Dimethadione embryotoxicity in the rat is neither correlated with maternal systemic drug concentrations nor embryonic tissue levels.

Pregnant rats treated with dimethadione (DMO), the N-demethylated metabolite of the anticonvulsant trimethadione, produce offspring having a 74% incidence of congenital heart defects (CHD); however, the incidence of CHD has high inter-litter variability (40-100%) that presents a challenge when studying the initiating events prior to the presentation of an abnormal phenotype. We hypothesized that the variability in CHD incidence was the result of differences in maternal systemic concentrations or embryonic tissue concentrations of DMO. To test this hypothesis, dams were administered 300 mg/kg DMO every 12h from the evening of gestational day (GD) 8 until the morning of GD 11 (six total doses). Maternal serum levels of DMO were assessed on GD 11, 12, 13, 14, 15, 18 and 21. Embryonic tissue concentrations of DMO were assessed on GD 11, 12, 13 and 14. In a separate cohort of GD 12 embryos, DMO concentrations and parameters of growth and development were assessed to determine if tissue levels of DMO were correlated with these endpoints. Embryos were exposed directly to different concentrations of DMO with whole embryo culture (WEC) and their growth and development assessed. Key findings were that neither maternal systemic concentrations nor tissue concentrations of DMO identified embryos that were sensitive or resistant to DMO in vivo. Direct exposure of embryos to DMO via WEC also failed to show correlations between embryonic concentrations of DMO with developmental outcomes in vitro. We conclude that neither maternal serum nor embryonic tissue concentrations of DMO predict embryonic outcome.

Maternal rat serum concentrations of dimethadione do not explain intra-litter differences in the incidence of dimethadione-induced birth defects, including novel findings in foetal lung.

To investigate mechanisms of chemical-induced congenital heart defects (CHD) we have developed a rat model using dimethadione (DMO), the N-demethylated metabolite of the anticonvulsant, trimethadione (TMD). Dosing pregnant rats with 300mg/kg DMO every 12h from the evening of gestational day (GD) 8 until the morning of GD 11 (six total doses) produces a mean 74% incidence of CHD with inter litter variability ranging from 40 to 100%. The goal of this study was to determine if the variability in maternal serum concentrations of DMO on GD 14, a surrogate marker for total exposure, was related to the inter-litter differences in teratogenic outcomes. To test this hypothesis, pregnant rats were dosed as described above and serum levels of DMO assessed on GD 14. On GD 21, foetuses were collected by caesarean section, assessed for a number endpoints and the outcomes were correlated with the GD 14 serum concentrations of DMO. DMO exposure was associated with decreased foetal body weight, increased incidence of sternal defects and CHD, but these endpoints were not meaningfully correlated with maternal concentrations of DMO. Novel findings were decreased viability as measured one-hour following caesarean section, and delayed alveolar maturation. The major conclusions from these studies were first, that serum DMO concentrations on GD 14 did not predict teratogenicity, and second, delayed lung development may contribute to the decreased survival of foetuses at the time of caesarean section.

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.

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.

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.

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.

The effects of inhibitors and substrates of different types of cytochrome P450 isozymes on serum dimethadione/trimethadione ratio in rats in vivo.

Trimethadione(TMO) is regarded as a model drug for estimating the hepatic drug oxidative capacity in vivo. However, the P450 isozymes that are responsible for TMO N-demethylation have not been identified clearly yet. This study was designed to determine these P450 isozymes that participate in the TMO N-demethylation in vivo by employing several typical P450 inhibitors and substrates. Male Sprague-Dawley(SD) rats were pretreated with P450 inhibitors or substrates before TMO(100mg/kg, p.o.) treatment. Serum dimethadione(DMO)/TMO ratios were employed for the assessment of metabolic capacity toward TMO. Pretreatment with imidazole and acetone significantly decreased the DMO/TMO ratios in a dose related manner. Weaker inhibitory effects were observed with SKF525A. However, pretreatment with alpha-naphthoflavone, quinine, debrisoquine, triacetyloleandomycin and lauric acid did not affect the ratios. These results suggest that various forms of P450 are involved in TMO metabolism to some extent and that CYP2E1 is attributed to major P450 isozyme for TMO N-demethylation in vivo.

Effect of dimethadione administered intravenously on pancreatic secretion in dogs.

In order to detect both pancreatic excretion of dimethadione (DMO), a weak organic acid, and the effect of pancreatic DMO on secretin-stimulated pancreatic secretion, DMO was given intravenously to dogs with pancreatic fistulae at a dose of 50, 100 and 200 mg/kg. DMO was promptly excreted into pancreatic juice; the concentration decreased exponentially as it did in plasma at the highest dose of the compound. At equilibrium of DMO between pancreatic juice and plasma, the DMO concentration in the juice depended directly on that in plasma; the juice/plasma concentration ratios for DMO exceeded 1.0, ranging from 1.7 to 2.1. Pancreatic DMO caused a small but significant decrease in the water, bicarbonate and sodium secretion at non-equilibrium, and in the bicarbonate secretion at equilibrium. A decrease in the bicarbonate secretion may result largely from the buffer action of bicarbonate on protons provided by the undissociated form of DMO. The sum of both bicarbonate and chloride concentrations in pancreatic juice decreased with the increased DMO concentration in the juice, implying that DMO may compete with the secretion of bicarbonate and/or chloride across the apical membrane of the duct cell. Pancreatic DMO can act as a non-specific inhibitor of pancreatic water and electrolyte secretions.

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.

Improved method for the determination of trimethadione and its demethylated metabolite, dimethadione, in human serum by gas chromatography.

An improved gas chromatographic method, involving the use of a wide-bore capillary column, for the determination of trimethadione and its only demethylated metabolite, dimethadione, in human serum is described. The results indicate that both substances and the internal standard (maleinimide) were well separated with no tailing peak. The detection limit was 10 ng/ml for trimethadione and 50 ng/ml for dimethadione. This improved method is reliable in terms of sensitivity, selectivity and reproducibility for the simultaneous determination of both compounds in human serum.

Trimethadione tolerance test for one-point estimation of the severity of liver damage in cirrhotic patients.

We evaluated the adequacy of the trimethadione (TMO) tolerance test (the method of estimation from the serum dimethadione [DMO]/TMO ratio, DMO is only one metabolite of TMO, at 4 hours after oral administration of TMO) for estimating the severity of liver damage in 40 cirrhotic patients with and without hepatic encephalopathy. Serum dimethadione (DMO)/TMO ratios in a single blood sample after oral administration of TMO were significantly lower in cirrhotic patients with (0.07 +/- 0.02, p less than 0.05) or without (0.29 +/- 0.12, p less than 0.05) hepatic encephalopathy than in normal subjects (0.63 +/- 0.04). Serum DMO/TMO rations showed a good correlation with the following laboratory data: plasma indocyanine green retention at 15 minutes (r = -0.857, p less than 0.001), serum choline-esterase activity (r = 0.844, p less than 0.001), and albumin (r = 0.736, p less than 0.001). In cirrhotic patients with hepatic encephalopathy, the serum DMO/TMO ratio was found to be below 0.10, which was 16% of the normal level, by the TMO tolerance test. These findings suggest that the TMO tolerance test is a useful indicator of the hepatic parenchymal function in cirrhotic patients.

Trimethadione tolerance test for the quantitative assessment of liver function in rats.

The quantitative estimation of the hepatic functional volume in rats was attempted using the serum dimethadione (DMO)/trimethadione (TMO) ratio in a single blood sampling after oral administration of TMO, which we call the TMO tolerance test, in order to develop a means of pre-operatively assessing hepatic resectability. Serum DMO/TMO ratios correlated well with the total amount of the hepatic microsomal TMO-N-demethylase activity (enzyme activity) and with the remnant liver weight after 37% and 68% partial hepatectomy. These ratios increased after partial hepatectomy in parallel with the changes in the enzyme activity and the remnant liver weight. The results suggest that the quantitative functional changes of the remnant liver after hepatectomy in both CCl4-treated and untreated rats can be estimated pre-operatively by the TMO tolerance test.

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.

Effects of cimetidine on quinidine distribution and tissue pH in rats.

To elucidate the mechanism(s) of the decrease of the volume of distribution at steady state (Vdss) and the tissue-to-plasma concentration ratio (Kp) of quinidine after cimetidine treatment, the following were studied; (1) the effect of cimetidine on the tissue binding of quinidine in vitro, (2) the non-linear tissue distribution of quinidine and (3) the effect of cimetidine on tissue pH. The in vitro binding of quinidine to rat tissue homogenates was not affected by cimetidine treatment. The tissue distribution of quinidine in rats was linear from 1 to 5 micrograms/ml of plasma concentration except for lung. The plasma disappearance of 5,5-dimethyl-2,4-oxazolidinedione (DMO) after a 200 mg/kg intravenous injection was fitted to a two compartment open model. In the cimetidine-treated rats (50 mg/kg), the pharmacokinetic parameters of DMO, such as the plasma total body clearance (Cltot), Vdss and the rate constant at the terminal phase (beta) increased to 230, 110 and 210% of those of the non-treated rats, respectively. The intracellular pH calculated by Kp of DMO increased significantly in liver, spleen, intestine, brain, muscle and skin. This suggests that cimetidine decreased the tissue-to-plasma pH partition coefficient (q) of unbound quinidine in several tissues. The decreases of Vdss and Kp of quinidine by cimetidine was attributed to the decrease of q resulting from the increase of tissue pH.

Age-related changes in trimethadione oxidizing capacity.

The metabolism of trimethadione (TMO) following oral administration (4 mg kg-1) has been studied in 11 young male and 11 elderly male patients. The elimination half-life (h) of TMO was 11.8 +/- 1.4 (mean +/- s.e. mean) in the young and 23.5 +/- 2.17 in the elderly (P less than 0.01). Total body clearance (1 h-1 kg-1) was 41.4 +/- 2.8 in the young and 29.0 +/- 2.4 in the elderly (P less than 0.01). The apparent volume of distribution (1 kg-1) was 0.67 +/- 0.03 in the young and 0.65 +/- 0.04 in the elderly. Serum dimethadione (DMO)/TMO ratios at 4 h were 0.65 +/- 0.03 in the young and 0.46 +/- 0.03 in the elderly (P less than 0.01). These results suggest that N-demethylation of TMO is inversely related to age.

Rapid and simple HPLC determination of 5,5-dimethyl-2,4-oxazolidynedione in rabbit serum.

A new method for the determination of 5,5-dimethyl-2,4-oxazolidynedione (DMO) in rabbit serum by reversed phase-HPLC with UV detection is described. The determination of DMO is performed without derivatisation. The internal standard is 5,5-diethylbarbituric acid (barbital). The method is rapid and simple with sensitivity limit of 20 ng/mL, high recovery (above 92%) and is suitable for pharmacokinetic studies.

Influence of short-term water deprivation on kinetics of trimethadione and its metabolite in rats.

The effects of acute (24-, 48- or 72-hr) water deprivation on the disposition kinetics of trimethadione (TMO) and its only metabolite, dimethadione (DMO), and on the microsomal hepatic drug-oxidizing enzyme activities were investigated in male rats. The DMO/TMO ratios in the serum at 2 hr after intravenous administration of 100 mg/kg TMO were significantly decreased in 48- and 72-hr water-deprived rats, but in 24-hr water-deprived rats, the DMO/TMO ratios were not changed as compared to controls and food restrictions. In the 48- and 72-hr water-deprived rats, contents of cytochrome p-450 and activities of aminopyrine N-demethylase were significantly decreased. On the other hand, activities of aniline hydroxylase in these rats were significantly increased as compared to controls and food restrictions. These results suggest that the effects of water deprivation on drug metabolism not only depend on the time of water deprivation but also vary with the indicator substrate.