Reduction of temazepam to diazepam and lorazepam to delorazepam during enzymatic hydrolysis.

It has been previously reported that treatment of urinary oxazepam by commercial ß-glucuronidase enzyme preparations, from Escherichia coli, Helix pomatia and Patella vulgata, results in production of nordiazepam (desmethyldiazepam) artefact. In this study, we report that this unusual reductive transformation also occurs in other benzodiazepines with a hydroxyl group at the C3 position such as temazepam and lorazepam. As determined by liquid chromatography-mass spectrometry analysis, all three enzyme preparations were found capable of converting urinary temazepam into diazepam following enzymatic incubation and subsequent liquid-liquid extraction procedures. For example, when H. pomatia enzymes were used with incubation conditions of 18 h and 50 °C, the percentage conversion, although small, was significant--approximately 1% (0.59-1.54%) in both patient and spiked blank urines. Similarly, using H. pomatia enzyme under these incubation conditions, a reductive transformation of urinary lorazepam into delorazepam (chlordesmethyldiazepam) occurred. These findings have both clinical and forensic implications. Detection of diazepam or delorazepam in biological samples following enzyme treatment should be interpreted with care.

Diazepam pharmacokinetics after nasal drop and atomized nasal administration in dogs.

The standard of care for emergency therapy of seizures in veterinary patients is intravenous (i.v.) administration of benzodiazepines, although rectal administration of diazepam is often recommended for out-of-hospital situations, or when i.v. access has not been established. However, both of these routes have potential limitations. This study investigated the pharmacokinetics of diazepam following i.v., intranasal (i.n.) drop and atomized nasal administration in dogs. Six dogs were administered diazepam (0.5 mg/kg) via all three routes following a randomized block design. Plasma samples were collected and concentrations of diazepam and its active metabolites, oxazepam and desmethyldiazepam were quantified with high-performance liquid chromatography (HPLC). Mean diazepam concentrations >300 ng/mL were reached within 5 min in both i.n. groups. Diazepam was converted into its metabolites within 5 and 10 min, respectively, after i.v. and i.n. administration. The half lives of the metabolites were longer than that of the parent drug after both routes of administration. The bioavailability of diazepam after i.n. drop and atomized nasal administration was 42% and 41%, respectively. These values exceed previously published bioavailability data for rectal administration of diazepam in dogs. This study confirms that i.n. administration of diazepam yields rapid anticonvulsant concentrations of diazepam in the dog before a hepatic first-pass effect.

Biotransformation of diazepam in a clinically relevant flat membrane bioreactor model using primary porcine hepatocytes.

In vitro biotransformation of drug using commercial culture medium with serum may not be the ideal culture medium for clinical application in extracorporeal bioartificial liver support (BAL) systems. In these systems, patient's blood or plasma is plumbed to primary hepatocytes within a seeded bioreactor, creating interaction between plasma and seeded hepatocytes. To address this situation, we investigated the biotransformation potential of diazepam in primary porcine hepatocytes with a flat membrane bioreactor (FMB); we used human plasma exposure and serum-free media in organotypical double gel culture model for long-term culture. We investigated diazepam clearance and all major metabolites of diazepam, such as oxazepam, temazepam, and desmethyldiazepam, in conventional single gel and organotypical sandwich models and compared them to the FMB model. Diazepam elimination was higher in double gel cultures with exposure to both SF 3 medium conditions and plasma, when compared to the single gel model in a Petri dish. It was observed that in the FMB, diazepam elimination was stable at about 3 pg/h/cell in plasma and SF 3 exposure. Oxazepam synthesis in the bioreactor was approximately one quarter less than in the Petri dish, but there were no differences between N-desmethyldiazepam and temazepam synthesis in double gel culture. In the flat membrane bioreactor, there was no decrease in the biotransformation of diazepam in plasma exposure compared with the control group. Our results suggest that this plasma exposure bioreactor may offer a useful approach in clinical use of extracorporeal BAL, as well as for drug metabolite investigation into toxicological research.

A novel reductive transformation of oxazepam to nordiazepam observed during enzymatic hydrolysis.

beta-Glucuronidase is an enzyme often employed to de-conjugate beta-glucuronides during urinary drug testing for benzodiazepines. It is commonly accepted that use of beta-glucuronidase is a preferred method of hydrolysis over acid-catalyzed hydrolysis, which is known to induce benzodiazepine degradation and transformation. Literature to date, however, has not reported any cases of benzodiazepine transformation initiated by commercial beta-glucuronidase products. In this study, urine specimens containing either oxazepam or oxazepam glucuronide were incubated with beta-glucuronidase enzymes obtained from Escherichia coli, Helix pomatia, and Patella vulgata under various incubation conditions. After liquid-liquid extraction, the extract was analyzed by both liquid chromatography-tandem mass spectrometry and gas chromatography-mass spectrometry for the presence of benzodiazepines. All three enzyme preparations examined were capable of reducing oxazepam or oxazepam glucuronide into nordiazepam (desmethyldiazepam). Nordiazepam formation was positively correlated with incubation temperature, incubation time, oxazepam concentration, and enzyme concentration. Under all enzymatic hydrolysis conditions investigated, the percentage of nordiazepam formation is < 2.5% relative to the amount of oxazepam present in the system. The findings of this study have both clinical and forensic implications, and it is clear that the detection of nordiazepam in biological samples subjected to testing involving enzyme-catalyzed hydrolysis should be interpreted with care.

Two compartment model of diazepam biotransformation in an organotypical culture of primary human hepatocytes.

Drug biotransformation is one of the most important parameters of preclinical screening tests for the registration of new drug candidates. Conventional existing tests rely on nonhuman models which deliver an incomplete metabolic profile of drugs due to the lack of proper CYP450 expression as seen in human liver in vivo. In order to overcome this limitation, we used an organotypical model of human primary hepatocytes for the biotransformation of the drug diazepam with special reference to metabolites in both the cell matrix phase and supernatant and its interaction of three inducers (phenobarbital, dexamethasone, aroclor 1254) in different time responses (1, 2, 4, 8, 24 h). Phenobarbital showed the strongest inducing effect in generating desmethyldiazepam and induced up to a 150 fold increase in oxazepam-content which correlates with the increased availability of the precursor metabolites (temazepam and desmethyldiazepam). Aroclor 1254 and dexamethasone had the strongest inducing effect on temazepam and the second strongest on oxazepam. The strong and overlapping inductive role of phenobarbital strengthens the participation of CYP2B6 and CYP3A in diazepam N-demethylation and CYP3A in temazepam formation. Aroclor 1254 preferentially generated temazepam due to the interaction with CYP3A and potentially CYP2C19. In parallel we represented these data in the form of a mathematical model with two compartments explaining the dynamics of diazepam metabolism with the effect of these other inducers in human primary hepatocytes. The model consists of ten differential equations, with one for each concentration c(i,j) (i=diazepam, temazepam, desmethyldiazepam, oxazepam, other metabolites) and one for each compartment (j= cell matrix phase, supernatant), respectively. The parameters p(k) (k=1, 2, 3, 4, 13) are rate constants describing the biotransformation of diazepam and its metabolites and the other parameters (k=5, 6, 7, 8, 9, 10, 11, 12, 14, 15) explain the concentration changes in the two compartments.

Distribution of diazepam and nordiazepam between plasma and whole blood and the influence of hematocrit.

The binding of drugs to plasma proteins is important to consider when concentrations in whole blood (eg, in forensic toxicology) are compared with therapeutic and toxic concentrations based on the analysis of plasma or serum. The plasma to whole blood distribution of diazepam (D) and its major metabolite nordiazepam (ND) was investigated under in vitro and ex vivo conditions. Studies in vitro were done by spiking whole blood with D and ND to give concentrations ranging from 0.1 to 1.0 microg/g. Venous blood was also obtained from hospital blood donors (n = 66) after informed consent. The hematocrit, hemoglobin, and water content of blood specimens were determined by routine procedures before D and ND were added to produce target concentrations of approximately 0.5 microg/g for each substance. The ex vivo work was done with blood specimens from hospital outpatients who were being medicated with D. Concentrations of D and ND were determined in body fluids by capillary column gas chromatography after adding prazepam as internal standard and solvent extraction with butyl acetate. The method limit of quantitation was 0.03 microg/g for both D and ND. The concentrations of D and ND were highest in plasma and lowest in erythrocytes. The plasma/blood (P/B) distribution ratios did not depend on drug concentration between 0.1 and 1.0 microg/g. The mean P/B ratios were 1.79:1 for D and 1.69:1 for ND when hematocrit was 45%. Furthermore, the P/B ratio for D (y) was positively correlated with blood hematocrit (x) and the regression equation was y = 0.636 + 0.025x (r = 0.86, P < 0.001). A similar strong association was found between the P/B ratio and hematocrit for ND (r = 0.79). P/B ratios of D and ND, blood hematocrit, hemoglobin, and the water content differed between sexes (P < 0.001). The overall mean P/B ratios for D and ND were 1.69 +/- 0.097 (+/- SD) and 1.62 +/- 0.08 (P < 0.001, n = 66) respectively when the mean hematocrit was 42.9 +/- 3.4 (+/- SD). For forensic purposes, it would be better to forgo making any conversion of a drug concentration measured in whole blood to that expected in plasma or serum; instead, therapeutic and toxic concentrations should be established for the actual specimens received.

SPME-LC with UV detection to study delorazepam-serum albumin interactions.

Solid phase microextraction coupled to high performance liquid chromatography with UV detection (SPME/LC-UV) has been employed to study the binding of delorazepam to human serum albumin (HSA) and bovine serum albumin (BSA). The procedure could also be potentially extended to the measurement of partition coefficients between a wide variety of semi- or non-volatile compounds and matrices. The method is solvent free, simple, fast, and drawbacks of the conventional analytical techniques are avoided. Moreover, the matrix did not interfere with the measurement by binding to the fibre and the amount extracted by the fibre was negligibly small; thus it did not disturb the delorazepam-protein binding.

The simultaneous determination of diazepam and its three metabolites in dog plasma by high-performance liquid chromatography with mass spectroscopy detection.

A fast, sensitive and specific LC/MS/MS method for the simultaneous determination of diazepam and its three metabolites, oxazepam, temazepam and desmethyldiazepam, in dog plasma is described. The method consists of an automated 96-well solid phase extraction procedure and electrospray LC/MS/MS analysis. D(5)-Diazepam is used as the internal standard for all the compounds. Intra-day and inter-day assay coefficients of variations are less than 12.7%. The lower limit of quantitation (LLOQ) is 1 nM for each analyte, based on 0.1 ml aliquots of dog plasma. The analytical run time was 5 min. Linearity is observed over the range of 1--500 nM. This method has been used to support the discovery of pharmacokinetic studies.

Sigmoidal kinetic model for two co-operative substrate-binding sites in a cytochrome P450 3A4 active site: an example of the metabolism of diazepam and its derivatives.

Cytochrome P450 3A4 (CYP3A4) plays a prominent role in the metabolism of a vast array of drugs and xenobiotics and exhibits broad substrate specificities. Most cytochrome P450-mediated reactions follow simple Michaelis-Menten kinetics. These parameters are widely accepted to predict pharmacokinetic and pharmacodynamic consequences in vivo caused by exposure to one or multiple drugs. However, CYP3A4 in many cases exhibits allosteric (sigmoidal) characteristics that make the Michaelis constants difficult to estimate. In the present study, diazepam, temazepam and nordiazepam were employed as substrates of CYP3A4 to propose a kinetic model. The model hypothesized that CYP3A4 contains two substrate-binding sites in a single active site that are both distinct and co-operative, and the resulting velocity equation had a good fit with the sigmoidal kinetic observations. Therefore, four pairs of the kinetic estimates (KS1, kalpha, KS2, kbeta, KS3, kdelta, KS4 and kgamma) were resolved to interpret the features of binding affinity and catalytic ability of CYP3A4. Dissociation constants KS1 and KS2 for two single-substrate-bound enzyme molecules (SE and ES) were 3-50-fold greater than KS3 and KS4 for a two-substrate-bound enzyme (SES), while respective rate constants kdelta and kgamma were 3-218-fold greater than kalpha and kbeta, implying that access and binding of the first molecule to either site in an active pocket of CYP3A4 can enhance the binding affinity and reaction rate of the vacant site for the second substrate. Thus our results provide some new insights into the co-operative binding of two substrates in the inner portions of an allosteric CYP3A4 active site.

Role of cDNA-expressed human cytochromes P450 in the metabolism of diazepam.

The metabolic conversion of diazepam (DZ) to temazepam (TMZ, a C3-hydroxylation product of DZ) and N-desmethyldiazepam (NDZ, an N1-demethylation product of DZ) was studied using cDNA-expressed human cytochrome P450 (CYP) isozymes 1A2, 2B6, 2C8, 2C9, 2C9R144C, 2E1, 3A4, and 3A5 and human liver microsomes from five organ donors. Of the CYPs examined, 3A5, 3A4, and 2B6 exhibited the highest enzymatic activities with turnovers ranging from 7.5 to 12.5 nmol of product formed/min/nmol for the total metabolism of DZ, while 2C8, 2C9, and 2C9R144C showed lesser and moderate activities. 1A2 and 2E1 produced insignificant amounts of metabolites of DZ. The regioselectivity of CYPs was determined, and 2B6 was found to catalyze exclusively and 2C8, 2C9, and 2C9R144C preferentially the N1-demethylation of DZ to form NDZ. 3A4 and 3A5 catalyzed primarily the C3-hydroxylation of DZ, which was more extensive than the N1-demethylation. The ratios of TMZ to NDZ formed in the metabolism of DZ by 3A4 and 3A5 were approximately 4:1. Enzyme kinetic studies indicated that 2B6- and 2C9-catalyzed DZ metabolism followed Michaelis-Menten kinetics, whereas 3A4 and 3A5 displayed atypical and non-linear curves in Lineweaver-Burk plots. Human liver microsomes converted DZ to both TMZ and NDZ at a ratio of 2:1. Our results suggest that hepatic CYP3A, 2C, and 2B6 enzymes have an important role in the metabolism of DZ by human liver.

Reduction of oxazepam to desmethyldiazepam by human intestinal bacteria.

The biotransformation of oxazepam by Bifidobacterium bifidum was studied. The major metabolite was purified by chromatographic methods and found to be desmethyldiazepam using NMR, IR and other physicochemical data.

Absorption of diazepam after its rectal administration in dogs.

A cross-over study was performed in 6 healthy mixed-breed dogs and 4 healthy Beagles. Diazepam was administered per rectum to Beagles (0.5 mg/kg of body weight) and mixed-breed dogs (2 mg/kg), and IV (0.5 mg/kg) to both groups of dogs. Each dog received the drug by both routes, with a 1-week washout period between dosages. After diazepam administration, blood samples were collected to measure plasma concentration of diazepam and its active metabolites, desmethyldiazepam and oxazepam, by use of reverse-phase high-performance liquid chromatography (HPLC). Systemic availability was assessed by comparing the area under the curve for diazepam metabolites for each route of administration. Mean (+/- SD) diazepam concentrations in plasma after rectal administration were low in comparison with those obtained after IV administration, with systemic availability of only 7.4 (+/- 5.9) and 2.7 (+/- 3.2)% for the high and low dose, respectively. However, diazepam was converted to its metabolites within minutes after administration. Accounting for the total concentration of benzodiazepines (diazepam plus desmethyldiazepam and oxazepam) in plasma, systemic availability was 79.9 (+/- 20.7) and 66.0 (+/- 23.8)% for the high and low dosage, respectively. After IV administration, diazepam concentration decreased, with a half-life of only 14 to 16 minutes, but desmethyldiazepam and oxazepam concentrations decreased more slowly, with a half-life of 2.2 to 2.8 hours and 3.5 to 5.1 hours, respectively. Each of the metabolites is reported to have anticonvulsant activity. After rectal administration of the high dose, mean total benzodiazepine concentration was above 1.0 microgram/ml within 10 minutes and was maintained above this concentration for at least 6 hours. We conclude that diazepam is absorbed after rectal administration in dogs, and that the pharmacologic effects are probably caused by the active metabolites, not the parent drug. Samples also were analyzed by use of a nonspecific commercial benzodiazepine fluorescence polarization immunoassay (FPIA). Correlation between the FPIA and HPLC assay was strongest for diazepam (R2 = 0.84), weak for desmethyldiazepam (R2 = 0.09), and nonexistent for oxazepam. We conclude from a comparison of assays that HPLC is preferred over the FPIA method for measuring benzodiazepines in dogs.

Interethnic difference in omeprazole's inhibition of diazepam metabolism.

OBJECTIVES: To compare the effect of omeprazole, a substrate and inhibitor of CYP2C19, on diazepam metabolism in white and Chinese subjects. SUBJECTS AND METHODS: The study, which took place at a clinical research center in a University Hospital, was designed as a double blind, crossover, two-stage study; each stage lasted 21 days and was separated by 4 weeks. Subjects were eight white and seven Chinese men who were extensive metabolizers of debrisoquin and mephenytoin. The subjects received, in a randomized order, omeprazole, 40 mg/day, and placebo for 21 days, followed by a 10 mg oral dose of diazepam. Diazepam and desmethyldiazepam plasma concentrations were determined by HPLC during a 26-day period after diazepam administration. RESULTS: In white subjects omeprazole treatment decreased diazepam clearance by 38% +/- 4.4% and increased desmethyldiazepam area under the plasma concentration-time curve (AUC) by 42.4% +/- 7.0%. In contrast, diazepam oral clearance decreased by only 20.7% +/- 7.3% and desmethyldiazepam AUC decreased by 25.4% +/- 4.6% in the Chinese group. The decrease in diazepam clearance and the prolongation in diazepam and desmethyldiazepam elimination half-lives after administration of omeprazole were significantly greater in the white group than in the Chinese group (p < 0.03, p < 0.001, and p < 0.004, respectively). In the absence of omeprazole, diazepam oral clearance was marginally greater (mean +/- SEM) (34.4 +/- 2.8 ml/min versus 25.2 +/- 3.5 ml/min, p = 0.057, respectively) and the AUC of desmethyldiazepam was significantly lower (8794 +/- 538 micrograms/L.hr versus 16,358 +/- 2985 mg/L.hr, p = 0.04, respectively) in the white subjects compared with the Chinese subjects. CONCLUSION: The extent of the inhibitory effect of omeprazole on diazepam metabolism is dependent on ethnicity. Further studies are needed to determine the mechanism responsible for this phenomenon.

Behavioral and metabolic consequences of neonatal exposure to diazepam in rat pups.

The short-term consequences of a neonatal exposure to diazepam (DZP) on neurobehavioral development and postnatal changes in local cerebral metabolic rates for glucose (LCMRglcs) in selected regions were studied in rats. Rat pups received a daily subcutaneous injection of 10 mg/kg DZP or of the dissolution vehicle from Postnatal Day 2 (P2) to 21 (P21). DZP did not affect the static righting reflex tested at P4 but increased suspension time at P10 and time to complete a 180 degrees pivoting on an inclined plane at P9. In a locomotor coordination test performed at P20, swimming or climbing on a vertical pole was not affected by DZP while the drug impaired the ability of the rat to place its hind-paws on the horizontal platform after climbing. Likewise, DZP induced marked decreases (19-45%) in LCMRglcs in most structures studied at P10, P14, and P21. The results of the present study show that neonatal DZP treatment induces motor deficits that appear to be quite subtle, to concern mainly posture and body balance. They are not apparent in tasks such as swimming or climbing but become obvious in more difficult tasks such as achieving a horizontal quadruped position on a platform after a climbing phase. Decreases in cerebral energy metabolism appear to be mainly located in areas controlling posture and body balance and are partly correlated with the locomotor impairments recorded in the present study.

Diazepam metabolism by human liver microsomes is mediated by both S-mephenytoin hydroxylase and CYP3A isoforms.

1. The primary metabolism of diazepam was studied in human liver microsomes in order to investigate the kinetics and to identify the cytochrome P450 (CYP) isoforms responsible for the formation of the main diazepam metabolites, temazepam and N-desmethyldiazepam. 2. The formation kinetics of both metabolites were atypical and consistent with the occurrence of substrate activation. A sigmoid Vmax model equivalent to the Hill equation was used to fit the data. The degree of sigmoidicity was greater for temazepam formation than for N-desmethyldiazepam formation, so that the ratio of desmethyldiazepam:temazepam formation increased as the substrate (diazepam) concentration decreased. 3. alpha-Naphthoflavone activated both reactions but with a greater effect on temazepam formation than on N-desmethyldiazepam formation. In the presence of 25 microM alpha-naphthoflavone the kinetics for both pathways were approximated by Michaelis-Menten kinetics. 4. Studies with a series of CYP isoform selective inhibitors and with an inhibitory anti-CYP2C antibody indicated that temazepam formation was carried out mainly by CYP3A isoforms, whereas the formation of N-desmethyldiazepam was mediated by both CYP3A isoforms and S-mephenytoin hydroxylase.

Radioiodinated nordiazepam analog for in vivo assessment of benzodiazepine receptors by single photon emission tomography.

2'-Iodo-nordiazepam (2'-IND), a nordiazepam analog iodinated at the 2'-position of the C-5 phenyl ring, was synthesized and evaluated as a potential radiopharmaceutical for investigating brain benzodiazepine receptors by SPECT. [125I]2'-IND was synthesized by the halogen exchange reaction and purified by HPLC. In an in vitro competitive binding study using [3H]diazepam and rat cortical synaptosomol membranes, 2'-IND showed an almost equal affinity for benzodiazepine receptors as diazepam. In a saturation binding study using rat cortical synaptosomal membranes, 2'-IND displayed a Kd of 1.10 nM and a Bmax of 1.87 pmol/mg protein. Biodistribution and metabolism studies in mice showed that [125I]2'-IND exhibited rapid and high accumulation in the brain, and that the cerebral uptake and distribution of this compound occurred in the intact form. Furthermore, the administration of diazepam and flumazenil reduced cortical uptake by approx. 20%, suggesting that the uptake of 2'-IND occurred at least partly in association with benzodiazepine receptors.

Kinetics of sequential metabolism. I. Formation and metabolism of oxazepam from nordiazepam and temazepam in the perfused murine liver.

In murine liver, temazepam (TZ) and nordiazepam (NZ) are mainly metabolized via N-demethylation and C3-hydroxylation, respectively, to form a common metabolite, oxazepam (OZ), which is then glucuronidated. With these precursors, we tested the hypotheses that the sequential metabolism of a primary metabolite (OZ) is less than that of the preformed metabolite and is dependent on the effective intrinsic clearance (unbound fraction x intrinsic clearance) of its precursor, as predicted by the parallel tube and dispersion models of hepatic drug clearances. Mouse livers were perfused with tracer concentrations of [14C]-NZ, [14C]-TZ and [3H]NZ in a single-pass fashion (2.5 ml/min). The steady-state extraction ratio (E) of [3H]NZ, [14C]NZ and [14C]TZ were 0.29, 0.40 and 0.49, respectively (P < .01), whereas the fractional metabolism (formation rate/total elimination rate of drug) of [3H]-NZ, [14C]NZ and [14C]TZ to form OZ was 0.39, 0.79 and 0.68, respectively. Values of E of [3H]NZ and [14C]NZ and fractional metabolism for OZ formation had differed because of a kinetic isotope effect (around 3.5) that affected the C3-hydroxylation of [3H]NZ. The extraction ratios of OZ (E[OZ,P]) arising from [14C]-NZ and [14C]TZ were both 0.056, and were less than that for preformed OZ (E[OZ]), previously found to be 0.125. The parameter E[OZ,P] was poorly correlated with the extraction ratio of the precursor, was overestimated by the parallel tube and dispersion models, but was highly correlated with the effective intrinsic clearance of the precursor (unbound fraction x intrinsic clearance).(ABSTRACT TRUNCATED AT 250 WORDS)

Kinetics of sequential metabolism. II. Formation and metabolism of nordiazepam and oxazepam from diazepam in the perfused murine liver.

Pharmacokinetic theory dictates that the extent of ensuing metabolism of a formed metabolite during drug transit through the liver is influenced by the number of consecutive reactions required for its genesis and the total intrinsic clearances of the precursors. This hypothesis was tested in the perfused murine liver by examining the successive conversion of the precursor diazepam (DZ) to its primary metabolite nordiazepam (NZ), and then the secondary metabolite oxazepam (OZ) and, finally, the tertiary metabolite, the oxazepam glucuronides. The concomitant C3-hydroxylation of DZ to temazepam, which can also be N-demethylated to form OZ, was minimal. The hepatic extraction ratios of NZ (E[NZ,DZ]) and OZ (E[OX,DZ]) after administration of [14C]DZ were compared to those obtained previously from [14C]NZ (E[NZ] and E[OZ,NZ]) and [3H]OZ (E[OZ]). The ability of three hepatic clearance models, the well-stirred, parallel-tube and dispersion models, to predict the experimental E[NZ,DZ] and E[OZ,DZ] was evaluated. DZ was highly extracted by the murine liver (E[DZ] = 0.95). The metabolism of NZ, generated in situ from DZ, was greater than that of preformed NZ (E[NZ,DZ] = 0.51; E[NZ] = 0.4), whereas E[OZ,DZ] (0.066) was similar to E[OZ,DZ] (0.056) and less than E[OZ] (0.0125). The unexpected observation of E[NZ,DZ] > E[NZ] may be explained by the coupling of N-demethylation and C3-hydroxylation/glucuronidation reactions or by a sequestration of hydrophobic substrates within the enzymic space, favoring sequential metabolism of products formed in situ. The atypical kinetic behavior of generated NZ may have also influenced the ensuing metabolic fate of its product, OZ, such that E[OZ,NZ] approximately E[OZ,DZ].(ABSTRACT TRUNCATED AT 250 WORDS)

Inhibitory effect of diltiazem on diazepam metabolism in the mouse hepatic microsomes.

In order to elucidate the drug interaction between diltiazem and diazepam, the effect of diltiazem on the N-demethylation of diazepam in the mouse hepatic microsomes was investigated. Kinetic study showed that diltiazem noncompetitively inhibited the N-demethylation of diazepam with inhibition constant (Ki) value of 247.8 microM, indicating that diltiazem exhibits an inhibitory effect on the hepatic oxidative metabolism of diazepam. It was therefore suggested that diltiazem may impair the metabolism of diazepam in vivo.

Studies of "endogenous" benzodiazepine in human hepatic encephalopathy.

As will be discussed by Dr E.A. Jones based on observations in animal models of hepatic encephalopathy (HE) in 1984 we commenced our studies on the possible role of "endogenous" benzodiazepines (BZs) in human HE in 1986. Unlike animals our initial studies in humans with HE were complicated by the frequent intake of prescription BZs by these patients. Re-education of our staff on the appropriate use of BZs in patients with liver disease and a carefully devised system to exclude patients who have received prescription BZs in the 3 months preceding hospital admission was instigated before our studies could commence. Initially, we examined CSF of patients with HE for the presence of BZ-like activity using a radiometric assay. Our findings of significantly increased activity have since been confirmed by other investigators. Subsequently we discovered fairly large quantities of BZs in blood and urine of these patients, the level of which correlated with the degree of severity of HE. The ultimate finding that this BZ-like activity was due to diazepam, desmethyldiazepam and some other 1-4 benzodiazepine compounds again raised the possibility that our findings were due to occult ingestion of commercially synthesized BZs even though similar findings were made in animal models of HE. Concurrently with this work it became apparent that the food cycle contains trace amounts of the same BZs. However, the levels of "natural BZs" in food cannot yet explain the high levels of BZs seen in patients with HE. The source for this high level of BZs is currently our main area of research.