Introduction

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Ketoconazole is an imidazole-piperazine compound that is effective against a wide range of fungal pathogens (Como and Dismukes, 1994; Kauffman and Carver, 1997). This commonly prescribed antimycotic is a potent inhibitor of cytochrome P450-3A activity, a property that affords the potential for clinically relevant interactions with the many substrates of this enzyme. As expected, in addition to its primary use, ketoconazole significantly decreases the in vivo clearance of cyclosporine (Gomez et al., 1995), terfenadine (Honig et al., 1993), benzodiazepines (Olkkola et al., 1994, von Moltke et al., 1996; Wright et al., 1997) and several other compounds that are biotransformed by the P450-3A isoforms.

Triazolam, a short-acting triazolobenzodiazepine, is metabolized by oxidation to -hydroxytriazolam and 4-hydroxytriazolam in the liver and in the gastrointestinal tract by cytochrome P450-3A (Kronbach et al., 1989). von Moltke et al. (1996) have shown that ketoconazole is a highly potent inhibitor of both hydroxylation reactions in vitro and in vivo. In addition, Varhe et al. (1994) have shown that both ketoconazole and itraconazole increased the AUC of triazolam >20-fold and prolonged the elimination half-life 7-fold.

In a recent study, we found that ketoconazole was a highly potent inhibitor of triazolam biotransformation in vitro compared with other potential inhibitors of P450-3A isoforms (von Moltke et al., 1996). In this same study, the coadministration of ketoconazole and triazolam in human volunteers significantly potentiated triazolam-induced deficits in digit-symbol substitution and word recall test performance and increased electroencephalographic beta activity. However, examination of pharmacokinetic and pharmacodynamic data revealed that the degree of impairment produced by triazolam in the presence of ketoconazole was less than would be predicted based on the plasma levels of triazolam alone. The present study was undertaken to determine whether the blunted effect of triazolam in the presence of ketoconazole could be due to an interaction of ketoconazole at the -aminobutyric acid_A receptor benzodiazepine site. To determine this, we studied in vitro benzodiazepine receptor binding as well as open-field activity in an experimental model.

	Methods

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Materials. Male CD-1 mice, 6 to 8 weeks of age, were purchased from Charles River Laboratories (Wilmington, MA), maintained on a 12-hr light/dark cycle and given food and water ad libitum. [³H]Flunitrazepam (specific acitivity, 71 Ci/mmol) and [³H]flumazenil (specific acitivity, 81 Ci/mmol) were purchased from New England Nuclear (Boston, MA). Triazolam and its metabolites, ketoconazole, paroxetine, buspirone and itraconazole were generously donated by their respective pharmaceutical maufacturers. All other reagents were obtained from standard commercial sources.

Drug administration. Triazolam and ketoconazole were dissolved in PEG 400/saline (1:1) or PEG 400, respectively, and administered intraperitoneally. Vehicle-treated mice received either PEG 400/saline (1:1) or PEG 400 alone depending on the experimental protocol.

Open-field activity. Activity for all groups, including distance traveled, rears and stereotypy, was assessed in 10-min intervals for 50 min in an Omnitech Digiscan apparatus (Columbus, OH). Ketoconazole (50 mg/kg) or vehicle was administered intraperitoneally 60 min before triazolam (0.05, 0.1 or 0.2 mg/kg) or vehicle. Behavioral testing began 10 min after the triazolam injection. Between each run, the interior of the activity chamber was cleaned with 70% ethanol and dried. All testing occurred between 9:00 a.m. and 12:00 p.m. Averages of the log(parameter + 1) for the total 50-min test period were computed. Log transformation of data was used because the data were not normally distributed.

[³H]Flunitrazepam binding. Benzodiazepine binding in vitro was performed in mouse cortical synaptosomes (P₂) as previously described using [³H]flunitrazepam (Miller et al., 1988). Briefly, samples (50 µg of protein) were incubated with [³H]flunitrazepam (0.03-30 µM) in the presence (total binding) or absence (nonspecific binding) of flumazemil (10 µM) for 60 min at 4°C. Incubations were terminated by filtration and filters were subsequently washed and counted.

Triazolam concentrations. Animals were injected intraperitoneally with ketaconazole (50 mg/kg) or vehicle (PEG 400) 60 min before triazolam (0.3 mg/kg). Animals were killed 30 min after the second injection. Cortical and liver tissues were weighed and homogenized in 1 ml pf distilled water with a Polytron (Brinkmann, Lucerne, Switzerland) on setting 7 for 10 to 15 sec. Trunk blood samples were allowed to clot, and the serum was separated. Triazolam concentrations were determined by gas chromatography with electron capture detection as previously described (von Moltke et al., 1996).

Ketoconazole concentrations. Animals received 100 mg/kg ketoconazole 45 min before death (n = 12). Cortical and liver tissues were weighed and homogenized in 1 ml of distilled water with a Polytron (Brinkmann) on setting 7 for 10 to 15 sec. Trunk blood samples were allowed to clot, and the serum was separated. Ketoconazole concentrations were determined by HPLC as previously described (von Moltke et al., 1996).

Triazolam biotransformation in vitro. Microsomes were prepared from livers obtained from male CD-1 mice as described previously (von Moltke et al., 1994, 1993). Varying concentrations of triazolam (0-1500 µM) were incubated with microsomes, cofactors and an NADPH-regenerating system (von Moltke et al., 1996). Reactions were stopped after 20 min, and concentrations of -OH- and 4-OH-triazolam were determined by HPLC. Rates of formation of the two metabolites were expressed in units of nmol of product formed/min/mg of protein. In a second study, a fixed concentration of triazolam (250 µM) was incubated with ketoconazole in concentrations ranging from 0 to 2.5 µM.

Data analysis. Data from binding studies were analyzed using the RADLIG program (version 4.0). Enzyme kinetic parameters were determined by nonlinear regression analysis of untransformed data (von Moltke et al., 1993, 1994, 1996). Comparisons among groups were performed using analysis of variance with Student-Newman-Keuls post-hoc test or Dunnett's post-hoc test.

	Results

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In vitro receptor binding. Flunitrazepam binding to mouse cortical synaptosome labeled receptors was best described by a single site with a K_d value of 2.24 nM and a B_max value of 3.51 pmol/mg protein (fig. 1). Binding of 5 nM [³H]flunitrazepam was displaced by triazolam (0.05-100 nM) in a dose-dependent manner with a mean IC₅₀ value of 0.85 ± 0.08 nM and a mean K_i value of 0.50 ± 0.04 nM. With the addition of ketoconazole (1, 3 or 9 µM), the mean IC₅₀ value of triazolam was increased to 1.11, 1.58 and 5.73 nM, respectively, whereas mean [³H]flunitrazepam maximal specific binding was decreased by 42%, 69% and 89% (fig. 2a). The addition of ketoconazole shifted the K_i value of triazolam slightly to the right. In a separate series of experiments, it was determined that the reduction of [³H]flunitrazepam binding by ketoconazole was most likely due to a competitive interaction of this ligand for the benzodiazepine site labeled by flunitrazepam (fig. 2b). Ketoconazole competitively displaced [³H]flunitrazepam in a concentration-dependent manner. Ketoconazole concentrations ranged from 0.01 to 500 µM with a mean IC₅₀ value of 1.6 ± 0.1 µM and a mean K_i value of 1.17 ± 0.08 µM.

View larger version (16K): [in this window] [in a new window]   Fig. 1.   Representative saturation plot of [3H]flunitrazepam binding to cortical benzodiazepine receptors. Inset, Scatchard plot of the same data set. [3H]flunitrazepam (0.03-30 nM) was added to thoroughly washed cortical synaptosomes and incubated in the presence (total binding) or absence (nonspecific binding) of 10 µM flumazenil for 60 min at 4°C in 50 mM Tris buffer (pH 7.4). Each concentration was assayed in triplicate. Analysis of ligand binding was performed using the RADLIG program (version 4.0). Nonlinear curves fit to each data set were computed to determine both apparent affinity (Kd) and receptor density (Bmax). The average Kd value was 2.24 ± 0.32 nM (n = 5), which was used in later analyses to determine the Ki value for both triazolam and ketoconazole (see fig. 2).

View larger version (14K): [in this window] [in a new window]   Fig. 2.   Representative graph illustrating displacement of 5 nM [3H]flunitrazepam binding. Triazolam (TRZ) and/or ketoconazole (KET) was added to tubes containing cortical synaptosomes and 5 nM [3H]flunitrazepam and incubated for 60 min at 4°C in 50 mM Tris buffer (pH 7.4). Nonspecific binding of flunitrazepam was determined in the presence of 10 µM flumazenil. a, Triazolam, at concentrations of 0.05 to 100 nM, displaced [3H]flunitrazepam in a dose-dependent manner with a mean IC50 value of 0.85 ± 0.08 nM and a mean Ki value of 0.50 ± 0.04 nM. Ketoconazole at 1, 3 and 9 µM inhibited the displacement of [3H]flunitrazepam by triazolam and slightly shifted the Ki value of triazolam to the right. b, Ketoconazole, at concentrations from 0.01 to 500 µM, displaced [3H]flunitrazepam in a dose-dependent manner with a mean IC50 value of 1.6 ± 0.1 µM and a mean Ki value of 1.17 ± 0.08 µM.

Open-field activity. Triazolam significantly decreased (P < .05) all three open-field parameters measured (distance traveled, number of rears and number of stereotypies) in a dose-dependent manner (fig. 3). Ketoconazole alone (50 mg/kg) had no effect on the average distance traveled or the average number of stereotypies but did significantly decrease the average number of rears (P < .05). Coadministration of ketoconazole (50 mg/kg) and triazolam (0.1 mg/kg) significantly decreased all three open-field parameters measured (P < .05). The combination of drugs yielded significantly lower activity than either drug alone (P < .05).

View larger version (20K): [in this window] [in a new window]   Fig. 3.   Effect of triazolam (TRZ) and/or ketoconazole (KET) on open-field activity. Data from (a) distance traveled, (b) number of rears and (c) number of stereotypies was recorded at 10-min intervals for a total of 50 min. Triazolam (0.05, 0.1 or 0.2 mg/kg) or vehicle was administered 60 min after ketoconazole (50 mg/kg) or vehicle administration (n  8). Behavioral testing began 10 min after the second injection. Average values of the log(parameter value + 1) for the test period were computed. Log transformation of data was used because the underlying distributions were not normal. Bars represent mean response ± S.E.M. Significant differences from control are indicated (* P < .05) as determined by analysis of variance and Student-Newman-Keuls post-hoc test.

Triazolam concentrations. Triazolam concentrations in both brain and serum were significantly increased (P < .05) in animals receiving 50 mg/kg ketoconazole (table 2). Ratios of brain to serum concentrations or liver to serum concentrations were unchanged with ketozonazole adminstration.

Ketoconazole concentrations. Average ketoconazole concentrations for serum, liver and brain were 80.9 µg/ml, 137.1 µg/g and 25.4 µg/g, respectively. The mean brain/serum ratio was 0.31, and the mean liver/serum ratio was 1.88.

Triazolam biotransformation in vitro. Both -OH- and 4-OH-triazolam were formed by mouse liver microsomes (fig. 4). Data points consisting of reaction velocity vs. substrate concentrations were best described by a modification of the Michaelis-Menten equation in which a sigmoidal shape is incorporated by inclusion of an exponent (von Moltke et al., 1993, 1994, 1996). The parameters for -OH-triazolam formation were V_max = 5.39 nmol/min/mg protein and S_0.5 = 34 µM. The parameters for 4-OH-triazolam formation were V_max = 14.1 nmol/min/mg protein and S_0.5 = 154 µM. Comparing the V_max/S_0.5 ratios (intrinsic clearance) for the two pathways indicates that 63% of net clearance was accounted for by -OH-triazolam formation and 37% by 4-OH-triazolam formation. However, it should be emphasized that the V_max/S_0.5 ratio represents only an approximate estimate of intrinsic clearance when the kinetic profile is modified by an exponent, as is the case with many substrates of P450-3A (Schmider et al., 1996).

View larger version (18K): [in this window] [in a new window]   Fig. 4.   Triazolam biotransformation in mouse liver microsomes in vitro. Varying concentrations of triazolam (0-1500 µM) were incubated with microsomes, cofactors and an NADPH-regenerating system. Reactions were stopped after 20 min and concentrations of -OH- and 4-OH-triazolam were determined by HPLC. Lines represent functions of best fit consistent with a Michaelis-Menten equation modified by an exponent. Rates of formation of the two metabolites are expressed in units of nmol of product formed/min/mg protein. Insert, enlarged view of metabolite formation in the presence of 0 to 100 µM triazolam. See text for enzyme kinetic parameters.

View larger version (23K): [in this window] [in a new window]   Fig. 5.   Effect of ketoconazole on triazolam metabolite formation in mouse liver microsomes in vitro. A fixed concentration of triazolam (250 µM) was incubated with ketoconazole in concentrations ranging from 0 to 2.5 µM. Reactions were stopped after 20 min, and concentrations of -OH- and 4-OH-triazolam were determined by HPLC. Reaction velocities were expressed as a ratio (in percent) relative to the velocity with no ketoconazole present. Ketoconazole inhibited the formation of both metabolites with IC50 values of 0.039 and 0.038 µM, respectively.

	Discussion

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Previously, we found that ketoconazole competitively inhibited the biotransformation of triazolam in vitro (von Moltke et al., 1996). Enhanced pharmacodynamic effects of triazolam with ketoconazole coadministration in the same study indicate that the pharmacokinetic interaction of these two compounds is likely of clinical importance, as had been reported previously (Varhe et al., 1994). However, the pharmacodynamic enhancement demonstrated by von Moltke et al. (1996) was less than would be predicted based on the increased plasma levels alone. The relationship of plasma triazolam concentration to clinical response was consistent with a reduced sensivity to triazolam with ketoconazole coadministration. The present data demonstrate that ketoconazole inhibits triazaolam displacement of flunitrazepam binding in a concentration-dependent manner in vitro. This modulation of triazolam binding is most likely due to competition of this ligand for the benzodiazepine site. Based on its minimal benzodiazepine agonist activity, however, it is probable that ketoconazole is acting as a neutral ligand or a very weak agonist at the benzodiazepine receptor site. This appears to be a specific property of ketoconazole, since itraconazole, a close structural analog, had only a small effect that was not concentration-dependent. The same concentrations of itraconazole, buspirone, dizocilpine or paroxetine did not result in a similar percent receptor occupancy (>90%) as ketoconazole.

Open-field behavior was evaluated as a pharmacodynamic parameter due to its noninvasive nature, simplicity of measurement and availability of data regarding acute effects of triazolam (Lopez et al., 1988). Horizontal activity, rears and stereotypy have been previously shown to be the most sensitive and reliable measures following benzodiazepine administration. All three parameters were significantly decreased in triazolam-treated animals. Coadministration of ketoconazole and triazolam further decreased all three open-field parameters measured. This potentiated pharmacodynamic effect is similar to that demonstrated in clinical studies by von Moltke et al. (1996).

Pharmacokinetic studies were consistent with pharmacodynamic data. The coadministration of ketoconazole and triazolam significantly increased triazolam concentrations in brain, liver and serum relative to the administration of triazolam alone. This is consistent with the decrease in clearance demonstrated previously in healthy human volunteers (von Moltke et al., 1996). There was no significant difference in the brain/serum and liver/serum ratios for triazolam in the two treatment groups.

In vitro biotransformation by mouse liver microsomes yielded two metabolites, -OH- and 4-OH-triazolam, formed by parallel hydroxylations at two positions on the molecule. This is consistent with our prior study in human liver microsomes (von Moltke et al., 1996). The major role of cytochrome P450-3A isoforms in mediating these two reactions is well established (Kronbach et al., 1989). In addition, biotransformation of a structural analog of triazolam, alprazolam, is similarly mediated by P450-3A isoforms and yields analogous metabolites (von Moltke et al., 1993, 1994).

Ketoconazole was a highly potent inhibitor of triazolam biotransformation by mouse liver microsomes in vitro. This is consistent with prior studies showing that ketoconazole is a potent and relatively selective inhibitor of cytochrome P450-3A isoforms at clinically relevant concentrations (Baldwin et al., 1995; Halpert, 1995; Newton et al., 1995). In addition, von Moltke et al. (1996) showed ketoconazole inhibition of triazolam biotransformation in human liver microsomes. This inhibition of metabolite formation explains the increased triazolam concentrations found after coadministration with ketoconazole.

Kinetic studies of ketoconazole clearly indicated that this compound is present in brain tissue after systemic administration, with a mean brain/serum ratio of 0.31. Although ketoconazole is highly lipophilic, brain uptake is probably restricted, at least in part, by plasma protein binding. Because ketoconazole is extensively bound to plasma components in humans (Como and Dismukes, 1994), it is unlikely that brain/plasma ratios as high as 0.3 would be reached in humans. Nevertheless, the ketoconazole K_i value for benzodiazepine receptor binding averaged 1.2 µM. The usual therapeutic plasma concentration range for ketoconazole is 2 to 10 µM (Como and Dismukes, 1994). Therefore, it is possible that ketoconazole concentrations in human brain may reach sufficient levels to impair receptor binding of benzodiazepine agonists. This may explain why potentiation of benzodiazepine agoinst effects of triazolam with coadmininstration of ketoconazole was less than expected based on the magnitude of increase of triazolam plasma levels (von Moltke et al., 1996).

The ability of a nonbenzodiazepine compound to displace benzodiazepines from their binding sites is not novel. It has long been known that the triazolopyridazines and the -carboline-3-carboxylic acid esters, among others, are recognized by the benzodiazepine receptor (Nielson and Braestrup, 1980; Squires et al., 1979). Ketoconazole is unique, however, in that it appears to be the only compound within its class to exhibit this ability. Additional studies should further characterize the clinical implications of benzodiazepine receptor occupancy by ketoconazole.