Trimethadione Research Articles

Vibrational analysis, NBO analysis, NMR, UV–VIS, hyperpolarizability analysis of Trimethadione by density functional theory

Trimethadione (TMD) is an anticonvulsant drug widely used against absences seizures. We have characterised the TMD by various spectra including UV–VIS, IR, Raman, GC–MS and NMR. In this work, we made use of Density Functional Theory (DFT) B3LYP method with 6-31G (d, p) basis set, to calculate the molecular structure of TMD, and predicted its infrared, Raman and ultraviolet spectra for the first time. FT-IR and FT-Raman spectra were recorded in the region 4000–400 cm−1 and 3500–50 cm−1, respectively. The vibrational frequencies were calculated and scaled values were compared with the experimental FT-IR and FT-Raman spectra. The observed and calculated frequencies are found to be in good agreement. The complete assignments were performed on the basis of the total energy distribution (TED) of the vibrational modes. The optimized geometry parameters were calculated. NMR chemical shifts of the molecule were calculated using the gauge independent atomic orbital (GIAO) method. The predicted first hyperpolarizibility also shows that the molecule might have convincingly good nonlinear optical (NLO) activities. The calculated HOMO-LUMO energy gap discloses that charge transfer occurs within the molecule.

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.

Developmental cardiotoxicity effects of four commonly used antiepileptic drugs in embryonic chick heart micromass culture and embryonic stem cell culture systems

Antiepileptic drugs (AEDs) are commonly used drugs in pregnant women with epilepsy. Prenatal exposure to AEDs increases the risk of major or minor congenital malformation during embryonic development. The precise mode of action and intracellular mechanisms of these AEDs during embryonic development remains unclear. To determine relative teratogenic risk of AEDs, four AED drugs including valproic acid (VPA), phenytoin (PHT), phenobarbital (PB), and trimethadione (TMD) were tested using two in vitro systems (the embryonic chick heart micromass (MM) culture and the in vitro differentiating mouse embryonic stem cells into cardiomyocytes culture systems). Cardiomyocyte cultures (the heart MM and ES cell-derived cardiomyocytes) were treated with or without different concentrations of PHT, PB, TMD (10–100μM), and VPA (100–2000μM). 5-Fluorouracil (5-FU) (1–10μM) and l-ascorbic acid (10–1000μM) were used as positive and negative controls. It was found that these four commonly used AEDs and 5-FU tested have the potential to inhibit embryonic heart cell differentiation (p<0.05) without inducing any cytotoxicity. VPA at higher concentrations (⩾800μM), and 5-FU at all doses proved to be cytotoxic in the differentiating ES cell culture system. The results demonstrated in this study suggest that the use of these four commonly prescribed AEDs during pregnancy may have an effect on embryonic heart development, and may be associated with congenital cardiovascular defects.

Potassium Depolarization and Raised Calcium Induces α-Synuclein Aggregates

α-Synuclein is the key aggregating protein in Parkinson's disease (PD), which is characterized by cytoplasmic protein inclusion bodies, termed Lewy bodies, thought to increase longevity of the host neuron by sequestering toxic soluble α-synuclein oligomers. Previous post-mortem studies have shown relative sparing of neurons in PD that are positive for the Ca(2+) buffering protein, calbindin, and recent cell culture and in vitro studies have shown that α-synuclein aggregation can be induced by Ca(2+). We hypothesized that depolarization with potassium resulting in raised Ca(2+) in a PD cell culture model will lead to the formation of α-synuclein protein aggregates and that the intracellular Ca(2+) buffer, BAPTA-AM, may suppress their formation. Live cell fluorescence microscopy was performed to monitor changes in intracellular free calcium in HEK293T, SH-SY5Y neuroblastoma or stably transfected HEK293T/α-synuclein cells. Raised intracellular free Ca(2+) was consistently observed in cells treated with KCl, but not controls. Immunohistochemistry analysis on cells 48-72h after K(+) treatment revealed two subsets of cells with either large (>2μm), perinuclear α-synuclein aggregates or multiple smaller (<2μm), cytoplasmic accumulations. Cells pre-treated with varying concentrations of trimethadione (TMO), a calcium channel blocker, showed suppression of the Ca(2+) transient following KCl treatment and no α-synuclein aggregates at TMO concentrations >5μM. Quantitative analysis revealed a significant increase in the number of cells bearing α-synuclein cytoplasmic inclusions in both HEK293T/α-synuclein and SHSY-5Y cells when transient intracellular raised Ca(2+) was induced (p=0.001). BAPTA-AM pre-loading significantly suppressed α-synuclein aggregates (p=0.001) and the intracellular free Ca(2+) transient. This study indicates that raised intracellular Ca(2+) mediated by K(+) depolarization can lead to α-synuclein aggregation.

Probiotic strain Lactobacillus casei does not markedly alter rat liver microsomal cytochromes P450

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.

Postnatal closure of membranous ventricular septal defects in Sprague-Dawley rat pups after maternal exposure with trimethadione.

Congenital membranous ventricular septal defects (VSD) have been shown to close during postnatal development in rats [Solomon et al., Teratology 55:185-194, 1997]. Although they may differ in size, spontaneous and treatment-related VSD are histologically similar; however, the postnatal fate of treatment-induced VSD is not known. The objective of this study was to determine if treatment-induced VSD persist throughout postnatal development. Groups of 40 female rats were given oral doses of trimethadione (TMD) at 400 mg/kg/day (200 b.i.d.) or 600 mg/kg/day (300 b.i.d.) on Gestation Days (GD) 9 and 10. Twenty dams in each group were designated for Cesarean section and 20 were allowed to deliver and rear their offspring to Postnatal Day (PND) 21. The integrity of the ventricular septum was evaluated in fetuses (GD 21) and pups (PND 21). The incidence of membranous VSD was 0.6, 7.6, and 49.8% per litter in the Control, 400, and 600 mg/kg groups, respectively, on GD 21. Both the incidence and severity of VSD increased with dose. The VSD at 400 mg/kg were small in size and initially detected by the presence of blood flowing through the defect from the closed right ventricle. In the 600 mg/kg dose group, the VSD, although still membranous, were larger and more readily detected without the need to examine the blood flow. At 600 mg/kg, not only were the VSD larger than those in the Control or the 400 mg/kg group, 10.1% per litter of the affected fetuses had other vessel anomalies associated with the VSD, which were incompatible with pup survival. On PND 21, VSD was noted in 0.3, 0, and 6.4% per litter evaluated in the Control, 400, and 600 mg/kg groups, respectively. This demonstrates that the small, isolated treatment-related VSD can resolve postnatally; however, the closure of the larger or more severe VSD may be prolonged or may not occur at all. Although TMD exposure reduced group mean fetal weights at both dose levels, there was no difference between the mean weight of fetuses with VSD and those fetuses without VSD in the same group. Treatment-induced VSD close postnatally, and appears to be a delay in cardiac development not associated with fetal weight. The timing of closure and survivability during closure is dependent on the severity of the VSD. Further characterization of the two sizes of VSD may provide diagnostic clarity; however, the current data support the smaller VSD as a variation with no significant impact on viability and growth, and the more severe VSD to be a malformation.

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

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. We used human liver microsomes and human recombinant CYPs expressed in human B-lymphoblast cells and baculovirus-infected insect cells. 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. 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?

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). 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. 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. 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.

In vivo and in vitro trimethadione oxidation activity of the liver from various animal species including mouse, hamster, rat, rabbit, dog, monkey and human.

Trimethadione (TMO) has the properties required of a probe drug for the evaluation of hepatic drug-oxidizing capacity and, in this study, we have summarized the in vivo and in vitro metabolism of TMO in various animal species including mouse, hamster, rat, rabbit, dog, monkey and human. In the in vivo study, the plasma TMO level was measured after intravenous or oral (human) administration of TMO at a dose of 4 mg/kg to various animal species. The rate of TMO metabolic clearance in these animal species in vivo was in the order mouse > hamster > rat > rabbit > dog > monkey > human. In the in vitro study, species differences were observed in the cytochrome P450 (P450) content and drug-oxidizing enzyme activity. The content of P450 was monkey> mouse > dog > rabbit > hamster > rat > human. On the other hand, TMO N-demethylation was in the order mouse > hamster > rat > rabbit > dog > monkey > human. There was a good correlation between the mean total body clearance of TMO (in vivo) and the mean TMO N-demethylase activity (in vitro) (y=1.7 x +0.11, r=0.965, P < 0.001). These results show that TMO is a probe agent with metabolic and pharmacokinetic characteristics making it attractive for the in vivo and in vitro characterization of metabolic activity in various animal species.

Age-related changes in hepatic drug-oxidizing activity using trimethadione as a probe drug in human

Objective: The purpose of this study was to examine the changes in trimethadione (TMO) metabolism over seven stages from neonates to elderly adults. Methods: The subjects were divided into seven groups according to age: neonates (<4 weeks; n=5), infants (<12 months; n=12), children (<10 years; n=21), adolescents (<20 years; n=3), young adults (<40 years; n=20), mature adults (<65 years; n=20) and elderly adults (⩾65 years; n=40). The metabolism of TMO following oral administration (4 mg/kg) was studied in 21 healthy volunteers and 100 patients. Results: The serum albumin, aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels were always within normal ranges. The serum dimethadione (DMO) versus TMO ratio (DMO/TMO) at 4 h after administration of TMO was low in neonates (mean±S.E.M.: 0.12±0.01). In infants, however, it reached approximately 95% of the peak observed in adolescents (0.64±0.09), and gradually decreased thereafter. Conclusion: These findings indicate that the metabolic rate of TMO varies according to the stage in life. Using TMO as a probe drug may be of value in determining changes in overall hepatic drug-oxidizing activity throughout the human life-span, thereby allowing evaluation of drug–drug interactions and clinically significant risks associated with drug therapy.

Pharmacologically induced embryonic dysrhythmia and episodes of hypoxia followed by reoxygenation: a common teratogenic mechanism for antiepileptic drugs?

Antiepileptic drugs (AEDs), such as phenytoin (PHT), carbamazepine (CBZ), trimethadione (TMD), and phenobarbital (PB), have all been associated with a similar pattern of malformations, as well as growth retardation and developmental delay. Valproic acid (VPA) has been associated with a different pattern of malformations. Recent studies suggest that PHT's fetal adverse effect is related to its membrane stabilizing pharmacological properties (blockage of voltage-dependent ion channels). During a restricted sensitive period, this results in induction of concentration-dependent bradyarrhythmia in the embryo and episodes of hypoxia/reoxygenation. The aim of this study was to compare the potential of PHT, CBZ, PB, TMD, and dimethadione (DMD; the active metabolite of TMD) to cause bradyarrhythmias. All of these AEDs exert mainly their pharmacological effect via blockage of ion channels. VPA and vigabatrin (VGB), which are pharmacologically active mainly by other mechanisms, were also tested. C57 Bl/6J mouse embryos were cultured in vitro on gestation day 10 in vitro (in 20% rat serum). The drugs were suspended in either water or dimethylsulfoxide and administered into the culture medium in increasing concentrations up to 20 times the human therapeutic plasma concentration. A scoring system was employed in order to rank the drugs based on their potential to cause bradycardia, ventricular arrhythmia, and cardiac arrest in relation to human therapeutic concentrations. Based on this system, the drugs were ranked as follows: DMD = PHT >> PB = CBZ > TMD = VPA >> VGB (no potential). The results correlate well with the available clinical/experimental data of the tested AED's potential to induce hypoxia-related fetal adverse effects, such as oral clefts, distal limb defects, growth retardation, and developmental delay. The results support the idea that adverse fetal effects after in utero exposure to PHT, PB, CBZ, and TMD (via the active metabolite DMD) are initiated via a common pharmacological mechanism: blockage of ion channels in the developing heart in the early embryo resulting in bradyarrhythmias, hemodynamic alterations, and hypoxia/reoxygenation damage.

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 andthen decreased gradually until reaching the level for themature,adult dog.The elimination half-life (t½) 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 d the study. However, the Vd of TMO was only high during week 1. 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 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.

Valproate induced lupus-like syndrome

Valproate (VPA) is one of most widely used antiepileptic drugs (AEDs) and its side effects include hepatic toxicity, hemorrhagic tendency, drowsiness, nausea, vomitting, encephalopathy, and Reye's syndrome (Coulter and Allen, 1980; Gerber et al., 1979; Lance and Anthony, 1977). Drug-induced systemic lupus erythematosus (SLE), has been reported after treatment with various AEDs, especially phenytoin (PHT), ethosuximide (ESM), trimethadione (TMD), primidone (PRM) and carbamazepine (CBZ) but very rarely with VPA (Asconape et al., 1994; Bleck and Smith, 1990; Drovy and Korczyn, 1993). We report here an adolescent boy with VPA-induced lupus-like syndrome.

18] Trimethadione: Metabolism and assessment of hepatic drug-oxidizing capacity

Publisher Summary This chapter discusses the metabolism and assessment of hepatic drug-oxidizing capacity using Trimethadione. Measurement of the activity of drug-metabolizing enzymes of the liver, which largely depends on cytochrome P450, is essential in evaluating the capacity of oxidative drug metabolism in liver disease. Trimethadione (TMO), an antiepileptic drug, may be a more suitable candidate for estimating hepatic drug-oxidizing activity. It is rapidly absorbed from the gastrointestinal tract, distributed into total body fluids, and is extensively N-demethylated to dimethadione (DMO) by P450-dependent monooxygenases in liver microsomes. The pharmacokinetic properties of TMO enable the determination of hepatic microsomal function with a single blood sample. The chapter discusses that the TMO may be used as a probe drug for the rapid determination of the hepatic drug-oxidizing capacity and the functional reserve mass of the liver.

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.

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.

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)