Chromosomal investigations in epileptic children during long-term therapy with phenytoin or primidone

Summary In epileptic children the long-term therapy with anticonvulsant drugs is absolutely necessary. However, anticonvulsant drugs have been suspected to be mutagenic and teratogenic. To investigate this problem metaphase chromosome observations were performed using short-time culture of peripheral blood lymphocytes from twenty children. Ten of the children had been treated with phenytoin and the other ten with primidone on monotherapy. The long-term administration of anticonvulsant drugs was monitored by measurement of the serum concentrations of phenytoin and primidone, by seizure anamnesis, and by repeated EEG investigations. Analyzing 100 mitoses from each proband, we found no increase of structural or numerical aberrations in our patients compared with six controls. In adults, however, anticonvulsant drugs have been found to cause structural aberrations and chromosomal damage. The absence of these lesions in children may reflect the higher efficiency of DNA-repair in local DNA-damage.Dedicated to Prof. Dr. H. Schoenenberg, Aachen, on his 65th birthdayThis work contains parts of the M.D. theses of U.M. and E.M.     

Disturbance of Calcium Metabolism by Anticonvulsant Drugs

A survey of calcium metabolism in epileptic patients in a residential centre showed a subnormal serum calcium level in 22·5% of patients and a raised alkaline phosphatase in 29%. Hypocalcaemia was related to high dosage of anticonvulsant drugs, to multiple drug therapy, and to the use of individual anticonvulsant drugs in the following order, with decreasing order of importance: pheneturide, primidone, phenytoin, phenobarbitone. Subnormal serum calcium levels occurred more commonly in patients with a raised liver alkaline phosphatase isoenzyme than in those whose phosphatase was mainly of bone origin.Preliminary results of treatment with calciferol suggested that the disturbance of calcium metabolism was the result of vitamin D deficiency. It is possible that anticonvulsant drugs accelerate the breakdown of vitamin D by liver enzyme induction.     

The Effect of Time of Administration of Phenytoin on its Serum Levels and Pharmacodynamic Parameters in Patients of Generalised Tonic Clonic Seizures

The role of the time of administration of phenytoin on its serum levels and pharmacodynamic parameters was studied in two comparable groups of patients of grand mal_epilepsy stabilized on serum phenytoin therapy. In these patients, phenytoin was advised to be taken as a single daily dose either at 08.00 (Group M) or 20.00 (Group E) every day. A serum phenytoin level profile over 24 hours was obtained and all patients were clinically followed up for six months. Maximum serum concentration (C-max) of phenytoin was achieved significantly faster (p < 0.001) in patients of Group E. The total number of serum samples having toxic concentrations and also the incidence and severity of toxic effects of phenytoin was less in Group E. The study suggests that bedtime administration of phenytoin in patients of grand mal–epilepsy may lead to faster absorption of the drug and less side effects as compared with drug administration in the morning.     

Incidence of Anticonvulsant Osteomalacia and Effect of Vitamin D: Controlled Therapeutic Trial

The bone mineral content (B.M.C.) in both forearms (related to total body calcium) was measured by photon absorptiometry for a controlled therapeutic trial in a representative sample of epileptic outpatients, comprising 226 patients treated with one or two major anticonvulsant drugs (phenytoin, phenobarbitone, primidone).Initially the mean B.M.C. value for all epileptic patients was 87% of normal. During treatment with 2,000 international units of vitamin D₂ daily for three months an average B.M.C. increase of 4% was found, whereas the B.M.C. values remained unchanged in the placebo group and in the control groups. The incidence of hypocalcaemia and raised serum alkaline phosphatase was 12% and 43% respectively. The biochemical indices of osteomalacia were related to B.M.C. These results indicate that epileptic patients should be closely supervised for the occurrence of anticonvulsant osteomalacia, and, possibly, receive prophylactic treatment with vitamin D.     

Robust isocratic high performance liquid chromatographic method for simultaneous determination of seven antiepileptic drugs including lamotrigine, oxcarbazepine and zonisamide in serum after solid-phase extraction

A rapid, simple and robust method is presented for the simultaneous determination of seven antiepileptic drugs (AEDs), including primidone, phenobarbital, phenytoin, carbamazepine with its two major metabolites carbamazepine-10,11-epoxide and carbamazepine-10,11-(trans)-dihydrodiol and the new AEDs lamotrigine, hydroxycarbazepine (active metabolite of oxcarbazepine) and zonisamide in serum by high performance liquid chromatography (HPLC)-diode array detector (DAD). After solid-phase extraction, separation is achieved on an Alltima 3C18 analytical column using isocratic elution with a mixture of acetonitrile, methanol and phosphate buffer at 45 degrees C. The method is exhaustively validated, including experimental design in combination with statistical evaluation (ANOVA) to study the robustness of chromatography and sample preparation. Commonly co-administered antiepileptic drugs do not interfere with the method. Intra-day precision (RSD<1.9%), linearity, lower limit of quantitation (LOQ<0.065 mg/l) and robustness make the method suitable for daily therapeutic drug monitoring and pharmacokinetic studies.     

Antiepileptic drugs: Impacts on human serum paraoxonase‐1

Serum paraoxonase (PON1) is a key enzyme related to high‐density lipoprotein (HDL)‐cholesterol particle. It can prevent the oxidation of low‐density lipoprotein (LDL) and HDL. The present article focuses on the in vitro inhibition role of some antiepileptic drugs (AEDs) such as valproic acid, gabapentin, primidone, phenytoin, and levetiracetam on human paraoxonase (hPON1). Therefore, PON1 was purified from human serum with a specific activity of 3976.36 EU/mg and 13.96% yield by using simple chromatographic methods. The AEDs were tested at various concentrations, which showed reduced in vitro hPON1 activity. IC₅₀ values for gabapentin, valproic acid, primidone, phenytoin, and levetiracetam were found to be 0.35, 0.67, 0.87, 6.3, and 53.3 mM, respectively. K_i constants were 0.261 ± 0.027, 0.338 ± 0.313, 0.410 ± 0.184, 10.3 ± 0.001, and 43.01 ± 0.003 mM, respectively. Gabapentin exhibited effective inhibitory activity as compared with the other drugs. The inhibition mechanisms of all compounds were noncompetitive.     

Microassay for primidone and its metabolites phenylethylmalondiamide,phenobarbital and p-hydroxyphenobarbital in human serum,saliva, breast milk and tissues by gas chromatography—mass spectrometry using selected ion monitoring

A method for the quantitative determination of primidone and its metabolites phenobarbital, phenylethylmalondiamide (PEMA) and hydroxyphenobarbital (free and conjugated) in serum, urine, saliva, breast milk and tissue has been developed. Following the addition of the methyl analogues of primidone, phenobarbital and PEMA as internal standards and of saturated ammonium sulphate, the samples (5–100 μl) were extracted twice with ethyl acetate—benzene (20:80). The extracts were divided into two equal portions; one portion was ethylated by Greeley's method for the analysis of primidone, phenobarbital and hydroxyphenobarbital, while the other was trimethylsilylated for the analysis of primidone and PEMA. A gas chromatographic—mass spectrometric system was used for the analysis of the derivatized extracts. Linear calibration curves were obtained in the concentration range studied (between 100 ng/ml and 30 μg/ml). The recoveries of the drugs were between 80 and 93%. The relative standard deviations were between 3.2 and 5.9% (100-μl serum samples containing 1 μg/ml of the drugs). The lower detection limits were found to be between 1.4 and 3.7 ng/ml using serum samples of 100 μl.These methods have been applied to the study of the placental transfer and neonatal disposition of primidone and its metabolites in the human.     

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.     

Therapeutic monitoring of anticonvulsant drugs in psychiatric patients: rapid, simultaneous gas-chromatographic determination of six commonly used anticonvulsants without interference from other drugs

A simple, sensitive and precise gas-chromatographic method for simultaneous extraction, derivatization and determination of methsuximide, ethosuximide, diphenylhydantoin, carbamazepine, phenobarbital and primidone in the presence of other drugs has been described. The method is especially useful for drug monitoring in patients on multiple anticonvulsant therapy while also on combination therapy with psychotropic drugs. It overcomes the analytical interferences between mephenytoin and phenobarbital; methsuximide and primidone; kemadrin and primidone; cholesterol and primidone; prolixin, haldol and other drugs; encountered in other methods using underivatized, trimethylsilylated or methylated drugs. As little as 0.5 microgram/ml of a drug can be determined and if needed the method can be scaled down to 0.3 ml plasma. The method yielded recoveries of 97-103% with standard deviations of 0.7-1.8. For a constant check of the precision, an internal quality control using daily analysis of a sample from a frozen plasma pool supplemented with known concentrations of the anticonvulsants was used. The method is suitable for use in routine clinical laboratory.     

Phenytoin absorption in volunteers receiving selected enteral feedings.

Phenytoin absorption is reportedly significantly altered in the presence of continuously administered enteral feedings, resulting in subtherapeutic serum phenytoin concentrations and loss of seizure control. We administered 500 mg of phenytoin as the suspension to five volunteers who were not receiving enteral feeding, again while they ingested protein hydrolysate enteral feedings hourly, and again during hourly ingestions of meat-base enteral feeding. Serum phenytoin concentrations, measured 3, 6, 9, 12, and 24 hours after phenytoin ingestion, were lowest with protein hydrolysate feedings. Mean serum phenytoin concentrations were consistently higher with the meat-base feeding than with the protein hydrolysate formula, although levels did not reach those of the control period. These data are in keeping with our previous observation that it is easier to attain therapeutic serum phenytoin concentrations in patients receiving a meat-base enteral feeding than in those receiving a protein hydrolysate formula.     

CYP2C polymorphisms, phenytoin metabolism and gingival overgrowth in epileptic subjects

Previous studies suggested that the onset of phenytoin-induced gingival overgrowth depended on serum phenytoin concentration. Cytochrome P450 2C (CYP2C) plays an important role in phenytoin metabolism. Recently, single nucleotide polymorphisms in the coding region of CYP 2C influencing phenytoin metabolism were identified. The purpose of the present study was to see if CYP 2C polymorphisms might relate to the onset and severity of phenytoin-induced gingival overgrowth. Twenty-eight epileptic patients taking phenytoin aged 15 to 75 (mean age: 42.2 years old, 20 males and 8 females) and 56 unrelated healthy subjects aged 30 to 48 (mean age: 36.8 years old, 48 males and 8 females) were examined for CYP 2C polymorphisms. All epileptic subjects were examined for the degree of gingival overgrowth, daily phenytoin dose and serum phenytoin concentration. The results indicated about 7% of the subjects including epileptic and healthy subjects examined were positive for CYP 2C9*3. However, the degree of gingival overgrowth did not directly correlate with CYP 2C polymorphisms. Nevertheless, the subjects with severer gingival overgrowth exhibited significantly higher serum phenytoin concentration, indicating that phenytoin metabolism is an important determinant for the severity of the disease. Additionally, CYP 2C9*3 carriers exhibited significantly higher serum drug concentration to drug dose. Therefore, we concluded although the gene analysis is not directly related to diagnose the disease itself, it can be utilized in estimating serum phenytoin concentration from drug dose, which in turn serves to predict the future development and clinical course of the disease.     

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.     

Anticonvulsants and thyroid function.

Serum total and free thyroid hormone concentrations were estimated in 42 patients with epilepsy taking anticonvulsants (phenytoin, phenobarbitone, and carbamazepine either singly or in combination). There was a significant reduction in total thyroxine (TT4), free thyroxine (FT4), and free triiodothyronine (FT3) in the treated group compared with controls. Free hormone concentrations were lower than total hormone concentrations, suggesting that increased clearance of thyroid hormones occurs in patients receiving anticonvulsants. Detailed analysis indicated that phenytoin had a significant depressant effect on TT4, FT4, FT3, and reverse T3 (rT3). Phenobarbitone and carbamazepine had no significant main effects, but there were significant interactions between phenytoin and carbamazepine for TT4 and FT4. phenobarbitone and carbamazepine for FT3, and phenytoin and phenobarbitone for rT3.     

Phenytoin-valproate interaction: importance of saliva monitoring in epilepsy.

Sodium valproate is often used with phenytoin when epilepsy cannot be controlled by a single drug. Sodium valproate depresses phenytoin protein binding and so invalidates plasma phenytoin monitoring as a means of determining precise phenytoin dosage requirements. Plasma and saliva phenytoin and plasma valproate concentrations were measured in 42 patients with epilepsy receiving both drugs. Phenytoin protein binding was also measured by ultrafiltration in 19 of these patients and 19 patients taking phenytoin alone. Saliva phenytoin concentration bore the same close correlation to unbound (therapeutically active) phenytoin in patients receiving both drugs as it did in patients receiving phenytoin alone, whereas plasma total phenytoin did not. The same therapeutic range for saliva phenytoin (4-9 mumol/1; 1-2 microgram/ml) was therefore valid in both groups. The depression of phenytoin binding was directly related to the plasma concentration of valproate both in random samples taken from the 42 patients and in samples taken throughout the day in two of these patients. This was confirmed in vitro. Even when the concentration of valproate is known the degree of binding cannot be predicted. Saliva rather than plasma monitoring of phenytoin treatment is therefore valuable in the presence of valproate and with reduced phenytoin binding from any cause.     

The necessity of drug level monitoring in anticonvulsant drug therapy.

A survey of 'steady-state' serum levels of anticonvulsant drugs from 221 epileptic patients at a university hospital was conducted. Serum concentrations of phenobarbital, primidone, and diphenylhydantoin were determined by a gas chromatographic method. Sixty-five percent (130) of the patients receiving diphenylhydantoin had levels below the therapeutic range of 10-20mug/ml. Subtherapeutic levels appear to be due to inadequate dosage adjustment. Only 25% (33) of the patients receiving phenobarbital had levels below the therapeutic range. Serum levels of diphenylhydantoin or phenobarbital could not be predicted from dosage. Most patients received two or more drugs. Over 10% of the patients had potentially toxic levels of anticonvulsant drugs. High levels of diphenylhydantoin were easily recognized clinically but high levels of phenobarbital were not.     

Liquid chromatographic assay in plasma of one of the members of a new series of anticonvulsants: -3-hydroxy-3-ethyl-3-phenylpropionamide

A method for the simultaneous determination of HEPP ( -3-hydroxy-3-ethyl-3-phenylpropionamide), a member of a new homologous series of phenylamide-derivative anticonvulsants, with six other antiepileptic drugs (ethosuximide, primidone, phenobarbital, phenytoin, carbamazepine and clonazepam) in plasma by high-performance liquid chromatography is described. These drugs are extracted from plasma by adding an equal volume of acetonitrile. An aliquot of the extract is then injected on a reversed-phase column with a acetonitrile-methanol-phosphate buffer mobile phase. The total time required for the whole analytical process, including the plasma pretreatment and chromatography, is approximately 30 min. The assay method is simple, rapid and reproducible, and therefore considered suitable for routine use in clinical investigations monitoring HEPP simultaneously with common antiepileptic drugs.     

Effect of topical phenytoin on burn wound healing in rats

To evaluate the effect of phenytoin on burn wounds and to compare the effect of the combination of topical phenytoin preparation in dexamethasone treated burn wounds in rats, partial thickness thermal burn wounds were inflicted upon five groups of six rats each. Group I was assigned as control, Group II received the standard silver sulphadiazine, Group III was given topical phenytoin and Group IV received injection dexamethasone, Group V received the combination of the phenytoin and the dexamethasone. The parameters observed were epithelialization period, percentage of wound contraction and histopathological analysis as indicative of the process of healing. Phenytoin group showed significant improvement in burn wound contraction in comparison to standard silver sulphadiazine group, the combination group of topical phenytoin and dexamethasone also showed significant contraction compared to dexamethasone group. The period of epithelialization also decreased significantly in groups II, III and V. In conclusion, phenytoin promotes burn wound healing as evidenced by decrease in period of epithelialization and faster wound contraction.     

Contributions of CYP2C9/CYP2C19 genotypes and drug interaction to the phenytoin treatment in the Korean epileptic patients in the clinical setting

We examined the contribution of CYP2C9 and CYP2C19 genotypes and drug interactions to the phenytoin metabolism among 97 Korean epileptic patients to determine if pharmacogenetic testing could be utilized in routine clinical practice. The CYP2C9 polymorphism is a wellknown major genetic factor responsible for phenytoin metabolism. The CYP219 polymorphism, with a high incidence of variant alleles, has a minor influence on phenytoin treated Koran patients. Using a multiple regression model for evaluation of the CYP2C9 and CYP2C19 genotypes, together with other non-genetic variables, we explained 39.6% of the variance in serum phenytoin levels. Incorporation of genotyping for CYP2C9 and CYP2C19 into a clinical practice may be of some help in the determination of phenytoin dosage. However, because concurrent drug treatment is common in patients taking phenytoin and many environmental factors are likely to play a role in drug metabolism, these factors may overwhelm the relevance of CYP polymorphisms in the clinical setting. Further investigations with an approach to dose assessment that includes comprehensive interpretation of both pharmacogenetic and pharmacokinetic data along with understanding of the mechanism of drug interactions in dosage adjustment is warranted.     

Liquid chromatographic assay in plasma of one of the members of a new series of anticonvulsants: d,l-3-hydroxy-3-ethyl-3-phenylpropionamide

A method for the simultaneous determination of HEPP (d,l-3-hydroxy-3-ethyl-3-phenylpropionamide), a member of a new homologous series of phenylamide-derivative anticonvulsants, with six other antiepileptic drugs (ethosuximide, primidone, phenobarbital, phenytoin, carbamazepine and clonazepam) in plasma by high-performance liquid chromatography is described. These drugs are extracted from plasma by adding an equal volume of acetonitrile. An aliquot of the extract is then injected on a reversed-phase column with a acetonitrile-methanol-phosphate buffer mobile phase. The total time required for the whole analytical process, including the plasma pretreatment and chromatography, is approximately 30 min. The assay method is simple, rapid and reproducible, and therefore considered suitable for routine use in clinical investigations monitoring HEPP simultaneously with common antiepileptic drugs.