A high-throughput LC-MS/MS method for determination of flunarizine in human plasma: Pharmacokinetic study with different doses.

A high-throughput and sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method has been developed and validated for the determination of flunarizine in human plasma. Liquid-liquid extraction under acidic conditions was used to extract flunarizine and flunarizine-d8 from 100 µL human plasma. The mean extraction recovery obtained for flunarizine was 98.85% without compromising the sensitivity of the method. The chromatographic separation was performed on Hypersil Gold C18 (50 × 2.1 mm, 3 µm) column using methanol-10 mm ammonium formate, pH 3.0 (90:10, v/v) as the mobile phase. A tandem mass spectrometer (API-5500) equipped with an electrospray ionization source in the positive ion mode was used for detection of flunarizine. Multiple reaction monitoring was selected for quantitation using the transitions, m/z 405.2 → 203.2 for flunarizine and m/z 413.1 → 203.2 for flunarizine-d8. The validated concentration range was established from 0.10 to 100 ng/mL. The accuracy (96.1-103.1%), intra-batch and inter-batch precision (CV ≤ 5.2%) were satisfactory and the drug was stable in human plasma under all tested conditions. The method was used to evaluate the pharmacokinetics of 5 and 10 mg flunarizine tablet formulation in 24 healthy subjects. The pharmacokinetic parameters Cmax and AUC were dose-proportional.

Determination of oxatomide in human plasma by high-performance liquid chromatography-electrospray ionization mass spectrometry.

A rapid, sensitive and specific high-performance liquid chromatography-electrospray ionization mass spectrometry (LC-ESI-MS) method has been developed and validated for the determination of oxatomide in human plasma. Flunarizine hydrochloride was employed as the internal standard (IS). The analytes were chromatographically separated on a Shimadzu Shim-pack VP-ODS C18 column (250 x 2.0 mm i.d.) with a mobile phase consisting of methanol and aqueous ammonium acetate solution (10 mm, pH 4.0; 85:15, v/v). Detection was performed on a single quadrupole mass spectrometer using an electrospray ionization interface with the selected-ion monitoring (SIM) mode. The method showed excellent linearity (r = 0.9995) over the concentration range of 0.5-500 ng/mL with good accuracy and precision. The intra- and inter-batch precisions were within 10% relative standard deviation. The recoveries were more than 90%. The validated method was successfully applied to a preliminary pharmacokinetic study of oxatomide in Chinese healthy male volunteers.

In vitro plasma protein binding determination of flunarizine using equilibrium dialysis and liquid chromatography-tandem mass spectrometry.

A highly sensitive method was developed and validated for determining the free fraction of flunarizine in human plasma. Equilibrium dialysis was used for the separation of free (unbound) drug and liquid chromatography/tandem mass spectrometry (LC-MS/MS) was used for quantitation. Post-dialysis plasma or buffer samples of 0.2 mL were extracted using a liquid-liquid extraction procedure and analyzed using a high performance liquid chromatography electrospray tandem mass spectrometer system. The compounds were eluted isocratically on a Supelco Supelcosil ABZ+Plus column, ionized using a positive ion atmospheric pressure electrospray ionization source, and analyzed using multiple reaction monitoring. The ion transitions monitored were m/z 405-->203 for flunarizine and m/z 409-->207 for flunarizine-d4 (internal standard, IS). The chromatographic run time was 3.5 min per injection, with retention times of 2.1 min for both flunarizine and IS. The calibration curve for flunarizine was linear over the concentration range of 0.25-2000 ng/mL (r(2)>0.9989) in the combined matrix of human plasma and isotonic sodium phosphate buffer (1:1, v/v) with the lower limit of quantitation of 0.25 ng/mL. The inter-assay coefficient of variability (CV) for the quality control samples was less than 13.5%, and the inter-assay percent nominal was greater than 98.2%. In vitro protein binding of flunarizine was determined at concentrations of 5, 10 and 100 microg/mL using the validated method. Flunarizine was extensively bound to plasma protein with a 0.083+/-0.005% overall percent free drug in plasma and a CV value less than 7.8%. This validated method will be used for the ex vivo assessment of flunarizine protein binding in human plasma from a drug-drug interaction clinical study.

Pharmacokinetic profile of topical flunarizine in rabbit eye and plasma.

The purpose of this study was to determine the aqueous humor, cornea, iris-ciliary body, retina and plasma levels and pharmacokinetics of a new topical formulation containing 0.05% flunarizine upon single drop application. Albino rabbits were used and tissue samples were collected at 15, 30, 60, 120 and 240 min after instillation. Drug concentrations in ocular tissues and plasma were measured by gas chromatography assay. After single dose application peak levels of drug were achieved at 15 min in cornea and at 30 min in aqueous humor, iris-ciliary body and retina. Unilateral topical flunarizine caused a significant reduction of intraocular pressure in rabbits. The pharmacokinetic profile showed a good ocular bioavailability of the drug providing that the new topical formulation containing 0.05% flunarizine reach the target tissues at effective concentrations and therefore may be use in the treatment of glaucoma.

Does percent reduction in seizure frequency correlate with plasma concentration of anticonvulsant drugs? Experience with four anticonvulsant drugs.

OBJECTIVE: To investigate the relationship between the percentage reduction in seizure frequency in patients with epilepsy and plasma concentrations after oral administration of 4 anticonvulsant drugs. METHODS: Patients with a minimum of 25% reduction in their seizure frequency from their baseline value were declared responders. The percentage reduction in seizure frequency was plotted against plasma concentrations with use of pharmacodynamic models (linear, log-linear, Emax, and sigmoidal Emax models). In addition to pharmacodynamic models, a logistic regression model was also fitted to the concentration-response data, with a value of 1 for responders and 0 for nonresponders. RESULTS: The concentration-effect relationship could not be adequately described either by the pharmacodynamic models or by the logistic regression analysis. CONCLUSIONS: Based on the results obtained from both pharmacodynamic models and logistic regression analysis the percentage reduction in seizure frequency may not be a true surrogate marker for anticonvulsant drugs to establish a pharmacodynamic relationship with plasma concentrations.

A limited sampling method for the estimation of flunarizine area under the curve (AUC) and maximum plasma concentration (Cmax).

A limited sampling model has been developed for flunarizine following a 30 mg oral dose in epileptic patients who were receiving phenytoin or carbamazepine or both, to estimate the area under the curve (AUC) and maximum plasma concentration (Cmax). The model was developed using training data sets from 30, 20, 15, or 10 patients at one or two time points. The equations describing the models for AUC using two time points (3 and 24h) and Cmax for the training data set of 30 subjects were AUCpredicted = 11.1 C3h + 121.4 C24h - 157 (r = 0.80) Cmax(predicted) = 0.036 AUC + 42.9 (r = 0.74) The model was validated on 64 patients who received flunarizine orally. The model provided reasonably good estimates for both AUC and Cmax. The mean predicted AUC of flunarizine was 1230 +/- 717 ng h mL-1, whereas the observed AUC was 1203 +/- 900 ng h mL-1. The bias of the prediction was 2% and precision was 28%. The mean predicted Cmax of flunarizine was 86 +/- 32 ng mL-1 as compared to an observed mean Cmax of 90 +/- 42 ng mL-1. The bias and precision of the prediction were 4% and 24%, respectively. The method described here may be used to estimate AUC and Cmax for flunarizine without detailed pharmacokinetic studies.

Rising dose study of safety and tolerance of flunarizine.

In a recent NIH-sponsored parallel-group placebo-controlled blinded study of flunarizine for the treatment of partial-onset seizures, the flunarizine serum concentration was controlled to a constant level among patients in order to reduce response variability. Flunarizine was found to exhibit modest anti-epileptic efficacy. A potential criticism of this study is that the chosen controlled concentration was too low to determine optimal efficacy. As a participating center in this study we investigated the effect of higher doses of open-label flunarizine on seizure frequency in 16 patients with refractory partial seizures. Following the completion of the blinded placebo/flunarizine phase, all patients were initiated at the flunarizine dose calculated to result in a serum concentration of 60 ng.ml-1. The dose was subsequently increased each 8-12 weeks to a maximum of 2.7 times the initial dose. On the initial maintenance flunarizine dose, seizure control was improved, with an average seizure reduction of 47% compared to pre-blinded-phase baseline. When higher doses were administered, adverse reactions were more common yet improved seizure control did not occur in most patients. These findings complement those of the concentration-controlled NIH study and suggest that appropriate flunarizine doses were utilized in that study.

Increasing plasma concentration tolerability study of flunarizine in comedicated epileptic patients.

Twelve patients with intractable partial seizures [4 receiving carbamazepine (CBZ), 4 phenytoin (PHT), and 4 both] entered a study of the tolerability of flunarizine (FNR) at specified plasma concentrations. After an 8-week baseline period, a single-dose pharmacokinetic study was performed for each patient to calculate a loading dose and maintenance dosage necessary to achieve a target plasma FNR concentration of 30 ng/ml. The first 8 patients received the loading dose (as divided doses) during a 1-week hospitalization and the maintenance dosage for the ensuing 8 weeks. These patients proceeded to treatment periods with target concentrations of 60 and then 120 ng/ml, using doses based on an assumed linear relation between dose and plasma concentration. The last 4 patients were studied only at the 120- ng/ml target level. Results indicated that this procedure successfully approximated target levels of 30 and 60 ng/ml, but observed concentrations in the last period exceeded the 120-ng/ml target level and continued to increase with time, often necessitating a dosage reduction owing to intolerability. Calculated doses for a given target concentration varied by a factor of 12. The most frequently reported adverse experiences were sedation and increased fatigue; reports of dizziness, headache, and lethargy were also common. Based on this study, a target concentration of at least 60 but < 120 ng/ml is recommended for a controlled clinical trial of the antiepileptic efficacy of FNR.

Double-blind placebo-controlled trial of flunarizine as add-on therapy in refractory childhood epilepsy.

Flunarizine (FLN) has been suggested as an add-on treatment in drug-resistant epilepsy patients. In view of the discordant experiences and of the paucity of controlled trials in children, we studied its effectiveness in 20 patients aged 6 to 18 years (10 males and 10 females), affected by drug-resistant epilepsy. 14 had symptomatic generalized epilepsy (the Lennox-Gastaut syndrome in 10; other forms in 4); 3 had cryptogenic generalized epilepsy (the Lennox-Gastaut syndrome in 2; myoclonic absences epilepsy in 1); 3 had symptomatic partial epilepsy (temporal lobe epilepsy). 7 of them were withdrawn: only 1 because of side effects. An initial four-month baseline pretrial period was followed by two four-month periods of administration of FLN or a placebo, under double blind conditions, in a randomized sequence. Preexisting antiepileptic (AEDs) medication was maintained at a constant dose throughout the study. FLN was administered as drops in a single evening dose of 5 mg (patients less than 10 years) or 10 mg. (patients greater than 10 years). During the pretrial phase, after phase 1 and phase 2, a waking EEG was recorded and blood samples were taken for hematology, hepatic-function tests, and AED serum levels. The evaluation of the activity of FLN was based on the total number of seizures. A 30-60% reduction in seizure frequency was found in 5 out of the 13 patients completing the trial (no changes occurred in the remainders). This result did not appear to be due to changes in the plasma levels of the AEDs. No significant differences were seen in the EEG paroxysmal activity in the three phases of the study. Side effects were rare. The serum FLN levels ranged between 16.4 and 109 ng/ml. It seems that the antiepileptic properties of FLN need further validation, particularly in childhood.

Flunarizine plasma concentrations and side effects in migraine patients.

Flunarizine plasma concentrations and side effects were evaluated in migraine patients during a 3 month course of prophylactic treatment. Plasma concentrations did not correlate with daily dose (in mg/kg). Mean flunarizine levels were higher in patients showing sleepiness or sedation. Weight gain was independent of plasma concentrations. Future clinical trials of flunarizine should be supported by drug monitoring in order to clarify the relationship between plasma levels and drug effects.

Streptokinase treatment versus calcium overload blockade in experimental thromboembolic stroke.

Thromboembolic brain ischemia was produced in dogs using an autologous blood clot model. The effect of postembolic treatment with flunarizine and streptokinase on hemispheric cerebral metabolic rate for oxygen (CMRO2), oxygen extraction ratio (OER), and cerebral blood flow (CBF) was studied by positron emission tomography (oxygen-15 technique) 24 hours after the insult. We studied five groups of experimental dogs and compared them with a control group of nonembolized dogs. Group I received no treatment, Group II was treated locally with 500,000 IU streptokinase starting 30 minutes after the insult, Group III received streptokinase locally 30 minutes after the insult and 0.1 mg/kg i.v. flunarizine immediately after the insult and 2 hours later, Group IV received flunarizine as Group III, and Group V was orally pretreated with 0.5 mg/kg/day flunarizine during 2 weeks preceding embolization. Compared with the contralateral hemisphere, in the embolized hemisphere a significant reduction of CMRO2 (-25% to -40%) and CBF in normocapnia (-35%) and hypercapnia (-50%) was observed in Groups I, II, and V. In Groups III and IV, CMRO2, OER, and CBF of the embolized hemisphere were within the normal range during normocapnia and hypercapnia; the extent of the ischemic lesions was markedly less than in the other groups of experimental dogs. We conclude that flunarizine treatment after experimental thromboembolic stroke had a favorable influence on brain tissue. Chronic preventive flunarizine treatment failed to have a beneficial effect.

Tolerance and pharmacokinetics of flunarizine after cardiac arrest. The Cerebral Resuscitation Study Group.

Neuronal calcium overloading after complete ischemia-anoxia of the brain might be the primary process initiating chemical cascades which lead to cell death. According to this hypothesis calcium-entry blocking agents act on the final common pathway of brain damage. Flunarizine, a selective calcium-entry blocker (without influence on heart rate and on cardiac contractile force), was administered to 12 unconscious patients, recovering from cardiac arrest (CA) of cardiac origin, according to a strict dose-range infusion protocol. Blood-pressure and heart rate (HR) were recorded before, during (t = 10 min, 20 min) and after (t = 30 min, 2 h, 4 h, 6 h, 8 h) each flunarizine infusion (maximum 4 infusions). A significant, although not clinically relevant, decrease in heart rate was noted during the first infusion. Systolic (SBP) and diastolic blood pressure (DBP) also decreased during the infusion without reaching statistical significance. Plasma levels of flunarizine were determined before and after each infusion (t = 15 min, 30 min, 1 h, 2 h, 4 h, 6 h, 8 h, 12 h). Flunarizine plasma concentrations declined very rapidly after cessation of each infusion. Sequential half-lives were in the order of 11-19 min and 5-7 h, and primarily reflect rates of distribution between the systemic circulation and the rapidly equilibrating tissues such as the brain. No substantial accumulation of flunarizine was described and plasma levels were proportional to the give dose. Therefore, flunarizine pharmacokinetics can be considered as linear for doses up to 50 mg.

Double-blind placebo-controlled trial with flunarizine in therapy-resistant epileptic patients.

The anticonvulsant efficacy and side-effect liability of flunarizine (15 mg/day) was investigated in a randomized, double-blind, placebo-controlled, crossover design in 30 outpatients with drug-resistant complex partial seizures. Flunarizine or placebo was added to the preexisting medication and each patient was followed up for 10 months. At the end of the study data from 22 patients were available for evaluation. In patients taking first flunarizine and then placebo, plasma levels of flunarizine were still detectable at the end of the 4 months' placebo phase. In the group of 13 patients starting therapy with placebo, a significant seizure frequency reduction was observed during the flunarizine period in 11 patients, whereas one patient showed no change and seizure frequency increased in another patient. Two patients had a 50% reduction in seizure frequency. Flunarizine was well tolerated and few side effects were noted.

Neuroendocrinological evidence of an anti-dopaminergic effect of flunarizine.

The effect of a one month treatment with flunarizine (5 mg/day) on pituitary responsiveness to gonadotrophin-releasing hormone (GnRH), thyrotrophin-releasing hormone (TRH) and arginine infusion was assessed in 17 adolescents (11 M, 6 F) treated with the drug to prevent migraine attacks. Basal prolactin concentrations as well as the prolactin response to TRH were significantly (p less than 0.05) increased after flunarizine treatment. Flunarizine had no effect on the folliclestimulating and luteinising hormone response to GnRH stimulation, growth hormone response to arginine infusion or thyrotrophin response to TRH stimulation. Our data suggest that flunarizine may interfere with the hypothalamic-pituitary-prolactin axis decreasing dopaminergic inhibitory tonus.

Clinical treatment of epilepsy with calcium entry blockers.

In view of the known role of Ca2+ in the paroxysmal depolarisation shifts of epileptic neurons, the possibility arises that certain Ca2+ entry blockers possess antiepileptic activity. The only drug of the class which readily passes blood-brain barrier is flunarizine. This is effective in experimental models of epilepsy and produced significant seizure reduction in two trials in therapy-resistant patients. Efficacy was maintained without development of tolerance. The safety of the drug has already been established in a great number of subjects treated for a long time for other diseases. It has few and mild side effects at therapeutic blood levels.