Identification of human cytochrome P450 isoforms involved in the metabolism of brotizolam

Article abstract of DOI:10.1080/004982597240082

1. To identify the cytochrome P450 (CYP) isoenzyme(s) responsible for the major metabolic pathways of brotizolam in man, we examined the metabolism of brotizolam using human liver microsomes and microsomes expressing individual human CYP isoenzymes (CYP1A1, 1A2, 2A6, 2B6, 2C9, 2C19, 2D6, 2E1, 3A4). 2. Brotizolem was metabolized to α-OH- and 6-OH-brotizolam by human liver microsomes (n = 3). V(max) for α- and 6-hydroxylation of brotizolam were 1470 ± 259 and 8983 ± 7740 pmol/min/mg protein respectively, and the corresponding K(m) were 49 ± 9.3 and 595 ± 580 μM respectively. 3. Among CYP inhibitors examined (furafylline, sulphaphenazole, quinidine, ketoconazole and cimetidine), ketoconazole showed the most potent inhibitory effect on brotizolam metabolism by human liver microsomes. K₁ of ketoconazole for α- and 6-hydroxylation were 0.05 and 0.07 μM respectively. 4. When incubated with microsomes expressing individual human CYP isoenzymes (CYP1A1, 1A2, 2A6, 2B6, 2C9, 2C19, 2D6, 2E1, 3A4), brotizolam was metabolized only by CYP3A4. 5. Brotizolam metabolism in human liver microsomes was almost completely inhibited by anti-CYP3A4 antiserum. 6. These results suggest that CYP3A4 is predominantly responsible for both α- and 6-hydroxylation of brotizolam in human liver microsomes.

Full text of DOI:10.1080/004982597240082

xenobiotica , 1997, vol. 27, no. 9, 913±922
Identi®cation of hum an cytochrome P450 isoforms
involved in the metabolism of brotizolam
C. SENDA, W. KISHIMOTO, K. SAKAI, A. NAGAKURA
and T. IGARASHI*
Department of Biochemistry, Kawanishi Pharma Research Institute, Nippon
Boehringer Ingelheim Co., Kawanishi, Hyogo, Japan
Received 3 March 1997
1. To identify the cytochrome P450 (CYP) isoenzyme(s) responsible for the major
metabolic pathways of brotizolam in man, we examined the metabolism of brotizolam using
human liver microsomes and microsomes expressing individual human CYP isoenzymes
(CYP1A1, 1A2, 2A6, 2B6, 2C9, 2C19, 2D6, 2E1, 3A4).
a
2. Brotizolam was metabolized to -OH- and 6-OH-brotizolam by human liver
a
for - and 6-hydroxylation of brotizolam were 1470³259 and
microsomes (n ¯ 3). V
max
8983³7740 pmol min mg protein respectively, and the corresponding K were 49³9 3
±
}
}
m
lm
and 595³580
respectively.
3. Among CYP inhibitors examined (furafylline, sulphaphenazole, quinidine, ketocon-
azole and cimetidine), ketoconazole showed the most potent inhibitory eåect on brotizolam
a
metabolism by human liver microsomes. K of ketoconazole for - and 6-hydroxylation
i
±
were 0 05 and 0 07
±
lm
respectively.
4. When incubated with microsomes expressing individual human CYP isoenzymes
(CYP1A1, 1A2, 2A6, 2B6, 2C9, 2C19, 2D6, 2E1, 3A4), brotizolam was metabolized only
by CYP3A4.
5. Brotizolam metabolism in human liver microsomes was almost completely inhibited
by anti-CYP3A4 antiserum.
a
6. These results suggest that CYP3A4 is predominantly responsible for both - and
6-hydroxylation of brotizolam in human liver microsomes.
Introduction
Brotizolam
(2-bromo-4-(o-chlorophenyl)-9-methyl-6H-thieno[3,2f ]-s-tri-
azolo[4,3-a] [1,4] diazepine, BRT ; ®gure 1) is a triazolothienodiazepine widely
used as a hypnotic agent (Sanchez-Martinez and Landa-Palos 1982). The drug is
!
absorbed well after oral administration, but
1 % of dosed BRT is excreted in the
urine as the unchanged form (Bechtel et al. 1986a) ; and the remainder is excreted as
metabolites hydroxylated at various sites in the molecule with subsequent conjugate
formation (Bechtel et al. 1986b). Bechtel et al. (1986b) identi®ed the two main
metabolites in man, WE964 and WE1061, as 2-bromo-4-(2-chlorophenyl)-9-
hydroxymethyl-6H-thieno-[3,2-f ]-1,2,4-triazolo-[4,3-a]-1,4-diazepine
(a-OH-
BRT ; ®gure 1) and 2-bromo-4-(2-chlorophenyl)-6-hydroxy-9-methyl-6H-thieno-
[3,2-f ]-1,2,4-triazolo-[4,3-a]-1,4-diazepine (6-OH-BRT ; ®gure 1) respectively.
They suggested that the route of systemic elimination of BRT in man is
predominantly via these two hydroxylated metabolites.
It is well known that cytochrome P450 (CYP) plays an important role in the
metabolic disposition of a variety of drugs and foreign compounds, and that it
consists of multiple isoforms with diåerent but broad and often overlapping
substrate speci®cities (Horai and Ishizaki 1988, Gonzalez 1992, Guengerich 1994).
* Author for correspondence.
±
’
0049±8254}97 $12 00
1997 Taylor & Francis Ltd

914
C. Senda et al.
a
Figure 1. Metabolism of BRT to its two principal metabolites, -OH-BRT and 6-OH-BRT.
In the case of benzodiazepines used as hypnotic agents (i.e. triazolam and
midazolam), CYP3A4 plays an important role in their metabolism in vitro
(Kronbach et al. 1989). Furthermore, an in vivo study suggested that alprazolam,
another benzodiazepine, is also metabolized by CYP3A4 (Yasui et al. 1996).
Although BRT structurally resembles benzodiazepines, it has a thieno ring in place
of the fused benzene ring of benzodiazepines such as triazolam, midazolam and
alprazolam. Therefore, the possibility exists that the CYP isoenzyme mainly
responsible for BRT metabolism is diåerent from that in the case of benzo-
diazepines. The aim of this study was to identify the CYP isoenzyme(s) responsible
for the metabolism of BRT.
M aterials and m ethods
Chemicals and reagents
a
BRT and -OH-BRT were synthesized by Boehringer Ingelheim Co., Germany, and 6-OH-BRT
was synthesized by the Analytical Department, Nippon Boehringer Ingelheim. These three chemicals
"
b
98 %) available. -Nicotinamide adenine dinucleotide phosphate
were of the highest purity
(
d
[tetrasodium salt] (NADPH), -glucose 6-phosphate [monosodium salt] (G6P), glucose 6-phosphate
dehydrogenase [type VII from yeast] (G6PDH), sulphaphenazole, cimetidine and diazepam were
purchased from Sigma Chemical Co. (St Louis, MO, USA). Ketoconazole, quinidine, and furafylline
were from Funakoshi (Tokyo, Japan), Wako Pure Chemical Industries (Osaka, Japan), and Daiichi Pure
Chemicals Co. (Tokyo, Japan) respectively. Human anti-CYP3A4 antiserum, which is a commercially
available rabbit antiserum raised against rat liver CYP3A2, was from Daiichi Pure Chemicals Co.

Brotizolam metabolism by human CYP3A4
915
Figure 2. Chromatographic separation of BRT metabolites formed by human liver microsomes. Units
l
of the y-axes are V, UV detector.
(Tokyo, Japan). This polyclonal antibody was used for studying the inhibition of the human CYP3A4
enzyme activity because of no inhibitory eåect against other human CYPs (i.e. CYP1A1, 1A2, 2A6, 2B6,
2C9, 2D6 and 2E1: data on lea¯et). Microsomes expressing human CYP1A1, 1A2, 2A6, 2B6, 2C9, 2C19,
2D6, 2E1 and 3A4, which were prepared from the human B lymphoblastoid cell line (GENTEST Co.,
USA), were all purchased from Daiichi. Human liver microsomes were obtained from Iwai Chemical
Co. (pooled Batch 2 : Osaka, Japan) or K.A.C. (HHM0163 and HHM0215: Kyoto, Japan). All other
chemicals and reagents were of analytical grade.
Kinetic analysis of BRT metabolism in human liver microsomes
For kinetic analysis of the metabolism of BRT in human liver microsomes, BRT (®nal concentrations
±
±
lm
NADPH-generating system (3 m G6P, 1 unit ml G6PDH, 1 2 m NADPH and 3 3 m MgCl ). The
m
phosphate buåer (pH 7 4) and an
10, 25, 50, 100 or 250
) was preincubated for 5 min with 0 1
±
±
m
m
m
}
#
reactions were initiated by addition of human liver microsomes (1 mg microsomal protein ml) in a ®nal
°
volume of 0 5 ml, and the mixture was incubated for 10 min at 37 C. An aliquot of the reaction mixture
(200 l) was mixed with 5 l 70 % perchloric acid (PCA) and placed on ice to terminate the reaction.
}
±
l
l
Eåect of speci®c CYP inhibitors on BRT metabolism in human liver microsomes
The eåects of various speci®c CYP isoenzyme inhibitors, furafylline (2 or 10
lm
), sulphaphenazole
±
(0 5 or 3
±
), ketoconazole (0 25 or 0 5
±
lm
lm
lm
lm
), and cimetidine (10 or 50 ), were
), quinidine (2 or 20
examined at the concentrations recommended in the literature (Newton et al. 1995, Wrighton and Ring
lm
1994). Incubation mixtures containing BRT (25, 50, 100 or 250 ), an inhibitor, and an NADPH-
generating system in 0 1 phosphate buåer (pH 7 4) were preincubated for 5 min. The reactions were
±
initiated by addition of human liver microsomes (0 5 mg) in a ®nal volume of 0 5 ml. After incubation for
±
m
±
±
°
10 min at 37 C, reactions were terminated as described above.
BRT metabolism by microsomes expressing individual human CYP isoenzymes
Microsomes expressing human CYP1A1, 1A2, 2A6, 2B6, 2C9, 2C19, 2D6, 2E1 or 3A4 (1 mg) were
°
± ±
m
BRT and an NADPH-generating system for 4 h at 37 C in 1 0 ml 0 1 phosphate
lm
incubated with 1
±
buåer (pH 7 4). For CYP2A6 or 2C9, phosphate buåer was replaced with 50 m Tris-HCl buåer
(pH 7 4) because the activity of these isoenzymes is decreased by phosphate. Aliquots of 200 l of reaction
m
±
l
l
mixture were taken at appropriate times and the reaction was terminated by addition of 600 l ice-cold
methanol.
Inhibition of BRT metabolism by anti-CYP3A4 antiserum
Immunoinhibition of BRT metabolism in human liver microsomes was examined with anti-CYP3A4
antiserum, which shows no cross reactivity with CYP1A1, 1A2, 2A6, 2B6, 2C9, 2D6 or 2E1 (see
l
Materials and methods). Ten or 1000 g IgG of the antiserum diluted with non-immunized rabbit

916
C. Senda et al.
a
Figure 3. Typical Michaelis±Menten plots of -OH-BRT (A) and 6-OH-BRT (B) formation in human
lm
liver microsomes (HHM0215). The BRT concentration range was 10±250
. Lines indicate the
best ®t of the Michaelis±Menten equation to the data. The inset in each ®gure is the Eadie±Hofstee
plot. Lines were estimated by least-squares linear regression.
±
serum was added to human liver microsomes (0 25 mg protein) and the mixture was preincubated at room
temperature for 30 min. A control reaction was run simultaneously using non-immune rabbit serum
lm
instead of the antiserum. After the preincubation, BRT (10 ), an NADPH-generating system, and
0 1 phosphate buåer (®nal incubation volume of 1 0 ml) were added. The reactions were performed at
±
37 C for 60 min. Aliquots of 200 l were taken at 1, 15, 30 and 60 min and the reaction was terminated
±
m
°
by addition of 5 l 70 % PCA.
l
l

Brotizolam metabolism by human CYP3A4
917
Table 1. Michaelis±Menten parameters for BRT metabolism in human liver microsomes.
a
-OH-BRT formation
6-OH-BRT formation
Human
liver
microsomes (pmol min mg protein)
V
}
K
V
K
m
max
m
max
lm
lm
)
(
)
V
K
}
(pmol min mg protein)
}
(
V
K
}
}
}
max
m
max
m
±
±
14 1
±
18 0
±
20 8
Batch 2
HHM0163
HHM0215
1820
1390
1200
62
41
41
29 5
19 890
4340
2720
1413*
241
131
±
33 5
±
27 6
±
±
±
±
17 7
±
2 7
Mean
SE
1470
259
49 0
30 2
8983
7740
595 0
±
9 3
±
2 5
±
580 1
All data are means from ®ve experiments.
* This value also shows the mean from ®ve, of which two are substantially diåerent from other three
lm
values (121, 351, 366
).
Hplc analysis of BRT and its metabolites
BRT metabolites were determined by an hplc method. The system consisted of a controller (Model
715, Gilson, USA), pump (LC-6A, Shimadzu, Japan), autoinjector (SIL-6B, Shimadzu), UV-VIS
detector (Model 486, Waters, USA), column oven (CTO-6A, Shimadzu), and reverse-phase C18 column
±
l
(Superspher RP-Select B, 125¬4 mm, 5 m, Merck, Japan). A ¯ow rate of 1 0 ml per min mobile phase
°
(50 m ammonium acetate methanol acetonitrile, 45 50 5), and a column temperature of 40 C were
used. The metabolites were determined by measuring their absorbance at 240 nm ; the retention times of
m
}
}
} }
±
-OH-BRT and 6-OH-BRT were 3 6 and 3 9 min respectively. After termination of the reaction, each
sample was mixed with 5 l diazepam (0 1 m ) as an internal standard and centrifuged at 15 000 g for
±
a
±
l
m
l
2 min. The resultant supernatant (170 l) was transferred to another tube and mixed with 50 l 1
MOPS and 5 l saturated KOH solution to adjust to pH 7 0. After centrifugation, 50 l supernatant was
l
m
±
l
l
injected directly into the hplc system. A calibration curve was prepared based on the peak heights of
±
standard samples (0 1±3 0
±
lm
).
For the determination of a low concentration of BRT (1
lm
), a more sensitive hplc method was
employed. This utilized a column-switching technique using a valve actuator (817, Gilson) and an
enrichment column (Pelliguard LC-8, 2 cm, Supelco, Japan). Superspher RP-Select B was used as the
±
±
analytical column. A ¯ow rate of 1 0 ml per min mobile phase (40 % acetonitrile in 0 03 % PCA solution
for 4 min, followed by a linear gradient to 60 % acetonitrile by 7 min then isocratic 60% acetonitrile for
1 min) was used, and elution of BRT was monitored by measuring the absorbance at 210 nm with a
variable-wavelength UV detector (Model 486, Waters). Under these conditions the retention time of
±
BRT was approximately 4 7 min. Amounts of BRT were determined from a linear calibration plot based
±
on the peak height of standard samples (0 25±25 n ).
m
Kinetic analysis
Apparent K (Michaelis±Menten constant) and V
(maximum rate of metabolite formation) were
estimated by ®tting data to the Michaelis±Menten equation using the enzyme kinetic program
m
max
`Pharmacologic Calculation System ’ (Tallarida and Murray 1986). Apparent K for ketoconazole,
cimetidine and quinidine were similarly calculated with this program.
i
Results
In human liver microsomes, BRT was metabolized to two hydroxylation
derivatives (a-OH-BRT and 6-OH-BRT ; ®gure 1), which were separated and
determined by hplc (®gure 2). Formation of these two metabolites proceeded
linearly with respect to incubation time up to 30 min (data not shown).
The formation of these metabolites in human liver microsomes (HHM0215)
depended on the substrate concentration (®gure 3), and the data ®tted simple
Michaelis±Menten kinetics well. The kinetic parameters obtained with three
preparations of human liver microsomes (Batch 2, HHM0163 and HHM0215) are
shown in table 1. Eadie±Hofstee plots (®gure 3) were ®tted by least squares linear
regression analysis.
The eåects of several speci®c CYP inhibitors on the two major oxidation
reactions of BRT are shown in ®gure 4. Ketoconazole markedly inhibited the

918
C. Senda et al.
Figure 4. For legend see facing page.

Brotizolam metabolism by human CYP3A4
919
Table 2.
K for inhibition of the formation of BRT metabolites in human liver microsomes.
i
lm
)
K
(
i
a
Inhibitor
-OH-BRT formation 6-OH-BRT formation
±
±
± ±
274 25³38 65
± ±
85 75³46 49
± ±
0 07³0 04
Cimetidine
Quinidine
Ketoconazole
178 13³9 72
±
±
43 23³29 80
±
±
0 05³0 01
Each value represents the mean³SE of values obtained with three diåerent preparations of
microsomes (Batch 2, HHM0163, HHM0215).
Figure 5. Metabolism of BRT by microsomes expressing human CYP isoenzymes: CYP1A1 (D );
CYP1A2 (* ); CYP2A6 (^); CYP2B6; (x ); CYP2C9 ( ); CYP2C19 (+ ); CYP2D6 ; (_ );
V
lm
CYP2E1 (y ); CYP3A4 (E ). Each microsomal fraction was incubated for 4 h with 1
BRT and
an NADPH-generating system. BRT concentration was measured at the indicated time after the
start of incubation. These data were from a single experiment because of the low amount of human
liver microsomes available.
±
formation of both a-OH- and 6-OH-BRT in a dose-dependent manner, with K 0 05
i
and 0 07 lm respectively. Quinidine and cimetidine were slightly inhibitory.
±
±
Furafylline (2, 10 lm ) and sulphaphenazole (0 5, 3 lm ) had no eåect (data not
shown). The apparent K of ketoconazole, cimetidine and quinidine in the three
i
diåerent preparations of human liver microsomes are given in table 2.
a
Figure 4. Lineweaver±Burk plots of the eåects of inhibitors on -OH- and 6-OH-BRT formation by
human liver microsomes. Human liver microsomes (HHM0215, 1 0 mg ml) were incubated for
±
}
lm
10 min with an NADPH-generating system and BRT at a concentration of 25, 50, 100 or 250
±
±
lm
lm
(+ );
in the absence (D ) or the presence of an inhibitor : ketoconazole, 0 25
lm lm lm lm
(+ ). The lines were
(_ ) or 0 5
quinidine, 2
(_ ) or 20
(+ ); cimetidine, 10
(_ ) or 50
determined by non-linear least-squares regression. These data were from a single experiment
because of the low amount of human liver microsomes available.

920
C. Senda et al.
Figure 6. Dose-dependent inhibition of BRT metabolism in human liver microsomes with human
l
CYP3A4 antiserum. Human liver microsomes were preincubated with 0 (D ), 10 g IgG (_ ) or
±
lm
1 0 mg IgG (+ ) antiserum and then incubated with 10
BRT in the presence of an NADPH-
generating system at 37 C for 30 min. Data were from a single experiment because of the low
°
amount of human liver microsomes available.
The catalytic activities of individual human CYPs expressed in microsomes were
examined. We decided to measure the decrease of BRT in this study, because the
production of metabolites during the early phase of incubation with 1 lm BRT
might be lower than the detection limit. BRT was decreased only by incubation with
CYP3A4 (®gure 5). Other CYPs (CYP1A1, 1A2, 2A6, 2B6, 2C9, 2C19, 2D6 and
2E1) did not metabolize BRT.
To con®rm identity of the microsomal enzyme responsible for the formation of
the major metabolites a-OH-BRT and 6-OH-BRT, the eåect of anti-CYP3A4
antiserum was investigated (®gure 6). During incubation for 30 min, 1 mg IgG ml
}
of anti-CYP3A4 antiserum inhibited the a-OH and 6-OH pathways to the extent of
97 and 86 % respectively.

Brotizolam metabolism by human CYP3A4
921
Discussion
We examined the metabolism of a triazolothienodiazepine drug, BRT, using
human liver microsomes as well as microsomes expressing individual human CYP
isoenzymes, in order to identify the CYP isoenzyme(s) involved in the major
metabolic pathways of BRT, i.e. a- and 6-hydroxylation. With three diåerent
preparations of human microsomes, the Eadie±Hofstee plots were linear (®gure 3)
in all cases, suggesting that a single CYP isoenzyme catalyses the hydroxylation of
BRT. Among various speci®c CYP inhibitors examined, ketoconazole extensively
±
±
inhibited both a- and 6-hydroxylation of BRT (K ¯ 0 05 and 0 07 lm respectively)
i
(®gure 4). Although high concentrations ( 10 lm) of ketoconazole inhibit reactions
"
mediated by several CYP3A species (Newton et al. 1995), low concentrations
!
(
1 lm) of ketoconazole speci®cally inhibit CYP3A4 (Maurice et al. 1992, Baldwin
±
±
et al. 1995). The concentration used in our study (0 25 or 0 5 lm), therefore, implies
a speci®c eåect of ketoconazole on CYP3A4. In the experiment using expressed
human CYPs, only CYP3A4 exhibited substantial activity for the a- and 6-
hydroxylation of BRT (®gure 5). These data suggest that CYP3A4 is the isoenzyme
predominantly responsible for the main metabolic pathways of BRT. The
immunoinhibition study using anti-CYP3A4 antiserum con®rmed this (®gure 6).
Alprazolam and triazolam have similar structures to BRT, and their main
metabolic pathways of a- and 4-hydroxylation were reported to be catalysed by
CYP3A4. The K of ketoconazole for the 4-hydroxylation of alprazolam was
i
0 046 lm (von Moltke et al. 1994) and the K of ketoconazole for a- and 4-
±
i
hydroxylation of triazolam were 0 006 and 0 023 lm respectively (von Moltke et al.
±
±
1996). These K are lower than the therapeutic plasma level of ketoconazole
i
(Badcock et al. 1987), so metabolic inhibition by ketoconazole would be expected in
clinical usage. In fact, the peak plasma concentration and AUC (area under the
concentration±time curve) of triazolam increased up to 3- and 22-fold respectively
when the drug was administered concurrently with ketoconazole to healthy
volunteers (Varhe et al. 1994). In our study, the K of ketoconazole for a-OH and 6-
i
±
±
OH-BRT formation were 0 05 and 0 07 lm respectively. Thus, the inhibitory eåect
of ketoconazole on BRT metabolism is less than that on triazolam metabolism.
Nevertheless, it seems necessary to monitor the changes in plasma levels of BRT in
patients when ketoconazole is administered at the same time.
Cimetidine slightly inhibited BRT hydroxylation (®gure 4, table 2). This H -
#
blocker is a CYP3A4 inhibitor, but its aænity is lower than that of azole derivatives
such as ketoconazole (Wrighton and Ring 1994, von Moltke et al. 1996).
Quinidine, a speci®c inhibitor of CYP2D6, caused weak inhibition of BRT
metabolism (®gure 4, table 2), but no metabolic activity towards BRT was observed
with expressed CYP2D6 (®gure 5). Metabolism of quinidine itself is thought to be
mediated by CYP3A4 (Guengerich et al. 1986), and quinidine also showed a slight
inhibition of testosterone 6b-hydroxylation (Newton et al. 1995). Hydroxylation of
both alprazolam and triazolam mediated by CYP3A4 was weakly inhibited by
quinidine (von Moltke et al. 1994, 1996). These results indicate that quinidine is not
only a speci®c inhibitor of CYP2D6 but also a substrate of CYP3A4. Therefore, the
inhibition of BRT by quinidine could be explained by competition for CYP3A4.
In conclusion, we have demonstrated that formation of the two major metabolites
of BRT, a-OH-BRT and 6-OH-BRT, in human liver microsomes is catalysed
predominantly by a single CYP isoenzyme, 3A4.

922
Brotizolam metabolism by human CYP3A4
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