Influence of Ivabradine on the Anticonvulsant Action of Four Classical Antiepileptic Drugs Against Maximal Electroshock-Induced Seizures in Mice

Although the role of hyperpolarization-activated cyclic nucleotide-gated (HCN) channels in neuronal excitability and synaptic transmission is still unclear, it is postulated that the HCN channels may be involved in seizure activity. The aim of this study was to assess the effects of ivabradine (an HCN channel inhibitor) on the protective action of four classical antiepileptic drugs (carbamazepine, phenobarbital, phenytoin and valproate) against maximal electroshock-induced seizures in mice. Tonic seizures (maximal electroconvulsions) were evoked in adult male albino Swiss mice by an electric current (sine-wave, 25 mA, 0.2 s stimulus duration) delivered via auricular electrodes. Acute adverse-effect profiles of the combinations of ivabradine with classical antiepileptic drugs were measured in mice along with total brain antiepileptic drug concentrations. Results indicate that ivabradine (10 mg/kg, i.p.) significantly enhanced the anticonvulsant activity of valproate and considerably reduced that of phenytoin in the mouse maximal electroshock-induced seizure model. Ivabradine (10 mg/kg) had no impact on the anticonvulsant potency of carbamazepine and phenobarbital in the maximal electroshock-induced seizure test in mice. Ivabradine (10 mg/kg) significantly diminished total brain concentration of phenytoin and had no effect on total brain valproate concentration in mice. In conclusion, the enhanced anticonvulsant action of valproate by ivabradine in the mouse maximal electroshock-induced seizure model was pharmacodynamic in nature. A special attention is required when combining ivabradine with phenytoin due to a pharmacokinetic interaction and reduction of the anticonvulsant action of phenytoin in mice. The combinations of ivabradine with carbamazepine and phenobarbital were neutral from a preclinical viewpoint.     

Antiepileptic drugs increase plasma levels of 4beta-hydroxycholesterol in humans: evidence for involvement of cytochrome p450 3A4

The major cholesterol oxidation products in the human circulation are 27-hydroxycholesterol, 24-hydroxycholesterol, and 7alpha-hydroxycholesterol. These oxysterols are formed from cholesterol by specific cytochrome P450 enzymes, CYP27, CYP46, and CYP7A, respectively. An additional oxysterol present in concentrations comparable with 7alpha- and 24-hydroxycholesterol is 4beta-hydroxycholesterol. We now report that patients treated with the antiepileptic drugs phenobarbital, carbamazepine, or phenytoin have highly elevated levels of plasma 4beta-hydroxycholesterol. When patients with uncomplicated cholesterol gallstone disease were treated with ursodeoxycholic acid, plasma 4beta-hydroxycholesterol increased by 45%. Ursodeoxycholic acid, as well as the antiepileptic drugs, are known to induce cytochrome P450 3A. Recombinant CYP3A4 was shown to convert cholesterol to 4beta-hydroxycholesterol, whereas no conversion was observed with CYP1A2, CYP2C9, or CYP2B6. The concentration of 4alpha-hydroxycholesterol in plasma was lower than the concentration of 4beta-hydroxycholesterol and not affected by treatment with the antiepileptic drugs or ursodeoxycholic acid. Together, these data suggest that 4beta-hydroxycholesterol in human circulation is formed by a cytochrome P450 enzyme.     

Cloud-point extraction for the determination of the free fraction of antiepileptic drugs in blood plasma and saliva

A method for the determination of the free fraction of antiepileptic drugs in plasma and saliva was developed. The separation of free and protein-bound fractions was obtained by cloud-point extraction under optimum conditions of pH, surfactant type, and concentration. It is shown that the free fraction of carbamazepine and of phenobarbital in plasma coincides with the drug concentration in saliva. The dependence of the free fraction in plasma on the administered dose was studied. The influence of the simultaneous administration of the two drugs on their free concentrations was revealed: In the presence of carbamazepine the free fraction of phenobarbital is decreased markedly whereas phenobarbital does not influence the behavior of carbamazepine.     

Effect of delta sleep-inducing peptide, anticonvulsant preparations and nicotinamide on generalized seizure activity

The influence of delta-sleep inducing peptide (DSIP) upon seizures induced by corazol, bicuculline, picrotoxin, strychnine, thiosemicarbazide were investigated in experiments on F1(CBA X C57 BL/6) mice. It was shown that DSIP increased the latency of first seizure manifestation which were induced by corazol, bicuculline and picrotoxin and also resulted in a suppression of seizure severity of corazol and bicuculline induced seizures. Anticonvulsant action of DSIP was evident under the condition of the mild severity seizures development. The effect of DSIP was mostly pronounced in range of its doses from 10 to 100 mcg/kg. DSIP when combined with phenobarbital, carbamazepine, diphenylhydantoin or nicotinamide enhanced the antiepileptic effects of these anticonvulsant drugs.     

Effects of anticonvulsants on cholinergic and GABAergic properties in the neuronal cell clone NG108-15

The effects of anticonvulsant drugs on growth, cholinergic, and GABAergic properties were examined in the neuronal cell clone NG108-15. Cells were exposed for 4 days to valproic acid, phenobarbital, phenytoin, or carbamazepine in concentrations equivalent to therapeutic free levels in human serum. Experiments were also performed with varying concentrations of a recently proposed antiepileptic, gamma-vinyl GABA. Of these five anticonvulsants, cell growth (total protein and cell counts) was decreased with valproic acid and phenytoin but only valproic acid and gamma-vinyl GABA altered neurotransmitter markers. Therapeutic concentrations of valproic acid increased choline acetyltransferase activity to 142% of control but had no effect on either the activity of glutamate decarboxylase or the level of GABA. The effects of a higher (toxic) concentration of valproic acid (200 g/ml) were similar to those induced by the differentiating agent dibutyryl cyclic AMP: both decreased cell growth, enhanced the activity of choline acetyltransferase and reduced the activity of glutamate decarboxylase. Gamma-vinyl GABA had no effect on cholinergic markers but, at 1300 g/ml, increased GABA levels to 135% of control despite the reduction of glutamate decarboxylase to 68% of control. In the NG108-15 cell clone, anticonvulsants have varying effects on cell growth, differentiation, and neurotransmitter systems. Our findings do not support the proposal that the mechanism of action for valproic acid, phenobarbital, phenytoin, and carbamazepine is via alteration of GABA levels.     

Mexiletine and its Interactions with Classical Antiepileptic Drugs: An Isobolographic Analysis

Using the mouse maximal electroshock test, the reference model of tonic–clonic seizures, the aim of the present study was to determine the type of interaction between mexiletine (a class IB antiarrhythmic drug) and classical antiepileptics: valproate, carbamazepine, phenytoin, and phenobarbital. Isobolographic analysis of obtained data indicated antagonistic interactions between mexiletine and valproate (for fixed ratio combinations of 1:1 and 3:1). Additivity was observed between mexiletine and valproate applied in proportion of 1:3 as well as between mexiletine and remaining antiepileptics for the fixed ratios of 1:3, 1:1, and 3:1. Neither motor performance nor long-term memory were impaired by mexiletine or antiepileptic drugs regardless of whether they were administered singly or in combination. Mexiletine did not significantly affected brain concentrations of carbamazepine, phenobarbital or phenytoin. In contrast, the antiarrhythmic drug decreased by 23 % the brain level of valproate. This could be, at least partially, the reason of antagonistic interaction between the two drugs. In conclusion, the observed additivity suggests that mexiletine can be safely applied in epileptic patients treated with carbamazepine, phenytoin or phenobarbital. Because of undesirable pharmacodynamics and pharmacokinetic interactions with valproate, mexiletine should not be used in such combinations.

     

Anticonvulsant properties of the cerebrospinal fluid during activation of the antiepileptic system of the brain

Cerebrospinal fluid (CSF) was obtained after 30-40 sessions of daily electrical stimulation of the cat cerebellum vermis. The intraventricular injection of CSF (10 microliters) to Wistar rats increased the latent period of initial seizure manifestations, significantly reduced the number of animals with seizures and reduced the severity of seizures induced by korazol injection (40 mg/kg). Analogous seizure changes were observed in rats after intraventricular injection of CSF (10 microliters) from cats subject to 3-10 electroshock seizure fits. Intraventricular injection of CSF (250 microliters) obtained from cats after electroshock to cats with strychnine-induced epileptic foci in the brain cortex led to the suppression of the epileptic activity. The conclusion was made that different ways of antiepileptic system activation cause the accumulation of endogenous antiepileptic substances in CSF.     

Amino Acid Levels in the Cerebrospinal Fluid of Newly Diagnosed Epileptic Patients: Effect of Vigabatrin and Carbamazepine Monotherapies

Abstract: We studied the CSF amino acid levels of 42 patients with newly diagnosed epilepsy before treatment with antiepileptic medication and during monotherapy with either vigabatrin or carabamzepine. The present study shows that patients with newly diagnosed epilepsy have elevated levels of the excitatory amino acid glutamate in CSF. Vigabatrin monotherapy effectively prevents the appearance of seizures in patients with high baseline CSF glutamate levels. In these patients, vigabatrin not only elevates the levels of γ-aminobutyric acid, but also decreases the elevated levels of glutamate in CSF, which may also be important to the antiepileptic efficacy of vigabatrin. Patients with low CSF glutamate levels did not benefit from vigabatrin-induced changes in amino acid levels and successful monotherapy with carbamazepine did not affect CSF amino acid levels. The elevation of γ-aminobutyric acid is thus not the only way to achieve seizure control and there are several factors underlying the generation and control of seizures. Follow-up of the patients with high baseline glutamate CSF levels will show if the observed abnormalities are related to the severity of epilepsy in individual patients and if early treatment with vigabatrin of these patients could prevent the development of intractable epilepsy.     

Alterations in CSF GABA levels and seizure susceptibility developing during repeated administration of pentetrazole in dogs. Effects of γ-acetylenic GABA,valproic acid and phenobarbital

Daily administration of convulsive doses of pentetrazole in dogs resulted in a decrease in the seizure threshold and development of increasingly severe clonic-tonic convulsions. Concomitantly, the concentration of γ-aminobutyric acid (GABA) in cisternal cerebrospinal fluid (CSF) was markedly reduced, whereas plasma GABA levels were not altered. When re-tested after a 3-week resting period, animals were found to have retained their increased seizure sensitivity and reduction in CSF GABA levels. γ-Acetylenic GABA and phenobarbital in doses antagonizing the establishment of increased convulsive sensitivity in response to repeated pentetrazole injections also counteracted the fall in CSF GABA. Valproic acid proved less effective to influence the convulsive response of continued pentetrazole administration. The data suggest that a functional deficit in the GABA system may underlie the persistent changes in seizure susceptibility observed.     

The Influence of Bicuculline-Induced Seizures on Free Fatty Acid Concentrations in Cerebral Cortex, Hippocampus, and Cerebellum

Abstract: Using ventilated rats maintained on N₂O-O₂ (70:30, vol/vol) we induced continuous seizures with i.v. bicuculline and analysed free fatty acids (FFA) in cerebral cortex, hippocampus, and cerebellum after seizure durations of 1–120 min. In the cerebral cortex, peak FFA concentrations were observed after 5 min, with a threefold increase in total FFA content. The values then remained unchanged for the next 15-20 min, but decreased thereafter. At 60 and 120 min, total FFA contents were only moderately increased above control. In the initial period, arachidonic acid increased about 10-fold and stearic acid 2- to 3-fold, with little change in palmitic acid and linoleic acid concentrations. At all times, the docosahexenoic acid concentration was markedly increased. Following its massive accumulation at 1 min, arachidonic acid gradually decreased in concentration. Pretreatment of animals with indomethacin did not alter this behaviour. After 20 and 120 min of seizure activity, changes in total and individual FFA concentrations in the hippocampus were similar to those observed in the cerebral cortex. The cerebellum behaved differently. Thus, at 20 min the only significant change was a 5- to 10-fold increase in arachidonic acid concentration and, after 120 min, total and individual FFA concentrations were similar to control values. Furthermore, since the control values for arachidonic acid were much lower in the cerebellum, the 20-min values were only about 20% of those observed in the cerebral cortex and the hippocampus.     

Comparisons of estradiol,LH and FSH patterns in pregnant and nonpregnant beagle bitches

To characterize plasma estradiol, LH and FSH patterns of secretion during the bitch estrous cycle, blood samples were obtained daily from 15 days before until 135 days after the LH surge in 10 pregnant and 10 nonpregnant beagle bitches. After an initial increase between days 15 and 10 and an expected proestrous peak, estradiol concentrations increased again from days 9-12 (corresponding to cytological metestrus) from basal values observed around day 9 after the LH surge, and remained significantly elevated throughout the luteal phase both in pregnant and nonpregnant animals. Concomitantly with the end of the luteal phase, plasma concentrations of estradiol returned to basal values in both groups. During the mid- to late-luteal phase, mean basal LH secretion was significantly elevated throughout in the pregnant relative to the nonpregnant animals. However, in nonpregnant animals, pulsatility was increased and peaks of higher amplitude were observed. The plasma FSH profiles, determined by a specific homologous RIA, differed significantly between pregnant and nonpregnant bitches during the last two-thirds of the luteal phase with a mean FSH level more elevated during pregnancy. The FSH level then decreased around parturition and low concentrations during lactation period were observed. The FSH concentrations remained steady in nonpregnant luteal phases from early luteal phase through mid-anestrus. The differences in pregnant and nonpregnant LH and FSH concentrations suggest pregnancy differences in regulation of the corpus luteum. Finally, the elevated estradiol concentrations observed during the luteal phase of both pregnant and nonpregnant animals suggest that an ovarian production of estrogens may be involved in overall corpus luteum regulation in dogs as in other species.     

Multi-Indication Carbamazepine and the Risk of Severe Cutaneous Adverse Drug Reactions in Korean Elderly Patients: A Korean Health Insurance Data-Based Study

Objective

To evaluate the risk of severe cutaneous adverse drug reactions (SCAR) after exposure to multi-indication antiepileptic drugs for in Korean elderly patients.

Methods

We used a nationwide database from the Korean Health Insurance Review and Assessment Service claims constructed for the monitoring of drug utilization among the entire Korean elderly population from January 2005 to June 2006. We identified cases of SCARs among inpatients aged ≥65 years and those newly diagnosed with erythema multiforme according to the International Classification of Diseases, 10th revision code (L51). Each case was matched to four controls for gender, age, and the first hospitalization date as the index date. The use of carbamazepine, gabapentin, lamotrigine, topiramate, phenobarbital, phenytoin, and valproate during a 60-day period before the index date was compared. A conditional logistic regression analysis was performed to calculate the odds ratios (OR) and 95% confidence intervals (CI) of SCARs for antiepileptic drug.

Results

We identified 286 cases of SCAR and 1,144 matched controls. Among the 25 patients who were prescribed antiepileptic drugs within 60 days of the index date. There were 11 cases (3.8%) of severe ocular manifestations, and most elderly patients were first-time or short-term users of antiepileptic drugs. Among the 10 cases of carbamazepine use, only 2 cases were prescribed carbamazepine for seizure. All antiepileptic drugs were associated with an increased SCAR risk (adjusted OR = 3.42, 95% CI: 1.75–6.63). The SCAR risk was highest in patients treated with carbamazepine (adjusted OR = 10.39, 95% CI: 2.64–40.86, for multi-indication; adjusted OR = 6.84, 95% CI: 1.55–30.10, for neuropathic pain).

Conclusion

Carbamazepine use was associated with a nearly 10-fold increase in severe cutaneous drug reactions in Korean elderly patients. This association was consistently high with SCAR patients who received carbamazepine for neuropathic pain.     

Micellar electrokinetic capillary chromatography for therapeutic drug monitoring of zonisamide

The simultaneous determination of zonisamide, a new type of antiepileptic drug, and the typical antiepileptic drugs phenobarbital, phenytoin and carbamazepine in human serum was developed using micellar electrokinetic capillary chromatography (MECC) with a diode array detector. A high correlation was revealed between the zonisamide levels in human serum obtained by MECC and those obtained by high-performance liquid chromatography (r=0.981). The serum levels of phenobarbital, phenytoin and carbamazepine determined by MECC were almost equal to those obtained by fluorescence polarization immunoassay. The reproducibility of separation and quantification with MECC analysis was appropriate for the intra- and inter-day assay coefficients. Therefore, the MECC method established here could provide a simple and efficient therapeutic drug monitoring method for antiepileptic drugs in patients, especially those treated with a combination of zonisamide and other antiepileptic drugs.     

Evidence for involvement of the astrocytic benzodiazepine receptor in the mechanism of action of convulsant and anticonvulsant drugs

The anticonvulsant drugs carbamazepine, phenobarbital, trimethadione, valproic acid and ethosuximide at pharmacologically relevant concentrations inhibit [3H]diazepam binding to astrocytes in primary cultures but have much less effect on a corresponding preparation of neurons. Phenytoin as well as pentobarbital (which is not used chronically as an anticonvulsant) are equipotent in the two cell types. The convulsants picrotoxinin and pentylenetetrazol, the convulsant benzodiazepine RO 5-3663 and the two convulsant barbiturates DMBB and CHEB similarly inhibit diazepam binding to astrocytes but have little effect on neurons. On the basis of these findings it is suggested that these convulsants and anticonvulsants owe at least part of their effect to an interaction with the astrocytic benzodiazepine receptor, perhaps by interference with a calcium channel.     

Effect of anticonvulsant drugs on plasma total cholesterol, high-density lipoprotein cholesterol, and apolipoproteins A and B in children with epilepsy

The effect of long-term anticonvulsant drug therapy with phenobarbital, phenytoin, carbamazepine, primidone, and valproic acid in epileptic children on plasma total cholesterol and high-density lipoprotein cholesterol (HDLC) was studied. Except valproic acid, all the drugs significantly increased the total cholesterol and HDLC, but the effect was more pronounced with HDLC. Among the subfractions of HDLC, almost all the increase due to drug therapy were in the HDLC-2 fraction. Treatment with antiepileptic drugs had no effect on HDLC-3. Apolipoprotein-A levels were significantly higher with drug therapy, but no effect was seen in the apolipoprotein-B levels. Plasma concentration of total cholesterol, HDLC, or its components was unaffected with valproic acid therapy.     

Hormonal regulation of retinoic acid-binding proteins in the mammary gland.

A cytosolic retinoic acid-binding (RAB) protein that sediments specifically as a 2S component on sucrose density gradients was detected in the mammary glands of virgin, pregnant and lactating rats. Mammary cytosol from pregnant rats contained significantly higher concentrations of cytosolic RAB protein than did cytosol from either virgin or lactating rats. The glands of pregnant animals exhibited increased concentration of cytosolic RAB protein during the first 5 days of pregnancy, and a steady decline was observed thereafter. The concentration of cytosolic RAB protein dropped to the value observed during lactation on the day 20 of pregnancy. Moreover, throughout lactation, low concentrations of cytosolic RAB protein were maintained. Daily treatment of virgin and lactating animals with 5 micrograms of oestradiol-17 beta for 1 week increased cytosolic RAB protein to concentrations comparable with those seen in pregnant rats. Progesterone, however, did not affect the mammary cytosolic RAB protein content of virgin rats. These results suggest hormonal involvement in the regulation of cytosolic RAB protein concentration of mammary gland during differentiation.     

Cholesterol synthesis in the perfused liver of pregnant hamsters

Pregnancy is a risk factor for the development of cholesterol gallstones. In pregnant women, biliary cholesterol saturation and secretion are increased. To investigate whether this was due to increased cholesterol synthesis, we studied hepatic cholesterol synthesis in Syrian Golden hamsters. Female controls and animals 10- to 14-days pregnant were studied. The studies were performed in the in situ perfused hamster liver. Cholesterol synthesis was determined by measuring the incorporation of 3H2O added to the perfusate into hepatic, perfusate, and bile cholesterol during a 90-min period. In both pregnant groups, bile flow decreased significantly, but biliary cholesterol concentration increased only in the 14-day pregnant group. The cholesterol synthesis rate averaged (mean +/- SD) 172 +/- 27, 127 +/- 37, and 552 +/- 79 nmol X hr-1 X g liver-1 in controls, 10-day, and 14-day pregnant animals, respectively. The 14-day pregnant animals secreted a markedly higher fraction (47.3 +/- 11.3 vs. 11.1 +/- 13.4%; P less than 0.01) of newly synthesized cholesterol into bile but not into perfusate. Chenodeoxycholate, but not cholate, synthesis rate was decreased in both pregnant groups. We conclude from our studies that hepatic cholesterol synthesis increases towards the end of pregnancy in the hamster and that more newly synthesized cholesterol is secreted into bile at that time. This could at least partially explain the increased biliary cholesterol saturation and secretion observed in women in the third trimenon, and explain pregnancy as a risk factor in the development of cholesterol gallstones.     

The effect of naloxone administration on pregnancy-associated seizures

Pregnant mice are more susceptible to flurothyl-induced seizures than are non-pregnant controls. The possibility that the well-known increase in beta-endorphin concentration which accompanies pregnancy was involved in this effect was examined by testing whether naloxone administration could block the increased seizure susceptibility. Pregnant female, control female and male C3H mice were treated with 5-50 mg/kg naloxone 5 min before flurothyl seizure testing. Naloxone markedly increased clonic seizure susceptibility in all three groups at a dose of 50 mg/kg, but had little effect at lower doses. In contrast, naloxone had differential effects on myoclonic seizures in pregnant and control female mice, being anticonvulsant in the controls, but proconvulsant in the pregnant mice. A role for endogenous opiates is unlikely in mediating clonic seizures in pregnant mice, but may be involved in myoclonic seizures.     

Fetal development, and placental and maternal plasma concentrations of progesterone in the little brown bat (Myotis lucifugus)

Progesterone concentrations measured in plasma samples from 280 bats captured during pregnancy or early lactation were related to fetal attributes indicative of stage of pregnancy. Fetal weight increased exponentially from 40 mg at crown-rump length of 6 mm to 2000 mg at 23 mm (term). Fetal weights at term accounted for up to 35% of the weight of intact pregnant animals. Progesterone concentrations increased from less than 5 ng/ml at 2 mm estimated crown-rump length to plateau values of approximately 65 ng/ml (geometric means) from 16 mm crown-rump length until the most advanced stages of pregnancy. Mean concentration in 8 post-partum bats, most of which were actively lactating, was 8.4 ng/ml; 11.6 ng/ml was measured in one animal that was carrying a wet neonate when sampled yet was still pregnant when captured 5 h earlier. Placental concentrations of progesterone ranged from 43 to 964 ng/g wet weight of tissue and mean values increased in a similar fashion though were about 4-fold greater than changes in plasma concentrations of the steroid. The concentrations in placental tissue were at least 15- to 20-fold higher than could be expected from blood contamination, indicating that placental steroidogenesis is likely to occur in this species.     

Apparent increase in type I 5'-deiodinase activity induced by antiepileptic medication in mentally retarded subjects

BACKGROUND/OBJECTIVES: Thyroid function measurements in 3 mentally retarded patients treated with antiepileptic drugs (phenytoin or carbamazepine) showed normal thyroid-stimulating hormone (TSH) responses in spite of markedly low levels of total thyroxine (T(4)), triiodothyronine (T(3)), and free thyroxine (FT(4)) concentrations; free triiodothyronine (FT(3)), as well as mean thyroxine-binding globulin (TBG) concentrations were normal. The objective of the present investigations was to determine if antiepileptic medication in these patients contributed to the disparate TSH and thyroid hormone (TH) levels. METHODS: Thyroid tests and other laboratory parameters were measured by conventional techniques. RESULTS: Circulating TH changes noted in retarded patients were similar to those observed in control subjects receiving carbamazepine alone. Reverse T(3) (rT(3)) levels in all patients were either undetectable or below the normal range. CONCLUSIONS: As type I 5'-deiodinase has a higher affinity for rT(3) than T(4), an increased activity of this enzyme would enhance rT(3) deiodination and reduce serum rT(3) concentration whereas enhanced T(4) deiodination would aid in normalizing intracellular FT(3) concentration. The finding of normal serum FT(3) concentration was consistent with normal TSH response and clinical euthyroidism in both retarded and control subjects. While phenytoin-induced increase in type I 5'-deiodinase has been previously noted, the present studies demonstrate a similar effect of carbamazepine on 5'-deiodinase.