![[Feedback]](/icons/banner/feedbackACT.gif){kind=icon}
![[For Subscribers]](/icons/banner/subscriberACT.gif){kind=icon}
![[Archive]](/icons/banner/archiveACT.gif){kind=icon}
![[Search]](/icons/banner/searchACT.gif){kind=icon}
![[Contents]](/icons/banner/tocACT.gif){kind=icon}
|
|
|
|
|
||
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
ABSORPTION, DISTRIBUTION, METABOLISM, AND EXCRETION
Department of Medicine, Division of Clinical Pharmacology, Indiana University School of Medicine, Indianapolis, Indiana
Received January 27, 2003; accepted March 28, 2003.
 |
Abstract |
|---|
 Top Abstract Materials and MethodsResultsDiscussionReferences |
|---|
94 µM
in HLMs and 234 µM in CYP2B6). In a panel of 11 HLMs,
8-hydroxyefavirenz and 8,14-dihydroxyefavirenz formation rates from efavirenz
(10 µM) correlated significantly with the activity of CYP2B6 and CYP3A.
N,N',N"-Triethylenethiophosphoramide (thioTEPA; 50
µM) inhibited the formation rates of 8-hydroxyefavirenz and
8,14-dihydroxyefavirenz from efavirenz (10 µM) by ≥60% in HLMs) and
CYP2B6, with Ki values < 4 µM. In conclusion, CYP2B6
is the principal catalyst of efavirenz sequential hydroxylation. Efavirenz
systemic exposure is likely to be subject to interindividual variability in
CYP2B6 activity and to drug interactions involving this isoform. Efavirenz may
be a valuable phenotyping tool to study the role of CYP2B6 in human drug
metabolism.
40–55 h after repeated dosing)
(Smith et al., 2001
) allows a
durable, long-lasting reduction in HIV RNA after once-a-day dosing (600 mg)
(Staszewski et al., 1999),
presenting an advantage for treatment compliance and efficacy. Efavirenz is
widely distributed in body compartments and is likely to be effective in
protected tissues such as the central nervous system and testes
(Taylor et al., 2001;
Wynn et al., 2002). Recent
clinical trials suggest that efavirenz has favorable effects on lipid profiles
(McComsey et al., 2003), which
make it an important drug to replace (switch from) protease inhibitorbased
therapy to minimize the consequences of hyperlipidemia.
However, the use of efavirenz is associated with variable response:
genotypic viral resistance and failure of therapy in some patients
(Marzolini et al., 2001;
Langmann et al., 2002) and CNS
adverse effects in a substantial number of patients (up to 50%)
(Marzolini et al., 2001).
Studies have shown that the plasma concentrations of efavirenz predict its
efficacy and CNS adverse effects: patients with subtherapeutic plasma
concentrations (<1 mg/l) appear to be at greater risk for development of
drug resistance and treatment failure
(Marzolini et al., 2001;
Langmann et al., 2002), and
those with plasma concentrations over 4 mg/l are at increased risk of CNS side
effects (Marzolini et al.,
2001). Therefore, it appears important to maintain maximum virally
suppressive efavirenz concentrations that will prevent the emergence of
resistance, while also ensuring an adverse event profile that is not only
safe, but does not significantly compromise overall quality of life. Achieving
this goal has become increasingly difficult, in part because the
pharmacokinetics of efavirenz varies widely among individuals
(Marzolini et al., 2001;
Smith et al., 2001), and
probably for this reason, the severity of drug interactions with efavirenz is
variable and unpredictable (Smith et al.,
2001; Lopez-Cortes et al.,
2002). The propensity for drug interactions with efavirenz is
particularly very high, which needs due attention. Efavirenz is always used in
combination therapy, frequently in the presence of herbal and nutritional
supplements or in concert with drugs directed at the treatment of
opportunistic infections and other HIV-related disorders. Efavirenz, being a
substrate (Mutlib et al.,
1999; Smith et al.,
2001), an inhibitor (Von
Moltke et al., 2001), and an inducer of cytochromes P450 (P450s)
(Mouly et al., 2002), exhibits
multiple interactions with the P450 system. Evaluation of drug interactions
with efavirenz is further complicated because efavirenz is reported to enhance
its own metabolism during repeated administration
(Smith et al., 2001). To
predict and identify factors that alter efavirenz pharmacokinetics, drug
interaction, and response, it is important to thoroughly understand the
mechanisms underlying intersubject variability in efavirenz systemic
exposure.
Efavirenz is extensively metabolized in humans by the P450 system to
inactive hydroxylated metabolites that include 8- and 7-hydroxyefavirenz, with
subsequent urinary and biliary excretion of these metabolites after
conjugation (mainly glucuronidation)
(Mutlib et al., 1999). It
follows that variable activity of the specific P450s involved in efavirenz
metabolism may account for the substantial interindividual variability in
efavirenz pharmacokinetics. The safe and effective use of this drug largely
depends on our ability to unequivocally identify the P450s involved and
determine the precise contribution of each isoform in efavirenz metabolism.
The product label of efavirenz [Product Information of Efavirenz (Sustiva),
Bristol-Myers Squibb Company, April 2002] and review articles
(Smith et al., 2001) implicate
CYP3A and CYP2B6 in efavirenz metabolism, but there have been no published
data that comprehensively address the contribution of these or any other
enzymes in vitro or in vivo. According to Mouly et al.
(2002), there was no
correlation between efavirenz systemic exposure and hepatic CYP3A activity in
humans. It is also important to note that clarithromycin, a known inhibitor of
CYP3A in the gut wall and the liver
(Gorski et al., 1998), had
little effect on the elimination of efavirenz in healthy volunteers [Product
Information of Efavirenz (Sustiva), Bristol-Myers Squibb Company, April 2002].
Recently, Chen et al. (2003)
have shown that CYP2B6 is the principal catalyst of the in vitro metabolism of
DPC 963, another non-nucleoside reverse transcriptase inhibitor that is
structurally similar to efavirenz. All these data suggest that P450s (most
likely CYP2B6) other than CYP3A might be responsible for the human metabolism
of efavirenz.
The primary objective of the present study was to identify the mechanisms
underlying interindividual variability in efavirenz pharmacokinetic and drug
interactions. Using human liver microsomes (HLMs) and recombinant P450s, we
determined the primary and secondary metabolism of efavirenz and the specific
P450s involved. We also tested the utility of efavirenz as a novel substrate
probe of CYP2B6. This enzyme system does play an important role in the
metabolism of a growing list of frequently prescribed drugs and other
chemicals (Ekins and Wrighton,
1999; Wang and Halpert,
2002), but it has been studied less because of unavailability of a
specific and safe substrate reaction marker that will allow prediction of in
vivo activity from in vitro studies.
|
Materials and Methods |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
-NADP were purchased from Sigma-Aldrich (St. Louis,
MO). Sulfaphenazole and furafylline were obtained from Ultrafine Chemicals
(Manchester, UK). thioTEPA was purchased from the U.S. Pharmacopeia
(Rockville, MD). Omeprazole was a generous gift from Dr. Tommy Anderson
(Clinical Pharmacology, Astra Hässle AB, Mölndal, Sweden). All other
chemicals and solvents were of HPLC grade.
Microsomal Preparations. The HLMs that we used were prepared from
human liver tissues that were medically unsuitable for transplantation.
Microsomal fractions were prepared by ultracentrifugation using standard
protocols as described elsewhere (Desta et
al., 1998), and protein concentrations were determined by the
Bradford method (Bradford,
1976) using bovine serum albumin as a standard. The microsomal
pellets were suspended in a reaction buffer to a protein concentration of 10
mg/ml (stock) and were kept at –80°C until used. Additional HLMs,
baculovirus-insect cell-expressed human P450s (1A2, 2A6, 2B6, 2C8, 2C9, 2C19,
2D6, 2E1, 3A4, and 3A5) (with oxidoreductase) and cytochrome
b5 were purchased from BD Gentest (Woburn, MA). The
microsomal preparations were stored at –80°C until used. The total
P450 content, protein concentrations, and specific activity of each P450
isoform were as supplied by the manufacturer.
Incubation Conditions and HPLC Assay. Pilot incubation experiments were performed in HLMs to identify potential oxidative metabolites and to optimize conditions for incubation and HPLC analysis. Efavirenz was dissolved in methanol and serially diluted in methanol to the required concentration. Any methanol was removed by drying in a speed vacuum before reconstituting the residue with phosphate buffer and addition of other incubation components. For the pilot studies, efavirenz (10 µM) and a NADPH-generating system (13 mM NADP, 33 mM glucose 6-phosphate, 33 mM MgCl2, and 0.4 U/ml glucose 6-phosphate dehydrogenase) in potassium phosphate buffer (pH 7.4) were allowed to equilibrate for 5 min at 37°C. After the reaction was initiated by adding 25 µl of HLMs (10 mg protein/ml) and incubated for 30 min at 37°C (final volume, 250 µl), the reaction was terminated by placing tubes on ice and immediately adding 500 µl of acetonitrile. Ritonavir (50 µl of 0.01 mg/ml) was added as an internal standard to the incubation sample, vortexmixed, and centrifuged at 14,000 rpm for 5 min in an Eppendorf model 5415C centrifuge (Brinkmann Instruments, Westbury, NY). The supernatant was removed to a clean tube and extracted twice using ethyl acetate under alkaline pH (0.5 ml of 0.5 M NaOH, adjusted to pH = 10 using 1% phosphoric acid). The organic phase was removed and evaporated to dryness. The residue was reconstituted with 150 µl of mobile phase, and 100 µl was injected onto the HPLC system (see below). Negative control incubations were run in parallel that included exclusion of efavirenz, a NADPH-generating system, or microsomes (bovine serum albumin was used instead) from the incubation mixture.
An HPLC assay method with UV detection was developed for the quantification of efavirenz and its metabolites. The HPLC system consisted of a Waters (Milford, MA) model 515 pump, model 717 autosampler, and model 490 programmable absorbance UV detector. The separation system consisted of a Zorbax SB-C18 column (150 x 4.6 mm, 3.5-µm particle size; Phenomenex, Torrance, CA), a Luna C18 Guard column (30 x 4.6 mm, 5 µm; Phenomenex), and a mobile phase composed of 55% 10 mM KH2PO4 (adjusted to pH 2.4 with 1% phosphoric acid) and 45% (v/v) acetonitrile (flow rate, 0.8 ml/min). The column eluate was monitored by UV detection at 245 nm. Efavirenz and its metabolites were quantified by using the ratio of peak area of the metabolite to peak area of internal standard and calibration curves that were constructed using known efavirenz concentrations. Although we recognize that standard curves constructed using synthetic metabolite references provide optimal quantification of efavirenz metabolism, we used efavirenz standard curves for quantification because we had initially no access to the metabolites of efavirenz or the amounts were not enough to construct repeated standard curves. Of note, when equimolar concentrations (10 µM) of 8-hydroxyefavirenz and efavirenz were directly injected to the HPLC system, the difference in UV activity between 8-hydroxyefavirenz and efavirenz was minimal (5%). At 1 and 10 µM efavirenz, the interday and intraday coefficient of variance of the assay was less than 10% and 4%, respectively.
Using the incubation and HPLC assay conditions described above, three well separated chromatographic HPLC peaks were noted in the incubation mixture consisting of efavirenz, a NADPH-generating system, and HLMs (but not in the negative control experiments). Linearity in the formation rate of these metabolites was established with regard to microsomal protein concentrations and incubation time. Efavirenz (10 µM) was incubated in HLMs (0–1.5 mg protein/ml) and a NADPH-generating system at 37°C across a range of incubation times (0–75 min). Based on the results obtained, linear conditions for efavirenz incubation consisted of 30-min incubation and a final protein concentration of 0.5 mg/ml, and were used in subsequent experiments unless otherwise stated.
Identification of Efavirenz Metabolites. We used two approaches to
obtain structural evidence for the metabolites of efavirenz in microsomal
incubates. First, retention times of efavirenz metabolite peaks after
injection of microsomal incubates of efavirenz into the HPLC system were
compared with that of reference metabolite standard (8-hydroxyefavirenz). The
retention time of 8-hydroxyefavirenz was determined after direct injection or
after adding it to incubation mixture that did not contain active microsomes.
Second, we used liquid chromatography/mass spectrometry (LC/MS) analysis.
Efavirenz (10 µM) was incubated with NADPH and HLMs for 30 min at 37°C,
and the reaction was terminated and processed as described above. Peaks of
interest were separated on a Luna 3-µm C18 –2 column (100 x
2.00 mm i.d.; Phenomenex). The eluate was delivered at a rate of 0.2 ml/min
without splitting to an electrospray ionization probe. LC/MS analysis was
performed in the extracted samples to determine the MS fragmental pattern. The
LC/MS was performed using a Thermo Finnigan (San Jose, CA) AQA mass
spectrometer equipped with a Thermo Finnigan HPLC system, which consisted of
an autosampler (AS3000), binary pump (P4000), and photodiode array detector
(UV6000LP). Mass spectrometer settings were as follows: cone voltage, 29 V;
probe voltage, 4.00 V; and probe temperature, 350°C. A gradient elution
profile was used to separate the metabolites. Initial mobile phase was 75%
ammonium formate (adjusted to pH 3.5 with formic acid) with 25% acetonitrile.
One minute after the start of the run, the acetonitrile percentage was
increased linearly over 19 min to 80% and held for 8 min. Then, initial mobile
phase conditions were resumed after 4 min. The column was allowed to
equilibrate for 5 min between injections. Data were collected in negative
electrospray ionization mode (single ion monitoring scan). The MS
fragmentation patterns obtained were compared with those from urinary
metabolite profiles of efavirenz described in humans
(Mutlib et al., 1999) and with
those of synthetic standards of efavirenz and its metabolite.
Kinetic Analyses in HLMs. Kinetic studies for efavirenz metabolism were conducted in two different human liver microsomal preparations. Efavirenz (0.1–200 µM) was incubated in duplicate for 30 min at 37°C with HLMs (0.5 mg protein/ml) and a NADPH-generating system, and the reaction was terminated and processed as described above. The formation rates of metabolites versus efavirenz substrate concentrations were fit to hyperbolic and nonhyperbolic enzyme kinetic models to estimate apparent kinetic parameters (e.g., Km and Vmax; see Data Analysis).
Correlation Experiments. Efavirenz (10 µM) was incubated with
microsomes from 11 different livers to test the correlation of efavirenz
metabolism with the activity of individual P450s. The total P450 contents,
oxidoreductase, and activity of each P450 isoform were as supplied by the
supplier (BD Gentest). Isoform-specific reaction markers were used to
determine the activity of each P450: phenacetin O-deethylase
(CYP1A2), coumarin 7-hydroxylase (CYP2A6), S-mephenytoin
N-demethylation (CYP2B6), paclitaxel 6
-hydroxylase (CYP2C8),
diclofenac 4'-hydroxylation (CYP2C9), S-mephenytoin 4'-hydroxylation
(CYP2C19), bufuralol 1'-hydroxylation (CYP2D6), chlorzoxazone 6-hydroxylation
(CYP2E1), testosterone 6-hydroxylation (CYP3A), and lauric acid
12-hydroxylation (CYP4A) (see
http://www.gentest.com).
The correlation coefficients between the formation rates of efavirenz
metabolites and the activity of each P450 isoform in different HLMs were
calculated by nonparametric regression analysis (Spearman's rank correlation
test) with GraphPad Prism Software Version 3.1 (GraphPad Software Inc., San
Diego, CA). p < 0.05 is considered statistically significant.
Inhibition Studies. The rates of efavirenz (10 µM) metabolism in
HLMs were evaluated in the absence (control) and presence of the following
known isoform-specific inhibitors: furafylline (20 µM) for CYP1A2,
sulfaphenazole (20 µM) for CYP2C9, omeprazole (10 µM) for 2C19,
quinidine (1 µM) for CYP2D6, troleandomycin (50 µM) and ketoconazole (1
µM) for CYP3A, diethyldithiocarbamate (50 µM) for CYP2E1, and thioTEPA
(50 µM) for CYP2B6. The specific conditions used with these inhibitors have
been described in detail in earlier publications
(Desta et al., 1998;
Rae et al., 2002). Efavirenz
was incubated in HLMs (0.5 mg/ml) with or without P450 isoform-specific
inhibitor and with a NADPH-regenerating system for 30 min. Troleandomycin,
furafylline, and diethyldithiocarbamate were preincubated with a
NADPH-regenerating system and HLMs for 15 min at 37°C before initiation of
the reaction by addition of efavirenz and further incubation for 30 min. The
inhibition (percentage) of metabolite formation rate by P450 isoform-specific
inhibitors was calculated by comparing the inhibited activity with uninhibited
controls (without inhibitors).
The inhibition data identified thioTEPA as the only potent inhibitor of efavirenz metabolism (with no marked effects by other inhibitors). Subsequent experiments were designed to further characterize this inhibition. A single concentration of efavirenz (10 µM) was incubated without or with multiple thioTEPA concentrations (1–25 µM) in HLMs and a NADPH-generating system. The data obtained from this experiment were used to calculate IC50 and to simulate appropriate ranges of efavirenz and thioTEPA concentrations to construct Dixon plots to estimate Ki values for the inhibition of efavirenz metabolism. Efavirenz concentration ranged from 5 to 25 µM and thioTEPA ranged from 0 to 10 µM. The inhibition data were modeled using appropriate enzyme inhibition equations (see Data Analysis).
An earlier report (Rae et al.,
2002) suggests that thioTEPA inhibits S-mephenytoin
N-demethylation, an in vitro substrate probe of CYP2B6
(Ko et al., 1998), in a
time-dependent manner. Thus, the time-dependent inhibition of efavirenz
metabolism by thioTEPA was also tested. thioTEPA (5 µM) was incubated with
HLMs and a NADPH-generating system for 0 to 30 min before the reaction was
initiated by addition of efavirenz (10 µM) and further incubated for 30
min. To determine whether thioTEPA caused metabolite intermediate complex
(MIC) formation, thioTEPA (50 µM) was incubated in pooled HLMs (1 mg/ml)
and recombinant human CYP2B6 (200 pmol/ml). MIC formation was characterized by
dual wavelength spectroscopy (Uvikon 933 double beam UV/VIS Spectrophotometer;
Research Instruments International, San Diego CA) by scanning from 380 to 500
nm as described in detail elsewhere (Jones
et al., 1999).
Metabolism of Efavirenz by Recombinant Human P450 Isoforms. To further identify the specific P450 isoforms catalyzing the metabolism of efavirenz, recombinant human CYP 1A2, 2A6, 2B6, 2C8, 2C9, 2C19, 2D6, 2E1, 3A4, or 3A5 (13 pmol each) was incubated with efavirenz (1, 10, and 100 µM) and a NADPH-generating system (same composition as above) at 37°C for 30 min. All other incubation conditions and assay of the metabolites were the same as described for HLMs above. The rates of formation of efavirenz metabolites as well as the amount of efavirenz remaining in the microsomal incubates after 30-min incubation relative to the initial concentrations added were calculated.
For those P450s showing activity toward efavirenz metabolism, full kinetic analyses were performed. Efavirenz (0–200 µM) was incubated with 13 µl of 1 nmol of P450/ml (recombinant human CYP2B6, CYP1A2, CYP3A4, and CYP3A5). To test whether the presence of cytochrome b5 modifies the kinetics of efavirenz metabolism by CYP2B6, 0 to 150 µM efavirenz was incubated with CYP2B6 (13 µl of 1 nmol of CYP2B6/ml) that was expressed with cytochrome b5. The kinetic parameters were obtained using hyperbolic and nonhyperbolic enzyme kinetic equations (see Data Analysis).
Sequential Metabolism of Efavirenz. Our LC/MS data indicated that
one (MIII) of the three efavirenz metabolite peaks is consistent with a
dihydroxylated metabolite of efavirenz, suggesting that it might represent a
secondary metabolite. Thus, we conducted experiments to systematically test
this possibility. First, we determined the time course for the formation of
each metabolite from efavirenz in HLMs. Efavirenz (5 µM) was incubated in
HLMs (0.5 mg/ml) and a NADPH-generating system (final volume, 250 µM) at
37°C. Reaction was terminated at time = 0 and after 5, 10, 15, 20, 30, 45,
60, and 90 min of incubation. Second, we tested whether MIII might be formed
when 8-hydroxyefavirenz is incubated in HLMs. Efavirenz is believed to undergo
relatively rapid 8-hydroxylation in humans
(Mutlib et al., 1999), raising
the possibility that the secondary metabolite is formed from efavirenz through
step-wise hydroxylation with 8-hydroxyefavirenz as an intermediate. Thus,
8-hydroxyefavirenz (10 µM) was incubated with HLMs (0.5 mg/ml) and a
NADPH-generating system at 37°C for 30 min, and reaction was terminated by
addition of 500 ml of acetonitrile. Appropriate negative controls and
efavirenz (2.5 µM) incubation experiments were also performed in parallel.
Extraction protocols and HPLC assay were similar to those described above for
efavirenz. The retention time of the metabolite peak formed from
8-hydroxyefavirenz microsomal incubation was compared with those produced when
efavirenz was used as a substrate. LC/MS analysis was performed according to
the protocol described for efavirenz metabolites (see above). These data
suggested that dihydroxyefavirenz is formed when both efavirenz and
8-hydroxyefavirenz were used as substrates. Third, since our studies with
recombinant human P450s indicated that the formation of the dihydroxyefavirenz
from efavirenz is dependent on CYP2B6, we tested whether the same enzyme also
catalyzes the conversion of 8-hydroxyefavirenz to dihydroxyefavirenz.
8-Hydroxyefavirenz (2.5 µM) was incubated in HLMs or a panel of
cDNA-expressed human P450s (13 pmol each) and a NADPH generating system for 30
min at 37°C. The formation rates of dihydroxyefavirenz and the
disappearance of 8-hydroxyefavirenz from microsomal incubate were quantified.
Fourth, we determined full kinetic analysis for the formation of
dihydroxyefavirenz from 8-hydroxyefavirenz in HLMs and CYP2B6.
8-Hydroxyefavirenz (0–200 µM) was incubated in HLMs (0.5 mg /ml) (or
CYP2B6, 52 pmol of P450/ml) and a NADPH-generating system. The formation rates
of dihydroxyefavirenz versus 8-hydroxyefavirenz concentrations were fit to
appropriate enzyme kinetic models to estimate kinetic parameters (see Data
Analysis).
Data Analysis. Estimates of kinetic constants were obtained by nonlinear regression analysis using WinNonlin Software Version 4.0 (Pharsight, Mountain View, CA). The simple single-site Michaelis-Menten equation (V = Vmax · C/(Km + C), Hill equation (V = Vmax · Cn/(C50 + Cn), or substrate inhibition equation (V = Vmax/(1 + Km)/C + C/Ksi) was fitted to formation rates (V) of efavirenz metabolites versus efavirenz (or 8-hydroxyefavirenz) concentrations (C). The apparent maximum formation rate (Vmax) and apparent substrate concentration resulting in 50% of Vmax, Hill coefficient (n), and substrate inhibition constant (Ksi) were calculated. In vitro intrinsic clearances (Clint) were given as Vmax/Km or Vmax/Kmn). Inhibition constants (Ki values) were estimated from the inhibition data using nonlinear regression for the noncompetitive inhibition model using WinNonlin. The models that best fit were selected based on the dispersion of residuals and standard errors of the parameter estimates. Kinetic constants were given as mean ± S.D. (or ±S.E. of parameter estimates).
|
Results |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
|
The identity of the metabolite peaks shown in
Fig. 1 was confirmed by
comparison of LC retention times with synthetic reference compounds and LC/MS
analysis. When we compare the HPLC retention times of efavirenz metabolite
peaks in microsomal incubates with that of 8-hydroxyefavirenz, the retention
time of MI (15.80 ± 0.04 min) coeluted with that of 8-hydroxyefavirenz
(15.80 ± 0.01 min). The LC/MS fragmentation patterns were compared with
previously published fragmentation patterns of efavirenz metabolism in vivo
(Mutlib et al., 1999). The
major metabolite peak (MI) obtained following incubation of efavirenz with
HLMs (LC retention time, 18 min) had characteristic ions at 330
m/z, 286 m/z, and 258.0
m/z, consistent with the fragmentation pattern of
8-hydroxefavirenz. A smaller peak (MII) from efavirenz incubation with a
retention time of 17.2 min showed characteristic ions at 258.0
m/z and 330 m/z that concur with
7-hydroxyefavirenz. MIII had characteristic ions at 346 m/z,
302 m/z, and 274 m/z, consistent with
8,14-dihydroxyefavirenz. In this in vitro study, 8-hydroxylation to
8-hydroxyefavirenz (MI), 7-hydroxylation to 7-hydroxyefavirenz (MII), and
8,14-dihydroxyefavirenz (MIII) were identified as efavirenz oxidative
metabolites.
In Fig. 2, the time course
for the formation of efavirenz metabolites is depicted. The formation of
8,14-dihydroxyefavirenz showed a significant lag-time relative to the
formation of 8-hydroxyefavirenz. These data, together with the results from
LC/MS analysis, suggest that 8,14-dihydroxyefavirenz is a secondary metabolite
of efavirenz. It is unlikely that 8- and 14-hydroxylation occurs
simultaneously. There are two possible routes for the formation of
8,14-dihydroxyefavirenz from efavirenz: 1) efavirenz undergoes
14-hydroxylation first, followed by 8-hydroxylation of the product, or 2)
8,14-dihydroxyefavirenz is formed via a step-wise hydroxylation of efavirenz,
with 8-hydroxyefavirenz as an intermediate. Although it was not determined
experimentally, there are reasons to believe that the first possibility is
unlikely. In vivo studies indicate that 8-hydroxyefavirenz is the main
metabolite and is rapidly formed from efavirenz in humans, but there is no
evidence that 14-hydroxyefavirenz is formed
(Mutlib et al., 1999). To test
the second possibility, we incubated 8-hydroxyefavirenz (2.5 µM) in HLMs
(HL9) and monitored the formation of 8,14-dihydroxyefavirenz. Indeed,
8-hydroxyefavirenz was efficiently metabolized to 8,14-dihydroxyefavirenz
(V = 52.7 pmol/min/mg protein). Collectively, our data provide
evidence that 8,14-dihydroxyefavirenz is formed from efavirenz by step-wise
hydroxylation; i.e., first 8-hydroxyefavirenz is formed, followed by further
14-hydroxylation. A significant quantity of both the primary and the secondary
metabolites was detected when efavirenz was used as a substrate
(Fig. 1); the formation of
8,14-dihydroxyefavirenz (but not that of 8-hydroxyefavirenz) behaved as a
secondary metabolite because the time course for its formation was
characterized by a significant lag-time; and, more importantly,
8,14-dihydroxyefavirenz was formed efficiently when 8-hydroxyefavirenz was
used as a substrate. Assuming the metabolites have similar UV-absorbance
intensity at equimolar concentrations, the data in
Fig. 1 and the time course
profile (Fig. 2) suggest that
the 8-hydroxylation pathway (formation of 8-hydroxyefavirenz + its sequential
metabolism to 8,14-dihydroxyerfavirenz) represents a major route of efavirenz
metabolism (over 92% of efavirenz oxidation), whereas the contribution of
7-hydroxylation appears to be small (<8%).
|
Kinetic Analysis in HLMs. Kinetic analysis of efavirenz metabolite formation rates was performed in two different HLM preparations. The kinetic parameters estimated are summarized in Table 1. The kinetic profiles of efavirenz metabolism to 8-hydroxyefavirenz in HLMs (HL9 and HL9/14/99) are shown in Fig. 3. The formation rate of 8-hydroxyefavirenz revealed sigmoidal saturation curves (Fig. 3, left panel) that were fit to a Hill equation (n = 1.33 in HL9 and 1.72 in HL09/14/99) (Table 1). The Eadie-Hofstee plots [(V) and V/S (substrate concentration)] of this metabolite showed curvilinear relationships (Fig. 3, right panel), indicating positive cooperativity. The formation rate of 7-hydroxyefavirenz in HLMs was characterized by a hyperbolic saturation curve and an Eadie-Hofstee plot that was linear (data not shown), suggesting the involvement of a single enzyme or more than one enzyme with similar affinity. The in vitro intrinsic clearance (CLint) for the formation of 8-hydroxyefavirenz was 17- and 14-fold higher in HL9 and HL9/14/99, respectively, than that for 7-hydroxyefavirenz. Of note, the CLint for the formation of 8-hydroxyefavirenz described here is clearly underestimated because it does not account for the sequential metabolism. The fold difference in CLint between the formation rate of 7-hydroxyefavirenz and the total 8-hydroxylation pathway (8-hydroxyefavirenz + 8,14-dihydroxyefavirenz) is therefore expected to be larger than that provided here.
|
|
The formation rate of 8,14-dihydroxefavirenz versus 8-hydroxyefavirenz concentrations was characterized by an initial rapid increase at lower substrate concentrations followed by a progressive decline at higher substrate concentrations (Fig. 4). Comparison of the goodness-of-fit values generated from these data indicates that a substrate inhibition equation enzyme model provided a better fit than did other models. The corresponding Eadie-Hofstee plot indicated a "hook" in the upper region of this plot (Fig. 4, inset), which is characteristic of substrate inhibition. The Km (µM), Vmax (pmol/min/mg protein), and Ksi (µM) estimated from these data, respectively, were 0.78 µM, 27.66 pmol/min/mg protein, and 94 µM. When efavirenz was used as a substrate, the formation of 8,14-dihydroxyefavirenz in HLMs showed kinetic profiles essentially similar to that described when 8-hydroxyefavirenz was used as a substrate: 6.6 µM, 66 pmol/min/mg protein, and 278 µM in HL and HL9/14/99, and 2.2 µM, 30 pmol/min/mg protein, and 91 µM in HL9. Although these data provided valuable information on what might be expected with respect to the formation of 8,14-dihydroxyefavirenz from efavirenz, the fact that 8,14-dihydroxyefavirenz is formed from efavirenz by a two-step reaction would mean that the kinetic parameters generated for the formation of this metabolite using efavirenz as a substrate are likely to be compromised by the rate of the first hydroxylation (8-hydroxylation) and the second hydroxylation (14-hydroxylation).
|
Correlation Analysis. Figure 5 shows the rate of efavirenz (10 µM) metabolism in a panel of 11 characterized HLMs. We assessed the formation rates of 8-hydroxyefavirenz and 8,14-dihydroxyefavirenz independently and as a summation. The average formation rates (pmol/min/mg protein) of efavirenz metabolism to 8-hydroxyefavirenz, 7-hydroxyefavirenz, 8,14-dihydroxyefavirenz, and 8-hydroxyefavirenz + 8,14-dihydroxyefavirenz in a panel of 11 HLMs were 38.7 ± 24.5 (range, 10.8–83.1; 7.7-fold), 2.8 ± 1.9 (range, 0–4.4), 9.5 ± 9.7 (range, 2.4–13.9; 13.4-fold), and 48.3 ± 33.9 (range, 13.6–114.1; 8.4-fold), respectively. 7-Hydroxyefavirenz was not detected in one of the HLMs, but in the 10 HLMs in which this metabolite was detected, variability between HLM preparations was low (3.5-fold). The summation of 8-hydroxyefavirenz and 8,14-dihydroxyefavirenz formation rates was significantly correlated with the formation rate of 8-hydroxyefavirenz (r = 0.999, p = 0.0001) and 8,14-dihydroxyefavirenz (r = 0.83, p < 0.0047). 7-Hydroxyefavirenz formation rates did not correlate with the other metabolites (r < 0.2; p > 0.7). In the panel of HLMs tested, 17.1 ± 5.3% (range, 8.8–28.8; 3.3-fold) of the total rate was accounted for by 8,14-dihydroxyefavirenz formation from 8-hydroxyefavirenz, while 82.9 ± 5.3% (range, 71.2-to 91.1; 1.3-fold) was accounted for by the remaining 8-hydroxyefavirenz formation rate. Assuming that 8,14-dihydroxyefavirenz is not further metabolized, it seems that the formation rates of 8-hydroxyefavirenz might be underestimated by <20%.
|
|
Metabolism of Efavirenz by Recombinant Human P450s. Three
concentrations of efavirenz (1 µM, 10 µM, and 100 µM) were incubated
with a panel of recombinant P450s. At 1 µM efavirenz, CYP2B6 significantly
reduced the concentration of efavirenz in the microsomal incubates (by about
96%) relative to other isoforms or incubations that do not contain microsomes
(instead, bovine serum albumin was used) (data not shown). The formation rates
(pmol of product/min/pmol P450) of 8-hydroxyefavirenz and
8,14-dihydroxyefavirenz after incubation of efavirenz (10 µM) with P450s
are shown in Fig. 6 (left
panel). Except for CYP2B6, none of the P450s tested catalyzed the formation of
8,14-dihydroxyefavirenz. CYP2B6 formed 8-hydroxyefavirenz at the highest rate.
CYP3A5, CYP1A2, and CYP3A4 also catalyzed the formation of 8-hydroxyefavirenz,
but when compared with CYP2B6, the involvement of these isoforms in the
formation of 8-hydroxyefavirenz was low (10.2-, 8.4-, and 24-fold lower,
respectively). At 100 µM efavirenz (Fig.
6, right panel), the following isoforms contributed to the
formation rates (pmol/min/pmol P450) of 8-hydroxyefavirenz: CYP2B6, CYP3A5,
CYP1A2, CYP 3A4, CYP2A6, and CYP2C9. These data show that CYP2B6 is 11.8-,
5.2-, and 30-fold more catalytically active than CYP1A2, CYP3A5, and CYP3A4,
respectively, toward the formation of 8-hydroxyefavirenz. CYP2B6 was also the
main catalyst of 8,14-dihydroxyefavirenz formation, with small contribution of
CYP2C9. Although CYP2B6 remained the main isoform responsible for efavirenz
metabolism at all substrate concentrations tested, the pattern of metabolite
formation varied with increasing substrate concentration. For example,
8,14-dihydroxyefavirenz was formed more efficiently at lower substrate
concentrations, whereas 8-hydroxyefavirenz was predominant with increasing
concentration of efavirenz [the formation rates of 8,14-dihydroxyefavirenz by
CYP2B6 were decreased (by 53%) from 0.496 ± 0.039 pmol/min/pmol
P450 at 10 µM to 0.262 ± 0.014 pmol/min/pmol P450 at 100 µM].
These data were essentially similar to the data observed in HLMs. At any of
the concentrations of efavirenz used, none of the recombinant P450s tested
formed 7-hydroxyefavirenz.
|
Full kinetic analysis was performed for the formation rate of 8-hydroxyefavirenz from efavirenz by recombinant human CYP2B6 (Fig. 7). The formation rates of 8-hydroxyefavirenz versus efavirenz concentration fit better to a single-site Michaelis-Menten equation. The respective data obtained are listed in Table 3. In HLMs, the formation of 8-hydroxyefavirenz was characterized by a sigmoidal curve (Fig. 3). Fitting the data obtained from recombinant CYP2B6 to the Hill equation did not improve the estimation (Hill coefficient, 1.19 ± 0.19; n = 3 experiments in duplicate determinations). The Km values obtained in recombinant CYP2B6 (12.4 ± 1.8 µM) (Table 3) were close to that obtained from HLMs (average from the two HLMs, 20.16 µM) (Table 1), suggesting that this same enzyme is catalyzing efavirenz metabolism in both preparations. Besides CYP2B6, CYP1A2, CYP3A5, and CYP3A4 showed some activity toward the formation of 8-hydroxyefavirenz (Fig. 6). Thus, we examined the kinetics for the formation rate of 8-hydroxyefavirenz in CYP1A2, CYP3A5, and CYP3A4 and compared the data with the formation rate in CYP2B6 (Fig. 8). The respective kinetic parameters derived from fitting the data to a single-site Michaelis-Menten equation are shown in Table 3. The Km values for the formation of 8-hydroxyefavirenz by CYP1A2, CYP3A5, and CYP3A4 were comparable to that obtained with CYP2B6, but the Vmax values obtained from CYP1A2, CYP3A5, and CYP3A4 were much smaller compared with those obtained in CYP2B6. Accordingly, the in vitro intrinsic clearance (µl/min/pmol P450) for the formation of 8-hydroxyefavirenz in CP2B6 was 5.8-, 13.5-, and 60-fold higher than that of CYP1A2, CYP3A5, and CYP3A4, respectively. CYP2B6 is the main enzyme that catalyzes the formation of 8-hydroxyefavirenz from efavirenz, but this metabolite is subject to additional metabolism by the same enzyme. The CLint for the formation of 8-hydroxyefavirenz by CYP2B6 (and the fold difference) would be larger than that listed in Table 3 when corrected for sequential metabolism.
|
|
|
The data in Fig. 6 suggested to us that CYP2B6 is the dominant enzyme responsible for the formation of 8,14-dihydroxyefavirenz from efavirenz (Fig. 6), and we have evidence that this metabolite is also formed in HLMs when 8-hydroxyefavirenz is used as a substrate. We tested whether CYP2B6 catalyzes this reaction when 8-hydroxyefavirenz was used as a substrate. As shown in Fig. 9, CYP2B6 catalyzed the formation of 8,14-dihydroxyefavirenz from 8-hydroxyefavirenz at the highest rate. Other enzymes also showed some activity, but at very low rate (>29-fold lower). Accordingly, CYP2B6 (but not other enzymes) decreased the amount of 8-hydroxyefavirenz remaining in microsomal incubate by 88%. Our data from HLMs indicate that the formation of 8,14-dihydroxyefavirenz exhibit substrate inhibition (Fig. 4). Consistent with these data, the kinetics for the formation of 8,14-dihydroxyefavirenz from 8-hydroxyefavirenz was characterized by the substrate inhibition equation (Fig. 10). When the substrate-velocity curve of 8,14-dihydroxyefavirenz was modeled using the substrate inhibition model, the following kinetic parameters were estimated: Km, 2.12 µM; Vmax, 8.5pmol/min/pmol P450; and Ksi, 233.6 µM.
|
|
Studies with CYP3A have provided useful insight into the mechanism by which
substrate inhibition might occur. Cytochrome b5 may
modulate the efficiency of CYP3A-mediated substrate inhibitions through
enhancement of the efficiency of CYP3A4-reactive oxygen production and, thus,
the availability of heme bound oxygen for the reaction and increasing the
partition of the iron-oxygen complex toward substrate oxidation
(Schrag and Wienkers, 2001,
and references therein). Since the kinetic profiles for the formation of
8,14-dihydroxyefavirenz from efavirenz or 8-hydroxyefavirenz were similar, we
determined the kinetics of 8-hydroxyefavirenz and 8,14-dihydroxyefavirenz
formation from efavirenz in CYP2B6, which was enriched with cytochrome
b5. We wished to test whether cytochrome
b5 might reverse the substrate inhibition observed for
8,14-dihydroxyefavirenz or explain differences in the kinetics observed in
HLMs and CYP2B6 with respect to the formation of 8-hydroxyefavirenz. As shown
in Table 3, cytochrome
b5 decreased the Km and
Vmax values for the formation of 8-hydroxyefavirenz, but
CLint remained similar to that without cytochrome
b5. The formation of 8,14-dihydroxyefavirenz was not
influenced in a significant way by cytochrome b5.
Chemical Inhibition of Efavirenz Metabolism. To further probe the
P450 isoforms participating in efavirenz metabolism, 10 µM efavirenz was
incubated with P450 isoform-specific inhibitors in HLMs. As shown in
Table 2, thioTEPA (50 µM)
inhibited the rates of 8-hydroxyefavirenz and 8,14-hydroxyefavirenz formation
by 65% and 75%, respectively. The effect of the other inhibitors tested on the
formation of 8-hydroxyefavirenz and 8,14-hydroxyefavirenz was < 20%. The
formation rates of 7-hydroxyefavirenz were modestly inhibited by
sulfaphenazole, omeprazole, and troleandomycin, weakly inhibited by
ketoconazole and quinidine, and activated by thioTEPA
(Table 4). The inhibition
profiles in HLMs were compared with those inhibition data in recombinant human
CYP2B6 (Table 4). Consistent
with the HLMs, thioTEPA (50 µM) was the only inhibitor of the formation
rates of 8-hydroxyefavirenz (by 60%) and 8,14-dihydroxyefavirenz (by 78%) in
CYP2B6. Similarly, thioTEPA (0–25 µM) inhibits the formation of
8-hydroxyefavirenz and 8,14-hydroxyefavirenz in a concentration-dependent
manner (IC50, 10 µM and 5 µM, respectively). It seems that
8,14-hydroxyefavirenz is more susceptible to inhibition (by 12.4–18.8%
at 2.5–25 µM inhibitor) by thioTEPA than is 8-hydroxyefavirenz.
Compared with microsomal incubations without inhibitor, thioTEPA increased the
rate of 7-hydroxyefavirenz at low concentrations (1–10 µM) by over
18% (peak 31%), whereas it showed a 49% decrease at 25 µM (data not shown).
We do not believe that the increase we noted was due to coelution of thioTEPA
or its metabolites with 7-hydroxyefavirenz because no peak was observed at the
retention time of 7-hydroxyefavirenz when thioTEPA was incubated alone
(without efavirenz).
|
To estimate Ki values for the inhibition of efavirenz
metabolism by thioTEPA, a range of substrate concentrations (1–25 µM)
was incubated without or with thioTEPA (2.5–10 µM) in HLMs.
Representative Dixon plots for the inhibition of 8-hydroxyefavirenz and
8,14-hydroxyefavirenz formation rate from efavirenz by thioTEPA are shown in
Fig. 11. The
Ki values estimated by nonlinear regression using a
noncompetitive enzyme inhibition model were 3.0 µM and 2.5 µM for the
formation of 8-hydroxyefavirenz and 8,14-hydroxyefavirenz, respectively, and
these values fall within the range of therapeutic concentrations reported for
thioTEPA (1.1–18.6 µM) during a 4-day intravenous infusion at a dose
of 400 to 800 mg/m2 (Kennedy et
al., 1995). No Dixon plot was presented for 7-hydroxyefavirenz
because thioTEPA showed activation at the substrate concentrations tested
(data not shown). The inhibition of efavirenz metabolism in HLMs was
time-dependent; i.e., the rate of metabolism of efavirenz markedly decreased
during preincubation of thioTEPA with HLMs and the NADPH-generating system
(Fig. 12) and concurs with our
earlier report with S-mephenytoin N-demethylation
(Rae et al., 2002), a probe of
CYP2B6 (Ko et al., 1998).
Experiments were performed to assess whether this inhibition of thioTEPA is
mediated by metabolite-intermediate complex. thioTEPA did not form any
detectable MIC in pooled human liver microsomes or cDNA-expressed CYP2B6 (data
not shown). It is possible that the time-dependent effect of thioTEPA on
CYP2B6 activity is due to its effect on CYP2B6 apoprotein. Of note, Hollenberg
and coworkers have identified numerous compounds that inactivate CYP2B6 by
interfering with apoprotein (e.g., Chun et
al., 2000; Kent et al.,
1999).
|
|
|
Discussion |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
|
That 8-hydroxyefavirenz is the major metabolite of efavirenz in vitro is
consistent with human studies in which this metabolite has been reported to be
the principal metabolite of the drug
(Mutlib et al., 1999).
Glucuronide and sulfate conjugates of 7-hydroxyefavirenz have been identified
in human urine (Mutlib et al.,
1999), but the contribution of 7-hydroxylation to the overall
clearance of efavirenz is likely to be small because the average
CLint estimated from two HLMs for 7-hydroxylation reaction was
>15-fold lower than that for 8-hydroxylation reaction. In addition to the
two primary metabolites, we also identified 8,14-dihydroxyefavirenz as a
secondary metabolite of efavirenz. According to Mutlib et al.
(1999),
8,14-dihydroxyefavirenz glucuronide has been detected in human plasma and
urine. These authors proposed that efavirenz undergoes step-wise hydroxylation
to form 8-hydroxyefavirenz and then 8,14-dihydroxyefavirenz in vivo
(Mutlib et al., 1999). We have
shown that 8-hydroxyefavirenz was efficiently metabolized in vitro to
8,14-dihydroxyefavirenz. The time course for the formation of
8,14-dihydroxyefavirenz from efavirenz in vitro (present study) and in vivo
(Mutlib et al., 1999)
indicates a significant lag-time relative to that of 8-hydroxyefavirenz. Our
findings provide direct evidence that 8,14-dihydroxyefavirenz is a secondary
metabolite of efavirenz in that its formation proceeds stepwise from
efavirenz, with 8-hydroxyefavirenz as intermediate. Based on our in vitro
results (and assuming no further metabolism of 8,14-dihydroxyefavirenz and
similar UV-absorbance intensity between the two metabolites), we estimate that
17% of 8-hydroxyefavirenz formed from efavirenz is further oxidized to
8,14-dihydroxyefavirenz in vitro. The fraction is likely to be small in vivo,
where 8-hydroxyefavirenz might undergo rapid glucuronidation
(Mutlib et al., 1999),
although this claim needs experimental testing. Since the main source of
8,14-dihydroxyefavirenz in vitro seems to be 8-hydroxyefavirenz, the overall
metabolism of efavirenz via the 8-hydroxylation route will be the summation of
the formation of 8-hydroxyefavirenz (measured) and its subsequent
14-hydroxylation. When corrected for sequential metabolism, we would then
predict substantially higher estimates of CLint for the overall
8-hydroxylation than those listed in Tables
1 and
3. Our in vitro data allow us
to estimate that the contribution of the total 8-hydroxylation pathway to the
overall efavirenz oxidative metabolic clearance is >90%, with
7-hydroxylation playing a minor role.
The oxidation of efavirenz to 8-hydroxyefavirenz in HLMs is characterized
by a sigmoidal substrate-velocity curve
(Fig. 3) (Hill coefficient of
1.33–1.7), unlike the kinetic data obtained in recombinant human P450s,
which were characterized by a hyperbolic Michaelis-Menten equation
(Fig. 7). A similar kinetic
profile has been observed with another CYP2B6-mediated bupropion hydroxylation
in HLMs (Faucette et al.,
2000). Other recombinant CYP2B6-mediated reactions seem to depend
on the substrate probe used: no autoactivation of efavirenz hydroxylation
(present data) and bupropion hydroxylation
(Faucette et al., 2000), or
allosteric kinetics of 7-ethoxy-4-fluoromethyl coumarin
O-deethylation (Ekins et al.,
1997) and testosterone 16-hydroxylation
(Ekins et al., 1998). The cause
for the atypical kinetics for the formation of 8-hydroxyefavirenz from
efavirenz in HLMs (but not in other P450s) is unclear. Differences in membrane
lipid composition, NADPH-P450 oxidoreductase, and cytochrome
b5 between the two systems could contribute to this.
Moreover, although our in vitro data implicate CYP2B6 as the major enzyme
catalyzing the sequential metabolism of efavirenz, the participation of P450s
other than CYP2B6 in HLMs cannot be excluded.
The formation of 8,14-dihydroxyefavirenz from efavirenz and
8-hydroxyefavirenz in HLMs and recombinant P450s exhibited substrate
inhibition. Similar substrate inhibition profiles have been observed
previously with CYP3A-mediated triazolam
(Schrag and Wienkers, 2001)-
and midazolam (Gorski et al.,
1994)-regioselective hydroxylation, which are suggestive of
multiple substrate-binding sites (or multiple regions within a single active
site). To our knowledge, this is the first report of CYP2B6-mediated substrate
inhibition. Although this observation may have no clinical relevance because
it does not affect the initial 8-hydroxylation of efavirenz and because the
expected in vivo steady-state concentrations of the substrate (efavirenz or
8-hydroxyefavirenz) after standard doses of efavirenz
(Smith et al., 2001) are much
lower than the substrate inhibition constants we obtained here, it may offer
insight into the characteristics of the enzyme.
Although our inhibition data (Table 4) implicate multiple enzymes in the 7-hydroxylation of efavirenz, the specific enzymes responsible remain unclear, because we were unable to further confirm them with experiments involving recombinant P450s and correlation analysis. It may be that the sensitivity of the assay did not allow us to measure 7-hydroxyefavirenz when recombinant P450s were used, or this metabolite is catalyzed by P450s other than those tested here or through other NADPH-dependent reactions. Because the contribution of 7-hydroxylation to the overall clearance of efavirenz is likely to be small, no further attempt was made to identify the P450s involved.
We provide strong evidence that efavirenz 8-hydroxylation and the
subsequent oxidation to 8,14-dihydroxyefavirenz is predominantly catalyzed by
CYP2B6. First, the formation rates of these metabolites from efavirenz were
potently inhibited by thioTEPA (Table
4), a specific inhibitor of CYP2B6
(Rae et al., 2002). Second,
CYP2B6 formed 8-hydroxyefavirenz and 8,14-dihydroxyefavirenz from efavirenz
(Fig. 6) [and
8,14-dihydroxyefavirenz from 8-hydroxyefavirenz
(Fig. 9)] with the highest
specific activity. Third, the Km value of
8-hydroxyefavirenz derived from HLMs (20.2 µM)
(Table 1) was similar to that
observed in CYP2B6 (12.4 µM) (Table
3). Fourth, the formation rates of 8-hydroxyefavirenz (or
8,14-dihydroxyefavirenz) correlated significantly with the activity of CYP2B6
in a panel of HLMs (Table 2).
We also noted that recombinant human CY1A2, CYP3A5, and CYP3A4 formed
8-hydroxyefavirenz from efavirenz, but the contributions of these isoforms to
efavirenz metabolism appear minor: 1) the CLint for
8-hydroxyefavirenz formation by CYP1A2, CYP3A5, and CYP3A4 was 5.8-, 13.5-,
and 60-fold lower, respectively (fold difference is larger when sequential
metabolism is taken in to account), than that obtained in recombinant human
CYP2B6 (Table 3); 2) a
CYP1A2-specific inhibitor (furafylline) and CYP3A-specific inhibitors
(ketoconazole and troleandomycin) did not inhibit the rates of formation of
8-hydroxyefavirenz or 8,14-dihydroxyefavirenz in HLMs and CYP2B6
(Table 4); and 3) the
disappearance of efavirenz (1 µM) from microsomal incubates was catalyzed
by CYP2B6 (but not by coincubation with recombinant human CYP3A4, CYP3A5, or
CYP1A2). The significant correlation we observed between the activity CYP3A
and efavirenz 8-hydroxylation in the panel of HLMs tested may not be due to
the actual involvement of CYP3A in efavirenz metabolism. Because our
inhibition and recombinant experiments do not support a significant role of
CYP3A in efavirenz metabolism, the observed significant correlation between
efavirenz metabolism and CYP3A is probably derived from the significant
correlation between the activity of CYP3A and CYP2B6 (Spearman r =
0.72; p = 0.0234) in the bank of human livers tested. Recently, Mouly
et al. (2002) reported that
there was no correlation between efavirenz systemic exposure and hepatic CYP3A
activity in healthy volunteers. It is also important to note that
clarithromycin, a known inhibitor of CYP3A in the gut wall and the liver
(Gorski et al., 1998), had
negligible effect on the elimination of efavirenz in humans (Bristol-Myers
Squibb Company, 2002). Besides, certain inhibitors and inducers of CYP3A had
no or minimal effect on the elimination of efavirenz in humans [Product
Information of Efavirenz (Sustiva), Bristol-Myers Squibb Company, April
2002].
Collectively, our in vitro data together with in vivo evidence from the
literature strongly suggest that CYP2B6 is the principal catalyst of efavirenz
metabolism (Fig. 13), which
may have important implications for HIV/AIDS therapy. In human livers in
vitro, wide interindividual variability in the expression of CYP2B6 is seen at
the level of mRNA (Chang et al.,
2003), protein (Code et al.,
1997; Stresser and Kupfer,
1999; Faucette et al.,
2000; Lang et al.,
2001), and catalytic activity
(Ekins et al., 1998;
Faucette et al., 2000). This
variability is probably due to effects of genetic polymorphisms of CYP2B6
(Ariyoshi et al., 2001;
Lang et al., 2001) or exposure
to drugs that are inducers (Gervot et al.,
1999; Fauchette et al., 2001;
Sueyoshi and Negishi, 2001) or
inhibitors (Hesse et al.,
2001; Rae et al.,
2002) of CYP2B6. If our in vitro data can be extrapolated to in
vivo conditions, we would expect that variability in efavirenz
pharmacokinetics and drug interactions is primarily a reflection of the large
interindividual differences in the activity of CYP2B6. It is now believed that
8% of the drugs on the market are fully or partially metabolized by
CYP2B6 (Treger and Stoll,
2002), and it represents 6% of the total liver P450 content
(Stresser and Kupfer, 1999),
in contrast to earlier estimates that it represented less than 1%
(Shimada et al., 1994).
Despite this and the identification of a growing list of clinically important
drugs, environmental chemicals, and endogenous substances as substrates of
CYP2B6 in vitro (Ekins and Wrighton,
1999; Wang and Halpert,
2002), it remains difficult to determine or predict its clinical
consequences because of the unavailability of a specific and safe probe to
measure the activity of the enzyme in vivo. Our data indicate that efavirenz
8-hydroxylation is a specific in vitro reaction marker of CYP2B6 and may have
utility as a phenotyping tool to study the role of this enzyme in human drug
metabolism.
|
Footnotes |
|---|
ABBREVIATIONS: HIV, human immunodeficiency virus; AIDS, acquired immunodeficiency syndrome; CNS, central nervous system; P450, cytochrome P450; HLM, human liver microsome; HPLC, high-performance liquid chromatography; LC/MS, liquid chromatography/mass spectrometry; MIC, metabolite intermediate complex; Ksi, substrate inhibition constant.
Address correspondence to: Dr. Zeruesenay Desta, Assistant Professor of Medicine and Pharmacology and Toxicology, Indiana University School of Medicine, Department of Medicine, Division of Clinical Pharmacology, 1001 West 10th Street, WD Myers Bldg., W7123, Indianapolis, IN 46202. E-mail: zdesta{at}iupui.edu
|
References |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
Ariyoshi N, Miyazaki M, Toide K, Sawamura Y, and Kamataki T (2001) A single nucleotide polymorphism of CYP2B6 found in Japanese enhances catalytic activity by autoactivation. Biochem Biophys Res Commun 281: 1256–1260.[CrossRef][Medline]
Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72: 248–254.[CrossRef][Medline]
Chang TK, Bandiera SM, and Chen J (2003) Constitutive
androstane receptor and pregnane X receptor gene expression in human liver:
interindividual variability and correlation with CYP2B6 mRNA levels.
Drug Metab Dispos 31:
7–10.
Chen H, Chen W, Gan LS, and Mutlib AE (2003)
Metabolism of
(S)-5,6-difluoro-4-cyclopropylethynyl-4-trifluoromethyl-3,4-dihydro-2(1H)-quinazolinone,
a non-nucleoside reverse transcriptase inhibitor, in human liver microsomes.
Metabolic activation and enzyme kinetics. Drug Metab
Dispos 31:
122–132.
Chun J, Kent UM, Moss RM, Sayre LM, and Hollenberg PF
(2000) Mechanism-based inactivation of cytochromes P450 2B1 and
P450 2B6 by 2-phenyl-2-(1-piperidinyl)propane. Drug Metab
Dispos 28:
905–911.
Code EL, Crespi CL, Penman BW, Gonzalez FJ, Chang TK, and Waxman DJ
(1997) Human cytochrome P4502B6: interindividual hepatic
expression, substrate specificity and role in procarcinogen activation.
Drug Metab Dispos 25:
985–993.
Desta Z, Kerbusch T, Soukhova N, Richard E, Ko JW, and Flockhart DA
(1998) Identification and characterization of human cytochrome
P450 isoforms interacting with pimozide. J Pharmacol Exp
Ther 285:
428–437.
Ekins S, Vandenbranden M, Ring BJ, Gillespie JS, Yang TJ, Gelboin
HV, and Wrighton SA (1998) Further characterization of the
expression in liver and catalytic activity of CYP2B6. J Pharmacol
Exp Ther 286:
1253–1259.
Ekins S, Vandenbranden M, Ring BJ, and Wrighton SA (1997) Examination of purported probes of human CYP2B6. Pharmacogenetics 7: 165–179.[CrossRef][Medline]
Ekins S and Wrighton SA (1999) The role of CYP2B6 in human xenobiotic metabolism. Drug Metab Rev 31: 719–754.[CrossRef][Medline]
Faucette S, Lindley C, Hawke R, Shord S, Clarke M, and LeCluyse E (2001) Effects of cytochrome P450 (CYP) P450 3A inducers on CYP2B6 catalytic activity. Clin Pharmacol Ther 69: 10, Abstr. PI-36.
Faucette SR, Hawke RL, LeCluyse EL, Shord SS, Yan B, Laethem RM,
and Lindley CM (2000) Validation of bupropion hydroxylation as a
selective marker of human cytochrome P450 2B6 catalytic activity.
Drug Metab Dispos 28:
1222–1230.
Gervot L, Rochat B, Gautier JC, Bohnenstengel F, Kroemer H, de Beradinis V, Martin H, Beaune P, and de Waziers I (1999) Human CYP2B6: expression, inducibility and catalytic activities. Pharmacogenetics 9: 295–306.[Medline]
Gorski JC, Hall SD, Jones DR, Vandenbranden M, and Wrighton SA (1994) Regioselective biotransformation of midazolam by members of the human cytochrome P450 3A (CYP3A) subfamily. Biochem Pharmacol 47: 1643–1653.[CrossRef][Medline]
Gorski JC, Jones DR, Haehner-Daniels BD, Hamman MA, O'Mara EM Jr, and Hall SD (1998) The contribution of intestinal and hepatic CYP3A to the interaction between midazolam and clarithromycin. Clin Pharmacol Ther 64: 133–143.[CrossRef][Medline]
Hesse LM, Von Moltke LL, Shader RI, and Greenblatt DJ
(2001) Ritonavir, efavirenz and nelfinavir inhibit CYP2B6
activity in vitro: potential drug interactions with bupropion.
Drug Metab Dispos 29:
100–102.
Jones DR, Gorski JC, Hamman MA, Mayhew BS, Rider S, and Hall SD
(1999) Diltiazem inhibition of cytochrome P-450 3A activity is
due to metabolite intermediate complex formation. J Pharmacol Exp
Ther 290:
1116–1125.
Kennedy MJ, Armstrong DK, Huelskamp AM, Ohly K, Clarke BV, Colvin OM, Grochow LB, Chen TL, and Davidson NE (1995) Phase I and pharmacologic study of the alkylating agent modulator novobiocin in combination with high-dose chemotherapy for the treatment of metastatic breast cancer. J Clin Oncol 13: 1136–1143.[Abstract]
Kent UM, Yanev S, and Hollenberg PF (1999) Mechanism-based inactivation of cytochromes P450 2B1 and P450 2B6 by n-propylxanthate. Chem Res Toxicol 12: 317–322.[CrossRef][Medline]
Ko JW, Desta Z, and Flockhart DA (1998) Human
N-demethylation of (S)-mephenytoin by cytochrome P450s 2C9 and 2B6.
Drug Metab Dispos 26:
775–778.
Lang T, Klein K, Fischer J, Nussler AK, Neuhaus P, Hofmann U, Eichelbaum M, Schwab M, and Zanger UM (2001) Extensive genetic polymorphism in the human CYP2B6 gene with impact on expression and function in human liver. Pharmacogenetics 11: 399–415.[CrossRef][Medline]
Langmann P, Weissbrich B, Desch S, Vath T, Schirmer D, Zilly M, and Klinker H (2002) Efavirenz plasma levels for the prediction of treatment failure in heavily pretreated HIV-1 infected patients. Eur J Med Res 7: 309–314.[Medline]
Lopez-Cortes LF, Ruiz-Valderas R, Viciana P, Alarcon-Gonzalez A, Gomez-Mateos J, Leon-Jimenez E, Sarasanacenta M, Lopez-Pua Y, and Pachon J (2002) Pharmacokinetic interactions between efavirenz and rifampicin in HIV-infected patients with tuberculosis. Clin Pharmacokinet 41: 681–690.[CrossRef][Medline]
Marzolini C, Telenti A, Decosterd LA, Greub G, Biollaz J, and Buclin T (2001) Efavirenz plasma levels can predict treatment failure and central nervous system side effects in HIV-1-infected patients. AIDS 15: 71–75.[CrossRef][Medline]
McComsey G, Bhumbra N, Ma JF, Rathore M, and Alvarez A
(2003) Impact of protease inhibitor substitution with efavirenz
in HIV-infected children: results of the First Pediatric Switch Study.
Pediatrics 111:
e275–e281.
Mouly S, Lown KS, Kornhauser D, Joseph JL, Fiske WD, Benedek IH, and Watkins PB (2002) Hepatic but not intestinal CYP3A4 displays dose-dependent induction by efavirenz in humans. Clin Pharmacol Ther 72: 1–9.[CrossRef][Medline]
Mutlib AE, Chen H, Nemeth GA, Markwalder JA, Seitz SP, Gan LS, and
Christ DD (1999) Identification and characterization of efavirenz
metabolites by liquid chromatography/mass spectrometry and high field NMR:
species differences in the metabolism of efavirenz. Drug Metab
Dispos 27:
1319–1333.
Rae JM, Soukhova NV, Flockhart DA, and Desta Z (2002)
Triethylenethiophosphoramide is a specific inhibitor of cytochrome P450 2B6:
implications for cyclophosphamide metabolism. Drug Metab
Dispos 30:
525–530.
Schrag ML and Wienkers LC (2001) Triazolam substrate
inhibition: evidence of competition for heme-bound reactive oxygen within the
CYP3A4 active site. Drug Metab Dispos
29:
70–75.
Shimada T, Yamazaki H, Mimura M, Inui Y, and Guengerich FP
(1994) Interindividual variations in human liver cytochrome P-450
enzymes involved in the oxidation of drugs, carcinogens and toxic chemicals:
studies with liver microsomes of 30 Japanese and 30 Caucasians. J
Pharmacol Exp Ther 270:
414–423.
Smith PF, DiCenzo R, and Morse GD (2001) Clinical pharmacokinetics of non-nucleoside reverse transcriptase inhibitors. Clin Pharmacokinet 40: 893–905.[CrossRef][Medline]
Staszewski S, Morales-Ramirez J, Tashima KT, Rachlis A, Skiest D,
Stanford J, Stryker R, Johnson P, Labriola DF, Farina D, et al.
(1999) Efavirenz plus zidovudine and lamivudine, efavirenz plus
indinavir and indinavir plus zidovudine and lamivudine in the treatment of
HIV-1 infection in adults. Study 006 Team. N Engl J
Med 341:
1865–1873.
Stresser DM and Kupfer D (1999) Monospecific
antipeptide antibody to cytochrome P-450 2B6. Drug Metab
Dispos 27:
517–525.
Sueyoshi T and Negishi M (2001) Phenobarbital response elements of cytochrome P450 genes and nuclear receptors. Annu Rev Pharmacol Toxicol 41: 123–143.[CrossRef][Medline]
Taylor S, Reynolds H, Sabin CA, Drake SM, White DJ, Back DJ, and Pillay D (2001) Penetration of efavirenz into the male genital tract: drug concentrations and antiviral activity in semen and blood of HIV-1-infected men. AIDS 15: 2051–2053.[Medline]
Treger JM and Stoll S (2002) Cytochrome P450: their impact on drug treatment. Hosp Pharm 167–173.
Von Moltke LL, Greenblatt DJ, Granda BW, Giancarlo GM, Duan SX, Daily JP, Harmatz JS, and Shader RI (2001) Inhibition of human cytochrome P450 isoforms by nonnucleoside reverse transcriptase inhibitors. J Clin Pharmacol 41: 85–91.[Abstract]
Wang Q and Halpert JR (2002) Combined
three-dimensional quantitative structure-activity relationship analysis of
cytochrome P450 2B6 substrates and protein homology modeling. Drug
Metab Dispos 30:
86–95.
Wynn HE, Brundage RC, and Fletcher CV (2002) Clinical
implications of CNS penetration of antiretroviral drugs. CNS
Drugs 16:
595–609.[CrossRef][Medline]
This article has been cited by other articles:
|
|
 |
|
  M. Zhu, S. Kaul, P. Nandy, D. M. Grasela, and M. Pfister Model-Based Approach To Characterize Efavirenz Autoinduction and Concurrent Enzyme Induction with Carbamazepine Antimicrob. Agents Chemother., June 1, 2009; 53(6): 2346 - 2353. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H.-l. Lin, H. Zhang, and P. F. Hollenberg Metabolic Activation of Mifepristone [RU486; 17{beta}-Hydroxy-11{beta}-(4-dimethylaminophenyl)-17{alpha}-(1-propynyl)-estra-4,9-dien-3-one] by Mammalian Cytochromes P450 and the Mechanism-Based Inactivation of Human CYP2B6 J. Pharmacol. Exp. Ther., April 1, 2009; 329(1): 26 - 37. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T W Mahungu, M A Johnson, A Owen, and D J Back The impact of pharmacogenetics on HIV therapy Int J STD AIDS, March 1, 2009; 20(3): 145 - 151. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Jeong, P. D. Nguyen, and Z. Desta Comprehensive In Vitro Analysis of Voriconazole Inhibition of Eight Cytochrome P450 (CYP) Enzymes: Major Effect on CYPs 2B6, 2C9, 2C19, and 3A Antimicrob. Agents Chemother., February 1, 2009; 53(2): 541 - 551. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 N. N. Bumpus and P. F. Hollenberg Investigation of the Mechanisms Underlying the Differential Effects of the K262R Mutation of P450 2B6 on Catalytic Activity Mol. Pharmacol., October 1, 2008; 74(4): 990 - 999. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. S. Rhee and D. J. Greenblatt Pharmacologic Consideration for the Use of Antiretroviral Agents in the Elderly J. Clin. Pharmacol., October 1, 2008; 48(10): 1212 - 1225. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Kwara, M. Lartey, K. W. Sagoe, F. Xexemeku, E. Kenu, J. Oliver-Commey, V. Boima, A. Sagoe, I. Boamah, D. J. Greenblatt, et al. Pharmacokinetics of Efavirenz When Co-administered With Rifampin in TB/HIV Co-infected Patients: Pharmacogenetic Effect of CYP2B6 Variation J. Clin. Pharmacol., September 1, 2008; 48(9): 1032 - 1040. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 N. Oezguen, S. Kumar, A. Hindupur, W. Braun, B. K. Muralidhara, and J. R. Halpert Identification and Analysis of Conserved Sequence Motifs in Cytochrome P450 Family 2: FUNCTIONAL AND STRUCTURAL ROLE OF A MOTIF 187RFDYKD192 IN CYP2B ENZYMES J. Biol. Chem., August 1, 2008; 283(31): 21808 - 21816. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. Ji, B. Damle, J. Xie, S. E. Unger, D. M. Grasela, and S. Kaul Pharmacokinetic Interaction Between Efavirenz and Carbamazepine After Multiple-Dose Administration in Healthy Subjects J. Clin. Pharmacol., August 1, 2008; 48(8): 948 - 956. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. Kim, K.-B. Kim, H.-Y. Ku, S.-J. Park, H. Choi, J.-K. Moon, B.-S. Park, J.-H. Kim, S. S. Yea, C.-H. Lee, et al. Identification and Characterization of Potent CYP2B6 Inhibitors in Woohwangcheongsimwon Suspension, an Herbal Preparation Used in the Treatment and Prevention of Apoplexy in Korea and China Drug Metab. Dispos., June 1, 2008; 36(6): 1010 - 1015. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. H. Hofmann, J. K. Blievernicht, K. Klein, T. Saussele, E. Schaeffeler, M. Schwab, and U. M. Zanger Aberrant Splicing Caused by Single Nucleotide Polymorphism c.516G>T [Q172H], a Marker of CYP2B6*6, Is Responsible for Decreased Expression and Activity of CYP2B6 in Liver J. Pharmacol. Exp. Ther., April 1, 2008; 325(1): 284 - 292. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. L. DiGiacinto, K. M. Chan-Tack, S. M. Robertson, K. S. Reynolds, and K. A. Struble Are Literature References Sufficient for Dose Recommendations? An FDA Case Study of Efavirenz and Rifampin J. Clin. Pharmacol., April 1, 2008; 48(4): 518 - 523. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. D. Luber Pharmacokinetic Considerations for Selection of HAART in Treatment-Naive HIV-Infected Individuals, 2008: More Than It Seems? J Int Assoc Physicians AIDS Care (Chic Ill), March 1, 2008; 7(1_suppl): 10 - 16. [Abstract] [PDF]
|
|
|
|
|
|
R. L. Walsky and R. S. Obach A Comparison of 2-Phenyl-2-(1-piperidinyl)propane (PPP), 1,1',1''-Phosphinothioylidynetrisaziridine (ThioTEPA), Clopidogrel, and Ticlopidine as Selective Inactivators of Human Cytochrome P450 2B6 Drug Metab. Dispos., November 1, 2007; 35(11): 2053 - 2059. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Kumar, Y. Zhao, L. Sun, S. S. Negi, J. R. Halpert, and B. K. Muralidhara Rational Engineering of Human Cytochrome P450 2B6 for Enhanced Expression and Stability: Importance of a Leu264->Phe Substitution Mol. Pharmacol., November 1, 2007; 72(5): 1191 - 1199. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. E. Estes, K. H. Busse, and S. R. Penzak Pharmacogenetic Considerations in the Management of HIV Infection Journal of Pharmacy Practice, June 1, 2007; 20(3): 234 - 245. [Abstract] [PDF]
|
|
|
|
|
|
C. J. O'Donnell, K. Grime, P. Courtney, D. Slee, and R. J. Riley The Development of a Cocktail CYP2B6, CYP2C8, and CYP3A5 Inhibition Assay and a Preliminary Assessment of Utility in a Drug Discovery Setting Drug Metab. Dispos., March 1, 2007; 35(3): 381 - 385. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. R. Faucette, T.-C. Zhang, R. Moore, T. Sueyoshi, C. J. Omiecinski, E. L. LeCluyse, M. Negishi, and H. Wang Relative Activation of Human Pregnane X Receptor versus Constitutive Androstane Receptor Defines Distinct Classes of CYP2B6 and CYP3A4 Inducers J. Pharmacol. Exp. Ther., January 1, 2007; 320(1): 72 - 80. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. K. Blievernicht, E. Schaeffeler, K. Klein, M. Eichelbaum, M. Schwab, and U. M. Zanger MALDI-TOF Mass Spectrometry for Multiplex Genotyping of CYP2B6 Single-Nucleotide Polymorphisms Clin. Chem., January 1, 2007; 53(1): 24 - 33. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. L. Walsky, A. V. Astuccio, and R. S. Obach Evaluation of 227 Drugs for In Vitro Inhibition of Cytochrome P450 2B6. J. Clin. Pharmacol., December 1, 2006; 46(12): 1426 - 1438. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K.-H. Liu, M.-G. Kim, D.-J. Lee, Y.-J. Yoon, M.-J. Kim, J.-H. Shon, C. S. Choi, Y. K. Choi, Z. Desta, and J.-G. Shin Characterization of Ebastine, Hydroxyebastine, and Carebastine Metabolism by Human Liver Microsomes and Expressed Cytochrome P450 Enzymes: Major Roles for CYP2J2 and CYP3A Drug Metab. Dispos., November 1, 2006; 34(11): 1793 - 1797. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. C. T. Casabar, A. D. Wallace, E. Hodgson, and R. L. Rose Metabolism of Endosulfan-{alpha} by Human Liver Microsomes and Its Utility as a Simultaneous in Vitro Probe for CYP2B6 and CYP3A4 Drug Metab. Dispos., October 1, 2006; 34(10): 1779 - 1785. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. J. Gardiner and E. J. Begg Pharmacogenetics, Drug-Metabolizing Enzymes, and Clinical Practice Pharmacol. Rev., September 1, 2006; 58(3): 521 - 590. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
U. M. Kent, H.-l. Lin, K. R. Noon, D. L. Harris, and P. F. Hollenberg Metabolism of Bergamottin by Cytochromes P450 2B6 and 3A5 J. Pharmacol. Exp. Ther., September 1, 2006; 318(3): 992 - 1005. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H.-K. Lee, J.-K. Moon, C.-H. Chang, H. Choi, H.-W. Park, B.-S. Park, H.-S. Lee, E.-C. Hwang, Y.-D. Lee, K.-H. Liu, et al. STEREOSELECTIVE METABOLISM OF ENDOSULFAN BY HUMAN LIVER MICROSOMES AND HUMAN CYTOCHROME P450 ISOFORMS Drug Metab. Dispos., July 1, 2006; 34(7): 1090 - 1095. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
N. N. Bumpus, U. M. Kent, and P. F. Hollenberg Metabolism of Efavirenz and 8-Hydroxyefavirenz by P450 2B6 Leads to Inactivation by Two Distinct Mechanisms J. Pharmacol. Exp. Ther., July 1, 2006; 318(1): 345 - 351. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. M. Hesse, D. J. Greenblatt, L. L. von Moltke, and M. H. Court Ritonavir has minimal impact on the pharmacokinetic disposition of a single dose of bupropion administered to human volunteers. J. Clin. Pharmacol., May 1, 2006; 46(5): 567 - 576. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
N. N. Bumpus, C. Sridar, U. M. Kent, and P. F. Hollenberg THE NATURALLY OCCURRING CYTOCHROME P450 (P450) 2B6 K262R MUTANT OF P450 2B6 EXHIBITS ALTERATIONS IN SUBSTRATE METABOLISM AND INACTIVATION Drug Metab. Dispos., June 1, 2005; 33(6): 795 - 802. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Zukunft, T. Lang, T. Richter, K. I. Hirsch-Ernst, A. K. Nussler, K. Klein, M. Schwab, M. Eichelbaum, and U. M. Zanger A Natural CYP2B6 TATA Box Polymorphism (-82T-> C) Leading to Enhanced Transcription and Relocation of the Transcriptional Start Site Mol. Pharmacol., May 1, 2005; 67(5): 1772 - 1782. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H.-l. Lin, U. M. Kent, and P. F. Hollenberg The Grapefruit Juice Effect Is Not Limited to Cytochrome P450 (P450) 3A4: Evidence for Bergamottin-Dependent Inactivation, Heme Destruction, and Covalent Binding to Protein in P450s 2B6 and 3A5 J. Pharmacol. Exp. Ther., April 1, 2005; 313(1): 154 - 164. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 G. H. Wynn, K. L. Cozza, M. J. Zapor, G. W. Wortmann, and S. C. Armstrong Antiretrovirals, Part III: Antiretrovirals and Drugs of Abuse Psychosomatics, February 1, 2005; 46(1): 79 - 87. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. Lang, K. Klein, T. Richter, A. Zibat, R. Kerb, M. Eichelbaum, M. Schwab, and U. M. Zanger Multiple Novel Nonsynonymous CYP2B6 Gene Polymorphisms in Caucasians: Demonstration of Phenotypic Null Alleles J. Pharmacol. Exp. Ther., October 1, 2004; 311(1): 34 - 43. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Z. Desta, B. A. Ward, N. V. Soukhova, and D. A. Flockhart Comprehensive Evaluation of Tamoxifen Sequential Biotransformation by the Human Cytochrome P450 System in Vitro: Prominent Roles for CYP3A and CYP2D6 J. Pharmacol. Exp. Ther., September 1, 2004; 310(3): 1062 - 1075. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Turpeinen, R. Nieminen, T. Juntunen, P. Taavitsainen, H. Raunio, and O. Pelkonen SELECTIVE INHIBITION OF CYP2B6-CATALYZED BUPROPION HYDROXYLATION IN HUMAN LIVER MICROSOMES IN VITRO Drug Metab. Dispos., June 1, 2004; 32(6): 626 - 631. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. Xun and C. M. Webster A Monooxygenase Catalyzes Sequential Dechlorinations of 2,4,6-Trichlorophenol by Oxidative and Hydrolytic Reactions J. Biol. Chem., February 20, 2004; 279(8): 6696 - 6700. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. Richter, T. E. Murdter, G. Heinkele, J. Pleiss, S. Tatzel, M. Schwab, M. Eichelbaum, and U. M. Zanger Potent Mechanism-Based Inhibition of Human CYP2B6 by Clopidogrel and Ticlopidine J. Pharmacol. Exp. Ther., January 1, 2004; 308(1): 189 - 197. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
V. Lamba, J. Lamba, K. Yasuda, S. Strom, J. Davila, M. L. Hancock, J. D. Fackenthal, P. K. Rogan, B. Ring, S. A. Wrighton, et al. Hepatic CYP2B6 Expression: Gender and Ethnic Differences and Relationship to CYP2B6 Genotype and CAR (Constitutive Androstane Receptor) Expression J. Pharmacol. Exp. Ther., December 1, 2003; 307(3): 906 - 922. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
 |
 |
 |
|
 |
 |
 |
