Involvement of cytochrome P450 2C9, 2E1 and 3A4 in trimethadione N-demethylation in human microsomes.

BACKGROUND AND OBJECTIVES: Trimethadione (TMO), an antiepileptic drug, may be used as a candidate for estimating hepatic drug-oxidizing activity. While TMO metabolism is mainly catalysed by CYP2C9, CYP2E1 and CYP3A4 the contribution of the different isoforms is unclear. In this study, we determined the percentage contribution of the three CYPs (CYP2C9, CYP2E1 and CYP3A4) to TMO N-demethylation. METHOD: We used human liver microsomes and human recombinant CYPs expressed in human B-lymphoblast cells and baculovirus-infected insect cells. RESULTS: The mean Km, Vmax and Vmax/Km values of TMO N-demethylation in human microsomes were 3.66 (mm), 503 (pmol/min/mg) and 2.61 (mL/h/mg), respectively. In the microsomes from human B-lymphoblast cells or baculovirus-infected insect cells, CYP 2C9, CYP 2E1 and CYP3A4 exhibited similar Km and higher Vmax in baculovirus-infected insect cells than B-lymphoblast cells. In baculovirus-infected insect cells, CYP2C9, CYP2E1 and CYP3A4 exhibited activities of 32, 286 and 77 pmol/min/pmol CYP, respectively. No CYP activity catalysed by CYP1A2 and 2D6 were detected in the two human cDNA expressed CYP isoforms. CONCLUSION: TMO is metabolized not only by CYP2E1 but also CYP3A4 and CYP2C9. The order of this metabolism is as follows: CYP2E1 >> CYP3A4 > CYP2C9.

Embryonic arrhythmia by inhibition of HERG channels: a common hypoxia-related teratogenic mechanism for antiepileptic drugs?

PURPOSE: There is evidence that drug-induced embryonic arrhythmia initiates phenytoin (PHT) teratogenicity. The arrhythmia, which links to the potential of PHT to inhibit a specific potassium channel (Ikr), may result in episodes of embryonic ischemia and generation of reactive oxygen species (ROS) at reperfusion. This study sought to determine whether the proposed mechanism might be relevant for the teratogenic antiepileptic drug trimethadione (TMO). METHODS: Effects on embryonic heart rhythm during various stages of organogenesis were examined in CD-1 mice after maternal administration (125-1,000 mg/kg) of dimethadione (DMO), the pharmacologically active metabolite of TMO. Palatal development was examined after administration of a teratogenic dose of DMO and after simultaneous treatment with DMO and a ROS-capturing agent (alpha-phenyl-N-tert-butyl-nitrone; PBN). The Ikr blocking potentials of TMO and DMO were investigated in HERG-transfected cells by using voltage patch-clamping tests. RESULTS: DMO caused stage-specific (gestation days 9-13 only) and dose-dependent embryonic bradycardia and arrhythmia at clinically relevant maternal plasma concentrations (3-11 mM). Hemorrhage in the nasopharyngeal part of the embryonic palate (within 24 h) preceded cleft palate in fetuses at term. Simultaneous treatment with PBN significantly reduced the incidence of DMO-induced cleft palate, from 40 to 13%. Voltage patch-clamping studies showed that particularly DMO (70% inhibition), but also TMO, had Ikr blocking potential at clinically relevant concentrations. CONCLUSIONS: TMO teratogenicity, in the same way as previously shown for PHT, was associated with Ikr-mediated episodes of embryonic cardiac arrhythmia and hypoxia/reoxygenation damage.

The clinical importance of the trimethadione tolerance test as a method for quantitative assessment of hepatic functional reserve in patients with biliary atresia.

BACKGROUND: The trimethadione (TMO) tolerance test was performed to evaluate its usefulness in the assessment of hepatic functional reserve in patients with biliary atresia. METHOD: Nineteen patients with biliary atresia after hepatic portoenterostomy (age range: 2 months to 25 years; sex: 6 males and 13 females) were studied. The study was performed in the morning after a 12-h fast. TMO was given orally, at a dose of 4 mg/kg, with 5 mL of 5% glucose 2 h before breakfast. Blood samples (0.5 mL) were collected to determine serum TMO and dimethadione (DMO), a metabolite of TMO, levels 4 h after the administration of TMO. TMO and DMO were measured by a gas-liquid chromatographic method. RESULTS: A higher total bilirubin level (over 1 mg/dL) in patients with jaundice was reflected in the smaller serum DMO/TMO ratio 4 h after the oral administration of TMO. In addition, these patients with total bilirubin levels of 1 mg/dL or less had a significantly lower DMO/TMO ratio than the control group (healthy subjects). The serum DMO/TMO ratio showed a close correlation with the Child-Pugh score, which is used for overall evaluation of severity of cirrhosis and Mayo risk scores for primary biliary cirrhosis in adults (0.856, P < 0.01 and 0.788, P < 0.01, respectively). The TMO tolerance test shows the benefit of performing a relatively early test of dynamic liver function to evaluate hepatic functional reserve in pre- and post-operative biliary atresia patients.

Cytochrome P450 2E1: its clinical and toxicological role.

Cytochrome (CYP) P450 2E1 is clinically and toxicologically important and it is constitutively expressed in the liver and many other tissues. In contrast to many other CYP isoenzymes, indisputable evidence for a functionally important polymorphism of CYP2E1 in the human population is lacking. CYP2E1 metabolizes a wide variety of chemicals with different structures, in particular small and hydrophobic compounds, including potential cytotoxic and carcinogenic agents. In addition, chlorzoxazone and trimethadione metabolism are good CYP2E1 probes for liver disease in vivo and in vitro. In the future, methods for fully analysing the function of CYP2E1 using knockout mice will be established. This article reviews recent advances in our understanding of the role of human CYP2E1 in drug metabolism.

The effects of organic solvents on trimethadione n-demethylation in rats.

Many organic solvents are frequently used as support solvents to dissolve chemicals in the study concerning drug metabolism mediated by cytochrome P450. However, some organic solvents used as the support solvents affect the chemical's metabolism. It has been reported that some organic solvents are metabolized by CYP2E1 or inhibit its enzymatic reaction. In this study we investigated the effects of organic solvents, such as acetonitrile (AN), dimethylsulfoxide (DMSO), ethanol (EtOH), methanol (MeOH), polyethylene glycol (PEG) and propylene glycol (PG) on TMO (trimethadione) metabolism, which is mainly mediated by CYP2E1 in the rat. In the in vivo study, male SD rats were pretreated with an organic solvent intraperitoneally at a dosage of 0.5, 1 or 2 mmol/kg 1 hour before TMO administration orally at the dose of 4 mg/kg. After 2 hours, serum concentrations of TMO and DMO were determined by gas chromatography/flame detection (CG/FTD) and the serum DMO/TMO ratio was employed for assessment of the metabolic capacity of TMO. In the in vitro study, hepatic microsomal fraction was used as an enzyme source of TMO N-demethylase and enzyme activities were determined by the production of DMO. Pretreatment with DMSO and PG decreased the DMO/TMO ratio in a dose-related manner in vivo study. Furthermore, in vitro study TMO N-demethylase activity was inhibited by DMSO, EtOH and PG with different potency in a concentration related manner. However, no remarkable effects were observed by AN or PEG both in vivo and in vitro study. These results indicated that there are variations in the inhibitory effects of these organic solvents on CYP2E1-mediated metabolism and AN and PEG will be useful solvents to dissolve chemicals in the metabolic study mainly mediated by CYP2E1.

Trimethadione metabolism and microsomal monooxygenases in untreated and phenobarbital-treated rhesus monkeys.

The contribution of induced cytochrome P450 (P450) isozymes (CMLa; CYP2B, CMLb; CYP2A and CMLc; CYP3A) and related enzymes to trimethadione (TMO) metabolism in phenobarbital-treated rhesus monkey were investigated. The animals received a single dose of TMO (4 mg kg-1) and plasma samples were withdrawn before this administration and again at 0.08, 0.25, 0.5, 1 and 2 h later. Phenobarbital-treatment (20 mg kg-1 day-1 for 3 days; i.p.) significantly increased the plasma dimethadione (DMO)/TMO ratios at 0.08, 0.5, 1 and 2 h one's appropriate controls. Phenobarbital treatment also increased the P450 content (1.7-fold) and activity of aniline p-hydroxylase (1.3-fold), p-nitroanisole O-demethylase (1.8-fold) and benzphetamine N-demethylase (2.3-fold). The content of CMLa, CMLb and CMLc were increased about 12.8, 2.3 and 2.7-fold by phenobarbital pretreatment, respectively. The activity of TMO N-demethylation was inhibited by anti-P450 CMLa and anti-P450 CMLb. However, the anti-P450 CMLc antibody had no effect on this activity in liver microsomes. The results of both in vivo and in vitro studies of the effects of phenobarbital treatment on TMO metabolism indicate that these effects may be attributed to the induction of CMLa. These findings suggest that plasma DMO/TMO ratio in a single blood sampling after TMO administration is very useful for determination the degree of hepatic induction in clinical study.

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 metabolism by human liver cytochrome P450: evidence for the involvement of CYP2E1.

1. Caucasian liver samples were used in this study. N-demethylation of trimethadione (TMO) to dimethadione (DMO) was monitored in the presence of chemical inhibitors of CYPs, such as fluconazole, quinidine, dimethyl-nitrosamine, acetaminophen, phenacetin, chlorzoxazone and mephenytoin. Trimethadione N-demethylation was selectively inhibited by dimethylnitrosamine and chlorzoxazone (> 50%) and weakly inhibited by tolbutamide (12%) and fluconazole (22%), whereas other inhibitors showed no effect. This result suggested that TMO metabolism to DMO is mainly mediated by CYP2E1 and marginally by CYP2C and CYP3A4. 2. Fifteen human livers were screened and interindividual variability of TMO N-demethylation activity was 3-fold. Chlorzoxazone 6-hydroxylation activity was also measured and both activities were significantly correlated (r=0.735, p < 0.01). 3. DMO production by human cDNA expressed CYP enzymes was observed mainly for CYP2E1 (10.8 nmol/tube), marginally for CYP2C8 (0.22 nmol/tube) and not detectable for other CYP enzymes. 4. These results indicate that TMO metabolism is primarily catalysed by CYP2E1 and that trimethadione would be a suitable selective probe drug for the estimation of human CYP2E1 activity 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.

Influence of partial hepatectomy in dogs on trimethadione metabolism and microsomal monooxygenases.

1. The recovery of trimethadione (TMO) metabolism and its association with liver weight and the activity of TMO N-demethylase have been reported in rat following partial (68%) hepatectomy. In the present study, we examined the effect of liver regeneration on hepatic P450 isozymes and TMO metabolism in dog. 2. The ratio of dimethadione (DMO), being the only TMO metabolite, to TMO at 2 h after i.v. injection of TMO (4 mg/kg) fell to 80% of that in the preoperative animals by 24 h after hepatectomy. The DMO/TMO ratio gradually recovered from days 7 to 14, and by day 21 after hepatectomy it had increased to about 25%. At 28 days post-hepatectomy the ratio had returned to preoperative levels. 3. The activity of benzphetamine N-demethylase, TMO N-demethylase, p-nitro-anisole O-demethylase and aniline hydroxylase increased 3 days post-hepatectomy, exhibiting levels 4.77, 3.45, 1.51 and 1.91 times greater respectively than that of the preoperative liver in the same animal. Two weeks post-hepatectomy these activities had returned to normal. The activity of the 16 beta- and 2 beta-hydroxylation of testosterone was unchanged. However, the activity of 6 beta-hydroxylase decreased 7 days post-hepatectomy, while 16 alpha-hydroxylation had increased at 3 and 7 days post-hepatectomy compared with controls. 4. The changes in liver weight were nearly restored to preoperative levels 7 days post-hepatectomy. 5. Although the P450 content was unchanged from days 1 to 7 post-hepatectomy, it had decreased by 30% at day 14 and by 20% at day 28. The P4502B11 content 3, 7 and 14 days post-hepatectomy had increased 8, 10 and 2 times respectively, while the P4503A12 content at 7 and 14 days decreased by 30 approximately 50% compared with that of the pre-operative liver. 6. The data presented above do not reveal any relationship between P4502B11 induction and liver regeneration. The reason for such a change is unknown, therefore further investigation needs to be carried out.

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.

Trimethadione metabolism, a useful indicator for assessing hepatic drug-oxidizing capacity.

The metabolism of trimethadione (TMO), a useful indicator of hepatic drug-oxidizing capacity in rats and humans, was studied using 14 different forms of rat cytochrome P450 (CYP1A1, 1A2, 2A1, 2A2, 2B1, 2B2, 2C6, 2C7, 2C11, 2C12, 2C13, 2E1, 3A2 and 4A2) and three forms of human cytochrome P450 (CYP1A2, 2C and 3A4). TMO N-demethylation was increased by treating rats with phenobarbital. CYP2C11 and 2B1 had high TMO N-demethylase activity, but 1A1 and 1A2 had low activity. Antibodies raised to CYP2C11 and 2B1/2 inhibited TMO N-demethylation in hepatic microsomes of untreated and phenobarbital-treated rats, respectively. In a reconstituted system, human CYP3A4 and 2C produced efficiently dimethadione (DMO), but CYP1A2 did not catalyse TMO N-demethylation. Antibodies raised to CYP3A2 and 2C11 inhibited TMO N-demethylation in human hepatic microsomes. These results indicated that the N-demethylation of TMO is catalysed mainly by CYP2C11 and 2B1 in rat hepatic microsomes, and that human CYP3A4 and an unspecified isoform of the 2C subfamilies contribute to TMO N-demethylation in human liver.

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

1. Trimethadione (TMO) has the properties required of probe drugs for the evaluation of hepatic oxidizing capacity in vivo. 2. TMO is demethylated to dimethadione (DMO), its only metabolite, in the liver after oral administration. 3. In rats with various types of hepatic intoxicated-, induced- and partially hepatectomized-rats, the serum DMO/TMO ratios, which were measured on blood samples obtained by a single collection 2 hr after oral administration of TMO, correlated well with the degree of hepatic damage or induction. 4. 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 the hepatic oxidizing capacity.

The effects of diltiazem on hepatic drug metabolizing enzymes in man using antipyrine, trimethadione and debrisoquine as model substrates.

Six healthy male subjects were given single oral doses of antipyrine (7 mg kg-1), trimethadione (4 mg kg-1) and debrisoquine (10 mg) before and during diltiazem treatment (30 mg three times daily orally for 8 days). Antipyrine clearance decreased from 33.7 +/- 9.1 to 22.5 +/- 4.9 ml min-1 (P less than 0.05, mean +/- s.e. mean) after diltiazem treatment without any significant change in apparent volume of distribution (0.59 +/- 0.06 to 0.60 +/- 0.04 1 kg-1), resulting in an increase in antipyrine elimination half-life from 13.4 +/- 4.8 to 19.7 +/- 3.2 h (P less than 0.05). The formation clearance of antipyrine to 4-hydroxyantipyrine was decreased significantly from 10.8 +/- 2.7 to 6.6 +/- 2.7 ml min-1 (P less than 0.05), while that to 3-hydroxymethylantipyrine and norantipyrine was not altered by diltiazem. The metabolic ratio of debrisoquine (urinary excretion of debrisoquine/4-hydroxydebrisoquine) was increased significantly from 0.70 +/- 0.05 to 1.95 +/- 0.20 (P less than 0.05), while that of trimethadione (serum concentration of dimethadione/trimethadione) was not changed significantly (0.48 +/- 0.08 vs 0.41 +/- 0.06) after diltiazem treatment. Diltiazem selectively inhibits cytochrome P-450 isoenzymes.

Effect of dimethadione derived from repeated oral administration of trimethadione on pancreatic secretion in dogs.

The effect of the weak organic acid of dimethadione (DMO) on secretin-stimulated pancreatic secretion was studied with repeated oral administration of trimethadione (TMO), the precursor of DMO, to dogs at a dose of 10 to 160mg/kg/day for a period of 14 days. The bicarbonate concentration in pancreatic juice at a steady state decreased significantly, reflecting a close correlation with the dose of TMO and DMO concentrations in plasma and pancreatic juice. The maximal decrement from the control of cases of no TMO administration was 18.8 mEq/l (12.1% of the control level). The chloride concentration in pancreatic juice showed a reciprocal relation to the bicarbonate concentration. The sum of both anion concentration was constant, irrespective of the dose of TMO. The average carbon dioxide tension of pancreatic juice in all doses of TMO was lower than that of the control, but differences were not statistically significant. The pH, flow rate, sodium and potassium concentrations in pancreatic juice at a steady state did not differ significantly in relation to the dose of TMO. These findings suggest that repeated oral administration of TMO cause a significant decrease in bicarbonate concentration in pancreatic juice, resulting probably from the buffer action of bicarbonate on protons provided from the undissociated form of DMO.