Changes in the enzymatic activities of beagle liver during maturation as assessed both in vitro and in vivo.

1. We have examined changes in caffeine and trimethadione (TMO) metabolism in vivo, agents which are used as probe drugs. In this study the total body clearance (Cl) of caffeine and TMO was low 1 week after birth (week 1), increased rapidly from week 3, peaked and then decreased gradually until reaching the level for the mature, adult dog. The elimination half-life (t1/2) of caffeine and TMO was prolonged during week 1; however, it then gradually became shorter. Gradually it became longer and reached the level for the adult dog. The apparent volume of distribution (Vd) of caffeine did not change throughout the study. However, the Vd of TMO was only high during week 1. 2. The in vitro changes in a variety of typical substrates for seven different cytochrome P450 (CYP) isozymes were examined. In this study three different patterns of metabolism can be identified: (1) activity is low immediately after birth, increases, peaks and then decreases to the adult dog level (p-nitroanisole; CYP1A1, caffeine; CYP1A2, benzphetamine; CYP3A/2B(?), aniline; 2E1 and TMO; CYP2C9/2E1/3A4); (2) activity generally increases rapidly soon after birth, continues to increase, peaks and then gradually decreases to the adult level (phenytoin; CYP2C9); and (3) activity is high (about the same level as the adult) immediately after birth, decreases and then gradually increases to the adult level (erythromycin; CYP3A4/5). 3. The results of these in vivo and in vitro studies suggest that changes in enzyme activity are due to differences in P450 isoenzymes during development.

Trimethadione as a probe drug to estimate hepatic oxidizing capacity in humans.

Trimethadione (TMO) has the properties required of probe drugs for the evaluation of hepatic drug-oxidizing capacity in humans in vivo. TMO is demethylated to dimethadione (DMO), its only metabolite, in the liver after oral administration. Involvement of two cytochrome P450's--CYP2C9 and 3A4--in TMO metabolism has been seen in humans, but involvement of 1A2 is not clearly established. In humans with various types of liver disease and hepatectomy, the serum DMO/TMO ratios, which were measured on blood samples obtained by a single collection 4 hr after oral administration of TMO, correlated well with the degree of hepatic damage. This finding suggests that TMO may be used as a probe drug in the rapid determination of the functional reserve mass of the liver as well as hepatic drug-oxidizing capacity in humans in vivo.

Trimethadione N-demethylation by rat liver CYP2E1 in vitro.

Trimethadione (TMO) is a model drug utilized for estimation of hepatic metabolism in clinical studies, and it was reported that TMO N-demethylase activity was inhibited by CYP2E1 inhibitors and substrates in rat in vivo. This study was performed to investigate the involvement of the CYP2E1 subfamily on TMO N-demethylation in vitro and to clarify these inhibitory mechanisms. The effects of acetone (AC), imidazole (IM) and N-nitrosodimethylamine (NDA) on TMO N-demethylation were studied in vitro. Rat hepatic microsomal fractions were employed as the enzyme source of TMO N-demethylase and the activity was determined by the production of dimethadione (DMO). DMO was analyzed by a GC/FTD equipped with a narrow-bore capillary column. TMO N-demethylation was biphasic by the graphic analysis of Eadie-Hofstee plots; this suggests the involvement of at least two enzymes in TMO metabolism in the rat. The kinetic parameters for the formation of DMO were analyzed graphically using double-reciprocal plots. The apparent K(m1), K(m2) and Vmax1, Vmax2 values for DMO formation were 4, 20 mM and 182, 595 pmol/mg protein/min, respectively. AC and IM inhibited TMO N-demethylase activity competetively. However, mixed inhibition kinetics was observed by NDA. Furthermore, TMO N-demethylase activity was inhibited by antiserum to CYP2E1 by 62% and CYP3A2 by 46%. These results indicate that the CYP2E1 subfamily is the major enzyme involved in TMO N-demethylation in rat in vitro although the CYP3A2 is also involved in this transformation.

Clinical significance of the trimethadione tolerance test in chronic hepatitis: a useful indicator of hepatic drug metabolizing capacity.

Trimethadione (TMO) was chosen as an indicator of quantitative hepatic microsomal function, and its pharmacokinetics were studied in 52 patients with chronic hepatitis. Findings in these patients were compared with those for 26 healthy subjects and 13 patients with renal failure. Patients with chronic hepatitis, but not those with renal failure, showed significant reduction in clearance (CL) and prolongation of half-life (t1/2), and the extent of abnormalities was found to reflect the severity of histologic changes in liver tissue. The serum dimethadione (DMO)/TMO ratio 4 h after the administration of TMO altered in parallel with the CL and t1/2 of TMO, and abnormalities in this simple ratio were also related to the histologic severity of changes in the liver tissue. A low DMO/TMO ratio (< 0.4) was associated with advanced histologic changes (chronic active hepatitis with bridging or chronic active hepatitis with cirrhosis), whereas a high DMO/TMO ratio (> 0.4) was associated with mild histologic changes (chronic persistent hepatitis or chronic active hepatitis) (sensitivity, 0.81; specificity, 0.86). These results indicate that the DMO/TMO ratio, which can be obtained from a single blood sampling, reflects the histologic severity of changes in tissue liver, and that the TMO tolerance test is a useful indicator of quantitative liver function.

Pharmacokinetic study of trimethadione and its metabolite in blood, liver and brain by microdialysis in conscious, unrestrained rats.

A microdialysis method has been developed in the past two decades to determine levels of drug and endogenous compounds in several organs under physiological conditions. In this study, we determined the pharmacokinetics of the model drug, trimethadione (TMO), and its only metabolite, dimethadione (DMO), in liver, blood and brain by the microdialysis method in freely-moving rats. Sampling times were extended up to 24 hours. The construction of a newly developed microdialysis probe for liver and blood is described. The elimination patterns of TMO in liver, blood and brain dialyzates were almost identical and the calculated t1/2 was approximately 3 hr in each sample. However, in brain, tmax was delayed compared with the others while the relative concentration of DMO (AUC0-24h) was lower in brain compared with liver and blood. These studies suggest that this concurrent and successive microdialysis sampling method will not only be a useful tool for pharmacokinetic and drug metabolism studies in the organs of small animals but will also decrease the number of experimental animals needed for a study.

Changes in caffeine, lidocaine and trimethadione metabolism in carbon tetrachloride-intoxicated rats as assessed by a "cocktail" study.

We investigated the possibility of predicting liver damage from changes in the serum concentrations of caffeine (10 mg/kg), lidocaine (4 mg/kg) and trimethadione (4 mg/kg), which are metabolized catalysed by different cytochrome P450 (P450) and/or are dependent on blood flow, in rats with carbon tetrachloride (CCl4: 0.25 ml/kg)-induced liver injury using a strategy referred to as a "cocktail" study. These 3 probe drugs were simultaneously administered intravenously. The half-lives (t1/2) of caffeine, lidocaine and trimethadione were significantly longer in the CCl4-treated group than in oil-treated controls, but no significant differences were observed in mean apparent volumes of distribution (Vd). Serum total body clearance (CL) values of all three drugs were markedly reduced in CCl4-treated animals. In rats with liver damage, the production of 3 metabolites (theobromine, paraxanthine and theophylline) of caffeine in addition to the only metabolite (dimethadione) of trimethadione after intravenous administration of probe drugs were significantly reduced compared to those of controls. These findings suggest that each probe drug, metabolized by different or partially overlapping P450, is useful in evaluating drug-oxidizing capacity in liver disease.

Dual effects of a novel thienodiazepine platelet-activating factor antagonist, on drug-oxidizing enzymes in beagle dog.

1. We have examined the effects of (S)-(+)-6-(2-chlorophenyl)-3-cyclopropanecarbonyl-8, 11-dimethyl-2,3,4,5-tetrahydro-8H-pyrido[4',3':4,5]thieno[3, 2-f][1, 2, 4]triazolo[4, 3-a][1, 4]diazepine (E-6123), a novel thienodiazepine platelet-activating factor antagonist, on drug-oxidizing capacity in beagle dog, using antipyrine (AP) and trimethadione (TMO) as two model substrates. 2. The plasma half-life (t1/2) and area under the curve (AUC) of AP (0.5 mg/kg, i.v. injection) increased in a dose-dependent manner after a single oral dose of E-6123 (0.2, 1 or 10 mg/kg), whereas the total body clearance (Cl) of AP was decreased, and the apparent volume of distribution (Vd) was unchanged. 3. The pharmacokinetic parameters (t1/2, Cl and AUC) of the metabolism of TMO (4 mg/kg, i.v.) after repeated oral administration of E-6123 (10 mg/kg for 7 days) were not significantly changed in comparison with findings in control dog. The ratio of dimethadione (DMO), being the only TMO metabolite, to TMO in plasma after i.v. administration of TMO in E-6123-treated dog was increased only 5 and 15 min after the final dose, but was not changed at other sampling times (0.5, 1, 2 4, 6, 8 and 12 h). 4. The content of b5, the activity of p-nitroanisole O-demethylase and benzphetamine N-demethylase were significantly increased, compared with controls, by repeated E-6123 treatment. However, aniline hydroxylase activity was not significantly changed. 5. Content of P450 2B was significantly increased in E-6123 treated dog, while that of 3A was not.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of E-5110, a novel non-steroidal anti-inflammatory drug, on trimethadione metabolism as an indicator of hepatic drug-oxidizing capacity in beagle dog.

1. We examined the effects of N-methoxy-3-(3,5-di-tert-butyl-4- hydroxybenzylidene pyrrolidin-2-one (E-5110), a novel non-steroidal anti-inflammatory drug, on the pharmacokinetics of trimethadione (TMO) and characterized the P450 isozymes involved in the metabolism of TMO in beagle dog. 2. In the E-5110-treated dog (50 mg/kg/day for 7 days: oral) the plasma half-life (t1/2) and the area under the curve (AUC) of TMO (4 mg/kg, i.v.) in vivo were decreased, and total body clearance (CL) was increased; the apparent volume of distribution (Vd) was relatively unchanged. 3. Contents of P450 and b5, and the activity of p-nitroanisole O-demethylase and benzphetamine N-demethylase in vitro were significantly increased compared with controls by repeated E-5110 treatment in dog. 4. Contents of CYP2B and 3A were increased by E-5110 pretreatment in dog. 5. TMO N-demethylation was inhibited by the anti-CYP2B and 3A IgG fractions in liver microsomes obtained from the E-5110-treated dog. 6. Results of both the in vivo and in vitro studies of the effects of E-5110 treatment in dog on TMO indicate that these effects may be attributed to the induction of CYP2B and 3A.

Comparison of hepatic drug-oxidizing activity after simultaneous administration of two probe drugs, caffeine and trimethadione, to human subjects.

Pharmacokinetic interactions between caffeine 2 mg/kg and trimethadione 4 mg/kg were evaluated in 10 healthy volunteers. Whether administered alone or together, the total body clearance (CL), the apparent volume of distribution (Vd) and half-life (t1/2) of caffeine and trimethadione were the same, however, there was a weak correlation between the CL of caffeine and trimethadione [alone: r = 0.51 (P < 0.05); coadministered: r = 0.56 (P < 0.05)]. There were also weak correlations between the CL of trimethadione and the area under the serum concentration-time curves (AUC) of theobromine (r = -0.61, P < 0.05), paraxanthine (r = -0.69, P < 0.05) and theophylline (r = -0.60, P < 0.05), when the two drugs were administered alone. After combined administration, the correlation between the CL of trimethadione and the AUCs of the metabolites of caffeine were as follows: theobromine r = -0.63 (P < 0.05); paraxanthine r = -0.68 (P < 0.05); theophylline r = -0.65 (P < 0.05). These findings suggest that caffeine and trimethadione metabolism in healthy subjects is mediated by only in part by a form(s) of P450 enzymes involved.

Simplified test for determination of drug-oxidizing capacity in rats with chemical-induced liver injury using caffeine and trimethadione as model drugs.

We examined the possibility of predicting the extent of hepatic drug-oxidizing capacity by determination of caffeine, trimethadione and their metabolites in three groups of rats with chemically induced liver injuries. Trimethadione (4 mg/kg) and caffeine (10 mg/kg) were simultaneously administered as two probe drugs. In rats with chemically induced liver injuries pretreated with carbon tetrachloride (CCl4: 0.25 ml/kg), alpha-naphthylisothiocyanate (ANIT: 40 mg/kg), or D-galactosamine (GalN: 400 mg/kg), the half-life (t1/2) of caffeine and trimethadione was significantly (P less than 0.01) prolonged compared to those of control groups total body clearance as dramatically reduced (P less than 0.01), whereas apparent volumes of distribution (Vds) were increased in ANIT and GalN groups. In rats with liver damage the production of three metabolites (theobromine, paraxanthine and theophylline) of caffeine as well as the only metabolite (dimethadione) of trimethadione after oral administration of both drugs were significantly decreased compared to those of controls. In rats with liver injuries, total body clearance of caffeine and trimethadione showed a strong correlations (r = 0.99, P less than 0.01); also total body clearance of caffeine correlated well with the ratio of dimethadione/trimethadione after 1, 2, and 4 hr of trimethadione administration (r = 0.93, P less than 0.01; r = 0.97, P less than 0.01 and r = 0.97, P less than 0.01 respectively). Besides, total body clearance of caffeine also correlated well (coefficients ranging from 0.74 to 0.96; P less than 0.01) with the ratio of theobromine/caffeine, paraxanthine/caffeine and theophylline/caffeine after 1, 2 and 4 hr) of caffeine administration.(ABSTRACT TRUNCATED AT 250 WORDS)

The effect of diltiazem on hepatic drug oxidation assessed by antipyrine and trimethadione.

The effect of pretreatment for 3 days with diltiazem 60 mg three times a day on the pharmacokinetics of 500-mg antipyrine and 250-mg trimethadione was studied in six healthy male subjects. Diltiazem decreased the total body clearance from 34.0 +/- 8.0 to 28.6 +/- 6.1 mL/min (P less than .01), and prolonged the elimination half-life from 12.6 +/- 3.0 to 14.3 +/- 2.5 hours (P less than .01) of antipyrine without any changes in volume of distribution. The cumulative renal excretion (% dose) of antipyrine was significantly increased from 2.23 +/- 0.73 to 2.78 +/- 0.83% (P less than .05). Clearances of production for three major antipyrine metabolites, norantipyrine (4.31 +/- 1.64 to 3.50 +/- 1.28 mL/min, P less than .01), 3-hydroxymethylantipyrine (4.67 +/- 1.63 to 3.82 +/- 1.34 mL/min, P less than .01) and 4-hydroxyantipyrine (10.47 +/- 3.41 to 8.16 +/- 2.82 mL/min, P less than .01) were reduced significantly by diltiazem. On the other hand, diltiazem did not produce any significant changes in pharmacokinetic parameters of trimethadione and plasma concentration ratio, oxidative major metabolite of trimethadione to trimethadione itself. These results suggest that other drugs metabolizing the same hepatic oxidative pathways as antipyrine, may be influenced by diltiazem.

The effect of roxatidine acetate and cimetidine on hepatic drug clearance assessed by simultaneous administration of three model substrates.

The effect of pretreatment for 7 days with either roxatidine acetate 75 mg twice daily or cimetidine 200 mg four times daily on the kinetics of antipyrine (AP), trimethadione (TMO) and indocyanine green (ICG) was studied in seven healthy, male, nonsmoking subjects. After pretreatment with cimetidine, the plasma clearances (CL) of AP and TMO were significantly lower and the elimination half-life (t1/2) of AP was significantly increased. The volumes of distribution (V) of AP and TMO were not affected. After roxatidine acetate, the pharmacokinetics of AP and TMO were unchanged. The cumulative renal excretion (% dose) and formation clearance of 3-hydroxymethyl-3-nor-antipyrine (NORA) were lowered by cimetidine treatment, but not following the administration of roxatidine acetate. ICG clearance was not changed significantly by either pretreatment. The results of this study show that roxatidine acetate does not impair the metabolism of three model substrates used to assess hepatic drug clearance.

The metabolism and excretion of trimethadione in patients with percutaneous transhepatic biliary drainage and renal dysfunction.

The metabolism and excretion of trimethadione (TMO) following an oral dose of 4 mg/kg has been examined in patients with percutaneous transhepatic biliary drainage (PTBD) and renal dysfunction. Biliary excretion as the total amount of TMO and its metabolite, dimethadione (DMO) was 2.0% of the dose during 0 to 48 h after TMO administration in patients with PTBD. Total urinary excretion (0-48 h) was 2.8% and 3.0% of the dose in healthy volunteers and patients with renal dysfunction, respectively. The serum DMO/TMO ratio at 4 h after oral dosing in patients of PTBD and renal dysfunction was not significantly changed in comparison with the ratio reported previously in healthy volunteers. The elimination half-life of TMO was also not altered in patients with PTBD in comparison with that reported previously in volunteers. These results suggest that metabolism and urinary and biliary excretion of TMO are not changed in patients with PTBD and renal dysfunction.

Comparison of trimethadione and antipyrine as indicators of oxidative drug metabolizing capacity in man.

Ten healthy male volunteers were given trimethadione (TMO) 4 mg/kg and antipyrine (AP) 500 mg alone or concomitantly to determine whether the metabolism of the drugs was mediated by the same or closely related forms of cytochrome P-450. Whether administered alone or together the clearance (CL) and half-life (t 1/2) of TMO and AP were the same, and there was a good correlation between the CL and t 1/2 of TMO and AP (alone r = 0.755 and 0.623, respectively; coadministered r = 0.771 and 0.503, respectively). Excretion of AP and its main metabolite and the clearance for production of AP metabolites after AP was administered alone were not significantly different when TMO and AP were taken together. When the two drugs were administered alone or coadministered, the correlation between the CL of TMO and the excretion of 3-hydroxymethyl-3-norantipyrine (NORA) was close (alone r = 0.734, coadministered r = 0.749). The correlation between the CL of TMO and CLm of NORA when TMO and AP were given alone or concomitantly was 0.762 and 0.772, respectively. The findings suggest that TMO metabolism and the formation of NORA in healthy subjects are mediated by a closely related form(s) of the cytochrome P-450 system.

Sex-related differences in hepatic drug-oxidizing capacity of streptozotocin-induced diabetic rats.

Male and female animal models of diabetes were prepared by treating rats with streptozotocin (STZ). Trimethadione (TMO) metabolism was depressed in male but increased in female diabetic rats. The insulin treatment normalized rats of both sexes. Serum dimethadione/TMO ratios at 2 h correlated with the elevated blood glucose levels in male and female control, the STZ-induced diabetic and the insulin-treated diabetic rats. Treatment with STZ in male rats affected the metabolism of antipyrine, decreasing urinary excretion of norantipyrine (NORA) urine but increasing elimination of 4-hydroxyantipyrine (OHA). In female diabetic rats, the amounts of the three major metabolites, NORA, OHA and 3-hydroxymethyl-3-norantipyrine and the total (conjugate + free) were increased compared to the control. In the insulin-treated groups, these changes were normalized. In conclusion, our study showed that the effect of STZ-induced diabetes on drug metabolism varies with the sex and the drugs used. Insulin normalized all these diabetic changes.

Induction of cytochrome P-450 and related drug-oxidizing activities in muscone (3-methylcyclopentadecanone)-treated rats.

In the present study, we investigated the effects of muscone on both in vitro and in vivo parameters of the hepatic microsomal drug-metabolizing enzyme system and other enzyme activities in rats. In the in vivo study, the serum dimethadione (DMO)/trimethadione (TMO) ratios at 2 hr after oral administration of TMO (100 mg/kg) were significantly increased in both male and female rats treated with 75 and 150 but not 40 mg muscone/kg. Antipyrine metabolite profile in 24 hr urine of rats pretreated with muscone (150 mg/kg) was examined. The results showed that the excretion of norantipyrine was significantly increased as compared to the control group. In the in vitro study, we found that the content of cytochrome P-450, and activities of aminopyrine, N-demethylase, aniline hydroxylase and delta-aminolevulinic acid (ALA) synthetase were significantly increased as compared to the controls in both male and female rats treated with muscone (75 and 150 mg/kg). This type of induction of the hepatic metabolizing enzymes was similar to that seen after treatment with a prototype drug, phenobarbital.

Trimethadione metabolism in patients with normal liver and in patients with chronic liver disease.

Serum dimethadione (DMO)/trimethadione (TMO) ratios after oral administration of TMO have been investigated in 10 patients with normal livers, 8 patients with hepatoma and 8 patients with hepatoma and cirrhosis. Serum concentration ratios of DMO to TMO at 4 h after oral administration of TMO in patients with chronic liver disease were significantly decreased by 27% for those with hepatoma and 52% for those with hepatoma and cirrhosis. Serum DMO/TMO ratios at 4 h correlated well with liver function characteristics (total protein r = 0.741, plasma albumin r = 0.826, total bilirubin r = -0.725, cholinesterase r = 0.853) as well as with pharmacokinetic parameters (total body clearance r = 0.852, half-life r = -0.636) in both patients with normal livers and patients with chronic liver disease. This study suggests that serum DMO/TMO ratios in a blood sample obtained by a single collection after an oral administration of TMO might provide a clinically useful index of the hepatic drug-oxidizing capacity in an individual patient with chronic liver disease without determining the liver function characteristics or the pharmacokinetic parameters.