current was observed.

View larger version (23K): [in this window] [in a new window]   Fig. 3. The effects of NNC 55-0396 on T-type and HVA Ca2+ channels. A, current traces of T-type Ca2+ currents in an HEK 293/1G cell before and after application of 8 µM NNC 55-0396. The holding potential was -70 mV, and the test potential was -20 mV. B, current traces of HVA Ca2+ currents in an INS-1 cell before and after 100 µM NNC 55-0396. The currents were recorded at 10 mV for 200 ms when held at -40 with perforated whole-cell patch-clamp. The traces were filtered at 1 kHz. C, Ca2+ current was inhibited by 10 µM nifedipine in an INS-1 cell. The currents were measured at 10 mV before and after nifedipine (Nif) administration with a holding potential of -70 mV. D, time-dependent effects of NNC 55-0396 on HVA and T-type Ca2+ currents. The open circles represent relative currents of HVA Ca2+ channels recorded in INS-1 cells before and after 100 µM NNC 55-0396 administration (n = 6), whereas the solid circles represent relative currents recorded in HEK 293/1G cells before and after 8 µM NNC 55-0396 administration (n = 4). NNC 55-0396 was applied to the bath 3 min after the beginning of the recording. E, dose-dependent inhibitory effects of NNC 55-0396 (open circles, n = 6) and mibefradil (solid circles, n = 4) on T-type Ca2+ currents recorded in HEK/1G cells. The smooth lines are consistent with the Hill equation, with IC50 values of 6.8 and 10.08 µM for NNC 55-0396 and mibefradil, respectively. The Hill slopes are -2.39 and -4.26 for NNC 55-0396 and mibefradil, respectively. F, comparison of current densities (pA/pF) among HEK/1G cells incubated in various concentrations of NNC 55-0396 for 30 to 60 min. The peak-current amplitudes were measured at -20 mV when held at -70 mV (n = 7–11 for each set of data). The asterisk represents p < 0.01 of the data compared with the control (0 nM NNC 55-0396).

The inhibitory potency of NNC 55-0396 on T-type Ca²⁺ currents in HEK 293/₁G was also compared with that of mibefradil, as shown in Fig. 3E. By fitting the data with a sigmoid dose-response relationship equation, NNC 55-0396 and mibefradil blocked T-type Ca²⁺ current with IC₅₀ values of 6.8 and 10.08, respectively. This result suggests that NNC 55-0396 retains potency similar to mibefradil as an inhibitor of T-type Ca²⁺ current.

Long-term exposure of HEK/₁G cells to NNC 55-0396 showed a decrease in current density of T-type Ca²⁺ channels. Using whole-cell patch-clamp, we measured T-type Ca²⁺ current density in the HEK/₁G cells that had bathed in the extracellular solutions containing 0, 10, 100, and 1000 nM NNC 55-0396 for 30 to 60 min (Fig. 3F). All peak-current amplitudes and slow capacitances were obtained during the first minute after breaking in; thus, the effect of the frequency-dependent inhibitory effect was minimal. Our data showed a slow yet more potent inhibitory effect of NNC 55-0396 on the T-type Ca²⁺ currents.

Figure 4A illustrates that there was no significant difference in the conductance-voltage relationship (G/V) curves of T-type Ca²⁺ currents between the control cells and cells incubated with 8 µM NNC 55-0396. The conductance (G) was calculated from the current (I) divided by the driving force (V_drive = V_membrane - V_reversal), where the V_reversal was hypothetically assigned as 70 mV. Curves were generated by fitting the data with the Boltzmann equation. The V_1/2 and k values are -32.61 ± 1.0 and 4.16 ± 0.9 for the controls (n = 3) and -32.12 ± 0.8 and 4.33 ± 0.73 for cells incubated with NNC 55-0396 (n = 3), respectively. The steady-state inactivation curves of T-type Ca²⁺ current were also generated with the Boltzmann equation (Fig. 4B). The V_1/2 values are -59.21 ± 0.38 and -62.84 ± 0.94 for the controls (n = 4) and cells incubated with NNC 55-0396 (n = 6), respectively. The slope k values are 4.92 ± 0.33 with a 95% confidence interval between 4.16 to 5.69 for the controls and 7.95 ± 0.72 with a 95% confidence interval between 6.31 to 9.58 for cells incubated with NNC 55-0396, respectively. Therefore, the steady-state inactivation curve of T-type Ca²⁺ current is flattened when NNC 55-0396 is present.

View larger version (13K): [in this window] [in a new window]   Fig. 4. Effects of NNC 55-0396 on voltage-dependent activation (G/V) and steady-state inactivation of T-type Ca2+ channel. A, voltage-dependent activation (G/V) curves of T-type Ca2+ channel with (solid circles) and without (open circles) the presence of 8 µM NNC 55-0396 were fit by the Boltzmann equation (n = 3). B, steady-state inactivation curves of T-type Ca2+ channel with (solid circles) and without (open circles) the presence of 8 µM NNC 55-0396 were fit by the Boltzmann equation (n = 6). Steady-state inactivation was determined by applying a prestimulating pulse of 1.5 s at various voltages immediately before the test pulse at -20 mV. For both A and B, currents were recorded in HEK 293/1G cells, and the cell membrane was held at -70 mV.

State-Dependent Block of T-Type Ca²⁺ Current by NNC 55-0396. The inhibitory mechanism of NNC 55-0396 on T-type Ca²⁺ current was further investigated by testing the effect of changing the holding potential and stimulation frequency. As shown in Fig. 5A, in the absence of NNC 55-0396, changing the holding potential from -120 to -80 mV did not alter the peak amplitude of T-type Ca²⁺ current in an HEK 293/₁G cell, elicited by depolarizations to -10 mV every 10 s. In contrast, after the addition of 8 µM NNC 55-0396, switching the membrane potential from -80 to -120 mV caused an increase in the current amplitude, suggesting a partial relief of block at the more hyperpolarized holding potential. Similarly, increasing the pulse rate from 0.05 to 0.5 Hz had little effect on current amplitude without NNC 55-0396, whereas the rate of inhibition of T-type Ca²⁺ current was accelerated by increasing the frequency of stimulation in the presence of the drug (Fig. 5B). Similar results of both voltage-dependent and frequency-dependent blockade were observed in four experiments.

View larger version (15K): [in this window] [in a new window]   Fig. 5. State-dependent inhibition of T-type Ca2+ channels by NNC 55-0396. A, whole-cell Ca2+ currents were measured at -10 mV when held at -120 and -80 mV, as indicated by the bar above the graph. The pulses were applied every 10 s. B, whole-cell Ca2+ currents were measured at -10 mV when held at -70 mV at stimulation frequencies of 0.05 and 0.5 Hz, as indicated by the bar above the graph. NNC and the arrows indicate the time when NNC 55-0396 was perfused into the bath.

Mass Spectrometric Analysis of NNC 55-0396-Treated INS-1 Cells. Previously, we found that mibefradil blocked both T-type and HVA Ca²⁺ current in INS-1 cells (Wu et al., 2000). After entering the cells, mibefradil is broken down into metabolites. One of them is des-methoxyacetyl mibefradil, which exerts an inhibitory effect on HVA Ca²⁺ channels by acting on the channels from inside the cell. To investigate why NNC 55-0396 and mibefradil have different effects on HVA Ca²⁺ channels, we used mass spectrometric analysis to examine whether des-methoxyacetyl mibefradil was produced intracellularly when the cells were treated with NNC 55-0396. Figure 6A shows a section of mass spectrum of molecules in INS-1 cells that had been incubated with NNC 55-0396 for 20 min. Notably, no des-methoxyacetyl mibefradil (which peaks at 424 m/z) was detected in this preparation. This finding suggests that, unlike mibefradil, the compound NNC 55-0396 is not hydrolyzed into des-methoxyacetyl mibefradil. Thus, NNC 55-0396 does not inhibit HVA Ca²⁺ channels as mibefradil does. The amount of NNC 55-0396 (which peaks at 492 m/z), however, increased in the cells for longer periods than 10 min as shown in Fig. 6B.

View larger version (17K): [in this window] [in a new window]   Fig. 6. Mass spectrometric analysis of INS-1 cells treated with NNC 55-0396. A, no ester side chain hydrolytic metabolite (peak is predicted at 424 m/z) was detected in INS-1 cells after 30 min of incubation with 20 µM NNC 55-0392, which peaked at 492 m/z. B, time-related relative intensity of NNC 55-0396 in INS-1 cells. Mass spectrometry was performed on the cells that had been incubated in 20 µM NNC 55-0396 for various time periods and then washed three times. The relative intensity is presented as the ratio of the energy power of the laser between the NNC 55-0396 found inside the cells and the standard solution containing 50 µM NNC 55-0396 (n = 4).

Accessibility of NNC 55-0396 on the T-Type Ca²⁺ Channel. The slow onset of NNC 55-0396 inhibition on T-type Ca²⁺ current and the increasing NNC 55-0396 accumulation in the cells with time suggest that this compound dissolves in or passes through the plasma membrane to exert its effect. To test this hypothesis, we examined the reversibility of the NNC 55-0396 blockade of T-type Ca²⁺ current. Whole-cell currents were evoked in HEK 293/₁G cells by test pulses to -10 mV from a holding potential of -70 mV. After establishing a steady current, small volumes of 8 µM NNC 55-0396 were delivered in close proximity to the recording cell with a quartz capillary positioned by a micromanipulator. The drug was washed out after more than a 50% inhibition of T-type Ca²⁺ current was achieved. As shown in Fig. 7A, the blockade of the T-type Ca²⁺ current by NNC 55-0396 was poorly reversible during a 10-min washout period in these experiments. Since the NNC 55-0396 blocks T-type Ca²⁺ current at a relatively slow rate and is poorly reversible by washing out, the drug binding site of the channel may be within transmembrane or intracellular domains of the channel.

View larger version (15K): [in this window] [in a new window]   Fig. 7. Washout and intracellular application of NNC 55-0396. A, inhibitory effect of 8 µM NNC 55-0396 on HEK/1 G T-type Ca2+ currents was not reversed after perfusion with the drug-free extracellular solution (n = 3). B, effect of intracellular application of NNC 55-0396 on HEK/1G T-type Ca2+ currents. The currents were recorded by the whole-cell patch-clamp with a pipette solution containing 8 µM NNC 55-0396 (solid circles, n = 10) or a drug-free solution (open circles, n = 6). All recordings were conducted at a holding potential of -70 mV and a test pulse of -20 mV.

If the site of action of NNC 55-0396 is on the intracellular side of the membrane, we would expect that intracellular perfusion of the drug would effectively block T-type Ca²⁺ current. To test this possibility, whole-cell currents were recorded in HEK 293/₁G cells with 8 µM NNC 55-0396 added to the pipette solution. As shown in Fig. 7B, there was no significant difference between the current in HEK 293/₁G cells with (n = 10) and without (n = 6) NNC 55-0396 included in the pipette solution. For example, at the 10-min timepoint, the p value is 0.12 with two-tailed unpaired Student's t test analysis. These data are inconsistent with the idea that the site of action of NNC 55-0396 is on the cytoplasmic face of the T-type Ca²⁺ channel. Thus, NNC 55-0396 appears to exhibit a relatively slow and wash-resistant binding to a site, perhaps within the transmembrane domains of the channel.

	Discussion

Top Abstract Materials and Methods Results Discussion References

 
Mibefradil blocks both T-type Ca²⁺ and HVA Ca²⁺ channels. Although mibefradil appears to act directly on T-type channels, we previously reported that its effect on HVA channels is not direct but instead involves cell permeation and hydrolysis to an active metabolite that acts from the cytoplasmic side of the membrane (Wu et al., 2000). In the present study, we developed a new compound, NNC 55-0396, that has an inhibitory effect on T-type Ca²⁺ current in HEK/₁G cells but is not hydrolyzed to an active metabolite and does not block HVA Ca²⁺ currents in INS-1 cells. Thus, NNC 55-0396 is a selective inhibitor for T-type Ca²⁺ channels. Our data also suggest that the effect of NNC 55-0396 on T-type channels is state-dependent; i.e., block is partially relieved by membrane hyperpolarization and enhanced by high-frequency channel activation.

Our previous findings (Wu et al., 2000) suggest that the methoxyacetyl group of mibefradil is a critical determinant for binding to HVA channels since its hydrolysis is the critical step in the binding of the compound to HVA channels. In contrast, this moiety may play only a modest role in interacting with the receptor domain of T-type Ca²⁺ channels since all three alternations—NNC 55-0395, NNC 55-0396, and NNC 55-0397—retain the capability of blocking T-type Ca²⁺ current, although with varying potency. Our study indicates that modification in the methoxyacetyl group of mibefradil can improve its selectivity for T-type Ca²⁺ channels over the HVA Ca²⁺ channels without sacrificing the potency of T-type Ca²⁺ channel antagonism.

The primary objective of this study is to examine the structure-selectivity relationship of a small group of mibefradil derivatives. Therefore, we chose HEK/₁G as a clean model system. We do not know how effectively NNC 55-0396 will block other T-type Ca²⁺ channels, nor do we know its potency on ₁G when other auxiliary subunits are present. However, we speculate that NNC 55-0396 will block ₁H and ₁I, for it has been shown that the IC₅₀ values of mibefradil blocking ₁G, ₁H, and ₁I are similar (Martin et al., 2000). In summary, our findings represent an important step toward development of a specific T-type Ca²⁺ inhibitor and thus have potentially important scientific and therapeutic implications.

	Acknowledgements

 
We thank Dr. B. Z. Peterson for constructive criticism on this work. The mass spectrometric analyses were carried out in the Louisiana State University Health Sciences Center New Orleans Core Laboratories (New Orleans, LA).

	Footnotes

 
Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.

DOI: 10.1124/jpet.103.060814.

This study was supported by American Heart Association Grant 0151047B (to M.L.) and by Canadian Institutes of Health Research Grant MT13485 (to D.S.R.).

ABBREVIATIONS: HVA, high-voltage-activated; HEK, human embryonic kidney; NNC 55-0395, (1S,2S)-2-(2-(N-[(3-benzimidazol-2-yl)propyl]-N-methylamino)ethyl)-6-fluoro-1,2,3,4-tetrahydro-1-isopropyl-2-naphtyl valeroate dihydrochloride); 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; NNC 55-0397, (1S,2S)-2-(2-(N-[(3-benzimidazol-2-yl)propyl]-N-methylamino)ethyl)-6-fluoro-1,2,3,4-tetrahydro-1-isopropyl-2-naphtyl isobutyrate dihydrochloride; PBS, phosphate-buffered saline; EI, electron impact; DMSO, dimethyl sulfoxide; G, conductance; MALDI-TOF, matrix-assisted laser desorption ionization/time of flight instrument.

Address correspondence to: Dr. Ming Li, Department of Pharmacology SL-83, 1430 Tulane Avenue, New Orleans, LA 70112. E-mail: mli{at}tulane.edu

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