![[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}
|
|
|
|
|
||
Vol. 282, Issue 1, 294-300, 1997
Storr Liver Unit (D.S., A.M.B., L.N., M.M.), Department of Medicine, University of Sydney, Westmead Hospital, Westmead, NSW 2145, and Department of Pharmacology (D.S.), University of Sydney, NSW 2006, Australia
 |
Abstract |
|---|
 Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
|
|---|
The antihypertensive agent diltiazem (DTZ) impairs hepatic drug
metabolism by inhibition of cytochrome P450 (CYP). The accumulation of
DTZ metabolites in serum occurs during prolonged therapy and leads to
decreased DTZ elimination. Thus, DTZ metabolites may contribute to CYP
inhibition. This study assessed the role of human CYPs in microsomal
DTZ oxidation and the capacity of DTZ metabolites to inhibit specific
CYP activities. DTZ N-demethylation varied 10-fold in microsomal
fractions from 17 livers (0.33-3.31 nmol/mg of protein/min). DTZ
oxidation was correlated with testosterone 6
-hydroxylation (r = 0.82) and, to a lesser extent, tolbutamide hydroxylation (r = 0.59) but not with activities mediated by CYP1A2 or CYP2E1. CYP3A4 in
lymphoblastoid cell microsomes catalyzed DTZ N-demethylation but CYP2C8
and CYP2C9 were also active (~20% and 10% of the activity supported
by CYP3A4); seven other CYPs produced little or no N-desmethyl DTZ from
DTZ. The CYP3A4 inhibitors ketoconazole and troleandomycin decreased
microsomal DTZ oxidation, but inhibitors or substrates of CYP2C, CYP2D
and CYP2E1 produced no inhibition. Some inhibition was produced by
-naphthoflavone, a chemical that inhibits CYP1As and also interacts
with CYP3A4. In further experiments, the capacities of DTZ and three
metabolites to modulate human CYP 1A2, 2E1, 2C9 and 3A4 activities were
evaluated in vitro. DTZ and its N-desmethyl and
N,N-didesmethyl metabolites selectively inhibited CYP3A4 activity,
whereas O-desmethyl DTZ was not inhibitory. The IC50 value
of DTZ against CYP3A4-mediated testosterone 6-hydroxylation
(substrate concentration, 50 µM) was 120 µM. The N-desmethyl
(IC50 = 11 µM) and N,N-didesmethyl (IC50 = 0.6 µM) metabolites were 11 and 200 times, respectively, more potent.
From kinetic studies, N-desmethyl DTZ and N,N-didesmethyl DTZ were
potent competitive inhibitors of CYP3A4 (Ki = ~2 and 0.1 µM, respectively). CYP3A4 inhibition was enhanced when
DTZ and N-desmethyl DTZ underwent biotransformation in
NADPH-supplemented hepatic microsomes in vitro, supporting
the contention that inhibitory metabolites may be generated in
situ. These findings suggest that N-demethylated metabolites of
DTZ may contribute to CYP3A4 inhibition in vivo, especially
under conditions in which N-desmethyl DTZ accumulates, such as during
prolonged DTZ therapy.
| |
Introduction |
|---|
|
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
|
|---|
The calcium channel antagonist
DTZ has been shown to inhibit mammalian CYPs (Renton, 1985
) and to
precipitate pharmacokinetic interactions with drugs such as
carbamazepine (Brodie and Macphee, 1986), cyclosporin A (Combalert
et al., 1989) and nifedipine (Toyosaki et al.,
1988). Because CYP3A4 participates in the oxidation of these drugs, it
seems likely that this enzyme may be a target for inhibition by DTZ.
Although there is evidence from animal studies that CYP3As may be
involved in DTZ metabolism, the contribution of CYPs to the reaction in
human liver has not been evaluated. Furthermore, the selectivity of DTZ
as an inhibitor of major CYP reactions in human liver remains to be
established.
Apart from the inhibitory effects of DTZ on drug oxidation, evidence
from the literature shows that DTZ metabolites accumulate during
prolonged therapy and may contribute to CYP inhibition (Abernethy and
Montamat, 1987; Montamat and Abernethy, 1987). In this study, we also
investigated the capacity of DTZ and its oxidized metabolites (fig.
1) to inhibit the activities of four major human CYPs
and determined the role of hepatic biotransformation in the generation
of DTZ metabolites with greater inhibitory potency toward CYP enzymes.
|
The principal findings to emerge from the present study were that DTZ N-demethylation is catalyzed extensively by CYP3A4, probably the major drug oxidase in human liver, and that DTZ is also a preferential inhibitor of CYP3A4 activity; DTZ did not inhibit reactions mediated by CYPs 1A2, 2C9 or 2E1. Importantly, both N-desmethyl and N,N-didesmethyl DTZ were significantly more potent than DTZ as inhibitors of CYP3A4. Kinetic studies indicated that the two metabolites elicited inhibition of CYP3A4 activity by a competitive mechanism. In vitro biotransformation of DTZ in human microsomes led to an increase in the observed extent of inhibition of CYP3A4 activity, as did the incubation of N-desmethyl DTZ with NADPH-fortified microsomes. Taken together, these results suggest that CYP3A4 contributes extensively to DTZ N-demethylation in human liver and that the N-demethylated metabolites produced may accumulate during chronic DTZ therapy and lead to inhibition of the enzyme.
| |
Materials and Methods |
|---|
|
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
|
|---|
Chemicals. DTZ HCl (d-cis-isomer), 7-ethylresorufin (also termed resorufin-7-ethyl ether or 7-ethoxyresorufin), tolbutamide, N-nitrosodimethylamine, 7-ethoxycoumarin and biochemicals were purchased from Sigma Aldrich (Castle Hill, NSW, Australia). DTZ metabolites were gifts from Tanabe Seiyaku Company (Osaka, Japan). [4-14C]Testosterone (specific activity, 57-59 mCi/mmol), Hyperfilm-MP and scintillation fluid (ACS-II) were purchased from Amersham Australia (North Ryde, NSW, Australia). Steroid metabolites were obtained from Steraloids (Wilton, NH) or the Medical Research Council Steroid Reference Collection (Queen Mary's College, London, UK). Aniline was obtained from Ajax Chemicals (Sydney, Australia) and redistilled from zinc dust before use. Analytical reagent and HPLC grade solvents and other chemicals were from Ajax or Rhone-Poulenc (Sydney, Australia). Silica gel 60 F254 plates for TLC were from Alltech (Sydney, Australia).
Liver donors and preparation of microsomal fractions.
Ethical approval for experiments on human tissue was granted by the
Human Ethics committee of the Western Sydney Area Health Service.
Tissue was obtained under the organ donation scheme from accident
victims, through either the Queensland or Australian Liver Transplant
Programs (Princess Alexandria Hospital, Brisbane, Queensland, and Royal
Prince Alfred Hospital, Sydney, NSW, Australia, respectively). In most
cases, samples were excess tissue from adult donors used in the
transplantation of pediatric recipients, but several samples were from
the normal margin of tissue taken for biopsy during liver resection.
Tissue was obtained from the operating theater, perfused with ice-cold
ViaSpan solution (Belzer University of Wisconsin solution; DuPont,
Wilmington, DE), packed in ice and transported to the laboratory where
it was snap-frozen in liquid nitrogen. Washed microsomes were prepared
by centrifugation and were resuspended in 50 mM potassium phosphate
buffer, pH 7.4, containing 1 mM EDTA and 20% glycerol for storage at
70°C (Murray et al., 1986). Seventeen individual livers
were used in this study (table 1). Microsomal protein
contents were determined according to the Lowry procedure (Lowry
et al., 1951); bovine serum albumin was used as the
standard.
|
Cell microsomes containing cDNA-derived human CYPs.
Microsomes from human lymphoblastoid cell lines (AHH-1
TK+/
) in which cDNA-derived human CYPs (1A1, 1A2, 2A6,
2B6, 2C8, 2C9, 2D6, 2E1, 3A4 and 4A11) had been expressed were
purchased from Gentest Corp. (Woburn, MA). Control microsomes were
prepared from untransfected AHH-1 TK+/ cells. Protein
contents of the preparations were 10 mg/ml; CYP contents were
determined by the supplier (table 2).
|
|
Assays of human microsomal CYP activities.
Testosterone
hydroxylation (0.15 mg of protein/0.4 ml incubation;
14C-testosterone 50 µM, 0.18 µCi) was measured as
previously described (Murray, 1992). Reactions were conducted in
potassium phosphate buffer (0.1 M, pH 7.4) at 37°C for 2.5 min.
Radioactive products were extracted from incubations, separated by TLC,
located by autoradiography and quantified according to methods outlined
previously (Murray, 1992).
.
Briefly, initial Lineweaver-Burk (1/V vs. 1/S) and Dixon
(1/S vs. I) analyses were followed by the construction of
primary replots (slopes of lines in the Lineweaver-Burk and Dixon plots
as a function of I or 1/S, respectively). The appearance of the replots
can be used to characterize the nature of the inhibition, and
Ki values are derived from the same analysis (Segel, 1975).
Aniline 4-hydroxylation in human liver microsomes (1.6 mg of
protein/0.6 ml incubation; substrate concentration 5 mM) was measured
as described previously (Murray and Ryan, 1982). Reactions were
conducted in potassium phosphate buffer (0.1 M, pH 7.4) at 37°C for
12 min. 4-Aminophenol formation was detected by the formation of a blue
complex with alkaline phenol and quantified at 610 nm.
N,N-Dimethylnitrosamine N-demethylation in human hepatic microsomes
(2.5 mg of protein/1.0 ml incubation; substrate concentration 4 mM) was
measured as described by Peng et al. (1983). Reactions were
conducted in potassium phosphate buffer (0.1 M, pH 7.4) at 37°C for
20 min.
7-Ethylresorufin O-deethylation activity (0.5 mg of protein/2 ml
incubation, substrate concentration 2.5 µM) was measured in
Tris · HCl buffer (0.1 M, pH 7.8) using the continuous
spectrofluorometric procedure of Prough et al. (1978).
Resorufin formation was monitored at the excitation/emission wavelength
pair of 560/580 nm.
Tolbutamide 4-hydroxylation activity (0.3 mg of protein/0.4 ml
incubation, tolbutamide 300 µM) was measured using the method of
Knodell et al. (1987). Reactions were conducted in potassium phosphate buffer (0.1 M, pH 7.4) at 37°C for 15 min. After the addition of HCl, the products were extracted with diethyl ether and the
residue was evaporated, reconstituted in acetonitrile and applied to a
Beckman Ultrasphere C18 column (5 µm, 25 cm × 4.6 mm i.d., Beckman Instruments Inc., San Ramon, CA). The mobile phase was
0.05% phosphoric acid, pH 2.6/acetonitrile (3:2). Retention times were
4.14 min for 4-hydroxytolbutamide, 9.25 min for chlorpropamide (internal standard) and 11.53 min for tolbutamide.
Oxidation of DTZ in human hepatic and lymphoblastoid cell
microsomes.
DTZ oxidation was determined in human hepatic
microsomes at a substrate concentration of 100 µM, except in the
determination of kinetic parameters, where the substrate range was 5 to
200 µM. Incubations (0.15 mg of protein/400 µl) were conducted in potassium phosphate buffer (0.1 M, pH 7.4) containing EDTA (1 mM) at
37°C for 10 min. Preliminary studies established that product formation was linear over the time indicated. Reactions were terminated by freezing in an acetone/dry ice bath. After subsequent thawing, the
internal standard was added (imipramine, 30 nmol), and the solution was
basified by the addition of 1 g of NaHCO3 and then extracted with ethyl acetate (4 ml). The organic phase (3.8 ml) was
extracted with HCl (0.01 M, 200 µl), which was then evaporated under
a stream of nitrogen and dissolved in 200 µl of 2 mM HCl (Hussain
et al., 1992) for separation by HPLC on an Ultrasphere C18 column (5 µm, 25 cm × 4.6 mm i.d.). The mobile
phase was 0.05 M KH2PO4 (pH 2.9), acetonitrile
and triethylamine (60:38.8:0.2), the flow rate was 1.5 ml/min and the
detection wavelength was 238 nm (Ascalone et al., 1994).
Retention times were 3.1 min for N,N-didesmethyl DTZ and O-desmethyl
DTZ, 3.6 min for N-desmethyl DTZ, 5.1 min for deacetyl DTZ, 7.8 min for
DTZ and 11.6 min for imipramine (fig. 2).
|
) because this activity
is known to be catalyzed by a number of individual CYPs (Chang et
al., 1993). Reactions contained 0.4 mg of microsomal protein and
400 µM ethoxycoumarin.
In further experiments to assess the effect of DTZ or N-desmethyl DTZ
biotransformation on CYP3A4 activity, these agents were incubated with
NADPH-fortified microsomes before transfer to tubes containing
14C-testosterone. Reactions were then continued for the
usual 2.5 min, after which radioactive metabolites were extracted and
separated as before.
Data analysis and statistics.
Values are presented
throughout as mean ± S.E.M. of triplicate estimates; except where
stated otherwise. Differences between means of several treatments were
detected using analysis of variance and Dunnett's q
-test.
| |
Results |
|---|
|
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
|
|---|
Interindividual variation in DTZ oxidation in human liver. Microsomal fractions from 17 individual human livers were assessed in this study. Where available, the recent drug histories of the donors are also indicated in table 1. Three of the individuals were cigarette smokers. As shown in table 1, the individual variation in DTZ (100 µM) oxidation to its major metabolite N-desmethyl DTZ was considerable (range, 0.33-3.31 nmol/mg of protein/min; n = 17).
A kinetic analysis of DTZ N-demethylation was undertaken in three human hepatic fractions (HL27, HL30 and HL31). From Hanes-Woolf plots, the Michaelis constant (Km) for the N-demethylation reaction was calculated to be 23 ± 10 µM. Maximal reaction velocities (Vmax) for the reactions were also determined and found to be 5.36, 1.65 and 0.99 nmol/mg of protein/min for HL27, HL30 and HL31, respectively. The Hanes-Woolf plot for DTZ N-demethylation carried out in HL31 is shown in figure 3.
|
Correlation of DTZ N-demethylation with other microsomal
oxidations.
As shown in table 3, in
microsomal fractions from 17 individual livers, rates of DTZ
N-demethylation were well correlated with CYP3A4-mediated testosterone
6-hydroxylation and, to a lesser extent, CYP2C-mediated tolbutamide
hydroxylation (r = 0.82; P < .001; r = 0.59; P < .05, respectively, fig. 4). In contrast, correlations
between DTZ oxidation and aniline 4-hydroxylation, 7-ethylresorufin
O-deethylation and N,N-dimethylnitrosamine N-demethylation did not
attain statistical significance. In this subset of 17 microsomal
fractions, testosterone 6-hydroxylation (CYP3A4) and tolbutamide
hydroxylation (CYP2C) activities were correlated (r = 0.61; P < .01), as were the hydroxylations of tolbutamide and aniline (CYP2C
and CYP2E1, respectively; r = 0.65, P < .05, n = 14) and the oxidations of aniline and
N,N-dimethylnitrosamine N-demethylation; correlations between other
pairs of substrate oxidation activities were not significant. Due to
limitations of sample availability, aniline 4-hydroxylation and
N,N-dimethylnitrosamine N-demethylation activities were determined on
only 14 of the microsomal fractions.
|
Modulation of DTZ oxidation in human liver by chemical agents.
The modulatory effects of chemical substrates or inhibitors of specific
human CYP enzymes on DTZ N-demethylation were evaluated. It is evident
from the data in figure 5 that the most pronounced effects were produced by the inhibitors of CYP3A4. Thus, ketoconazole (25 µM) and troleandomycin (500 µM) decreased DTZ oxidation to ~10% of control activity; an IC50 value of 0.13 µM was
determined for ketoconazole. -Naphthoflavone, generally considered
to be a CYP1A2 inhibitor but also known to interact as an activator of
CYP3A4, decreased DTZ oxidation activity in microsomal fractions from
three individual livers to ~50% of control when incorporated into
incubations at a concentration of 100 µM (fig. 5). Tolbutamide (300 µM; 2C8/9 substrate), sulfaphenazole (50 µM; 2C9 inhibitor), debrisoquine sulfate (100 µM; 2D6 substrate) and acetaminophen (1 mM;
substrate for 1A2 and 2E1) were essentially without effect on
microsomal DTZ N-demethylation.
|
N-Demethylation of DTZ by cDNA-derived human CYPs.
To
investigate further the participation of human CYPs in DTZ
N-demethylation, the capacities of cDNA-derived CYPs to support the
reaction in lymphoblastoid cell microsomes were assessed (100 µM).
Thus, CYPs 3A4, 2C8 and 2C9 catalyzed DTZ oxidation to N-desmethyl DTZ.
CYP3A4 produced 9.92 pmol of product/hr/pmol of CYP, whereas 2C8 and
2C9 generated the metabolite at ~20% and ~10%, respectively, of
the rate exhibited by 3A4. CYPs 1A1, 1A2, 2A6, 2B6, 2D6, 2E1 and 4A11
exhibited little or no activity in DTZ N-demethylation (table 2).
7-Ethoxycoumarin O-deethylation activities catalyzed by nine of the
preparations were determined (range, 0.25-32 pmol/hr/pmol of P450)
because this reaction is mediated by a number of CYPs and can be used
for comparative purposes (Chang et al., 1993).
Inhibition of CYP3A4-dependent steroid 6-hydroxylation by DTZ
and its metabolites.
Of the four compounds tested, three
were found to be inhibitory. The parent drug DTZ exhibited an
IC50 value of 120 µM in microsomes (data obtained with
three separate fractions). N-Desmethyl DTZ and N,N-didesmethyl DTZ
were, respectively, 11- and 200-fold more potent (IC50
values of 11 and 0.6 µM) than DTZ as an inhibitor of CYP3A4 activity;
the O-demethylated metabolite of DTZ did not inhibit the activity. The
mechanism of inhibition of CYP3A4 activity by these two metabolites was
assessed in kinetic experiments. As shown in figures 6
and 7, both N-desmethyl DTZ and N,N-didesmethyl DTZ were
competitive inhibitors of CYP3A4-mediated testosterone 6-hydroxylation. Thus, Ki values for
N-desmethyl DTZ were 2.2 and 2.5 µM in microsomes from HL26 and HL27,
respectively, and the corresponding values for N,N-didesmethyl DTZ were
0.07 and 0.10 µM, respectively).
|
|
Potentiation of CYP3A4 inhibition during DTZ and N-desmethyl DTZ
biotransformation in human hepatic microsomes.
From the foregoing
experiments it was apparent that DTZ underwent CYP-mediated oxidation
to N-desmethyl DTZ and that this metabolite is an effective inhibitor
of CYP3A4 activity. Subsequent studies assessed the effect of DTZ
metabolism on the extent of CYP3A4 inhibition in microsomal
incubations. Thus, at a concentration of 100 µM, with preincubation
times up to 17.5 min, NADPH-supported microsomal DTZ biotransformation
enhanced the extent of inhibition of testosterone 6-hydroxylation
(fig. 8). Under such conditions, the concentration of
N-desmethyl DTZ formed during the course of the incubation was ~6.5
µM. Therefore, because N-desmethyl DTZ was an effective inhibitor of
testosterone 6-hydroxylation (IC50 = 12 µM), it is
feasible that this metabolite was responsible for the enhanced
inhibition of CYP3A4 activity.
|
-hydroxylation was
enhanced.
| |
Discussion |
|---|
|
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
|
|---|
The results of the present study implicate CYP3A4, and perhaps
other members of the CYP3A subfamily, as the major enzyme active in DTZ
N-demethylation in human liver. The supporting evidence can be
summarized as follows: (1) the correlation between rates of
CYP3A4-dependent testosterone 6-hydroxylation and DTZ
N-demethylation was highly significant, (2) the CYP3A4 inhibitors
ketoconazole and troleandomycin inhibited DTZ oxidation potently and
(3) of the 10 cDNA-derived CYPs in lymphoblastoid cell microsomes that were evaluated, CYP3A4 was the most active catalyst of DTZ
N-demethylation. A good correlation between CYP2C9-mediated tolbutamide
hydroxylation and DTZ N-demethylation was also observed in human
hepatic microsomes (although the correlation between testosterone
6-hydroxylation and tolbutamide hydroxylation was significant in the
subset of livers used in the present investigation). Notwithstanding
this point, cDNA-derived CYPs 2C8 and 2C9 supported DTZ oxidation in lymphoblastoid cell microsomes. Thus, there is some evidence that CYPs
from the 2C subfamily may participate in the oxidation of DTZ in liver,
but they may be minor catalysts of the reaction.
CYP3A protein is quantitatively significant in human liver.
Shimada et al. (1994) documented that 30% to 50% of total
CYP in human hepatic microsomes was immunoreactive with an anti-CYP3A IgG. It is also apparent from the recent literature that CYP3A4 is
involved in the enzymatic oxidation of an increasing list of drugs,
including midazolam (Kronbach et al., 1989), alfentanil (Yun
et al., 1992), lansoprazole (Pichard et al.,
1995) and docetaxel (Marre et al., 1996). CYP3A4 also
activates carcinogens and mutagens, like aflatoxin B1 and
sterigmatocystin (Shimada and Guengerich, 1989; Shimada et
al., 1989), to the toxic species. Certainly, impaired elimination
of drugs that are metabolized extensively by CYP3A4, resulting in their
accumulation in serum could lead to adverse effects, including enhanced
therapeutic effect.
The potential for DTZ to inhibit CYP-mediated drug oxidation has
been known for some time, and clinically significant pharmacokinetic interactions have been reported during concurrent drug therapy (Brodie
and Macphee, 1986). However, the specificity of the interaction of DTZ
with human CYPs has not been completely characterized. One of the major
findings from the present study is that DTZ is a selective inhibitor of
CYP3A4, which is involved in the enzymic oxidation of many therapeutic
agents. Thus, it was found that the 6-hydroxylation of testosterone
in human hepatic microsomes, a reaction characteristic of CYP3A4
(Waxman et al., 1988), was inhibited by DTZ
(IC50 = 120 µM). The relative affinity of CYP3A4 for DTZ
and testosterone is reflected by the respective
Km values of 23 µM (present study; fig. 3) and
55 µM (present study; data not shown). In a previous study, we
determined a Km value of 94 µM for
testosterone 6-hydroxylation in human liver (Murray et al., 1994). These data suggest that testosterone has a somewhat lower affinity than DTZ for CYP3A4 and therefore are consistent with
the inhibition of CYP3A4 that was observed in the present study. In
contrast, 7-ethylresorufin O-deethylation, catalyzed by CYP1A2
(Tassaneeyakul et al., 1993); tolbutamide 4-hydroxylation, catalyzed by CYP2C9 (Knodell et al., 1987); and aniline
4-hydroxylation, catalyzed principally by CYP2E1 (Wrighton and Stevens,
1992), were refractory to inhibition by DTZ.
The administration of DTZ by multiple dosage regimen leads to a
prolongation of the half-life of DTZ (Montamat and Abernethy, 1987).
Accordingly, the present study also evaluated the role of DTZ
metabolites in CYP inhibition. Of the three DTZ metabolites examined,
N-desmethyl and N,N-didesmethyl DTZ were 11- and 200-fold, respectively, more potent than DTZ as inhibitors of CYP3A4; O-desmethyl DTZ was not inhibitory. The selectivity of inhibition by these metabolites was similar to that of the parent compound. Thus, neither
N-desmethyl nor N,N-didesmethyl DTZ inhibited activities associated
with CYPs 1A2, 2E1 or 2C9. The mechanism by which the two metabolites
modulate CYP3A4 activity was explored in the present study using a
kinetic approach. It was evident that both metabolites were competitive
inhibitors of CYP3A4 activity: N-desmethyl exhibited a
Ki value of ~2 µM, and the N,N-didesmethyl
analog exhibited a Ki value of ~0.1 µM.
Thus, it is clear that both metabolites, especially the N,N-didesmethyl
compound, have high capacity to interact with the enzyme. Indeed, from
a comparison of these Ki values with the
Km value for testosterone 6-hydroxylation, it appears that CYP3A4 has ~25- and ~500-fold greater affinity for the
N-desmethyl and N,N-didesmethyl metabolites than for the substrate. Thus, these in vitro kinetic data provide a basis for
interpretation of the considerable capacity of the metabolites to
inhibit CYP3A4 activity in vivo. It should be borne in mind
that the inhibition of CYP3A4 by DTZ metabolites, although quite
potent, is reversible and should be distinguished from processes such
as metabolite-intermediate complexation and mechanism-based
inactivation. Inhibition processes of that type are characterized by
the generation of destructive metabolites that can produce long-term
inhibition of CYP enzymes (Murray and Reidy, 1990). However, the
formation of drug metabolites that are potent reversible CYP inhibitors
may be a more widespread phenomenon. This possibility requires
continuing evaluation in further studies.
Preincubation of DTZ or N-desmethyl DTZ with NADPH-fortified
microsomes significantly increased the extent of inhibition of testosterone 6-hydroxylation. N-Desmethyl DTZ concentrations of
~6.5 µM were detected and appeared to be increasing in a linear fashion (not shown); such concentrations of N-desmethyl DTZ are sufficient to produce substantial inhibition of CYP3A4. The more potent
N,N-didesmethyl metabolite was also detected in incubations containing
N-desmethyl DTZ. It is therefore possible that the N,N-didesmethyl DTZ
detected in serum in previous studies (Sugawara et al.,
1988) may be formed in liver from its precursor N-desmethyl DTZ.
In summary, this study has found that of the four major human CYPs, only CYP3A4 is inhibited by DTZ and its major hepatic metabolites in vitro. The other major CYPs, 1A2, 2E1 and 2C9, are not affected by DTZ, so pharmacokinetic interactions between DTZ and drugs metabolized by these CYPs are unlikely. In vitro biotransformation of DTZ by CYP3A4 produces N-demethylated metabolites that are potent inhibitors of the activity of the enzyme. These metabolites are likely to be responsible for the impaired elimination of DTZ under multiple dosing regimen in vivo.
| |
Acknowledgments |
|---|
The assistance of the Australian and Queensland Liver Transplant Programs in obtaining human liver is gratefully acknowledged. We thank Tanabe Seiyaku Co. (Osaka, Japan) for their generous gifts of authentic DTZ metabolites and Dr. H. Smith (ICI, Australia) for assistance in obtaining these compounds.
| |
Footnotes |
|---|
Accepted for publication March 20, 1997.
Received for publication November 15, 1996.
1 This work was supported by a grant from the National Health and Medical Research Council of Australia.
Send reprint requests to: Dr. Michael Murray, Department of Medicine, Westmead Hospital, Westmead, NSW 2145, Australia.
| |
Abbreviations |
|---|
DTZ, diltiazem; HPLC, high pressure liquid chromatography; CYP, cytochrome P450; TLC, thin-layer chromatography.
| |
References |
|---|
|
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
|
|---|
-hydroxylation activity by cyclophosphamide and ifosfamide in vitro.
J. Pharmacol. Exp. Ther.
270: 645-649, 1994-hydroxylase cytochrome P-450 enzyme.
Arch. Biochem. Biophys.
263: 424-436, 1988[Medline].This article has been cited by other articles:
|
|
 |
|
  P. Zhao, C. A. Lee, and K. L. Kunze Sequential Metabolism Is Responsible for Diltiazem-Induced Time-Dependent Loss of CYP3A Drug Metab. Dispos., May 1, 2007; 35(5): 704 - 712. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M Iwase, N Kurata, R Ehana, Y Nishimura, T Masamoto, and H Yasuhara Evaluation of the effects of hydrophilic organic solvents on CYP3A-mediated drug-drug interaction in vitro Human and Experimental Toxicology, December 1, 2006; 25(12): 715 - 721. [Abstract] [PDF]
|
|
|
|
 |
|
 M. Jerling, B.-L. Huan, K. Leung, N. Chu, H. Abdallah, and Z. Hussein Studies to Investigate the Pharmacokinetic Interactions Between Ranolazine and Ketoconazole, Diltiazem, or Simvastatin During Combined Administration in Healthy Subjects J. Clin. Pharmacol., April 1, 2005; 45(4): 422 - 433. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
E. Molden, A. Asberg, and H. Christensen Desacetyl-Diltiazem Displays Severalfold Higher Affinity to CYP2D6 Compared with CYP3A4 Drug Metab. Dispos., January 1, 2002; 30(1): 1 - 3. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. Kosuge, Y. Jun, H. Watanabe, M. Kimura, M. Nishimoto, T. Ishizaki, and K. Ohashi Effects of CYP3A4 Inhibition by Diltiazem on Pharmacokinetics and Dynamics of Diazepam in Relation to CYP2C19 Genotype Status Drug Metab. Dispos., October 1, 2001; 29(10): 1284 - 1289. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y. Naritomi, S. Terashita, S. Kimura, A. Suzuki, A. Kagayama, and Y. Sugiyama Prediction of Human Hepatic Clearance from in Vivo Animal Experiments and in Vitro Metabolic Studies with Liver Microsomes from Animals and Humans Drug Metab. Dispos., October 1, 2001; 29(10): 1316 - 1324. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. F. McGinnity, A. J. Parker, M. Soars, and R. J. Riley Automated Definition of the Enzymology of Drug Oxidation by the Major Human Drug Metabolizing Cytochrome P450s Drug Metab. Dispos., November 1, 2000; 28(11): 1327 - 1334. [Abstract] [Full Text]
|
|
|
|
 |
|
 J.R. Cockcroft and I.B. Wilkinson Cholesterol reduction, statins and the cytochrome P-450 system Eur. Heart J., September 2, 2000; 21(18): 1555 - 1556. [PDF]
|
|
|
|
|
|
E. Molden, H. Christensen, and R. B. Sund Extensive Metabolism of Diltiazem and P-Glycoprotein-Mediated Efflux of Desacetyl-Diltiazem (M1) by Rat Jejunum In Vitro Drug Metab. Dispos., February 1, 2000; 28(2): 107 - 109. [Abstract] [Full Text]
|
|
|
|
|
|
B. Ma, T. Prueksaritanont, and J. H. Lin Drug Interactions with Calcium Channel Blockers: Possible Involvement of Metabolite-Intermediate Complexation with CYP3A Drug Metab. Dispos., February 1, 2000; 28(2): 125 - 130. [Abstract] [Full Text]
|
|
|
|
|
|
R. S. Obach Prediction of Human Clearance of Twenty-Nine Drugs from Hepatic Microsomal Intrinsic Clearance Data: An Examination of In Vitro Half-Life Approach and Nonspecific Binding to Microsomes Drug Metab. Dispos., November 1, 1999; 27(11): 1350 - 1359. [Abstract] [Full Text]
|
|
|
|
 |
|
 S. Ekins, G. Bravi, J. H. Wikel, and S. A. Wrighton Three-Dimensional-Quantitative Structure Activity Relationship Analysis of Cytochrome P-450 3A4 Substrates J. Pharmacol. Exp. Ther., October 1, 1999; 291(1): 424 - 433. [Abstract] [Full Text]
|
|
|
|
|
|
S. Ekins, G. Bravi, S. Binkley, J. S. Gillespie, B. J. Ring, J. H. Wikel, and S. A Wrighton Three- and Four-Dimensional Quantitative Structure Activity Relationship Analyses of Cytochrome P-450 3A4 Inhibitors J. Pharmacol. Exp. Ther., July 1, 1999; 290(1): 429 - 438. [Abstract] [Full Text]
|
|
|
|
|
|
M. A. Gibbs, K. L. Kunze, W. N. Howald, and K. E. Thummel Effect of Inhibitor Depletion on Inhibitory Potency: Tight Binding Inhibition of CYP3A by Clotrimazole Drug Metab. Dispos., May 1, 1999; 27(5): 596 - 599. [Abstract] [Full Text]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
, 10 µM; 
, 20 µM; 
, 50 µM; 
, 100 µM; 
, 200 µM.
B, Dixon plot slopes vs. reciprocal testosterone
concentrations. Units are substrate and inhibitor concentrations, µM;
V, nmol/min/mg of protein.
