Influence of naloxone on the increased sensitivity to propofol during hypovolemia in the rat.

OBJECTIVE: Hypovolemia has been shown to decrease the dose requirement for propofol. This increased effect has been explained partially by an increased end organ sensitivity to the anesthetic effect of propofol. We used the opioid blocking agent naloxone to test the hypothesis that endogenous opioids may be involved in this increased sensitivity. SUBJECTS: Thirty-two chronically instrumented rats were assigned randomly to either the hypovolemia (n = 16) or the control (n = 16) group. INTERVENTIONS: After pretreatment of each rat in the two groups with either intravenous saline (n = 8) or naloxone (3 mg/kg; n = 8), an intravenous infusion of propofol (150 mg x kg(-1) x hr(-1)) was given until 5 secs of electrical suppression of the electroencephalographic signal was observed. Return of righting reflex was used to assess depth of anesthesia, and the propofol blood concentration was determined simultaneously with high-performance liquid chromatography. MEASUREMENTS AND MAIN RESULTS: The mean propofol blood concentrations at the return of righting reflex were significantly lower in the hypovolemic animals compared with the controls within both naloxone-treated (2.1 +/- 0.2 microg/mL vs. 2.9 +/- 0.2 microg/mL; p < .01) and saline-treated (2.2 +/- 0.1 vs. 3.0 +/- 0.2 microg/mL; p < .01) rats. The mean concentrations were not different between the saline- and naloxone-treated rats either within the hypovolemic group or within the control group. CONCLUSIONS: The results of our study indicate that it is unlikely that the increased end organ sensitivity to propofol during hypovolemia is mediated by endogenous opioids, because it was not reversed by naloxone.

Metoprolol-paroxetine interaction in human liver microsomes: stereoselective aspects and prediction of the in vivo interaction.

This study in human liver microsomes was undertaken to establish whether paroxetine stereoselectively inhibits the oxidative metabolism of metoprolol in vitro, and whether the in vivo observed magnitude of the paroxetine-metoprolol interaction was predictable from these in vitro data. Two distinct approaches were used: inhibitory effect of paroxetine on 1) the formation of alpha-hydroxymetoprolol and O-desmethylmetoprolol from the individual metoprolol enantiomers and 2) on the depletion of the enantiomers from the incubation mixture. Nonspecific binding of both metoprolol and paroxetine to human liver microsomes was also investigated. Whereas metoprolol displayed negligible binding, paroxetine was extensively bound to microsomal proteins. This was taken into account in order to obtain unbiased K(i) values and unbound concentrations of paroxetine. In the substrate depletion experiments, the intrinsic clearance (CL(int)) of (R)-metoprolol was larger than that of (S)-metoprolol. Paroxetine caused a concentration-dependent decrease in CL(int) of both enantiomers and abolished the stereoselectivity. In the metabolite formation experiments paroxetine did not stereoselectively affect alpha-hydroxylation, but preferentially inhibited the O-demethylation of the (R)-enantiomer versus the (S)-enantiomer. The use of unbound paroxetine concentrations in the two in vitro methods yielded comparable predicted increases in area under the curve (1.7-1.9 and 2.2-2.5 for (S)- and (R)-metoprolol, respectively) but underestimated the in vivo observed changes of about 7- and 10-fold, respectively. In conclusion, this study showed that paroxetine abolishes the stereoselective metabolism of metoprolol due to a stereoselective inhibition of the O-demethylation toward (R)-metoprolol. Furthermore, the extent of the in vivo metoprolol-paroxetine interaction was substantially underestimated by either one of the two in vitro approaches used when a competitive mechanism was assumed.

Development and validation of a high-performance liquid chromatographic method for the determination of gamma-hydroxybutyric acid in rat plasma.

A method for the determination of gamma-hydroxybutyric acid (GHB) in rat plasma was developed using solid-phase extraction (SPE) and high-performance liquid chromatography (HPLC) with UV detection. GHB was isolated from plasma using strong anion-exchange SPE columns. The chromatographic separation was performed on a C18 Aqua column. The lower limit of quantification was 10 microg/ml using 60 microl of plasma. The linearity of the calibration curves was satisfactory as indicated by correlation coefficients of >0.990. The within-day and between-day precision were <10% (n=24), the accuracy was nearly 101%. Plasma concentrations in rats after GHB infusion determined by HPLC-UV were compared with the corresponding concentrations determined with a validated gas chromatographic-mass spectrometric method by orthogonal distance regression. A good correlation was observed and a t-test indicated no significant differences from 0 and 1 for the intercept and slope, respectively.

Relationship between gamma-hydroxybutyrate plasma concentrations and its electroencephalographic effects in the rat.

In view of the potential interest in an objective parameter for the depth of coma in intoxications with the recreational drug gamma-hydroxybutyrate (GHB), we have studied the relationship between the plasma concentrations and the electroencephalographic (EEG) changes induced by GHB in the rat. Fifteen rats randomly received either 150 (n = 3), 200 (n = 6) or 300 mg kg(-1) (n = 6) GHB over 5 min, followed by a supramaximal dose of 450 mg kg(-1) over 5 min at the end of the experiment. Plasma concentrations were determined with HPLC. The EEG was continuously recorded and the amplitude in the 15.5-30 Hz frequency band was quantified using aperiodic analysis. The plasma concentration-time profiles were fitted to a two-compartment model with Michaelis-Menten elimination. The pharmacokinetic parameters Vmax, Km and the apparent volume of distribution (Vd) proved to be independent of the dose and the mean pooled values were Vmax 2068 +/- 140 microg min(-1) kg(-1), Km 58 +/- 16 microg mL(-1) and Vd 476 +/- 12 mL kg(-1). The EEG amplitude in the 15.5-30 Hz frequency band displayed a monophasic inhibition and the effect-plasma concentration curve showed hysteresis. This hysteresis between EEG effect and plasma concentrations was minimized by simultaneous calculation of hypothetical effect-site concentrations and fitting the effect vs effect-site concentration curve to a sigmoid inhibitory Emax model. The descriptors of this Emax model (Emax, EC50, k(e,0), gamma and E0) were independent of the dose with an equilibration half-life t1/2k(e,0) of 5.6 +/- 0.3 min (mean value of the pooled results of the 5-min treatment groups). To investigate the origin of this hysteresis, a dose of 600 mg kg(-1) GHB was infused over either 45 or 60 min each in three animals. The hysteresis was much less pronounced with 45 min than with 5 min and was absent with 60-min infusions. This indicated that the hysteresis was due to a distribution delay between the central compartment and the effect site. This study showed that the concentration-effect relationship of GHB could be characterized in individual rats using aperiodic analysis in the 15.5-30 Hz frequency band.

Effect of selective serotonin reuptake inhibitors on the oxidative metabolism of propafenone: in vitro studies using human liver microsomes.

Propafenone is mainly metabolized by CYP2D6 to form 5-hydroxypropafenone (5-OHP) and to a minor extent by CYP1A2 and CYP3A4 to form N-depropylpropafenone (N-DPP). The in vitro inhibitory effect of selective serotonin reuptake inhibitors (SSRIs) on the formation of both metabolites was studied, using human liver microsomes. The 5-OHP formation from racemic propafenone and from its individual enantiomers followed one-enzyme Michaelis-Menten kinetics. Incubation with the racemate yielded a mean Vmax of 64 pmol x min(-1) x mg(-1) and a mean Km of 0.12 microM (N = 3). Stereoselectivity in Vmax and Km values was observed, with (S)-propafenone displaying higher Km and Vmax values. N-DPP formation from racemic propafenone followed one-enzyme Michaelis-Menten kinetics and yielded a mean Vmax of 403 pmol x min(-1) x mg(-1) and a mean Km of 116 microM (N = 3). No stereoselectivity in propafenone N-dealkylation was observed. The influence of SSRIs and quinidine, a prototypical CYP2D6 inhbitor, on propafenone 5-hydroxylation was investigated. Quinidine was the most potent inhibitor, followed by fluoxetine, norfluoxetine, and paroxetine. Sertraline, desmethylsertraline, and fluvoxamine had only a moderate inhibitory effect, whereas citalopram displayed slight or no inhibition when racemic propafenone was used as substrate. Mean Ki values of quinidine, fluoxetine, norfluoxetine, and paroxetine were 0.13, 0.33, 0.55, and 0.54 microM, respectively (N = 3). Quinidine and paroxetine were also tested as inhibitors using the individual enantiomers, but no stereoselectivity was observed. Among the SSRIs tested, only fluvoxamine substantially inhbited propafenone N-dealkylation with a mean IC50 of 7.0 microM (N = 3). There was a more pronounced inhibitory effect of fluvoxamine on (R)-propafenone than on (S)-propafenone N-dealkylation. In conclusion, these in vitro data suggest that an in vivo interaction between propafenone and the SSRIs, fluoxetine and paroxetine, can be expected, which can lead to clinically relevant beta-blockade and an increased risk of side effects in the central nervous system. An interaction with fluvoxamine may be of importance in poor metabolizers for CYP2D6.

Paroxetine affects metoprolol pharmacokinetics and pharmacodynamics in healthy volunteers.

OBJECTIVE: To investigate the effect of multiple-dose paroxetine intake on the stereoselective pharmacokinetics and the pharmacodynamics of metoprolol. METHODS: We conducted an open trial with two sessions in eight healthy male volunteers. Racemic metoprolol (100 mg single oral dose) was administered before and after paroxetine treatment (20 mg/day for 6 days). The (R)- and (S)-metoprolol pharmacokinetics, metoprolol metabolic ratio (MR), exercise heart rate and blood pressure were assessed for 12 (pharmacodynamic data) to 24 (pharmacokinetic data) hours after each metoprolol intake. RESULTS: Paroxetine treatment increased the mean area under the plasma concentration-time curve extrapolated to infinity (AUC) of (R)- and (S)-metoprolol significantly (169 to 1,340 ng x h/mL [P < .001] and 279 to 1,418 ng x h/mL [P < .001], respectively), with an approximately twofold increase in both maximum plasma concentration and terminal elimination half-life. Furthermore, the (S)/(R) AUC ratio was significantly decreased, from 1.72 to 1.07 (P < .001). The mean metoprolol MR was significantly increased, from 0.17 to 5.69 (P < .05). The AUC of the metoprolol-induced decrease in exercise heart rate versus time curve was increased, with 46% (P < .01) after multiple-dose paroxetine intake, reaching significance from 6 hours after metoprolol intake, illustrating a more sustained beta-blockade. Similar results were obtained for the effect on exercise systolic blood pressure. Multiple-dose metoprolol administration combined with paroxetine can lead to an accumulation of the beta-blocking (S)-enantiomer of metoprolol, possibly resulting in unacceptable bradycardia, loss of cardioselectivity, or both. CONCLUSION: Multiple-dose paroxetine intake affects both metoprolol pharmacokinetics and pharmacodynamics and suggests that when paroxetine is added to an ongoing metoprolol therapy, caution is warranted and a reduction of the metoprolol dose may be required to prevent undesired adverse effects.

Influence of hypovolemia on the pharmacokinetics and the electroencephalographic effect of propofol in the rat.

BACKGROUND: Hypovolemia decreases the dose requirement for anesthetics, but no data are available for propofol. As it is impossible to study this in patients, a rat model was used in which the influence of hypovolemia on the pharmacokinetics and pharmacodynamics of propofol was investigated. METHODS: Animals were randomly allocated to either a control (n = 9) or a hypovolemia (n = 9) group, and propofol was infused (150 mg x kg(-1) x h(-1)) until isoelectric periods of 5 s or longer were observed in the electroencephalogram. The changes observed in the electroencephalogram were quantified using aperiodic analysis and used as a surrogate measure of hypnosis. The righting reflex served as a clinical measure of hypnosis. RESULTS: The propofol dose needed to reach the electroencephalographic end point in the hypovolemic rats was reduced by 60% (P < 0.01). This could be attributed to a decrease in propofol clearance and in distribution volume. Protein binding was similar in both groups. To investigate changes in end organ sensitivity during hypovolemia, the electroencephalographic effect versus effect-site concentration relation was studied. The effect-blood concentration relation was biphasic, exhibiting profound hysteresis in both hypovolemic and control animals. Semiparametric minimization of this hysteresis revealed similar equilibration half-lives in both groups. The biphasic effect-concentration relation was characterized by descriptors showing an increased potency of propofol during hemorrhage. The effect-site concentration at the return of righting reflex was 23% (P < 0.01) lower in the hypovolemic animals, also suggesting an increased end organ sensitivity. CONCLUSIONS: An increased hypnotic effect of propofol occurs during hypovolemia in the rat and can be attributed to changes in both pharmacokinetics and end organ sensitivity.

Is vitreous humour useful for the interpretation of 3,4-methylenedioxymethamphetamine (MDMA) blood levels? Experimental approach with rabbits.

As drug instability and redistribution are factors known to affect the interpretation of post-mortem blood levels, we questioned whether post-mortem vitreous humour concentrations could be useful as predictors for the MDMA load at the time of death. In a first series of in vivo experiments using rabbits, 3,4-methylenedioxymethamphetamine (MDMA) concentrations in plasma, blood and vitreous humour were studied as a function of time after intravenous (i.v.) administration of MDMA. Equilibration between the vascular compartment and vitreous humour was attained about 1 h after i.v. MDMA administration. In a second series of experiments, the post-mortem stability of MDMA in vitreous humour in relation to ambient temperature was investigated. Post-mortem MDMA concentrations in vitreous humour were closer to the ante-mortem blood levels when compared to cardiac blood samples. These preliminary investigations in the rabbit model indicate that measurements of vitreous humour concentrations could also be of interest for predicting the blood concentration at the time of death in humans.

In vitro study on the involvement of CYP1A2, CYP2D6 and CYP3A4 in the metabolism of haloperidol and reduced haloperidol.

OBJECTIVE: To investigate in vitro which CYP isoforms (CYP1A2, CYP2D6 and CYP3A4) are involved in the biotransformation of haloperidol (HAL) and reduced haloperidol (RHAL). METHODS: The biotransformation of HAL and RHAL is evaluated by measuring HAL and RHAL remaining after incubation with human liver microsomes and with supersomes from human baculovirus-infected cells expressing human P(450) isoforms. The influence of chemical- and immuno-inhibition of specific isoforms on the disappearance of HAL and RHAL was also studied. RESULTS: After 60-min incubation of 2 microM and 20 microM HAL or RHAL with human liver microsomes, for HAL, 58% and 64%, respectively, remained in the incubation mixture, for RHAL, 53% and 66%, respectively. Ketoconazole had the most pronounced inhibitory effect on the biotransformation of both substrates, while for quinidine and furafylline there was only a weak or no influence. Anti-CYP3A4 antibodies inhibited strongly the biotransformation of HAL and RHAL, while the influence of anti-CYP2D6 antibodies was much less pronounced. After incubation with supersomes of recombinant CYP3A4, HAL and RHAL disappeared rapidly; disappearance was slow after incubation with CYP2D6 supersomes, and negligible with CYP1A2 supersomes. CONCLUSION: The results show that CYP3A4 is the most important CYP isoenzyme involved in the biotransformation of HAL and RHAL, and that the metabolism by CYP2D6 is only a minor pathway; CYP1A2 has no or only a negligible influence.

Effects of smoking, CYP2D6 genotype, and concomitant drug intake on the steady state plasma concentrations of haloperidol and reduced haloperidol in schizophrenic inpatients.

The effects of smoking, CYP2D6 genotype, and concomitant use of enzyme inducers or inhibitors on the steady state plasma concentrations of haloperidol (HAL) and reduced haloperidol (RHAL) were evaluated in 92 schizophrenic inpatients. All but three of these patients received concomitant medication, in many cases with drugs potentially interacting with HAL. Of the 92 patients, 63 were treated orally with HAL in a daily dose of 0.4 to 50 mg; 29 patients were treated intramuscularly with a daily equivalent dose of HAL decanoate (expressed as HAL) of 1.8 to 17.9 mg. A wide interindividual variation in HAL dose and in steady state plasma concentrations of HAL and RHAL was observed. In the patients treated orally, the daily oral dose was about 4 times higher and the dose-normalized HAL (but not RHAL) plasma concentrations were significantly lower in smokers (n = 40) than in nonsmokers (n = 23) (p < 0.01). The dose-normalized RHAL (but not HAL) plasma concentrations and the RHAL/HAL ratio were significantly higher in poor metabolizers (PMs) than in extensive metabolizers (EMs). There was a trend toward an effect of potentially interacting drugs (inducers or inhibitors) on dose, dose-normalized HAL and RHAL plasma concentrations, and the RHAL/HAL ratio. In the patients treated intramuscularly, the dose-normalized HAL (but not RHAL) plasma concentrations were significantly lower in smokers than in nonsmokers, but no differences in doses were observed. This naturalistic study of modest sample size in a polymedicated population shows an effect of smoking and CYP2D6 genotype (and to a lesser extent, of interacting drugs) on the kinetics of HAL.

Influence of hypovolemia on the pharmacokinetics and the electroencephalographic effect of etomidate in the rat.

The influence of hypovolemia (removal of 30% of the blood volume) on the pharmacokinetics and pharmacodynamics of etomidate was investigated in the rat. Chronically instrumented animals were randomly allocated to either a control (n = 9) or a hypovolemia (n = 9) group, and etomidate was infused (50 mg/kg/h) until isoelectric periods of 5 s or longer were observed in the electroencephalogram. The changes observed in the electroencephalogram were quantified using aperiodic analysis in the 2.5- to 7.5-Hz frequency band and used as a surrogate measure of hypnosis. The righting reflex was used as a clinical measure of hypnosis. The etomidate dose that had to be infused to reach the electroencephalographic endpoint was almost 40% lower (p <.01) in the hypovolemic animals than in the control animals. This difference could be attributed to a decrease in clearance (-20%; p =.06) and distribution volume (-30%; p <.01) of etomidate. Protein binding was similar in both groups. To investigate changes in end organ sensitivity during hypovolemia, the electroencephalographic effect-versus-effect-site concentration relationship was studied. The effect-plasma concentration relationship was biphasic, exhibiting profound hysteresis in both hypovolemic and control animals. Semiparametric minimization of this hysteresis revealed similar equilibrium half-lives in both groups, and the biphasic effect-concentration relationship was characterized nonparametrically by descriptors. With these descriptors, a slightly increased potency of etomidate during hemorrhage was observed. The concentration at the return of righting reflex was 16% (p <.05) lower in the hypovolemic animals. In conclusion, an increased hypnotic effect of etomidate was observed during hypovolemia that is mainly attributed to pharmacokinetic changes. Our data also suggest a small increase in central nervous system sensitivity for etomidate in hypovolemic animals.

Relationship between etomidate plasma concentration and EEG effect in the rat.

PURPOSE: The effect-plasma concentration relationship of etomidate was studied in the rat using electroencephalographic changes as a pharmacodynamic parameter. METHODS: Etomidate was infused (50 mg/kg/h) in chronically instrumented rats (n=6) until isoelectric periods of 5 s or longer were observed in the electroencephalogram (EEG). The EEG was continuously recorded during the experiment and frequent arterial blood samples were taken for determination of etomidate plasma concentrations. The changes observed in the raw EEG signal were quantified using aperiodic analysis in the 2.5-7.5 Hz frequency band. The return of the righting reflex was used as another parameter of anesthesia. RESULTS: A mean dose of 8.58+/-0.41 mg/kg needed to be infused to reach the end point of 5 s isoelectric EEG. The plasma concentration time profiles were most adequately fitted using a three-exponential model. Systemic clearance, volume of distribution at steady-state and elimination half-life averaged 93+/-6 ml/min/kg, 4.03+/-0.24 l/kg and 59.4+/-10.7 min respectively. The EEG effect-plasma concentration relationship was biphasic exhibiting profound hysteresis. Semi-parametric minimization of this hysteresis revealed an equilibration half-life of 2.65+/-0.15 min, and the biphasic effect-concentration relationship was characterized nonparametrically by descriptors. The effect-site concentration at the return of the righting reflex was 0.44+/-0.03 microg/ml. CONCLUSIONS: The results of the present study show that the concentration-effect relationship of etomidate can be characterized in individual rats using aperiodic analysis in the 2.5-7.5 Hz frequency band of the EEG. This characterization can be very useful for studying the influence of diseases on the pharmacodynamics of etomidate in vivo.

Inhibition of CYP2C9 by selective serotonin reuptake inhibitors: in vitro studies with tolbutamide and (S)-warfarin using human liver microsomes.

OBJECTIVE: To investigate the in vitro potential of selective serotonin reuptake inhibitors (SSRIs) to inhibit two CYP2C9-catalysed reactions, tolbutamide 4-methylhydroxylation and (S)-warfarin 7-hydroxylation. METHODS: The formation of 4-hydroxytolbutamide from tolbutamide and that of 7-hydroxywarfarin from (S)-warfarin as a function of different concentrations of SSRIs and some of their metabolites was studied in microsomes from three human livers. RESULTS: Both tolbutamide 4-methylhydroxylation and (S)-warfarin 7-hydroxylation followed one enzyme Michaelis-Menten kinetics. Kinetic analysis of 4-hydroxytolbutamide formation yielded a mean apparent Michaelis-Menten constant (Km) of 133 microM and a mean apparent maximal velocity (Vmax) of 248 pmol x min(-1) x mg(-1); formation of 7-hydroxywarfarin yielded a mean Km of 3.7 microM and a mean Vmax of 10.5 pmol x min(-1) x mg(-1). Amongst the SSRIs and some of their metabolites tested, only fluvoxamine markedly inhibited both reactions. The average computed inhibition constant (Ki) values and ranges of fluvoxamine when tolbutamide and (S)-warfarin were used as substrate, were 13.3 (6.4-17.3) microM and 13.0 (8.4-18.7) microM, respectively. The average Ki value of fluoxetine for (S)-warfarin 7-hydroxylation was 87.0 (57.0-125) microM. CONCLUSION: Amongst the SSRIs tested, fluvoxamine was shown to be the most potent inhibitor of both tolbutamide 4-methylhydroxylation and (S)-warfarin 7-hydroxylation. Fluoxetine, norfluoxetine, paroxetine, sertraline, desmethylsertraline, citalopram, desmethylcitalopram had little or no effect on CYP2C9 activity in vitro. This is consistent with in vivo data indicating that amongst the SSRIs, fluvoxamine has the greatest potential for inhibiting CYP2C9-mediated drug metabolism.

The influence of hemorrhagic shock on the pharmacokinetics and the analgesic effect of morphine in the rat.

The influence of hemorrhagic shock (removal of 30% of the blood volume) on the pharmacokinetics and the analgesic effect of morphine was investigated in conscious rats. Plasma concentrations of morphine after a bolus injection (5 mg/kg) are higher in the shock animals, which is attributed to a small decrease in clearance (-22%; P > 0.05) and a significant decrease in distribution volume (-33%; P < 0.05) of the drug. The areas under the plasma concentration-time curve of the metabolite morphine-3-glucuronide (M3G) are significantly higher (+237%; P < 0.01) in the shock rats, which is probably explained by a decreased distribution and renal excretion. The analgesic effect of morphine was evaluated using the tail-flick test during a continuous infusion (10 mg/kg/h) with measurement of the plasma concentrations of morphine and M3G. Data from these experiments show higher plasma concentrations of morphine (+33%; P < 0.05) and M3G (+66%; P > 0.05) during shock, and a significantly increased analgesic effect (+43%; P < 0.05). Our data suggest that the increased analgesic effect of morphine during hemorrhagic shock can most likely be explained by pharmacokinetic changes resulting in higher morphine concentrations.

In-vitro characterization of the cytochrome P450 isoenzymes involved in the back oxidation and N-dealkylation of reduced haloperidol.

In-vitro studies were performed using human liver microsomes and c-DNA-expressed human P450 isoforms to identify the cytochrome P450 isoenzyme(s) involved in the back oxidation and N-dealkylation of reduced haloperidol. Back oxidation and N-dealkylation of reduced haloperidol were assessed by measuring the formation of haloperidol and 4-(4-chlorophenyl)-4-hydroxypiperidine (CPHP), respectively. The haloperidol and CPHP formation rates as a function of substrate concentration, measured in three livers, followed monophasic enzyme kinetics. For haloperidol formation Km values ranged from 51-59 microM, and Vmax values from 190-334 pmol mg(-1) min(-1); for CPHP formation Km values were 44-49 microM, and Vmax values 74-110 pmol mg(-1) min(-1). Haloperidol and CPHP formation rates in the nine liver preparations were significantly correlated with dextromethorphan N-demethylase activity (a marker of CYP3A4 activity), but not with the CYP2D6, CYP1A2 and CYP2C9 activity. Ketoconazole and troleandomycin, inhibitors of CYP3A4, inhibited competitively both haloperidol and CPHP formation, with a Ki value lower than 0.2 microM for ketoconazole and lower than 0.3 microM for troleandomycin. Sulphaphenazole (CYP2C9), furafylline (CYP1A2) and quinidine and paroxetine (CYP2D6) gave only little inhibition (IC50 > 60 microM). CPHP and haloperidol formation were, moreover, enhanced by alpha-naphthoflavone, an effect known for CYP3A4 mediated reactions. Anti-CYP3A4 antibodies strongly inhibited haloperidol and CPHP formation, whereas CYP2D6 antibodies did not. Among the recombinant human CYP isoforms tested, CYP3A4 exhibited the highest activity with respect to haloperidol and CPHP formation rates, with no detectable effect of CYP1A2, CYP2D6 and CYP2C9. These results strongly suggest that back oxidation and N-dealkylation of reduced haloperidol in human liver microsomal preparations are mediated by CYP3A4.

The oxidative metabolism of metoprolol in human liver microsomes: inhibition by the selective serotonin reuptake inhibitors.

OBJECTIVE: Biotransformation of metoprolol to alpha-hydroxymetoprolol (HM) and O-demethylmetoprolol (ODM) is mediated by CYP2D6. The selective serotonin reuptake inhibitors (SSRIs) are known to inhibit CYP2D6. The aim was to study in vitro the potential inhibitory effect of SSRIs on metoprolol biotransformation. METHODS: Using microsomes from two human livers, biotransformation of metoprolol to alpha-hydroxymetoprolol (HM) and O-demethylmetoprolol (ODM) as a function of the concentrations of the SSRIs and of some of their metabolites was studied. RESULTS: The kinetics of the formation of both metabolites are best described by a biphasic enzyme model. The estimated values of Vmax and kM for the high affinity site are for the alpha-hydroxylation in human liver HL-1 32 pmol mg(-1) min(-1) and 75 micromol x l(-1) respectively, and in human liver HL-9 39 pmol mg(-1) x min(-1) and 70 micromol x l(-1) respectively; for the O-demethylation in HL-1 131 pmol mg(-1) min(-1) and 95 micromol x l(-1) respectively, and in HL-9 145 pmol mg(-1) min(-1) and 94 micromol x l(-1) respectively. Quinidine is for both pathways a potent inhibitor of the high-affinity site, with K(i) values ranging from 0.03 to 0.18 micromol x l(-1). Fluoxetine, norfluoxetine and paroxetine are likewise potent inhibitors, with Ki values ranging from 0.30 to 2.1 micromol x l(-1) fluvoxamine, sertraline, desmethylsertraline, citalopram and desmethylcitalopram are less potent inhibitors, with K(i) values above 10 micromol x l(-1). CONCLUSION: The rank order of the SSRIs for inhibition of metoprolol metabolism is comparable to that reported in the literature for other CYP2D6 substrates, with fluoxetine, norfluoxetine and paroxetine being the most potent. These findings need further investigation to determine their clinical relevance.

Comparison of two high-performance liquid chromatographic methods for monitoring plasma concentrations of haloperidol and reduced haloperidol.

Two high-performance liquid chromatographic methods for monitoring haloperidol (HAL) and reduced haloperidol (RHAL) plasma concentrations were compared. In one method ultraviolet detection and a C18 column were used (UV method); in the other method electrochemical detection and a CN-column were used (EC method). Both methods are accurate and precise. For plasma samples spiked with HAL or RHAL, an excellent correlation was observed between the concentrations of HAL and RHAL found with both methods (r < or = 0.99, p < 0.01). However, for plasma obtained from patients treated with HAL the correlation between the two methods was poor (r > or = 0.71, p < 0.01). The main reason for the discrepancy between the two methods is probably interference of comedications or their metabolites, mostly in the EC method. Although the quantitation limit of the UV method (2 ng/ml for HAL and RHAL) is higher than that of the EC method (0.5 ng/ml for HAL and RHAL), the UV method is to be preferred for monitoring plasma levels in psychiatric patients because there is less interference from comedication.

Diagnostic performance of the EMIT-tox benzodiazepine immunoassay, FPIA serum benzodiazepine immunoassay, and radioreceptor assay in suspected acute poisoning.

We evaluated the diagnostic performance of the EMIT-tox serum benzodiazepine assay adapted to a Hitachi 717 analyzer (EMIT), the Abbott ADx serum benzodiazepine fluorescence polarization immunoassay (FPIA), and a radioreceptor assay (RRA) in 113 patients with suspected acute poisoning. The reference method was high-performance liquid chromatography with ultraviolet detection after solid-phase extraction. For the discrimination between negative and positive samples, the areas under the receiver-operating characteristic (ROC) curves were 0.976, 0.991, and 0.991 for EMIT (cutoff, 50-ng/mL diazepam), FPIA (cutoff, 12-ng/mL nordiazepam), and RRA (cutoff, 50-ng/mL diazepam), respectively. For the discrimination between non-toxic and toxic concentrations, the areas under the ROC curves were 0.896, 0.893, and 0.933, respectively. EMIT (with the cutoff lowered to 50 ng/mL), FPIA, and RRA can be reliably used to screen for the presence of benzodiazepines in serum, but in many cases they cannot discriminate between toxic and nontoxic concentrations.

Characterization of the cytochrome P450 isoenzymes involved in the in vitro N-dealkylation of haloperidol.

AIMS: The present study was carried out to identify the cytochrome P450 isoenzyme(s) involved in the N-dealkylation of haloperidol (HAL). METHODS: In vitro studies were performed using human liver microsomes and c-DNA-expressed human P450 isoforms. N-dealkylation of HAL was assessed by measuring the formation of 4-(4-chlorophenyl)-4-hydroxypiperidine (CPHP). RESULTS: There was a tenfold variation in the extent of CPHP formation amongst the nine human liver microsomal preparations. The CPHP formation rates as a function of substrate concentration, measured in three livers, followed monophasic enzyme kinetics. Km and Vmax values ranged respectively from 50 to 78 microM and from 180 to 412 pmol mg-1 min-1 CPHP formation rates in the nine liver preparations were significantly correlated with dextromethorphan N-demethylase activity (a marker of CYP3A4 activity), but not with the activity of dextromethorphan O-demethylase (CYP2D6), phenacetin O-deethylase (CYP1A2) or tolbutamide hydroxylase (CYP2C9). Ketoconazole, an inhibitor of CYP3A4, inhibited competitively CPHP formation (Ki=0.1 microM), whereas sulphaphenazole (CYP2C9), furafylline (CYP1A2) and quinidine (CYP2D6) gave only little inhibition (IC50 > 100 microM). CPHP formation was, moreover, enhanced by apha-naphtoflavone, an effect common to CYP3A4 mediated reactions. Anti-CYP3A4 antibodies strongly inhibited CPHP formation, whereas no inhibition was observed in the presence of CYP2D6 antibodies. Among the recombinant human CYP isoforms tested, CYP3A4 exhibited the highest activity with respect to CPHP formation rate, with no detectable effect of other CYP isoforms (CYP1A2, CYP2D6 and CYP2C9). HAL inhibited dextromethorphan O-demethylase (CYP2D6) with IC50 values between 2.7 and 8.5 microM, but not (IC50 > 100 microM) dextromethorphan N-demethylase (CYP3A4), phenacetin O-deethylase (CYP1A2) or tolbutamide hydroxylase (CYP2C9). CONCLUSIONS: These results strongly suggest that the N-dealkylation of HAL in human liver microsomal preparations is mediated by CYP3A4.

High performance liquid chromatography determination of dextromethorphan and its metabolites in urine using solid-phase extraction.

A high performance liquid chromatography assay coupled with fluorescence detection was developed for the determination of dextromethorphan and its metabolites in urine. The products and the internal standard, pholcodine, were separated on an Alltima C18, 5 microns column (250 x 4.6 mm), using a mobile phase containing sodium dodecyl sulphate (1 mM) in a mixture of acetonitrile-sodium dihydrogen phosphate (0.01 M) 40.5:59.5, v/v) (pH* = 2.5). A novel solid-phase extraction procedure with strong cation exchange, non end-capped, Isolute SCX cartridges allows good recovery of the products (mean 85% or more). For all analytes, the assay is sensitive (LOQ 25 ng ml-1, using 200 microliters urine), reproducible (RSD < 15%) and accurate (< 15% deviation of the nominal value) over the range evaluated. This method can be used to measure dextromethorphan and its metabolites to phenotype individuals as poor or extensive metabolizers of drugs metabolized by CYP2D6.