Inhibition of lipid peroxide decomposition by compounds which bind with cytochrome p-450

The kinetics of lipid peroxide decomposition catalysed by microsomal enzymes and inhibited by SKF-525 A, hexobarbital, phenobarbital and aniline were investigated. The results indicate that the in vitro interaction of hexobarbital and SKF-525 A (type I binding compounds) with microsomal cytochrome p-450 inhibits the peroxidase activity while the in vitro interaction of aniline (type II binding compound) only slightly affect the peroxidase activity. It is suggested that LAHPO and type I binding compounds are competing for the hydrophobic binding site on cytochrome p-450, while type II binding compounds such as aniline negate electron transfer non-competitively by combining with the heme.     

Changes in Regional Brain Levels of Amino Acid Putative Neurotransmitters After Prolonged Treatment with t he Anticonvulsant Drugs Diphenylhydantoin, Phenobarbitone, Sodium Valproate, Ethosuximide, and Sulthiame in the Rat

The effect of prolonged treatment (10 days) with the anticonvulsant drugs diphenylhydantoin (DPH), phenobarbitone, sodium valproate, ethosuximide and sulthiame, both singly and in combination, on regional rat brain amino acid neurotransmitter concentrations (GABA, glutamate, aspartate and taurine) were assessed. DPH had a major effect in the cerebellum and hypothalamus in that it significantly reduced cerebellar GABA, taurine and aspartate and hypothalamic GABA and aspartate. Sodium valproate significantly elevated GABA and taurine in most regions. Aspartate and glutamate were less affected. Phenobarbitone significantly elevated GABA concentrations in all brain regions, while taurine concentration was only elevated in the cerebral cortex. Ethosuximide induced changes were small compared to the other anticonvulsants while sulthiame produced complex changes. Anticonvulsant drugs administered in combination resulted in complex changes, suggesting that their mode of action is different.     

Spectral studies on drug-cytochrome P-450 interaction in isolated rat liver cells

Various drugs including hexobarbital, lidocaine and nortriptyline were added to suspensions of liver cells isolated from untreated and phenobarbital-treated male rats. Upon drug addition, there was a fast binding to cytochrome P-450, as revealed by the appearance of a rapidly growing type I spectral change in the difference spectrum. When this had reached optimal magnitude, an absorption peak at 437 nm could often be seen to appear and quickly disappear, followed by yet another increase in absorption at about 446 nm; the latter and the type I spectral change then rapidly disappeared. These spectral changes were most pronounced with liver cells from phenobarbital-treated rats which contained markedly increased levels of cytochrome P-450. Also the rate of hexobarbital binding to cytochrome P-450 seemed to be increased after phenobarbital pretreatment. Finally, evidence was obtained that the major part of cytochrome P-450 in the isolated liver cells is present in an oxidized, non-substrate-bound form.     

Antiepileptic Agents Affect Hypothalamic β-Endorphin Concentrations

beta-Endorphin, Met-enkephalin, substance P, and somatostatin concentrations were evaluated in the hypothalami of rats treated either acutely or chronically (15 days) with sodium valproate, diphenylhydantoin, phenobarbital, or ethosuximide. All of these drugs, with the exception of ethosuximide, induced significant decreases in beta-endorphin concentrations after acute treatment, while only sodium valproate induced a decrease after chronic treatment. The acute and chronic effects of sodium valproate were also produced by aminooxyacetic acid, an inhibitor of gamma-aminobutyric acid (GABA) transaminase, while another GABA transaminase inhibitor, ethanolamine-O-sulphate, and THIP, a GABA receptor agonist, were effective after acute administration. Metenkephalin, substance P, and somatostatin concentrations were never affected by the drugs used. The present results, indicating that antiepileptic agents specifically decrease beta-endorphin concentrations, seem to correlate well with the capacity of these agents to blunt the epileptic activity of the peptides tested. Moreover, our data suggest that GABA may be involved in the anticonvulsant-induced reduction of beta-endorphin concentrations.     

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.     

Nonsubstrate induction of a soluble bacterial cytochrome P-450 monooxygenase by phenobarbital and its analogs

A soluble, cytochrome P-450-dependent fatty acid hydroxylase--epoxidase complex from Bacillus megaterium ATCC 14581 can be induced more than 100-fold by the addition of phenobarbital or one of its analogs (hexobarbital) to the growth medium. These barbiturate inducers are apparently not substrates for the enzyme nor do they activate the monooxygenase in the cell-free system. The induction efficiency of both phenobarbital and hexobarbital can be significantly increased with respect to monooxygenase activity by autoclaving the inducer in the growth medium rather than by adding it to the medium after autoclaving. Turnover numbers of about 3 000 nmoles of substrate oxygenated per min per nmole of P-450 were obtained in crude cell-free preparations obtained from maximally induced cultures. Our data indicate that products formed by heating phenobarbital or hexobarbital in the growth medium are significantly better inducers of monooxygenase activity than are the unaltered drugs.     

Effects of prenatal X-irradiation: studies on the mechanism of X-irradiation-induced inhibition of microsomal enzyme development in rat liver

Exposure of pregnant rats to 25 R of x-irradiation on the fourteenth gestation day has produced in the male offspring an impairment of the development of the hepatic microsomal enzyme system which metabolizes hexobarbital. However, irradiation did not suppress the increase of enzyme activity brought about by the administration of chemical inducers (pheno-barbital). Actinomycin, on the other hand, inhibited to varying degrees both the ontogenic and phenobarbital-induced increases in enzyme activity. The effects on the enzyme system have been supported by in vivo measurements of the duration of hexobarbital hypnosis. The ontogenic increase in enzyme activity is hormone-dependent, while that following phenobarbital administration is independent of hormonal regulation as evidenced by the response in hypophysectomized or sexually immature animals. It is concluded from these results that the inhibitory effect of x-irradiation on the hepatic enzyme system is mediated through an action on the hormonal regulation of enzyme activity. Evidence for this hypothesis is discussed.     

Extent and duration of drug-induced stimulation of renal excretion of p-aminohippuric acid]

Repeated pretreatment with p-aminohippuric acid (PAH), probenecide, cyclopenthiazide, and phenobarbital stimulates the renal excretion of PAH. For an interval of at least 6 hrs following i.p. application pretreated rats excrete more PAH excretion than the controls. All the drugs studied were found to stimulate renal PAH excretion within a period of 3 hrs by 50-60% of the control value. The required duration of pretreatment varies with the substance used. With cyclopenthiazide, the excretion of PAH is demonstrable for 2-3 weeks. Phenobarbital has a brief stimulating action. Correlations of a binding of drugs to structures of their tubular cell and the length of the stimulating action are discussed.     

Effect of anticonvulsants on human chromosomes. 2. In vitro studies

The effects of 3 anticonvulsant drugs (diphenylhydantoin, ethosuximide, and phenobarbital) on human peripheral lymphocytes in vitro were studied. The rate of chromosomal aberrations induced by the 3 anticonvulsants was significantly increased from the first concentration analyzed, similar to half the therapeutic serum concentration. These findings are compared with other previous reports.     

Benzodiazepine Receptors on Primary Cultures of Mouse Astrocytes

Benzodiazepines bind to glial membranes on a single type of site, with a high affinity (KD = 5 x 10(-9) M) on about 100 fmol of sites per mg protein. The number of binding sites is increased when the membranes are treated with Triton X-100. Antiepileptic drugs such as clonazepam and phenobarbital and hypnotic drugs such as Ro-11-3128 and Ro-11-6896 are able in pharmacological concentrations to displace [3H]flunitrazepam from its glial binding sites.     

Anticonvulsant drugs and the genetically epilepsy-prone rat

Anticonvulsant drugs were evaluated in members of two colonies of genetically epilepsy-prone rats (GEPR). Virtually all of the animals in the first colony experience a wild running fit that terminates in a generalized clonic convulsion when they are stimulated by sound. According to our convulsion intensity scoring system, these animals have an audiogenic response score (ARS) of 3 and the colony is designated the GEPR-3 colony. In the second colony, more than 95% of the animals experience a wild running phase terminating in a tonic extensor convulsion when they are stimulated by sound. That is, they have an ARS of 9 and the colony is designated the GEPR-9 colony. All of the established antiepileptic drugs that were tested produced anticonvulsant effects in the GEPR. Three tricyclic antidepressant agents acted as anticonvulsants in doses substantially lower than the toxic doses that produced spontaneous convulsions. Two of the established anticonvulsants, phenobarbital and ethosuximide, produced anticonvulsant effects in very similar doses in members of GEPR-3 and GEPR-9 colonies. Valproic acid produced an anticonvulsant effect in GEPR-3 in significantly lower doses than in GEPR-9. Carbamazepine, phenytoin, imipramine, amitriptyline, and desipramine produced anticonvulsant effects in essentially equimolar doses and in each case the protective dose was significantly lower in GEPR-9 than in GEPR-3 colonies. GEPR did not experience the convulsive effects of imipramine, amitriptyline, and desipramine at lower doses than did control animals. Thus, these epilepsy-prone animals are no more likely to experience convulsions in response to overdose of one of these three drugs than are nonepileptic subjects.     

Competition between hydrocarbon and barbiturate for spectral binding to hepatic cytochrome P-450. Inferences concerning spin state of the enzyme

The substrates hexobarbital and ethylbenzene have been shown to compete for the spectral binding site of phenobarbital-induced rat hepatic microsomal cytochrome p-450. The two substrates produce different delta Absmax values, and the presence of one substrate does not affect the delta Absmax of the other substrate and vice versa. The respective binding constants for the two substrates are similarly unaffected. The conclusion drawn from these observations is that, over the concentration ranges studied, there is no change in the availability of the enzyme as a result of substrate addition; the difference in delta Absmax apparently being due to varying abilities of different substrates to bring about a spin shift in the enzyme. Evidence is presented to indicate that differences between enzymes from untreated male rats and phenobarbital-treated male rats are attributable to differences in the enzyme itself and not to changes in the nature of the membrane brought about by phenobarbital administration, at least insofar as heat entropy compensation is concerned. The enthalpy-entropy compensation observed in the binding of a homologous series of barbiturates to the microsomal membrane as determined from the membrane concentration dependence of their binding constants is shown to agree surprisingly well with the direct determination performed by Sitar and Mannering.     

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.     

Effect of praseodymium chloride on liver microsomal enzymes of rats

A single i.v. dose (5 mg/kg) of a light lanthanon, praseodymium, prolonged the duration of hexobarbital-induced sleep and zoxazolamine-induced paralysis, as well as it modified pharmacokinetic parameters of hexobarbital and zoxazolamine, in rats. Half-lives (t1/2) and area under the curve (AUC) were increased, while elimination coefficient (beta) and clearance (Cl) were decreased. However, in daily doses of 1 mg/kg i.p. for 15 days, praseodymium did not alter pharmacological effects and pharmacokinetic parameters. The in vitro hydroxylation of hexobarbital and zoxazolamine by liver microsomes was inhibited when the animals were treated previously with a single i.v. dose (5 mg/kg) of praseodymium chloride. In these animals, the amount of cytochromes P-450 and b5 were reduced significantly, whereas that of NADPH-cytochrome c reductase remained unchanged. The pretreatment of animals with phenobarbital normalized the microsomal enzyme impairment caused by praseodymium.     

Spectrophotometric and radiometric studies on the in vivo binding of phenobarbital to hepatic microsomes.

We have examined hepatic microsomes prepared from phenobarbital-treated animals for the presence of the inducer and found that significant concentrations of the drug remain bound even after a number of washings. The bound drug is at least 70% unmetabolized and interferes with the in vitro binding of ethylmorphine and phenobarbital to the type I binding site but is not bound to the type I site since the K_s is 0.2–6.4 μm whereas the K_s for type I binding of phenobarbital is 103 μm. The high affinity site was not observed in microsomes from control animals either spectrophotometrically or radiometrically. The bound drug can be removed by bovine serum albumin to give a reverse pseudo-type I spectrum with a peak at 412 nm rather than the 425 nm observed for the trough of the typical type I spectrum. These data suggest that induction with phenobarbital may alter the spectral properties of cytochrome P-450 and hence care should be taken in comparing spectral data between microsomes from phenobarbital-treated and control animals.     

Circadian Rhythm of the Liver of Male Rats Pre-Treated with Phenobarbital—ii. Hexobarbital Sleeping Time and Lipids Content in Liver and Serum

The circadian rhythm of hexobarbital sleeping time and lipids content in liver and serum were studied in 226 male Sprague-Dawley rats pretreated daily at 0800-0900 with 70 mg/kg (study 1 or 3) or 50 mg/kg (study 2) phenobarbital (PB) orally for 7 days. Thereafter, eight (study 1) or five (study 2 and 3) rats each were studied at 4-hr intervals at 1000, 1400, 1800, 2200, 0200, 0600 and 1000 through the following day. The lighting schedule in the colony was 12:12 ± light:dark (light from 0600 to 1800). The hexobarbital sleeping times of PB-pretreated rats were generally shortened compared to the controls and no circadian rhythm was observed. PB-treatment increased slightly the liver content of cholesterol, and significantly that of triglycerides and phospholipids. Liver cholesterol and phospholipids showed circadian rhythms with peaks during the dark phase. No circadian rhythm of liver triglycerides existed. In serum, levels of triglycerides and phospholipids were slightly lowered by PB-treatment, while levels of cholesterol and beta-lipoprotein were not influenced. Serum values did not exhibit circadian rhythms.     

Induction of the microsomal monooxygenase system of rat liver by combined administration of testosterone and phenobarbital

A comparative study of the ability of phenobarbital, testosterone and their combination to induce the liver microsomal monooxygenase system after 9-day administration of these compounds to intact male and female rats was carried out. It was shown that administration of testosterone does not increase the level of cytochromes P450 and b5 in the livers of male and female rats. However, after a combined administration of the two compounds testosterone significantly enhances the inducing effects of phenobarbital (i. e. superinduction) in female rats; no such effect was observed in the livers of male rats. The rates of oxidation of hexobarbital, ethylmorphine and testosterone by liver microsomes are also increased after a combined administration of the two inducers. However, the additive effects of the two substances on substrate oxidation are observed when the latter was calculated per mole of cytochrome P450. An administration of testosterone to male rats does not result in an increase of the rate of hexobarbital and testosterone oxidation by isolated liver microsomes.     

Hippocampal afterdischarges in rats. I. Effects of antiepileptics

Hippocampal afterdischarges (ADs) are considered to be a model of complex partial seizures. To study the pharmacology of these ADs, stimulation electrodes were implanted into the dorsal hippocampus of 33 male Wistar rats. Stimulation (15-s series of monophasic rectangular pulses with a duration of 1 ms and frequency of 8 Hz) was applied four times with interstimulation intervals of 15 min. Drugs (carbamazepine 50 and 100 mg/kg; clonazepam 0.2 and 0.5 mg/kg; ethosuximide 125 and 250 mg/kg; phenobarbital 40 and 80 mg/kg) as well as solvent and isotonic saline were injected intraperitoneally 2 min after the cessation of the first AD. Duration of AD, of the latent period between AD and recurrent AD and duration of recurrent AD and the number of wet dog shakes were measured. ADs were markedly shortened by both doses of clonazepam and phenobarbital and by the higher dose of carbamazepine. The action of ethosuximide was negligible. Wet dog shakes were influenced in the same way as AD duration. Recurrent ADs were more sensitive to antiepileptics than ADs and wet dog shakes.     

Occupational exposure to anaesthetic gases and urinary excretion of D-glucaric acid

In order to ascertain whether the urinary excretion of D-glucaric acid (DGA) might be a suitable biomarker of effect in monitoring workers exposed to anaesthetic gases, we measured DGA before and after an operating session (and, in some workers, before and after a 2-week vacation) in 229 workers of surgical units and in 229 controls. In the former, we also measured urinary levels of nitrous oxide (N₂O) and isofiurane after at least 4 h of exposure. For all subjects, information on age, smoking habits, daily intake of alcohol, coffee, and drugs, history of liver or kidney disease was collected. Study subjects were ranked according to: exposure (class 0: subjects not exposed; class 1: N₂     

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.