Pituitary-dependent masculinization of hepatic hexobarbital hydroxylase in Crl:CD-1(ICR)BR mice

The sexual dimorphism in hepatic drug metabolism found in Crl:CD-1 mice is due to the normally repressive effects of testicular androgens on the activities of hepatic monooxygenases. The ability of testosterone to elevate the Michaelis constant (Km) and reduce the maximum velocity (Vmax) of hepatic hexobarbital hydroxylase is dependent upon the pituitary, so that in the hypophysectomized mouse androgens have no repressive effects on the activities of hepatic monooxygenases.     

The NADPH-dependent cytochrome P-450 reduction in liver microsomes of rats of different ages with and without phenobarbital pretreatment.

The activity of NADPH-cytochrome P-450 reductase in liver microsomes of 10- to 60-day-old rats was determined. Neither the half life time of cytochrome P-450 reduction nor the absolute amount of cytochrome P-450 reduced per time unit depend on age. Phenobarbital pretreatment enhances the reduction rate in all age groups. The addition of hexobarbital or ethylmorphine to microsomal suspension accelerates the reduction of cytochrome P-450 in some age groups only. Age differences corresponding to developmental changes in drug-metabolizing activities are not detectable. The NADPH-cytochrome P-450 reductase seems to be not responsible for the age dependence of drug metabolism.     

Biphasic effect of methadone on hepatic drug metabolism

When methadone HCl (30 mg/kg, po) was given acutely to mice, it was found to inhibit drug metabolism as evidenced by a prolongation of hexobarbital sleeping time and zoxazolamine paralysis time. Pharmacokinetic studies revealed that this acute dose of the narcotic analgesic could also prolong the plasma half-life of aminopyrine without any change in its volume of distribution. When added to the incubation mixture containing 10,000 g mouse liver supernatant fraction and a complete system for measuring aminopyrine N-demethylase or aniline hydroxylase, methadone showed a dose-dependent inhibition of the enzymes; the former enzyme was inhibited to a greater extent than the latter one. However, subacute treatment of mice with methadone HCl (30 mg/kg, po, twice daily for 3 days) resulted in increases in liver weight, microsomal protein, and cytochrome P-450 content in consonant with the increased activities of four hepatic drug-metabolizing enzymes: aminopyrine N-demethylase, aniline hydroxylase, p-nitroanisole, O-demethylase, and benzphetamine N-demethylase. Moreover, both hexobarbital sleeping time and zoxazolamine paralysis time were shortened. The plasma half-life of aminopyrine was decreased. These changes were prevented by simultaneous administration of puromycin diHCl (80 mg/kg, ip). Methadone thus seems to act in a manner very similar to that of propoxyphene or SKF-525A, acting as a potent inhibitor of hepatic drug metabolism when given acutely and as an inducer when given subacutely.     

Increase of hexobarbital sleeping time and inhibition of drug metabolism by the major metabolite of diphenylhydantoin

The major metabolite of diphenylhydantoin, 5-(p-hydroxyphenyl)-5-phenylhydantoin, has been shown to inhibit hexobarbital and ethylmorphine metabolism by a rat liver homogenate, and to markedly prolong hexobarbital sleeping time in mice. Such cross-inhibition of metabolism among drugs which are detoxified by formation of hydroxylated metabolites represents an example of a drug-drug interaction which must be taken into account when describing the behavior of drugs in the body.     

Enhancement of hexobarbital-induced sleep by lysine and its metabolites

Lysine has been shown to be metabolized in the rat brain to pipecolic acid which is a precursor of piperidine. Lysine and its proposed metabolites in this pathway were studied for the first time for their effect on the sleeping time induced by hexobarbital in the rat. Only $\text{L}$-lysine and $\text{D}$-lysine were found to prolong sleeping time significantly without toxic effect. A 3-day pretreatment with $\text{L}$-lysine produced an even more profound sleep prolongation. In most cases sleep enhancement was accompanied by a significant shortening of the time of sleep onset. Quantification of brain hexobarbital levels in the control and treated rats indicates that prolongation of sleeping time was not produced by inhibition of hexobarbital metabolism. The sleep prolonging effect of lysine, therefore, may be a direct action of lysine, or the metabolite(s) derived $\text{in}$$\text{vivo}$ from lysine, on the central nervous system.     

Alcohol-hexobarbital interaction in rats under acute stress.

In male Sprague-Dawley rats, one hour stress (unilateral hindleg ligature) and 3 g/kg ethanol (oral intubation) in combination inhibited $\text{in vitro}$ liver hexobarbital (HB) metabolism to a greater extent than either treatment alone. These treatments produced analogous effects on plasma HB disappearance $\text{in vivo}$. Ethanol alone or in combination with stress also increased HB sleep time. But stress alone or with ethanol reduced the HB sleep time, results which suggest that sleep time is not a reliable index of metabolism in stressed rats. This study shows that the effects of acute stress and alcohol on HB metabolism are additive.     

Liver extract effect on liver microsomal system and on an experimental model of intoxication]

A lyophilized liver extract (FLR) lengthens hexobarbital action in control rats ; it decreases slightly the induction of hepatic microsomal enzymes and normalizes diphenylhydantoin protection against electroshock seizure in phenobarbital-treated animals ; in the event of a CC14 intoxication, this extract reestablishes values close to normal for mebubarbital metabolism in mice and for BSP clearance in rats. FLR could act in regularizing drug metabolism.     

The influence of propranolol on the circadian fluctuations in the duration of the hypnogenic effect of diazepam and hexenal]

The influence of propranolol on the circadian variations of duration of the anaesthetic effect of diazepam and hexobarbital sodium were studied at 2-hour intervals in two separate round-the-clock observations on mice. Propranolol significantly changed the daily course of the anaesthetic effect of diazepam, caused its inversion and reduced the mean duration of hexobarbital anaesthesia. The observed antagonism between propranolol and hypnotics with varying anaesthetic properties could be attributed to its ability to interfere with both the cerebral neuromediator sleep regulating and beta-adrenoblocking systems but not to its specific blocking effects on pineal functions.     

Metabolism of diphenyloxazole (PPO) by mouse liver microsomes.

2, 5-Diphenyloxazole (PPO) is an inducer and inhibitor of aryl hydrocarbon hydroxylase. We report that PPO is itself metabolized to an alkali-extractable metabolite with intense fluorescence. The fluorescence spectra of excitation and emission indicate peaks at 345 nm and 510 nm, respectively. The reaction is linear with respect to time and enzyme concentration. NADPH is required for activity and the reaction is inhibited by carbon monoxide and 7, 8-benzoflavone but not by SKF-525A or hexobarbital. The intensity of fluorescence produced is similar to that of benzo (a) pyrene. PPO may be a useful model compound in studies of drug metabolism by the mixed function oxidase.     

Visualizing drug efficacy in vivo

Many enzymes are therapeutic targets for drug discovery, whereas other enzymes are important for understanding drug metabolism and pharmacokinetics during compound testing in animals. Testing of drug efficacy and metabolism in an animal model requires the measurement of disease endpoints as well as assays of enzyme activity in specific tissues at selected time points during treatment. This requires the removal of tissue and biochemical assays. Techniques to noninvasively assess drug effects on enzyme activity using imaging technology would facilitate understanding of drug efficacy, pharmacokinetics, and drug metabolism. Using a commercially available cytochrome P-450 3A substrate whose oxidized product is a luciferase substrate, we show for the first time that cytochrome P-450 enzyme activity can be measured in vivo in real time by bioluminescent imaging. This imaging approach could be applicable to study drug effects on therapeutic target enzymes, as well as drug metabolism enzymes.     

Changes in mixed-function oxidase system in the perfused liver of the cold-acclimated rat

Changes in the hepatic cytochrome P-450-dependent drug-metabolizing system were studied in perfused livers obtained from cold-acclimated male Wistar rats after 30 days of cold exposure (4C) when using hexobarbital as a substrate. In fasted animals the cold-acclimated rats showed higher levels of hexobarbital metabolic rates compared to control rats, but there was no significant difference in fed animals. The maximum rates of hexobarbital metabolism produced by xylitol perfusion were also significantly higher in the perfused liver of cold-acclimated rats. It was concluded that the function of the cytochrome P-450 system for hexobarbital in cold-acclimated rats changed due to both an increase in the activity of the cytochrome P-450 system and to changes in regulation of the cytochrome P-450 system by the supply of reducing equivalents.     

Development of a highly sensitive in vitro assay method for biological activity of endotoxin contamination in biological products.

The pyrogen test in rabbits has been replaced by the bacterial endotoxin test. The endotoxin test, however, showed a considerable discrepancy with pyrogenicity and was, therefore, assumed to have an efficacy limitation in directly predicting harmful biological effects of endotoxin. We developed a sensitive in vitro assay method by making use of tumour necrosis factor alpha (TNF-alpha) induction in RAW264.7 cells, which showed a fine correlation with pyrogenicity in rabbits. RAW264.7 cells maintained by serial subculture under an endotoxin-free condition have gained the similar level of sensitivity as the endotoxin test to allow extensive dilutions of a drug for eliminating adverse effects on the cells. The in vitro TNF-alpha induction assay was shown to be capable to detect quantitatively a synergistic effect of a drug and endotoxin. The synergy is assumed necessary to be taken into consideration to define the limit value for the endotoxin test for guaranteeing the similar level of safety as by the pyrogen test.     

Recombinant tumor necrosis factor depresses cytochrome P450-dependent microsomal drug metabolism in mice

The effect of recombinant tumor necrosis factor on liver cytochrome P450 and related drug metabolism enzymes was investigated. Treatment of mice with tumor necrosis factor caused a marked depression of cytochrome P450 and some drug metabolizing enzymes (ethoxycoumarin deethylase and arylhydrocarbon hydroxylase) in the liver and many other organs. This effect was maximal 24-48 h after treatment and was dependent on the dose of tumor necrosis factor administered. Depression of liver drug metabolizing enzymes was also observed in the endotoxin-resistant C3H/HeJ strain of mice, thus ruling out that this effect may be due to minor endotoxin contamination of recombinant tumor necrosis factor. These data indicate that depression of liver drug metabolism might be an important side effect of tumor necrosis factor, and suggest a role for this macrophage product as an endogenous regulator of liver metabolism.     

Hormonal disturbances and effect of pharmaca.

The hormonal milieu of the organism may exert certain influences on the activity and lasting of the effect of some drugs. This was demonstrated in female rats with the example of a steroid hormone (estrone) and a short acting barbiturate derivative (hexobarbital). Estrus produced with estrone is not to be influenced by ovariectomy. Hyperthyrosis lead to the cumulation of estrogens in active form and lengthened their effect. Hypothyrosis accelerated estrogen metabolism inducing a relatively ephemer effect of estrone. Hexobarbital metabolism is faster in ovariectomised rats as well as in thyroidectomised ones than in controls. The catatoxic effect of testosterone is bringing about a short anesthesia. Hyperthyrosis reduced the effect of hexobarbital to one fourth of the control values. A special care is necessary in administering drugs when endocrine disorders are present. Individual dosages of the pharmaca may be taken into consideration to adapt the right dosages to the hormonal disturbances.     

Endotoxin removal by charge-modified filters.

The effects of positively charged nylon and depth (cellulose-diatomaceous earth) filters on endotoxin removal from various solutions were evaluated. The charged filter media removed significant amounts of Escherichia coli and natural endotoxin from tap water, distilled water, sugars, and NaCl solutions; no significant removal of endotoxin was observed with negatively charged filter media. The extent of removal was influenced by pH, the presence of salts, and organic matter. Such media may be useful for the control of endotoxins in raw-product water or solutions used to prepare parenteral drug products or in other fluids where endotoxin control is desired.     

Endotoxin removal by charge-modified filters

The effects of positively charged nylon and depth (cellulose-diatomaceous earth) filters on endotoxin removal from various solutions were evaluated. The charged filter media removed significant amounts of Escherichia coli and natural endotoxin from tap water, distilled water, sugars, and NaCl solutions; no significant removal of endotoxin was observed with negatively charged filter media. The extent of removal was influenced by pH, the presence of salts, and organic matter. Such media may be useful for the control of endotoxins in raw-product water or solutions used to prepare parenteral drug products or in other fluids where endotoxin control is desired.     

Prevention of the toxicity of erythromycin estolate in the perfused rat liver by endotoxin

The possibility that endotoxin pretreatment could prevent the hepatotoxic effects of erythromycin estolate (EE) was investigated using the isolated perfused rat liver. The addition of E. coli endotoxin (25 micrograms/ml) to the perfusate, 30 min prior to EE administration at 150 or 200 microM, significantly ameliorated the decreases in bile and perfusate flow caused by either concentrations of the drug in control liver preparations. This phenomenon was also studied using liver isolated from rats pretreated in vivo with endotoxin for three days. In these preparations, EE at both concentrations did not alter bile flow and caused reductions of perfusate flow which were far less than those observed in untreated control livers. Furthermore, in livers from endotoxin-treated rats EE induced less reduction of bile acid excretion and, at 150 microM, it did not increase the bile to perfusate ratio of sucrose seen in control preparations after the drug, which may be an expression of altered hepatocytic membrane permeability. Since it is known that both endotoxin and EE interact with membranes, it is suggested that the "protective" effects of endotoxin may occur at the membrane level.     

Pharmacokinetics of Hexobarbital,Sulphadimidine and Chloramphenicol in Neonatal and Young Pigs

Half-life and apparent specific volume of distribution of hexobarbital, sulphadimidine and chloramphenicol were investigated in newborn, 1, 3, 5 and 8 weeks old pigs. Hexobarbital sleeping time and plasma concentration of hexobarbital at recovery were measured in the same age groups. The half-life of hexobarbital and chloramphenicol was long in newborn pigs but decreased fast during the first week after birth. From 1 to 8 weeks after birth the decrease was less pronounced. The half-life of sulphadimidine increased during the first 3 weeks of life, but in 1 and 3 weeks old pigs the amount of N4-acetylated sulphadimidine in plasma at 200 min. after the injection was higher than in the newborn pigs. The apparent specific volume of distribution of hexobarbital, sulphadimidine and chloramphenicol was changed in different ways from birth to 8 weeks of age. The hexobarbital sleeping time was very long in the newborn pigs and decreased until 3 weeks of age. The concentration of hexobarbital in plasma at recovery was unchanged from birth to 8 weeks of age. The concentration of chloramphenicol metabolites in plasma 100 min. after the injection increased very fast during the 8 weeks of observation. The concentration of N4-acetylated sulphadimidine in plasma at 200 min. after the injection increased from birth to 1 week of age, then it decreased. The data are stressing that the neonatal pig is a convenient model for pharmacokinetic testing of drugs used as pharmacotherapeutics in neonatal life.     

Enantiospecific quantification of hexobarbital and its metabolites in biological fluids by gas chromatography/electron capture negative ion chemical ionization mass spectrometry.

A highly sensitive and specific assay based on gas chromatography/electron capture negative ion chemical ionization mass spectrometry has been developed for the analysis of the enantiomers of hexobarbital and its major metabolites in human urine and plasma. S-(+)-(5-2H3)hexobarbital and R-(-)-(5-2H3)hexobarbital were synthesized for clinical studies along with (+/-)-(1,5-2H6)hexobarbital and the deuterated major metabolites for use as internal and reference standards. Hexobarbital enantiomers and their metabolites were analyzed after pentafluorobenzyl and trimethylsilyl derivatization, following solid-phase extraction from plasma and urine. Intense negative ion spectra were observed for all of the derivatives. The base peak in the spectra corresponded to the M-pentafluorobenzyl anion [M-PFB]- except for 1,5-dimethylbarbituric acid, where M-. was the most abundant ion. The applicability of the method was demonstrated by following the plasma concentration-time profiles and urinary excretion in a male extensive metabolizer of mephenytoin who was given a pseudoracemic oral dose of hexobarbital containing equal 50 mg amounts of S-(+)-2(H0)hexobarbital and R-(-)-(2H3)hexobarbital. Marked stereoselective disposition was observed, with the R-(-)-enantiomer being more efficiently metabolized, primarily by alicyclic oxidation and ring cleavage.     

Expression of a human liver cytochrome P-450 protein with tolbutamide hydroxylase activity in Saccharomyces cerevisiae

The human liver cytochrome P-450 (P-450) proteins responsible for catalyzing the oxidation of mephenytoin, tolbutamide, and hexobarbital are encoded by a multigene family (CYP2C). Although several cDNA clones and proteins related to this "P-450MP" family have been isolated, assignment of specific catalytic activities remains uncertain. Sulfaphenazole was found to inhibit tolbutamide hydroxylation to a greater extent than mephenytoin or hexobarbital hydroxylation. The inhibition by sulfaphenazole was competitive for tolbutamide and hexobarbital hydroxylation but with much different Ki values (5 vs 480 microM, respectively). Inhibition of mephenytoin hydroxylase was not competitive. The results suggest that different P-450 proteins in the P450MP family may be involved in the metabolism of these compounds. A cDNA clone (MP-8) related to the P-450MP family, isolated from a bacteriophage lambda gt11 human liver library, was expressed in Saccharomyces cerevisiae by using the pAAH5 expression vector. Yeast transformed with pAAH5 containing the MP-8 sequence (pAAH5/MP-8) showed a ferrous-CO spectrum typical of the P-450 proteins. Immunoblotting with anti-P450MP revealed that pAAH5/MP-8 microsomes contained a protein with an Mr similar to that of P-450MP-1 (approximately 48,000) that was not present in microsomes from yeast transformed with pAAH5 alone (1.7 X 10(4) molecules of the expressed P-450 per cell). Microsomes from pAAH5/MP-8 contained no detectable mephenytoin 4'-hydroxylase activity but were more active in tolbutamide hydroxylation, on a nanomoles of P-450 basis, than human liver microsomes. The pAAH5/MP-8 microsomes also contained hexobarbital 3'-hydroxylase activity, although the enrichment compared to liver microsomes was not great with respect to the tolbutamide hydroxylase activity.(ABSTRACT TRUNCATED AT 250 WORDS)