Proteomic and Bioinformatics Analysis of Membrane Lipid Domains after Brij 98 Solubilization of Uninduced and Phenobarbital-Induced Rat Liver Microsomes: Defining the Membrane Localization of the P450 Enzyme System.

The proteomes of ordered and disordered lipid microdomains in rat liver microsomes from control and phenobarbital (PB)-treated rats were determined after solubilization with Brij 98 and analyzed by tandem mass tag (TMT)-liquid chromatography-mass spectrometry (LC-MS). This allowed characterization of the liver microsomal proteome and the effects of phenobarbital-mediated induction, focusing on quantification of the relative levels of the drug-metabolizing enzymes._The microsomal proteome from control rats was represented by 333 (23%) proteins from ordered lipid microdomains, 517 (36%) proteins from disordered lipid domains, and 587 (41%) proteins that uniformly distributed between lipid microdomains. Most enzymes related to drug metabolism were mainly localized in disordered lipid microdomains. However, cytochrome P450 (CYP) 1A2, multiple forms of CYP2D, and several forms of UDP glucuronosyltransferases (UGT) 1A1 and 1A6) localized to ordered lipid microdomains. Other drug-metabolizing enzymes, including several forms of cytochromes P450, were uniformly distributed between the ordered and disordered regions. The redox partners, NADPH-cytochrome P450 reductase and cytochrome b5, localized to disordered microdomains. PB induction resulted in only modest changes in protein localization. Less than five proteins were variably associated with the ordered and disordered membrane microdomains in PB and control microsomes. PB induction was associated with fewer proteins localizing in the disordered membranes and more being uniformly distributed or localized to ordered domains. Ingenuity Pathway Analysis (IPA) was used to ascertain the effect of PB on cellular pathways, resulting in attenuation of pathways related to energy storage/utilization and overall cellular signaling and an increase in those related to degradative pathways. SIGNIFICANCE STATEMENT: This work identifies the lipid microdomain localization of the proteome from control and phenobarbital-induced rat liver microsomes. Thus, it provides an initial framework to understand how lipid/protein segregation influences protein-protein interactions in a tissue extract commonly used for studies in drug metabolism and uses bioinformatics to elucidate the effects of phenobarbital induction on cellular pathways.

Concentration of antiepileptic drugs in persons with epilepsy: a comparative study in serum and saliva.

AIM OF THE STUDY: The monitoring of antiepileptic drugs (AEDs) in clinical setting is important for measuring the efficacy of drugs and their safety and in personalizing drug therapy. We investigated the levels of AED, carbamazepine (CBZ), phenytoin (PHT) and phenobarbital (PHB), to understand their association in saliva compared with those in serum during the therapy. MATERIALS AND METHODS: In this study, we performed a prospective study of 116 persons with epilepsy (PWE; mean age 26.90 ± 11.83 years). Serum and saliva samples were collected at trough levels from the patients, who were under the treatment of CBZ, PHT and PHB either alone or in combination of these drugs for at least three months. The drug levels were assessed by high-performance liquid chromatography. RESULTS AND CONCLUSIONS: The number of males (n = 88; 75.86%) was higher than females (n = 28; 24.14%) among the recruited patients. The intake of CBZ, PHT and PHB was observed in 49.14%, 68.10% and 38.79% of PWE, respectively. The levels of these AEDs showed a significant correlation (p < 0.05) between serum and saliva. Interestingly, the levels of mono-therapy or bi-therapy showed a significant association (p < 0.05) between serum and saliva, however, there was no significant association in case of poly-therapy. This is the first report in the Indian population on simultaneous estimation of the three commonly used AEDs, such as CBZ, PHT and PHB in serum and saliva implicating their associations, either in mono-therapy or bi-therapy in PWE.

Clinical Pharmacology of Phenobarbital in Neonates: Effects, Metabolism and Pharmacokinetics.

Phenobarbital is an effective and safe anticonvulsant drug introduced in clinical use in 1904. Its mechanism of action is the synaptic inhibition through an action on GABAA. The loading dose of phenobarbital is 20 mg/kg intravenously and the maintenance dose is 3 to 4 mg/kg by mouth. The serum concentration of phenobarbital is up to 40 µg/ml. Nonresponders should receive additional doses of 5 to 10 mg/kg until seizures stop. Infants with refractory seizures may have a serum concentration of phenobarbital of 100 µg/ml. Phenobarbital is metabolized in the liver by CYP2C9 with minor metabolism by CYP2C19 and CYP2E1. A quarter of the dose of phenobarbital is excreted unchanged in the urine. In adults, the half-life of phenobarbital is 100 hours and in term and preterm infants is 103 and 141 hours, respectively. The half-life of phenobarbital decreases 4.6 hours per day and it is 67 hours in infants 4 week old.

Drug concentration in saliva.

It is possible to predict plasma concentrations of drugs by measurement in saliva, obviating the need for venipuncture. Using a selection of weakly acidic and basic drugs, we have found this prediction reliable for drugs largely nonionized at normal plasma pH (phenytoin, phenobarbital, antipyrine) but unreliable for ionized drugs (chlorpropramide, tolbutamide, propranolol, meperidine). Deliberate alteration of saliva flow rate and pH using different stimuli have produced twofold changes in saliva drug concentrations. Wide interindividual variability of saliva pH is the likely explanation for the inconstancy of saliva to plasma concentration ratios for ionized drugs.

Pentobarbital sodium and attack behavior in male Siamese fighting fish.

An experiment was undertaken to determine the effects of pentobarbital sodium on intraspecific attack behavior in male Siamese fighting fish in an attempt to extend earlier findings with chlordiazepoxide and secobarbital sodium. Pairs of fish fought while immersed in 20 microgram/ml or 40 microgram/ml pentobarbital sodium or plain water. The 40 microgram/ml group showed significantly less attack (e.g., biting, jaw locking) than either control or low dose groups without producing a change in general arousal. Quasisexual behavior, seen in an earlier chlordiazepoxide study, did not occur in the present study.

The effect of phenytoin and ethosuximide on primidone metabolism in patients with epilepsy.

Little is known about the influence of phenytoin and ethosuximide on primidone. Therefore we studied three groups of patients: 28 receiving primidone alone, 16 on comedication of primidone with phenytoin and 9 on primidone plus ethosuximide. Antiepileptic drug determinations were done with Kupferberg's gas chromatographic method. The results show that the addition of phenytoin--but not ethosuximide--does increase the plasma concentration of phenobarbital derived from primidone but not of primidone itself. The phenobarbital/primidone plasma concentration ratio is with 4.2 +/- 0.7 (+/- S.E.) significantly (P less than 0.001) higher in patients receiving primidone and phenytoin as compared to those on primidone alone (1.6 +/- 0.2) or together with ethosuximide (1.4 +/- 0.7). The effect of phenytoin occurs and persists for several days after the steady state plasma concentration of phenytoin has been reached. This effect is probably not due to induction of enzymes hydroxylating primidone but rather to inhibition of the metabolism and/or excretion of phenobarbital. A case of phenobarbital intoxication due to addition of phenytoin to primidone medication is described in detail.

Secretion of drugs by the parotid glands of rats and human beings.

The following drugs have been demonstrated to be secreted by the parotid glands of rats and human beings: amobarbital, chlorpromazine, codeine, glutethimide, meprobamate, pentobarbital, phenobarbital, and secobarbital. Methadone could not be detected in the parotid saliva of either rats or human beings, and morphine has been demonstrated only in parotid saliva of rats.

Quantitation and comparison of phenobarbital levels in the plasma, saliva, and cerebrospinal fluid of the rat, and the demonstration of an alteration of drug passage by ethanol.

In this study, the ability and extent of three biological fluids--plasma, saliva, and cerebrospinal fluid--to compartmentalize intravenously administered phenobarbital was examined and correlated. The three fluid compartments show markedly different levels of phenobarbital, though this probably does not reflect qualitative differences in the barriers that separate them, but rather in the nature of the compartments themselves. In addition to the quantitation and correlation of drug levels in the various compartments, intravenous administration of 400 mg/kg ethanol following the intravenous administration of 20 mg/kg phenobarbital was shown to alter the passage of phenobarbital into the different fluid compartments, causing a significant increase in the phenobarbital level of cerebrospinal fluid as compared to controls receiving no ethanol. Though the effect seen in the cerebrospinal fluid is significant, while the effect in saliva is not (though the trend was present), it is felt that the action of ethanol to alter drug passage is a non-specific effect on the vasculature. This finding of altered drug passage may help explain the observed synergistic interaction of ethanol and various sedative drugs.

Effects of encapsulation of primidone on its oxidative metabolism in rats.

The aim of this study was to evaluate the influence of primidone (PRM) nanoencapsulation on its metabolism. Suspensions of PRM powder and PRM-loaded poly-epsilon-caprolactone nanocapsules were administered orally in the same way to rats. Primidone-loaded poly-epsilon-caprolactone nanocapsules were prepared according to the interfacial deposition technique. Free PRM suspensions were obtained by addition of PRM powder to a suspension of 0.212% carboxymethylcellulose CMC 12H in water. The dose was 20 mg/kg, n = 6, for each experiment. Urinary and faecal levels of PRM and of its three major metabolites, phenylethylmalonamide (PEMA), phenobarbital (PB), and p-hydroxyphenobarbital (p-HO-PB), were determined. Concentrations were evaluated by high-performance liquid chromatography (HPLC) according to a validated analytical method. After PRM nanocapsule administration, non-metabolised PRM urinary levels were increased compared to those observed after administration of a suspension of primidone powder (43.7+/-8.8% after PRM-loaded nanocapsule and 37.7+/-8.1% after free PRM administration). For phenylethylmalonamide, no difference was observed in urinary excretion in the two cases. For two of the oxidised metabolites, PB and p-HO-PB, excretion was delayed and shortened. The amount of these oxidised metabolites was lowered from 0.95% after free PRM administration to 0.25% after PRM-loaded nanocapsule administration. No difference was noted in non-metabolised primidone excretion in faeces. These results suggest that primidone-loaded nanocapsules could be used as a vehicle for oral primidone administration in order to minimise the phenobarbital metabolic pathway.

Longitudinal study of phenobarbital in serum, cerebrospinal fluid, and saliva in the dog.

Serial assays for phenobarbital were done on the blood serum, cerebrospinal fluid (CSF), and saliva from three epileptic and two clinically normal dogs that were given various oral doses of the drug. In all dogs, the three fluids showed considerable daily fluctuations of phenobarbital concentrations, even after several weeks at the same dosage. The average ratio for CSF/serum was 0.53 and 0.39 for saliva/serum. A highly significant (P less than 0.001) correlation coefficient occurred between CSF and serum (0.95) and between CSF and saliva (0.93); although in some dogs at particular dosages, the CSF and saliva were poorly correlated. Dose-related curves demonstrated a gradually increased phenobarbital concentration in the three fluids up to dosages of 9.0 mg/kg of body weight. The present study suggests that multiple serum or saliva samples should be assayed to account for daily fluctuations; saliva can be a good indicator of unbound phenobarbital concentration in CSF.

[Drug permeation through synthetic lipid membranes. Part 10: The effects of diffusion layers on the permeation process (author's transl)]. / Arzneimittelpermeation durch künstliche Lipoidmembranen. 10. Mitteilung: Einfluss von Diffusionsschichten auf den Permeationsprozess.

Two different processes take their courses during the permeation of substances through lipid membranes. The substance must pass across the diffusion layers on the membranes and also across the lipid barrier proper. The effects of these two processes on the permeation vary with the kind of the membrane and of the substance as well as with the diffusion layer thickness which is determined by the intensity of movement. Correlations have been established which permit to calculate diffusion layers, membrane permeation coefficients, and also diffusion coefficients in the diffusion layers. Thus have been provided the prerequisites for the comparison of results obtained on using different model parameters.

Metabolism of dimethoxymethyl phenobarbital (eterobarb) in patients with epilepsy.

The metabolism of dimethoxymethyl phenobarbital (DMMP) was investigated in 6 epileptic patients using deuterium-labeled drug measured by integrated gas chromatography/mass spectrometry. Following an oral dose of 120 mg, none of the parent compound appeared in serum, urine, or saliva in spite of a measurement sensitivity of less than 0.05 nmol/ml. A metabolite, monomethoxymethyl phenobarbital (MMP), was detected in all patients, with peak serum concentrations averaging 1.96 nmol/ml at 1.3 hours. Phenobarbital was the major metabolite detected, and it reached peak levels in 6 to 12 hours. Two of the patients were continued on DMMP therapy for several months and then administered a pulse dose of deuterated drug. Again no DMMP was detectable. Steady-state drug administration had no effect on the kinetics of metabolism of either MMP or phenobarbital although peak serum concentrations were lower for both metabolites. In the urine of 5 patients an unidentified labeled fragment was detected that does not appear in urine from patients taking phenobarbital. From these pharmacokinetic studies it appears likely that the anticonvulsant activity of DMMP results from its metabolic conversion to phenobarbital.

Estimation of plasma unbound phenobarbital concentration by using mixed saliva.

Relationships among plasma total (Ct), plasma protein unbound (Cf) and mixed salivary (Cs) concentrations of phenobarbital (PB) were studied in 29 epileptic patients. A highly significant correlation was observed between Ct and Cf. Although a significant correlation was observed between Cf and Cs, Cs/Cf ratio was only 0.63. When Cs was corrected by using pKa (7.41) and pH of saliva to estimate free PB concentration (Cest f), a highly significant correlation was obtained between Cf and Cest f, and Cest f/Cr ratio was 0.96. The temporal course of Cest f obtained from Cs after an oral single dose of 50 mg PB in a healthy adult male volunteer was approximated as a triexponential equation. The peak time was 2 hr after ingestion and apparent half life was 66 hr. The prediction of minimum concentrations at steady state on the basis of these parameters corresponded well with actually obtained Cest f following repetitive administration of PB.

The interaction of albumin and drugs with two haemofiltration membranes.

The sorption of phenobarbitone sodium, barbitone sodium and fluconazole onto haemofiltration membranes made from polysulphone or a co-polymer of polyamide and polyvinylpyrrolidone was investigated in the presence and absence of albumin. The sorption of albumin was also followed in the presence of phosphate-buffered saline. Drug binding to the membrane was found to be reversible. Knowledge of the lipophilicity of the drug and hydrophobic/hydrophilic nature of the membrane did not allow successful prediction of the extent of binding of all the drugs; nor did knowledge of the extent of ionization of the drug and the charge of the membrane. Albumin bound to the polysulphone membrane in a manner that suggested the surface area to which it was binding was around 10 times greater than reported. In the presence of albumin there was a larger coefficient of variation in the binding of drugs to both membranes. The presence of albumin significantly decreased the binding of fluconazole, but not the other drugs, to the polysulphone membrane; however, albumin had no effect on the binding of any of these drugs to the polyamide membrane. We conclude that the binding of drugs to haemofiltration membranes cannot be simply predicted from knowledge of the hydrophilic/hydrophobic nature or charge of the drug and membrane, nor from the protein binding of the drug.

Diphenylhydantoin, phenobarbital, and primidone in saliva, plasma, and cerebrospinal fluid.

Diphenylhydantoin, primidone, and phenobarbital were determined in saliva and plasma of 164 patients by gas-liquid chromatography. The saliva ratio was about one-tenth in patients on diphenylhydantoin, 0.32-0.38 on phenobarbital alone and with other drugs, 0.97 and 0.96 on primidone alone and with other drugs. The S/P ratio of phenobarbital was similar in patients treated with primidone alone or with co-medication. For diphenylhydantoin and primidone, the S/P and CSF/plasma ratio were similar; for phenobarbital the S/P ratio was lower due to the difference in pH of saliva and CSF. Thus the concentration in saliva serves as a measure of the nonprotein-bound or free concentration in plasma with the advantage that saliva is easy to obtain. Co-medication does not change the S/P ratio for the three drugs studied. The high correlation between levels in plasma and in saliva allows the plasma levels to be predicted from the concentration in saliva.