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Caffeine (1,3,7-trimethylxanthine, CA) is a compound usually contained in beverages such as coffee and tea. CA is known to pharmacologically antagonize ethanol. Thus, the simultaneous intake of CA and ethanol takes place very commonly. Sometimes, toxic interactions also occur following combined intake of these two compounds. In this study, we conducted in vitro experiments to examine the interaction between ethanol and CA at high concentrations by observing mixed-function oxidation reaction using human liver microsomes. After incubation of the mixtures, CA and its three main metabolites theobromine (3,7-dimethylxanthine, TB), paraxanthine (1,7-dimethylxanthine, PX), and theophylline (1,3-dimethylxanthine, TP) were measured by high-performance liquid chromatography with ultraviolet detection. As results, the production of PX, the main metabolite of CA, was consistently inhibited by 24%–53% (P < 0.05) by high ethanol concentrations (20, 40, and 60 mM), but that of TP or TB was not. These results suggest that pharmacological or toxicological effects of CA may be enhanced by simultaneous use of ethanol and CA in humans.

Imipramine is a tricyclic antidepressant that is widely used for the treatment of major depression. There is a possibility that toxic interactions occur following combined use of imipramine and intake of alcohol. In this study, we investigated the in vitro interaction between ethanol and imipramine at high concentrations by observing a mixed-function oxidation reaction using human liver microsomes. Imipramine and its three main metabolites (desipramine; 2-hydroxyimipramine, 2-OHI; 2-hydroxydesipramine, 2-OHD) were measured by high-performance liquid chromatography with ultraviolet detection. The production of 2-OHD, the main metabolite of imipramine, was significantly inhibited, by 15%–50% (P < 0.05), by ethanol, but that of desipramine or 2-OHI was not. These results suggest that enhanced toxicity is attained by simultaneous use of ethanol and high dose of imipramine in the human body.

A simultaneous determination of 20 antidepressant drugs (imipramine, amitriptyline, desipramine, trimipramine, nortriptyline, clomipramine, amoxapine, lofepramine, dosulepin, maprotiline, mianserin, setiptiline, trazodone, fluvoxamine, paroxetine, milnacipran, sulpiride, tandspirone, methylphenidate and melitracen) in human plasma was developed using LC/MS with sonic spray ionization (SSI) method. These drugs showed good separation and sensitivity by LC–MS using an Inertsil C-8 column with methanol:10 mM ammonium acetate (pH 5.0):acetonitrile (70:20:10) as mobile phase at 0.10 mL/min at 35 °C. Solid-phase extraction of these drugs added to the human plasma was performed with an Oasis ® HLB cartridge column. Recovery and limit of detection of these drugs were between 69 and 102% and between 0.03 and 0.63 μg/mL, respectively. The present procedure offers an easier and more convenient screening method for antidepressants, and will be useful for forensic toxicology investigations.

A rapid and simple method for determination of oxazolobenzodiazepines (oxazolam, haloxazolam, mexazolam, cloxazolam, and flutazolam) in sera was developed by gas chromatography (GC) using nitrogen-phosphorus detection (NPD) and a methyl silicone fused-silica wide-bore capillary (DB-1) column. Underivatized oxazolobenzodiazepine drugs gave relatively low sensitivity, because of decomposition during their passage through the column. On the other hand, on-column methylation of the oxazolobenzodiazepines with 0.4 mM trimethylanilium hydroxide in methanolic solution resulted in higher sensitivity; detection was possible at levels at least two to eight times lower than those without derivatization. The average recoveries of five oxazolobenzodiazepines from 0.5-ml volumes of sera containing 1.0 or 0.1 μg of each drug were 88–101% and 66–106%, respectively. The limits of detection of methylated oxazolobenzodiazepines by wide-bore capillary GC-NPD were 50–100pg on column. This method could be used for determination of the drugs in actual serum specimens with concentrations as low as 25–70ng/ml.

A high-performance liquid chromatographic (HPLC) method has been developed for the simultaneous analysis of the local anesthetic amide drugs, bupivacaine, mepivacaine and ropivacaine, belonging to the pipecoloxylidide group using a C18 reversed-phase column (150 × 4.6 mm I.D.) filled with 5-μm particles and attached to a UV detector. The mobile phase was composed of acetonitrile–methanol–30 mM NaH2PO4 (pH 5.6) (100:100:300, v/v/v) and the flow rate was 1 ml/min. The absorbance of the eluate was monitored at 210 nm. The retention times of the three compounds were: 4.6 min (mepivacaine), 9.7 min (ropivacaine) and 16.4 min (bupivacaine). With this sample preparation method, good and consistent recoveries of the three compounds were obtained: 88–91% for mepivacaine, 87–89% for ropivacaine and 88–91% for bupivacaine. The limit of quantification for three compounds in human serum was 2 ng/ml for mepivacaine, 5 ng/ml for bupivacaine and ropivacaine. This method may be useful in clinical and forensic applications for the determination or identification of the local anesthetic drugs: bupivacaine, mepivacaine or ropivacaine.

A combination of the general anesthetic propofol and epidural anesthesia with a local anesthetic is widely used. The metabolism of ropivacaine and that of lidocaine are mediated by similar P450 isoforms. Previously, propofol was found to inhibit the metabolism of lidocaine in vitro. Here we investigated whether propofol inhibits the metabolism of ropivacaine using human liver microsomes in vitro. Ropivacaine (6.0 micromol.l(-1)) as the substrate and propofol (1-100 micromol.l(-1)) were reacted together using human microsomes. The concentrations of ropivacaine and its major metabolite 2',6'-pipecoloxylidide (PPX) were measured using high-performance liquid chromatography. The metabolic activity of ropivacaine was reflected in the production of PPX. The inhibitory effects of propofol on ropivacaine metabolism were observed to be dose-dependent. The IC50 of propofol was 34.9 micromol.l(-1). Propofol shows a competitive inhibitory effect on the metabolism of ropivacaine (i.e., PPX production mediated by CYP3A4) in human CYP systems in vitro.

The urinary metabolites of 5-methoxy-N,N-diisopropyltryptamine (5-MeO-DIPT) in humans have been investigated by analyzing urine specimens from its users. For the unequivocal identification and accurate quantification of its major metabolites, careful analyses were conducted by gas chromatography/mass spectrometry, liquid chromatography/mass spectrometry, and liquid chromatography-tandem mass spectrometry, using authentic standards of each metabolite synthesized. Three major metabolic pathways were revealed as follows: 1) side chain degradation by O-demethylation to form 5-hydroxy-N,N-diisopropyltryptamine (5-OH-DIPT), which would be partly conjugated to its sulfate and glucuronide; 2) direct hydroxylation on position 6 of the aromatic ring of 5-MeO-DIPT, and/or methylation of the hydroxyl group on position 5 after hydroxylation on position 6 of the aromatic ring of 5-OH-DIPT, to produce 6-hydroxy-5-methoxy-N,N-diisopropyltryptamine (6-OH-5-MeO-DIPT), followed by conjugation to its sulfate and glucuronide; and 3) side chain degradation by N-deisopropylation, to the corresponding secondary amine 5-methoxy-N-isopropyltryptamine (5-MeO-NIPT). Of these metabolites, which retain structural characteristics of the parent drug, 5-OH-DIPT and 6-OH-5-MeO-DIPT were found to be more abundant than 5-MeO-NIPT. Although the parent drug 5-MeO-DIPT was detectable even 35 h after dosing, no trace of its N-oxide was detected in any of the specimens examined.

Triazolam is widely used as an ultrashort-acting anxiolytic drug and hypnosedatives and its effect appears at very low doses. Ethanol is used as a social drug worldwide. Sometimes, toxic interactions occur following combined administration of these two drugs. In this study, we have investigated the interaction between alcohol and triazolam in vitro.The interaction effects between alcohol and triazolam were examined by a mixed-function oxidation reaction using a human liver microsomal preparation. Triazolam and its two metabolites (alpha-hydroxytriazolam: alpha-OH triazolam, 4-hydroxytriazolam: 4-OH triazolam) were measured by HPLC/UV.The production of alpha-OH triazolam and 4-OH triazolam was shown to be weakly inhibited by 13-29% (p0.05) and 8-14%,respectively, by ethanol (20-80 mM).These results using a human liver microsomal preparation show that the formation of both metabolites of triazolam is weakly inhibited by ethanol. Toxic levels may be reached by simultaneous administration of ethanol and triazolam.

Summary 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.

Relatively selective in vivo substrate probes have been developed for several major CYP isoforms involved in oxidative drug metabolism. There are basically two in vivo methods for identifying the phenotype. One method, the selective (CYP-specific) phenotyping method, involves administering one single probe drug, whereas the other is a mixed phenotyping or "cocktail" method involving the simultaneous administration of multiple probe drugs, specific for the individual P450. At present, caffeine and chlorzoxazone are used most often as probe drugs for CYP1A2 and CYP2E1, respectively, but these are not necessarily the best probe drugs. Of the potential probe drugs for CYP2C9, CYP2C19, CYP2D6 and CYP3A4, none is really useful. Despite current limitations, the cocktail method for obtaining information about multiple CYP activities in a single experimental session is likely to be more widely used as a screening or phenotyping method for humans in the future.

This review focuses on the toxicological interactions between alcohol (ethanol) and psychiatric drugs (antidepressants and antipsychotics), including those leading to fatal poisoning. Acute or chronic ingestion of alcohol when combined with psychiatric drugs may lead to several clinically significant toxicological interactions. The metabolism of these drugs is generally but not always delayed by acute alcohol ingestion. Drugs undergoing metabolism may also show increased metabolic clearance with chronic alcohol ingestion. Therefore, the net effect may be influenced by internal (e.g. disease, age, gender), external (e.g. environment, diet) and pharmacokinetic (e.g. dose, timing of ingestion, gastrointestinal absorption, distribution and elimination) factors. Cases of fatal poisoning involving coadministration of psychiatric drugs, alcohol and other drugs prompted this review.

A method for the determination of flunitrazepam (FNZ) and 7-aminoflunitrazepam (7-AFNZ) in human serum was developed with ion trap gas chromatography (GC)-tandem mass spectrometry. The 7-AFNZ was derivatizated with 50 microl trifluoroacetic anhydride (TFAA), 60 degrees C-20 min. EI mass spectra and tandem mass spectra of FNZ and 7-AFNZ-TFA were m/z 238, 239, 266, 286, 294, 312, 313(M(+)), m/z 350, 351, 360, 378, 379(M(+)), m/z 238, 239, 240 (precursor ion m/z 286, collision energy 1.5 V), and m/z 239, 254, 264, 336 (precursor ion m/z 351, collision energy 1.8 V), respectively. The detection limits of full scan EI mass spectrometry and tandem mass spectrometry for FNZ and 7-AFNZ in human serum were ca. 200 ng/ml, 60 ng/ml, 15 ng/ml and 1 ng/ml, respectively.

Hypervariable segments of mitochondrial DNA (mtDNA) (HV1 and HV2) were analyzed in Klinefelter's syndrome and compared to normal population data. One pair of samples consisting of a Japanese mother and affected son with Klinefelter's syndrome (involved in a criminal case), and seven unrelated DNA samples from Caucasian Klinefelter males (two involved in criminal cases and five diagnosed) were collected in Japan and the United States. The diagnosis of Klinefelter's syndrome was established previously by multiplex XY-STR typing detecting two X alleles and one Y allele in the samples. Haplotype analysis of the mtDNA sequence in Klinefelter males was found to be identical, unique, and specific, as it was not found in the normal population. Astonishingly, family data exhibited that the haplotype of the mtDNA in the son was apparently different from the mother's, suggesting that the mtDNA of Klinefelter male would not be inherited from mother to son. Our data indicate that possible interaction of the sex chromosome and the mtDNA exists, and suggests that the specific mtDNA haplotype could cause the abnormal cell to fertilize and reproduce itself.

In this study, we have investigated the relationship between lidocaine metabolism and premedication, i.e., psychotropic and anti-anxiety agents (diazepam, midazolam), hypnotics (pentobarbital, thiamylal), depolarizing muscular relaxants (vecuronium, pancuronium and suxamethonium), an active anti-hypertensive (clonidine) and an H2 receptor antagonist (cimetidine) using rat hepatic microsomes in vitro. Lidocaine metabolism was noncompetitively inhibited by midazolam (Ki=29.0 mM). Thilamylal was a moderate competitive inhibitor of lidocaine metabolism (Ki=77.8 mM). Pentobarbital, diazepam and cimetidine weakly inhibited lidocaine metabolism formation in a concentration-dependent manner at high substrate concentrations. On the other hand, vecuronium, pancuronium, suxamethonium and clonidine did not inhibit lidocaine metabolism over the therapeutic range. These results show that the interaction between lidocaine and midazolam and thiamylal, catalyzed by a similar cytochrome P450, is of potential importance in toxicological and clinical studies.

Objective : New international criteria for the estimation of the postmortem interval (PMI) in autopsy cases could be useful. At present, there is no published information comparing differences between people of different ethnic origin. To address this, we have carried out an investigation to see if there is any ethnic difference in the postmortem interval in autopsy cases involving Myanmar and Japanese populations estimated using the vitreous potassium (K + ) level in aqueous humor. Results : Estimation of the PMI from the vitreous K + level was investigated using 82 known PMI autopsy samples collected from various sources and backgrounds (e.g., age, sex, cause of death and ambient temperature) from Myanmar and Japanese subjects using a Copenhagen Radiometer (ABL system). The present study shows that raised vitreous K + levels (mean of right and left eyes) from 1 to 60 hr after death are linearly correlated with increased PMI and the regression line follows that of the vitreous K + levels correlated with PMI and show no significant difference between race (Myanmar, r = 0.93 and Japanese, r = 0.91), eyes (right, r = 0.92 and left, r =0.93), sex (male, r = 0.91 and female, r = 0.93) and age ( r = 0.89). The PMI from the vitreous K + levels of Myanmar and Japanese and their 95% limits of confidence was ±13.8 and ±12.7 hr, respectively. Conclusion: The above result shows that measurement of the vitreous K + level of aqueous humor helps in theestimation of PMI and it appears that there is no significant difference between the two ethnic groups studied. In addition, estimation of the vitreous K + level may be a useful international criterion for estimation of the PMIinautopsy cases.

Background: We review recent findings on the toxicological interactions between alcohol (ethanol) and benzodiazepines, and the combined use of benzodiazepines and alcohol in fatal poisoning. Acute ingestion of alcohol combined with benzodiazepines is responsible for several toxicological interactions that can have significant clinical implications. In general, metabolism of these drugs is delayed when combined with acute alcohol ingestion although some reports suggest otherwise. Alternately, the drugs metabolized during chronic alcohol ingestion have an increased clearance. The net effect may also be influenced by internal (e.g., disease, age) and external (e.g., environment, diet) factors. Fatal poisoning involving coadministration of alcohol and benzodiazepine, especially triazolam, continues to be a serious problem.

UNLABELLED There is no report concerning oral clonidine's effects on epidural lidocaine in children. Therefore, we performed a study to assess the concentrations of plasma lidocaine and its major metabolite (monoethylglycinexylidide [MEGX]) in children receiving continuous thoracic epidural anesthesia after oral clonidine premedication. Ten pediatric patients, aged 1-9 yr, were randomly allocated to the Control or Clonidine 4 microg/kg group (n = 5 each). Anesthesia was induced and maintained with sevoflurane in oxygen and air (FIO2 40%). Epidural puncture and tubing were carefully performed at the Th11-12 intervertebral space. An initial dose of 1% lidocaine (5 mg/kg) was injected through a catheter into the epidural space, followed by 2.5 mg x kg(-1) x h(-1). Plasma concentrations of lidocaine and MEGX were measured at 15 min, 30 min, and every 60 min for 4 h after the initiation of continuous epidural injection. The concentrations of lidocaine and MEGX were measured using high-pressure liquid chromatography with ultraviolet detection. Hemodynamic variables were similar between members of the Control and Clonidine groups during anesthesia. The Clonidine group showed significantly smaller lidocaine concentrations (p < 0.05) and the concentration of MEGX tended to be smaller in the plasma of the Clonidine group for the initial 4 h after the initiation of epidural infusion. In conclusion, oral clonidine preanesthetic medication at a dose of 4 microg/kg decreases plasma lidocaine concentration in children. IMPLICATIONS Oral clonidine decreases the plasma lidocaine concentration in children. Our finding may have clinical implications in patients receiving continuous epidural anesthesia. Additionally, perhaps an additional margin of safety regarding lidocaine toxicity is gained through the use of oral clonidine in children who will receive epidural lidocaine.

In the present study, small volumes of plasma were used for the measurement of bromvalerylurea (BVU), its metabolite, 3-methylbutyrylurea (MVU), and bromide in carbon tetrachloride (CCl4)-treated rats by HPLC-UV and energy dispersive X-ray spectrometry. A liquid-liquid extraction system was also investigated. BVU and MVU were extracted from 100 microl plasma samples in a single-step involving deproteination with 1 M hydrochloric acid using ethenzamide as internal standard. Samples were separated by HPLC in an acetonitrile-8 mM potassium dihydrogenphosphate buffer (35:65, v/v) mobile phase at a flow-rate of 0.4 ml/min on a 15 cm octadecylsilyl column at room temperature. Analytes were detected at a wavelength of 210 nm. The limits of quantitation for BVU, MVU and bromide are 0.1, 0.1 and 50 microg/ml, respectively. The intra-day accuracies over the range of concentrations were 95.8 to 121.1%, 97.2 to 119.7% and 96.2 to 105.8% for BVU, MVU and bromide, respectively. The inter-day accuracies were 97.7 to 115.1%, 98.3 to 111.6% and 98.3 to 102.9% for BVU, MVU and bromide, respectively. The absolute recoveries using tert.-butyl methyl ether are 96-98% for BVU and 95-98% for MVU. The decline in the plasma concentrations of BVU in olive oil-treated rats fitted a one-compartment model and the plasma MVU level reached a peak at around 1.5-2 h and then decreased gradually. The elimination of BVU in CCl4 (1 ml/kg)-treated rats was delayed and MVU production was less than that in the olive oil-treated group. However, there was no difference in the plasma levels of bromide between CCl4-treated rats and control rats. rights reserved.

In order to establish an analytical method for the determination of phenobarbital (PB), phenytoin (PPH) and their hydroxylated metabolites in hair, animal model experiments were performed. Five male dark-agouti pigmented rats, aged 5 weeks, were intraperitoneally and orally administered PB or PPH independently at 25 mg/kg once a day for 5 successive days. The growing back hair was collected 15d after the first administration. Four typical extraction methods, using NH4OH-methanol-acetone, TFA-methanol-acetone, 1M sodium hydroxide and proteinase K, were evaluated using the rat hair samples containing PB or PPH. Methanol-acetone-NH4OH (10: 10: 1) was the best extraction method from all aspects, such as high extraction efficiency and low noise. The analytes in the extract were methylated in acetonitrile with 20% tetramethylammonium hydroxide and methyliodide at 70 degrees C for 10 min. After purification with Bond Elut Certify, the methylated products were analyzed by GC-MS. From rat hair, PB, p-hydroxy PB, PPH and p-hydroxy PPH were detected at average concentrations of 26.9, trace, 4.2 and 0.4 ng/mg with an intraperitoneal (i.p.) injection, and at 30.9, trace, 4.0 and 0.4 ng/mg with oral administration, respectively. There was little difference in hair concentrations between i.p. injection and oral administration. This method was applied to the head hair of two patients who orally took toxic amounts of PB and two volunteers who orally took 100 mg of PPH daily for 5 d. The hair concentrations of PB in the two patients were 16.2 and 14.7 ng/mg, and those of PPH in the two volunteers were 3.3 and 0.1 ng/mg.

The cytochrome P450 (P450 or CYP) monooxygenases, CYP2D6, CYP2C19 and CYP2C9, display polymorphism. CYP2D6 and CYP2C19 have been studied extensively and, despite their low abundance in the liver, they have been found to catalyze the metabolism of many drugs. CYP2D6 has many allelic variants, whereas CYP2C19 has only seven. Most variants are translated into inactive, truncated proteins or fail to express any protein. There is, as yet, no clear information about the other CYPs (CYP1A1, CYP2A6, CYP2B6, CYP2E1 and CYP3A4/5) polymorphism.

Acetaminophen (paracetamol) is one of the most commonly used over-the-counter medications. Taken in doses greater than 150 mg/kg/day (>10 g), it usually causes acute liver failure. The authors review mainly the management of acetaminophen toxicity in both users and nonusers of alcohol. Chronic alcoholics are a special subgroup, who risk serious toxicity when taking acetaminophen, even in therapeutic doses. The acetaminophen-alcohol interaction is complex, because acute and chronic ethanol have opposite effects. This review also considers physiological and clinical changes, as well as the diagnosis and treatment of acetaminophen poisoning.

The cytochrome P450 (P450 or CYP) monooxygenases, CYP2D6, CYP2C19, CYP2E1 and CYP2C9, and non-P450 monooxygenases, N-acetyltransferase, thioprine methyltransferases and dihydropyrimidine dehydrogenase, all display polymorphism. CYP2D6 and CYP2C19 have been studied extensively and, despite their low abundance in the liver, they have been found to catalyse the metabolism of many drugs. CYP2D6 has many allelic variants, whereas CYP2C19 has only two. Most variants are translated into inactive, truncated proteins or fail to express protein. There is, as yet, no clear information about CYP2E1 polymorphism. In addition, genetic differences in certain foreign-compound metabolizing enzymes, such as Phase II enzymes, have been shown to be associated with an increased risk of developing environmentally and occupationally related diseases such as cancer. When two drugs that are substrates of a polymorphic CYP enzyme are administered concomitantly during drug therapy, each will compete for that enzyme and competitively inhibit the metabolism of the other substrate. This can result in toxicity. Patients who are poor metabolizers (PMs), extensive metabolizers (EMs) and ultrarapid metabolizers (URMs) can be identified. Having such information will help in determining the appropriate dosage of certain drugs when treating patients with an inherited abnormality of a drug-metabolizing enzyme. In view of the remarkable progress in this particular field, it is to be expected that more genetic polymorphisms will be discovered in the near future.

The reports of interactions between benzodiazepines (BZPs) and other drugs (e.g., antidepressants, selective serotonin reuptake inhibitors, antiulcer drugs, antiepileptic drugs, macrolide antibiotics) during their combined use are reviewed. In general, metabolism of BZPs is delayed when combined with a number of other drugs but some reports have suggested otherwise. In recent years, the cytochrome P450 (P450 or CYP) isoenzyme that catalyses the metabolism of BZPs has also been identified. BZPs are mainly catalysed by CYP3A4. When published reports are studied, it appears necessary to be exceptionally careful about interactions mainly between BZPs and selective serotonin reuptake inhibitors, cimetidine, antiepileptic drugs, macrolide antibiotics and antimycotics. More information is necessary to identify individuals at greatest risk of drug interactions and adverse events.

In this review I have attempted to summarize gender differences in pharmacokinetics involving the cytochrome P450 (CYP) isozymes of young and mature adults, excluding the effects of the menstrual cycle, use of oral contraceptives and pregnancy. Sex differences in drug metabolism and elimination are mainly related to steroid hormone levels. CYP3A4, responsible for the metabolism of over 50% of therapeutic drugs, exhibits higher activity in women than in men. Nonetheless, the absence of a sex difference has been reported by some workers. The activity of several other CYP (CYP2C19, CYP2D6, CYP2E1) isozymes and the conjugation (glucuronidation) activity involved in drug metabolism may be higher in men than in women. Drug metabolism in women is affected by sex-specific factors (menopause, pregnancy and menstruation) in addition to the cigarette smoking, drug ingestion and alcohol consumption that are more commonly observed factors in men. Furthermore, they are affected by physiological factors such as drug absorption, protein binding and elimination. Thus, careful attention should be paid to the side-effects and toxicity arising from sex differences in drug metabolism in clinical situations. Although there are specific ethical considerations regarding carrying out drug trials in women, the relationship between the side-effects and toxicity that may be influenced by hormones during drug metabolism and drug treatment needs further study.

Drug-drug interactions have become an important issue in health care. It is now realized that many drug-drug interactions can be explained by alterations in the metabolic enzymes that are present in the liver and other extra-hepatic tissues and many of the major pharmacokinetic interactions between drugs are due to hepatic cytochrome P450 (P450 or CYP) enzymes being affected by previous administration of other drugs. After coadministration, some drugs act as potent enzyme inducers, whereas others are inhibitors. However, reports of enzyme inhibition are very much more common. Understanding these mechanisms of enzyme inhibition or induction is extremely important in order to give appropriate multiple-drug therapies. In the future, it may help to identify individuals at greatest risk of drug interactions and adverse events.

Recent reports of interactions between alcohol and benzodiazepines, tricyclic and tetracyclic antidepressants during their acute concomitant use are reviewed. Acute ingestion of alcohol (ethanol) with tranquilizers or hypnotics is responsible for several pharmacokinetic interactions that can have significant clinical implications. In general, metabolism of these drugs is delayed when combined with alcohol but some reports have suggested otherwise. The amount of alcohol consumed, the presence or absence of liver disease, and differences in the dosage and administration of these drugs may account for the observed discrepancies. In recent years, the cytochrome P450 (P450 or CYP) isoenzyme that catalyses the metabolism of these drugs has also been identified. However, since changes in the pharmacogenetic metabolism of benzodiazepines and tricyclic and tetracyclic antidepressants are mainly governed by CYP2C19 and CYP2D6, caution is needed when used together with alcohol.

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 (

Very few studies have been carried out looking at how the effects of drugs and their toxicity in humans change during their lifespan (developing and ageing). The purpose of this study is to review the literature on the changes in probe-drug metabolism, classified by cytochrome P450 (P450 or CYP) at five stages in life: neonates4 weeks, infants12 months, children19 years, young/mature adults 20-64 years, and elderly adults65 years. The main probe drugs include caffeine and theophylline, whose metabolism is catalysed by CYP1A2, tolbutamide, phenytoin and ibuprofen, catalysed by CYP2C9, amitriptyline and nortriptyline, catalysed by CYP2C19, acetaminophen, catalysed by CYP2E1 and lidocaine, midazolam and terfenadine, catalysed by 3A3/4. From the published in vivo studies two different patterns of drug metabolism can be identified: (i) activity is low immediately after birth, increases, then peaks at the young/mature adult level and, finally, decreases in old age (drugs catalysed by CYP1A2, CYP2C9, CYP2C19, CYP2D6 and CYP3A3/4) and (ii) activity increases rapidly after birth to reach a level equivalent to that in the young/mature adult, then gradually decreases and finally decreasing faster in old age (drugs catalysed by CYP2E1). Further study of the changes in P450 with age is warranted to help prevent adverse reactions and to guide us in tailoring therapy better for the individual patient.

Liver disease is associated with reduced metabolic capacity for drugs that are metabolized by oxidative biotransformation. Three cytochrome P450 (P450 or CYP) gene families in liver microsomes (CYP 1, CYP2 and CYP3) appear to be responsible for much of the drug metabolism that takes place. The genetic polymorphism of the CYPs responsible for debrisoquine/ sparteine (CYP2D6) metabolism and S-mephenytoin (CYP2C19) metabolism has been well documented, but information on the impairment of each isoform in liver disease is still limited. There are two types of hepatic P450 function tests. One type consists of non-genetic P450 function tests (CYP1A2, 2A6, 2C9/10, 2E1 and 3A3/4), and probe drugs include caffeine, catalysed by CYP1A2, coumarin by CYP2A6, phenytoin by CYP2C6, chlorzoxazone by CYP2E1, and nifedipine, erythromycin and lidocaine by CYP3A3/4. The second type of genetic P450 function tests (CYP2C19 and CYP2D6) involves probe drugs such as S-mephenytoin, catalysed by CYP2C19, and debrisoquine and sparteine, catalysed by CYP2D6. The metabolism of the probe drugs used in non-genetic P450 function tests in patients with liver disease falls into two categories: reduced (CYP1A2, CYP2C, 2E1 and 3A) and unchanged (CYP2C). In genetic P450 function tests there seems to be a lesser degree of inhibition in poor metabolizers (PMs) than extensive metabolizers (EMs) among patients with liver disease. There have been very few reports on changes in metabolism of the probe drugs used in genetic P450 function tests in liver disease. In this paper the subject is reviewed.

Drug-metabolizing cytochrome P450 enzymes, the major phase I enzymes, are active in human liver already at very early stages of intrauterine development, although presumably at fairly low concentrations and in low numbers. During maturation, these enzymes go through various developmental programmes towards adulthood. The major increase both in abundance as well as in number of different enzymes takes place after birth, probably during the first year of life. Detailed information concerning these developmental changes is still limited. The major drug-metabolizing P450 enzymes appear to be primarily members of the CYP3A subfamily in all stages of development. The balance between different members of this subfamily, however, undergoes significant switches from the foetal predominant CYP3A7 to the major adult form CYP3A4. The ontogeny of the other cytochrome P450 enzymes is less well characterized, but the major switch-on appears to occur mainly after birth. Developmental expression of P450 enzymes is one of the key factors determining the pharmacokinetic status of developing individuals both pre- and postnatally.

肝予備能評価法として我々が用いているtrimethadione (TMO) 負荷試験が, 肝硬変例における肝障害についての臨床的な重症度の評価に有効か否かを判定するために, Child-Pugh基準 (Pugh score) と対比して検討した.TMO経口投与4時間後のTMOと, その唯一の代謝物dimethadione (DMO) の血中濃度比 (DMO/TMO値) は, 臨床的な肝障害重症度を判定するPughscoreによる分類のA群0.33±0.07, B群0.26±0.10, C群0.11±0.06で, 各群間に有意差を認めた (p

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.

A fatal overdose involving case by 5-methoxy- N , N -diisopropyltryptamine (5-MeO-DIPT) is reported. 5-MeO-DIPT and its two metabolites, 5-hydroxy- N , N -diisopropyltryptamine (5-OH-DIPT) and 5-methoxy- N -isopropyltryptamine (5-MeO-NIPT), were identified by LC–MS. The level of 5-MeO-DIPT, 5-OH-DIPT and 5-MeO-NIPT in blood and urine was 0.412, 0.327 and 0.020μg/ml, and 1.67, 27.0 and 0.32μg/ml, respectively. These blood and urine levels were higher than published data for such poisoning.

A high-performance liquid chromatographic method has been developed for the forensic analysis of 10 frequently used barbiturates (BARs) (allobarbital, amobarbital, barbital, cyclobarbital, hexobarbital, metharbital, pentobarbital, phenobarbital, secobarbital and thiopental) using a recently developed reversed-phase column packed with 2-micron particles. The results show that the new ODS column packing gives higher sensitivity and a shorter analysis time than the conventional ODS column packing when applied to the analysis of biological samples.

A high-performance liquid chromatographic method has been developed for the simultaneous analysis of twelve frequently used benzodiazepines (BZPs) (bromazepam, clonazepam, chlordiazepoxide, estazolam, etizolam, flutazoram, haloxazolam, lorazepam, nitrazepam, oxazolam, triazolam and diazepam, internal standard) by using commercially available 2 or 5 microns particle size reversed-phase columns and a microflow cell-equipped ultraviolet detector. The separation was achieved using a C18 reversed-phase column (condition 1: 100 x 4.6 mm I.D., particle size 2 microns, TSK gel Super-ODS: conditon 2: 100 x 4.6 mm I.D., particle size 5 microns, Hypersil ODS-C18). The mobile phase was composed of methanol-5 mM NaH2PO4 (pH 6) (45:55, v/v), and the flow-rate was 0.65 ml/min (condition 1 and 2). The absorbance of the eluent was monitored at 254 nm. Retention times under condition 1 were shorter than those of condition 2. When the twelve benzodiazepines were determined, sensitivity and limits of quantification were about four to ten times better under condition 1 than under condition 2. The rate of recovery and linearity in condition 1 were approximately the same as those in condition 2. These results show that a new ODS filler with a particle size of 2 microns was more sensitive, provided better separation and was more rapid than that with conventional ODS filler.

肝移植に伴う肝阻血障害が移植肝ミクロソーム機能におよぼす影響について, タクロリムスとトリメタジオン (TMO) の薬物動態により検討した. 雑種成犬を用いた同所性全肝移植後第1日と第7日に, タクロリムスとTMOの薬物動態を調べ, 無処置犬を対照群として比較した. タクロリムスは0.3mg/kgを30分かけて静注し, 全血中濃度の変化を測定した. TMOは4mg/kgを急速静注し, 血清中のTMOとその代謝物であるジメタジオン (DMO) の変化を測定した. 両群とも薬物動態を調べる日以外はタクロリムス0.3mg/kg/日を経口的に投与した. TMOのクリアランスと投与2時間後の血清中DMO/TMO比は, 肝移植群で移植後第1日に低下していたが第7日には両群間に差を認めなかった. タクロリムスのクリアランス, 投与24時間後の血中濃度は移植後第1日, 第7日とも両群間に差を認めなかった. TMOの代謝はタクロリムスの代謝に比べて肝阻血による障害を強くうけることが示された.

肝ミクロゾーム分画で代謝される抗てんかん薬のtrimethadione (TMO)を用いた負荷試験が,肝のいかなる組織形態学所見を反映しているか検討した.対象は肝硬変(LC) 16例,慢性肝炎(CH)と肝線維症の各2例の20例である.術前の肝CT像より算出した肝容積と肝組織の光学顕微鏡所見の画像解析により肝実質比(実質/実質+間質)や単位体積あたりの細胞数を求めた.それらの指標より肝実質量(肝容積×実質比),肝細胞量(肝容積×単位体積あたりの肝細胞数),肝実質細胞総量(肝容積×実質比×単位体積あたりの細胞数)を算出した.TMO負荷試験値と肝実質量や肝細胞量とは,それぞれ高い相関(r=0.771, r=0.809; p

Nowadays, many of the DNA sequences of ABO blood group system have been disclosed to perform DNA testing, and one-base mutations or variations among different ABO genotypes could be detected for genotyping. For this purpose, single-strand conformation polymorphism (SSCP), which detects only one-base difference between different genotypes, is applied to detect ABO genotypes and is one of the choices among ABO genotyping technology. For practical application in forensic DNA testing, we tried to apply SSCP method using high-resolution ALF express DNA sequencer (Pharmacia Biotech). High-resolution SSCP is extremely sensitive and reliable that almost all cases of trace evidence obtained from criminal investigation were able to be genotyped visually. The denatured single-stranded amplicons were electrophoresed in sequencing gel, analyzed by laser detector, and visualized the peak patterns of chromatogram. With this method, we are able to classify ABO genotypes into 15 groups and additional subtypes. In addition, analysis of ABO genotypes of 400 DNA samples extracted from individuals living in Japan, Mongolia, and Colombia revealed a remarkable regional difference in allele frequency of A101 versus A102, and OA vs. OG.

The clear evidence that curious fashion of mitochondrial DNA (mtDNA) inheritance was reported in sex chromosomal aberration. Human mtDNA is located outside the nucleus of the cell producing energy for the cell. In the recent years, mtDNA is being used in forensic DNA identification because each cell has many copies of mitochondria, and is easy to be amplified by PCR. Human mtDNA has polymorphic sites in hypervariable regions (HVR) I, II and III. However, human mtDNA evokes some problems, such as heteroplasmic point mutations and questionable maternal inheritance. To investigate this phenomenon, we had a chance to perform a mtDNA testing on 1-month-old infant, diagnosed as Turner Syndrome which was confirmed to be 45XO karyotype by X-STR testing. As a result, two remarkable evidences were obtained. The first, an intermarriage, is seemed to be related in the genesis of the sex chromosomal aberration. The second, the exact maternal inheritance of mtDNA was not maintained in Turner syndrome, as well as in Klinefelter syndrome reported by us previously. In these cases, although paternal influence of mtDNA inheritance was deniable, mutations of mother's mtDNA were excluded or repaired to the usual type in child's mtDNA.

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.

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.

1. Caffeine (CA) metabolism in animal and man is mainly catalysed by P4501A2. Lidocaine (LID) and phenytoin (PHT) are metabolized by P4503A2 and 2B1/2 in animals and 3A4 and 2C9 in man, respectively. 2. We investigated the possibility of predicting liver damage from changes in blood concentrations after simultaneous i.v. administration (cocktail study) of the three probe drugs CA (10 mg/kg), LID (4 mg/kg) and PHT (4 mg/kg), and their main metabolites, paraxanthine (PX), monoethylglycinexylide (MEGX) and 5-(p-hydroxyphenyl)-5-phenylhydantoin (p-HPPH) respectively. 3. The metabolism of CA, LID and PHT and production of their metabolites (PX, MEGX and p-HPPH) in the carbon tetracholride (CCl4 0.25 and 0.5 ml/kg)-treated rat were reduced in comparison with the control group. 4. The ratios (PX/CA and p-HPPH/PHT) of CA and PHT to the serum levels of the their metabolites 2 h after i.v. administration of three drugs to the CCl4-treated rat were significantly reduced. 5. The correlation coefficients among CLs of CA, LID and PHT, and the PX/CA and p-HPPH/PHT ratios in rat pretreated with different doses of CCL4 are very high. 6. These results suggest that different estimates of hepatic oxidizing capacity, catalysed mainly by P4501A2, 2C and 3A, may be related to the extent of liver disease in the CCl4-intoxicated rat. Therefore a 'cocktail' is not in fact needed to assess hepatic damage in the CCl4-intoxicated rat.

A sensitive method for the simultaneous determination of quazepam and two of its metabolites, 2-oxoquazepam and 3-hydroxy-2-oxoquazepam, in human urine was developed using gas chromatography–mass spectrometry (GC/MS) with an Rtx-5MS capillary column. The quazepam and its metabolites were extracted from human urine using a simple solid-phase extraction Oasis ® HLB cartridge column, and the 3-hydroxy-2-oxoquazepam was derivatised using BSTFA/1%TMCS and pyridine at 60°C for 30min. The mass spectrometric detection of the analytes was performed in the full scan mode, m / z 60–480, and selected ion monitoring (SIM) mode, m / z 386, for quazepam; m / z 342, for 2-oxoquazepam; m / z 429, for 3-hydroxy-2-oxoquazepam-TMS; and m / z 284, for alprazolam-d5 (internal standard), by electron ionization. The calibration curves of quazepam and its metabolites in urine showed good linearity in the concentration range of 2.5–500ng/0.2ml of urine. The average recoveries of quazepam and its metabolites from 0.2ml of urine containing 500ng and 50ng of each drug were 71–83% and 88–90%, respectively. The limits of detection of quazepam, 2-oxoquazepam and 3-hydroxy-2-quazepam in urine by the selected ion monitoring mode were 0.096–0.37ng/ml. This method would be applicable to other forensic biological materials containing low concentrations of quazepam and its metabolites.

To examine the effects of cyclosporin A (CsA) on renal cytochrome P450 forms, CsA was administered to rats, and the renal levels of P450 were determined by immunoblotting. CsA treatment for 17 days increased total renal P450 content by 40% with a concomitant elevation of the omega- and (omega-1)-hydroxylation activities of lauric acid. Arachidonic acid omega-hydroxylation activity was also induced 2-fold by treatment with CsA for 17 days. Among the P450 forms, CYP4A2 was induced significantly, whereas CYP2C23, CYP4A1 and CYP4A8 were unaffected. These changes were accompanied by slight but significant increases in blood urea nitrogen and systolic blood pressure. These data suggest that CsA increased arachidonic acid omega-hydroxylation activity by the induction of CYP4A2. The specific induction of CYP4A2 may be related to CsA-induced nephrotoxicity and elevated blood pressure, because omega-hydroxyarachidonic acid is a potent vasoconstrictor.

It has been reported that caffeine (CA) metabolism in humans is catalyzed by cytochrome P450(CYP)1A2. Recently, we reported that human CYP3A4 and 2C, but not 1A2, contribute to trimethadione (TMO) N-demethylation in the human liver. In this study we simultaneously administered two indicators, CA (2 mg/kg) and TMO (4 mg/kg), which have different patterns of metabolism, to patients with cholelithiasis (control) or cirrhosis. The serum levels of CA and TMO, and their individual metabolites, were determined by high-performance liquid chromatography and gas chromatography, respectively. The metabolism of CA and TMO was significantly inhibited in patients with cirrhosis as compared with controls. The ratios of serum paraxanthine (PX)/CA and DMO/TMO at 4 h after administration of the two probe drugs were also significantly decreased in patients with cirrhosis. The PX/CA ratios of the smoking patients with cirrhosis were higher than those of non-smokers. No differences in serum DMO/TMO ratios were observed between the smokers and non-smokers. These results suggest that different estimations of hepatic oxidizing capacity, catalysed mainly by CYP2C and 3A4, are possible in humans.

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.

肝硬変併存肝癌例の耐術評価に用いられている肝機能検査が, いかなる肝の形態的所見を反映しているか, 画像解析装置を用いて検討した. 肝硬変併存肝癌16例の肝実質の数か所から得た組織切片から単位体積あたりの肝細胞数と肝実質比を測定した. それにCT像から求めた肝容積とを組み合わせて肝細胞量や肝実質量および肝細胞総量を算出して形態的所見の定量化を試みた. さらに, これらの指標とICG負荷試験値 (ICGR15) や一般肝機能検査値とを比較検討した. ICGR15と, 実質比や単位体積あたりの細胞数および細胞量とは, r=-0.506 (p