flunarizine and dotarizine

Serum albumin protects against cell death elicited by various cytotoxic agents; however, conflicting views on the protective mechanism still remain. Hence, we have studied the ability of serum albumin to prevent apoptosis of human neuroblastoma SH-SY 5 Y cells elicited by four compounds known to release Ca(2+) from the endoplasmic reticulum, i.e. dotarizine, flunarizine, thapsigargin and cyclopiazonic acid. Spontaneous basal apoptosis, after 24 h incubation in Dulbecco's Modified Eagle Medium (DMEM) containing 10% serum, was 5%. Dotarizine (30--50 microM) enhanced basal apoptosis to 18--43%, flunarizine (30--50 microM) to 15%, thapsigargin (1--10 microM) to 21--35%, and cyclopiazonic acid (100 microM) to 10%. Serum deprivation augmented basal apoptosis to 20%. Under serum-free medium, 30 microM dotarizine or flunarizine drastically enhanced apoptosis to 63% and 68%, respectively; the increase was milder with 1 microM thapsigargin (37%) and 30 microM cyclopiazonic acid (27%). In serum-free medium, albumin (29 or 49 mg/ml) fully prevented the apoptotic effects of dotarizine, flunarizine and cyclopiazonic acid. The four compounds increased the cytosolic Ca(2+) concentration ([Ca(2+)](c)) in fluo-4 loaded cells; such increase developed slowly to reach a plateau after several minutes, followed by a slow decline. Albumin did not modify the kinetic parameters of such increase. In the absence of serum, dotarizine, flunarizine, thapsigargin, and cyclopiazonic acid caused mitochondrial depolarization in tetramethylrhodamine ethyl ester (TMRE)-loaded cells; depolarization was inhibited by cytoprotective concentrations of albumin. These results suggest that albumin protects cells from entering into apoptosis by preventing mitochondrial depolarization. They also suggest that inhibition of mitochondrial depolarization might become a target to develop new anti-apoptotic compounds with therapeutic neuroprotective potential in stroke, Alzheimer's disease, and other neurodegenerative diseases.

Topics: Apoptosis; Benzhydryl Compounds; Calcium; Calcium Channel Blockers; Cell Line, Tumor; Culture Media, Serum-Free; Dose-Response Relationship, Drug; Endoplasmic Reticulum; Enzyme Inhibitors; Flunarizine; Humans; Membrane Potentials; Mitochondria; Neuroblastoma; Piperazines; Serum Albumin, Bovine; Thapsigargin; Time Factors

The novel antimigraineur, dotarizine, inhibited 5-HT (5 hydroxytryptamine)-evoked contractions of rabbit vertebral, aorta, femoral and mesenteric arteries, with IC(50)s of 1.35, 1.40, 0.52 and 1.09 microM, respectively. Flunarizine had little effect on these contractions, while ketanserin was more potent (IC(50)s of 0.17 microM for vertebral, 0.22 microM for aorta, 0.05 microM for femoral and 0.03 microM for mesenteric arteries). At 10 microM, dotarizine caused 40% blockade of K(+)-evoked contractions of rabbit aorta, and 70% inhibition of 5-HT-evoked responses; these values were 30% and 20% for 10 microM flunarizine. Contractions of rabbit aorta elicited by noradrenaline, angiotensin II or prostaglandin F(2alpha) were not affected by 10 microM dotarizine or flunarizine. Ketanserin shifted to the right, in parallel, the concentration-response curves for 5-HT in rabbit aorta; however, dotarizine caused a non-competitive type of blockade, increasing the maximum 5-HT contraction at 30 nM and decreasing it at 3 and 30 microM. K(+)-evoked contractions of rabbit aorta were halved by 3 microM dotarizine in a voltage-independent manner; flunarizine caused a delayed-type, non-reversible post-drug blockade, and exhibited some voltage-dependence. Blockade by nifedipine was voltage-dependent and fully reversible. Ca(2+)-evoked contractions of depolarised bovine middle cerebral arteries were blocked by 1--3 microM dotarizine in a non-surmountable manner. Contraction of these vessels evoked by electrical stimulation was blocked 50% and 70% by 1 and 3 microM dotarizine, respectively. Dotarizine (1--3 microM) also inhibited to a similar extent the K(+)-evoked [(3)H]noradrenaline release from cultured rat sympathetic neurones. These data suggest that the mechanism of blockade by dotarizine of cerebral vessels contractility has three components: (i) presynaptic inhibition of noradrenaline release; (ii) blockade of postsynaptic vascular 5-HT receptors; (iii) blockade of Ca(2+)entry into the vascular smooth muscle cell cytosol. The compound does not affect the vascular receptors for noradrenaline, angiotensin II or prostaglandin F(2alpha).

Topics: Animals; Benzhydryl Compounds; Blood Vessels; Calcium Channel Blockers; Cattle; Cerebrovascular Circulation; Electric Stimulation; Electrophysiology; Flunarizine; In Vitro Techniques; Male; Middle Cerebral Artery; Migraine Disorders; Muscle Contraction; Muscle, Smooth, Vascular; Norepinephrine; Piperazines; Rabbits; Serotonin; Serotonin Antagonists; Vasodilator Agents

In experiments on Wistar and Long Evans rats, using behavioral methods for passive (step-down and step-through) and active (shuttle-box two-way avoidance with punishment reinforcement) the newly synthesized diphenyl-methyl-piperazine derivative with Ca2+ and 5-HT antagonistic action dotarizine (DOT) administered repeatedly at oral doses of 50 and 10 mg/kg in some cases improve memory process. Under the same experimental conditions the chemically related to dotarizine Ca2+ antagonist flunarizine significantly facilitated retention. In old (Long Evans and Wistar) rats DOT in large dose decreases values of learning criterion. Probably this is a manifestation of the inherent to drugs with nootropic action "therapeutic window". Earlier investigations of the same and other authors suggest the participation of serotonergic neurotransmission in the mechanism of the memory effects of the drug DOT.

Topics: Animals; Avoidance Learning; Benzhydryl Compounds; Calcium; Calcium Channel Blockers; Cinnarizine; Drug Interactions; Flunarizine; Male; Memory; Piperazines; Rats; Rats, Long-Evans; Rats, Wistar; Serotonin Antagonists; Serotonin Receptor Agonists

Dotarizine (a novel piperazine derivative with antimigraine properties) and flunarizine (a Ca2+ channel antagonist) were compared concerning: first, their ability to cause chromaffin cell damage in vitro; second, the possible correlation of their octanol/water partition coefficients and those of another 28 compounds (i.e., Ca2+ channel antagonists, blockers of histamine H1 receptors, antimycotics, beta-adrenoceptor antagonists, neuroleptics), with their ability to cause cell damage; third, their capacity to protect the cells against the damaging effects of veratridine; and fourth, their capabilities to enhance the basal cytosolic Ca2+ concentration in fura-2-loaded single chromaffin cells, or to modify the pattern of [Ca2+]i oscillations elicited by veratridine. After 24-h exposure to 1-30 microM dotarizine, the viability of bovine adrenal chromaffin cells (measured under phase contrast or as lactate dehydrogenase, released into the medium) was similar to that of control, untreated cells; at 100 microM, 80% lactate dehydrogenase release was produced. At 1-3 microM flunarizine caused no cell damage; however 10 microM caused 20% lactate dehydrogenase release and 30 and 100 microM over 90% lactate dehydrogenase release. The time course of cell damage was considerably faster for flunarizine, in comparison to dotarizine. Out of 30 molecules tested (at 10 microM), having different octanol/water partition coefficients (log P), dotarizine was among the molecules causing no cell damage; flunarizine caused 20% cell loss, lidoflazine and verapamil over 50% cell loss, and penfluridol, draflazine, astemizole or nifedipine over 80% cell loss. No correlation was found between log P and cytotoxicity. Both dotarizine (10-30 microM) and flunarizine (3-10 microM) provided protection against veratridine-induced cell death; however, at 30 microM dotarizine afforded a pronounced protection while flunarizine enhanced the cytotoxic effects of veratridine. Dotarizine (30 microM) (but not flunarizine) caused a prompt transient elevation of the basal [Ca2+]i. Both compounds abolished the K+-induced increases of [Ca2+]i as well as the oscillations of [Ca2+]i induced by veratridine. The blocking effects of dotarizine were readily reversed after washout, while those of flunarizine were long-lasting. These differences might be relevant to the clinical use of dotarizine as an antimigraine drug.

Topics: Animals; Benzhydryl Compounds; Calcium; Calcium Channel Blockers; Cattle; Cell Death; Cell Membrane; Cell Survival; Chromaffin Cells; Cytosol; Flunarizine; Lipid Bilayers; Piperazines; Potassium; Veratridine

1. The novel antimigraineur, dotarizine (30 microM), increased cytosolic Ca2+ concentration, [Ca2+]c, in fura-2-loaded bovine adrenal chromaffin cells. This increase was transient, reached a peak in about 2 - 5 min (0.53+/-0.07 microM; n=19) and then declined to basal levels over a further 5 min period. 2. This transient rise of [Ca2+]c was mimicked by 1 microM thapsigargin and by 30 microM cyclopiazonic acid (CPA), but not by 30 microM flunarizine. Both thapsigargin and CPA occluded the effects of dotarizine and vice versa. 3. All three compounds suppressed the transient [Ca2+]c rises induced by caffeine (10 mM, 10 s); blockade induced by thapsigargin was irreversible and that induced by CPA and dotarizine was reversible. 4. Of the three compounds, only dotarizine blocked reversibly the [Ca2+]c spikes induced by short pulses of high K+ (70 mM, 5 s), suggesting that dotarizine blocks voltage-dependent Ca2+ channels but CPA and thapsigargin do not. 5. Dotarizine caused a gradual and reversible depletion of endoplasmic reticulum (ER) Ca2+ in chromaffin cells transfected with ER-targeted aequorin. CPA had a similar effect. 6. These data show that dotarizine shares with thapsigargin and CPA the ability to deplete Ca2+ in the ER; this novel action of dotarizine could be relevant to its prophylactic effects in migraine. Unlike thapsigargin and CPA, however, dotarizine additionally and reversibly blocks Ca2+ entry through voltage-dependent Ca2+ channels.

Topics: Adrenal Medulla; Animals; Benzhydryl Compounds; Caffeine; Calcium; Cattle; Cells, Cultured; Chromaffin Cells; Flunarizine; Indoles; Migraine Disorders; Piperazines; Potassium; Thapsigargin; Vasodilator Agents

In experiments on rats, we studied the changes in stereotypy induced by apomorphine (2.5 mg/kg i.p.) or amphetamine (2 mg/kg i.p.) and in haloperidol (1 mg/kg i.p.) catalepsy in rats treated with dotarizine (25 mg/kg orally), flunarizine (25 mg/kg) or vehicle for 10 days. Dotarizine did not induce any significant changes in the intensity and duration of apomorphine- or amphetamine-induced stereotypy nor in haloperidol-induced catalepsy. The flunarizine-induced changes in the behavioral effects of apomorphine, amphetamine and haloperidol suggest the decrease of striatal dopaminergic neurotransmission, whereby the risk of occurrence of extrapyramidal side effects of the drug when used in clinical practice. Dotarizine is not associated with such a risk.

Topics: Administration, Oral; Animals; Area Under Curve; Benzhydryl Compounds; Calcium Channel Blockers; Catalepsy; Corpus Striatum; Dopamine Antagonists; Flunarizine; Haloperidol; Injections, Intraperitoneal; Male; Piperazines; Rats; Rats, Wistar; Stereotypic Movement Disorder; Synaptic Transmission

Behavioral and nociceptive effects of dotarizine (DOT) and other substances acting on migrainous attacks and nitric oxide (NO) metabolism were studied in comparative experiments on rats. Behavioral effects were evaluated by the changes induced in ambulations and rearings of rats in the Opto-Varimex apparatus; effects on nociception were determined by the changes of pain threshold in growing mechanical pressure on one of the rat paw. The data showed that (1) NO did not participate directly in the mechanism of the behavioral actions of DOT. A role could be ascribed to the modulating influence of DOT on the changes in NO formation induced by other agents; (2) the NO system did not participate in the mechanisms of the responses to the painful mechanical pressure on the rat paw; (3) the behavioral effects of the substances with facilitating or inhibitory action on the migrainous process (m-CPP and ergotamine) and the influence of substances proved to affect NO formation (L-arginine, histamine, L-NAME) on these effects suggest a role for NO as a modulating but not a basic factor in the mechanisms of action of these pro- and antimigrainous substances; and (4) the behavioral effects of DOT were similar to the effects of the antimigrainous drug ergotamine and different from the promigrainous drug meta-chlorophenyl-piperazine (m-CPP)--which suggest an antimigrainous activity of dotarizine.

Topics: Animals; Arginine; Benzhydryl Compounds; Calcium Channel Blockers; Ergotamine; Flunarizine; Histamine; Male; Motor Activity; NG-Nitroarginine Methyl Ester; Nitric Oxide; Pain Threshold; Piperazines; Rats; Serotonin Receptor Agonists; Time Factors; Vasodilator Agents

Repetitive application of short depolarizing K+ pulses (70 mM K+, 2 mM Ca2+ Krebs-HEPES solution, for 10 s every 5 min) produced reproducible catecholamine secretory responses from superfused bovine chromaffin cells. At 10 microM for 15 min, the piperazine derivatives dotarizine, flunarizine and lidoflazine inhibited secretion by around 90%; cinnarizine halved the secretory response. Recovery of secretion after 30-min washout with Krebs-HEPES solution amounted to 75% in the case of dotarizine, 8% for flunarizine, 46% for lidoflazine and 21% for cinnarizine. The benzothiazol derivatives (10 microM) (+)-S-lubeluzole and R91154 (the (-)-R-enantiomer of lubeluzole) blocked the response by 75%; sabeluzole inhibited secretion by only 34% and R56865 (N-[1-(4-(4-fluorophenoxy)butyl]-4-piperidinyl-N-methyl-2-benzo-thiaz olamine) by 61%. Recoveries were around 70% in the case of these four benzothiazol derivatives. The diphenylbutyl-piperazine derivatives fluspirilene and penfluridol inhibited secretion by over 80%; no recovery was produced after 30-min washout. The inhibition of secretion was time dependent, as the recovery of the response was. Blockade of secretion by dotarizine and flunarizine occurred even in the absence of intermittent K+ stimulations of the cells. No obvious correlation was seen between the octanol/water partition coefficients of the ten compounds tested (that ranged between 6 and 4.61), the rate and extent of blockade of secretion, and the recovery of the secretory response upon washout. Rather than non-specific actions on ion channels (and secretion) due to their high lipophilicity, we believe that blockade of various Ca2+ channels relates to their binding properties to specific channel micro and macrodomains, as the case might be for 'narrow' (omega-conotoxin GVIA) and 'wide-spectrum' (omega-conotoxin MVIIC) peptide toxins.

Topics: Animals; Benzhydryl Compounds; Calcium Channel Blockers; Catecholamines; Cattle; Chromaffin Cells; Flunarizine; Piperazines; Potassium; Secretory Rate

In strips of pig coronary arteries incubated in oxygenated Krebs-bicarbonate solution at 37 degrees C, dotarizine blocked the phasic contractions evoked by 5-HT (0.5 microM) or K+ depolarization (35 mM K+) with an IC50 of 0.22 and 3.7 microM, respectively. Flunarizine inhibited both types of contractions with IC50 values of 1.7 microM for 5-HT and 2.4 microM for K+ responses. In Xenopus oocytes injected with in vitro transcribed RNA encoding for 5-HT2A or 5-HT2C receptors, 5-HT (100 nM for 20 s) applied every 10 min caused, in both cases, a reproducible inward current through Ca2(+)-activated Cl- channels (ICl). Dotarizine inhibited the 5-HT2A response in a concentration-dependent manner, with an IC50 of 2.2 nM. In contrast, the 5-HT2C response was unaffected by 1 microM dotarizine and blocked around 62% by 10 microM of this drug. The ICl activated either by intracellular injection of inositol 1,4,5-trisphosphate (IP3) in oocytes or by direct photorelease of Ca2+ in DM-nitrophen-injected oocytes was unaffected by 10 microM dotarizine. It is concluded that dotarizine blocks 5-HT2A receptors with a high affinity; the compound is devoid of intracellular effects on any further steps of the transduction pathway (i.e., IP3 receptor). Contrary to flunarizine that blocks equally well the serotonergic and the K+ vascular responses, dotarizine exhibits 17-fold higher affinity for vascular 5-HT receptors. These findings might be relevant to an understanding of the mechanism involved in the use of dotarizine and flunarizine as prophylactic agents in migraine.

Topics: Animals; Arteries; Benzhydryl Compounds; Calcium Channel Blockers; Coronary Vessels; Female; Flunarizine; Male; Microinjections; Oocytes; Piperazines; Receptors, Serotonin; Serotonin Antagonists; Swine; Vasoconstriction; Vasodilator Agents; Xenopus laevis

Dotarizine and Flunarizine are piperazine derivatives considered to be effective compounds for the treatment of various cerebrovascular disorders. In the present study the influence of these two drugs on changes in cerebral vessel diameter and blood flow velocity were measured and compared utilising transcranial Doppler sonography during hyperventilation in anaesthetized cats. Drugs were administered in 15 min intravenous infusions at a dose of 0.05 mg/kg/min. This investigation revealed that the 15 min intravenous administration of both compounds abolished the cerebral vasoconstrictor effects of hyperventilation and due to vasodilator effects they increased blood flow velocity to initial values. No statistically significant differences were found between the vasodilator effects of Dotarizine and Flunarizine. Results obtained suggest that Dotarizine, a novel piperazine derivative, has similar vasodilator and Ca2+ channel blocking effects on cerebrovascular reactivity compared to the widely clinically applied Flunarizine.

Topics: Animals; Benzhydryl Compounds; Blood Flow Velocity; Blood Pressure; Calcium Channel Blockers; Cats; Cerebrovascular Circulation; Flunarizine; Hyperventilation; Male; Piperazines; Vasoconstriction

1. Dotarizine is a novel piperazine derivative structurally related to flunarizine that is currently being evaluated in clinical trials for its antimigraine and antivertigo effects. This clinical profile may be related to its Ca2+ antagonist properties. Therefore, the actions of both compounds as calcium antagonists were compared in bovine chromaffin cells. 2. Dotarizine and flunarizine blocked 45Ca2+ uptake into K+ depolarized chromaffin cells (70 mM K+/0.5 mM Ca2+ for 60 s) in a concentration-dependent manner, with IC50s of 4.8 and 6.7 microM, respectively. 3. Dotarizine and flunarizine also inhibited the whole-cell Ca2+ and Ba2+ currents (ICa, IBa) in voltage-clamped chromaffin cells, induced by depolarizing test pulses to 0 mV, during 50 ms, from a holding potential of -80 mV. Blockade exhibited IC50s of 4 microM for dotarizine and 2.2 microM for flunarizine. Dotarizine increased the rate of inactivation of ICa and IBa; inhibition of whole-cell currents was use-dependent. 4. Transient increases of the cytosolic Ca2+ concentration, [Ca2+]i, produced by K+ stimulation (70 mM K+ for 5 s) of single fura-2-loaded chromaffin cells, were also inhibited by dotarizine and flunarizine with IC50s of 1.2 and 0.6 microM, respectively. Upon washout of dotarizine, the [Ca2+]i increases recovered fully after 5-10 min. In contrast, the responses remained largely inhibited 10 min after washing out flunarizine. 5. Catecholamine release induced by K+ stimulation (10-s pulses of 70 mM) was inhibited by dotarizine with an IC50 of 2.6 microM and by flunarizine with an IC50 of 1.2 microM. The blocking effects of both compounds developed slowly, and was fully established after 20-30 min of superfusion. While blockade by dotarizine quickly reversed upon its washout, that of flunarizine lasted even 25 min after washing out.6. Catecholamine release from electroporated chromaffin cells triggered by 10 micro M Ca2+ was not affected by 10 micro M dotarizine or flunarizine.7. Overall, the results suggest that dotarizine behaves as a Ca2+ antagonist in bovine chromaffin cells. It exhibits a potency similar to flunarizine in blocking Ca2+ entry, Ca2+ channels, Cai2+ signals and secretion. The dotarizine effects are readily reversible suggesting that in contrast to flunarizine, it does not accumulate in cells. Dotarizine is devoid of intracellular effects on the secretory machinery. All its blocking effects on Ca2+ entry, [Ca2+]i rise and secretion are probably due to blockade of vario

Topics: Animals; Barium; Benzhydryl Compounds; Calcium; Calcium Channel Blockers; Calcium Radioisotopes; Catecholamines; Cattle; Cells, Cultured; Chromaffin System; Egtazic Acid; Flunarizine; Fura-2; Piperazines; Potassium

The diphenyl-methyl-piperazine derivatives with Ca(2+)-antagonistic effect dotarizine (DOT), Fl-6020 and flunarizine were investigated in experiments on rats. The substances tested were administered repeatedly at an oral dose of 50 mg/kg. Behavioral methods were used to study the exploratory activity when the animals were placed in an environment that was unfamiliar to them (the chamber of the Opto Varimex apparatus), the elevated plus-maze method for examining the effect on anxiety, and the method of recording changes in motor activity (using the Automex II apparatus). DOT was found to increase motor activity and to have an anxiolytic effect. Combination of DOT--a compound with Ca(2+)--and 5-HT2-receptor antagonistic action--and the 5-HT-receptor agonists and antagonists used (buspirone, NAN190, pindolol, ritanserin and ondansetron) resulted in such changes in the development of habituation and in anxiety, which suggest that the modulating effects of DOT depend but partly on its typical interaction with the 5-HT2 receptor. Apparently, the Ca(2+)-antagonistic action of DOT plays a definite role, changing its biological activity depending on the 5-HT receptor subtype at the level of which the interaction is taking place.

Topics: Animals; Behavior, Animal; Benzhydryl Compounds; Calcium Channel Blockers; Drug Combinations; Exploratory Behavior; Flunarizine; Male; Motor Activity; Piperazines; Rats; Rats, Wistar; Receptors, Serotonin; Serotonin Antagonists