ZD7288 and mibefradil inhibit the myogenic heartbeat in Daphnia magna indicating its dependency on HCN and T-type calcium ion channels.

Daphnia magna heartbeat is myogenic-originating within the animal's heart. However, the mechanism for this myogenic automaticity is unknown. The mechanism underlying the automaticity of vertebrate myogenic hearts involves cells (pacemaker cells), which have a distinct set of ion channels that include hyperpolarization activated cyclic nucleotide-gated (HCN) and T-type calcium ion channels. We hypothesized that these ion channels also underlie the automatic myogenic heartbeat of Daphnia magna. The drugs, ZD7288 and mibefradil dihydrochloride, block HCN and T-type calcium ion channels respectively. Application of these drugs, in separate experiments, show that they inhibit the heartbeat of Daphnia magna in a dose-dependent manner. Calculation of the percent difference between the heart rate of pretreatment (before drug application) and heart rate following drug application (post-treatment) allowed us to graph a dose-response curve for both ZD7288 and mibefradil, revealing that ZD7288 produces a greater effect on decreasing heart rate. This indicates the HCN ion channels play a foremost role in generating Daphnia magna heartbeat. Our results show conclusively that HCN and T-type calcium ion channels underlie the automatic myogenic heartbeat in Daphnia magna-and suggest a conserved mechanism for generating myogenic heartbeat within the animal kingdom. Thus, Daphnia magna represents a credible model system for further exploration of cardiac physiology.

The roles of T-type calcium channel in the development of neuropathic pain following chronic compression of rat dorsal root ganglia.

This study aimed to elucidate the role of T-type calcium channels in the nociceptive signal transmission at the spinal level. The chronic compression of dorsal root ganglion (CCD) rat model was adopted. Three doses (50, 100 and 200 microg in groups Mib50, Mib100 and Mib200, respectively) of specific T-type Ca2+ channel inhibitors mibefradil (Mib) or normal saline (NS) were intrathecally administered on the 5th day after the CCD model had been established. The paw withdrawal latency from a noxious thermal stimulus and paw withdrawal mechanical threshold of von Frey filament was used to measure the thermal hyperalgesia and tactile allodynia, respectively. Lumbar spinal cords of the rats isolated on the 5th day after the operation were prepared to measure the mRNA expression of T-type (Cav3.1, Cav3.2 and Cav3.3) calcium channel with RT-PCR methods. The results demonstrated that CCD rats produced reliable thermal hyperalgesia and tactile allodynia after surgery. The intrathecal administration of Mib significantly suppressed thermal hyperalgesia and allodynia in CCD rats (p< 0.01), and the inhibitory effect lasted for 2 h. However, only Cav3.2 and Cav3.3 T-type calcium channel mRNA were detected in the lumbar spinal cord of rats, and there were no Cav3.1 calcium channels. Compared with native and sham groups, the Cav3.2 and Cav3.3 calcium channel mRNA expression increased significantly (p < 0.05). These data support the view that spinal T-type calcium (Cav3.2 and Cav3.3 but not Cav3.1) channels may play an important role in the pathogenesis of neuropathic pain.

A selective T-type Ca2+ channel blocker R(-) efonidipine.

Recently, novel compound R(-) efonidipine was reported to selectively block low-voltage-activated (LVA or T-type) Ca2+ channels in peripheral organs. We examined how R(-) efonidipine acts on T-type and high-voltage-activated (HVA) Ca2+ channels in mammalian central nervous system (CNS) neurons. Furthermore, we compared the effects of R(-) efonidipine with those of flunarizine and mibefradil on both T-type and HVA Ca2+ channels in rat hippocampal CA1 neurons by using the nystatin perforated-patch clamp technique. Flunarizine and mibefradil nonselectively inhibited both T-type and HVA Ca2+ channels, though the dose-dependent blocking potency of flunarizine on T-type Ca2+ channels was slightly stronger than that of mibefradil. In contrast, R(-) efonidipine inhibited only T-type Ca2+ channels and did not show any effect on HVA Ca2+ channels. The inhibitory actions of R(-) efonidipine or flunarizine were similar on both Ba2+ and Ca2+ current components passing through T-type Ca2+ channels. In addition, flunarizine but not R(-) efonidipine inhibited voltage-dependent Na+ channels and Ca2+-activated K+ channels. Thus, it appears that R(-) efonidipine is a selective blocker for T-type Ca2+ channels. It could be used as a pharmacological tool in future studies on T-type Ca2+ channels.

Timed sequential therapy of the selective T-type calcium channel blocker mibefradil and temozolomide in patients with recurrent high-grade gliomas.

Background: Mibefradil (MIB), previously approved for treatment of hypertension, is a selective T-type calcium channel blocker with preclinical activity in high-grade gliomas (HGGs). To exploit its presumed mechanism of impacting cell cycle activity (G1 arrest), we designed a phase I study to determine safety and the maximum tolerated dose (MTD) of MIB when given sequentially with temozolomide (TMZ) in recurrent (r)HGG. Methods: Adult patients with rHGG ≥3 months from TMZ for initial therapy received MIB in 4 daily doses (q.i.d.) for 7 days followed by standard TMZ at 150-200 mg/m2 for 5 days per 28-day cycle. MIB dose escalation followed a modified 3 + 3 design, with an extension cohort of 10 patients at MTD who underwent 3'-deoxy-3'-18F-fluorothymidine (18F-FLT) PET imaging, to image proliferation before and after 7 days of MIB. Results: Twenty-seven patients were enrolled (20 World Health Organization grade IV, 7 grade III; median age 50 y; median KPS 90). The MTD of MIB was 87.5 mg p.o. q.i.d. Dose-limiting toxicities were elevation of alanine aminotransferase/aspartate aminotransferase (grade 3) and sinus bradycardia. The steady-state maximum plasma concentration of MIB at the MTD was 1693 ± 287 ng/mL (mean ± SD). 18F-FLT PET imaging showed a significant decline in standardized uptake value (SUV) signal in 2 of 10 patients after 7 days of treatment with MIB. Conclusions: MIB followed by TMZ was well tolerated in rHGG patients at the MTD. The lack of toxicity and presence of some responses in this selected patient population suggest that this regimen warrants further investigation.

Indispensable role of the voltage-gated calcium channels in the procognitive effects of angiotensin IV.

BACKGROUND: Voltage-gated calcium channels (VGCCs) play a major role in brain functioning, including that of cognition-related structures such as cerebral cortex and hippocampus. Cellular mechanisms underlying learning and memory enhancing effect of the neuropeptide angiotensin IV (Ang IV) have been linked to VGCCs but only in respect of its long-term potentiation (LTP)-inducing effect. OBJECTIVE: To assess behaviorally effects of L- and T-type VGCCs blocking drugs in low, behaviorally inactive, doses on Ang IV facilitation of recall of aversively (foot-shock) and appetitively (curiosity for novelty) motivated behaviors. METHODS: About 240 male Wistar rats were used. All animals received oral (p.o.) dose of nimodipine (12mg/kg) or mibefradil (1mg/kg) dissolved in saline or saline alone followed by an intracerebroventricular (i.c.v.) injection of 1nmol of Ang IV dissolved in 2µl of normal saline or saline alone 15min later. Groups of about 10 rats, separate for each experiment, were tested for recall of aversively (inhibitory avoidance, IA) and appetitively (object recognition, OR) reinforced behaviors. To verify lack of unspecific motor and emotional effects of our treatments separate groups of rats were tested in open field (OF) and elevated 'plus' maze (EPM), respectively. RESULTS: Both, nimodipine and mibefradil prevented recall facilitating effects of subsequently injected Ang IV. The peptide as well as both VGCCs blocking drugs had no (OF), or only negligible (EPM) effects on motor performance and emotionality of rats. CONCLUSION: The data support a notion about key role of the functional VGCCs in neuronal procognitive effects of Ang IV.

Chronic intrathecal infusion of T-type calcium channel blockers attenuates CaV3.2 upregulation in nerve-ligated rats.

OBJECTIVE: T-type channel (TCC) CaV3.2 plays a pivotal role in pain transmission. In this study, we examined the effects of intrathecal TCC blockers on CaV3.2 expression in a L5/6 spinal nerve ligation (SNL) pain model. The neurotoxicity of TCC blockers were also evaluated. METHODS: 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. RESULTS: 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.

The angiographic and clinical benefits of mibefradil in the coronary slow flow phenomenon.

OBJECTIVES: The aim of the study was to assess the angiographic and clinical benefits of the calcium T-channel blocker, mibefradil, in the coronary slow flow phenomenon (CSFP). BACKGROUND: The CSFP is characterized by delayed vessel opacification on angiography (Thrombolysis In Myocardial Infarction [TIMI]-2 flow) in the absence of obstructive epicardial coronary disease and is often associated with recurrent chest pain. METHODS: A total of 10 CSFP patients (46 +/- 9 years) underwent angiography before and 30 min after 50 mg mibefradil; off-line blinded analysis of angiographic data included comparisons of epicardial vessel diameter, TIMI flow grade and TIMI frame count. We also performed a randomized, double-blind, placebo-controlled, cross-over study to examine the long-term efficacy of mibefradil 100 mg/day on the frequency of total angina, prolonged angina (i.e., persisting >20 min) episodes, and sublingual nitrate consumption, during consecutive one-month treatment periods in 20 patients (age 51 +/- 12 years) with the CSFP. RESULTS: Without changing epicardial vessel diameter or rate-pressure product, mibefradil reduced the number of vessels exhibiting TIMI-2 flow from 18 to 5; furthermore, mibefradil significantly improved the TIMI frame count only in those vessels exhibiting TIMI-2 flow (28 +/- 18%, p < 0.005). Compared with placebo, mibefradil significantly reduced total angina frequency by 56% (p < 0.001), prolonged episodes of angina by 74% (p < 0.001), and sublingual nitrate consumption by 59% (p < 0.01); furthermore, mibefradil improved physical quality of life as assessed by the Health Outcome Study Short Form-36. CONCLUSIONS: These angiographic and clinical improvements produced by mibefradil support a microspastic pathogenesis of the CSFP.

Nonlinear kinetics and pharmacologic response to mibefradil.

BACKGROUND: Increasing oral doses of mibefradil (10 to 320 mg) decrease its apparent oral clearance; however, intravenous doses up to 80 mg do not reduce its systemic clearance. This study aimed to understand the mechanisms underlying the zero-order kinetics of mibefradil. METHODS: A group of 10 normotensive volunteers received 50 mg/day oral mibefradil for 8 days and, on days 1 and 8, 5 mg deuterated mibefradil by infusion. Ten additional volunteers observed the same protocol with a daily oral dose of 100 mg mibefradil. Serial blood samples were withdrawn, and mibefradil plasma concentrations were assayed by liquid chromatography-mass spectrometry. Blood pressure and heart rate were measured for 4 hours, and an ECG was performed 2 hours after drug administration. RESULTS: Repeated oral administration of 50 mg mibefradil generated zero-order kinetics secondary to a decrease in mibefradil systemic clearance. Compared with the 50-mg dose, single and repeated oral doses of 100 mg further decreased mibefradil clearance. Mibefradil bioavailability was not affected by increasing mibefradil doses. Mean diastolic blood pressure was decreased by single and repeated doses of 100 mg to the same extent. Repeated doses of 100 mg reduced heart rate and prolonged the PR and QTc, changes that were associated with mibefradil plasma concentrations. CONCLUSIONS: Repeated doses of 50 mg or doses of 100 mg mibefradil generated zero-order kinetics secondary to a decrease in hepatic extraction of the drug. Zero-order kinetics did not affect the response-concentration relationship of mibefradil.

Diltiazem and mibefradil increase the plasma concentrations and greatly enhance the adrenal-suppressant effect of oral methylprednisolone.

OBJECTIVE: To examine the possible interaction of the calcium channel blockers diltiazem and mibefradil with orally administered methylprednisolone. METHODS: In this randomized, double-blind, placebo-controlled, three-phase crossover study, nine healthy SUBJECTS received 60 mg diltiazem three times a day, 50 mg mibefradil once a day, or placebo orally for 3 days. On day 3, each subject received a 16-mg oral dose of methylprednisolone. Plasma concentrations of methylprednisolone and cortisol were determined by HPLC up to 47 hours. RESULTS: Compared with placebo, diltiazem and mibefradil increased the total area under the plasma concentration-time curve of methylprednisolone [AUC(0-infinity)] 2.6-fold (P < .001) and 3.8-fold (P < .001), the peak plasma concentration 1.6-fold (P < .001) and 1.8-fold (P < .001), and the elimination half-life 1.9-fold (P < .001) and 2.7-fold (P < .001), respectively. The nighttime exposure to methylprednisolone [AUC(12-23)] was increased 28.2-fold (P < .01) and 72.1-fold (P < .001) by diltiazem and mibefradil, respectively, and correlated negatively (r = -0.81, P < .001) with the morning plasma cortisol concentration (measured at 8 AM, 23 hours after the administration of methylprednisolone). During the diltiazem phase, the morning plasma cortisol concentration was 12% of that during the placebo phase (P < .001); during the mibefradil phase, the morning plasma cortisol concentration was 2% of that during the placebo phase (P < .001). CONCLUSIONS: Coadministration of diltiazem or mibefradil with methylprednisolone resulted in increased plasma concentrations and a greatly enhanced adrenal-suppressant effect of oral methylprednisolone. Care should be taken if methylprednisolone is coadministered with a potent CYP3A4 inhibitor for a long period.

Mibefradil but not isradipine substantially elevates the plasma concentrations of the CYP3A4 substrate triazolam.

BACKGROUND: The calcium channel blockers mibefradil and isradipine inhibit CYP3A4 in vitro. However, their in vivo inhibitory effects on CYP3A4 are not known in detail, although mibefradil was recently withdrawn from the market because of serious drug interactions. METHODS: The effects of mibefradil and isradipine on the pharmacokinetics and pharmacodynamics of oral triazolam, a model substrate of CYP3A4, were studied in a randomized, double-blind crossover study with three phases. Nine healthy subjects took 50 mg mibefradil, 5 mg isradipine, or placebo orally once a day for 3 days. On day 3, each subject received a single 0.25 mg oral dose of triazolam. Thereafter, blood samples were collected up to 18 hours, and pharmacodynamic effects of triazolam were measured up to 8 hours. RESULTS: Mibefradil increased the total area under the plasma triazolam concentration-time curve [AUC(0 - infinity)] 9-fold compared with placebo (P < .001). The peak plasma concentration of triazolam was increased 1.8-fold (3.4+/-0.1 ng/mL versus 1.8+/-0.2 ng/mL [mean +/- SEM]; P < .001), and the elimination half-life (t 1/2) was increased 4.9-fold (18.5+/-1.9 hours versus 4.0+/-0.5 hours; P < .001) by mibefradil. In addition, mibefradil was associated with increased pharmacodynamic effects of triazolam. In contrast to mibefradil, isradipine reduced the AUC(0 - infinity) and t 1/2 of triazolam by about 20% (P < .05) and had no significant effects on the pharmacodynamics of triazolam. CONCLUSION: Mibefradil but not isradipine markedly increases the plasma concentrations of triazolam and thereby enhances and prolongs its pharmacodynamic effects, consistent with potent inhibition of CYP3A4.

Reversal of experimental neuropathic pain by T-type calcium channel blockers.

Experimental nerve injury results in exaggerated responses to tactile and thermal stimuli that resemble some aspects of human neuropathic pain. Neuronal hyperexcitability and neurotransmitter release have been suggested to promote such increased responses to sensory stimuli. Enhanced activity of Ca(2+) current is associated with increased neuronal activity and blockade of N- and P-types, but not L-type, calcium channels have been found to block experimental neuropathic pain. While T-type currents are believed to promote neuronal excitability and transmitter release, it is unclear whether these channels may also contribute to the neuropathic state. Rats were prepared with L(5)/L(6) spinal nerve ligation, and tactile and thermal hypersensitivities were established. Mibefradil or ethosuximide was administered either intraperitoneally, intrathecally (i.th.), or locally into the plantar aspect of the injured hindpaw. Systemic mibefradil or ethosuximide produced a dose-dependent blockade of both tactile and thermal hypersensitivities in nerve-injured rats; responses of sham-operated rats were unchanged. Local injection of mibefradil also blocked both end points. Ethosuximide, however, was inactive after local administration, perhaps reflecting its low potency when compared with mibefradil. Neither mibefradil nor ethosuximide given i.th. produced any blockade of neuropathic behaviors. The results presented here suggest that T-type calcium channels may play a role in the expression of the neuropathic state. The data support the view that selective T-type calcium channel blockers may have significant potential in the treatment of neuropathic pain states.

Comparative effects of mibefradil and nifedipine gastrointestinal transport system on autonomic function in patients with mild to moderate essential hypertension.

BACKGROUND: L-type dihydropyridine calcium channel blockers (CCBs) have been implicated in increased cardiovascular events in patients with hypertension, perhaps due to adverse effects on autonomic nervous system (ANS) function. Blockade of T-type calcium channels may limit ANS dysfunction by inhibition of T channel-mediated neuroendocrine effects. OBJECTIVE AND DESIGN: This double-blind, parallel group study compared the effect of nifedipine gastrointestinal transport system (GITS) (L-type CCB) versus mibefradil (T-type CCB) on ANS function in patients with mild-moderate essential hypertension. METHODS: Sixteen patients (10 male, 6 female; age 57.2 +/- 2.3 years), diastolic blood pressure (DBP) < 95 mmHg were randomized to nifedipine 30 mg daily or mibefradil 50 mg daily (2 weeks), then nifedipine 60 mg daily or mibefradil 100 mg daily (4 weeks). Sympathetic nervous system activity (SNSA) was assessed using norepinephrine kinetics. Parasympathetic nervous system activity (PSNA) was assessed from 24 h Holter recordings of heart rate variability (HRV). Non-invasive baroreflex sensitivity (BRS) provided integrated assessment of ANS. RESULTS: Patient groups were well matched at baseline. Achieved DBP was lower in patients treated with mibefradil compared with nifedipine, (83.4 +/- 1.7 versus 95.25 +/- 3.3 mmHg). There were no significant differences in SNSA and BRS between groups, however the root mean square of successive differences and high frequency power (HFP) were increased in mibefradil compared with nifedipine-treated patients [(+ 1.07 +/- 1.6 versus -3.36 +/- 1.2 ms, P < 0.05) and (+ 0.28 +/- 0.1 versus -0.23 +/- 0.1 ms2, P < 0.01), respectively]. Furthermore, Ln HFP/Ln total power was increased from week 0 to week 6 in the mibefradil-treated group, (0.71 +/- 0.02 versus 0.74 +/- 0.03, P = 0.046). CONCLUSION: No differences existed between effect of L- and T-type CCBs on SNSA and BRS. However, T-type CCBs increased PSNA, independent of achieved changes in heart rate.

Comparative pharmacokinetic interaction profiles of pravastatin, simvastatin, and atorvastatin when coadministered with cytochrome P450 inhibitors.

Three-hydroxy-3-methylglutaryl coenzyme-A reductase inhibitors (statins) are first-line treatments for hypercholesterolemia. Although exceedingly well tolerated, treatment with statins incurs a small risk of myopathy or potentially fatal rhabdomyolysis, particularly when coadministered with medications that increase their systemic exposure. Studies compared the multiple-dose pharmacokinetic interaction profiles of pravastatin, simvastatin, and atorvastatin when coadministered with 4 inhibitors of cytochrome P450-3A4 isoenzymes in healthy subjects. Compared with pravastatin alone, coadministration of verapamil, mibefradil, or itraconazole with pravastatin was associated with no significant changes in pravastatin pharmacokinetics. However, concomitant verapamil increased the simvastatin area under the concentration:time curve (AUC) approximately fourfold, the maximum serum concentration (C(max)) fivefold, and the active metabolite simvastatin acid AUC and C(max) approximately four- and threefold, respectively (all comparisons p <0.001). Similar (greater than fourfold) important increases in these parameters and a >60% increase in the serum half-life (p = 0.03) of atorvastatin were observed when coadministered with mibefradil. The half-life of atorvastatin also increased by approximately 60% (p = 0.052) when coadministered with itraconazole, which elicited a 2.4-fold increase in the C(max) of atorvastatin and a 47% increase in the AUC (p <0.001 for C(max) and AUC). Clarithromycin significantly (p <0.001) increased the AUC (and C(max)) of all 3 statins, most markedly simvastatin ( approximately 10-fold increase in AUC) and simvastatin acid (12-fold), followed by atorvastatin (greater than fourfold) and then pravastatin (almost twofold). Pravastatin has a neutral drug interaction profile relative to cytochrome P450-3A4 inhibitors, but these substrates markedly increase systemic exposure to simvastatin and atorvastatin.

Effect of mibefradil on CYP3A4 in vivo.

Mibefradil, a calcium channel blocker, was removed from the market because of adverse drug interactions with coadministered CYP3A4 substrates. This study examined the effect of mibefradil on the activity of hepatic and intestinal CYP3A4 in vivo, employing the erythromycin breath test (EBT) and oral midazolam pharmacokinetics. This was a two-period, single-blind, placebo-controlled crossover study in which 8 male volunteers were randomized to the order of receiving placebo and a single 100-mg oral dose of mibefradil. Oral midazolam was coadministered with intravenous [14C N-methyl] erythromycin 1 hour after mibefradil/placebo administration. The EBT was performed 20 minutes following erythromycin administration. Blood and urine were collected during the 36 hours following probe drug administration for analysis of midazolam pharmacokinetics. Coadministration of mibefradil increased the Cmax of midazolam 3-fold, the AUC 8- to 9-fold, and the t1/2 4-fold. Mibefradil coadministration decreased the amount of exhaled 14CO2 in 6 of 8 subjects, with a mean decrease of 25%. It was concluded that a single oral dose of mibefradil significantly inhibits CYP3A4 in intestine and liver. These data support that adverse drug interactions involving mibefradil reflect inhibition of CYP3A4 in intestine and liver. Also, they suggest that the EBT, while a valid probe of in vivo hepatic CYP3A4 activity, is a single time point measurement and may be less sensitive than oral midazolam pharmacokinetics in detecting CYP3A4 inhibition.

Pharmacokinetic and pharmacodynamic aspects of concomitant mibefradil-digoxin therapy at therapeutic doses.

This study investigated the effect of mibefradil on digoxin pharmacokinetics an pharmacodynamics. Following a loading dose of digoxin (0.375 mg, three times, day 1), 0.375 mg was administered once daily to 40 healthy subjects (days 2-15). Mibefradil was administered daily at 50 mg, 100 mg, or 150 mg (days 9-15). With co-administration of 50 mg or 100 mg mibefradil (the recommended doses), mean digoxin Cmax values increased 1.19- and 1.32-fold, respectively; Cmin values were 0.95- and 1.04-fold, respectively; mean AUC0-24 h increased 1.05- and 1.11-fold, respectively; and the total amount of digoxin excreted in urine remained unchanged. Digoxin monotherapy produced modest but transient prolongations of PQ interval, small decreases in heart rate, and no changes in blood pressure. With the addition of mibefradil, no effects on trough blood pressure or cardiac index were observed, but there was a further increase in PQ interval and decrease in heart rate. In a previous study, mibefradil had no significant effect on trough plasma digoxin concentration in patients with congestive heart failure and ischemia. Therefore, while the vast majority of patients should not need their digoxin dosages adjusted when given mibefradil, an occasional patient may require dose reductions based on clinical response and plasma digoxin.

Mibefradil reduces blood glucose concentration in db/db mice.

OBJECTIVE: Numerous recent studies suggest that abnormal intracellular calcium concentration ([Ca2+]i) is a common defect in diabetic animal models and patients. Abnormal calcium handling is an important mechanism in the defective pancreatic ß-cell function in type 2 diabetes. T-type Ca2+ channel antagonists lower blood glucose in type 2 diabetes, but the mechanism remains unknown. METHODS: We examined the effect of the Ca2+ channel antagonist mibefradil on blood glucose in male db/db mice and phenotypically normal heterozygous mice by intraperitoneal injection. RESULTS: Mibefradil (15 mg/kg, i.p., b.i.d.) caused a profound reduction of fasting blood glucose from 430.92±20.46 mg/dl to 285.20±5.74 mg/dl in three days. The hypoglycemic effect of mibefradil was reproduced by NNC 55-0396, a compound structurally similar to mibefradil but more selective for T-type Ca2+ channels, but not by the specific L-type Ca2+ channel blocker nicardipine. Mibefradil did not show such hypoglycemic effects in heterozygous animals. In addition, triglycerides, basal insulin and food intake were significantly decreased by mibefradil treatment in the db/db mice but not in the controls. Western blot analysis, immunohistochemistry and immunofluorescence staining showed a significantly increased expression of T-type Ca2+ channel α-subunits Cav3.1 and Cav3.2 in liver and brain tissues from db/db mice compared to those from heterozygous animals. CONCLUSIONS: Collectively, these results suggest that T-type Ca2+ channels are potential therapeutic targets for antidiabetic drugs.

Upregulation of T-type Ca2+ channels in primary sensory neurons in spinal nerve injury.

STUDY DESIGN: Painful behavior testing, whole-cell patch clamp recordings, and PCR analysis were served to test the influence of T-type Ca channels in spinal nerve-injured rats. OBJECTIVE: To determine the changes of T-type Ca channels in dorsal root ganglion (DRG) neurons of different sizes and the contribution to neuronal firing and painful behavior in neuropathic pain induced by nerve injury. SUMMARY OF BACKGROUND DATA: T-type and high-voltage-activated Ca channels play an important role in the transmission of nociceptive signals, especially in neuronal hyperexcitability in neuropathic pain. However, little is known about how nerve injury affects T-type Ca channels in DRG neurons of different sizes. METHODS: The effect of intrathecal administration of mibefradil in nerve-ligated rats was examined by painful behavior testing and current clamp. The changes of T-type Ca channels in DRG neurons caused by spinal nerve ligation were determined by RT-PCR analysis and voltage clamp. RESULTS: Spinal nerve injury significantly increased current density of T-type Ca channels in small DRG neurons. In addition, nerve injury significantly increased the percentage of T-type Ca channels in medium and large DRG neurons. Nerve injury significantly increased the mRNA levels of Cav3.2 and Cav3.3 in DRGs. Block of T-type Ca channels on mibefradil administration significantly normalized painful behavior and hyperexcitability in neuronal firing in spinal nerve-injured rats. CONCLUSION: Our study first indicated the upregulation of functional T-type Ca channels in DRG neurons of different sizes and the changes in different subtypes of T-type Ca channels by spinal nerve injury. Considering the effect of blocking T-type Ca channels in painful behavior and abnormal neuronal firing in rats with nerve injury, our results suggest that T-type Ca channels are potential therapeutic targets for the treatment of spinal nerve ligation-induced neuropathic pain.

Effects of T-type calcium channel blockers on cocaine-induced hyperlocomotion and thalamocortical GABAergic abnormalities in mice.

RATIONALE: Repetitive cocaine exposure has been shown to induce GABAergic thalamic alterations. Given the key role of T-type (Ca(V)3) calcium channels in thalamocortical physiology, the direct involvement of these calcium channels in cocaine-mediated effects needs to be further explored. OBJECTIVE: The objective of this study was to investigate the effect of T-type calcium channel blockers on acute and repetitive cocaine administration that mediates thalamocortical alterations in mice using three different T-type blockers: 2-octanol, nickel, and mibefradil. METHODS: During in vitro experiments, whole-cell patch-clamp recordings were conducted in ventrobasal (VB) thalamic neurons from mice treated with acute repetitive cocaine administration (3 x 15 mg/kg, i.p., 1 h apart), under bath application of mibefradil (10 µM), 2-octanol (50 µM), or nickel (200 µM). After systemic administration of T-type calcium channel blockers, we evaluated locomotor activity and also recorded GABAergic neurotransmission onto VB neurons in vitro. RESULTS: Bath-applied mibefradil, 2-octanol, or nickel significantly reduced both GABAergic neurotransmission and T-type currents of VB neurons in cocaine-treated mice. In vivo i.p. pre-administration of either mibefradil (20 mg/kg and 5 mg/kg) or 2-octanol (0.5 mg/kg and 0.07 mg/kg) significantly reduced GABAergic mini frequencies onto VB neurons. Moreover, both mibefradil and 2-octanol were able to decrease cocaine-induced hyperlocomotion. CONCLUSION: The results shown in this study strongly suggest that T-type calcium channels play a key role in cocaine-mediated GABAergic thalamocortical alterations, and further propose T-type channel blockers as potential targets for future pharmacological strategies aimed at treating cocaine's deleterious effects on physiology and behavior.

Mibefradil reduces blood glucose concentration in db/db mice

OBJECTIVE: Numerous recent studies suggest that abnormal intracellular calcium concentration ([Ca2+]i) is a common defect in diabetic animal models and patients. Abnormal calcium handling is an important mechanism in the defective pancreatic β-cell function in type 2 diabetes. T-type Ca2+ channel antagonists lower blood glucose in type 2 diabetes, but the mechanism remains unknown. METHODS: We examined the effect of the Ca2+ channel antagonist mibefradil on blood glucose in male db/db mice and phenotypically normal heterozygous mice by intraperitoneal injection. RESULTS: Mibefradil (15 mg/kg, i.p., b.i.d.) caused a profound reduction of fasting blood glucose from 430.92±20.46 mg/dl to 285.20±5.74 mg/dl in three days. The hypoglycemic effect of mibefradil was reproduced by NNC 55-0396, a compound structurally similar to mibefradil but more selective for T-type Ca2+ channels, but not by the specific L-type Ca2+ channel blocker nicardipine. Mibefradil did not show such hypoglycemic effects in heterozygous animals. In addition, triglycerides, basal insulin and food intake were significantly decreased by mibefradil treatment in the db/db mice but not in the controls. Western blot analysis, immunohistochemistry and immunofluorescence staining showed a significantly increased expression of T-type Ca2+ channel α-subunits Cav3.1 and Cav3.2 in liver and brain tissues from db/db mice compared to those from heterozygous animals. CONCLUSIONS: Collectively, these results suggest that T-type Ca2+ channels are potential therapeutic targets for antidiabetic drugs. .

In vivo approach for the evaluation of mechanism-based inhibition of cytochrome P450 3A in rats.

1. There have been no reports showing that the area under the concentration-time curve (AUC) of a probe drug is elevated due to mechanism-based inhibition (MBI) of drug-metabolizing enzymes in animals. This study ascertained that mechanism-based inhibitors reported to induce drug-drug interactions (DDIs) in humans also caused MBI in rats. 2. Midazolam (MDZ), mainly metabolized by cytochrome P450 3A in rats, and mibefradil, which showed the most intense time-dependent inhibition among the inhibitors tested, were selected as the probe and the inhibitor, respectively. Following pretreatment of mibefradil at 24 h before MDZ administration in rats, the C(max) and AUC values of MDZ were significantly elevated in comparison with the control. The free plasma concentration of mibefradil was substantially lower than the IC(50) value observed in the in vitro inhibition study, suggesting that the DDI was due to MBI. 3. It is concluded that the evaluation of MBI in rats in vivo in combination with in vitro data using human enzymes could be useful to evaluate risk in clinical studies.