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Adriano B. L. Tort . Oscar P. Dall’Igna .
Ricardo V. de Oliveira . Carlos E. A. Mantese .
Paulo Fett . Márcio W. S. Gomes . Juliana Schuh .
Diogo O. Souza . Diogo R. Lara Atypical antipsychotic profile of flunarizine in animal models Received: 12 April 2004 / Accepted: 29 May 2004 / Published online: 28 July 2004 Abstract Rationale: Flunarizine is known as a calcium channel blocker commonly used in many countries to treatmigraine and vertigo. Parkinsonism has been described as Atypical antipsychotics were an important advance in the one of its side-effects in the elderly, which is in agreement treatment of schizophrenia and other psychotic disorders with its recently characterized moderate D2 receptor (Meltzer et al. ). Their main advantages include better tolerability, especially regarding extrapyramidal symp- evaluation of flunarizine as a potential antipsychotic.
toms, efficacy in a wider range of symptoms (Volavka et Methods: We evaluated the action of orally administered al. and increase in quality of life (Karow and Naber flunarizine in mice against hyperlocomotion induced by ). However, there are concerns about metabolic and amphetamine and dizocilpine (MK-801) as pharmacolo- cardiovascular side-effects that may affect morbidity and gical models of schizophrenia, induction of catalepsy as a mortality of patients (Meltzer et al. ), added to the measure for extrapyramidal symptoms and impairment high cost of treatment, making them inaccessible for many induced by dizocilpine on the delayed alternation task for patients, particularly in developing countries. Furthermore, working memory. Results: Flunarizine robustly inhibited except for injectable depot risperidone, atypical antipsy- hyperlocomotion induced by both amphetamine and chotics are not yet available in long-acting formulations, dizocilpine at doses that do not reduce spontaneous which facilitate adhesion to treatment.
locomotion (3–30 mg/kg). Mild catalepsy was observed Flunarizine, a piperazine derivative with chemical at 30 mg/kg, being more pronounced at 50 mg/kg and structure similar to the neuroleptic trifluoperazine, is a 100 mg/kg. Flunarizine (30 mg/kg) improved dizocilpine- non-selective T-type, N-type and L-type calcium channel induced impairment on the delayed alternation test.
blocker, which has long been used in some countries for Conclusions: These results suggest a profile comparable the treatment of migraine, vertigo and cerebrovascular to atypical antipsychotics. The low cost, good tolerability disorders (Todd and Benfield ; Leone et al. ; and long half-life (over 2 weeks) of flunarizine are Schmidt and Oestreich Flunarizine is usually well possible advantages for its use as an atypical antipsychot- tolerated, but clinical reports showed aggravation and ic. These results warrant clinical trials with flunarizine for inducement of extrapyramidal motor signs secondary to chronic treatment with flunarizine, mainly in elderlypatients (Chouza et al. ; Brücke et al. Keywords Flunarizine . Amphetamine . Dizocilpine .
Accordingly, animal studies suggested that this side-effect Locomotion . Antipsychotic . Schizophrenia could be due to moderate striatal D2 receptor antagonism(Pani et al. ; Ambrosio and Stefanini Kariya etal. ; Haraguchi et al. which was in the low to A. B. L. Tort . O. P. Dall’Igna . R. V. de Oliveira .
moderate micromolar range. In humans, this was con- C. E. A. Mantese . P. Fett . M. W. S. Gomes . J. Schuh .
firmed by Brücke et al. ), who found around 50% of D2 receptor blockade in a SPECT study in patients Departamento de Bioquímica, ICBS, UFRGS, chronically treated with flunarizine. Thus, based on its in vivo D2 receptor occupancy and in vitro affinity, fluna- rizine can be categorized as a dopamine D2 receptor antagonist of moderate affinity, in the range between Av. Ipiranga, 6681-Prédio 12 A, Caixa Postal 1429, olanzapine and clozapine (Seeman et al. ), therefore sharing the main mechanism of atypical antipsychotics, e-mail: [email protected]: +55-51-33203612 according to Seeman’s proposal (Seeman and Tallerico Kapur and Seeman ). Importantly, extrapyra- midal symptoms typically appear after at least 6 months oftreatment with flunarizine, which can be explained by its Mice were orally treated at 8:00 a.m. with vehicle or flunarizine atdifferent doses (1.0, 3.0, 10.0, 30.0 mg/kg). Three hours later, long-half life (around 15–20 days) (Kariya et al. spontaneous locomotor activity was recorded for 1 h, followed by IP leading to its accumulation due to daily administration up injection with dizocilpine (0.25 mg/kg) or amphetamine (5 mg/kg) to the point when dopaminergic activity is excessively and further recording for 3 h. A control group with oral vehicle inhibited. Also, all patients who experienced extrapyrami- (water) and IP saline was also included. For all injections (oral andIP), a volume of 10 ml/kg was administered.
dal symptoms in the literature were older than 55 years, To assess locomotor activity, mice were randomly allocated to when the physiological dopaminergic tone is decreased individual triangular boxes (50 cm×30 cm×30 cm, 50 cm high) with rounded corners, placed on the floor of a soundproof and diffusely Despite these findings, flunarizine has not been illuminated room. Locomotor activity of eight mice was recorded proposed for the treatment of psychotic disorders or simultaneously by a video-computerized system, with imageanalysis at four frames per second. The software (programmed by adequately tested in pre-clinical studies aiming at its ABL Tort) tracked the animals by distinguishing their white color putative antipsychotic actions. However, flunarizine, from the black background of the floor, registering X and Y among other calcium channel blockers, has already been horizontal coordinates. The method was set to examine horizontal used as a pharmacological tool to study the role of calcium locomotor activity, ignoring small movements, such as breathing,head and tail actions, and tremors. Animals had not been previously channels in the effects of amphetamine and NMDA habituated to the boxes and were observed for a total of 4 h (1 h receptor antagonists, which are pharmacological models habituation, and 3 h after IP injection), with data divided into 10 min with predictive validity for antipsychotics in pre-clinical studies. It was observed that flunarizine produced asignificant inhibitory effect against behaviors induced bythe indirect dopaminergic agonist amphetamine in rodents Mice were orally treated with flunarizine at different doses (3.0, Barrett ; Hori et al. and a borderline inhibitory 10.0, 30.0, 50.0 and 100.0 mg/kg) or vehicle, and had their catalepsy effect against the NMDA receptor antagonist PCP (Grebb time determined 3 h and 6 h later. Mice treated with haloperidol 1 mg/kg PO were used as positive controls. Catalepsy time was prevented, whereas haloperidol potentiated, the EEG measured after mice forepaws were placed over a horizontal glass effects of PCP (Popoli et al. Feinberg and Campbell bar (0.6 cm diameter), elevated 6 cm from the floor. The time micemaintained both forepaws over the bar and both hindpaws on the Importantly, in all these studies flunarizine has been ground was recorded with a cut-off time of 180 s, allowing three administered up to 30 min before testing, not taking into immediate attempts to replace the animal in cataleptic position account the 2–4 h period to reach peak serum levels within the first 10 s. Mice that kept their paws over the bar, but showed active body or head movements were also not considered ascataleptic. The experimenter was blind to drug treatment.
In this study we investigated the profile of flunarizine as an atypical antipsychotic. To this end, we evaluated theeffect of orally administered flunarizine on hyperactivity induced by systemic administration of the NMDA receptorantagonist dizocilpine (MK-801) and the indirect dopa- Delayed alternation performance was assessed in the T-maze task.
mine agonist amphetamine as pharmacological models of The starting arm is 60 cm long, each side arm is 30 cm long, and schizophrenia. The motor side-effects of flunarizine were both are 20 cm high and 10 cm wide, and the test was performed in adimly illuminated room.
also evaluated by testing the potency to reduce spontane- Mice were deprived from food until they achieved 80% of their ous locomotor activity and to induce catalepsy. Finally, initial weight. Then they were habituated in the T-maze for 4 days, cognitive impairment induced by dizocilpine on the receiving a food reward (Nescau cereal) at the end of the goal arms.
delayed alternation task was used as a measure of working In this habituation period, each mouse was placed in the start arm of the maze and permitted to explore it freely for 10 min, with the twoopen “goal” arms baited.
After these adaptation sessions, mice were trained as follows. In the first trial, food reward was presented in both goal arms. During the next 15 trials, the arm opposite to the one the animal had enteredon the previous trial was baited with food reward, except when theanimal had gone to the empty arm on the last trial. In this case, the food was left in the same place and the baited side was changed onlyafter the animal had alternated. Sliding doors were used to keep the Experiments were performed with male adult albino mice (CF1) animal for a 10 s inter-trial interval in the starting arm, and to purchased from Fundação Estadual de Pesquisa em Saúde (FEPS) confine the mouse into the goal arm for 20 s, once it had entered in when 21 days old and maintained in our own animal facilities under it. This training continued until the animal reached a criterion of at controlled environment (23±2°C, 12 h light/dark cycle, lights on at least 11 correct choices (score) in 15 trials on 3 consecutive days. A 7:00 a.m. with free access to standard food and water) up to 3–4 maximum of 10 blocks of 15 trials (10 days) was given to each months old (35–45 g). All behavioral experiments were in mouse. Animals that failed to reach the criterion in these training accordance with the Guidelines for Animal Care of our university.
Different groups of animals were used in the distinct experiments.
In the day after they matched the criterion, they received flunarizine (10 mg/kg or 30 mg/kg) or vehicle PO and after 3 hthey were tested (15 trials). This first score was considered as pre-dizocilpine. As soon as this session was over, they received dizocilpine (0.4 mg/kg IP) and after 30 min they were retested. Thissecond testing session was called post-dizocilpine.
Dizocilpine maleate and d-amphetamine sulfate were purchased from Sigma (St Louis, Mo., USA) and were dissolved in fresh saline(0.9% NaCl) for acute administrations. Commercially availablesolutions for oral use of flunarizine (Flunarin, Asta Medica) andhaloperidol (Haldol, Janssen) were used.
Fig. 2 Flunarizine inhibits hyperlocomotion induced by dizocilpinein mice. Flunarizine was orally administered to male adult albino Comparisons of locomotor activities at different time points were mice 3 h before spontaneous locomotor recording in a computerized analyzed using General Linear Model (GLM) repeated measure system. After 1-h habituation, mice were injected with 0.25 mg/kg (drug treatment versus time) with time as the repeated measure.
dizocilpine or saline IP and locomotion was recorded for 3 h (n=6 Duncan’s post hoc was used to determine differences among specific per group). Results shown as mean±SEM. Statistics (two-way groups. Catalepsy time and delayed alternation task performance ANOVA with time as the repeated measure): no difference between were analyzed using the Kruskal–Wallis followed by the Mann– groups at 0–60 min interval; saline > flu3=flu10 > flu30 at 70– Whitney U-test due to cut-off time. A value of P<0.05 was considered statistically significant.
statistically different from saline controls (P=0.08). Flu- narizine caused catalepsy in a dose-dependent fashion,with no or minimal catalepsy up to 30.0 mg/kg (at 6 h: Z= −2.747; P=0.006), whereas the higher doses of 50.0 mg/kg and 100.0 mg/kg produced consistent catalepsy at both 3 h P<0.001], with sal=1.0>3.0=10.0=30.0 mg/kg [between (Z=−3.724 for 50.0 mg/kg and −3.832 for 100.0 mg/kg; groups: F(5,36)=7.205; P<0.001] (Fig. Against P<0.001) and 6 h (Z=−3.592 for 50.0 mg/kg and −3.622 dizocilpine, flunarizine presented a dose-time-dependent for 100.0 mg/kg; P<0.001) after oral injections, but still inhibition of the hyperlocomotion induced by this NMDA less than 1 mg/kg haloperidol (at 3 h: Z=−4.310 and at 6 h receptor antagonist [F(68,374)=7.779; P<0.001], with sal >3.0=10.0>30.0 mg/kg [between groups: F(4,22)=9.008; In the delayed alternation task, flunarizine 30 mg/kg attenuated the impairment provoked by dizocilpine (Z= Regarding motor side-effects, considering the data of −1.983; P<0.05), while the dose of 10 mg/kg did not the 1 h habituation period in both trials, flunarizine 30 mg/ kg presented, if anything, a mild inhibition of spontaneous locomotion (about 18% reduction), which was not Fig. 1 Flunarizine inhibits hyperlocomotion induced by amphet-amine in mice. Flunarizine was orally administered to male adultalbino mice 3 h before spontaneous locomotor recording in acomputerized system. After 1-h habituation, mice were injected with Fig. 3 Effect of flunarizine and haloperidol on catalepsy. Catalepsy 5 mg/kg amphetamine or saline IP and locomotion was recorded for time was determined 3 h and 6 h after treatment with vehicle, 3 h (n=6 per group). Results shown as mean±SEM. Statistics (two- flunarizine or haloperidol PO. A cut-off time of 180 s was used. n=8 way ANOVA with time as the repeated measure): no difference for all groups. Data presented as medians and interquartile range. * between groups at 0–60 min interval; sal=flu1 > flu3=flu10=flu30 at Denotes statistically significant (P<0.05) difference from control synaptically by A1 receptors, which inhibit dopaminerelease, as well as post-synaptically by decreasing D2receptor affinity via A – and Souza ). Nevertheless, these combined mechan-isms seem not to excessively decrease dopaminergicactivity based on its much lower potency to producesignificant catalepsy and hypolocomotion, which is at least1 order of magnitude distant from the effective dosesagainst amphetamine and dizocilpine induced hyperloco-motion. A similar pattern has been observed forolanzapine (Ninan and Kulkarni Age (especially >70 years old) was found to be a risk factor for developing extrapyramidal symptoms withflunarizine (Brücke et al. similarly to antipsycho-tics. This profile is probably due to the ontogenetic decay Fig. 4 Flunarizine attenuates dizocilpine induced impairment in the of dopaminergic tone (Brücke et al. To our delayed alternation task. In mice previously trained to perform a knowledge, there is no report of extrapyramidal effect of delayed alternation task, flunarizine (10 mg/kg or 30 mg/kg) or flunarizine in patients younger than 55 years old. Long- vehicle PO was orally administered to male adult albino mice 3 hbefore testing 15 trials (pre). As soon as this session was over, they term use (usually more than 6 months) was another risk received dizocilpine (0.4 mg/kg IP) and after 30 min they were factor, which is not unexpected considering flunarizine’s retested (post). n=8 for all groups, * denotes statistically significant long half-life (more than 2 weeks). This characteristic has (P<0.05) difference from its respective pre-test control, # denotes been consistently overlooked in clinical practice, since it is statistically significant (P<0.05) difference from saline post-test normally prescribed at daily intakes. With such long half-life, rats treated daily with flunarizine presented an almostlinear accumulation of the drug in plasma and striatum (Kariya et al. indicating that dose reduction orlonger intervals between intakes should be considered to The present work showed that flunarizine potently avoid motor side-effects. Apart from this side effect after inhibited hyperlocomotion induced by amphetamine and long-term use, flunarizine is well tolerated even by the dizocilpine, two models with predictive validity for antipsychotics, at doses that produced no hypolocomotion Glutamate NMDA receptor antagonists, such as phen- and cataleptic behavior, a characteristic suggestive of cyclidine and dizocilpine, have also been used as a atypical antipsychotics (Ninan and Kulkarni ). Flu- pharmacological model for schizophrenia, producing both narizine also improved dizocilpine-induced impairment in hyperlocomotion and cognitive deficits in rodents (Ninan the delayed alternation test at 30 mg/kg, a dose that caused and Kulkarni ). Of note, typical antipsychotics only mild catalepsy. Such profile was observed with a 3-h typically inhibit hyperactivity induced by NMDA receptor pretreatment interval, which is more suitable to the antagonists at doses that inhibit spontaneous activity per pharmacokinetic profile of flunarizine, in contrast with se, contrary to atypical antipsychotics (O’Neill and Shaw previous studies, which typically administered flunarizine ). Flunarizine produced a substantial dose-dependent 15–30 min before the experiments (Grebb ; Sukho- effect in this model without significantly inhibit sponta- neous locomotion. Flunarizine was also able to attenuate Among animal models to identify novel compounds the cognitive impairment induced by dizocilpine in the with potential antipsychotic action, the indirect dopamine delayed alternation test for working memory, which is agonist amphetamine has been the most used pharmaco- thought to assess frontal lobe function (Le Marec et al.
logical strategy for decades (Ellenbroek This model ). These results, therefore, further suggest an atypical has gained further merit after direct evidence of increased profile of flunarizine, which may count with the contribu- dopaminergic activity in a high proportion of schizo- tion of other mechanisms of action, such as sodium phrenic patients (for review, see Kapur Flunarizine channel blockade, which may also inhibit the effects of potently inhibited amphetamine induced locomotion with- NMDA receptor antagonists (Farber et al. out a gradual dose response, since 1 mg/kg was ineffective Based on its pharmacological profile in clinical practice and the doses of 3, 10 and 30 mg/kg were equally (regarding tolerability) and in these models, flunarizine effective. Given the complex mechanism of action fluna- has putative antipsychotic action without major motor rizine, perhaps this effect may not be ascribed solely to its side-effects, similarly to atypical antipsychotics. More- D2 receptor antagonist properties. Inhibition of calcium over, after target symptoms have improved, flunarizine has and sodium channels by flunarizine (Holmes et al. the potential to be orally administered weekly or twice a Velly et al. ; Pauwels et al. can inhibit month, which could considerably improve typically poor catecholamine release. Also, a possible increase in aden- treatment compliance in psychotic patients (Perkins osine (Phillis et al. ; Popoli et al. ) by flunarizine The long half-life also may prevent abrupt exacerbation of treatment can also attenuate dopaminergic activity pre- symptoms in the case of abandoning the treatment.
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