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Many bone diseases such as osteoporosis and periodontitis are caused by hyperactivation of osteoclasts. Calcium (Ca2+ ) signals are crucial for osteoclast differentiation and function. Thus, the blockade of Ca2+ signaling may be a strategy for regulating osteoclast activity and has clinical implications. Flunarizine (FN) is a Ca2+ channel antagonist that has been used for reducing migraines. However, the role of FN in osteoclast differentiation and function remains unknown. Here, we investigated whether FN regulates osteoclastogenesis and elucidated the molecular mechanism. FN inhibited osteoclast differentiation along with decreased expression of nuclear factor of activated T cells, cytoplasmic 1 (NFATc1), and attenuated osteoclast maturation and bone resorption. FN inhibition of osteoclast differentiation was restored by ectopic expression of constitutively active NFATc1. FN reduced calcium oscillations and its inhibition of osteoclast differentiation and resorption function was reversed by ionomycin, an ionophore that binds Ca2+ . FN also inhibited Ca2+ /calmodulin-dependent protein kinase IV (CaMKIV) and calcineurin leading to a decrease in the cAMP-responsive element-binding protein-dependent cFos and peroxisome proliferator-activated receptor-γ coactivator 1ß expression, and NFATc1 nuclear translocation. These results indicate that FN inhibits osteoclastogenesis via regulating CaMKIV and calcineurin as a Ca2+ channel blocker. In addition, FN-induced apoptosis in osteoclasts and promoted osteogenesis. Furthermore, FN protected lipopolysaccharide- and ovariectomy-induced bone destruction in mouse models, suggesting that it has therapeutic potential for treating inflammatory bone diseases and postmenopausal osteoporosis.

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Gap junctions (GJs), which consist of proteins called connexins, are intercellular channels that allow the passage of ions, second messengers, and small molecules. GJs and connexins are considered as emerging therapeutic targets for various diseases. Previously, we screened numerous compounds using our recently developed iodide yellow fluorescent protein gap junctional intercellular communication (I-YFP GJIC) assay and found that flunarizine (FNZ), used for migraine prophylaxis and as an add-on therapy for epilepsy, inhibits GJIC in LN215 human glioma cells. In this study, we confirmed that FNZ inhibits GJIC using the I-YFP GJIC assay. We demonstrated that FNZ inhibits GJ activities via a mechanism that is independent of calcium channels and dopaminergic D2, histaminergic H1, or 5-HT receptors. In addition, we showed that FNZ significantly increases connexin 43 (Cx43) phosphorylation on the cell surface, but does not alter the total amount of Cx43. The beneficial effects of FNZ on migraines and epilepsy might be related to GJ inhibition.

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In a previous study we ascertained the presence of sigma1 and sigma2 recognition sites in the rabbit iris-ciliary body, an ocular structure involved in aqueous humor production and drainage. We characterized the sigma1 sites using the preferential ligand (+)-pentazocine, which caused a significant reduction of intraocular pressure (IOP). In the present study, flunarizine, a calcium channel blocker with a complex pharmacological profile, bound to sigma1 sites expressed in the iris-ciliary body with moderate affinity (K(i) = 68 nM). Unilateral topical flunarizine (0.01-0.1%) caused a dose-related reduction of IOP in ocular normotensive rabbits and in the alpha-chymotrypsin model of ocular hypertension, without altering the IOP of the contralateral eye. This activity was blocked by the sigma1 site antagonist NE-100 [N,N-dipropyl-2-[4-methoxy-3-(2-phenylethoxy)phenyl]ethylamine HCl] which, by itself, had no effect on IOP. Detection of flunarizine in rabbit iris-ciliary body homogenates, after topical instillation, showed that it adequately penetrates the rabbit eye. To investigate mechanisms that may contribute to ocular hypotension induced by sigma1 agonists, we carried out in vitro studies on the isolated rabbit iris-ciliary body. Flunarizine (IC50 = 5. 96 nM) and (+)-pentazocine (IC50 = 3. 81 nM) inhibited [3H]norepinephrine release. Moreover, flunarizine (IC50 = 6.34 nM) and (+)-pentazocine (IC50 = 27.26 nM) also antagonized isoproterenol-induced cAMP accumulation. The action of flunarizine and (+)-pentazocine was sensitive to NE-100 antagonism; however, this latter compound partially prevented their effect on [3H]norepinephrine and cAMP accumulation. These findings indicate that flunarizine and (+)-pentazocine interact with ocular sigma1 sites and may prove effective in the control of ocular hypertension.

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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.

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The oxidative metabolism of cinnarizine [(E)-1-(diphenylmethyl)-4-(3-phenyl-2-propyl)piperazine, CZ] and flunarizine [(E)-1-[bis(4-fluorophenyl)methyl]-4-(3-phenyl-2-propyl)piperazine, FZ] was examined in microsomes from lymphoblastoid cells that expressed human cytochrome P450 (CYP) enzymes. Among 10 kinds of CYP enzymes examined, only CYP2D6 catalyzed p-hydroxylation of the cinnamyl phenyl ring of CZ (C-2 formation) and FZ (F-2 formation), and only CYP2B6 exhibited activity for p-hydroxylation (C-4 formation) of the diphenylmethyl group of CZ at a substrate concentration of 50 microM. On the other hand, CYP2C9 together with CYP1A1, -1A2 and/or -2A6 mediated N-desalkylation at the 1- and 4-positions of the piperazine ring of the two drugs that formed C-1 and C-3 from CZ and F-1 and F-3 from FZ, respectively, whereas CYP2C8, -2C19, -2E1 or -3A4 did not show detectable activity for these reactions under the conditions used. We then examined kinetics for the oxidative metabolism of CZ and FZ using CYP2B6 and -2D6 that have considerable activities. CYP2D6 with Km values of 2 to 4 microM had intrinsic clearance values (Vmax/Km) of 0.31 and 0.14 ml/min/nmol CYP for C-2 and F-2 formation, respectively, while CYP2B6 with a Km value of 17 microM exhibited the clearance value of 0.10 ml/min/nmol CYP for C-4 formation. These results suggest that CYP2D6 mainly mediates p-hydroxylation of the cinnamyl phenyl rings of CZ and FZ, and CYP2B6 mediates that of the diphenylmethyl group of CZ.

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Potentialities of cinnarizine [1-(diphenylmethyl)-4-(3-phenyl-2-propenyl)piperazine, CZ] and its fluorine derivative flunarizine [1-[bis(4-fluorophenyl)-methyl]-4-(3-phenyl-2-propenyl)piperazine, FZ] to induce parkinsonism as an adverse effect were evaluated pharmacokinetically and pharmacodynamically in rats. In multiple-dose experiments, CZ or FZ was given to rats at a daily dose of 20 mumol/kg for 1, 5, 10, 15, and 30 days, and CZ, FZ, and the ring-hydroxylated metabolites of their cinnamyl moiety [1-(diphenylmethyl)-4-[3-(4'-hydroxyphenyl)-2-propenyl]piperazine, C-2 and 1-[bis(4-fluorophenyl)methyl]-4-[3-(4'- hydroxyphenyl)propenyl]piperazine, F-2] in the plasma and striatum were determined 24 hr after the final dose. Plasma and striatum concentrations of the above compounds except for FZ reached steady state after 10 doses, but their concentrations of FZ continued to increase throughout the experiments. The concentrations obtained after the 30 doses were in the order of FZ > F-2 > CZ > C-2 for the plasma and of F-2 > FZ > CZ > C-2 for the striatum. The ratios of striatum to plasma concentrations of C-2 and F-2 were 2.4 and 3 times higher than those of the parent drugs. Binding affinities of CZ, FZ, and their 10 metabolites for rat striatal dopamine D-2 receptors (D2-R) were assessed by competitive radioligand-binding studies using [3H]-N-[(2RS,3RS)-1-benzyl-2-methyl-3-pyrrolidinyl]-5-chloro-2-met hoxy- 4-methylamino-benzamide ([3H]-YM-09151-2). The IC50s calculated from their Ki values were in the order of F-2 < C-2 < FZ < CZ < C-4 << F-1, indicating that C-2 and F-2 exhibit higher affinities for D2-R than the parent drugs, whereas affinities of other metabolites were 1 to 2 orders of magnitude less than those of C-2 and F-2. These results suggest some important roles of C-2 and F-2 in the development of parkinsonism as active metabolites during chronic medication with CZ and FZ, respectively.

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A simple, stability-indicating liquid chromatographic method has been developed for the assay of flunarizine dihydrochloride in the presence of its acid-induced degradation product. A Bondapak-C18 column was used with a mobile phase consisting of methanol-water (75:25, v/v) containing 0.5% w/v sodium chloride and 0.2% v/v triethanolamine adjusted to pH 6.6 with 30% hydrochloric acid at a flow rate 2 ml min-1. Quantitation was achieved with UV detection at 254 nm based on peak area or peak height ratios. The proposed method was successfully applied to the determination of the drug in laboratory-prepared mixtures in the presence of its degradation product and in capsules. Moreover, the method was utilized to investigate the kinetics of the degradation process at different temperatures and the apparent first-order rate constant, half-life and activation energy calculated.

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Oxidative metabolism of cinnarizine (CZ) and its fluorine derivative flunarizine (FZ), both of which are selective calcium entry blockers, was examined in human liver microsomes. The ring-hydroxylations and the N-desalkylations constituted primary metabolic pathways in microsomal metabolism of CZ and FZ. Among these pathways, the ring-hydroxylase (p-hydroxylation) activities at the cinnamyl moiety of both drugs were highly correlated with debrisoquine 4-hydroxylase activity and CYP2D6 content. Quinidine, a selective inhibitor of CYP2D6, suppressed the ring-hydroxylase activities of CZ and FZ. These results suggest that CYP2D6 is involved in the ring-hydroxylation of the cinnamyl moiety of both CZ and FZ in human liver microsomes.

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We have examined the partitioning/transfer of the Ca2+ antagonist flunarizine from the aqueous phase into phospholipid bilayers. We show that the binding of the cationic amphiphilic drug flunarizine to phospholipid bilayers displays traditional linear concentration-dependent characteristics once unmasked of electrostatic effects. The coefficient for the binding/partitioning of flunarizine to phosphatidylcholine was found to be 28700 M-1, supporting the notion that this drug may be particularly membrane-active. The thermodynamics of the partitioning/transfer process have also been studied using high-sensitivity titration calorimetry. Binding was found to be predominantly enthalpy-driven with only a small entropic contribution; delta H = -22.1 kJ.mol-1 (-5.3 kcal.mol-1) at 27 degrees C. This is in conflict with established ideas of entropy-driven partitioning of drugs into phospholipid membranes as a result of the 'hydrophobic effect'. The strong enthalpic nature of binding is interpreted as being indicative of strong lipophilic interactions between the drug and the phospholipid phase.

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1. The biotransformation of 3H-flunarizine ((E)-1-[bis(4-fluorophenyl)methyl]-4-(3-phenyl-2-propenyl)piperazine dihydrochloride, FLUN) was studied in subcellular liver fractions (microsomes and 12,000 g fraction) and in suspensions or primary cell cultures of isolated hepatocytes of rats, dogs and man. The major in vitro metabolites were characterized by h.p.l.c. co-chromatography and/or by mass spectrometric analysis. 2. The kinetics of FLUN metabolism was studied in microsomes of dog and man. The metabolism followed linear Michaelis-Menten kinetics over the concentration range 0.1-20 microM FLUN. 3. A striking sex difference was observed for the in vitro metabolism of FLUN in rat. In male rats, oxidative N-dealkylation at one of the piperazine nitrogens, resulting in bis(4-fluorophenyl) methanol, was a major metabolic pathway, whereas aromatic hydroxylation at the phenyl of the cinnamyl moiety, resulting in hydroxy-FLUN, was a major metabolic pathway in female rats. In incubates with hepatocytes, these two metabolites were converted to the corresponding glucuronides. 4. In human subcellular fractions, aromatic hydroxylation to hydroxy-FLUN was the major metabolic pathway. In primary cell cultures of human hepatocytes, oxidative N-dealkylation at the 1- and 4-piperazine nitrogen and glucuronidation of bis(4-fluorophenyl)methanol were observed. The in vitro metabolism of FLUN in humans, resembled more than in female rats and in dogs than that in male rats. 5. The present in vitro results are compared with data of previous in vivo studies in rats and dogs. The use of subcellular fractions and/or isolated hepatocytes for the study of species differences in the biotransformation of xenobiotics is discussed.

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The effects of KB-2796, a new diphenylpiperazine analogue, on [3H]nitrendipine ([3H]NTD) binding, KCl-induced contraction and 45Ca influx has been examined in dog vascular smooth muscle, and compared with those of other diphenylpiperazines. In the binding study, [3H]NTD was found to bind with a high affinity to a single class of sites on aortic membranes (Kd = 0.41 nM and Bmax = 31 fmol (mg protein)-1). KB-2796 inhibited specific [3H]NTD binding in a concentration-dependent manner, with a Ki value of 0.34 microM. The other diphenylpiperazine derivatives such as flunarizine and cinnarizine also inhibited binding in the same manner. Also, in the contraction study, all the diphenylpiperazines antagonized the 50 mM KCl-induced contraction of isolated mesenteric arteries concentration-dependently. The IC50 values of the compounds for KCl-induced contraction correlated strongly with the respective Ki values obtained in the [3H]NTD binding study. In the 45Ca influx study, KB-2796 also effectively inhibited KCl-induced 45Ca influx in mesenteric arteries, with an IC50 value of 0.14 microM. This was close to the IC50 value found in the KCl-induced contraction study. These findings suggest that calcium antagonism by KB-2796 is responsible for its vasorelaxing action in vascular smooth muscle.

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Flunarizine dose dependently inhibits the binding of both [3H]spiperone and [3H]SCH 23390 with Ki values of 112 +/- 9 and 532 +/- 39 nM, respectively. The inhibition of [3H]spiperone binding by flunarizine was competitive as revealed by the Schild plot. The results indicate that flunarizine is a fairly potent dopamine D2 receptor antagonist and offer a possible explanation for the extrapyramidal side-effects observed in patients treated with the drug.

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The partition coefficients (Kp) of three prototype Ca2+ antagonists, nitrendipine, (-)-desmethoxyverapamil and flunarizine were determined in native synaptic plasma membranes (SPM) isolated from sheep brain cortex and in liposomes prepared with the total lipids extracted from the membranes. We found that at 25 degrees and at 5 x 10(-6) M drug concentration the Kp values of the drugs for native SPM are higher than those obtained for liposomes, and are of the order of 334 +/- 53, 257 +/- 36 and 23 X 10(3) for nitrendipine, (-)desmethoxyverapamil and flunarizine, respectively, whereas the Kp values in liposomes are 190 +/- 41, 118 +/- 10 and 6 x 10(3) for the same drugs. The results suggest that the presence of membrane proteins favors the incorporation of the drugs in the membranes. Furthermore, the Kp values of the three Ca2+ antagonists studied increase with temperature in native membranes, but not in liposomes. It is concluded that the physical partitioning in membranes of drugs which act on Ca2+ channels may play some role in the mechanism of interaction of these drugs with the Ca2+ channel proteins.

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1. The effects of flunarizine, (+)-PN 200-110 and nifedipine on [3H]-(+)-PN 200-110 specific binding were investigated in intact rat mesenteric arteries bathed in physiological solution or in KCl-depolarizing solution, and in a membrane fraction from rat mesenteric arteries. 2. Unlabelled dihydropyridines, (+)-PN 200-110 and nifedipine, inhibited [3H]-(+)-PN 200-110 specific binding concentration-dependently in polarized as well as in depolarized intact arteries. The Ki value of (+)-PN 200-110 was decreased in arteries bathed in KCl-depolarizing solution compared to arteries bathed in physiological solution, while the Ki value of nifedipine was not significantly changed. Ki values measured in depolarized arteries were close to the IC50 values (concentrations inhibiting by 50% the KCl-contraction of rat mesenteric artery). 3. Flunarizine (10(-6) M) was unable to displace the specific binding of [3H]-(+)-PN 200-110 in intact arteries bathed in physiological solution. At 10(-7) M-10(-6) M, it inhibited the binding in depolarized arteries, suggesting that prolonged depolarization is required for the interaction of flunarizine with the dihydropyridine receptor. 4. In a membrane fraction isolated from rat mesenteric arteries, (+)-PN 200-110, nifedipine and flunarizine were all able to displace completely the specific binding of [3H]-(+)-PN 200-110. Displacement curves were parallel and Hill coefficients were close to unity. Ki values were close to the values obtained in depolarized intact arteries. 5. These results revealed a good correlation between the data obtained from binding tests and from pharmacological studies for dihydropyridine calcium entry blocking drugs, taking into account the time-dependence associated with their action on KCl-contraction compared to their binding properties. There was an important discrepancy between the concentrations of flunarizine active in binding studies and those active in pharmacological studies, which could be accounted for by the existence of multiple binding sites for calcium entry blockers in calcium channels.

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The pH-dependent 45Ca binding to phosphatidylserine monolayers was investigated. Ca binding increased with increasing pH. Between pH 10 and pH 11 a steep increase of Ca binding could be observed. This increase was interpreted to be due to complex Ca binding opposed to ionic binding at low pH. Flunarizine added to the spreading solution of the monolayer dose dependently displaced up to 100% Ca at pH 5 independently of phospholipid packing. At pH 11 less than 20% of Ca could be displaced by flunarizine. Intermediate results were found at pH 7. Flunarizine displaced less Ca from dense than from loosely packed monolayers at pH 7. The results suggest two binding states of flunarizine: ionic binding at low pH and apolar binding at high pH. The latter is much less effective in displacing Ca from phosphatidylserine monolayers. The Ca displacing properties of charged flunarizine may prevent a deleterious phospholipid reorientation within the membrane induced by the intracellular Ca rise during ischemia.

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Various kinds of calmodulin-interacting agents have been proved to suppress tumor promotion in vivo. In this study, we further demonstrate that flunarizine and dehydroepiandrosterone, which are proved to interact with calmodulin, showed antitumor-promoting activity in two-stage carcinogenesis of mouse skin. These results indicate that Ca2+/-calmodulin system, may play an important role in tumor promotion.

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