Hexobarbital Research Articles

Automated pKa Determination of Soluble and Sparingly Soluble Drugs by Capillary Zone Electrophoresis

Capillary zone electrophoresis (CZE) was used for the pKa determination of several acidic drugs. The methodology relies on the measurement of the effective mobility of ionic species in electrophoretic buffers prepared at different pH values. The validation of the assay was achieved by comparing the pKa obtained by CZE with literature values obtained by standard techniques for a variety of soluble drugs (sulphadimethoxine, tolbutamide, nitrofurantoin, sulphaphenazole, ibuprofen, hexobarbitone) whose pKa values ranged from 4 to 8. The pKa values determined by CZE correlated with literature values (slope of 0.99 and coefficient of correlation of 0·995 as determined by linear regression). The main advantage of the CZE methodology over conventional techniques was the low amount of material needed (∼1 mg to prepare a stock solution) and the low concentration required (50–100 μm in this work) for pKa determination. Using eight non-steroidal anti-inflammatory drug candidates as model compounds, we were able to determine the pKa of these sparingly soluble compounds in 100% aqueous solutions. The values obtained by CZE for these compounds were compared with the apparent pKa values obtained by potentiometry in 50% methanolic solutions. Using this latter technique, three of the eight compounds tested could not have their pKa determined because of precipitation problems. The pKa values of the five remaining compounds determined by potentiometry were consistent with those determined by CZE. This work has shown that CZE can be a powerful automated technique for pKa determination and one of its major advantages is its application to the pKa analysis of sparingly soluble compounds.

Stereoselective hexobarbital 3′‐hydroxylation by CYP2C19 expressed in yeast cells and the roles of amino acid residues at positions 300 and 476

We examined the enzymatic function of recombinant CYP2C19 in enantiomeric hexobarbital (HB) 3'-hydroxylation, and searched the roles of amino acid residues, such as Phe-100, Phe-114, Asp-293, Glu-300, and Phe-476 of CYP2C19 in the stereoselective HB 3'-hydroxylation, using a yeast cell expression system and site-directed mutagenesis method. CYP2C19 wild-type exerted substrate enantioselectivity of (R)-HB>>(S)-HB and metabolite diastereoselectivity of 3'(R)<3'(S) in 3'-hydroxylation of HB enantiomers. The substitution of Asp-293 by alanine failed to yield an observable peak at 450 nm in its reduced carbon monoxide-difference spectrum. CYP2C19-E300A and CYP2C19-E300V with alanine and valine, respectively, in place of Glu-300 exerted total HB 3'-hydroxylation activities of 45 and 108%, respectively, that of the wild-type. Interestingly, these two mutants showed substrate enantioselectivity of (R)-HB<(S)-HB, which is opposite to that of the wild-type, while metabolite diasteroselectivity remained unchanged. The replacement of Phe-476 by alanine increased total HB 3'-hydroxylation activity to approximately 3-fold that of the wild-type. Particularly, 3'(S)-OH-(S)-HB-forming activity elevated to 7-fold that of the wild-type, resulting in the reversal of the substrate enantioselectivity. In contrast, the substitution of phenylalanine at positions 100 and 114 by alanine did not produce a remarkable change in the total activity or the substrate enantioselectivity. These results indicate that Glu-300 and Phe-476 are important in stereoselective oxidation of HB enantiomers by CYP2C19.

Combined effect of propranolol with nifedipine or with diltiazem on rat liver monooxygenase activities

The effects of two Ca 2+ antagonists nifedipine (NF) and diltiazem (DL) and of the nonselective β-adrenergic blocking agent propranolol (PR) on the hexobarbital (HB) sleeping time and on the activity of some liver drug-metabolizyng enzyme systems in male Wistar rats were studied. Two h after single oral administaration PR (50 mg/kg) did not change HB sleeping time, while NF (50 mg/kg) and DL (30 mg/kg) prolonged it by 171.2 and 99.6%, respectively. Coadmistration of PR with DL or with NF significantly prolonged HB sleep by 240.7 and 129%, respectively. Only NF increased aniline 4-hidroxylase (AH) activity (by 92%) and the total P-450 content (by 24%). PR and NF increased cytochrome b5 content and this effect was also observed with the combinations PR+NF (by 109%) and PR+DL (by 102%). The NADPH cytochrome P-450 reductase activity was significantly decreased by NF and DL and after their combination with PR. The ethymorphine- N-demethylase (EMND) and amidopyrine- N-demethylase (APND) activities were not changed. The effects of PR, NF and DL administrated alone or in combination on liver oxidative metabolism are considered as possible mechanisms of drug interactions.

19] Antipyrine, theophylline, and hexobarbital as in vivo P450 probe drugs

This chapter focuses on antipyrine (AP), hexobarbital (HB), and theophylline (TH) as probe drugs for in vivo characterization of specific P450 enzymes. AP is the commonly used substrate in estimating the influence of all kinds of endogenous and exogenous factors on P450 enzyme activities. AP, HB, and TH can be used in experimental animals and in humans without side effects at the dose levels required for determination of P450 enzyme activity. Absorption of AP, TH, and HB from an oral solution is rapid and complete. They have been used alone, but also in combination with each other or other probe drugs. The chapter describes the methods that are reliable in estimating the pharmacokinetics of AP, TH, and HB. These model substrates have been applied very extensively in studying the influence of environmental and disease conditions on P450 enzyme activities both in humans and in experimental animals. Thereby, much insight has been obtained in the factors governing overall oxidative drug-metabolizing enzyme activity in vivo . Studies with other probe drugs, which are more specific for single P450 enzymes, are needed to elucidate selectivity in altered enzyme activity.

Effect of nifedipine, verapamil and diltiazem on the enzyme-inducing activity of phenobarbital and beta-naphthoflavone

1. 1. The effects of three calcium antagonists (CA), nifedipine (NF), verapamil (V) and diltiazem (DL) on the enzyme-inducing activity of phenobarbital (PB) and β-naphthoflavone (β-NF) were measured by hexobarbital (HB) sleeping time, benzphetamine-N-demethylase (BND) ethoxycourmarin-O-deethylase (ECOD) and ethoxyresorufin-O-deethylase (EROD) activities, as well as by the cytochrome P-450 and cytochrome b5 content. 2. 2. NF, V and DL at oral doses of 100, 80 and 60 mg/kg administered 1 hr before PB additionally shortened the HB sleeping time. 3. 3. Single administration of calcium antagonists on the background of developed PB-provoked enzyme induction prolonged the PB-shortened hexobarbital sleeping time. 4. 4. Three day administration of NF, V and DL plus PB slightly decreased the enzyme-inducing effect of PB on BND activity and cytochrome P-450 and cytochrome b5 content. 5. 5. NF and V administered only once after PB-provoked enzyme induction increased the PB enzyme-inducing effect on BND activity and on the content of cytochrome P-450. DL decreased the PB-induced BND activity and cytochrome P-450 and cytochrome b5 content. 6. 6. The calcium antagonists studied affected the enzyme-inducing activity of β-NF to a lesser extent when compared to PB. Only NF was found to potentiate the enzyme-inducing effect of β-NF on the EROD activity and on the cytochrome P-450 content.

Correlation between cytochrome p450 2b1 induction by barbiturates and toxicity in the chick embryoin ovo

Using the highly sensitive probe‐substrate pentoxyresorufin towards cytochrome P450 2B1 isozymes, the relationship between the induction of this set of hemoproteins by barbiturates and toxicity was studied. Each chemical, allobarbital (AB), amobarbital (AM), barbital (BA), butobarbital (BB), buthetal (BU), cyclobarbital (CB), heptobarbital (HE), hexobarbital (HX), mephobarbital (ME), pentobarbital (PA), pentotal (PT), phenobarbital (PB), secobarbital (SE) and vinbarbital (VB) was injected in the chick embryo at the 16–18 day of embryonic incubation in equivalent doses (50, 25, 12.5, 6.25 and 3.12% of the respective LD50) and the mixed function monooxygenase activity examined in purified hepatic microsomes. For each barbiturate, linear regression was performed for both the inductive and toxic slope related to P450 2B1‐like activity in order to evaluate the IC100 in μmol/egg [the dose producing a 100 % increase of pentoxyresorufin dealkylation (PROD) during the induction phase] and the TC100, (the dose prod...

Effects of furazolidone on duration of righting reflex loss induced with hexobarbital and zoxazolamine in the rat.

Effects of furazolidone (FZ) on the sleeping time induced with hexobarbital (HEX) and paralysis time induced by zoxazolamine (ZOX) were investigated by measuring the length of time required to recover from righting reflex loss in rats after oral administration of FZ at doses of 50, 100, 200 and 400 mg/kg/day for 4 successive days. Administration of 50 mg/kg to rats of both sexes induced no effect on the HEX sleeping time, but of 100 mg/kg FZ or more induced prolongation of sleeping time dose-dependently. In female rats, HEX sleeping time of the control group was twice that of the male rats, but HEX sleeping time after receiving FZ above 200 mg/kg was approximately the same as in the male rats. ZOX paralysis time exhibited no sex differences in the control rats, and it was significantly prolonged by FZ at a dose of 100 mg/kg or more. No significant differences in blood levels of HEX and ZOX at the time of recovery were found between the control and FZ treated rats, suggesting that FZ produced prolongation of the drug effects was due to the maintenance of the blood levels rather than the change in the sensitivities of rats at the receptor sites. Body weight gains were inhibited in the rats treated with FZ at doses over 100 mg/kg. Cytochrome P-450 content in hepatic microsomes in the rats which received 100 mg/kg FZ were slightly increased. It is suggested that successive oral administration of FZ to rats at high doses impaired drug clearance and this resulted in the prolongation of HEX sleeping and ZOX paralysis times.

On the enzyme-inducing action of calcium antagonists.

The effects of three calcium antagonists, nifedipine (NF), verapamil (V) and diltiazem (DL), on rat liver monooxygenases were studied. The drugs were administered in oral doses of 50, 40 and 30 mg/kg daily for 3 weeks in male Wistar rats. NF and V shortened the hexobarbital (HB) sleeping time and increased benzphetamin-N-demethylase (BND), ethylmorphine-N-demethylase (EMND), aniline hydroxylase (AH), ethoxycoumarine-O-deethylase (ECOD), ethoxyresorufin-O-deethylase (EROD) and NADPH-cytochrome c reductase activities and the content of cytochrome P-450 and microsomal heme, but did not change the content of cytochrome b5. The data suggest that these calcium antagonists exert an enzyme-inducing effect on the hepatic monooxygenases. DL significantly increased only the EROD and NADPH-cytochrome c reductase activities and shortened HB sleeping time to a lesser extent, suggesting a weaker enzyme-inducing effect as compared to NF and V. The three drugs increased the delta-aminolevulinic acid (ALA) synthetase activity and decreased heme oxygenase (HO) activity. The increased cytochrome P-450 content is probably due to the increased synthesis and the decreased breakdown of this hemoprotein.

Effects of nitrogen compounds with hexobarbital induced sleep in Swiss Albino mice

Many h e t e r o c y c l i c n i t r o g e n compounds a r e u t i l i z e d as i n d u s t r i a l s o l v e n t s and s y n t h e t i c i n t e r m e d i a t e s . P re sence of a l k y l s u b s t i t u t e d p y r i d i n e s in food f l a v o r s , and foods have been i d e n t i f i e d (Suyama and Adachi 1980). B u t t e r y e t a l . (1977) s e p a r a t e d 12 d i f f e r e n t p y r i d i n e s from r o a s t e d lamb. A d d i t i o n a l l y , p y r i d i n e compounds a r e formed endogenous ly in the o rgan i sms by the r e a c t 8 between amino a c i d s and c a r b o n y l compounds w i th ammonia (Smith 1976). C e r t a i n p y r i d i n e s l i k e 4-amino p y r i d i n e and a n a l o g u e s a r e used as r e v e r s a l a g e n t s f o r n o n d e p o l a r i z i n g muscle r e l a x a n t s , and a l s o i n neuromusc u l a r d i s o r d e r s ( B i e s s e l s e t a l . 1984 and 1985). I n s p i t e of the wide o c c u r r e n c e and u t i l i t y of n i t r o g e n c o n t a i n i n g c h e m i c a l s , l i t t l e d a t a i s a v a i l a b l e on t h e i r t o x i c e f f e c t s and i n t e r a c t i o n s wi th b i o c h e m i c a l and p h y s i o l o g i c a l p r o c e s s e s in mammalian s p e c i e s . There f o r e , t h i s i n v e s t i g a t i o n was conduc ted to e v a l u a t e in v ivo e f f e c t s of n i t r o g e n c o n t a i n i n g c h e m i c a l s on the d u r a t i o n of s l e e p induced w i th a commonly used s e d a t i v e h y p n o t i c h e x o b a r b i t a l . The h e x o b a r b i t a l i s known to be e x c l u s i v e l y m e t a b o l i z e d by the microsomal mixed f u n c t i o n o x i d a s e sys tem in mammalian l i v e r ( G i l l e t t e e t a l . 1973) and any chemica l i n t e r f e r i n g wi th i t s me t a bo l i s m may produce changes in i t s p h a r m a c o l o g i c a l r e s p o n s e s .

Relationship between the metabolism of antipyrine, hexobarbital and theophylline in patients with liver disease as assessed by a ‘cocktail’ approach

Antipyrine (AP), hexobarbital (HB) and theophylline (TH) were administered simultaneously ('cocktail' design) to 24 patients with various types of liver disease. Clearance (Cl) of AP, HB and TH and formation clearance of the AP-metabolites 3-hydroxymethylantipyrine (HMA), norantipyrine (NORA) and 4-hydroxyantipyrine (OHA) were determined and correlation coefficients and orthogonal least-squares regression lines calculated between the clearance and formation clearance parameters. The results were compared with those obtained in a study in which the same 'cocktail' was administered to 26 healthy control subjects. In the patients ClAP, ClHB and ClTH were 23.0 +/- 14.3 ml min-1, 206 +/- 128 ml min-1 and 39.9 +/- 26.1 ml min-1 respectively. All values were considerably lower than those found in the control subjects. With regard to AP metabolism preferential impairment of NORA formation was observed. Relatively high correlation coefficients were found between ClAP, ClHB and ClTH, which suggests, like the results of orthogonal regression analysis, a strong correlation between total metabolism of these probe drugs. Therefore it is likely that impairment in oxidation in patients with liver disease not only leads to reduction in clearance but also to reduced substrate selectivity of cytochrome P-450 isozymes.

Isolation of hepatic drug metabolism inhibitors from the Rhizomes ofCurcuma zedoaria

The methanolic extract of the Rhizome ofCurcuma zedoaria exhibited a significant prolongation of hexobarbital (HB)-induced hypnosis. Through liquid chromatography of an ether soluble fraction, monitoring by bioassay, three sesquiterpenes, germacrone(A), curzerenone(B) and germacrone epoxide(C) were isolated as active constituents. A single treatment (100–200mg/kg, i.p.) of each compound showed not only a significant prolongation of HB-induced sleeping time but also a significant inhibition of amino-pyrine N-demethylase activity in mice, and further exhibited a typical type I binding spectra with oxidized rat hepatic cytochrome P-450 induced by phenobarbital. All of the compounds provoked a sleep episode at a subhypnotic dose of HB, implying that they possess CNS depressant properties.

Relationship between the metabolism of antipyrine, hexobarbitone and theophylline in man as assessed by a 'cocktail' approach.

1. Three model substrates for the characterization of drug oxidation activity, antipyrine (AP), hexobarbitone (HB) and theophylline (TH), were administered to 26 healthy volunteers on two different occasions: in the first experiment a combination of AP (250 mg) and HB (250 mg) was given and in the second experiment TH (150 mg) was added to the former combination. 2. Plasma concentrations of AP, HB and TH and urinary excretion of TH and the three main metabolites of AP (3-hydroxymethylantipyrine: HMA, norantipyrine: NORA and 4-hydroxyantipyrine: OHA) were determined and the intrinsic clearance (CLint) of the three substrates and the clearance to the formation of AP metabolites were calculated. 3. The correlation coefficients between CLHB and CL-greater than metabolites of AP were highest for CL-greater than HMA and CL-greater than NORA (greater than 0.80) and lowest for CL-greater than OHA (0.63). High correlation coefficients also were found between CLTH and CL-greater than OHA (0.89) and CL-greater than HMA (0.80). 4. Ideal relationships, defined by a slope of the orthogonal regression line equal to unity, did exist between CLHB and CL-greater than HMA as well as CL-greater than NORA and between CLTH and CLAP as well as CL-greater than OHA. 5. Based on the results of correlation and regression analysis it can be concluded that isozymes of the cytochrome P-450 system responsible for the oxidation of HB and formation of HMA and NORA are very closely related and also that isozymes responsible for the oxidation of TH and formation of OHA show a very close relation. 6. With this strategy of simultaneous administration of substrates ('cocktail' approach) it seems possible to characterize and correlate activities of different P-450 isozymes and to investigate their in vivo substrate selectivity without the disturbing influence of intra-individual variation in drug oxidation.

Disposition of hexobarbital: 15 years of an intriguing model substrate.

(1988). Disposition of hexobarbital: 15 years of an intriguing model substrate. Drug Metabolism Reviews: Vol. 19, No. 2, pp. 109-164.

Correlation of apparent intrinsic clearances of simultaneously administered S (+) and d 3R (−) hexobarbital in the rat

Pseudoracemic hexobarbital (HB), consisting of equal molar fractions of 5 (+) HB and deuterium-labeled R (−) HB, d 3 R (−) HB, was administered orally to rats in a dose of 50 mg/kg. Concentrations of both enantiomers in blood were measured by an enantioselective mass fragmentographic assay. Clearance data of 5 (+) HB and d 3 R (−) HB were correlated in untreated rats, and in rats pretreated with 3-methylcholanthrene (MC), carbon tetrachloride (CCl 4), and different doses of phenobarbital (PB). Although in the different groups some variation in the clearance ratio of 5 (+) HB over d 3 R (−) HB was found, the clearance of 5 (+) HB was generally up to a factor of five higher than the clearance of d 3 R (−) HB, except for the CCl 4-treated rats. From the present data it can be tentatively concluded that S (+) HB and R (−) HB are metabolized by similar (PB-inducible) cytochrome P-450s in control and PB- and MC-pretreated rats and that clearance data obtained with racemic HB following different pretreatments may be employed as a reflection of (PB-inducible) cytochrome P-450 activity.

Pharmacokinetics of orally administered hexobarbital in plasma and saliva of healthy subjects.

Hexobarbital (HB) concentrations were determined in plasma and saliva of 8 healthy subjects, following oral administration of 500 mg HB-Na. Mean plasma half-lives were 3.2 +/- 0.1 h, and salivary half-lives 3.3 +/- 0.2 h. Mean plasma clearance was 22.9 +/- 2.3 1 h-1. There was a linear relationship between HB concentrations in saliva and plasma (r = 0.92). Mean salivary levels were 34 per cent of plasma levels. Salivary pH was constant throughout the experiment, 7.06 +/- 0.09. There was an inconsistent tendency of the saliva over plasma ratios to increase as a function of time. The percentage of protein binding calculated from saliva over plasma ratios was in reasonable agreement with in vitro data of equilibrium dialysis, 64.1 +/- 2.6 per cent and 65.9 +/- 0.8 per cent, respectively. The experiment was repeated in 4 subjects, and considerable intraindividual differences were shown to exist in saliva over plasma ratio, half-lives, and protein binding. It was concluded that HB elimination half-lives can relatively accurately be determined from salivary concentrations. Oral plasma clearance can only be estimated if the individual saliva over plasma ratios are known; this would require the taking of at least one blood sample during the experiment. When employing HB as a model substrate for drug metabolizing enzyme activity in vivo, the determination of its pharmacokinetic parameters, particularly oral plasma clearance as a reflection of cytochrome P-450 activity, cannot be achieved by taking saliva samples only.

Disposition of allylic oxidation pathway metabolites of racemic hexobarbital in the rat.

The pharmacokinetics in blood of the major metabolites of hexobarbital (HB), 3'-hydroxyhexobarbital (OH-HB) and 3'-ketohexobarbital (K-HB) were studied in rats. In addition urinary excretion of OH-HB and K-HB and 1,5-dimethylbarbituric acid (DMBA) was determined. Half-lives of OH-HB and K-HB were slightly longer than that of the parent drug. Urinary recovery of OH-HB, K-HB and DMBA following i.a. administration of OH-HB (75%) was more complete than the recovery following i.a. administration of K-HB (52%). Most probably further metabolism of K-HB takes place. Of K-HB, 41% was excreted renally, and 3.4% of K-HB reverted back to OH-HB. Of OH-HB, about 45% was excreted renally, following p.o. or i.a. administration. Since about 10% of both OH-HB and K-HB was converted to DMBA, it seems that the epoxide-diol pathway as proposed for HB also plays a minor role in the metabolism of OH-HB and K-HB. It is further concluded that measuring allylic pathway oxidation metabolites of HB does not improve the usefulness of HB as a model compound in the assessment of the activity of oxidative drug metabolizing activity.

Effect of beta-cyclodextrin on sleeping time induced by barbituric acid derivatives in mice.

An investigation was carried out on the effect of β-cyclodextrin (β-CD), which is an excellent complexing agent, on the hypnotic potency of four kinds of barbituric acid derivatives (BAD) after intravenous and intraperitoneal administration in mice. Among BAD, hexobarbital (HBA), pentobarbital (PBA), phenobarbital (PhBA) and thiopental (TPA) were chosen. To evaluate the hypnotic potency, the sleeping lag (the time until the onset of the righting reflex from administration) and the sleeping time (the time until recovery from the loss of righting reflex) were determined. In the cases of intravenous administration at the dose of 215.3μmol/kg weight, the sleeping times were significantly shortened, but the sleeping lags were not affected by the simultaneous administration of β-CD equivalent (in molar amount) to BAD. However, the brain concentration of BAD at the time of awakening was little affected by the simultaneous administration of β-CD. The solubility of HBA in pH 7.4 phosphate buffer solution with rat serum increased sigmoidally with increasing amount of β-CD. The present results suggest that the shortening in sleeping time may be due to a decrease in the distribution of BAD to the brain, at least partly as a result of complex formation of BAD with β-CD, resulting in a shortening of the BAD-induced sleeping time.

A comparative investigation in the rat of the anesthetic effects of the isomers of two barbiturates

The sensitivities in the brain of the barbiturates N-methyl-cyclobarbital (MCB) and hexobarbital (HEX) were determined by an anesthesia threshold in male rats. The criterion was a burst suppression in the EEG of1 s or more (the ‘silent second’). The dose needed to induce the criterion was used as the threshold. Available for testing were with MCB the isomers and the racemate and with HEX the isomers and various combinations of them. In experiment 1 the 4 isomers and the racemates were all tested in the same group of animals. In experiment 2 various combinations of HEX were tested. With HEX the (+)-S-isomer and with MCB the (−)-R-isomer was the most potent one. The differences in anesthetic action between the isomers were largest with HEX. During induction of anesthesia no jerks were recorded with (−)-R-MCB. All the other isomers induced jerks. The only significant regression found, when all isomers were tested against each other, was a negative one between (−)-R-MCB and (−)-R-HEX ( r=−0.79). No significant relations were found between the isomers and the corresponding racemate. Experiments with the racemate of MCB and with various combinations of the isomers of HEX indicated that a potentiation appeared when the isomers were used together.

Effect of spices on hepatic microsomal enzyme function in mice

The effect of twenty two spices on liver microsomal monooxygense activity was tested as measured by alteration of hexobarbital (HB) narcosis and strychnine mortality in mice. Oral administration of seven spices for 7 consecutive days caused a significant shortening of the duration of HB-induced sleeping time. The treatment of mice with a singlei.p. injection of 9 spices resulted in a significant prolongation of the sleeping time. White pepper, dill and fennel reduced the toxicity of strychine. These results strongly indicated that some spices might affect the activity of liver microsomal drug metabolizing enzyme (DME) function.

Comparison of hepatic drug-metabolizing enzymes induced by 3-methylcholanthrene and phenobarbital between pre- and postnatal rats

Effects of 3-methylcholanthrene (3MC) and phenobarbital (PB) on the hepatic drug-metabolizing enzyme system in fetal liver of rats were investigated. Intraperitoneal administration of 3MC (25 mg/kg, 72 and 48 hr before death) to pregnant rats significantly increased hexobarbital (HB) and aminopyrine (AM)-metabolizing activities in fetuses on the 21st day of gestation to 148.0 and 150.6% of control fetuses, respectively. In contrast, HB and AM-metabolizing activities in 4-day-old neonates and mothers were decreased by administration of 3MC on the 21st day of gestation. Benzo[a]pyrene (BP)-metabolizing activity, NADPH-cytochrome c reductase activity, and cytochrome P-450 content in 3MC-treated fetuses were significantly increased to 2143.6, 137.6, and 323.8% of the control, respectively. Following 3MC administration, the maximum absorption of the cytochrome P-450-CO difference spectra in liver microsomes of fetuses was observed at 449–450 nm. The induction profile following 3MC administration in the fetal livers was different from that in the neonatal and the maternal livers. On the other hand, intraperitoneal administration of PB (60 mg/kg, 72, 48, and 24 hr before death) significantly increased HB, AM, and BP-metabolizing activities in fetal livers to 263.7, 231.0, and 151.2% of the respective controls. The profile induced by PB in the fetal livers was similar to that in maternal livers. These results suggest that HB and AM-metabolizing enzymes in fetal livers treated with 3MC or PB possess the capacity to be induced, and the responsiveness of the drug-metabolizing enzyme system to 3MC during the prenatal stage may differ from the postnatal stage.