Comparison of mibefradil and derivative NNC 55-0396 effects on behavior, cytochrome P450 activity, and tremor in mouse models of essential tremor

Abstract

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], is a mibefradil derivative that retains potent in vitro T-type calcium channel antagonist efficacy. We compared the two compounds for behavioral toxicity, effects on cytochrome P450 activity, and efficacy against tremor in the γ-aminobutyric acid type A (GABAA) receptor subunit α1-null mouse, and the harmaline tremor model of essential tremor in wild-type mice. NNC 55-0396 was better tolerated than mibefradil in the horizontal wire test of sedation/motor function, with 3/6 failing at 300 and 30mg/kg respectively. To assess for a potential interaction with harmaline, mice were given the drugs, followed by harmaline or vehicle, and tested 30min later in the inverted wire grid test. Mibefradil exacerbated, whereas NNC 55-0396 ameliorated harmaline-induced test deficits. In mouse liver microsomes, NNC 55-0396 was a less potent inhibitor of harmaline O-demethylation than mibefradil (Ki: 0.95 and 0.29μM respectively), and also less potent at inhibiting testosterone 6-β-hydroxylation (Ki: 0.71 and 0.12μM respectively). In the GABAA α1-null model, NNC 55-0396 but not mibefradil, (each at 20mg/kg), suppressed tremor while NNC 55-0396 at 12.5mg/kg suppressed harmaline-induced tremor by half by 20-100min, whereas mibefradil at the same dose did not significantly affect tremor. In contrast to mibefradil, NNC 55-0396 is well tolerated and suppresses tremor, and exerts less cytochrome P450 inhibition. These results suggest potential clinical utility for NNC 55-0396 or similar derivatives as a T-type calcium antagonist.

Fig. 1

Structures of mibefradil and NNC 55-0396.

Fig. 2

Effect of drugs on retention time by mice on an inverted wire grid. Mice were pre-treated with saline vehicle (V), mibefradil (M), 20 mg/kg, or NNC 55-0396 (N), 20 mg/kg i.p., then 30 min later administered saline vehicle (n = 10, 14, 7 respectively) or harmaline, 20 mg/kg s.c. (n = 7, 11, 8 respectively). The duration of time mice held on to an inverted wire grid was assessed 30 min after vehicle or harmaline. Unless otherwise indicated, comparisons are with the vehicle-pre-treated subgroup of the same group. Means and S.E.M.s are shown. ** P < 0.01 *** P < 0.001.

Fig. 3

Comparison of mibefradil and NNC 55-0396 effects on harmaline metabolism. A. Effect of mibefradil and NNC 55-0396 at various concentrations on harmaline O-demethylation to harmalol by mouse liver microsomes, as measured by the area under the curve (AUC) from HPLC chromatograms. The K_i of mibefradil inhibition of harmaline demethylation is less than that of NNC 55-0396 (P = 0.02). B. Effect of the mibefradil metabolite Ro 40-5966 on harmaline O-demethylation to harmalol. This K_i is also less than of NNC 55-0396 (P = 0.02), indicating that mibefradil and its primary metabolite are more prone to inhibit harmaline metabolism than NNC 55-0396. Each K_i, based on duplicate determinations, is expressed as the mean ± S.D.

Fig. 4

Effect of mibefradil and NNC 55-0396 at various concentrations on testosterone 6-β-hydroxylation by mouse liver microsomes, as measured by the area under the curve (AUC) from HPLC chromatograms. The Ki of mibefradil inhibition is less than that of NNC 55-0396 (P = 0.01). Each Ki is based on duplicate determinations and expressed as the mean ± S.D.

Fig. 5

Effect of NNC 55-0396 and mibefradil on tremor in the GABAA receptor α1 subunit-null mouse model. A. Spectral motion power in an α1-null mouse before and after saline vehicle. During a 30-second suspension by the tail, a tremor-associated motion power peak at 22–27 Hz occurs. After vehicle administration and retest 30 min later, a comparable tremor peak is reproduced. B. Before and after administration of NNC 55-0396, 40 mg/kg, i.p., in an α1-null mouse. On retest, NNC 55-0396 suppressed the tremor motion power peak that was present in baseline. C. Tremor after saline vehicle or NNC 55-0396, 20 mg/kg (n = 6 each group), or vehicle or mibefradil 20 mg/kg (n = 5 each group), expressed as a percentage of baseline tremor power. NNC 55-0396, but not mibefradil, significantly reduced tremor on re-test. Means and S.E.M.s are shown. Comparisons are with vehicle group, Student's t test. * P < 0.05 ** P < 0.01 *** P < 0.001.

Fig. 6

Effect of NNC 55-0396 and mibefradil on tremor in the harmaline mouse model. A. Example motion spectra in mice given either harmaline, 20 mg/kg, plus saline vehicle or harmaline plus NNC 55-0396, 20 mg/kg. Whereas the control mouse exhibits a characteristic tremor-associated motion power peak at 10–16 Hz, the drug-treated mouse displays much less tremor-associated motion power. B. Motion power in the tremor bandwidth (10–16 Hz) as a percentage of overall 0–33 Hz power, during pre-harmaline baseline and at 20–60, 60–100 min after administration of vehicle or NNC 55-0396, 12.5 mg/kg or 20 mg/kg (n = 5 each group). C. Motion power during pre-harmaline baseline and at 20–60, 60–100 min after vehicle or mibefradil, 12.5 mg/kg (n = 6 each group). A dose of 20 mg/kg could not be utilized due to the behavioral interaction with harmaline depicted in Fig. 2. NNC 55-0396 suppressed tremor effectively whereas a comparable dose of mibefradil had no significant effect. Means and S.E.M.s are shown.